Резюме книги для 27th Конгресс Европейской ассоциации гематологии

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Presidential Symposium

S100: QUIZARTINIB PROLONGED SURVIVAL VS PLACEBO PLUS INTENSIVE INDUCTION AND CONSOLIDATION THERAPY FOLLOWED BY SINGLE-AGENT CONTINUATION IN PATIENTS AGED 18-75 YEARS WITH NEWLY DIAGNOSED FLT3-ITD+ AML

H. Erba 1,* , P. Montesinos 2 , R. Vrhovac 3 , E. Patkowska 4 , H.-J. Kim 5 , P. Zak 6 , P.-N. Wang 7 , T. Mitov 8 , J. Hanyok 9 , L. Liu 9 , A. Benzohra 9 , A. Lesegretain 9 , J. Cortes 10 , A. Perl 11 , M. Sekeres 12 , H. Dombret 13 , S. Amadori 14 , J. Wang 15 , M. Levis 16 , R. Schlenk 17

1 Duke Cancer Institute, Durham, NC, United States of America; 2 La Fe University and Polytechnic Hospital, Valencia, Spain; 3 University Hospital Centre Zagreb, Zagreb, Croatia; 4 Institute of Hematology and Blood Transfusion, Warsaw, Poland; 5 The Catholic University of Korea, Seoul St. Mary’s Hospital, Seoul, South Korea; 6 University Hospital Hradec Kralove, Hradec Kralove, Czechia; 7 Chang Gung Medical Foundation, Linkou, Taiwan; 8 Daiichi Sankyo UK Ltd, Uxbridge, United Kingdom; 9 Daiichi Sankyo, Inc, Basking Ridge, NJ; 10 Augusta University Medical Center, Augusta, GA; 11 University of Pennsylvania, Philadelphia, PA; 12 University of Miami Health System, Miami, FL, United States of America; 13 Saint Louis Hospital, University of Paris, Paris, France; 14 Tor Vergata Polyclinic Hospital Rome, Rome, Italy; 15 Institute of Hematology and Blood Diseases Hospital, Tianjin, China; 16 Johns Hopkins University, Baltimore, MD, United States of America; 17 Heidelberg University Hospital and German Cancer Research Center, Heidelberg, Germany

Background: Quizartinib (Quiz) is an oral, highly potent, and selective type II FLT3 inhibitor with single-agent activity in relapsed/refractory FLT3 –internal tandem duplication positive ( FLT3 -ITD+) acute myeloid leukemia (AML). This is the first report of the global, randomized, double-blind, placebo (PBO)-controlled phase 3 QuANTUM-First trial ( NCT02668653 ).

Aims: QuANTUM-First aimed to determine if the addition of Quiz to standard induction (IND) and post remission (including allogeneic hematopoietic cell transplant [allo-HCT]) in first complete remission [CR1]) consolidation followed by single-agent continuation therapy for up to 3 years improved survival compared with chemotherapy alone in patients (pts) with newly diagnosed FLT3 -ITD+ AML.

Methods: Pts aged 18-75 y with newly diagnosed AML were centrally screened for FLT3 -ITD prior to initiation of IND with cytarabine 100 mg/m 2 /day (200 mg/m 2 /day if institutional standard) for 7 days and anthracycline (daunorubicin 60 mg/m 2 /day or idarubicin 12 mg/m 2 /day) for 3 days. Pts at 193 sites in 26 countries who were FLT3 -ITD+ provided informed consent and were randomized to Quiz (40 mg/day days 8-21) or PBO and were stratified by region (North America, Europe, and Asia/Other regions), age (<60 y, ≥60 y), and white blood cell count (<40×10 9 /L, ≥40×10 9 /L) at diagnosis. A second IND was allowed if residual AML was noted at the post-IND marrow exam. Pts who achieved CR or CR with incomplete hematologic recovery (CRi) received up to 4 cycles of high-dose cytarabine plus Quiz (40 mg/day) or PBO and/or allo-HCT followed by up to 3 y of continuation therapy with Quiz (30-60 mg/day) or PBO. The primary endpoint was overall survival (OS).

Results: Between September 2016 and August 2019, 3468 pts were screened, and 539 pts with FLT3 -ITD+ AML were randomized to Quiz (n=268) or PBO (n=271). The median age was 56 y (range, 20-75 y). Baseline pt and disease characteristics, including FLT3 -ITD variant allele frequency, were balanced between the 2 arms. At data cutoff (August 2021), the median follow-up was 39.2 months and 58 pts remained on continuation therapy. OS was significantly longer in the Quiz arm than the PBO arm (hazard ratio [HR], 0.776; 95% CI, 0.615-0.979; 2-sided P =.0324). Median OS was 31.9 mo with Quiz vs 15.1 mo with PBO (Figure). CR/CRi rates were 71.6% and 64.9%, respectively. Allo-HCT in CR1 was performed in 157 pts (Quiz, 31%; PBO, 27%). When censored for allo-HCT, OS trended longer with Quiz vs PBO (HR, 0.752; 95% CI, 0.562-1.008; 2-sided P =0.055). Relapse-free survival was longer with Quiz than PBO (HR, 0.733; 95% CI, 0.554-0.969). Although rates of grade ≥3 adverse events (AEs) were similar across arms, grade ≥3 neutropenia was more frequent in the Quiz arm (18.1% vs 8.6%). Discontinuations due to AEs occurred in 20.4% of Quiz and 8.6% of PBO pts. A total of 56 treatment-emergent AEs were associated with a fatal outcome (Quiz, 11.3%; PBO, 9.7%), mostly due to infections. Grade 3/4 electrocardiogram QT prolonged occurred in 3.0% of Quiz vs 1.1% of PBO pts.

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Summary/Conclusion: These pivotal findings show that the addition of Quiz to standard chemotherapy and up to 3 years of continuation therapy yielded statistically significant and clinically meaningful improvements to OS in adults with newly diagnosed FLT3 -ITD+ AML up to age 75 y. The manageable safety profile further supports use of Quiz in combination with standard therapy, including allo-HCT, in FLT3 -ITD+ AML.

S101: GENETIC AND EPIGENETIC FACTORS DRIVING PRIMARY MEDIASTINAL B-CELL LYMPHOMA PATHOGENESIS AND OUTCOME

D. Noerenberg 1,* , F. Briest 1 , C. Hennch 1 , K. Yoshida 2,3 , J. Nimo 1 , R. Hablesreiter 1 , Y. Takeuchi 2 , D. Sasca 4 , H. Ueno 2 , L. Mansouri 5 , Y. Inoue 2 , L. Wiegand 1 , A. M. Staiger 6,7 , B. Casadei 8,9 , M. Ziepert 10 , F. Asmar 11 , P. Korkolopoulou 12 , M. Kirchner 13 , P. Mertins 13 , J. Weiner 14 , E. Toth 15 , T. Weber 16 , A. Warth 17 , T. Schneider 18 , R.-M. Amini 19 , W. Klapper 20 , M. Hummel 21,22 , V. Poeschel 23 , G. Kanellis 24 , A. Rosenwald 25 , G. Held 23,26 , E. Campo 27,28,29 , K. Stamatopoulos 5,30 , I. Anagnostopoulos 21,25 , L. Bullinger 1,22 , N. Goldschmidt 31 , P. L. Zinzani 9,32 , C. Bödor 33 , R. Rosenquist 5,34 , T. P. Vassilakopoulos 35 , G. Ott 6 , S. Ogawa 2,36,37 , F. Damm 1,22

1 Department of Hematology, Oncology and Cancer Immunology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany; 2 Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3 Wellcome Trust Sanger Institute, Hinxton, United Kingdom; 4 Department of Hematology, Oncology, and Pulmonary Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany; 5 Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 6 Department of Clinical Pathology, Robert-Bosch-Krankenhaus; 7 Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology Stuttgart, and University of Tuebingen, Stuttgart, Germany; 8 Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna; 9 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy; 10 Institute of Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany; 11 Department of Hematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 12 First Department of Pathology, National and Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece; 13 Core Unit Proteomics, Berlin Institute of Health, Charité - Universitätsmedizin Berlin and Max-Delbrück-Center for Molecular Medicine; 14 Core Unit Bioinformatics, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany; 15 National Institute of Oncology, Budapest, Hungary; 16 Department of Internal Medicine IV, Haematology and Oncology, University Hospital Halle (Saale), Martin-Luther-University Halle-Wittenberg, Halle; 17 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; 18 National Institute of Oncology, Budapest, Hungary; 19 Department of Immunology, Genetics and Pathology, Uppsala University and University Hospital, Uppsala, Sweden; 20 Department of Pathology, Hematopathology Section and Lymph Node Registry, Universitätsklinikum Schleswig-Holstein, Kiel; 21 Department of Pathology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin; 22 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg; 23 Department of Internal Medicine 1 (Oncology, Hematology, Clinical Immunology, and Rheumatology), Saarland University Medical School, Homburg/Saar, Germany; 24 Department of Hematopathology, Evangelismos General Hospital, Athens, Greece; 25 Institute of Pathology, University of Würzburg and Comprehensive Cancer Center (CCC) Mainfranken, Würzburg; 26 Department Internal Medicine I, Westpfalzklinikum Kaiserslautern, Kaiserslautern, Germany; 27 Centro de Investigacion Biomedica en Red en Oncologia (CIBERONC), Madrid; 28 Hospital Clinic of Barcelona, University of Barcelona; 29 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 30 Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 31 Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 32 Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 33 HCEMM-SE Momentum Molecular Oncohematology Research Group, 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary; 34 Clinical Genetics, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden; 35 Department of Hematology and Bone Marrow Transplantation, National and Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece; 36 Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan; 37 Department of Medicine, Centre for Haematology and Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden

Background: Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive lymphoma affecting predominantly young female patients. Previous studies in this rare entity have focused on single genes or were limited in cohort size.

Aims: To unravel the underlying genetic pathogenesis and its impact on outcome, we embarked on a comprehensive large-scale genetic investigation.

Methods: Specimens of 486 previously untreated PMBCL patients were analyzed by paired tumor/normal whole-genome (WGS, n=14), whole-exome (WES, n=78) and targeted sequencing (TS, n=486). To understand the consequences of highly recurrent mutations in the chromatin-modifying gene ZNF217 , we conducted functional and multi-omics analyses in CRISPR/Cas9 engineered cell lines.

Results: WGS/WES revealed a complex genomic landscape in PMBCL with a median of 85 structural variants, a mutational burden of 5 mutations/Mb, 12 mutated coding candidate driver genes (CDG) and 4 focal somatic copy-number aberrations per sample (Fig.1 a ). Besides known targets, significant breakpoints were identified in genes previously not implicated in B-lymphomagenesis such as TOX and TP73 (36% and 21%). In addition, non-coding mutations clustered within the PAX5 enhancer region. With the identification of 50 recurrently mutated CDGs, we significantly expand the repertoire of known PMBCL drivers. The 10 most frequently mutated CDGs were SOCS1 (86%), B2M (67%), ITPKB (64%), ACTB (58%), STAT6 (58%), IGLL5 (56%), TNFAIP3 (53%), NFKBIE (49%), GNA13 (47% ), and ZNF217 (36% ), respectively. The operative mutational processes were attributed to aging, AID/APOBEC activity, defective MMR, and an unexpected infidelity of DNA-Polymerase ε. Next, we performed TS in 486 samples using a PMBCL-specific 106-gene panel. Recurrent lesions in 25 epigenetic modifiers were found in >90%, with ZNF217 being among the most frequently mutated genes (Fig.1b). After knockdown of ZNF217 in Karpas1106P and L428 cells, we demonstrated altered proliferation, migration, and apoptosis. Using mass spectrometry, we showed that ZNF217 is acting in a LSD1, CoREST and HDAC containing histone modifier complex. Accordingly, knockout of ZNF217 led to global changes in chromatin accessibility with an enrichment of differentially accessible motifs for crucial lymphoma-associated transcription factors, especially of the NF-κB, BATF/AP1, and IRF family, but also of CTCF, a major regulator of global 3D chromatin architecture. Resulting gene expression was characterized by changes in interferon-responsive genes and inflammation-associated transcription (Fig 1 c ). Clinical data were available for 329 cases, including 84 cases from clinical trials. Multivariate analysis using an IPI-corrected Cox regression model was performed. The estimated 5-year PFS and OS were 77% and 86%. Among the genetic lesions with the strongest association for poor outcome, we identified patients with mutated CD58 having a significantly shorter survival (PFS: HR 2.96; p<.001; OS: HR 2.55; p=.006). In contrast, mutated DUSP2 indicated longer survival (PFS: HR 0.28; p=.002; OS: HR 0.15; p=.011) (Fig1 d ). Notably, DUSP2 mutated patients (25%) showed a similar outcome for CR rate, PFS and OS when comparing CHOP-like and intensified treatment regimens, suggesting no further benefit from treatment intensification in this very-low risk patient population.

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Summary/Conclusion: Here, we present the genetic landscape of PMBCL highlighting a previously underappreciated role of chromatin modifying genes, identify novel treatment targets and provide a solid basis for guiding precision medicine approaches.

S102: COMPREHENSIVE GENOME CHARACTERIZATION REVEALS NEW SUBTYPES AND MECHANISMS OF ONCOGENE DEREGULATION IN CHILDHOOD T-ALL

P. Pölönen 1,* , A. Elsayed 1,2 , L. Montefiori 1 , S. Kimura 1 , J. Myers 3 , D. Hedges 3 , J. Xu 4 , Y. Hui 3 , Z. Cheng 3 , Y. Fan 3 , Y. Chang 1 , R. Shraim 5 , M. Devidas 6 , S. Winter 7 , K. Dunsmore 8 , J. J. Yang 9 , T. L. Vincent 10 , K. Tan 10,11,12,13,14 , C. Chen 10 , H. Newman 15 , M. Loh 16 , E. Raetz 17 , S. P. Hunger 18 , E. Rampersaud 3 , T.-C. Chang 3 , G. Wu 3 , S. Pounds 2 , C. G. Mullighan 1,19 , D. T. Teachey 5,12,20

1 Pathology; 2 Biostatistics; 3 Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis; 4 Perelman School of Medicine at the University of Pennsylvania; 5 Department of Pediatrics and the Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia; 6 Global Pediatric Medicine, St. Jude Children’s Research Hospital, Memphis; 7 Minnesota Research Institute and Cancer and Blood Disorders Program, Children’s Minnesota Research Institute, Minneapolis; 8 University of Virginia Children’s Hospital, Charlottesville; 9 Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis; 10 Division of Oncology and Center for Childhood Cancer Research; 11 Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia; 12 Perelman School of Medicine; 13 Institute for regenerative medicine; 14 Penn Epigenetics Institute, University of Pennsylvania; 15 Division of Oncology and Center for Childhood Cancer, Children’s Hospital of Philadelphia, Philadelphia; 16 Department of Pediatrics, Benioff Children’s Hospital and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco; 17 Department of Pediatrics and Perlmutter Cancer Center, NYU Langone Health, New York; 18 Department of Pediatrics and the Center for Childhood Cancer Research, Children’s Hospital of Philadelphia and The Perelman School of Medicine at The University of Pennsylvania, Philadelphia; 19 Hematological Malignancies Program, St. Jude Children’s Research Hospital, Memphis; 20 Divisions of Hematology and Oncology, Children’s Hospital of Philadelphia, Philadelphia, United States of America

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy including leukemias of early T cell precursor acute lymphoblastic leukemia (ETP-ALL) and transformed thymocytes. Prior genomic studies of T-ALL had limited cohort size, excluded refractory disease and focused on alterations in coding parts of the genome.

Aims: Investigate the genomic basis of T-ALL by identifying both coding and non-coding alterations and defining T-ALL subtypes.

Methods: We performed whole-genome sequencing (WGS), whole-exome sequencing (WES), RNA-sequencing of 1,313 cases enrolled on the Children’s Oncology Group AALL0434 trial.

Results: Uniform Manifold Approximation and Projection (UMAP) and gene expression clustering analyses of RNA-seq data identified 16 subtypes, of which 4 have not been reported previously. Furthermore, we could divide existing subtypes into smaller subgroups with common subtype-defining alterations, such as structural variation (SV) and copy number variation (CNV). TLX1 activation was linked with TLX1-TCR SVs and deletions in the TLX1 chromosomal domain, whereas TLX3 deregulation was associated with TLX3-BCL11B enhancer hijacking, but also through TLX3-TCR, TLX3-CDK6 rearrangements. We discovered two separate NKX2-1 deregulated groups, one characterized by TCR rearrangements and RPL10 mutations and the other by NKX2-1 CNVs, chromosome 14 chromothripsis, or MYB-TCR rearrangements. We also observed a distinct group of 9 cases aged 1-2 years, with recurrent STAG2-LMO2 rearrangements and a patient with inactivating STAG2 mutation and CELF1 enhancer hijacking by LMO2 . Moreover, we found a group of 22 cases that were highly enriched for ETP-ALL with the following hallmark lesions: BCL11B enhancer amplification, BCL11B locus SVs involving enhancer hijacking of ARID1B, CCDC26, and novel CD34+ enhancer hijacking of lincRNA locus in chromosome 6.

Gene expression-based clustering was unable to stratify patients based on TAL1, TAL2, LMO1, LMO2, LYL1 expression, and their respective activation mechanisms. However, WGS enabled further characterization of these patients, by identifying several types of activation mechanisms and co-occurring alterations for each oncogene. We identified canonical events, such as TAL1-STIL fusions, TCR rearrangements, and activation by TAL1/LMO1/LMO2 regulatory region indels, but also novel events, such as CD34 specific enhancer duplications downstream of TAL1 and BCL11B enhancer hijacking by LMO2 . We also observed two smaller clusters with TAL1/LMO2 activation, where one with 39 cases was associated with TAL1/LMO2 activation and RPL10 mutations and the other with 8 cases had refractory disease (Day 29 MRD >5%). HOXA gene expression-associated subtypes were defined by fusions involving KMT2A or MLLT10 and PICALM/DDX3X or HOXA9-TCR SVs. Furthermore, our analysis revealed segregation of patients by HOXA13 or ZFP36L2 rearrangements and NUP98/NUP214 fusions. Interestingly, HOXA locus breakpoints involving HOXA13 and HOXA9 were in different chromatin compartments and were associated with mutually exclusive activation of either HOXA13 or other HOXA genes. Interestingly, HOXA13 -deregulation was associated with ETP-ALL and frequent BCL11B enhancer hijacking, whereas HOXA9 breakpoints typically involved TCRg and were non-ETP.

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Summary/Conclusion: Large-scale analysis of all children enrolled on the AALL0434 study has identified new subtype-defining lesions in T-ALL, including candidate novel enhancer hijacking events and enhancer duplications that are likely to result in oncogene deregulation in T-ALL.

S103: EFFICACY AND SAFETY OF ARI0002H, AN ACADEMIC BCMA-DIRECTED CAR-T CELL THERAPY WITH FRACTIONATED INITIAL THERAPY AND BOOSTER DOSE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

C. Fernandez De Larrea 1,* , V. González-Calle 2 , A. Oliver-Caldés 1 , V. Cabañas 3 , P. Rodríguez-Otero 4 , M. Español-Rego 1 , J. L. Reguera 5 , L. López-Corral 2 , B. Martin-Antonio 1 , B. Paiva 4 , S. Inogés 4 , L. Rosiñol 1 , A. López-Díaz de Cerio 4 , N. Tovar 1 , M. López-Parra 2 , L. G. Rodríguez-Lobato 1 , A. Sánchez-Salinas 3 , S. Varea 1 , V. Ortiz-Maldonado 1 , J. A. Pérez Simón 5 , F. Prósper 4 , M. Juan 1 , J. M. Moraleda 3 , M. V. Mateos 2 , M. Pascal 1 , A. Urbano-Ispizua 1

1 Hospital Clínic de Barcelona. IDIBAPS. University of Barcelona, Barcelona; 2 Hospital Universitario de Salamanca, Instituto de Investigacion Biomedica de Salamanca (IBSAL), Centro de Investigación del Cancer (IBMCC-USAL, CSIC), Salamanca; 3 Hospital Clínico Universitario Virgen de la Arrixaca. IMIB-Arrixaca. University of Murcia, Murcia; 4 Clínica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA), IDISNA, CIBERONC Pamplona, Pamplona; 5 Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS/CSIC/CIBERONC), University of Sevilla, Sevilla, Spain

Background: ARI0002h is a lentiviral autologous CAR T-cell product with a 4-1BB co-stimulatory domain and a humanized single chain variable fragment targeting BCMA. In pre-clinical studies, this academic CAR-T has demonstrated potent in vitro and in vivo activity.

Aims: We report the safety and efficacy results of the CARTBCMA-HCB-01 multicenter clinical trial for patients with relapsed/refractory multiple myeloma (RRMM) ( NCT04309981 ) who received ARI0002h in 5 Spanish centers.

Methods: Patients (pts) aged 18-75 years old with RRMM were eligible for this study if they had measurable disease, received ≥2 prior regimens, including a proteasome inhibitor, an immunomodulatory drug and an anti-CD38 antibody, and were refractory to the last line of treatment. Bridging therapy was allowed after apheresis. Cyclophosphamide (300 mg/m 2 ) and fludarabine (30 mg/m 2 ) were used as lymphodepletion regimen. The targeted dose was 3x10 6 /kg CAR+ cells and was administered in a fractionated manner (10%/30%/60%), with at least 24h between infusions. A second dose of 3x10 6 CAR+ cells/kg was planned at least 4 months after the first dose in pts who achieved any grade of response any response and had not or serious complications after the first administration. Primary objectives were overall response rate (ORR; at least partial response -PR-) within 3 months of the first infusion and rate of cytokine release syndrome (CRS) and/or neurological toxicity in the first 30 days. Response was assessed as per IMWG criteria and bone marrow minimal residual disease (MRD) was analyzed by next-generation flow (NGF).

Results: As of February 9 th 2022, 35 pts (median age 61 years) with RRMM were included in the trial. Four pts could not receive ARI-0002h due to MM progression and one died of infection. Therefore, 30 pts received ARI0002h cells (modified intention-to-treat population), of which 47% received bridging therapy. Median CAR-T cell production time was 11 days (range 9-14) with a 100% manufacture success.

Median follow-up after ARI0002h administration for surviving pts was 16 months. The ORR of 30 evaluable pts was 100%, with a stringent complete remission (sCR) plus very good partial response (VGPR) rate of 90%. Median time to first response was one month. Of 26 MRD-evaluable pts at day +100, 92% were MRD-negative in bone marrow by NGF. 53% of patients were alive and without progression at 16 months. Median overall survival (OS) was not reached and the 16-month OS rate was 80% (Figure 1).

AEs reported in >70% of pts were CRS (87%; grade [gr] 3/4 0%; gr 1 73%), neutropenia (97%; gr 3/4 100%), anemia (85%; gr 3/4 43%), and thrombocytopenia (79%; gr 3/4 70%). Median duration of CRS was 4 days (range 1-12). No CAR-T cell-related neurotoxicity cases were reported. Tocilizumab and corticosteroids were administered in 76% (mainly for persistent grade 1 CRS) and 12% of pts, respectively.

ARI0002h cells demonstrated peak expansion on day 14 (range 7 days-6 months). 24 out of 28 eligible pts (86%) received the second dose (range 1.2-3x10 6 CAR+ cells/kg). Median time after first infusion was 4 months and 38% received a second lymphodepletion regimen. No relevant toxicities after second infusions were reported. 7 pts (29%) improved their response after reinfusion.

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Summary/Conclusion: ARI0002h is the first European academic CART for RRMM that has demonstrated deep and durable responses and a favorable safety profile, including the absence of neurotoxicity and the feasibility of a second booster dose.

S104: RBPS DYSREGULATION CAUSE HYPER-NUCLEOLI AND RIBOSOME GAIN-OF-FUNCTION DRIVING BONE MARROW FAILURE

P. Aguilar-Garrido 1,* , M. Velasco 1 , M. Hernández Sánchez 2 , M. Á. Navarro Aguadero 1 , P. Malaney 3 , M. JL Aitken 3 , X. Zhang 3 , K. H Young 3 , R. Duan 3 , P. Hu 3 , S. Kornblau 3 , A. Fernández 1 , A. Ortiz 1 , Á. Otero-Sobrino 1 , P. J. de Andrés 2 , D. Megías 1 , M. Pérez 1 , J. Gómez 1 , G. Mata 1 , J. Martínez López 1 , S. Post 3 , M. Gallardo 1

1 CNIO; 2 UCM, Madrid, Spain; 3 MD Anderson, Houston, United States of America

Background: Nucleoli and ribosome cross-talk regulates cell translation capacity. Its dysregulation and impairment drive ribosome and nucleolus stress (NS), related to the biological mechanism of cancer. Ribosomopathies are a group of diseases characterized by ribosome defects leading to complex syndromes that include bone marrow failure.

hnRNP K is an RNA binding protein (RBP) in charge of processing nascent RNAs (nucleoli) into mature mRNAs (ribosome). Our research found a novel ribosome gain-of-function ribosomopathy phenotype by hyper-nucleoli generation due RBP hnRNP K dysregulation.

Aims: We aim to elucidate how hnRNP K dysregulation impact on haematopoietic stem cell biology.

Methods: Hnrnpk overexpression was established in MEFs using CRISPR/SAM (Konermann S. et al, Nature). Global protein synthesis was tested using a Click-iT OPP and proteasome function was evaluated by Proteasome 20S activity NS hallmarks were analyzed by confocal microscopy evaluating Ncl, NS sensor marker. To evoke NS in our cells, we used Actinomycin D insult. Cell cycle FACS analysis (DAPI) and senescence assays (β-galactosidase staining) were performed. Molecular mechanism underlying was elucidated by qRT-PCR and WB (p21, p16, c-Myc, and mTor). To study the impact of hnRNP K overexpression in vivo , we developed an inducible tamoxifen mouse model activated 30-60 days after birth ( Hnrnpk Tg-Ubc-creERT2 ). Survival was evaluated by Kaplan-Meier, and phenotype described by symptoms, signs, CBC and bone marrow IHC panel (CD34, Gr1, B220, MPO).

Results: Hnrnpk overexpressing cells led to an increment in protein and gene expression of Ncl, mTor and c-Myc (Figure 1A-B). Moreover, we found an increase in global protein synthesis (Figure 2C-D). Nevertheless, the elevation of hnRNP K inversely correlated with the proteasome function, which dropped significantly (Figure 1E). NS induction promoted higher hnRNP K expression. Additionally, hnRNP K overexpressing cells showed NS hallmarks associated with an increase of the number of nucleoli, and total area of the nucleoli and nucleus (Figure 1F-G). Cell cycle analysis confirmed an increment of arrested G2/M phase cells (Figure 1H-I), linked to an increment in p21 and p16 levels all leading towards a senescent cell phenotype (Figure 1J-L).

Hnrnpk Tg-Ubc-creERT2 mice had widespread Hnrnpk overexpression (Figure 1M) and a reduction in lifespan (Figure 1N), mainly due to dysplastic and bone marrow failure phenotype, with dramatic reduction of CD34 and b-cells, leukopenia, anaemia and thrombocytopenia. (Figure 1O-Q).

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Summary/Conclusion: The overexpression of hnRNP K drives to an increase in nucleoli activity, leading to ribosome biogenesis and higher global translation by the regulation of molecules involved in both systems: Ncl, c-Myc or mTor. Our work found that hnRNP K overexpression in vivo drives a bone marrow failure phenotype, promoting the exhaustion of haematopoietic stem cells by ribosome dysregulation that triggered cell senescence.

Of note, this is the first time reported that a nucleoli/ribosome-gain-of-function induce bone marrow failure ribosomopathies phenotype.

This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140), Cancer Research UK [C355/A26819], FC AECC and AIRC under the Accelerator Award Program and National Cancer Institutes of Health Award (R01CA207204, SMP) Leukemia and Lymphoma Society (6577-19, SMP).

Oral Sessions

S105: IN VIVO PDX CRISPR/CAS9 SCREENS REVEAL MUTUAL THERAPEUTIC TARGETS TO OVERCOME HETEROGENEOUS ACQUIRED CHEMO-RESISTANCE

A.-K. Wirth 1,* , L. Wange 2 , S. Vosberg 3 , A. K. Jayavelu 4 , W Enard 2 , T Herold 5 , I Jeremias 1

1 Apoptosis in Hematopoietic stem cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Munich; 2 Anthropology and Human Genomics, Faculty of Biology, Ludwig Maximilian University (LMU), Martinsried, Germany; 3 Clinical Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; 4 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried; 5 Department of Medicine III, and Laboratory for Leukemia Diagnostics, Ludwig Maximilian University (LMU), Munich, Germany

Background: Acquired resistance to conventional polychemotherapy regimens leads to relapse and poor prognosis, and remains a major unmet clinical need.

Aims: To identify therapeutic options to overcome acquired chemo-resistance.

Methods: We studied acute lymphoblastic leukemia (ALL) as model disease and combined long-term in vivo treatment in orthotopic patient-derived xenograft (PDX) models with multi-omics profiling and functional genomic CRISPR/Cas9 screens.

Results: We adapted conventional chemotherapeutic protocols to allow treatment of mice for up to 18 consecutive weeks, using a combination of the widely used drugs cyclophosphamide and vincristine. Three luciferase-transgenic PDX models were monitored by repetitive in vivo imaging. Polychemotherapy strongly reduced PDX ALL cells within the first weeks, proving initial sensitivity of the sample. Under continuous treatment, tumor load persisted in mice at the level of minimal residual disease for several weeks, until tumors resumed growth despite treatment, indicating acquired resistance.

In an exemplary PDX model, eight resistant derivatives were generated in replicate mice and characterized individually. Genomic profiling revealed profound genomic heterogeneity between distinct derivatives; individual resistant derivatives acquired different copy number alterations in regions associated with resistance and distinct point mutations, e.g. in TP53. In contrast to genomic heterogeneity, transcriptome and proteome profiling identified a group of genes differentially expressed between sensitive and resistant cells, but similar across all derivatives.

To gain insights into underlying mechanisms and to identify therapeutic targets to overcome acquired resistance, a customized CRISPR/Cas9 in vivo dropout screen was performed in individual resistant PDX derivatives, to test the relevance of around 200 candidate genes under treatment. Among others, sgRNAs targeting BCL2 , BRIP1 or COPS2 dropped out in the in vivo screen specifically during treatment; single knockout experiments confirmed that knockout of either BCL2, BRIP1 or COPS2 re-sensitized PDX ALL cells towards chemotherapy. Of direct translational relevance, treatment of mice with the BCL2 inhibitor ABT-199 sensitized resistant PDX cells towards polychemotherapy in vivo. Interestingly, BCL2 inhibition restored treatment response in resistant derivatives independently from the highly diverse underlying genetic alterations, e.g., in clones with and without mutation in TP53 .

Summary/Conclusion: Taken together, we established a highly clinically relevant PDX in vivo model of acquired resistance to conventional chemotherapy. Using this model, we demonstrate that heterogeneous genomic alterations evolved in parallel in replicate mice, which could be overcome by a single therapeutic approach to re-sensitize tumors towards conventional chemotherapy.

S106: UBTF-ATXN7L3 GENE FUSION DUE TO 17Q21.31 DELETION DEFINES NOVEL HIGH-RISK ALL SUBTYPE AMENABLE TO MRD-BASED TREATMENT INTENSIFICATION

L. Bastian 1,* , A. Hartmann 1 , T. Beder 1 , S. Hänzelmann 1 , J. Kässens 1 , M. Bultmann 1 , M. P. Höppner 2 , S. Franzenburg 2 , M. Wittig 2 , A. Franke 2 , I. Nagel 3 , M. Spielmann 3 , N. Reimer 4 , H. Busch 4 , S. Schwartz 5 , B. Steffen 6 , A. Viardot 7 , K. Döhner 7 , M. Kondakci 8 , G. Wulf 9 , K. Wendelin 10 , A. Renzelmann 11 , A. Kiani 12 , H. Trautmann 1 , M. Neumann 1 , N. Gökbuget 6 , M. Brüggemann 1 , C. Baldus 1

1 Department of Medicine II, Hematology and Oncology, University Medical Center Schleswig-Holstein, Kiel; 2 Institute for Clinical Molecular Biology, Kiel University, Kiel; 3 Institute of Human Genetics, University Medical Center Schleswig-Holstein, Kiel and Lübeck, Kiel and Lübeck; 4 Medical Systems Biology Group and Institute for Cardiogenetics, University of Lübeck, Lübeck; 5 Department of Hematology, Oncology and Tumor Immunology (Campus Benjamin Franklin), Charité - Universitätsmedizin Berlin, Berlin; 6 Department of Medicine II, Hematology/Oncology, Goethe University Hospital, Frankfurt / Main; 7 Department of Internal Medicine III, University Hospital Ulm, Ulm; 8 Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Düsseldorf; 9 Department of Hematology and Oncology, University Hospital Göttingen, Göttingen; 10 Medical Department V, Hospital Nürnberg, Paracelsus Medizinische Privatuniversität, Nürnberg; 11 Medical Department Oncology and Hematology, University Medical Center Oldenburg, Oldenburg; 12 Department of Medicine IV, Hematology/Oncology, Klinikum Bayreuth, Bayreuth, Germany

Background: Response to induction chemotherapy assessed by quantification of minimal residual disease (MRD) is the strongest independent prognosticator in B precursor acute lymphoblastic leukemia (BCP-ALL). Molecular underpinnings of MRD poor response are insufficiently understood.

Aims: We aimed to identify novel high-risk subtypes in adult BCP-ALL as cell-intrinsic determinants of MRD poor response.

Methods: Adult BCP-ALL patients (n=565) were treated according to pediatric inspired protocols of the German Acute Lymphoblastic Leukemia Study group (GMALL) and profiled for integrative analyses by RNA-Seq (n=565), SNP-arrays (n=115), whole exome sequencing (WES; n=84) and whole genome sequencing (WGS; n=3).

Results: Concordance between transcriptomic and genomic profiles was used to allocate samples to one of 15 established molecular driver subgroups (Figure A). Unsupervised clustering of gene expression from the remaining samples revealed a distinct cluster (n=12/565, 2.1%) with an in-frame gene fusion between upstream binding transcription factor ( UBTF ) and ataxin-7-like protein 3 ( ATXN7L3 ) as exclusive event in these patients. Both fusion partners are in direct neighborship at 17q21.31. WGS revealed a 10.08 kb genomic deletion which truncated UBTF at exon 17/21 and comprised most of the intergenic region between both genes (Figure B). UBTF-ATXN7L3 rearranged cases frequently harbored 1q gains (n=5/7). Further genomic profiling showed a remarkable paucity of additional cooperating events compared to other molecular subtypes, supporting a prominent driver function of the newly identified fusion. UBTF and ATXN7L3 are global epigenetic regulators involved in transcriptional control. Both genes were highly expressed across the entire cohort. The gene fusion was associated with a marked increase of Caudal Homeobox 2 ( CDX2 ) expression. Analysis of functional modules related CDX2 to upregulated HOXA9 and MEIS1 , described essential co-regulators of KMT2A-driven leukemogenesis. NTRK3 expression was also strongly upregulated, suggesting a possible rationale for specific inhibitors.

UBTF-AXTN7L3 rearranged patients were older, more frequently female and presented with normal leukocyte counts, low bone marrow infiltration and pro-B immunophenotypes or common ALL with reduced CD10 expression. Response to induction chemotherapy in evaluable patients (n=11) was poor with only 3 patients achieving MRD negativity after consolidation I compared to n=271/402 (67%; p=0.019) in the remaining cohort. Four patients suffered either cytologic (n=2) or molecular (n=2) relapse. Immunotherapeutic treatment intensification using blinatumomab (n=5) or inotuzumab ozogamizin (n=1) and / or allogenic stem cell transplantation (n=7) in MRD poor responders or relapsed cases resulted in an overall survival probability of 80% (+/- 12%) vs. 73% (+/- 2%; p=0.07) in the remaining cohort. Heterogeneous MRD responses were observed for other molecular subtypes (poor: ZNF384 - 48.2% MRD neg., p=0.056; Ph-like - 54.0% MRD neg., p=0.003; KMT2A - 55.8% MRD neg., p=0.127 / good: High Hyperdiploid - 90.9% MRD neg., p=0.01; TCF3-PBX1 - 94.1% MRD neg., p=0.016) indicating how molecular drivers affect chemo-sensitivity in adult BCP-ALL.

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Summary/Conclusion: Molecular driver alterations determine sensitivity to induction chemotherapy in adult BCP-ALL. UBTF-ATXN7L3 ALL represents a novel subtype with poor induction chemotherapy response which could be successfully salvaged by MRD-based treatment intensification using immunotherapeutic strategies.

S107: SOD2 PROMOTES ACUTE LEUKEMIA ADAPTATION TO AMINO ACID STARVATION THROUGH THE N-DEGRON PATHWAY

N. K. Ibrahim 1,* , S. Schreek 1 , B. Cinar 1 , L. Loxha 1 , B. Fehlhaber 1 , J.-P. Bourquin 2 , B. Bornhauser 2 , C. Eckert 3 , G. Cario 4 , M. Forster 5 , M. Stanulla 1 , A. Gutierrez 6 , L. Hinze 1

1 Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany; 2 Department of Pediatric Hematology and Oncology, University Children’s Hospital Zurich, Zurich, Switzerland; 3 Department of Pediatric Hematology and Oncology, Charité Universitätsmedizin, Berlin; 4 Department of Pediatrics I, Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein; 5 Institute of Clinical Molecular Biology, Kiel, Germany; 6 Division of Pediatric Hematology and Oncology, Boston Children’s Hospital, Boston, United States of America

Background: The ability of cells to tolerate amino acid starvation is fundamental for survival under cellular stress conditions. Some cancer cells are vulnerable to asparagine depletion, which is exploited therapeutically using asparaginase. However, the mechanisms of adaptation to amino acid starvation in leukemia cells remain incompletely understood.

Aims: We recently performed a genome-wide CRISPR/Cas9 loss-of-function screen in the resistant T-ALL cell line CCRF-CEM to identify molecular pathways that promote asparaginase resistance. We found that Wnt-dependent stabilization of proteins (Wnt/STOP) induces a profound therapeutic vulnerability to asparaginase in acute leukemias and colorectal cancers (Hinze et. al., 2019; Hinze et al., 2020). Another unrelated gene on the top of the screen included SOD2 , a mitochondrial superoxide dismutase. Intriguingly, to date, SOD2 activity has not been linked to a cellular amino acid starvation response, whose biologic basis we thus sought to further investigate.

Methods: To evaluate the significance of SOD2 in mediating an asparaginase response, we employed genetic epistasis experiments as well as phenotypic assays including short hairpin RNA (shRNA) mediated knockdowns, quantitative PCRs, Western blots, amino acid starvation, and viability assays.

Results: Knockdown of SOD2 (shSOD2) resulted in a profound sensitivity to asparaginase in several T-ALL and B-ALL cell lines ( p <0.0001), and an increase in apoptosis, as assessed by caspase 3/7 activity ( p <0.001). The sensitization was rescued by either overexpressing SOD2 cDNA ( p <0.0001), or by adding the functional SOD2 mimetic MnTBAP ( p <0.01). Of note, shSOD2 mediated sensitization was selective to asparaginase, as it could not be observed for other commonly used chemotherapeutic agents including vincristine, doxorubicin, dexamethasone, and 6-mercaptopurine ( p =ns). Due to the selectivity to asparagine depletion, we then investigated whether SOD2 inhibition mediates a broader amino acid starvation response. Indeed, culturing SOD2 -inhibited T-ALL cells in the absence of essential amino acids (EAA) or non-EAA, induced a significant decrease in cell viability ( p <0.05). Sensitization appeared to be specific to the SOD2 isoform, and distinct from known SOD2-associated pathways including reactive oxygen species, cell cycle changes, alterations of mTOR signaling, or glutamine anaplerosis. To better understand the molecular underpinnings of SOD2 in regulating an amino acid starvation response, we leveraged the Bioplex Interactome database (Huttlin et al., 2020), and identified UBR2, an E3 ubiquitin ligase in the N-degron pathway, as a unique binding partner of SOD2. Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation (Yang et al., 2010). Indeed, SOD2 and UBR2 were co-immunoprecipitated, suggesting the formation of a complex that can drive proteasome-dependent protein catabolism. In line, inhibition of SOD2 significantly decreased ubiquitin levels, suggesting that SOD2 positively regulates catabolic protein degradation through the N-degron pathway to promote cancer cell fitness in amino acid starved conditions.

Summary/Conclusion: The interaction of SOD2 and the N-degron pathway represents a previously unknown molecular adaptation of cancer cells in response to amino acid starvation. These results serve as a strong proponent for an in-depth characterization of the N-degron pathway in mediating leukemia cell fitness upon amino acid starvation and thus provide a basis for therapeutic intervention in refractory leukemias.

S108: PEDIATRIC T- ALL RELAPSE: CONSTITUTIONAL CANCER PREDISPOSITION AND HYPERMUTATATOR PHENOTYPES

P. Richter-Pechanska 1 2,* , J. Kunz 1 2 , T. Rausch 2 3 , B. Erarslan-Uysal 1 2 , B. Bornhauser 4 , V. Frismantas 4 , Y. Assenov 5 , M. Zimmermann 6 , M. Happich 1 , C. von Knebel-Doeberitz 1 , N. von Neuhoff 7 , R. Koehler 8 , M. Stanulla 6 , M. Schrappe 9 , G. Cario 9 , G. Escherich 10 , R. Kischner-Schwabe 11 , C. Eckert 11 , S. Avigad 12 , S. Pfister 5 13 , M. Muckenthaler 1 2 , J.-P. Bourquin 4 , J. Korbel 2 3 , A. Kulozik 1 2 5

1 University Hospital Heidelberg; 2 MMPU; 3 EMBL, Heidelberg, Germany; 4 University Children’s Hospital, Zürich, Switzerland; 5 DKFZ, Heidelberg; 6 Hannover Medical School, Hannover; 7 University Hospital, University of Duisburg-Essen, Essen; 8 University Heidelberg, Heidelberg; 9 University Hospital Schleswig-Holstein, Kiel; 10 University Medical Center Hamburg-Eppendorf, Hamburg; 11 Charite, Berlin, Germany; 12 Schneider Children’s Medical Center of Israel, Petah Tikva, Israel; 13 KiTZ, Heidelberg, Germany

Background: Relapse is the main cause of death from pediatric acute precursor T-cell leukemia (T-ALL), but the underlying mechanisms of disease evolution from initial disease to relapse remain incompletely understood and show remarkable interpatient heterogeneity.

Aims: As cross-sectional studies failed to identify unifying determinants of relapse, we adopted a longitudinal strategy and performed multi-omic analyses in 13 matched pairs of initial diagnosis and relapse samples and their matched PDXs. We extended this set by WES and methylome analyses in an additional cohort of 25 matched DNA samples from patient cells collected at initial diagnosis, remission, and relapse.

Methods: Thirty-eight patients were recruited from the ALL‐BFM 2000/2009, CoALL97/03/09, and ALL‐REZ BFM 2002 trials or from Schneider Children’s Medical Center of Israel at time points of initial diagnosis, remission, and relapse. Material from 13 matched pairs of PDXs (RNA, cells) was used for multi-omic analyses, including DNA-Seq (WES), RNA-Seq, ATAC-Seq and methylation analysis with EPIC arrays.

Results: Based on the profile of SNVs and InDels we distinguished 18 (47%) type-1 (derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). We observed stronger remodeling on the way to type 2 than to type 1 relapses reflected by more evident changes in methylation, chromatin accessibility and gene expression. At the time of relapse, 3/20 type 2 patients exhibited a hypermutator phenotype, probably caused by gains of mutations in TP53 , BLM and BUB1B combined with PMS2 . Moreover, type 2 T-ALLs were predominantly TAL1-driven (4/8) in contrary to type 1 (0/5). T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. The fraction of subclonal mutations of those T-ALLs that later developed into a type-2 relapse was significantly higher already at the time of initial diagnosis than in those T-ALLs that later developed into a type-1 relapse (p=0.0387; Fisher’s exact), a difference that became even more pronounced at the time of relapse (p<0.0001). On the other hand, relapse type 1 T-ALLs exhibited overexpression of IL7R , its ligand HGF , and repressors of cytokine signaling ( SOCS1, SOCS2, SOCS3 ) which regulates the IL7R pathway via negative feedback loop. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. Moreover, an analysis of remission samples revealed a significant enrichment of mutations in constitutional cancer predisposition genes (CPG) in type 2 patients, thus indicating fundamental differences between these two groups of patients. In both types of relapse, we observed known and novel drivers of drug resistance including MDR1 and MVP and NT5C2 .

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Summary/Conclusion: In sum, our comprehensive analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia. Leukemias of patients with type-1 relapses were often characterized by upregulation of the IL7R pathway, whereas type-2 relapses were characterized by (i) an enrichment of TAL-1 fusion, (ii) and of constitutional mutations in CPG, (iii) divergent genetic and epigenetic remodeling, and (iv) an enrichment of somatic hypermutator phenotypes.

S109: ONCOGENIC DEUBIQUITINATION CONTROLS TYROSINE KINASE SIGNALING AND THERAPY RESPONSE IN ACUTE LYMPHOBLASTIC LEUKEMIA

P. Ntziachristos 1,* , Q. Jin 2 , B. Gutierrez 3

1 Ghent University, Ghent, Belgium; 2 MD Anderson, Houston; 3 Northwestern University, Chicago, United States of America

Background: Dysregulation of kinase signaling pathways via mutations favors tumor cell survival and resistance to therapy and it is common in cancer. Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell acute lymphoblastic leukemia (T-ALL). Here, we reveal a novel mechanism of post-translational regulation of kinase signaling and nuclear receptor activity via deubiquitination in acute leukemia.

Aims: This study aims at 1) characterizing the function of an oncogenic complex composed by two deubiquitinating enzymes in in vitro and in vivo leukemia systems and 2) testing the association of deubiquitinase activity with resistance to therapy in acute lymphoblastic leukemia.

Methods: We use genetic mouse and human:mouse xenograft models of T-cell leukemia, biochemical studies (quantitative global proteomics, phosphoproteomics and ubiquitination analysis) and high-throughput molecular biology (chromatin conformation capture (HiC), chromatin accessibility (ATAC-Seq) and gene expression (RNA-Seq)) analyses.

Results: We observed that the ubiquitin specific protease 11 (USP11) is highly expressed in lymphoblastic leukemia and associates with poor prognosis in this disease. USP11 ablation inhibits leukemia growth in vitro and in vivo , sparing normal hematopoiesis and thymus development, suggesting that USP11 could be a therapeutic target in leukemia. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK). Deubiquitination of LCK controls its activity, thereby altering T cell receptor signaling. Impairment of LCK activity leads to increased expression of the glucocorticoid receptor transcript, culminating into transcriptional activation of pro-apoptotic target genes, and sensitizes cells to glucocorticoids in T cell leukemia patient samples. The transcriptional activation of pro-apoptotic target genes, such as BCL2L11 , is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Pharmacological inhibition of USP7 or genetic knockout of USP7 in combination treatment of glucocorticoid displayed improved anti-T-ALL efficacy in vivo .

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Summary/Conclusion: Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell leukemia.

S110: A NOVEL AND SUCCESSFUL CD7 GENE KNOCKOUT CAR-T CELL THERAPY FOR RELAPSED OR REFRACTORY T-CELL HEMATOLOGIC MALIGNANCIES

J. Yang 1 , J. Li 1 , X. Zhang 1 , L. Qiu 1 , P. Lu 1,*

1 Hebei Yanda Lu Daopei Hospital, Langfang, China

Background: T cell malignancies represent a group of hematologic cancers with high relapse and mortality rates. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T due to unintended CAR-T fratricide. Here, we develop a fratricide-resistant anti-CD7 CAR-T modified by CD7 ablation through CRISPR/CAS9 gene editing (KO7CAR).

Aims: In a phase I clinical trial, we explored the efficacy and safety of KO7CAR T-cells for relapsed or refractory (R/R) T-cell malignancies ( NCT04916860 & NCT04938115 ).

Methods: Peripheral blood mononuclear cells were collected from patients (n=13) or the transplant donor (n=2) by leukapheresis. CD7-ablated CAR T cells (KO7CAR) were derived by electroporation of bulk T cells with CD7-targeting Cas9-gRNA RNP 24 hours before 7CAR transduction. This KO7CAR is a second-generation CAR-T with the co-stimulatory domain of 4-1BB and CD3ζ targeting CD7. Intravenous fludarabine (30mg/m 2 /d) and cyclophosphamide (300mg/m 2 /d) were given to all patients on day -5 to day -3 prior to KO7CAR-T cells infusion.

Results: From Oct. 2020 to Oct. 2021, 15 patients with T-cell acute lymphoblastic leukemia (n=10), T-cell lymphoblastic lymphoma (n=3), and mixed phenotype acute leukemia (n=2) were enrolled and received KO7 CAR-T cells (Table 1). The median age was 28 (8-46) years old. Four patients had prior hematopoietic stem cell transplantation (HSCT). At enrollment, 10 patients had bone marrow (BM) blasts >5% by morphology, and 10 patients had the extramedullary disease (EMD, diffuse involvement, n=8, and bulky mediastinal masses, n=2). Both patient- and donor-derived KO7CAR-T cells were successfully generated with a transduction efficiency of 59.0% (22.5%-97.4%). A single dose of KO7CAR-T cells was infused to patients at low dose (1.5~5x10 5 cells/kg, n=8), medium dose (1x10 6 cells/kg, n=6) or high dose (2x10 6 cells/kg, n=1).

On day 28, 15/15 (100%) patients achieved minimal residual disease (MRD) negative complete remission (CR). Among the 10 patients with EMD, 7 achieved EMD CR on day 30, 2 achieved partial response (PR), and 1 who relapsed post 2 nd transplant had no response on day 35 then withdrew. Up to data cutoff Feb.10, 2022, the median follow-up time was of 309 days (35~407 days). About 2 months post KO7CAR, 12 patients bridged into allogeneic HSCT, and all remained progression-free after a median time of 253 (30~388) days after HSCT except for 1 who relapsed on day 147 then died from intracerebral hemorrhage on day 249. The other 2 patients without subsequent HSCT (all had a prior transplant) died from infection on day 78 and GVHD on day 103, respectively, post KO7CAR.

Mild cytokine release syndrome (CRS, ≤grade II) occurred in 10/15 (66.7%) patients, and 5/15(33.3%) patients had grade III CRS. One patient had grade I neurotoxicity, and 2 had grade III/IV neurotoxicity. All was controlled after the administration of corticosteroids and/or tocilizumab.

Following infusion, the median peak of circulating KO7CAR-T cells was 1.77×10 5 (0.279~14.3×10 5 ) copies/μg genomic DNA which occurred around day 20 (day10~ day25) and 63.47% (23.1%~94.18%) occurring on day15 (day 9~day25) by q-PCR and flow cytometry respectively.

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Summary/Conclusion: This study demonstrated KO7CAR-T therapy had a high efficacy for CD7+ T-cell malignancies even for those who relapsed post-transplant. Safety was manageable, however, more data on additional patients and longer observation time are needed to evaluate the efficacy of KO7 CAR-T products further.

S111: REPEATED INFUSIONS OF ESCALATING DOSES OF EXPANDED AND ACTIVATED AUTOLOGOUS NATURAL KILLER CELLS IN MINIMAL RESIDUAL DISEASE-POSITIVE PH+ ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS. A GIMEMA PHASE 1 TRIAL

G. F. Torelli 1,* , S. Chiaretti 1 , N. Peragine 1 , W. Barberi 1 , L. Santodonato 2 , G. D’Agostino 2 , E. Abruzzese 3 , M. I. Del Principe 4 , A. Mancino 5 , M. Matarazzo 1 , M. S. Bafti 1 , M Mancini 1 , M. Messina 5 , L. Castiello 2 , A. Guarini 1 , R. Foà 1

1 Hematology, Department of Translational and Precision Medicine, Sapienza University; 2 FaBioCell Cell Factory, Istituto Superiore di Sanità; 3 Hematology, Sant’Eugenio Hospital, ASL Roma 2 , Tor Vergata University; 4 Hematology, Department of Biomedicine and Prevention, Tor Vergata University; 5 Fondazione GIMEMA Onlus, Rome, Italy

Background: Due to age and co-morbidities, many Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients are ineligible to undergo high-dose chemotherapy or allogeneic transplant as consolidation treatment. Our group reported the promising results of the chemo-free scheme D-ALBA based on dasatinib/blinatumomab in induction/consolidation, underlying the potential role of immunotherapy in this setting (Foà et al, NEJM 2020;383:1616-23).

Considerable interest has been raised by natural killer (NK) cells. We developed a GMP protocol for NK cell ex vivo expansion in the presence of IL-2 and IL-15, and report the results of a phase 1 protocol of adoptive immunotherapy with activated and expanded autologous NK cells for Ph+ ALL patients in complete hematologic remission (CHR) but with persistent/recurrent minimal residual disease (MRD) ≥60 years or ineligible for other post-CHR treatment modalities.

Aims: The primary endpoint was to determine the maximum tolerated dose of NK cells and the recommended dose for subsequent studies. Secondary endpoints were the assessment of safety and tolerability of the treatment, the immunologic modifications induced by the procedure and the clinical response to treatment.

Methods: The planned 6 patients were enrolled: 5 in 1 st CHR and 1 in 2 nd CHR. Patients underwent repeated infusions (maximum 5) of escalating doses of NK cells, ranging from 1x10 6 to 5x10 7 /kg of body weight (BW). No conditioning therapies were administered before the infusion; patients were allowed to continue tyrosine kinase inhibitors. Patients underwent a comprehensive MRD monitoring by Q-RT-PCR with a one-year follow-up. Immunophenotypic analysis on the NK cell product was performed before and after the expansion. Intracellular cytokine production and PBMC cytotoxic activity against K562 cells, allogeneic and autologous blasts were evaluated after expansion and at time 0 and 7 days from each NK cell infusion.

Results: NK cells presented a 12.3-fold ex vivo expansion. Expanded cells showed an increased expression of activating receptors and measurable cytotoxicity against primary allogeneic and autologous blasts. One patient received a maximum NK cell dose of 5x10 6 cells/kg, 2 patients 1x10 7 cells/kg and 3 5x10 7 cells/kg/BW. No patient experienced infusion-related toxicities. Two adverse events were recorded (grade 1 and 2), both judged not treatment-related, that resolved after TKI suspension. The higher cell dose infusion resulted in a significantly increased expression of natural cytotoxicity receptors, a greater cytokine production by NK, T and NKT cells, and in an increased capacity of PBMC to lyse K562 cells. These modifications appear persistent over time.

At a 1-year follow-up from the last infusion, 5/6 patients are alive in CHR (Table 1). The MRD levels reduced over time and 4/6 patients reached a complete molecular response (CMR) or a positive-not-quantifiable (PNQ) status during the study period. At a median follow-up of 30.8 months from the last infusion, the 5 patients who received the NK treatment in 1 st CHR are still in CMR or PNQ, though 1 patient required additional treatment. The patient in 2 nd CHR at the time of the infusions showed a rise in MRD and died of disease progression.

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Summary/Conclusion: This phase 1 study demonstrates that autologous NK cells can be efficiently expanded ex vivo from MRD-positive Ph+ ALL patients in CHR. The infusion of these expanded cells is safe and induces a marked in vivo host immune response, suggesting that this approach represents a tolerable and feasible model worthy of being investigated in larger clinical studies.

S112: TISAGENLECLEUCEL IN PEDIATRIC AND YOUNG ADULT PATIENTS (PTS) WITH RELAPSED/REFRACTORY (R/R) B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL): FINAL ANALYSES FROM THE ELIANA STUDY

S. Rives 1,* , S. L. Maude 2 , H. Hiramatsu 3 , A. Baruchel 4 , P. Bader 5 , H. Bittencourt 6 , J. Buechner 7 , T. Laetsch 2 , B. De Moerloose 8 , M. Qayed 9 , H. E. Stefanski 10 , K. L. Davis 11 , P. L. Martin 12 , E. Nemecek 13 , C. Peters 14 , G. Yanik 15 , A. Balduzzi 16 , N. Boissel 17 , S. L. Khaw 18 , J. Krueger 19 , J. Levine 20 , S. Davies 21 , G. D. Myers 22 , A. Yeo 23 , D. O’Donovan 24 , R. Ramos 23 , M. Pulsipher 25 , S. Grupp 2

1 Department of Pediatric Hematology – Oncology, Hospital Sant Joan de Déu Barcelona, and Institut de Recerca Sant Joan de Déu, Barcelona, Spain; 2 Division of Oncology, Center for Childhood Cancer Research and Cancer Immunotherapy Program, Children’s Hospital of Philadelphia and Department of Pediatrics, Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 3 Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 4 University Hospital Robert Debré (APHP) and Université de Paris, Paris, France; 5 Division of Stem Cell Transplantation and Immunology, Hospital for Children and Adolescents, University Hospital Frankfurt, Frankfurt, Germany; 6 Department of Pediatrics, Faculty of Medicine, University of Montreal, and the Hematology Oncology Division and Charles-Bruneau Cancer Center, Centre Hospitalier Universitaire Sainte-Justine Research Centre, Montreal, QC, Canada; 7 Department of Pediatric Hematology and Oncology, Oslo University Hospital, Oslo, Norway; 8 Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium; 9 Aflac Cancer and Blood Disorders Center, Emory University, Atlanta, GA; 10 National Bone Marrow Donor Program, Be the Match, Division of Pediatric Blood and Marrow Transplant, University of Minnesota, Minneapolis, MN; 11 Division of Hematology, Oncology and Stem Cell Transplant, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA; 12 Pediatric Transplant and Cellular Therapy, Duke University Medical Center, Durham, NC; 13 Oregon Health and Science University, Portland, OR, United States of America; 14 Stem Cell Transplantation Unit, St. Anna Children’s Hospital, Vienna, Austria; 15 Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, MI, United States of America; 16 Clinica Pediatrica Università degli Studi di Milano Bicocca, Fondazione MBBM, Ospedale San Gerardo, Monza, Italy; 17 Saint-Louis Hospital (APHP) and Université de Paris, Paris, France; 18 Children’s Cancer Centre, Royal Children’s Hospital and Murdoch Children’s Research Institute, Parkville, VIC, Australia; 19 Division of Haematology/Oncology/Bone Marrow Transplantation, Department of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 20 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY; 21 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 22 Children’s Mercy Hospital and Clinics, Kansas City, MO; 23 Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States of America; 24 Novartis Pharmaceuticals Corporation, Dublin, Ireland; 25 Division of Pediatric Hematology and Oncology, Intermountain Primary Children’s Hospital, Huntsman Cancer Institute at the University of Utah, Salt Lake City, UT, United States of America

Background: Pediatric and young adult pts with R/R B-ALL experience a treatment journey characterized by diminishing likelihood of cure and increasing morbidity. Tisagenlecleucel is an autologous CD19-directed chimeric antigen receptor (CAR) T-cell therapy approved for use in pediatric and young adults with B-ALL and adults with B-cell lymphomas. Tisagenlecleucel provided high rates of remission (>80%) in children and young adults with R/R B-ALL in ELIANA, with 62% of responders remaining relapse-free at 24 mo (Grupp et al, Blood , 2018).

Aims: Here, we report the final efficacy and safety analyses in pts followed up to 5.9 years post-tisagenlecleucel infusion.

Methods: ELIANA ( NCT02435849 ) was a pivotal, Phase II, open-label, multicenter, global study of tisagenlecleucel in pediatric and young adult pts with R/R B-ALL. Pts received a single infusion of tisagenlecleucel at 0.2-5.0×10 6 CAR+ viable T cells/kg body weight for pts ≤50 kg and 0.1-2.5×10 8 CAR+ viable T cells for pts >50 kg. Endpoints included overall remission rate (ORR) within 3 mo, relapse-free survival (RFS), duration of remission (DOR), overall survival (OS), persistence of B-cell aplasia, and short- and long-term safety events.

Results: Results : As of September 24, 2021, 97 pts were enrolled and 79 pts (81%) received tisagenlecleucel. Median time from infusion to data cutoff was 5.5 y; 64 pts had ≥5 y of follow-up. At study entry, the median age was 11 y (range, 3-24). Pts were heavily pretreated with a median of 3 prior lines of therapy (range, 1-8) and 61% had a history of prior stem cell transplant (SCT). ORR (complete remission [CR] or CR with incomplete hematologic recovery within 3 mo after infusion) was 82% (95% CI, 72-90). Among pts in remission (CR/CRi), the 5y RFS rate was 49% (95% CI, 34-62), and the median RFS was not reached ( Figure , 46.8 mo when censoring for SCT; n=15). The median time to B-cell recovery was 38.6 mo (95% CI, 23-not reached) and the probability of B-cell aplasia at 6 mo and 12 mo was 83% (95% CI, 71-91) and 71% (95% CI, 57-82), respectively. Pts with B-cell recovery (<6 mo, n=10; 6-12 mo, n=4; >12 mo, n=7) experienced a 2y cumulative incidence of relapse of 25.2% (with SCT treated as a competing risk). Among all pts, the 5y EFS and OS rates were 42% (95%CI, 29-54) and 55% (95% CI, 43-66), respectively. There were no significant differences in any efficacy endpoint between pediatric (<18 y; n=65) and young adult (≥18 y; n=14) pts. No new or unexpected AEs were reported during long-term follow-up. Among pts in remission, the most commonly reported grade ≥3 AEs occurring >1 y post-infusion were infection (20%) and cytopenias (6%). Ten (14%) pts in remission experienced long-term cytopenias persisting for >1 y; however, none of these pts experienced cytopenias persisting for >5 y (median 2 y; range, 1.1-5y). Eighty-two percent of pts received IVIG any time post-infusion.

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Summary/Conclusion: This >5 y follow-up study demonstrates continued durable efficacy of tisagenlecleucel without late adverse effects in heavily pretreated pediatric and young adult pts with R/R B-ALL. Tisagenlecleucel continues to be a potentially curative treatment option for pediatric and young adult patients with R/R B-ALL.

S113: NATIONAL PEGASPARGASE-MODIFIED RISK-ORIENTED PROGRAM FOR PHILADELPHIA-NEGATIVE ADULT ACUTE LYMPHOBLASTIC LEUKEMIA/LYMPHOBLASTIC LYMPHOMA (PH− ALL/LL). GIMEMA LAL 1913 FINAL RESULTS.

R. Bassan 1,* , S. Chiaretti 2 , I. Della Starza 3 , O. Spinelli 4 , A. Santoro 5 , L. Elia 3 , M. S. De Propris 3 , A. M. Scattolin 1 , F. Paoloni 6 , M. Messina 7 , E. Audisio 8 , L. Marbello 9 , E. Borlenghi 10 , P. Zappasodi 11 , C. Vetro 12 , G. Martinelli 13 , D. Mattei 14 , N. Fracchiolla 15 , M. Bocchia 16 , P. De Fabritiis 17 , M. Bonifacio 18 , A. Candoni 19 , V. Cassibba 20 , P. Di Bartolomeo 21 , G. Latte 22 , S. Trappolini 23 , A. Guarini 24 , A. Vitale 3 , P. Fazi 6 , M. Vignetti 6 , A. Rambaldi 4 , R. Foà 3

1 Hematology, Ospedale dell’Angelo, Venice; 2 Translational and Precision Medicine, Sapienza Univesrity of Rome; 3 Translational and Precision Medicine, Sapienza University of Rome, Rome; 4 UOC Ematologia, ASST-Papa Giovanni XXIII, Bergamo; 5 Divisione di Ematologia con UTMO, Ospedali Riuniti Villa Sofia-Cervello, Palermo; 6 GIMEMA Data Center; 7 GIIMEMA Data Center, Fondazione GIMEMA – Franco Mandelli Onlus, Rome; 8 Ematologia, Città della Salute, Torino; 9 Hematology, Niguarda Ca’ Granda Hospital, Milan; 10 Hematology, Spedali Civili, Brescia, Italy, Spedali Civili, Brescia; 11 Hematology, Foundation IRCCS Policlinico San Matteo, Pavia; 12 General Surgery and Medical-Surgical Specialties, University of Catania, Catania; 13 Institute of Hematology, L. and A. Seràgnoli, Bologna; 14 Hematology, Ospedale S. Croce, Cuneo, Italy, Cuneo; 15 UOC Oncoematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milan; 16 Hematology Unit, Azienda Ospedaliera Universitaria Senese, Siena; 17 Hematology Division, S. Eugenio Hospital, Rome; 18 Ospedale Policlinico “G.B. Rossi”, University of Verona, Verona; 19 Clinica Ematologica, Azienda Sanitaria Universitaria Integrata di Udine, Udine; 20 Divisione di Ematologia, Ospedale Civile, Bolzano; 21 Oncology Hematology, Ospedale Civile, Pescara; 22 Hematology, S. Franceso Hospital, Nuoro; 23 Clinica di Ematologia, Azienda Ospedaliero - Universitaria Ospedali Riuniti Umberto I, Ancona; 24 Molecular Medicine, Sapienza University of Rome, Rome, Italy

Background: Pediatric-inspired chemotherapy is standard of care for younger adults with Ph− ALL/LL. An essential component of these regimens is pegaspargase, here incorporated into a national treatment program for patients 18-65 years.

Aims: To assess in the GIMEMA Phase 2 LAL 1913 study the feasibility and efficacy of a pegaspargase-containing induction and consolidation regimen sustaining a risk-oriented strategy for adult Ph− ALL/LL (ClinicalTrials.gov ID NCT02067143 ).

Methods: Our prior, reference 8-block chemotherapy protocol (Blood Cancer J 2020;10:119) was modified to include pegaspargase 2000 IU/m 2 at courses 1 (d10), 2 (d8), 5 (d3, with HD-MTX) and 6 (d8), with dose reductions in patients >55 years (pegaspargase 1000 IU/m 2 ). Serum drug activity was not assessed in this study. Responders were risk-stratified for allogeneic stem cell transplantation (SCT) or maintenance according to a mixed risk model based on WBC count, immunophenotype, genetics and post-remission molecular minimal residual disease (MRD): patients with high-risk (HR) features or MRD ≥ 10 -4 at weeks 10-16 or positive at week 22 were eligible to SCT; standard-risk (SR) patients were eligible to maintenance.

Results: Two hundred and three patients entered the study (median age 39.8 years; 139 B- and 64 T-phenotype). The complete remission (CR) rate was 91% (100% in T-ALL/LL), with a 3-year cumulative relapse incidence and non-relapse mortality of 24.2% and 12.6%, respectively; 60 patients underwent a SCT. Overall (OS), event-free (EFS) and disease-free (DFS) survival were 66.7% (95% CI, 60.1-74.1%), 57.7% (95% CI, 51.0-65.3%) and 63.3% (95% CI, 56.3-71.1%) at 3 years. HR class (n=95) and LL diagnosis (n=20) did not affect prognosis. T-cell phenotype (CR 100%, P=0.001; EFS 67.1%, P=0.038), age 18-40 years (EFS 72.6%, P<0.0001) and MRD <10 -4 after courses 1 (55%: DFS 77.9%, P=0.023) and 3 (79%: DFS 75.2%, P=0.048) were prognostically favorable. One hundred and eighty-seven patients had pegaspargase at course 1 (92.1%, 11 delayed, 3 reduced), 154 at course 2 (84.6%; 11 delayed, 12 reduced), 110 at course 5 (83.9%; 2 delayed, 11 reduced) and 73 at course 6 (68.8%; 3 delayed, 7 reduced). Dose reductions and delays were related to high-risk profile (liver dysfunction/steatosis, obesity etc.) or treatment toxicity. Toxicity of grade 2 or more was mainly observed at course 1 (hepatic 12.8%, coagulation/thrombosis 3.2% [enoxaparin prophylaxis recommended with platelets >30-50], pancreatic 1.6%), contributing to an induction death in 3 patients (1.4%), but was rare afterwards.

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Summary/Conclusion: This pegaspargase-based ALL regimen was safely applicable to the majority of study patients, resulting in 3-year OS, EFS and DFS rates >50% in a patient population aged 18-65. The results were more favorable in patients up to the age of 55, especially in those aged 18-40 years, and in those who achieved maximum MRD response regardless of age (Figure). Subsequently, a pegaspargase dosing algorithm based on patient age, body mass index, hepatosteatosis and selected toxicities at first or prior drug exposure was developed to minimize toxicity, and was used in a successor GIMEMA trial of sequential chemotherapy-blinatumomab for CD19+ adult B-ALL (EHA Congress 2021, abstract S114).

S114: PONATINIB AND BLINATUMOMAB FOR PATIENTS WITH PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA: UPDATED RESULTS FROM A PHASE II STUDY

N. Short 1,* , H. Kantarjian 1 , M. Konopleva 1 , N. Jain 1 , F. Ravandi 1 , X. Huang 2 , W. Macaron 1 , W. Wierda 1 , G. Borthakur 1 , T. Kadia 1 , K. Sasaki 1 , G. Issa 1 , G. Montalban-Bravo 1 , Y. Alvarado 1 , G. Garcia-Manero 1 , C. Dinardo 1 , J. Thankachan 1 , R. Delumpa 1 , E. Mayor 1 , W. Deen 1 , A. Milton 1 , J. Rivera 1 , L. Waller 1 , C. Loiselle 1 , R. Garris 1 , E. Jabbour 1

1 Department of Leukemia; 2 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Ponatinib and blinatumomab are both highly effective therapies for Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). The combination of these two agents may offer an effective chemotherapy-free strategy in these patients (pts).

Aims: We evaluated the efficacy and safety of ponatinib and blinatumomab in pts with newly diagnosed (ND), relapsed/refractory (R/R) Ph+ ALL or CML in lymphoid blast phase (CML-LBP). For pts with ND Ph+ ALL, the primary endpoint was the complete molecular response (CMR) rate. For pts with R/R Ph+ ALL, the primary endpoint was the CR/CRi rate. Secondary endpoints included safety, event-free survival (EFS) and overall survival (OS).

Methods: In this phase II study, adults with ND Ph+ ALL, R/R Ph+ ALL, or CML-LBP were eligible. Pts were required to have a performance status of ≤2, total bilirubin ≤2x the upper limit of normal (ULN), and ALT and AST ≤3x the ULN. Pts with uncontrolled cardiovascular disease or clinically significant central nervous system (CNS) comorbidities (except for CNS leukemia) were excluded. Pts received up to 5 cycles of blinatumomab as a continuous infusion at standard doses. Ponatinib 30mg daily was given during cycle 1 and was decreased to 15mg daily once CMR was achieved. After 5 cycles of blinatumomab, ponatinib was continued for at least 5 years. Twelve doses of prophylactic IT chemotherapy with alternating cytarabine and methotrexate were administered.

Results: Between 2/2018 to 1/2022, 55 pts were treated (35 with ND Ph+ ALL, 14 with R/R Ph+ ALL and 6 with CML-LBP). Baseline characteristics are shown in Table 1 .

Among the 35 pts with ND Ph+ ALL, 12 were in CR at enrollment (including 2 pts in CMR). 22 of the 23 evaluable pts (96%) achieved CR/CRi. One pt died on day 18 from intracranial hemorrhage in the setting of chemotherapy administered prior to enrollment. After one cycle, 21/33 pts (64%) achieved CMR, and 28/33 pts (85%) achieved CMR at any time. 11 of 15 tested pts (73%) also became MRD-negative by an NGS assay with sensitivity of 1x10 -6 .

CR/CRi was achieved in 12/13 (92%) evaluable pts with R/R Ph+ ALL. CMR was achieved in 10 pts (71%) after cycle 1 and in 11 pts (79%) overall. 5 of 6 pts with CML-LBP achieved CR/CRi, and 1 pt achieved PR as best response. 2 pts (40%) achieved CMR.

In the ND Ph+ ALL cohort, 1 of 34 pts who received at least 1 full cycle died in CR; the other 33 are in ongoing hematologic remission. Only one pt underwent stem cell transplant (SCT) in first remission for persistently detectable BCR/ABL1 transcripts. Among 13 responding pts in the R/R Ph+ ALL cohort, 6 proceeded to SCT, 4 did not undergo SCT and subsequently relapsed, 1 died in CR, and 2 are in ongoing remission without SCT. In the CML-LBP cohort, 3 of the 5 responding pts subsequently relapsed.

The median follow-up is 11 months (range, 1-46+). For ND Ph+ ALL, the 2-year EFS and OS are both 93% (Figure 2 ). There were no relapses or leukemia-related deaths in this cohort. In the R/R Ph+ ALL cohort, the 2-year EFS rate was 42% and the 2-year OS rate was 61%. In the CML-LBP cohort, the 2-year EFS was 33% and the 2-year OS was 60%.

The treatment was well-tolerated, and most toxicities were grade 1-2 and consistent with the known toxicities of the two agents. Two pts discontinued ponatinib due to toxicity (1 due to stroke and 1 due to DVT). One pt discontinued blinatumomab due to persistent grade 2 tremor.

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Summary/Conclusion: The chemotherapy-free regimen of simultaneous ponatinib and blinatumomab is safe and effective in pts with Ph+ ALL. For pts with ND Ph+ ALL, SCT does not appear to be needed in first remission.

S115: MUTANT NPM1 BINDS CHROMATIN AND COOPERATES WITH MLL1 TO REGULATE ONCOGENIC TRANSCRIPTION

H. Uckelmann 1,* , S. Armstrong 1 , E. Haarer 1 , E. Wong 1 , C. Hatton 1 , F. Perner 1 , C. Marinaccio 1 , C.-W. Chen 2

1 Pediatric Oncology, Dana-Farber Cancer Institute, Boston; 2 Department of Systems Biology, Beckman Research Institute, City of Hope, United States of America

Background: The dysregulation of stem cell and self-renewal associated genes is a common phenomenon during leukemia development. In acute myeloid leukemia (AML) around 50 % of cases express high levels of HOXA cluster genes and MEIS1 . Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncogene that is mislocalized from the nucleolus to the cytoplasm. Hence, most studies of NPM1c have focused on a potential cytoplasmic role. However, it remains unclear how NPM1c expression in hematopoietic cells leads to its characteristic gene expression pattern. Furthermore, NPM1c AMLs are highly sensitive to the disruption of the MLL1 histone methyltransferase complex. Small molecule inhibitors that block the interaction between MLL1 and its adaptor protein Menin have been shown to impair binding of MLL1 to a subset of its target genes and to inhibit leukemia cell proliferation and self renewal. Several MLL1-Menin inhibitors are currently in Phase I/II clinical trials and show promising activity in patients with NPM1c AML. The effectiveness of these molecules in NPM1c AML prompts the question whether NPM1c and the wildtype MLL complex cooperate directly on chromatin to drive leukemic self-renewal.

Aims: In this study we investigated the potential role of NPM1c in regulating oncogenic transcription on chromatin and the interplay between NPM1c and the histone methyltransferase complex KMT2A (MLL1).

Methods: We used an endogenously degrader tagged NPM1c leukemia cell line that allows rapid small molecule induced degradation to show that NPM1c occupies specific chromatin targets in AML. To characterize the effects of NPM1c degradation on the chromatin landscape and transcriptional output at genomic loci that are bound by NPM1c we used ChIPseq, PROseq and nascent RNAseq methods.

Results: Our results show that endogenous NPM1c directly binds to chromatin at specific target genes, such as HOXA9 and MEIS1, which are highly expressed in NPM1c patient samples. The loss of NPM1c from its targets leads to specific alterations in active chromatin marks and RNA Polymerase II (Pol II) chromatin occupancy which are accompanied by rapid changes in gene expression as well as Pol II transcriptional activity. The recruitment of NPM1c to chromatin is dependent on the nuclear exporter CRM1 as well as one of the acidic domains of NPM1c. We further show that NPM1c is lost from specific loci after treatment with small molecules that disrupt the MLL1-Menin complex interaction thus functionally linking targeted epigenetic therapy and NPM1c function.

Summary/Conclusion: Overall, we demonstrate that NPM1c directly regulates a network of leukemia self-renewal associated genes through direct chromatin interaction. We further found that NPM1c acts in collaboration with the MLL1 complex and define the mechanism by which MLL1-Menin small molecule inhibitors produce clinical responses in patients with NPM1 -mutated AML.

S116: CELLULAR AND MOLECULAR MECHANISMS OF EVI1-EXPRESSING MLL-REARRANGED ACUTE MYELOID LEUKEMIA

Hugues-Etienne Châtel-Soulet 1 , Sabine Juge 1 , Ana Luisa Pereira 2 , Frederik Otzen Bagger 3 , Alexandar Tzankov 4 , Mineo Kurokawa 5 , Athimed El Taher 6 , Jonathan Seguin 1 , César Nombela Arrieta 2 , Juerg Schwaller 1

1 Biomedicine, University Children’s Hospital, Basel, Switzerland; 2 Medical Oncology & Hematology, University Hospital, Zürich, Switzerland; 3 Center for Genomic Medicine, University Hospital, Kopenhagen, Denmark; 4 Institute for Pathology, University Hospital, Basel, Switzerland; 5 Hematology & Oncology,The University of Tokyo, Tokyo, Japan; 6 Biomedicine, University of Basel, Basel, Switzerland

Background: Expression of a doxycycline (DOX)-inducible acute myeloid leukaemia (AML)-associated iMLL-AF9 fusion transgene in long-term haematopoietic stem cells (LT-HSC) can lead to an invasive and chemo-resistant disease expressing the transcription factor EVI1. High EVI1 expression has been suggested as marker of poor outcome in AML patients even without rearrangements of the EVI1 locus at 3q26.

Aims: We addressed the association between EVI1 expression, the cellular origin and poor disease outcome in AML driven by the iMLL-AF9 fusion gene.

Methods: The role of EVI1 expression was studied in iMLL-AF9 transgenic mice carrying an Evi1-IRES GFP reporter in vitro using flow cytometry, colony formation and RT-qPCR assays, ex vivo with high-resolution bone marrow (BM) imaging, and in vivo, by transplantation of enriched naïve Evi1+ iMLL-AF9 hematopoietic stem and progenitor cells (HSPC) into irradiated recipients on DOX. Haematopoiesis of symptomatic mice was analysed by flow cytometry and histology. For mechanistic studies, single cell and bulk RNA sequencing was performed on enriched HSPC or BM samples from diseased mice.

Results: Analysis of BM cells from Evi1-IRES-GFP reporter mice revealed that not only the mostly quiescent LT-HSC but also fractions of the more proliferating multipotent progenitors (MPP1-3) express abundant Evi1 (“Evi1high”). Induction of the iMLL-AF9 fusion did not result in significant changes in numbers of Evi1+ cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments. However, in colony assays, Evi1high iMLL-AF9 cells retained a more immature phenotype and produced more colonies with an invasive morphology than Evi1low cells (n=11, p<0.05). While Evi1 expression did not influence disease induction upon transplantation of LT-HSC, recipients of Evi1+ MPP1 cells developed AML earlier than Evi1- MPP1 (n=11, 79 vs. 269d, p<0.05). Disease induced by Evi1+ cells presented with more extensive leukemic organ infiltration than Evi1- AML. Evi1 expression also correlated with in vitro Ara-C resistance. We also examined whether some exogenous factors may increase AML susceptibility by expanding the Evi1+ HSPC. Although a single injection of recombinant mouse thrombopoietin (TPO) only increased the number of LT-HSC and not of MPP1, the Evi1high cell fraction was enlarged in both compartments (LT-HSC: 23 vs. 50%; MPP1: 22 vs. 47%; n=29, p<0.0001) supported by high-resolution imaging. Interestingly, increased TPO induced HSPC cycling was confined to the Evi1high cell population (n=3, p<0.05). Transplantation of TPO-treated iMLL-AF9 LT-HSC or MPP1 resulted in a significantly faster induction of Evi1+ AML than controls (n=19, MPP1: 35 vs. 79d, p<0.001; LT-HSC: 41 vs. 90d, p<0.001). To better understand mechanisms of aggravated AML after TPO mediated expansion of Evi1+ HSCP we performed multiplexed single cell RNA sequencing of highly enriched HSPC cells in iMLL-AF9 and control mice. While we observed no changes of cellular cluster organisation 2 days after TPO injection, we found some differentially expressed genes in TPO-stimulated cycling iMLL-AF9 HSPC, including potential stemness regulators.

Summary/Conclusion: Our results suggest that expansion of Evi1-expressing HSPC by exogenous factors can result in a more aggressive MLL-AF9-driven AML. Ongoing data exploration and validation may characterize aberrantly expressed genes in TPO-stimulated Evi1+ iMLL-AF9-expressing HSPC as potential therapeutic targets to impair stemness of AML cells.

S117: EVI1 DRIVES LEUKEMOGENESIS THROUGH ABERRANT ERG ACTIVATION

J. Schmoellerl 1,* , I. Barbosa 1 , M. Minnich 1 , F. Andersch 1 , L. Smeenk 2 , M. Havermans 2 , T. Eder 3 , T. Neumann 1 , J. Jude 1 , M. Fellner 1 , A. Ebert 1 , M. Steininger 1 , R. Delwel 2 , F. Grebien 3 , J. Zuber 1

1 Research Institute of Molecular Pathology (IMP), Vienna, Austria; 2 Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 3 Institute for Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria

Background: Chromosomal rearrangements leading to overexpression of EVI1 ( MECOM ) on chromosome 3q26 define a distinct subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and a 2-year survival of <10%. While genetic events driving aberrant expression of EVI1 are increasingly understood, the molecular functions of EVI1 that drive leukemogenesis are unclear, which has so far precluded the development of targeted therapeutics.

Aims: We aimed to elucidate transcriptional programs that are maintained by aberrant EVI1 expression and to systematically identify vulnerabilities of EVI1-driven AML.

Methods: We developed a panel of mouse models that recapitulate phenotypic and transcriptional hallmarks of patients suffering from EVI1-driven AML, allow tetracycline-controllable EVI1 expression and the functional interrogation of genetic targets using CRISPR/Cas9. We mapped transcriptional programs upon acute EVI1 repression in vivo and in vitro, profiled global EVI1 chromatin occupancy in human AML cell lines and primary patient-derived AML cells and performed comparative genome-wide CRISPR/Cas9-based loss-of-function screens in murine and human EVI1-driven AML.

Results: Integration of these datasets revealed a conserved core of genes that is transcriptionally regulated by EVI1 in murine and human AML, among which we identified the ETS transcription factor ERG as the only dependency that is highly selective for EVI1-driven AML. Suppression of ERG specifically triggered cellular differentiation and apoptosis of EVI1-driven leukemia cells while other AML cell lines were unaffected. Strikingly, ectopic expression of ERG was sufficient to functionally rescue loss of EVI1 in EVI1-driven AML cells, suggesting that the major oncogenic function of EVI1 in AML is the aberrant activation of ERG.

Summary/Conclusion: Interfering with the EVI1/ERG regulatory axis may provide entry points for the development of rational targeted therapies that are urgently needed for this group of AML patients.

S118: IDENTIFICATION OF DIRECT TRANSCRIPTIONAL TARGET GENES OF NUP98-KDM5A REVEALS REGULATORY NETWORKS IN ACUTE MYELOID LEUKEMIA

S. Troester 1,* , J. Schmoellerl 2 , T. Eder 1 , G. Manhart 1 , G. Winter 3 , J. Zuber 2 , F. Grebien 1

1 Institute for Medical Biochemistry, University for Veterinary Medicine Vienna; 2 Research Institute of Molecular Pathology (IMP); 3 Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Vienna, Austria

Background: Oncogenic fusion proteins involving the Nucleoporin 98 ( NUP98 ) gene are recurrently found in acute myeloid leukemia (AML) with a particular prevalence in pediatric patients. A chromosomal rearrangement resulting in the fusion of NUP98 to the gene encoding the lysine-specific demethylase 5A ( KDM5A ) is the most frequent NUP98-fusion in infant leukemia and is associated with particularly poor prognosis. The urgent need for the development of tailored treatments requires a better understanding of the effects of NUP98-KDM5A on the deregulation of gene expression programs. Although it has been shown that oncogenic NUP98-fusion proteins act as transcriptional regulators, it is unclear if and how NUP98-KDM5A directly regulates gene expression to drive leukemia.

Aims: In this study, we aimed to identify immediate critical effectors of the NUP98-KDM5A fusion protein and to characterize the transcriptional programs through which they regulate the development and maintenance of NUP98-KDM5A-driven AML.

Methods: We conducted a genome-scale CRISPR/Cas9 loss-of-function screen in a NUP98-KDM5A-driven murine AML cell line to unravel functional genetic dependencies that could be exploited to target leukemia cells. In parallel, we developed a new model for degradation tag (dTAG)-mediated ligand-induced degradation of the NUP98-KDM5A protein to gain a detailed understanding of direct transcriptional effects of NUP98-fusion-dependent gene regulation. We used this model to measure immediate changes in transcription upon acute NUP98-KDM5A degradation by nascent RNA-seq (SLAM-seq). An inducible shRNA system was used to assess the requirements of direct NUP98-KDM5A target genes for leukemia cell growth and to measure global gene expression changes by RNA-seq.

Results: Analysis of the CRISPR/Cas9 screen identified 4105 genes that are required for the proliferation and survival of NUP98-KDM5A-driven AML cells. Complete loss of the dTAG-NUP98-KDM5A fusion protein was achieved within one hour after ligand addition, resulting in cell cycle arrest, terminal differentiation and apoptosis of leukemia cells. Global analysis of nascent mRNA expression by SLAM-seq revealed 45 immediate NUP98-KDM5A target genes, as their transcription was significantly downregulated upon fusion protein degradation. Among these genes, 12 were classified as essential factors for NUP98-KDM5A cell growth from the CRISPR/Cas9 screen. This list included known target genes of NUP98-fusion proteins, such as members of the Hoxa gene cluster, but also other transcription factors, enzymes and RNA binding proteins. shRNA-mediated knockdown of the 12 candidate genes confirmed their essentiality for NUP98-KDM5A leukemia cell proliferation. Through RNA-seq studies, we found that the knockdown of a small subset among the 12 candidate genes was able to recapitulate global patterns of gene deregulation that are induced by NUP98-KDM5A knockdown.

Summary/Conclusion: Using a combination of CRISPR/Cas9 screening and ligand-induced degradation, we identified direct transcriptional target genes of NUP98-KDM5A that are functionally essential in AML. Multi-layered investigations of the interplay between the members of this small network of genes using a variety of models including primary patient samples will allow us to further dissect their role in the regulation of aberrant gene expression in NUP98-KDM5A-expressing leukemia cells and might identify novel therapeutic targets.

S119: CEBPA AND TET2 MUTATIONS COOPERATE TO INDUCE AGGRESSIVE AML VIA GATA-2 DOWNREGULATION

E. Heyes 1,* , A. S. Wilhelmson 2 3 4 , A. Wenzel 2 3 4 , M. B. Schuster 2 3 4 , M. Ali 3 4 , T. D’Altri 2 3 4 , T. Eder 1 , G. Manhart 1 , E. Rzepa 1 , L. Schmidt 1 , M. Meggendorfer 5 , T. Haferlach 5 , G. Volpe 6 , C. Nerlov 7 , J. Frampton 6 , K. Jae Won 3 4 , F. Grebien 1 , B. Porse 2 3 4

1 Institute of Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria; 2 The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences; 3 Biotech Research and Innovation Center (BRIC); 4 Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 5 MLL Munich Leukemia Laboratory, München, Germany; 6 Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham; 7 MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

Background: The transcription factor CCAAT-enhancer-binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is mutated in 10-15 % of Acute Myeloid Leukemia (AML) patients. N-terminal frameshifts represent the predominant type of lesions and ablate the expression of the full-length protein p42, leading to overexpression of the shorter isoform p30. Mutations in the C-terminal basic-region leucine zipper (bZip) can disrupt the DNA-binding ability of C/EBPα. AML patients harbor mono- or biallelic CEBPA mutations (CEBPAmo or CEBPAbi). The most commonly co-occurring mutations are loss-of-function mutations in the methylcytosine dioxygenase TET2, resulting in adverse overall survival. We hypothesized that combinatorial effects of CEBPA mutations together with TET2 loss specifically rewire transcriptional and epigenetic circuitries in AML cells, thereby strongly influencing disease outcome.

Aims: We aimed to elucidate the molecular mechanisms behind cooperative effects of CEBPA and TET2 mutations through state-of-the-art transcriptomic and epigenomic analyses of relevant in vitro and in vivo models as well as data from AML patients.

Methods: We used the CRISPR/Cas9 technology to introduce Tet2 mutations in murine cell lines expressing only p30 ( Cebpa p30/p30 ) or mimicking biallelic CEBPA mutations ( Cebpa p30/C-mut. ) to study the functional cooperation of these mutations in vitro. A Cebpa -/p30 Tet2 -/ - mouse model was used to study effects of Tet2 loss in CEBPA mutated AML. We performed RNA-, C/EBPα-ChIP-, ATAC-, Bisulfite-, CUT&RUN and CRISPR/Cas9-mediated enhancer screens to generate a comprehensive dataset for in-depth comparative analysis and correlation with relevant patient data from the beatAML collection.

Results: Integration of transcriptomic and epigenomic data from in vitro and in vivo models of CEBPA-TET2 co-mutated AML, in combination with gene expression analyses in AML patients, identified the transcription factor GATA2 as a conserved target of the CEBPA-TET2 axis. p30 and TET2 were strongly bound to the -77 kb enhancer of the Gata2 gene, and CRISPR/Cas9-induced enhancer deletions diminished Gata2 expression. Furthermore, TET2 loss reduced chromatin accessibility and increased DNA methylation of the Gata2 promoter, resulting in decreased Gata2 mRNA levels. RNAi-mediated silencing revealed a dose-dependent effect of Gata2 expression on leukemia cell fitness in vivo . Reduction of Gata2 levels by 25-50 % provided a strong competitive advantage to Cebpa p30/p30 cells while near-complete downregulation of Gata2 expression (>75 %) dramatically reduced cellular fitness. Finally, treatment with the demethylating agent 5-azacytidine restored Gata2 expression in an AML model with Cebpa and Tet2 mutations and caused a significant survival benefit.

Summary/Conclusion: The datasets generated from these models enable deeper insights into the epigenetic and transcriptomic changes that depend on CEBPA and TET2 mutations in a physiologically relevant mutational context. Our results reveal that mutational disruption of CEBPA and TET2 results in down-regulated GATA2 expression, causing aggressive AML. We propose a mechanism in which C/EBPα p30 mediates recruitment of TET2 to regulatory regions in the GATA2 gene to maintain its expression. Conversely, loss of TET2 leads to reduced GATA2 levels, which is restored by 5-azacytidine treatment. Thus, interference with the C/EBPα-TET2 axis may provide entry points for the development of rational targeted therapies in AML patients with these mutations.

S120: ACUTE MYELOID LEUKEMIA REPRESENTS A FERROPTOSIS-SENSITIVE CANCER ENTITY RAISING THE POSSIBILITY FOR NOVEL TARGETING STRATEGIES

A. Narr 1 2 3,* , H. Alborzinia 1 2 , E. Donato 1 2 , F. Vogel 4 , T. Boch 1 , A.-M. Leppä 1 2 , A. Waclawiczek 1 2 , S. Renders 1 2 , A. Schulze 4 , A. Trumpp 1 2

1 Division of Stem Cells & Cancer, German Cancer Research Center (DKFZ); 2 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH); 3 University Heidelberg; 4 Division of Tumor Metabolismus und Microenvironment, German Cancer Research Center (DKFZ), Heidelberg, Germany

Background: Despite advances in the treatment of Acute Myeloid Leukemia (AML), the 5-year patient survival rate remains poor with 15-20%. The majority of patients develop recurrence partly due to resistance mechanisms to classical cell death programs. Exploiting new forms of cell death therefore may allow novel therapeutic options to limit survival or occurrence of resistant clones. Ferroptosis has been identified as non-apoptotic and iron-dependent form of cell death, characterized by an excessive ROS-induced peroxidation of cell membranes. Recently it has been implicated with a higher sensitivity in cancer stem cells and drug-resistant cancer cells, potentially making ferroptosis-inducing (FIN) therapies a promising option.

Aims: We aim to elucidate whether AML represents a ferroptosis-sensitive cancer entity and is susceptive for potential FIN-therapies.

Methods: IC 50 values of ferroptosis inducers were determined and used in combination with the TCGA-LAML and a pan-cancer CRISPR screen dataset (CERES) to describe ferroptosis sensitivity of AML. A drug screen with known ROS-inducers was performed in 9 AML cell lines to identify ferroptosis induction. To mechanistically characterize ferroptotic cell death by the identified drug on the genetic and metabolic level, we performed SLAM-RNA-, RNA-Sequencing, Mass Spectrometry and Metabolite Tracing (Cystine/Ser/Gln) in HL-60 cells in vitro and in vivo . Genetic (CRISPR-Cas9-KO) and chemical inhibition were used to characterize the role of identified hits and pathways, aiming to design new synergistic combination therapies.

Results: We find that AML belongs to the most dependent cancer entities for various ferroptosis-associated genes and represents a ferroptosis-sensitive cancer entity. Furthermore, a 12-gene-ferroptosis-signature allows us to predict the Overall Survival of AML patients and shall help to identify a potential cohort for FIN-therapies. By screening known ROS-inducing drugs for ferroptosis induction, we identify one drug capable of inducing lipid peroxidation and subsequently ferroptosis in multiple AML and other tumor cell lines. Notably, in detail characterization of the drug’s mechanism on the genetic and metabolic level reveal the activity of the iron-metabolism gene HMOX-1 as essential for ferroptosis induction. Mechanistically, we further find that this drug directly inhibits GPX-4, one of the major ferroptosis-suppressive regulators, and facilitates its degradation. Furthermore, we demonstrate that drug treatment results in an upregulation of different metabolic pathways involved in glutathione (GSH) synthesis; namely (1) SLC7A11-mediated Cystine-uptake, (2) transsulfuration pathway as well as (3) glutaminolysis. Genetical and chemical inhibition of these pathways together with drug treatment in in vitro as well as in vivo studies shows strong synergistic effects marking them as new targetable vulnerabilities in AML. Using these insights of the drug’s mechanism of action, we design a synergistic combination therapy and demonstrate its tumor-eradicating capacity in AML cell lines ( in vitro & in vivo ) and in primary de novo as well as relapse AML patient samples ( ex vivo).

Summary/Conclusion: These findings classify AML as ferroptosis sensitive and reveal the capacity of this drug to induce ferroptosis. Detailed characterization of the drug’s mechanism on the genetic and metabolic level allows us to design an effective combination therapy exploiting newly identified vulnerabilities in AML. These results provide a rational basis for the additional pre-clinical and subsequent clinical evaluation of FIN-therapies in AML patients.

S121: CD123-CD33 COMPOUND CAR-T CELLS WITH NOVEL ANTIGEN BINDING DOMAINS PROVIDE A NEW HOPE FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA

Z. Wang 1,* , Y. LU 1 , S. Qiu 1 , M. Wang 1 , D. Xiong 1 , J. Wang 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Tianjin Key Laboratory of Cell Therapy for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology and Blood Diseases Hospital, TianJin, China

Background: CAR-T therapy needs to be optimized to treat myeloid malignancies. Antigen-escape mediated relapse after CAR-T treatment is the vital mechanism responsible for the nondurable response. Targeting CD33 and CD123 simultaneously could cover almost all patients with AML. The off-target cytotoxicity of CD123 CAR-T and CD33 CAR-T, especially on HSPCs, has always been concerned. Several researchers have explored this issue and the conclusions are inconsistent in different studies.

Aims: To improve the outcome of relapse and/or refractory acute myeloid leukemia and explore the influence of CD123-CD33 CAR-T on hematopoiesis.

Methods: We develpoed a panel of anti-CD123 monoclonal antibodies (mAbs) using hybridoma technology, named 6E11, 8D7, 12H7 and 13C3 and incorporated each of the four scFvs into 4-1BB/CD3ζ signaling domain to generate 4 second generation CARs. T cells were infected with lentiviral supernatants to generate CAR-T cells. Comparative analysis were made from the expression property of CAR protein, in vitro and in vivo anti-leukemia efficacy to screen out the best candidate CD123 CAR. Next, we generated a CD123-CD33 dual targeting CAR in a compound way. We established an escape-simulation model to explore whether the compound CAR-T could reduce the risk of relapse associated with antigen loss. Flowcytometry analysis, CFU test and single cell RNA sequecing methods were jointly used to explore the influence of our CD123-CD33 CAR-T on hematopoiesis.

Results: CD123 CAR constructs with four different novel scFvs differed in CAR protein expression ratio. A changing trend of CAR proteins from dropping to rising after encountering target cells was observed in 3 CAR-T cell group except in 8D7 CAR-T. 13C3 CAR was the most suitable construct from the perspective of basic and dynamic expression. 123CAR-T cells with different scFvs mediated variable anti-leukemia effect in vitro and in vitro.

123CAR and 33CAR proteins expressed efficiently on the compound CAR-T cell surface without codon optimization. The compound CAR-T showed superiority in the treatment of antigen-escape mouse model.

FCM analysis showed no significant difference of residual CD34 + cells proportion between NCT cell group and compound CAR-T group. scRNA-seq analysis showed that the most undifferentiated HSC population barely changed among each group. GSEA revealed that the HSC population in CAR-T groups had no difference in the enrichment of quiescence, cell cycle, G2M checkpoint related genes compared with that of NCT group. In addition, no downregulation/enrichment of TGF-beta signaling pathway/ PI3K-AKT-mTOR signaling pathway was observed in compound CAR-T group compared with NCT group.The CFU assay demonstrated that there were still quite numbers of hematopoietic colonies formed in compound CAR-T treatment groups.

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Summary/Conclusion: Taken together, in this study we developed a panel of second generation CD123 CARs with novel scFvs on the basis of 4 novel anti-CD123 mAbs. We proved that scFv sequence has a significantly impact on CAR expression both originally and dynamically and further affects CAR-T function, addressing the importance of scFv development in CAR-T therapy refining. The compound CAR-T in our study minimize the risk of antigen-loss mediated escape. Notably, wildtype DNA sequence of cCAR without codon optimization brings no potential risk such as immunogenic complications. scRNA-seq and CFU assay convincingly demonstrated that the CD123-CD33 compound CAR-T had limited toxicity on hematopoiesis. Thus, the CD123-CD33 compound CAR-T with novel scFvs provides new hope for R/R AML patients.

S122: ROCK INHIBITORS TARGET SRSF2 LEUKEMIA BY DISRUPTING CELL MITOSIS AND NUCLEAR MORPHOLOGY

M. Su 1 2,* , T. Fleisher 3 , I. Grosheva 4 , M. Horev 4 , M. Olszewska 5 , B. Haim 6 , A. Plotnikov 6 , S. Carvalho 6 , Y. Moskovitz 3 , M Minden 7 , N. Chapal-Ilani 3 , E. Papapetrou 8 , N. Dezorella 9 , T. Cheng 10 , N. Kaushansky 3 , B Geiger 4 , L. Shlush 11 12 13

1 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; 2 Molecular and Cellular Biology, Weizmann Institute of Science, Rehovot; 3 Molecular and Cellular Biology, Weizmann Institute of Science; 4 Immunology, Weizmann Institute of Science, Rehovot, Israel; 5 Icahn School of Medicine at Mount Sinai, New York, United States of America; 6 Wohl Institute for Drug Discovery, Weizmann Institute of Science, Rehovot, Israel; 7 University Health Network (UHN), Toronto, Canada; 8 Icahn School of Medicine at Mount Sinai, New York, United States of America; 9 Electron Microscopy Unit, Weizmann Institute of Science, Rehovot, Israel; 10 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China, Tianjin, China; 11 Molecular and Cellular Biology, Weizmann Institute of Science, Rehovot; 12 Division of Hematology, Rambam Healthcare Campus, Haifa; 13 Molecular hematology clinic, Maccabi Healthcare, Tel Aviv, Israel

Background: Spliceosome machinery mutations are common early mutations in myeloid malignancies, however effective targeted therapies against them are still lacking in clinical settings.

Aims: Exploring safe and efficient methods to target hematopoietic cells carrying SRSF2 mutations might be a powerful tool not just for leukemia prevention and treatment, but also to understand the mechanisms behind SRSF2 mutations.

Methods: We generated five SRSF2 Mut (P95H) isogenic cell lines with CRISPR/CAS9 system and performed high throughput drug screen with 3988 compounds in a single dose. After we narrowed down our targets, dose response assay was performed on four different ROCK inhibitors (ROCKi). The leading ROCKi compound was validated in vivo with SRSF2 Mut AML xenograft models. Next, we aimed at understanding why ROCKi target SRSF2 Mut cells and used proteomics, gene expression and imaging of the cytoskeletal system to study the effect of ROCKi on Mut and WT cells.

Results: In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified ROCK inhibitors (ROCKi) as selective inhibitors of SRSF2 Mut in MOLM14 and AML2 cells. To study the efficacy of RKI-1447 on human samples, we conducted six AML patient-derived xenograft (PDX) experiments, with SRSF2 Mut samples. In four out of the six primary AML samples, RKI-1447 significantly reduced engraftment compared to the untreated group and inhibited the engraftment of both leukemic blasts and pre leukemic-HSPCs (preL-HSPCS). RKI-1447 was not toxic to mice nor human cells. ROCKi induced mitotic catastrophe (G2M arrest and multipolar spindles) through their apparent effects on microtubules and nuclear organization, and leading to cell death. Confocal imaging and transmission electron microscopy (TEM) data revealed that SRSF2 mutations induce deep nuclear indentation and segmentation, which is reminiscent of the nuclear shape of Pelger–Huët anomaly (PHA). The nuclear volume and area were significantly higher in SRSF2 Mut compared with WT MOLM14 cells regardless of ROCKi. To investigate why SRSF2 Mut cause the structural changes, we looked into the cytoplasmic intrusions that segment the nuclei, and the structures that connect the lobes of the nuclei by TEM. This examination revealed enrichment of microtubule bundles inside the nuclear indentations. More importantly, these microtubules were located at the tip of the cytoplasmic intrusions suggesting that they take an active role in the nuclear segmentation process. After exposure to RKI-1447, the cytoskeletal and nuclear morphological abnormalities of SRSF2 Mut are augmented to a level which are incompatible with cell survival.

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Summary/Conclusion: We believe it is the first report describing in high resolution the changes in the nucleus after SRSF2 mutations are introduced. The mechanisms we identified are most probably relevant to other SMMs and to the dysplastic phenotype observed in MDS and AML. Accordingly, our findings have wide implications as ROCKi might be even more useful than describe here. Altogether, we provide data from many directions that ROCKi should be tested in clinical trials.

S123: DECODING TRANSCRIPTOMIC AND EPIGENETIC CONSEQUENCES OF STRUCTURAL VARIANTS IN CK-AML AT SINGLE-CELL RESOLUTION

A.-M. Leppä 1,* , K. Grimes 2 , H. Jeong 2 , T. Boch 1 , D Karpova 1 , A. Jauch 3 , F. Grünschläger 1 , A. Dolnik 4 , L. Bullinger 4 , A. Krämer 5 , A. D. Sanders 2 , J. O. Korbel 2 , A. Trumpp 1

1 Division of Stem Cells and Cancer, German Cancer Research Center; 2 Genome Biology Unit, European Molecular Biology Laboratory; 3 Institute of Human Genetics, University of Heidelberg, Heidelberg; 4 Hämatologie, Onkologie und Tumorimmunologie, Charité Universitätsmedizin Berlin, Berlin; 5 Molecular Hematology/Oncology, German Cancer Research Center, Heidelberg, Germany

Background: Acute myeloid leukemia (AML) with complex karyotype (CK) is a heterogenous AML subgroup with dismal response to standard treatment. CK-AML has remained poorly characterized at the molecular level, with mainstream techniques struggling to unravel the complexity of the structural variants (SVs) and link them to phenotypic characteristics. To improve our understanding of molecular dynamics in CK-AML, we established an integrated single cell multi-omics approach that combines the analysis of SVs and nucleosome occupancy (NO) profiling (scNOVA) with concurrent immunophenotypic and transcriptomic profiling (CITE-seq).

Aims: To characterize the genetic and non-genetic intra-patient heterogeneity at single cell resolution in CK-AML.

Methods: For scNOVA analysis, blasts from four CK-AML patients were cultured in the presence of BrdU for the duration of one cell division, followed by single-cell template strand-sequencing (Strand-seq). For CITE-seq analysis, cells were stained with 38 antibody-derived tags for combined analysis of single cell transcriptome and immunophenotype. For functional studies, xenografts of the same CK-AML patients were generated by injection of AML cells into the femoral BM cavity of sublethally irradiated NOD. Prkdc scid . Il2rg null (NSG) mice.

Results: To characterize cellular hierarchies together with epigenetic, transcriptomic, and surface protein features of CK-AML, we applied an integrated single-cell analytical framework, termed scNOVA-CITE, on over 15,000 leukemic cells from four diagnostic CK-AML patient samples. We detected complex clonal evolution patterns driven by simple and complex SVs in all patients. Three out of four patients harbored SVs leading to loss of one TP53 wild-type allele, often accompanied by loss-of-function mutations in the remaining allele. One patient showed parallel evolution of TP53-deleted and TP53 wild-type subclones in the same sample, with the latter clone harboring a lower abundance of SVs compared to the TP53-deleted cells. Moreover, based on NO we inferred lowest activity of TP53 pathway in the cells with TP53 aberrations, emphasizing the role of TP53 in intra-patient cytogenetic evolution as well as inter-patient heterogeneity. Thereby, we unraveled novel single-cell SV landscapes in CK-AML and explored their epigenetic impact.

To assess in depth the functional outcomes of the genetic complexity, we also characterized the transcriptome and cell surface proteome using scNOVA-CITE. Two patients showed high genetic intra-patient heterogeneity and harbored multiple subclones at diagnosis with unique transcriptomic features. When transplanted into NSG mice, we detected predominantly monoclonal engraftment and strongly reduced genomic instability in the respective patient-derived xenografts. The engrafting clones could be traced back to a small subclone in the original patient samples. Molecular analysis of the subclones in the diagnostic samples by scNOVA-CITE revealed converging characteristics leading to stem-like phenotypes. Taken together, these data indicate that cytogenetic evolution resulting in stemness phenotype in CK-AML is advantageous for leukemic stem cell expansion in mice, unfolding novel ways to study regulation of stemness and to identify potential therapeutic targets.

Summary/Conclusion: By developing scNOVA-CITE, we established an integrative single-cell multi-omics framework allowing to characterize genetic, epigenetic, transcriptomic and cell surface proteomic properties of CK-AML patient cells. We provide unique insights into the genetic complexity of CK-AML, and evidence of the resulting functional outcomes.

S124: BONE MORPHOGENETIC PROTEIN 2 TRIGGERS OFF AN IMMUNOSUPPRESSIVE EFFECT OF ΓΔ T CELLS IN ACUTE MYELOID LEUKEMIA

S. Liang 1 2,* , T. Dong 1 , K. Yue 1 , H. Gao 1 , N. Wu 1 , R. Liu 1 , J. Liu 1 , X.-J. Huang 1 2

1 Peking University People’s Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key laboratory of Hematopoietic Stem Cell Transplantation; 2 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking university, Beijing, China

Background: Description of immune landscapes that indulge or restrain survival and proliferation of malignant cells is critical to the improvement of therapeutic strategies. Acute myeloid leukemia (AML) remains a severe life-threatening malignancy and often confronts treatment dilemma in clinic. Although γδ T cells exhibit independent and potent cytotoxicity against leukemic cells in vitro and in the mouse models, efficacy of γδ T cell-based immunotherapy on AML patients has seemed unsatisfying so far. How the anti-AML capacity of γδ T cells is suppressed in vivo is unknown.

Aims: In this study, we aim to dissect the abnormal changes and functions of intrinsic γδ T cells in the context of AML and dig out the correlated factors in the AML tumor environment.

Methods: 1. Immunophenotyping analyses of γδ T cells in bone marrows from newly diagnosed AML patients (n = 62) were detected using flow cytometry, compared with healthy donors (n=51). The levels of TGF-β family members in the supernatants of bone marrows were detected by ELISA kits. 2. Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were stimulated with AML blasts and morphogenetic protein 2 (BMP2) to validate clinical phenotypes. Functional experiments were performed to depict the role of a BMP2-induced subset of γδ T cells in the anti-AML activity of effector γδ T cells. 3. To confirm the findings described above, we established human CD34 + cells-implanted mice models followed by injection with luciferase and GFP co-expressing AML cells.

Results: The proportions of total γδ T cells and Vδ1 + fraction were not significantly different between AML patients and donors. In contrast, the percentage of Vδ2 + T cells was markedly decreased in AML patients. Meanwhile, a key activating receptor of γδ T cells, NKG2D, was dramatically decreased and regulatory receptors CD25 and PD-1 were increased on Vδ2 + T cells in AML patients. The expansion capacity of Vδ2 + T cells was profoundly impaired in AML patients. The functional impairments of γδ T cells were accompanied with an abnormally increased levels of BMP2, rather than TGF-β, BMP4 and ActivinA, in AML patients. Furthermore, we demonstrated that BMP2 directly induced an aberrant γδ T cells subset expressing Vδ2 + CD25 + CD127 low (abbreviated as BMP2-Vδ2) in PBMCs from healthy donors. Consistently, BMP2-Vδ2 cells were significantly increased in the bone marrows of AML patients compared with donors. The frequencies of BMP2-Vδ2 cells correlated to soluble BMP2 levels and the disease status. Distinct from effector Vδ2 cells, BMP2-Vδ2 not only lost the ability of killing AML cells, but also was able to reduce the anti-AML activity of effector Vδ2 cells after co-cultures. In AML-loaded humanized mice, treatment with BMP2 induced the occurrence of BMP2-Vδ2 cells and reduced the survival rate of mice. Injection of BMP2-Vδ2 cells significantly attenuated the activity of effector Vδ2 cells on the elimination of AML cells in mice. Together, these results demonstrated the immunoregulatory effect of BMP2 on the phenotype and function of γδ T cells in AML microenvironment.

Summary/Conclusion: We first reported that dysregulated BMP2 in AML triggered an immunosuppressive subset of γδ T cells, leading to the impairment of anti-AML function of effector γδ T cells. Our findings provide a novel insight into the mechanisms of immunosuppression in the context of leukemia and suggest potential targets for the treatment of AML and other hematopoietic malignancies.

S125: 10-DAY DECITABINE VS. CONVENTIONAL CHEMOTHERAPY (“3 + 7”) FOLLOWED BY ALLOGRAFTING (HSCT) IN AML PATIENTS ≥60 YEARS: A RANDOMIZED PHASE III STUDY OF THE EORTC LEUKEMIA GROUP, GIMEMA, CELG, AND GMDS-SG

M. Lübbert 1,* , P. Wijermans 2 , M. Kicinski 3 , S. Chantepie 4 , W. van der Velden 5 , R. Noppeney 6 , L. Griskevicius 7 , A. Neubauer 8 , M. Crysandt 9 , R. Vrhovac 10 , M. Luppi 11 , S. Fuhrmann 12 , E. Audisio 13 , A. Candoni 14 , O. Legrand 15 , R. Foà 16 , G. Gaidano 17 , D. van Lammeren-Venema 2 , E. F. Posthuma 18 , M. Hoogendoorn 19 , A. Giraut 3 , M. Stevens-Kroef 20 , J. H. Jansen 21 , E. Ammatuna 22 , J.-P. Vilque 4 , R. Wäsch 1 , H. Becker 1 , N. Blijlevens 5 , U. Dührsen 6 , F. Baron 23 , S. Suciu 3 , S. Amadori 24 , A. Venditti 24 , G. Huls 22

1 Department of Hematology, Oncology and Stem Cell Transplantation, Faculty of Medicine, University Medical Center Freiburg, University of Freiburg, Freiburg, Germany; 2 Department of Hematology, Haga Teaching Hospital, The Hague, Netherlands; 3 EORTC Headquarters, Brussels, Belgium; 4 Institut d’Hématologie de Basse Normandie, Centre Hospitalo-Universitaire de Caen, Caen, France; 5 Department of Hematology, Radboud University Medical Centre, Nijmegen, Netherlands; 6 Klinik für Hämatologie, Universitätsklinikum Essen, Essen, Germany; 7 Department of Hematology, Oncology and Transfusion Medicine Center, Vilnius University Hospital Santaros Klinikos, Vilnius University, Vilnius, Lithuania; 8 Department of Internal Medicine, Hematology, Oncology and Immunology, Philipps University Marburg and University Hospital Gießen and Marburg, Campus Marburg, Marburg; 9 Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Medical Faculty, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), Aachen, Germany; 10 Department of Haematology, University Hospital Centre Zagreb, Zagreb, Croatia; 11 Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell’Adulto, University of Modena and Reggio Emilia, Azienda Ospedaliera Universitaria, Modena, Italy; 12 Department of Hematology and Oncology, HELIOS Hospital Berlin-Buch, Berlin, Germany; 13 SC Ematologia Città della Salute e della Scienza Torino, Torino; 14 Clinica Ematologica Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy; 15 Service d’Hématologie Clinique et de Thérapie cellulaire, Hôpital Saint Antoine, APHP, Paris, France; 16 Ematologia, Dipartimento di Medicina Traslazionale e di Precisione, “Sapienza” Università di Roma, Rome; 17 Division of Hematology, Department of Translational Medicine, Università del Piemonte Orientale and Azienda Ospedaliero-Universitaria Maggiore della Carità, Novara, Italy; 18 Department of Internal Medicine, Reinier de Graaf Hospital, Delft; 19 Department of Hematology, Medical Center Leeuwarden, Leeuwarden; 20 Radboud University Medical Center, Nijmegen, Netherlands; 21 Dept laboratory Medicine, Lab Hematology, Radboud University Medical Center, Nijmegen; 22 University Medical Center Groningen, Groningen, Netherlands; 23 University of Liège, Liège, Belgium; 24 Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy

Background: Older, fit AML patients (pts) treated with induction chemotherapy (IC) have poor long-term survival unless HSCT is performed. DNA-hypomethylating agents have become the backbone of AML therapy in pts unfit for IC. Promising outcomes have been reported for the 10-day decitabine (DEC) schedule, suggesting it may be a better treatment prior to HSCT as compared to IC.

Aims: To compare efficacy and safety of 10-day DEC followed by allografting to IC followed by allografting in older fit AML pts.

Methods: This was an international open-label randomized phase III trial ( NCT02172872 ). Key inclusion criteria were newly diagnosed AML, age ≥60 years, eligible for IC, WHO performance status 0-2. DEC was administered 10 days consecutively in cycle 1 (20 mg/m 2 ), 10 or 5 days in subsequent cycles (depending on bone marrow blast clearance at day 28). IC treatment was daunorubicin 60 mg/m 2 x 3 days, cytarabine 200 mg/m 2 x 7 days, followed by 1-3 additional chemotherapy cycles. Pts who had an HLA-matched donor and at least stable disease were encouraged to undergo HSCT after ≥1 treatment cycle. Pts from the DEC arm not receiving HSCT could continue DEC treatment. The primary endpoint was overall survival (OS). Pts were randomized 1:1, stratified by de novo AML vs. secondary AML, age (60-64 vs 65-70 vs ≥70 yrs), and institution. The statistical design aimed to detect a hazard ratio (HR) for OS of 0.75 (HR<1 indicates longer survival for DEC), requiring 441 deaths (one-sided alpha 0.025, 85% power). Due to the slow accumulation of deaths, the final analysis was performed with a clinical cut-off (CCO) date June 30, 2021, following the Data Monitoring Committee recommendation.

Results: Between 12/2014 and 8/2019, 606 pts were randomized, 303 in each arm. Median follow-up was 4.0 yrs. Median age was 68 yrs (range 60-81), 34% of pts were ≥ 70 yrs old and 57% were male, 21% and 32% had good and adverse ELN 2017 risk profile, respectively. A median of 3 DEC cycles (Q1-3: 2-5) and 2 IC cycles (Q1-3: 1-2) were administered. The CR/CRi rate was 48% with DEC and 61% with IC. HSCT as part of the protocol was performed in 122 pts (40%, 30 of them not in CR/CRi) from the DEC and 118 (39%, 11 of them not in CR/CRi) from the IC arm, and in 52% in both arms at any time. By the CCO, 423 deaths occurred. The OS was not significantly different between DEC and IC groups (HR=1.04, 95% confidence interval [CI]: 0.86-1.26; 2-sided p=0.68). The median OS was 15 months (95% CI: 13-18) in the DEC and 18 months (95% CI: 14-22) in the IC group. The OS rates (%) after 1, 2, 3 and 4 years for the DEC and IC groups were 58 vs 59, 38 vs 40, 30 vs 33, and 26 vs 30, respectively. In age subgroups, the estimated HR for OS for DEC vs IC was 1.34 (99% CI: 0.79-2.28) for pts aged 60-64, 1.14 (99% CI: 0.77-1.69) for pts aged 65-69, and 0.84 (99% CI: 0.55-1.26) for pts aged ≥70 yrs (p-value for trend: 0.058). Notable differences in the incidence of grade 3-5 adverse events (%) reported (before HSCT) were: febrile neutropenia (37% for DEC vs 57% for IC), decrease in platelets (24% for DEC vs 32 % for IC), oral mucositis (2% for DEC vs 10% for IC), diarrhea (1% for DEC vs 8% for IC), decrease in neutrophils (19% for DEC vs 13% for IC). The 30-day mortality rate was 3.6% for DEC and 6.4% for IC. The incidence of grade 5 treatment-related adverse events after HSCT was comparable in both treatment arms (25% for DEC and 22% for IC).

Summary/Conclusion: Treatment with DEC resulted in a similar OS and HSCT rate but a better safety profile compared to IC in older AML pts ≥ 60 yrs, eligible for IC.

S126: GEMTUZUMAB-BASED INDUCTION CHEMOTHERAPY COMBINED WITH MIDOSTAURIN FOR FLT3 MUTATED AML. RESULTS FROM THE NCRI AML19 “MIDOTARG” PILOT TRIAL

N. Russell 1,* , C. Wilhelm-Benartzi 2 , J. Othman 3 , R. Dillon 3 , N. Potter 3 , J. Jovanovic 3 , A. Gilkes 4 , L. M. Batten 2 , J. Canham 2 , E. L. Hinson 2 , P. Kottaridis 5 , J. Cavenagh 6 , C. Arnold 7 , M. Dennis 8 , S. Knapper 4

1 Haematology, Guy’s and St Thomas’ NHS Foundation Trust, London; 2 Centre for Trials Research, Cardiff University, Cardiff; 3 Department of Medical and Molecular Genetics, Kings College London, London; 4 School of Medicine, Cardiff University, Cardiff; 5 Haematology, University College London Hospitals NHS Foundation Trust; 6 Department of Haematology, St Bartholomew’s Hospital, London; 7 Haematology, Belfast City Hospital, Belfast; 8 Haematology, The Christie NHS Foundation Trust, Manchester, United Kingdom

Background: Following the RATIFY study Midostaurin in combination with “7 + 3” like chemotherapy has become the standard of care for patients with newly diagnosed FLT3 mutated AML. The ALFA 0701 trial also suggested a benefit for Gemtuzumab Ozogamicin (GO) in FLT3 mutated AML however the combination of GO-based induction with Midostaurin has not been formally assessed.

Aims: To assess the safety of midostaurin in combination with Gemtuzumab and intensive induction therapy and to assess impact of the combination on MRD kinetics

Methods: The NCRI AML19 trial randomised patients to receive DA 3 + 10 (Daunorubicin 60mg/m2 on days 1,3,5 plus AraC 100mg/m 2 bd on days 1-10) plus a single dose of GO 3mg/m 2 on day 1 (DAGO1) or 2 doses (3mg/m 2 , maximum 5mg) on days 1 and 4 (DAGO2) plus 50mg bd of midostaurin (m) for 14 days following completion of chemotherapy. Eligibility included age 18-60 years with a FLT3 -ITD or TKD mutation. Enhanced pharmacovigilance (PV) was performed for four weeks following the first induction course. Midostaurin was also given following second induction (DA 3 + 8 without GO) and 2 courses of HDAC consolidation and as maintenance for 12 cycles in non-transplanted patients. From November 2020 to November 2021, 77 patients were enrolled into the Midotarg pilot receiving DAGO1m (n=39) or DAGO2m (n=38). 59 had a FLT3 ITD and 22 a FLT3 -TKD (and 4 had both). 59 patients have completed course 1 and are evaluable. RT-qPCR MRD monitoring for patients with an NPM1 mutation (n=48) was performed following each cycle of chemotherapy. A descriptive comparison is presented here of toxicity and MRD kinetics with FLT3 mutated patients receiving DAGO1 or DAGO2 without Midostaurin in the same trial.

Results: Treatment compliance in course 1 was 88% for DAGO1m and 100% for DAGO2m. One patient did not receive any Midostaurin because of colitis during induction. Dose interruption for QTc prolongation occurred in 1 patient. A total of 17 SAEs (CTC grade 3 or greater) were reported (GO1, n=11, GO2 n= 6). Day 60 mortality was 0%. No cases of VOD were reported. Blood count recovery was not delayed. Time to neutrophil recovery to 1 x 10 9 /L was 31 and 32 days with DAGO1m and DAGO2m compared to 31 and 32 days in DAGO1 and DAGO2 .. Time to platelet recovery to 100 x 10 9 /L was 28 and 29 days with DAGO1m and DAGO2m respectively compared to 30 days in DAGO1 and DAGO2.

Complete remission (CR plus CRi) was achieved in 51/59 (88%) evaluable patients who have completed induction with DAGOm. In 44 evaluable patients in remission with NPM1 mutation, 34 (74%) were MRD negative in the peripheral blood after course 2. This compares with 63% in 54 evaluable patients with DAGO only. Bone marrow NPM1 transcript levels after courses 1 to 4 were compared with FLT3 mutated patients receiving DAGO1 and DAGO2 without Midostaurin (Figure 1) with a higher proportion of patients becoming MRD negative from course 2 onwards and 81% being MRD negative after course 4.

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Summary/Conclusion: The addition of midostaurin to DAGO using a single or fractionated dose of GO was well tolerated with no evidence of increased toxicity, high response rate and with encouraging clearance of NPM1 mutant transcripts. A randomised study of DAm versus DAGOm in FLT3 mutated AML is planned

S127: QUIZARTINIB WITH DECITABINE AND VENETOCLAX (TRIPLET) IS ACTIVE IN PATIENTS WITH FLT3-ITD MUTATED ACUTE MYELOID LEUKEMIA - A PHASE I/II STUDY

M. Yilmaz 1,* , M. Muftuoglu 1 , H. Kantarjian 1 , C. DiNardo 1 , T. Kadia 1 , G. Borthakur 1 , N. Pemmaraju 1 , N. Short 1 , Y. Alvarado 1 , A. Maiti 1 , L. Masarova 1 , G. Montalban Bravo 1 , S. Loghavi 2 , K. Patel 2 , S. Kornblau 1 , E. Jabbour 1 , G. Garcia-Manero 1 , M. Andreeff 1 , N. Daver 1

1 Leukemia; 2 Hematopathology, MD Anderson Cancer Center, Houston, United States of America

Background: Quizartinib (QUIZ), a potent 2nd generation FLT3 inhibitor (FLT3i) demonstrated synergy with venetoclax (VEN) in AML cell lines and PDX models (Mali Haematologica 2020).

Aims: To evaluate the safety and efficacy of Decitabine (DAC) + VEN + QUIZ triplet in patients (pts) with newly diagnosed (ineligible for intensive induction chemotherapy) or relapsed/refractory (R/R; up to 5 prior chemotherapies) FLT3 ITD mutated AML.

Methods: All pts received 10 days of DAC (20 mg/m2) in Cycle 1. Pts underwent day 14 bone marrow (BM) biopsy, and VEN (400 mg/day starting from day1) was put on hold in pts with BM blasts ≤ 5% or aplasia. Those with day14 BM blast >5% continued VEN for 21 days during cycle 1. In subsequent cycles, DAC was reduced to 5 days. QUIZ (30 or 40 mg/day) was administered daily continuously.

Results: Overall 28 pts were enrolled and evaluable at the time of this report. Of 23 pts with R/R AML (median 3 prior Rx, 78% with ≥1 prior FLT3i including prior gilteritinib in 70%, and 39% had a prior alloSCT), 78% achieved CRc (3 CR, 15 CRi) with 6/16 and 5/18 responders achieving FLT3-PCR and multicolor flow cytometry negativity, respectively. The CRc rates were 75% and 72% in pts who received prior gilteritinib and prior HMA+VEN, respectively. 60-day mortality rate was 5%. Of 5 pts with newly diagnosed AML (median age 69), all achieved CRc (2 CR, 3 CRi) with 4/5 and 2/4 responders achieving FLT3-PCR and MFC negativity, respectively. 60-day mortality in frontline was 0.

RAS/MAPK mutations appear to drive both primary and secondary resistance. Pts with underlying RAS/MAPK mutations had the lowest response rates, at 40% compared to 94% in those without. None of the six pts who achieved a durable remission (> 6months) had RAS/MAPK mutations at baseline ( Figure A ). 4 of 16 (25%) of pts who relapsed (< 6 months of remission) or were refractory to this triplet regimen had baseline RAS/MAPK mutations. We had pre- and post-treatment NGS data from 8 pts who had a response but then relapsed ( Figure B ). Emergent RAS/MAPK mutations were noted in 37% of relapses (3/8), while emergent FLT3 F691L gatekeeper mutations was noted in 25% of relapses (2/8). Interestingly, there were no emergent FLT3 TKD mutations.

No pts developed a dose limiting toxicity (DLT) with 30 mg/day quizartinib, however with the 40mg/day quizartinib 2 pts developed hematologic DLT (grade 4 neutropenia with a <5% cellular bone marrow lasting ≥42 days). Hence, quizartinib 30 mg/day dose was determined to be the recommended phase 2 dose for the triplet. Most common Grade 3/4 non-hematologic toxicities included lung infections (42%) and neutropenic fever (30%). No QTcF prolongations >480 msec were noted.

With a median follow-up (f/u) of 13 months, the median OS was 7.6 months in R/R cohort. Median OS in prior Gilt exposed pts was 6.3 months and ≥1 prior FLT3i exposed pts was 6.3 months. 8/18 R/R pts (including 5/8 prior Gilt exposed pts) underwent ASCT with a median OS of 19 vs 8 months in pts who underwent ASCT versus not (p=0.26). Of the 5 frontline responding pts median OS was 14.5, 2 were alive in CR, 1 died in CR1 post-ASCT, 2 died due to progressive disease at the last f/u.

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Summary/Conclusion: DAC + VEN + QUIZ is active in R/R FLT3-ITD mutated AML pts, with CRc rates of 78% and the median OS of 7.6 months. The high response rate was maintained in prior Gilteritinib exposed pts. Interestingly, RAS/MAPK mutations but not emergent TKD mutations were associated with primary and secondary resistance to the triplet. Accrual continues and updated clinical, NGS and mass cytometry (CyTOF) data will be presented.

S128: A RANDOMISED COMPARISON OF CPX-351 AND FLAG-IDA IN HIGH RISK ACUTE MYELOID LEUKAEMIA. RESULTS FROM THE NCRI AML19 TRIAL

N. Russell 1,* , C. Wilhelm-Benartzi 2 , S. Knapper 3 , L. Batten 2 , J Canham 2 , E. Hinson 2 , U. Malthe Overgaard 4 , J. Othman 5 , R. Dillon 5 , P. Mehta 6 , P. Kottaridis 7 , J. Cavenagh 8 , C. Hemmaway 9 , C. Arnold 10 , M. Dennis 11

1 Guy’s and St Thomas’ NHS Foundation Trust, Department of Haematology, London; 2 Centre for Trials Research; 3 School of Medicine, Cardiff University, Cardiff, United Kingdom; 4 Copenhagen University Hospital, Copenhagen, Denmark; 5 Department of Medical and Molecular Genetics, Kings College London, London; 6 University Hospitals of Bristol and Weston NHS Trust, Bristol; 7 University College London Hospitals NHS Foundation Trust; 8 Department of Haematology, St Bartholomew’s Hospital, London, United Kingdom; 9 Aukland Hospital, Aukland, New Zealand; 10 Belfast City Hospital, Belfast; 11 The Christie NHS Foundation Trust. On behalf of the NCRI AML Working Group, Manchester, United Kingdom

Background: Liposomal daunorubicin and cytarabine (CPX-351) has shown a survival advantage for older patients (>60years) with secondary AML compared to 3 + 7 chemotherapy however in younger patients there is a lack of randomised evidence for benefit. We have previously reported improved survival with FLAG-Ida treatment as treatment intensification for younger patients identified with high risk (HR) AML following induction therapy and for patients with secondary AML (Burnett AK et al. Leukemia. 2018 Dec;32(12):2693-2697) and considered this regimen an appropriate comparator for trials in younger patients.

Aims: We compared CPX-351 with FLAG-Ida in a randomised fashion in patients who were either HR at trial entry based on cytogenetics or identified as HR following induction or at relapse.

Methods: The AML19 trial (ISRCTN78449203) randomised CPX-351 vs FLAG-Ida in 635 patients mainly <60 years with HR AML or MDS (>10% blasts) (median age 53.6 yrs). Three groups of HR patients were randomised 2:1 in favour of CPX with the aim of proceeding to allogeneic transplant. Group 1 (n=195) had known adverse risk cytogenetics (Grimwade et al, Blood 2010,116, 354) and were randomised at diagnosis between 4 courses of CPX-351 and 2 courses of FLAG-Ida followed by MACE/MidAC consolidation. Group 2 (n=263) were HR by validated risk score, had FLT3 -ITD without an NPM1 mutation, had refractory disease and were randomised after induction course 1. Group 3 (n=177) were randomised after course 2 if they had persisting MRD at the time of relapse. Here we present results for Group 1. Group 1 was not powered to claim statistical significance; therefore, these results are intended to be exploratory and hypothesis generating.

Results: Group 1 included 49.2% with de novo AML 20.3% patients with secondary AML and 30.5% with HR MDS.

The Overall response rate(CR/CRi) was 64.8% for CPX-351 and 74.4% for FLAG-Ida (univariate OR:0.57, 95%CI 0.30-1.10, p=0.09). Overall survival (OS) at 3 years was 32% and 24%, median OS was 13.3 months vs 10.2 months (univariate HR:0.83, 95%CI 0.58-1.18, p=0.3) for CPX -351 and FLAG-Ida respectively (Figure 1a). Event free survival (EFS) was not significantly different (HR:0.91 95%CI: 0.50-1.64, p=0.76). Relapse free survival (RFS) at 3 years was 43% and 28%, median RFS was 22.1 months vs 14 months (univariate HR:0.66, 95%CI 0.41-1.06, p=0.09) for CPX -351 and FLAG-Ida respectively (Figure 1b). RFS was significant when adjusting for NPM1 mutation status or FLT3 mutation status using multivariable cox regression model with RFS being better with CPX-351 compared to FLAG-Ida (HR:0.58, 95% CI0.36-0.95, p=0.03). Median duration of remission favoured CPX and was 319.5 days vs 167 days (p=0.046) for CPX vs FLAG-Ida respectively. Haematological toxicity was greater in course 1 with CPX-351 with platelet recovery to 100x10 9 /L at 34.5 days versus 29 days (p<0.001) and neutrophil recovery to 1.0x10 9 /L at 32 days vs 30 days.(p=0.12). Day 30 and day 60 mortality were not significantly different between arms with 4.8% vs 7.3% (p=0.46) and 12.4% vs 11.0% (p=0.77) for CPX-351 and FLAG-Ida respectively.Compliance was better with CPX-351 with 90.7% vs 83.0% receiving the scheduled course 1 dose. More patients receiving CPX-351 were transplanted (50.5% vs 41.5%) with the median number of courses given prior to transplant 2 in both arms.

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Summary/Conclusion: In patients with adverse cytogenetics CPX-351 did not improve response, OS or EFS compared to FLAG-Ida but was associated with better duration of remission and RFS. Further follow-up is needed to determine the clinical significance of those differences.

S129: TAKEAIM LEUKEMIA- A PHASE 1/2A STUDY OF THE IRAK4 INHIBITOR EMAVUSERTIB (CA-4948) AS MONOTHERAPY OR IN COMBINATION WITH AZACITIDINE OR VENETOCLAX IN RELAPSED/REFRACTORY AML OR MDS

G. Garcia-Manero 1,* , E. S. Winer 2 , D. J. DeAngelo 2 , S. Tarantolo 3 , D. A. Sallman 4 , J. Dugan 5 , S. Groepper 6 , A. Giagounidis 6 , K. Götze 7 , K. H. Metzeler 8 , C.-C. Li 9 , L. Zhou 9 , E. Martinez 9 , M. Lane 9 , R. von Roemeling 9 , M. Bohme 9 , A. S. Kubasch 10 , A. Verma 11 , U. Platzbecker 10

1 Luekemia, MD Anderson Cancer Center, Houston; 2 Dana-Faber Cancer Institute, Boston; 3 Nebraska Cancer Specialist, Omaha; 4 Moffitt Cancer Center, Tampa; 5 Novant Health Cancer Institute, Forsyth Medical Center, Winston-Salem, United States of America; 6 Marien Hospital/ Univ. of Dusseldorf Germany, Dusseldorf; 7 Faculty of Medicine Technical University of Munic, Munich; 8 Hematology, Cellular Therapy and Hemostaseology, University Hospital Leipzig, Leipzig, Germany; 9 Curis, Lexington, United States of America; 10 University Hospital Leipzig, Leipzig, Germany; 11 Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, United States of America

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) and FLT3. IRAK4 is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of IRAK4 and have been associated with disease progression and poor prognosis of high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML).

Aims: Assessment of safety, clinical activity, and Recommended Phase 2 Dose (RP2D) of emavusertib as monotherapy or in combination with azacitidine (AZA) or venetoclax (VEN).

Methods: This is an open-label, phase 1/2a dose escalation and cohort expansion trial ( NCT04278768 ). In phase 1 Dose Escalation, patients with R/R AML or HR-MDS are treated with emavusertib monotherapy. Phase 1b includes 2 arms of combination therapy: emavusertib + AZA and emavusertib + VEN. The primary objectives of this study are to assess the safety, clinical activity, and identify the RP2D of emavusertib as monotherapy or in combination with AZA or VEN in R/R AML or HR-MDS. The Phase 2a Dose Expansion includes patients for emavusertib monotherapy: R/R AML with FLT3 mutation, or AML and HR-MDS R/R to HMA with U2AF1 or SF3B1 mutations.

Results: As of December 16 th , 2021, 49 patients have been treated in the phase 1 portion, of whom 43 started by September 30 th , allowing 2 on-study disease assessments. The median number of prior therapies was 2 (range 1-5). Four monotherapy dose levels of emavusertib were tested (200 to 500 mg orally BID). No dose-limiting toxicities were observed at 200 mg and 300 mg BID. No Grade 4 or 5 treatment-related AEs (TRAEs) were reported, and all the TRAEs were manageable. Reversible, manageable Grade 3 rhabdomyolysis occurred in 1/26 (4%) patients at 300 mg BID, 2/17 (12%) at 400 mg BID, and 1/3 (33%) at 500 mg BID. RP2D was determined as 300 mg BID. Of 43 patients starting before Sept 30 th , 2021, 14 had SF3B1 , U2AF1 or FLT3 mutations and demonstrated more promising efficacy. In the 5 evaluable AML patients with spliceosome mutations, 40% reached CR/CRh (1 CR, 1 CRh), both with study duration >6 months. In the 7 spliceosome-mutated HR-MDS patients, 57% reached marrow CR, including 1 with RBC transfusion independence and 1 proceeding to HSCT. One of the three FLT3 -mutated AML reached CR, and 2 became FLT3 -negative. Among the 29 patients without SF3B1/U2AF1/FLT3 mutations, 1 reached CR and 2 PR. Phase 1b and Phase 2a are ongoing. RNA-seq on selected samples showed decrease in relative expression of IRAK4-long isoforms with response to emavusertib.

Summary/Conclusion: Emavusertib is well tolerated and effective in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1/SF3B1/FLT3 mutations. No dose-limiting myelosuppression was reported, suggesting emavusertib may be a candidate for combination therapy. Accrual of Phases 1b and 2a is ongoing.

S130: NPM1 MUTATED AML: IMPACT OF CO-MUTATIONAL PATTERNS - RESULTS OF THE EUROPEAN HARMONY ALLIANCE

A. Hernández-Sánchez 1 2,* , Á. Villaverde-Ramiro 2 , J. Martínez Elicegui 2 , T. González 2 3 , A. Benner 4 , E. Sträng 5 , G. Castellani 6 , C. A. Heckman 7 8 , J. Versluis 9 , M. Abáigar 2 3 , M. Sobas 10 , R. Azibeiro 1 2 , L. Tur 11 , P. J. Valk 9 , K. H. Metzeler 12 , R. Ayala 13 , D. Dall’Olio 6 , J. Tettero 14 , J. Martínez-López 13 , H. Dombret 15 , M. Pratcorona 16 , F. Damm 5 , K. I. Mills 17 , J. Mayer 18 , C. Thiede 19 , M. T. Voso 20 , G. F. Sanz 21 22 , F. Calado 23 , K. Döhner 24 , V. I. Gaidzik 25 , M. Heuser 26 , T. Haferlach 27 , A. T. Turki 28 29 , D. Reinhardt 28 , R. Villoria Medina 11 , M. van Speybroeck 30 , R. Schulze-Rath 31 , M. Barbus 32 , J. E. Butler 33 , J. M. Hernández Rivas 1 2 3 , B. J. Huntly 34 , G. J. Ossenkoppele 14 35 36 , H. Döhner 24 , L. Bullinger 5

1 Hospital Universitario de Salamanca; 2 Instituto de Investigación Biomédica de Salamanca (IBSAL); 3 Centro de Investigación del Cáncer (CIC), Salamanca, Spain; 4 German Cancer Research Center (DKFZ), Heidelberg; 5 Charité Universitätsmedizin Berlin, Berlin, Germany; 6 University of Bologna, Bologna, Italy; 7 University of Helsinki; 8 Institute for Molecular Medicine Finland, Helsinki, Finland; 9 Erasmus University Medical Center Cancer Institute, Rotterdam, Netherlands; 10 Wroclaw Medical University, Wroclaw, Poland; 11 GMV Innovating Solutions, Valencia, Spain; 12 University of Munich, Munich, Germany; 13 Hospital Universitario; 12 de Octubre, Madrid, Spain; 14 Amsterdam University Medical Center, Amsterdam, Netherlands; 15 EA3518 Leukemia Translational Laboratory, Paris, France; 16 Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 17 Queens University Belfast, Belfast, United Kingdom; 18 University Hospital Brnoand Masaryk University, Brno, Czechia; 19 University of Technics Dresden Medical Dept., Dresden, Germany; 20 University of Rome “Cattolica S. Cuore”, Rome, Italy; 21 Hospital Universitario y Politécnico La Fe; 22 Instituto de Salud Carlos III (CIBERONC), Valencia, Spain; 23 Novartis, Oncology Region Europe, Basel, Switzerland; 24 University Hospital of Ulm; 25 Ulm University Hospital, Ulm; 26 Hannover Medical School, Hannover; 27 MLL Munich Leukemia Laboratory, Munich; 28 Essen University Hospital; 29 West-German Cancer Center, Essen, Germany; 30 Janssen Pharmaceutica N.V., Beerse, Belgium; 31 Bayer Pharma AG, Berlin; 32 AbbVie Germany GmbH & Co. KG, Wiesbaden; 33 Bayer AG, Berlin, Germany; 34 Wellcome - MRC Cambridge Stem Cell Institute, Cambridge, United Kingdom; 35 VU University Medical Center; 36 Cancer Center Amsterdam, Amsterdam, Netherlands

Background: Acute myeloid leukemia (AML) is a heterogeneous disease in terms of clinical features, outcomes and genetics. While mutations of NPM1 are usually considered as a favorable prognostic marker, the vast majority of the patients carry several co-mutations that might influence the prognosis. Therefore, a better understanding of the NPM1 mut AML mutational landscape is warranted. The large cohort of AML patients collected within the European HARMONY Alliance provides an excellent basis for this purpose.

Aims: To identify clinically significant co-mutational patterns in NPM1 mut AML in order to establish a revised risk stratification model.

Methods: From the HARMONY Alliance AML database, a total of 1001 NPM1 mut intensively treated patients were selected. Clinically significant co-mutations were evaluated using graphical patterns created with the Gephi tool and confirmed by detailed survival analysis using Kaplan-Meier and Cox regression models. Finally, a novel multi-state risk stratification model for NPM1 mut AML was established.

Results: The study population of 1001 NPM1 mut AML patients included 57% females and median age was 53 years. Regarding ELN2017 classification, 68% of patients were classified into the favorable, 29% intermediate and 3% adverse risk groups. The most frequent co-mutations were DNMT3A (54%), followed by FLT3 -ITD (38%). In total, 24% of patients presented with a high allelic mutant-to-wildtype ratio ≥0.5 ( FLT3 -ITD high ) while 14% had low allelic ratio <0.5 ( FLT3 -ITD low ). Other frequent co-mutations were NRAS (21%), TET2 (20%) and PTPN11 (15%).

The triple mutation pattern of NPM1 mut + FLT3 -ITD high + DNMT3A mut identified a subgroup with adverse prognosis (2-year OS of 25%), similar to NPM1 mut + TP53 mut . The combination of FLT3 -ITD low + DNMT3A mut or FLT3 -ITD high + DNMT3A wt was associated with intermediate prognosis (2-year OS of 45% and 53% respectively). Notably, mutations of NRAS , KRAS , PTPN11 or RAD21 were identified to be associated with better OS. However, in the context of NPM1 mut + DNMT3A mut these mutations did not affect the prognosis when a FLT3 -ITD was present. This information is summarized in a 3-category risk classification model ( Figure 1 ).

The revised NPM1 mut favorable group presented with a 2-year OS of 73%, while for intermediate and adverse groups the OS was 54% and 27% respectively (p<0.001). Regarding relapse free survival (RFS), the median was not reached in the favorable group, while it was 23 months for intermediate and 6 months for adverse group (p<0.001). It should be noted that 171 patients in the NPM1 mut intermediate group would be considered as favorable according to the ELN2017 criteria, as well as 162 patients in the NPM1 mut adverse group were previously classified as intermediate risk. Therefore, our model was able to reclassify 33% of NPM1 mut AML patients in comparison to ELN2017 criteria.

Multivariate analysis of OS in NPM1 mut AML identified the following independent prognostic factors: NPM1 mut model (taking favorable group as reference, HR 1.6 for intermediate and HR 2.7 for adverse group, p<0.001); secondary or therapy-related AML (HR 1.8, p<0.001), WBC at diagnosis >100x10 3 /μL (HR 1.5, p<0.001) and age >60 years (HR 1.4, p<0.001).

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Summary/Conclusion: Analysis of large NPM1 mut AML cohorts allows the discovery of co-mutation patterns associated with prognostic outcome. In accordance, we propose a new genetic stratification model for NPM1 mut AML that identifies 3 groups with different OS and RFS. This model improves ELN2017 criteria as it is able to correctly reclassify 33% of NPM1 mut AML patients.

S131: CLINICAL IMPLICATIONS OF SECONDARY-AML TYPE MUTATIONS IN PATIENTS WITH DE NOVO ACUTE MYELOID LEUKEMIA

K. Sun 1,* , C.-H. Tsai 1 , M.-Y. Lo 1 , M.-H. Tseng 1 , Y.-Y. Kuo 2 , M.-C. Liu 3 , C.-C. Lin 1 , C.-L. Cheng 1 , S.-J. Wu 1 , C.-Y. Chen 1 , B.-S. Ko 1 4 , M. Yao 1 , W.-C. Chou 1 , H.-A. Hou 1 , H.-F. Tien 1 1

1 Hematology; 2 Oncology; 3 Pathology, National Taiwan University Hospital; 4 Hematology, National Taiwan University Cancer center, Taipei, Taiwan

Background: More and more gene mutations have been identified in patients with de novo acute myeloid leukemia (AML). Among them, a set of gene mutations, including SRSF2 , ZRSR2 , SF3B1 , U2AF1 , ASXL1 , EZH2 , STAG2 , and BCOR mutation, are categorized as secondary AML (sAML)-type mutations, for their distinct distribution in secondary AML, compared to primary AML (Lindsley et al, Blood 2015), but the reports regarding the prognostic impact have been scanty, especially in younger patients.

Aims: In this study, we aimed to explore the clinical significance and prognostic implication of sAML-type mutations in non-M3 AML patients.

Methods: We consecutively enrolled 921 de novo non-M3 AML patients; 368 were 60 years or older (older patients) and 553 were younger. Patients with an antecedent history of hematologic diseases, or therapy-related AML were excluded. sAML-type mutations were identified by targeted next-generation sequencing of 54 myeloid malignancies related gene mutations.

Results: A total of 243 (26.4%) patients harbored sAML-type mutations (ST group), 40.2% in older patients and 17.2% in younger ones. Patients in the ST group were significantly older, had a lower WBC count, peripheral blast count and lactate dehydrogenase level at diagnosis. sAML-type mutations were negatively correlated with inv(16), monosomy 17, and complex karyotype, but positively associated with 2017 European LeukemiaNet (ELN)-defined unfavorable-risk genetic category.

Among the patients receiving standard chemotherapy (n=686, 74.5%), the ST group had a significantly lower CR rate (62.9% vs. 81.5%, P< 0.01), especially among younger patients (70.8% vs. 86.7%, P<0.01), but only a trend in older patients (49.0% vs. 59.6, P=0.17). With a median follow-up of 4.7 years, patients in the ST group had a shorter overall survival (OS, median, 2.1 years vs . not reached, P< 0.01) and disease-free survival (DFS, median, 0.4 years vs . 0.9 years, P< 0.01) than the non-ST group. Subgroup analyses showed that sAML-type mutations conferred a significantly poorer DFS (median, 0 years vs. 0.5, P=0.03) and a trend of shorter OS (0.8 years vs . 1.1 years, P=0.08) in older patients, and a significantly worse OS (not reached vs. not reached, P=0.03) and a trend of shorter DFS (0.7 years vs . 1.0 years, P=0.16) in younger patients. The numbers of sAML-type mutations had prognostic impacts on both OS (median, 5.8 years vs. 1.5 vs . 1.3 for patients with 0, 1, and ≥2 mutations, respectively, P<0.01) and DFS (median, 0.9 years vs . 0.6 vs. 0 for those with 0, 1, and ≥2 mutations, respectively, P< 0.01). Intriguingly, these findings were valid among both the younger and older patients. Furthermore, the ELN-defined intermediate-risk patients with sAML-type mutations had similar poor OS and DFS to the ELN unfavorable-risk patients in total cohort (Figure 1), as well as in the older and younger patients. Among the patients with sAML-type mutations, allogeneic hematopoietic stem cell transplantation did improve their outcome (median OS 3.0 vs. 0.8 years, P< 0.01).

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Summary/Conclusion: AML patients with sAML-type mutations had distinct clinical features and poorer outcomes. Incorporating sAML-type mutations can further refine the 2017 ELN risk stratification. It is suggested that AML patients with sAML-type mutations receive more intensive treatment, such as allo-HSCT, and/or novel therapies.

S132: TOLERABILITY AND EFFICACY OF THE FIRST-IN-CLASS ANTI-CD47 ANTIBODY MAGROLIMAB COMBINED WITH AZACITIDINE IN FRONTLINE PATIENTS WITH TP53-MUTATED ACUTE MYELOID LEUKEMIA: PHASE 1B RESULTS

N. G. Daver 1,* , P. Vyas 2 , S. Kambhampati 3 , M. M. Al Malki 4 , R. Larson 5 , A. Asch 6 , G. Mannis 7 , W. Chai-Ho 8 , T. Tanaka 9 , T. Bradley 10 , D. Jeyakumar 11 , E. Wang 12 , G. Xing 13 , M. Chao 13 , G. Ramsingh 13 , C. Renard 13 , I. Lal 13 , J. Zeidner 14 , D. Sallman 15

1 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2 University of Oxford, Oxford, United Kingdom; 3 Healthcare Midwest, Kansas City; 4 City of Hope National Medical Center, Duarte; 5 University of Chicago, Chicago; 6 University of Oklahoma, Oklahoma City; 7 Stanford University, Stanford; 8 University of California Los Angeles, Los Angeles; 9 University of California San Diego, San Diego; 10 University of Miami, Miami; 11 University of California Irvin, Irvine; 12 Roswell Park Comprehensive Cancer Center, Buffalo; 13 Gilead Sciences, Inc., Foster City; 14 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill; 15 Moffitt Cancer Center, Tampa, United States of America

Background: Patients (pts) with TP53 -mutated acute myeloid leukemia (AML) have a poor prognosis, with limited responses to currently available therapies and low survival outcomes, representing a significant unmet medical need. Magrolimab is a monoclonal antibody blocking CD47, a “don’t eat me” signal overexpressed on cells in cancers such as AML. This blockade induces phagocytosis of tumor cells and is synergistic with azacitidine (AZA) via upregulation of “eat me” signals.

Aims: To report tolerability and efficacy data from a phase 1b trial of magrolimab + AZA in frontline pts with TP53 -mutated AML unsuitable for intensive chemotherapy ( NCT03248479 ).

Methods: Frontline pts with AML not suitable for intensive chemotherapy received magrolimab IV starting with a priming dose (1 mg/kg) followed by ramp-up to 30 mg/kg QW or Q2W as the maintenance dose. AZA 75 mg/m 2 was given IV or SC on days 1-7 of each 28-day cycle. Primary endpoints were safety/tolerability and complete remission (CR) rate by European LeukemiaNet (ELN) 2017 criteria.

Results: 72 pts with TP53 -mutated AML were treated (Table). Common all-grade treatment-emergent adverse events (TEAEs) were constipation (52.8%), diarrhea (47.2%), febrile neutropenia (45.8%), nausea (43.1%), fatigue (37.5%), decreased appetite (37.5%), thrombocytopenia (31.9%), peripheral edema (30.6%), and cough (30.6%). Most common grade ≥3 TEAEs were febrile neutropenia (37.5%), anemia (29.2%; grade 3, 26.4%; grade 4, 2.8%), thrombocytopenia (29.2%), pneumonia (26.4%), and neutropenia (20.8%). Grade 3 hemolysis was reported in 1 pt (1.4%); no grade 4 hemolysis was reported. Objective response rate by intent to treat was 48.6% (CR, 33.3%; CR with incomplete hematologic recovery [CRi]/CR with partial hematologic recovery [CRh], 8.3%; morphologic leukemia-free state [MLFS], 1.4%; partial remission, 5.6%). Stable disease and progressive disease (PD) were reported in 16.7% and 5.6% of pts, respectively; 30- and 60-day mortality rates were 8.3% and 18.1%, respectively. Response assessments were unavailable in an additional 4.2% of pts who discontinued due to AEs and 6.9% due to other reasons, prior to the cycle 3 day 1 assessment. Median time to CR/CRi was 2.2 months (range, 1.7-7.2 months) and to CR was 3.0 months (range, 1.8-9.6 months); 14 of 31 (45.2%) evaluable pts with CR/CRi/CRh/MLFS achieved negative MRD by flow cytometry (investigator reported). Of 24 pts with CR, 8 had a longitudinal TP53 variant allele frequency (VAF) assessment and 5 of 8 (63%) had VAF decreased to ≤5%. Treatment was stopped due to stem cell transplant (9 [12.5%]), PD (26 [36.1%]), death (8 [11.1%]), AE (13 [18.1%]), and other (14 [19.4%]). Median durations of CR and CR/CRi were 7.7 months (95% CI, 4.7-10.9 months) and 8.7 months (95% CI, 5.3-10.9 months), respectively. Median overall survival (OS) in 72 pts was 10.8 months (95% CI, 6.8-12.8 months) (figure), with median follow-up of 8.3 months.

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Summary/Conclusion: In high-risk frontline pts with TP53 -mutated AML unsuitable for intensive chemotherapy, magrolimab + AZA showed durable responses and encouraging OS in a single-arm study. A Phase 3 trial of this combination vs standard of care in TP53 -mutated AML (ENHANCE-2; NCT04778397 ) is ongoing.

S133: OFF-THE-SHELF CD33 CAR-NK CELL THERAPY FOR RELAPSE/REFRACTORY AML: FIRST-IN-HUMAN, PHASE I TRIAL

R. Huang 1,* , Q. Wen 1 , X. Wang 1 , H. Yan 1 , Y. Ma 1 , M. Wang 1 , X. Han 1 , L. Gao 1 , L. Gao 1 , C. Zhang 1 , X. Zhang 1

1 Medical Center of Hematology, Xinqiao Hospital of Army Medical University, Chongqing, China

Background: The primary results of CAR-T therapy for patients with R/R AML has shown limited efficacy and severe side-effect. One of the main challenges is that current targets for myeloid malignancies are either widely expressed on healthy hemopoietic stem cells such as CD33 or specific for a group of tumor cells presented as Lewis Y antigen, which could cause lasting bone marrow depression induced by “on target off tumor” side-effect or target negative relapse. Therefore, to receive a balance, we designed a CD33 CAR to recognize AML cells and using NK cells to replace T cells as the carrier to eliminate tumor cells. The CD33 CAR NK cells have combined the wide-expression advantage of CD33 target and the safety of NK cells.

Aims: To evaluate the safety and primary efficacy of CD33 CAR NK cells

Methods: 5 qualified subjects with R/R AML aged between 18 and 65 years-old were enrolled and received round(s) of infusion of anti-CD33 CAR NK cells (6×10 8 , 1.2×10 9 or 1.8×10 9 cells per round after the precondition with Fludarabine (30mg/m 2 ) and Cytoxan 300-500mg/m 2 for 3 days to 5 days, determined by tumor burden at baseline. We investigated the response rate at D28 and treatment related side-effect after the CAR NK cell infusion and the long-term efficacy.

Results: As of data cut (February 26, 2021), 5 pts have finished CAR NK cells infusion. The median age was 43 (18-65) years-old and the median tumor burden before infusion is 31% (21%-77.5%). 4 of 5 patients have received MRD negetive CR at day 28 assessment. In dose group one, three patients have received 3 rounds of CAR NK cells (6×10 8 , 1.2×10 9 and 1.8×10 9 cells) with the interval of 7 days after last round, and only patient 1 developed grade 1 CRS represented as fever after the infusion of 1.8×10 9 CAR NK cells and alleviated within 24h after symptomatic treatment. Patient 1 and patient 2 received MRD negetive CR at day 14 and 21, but patient 2 relapsed at day 43 after first round infusion. In dosage group 2, patient 4 and patient 5 both received one dose of 1.8 ×10 9 cells CD33 CAR NK cells after precondition, patient 5 developed grade 2 CRS presenting as lasting fever for 6 days after infusion and alleviated after 5mg Dexamethasone I.V., both patient 4 and patient 5 recieved MRD negetive CR at day 28 after infusion. At the time of this abstract being uploaded, three patients remain MRD negetive CR.

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Summary/Conclusion: Our primary data of the phase I trial have proved the primary efficacy and safety of CD33 CAR NK cells for patients with R/R AML. The efficacy needs expanded samples and longer follow up.

S134: INTRA-PATIENT FUNCTIONAL HETEROGENEITY OF AML DETERMINES FIRST-LINE TREATMENT RESPONSE

Y. Severin 1,* , Y. Festl 1 , M. Roiss 2 , T. Benoit 3 , T. Heinemann 1 , R. Wegmann 1 , A.-K. Kienzler 3 , M. Bissig 3 , M. Scharl 3 , M. Manz 3 , A. M. Müller 3 , B. Snijder 1

1 Institute of Molecular Systems Biology, ETH Zurich; 2 Medical clinic of Oncology and Haematology, University Hospital Zurich; 3 Medical clinic of Oncology and Haematology, University Hospital Zurich, Zürich, Switzerland

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with limited treatment options and poor long-term survival. AML is characterized by a fast clonal expansion of premature myeloid cells with highly variable combinations of chromosomal aberrations and somatic mutations. Patient-to-patient variability is further complicated by the fact that AML partially recapitulates myeloid maturation with a rare leukemic stem cell population at the top of the hierarchy. Although eradication of bulk tumor cells is necessary, matched targeted therapies against the AML stem cell compartment are likely essential for a long lasting cure of patients. Despite this large tumor heterogeneity in de-novo AML patients, most patients receive the same standard-of-care treatment resulting in large differences of treatment success and overall survival. Functional ex vivo drug-response profiling offers a possible route to tailor individual treatments for AML patients. However, the intra-patient functional heterogeneity of divergent AML cell subpopulations, and their contribution to clinical outcome, have not yet been systematically studied.

Aims: This study has two aims: 1) To analyze the intra-patient functional heterogeneity of AML blasts, and their contribution to the response to first-line treatment. 2) To improve the clinical response predictions from functional ex vivo drug response profiling by explicit analysis of functional tumor heterogeneity.

Methods: In this prospective, non-interventional study, 180 AML patient biopsies were collected with written informed consent (from 44 patients with newly diagnosed AML undergoing intensive induction chemotherapy). Patient matched bone marrow and blood samples were obtained for each donor at three different time points across the course of the treatment. For each sample, we perform ex vivo drug-response profiling analyzed by multiplexed immunofluorescence, automated microscopy, and deep learning-based morphological profiling to determine drug sensitivities on a single-cell level. The drug library includes 100 distinct drugs and drug combinations, including standard of care induction chemotherapy, and single-cell drug sensitivity data is analyzed as a function of cellular clone and blast maturation state. The drug response data is integrated with patient-matched serum cytokine profiling, RNA-sequencing, and clinical data, allowing to identify the cellular subpopulations whose functional response is predictive of treatment response.

Results: Our workflow excelled at predicting patient responses to first line treatment, with an overall predictive power of 85% accuracy and a diagnostic odds ratio of 47. Molecular and morphology-based phenotyping of cancer subpopulations strengthened clinical associations and revealed blast maturity as well as immune activation to be strong predictors of treatment success and the risk of chemotherapy-related infectious complications. Furthermore, the screening of 100 unique drugs and combinations enabled individualized ex vivo treatment predictions, suggesting more potent treatment options than those currently deployed.

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Summary/Conclusion: Maturation-associated intra-tumor drug response heterogeneity is a strong predictor of clinical response to first-line intensive induction chemotherapy in AML. Our multiplexed ex vivo drug screening framework is highly flexible, allowing us to discover, molecularly describe, and target clinically relevant AML subpopulations on a personal basis, leading to improved personalized treatment recommendations.

S135: CRISPR/CAS9 EDITING REVEALS DEPENDENCE OF HUMAN RICHTER SYNDROME AND MURINE CLL CELLS ON SIGNALS FROM B CELL RECEPTOR, CXCR4 RECEPTOR AND MACROPHAGES BUT NOT FROM TOLL-LIKE RECEPTORS IN VIVO

C. Martines 1,* , S. Chakraborty 1 , V. Guastafierro 1 , M. Vujovikj 2 , T. Vaisitti 3 , S. Deaglio 3 , L. Laurenti 4 , A. Dimovski 2 , D. Efremov 1

1 Molecular Hematology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 2 Research Center for Genetic Engineering and Biotechnology, Macedonian Academy of Sciences and Arts, Skopje, North Macedonia, Republic of; 3 Functional Genomics Unit, Department of Medical Sciences, University of Turin, Torino; 4 Department of Hematology, Catholic University Hospital “A. Gemelli”, Rome, Italy

Background: Numerous signals from the microenvironment have been identified that can increase the survival or induce the proliferation of chronic lymphocytic leukemia (CLL) cells in vitro . These signals typically represent various secreted or cell surface ligands that are expressed by different cell types present in the lymph node tumor microenvironment, such as T cells, macrophages and stromal cells, or molecules that would be expected to be released by apoptotic cells, such as apoptosis associated autoantigens or CpG-unmethylated mitochondrial DNA. However, the extent to which these different signals contribute to the growth and survival of the leukemic cells in vivo has still not been fully established.

Aims: To determine the relevance of signals from Toll-like receptors (TLRs), the B cell receptor (BCR) and the chemokine receptor CXCR4 for the growth of murine Eμ-TCL1 CLL cells and human Richter Syndrome (RS) patient-derived xenograft (PDX) cells in vivo .

Methods: To understand the impact of pharmacological inhibition of the TLR pathway, immunocompetent C57BL/6 or immunodeficient NSG mice were transplanted with murine Eμ-TCL1 CLL or human RS-PDX cells, respectively, and were treated with an inhibitor of the kinase IRAK4 or vehicle control. To determine the effects of genetic disruption of the TLR, BCR and CXCR4 pathways, the IRAK4, IgM heavy chain (IGHM) and CXCR4 genes were disrupted by CRISPR/Cas9 editing in the murine Eμ-TCL1 CLL cells or the human RS-PDX lines RS9737, RS1316 and IP867/17 prior to transplantation. The effects of genetic disruption of these pathways on the growth of the malignant cells in vivo were investigated by analyzing the proportion of mutant and wild type alleles in different anatomical compartments of the transplanted mice.

Results: Treatment with the IRAK4 inhibitor significantly prolonged the survival of C57BL/6 mice transplanted with murine Eμ-TCL1 CLL cells and significantly delayed the growth of the human RS-PDX lines RS9737 and RS1316 xenografted in immunodeficient NSG mice. However, genetic disruption of IRAK4 in the human RS-PDX lines RS9737, RS1316 and IP867/17 or of the TLR adaptor MyD88 in the murine Eμ-TCL1 CLL cells did not result in negative selection of these cells in vivo , suggesting that these tumors do not receive or do not rely on TLR signals for their growth and that the therapeutic activity of the IRAK4 inhibitor is not caused by disruption of TLR signaling in the malignant cells themselves. In contrast, genetic disruption of the IGHM or CXCR4 gene was associated with significantly reduced growth of these cells compared to their wild type counterparts and resulted in almost complete disappearance of the mutated cells at later timepoints following transplantation. Analysis of the effects of the IRAK4 inhibitor on other cell types from the tumor microenvironment revealed a significant reduction in the number of macrophages, which in co-culture experiments were shown to strongly protect human Richter syndrome and murine Eμ-TCL1 leukemia cells from spontaneous apoptosis.

Summary/Conclusion: These data provide evidence that signals from the BCR, CXCR4 and macrophages support the growth and survival of CLL and RS cells in vivo and argue against a role for TLR signals in the pathogenesis of CLL. In addition, they suggest that targeting the TLR pathway may potentially provide a therapeutic benefit in CLL, but that this benefit would be derived from inhibition of TLR signaling in monocytes and macrophages rather than inhibition of TLR signaling in the malignant cells themselves.

S136: CONSTITUTIVE VLA-4 ACTIVATION IN CHRONIC LYMPHOCYTIC LEUKEMIA. THE OTHER SIDE OF BCR AUTONOMOUS SIGNALING.

E. Tissino 1,* , R. Bomben 1 , P. C. Maity 2 , F. Pozzo 1 , G. Forestieri 3 , A. Nicolò 2 , A. Härzschel 4 , T. Bittolo 1 , F. M. Rossi 1 , M. Datta 2 , F. Zaja 5 , G. Capasso 1 , G. D’Arena 6 , A. Chiarenza 7 , F. Di Raimondo 7 , H. Jumaa 2 , D. Rossi 3 , G. Del Poeta 8 , T. N. Hartmann 4 , A. Zucchetto 1 , V. Gattei 1

1 Clinical and Experimental Onco-Hematology, Centro di Riferimento Oncologico, Aviano, Italy; 2 Institut für Immunologie, Universitätsklinikum Ulm, Ulm, Germany; 3 Division of Hematology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 4 Department of Internal Medicine, Faculty of Medicine, University Medical Center Freiburg, University of Freiburg, Freiburg, Germany; 5 Dipartimento di Scienze Mediche e Chirurgiche e della Salute, University of Trieste, Trieste; 6 IRCCS Centro Di Riferimento Oncologico Della Basilicata, IRCCS Centro Di Riferimento Oncologico Della Basilicata, Rionero In Vulture; 7 Divisione di Ematologia, Ospedale Ferrarotto, A.O.U. Policlinico-OVE, Università di Catania, Catania; 8 Division Hematology, S.Eugenio Hospital and University of Tor Vergata, Rome, Italy

Background: In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator and key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. In CLL, beside the canonical antigen (Ag)-dependent mechanism, BCR signaling has been recently demonstrated to occur via an autonomous Ag-independent manner.

Aims: To investigate the role of autonomous BCR signaling on constitutive VLA-4 activation state in CLL.

Methods: VLA-4 activation/affinity was determined by flow cytometry (FC) using the conformation-sensitive anti-CD29 mAb HUTS21 and/or by “real-time” (FC) measuring the binding of the VLA-4 ligand LDV-FITC as reported (Tissino et al, J Exp Med, 2018) in: i) 1,984 consecutive CLL all with IGHV gene mutations/BCR features available; ii) sequential samples (0, 14, 30, 60 90 days) from CLL patients (n=26) treated in vivo with ibrutinib (IB) in real-world and from a clinical trial ( NCT02827617 ). HUTS21 staining was also performed in the presence of: plasma depletion/replacement, soluble (s) VCAM-1, fibronectin (FN) and blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). BCR signaling were investigated by Ca ++ influx assay in a 4-OH tamoxifen (4-OHT)-inducible murine TKO cell model (Dühren-von Minden M et al, Nature, 2012).

Results: Out of 1,984 CLL, 1,070 (54%) expressed CD49d (cutoff 30%) and, among them, 250/1,070 (23%) were HUTS21+ (cutoff 20%), indicating an activated VLA-4 conformation. HUTS21 staining was: i) impaired by depletion of plasma from whole blood samples, and reconstituted by specific plasma components (sVCAM-1, FN); ii) impaired by pre-incubation with anti-CD49d HP1/2 blocking mAbs before addition of plasma, sVCAM-1 and FN.

sVCAM-1 was higher in CD49d+ vs CD49d- CLL (p<0.0001); among CD49d+ cases, sVCAM-1 was lower in HUTS21+ (i.e. VLA-4 activated) cases (p=0.0096), suggesting ligand sequestration by activated surface VLA-4.

CLL with mutated IGHV expressed higher levels of activated VLA-4 compared to unmutated IGHV CLL (p=0.001). Higher levels of activated VLA-4 were found in CLL using the IGHV3 and IGHV4 families, compared to cases using the IGHV1 family (p=0.043 and p=0.004). Finally, analysis of BCR stereotypy highlighted higher VLA-4 activation levels in CLL from subset#2 compared to CLL from subset#1 (p=0.02).

To validate these data, murine TKO cells, expressing high VLA-4 levels, were transfected with different BCRs derived from 4 CLL with high level of constitutively activated VLA-4 (TKO-high) and 4 CLL with low level of constitutively activated VLA-4 (TKO-low). Compared to TKO-low cells, TKO-high cells showed a higher autonomous Ca ++ influx (p=0.03), and consistently higher VLA-4 affinity (p=0.01). IB treatment impaired both BCR autonomous signaling and VLA-4 affinity. Notably, anti-IgM stimulation induced high Ca ++ influx and high VLA-4 affinity state in both TKO-high and TKO-low, irrespective of IB treatment.

According to TKO data, a decreased constitutive VLA-4 activation was observed in CLL cells collected at pre-treatment and at day 14, 30, 60 and 90 from patients on IB, confirming an IB-dependent impairment of VLA-4 activation via BCR signal.

Summary/Conclusion: The presence of a constitutively activated form of VLA-4 is observed in a fraction of CD49d+ CLL, due to a continuous VLA-4 inside-out stimulation derived from autonomous BCR signaling.

S137: SMALL EXTRACELLULAR VESICLES IN THE LEUKEMIA MICROENVIRONMENT SUSTAIN CLL PROGRESSION BY HAMPERING T CELL-MEDIATED ANTI-TUMOR IMMUNITY

E. Gargiulo 1,* , E. Viry 1 , P. E. Morande 1 2 , A. Largeot 1 , S. Gonder 1 3 , F. Xian 1 4 , N. Ioannou 5 , M. Benzarti 1 3 , F. Kleine Borgmann 1 3 6 , M. Mittelbronn 1 3 7 , G. Dittmar 1 3 , P. V. Nazarov 1 , J. Meiser 1 , B. Stamatopoulos 8 , A. G. Ramsay 5 , E. Moussay 1 , J. Paggetti 1

1 Luxembourg Institute of Health, Luxembourg, Luxembourg; 2 Instituto de Medicina Experimental, Buenos Aires, Argentina; 3 University of Luxembourg, Luxembourg, Luxembourg; 4 University of Vienna, Vienna, Austria; 5 King’s College London, London, United Kingdom; 6 Centre Hospitalier de Luxembourg, Luxembourg; 7 Laboratoire national de santé, Dudelange, Luxembourg; 8 Université Libre de Bruxelles, Brussels, Belgium

Background: Small extracellular vesicles (sEV) are nano-sized particles released by every cell and found in all biofluids. Given their composition and abundance, sEV are commonly involved in cell-to-cell communication through the transfer of genetic material and proteins. Furthermore, sEV possess direct functions carried out by sEV-ligands capable to affect the biological functions of targeted cells. In cancer, tumor-derived sEV are involved in the re-education of microenvironment (ME) cells promoting tumor proliferation, immune escape and metastasis. We previously demonstrated that leukemia-derived sEV are involved in the re-education of surrounding cells and increased immune escape. Indeed, chronic lymphocytic leukemia (CLL)-derived sEV induce stromal cell conversion into cancer-associated fibroblasts (Paggetti et al ., Blood, 2015), and modulate PD-L1 expression in monocytes (Haderk et al . Science Immunology, 2017).

Aims: The goal of the present work was to characterize leukemia ME-derived sEV (LME-sEV) and to evaluate their role in the disease development and progression in vivo .

Methods: To obtain a biological representation of sEV in CLL microenvironment, we isolated LME-sEV directly from spleens of leukemic mice, obtaining a complex mix of sEV released by both CLL and ME cells alike. Small EV characterization was performed using a wide range of techniques, including qPCR, mass spectrometry and single sEV flow cytometry (FC). The effect on target cells was evaluated both ex vivo and in vivo using high-throughput techniques, FC, qPCR and cytotoxic assay. Small EV impact on CLL development in vivo was evaluated by generating a novel preclinical mouse model in which sEV release is genetically impaired due to Rab27a/b knock-out. Finally, we analyzed the expression of sEV-related genes in a cohort of 144 CLL patients using qPCR followed by regression analysis.

Results: LME-sEV showed a distinct proteome (A ) and RNA contents compared to healthy counterparts (HCME-sEV), including miRNA enriched in the plasma of CLL patients. Furthermore, FC-based immune checkpoint (ICP) screening showed the presence of multiple ICP ligands anchored on CLL-derived sEV (CD20 + subset of LME-sEV) (B ), while high expression of the corresponding ICP receptors was found on T cells from matching LME. We also found that LME-sEV are internalized by different T cell subsets, thus we performed in vivo and ex vivo functional studies to assess sEV impact on T cells. High-throughput analysis of cells isolated from spleens of control mice treated with LME-sEV revealed considerable physiological changes mainly in CD8 + T cells. Indeed, CD8 + T cells showed alterations in their transcriptome, proteome and metabolome leading to cell exhaustion, decreased functions and survival. In line with this, absence of sEV dramatically delayed CLL progression in vivo . This effect was due to CLL inability to escape immune surveillance in absence of sEV and this was rescued by LME-sEV treatment (C ). Finally, we identified a consistent sEV gene signatures in CLL patients correlating with treatment-free survival, overall survival, and with unfavorable clinical parameters routinely used in CLL diagnosis and prognosis (D ).

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Summary/Conclusion: By using different preclinical murine models and strategies, our results demonstrated for the first time that sEV in CLL ME play a key pro-tumoral role in leukemia development by negatively affecting the anti-tumor immune response. Furthermore, high expression of sEV-related genes correlated with poor survival and clinical parameters in CLL patients, suggesting sEV profiling as prognostic tool in CLL.

S138: MICROENVIRONMENT-REGULATED TRANSCRIPTIONS FACTORS AHR AND HIF-1Α EXPRESSION IN TREGS PROMOTE CLL PROGRESSION BY IMPAIRING CD8+ T-CELL MEDIATED ANTI-TUMOR IMMUNITY

G. Pagano 1,* , M. Wierz 1 , I. Fernandez Botana 1 , S. Gonder 1 , E. Gargiulo 1 , E. Moussay 1 , J. Paggetti 1

1 Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg

Background: Chronic Lymphocytic Leukemia (CLL) progression is highly dependent on complex interactions between tumor cells and the tumor microenvironment (TME). Indeed, CLL cells can modify stromal cells and immune cells to promote the survival of the leukemic clone and to escape from the immune system surveillance. Within the TME, regulatory T cells (Tregs) represent a subtype of CD4+ T cells with immunosuppressive abilities, causing the evasion of cancer cells from the immune system. We previously characterized extensively the immune microenvironment of pre-clinical CLL mouse models using mass cytometry, and we described a significant increase in the Tregs subsets with an enhanced immunosuppressive and activated phenotype compared to non-leukemic animals (Wierz et al., Blood, 2018). Interestingly, TIGIT+ Tregs are more immunosuppressive than their TIGIT- Treg counterparts and express higher levels of several transcription factors, including Ahr and Hif1α (Joller et al., Immunity, 2014), both involved in the cellular response to microenvironment-mediated stimuli.

Aims: The aim of the present study is to investigate the role of AHR and HIF-1α in the suppressive ability of Tregs during CLL development.

Methods: We generated conditional knock out mice (cKO) lacking Ahr or Hif1a genes exclusively in Tregs ( Foxp3 YFP-Cre Ahr fx/fx and Foxp3 YFP-Cre Hif1a fx/fx mice). We then performed adoptive transfer (AT) of CLL cells obtained from diseased Eµ-TCL1 mice into cKO and control mice. In order to decipher the mechanism by which AHR and HIF-1α pathways in Tregs affect CLL progression, we analyzed the splenic TME of recipient mice and evaluated immune checkpoint expression and cytokine production. Finally, we evaluated the suppression ability of the regulatory T cells with ex vivo suppression assays.

Results: Generated cKO mice showed no sign of abnormalities or autoimmune phenotypes, and immune cells phenotyping revealed no major differences. However, we showed that CLL growth in cKO mice was drastically delayed compared to the control mice (A ). Interestingly, this decrease was mitigated when CD8+ T cells were depleted. The analysis of the splenic TME in recipient cKO mice revealed an increase in IL-17 and TNF-α production, two major T-cell cytokines, in CD4+ T cells as compared to their WT counterparts. We also measured the expression of immune checkpoints and activation markers in the different T cell subpopulations and observed that Tregs lacking AHR or HIF-1α show decreased levels of the immune checkpoints CTLA-4 and TIGIT, two key proteins for the suppressive functions of Tregs.

Ex vivo suppression assays demonstrated an increased proliferation of CD8+ T cells in the presence cKO Tregs (B-C). This result confirmed the decreased suppressive ability of Tregs in absence of the two transcription factors, explaining the observed delay in CLL progression in cKO mice.

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Summary/Conclusion: Altogether, these results indicate that the TME-regulated transcription factors AHR and HIF-1α in Tregs are crucial for CLL development by promoting escape to anti-tumor immune response, and therefore represent potential therapeutic targets during CLL progression.

S139: BCOR DELETION SUSTAINS NOTCH1 SIGNALLING ACTIVATION TO ACCELERATE CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PROGRESSION TOWARD RICHTER TRANSFORMATION IN MICE

C. Rompietti 1,* , D. Sorcini 1 , F. De Falco 1 , E. Dorillo 1 , F. M. Adamo 1 , E. C. Silva Barcelos 1 , A. Stella 1 , A. Scialdone 1 , A. Esposito 1 , R. Arcaleni 1 , B. Bigerna 2 , G. Martino 3 , L. Moretti 2 , M. G. Mameli 2 , C. Geraci 1 , L. Sandoletti 1 , A. Cipiciani 1 , E. Rosati 4 , B. Falini 1 , P. Sportoletti 1

1 Medicine and Surgery, University of Perugia (Center for Hemato-Oncology Research); 2 Hospital of Perugia, Perugia; 3 Pathology Unit, Azienda Ospedaliera Santa Maria di Terni, University of Perugia, Terni; 4 Medicine and Surgery, University of Perugia, Perugia, Italy

Background: BCL6 co-repressor (BCOR) is a transcription factor involved in various biological processes including lymphoid development. BCOR disruptive mutations were found in up to 2% of CLL and frequently associated with aberrations of NOTCH1 , one of the most common genetic alterations with poor prognosis in CLL and Richter transformation (RT). Recent evidence also indicates that BCOR is involved in NOTCH signalling suppression during embryogenesis. These data indicate the need for further investigation on the role of BCOR mutation and its interplay with NOTCH1 in CLL pathogenesis.

Aims: We aim to elucidate the impact of Bcor deficiency in CLL and RT, focusing on the role of active NOTCH1 signalling.

Methods: We used a conditional knockout mouse of Bcor (Sportoletti et al, Leukemia 2021) crossed with CD19-Cre mice, to specifically delete Bcor in B-cells, and with the Eμ-TCL1 mouse model of CLL. Mice were characterized for disease phenotype (using flow-cytometry and histological analyses), overall survival and drug response. NOTCH1 activity was assessed by western blot (WB) for the NOTCH1-intracellular domain (NOTCH1-IC) and HES1 expression.

Results: B-cell restricted loss of Bcor in Eµ-TCL1 mice significantly expanded leukemic CD5+CD19+ cells in the peripheral blood (PB; p <0.05), spleen ( p <0.01), bone marrow (BM; p <0.001), compared to Eµ-TCL1 control mice. No leukemic cells were found in BcorCD19Cre+ mice. Cellular changes resulted in a poorer survival of double mutant mice compared to single-mutant and wild-type (WT) controls (median survival of 334 days vs unreached in BcorCD19Cre+;Eμ-TCL1 vs the other groups, respectively). Adoptive transfer of spleen cells (CD5+CD19+ leukemic burden >50%) resulted in a more rapidly lethal disease in recipient of BcorCD19Cre+;Eμ-TCL1 compared to Eμ-TCL1 cells (median survival of 37.5 vs 74 days, respectively, p <0.05; Figure1 A ). At necropsy, double mutant mice presented massive splenomegaly, whose histopathological examination revealed a diffuse infiltration by high-mitotic blastoid cells, significantly increased in size, defining a high-grade lymphoid malignancy distinct from the leukemia of Eμ-TCL1 counterparts (Fig.1 B ). In order to gain mechanistic insight relating to the phenotypic observations, we measured NOTCH1 activity. BcorCD19Cre+;Eμ-TCL1 splenic CD5+CD19+ cells showed significantly increased levels of the active NOTCH1-IC and the up-regulation of its direct target HES1, compared to leukemic cells from Eμ-TCL1 mice and CD19+ cells from non-leukemic BcorCD19Cre+ and WT mice used as control (Fig.1 C ). In vivo treatment with the NOTCH1 inhibitor Bepridil resulted in a significant growth delay of CD5+CD19+ cells in the PB of mice transplanted with BcorCD19Cre+;Eμ-TCL1 leukemic cells compared to vehicle. At sacrifice, Bepridil caused a significant reduction of spleen dimensions and CD5+CD19+ cellularity, associated with reduced levels of active NOTCH1-IC, c-MYC and HES1 compared to vehicle (28%, 54% and 38% reduction, respectively). NOTCH1 inhibition significantly improved the survival of diseased mice (median survival of 39 vs 33 days in Bepridil vs vehicle, respectively; N=4, p <0.05).

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Summary/Conclusion: We showed for the first time the tumour suppressor activity of Bcor in a CLL mouse model, ultimately leading to transformation towards a high-grade lymphoma, mimicking human RT. Mechanistically, we implied NOTCH1 signalling activation in Bcor loss mediated tumorigenesis with potential for targeted treatment for high-risk CLL and RT patients.

S140: SINGLE-CELL MULTIOMICS ANALYSES REVEAL COMPLEX INTRA-PATIENT HETEROGENEITY IN RELAPSED CLL FOLLOWING VENETOCLAX THERAPY

R. Thijssen 1 2,* , L. Tian 1 2 , C. Flensburg 1 2 , M. A. Anderson 1 2 3 4 , A. Jarratt 1 2 , H. Peng 1 2 , I. Majewski 1 2 , C. Tam 3 4 , J. Seymour 3 4 , P. Blombery 3 4 , M. Ritchie 1 2 , D. Huang 1 2 , A. Roberts 1 2 3 4

1 The Walter and Eliza Hall Institute of Medical Research; 2 Medical Biology, University of Melbourne; 3 Clinical Haematology, Royal Melbourne Hospital and Peter MacCallum Cancer Centre; 4 Medicine, University of Melbourne, Melbourne, Australia

Background: Venetoclax (VEN) is the first approved-in-class BH3-mimetic therapy that inhibits pro-survival BCL2 to induce apoptosis. It is now a standard of care for patients with chronic lymphocytic leukaemia (CLL) and acute myeloid leukaemia. Despite high remission rates, secondary resistance remains problematic with disease progressing on continuous therapy in most patients. While previous studies by ourselves and others demonstrated a BCL2 mutation (Blombery et al., 2019 Cancer Disc) or MCL1 amplification (Guièze et al., 2019 Cancer Cell) confer resistance in patient samples, it is clear that these only account for a fraction of tumour cells at disease progression and are not present in all leukaemias. Given that these changes only provide partial explanations, other mechanisms must operate to subvert the action of VEN.

Aims: To uncover why VEN fails in patients by assessing heterogeneity, distinguishing cells with known changes (e.g., BCL2 mutation, MCL1 amplification) and to elucidate what is happening in the cells not harbouring these alterations.

Methods: We applied a single-cell (sc) sequencing method that simultaneously measures gene expression and full-length transcripts to determine genotype on samples from patients with progressive CLL with long term follow-up on trials of continuous VEN therapy (Figure 1).

Results: By applying scRNA-seq to 161,499 tumour cells, we discovered a high degree of inter- and intra-patient heterogeneity. The majority of CLL cells displayed an altered transcriptional profile at relapse compared to before VEN treatment. 8/13 VEN-relapse samples demonstrated a complex sub-clonal architecture (Figure 1).

By applying scLong-read sequencing, we detected BCL2 mutations in 4/13 samples at relapse. We also identified subclones with loss of NOXA or BAX , but overall, alterations in genes for the pro-apoptotic proteins were uncommon. In contrast, altered expression of the pro-survival genes was common and high MCL1 expression was seen in most of the CLL cells in 11/13 relapses. This could only be fully explained by MCL1 amplification in 1 patient and partially in 2 (Figure 1).

At VEN resistance, we identified near universal activation of NF-ΚB in circulating tumour cells and this was highly correlated with MCL1 expression. We next demonstrated that the MCL1 locus was transcriptionally targeted by NF-ΚB accounting for the MCL1 increase. NF-ΚB activation and associated high MCL1 was also observed in cells harbouring the BCL2 mutations.

Strikingly, NF-ΚB activation (and consequent increased MCL1 ) dissipated after VEN cessation and commencement of a BTK inhibitor as the leukaemic cells substantially recovered their in vitro sensitivity to VEN. Consistent with the hypothesis that VEN was driving these changes, NF-ΚB activation (and high MCL1 ) was not detected in a patient (CLL26) with disease relapse 3 years after ceasing VEN in CR. The disease in this patient responded promptly to VEN reinduction.

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Summary/Conclusion: Taken together, our findings provide a much clearer understanding of resistance to VEN and provide impetus for improved VEN-based clinical management of CLL patients. Given the multiple ways a CLL population in a patient can become resistant to ongoing VEN, it seems unlikely that simply adding other drugs at relapse will be durably effective in most patients. The data pinpoint NF-ΚB activation as a biomarker for in vivo VEN-resistance and provide a specific biological rationale for ceasing VEN in deep response, as is currently being used in time-limited and explored in response-directed regimens.

S141: ELICITING ANTI-TUMOR T CELL ACTIVITY IN CHRONIC LYMPHOCYTIC LEUKEMIA WITH BISPECIFIC ANTIBODY-BASED COMBINATION THERAPY

D. Papazoglou 1,* , L. Ysebaert 2 , N. Ioannou 1 , B. Apollonio 1 , P. Patten 3 , S. Herter 4 , M. Bacac 4 , A. Deutsch 5 , C. Klein 4 , A. Vardi 6 , A. Quillet-Mary 2 , A. Ramsay 1

1 Hemato-oncology, King’s College London, London, United Kingdom; 2 Centre de Recherches en Cancérologie de Toulouse, Toulouse, France; 3 King’s Health Partners, King’s College Hospital, London, United Kingdom; 4 Roche Innovation Center Zurich, Roche Pharma Research and Early Development, Zurich, Switzerland; 5 Medical University of Graz, Graz, Austria; 6 Hematology Department and HCT Unit, G. Papanikolaou Hospital, Thessaloniki, Greece

Background: Identifying effective combination immunotherapy could offer hope to chronic lymphocytic leukemia (CLL) patients who relapse on previous lines of therapy. Although prior studies have described an exhausted and pro-tumor T cell state, there may be potential to stimulate anti-CLL cytolytic T cell activity with novel therapy.

Aims: Investigate the ability of the CD20-targeted T-cell-engaging bispecific antibody glofitamab (CD20-TCB) to overcome T cell exhaustion and tumor microenvironment (TME)-mediated immunosuppression in CLL.

Methods: We pre-clinically investigate CD20-TCB efficacy in CLL utilizing immune and TME model assays that include in vitro functional T cell assays, patient-derived xenografts and lymph node organotypic models.

Results: Our previous studies of the CLL lymph node (LN) TMEs suggested that low numbers of infiltrated CD8 + T cells likely contribute to the generation of a “cold” TME that could represent a barrier to effective therapy. Here, we show that CD20-TCB treatment induced the activation, proliferation and migration of previously exhausted patient CD4 + and CD8 + T cells and triggered the release of immunostimulatory cytokines related to immune cell recruitment and cytotoxic function. Additionally, we detected enhanced cytolytic (perforin + ) immune synapse activity in CD20-TCB-treated cultures which correlated with high levels of T cell-mediated CLL cell death. Importantly, bispecific antibody treatment also induced high levels of anti-CLL T cell activity in both ibrutinib responding and refractory patient samples.

Given the emerging evidence that endothelial cells (ECs) that reside within the TME can possess immunomodulatory activity, we next evaluated CD20-TCB-mediated T cell responses in the presence of this stromal cell compartment. We established triple culture assays that allowed patient T and CLL cells to be cultured with CLL-activated ECs (CLL-ECs). These assays revealed that TCB-activated T cells in the presence of CLL-ECs showed a reduced ability to kill target CLL cells compared to healthy ECs. Flow cytometric and multicolor confocal microscopy analysis revealed that CLL-ECs in vitro and in situ expressed increased levels of ICAM-1 and PD-1 ligands that we demonstrated contribute to T cell suppression. In order to overcome this TME-mediated inhibition and enhance anti-CLL CD20-TCB activity, we next investigated combination immunotherapy. Combination of CD20-TCB with a CD19-4-1BBL fusion protein (tumor-targeted co-stimulatory drug) resulted in enhanced CLL cell killing in both T:CLL and triple T:CLL-EC culture assays.

Patient-derived xenograft models further confirmed the ability of bispecific antibody combination therapy to induce higher levels of anti-CLL T cell activity compared to monotherapy. Additionally, the pairing of CD20-TCB with CD19-4-1BBL promoted the proliferation and activation of patient T cells within splenic TMEs. Finally, we established 3D organotypic cultures of excess diagnostic LN biopsies that preserve the intact TME cellular composition and spatial organization. LNs of treatment naïve, as well as ibrutinib and venetoclax relapsed CLL patient samples, were treated ex vivo with CD20-TCB combination for subsequent 3D image analysis. Importantly, combination immunotherapy triggered improved anti-tumor T cell killing function and promoted an inflammatory cytokine-rich “hot” TME.

Summary/Conclusion: Collectively, this preclinical study supports the concept of triggering cytolytic T cell activity in CLL with bispecific antibody combination immunotherapy that could help overcome T cell exhaustion and the immunosuppressive TME.

S142: DECIPHERING THE COMPLEXITY OF T CELLS IN BLOOD AND LYMPH NODES OF PATIENTS WITH CLL BY INTEGRATIVE SINGLE-CELL RNA-SEQ AND MASS CYTOMETRY ANALYSES

L. Llaó Cid 1 , J. Wong 1 , M. Wierz 2 , Y. Paul 1 , S. Gonder 2 , I. Fernandez Botana 2 , T. Roider 3 , S. Dietrich 3 , D. Colomer 4 , P. Lichter 1 , M. Zapatka 1 , E. Moussay 2 , J. Paggetti 2 , M. Seiffert 1,*

1 Molecular Genetics, B060, German Cancer Research Center, Heidelberg, Germany; 2 Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg; 3 Department of Medicine V, University Clinic Heidelberg, Heidelberg, Germany; 4 Hematopathology Unit, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

Background: Abnormal distribution and impaired function of T cells are key features of chronic lymphocytic leukemia (CLL), a malignancy of mature B lymphocytes that develops in secondary lymphoid tissue and blood. These defects have been linked to failure of immune control and resistance to immunotherapy including immune checkpoint or CAR T-cell therapy. A better characterization of T cells and their loss of function in CLL will help to improve such treatment approaches.

Aims: The goal of this study was an in-depth characterization of the T-cell compartment in blood and tissue samples of patients and mouse models with CLL to gain insights into the spectrum of phenotypes and transcriptional programs of T cells and the underlying mechanisms of their development.

Methods: We performed mass cytometry (CyTOF) with a panel of 35 antibodies to characterize T cells in blood (n=8), bone marrow (n=3), and lymph nodes (n=21) of CLL patients, as well as reactive lymph nodes of non-tumor patients (n=13). We further generated single-cell transcriptome and T-cell receptor sequencing data of T cells from lymph nodes of CLL patients (n=5) and spleen samples of the Eµ-TCL1 mouse model (n=3), and performed integrative analyses of all data sets.

Results: CyTOF analysis allowed for the identification and quantification of 15 clusters of CD4 + and 14 clusters of CD8 + T cells in blood, lymph node and bone marrow samples. T cells in blood and bone marrow were clearly distinct from lymph node derived cells, and several cell subsets showed a positively correlated abundance. A comparative analysis revealed an accumulation of several regulatory T-cell subsets, as well as T cells harboring an exhausted and dysfunctional phenotype in CLL lymph nodes.

Single-cell transcriptome and T-cell receptor sequencing demonstrated the presence of clonally expanded and gradually exhausted T-cell clusters in human and murine CLL, and provided insights into the transcriptional programs and regulatory networks of these cell subtypes. Using CellPhoneDB (Efremova et al., Nature Protocols, 2020) and CellChat (Jin et al., Nature Communications, 2021) repositories, we identified novel ligand-receptor-interactions between CLL and T-cell clusters with impact on anti-tumor immune control which are currently tested in preclinical models for intervention therapy.

Summary/Conclusion: Altogether, our study provides a detailed characterization of the T-cell compartment in CLL that helps us to understand T-cell exhaustion and suggests novel targets to improve immunotherapy for patients with CLL and likely also other malignancies.

MZ, EM, JP and MS share senior authorship.

S143: TRANSCRIPTOMIC CHARACTERIZATION OF MRD RESPONSE AND NON-RESPONSE IN PATIENTS TREATED WITH FIXED-DURATION VENETOCLAX-OBINUTUZUMAB

O. Al-Sawaf 1 2 3,* , H. Y. Jin 4 , C. Zhang 1 , Y. Choi 4 , S. Balasubramanian 4 , S. Robrecht 1 , A. Kotak 5 , N. Chang 5 , A.-M. Fink 1 , E. Tausch 6 , C. Schneider 6 , M. Ritgen 7 , K.-A. Kreuzer 1 , B. Chyla 8 , J. Paulson 4 , B. Eichhorst 1 , S. Stilgenbauer 6 , Y. Jiang 4 , M. Hallek 1 , K. Fischer 1

1 Department I of Internal Medicine and Center of Integrated Oncology Aachen Cologne Bonn Duesseldorf, University Hospital of Cologne, Cologne, Germany; 2 Cancer Institute, University College London; 3 Francis Crick Institute, London, United Kingdom; 4 Genentech Inc., South San Francisco, CA, United States of America; 5 Roche Products Ltd, Welwyn Garden City, United Kingdom; 6 Department III of Internal Medicine, Ulm University, Ulm; 7 Department II of Internal Medicine, University of Schleswig Holstein, Kiel, Germany; 8 AbbVie Inc., North Chicago, IL, United States of America

Background: Minimal residual disease (MRD) is a key surrogate for the depth of remission of CLL. The highest rates of undetectable MRD have been observed with regimens using the BH3 mimetic venetoclax. In CLL14, most patients (pts) receiving venetoclax-obinutuzumab (Ven-Obi) had undetectable MRD (uMRD <10 -4 ) at the end of treatment (EoT). However, a subgroup of pts remained MRD positive, regardless of TP53 aberrations or unmutated IGHV status. The biological drivers of response or non-response have so far not been elucidated.

Aims: This study explores whether differential transcriptomic profiles can discriminate between pts with vs without deep MRD remissions, particularly after Ven exposure. This might eventually allow for biology-informed treatment development for pts in this high-risk disease setting.

Methods: NGS-based MRD measurements (clonoSEQ) from peripheral blood (PB) at follow-up month 3 were grouped into uMRD <10 -6 (MRD6), 10 -6 ≤ and <10 -4 (MRD4/5), and detectable MRD ≥10 -4 (MRD+). Pre-treatment CD19-enriched PB samples, and samples collected at relapse from a subset of pts, were subjected to bulk RNAseq on the Illumina NovaSeq platform. Transcriptomic clustering, linear modeling, differential expression (DE), and gene set enrichment analyses were run using the R packages Seurat, DESeq2, fGSEA, and GSVA. Top DE genes were selected based on P-values <0.05 and log2-fold-change >0.5, comparing MRD+ and MRD6 populations.

Results: Within the intention-to-treat (ITT) population after a median observation time of 65.4 months, pts with MRD6 had a significantly longer PFS than pts who had MRD4/MRD5 ( A ). Pre-treatment RNAseq data were available for 405 of the 432 pts in the ITT population (202 Clb-Obi, 203 Ven-Obi), and at relapse for 41 pts (14 Clb-Obi and 27 Ven-Obi). The pre-treatment transcriptomic profile correlated with the IGHV status, trisomy 12, and del13q ( B ). No clustering according to MRD status was observed, suggesting that individual pathways/genes rather than global differences are associated with MRD response. Individual gene-level analysis of pre-treatment cohorts showed that MRD+ status was associated with resistance markers such as multi-drug response (MDR) gene or ABCB1, but MRD6 was associated with pro-apoptotic BCL2L11 (BIM) ( C ). Hallmark apoptotic pathways (P53 and Apoptosis), as well as canonical oncogenic pathways (MYC, mTORC1, TNFɑ/NFκB), were enriched in MRD6 pts in both Ven-Obi and Clb-Obi-treated pts ( D ). In contrast, inflammatory pathway gene sets (Inflammatory response, IFNγ response, IL2/STAT5) were enriched in MRD+ pts in the Ven-Obi arm, suggesting their role in Ven resistance. Gene sets enriched at progression compared to baseline were mostly related to cell proliferation and oncogenic signaling ( E ). Moreover, in a GSVA analysis, inflammatory pathway gene sets were also markedly upregulated at progression compared to baseline, providing an orthogonal validation that inflammatory signaling might be associated with resistance to venetoclax therapy ( Fig F ). Common leading edge genes included IL2RB, LCP2, BST2, IRF7, CASP3, CD86, IRF8, and NCOA3.

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Summary/Conclusion: This analysis of a large, prospective CLL RNAseq dataset confirms that global transcriptomic profiles cluster according to IGHV, trisomy 12 and del13q. Response and non-response to therapy, as assessed by NGS-based MRD status, was associated with a distinct transcriptomic profile that was characterized by upregulated oncogenic pathways and inflammatory signaling, respectively. These data suggest possible biological vulnerabilities that could be leveraged to overcome resistance to venetoclax.

S144: IMMUNE RESTORATION AND SYNERGISTIC ACTIVITY WITH FIRST-LINE (1L) IBRUTINIB (IBR) PLUS VENETOCLAX (VEN): TRANSLATIONAL ANALYSES OF CAPTIVATE PATIENTS WITH CLL

I. Solman 1,* , R. Singh Mali 2 , L. Scarfo 3 , M. Choi 4 , C. Moreno 5 6 , A. Grigg 7 , J. P. Dean 1 , E. Szafer-Glusman 1

1 Pharmacyclics LLC, an AbbVie Company, South San Francisco, CA; 2 AbbVie, North Chicago, IL, United States of America; 3 Ospedale San Raffaele, Milan, Italy; 4 University of California San Diego, San Diego, CA, United States of America; 5 Hospital de la Santa Creu I Sant Pau, Autonomous University of Barcelona; 6 Josep Carreras Leukaemia Research Institute, Barcelona, Spain; 7 Austin Hospital, Heidelberg, VIC, Australia

Background: Ibr and Ven work synergistically through their distinct and complementary modes of action: BTK inhibition by Ibr decreases levels of anti-apoptotic MCL-1 and BCL-XL proteins, but not BCL-2, and therefore increases sensitization to BCL-2 inhibition by Ven in vivo . Single agent Ibr has been shown to normalize CLL-associated immune cell alterations in number and function but the impact of the Ibr plus Ven combination (I+V) on immune cells has not been evaluated.

Aims: To 1) confirm BCL-2 sensitization by single-agent Ibr and 2) monitor changes in the cellular immune profile of patients (pts) treated with I+V in CAPTIVATE ( NCT02910583 ), a multicenter phase 2 study evaluating 1L I+V treatment (tx) in CLL and SLL.

Methods: Ibr (420mg po daily) effects on anti-apoptotic proteins were evaluated by flow cytometry with samples from 4 previously untreated pts with CLL treated for 1 cycle (28 days). Immune restoration was evaluated in 79 previously untreated pts with CLL enrolled in the CAPTIVATE Minimal Residual Disease (MRD) cohort. Pts received 3 cycles of Ibr, followed by 12 cycles of I+V (Ibr 420 mg/d orally; Ven ramp-up to 400 mg/d orally). Pts who met criteria for confirmed undetectable MRD (Confirmed uMRD) were then randomized 1:1 to placebo fixed-duration (FD) tx or Ibr at cycle 16; pts who did not meet Confirmed uMRD criteria (uMRD Not Confirmed) were randomized 1:1 to Ibr or I+V. Cryopreserved peripheral blood mononuclear cells at baseline (pre-dose cycle 1) and day 1 of cycles 4, 7, 16, 20, 23, and 29 in each of the 4 arms were analyzed by high-dimensional flow cytometry (34-color panel). Immune cell subset counts of B cells, T cells, monocytes, dendritic cells (DCs), myeloid-derived suppressor cells, natural killer cells, and innate lymphoid cells were calculated and compared to those of 20 age-matched healthy donors. Median changes from baseline are reported.

Results: In pts treated with single-agent Ibr for 1 cycle, MCL-1, BCL-XL and BCL-2 expression decreased by 74%, 95% and 10%, respectively, in lymph node emigrant (CD5 + CXCR4 dim ) CLL cells, confirming increased BCL-2-dependence and corresponding inhibitor sensitization. In CAPTIVATE pts treated with I+V, a rapid and significant decrease in circulating CLL cells was observed within the first 3 cycles of Ven tx initiation ( Fig 1A , cycle 7). From cycle 16 and onwards, Confirmed uMRD pts had CLL cell counts similar to healthy donors (≤0.8 CLL cell/mL), while in uMRD Not Confirmed pts counts were 1.5 to 22.9 CLL cell/mL. Subsequently, normal B-cell count restoration occurred in Confirmed uMRD pts randomized to placebo (FD tx) with a recovery to levels similar to healthy donors (+332% at cycle 29; Fig 1B ). Across all arms, abnormal counts of T cell subsets, classical monocytes and conventional DCs characteristic of CLL were normalized to healthy donor levels (‒49%, +101% and +91% respectively) within the first 6 months of tx and were maintained thereafter regardless of randomized tx. Plasmacytoid DCs counts recovered by cycle 20 (+598%) in all 4 arms.

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Summary/Conclusion: Sensitization to BCL-2 inhibitor effects by Ibr supports the synergistic activity of I+V. The FD regimen (Confirmed uMRD, placebo arm) of I+V effectively eradicated CLL cells to healthy donor levels and enabled sustained regeneration of normal B cell counts in contrast to ongoing therapy in the remaining tx arms. Combination I+V also allowed for normalization of other critical immune cells, including T cell subsets, classical monocytes, and conventional and plasmacytoid DCs. These data demonstrate promising evidence of immune restoration with FD I+V tx.

S145: THE COMBINATION OF IBRUTINIB PLUS VENETOCLAX RESULTS IN A HIGH RATE OF MRD NEGATIVITY IN PREVIOUSLY UNTREATED CLL: THE RESULTS OF THE PLANNED INTERIM ANALYSIS OF THE PHASE III NCRI FLAIR TRIAL

P. Hillmen 1,* , A. Pitchford 2 , A. Bloor 3 , A. Pettitt 4 , P. Patten 5 , F. Forconi 6 , A. Schuh 7 , C. Fox 8 , N. Elmusharaf 9 , S. Gatto 10 , B. Kennedy 11 , J. Gribben 12 , N. Pemberton 13 , O. Sheehy 14 , G. Preston 15 , D. Howard 16 , A. Hockaday 2 , D. Cairns 2 , S. Jackson 2 , N. Greatorex 2 , N. Webster 17 , S. Dalal 17 , J. Shingles 17 , K. Cwynarski 18 , S. Paneesha 19 , D. Allsup 20 , A. Rawstron 17 , T. Munir 21

1 St James’s University Hospital; 2 Leeds Cancer Research UK Clinical Trials Unit, Leeds Institute of Clinical Trials Research, Leeds; 3 University of Manchester, Manchester; 4 Clatterbridge Cancer Centre NHS Foundation Trust, Liverpool; 5 King’s College Hospital, London; 6 University of Southampton, Southampton; 7 Oxford University Hospital NHT Trust, Oxford; 8 Nottingham University Hospitals NHS Trust, Nottingham; 9 Wales Teaching Hospital; 10 University Hospital of Wales, Cardiff; 11 Leicester Royal Infirmary, Leicester; 12 St Bartholomew’s Hospital, London; 13 Worcestershire Acute Hospitals NHS Trust, Worcester; 14 Belfast City Hospital, Belfast; 15 Aberdeen Royal Infirmary, Aberdeen; 16 Roche, Reading; 17 HMDS, Leeds Cancer Centre, Leeds; 18 University College London, London; 19 Birmingham Heartlands Hospital, Birmingham; 20 Castle Hill Hospital, Hull; 21 UK NCRI CLL sub-group, Leeds, United Kingdom

Background: Ibrutinib (I) and venetoclax (V) improve outcome in CLL. I rarely eradicates measurable residual disease (MRD), whereas V (alone or with anti-CD20) can eradicate MRD permitting time limited therapy. Small studies suggest synergy between I and V, as I+V results in MRD negativity in many patients (pts).

Aims: The primary aim was to compare the MRD eradication rate between I and I+V. Key secondary aims were IWCLL overall (ORR), complete response (CR) and safety.

Methods: FLAIR (ISRCTN01844152), a phase III, randomised, controlled trial for previously untreated CLL requiring therapy by IWCLL criteria. Pts >75 yrs or with >20% 17p del were excluded. FLAIR was adapted in July 2017 to add two arms, I monotherapy and I+V. I was given at 420mg/day. For I+V, V was added after two months of I with dose escalation to 400mg/day over 5 weeks. The duration of therapy (DOT) was defined by MRD with treatment for up to 6 years. MRD was assessed centrally by flow cytometry, MRD negativity was defined as <1 CLL cell in 10,000 leucocytes (IWCLL criteria), was assessed in peripheral blood (PB) and bone marrow (BM) at 9 months post-randomisation, in PB at 12 months and then 6 monthly. When PB was MRD negative, this was repeated after 3 months and then in both PB & BM 3 months later. If PB & BM were negative the time to MRD negativity was calculated (treatment start to first MRD negative PB) and DOT was twice this. The earliest therapy could stop was 2 years post-randomisation. A formal interim analysis was performed when 50% pts in I and I+V arms had reached 2 yrs post-randomisation and a p-value of <0.005 was statistically significant.

Results: 523 pts were randomised to I or I+V. We report the interim analysis in the first 274 pts (I [n=138] and I+V [n=136]) reaching 2 yrs post-randomisation from 83 UK Centres from 13/07/17 to 15/03/19. 72.3% male, median age 63 yrs (34.3% >65yo) and 40.9% Binet C. IGHV were available for 256 (93.4%) pts - 48.2% IGHV unmutated (≥98% homology to germline), 45.3% IGHV mutated and 9.1% Subset 2. Hierarchical FISH testing revealed 16.1% 11q del, 19% trisomy 12, 21.9% normal and 36.9% 13q del with 6.2% failed. The arms were well-balanced for all variables. For I+V arm, MRD negativity was achieved within 24 months in BM in 89/136 (65.4%) and PB in 97/136 (71.3%) compared to no pts for I (p<0.0001). MRD negativity for I+V in BM within 24 months was 51/64 (79.7%) for IGHV unmutated and 31/55 (56.4%) for IGHV mutated. At 9 months post-randomisation 49/136 (36%) I+V pts were MRD negative in BM and 56/136 (41.2%) negative in PB compared to 0/138 with I (p<0.0001). ORR at 9 months in 120/136 (88.2%) I+V pts and 119/138 (86.2%) I pts (p=0.6157). At 9 months CR in 81/136 (59.6%) for I+V and 11/138 (8%) for I (p<0.0001). For I+V CR at any time was 93.4%. At 24 months, 54/136 (39.7%) stopped I+V due to meeting MRD stopping criteria.

SAEs were reported in 41.5% I+V and 38.2% I pts. Infectious SAEs 14.8% vs 19.9% and cardiac SAEs 11.9% vs 8.1% of pts for I+V & I respectively. Laboratory TLS was reported in 6/136 (4.4%) of I+V pts and none with I. There were no cases of clinical TLS. Most frequent any grade AEs within 12 months of randomisation differing between I+V & I were diarrhoea (52.6% I+V pts, 29.4% I), anaemia (28.9% vs 16.9%), leucopenia (36.3% vs 8.8%), thrombocytopenia (23.7% vs 14%). Leucopenia was the only grade ≥3 SAE reported in >10% of pts (27.4% I+V and 5.1% I).

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Summary/Conclusion: Ibrutinib plus venetoclax is an effective and well tolerated combination resulting in a high rate of MRD negativity in blood (71.9%) and marrow (65.4%) in the first 2 years of treatment.

S146: VENETOCLAX IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P DELETION: 6-YEAR FOLLOW-UP AND GENOMIC ANALYSES IN A PIVOTAL PHASE 2 TRIAL

S. Stilgenbauer 1,* , E. Tausch 1 , A. W. Roberts 2 , M. S. Davids 3 , B. Eichhorst 4 , M. Hallek 4 , P. Hillmen 5 , C. Schneider 1 , S. Böttcher 6 , R. Popovic 7 , M. T. Ghanim 7 , M. Moran 7 , W. J. Sinai 7 , X. Wang 7 , N. Mukherjee 7 , B. Chyla 7 , W. G. Wierda 8 , J. F. Seymour 2

1 Division of CLL, Internal Medicine III, Ulm University, Ulm, Germany; 2 Peter MacCallum Cancer Centre, Royal Melbourne Hospital, and University of Melbourne, Melbourne, Australia; 3 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, United States of America; 4 Department of Internal Medicine, Center of Integrated Oncology Köln Bonn, University Hospital of Cologne, Cologne, Germany; 5 Leeds Teaching Hospitals, NHS Trust, Leeds, United Kingdom; 6 Division of Internal Medicine, Medical Clinic III-Hematology, Oncology and Palliative Medicine, Rostock University Medical Center, Rostock, Germany; 7 AbbVie Inc, North Chicago, IL; 8 The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America

Background: Patients (pts) with chronic lymphocytic leukemia (CLL) with del(17p) and/or mutated TP53 have adverse prognosis and are a population of interest for improving outcomes. In a pivotal Phase 2 study ( NCT01889186 ) evaluating venetoclax (Ven; a selective oral BCL2 inhibitor) monotherapy in del(17p) CLL (N=158), overall response rate (ORR) was 77% (median time on study, 26.6 mo; Stilgenbauer. J Clin Oncol. 2018;17:1973).

Aims: To report final analyses after 5 y from last pt enrolled, including post-hoc subgroup analyses of prognostic markers.

Methods: Adults with R/R or previously untreated del(17p) CLL received Ven 400 mg (via ramp-up) orally daily until progressive disease (PD) or intolerance. Main cohort comprised pts with R/R CLL; safety expansion cohort included pts with R/R or untreated CLL. Primary endpoints were ORR (main) and safety (safety expansion). Peripheral blood (PB) and bone marrow (BM) were analyzed pre-Ven for somatic mutations via targeted next-generation sequencing (NGS). Minimal residual disease (MRD) was assessed by flow cytometry, ASO-PCR, and/or clonoSEQ NGS; undetectable MRD (uMRD) is reported at <10 −4 .

Results: In all, 158 pts received Ven (main, n=107; safety expansion, n=51). Data cutoff was 15 December 2020. Pts had median 2 (range, 0−10) prior lines of therapy (LOT); 5 had untreated CLL (first-line [1L]). Common mutations (n/N) were TP53 (113/138 [82%]; 35/113 [31%] had >1), SF3B1 (28/137 [20%]) NOTCH1 (22/137 [16%]), ATM (11/120 [9%]), and BIRC3 (6/120 [5%]); 93/115 (81%) had unmutated IGHV and 118/158 (75%) had ≥20% del(17p). Median time on Ven was 27.4 (range, 0–79.3) mo. At study close, 77 pts were alive; 26 remained on Ven. No new safety signals were observed.

With 70 mo median follow-up (f/u), investigator-assessed ORR was 77% (95% CI, 70−84); 21% achieved complete remission (CR)/CR with incomplete blood count recovery (CRi). Median duration of response was 39.3 mo (95% CI, 31.1−50.5); 28% had ongoing response at 60 mo. Of 61 evaluable pts with a PB MRD assessment, 25% had uMRD at ~2 y (24−30 mo). Median progression-free survival (mPFS) was 28.2 mo (95% CI, 23.4−37.6; Figure ). Median overall survival (mOS) was 62.5 mo (95% CI, 51.7−NR; 5-y PFS and OS rates, 24% and 52%). Of 5 1L pts, 4 were alive and progression-free. In pts with CR/CRi (n=37), mPFS was 62.2 mo (95% CI, 53.1−NR); in pts with partial remission (PR)/nodular PR (n=85), mPFS was 27.6 mo (95% CI, 22.8−38.0). Overall, 98/158 (62%) pts had PD, including 24/158 (15%) with Richter transformation. Of pts with PD (n=98) or whose disease was refractory to Ven (n=11), 73 received another LOT, most commonly ibrutinib (n=41); mOS from ibrutinib initiation was 28.0 mo.

In pts with both del(17p) and TP53 mutation (n=111) vs those with either (n=19), ORR was 75% vs 79%; mPFS was 27.4 vs 22.8 mo ( P =.8). In pts with mutated (n=22) vs unmutated IGHV (n=93), mPFS was 40.4 vs 26.9 mo ( P =.11). No significant difference in ORR, PFS, or OS was observed in pts with vs without ≥20% del(17p), >1 TP53 mutation, NOTCH1 , ATM , or BIRC3 mutations; mPFS was shorter in pts with mutated SF3B1 (n=28) vs without (n=109; 16.4 vs 30.2 mo [ P =.0071]). Multivariate analysis is ongoing.

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Summary/Conclusion: At end of study (median f/u, 70 mo), 48% of pts were alive, 24% were progression-free, and 16% remained on Ven, confirming the long-term activity of Ven in this high-risk population with del(17p) CLL and median 2 prior LOT. Except SF3B1 mutation, other adverse features (eg, >1 TP53 mutation, NOTCH1 mutations, unmutated IGHV) did not influence outcomes with Ven treatment in this cohort.

S147: PIRTOBRUTINIB, A HIGHLY SELECTIVE, NON-COVALENT (REVERSIBLE) BTK INHIBITOR IN PREVIOUSLY TREATED CLL/SLL: UPDATED RESULTS FROM THE PHASE 1/2 BRUIN STUDY

A. R. Mato 1,* , J. M. Pagel 2 , C. C. Coombs 3 , N. N. Shah 4 , N. Lamanna 5 , T. Munir 6 , E. Lech-Maranda 7 , T. A. Eyre 8 , J. A. Woyach 9 , W. G. Wierda 10 , C. Y. Cheah 11 , J. B. Cohen 12 , L. E. Roeker 1 , M. R. Patel 13 , B. Fakhri 14 , M. A. Barve 15 , C. Tam 16 , D. Lewis 17 , J. N. Gerson 18 , A. J. Alencar 19 , C. Ujjani 20 , I. Flinn 21 , S. Sundaram 22 , S. Ma 23 , D. Jagadeesh 24 , J. Rhodes 25 , J. Taylor 19 , O. Abdel-Wahab 1 , P. Ghia 26 , S. J. Schuster 18 , D. Wang 2 , B. Nair 2 , E. Zhu 2 , D. E. Tsai 2 , M. S. Davids 27 , J. R. Brown 27 , W. Jurczak 28

1 Memorial Sloan Kettering Cancer Center, New York; 2 Loxo Oncology at Lilly, Stamford; 3 University of North Carolina at Chapel Hill, Chapel Hill; 4 Medical College of Wisconsin, Milwaukee; 5 Herbert Irving Comprehensive Cancer Center, Columbia University, New York, United States of America; 6 Haematology, St. James’s University Hospital, Leeds, United Kingdom; 7 Institute of Hematology and Transfusion Medicine, Warsaw, Poland; 8 Oxford University Hospitals NHS Foundation Trust, Churchill Cancer Center, Oxford, United Kingdom; 9 The Ohio State University Comprehensive Cancer Center, Columbus; 10 MD Anderson Cancer Center, Houston, United States of America; 11 Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 12 Winship Cancer Institute, Emory University, Atlanta; 13 Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota; 14 University of California San Francisco, San Francisco; 15 Mary Crowley Cancer Research, Dallas, United States of America; 16 Peter MacCallum Cancer Center, Royal Melbourne Hospital, and University of Melbourne, Melbourne, Australia; 17 Plymouth Hospitals NHS Trust - Derriford Hospital, Plymouth, United Kingdom; 18 Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia; 19 University of Miami Miller School of Medicine, Miami; 20 Fred Hutchinson Cancer Research Center, Seattle; 21 Sarah Cannon Research Institute, Nashville; 22 Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York; 23 Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago; 24 Cleveland Clinic, Cleveland; 25 Northwell Health Cancer Institute, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell Health, New Hyde Park, United States of America; 26 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 27 Dana-Farber Cancer Institute and Harvard Medical School, Boston, United States of America; 28 Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland

Background: Covalent BTK inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but many patients (pts) will require additional treatment. Pirtobrutinib is a highly selective, non-covalent (reversible) BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency.

Aims: BRUIN is a phase 1/2 multicenter study ( NCT03740529 ) of oral pirtobrutinib monotherapy in pts with advanced B-cell malignancies who have received ≥2 prior therapies.

Methods: Pirtobrutinib was dose escalated in a standard 3 + 3 design in 28-day cycles. The primary objective for phase 1 was to determine the recommended phase 2 dose (RP2D) and the primary objective of phase 2 was overall response rate (ORR); secondary objectives included duration of response, progression-free survival, overall survival, safety and tolerability and pharmacokinetics. Response was assessed every 8 weeks from cycle 3, and every 12 weeks from cycle 13 and was measured according to the iwCLL 2018 criteria, including PR with lymphocytosis (PR-L). Safety was assessed in all pts.

Results: As of 27 September 2020, 323 pts with B-cell malignancies (170 CLL/SLL, 61 MCL, 26 WM, 26 DLBCL, 13 MZL, 12 FL, 9 RT, and 6 other) were treated on 7 dose levels (25-300mg QD). Among the 170 pts with CLL/SLL, the median age was 69 (36-88) years. Median number of prior lines of therapies was 3 (1-11). Majority of the CLL/SLL pts had received a prior BTKi (86%), an anti-CD20 antibody (90%), or a chemotherapy (82%). High risk molecular features such as 17p deletion, TP53 mutation, and unmutated IGHV were present in 25% (20/81), 30% (27/91), and 88% (71/81) of pts, respectively. No dose-limiting toxicities (DLTs) were reported and maximum tolerated dose (MTD) was not reached (n=323). 200mg QD was selected as the RP2D. Fatigue (20%), diarrhea (17%) and contusion (13%) were the most frequent treatment-emergent adverse events (TEAEs) regardless of attribution or grade seen in ≥10% of pts (n=323). The most common adverse event of grade ≥3 was neutropenia (10%). Treatment-related hemorrhage and hypertension occurred in 5 (2%) and 4 (1%) pts, respectively. 1% pts discontinued due to TEAEs. 139 CLL/SLL pts were efficacy-evaluable with a median follow up time of 6 months (0.16-17.8+). The ORR was 63% (95% CI 55-71) among the 139 efficacy evaluable pts with 69 PRs (50%), 19 PR-Ls (14%), 45 SDs (32%), and 1 PD (1%), and 5 (4%) discontinued prior to first response assessment. Among the 121 BTKi pretreated pts, the ORR was 62% (95% CI 53-71). Responses deepened over time with an ORR of 86% among the pts with at least 10 months follow-up. ORR was similar in pts who discontinued prior BTKi due to progression (67%), or adverse events or other reasons (52%). Of the 88 responding pts, all except 5 remained on therapy (4 progressed and 1 achieved a PR and electively discontinued treatment to undergo transplant). The longest-followed responding patient had been on treatment for 17.8+ months.

Summary/Conclusion: Pirtobrutinib demonstrated promising efficacy in heavily pretreated CLL/SLL pts following multiple prior lines of therapy and in pts with BTK C481 mutations. Pirtobrutinib was well tolerated and exhibited a wide therapeutic index. Updated data, including approximately 100 new pts with CLL and an additional 10 months since the prior data cut will be presented.

S148: VENETOCLAX-OBINUTUZUMAB FOR PREVIOUSLY UNTREATED CHRONIC LYMPHOCYTIC LEUKEMIA: 5-YEAR RESULTS OF THE RANDOMIZED CLL14 STUDY

O. Al-Sawaf 1 2 3,* , C. Zhang 1 , S. Robrecht 1 , A. Kotak 4 , N. Chang 4 , A.-M. Fink 1 , E. Tausch 5 , C. Schneider 5 , M. Ritgen 6 , K.-A. Kreuzer 1 , B. Chyla 7 , B. Eichhorst 1 , Y. Jiang 8 , S. Stilgenbauer 5 , M. Hallek 1 , K. Fischer 1

1 Department I of Internal Medicine and Center of Integrated Oncology Aachen Cologne Bonn Duesseldorf, University Hospital of Cologne, Cologne, Germany; 2 Cancer Institute, University College London; 3 Francis Crick Institute, London; 4 Roche Products Ltd, Welwyn Garden City, United Kingdom; 5 Department III of Internal Medicine, Ulm University, Ulm; 6 Department II of Internal Medicine, University of Schleswig Holstein, Kiel, Germany; 7 AbbVie Inc., North Chicago, IL; 8 Genentech Inc., South San Francisco, CA, United States of America

Background: One-year fixed-duration venetoclax-obinutuzumab (Ven-Obi) has demonstrated significant improvement of progression-free survival (PFS) as compared to chlorambucil-obinutuzumab (Clb-Obi) in patients with previously untreated chronic lymphocytic leukemia (CLL) and coexisting conditions in the CLL14 trial. As high rates of undetectable minimal residual disease (uMRD) suggested deep remissions, long-term efficacy data including patients with high-risk disease is of particular interest.

Aims: The aim of this report is to provide updated efficacy and safety data from the ongoing follow-up of the CLL14 study with all patients being off study treatment for ≥ 4 years.

Methods: Patients with previously untreated CLL and coexisting conditions were randomized 1:1 to 12 cycles of venetoclax with 6 cycles of obinutuzumab or 12 cycles of chlorambucil with 6 cycles of obinutuzumab. The primary endpoint was investigator-assessed PFS. Secondary endpoints included safety, rates of MRD response (measured every 3-6 months up to 9 years after last patient enrolment), time to next treatment (TTNT) and overall survival. Follow-up is ongoing, all patients are off study treatment.

Results: Of the 432 enrolled patients, 216 were randomly assigned to receive Ven-Obi and 216 to receive Clb-Obi. With a current median follow-up of 65.4 months (interquartile range 52.6-69.4), PFS remained significantly superior for Ven-Obi compared to Clb-Obi (median not reached [nr] vs 36.4 months; hazard ratio [HR] 0.35 [95% CI 0.26-0.46], p<0.0001). At 5 years after randomization, the estimated PFS rate was 62.6% after Ven-Obi and 27.0% after Clb-Obi. Overall, 52 cases of progressive disease (PD) with 28 required second-line treatments occurred in the Ven-Obi arm and 132 with 86 second-line treatments in the Clb-Obi arm. TTNT was significantly longer after Ven-Obi (5-year TTNT 72.1% vs 42.8%; HR 0.42, 95% CI 0.31-0.57, p<0.0001). In both arms, the majority of next-line therapies were BTK inhibitors (54.3% in the Ven-Obi arm, 47.1% in the Clb-Obi arm). The PFS and TTNT difference was maintained across all risk groups, including patients with TP53 mutation/deletion (5-year PFS 40.6% vs 15.6%; 5-year TTNT 48.0% vs 20.8%) and unmutated IGHV status (5-year PFS 55.8% vs 12.5%; 5-year TTNT 66.2% vs 25.1%). A multivariable analysis indicated 17p deletion and high disease burden as independent prognostic factors for PFS in patients treated with Ven-Obi.

Four years after treatment completion, 39 (18.1% of the intention-to-treat population) patients in the Ven-Obi arm still had uMRD (<10 -4 by NGS in peripheral blood), 27 (12.5%) had low (L)-MRD (≥ 10 -4 and < 10 -2 ) and 41 (19.0%) high (H)-MRD (≥ 10 -2 ), compared to 4 (1.9%) uMRD, 13 (6.0%) L-MRD and 24 (11.1%) H-MRD in the Clb-Obi arm.

Overall, 40 deaths were reported in the Ven-Obi arm (8 PD related) and 57 in the Clb-Obi arm (23 PD related); at 5 years after randomization the estimated OS rate was 81.9% in the Ven-Obi arm and 77.0% in the Clb-Obi arm (HR 0.72 [0.48-1.09], p=0.12). Second primary malignancies were reported in 44 (20.8%) patients in the Ven-Obi arm and 32 (15.0%) in the Clb-Obi arm. No new safety signals were observed.

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Summary/Conclusion: These data confirm that over 60% of patients who had received 1-year fixed-duration Ven-Obi have remained in remission four years after end of therapy. The majority of patients treated with Ven-Obi still have not required a second line of CLL therapy. Hence, the 1-year Ven-Obi regimen continues to be an effective fixed-duration option for patients with CLL and coexisting conditions, also in the context of high-risk disease.

S149: LONG TERM OUTCOMES OF IFCG REGIMEN FOR FIRSTLINE TREATMENT OF PATIENTS WITH CLL WITH MUTATED IGHV AND WITHOUT DEL(17P)/TP53 MUTATION

N. Jain 1,* , P. Thompson 1 , J. Burger 1 , A. Ferrajoli 1 , K. Takahashi 1 , Z. Estrov 1 , G. Borthakur 1 , P. Bose 1 , T. Kadia 1 , N. Pemmaraju 1 , K. Sasaki 1 , M. Konopleva 1 , E. Jabbour 1 , N. Garg 2 , X. Wang 3 , R. Kanagal-Shamanna 4 , K. Patel 4 , W. Wang 4 , S. Wang 4 , J. Jorgensen 4 , W. Lopez 1 , A. Ayala 1 , W. Plunkett 5 , V. Gandhi 5 , H. Kantarjian 1 , S. O’Brien 6 , M. Keating 1 , W. Wierda 1

1 Leukemia; 2 Radiology; 3 Biostatistics; 4 Hematopathology; 5 Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston; 6 Chao Family Comprehensive Cancer Center, University of California Irvine Medical Center, Orange, United States of America

Background: Chemoimmunotherapy with FCR has been an effective treatment for patients (pts) with CLL. Pts with mutated IGHV ( IGHV -M) have favorable long-term outcomes after receiving FCR. We designed an investigator-initiated, phase 2 trial with ibrutinib, fludarabine, cyclophosphamide, and obinutuzumab (iFCG) for previously untreated pts with IGHV -M CLL ( NCT02629809 ). We report here long-term outcomes with a median follow-up of 56.8 months.

Aims: Investigate the role of combined chemoimmunotherapy and targeted therapy in CLL.

Methods: Eligibility included age ≥18, IGHV -M, no del(17p)/ TP53 mutation. Pts received 3 courses of iFCG. Pts achieving CR/CRi with undetectable MRD (U-MRD) in bone marrow (4-color flow-cytometry, sensitivity 10 -4 ) after 3 courses of iFCG received ibrutinib with obinutuzumab (iG) for 3 cycles, followed by ibrutinib monotherapy for 6 months. All other pts received iG for 9 cycles (C4-12). Pts with marrow U-MRD at end of Cycle 12 stop all therapy. Response assessment was per 2008 iwCLL criteria with BM and CT scans every 3 months during the first year. After completion of all therapy, pts were followed by exam, blood counts and peripheral blood MRD every 6 months. NGS MRD (sensitivity 10 -6 ) was performed in bone marrow in pts with available samples.

Results: 45 pts initiated treatment. Median age was 60 [range, 25-71]. 69% had del(13q). After three cycles of iFCG, 39/45 (87%) pts achieved marrow U-MRD. Responses improved with continued therapy with 40/45 (89%) and 41/45 (91%) achieving marrow U-MRD after Cycles 6 and 12, respectively. Overall, 44/45 (98%) pts achieved marrow U-MRD as best response at any time during the study.

The 5-year PFS and OS are 97.7% (95% CI 94–100%) and 97.8% (95% CI 94–100%), respectively. No pt had CLL progression or Richter transformation. One pt developed therapy-related MDS; this pt is being monitored for 38+ months without any therapy for MDS with normal blood counts. The sole event noted on both the PFS and OS curve is a pt death from heart failure.

41/45 pts completed 12 cycles of treatment (4 pts came off study prior to C12). All 41 pts achieved marrow U-MRD4 by flow-cytometry and per protocol, all 41 pts discontinued ibrutinib. After a median follow-up of 44.2 mos post-discontinuing ibrutinib, 6 pts had an MRD recurrence (defined as 2 consecutive values of ≥0.01% in peripheral blood by flow cytometry) at a median of 27.2 mos (range, 20.7-49.0 mos) after stopping all therapy. All 6 pts are being monitored with no clinical progression or active therapy; notably, all 6 pts were MRD+ at 10 -6 by marrow NGS after the completion of 3 cycles of iFCG and 4/5 (1 missed sample) were MRD+ at 10 -6 by marrow NGS after the completion of Cycle 12.

Of the 16 pts who were marrow NGS MRD+ at 10 -6 after 3 cycles of iFCG, 6/16 had an MRD recurrence in blood in follow-up vs . 0/20 who were NGS MRD negative/indeterminate (p = 0.004). Of the 12 pts who were marrow NGS MRD+ at 10 -6 after Cycle 12, 4/12 had an MRD recurrence in blood in follow-up vs . 1/26 who were NGS MRD negative/indeterminate (p = 0.02).

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Summary/Conclusion: The iFCG regimen, using only 3 cycles of chemotherapy (as opposed to 6 cycles of chemoimmunotherapy) achieves a very high rate of U-MRD in previously-untreated pts with CLL with IGHV -M CLL. No pt had disease progression with a median follow-up of close to 5 years. The 5-year PFS is 97.7%; this is favorable compared to 5-year PFS of ≈65% with FCR (CLL10), ≈70% with ibrutinib (A041202 trial), and 81% with ibrutinib (RESONATE-2) for IGHV-M CLL. Not unexpectedly, MRD recurrence during follow-up correlated with MRD positivity by NGS during therapy.

S150: BIOLOGY AND FUNCTION OF CIRCULAR PCMDT1 IN CHRONIC MYELOID LEUKEMIA IN BLAST CRISIS (CML-BC)

A. Prathap Urs 1,* , D. Papaioannou 2 , R. Buisson 3 , S. Khanal 1 , M. Karunasiri 1 , S. Kauppinen 4 , A. Dorrance 1 5 , R. Garzon 1 5

1 Comprehensive Cancer Center, The Ohio State University, Columbus; 2 NYU Langone Health, Laura and Isaac Perlmutter Cancer Center, New York; 3 Department of Biological Chemistry, University of California, California, United States of America; 4 Department of Clinical Medicine, Aalborg University, Copenhagen, Denmark; 5 Department of Internal Medicine, The Ohio State University, Columbus, United States of America

Background: The prognostic and biologic significance of circular RNAs (circRNAs) in patients with acute myeloid leukemia (AML) has been reported. Circular PCMTD1 ( cPCMTD1 ) was among the circRNAs that were prognostic in this disease.

Aims: To study the functional role of cPCMTD1 in leukemias.

Methods: We used RNase H-recruiting, locked nucleic acid-modified oligonucleotides (gapmers) for knock-down (KD) experiments. Cell cycle analyses were performed with bromodeoxyuridine and 7-actinomycin D staining. Chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU) were used for DNA fiber assays.

Results: To study whether c PCMTD1 is important for leukemia, we KD-c PCMTD1 using gapmers that specifically degraded the circular transcript without affecting the levels of the linear PCMTD1 . Among the 8 AML cell lines that were tested, we found that depletion of the cPCMTD1 led to a decrease in the proliferating fraction and potent G2/M blockade of only K-562 and LAMA-84 CML-BC cells. In contrast, linear PCMTD1 -KD did not have any effect on cell cycle. Mass cytometry (CyTOF) experiments validated the cell cycle blockade after cPCMTD1 -KD and identified an increase of H2AX phosphorylation. We validated this finding with western blots and could also show that cPCMDT1 -KD led to an increase in the phosphorylation of the ATM, ATR, CHK1, DNA-PK and RPA32 proteins. Further, DNA fiber assays detected a global decrease in the length of CldU- and IdU-labeled fibers as well as of the IdU/CldU ratio in the cPCMTD1 -KD cells compared to controls. These results were confirmed by COMET assays. Taken together these data indicate that cPCMTD1 -KD causes impaired DNA replication in CML-BC. Then, we examined whether we found in CML-BC cell lines was also true for patients. Thus, we measured cPCMDT1 expression in a panel of CML patients in chronic phase (n=15), accelerated phase (n=4) and blast crises (n=7) and we found a significant increase of cPCDMT1 in CML-BC with respect to chronic and accelerated phases. Next, we performed cPCMDT1 -KD in CML-BC patient samples and found increase of H2AX phosphorylation. Like the cell line data, cPCMDT1 -KD in 3 samples from AML patients had no functional effect. Finally, after confirming that c PCMDT1 is most abundant in the cytoplasm, in silico analysis and polysome profiling experiments indicated that cPCMDT1 encodes for a small protein. We generated a custom antibody that detected the predicted protein of 30kD, which was depleted after cPCMDT1 -KD. Immunoprecipitation experiments followed by mass spectrometry analysis using our custom antibody showed a strong and specific enrichment of the BTR complex (BLM, TOP3A and RMI1), which is implicated in DNA replication and repair. cPCMDT1 -KD had no effect on the total amount of each of the three proteins but reduced their interaction and the amount of the formed BTR complex. To validate the therapeutic potential of this strategy we developed a CML-BC PDX model by transplanting a CML-BC patient sample into NSGS mice. This PDX is very aggressive, and mice die from disease by 20 weeks. We are currently treating these mice in vivo with lipid nanoparticle anti-c PCMDT1 gapmers. Results will be updated in the meeting.

Summary/Conclusion: cPCMDT1 is a circRNA with protein-coding potential that is over-expressed in CML-BC. cPCDMT1 is critical for the proliferation of CML-BC through the regulation of DNA replication and may represent a novel target for therapy.

S151: HIGH-THROUGHPUT EVALUATION OF THE POTENTIAL OF CANCER DRUGS TO ENHANCE NATURAL KILLER CELL IMMUNOTHERAPY IN CHRONIC MYELOID LEUKEMIA.

P. Nygren 1 2,* , J. Bouhlal 1 2 , E. Laajala 1 2 , A. Ianevski 3 , J. Klievink 1 2 , H. Lähteenmäki 1 2 , K. Saeed 1 2 , D. Lee 4 , T. Aittokallio 3 5 6 , O. Dufva 1 2 , S. Mustjoki 2 7

1 Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital; 2 Translational Immunology Research Program; 3 Institute for Molecular Medicine Finland, FIMM, HiLIFE, University of Helsinki, Helsinki, Finland; 4 Division of Hematology, Oncology, and BMT, Nationwide Children’s Hospital, Columbus, United States of America; 5 Institute for Cancer Research, Department of Cancer Genetics, Oslo University Hospital; 6 Centre for Biostatistics and Epidemiology (OCBE), Faculty of Medicine, University of Oslo, Oslo, Norway; 7 Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital, Helsinki, Helsinki, Finland

Background: Natural killer (NK) cell immunotherapies are promising novel cancer treatments and complete remission has been achieved in patients with relapsed/refractory myeloid leukemias. The significance of NK cells in chronic myeloid leukemia (CML) patients has been highlighted in several studies. CML patients with a higher percentage of mature NK cells have increased relapse-free survival after treatment discontinuation. NK KIR receptor profiles have also been associated with the achievement of remission in response to tyrosine kinase inhibitor (TKI) therapy. Moreover, complex interactions between TKIs and NK cell function have been discovered in CML. For example, dasatinib and imatinib have been suggested to enhance NK cell cytotoxicity through regulation of activating and inhibitory receptors. NK cell responses are usually short-lived and attempts have been made to enhance their function against malignant cells using cytokines, bi- and tri-specific antibodies, and small molecule inhibitors. However, a large-scale drug screening evaluating the combinatorial effects of NK cells and cancer drugs has not been previously conducted.

Aims: To evaluate the potential of small molecule cancer drugs to synergize with NK cell immunotherapy in CML and to discover which drugs can enhance or inhibit cytotoxicity in CML cells.

Methods: A high-throughput drug sensitivity and resistance testing (DSRT) screen was used to evaluate the effect of 528 investigational and approved cancer drugs on the cytotoxicity of NK cells. CML cells (K562) expressing luciferase and primary expanded NK cells were co-cultured for 24 hours on 384 well drug plates. Target cell viability was measured using a luciferase-based readout, and results were run through a custom pipeline to calculate differential drug sensitivity scores (dDSS) between co-cultured cells and target cells alone, for each drug. Most promising drugs with highest and lowest dDSS were selected for further analysis with single-cell RNA sequencing to determine their mechanism of action.

Results: Out of 528 screened drugs, 161 had an inhibitory effect on cytotoxicity (dDSS < -2.5), 325 had no effect, and 42 had an enhancing effect (dDSS > 2.5). The SMAC mimetics birinapant and LCL-161 were the most potent enhancers of NK cell cytotoxicity and had no effect on target cells alone. Individual drugs with other mechanisms, such as PKC activators, were also effective enhancers of NK cytotoxicity. On the contrary, cytotoxicity-inhibiting drugs included previously known immunosuppressive drugs dexamethasone and prednisolone, as well as novel candidates, such as sotrastaurin, a PKC inhibitor.

Single-cell RNA sequencing of drug-treated co-cultures revealed the transcriptomic phenotypes of NK cell and target cells under SMAC mimetic treatment. NF-kB target genes such as BIRC3, NFKB2, MHC II genes, and the T/NK cell-recruiting chemokines CXCL9, CXCL10, and CXCL11 were activated in target cells exposed to both birinapant and NK cells, compared to target cells exposed to NK cells alone (p value adj < 0.05). These data suggest that SMAC mimetics in combination with NK cells may induce an immune-inflamed phenotype in addition to sensitizing CML cells to NK cell cytotoxicity.

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Summary/Conclusion: We discovered novel drug classes, which had both activating and inhibitory effects on the NK cell cytotoxicity against CML cells in vitro. Defining NK cell - drug - CML cell interactions in a high-throughput setting provides a framework for future combination immunotherapies to further improve treatment-free remission in CML.

S152: SETD2/H3K36ME3 DEFICIENCY SUSTAINS GENOMIC INSTABILITY AND ENHANCES CLONOGENIC POTENTIAL OF CHRONIC MYELOID LEUKEMIA (CML) PROGENITORS

M. Mancini 1,* , S. De Santis 2 , C. Monaldi 2 , S. Bruno 2 , F. Castagnetti 2 , G. Gugliotta 1 , A. Iurlo 3 , M. Cerrano 4 , S. Galimberti 5 , S. Balducci 5 , F. Stagno 6 , G. Rosti 7 , M. Cavo 8 , S. Soverini 2

1 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; 2 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna; 3 UO Onco-ematologia, Fondazione IRCCS Ca’ Granda - Ospedale Policlinico, Milano; 4 A.O.U. Citta della Salute e della Scienza di Torino, Torino; 5 Clinical and Experimental Medicine, Hematology, University of Pisa, Pisa; 6 Ematologia Universitaria e Trapianto Midollo Osseo, Ospedale Gaspare Rodolico, Catania; 7 Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST Srl Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Meldola (FC); 8 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy

Background: Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease and results in BCR-ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem cells and may underlie their persistence. We have recently reported that virtually all CML patients (pts) in BC display loss of function of SETD2, an enzyme that trimethylates histone H3 lysine 36 (H3K36me3). SETD2 is a tumor suppressor implicated in several neoplastic conditions. H3K36me3 is required for homologous recombination (HR) repair of double strand breaks and for DNA mismatch repair (MMR).

Aims: We investigated SETD2/H3K36me3 status in CD34+ progenitors of CP CML pts and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models.

Methods: CD34+ progenitor cells were isolated from 20 newly diagnosed CP CML pts and screened for SETD2 and H3K36me3 by Western blotting (WB). SETD2-proficient (LAMA84) and -deficient (KCL22) CML cell lines were also studied. SETD2 knock-down and SETD2 forced expression in CD34+ progenitors and cell lines were performed by RNAi and nucleofection, respectively. DNA damage and DNA repair activation were assessed by WB and immunofluorescence (IF). Clonogenic capacity was evaluated by clonogenic assays.

Results: Loss of SETD2 protein expression and function (the latter assessed using loss of H3K36me3 as surrogate marker) were detected by WB in the CD34+ cell fraction of 20/20 newly diagnosed CP CML pts, as compared to the corresponding total mononuclear cell fraction or to the CD34+ fraction obtained from a pool of healthy donors. To investigate whether SETD2/H3K36me3 loss impinges on the activation and proficiency of HR, we used UV rays to induce DNA damage in SETD2 siRNA-depleted LAMA 84 (SETD2-proficient) cells. Compared to control cells, cells silenced for SETD2 displayed an increase in the expression of the DNA damage maker γH2AX associated with a loss of RAD51 (HR) and MSH6 (MMR) repair foci. To confirm the role of SETD2 as a tumor suppressor implicated in maintaining genomic stability in CML, we transfected KCL22 (SETD2-deficient) cells with an ectopic SETD2 plasmid. SETD2 forced expression induced more than 50% reduction in cell doubling time and a significant reduction in clonogenic potential. Moreover, SETD2 overexpression was able to restore DNA damage response, as demonstrated by WB and immunofluorescence detection of H2AX phosphorylation, RAD51 (HR) foci, THEX1 (DNA replication) and MSH6 (MMR) observed after UV exposure.

In line with the effects of SETD2 deficiency/overexpression observed in cell line models, CML progenitor cells displaying SETD2/H3K36me3 deficiency showed hyper-phosphorylation of H2AX and the overexpression of RAD51 (HR) and MSH6 (MMR) did not result in DNA repair foci. Moreover, forced overexpression of SETD2 restored proliferation control in CD34+ cells from 5 CP CML pts, since after nucleofection clonogenic assays showed a >50% reduction in clonogenic potential.

Summary/Conclusion: SETD2/H3K36me3 deficiency is a novel, BCR-ABL1-independent mechanism of genetic instability in CML. SETD2 behaves like a true tumor suppressor in CD34+ progenitor cells of CP CML pts. Supported by AIRC IG 2019 grant (23001) and Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.

S153: DECRYPTING THE ROLE OF HSP90Α AND Β ISOFORMS TO OVERCOME RESISTANCE IN BCR-ABL1 LEUKEMIA

M. Vogt 1 2,* , N. Dienstbier 1 , J. Schliehe-Diecks 1 , K. Scharov 1 , J.-W. Tu 1 , P. Gebing 1 , J. Hogenkamp 1 , D. Picard 1 , T. Lenz 3 , K. Stühler 3 4 , R. Wagener 1 5 , A. Pandyra 1 5 , J. Hauer 6 , U. Fischer 1 5 , A. Borkhardt 1 5 , S. Bhatia 1 5

1 Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich Heine University Düsseldorf, University Hospital; 2 DSO - Düsseldorf school of oncology; 3 Molecular Proteomics Laboratory, Biological-Medical Research Center, Heinrich Heine University Düsseldorf; 4 Institute of Molecular Medicine, Proteome Research, Medical Faculty and University Hospital, Heinrich Heine University Düsseldorf; 5 German Cancer Consortium (DKTK), partner site Essen/Düsseldorf, Düsseldorf; 6 Department of Pediatrics, Technical University of Munich, School of Medicine, Munich, Germany

Background: The BCR-ABL1 fusion protein, which results from the chromosomal translocation t(9;22)(q34;q11) is detected in over 95% of CML, 25% of adult BCP-ALL and 3-5% of childhood BCP-ALL patients. The development of tyrosine kinase inhibitors (TKi) has enabled targeted treatment, however development of resistance is common and the BCR-ABL1 leukemia subtype is still associated with poor prognosis. Among the chaperone proteins involved in maintaining the homeostasis of cancerous cells, heat shock protein 90 (HSP90) have been widely studied, due to its crucial involvement in stabilizing a variety of oncogenic proteins, including BCR-ABL1 and STAT3/5. Therefore, inhibition of HSP90 can be an effective alternative approach to target TKi resistant and BCR-ABL1 T315I mutant expressing cells. In the past, over 17 HSP90 inhibitors (HSP90i) have been evaluated in clinical trials, however associated resistances (e.g. HSR) and dose limited toxicity have thus far precluded their clinical approval.

Aims: In mammalian cells, there are two predominant cytosolic isoforms of HSP90, a stress-inducible HSP90α and a constitutively expressed HSP90β isoform. In the past, the distinct roles of the isoforms were studied by applying pan- and isoform-targeted HSP90i. We aimed to decrypt the role of cytosolic HSP90 isoforms using genetically edited models and to identify novel therapeutic vulnerabilities to target therapy refractory BCR-ABL1+ leukemia.

Methods: To study the kinetic implications of the resistance mechanism associated with pharmacological targeting of HSP90, we generated a transient knockdown (Kd) and a stable CRISPR-Cas9 based knockout (KO) model of HSP90α/β in BCR-ABL1+ cells. The edited cells were analyzed in ex vivo functional assays (e.g. IF staining and WB) and their transplantation efficiency was determined in NSG mice. Global transcriptomic and proteomic profiling was performed in conjunction with high throughput drug screening (HTDS) to identify therapeutic vulnerabilities upon loss of HSP90α/β.

Results: HSP90α/β-KO or -Kd did not affect the expression of other HSP90 paralogues (TRAP1 and GRP94). Only HSP90β-KO cells displayed hyperactive pro-survival HSR. Other reported HSP90 isoform-dependent clients, such as Survivin, CDK4 and CDK6 were also not affected upon KO or Kd of HSP90α/β. As HSP90 is involved in stabilizing, proper folding and subcellular localization of BCR-ABL1 protein, immunofluorescence imaging demonstrated two fold higher abundance of BCR-ABL1 foci (cytoplasmic/nucleocytoplasmic region) in HSP90α KO cells, with hyperactive BCR-ABL1 and downstream signaling (pBCR-ABL and pSTAT5a). When KO cells were transplanted into NSG mice (n=5 mice per arm) the engraftment of the HSP90α-KO cells was significantly reduced with a prolonged overall survival of the animals (19 days, p=0.0083) as compared to HSP90β-KO and control group. To find possible target (s) in order to circumvent resistance associated with the use HSP90i, we performed HTDS with HSP90α/β KO cells, which revealed distinct sensitivities toward certain classes of inhibitors. One of these classes was CDK7i, which was differentially active in HSP90α KO cells. These results can be explained by our global transcriptomic results, which revealed hyperactive androgen receptor signaling in HSP90α KO cells (GSEA), which is known to be a prognostic marker for CDK7 inhibition.

Summary/Conclusion: I n vivo targeting of HSP90α is a promising approach to target BCR-ABL1+ leukemia cells. Moreover, the combination with CDK7i may help to overcome the resistance associated with clinical use of pan HSP90i.

S154: IMATINIB-RESISTANT CLONES ISOLATED FROM A MODEL OF BLAST CRISIS OF CHRONIC MYELOID LEUKAEMIA DIFFER IN MUTATIONS IN BCR::ABL1 AND OTHER CANCER RELATED GENES AND IN THEIR SENSITIVITY TO BH3-MIMETICS

A. Laznicka 1 2,* , N. Curik 1 3 , V. Polivkova 1 , J. Koblihova 1 , P. Burda 1 3 , A. Dolnikova 3 , E. Pokorna 3 , C. Salek 1 , H. Klamova 1 , D. Srbova 1 , P. Klener 3 4 , K. Machova Polakova 1 3

1 Institute of Hematology and Blood Transfusion; 2 Second Faculty of Medicine, Charles University; 3 Institute of Pathological Physiology, First Faculty of Medicine, Charles University; 41 st Dep. of Medicine – Dep. of Hematology, First Faculty of Medicine, Charles University, and General University Hospital, Prague, Czechia

Background: The treatment of blast crisis of chronic myeloid leukaemia (BC-CML) remains difficult and the outcome of patients is poor.

A dysregulated apoptosis leads to survival of malignant cells. BH3-mimetics inhibit antiapoptotic proteins of BCL-2 family. These drugs have been recently introduced in the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. This work assumed that these drugs may have a treatment potential for myeloid and/or lymphoid BC-CML.

Aims: The aim is to test the sensitivity of imatinib-resistant (IR) clones of KCL-22, model of BC-CML, and primary blast cells to BH3-mimetics and describe a mechanism of sensitivity/resistance using protein analysis of apoptotic pathways.

Methods: Isolated IR-clones (n=10) of KCL-22 were characterized by NGS and cultivated with 4µM imatinib (IM) and dilution series of BH3-mimetics (venetoclax – anti-BCL-2, S63845 – anti-MCL-1, A-1155463 - anti-BCL-XL) for 72 hours. IC 50 was determined based on proliferation. Protein expression of BCL-2 family (n=10) was conducted in 4 IR-clones cultivated with IM and with/without BH3-mimetics. For in vivo experiments NOD-SCID-gamma mice (n=16) were subcutaneously injected with 10 6 cells of BCR::ABL1-T315I clone and divided into control group and treated groups. Primary cells of patients with BC-CML (n=4) were exposed to IM and BH3-mimetics for 7 days and LC 50 was estimated.

Results: IR-clones of KCL-22 differ in their sensitivity to BH3-mimetics (Table 1). The majority of clones (8/10) including 4 clones with T315I were sensitive to MCL-1 inhibitor. A reduced sensitivity was observed in the T315I clone carrying mutated GATA2 and other clone with mutation Y253H and mutated BCOR. Moreover, two T315I clones were sensitive to venetoclax, but other T315I clones with mutations in other cancer related genes showed insensitivity (n=2) or decreased sensitivity (n=1). Interestingly, two T315I clones with mutations in other cancer related genes insensitive to venetoclax were sensitive to BCL-XL inhibitor and in general, it seems that clones insensitive to venetoclax were sensitive to BCL-XL inhibitor and vice versa.

Protein analysis of parental KCL-22IR cells and 4 clones showed a high expression of BCL-2 and BCL-XL and undetectable MCL-1 expression. Except for the T315I clone (B8, Table 1), other analysed clones expressed MCL-1 after exposure to BH3-mimetics. The sensitivity of T315I clone to anti-MCL-1 can be explained by detection of cBAX (an active form of apoptotic effector) after exposure to the inhibitor. The parental cell line showed dissociation of antiapoptotic complex MCL-1/BIM after exposure to MCL-1 inhibitor. The released BIM accelerates the apoptosis as an apoptotic activator.

In vivo analysis revealed a decreased tumour growth of xenografted T315I clone (B8) in IM, venetoclax or combined treatment compared to control. An additive effect of dual therapy against monotherapy was not observed. This can be explained by IM inhibition of non-mutated BCR::ABL1 in KCL-22IR carrying 2 Ph chromosomes.

LC 50 analysis of primary blasts (4 patients) showed anti-MCL-1 to be the most potent inhibitor. Mutations in other cancer related genes and/or changes in karyotype increased LC 50 for venetoclax and BCL-XL inhibitor or venetoclax, respectively.

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Summary/Conclusion: The preliminary data of this study showed that the MCL-1 inhibitor S63845 seems to be the most potent BH3-mimetic to induce apoptosis in BC-CML. The combined therapy of TKI and BH3-mimetics should reflect mutation status of BCR::ABL1 and other cancer related genes.

S155: EFFICACY AND SAFETY RESULTS FROM ASCEMBL, A PHASE 3 STUDY OF ASCIMINIB VS BOSUTINIB IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE AFTER ≥2 PRIOR TYROSINE KINASE INHIBITORS: WK 96 UPDATE

D. Rea 1,* , A. Hochhaus 2 , M. J. Mauro 3 , Y. Minami 4 , E. Lomaia 5 , S. Voloshin 6 , A. Turkina 7 , D.-W. Kim 8 , J. F. Apperley 9 , J. E. Cortes 10 , A. Abdo 11 , L. M. Fogliatto 12 , D. D. H Kim 13 , P. le Coutre 14 , S. Saussele 15 , M. Annunziata 16 , T. P. Hughes 17 , N. Chaudhri 18 , L. Chee 19 , V. García-Gutiérrez 20 , K. Sasaki 21 , S. Kapoor 22 , A. Allepuz 23 , S. Quenet 23 , V. Bédoucha 23 , C. Boquimpani 24

1 Hôpital Saint-Louis, Paris, France; 2 Universitätsklinikum Jena, Jena, Germany; 3 Memorial Sloan Kettering Cancer Center, New York, United States of America; 4 National Cancer Center Hospital East, Kashiwa, Japan; 5 Almazov National Medical Research Centre; 6 Russian Research Institute of Hematology and Transfusiology, St. Petersburg; 7 National Medical Research Center for Hematology, Moscow, Russia; 8 Uijeongbu Eulji Medical Center, Geumo-dong, Uijeongbu-si, South Korea; 9 Centre for Haematology Imperial College London, London, United Kingdom; 10 Georgia Cancer Center, Augusta, United States of America; 11 Instituto do Câncer do Estado de São Paulo (ICESPSP), São Paulo; 12 Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Brazil; 13 Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada; 14 Charité–Universitätsmedizin Berlin, Berlin; 15 III. Medizinische Klinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany; 16 Division of Hematology, AORN Cardarelli, Naples, Italy; 17 South Australian Health and Medical Research Institute and University of Adelaide, Adelaide, Australia; 18 King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia; 19 Peter MacCallum Cancer Center and The Royal Melbourne Hospital, Victoria, Australia; 20 Servicio de Hematología, Hospital Universitario Ramón y Cajal (IRYCIS), Madrid, Spain; 21 The University of Texas MD Anderson Cancer Center, Houston; 22 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 23 Novartis Pharma AG, Basel, Switzerland; 24 HEMORIO, State Institute of Hematology Arthur de Siquiera Cavalcanti, Rio de Janeiro, Brazil Oncoclínica Centro de Tratamento Oncológico, Rio de Janeiro, Brazil

Background: Asciminib is the 1st BCR::ABL1 inhibitor to S pecifically T arget the A BL M yristoyl P ocket (STAMP). In the ASCEMBL primary analysis, asciminib had superior efficacy and better safety/tolerability vs bosutinib (BOS) in patients (pts) with chronic myeloid leukemia in chronic phase (CML-CP) after ≥2 prior tyrosine kinase inhibitors (TKIs). Major molecular response (MMR) rate at wk 24 was 25.5% on asciminib vs 13.2% on BOS; the difference in MMR rates after adjusting for baseline major cytogenetic response (MCyR) was 12.2% (95% CI, 2.19%-22.30%; 2-sided P =.029). Fewer grade ≥3 adverse events (AEs) and AEs leading to treatment discontinuation occurred on asciminib vs BOS. After a median follow-up of 2.3 years (16.5 months’ additional follow-up since the primary analysis), we report updated efficacy and safety results (cutoff: October 6, 2021).

Aims: The key secondary objective was to compare MMR rate at wk 96 on asciminib vs BOS.

Methods: Eligible pts provided informed consent, were adults with CML-CP after ≥2 prior TKIs, with intolerance or lack of efficacy per 2013 European LeukemiaNet recommendations. They were randomized 2:1 to asciminib 40 mg twice daily or BOS 500 mg once daily, stratified by baseline MCyR status (Ph+ metaphases ≤35%).

Results: 233 pts were randomized to asciminib (n=157) or BOS (n=76). At cutoff, treatment was ongoing in 84 (53.5%) and 15 (19.7%) pts, respectively; the most common reason for discontinuation was lack of efficacy in 38 (24.2%) and 27 (35.5%) pts, respectively.

MMR rate at wk 96 (per ITT) was 37.6% on asciminib and 15.8% on BOS, meeting the key secondary objective. The difference after adjusting for baseline MCyR was 21.7% (95% CI, 10.5%-33.0%; 2-sided P =.001). Preplanned subgroup analyses showed that MMR rate at wk 96 was consistently higher with asciminib than BOS in all demographic and prognostic subgroups, including all prior lines of TKI therapy, and regardless of the reason for discontinuation of the last TKI (Figure).

At wk 96, more pts on asciminib than BOS had BCR::ABL1 IS ≤1% (45.1% vs 19.4%) (Table). Responses were durable, with a probability (95% CI) of maintaining MMR and BCR::ABL1 IS ≤1% for ≥72 wk of 96.7% (87.4%-99.2%) and 94.6% (86.2%-97.9%), respectively, on asciminib and 92.9% (59.1%-99.0%) and 95.0% (69.5%-99.3%), respectively, on BOS. Median time to treatment failure was 24 months on asciminib and 6 months on BOS.

Median duration (range) of exposure was 103.1 (0.1-201.1) wk on asciminib and 30.5 (1.0-188.3) wk on BOS. Despite asciminib’s longer duration of exposure, its safety/tolerability continued to be better than that of BOS (Table). Fewer pts on asciminib than BOS had AEs leading to treatment discontinuation (7.7% vs 26.3%). No new on-treatment deaths were reported since the primary analysis. Most frequent (>10%) grade ≥3 AEs on asciminib vs BOS were thrombocytopenia (22.4%, 9.2%), neutropenia (18.6%, 14.5%), diarrhea (0%, 10.5%), and increased alanine aminotransferase (0.6%, 14.5%).

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Summary/Conclusion: After >2 years of follow-up, asciminib continued to show clinically and statistically significant, superior efficacy and better safety/tolerability vs BOS. Responses were durable, and MMR was more than double on asciminib than BOS. The difference in MMR rates between the 2 arms increased from 12.2% at wk 24 to 21.7% at wk 96. A higher proportion of pts had BCR::ABL1 IS ≤1%, a milestone response in later lines that is associated with improved long-term survival. These results further support the use of asciminib as a new CML therapy, with the potential to transform standard of care.

S156: INTERNATIONAL, PROSPECTIVE STUDY COMPARING NILOTINIB VERSUS IMATINIB WITH EARLY SWITCH TO NILOTINIB TO OBTAIN SUSTAINED TREATMENT-FREE REMISSION IN PATIENTS WITH WITH CHRONIC MYELOID LEUKEMIA

F. Pane 1,* , F. Castagnetti 2 , L. Luciano 1 , A. Russo Rossi 3 , E. Abruzzese 4 , R. Bassan 5 , G. Binotto 6 , G. Caocci 7 , G. Cimino 8 , P. Fazi 9 , A. Gozzini 10 , M. Lunghi 11 , R. Marasca 12 , B. Martino 13 , M. Bonifacio 14 , F. Cavazzini 15 , F. Paoloni 16 , G. Saglio 17 , S. Sica 18 , A. Tafuri 19 , D. Vallisa 20 , M. Vignetti 9 , P. Westerweel 21 , G. Rosti 22 , M. Breccia 23

1 Dpt. of Clinical Medicine and Surgery, University of Naples, Naples; 2 Department of Experimental, Diagnostic, and Specialty Medicine, University of Bologna, Bologna; 3 Hematology and Transplantation Unit, University of Bari, Bari; 4 Division of Hematology, Ospedale S. Eugenio, Rome; 5 Hematology Unit, Ospedale Dell’Angelo, Venezia-Mestre; 6 Hematology Unit, University of Padova, Padova; 7 Department of Medical Sciences and Public Health, Hematology, Businco Hospital, University of Cagliari, Cagliari; 8 UOC Ematologia e trapianto, S.Maria Goretti Hospital, Latina; 9 GIMEMA Data Center, Fondazione GIMEMA Franco Mandelli Onlus, rome; 10 Department of Cellular Therapy and Transfusional Medicine, AUO Careggi, Florence; 11 Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara; 12 Section of Hematology, Department of Medical Sciences, University of Modena and Reggio Emilia, Modena; 13 Division of Hematology, Azienda Ospedaliera ‘Bianchi Melacrino Morelli’, Reggio Calabria; 14 Section of Hematology, Department of Medicine, University of Verona, Verona; 15 Hematology Section, Department of Medical Sciences, University of Ferrara, University Hospital Arcispedale S. Anna, Ferrara; 16 Biostatistic Central Office, Fondazione GIMEMA Franco Mandelli Onlus, Rome; 17 Hematology Division, University of Turin, Turin; 18 Ematologia, FONDAZIONE POLICLINICO UNIVERSITARIO AGOSTINO GEMELLI IRCCS, Roma; 19 Department of Clinical and Molecular Medicine, Hematology, Sant’Andrea University HospitalSapienza University of Rome, Rome; 20 Unit of Hematology, Department of Oncology and Hematology, Guglielmo da Saliceto Hospital, Piacenza, Italy; 21 Internal Medicine, Albert Schweitzer Hospital, Dordrecht, Netherlands; 22 Hematology Unit, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola; 23 Translational medicine OUC Hematology, La Sapienza university, Rome, Italy

Background: Treatment free remission (TFR) is one of the most important goals of the CML treatment but, so far, the best treatment to reach this aim is still undefined. It is widely accepted that a sustained deep molecular remission (DMR) is the pre-requisite to discontinue TKI, and it is expected that in patients who start treatment with imatinib (IMA) and fail early molecular remission (EMR), a switch to a second generation TKI may improve the probability of achieving a DMR

Aims: We launched in November 2016 an international, prospective, interventional, randomized, two arms, study to evaluate both the depth of the molecular response and the rate of TFR in newly diagnosed CP-CML patients treated with a second generation TKI (Nilotinib, NIL) or with IMA followed by switching to NIL in absence of optimal response (defined according the ELN 2013 criteria (Clinical Trial number 02602314).

Methods: The patients are randomized 1:1 between NIL and IM according to Sokal risk score (high versus intermediate/low risk) and country. All the patients who obtain a residual disease reduction greater than 4.0 logs (MR4.0) within the first three years of treatment and maintain this level of response up to the end of the fourth years of therapy qualify for the discontinuation phase of the study. The study has two primary end-points: a) the rates of molecular response (MR4.5) at 24 months, and b) the rate of patients who remain in sustained treatment free remission (≥MR3.0) without molecular relapse 12 months after entering the TFR phase. The molecular relapse is defined as loss of MMR or confirmed loss of MR3.0 (figure).

Results: From November 2016 to January 2021, 457 patients with newly diagnosed CP-CML patients were enrolled into the study and 448 of these (228 and 220 randomized to the NIL and IMA arms, respectively) were evaluable (mean age 54.2 yrs - range 19.4 – 85.8). At baseline, 183 (40.8%), 191 (42.6%) and 72 (16.1%) patients were classified as low, intermediate, or high-risk Sokal, respectively, while 278 (62.3%), 128 (28.7%) and 40 (9.0%) had a low, intermediate, or high-risk ELTS risk score. The median follow-up of the whole cohort of patients is 30.4 mo. Fifthy-six (25.4%) of the 220 patients of the IMA arm did not fulfill the ELN criteria for optimal response within the first 12 mo. of treatment and, according to the protocol, switched to NIL therapy. At the last analysis of the protocol database (February 2022), 59 patients had had stopped the protocol treatment since their decision, death (24), toxicity (23), progression (9), uncontrolled second neoplasia (2) or protocol violation (1), 69 patients had not reach 24 mo. of follow-up and other 15 had missing data. Of the remaining 304 patients, 35 showed non optimal response to therapy. At the 24 mo. of follow-up, 76 of the 322 patients with an available molecular response (23.6%), reached a MR4.5 response that showed a significantly higher frequency within the patients randomized to the NIL arm (48 vs 28; p=0.015) (first primary co-endpoint of the study).

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Summary/Conclusion: This is the first and, so far, the unique prospective study comparing not only the rate of DMR but, more important, also the rate of TFR according to treatment: a second generation TKI frontline vs IMA frontline followed by the same second generation in case of non-optimal response. The analysis of the first co-primary endpoint indicates that, despite the early switch in the IMA randomized patients, NIL therapy is more effective to induce DMR. Subsequent analysis will clarify whether the higher rates of DMR in the NIL arm may translate into a higher rate of TFR.

S157: BCR::ABL1 DIGITAL PCR IDENTIFIES CHRONIC PHASE CML PATIENTS SUITABLE FOR AN EARLY TKI DISCONTINUATION ATTEMPT: A PATIENT-LEVEL META-ANALYSIS

C. Kockerols 1,* , S. Dulucq 2 , S. Bernardi 3 , M. Farina 3 , I. Civettini 4 , G. Colafigli 5 , S. Mori 6 , P. Valk 7 , F.-X. Mahon 8 , C. Gambacorti-Passerini 4 9 , F.-E. Nicolini 10 , M. Breccia 5 , D. Russo 3 , P. E. Westerweel 1

1 Internal Medicine, Albert Schweitzer Hospital, Dordrecht, Netherlands; 2 Laboratory of Hematology, Hôpital Haut Lévêque, University hospital of Bordeaux, Pessac, France; 3 Clinical and Experimental Sciences, Hematology; 2 and Bone Marrow Transplant Center, University of Brescia, ASST Spedali Civili of Brescia, Brescia; 4 University of Milano Bicocca, Monza; 5 Translational and Precision Medicine, Az. Policlinico Umberto I-Sapienza University, Rome; 6 University of Milano-Bicocca, Monza, Italy; 7 Molecular Biology, Erasmus University Medical Center, Rotterdam, Netherlands; 8 Hematology, Institut Bergonié, Bordeaux, France; 9 Hematology, S. Gerardo Hospital, Monza, Italy; 10 Hematology, Centre Léon Bérard and CRCL, Lyon, France

Background: Digital PCR (D-PCR) is an emerging technique that delivers a highly accurate BCR::ABL1 quantification, even in CML patients with low residual disease. This is crucial in the context of treatment-free remission (TFR) for the selection of patients who may successfully discontinue TKI therapy. However, it is unclear how its prognostic value relates to time variables such as treatment duration prior to the TFR attempt. Current guidelines suggest aiming for a TKI treatment duration >6 years to increase TFR success rate.

Aims: In current analysis, we aimed to assess the prognostic value of BCR::ABL D-PCR in relation to prior TKI treatment duration.

Methods: We performed an Individual Patient Data Meta-Analysis (IPD-MA) combining data from different study cohorts in which BCR::ABL1 was assessed by D-PCR prior to TKI discontinuation. Eligibility criteria included age ≥18 years and CML diagnosed in chronic phase. Data of the participating studies were pooled and stratified based on D-PCR and/or treatment duration. BCR::ABL1 D-PCR was dichotomized based on each study-defined prediction cut-off. Strata were assessed for molecular relapse (MolR) with Kaplan-Meier estimates and with cox regression analysis including a frailty term for correction for between-study heterogeneity and including confounding variables: age at diagnosis, gender, Sokal score, TKI generation, treatment duration, DMR duration (model 1) and BCR::ABL1 transcript type (model 2). MolR was defined as a BCR::ABL1 >0,1%IS or a 1-log BCR::ABL1 increase in two consecutive analyses. Patients were censored at last follow-up.

Results: For this meta-analysis, data were combined from five cohorts: four published cohorts (STIM2 cohort [Nicolini et al .], n=175; ISAV cohort [Diral et al. ], n=107; Bernardi et al ., n=111; Colafigli et al ., n=50) and 1 unpublished cohort (Dutch cohort; n=40). The pooled dataset comprised 483 patients (Table).

A total of 205 patients (42%) experienced MolR with a median time to relapse of 3 months. Median follow-up duration for TFR patients was 27 months. MolR patients had a significantly shorter treatment duration prior to TKI discontinuation (6,7 vs 7,9 years, p=0.006) and more often presented a BCR::ABL1 D-PCR above the study-defined prediction cut-off (34% vs 19%, p<0.001). Interestingly, the median treatment durations were almost identical in patients with a BCR::ABL1 below or above the cut-off (7.0 vs 7.5 years, p = 0.470).

The probability of MolR at 24 months was 38% versus 58% for patients with a D-PCR BCR::ABL1 below versus above the prediction cut-off (p <0.001). In the cox regression analysis, the HR of D-PCR BCR::ABL1 below the cut-off for MolR was 0.48 (95% CI 0.35-0.66, p<0.001). Treatment duration and BCR::ABL1 transcript type also remained independent predictors, but TKI generation and DMR duration did not.

When stratifying into 4 groups based on the D-PCR result and treatment duration, patients with a TKI treatment for ≥6 years and low D-PCR result had the lowest MolR rate (33% at 24 months, figure). Patients treated <6 years and with a low D-PCR result had a rate of 48% at 24 months, while patients treated <6 years and a D-PCR result above the cut-off had the highest MolR rate of 72% at 24 months.

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Summary/Conclusion: These combined patient-level data of multiple CML cohorts further support the independent prognostic value of BCR-ABL1 D-PCR for TFR success rate. Importantly, patients with a TKI treatment duration <6 years and a low D-PCR result were found to have a clinically acceptable MolR rate (48%).

S158: FINAL RESULT OF TKI DISCONTINUATION TRIAL WITH DASATINIB FOR SECOND ATTEMPT OF TREATMENT FREE REMISSION AFTER FAILING FIRST ATTEMPT WITH IMATINIB: TREATMENT-FREE REMISSION ACCOMPLISHED BY DASATINIB

D. Kim 1,* , E. Atenafu 2 , D. Forrest 3 , I. Bence-Bruckler 4 , L. Savoie 5 , M.-M. Keating 6 , L. Busque 7 , R. Delage 8 , A. Xenocostas 9 , E. Liew 10 , P. Laneuville 11 , K. Paulson 12 , T. Stockley 13 , J. Lipton 1 , B. Leber 14

1 Princess Margaret Cancer Centre, Toronto, Canada; 2 Statistics, Princess Margaret Cancer Centre, Toronto; 3 Vancouver General Hospital, Vancouver; 4 Ottawa Hospital Research Institute, Ottawa; 5 University of Calgary, Calgary; 6 Queen Elizabeth II Health Sciences Centre, Halifax; 7 Hôpital Maisonneuve-Rosemont, Montreal; 8 Centre Universitaire d’Hématologie et d’Oncologie de Québec, Quebec; 9 London Health Sciences Centre, London; 10 University of Alberta, Edmonton; 11 McGill University Health Centre, Montreal; 12 CancerCare Manitoba, Winnipeg; 13 Division of Clinical Laboratory Genetics, University Health Network, Toronto; 14 Juravinstki Cancer Centre, Hamilton, Canada

Background: The Canadian tyrosine kinase inhibitor (TKI) discontinuation (DISC) trial evaluated if Dasatinib (DA) therapy can lead to a successful second treatment-free remission (TFR2) after failing a Imatinib (IM) DISC for first TFR (TFR1) attempt. We previously reported that 1) The 12-month molecular relapse free survival (mRFS) rate for TFR1 was 58.0%; 2) doubling time (DT) at 2 months after IM DISC correlates with TFR1 failure.

Aims: Here, we report the final result of TFR2 rate after DA DISC. The null hypothesis was a TFR2 rate of 17.5% while the alternative hypothesis was a TFR2 rate of 35.0%, and the study was designed to reject our null hypothesis if > 28% of pts remain in TFR2 after DA DISC.

Methods: This prospective clinical trial (BMS CA180-543, NCT#02268370) had 3 phases: 1) IM DISC phase, 2) DA rechallenge phase, 3) DA DISC phase. Key inclusion criteria included: 1) CML in chronic phase at original diagnosis, 2) total duration of IM therapy of minimum 3 years, 3) total duration of MR 4.5 or deeper response over 2 years. Molecular relapse was defined as an increase in BCR-ABL qPCR > MR4 on 2 consecutive occasions, or a single increase in BCR-ABL qPCR > MR3. DA treatment was started at 100mg once daily after molecular relapse was confirmed, and continued for at least 12 months after achieving ≥ MR4 until DISC for TFR2.

Results: The study was launched on March 2015 and completed all participants’ planned visits as of Feb 2022 with a median follow-up duration of 27.5 months (range 2-51 months).

1) In the IM DISC phase, 58 of 131 pts (44.3%) experienced molecular relapse with a median onset of 3.53 months, thus the remaining 73 pts achieved TFR1 (55.7%): The mRFS rate at 12 months was 56.8% (95% CI, 47.8-64.8 %). Distribution of monthly DT in 6 months is presented in Fig A .

2) In the DA rechallenge phase, out of the 58 pts who failed TFR1, 51 pts received DA. At 3 months, 98.0%, 87.9%, and 75.4% of pts achieved MMR, MR4 and MR4.5. The halving time (HT) after DA re-therapy is summarized in Fig B . Notably, HT at 2 and 3 months was 10.6 and 10.7 days, consistent with rapid reduction of BCR-ABL qPCR in first 3 months.

3) In the DA DISC phase, 35 pts who attained MR4 or deeper response for ≥ 12 months discontinued DA for a TFR2 attempt. Out of 35 pts, only 4 pts (11.4%) has maintained the molecular response at last follow-up, while the remaining 31 pts lost the molecular response with a median 3.65 months. The actuarial mRFS rate at 6 and 12 months was 22.9% (10.8-37.6%) and 10.0% (2.7-23.1%). Monthly DT within 6 months after DA DISC is presented in Fig C . Based on the final result of 11.4% TFR2 rate, we conclude that 12 months’ DA re-therapy could not improve TFR2 rate significantly.

4) For DT after IM DISC, average DT at 2/3 months was much shorter in those lost molecular response (10.6 / 10.7 days) than those who did not (23.5 / 30.1 days). For DT after DA DISC, while average DT was 7.6 and 14.3 days at 2/3 months in overall population, it was much shorter in those lost molecular response within 6 months (3.2 and 13.3 days at 2/3 months).

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Summary/Conclusion: The final result indicates that re-challenge with DA after TFR1 failure with IM DISC is effective in restoring deep molecular response as most cases rapidly regained at least MR4. However, 12 months’ DA re-therapy does not significantly improve TFR2 rate. Further studies should consider increasing MR4 duration before TFR2 attempt, adding other therapeutics and refining risk factor for TFR2 failure.

S159: QUÉBEC CML RESEARCH GROUP ANALYSIS OF TREATMENT PATTERNS IN CHRONIC MYELOGENOUS LEUKEMIA: SWITCHING IS DRIVEN BY INTOLERANCE AND SIMILAR ACROSS TYROSINE KINASE INHIBITORS AND LINES OF TREATMENT

L. Busque 1 2,* , M. Harnois 2 , N. Szuber 1 2 , R. Delage 2 3 , L. Mollica 1 2 , H. Olney 2 4 , P. Laneuville 2 5 , S. Sirhan 2 6 , G. Cournoyer 2 7 , I. Chamakhi 2 8 , M. Lalancette 2 3 , D. Talbot 2 9 , V. Éthier 2 10 , P. Desjardins 2 11 , S. Assouline 2 6

1 Hematology, Hôpital Maisonneuve-Rosemont, Université de Montréal; 2 Groupe Québécois de Recherche en LMC-NMP, GQR-LMC-NMP, Montreal; 3 Hematology, Centre Hospitalier Universitaire de Québec (CHUQ), Université Laval, Québec; 4 Hematology, Centre Hospitalier Universitaire de Montréal (CHUM), Université de Montréal; 5 Hematology, McGill University Health Center (MUHC), McGill University; 6 Hematology, Jewish General Hospital, McGill University, Montreal; 7 Hemato-oncology, Hôpital regional de St-Jérôme, St-Jérôme; 8 Hemato-oncology, Hôpital du Sacré-Cœur de Montréal, Université de Montréal, Montreal; 9 Hemato-oncology, Hôpital de la Cité-de-la-Santé, Laval; 10 Hemato-oncology, Centre Hospitalier Universitaire de Sherbrooke (CHUS), Université de Sherbrooke, Sherbrooke; 11 Hemato-oncology, Hôpital Charles-Lemoyne, Université de Sherbrooke, Greenfield Park, Canada

Background: In 2001, imatinib was the first tyrosine kinase inhibitor (TKI) approved for the treatment of chronic myelogenous leukemia (CML) which translated into a revolution in the management of this disease. The arsenal of TKIs was progressively reinforced with the addition of other TKIs such as dasatinib, nilotinib, bosutinib, ponatinib and recently asciminib. Two decades of clinical trials have provided critical insight into differential efficacies and specific toxicity profiles of these TKIs supporting the development of guidelines such as the European Leukemia Net (ELN) and the national comprehensive cancer network (NCCN). Real-world evidence studies provide unique and complementary information on treatment patterns, efficacy, side effects and may help identify unmet medical needs of CML patients

Aims: This study was aimed at studying frequency of TKI switching, reason for switch, duration of treatment without switching as a function of line of treatment and specific TKI using the real-world data of the Québec registry created in 2009

Methods: Patients with Philadelphia positive (Ph+) CML were recruited with informed consent in the Québec registry. 795 patients were included in this analysis. Data from the registry were extracted July 31 ist 2021. Switching was defined as a change of a specific TKI to another. Reasons to initiate switching were categorized as resistance (primary and secondary) or intolerance (hematological or non-hematological). Standard statistical methods were used to evaluated bivariate and continuous variables. Kaplan-Meier curve were used to evaluate overall survival and survival without switching (JMP® Pro 14.1 software, SAS Institute, Cary, NC, USA).

Results: At the time of data collection, the median time of follow-up was 7.5 years. Proportion of switching per line of treatment: 1 st : 357/795 (44.9%); 2 nd : 157/357 (43,9%); 3 rd : 59/157 (37,6%); 4 th :23/59 (39%). The reason for switching (ratio intolerance/resistance) for each line were: 1 st : 1.33; 2 nd : 4.3; 3 rd : 2.4; 4 th : 2.7. 20 patients switched serially across all lines for intolerance and only 3 for serial resistance. Survival without switching was evaluated using a Kaplan-Meier curve by line of treatment and by specific TKI (Figure 1). The survival without switching was similar for patients on imatinib, dasatinib or nilotinib in first line as in second line. In third line, there was no difference between imatinib, nilotinib, dasatinib or bosutinib although numbers of patients were small. We then compared the survival from diagnosis of patients that remained in first or second line versus the patients that reached third or more lines of treatment. Although the mean age at diagnosis and SD were identical between the two groups (54,8 years and SD 15,2) there was a statistically significant difference in survival in favor of patients needing only 1 or 2 lines of treatment (P= 0.0254).

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Summary/Conclusion: We demonstrate: (i) that switching of TKI is frequent and mainly driven by intolerance in all lines of treatment; (ii) serial intolerance is 6.6 time more frequent than serial resistance suggesting a class effect for intolerance in some patients; (iii) all TKIs have a similar «retention level» in all lines of treatment; (iv) that patients necessitating 3 or more lines of treatment have a survival disadvantage. Our results suggest that one of the most important unmet medical need in CML management is availability of better tolerated drugs

S160: MOLECULAR DETERMINANTS OF DISEASE PROGRESSION AFTER HYPOMETHYLATING AGENT THERAPY IN RAS PATHWAY MUTANT CHRONIC MYELOMONOCYTIC LEUKEMIA AT THE SINGLE-CELL LEVEL

G. Montalban-Bravo 1,* , F. Ma 2 , I. Ganan-Gomez 1 , R. Kanagal-Shamana 3 , V. Adema 1 , N. Thongon 1 , H. Yang 1 , K. A. Soltysiak 1 , C. Bueso-Ramos 3 , H. Kantarjian 1 , G. Garcia-Manero 1 , S. Colla 1

1 Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 2 Molecular Biology Institute, University of California, Los Angeles; 3 Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Most patients (pts) with chronic myelomonocytic leukemia (CMML) have incomplete or transient responses to hypomethylating agent (HMA) therapy. CMML cases driven by mutations in RAS pathway signaling genes or ASXL1 have a higher risk of failure and progression to acute myeloid leukemia (AML). Development of effective alternative therapies has been delayed, owing to an incomplete understanding of how different hematopoietic populations contributes to disease maintenance and progression.

Aims: We aimed to dissect the cellular and molecular mechanisms underpinning CMML maintenance and progression in RAS mutant CMML.

Methods: We performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin - ) CD34 + hematopoietic stem and progenitor cells (HSPCs) and BM mononuclear cells (MNCs) isolated from RAS pathway mutant CMML pts (n=5 and 6, respectively) and age-matched healthy donors (HD; n=2 and 3, respectively). CMML samples were obtained at the time of diagnosis and HMA failure. Additionally, we performed scATAC-seq analysis of Lin - CD34 + HPSCs isolated at the times of diagnosis and progression (n=1).

Results: Our analysis revealed that CMML HSPCs had a predominantly granulomonocytic differentiation route with increased frequencies of myeloid-monocytic progenitors, at the expense of hematopoietic stem cells (HSCs) (Fig 1a), and upregulated expression of genes involved in the oxidative phosphorylation, type I interferon (IFN) and IFNg pathways. Consistent with these results, scRNA-seq analysis of MNCs revealed expanded populations of myelomonocytic progenitors and monocytes and upregulated expression of genes involved in IFNg response and NF-kB activation (Fig 1b), along with upregulation of the NF-kB transcriptional effector BCL2A1 (Fig 1c). Assessment of ligand-receptor interactions using the CellPhoneDB repository identified that CMML monocytes established a high number of cell-cell interactions (n=638) with dendritic cells, NK cells, and HSPCs via chemokines, cytokines, and inhibitory molecules known to induce NF-kB signaling and NK-cell exhaustion. Disease progression was associated with expansion of lympho-myeloid progenitors (LMPPs) (Fig 1d) characterized by the highest levels of IFNg response, NF-kB survival signaling, and cell cycle regulators. scATAC-seq of Lin - CD34 + confirmed higher activity of transcriptional factors associated with monocytic differentiation and NF-kB signaling (Fig 1e-f). Accordingly, scRNA-seq analysis of MNCs showed increased frequencies of HSPCs and myelomonocytic precursors, a reduction of T cells (Fig 1f), and emergence of a monocyte population characterized by the highest expression of NF-kB signaling and its effectors MCL1 and BCL2A1 . BCL2A1 protein expression at progression was confirmed by immunohistochemistry (Fig 1g). CellPhoneDB analysis identified a high number of cell-cell interactions (n=2978) involving cytokines, chemokines, and surface proteins known to elicit NF-kB activation and immune evasion between expanded monocytes, LMPPs, myelomonocytic precursors, and immune cells during progression.

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Summary/Conclusion: Our data suggests that CMML is maintained through metabolically active HSPCs, which leads to monocytes’ reprograming and survival through NF-kB signaling activation. We showed that disease progression arises from the expansion of NF-kB dependent immature myeloid progenitors, which leads to therapy resistance and immune evasion. This study has implications for the development of therapies targeting downstream effectors of NF-kB–mediated survival pathway to overcome treatment failure. In vitro validation is ongoing.

S161: ZRSR2 AND TET2 MUTATIONS PROMOTE MDS BY DYSREGULATING GENE EXPRESSION AND ABERRANT ALTERNATIVE SPLICING IN MICE

C. Garcia-Ruiz 1,* , C. Martínez-Valiente 1 , A. Liquori 1 , A. Gutiérrez-Adán 2 , J. Cervera 3 , A. Sanjuan-Pla 1

1 Hematology Research Group, IIS La Fe, Valencia; 2 Animal Reproduction Department, INIA, Madrid; 3 Genetics Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain

Background: Mutations in splicing factors and epigenetic regulators are the most frequent genetic alterations in patients with myelodysplastic syndromes (MDS). The minor spliceosome factor ZRSR2 and the epigenetic regulator TET2 appear significantly associated in MDS patients. However, the functional impact of such mutations in the hematopoietic system and MDS have been scarcely studied. To address this question, we established a murine model ( Zrsr2 m/m Tet2 −/− ) carrying mutations in both genes, which exhibited signs compatible with MDS disease in mice. However, the molecular disease mechanism has not yet been elucidated.

Aims: To interrogate the impact of ZRSR2 and TET2 mutations in gene expression and alternative splicing in the context of hematopoiesis and MDS.

Methods: Whole transcriptome sequencing was performed to investigate changes in gene expression and alternative splicing patterns. Lin - Sca-1 + c-kit + (LSK) cells (50,000 per sample) were sorted from pools of 3 mice, and mRNA was sequenced in an Illumina NovaSeq 6000 platform. Differential gene expression was determined using DESeq2 with adjusted P-value ≤ 0.05, log2FoldChange ≥ 0. Functional enrichment from differentially spliced genes was identified with KEGG, and the enrichment cut-off p-value was set as adjusted P-value ≤ 0.05. Relevant alternative splicing events were validated by RT-PCR.

Results: RNA-seq analysis of Zrsr2 m/m Tet2 −/− cells revealed 2952 differentially expressed genes (DEG), compared to 571 in Zrsr2 m/m and 1203 in Tet2 −/− . From 2952, 1327 were up-regulated and 1625 were downregulated. Interestingly, relevant genes for hematopoietic lineage specification were significantly dysregulated. In particular, genes related to lymphoid lineage (Flt3, Notch1, and Tlr7) were downregulated, while those related to myeloid lineage (Ccl9, Mpo, Ms4a6b, and Prtn3) and megakaryocytic-erythroid lineage (Pdfgrb, Itgb3, Optn, and Tgfbr3) were upregulated. This suggests an early myeloid and megakaryocyte-erythroid priming of LSK towards the production of cells from these lineages in Zrsr2 m/m Tet2 −/− mice. Enrichment analysis of DEG using Go enrichment analysis identified ribosome function, inflammation, and migration/motility processes as the most significantly altered. Further, KEGG pathway enrichment pointed ribosome, the MAPK family, and pro-inflammatory pathways as significantly enriched in Zrsr2 m/m Tet2 −/− cells. Finally, KEGG pathway enrichment of alternative splicing targets identified the MAPK family and the Fanconi anemia pathway as the most altered targets in Zrsr2 m/m Tet2 −/− cells. Importantly, a total of 9 genes related to the MAPK pathway were identified as mis-spliced, from which Dusp1, Tgfbr2, and Fgf11 were validated in this study.

Summary/Conclusion: In this study, we broaden our previous report and show that concurrent mutations in Zrsr2 and Tet2 dysregulate normal gene expression and cause aberrant mRNA splicing. Gene expression analysis identified ribosome function, inflammation, and migration/motility as the most altered processes in Zrsr2 m/m Tet2 −/− cells. Alternative splicing analysis identified the MAPK and the Fanconi Anemia pathway as key targets of aberrant splicing. All in all, gene expression dysregulation and aberrant mRNA splicing disturb important biological pathways and drive the molecular pathomechanism in Zrsr2 m/m Tet2 −/− mice.

S162: SOMATIC GENETIC LANDSCAPE IN GATA2 DEFICIENCY PATIENTS

L. Largeaud 1 2,* , M. Collin 3 , N. Monselet 4 , F. Vergez 5 , L. Larcher 6 , P. Hirsch 7 , N. Duployez 8 , J. Bustamante 9 , C. Bellanné-Chantelot 10 , J. Donadieu 11 , F. Sicre de Fontbrune 12 , M. Nolla 13 , C. Fieschi 14 , F. Delhommeau 7 , E. Delabesse 15 , M. Pasquet 13

1 Clinical Haematology laboratory, Toulouse Hospital; 2 UMR1037, Cancer Research Center of Toulouse, Toulouse, France; 3 Haematology Department, Institute of Cellular Medicine Newcastle University, Newcastle, United Kingdom; 4 Bureau des essais cliniques, Claudius Rigaud Institut; 5 Haematology Department, Toulouse Hospital, Toulouse; 6 Clinical Haematology laboratory, Saint Louis Hospital APHP; 7 Clinical Haematology laboratory, Saint-Antoine Hospital, APHP, Paris; 8 Clinical Haematology laboratory, CHU Lille, Lille; 9 Unité centre d’études des déficits immunitaires, Necker Hospital APHP; 10 Centre de génétique moléculaire et chromosomique, Hôpital Pitié-Salpêtrière, APHP; 11 Haematology Department, Trousseau Hospital, APHP; 12 Service d’hématologie greffe, Saint-Louis Hospital, APHP, Paris; 13 Service d’hématologie et immunologie pédiatrique, Toulouse Hospital, Toulouse; 14 Immunologie clinique, Saint-Louis Hospital, APHP, Paris; 15 Clinical Hematology laboratory, Toulouse Hospital, Toulouse, France

Background: Heterozygous germline GATA2 mutations strongly predispose to myeloid malignancies, immunodeficiency, and/or lymphedema. The progression towards haematological diseases seems to be correlated with the acquisition of molecular and cytogenetic abnormalities.

Aims: Our study therefore focuses on the biological characterization of the different progression stages of germline GATA2 deficiency patients and their correlation with clinic features.

Methods: We describe here a cohort of 78 patients (62 from the French-Belgian cohort and 16 British patients). Molecular analysis by next generation sequencing (targeting 90 genes frequently mutated in myeloid malignancies with a sensitivity = 1%) was performed in 76 patients. Cytogenetic analyses were determined for 76 patients.

Results: Median age of our cohort was 21.8 years [6months–62years]. 44 had missense mutations, 32 had null mutations, one intronic and one synonymous mutation. Our results showed a trend toward a younger age at diagnosis in patients harboring null mutations compared to patients with missense mutations (13 vs 17 years, p=0,086) and more chronic infections during follow-up (74% vs 25%, p<0,001).

55 karyotypic abnormalities were identified including monosomy 7 (29%), trisomy 8 (16%) and der (1;7) (9%). Moreover, 141 somatic mutations were identified targeting STAG2 (38%), ASXL1 (13%), SETBP1 (8%), EZH2 (4%) and RUNX1 (3%) genes.

To better stratify patients, we defined 3 spectra based on morphological bone marrow analysis: 12 (15%) patients had spectrum 0 (normal bone marrow), 47 (60%) had spectrum 1 (hypoplastic marrow and/or low-grade myelodysplasia (MDS)) and 19 (25%) were classified as spectrum 2 (MDS with excess blasts, AML and CMML). We found a genotype-phenotype correlation: spectrum 1 is more associated with null mutations and spectra 0 and 2 with missense mutations (p=0.023). Patients in spectrum 0 exhibited no acquired molecular and karyotypic abnormalities. Spectrum 2 was enriched with mutations of SETBP1 , RAS pathway genes, and RUNX1 as well as more other cytogenetic abnormalities excluding -7, tri8 and der(1;7) (p<0,001). The proportion of monosomy 7 was higher in spectrum 2 than in spectrum 1 without reaching the significance level (47% vs 28%).

Conversely, STAG2 mutations were mainly found in spectrum 1. 53 STAG2 mutations were identified in 25 patients (1 to 8 mutations per patient), mostly at low mutated cell fraction (median: 6%). Follow-up of 3 patients with STAG2 clones in spectrum 1 showed no progression toward spectrum 2. Moreover, clonal hierarchy of 3 patients with STAG2 mutations in spectrum 2 showed that STAG2 mutations were subclonal events. Our results suggested that STAG2 mutation did not act as driver and could be related to clonal hematopoiesis. Interestingly, Stag2 KO mice showed an enrichment of GATA2 binding motif (Ochi et al. Cancer Discov 2020) which could lead to an increase of GATA2 activity. We speculate that in GATA2 deficiency syndrome, STAG2 mutations could induce a mechanism of indirect Somatic Genetic Rescue (SGR) by compensating the loss of GATA2 activity induced by the mutated allele.

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Summary/Conclusion: This study reported that STAG2 mutations were recurrent in GATA2 deficiency clonal hematopoiesis. Some genetic abnormalities are associated with the leukemic transformation stage such as SETBP1 , RAS pathway genes and other cytogenetic abnormalities. An improved ability to identify patients with high risk of developing leukemic transformation has the potential to improve clinical outcomes and help clinicians determine the optimal timing of bone marrow transplantation.

S163: EFFICACY OF JAK 1/2 INHIBITION IN MURINE IMMUNE BONE MARROW FAILURE

E. Groarke 1,* , X. Feng 1 , N. Aggarwal 1 , A. L. Manley 1 , Z. Wu 1 , S. Gao 1 , B. Patel 1 , J. Chen 1 , N. Young 1

1 Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, United States of America

Background: Immune aplastic anemia (AA) is a severe blood disorder characterized by cytotoxic T-lymphocyte mediated stem cell destruction. Therapies including hematopoietic stem cell transplant and immunosuppression are effective but entail costs and risks, and are not effective or possible in all patients. The Janus Kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T cell activation and inhibits production of interferon gamma and tumor necrosis factor alpha in models of graft-versus-host disease.

Aims: Assess RUX in murine immune AA for potential therapeutic benefit.

Methods: In a murine major histocompatibility complex mismatched C67BL/6(B6) to CByB6F1 lymph node (LN) cell infusion AA model, and a C.B10 minor histocompatibility antigen mismatched B6 to C.B10 LN cell infusion AA model, RUX was administered as a food additive (Rux-chow), which achieves therapeutic levels in 48-72 hours after administration. Control BMF mice received chow without RUX. Animals were fed with Rux-chow two days before LN cell infusion as a prophylaxis (BMF+RUXD-2), or two days after LN cell infusion as therapy (BMF+RUXD+2). Recipient mice were either bled and euthanized at day 14 following LN infusion to collect tissues or were kept for 56 days to record animal survival. Blood counts were measured biweekly. Samples for flow cytometry, histology, and gene expression assays were collected.

Results: In both AA murine models, RUX attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, and prevented mortality when used either prophylactically or therapeutically. Treated mice had significantly higher blood counts (neutrophils, red blood cells, hemoglobin, platelets) as well as bone marrow (BM) and RBM (residual BM, excluding T-cells), and cellularity relative to control BMF mice. RUX suppressed infiltration, proliferation and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. All mice who received RUX were alive at 56 days while control BMF mice all died (Figure 1). With the exception of two mice with low RBC at day 56, discontinuing Rux-chow at day 28 or day 42 did not affect animal blood counts measurements, nor survival. Gene expression in mice who received RUX revealed downregulated T cell function and JAK/STAT pathway-related genes ( Stat1 , Stat3 , Stat4 , Fas , Ly6a , Infg , Gzmb , Gzma , Gzmk , Infgr1 , Il2rb , Il2rg , and Lag3 ). On network analysis of differentially expressed genes in downregulated pathways, Stat1 and Ifn-g genes were at the center of the network, connecting immune responses and cell cycle pathways. When toxicity was assessed, RUX exerted modest suppression of lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice, but the drug showed impressive clinical efficacy despite this.

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Summary/Conclusion: RUX showed striking therapeutic efficacy, improving blood counts and prolonging survival in two different BMF murine models. In patients, clonal T cell expansion and activation, IFN-g and TNF-α upregulation, and Fas mediated cell death, lead to severe BM destruction and the development of AA. Our study demonstrated that JAK 1/2 inhibition with RUX produced its suppressive effects by inhibiting T cell activation, reducing inflammatory cytokine secretion, and limiting FasL/Fas-mediated BM destruction. Given these results, JAK 1/2 inhibition with RUX is an exciting and novel potential therapy for BMF patients based on a well characterized mechanism of action.

S164: CLONAL DYNAMICS, IMMUNE PHENOTYPES, AND TARGETS OF BONE MARROW-INFILTRATING T CELLS IN ACQUIRED APLASTIC ANEMIA

A. Ben Hamza 1,* , C. Welters 1 , M. Brüggemann 2 , K. Dietze 1 , L. Bullinger 1 3 , T. H. Brümmendorf 4 5 , J. Strobel 6 , H. Hackstein 6 , K. Dornmair 7 , F. Beier 4 5 , L. Hansmann 1 3

1 Department of Hematology, Oncology and Cancer Immunology, Charité - Universitätsmedizin Berlin, Berlin; 2 Department of Medicine II, Hematology and Oncology, University Hospital Schleswig Holstein, Kiel; 3 German Cancer Consortium (DKTK), partner site Berlin, and German Cancer Research Center (DKFZ), Heidelberg; 4 Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University; 5 Center for Integrated Oncology, Aachen Bonn Cologne Düsseldorf (CIO ABCD), Aachen; 6 Department of Transfusion Medicine and Haemostaseology, University Hospital of Erlangen, Friedrich Alexander University Erlangen-Nürnberg (FAU), Erlangen; 7 Institute of Clinical Neuroimmunology, Biomedical Center and University Hospital, Ludwig-Maximilian-University Munich, Munich, Germany

Background: Acquired aplastic anemia is a hematological disease characterized by hypocellular bone marrow with insufficient hematopoiesis and pancytopenia. High response rates to immunosuppressive therapy with anti-thymocyte-globulin and cyclosporin A suggest a key role for T cells in disease pathogenesis. However, bone marrow-infiltrating clonal T cell expansion, associated immune phenotypes, and T cell targets remain poorly understood.

Aims: To defined clonal T cell expansion, associated immune phenotypes, and potential T cell targets in bone marrow specimens of aplastic anemia patients before and after immunosuppressive therapy.

Methods: Within bone marrow of 15 patients with acquired aplastic anemia, we determined T cell clone-associated immune phenotypes by multi-parameter flow cytometry single cell index sorting for subsequent T cell receptor (TCR) αβ and cytokine/transcription factor sequencing. T cell clones of 10 patients were monitored before and after immunosuppressive therapy (median time span: 9 months) by TCRβ repertoire sequencing. Twenty-seven TCRs of expanded clones were re-expressed in reporter cell lines to determine recognition of autologous hematopoietic precursor cells.

Results: We detected oligoclonal T cell expansion in all patients and 92.9% of all expanded clones were CD8 + , while only 9.6% were CD4 + . Frequencies of dominant T cell clones varied between patients (1.1-20.6% of CD8 + T cells). Expanded CD8 + clones were almost exclusively CCR7 - PD1 - TIM3 - CD39 - and frequently expressed cytokines and transcription factors associated with cytotoxic effector differentiation ( IFNG, GZMB, PRF1, TBX21 ). More than 50% of the CD45RA + clones (T EMRA ) were CD57 + , while CD45RA - clones (T EM ) were mainly CD28 + and smaller in size. While T EM clones showed similar frequencies in bone marrow and peripheral blood, T EMRA displayed higher localization-dependent differences in size. Interestingly, almost all (91.1%) expanded clones with frequencies >1% of all T cells persisted in the bone marrow after immunosuppressive therapy independent of clinical response. Similarly, highly expanded CD4 + clones, albeit low in numbers, were stable in frequencies before and after therapy. To determine targets of CD8 + T cell clones with strong expansion, we re-expressed twenty-seven TCRs of eight patients and found five to be specific for immunodominant epitopes of cytomegalovirus. Strikingly, two TCRs recognized hematopoietic progenitor cells expanded from CD34-enriched autologous bone marrow.

Summary/Conclusion: We tracked T cell clonotypes and associated immune phenotypes under immunosuppressive therapy at the single cell level. Expanded CD8 + clones showed cytotoxic effector differentiation and persisted in the bone marrow after immunosuppressive therapy even in situations of therapeutic response, challenging the hypothesis that clinical response follows the decline of highly expanded autoreactive T cell clones. We identified two expanded T cell clones that recognized hematopoietic progenitor populations providing experimental proof of concept that expanded autoreactive T cell clones can be critically involved in aplastic anemia pathogenesis.

FB and LH contributed equally.

S165: INTEGRATED GENETIC DIAGNOSTICS OF PATIENTS WITH EARLY ONSET OF DE NOVO MYELODYSPLASTIC SYNDROMES.

E. Attardi 1,* , L. Tiberi 2 , D. Formicola 3 , R. Artuso 3 , V. Santini 1

1 MDS Unit, Hematology, AOU Careggi - Department of Experimental and Clinical Medicine; 2 Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence; 3 Medical Genetics Unit, Meyer Children’s University Hospital, Florence, Italy

Background: Cytogenetic and molecular alterations determine myelodysplastic syndrome (MDS) prognosis and must be evaluated during disease history. Relevance of germline (GM) predisposition in MDS was stressed in WHO classification, but its actual incidence is probably underestimated. Recently, the widespread availability of large-scale genomic sequencing techniques has facilitated the investigation of GM variants, and the identification of novel GM variants predisposing to MDS is expected. In de novo MDS patients (pts) with an early onset of the disease (age ≤55 yrs) when no syndromic signs drive the genetic assessment, whole exome sequencing (WES) analysis of bone marrow (BM) and saliva can constitute an optimal diagnostic procedure, improving the performance of targeted next-generation sequencing (t-NGS) approach.

Aims: We adopted integrated diagnostics to define the cytogenetic profile, and WES to investigate possible GM predisposition of a cohort of 30 de novo MDS pts with an unusual young age at disease onset (≤55 yrs). Standard karyotype (SK) was paralleled by low coverage whole genome sequencing (lc-WGS) on plasma cell-free DNA (cf-DNA), as non-invasive screening test for detection of structural chromosome abnormalities.

Methods: Patient BM-DNA / peripheral blood DNA (PB-DNA) was analysed by WES and compared to DNA extracted from saliva (S-DNA) samples as control for GM variants. To identify high-confidence MDS variants, we attributed an internal deleteriousness score to define variants, from “of uncertain significance” to “pathogenic” (ACMG guidelines). Variants were suspected as GM where variant allele frequency was > 0.3 and had been confirmed for genes detected in both S-DNA and BM/PB-DNA. To confirm the somatic nature of some variants, deep t-NGS of 27 myeloid custom panel gene was applied in 26/30 MDS cases on BM-DNA. SK and lc-WGS (0.2x) on plasma cf-DNA were assessed in 27/30 pts.

Results: The small cohort of “young” MDS pts here analysed (Table 1). The majority of pts, 17/30 (57%) presented at least one high-confidence variant evaluated as GM for MDS. A total of 26 GM variants were recognized. Patients could be grouped on the basis of the type of variant as follows: 11/30 pts (37%) presented variants in genes involved in DNA repair defects and cancer predisposition ( ATM, ATR, FANCA, FANCM, PARN, BRCA1, BRCA2, CHEK2 ); 4/30 (13%) had variants involved in genetic predisposition to myeloid neoplasm - GATA2, ANKRD26 and RBBP6 - the latter classically associated to MPN familial cases; one presented compound heterozygous variants in SBDS ; 4 presented variants related to hereditary red blood cell defects. In our cohort, we identified cases with a complex mode of inheritance (4/17) as well as high confidence GM variants in 4 pts in 2 new interesting cancer predisposition genes: RBBP6 and PARN . Lc-WGS of plasma cf-DNA confirmed all the cytogenetic alterations found by SK (8/27) and identified a 21q22.12 deletion involving RUNX1 locus in a patient, not revealed by SK.

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Summary/Conclusion: By means of an integrated diagnostics, defined as the convergence of diagnostic techniques with advanced information technology (WES, t-NGS, lc-WGS), as a proof of principle, it was possible to fully characterize the genetic asset of a particular subgroup of 30 MDS cases. We also demonstrated that lc-WGS of plasma cf-DNA has an excellent sensitivity allowing to perform periodic cytogenetic evaluations in MDS pts without invasive maneuvers. We showed that, by the use of WES, GM alterations were demonstrated in a small cohort of “young” non-syndromic MDS pts in 57% of cases, a proportion unexpectedly high.

S166: MAGROLIMAB IN COMBINATION WITH AZACITIDINE FOR PATIENTS WITH UNTREATED HIGHER-RISK MYELODYSPLASTIC SYNDROMES (HR MDS): 5F9005 PHASE 1B STUDY RESULTS

D. A. Sallman 1,* , M. M. Al Malki 2 , A. S. Asch 3 , E. S. Wang 4 , J. G. Jurcic 5 , T. J. Bradley 6 , I. W. Flinn 7 , D. A. Pollyea 8 , S. N. Kambhampati 9 , T. N. Tanaka 10 , J. F. Zeidner 11 , G. Garcia-Manero 12 , D. Jeyakumar 13 , L. Gu 14 , A. Tan 14 , M. Chao 14 , C. O’Hear 14 , I. Lal 14 , P. Vyas 15 , N. Daver 12

1 Moffitt Cancer Center, Tampa; 2 City of Hope National Medical Center, Duarte; 3 Stephenson Cancer Center, Oklahoma University Health, Oklahoma City; 4 Roswell Park Comprehensive Cancer Center, Buffalo; 5 Columbia University Medical Center, New York; 6 Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami; 7 Tennessee Oncology, Nashville; 8 University of Colorado School of Medicine, Denver; 9 Sarah Cannon Research Institute, Kansas City; 10 University of California San Diego Moores Cancer Center, San Diego; 11 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill; 12 The University of Texas MD Anderson Cancer Center, Houston; 13 University of California Irvine, Orange; 14 Gilead Sciences, Inc., Foster City, United States of America; 15 Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

Background: Magrolimab is a monoclonal antibody that blocks CD47, a “don’t eat me” signal overexpressed on cancer cells. CD47 blockade by magrolimab induces macrophage-mediated phagocytosis of tumor cells and is synergistic with azacitidine (AZA) in the upregulation of “eat me” signals. A high unmet need exists to build on current standard-of-care AZA frontline therapy to increase efficacy while maintaining a tolerable safety profile in patients (pts) with HR MDS.

Aims: To report the final safety/tolerability and efficacy data from a phase 1b trial of magrolimab + AZA in pts with untreated HR MDS ( NCT03248479 ).

Methods: Pts with previously untreated intermediate, high, or very high–risk MDS per the Revised International Prognostic Scoring System (IPSS-R) received magrolimab IV as a priming dose (1 mg/kg) followed by ramp-up to a 30-mg/kg QW or Q2W maintenance dose. AZA 75 mg/m 2 was administered IV or SC on days 1-7 of each 28-day cycle. Primary endpoints were safety/tolerability and complete remission (CR) rate.

Results: A total of 95 pts (median age, 69 y [range, 28-91 y]) were treated. IPSS-R risk was intermediate, high, or very high in 27%, 52%, and 21% of pts. MDS was therapy related in 22% of pts; 26% had a TP53 mutation, and 62% had poor-risk cytogenetics. Median number of cycles was 6 (range, 1-27). The most common treatment-emergent adverse events (TEAEs) included constipation (68%), thrombocytopenia (55%), anemia (52%), neutropenia (47%), nausea (46%), and diarrhea (44%). The most common grade 3/4 TEAEs included anemia (47%), neutropenia (46%), thrombocytopenia (46%), and white blood cell count decreased (30%). Six pts discontinued treatment due to AEs. The 60-day mortality rate was 2%. Median hemoglobin change from baseline (BL) at first postdose sample was −0.7 g/dL (range, −3.1 to 2.4 g/dL). CR and objective response (OR) rates were 33% and 75%, with 31% of OR-evaluable pts with abnormal cytogenetics at BL having cytogenetic CR. Median time to first OR, duration of CR (DCR), duration of OR, and progression-free survival (PFS) were 1.9, 11.1, 9.8, and 11.6 mo. Overall survival (OS) rates at 12 and 24 mo were 75% and 52%; median OS was not reached (NR) with 17.1 mo of follow-up (figure). In pts evaluated with sequential whole-exome sequencing with a variant allele frequency (VAF) cutoff of 5%, 3 of 3 pts with TP53 mutation who achieved CR had TP53 VAF <5% by cycle 5 day 1. Favorable outcomes were observed both in pts with TP53 mutation (CR rate, 40%; median OS, 16.3 mo) and wild-type TP53 (CR rate, 31%; median OS, NR) (table).

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Summary/Conclusion: Magrolimab + AZA was well tolerated with promising efficacy in pts with untreated HR MDS, including those with TP53 -mutated and –wild-type disease. A phase 3 trial of magrolimab/placebo + AZA (ENHANCE: NCT04313881 ) is ongoing.

S167: PREDICTION OF RELAPSE AFTER ALLOGENEIC STEM CELL TRANSPLANTATION USING INDIVIDUALIZED MEASURABLE RESIDUAL DISEASE MARKERS; THE PROSPECTIVE NORDIC STUDY NMDSG14B

M. Tobiasson 1,* , T. Pandzic 2 , J. Illman 3 , L. Nilsson 4 , S. Weström 2 , K. Sollander 2 , E. Ejerblad 5 , A. Olsnes Kittang 6 , G. Olesen 7 , O. Werlenius 8 , A. Björklund 9 , J. Wiggh 1 , C. lindholm 1 , F. Lorentz 10 , B. Rasmussen 11 , J. Cammenga 12 , D. Weber 13 , D. Grönnås 14 , M. Dimitriou 15 , S. Kytölä 16 , G. Walldin 15 , P. Ljungman 9 , K. Groenbeck 17 , S. Mielke 9 , S. E. Jacobsen 15 , F. Ebeling 3 , L. Cavelier 2 , L. Smidstrup Friis 17 , I. Dybedal 18 , E. Hellström-Lindberg 1

1 Hematology, Karolinska University Hospital, Stockholm; 2 Department of Immunology, Genetics and Pathology, Science for life, Uppsala, Sweden; 3 Hematology, Helsinki University Hospital, Helsinki, Finland; 4 Hematology, Lund University Hospital, Lund; 5 Hematology, Akademiska sjukhuset, Uppsala, Sweden; 6 Hematology, Haukeland University Hospital, Bergen, Norway; 7 Hematology, Arhus University Hospital, Århus, Denmark; 8 Hematology, Sahlgrenska University Hospital, Gothenburg; 9 Centre for allogeneic stem cell transplantation, Karolinska University Hospital, Stockholm; 10 Hematology, Norrlands University Hospital, Umeå; 11 Hematology, Örebro University Hospital, Örebro; 12 Hematology, Universitetssjukhuset, Linköping, Sweden; 13 Hematology, Odense University Hospital, Odense, Denmark; 14 Institution for environmental medicine; 15 Centre for hematology and regenerative medicine, Institution for medicine, Huddinge, Karolinska Institute, Stockholm, Sweden; 16 HUS Diagnostic Center, HUSLAB, Helsinki University Hospital, Helsinki, Finland; 17 Hematology, Rigshospitalet, Copenhagen, Denmark; 18 Hematology, Oslo University Hospital, Oslo, Norway

Background: One third of patients with myelodysplastic syndrome (MDS) relapse after allogeneic stem cell transplantation (HCT). Early detection of impending relapse would enable pre-emptive treatment and potentially reduce relapse risk but is limited by the lack of sensitive markers for measurable residual disease (MRD). We developed a pipeline where patient-specific mutations, as determined by a myeloid next generation sequencing (NGS) panel are tracked using digital droplet PCR (ddPCR).

Aims: To evaluate if personalized MRD detection by ddPCR can predict clinical relapse earlier than conventional methods.

Methods: The prospective study ( NCT02872662 ) enrolled patients with MDS, MDS/MPN or MDS-AML with < 30% marrow blasts undergoing HCT. Patients were included before HCT, and serial bone marrow (BM) samples were collected every third month post-HCT for 2 years. Peripheral blood (PB) samples were collected monthly. MRD results were not available for the treating physician.

Results: We screened 286 pts between 2016 and 2020, whereof 20 were excluded mainly due to lack of genetic aberration or no HCT performed. 266 pts were included from 12 HCT centers. Median age was 64 (18-78) years and 59% were male. Myeloid panel NGS screening identified a median of 2 (0-9) mutations. The most common mutations were TET2 (n=85), ASXL1 (n=73) and SRSF2 (n=59).

Median time of follow up was 886 (4-1934) days. Sixty pts relapsed after a median of 189 (53-1281) days and 46 died due to non-relapse mortality after a median of 121 (4-1036) days. Remaining pts (n=160) were in continuous complete remission (CCR) after a median follow-up of 1053 (479-1934) days. Estimated 1 and 2y overall survival was 79%, and 71%, respectively, while estimated 1 and 2y relapse-free survival (RFS) was 75% and 66%, respectively.

MRD data was missing in 46 pts; no post-HCT samples available (n=15), no mutation detected (n=14) and difficulties to design ddPCR primers (n=11). 221 pts were available for MRD analysis with a median number of 4 (0-13) and 5 (0-23) samples from BM and PB, respectively.

Of 53 clinical relapses with MRD results available, 42 were preceded by pos MRD (>0.1%) with a median of 70 (range 20-425) days between first pos MRD and clinical relapse. For the 11 remaining pts, 8 were inadequately sampled with a median time of 189 (82-397) days between last sampling and clinical relapse. One patient had an extramedullary relapse only.

Of 31 pts who died without relapse, 19 were consistently MRD neg, while 5 were borderline positive (MRD > 0.1% and <0.5%) during the first 100 days but negative thereafter. Four MRD+ patients died without clinical relapse. Three pts were initially MRD+ but turned negative, all of which had chronic GVHD (cGVHD).

Of 136 CCR patients, 94 were consistently MRD neg; 26 were borderline pos (MRD > 0.1% and <0.5%) during the first 100 days followed by neg samples; 16 were MRD positive (either > 0.5% during the first 100d or > 0.1% after 100d) of which 10 had a transition from pos to neg samples (all had cGVHD); one patient was treated for a molecular relapse detected by clinical routine method (FISH) and five patients were MRD positive at time of last follow-up.

MRD used as a time-dependent co-variate was negatively associated with RFS (HR 7.1, p<0.01). Estimated cumulative incidence of relapse and non-relapse mortality 2y after pos MRD was 60% and 7% respectively (see figure).

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Summary/Conclusion: We report the development of a highly functional personalized MRD pipeline based on patient-specific mutations showing a high sensitivity to predict relapse and relapse-free survival.

S168: ERYTHROPOIETIN STIMULATION AGENTS SIGNIFICANTLY IMPROVES OUTCOME IN LOWER RISK MDS.

H. Garelius 1,* , A. Smith 2 , T. Bagguley 2 , A. Taylor 2 , P. Fenaux 3 , D. Bowen 4 , A. Symeonidis 5 , M. Mittelmann 6 , R. Stauder 7 , J. Čermák 8 , G. Sanz 9 , S. Langemeijer 10 , L. Malcovati 11 , U. Germing 12 , R. Itzykson 13 , A. Guerci-Bresler 14 , D. Culligan 15 , I. Kotsianidis 16 , K. Koinig Mag 17 , C. van Marrewijk 18 , S. Crouch 2 , T. de Witte 19 , E. Hellström-Lindberg 20

1 Section of Hematology, Specialist Medicine, Sahlgrenska University hospital, Göteborg, Sweden; 2 Department of Health Sciences, University of York, York, United Kingdom; 3 Service d’Hematologie, Hôpital Saint-Loius, Assistance Publique des Hopitaux de Paris (AP-HP) and Universite Paris; 7 , Paris, France; 4 St.James’s Institute of Oncology, Leeds Teaching Hospitals, Leeds, United Kingdom; 5 Department of Medicine, Division of hematology, University of Patras Medical Scholl, Patras, Greece; 6 Department of Medicine, Tel Aviv Sourasky (Ichilov) Medical Center and Sackler Medical Faculty, Tel Aviv University, Tel Aviv, Israel; 7 Department of Internal Medicine V (Haematology and Oncology), Innsbruck Medical University, Innsbruck, Austria; 8 Dep. of Clinical Hematology, Institute of Hematology & Blood Transfusion, Prague, Czechia; 9 Department of Haematology, Hospital Universitario y Politécnico La Fe, Valencia, Spain; 10 Department of Hematolog, Radboud university medical center, Nijmegen, Netherlands; 11 Department of Hematology Oncology, Fondazione IRCCS Policlinico San Matteo,University of Pavia, Pavia, Italy; 12 Department of Haematology, Oncology and Clinical Immunology, Universitätsklinik Düsseldorf, Düsseldorf, Germany; 13 Service d’Hématologie, Hôpital Saint-Louis, Assistance Publique des Hôpitaux de Paris (AP-HP) and Université Paris; 7 , Pari, Paris; 14 Service d’Hématologie, Centre Hospitalier Universitaire Brabois Vandoeuvre, Nancy, France; 15 Department of Haematology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom; 16 Dep. of Hematology, Democritus University of Thrace Medical School, University Hospital of Alexandroupolis, Alexandroupolis, Greece; 17 Department of Internal Medicine V (Hematology and Oncology), Medical University Innsbruc, Innsbruck, Austria; 18 Department of Haematology, Radboud university medical center; 19 Department of Tumor Immunology - Nijmegen Center for Molecular Life Sciences, Radboud university medical cente, Nijmegen, Netherlands; 20 Department of Medicine, Div. Hematology, Karolinska Institutet, Stockholm, Sweden

Background: The EUMDS Registry started in 2008 as a prospective, non-interventional longitudinal study, enrolling newly diagnosed patients with IPSS low or intermediate-1 MDS from 16 European countries and Israel.

Aims: The aim of the present analysis was to see how treatment with or without Erythropoietin Stimulating Agents (ESAs) and/or red blood cell transfusions (RBCT) impact overall survival (OS) and quality of life (QoL).

Methods: Patient management was recorded electronically every 6 months (“visit”) in a central database, including treatment, transfusions, blood values, and health related quality of life (HRQoL) using the EQ-5D 3-Level index and Visual Analog Scale (VAS). Patients were eligible to be included in the analyses if their hemoglobin was recorded as less than <10 g/dl at a visit. To overcome potential confounding by non-random allocation of ESA treatment, propensity score matching was performed to ensure that treated and untreated patients had similar characteristics. Only patients with comparable propensity scores were included in the analyses to estimate the effects of ESA treatment on outcomes using standard time to event

analyses; OS was estimated from the first visit a Hb value of <10g/dl was recorded. OS was examined for patients treated with ESA stratified by their transfusion status prior to commencing ESA treatment (no RBCT, <4 units, ≥4 units).

Patients were separated into 4 groups at each clinical visit, depending on the treatment received in the interval leading up to that visit; no ESA nor RBCT, ESA only, ESA and RBCT and RBCT only. HRQoL at each visit according to the treatment status was summarized for patients who had completed a questionnaire at visit 1 and 2; mean values were examined by treatment group.

Results: Of 2562 patients registered by November 2021, 2448 were diagnosed before July 2019 and included in the analysis; these patients were divided into two groups: ESA untreated (n=1265) and ESA treated (n=1183). Patients whose Hb remained above 10g/dl were excluded leaving 529 untreated patients and 749 ESA treated; after propensity score matching was applied two comparable groups were produced: ESA untreated (n= 426) and ESA treated (n= 742). Median OS from reaching the eligibility criteria in the ESA treated vs untreated groups were 44.9 and 34.8 months respectively (Fig 1a), giving a clear survival advantage to the ESA-treated group. (p<0.003). In the ESA-treated group, OS was poorer in those who had been transfused prior to commencing ESA (Fig 1b, p<0.001).

Fig 1c shows the number of patients at each visit who had been treated with transfusions or ESA; 647/1278 had received neither at visit 1, the figure shows the “flow” of patients by treatment for the first 6 visits. HRQoL was examined for the 695 patients who had completed a questionnaire at both visit 1 and 2 up to visit 6; differences were seen by treatment (Fig 1d). Patients who had received no treatment reported, on average, the highest mean HRQoL, in contrast, patients who had RBCT had the lowest (p<0.001).

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Summary/Conclusion: This unique large prospective registry study clearly shows a significant survival advantage for lower-risk MDS patients exposed to ESA treatment at onset of anemia (Hb <10g/dL) but before onset of transfusion therapy, strongly supporting recommendations to start ESA treatment early. The effect on patients with an early transfusion need warrants further studies. Moreover, ESA exposure is associated with maintained QoL, while RBCT development with or without ESA exposure is associated with significantly deterioration in QoL.

S169: CLINICAL AND MOLECULAR MARKERS FOR PREDICTING RESPONSE TO ROMIPLOSTIM TREATMENT IN LOWER-RISK MYELODYSPLASTIC SYNDROMES

A. S. Kubasch 1 2 3,* , A. Giagounidis 2 3 4 , G. Metzgeroth 5 , A. Jonasova 6 , R. Herbst 7 , J. M. T. Diaz 8 , B. De Renzis 9 , K. S. Götze 2 3 10 , M.-L. Huetter-Kroenke 11 , M.-P. Gourin 12 , B. Slama 13 , S. Dimicoli-Salazar 14 , P. Cony-Makhoul 15 , K. Laribi 16 , S. Park 17 , K. Jersemann 18 , D. Schipp 19 , K. H. Metzeler 20 , O. Tiebel 21 , K. Sockel 2 22 , S. Gloaguen 2 3 , A. Mies 22 , F. Chermat 23 , C. Thiede 22 , R. Sapena 23 , R. F. Schlenk 24 25 , P. Fenaux 3 23 26 , U. Platzbecker 2 3 20 , L. Ades 3 23 26

1 Department of Hematology, Cellular Therapy and Hemostaseology, University Hospital Leipzig; 2 German MDS Study Group (D-MDS); 3 The European Myelodysplastic Syndromes Cooperative Group (EMSCO), Leipzig; 4 Department of Oncology, Hematology and Palliative Care, Marien Hospital, Düsseldorf; 5 Department of Hematology and Oncology, University Medical Centre, Mannheim, Germany; 61 st Medical Department - Hematology, General Hospital, Prague, Czechia; 7 Medizinische Klinik III, Klinikum Chemnitz, Chemnitz, Germany; 8 Department of Hematology and Oncology, CHU de Poitiers, Poitiers; 9 Service d’Hématologie Clinique Adulte, Clermont Ferrand, France; 10 Department of Medicine III, Technical University of Munich, Munich; 11 Department of Hematology, Oncology, and Tumor Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany; 12 CHU Limoges, Limoges; 13 Service d’Hématologie, Centre Hospitalier d’Avignon, Avignon; 14 University Hospital Bordeaux, Pessac; 15 Centre Hospitalier Annecy-Genevois, Pringy; 16 Centre Hospitalier Du Mans, Le Mans; 17 Department of Hematology, CHU Grenoble, Grenoble, France; 18 GWT-TUD GmbH, Dresden; 19 DS-Statistics, Rosenthal-Bielatal; 20 Department of Hematology, Cellular Therapy and Hemostaseology, Leipzig University Hospital, Leipzig; 21 Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty, Technical University Dresden; 22 Department of Internal Medicine I, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany; 23 Groupe Francophone des Myélodysplasies, Paris, France; 24 Department of Internal Medicine V, University Hospital of Heidelberg; 25 NCT-clinical trials office, German Cancer Research Center, Heidelberg, Germany; 26 Hématologie Clinique, Hôpital Saint-Louis, Paris, France

Background: In about half of patients with lower-risk (LR) myelodysplastic syndromes (MDS), thrombocytopenia is present at the time of diagnosis, being associated with shortened survival and a higher risk of progression to acute myeloid leukemia (AML). Romiplostim (ROM), a thrombopoietin receptor agonist (TPO-RA), has shown safety and clinical efficacy in prospective trials in LR-MDS. Post-hoc analyses have demonstrated hematologic improvement of platelets (HI-P) after ROM treatment contingent on endogenous thrombopoietin (TPO) levels and platelet transfusion events (PTE) (Sekeres et al. BJH 2014).

Aims: The prospective EUROPE multicenter phase 2 trial ( NCT02335268 ) investigated the predictive value of biomarkers like endogenous TPO levels, PTE and molecular markers on the clinical efficacy of single-agent ROM treatment within the `European Myelodysplastic Syndromes Cooperative Group` (EMSCO) network. Patients with IPSS low or intermediate 1 risk were eligible if baseline bone marrow blast count was <5% (central morphology) and platelet count was ≤30 Gpt/L or ≤50 Gpt/L in case of a bleeding history.

Methods: According to a previously published model of response to TPO-RA (Sekeres et al. BJH 2014), patients were assigned into two different cohorts at the time of screening based on previous PTE and centrally assessed TPO serum levels (cohort A: TPO<500 ng/l and PTE<6 units/past year; cohort B: TPO>500 ng/l, and/or PTE 3 6 units/past year). The primary efficacy endpoint was the rate of HI-P according to IWG 2006 criteria lasting for 3 8 weeks. ROM was initiated at a dose of 750 μg weekly by subcutaneous injection, the dose was adjusted based on the patient`s platelet counts.

Results: From 2015 to 2019, a total of 77 patients were included at 29 different trial sites in Germany, France and the Czech Republic. Regarding the primary endpoint, 32 out of 77 (42%) responded (HI-P) with a numerically higher response rate in cohort A (47%, n=24) vs. cohort B (31%, n=8) (p=0.2953). At 16 weeks of ROM treatment, three (4%) and seven (9%) patients had additional neutrophil (HI-N) and erythroid (HI-E) responses, respectively. None of the patients achieved trilineage responses (HI-P, HI-E and HI-N). Median duration of response was significantly longer for patients in cohort A (351 days) compared to cohort B (315 days) (p=0.006, log-rank-test). Mutated SRSF2 was significantly more frequent in responders (41%) compared to non-responders (16%) (p=0.018, Fisher’s exact test). In patients with an SRSF2 mutation, the probability to achieve HI-P was 65% compared to 33% inpatients with SRSF2 wildtype (Figure 1A). Comparing responders vs. non-responders, we found no significant changes of variant allelic burden of variants detected pre- and post-ROM (Figure 1B). Finally, we developed a response prediction model to ROM therapy with the aim to improve personalized patient stratification in the future. The percentage of correctly predicted HI-P was highest for the model, which included the variables platelet count, SRSF2 mutation status and the hemoglobin level using the threshold of 11.4 g/dl and resulted in an overall accuracy of 70 % for a correct ROM response prediction (Figure 1A).

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Summary/Conclusion: In conclusion, this prospective study confirms the efficacy and overall safety of ROM in this subgroup of LR-MDS patients with thrombocytopenia. To avoid overfitting of variables and to confirm our results, the here presented response prediction model needs to be validated in an external independent cohort.

* U.P. and L.A. contributed equally to this study as senior authors

S170: DYNAMIC INTERPLAY BETWEEN TUMOR AND MICRO-ENVIRONMENT DURING MYELOMA DISEASE PROGRESSION

M. C. Köse 1,* , I. Bergiers 2 , M. Malfait 3 , B. Heidrich 4 , D. De Maeyer 2 , N. Fourneau 2 , B. Verbist 2 , J. Van Houdt 2 , G. Vanhoof 2 , R. Verona 4 , M. Delforge 5 , J. Depaus 6 , N. Meuleman 7 , J. Van Droogenbroeck 8 , P. Vlummens 9 , C. J. Heuck 4 , N. Bahlis 10 , J. Caers 1 , T. Casneuf 2

1 Laboratory of Hematology, University of Liege, Liege; 2 Janssen Research & Development, Beerse; 3 Department of Applied Mathematics, Computer Science and Statistics, University of Ghent, Ghent, Belgium; 4 Janssen Research & Development, Spring House, United States of America; 5 University Hospital Leuven, Leuven; 6 Department of Hematology, CHU UCL Namur, Yvoir; 7 Service d’Hématologie, Université Libre, Brussels; 8 Departmentof Haematology, AZ Sint-Jan Brugge-Oostende AV, Brugge; 9 Department of Clinical Hematology, Ghent University Hospital, Ghent, Belgium; 10 Department of Hematology and Oncology, University of Calgary, Calgary, Canada

Background: Multiple Myeloma (MM) is an incurable plasma cell (PC) malignancy that evolves from two premalignant stages: Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). The disease progression has been characterized to be driven by intrinsic genomic events in the myeloma cells and by gradual dysregulation of the immune system.

Aims: We investigated how the interplay between tumor cells with their microenvironment and the underlying complex and dynamic immune biology evolve during this process.

Methods: Single cell multi-omics profiling, including RNA, B-cell receptor (BCR) and antibody barcode-tagged 10x sequencing, was conducted on human bone marrow (BM) aspirates collected at 6 Belgian centers from 4 cohorts: 31 healthy elderly and 28 MGUS, 32 SMM and 32 newly diagnosed MM. Mononuclear cell isolation, freezing and transport to central facilities was optimizedand data were integrated and filtered using Scanpy and Scirpy. The main immune cell types were identified from the RNA and antibody data using SingleR. Further functional subtyping was done using Leiden clustering. Differential pathway expression analysis was performed with Muscat and FGSEA.

Results: From the tumor cell transcriptomes, our analyses confirmed the previously documented myeloma molecular hallmarks, such as MYC and IFN-a signaling, cell proliferation, energy metabolism and oxidative phosphorylation. Evidence was found for transcriptomic similarities and within-and between-patient malignant PC transcriptomic heterogeneity, as well as the existence of multiple transcriptomic clones in several patients. We observed a positive correlation between the antigen processing mechanism in the PCs with IFN response, suggesting that this mechanism associates with initiation of the immune recognition and activation against the tumor.The gradually increasing differential gene expression was also observed in the immune microenvironment: dysregulation of signaling pathways initiates early in MGUS and spreads throughout the various cell types surrounding the tumor cells. Cell population shifts were also found. In the CD1C+ DCs, that play a role in cancer immune control, a functional shift was observed that correlated with disease progression towards a more mature and antigen presenting phenotype with higher levels of CD83, HBEGF, MCL1 and CXCL16 as well as increased TNF-a pathway. Similarly, a shift was observed in the macrophage population, toward M1 state showing high IFN response along with expression of MS4A4A, STAT1, TNFSF13B and TRAIL in more severe disease. Interestingly, in the CD8+ T cells, we detected a pre-dysfunctional subpopulation with high expression of GZMK, activation markers CD69, CCL4, CXCR4 and genes associated with T cell pre-dysfunctionality NR4A2, RGS1, TOX and TIGIT, that was found to be associated with progression (Figure). In the CD4+ cytotoxic T cells, a proportion change was observed with more severe disease.

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Summary/Conclusion: With the atlas of healthy, precursor and active MM patient BM samples, we generated a comprehensive and granular view of the various cell types involved in disease progression and provide evidence for early and gradually increasing immune dysregulation and activation of oncogenic driver pathways. Our data evidence the co-divergence and reciprocal stimulation of transcriptomes of tumor and microenvironment and support the postulation of microenvironment as a central modulator of cancer cell growth, survival and metastasis.

S171: MOLECULAR CLUSTERS OF IGM MONOCLONAL GAMMOPATHIES PRESENT DISTINCT BIOLOGIC, IMMUNE AND METABOLIC FEATURES

P. Mondello 1,* , J. Paludo 1 , J. Novak 1 , K. Wenzl 1 , S. Jalali 1 , J. Krull 1 , E. Braggio 2 , S. Dasari 3 , M Manske 1 , J. Abeykoon 1 , S. Vivekananda 1 , P. Kapoor 1 , A. Paulus 4 , C. Reeder 2 , S. Ailawadhi 4 , A. Chanan-Khan 4 , R. Kyle 1 , M. Gertz 1 , Z.-Z. Yang 1 , A. Novak 1 , S. Ansell 1

1 Medicine, Mayo Clinic, Rochester; 2 Medicine, Mayo Clinic, Phoenix; 3 Bioinformatics, Mayo Clinic, Rochester; 4 Medicine, Mayo Clinic, Jacksonville, United States of America

Background: IgM MGUS and Waldenstrom Macroglobulinemia (WM) represent a wide range of conditions whose management varies from observation to immunochemotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions.

Aims: To shed light on the biology that may account for the clinical differences, we performed the first comprehensive multi-omics analysis of IgM monoclonal gammopathies.

Methods: We used bone marrow (BM) CD19 + CD138 + sorted cells and matched BM plasma from 32 pts (7 IgM MGUS, 25 WM) and 5 healthy controls to perform whole exome sequencing, RNA-seq, proteomic and metabolomic analysis. 7 matched WM and 4 IgM MGUS pts were also evaluated using mass cytometry (CyTOF).

Results: Applying principal component analysis to gene expression profiling, most of WM pts clustered together, while a small subset of them grouped separately with MGUS pts, suggesting a biologic dichotomy within WM. The controls formed a group distinct from most WM and MGUS pts. Fig1A We then applied a non-negative matrix factorization consensus clustering to the gene expression data and identified three robust clusters. Cluster 1 (C1) included only pts with WM, cluster 2 (C2) included pts with both WM and MGUS, and cluster 3 (C3) included all normal controls as well as a small number of WM and MGUS pts. Fig1B-C When mutations commonly identified in WM were analyzed, there was no difference among the three groups (excluding controls) in mutation burden of MYD88 L265P and CXCR4 . Interestingly, aberrant expression of TNFAIP3 was a distinct feature of C1 as deletion of 6q (which encodes for TNFAIP3 ) and TNFAIP3 mutations were each significantly enriched in C1 (47%) compared to C2 (0%) and C3 (20%; p=0.04). Individual clusters associated with specific transcriptional signatures and clinical features. While C1 displayed enrichment of RNA processing, downregulation of inflammatory pathways and aggressive clinical behavior, C2 showed increased inflammatory signaling and senescence with indolent clinical behavior. C3 had intermediate features with combined proliferative and antigen response signatures. Fig1D In accordance with transcriptomics, the proteomic hallmark of C1 was upregulation of proteins involved in proliferation (eg AKT, MAPK) and downregulation of inflammatory proteins (eg IL4, IL10) while the opposite was observed in C2. Once more, C3 confirmed intermediate features with combined upregulation of proliferation and inflammatory proteins. The metabolism was rewired towards mitochondrial anabolism in C1 and C3, while towards glycolysis in C2. Accordingly, C1 and C3 showed undetectable concentrations of 3-hydroxybutyric acid as opposed to C2 which had increased levels of lactic acid, as end products of fatty acid oxidation and glycolysis respectively. Next, we explored whether C1 and C2 displayed a distinct immune profile. tSNE analysis showed that CD4 + T cells were more abundant in C2 compared to C1 while the opposite was observed for CD8 + T cells. Among CD4 + T cells, activated follicular helper (T FH ; p=0.02) and regulatory (T reg ; p=0.008) cells were predominantly expressed in C2. In contrast, C1 showed a higher expression of senescent T effector memory (T EM ) cells. Fig1E In support of this, SPADE clustering analysis identified three clusters including T FH , T reg and T EM cells.

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Summary/Conclusion: We have identified three molecular clusters in IgM monoclonal gammopathies with distinct clinical, proteomic, metabolomic and immune features, suggesting a potential biologic classification that may have therapeutic implications.

S172: FUNCTIONAL SUBSETS OF PLASMA CELLS ASSOCIATED WITH AMYLOID PRODUCTION AND

X. Wang 1 2 3,* , H. Han 1 3 , J. Sun 3 4 , Q. Wang 2 , X.-M. Gao 1 3 , K.-N. Shen 1 3 , L. Zhang 1 3 , Y. Zhao 2 , X.-X. Cao 1 3 , M. Qian 5 , Y. Chen 2 , J. Li 1 3

1 Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; 2 The State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College; 3 State Key Laboratory of Complex Severe and Rare Diseases; 4 Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; 5 School of Mathematical Sciences, Peking University, Beijing, China

Background: Light chain amyloidosis (AL amyloidosis, AL) is a life-threatening plasma cell dyscrasia characterized by misfolded monoclonal immunoglobulin light chain production by pathogenic bone marrow plasma cells (BMPCs), leading to irreversible damage in multiple organs. Until now, the pathogenic BMPCs in AL haven’t been elucidated, let alone the functional roles of BMPC subsets.

Aims: We aimed to identify intra- and inter-individual heterogeneities in BMPCs, especially those related to AL development, light chain production, organ tropism, and chemotherapy response.

Methods: Here, we conducted single-cell RNA sequencing and image flow cytometry analysis of BMPCs from patients with AL (n=3), compared with that from monoclonal gammopathy of undetermined significance (MGUS) (n=2) and healthy controls (n=21). All the subjects provided informed consent.

Results: We identified 7 functional subsets of BMPCs in AL, MGUS or healthy controls (Fig. 1a). These subsets had over-lapping but distinct functions, including DNA repair, cell proliferation, drug response, osteoclast differentiation, and immunoglobulin production (Fig. 1b). Subsets enriched in AL showed up-regulation of amyloidosis-associated genes (Fig. 1c), such as the amyloid-beta binding protein-encoding Apolipoprotein E (APOE), and showed plasmablastic morphology (Fig. 1d). Subsets defined by aberrant light chain production up-regulated the pathways related to neutrophil degranulation, transportation to and modifications in the Golgi apparatus, and asparagine N-linked protein glycosylation. High expression of Cyclin D1 (CCND1), CD79A, and V-Set Pre-B Cell Surrogate Light Chain 3 (VPREB3) were observed in the predominant subset of AL predicted sensitive to venetoclax, while Cyclin D2 (CCND2), S100 Calcium Binding Protein A6 (S100A6), Cystatin C (CST3) were up-regulated in that of AL predicted resistant (Fig. 1e). In an independent cohort of bulk RNA sequencing (n=29), clinical subgroups of patients with AL were defined by the proportion of functional subsets, including 2 major subgroups that were consistent with the aforementioned AL with differential sensitivity to venetoclax (Fig. 1f). Co-expression of CCND1, CD79A, and VPREB3, and a larger predominant subset, were in the venetoclax-sensitive subgroup. The up-regulation of CCND2 and amyloidosis-associated genes, including S100A6 and CST3, were in the venetoclax-resistant subgroup (Fig. 1g).

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Summary/Conclusion: These functional subsets of BMPCs provide mechanistic insights into AL pathogenesis, light chain production, and chemotherapy response. These results suggest potential avenues for the exploration of clinical subgroups among AL patients.

S173: T-CELL ACTIVATION AND MYELOMA CELL KILLING CONFIRM THE MODE OF ACTION OF RG6234, A NOVEL GPRC5D T-CELL ENGAGING BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

I. Dekhtiarenko 1,* , I. Lelios 2 , J. Attig 2 , N. Sleiman 2 , D. Lazzaro 2 , I. Clausen 2 , N. Gräfe 3 , H.-J. Helms 2 , E. Schindler 2 , S. Belli 2 , T. Fauti 1 , J. Eckmann 3 , P. Umana 1 , W. Jacob 3 , M. Schneider 2 , C. Hasselbalch Riley 4 , M. Hutchings 4 , S.-S. Yoon 5 , Y Koh 5 , S. Manier 6 , T. Facon 6 , S. J. Harrison 7 , J. Er 7 , F. Volzone 8 , A. Pinto 8 , C. Montes 9 , E. M. Ocio 9 , A. Alfonso-Pierola 10 , P. Rodríguez Otero 10 , F. Offner 11 , A. Guidetti 12 , P. Corradini 12 , C. Titouan 13 , C. Hulin 13 , C. Touzeau 14 , P. Moreau 14 , R. Popat 15 , S. Leong 15 , R. Mazza 16 , C. Carlo-Stella 16 , A.-M. E. Bröske 3

1 Roche Pharma Research and Early Development, Roche Innovation Center Zurich, Zurich; 2 Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland; 3 Roche Pharma Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany; 4 Rigshospitalet, Copenhagen, Denmark; 5 Seoul National University College of Medicine, Seoul, South Korea; 6 Lille University Hospital, Lille, France; 7 Peter MacCallum Cancer Center and Royal Melbourne Hospital, and Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia; 8 Istituto Nazionale dei Tumori, Fondazione Pascale, IRCCS, Napoli, Italy; 9 Hospital Universitario Marques de Valdecilla (IDIVAL), Universidad de Cantabria, Santander; 10 Clinica Universidad de Navarra, Navarra, Spain; 11 Universitair Ziekenhuis Gent, Gent, Belgium; 12 Istituto Nazionale dei Tumori, Milano, Italy; 13 CHU de Bordeaux, Bordeaux; 14 CHU de Nantes, Nantes, France; 15 University College London Hospitals NHS Foundation Trust, London, United Kingdom; 16 Department of Biomedical Sciences, Humanitas University and Department of Oncology and Hematology, IRCCS Humanitas Research Hospital, Milano, Italy

Background: RG6234 is a novel T-cell engaging bispecific antibody targeting G protein-coupled receptor 5D (GPRC5D) with a unique 2:1 format. GPRC5D is highly expressed on multiple myeloma (MM) cells and concurrent binding of RG6234 to GPRC5D and CD3 on T cells results in immunological synapse formation and potent T-cell directed tumor cell killing. An ongoing Phase 1 dose-escalation study ( NCT04557150 ) is investigating the safety, clinical activity, pharmacodynamics (PD), and pharmacokinetics of RG6234 monotherapy in patients (pts) with relapsed/refractory MM (RRMM). Clinical activity was observed during dose escalation, and the safety profile was manageable (Riley et al. EHA 2022).

Aims: Here, we present preliminary clinical biomarker data highlighting PD effects after intravenous (IV) administration that confirm the mechanism of action and high potency of RG6234.

Methods: Exploratory biomarker analyses included data from pts treated with RG6234 doses ranging from 0.006mg to 4.8mg in dose escalation. RG6234 was administered as an IV infusion under a step-up dosing regimen, reaching the target dose not later than 2 weeks after the priming dose. Peripheral biomarkers were evaluated using whole blood flow cytometry (n=28), plasma cytokine Protein Simple ELLA (n=33), and plasma sBCMA Protein Simple ELLA (n=26). MM cells were assessed at baseline and on-treatment by bone marrow (BM) aspirate flow cytometry and by BM biopsy CD138/CD8 immunohistochemistry. Informed consent was obtained from participating pts. The clinical cut-off date for the current analysis was January 31, 2022.

Results: PD changes were observed in peripheral blood at all tested doses. Cytokines (IFNg, TNFa, CXCL10, IL6, IL10, IL2, IL8) and sCD25 peaked at 4 to 24 hours (h) following the first administration, while cytokine peak magnitudes decreased at subsequent administrations. Cytokine release was followed by transient reduction of circulating T cells at 4 h after infusion, with partial recovery of peripheral T-cell counts by Day 8 after first administration. Elevation of sCD25 and IFNg in plasma (~3.2 and 33 median fold change from baseline, respectively) together with increase in T-cell proliferation (~4 median fold increase of Ki67+CD8+ T-cells) within 72 h post first infusion indicated T-cell activation. Analysis of a limited number of paired baseline and on-treatment BM biopsies (n=13) revealed that the density of CD8+ tumor-infiltrating T cells increased upon treatment in responders, indicating T-cell recruitment towards the tumor. RG6234 induced rapid depletion of MM cells, as demonstrated by a decrease of sBCMA in the plasma of responding pts already 8 days after the first administration (median 33.5% reduction from baseline in responding pts; n=15). Moreover, at the end of Cycle 1, the majority of pts (14/15) had <1% of MM cells in BM based on flow cytometry readout. GPRC5D expression was detected at baseline in all pts with evaluable bone marrow aspirate and >20 detectable MM cells (n=16). Updated exploratory biomarker data will be presented.

Summary/Conclusion: Cytokine release, T-cell activation, BM infiltration, and MM cell depletion are early PD changes seen after treatment with RG6234 and precede clinical responses. These PD changes indicate that RG6234 leads to T-cell engagement in the BM of pts with RRMM and clearly demonstrate rapid and effective T-cell mediated anti-MM activity.

S174: HIGH LEVEL OF CIRCULATING TUMOUR DNA AT DIAGNOSIS CORRELATES WITH DISEASE SPREADING AND DEFINES MULTIPLE MYELOMA PATIENTS WITH POOR PROGNOSIS

M. Martello 1,* , A. Poletti 1 , D. Bezzi 2 , E. Borsi 1 , B. Taurisano 1 , V. Solli 1 , S. Armuzzi 1 , I. Vigliotta 1 , G. Mazzocchetti 1 , I. Pistis 3 , L. Pantani 3 , S. Rocchi 1 , K. Mancuso 1 , P. Tacchetti 3 , I. Rizzello 1 , M. Cavo 1 , E. Zamagni 1 , C. Nanni 2 , C. Terragna 3

1 IRCCS - Azienda Ospedaliero Universitaria di Bologna - Department of Experimental, Diagnostic and Specialty Medicine - University of Bologna; 2 IRCCS - Azienda Ospedaliero Universitaria di Bologna - Department of Nuclear Medicine; 3 IRCCS - Azienda Ospedaliero Universitaria di Bologna, Bologna, Italy

Background: Multiple Myeloma (MM) is a plasma cell (PC) disorder characterized by the presence of skeletal involvement at the time of diagnosis, as detected by MRI and/or FDG PET/CT, in most of the patients. The patchy nature of the disease is probably related to the ability of fitter clones to spread into peripheral blood reaching distant sites, where favourable microenvironment conditions might promote clones’ seeding. Recently, cell-free DNA (cfDNA) has been proven to resume the heterogeneity of spatially distributed clones. However, it has to be determined to which extent cfDNA correlates with disease distribution and its possible implications with patients’ outcome. Moreover, the potential of cfDNA to track the evolutionary dynamics and the heterogeneity of MM, possibly anticipating the emergence of therapy resistant residual cells, remains to be confirmed.

Aims: Aim of this study is to quantitatively and qualitatively evaluate cfDNA at diagnosis and during follow-up in correlation with imaging data to possibly define the integration of this approach with molecular bone marrow (BM) and whole-body residual disease assessment.

Methods: A total of 88 newly diagnosed MM patients were screened at baseline with 18F-FDG PET/CT, and molecularly assessed by Ultra Low Pass-Whole Genome Sequencing (ULP-WGS). In a subgroup of 22 patients, cfDNA was monitored monthly, whereas PET/CT was reassessed after induction therapy to evaluate metabolic tumor response. For each pts, ULP-WGS was used to characterize both the neoplastic PC clone(s) in the BM (gDNA) and the cfDNA from peripheral blood. Data were analysed by ichorCNA and Clonality R packages.

Results: At diagnosis, the cfDNA tumor fraction (TF) was significantly lower as compared to gDNA TF [median (M) TF: 4.4 vs. 59.7%, respectively]. Nevertheless, high cfDNA TF levels (> 4.4% cfDNA TF values; range: 4.4-84.3%) correlated with high gDNA TF levels (>65.7% gDNA TF values; range: 65.7-96.7%). This observation was further confirmed by a significant correlation between cfDNA TF and the percentage of BM CD138/CD38 positive plasma cells (r=0.47; p<.0001).

Interestingly, patients with high cfDNA TF at baseline were more likely to present with extramedullary disease (EMD) and a higher number of focal lesions, and also featured a more active tumour metabolism, as compared to pts with low TF (EMD 4/44=9% vs. 1/44=2.3%, p=ns; M n. PET lesions: 1.7 vs. 2.5, p= 0.003; SUVmax: 5.2 vs. 9.6, p = 0.01).

Despite an overall concordance between cfDNA and BM genomic profiles (80/88=90.9%), those patients with high cfDNA TF showed more frequently evidence of spatial heterogeneity, as highlighted by a substantial divergence of copy number alterations profiles between cfDNA and gDNA (7/8=87.5% of pts with divergent profiles). Finally, high cfDNA TF at diagnosis predicted for poorer prognosis as compared with low cfDNA TF (PFS at 20 months: 67% vs. 86%, p=0.05; OS at 20 months: 90% vs. 100%, p=0.04).

Interestingly, after bortezomib-based induction therapy, imaging data and cfDNA TF levels were concordant; notably, in 4/10 (40%) patients with median SUVmax 5.8 and >2 PET lesions, cfDNA TF was still detectable (>1% TF), thus corroborating a possible role for cfDNA in monitoring response to therapy.

Summary/Conclusion: In conclusion, patients with high cfDNA TF displayed imaging data that overall suggested a higher propensity to a metastatic spread of the disease, which finally correlated with poorer prognosis. Future studies will be addressed to exploit the use of cfDNA in disease monitoring.

Acknowledgements: AIRC IG2019, AIL BOLOGNA ODV

S175: ATLAS: A PHASE 3 RANDOMIZED TRIAL OF CARFILZOMIB, LENALIDOMIDE, AND DEXAMETHASONE VERSUS LENALIDOMIDE ALONE AFTER STEM-CELL TRANSPLANT FOR MULTIPLE MYELOMA

D. Dytfeld 1,* , T. Wróbel 2 , K. Jamroziak 3 , T. Kubicki 1 , P. Robak 4 , A. Walter-Croneck 5 , J. Czyż 6 , A. Tyczyńska 7 , A. Druzd-Sitek 8 , K. Giannopoulos 9 , A. Nowicki 1 , T. Szczepaniak 1 , A. Łojko-Dankowska 1 , M. Matuszak 1 , L. Gil 1 , B. Puła 10 , J. Rybka 2 , M. Majcherek 2 , L. Usnarska-Zubkiewicz 10 , L. Szukalski 11 , A. Końska 10 , J. M. Zaucha 7 , J. Walewski 8 , D. Mikulski 4 , O. Czabak 5 , T. Robak 4 , U. Grzybowska 1 , O. B. Lahoud 12 , J. A. Zonder 13 , K. Griffith 14 , A. Stefka 15 , A. Major 15 , B. A. Derman 15 , A. J. Jakubowiak 15

1 Poznan University of Medical Sciences, Poznan; 2 Wroclaw Medical University, Wroclaw; 3 Medical University of Warsaw, Warsaw; 4 Medical University of Lodz, Lodz; 5 University of Medical Sciences in Lublin, Lublin; 6 Collegium Medicum, Bydgoszcz; 7 Medical University of Gdansk, Gdansk; 8 Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw; 9 Medical University of Lublin, Lublin; 10 Institute of Hematology and Blood Transfusion, Warsaw; 11 Medical University of Bydgoszcz, Bydgoszcz, Poland; 12 Memorial Sloan Kettering Cancer Center, New York; 13 Karmanos Cancer Institute, Detroit; 14 University of Michigan, Ann Arbor; 15 University of Chicago, Chicago, United States of America

Background: Treatment following autologous stem cell transplantation (ASCT) for multiple myeloma (MM) remains an area of active investigation. We have shown that extended post-ASCT treatment with carfilzomib, lenalidomide, and dexamethasone (KRd) after KRd induction improved the depth and duration of response (Jasielec et al, Blood 2020), suggesting a benefit of post-ASCT KRd therapy.

Aims: We report the results of ATLAS, a multicenter, international, open-label randomized phase 3 study to determine the efficacy and safety of post-ASCT KRD vs standard lenalidomide (R) maintenance ( NCT02659293 ).

Methods: Newly-diagnosed MM patients (pts) who received any induction therapy for up to 12 months (mo) followed by single ASCT and achieved at least stable disease within 100 days afterward were randomized to receive either KRd or R, stratified by post-transplant response (≥VGPR vs <VGPR) and cytogenetic risk [standard risk (SR) vs high [HR: presence of t(4;14), t(14;16), or del(17p)]. Pts randomized to KRd received carfilzomib 36 mg/m 2 on days (D) 1,2,8,9,15,16 for 4 cycles (C) then D1,2,15,16 starting C5; R 25 mg D1-21, and dexamethasone 20 mg D1,8,15,22 in 28-day cycles. KRd pts with SR who reached IMWG-defined MRD-negativity after C6 de-escalated therapy to R alone after C8 (KRd->R); the rest continued KRd through C36 followed by R alone until progression. Pts randomized to R received lenalidomide 10 mg C1-3 and then 15 mg daily. The primary endpoint was progression free survival (PFS) rate: based on historical PFS rates, a sample size of 180 Pts was calculated to provide 85% power with 2-sided alpha 0.05. Secondary endpoints included minimum residual disease (MRD) negativity response rates and treatment-related adverse events.

Results: 180 pts were enrolled (R n=87; KRd n=93) through 10/21/20; data cutoff was 12/31/21. Pt characteristics in the KRd and R arms were balanced for median age (58 vs 59 yrs), ≥VGPR (88% vs 92%), and HR (23% vs 21%). After 6 cycles, 47% pts in the KRd arm and 29% in the R arm achieved MRD-negativity (p=0.017). 34 KRd pts eligible for de-escalation converted to R alone after C8 and were analyzed on the KRd arm per intention-to-treat. At median follow-up of 33.8 mo, 23 pts (25%) on the KRd arm and 38 pts (44%) on the R arm progressed; estimated median PFS was 59.0 mo for KRd vs 41.4 mo for R (Hazard Ratio 0.56, logrank p=0.026). At cutoff, 90% of KRd and 87% of R pts were alive; no deaths were treatment-related. All-grade toxicities were generally comparable between arms. The most common grade 3+ AEs and those of special interest were neutropenia (KRd 47%; R 59%), thrombocytopenia (KRd 13%; R 7%), infections (KRd 15%; R 6%), cardiovascular toxicities (KRd 4%, R 5%), and secondary malignancies (KRd 2, R 2).

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Summary/Conclusion: This is the first randomized phase 3 trial demonstrating superior PFS with extended post-transplant KRd therapy compared to R maintenance. Therefore, MRD/risk-adapted post-ASCT extended KRd treatment may represent a new standard of care.

S176: DARATUMUMAB CARFILZOMIB LENALIDOMIDE AND DEXAMETHASONE AS INDUCTION THERAPY IN HIGH-RISK TRANSPLANT ELIGIBLE NEWLY DIAGNOSED MYELOMA PATIENTS: RESULTS OF THE PHASE 2 STUDY IFM 2018-04

C. Touzeau 1,* , A. Perrrot 2 , C. Hulin 3 , S. Manier 4 , M. Macro 5 , M.-L. Chretien 6 , L. Karlin 7 , O. Decaux 8 , C. Jacquet 9 , M. Tiab 10 , X. Leleu 11 , L. Planchce 12 , H. Avet-Loiseau 2 , P. Moreau 13

1 CHU Nantes - Hotel Dieu, CHU Nantes - Hotel Dieu, Nantes; 2 Hematology, IUCT Oncopole, Toulouse; 3 Hematology, CHU Bordeaux, Bordeaux; 4 Hematology, CHU Lille, Lille; 5 Hematology, CHU Caen, Caen; 6 Hematology, CHU Dijon, DIjon; 7 Hematology, CHU Lyon, Lyon; 8 Hematology, CHu Rennes, Rennes; 9 Hematology, CHU Nancy, Nancy; 10 Hematology, CHD, La Roche sur Yon; 11 Hematology, CHU Poitiers, Poitiers; 12 Biostatistic; 13 Hematology, CHU Nantes, Nantes, France

Background: High-risk (HR) cytogenetic is associated with poor outcome in transplant eligible (TE) newly diagnosed myeloma multiple myeloma (NDMM). The triplet combination carfilzomib lenalidomide and dexamethasone (KRD) plus transplantation demonstrated high efficacy with favorable safety profile in TE-NDMM patients (FORTE). The addition of daratumumab (Dara) to frontline therapy also improved response rate and progression free-survival in TE-NDMM patients (CASSIOPEIA, GRIFFIN). Double transplant also improved outcome of HR TE NDMM patients (EMN02, STAMINA).

Aims: The phase 2 trial 2018-04 from the Intergroupe Francophone du Myelome (IFM) is evaluating an intensive strategy with Dara-KRD induction and consolidation plus double transplant in HR TE NDMM ( NCT03606577 ).

Methods: HR MM was defined by the presence of del17p, t(4;14) and/or t(14;16). Stategy includes Dara-KRD induction (6 cycles), autologous stem cell transplantation (ASCT), Dara-KRD consolidation (4 cycles), second ASCT, Dara-lenalidomide maintenance. The primary endpoint was the feasibility of this intensive strategy. Here, we report efficacy and safety analysis of Dara-KRD induction.

Results: Fifty patients with previously untreated NDMM were included from july 2019 to march 2021 in 11 IFM centers Median age was 57 (range 38 -65). ISS stage 3 was present in 12 (24%) patients. Based on inclusion criteria, all patients had HR cytogenetic, including 17p deletion (n=20, 40%), t(4;14) (n=26, 52%) or t(14;16) (n=10,20%). Forty-six patients completed Dara-KRD induction. Two patients discontinued treatment due to severe adverse event (COVID-19 infection, n=1; drug-induced hepatitis, n=1) and 2 patients discontinued treatment due to disease progression. Grade 3-4 treatment related adverse event (>5% of patients) were neutropenia (38%), anemia (14%), thrombocytopenia (8%), infection (6%), renal insufficiency (6%) and deep-vein thrombosis (6%). Two patients (6%) experienced stem-cell collection failure. Overall response rate was 96%, including 92 % ≥ very good partial response. Among 37/46 evaluable patients post induction, Minimal Residual Disease negativity rate (NGS, 10- 5 ) was 62%.

Summary/Conclusion: Dara-KRD as induction prior ASCT is safe and allows deep responses in TE NDMM patients with high-risk cytogenetic profile. IFM 2018-04 study is ongoing and longer follow-up is needed to evaluate safety and efficacy of the overall strategy with Dara-KRD induction and consolidation plus double transplant in this subset of HR patients.

S177: EVALUATING SERUM FREE LIGHT CHAIN RATIO AS A BIOMARKER FOR MULTIPLE MYELOMA

T. Akhlaghi 1,* , K. Maclachlan 2 , N. Korde 2 , S. Mailankody 2 , A. Lesokhin 2 , H. Hassoun 2 , S. X. Lu 2 , D. Patel 2 , U. Shah 2 , C. Tan 2 , A. Derkach 3 , O. Lahoud 4 , H. J. Landau 4 , G. L. Shah 4 , M. Scordo 4 , D. J. Chung 4 , S. A. Giralt 4 , S. Z. Usmani 2 , O. Landgren 5 , M. Hultcrantz 2

1 Department of Internal Medicine, Icahn School of Medicine, Mount Sinai Morningside and West; 2 Myeloma Service, Department of Medicine; 3 Department of Epidemiology and Biostatistics; 4 Adult Bone Marrow Transplant Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York; 5 Myeloma Service, Sylvester Comprehensive Cancer Center, University of Miami, Miami, United States of America

Background: In 2014, the definition of multiple myeloma (MM) was updated to include serum free light chain (FLC) ratio ≥100 as a myeloma defining biomarker, based on retrospective data indicating a 2-year progression rate of 80% and a median time to progression (TTP) of 12 months associated with this marker. As a result, patients previously diagnosed as smoldering MM (SMM), were considered to have MM requiring treatment if they had a FLC ratio ≥100. However, two recent studies have reported lower 2-year progression rates, 30-44%, and a longer median TTP of 40 months in patients with FLC ratio ≥100, raising concern about the FLC criteria.

Aims: To assess the risk of progression in patients with SMM and a FLC ratio ≥100.

Methods: We performed a retrospective analysis of patients diagnosed with SMM at Memorial Sloan Kettering Cancer Center between January 2000 and December 2017. Diagnosis of SMM and progression to active MM requiring therapy was defined according to the International Myeloma Working Group (IMWG) criteria at the time of diagnosis. Kaplan-Meier method was used to assess TTP and generate survival curves, with log-rank test for comparison between groups.

Results: A total of 438 patients were included in the study, with a median follow-up time of 52 months. While all patients with FLC ≥100 (n=66) had elevated involved FLC levels, 35 (53%) had an involved FLC concentration >100 mg/L. Per current diagnostic criteria, we only included patients with an involved FLC concentration >100 mg/L in the FLC ratio ≥100 group and found a median TTP of 31 months (95% confidence interval [CI] 16-59 months) and a 2-year progression rate of 49% (CI 28-63%). In a sensitivity analysis including all 66 cases with FLC ratio ≥100 (independent of involved FLC concentration), we found the median TTP to be 41 months (CI 30-72 months), compared to 101 months for those with a FLC ratio <100 (CI 78-127 months; p<0.0001). Furthermore, the risk of progression within 2 years was 35% (CI 22-46%) compared to 18% (CI 14-23%; p<0.0001) for those with a FLC ratio <100.

Of note, 22 patients with a FLC ratio ≥100 were monitored expectantly for >4 years, among whom 12 patients had involved FLC levels >100 mg/L. Ten patients (7 with involved FLC level >100 mg/L) were followed over a period ranging from 4 to 8.5 years before eventually progressing, and 12 patients (5 with an involved FLC level >100 mg/L) were followed between 4 and 8 years and did not progress during the study period.

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Summary/Conclusion: While FLC ratio ≥100 is associated with a high risk of progression in patients with SMM, it does not infer an imminent risk of progression, defined by the IMWG as median TTP of 12 months and 2-year progression rate of at least 80%. On the contrary, select patients with FLC ratio ≥100 can be followed for many years without progressing and some may never progress despite long-term follow-up. These findings suggest that in patients where FLC ratio ≥100 is the only myeloma-defining event, other high-risk features as well as the evolution of FLCs over time should be considered in the decision to start a patient on treatment.

S178: SAFETY AND EFFICACY OF BELANTAMAB MAFODOTIN IN COMBINATION WITH RD IN NEWLY DIAGNOSED, TRANSPLANT INELIGIBLE MULTIPLE MYELOMA PATIENTS: A PHASE 1/2 STUDY BY THE HELLENIC SOCIETY OF HEMATOLOGY

E. Terpos 1,* , M. Gavriatopoulou 1 , I. Ntanasis-Stathopoulos 1 , P. Malandrakis 1 , D. Fotiou 1 , N. Kanellias 1 , S. Gkolfinopoulos 2 , K. Manousou 2 , E. Kastritis 1 , M.-A. Dimopoulos 1

1 Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 2 Health Data Specialists, Dublin, Ireland

Background: Belantamab mafodotin (belamaf), a multi-modal antibody-drug conjugate targeting BCMA, has shown efficacy and tolerability in pretreated patients (pts) with relapsed/refractory multiple myeloma. In pts refractory to immunomodulatory drugs, proteasome inhibitors, and antiCD38, belamaf plus pomalidomide and dexamethasone induced a very good partial response (VGPR) or better of 69.2% and a median progression-free survival of 16.2 months (Trudel S, ASH 2021). Preclinical evidence suggests that belamaf plus lenalidomide enhances antimyeloma activity, with no overlapping toxicities.

Aims: To evaluate the safety and efficacy of belamaf plus lenalidomide and dexamethasone (Rd) in transplant-ineligible (TI) pts with newly diagnosed multiple myeloma (NDMM).

Methods: The ongoing, prospective, open-label, 2-part, phase 1/2 BelaRd study ( NCT04808037 ) aims to enroll 66 pts with TI NDMM from a Greek center. Eligible are adult pts with Eastern Cooperative Oncology Group status 0–2 and adequate organ function. Part 1 (dose selection) evaluates the safety/tolerability of 3 belamaf doses (2.5, 1.9, and 1.4 mg/kg on Day 1 of every other 28-day cycle) plus Rd (each dose regimen administered to a cohort of 6 pts) over ≥4 weeks of follow up; subsequently, an additional 6 pts are enrolled in each dose cohort to establish the recommended phase 2 dose (RP2D). Part 2 (dose expansion) evaluates the safety and clinical activity of belamaf RP2D plus Rd in 30 additional pts. This descriptive analysis presents the safety data for all Part 1 pts and the efficacy data for all Part 1 pts with ≥2 post-baseline efficacy assessments by the cut-off date (14/01/2022).

Results: Of 36 pts included, 35 (97.2%) continued study treatment by the cut-off date, and 1 (2.8%) had died due to a belamaf unrelated adverse event (AE). The pt median age was 72.5 years (range 64.0–86.0). Of pts with available data (30 [83.3%]), pts at revised International Staging System stages I, II, and III were 6 [20.0%], 21 [70.0%], and 3 [10.0%], respectively, and 3 (10.0%) had high-risk cytogenetics (i.e., del17p13, t(4;14), t[14;16]). Median duration of therapy was 4.2 months (range 0.5–11.9) and median number of cycles reached was 5.0 (range 1.0–11.0). Twenty-two (61.1%) pts experienced at least one grade (Gr) 3–4 AE. One (2.8%) pt experienced a Gr 5 AE (pneumonia), unrelated to belamaf. Most common (≥ 10.0% of pts) Gr 3–4 AE were fatigue (13 [36.1%] pts), visual acuity reduced (6 [16.7%] pts), and rash (5 [13.9%] pts). Gr 3–4 thrombocytopenias and infections were not reported, as were any Gr infusion-related reactions (Table). Pts with Gr 1–2 ocular symptoms, visual acuity reduced, and keratopathy, were 27 (75.0%), 21 (58.3%), and 18 (50.0%), respectively. Pts with Gr 3–4 ocular symptoms, visual acuity reduced, and keratopathy were 0 (0.0%), 5 (13.9%) and 0 (0.0%), respectively. Of all pts, 28 (77.8%) were evaluable for efficacy (Table). At a median follow up of 4.2 months (range 0.5–11.9), the overall response rate was 96.4% (27/28 pts; complete response [CR]: 14.3% [4/28 pts]; VGPR: 35.7% [10/28 pts]; partial response [PR]: 46.4% [13/28 pts]); no disease progression was reported. Of pts achieving VGPR, 6/10 (60.0%) had negative status serum/urine immunofixation electrophoresis. Median time to PR or better was 1.0 months (range 0.9–2.0).

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Summary/Conclusion: In pts with TI NDMM, belamaf every 2 months plus Rd showed an improved safety profile, especially at lower doses. Rapid and deep hematological responses were recorded.

S179: RANDOMIZED COMPARISON BETWEEN KRD AND KTD INDUCTION, FOLLOWED BY K MAINTENANCE OR OBSERVATION IN TRANSPLANT NON-ELIGIBLE PATIENTS WITH NDMM (AGMT-MM02 TRIAL)

H. Ludwig 1,* , T. Melchardt 2 , S. Sormann 3 , N. Zojer 4 , J. Andel 5 , B. Hartmann 6 , C. Tinchon 7 , E. Gunsilius 8 , K. Podar 9 , A. Egle 2 , W. Willenbacher 8 10 , E. Wöll 11 , M. Schreder 4 , R. Ruckser 12 , B Bozic 12 , M.-T. Krauth 13 , A. Petzer 14 , C Schmitt 15 , S. Machherndl-Spandl 16 , H. Agis 17 , M. Fillitz 18 , W. Pönisch 19 , S. Knop 20 , B. Paiva 21 , R. Greil 2

1 Wilhelminen Cancer Research Institute, Vienna; 2 Department of Internal Medicine III with Haematology, Medical Oncology, Haemostaseology, Infectiology and Rheumatology, Oncologic Center, Salzburg Cancer Research Institute – Laboratory for Immunological and Molecular Cancer Research (SCRI-LIMCR), Paracelsius Medical University, Cancer Cluster Salzburg, Salzburg; 3 Department of Internal Medicine, University Clinic Graz, Graz; 4 Department of Medicine I, Clinic Ottakring, Vienna; 5 Department of Internal Medicine IIr, Pyhrn-Eisenwurzen Klinikum Steyr, Steyr; 6 Department of Internal Medicine II, LKH Rankweil, Rankweil; 7 Department of Internal Medicine, LKH Hochsteiermark, Leoben; 8 Department of Internal Medicine V, Medical University Innsbruck, Innsbruck; 9 Department of Internal Medicine II, University Hospital Krems, Karl Landsteiner University of Health Sciences, Krems; 10 Oncotyrol, Center for personalized Cancer Medicine, Innsbruck; 11 Department of Internal Medicine, KH Zams, Zams; 12 Department of Medicine II, Clinic Donaustadt; 13 Department of Internal Medicine I, Division Hematology & Hemostaseology, Medical University Vienna, Vienna; 14 Department of Internal Medicine I, Ordensklinikum Linz Barmherzige Schwestern; 15 Clinic for Internal Medicine; 3 , Kepler University Clinic Linz; 16 Ordensklinikum Linz Elisabethinen, Linz; 17 Department of Internal Medicine I, Division of Oncology, Medical University Vienna; 18 Department of Internal Medicine, Hanusch Krankenhaus, Vienna, Austria; 19 Medical Clinic and Policlinic I, University Clinic Leipzig, Leipzig; 20 Medical Clinic and Policlinic II, University Clinic Würzburg, Würzburg, Germany; 21 Clinica Universidad de Navarra, Pamplona, Spain

Background: Carfilzomib (K)-based combinations have been established as effective frontline and relapse regimens in pts with multiple myeloma (MM).

Aims: In this randomized phase II trial we evaluated the impact of either Revlimid (R) or Thalidomide (T) as combination partner for K and dexamethasone (KRd or KTd) on outcome in pts with newly diagnosed MM (NDMM) not eligible for autologous transplantation (TNE). Further, we evaluated the role of one year K maintenance therapy compared to observation.

Methods: One hundred twenty two pts have been enrolled (ITT population). Median age was 75 yrs, ISS stage I/II/III: 29 (23.8%)/48 (39.3%)/45 (36.9%), ECOG stage 0/1: 64 (52.5%) / 58 (47.5%). t(4;14) ± del17p was noted in 15 (16.3%) of 92 pts with results available. Pts were randomized to 9 cycles of KRd or KTd, and 107 pts received at least one full cycle. Carfilzomib (K) was started with 20mg/m 2 at d 1 of cycle 1, and was continued with 27mg/m 2 for the first 2 cycles (d 1 + 2, 8 + 9, 15 + 16 schedule); followed by K administration at 57mg/m 2 once weekly for a 28 d cycle. Thalidomide 100mg/d (50mg in pts >75 yrs of age), d 1-28, or Revlimid 25mg/d (15mg in pts 3 75 yrs of age) d 1-21. Dexamethasone 40mg (20mg in pts 3 75 yrs of age) once/week. After induction, pts with 3 SD were randomized to K maintenance (d 1 and 15) for 12 cycles or observation. MRD was assessed by NGF with a sensitivity of 10 -6 in pts with ≥VGPR. Survival estimates were calculated according to Kaplan-Meier and survival curves were compared with the log-rank test. PFS and OS results presented are given for the ITT population. This trial is registered on clinicaltrials.gov ( NCT02891811 ).

Results: Median follow-up was 25.3 mos, 15 pts discontinued therapy within the first cycle due to patient (3) or investigator (1) decision, AE/toxicity (8), death or progressive disease (2) or other reason (1). Overall response rate was 91.3% in the entire group with available data (n=115). Results for sCR, CR, VGPR, PR, and ORR for KRd and KTd were similar between both groups (7.3%/10.0%, 27.3%/33.3%, 38.2%/35.0%, 14.5/16.7%, 87.3%/95.0%, respectively). Minor response was noted in 4 (3.5%), stable disease in 5 (4.3%) and progressive disease in 1 (0.8%) pts.

PFS (median 26.9 and 23.5 mos, p=0.832) and OS (not reached vs 52.2 mos, p=0.398) were similar between the KRd and KTd group, respectively. The OS rate at 36 mos was 82% in both groups. MRD testing was performed in 57 pts at time of CR/VGPR. Of those, 43.9%, (20.5% of the ITT group) pts were found to be MRD neg . PFS was significantly longer in MRD neg vs. MRD pos pts (p=0.003). Seventy six pts were randomized to K maintenance therapy or observation. Median PFS was numerically higher in the pts with K maintenance treatment (median 33.0 vs 24.0, p=0.714), but the difference was not statistically significant. Data on OS are not mature yet (only 9 events).

Grade 3/4 hematologic AEs were anemia (4.1%), leukopenia (0.8%), thrombocytopenia (7.4%), while non-hematologic grade 3/4 AEs were infection (20.5%), GI-disorders (7.4%), hypertension (7.4%), renal and cardiac impairment/failure (6.6% and 8.2% respectively).

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Summary/Conclusion: Our data show similar high efficacy of KRd and KTd in elderly NTE NDMM pts, including no difference in ORR (KRd and KTd, 87.3% and 95%, respectively), PFS and OS. Overall survival rate at 3 yrs was 82%. Median PFS was significantly longer in MRD neg pts. PFS was numerically, but not statistically longer in pts on K maintenance vs observation. Treatment was associated with an acceptable tolerance profile.

S180: RG6234, A NOVEL GPRC5D T-CELL ENGAGING BISPECIFIC ANTIBODY, INDUCES RAPID RESPONSES IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA: PRELIMINARY RESULTS FROM A FIRST-IN-HUMAN TRIAL

C. Hasselbalch Riley 1,* , M. Hutchings 1 , S.-S. Yoon 2 , Y. Koh 2 , S. Manier 3 , T. Facon 3 , S. J. Harrison 4 , J. Er 4 , F. Volzone 5 , A. Pinto 5 , C. Montes 6 , E. M. Ocio 6 , A. Alfonso-Pierola 7 , P. Rodriguez Otero 7 , F. Offner 8 , A. Guidetti 9 , P. Corradini 9 , C. Titouan 10 , C. Hulin 10 , C. Touzeau 11 , P. Moreau 11 , R. Popat 12 , S. Leong 12 , R. Mazza 13 , A.-M. E. Bröske 14 , I. Dekhtiarenko 15 , H.-J. Helms 16 , S. Belli 16 , T. Vardar 16 , T. Fauti 15 , J. Eckmann 14 , T. Moore 14 , M. Schneider 16 , W. Jacob 14 , M. Weisser 14 , C. Carlo-Stella 13

1 Rigshospitalet, Copenhagen, Denmark; 2 Seoul National University College of Medicine, Seoul, South Korea; 3 Lille University Hospital, Lille, France; 4 Peter MacCallum Cancer Center and Royal Melbourne Hospital, and Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia; 5 Istituto Nazionale dei Tumori, Fondazione Pascale, IRCCS, Napoli, Italy; 6 Hospital Universitario Marques de Valdecilla (IDIVAL), Universidad de Cantabria, Santander; 7 Clinica Universidad de Navarra, Navarra, Spain; 8 Universitair Ziekenhuis Gent, Gent, Belgium; 9 Istituto Nazionale dei Tumori, Milano, Italy; 10 CHU de Bordeaux, Bordeaux; 11 CHU de Nantes, Nantes, France; 12 University College London Hospitals NHS Foundation Trust, London, United Kingdom; 13 Department of Biomedical Sciences, Humanitas University and Department of Oncology and Hematology, IRCCS Humanitas Research Hospital, Milano, Italy; 14 Roche Pharma Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany; 15 Roche Pharma Research and Early Development, Roche Innovation Center Zurich, Zurich; 16 Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland

Background: Patients (pts) with relapsed/refractory multiple myeloma (RRMM) are in need of effective treatment options. RG6234 is a T-cell engaging bispecific antibody targeting GPRC5D with a novel 2:1 format. NCT04557150 is an ongoing first-in-human Phase 1 trial. We present initial results from the dose-escalation part using intravenous (IV) administration.

Aims: To characterize the safety of RG6234 and to identify a recommended Phase 2 dose (RP2D), and to characterize the pharmacokinetics, pharmacodynamics, and preliminary clinical activity.

Methods: Eligible pts with RRMM received RG6234 in escalating weekly step-up doses followed by a q2w regimen at a peak ‘target dose’ for up to 1 year. A mCRM model was used for overdose control. Adverse events (AEs) were graded per CTCAE v5.0 and cytokine release syndrome (CRS) per ASTCT criteria (Lee et al. 2019). Response was assessed by the investigator according to IMWG criteria. All pts provided informed consent.

Results: As of January 31, 2022, 41 pts had received RG6234 IV (0.006mg to 10mg). Median age was 63 years (range: 27 to 78) and 61% had R-ISS stage II or III at study entry. High-risk cytogenetics (t(4;14), t(14;16), del(17p), 1q21 gain) were present in 17/29 (58%) evaluable cases. Median number of prior therapies was 5 (range: 2 to 15). Pts were triple-class refractory in 67% of cases and penta-class refractory in 36%. Six pts (14.6%) had prior BCMA-directed therapy.

CRS occurred in 85.4% of pts (G1 56.1%, G2 24.4%, G3 2.4%). CRS was generally confined to the first cycle, with a median time to onset of 4.2 hours (range: 0 to 64.2), and 14 pts (34%) requiring tocilizumab. Three pts (7.3%) experienced CNS toxicity related to RG6234 (G1 and G3 headache; G1 confusion). G3/4 neutropenia, thrombocytopenia, and anemia occurred in 9.8%, 19.5%, and 12.2% of pts, respectively.

Infections were reported in 46.3% of pts (≥G3 14.6%). One patient died of an E. coli sepsis not considered related to RG6234.

AEs probably related to target expression included skin-related AEs in 66% of pts (G3 7.3%), dysgeusia/ageusia (36.6%, all G1/2), dry mouth (36.6%, all G1/2), dysphagia (17.1%, all G1/2), and nail changes (12.2%, all G1/2).

No discontinuations due to RG6234-related AEs occurred. The maximum tolerated dose (MTD) was not reached.

Within the tested target-dose range, RG6234 serum exposure generally showed a dose-proportional increase, with a preliminary IV terminal T ½ of ～5 days (mean value, n=22). At the predicted active-dose range (>0.162mg), RG6234 induced profound and rapid reductions in serum free light chains (FLC) and sBCMA. After the first treatment cycle, 93% of evaluable pts (14/15) had <1% of multiple myeloma cells in bone marrow based on flow cytometry.

Overall response rate in 34 efficacy evaluable pts across all doses was 68%, and 50% achieved a VGPR or better response, including 2/6 BCMA pre-treated pts. Responses occurred rapidly, with a median time to first response of 1.3 months (95% CI: 1.1, 1.6).

Median follow-up was 3.5 months (range: 0.03 to 11.7). Treatment was ongoing for 58.5% of pts at the time of the data cut. Duration of response was not mature. Response was ongoing in 18/23 (78.3%) responding pts, with a longest duration of response of 10 months.

Summary/Conclusion: RG6234, a novel T-cell engaging bispecific antibody targeting GPRC5D, demonstrates compelling clinical activity with manageable safety in heavily pretreated pts with RRMM. Dose escalation via IV and subcutaneous routes continues, and data will be updated.

S181: MODAKAFUSP ALFA (TAK-573): UPDATED CLINICAL, PHARMACOKINETIC (PK), AND IMMUNOGENICITY RESULTS FROM A PHASE 1/2 STUDY IN PATIENTS (PTS) WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM)

J. L. Kaufman 1,* , S. Atrash 2 , S. A. Holstein 3 , O. Nadeem 4 , D Benson 5 , K. Suryanarayan 6 , Y. Liu 7 , C. Li 8 , L. Yang 8 , X. Parot 8 , D. T. Vogl 9

1 Winship Cancer Institute of Emory University, Atlanta; 2 Haematology and blood disorders, Levine Cancer Institute, Charlotte; 3 University of Nebraska Medical Center, Omaha; 4 Dana-Farber Cancer Institute Boston, Boston; 5 Hematology, Ohio State University Comprehensive Cancer Center, Columbus; 6 Oncology Clinical Research; 7 Statistical and Quantitative Sciences; 8 Takeda Development Center Americas, Inc. (TDCA), Lexington; 9 Hematologic Malignancies and Bone Marrow Transplant Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, United States of America

Background: Modakafusp alfa (TAK-573) is a first-in-class immunocytokine designed to deliver attenuated interferon alpha-2b to CD38+ cells. In this phase 1/2 study ( NCT03215030 ), the maximum tolerated dose of modakafusp alfa was defined as 3 mg/kg every 4 weeks (Q4W); preliminary data from 29 pts treated at 1.5 mg/kg Q4W (5 in dose escalation, 24 in dose expansion) showed single-agent, anti-myeloma activity with an overall response rate (ORR) of 38% after a median follow-up of 4.2 months (mos) (Vogl ASH 2021, #898).

Aims: We present a clinical update on the efficacy and safety of modakafusp alfa 1.5 mg/kg Q4W after a median follow-up of 4.5 mos. We also present PK, immunogenicity, and dose-exposure-response data from the overall study.

Methods: Eligible pts had received ≥3 prior lines of treatment. Pts received modakafusp alfa as a 1–4-hour IV infusion at 10 dose levels from 0.001–6 mg/kg at weekly (cycles 1–2 only; 0.001–0.75 mg/kg), every-2-week (0.2–0.3 mg/kg), every-3-week (Q3W; 0.4–0.75 mg/kg), or Q4W (0.75–6 mg/kg) dosing intervals; an expansion cohort was opened at 1.5 mg/kg Q4W after anti-myeloma activity was observed in 3/5 pts in the escalation cohort at that dose. Serum samples prepared from blood were used for PK and anti-modakafusp alfa antibody (ADA) assessments. Modakafusp alfa concentrations were determined via a validated enzyme-linked immunosorbent assay; ADA were detected using a validated bridging electrochemiluminescence assay.

Results: As of Oct 2021, 30 pts had received modakafusp alfa 1.5 mg/kg Q4W. The median number of prior lines was 7 (range 3–16); 90% of pts were refractory to an anti-CD38 monoclonal antibody (mAb), while 37% were exposed to an anti-B-cell maturation agent (BCMA). Grade (G) 3–4 treatment-emergent adverse events (TEAEs) were reported in 25 pts (83%); the most frequent G3–4 TEAEs were neutropenia in 19 (63%), thrombocytopenia in 13 (43%), leukopenia in 12 (40%), anemia in 9 (30%), and decreased lymphocyte count in 9 (30%) pts. As reported previously, 1 pt in the 1.5 mg/kg Q4W cohort had a G3 bleeding event, while 3 pts had G3 infections. Among all pts, the ORR was 40% (complete response, 7%; very good partial response, 20%; partial response, 13%); among anti-CD38 mAb-refractory and anti BMCA-exposed pts, the ORR was 37% and 27%, respectively. Median progression-free survival was 6 mos and median duration of response had not been reached (Kaplan-Meier estimate, 91% at 6 mos). Based on pooled available data for the Q3W and Q4W cohorts (all doses) within the escalation and expansion phases, there was a strong trend for a dose-exposure-response relationship for ORR, with the apparent inflection point at 1.5 mg/kg Q4W dosing. No apparent relationship between dose-exposure-response and G3/4 thrombocytopenia or neutropenia was observed across all doses, whereas a correlation between dose-exposure-response and incidence of infusion-related reactions across the 0.4–6 mg/kg Q3W and Q4W dosing cohorts was found. There was an apparent non-linear (more than dose proportional) increase in exposure in the dose range of 0.1–3 mg/kg with a geometric mean terminal half-life of 13 hours in serum for modakafusp alfa 1.5 mg/kg. Based on the available data, 14% and 60% of pts were ADA positive at baseline and post-treatment, respectively.

Summary/Conclusion: Modakafusp alfa has a novel mechanism of action and has shown encouraging activity with a manageable safety profile in pts with RRMM. Characterization of the potential clinical impact of immunogenicity is ongoing. The optimal dose for single-agent modakafusp alfa will be further investigated in a phase 2 study.

S182: TALQUETAMAB, A G PROTEIN-COUPLED RECEPTOR FAMILY C GROUP 5 MEMBER D X CD3 BISPECIFIC ANTIBODY, IN RELAPSED/REFRACTORY MULTIPLE MYELOMA: UPDATED EFFICACY AND SAFETY RESULTS FROM MONUMENTAL-1

M. C. Minnema 1,* , A. Krishnan 2 , J. G. Berdeja 3 , A. Oriol 4 , N. W. van de Donk 5 , P. Rodríguez-Otero 6 , D. Morillo 7 , M.-V. Mateos 8 , L. J. Costa 9 , J. Caers 10 , D. Vishwamitra 11 , J. Ma 11 , S. Yang 11 , B. W. Hilder 11 , J. Tolbert 11 , J. D. Goldberg 12 , A. Chari 13

1 University Medical Center Utrecht, Utrecht, Netherlands; 2 City of Hope Comprehensive Cancer Center, Duarte, CA; 3 Sarah Cannon Research Institute and Tennessee Oncology, Nashville, TN, United States of America; 4 Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 5 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 6 Clínica Universidad de Navarra, Navarra; 7 Hospital Universitario Fundación Jiménez Díaz, Madrid; 8 University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain; 9 University of Alabama at Birmingham, Birmingham, AL, United States of America; 10 University of Liège, Liège, Belgium; 11 Janssen Research & Development, Spring House, PA; 12 Janssen Research & Development, Raritan, NJ; 13 Mount Sinai School of Medicine, New York, NY, United States of America

Background: G protein-coupled receptor family C group 5 member D (GPRC5D) is a promising therapeutic target for multiple myeloma (MM) immunotherapy. GPRC5D has limited expression in healthy human tissue but is highly expressed on malignant plasma cells. Talquetamab (JNJ-64407564) is a first-in-class, GPRC5D x CD3 bispecific IgG4 antibody that induces T cell–activated lysis of GPRC5D+ MM. MonumenTAL-1 is a phase 1 trial evaluating the safety and preliminary efficacy of talquetamab in patients with relapsed/refractory MM (RRMM) ( NCT03399799 ).

Aims: We report updated results from MonumenTAL-1 with additional patients and longer follow-up.

Methods: Patients with RRMM or who were intolerant to standard therapies were eligible; prior treatment with B-cell maturation antigen-directed therapies was allowed. The primary objectives were to identify the recommended phase 2 doses (RP2Ds) (part 1) and assess the safety and tolerability of talquetamab at the RP2Ds (part 2). Based on the collective safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) data, 2 RP2Ds of talquetamab were identified: 405 μg/kg SC QW (n=30) and 800 μg/kg SC Q2W (n=44). To mitigate severe cytokine release syndrome (CRS), step-up dosing was used. Administration of required premedications were limited to step-up doses and the first full dose of talquetamab. Adverse events (AEs) were graded by CTCAE v4.03, and CRS events were graded per Lee et al 2014 criteria. Responses were assessed by the investigators as per International Myeloma Working Group criteria.

Results: As of Jan 17, 2022, patients in the 405 μg/kg /800 μg/kg groups, respectively, received a median of 6/5 prior lines of therapy, 100%/98% were triple-class (TC) exposed, and 77%/75% were TC refractory, and median follow-up (range) was 11.7 (1.0–21.2)/4.2 (0.7–13.7) months. AEs were mostly grade 1 or 2, and cytopenias and CRS were the most commonly reported AEs. Cytopenias, including neutropenia (67%/36%; grade 3/4: 53%/23%), were mostly limited to step-up and cycle 1–2 doses, reversible, and generally resolved within 1 week. CRS (77%/80%; grade 3: 3%/0%) were mostly reported during step-up dosing. Infections were reported in 47%/34% (grade 3/4: 7%/9%) of patients. 83%/75% of patients reported skin-related and nail disorder AEs (most commonly skin exfoliation: 37%/39% [all grade 1 and 2]). Dysgeusia (63%/57%) was generally mild and managed with dose adjustments. Response-evaluable patients had overall response rates of 70% (21/30 patients)/64% (28/44 patients). Very good partial response or better rates were 57%/52%, and median time to first confirmed response (range) was 0.9 (0.2–3.8)/1.2 (0.3–6.8) months. Median duration of response will be reported. No deaths from drug-related AEs were reported. Both RP2Ds showed comparable PK and PD profiles.

Summary/Conclusion: Both RP2Ds of talquetamab appear tolerable with comparable safety and PK profiles. Talquetamab demonstrated highly promising efficacy as a novel, first-in-class therapy for heavily pretreated patients with RRMM.

S183: NOVEL COMBINATION IMMUNOTHERAPY FOR THE TREATMENT OF RELAPSED/REFRACTORY MULTIPLE MYELOMA: UPDATED PHASE 1B RESULTS FOR TALQUETAMAB (A GPRC5D X CD3 BISPECIFIC ANTIBODY) IN COMBINATION WITH DARATUMUMAB

N. W. van de Donk 1,* , N. Bahlis 2 , M.-V. Mateos 3 , K. Weisel 4 , B. Dholaria 5 , A. L. Garfall 6 , H. Goldschmidt 7 , T. G. Martin 8 , D. Morillo 9 , D. E. Reece 10 , D. Hurd 11 , P. Rodríguez-Otero 12 , M. Bhutani 13 , A. D’Souza 14 , A. Oriol 15 , E. Askari 16 , J. F. San-Miguel 17 , K. M. Kortüm 18 , D. Vishwamitra 19 , S. Xin Wang Lin 19 , T. J. Prior 19 , L. Vandenberk 20 , M.-A. D. Smit 21 , J. D. Goldberg 22 , R. Wäsch 23 , A. Chari 24

1 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 2 Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, AB, Canada; 3 University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain; 4 University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 5 Vanderbilt University Medical Center, Nashville, TN; 6 Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 7 University Hospital Heidelberg, Internal Medicine and National Center for Tumor Diseases, Heidelberg, Germany; 8 University of California, San Francisco, San Francisco, CA, United States of America; 9 Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain; 10 Princess Margaret Cancer Centre, Toronto, ON, Canada; 11 Section on Hematology and Oncology, Department of Internal Medicine, Comprehensive Cancer Center of Wake Forest University, Winston-Salem, NC, United States of America; 12 Clínica Universidad de Navarra, Navarra, Spain; 13 Levine Cancer Institute/Atrium Health, Charlotte, NC; 14 Medical College of Wisconsin, Milwaukee, WI, United States of America; 15 Institut Català d’Oncologia and Institut Josep Carreras and Hospital Germans Trias i Pujol, Badalona, Barcelona; 16 Department of Hematology, Fundacion Jimenez Diaz University Hospital, Madrid; 17 Clínica Universidad de Navarra, CCUN, CIMA, CIBERONC, IDISNA, Pamplona, Spain; 18 Medizinische Klinik II, Universitätsklinikum Würzburg, Würzburg, Germany; 19 Janssen Research & Development, Spring House, PA, United States of America; 20 Janssen Research & Development, Beerse, Belgium; 21 Janssen Research & Development, Los Angeles, CA; 22 Janssen Research & Development, Raritan, NJ, United States of America; 23 University of Freiburg, Freiburg, Germany; 24 Mount Sinai School of Medicine, New York, NY, United States of America

Background: Talquetamab (tal; JNJ-64407564) is a first-in-class, bispecific IgG4 antibody that binds both to G protein-coupled receptor family C group 5 member D (GPRC5D), a receptor highly expressed on malignant plasma cells but with limited expression in healthy tissue, and CD3 to mediate T-cell–activated lysis of GPRC5D+ multiple myeloma (MM) cells. Daratumumab (dara) is an anti-CD38 mAb with direct on-tumor and immunomodulatory actions. Initial clinical results from the phase 1b multicohort TRIMM-2 study identified the recommended phase 2 doses (RP2Ds) of tal as 400 μg/kg weekly or 800 μg/kg Q2W and support the combination of tal + dara for the treatment of RRMM, with manageable safety, no overlapping toxicities, and promising efficacy.

Aims: Here we report updated results for both RP2Ds of tal + dara in TRIMM-2 with additional patients (pts) and longer follow-up.

Methods: Eligible MM pts (aged ≥18 years) had received ≥3 prior lines of therapy (LOT; including a PI and IMiD) or were double refractory to a PI and an IMiD, and could not have received anti-CD38 therapy within 90 days. Pts received dara SC 1800 mg per approved schedule and tal (400 μg/kg weekly or 800 μg/kg Q2W) with step-up dosing. The primary objectives were to identify the RP2D(s) of tal for combination therapy and evaluate safety of the combination. AEs were graded per CTCAE v5.0; cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) were graded per ASTCT guidelines. Responses were assessed by IMWG criteria.

Results: At data cutoff (Jan 13, 2022, N=46), median follow-up was 4.0 months (range 0.4-16.3), median age was 65 years (range 47-81), and 48% were female. Pts received a median of 5 prior LOT (range 2-16); 83% were triple-class exposed, 61% penta-drug exposed, 37% anti-BCMA non–CAR-T exposed, and 4% anti-BCMA CAR-T exposed. 96% of pts had ≥1 AE (gr 3/4: 67%). The most frequently reported AEs (≥30% across tal + dara cohorts) were CRS (65%; all gr 1/2; median time to onset: 2 days; median duration: 2 days), dysgeusia (57%), thrombocytopenia (35%; gr 3/4: 20%), anemia (39%; gr 3/4: 20%), and dry mouth (44%). Infections occurred in 50% of pts (gr 3/4: 13%). Skin disorders were reported in 72% of pts (gr 3/4: 11%): skin exfoliation in 26% (all gr 1/2) and nail disorders in 11% (all gr 1/2). Two ICANS events were reported in the 800 Q2W group (both gr 1 and resolved within 1 day). 3 pts discontinued due to AEs. Response rates were consistent across both RP2Ds supporting their equivalence (Table). Median time to first response across dosing cohorts was 0.95 months (range 0.9-9.7); median duration of response was not reached. Upregulation of CD38+/CD8+ T cells and proinflammatory cytokines was observed with tal + dara, supporting potential synergy of the combination in pts with prior anti-CD38 exposure. Updated results will be presented.

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Summary/Conclusion: Longer follow-up with additional patients shows comparable efficacy and safety across both RP2Ds, with no new safety signals, strengthening the benefit-risk profile of tal + dara as a novel immunotherapy-based approach for heavily pretreated pts with RRMM.

S184: EVALUATING TECLISTAMAB IN PATIENTS WITH RELAPSED/ REFRACTORY MULTIPLE MYELOMA FOLLOWING EXPOSURE TO OTHER B-CELL MATURATION ANTIGEN (BCMA)-TARGETED AGENTS

C. Touzeau 1,* , A. Krishnan 2 , P. Moreau 1 , A. Perrot 3 , S. Z. Usmani 4 , S. Manier 5 , M. Cavo 6 , C. Martinez-Chamorro 7 , A. Nooka 8 , T. Martin 9 , L. Karlin 10 , X. Leleu 11 , N. Bahlis 12 , B. Besemer 13 , L. Pei 14 , R. Verona 15 , S. Girgis 15 , C. Uhlar 15 , R. Kobos 14 , A. Garfall 16

1 University Hospital Hôtel-Dieu, Nantes, France; 2 City of Hope Comprehensive Cancer Center, Duarte, United States of America; 3 Centre Hospitalier, Universitaire de Toulouse, Service d’Hematologie, Toulouse, France; 4 Levine Cancer Institute/Atrium Health, Charlotte, United States of America; 5 University of Lille, Lille, France; 6 IRCSS Aziena Ospedaliero-Universitaria di Bologna, Bologna University School of Medicine, Bologna, Italy; 7 University Hospital Quirónsalud, Pozuelo de Alarcón, Madrid, Spain; 8 Winship Cancer Institute, Emory University, Atlanta; 9 University of California, San Francisco, San Francisco, United States of America; 10 Service d’Hématologie Clinique, Centre Hospitalier Lyon Sud, Pierre-Bénite; 11 Centre Hospitalier Universitaire de Poitiers, Poitiers, France; 12 Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada; 13 University of Tuebingen, Tuebingen, Germany; 14 Janssen Research & Development, Raritan; 15 Janssen Research & Development, Spring House; 16 Abramson Cancer Center, Perelman School of Medicine, Philadelphia, United States of America

Background: Teclistamab (JNJ-64007957) is a T cell redirecting bispecific antibody that targets both B-cell maturation antigen (BCMA) and CD3 receptors to induce T cell mediated cytotoxicity of BCMA-expressing myeloma cells. MajesTEC-1 is an open-label, multicohort, phase 1/2 study evaluating teclistamab in patients (pts) with relapsed/ refractory multiple myeloma (RRMM) previously treated with ≥3 prior lines of therapy (LOT). An overall response rate (ORR) of 62.0% in pts with no prior anti-BCMA treatment (tx) was previously reported in a pooled analysis from phase 1 and phase 2 cohort A at a median follow-up of 7.8 mo.

Aims: We report efficacy and safety results of teclistamab from cohort C, which enrolled patients who had prior exposure to anti-BCMA treatment.

Methods: Eligible pts (age ≥18 y) had multiple myeloma (MM) per IMWG criteria and were previously treated with ≥3 prior LOT, including a PI, IMiD, anti-CD38 antibody, and anti-BCMA treatment (chimeric antigen receptor T cell therapy [CAR-T] or Ab drug conjugate [ADC]). Pts were enrolled using a Simon’s stage design to receive weekly subcutaneous teclistamab 1.5 mg/kg (step-up doses of 0.06 and 0.3 mg/kg). ORR per IMWG 2016 criteria was the primary endpoint. All AEs were graded per CTCAE v4.03; immune effector cell–associated neurotoxicity syndrome (ICANS) and cytokine release syndrome (CRS) were graded per ASTCT guidelines.

Results: In cohort C, 38 pts (median age 63.5 y [range 32–82]; 63% male) received teclistamab (median prior LOT 6 [range 3–14]) at the data cutoff date of Sep 7, 2021. Of the 38 patients, 25 (66%) were refractory to an anti-BCMA treatment, and 32 (84%) were refractory to last LOT. Among 25 efficacy-evaluable patients, 16 (64%) received prior ADC, 11 (44%) received prior CAR-T, and 2 pts received both. The ORR was 40% (95% CI, 21–61) at a median follow-up of 6.9 mo (range 0.7–8.7). Complete response or better were observed in 5 pts (20%). In ADC-exposed and CAR-T-exposed pts, the ORR was 38% (95% CI, 15–65) and 45% (95% CI, 17–77), respectively. Responses were rapid in most, with deepening of responses over time in 7 of 25 pts. While the median duration of response was not reached, median time to first response was 1.2 mo (range, 0.2–4.9) and to best response was 2.1 mo (range, 1.1–5.7). No new safety concerns were observed, and the safety profile was comparable with that of BMCA tx-naive pts. Infections were reported in 16 pts (42%; grade 3/4, 26%). Most common AEs (n=38) were CRS (63%; all grade 1/2; median time to CRS onset: 3 d [range, 2–6], duration of CRS: 2 d [range, 1–4]), thrombocytopenia (42%; grade 3/4, 29%), neutropenia (55%; grade 3/4, 50%), lymphopenia (40%; grade 3/4, 37%), and anemia (39%; grade 3/4 29%), Grade 3 ICANS was reported in 1 pt which resolved with supportive care and the pt remained on tx. Anti-teclistamab Abs were not detected in any pts. Baseline BCMA expression levels were comparable with those reported in BCMA tx-naive pts. Updated efficacy and safety results will be presented for 40 pts.

Summary/Conclusion: These preliminary results observed with serial targeting of BCMA with teclistamab following ADC or CAR-T tx suggest a promising ORR with early responses that deepen over time. Additionally, a well-tolerated safety profile was observed in patients treated with anti-BCMA tx.

S185: CARTITUDE-2 COHORT B: UPDATED CLINICAL DATA AND BIOLOGICAL CORRELATIVE ANALYSES OF CILTACABTAGENE AUTOLEUCEL IN PATIENTS WITH MULTIPLE MYELOMA AND EARLY RELAPSE AFTER INITIAL THERAPY

M. E. Agha 1,* , N. W. van de Donk 2 , A. D. Cohen 3 , Y. C. Cohen 4 , S. Anguille 5 , T. Kerre 6 , W. Roeloffzen 7 , J. M. Schecter 8 , K. C. De Braganca 8 , H. Varsos 8 , P. Mistry 9 , T. Roccia 9 , E. Zudaire 10 , C. Corsale 8 , M. Akram 11 , D. Geng 11 , T Nesheiwat 11 , L. Pacaud 11 , P. Sonneveld 12 , S. Zweegman 2

1 UPMC Hillman Cancer Center, Pittsburgh, PA, United States of America; 2 Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 3 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, United States of America; 4 Tel-Aviv Sourasky (Ichilov) Medical Center and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 5 Vaccine and Infectious Disease Institute, University of Antwerp, Edegem, Belgium, Center for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Edegem, Belgium; 6 University Hospital Ghent, Ghent, Belgium; 7 University Medical Center Groningen, Groningen, Netherlands; 8 Janssen R&D, Raritan, NJ, United States of America; 9 Janssen R&D, High Wycombe, United Kingdom; 10 Janssen R&D, Spring House, PA, United States of America; 11 Legend Biotech USA, Piscataway, NJ, United States of America; 12 Erasmus MC University and Medical Center, Rotterdam, Netherlands

Background: CARTITUDE-2 ( NCT04133636 ) is a phase 2, multicohort study evaluating the efficacy and safety of the chimeric antigen receptor (CAR) T-cell therapy ciltacabtagene autoleucel (cilta-cel) in patients (pts) with multiple myeloma (MM). Cohort B enrolled pts who had early relapse after initial therapy. These pts have functionally high-risk disease, as early relapse following autologous stem cell transplantation (ASCT) is associated with poor prognosis, representing an unmet medical need.

Aims: To present updated results from CARTITUDE-2 cohort B.

Methods: All pts provided informed consent. Pts had MM, received 1 prior line of therapy (proteasome inhibitor and immunomodulatory drug required), had disease progression per International Myeloma Working Group criteria (either ≤12 months after ASCT or ≤12 months after initiation of anti-myeloma therapy for pts not treated with ASCT), and were naive to CAR-T or other anti-B-cell maturation antigen therapies. After lymphodepletion, a single cilta-cel infusion was administered at a target dose of 0.75×10 6 CAR+ viable T cells/kg. Efficacy and safety were evaluated. The primary endpoint was minimal residual disease (MRD) negativity at 10 -5 . Patient management strategies were used to minimize risk of movement and neurocognitive adverse events (MNTs). Pharmacokinetic (PK) analyses, including C max and T max of CAR+ T-cell transgene levels in blood are being performed. Additional assessments include levels of cytokine release syndrome (CRS)-related cytokines (eg, IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with immune effector cell-associated neurotoxicity syndrome (ICANS), and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS.

Results: 19 pts (median age: 58.0 years [range: 44-67]; 74% male) received cilta-cel as of January 2022; 79% received prior ASCT. Median follow-up was 13.4 months (range: 5.2-21.7). Overall response rate was 100.0%, with 90% of pts achieving ≥complete response, and 95% achieving ≥very good partial response. Median time to first response was 0.95 months (range: 0.9-9.7) and median time to best response was 5.1 months (range: 0.9-11.8). Among 15 MRD-evaluable pts, 14 (93%) achieved MRD 10 -5 negativity. Median duration of response was not reached. The 12-month progression-free survival rate was 90%; 12-month event-free rate was 88.9%. CRS occurred in 16 (84.2%) pts (1 grade 4); median time to onset was 8 days (range: 5-11) and all events resolved. ICANS (grade 1) and MNT (grade 3, previously reported) occurred in 1 pt each. 1 pt died post cilta-cel (progressive disease, day 158). Preliminary PK data showed peak expansion of CAR-T cells on day 13.1 (range: 8.96-209.9); median persistence was 76.9 days (range: 40.99-221.8).

Summary/Conclusion: This functionally high-risk pt population with early relapse after initial therapy experienced deep and durable responses with manageable safety following a single cilta-cel infusion. Cilta-cel led to responses in pts with ineffective or insufficient response to ASCT. Follow-up is ongoing and responses continue to deepen. We will present updated and detailed PK, cytokine, and CAR-T subset analyses as well as clinical correlation to provide insight into biological correlates of efficacy and safety in these pts.

S186: UPDATED RESULTS OF A MULTICENTER FIRST-IN-HUMAN STUDY OF BCMA/CD19 DUAL-TARGETING FAST CAR-T GC012F FOR PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM)

J. Du 1,* , H. Jiang 1 , B. Dong 2 , L. Gao 3 , L. Liu 4 , J. Ge 5 , A. He 6 , L. Li 1 , J. Lu 1 , X. Chen 2 , M. Sersch 7 , H. Zhang 7 , L. Shen 7 , J. Liu 7 , W. Fu 1

1 Changzheng Hospital, Shanghai; 2 Xijing Hospital, Xian; 3 Chongqing Xinqiao hospital, Chongqing; 4 Tangdu Hospital, Xian; 5 The first affiliated hospital of Anhui medical University, Anhui; 6 The second affiliated hospital of Xian Jiaotong University, Xian; 7 Gracellbiotechnologies Ltd, Shanghai, China

Background: GC012F is a B cell maturation antigen (BCMA) and CD19 dual-targeting CAR-T developed on the novel FasT CAR-T platform enabling 22-36h manufacturing designed to improve depth of response and overall efficacy. Data was presented at ASCO and EHA 2021 for initial 19 pts. Here we present updated data for study ( NCT04236011 ; NCT04182581 ) with longer follow up and 9 additional pts treated (total n=28) in 3 different dose levels.

Aims: The study aims to assess safety and preliminary efficacy for FAST-CAR GC012F in RRMM patients.

Methods: From October 2019 to November 2021, 28 heavily pretreated RRMM pts (age 27-76) with a median of 5 prior lines (range 2-9) were treated on this single-arm, open label, multicenter Investigator Initiated Trial receiving a single infusion of GC012F. 89.3% (25/28) were high risk (HR- mSMART), 8 pts had EM disease, 3 had never achieved a CR including after transplant, 1 pts presented with plasma cell leukemia, 24/28 pts were refractory to last therapy, 3 pts primary refractory. 9/28 pts had received prior anti-CD38, 27/28 pts prior IMiDs. 26/28 pts were refractory to PI, 26/28 pts to IMiDs. After lymphodepletion over 2-3 days (30 mg/m 2 /d, 300mg/ m 2 /d Flu/Cy) GC012F was administered as single infusion at 3 dose levels: 1x10 5 /kg (DL1) n=2, 2x10 5 /kg (DL2) n=10 and 3x10 5 /kg (DL3) n=16.

Results: As of Jan 26 th 2022 cut-off, 28 pts - median follow-up (f/u) 6.3 mths (1.8-29.9) - had been evaluated for response. Overall response rate (ORR) in DL1 was 100% (2/2)- DL 2 -80% (8/10) DL 3 -93.8% (15/16) with 27 pts MRD negative by flow cytometry (sensitivity 10 -4 -10 -6 ). 100% of MRD assessable pts (27/27) achieved MRD negativity. One patient out of 28 could not get assessed. At d28, 21/24 assessable patients were MRD negative (81.5%), 4/28 pts could not get d28 MRD assessment f/u due to COVID-19 restrictions however were assessed at a later timepoint. To date best response is MRD- sCR in 21/28 patients (75.0%) across all dose levels. Some pts after short f/u show responses that are still deepening. Cytokine Release Syndrome (CRS) was mostly low grade: gr 0 n=3 (10.7%), gr 1-2 n=23 (82.1%), gr 3 n=2 (7.1%) – no gr 4/5 CRS and no ICANs were observed (Graded by ASBMT criteria). Median duration of CRS was 3 d (1-8 d). PK results showed no difference amongst dose levels DL1 to DL3. Overall, CAR-T median Tmax was 10 d (range 8-14 d), median peak copy number (Cmax) was 97009 (16,011-374,346) copies /ug DNA with long duration of persistence of up to d793 (data cut-off). CAR-T geometric mean AUC 0-28 for DL1, DL2 and DL3 were 468863, 631540 and 581620 copies/ug DNA×day, respectively. Pts continue to be monitored for safety and efficacy including DOR.

Summary/Conclusion: BCMA-CD19 dual FasT CAR-T GC012F continues to provide deep and durable responses with a favorable safety profile in additional RRMM pts across all dose levels demonstrating a very high MRD negativity rate including in pts refractory to anti-CD38, PI and IMIDs. Based on these promising results GC012F is being studied in earlier lines of therapy as well as additional indications.

S187: UPDATED PHASE 1/2 DATA OF SAFETY AND EFFICACY OF CT103A, FULLY HUMAN BCMA-DIRECTED CAR T CELLS, IN RELAPSED/REFRACTORY MULTIPLE MYELOMA

C. Li 1,* , D. Wang 1 , Y. Song 2 , J. Li 3 , H Huang 4 , B. Chen 5 , J. Liu 6 , K. Hu 7 , H. Ren 8 , X. Zhang 9 , Z. Li 10 , D. Zou 11 , Q. Yin 2 , L. Chen 12 , Y. Hu 4 , Y. Xu 5 , Q. Cheng 6 , Y. Guo 7 , Y. Dong 13 , L. Gao 9 , S. Chen 14 , A. Xu 15 , S. Cai 15 , M. Wu 15 , J. Guo 15 , Z. Yao 15 , W. Wang 15 , J. Wang 1 , L. Chen 1 , J. Zhou 1 , L. Qiu 11

1 Department of Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan; 2 Department of Hematology, Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Zhengzhou; 3 Department of Hematology, Pukou CLL Center, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing; 4 Department of Hematology, Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou; 5 Department of Hematology, Nanjing University Medical School, the Affiliated Nanjing Drum Tower Hospital, Nanjing; 6 Department of Hematology, The Third Xiangya Hospital of Central South University, Changsha; 7 Department of Hematology, Beijing Boren Hospital; 8 Department of Hematology, Peking University First Hospital, Beijing; 9 Department of Hematology, Medical Center of Hematology, Xinqiao Hospital, State Key Laboratory of Trauma, Burn and Combined Injury, Army Medical University, Chongqing; 10 Department of Hematology, Affiliated Hospital of Xuzhou Medical University, Xuzhou; 11 Department of Hematology, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Blood Diseases Hospital & Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin; 12 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Nanjing; 13 Department of Hematology, Peking University First Hospital, Beijing; 14 Department of Hematology; 15 Nanjing IASO Biotherapeutics Ltd, Nanjing, China

Background: CT103A, a fully human BCMA-directed CAR-T therapy, showed excellent safety and promising efficacy in the ongoing phase 1/2 FUMANBA-1 study (ChiCTR1800018137, NCT05066646 ) in patients with relapsed/refractory multiple myeloma (Wang, ASH, 2021).

Aims: We report updated data from the FUMANBA-1 study with more subjects and a longer duration of follow-up.

Methods: Enrolled patients had received three or more prior lines of anti-myeloma therapy, including a proteasome inhibitor and an immunomodulatory agent, and were required to be refractory to the last line of therapy. CT103A was administered at RP2D of 1.0×10 6 CAR + T cells/kg. Lymphodepletion with cyclophosphamide (500 mg/m 2 ) and fludarabine (30 mg/m 2 ) was used for 3 consecutive days. Study objectives included safety and efficacy profile. Pharmacokinetics, pharmacodynamics, and immunogenicity were also explored. MRD in bone marrow aspirate was detected by Euroflow TM standardized flow cytometry with the sensitivity of 10 -5 . Adverse events (AEs) were graded using CTCAEv5.0, except that CRS and ICANS were graded by ASTCT criteria.

Results: As of Jan 21, 2022, 79 patients received CT103A with the median follow-up of 9.6 months (range 1.2, 40.0). 28 (35.4%) patients had high-risk cytogenetic abnormality defined as del(17p), t(4;14), or t(14;16), and 11 (13.9%) had extramedullary plasmacytomas. Patients received a median of 5 (range 3, 23) prior lines of therapy. Notably, 13 (16.5%) had received prior non-human BCMA-targeted CAR-T cell therapy.

ORR was 94.9% (75/79), with 55 (69.6%) patients achieving CR/sCR, and 71 (89.9%) achieving ≥VGPR. Median TTR was 16 days (range 11, 123), and the median time to CR/sCR was 95 days (range 14, 557). For 13 patients who had prior CAR-T therapy, ORR was 98.5%, with 6 (46.2%) achieving CR/sCR. For 11 patients with extramedullary disease at baseline, ORR was 100%, with 9 (81.8%) achieving CR/sCR. All patients with CR/sCR were MRD-negative. The median duration of MRD negativity was not reached. The probability of MRD negativity at month 12 was 88.7% (95% CI 65.9%, 96.6%).

CRS occurred in 75 (94.9%) patients, of which only 2 (2.5%) had grade 3 CRS. The median time to onset of CRS was six days (range 1, 12) with a median duration of 5 days (range 2, 30). ICANS occurred in only 2 (2.5%) patients, of which one was grade 1, and the other was grade 2. All CRS were resolved, with the use of tocilizumab in 19 (25.3%) patients and steroids in 47 (62.7%). The most common ≥ grade 3 treatment-related AEs were hematological toxicities, including neutropenia (78.5%), leukopenia (74.7%), lymphopenia (55.7%), thrombocytopenia (54.4%), and anemia (46.8%).

CT103A reached the peak at a median of 12 days (range 0, 26) after infusion and were still detectable in 40 of 61 evaluable patients (65.6%) at month 6 and 13 of 21 (61.9%) at month 12. Soluble BCMA decreased to the lower limit of quantification (LLOQ) at a median of 2 months and can maintain bellow LLOQ over 24 months. 11 (13.9%) patients were positive for anti-drug antibodies, of whom 1/76 (1.3%) developed anti-drug antibodies at month 3, 5/43 (11.6%) at month 6 and 5/16 (31.3%) at month 12. Among these, neutralizing antibodies were only identified in one patient (at month 12).

Summary/Conclusion: Updated data from the Phase 1/2 FUMANBA-1 study continue to show encouraging efficacy of CT103A with a favorable safety profile in relapsed/refractory multiple myeloma. CT103A led to a deepening and durable response with robust expansion and prolonged persistence. Patients with prior BCMA CAR-T therapy could still benefit from CT103A.

S188: TECLISTAMAB IN COMBINATION WITH DARATUMUMAB, A NOVEL, IMMUNOTHERAPY-BASED APPROACH FOR THE TREATMENT OF RELAPSED/REFRACTORY MULTIPLE MYELOMA: UPDATED PHASE 1B RESULTS

P. Rodriguez Otero 1,* , A. D’Souza 2 , D. Reece 3 , N. W. van de Donk 4 , A. Chari 5 , A. Krishnan 6 , T. Martin 7 , M. V. Mateos 8 , D. Morillo 9 , D. Hurd 10 , L. Rosinol 11 , A. S. Balari 12 , R. Wäsch 13 , D. Vishwamitra 14 , S. X. Wang Lin 14 , T. Prior 14 , L. Vandenberk 15 , M.-A. D. Smit 16 , A. Oriol 17 , B. Dholaria 18

1 University of Navarra, Navarra, Spain; 2 Medical College of Wisconsin, Milwaukee, WI, United States of America; 3 Princess Margaret Cancer Centre, Toronto, ON, Canada; 4 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 5 Mount Sinai School of Medicine, New York, NY; 6 City of Hope Comprehensive Cancer Center, Duarte, CA; 7 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA, United States of America; 8 University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca; 9 Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain; 10 Comprehensive Cancer Center of Wake Forest Baptist Health, Winston-Salem, NC, United States of America; 11 Hospital Clínic, Institut d’investigacions Biomèdiques August Pi i Sunyer, Barcelona; 12 Institut Català d’Oncologia – Hospitalet, IDIBELL, University of Barcelona, Barcelona, Spain; 13 Freiburg University Medical Center, Freiburg, Germany; 14 Janssen Research and Development, Spring House, PA, United States of America; 15 Janssen Research & Development, Antwerp, Belgium; 16 Janssen Research & Development, Los Angeles, CA, United States of America; 17 Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 18 Vanderbilt University Medical Center, Nashville, TN, United States of America

Background: Teclistamab (JNJ-64007957) is a B-cell maturation antigen (BCMA) × CD3 T-cell redirecting bispecific antibody currently under investigation in patients with relapsed/refractory multiple myeloma (RRMM). Daratumumab is a CD38-targeting monoclonal antibody with direct on-tumor and immunomodulatory mechanisms of action. The preliminary results from the phase 1b multicohort TRIMM-2 study showed tolerable safety with no overlapping toxicities, and encouraging efficacy, supporting the combination of teclistamab with daratumumab for the treatment of RRMM.

Aims: We report updated results from the TRIMM-2 study with additional patients and longer follow-up.

Methods: Eligible patients were ≥18 years of age with a MM diagnosis and previously treated with ≥3 prior lines of therapy (including a proteosome inhibitor [PI] and immunomodulatory drug [IMiD]) or were double-refractory to a PI and IMiD. Patients who had received anti-CD38 therapy ≤90 days prior were excluded. Written informed consent was obtained from all eligible patients. Patients received subcutaneous (SC) daratumumab 1800 mg per approved schedule and teclistamab SC 1.5–3 mg/kg once weekly or every 2 weeks. Primary objectives of the study were to identify the recommended phase 2 dose for the teclistamab and daratumumab combination and to assess safety of the combination. Responses were assessed by IMWG criteria. Adverse events (AEs) were graded per CTCAE v5.0, except for cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), which were graded per ASTCT guidelines.

Results: At the Jan 13, 2022 data cutoff, the median follow-up was 7.2 months (range 0.1–16.6). Among the safety population (N=46), 52% were females, and the median age was 67 years (range 50–79). Patients received a median of 6 prior lines of therapy (range 2–17); 74% of patients were triple-class exposed; 63% were penta-drug exposed, and 15% were anti-BCMA exposed. Overall, 91% of patients had ≥1 AE of any grade; 78% had grade 3/4 AEs. The most common AE was CRS (61%; all grade 1/2); median time to onset was 2 days and median duration was 2 days. Other AEs included neutropenia (54%; grade 3/4 50%), anemia (46%; grade 3/4 28%), thrombocytopenia (33%; grade 3/4 28%), and diarrhea (33%; grade 3/4 2%). Infections occurred in 29 patients (63%; grade 3/4 28%). One patient had grade 1 ICANS that was fully resolved. Among 37 response-evaluable patients, the overall response rate was 78% (29/37); 27 patients (73%) had very good partial response (VGPR) or better (Table). While the median duration of response was not reached, median time to first response across dosing cohorts was 1.0 month (range 0.9–2.8). Upregulation of CD38 + /CD8 + T cells and proinflammatory cytokines was observed after teclistamab dosing in combination with daratumumab, supporting potential synergy of the combination in patients with prior anti-CD38 exposure. Updated results will be presented.

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Summary/Conclusion: Teclistamab in combination with daratumumab is a novel immunotherapy approach that may yield improved clinical efficacy in heavily pretreated patients with RRMM.

S189: EARLY, DEEP, AND DURABLE RESPONSES, AND LOW RATES OF CRS WITH REGN5458, A BCMAXCD3 BISPECIFIC ANTIBODY, IN A PHASE 1/2 FIRST-IN-HUMAN STUDY IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

J. A. Zonder 1 , J. Richter 2,* , N. Bumma 3 , J. Brayer 4 , J. E. Hoffman 5 , W. I. Bensinger 6 , K. L. Wu 7 , L. Xu 8 , D. Chokshi 8 , A. Boyapati 8 , D. Cronier 8 , Y. Houvras 8 , K. Rodriguez Lorenc 8 , G. S. Kroog 8 , M. V. Dhodapkar 9 , S. Lentzsch 10 , D. Cooper 11 , S. Jagannath 2

1 Karmanos Cancer Institute, Detroit; 2 Icahn School of Medicine at Mount Sinai, New York; 3 The Ohio State University Comprehensive Cancer Center, Columbus; 4 H. Lee Moffitt Cancer Center, Tampa; 5 University of Miami Health System, Miami; 6 Swedish Center for Blood Disorders and Stem Cell Transplants, Seattle, United States of America; 7 Ziekenhuis Netwerk Antwerpen Stuivenberg, Antwerp, Belgium; 8 Regeneron Pharmaceuticals, Inc, Tarrytown; 9 Emory University School of Medicine, Atlanta; 10 Columbia University Medical Center, New York; 11 Rutgers Cancer Institute of New Jersey, New Brunswick, United States of America

Background: Despite recent advances in treatment options, multiple myeloma (MM) remains incurable. REGN5458 is a BCMAxCD3 bispecific antibody currently under investigation in relapsed/refractory MM (RRMM) in an ongoing Phase 1/2 trial ( NCT03761108 ). Preliminary data suggest that REGN5458 has a manageable safety profile with early, deep and durable responses in heavily pretreated patients (pts).

Aims: Here we describe updated safety, overall response and response durability in pts treated with REGN5458 in the Phase 1 portion of this ongoing trial.

Methods: The Phase 1 primary objectives are to assess safety, tolerability and occurrence of dose-limiting toxicities of REGN5458 and to determine a recommended Phase 2 dose regimen (RP2DR). Key secondary objectives include: assessment of objective response rate as determined by the investigator, duration of response (DOR) and minimal residual disease status; pharmacokinetic evaluation; and characterization of immunogenicity. Pts with progressive RRMM, who are double- or triple-refractory, or intolerant to prior lines of systemic therapy including a proteasome inhibitor, immunomodulatory agent, and anti-CD38 antibody are treated with REGN5458 monotherapy following a modified 3 + 3 dose-escalation design (4 + 3). Treatment consists of 16 weekly infusions of REGN5458 followed by q2w dosing until disease progression. Response assessments are measured using modified International Myeloma Working Group criteria.

Results: At data cut-off (September 30, 2021), 73 pts were treated with REGN5458 in the dose escalation cohort with full doses ranging from 3–800 mg. Median age at enrollment was 64 years (range, 41–81) and 20.5% pts were ≥75 years. As per Revised International Staging System, stage was 1, 2 or 3 in 15.0%, 57.5% and 23.3% of pts respectively. Pts had a median of 5 prior lines of systemic therapy (range, 2–17) with 38.4% of pts being penta-refractory (Table). Median duration of follow-up was 3.0 months (range, 0.7–22.1).

Treatment-emergent adverse events (TEAE) were reported in 73 pts (100%), Grade (Gr) 3 in 31 pts (42.5%), Gr 4 in 24 pts (32.9%). The most common Gr 3/4 TEAEs were hematologic (39.0%). The most frequent TEAEs were fatigue in 33 pts (45.2%), Gr 1/2 in 31 pts (42.5%), Gr 3 in 2 pts (2.7%); cytokine release syndrome (CRS) in 28 pts (38.4%), Gr 1 in 25 pts (34.2%), Gr 2 in 3 pts (4.1%). No pt had Gr ≥3 CRS or discontinued treatment due to CRS. There were no Gr ≥3 neurotoxicity events. Nausea was reported in 24 pts (32.9%), Gr 1 in 17 pts (23.3%), Gr 2 in 7 pts (9.6%).

Responses were observed at all dose levels. Across all dose levels, 86.5% (n=32/37) of all responders achieved at least a very good partial response and 43.2% (n=16) of responders had a complete response (CR) or stringent CR. Amongst pts treated at the 200–800 mg dose levels, the response rate was 75.0% (n=18/24). The Kaplan–Meier estimated probability of responders being in response for 8 months or more was 90.2% (95% confidence interval: 72.6–96.7), and median DOR was not reached.

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Summary/Conclusion: REGN5458 shows a manageable safety and tolerability profile, with Gr 2 CRS in only 4.1% of pts and no Gr ≥3 CRS or neurotoxicity events. No new safety signals were observed during the additional follow-up period. Early, deep and durable responses were seen in triple- to penta-refractory pts with RRMM, with a 75.0% response rate at the combined 200–800 mg dose levels. The Phase 2 portion of the study is currently recruiting.

S190: ASXL1 MUTATIONS ACCELERATE BONE MARROW FIBROSIS VIA EGR1-TNFA AXIS MEDIATED INFLAMMATION AND FIBROCYTE GENERATION IN MYELOPROLIFERATIVE NEOPLASMS

Z. Shi 1 2,* , J. Liu 1 , Y. Zhao 3 , L. Yang 1 , Y. Cai 1 , P. Zhang 4 , Z. Xu 1 , T. Qin 1 , S. Qu 1 , L. Pan 1 , J. Wu 1 , X. Yan 1 , Z. Li 3 , W. Zhang 1 , Y. Yan 1 , H. Huang 1 , G. Huang 5 , B. Li 6 , X. Wu 7 8 , Z. Xiao 1

1 MDS/MPN center, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin; 2 Department of Hematology, the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing; 3 Department of Cell Biology, Tianjin Medical University; 4 pathology center, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin, China; 5 Divisions of Pathology and Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States of America; 6 MDS/MPN center, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College; 7 Department of Cell Biology, Tianjin Medical University; 8 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin, China

Background: Apart from the central role of activated JAK/STAT signaling pathway, ASXL1 mutations are the most recurrent additional mutations in myeloproliferative neoplasms (MPNs) and occurred much more common in myelofibrosis (MF) than essential thrombocythemia (ET) and polycythemia vera (PV) patients. However, the mechanism of the association with ASXL1 mutations and bone marrow (BM) fibrosis remains unknown.

Aims: To investigate the role of ASXL1 mutations in MF acceleration and explore the underlying mechanism.

Methods: We studied the clinical data of 302 consecutive MF patients in our hospital. For mouse model study, Vav1 -Cre mice, Asxl1 flox/flox and Jak2 V617F/+ knockin alleles were used to achieve hematopoietic cell-specific Jak2 V617F/+ /Asxl1 flox/flox ( Asxl1-/-Jak2 VF ), Jak2 V617F/+ ( Jak2 VF ), and Asxl1 flox/flox ( Asxl1-/ -) mice. Integrated analysis of RNAseq, transposase-accessible chromatin using sequencing (ATAC-Seq) and chromatin immunoprecipitation (ChIP)-seq were performed to explore the underlying mechanism.

Results: 98 of 302 (32.5%) patients harbored ASXL1 mutations. ASXL1 mutations are correlated with higher monocyte counts, increasing CD34 + cells in peripheral blood (PB), larger spleen sizes, and higher MF grades. Consistent with clinical findings, Asxl1-/-Jak2 VF mice showed lower platelet counts, higher monocyte counts and c-kit + cells in PB compared with Jak2 VF mice. Hematopoietic stem and progenitor cells (HSPCs) compartment analysis revealed increased granulocyte/macrophage progenitors (GMPs), and megakaryocyte /erythroid progenitors (MEPs) in spleens of Asxl1-/-Jak2 VF , not in BM compared with Jak2 V617F mice ( Figure 1A ), in line with increased spleen weights of Asxl1-/-Jak2 VF mice ( Figure 1B ). Histology analysis revealed reticulin infiltration in BM of Asxl1-/-Jak2 VF mice, not in other age-matched genotypes.

Both ASXL1 MT MF patients and Asxl1-/-Jak2 VF mice showed increased monocytes/macrophages in BM ( Figure 1C-D ), accounting for more severe inflammation in ASXL1 MT MF patients and mouse models. In vitro differentiation assay revealed monocyte/ macrophage differentiation bias of Asxl1-/-Jak2 VF HSPCs (Figure 1E ). In fibrosis driving cells analysis, ASXL1 MT MF patients showed comparable mesenchymal stromal cells (MSCs) derived myofibroblasts while increased monocyte derived fibrocytes in BM compared with ASXL1 WT MF patients ( Figure 1F ). In vitro fibrocyte differentiation assay ( Figure 1G ) and flow cytometry analysis confirmed increased fibrocytes in BM of Asxl1-/-Jak2 VF mice compared with other genotypes.

Mechanismly, Asxl1 deletion in Jak2 VF HSPCs leads to enhancer activation of polycomb group (PcG) target genes, such as Egr1 (Figure 1H ). The upregulation of Egr1 , which was validated in mouse models and patients (Figure 1I-J), in turn accounts for increased HSPCs commitment to monocyte/macrophage lineage (Figure 1K ). Moreover, Egr1 induces the activation of Tnfa (Figure 1L ) and thereby further drives the differentiation of monocytes to fibrocytes (Figure 1M ). Accordingly, combined TNFR antagonist (R-7050) with ruxolitinib significantly reduces fibrocytes production in vitro in Asxl1-/-Jak2 VF mice and ASXL1 MT MF patients (Figure 1N-O ).

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Summary/Conclusion: Our study illustrated the crucial role of ASXL1 mutation in MPN phenotypes and BM fibrosis. ASXL1 mutations activate the EGR1-TNFA axis in MPNs, leading to monocyte/macrophage mediated inflammation and fibrocyte induced BM fibrosis. Ruxolitinib along with TNFR antagonist may ameliorate fibrocytes, providing an attractive theoretical approach for antifibrosis treatment.

S191: CALRETICULIN-MUTATED HEMATOPOIETIC CELLS ARE VULNERABLE TO THE COMBINED INHIBITION OF THE PROTEASOME AND THE IRE1A-XBP1 AXIS OF THE UNFOLDED PROTEIN RESPONSE

J. S. Jutzi 1,* , A. E. Marneth 1 , M. J. Jimenez-Santos 2 , A. Guerra-Moreno 1 , S. A. Myers 3 4 , S. A. Carr 3 , P. van Galen 1 , F. Al-Shahrour 5 , A. S. Nam 6 , A. Mullally 1

1 Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Boston, United States of America; 2 Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 3 The Broad Institute of MIT and Harvard, Cambridge; 4 Center for Autoimmunity and Inflammation, La Jolla Institute for Immunology, La Jolla; 5 Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid; 6 Weill Cornell Medicine, New York, United States of America

Background: Mutations in the endoplasmic reticulum (ER) chaperone calreticulin ( CALR ) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). These mutant CALR proteins have impaired chaperone function. Concordant with this, transcriptional upregulation of the unfolded protein response (UPR) has been reported in patients with CALR-mutated MPN. However, it is not understood how CALR-mutated cells counter-balance ER stress resulting from impaired chaperone function. Despite the frequency of CALR mutations in MPN, there are currently no treatment strategies to preferentially target CALR-mutant cells over healthy cells.

Aims: To determine the mechanisms by which mutant CALR cells alleviate ER stress and to exploit these mechanisms selectively by pharmacological intervention in vivo .

Methods: Long-term HSCs (LT-HSCs) from Calr Δ 52 knockin mice were isolated for RNA-Seq. Calr Δ 52 bone marrow (BM) was differentiated ex vivo , subsequently enriched for megakaryocytes, and subjected to quantitative proteomic analysis. Pathway analyses of CALR -mutant megakaryocyte progenitors (MkPs) were performed on Genotyping of Transcriptome (GoT) data from patients with Essential Thrombocytosis (ET). Calr Δ 52 VAVCre mice and chimeric transplanted Calr Δ 52 mice were treated with a proteasome inhibitor, an IRE1a inhibitor, or both.

Results: To identify dysregulated pathways in Calr Δ 52/ Δ 52 LT-HSCs, we performed RNA-seq and found that Xbp1s downstream of IRE1a and the proteasome pathway was significantly upregulated in Calr Δ52/Δ52 animals. We confirmed elevated XBP1s protein level in heterozygous Calr Δ52 knockin mice by flow cytometry. Since elevated and abnormal megakaryopoiesis are hallmarks of CALR-mutant MPN, we performed quantitative proteomics on megakaryocyte-enriched cells from Calr Δ52 mice. We confirmed upregulation of both proteasome and ER chaperone proteins in Calr -mutant cells. To verify these findings in human MPN, we interrogated a published GoT data set and found that in addition to XBP1s, the proteasome pathway was also differentially upregulated in CALR -mutated patient MkPs compared to the wildtype MkPs (p<2e-16).

To investigate the functional consequences of IRE1a or proteasome inhibition, we treated CALR-mutant hematopoietic cell lines with KIRA6 (IRE1a inhibitor) and/or bortezomib (proteasome inhibitor) and found CALR -mutant cells to be differentially sensitive as compared to isogenic controls. We subsequently found that treatment with bortezomib leads to the accumulation of misfolded and ubiquitinylated proteins in mutant Calr BM, resulting in pro-apoptotic priming. In vivo treatment of Calr Δ52 /+ mice with bortezomib resulted in a significant reduction in platelets. Finally, we combined IRE1a and proteasome inhibition in a chimeric BM transplantation in vivo model using CD45.2 Calr Δ52 /+ MxCre GFP and CD45.1 wild-type competitor cells. Following combination therapy, we found a significant reduction in Calr -mutant donor chimerism, specifically in LT-HSCs and platelets, accompanied by reductions in LT-HSC frequency and platelet count.

Summary/Conclusion: In summary, we have found that disrupted proteasis in CALR-mutated MPN cells results in a dependency on the IRE1-XPB1 axis of the UPR and proteasome activity. Using pre-clinical MPN models, we found that combined inhibition of both pathways in vivo normalizes important features of MPN and preferentially targets Calr Δ 52 over wild-type cells. These findings highlight combined inhibition of IRE1 and the proteasome as a promising therapeutic strategy in CALR -mutant MPN.

S192: SINGLE-CELL RNA PROFILING OF MYELOFIBROSIS PATIENTS REVEALS PELABRESIB-INDUCED DECREASE OF MEGAKARYOCYTIC PROGENITORS AND NORMALIZATION OF CD4+ T CELLS IN PERIPHERAL BLOOD

O. Zavidij 1,* , N. J. Haradhvala 2 , R. Meyer 1 , J. Cui 1 , S. Verstovsek 3 , S. Oh 4 , A. Mead 5 , P. Taverna 1

1 Constellation Pharmaceuticals a MorphoSys Company; 2 Harvard Graduate Program in Biophysics, Harvard University, Boston, MA; 3 Leukemia Department, University of Texas MD Anderson Cancer Center, Houston, TX; 4 Hematology Division, Washington University, St Louis, MO; 5 NIHR Biomedical Research Centre, University of Oxford, Oxford, United States of America

Background: Abnormal differentiation of the megakaryocytic (MK) lineage in conjunction with overproduction of proinflammatory cytokines (Ck), resulting in bone marrow fibrosis, anemia, extramedullary hematopoiesis and often hepatosplenomegaly, are the main biological and clinical characteristics of myelofibrosis (MF). Bromodomain and extraterminal domain (BET) proteins play a key role in the regulation of neoplastic myeloproliferation and proinflammatory Ck production. Pelabresib (CPI-0610) is a potent, selective BET inhibitor under investigation in MF patients (pts) as monotherapy and in combination with the Janus kinase inhibitor (JAKi) ruxolitinib (RUX) in the ongoing MANIFEST Phase 2 study ( NCT02158858 ). Arm 1: pelabresib as monotherapy in RUX-intolerant, ineligible or refractory MF pts; Arm 2: pelabresib in combination with RUX as an ‘add-on’ in MF pts with suboptimal response to RUX; Arm 3: pelabresib combination therapy with RUX in JAKi-naïve MF pts. Pelabresib as monotherapy and in combination with RUX improved spleen volume reduction, symptoms and hemoglobin levels (Talpaz M, ASH 2020). The effects of pelabresib on hematopoietic stem/progenitor cells (HSPCs) and immune cell populations in MF pts are presented here.

Aims: We aimed to characterize the cellular composition and transcriptional alterations occurring in peripheral blood (PB) obtained from MF pts enrolled in the MANIFEST trial.

Methods: We performed single-cell RNA sequencing on 234,904 CD34 + HSPCs and 135,970 CD34 - mature PB cells (Figure). Cells were obtained from a random pool of 20 pts (Arm 1 n=5, Arm 2 n=8, Arm 3 n=7), which included a baseline sample (BL) and samples collected during treatment (range: C3 through C12) for each pt. Mobilized PB cells from healthy donors (HD) were used as a control (n=11).

Results: Analysis of CD34 + HSPCs at BL demonstrated a statistically significant increase of MK, neutrophilic and erythroid progenitors in MF pts compared with HDs and decreased numbers of myeloid and B cell lineage progenitors. Pelabresib as monotherapy and in combination with RUX led to a significant reduction of MK-, neutrophilic and erythroid progenitors as compared with BL in all pts analyzed.

Analysis of CD34 - cells from MF pts identified a significantly lower proportion of CD4 + T cells and increased numbers of erythroid cells at BL compared with HD. Individual pts also exhibited reduction in natural killer cells and CD16+ monocytes as well as elevated MK lineage cells. Pelabresib as monotherapy and in combination with RUX increased the proportion of CD4 + T cells, and more importantly, reduced MK lineage cells compared with BL in both treatment-naïve and RUX relapsed/refractory (r/r) pts.

In MF pts at BL, a larger spleen volume was observed in pts with lower numbers of CD4 + T cells and increased numbers of MK- and myeloid CSF3R + cells.

Updated results of the cell composition analysis and transcriptional reprogramming during pelabresib treatment, as well as correlation with effects on cytokine plasma levels and clinical efficacy data, will be presented.

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Summary/Conclusion: The single-cell profiling of a subset of MF pts enrolled in the MANIFEST study suggests that pelabresib alone and in combination with RUX induces an improvement of the myeloid-lymphoid imbalance in both JAKi-naïve and r/r MF pts. The observed effects of pelabresib on HSPCs from MF pts confirm the ex vivo activity on erythroid and MK differentiation of stem cells (Keller P, EHA 2021; Verstovsek S, ASH 2021) and implies a potential disease-modifying effect warranting further investigation.

S193: GENOMIC AND FUNCTIONAL IMPACT OF TP53 INACTIVATION IN JAK2V617F MYELOPROLIFERATIVE NEOPLASMS: A TRANSGENIC MOUSE MODEL APPROACH.

P. GOU 1 , D. LIU 1,* , E. Lauret 2 , N. Maslah 3 , V. Montcuquet 4 , V. meignin 5 , J.-J. Kiladjian 6 , B. Cassinat 3 , S. Giraudier 3

1 U1131, 25Université de Paris, Institut Cochin, INSERM U1016, CNRS UMR 8104, INSERM; 3 U1131, INSERM Univrsité de Paris and APHP; 4 Animal Facility Unit, Université de Paris; 5 Histo-pathological department, Hôpital Saint Louis; 6 Centre Investigations Cliniques, Hôpital Saint-Louis, Paris, France, INSERM Univrsité de Paris and APHP, Paris, France

Background: MPN are characterized by increased proliferation of myeloid cells and a risk of transformation to AML. MPN are due to the acquisition of mutations (JAK2, CALR, MPL) leading to JAK2 pathway activation but cooperating mutations involved in MPN evolution have been described. Mutations of TP53 in JAK2V617F patients are linked to AML transformation, illustrated by development of leukemias after retroviral overexpression of JAK2V617F in TP53 inactivated (KO) cells transplanted in irradiated animals, thus a possible cooperation between TP53 inactivation and JAK2 signaling in transformation process has been postulated. However, JAK2V617F signaling has also been reported as able to reduce p53 expression per se suggesting a redundant role of p53 inactivation in JAK2V617F cells.

Aims: Determine the role of TP53 in JAK2V617F induced genetic and phenotypic modifications.

Methods: To better understand the role of TP53 in MPN phenotype and evolution in steady state hematopoiesis, we developed a transgenic model of vav-cre induced JAK2V617F expression in TP53 knock-out (KO) animals and analyzed phenotype, survival, genetic modifications using stem cell compartments RNA-Seq and response to therapy of this triple transgenic mice.

Results: JAK2V617F vav-inductible-Tg/TP53 KO mice were generated to recombine JAK2V617F at an endogenous level in TP53 inactivated hematopoietic cells.

During follow-up, JAK2V617F/TP53KO mice developed the same phenotype than JAK2V617F transgenic mice without leukemia. In competitive repopulations with grafts with different ratio of JAK2V617F, JAK2V617F/TP53KO and wild type cells, we observed that JAK2V617F/TP53KO cells outcompete JAK2V617F cells.

In order to better define TP53-dependent and independent pathways linked to these phenotypes, LT-HSC, ST-HSC, MPP, CMP, MEP and GMP from normal, JAK2V617F and JAK2V617F/TP53 KO mice were cell sorted and RNA-Seq analysis was performed. Principal component analysis demonstrated that JAK2V617F/TP53KO cells were closer to normal cells than JAK2V617F cells whatever the (stem) cell compartment, illustrating that JAK2V617F-induced modifications are largely p53 dependent. Further analysis demonstrated that approximatively half of JAK2V617F deregulated genes in the different compartments are TP53 dependent including the IFN pathways. In order to validate this finding, mice repopulated with a mix of WT and JAK2V617F (either p53 KO or wild-type) cells were treated for 8 weeks with recombinant murine pegylated IFN-a. JAK2V617F reconstituted animals entered in complete hematological remission while JAK2VF/TP53KO reconstituted animals did not, illustrating that loss of TP53 in this context induced resistance to IFN-a.

On the other hand, since MPN develop in the same way in JAK2V617F-only and in JAK2V617F/TP53KO mouse, this suggests that JAK2V617F-specific pathways also found differentially expressed in JAK2V617F/TP53KO are linked to the MPN phenotype. KEGG and GO analysis demonstrated that these genes were mainly implicated in cytokine response, cell proliferation, differentiation, and leukemia evolution illustrating that the development of MPN and its possible risk of transformation in this mouse model is largely TP53 independent.

Summary/Conclusion: Taken together, our results show that a large part of genetic modifications induced by JAK2V617F mutation are p53 dependent but MPN phenotype is not, TP53 loss is insufficient to induce quick leukemic transformation in steady-state hematopoiesis in Jak2 V617F MPN despite it increases LT-HSC cell proliferation and finally that TP53 loss could be involved in IFN resistance in MPN.

S194: INTEGRATIVE CLINICAL PROTEOTYPING AND DRUG RESPONSE PROFILING IDENTIFIES TARGETABLE BIOLOGY UNDERLYING MYELOPROLIFERATIVE NEOPLASMS

M. H. Wildschut 1 2 3,* , M. van Oostrum 2 , J. Settelmeier 2 4 , J. Mena 1 , C. Dördelmann 5 , Y. Festl 1 , A. Ring 3 , R. C. Skoda 6 , M. Lopes 5 , B. Wollscheid 2 4 , B. Snijder 1 , A. Theocharides 3

1 Institute of Molecular Systems Biology, Department of Biology; 2 Institute of Translational Medicine, Department of Health Science and Technology, ETH Zurich; 3 Department of Medical Oncology and Hematology, Division of Hematology, University Hospital Zurich, Zurich; 4 Swiss Institute of Bioinformatics, Lausanne; 5 Insititute of Molecular Cancer Research, University of Zurich, Zurich; 6 Department of Biomedicine, Experimental Hematology, University Hospital Basel and University of Basel, Basel, Switzerland

Background: Myeloproliferative neoplasms (MPN) are a family of hematopoietic stem cell diseases characterized by frequent driver mutations in Calreticulin (CALR) and JAK2. While patients with essential thrombocythemia (ET) have a more favorable outcome, the prognosis of patients with advanced myelofibrosis (MF) is particularly poor with limited therapeutic options.

Aims: Here, we set out to elucidate biology underlying MPN and translate this into vulnerabilities that can be exploited by targeted therapies. By integration of proteomic analyses and drug response profiling we aimed to correlate MPN protein alterations directly to targeted drug responses, and subsequently validate pathway alterations using dedicated follow-up experiments.

Methods: We gathered extended MPN patient and matched healthy donor (HD) cohorts (n = 119) and a follow-up MF-specific cohort (n = 44) to investigate both general and subgroup-specific alterations. We performed proteomics on isolated granulocytes, hematopoietic stem and progenitor cells (HSPCs), and T-cells, and drug screening on peripheral blood mononuclear cells (PBMCs). For follow-up experiments, we established a CALR mutant-specific antibody for use in high-content immunofluorescence imaging.

Results: We identify a specific signature of disease- and mutation-specific protein alterations present in MPN patients by machine learning. Among the identified alterations, we find strong upregulation of proteins belonging to the MCM complex, a helicase involved in DNA replication, to be specifically present in a subgroup of MF patients that are clinically characterized by a worse prognosis. In these patients, a proteomic signature related to DNA replication and cell cycling is found to be elevated across blood cell types. We proof that cells from these patients are a) characterized by high proliferation rates, and b) respond well to drugs targeting this process (e.g. topoisomerase inhibitors). Furthermore, we find upregulation of proteins involved in endoplasmic reticulum (ER) stress to be significantly correlated to the CALR mutation burden across HSPCs, granulocytes, and T-cells. Using an advanced imaging approach we show that on a single cell level, the amount of ER stress corresponds to the level of CALR mutant protein expression. This results in a higher sensitivity of CALR-mutated cells to drugs targeting ER stress or the corresponding unfolded protein response (UPR).

Summary/Conclusion: Using large-scale MPN patient cohorts, we are able to elucidate biology underlying MPN disease. We here show specific proteomic signatures of proliferation and ER stress to be present in subsets of MPN patients and translate this to specific biological alterations and correlated therapeutic vulnerabilities. Our findings can be instrumental to both improved stratification of response and discovery of novel targeted therapeutics for MPN patients.

S195: MOMENTUM: PHASE 3 RANDOMIZED STUDY OF MOMELOTINIB (MMB) VERSUS DANAZOL (DAN) IN SYMPTOMATIC AND ANEMIC MYELOFIBROSIS (MF) PATIENTS PREVIOUSLY TREATED WITH A JAK INHIBITOR

S. Verstovsek 1,* , A. Vannucchi 2 , A. Gerds 3 , H. K. Al-Ali 4 , D. Lavie 5 , A. Kuykendall 6 , S. Grosicki 7 , A. Iurlo 8 , Y. T. Goh 9 , M. Lazaroiu 10 , M. Egyed 11 , M. L. Fox 12 , D. McLornan 13 , A. Perkins 14 , S.-S. Yoon 15 , V. Gupta 16 , J.-J. Kiladjian 17 , R. Donahue 18 , J. Kawashima 18 , R. Mesa 19

1 The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America; 2 Center Research and Innovation of Myeloproliferative Neoplasms, AOU Careggi, University of Florence, Florence, Italy; 3 Cleveland Clinic Department of Hematology and Medical Oncology, Avon, OH, United States of America; 4 University Hospital of Halle, Halle, Germany; 5 Hadassah Hebrew University Medical Center, Jerusalem, Israel; 6 Moffitt Cancer Center, Tampa, FL, United States of America; 7 Medical University of Silesia, Katowice, Poland; 8 Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 9 Singapore General Hospital, Singapore, Singapore; 10 Policlinica de Diagnostic Rapid Brasov, Brasov, Romania; 11 Somogy County Mór Kaposi General Hospital, Kaposvár, Hungary; 12 Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain; 13 Guy’s and Saint Thomas’ NHS Foundation Trust, London, United Kingdom; 14 Monash University, Melbourne, Australia; 15 Seoul National University Hospital, Seoul, South Korea; 16 Princess Margaret Cancer Centre, Toronto, ON, Canada; 17 Saint-Louis Hospital (AP-HP), Paris, France; 18 Sierra Oncology, Inc., San Mateo, CA; 19 UT Health San Antonio Cancer Center, San Antonio, TX, United States of America

Background: MMB, a novel oral ACVR1/ALK2 and JAK1/2 inhibitor, showed clinical activity on MF symptoms, red blood cell (RBC) transfusion requirements (anemia), and spleen volume in the SIMPLIFY trials.

Aims: This pivotal phase 3 study of MF patients (pts) previously treated with a JAK inhibitor (JAKi) tested MMB vs DAN on key symptom, anemia, and spleen volume endpoints at 24 weeks (wks).

Methods: Eligibility: Primary or post-ET/PV MF; DIPSS high risk, Int-2, or Int-1; MF Symptom Assessment Form Total Symptom Score (MFSAF TSS) ≥10; hemoglobin (Hgb) <10 g/dL; prior JAKi for ≥90 days, or ≥28 days if RBC transfusions ≥4 units in 8 wks or Gr 3/4 thrombocytopenia, anemia, or hematoma; palpable spleen ≥5 cm. Stratification: TSS (≥22 vs <22), palpable spleen (≥12 cm vs <12 cm), and RBC units transfused (0, 1-4, and 5+). JAKi taper and washout was ≥21 days. Randomization: 2:1 to MMB 200 mg QD plus DAN placebo or DAN 600 mg QD plus MMB placebo for 24 wks, after which pts could receive open-label MMB. Assessments: Pt reported symptoms using a daily eDiary and spleen volume by MRI or CT. The primary endpoint was TSS response (≥50% reduction from baseline [BL]) rate at wk 24. Secondary endpoints, assessed sequentially at wk 24, were RBC transfusion independence (TI) rate, splenic response rate (SRR; ≥25% reduction in volume from BL), change from BL in TSS, SRR (≥35% reduction from BL) and rate of zero transfusions since BL. Informed consent was obtained from all participants.

Results: 94 of 130 (72%) MMB pts and 38 of 65 (58%) DAN pts completed the 24-wk randomized treatment (RT) phase. Median BL TSS were 28 (MMB) and 26 (DAN), Hgb were 8.1 (MMB) and 7.9 (DAN) g/dL, and platelets were 97 (MMB) and 94 (DAN) x10 9 /L. BL TI was 13% (MMB) and 15% (DAN). BL mean spleen volume was 2367 (MMB) and 2288 (DAN) cm 3 . Prior JAKi was ruxolitinib in 195 pts (100%) and fedratinib in 9 pts (5%); mean duration of prior JAKi was 134 weeks. All primary and key secondary endpoints were met (Table). Most common Gr ≥3 treatment-emergent adverse events (TEAEs) in the RT phase of the study were thrombocytopenia (MMB, 22%; DAN, 12%) and anemia (MMB, 8%; DAN, 11%). Gr ≥3 infections occurred in 15% of MMB and 17% of DAN pts. Peripheral neuropathy (PN) occurred in 5 (4%) of MMB (all Gr ≤2) and 1 (2%) of DAN (Gr ≤2) pts in the RT phase, and none discontinued study drug due to PN. Overall, TEAEs led to study drug discontinuation in 18% of MMB and 23% of DAN pts, and serious TEAEs were reported in 35% of MMB and 40% of DAN pts, in RT phase. A trend toward improved OS up to wk 24 was seen with MMB vs DAN (HR=0.506, p=0.0719).

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Summary/Conclusion: In symptomatic and anemic MF pts, MMB was superior to DAN for symptom responses, transfusion requirements, and spleen responses with comparable safety and favorable survival. MMB may address a critical unmet need, particularly in MF pts with anemia. NCT04173494 .

S196: ROPEGINTERFERON ALFA-2B ACHIEVES PATIENT-SPECIFIC TREATMENT GOALS IN POLYCYTHEMIA VERA: FINAL RESULTS FROM THE PROUD-PV/CONTINUATION-PV STUDIES

H. Gisslinger 1,* , C. Klade 2 , P. Georgiev 3 , D. Krochmalczyk 4 , L. Gercheva-Kyuchukova, M. Egyed 6 , P. Dulicek 7 , A. Illes 8 , H. Pylypenko 9 , L. Sivcheva 10 , J. Mayer 11 , V. Yablokova 12 , V. Empson 2 , K. Krejcy 2 , H. Hasselbalch 13 , R. Kralovics 14 , J.-J. Kiladjian 15

1 Department of Internal Medicine I, Division of Hematology and Blood Coagulation, Medical University Vienna; 2 AOP Orphan Pharmaceuticals GmbH, Vienna, Austria; 3 Medical University of Plovdiv, Plovdiv, Bulgaria; 4 Teaching Unit of the Hematology Department, University Hospital in Krakow, Krakow, Poland; 5 Clinical Hematology Clinic, Multiprofile Hospital for Active Treatment “Sveta Marina”, Varna, Bulgaria; 6 Department of Internal Medicine II, Kaposi Mor County Teaching Hospital, Kaposvar, Hungary; 7 Department of Clinical Hematology, University Hospital Hradec Kralove, Hradec Kralove, Czechia; 8 Department of Hematology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; 9 Department of Hematology, Regional Treatment and Diagnostics Hematology Centre, Cherkasy Regional Oncology Centre, Cherkasy, Ukraine; 10 First Department of Internal Medicine, Multiprofile Hospital for Active Treatment - HristoBotev, Vratsa, Bulgaria; 11 Clinic of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czechia; 12 Department of Hematology, Yaroslavl Regional Clinical Hospital, Yaroslavl, Russia; 13 Department of Hematology, Zealand University Hospital, University of Copenhagen, Roskilde, Denmark; 14 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; 15 Centre d’Investigations Cliniques, INSERM, CIC1427, Université de Paris, AP-HP, Hôpital Saint-Louis, Paris, France

Background: Treatment of polycythemia vera (PV) aims to prevent thromboembolic complications, reduce the risk of progression to acute leukemia or myelofibrosis - of particular concern to patients - and ameliorate the symptom burden; specifically, to improve quality of life, therapy should address the most clinically important symptoms while reducing phlebotomies to avoid iron-deficiency symptoms. Long-term efficacy and safety of ropeginterferon alfa-2b have been demonstrated in PROUD-PV/CONTINUATION-PV; the final analysis applied a patient-focused approach.

Aims: To analyze the patient-relevant benefit of ropeginterferon alfa-2b versus hydroxyurea (HU)/best available treatment (BAT) over 6 years.

Methods: Patients diagnosed with PV according to WHO 2008 criteria who were cytoreduction-naïve or hydroxyurea pre-treated and gave written informed consent were randomized 1:1 to ropeginterferon alpha-2b or control treatment (HU) for one year in PROUD-PV. In CONTINUATION-PV, control arm patients could switch from HU to BAT. Patient-reported PV symptom burden was assessed based on adverse events documented in the patient diary and recorded at each visit; items defined in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-SAF TSS; fatigue, concentration problems, early satiety, inactivity, night sweats, itching, bone pain, abdominal discomfort, weight loss, and fevers) and medical synonyms were evaluated post-hoc. Efficacy assessments included Kaplan-Meier analysis of event-free survival, phlebotomy need and JAK2 V617F allele burden. Analyses were conducted on the CONTINUATION-PV full analysis set over 6 years of treatment.

Results: The full analysis set comprised 95 patients in the ropeginterferon alfa-2b arm and 74 in the control arm.

Patient-reported symptoms defined in the MPN-SAF TSS were present in a small minority (9.5%) of patients per arm at baseline (up to Week 4 of treatment) in this early-stage PV population. Occurrence of the defined symptoms remained low over long-term treatment, reported in 15.7% of patients in the ropeginterferon alfa-2b arm and 20.7% in the control arm during the 6 th year of treatment.

No phlebotomies were required to maintain hematocrit <45% in the 6 th year of treatment in 81.4% of patients receiving ropeginterferon alfa-2b compared with 60.0% of patients in the control arm (p=0.005).

Depletion of the JAK2 V617F alle burden, which may lower the risk of progression to myelofibrosis, was observed in ropeginterferon alfa-2b treated patients; JAK2 V617F allele burden <1% at 6 years was achieved in 19/92 (20.7%) patients in the ropeginterferon alfa-2b arm with baseline allele burden >10%. One patient met this threshold in the control arm (1/70 [1.4%]; p=0.0001).

Event-free survival (risk events: disease progression, death and thromboembolic events) over ≥6 years of treatment was significantly higher among ropeginterferon alfa-2b treated patients than the control group (risk events reported in 5/95 vs. 12/74 patients, respectively; p=0.04 [Log-Rank]; Fig 1).

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Summary/Conclusion: Long-term ropeginterferon alfa-2b therapy fulfils treatment goals important to patients with PV: a good quality of life as indicated by a low symptom burden and phlebotomy requirement, the potential to influence myelofibrosis risk, and better event-free survival versus BAT.

S197: NAVITOCLAX PLUS RUXOLITINIB IN JAK INHIBITOR-NAÏVE PATIENTS WITH MYELOFIBROSIS: PRELIMINARY SAFETY AND EFFICACY IN A MULTICENTER, OPEN-LABEL PHASE 2 STUDY

F. Passamonti 1,* , J. Foran 2 , A. Tandra 3 , V. De Stefano 4 5 , M. Laura Fox 6 , A. Mattour 7 , M. F. McMullin 8 , A. C. Perkins 9 , G. Rodriguez-Macías 10 , H. Sibai 11 , Q. Qin 12 , J. Potluri 12 , J. How 13

1 Department of Medicine and Surgery, University of Insubria, Varese, Italy; 2 Mayo Clinic, Jacksonville, FL; 3 Indiana Blood and Marrow Transplant, Indianapolis, United States of America; 4 Section of Hematology, Department of Radiological and Hematological Sciences, Catholic University; 5 Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy; 6 Department of Hematology, Hospital Universitari Vall d’Hebron, Experimental Hematology, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Hospital Campus, Barcelona, Spain; 7 Henry Ford Hospital, Detroit, MI, United States of America; 8 Centre for Medical Education, Queenʼs University Belfast, Belfast, United Kingdom; 9 Australian Centre for Blood Diseases, Monash University and The Alfred Hospital, Melbourne, Australia; 10 Department of Hematology, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 11 Medical Oncology and Hematology, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada; 12 AbbVie Inc, North Chicago, IL, United States of America; 13 Division of Hematology, McGill University Health Center, Montreal, Canada

Background: Ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, is the current standard of care for patients with myelofibrosis (MF) that improves disease symptoms in approximately 40% of patients with limited impact on disease biology. Many patients lose response over time, highlighting an unmet need for novel therapies. Navitoclax is a first-in-class, oral, small molecule that binds with high affinity to BCL-XL and BCL-2, key pro-survival proteins in the apoptotic pathway, and has a synergistic effect when used in combination with JAK inhibitors to enhance malignant cell death in MF. This ongoing, open-label, multicenter, phase II trial ( NCT03222609 ) is evaluating the efficacy and safety of navitoclax with or without ruxolitinib in patients with MF and previously published data suggest clinically meaningful activity of the combination in patients with prior ruxolitinib failure (Pemmaraju et al., ASH 2020). Here, we report results from patients who were JAK inhibitor-naïve and treated with navitoclax and ruxolitinib combination therapy.

Aims: N/A

Methods: Enrolled patients had primary or secondary MF with splenomegaly (DIPSS ≥ Intermediate-1) and did not receive prior therapy with JAK-2 or bromodomain and extraterminal motif (BET) inhibitors. Patients initiated navitoclax at 100 mg once per day (QD) or 200 mg QD if baseline platelet count was ≤150 × 10 9 /L or >150 × 10 9 /L, respectively. Ruxolitinib was orally administered twice daily at a starting dose based on baseline platelet count as per the local ruxolitinib label. The primary endpoint was spleen volume reduction of ≥35% (SVR 35 ) from baseline at week 24. SVR 35 was assessed at weeks 12, 24, and once every 24 weeks thereafter. Key secondary endpoints were ≥50% reduction in total symptom score (TSS 50 ), bone marrow fibrosis reduction, and anemia response (per International Working Group criteria). Adverse events (AEs) were monitored throughout the study.

Results: As of Oct 04, 2021, 32 patients received navitoclax plus ruxolitinib. The median duration of follow-up was 6.1 (range, 1.9 to 18.6) months. Twenty-eight (88%) patients received a navitoclax dose of 200 mg, and 4 (13%) received 100 mg OD. The median age was 69 years (range, 44 to 83), median spleen volume was 1889.08 cm 3 (range, 645.6 to 7339.6), and 38% had at least 1 prior line of therapy.

The median navitoclax and ruxolitinib exposures were 24.1 (range, 5.1 to 80.9) and 24.1 (5.1 to 80.9) weeks, respectively. Thirty-one (97%) patients experienced ≥1 AE, 25 (78%) had Grade ≥3 AEs, and 6 (19%) had serious AEs. The most frequent Grade ≥3 AEs were anemia (34%), thrombocytopenia (31%), and neutropenia (19%). Three (9%) and 2 (6%) patients experienced an AE that led to navitoclax and ruxolitinib discontinuation, respectively, and 2 (6%; 1 progressive disease, 1 cardiac disorder unrelated to navitoclax) AEs led to death ≤30 days after the last dose of navitoclax. SVR 35 was achieved by 52% of evaluable patients at week 24 (SVR 35 in DIPSS Intermediate-2, 50%; High-risk, 33%) and by 76% at any time on the study (Table). The median time to first SVR 35 was 12.1 (range, 11 to 47) weeks.

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Summary/Conclusion: The combination of navitoclax and ruxolitinib was well tolerated and demonstrated early and robust reductions in spleen volume, anemia, and BM fibrosis in patients without prior JAK-2 inhibitor exposure. SVR 35, anemia, and bone marrow fibrosis improved over time.

S198: BET INHIBITOR PELABRESIB (CPI-0610) COMBINED WITH RUXOLITINIB IN PATIENTS WITH MYELOFIBROSIS — JAK INHIBITOR-NAÏVE OR WITH SUBOPTIMAL RESPONSE TO RUXOLITINIB — PRELIMINARY DATA FROM THE MANIFEST STUDY

J. Mascarenhas 1,* , M. Kremyanskaya 1 , A. Patriarca 2 , C. Harrison 3 , P. Bose 4 , R. K. Rampal 5 , F Palandri 6 , T. Devos 7 , F. Passamonti 8 , G. Hobbs 9 , M. Talpaz 10 , A. Vannucchi 11 , J.-J. Kiladjian 12 , S. Verstovsek 13 , R. Hoffman 1 , M. E. Salama 13 , D. Chen 14 , P. Taverna 15 , A. Chang 15 , G. Colak 15 , S. Klein 15 , V. Gupta 16

1 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America; 2 Hematology Unit, Department of Translational Medicine, University of Eastern Piedmont and AOU Maggiore della Carità, Novara, Italy; 3 Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; 4 MD Anderson Cancer Center, Houston, TX; 5 Memorial Sloan-Kettering Cancer Center, New York, NY, United States of America; 6 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy; 7 University Hospitals Leuven and Laboratory of Molecular Immunology (Rega Institute), KU Leuven, Leuven, Belgium; 8 University of Insubria, Varese, Italy; 9 Division of Hematology/Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 10 University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, United States of America; 11 Azienda Ospedaliero-Universitaria Careggi, University of Florence, Florence, Italy; 12 Hôpital Saint-Louis, Université de Paris, Paris, France; 13 Leukemia Department, University of Texas MD Anderson Cancer Center, Houston, TX; 14 Mayo Clinic, Rochester, MN; 15 Constellation Pharmaceuticals a MorphoSys Company, Boston, MA, United States of America; 16 Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada

Background: Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis, anemia, splenomegaly and constitutional symptoms. Progressive BM fibrosis results from aberrant megakaryopoiesis and proinflammatory cytokine expression, two processes that are tightly regulated by bromodomain and extraterminal domain (BET) protein-mediated expression of genes (e.g. NF-κB , MYC and BCL-2 ) and often lead to myeloproliferation and cytopenias. While many MF patients (pts) receive the Janus kinase inhibitor (JAKi) ruxolitinib (RUX) as the current standard of care, the depth and durability of responses, along with the percentage of pts that achieve ≥35% spleen volume reduction from baseline (BL; SVR35) and ≥50% total symptom score reduction from BL (TSS50) are limited; thus, a significant unmet medical need exists. Pelabresib (CPI-0610) is an oral, small-molecule, investigational BET inhibitor that downregulates NF-κB gene expression and other relevant genes involved in MF disease pathways.

Aims: Data (Sep 2021 data cut) on the safety and efficacy of pelabresib in combination with RUX in pts with MF from Arms 2 and 3 of the ongoing, open-label Phase 2 MANIFEST study ( NCT02158858 ). In Arm 2, MF pts with suboptimal response to RUX are treated with pelabresib as ‘add-on’ to RUX (Arm 2A: transfusion dependent [TD]; Arm 2B: non-TD). In Arm 3, JAKi-naïve MF pts are treated with pelabresib in combination with RUX.

Methods: The primary endpoints are SVR35 at Week (Wk) 24 for Arm 3 and Arm 2B (non-TD cohort) and TD to transfusion independence (TI) in Arm 2A (TD cohort). The key secondary endpoint is TSS50 per Myelofibrosis Symptom Assessment Form v4.0 at Wk 24; in Arm 2A (TD cohort), SVR35 is an additional key secondary endpoint. BM biopsies to assess BM fibrosis and safety data are also evaluated.

Results: At Wk 24 in Arm 3 (N=84), 68% (57/84) pts achieved SVR35 (median change: –50%; Figure), and 56% (46/82) pts achieved TSS50 (median change: –59%). Twenty-four percent of pts had a mean hemoglobin increase ≥1.5 g/dL from BL over 12 weeks without transfusions. At Wk 24 in Arm 2 (N=86), 20% (16/81) pts achieved SVR35 (median change: –18%; Figure), and 37% (30/81) pts achieved TSS50 (median change: –47%). In Arm 2A, the TD to TI rate was 16% (6/38).

BM fibrosis improvement by ≥1 grade was achieved in 31% (16/52) and 25% (9/36) pts in Arm 3 and 2, respectively. Further central review and exploratory analyses of BM pathology from a more mature data set, including clinical correlations, will be presented.

The most common hematologic treatment-emergent adverse event (TEAE) of any grade was thrombocytopenia, reported in 52% (≥Grade 3: 12%) and 52% (≥Grade 3: 33%) pts in Arm 3 and 2, respectively. Anemia was reported in 42% (≥Grade 3: 35%) and 27% (≥Grade 3: 19%) pts in Arm 3 and 2, respectively. Low-grade gastrointestinal TEAEs and respiratory infections in Arm 3 and 2 were observed but rarely a reason for treatment discontinuation.

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Summary/Conclusion: Based on these interim Phase 2 data, pelabresib in combination with RUX, in both RUX treatment-naïve and -experienced pts with MF, resulted in splenic and symptom responses and BM fibrosis improvement, and appeared to be well tolerated. Based on data from MANIFEST Arm 3, the randomized, double-blind, active-control Phase 3 MANIFEST-2 study was initiated to further evaluate the safety and efficacy of pelabresib in combination with ruxolitinib in JAKi treatment-naïve pts with MF ( NCT04603495 ).

S199: A NATIONAL RETROSPECTIVE COHORT STUDY OF MPN-SVT: RESULTS FROM THE UK MYELOPROLIFERATIVE NEOPLASMS ASSOCIATED SPLANCHNIC VEIN THROMBOSIS (MASCOT) REGISTRY

R. Hargreaves 1,* , M. Subhan 2 , S. Alimam 1 2 , S. Shapiro 3 4 , A. Robinson 5 , M. Carter 5 , A. Godfrey 5 , M. Ul-Haq 6 , M. Jain 6 , G. Greenfield 7 , A. McGregor 8 , H. K. Hussein 9 , D. Tripathi 10 , H. Network 11 , A. Doyle 12 , K. White 12 , N. Curto-Garcia 12 , D. Patch 13 , M. Sekhar 1 2

1 Haematology, Royal Free Hospital NHS Foundation Trust; 2 Haematology, University College London Hospitals NHS Foundation Trust, London; 3 Haematology, Oxford University Hospitals NHS Foundation Trust, NIHR Biomedical Research Centre; 4 Radcliffe Department of Medicine, University of Oxford, Oxford; 5 Haematology, Cambridge University Hospitals NHS Foundation Trust, Cambridge; 6 Haematology, Leeds Teaching Hospitals NHS Trust, Leeds; 7 Patrick G Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast; 8 Haematology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne; 9 Haematology; 10 Hepatology, University Hospitals Birmingham NHS Foundation Trust, Birmingham; 11 Haematology Specialty Trainee Audit and Research, HaemSTAR Network, Nationwide; 12 Haematology, Guy’s and St Thomas’ NHS Foundation Trust; 13 Hepatology, Royal Free Hospital NHS Foundation Trust, London, United Kingdom

Background: Patients with myeloproliferative neoplasms (MPN) are at increased risk of splanchnic vein thrombosis (SVT), defined as abdominal thrombosis in portal, splenic, mesenteric or hepatic veins. MPN-SVT carries a significant mortality and morbidity burden and poses clinical management challenges. Detailed studies of this patient population are lacking.

Aims: To establish a national web-based clinical registry for MPN-SVT (MASCOT Registry) and use this to investigate demographics, clinical features, co-morbidities, outcome & UK treatment practices for MPN-SVT.

Methods: The MASCOT Registry is a UK-wide retrospective cohort of patients with MPN-SVT from 9 large haematology/hepatology centres. Participating centres entered pseudo-anonymised data for patients with MPN-SVT. New and historical cases were added; demographic, radiologic, clinical and outcome data were collected.

Results: 232 patients with MPN-SVT were registered on the online database between May 2019 and January 2022. 1 centre registered 92 cases (40%) and 2 centres each registered 39 cases (17%) with the remaining 62 cases (27%) from 6 centres. 57% of patients were female and 43% male and the age of SVT onset was 49 years or less in 70% of patients. Median follow up was 7.3 years (range 35 days-31.8 years). SVT was diagnosed in 2009 or later in 72%.

MPN was diagnosed first in 30% of patients, SVT first in 42% of patients and simultaneous diagnosis in 28%. Figure 1 shows the subtypes of MPN associated with SVT.

Multiple splanchnic vein thromboses were observed in 42% of patients. Of those with single vein thrombosis, the portal vein was affected in 26% and the hepatic vein in 23%.

Initial anticoagulation treatment was in line with local policy. TIPSS (transjugular intrahepatic portosystemic shunt) was performed in 21% and thrombolysis in 7%. Warfarin only was the preferred long-term anticoagulant (53%).

Cytoreduction at registration comprised 44% of patients on hydroxycarbamide, 18% on Pegylated interferon, 16% on ruxolitinib and 8% on no therapy.

37 patients required major abdominal surgery and 17 received liver transplants.

71 patients (33%) suffered from other non-SVT thromboses (34% pre-SVT, 9% simultaneously and 57% post-SVT), comprising 84 thrombotic events (60 venous, 24 arterial). Grade 3+ haemorrhage was observed in 60 patients (28%), of which the majority (48%) occurred within the first year of SVT diagnosis.

111 patients (51%) were re- image d at least once within 12 months post-SVT, demonstrating clot extension in 12%, recanalization in 25% and stable appearances in 54%. Overall survival was 88% with 14 deaths, of which 6 were disease-related. Median age at death was 72 years and death occurred at a median of 12 years post SVT diagnosis.

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Summary/Conclusion: This is one of the largest cohort studies of patients with MPN-SVT to date with over 70% diagnosed in the last 13 years. Similar to previous published studies, our evidence corroborates the female predominance and relatively young age at diagnosis.

Our data highlight the significant morbidity and mortality burden incurred by patients with MPN-SVT, including a substantially lower rate of re-canalisation compared to a recent meta-analysis of all patients with SVT and high rates of thrombosis recurrence.

We have highlighted variations in clinical practice including choice of anticoagulant, preferred cytoreductive agent and use of thrombolysis and TIPSS.

Future studies to further understand this patient population should be focused on access to optimal care, the aetiology of thrombosis, optimum anticoagulation management and strategies to minimise bleeding.

S200: IMPROVED OVERALL SURVIVAL WITH FIRST-LINE BRENTUXIMAB VEDOTIN PLUS CHEMOTHERAPY IN PATIENTS WITH STAGE III/IV CLASSICAL HODGKIN LYMPHOMA: 6-YEAR ANALYSIS OF ECHELON-1

M. Hutchings 1,* , S. M. Ansell 2 , D. J. Straus 3 , J. M. Connors 4 , W. S. Kim 5 , A. Gallamini 6 , R. Ramchandren 7 , J. W. Friedberg 8 , R. Advani 9 , A. M. Evens 10 , P. Smolewski 11 , K. J. Savage 4 , N. L. Bartlett 12 , H.-S. Eom 13 , J. S. Abramson 14 , C. Dong 15 , F. Campana 15 , K. Fenton 16 , M. Puhlmann 16 , J. Radford 17

1 Department of Haematology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark; 2 Division of Hematology, Mayo Clinic, Rochester, MN; 3 Department of Medicine, Lymphoma Service, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 4 BC Cancer Centre for Lymphoid Cancer and Department of Medical Oncology, Vancouver, Canada; 5 Division of Hematology-Oncology, Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 6 Research and Innovation, Antoine-Lacassagne Cancer Centre, Nice, France; 7 The University of Tennessee Graduate School of Medicine, Knoxville, TN; 8 James P. Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY; 9 Department of Medicine, Division of Oncology, Stanford University, Stanford, CA; 10 Division of Blood Disorders, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United States of America; 11 Department of Experimental Hematology, Medical University of Lodz, Lodz, Poland; 12 Washington University School of Medicine Siteman Cancer Center, St Louis, MO, United States of America; 13 Center for Hematologic Malignancy, National Cancer Center, Goyang, South Korea; 14 Massachusetts General Hospital, Boston, MA; 15 Takeda Development Center Americas, Inc. (TDCA), Lexington MA; 16 Seagen Inc., Bothell, WA, United States of America; 17 The University of Manchester and the Christie NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom

Background: In classical Hodgkin lymphoma (cHL), improved overall survival (OS) with first-line treatment using new combinations has seldom been observed compared with existing approaches. Five-year data from the randomized phase 3 ECHELON-1 study ( NCT01712490 ) supported long-term progression-free survival (PFS) with brentuximab vedotin, doxorubicin, vinblastine, and dacarbazine (A+AVD) versus doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), in patients (pts) with previously untreated stage III/IV cHL, regardless of interim positron emission tomography (PET) status. The long-term safety profile of A+AVD was manageable, and numerically fewer secondary malignancies were reported versus ABVD, as well as a greater number of pregnancies (Straus et al, Lancet Haematol 2021).

Aims: To report the pre-specified OS analysis from ECHELON-1, as well as relevant long-term safety data, after approximately 6 years of follow-up (cut-off June 1, 2021).

Methods: Pts were randomized 1:1 to receive up to 6 cycles of A+AVD (n=664) or ABVD (n=670) on day 1 and 15, every 28 days. PFS per investigator was reported for long-term follow-up. The key secondary end point was OS, which was an event-driven, pre-specified, alpha-controlled analysis in the intention-to-treat population, with a prespecified interim analysis after 103 deaths. Analysis of OS in prespecified subgroups was exploratory and was not adjusted for multiplicity. Informed consent was obtained for all pts.

Results: In total, 39 OS events in the A+AVD arm and 64 in the ABVD arm had occurred at a median follow-up of 73 months, significantly favoring A+AVD (hazard ratio [HR] 0.590; 95% CI 0.396–0.879; p=0.009). There was a consistent OS benefit for A+AVD vs ABVD across prespecified subgroups, including: stage III (HR: 0.863; 95% CI 0.452–1.648) and stage IV disease (HR: 0.478; 95% CI 0.286–0.799) at diagnosis, pts who were PET-negative at cycle 2 (PET2)-negative (HR: 0.583; 95% CI 0.338–0.856) and PET2-positive (HR: 0.163; 95% CI 0.037–0.717), pts aged <60 years (HR: 0.509; 95% CI 0.291–0.890), pts aged ≥60 years (HR: 0.829; 95% CI 0.469–1.466), and across all geographies, including Europe (HR: 0.783; 95% CI 0.467–1.315) and North America (HR: 0.327; 95% CI 0.153–0.699). The 6-year PFS estimate was 82.3% (79.1–85.0) vs 74.5% (70.8–77.7) with A+AVD vs ABVD, respectively (HR: 0.678; 95% CI: 0.532–0.863). The long-term safety profile of A+AVD was comparable to that of ABVD. In both A+AVD and ABVD groups, treatment-emergent peripheral neuropathy continued to resolve or improve, with 86% (379/443) and 87% (249/286) of cases in the A+AVD and ABVD arms either completely resolving (72% vs 79%) or improving (14% vs 8%) by last follow-up. Overall, 23 secondary malignancies in the A+AVD arm and 32 in the ABVD arm were reported. Pregnancies and live births were reported by a greater number of female pts in the A+AVD group vs the ABVD group (49 vs 28 and 42 vs 19, respectively) and there were no stillbirths reported during the study. There were no new safety signals.

Summary/Conclusion: There was a statistically significant 41% reduction in the risk of death with A+AVD vs ABVD, with a consistent OS benefit across prespecified subgroups. The long-term safety profile was manageable, consistent with prior reports. These data support A+AVD as a preferred option for previously untreated stage III and IV cHL.

S201: CAMIDANLUMAB TESIRINE: UPDATED EFFICACY AND SAFETY IN AN OPEN-LABEL, MULTICENTER, PHASE 2 STUDY OF PATIENTS WITH RELAPSED OR REFRACTORY CLASSICAL HODGKIN LYMPHOMA (R/R CHL)

C. Carlo-Stella 1,* , S. Ansell 2 , P. L Zinzani 3 , J. Radford 4 , K. Maddocks 5 , A. Pinto 6 , G. P. Collins 7 , V. Bachanova 8 , N. Bartlett 9 , I. Bence-Bruckler 10 , M. Hamadani 11 , J. Kline 12 , J. Mayer 13 , K. J. Savage 14 , R. Advani 15 , P. Caimi 16 , R.-O. Casasnovas 17 , T. Feldman 18 , B. Hess 19 , M. Bastos-Oreiro 20 , S. Iyengar 21 , S. Eisen 22 , Y. Negievich 22 , L. Wang 23 , J. Wuerthner 22 , A. F. Herrera 24

1 Department of Oncology and Hematology, Humanitas Clinical and Research Center - IRCCS and Humanitas University, Rozzano, Milano, Italy; 2 Division of Hematology, Mayo Clinic, Division of Hematology, Minnesota, United States of America; 3 Hematology, Institute of Hematology Seràgnoli University of Bologna, Bologna, Italy; 4 NIHR Clinical Research Facility, the Christie NHS Foundation Trust and University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom; 5 Division of Hematology, Ohio State University Medical Center, Columbus, United States of America; 6 Fondazione G. Pascale, IRCCS, Istituto Nazionale Tumori, Naples, Italy; 7 Oxford Cancer and Haematology Centre, NIHR Oxford Biomedical Research Centre, Churchill Hospital, Oxford, United Kingdom; 8 Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis; 9 Division of Oncology, Washington University School of Medicine in St. Louis, St. Louis, United States of America; 10 Ottawa-Hospital General Campus, The Ottawa Hospital-General Campus, Ottawa, Canada; 11 Medical College of Wisconsin, BMT & Cellular Therapy Program, Department of Medicine, Milwaukee; 12 Department of Medicine, The University of Chicago, Chicago, United States of America; 13 Internal Medicine, University Hospital and Masaryk University, Brno, Czechia; 14 Department of Medical Oncology, BC Cancer and University of British Columbia, Vancouver, Canada; 15 Division of Oncology, Department of Medicine, Stanford University, Stanford; 16 Cancer Center, Cleveland Clinic/Case Comprehensive Cancer Center, Cleveland, United States of America; 17 Department of Hematology, University Hospital F. Mitterrand and Inserm UMR 1231, Dijon, France; 18 John Theurer Cancer Center, Hackensack Meridian Health, Hackensack; 19 Division of Hematology and Medical Oncology, Department of Medicine, Medical University of South Carolina, Charleston, United States of America; 20 Haematology Department, Gregorio Marañón Health Research Institute, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 21 Royal Marsden Hospital, London, United Kingdom; 22 ADC Therapeutics SA, Epalinges, Switzerland; 23 ADC Therapeutics America, Inc, Murray Hill; 24 Department of Hematology & Hematology & Hematopoietic Cell Transplantation, City of Hope Comprehensive Cancer Center, Duarte, United States of America

Background: Camidanlumab tesirine (Cami), an antibody-drug conjugate comprising a human IgG1 anti-CD25 monoclonal antibody conjugated to a pyrrolobenzodiazepine (PBD) dimer, displayed antitumor activity and manageable toxicity in a phase 1 trial in lymphoma, including R/R cHL (Hamadani et al. 2021).

Aims: Present updated efficacy and safety data from a phase 2 study of Cami monotherapy in patients (pts) with R/R cHL ( NCT04052997 ).

Methods: Pts with R/R cHL and ≥3 prior systemic therapies, including brentuximab vedotin (BV) and anti-PD-1 (≥2 therapies if hematopoietic cell transplantation [HCT] ineligible), were enrolled. Interim data was reported (Herrera et al. 2020, Zinzani et al. 2021). The primary endpoint is overall response rate (ORR by 2014 Lugano criteria; central review). Secondary endpoints include duration of response (DOR), progression-free survival (PFS), and frequency and severity of adverse events (AEs). Pts received Cami 45 µg/kg (30-min infusion) on Day 1 of each 3-week cycle (2 cycles), then 30 µg/kg (subsequent cycles) for up to 1 year.

Results: As of Nov 1, 2021, enrollment was complete (N=117). Median age (range) was 37 years (19-87), 62% of pts were male, and 95% had an ECOG score of 0-1. Pts had received a median (range) of 6 (3-19) prior therapies and received a median (range) of 5.0 (1-15) Cami cycles. Fourteen pts (12.0%) withdrew to undergo HCT (of which 12 [10.3%] received HCT and were censored). In the all-treated population (N=117), ORR was 70.1% (82/117; 95% CI: 60.9-78.2), and 33.3% (39/117) had complete response (CR) ( Fig 1A ). At median (range) follow-up of 10.7 (1.2-25.2+) months (mos), the median (95% CI) DOR was 13.7 mos (7.4-14.7) for all responders, 14.5 (7.4-not reached, NR) mos and 7.9 (3.8-NR) mos for pts with CR or PR, respectively ( Fig 1B ). Median (95% CI) PFS was 9.1 (5.1-15.0) mos.

All-grade treatment-emergent AEs (TEAEs) in ≥25% of 117 pts were fatigue (45, 38.5%), maculopapular rash (MR, 38, 32.5%), pyrexia (35, 29.9%), nausea (32, 27.4%), and rash (31, 26.5%). Grade ≥3 TEAEs in ≥5% of pts were thrombocytopenia (11, 9.4%), anemia (10, 8.5%), hypophosphatemia (9, 7.7%), neutropenia (9, 7.7%), MR (8, 6.8%), and lymphopenia (6, 5.1%). TEAEs leading to Cami dose delay/reduction or discontinuation occurred in 66 (56.4%) and 32 (27.4%) pts, respectively. TEAEs considered PBD-associated were skin/nail reactions (any grade, 87, 74.4%; grade ≥3, 24, 20.5%), hepatobiliary test abnormalities (any grade, 34, 29.1%; grade ≥3, 8, 6.8%), and edema/effusion (any grade, 20, 17.1%; grade ≥3, 0).

TEAEs considered immune-related (IR) occurred in 38 (32.5%) pts. Grade ≥3 IR AEs (TEAEs and non-TEAEs; 10, 8.5%) included diabetic ketoacidosis/type 1 diabetes (2, 1.7%); drug-induced liver injury (2, 1.7%); tubulointerstitial nephritis (2, 1.7%); and aplastic anemia, autoimmune hemolytic anemia or hepatitis, and lichenoid keratosis (1 each, 0.9%).

Guillain–Barré syndrome (GBS)/polyradiculopathy was seen in 8 (6.8%) pts (Grade 2, n=2; grade 3, n=3; grade 4, n=3). In 4 pts, onset was after 2 Cami cycles (range: 2-7) and median (range) duration was 115 days (43-523). At data cutoff, 4 cases had recovered, and 4 had not recovered.

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Summary/Conclusion: With median follow-up of 10.7 mos, Cami demonstrated an ORR of 70.1% (CR: 33.3%) in heavily pretreated R/R cHL after BV and PD-1 blockade failure, with an encouraging median DOR of 13.7 mos and median PFS of 9.1 mos. Safety is consistent with prior findings, including similar incidence rates of GBS/polyradiculopathy.

S202: CHILDBEARING AMONG CLASSICAL HODGKIN LYMPHOMA SURVIVORS TREATED WITH BEACOPP AND ABVD IN SWEDEN, DENMARK, AND NORWAY

J. P. Entrop 1,* , C. E. Weibull 1 , K. E. Smedby 1 , L. H. Jakobsen 2 3 , A. K. Øvlisen 2 , D. Molin 4 , I. Glimelius 4 , A. Marklund 5 , K. B. Smeland 6 , T. C. El-Galaly 2 , S. Eloranta 1

1 Clinical Epidemiology Division, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden; 2 Department of Hematology, Clinical Cancer Research Center, Aalborg University Hospital; 3 Department of Mathematical Sciences, Aalborg University, Aalborg, Denmark; 4 Department of Immunology, Genetics and Pathology, Unit of Experimental and Clinical Oncology, Uppsala University, Uppsala; 5 Department of Reproductive Medicine, Division of Gynecology and Reproduction, Karolinska University Hospital, Stockholm, Sweden; 6 Department of Oncology, Oslo University Hospital, Oslo, Norway

Background: Recent studies have shown decreased childbearing rates in some classical Hodgkin lymphoma (cHL) survivors compared to the general population. This decrease is hypothesized to be caused by administered intensive chemotherapy. Understanding if currently used treatment protocols lead to reduced post-treatment childbirth potential is important as differential impact on childbearing may influence treatment decisions for younger patients. Firm conclusions from previous population-based studies have been hampered by low power for specific treatment contrasts.

Aims: To increase understanding of differences in childbearing patterns among male and female HL survivors treated with ABVD and BEACOPP in a study combining population-based register data from Sweden, Denmark, and Norway.

Methods: In this cohort study 2,937 individuals aged 18-40 years with a recorded diagnosis of cHL in the Swedish lymphoma register (SLR), the Danish lymphoma registry (LYFO), or the clinical database at Oslo University Hospital (OUH) between 1995 and 2019 were included. Information on first-line chemotherapy treatment was available in the registers. Treatment regimens were classified as ABVD, 6-8 BEACOPP, or other. The SLR, LYFO, and OUH databases were linked to national medical birth registers to obtain information on childbirths. Follow-up started 9 months after cHL diagnosis and was accrued until date of first childbirth, death, or administrative censoring (December 2017, 2018, or 2019 for Norway, Sweden, and Denmark, respectively) or after 10 years. Females were additionally censored at date of relapse or stem cell transplantation due to the limited accessibility of assisted reproductive techniques for female patients experiencing one of those events. Stratified Cox models allowing for effect modification between sex and treatment and adjusted for stage, age at diagnosis, year of diagnosis, parity, and performance status were used to estimate hazard ratios (HRs). Flexible parametric survival models were used to estimate marginal childbirth rates and standardized cumulative incidence of childbirth across treatment groups. A comprehensive study analysis plan been published and pre-registered on the Open Science Framework ( https://osf.io/eumy5/ ).

Results: In summary, 75% of HL patients were treated with ABVD and 10.8% with 6-8 BEACOPP. An additional 14.2% received other treatments or had missing treatment information and were excluded from further analyses. The rate of first childbirth per 1,000 person-years for males and females was 45.5 and 50.0 respectively in the ABVD group, and 23.8 and 43.5 in the 6-8 BEACOPP group. The adjusted HR of first childbirths for males and females, comparing patients treated with 6-8 BEACOPP to ABVD were 0.54 (95% CI 0.37-0.79) and 0.92 (95% CI 0.61-1.39) respectively. Plots of the cause-specific cumulative incidence functions (CIF) of childbirth show a constantly lower proportion of male patients with childbirths over time since HL diagnosis comparing patients treated with 6-8 BEACOPP and ABVD. However, this difference was not present among female patients. Adjusted marginal estimates of the cause-specific CIF of childbirth showed the same pattern.

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Summary/Conclusion: BEACOPP treatment is associated with decreased childbearing rates in male but not female HL patients. Infertility counselling for this group is advisable.

S203: ABSCOPAL EFFECT OF RADIOTHERAPY AND NIVOLUMAB IN RELAPSED OR REFRACTORY HODGKIN LYMPHOMA: PRE-PLANNED INTERIM ANALYSIS OF THE INTERNATIONAL GHSG PHASE II AERN TRIAL

P. Bröckelmann 1 2 3 4,* , I. Bühnen 3 , J. Zijlstra 5 , A. Fossa 6 , J. Meissner 7 , S. Mathas 8 , J. Rosenbrock 9 , C. Kobe 10 , M. Fuchs 3 , A. Plütschow 3 , S. Marnitz 9 , P. Borchmann 11 , A. Engert 11 , C. Baues 9

1 Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), University Hospital of Cologne; 2 Max Planck Institute for the Biology of Ageing; 3 German Hodgkin Study Group (GHSG), University of Cologne; 4 Mildred Scheel School of Oncology Aachen Bonn Cologne Düsseldorf (MSSO ABCD), Cologne, Germany; 5 Amsterdam UMC, Department of Hematology, Cancer Center Amsterdam, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 6 Department of Oncology, Oslo University Hospital, Oslo, Norway; 7 Faculty of Medicine and University Hospital Heidelberg, Department V of Internal Medicine, University of Heidelberg, Heidelberg; 8 Department of Hematology, Oncology and Tumor Immunology, Charité – Universitätsmedizin Berlin, and Experimental and Clinical Research Center (ECRC), Berlin; 9 University Hospital Cologne, Department of Radiation Oncology and Cyberknife Center; 10 Department of Nuclear Medicine; 11 Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), University of Cologne, Faculty of Medicine, Cologne, Germany

Background: Failure of anti-PD1 checkpoint blockade in relapsed or refractory Hodgkin lymphoma (r/r HL) constitutes an unmet medical need. It has been postulated that anti-PD1 and localized radiotherapy (RT) may work synergistically to induce a systemic anti-lymphoma effect resulting in distant and systemic (“abscopal”) response outside the RT field.

Aims: We aimed to formally investigate safety and efficacy of nivolumab and RT in r/r HL failing anti-PD1 treatment in a proof-of-concept study.

Methods: AERN is an ongoing international investigator-sponsored two stage phase II trial enrolling adult patients with histologically confirmed r/r HL ( NCT03480334 ). All patients must have failed anti-PD1 treatment by either showing progressive disease (PD) or stable disease (SD) >6 months as best response at the time of enrollment. Presence of two distinct FDG-avid lesions with one eligible for 20Gy localized RT and the other >5cm apart and outside the prescribed 95%isodose for measurement of an abscopal response must be confirmed by the GHSG reference panel. After enrollment patients receive 240mg nivolumab 2-weekly and 20Gy RT in 2Gy fractions is started on day 6 after the first infusion on trial. Treatment is continued until PD or unacceptable toxicity or for a maximum of 1.5 years. The primary endpoint is abscopal response rate (ARR) at first interim restaging after six infusions, secondary endpoints include objective response rate (ORR), progression-free survival (PFS), overall survival and safety. Target enrollment are nine and 20 patients in a Simon’s two-stage desig. A pre-planned interim analysis was conducted after completion of stage I and is reported herein.

Results: Of 11 patients enrolled into stage I of the trial, two terminated trial participation early (screening failure, withdrawal of informed consent) and nine patients at a median age of 34 (range 25-82) years were evaluable for stage I. All patients presented with stage III/IV and a median of 4 (3-15) prior lines of treatment. While all patients had received prior anti-PD1 antibodies (100%), the majority also received brentuximab vedotin (89%) and high-dose chemotherapy plus autologous stem-cell transplantation (ASCT, 56%). Trial treatment was well tolerated. The most frequently reported °1/2 toxicities were those affecting the skin (55%), one case of °3 gastrointestinal and no °4/5 toxicities. Patients received a median of 19 (7-36) nivolumab infusions with a median treatment duration of 9 (3-18) months. At first interim restaging, five patients had an abscopal response (ARR 56%, 95%CI 16.9-74.9%) by central response assessment and 56% achieved a PR (ORR 56%). Most patients experienced a reduction in disease burden at first interim restaging with a mean decrease of SPD -36.3% (0 to -81.2%). Four out of five patients discontinuing treatment experienced PD at the last restaging. With a median observation time of 15.5 months, the 1-year PFS was 42.3% and all patients were alive at data cut off.

Summary/Conclusion: Treatment with nivolumab and 20Gy RT at a single lesion was well tolerated and led to an abscopal response in the majority of patients (56%) previously failing anti-PD1 treatment for r/r HL. An ORR of 56% and 1-year PFS of 42.3% further highlight the potential of this innovative treatment strategy. After meeting the primary efficacy endpoint in this pre-planned interim analysis, AERN trial enrollment is currently continuing internationally.

S204: PEMBROLIZUMAB IN CHILDREN AND YOUNG ADULTS WITH NEWLY DIAGNOSED CLASSICAL HODGKIN LYMPHOMA WITH SLOW EARLY RESPONSE TO FRONTLINE CHEMOTHERAPY: THE PHASE 2, OPEN-LABEL, KEYNOTE-667 STUDY

L. Vinti 1,* , S. Daw 2 , C. Sabado Alvarez 3 , F. Fagioli 4 , A. Beishuizen 5 , G. Michel 6 , M. L. Moleti 7 , M. Cepelova 8 , A. Thorwarth 9 , C. Rigaud 10 , D. Plaza Lopez de Sabando 11 , J. Landman Parker 12 , Y. Zhu 13 , P. Pillai 13 , A. Nahar 13 , C. Mauz-Koerholz 14 15

1 IRCCS Ospedale Pediatrico Bambino, Gesu, Italy; 2 University College London Hospitals NHS Foundation Trust, London, United Kingdom; 3 Hospital Universitari Vall d Hebron, Barcelona, Spain; 4 Ospedale Infantile Regina Margherita, Turin, Italy; 5 Princess Máxima Centrum, Utrecht, Netherlands; 6 CHU de Marseille Hopital de la Timone Enfants, Marseille, France; 7 Universita degli Studi di Roma La Sapienza, Rome, Italy; 8 Fakultni nemocnice v Motole, Prague, Czechia; 9 Charite-Universitaetsmedizin Berlin Campus Virchow-Klinikum, Berlin, Germany; 10 Gustave Roussy Cancer Campus, Villejuif, France; 11 Hospital Universitario La Paz, Madrid, Spain; 12 Hopital d’Enfants Armand Trousseau, Paris, France; 13 Merck Sharp & Dohme, Corp., a subsidiary of Merck & Co., Inc., Kenilworth, United States of America; 14 Justus-Liebig University of Giessen, Giessen; 15 Medical Faculty, Martin-Luther-University of Halle-Wittenberg, Halle, Germany

Background: Patients with classical Hodgkin lymphoma (cHL) with slow early response (SER) to initial chemotherapy (chemo) are at higher risk of relapse, and the burden of late organ toxicities may be higher after dose intensification and radiotherapy (RT). The phase 2, open-label KEYNOTE-667 ( NCT03407144 ) study is evaluating the efficacy and safety of pembrolizumab (pembro) plus chemo in patients with cHL and SER to frontline chemo.

Aims: To present results of an interim analysis of efficacy and safety in patients with high risk cHL (Group 2) who were SER.

Methods: In Group 2, eligible patients aged 3-17 (children) or 18-25 years (young adults) with newly-diagnosed stage IIEB, IIIEA, IIIEB, IIIB, IVA, or IVB cHL were enrolled to receive induction with vincristine, etoposide, prednisone/prednisolone, doxorubicin (OEPA) for 2 cycles. Response assessment after induction (early) and consolidation (late) therapy was done by PET/MRI/CT. After induction treatment, patients with rapid early response (RER) received non-study therapy and patients with SER received consolidation with 4 cycles cyclophosphamide, vincristine, prednisone/prednisolone, dacarbazine (COPDAC-28) plus pembro 2 mg/kg up to 200 mg IV Q3W (3-17 years of age) or 200 mg IV Q3W (18-25 years of age). After consolidation therapy, patients with PET-positivity (Deauville score 4-5) received modified involved-site RT to late PET-positive residua; RT was omitted in patients with PET-negativity. All patients with SER received maintenance pembro Q3W, for a total of 17 doses. Safety analyses included all patients with SER treated with pembro, while efficacy analyses was based on all patients who had completed late response assessment. All patients provided informed consent. The primary endpoint was ORR by BICR per Cheson 2007 IWG criteria in patients with SER. Secondary endpoints included rate of PET-negativity after consolidation, details of RT, and safety. Data cut-off was Nov 22, 2021.

Results: At data cut-off, median (range) follow-up was 9.6 mo (2.5-21.2). A total of 30 patients with high-risk cHL with SER were included. Median (range) age was 15y (6-19), 13 (43%) had bulky disease, 19 (63%) had Ann Arbor stage IV disease. A total of 6 (20%) patients had completed, and 24 (80%) were ongoing on treatment, with median time on treatment of 3.3 mo (range, 0-11.8). Of 30 patients, 25 (83%) had a late response assessment, of which 17 (68%) were PET-negative by BICR; 18 (72%) PET-negative by investigator. All cause AEs occurred in 23 (77%) patients, with 14 (47%) having a treatment-related AE. Grade ≥3 AEs occurred in 6 (20%) patients, with 3 (10%) having an SAE. Grade ≥3 treatment-related AEs occurred in 2 (7%) patients. One (3%) pt had a grade 2 immune-mediated AE of hypothyroidism.

Summary/Conclusion: In pediatric patients with high-risk cHL and SER to standard OEPA induction, pembro in combination with COPDAC 28 consolidation therapy was well tolerated and resulted in 68% of patients having a PET-negative response at end of chemo, and being spared RT. This early data suggests that addition of pembro potentially may have the ability to augment responses in this high-risk subgroup of patients.

S205: ZANUBRUTINIB + OBINUTUZUMAB (ZO) VS OBINUTUZUMAB (O) MONOTHERAPY IN PATIENTS (PTS) WITH RELAPSED OR REFRACTORY (R/R) FOLLICULAR LYMPHOMA (FL): PRIMARY ANALYSIS OF THE PHASE 2 RANDOMIZED ROSEWOOD TRIAL

P. L. Zinzani 1,* , J. Mayer 2 , R. Auer 3 , F. Bijou 4 , A. C. de Oliveira 5 , C. R. Flowers 6 , M. Merli 7 , K. Bouabdallah 8 , P. S. Ganly 9 , Y. Song 10 , H. Zhang 11 , R. Johnson 12 , A. M García-Sancho 13 , M. Provencio 14 , M. Trněný 15 , S. Yuen 16 , H. Tilly 17 , E. Kingsley 18 , G. Tuyman 19 , S. E. Assouline 20 , E. Ivanova 21 , P. Kim 22 , J. Huang 22 , R. Delarue 21 , J. Trotman 23 24

1 Institute of Hematology “Seràgnoli”, University of Bologna, Bologna, Italy; 2 Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital, Brno, Czechia; 3 St. Bartholomew’s Hospital, Barts Health NHS Trust, London, United Kingdom; 4 Institut Bergonié, Bordeaux, France; 5 Institut Catala d’Oncologia (ICO) Hospital Duran I Reynals, Hospital, Barcelona, Spain; 6 Department of Lymphoma/Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America; 7 Hematology, University Hospital “Ospedale di Circolo e Fondazione Macchi” - ASST Sette Laghi, University of Insubria, Varese, Italy; 8 Hôpital Haut-Lévêque, CHU Bordeaux, Pessac, France; 9 Department of Haematology, Christchurch Hospital, Christchurch, New Zealand; 10 Beijing Cancer Hospital, Beijing; 11 Tianjin Medical University Cancer Institute & Hospital, Tianjin, China; 12 St. James’s University Hospital Trust, Leeds, United Kingdom; 13 Hospital Universitario de Salamanca, Salamanca; 14 Hospital Universitario Puerta de Hierro — Majadahonda, Madrid, Spain; 15 Vseobecna fakultní nemocnice v Praze, Prague, Czechia; 16 Calvary Mater Newcastle, Waratah, New South Wales, Australia; 17 Centre Henri-Becquerel, Rouen, France; 18 Comprehensive Cancer Centers of Nevada, Las Vegas, NV, United States of America; 19 Department of Chemotherapy of Hemoblastosis, Blokhin Russian Cancer Research Center, Moscow, Russia; 20 Jewish General Hospital, Montreal, Canada; 21 BeiGene Switzerland GmbH, Basel, Switzerland; 22 BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 23 Concord Repatriation General Hospital; 24 Department of Haemotology, University of Sydney, Concord, New South Wales, Australia

Background: FL is the most common type of indolent non-Hodgkin lymphoma. Approved treatment options are limited for pts with R/R FL. In a phase 1b trial ( Blood Adv . 2020;4(19):4802-4811), ZO was found to be tolerable and associated with early signal of efficacy.

Aims: To present a primary analysis of ROSEWOOD (BGB-3111-212; NCT03332017 ), a phase 2, randomized study designed to assess efficacy and safety of ZO vs O in pts with R/R FL.

Methods: Pts with R/R FL who received ≥2 lines of therapy, including an anti-CD20 antibody and an alkylating agent, were randomized 2:1 to receive either ZO or O. O was given in both arms on Days 1, 8, and 15 of Cycle 1, Day 1 of Cycles 2-6, and then every 8 weeks up to 20 doses maximum. Z (160 mg twice daily) was given until progressive disease (PD) or unacceptable toxicity; pts with confirmed PD or no response within 12 months in the O arm were allowed to crossover to ZO. Primary endpoint was overall response rate (ORR) by independent central review. Secondary endpoints included complete response rate (CRR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Exploratory endpoint included ORR by investigator after crossover. Primary analysis cutoff was October 8, 2021. All pts gave informed consent.

Results: A total of 217 pts were randomized to ZO (n=145) or O (n=72). Median study follow-up was 12.5 mo; median age was 64 yrs. Incidence of high FL International Prognostic Index score was 53% (ZO) and 51% (O). Pts received a median of 3 prior lines of therapy, with 28% (ZO) and 25% (O) of pts receiving >3 lines. Proportion of pts refractory to rituximab, refractory to the most recent line of therapy, or with PD within 24 mo of initiation of first-line therapy was 54%, 32%, and 35% with ZO and 50%, 40%, and 42% with O, respectively. The study met its primary endpoint: ORR was 68.3% with ZO vs 45.8% with O (p=0.0017). CRR was 37.2% (ZO) vs 19.4% (O); 18-mo DOR rate was 70.9% (ZO) vs 54.6% (O); and median PFS was 27.4 mo (ZO) vs 11.2 mo (O; hazard ratio [HR], 0.51 [95% CI, 0.32-0.81], p=0.0040). Median time to new anti-lymphoma therapy or crossover was not evaluable (ZO; NE) vs 12.1 mo (O; HR, 0.37 [95% CI, 0.23-0.60], p<0.0001). ORR for 29 pts who crossed over to ZO was 24.1%. Median OS was NE; 18-mo OS probability was 85.4% (ZO) vs 72.6% (O). Most common any-grade adverse events (AEs) with incidence >10% in the ZO arm were thrombocytopenia (34.3%), neutropenia (27.3%), diarrhea (16.1%), fatigue (14.0%), constipation (13.3%), cough (11.9%), pyrexia (11.2%), and dyspnea (10.5%). Grade ≥3 AEs with incidence >5% with ZO were neutropenia (22.4%) and thrombocytopenia (14.0%); incidence of atrial fibrillation was 0.7% and major hemorrhage was 1.4%. Incidence of treatment-emergent AEs leading to death was 5.6% (ZO) and 9.9% (O).

Summary/Conclusion: ZO demonstrated superior efficacy to O in treatment of pts with R/R FL. ZO had a favorable benefit-risk profile and represents a potential combination therapy for pts with R/R FL.

S206: OBINUTUZUMAB PLUS CHEMOTHERAPY DEMONSTRATES LONG-TERM BENEFIT OVER RITUXIMAB PLUS CHEMOTHERAPY IN PATIENTS WITH PREVIOUSLY UNTREATED FOLLICULAR LYMPHOMA: FINAL ANALYSIS OF THE GALLIUM STUDY

W. Townsend 1,* , W. Hiddemann 2 , C. Buske 3 , G. Cartron 4 , D. Cunningham 5 , M. J. Dyer 6 , J. Gribben 7 , E. Phillips 8 , M. Dreyling 2 , J. F. Seymour 9 , A. Grigg 10 , T.-Y. Lin 11 , X.-N. Hong 12 , D. Kingbiel 13 , T. G. Nielsen 13 , A. Knapp 13 , M. Herold 14 , R. Marcus 15

1 Cancer Research UK and UCL Cancer Trials Centre, University College Hospitals London, London, United Kingdom; 2 Ludwig-Maximilians-University Hospital Munich, Munich; 3 Universtitätsklinikum Ulm, Ulm, Germany; 4 CHU Montpellier, Montpellier, France; 5 Royal Marsden Hospital, Sutton; 6 Ernest and Helen Scott Haematological Research Institute, University of Leicester, Leicester; 7 Barts Cancer Institute, Queen Mary University of London, London; 8 University of Manchester, The Christie Hospital and National Institutes of Health Research Manchester Biomedical Research Centre, Manchester, United Kingdom; 9 Peter MacCallum Cancer Centre and the Royal Melbourne Hospital, Melbourne; 10 Austin Hospital, Austin, Australia; 11 Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, and Collaborative Innovation Center for Cancer Medicine, Guangzhou; 12 Fudan University Shanghai Cancer Center, Shanghai, China; 13 F. Hoffmann-La Roche Ltd, Basel, Switzerland; 14 HELIOS-Klinikum Erfurt, Erfurt, Germany; 15 Kings College Hospital, London, United Kingdom

Background: Immunochemotherapy with rituximab plus chemotherapy (R-chemo) has significantly improved outcomes for patients (pts) with previously untreated follicular lymphoma (FL). However, most pts still experience disease relapse, progression, or death. Obinutuzumab (G) is a type II anti-CD20 monoclonal antibody with enhanced direct cell killing, antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis versus R. The safety and efficacy of G-chemo versus R-chemo was assessed in pts with previously untreated, advanced stage FL in the randomized, Phase III GALLIUM ( NCT01332968 ) study. In the primary analysis, G-chemo demonstrated a significant improvement in progression-free survival (PFS) versus R-chemo, with a manageable safety profile (Marcus, et al. 2017); this efficacy benefit was maintained after 5 years of observation (Townsend, et al. 2020).

Aims: To report the final analysis of the GALLIUM study after a median observation time of 8 years.

Methods: Pts aged ≥18 years with previously untreated histologic Grade 1–3a FL requiring treatment were enrolled. Pts were randomized 1:1 to receive G 1000mg intravenously (IV; Days [D]1, 8 and 15 of Cycle 1 and D1 of subsequent cycles) or R 375mg/m 2 IV (D1 of each cycle) plus chemo for 6 or 8 cycles depending on the chemo backbone selected at each institution (cyclophosphamide, doxorubicin, vincristine, and prednisolone [CHOP]; cyclophosphamide, vincristine, and prednisolone [CVP]; or bendamustine). Pts attaining a complete or partial response received maintenance with the same antibody every 2 months for 2 years or until disease progression (PD). The primary endpoint was investigator-assessed PFS; secondary endpoints included time-to-next anti-lymphoma treatment (TTNLT), overall survival (OS) and incidence of adverse events (AEs). All pts provided written informed consent.

Results: 1202 pts with FL were enrolled (G-chemo, n=601; R-chemo, n=601). As of July 30, 2021, median observation time was 8 years. Seven-year PFS was improved with G-chemo (63.4%) versus R-chemo (55.7%; hazard ratio [HR], 0.77; 95% confidence interval [CI]: 0.64–0.93; p=0.006; Figure ). TTNLT was also improved with G-chemo versus R-chemo (HR, 0.71; 95% CI: 0.58–0.87; p=0.001); the proportion of pts who had not started their next treatment at 7 years was 74.1% and 65.4%, respectively. Disease transformation was observed in 4.2% of pts with G-chemo and 5.0% of pts with R-chemo. Seven-year OS was similar in both arms, 88.5% with G-chemo versus 87.2% with R-chemo (HR, 0.86; 95% CI: 0.63–1.18; p=0.36). Seventy-five pts in the G-chemo arm and 86 pts in the R-chemo arm had died, most commonly due to PD (4.2% and 6.0%, respectively). The incidence of serious AEs (SAEs) was 48.9% with G-chemo (28.2% and 24.4% during induction and maintenance, respectively) and 43.4% with R-chemo (24.6% and 21.7%, respectively). Rates of fatal AEs were similar with G-chemo (4.4%) and R-chemo (4.5%); pneumonia was the most common fatal AE (0.8% [n=5] and 0.2% [n=1], respectively) and SAE (5.9% [n=35] and 6.2% [n=37], respectively). Second malignancies occurred in 13.1% of pts in the G-chemo arm and 9.9% of pts in the R-chemo arm. After adjusting for observation time, rates were similar between arms. These safety findings are consistent with previous analyses.

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Summary/Conclusion: After a median observation time of 8 years, a meaningful improvement in PFS was maintained with G-chemo versus R-chemo in pts with previously untreated FL, confirming the role of G-chemo as a standard of care for the first-line treatment of pts with FL.

S207: EFFICACY AND SAFETY OF A THIRD GENERATION CD20 CART (MB-106) FOR TREATMENT OF RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA (FL)

M. Shadman 1,* , C. Yeung 1 , M. Redman 2 , S. Y. Lee 2 , D. H. Lee 2 , S. Ra 2 , D. Qian 2 , C. Ujjani 1 , B. Dezube 3 , C. Poh 1 , E. H. Warren 1 , A. Chapuis 1 , D. Green 1 , A. Cowan 1 , R. Cassaday 1 , H.-P. Kiem 1 , V. Chow 1 , J. Gauthier 1 , C. Turtle 1 , R. Lynch 1 , S. Smith 1 , A. Gopal 1 , D. Maloney 1 , B. Till 1

1 Fred Hutchinson Cancer Research Center / University of Washington; 2 Fred Hutch, Seattle; 3 Mustang Bio, Worcester, United States of America

Background: Follicular lymphoma (FL) is incurable and novel treatments with high efficacy and low toxicity are needed. Chimeric antigen receptor T-cells (CAR-T) targeting CD19 are effective, and axicabtagene ciloleucel is currently approved by FDA for treatment of patients (pts) with relapsed FL, but toxicities like cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) may limit its use. MB-106 is a fully human 3rd-generation CD20-targeted CAR-T product with both 4-1BB and CD28 costimulatory domains.

Aims: We present the results of the FL cohort from our ongoing phase I/II clinical trial investigating MB-106 for B-cell lymphoma/CLL.

Methods: Pts with R/R B-cell malignancies including FL were eligible after confirmation of CD20 expression. Prior treatment with CD19 CAR-T is permitted. Lymphodepletion (LD) consists of cyclophosphamide (Cy) ± fludarabine (Flu). CAR-T cells are administered at one of 4 dose levels (DL): DL1: 3.3x10 5 , DL2: 1x10 6 , DL3: 3.3x10 6 , DL4: 1x10 7 CAR T cells/kg. A continual reassessment method dose escalation design was used to find the maximally tolerated dose. CAR-T was infused in the outpatient setting except for the first pt of each dose cohort (overnight observation). Initial treatment response is assessed on day 28 and best response is reported here. CRS and ICANS are graded per ASTCT.

Results: Between Dec 2019 and Dec 2021, 16 pts with FL were treated and had day 28 assessment. Median age was 61.5 years (range: 45 – 81). High-risk features included pts with progression of disease within 24 months of first-line chemotherapy (POD24) (n=11; 69%), history of histologic transformation (n=3; 19%), prior treatment with a CD19 targeted CAR-T (n=1; 6%). 6 pts (37.5%) had a PI3 kinase inhibitor. Median time between leukapheresis and LD was 15 days (range: 9-21) and 2 pts received bridging therapy with lenalidomide (1) and high-dose corticosteroids (1). All DLs were reached (DL0=1, DL1=1, DL2=4, DL3=7, DL4=2), with no dose-limiting toxicities. All CRS events were grade 1 (n=4; 25%) or grade 2 (n=1; 6%), with no grade ≥ 3 CRS events. There was no occurrence of ICANS of any grade. No pts had tumor lysis syndrome or Gr 3-4 infections. In the first 28 days, thrombocytopenia (Gr 3-4: 12.5%) and neutropenia (Gr 3-4: 94%) were common but there were no bleeding complications, and the rate of febrile neutropenia was 25%. Overall response (ORR) rate was 94% (15/16) and a complete response (CR) rate was 75% (12/16). Pts who received DL3 or DL4, had an ORR 100% and CR rate of 89%. The single patient who had previously been treated with a CD19 targeted CAR-T achieved a CR. With median follow-up of 10 months, one patient died, from complications of myelodysplastic syndrome (MDS) while still in remission; the MDS was attributed to the prior treatments (7 lines of chemo-based therapy and radioimmunotherapy). Most of the remissions are ongoing and 4 pts have relapsed during the follow-up median 7 months after treatment (range 3-10.5). CAR T cells were detectable by flow cytometry at last available timepoint (up to 2 years) for 15 of 16 patients. One patient had undetectable CAR T cells by day 201.

Summary/Conclusion: Treatment with MB-106, a third generation CD20 targeting CAR-T, resulted in high ORR and CR rates and CAR-T persistence in FL pts and was associated with favorable safety profile with no occurrence of Gr 3 or Gr 4 CRS and no ICANS event of any grade. A multicenter trial for treatment of B-cell malignancies including FL will start enrollment in 2022.

S208: EFFICACY AND SAFETY OF ZANDELISIB ADMINISTERED BY INTERMITTENT DOSING (ID) IN PATIENTS WITH RELAPSED OR REFRACTORY (R/R) FOLLICULAR LYMPHOMA: PRIMARY ANALYSIS OF THE GLOBAL PHASE 2 STUDY TIDAL

A. Zelenetz 1,* , W. Jurczak 2 , V. Ribrag 3 , K. Linton 4 , G. Collins 5 , J. Lopéz-Jiménez 6 , N. Reddy 7 , A. Mengarelli 8 , T. Phillips 9 , G. Musuraca 10 , O. Sheehy 11 , J. Li 12 , W. Xu 12 , M. Azoulay 13 , R. Ghalie 12 , P. L. Zinzani 14

1 Memorial Sloan Kettering Cancer Institute, New York City, United States of America; 2 Maria Sklodowska Curie National Research Institute of Oncology, Krakow, Poland; 3 Institut Gustave Roussy, Villejuif, France; 4 Manchester Cancer Research Centre, Manchester; 5 GenesisCare, Oxford, United Kingdom; 6 Hospital Universitario Ramon y Cajal, Madrid, Spain; 7 Vanderbilt University, Nashville, United States of America; 8 Regina Elena National Cancer Institute, Roma, Italy; 9 University of Michigan Health System, Ann Arbor, United States of America; 10 Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori I.R.S.T., Meldola, Italy; 11 Belfast Health and Social Care Trust, Belfast, United Kingdom; 12 MEI Pharma, Inc., San Diego; 13 Kyowa Kirin Co., Princeton, United States of America; 14 University of Bologna, Bologna, Italy

Background: Zandelisib, a PI3Kδ inhibitor with high target-binding affinity, is administered by intermittent dosing (ID) on days 1 to 7 of 28-day cycles to potentially enable regulatory T-cell repopulation and lower the risk of immune-related adverse events (irAEs) associated with continuous PI3Kδ inhibition. In a phase 1b study in 37 patients (pts) with R/R FL, zandelisib administered daily for two 28-day cycles for tumor debulking then on ID achieved an overall response rate (ORR) of 87% (78% as single agent and 95% with rituximab), a median duration of response (DOR) not reached at median follow-up of 16.9 months, and a low rate (<10%) of grade 3 irAEs (Pagel et al. ICML 2021; #113).

Aims: We conducted the TIDAL study ( NCT03768505 ) to further evaluate in a global trial the efficacy and safety of zandelisib in larger group of pts with R/R FL and marginal zone lymphoma (MZL). The MZL cohort is still enrolling and not reported here.

Methods: Eligible pts ≥18 years with FL Grade I-IIIA, ECOG performance status 0-1, progressive disease after ≥2 prior therapies including an anti-CD20 antibody and chemotherapy, and no prior PI3Kδ inhibitor, provided consent and then received zandelisib 60 mg daily for two 28-day cycles then on ID. Another study arm evaluating zandelisib 60 mg daily continuously was closed to enrollment early and is not reported here. The planned sample size in FL was 120 pts on ID, with the primary efficacy population (PEP) pre-defined as the first 91 pts treated and the safety population consisting of all FL pts treated. The primary efficacy endpoint was ORR as assessed by independent review using the Lugano criteria and analyzed 6 months after completing enrollment in the PEP.

Results: 121 FL pts were enrolled. In the PEP (N=91 pts), the median number of prior therapies was 3 (range 2-8), 21 pts (23%) have received prior stem cell transplant, 42 pts (46%) were refractory to last therapy, 31 pts (34%) had tumors ≥5 cm, and 51 pts (56%) were POD24. The ORR was 70.3% (N=64) (95% CI 59.8-79.5%), with 32 pts (35.2%) achieving a complete response (CR). Responses occurred early, with 85.5% (N=56) achieved in the first 2 cycles of therapy and 75% of CRs (N=24) achieved the first 4 cycles. The data are still immature to estimate accurately the DOR. With a median follow-up of 9.4 months (range 0.8-24) in the safety population of 121 pts, 12 pts (9.9%) discontinued therapy due to any drug-related AE. Grade 3 AEs of special interest (AESI) were diarrhea in 6 pts (5%), colitis in 2 (1.7%), cutaneous rash in 4 (3.3%), stomatitis in 3 (2.5%), and 1 (0.8%) each for AST and ALT elevation, and non-infectious pneumonitis. Grade 3 AESIs primarily (15 of 18, 83%) occurred in cycles 1-3, during daily dosing, with only 3 cases reported on ID in Cycles ≥4.

Summary/Conclusion: Zandelisib on ID achieved high ORR (70.3%) and CR rate (35.2%) in heavily pretreated R/R FL pts, and was associated with a low rate (<10%) of grade 3 AESI and discontinuations due to AEs, results comparable to the Phase 1b study. Longer follow-up is needed to estimate median DOR. This profile supports evaluation of zandelisib as a single agent and in combination regimens in various B-cell malignancies, both in R/R disease and in earlier lines of therapy. Zandelisib plus rituximab vs chemoimmunotherapy is being evaluated in the phase 3 study COASTAL in R/R FL and MZL ( NCT04745832 ).

S209: PRIMARY RESULTS FROM THE PHASE 3 SHINE STUDY OF IBRUTINIB IN COMBINATION WITH BENDAMUSTINE-RITUXIMAB (BR) AND R MAINTENANCE AS A FIRST-LINE TREATMENT FOR OLDER PATIENTS WITH MANTLE-CELL LYMPHOMA

M. L. Wang 1,* , W. Jurczak 2 , M. Jerkeman 3 , J. Trotman 4 , P. L. Zinani 5 , D. Belada 6 , C. Boccomini 7 , I. W. Flinn 8 , P. Giri 9 , A. Goy 10 , P. A. Hamlin 11 , O. Hermine 12 , J.-Á. Hernández-Rivas 13 , X. Hong 14 , S. J. Kim 15 , D. Lewis 16 , Y. Mishima 17 , M. Özcan 18 , G. F. Perini 19 , C. Pocock 20 , Y. Song 21 , S. E. Spurgeon 22 , J. M. Storring 23 , J. Walewski 24 , J. Zhu 21 , R. Qin 25 , T. Henninger 25 , S. Deshpande 25 , A. Howes 25 , S. Le Gouill 26 , M. Dreyling 27

1 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2 Maria Sklodowska-Curie National Research Institute of Oncology, Karkow, Poland; 3 Skane University Hospital and Lund University, Lund, Sweden; 4 Concord Repatriation General Hospital, University of Sydney, Sydney, Australia; 5 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale Università di Bologna, Bologna, Italy; 64 th Department of Internal Medicine - Haematology, Charles University, Hospital and Faculty of Medicine, Hradec Králové, Czechia; 7 SC Ematologia, AOU Città della Salute e della Scienza di Torino - Presidio Molinette, Torino, Italy; 8 Sarah Cannon Research Institute and Tennessee Oncology, Nashville, United States of America; 9 Royal Adelaide Hospital, Adelaide, Australia; 10 John Theurer Cancer Center, Hackensack; 11 Memorial Sloan Kettering Cancer Center, New York, United States of America; 12 Department of Hematology, Hôpital Necker, Assistance Publique - Hôpitaux de Paris and Institut Imagine, Université de Paris, INSERM UMR1183, Paris, France; 13 Department of Hematology, Hospital Universitario Infanta Leonor, Universidad Complutense, Madrid, Spain; 14 Fudan University Shanghai Cancer Center, Shanghai, China; 15 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 16 University Hospitals Plymouth NHS Trust, Plymouth, United Kingdom; 17 Department of Hematology Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research, Toyo, Japan; 18 Ankara University School of Medicine, Ankara, Turkey; 19 Hospital Israelita Albert Einstein, Sao Paulo, Brazil; 20 Kent and Canterbury Hospital, Canterbury, United Kingdom; 21 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Lymphoma, Peking University Cancer Hospital & Institute (Beijing Cancer Hospital), Beijing, China; 22 Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, United States of America; 23 The Research Institute of the McGill University Health Centre, McGill University, Montreal, Canada; 24 Maria Sklodowska-Curie National Research Institute of Oncology, Warszawa, Poland; 25 Janssen Research & Development, Raritan, United States of America; 26 Institut Curie comprehensive cancer center, Paris, France; Hospitalier Universitaire de Nantes, Centre de Recherche en Cancérologie et Immunologie Nantes Angers, INSERM, Université de Nantes, Nantes, France; 27 Department of Medicine III, Klinikum der Universität München, LMU, Munich, Germany

Background: Elderly patients (pts) with mantle-cell lymphoma (MCL) are unsuitable for intensive chemotherapy or transplantation due to excessive toxicities. Single-agent ibrutinib (Ibr), a first-in-class, oral Bruton’s tyrosine kinase inhibitor (BTKi), has transformed the care of pts with relapsed or refractory MCL with durable activity.

Aims: We conducted a phase III trial (SHINE; NCT01776840 ) to evaluate combining Ibr with a standard chemoimmunotherapy (BR) and R maintenance in older pts with untreated MCL.

Methods: Pts aged ≥ 65 years, enrolled between May 2013 and November 2014 from 183 sites across all geographical regions, were stratified by simplified MIPI score (low vs intermediate vs high risk) and were randomized 1:1 to Ibr (560 mg orally daily) or placebo (Pbo), plus 6 cycles of B (90 mg/m 2 ) and R (375 mg/m 2 ). Pts who achieved an objective response received R maintenance, administered every 8 weeks for up to 12 additional doses in both arms. Ibr and Pbo were administered until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS) assessed by the investigators.

Results: A total of 523 pts were randomized to Ibr + BR (n = 261) or Pbo + BR (n = 262). Median age was 71 years (range, 65–87), 65.6% of pts had low/intermediate simplified MIPI, and 8.6% had blastoid/pleiomorphic histology. At the primary analysis, median follow up was 84.7 months. The primary endpoint was met as PFS was significantly improved in the Ibr arm vs the Pbo arm (hazard ratio, 0.75; one-sided P = 0.011) (Figure). Median PFS was 80.6 months with Ibr in combination with BR and R maintenance, a 50% improvement over Pbo in combination with BR and R maintenance (median PFS of 52.9 months). The complete response rate was 65.5% in the Ibr arm and 57.6% in the Pbo arm ( P = 0.0567). There was no difference in overall survival between treatment arms ( P = 0.648). Time to next treatment was longer in the Ibr arm compared with the Pbo arm ( P < 0.001). Fifty-two (19.9%) and 106 (40.5%) pts received subsequent anti-lymphoma therapy in the Ibr and Pbo arms, respectively; 41/106 (38.7%) received a second-line BTKi in the Pbo arm.

Rates of grade 3 or 4 treatment-emergent adverse events were 81.5% and 77.3% in the Ibr and Pbo arms, respectively. Of adverse events of clinical interest for BTKis, atrial fibrillation was reported in 13.9% and 6.5% of pts in the Ibr and Pbo arms, respectively. Rates of major hemorrhage, hypertension, arthralgia, and secondary primary malignancies were similar in both arms. Quality of life was also similar in both arms.

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Summary/Conclusion: This phase III study in untreated MCL demonstrated that Ibr combined with BR and R maintenance significantly improved PFS compared with standard chemoimmunotherapy, with a median PFS of 6.7 years. The safety profile was consistent with the known profiles of the individual drugs.

S210: CAR T-CELLS ASSOCIATED ACUTE TOXICITY IN B-CELL NON-HODGKIN LYMPHOMA: REAL-WORLD STUDY FROM THE DESCAR-T REGISTRY

P. SESQUES 1,* , R. DI BLASI 2 , S. LE GOUILL 3 , G. CARTRON 4 , G. MANSON 5 , D. BEAUVAIS 6 , F. LE BRAS 7 , F. X. GROS 8 , S. CHOQUET 9 , P. BORIES 10 , M. T. RUBIO 11 , R. O. CASASNOVAS 12 , L. BOUNAIX 13 , M. MOHTY 14 , M. JORIS 15 , J. ABRAHAM 16 , C. CASTILLA LLORENTE 17 , M. LOSCHI 18 , S. CARRAS 19 , A. CHAUCHET 20 , L. DRIEU LA ROCHELLE 21 , J. ZERBIT 22 , O. HERMINE 23 , S. GUIDEZ 24 , T. GASTINNE 25 , J. J. TUDESQ 26 , P. FOGARTY 27 , F. BROUSSAIS 28 , F. MORSCHHAUSER 29 , R. HOUOT 30 , C. THIEBLEMONT 31 , E. BACHY 1

1 CHU LYON SUD, LYON; 2 Hematology, Assistance Publique Hôpitaux de Paris; 3 Hematology, INSTITUT CURIE, PARIS; 4 HEMATOLOGY, Département d’Hématologie clinique, CHU de Montpellier, MONTPELLIER; 5 CHU de Rennes, RENNES; 6 Hématologie clinique, CHU de Lille, LILLE; 7 Lymphoid Malignancies, CRETEIL; 8 Hématologie Clinique et Thérapie cellulaire, MERIGNAC; 9 APHP la Pitié Salpêtriere, PARIS; 10 Hematology Laboratory, Onco-occitanie Network, TOULOUSE; 11 Service Hématologie, NANCY; 12 Department of Hematology, DIJON; 13 Department of Hematology, Clermont-Ferrand University Hospital, CLEMRONT FERRAND; 14 Hôpital St Antoine, PARIS; 15 Hematology department, CHU Amiens, AMIENS; 16 CHU DE LIMOGES - HOPITAL DUPUYTREN, LIMOGES; 17 GUSTAVE ROUSSY CANCER CAMPUS GRAND PARIS, VILLEJUIF; 18 CHU NICE, NICE; 19 CHU DE GRENOBLE, GRENOBLE; 20 CHU JEAN MINJOZ, BESANCON; 21 CHU BRETONNEAU, TOURS; 22 APHP HOPITAL COCHIN; 23 APHP HOPITAL NECKER, PARIS; 24 CHU DE POITIERS - HOPITAL DE LA MILETRIE, POITIERS; 25 Nantes University Hospital Clinical Hematology, NANTES; 26 Hematology Department, Montpellier University Hospital, Univ Montpellier, MONTPELLIER; 27 biostatistics; 28 LYSARC, LYON; 29 Maladies du Sang CHRU Lille, LILLE; 30 CHU de Rennes, Université de Rennes, INSERM U1236, EFS, RENNES; 31 Saint Louis Hospital, PARIS, France

Background: Cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) are common immune-related toxicities associated with chimeric antigen receptor (CAR)–T-cell therapy. Their clinical manifestations can be severe and potentially life threatening.

Aims: Here, we report the French experience of CAR-T toxicity, we specifically addressed the modifiable risk factors for toxicity and we assessed management toxicity in real-world population.

Methods: We conducted a study in a large cohort of R/R aggressive B-cell lymphoma patients (pts) treated with commercial products. All data were collected through the French DESCAR-T registry. CRS/ICANS were graded prospectively (ASTCT grade scale). Regarding previously validated predictive score of CRS and ICANS in the literature, EASIX score (LDHxCreatinine/Platelets), modified-EASIX score (m-EASIX: CRPxCreatinine/Platelets) and simplified-EASIX score (s-EASIX: LDH/Platelets) (Pennisi et al., 2021) were assessed in our cohort.

Results: A total of 705 pts were included for the analysis of toxicity with a median follow-up of 12 months (range: 0.2-39). Notably, 74 pts (11%) pts had an ECOG/PS ≥ 2 before lymphodepletion. CRS of any grade occurred in 587 pts (83.3%) including 62 (10.5%) with grade 3 or higher (grade 3+). The median time from the infusion to the onset of CRS was 2 days (range: 0-34); the median time to resolution was 6 days (range: 1-30). ICANS of any grade occurred in 289 pts (41%) including 78 (27%) with grade 3+. The median time from the infusion to the onset was 6 days (range: 0-379); the median time to resolution was 7 days (range: 1-100). Most patients (94.5%) with ICANS had previously experienced CRS. Only 89 pts (29.9%) required admission to intensive care unit. Tocilizumab was the most common treatment for toxicity (411 pts, 68%) and 272 pts (45%) received corticosteroids. Interestingly, 38% of patients with CRS grade 1, 53 % with grade 2 and 65% with grade 3+ developed ICANS subsequently.

In multivariate analysis, the parameters which retained statistical significance to predict any CRS were Axi-cel ([OR] = 2.79, 95% CI 1.75-4.45; p <0.0001) and elevated LDH (>upper limit normal; [OR] = 1.69, 95% CI 1.03-1.63; p = 0.03). Bulky mass > 5 cm ([OR] = 2.95, 95% CI 1.60-5.45; p = 0.0005), age <65 years ([OR] = 0.42, 95% CI 0.20-0.86; p = 0.01) and higher s-EASIX score ([OR] = 1.38, 95% CI 1.15-1.67; p = 0.0005) increased the risk of developing grade 3+ CRS. Parameters which retained statistical significance to predict any ICANS were Axi-cel ([OR] = 3.95, 95% CI 2.57-6.06; p <0.0001), ECOG ≥2 ([OR] = 2.10, 95% CI 1.26-3.51; p = 0.004), age ≥65 ([OR] = 3.17, 95% CI 2.10-4.79; p <0.0001) and higher s-EASIX score ([OR] = 1.20, 95% CI 1.03-1.40; p = 0.01). Axi-cel ([OR] = 13.1, 95% CI 3.98-43.3; p <0.001), ECOG ≥2 ([OR] = 0.04, 95% CI 1.007-4.38; p = 0.04) and higher s-EASIX score ([OR] = 1.35, 95% CI 1.08-1.67; p = 0.0067) increased the risk of developing grade 3+ ICANS. No survival impact (OS and PFS) was detected for pts who experimented any grade or grade 3+ of ICANS or CRS (Figure 1). Importantly, cumulative dose and duration of tocilizumab or corticosteroids were not significantly associated with adverse PFS or OS.

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Summary/Conclusion: In this large study, robust biological and clinical predictive factors of CRS and ICANS were identified. A propensity-score matched comparison of axi-cel and tisa-cel for toxicities will be performed for the EHA congress. Neither grade 3+ CRS, ICANS, nor Tocilizumab or corticosteroids use for CAR-T toxicity had negative impact on survival.

S211: CLINICAL AND PATIENT-REPORTED OUTCOMES IN A PHASE 3 STUDY OF AXICABTAGENE CILOLEUCEL (AXI-CEL) VS STANDARD-OF-CARE IN ELDERLY PATIENTS WITH RELAPSED/REFRACTORY LARGE B-CELL LYMPHOMA (ZUMA-7)

A. Sureda 1,* , J. Westin 2 , F. L. Locke 3 , M. Dickinson 4 , A. Ghobadi 5 , M. Elsawy 6 , T. van Meerten 7 , D. B. Miklos 8 , M. Ulrickson 9 , M.-A. Perales 10 , U. Farooq 11 , L. Wannesson 12 , L. Leslie 13 , M. J. Kersten 14 , C. A. Jacobson 15 , J. M. Pagel 16 , G. Wulf 17 , P. Johnston 18 , A. P. Rapoport 19 , L. I. Gordon 20 , Y. Yang 21 , A. Peng 21 , L. Du 21 , J. T. Snider 21 , J. Shah 21 , M. Schupp 21 , P. Cheng 21 , C. To 21 , O. O. Oluwole 22

1 Hematology Department, Institut Català d’Oncologia-Hospitalet, IDIBELL, Universitat de Barcelona, Barcelona, Spain; 2 The University of Texas MD Anderson Cancer Center, Houston; 3 Moffitt Cancer Center, Tampa, United States of America; 4 Peter MacCallum Cancer Centre, Royal Melbourne Hospital and the University of Melbourne, Melbourne, Victoria, Australia; 5 Washington University School of Medicine, St Louis, United States of America; 6 Division of Hematology, Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; 7 University Medical Center Groningen, on behalf of HOVON/LLPC, Groningen, Netherlands; 8 Stanford University School of Medicine, Stanford; 9 Banner MD Anderson Cancer Center, Gilbert; 10 Memorial Sloan-Kettering Cancer Center, New York; 11 University of Iowa, Iowa City, United States of America; 12 Istituto Oncologico della Svizzera Italiana, Bellinzona, Switzerland; 13 John Theurer Cancer Center, Hackensack, United States of America; 14 Amsterdam UMC, University of Amsterdam, on behalf of HOVON/LLPC, Amsterdam, Netherlands; 15 Dana-Farber Cancer Institute, Boston; 16 Swedish Cancer Institute, Seattle, United States of America; 17 University Medicine Göttingen, Göttingen, Germany; 18 Mayo Clinic, Rochester; 19 University of Maryland School of Medicine and Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore; 20 Northwestern University Feinberg School of Medicine, Chicago; 21 Kite, a Gilead Company, Santa Monica; 22 Vanderbilt University Cancer Center, Nashville, United States of America

Background: The median age at large B-cell lymphoma (LBCL) diagnosis is 66 years, and older patients with relapsed/refractory (R/R) LBCL are at risk of inferior outcomes, increased toxicity, and inability to tolerate second-line standard-of-care (SOC) treatment (Di M, et al. Oncologist. 2021). Further, second-line SOC treatment is often associated with poor health-related quality of life (Lin V, et al. J Clin Oncol . 2020;38:e20070). In the global Phase 3, randomized ZUMA-7 study, axi-cel, an autologous anti-CD19 CAR T-cell therapy, significantly improved event-free survival (EFS; hazard ratio [HR], 0.398, P<0.0001; median 8.3 vs 2 months, respectively) compared with second-line SOC in R/R LBCL (Locke FL, et al. N Engl J Med. 2022;386:640-654).

Aims: Here we report results of a planned subgroup analysis of the ZUMA-7 study assessing outcomes, including patient-reported outcomes (PROs), of second-line axi-cel vs SOC in patients aged ≥65 years.

Methods: Patients with ECOG performance status 0-1 and R/R LBCL ≤12 months after first-line chemoimmunotherapy were randomized 1:1 to axi-cel or SOC (2-3 cycles of platinum-based chemoimmunotherapy; patients with partial or complete response [CR] proceeded to high-dose therapy with autologous stem cell transplantation). PRO instruments, including the EORTC QLQ-C30 (Global Health and Physical Functioning) and the EQ-5D-5L visual analog scale (VAS), were administered at baseline (prior to treatment), Day 50, Day 100, Day 150, and Month 9, then every 3 months up to 24 months or time of EFS event, whichever occurred first. The quality-of-life analysis set included all patients who had a baseline PRO and ≥1 completed measure at Day 50, 100, or 150. A clinically meaningful change was defined as 10 points for each EORTC QLQ-C30 score and 7 points for EQ-5D-5L VAS score.

Results: The data cutoff for this analysis was March 18, 2021 and included 51 axi-cel and 58 SOC patients with median ages of 70 years (range, 65-80) and 69 years (range, 65-81), respectively. At baseline, more axi-cel vs SOC patients had high-risk features, including second-line age-adjusted International Prognostic Index 2-3 (53% vs 31%) and elevated lactate dehydrogenase (61% vs 41%). EFS was superior with axi-cel vs SOC (HR, 0.276, P <0.0001), with higher CR rates (75% vs 33%). Grade ≥3 treatment-emergent adverse events (AEs) occurred in 94% and 82% of axi-cel and SOC patients, respectively, and Grade 5 treatment-related AEs occurred in 0 and 1 patient. In the quality-of-life analysis set comprising 46 axi-cel and 42 SOC patients, there were statistically significant and clinically meaningful differences in mean change of scores from baseline at Day 100 favoring axi-cel for EORTC QLQ-C30 Global Health ( P <0.0001) and Physical Functioning ( P =0.0019) and EQ-5D-5L VAS ( P <0.0001). For all 3 domains, scores also favored ( P <0.05) axi-cel over SOC at Day 150. The mean estimated scores numerically returned to or exceeded baseline scores earlier in the axi-cel arm (by Day 150) but never equaled or exceeded baseline scores by Month 15 in the SOC arm.

Summary/Conclusion: Axi-cel demonstrated superiority over second-line SOC in patients ≥65 years with significantly improved EFS and a manageable safety profile. Axi-cel also showed meaningful improvement in quality of life over SOC, measured by multiple validated PRO instruments, with suggested faster recovery to pretreatment quality of life. The superior clinical outcomes and patient experience with axi-cel over SOC should help inform treatment choices in second-line R/R LBCL for patients ≥65 years.

S212: PHASE I STUDY OF YTB323, A CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELL THERAPY MANUFACTURED USING T-CHARGE™, IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA

M. Dickinson 1,* , M. Kwon 2 , J. Briones 3 , U. Jäger 4 , K. Kato 5 , E. Bachy 6 , D. Blaise 7 , N. Boissel 8 , N. Shah 9 , M Frigault 10 , P. Riedell 11 , L. Shune 12 , T. Teshima 13 , X. Zhu 14 , E. Orlando 14 , L. Yi 15 , J. Davis 16 , E. Bleickardt 16 , I. Flinn 17 , P. Barba 18 , P. Barba 18

1 Peter MacCallum Cancer Centre and Royal Melbourne Hospital and the University of Melbourne, Melbourne, Australia; 2 Department of Hematology, Hospital General Universitario Gregorio Marañón, Institute of Health Research Gregorio Marañón, Madrid; 3 Hematology Department, Hospital Santa Creu i Sant Pau, Barcelona, Spain; 4 Clinical Division of Hematology and Hemostaseology, Department of Medicine I, Vienna General Hospital – Medical University of Vienna, Vienna, Austria; 5 Department of Hematology, Oncology and Cardiovascular Medicine, Kyushu University Hospital, Fukuoka, Japan; 6 Hematology department, Hospices Civils de Lyon and Université Claude Bernard Lyon; 1 , Lyon; 7 Département d’Hématologie, Programme de Transplantation et de Thérapie Cellulaire, Centre de Recherche en Cancérologie de Marseille, Aix-Marseille University, Institut Paoli Calmettes, Marseille; 8 Hematology Adolescent and Young Adult Unit, Saint-Louis Hospital, Paris, France; 9 Medical College of Wisconsin, Milwaukee; 10 Massachusetts General Hospital, Massachusetts; 11 University of Chicago, Chicago; 12 Division of Hematologic Malignancies and Cellular Therapeutics, University of Kansas Medical Center, Kansas City, United States of America; 13 Department of Hematology, Hokkaido University Hospital, Sapporo, Japan; 14 Novartis Institutes for BioMedical Research; 15 Novartis Pharmaceuticals Corporation, Cambridge; 16 Novartis Pharmaceuticals Corporation, East Hanover; 17 Sarah Cannon Research Institute and Tennessee Oncology, Nashville, United States of America; 18 Hematology Department, Hospital Universitari Vall d’Hebrón, Universitat Autònoma de Barcelona, Barcelona, Spain

Background: CD19-directed CAR-T cell therapies (tx) demonstrate efficacy in patients (pts) with B-cell malignancies. However, despite positive outcomes with currently approved CAR-T cell tx, many patients fail to respond or relapse after initial response. YTB323 is an autologous CD19-directed CAR-T cell tx generated by the innovative T-Charge™ platform, which demonstrates high potency, preserves T-cell stemness in the final product, and takes <2 d to manufacture.

Aims: T-Charge™ is expected to prolong CAR-T cell persistence and yield higher response rates and durability.

Methods: These data from the ongoing Phase I, multicenter, dose-escalation study ( NCT03960840 ) focus on safety and efficacy of YTB323 in adults with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Eligible pts had measurable disease at enrollment, ECOG 0-1, and r/r DLBCL after ≥2 lines of prior tx. Pts received single-dose YTB323 at targeted dose levels (DL) 1 (2.5×10 6 CAR+ cells), DL2 (12.5×10 6 CAR+ cells), DL3 (25×10 6 CAR+ cells), or DL4 (40×10 6 CAR+ cells). Results presented here focus on DL1 and DL2. The primary endpoints are to identify a recommended dose for subsequent trials and characterize the safety and dose-limiting toxicities. Secondary endpoints include overall response rate (ORR) by local investigator assessment and cellular kinetics.

Results: As of August 20, 2021, 20 pts with r/r DLBCL received YTB323: 4 at DL1 and 16 at DL2. Median age was 65 y, 65% received 2 prior lines of tx, and 35% had prior SCT. The median follow-up for pts in DL1 and DL2 was 20 and 7 months, respectively. Of 20 pts evaluable for safety, all experienced at least 1 adverse event (AE) of any Grade (Gr) and 80% at least 1 AE Gr ≥3. One pt (25%) experienced Gr 1 cytokine release syndrome (CRS) at DL1, and 5 (31%) pts experienced CRS at DL2, including 4 (25%) Gr 1 or 2 and 1 (6%) Gr 4 (Lee et al, 2014), which met protocol defined DLT criteria at DL2. Tocilizumab and corticosteroids were used for CRS management in 4 (80%) and 2 (40%) pts at DL2, respectively. Median time to CRS onset was 10 d (range, 1-17 d). Five pts (25%) had neurological events (NE), of which the majority were Gr 1. Median time to onset and resolution of NE was 7 and 12 d, respectively. Five pts died on trial (beyond 30 d); 3 pts died due to disease progression (1 at DL1, 2 at DL2), and 1 pt each due to sepsis (1 at DL1) and intestinal hemorrhage (1 at DL2). At DL1, ORR and complete response (CR) rate were both 75%. At DL2, ORR and CR rates were 81% and 75%, respectively. Of the 15 pts who received YTB323 at DL2 at least 3 mo prior to the data cutoff, CR rate at mo 3 was 73% (95% CI, 44.9%-92.2%) ( Figure ). Median time to peak YTB323 expansion was ~16 d and coincided with cytokine peak. YTB323 expansion (C max and AUC 0-28d ) with 12.5×10 6 CAR+ cells (DL2) was comparable to a median tisagenlecleucel dose of 312×10 6 CAR+ cells in pts with DLBCL, a 25-fold lower median dose (Awasthi R, et al. 2020). Limited data indicate that CAR expression was detectable by flow cytometry for at least 9 mo at DL2. T-Charge™ allowed preservation of CD4 and CD8 naive/stem memory T cells in the final product according to flow cytometry. Bulk RNAseq analysis demonstrated that YTB323 retained a naive stem-like gene signature.

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Summary/Conclusion: YTB323 is a potent new CAR-T cell tx with distinct cellular kinetics, encouraging early efficacy results across DL1 and DL2, and a manageable safety profile. Updated results will be presented at the meeting along with a recommended dose for subsequent trials.

S213: EFFICACY AND SAFETY OF HUMANIZED VERSUS MURINIZED CD19 AND CD22 CAR-T CELL COCKTAIL THERAPY FOR REFRACTORY/RELAPSED B-CELL LYMPHOMA

L. Huang 1 2 , J. Li 2 , J. Yang 2 , X. Zhang 2 , M. Zhang 2 , J. He 2 , G. Zhang 2 , Y. Su 2 , W. Li 2 , H. Wang 2 , P. Lu 1 2,*

1 Tsinghua University School of Medicine-Lu Daopei Institute of Hematology, Beijing; 2 Hebei Yanda Lu Daopei Hospital,., langfang, China

Background: CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has demonstrated about 50~60% of response rate in relapsed or refractory (R/R) B-cell non-Hodgkin lymphoma. However, antigen-escape relapse has emerged as a major challenge for long-term disease control after CD19-targeted therapies. In our previous study, we described a dual-targeted of CD19 and CD22 CAR-T cell cocktail therapy, whichyielded 93.3% of complete remission (CR) rate in R/R B-cell acute lymphoblastic leukemia and could reduce the risk of CD19-negative relapse.

Aims: A phase I/II clinical trial ( NCT05206071 ) was designed to explore the safety and efficacy of CAR19/22 T-cell cocktail therapy for patients with R/R aggressive B-cell lymphoma.

Methods: Eligible R/R aggressive B-cell lymphoma patients were enrolled from July 2020 toDecember 2021. All patients received fludarabine 30 mg/m2/d and cyclophosphamide 250 mg/m2/d for 3 consecutive days (day −5 to day −3) followed by cocktail CAR-T cell infusion. CAR-T-cell-related cytokine release syndrome (CRS) was graded to assess the safety. PET-CT was performed on day 28 and 3rd month to evaluate the response rate.

Results: A total of 26 eligible patients received CAR19/22 cocktail infusion with a median age of 46 years old (4-75) and a median of 3 prior lines of therapies (2-5 lines). Of 26 patients, 4 were Burkitt lymphoma, 2 were follicular lymphoma (grade 3a), and the rest were diffuse large B-cell lymphoma. Besides, 7 patients were diagnosed with double-hit lymphoma and 4 patients were relapsed after previous CD19 CAR-T cell therapy.

Fourteen patients received the murinized CAR-T cells at a median dose of 2.18×10 6 /kg (1-3×10 6 /kg) CAR19 and 1.75×10 6 /kg (1-3×10 6 /kg) CAR22, while 12 patients received the does of the humanized CAR-T cells consisting of 2×10 6/ kg CAR19 and 5×10 5 /kg CAR22.

At the day 28 assessment, 12/12 (100%) patients received the humanized CAR-T cells achieved overall response, including 9/12 (75%) CR and 3/12 (25%) partial remission (PR), while the overall response rate (ORR) and CR rate of the murinized group was 85.71% (12/14) and 42.86% (6/14) respectively. Among of them, all 4 Burkitt lymphoma patients in both groups achieved CR. In 3 months evaluation, 7/10 (70%,2 cases received infusion for less than 3 months) patients in the humanized group maintained in CR, which was higher than the murinized group with CR rate of 5/14 (35.71%). (Figure 1A-B). At a median follow-up of 291 days (range, 31 to 544), patients in the humanized group had a favorable progression-free survival (PFS) than those in the murinized (1-year PFS of 67.9% vs. 26.8%), although there was no statistical significance (p=0.08) due to the limited number of patients enrolled (Figure 1E-F). Achieving CR on day 28 (HR: 0.21, 95% CI: 0.06-0.72; P=0.012) and maintaining CR till the 3rd month (HR: 0.18, 95% CI: 0.06-0.59; P=0.004) were found as independent prognostic factors associated with favorable PFS. Maintaining CR till the 3rd month (HR: 0.10, 95% CI: 0.01-0.61; P=0.012) and non-double hit (HR: 0.11, 95% CI: 0.02-0.76, P=0.024) predicted a longer overall survival (OS) (Figure 1G-L). Despite impressive CR rates wereachieved, majority of patients had mild CRS (grade 1-2) in both group. Only 2patients experienced grade 3 CRS, and 2 patients developed grade 3 neurotoxicity.

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Summary/Conclusion: This clinical trial demonstrates promising efficacy and safety of CD19/CD22 CAR-T cocktail therapy for R/R aggressive B-cell lymphoma, and patients in the humanized group showed better results than those with murinized, although this not a randomized trial.

S214: PHASE 1/2 STUDY OF ANBAL-CEL, NOVEL ANTI-CD19 CAR-T THERAPY WITH DUAL SILENCING OF PD-1 AND TIGIT IN RELAPSED OR REFRACTORY LARGE B CELL LYMPHOMA

W. S. Kim 1,* , S. J. Kim 1 , S. E. Yoon 1 , J. R. Kim 2

1 Hematology and Oncology, Samsung Medical Center, Seoul; 2 Clinical Development, Curocell Therapeutics, DaeJeon, South Korea

Background: Anbal-cel is a novel 2 nd generation autologous CD19 CAR-T cell therapy which has been knock-downed for PD-1 and TIGIT using OVIS platform. Anbal-cel demonstrated the eradication of CD19 positive tumor cells in vitro and in vivo better than conventional CD19 CAR-T cells. The knock-down of PD-1 and TIGIT at CD19 CAR-T cells exerts the superior T-cell functionality by delaying the exhaustion of CAR-T cells.

Aims: This phase 1 dose escalation part ( NCT04836507 ) was to evaluate the safety (DLT, MTD), PK and preliminary efficacy (objective response rate and duration of response) in patients with r/r LBCL. Anbal-cel was manufactured at GMP facility with fresh leukapheresis product.

Methods: Patient was administered as a single intravenous dose at dose level 1 (2x10 5 cells/kg), dose level 2 (7x10 5 cells/kg) or dose level 3 (2x10 6 cells/kg). Lymphodepletion with cyclophosphamide (500mg/m 2 ) and fludarabine (30mg/m 2 ) was performed for 3 days prior to Anbal-cel infusion.

Results: As of Jan 17 2022, 9 patients with r/r DLBCL were infused with Anbal-cel; 4pts at DL1, 3pts at DL2 and 2pts at DL2. Median age was 54 (range 26-71); all patients received 2 or more prior lines of therapy and 44% (4/9) received ≥4 prior line of treatment before the study. 78% (7/9) patients were refractory to their last treatment. 67% (6/9) of patients were at IPI 3-4 and 44% (4/9) of patients had bulky disease. No patient experienced DLT during the study. Of the 9 patients, 5 (56%) experienced CRS; 4 (44%) were grade 1 or 2 and one patient experienced grade 3 CRS. Median time to onset of CRS was 7 days (range, 1-16) with median duration of 4 days (range, 0-18). One patient dosed at DL3 experienced grade 2 ICANS, time to onset of ICANS was 7 days and lasted for 13 days. This patient had prior CNS involvement history before the study. Most commonly reported grade 3/4 AEs were neutrophil count decrease (6/9, 67%), anemia (5/9, 56%), thrombocytopenia (2/9, 22%), platelet count decrease (2/9, 22%) and no infection was reported. Complete response rate (CRR) was 78% and complete responses were observed at the lowest dose level and from patients expressing less than 10% CD19 at IHC; 3 complete responses (CR) at DL1, 2 CRs at DL2 & DL3 respectively. Dose-dependent CRC01 expansion was observed; median T max was 15.4, 15.8, 11.0 days at DL1, DL2 & DL3 each; median C max was 18,003, 30,103, 53,688 copies/ug gDNA at DL1, DL2 & DL3 each; median AUC 0-28day was 679,125, 1,110,108, 2,852,235 copies/ug gDNA at DL1, DL2 & DL3 respectively.

Summary/Conclusion: Anbal-cel demonstrated promising efficacy and tolerable safety profile in this dose escalation study. Based on this phase 1 study, phase 2 patient enrollment will be commenced in Mar 2022 to evaluate the response rate, duration of response of CRC01 as well as safety. In addition, various biomarker studies are planned to investigate the differential mode of action of Anbal-cel during phase 2 study.

S215: GEMSTONE-201: PRE-PLANNED PRIMARY ANALYSIS OF A MULTICENTER, SINGLE-ARM, PHASE 2 STUDY OF SUGEMALIMAB IN PATIENTS WITH RELAPSED OR REFRACTORY EXTRANODAL NATURAL KILLER/T CELL LYMPHOMA (R/R ENKTL)

H. Huang 1,* , R. Tao 2 , Y. Yang 3 , H. Cen 4 , H. Zhou 5 , Y. Guo 6 , L Zou 7 , J. Cao 8 , Y. Huang 9 , J. Jin 10 , L. Zhang 11 , H. Yang 12 , X. Xing 13 , H. Zhang 14 , Y. Liu 15 , K. Ding 16 , X. Zhu 17 , T. Fang 17 , H. Dai 17 , Q. Qi 17 , J. Yang 17

1 Sun Yat-sen University Cancer Center, Guangzhou; 2 Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai; 3 Fujian Cancer Hospital & Fujian Medical University Cancer Hospital, fuzhou; 4 Guangxi Cancer Hospital and of Guangxi Medical University Affiliated Cancer Hospital, nanning; 5 Hunan Cancer Hospital, changsha; 6 Shanghai East Hospital, Tongji University School of Medicine, Shanghai; 7 State Key Laboratory, Cancer Center, West China Hospital of Sichuan University, chengdu; 8 Fudan University Shanghai Cancer Center, Shanghai; 9 The Affiliated Cancer Hospital of Guiyang Medical University, guiyang; 10 First Affiliated Hospital, College of Medicine, Zhejiang University, hangzhou; 11 Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, wuhan; 12 Zhejiang Cancer Hospital, hangzhou; 13 Liaoning Cancer Hospital & Institute, Shenyang; 14 Tianjin Medical University Cancer Institute & Hospital, tianjin; 15 Henan Provincial Cancer Hospital, zhengzhou; 16 Anhui Provincial Cancer Hospital, Hefei; 17 CStone Pharmaceuticals (Su Zhou) Co., Ltd., Shanghai, China

Background: R/R ENKTL is a rare and aggressive type of non-Hodgkin’s lymphoma. Responses to chemotherapy after failure of prior asparaginase-based regimen were not durable with a median OS of < 7 months (mos) and 1-year OS rate of < 20% (Lim et al, Ann Oncol 2017; Bellei et al, Haematologica 2018). The only targeted therapy approved in China for R/R peripheral T cell lymphoma (ENKTL included) showed an ORR of 18.8% and a CR rate of 6.3% (Shi et al, Ann Oncol 2015).

Aims: Here, we present the primary analysis from GEMSTONE-201, the largest registrational study reported to date to evaluate an anti-PD-L1 mAb in R/R ENKTL. Sugemalimab (Suge) received breakthrough therapy designation from US FDA in 2021 for adult R/R ENKTL patients (pts) based on preliminary data of this study.

Methods: Pts with ECOG PS of 0 or 1 and histologically confirmed ENKTL who failed prior asparaginase-based regimen were enrolled. Pts accepted suge at 1200 mg intravenously every 3 weeks, for up to 24 mos until progression, death, or withdrawal from study. The primary endpoint was ORR (CR+PR) assessed by independent radiological review committee (IRRC) per Lugano 2014 criteria. Key secondary endpoints included investigator-assessed ORR, CR and PR rate, DoR assessed by IRRC and investigators, and safety.

Results: As of the data cutoff date, 10 November 2021, 80 pts were enrolled and treated (median follow-up of 13.4 mos). Median age was 48 years (range 29-74); 64% were males; 74% had ECOG PS of 1 at baseline; 68% had stage IV disease; about half (49%) received ≥ 2 lines of prior systemic therapy. The median duration of treatment was 5.2 mos (range 0.7-37.4); 23 pts remained on treatment. Among the 78 evaluable pts as per IRRC, ORR was 46.2% (95% CI: 34.8%, 57.8%); 29 (37.2%) pts achieved CR; median DoR was not reached (NR); 12-mo DoR rate was 86%. Investigator’s assessments in 79 evaluable pts were consistent with IRRC results, i.e., ORR of 45.6% (95% CI: 34.3%, 57.2%), 24 (30.4%) pts with CR, and median DoR of NR. The 1- and 2-year OS rates were 68.6% and 54.6%, respectively; median OS was NR (range 0.9-37.2+ mos).

Of all pts, 96% (n = 77) had at least one AE. The most common AEs were pyrexia and WBC decreased (n = 24 each, 30%). Grade ≥ 3 AEs occurred in 31 (38.8%) pts. Suge-related AEs occurred in 61 (76%) pts and were mostly (60%) Grade 1 or 2. The most common immune-related AE assessed by sponsor was hypothyroidism (n = 13, 16%). SAEs occurred in 18 (23%) pts; 5 (6%) pts had suge-related SAEs which had all been resolved (1 with sequelae). Fatal AEs occurred in 5 (6%) pts and none were suge-related as assessed by investigators.

Summary/Conclusion: Suge has demonstrated deep and durable anti-tumor activity in R/R ENKTL pts, with a high CR rate and a promising OS benefit trend comparing to historical data. Suge had a well-tolerated safety profile and no new safety signals were detected. Primary analysis indicates that suge could provide a new treatment option to R/R ENKTL pts.

S216: CLINICAL ACTIVITY OF CC-99282, A CEREBLON E3 LIGASE MODULATOR (CELMOD) AGENT, IN PATIENTS (PTS) WITH RELAPSED/REFRACTORY NON-HODGKIN LYMPHOMA (R/R NHL) – RESULTS FROM A PHASE 1, OPEN-LABEL STUDY

J.-M. Michot 1,* , J. C. Chavez 2 , C Carpio 3 , S. Ferrari 4 , T. A. Feldman 5 , D. Morillo 6 , J. Kuruvilla 7 , A. Pinto 8 , V. Ribrag 1 , E Bachy 9 , T. J. Buchholz 10 , S. Carrancio 11 , W.-C. Chou 12 , C. Guarinos 13 , F. Wu 14 , S. Li 15 , P. Patah 16 , M. Pourdehnad 17 , L. Nastoupil 18

1 Département d’Innovation Thérapeutique et d’Essais Précoces (DITEP), Gustave Roussy Institute of Cancer, Villejuif, France; 2 Department of Malignant Hematology, Moffitt Cancer Center, University of South Florida, Tampa, United States of America; 3 Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), University Hospital Vall d’Hebron, Autonomous University of Barcelona (UAB), Barcelona, Spain; 4 Hematology and Bone Marrow Transplant Unit, ASST Papa Giovanni XXIII, Bergamo, Italy; 5 Lymphoma Division, John Theurer Cancer Center at Hackensack Meridian Health, Hackensack, United States of America; 6 Department of Hematology, University Hospital Fundación Jiménez Díaz, Madrid, Spain; 7 Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada; 8 Hematology-Oncology & Stem Cell Transplantation Unit, National Cancer Institute, Fondazione G. Pascale, IRCCS, Naples, Italy; 9 Department of Hematology, Hospices Civils de Lyon, Lyon, France; 10 Early Clinical Development, Oncology, Bristol Myers Squibb, San Francisco; 11 Oncogenesis (ONC) Thematic Research Center (TRC), Bristol Myers Squibb, San Diego; 12 Early Development Predictive Sciences, Bristol Myers Squibb, Cambridge, United States of America; 13 ONC TRC-Celgene Institute for Translational Research Europe (CITRE), Bristol Myers Squibb, Seville, Spain; 14 Clinical Pharmacology & Pharmacometrics, Bristol Myers Squibb, Summit; 15 Global Biometric Sciences, Bristol Myers Squibb, Berkley Heights; 16 Early Clinical Development, Bristol Myers Squibb, Lawrenceville; 17 Early Clinical Development, Hematology/Oncology and Cell Therapy, Bristol Myers Squibb, San Francisco; 18 Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: CC-99282 is a novel, oral small molecule CELMoD ® agent that co-opts cereblon to induce targeted degradation of Ikaros/Aiolos, transcription factors critical for development of B-cell malignancies. Compared with immunomodulatory drugs (IMiD ® ) and other CELMoD agents, CC-99282 had similar immunostimulatory effects and 10- to 100-fold stronger antiproliferative and apoptotic activity in preclinical models of diffuse large B-cell lymphoma (DLBCL).

Aims: To evaluate the safety, tolerability, maximum tolerated dose, pharmacokinetics (PK), and preliminary efficacy of CC-99282 in pts with R/R NHL.

Methods: CC-99282-NHL-001 ( NCT03930953 ) is a 2-part multicenter first-in-human study comprising dose escalation of CC-99282 monotherapy (part A) and expansion with or without combination partners (part B). Part A includes pts with R/R DLBCL or follicular lymphoma (FL) who had progressed after ≥2 lines of therapy, including IMiD/CELMoD agents and chimeric antigen receptor T-cell (CAR-T) therapy, and pts with R/R DLBCL who received ≥1 line of standard therapy and are unfit for transplant. Pts receive oral CC-99282 0.2, 0.4, 0.6, or 0.8 mg once daily on 3 intermittent dosing schedules of 28-day cycles, with ≥3 pts per dosing cohort.

Results: As of Oct 6, 2021, 50 pts were treated in part A (38 DLBCL, 12 FL; median age 66y; 58% male); 17 pts (34%) were ongoing and 26 pts (52%) discontinued due to progressive disease. Grade 3/4 CC-99282–related adverse events were reported in 32 pts (64%); the most common were neutropenia (29 pts [58%]), thrombocytopenia (4 pts [8%]), and anemia (4 pts [8%]). Correlation analysis showed that grade 4 neutropenia in the first month was more strongly associated with number of prior lines of alkylating agent and anti-CD20 therapy than with CC-99282 dose level or schedule. Neutropenia was manageable with CC-99282 dose modifications and granulocyte colony–stimulating factors. All dose-limiting toxicities were hematologic. The maximal recommended doses for schedules of interest were 0.6 mg on 7/14 days and 0.4 mg on 14/28 days. For doses ≥0.4 mg on schedules of interest, the overall response rate was 42% (15/36 evaluable pts; 6 FL, 9 DLBCL), with complete responses in 6 pts and partial responses in 9 pts. Responders included pts previously treated with CAR-T therapy and/or IMiD/CELMoD agents. Responses were durable, with median durations of 239 days (range 48–587; median follow-up [mFUP] 247 days [range 21–690]) and 112 days (range 63–414; mFUP 121 days [range 22–464]) for 7/14- and 14/28-day schedules, respectively. CC-99282 was absorbed rapidly and had a prolonged terminal half-life (median ~50 hours at doses ≥0.4 mg), with considerable distribution into the peripheral compartment. Increase in plasma CC-99282 and degradation of Ikaros/Aiolos in peripheral T cells occurred in a dose-dependent manner, and maximum degradation (>90%) occurred by day 4 of treatment at doses ≥0.4 mg. Within 2 weeks of initiating CC-99282, peripheral T-cell subsets showed a significant shift toward a more activated phenotype ( P <0.05). Analysis of mutant circulating tumor DNA levels showed rapid reductions that correlated with response to CC-99282, suggesting strong tumor cell–intrinsic activity.

Summary/Conclusion: CC-99282 monotherapy showed a manageable safety profile and demonstrated promising efficacy in heavily pretreated pts with R/R NHL. PK and pharmacodynamic data were consistent with robust and rapid CC-99282 antitumor activity. This study is ongoing, with patients actively being enrolled in the monotherapy and CC-99282+rituximab combination expansion cohorts.

S217: PRELIMINARY ANALYSIS OF THE PHASE II STUDY USING TOLINAPANT (ASTX660) MONOTHERAPY IN 98 PERIPHERAL T-CELL LYMPHOMA AND 51 CUTANEOUS T-CELL LYMPHOMA SUBJECTS WITH RELAPSED REFRACTORY DISEASE.

J.-M. Michot 1 , A. Mehta 2 , F. Samaniego 3 , E. Bachy 4 , P. L. Zinzani 5 , A. Prica 6 , G. P. Colins 7 , V Ribrag 1,* , N. Wagner-Johnston 8 , D. El-Sharkawi 9 , O. A. O’Connor 10 , R. Wilcox 11 , L. Wang 12 , L. Wilson 12 , M. Sims 13 , J. A. Taylor 12 , H. N. Keer 12 , F. Foss 14

1 Department of Hematology, Institut Gustave Roussy, Villejuif, France; 2 University of Alabama at Birmingham, Birmingham; 3 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 4 Hematology Department, Lyon Sud Hospital, Lyon, France; 5 Institute of Hematology “L. e A. Seràgnoli”, Bologna, Italy; 6 Princess Margaret Cancer Centre, Toronto, Ontario, Canada; 7 Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 8 Johns Hopkins University, The Sidney Kimmel Comprehensive Cancer Center, Baltimore, United States of America; 9 The Royal Marsden NHS Foundation Trust, Surrey, United Kingdom; 10 University of Virginia, Charlottesville; 11 University of Michigan Medical School, Ann Arbor; 12 Astex Pharmaceuticals, Inc., Pleasanton, United States of America; 13 Astex Pharmaceuticals, Cambridge, United Kingdom; 14 Yale Cancer Center, New Haven, United States of America

Background: There are limited treatment options for patients with Peripheral T-cell lymphoma (PTCL) and Cutaneous T-cell lymphoma (CTCL), especially when front line therapy has failed. Tolinapant (ASTX660) is a novel oral non-peptidomimetic, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which also induces necroptosis in T-cell lymphoma models (Ferrari et al ., Blood Advances, 2021). Tolinapant is being evaluated in a first-in-human ongoing Phase I/II study in subjects with advanced solid tumors and lymphoma (ClinicalTrials.gov NCT02503423 ). Phase I results were previously reported (Mita et al. Clin Cancer Res, 2020) and the recommended phase 2 dosing (RP2D) was established. Initial results for Phase II were previously reported at EHA 2019 (Mehta et al ., EHA 2019, # PS1073).

Aims: Here we report the preliminary efficacy and safety analysis for the Phase 2 PTCL and CTCL cohorts.

Methods: This is a single-arm open-label Phase II study. To be eligible, subjects must have evidence of documented progressive disease and received at least two prior systemic therapies. Subjects received treatment with tolinapant at the RP2D 180 mg/day on Days 1 to 7, and 15 to 22 in a 28-day cycle. The primary endpoint is best overall response rate (ORR) as assessed by the investigator according to either the Lugano criteria (PTCL) or Global Assessment (CTCL). Adverse events (AEs) are assessed per CTCAE v4.03. The efficacy data set is based on subjects who had tumor evaluation at baseline and at least 1 post-treatment tumor evaluation visit, unless they died or stopped treatment earlier due to clinical progression or toxicity. The safety data set is based on all subjects that received at least one dose of tolinapant.

Results: As of the data cut of January 5, 2022, there were 98 subjects with PTCL and 51 subjects with CTCL that received drug and 98 and 50 subjects that were evaluable respectively. The study is currently closed to enrollment with a minimum of 6 months follow-up on all subjects at the time of the data cut. Subject characteristics: median (range) age PTCL 62.5 (27, 82) and CTCL 62 (24,87), median number of previous therapies PTCL 3 (0-8) and CTCL 6 (1-10). Among all subjects, the most common related AEs of any grade (≥ 15%) were: lipase elevation (35%), amylase elevation (25%), rash (combined listings) (24%), ALT elevation (15%), and AST elevation (15%). Related AEs ≥ Grade 3 (≥ 5%) were: lipase elevation (15%), rash (combined listings) (9%), and amylase elevation (7%). Pancreatitis was identified in 2 subjects (1%) (both Grade 4). There were no related ≥ Grade 3 AEs for diarrhea, nausea or vomiting; for related Grade 2 AEs there was a 5% incidence of diarrhea and 1% incidence of nausea/vomiting.

The ORR for PTCL is 22%, including 9 complete responses (CRs) and 12 partial responses (PRs). The ORR in CTCL is 26% including 2 CRs and 11 PRs. The median durability of response for PTCL is 133 (Q1-Q3; 69 - 280) days and for CTCL is 148 (Q1-Q3; 103 - 294) days. Pharmacodynamic and correlative analysis is ongoing with preliminary analysis suggesting an immunomodulatory antitumoral effect of tolinapant (Ferrari et al ., Blood Advances, 2021).

Summary/Conclusion: In this Phase II study, the novel oral agent tolinapant has shown meaningful clinical activity against PTCL and CTCL with a manageable safety profile. These results support the continued development of tolinapant for the treatment of R/R PTCL and CTCL. A drug combination study using tolinapant in R/R PTCL is being developed.

S218: A PHASE I/II STUDY OF GOLIDOCITINIB, A SELECTIVE JAK1 INHIBITOR, IN REFRACTORY OR RELAPSED PERIPHERAL T CELL LYMPHOMA

W.-S. Kim 1,* , D.-H. Yoon 2 , Y. Song 3 , H. Yang 4 , J. Cao 5 , D. Ji 5 , Y. Koh 6 , H. Jing 7 , H.-S. Eom 8 , J.-Y. Kwak 9 , W.-S. Lee 10 , J.-S. Lee 11 , H.-J. Shin 12 , J. Jin 13 , M. Wang 14 , J. Li 14 , X. Huang 14 , X. Deng 14 , Z. Yang 14 , J. Zhu 3

1 Department of Hematology and Oncology, Samsung Medical Center; 2 Department of Oncology - Hematologic Cancer & BMT center, Asan Medical Center, Seoul, South Korea; 3 Department of Lymphoma, Peking University Cancer Hospital, Beijing; 4 Department of Lymphoma, Zhejiang Cancer Hospital, Hangzhou; 5 Department of Oncology, Fudan University Shanghai Cancer Center, Shanghai, China; 6 Department of Hemato-Oncology Center, Seoul National University Hospital, Seoul, South Korea; 7 Department of Hematology, Peking University Third Hospital, Beijing, China; 8 Center for Hematologic Malignancy, National Cancer Center, Goyang; 9 Department of Hemato-Oncology Center, Chonbuk National University Hospital, Jeonju; 10 Department of Hemato-Oncology Center, Inje University Busan Paik Hospital, Busan; 11 Department of Hematology & Medical Oncology, Seoul National University Bundang Hospital, Seongnam; 12 Department of Hemato-Oncology Center, Pusan National University Hospital, Busan, South Korea; 13 Department of Hematology, The First Affiliated Hospital of Zhejiang University, Hangzhou; 14 Clinical Development, Dizal Pharmaceutical, Shanghai, China

Background: Peripheral T cell lymphoma (PTCL) is a group of heterogeneous T cell lymphomas. Patients who relapse from or are refractory to 1 st line therapy face dismal prognosis. The response rates to commonly used 2 nd line agents such as histone deacetylase inhibitors are below 30%. Immunotherapies, such as anti-PD1 antibodies, may induce hyperprogression in certain PTCL subtypes. Hence, r/r PTCL patients urgently need better therapies.

Aims: Preclinical data shows JAK/STAT pathway may mediate the pathogenesis of PTCL, making it a promising target. Golidocitinib (DZD4205) is an orally available, potent, JAK1 specific inhibitor, demonstrating profound anti-tumor activities in T lymphoma cells in vitro and tumor xenograft in vivo . Here we report the preliminary data from an ongoing phase I/II study ( NCT04105010 ) of Golidocitinib in r/r PTCL.

Methods: The study included two parts: Part A (dose escalation) and Part B (dose expansion). In Part A, patients with r/r PTCL were enrolled and received Golidocitinib at different doses (150 mg or 250 mg, QD) to determine the recommended phase II dose (RP2D). Evaluation of safety and efficacy were performed by investigators per CTCAE and Lugano criteria, respectively. Part B is a single-arm, pivotal study, where patients with r/r PTCL will receive Golidocitinib at the RP2D till disease progression or intolerance.

Results: As of May 31, 2021, a total of 51 patients enrolled in Part A and received Golidocitinib at 150 mg (n = 35) or 250 mg (n = 16). Patient characteristics: median age (range): 61.0 years (29-79); median prior systemic therapies (range): 2 lines (1-8). Ten patients (19.6%) had undergone hematopoietic stem cell transplantation. Fifteen patients (29.4%) had bone marrow involvement at baseline. Histological subtypes included PTCL-NOS (41.2%), AITL (39.2%), ALCL ALK- (7.8%), NKTCL (7.8%), and MEITL (3.9%).

At the data cut-off (DCO), 49 patients completed at least one post-treatment Lugano assessment, of whom 21 (42.9%) achieved tumor response, including 11 complete responses (CRs, 22.4%) and 10 partial responses (20.4%). Tumor response was observed in various subtypes, including AITL (13/20), PTCL-NOS (5/19), ALCL ALK- (2/4) and NKTCL (1/4). At the DCO, the median duration of response (DoR) was not reached, and the longest DoR was > 14 months.

Forty-eight patients (94.1%) experienced treatment emergent adverse events (TEAEs), of whom 30 (58.8%) experienced ≥ grade 3 TEAEs. Per investigators’ assessment, 20 patients (39.2%) experienced ≥ grade 3 TEAEs possibly related to the drug. The most common (≥ 10%) ≥ grade 3 TEAEs were neutropenia (29.4%), thrombocytopenia (15.7%) and pneumonia (11.8%). The majority of TEAEs were reversible or clinically manageable with dose modifications.

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Summary/Conclusion: Golidocitinib shows good safety and promising anti-tumor efficacy in r/r PTCL, indicating its potential as a therapeutic option for this unmet medical need.

S219: FIRST CLINICAL STUDY OF THE ANTI-SIGNAL REGULATORY PROTEIN-ALPHA (SIRPΑ) ANTIBODY CC-95251 COMBINED WITH RITUXIMAB IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) NON-HODGKIN LYMPHOMA (NHL)

P. Strati 1,* , E. Hawkes 2 , N. Ghosh 3 , J. Tuscano 4 , Q. Chu 5 , M. A. Anderson 6 , A. Patel 7 , M. R. Burgess 7 , K. Hege 7 , S. Chhagan 7 , S. Boyanapalli 7 , T. Day 7 , F. Shen 7 , A. Mehta 8

1 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2 Austin Health–Austin Hospital, Heidelberg, Australia; 3 Levine Cancer Institute, Charlotte; 4 University of California, Davis, Sacramento, United States of America; 5 Cross Cancer Institute, Edmonton, Canada; 6 Peter MacCallum Cancer Centre, Melbourne, Australia; 7 Bristol Myers Squibb, Princeton; 8 University of Alabama at Birmingham, Birmingham, United States of America

Background: CD47 is a cell-surface ligand overexpressed in various malignancies that binds to SIRPα on effector macrophages to promote tumor cell evasion of phagocytosis. Blockade of this CD47–SIRPα interaction enhances phagocytosis mediated by tumor-targeting antibodies, such as rituximab (ritux). Agents targeting CD47 combined with ritux have demonstrated promising clinical activity in R/R NHL; however, the broad expression of CD47 potentially acts as an antigen sink, reducing anti-tumor activity, and leads to on-target, off-tumor toxicities, including hemolytic anemia. CC-95251 is a novel, fully human immunoglobulin G1 antibody (Ab) that binds to SIRPα on macrophages to potently block the CD47–SIRPα interaction.

Aims: The primary objectives of this multicenter, open-label, phase 1, dose-escalation and dose-expansion study were to evaluate the safety and tolerability of escalating doses of CC-95251 + ritux and to define the maximum tolerated dose (MTD) and/or recommended phase 2 dose for the combination in patients (pts) with CD20+ R/R NHL ( NCT03783403 ). Here, we report interim results.

Methods: Pts were treated in 28-day cycles (C) with CC-95251 administered intravenously at doses of 3, 10, or 20 mg/kg every week (QW) and with ritux 375 mg/m 2 on days (D) 1, 8, 15, and 22 of C1, D1 of C2–5, and D1 of every other cycle C6–24 until disease progression or unacceptable toxicity.

Results: As of 5 August 2021, 17 pts had received ≥ 1 dose of CC-95251 + ritux. Median age was 67 (range 30–84) years. Pts had received a median of 3 (range 1–7) prior systemic therapies, with 100% of pts having a prior history of anti-CD20 Ab exposure. Enrolled tumor types included R/R diffuse large B-cell lymphoma in 13 (77%) pts, follicular lymphoma in 2 (12%), and mantle cell lymphoma and marginal zone lymphoma in 1 (6%) pt each. Twelve (71%) pts had disease refractory to any prior line of therapy (LOT), including 10 (59%) refractory to any anti-CD20 Ab-containing regimen, and 9 (53%) to their last LOT. Pts received a median of 4 (range 1–14) cycles of CC-95251. Median duration of treatment was 16.4 (range 2.1–53.1) weeks. There was 1 CC-95251 dose reduction, and 6 (35%) pts experienced ≥ 1 treatment-emergent adverse event (TEAE) leading to CC-95251 dose interruption. MTD has not been reached. Neutropenia and infection were the most common TEAEs for any grade (71% and 59% respectively) and grade ≥ 3 (59% and 29% respectively). Treatment-related adverse events of grade ≥ 3 included neutropenia in 9 pts (53%) and infection in 2 pts (12%); no treatment-related anemia or deaths were reported. Overall response rate in the efficacy evaluable population was 56% (9/16), with 4 (25%) pts achieving a complete response (CR). Of 9 pts refractory to any prior anti-CD20 Ab-containing regimen, 2 pts achieved a CR and 2 pts had stable disease. Median time to response was 7.9 weeks. Duration of response ranged 7.4–28.1 weeks with a CR ongoing in 1 pt. In this dose-escalation study, CC-95251 demonstrated dose-proportional increases in exposure at doses > 3 mg/kg QW and full receptor occupancy at doses ≥ 3 mg/kg.

Summary/Conclusion: CC-95251 + ritux demonstrated a manageable safety profile and promising efficacy in pts with heavily pretreated CD20+ R/R NHL. The study is currently enrolling in the dose-expansion phase.

S220: GLOFITAMAB INDUCES DURABLE COMPLETE REMISSIONS AND HAS FAVORABLE SAFETY IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA AND ≥2 PRIOR THERAPIES: PIVOTAL PHASE II EXPANSION RESULTS

M. Dickinson 1,* , C. Carlo-Stella 2 , F. Morschhauser 3 , E. Bachy 4 , P. Corradini 5 , G. Iacoboni 6 , C. Khan 7 , T. Wróbel 8 , F. Offner 9 , M. Trněný 10 , S.-J. Wu 11 , G. Cartron 12 , M. Hertzberg 13 , A. Sureda 14 , D. Perez-Callejo 15 , L. Lundberg 15 , J. Relf 16 , E. Clark 16 , K. Humphrey 16 , M. Hutchings 17

1 Peter MacCallum Cancer Centre, Royal Melbourne Hospital and The University of Melbourne, Melbourne, VIC, Australia; 2 Humanitas University and IRCCS Humanitas Research Hospital, Milan, Italy; 3 Hôpital Claude Huriez and CHU de Lille, Lille; 4 Centre Hospitalier Lyon-Sud, Lyon, France; 5 Università degli Studi di Milano and Fondazione Istituti di Ricovero e Cura a Carattere Scientifico (IRCSS) Istituto Nazionale dei Tumori, Milan, Italy; 6 Vall d’Hebron University Hospital, Barcelona, Spain; 7 Allegheny Health Network, Pittsburgh, PA, United States of America; 8 Uniwersytet Medyczny we Wrocławiu, Wroclaw, Poland; 9 Universitair Ziekenhuis Gent, Ghent, Belgium; 10 Charles University Hospital, Prague, Czechia; 11 National Taiwan University Hospital, Taipei, Taiwan; 12 CHU de Montpellier, Montpellier, France; 13 Prince of Wales Hospital and University of New South Wales, Sydney, NSW, Australia; 14 Institut Català d’Oncologia Hospital, Barcelona, Spain; 15 F. Hoffmann-La Roche Ltd, Basel, Switzerland; 16 Roche Products Ltd, Welwyn Garden City, United Kingdom; 17 Rigshospitalet, Copenhagen, Denmark

Background: Glofitamab is a T-cell engaging bispecific antibody (Ab) with a novel 2:1 configuration that confers bivalency for CD20 (B cells) and monovalency for CD3 (T cells). In a Phase I/II study ( NCT03075696 ), escalating glofitamab doses were highly active and well tolerated in patients with relapsed/refractory (R/R) B-cell lymphomas, with obinutuzumab pretreatment (Gpt) and Cycle (C) 1 step-up dosing providing effective cytokine release syndrome (CRS) mitigation.

Aims: We present pivotal Phase II expansion results in patients with R/R diffuse large B-cell lymphoma (DLBCL) and ≥2 prior therapies.

Methods: All patients had DLBCL (DLBCL not otherwise specified [NOS], high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, or transformed follicular lymphoma) and had received ≥2 prior regimens, including ≥1 anti-(a) CD20 Ab and ≥1 anthracycline. Intravenous (IV) Gpt (1000mg) was given 7 days before the first glofitamab dose. IV glofitamab was then given as step-up doses on Day (D) 1 (2.5mg) and D8 (10mg) of C1 and at the target dose (30mg) on D1 of C2–12 (21-day cycles). The primary endpoint was complete response (CR) rate assessed by Independent Review Committee (IRC) using Lugano 2014 criteria. CRS was assessed using ASTCT criteria. All patients provided informed consent.

Results: As of September 14, 2021, 107 patients had received ≥1 dose of study treatment (median age: 66 years [21–90]; Ann Arbor stage III–IV disease: 74%; IPI score ≥3: 54%; DLBCL NOS: 74%). Median prior therapies was 3 (2–7); 59% had ≥3 prior therapies and 35% had received prior CAR T-cells (CAR-Ts). Most patients were refractory to a prior aCD20 Ab-containing regimen (85%) and to their most recent regimen (85%). Many were refractory to their initial therapy (59%) and to prior CAR-Ts (32%). After a median follow-up of 9 months (0.1–16), overall response and CR rates by IRC were 50.0% and 35.2%, respectively. CR rates were consistent in patients with and without prior CAR-Ts (32% vs 37%). Median time to CR was 42 days (95% CI: 41–48). The majority of CRs (33/38; 87%) were ongoing at data cut. An estimated 84% of complete responders and 61% of responders remained in response at 9 months. At data cut, the projected 12-month overall survival rate was 48%, and 92% of complete responders were alive. These results are consistent with earlier Phase I data in 100 patients treated with target glofitamab doses ≥10mg (CR rate: 34%; estimated 20-month CR rate in complete responders: 72%). CRS occurred in 68% of patients, was primarily associated with the initial doses, and was mostly Grade (Gr) 1 (51%) or Gr 2 (12%); Gr 3 (3%) and Gr 4 (2%) events were uncommon. All but 2 CRS events were resolved at data cut. Glofitamab-related neurologic adverse events (AEs) potentially consistent with immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in 3 patients (all Gr 1–2). No glofitamab-related Gr 5 (fatal) AEs occurred. Glofitamab-related AEs leading to discontinuation were uncommon (3 patients, 3%).

Summary/Conclusion: Fixed-duration glofitamab induces durable complete remissions and has favorable safety in patients with R/R DLBCL and ≥2 prior therapies, including those with prior exposure to CAR-Ts. Glofitamab is a promising new therapy for patients with heavily pretreated and/or highly refractory DLBCL.

S221: BORTEZOMIB TO R-DHAP COMPARED TO R-DHAP IN RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA ELIGIBLE TO STEM CELL TRANPLANTATION: FINAL RESULTS OF PHASE II RANDOMIZED FIL-VERAL12

A. Chiappella 1,* , M. Balzarotti 2 , B Botto 3 , A. Castiglione 4 , F. Cavallo 5 , S. V. Usai 6 , M. Zanni 7 , C. Califano 8 , F. Re 9 , C. Ghiggi 10 , A. Olivieri 11 , P. Corradini 12 , A. M. Liberati 13 , S. Volpetti 14 , M. G. Michieli 15 , A Tucci 16 , G. Gaidano 17 , M. Tani 18 , F. Ciambelli 19 , G. Musuraca 20 , D. Vallisa 21 , F. Merli 22 , A. L. Molinari 23 , A. Tafuri 24 , V. R. Zilioli 25 , D. Marino 26 , C. Stelitano 27 , S. A. Pileri 28 , G. Ciccone 4 , U. Vitolo 29

1 Hematology, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milano; 2 Hematology, Humanitas Cancer Center, Istituto Clinico Humanitas, Rozzano; 3 Hematology, AOU Città della Salute e della Scienza; 4 Unit of Clinical Epidemiology, AOU Città della Salute e della Scienza e CPO Piemonte; 5 Hematology, Department of Molecular Biotechnologies and Health Sciences, Università degli studi e AOU Città della Salute e della Scienza, Torino; 6 Hematology, Ospedale Businco, Cagliari; 7 Hematology, AO SS. Antonio e Biagio e Cesare Arrigo, Alessandria; 8 Oncology and Hematology, Presidio ospedaliero “A. Tortora”, Pagani; 9 Hematology, Ospedale Policlinico San Martino S.S.R.L. - IRCCS per l Oncologia, Parma; 10 Hematology, Ospedale Policlinico San Martino S.S.R.L. - IRCCS per l Oncologia, Genova; 11 Hematology, AOU Ospedali Riuniti, Ancona; 12 Chair of Hematology, Università degli Studi di Milano e Hematology, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milano; 13 Oncology and Hematology, AO. S. Maria, Terni; 14 Hematology, Azienda Sanitaria Universitaria Friuli Centrale, Udine; 15 Oncology and Hematology, IRCCS Centro di Riferimento Oncologico, Aviano; 16 Hematology, ASST Spedali Civili, Brescia; 17 Hematology and Department of Translational Medicine, Università del Piemonte Orientale e AOU Maggiore della Carità, Novara; 18 Hematology, Ospedale delle Croci, Ravenna; 19 Oncology, AO S. Antonio Abate, Gallarate; 20 Hematology, IRCCS Istituto Romagnolo per lo studio dei Tumori “Dino Amadori” – IRST S.R.L., Meldola; 21 Hematology, Ospedale Guglielmo da Saliceto, Piacenza; 22 Hematology, Azienda Unitа Sanitaria Locale-IRCCS - Arcispedale Santa Maria Nuova, Reggio Emilia; 23 Hematology, Ospedale degli Infermi, Rimini; 24 Hematology, AO Sant Andrea, Roma; 25 Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milano; 26 Oncology, IRCCS Istituto Oncologico Veneto, Padova; 27 Hematology, Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio Calabria; 28 Haematopathology, Istituto Europeo di Oncologia IRCCS, Milano; 29 Hematology, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy

Background: The standard treatment in patients with diffuse large-B cell lymphoma (DLBCL) relapsed/refractory (R/R) after first line therapy is a cisplatin-containing regimen followed by consolidation with high-dose chemotherapy and autologous stem cell transplantation (auto-SCT) in responsive ones after induction. Bortezomib had proven activity in aggressive lymphomas.

Aims: On these bases, the Fondazione Italiana Linfomi designed the FIL-VERAL12 trial, aimed at evaluating whether the addition of bortezomib to rituximab-cisplatin-cytarabine-dexametasone (BR-DHAP) increases complete response rate (CR, according to Lugano 2007 criteria) prior auto-SCT compared to standard R-DHAP.

Methods: FIL-VERAL12 was a prospective, multicenter, two-arm randomized phase II trial ( NCT01805557 ).The primary study endpoint was CR after 4 courses of R-DHAP or BR-DHAP, assuming a 30% CR for the standard arm and a 50% CR in experimental arm. Inclusion criteria were: patients aged 18-65 years eligible to high-dose therapy, with R/R DLBCL after first line chemoimmunotherapy. Patients were stratified by relapsed or refractory and randomized 1:1 to receive: a) the standard salvage therapy R-DHAP every 28 days for 4 cycles and b) subcutaneous 1.5 mg/ms bortezomib on days 1 and 4 of each 4-week cycle in addition to the same regimen.

Results: From January 2013 to November 2018, 114 patients were screened, and 107 patients that fulfilled the inclusion criteria were enrolled into the trial and randomized to receive R-DHAP or BR-DHAP (54 patients in R-DHAP, 53 in BR-DHAP). Principal clinical characteristics were: median age 57 years (IQR: 48;62); stage III/IV 83 patients (78%); International Prognostic Index (IPI) risk >2 37 (35%). All patients received rituximab and anthracycline-based regimens as first line treatment. Considering the time at relapse, 53 patients (50%) were registered as relapsed (median time at relapse 10.8 months, IQR: 6.9;20.9) and 54 (50%) as refractory (0.9 months, IQR 0.52;1.3). 52 (49%) patients completed the planned 4 cycles of therapy; 55 did not, due to progressive disease in 42, adverse events or clinician decision in 13. At the end of the 4 courses, the pre-auto-SCT response was: CR 29 (27%), Partial Response (PR) 9 (17%); according to arm of randomization, the primary end point was not met, with CR 28% for R-DHAP and 26% for BR-DHAP (p-value 0.563). Fifty patients (44%) performed a consolidation with SCT, 24 in R-DHAP arm and 26 in BR-DHAP arm; auto-SCT was performed in 39 patients and allo-SCT in 11. The addition of bortezomib to standard R-DHAP did not impact the mobilization, with a median number of CD34+ collected of 6.43 x 10^6 cells CD34/kg (IQR: 4.40;9.11) in R-DHAP and 6.78 x 10^6 cells CD34/kg (IQR: 5.00;9.68) in BR-DHAP. Sixty patients died: 49 (82%) due to lymphoma, 1 due to toxicity, 3 due to transplant related mortality, 7 due to other causes. The incidence of adverse events was similar in the two arms, with grade 3-4 haematological toxicities in 96 patients (90%), g3-4 infection in 5 (5%), g3-4 neurotoxicity in 4 (4%). At a median follow-up of 50 months, 2-years PFS was 29% (95%CI: 19.94;41.83) and 41% (27.67;53.84) for R-DHAP and BR-DHAP, respectively; HR 0.65 (0.41;1.02) p 0.062; 2-years OS was 43% (28.98;56.30) and 52% (37.80;64.56) for R-DHAP and BR-DHAP, respectively; HR 0.74 (0.44;1.23) p 0.244.

Summary/Conclusion: In the FIL-VERAL12 phase II randomized trial, the addition of bortezomib to R-DHAP did not improve the CR rate pre-auto-SCT of R/R DLBCL patients eligible to high-dose chemotherapy plus SCT; a numerically higher 2-year PFS rate was observed in BR-DHAP arm.

S222: MATURE T AND NK CELL LYMPHOMAS CLASSIFIED ACCORDING TO 2016 WHO CLASSIFICATION. A REPORT OF THE INTERNATIONAL PROSPECTIVE T-CELL PROJECT 2.0.

M. Federico 1,* , Y. Stepanishyna 2 3 , T. Skrypets 2 4 , C. S. Chiattone 5 , M. H. Prince 6 , A. Pavlovsky 7 , A. Lymboussakis 1 , M. Manni 1 , M. Civallero 1 , C. A. de Souza 8 , E. A. Hawkes 9 , L. Fiad 10 , R. Nair 11 , I. Kriachok 12 , F. Hitz 13 , O. Kostina 14 , C. Tomuleasa 15 , A. Guarini 16 , S. Luminari 1 17

1 Surgical, Medical and Dental Department of Morphological Sciences related to Transplant, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia; 2 TCP 2 Trial Office, TCP 2 Trial Office, Modena, Italy; 3 Department of Bone Marrow Transplant, National Cancer Institute, Kiev, Ukraine; 4 Clinical and Experimental Medicine (CEM), University of Modena and Reggio Emilia, Modena, Italy; 5 Santa Casa Medical School, Santa Casa Medical School, Sao Paulo, Brazil; 6 Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia; 7 Haematology, Fundaleu, Buenos Aires, Argentina; 8 Universidade de Campinas (UNICAMP), Universidade de Campinas (UNICAMP), Campinas, Brazil; 9 School of Public Health and Preventive Medicine Monash University and Olivia Newton John Cancer Research Institute, Austin Health, Melbourne, Australia; 10 Hospital Italiano La Plata, Hospital Italiano La Plata, Buenos Aires, Argentina; 11 TATA Medical Center, TATA Medical Center, Kolkata, India; 12 Department of Oncohematology, National Cancer Institute, Kiev, Ukraine; 13 Department of Oncology/Haematology, Cantonal Hospital, St Gallen, Switzerland; 14 North Estonia Medical Centre, North Estonia Medical Centre, Tallinn, Estonia; 15 Ion Chiricuta Oncology Institute, Ion Chiricuta Oncology Institute, Cluj Napoca, Romania; 16 Haematology and Cell Therapy Unit, IRCCS-Istituto Tumori ‘Giovanni Paolo II’, Bari; 17 Hematology Unit, Azienda Unità Sanitaria Locale IRCCS, Arcispedale Santa Maria Nuova IRCCS, Reggio Emilia, Italy

Background: Mature T and NK-cell lymphomas represent a heterogeneous group of rare lymphoid disorders arising from mature T cells of post-thymic origin. In 2018 the International T-cell non-Hodgkin’s Lymphoma Study Group launched the T-cell Project 2.0. Here we report the distribution of cases of PTCLs registered in the study, based on the diagnosis made locally according to the WHO 2016 classification, and some preliminary information on treatment and outcome.

Aims: The aim of the study is a better understanding of this group of rare disorders, capturing a real-life snapshot of the evolving landscape of T-cell lymphoma biology, treatment strategies, and outcome.

Methods: The T-cell Project 2.0 (ClinicalTrials.gov Identifier: NCT03964480 ) is a prospective, longitudinal, international, observational study of patients with PTCLs.

Results: Between October 2018 and February 2022, 901 patients with newly diagnosed PTCL were registered by 94 active centers across 17 countries.

Distribution of cases according to different subtypes is reported in Figure 1. Overall, the most frequent 6 subtypes account for 93% of cases, and the remaining 7% was represented by few cases of 12 different subtypes. Of note, only 9 cases have been classified according to entities not considered in the previous WHO 2008 classification.

With respect to clinical presentation, the median age at diagnosis was 56 years (18-93), 57.4% of the patients were male, the presence of systemic symptoms was reported in 31% of cases, 8.9% had ECOG-PS 3-4, 71% advanced disease and 35.4% bone marrow involvement.

At time of data lock, 831 cases had information on initial therapy. Overall, 731 patients (88%) were treated with combination chemotherapy and 125 (15%) were consolidated with high dose therapy and stem cell transplantation.

After a median follow-up of 20 months, the 2-year PFS and OS were 49% and 57%, respectively. For patients with PTCL-NOS, AITL, ENKTL, ALCL ALK- and ALCL ALK+, a comparison in terms of PFS was performed for those enrolled in the TCP2 and in the previous TCP1. Interestingly, a statistically significant difference (p=0.009) was observed, with 2-year PFS of 50% and 45% for patients enrolled in the TCP2 and TCP1, respectively.

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Summary/Conclusion: Regardless the difficulties linked to the COVD-19 pandemic, the TCP2 is recruiting very well and allow us in better understanding the outcome of patients with PTCL classified according to the 2016 WHO in the real world. Moreover, our data show that some improvement in the curability of patients affected by PTCL is emerging.

S223: LATE CARDIOVASCULAR TOXICITY AFTER HIGH-DOSE CHEMOTHERAPY FOR LYMPHOMA: A DANISH POPULATION-BASED STUDY

S. Husby 1,* , J. Baech 2 , T. Trab 2 , J. Gørløv 3 , J. M. Jørgensen 4 , S. Gudbrandsdottir 5 , M. T. Severinsen 2 , K. Grønbæk 3 , T. S. Larsen 6 , P. Brown 3 , L. H. Jakobsen 2 , K Kragholm 7 , T. C. El-Galaly 2

1 Hematology, Aalborg University hospital, Copenhagen N; 2 Hematology, Aalborg University hospital, Aalborg; 3 Hematology, Rigshospitalet, Copenhagen N; 4 Hematology, Aarhus University Hospital, Aarhus; 5 Hematology, Zealand University Hospital, Roskilde; 6 Hematology, Odense University Hospital, Odense; 7 Cardiology, Aalborg University hospital, Aalborg, Denmark

Background: Salvage chemotherapy followed by high-dose chemotherapy (HDT) and autologous stem cell transplantation (ASCT) are standard treatments for younger patients with relapsed/refractory aggressive diffuse large B-cell lymphoma (DLBCL) and Hodgkin’s lymphoma (HL) as well as for consolidation in 1 st line treatment for mantle cell lymphoma (MCL) and peripheral T-cell lymphoma (PTCL). Congestive heart failure (CHF) is a known late complication to lymphoma therapy, but it is unclear how much HDT adds to CHF risk. Delineating late complications to HDT/ASCT is increasingly important due to the availability of alternatives such as CAR-T therapy.

Aims: To investigate how HDT/ASCT treatment influences the risk of developing congestive heart failure in a population-based nationwide setting.

Methods: Data were obtained from the nationwide Danish lymphoma registry and the Danish National Patient Register. DLBCL, MCL, HL, and PTCL patients treated with HDT between 2001 and 2017 and without CHF prior to HDT were included. Patients were compared to two populations: 1) A matched population of individuals from the general population without prior CHF matched on age and sex in a 1:5 ratio, and 2) A population of lymphoma patients (HL or DLBCL) in clinical remission two years after standard 1 st line treatment without HDT. The latter group was chosen due to very low lymphoma-specific mortality. CHF was defined by ICD-10 outpatient clinic and hospital discharge diagnosis codes. The Aalen-Johansen estimator was used to compute the cumulative risks treating death as competing event. This analysis was conducted for CHF and cardiovascular diseases in general, separately (CVD; ischemic heart disease, atrial fibrillation/flutter, ventricular fibrillation/flutter, and CHF). Crude and adjusted cause-specific hazard ratios (HR) were obtained using Cox regression and adjustments were made for age and sex.

Results: A total of 958 HDT patients were identified (35% DLBCL, 28% MCL, 21% PTCL, and 16% HL) with a median follow-up of 7.9 years. Almost all patients were previously treated with anthracycline (97.8%).

The risk of both CHF and CVD was significantly increased in patients treated with HDT compared to a matched background population (p<0.001; Fig. 1a, 1b). The risk of CHF was more pronounced in HDT-treated males compared to females (10-year incidence 7.8% vs 3.8%, respectively, Fig. 1c and Fig. 1d ); however, the risk was significantly increased for both sexes (p<0.001 for both).

Multivariable Cox regression adjusted for sex and age confirmed the increased risk of CHF for HDT patients. The risk was increased both when compared to the matched background population (adjusted HR 6.40) and to lymphoma patients not treated with HDT (adjusted HR 2.54). This is intriguing since HDT patients are generally considered more fit than unselected lymphoma patients. Comparison of comorbidities between the groups is ongoing.

In the HDT group, the major risk factors for CHF were older age at the time of HDT (HR 1.20 per decade), and male sex (HR 2.15).

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Summary/Conclusion: The risk of CHF and CVD in general is significantly increased for HDT treated patients compared to both a matched background population and patients treated with 1 st line therapy without consolidating HDT, despite likely selection of healthy patients to HDT. Major risk factors were male sex and age. These findings are important when considering the possible use of new therapies such as CAR-T cell therapy which have a different safety profile.

S224: PRELIMINARY RESULTS OF A PHASE II STUDY OF ORELABRUTINIB IN COMBINATION WITH ANTI-PD-1 MONOCLONAL ANTIBODY IN REFRACTORY OR RELAPSED PRIMARY CNS LYMPHOMA

Y. Zhang 1,* , W. Wang 1 , D. Zhao 1 , Y. Zhang 2 , W. Zhang 1 , D. Zhou 1

1 Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College; 2 Department of Hematology, Beijing Longfu Hospital, Beijing, China

Background: The survival outcomes of patients with relapsed/refractory (R/R) primary central nervous system lymphoma (PCNSL) remain extremely poor and there are no approved therapies or widely accepted “standard-of-care” approaches. Previous studies showed that Bruton tyrosine kinase (BTK) inhibitor and anti-programmed death protein-1(PD-1) monoclonal antibody have significant activities in R/R PCNSL, respectively. The combination of BTK inhibitor and anti-PD-1 monoclonal antibody demonstrated synergistic effects both in vivo and in vitro in diffuse large B cell lymphoma, but no clinical data is currently available for PCNSL. Orelabrutinib is a new-generation BTK inhibitor with high CSF concentration and sintilimab is a new-generation anti-PD-1 monoclonal antibody.

Aims: To evaluate the safety and efficacy of orelabrutinib combined with sintilimab in patients with R/R PCNSL.

Methods: The prospective, multicenter, single-arm phase II study ( NCT04899427 ) enrolled immunocompetent adult patients with R/R PCNSL and eligible organ functions. The patients received once daily orelabrutinib (150mg) in combination with sintilimab (200mg on day 1 of each cycle) every 3 weeks per cycle up to two years or until disease progression, intolerable toxicity, or death. The primary objective was the overall response rate (ORR; defined as partial response [PR] or better) after 4 cycles. Other endpoints mainly included 1-year progression free survival (PFS) rate, time to response (TTR) and safety.

Results: The data cut-off date was February 25, 2022. Thirteen patients were enrolled from March 2021 to January 2022, with a median follow-up of 7.0 (1.5-10.5) months. Median age was 61 yr (range 48 to 71) and 6 (46.1%) were male. The median lines of prior treatment were 2 (range 1 to 4). All the patients were high-dose methotrexate treated, seven of those (53.8%) were refractory to the last treatment. Ten patients completed 4 cycles of the experimental regimen while 3 patients ended of treatment in the first 2 cycles due to disease progression. The toxicities were quite mild, with one grade 3 adverse event (AE) of interstitial pneumonitis related pneumocystis carinii infection. No other Grade 3-4 hematological or non-hematological AE was reported.

All patients were evaluable for response. The ORR was 61.5%, and 4 patients achieved complete remission (CR), 1 CRu, and 3 PR. The median TTR was 6 weeks (2 to 4 cycles). No relapses were observed in patients who achieved ORR after 4 cycles. Only 1 patient died of disease progression (Fig. 1). The estimated median1-year PFS rate was 67.7% (Fig. 2). Plasma and CSF sample from 4 of 13 patients were collected at 2 h after 15 days orelabrutinib administration. The median CSF concentration of orelabrutinib was 28.7ng/ml (rang 11.8 ng/ml to 52.7 ng/ml). The median CSF/plasma free ratio was 59.8%(rang 46.09% to 86.67%).

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Summary/Conclusion: The combination of orelabrutinib and sintilimab showed a high ORR and a rapid onset of response in patients with r/r PCNSL with well-tolerated toxicities. Although preliminary, these results supported the use of BTK inhibitor plus anti-PD-1 monoclonal antibody. More clinical data will be updated from this ongoing study.

S225: CIRCULATING TUMOR DNA IS A PROGNOSTIC BIOMARKER AT BASELINE AND IMPROVES THE ACCURACY OF INTERIM PET IN CLASSIC HODGKIN LYMPHOMA

M. C. Pirosa 1,* , A. Bruscaggin 1 , L. Terzi di Bergamo 1 , M. Salehi 1 , K. Pini 1 , V. Spina 1 , S. Bocchetta 1 , A. Condoluci 1 , G. Forestieri 1 , D. Piffaretti 1 , J. Marques De Almeida 1 , S. Annunziata 2 , F. Bergesio 3 , E. Borsatti 4 , P. Bulian 5 , S. Chauvie 3 , C. Marco 6 , D. T. Martina 7 , G. Bernhard 8 , M. Kurlapski 9 , A. Moccia 10 , R. Moia 11 , A. Rinaldi 12 , M. Rodari 13 , G. Romanowicz 14 , G. M. Sacchetti 15 , A. Stasia 8 , A. Stathis 10 , G. Stüssi 8 , I. Zangrilli 16 , L. M. Larocca 17 , A. Pinto 18 , A. Santoro 7 , F. Cavalli 1 , E. Zucca 10 , V. Gattei 19 , J. M. Zaucha 9 , C. Carlo-Stella 7 , S. Hohaus 16 , G. Gaidano 11 , L. Ceriani 6 , D. Rossi 1

1 Division of Experimental Hematology, Institute of Oncology Research, Bellinzona, Switzerland; 2 Institute of Nuclear Medicine, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome; 3 Department of Medical Physics, Santa Croce e Carle Hospital, Cuneo; 4 Nuclear Medicine, IRCCS CRO; 5 Clinical and Experimental Onco Hematology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy; 6 Clinic of Nuclear Medicine and Molecular Imaging, Imaging Institute of Southern Switzerland, Bellinzona, Switzerland; 7 Humanitas Cancer Center, Humanitas Clinical and Research Center, Milan, Italy; 8 Division of Hematology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 9 Department of Hematology and Transplantology, Medical University of Gdańsk, Gdańsk, Poland; 10 Clinic of Medical Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 11 Division of hematology, University of Eastern Piedmont, Novara, Italy; 12 Genomics Facility, Institute of Oncology Research, Bellinzona, Switzerland; 13 Unit of Nuclear Medicine, Humanitas Clinical and Research Center, Milan, Italy; 14 Department of Nuclear Medicine, Medical University of Gdańsk, Gdańsk, Poland; 15 Nuclear Medicine, “Maggiore della Carità ” Hospital, Novara; 16 Department of diagnostic imaging, oncological radiotherapy and hematology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS; 17 Division of Pathology, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome; 18 Hematology‐Oncology and Stem Cell transplantation Unit, National Cancer Institute, Fondazione “G. Pascale” IRCCS, Naples; 19 Clinical and Experimental Onco Hematology Unit, IRCCS CRO, Aviano, Italy

Background: Classic Hodgkin lymphoma (cHL) has few pre-treatment prognostic biomarkers. Circulating tumor DNA (ctDNA) is a metrics of tumor volume and inflammation, which are both prognostic in cHL, it improves the accuracy of treatment response assessment, which is an unmet need at the interim timepoint in cHL, and it allows to accurately genotype the disease, which has technical hurdles if done of the tumor biopsy in cHL.

Aims: The study aims at addressing the following questions: i) is pre-treatment ctDNA load a metrics that captures both tumor and inflammation extents in a single, measurable and radiation-free test? Can the integration of ctDNA with interim PET (iPET) improve accuracy of the sole iPET in predicting treatment outcome? Can ctDNA identify molecular groups with phenotype- and outcome-associated signatures?

Methods: IOSI-EMA003 ( NCT03280394 ) is a prospective, observational, multi-center study. The study recruited adult patients with previously untreated cHL. Blood samples were collected during staging and disease response assessment at the same time of PET scan acquisition. PET scans were centrally and blindly reviewed by a panel of nuclear medicine physicians. ctDNA was genotyped and quantified by phased variant-enhanced-LyV4.0 ctDNA CAPP-seq assay. Interim molecular response (iMR) was defined as lack tumor reporters in cfDNA collected at the interim timepoint (sensitivity 10 -4 ).

Results: A total of 215 patients were recruited. Median follow-up was 30 months. The full analysis dataset was divided into training (N=135) and validation (N=80) cohorts by using a random sample procedure. In the training cohort, ctDNA load directly associated with inflammation (B symptoms, elevated ESR), but not with disease burden: total metabolic tumor volume (TMTV), GHSG stage. The optimized threshold of pretreatment ctDNA load to stratify PFS was 850 hGE/mL of plasma (Figure 1A). In multivariable analysis, pretreatment ctDNA levels remained prognostic for PFS when controlling for either GHSG stage, TMTV, B symptoms or ESR. The validation cohort confirmed that patients with higher pretreatment ctDNA levels had inferior PFS (Figure 1B). Among intermediate and advanced stage patients, the PFS of patients with positive iPET was significantly inferior (Figure 1C). iMR was achieved in 54% patients. Patients who did not achieved iMR had a lower PFS. iMR combined with iPET had a statistically significant superior accuracy for the anticipation of progression or relapse compared to iMR or iPET alone (Figure 1D). To identify molecular subgroups within cHL, we focused on fragmentation profiles of cfDNA. cfDNA fragmentome can comprehensively represent both genomic and chromatin characteristics. Pre-treatment cfDNA fragmentation patterns were characterized by a more prominent mononucleosomal fragments abundance in 51% of patients (mono-nucleosomal cluster), whereas 33% of patients had a more prominent shift towards submono-nucleosomal fragment lengths (submono-nucleosomal cluster) (Figure 1E). Patients belonging to the submono-nucleosomal cluster had different clinical and biological nuances, including advanced stage, B-symptoms, elevated ESR, higher TMTV and total lesion glycolysis, higher mutation load, higher circulating immune suppressive cytokines and chemoattractants for monocytes and T h -cells. Accordingly, patients with submono-nucleosomal cluster had a lower PFS.

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Summary/Conclusion: ctDNA is a validated prognostic biomarker of cHL, can improve the accuracy of interim response assessment and provide the bases for a molecular classification of cHL.

S226: LOSS OF NR4A1 IS ASSOCIATED WITH HIGHER EXPRESSION OF IMMUNE CHECKPOINT COMPONENTS AND REDUCED T CELL-MEDIATED LYMPHOMA CELL KILLING IN AGGRESSIVE LYMPHOMA

K. Pansy 1,* , K. Fechter 1 , A. Arra 2 , M. Szmyra 1 , S. Haingartner 1 , A. Ramsay 3 , H. Greinix 1 , C. Beham-Schmid 4 , P. Neumeister 1 , A. Deutsch 1

1 Division of Hematology, Medical University of Graz, Graz, Austria; 2 Clinic for Experimental Pedriatics, Otto-von-Guericke-Universität, Magdeburg, Germany; 3 Lymphoma Immunology research group, Kings ‘s College, London, United Kingdom; 4 Institute of Pathology, Medical University of Graz, Graz, Austria

Background: Aggressive lymphomas represent the most common type of lymphoid malignancies with a five-year survival rate of 60%. Despite effective initial treatment, one-third of all patients will experience a relapse, warranting more research to discover novel therapeutic strategies. We recently detected a significant lower expression of NR4A1 that correlates with a poor lymphoma-specific survival. Furthermore, ectopic expression of NR4A1 induces apoptosis in vitro and suppresses lymphoma growth in xenografts indicating its tumor suppressive properties.

Aims: The aim of this study was to comprehensively study the function of Nr4a1 loss in lymphomagenesis.

Methods: Therefore, we intercrossed the EµMyc lymphoma mouse model with the Nr4a1-/ - mouse and monitored them until the onset of disease. Furthermore, we transplanted lymphoma cells of EµMyc Nr4a1-/ - and EµMyc Nr4a1+/+ mice into immune-competent C57BL/6 mice and immune-deficient Fox Chase SCID beige mice. Furthermore, we determined the expression levels of immune checkpoint components in biopsies of our human diffuse large B cell lymphoma (DLBCL) cohort and correlated them to NR4A1 content. Finally, to investigate the immune-regulatory function of Nr4a1 , we performed co-culture cytotoxicity assays using OVA 257-264 peptide-pulsed EµMyc Nr4a1+/+ and EµMyc Nr4a1-/ - lymphoma cells and Ctla4+/+ and Ctla4-/ - OVA targeting CD8 + T cells derived from OT-I mice and measured T cell-mediated lymphoma cell lysis via flow cytometry, respectively.

Results: We observed that the loss of Nr4a1 leads to an accelerated lymphomagenesis in vivo , concomitant with increased expression of immune checkpoint components of the Pd1-Pdl1-Pdl2- and Ctla4-Cd80-Cd86-axes. Immuno-competent, but not immune-deficient mice, transplanted with Nr4a1 -deficient lymphoma cells exhibited rapid lymphoma development, reduced survival, and upregulation of the immune checkpoint components like in the primary model. Importantly, low NR4A1 expression correlated with high expression of immune checkpoint components in biopsies of our human DLBCL cohort largely resembling our mouse data. To unravel the impact of Nr4a1 on T cell-mediated lymphoma cell killing and on the regulation of the Ctla-4-Cd80-Cd86-axis of aggressive lymphomas, we performed co-culture cytotoxicity assays using OVA peptide-pulsed EµMyc Nr4a1+/+ and EµMyc Nr4a1-/ - lymphoma cells and Ctla4+/+ and Ctla4-/ - OVA targeting CD8 + T cells. In these experiments, we observed a massively diminished lymphoma cell killing in the EµMyc Nr4a1-/ - setting, when we used Ctla4+/+ OT-1 CD8+ T cells. Interestingly, when we used Ctla4-/ - OT-1 CD8+ T cells for the cytotoxicity assays, lymphoma cell killing was enhanced in the Nr4a1-/ - setting compared to that using Ctla4+/+ OT-1 CD8+ T cells.

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Summary/Conclusion: Our data suggest that Nr4a1 plays a critical role in regulating the licensing of immune evasion in aggressive lymphomas by regulating immune checkpoint expression. Thus, it might act as a promising target to restore anti-lymphoma immune responses.

S227: SBNO2 IS A SPECIFIC DEPENDENCY OF STAT3-DRIVEN T-CELL MALIGNANCIES

T. Brandstoetter 1,* , B. Maurer 1 , J. Schmoellerl 2 , S. Kollmann 1 , J. Huuhtanen 3 4 , R. Grausenburger 1 , S. Mustjoki 3 5 , J. Zuber 2 , V. Sexl 1

1 Dept. of Biomedical Sciences at the Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna; 2 Research Institute of Molecular Pathology, Vienna, Austria; 3 Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, Helsinki; 4 Department of Computer Science, Aalto University, Espoo; 5 Translational Immunology Research program, University of Helsinki, Helsinki, Finland

Background: The transcription factor signal transducer and activator of transcription 3 (STAT3) plays important roles in regulating the survival, growth and differentiation of hematopoietic cells, particularly within the lymphoid lineage. Recently, gain-of-function mutations within the SH2 domain of STAT3 have been identified in patients suffering from T-cell large granular lymphocytic leukemia (T-LGLL). The most frequently found hyperactivating mutation represents STAT3 Y640F .

Aims: Transcription factors are notoriously difficult to target, hence a better understanding of STAT3-dependent transcriptional co-factors and their molecular targets is critical to identify novel therapeutic approaches.

Methods: To unravel the molecular mechanisms behind STAT3 Y640F -induced malignancies, we developed a murine in vitro model that recapitulates common transcriptional features that are found in CD8 + transformed T-LGLL cells from patients harbouring STAT3 mutations. Gene expression analyses and chromatin occupancy profiling of mutated STAT3 Y640F identified a core set of direct transcriptional targets, which were interrogated for their functional relevance in genome-wide CRISPR/Cas9-based loss-of-function screens.

Results: Expression of STAT3 Y640F blocked differentiation of murine hematopoietic stem and progenitor cells (mHSPC), enhanced their self-renewal capacity and increased proliferation in comparison to wild type STAT3 expressing cells. Mechanistically, the STAT3 Y640F mutation led to increased and prolonged activation of STAT3 signalling and decreased their dependence on cytokine stimulation. In contrast to mHSPC expressing wild-type STAT3, the Y640F-mutated variant resulted in a strong inflammatory gene expression signature that was characterized by overactivation of TNF alpha/Interferon gamma pathways, and is conserved in T-LGLL patients harbouring STAT3 mutations. Through genome-wide loss-of-function screens using CRISPR/Cas9 we identified that the transcriptional co-regulator strawberry notch homolog 2 (SBNO2) is an essential direct transcriptional target of STAT3 Y640F in transformed mHSPC. SBNO2 was overexpressed in both, STAT3 Y640F -transformed mHSPC (versus STAT3 WT ) and T-LGLL patients harbouring STAT3 mutations (versus T-LGLL patients with wild type STAT3). As seen with loss of STAT3-Y640F expression, loss of SBNO2 also impaired proliferation of transformed mHSPC. Strikingly, global analyses of gene dependency datasets revealed that various human T-cell malignancies that are driven by aberrant STAT3 signalling depend on SBNO2 expression.

Summary/Conclusion: Together, our data show that the STAT3/SBNO2 axis could be a promising therapeutic intervention site in STAT3 driven T-cell malignancies.

S228: ACTIVATED SUMOYLATION RESTRICTS MHC CLASS I ANTIGEN PRESENTATION TO CONFER IMMUNE EVASION IN LYMPHOMA

U. M. Demel 1,* , M. Böger 1 , S. Yousefian 2 , C. Grunert 1 , P. Hotz 3 , S. Haas 2 , S. Müller 3 , M. Wirth 1 , M. Schick 1 , U. Keller 1

1 Charité Universitätsmedizin Berlin; 2 Max Delbrück Center for Molecular Medicine, Berlin; 3 Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt, Germany

Background: During the last decade, the implementation of immunotherapies has revolutionized cancer treatment. However, despite the striking success, often only subgroups of patients respond, and loss of the MHC-I antigen presentation machinery (APM) has been identified as a frequent cause of primary and acquired resistance to immunotherapies. Thus, strategies to restore these pathways may enhance the efficacy of immunotherapies. The post-translational protein modification SUMOylation (SUMO) is a crucial regulatory mechanism in the cellular stress response. Activated SUMOylation is regarded as a hallmark of aggressive cancers. Notably, a selective small molecule inhibitor of SUMOylation (SUMOi), subasumstat, is currently tested in clinical trials.

Aims: We aimed to identify the implications of activated SUMOylation for lymphoma immune evasion, and to develop a pharmacological intervention strategy to improve cancer immunotherapies.

Methods: A multi-OMICs approach was used to interrogate SUMO-driven immune evasion mechanisms in lymphoma. In vitro findings were informed by in vivo studies in lymphoma xenograft models and syngeneic murine tumor models. Single-cell RNA sequencing (CITE-seq) was applied to decipher in vivo SUMOi-driven changes in blood and immune cell subsets.

Results: Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved pathway of MYC-induced SUMOylation, which attenuated the immunogenicity of lymphoma cells. While activated SUMOylation repressed the MHC-I antigen processing and presentation machinery (APM) and enabled lymphoma cells to evade CD8 + T-cell immunosurveillance, pharmacological inhibition of SUMOylation restored T-cell mediated tumor killing. Importantly, SUMOi amplified the activation of the STAT1 pathway in response to IFNg, which is one of the key regulators of cytokine-induced MHC-I expression. Beyond this, we identified a previously unknown mechanism of SUMO-mediated basal repression of MHC-I and established SAFB, a repressor of immune regulators, as a SUMO-regulated mediator of MHC-I suppression that mechanistically links SUMOylation to MHC-I repression. We thus showed that activated SUMOylation converted lymphoma cells into a less immunogenic state that was restored by pharmacological SUMO inhibition, enhancing the susceptibility of lymphoma cells to CD8 + T-cell mediated killing. Moreover, we revealed that activated SUMOylation was associated with lower abundance and activity of tumor-infiltrating T-cells in vivo , proposing SUMOi as a novel therapeutic strategy to enhance efficacy of immunotherapies. Moreover, by means of single cell RNA-seq analysis we accounted SUMOi with a key function in altering the global immune landscape.

Summary/Conclusion: In summary, we show that activated SUMOylation allows tumor cells to evade anti-tumor immunosurveillance and establish SUMOi as rational therapeutic strategy for enhancing the efficacy of immunotherapies in lymphoma and other aggressive cancers.

S229: CHARACTERISATION OF TUMOR MICROENVIROMENT REMODELLING IN THE PROGRESSION OF AITL WITH SINGLE-CELL RNA SEQUENCING AND IMAGING MASS CYTOMETRY

H. Jin 1,* , X. Lu 1 , L. Fan 1 , L. Cao 1 , W. Zhang 1 , J. Li 1 , Z. Wu 1

1 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China

Background: Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL). AITL is an aggressive malignancy with a poor prognosis, and its clinical manifestations vary greatly among individuals. The current chemotherapy regimens based on anthracycline show limited efficacy, and there is no best rescue treatment for patients with relapsed and refractory (RR) AITL. In addition, the lack of optimal AITL models in vitro greatly limits the basic research on the mechanism of disease occurrence and progression, and also hinders the development of new drugs and preclinical trials.

Aims: Our study aims to deeply analyze the tumor heterogeneity and microenviroment remodelling in the progression of AITL at single cell resolution, discovering key molecules of drug resistance and potential theraputic targets.

Methods: We detected fresh lymph node samples from initail diagnosed (ID) and relapsed and refractory (RR) AITL patients using single-cell RNA sequencing (scRNA-seq), combined with imaging mass cytometry (IMC) and whole exome sequencing. scRNA-seq was performed to compare the differential transcriptome expression patterns in ID- and RR-AITL samples. IMC was performed to analyze the spatial position relationship and protein expression characteristics of different subgroups in the tumor microenvironment of AITL. Immunofluorescence staining was conducted to detect the expression of relative markers on tissue microarrays in patients with AITL. In addition, AITL patient-derived organoid model was established to study the regulatory role of YY1 and its inhibitors in relapsed and refractory AITL.

Results: We found that RR-AITL samples exhibited significant differences in the tumor microenvironment compared with ID patients. ScRNA-seq analysis revealed that transcription factor YY1 was significantly highly expressed in follicular helper T cells (Tfh) of RR-AITL patients, which promoted the proliferation and drug resistance of AITL cells (Fig A-D), consistent with the results of IMC (Fig E,F). The proportion of CD8+ T cells in the RR-AITL sample was reduced, while the proportion of Treg was increased, as well as the depletion of T cells (Fig G). Furthermore, the stemness of B cells in RR-AITL was enhanced and exhibits significant malignant characteristics (Fig H). We also found decreased interaction in RR-AITL samples. B cells and myeloid subgroups may play important roles in the progression of AITL (Fig I,J). As shown in Fig K, EBV+ B cells exhibited a wider and more distribution in RR-AITL (Fig L). Interestingly, EBV+ endothelial cells were presented in both ID- and RR- samples, while the spatial analysis showed that the distance of EBV+ endothelial cells to B cells in RR-AITL was obviously greater than in ID group, and the distance of Tfh to B cells also showed similar result (Fig M,N). Moreover, for the first time, we established AITL patient-derived organoid models that can be stablely cultured in vitro. On this basis, we could further clarify the important roles of transcription factor YY1 in the drug resistance of AITL, evaluate the cytotoxic effect of YY1 inhibitor NP-001 on AITL tumor cells.

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Summary/Conclusion: In conclusion, our study revealed the differences between initial diagnosed and relapsed /refractory AITL in terms of tumor microenvironment, single-cell transcriptomes, the spatial distributions of different clusters and their interactions features. YY1 may serve as an novel target for drug resistance for RR-AITL patients. These findings may provide a theoretical foundation for improving the clinical treatment of AITL.

S230: HUMAN ΒETA-DEFENSIN 2 TREATMENT MODULATES THE INTESTINAL MICROBIOME AND THE ALLOGENEIC T CELL RESPONSE TO LIMIT ACUTE GRAFT-VERSUS-HOST DISEASE

T. Rückert 1,* , G. Andrieux 2 3 , M. Boerries 2 3 , N. M. Woessner 4 5 6 , S. Minguet 4 5 7 , S. Doetsch 4 5 , K. Aumann 8 , M Schiff 1 , L. M. Braun 1 , E. Haring 1 4 , B. A. Siranosian 9 , A. S. Bhatt 9 10 , P. Nordkild 11 , J. Wehkamp 12 , B. A. H. Jensen 13 , J. Duyster 1 3 , R. Zeiser 1 3 14 , N. Köhler 1 14

1 Department of Medicine I; 2 Institute of Medical Bioinformatics and Systems Medicine, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg; 3 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg; 4 Faculty of Biology; 5 Signalling Research Centres BIOSS and CIBSS; 6 Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg; 7 Institute for Immunodeficiency, Center for Chronic Immunodeficiency (CCI); 8 Institute of Surgical Pathology, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; 9 Department of Genetics; 10 Department of Medicine (Hematology, Blood and Marrow Transplantation), Stanford University, Stanford, United States of America; 11 Defensin Therapeutics, Copenhagen, Denmark; 12 Department of Internal Medicine I, University Hospital Tübingen, Tübingen, Germany; 13 Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; 14 CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy option for patients with hematological malignancies. Acute graft-versus-host disease (GVHD) is primarily driven by allogeneic donor T cells and remains a serious cause of morbidity and mortality post allo-HCT. In particular, acute GVHD of the gastrointestinal tract (GI GVHD) is associated with a high mortality rate and treatment options are limited. Human β-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide, which is induced by inflammatory stimuli and possesses both antimicrobial and immunomodulatory functions.

Aims: In this study, we aimed to investigate the expression of beta defensins in human and murine GI GVHD and the therapeutic effect of recombinant hBD-2 on acute GVHD development in mouse models.

Methods: To investigate the functional role of hBD-2 post allo-HCT, we employed established murine GVHD and graft-versus-leukemia (GVL) models and analyzed the allogeneic T cell response using transcriptome and kinome profiling. Shotgun metagenomic sequencing was used to examine the effect of hBD-2 on the intestinal microbiome. Furthermore, we studied beta-defensin expression in two independent acute GVHD patient cohorts and in mice with acute GVHD.

Results: We found that expression of murine beta-defensin 4 (mBD-4), the murine orthologue for hBD-2, was reduced in the colon and ileum of mice developing acute GVHD. Oral treatment of mice with recombinant hBD-2 post allo-HCT reduced weight loss and acute GVHD severity and mortality. Furthermore, hBD-2 treatment affected the intestinal microbial composition, including a shift towards higher abundance of Bacteroides species in hBD-2 treated compared to vehicle treated mice. The changes in the microbiome resulted in reduced neutrophil infiltration of the ileum during acute GVHD induction in hBD-2 treated mice. Additionally, hBD-2 dampened pro-inflammatory Th1 cytokine production (TNF, IFN-gamma) by allogeneic T cells in vivo , while preserving the beneficial GVL effect in two different leukemia models. Mechanistically, oral hBD-2 treatment decreased alloreactive T cell infiltration and the expression of genes involved in T cell receptor (TCR) signaling in the ileum of mice with acute GVHD. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation and metabolism by reducing proximal TCR signaling. In patients with acute GI GVHD, intestinal hBD-2 expression was inadequately induced in response to inflammation at the mRNA and protein level when compared to healthy subjects and patients with ulcerative colitis.

Summary/Conclusion: In conclusion, our study demonstrates that hBD-2 reduces acute GVHD severity, likely through its effects in shaping the intestinal microbiota, reducing neutrophil infiltration in the ileum and dampening allogeneic T cell responses. Both human and murine acute GVHD are characterized by a lack of intestinal beta-defensin induction and recombinant hBD-2 represents a potential novel therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

S231: GLUCOCORTICOID AND GLYCOLYSIS INHIBITORS COOPERATIVELY ABROGATE ACUTE GRAFT-VERSUS-HOST DISEASE

Q. wen 1 , Z. xu 1,* , Y. wang 1 , M. lv 1 , Y. song 1 , Z. lv 1 , T. xing 1 , L. xu 1 , X. zhang 1 , X. huang 1 , Y. kong 1

1 Peking University Institute of hematology, Beijing, China

Background: Although glucocorticoids(GCs) are the standard first-line therapy for acute graft-versus-host disease(aGvHD), approximately 50% of aGvHD patients have no response to GCs. The role of T cell metabolism in murine aGvHD has been recently reported. Pharmacological inhibition of glycolysis by targeting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3) ameliorates aGvHD. However, the question of whether glycolysis is required for the function of activated T cells and whether abnormal glycolysis is involved in the occurrence of human aGvHD need to be elucidated. Moreover, the issue as to whether GCs and metabolism regulators can cooperatively suppress T cell alloreactivity and ameliorate aGvHD remains to be elucidated.

Aims: The study aimed to analyse the glucose metabolism profiles of activated T cells in aGvHD patients, and to evaluate the roles of PFKFB3-stimulated glycolysis in alloantigen-activated T cells and aGvHD induction. Furthermore, the study was performed to explore the issue of whether GCs could restore activated T cells by regulating glycolysis and the effect of GCs combined with glycolysis inhibitors on activated T cells.

Methods: In this prospective nested case–control study, a total of 15 aGvHD patients at diagnosis and 15 matched non-aGvHD patients. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. Subsequently, to assess whether functional T cell activation is caused by the regulation of PFKFB3, we used lentivirus transduction for the genetic regulation of PFKFB3 in vitro and in a humanized xenogeneic aGvHD model. The effect of GCs and glycolysis inhibitors on activated T cells was further explored in vitro and in a humanized murine model of aGvHD and graft versus leukaemia (GVL). In addition, to evaluate the synergistic effect of GCs, 3PO or their combination, combination index (CI) studies were performed by using the Chou-Talalay method for drug interactions.

Results: Increased glycolysis, which was characterized by elevated PFKFB3, as well as higher rates of glucose consumption and lactate production in aGvHD T cells. Importantly, in vitro treatment with glycolysis inhibitor 3PO improved the activity of T cells derived from aGvHD patients through down-regulating glycolytic activity of T cells. Genetic upregulation of PFKFB3 induced T cell proliferation and differentiation into proinflammatory T cells. In a humanized mouse model, PFKFB3-overexpressing T cells aggravated aGvHD, as characterized by high aGvHD clinical scores, pathological scores and rapid lethality. Importantly, our integrated data from patient samples in vitro showed that GCs combined with 3PO decreased proinflammatory T cells and T cell proliferation, reduced glucose consumption, lactate production and PFKFB3 expression compared with the single GCs group. Notably, the average CI values of GCs and 3PO were 0.364 for the synergistic inhibition of IFN-γ synthesis and 0.475 for the synergistic inhibition of proliferation, respectively. In a humanized murine model of aGvHD and GVL demonstrated that GCs and 3PO cooperatively reduced the alloreactivity of T cells and ameliorate aGvHD without a loss of GVL effects.

Summary/Conclusion: Our data indicated that glycolysis is critical for T cell activation and the induction of human aGvHD. GCs combined with glycolysis inhibitors demonstrated synergistic effects on reducing T cell alloreactivity and ameliorating aGvHD by regulating T cell glycolysis in vitro and in vivo . Thus, GCs combined with glycolysis inhibitors promise to be a novel therapeutic strategy for aGvHD patients.

S232: EXPANDING THE REPERTOIRE OF HLA CLASS I-RESTRICTED MINOR HISTOCOMPATIBILITY ANTIGENS FOR IMMUNE MONITORING AND MODULATION AFTER ALLOGENEIC STEM CELL TRANSPLANTATION

K. Fuchs 1,* , M. van de Meent 1 , W. Honders 1 , C. van Bergen 1 , J. F. Falkenburg 1 , M. Griffioen 1

1 Hematology, LUMC, Leiden, Netherlands

Background: Allogeneic stem cell transplantation is given as curative treatment for hematological malignancies, but patients face the risk of relapse of their malignancy as well as Graft-versus-Host Disease. After transplantation, donor T cells recognize polymorphic peptides presented by HLA surface molecules on the patient’s cells. These polymorphic peptides, called minor histocompatibility antigens, are caused by genetic differences in single nucleotide polymorphisms (SNPs) between patient and donor. Dependent on whether the antigen is presented on tumor cells or healthy non-hematopoietic tissues of the patient, donor T cells may induce the favourable Graft-versus-Leukemia effect or Graft-versus-Host-Disease, respectively.

Aims: The aim is to identify the dominant repertoire of minor histocompatibility antigens in seven common HLA class I molecules for immune monitoring and modulation after allogeneic stem cell transplantation.

Methods: Donor T cells isolated from patients after allogeneic stem cell transplantation are tested for recognition of a new panel of 191 selected B cell lines, which are sequenced in the 1000 Genomes Project. This panel enables the inclusion of seven common HLA class I molecules and increases SNP coverage to 11 million (MAF > 0.01). SNPs that strongly associate with T cell recognition are subsequently validated to encode minor histocompatibility antigens.

Results: Using an optimized approach for whole genome association scanning, more than 80 new minor histocompatibility antigens have been found that are presented by seven common HLA class I molecules. Antigens targeted in immune responses after transplantation were often shared between patients, and about 25-30% of antigens were translated in other reading frames than human proteins with known function. Furthermore, the extended repertoire was investigated for T cell frequency and tissue distribution of antigen-encoding genes, pointing towards several antigens as potential targets for immunotherapy.

Summary/Conclusion: In conclusion, despite many SNP mismatches between patients and donors, our data demonstrate that the repertoire of minor histocompatibility antigens is confined. As the antigens were identified by an unbiased forward strategy (T cell-to-antigen), our collection provides relevant insight into the various sources of antigens in annotated and unannotated reading frames, which is essential for accurate antigen prediction by reverse strategies (antigen-to-T cell). Furthermore, we more than doubled the number of minor histocompatibility antigens which are fundamental to predict, follow or manipulate immune responses after allogeneic stem cell transplantation to improve clinical outcome of transplanted patients.

S233: THERAPEUTIC GENE EDITING OF T CELLS CORRECTS CTLA4 INSUFFICIENCY.

T. Fox 1,* , B. Houghton 1 , L. Petersone 1 , N. Edner 1 , O. Preham 1 , E. Waters 1 , C. Hinze 1 , A. McKenna 1 , C. Williams 1 , A. Kennedy 1 , A. Pesenacker 1 , P. Genovese 2 , L. Walker 1 , S. Burns 1 , D. Sansom 1 , C. Booth 1 , E. Morris 1

1 University College London, London, United Kingdom; 2 Boston Children’s Hospital, Boston, United States of America

Background: Heterozygous mutations in CTLA4 result in an inborn error of immunity (IEI) (also known as primary immunodeficiency) with a severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic stem cell transplantation. The mutational landscape and requirement for tight regulation of CTLA4 make viral gene addition approaches unappealing. Gene editing strategies permit alteration of CTLA4 while retaining the endogenous gene control machinery.

Aims: We set out to devise a CRISPR/Cas9/AAV6 gene editing strategy to correct CTLA4 insufficiency in T cells.

Methods: We designed several homology directed repair (HDR) editing strategies that would correct the genetic defect. We first assessed correction of an individual point mutation. We then evaluated several universal strategies that enable correction of most disease-causing mutations with a single edit; the first that inserts the CTLA4 cDNA in exon 1, and a second that inserts the CTLA4 cDNA at the 3’end of the first intron of CTLA4. All AAV6 HDR donor templates included a GFP reporter gene to enable easy identification of the edited cells.

Results: Superior editing efficiencies were obtained with the intronic approach compared to the other editing strategies and this strategy was then further evaluated. CTLA4 function and expression kinetics were assessed following editing using flow cytometry-based assays. Functional studies using CTLA4 transendocytosis (TE) assays, demonstrated restoration of CD80 and CD86 internalization in the edited CD4+ T cells. Following gene editing, transgene expression kinetics were comparable to healthy control CD4+ T cells. Gene editing of T cells isolated from patients with CTLA4 insufficiency restored CTLA4 expression and rescued transendocytosis of CD80 and CD86 in vitro . Using a similar approach, gene corrected T cells from CTLA4 -/- mice engrafted in immunodeficient mice at clinically relevant frequencies and tail vein bleeds were performed 1, 3 and 4 weeks post adoptive transfer. In the mice which received the GFP+ edited cells, a stable population of GFP+ cells was detectable at all timepoints demonstrating in vivo persistence as well as genetic stability. All mice were sacrificed 4 weeks after cell transfer. To assess lymphoproliferation, the cellularity of peripheral lymph nodes and spleen weight were analyzed. Spleen and lymph node size, lymph node cell counts, and spleen weight were all significantly lower in mice which received the edited cells (n=5) compared to non-edited controls (n=5) and there was no significant difference in these parameters between mice who had received edited CTLA4 -/- T cells and those who received WT T cells (n=4). Analysis of lymph node and spleen cells confirmed persistence of CTLA4 expression in the edited (GFP+) cells and revealed that a higher proportion of Treg than Tconv had been successfully edited.

Summary/Conclusion: Together these data demonstrated that CTLA4 edited T cells survived in vivo , expressed CTLA4 and were able to control the clinical phenotype of CTLA4 insufficiency, providing a powerful proof-of-principle of our T cell GT approach. Our data provide proof-of-concept that gene editing can restore CTLA4 function in T cells demonstrating the potential of this approach to treat CTLA4 insufficiency. A similar approach could be used in other IEIs that are caused by multiple heterozygous mutations.

S234: CLINICAL-GRADE MBIL21/4-1BBL EXPANDED NK CELLS EXHIBIT STRONGER COMPETENCE COMPARED WITH PRIMARY NK CELLS AGAINST HCMV INFECTION

Q.-N. Shang 1 2 3,* , X.-X. Yu 1 2 3 , Z.-L. Xu 1 2 , T.-T. Han 1 2 , J. Xie 1 2 , Z.-Y. Fan 1 2 , M. Zhao 1 2 , X.-H. Cao 1 2 , X.-F. Liu 1 2 3 , Y.-H. Chen 1 2 , M. Lv 1 2 , Y.-Q. Sun 1 2 , Y.-J. Chang 1 2 , Y. Wang 1 2 , L.-P. Xu 1 2 , X.-H. Zhang 1 2 , K.-Y. Liu 1 2 , X.-Y. Zhao 1 2 , X.-J. Huang 1 2 3

1 Peking University People’s Hospital; 2 Peking University Institute of Hematology; 3 Peking-Tsinghua Center for Life Sciences, Beijing, China

Background: Cytomegalovirus (CMV) infection remains a common complication and leads to high mortality in subjects who undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). Human NK cells are the first lymphocyte recovering after allo-HSCT. Previous studies indicated the early NK cell reconstitution may be protective against development of HCMV infection post HSCT. Our previous data also showed that rapid reconstitution of IFN-γ secreted NK cells at day 15 post transplantation predicted lower HCMV reactivation. However, little is currently known about whether and how NK cells are responsible for preventing HCMV infection post transplantation. NK cell adoptive transfer is a promising method for cancer immunotherapy. Ex vivo mbIL21/4-1BBL expanded NK cells exhibited high cytotoxicity against leukemia cells. Nevertheless, whether expanded NK cells owned higher anti-HCMV function compared with primary NK cells in vitro and in vivo were still unknown.

Aims: To investigate whether expanded NK cells owned higher anti-HCMV function compared with primary NK cells in vitro and in vivo and the efficacy of adoptive NK cells infusion to patients post HSCT to prevent HCMV infection.

Methods: NK cells were firstly ex vivo expanded by K562-mbIL21/4-1BBL feeder cells. We compared not only the cytotoxicity against HCMV infected fibroblast but also the competence of inhibiting HCMV propagation and reducing HCMV infection between ex vivo expanded NK cells and primary NK cells. Then primary NK cells and expanded NK cells were adoptive infused to HCMV-infected humanized mice to investigate the ability to eliminate HCMV infection in vivo . At last, 20 patients post HSCT were enrolled in our clinical trial to explore the safety and efficacy of adoptive infusion of K562-mbIL21/4-1BBL expanded NK cells to protect from HCMV infection.

Results: 1. Most activating receptors, cell adhesion molecule receptors and chemokine receptors exhibited enhanced expression on expanded NK cells (figure 1a). Expanded NK cells showed stronger cytotoxicity against HCMV infected fibroblasts and enhanced abilities to inhibit HCMV propagation compared with primary NK cells in vitro (figure 1b).

2. Both expanded NK cells and primary NK cells showed the ability to migrate to the spleen, liver, and lung and persisted in these target organs in HCMV-infected humanized mice. On day 14 post infusion, expanded NK cells showed higher percentage in tissues than primary NK cells. Moreover, mice with expanded NK cell infusion exhibited more effective HCMV elimination compared with primary NK cell infusion.

3. The cumulative incidence of HCMV and refractory HCMV infection for patients in NK infusion cohort were significantly reduced compared with control cohort (figure 1c). Total HCMV persisting time was shortened (14 (5-24) days vs. 17 (7-56) days, p=0.013). What is more, the absolute number of NK cells was higher for patients in NK cell infusion group than that in control group. Higher quantitative reconstitution, more matured NK cell phenotypes and stronger function were determined in patients with expanded NK cell infusion than those in control group.

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Summary/Conclusion: Based on in vitro and in vivo studies, our data demonstrated that adoptive NK cells infusion exhibited stronger activities compared with primary NK cells against HCMV infection.

S235: A TWO-PART, SINGLE- AND TWO-ARM RANDOMIZED, OPEN-LABEL STUDY TO EVALUATE THE SAFETY, TOLERABILITY AND PHARMACOKINETICS OF THE S1P RECEPTOR MODULATOR KRP203 IN SUBJECTS WITH HEMATOLOGICAL MALIGNANCIES

S. dertschnig 1,* , J. finke 2 , D. heim 3 , U. schanz 4 , E. holler 5 , U. holtick 6 , G. socié 7 , T. teshima 8 , C. bucher 9 , J. passweg 10

1 Priothera SAS, Saint Louis, France; 2 Heamtology, Oncology and SCT, University Freiburg, Freiburg, Germany; 3 Hematology, University Hosptial Basel, Basel; 4 University Hospital Zürich, Zürich, Switzerland; 5 Klinik und Poliklinik Innere Medizin III, Unuversitätsklinikum Regensburg, Regensburg; 6 Klinik I für Innere Medizin, Köln, Germany; 7 Saint Louis Hospital, Paris, France; 8 Hokkaido University, Faculty of Medicine, Sapporo, Japan; 9 Universtity Hospital Basel; 10 University Hosptial Basel, Basel, Switzerland

Background: The success of allogeneic hematopoietic stem cell transplantation (HSCT) is limited by disease relapse. Alloreactive donor T cells have the potential to prevent relapse by the graft-versus-leukemia (GVL) response. The GVL response is essential, however, the same allo-T cells cause acute graft-versus-host disease (aGVHD). T cell trafficking out of lymphoid organs is a major step in raising peripheral immune responses and is regulated by sphingosine-1-phosphate (S1P) gradients. Pre-clinical data showed that pharmacological modulation of S1P receptor (S1PR) signalling by KRP203 sequesters T cells in lymphoid organs and prevents their egress to aGVHD target sites. In murine models, KRP203 efficiently reduced aGVHD. Because T cell function is not suppressed by KRP203, GVL was maintained, resulting in improved survival. Here, we show data of the first clinical trial investigating the safety and tolerability of KRP203 in patients with hematological malignancies undergoing HSCT.

Aims: The aim of this study was to evaluate the safety, tolerability and pharmacokinitecs of a S1P receptor modulator towards a novel treatment approach that aims at reducing GvHD while maintaining Graft versus leukemia.

Methods: This multicentric, phase Ib, prospective, open label, two-part study evaluated safety, tolerability and pharmacokinetics of KRP203 in intermediate to high-risk patients undergoing HSCT for hematological malignancies. Three treatment arms were investigated: 3mg KRP203+CsA/MTX, 1mg KRP203+CsA/MTX and 3mg KRP203+Tac/MTX. KRP203 was administered from day 1 until day 111 and HSCT was performed on day 11.

Results: KRP203 was safe and well-tolerated in these fragile patients. Upon conditioning and KRP203 treatment absolute lymphocyte counts (ALC) were reduced to about 0.2x10 9 /L, engrafted normally and during KRP203 exposure ALC stabilized at <0.7x10 9 /L in all three treatment arms, well below pre-HSCT levels. The treatment effect on ALC counts resolved when KRP203 was stopped on day 111, resulting in blood ALC to pre-HSCT levels. CD4 + and CD8 + T cells, were reduced in peripheral blood in response to KRP203 treatment. CD4 + T cells remained below pre-HSCT level (<100 cells/µl) during the treatment period. CD8+ T cell counts recovered more rapidly, also during treatment. Overall survival probability was highest for subjects in the 3mdg KRP203+CsA arm with the first death reported on day 532. Overall survival at 1 year was 100% in the 3mg KRP203+CsA, 67% in the 1mg KRP203+CsA and 36% in the 3mg KRP203+Tac arm. The median time to relapse was 749 days. 2/6 subjects in 1mg KRP203+CsA, 4/10 subjects in 3mg KRP203+CsA and 4/7 subjects in 3mg KRP203+Tac arm relapsed. The median time to any aGVHD was 55 days. 22% of subjects presented with grade III-IV aGVHD. The median time to any chronic GVHD was 174 days. 57% of subjects presented with moderate and 9% with severe chronic GVHD.

Summary/Conclusion: KRP203 is safe and well tolerated and shows promising early clinical outcomes with a limited number of relapses, acute and chronic GVHD. These data support the initiation of a phase 2b trial investigating KRP203 as an adjunctive and maintenance treatment as an adjunctive and maintenance treatment to increase leukemia free survival in adult AML patients undergoing allo-HSCT.

S236: RANDOMIZED MULTICENTER PHASE III STUDY OF HAPLO VERSUS HLA-MATCHED UNRELATED DONOR (UD) ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION (ALLO HSCT) FOR PATIENTS OLDER THAN 55 YEARS

S. Harbi 1 , J.-M. Boher 2 , E. Forcade 3 , P. Chevallier 4 , R. Peffault De Latour 5 , F. Malard 6 , S. Francois 7 , A. Charbonnier 8 , E. Hermet 9 , C. E. Bulabois 10 , A. Huynh 11 , A. Berceanu 12 , T. Cluzeau 13 , M. T. Rubio 14 , S. Furst 1 , R. devillier 15 , M. Mohty 6 , D. Blaise 1,*

1 hematology; 2 DRCI, Institut Paoli Calmettes, Marseille; 3 hematology, CHU, Bordeaux; 4 hematology, CHU, Nantes; 5 Transplant Unit, Hopital Saint Louis; 6 hematology, Hopital Saint Antoine, Paris; 7 hematology, CHU, Angers; 8 hematology, CHU, Amiens; 9 hematolgy, CHU, Clermont Ferrand; 10 hematolgy, CHU, Grenoble; 11 hematology, CHU, Toulouse; 12 hematology, CHU, besancon; 13 hematology, CHU, Nice; 14 hematology, CHU, Nancy; 15 hematolgy, Institut Paoli Calmettes, Marseille, France

Background: In most situations where allo HSCT is wished a matched sibling donor (MSD) is lacking. The respective advantages of alternative donor such as UD or HaploD are yet to be precisely assessed notably in older population including the search and identification process.

Aims: We performed a prospective, multicenter, open-label, randomized controlled trial ( NCT02623309 ) comparing Haplo versus UD search strategies for allo HSCT in pts with hematological malignancies older than 55 years after the absence of MSD was established.

Methods: The primary objective was the comparison of Chronic Graft-versus-Host Disease-free and Relapse-free survival (cGRFS) from time of randomization. Conditioning was RIC based on fludarabine (150 mg/m 2 ), IV busulfan (260 mg/m 2 ) for haplo HSCT with the adjunction of thiotepa (5 mg/kg) for UD-HSCT. Pts receiving Haplo-SCT and UD-HSCT received GVHD prophylaxis based on HD-PTCy (100 mg/kg) and ATG (5 mg/kg), respectively. Additionnal prophylaxis consisted in CSA and mycophenolate mofetil for all patients (pts).

Results: 108 pts were enrolled, and 106 pts were analyzed with a median follow-up of 27 (14-34) months. Median age was 65 years (55-70). Diseases were myeloid malignancies in 84 pts (79%). DRI was low, intermediate and high in 5(5%), 59(55%) and 42(40%),respectively. 55 and 51 pts were assigned to Haplo and UD group respectively. In case an alternative donor was not identified in due time, a search for another donor was allowed.

Of the 106 pts, 77 (73%) pts proceeded to transplant (40 (73%) and 37 (73%) pts in Haplo and UD group respectively). Fifteen (27%) pts in Haplo group did not because of progression (n=9), patient contraindication (n=5), absence of donor (n=1). Likewise 14 (27%) pts in UD group were not transplanted because of progression (n=8), patient contraindication (n=6). Overall median time from randomization to allo-SCT was 76 (21-179) and 95 (37-310) days in haplo and UD group respectively (P=NS). Thity-one (56%) and 26 (51%) pts actually were transplanted according to randomization while 9 (16%) and 11 (22%) pts were transplanted from a UD or a HaploD in haplo and UD search group respectively. Median time from randomization to allo-SCT was 81 (21-288) when the initial desired donor was found and 117 (38-310) days when another donor type was to be searched (p=0,04). In an intent-to-treat analysis from date of randomization, 2-year GRFS, PFS and OS did not differ between the two groups (Haplo vs UD search group: 29% vs 37%, p=0.22; 45 vs 49 %, p=0.56; 50 vs 59%, p=0.47).

Overall 42 and 35 pts were actually transplanted from a haploidentical and unrelated donor, respectively whatever the randomization arm. With a median follow up of 24 months after transplant 2-year GRFS and PFS from transplant did not differ between Haplo and UD HSCT: 40% vs 34%, p=0.66; 48%vs 45%, p=0.78). No statistical difference was documented for G2-4 aGVHD, severe cGVHD, NRM and relapse probabilities.

Summary/Conclusion: This trial from alternative donor search initiation time-point establishes for the first time that the search for a HaploD or UD conduct to the same outcomes in older pts. However, although 77% of the patients can achieved a transplant with either strategies, only half of the original pts will receive a transplant from the prospected donor type. Outcomes being similar, this may invite to initially perform search for both type of donors to avoid delay with secondary searches.

S237: ORCA-T, AN ENGINEERED ALLOGRAFT, RESULTS IN HIGH GVHD-FREE AND RELAPSE-FREE SURVIVAL FOLLOWING MYELOABLATIVE CONDITIONING FOR HEMATOLOGICAL MALIGNANCIES

E. Meyer 1,* , A. Pavlova 1 , A. Gandhi 2 , R. Hoeg 3 , C. Oliai 4 , R. Mehta 5 , S. Srour 5 , J. McGuirk 6 , E. Waller 7 , N. Fernhoff 8 , M. S. Killian 8 , J. Mcclellan 8 , A. Putnam 8 , B. Shaw 9 , M. Abedi 10 , R. Negrin 11

1 BLOOD AND MARROW TRANSPLANTATION AND CELLULAR THERAPY, Stanford Hospital and Clinics, Stanford, CA; 2 Department of Medicine, Division of Hematology/Medical Oncology, Oregon Health and Science University, Portland, OR; 3 Department of Medicine, Division of Bone Marrow Trasnplant, University of California, Davis, Comprehensive Cancer Center, Davis, CA; 4 Department of Medicine, Blood and Bone Marrow Transplant Program, University of California, Los Angeles, Los Angeles, CA; 5 Department of Stem Cell Transplantation and Cellular Therapy, Division of Cancer Medicine, MD Anderson Cancer Center, Houston, TX; 6 Department of Hematologic Malignancies and Cellular Therapeutics, University of Kansas Medical Center, Kansas City, KS; 7 Bone Marrow and Stem Cell Transplant Center, Winship Cancer Institute of Emory University, Atlanta, CA; 8 Orca Bio, Menlo Park, CA; 9 Department of Medicine, Division of Hematology and Oncology, BMT Program, Medical College of Wisconsin, Wilwaukee, WI; 10 Department of Medicine, Division of Bone Marrow Transplant, University of California, Davis Medical Center, Davis, CA; 11 Department of Blood and Marrow Transplantation and Cellular Therapy, Stanford Hospital and Clinics, Stanford, CA, United States of America

Background: Rates of graft versus host disease (GVHD) and non-relapse mortality (NRM) following myeloablative allogeneic hematopoietic stem cell transplant (MA-alloHSCT) remain unacceptably high. Strategies to reduce GVHD and NRM have been compromised by limited efficacy or increased risk of infection and relapse, emphasizing the need for new approaches that holistically improve outcomes.

Orca-T is a high-precision, allogeneic investigational cell therapy product comprised of stem and immune cells that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses, reducing the need for pharmacologic GVHD prophylaxis. Orca-T is produced in a central GMP facility and has been successfully scaled to clinical centers throughout the U.S.

Aims: The aim of these studies was to evaluate the safety and efficacy of Orca-T in patients with hematologic malignancies.

Methods: As of 28 February 2022, 138 patients with high-risk hematologic malignancies have received Orca-T in a single-center Phase 1-2 study ( NCT01660607 , n=41) and a multicenter Phase 1b study ( NCT04013685 , n=97) and have ≥ 100 days of follow-up. Informed consent was obtained from all transplant recipients and donors, and the studies received IRB approval from participating institutions. Orca-T was produced from G-CSF-mobilized peripheral blood (PB) from matched related donors (n=72), matched unrelated donors (n=62), or mismatched unrelated donors (MMUD, n=4). Median follow-up for recipients was 300 days (range: 27-1941). Median age was 49 years, and diagnoses included AML (43%), ALL (27%), MDS (10%), myelofibrosis (7%), and CML (6%). Patients received myeloablative conditioning (busulfan-based, n=109; TBI-based, n=27; BCNU, n=2) followed by GVHD prophylaxis with either single-agent tacrolimus (tac, n=127), sirolimus (n=7), or tac plus mycophenolate (n=4, MMUD). A contemporaneous CIBMTR-based control arm was obtained that consisted of patients with similar diagnoses who received myeloablative alloHSCT from a PB source followed by tac/methotrexate PPX.

Results: Orca-T was successfully manufactured, distributed, and infused for all patients enrolled. Overall time from donor centers to recipient centers was under 60 hours in all cases. Median time to neutrophil engraftment was 13 days. The rates of grade ≥ 3 acute GVHD in the first 180 days and moderate to severe chronic GVHD through 1 year were low with Orca-T at 4% and 5%, respectively. NRM was infrequent at 4% through 1 year. Orca-T exhibited GRFS of 71% & OS of 90% at 1 year. No formal comparison to the CIBMTR cohort was performed, and a Phase 3 study has been initiated to confirm these findings. Longitudinal immune reconstitution data was collected and will be presented. Clinical data is summarized in Table 1.

*MAGIC Criteria **NIH Consensus Grading

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Summary/Conclusion: Results from patients treated with Orca-T, a high-precision Treg-engineered donor product, suggest a reduction in cGVHD, improved GRFS, and low toxicity relative to historic data. Orca-T manufacturing was accomplished with consistent and reliable cell manufacturing and distribution across a wide geographic area. A multicenter randomized-control trial phase 3 trial comparing Orca-T to SOC has been initiated.

S238: MATCHED RELATED VERSUS UNRELATED VERSUS HAPLOIDENTICAL DONORS FOR ALLOGENEIC TRANSPLANTATION IN AML PATIENTS ACHIEVING FIRST COMPLETE REMISSION AFTER TWO INDUCTION COURSES: A STUDY FROM THE ALWP/EBMT

A. Nagler 1,* , M. Labopin 2 , S. Mielke 3 , J. Passweg 4 , D. Blaise 5 , T. Gedde-Dahl 6 , J. J. Cornelissen 7 , U. Salmenniemi 8 , I. Yakoub-Agha 9 , P. Reményi 10 , G. Socié 11 , G. Van Gorkom 12 , H. Labussière-Wallet 13 , X.-J. Huang 14 , M. Thérèse Rubio 15 , J. L Byrne 16 , C. Craddock 17 , L. Griskevicius 18 , F. Ciceri 19 , M. Mohty 20

1 Hematology Division, Sheba Medical Center, Ramat-Gan, Israel; 2 EBMT ALWP office, Hôpital Saint-Antoine, Paris, France; 3 Hematology, Karolinska University Hospital; 4 Department of Cell Therapy and Allogenic Stem Cell Transplantation (CAST), Department of Laboratory Medicine (LabMED), Karolinska University Hospital and Institutet, Karolinska Comprehensive Cancer Center, Stockholm, Sweden; 5 Programme de Transplantation & Therapie Cellulaire, Centre de Recherche en Cancérologie de Marseille, Paoli Calmettes, Marseille, France; 6 Hematology Dept., Section for Stem Cell Transplantation, Oslo University Hospital, Rikshospitalet, Clinic for Cancer Medicine, Oslo, Norway; 7 Department of Hematology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, Netherlands; 8 Stem Cell Transplantation Unit, HUCH Comprehensive Cancer Center, Helsinki, Finland; 9 CHU de Lille LIRIC, INSERM U995, Université de Lille, Lille, France; 10 Haematology and Stem Cell Transplant, Dél-pesti Centrumkórház – Országos Hematológiai és Infektológiai Intézet, Budapest, Hungary; 11 Hematology – BMT, Hopital St. Louis, Paris, France; 12 Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, Netherlands; 13 Service Hematologie, Centre Hospitalier Lyon Sud, Lyon, France; 14 Institute of Haematology, Xicheng District, Peking University People´s Hospital, Beijing, China; 15 CHRU BRABOIS, Vandoeuvre les Nancy, Nancy, France; 16 Nottingham University, Hucknall Road, Nottingham; 17 Haematology, University Hospital Birmingham NHSTrust, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, United Kingdom; 18 Haematology, Oncology & Transfusion Center, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania; 19 Hematology and BMT, San Raffaele Scientific, Milan, Italy; 20 EBMT ALWP office Paris, Saint Antoine Hospital, Paris, France

Background: Transplantation (HSCT) outcome is significantly inferior in AML patients (pts) who required 2 rather than 1 induction course to achieve CR1, with a higher relapse (RI) and lower leukemia-free (LFS) and overall survival (OS) and lower graft-versus-host disease (GVHD)-free, relapse-free survival (GRFS).

Aims: To allocate the best donor for HSCT in AML pts achieving CR1 after 2 inductions comparing matched related sibling donors (MSD) with 9-10/10 HLA compatible unrelated donors (UD) and non-T cell depleted haploidentical (Haplo) donors, respectively.

Methods: This was a retrospective analysis including adult pts aged ≥18 years with AML undergoing HSCT while in CR1 achieved after 2 inductions during the period 2010-2020. Allografts were from MSD, UD or Haplo donors. Multivariate analysis (MVA) adjusting for potential confounding factors was performed using a Cox’s proportional-hazards regression model for main outcomes.

Results: 1295 pts were included: MSD (n=428), UD 10/10 (n=554), UD 9/10 (n=135), Haplo (n=178). Median follow up was 48.4, 46.0,40.5 and 35.2 months, respectively. Median age was 52.1, 53.8, 52.2 and 49.5 years, respectively. The 4 groups did not differ with respect to pts’ gender, type of AML, cytogenetics, and performance status. Haplo transplants were performed more recently, with more BM grafts and lower frequency of pt CMV seropositivity. Post-transplant cyclophosphamide (PTCy) was the main anti GVHD prophylaxis in HSCT from Haplo donors, while in vivo T-cell depletion was more frequently used in transplants from UD. Myeloablative conditioning was less frequently used in transplants from UD and time from diagnosis to HSCT was shorter in transplants from MSD. Cumulative incidence of ANC >0.5x109/L at day 30 was 98.1%, 98.5%, 95.5% and 92.1%, respectively (global P value=0.03). Univariate analysis of 2- and 5-year outcomes after transplantation are given in Table 1. On MVA, acute (a) GVHD II-IV but not III-IV was higher in all groups compared to MSD: UD 9/10, HR=2.53; 95% CI 1.68-3.82, P<0.0001; UD 10/10, HR=1.96; 95% CI 1.42-2.69, P<0.0001 and Haplo HR=2.15; 95% CI 1.41-3.3, P=0.0004. Incidence of extensive chronic (c) GVHD was significantly higher in UD 9/10 (HR=2.52; 95% CI 1.55-4.11, P=0.0002) and UD 10/10 (HR=1.48; 95% CI 1.03-2.13, P=0.036) and cGVHD all grades was higher in UD 9/10 vs MSD (HR=1.77; 95% CI 1.26-2.49, P=0.0009), while it did not differ in Haplo transplants. Non-relapse mortality (NRM) was higher in HSCT from UD 10/10, 9/10 and Haplo donors compared with those from MSD (HR= 1.75; 95% CI 1.16-2.63, P= 0.007, HR =2.22; 95% CI 1.29-3.83, P= 0.004 and HR=2.53; 95% CI 1.47-4.34, P=0.0008), respectively. RI, LFS and OS did not differ significantly between donor types: RI HR=0.84, P=0.18; HR=0.9, P=0.59 and HR=0.76, P=0.17 in UD 10/10, UD 9/10 and Haplo vs MSD, respectively. LFS HR=1.02, P=0.87; HR=1.15, P=0.35 and HR=1.11, P=0.49 and OS HR=1.12, P=0.3, HR=1.27, P=0.14 and HR=1.33, P=0.081, respectively. Finally, GRFS was lower in HSCT from UD 9/10 (HR=1.56, 95% CI 1.20-2.03, P<0.001) but not in those from UD 10/10 (HR=1.13, P=0.22) and Haplo donors (HR=1.12, P=0.43) compared to MSD, respectively. Main cause of death in all groups was original disease, followed by infections, and GVHD.

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Summary/Conclusion: In AML patients undergoing allogeneic transplantation in CR1 achieved after two induction courses which has been shown to be a poor prognostic factor, and those who do not have an MSD, HLA matched UD and Haplo donors are comparable alternatives. 40-50% of these pts achieved long term LFS.

S239: COMPARABLE LONG-TERM OUTCOMES BETWEEN UPFRONT HAPLOIDENTICAL AND IDENTICAL SIBLING DONOR HSCT IN APLASTIC ANEMIA: A NATIONAL REGISTRY-BASED STUDY

Z. xu 1,* , L. xu 1 , D. wu 2 , S. wang 3 , X. zhang 4 , R. xi 5 , S. gao 6 , L. xia 7 , J. yang 8 , M. jiang 9 , X. wang 10 , Q. liu 11 , J. chen 2 , M. zhou 3 , X. huang 1

1 Peking university institute of hematology, Beijing; 2 The First affiliated Hospital of Soochow University, Soochow; 3 Guangzhou First People’s Hospital, Guangzhou; 4 Xinqiao Hospital affiliated to Third Military Medical University, Chongqing; 5 General Hospital of Lanzhou Military Region of PLA, Lanzhou; 6 The First Hospital of Jilin University, Changchun; 7 Xiehe Hospital; 16 affiliated to Huazhong University of Science and Technology, Wuhan; 8 Changhai Hospital affiliated to Second Military Medical University, Shanghai; 9 The First affiliated Hospital of Xinjiang Medical University, Urumchi; 10 Shandong Provincial Hospital, Jinan; 11 Nanfang Hospital affiliated to Southern Medical University, Guangzhou, China

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a curative option for severe aplastic anemia (SAA), and transplantation from identical sibling donors (ISDs) has been recommended as a first-line treatment.

Aims: Haploidentical donor (HID) transplantation for SAA has made great advances; thus, an increased role of HID-SCT in SAA should be considered if an ISD is unavailable.

Methods: We performed a national registry-based analysis comparing long-term outcomes in the upfront HID or upfront ISD SCT setting.

Results :

A total of 342 SAA patients were enrolled, with 183 patients receiving HID SCT and 159 receiving ISD SCT.

The estimated 9-year overall survival (OS) and failure-free survival (FFS) were 87.1±2.5% and 89.3±3.7% (P = 0.173) and 86.5±2.6% vs. 88.1±3.8% (P = 0.257) for patients in the HID and ISD SCT groups, respectively. Transplantation from HID or ISD SCT has greatly improved quality of life (QoL) levels post-HSCT compared to pre-HSCT. The occurrence of chronic graft versus host disease was the only identified adverse factor affecting each subscale of QoL. Physical and mental component summaries in adults as well as physical, mental, social, and role well-being in children were all similar between HID and ISD SCT at 5-year points. At the last follow-up, the proportion of returning to society was comparable between the HID and ISD groups, showing 78.0% versus 84.6% among children and 74.6% versus 81.2% among adults.

A total of 342 SAA patients were enrolled, with 183 patients receiving HID SCT and 159 receiving ISD SCT. The estimated 9-year overall survival (OS) and failure-free survival (FFS) were 87.1±2.5% and 89.3±3.7% (P = 0.173) and 86.5±2.6% vs. 88.1±3.8% (P = 0.257) for patients in the HID and ISD SCT groups, respectively. Transplantation from HID or ISD SCT has greatly improved quality of life (QoL) levels post-HSCT compared to pre-HSCT. The occurrence of chronic graft versus host disease was the only identified adverse factor affecting each subscale of QoL. Physical and mental component summaries in adults as well as physical, mental, social, and role well-being in children were all similar between HID and ISD SCT at 5-year points. At the last follow-up, the proportion of returning to society was comparable between the HID and ISD groups, showing 78.0% versus 84.6% among children and 74.6% versus 81.2% among adults.

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Summary/Conclusion: These data suggest the recommendation for upfront transplantation with a haploidentical donor for SAA patients in the absence of an ISD, especially in experienced transplant centers.

S240: CD7 CHIMERIC ANTIGEN RECEPTOR T CELLS BRIDGING TO ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IMPROVED DISEASE-FREE SURVIVAL IN REFRACTORY/RELAPSED T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Z. Li 1,* , X. Wang 1 , X. Wen 1 , T. Xu 1 , Y. Song 1 , T. Wu 1

1 BEIJING GOBROAD BOREN HOSPITAL, Beijing, China

Background: Our previous clinical study has shown that the patients with refractory/relapsed T-cell acute lymphoblastic leukemia (r/r T-ALL) have achieved 90% complete remission (CR) with donor-derived CD7 chimeric antigen receptor T cells (CART), but some cases have developed donor-recipient mixed chimerism and remarkable pancytopenia (Pan J. et al. JCO 2021). It is crucial to quickly bridge to allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematopoietic reconstitution.

Aims: In current study, the safety and efficacy of allo-HSCT in r/r T-ALL after CD7-CART are investigated.

Methods: From February 2018 to December 2021, total 57 patients with r/r T-ALL who underwent allo-HSCT in our hospital were included. The median age was 13(4-69) years old. Extramedullary disease (EMD) was found in 32 patients (56.1%). Somatic gene mutations (NOTCH 18, IL7R 3, JAK 2, FBXW7 2, WT1 2, NRAS 2) were detected in 17 patients. Eighteen patients who were resistant to chemotherapy received CD7-CART (CART group) before transplant, and all patients except one achieved CR. Thirty-nine r/r T-ALL patients were managed with chemotherapy before transplant (non-CART group), and 25 patients achieved CR (CR arm), 14 patients were in non-remission (NR) (NR arm) before transplantation. Conditioning regimens in CART group were either busulfan/fludarabine (n=12) or TBI/fludarabine (n=6). Conditioning regimens in non-CART group were either busulfan/fludarabine (n=11) or TBI/ fludarabine (n=28). ATG was used for haploidentical or unrelated transplants. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for GVHD prophylaxis.

Results: In CART group, the median time of neutrophils and platelets engraftment were 15 (10-22) and 18 (8-50) days. With the median follow-up 283 (14-496) days, 13 patients survived and 5 patients died (infection 4, cerebral hemorrhage 1). Three of 18 (16.7%) patients relapsed (CD7- 1, CD7 + 2) after transplantation and have been receiving treatment so far. One-year overall survival (OS) and disease-free survival (DFS) were 77.8% and 61.2%. Transplantation-related mortality (TRM) was 16.6%. In non-CART group, the median time of neutrophils and platelets engraftment were 15 (10-22) and 15 (8-28) days. Total 25 patients survived and 14 cases died (relapse 3, infection 4, GVHD 1, multiple organ failure 6). Fifteen of 39 (38.5%) patients relapsed. In CR arm, with the median follow-up 637(9-1357) days, one-year OS and DFS were 71.0% and 59.2%. In NR arm, with the median follow-up 517 (7-513) days, one-year OS and DFS were 59.2% and 40%. TRM was 28.2%.

Summary/Conclusion: Donor-derived CD7-CART followed by allo-HSCT has achieved similar survival (OS, DFS) and lower relapse rate and TRM compared with chemotherapy followed by allo-HSCT in r/r T-ALL. CD7-CART may result in better disease control which will translate into less disease recurrence after transplant.

S241: NON-RESTRICTIVE DIET DOES NOT INCREASE GASTROINTESTINAL INFECTIONS AND FEBRILE NEUTROPENIA IN PATIENTS WITH NEUTROPENIA AFTER STEM CELL TRANSPLANTATION: DATA FROM A MULTICENTRE, RANDOMIZED TRIAL

F. Stella 1,* , V. Marasco 2 , G. Levati 2 , A. Guidetti 1 , A. Chiappella 2 , G. Perrone 2 , M. Pennisi 2 , C. Tecchio 3 , N. Mordini 4 , G. Ferrara 2 , G. Gobbi 2 , L. Saracino 2 , C. Carniti 2 , P. Corradini 1 2

1 Dept. of Oncology and Hematology, Università degli studi di Milano; 2 Division of Hematology and Bone Marrow Transplant, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan; 3 Department of Medicine, University of Verona, Verona; 4 Division of hematology, Azienda Ospedaliera S. Croce e Carle, Cuneo, Italy

Background: Infections represent a major cause of morbidity and mortality during neutropenia, especially after hematopoietic stem cell transplantation (HSCT). Multiple preventive measures, including low microbial diet, have been adopted to reduce these complications. However, the effect of a protective diet (PD) in this setting has never been explored prospectively and evidence-based results lack. Conversely, there is evidence that PD could negatively affect the quality of life and that a prolonged fasting duration can increase the incidence of acute gastrointestinal graft-versus-host disease (aGVHD) in allo-HSCT recipients.

Aims: To prospectively compare infection and aGVHD rates in patients receiving a non-restrictive diet (NRD) compared to PD after HSCT.

Methods: Since July 2016 we conducted a multicentre randomized interventional study comparing the use of a PD (Arm A) vs NRD (Arm B) in hematological patients aged≥18 years hospitalized to receive autologous or allogenic HSCT. Stratification for allo-HSCT patients was planned. Patients received the assigned diet during the entire period of neutropenia. For PD, foods cooked >80°C and/or thick peel fruit were considered diet-specific. For the NRD, raw fruit and vegetables (adherent to hospital hygiene standards) were considered diet-specific.

The primary objective was to demonstrate the lack of significant differences in incidence of infections grade >2 (according to CTCAE 4.0) and deaths during neutropenia between the two arms.

Secondary endpoints included assessment of gastrointestinal infections and fever of undetermined origin (FUO), change in body weight, length of hospitalization, overall survival estimated at 30 days and cumulative incidence of aGVHD within 100 days after allo-HSCT.

Results: Overall, 162 patients were analyzed at interim analysis, 80 patients in PD group and 82 in NRD group. The two arms were well balanced in terms of: sex, age, disease type, number of previous therapeutic lines, disease status at enrollment, antimicrobial prophylaxis, and reason for hospital admission. Moreover, 32 patients received an allo-HSCT, 17 in the PD and 15 in the NRD group respectively. Detailed patients’ characteristics are summarized in Table 1.

We did not observe a significant difference in terms of infections in the two randomized arms; infections grade >2 or death were reported in 35 (43.7%) patients in arm A and in 34 (41.5%) patients in arm B [relative risk RR=1.05, confidence interval (CI)95%=0.76-1.4]. The number of patients developing gastrointestinal infections and FUO during hospitalization was 8 (10%) vs 8 (9.8%) [RR=1.01, CI95%=0.57-1.53] and 32 (40%) vs 28 (34.1%) [RR=1.13, CI95%=0.82-1.54] in arm A and arm B, respectively. No differences in weight variations from admission to discharge were observed comparing arm A and arm B (mean 4.15kg vs 3.66kg, p=0.3). Average hospitalization length in the two arms was respectively 20.6 vs 21.5 days (p=0.4). No deaths were reported at day+30. For 32 allo-HSCT recipients, aGVHD grade ≥2 incidence at day +100 was 5% vs 1.2% in arm A and arm B, respectively (RR 1.65, CI95%=0.77-2.19).

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Summary/Conclusion: Results of this multicentre prospective trial show similar rate of infections and deaths between patients receiving a PD versus a NRD during neutropenia after HSCT. These data suggest that the use of NRD could be considered for transplanted patients without risks of more infective events.

S242: EARLY INFUSION OF MSCS IN THE PROPHYLAXIS OF GVHD AFTER HAPLO-HEMATOPOIETIC STEM-CELL TRANSPLANTATION

R. Huang 1,* , T. Chen 1 , S. Wang 2 , J. Wang 3 , Y. Su 4 , J. Liu 5 , Q. Wen 1 , P. Kong 1 , C. Zhang 1 , L. Gao 1 , L. Gao 1 , X. Zhang 1

1 Medical Center of Hematology, Xinqiao Hospital of Army Medical University, Chongqing, 2920th Hospital of Joint Logistics Support Force of People’s Liberation Army of China, Sichuan; 3 The Affiliated Hospital of Guizhou Medical University, Guiyang; 4 The General Hospital of Western Theater Command PLA, Sichuan; 5 The Third Xiangya Hospital of Central South University, Hubei, China

Background: After the efficiency of repeated infusion umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in preventing chronic graft versus host disease (GVHD) from 100 days after haploidentical hematopoietic stem-cell transplantation (haplo-HSCT) was proved in the last trial, we design a sequel clinical trial to further explore the early repeated infusion of UC-MSCs in GVHD prophylaxis.

Aims: To evaluate the efficacy of repeated infsion of MSCs in GVHD prophylaxis from D45 after haplo-HSCT.

Methods: 128 qualified subjects aged between 18 and 60 with haplo-HSCT in five transplant centers in Mid-West of China were enrolled and randomly assigned equally into the MSCs group and the control group. Patients in the MSCs group received 4 rounds infusion of UC-MSCs (1×10 6 cells/kg per two weeks from 45 days after transplantation). We investigated the incidence of GVHD and relapse free survival (GRFS), the incidence and severity of aGVHD and cGVHD and the accumulative relapse rate.

Results: The one-year GRFS in the MSCs group is 65.6% in the MSCs group, which is significantly higher than the control group (43.7%, P= 0.0129). The accumulative incidence of III-IV aGVHD and severe cGVHD of patients in the MSCs group significantly decreased, which is 3.1% and 6.3%, while the control group is 25.6% and 18.8% (P=0.0089 and P=0.0260). The MSCs infusion did not influence the accumulative relapse rate, which is 10.9% in both groups.

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Summary/Conclusion: In this trial, we proved that early repeated infusion of UC-MSCs could effectively improve the GRFS rate for patients after haplo-HSCT by decreasing the incidence and severity of GVHD without increasing the incidences of relapse for these patients.

S243: ANTIBODY AND T-CELL RESPONSE SIX MONTHS AFTER INITIATION OF BNT162B2 VACCINATION IN PATIENTS WITH MULTIPLE MYELOMA OR CHRONIC LYMPHATIC B-CELL LEUKEMIA COMPARED TO HEALTHY CONTROLS

L. D. Heftdal 1 2 3,* , L. Pérez-Alós 4 , S. R. Ham 2 , J. R. Madsen 4 , K. Fogh 5 , C. C. Kronborg 2 , A. P. Vallentin 1 3 , R. B. Hasselbalch 5 , S. R. Ostrowski 6 7 , R. Frikke-Schmidt 7 8 , E. Sørensen 6 , L. Hilsted 8 , H. Bundgaard 7 9 , K. Iversen 5 7 10 , P. Garred 4 7 , S. D. Nielsen 2 7 11 , K. Grønbæk 1 3 7

1 Department of Hematology; 2 Department of Infectious Diseases, Rigshospitalet, Copenhagen University Hospital; 3 Biotech Research and Innovation Centre, University of Copenhagen; 4 Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Rigshospitalet, Copenhagen University Hospital, Copenhagen; 5 Department of Cardiology, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev; 6 Department of Clinical Immunology, Section 2034, Rigshospitalet, Copenhagen University Hospital; 7 Department of Clinical Medicine, University of Copenhagen; 8 Department of Clinical Biochemistry; 9 Department of Cardiology, Rigshospitalet, Copenhagen University Hospital, Copenhagen; 10 Department of Emergency Medicine, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev; 11 Department of Surgical Gastroenterology and Transplantation, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark

Background: The ongoing COVID-19 pandemic has resulted in more than 419 million cases and more than 5.9 million deaths. Previous studies have indicated inferior responses to SARS-CoV-2 vaccination across different hematological diseases. Through this prospective cohort study, we examined the development and durability of anti-receptor binding domain (RBD) IgG after two doses of BNT162b2 in 179 patients with either multiple myeloma (MM) or Chronic Lymphatic B-cell Leukemia (B-CLL) six months after vaccination and compared to immunocompetent controls.

Aims: We aimed to investigate the durability of immune responses to COVID-19 vaccination in patients with MM or B-CLL compared to healthy controls, and to identify risk factors for humoral non-response, including type of diagnosis.

Methods: We measured anti-receptor binding domain (RBD) IgG after two doses of BNT162b2 in 179 patients (MM: n=78, B-CLL: n=101) and 179 age and sex matched healthy controls up to six months after first vaccination. Anti-RBD IgG levels and neutralizing capacity of antibodies were measured at first and second dose of BNT162b2 and two and six months after first dose. Humoral response was defined as anti-RBD IgG > 225 AU/mL with a neutralizing index ≥ 25%. Humoral non-response was defined as the absence of a humoral response. T-cell responses were assessed six months after the first dose using an ELISA-based interferon-gamma release assay. A positive T-cell response was defined as IFN-ɣ release > 200 mIU/mL. Data on diagnoses were obtained through medical records, and data on vaccination status were obtained from the Danish Vaccination Register.

Results: In patients with MM or B-CLL, the geometric mean concentration (GMC) of anti-RBD IgG increased from baseline 1.49 AU/mL (95% CI: 1.21-1.84) to three weeks after the first vaccine dose 15.10 AU/mL (95% CI: 9.39-24.29) and after receiving the second dose 1179.60 AU/mL (95% CI: 727.78-1919.85). From two to six months after first vaccine there was a significant decline in the GMC of anti-RBD IgG to 252.75 AU/mL (95% CI: 159.17-403.43). The mean neutralizing capacity in patients with MM or B-CLL was lower than in controls at all time points after the first vaccine dose. Six months after first vaccine dose, 79 of 179 (44.1%) patients with MM or B-CLL had a positive humoral response, while this was the case for 170 of 179 controls (95.0%), p<0.001.

Having MM or B-CLL was significantly associated with risk of humoral non-response. This was most pronounced in B-CLL patients who had an age and sex adjusted risk ratio (RR) of 12.25 (95% CI: 6.42-23.38, p< 0.001) of humoral non-response compared to healthy controls. For MM patients the RR was 4.65 (95% CI: 2.21-9.80, p< 0.001).

T-cell response was assessed in a subset of 48 patients with MM (n=28) or B-CLL (n=20) and 26 controls, six months after first vaccine dose. A total of 21 (43.8%) patients with MM (12/28) or B-CLL (9/20) and 14 (53.8%) controls had a positive T-cell response (p =0.56). Seven of 20 (35.0%) patients with MM or B-CLL who did not develop a humoral response, developed a T-cell response (MM: 3/8, B-CLL: 4/12), while 14 of 28 (50.0%) patients with MM or B-CLL who developed a humoral response developed a T-cell response (p =0.46, MM: 9/11, B-CLL: 5/8). In healthy controls 14 of 25 (56.0%) people who developed a humoral response also developed a T-cell response.

Summary/Conclusion: Humoral response to BNT162b2 was impaired in patients with MM or B-CLL compared to healthy controls. Both patients with MM and B-CLL were at higher risk of humoral non-response compared to healthy controls.

S244: EFFECT OF ANTI-SPIKE NEUTRALIZING MONOCLONAL ANTIBODIES ON COVID-19 PROGRESSION AND TIME TO VIRAL CLEARANCE IN PATIENTS WITH HEMATOLOGICAL MALIGNANCIES AND SARS-COV-2 INFECTION: THE GIMEMA EXPERIENCE

V. Marasco 1,* , A. Guidetti 1 2 , A. Piciocchi 3 , A. Candoni 4 , M. Bocchia 5 , R. Bruna 6 , P. Musto 7 , A. Visentin 8 , M. Turrini 9 , A. Tucci 10 , C. Selleri 11 , E. Crea 3 , P. Fazi 3 , F. Passamonti 12 , P. Corradini 1 2

1 Hematology and Bone Marrow Transplantation, Istituto Nazionale Dei Tumori di Milano; 2 School of Medicine, Università degli Studi di Milano, Milan; 3 GIMEMA Foundation, Rome; 4 Dipartimento di Medicina Specialistica, University of Udine, Udine; 5 Hematology Unit, University of Siena, Azienda Ospedaliero Universitaria Senese, Siena; 6 Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont and Ospedale Maggiore della Carità, Novara; 7 Department of Emergency and Organ Transplantation, “Aldo Moro” University School of Medicine and Unit of Hematology and Stem Cell Transplantation, AOU Consorziale Policlinico, Bari; 8 Hematology and Clinical Immunology unit, Department of Medicine, University of Padova, Padova; 9 Hematology, Ospedale Valduce, Napoli; 10 Department of Hematology, ASST Spedali Civili di Brescia, Brescia; 11 Hematology, Ospedale San Giovanni di Dio e Ruggi D’Aragona, Salerno; 12 Department of Medicine and Surgery, niversity of Insubria and ASST Sette Laghi, Ospedale di Circolo of Varese, Varese, Italy

Background: Patients with hematological malignancies (HM) infected with SARS-CoV-2 have a higher risk of developing severe coronavirus disease (COVID-19) with consequent death, due to immune system impairment. Anti-spike Neutralizing Monoclonal Antibodies (nMoAbs) are indicated for the treatment of paucisymptomatic COVID-19 patients, but evidence of safety and efficacy among HM subjects is still lacking.

Aims: To assess the efficacy of different nMoAbs approved by Agenzia Italiana del Farmaco (AIFA) on HM patients affected by paucisymptomatic SARS-COV-2.

Methods: Multicenter retrospective observational study at ten sites in Italy, which enrolled consecutive patients with SARS-CoV-2 infection and treated with nMoAbs from February 2020 to December 2021. Only HM subjects on treatment or in disease remission within 6 months from treatment discontinuation with paucisymptomatic SARS-COV-2 infection were included. nMoAbs approved by AIFA include Bamlanivimab, Bamlanivimab/Etesevimab, Casirivimab/Imdevimab, Sotrovimab, and Regdanvimab. The primary endpoint was to assess the time to SARS-CoV-2 molecular swab negativization. A comparison to an historical control not receiving nMoAbs was assessed. Secondary endpoints consisted in evaluation of hospitalization rate due to COVID-19, including intensive care unit (ICU) admission rate due to respiratory failure, and safety assessment.

Results: Overall 51 HM patients (median age 62 years; 35% women) were evaluated. Seventeen of them had non-Hodgkin lymphomas, 9 multiple myeloma, 6 chronic lymphocytic leukemia, 6 acute myeloid leukemia, 3 Hodgkin lymphoma, 2 acute lymphoblastic leukemia, 2 myeloproliferative neoplasm, 1 Waldenstrom macroglobulinemia and 5 had other HM diagnosis. Thirty-six patients were on active treatment, whereas 11 had completed their therapies within 6 months from nMoAbs administration, for 4 patients data were missing. In 7 subjects the last treatment was chemotherapy, in 19 immunotherapy with or without chemotherapy, in 9 target therapy, in 4 autologous stem cell transplantation, in 2 allogeneic stem cell transplantation, for 4 patients data were missing. Detailed description of patients’ characteristics is reported in table 1. Twenty-six patients were treated with Bamlanivimab/Etesevimab, 17 with Casirivimab/Imdevimab, 3 with Bamlanivimab, and 2 with Sotrovimab, for 3 patients data were missing. Median time to SARS-CoV-2 molecular swab negativization was evaluable in 41 subjects and was 17 days (min 5, IQR 12-26, max 174). This result compared well with the previous finding of 28 days reported in an historical group of HM patients not treated with nMoAbs. We did not find any subpopulation, according to age, diagnosis, period of infection or type of nMoAbs who achieved a major benefit from nMoAbs treatment. The rate of hospitalization due to COVID-19 progression was 19% (10/51), with an extremely low percentage of patients requiring ICU admission due to sever COVID-19 (2%,1/51). Most frequent side effects included chills (8%), diarrhea (6%), headache (2%), nausea (2%) and vomiting (2%).

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Summary/Conclusion: Among paucisymptomatic SARS-CoV-2 positive HM patients on active treatment or in disease remission within 6 months from treatment discontinuation, the administration of nMoAbs substantially reduced the time to swab negativization compared to an historical control of HM subjects. This treatment was also able to reduce the rate of hospitalization and death due to COVID-19 progression in this high risk group.

S245: ALTERATION OF BONE MARROW NICHE BY ALLOGENEIC IMMUNE REACTION AFTER HSCT

M Kimura 1,* , N. Asada 2 , M. Matsuda 1 , M. Abe 1 , W. Kitamura 1 , K.-I. Matsuoka 1 , H. Fujiwara 2 , M. Ono 3 , W. Ziyi 3 , T. Mukai 4 , Y. Morita 5 , Y. Maeda 1

1 Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science; 2 Hematology and Oncology, Okayama University Hospital; 3 Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama; 4 Immunology and Molecular Genetics; 5 Rheumatology, Kawasaki Medical School, Kurashiki, Japan

Background: Allogeneic stem cell transplantation (allo-SCT) has been considered as a curable therapy for hematologic diseases. Post-transplant myelosuppression is one of the life-threatening complications. Hematopoietic stem cell (HSC) functions are tightly regulated by a specialized microenvironment called “niche” in the bone marrow (BM) and we have identified differential contributions of perivascular stromal cells as HSC niche (Asada et al. Nat Cell Biol 2017). Recent studies indicate that BM niche can be targeted by allo-immune reaction but it remains unclear how the perivascular niche cells are involved in the mechanism of HSC dysfunction after allo-SCT.

Aims: In this study, we aim to investigate the mechanisms of the impairment of the niche cells by allo-immune reactions, and to explore a targetable pathway for myelosuppression after allo-SCT.

Methods: To evaluate the alterations in niche cells after allo-SCT, we utilized allo-SCT mice models (C3H/HeJ into C57BL/6, C57BL/6 into B6D2F1). Hematopoietic cells, including HSCs and mature cells, and perivascular stromal cells in BM were analyzed by flow cytometry (FACS) after transplantation. In imaging studies, we visualized perivascular stromal cells using Nestin-GFP (Nes-GFP), Myh11-Cre ER /tdTomato, LeptinR-Cre/tdTomato transgenic mice and observed them with confocal laser scanning microscopy. The gene expression analysis of niche factors was performed by real-time PCR. To assess the landscape of transcriptional remodeling in BM after allo-SCT, we performed single-cell RNA sequencing (scRNAseq) of BM cells.

Results: FACS analyses showed that Nes-GFP + stromal cells were dramatically reduced, and HSC recovery was severely impaired in allo-SCT mice compared to control mice at day 21 after SCT. Imaging studies also revealed that perivascular stromal cells are morphologically damaged in allo-SCT mice. Nes-GFP + stromal cells in allo-SCT mice presented a decreased expression of niche factors, including Cxcl12 and Scf, which are essential for HSC maintenance, indicating the dysfunction of niche cells. We confirmed the impaired niche function in allo-SCT mice by tandem transplantation analysis in vivo , in which the recovery of syngeneic HSCs was also impaired in damaged microenvironment in allo-SCT mice. Prevention of allo-reactive T cell expansion by post-transplant cyclophosphamide ameliorated the niche cell reduction along with improved recovery of HSCs. These results indicate that niche impairment caused by allo-immune reaction leads to HSC dysfunction. The scRNAseq of BM highlighted the elevated expression levels of inflammation response genes, including Cxcl9 and Cxcl10, and MHC classⅡ, together with the deceased expression of niche factors in niche cells in allo-SCT mice. These alterations seemed to be induced by proinflammatory cytokines such as interferon-gamma (INF-γ) derived from allo-T cells, as the increased number of T cells and high levels of INF-γ in BM were observed in allo-SCT mice. Additionally, evaluation of T cell localization by using imaging techniques of BM revealed that allo-T cells reside significant closer to LepR + niche cells in allo-SCT mice. Since activation of CXCR3, which is a receptor for chemokines Cxcl9 and Cxcl10, has been shown to induce chemotaxis of activated T cells, we treated allo-SCT mice with AMG487, an antagonist of CXCR3, resulted in an improved recovery of both perivascular niche cells and hematopoiesis in allo-SCT mice.

Summary/Conclusion: Collectively, allo-immune reaction severely damages perivascular HSC niche cells, leading to impaired recovery of hematopoiesis after allo-SCT.

S246: A HUMAN BONE MARROW ORGANOID FOR DISEASE MODELLING AND DRUG SCREENING IN BLOOD CANCERS

A. Khan 1 2,* , M. Colombo 2 , J. Reyat 1 , G. Wang 2 3 , A. Rodriguez-Romera 2 , W. X. Wen 2 3 , L. Murphy 2 , B. Grygielska 1 , C. Mahoney 4 , A. Stone 5 , A. Croft 4 , D. Bassett 6 , G. Poologasundarampillai 7 , A. Roy 8 , S. Gooding 8 , J. Rayes 1 , K. Machlus 5 , B. Psaila 2

1 Institute of Cardiovascular Sciences, University of Birmingham, Birmingham; 2 MRC Weatherall Institute of Molecular Medicine; 3 Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford; 4 Rheumatology Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom; 5 Vascular Biology Program, Boston Children’s Hospital, Department of Surgery, Harvard Medical School, Boston, United States of America; 6 Healthcare Technologies Institute, School of Chemical Engineering; 7 School of Dentistry, Institute of Clinical Sciences, University of Birmingham, Birmingham; 8 MRC Weatherall Institute of Molecular Medicine, Department of Paediatrics and National Institute of Health Research (NIHR) Oxford Biomedical Research Centre, University of Oxford, Oxford, United Kingdom

Background: Blood cancers are the most common cancer affecting children, and among the top 10 most common cancers in adults, in whom they remain largely incurable. In the era of advanced “omics”, a major bottleneck to the development of novel therapies is target prioritization and validation. There is a pressing need for experimental platforms that model the cellular and molecular complexity of the bone marrow niche, to enable mechanistic studies into the role of the bone marrow microenvironment in the initiation and propagation of blood cancers.

Aims: Organoid technologies have been transformative in other settings, but an organoid with adequate homology to native haematopoietic tissues has not yet been reported. We therefore sought to develop organoids that emulate the cellular, molecular and spatial architecture of human bone marrow.

Methods: A directed-differentiation protocol was devised in which human iPSCs formed mesodermal aggregates before hydrogel embedding and commitment to vascular and hematopoietic lineages. Multi-modal imaging and single cell RNA-sequencing were used to confirm cellular, molecular and architectural homology to native human hematopoietic tissues.

Results: We optimized hydrogel compositions and cytokine cocktails to support the differentiation of a 3D bone marrow perivascular niche. The resulting organoids contained distinct haematopoietic stem/progenitor cells (HSPCs), stromal and myeloid cellular subtypes including pro-platelet forming megakaryocytes and lumen-forming vasculature. 3D whole-organoid imaging revealed a vascular network with sinusoid-like vessels invested with MSCs/fibroblasts (Fig1A), and the extravasation of haematopoietic cells into vessel lumens. Megakaryocytes closely associated with vessels, and extended pro-platelet protrusions into the vasculature with remarkable similarity to native bone marrow. scRNAseq analysis confirmed transcriptional homology to human bone marrow cellular subtypes, and highlighted strong cell-cell communication networks and haematopoietic support from organoid stroma. We first explored the utility of the organoids to model myelofibrosis, an exemplar malignancy that involves cancer-induced bone marrow remodelling and where there is a need for improved models for target prioritization and novel therapies. Treatment of organoids with TGFβ resulted in a dose-dependent increase in hallmarks of fibrosis (Fig 1B), which was effectively reversed using pharmacological agents, enabling drug screening. Given their homology to native bone marrow, we hypothesized that the organoids may support engraftment of primary human cells. Indeed, cells from healthy donors and patients with blood cancers efficiently engrafted throughout the organoids. After 14 days, organoids engrafted with myelofibrosis but not healthy cells showed dramatic fibrotic remodelling (Fig 1C). The organoids also supported primary cells from other myeloid and lymphoid blood cancers, including those notoriously difficult to maintain in vitro – e.g. myeloma cells engrafted into organoids were >90% viable at 10 days following seeding, while rapidly dying within 48 hrs without organoid support. The ability to study primary cells ex vivo overcomes a huge hurdle to translational research in the myeloma field.

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Summary/Conclusion: This platform is an enabling technology for the interrogation of disease mechanisms in haematological disorders. Target identification and screening using a species-specific, 3D model that can incorporate primary patient cells will reduce dependence on animal models and may accelerate translational research.

S247: EMERGENCY GRANULOPOIESIS INDUCES A LYMPHOID TO MYELOID BIAS SWITCH IN A SUBSET OF HEMATOPOIETIC STEM CELLS

K. Vanickova 1 2,* , P. Danek 1 , M. Milosevic 3 , M. K. Adamcova 1 4 , N. Rai 5 , F. Matteini 6 7 , M. C. Florian 6 7 8 , J. D’Armiento 9 , J. Rohlena 3 , K. Rohlenova 3 , M. Alberich-Jorda 1 4

1 Department of Hemato-oncology, Institute of Molecular Genetics of the CAS, Prague; 4 ; 2 Faculty of Science, Charles University; 3 Institute of Biotechnology of the Czech Academy of Sciences; 4 Childhood Leukaemia Investigation Prague, Department of Pediatric Haematology and Oncology; 2 nd Faculty of Medicine, Charles University in Prague, University Hospital Motol, Prague, Czechia; 5 Department of Pediatrics, Columbia University, New York, United States of America; 6 Stem Cell Aging Group, Regenerative Medicine Program, The Bellvitge Institute for Biomedical Research (IDIBELL); 7 Program for Advancing the Clinical Translation of Regenerative Medicine of Catalonia, P-CMR[C], Barcelona; 8 Center for Networked Biomedical Research on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain; 9 Department of Anesthesiology, Columbia University, New York, United States of America

Background: Granulocytes represent the first line of defense against bacteria and fungi, thus their production needs to be adapted to specific demands. While daily production of granulocytes is sustained by steady-state granulopoiesis, upon infection this program switches to emergency granulopoiesis (EG), to ensure enhanced and accelerated granulocytic production. Recently, it was shown that unperturbed hematopoiesis is sustained by multipotent progenitors, however, during stress conditions, hematopoietic stem cells (HSCs) become active and responsible for coping with stress situations. Yet, whether and how HSCs play a role during EG is unknown.

Aims: The aim is to understand the role of HSCs at early stages of acute infection, and to determine the mechanisms that drive emergency granulopoiesis at the stem cell level.

Methods: The methods used in the project include single cell RNA sequencing, qRT-PCR, ELISA, whole-mount bone marrow microscopy, flow cytometry sorting and analysis, stem cell in vitro cultures, Wnt10b knockout mouse model and murine LPS administration.

Results: To understand the contribution of HSCs to the EG response, we performed single cell RNA sequencing analysis of sorted murine HSCs (Lin - c-Kit + Sca-1 + CD48 - CD150 + ) 4 hours after lipopolysaccharide (LPS) stimulation, which mimics a bacterial infection and activates EG in vivo . Strikingly, we observed radical changes in the HSC cluster composition between PBS control and LPS treated mice. Interestingly, these changing population dynamics were marked by alterations in HSC lineage bias, demonstrating that under EG there is an expansion of the myeloid-bias HSCs at expenses of the lymphoid-bias HSCs. Remarkably, we identified Procr (CD201) as a specific cell surface marker for this lymphoid to myeloid transition, allowing us to distinguish, sort and track the switch from CD201 + lymphoid-bias HSCs (present in PBS control) towards CD201 - myeloid-bias HSCs (present upon LPS stimulation). Further, the abundant CD201 - myeloid-bias HSC clusters were characterized by an inflammatory gene signature. Particularly interesting, was the elevated expression of Wnt10b. We verified that Wnt10b is upregulated in murine primary bone marrow cells upon LPS stimulation in vitro using qRT-PCR. Further, we demonstrated that Wnt10b is released in the bone marrow following LPS treatment in vivo using ELISA and whole-mount microscopy. We showed that Wnt10b activates the canonical Wnt/β-catenin signaling pathway and promotes myeloid differentiation of Lin - c-Kit + Sca-1 + cells in vitro . In addition, our preliminary data suggests that the EG response is impaired in Wnt10b KO mice following LPS stimulation in vivo .

Summary/Conclusion: In conclusion, we observed that HSCs actively take part in early stages of EG by activating an inflammatory signature associated with the switch from lymphoid to myeloid bias, therefore ensuring sufficient production of myeloid cells to fight the infection.

Acknowledgements: This work was partially supported by a GACR grant 22-18300S and institutional funding from the IMG CAS (RVO 68378050).

S248: THE ENDOSOMAL ADAPTOR PROTEIN MYCT1 CONTROLS ENVIRONMENTAL SENSING IN HUMAN HSCS

J. Aguade-Gorgorio 1,* , Y. Jami-alahmadi 1 , M. Kardouh 1 , I. Fares 1 , V. Calvanese 2 , H. Johnson 1 , M. Magnusson 3 , J. Shin 4 , H. Goodridge 4 , J. Wohlschlegel 1 , H. Mikkola 1

1 UCLA, Los Angeles, United States of America; 2 University College London, London, United Kingdom; 3 Lund University, Lund, Sweden; 4 Cedars Sinai, Los Angeles, United States of America

Background: Hematopoietic stem cells (HSC) integrate diverse environmental cues to maintain the balance between quiescence, self-renewal and differentiation throughout life. However, the molecular programs governing HSC stemness become dysregulated during culture, compromising HSC self-renewal and engraftment ability. Despite recent advances in optimizing culture conditions for human HSC, our ability to expand or modify functional human HSCs in culture for therapeutic use is still limited. Our data uncovered MYCT1 (Myc target 1) as a critical human HSC regulator that is selectively expressed in undifferentiated HSPCs and endothelial cells (EC) but becomes drastically downregulated during culture, concomitantly with loss of engraftment potential.

Aims: We aimed to understand the molecular programs controlled by MYCT1 that are essential for maintaining human HSC function.

Methods: We analyzed multiple RNAseq gene expression data sets of cultured and uncultured human HSPCs from developmental and postnatal hematopoietic tissues to identify genes associated with self-renewing HSCs. To assess the importance of MYCT1 expression in human HSC function we transduced fetal liver (FL) and cord blood (CB) HSPCs (CD34+CD38-CD90+ and GPI80+ in FL) with lentiviral shRNA vectors. We quantified the expansion of control or knockdown (KD) HSPCs at distinct time points, and determined engraftment ability at 6, 12, and 24 weeks after transplanting equal numbers of sorted HSPCs into conditioned immunodeficient mice.

To define MYCT1 localization and interactome we performed subcellular fractionation, immunofluorescence, and immunoprecipitation coupled with high-sensitivity mass spectrometry in endothelial cells and the HSC-like KG1 cell line. We assessed endocytosis by quantifying the internalization of fluorescently labeled dextran and transferrin by FACS in human HSPCs and ECs after MYCT1 KD or overexpression (OE). To determine the signaling and cellular consequences of MYCT1 loss we performed phospho-proteomics, western blot, and single cell RNAseq in ECs and/or human HSPCs. HSPC proliferation was quantified after MYCT1 KD or OE by CFSE dilution assay and monitoring of single cell divisions for 5 days.

Results: MYCT1 KD experiments revealed that MYCT1 is critical for human HSPC ex vivo expansion and engraftment after transplantation. We determined that MYCT1 is a membrane-associated protein localized in endosomes, where it interacts with vesicle trafficking components and signaling receptors with critical functions in HSC biology. MYCT1 KD in human ECs and HSPCs led to hyperactivation of endocytosis, a crucial regulatory step determining the responsiveness to extracellular cues and governing cell fate decisions. MYCT1 KD then led to widespread dysregulation of signaling pathways, including a hyperactivation Akt, and cellular functions, including defective proliferation, whereas MYCT1 overexpression had the opposite effect

Summary/Conclusion: Our data suggest that MYCT1 governs human HSC stemness and fate decisions by controlling the sensing of extracellular signals and their downstream signaling through endocytosis. As MYCT1 expression is downregulated in cultured HSPC, this work suggests that the inability to properly sense microenvironmental signals in cultured HSCs is a key mechanism contributing to culture-associated HSC dysfunction that will need to be overcome to improve transplantability of ex vivo expanded human HSCs.

S249: EX VIVO HEMATOPOIETIC STEM CELL (HSC) EXPANSION USING BONE-LINING REINVIGORATING MESENCHYMAL STROMAL CELLS (RMSCS)

S. Sood 1 2,* , L Klein 1 , P. Albert 1 , F. Pilz 1 , N. Schmitt 3 , D. Nowak 3 , M. Essers 1 2

1 Inflammatory Stress in Stem Cells; 2 HI-STEM, DKFZ, Heidelberg; 3 Department of Hematology and Oncology, Heidelberg University, Mannheim, Germany

Background: Bone marrow transplants (BMTs) have highlighted the Hematopoietic Stem Cell (HSC) potential to restore a new functional hematopoietic system in diseased recipients. However, a major roadblock for the clinical application and translational research on HSCs is our limited potential for ex vivo HSC expansion.

Aims: To develop an approach for improved ex vivo HSC expansion using novel bone-lining reinvigorating Mesenchymal Stromal Cells (rMSC)-based culture system.

Methods: Using a functional approach, we developed a robust pipeline for the fluorescence-activated cell sorting (FACS)-based isolation and ex vivo expansion of rMSCs from both murine and patient-individualized human samples. We tested the long-term HSC expansion potential in the rMSC-based co-culture system, phenotypically using FACS based proliferation analysis and functionally using limiting dilution and colony forming assays. Further, we performed transplantation experiments to study the hematopoietic reconstitution ability of the ex vivo expanded HSCs. Moreover, we expanded a single HSC over multiple cell divisions using our culture system to demonstrate bona fide HSC self-renewal potential of the rMSC-based system.

Results: We propose a potent ex vivo HSC expansion system based on novel bone lining-derived reinvigorating Mesenchymal Stromal Cells (rMSCs). Both bulk- and single-HSCs expanded long-term using the rMSC co-culture system maintained phenotypic stemness over multiple cell differentiation cycles and possessed functional bone marrow reconstitution capabilities upon transplant. Notably, our rMSC co-culture system outperformed existing alternatives for HSC expansion including systems using stromal cells, non-cellular coating factors, or different medium compositions. Further, our results highlight the reliable isolation and robust culture of human rMSCs using our experimental strategy, and the potential to also utilize the rMSC co-culture system for the ex vivo expansion of human HSCs.

Summary/Conclusion: We could demonstrate that our rMSC-based system for HSC expansion can play a pivotal role in research to reduce the number of mice used for ex vivo experiments. Moreover, our data also shows that the rMSC-based co-culture can be used for human HSC expansion, opening possibilities of applying this potent system for research on numerous diseases including immunodeficiencies and leukaemia.

S250: CHOLINERGIC SIGNALS PROMOTE THE QUIESCENCE OF NORMAL OR LEUKAEMIC STEM CELLS THROUGH THE ACTIVATION OF ALPHA 7-NICOTINIC RECEPTOR IN BONE MARROW MESENCHYMAL STROMAL CELLS

C. Fielding 1 2 3,* , A. García-García 1 2 3 , C. Korn 1 2 3 , S. Gadomski 1 2 3 4 5 , Z. Fang 1 2 3 , C. Kapeni 1 2 3 , J. L. Reguera 6 , J. A. Pérez-Simón 6 7 , B. Göttgens 1 3 , S. Méndez-Ferrer 1 2 3 8 9

1 Department of Haematology, University of Cambridge; 2 National Health Service Blood and Transplant, Cambridge Biomedical Campus; 3 Wellcome-MRC Cambridge Stem Cell Institiute, University of Cambridge, Cambridge, United Kingdom; 4 Skeletal Biology Section, National Institute of Dental and Craniofacial Research, Bethesda; 5 NIH-OxfordCambridge Scholars Program in partnership with Medical University of South Carolina, Charleston, United States of America; 6 Department of Haematology, University Hospital Virgen del Rocio, 41013 Sevilla, Spain; 7 National Health Service Blood and Transplant, NIH-OxfordCambridge Scholars Program in partnership with Medical University of South Carolina, Charleston, United States of America; 8 Instituto de Biomedicina de Sevilla (IBiS/CSIC); 9 Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, 41013 Sevilla, Spain

Background: Our recent research has suggested that the sympathetic nervous system, a master regulator or homeostasis and stress responses, regulates the activity of hematopoietic stem cells (HSCs) in different bone marrow niches. We recently showed that chemotherapy or irradiation, which are routinely used for cancer treatment or as conditioning regimens for HSC transplantation, increase the release of cholinergic signals by sympathetic neurons and osteolineage cells. As a result, not all HSCs become activated (which could cause their exhaustion), but some remain quiescent, thereby retaining their full potential. Cholinergic signals activate α7 nicotinic receptor (α7nAChR) in bone marrow mesenchymal stromal cells (BMSCs), which in turn preserve stem cell quiescence under proliferative stress (Nat Commun 13:543). In addition, α7nAChR signalling exhibits anti-inflammatory properties (PMID:32595942). However, whether this signalling pathway influences acute myeloid leukaemia (AML) development and response to treatment is unclear.

Aims: Investigate the influence of cholinergic signals on leukaemogenesis and therapy response in AML.

Methods: Analysis of bone marrow (BM) from mouse models lacking sympathetic cholinergic innervation or α7nAChR in BMSCs, transplanted with BM cells from an inducible MLL-AF9+ AML model (Cancer Cell 15:791) and treated with cytarabine. Flow cytometry, histology, FACS, qRT-PCR.

Results: We confirmed reduced density of sympathetic noradrenergic innervation in the BM of AML mice; however, interestingly, the cholinergic innervation, which also has sympathetic origin (Cell Stem Cell, in press) was preserved. To investigate the role of these fibres in AML development, we utilised a mouse model lacking sympathetic cholinergic innervation due to loss of the neurotrophic receptor GFRα2. AML developed faster in cholinergic-neural-deficient mice, compared with WT mice, transplanted with iMLL-AF9+ BM cells. This was associated with reduced quiescence of leukaemia-initiating cells (LIC) upon acute cytarabine treatment of mice lacking cholinergic skeletal innervation. This effect was more pronounced after repeated cytarabine treatment, leading to increased proliferation of LICs and augmented leukaemia burden at relapse.

Given that α7nAChR transduces the cholinergic regulation of normal HSCs by BMSCs, we utilised LepR-Cre;Chrna7f/f mice to conditionally delete this signalling pathway in leukaemic mice. Reproducing the observations in mice lacking cholinergic innervation, α7nAChR deletion in BMSCs accelerated AML development, and repeated treatment with cytarabine further increased LIC proliferation, spleen infiltration and leukaemia burden after relapse.

Summary/Conclusion: These results suggest that cholinergic signals increase the quiescence of normal or leukaemic stem cells through the activation of a7-nicotinic receptor in BMSCs and might influence AML response to chemotherapy.

S251: DISRUPTION OF SUCCINATE RECEPTOR SIGNALING PROMOTES MYELOPROLIFERATION AND ACUTE MYELOID LEUKAEMIA

V. Cuminetti 1,* , J. Konieczny 1 , A. Bernal 1 , A. Villatoro 1 , H. Taman 2 , M. Ristic 1 , N. Villaplana-Lopera 3 , R. H. Paulssen 2 , G. Giovinazzo 4 , A. Vik 5 , P. Garcia 3 , L. Arranz 1

1 IMB, Stem Cells Ageing and Cancer; 2 Genomics Support Center Tromsø, UiT The Arctic University of Norway, Tromsø, Norway; 3 Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom; 4 Pluripotent Cell Technology Unit, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain; 5 Department of Hematology, University Hospital of North Norway, Tromsø, Norway

Background: Unlike healthy haematopoietic stem cells (HSC), leukaemia stem cells (LSC) have high oxidative phosphorylation and hence targeting the tricarboxylic acid (TCA) cycle seems a promising therapeutic strategy in acute myeloid leukemia (AML) treatment. Accumulation of the TCA cycle oncometabolite succinate is associated with tumourigenesis, but its potential involvement in HSC function and leukaemia is unkown. Succinate stabilizes the oxygen-sensing transcription factor hypoxia-inducible factor (HIF)-1-α and activates interleukin (IL)-1β production in macrophages. Succinate may also be exported and has cell extrinsic effects through its receptor, succinate receptor-1 (SUCNR1), which may be pro- or anti-inflammatory.

Aims: To investigate the in vivo role and molecular mechanisms of succinate and its receptor in normal and malignant haematopoiesis.

Methods: To study the effect of succinate receptor on haematopoiesis, we characterized the immunophenotype of Sucnr1 -KO mice. To dissect the role of Sucnr1 on healthy haematopoiesis via cell-autonomous effects, we transplanted fl/Sucnr1/fl control or Mx1-Cre fl/Sucnr1/fl bone marrow (BM) cells into wild-type (WT) C57BL/6J mice, which were later injected with pIpC. To study the role of Sucnr1 through non-cell autonomous effects, we transplanted WT BM into Nes-Cre ERT2 fl/Sucnr1/fl recipients or fl/Sucnr1/fl controls, which were induced with tamoxifen. We characterized the effects of Sucnr1 deletion at the molecular level by bulk and single-cell RNA-sequencing (scRNA-seq), and aimed to uncover the molecular mechanisms mediating the effects of Sucnr1 ablation using in vivo tageting with drugs. To study the effect of succinate on leukaemic progression, we used two different mouse models, i.e. Mx1-Cre Nras G12D and MLL-AF9 knock-in (KI). In human AML, we quantified SUCNR1 expression in peripheral blood mononuclear cells (PBMC) from AML patients versus healthy volunteers, and treated xenograft recipients of CD34+ AML cells with succinate or vehicle.

Results: In vivo deletion of Sucnr1 induced myeloid bias and expansion of the haematopoietic stem and progenitor cell (HSPC) compartment. In vivo deletion of Sucnr1 from Nestin + stromal cells promoted expansion of long-term HSC (LT-HSC) versus intact recipients. Conversely, in vivo deletion of Sucnr1 from the haematopoietic system induced expansion of multipotent progenitors (MPP) versus intact haematopoietic cells. RNA-seq in HSPC from Sucnr1 -KO versus WT mice showed changes in genes associated with myeloid output, and pinpointed related pathways potentially involved. scRNA-seq confirmed expansion of LT-HSC in Sucnr1 -KO versus WT mice. In vivo treatments targeted signalling pathways at least partially responsible for the haematopoietic abnormalities in Sucnr1 -KO. We found higher content of succinate in the BM extracellular fluid of leukaemic Mx1-Cre Nras G12D . Succinate injection to xenograft recipients of CD34 + AML cells increased leukaemic output versus vehicle, promoted myeloid bias in Mx1-Cre Nras G12D mice and reduced survival in mice transplanted with BM of MLL-AF9 leukaemic donors. PBMC from AML patients expressed lower level of SUCNR1 versus healthy donors and BM nucleated cells from Mx1-Cre Nras G12D also expressed less Sucnr1 than their healthy counterparts.

Summary/Conclusion: We show a novel role for succinate and its receptor in the haematopoietic system and pinpoint this pathway as a novel means of communication within the BM HSC microenvironment, whose disruption may be involved in myeloid malignancy. These findings may help design new therapeutic strategies for AML patients.

S252: M2-POLARIZED MACROPHAGES CONTROL LEUKEMIC STEM CELL FATE BY PROMOTING METABOLIC REPROGRAMMING

I. Weinhäuser 1 2 3,* , D. A. Pereira-Martins 1 2 3 , J. R. Hilberink 1 , L. Y. Almeida 2 3 , D. R. A. Silveira 4 , L. Quek 4 , S. M Hogeling 1 , J. M. Mota 5 , E. Ammatuna 1 , A. R. Lucena-Araujo 6 , G. A. Huls 1 , E. M. Rego 7 , J. J. Schuringa 1

1 Experimental Hematology, University medical centre groningen, Groningen, Netherlands; 2 Center for Cell Based Therapy, University of Sao Paulo; 3 Internal Medicine, Medical School of Ribeirao Preto, Ribeirao Preto, Brazil; 4 Myeloid Leukaemia Genomics and Biology Group, Kings College London, London, United Kingdom; 5 Medical Oncology Service, Sao Paulo State Cancer Institute, Sao Paulo; 6 Department of Genetics, Federal University of Pernambuco, Recife; 7 Center for Cell Based Therapy, University of Sao Paulo, Sao Paulo, Brazil

Background: Being a crucial part of the tumor microenvironment (TME) in solid tumors, tumor-associated macrophages are often associated with poor prognosis (Bruni et al. Nat Rev Cancer 2020). Yet, in acute myeloid leukemia (AML) the role of macrophages remains unclear.

Aims: Here, we evaluated the impact of M2 macrophages in AML using a patient derived xenograft (PDX) model.

Methods: To do so, we first injected 1x10 5 peripheral blood (PB) derived M2 macrophages into NSGS mice and next transplanted notoriously difficult to engraft primary Acute Promyelocytic Leukemia (APL) cells (1 x 10 6 cells - n=7 different patient samples) via the retro-orbital vein.

Results: As a result, mice with co-injected human M2-macrophages developed full-blown leukemia with increased spleen weight in comparison with the control. Perhaps even more strikingly, ex vivo culture of APL and other favorable AML subtypes such as inv (16) or NPM1 mutant leukemic cells on M2-macrophages for 48h was sufficient to “train” these cells to engraft and induce fatal leukemia. Maintenance of self-renewal was shown in a secondary transplant assay and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays (LSC frequency: Control: 1/6x10 6 cells vs M2 pre-culture: 1/7.2x10 4 cells).

To better understand the biological changes induced on leukemic blast when exposed to M2 macrophages, we performed an RNA sequencing analysis comparing AML/APL samples at diagnosis to cells that were “trained” (48h) on M2-macrophages or on MS5 mesenchymal bone marrow stromal cells, as a control. Gene ontology and gene set enrichment analysis on the genes up regulated upon M2 co-culture were significantly enriched for oxidative phosphorylation (OxPhos) signatures. Evaluation of functional respiration using seahorse measurements, confirmed the increase of oxygen consumption rate (OCR, basal and maximum) in primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5. The increase of basal and maximum OCR suggested enhanced mitochondrial metabolism, which prompted us to determine whether macrophages, similar to MSC cells, can transfer mitochondria to primary AML cells (van der Vlist et al. Neuron 2022). Flow cytometry analysis revealed an efficient mitochondrial transfer from M2 macrophages to leukemic blasts, which was more efficient compared to mitochondrial exchange from MS5 cells. Treatment with the CPT1A inhibitor Etomoxir (50 µM), prevented the gain in functional respiration and enhanced proliferation of AML cells when those were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures. These results suggest increased fatty acid oxidation to drive the OXPHO-like state in AML.

Summary/Conclusion: Overall, we revealed that M2 macrophages can support leukemic growth of favorable AML subtypes that are commonly difficult to engraft in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter the biology of AML cells transforming a non-engraftable cell into a cell with high leukemic potential. In vitro we show that AML blast cells cultured on M2 macrophage adapt a more OxPhos-like state linked to intrinsic changes associated with increased uptake of mitochondria. In summary, our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions.

S253: THE SHEDDASE DOMAIN OF ADAM10 AUGMENTS THE INTERACTION OF LEUKEMIA CELLS WITH THE BONE MARROW NICHE IN VIVO AS SHOWN BY RECONSTITUTING PDX LEUKEMIA CELLS WITH CRISPR-CAS9-INDUCED KNOCKOUT

J. P. Schmid 1 2,* , E. Bahrami 1 , M. Becker 1 , A. K. Jayavelu 3 , A.-K. Wirth 1 , V. Jurinovic 1 4 5 , R. Öllinger 6 7 8 , R Rad 2 6 7 8 , B. Vick 1 2 , M. Mann 3 , T. Herold 1 5 , I. Jeremias 1 2 4

1 Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU); 2 German Cancer Consortium (DKTK), partner site Munich, Munich; 3 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried; 4 Department of Pediatrics, University Hospital, Ludwig Maximilian University (LMU); 5 Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich; 6 Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Technische Universität München; 7 Department of Medicine II, Klinikum rechts der Isar, Technische Universität München; 8 Institute of Molecular Oncology and Functional Genomics, Technische Universität München, Munich, Germany

Background: Tumor-microenvironment interactions are critically important determinants contributing to leukemia formation and maintenance. Interrupting the leukemia-bone marrow interaction represents an attractive therapeutic approach in acute leukemia (AL). Functional genomics significantly increases our understanding of the vulnerabilities and gene dependencies of individual tumors.

Aims: Here, we developed a CRISPR-Cas9 screening approach for functional analysis of surface molecules in patient-derived xenograft (PDX) AL models in vivo.

Methods: Size of CRISPR library was determined by genetic barcoding. Stable expression of fluorescently labelled Cas9 and sgRNA constructs in two PDX samples. Enrichment of double positive cells by MACS and injection into NSG mice. Gene depletion analysis using MAGeCK algorithm to screen and functional competitive in vivo assays to validate the candidates. Characterization of the ADAM10 KO or inhibitor (GI254023X) treated cells for engraftment capacity by homing assay, frequency of leukemic stem cells by competitive limiting dilution transplantation assay (LDTA), sensitivity towards routine chemotherapy by in vivo competitive chemotherapy trials in both lineages. Rescue assay by reconstitution of ADAM10 variants in functional competitive in vivo assays.

Results: When running a customized CRISPR-Cas9 screen targeting about 100 cell surface candidates in two AL PDX samples, several sample-specific, but also commonly depleted candidates were identified. CRISPR screen findings were confirmed on the level of single molecules, using a competitive molecular in vivo approach and testing the PDX cells with and without knockout in the same mouse. These experiments validated an essential function for the two well-known depleted candidates CXCR4 and ITGB1 in both PDX models in vivo . Of note, various members of the Solute Carrier Family (SLC) were among the list of drop-out candidates. ADAM10 was identified as a commonly depleted candidate in both PDX models. In vivo competitive experiments confirmed the essential role of ADAM10 in PDX models from 6 additional patients with either acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML), indicating a broad essential role of ADAM10 in both, ALL and AML, independent from their oncogenic-driver mutations and chromosomal abnormalities. Moreover, treating PDX cells with an ADAM10 chemical inhibitor resulted in significantly reduced engraftment capacity into the bone marrow (BM), indicating a role for ADAM10 in the early engraftment and homing process in the BM microenvironment. Knockout of ADAM10 reduced the frequency of leukemia stem cells, indicating that a relevant fraction of stem cells depends on ADAM10. ADAM10 KO ALL and AML PDX samples showed increased sensitivity towards routine chemotherapy treatments, indicating that inhibition of ADAM10 sensitizes AL towards conventional chemotherapy. When ADAM10 knockout cells were reconstituted with different recombinant ADAM10 variants, PDX in vivo experiments revealed that wildtype ADAM10 rescued the phenotype, while an ADAM10 variant lacking the enzymatic domain did not, highlighting the importance of the sheddase activity for ADAM10 function in leukemia maintenance.

Summary/Conclusion: In summary, we established CRISPR-Cas9 drop-out screens in PDX models in vivo as technology to explore patient-specific tumor dependencies. Our data revealed a yet unknown function of ADAM10 to maintain patient leukemic cells in the bone marrow microenvironment niche. ADAM10 thus represents an attractive future therapeutic target for the treatment of acute leukemia.

S254: CD4+ T CELL-DERIVED IL21 REGULATES STEM CELL FATE IN ACUTE MYELOID LEUKEMIA

V. Rubino 1 2 3,*

1 Department for BioMedical Research, University of Bern; 2 Department of Medical Oncology, Inselspital, Bern University Hospital; 3 Graduate School of Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland

Background: Acute myeloid leukemia (AML) is characterized by poor prognosis for patients, due to the very high rate of relapse, which occurs even after chemotherapy-induced complete remission. Therapy-insensitive AML cells, so-called leukemia stem cells (LSCs), are thought to be the main driver of relapse. In fact, high LSCs number and active LSC self-renewal programs are related to poor therapy response and adverse outcome in AML. Therefore, eliminating LSCs is an urgent, yet unmet, medical need. LSCs properties are crucially regulated and maintained also through interactions with the bone marrow microenvironment, which includes immune cells. Despite evidences of mutual interactions between AML cells and immune cells, their interplay is still poorly understood. A better understanding of these interactions might provide novel methods to effectively eradicate LSCs and achieve durable AML cure.

Aims: The aim of this study was to investigate the role of IL21/IL21R signaling in the so far unexplored context of AML. Our aims involved identification of IL21 source in murine and human AML and elucidation of the mechanisms how IL21/IL21R signaling regulates LSCs properties.

Methods: We used a combination of flow cytometry, ELISA and qRT-PCR to determine protein and mRNA expression of both IL21 and IL21R in primary AML samples and analyzed the correlation between IL21 serum levels with patients’ survival and complete remission rate. Colony-forming assays and short-term cultures were used to determine the in vitro effect of IL21 on primary normal and leukemic stem cells. In addition, patient-derived xenografts were employed to study IL21 effect on stem cell frequency and AML engraftment capacity.

We used various syngeneic murine AML models (e.g. MLL/AF9 and MLL/ENL) to study the functional role of IL21/IL21R signaling in vivo. We performed RNA-Seq, together with analysis of symmetric cell division, expression of differentiation markers, NF-kB signaling, stem cell frequencies by limiting-dilution secondary transplantation experiments to investigate the mechanisms how IL21 regulates LSCs.

Results: In human AML, serum IL21 was identified as an independent positive prognostic biomarker for overall survival (OS). Furthermore, high dose chemotherapy was more effective in patients with high levels of IL21 at diagnosis, as illustrated by a significantly higher rate of complete remission (CR rate: 81 vs 59 %) and prolonged OS (median survival: 915 vs. 364 days, HR: 1.8). Functionally, IL21 inhibited cell growth and clonogenic potential of primary AML stem and progenitor cells but not normal hematopietic stem cells (HSCs). In murine AML models, IL21/IL21R signaling in LSCs reduced stem cell frequency, prolonged survival of the mice and resulted in the downregulation of stemness-related and the up-regulation of differentiation-promoting pathways. In both human and murine AML, CD4 + T cells were identified as the main source of IL21.

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Иллюстрация — в первоисточнике ↗

Summary/Conclusion: In this work, we have identified IL21/IL21R signaling pathway as an important regulator of cell fate in human and murine LSCs, but not HSCs. We found that IL21 is a positive prognostic marker for OS and that higher serum IL21 levels correlate with better survival and higher rate of complete remission in patients that undergo chemotherapy. Our findings therefore suggest that promoting IL21/IL21R signaling on LSCs may be a novel approach to decrease stemness and increase differentiation in AML.

S255: EFFICACY AND SAFETY OF TISAGENLECLEUCEL IN PEDIATRIC AND YOUNG ADULT PATIENTS (PTS) WITH RELAPSED OR REFRACTORY (R/R) MATURE B-CELL NON-HODGKIN LYMPHOMA (NHL): THE PHASE II BIANCA STUDY

V. Minard-Colin 1,* , J. Buechner 2 , F. Locatelli 3 , B. Gonzalez Martinez 4 , B. J. Vormoor 5 , S. Cooper 6 , J. Krueger 7 , S. Napolitano 8 , A. Attarbaschi 9 , A. Baruchel 10 , S. Ghorashian 11 , M. L. Hermiston 12 , H. Hiramatsu 13 , M. Ifversen 14 , S. John 15 , S. L. Khaw 16 , T. A. O’Brien 17 , C. L. Phillips 18 , C. Diaz de Heredia 19 , D. Tomizawa 20 , K. Vettenranta 21 , A. S. Wayne 22 , S. Newsome 23 , R. Awasthi 24 , S. Redondo 25 , A. Masood 24 , S. L. Maude 26 , B. Burkhardt 27

1 Department of Pediatric and Adolescent Oncology, Gustave Roussy, Université Paris-Saclay, Villejuif, France; 2 Department of Pediatric Hematology and Oncology, Oslo University Hospital, Oslo, Norway; 3 Department of Pediatric Hematology and Oncology, IRCCS Ospedale Bambino Gesù Children’s Hospital, Sapienza, University of Rome, Rome, Italy; 4 Hospital Universitario La Paz, Madrid, Spain; 5 Prinses Maxima Centrum, Utrecht, Netherlands; 6 Johns Hopkins Comprehensive Cancer Center, Baltimore, MD, United States of America; 7 Division of Haematology/Oncology/Bone Marrow Transplantation, Department of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 8 Hematology-Oncology and Bone Marrow Transplantation Unit, Pediatric Department, and Monza and Brianza Foundation for Children and their Mums, San Gerardo Hospital, Lombardia, Monza, Italy; 9 St. Anna Kinderspital and Children’s Cancer Research Institute, Vienna, Austria; 10 University Hospital Robert Debré (APHP) and Université de Paris, Paris, France; 11 Great Ormond Street Hospital for Children NHS Trust Haematology Department, London, United Kingdom; 12 University of California San Francisco Benioff Children’s Hospital, San Francisco, CA, United States of America; 13 Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 14 Department of Pediatrics and Adolescent Medicine, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark; 15 University of Texas Southwestern Medical Center, Dallas, TX, United States of America; 16 Children’s Cancer Centre, Royal Children’s Hospital and Murdoch Children’s Research Institute, Parkville, VIC; 17 Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Australia; 18 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America; 19 Hospital Universitari Vall d’Hebron, Vall d’Hebron Institut de Recerca, Barcelona, Spain; 20 Children’s Cancer Center, National Center for Child Health and Development, Tokyo, Japan; 21 University of Helsinki, Helsinki, Finland; 22 Children’s Hospital Los Angeles, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States of America; 23 Novartis Pharma AG, Basel, Switzerland; 24 Novartis Pharmaceutical Corporation, East Hanover, NJ, United States of America; 25 Novartis Farmaceutica S.A., Madrid, Spain; 26 Division of Oncology, Center for Childhood Cancer Research and Cancer Immunotherapy Program, Children’s Hospital of Philadelphia and Department of Pediatrics, Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 27 Pediatric Haematology and Oncology, University Hospital Muenster, Muenster, Germany

Background: Chimeric antigen receptor (CAR)-T cell therapy (tx) targeting CD19 is approved for treatment of adult r/r large B-cell lymphoma (LBCL) and pediatric r/r B-cell acute lymphoblastic leukemia (ALL). Pediatric and young adult pts with r/r mature B-NHL have dismal prognoses, especially those with Burkitt lymphoma (BL), and limited clinical benefit from available tx.

Aims: Herein we report primary efficacy and safety outcomes from the global, multicenter, open-label, single-arm Phase II BIANCA trial ( NCT03610724 ).

Methods: Pts ≤25 y and ≥6 kg at screening with histologically confirmed CD19+ r/r mature B-NHL after ≥1 prior lines of tx were eligible following successful leukapheresis. Pts received optional bridging chemotherapy and fludarabine/cyclophosphamide or cytarabine/etoposide for lymphodepletion prior to a single intravenous injection of tisagenlecleucel (target dose range, 0.2-5x10 6 /kg bodyweight [pts ≤50 kg] or 0.1-2.5x10 8 [pts >50 kg] CAR+ viable T cells). The primary endpoint was overall response rate (ORR=complete response [CR]+partial response [PR]) by local investigator assessment in the efficacy analysis set (EAS), which excludes pts with pre-infusion CR. Secondary endpoints included progression-free survival (PFS), overall survival (OS), safety, and CAR-T cell kinetics.

Results: As of October 10, 2021, 33/34 pts enrolled received tisagenlecleucel (EAS, N=28). Median age was 13 y (range, 3-22 y), 70% were male, 55% had BL, and 45% had LBCL. Pts received a median of 2 prior tx (18% received prior stem cell transplant [SCT]), 85% had stage III/IV disease at initial diagnosis, 15% were primary refractory, 30% were refractory, and 55% had relapsed/progressed at study entry. Median time from enrollment to infusion was 35 d (range, 7-62 d). Median time from tisagenlecleucel infusion to data cutoff was 16 mo (range, 6-30 mo). Bridging chemotherapy was given to 94%. ORR was 32% (95% CI: 15.9-52.4); 7% had a CR. Subgroup analysis suggested that pts with BL had a lower ORR than pts with LBCL (20% vs 46%). Median PFS was 2.5 mo (95% CI: 1.1-2.9); median OS was 11.4 mo (95% CI: 3.4-not estimable); 12-mo estimated PFS and OS were 23% (95% CI: 8.9-40.3) and 47% (95% CI: 26.6-65.0), respectively. Of the 18 BL pts, 7 (39%) are alive post infusion (1 received allogeneic SCT, 1 surgery, 3 other tx, and 2 no other tx). There were no tx-related deaths; most tx-related grade (gr) ≥3 (94%) and serious (52%) adverse events occurred ≤8 w post infusion. A neurologic event (NE) occurred in 27%; 15% had a gr ≥3 NE, and none had gr 5 NE. Cytokine release syndrome (CRS; per Lee 2014 criteria) occurred in 70%, 9% had gr 3 CRS, and there was no gr ≥4 CRS. Median time to CRS onset and duration were 6 d (range, 1-27 d) and 5 d (range, 1-13 d), respectively. Only 1 pt died ≤30 d post infusion, due to disease progression. Geometric mean maximal expansion (C max ) across all pts was 5730 copies/μg, and median time to C m ax was 12.8 d (range, 2.5-21.9 d) by qPCR. Median CAR-T cell persistence in pts with CR or PR was 182 d (range, 20.9-562 d).

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Summary/Conclusion: Tisagenlecleucel demonstrated efficacy in pediatric and young adult pts with r/r mature B-NHL and comparable safety to that recorded in adults with DLBCL. The OS is encouraging; however, optimal positioning of tisagenlecleucel in the treatment of pediatric r/r B-NHL requires further exploration, especially for BL, which is highly aggressive and challenging to treat in the relapse setting.

S256: TABELECLEUCEL FOR EBV-DRIVEN POST-TRANSPLANT LYMPHOPROLIFERATIVE DISEASE FOLLOWING ALLOGENEIC HEMATOPOIETIC CELL OR SOLID ORGAN TRANSPLANT AFTER FAILURE OF RITUXIMAB ± CHEMOTHERAPY (ALLELE)

S. Prockop, MD 1,* , A. Beitinjaneh, MD 2 , S. Choquet, MD 3 , S. Dahiya, MD 4 , R. Dinavahi, MD 5 , R. Farah, MD 6 , L. Gamelin, MD, PhD 5 , A. Ghobadi, MD 7 , A. Mehta 5 , P. Nayak, MD 8 , R. Reshef, MD 9 , G. Satyanarayana, MD 10 , P. Stiff, MD 11 , W. Ye, PhD 8 , K. M. Mahadeo, MD, MPH 12

1 Boston Children’s Hospital/Dana Farber Cancer Institute, Boston; 2 University of Miami/Jackson Memorial Hospital, Miami, United States of America; 3 Sorbonne Université, Hôpital de la Pitié-Salpêtrière, Paris, France; 4 University of Maryland School of Medicine, Baltimore; 5 Atara Biotherapeutics, Thousand Oaks; 6 UPMC Hillman Cancer Center, Pittsburgh; 7 Washington University, Division of Oncology, St. Louis; 8 Atara Biotherapeutics, South San Francisco; 9 Columbia University Medical Center, New York; 10 Ingram Cancer Center, Vanderbilt Health, Nashville; 11 Loyola University Medical Center, Chicago; 12 MD Anderson Cancer Center, Houston, United States of America

Background: Tabelecleucel is an investigational, off-the-shelf, allogeneic Epstein–Barr Virus (EBV)-specific T-cell immunotherapy being explored in patients (pts) with EBV-driven post-transplant lymphoproliferative disease (EBV + PTLD). Median overall survival (OS) after rituximab (R) ± chemotherapy (C) failure is 0.7 months in EBV + PTLD post allogeneic hematopoietic cell transplant (HCT) (Sanz ASH 2021) and 4.1 months post solid organ transplant (SOT) after R+C failure (Dharnidharka ASH 2021), demonstrating an urgent need for therapies in this ultra-rare disease. Tabelecleucel has shown promising outcomes (Prockop JCI 2020). Additionally, an investigator-assessed objective response rate (ORR) of >60% with >80% 2-year OS (Prockop EBMT 2021, Prockop ATC 2021, Prockop ASH 2021) was seen in pts with relapsed/refractory (R/R) EBV + PTLD, with either complete or partial response to tabelecleucel. Safety data in >180 pts with tabelecleucel treated EBV + PTLD demonstrate tumor flare reaction (TFR) as the only identified risk (Atara Biotherapeutics, Data on file).

Aims: Report data from the ongoing Phase 3 ALLELE study ( NCT03394365 ).

Methods: ALLELE is a multicenter, open-label study investigating tabelecleucel in pts with EBV + PTLD post HCT after R failure (n=33) and post SOT after R±C failure (n=33). Pts receive tabelecleucel at 2 x 10 6 cells/kg on Days 1, 8 and 15 in 35-day cycles. Response is evaluated by investigator and by independent oncologic response adjudication (IORA; primary assessment) using Lugano Classification with LYRIC modification. Efficacy endpoints include ORR, duration of response (DOR), time to response (TTR), and OS. Pts are assessed post treatment for up to 5 years for survival.

Results: As of May 2021, 38 pts (14 HCT, 24 SOT) were evaluable by IORA and had the opportunity for 6 months follow-up. Median age was 52.9 (range, 3.2–81.5) years, 44.4%/47.2% of pts were high/intermediate risk per PTLD-adapted prognostic index, and median number of lines of prior systemic treatment was 1 (range, 1–5). R/R HCT and SOT pts received a median (range) of 3 (1–5) and 2 (1–6) cycles of tabelecleucel, respectively. ORR was 50.0% (19/38, 95% CI: 33.4, 66.6) overall, 50.0% (7/14, 95% CI: 23.0, 77.0) in HCT, and 50.0% (12/24, 95% CI: 29.1, 70.9) in SOT ( Table ). Overall, median TTR was 1.1 (0.7–4.7) months, 11 of 19 responders had DOR > 6 months, and median DOR was not reached ( Table ).

Median OS was 18.4 (95% CI: 6.9, NR) months overall, not yet reached for HCT, and 16.4 (95% CI: 3.5, NR) months for SOT. 1-year survival rates were 61.1% (95% CI: 42.9, 75.0) overall, 66.8% (95% CI: 32.4, 86.6) for HCT, and 57.4% (95% CI: 35.2, 74.5) for SOT. Median OS was NR (95% CI: 16.4, NR) for responders and 5.7 (95% CI: 1.8, 12.1) months for non-responders. Responders had a higher 1-year survival rate vs non-responders: 89.2% (95% CI: 63.1, 97.2) vs 32.4% (95% CI: 12.1, 54.9) ( Table ).

Serious treatment emergent AEs (TEAEs) and fatal TEAEs were reported in 57.1% and 7.1% of HCT and 62.5% and 16.7% of SOT pts respectively. No fatal TEAE was treatment related. There were no reports of TFR, infusion reactions, cytokine release syndrome, marrow rejection, or transmission of infectious diseases, and no events of graft vs host disease or organ rejection reported as related to tabelecleucel.

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Summary/Conclusion: Tabelecleucel Phase 3 data show clinically meaningful outcomes and promising ORR and OS in a pt population with poor survival and no approved therapies. Tabelecleucel was well tolerated without evidence of safety concerns typically observed with other adoptive T-cell therapies.

S257: A PHASE 1, FIRST-IN-HUMAN, DOSE-ESCALATION CLINICAL TRIAL OF MEMORY-ENRICHED CD30-CAR T-CELL THERAPY FOR THE TREATMENT OF RELAPSED OR REFRACTORY HODGKIN LYMPHOMA AND CD30+ T-CELL LYMPHOMA

A. C. Caballero 1,* , L. Escribà-Garcia 1 , R. Montserrat-Torres 1 , E. Escudero-López 1 , P. Pujol-Fernández 1 , C. Ujaldón-Miró 1 , I. Garcia-Cadenas 1 , A. Esquirol 1 , R. Martino 1 , J. Sierra 1 , C. Alvarez-Fernández 1 , J. Briones 1

1 Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

Background: Up to 30% of Hodgkin lymphoma (HL) patients are refractory or relapse (R/R) after first treatment and their prognosis is poor. We have developed a refined CD30-CAR-T (HSP-CAR30) targeting a proximal epitope within the CD30 molecule to overcome soluble CD30 and generated products enriched in memory T-cells to ensure efficient engraftment, persistence and enhancement of antitumor efficacy (Alvarez-Fernandez et al, 2021 ). Here, we report the results of our Phase 1 study evaluating HSP-CAR30 for the treatment of R/R HL and CD30 + T-cell non-Hodgkin lymphoma (T-NHL) ( NCT04653649 ).

Aims: Primary endpoints were to assess safety of HSP-CAR30 and to establish maximum tolerated dose (MTD) recommended for the following Phase 2. Secondary objectives include best response rates after infusion.

Methods: We conducted a phase 1 dose-escalation study in 11 patients with R/R HL or CD30 + T-NHL. HL patients were R/R to treatments including chemotherapy, brentuximab and anti-PD-1 antibodies, while T-NHL patients were R/R to at least 2 chemotherapy treatments. T-cells were transduced with a lentivirus encoding a second-generation 4-1BB costimulated CAR, containing a scFv directed against an epitope from the proximal non-cleavable part of CD30 protein. Three cell-dose levels were evaluated: DL1 (3x10 6 /kg), DL2 (5x10 6 /kg) and DL3 (10x10 6 /kg) CAR30 + T-cells.

Results: From February 2021 to December 2021, 11 patients (9 HL and 2 CD30 + T-NHL) were enrolled and underwent leukapheresis. Of these, 10 patients received HSP-CAR30: 3 patients at DL1, 3 at DL2 and 4 at DL3. Demographic characteristics and baseline disease features are summarized in the Table . Median age was 49.9 years (range 21–65). Median number of prior lines of treatment was 4.6 (range 3–7). One patient did not received treatment due to lack of T-cell expansion. All patients received LD before infusion, fludarabine/bendamustine in HL patients (n=8) and fludarabine/cyclophosphamide in T-NHL (n=2). Mean HSP-CAR30 expression was 94.79±3,38% (±SD). Memory T-cell subset comprised 93.07±4,8% (±SD) in CD4 + and 91.64±4,9% (±SD) in CD8 + . Mean time to HSP-CAR30 cell peak level across all doses was 29 days (range 6–98). CAR + T-cells were detectable by flow cytometry up to 11 months after infusion. HSP-CAR30 infusion was well tolerated; there were no dose limiting toxicities (DLTs). Relevant adverse events are shown in the Table . Grade 1 cytokine release syndrome (CRS) was observed in 6 (60%) patients. No patient developed neurotoxicity. Self-limited skin rash was seen in 4 (40%) patients. One patient with history of cytomegalovirus (CMV) infections had CMV pneumonia. Another patient developed pulmonary tuberculosis. At data cutoff (February 21 st , 2021), the median follow-up was 204 days (60–351). Best objective response was 100%, including 5 (50%) patients with complete response (CR), all with HL (DL1=1; DL2=3; DL3= 1). Three patients have died of progressive disease (2 T-NHL and 1 HL). There were no non-relapse mortality events. Median PFS and median overall survival (OS) was not reached. Six-month PFS for HL patients was 75%.

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Summary/Conclusion: This is the first European academic CART clinical trial evaluating a T-cell memory-enriched CART 30. Our Phase 1 study provides evidence for feasibility and safety of HSP-CAR30. Additionally, HSP-CAR30 has shown promising efficacy in heavily treated HL patients that is being explored in a phase 2 trial already started.

S258: LISOCABTAGENE MARALEUCEL (LISO-CEL) AS SECOND-LINE THERAPY FOR R/R LARGE B-CELL LYMPHOMA (LBCL) IN PATIENTS NOT INTENDED FOR HSCT: PRIMARY ANALYSIS FROM THE PHASE 2 PILOT STUDY

A. Sehgal 1,* , D. Hoda 2 , P. A. Riedell 3 , N. Ghosh 4 , M. Hamadani 5 , G. C. Hildebrandt 6 , J. E. Godwin 7 , P. Reagan 8 , N. Wagner-Johnston 9 , J. Essell 10 , R. Nath 11 , S. R. Solomon 12 , R. Champion 13 , E. Licitra 14 , S. Fanning 15 , N. Gupta 16 , R. Dubowy 17 , A. D’Andrea 18 , L. Wang 17 , L. I. Gordon 19

1 University of Pittsburgh Medical Center, Hillman Cancer Center, Pittsburgh; 2 Intermountain Healthcare, Loveland Clinic for Blood Cancer Therapy, Salt Lake City; 3 University of Chicago Comprehensive Cancer Center, Chicago; 4 Levine Cancer Institute, Atrium Health, Charlotte; 5 BMT & Cellular Therapy Program, Medical College of Wisconsin, Milwaukee; 6 Markey Cancer Center, University of Kentucky, Lexington; 7 Providence Cancer Center, Earle A. Chiles Research Institute, Portland; 8 University of Rochester Medical Center, Rochester; 9 Johns Hopkins Hospital, Baltimore; 10 Oncology Hematology Care, Cincinnati; 11 Banner MD Anderson Cancer Center, Gilbert; 12 Northside Hospital Cancer Institute, Atlanta; 13 Norton Cancer Institute, Louisville; 14 Astera Cancer Care, East Brunswick; 15 Prisma Health, Greenville; 16 Stanford Cancer Genetics Clinic, Palo Alto; 17 Bristol Myers Squibb, Seattle, United States of America; 18 Celgene, a Bristol-Myers Squibb Company, Boudry, Switzerland; 19 Northwestern University, Feinberg School of Medicine, Robert H. Lurie Comprehensive Cancer Center, Chicago, United States of America

Background: Patients with relapsed or refractory (R/R) LBCL after first-line treatment who are unable to undergo high-dose chemotherapy (HDCT) and hematopoietic stem cell transplantation (HSCT) have poor outcomes and limited treatment options.

Aims: PILOT ( NCT03483103 ) evaluated liso-cel, an autologous, CD19-directed chimeric antigen receptor (CAR) T cell product, as second-line treatment in patients with R/R LBCL not intended for HSCT.

Methods: Eligible patients were adults with R/R LBCL after first-line treatment who were not deemed candidates for HDCT and HSCT by their physician and met ≥ 1 frailty criteria as follows: age ≥ 70 years, Eastern Cooperative Oncology Group performance status (ECOG PS) of 2, diffusing capacity for carbon monoxide ≤ 60%, left ventricular ejection fraction < 50%, creatinine clearance < 60 mL/min, or alanine aminotransferase/aspartate aminotransferase > 2 × the upper limit of normal. Bridging therapy was allowed. Patients received lymphodepletion with cyclophosphamide and fludarabine, followed 2–7 days later by liso-cel infusion at a target dose of 100 × 10 6 CAR + T cells. Cytokine release syndrome (CRS) was graded per Lee 2014 criteria. Neurological events (NE) were defined as investigator-identified neurological adverse events related to liso-cel and graded using the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. The primary endpoint was objective response rate (ORR) per independent review committee; all patients had ≥ 6 months of follow-up from first response.

Results: Of 74 patients who underwent leukapheresis, 61 received liso-cel and 1 received nonconforming product (ie, product wherein one of the CD8 or CD4 cell components did not meet one of the requirements to be considered liso-cel). Common reasons for preinfusion dropout included death and loss of eligibility (5 each). For liso-cel–treated patients, median age was 74 years (range, 53–84; 79% ≥ 70 years) and 69%, 26%, and 5% met 1, 2, and 3 frailty criteria, respectively; 26% had ECOG PS of 2 and 44% had Hematopoietic Cell Transplantation-specific Comorbidity Index score ≥ 3. After first-line treatment, 54% were chemotherapy refractory, 21% relapsed within 12 months, and 25% relapsed after 12 months; 51% of patients received bridging chemotherapy. Median (range) on-study follow-up was 12.3 months (1.2–26.5). ORR and complete response rate were 80% and 54%, respectively ( Table ). Median duration of response and progression-free survival were 12.1 months and 9.0 months, respectively. Median overall survival has not been reached. The most frequent treatment-emergent adverse events (TEAE) were neutropenia (51%), fatigue (39%), and CRS (38%), with grade 3 CRS in 1 patient (2%) and no grade 4/5 CRS events. Any-grade NEs were seen in 31% (n = 19) of patients; grade 3 NEs occurred in 5% (n = 3) of patients and no grade 4/5 NEs were reported. Seven percent (n = 4) received tocilizumab only, 3% (n = 2) received corticosteroids only, and 20% (n = 12) received both tocilizumab and corticosteroids for treatment of CRS and/or NEs. Overall, grade ≥ 3 TEAEs occurred in 79% (n = 48) of patients, with grade 5 TEAEs in 2 patients (both due to COVID-19). Two patients (3%) had grade 3/4 infections and 15 (25%) had grade ≥3 neutropenia at Day 29.

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Summary/Conclusion: In the PILOT study, liso-cel as second-line treatment in patients with LBCL who met ≥ 1 frailty criteria and for whom HSCT was not intended demonstrated substantial and durable overall and complete responses, with no new safety concerns.

S259: DUAL ANTIGEN TARGETING WITH CO-TRANSDUCED CD19/22 CAR T CELLS FOR RELAPSED/REFRACTORY ALL

S. Ghorashian 1,* , G. Lucchini 2 , R. Richardson 3 , K. Nguyen 3 , C. Terris 3 , J. Yeung 3 , J. Chu 2 , L. Williams 2 , K. Ko 2 , C. Walding 4 , K. Watts 5 , S Inglott 1 , S. Adams 1 , E. Gravett 1 , K. Gilmour 6 , A. Lal 7 , S. Kunaseelan 7 , B. Popova 7 , A. Lopes 7 , Y. Ngai 7 , E. Kokalaki 8 , K. Rao 2 , R. Chiesa 2 , J. Silva 2 , K Mullanfiroze 2 , A. Lazareva 2 , D. Bonney 5 , R Wynn 5 , M. Pule 8 , R. Hough 4 , P. Amrolia 2

1 Haematology; 2 Bone Marrow Transplant, Great Ormond St Children’s Hospital; 3 Molecular and Cellular Immunology, UCL Great Ormond St Institute of Child Health; 4 Haematology, University College London Hospital NHS Trust, London; 5 Blood and Marrow Transplant, Royal Manchester Children’s Hospital, Manchester; 6 Cell Therapy and Immunology, Great Ormond St Children’s Hospital; 7 Cancer Research UK & UCL Cancer Trials Centre; 8 Autolus Ltd, London, United Kingdom

Background: CD19 negative escape is a major cause of relapse after CD19 CAR T cell therapy for relapsed/refractory (r/r) paediatric ALL and dual targeting of CD19/CD22 may overcome this. We have previously shown that AUTO1, a fast off rate autologous CD19 CAR T cell therapy was highly active in ALL with a favorable safety profile and excellent persistence (Ghorashian et al Nat.Med. 2019). Building on these properties, we developed AUTO1/22 in which autologous T cells are co-transduced with 2 different lentiviral vectors encoding our existing CD19 CAR and a novel CD22CAR designed to recognise targets with low antigen density. AUTO1/22 was evaluated in a Phase I study in children/young adults with r/rALL ( NCT02443831 ).

Aims: To determine the safety/biological efficacy of AUTO1/22

Methods: Patients with r/r B-ALL age < 25 ywho were ineligible for/relapsed after Tisagenlecleucel were recruited. Following fludarabine/cyclophosphamide lymphodepletion, patients received 1x10 6 /kg CAR + T cells. The presence of CAR T cells in the blood/bone marrow (BM) was assessed by flow cytometry + qPCR and BM MRD was assessed by IgH qPCR + flow cytometry. Primary end-points were incidence of grade 3-5 toxicity and the proportion of patients achieving MRD negative remission.

Results: Ten patients have been treated and 8 are evaluable with >1 month follow-up. The median age was 12 years and patients had a median of 3.5 prior lines of therapy (range 2-6). Five of 8 patients had relapsed post allogeneic SCT, 4 had received prior Blinatumomab/Inotuzumab and 3 had relapsed after prior Tisagenlecleucel. Prior to lymphodepletion, 2 patients had >5% BM disease, 5 had MRD between 10 -2 and 10 -5 and 1 was BM MRD negative. CAR T cell products had a central memory phenotype with predominance of CD19/22 double positive cells (median 59.3%) and balanced populations of CD19 and CD22 single positive cells (16% and 10.9% respectively).Cytokine release syndrome (CRS) occurred in 7/8 patients (grade 1 n=2 , grade 2 n=5 ) requiring Tocilizumab in 3 cases, but severe (≥ grade 3) CRS was not seen and no patients required ICU admission for CRS. Grade 1-2 ICANS was observed in 3 patients. One patient had delayed grade 4 leucoencephalopathy (MRI/brain biopsy were more indicative of fludarabine toxicity than CAR T related) and has ongoing neurological recovery. 7 patients had grade 3-4 cytopenia persisting beyond/recurring after day 28, requiring a CD34+ stem cell top up in 1 case. 5/8 patients had CD19CAR T cells and 3/8 patients had CD22CAR T cells detectable at last follow-up. 7 of 8 evaluable patients (88%) achieved MRD negative CR/CRi at 1 month post-infusion. One patient did not respond with CD19+ CNS relapse + MRD level BM disease at day 28. Of the 7 responding patients, 1 had frank CD19+CD22+ BM and extramedullary relapse at 3 months and 1 had emergence of MRD level disease at 10.5 months post infusion, in both cases associated with loss of CAR T cells. One other patient had early loss of CAR T cells with B cell recovery but ongoing MRD negative CR at 3 months post-infusion and remains in MRD negative CR on maintenance chemotherapy. Overall, at a median follow-up of 4.8 months, 5/8 patients remain in MRD negative CR at last follow-up.

Summary/Conclusion: We demonstrate that dual CD19/22 targeting CAR T cells generated by co-transduction show an acceptable safety profile, with robust expansion/persistence and early efficacy in a heavily pre-treated cohort. To date with limited follow-up we have not observed antigen negative relapse but longer follow up is needed.

S260: A MATCHED COMPARISON OF TISAGENLECLEUCEL AND AXICABTAGENE CILOLEUCEL CAR T CELLS IN RELAPSED OR REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA: A REAL-LIFE LYSA STUDY FROM THE FRENCH DESCAR-T REGISTRY

E. Bachy 1,* , S. Le Gouill 2 , P. Sesques 3 , R. Di Blasi 4 , M. Guillaume 5 , G. Cartron 6 , D. Beauvais 7 , L. Roulin 8 , F. X. Gros 9 , M. T. Rubio 10 , P. Bories 11 , J. O. Bay 12 , C. Castilla Llorente 13 , S. Choquet 14 , R.-O. Casasnovas 15 , M. Mothy 16 , S. Guidez 17 , M. Joris 18 , M. Loschi 19 , S. Carras 20 , J. Abraham 21 , A. Chauchet 22 , L. Drieu La Rochelle 23 , J. Zerbit 24 , O. Hermine 25 , T. Gastinne 26 , J. J. Tudesq 6 , E. Gat 27 , F. Broussais 27 , C. Thieblemont 28 , R. Houot 5 , F. Morschhauser 7

1 Hematology, Hospices Civils de Lyon, Pierre Bénite; 2 Institut Curie, Paris; 3 Hospices Civils de Lyon, Pierre Bénite; 4 AP-HP, Paris; 5 CHU Rennes, Rennes; 6 CHU Montpellier, Montpellier; 7 CHU Lille, Lille; 8 CHU Mondor, Paris; 9 CHU Bordeaux, Bordeaux; 10 CHU Nancy, Nancy; 11 CHU Toulouse, Toulouse; 12 CHU Clermont Ferrand, Clermont Ferrand; 13 GUSTAVE ROUSSY CANCER CAMPUS GRAND PARIS, Villejuif; 14 CHU Pitié Salprétrière, Paris; 15 CHU Dijon, Dijon; 16 CHU Saint Antoine, Paris; 17 CHU Poitiers, Poitiers; 18 CHU Amiens, Amiens; 19 CHU Nice, Nice; 20 CHU Grenoble, Grenoble; 21 CHU Limoges, Limoges; 22 CHU Besancon, Besancon; 23 CHU Tours, Tours; 24 APHP Cochin; 25 CHU Necker, Paris; 26 CHU Nantes, Nantes; 27 LYSARC, Lyon; 28 CHU Saint Louis, Paris, France

Background: Axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) have both demonstrated impressive clinical activity in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). In a previous propensity score matching (PSM) analysis (Bachy et al., ASH 2021) allowing for balanced comparison between axi-cel and tisa-cel outcomes, we reported on a prolonged progression-free survival (PFS) but a higher toxicity associated with axi-cel compared with tisa-cel. No OS difference was observed but the follow-up was short (6 months).

Aims: We aim at reporting on PSM analysis with longer follow-up and with additionnal patients treated with axi-cel or tisa-cel.

Methods: All patients treated in France with axi-cel or tisa-cel from the 1 st July 2018 to the 1 st October 2021 and included in the DESCAR-T registry were considered.

Propensity score matching (PSM) was used to create a balanced covariate distribution between a cohort of patients treated with tisa-cel and a cohort of patients treated with axi-cel. An exhaustive list of covariates was used for PSM: age, sex, LDH level, C reactive protein (CRP), time between last treatment and infusion, Eastern Cooperative Oncology Group (ECOG) performance status (PS), Ann Arbor stage, number of prior lines of treatment before CAR-T, bridging and response to bridging, prior stem cell transplant (SCT) either autologous or allogeneic, bulk assessed at lymphodepletion, centre, histological diagnosis. PSM was performed considering a 1:1 matching without replacement and with optimal matching applying a calliper width of the propensity score set at 0.1. Inverse probability of treatment weighting (IPTW) was used as another approach to further validate PSM analysis.

Results: 809 patients from 25 French centres with R/R DLBCL after at least 2 lines of previous therapy had a commercial CAR-T order with axi-cel or tisa-cel and were registered in DESCAR-T. Out of 809 patients with a CAR-T order, 60 were not infused due to progression or death between leukapheresis and lymphodepletion and 20 did not proceed to lymphodepletion for other reasons. Finally, 729 proceeded to lymphodepletion and CAR-T infusion. In the 1:1 matched population (N=418, 209 patients treated with tisa-cel and 209 patients treated with axi-cel), the best ORR/CRR was 66/42% versus 80/60% for patients treated with tisa-cel compared with axi-cel, respectively ( P <0.001 for both ORR and CRR comparisons). After a median FU of 11.7 months (95% CI, 10.5-12.0 months) the 1-yr PFS was 33% for tisa-cel and 47% for axi-cel (HR=1.65, 95% CI 1.26-2.18, P =0.0003). OS was also significantly longer following axi-cel infusion than following tisa-cel infusion (1-yr OS 63% versus 49%; HR=1.58, 95% CI, 1.13-2.21; P =0.0072). Similar findings were found using IPTW statistical approach. Grade 1-2 CRS were significantly more frequent with axi-cel than tisa-cel ( P =0.004) but no significant difference was observed for grade 3 or more CRS (9% versus 5% for tisa-cel and axi-cel respectively, P =0.130). Regarding ICANS, both all grades and severe (i.e. grade ≥3) ICANS were significantly more frequent with axi-cel than tisa-cel. 48% of patients experienced ICANS after axi-cel infusion compared to 22% after tisa-cel infusion. 29 patients (14%) presented a grade ≥3 ICANS with axi-cel compared with 6 (3%) only with tisa-cel.

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Summary/Conclusion: In conclusion, our matched-comparison study supports a higher efficacy but also a higher toxicity of axi-cel compared with tisa-cel in third or more treatment line for R/R DLBCL.

S261: SAFETY AND PRELIMINARY EFFICACY FINDINGS OF AUTO4, A TRBC1-TARGETTING CAR, IN RELAPSED/REFRACTORY TRBC1 POSITIVE SELECTED T CELL NON-HODGKIN LYMPHOMA

K. Cwynarski 1,* , E. Tholouli 2 , G. Iacoboni 3 , T. Menne 4 , D. Irvine 5 , L. Wood 6 , N. Balasubramaniam 7 , J. Shang 8 , M Zhang 8 , K. Duffy 9 , B. Huber 10 , M. Vinson 11 , W. Brugger 9 , M. Pule 12 13

1 Haematology, University College London, London; 2 Manchester Royal Infirmary, Manchester, United Kingdom; 3 VHIO Vall d’Hebron Hospital, Barcelona, Spain; 4 Freeman Hospital Newcastle, Newcastle; 5 University of Glasgow, Glasgow; 6 Cancer Clinical Trials Unit, University College London Hospitals; 7 Cancer Clinial Trials Unit, University College London, London, United Kingdom; 8 Autolus Therapeutics, Rockville, United States of America; 9 Clinical Development, Autolus Therapeutics, London, United Kingdom; 10 Autolus Therapeutics, Munich, Germany; 11 Autolus Therapeutics, London, United Kingdom; 12 Research & Development, Autolus Therapeutics; 13 University College London, London, United Kingdom

Background: Peripheral T cell lymphomas (PTCL) are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapy is limited by a lack of target antigens that discriminate malignant from normal T cells. Unlike B cell depletion, pan–T cell aplasia is prohibitively toxic. We recently described a targeting strategy based on the mutually exclusive expression of T cell receptor beta-chain constant domains 1 and 2 (TRBC1 and TRBC2) (Maciocia, PM. et al, Nat Med 2017) which can spare a proportion of the normal T cell compartment.

Aims: Here we describe early clinical findings of AUTO4, a TRBC1 directed autologous CAR T cell therapy, tested against relapsed/refractory (r/r) TRBC1+ PTCL.

Methods: NCT03590574 is multi-centre, single-arm study of AUTO4 with a phase I dose escalation component and a phase II expansion cohort. Here we report the initial findings of the phase I component. Biopsies from patients >18 years of age were screened for TRBC1-positive PTCL using next-generation sequencing. Four flat dose levels were explored: 25 x 10 6 , 75 x 10 6 , 225 x 10 6 , and 450 x 10 6 CAR T cells administered as a single dose. CAR T-cell products are generated using a semi-automated closed process. Patients received lymphodepletion with fludarabine (30mg/m 2 x4, day-6 to day-3) and cyclophosphamide (500mg/m 2 x2 on day-6 and day-5) (Flu/Cy) prior to AUTO4 infusion on Day 0. Primary endpoints were incidence of Grade 3 to 5 toxicity occurring within 60 days of AUTO4 infusion and the frequency of dose limiting toxicities within 28 days of AUTO4 infusion (DLT period). Overall response (CR+PR) rate post AUTO4 infusion by PET-CT (Lugano 2014 criteria) was a secondary endpoint.

Results: As of 09-FEB-2022, n=64 patients consented for screening of TRBC1-positive PTCL. N=24 samples were TRBC1-positive; 7 patients were screen failures including 1 patient who died during screening. 11 products were manufactured; one patient relapsed prior to AUTO4 infusion, and one patient screen failed after product manufacture. To date 9 patients have been treated with AUTO4. The median age in these 9 patients was 57 years (range 34 to 63 years). The T-cell lymphoma subtypes treated were PCTL-NOS (n=4), ALCL (n=1), and AITL (n=4). Two patients had prior stem cell transplantation. The median number of prior treatment lines was 3 (range 1-5). After lymphodepletion with Flu/Cy, 3 patients received 25 × 10 6 CAR T cells, 2 patients received 75 × 10 6 CAR T cells, 1 patient received 225 × 10 6 CAR T cells and 3 patients received 450 × 10 6 CAR T cells. No patient experienced any dose limiting toxicities. The median (range) number of CD3+ T-cells/µl in blood prior to lymphodepletion and at the end of the DLT period (Day 28) was 204 (94-698) and 123 (19-458), respectively. 3 patients (33%) experienced CRS (1 patient with Grade 1, 1 patient with Grade 2, and 1 patient with Grade 3). None of the patients experienced neurotoxicity/ICANS. The most common treatment-emergent adverse events were cytopenias (anemia and neutropenia). Of the 9 patients treated, 5 patients had achieved complete metabolic responses (CMR) by PET-CT at Month 1, one patient remains with a PR 6 months post AUTO4 infusion, and 3 patients did not respond. All 3 patients at the 450x10 6 cell dose achieved a CMR at Month 1.

Summary/Conclusion: AUTO4 has a tolerable safety profile in patients with r/r TRBC1+ peripheral T-cell lymphoma. Early data shows encouraging response rates. Updated data and longer follow up will be presented.

S262: THE COBALT-LYM STUDY OF CTX130: A PHASE 1 DOSE ESCALATION STUDY OF CD70-TARGETED ALLOGENEIC CRISPR-CAS9–ENGINEERED CAR T CELLS IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) T-CELL MALIGNANCIES

S. P. Iyer 1,* , R. A. Sica 2 , P. J. Ho 3 , B. Hu 4 , J. Zain 5 , A. Prica 6 , W.-K. Weng 7 , Y. H. Kim 8 , M. S. Khodadoust 9 , M. L Palomba 10 , F. M. Foss 11 , K. Tipton 12 , E. L. Cullingford 12 , Q. He 12 , A. Sharma 12 , S. M. Horwitz 10

1 Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston; 2 Department of Oncology, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, United States of America; 3 Institute of Haematology, Royal Prince Alfred Hospital, Camperdown, Australia; 4 Division of Hematology and Hematologic Malignancies, Huntsman Cancer Institute, Salt Lake City; 5 Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, United States of America; 6 Princess Margaret Cancer Centre, Toronto, Canada; 7 Division of Blood and Marrow Transplantation and Cellular Therapy; 8 Department of Dermatology; 9 Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford; 10 Memorial Sloan Kettering Cancer Center, New York; 11 Department of Dermatology, Yale School of Medicine, New Haven; 12 CRISPR Therapeutics, Cambridge, United States of America

Background: Overall survival (OS) in a subset of patients (pts) with T-cell lymphoma (TCL) has improved with front-line combination chemotherapy; however, R/R TCL pts continue to have very limited treatment options. For pts with R/R peripheral (PTCL) and transformed cutaneous TCL (CTCL), median OS is 1-2.5 and <5 yrs, respectively. Adapting autologous chimeric antigen receptor (CAR) T cell therapy for TCL continues to be challenging due to poor function of donor T cells, fratricide effect, and risk of infusing transduced malignant CAR T cells into pts. CTX130 TM is a first-in-class, CD70-targeting allogeneic (allo) CAR T therapy that may allow for CAR T therapy in pts whose own T cells are not ideal to manufacture auto CAR T cells. CD70 is a co-stimulatory protein with temporally limited expression on activated lymphocytes and is highly expressed in many TCLs. CTX130 is modified with CRISPR/Cas9-editing to eliminate expression of: 1) T-cell receptor (TCR) by TCR alpha constant disruption, 2) major histocompatibility complex class I expression by β2-microglobulin disruption, and 3) CD70 to mitigate fratricide and enhance performance.

Aims: Investigate safety and efficacy of CTX130 in pts with R/R TCL.

Methods: COBALT TM -LYM ( NCT04502446 ) is an open-label, multicenter, global study evaluating the safety and efficacy of CTX130 in pts ≥18 y with CD70+ (≥10% by immunohistochemistry) R/R TCL (PTCL or CTCL). Pts with PTCL and CTCL must have received ≥1 or ≥2 prior lines of systemic therapy, respectively. Pts received lymphodepleting chemotherapy (LDC) with fludarabine 30mg/m 2 and cyclophosphamide 500mg/m 2 for 3 days, followed by CTX130. Pts were treated with CTX130 at doses from 3x10 7 (dose level [DL]1) to 9x10 8 (DL4) CAR+ T cells. Pts could receive a second course of CTX130 if response was not achieved but had experienced clinical benefit or disease progression. The primary endpoint is safety (incidence of dose limiting toxicities [DLTs]). Key secondary endpoints include overall response rate (ORR, by Lugano and ISCL criteria for PTCL and CTCL, respectively), disease control rate (DCR; ≥stable disease [SD]), duration of response and OS.

Results: As of 6 Dec, 2021, 17 pts with TCL were enrolled; 15 received CTX130, were evaluable for a Day 28 assessment and are included in the analysis. Among the pts who received CTX130, median age was 67 y, 7 pts had PTCL, and 8 pts had CTCL. Median CD70 expression was 90% (range, 20-100%). Median follow up was 3.1 months. At DL ≥3, ORR was 71%, CR rate was 29% and DCR was 100% ( Table ). Responses were observed in PTCL (75% ORR at DL≥3) and CTCL (67% ORR at DL≥3) and across disease compartments. CTX130 had an acceptable safety profile across all DLs. There were no instances of graft versus host disease, tumor lysis syndrome, hemophagocytic lymphohistiocytosis, or infusion reactions with LDC or CTX130. There were no DLTs, no Grade (Gr) ≥3 cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS); Gr 1-2 CRS and ICANS were 47% and 20%. There was no increase in frequency or severity of CRS in pts who received second infusions of CTX130. 1 pt (7%) experienced a Gr ≥3 infection. There was a sudden death in 1 pt with William’s syndrome in the context of a lung infection.

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Summary/Conclusion: We have observed clinically meaningful responses, including CRs with CTX130, the first CAR T directed against the novel target, CD70. CTX130 has an acceptable safety profile in pts with heavily pretreated R/R TCL and will be investigated further in an expansion phase of the study.

S263: PHASE I OPEN-LABEL SINGLE ARM STUDY OF GPRC5D CAR-T CELLS (ORICAR-017) IN PATIENTS WITH RELAPTSED/REFRACTORY MULTIPLE MYELOMA (POLARIS)

M. Zhang 1 , Y. Hu 1 , X. Ding 2 , Y. Tang 2 , Y. Yang 2 , S. Chen 2 , H. Huang 1,*

1 Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou; 2 Oricell Therapeutics Co., Ltd, Shanghai, China

Background: G-protein–coupled receptor class 5-member D (GPRC5D), a type-C 7-pass transmembrane receptor protein, is predominantly expressed on malignant plasma cell phenotype, including most malignant plasma cells from pts with multiple myeloma (MM).

The autologous GPRC5D-directed CAR-T cell (OriCAR-017), owning to an additional proprietary Ori element, is the standard second generation CAR-T cell with improvement in expansion and durability of CAR-T cells post-transfusion.

Here we report initial results from the phase I open-label single arm study of GPRC5D CAR-T Cells (OriCAR-017) in pts with Relapsed/Refractory (R/R) MM ( NCT05016778 ).

Aims: The primary objective in the dose escalation phase was to evaluate safety and tolerability of OriCAR-017. Secondary endpoints included efficacy and pharmacokinetics (expansion and persistence of OriCAR-017.

Methods: Key enrolment criteria included adults with measurable MM, R/R or intolerant to established MM therapies, prior BCMA-targeted therapy allowed. Patient received lymphodepleting chemotherapy with Fludarabine 30mg/m 2 daily and cyclophosphamide 300mg/m 2 daily for 3 days followed by a single infusion of OriCAR-017. The trial followed a standard 3 + 3 design with the following dose cohorts: 1×10 6 /kg, 3×10 6 /kg and 6×10 6 /kg CAR + T cells.

Results: Eleven pts with R/R MM were enrolled and underwent apheresis during June 9, 2021 and February 25, 2022. Nine of them have completed OriCAR-017 infusion to date. 2 pts were suspended infusion due to rapid disease progression.

Of the 9 pts infused, median age of 65 years (range: 41-71) and a median of 6 (range 3-17) prior lines of therapy; 4 (44.4%) prior BCMA CART therapy. 3 (33.3%) pts with extramedullary plasmacytoma ≥ 1 and 7 (77.8%) pts with bone marrow plasma cells ≥60% at baseline. Five out of 6 pts had high-risk cytogenetic profiles, including 2 pts with del(17p).

No dose-limiting toxicities occurred. The most common treatment-related AEs were hematological toxicities. All pts experienced Cytokine Release Syndrome (CRS, with 8(88.9%) pts in G1, 1 (11.1%) patient in G2), no G3/4 CRS was observed. All CRS cases were rapidly relieved after conventional CRS intervention, including tocilizumab and steroids. No neurologic toxicities were reported to date.

All 9pts median follow up time was 118 days (range33—220). A 100.0% ORR were observed with 3 (33.3%) pts achieved CR/sCR, 3 pts (33.3%) were VGPR, 3 pts were PR (33.3%). 4 pts relapsed from BCMA CAR-T therapy had responses with 1 sCR, 2 VGPR, 1 PR. All 9 pts were Minimal Residual Disease (MRD) negative in the bone marrow by flow cytometry (sensitivity: 10 -5 ) at day 28 after infusion, and 6pts continued MRD negative at month 3 and 2 pts at month 6 after infusion.

Robust OriCAR-017 expansion in peripheral blood by using qPCR in 3 dose cohorts, median peak was 349866.1copies/ml (range 60837.5 – 2240358.5), median time to peak expansion was 10 days (range 7-14). 8 out of 9 patients detected CAR + cell in bone marrow at D28 after infusion with median 498048.3copies/ml (range 495.4- 2358538.6).

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Summary/Conclusion: In this phase I study, OriCAR-017 was safe and showed impressive efficacy among previously heavily treated R/RMM pts. Majority of AEs were transient, manageable, and reversible. 100% ORR and 100% MRD negative rate, along with favorable safety evidenced support OriCAR-017 could be a competitive therapy for pts with R/RMM. Furthermore, pts who had relapsed from BCMA CAR-T therapy may still benefit from OriCAR-017 treatment.

S264: PRELIMINARY ANALYSES OF A NON-GENE-EDITING ALLOGENTIC CAR-T IN CD19+ RELAPSED OR REFRACTORY NON-HODGIN’S LYMPHOMA

X. Wang 1 , L. Xue 1,* , S. Li 1 , Q. Fan 1 , K. Liu 2 , R. Jin 2 , X. Yang 1 , T. Wang 2 , L. He 2 , J. Li 2

1 Department of Hematology, The First Affiliated Hospital of USTC, Hefei; 2 Fundamenta Therapeutics Inc., Suzhou, China

Background: A novel non-gene-editing allogeneic CAR-T platform was developed on the base of intracellular retention of membrane proteins, and named ThisCART (This = T CR and/or H LA-I i ntracellular s equestered). CD19-directed ThisCART (ThisCART19A) cells are readily produced with a single lentiviral vector, encoding both a CD19-targeting CAR and an anti-CD3 single chain antibody with the KDEL peptide fused to its C-termini (Figure 1). ThisCART19A cells, deficient of surface TCR/CD3 complexes in extended culture, induced no xegoneic GvHD reaction in murine model. Similar to conventional CD19-directed CAR-T, they exhibited potent CD19-specific cytotoxicities both in vitro and in vivo .

Aims: To evaluate the safety, pharmacokinetics and clinical activity of ThisCART19A in patients with refractory or relapsed CD19 positive B cell malignancies.

Methods: A single-center, open-label, single-arm study ( NCT04384393 ) is ongoing to evaluate the safety, pharmacokinetics and clinical activity of ThisCART19A in patients with refractory or relapsed CD19 positive B cell malignancies, including acute or chronic lymphoblastic leukemia, non-Hodgkin’s lymphoma. Standard or enhanced lymphodepletion with fludarabine and cyclophosphamide was followed by infusion of ThisCART19A at a dose range of 0.2-60x10 6 cells/kg. The pharmacokinetics of ThisCART19A cell was assessed by flow cytometry or quantitative PCR.

Results: As of January 30th, 2022, eight evaluble subjects with aggressive or advanced non-Hodgkin’s lymphoma received ThisCART19A infusion (Table 1). Subject #3 had received two subsequent autologous CAR-T therapies and relapsed after each treatment. Adverse event profile is acceptable: no ≥3 grade of CRS or ICANS and no evidence of GvHD reaction. Two subjects experienced reversible Hemophagocytic lymphohistiocytosis with decreased lymphocytes and neutrophils. Dose-dependent CAR-T expansion was observed in six subjects pretreated with enhanced lymphodepletion. Lack of CAR-T expansion in the other two is likely due to the standard lymphodepletion or quick disease progress. PET/CT (≥28 days) scan demonstrated one partial response and five complete responses. Among these five, three remain in remission with one in continued follow-up over 8 months.

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Summary/Conclusion: ThisCART19A cells demonstrated potent CD19-dependent cytotoxicity and no xenogentic GvHD in murine models. In this preliminary analysis on evaluable lymphoma subjects, ThisCART19A exhibited an acceptable safety and an encouraging clinical response profiles, both comparable to those for marketed autologous products.

S265: RADIOMICS AND ARTIFICIAL INTELLIGENCE FOR IDENTIFICATION AND MONITORING OF SILENT CEREBRAL INFARCTS IN SICKLE CELL DISEASE: FIRST ANALYSIS FROM THE GENOMED4ALL EUROPEAN PROJECT

M. P. Boaro 1 , R. Biondi 2 , N. Biondini 2 , A. Collado Gimbert 3 , E. F. JM 4 , V. Pinto 5 , N. Romano 6 , V. Voi 7 , G. B. Ferrero 8 , M. Casale 9 , M. Cirillo 10 , G. Palazzi 11 , F. Cavalleri 12 , G. L. Forni 5 , G. Reggiani 1 , S. Perrotta 13 , M. Manu Pereira 14 , S. Zazo 15 , K. Marias 16 , M. De Montalembert 17 , P. Bartolucci 18 , E. van Beers 19 , F. Alvarez 20 , F. Cremonesi 21 , T. Sanavia 22 , P. Fariselli 22 , G. Castellani 23 , R. Manara 24 , R. Colombatti 1,*

1 Woman’s and Child’s Health, University of Padova, Padova; 2 Of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy; 3 Servei d’Oncologia i Hematologia Pediàtriques, Hospital Universitari Vall d’Hebron; 4 Radiology, Hospital Universitari Vall d’Hebron, Barcelona, Spain; 5 Centro della Microcitemia, Anemie Congenite e Dismetabolismo del Ferro; 6 Department of Diagnostic and Interventional Neuroradiology, Ospedali Galliera, Genova; 7 Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale San Luigi Gonzaga, Orbassano; 8 Dipartimento di Scienze Cliniche e Biologiche, University of Torino, Torino; 9 Donna, del Bambino e di Chirurgia Generale e Specialistica, Università̀ degli Studi della Campania “Luigi Vanvitelli”; 10 Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli, Napoli; 11 Dipartimento Integrato Materno Infantile, Azienda Ospedale-Università di Modena e Reggio Emilia; 12 Neurological Department, University of Modena, Modena; 13 Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Università̀ degli Studi della Campania “Luigi Vanvitelli”, Napoli, Italy; 14 Vall d’Hebron Research Institute, Barcelona; 15 Information Processing and Telecommunications Center, Universidad Politécnica de Madrid, Madrin, Spain; 16 Computational Medicine Laboratory, Institute of Computer Science, FORTH, Heraklion, Greece; 17 Department of Pediatrics, Hopital Necker, Paris; 18 Red Cell Genetic Disease Unit, Université Paris Est, Institut Mondor de Recherche Biomédicale (IMRB), Creteil, France; 19 Universitair Medisch Centrum Utrecht, Utrecht, Netherlands; 20 Universidad Politecnica Madrid, Madrid, Spain; 21 Datawizard s.r.l, Roma; 22 Medical Sciences, Division of Gastroenterology and Hepatology, Città della Salute e della Scienza di Torino, University of Turin, Torino; 23 Department of Physics, University of Bologna, Bologna; 24 Neuroscience, University of Padova, Padova, Italy

Background: The use of Artificial Intelligence (AI) for personalized medicine has recently guided improvements in the diagnostic pathway of many diseases. The EU Project GENOMED4ALL: “Genomics and Personalized Medicine for All through Artificial Intelligence in Haematological Diseases” aims at using European data of patients affected by Sickle Cell Disease (SCD) to find correlation between -omics data – and phenotype, seizing the opportunity to improve diagnostics through AI.

Silent Cerebral Infarcts (SCIs) are a significant cause of morbidity in SCD: they affect 25% of children by the age of 6 and 40% by the age of 18 with consequences on cognition, schooling, working capacity and quality of life. Hence, one of the aims of the SCD clinical case in GENOMED4ALL is the use of radiomics – quantitative method for the evaluation and interpretation of medical image s- and AI firstly to develop an automatic and uniform identification and characterization of SCI on MRIs, secondly, to correlate imaging data with other types of omics data in order to predict risk of recurrence

Aims: The first phase of the GENOMED4ALL project aimed at collecting anonymized MRI data available in Centers of the EuroBloodNet Network to develop an algorithm that could identify SCI on MRI from different sources, distinguishing them from other lesions

Methods: MRI protocol included 3D-T1, FLAIR and DWI sequences. Neuroradiological reports were checked for consistency. A stepwise segmentation (identification of lesion volume), pre-processing and extraction of MRIs was performed utilizing different open-source software: Lesion Segmentation Tools and UNet for segmentation, Freesurfer for brain extraction. To optimize SCI identification, the best software for segmentation was selected

Results: Six SCD expert centers participated in the first phase with 501 MRIs: 225 were classified as abnormal by the local neuroradiologists due to presence of SCI; 70% were pediatric MRIs. Different instruments were used in the centers: Philips 1.5 T (n.2), Siemens 1.5 T and 3 T (n.2), GE 3T (n.2).

A stepwise procedure allowed optimization of SCI identification, with distinction between SCI and non-clinically significant background (periventricular areas) or other white matter hyperintensities (transient glial maturation).

As shown in Figure 1, to segment SCI in FLAIR MRI, we used a pre-trained UNet [Li H, 2018] winner of the MICCAI challenge. Firstly, we extracted the brain registering the MNI152 on the T1 image . Then we applied the brain mask and a threshold taking only the largest component. The UNet performed the segmentation of hyperintensities. We removed the regions surrounded by less than 90% of White Matter or near the brain ventricles to remove non-SCI region (refinement). We then enlarged the remaining ones.

To date, segmentation on 303 exams from different centers showed very few false positive and some false negatives highlighting the need to take into account different technical characteristics (various equipment in different centers)

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Summary/Conclusion: SCD is a rare systemic disorder with extreme phenotypic variability. Radiomics and AI offer the opportunity to seize the potential of big dataset analysis to understand natural history and optimize diagnostics. SCI can be detected automatically from different datasets. Four more European Centers will add their MRI in the second phase, in order to increase variability and allow correlation of detailed Radiomics features with clinical-hematological variables and other omics data

S266: OXYGEN GRADIENT EKTACYTOMETRY-DERIVED BIOMARKERS ARE ASSOCIATED WITH THE OCCURRENCE OF ACUTE COMPLICATIONS IN SICKLE CELL DISEASE

M. Rab 1,* , C. Kanne 2 , C. Boisson 3 , J. Bos 1 , B. van Oirschot 1 , M. Houwing 4 , C. Renoux 5 , R. Schutgens 6 , M. Bartels 6 , A. Rijneveld 7 , E Nur 8 , M. Cnossen 4 , P. Joly 5 , R. Fort 9 , P. Connes 3 , R. van Wijk 1 , V. Sheehan 2 , E. van Beers 6

1 Central Diagnostic Laboratory-Research, University Medical Center Utrecht, Utrecht, Netherlands; 2 Department of Pediatrics, Emory University School of Medicine, Atlanta, United States of America; 3 Laboratory LIBM EA7424, University of Lyon; 1 , Lyon, France; 4 Department of Pediatric Hematology, Erasmus University Medical Center, Rotterdam, Netherlands; 5 Laboratory of Biochemistry and Molecular Biology, Hospices Civils de Lyon, Lyon, France; 6 Van Creveldkliniek, University Medical Center Utrecht, Utrecht; 7 Department of Hematology, Erasmus University Medical Center, Rotterdam; 8 Department of Hematology, Amsterdam University Medical center, Amsterdam, Netherlands; 9 Department of Internal Medicine, Hospices Civils de Lyon, Lyon, France

Background: Sickle cell disease (SCD) is a monogenetic disorder with a highly complex pathophysiology. There is an unmet need for robust reproducible biomarkers that can assess red blood cell (RBC) function and predict disease severity and complications.

Aims: To explore the association between oxygen gradient ektacytometry-derived biomarkers, and blood viscosity with incidence of major (acute) SCD-related complications.

Methods: Oxygen gradient ektacytometry measures RBC deformability continuously while the sample is gradually deoxygenated and subsequently reoxygenated, and identifies the oxygen tension at which sickling occurs. We examined associations between the occurrence of acute chest syndrome, cerebral infarction and vaso-occlusive crisis (VOC) and known biomarkers such as fetal hemoglobin, blood viscosity as well as exploratory oxygen gradient ektacytometry-derived biomarkers in an adult cohort of 50 individuals with SCD (HbSS or HbS/β o -thalassemia) and a pediatric cohort consisting of 177 children with SCD (HbSS or HbS/β o -thalassemia). A substantial number of subjects were on hydroxyurea therapy (64% of adults and 89% of children). Subjects that received a blood transfusion less than three months prior to measurements were excluded from the study. A logistic regression analysis was performed; odds ratios were adjusted for age and hydroxyurea therapy.

Results: In the adult cohort, for every 10 mmHg increase in Point of Sickling (PoS, the pO 2 tension where RBCs start to sickle, reflecting sickling tendency) the likelihood of >1 acute complication increased; the adjusted odds ratio (aOR) was 3.00 (p=0.015). For every 0.1 increase in EImax (reflecting RBC deformability at normoxia), the aOR was 0.33 (p=0.035, Table 1). In the pediatric cohort, for every 10 mmHg increase in PoS, the likelihood of >1 acute complication increased; the aOR was 1.65 (p=0.006). For every 0.1 increase in EImin (reflecting RBC deformability at hypoxia), the aOR was 0.50 (p=0.007). Fetal hemoglobin and blood viscosity levels were not associated with likelihood of multiple acute complications. However, fetal hemoglobin was associated with reduced likelihood of VOC in adults (aOR of 0.32 for every 10% increment, p=0.010) but not in children (aOR of 0.68, p=0.231, data not shown). In the adult cohort higher EImax was associated with reduced likelihood of VOC (aOR 0.31, p=0.029). There was a trend found for an association between higher PoS and greater likelihood of VOC (aOR 2.22, p=0.050), and no association for EImin. In the pediatric cohort only EImin was associated with VOC (aOR 0.68, p=0.036).

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Summary/Conclusion: These findings indicate that oxygen gradient ektacytometry generates novel clinically relevant biomarkers and provide a rationale for further development of these biomarkers in the evaluation of novel therapies, as part of clinical care, or clinical trial endpoints. In particular, in assessment of treatment strategies that do not target HbF induction, such as pyruvate kinase activators, voxelotor and l-glutamin, oxygen gradient ektacytometry can generate relevant biomarkers.

S267: PROPERDIN-BLOCKING ANTIBODIES ATTENUATE COMPLEMENT ALTERNATIVE PATHWAY ACTIVATION TRIGGERED BY CELL-FREE HEME IN SICKLE CELL DISEASE MODELS

T. Trovati Maciel 1 , R. Cofiell 2 , C. Carvalho 1 , S. Allali 1 , R. Rignault 1 , A. Bennet 3 , Y. Dai 3 , P. Tamburini 4 , C. Gasteyger 5 , O. Hermine 1 , S. K. Kim 2,*

1 Laboratory of Cellular and Molecular Mechanisms of Hematological Disorders and Therapeutical Implications, Imagine Institute, Inserm, Paris, France; 2 Translational Research, Alexion AstraZeneca Rare Disease, New Haven, CT; 3 Clinical Development and Translational Sciences, Alexion AstraZeneca Rare Disease, Boston, MA; 4 Research, Alexion AstraZeneca Rare Disease, New Haven, CT, United States of America; 5 Clinical Development and Translational Sciences, Alexion AstraZeneca Rare Disease, Zurich, Switzerland

Background: Complement activation may play an important role in sickle cell disease (SCD) pathophysiology. Heme has been identified as a trigger of complement activation in SCD patients and is present in excess during severe vaso-occlusive crises. Heme is also a DAMP (damage associated molecular pattern) and induces thrombo-inflammation through tissue factor expression.

ALXN1820 is a bi-specific VHH nanobody that simultaneously binds human albumin and properdin, thereby effectively and selectively inhibiting alternative pathway (AP) activation and formation of C3 and C5 convertases. The safety of ALXN1820 has been assessed in non-human primates and is currently being tested in a phase 1 study in healthy participants.

Aims: 1) To assess if complement activation and heme are functionally linked; 2) To investigate the therapeutic potential of ALXN1820 in in vitro SCD models and in in vivo SCD models using a mouse properdin-blocking monoclonal antibody (mAb), 14E1.

Methods: In vitro, complement inactivated C3b (iC3b) and C5b-9 deposition on red blood cells (RBCs) from patients with SCD and complement iC3b and C5b-9 deposition on the endothelial cell line HMEC-1 were assessed by flow cytometry after exposure to heme +/- ALXN1820.

In vivo , in Townes SCD mice, vaso-occlusion and hyper-hemolysis were induced by intravenous heme injection (50 µmol/kg) or hypoxia-reoxygenation (8% O 2 for 3h then 21% O 2 for 1h) in the presence and absence of 14E1 (40 mg/kg), a mouse-specific properdin inhibitor. Vaso-occlusion was measured by staining RBCs with immunofluorescence-labeled Ter-119 antibodies on lung and liver sections. C3b and C5b-9 deposition on SCD RBCs was assessed by flow cytometry. Markers of hemolysis (bilirubin, lactate dehydrogenase, free hemoglobin, free heme) were quantified in plasma using commercial assays.

Results: In RBCs from patients with SCD, cell-free heme triggered marked iC3b and C5b-9 deposition (Table). Deposition was blocked in the presence of ALXN1820 by >95% for iC3b and by >85% for C5b-9. HMEC-1 cells exposed to heme showed marked deposition of iC3b and C5b-9. In the presence of ALXN1820, deposition was blocked by >70% for iC3b and >85% for C5b-9. Pretreatment of mice with 14E1 markedly ameliorated vaso-occlusion and reduced both C3b and C5b-9 deposition on RBCs, and hemolysis biomarkers.

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Summary/Conclusion: These data strengthen the hypothesis that in SCD, cell-free heme is a potent trigger of complement AP activation, which can be blocked by targeting properdin. Investigating the efficacy and safety of anti-properdin ALXN1820 in patients with SCD is warranted as a novel approach to treatment of acute and chronic SCD complications.

S268: SAFETY, TOLERABILITY, AND PHARMACOKINETIC/PHARMACODYNAMIC RESULTS FROM PHASE 1 STUDIES OF GBT021601, A NEXT-GENERATION HBS POLYMERIZATION INHIBITOR FOR TREATMENT OF SICKLE CELL DISEASE

C. Brown 1,* , C. Key 2 , I. Agodoa 3 , J. Olbertz 3 , K. Duchin 3 , A. Barth 3 , E. Lisbon 3

1 Aflac Cancer and Blood Disorder Center of Children’s Healthcare of Atlanta and Department of Pediatrics, Emory School of Medicine, Atlanta; 2 ICON plc, San Antonio; 3 Global Blood Therapeutics, South San Francisco, United States of America

Background: Sickle cell disease (SCD) is caused by polymerization of sickle hemoglobin (HbS). Voxelotor is a first-in-class HbS polymerization inhibitor approved by the United States Food and Drug Administration for the treatment of SCD in adult and pediatric patients aged ≥4 years, and by the European Medicines Agency for the treatment of hemolytic anemia due to SCD in adult and pediatric patients aged ≥12 years as monotherapy or in combination with hydroxycarbamide. GBT021601 is a next-generation HbS polymerization inhibitor with improved pharmacokinetic (PK) properties that stabilizes Hb in the oxygenated state, thus inhibiting polymerization. GBT021601 has the potential to achieve higher Hb occupancies at lower doses than voxelotor, potentially reducing treatment burden and improving clinical outcomes for patients with SCD.

Aims: To explore the safety, tolerability, PK, and pharmacodynamics of GBT021601 in healthy volunteers and patients with SCD.

Methods: Two studies are in progress: (1) GBT021601-011: a phase 1, randomized, double-blind, placebo-controlled, parallel-group study of single ascending doses (SADs) and multiple ascending doses (MADs) of GBT021601 in healthy volunteers aged 18 to 55 years; and (2) GBT021601-012: a phase 1, single-arm, intrapatient, single-dose and MAD study in patients with SCD (homozygous HbS) aged 18 to 60 years. In the GBT021601-011 SAD phase, participants (n=8) were randomized 6:2 to receive a single oral dose of GBT021601 (50 to 2200 mg) or placebo. In the GBT021601-011 MAD phase, participants (n=10) were randomized 7:3 to 14 days of GBT021601 (3 days of loading doses followed by 11 days of once-daily maintenance doses) or placebo. Data from the 50 mg MAD cohort are reported in this analysis. In the GBT021601-012 study, patients with SCD (N=6) received a single 100 mg dose of GBT021601, followed by an 8-week washout, then 2 continuous MADs: once-daily maintenance doses of 50 mg (5 weeks) then 100 mg (3 weeks). Each MAD phase participant received 2 days of loading doses.

Results: In the GBT021601-011 50 mg MAD cohort, blinded safety data demonstrated that GBT021601 was well tolerated. Adverse events (AEs) were predominantly grade 1 and not related to treatment. Drowsiness, headache, and constipation were the most frequent AEs. Mean (SD) Hb occupancy of GBT021601, determined via PK analysis, was 25.8% (3.9%). In GBT021601-012, patients with SCD completed treatment with no AEs leading to study discontinuation. Three vaso-occlusive crises (grades 2 and 3) occurred in 2 patients and were deemed unrelated to GBT021601. After dosing for 8 weeks, the mean (SD) Hb occupancy achieved was 32.6% (9.4%), and the mean (SD) increase in Hb was 2.3 (0.9) g/dL. Markers of hemolysis decreased, with a mean (SD) change in absolute reticulocytes of –37.4% (17.3%), reticulocyte percentage of –54.0% (11.3%), indirect bilirubin of –6.6% (119.3%), and lactate dehydrogenase of –33.1% (15.4%). The terminal elimination half-life of GBT021601 from blood averaged about 10 days. Ektacytometry scans demonstrated improvements in red blood cell (RBC) health, and blood smears showed increased counts of RBCs with improved morphology (Figure); no changes in erythropoietin levels were observed.

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Summary/Conclusion: Multiple daily doses of GBT021601 were well tolerated in both studies. In patients with SCD, the GBT021601 100 mg maintenance dose led to a mean Hb occupancy >30%, increased Hb, reduced markers of hemolysis, and improved RBC health. These data highlight the potential of GBT021601 and support its further development.

S269: MACROPHAGE FUNCTIONAL AND METABOLIC REWIRING THROUGH PPAR/PGC1 MODULATION IMPROVES HEME/IRON-DRIVEN DEFECTIVE EFFEROCYTOSIS AND PROMOTES TISSUE DAMAGE RESOLUTION IN SICKLE CELL DISEASE

R. Sharma 1 , S. Z. Vance 1 , A. Antypiuk 1 , D. Manwani 2 , F. Vinchi 1,*

1 Iron Research Laboratory, New York Blood Center; 2 Pediatric Hematology, The Children’s Hospital at Montefiore, New York, United States of America

Background: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is contributed by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme, which is released from oxidized hemoglobin upon hemolysis and acts as damage-associated molecular pattern to stimulate macrophage pro-inflammatory phenotypic switching through TLR4 signaling activation and ROS production. Heme-induced M1 pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to organ damage typically associated with this disease.

Aims: While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties and whether this exacerbates tissue injury remain unexplored. Macrophage functions as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury.

Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages, to unveil the impact of hemolysis on macrophage functional properties.

Results: Our results demonstrate that, in addition to inflammatory activation, heme strongly alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme and iron excess drives defective efferocytosis of apoptotic neutrophils in vitro, in cultured bone-marrow-derived macrophages, and in vivo, in mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the transcription factor PPARγ and its coactivator PGC1α. These changes lead to reduced expression of efferocytic receptors and impaired mitochondrial biogenesis as well as mitochondrial dysfunction, and is associated with metabolic skewing. This results in limited recognition and engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4/10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. We further demonstrate that impaired phagocytic capacity and tissue damage are improved by hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment in SCD mice through phenotypic and functional macrophage rewiring. Interestingly, defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients’ plasma and rescued by hemoglobin/heme scavenging via haptoglobin and hemopexin and PPARγ/PGC1α modulation via PPARγ agonist or IL-4.

Summary/Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events.

S270: LONGER-TERM ANALYSIS OF EFFICACY OF LUSPATERCEPT VERSUS PLACEBO IN PATIENTS WITH TRANSFUSION-DEPENDENT BETA-THALASSEMIA ENROLLED IN THE BELIEVE STUDY

M. D. Cappellini 1,* , A. T. Taher 2 , J. B. Porter 3 , K. H. Kuo 4 , T. D. Coates 5 6 , E. Voskaridou 7 , G. L. Forni 8 , S. Perrotta 9 , A. Khelif 10 , A. Lal 11 , A. Kattamis 12 , A. Piga 13 , O. Hermine 14 15 , N. Holot 16 , F. Lersch 17 , J. K. Shetty 17 , S. Vodala 16 , J. Zhang 16 , D. Miteva 17 , V. Viprakasit 18

1 Fondazione IRCCS Ca’ Granda Policlinico Hospital, University of Milan, Milan, Italy; 2 Department of Internal Medicine, American University of Beirut Medical Center, Beirut, Lebanon; 3 University College London, University College London Hospitals, London, United Kingdom; 4 Division of Medical Oncology and Hematology, Department of Medicine, University Health Network, Division of Hematology, Department of Medicine, University of Toronto, Toronto, ON, Canada; 5 Children’s Center for Cancer and Blood Diseases, Children’s Hospital Los Angeles; 6 USC Keck School of Medicine, Los Angeles, CA, United States of America; 7 Thalassemia and Sickle Cell Center, Laiko General Hospital, Athens, Greece; 8 Centro della Microcitemia e Anemie Congenite e del Dismetabolismo del Ferro, Ospedale Galliera, Genoa; 9 Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Università della Campania, Luigi Vanvitelli, Naples, Italy; 10 Farhat Hached Teaching Hospital, Sousse University, Sousse, Tunisia; 11 University of California San Francisco Benioff Children’s Hospital, Oakland, CA, United States of America; 12 First Department of Pediatrics, National and Kapodistrian University of Athens, Athens, Greece; 13 University of Turin, Turin, Italy; 14 Department of Hematology, Hôpital Necker, Assistance Publique Hôpitaux de Paris, University Paris Cité; 15 INSERM U1163 and CNRS 8254, Imagine Institute, Université Sorbonne Paris Cité, Paris, France; 16 Bristol Myers Squibb, Princeton, NJ, United States of America; 17 Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland; 18 Siriraj Hospital, Mahidol University, Bangkok, Thailand

Background: Ineffective erythropoiesis and anemia are hallmarks of β-thalassemia. Luspatercept, a first-in-class erythroid maturation agent, has shown efficacy in the phase 3 BELIEVE ( NCT02604433 ) trial in patients (pts) with β-thalassemia requiring regular red blood cell transfusions (RBCT). However, longer-term analyses of efficacy are yet to be reported.

Aims: To observe and report the longer-term effect of luspatercept treatment on transfusion burden and liver iron concentration (LIC).

Methods: Pts ≥18 years of age with β-thalassemia or hemoglobin E/β-thalassemia (compound β-thalassemia mutation and/or multiplication of α-globin genes allowed) requiring regular RBCT (6–20 RBC units/24 wk before randomization with no transfusion-free period >35 d) were randomized 2:1 to luspatercept 1.0 mg/kg (titration up to 1.25 mg/kg allowed) or placebo subcutaneously every 3 wk. Response was defined as RBCT burden reduction of ≥33% (≥33% response) or ≥50% (≥50% response) from baseline (BL; ≥2 units) during a rolling 12- or 24-wk interval. LIC was determined by MRI.

Results: As of Jan 5, 2021, 125/224 (55.8%) pts randomized to luspatercept completed 144 wk of treatment and 6 (2.7%) pts completed 192 wk. The main reasons for treatment discontinuation were pt withdrawal (23.7% luspatercept vs 11.6% placebo), adverse events (10.3% vs 1.8%), and lack of efficacy (1.8% vs 7.1%). More pts receiving luspatercept vs placebo had ≥33% or ≥50% response during any rolling 12- or 24-wk interval up to the cutoff date (all P <0.0001); the number of responders receiving luspatercept was higher compared with previous data cuts (Table). The median (95% CI) longest duration of response for ≥33% and ≥50% responders (rolling 12-wk interval) in the luspatercept group increased from the primary data cut to 114.0 (107.0–137.0) and 99.0 (95.0–104.0) d, respectively (Table); median (95% CI) longest duration of response for placebo pts was 90.0 (86.0–94.0) and 86.0 (84.0–103.0) d, respectively. The median (range) total duration of response for ≥33% and ≥50% responders (rolling 12-wk interval) in the luspatercept group was 586.0 (84–1300) and 357.5 (84–1267) d, respectively, and 171.0 (84–627) and 169.0 (84–485) d in the placebo group. The 6 pts who completed 192 wk of luspatercept required on average 6.41 (SD 4.32) fewer RBC units during wk 145–192 compared with BL. Overall mean change from BL in transfusion window for ≥50% responders (any 24-wk interval) was increased by 9.88 d (SD 22.04, n=51). More pts receiving luspatercept (27/224 [12.1%]) than placebo (2/112 [1.8%]) achieved RBCT independence (RBC-TI) ≥8 wk ( P =0.0015), an increase from previous data cuts (Table). Median (95% CI) longest durations of RBC-TI were 72.0 (62.0–103.0) d for luspatercept and 71.5 (62.0–NA) for placebo, though the placebo result was driven by 2 pts. Longer-term treatment with luspatercept resulted in a decreasing trend in LIC compared with BL for ≥33% responders (rolling 12-wk interval) with a mean (SD) change from BL in LIC at wk 144 (n=25) of −3.73 (9.08) mg/g dry weight.

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Summary/Conclusion: Continued treatment with luspatercept resulted in more pts experiencing reduced RBCT burden and longer duration of response compared with previous data cuts. RBC units transfused decreased over the longer-term treatment period and the time between transfusions increased compared with BL for ≥50% responders. Longer-term luspatercept treatment also resulted in more pts experiencing RBC-TI ≥8 wk and a potential decreasing trend in LIC; LIC analysis is ongoing. These data indicate that pts continue to benefit from longer-term luspatercept treatment.

S271: BONE MARROW TFR2 DELETION IMPROVES THE THERAPEUTIC EFFECT OF ACTIVIN LIGAND TRAP RAP-536 IN Β-THALASSEMIC MICE

E. Tanzi 1 2 , S. M Di Modica 1 , J. Bordini 3 , V. Olivari 1 4 , M. Pettinato 1 , A. Pagani 1 , A. Campanella 3 4 , L. Silvestri 1 4 , A. Nai 1 4,*

1 Division of Genetics and Cell Biology, Ospedale San Raffaele; 2 University of Milan Bicocca; 3 Division of Experimental Oncology, Ospedale San Raffaele; 4 Vita-Salute San Raffaele University, Milan, Italy

Background: β-thalassemia is a disorder due to mutations in the β-globin gene, characterized by ineffective erythropoiesis (IE), anemia, and iron overload, whose treatment still requires improvement. Luspatercept, an activin receptor-ligand trap that promotes terminal erythroid differentiation downregulating the TGF-β pathway is a recently approved drug for treatment of transfusion dependent β-thalassemia (TDT) and in phase 3 trial for non TDT (NTDT). However, its precise mechanism of action remains to be elucidated and the possible connection with the erythropoiesis driving cytokine erythropoietin (EPO) is still controversial. In addition, Luspatercept is only partially effective in correcting anemia, thus the identification of strategies for enhancing its efficacy would be of clinical benefit.

The second transferrin receptor (TFR2) balances red blood cells (RBC) production with iron availability, contributing to the activation of the iron hormone hepcidin in the liver and acting as an EPO receptor partner in erythroid cells. We have demonstrated that bone marrow (BM) Tfr2 deletion increases erythroblast EPO sensitivity causing erythrocytosis in wild-type mice (Nai et al, Blood 2015) and improving IE, anemia, RBCs morphology and iron-overload in NTDT mice (Artuso et al, Blood 2018).

Aims: As we have previously demonstrated that the combined targeting of different pathophysiologic cues of the disease results in a synergistic benefit on thalassemic erythropoiesis and anemia (Casu, Pettinato, et al, Blood 2020), here we investigate whether BM Tfr2 genetic inactivation improves the efficacy of Luspatercept in a NTDT model ( Hbb th3/+ ).

Methods: Hbb th3/+ mice with BM Tfr2 deletion (Tfr2 BMKO /Hbb th3/+ ) and Hbb th3/+ controls were treated with RAP-536 (Celgene, BMS group), a murine analogue of Luspatercept, 10mg/kg, twice a week for 8 weeks. Complete blood count was evaluated every two weeks. At sacrifice, blood and organs were collected for full phenotypic analysis.

Results: As expected, BM Tfr2 deficiency strongly ameliorated anemia and IE of Hbb th3/+ mice (hemoglobin, Hb=13.48±0.19g/dL vs 8.65±0.24g/dL in controls, P <0.000001). RAP-536 moderately increased RBC count, Hb and hematocrit after 2 weeks of treatment, and the beneficial effect was maintained until the end of the protocol with a similar efficacy in Hbb th3/+ (Hb=9.54±0.33g/dL) and Tfr2 BMKO /Hbb th3/+ mice (Hb=14.48±0.50g/dL), without significant changes in MCV, MCH and reticulocyte count. Splenomegaly was not affected by RAP-536 in Hbb th3/+ mice, despite an amelioration of both spleen and BM IE, with reduced proportion of immature erythroblasts and increased mature RBCs. Erythroid differentiation was further improved in RAP-536-treated Tfr2 BMKO /Hbb th3/+ mice both in the spleen and in the BM, leading to a significant reduction of spleen size, to values of wt animals.

Hepatic and serum iron levels were reduced in mice lacking Tfr2 , likely because of high erythroid consumption, while RAP-536 did not affect iron homeostasis in both genotypes.

Summary/Conclusion: BM Tfr2 deletion and RAP-536 treatment had additive effect in correcting IE and anemia in β-thalassemic mice. Since RAP-536 has comparable therapeutic efficacy in Hbb th3/+ and Tfr2 BMKO /Hbb th3/+ animals, it improves erythropoiesis through mechanism(s) other than Tfr2 deletion, thus independent of EPO stimulation. Overall, our results add a piece of information on the mechanism of action of Luspatercept and suggest that the concomitant TFR2 targeting might represent a promising option for boosting the therapeutic efficacy of activin receptor ligand-traps.

S272: SAFETY AND PRELIMINARY PHARMACODYNAMIC EFFECTS OF THE FERROPORTIN INHIBITOR VAMIFEPORT (VIT-2763) IN PATIENTS WITH NON-TRANSFUSION-DEPENDENT BETA THALASSEMIA (NTDT): RESULTS FROM A PHASE 2A STUDY

A. Taher 1,* , A. Kourakli-Symeonidis 2 , A. Tantiworawit 3 , P. Wong 4 , P. Szecsödy 5

1 American University of Beirut Medical Center, Beirut, Lebanon; 2 University of Patras – Rio Regional University Hospital, Rio, Greece; 3 Chiang Mai University – Faculty of Medicine, Chiang Mai; 4 Naresuan University Hospital, Mueang Phitsanulok, Thailand; 5 Vifor Pharma AG, Glattburg, Switzerland

Background: NTDT is characterized by an imbalanced α/β-globin chain ratio, ineffective erythropoiesis, increased intestinal iron absorption and iron overload. Vamifeport inhibits ferroportin-mediated iron export into plasma and has been shown to decrease serum iron and transferrin saturation (TSAT) levels in relevant animal disease models and healthy volunteers.

Aims: The primary objective of this multicenter, Phase 2a, double-blind, randomized, placebo-controlled study ( NCT04364269 ) was to assess the safety and tolerability of vamifeport (given once [QD] or twice [BID] daily) versus placebo in patients with NTDT over a 12-week treatment period. A secondary objective was to assess preliminary efficacy of vamifeport versus placebo with respect to pharmacodynamic effects on iron parameters (serum iron, ferritin, hepcidin and TSAT) in these patients.

Methods: Adults (18–65 years) with documented NTDT (including β-thalassemia intermedia) who provided informed consent, were included. NTDT was defined as receipt of <5 units of red blood cells during the 24 weeks prior to randomization. Patients also required a mean baseline hemoglobin of ≤11 g/dL on two consecutive measurements ≥1 week apart within 6 weeks prior to randomization. Use of iron chelation therapy (ICT) within 4 weeks prior to randomization was not permitted; patients with TSAT <30%, serum ferritin <150 ng/mL and/or liver iron content ≤1 mg/g (<300 ng/mL/3 mg/g, respectively, for those recently on ICT) were also excluded. Patients received vamifeport at a dose of 60 mg if their body weight was 40–59 kg or 120 mg if their weight was 60–100 kg.

Results: Twenty-five patients were included (vamifeport QD n=9, BID n=12, placebo n=4; median age 42.0, 31.0 and 40.5 [range 18–61] years, respectively). Overall, 64% were male, 56% had a body weight <60 kg and 16% had received prior ICT. At baseline, mean (standard deviation [SD]) serum iron concentrations were 27.2 (11.6) µmol/L and mean TSAT was 76.2 (27.6)% for the population as a whole. Mean (SD) serum ferritin concentrations were 746.7 (1431.7) µg/L. There were no differences between groups in treatment-emergent adverse event (TEAE) rates (vamifeport QD 66%, BID 58%; placebo 75%). All TEAEs were mild or moderate intensity, and each was reported only once. There were no deaths or serious AEs (1 patient receiving vamifeport QD had an acute hemolytic event); one TEAE (increased creatine kinase and transaminase levels, not drug-related) led to drug discontinuation in the vamifeport BID group. There were also no clinically relevant changes in any assessed safety parameter. Serum iron concentrations decreased 3 h after first vamifeport dose (Day 1) in all vamifeport-treated patients (mean [SD] decrease: QD -11.3 [7.2] µmol/L; BID -17.0 [9.6] µmol/L) and were maintained below baseline levels at 3–4 h post vamifeport administration at each visit throughout the remaining treatment period. Mean (SD) TSAT also decreased at Day 1 (3 h post dose; QD -32.6 [19.6]%; BID -46.8 [22.6]%) and were below baseline levels 3–4 h post-dose at each subsequent visit in the vamifeport groups (Figure). There were no clinically meaningful changes from baseline in serum iron or TSAT levels in the placebo group, or in ferritin or hepcidin levels in any treatment group.

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Summary/Conclusion: In this 12-week Phase 2a study, vamifeport had a favorable safety and tolerability profile and showed promising target engagement and pharmacodynamic effects on serum iron and TSAT levels versus placebo in adults with NTDT.

S273: EVIDENCE OF NONINFERIORITY OF MITAPIVAT VERSUS SPLENECTOMY IN MURINE HEREDITARY SPHEROCYTOSIS

A. Matte’ 1,* , A. Recchiuti 2 , E. Federti 1 , P. A. Kosinski 3 , V. Riccardi 1 , I. Iatcenko 1 , L. Dang 3 , A. Iolascon 4 , R. Russo 4 , C. Brugnara 5 , A. Janin 6 , C. Leboeuf 6 , N. Mohandas 7 , L. De Franceschi 1

1 Dept of Medicine, University of Verona, Verona; 2 Dept of Medical, Oral, and Biotechnological Science, “G. d’Annunzio” University of Chieti, Chieti, Italy; 3 Agios Pharmaceuticals, Cambridge, United States of America; 4 Dept. of Molecular Medicine and Medical Biotechnology and CEINGE, University of Naples Federico II, Naples, Italy; 5 Dept of Laboratory Medicine, Boston Children’s Hospital - Harvard Medical School, Boston, United States of America; 6 University Diderot of Paris, Paris, France; 7 Laboratory of Red Cell Physiology, New York Blood Center, New York, United States of America

Background: Hereditary spherocytosis (HS) is the most common cause of inherited red cell membranopathy, due to mutations in genes encoding membrane or cytoskeletal proteins, including band 3, ankyrin, spectrin or band 4.2. Loss of membrane cohesion results in surface area loss and generation of spherocytic red cells with decreased cellular deformability and reduced red cell survival due to splenic sequestration. Relative PK deficiency has been observed in HS red cells (Andreas O et al. BJH 187: 386, 2019). Splenectomy is the gold standard treatment of anemia in patients with HS. However, the benefits of splenectomy in mild HS is still controversial and its cost/effectiveness might be underestimated. Using band 4.2 -/- mice, a well-established model of HS (Peters LL et al JCI 103: 1527, 1999), we recently show that mitapivat, an oral PK activator, ameliorates anemia, reduces chronic hemolysis and improves RBC features in 4.2 -/- mice (Matte A et al. EHA 2021).

Aims: To understand whether the oral pyruvate kinase activator, mitapivat, is not less efficacious than splenectomy in treatment of anemia in 4.2 -/- mice.

Methods: Splenectomized 4.2 -/- mice (2.5 months of age) were either treated with vehicle or mitapivat at dosages of 100 mg/kg/day over 4 months. An unsplenectomized group of 4.2 -/- mice treated with and without mitapivat was also analyzed. The following parameters were evaluated: hematologic parameters, markers of hemolysis, erythropoiesis and iron homeostasis.

Results: In band 4.2 -/- mice, splenectomy resulted in (i) amelioration of anemia with reduced reticulocyte count (Hb 13.3 ±0.5 g/dL, n =3 vs 11.9±0.6 g/dL, n =8; P <0.05; retics: 4.9±0.5%, n=3 vs 10.0±1.4%, n=8 P <0.05) and reduced hemolytic indices (LDH, total bilirubin) when compared to non-splenectomized 4.2 -/- mice. The decreased hemolysis was associated with reduction in serum erythropoietin (EPO). Mitapivat was non-inferior to splenectomy for amelioration of anemia (Hb 13.4±0.8 g/dL, n=11 vs 11.9±0.6 g/dL, n =8), reduction in reticulocyte count (retics: 7.8±1.4%, n=11 vs 10.0±1.4%, n=8 P <0.05) and EPO levels (401±123 U/L, n=3). Noteworthy, in splenectomized 4.2 -/- mice, mitapivat further improved Hb (Hb: 14.1±0.3 g/dL, n=3), reduced reticulocyte counts (retics: 3.6±0.2%, n=3) and EPO levels when compared to vehicle treated splenectomized 4.2 -/- mice.

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Summary/Conclusion: Our data indicate that mitapivat was at least as effective as splenectomy in treatment of anemia in a murine model of HS.

S274: A NOVEL SUBTYPE OF ANEMIA CAUSED BY MUTATIONS IN TFRC GENE

S. Colucci 1 2,* , V. Venturi 3 , F. Nicole 1 2 , D. Jové Solavera 3 , M. Zimon 4 , P. Richter-Pechanska 1 , G. Hernandez 3 5 , S. Unal 6 , F. Gumruk 6 , A. Diaz-Conradi 7 , L. Romero-Cortadellas 3 , X. Ferrer-Cortès 3 5 , M. Olivella 8 , M. Erlacher 9 , C. Niemeyer 9 , T. Wiesel 10 , R. Pepperkok 4 , M. D. Fleming 11 , A. E. Kulozik 1 2 , M. Sanchez 3 5 , M. U. Muckenthaler 1 2

1 Department of Pediatric Oncology, Hematology, and Immunology, Heidelberg University; 2 Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany; 3 Department of Basic Sciences, Iron metabolism: Regulation and Diseases, Universitat Internacional de Catalunya (UIC), Sant Cugat del Vallès, Spain; 4 European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 5 BloodGenetics S.L. Diagnostics in Inherited Blood Diseases, Esplugues de Llobregat, Spain; 6 Department of Pediatrics, Hacettepe University Faculty of Medicine, Ankara, Turkey; 7 Hospital de Nens de Barcelona, Barcelona; 8 Biosciences Department, Faculty of Sciences and Technology (FCT), University of Vic - Central University of Catalonia, Vic, Spain; 9 Department of Pediatrics and Adolescent Medicine, Medical Center, University of Freiburg, Freiburg; 10 Vestische Kinder- und Jugendklinik Datteln, University Witten/Herdecke, Datteln, Germany; 11 Department of Pathology, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

Background: Anemia affects ~25% of the world population and is frequently caused by iron deficiency as a consequence of malnutrition or inflammation. Only in rare cases anemia is due to mutations in genes responsible for balancing iron homeostasis. Among these, loss of function variants of MT2 cause iron-refractory iron-deficiency anemia (IRIDA); a disease characterized by inappropriately high levels of hepcidin. Hepcidin is a hepatic hormone that limits dietary iron uptake and iron release from intracellular stores and thus renders IRIDA patients resistant to iron therapies. In mice, a form of anemia resistant to iron supplementation is further caused by mutations in Transferrin Receptor 1 (TfR1; Tfrc gene). TfR1 internalizes iron-bound transferrin, a process fundamental for erythropoiesis. However, up to date, the sole human report describing a TfR1 mutation (p.Tyr20His) showed that it impairs the immunological compartment rather than erythroblast maturation. Here, we report pediatric patients with previously undescribed mutations in TFRC , diagnosed with microcytic hypochromic anemia, partially resistant to iron therapies.

Aims: Identification of novel disease alleles that cause anemia.

Methods: Whole exome sequencing (WES) of DNA of patients with anemia. Functional characterization of novel mutations identified in TFRC gene was assessed in HeLa cells overexpressing TfR1 wild-type and mutated by western blotting and wide-field microscopy.

Results: A 4-year old boy manifested symptoms of anemia and the analysis of hematological parameters revealed low levels of hemoglobin, MCV and MCH. Plasma iron, transferrin saturation and hepcidin were within physiological ranges. Unexpectedly, soluble TfR1 (sTfR1) levels were almost undetectable and the bone marrow smear revealed increased erythropoiesis and iron deficiency in this compartment. The condition of hypochromic microcytic anemia was persistent over years and could not be corrected by oral iron therapy. WES analysis identified a TFRC _c.941C>T homozygous mutation, encoding for TfR1 P314L . Relatives carrying the same mutation in heterozygosity were not anemic. This indicates that TFRC _c.941C>T mutation causes a recessive form of anemia. Overexpression of a construct expressing the TfR1 P314L mutant in HeLa cells showed a reduction in total and secreted TfR1, thus reflecting upon the observation of very low sTfR1 levels in the blood of the patient. We further show that lower TfR1 levels are likely due to a faster degradation rate of TfR1 P314L compared to TfR1 WT . In addition, the TfR1 P314L mutated protein showed impaired internalization of fluorescently labeled transferrin, most likely explaining the reduced iron levels observed in the bone marrow.

In addition to this index patient, 3 additional children of 2 independent families were identified with TFRC mutations and hypochromic microcytic anemia. The genetic analysis detected a TFRC _c.967C>G heterozygous mutation encoding for TfR1 P323A and a TFRC _c.934G>A homozygous mutation, encoding for TfR1 G312R . All three TFRC mutations are highly conserved across species and are located in the same extracellular loop of TfR1.

Summary/Conclusion: We identified a novel subtype of juvenile hereditary hypochromic microcytic anemia that is partially resistant to iron therapies and caused by missense mutations in the TFRC gene. Reduced endocytosis of iron-bound transferrin may contribute to insufficient iron supply for erythropoiesis and resistance to iron supplementation. In the clinical practice, our findings suggest to include TFRC sequencing analysis for patients with unexplained anemia.

S275: ALTERATION OF IRON HOMEOSTASIS THROUGH GENETIC AND PHARMACOLOGIC MODULATION OF FERROPORTIN MODIFIES MDS PATHOPHYSIOLOGY IN A PRECLINICAL MOUSE MODEL

S. Z. Vance 1 , A. Antypiuk 1 , R. Sharma 1 , F. Dürrenberger 2 , V. Manolova 2 , F. Vinchi 1,*

1 Iron Research Laboratory, New York Blood Center, New York, United States of America; 2 Vifor (International) Ltd, St. Gallen, Switzerland

Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it remains unclear whether and how iron excess is detrimental for MDS pathophysiology.

Aims: This study aimed at characterizing altered iron homeostasis in a preclinical MDS mouse model, understanding the molecular mechanisms underlying iron toxicity and unraveling the potential therapeutic value of pharmacologic iron restriction in this disease condition.

Methods: To this end, we took advantage of complementary approaches and analyzed the effect of iron overload obtained through genetic activation of the iron exporter ferroportin (FPN C326S ), and iron restriction achieved through the administration of the FPN inhibitor Vamifeport for 3 months in NUP98-HOXD13 MDS mice. Mice were analyzed at 6 months of age.

Results: MDS mice develop anemia and low white blood cell counts in the peripheral blood associated with a significant iron overload phenotype, hallmarked by low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. FPN C326S MDS mice show an aggravated iron phenotype, with further elevated NTBI and tissue iron accumulation and still inappropriately low hepcidin levels. Vamifeport administration in MDS mice reduced serum iron and NTBI levels and prevented tissue iron loading. While iron excess in FPN C326S MDS mice did not improve erythropoiesis and hematologic parameters in the peripheral blood, iron restriction by Vamifeport significantly ameliorated red blood cell maturation and hemoglobin levels in MDS mice. The improved ineffective erythropoiesis was associated with reduced oxidative stress and apoptosis in erythroid progenitors. Unexpectedly, we observed a major role of the iron status in myeloid expansion in MDS. The number of immature myeloid blasts and myeloid progenitors as well as inflammation markers in the bone marrow of MDS mice were aggravated by iron overload in FPN C326S MDS mice and attenuated by iron restriction in Vamifeport-treated MDS mice compared to control MDS animals. Myeloid bias, monitored as percentage bone marrow myeloid cells, was exacerbated by iron overload and alleviated by iron restriction. Overall, this translated into a faster and delayed transition to AML, respectively. Finally, iron showed a profound impact on the stem cell pool in MDS. Iron overload aggravated and iron restriction alleviated the formation of reactive oxygen species, accumulation of DNA damage and levels of the apoptosis marker annexin V in hematopoietic stem and progenitor cells (HSPCs). By reducing the intracellular labile iron pool, Vamifeport improved HSPC quality and survival, partially rescuing the stem cell pool size in MDS mice. The improved stem cell pool, reduced myeloid bias and attenuated anemia resulted in a significant survival increment in iron-restricted compared to control MDS mice.

Summary/Conclusion: Our results show for the first time in a preclinical mouse model of MDS that iron excess driven by ineffective erythropoiesis has pathological implication in transfusion-independent MDS. These effects are likely aggravated by transfusions causing additional iron overload. Finally, iron restriction achieved through pharmacologic FPN inhibition significantly improves MDS pathophysiology, uncovering the therapeutic potential of early prevention of NTBI formation in MDS.

S276: TRANSFERRIN RECEPTOR 2 TARGETING AMELIORATES DIFFERENT FORMS OF ANEMIA CAUSED BY CHRONIC INFLAMMATION

V. Olivari 1 2,* , M. R. Lidonnici 3 , E. Tanzi 1 4 , S. M. Di Modica 1 , F. Tiboni 3 , L. Silvestri 1 2 , G. Ferrari 2 3 , A. Nai 1 2

1 Regulation of Iron Metabolism Unit - Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute; 2 Vita-Salute San Raffaele University; 3 Gene Transfer into Stem Cell Unit, SR-Tiget, IRCCS San Raffaele Scientific Institute; 4 University of Milan Bicocca, Milan, Italy

Background: Anemia is a common complication of chronic inflammation. It is caused by an increased production of the iron-regulatory hormone hepcidin, that leads to iron restriction, and by decreased erythrocytes sensitivity to erythropoietin (EPO) stimulation. Anemia of Chronic Kidney Disease (CKD) is a form of anemia of inflammation, in which progressive renal damage, besides causing systemic inflammation and iron deficiency, impairs erythropoietin (EPO) production. Current EPO-based treatments may cause off-target effects on long term and often require iron supplementation, mainly for CKD.

Transferrin Receptor 2 (TFR2) modulates iron homeostasis in the liver, contributing to the transcriptional activation of hepcidin, and acts as a brake of EPO signaling in erythroid cells. Its selective deletion in the bone marrow (BM, Tfr2 BMKO ) enhances erythropoiesis both in wild-type and thalassemic mice.

Aims: We investigated whether Tfr2 targeting might represent a therapeutic option for different forms of anemia, able to enhance erythropoiesis in chronic inflammation and simultaneously increase iron availability in CKD.

Methods: Chronic sterile inflammation was induced by weekly subcutaneous injection of turpentine oil (100ul/20g body weight for 3 weeks) in Tfr2 BMKO mice. Mice were sacrificed 2 or 14 days after the last injection.

CKD was induced feeding Tfr2 BMKO mice an adenine-rich diet for 8 weeks. The same protocol was applied also in germline Tfr2 deficient ( Tfr2-ko ) animals to simultaneously inactivate erythroid and hepatic Tfr2 .

Complete blood count was periodically determined, and a complete phenotypic analysis was performed at sacrifice.

Results: Chronically inflamed Tfr2 BMKO mice maintained red blood cell count and hemoglobin levels higher than controls for the entire 5-week-long timespan, despite comparable inflammation.

In CKD mice, BM-specific Tfr2 inactivation enhanced erythropoiesis and delayed anemia until iron availability was sufficient: indeed, hemoglobin came back to control values at the end of the protocol. The drop in hemoglobin levels was prevented in Tfr2-ko mice, in which iron supply is increased. Renal damage was comparable among the experimental groups, excluding a differential effect of the diet.

Of note, BM-specific Tfr2 deletion boosted erythropoiesis and activated EPO-EPOR pathway without affecting EPO levels both in CKD and turpentine mice.

Summary/Conclusion: We proved that erythroid Tfr2 targeting ameliorates hematological parameters in murine models of anemia of inflammation and anemia of CKD. The beneficial effect is achieved enhancing erythroid EPO responsiveness, without affecting EPO levels per se. For this reason, an approach based on erythroid TFR2 targeting would avoid the side effects of the available therapies.

Moreover, we demonstrated that the concomitant inhibition of hepatic and erythroid Tfr2 in anemia of CKD would represent a therapeutic opportunity to simultaneously increase iron availability and erythroid responsiveness in a balanced manner.

Overall, our results prove that TFR2 targeting may represent a tunable therapeutic approach for different forms of anemia.

S277: HYPOFERREMIA CAUSED BY FERROPORTIN DOWNREGULATION IS REGULATED BY NFКB THROUGH HDAC-DEPENDENT MECHANISMS

O. Marques 1,* , N. Horvat 1 , M. C. Lai 2 , L. Zechner 1 , S. Colucci 1 , R. Sparla 1 , S. Zimmermann 3 , C. J. Neufeldt 4 , S. Altamura 1 , J. Zaugg 2 , K. Mudder 1 , J. D. Knopf 5 , M. K. Lemberg 5 , M. Pasparakis 6 , M. W. Hentze 2 , M. U. Muckenthaler 1

1 Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Universitatsklinikum Heidelberg; 2 European Molecular Biology Laboratory; 3 Department of Medical Microbiology and Hygiene; 4 Department of Molecular Virology, Universitatsklinikum Heidelberg; 5 ZMBH, University of Heidelberg; 6 CECAD, University of Cologne, Heidelberg, Germany

Background: Anemia of Inflammation (AI) is a highly prevalent comorbidity in patients with chronic inflammatory disorders. The underlying pathophysiology is partly explained by hypoferremia that results from iron sequestration in macrophages and the reduction of dietary iron uptake due to decreased iron export mediated by Ferroportin (Fpn). In response to inflammation, Fpn levels are decreased by hepcidin-mediated post-translational and/or transcriptional mechanisms, depending on the type of inflammatory pathway activated. Despite the fact that Fpn transcriptional repression is frequently observed in inflammatory conditions, the regulatory mechanisms involved remained unidentified.

Aims: To gain mechanistic insight how Fpn mRNA levels are regulated in response to inflammation.

Methods: We conducted a pharmacological and RNAi screen, targeting mediators of TLR2/6 signalling, in bone marrow-derived macrophages (BMDMs) stimulated with the TLR2/6-specific ligand FSL1. Changes in chromatin accessibility were evaluated by chromatin-accessibility Real-Time Polymerase Chain Reaction (CHART-PCR) and Assay for Transposase-Accessible Chromatin using sequencing (ATACseq). Data obtained in cell-based assays were validated in mice injected with FSL1.

Results: The screen in BMDMs together with validation experiments demonstrated that the FSL1 induced TLR2 response represses Fpn mRNA levels via MYD88 and TRAF6 signaling transducers. While FSL1 can activate independent JNK, p38 and NFκB signaling branches, we show that Fpn transcriptional repression is specifically dependent on alternative NFκB signaling involving the transcription factor RELB. This contrasts our findings for TLR4 stimulation by LPS, where reduced Fpn mRNA expression depends on classical NFκB signaling, involving the NFкB subunits RELA and p50.

Using ATACseq we identified Fpn genomic regions that are altered by inflammatory stimuli. These data show chromatin condensation around the Fpn transcription start site (TSS) upon FSL1 or LPS treatment. Since chromatin condensation is mainly promoted by histone deacetylases (HDACs), we next tested whether Fpn mRNA reduction upon inflammatory cues could be counteracted by treatment with HDAC inhibitors (HDACi). We demonstrate that pre-treatment with the pan-HDACi SAHA (Suberoylanilide Hydroxamic Acid) prevented chromatin remodelling at the Fpn locus and Fpn transcriptional repression in response to inflammation and RELB overexpression, suggesting that the effect of HDACs on Fpn responses occurs downstream of NFкB. In line with these data, NFκB pharmacological inhibition also prevented Fpn chromatin remodeling in the vicinity of the TSS. The findings obtained in BMDMs were extended in mice treated with SAHA prior to FSL1 injection, showing reduced hypoferremia and attenuation of splenic Fpn mRNA repression, while serum hepcidin levels remained unaltered.

Summary/Conclusion: These results identify NFκB as an important repressor of Fpn transcription in response to inflammatory signals involving chromatin remodeling in a genomic region near Fpn ’s TSS. Importantly, HDAC inhibition attenuates Fpn mRNA repression as well as the resulting hypoferremia in mice. Our findings generate molecular insight about how hypoferremia is generated and demonstrate that Fpn, like hepcidin, is controlled by HDACs.

S278: EVALUATION OF THE MAIN REGULATORS OF SYSTEMIC IRON HOMEOSTASIS IN PYRUVATE KINASE DEFICIENCY

A. Zaninoni 1 , R. Russo 2 3 , R. Marra 2 3 , I. Andolfo 2 3 , E. Fermo 1 , A. P. Marcello 1 , D. Consonni 4 , B. Eleni Rosato 2 3 , S. Martone 2 3 , B. Fattizzo 1 , W. Barcellini 1 , A. Iolascon 2 3 , P. Bianchi 1,*

1 Hematology Unit - Physiopathology of Anemias Unit, Foundation IRCCS Cà Granda Policlinico Milan, Milan; 2 Department of Molecular Medicine and Medical Biotechnology; 3 CEINGE – Advanced Biotechnologies, University Federico II, Naples; 4 Epidemiology Unit, Foundation IRCCS Cà Granda Policlinico Milan, Milan, Italy

Background: Iron loading anemias are characterized by ineffective erythropoiesis and iron overload. This group of anemias includes thalassemia syndromes, congenital dyserythropoietic anemias (CDA), and some forms of congenital hemolytic anemias. Among them pyruvate kinase deficiency (PKD) has been shown to develop iron overload also in absence of transfusions suggesting dyserythropoietic features. Moreover, severe forms can be misdiagnosed as CDA due to bone marrow abnormalities and ineffective erythropoiesis further supporting these evidences. The hormone erythroferrone (hERFE) is produced by erythroblasts in response to erythropoietin (EPO), and acts by suppressing hepcidin, thereby increasing iron absorption and mobilisation for erythropoiesis demand. The ERFE-hepcidin axis seems to play a crucial role in the pathogenesis of these disorders; an increased erythroferrone release by immature erythroid cells results in hepcidin suppression and secondary iron overload that could finally results in ineffective erythropoiesis and anemia.

Aims: To investigate the pathophysiological basis of iron overload in PKD.

Methods: We analysed the levels of hERFE, EPO, hepcidin, and soluble transferrin receptor (sTFR) in a large group of 41 PKD patients equally distributed by gender, age and severity. The results were analysed in comparison with two groups of patients affected by hemolytic anemia with overt dyserythropoiesis (42 patients with CDA type II) and with congenital hemolytic anemia due to RBC membrane defects (51 patients with hereditary spherocytosis [HS]), respectively.

Results: Demographic, hematologic, and biochemical features of the three groups of patients are reported in the Figure 1A. Among the PKD patients, 18/41 were <18 yrs, median Hb level at the time of the study was 9.05g/dL (range 5.5-14.5), 12 underwent splenectomy, 28 ever received at least three transfusions their life, 14 of them transfusion dependent (>6 tx/yrs). Mean ferritin levels at the time of the study were 546 ng/ml (range 59-4990), 15/41 patients requiring chelation therapy for iron overload developed also in absence of transfusions.

As expected, CDAII patients showed decreased hepcidin levels (3.74 ng/mL; n.v. 17.25, P<0.001) associated with increased erythropoietin (62.7 IU/L, n.v. 6.5, P=0.01) and hERFE (24.8 ng/mL, n.v. 1, P<0.0001). On the contrary, HS showed increased hepcidin, with less marked increased of ERFE (9.9 ng/mL, P=0.02) and EPO (36.4 IU/L, P=0.005). In PKD patients we observed decreased hepcidin levels (7.15 ng/mL, P=0.03)), increased hERFE (18 ng/mL, P<0.0001) and EPO (75.6 IU/L, P=0.009). Instead, sTFR was equally increased in the three groups of patients (Figure 1B).

Interestingly, by comparing the three groups of patients, PKD showed dyserythropoietic features as evidenced by the observation of intermediate values between HS and CDAII of hepcidin (P=0.007 PKD v CDAII and P=0.0002 PKD vs HS), hEFRE, and sTFR (Figure 1C).

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Summary/Conclusion: This study provides the first analysis of the main regulators of systemic iron homeostasis in PK deficiency compared either with the model of a structural RBC defect (HS) or with the typical model of dyserythropoietic anemia with ineffective erythropoiesis, such as CDAII. These data provide evidence of the dyserythropoietic features of PK deficiency, underlining the need of accurate diagnosis and paving the way of novel therapeutic approaches in PK deficiency.

S279: INCREASED RISK OF INFECTIONS IN INDIVIDUALS WITH HEMOCHROMATOSIS C282Y HOMOZYGOSITY AND IN INDIVIDUALS WITH HIGH OR LOW PLASMA IRON OR TRANSFERRIN SATURATION: A PROSPECTIVE STUDY OF 138252 INDIVIDUALS

M. Mottelson 1 2 3,* , A. Glenthøj 1 2 , B. Nordestgaard 2 3 4 5 , C. Ellervik 4 6 7 8 , S. Bojesen 2 3 4 5 , J. Helby 1 3

1 Department of Hematology, Copenhagen University Hospital - Rigshospitalet; 2 Department of Clinical Medicine, University of Copenhagen; 3 The Copenhagen General Population Study; 4 Department of Clinical Biochemistry, Copenhagen University Hospital - Herlev and Gentofte; 5 The Copenhagen City Heart Study, Copenhagen University Hospital – Bispebjerg and Frederiksberg, Copenhagen; 6 Department of Production, Researh, and Innovation, Region Zealand, Sorø, Denmark; 7 Department of Laboratory Medicine, Boston Children’s Hospital; 8 Department of Pathology, Harvard Medical School, Boston, MA, United States of America

Background: Plasma iron and transferrin saturation are measures of iron available in the blood stream. Plasma ferritin indicate body iron stores. Levels of plasma iron, transferrin saturation and ferritin are increased in hemochromatosis C282Y homozygous individuals.

Aims: We tested the hypothesis that high and low plasma iron and transferrin saturation are associated with increased risk of infections observationally and genetically through hemochromatosis C282Y homozygosity.

Methods: We studied 138,252 general population individuals. Plasma iron and transferrin saturation were measured in 136,656 and 136,599 individuals, respectively. 132,542 individuals were genotyped for the C282Y hemochromatosis variant. Individuals were followed prospectively for up to 28 years for hospital admissions and emergency room visits due to infectious diseases using the National Patient Register which covers all danish hospitals.

Results: During follow-up 31,199 individuals were hospitalized due to an infection. After multivariable adjustment including other risk factors for infections, individuals with the 5% lowest and 5% highest plasma iron levels had hazard ratios for any infection of 1.20 (95% CI:1.12-1.28;P<0.001) and 1.14 (95% CI:1.07-1.22;P<0.001) respectively, when compared to individuals with plasma iron levels between the 25 th -75 th percentile. Findings for transferrin saturation were similar. Hazard ratio for any infection in hemochromatosis C282Y homozygous individuals was 1.40 (95% CI:1.16-1.69; P<0.001) when compared to wildtype individuals. Risk of sepsis was especially high in C282Y homozygotes (hazard ratio 1.69;95% CI:1.05-2.73; P=0.03). Risk of any infection was increased even in C282Y homozygotes with normal levels of plasma iron, transferrin saturation or ferritin.

Summary/Conclusion: High and low plasma iron and transferrin saturation were associated with increased risk of infections. Hemochromatosis C282Y homozygotes had increased risk of infection even when they had normal levels of plasma iron, transferrin saturation or ferritin.

Current hemochromatosis treatment strategy centralized around ferritin normalization may not reduce risk of infection satisfactorily in patients with hereditary hemochromatosis genotype.

S280: B-CELL MALIGNANCIES TREATED WITH TARGETED DRUGS AND SARS-COV-2 INFECTION. A EUROPEAN HEMATOLOGY ASSOCIATION SURVEY (EPICOVIDEHA)

M. S. Infante 1 , J. Salmanton-García 2,* , A. Fernandez-Cruz 3 , F. Marchesi 4 , Z. RÁČIL 5 , M. Hanakova 6 , O. Jaksic 7 , B. Weinbergerova 8 , C. Besson 9 , R. Duarte 10 , F. Itri 11 , T. VALKOVIĆ 12 , A. Busca 13 , A. Guidetti 14 , A. GLENTHØJ 15 , G. Collins 16 , V. Bonuomo 17 , U. Sili 18 , G. Seval 19 , M. Machado 20 , A. Lopez-Garcia 21 , R. Cordoba 22 , O. Blennow 23 , G. Abu-zeinah 24 , S. Lemure 25 , A. Kulasekararaj 26 , I. Falces-Romero 27 , C. Cattaneo 28 , O. A. Cornely 29 , J.-A. Hernandez-Rivas 30 , L. Pagano 31

1 Hematology Department, University Hospital Infanta Leonor, MADRID, Spain; 2 Internal Medicine, University of Cologne, Excellence center for Medical Mycology, Cologne, Germany; 3 Internal Medicine, Hospital Universitario Puerta de Hierro, Majadahonda, Spain; 4 hematology and stem cell transplant Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy; 5 Institute of Hematology and Blood transfusion, Prague, Czechia; 6 Hematology Department, Institute of Hematology and Blood transfusion, Prague, Czechia; 7 Hematology Department, University Hospital Dubrava, Zagreb, Croatia; 8 Internal Medicine, Masaryk University and University Hospital Brno, Brno, Czechia; 9 Hematology Department, Centre Hospitalier de Versailles, Versailles, France; 10 hematology and stem cell transplant Unit, Hospital Universitario Puerta de Hierro, MADRID, Spain; 11 Hematology Department, San Luigi Gonzaga Hospital, Orbassano, Italy; 12 Croatian Cooperative Group for Hematological Diseases (CROHEM), University Hospital Centre, Rijeka, Croatia; 13 Stem Cell Transplant Center, AOU Cittá della Salute e della Scienza, Turin; 14 Hematology Department, Fondazione IRCCS Istituto Nazionale, Milan, Italy; 15 Hematology Department, Rigshospitalet University Hospital, Copenhagen, Denmark; 16 Hematology Department, NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom; 17 Hematology Department, University Hospital of Verona, Verona, Italy; 18 Hematology Department, Marmara University Hospital, Istanbul; 19 Hematology Department, Ankara University Hospital, Ankara, Turkey; 20 Clinical Microbiology and Infectious diseases, Hospital Universitario Gregorio Marañon; 21 Hematology Department, Fundación Jimenez Diaz University Hospital; 22 Hematology, Fundacíon Jimenez Diaz, Madrid, Spain; 23 Infectious diseases, Karolinska University Hospital, Stockolm, Sweden; 24 Hematology Department, Weil Cornell Medicine, New York, United States of America; 25 Hematology Department, CHU de Montpellier, Montpellier, France; 26 Hematology Department, King’s College Hospital, London, United Kingdom; 27 Hematology, University Hospital La Paz, MADRID, Spain; 28 Hematology, ASST-Ospedali Civili, Brescia, Italy; 29 Internal Medicine, University of Cologne, Cologne, Germany; 30 Hematology, University Hospital Infanta Leonor, Madrid, Spain; 31 Hematology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Roma, Italy

Background: Patients with lymphoproliferative diseases (LPD) appear particularly vulnerable to SARS-CoV-2 infection, partly because of the effects of the anti-neoplastic regimens (chemotherapy, signaling pathway inhibitors, and monoclonal antibodies) on the immune system. The real impact of COVID-19 on the life expectancy of patients with different subtypes of lymphoma and targeted treatment is still unknown.

Aims: The aim of this study is to describe and analyse the outcome of COVID-19 patients with underlying LPD treated with targeted drugs such as monoclonal antibodies (obinutuzumab, ofatumumab, brentuximab, nivolumab or pembrolizumab), BTK inhibitors (ibrutinib, acalabrutinib), PI3K inhibitors (idelalisib), BCL2 inhibitors (venetoclax) and IMIDs, (lenalidomide).

Methods: The survey was supported by EPICOVIDEHA registry. Adult patients with baseline CLL or non-Hodgkin Lymphoma (NHL) treated with targeted drugs and laboratory-confirmed COVID-19 diagnosed between January 2020 and January 2022 were selected.

Results: The study included 368 patients (CLL n=205, 55.7%; NHL n=163, 44.3%) treated with targeted drugs (Table 1). Median follow-up was 70.5 days (range 19-159). Most used targeted drugs were ITKs (51.1%), anti-CD20 other than rituximab (16%), BCL2 inhibitors (7.3%) and lenalidomide (7.9%). Of note, only 16.0% of the patients were vaccinated with 2 or more doses of vaccine at the onset of COVID-19.

Pulmonary symptoms were present at diagnosis in 244 patients (66.2%). Severe COVID-19 was observed in 47.8 % patients while 21.7% were admitted to to intensive care unit (ICU), being 55 (26.8%) CLL patients and 25 (15.3%) NHL patients. More comorbidities were reported in patients with severe-critical COVID-19 compared to those with mild- asymptomatic infection (p=0.002). This difference was relevant in patients with chronic heart diseases (p=0.005). Overall, 134 patients (36.4%) died. Primary cause of death was COVID-19 in 92 patients (68.7%), LPD in 14 patients (10.4%), and a combination of both in 28 patients (20.9%).Mortality was 24.2% (89/368) at day 30 and 34.5%(127/368) at day 200. After a Cox multivariable regression age >75 years (p<0.001, HR 1.030), active malignancy (p=0.011, HR 1.574) and admission to ICU (p<0.00, HR 4.624) were observed as risk factors. Survival in patients admitted to ICU was 33.7% (LLC 38.1%, NHL 24%). Mortality rate decreased depending on vaccination status, being 34.2% in not vaccinated patients, 15.9-18% with one or two doses, decreasing to 9.7% in patients with booster dose (p<0.001). There was no difference in OS in NLH vs CLL patients (p=0.344), nor in ITKs vs no ITKs treated patients (p=0.987). Additionally, mortality rate dropped from the first semester 2020 (41.3%) to last semester 2021 (25%).

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Summary/Conclusion: - Our results confirm that patients with B-malignancies treated with targeted drugs have a high risk of severe infection (47.8%) and mortality (36.4%) from COVID-19. - Presence of comorbidities, especially heart disease, is a risk factor for severe COVID-19 infection in our series. - Age >75 years, active malignancy at COVID-19 onset and ICU admission were mortality risk factors. - COVID-19 vaccination was a protective factor for mortality, even in this population with humoral immunity impairment. - The learning curve in the management of the infection throughout the pandemic and the development of COVID-19 treatments showed benefit in this particularly vulnerable population.

S281: PRIOR EXPOSURE TO IMMUNOSUPPRESSIVE AGENTS AND COMORBIDITIES ARE ASSOCIATED WITH WORSE OUTCOMES OF SARS-COV2 INFECTION IN PH-NEG CHRONIC MYELOPROLIFERATIVE NEOPLASMS: RESULTS OF EPICOVIDEHA SURVEY

M. Marchetti 1,* , J. Salmanton-García 2 , S. El-Ashwah 3 , M. V. Sacchi 1 , F. Marchesi 4 , Z. Ráčil 5 , M. Hanakova 6 , G. Zambrotta 7 , L. Verga 7 , F. Passamonti 8 , F. Itri 9 , J. Van Doesum 10 , S. Martìn-Pérez 11 , A. López-García 12 , J. Dávila-Valls 11 , R. Cordoba 13 , G. Abu-Zeinah 14 , G. Dragonetti 15 , C. Cattaneo 16 , V. Bonuomo 17 , L. Prezioso 18 , A. Glenthøj 19 , F. Farina 20 , R. Duarte 21 , O. Blennow 22 , O Cornely 23 , L. Pagano 24

1 Hematology & TMO Unit, Azienda Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Alessandria, Italy; 2 Department I of Internal Medicine, Excellence Center for Medical Mycology (ECMM), University of Cologne and University Hospital, Faculty of Medicine, Cologne, Germany; 3 Oncology Center, Mansoura University, Mansoura, Egypt; 4 Hematology and Stem Cell Transplant Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy; 5 Institute of Hematology and Blood Transfusion, Prague and Brno Hospital; 6 Institute of Hematology and Blood Transfusion, Prague Hospital, Prague, Czechia; 7 Hematology Unit, Azienda Ospedaliera San Gerardo; University Bicocca-Milan, Monza; 8 Division of Hematology, Department of Medicine and Surgery, University of Insubria, Varese; 9 Hematology Unit, Hospital San Luigi Gonzaga, Orbassano, Italy; 10 University Medical Center Groningen, University of Groningen, Groningen, Netherlands; 11 Hospital Nuestra Señora de Sonsoles, Ávila; 12 Health Research Institute IIS-FJD; 13 Fundacion Jimenez Diaz University Hospital, Madrid, Spain; 14 Division of Hematology and Oncology, Weill Cornell Medicine, New York, United States of America; 15 Hematology Unit, Fondazione Policlinico Universitario Agostino Gemelli - IRCCS, Rome; 16 Hematology Unit, ASST-Spedali Civili, Brescia; 17 Department of Medicine, Section of Hematology, Azienda Ospedaliera Universitaria Integrata, Verona; 18 Hematology and Bone Marrow Unit, University Hospital, Parma, Italy; 19 Department of Hematology, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark; 20 Hematology & TMO unit, IRCCS Ospedale San Raffaele, Milan, Italy; 21 Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain; 22 Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden; 23 Dpt I of Internal Medicine, ECMM, CECAD, Clinical Trials Centre Cologne (ZKS Köln), Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, German Centre for Infection Research (DZIF), Cologne, Germany; 24 Hematology Unit, Fondazione Policlinico Universitario Agostino Gemelli - IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy

Background: Philadelphia-negative chronic myeloproliferative neoplasms (MPN) typically incur high rates of thrombosis and infections and cytoreductive drugs may modulate such risks.

Aims: The present analysis aims at assessing the severity and outcomes of MPN facing coronavirus disease 2019 (COVID-19). Hence, we aimed to assess the impact of immunosuppressive agents and comorbidity burden in COVID-19 outcome.

Methods: The EPICOVIDEHA registry is an online survey ( www.clinicalsurveys.net ) that has collected since April 2020 until January 2022 5,445 cases of COVID-19 in individuals with baseline haematological malignancies (Salmanton-García et al, 2021 Hemasphere) The survey is promoted by the European Hematology Association - Infectious Diseases Working Party (EHA-IDWP) and has been approved centrally by the Institutional Review Board and Ethics Committee of Fondazione Policlinico Universitario A. Gemelli – IRCCS – Università Cattolica del Sacro Cuore, Rome, Italy (Study ID: 3226).

Results: Overall, 308 patients (5.6%) with MPN were observed for a median of 102 days (IQR: 21-223, range 22-97) after COVID-19 diagnosis. Median age at infection was 69 years (IQR: 58-77, range 22-97) and at least one comorbidity was reported from most of the individuals (62.6%, n = 193). A large portion of patients had a history of cardiopathy (n=109, 35.4%), diabetes (n=40, 15.9%), or chronic pulmonary disease (n=44, 14.3%). Myelofibrosis (MF) (n=140, 45.4%) was the most prevalent baseline malignancy, with 18 MF patients (12.9%) reporting 3 or more comorbidities. Out of the whole cohort, 72 patients (42.8% of MF) received immunosuppressige therapies including steroids, immunomodulatory drugs (IMiDs) or JAK-inhibitors.

Hospitalization and consecutive admission to intensive care unit was required for 187 (60.7%) and 45 (24%) patients, respectively. At multivariate logistic regression, hospital admission was predicted by age ≥70 years (OR 2.809; 95% CI 1.651-4.779), exposure to immunosuppressive therapies (OR 2.802; 95% CI 1.5380-5.103) and comorbidity burden.

During the study follow-up (median 101 days; range 21-222) 84 patients deceased after a median time of 14 days (IQR: 8-49, range 0-457) since COVID-19 diagnosis. The fatality rate (FR) decreased from 40.3% (50 out of 124) in the first two quarters of year 2020 to 15.8% (3 out of 19) in the first two quarters of year 2021 (p<0.05). Death was principally attributable to COVID-19 in 58 patients (69.0%) and contributable by COVID-19 in 15 (17.9%). FR was particularly high (54 out of 140, 38.6%) in MF patients and in patients receiving immunosuppressive agents (32 out of 86, 37%). Moreover, FR increased from 13.0% in individuals with no comorbidity to 36.0% and 62.1% in those with ≥2 or ≥3 comorbidities, respectively. More specifically, three comorbidities independently increased the FR: chronic cardiopathy (HR 1.653; 95%CI 1.017-2.687), chronic pulmonary disease (HR 1.847; 95% CI 1.097-3.109), and diabetes mellitus (HR 1.712; 95% CI 1.006-2.914). A heavy comorbidity burden, namely 3 or more comorbidities (HR 2.956; 95% CI 1.403-6.227), advanced age, namely ≥70 years (HR .809; 95% CI 1.651-4.779), myelofibrosis (HR 2.501; 95% CI 1.384-4.519), and ICU admission (HR 2.669; 95% CI 1.641-4.342) independently predicted FR.

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Summary/Conclusion: COVID-19 infection led to a particularly dismal outcome in patients exposed to immunosuppressive agents and in those with chronic heart or pulmonary diseases, or diabetes. These data allow to tailor future strategies for preventing severe COVID-19 in MPN patients.

S282: A RANDOMIZED CONTROLLED CLINICAL TRIAL DEMONSTRATES THAT PLASMA FROM CONVALESCENT AND VACCINATED DONORS IMPROVES OUTCOME OF COVID-19 IN PATIENTS WITH HEMATOLOGICAL DISEASE, CANCER OR IMMUNOSUPPRESSION

C. Müller-Tidow 1,* , M. Janssen 1 , U. Schäkel 1 , J. Gall 2 3 , A. Leo 4 , P. Stelmach 1 , J. Krisam 5 , L. Baumann 5 , J. Stermann 5 , U. Merle 6 , M. Zeier 7 , M. A. Weigand 8 , L. Bullinger 9 , J.-F. Schrezenmeier 9 , M. Bornhäuser 10 , N. Alakel 10 , O. Witzke 11 , T. Wolf 12 , M. J. Vehreschild 12 , S. Schmiedel 13 , M. M. Addo 13 , F. Herth 14 , M Kreutner 15 , P.-R. Tepasse 16 , B. Hertenstein 17 , M. Hänel 18 , A. Morgner 18 , M. Kiehl 19 , O. Hopfer 19 , M.-A. Wattad 20 , C. C. Schimanski 21 , C Celik 21 , T. Pohle 22 , M. Ruhe 22 , W. V. Kern 23 , A Schmitt 1 , M. Schmitt 1 , P. Dreger 1 , H.-M. Lorenz 1 , M. Souto-Carneiro 1 , N. Halama 24 , S. Meuer 4 , H.-G. Kräusslich 25 , B. Müller 25 , R. Bartenschlager 26 , J. Klemmer 1 , K. Kriegsmann 1 , R. F. Schlenk 1 2 3 , C. M. Denkinger 27

1 Department of Internal Medicine V, Heidelberg University Hospital; 2 NCT-Trial Center, National Center of Tumor Diseases, Heidelberg University Hospital; 3 German Cancer Research Center; 4 Institute for Clinical Transfusion Medicine and Cell Therapy Heidelberg; 5 Institute for Medical Biometry and Informatics, Ruprecht-Karls University Heidelberg; 6 Department of Internal Medicine IV, Heidelberg University Hospital,; 7 Department of Nephrology, University of Heidelberg, Heidelberg; 8 Department of Anaesthesiology, Heidelberg University Hospital, Heidelberg, Heidelberg; 9 Department of Hematology, Oncology and Tumor Immunology, Charité University Medicine, Campus Virchow Clinic, Berlin; 10 Department of Internal Medicine I, University Hospital and Faculty of Medicine Carl Gustav Carus of TU Dresden, Dresden; 11 Department of Infectious Diseases, West German Centre of Infectious Diseases, University Hospital Essen, University Duisburg-Essen, Essen; 12 Department of Internal Medicine, Infectious Diseases, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt am Main; 13 I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg; 14 Pneumology and Critical Care Medicine, Thoraxklinik, University of Heidelberg; 15 Center for Interstitial and Rare Lung Diseases, Thoraxklinik, University of Heidelberg, German Center for Lung Research (DZL), Heidelberg; 16 Department of Medicine B, Gastroenterology and Hepatology, University Hospital Münster, Münster; 17 Medical Department I, Klinikum Bremen-Mitte, Bremen; 18 Department of Internal Medicine III, Klinikum Chemnitz gGmbH, Chemnitz; 19 Department I of Internal Medicine, Frankfurt (Oder) General Hospital, Frankfurt (Oder); 20 Department of Hematology, Oncology, Palliative Care and Stem Cell Transplantation, Klinikum Hochsauerland GmbH, Meschede; 21 Department of Internal Medicine II, Klinikum Darmstadt GmbH, Darmstadt; 22 Department of Internal Medicine I, Klinikum Herford, Herford; 23 Department of Medicine II, Division of Infectious Diseases and Travel Medicine, University Medical Centre Freiburg, Freiburg; 24 Department of Medical Oncology, National Center for Tumor Diseases, Heidelberg University Hospital; 25 Department of Infectious Diseases, Virology, Heidelberg University Hospital; 26 Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital; 27 Division of Tropical Medicine, Department of Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany

Background: Therapy options are limited for COVID-19 patients with hematological disease, cancer, immunosuppression or advanced age. Even though no benefit was observed for convalescent plasma in unselected patients with COVID-19, retrospective data suggest that it could be effective in patients unable to mount a sufficient immune response upon SARS-CoV-2 infection. Plasma from vaccinated donors has not been systematically assessed for COVID-19 treatment.

Aims: We conducted a randomized clinical trial to address plasma efficacy in patients at high risk for an adverse outcome.

Methods: COVID-19 patients with confirmed SARS-CoV-2 infections and oxygen saturation <=94% were randomized ( NCT05200754 ). Patients received convalescent or vaccinated SARS-CoV-2 plasma in two bags (238 - 337 ml plasma each) from different donors on day 1 and 2 (PLASMA) or standard of care (CONTROL). Randomization was stratified according to four clinical patient groups, hematological/solid cancer (group-1), treatment or disease associated immunosuppression (group 2), high risk disease by standard parameters (group-3) or age >=75 years (group-4). Mechanically ventilated patients were not eligible. Plasma was obtained from donors with high level neutralizing activity (titer >=1:80) either after SARS-CoV-2 infection (convalescent) or after vaccination with at least two doses of mRNA vaccines (vaccinated). Crossover for the control group was allowed at day 10. The primary endpoint was time to improvement as two points on a seven-point ordinal scale or live discharge from the hospital (IMPROVEMENT) with prespecified analyses of subgroups (Janssen M, et al. Trials 2020 Oct 6;21(1):828).

Results: A total of 133 patients were randomized with 68 receiving PLASMA with a median age of 68 years (range 36-95) or CONTROL (n=65, of which n=10 (15.4%) crossed over at day 10) with a median age of 70 years (range 38-90). The distribution of the four predefined groups was group-1, n=53; group-2, n=18; group-3, n=35; and group-4, n=27. The intention to treat analysis revealed a non-significant shorter time to IMPROVEMENT for patients in PLASMA (median 12.5 days, 95%-CI [10; 16]) compared to patients in CONTROL (median 18 days, 95%-CI [11; 28]), hazard ratio 1.24, 95% confidence interval [0.83; 1.85], p=0.29). Overall, 27 patients died (PLASMA, n=12; CONTROL, n=15; p=0.80). Predefined subgroup analysis revealed a clinically significant benefit in patients with hematological malignancies, other cancers or immunosuppression (group-1, group-2, n=71). With a median time to improvement of 13 days (95%-CI [9; 19]) for PLASMA and 32 days (95%-CI [17; 57]) for CONTROL(HR 2.03, 95%-CI [1.17; 3.6], p=0.01). A sensitivity analysis revealed that IMPROVEMENT appeared to be seen even earlier with vaccinated (median 10 days, 95%-CI [8; 14]) compared to convalescent SARS-CoV-2 plasma (median 13 days, 95%-CI [6; 38]) and CONTROL. Within group-1 and group-2, six patients in PLASMA (18.2%) and 10 in CONTROL (28.6%) died. No significant differences in improvement were observed in group-3 and group-4 with a HR of 0.72 (95%-CI [0.41; 1.28], p=0.26). Within group-3 and group-4, six patients in PLASMA (18.8%) and five in CONTROL (16.7%) died. No previously unknown side effects of plasma therapy emerged within the trial.

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Summary/Conclusion: Plasma from convalescent and particularly vaccinated donors improved outcome of COVID-19 patients with an underlying hematological disease /cancer or other reasons of impaired immune response. Plasma did not improve outcome in immune-competent patients with other risk factors and/or older age.

S283: ERN-EUROBLOODNET EUROPEAN REGISTRY OF PATIENTS AFFECTED BY RED BLOOD CELL DISORDERS AND COVID-19

P. Velasco 1,* , F. Longo 2 , A. Piolatto 2 , E. J. Bardón-Cancho 3 4 5 , B. Ponce-Salas 6 , P. Flevari 7 , E. Voskaridou 8 , B. J. Biemond 9 , E Nur 9 10 , P. Delaporta 11 , T. Besse-Hammer 12 , A. Ruiz-Llobet 13 , S. Raso 14 , A. Spasiano 15 , M. E. Guerzoni 16 , D. Beneitez-Pastor 17 18 19 20 , L. Dedeken 21 , A. Pepe 22 , R. Rosso 23 , J. B. Kunz 24 , M. de Montalembert 25 , S. Campisi 26 , A. Glenthøj 27 , P. Gonzalez Urdiales 28 29 , F. S. Benghiat 30 , M.-A. Azerad 31 , C. J. Saunders 32 , T. Ferreira Faria 33 , T. Casini 34 , S. Bagnato 35 , A. Van de Velde 36 , V. Labarque 37 , E. Bertoni 38 , A. Van Damme 39 , M. D. Diamantidis 40 , R. Russo 41 42 , E. Stiakaki 43 , A Quota 44 , S. Christou 45 , M. J. Teles 46 47 48 49 50 , I. Lafiatis 51 , J.-L. Kerkhoffs 52 , M. Argüello Marina 53 , M. Lorite 54 , A. Rodriguez 55 56 , A. Iolascon 41 42 , A. T. Taher 57 , R. Colombatti 58 , N. Roy 59 , M. D. M. Mañú Pereira 60

1 pediatric oncology and hematology, Hospital Vall d’Hebron, Barcelona, Spain; 2 Department of Clinical and Biological Sciences, University of Torino, Orbassano (To), Italy; 3 Pediatric Hematology Unit, Hospital General Universitario Gregorio Marañón; 4 Facultad de Medicina, Universidad Complutense de Madrid; 5 Instituto Investigación Sanitaria Gregorio Marañón; 6 Onco-Hematología Infantil, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 7 Center of Excellence in Rare Haematological Diseases-Haemoglobinopathies, Laiko General Hospital; 8 Center of Excellence in Rare Haematological Diseases-Haemoglobinopathies, Laiko General Hospital, Athens, Greece; 9 Department of Hematology, Amsterdam University Medical Centers; 10 Department or Blood Cell Research, Sanquin Research, Amsterdam, Netherlands; 11 First Department of Pediatrics, National and Kapodistrian University of Athens, ‘AghiaSophia’ Children’s Hospital, Athens, Greece; 12 URC, CHU Brugmann, Brussels, Belgium; 13 Paediatric Haematology Department, Hospital Sant Joan de Déu, Universitat de Barcelona, Barcelona, Spain; 14 Department of Hematology and Rare Diseases, V Cervello, Azienda Ospedaliera Ospedali Riuniti Villa Sofia-Cervello, Palermo; 15 Malalttie Rare del Globulo Rosso, AORN A. Cardarelli, Naples; 16 Pediatric Unit,Department of Medical and Surgical Sciences of Mothers, Pediatric Unit,Department of Medical and Surgical Sciences of Mothers, Modena, Italy; 17 Translational ResearchGroup in Rare Anemia Disorders, Vall d’Hebron Institut de Recerca; 18 Hematology Department, Vall d’Hebron Hospital Universitari; 19 Experimental Hematology Group, Vall d’Hebron Institute of Oncology (VHIO); 20 Vall d’Hebron Hospital Universitari, ERN-EuroBloodNet, Barcelona, Spain; 21 Hôpital Universitaire des Enfants Reine Fabiola (ULB), Brussels, Belgium; 22 Fondazione Toscana G. Monasterio per la ricerca medica e di sanità pubblica, Pisa; 23 Thalassemia and Heamoglobinophaties, Policlinico Universitario G. Rodolico, Catania, Italy; 24 University Hospital, Heidelberg, Germany; 25 Hôpital Necker, Paris, France; 26 Ospedale Umberto I, Siracusa, Italy; 27 Department of Hematology, Rigshospitalet, Copenhagen, Denmark; 28 Pediatric oncology and hematology department, Hospital Universitario Cruces; 29 Instituto de investigaciónBiocruces Bizkaia, Barakaldo, Spain; 30 CUB Erasmus Hospital, Brussels; 31 Hematology, CHU Liège Citadelle site, Liège, Belgium; 32 Hospital dos Capuchos, Centro Hospitalar e Universitário Lisboa Central, Lisbon; 33 Pediatric Department, Hospital Fernando Fonseca, Amadora, Portugal; 34 Pediatric OncoHematology, Meyer Children Hospital, Florence; 35 Thalassemia, Ospedale Civile, Lentini, Italy; 36 Hematology, University Hospital Antwerp, Edegem; 37 Pediatric Hemato-Oncology, University Hospitals Leuven, Leuven, Belgium; 38 Oncohaematology and BMT Unit, ASST Spedali Civili di Brescia, Brescia, Italy; 39 Paediatric Hémato-oncology, Cliniques Universitaires Saint-Luc, Brussels, Belgium; 40 Thalassemia and Sickle Cell Disease Unit, Department of Hematology, General Hospital of Larissa, Larissa, Greece; 41 Department of Molecular Medicine and Medical Biotechnology, University Federico II; 42 CEINGE - Advanced Biotechnologies, Naples, Italy; 43 Pediatric Hematology-Oncology Department, Universityof Crete, University Hospital of Heraklion, Heraklion, Greece; 44 UOSD Thalassemia, V.Emanuele Hospital, Gela, Italy; 45 Thalassemia Clinic, Archbishop Makarios III Hospital, Nicosia, Cyprus; 46 Clinical Pathology, Centro Hospitalar e Universitário de São João; 47 Clinical Hematology, Centro Hospitalar e Universitário do Porto; 48 Anemia Working Group Portugal; 49 Institute for the HealthResearch and Innovation (i 3 S); 50 Hematology Working Group of the Portuguese Society of Clinical Pathology, Porto, Portugal; 51 General Hospital of Mytilini, Mytilini, Greece; 52 Hematology, HagaZiekenhuis, The Hague, Netherlands; 53 Hematology, Hospital Príncipe de Asturias, Madrid; 54 Department of pediatric Hematology, Hospital Son Espases, Palma de Mallorca; 55 Clinical Pharmacology Service, Hospital Universitari Vall d’Hebron; 56 Vall d’Hebron Institut de Recerca, Barcelona, Spain; 57 Division of Hematology and Oncology, Department of Internal Medicine, American University of Beirut Medical Center, Beirut, Lebanon; 58 UOC Pediatric Hematology Oncology, University of Padova, Padova, Italy; 59 Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 60 Translational Research in Child and Adolescent Cancer, Vall d’Hebron Institut de Recerca, Barcelona, Spain

Background: Patients with red blood cell disorders (RBCD), are likely to be at increased risk of complications from SARS-Cov-2 (Covid-19), but evidence in this population is scarce due to its low frequency and heterogeneous distribution.

Aims: ERN-EuroBloodNet, the European Reference Network in rare hematological disorders, established a European registry to determine the impact of COVID-19 on RBCD patients and identify risk factors predicting severe outcomes.

Methods: The ERN-EuroBloodNet registry was established in March 2020 by VHIR based on Redcap software in accordance with the Regulation (EU) 2016/679 on personal data. The local Research Ethics Committee confirmed that the exceptional case of the pandemic justifies the waiver of informed consent.

Eligible patients had confirmed RBCD and COVID-19. Data collected included demographics, diagnosis, comorbidities, treatments, and COVID-19 symptoms and management.

For analysis of COVID-19 severity, two groups were established 1) Mild: asymptomatic or mild symptoms without clinical pneumonia and 2) Severe: pneumonia requiring oxygen/respiratory support and/or admission to intensive care unit. Continuous variables were compared using the Wilcoxon rank-sum test or Kruskall Wallis test, while categorical variables were analyzed using the Chi-square test or Fisher’s Exact test. Relevant factors influencing disease or severity were examined by the logistic regression adjusted for age.

Results: As of February 25, 2022, 42 medical centers from 10 EU countries had registered 428 patients: 212 Sickle cell disease (SCD), 186 Thalassemia major and intermedia (THAL).

The mean age of SCD was lower (22y) than of THAL (39.4y). Splenectomy and comorbidities were higher in THAL (51.4% and 61,3%) than in SCD (16,3% and 46,8%) (p<0.001, p=0.004).

Age and BMI correlated with COVID-19 severity, as described in the general population (p=0.003, p<0.001). Fig 1 shows age distribution and COVID-19 severity by disease severity groups. The mean age for severe COVID-19 was lower in patients with severe SCD (SS/SB0 vs SC/SB+: 23y vs 67.5y) and THAL (major vs intermedia: 43.5 vs 51.3y) (p<0.001).

Potential risk factors such as elevated ferritin, current chelation or history of splenectomy did not confer additional risk for developing severe COVID-19 in any patient group. Only diabetes as a comorbidity correlated with severity grade in SCD (p=0.01) and hypertension in THAL (p=0.009). While severe COVID-19 infection in SCD was associated with both ACS (p<0.001) and kidney failure requiring treatment (p<0.001), this was not predicted by a history of previous ACS or kidney disease in steady state.

Overall, 14,6% RBC patients needed oxygen/respiratory support, 4% were admitted to ICU with an overall mortality rate of 1%, much lower than reported in other similar cohorts.

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Summary/Conclusion: Results obtained so far show that severe COVID-19 occurs at younger ages in more aggressive forms of SCD and THAL. Current preventive approaches focus on age over disease severity. Our data highlights the risk of severe COVID-19 infection in some young patients, particularly those with SS/SB0 SCD, suggesting that immunization should be considered in this pediatric group as well.

Results between similar sized cohorts of RBCD patients vary between each other and those presented here, highlighting the importance of collecting all of these small cohorts together to ensure adequate statistical power so that definitive risk factors can be reliably identified and used to guide management of patients with these rare disorders in the light of the ongoing pandemic.

S284: RISK FACTORS AND OUTCOMES OF PATIENTS WITH HAEMATOLOGICAL MALIGNANCY AND COVID-19: ONGOING REDUCTION IN MORTALITY DURING THE 3RD WAVE OF THE PANDEMIC

J. Maddox 1,*

1 Haematology, South Tees NHS Trust, Middlesborough, United Kingdom

Background: It was established early in the COVID-19 pandemic that patients with cancer, in particular haematological malignancy, had worse outcomes than non-cancer patients. There is a lack of recent data to describe how mortality in patients with haematology malignancy has changed with widespread vaccination and several effective treatments now being available.

Aims: Based in a large NHS Trust in the North-East of England, our aim was to identify all local patients with haematological malignancy who had contracted COVID-19 since the start of the pandemic. We then examined these patients in more detail to ascertain risk factors for mortality, and how this has changed over the course of the pandemic.

Methods: We included patients with an active diagnosis of haematological malignancy, or those who had received potentially curative treatment within the past 3 years. We excluded patients with pre-malignant conditions. Data up to the end of November 2021 found 213 eligible patients. Nearly all patients identified in the 1 st wave of the pandemic were identified from hospital testing, reflecting the lack of availability of widespread testing in the community. More recently, patients identified in the community have predominated.

Results: Overall mortality following COVID-19 infection was 21.6% at 4 weeks and 27.7% at 8 weeks after COVID-19 diagnosis. Mortality was highest in wave 1 (March – June 2020), decreasing in wave 2 (Sept 20 – March 2021) and again in wave 3 (May 2021 – current), with 4-week mortality figures of 44%, 26% and 8% respectively. It should be noted that widespread community testing was not available early in the pandemic so the recorded cases in wave 1 were sicker patients needing hospital care. Removing pillar 2 data still shows a reduction in 4-week mortality over the course of the pandemic, from 46% (wave 1) to 39% (wave 2) and 8% (wave 3).

Although official COVID-19 mortality figures only include deaths within 4 weeks of a confirmed infection, we note that the Kaplan-Myer mortality curve did not level out until 6-8 weeks after initial infection (figure 1). Overall mortality at 8 weeks was therefore higher at 61% (wave 1), 31% (wave 2) and 11% (wave 3).

The main risk factor for mortality was patient age, with 8-week mortality in age groups <60yr, 60-69yr, 70-79yr, 80-89yr and 90+yr being 2%, 26%, 30%, 51% and 86% respectively. Although men comprised the majority of detected cases (62%), mortality at four and eight weeks was near identical between sexes.

Although disease subgroups were relatively small, we found the highest COVID-related mortality in patients with CLL (43%) and MDS (42%). It is perhaps not surprising as these two disease groups also had the highest mean patient ages.

Patients receiving chemotherapy had no significant increase in 8-week mortality compared to those not receiving chemotherapy, 30% vs. 27% (p=ns). The presence of neutropenia was however a risk factor for mortality. Patients who were neutropenic at the time of infection had an 8-week mortality of 43%, compared to 25% in those who were not neutropenic (P=0.04).

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Summary/Conclusion: Overall, COVID-19 related mortality in patients with haematological malignancy has significantly declined over the course of the pandemic. The main risk factor for death is increased patient age, with neutropenia also being a risk factor.

S285: INHIBITION OF COMPLEMENT C1S WITH SUTIMLIMAB IN PATIENTS WITH COLD AGGLUTININ DISEASE (CAD): 2-YEAR FOLLOW-UP FROM THE CARDINAL STUDY

A. Röth 1,* , W. Barcellini 2 , S. D’Sa 3 , Y. Miyakawa 4 , C. M. Broome 5 , M. Michel 6 , D. J. Kuter 7 , B. Jilma 8 , T. H. A. Tvedt 9 , I. C. Weitz 10 , T. Sourdille 11 , J. Wang 11 , D. S. Vagge 12 , K. Kralova 13 , F. Shafer 14 , M. Wardecki 15 , M. Lee 14 , S. Berentsen 16

1 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 3 UCLH Centre for Waldenström’s Macroglobulinemia and Related Conditions, University College London Hospitals NHS Foundation Trust, London, United Kingdom; 4 Thrombosis and Hemostasis Center, Saitama Medical University Hospital, Saitama, Japan; 5 Division of Hematology, MedStar Georgetown University Hospital, Washington DC, United States of America; 6 Henri-Mondor University Hospital, Assistance Publique-Hôpitaux de Paris, UPEC, Créteil, France; 7 Division of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States of America; 8 Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria; 9 Section for Hematology, Department of Medicine, Haukeland University Hospital, Bergen, Norway; 10 Keck School of Medicine of USC, Los Angeles, CA; 11 Sanofi, Cambridge, MA, United States of America; 12 IQVIA, Bangalore, India; 13 Sanofi, Paris, France; 14 Sanofi, Bridgewater, NJ, United States of America; 15 Sanofi, Warsaw, Poland; 16 Department of Research and Innovation, Haugesund Hospital, Haugesund, Norway

Background: CAD is a rare chronic autoimmune hemolytic anemia characterized by classical complement pathway (CP)-mediated hemolysis. Sutimlimab is a first-in-class humanized monoclonal antibody that selectively inhibits C1s of the C1 complex, preventing CP activation, while leaving the alternative and lectin pathways intact. One-year interim follow-up from the CARDINAL study ( NCT03347396 ) have previously demonstrated that sutimlimab resulted in sustained improvements in hemolytic markers and quality of life.

Aims: To report 2-year sutimlimab efficacy and safety from the CARDINAL Part B extension.

Methods: CARDINAL was a Phase 3, open-label, single-arm study with a 26-week treatment period (Part A) and a 2-year extension (Part B) after the last patient (pt) finishes Part A. Sutimlimab was administered through intravenous infusions on Days 0 and 7, followed by biweekly dosing. Efficacy data through Week 131, the last data recording within the 2-year Part B period, are reported here. Efficacy endpoints included change from baseline in hemolytic markers, pharmacodynamic (PD) markers and blood transfusions. Quality of life (QOL) was assessed using the Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue Scale. Safety was recorded until end-of-study visit 9 weeks after their last dose; endpoints included incidence of treatment-emergent adverse event (TEAE) and serious TEAE (TESAE). Descriptive statistics, frequency, and percentage were used to analyze outcomes.

Results: Of the 24 pts enrolled in Part A, 22 completed Part A and entered Part B, with 19 (86.4%) pts completing Part B. Sutimlimab treatment improved mean (SD) hemoglobin (Hb) levels within one week; mean Hb remained >11 g/dL from Week 5–131 (baseline: 8.64 (1.67) ( Figure ). Mean total bilirubin was normalized from Week 3–131 ( Figure ). Mean FACIT-Fatigue scores improved within 1 week and remained ≥5 from Week 1–123 ( Figure ), consistent with a clinically meaningful change. Improvements in Hb, bilirubin, and FACIT-Fatigue correlated with normalization of C4 and near-complete inhibition of CP activity. Normalization of mean absolute reticulocyte count was observed alongside normalized haptoglobin levels and reductions in LDH. From Week 26–131, 15 (68.2%) pts remained transfusion-independent. All 22 pts experienced ≥1 TEAE; 12 (54.5%) pts experienced ≥1 TESAE. Serious infections were reported in 7 (31.8%) pts, including one pt with sepsis due to streptococcus pneumoniae. No meningococcal infections were reported. Three pts discontinued the study due to AEs (cyanosis and klebsiella pneumoniae (n=1); vitreous hemorrhage (n=1); cyanosis and gastrointestinal symptoms including erosive gastritis (n=1)). No pts developed systemic lupus erythematosus, serious hypersensitivity or anaphylaxis. Two pts died during the study (klebsiella pneumoniae (n=1); exacerbation of CAD (n=1) in a patient with a femoral neck fracture and complex medical history including myelodysplastic syndrome, approximately 1.5 months after receiving the last dose of sutimlimab).

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Summary/Conclusion: Sutimlimab, a first-in-class selective anti-C1s classical complement pathway inhibitor, maintained mean Hb levels >11g/dL, achieved sustained normalization of mean bilirubin, haptoglobin and reticulocyte count. Sutimlimab continued to improve FACIT-Fatigue scores, with no newly identified safety concerns at 2 years of treatment. This study has demonstrated that sutimlimab is an effective and well-tolerated long-term therapy for the management of chronic CAD through continued upstream inhibition of the classical CP.

S286: LONG-TERM EFFICACY AND SAFETY RESULTS FROM AN ONGOING OPEN-LABEL PHASE 2 STUDY OF PARSACLISIB FOR THE TREATMENT OF AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA)

W. Barcellini 1,* , I. Murakhovskaya 2 , L. Terriou 3 , F. Pane 4 , A. Patriarca 5 , K. Butler 6 , S. Moran 6 , S. Wei 6 , U. Jäger 7

1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2 Albert Einstein College of Medicine/Montefiore Medical Center Bronx, New York, United States of America; 3 Univ. Lille, Inserm, CHU Lille, Centre de Référence des Maladies Autoimmunes Systémiques Rares du Nord et Nord-Ouest de France (CeRAINO), INFINITE – Institute for Translational Research in Inflammation, Lille, France; 4 University of Naples “Federico II”, Naples; 5 University of Eastern Piedmont and AOU “Maggiore della Carità”, Novara, Italy; 6 Incyte Corporation, Wilmington, United States of America; 7 Medical University of Vienna, Vienna, Austria

Background: AIHA is a rare condition caused by autoantibody-mediated hemolysis of red blood cells. Few therapies beyond steroids and rituximab are available.

Aims: To report updated results from an ongoing multicenter, phase 2, open-label study of the phosphoinositide 3-kinase-δ inhibitor parsaclisib in patients (pts) with AIHA ( NCT03538041 ).

Methods: Pts ≥18 years old with warm AIHA (wAIHA), cold agglutinin disease (CAD), or mixed-type AIHA; hemoglobin (Hgb) 7–10 g/dL; and failure of ≥1 standard therapy were eligible. After informed consent, pts were treated with oral parsaclisib for 12 wk at a starting dose of 1.0 mg once daily (QD; cohort 1) or 2.5 mg QD (cohort 2). Increases to 2.5 mg QD were allowed in cohort 1 after 6 wk if no clinical response was achieved and/or transfusion was required; reductions to 1.0 mg QD were permitted in cohort 2 for tolerability issues. Concomitant corticosteroids (≤20 mg/d prednisone) were allowed. After 12 wk of treatment, pts responding on parsaclisib could continue into an extension period. Primary endpoints were efficacy (proportion of pts with complete response [CR; Hgb ≥12 g/dL] or partial response [PR; Hgb 10–12 g/dL or ≥2 g/dL increase from baseline, inclusive of CRs] at any visit from Wk 6–12) and safety (treatment-emergent adverse events [TEAEs]).

Results: As of Aug 5, 2021, 25 pts enrolled and received parsaclisib (cohort 1, n=10 [8 with dose increase]; cohort 2, n=15); 20 pts (80%) completed 12 wk of treatment. Sixteen (64%), 6 (24%), and 3 (12%) pts had wAIHA, CAD, and mixed AIHA, respectively. Mean (SD) age was 61.6 (17.0) years; 14 pts (56%) were female and 23 (92%) were White. Mean (SD) Hgb at baseline was 8.9 (0.8) g/dL, and 9 pts (36%) had transfusions in the past year. Mean parsaclisib exposure was 334 (range, 7–819) days. Overall, 8 pts (32%) achieved CR and 16 (64%) had PR at any visit from Wk 6–12. Among pts with wAIHA (n=16), 14 pts (88%) completed 12 wk of treatment; 8 (50%) and 12 (75%) achieved CR and PR, respectively, at any visit from Wk 6–12. Seventeen pts entered the extension period (12 with wAIHA). Increase in Hgb was sustained in the total cohort and among pts with wAIHA during the initial 12-wk treatment and extension periods ( Figure ).

During the 12-wk treatment period, TEAEs occurred in 21 pts (84%) overall. Six pts (24%) had grade (Gr) ≥3 TEAEs, with only neutropenia occurring in >1 pt (n=2 [8%]); 2 pts (8%) had serious AEs (SAEs). Seven pts (28%) had treatment-related AEs, with only pruritic rash reported in >1 pt (n=2 [8%]). Three pts discontinued during the 12-wk treatment period (AE [n=1; thrombocytopenia], lack of efficacy [n=1], withdrawal by subject [n=1]), and 2 pts were of unknown status.

During the extension period, 15/17 pts (88%) experienced ≥1 TEAE. Gr ≥3 AEs and SAEs were each reported in 9 pts (53%). Six pts (35%) had treatment-related AEs, with diarrhea and rash reported in >1 pt (n=2 [12%] each). One Gr 3 TEAE (psoriasis) and 3 SAEs (diarrhea [Gr 2], cytomegalovirus reactivation [Gr 2], psoriasis [Gr 3]) were deemed treatment related. Two (12%) pts had TEAEs leading to parsaclisib discontinuation. One fatal TEAE (acute respiratory failure in the extension period) was deemed unrelated to parsaclisib.

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Summary/Conclusion: Parsaclisib was generally well tolerated and resulted in Hgb improvements as early as Wk 2 that increased over 12 wk of treatment and were sustained through the extension period. Parsaclisib may be an effective oral treatment for AIHA, and a randomized, controlled phase 3 trial in wAIHA is now recruiting ( NCT05073458 ).

S287: FACTORS INFLUENCING AUTOLOGOUS LYMPHOCYTE COLLECTIONS FOR CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELLS – THE ROLE OF T-CELL SENESCENCE

V. Vucinic 1,* , T. Tumewu 1 , M. Brückner 1 , M. Jentzsch 1 , F. Ramdohr 1 , R. Buhmann 2 , Y. Remane 3 , S. Hoffmann 1 , M. Janz 4 , O. Penack 5 , G. Vogtmann 1 , E. Ruschpler 1 , L. Bullinger 5 , U. Keller 4 , M. Cross 1 , S. Schwind 1 , M. Herling 1 , G.-N. Franke 1 , E. Bach 1 , H. Reinhard 2 , U. Platzbecker 1

1 Medical Clinic and Policlinic for Hematology and Celltherapy; 2 Institute for Transfusion Medicine; 3 Pharmacy, University Leipzig, Leipzig Medical Center, Leipzig; 4 University of Berlin, Campus Benjamin-Franklin; 5 University of Berlin, Campus Virchow-Klinikum, Berlin, Germany

Background: The apheresis of autologous CD3+ lymphocytes is the first pivotal step in the production process of chimeric antigen receptor T-cells. A range of factors like previous cytotoxic therapies or disease status can influence the quality of collection but may also impact the fitness of T-lymphocytes. Repeated T-cell activation leads to progressive loss of expression of CD27 and CD28, receptors shown to be uniformly present on naïve CD4+ cells (van Leewen et al, J. Immunol, 2004).

Aims: To evaluate factors influencing both collection of autologous lymphocytes and the subsequent manufacturing of tisagenlecleucel with a special focus on T-cell senescence, defined as loss of CD27 and CD28.

Methods: Between February 2019 and October 2021, 59 collections were performed for subsequent CAR-T cell therapy with tisagenlecleucel in 51 patients with relapse/refractory (r/r) diffuse large B-cell lymphoma and one patient with refractory acute lymphoblastic leukemia.

The median age was 60.5 (range 17-80) years and 40 (77%) patients were male. The collections were performed on a Spectra Optia cell-separator by processing a median 3x total blood volume.

Results: The target numbers of CD3+ cells (>0.55 x10 9 ) were successfully collected within a single collection day in all patients but one. The median yield of CD3+ cells was 5.0 (0.4-31.9 x10 9 , with yields for CD3+CD4+ and CD3+C8 + 20.1 (range 2.4-102.8) x10 8 and 27.4 (range 2.41-266.8) x10 8 , respectively. The median CD4:CD8 ratio was 0.7 (0.08-5.3).

The yields for CD3+CD27+CD28+ and CD3+CD27-CD28- cells were 25.9 (range 1.1-93.0) x10 8 and 10.9 (range 0.2-183.8) x10 8 , respectively. We observed no difference in median collected CD3+ cell yields in patients with > vs ≤ 3 prior therapy lines (p=0.29), with or without prior treatment with bendamustine (p=0.42) or bone marrow infiltration (p=0.95).

56 collections underwent further manufacturing, with production according to specification in 38 (68%) collections. 18 collections resulted in production failure, 13 (72%) due to insufficient proliferation of T-cells, 1 (6%) because of microbiological contamination and 4 (22%) due to undeterminable test results for mycoplasms. The manufacturing failures did not associate with age >60 (p=0.16), sex (p=0.99), bone marrow infiltration (p=0.47) or >3 prior treatment lines (p=0.78). However, we did notice a trend regarding prior treatment with bendamustine (p=0.07).

Importantly, the collections resulting in production failure had significantly lower CD3+/CD27+/CD28+ counts of 16.1 (range 1.1-52.3) vs 33.2 (range 3.3-93.5) x10 8 (p<0.01), as well as a trend for lower CD3+/CD4+ counts with 15.9 (range 3.0-43.9) vs 24.5 (range 2.4-102.7) x10 8 (p=0.05). However, there were no differences in CD3+/CD8+ counts (p=0.19), CD4:CD8 ratio (p= 0.77) or CD3+/CD27-/CD28- cells (p=0.81; Figure 1).

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Summary/Conclusion: In our real-life cohort of lymphoma patients intended to undergo treatment with tisagenlecleucel, adequate numbers of CD3 cells could be collected in most cases irrespective of the number of prior therapies. We could observe a negative prognostic role of T-cell senescence on the manufacturing process. Further analyses will be required to determine measures appropriate for further optimization and improvement of collection outcomes.

S288: A 9 YEAR REVIEW OF BLOOD TRANSFUSION PRACTICE AND ADHERENCE TO NICE GUIDELINES AT A DISTRICT GENERAL HOSPITAL, UK

N. Clayden 1,* , E. O’Donovan 1

1 Haematology, Surrey and Sussex Healthcare Trust, Surrey, United Kingdom

Background: Blood transfusions are common medical practice in the UK, with approximately 2.1 million blood products being issued by UK blood services in 2020. The Serious Hazard of Transfusion (SHOT) scheme reported that the risk of death and serious harm related to transfusions was 1 in 53,193 and 1 in 15,142 in 2020, respectively. Multiple complications can occur as a result of blood transfusions, with the most common cause of death being Transfusion Associated Circulatory Overload (TACO). Although low risk, there has been an increase in deaths related to blood transfusions from 17 deaths in 2019, to 39 deaths in 2020, in the UK, with 81.6% of adverse reactions and events being due to preventable errors. This stresses the importance of safe transfusion practice.

Aims: To review the rates of blood transfusions from 2013 to 2021, and compare our adherence to NICE guidance. We analysed rates of transfusion, number of red blood cells (RBC) units per transfusion, haemoglobin levels and the incidence of iron deficiency anaemia (IDA) prior to transfusion.

Methods: Data was collected retrospectively from patients’ records and the transfusion laboratory database at Surrey and Sussex healthcare Trust. Adults (>16 years old) who received inpatient or outpatient RBC transfusions from 2013 to 2021 were included (n = 53,941). We looked in further detail at the RBC transfusions from the first 2 weeks of December from 2014 to 2021 (n=546).

Results: Figure 1 shows that there has been a significant decline in the number of RBC transfused from 2014 to 2021; an average gradient of -36.25 units per year (R 2 = 0.72). There has also been a decline in the average number of units per blood transfusion, with a downward trendline (gradient -0.15 RBC units/year, p= 0.001). There has been a significant increase in the percentage of single unit transfusions from 14% in 2014 to 65% in 2021; the trendline has a gradient of +7% / year (p=0.0009). The average haemoglobin at initiation of transfusion has remained relatively unchanged (69-78g/L), which is in line with NICE guidance. There is no improvement in transfusion of patients with IDA; defined as a low transferrin saturation (<20%). The percentage of these patients being transfused varying from 43% to 79%, with no significant trend over the years from 2014 to 2021 (p=0.71).

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Summary/Conclusion: The total number of RBC transfused has significantly decreased over 9 years. However, there is a slight rise from August 2020, which may have been a result of expansion of the hospital bed base from 697 to 800 between years 2018-2021. It also may have been impacted by the COVID-19 pandemic. There is a decrease in the amount of RBC units transfused per patient. We are also encouraged to see a significant increase in the number of single unit transfusions, in line with NICE Guidance. We found there was no improvement in the number of patients with IDA being transfused (average 64%). This indicates that, depending on the clinical scenario, some patients may be receiving unnecessary blood transfusions. They may benefit from receiving iron replacement as an alternative treatment, thereby minimising exposing patients unnecessarily to the risks associated with blood transfusion. Appropriate management of IDA needs further work in the trust, and we have initiated an IV iron service over the last 18 months to improve this. Limitations of this study include using two weeks of the year to extrapolate for each year and data may have been skewed over the last two years due to the COVID-19 pandemic.

S289: MOTIVATORS AND BARRIERS TO BLOOD DONATION AMONG POTENTIAL DONORS OF AFRICAN AND CAUCASIAN ETHNICITY

H. Fogarty 1 2,* , M. Sardana 3 , L. Sheridan 4 , P. Chieng 3 , S. Kelly 3 , N. Ngwenya 2 , C. Sheehan 2 , K. Morris 5 , E. Tuohy 2

1 Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland; 2 Department of Haematology, St James’s Hospital; 3 School of Medicine; 4 School of Pharmacy, Royal College of Surgeons in Ireland; 5 Irish Blood Transfusion Service, Dublin, Ireland

Background: Blood donors of minority ethnicity, especially those of African origin, are under-represented in many high-income Western countries, including in Ireland. Conversely, the rising number of patients with Sickle Cell Disease (SCD) in many European countries has resulted in increased demand for blood transfusion. Given the high risk of alloimmunization among haemoglobinopathy patients, extended red blood cell matching is now recommended. To increase the probability of a phenotypic match, donors and recipients should share the same racial and/or ethnic background. Hence, the discrepancy between the number of African blood donors and the number of SCD patients is creating a strain on the blood supply chain and recruitment of African donors has become a major priority for transfusion services worldwide.

Aims: A key step towards enhancing recruitment of blood donors is to first understand perceived barriers and facilitators to blood donation, which may differ across different demographic categories, including ethnicity. This study aims to explore 1) Barriers and 2) Motivators to blood donation and 3) Awareness of SCD among potential donors of diverse ethnic backgrounds in Ireland.

Methods: Following ethical approval, patients attending the National Sickle Cell Disease and Thalassemia service at St James’s Hospital, Dublin, were invited to share an anonymous, online, 33-item survey within their local communities to achieve snowball-sampling. Inclusion criteria were adults aged ≥18 years who had not previously donated blood in Ireland. Participants were asked to state their age, sex and ethnicity. Responses for each survey item were recorded on a four-point Likert scale: Strongly Agree, Agree, Disagree or Strongly Disagree. Survey data were collected from August-December 2021. Statistical analysis was conducted using GraphPad Prism 9.1 (GraphPad Software Inc., USA).

Results: 387 respondents completed the survey, including 311 non-donors (median age 25 years, 67% female). Ethnic backgrounds included: African (59%), Caucasian (25%), Asian (8%), Hispanic/Latino (3%), Middle Eastern (3%) and Multiracial (2%). The most common barrier overall was lack of information on blood donation, identified by 60%. African respondents were 3 times more likely to report lack of information (OR 3.1 CI 1.8-5.3, p<0.0001) and 5 times more likely to report malaria-related barriers than Caucasians (OR 5.1 CI 2.1-12.3, p=0.0002). Conversely, Caucasians were twice as likely to report fear of needles/pain (OR 2.1, CI 1.1-3.7, p=0.02). Motivators also varied by ethnicity, with African respondents 9 times more likely to donate to help someone within their own community (OR 9.3 CI 4.6-18.3, p<0.00001) and 5 times more likely to donate for religious motivators (OR 5.5 CI 2.9-10.4 p<0.0001) than Caucasians. Awareness of SCD was 5 times higher among African respondents (92% versus 66%, OR 5.3 CI 2.7-10.5, p<0.0001).

Summary/Conclusion: This study enhances our understanding of ethnic differences in barriers and motivators to blood donation. While some barriers are shared across ethnic groups, including lack of information, notable differences exist between Caucasian and African respondents. Given that many European countries experience difficulties in the provision of phenotypically matched blood for SCD patients, these data provide valuable insights to inform future campaigns aimed at recruitment of African donors. At a time when the importance of diversity and inclusivity are increasingly recognized in society, enhancing ethnic diversity in blood donors may reflect a positive force for healthcare and social equality.

S290: ATRA CAN CORRECT DEFECTIVE HIF-1Α/S1P AXIS-MEDIATED CYTOSKELETAL REORGANIZATION IN PROPLATELET FORMATION OF ITP

Q.-S. Huang 1 2,* , J. Xue 1 2 , F.-Q. Liu 1 2 , Q. Chen 1 2 , G.-C. Zhang 1 2 , X.-Y. Sun 1 2 , C.-C. Wang 1 2 , L.-P. Yang 1 2 , Y.-Y. Li 3 4 5 , Q.-F. Wang 3 4 5 , J. Peng 6 , M. Hou 6 , X.-J. Huang 1 2 7 8 , X.-H. Zhang 1 2 7 8

1 Peking University People’s Hospital; 2 National Clinical Research Center for Hematologic Disease; 3 Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China; 4 China National Center for Bioinformation; 5 University of Chinese Academy of Sciences, Beijing; 6 Department of Hematology, Shangdong Key Laboratory of Immunochematology, and Shandong Provincial Clinical Medicine Research Center for Hematology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan; 7 Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation; 8 Collaborative Innovation Center of Hematology, Beijing, China

Background: Bone marrow physiological hypoxia plays a crucial role in haematopoietic stem cell homeostasis. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. HIF-1α expression was decreased in the bone marrow of patients with immune thrombocytopenia (ITP), and HIF-1α activation was shown to enhance megakaryopoiesis in mice. Recent studies suggest that “inside-out” signalling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) ( Blood, 2013; J EXP MED, 2012 ). Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies ( Blood , 2021; Lancet haematology , 2017; Lancet haematology , 2021), could restore cytoskeletal reorganization and correct impaired PPF.

Aims: This study aimed to explore the role of hypoxia inducible factor-1α (HIF-1α) in proplatelet formation (PPF) and the underlying mechanisms of ATRA treatment in ITP patients.

Methods: Thirty consecutive patients with newly diagnosed ITP and 30 healthy donors were included in our study. MKs were isolated from bone marrow samples. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Confocal microscopy and transmission electron microscopy were used to observe the PPF and cytoskeleton structure of ITP MKs. An ITP mouse model was established to observe the therapeutic effects of ATRA in the PPF.

Results: In the present study, we observed that MKs displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF) in ITP patients. Targeted and untargeted metabolite profiling revealed a decreased sphingosine-1-phosphate (S1P) level in ITP. Downregulated sphingosine kinase 2 (SPHK2) expression in MKs accounted for the low level of S1P in ITP. S1P is essential for S1P receptor 1 (S1PR1) and Rac1 activation, Src family kinases (SFKs) activity, and subsequent cytoskeletal reorganization and PPF regulation. Moreover, we demonstrated that HIF-1α mediated SPHK2 activation and S1P production. Decreased HIF-1α levels were found in the MKs of patients with ITP, contributing to impaired PPF.

We then investigated the effect of ATRA on PPF in ITP patients. ATRA upregulated HIF-1α and SPHK2 expression, increased S1P production and corrected impaired PPF in vitro. In an ITP mouse model, ATRA alleviated thrombocytopenia and restored cytoskeletal reorganization. ATRA corrected impaired PPF by upregulating HIF-1α expression. The exposure of ITP MKs to selective RARα (AM580) or RARγ (BMS961) agonists did not change PPF. However, the treatment of ITP MKs with a selective RARβ agonist (CD2314) significantly increased PPF. Furthermore, we found that the effect of ATRA on enhancing PPF in ITP MKs was reversed in the presence of an RARβ antagonist (CD2665). These data suggest that impaired PPF in ITP MKs is corrected by ATRA in a RARβ-dependent manner.

Summary/Conclusion: Together, our data show that the HIF-1α/S1P axis mediates altered cytoskeletal reorganization and impaired PPF in ITP and suggest that ATRA correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP.

S291: PHASE I/II STUDY OF RILZABRUTINIB, AN ORAL BRUTON TYROSINE KINASE INHIBITOR, IN PATIENTS WITH IMMUNE THROMBOCYTOPENIA: LONG-TERM FOLLOW-UP

D. J. Kuter 1,* , M. Efraim 2 , Z. Kaplan 3 , J. Mayer 4 , P. Choi 5 , A. G. Jansen 6 , V. McDonald 7 , R. Baker 8 , R. Bird 9 , M. Garg 10 , J. Gumulec 11 , M. Kostal 12 , T. Gernsheimer 13 , W. Ghanima 14 , M. Yao 15 , A. Daak 16 , N. Cooper 17

1 Hematology Division, Massachusetts General Hospital, Harvard Medical School, Boston, United States of America; 2 Multiprofile Hospital for Active Treatment Sveta Marina EAD, Varna, Bulgaria; 3 Monash Medical Centre, Clayton, Australia; 4 Department of Internal Medicine, Hematology and Oncology, Masaryk University Hospital, Brno, Czechia; 5 The Canberra Hospital, Garran, Australia; 6 Erasmus MC, University Medical Center, Rotterdam, Netherlands; 7 Barts Health NHS Trust, The Royal London Hospital, London, United Kingdom; 8 Perth Blood Institute, Murdoch University, Perth; 9 Princess Alexandra Hospital, Woolloongabba, Australia; 10 Leicester Royal Infirmary, Leicester, United Kingdom; 11 Department of Hematooncology, University Hospital Ostrava and Faculty of Medicine University of Ostrava, Ostrava; 12 Fourth Department of Internal Medicine and Hematology, Faculty of Medicine, University Hospital of Hradec Kralove, Hradec Kralove, Czechia; 13 University of Washington Medical Center, Seattle, United States of America; 14 Østfold Hospital Foundation, Gralum, Norway; 15 Biostatistics, Sanofi US Services Inc., Bridgewater; 16 Sanofi Genzyme, Cambridge, United States of America; 17 Department of Medicine, Hammersmith Hospital, London, United Kingdom

Background: Immune thrombocytopenia (ITP) is an autoimmune disease associated with autoantibody-mediated platelet destruction and impaired platelet production, resulting in thrombocytopenia and high bleeding risk. In a global phase I/II trial ( NCT03395210 ) of heavily pretreated patients with long-standing disease, the Bruton tyrosine kinase inhibitor rilzabrutinib showed a rapid and durable platelet count increase and was well tolerated. We present results for patients initiating rilzabrutinib 400 mg BID who are continuing in the long-term extension (LTE).

Aims: Assess if the efficacy and safety of rilzabrutinib 400 mg BID continue to be maintained in LTE patients on rilzabrutinib ± concomitant medication.

Methods: Patients with 2 baseline platelet counts <30×10 9 /L were required to have responded to ≥1 prior ITP therapy but at baseline were unable to maintain an adequate response to prior/concomitant therapies. The main treatment period was 24 weeks; patients who achieved platelet counts ≥50×10 9 /L at ≥50% of the visits during the last 8 weeks of the main period were permitted to continue 400 mg BID in the LTE. Primary endpoints were safety and efficacy (≥2 consecutive platelet counts ≥50×10 9 /L and increased ≥20×10 9 /L from baseline without requiring rescue medication). Stable doses of concomitant ITP medication (thrombopoietin-receptor agonists [TPO-RA] and corticosteroids [CS]) were allowed for patients with inadequate platelet response. Patients who received rescue medication discontinued from the study. All patients provided informed consent.

Results: At baseline, the 45 patients initiating 400 mg BID in the main period had a median age of 49 years, median duration of ITP of 6.1 years, median platelet count of 15×10 9 /L, and a median of 4 unique prior therapies (24% prior splenectomy). A total of 15 patients (33%) received rilzabrutinib monotherapy, and 30 had concomitant therapy (TPO-RA n=13 [29%], CS n=12 [27%], TPO-RA + CS n=5 [11%]). Primary platelet response was achieved by 18 patients (40%): 6 on monotherapy and 12 on concomitant medication. Platelet counts of ≥50×10 9 /L, ≥30×10 9 /L, and ≥20×10 9 /L from baseline were maintained for a median of 71%, 95%, and 87% of weeks, respectively. As of 21Jan2022, 16/60 patients in the main study population had proceeded to LTE, of whom 11 were ongoing. Five LTE patients (31%) continued on rilzabrutinib monotherapy and 11 used concomitant medication (TPO-RA n=2 [13%], CS n=7 [44%], TPO-RA + CS n=2 [13%]; Figure); 1 patient used rescue medication. Median platelet count at LTE entry was 87×10 9 /L. In patients on rilzabrutinib monotherapy, the median platelet count was 68×10 9 /L, which was sustained at 6 months of follow-up (Table). In all LTE patients, platelet counts ≥50×10 9 /L, ≥30×10 9 /L, and ≥20×10 9 /L from baseline were maintained for a median of 88%, 100%, and 97% of weeks, respectively. In patients on rilzabrutinib ± concomitant medication, results were consistent at 3 and 6 months of follow-up (Table). Three patients (19%) experienced related treatment-emergent adverse events (TEAEs), including 1 grade 2 upper respiratory tract infection. Three patients (19%) discontinued rilzabrutinib due to a TEAE. Six patients had ≥1 bleeding event, none were treatment-related; no related serious adverse events or deaths. The average ITP-BAT bleeding scale score at 6 months showed no increase in bleeding in the LTE.

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Summary/Conclusion: With extended treatment duration, rilzabrutinib 400 mg BID showed durable clinical efficacy and was well tolerated in patients on rilzabrutinib ± concomitant medication.

S292: SUSTAINED RESPONSE OFF TREATMENT IN ELTROMBOPAG-TREATED PATIENTS WITH ITP WHO ARE REFRACTORY OR RELAPSED AFTER FIRST-LINE STEROIDS: PRIMARY ANALYSIS OF THE PHASE II TAPER TRIAL

N. Cooper 1,* , W. Ghanima 2 , N. Vianelli 3 , D. Valcárcel 4 , I. Yavaşoğlu 5 , A. Melikyan 6 , E. Yañez Ruiz 7 , J. Haenig 8 , J. Lee 9 , J. Maier 8 , N. Zolkin 10 , F. Zaja 11

1 Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, Hammersmith Hospital, London, United Kingdom; 2 Department of Medicine, Østfold Hospital Trust, Kalnes, Norway; 3 Scientific Institute for Research, Hospitalization and Healthcare (IRCCS), Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 4 Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), University Hospital Vall d’Hebron, Barcelona, Spain; 5 Department of Hematology, Adnan Menderes University, Aydin, Turkey; 6 National Research Center for Hematology, Moscow, Russia; 7 Hematology-Oncology Unit, Department of Internal Medicine, School of Medicine, Universidad de la Frontera, Temuco, Chile; 8 Novartis Pharma AG, Basel, Switzerland; 9 Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States of America; 10 IQVIA, St. Petersburg, Russia; 11 Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy

Background: Corticosteroids (CSs) are the standard 1 st -line treatment for primary immune thrombocytopenia (ITP); however, long-term CS use is associated with high relapse rates and considerable toxicity. Treatments that achieve a sustained response and reduce the need for long-term CS use are needed. Eltrombopag (EPAG) is a thrombopoietin receptor agonist (TPO-RA) indicated in Europe for the treatment of patients (pts) aged ≥1 year with primary ITP lasting ≥6 months who are refractory to other treatments (eg, CSs). Evidence suggests that a proportion of pts treated with TPO-RAs achieve sustained responses that are maintained after TPO-RA tapering and discontinuation; however, much of the evidence is retrospective with only a few prospective studies investigating sustained response off treatment (SRoT).

Aims: TAPER ( NCT03524612 ), a Phase II, open-label, prospective, single-arm study, aims to determine whether EPAG can induce SRoT in pts with ITP after 1 st -line CS failure.

Methods: Adult (≥18 years) pts with ITP who did not respond to or had relapsed after initial CS therapy, with platelet counts <30×10 9 /L and assessed as needing treatment, were included. Patients received a 50 mg/day starting dose of EPAG (25 mg/day for East/Southeast Asian pts; 12.5 mg/day for Japanese pts in Japan), which could be increased up to 75 mg/day (50 mg/day in Japan) if needed. The primary endpoint was the number (%) of pts with SRoT by Month 12; SRoT was defined as achieving a complete response (CR, ie, platelet count ≥100×10 9 /L), then maintaining a stable platelet count (no counts <70×10 9 /L) for 2 months, followed by successful EPAG tapering and discontinuation with platelet counts ≥30×10 9 /L and no bleeding events or rescue therapy. Patients with SRoT at Month 12 were followed for a further year. Secondary outcomes included SRoT duration and platelet count changes from baseline. Data to Month 12 are presented.

Results: N=105 pts were enrolled. The median (interquartile range [IQR]) age was 46 (30-65) years; 61% were female. In the 1 st 12 months, median (IQR) duration of exposure to EPAG was 5.6 (2.3-11.9) months and median (IQR) EPAG dose was 57.1 (37.5-69.0) mg/day. Overall, 89 pts (85%) achieved CR at least once and 65 pts (62%) maintained a platelet count ≥70×10 9 /L for 2 months after CR. EPAG tapering and discontinuation was achieved in 44 pts (42%). The primary endpoint was met, with 32 pts (30.5% [95% confidence interval, 21.9-40.2]; P <0.001 [H1: P>15%; alpha: 0.05]) achieving SRoT until Month 12 (Fig. 1); SRoT was maintained from last dose to Month 12 for a median (IQR) of 33.3 (25.7-45.3) weeks. The median (IQR) absolute increase in platelet counts from baseline at Month 12 was 77.0×10 9 /L (35.0-145.0). All-grade adverse events (AEs) occurred in 92/105 (88%) pts (grade ≥3 AEs: 33/105 [31%]). Treatment-related AEs occurred in 37 (35%) pts (8 [7.6%] grade ≥3). The most common all-grade AEs were headache (21% of pts with ≥1 event [grade ≥3: 1% of all pts]), thrombocytopenia (17% [10.5%]), and petechiae (11% [1%]). There were 4 deaths (none were considered treatment related): 3 were on-treatment (central nervous system hemorrhage [n=1], intracranial hemorrhage [n=1], metastases to peritoneum [n=1]) and 1 death (malignant neoplasm) occurred 238 days after last dose.

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Summary/Conclusion: Data from the TAPER study indicate that a significant proportion of pts experience sustained response following tapering and discontinuation of EPAG, even after a relatively short duration of exposure. Overall EPAG was well tolerated with no unexpected AEs.

S293: RISK OF FRACTURES IN ADULT PATIENTS WITH PRIMARY AND SECONDARY IMMUNE THROMBOCYTOPENIA: A DANISH NATIONWIDE COHORT STUDY

N. Mannering 1 2,* , D. L. Hansen 1 2 , G. Moulis 3 4 , W. Ghanima 5 6 , A. Pottegård 7 , H. Frederiksen 1 2

1 Department of Hematology, Odense University Hospital; 2 Clinical Institute, University of Southern Denmark, Odense, Denmark; 3 Department of Internal Medicin; 4 Clinical Investigation Center 1436, Team PEPSS, Toulouse University Hospital, Toulouse, France; 5 Department of Hematology, Østfold Hospital; 6 Institute for Clinical Medicine, University of Oslo, Oslo, Norway; 7 Department of Public Health, University of Southern Denmark, Odense, Denmark

Background: The mainstay of first line treatment in immune thrombocytopenia (ITP) has remained high-dose corticosteroids for decades. Although steroid treatment is recommended to be tapered quickly in ITP, previous data shows that during the first six weeks of primary ITP treatment the mean accumulated steroid dose was 2-3 g of prednisolone. In addition, patients may be exposed to repeated courses of corticosteroids during relapses due to its rapid effect on platelet count. Corticosteroids is a well-known risk factor for bone demineralization and osteoporosis that may lead to fractures, but it is unknown if patients with ITP suffer an increased risk of fractures.

Aims: In this study we investigated incidences of fractures in primary (pITP) and secondary ITP (sITP) compared to the general population.

Methods: Incident patients with ITP ≥18 years were identified in the nationwide Danish health registries during 1980-2016 by using the first registration of the designated codes 287.10 (ICD-8) or D.69.3 (ICD-10). Prevalent ITP and other thrombocytopenic conditions were excluded. Secondary ITP was classified when one or more associated diagnoses were registered any time before or up to 30 days after ITP diagnosis.

Each patient with ITP was age-sex matched with up to 40 comparators from the general population. Date of the first ITP registration marked start date of follow-up, and comparators were assigned the same start date. Incident fractures were identified using designated fracture registrations, and divided into five groups: hip and femoral, humeral, antebrachial, axial, and any of the before mentioned. All individuals were followed from start date until the first of the following: fracture, death, emigration, or end of study. Using these data, we calculated rates, incidence-rate-ratios (IRR) and cumulative incidences for fractures in patients vs. comparators after 1, 5 and 10 years, and end of study period.

Results: We identified 4,789 patients with pITP, 654 patients with sITP, and 217,370 comparators. Median age was 59.6 years for primary ITP and 57.3 for secondary ITP. Women constituted 54% of primary ITPs, and 64% of secondary ITP patients.

IRR in pITP was 1.15 [95% CI 1.04; 1.27] for any fracture, and 1.34 [1.09; 1.62] for axial fractures. For sITP, overall IRR was 1.37 [1.06; 1.74] for any fracture, and 1.65 [1.16; 2.29] for hip – and femoral fractures.

IRR was significantly elevated for any fracture during the first 5 years after diagnosis of pITP, but equalized hereafter. The IRR of hip – and femoral fractures in the first year after diagnosis was 2.04 [1.41; 2.86] in pITP, and 2.30 [1.32; 3.74] for axial fractures, however these differences also equalized over time. For sITP, the risk of any fracture was particularly elevated during the first year with an IRR of 2.65 [1.39; 4.63], as well as the risk of distal antebrachial fracture with an IRR of 3.16 [1.33; 6.46]. During year 2-5, IRR for hip – and femoral fracture was 1.92 [1.04; 3.26]. All remaining IRR-estimates were not statistically significant.

Cumulative incidence proportions showed similar trends (Figure) with largest differences during first years after diagnosis, but equalizing over time.

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Summary/Conclusion: The incidence of some fractures in ITP is increased during the first years after diagnosis compared to the general population. Clinical attention and action should be directed upon this matter.

Data on comorbidity, sub grouped cumulative incidences, and additional risk estimates will be presented with the abstract.

S294: LONG-TERM SAFETY AND EFFICACY OF CAPLACIZUMAB FOR ACQUIRED THROMBOTIC THROMBOCYTOPENIC PURPURA (ATTP): THE POST-HERCULES STUDY

M. Scully 1,* , J. de la Rubia 2 3 , K. Pavenski 4 5 , A. Metjian 6 , P. Knöbl 7 , F. Peyvandi 8 9 10 11 , S. Cataland 12 , P. Coppo 13 , J. A. Kremer Hovinga 14 , R. De Passos Sousa 15 , F. Callewaert 16 , S. Gunawardena 17 , J. Lin 17

1 Department of Haematology, University College London Hospitals, London, United Kingdom; 2 Hematology Department, Internal Medicine, School of Medicine and Dentistry, Catholic University of Valencia; 3 Hospital Doctor Peset, Valencia, Spain; 4 Departments of Medicine and Laboratory Medicine, St. Michael’s Hospital; 5 University of Toronto, Toronto, ON, Canada; 6 Division of Hematology, Department of Medicine, University of Colorado–Anschutz Medical Center, Denver, CO, United States of America; 7 Department of Medicine; 1 , Division of Hematology and Hemostasis, Medical University of Vienna, Vienna, Austria; 8 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 9 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center; 10 Fondazione Luigi Villa; 11 Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; 12 Department of Internal Medicine, Ohio State University, Columbus, OH, United States of America; 13 Department of Hematology, Reference Center for Thrombotic Microangiopathies (CNR-MAT), Saint-Antoine University Hospital, AP-HP, Paris, France; 14 Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; 15 Sanofi, Lisbon, Portugal; 16 Sanofi, Diegem, Belgium; 17 Sanofi, Cambridge, MA, United States of America

Background: The efficacy and safety of caplacizumab (CPLZ) for patients with acquired thrombotic thrombocytopenic purpura (aTTP; also known as immune-mediated TTP) was demonstrated in the Phase 3 HERCULES trial, with a 28-day follow-up period after end of treatment.

Aims: To evaluate long-term safety and efficacy of CPLZ in patients with aTTP, and safety and efficacy of repeated CPLZ use for aTTP recurrence.

Methods: In post-HERCULES ( NCT02878603 ), patients who completed the HERCULES trial were invited to attend twice-yearly visits over 3 years. Patients could receive open-label CPLZ with therapeutic plasma exchange (TPE) and immunosuppressive therapy (IST) in case of aTTP recurrence. Safety was assessed during the overall study period in the intention-to-observe (ITO) population (all enrolled patients; n=104). TTP-related events (TTP-related mortality, recurrence, or major thromboembolic events) were assessed in patients without a recurrence during HERCULES or prior to the start of post-HERCULES (efficacy ITO population, n=78). Safety and efficacy outcomes were also evaluated during recurrences (recurrence population, n=19), including patients treated at least twice with CPLZ (received CPLZ in HERCULES or twice in post-HERCULES; repeat-use population, n=9). All patients provided informed consent.

Results: Of 104 patients who enrolled, 75 had been treated with CPLZ with TPE+IST during HERCULES (CPLZ group) and 29 had been treated with TPE+IST only (placebo group). Incidence of adverse events (AEs) and serious AEs during the overall study period was similar between groups. Recurrence occurred in 11/75 patients (15%) in the CPLZ group and 8/29 (28%) in the placebo group. In the efficacy ITO population, TTP-related events occurred in 4/49 patients (8%) randomized to CPLZ versus 11/29 (38%) randomized to placebo; in patients randomized to CPLZ, relapse rates were similar between patients with (10%) versus without (7%) prior rituximab use. A total of 19 patients had ≥1 recurrence; 13 were treated with CPLZ (6/13 received concomitant rituximab during the first recurrence period). The first recurrence episode was resolved (12/13) or resolving (1/13) for all patients treated with CPLZ, including the 9 patients with repeat CPLZ use. All second recurrences (6/6) were resolved. One patient who was not treated with CPLZ in either HERCULES or post-HERCULES died as an outcome of recurrence. For the first recurrence period, the median (min, max) time to initial platelet count ≥150x10 9 /L with subsequent stop of TPE within 5 days was 7.0 (3, 24) days for patients treated with CPLZ (n=13) and 10.0 (5, 15) days for patients not treated with CPLZ (n=5); duration of TPE was 5.0 (2, 19) days (n=13) and 5.5 (4, 7) days (n=4), respectively; length of hospital stay was 7.0 (4, 23) days (n=11) and 10.0 (9, 11) days (n=3), respectively. The safety profile of CPLZ for recurrence was consistent with that observed in HERCULES (Table). In the repeat-use population (n=9), bleeding events were reported in 5/9 patients (56%) for the first recurrence; 2 patients had a serious treatment-related treatment-emergent AE: genitourinary bleeding (in 1 patient during the first recurrence) and gastrointestinal bleeding (in 1 patient during the second recurrence).

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Summary/Conclusion: The long-term safety profile of patients treated with CPLZ in combination with TPE+IST was generally similar to those who received IST+TPE only, with no observed increases in aTTP recurrence. Repeat use of CPLZ was efficacious, with no new safety concerns.

S295: CLINICAL RELEVANCE OF SPLEEN VOLUME AND PLATELET COUNT WITH BLEEDING EVENTS IN PATIENTS WITH ACID SPHINGOMYELINASE DEFICIENCY (ASMD)

M. Fournier 1 , J. Msihid 1 , A. Willemze 2 , F. Laredo 3 , R. Pulikottil-Jacob 4,*

1 Sanofi, Chilly-Mazarin, France; 2 Sanofi, Amsterdam, Netherlands; 3 Sanofi, São Paulo, Brazil; 4 Rare Disease, Health Economics and Value Assessment, Sanofi, Berkshire, United Kingdom

Background: Acid Sphingomyelinase Deficiency (ASMD) is an ultra-rare, lysosomal storage disease with a broad spectrum of clinical manifestations. Within that spectrum, ASMD type B and type A/B are often referred to as chronic ASMD. In Type 1 Gaucher disease, increased bleeding tendency is attributed to thrombocytopenia secondary to hypersplenism and/or bone marrow infiltration noticed in patients with splenomegaly and thrombocytopenia. However, ASMD patients have a bleeding tendency that is not proportionally attributed to any association or explanation; hence further research is needed to study the cause of bleeding among patients with ASMD.

Aims: To characterize the association between bleeding events (BE), spleen volume (SV), and platelet count by retrospectively analyzing data from a previously published prospective, multicenter, longitudinal study (MSC12840 [SPHINGO-001-00]) in patients with chronic ASMD (N=59; age: 7–64 years; 30 were < 18 years; male: 53%; up to 3 visits per patient with 6–11 years follow-up, 2001–2012).

Methods: This study retrospectively screened medical charts of 59 adults and pediatrics for BE specified under the hematopoietic body system and their respective SV and platelet count. SV (in multiple of normal, MN) and platelet count (in 10 3 /UL) were measured at baseline, year 1, and end of study [median years of observation (range), 10.2 years (4.7–11.1)]. As reported in the medical history captured at each visit, BE were selected with a series of keywords indicating hemorrhage, bleed, or transfusion post-medical review. To characterize their association with BE, SV and platelet counts were considered at the visit prior to the observation of the event. A general linear mixed model was performed using SV as a continuous or a class variable (<6, 6–15, and >15 MN). The associations between platelet counts (below 100 x 10 3 /UL) and SV were also explored.

Results: Overall, 59 patients were enrolled, 50 patients completed year 1 visit, and 32 patients completed the final visit (9 died, 9 discontinued, and 9 were lost to follow-up). Twenty-six patients reported BE across the two observed periods, i.e., baseline to 1 year and 1 year to final study visit. No BE was reported with SV <6 MN at any period; however, there was a trend for a higher event rate in patients with higher SV, per visit (Table 1). A higher SV of +5 MN or +10 MN was associated with a 1.8-times (odds ratio [95% confidence interval]: 1.85 [1.05, 3.29]) and 3.4-times (3.45 [1.10, 10.82]) increased risk of BE, respectively. Severe splenomegaly (>15 MN) was associated with a higher and non-significant risk of BE vs. moderate splenomegaly (6–15 MN; 2.54 [0.58, 11.08]). In addition, patients with low platelet counts (<100 x 10 3 /UL) had higher SV, per visit (Table 1). The p-value for the chi-square test for association between platelet count and SV across visits was 0.006; it was 0.058, 0.127, and 0.302 at baseline, year 1, and end of the study, respectively. There was a moderate negative correlation (r = –0.47) between platelet count and SV across visits.

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Summary/Conclusion: This analysis highlighted the association between SV and BE among patients with chronic ASMD. Since a statistically significant association between SV and thrombocytopenia was observed across visits, hypersplenism appears to be a contributor for the pathophysiology of bleedings among these patients. These associations help better understand the clinical relevance of SV measurements in ASMD. Prospective validation is warranted to confirm the results of these analyses.

S296: IN VITRO ANALYSIS OF THE IMPACT OF INHIBITORS ON THE PROCOAGULANT EFFECTS OF BYPASS AGENTS OR HEMOSTATIC FACTORS IN COMBINATION WITH EMICIZUMAB

A. Dos Santos Ortas 1,* , E. García Arias-Salgado 1 , E. Monzón Manzano 1 , P. Acuña 1 , M. T. Álvarez Roman 1 , M. Martín Salces 1 , M. I. Rivas Pollmar 1 , E. García Pérez 1 , M. Gutiérrez Alvariño 1 , A. Gonzalez Ceberino 1 , S. García Barcenilla 1 , N. Butta 1 , V. Jiménez Yuste 1

1 HOSPITAL UNIVERSITARIO LA PAZ, MADRID, Spain

Background: Emicizumab is a non-replacement treatment used in hemophilia A patients (PwHA). However, patients on prophylaxis with emicizumab exhibit mild-moderate bleeding phenotypes, thus Factor (F) VIII or bypassing agents (BPAs) are required to control breakthrough or perioperative bleeds. Increments of FIX levels, the main limiting factor for the FIXa-emicizumab-FX complex formation, also produce an increase of the emicizumab procoagulant effects similar to those obtained with FVIII or BPAs.

Aims: To elucidate the impact of FVIII inhibitors in the efficacy of this hemostatic response, we compared the in vitro procoagulant effects of BPAs and FIX products in samples from emicizumab-treated patients with or without inhibitors.

Methods: Blood from 21 patients on prophylaxis with emicizumab, 8 with inhibitor, was collected in tubes with CTI (Corn Trypsin Inhibitor) to prevent activation of the contact pathway.

Spiking with therapeutic doses of 100 IU/dl octocog-alfa (rFVIII), BPAs [1 µg/ml eptacog alfa activated (rFVIIa); 5 U/dl activated prothrombin complex concentrate (aPCC)] or 100 IU/dl of plasma-derived FIX (pFIX), two standard-half-life (SHL) rFIX (nonacog-alfa and nonacog-gamma), and one extended-half-life (EHL) rFIX (albutrepenonacog-alfa) were tested.

Clotting time (CT) was evaluated by Rotational Thromboelastometry (ROTEM) with whole blood, and Thrombin Peak by thrombin generation test (TGT) in plasma using low tissue factor concentration.

Results: Emizicumab-treated samples showed prolonged CT values (Fig.1A) and lower thrombin peak levels (Fig.1B) than healthy controls and no significant differences were observed between samples with and without inhibitors.

A ddition of 100 IU/dl of all FIX products normalized the CT values similarly to the addition of the same dose of rFVIII. Nonacog-alfa was more efficient producing similar procoagulant effects than the addition of 1μg/ml rFVIIa or 5 U/dl aPCC. Reduction of CT was similar in samples from PwHA without or with inhibitors in all assayed conditions (Fig.1A).

Higher thrombin peak values were also obtained by the addition of all the factors or BPAs tested by TGT but the procoagulant effect of all the recombinant FIX was significantly higher in samples with inhibitors (Fig.1B).

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Summary/Conclusion: Procoagulant effect of BPAs with emicizumab was similar with or without inhibitors whereas thrombin generation increment produced by rFIX was enhanced in the presence of inhibitors. These results provide a rationale for investigating the concomitant use of rFIX and emicizumab in PwHA with inhibitor in breakthrough bleeding and surgery.

Funding: ISCIII-FEDER (PI19/00631); CLS Behring; Catedra UAM-Roche; and Investigator-Initiated Reserach grant (no. IISR-2019-104361) from Baxalta GmbH, a wholly owned subsidiary of Takeda Pharmaceutical Comany Limited.

S297: ASSESSING THE COST-EFFECTIVENESS OF LONG-TERM PROPHYLAXIS STRATEGIES IN VON WILLEBRAND DISEASE

M. Wilson 1 , G. Castaman 2 , G. Escolar 3 , W. Miesbach 4 , S. Santos 5 , S. Yan 6,*

1 RTI Health Solutions, Research Triangle Park, NC, United States of America; 2 Center for Bleeding Disorders, Careggi University Hospital, Florence, Italy; 3 Department of Hematopathology, Centre Diagnostic Biomedic, Hospital Clinic, Barcelona, Spain; 4 Haemophilia Centre, Medical Clinic II, Institute of Transfusion Medicine, Goethe University Hospital, Frankfurt am Main; 5 CSL Behring, Hattersheim am Main, Germany; 6 CSL Behring, King of Prussia, PA, United States of America

Background: Von Willebrand disease (vWD) is the most common inherited bleeding disorder. Patients with vWD may experience excessive bleeding events resulting in morbidity, reduced quality of life, and a substantial economic burden.

Management strategies include von Willebrand factor concentrates either as on-demand treatment (ODT) of bleeds or long-term prophylaxis (LTP) to prevent bleeds. According to recent treatment guidelines, long-term prophylaxis is recommended in individuals with severe and frequent bleeds.

Aims: To assess the cost effectiveness of ODT and LTP treatment strategies in vWD patients with low, medium and high annual bleed rate (ABR), using pdVWF/FVIII 2,4:1 products (2,4:1 product A in the United Kingdom (UK) and 2,4:1 product B in Sweden).

Methods: A Markov structure considering risk of joint surgery and bleed rate was used to estimate the life years (LYs), quality-adjusted life years (QALYs), and costs of vWD treatment over a lifetime horizon. Treatment options included ODT or LTP with pdVWF/FVIII 2,4:1 or pdVWF/FVIII 1:1 in the UK and in Sweden. Product dosing for LTP and for ODT were obtained from each product’s summary of product characteristics or assumed equal to similar products where data are not available. Bleed risks (major and minor bleed) were estimated for each product in ODT and LTP using published prophylaxis studies and were used to estimate costs of product needed and to determine the probability of requiring joint surgery over time based on the progression of Pettersson Score. Resource use (inpatient and outpatient) costs for bleeds and joint surgery were obtained from standard country-specific costing sources. All costs are shown in 2021 GBP or Swedish Kr for the UK and Swedish analyses, respectively. Health-state utility weights and disutilities of bleeds were obtained from published literature. Annual discount rates for costs and outcomes were 3.5% for the UK and 3% for Sweden. One-way and probabilistic sensitivity analyses were conducted to evaluate the impact of parameter uncertainty. Each analysis considered a low, medium, and high ABR patient population.

Results: In the base case analyses (medium ABR) over a lifetime horizon, pdVWF/FVIII 2,4:1 LTP regimens were cost-effective compared with ODT. For pdVWF/FVIII 2,4:1 in the UK, LTP was both cost-saving (-£831,206 incremental cost) and more effective (6.14 incremental QALY) than ODT. Costs of bleed events more than offset the costs of prophylaxis. In Sweden with pdVWF/FVIII 2,4:1, LTP was also dominant (-8,841,901 kr; 6.52 QALY) compared with ODT. In comparisons of LTP regimens, pdVWF/FVIII 2,4:1 was dominant versus pdVWF/FVIII 1:1 in both the UK (-£2,307,370; 1.56 QALY) and in Sweden (-21,939,947 kr; 1.42 QALY). In comparison of ODT regimens, pdVWF/FVIII 2,4:1 was less expensive than pdVWF/FVIII 1:1 in the UK (-£1,250,369; equal QALY) and in Sweden (-7,923,741 kr; equal QALY) due to differences in product costs. Results were similar for comparisons in the high ABR populations. In probabilistic sensitivity analyses, pdVWF/FVIII 2,4:1 was dominant in over 95% of simulations in the UK and in over 85% of simulations in Sweden, and was cost-effective in 95% or more of simulations in both countries.

Summary/Conclusion: These results suggest that LTP with pdVWF/FVIII 2,4:1 is a cost-effective strategy compared with ODT, in both the UK and Sweden, for medium and high ABR vWD patients. pdVWF/FVIII 2,4:1 was shown to be cost-effective compared with pdVWF/FVIII 1:1 in the LTP setting, as well as less costly for patients treated ODT for both the UK and Swedish settings, respectively.

S298: CS585 IS A FIRST-IN-CLASS COMPOUND TARGETING THE IP RECEPTOR FOR PREVENTION OF THROMBOSIS WITHOUT INCREASED RISK OF BLEEDING

S. Lambert 1 , R. Adili 1 , P. Yalavarthi 1 , N. Rhoads 1 , B. Dahlof 2 3 , A. White 4 , N. Bergh 2 3 , M. Holinstat 1 5,*

1 Pharmacology, University of Michigan Medical School, Ann Arbor, United States of America; 2 Cereno Scientific; 3 Institute of Medicine, University of Gothenburg, Gothenburg, Sweden; 4 Medicinal Chemistry; 5 Internal Medicine, University of Michigan Medical School, Ann Arbor, United States of America

Background: Uncontrolled platelet activation leads to the formation of occlusive thrombi resulting in myocardial infarction, stroke, VTE and critical limb ischemia. There is an unmet need for more efficacious anti-thrombotics with less bleeding in this area. Our group recently identify a novel oxidized lipid in the blood that potently and selectively inhibits platelet activation through activation of the prostacyclin receptor.

Aims: Develop a first-in-class antiplatelet drug, CS585, that potently and selectively inhibits platelet reactivity and thrombosis without altering hemostasis and bleeding risk.

Methods: We developed a mimetic of the oxidized lipid shown to selectively bind to and activate the prostacyclin receptor. Using human platelets, we assessed the ability of CS585 to inhibit platelet activation by assessing 1)aggregometry, 2)adhesion under arterial flow, and 3) granule secretion and integrin activation using flow cytometry. We additionally assessed thrombosis in vivo in 2 different mouse models as well as bleeding. Finally, we assessed potential off-target effects using thromboelastography (TEG).

Results: CS585 potently inhibited both collagen and thrombin-induced platelet aggregation. This inhibition was confirmed by measuring inhibition of αIIbβ3 activation, α-granule secretion, and dense-granule secretion by flow cytometry. Selectivity was confirmed in human platelets by demonstrating a full reversal of inhibition when the IP receptor was pharmacologically blocked or genetically eliminated in IP receptor knockout mice. In vivo inhibition of injury-induced thrombosis in the small (laser-induced cremaster thrombosis model) and large (FeCl 3 -induced carotid artery thrombosis model) vessels by CS585 was demonstrated and no increased risk for bleeding was observed using the tail vein bleeding model. Finally, TEG experiments in human blood spiked with CS585 demonstrated no delay or decrease in clot strength confirming the tail vein bleeding assay experiments in the mouse.

Summary/Conclusion: We have shown for the first time that CS585, a first-in-class analog of an oxidized lipid in the blood potently and selectively activates the prostacyclin receptor resulting in inhibition of human and mouse platelet activation and thrombosis without an increased risk of bleeding. This discovery represents a new class of inhibitors for prevention of platelet activation and thrombosis and protection from myocardial infarction, stroke, VTE, and critical limb ischemia.

S299: ROLE OF RED BLOOD CELL MEMBRANE-DERIVED PARTICLES ON ENDOTHELIAL DAMAGES DURING ONSET STAGE OF DELAYED HEMOLYTIC TRANSFUSION REACTION

N.-P. Kim-Anh 1 2,* , L. Kiger 2 , X. Decrouy 3 , L. Bencheikh 1 2 , A. Habibi 4 , F. Pirenne 1 2 5 , L. Roumenina 6 , P. Bartolucci 2 4 5

1 EFS recherche, Etablissement Français du sang; 2 Team Pirenne; 3 Imaging platform, INSERM U955 IMRB; 4 Sickle cell referral center, Hôpital Henri-Mondor; 5 Université Paris Est Créteil, Créteil; 6 INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, Sorbonne Universités, Université de Paris, Paris, France

Background: Delayed hemolytic transfusion reaction (DHTR) is a life-threatening complication of red blood cell (RBC) transfusion in sickle cell disease (SCD) patients, leading to the increase of plasma hemoglobin (Hb) and heme which are responsible for endothelial damage and organs failure. By the development of an in vitro model reproducing endothelial damages at the early phase of DHTR (Nguyen et al, ASH 2018), our previous study suggested that RBC membrane-derived particles released during the onset stage of DHTR could be also involved in DHTR physiopathology.

Aims: In this study, we aim to determine the mechanism of endothelial damages induced by these RBC particles.

Methods: Human Umbilical Vein Endothelial Cells (HUVECs) cultured in flow condition for 24 hours were perfused by either serum only (decomplemented or not) or serum containing either whole hemolysate (sonicated RBCs) or different hemolysate components or RBCs membranes (ghosts) under shear stress 1 dyne/cm2. Annexin V and Eculizumab were used for complement studies.

Endothelial activation and damages were assessed by membrane CD54 (PECAM-1, Platelet endothelial cell adhesion molecule 1), CD106 (VCAM-1, Vascular Cell Adhesion Molecule-1) and actin network staining. The whole blood adhesion assays and platelets aggregation study on hemolysate-preconditioned HUVECs were performed in continuous flow.

Confocal microscopy was used to study interaction between HUVECs and hemoglobin (Hb) and RBC membrane-derived particles.

Results: Our fluidic model reproduced a hemolysate-induced pro-inflammatory phenotype and damages of HUVECs via NFkB signaling pathway in a TLR-4 independent manner. The most deleterious effects were observed in whole hemolysate conditions in which, an important quantity of RBC large-size particles (> 2 μm) mostly positive for C3 was detected. These effects on HUVECs were significantly reduced after their elimination by 14000 g centrifugation (Figure 1). Noteworthy, the suspension of ghosts induced a similar activation on HUVECs compared to whole hemolysate suggesting the major role of RBC membrane-derived particles in early hemolysis-induced endothelial damages.

Confocal microscopy for intracellular compartment of hemolysate-treated HUVECs demonstrated that while the CD235a+ Hb+ particles were only found in whole hemolysate conditions, the Hb was detected similarly in all conditions tested. Selective blocking of RBCs particles and Hb endocytosis did not affect hemolysate-induced HUVECs activation. These observations demonstrate that in the very early stage (≤4H) of intravascular hemolysis, erythrocytes large-size particles can be internalized in HUVECs, but their internalization is not necessary for endothelial activation effect.

Complement inactivation of serum or treatment of RBC particles by either Annexin V or Eculizumab could reduced their effects on endothelial activation suggesting a complement-dependent mechanism

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Summary/Conclusion: This study suggested for the first time the major role of RBCs large-size particles on endothelial damages at the onset of DHTR in a complement-dependent manner justifying the complement blocking treatment in certain DHTR patients. Furthermore, we have also demonstrated the endocytosis of free Hb by HUVECs, but its pathological impact is still unclear. This endocytosis would facilitate the Hb clearance during a massive hemolysis? These new understandings will allow a better and earlier management to preserve endothelial functions.

S300: HEALTH-RELATED QUALITY OF LIFE IN TRANSPLANT-INELIGIBLE REAL-LIFE MULTIPLE MYELOMA PATIENTS TREATED WITH BORTEZOMIB-MELPHALAN-PREDNISONE (VMP) VS. LENALIDOMIDE-DEXAMETHASONE (RD)

M. D’Agostino 1,* , S. Bringhen 1 , R. Ria 1 , F. Ciceri 1 , A. P. Falcone 1 , M. Michieli 1 , M. Grasso 1 , F. Pane 1 , M. Quaresima 1 , F. Cattel 2 , M. Mirabile 1 , F. Fioritoni 1 , M. T. Petrucci 1 , V. Cotugno 2 , A. Capra 1 , S. Pezzatti 1 , M. L. Mosca Siez 1 , M. Cantonetti 1 , G. Margiotta Casaluci 1 , P. Bertazzoni 1 , R. Floris 1 , M. Offidani 1 , G. Pietrantuono 1 , A. Evangelista 3 , M. Boccadoro 1 , A. Larocca 1

1 European Myeloma Network, (EMN), Italy; 2 S.C. Farmacia Ospedaliera, A.O.U. Città della Salute e della Scienza di Torino, Torino, Italy; 3 Unit of Clinical Epidemiology, A.O.U. Città della Salute e della Scienza di Torino and CPO Piemonte, Torino, Italy

Background: Multiple Myeloma (MM) is a chronic disease, and patients (pts) receive long-lasting treatment. Thus, the impact on health-related quality of life (HRQoL) may be burdensome, especially in elderly pts. Bortezomib-melphalan-prednisone (VMP) and lenalidomide-dexamethasone (Rd) represented standard-of-care treatments for transplant-ineligible (NTE) newly diagnosed (ND)MM pts before the introduction of daratumumab upfront. No prospective data are available comparing HRQoL in pts receiving VMP vs Rd in a randomized fashion.

Aims: We conducted an analysis of Patient Reported Outcomes (PROs) in the context of a randomized multicenter phase IV trial (Real MM Trial, NCT03829371 ; funded by the Italian Medicines Agency AIFA - Independent Research), to compare HRQoL differences associated with VMP vs Rd treatment in an unselected real-life MM population.

Methods: NTE NDMM pts were randomized to receive 9 VMP cycles vs continuous Rd according to standard practice. Pts gave written informed consent and were enrolled regardless of performance status, comorbidities, renal function, or baseline laboratory values. PROs were collected and analyzed using the validated EORTC QLQ-C30 scales and the EQ-5D-5L visual analog scale (VAS) instruments. PROs were collected at baseline, every 3 months during the first year, and every 6 months thereafter. The PROs analyses included pts from the interim analysis (median follow-up: 14.0 months) who had at least a baseline and a follow-up questionnaire available. Data through month 12 are reported. Change in HRQoL from baseline in VMP vs Rd pts was analyzed in a linear mixed model adjusted for International Myeloma Working Group (IMWG) frailty score and cytogenetic risk. Results are presented as least squares (LS) mean change from baseline.

Results: At the data cut-off (17-12-2021), 104 pts (56 in the VMP arm and 48 in the Rd arm) had available PROs and were eligible for the analysis. Overall, 46% of pts had >75 years and 40% were frail. No differences in terms of baseline characteristics and response rates were found in the VMP vs Rd arms. Mean baseline values of PROs reflected the deep impairment of pts’ HRQoL at MM diagnosis and were similar in the two arms. After the start of treatment, the different QLQ-C30 scales were analyzed. Global Health Status (GHS) was significantly worse with VMP vs Rd at 3 months (-3.3 vs +9.0; P =0.002), while from the 6-month time point onwards no differences can be found due to an improvement in GHS in the VMP arm ( Figure ). The physical functioning scale was worse in the VMP vs Rd arm at 3 (-7.8 vs +0.04; P =0.070) and 6 months (-8.2 vs +3.2; P =0.013), with no differences in later time points. The role functioning scale behaved similarly (-5.9 vs +5.2 at 3 months with VMP vs Rd; P =0.038). VMP also increased fatigue (+9.4 vs -2.7; P =0.015), nausea (+8.6 vs +2.8; P =0.04), and appetite loss (+12.0 vs -3.0; P =0.007) at 3 months, with an improvement in the symptoms thereafter. In both arms, pain decreased after treatment. No clear differences in the other QLQ-C30 scales were observed. The EQ-5D-5L VAS scale showed a significantly worse score in the VMP vs Rd arm at 3 months (-7.9 vs +1.7; P =0.005) and at 12 months (-0.6 vs +10.3; P =0.017).

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Summary/Conclusion: A comparison of PROs in real-life NTE NDMM pts treated with VMP vs Rd showed a worse HRQoL in the VMP arm early in the treatment course that improved thereafter. At 3 months, after treatment start, VMP was associated with worse GHS, physical functioning, role functioning, fatigue, nausea, appetite loss, and EQ-5D-5L VAS scores.

S301: GERMAN AMLCG-SURVIVORSHIP STUDY: QUALITY OF LIFE AND LIFE SATISFACTION IN AML LONG-TERM SURVIVORS

E. Telzerow 1,* , D. Görlich 2 , C. Sauerland 2 , A. S. Moret 1 , M. Rothenberg-Thurley 1 , F. H. A. Mumm 1 , S. Amler 2 3 , W. E. Berdel 4 , B. Wörmann 5 , U. Krug 6 , J. Braess 7 , P. Heussner 8 , W. Hiddemann 1 , K. Spiekermann 1 , K. H. Metzeler 9

1 Department of Medicine III and Comprehensive Cancer Center (CCC Munich LMU), University Hospital, LMU Munich, Munich; 2 Institute of Biostatistics and Clinical Research, University of Münster, Münster; 3 Current adress: Friedrich Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald; 4 Department of Medicine A, Hematology and Oncology, University of Münster, Münster; 5 Charité University Hospital Berlin, Berlin; 6 Department of Medicine; 3 , Hospital Leverkusen, Leverkusen; 7 Department of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg; 8 Departement of Internal Medicine, Hospital Garmisch-Partenkirchen, Garmisch-Partenkirchen; 9 Department of Medicine; 1 , Hematology and Cell Therapy, University Hospital Leipzig, Leipzig, Germany

Background: An increasing proportion of patients with acute myeloid leukemia (AML) become long-term survivors. Somatic and psycho-social outcomes in survivors are therefore becoming increasingly important, but little is known about the long-term effects of the disease and its treatment.

Aims: The primary aim of this study was to compare quality of life (QoL, measured by the FACT-G questionnaire) and general and health-related life satisfaction (gLS/hLS, measured by the FLZ-M questionnaire) of AML-LTS with normative data of German adults who were not diagnosed with AML.

Methods: We designed a comprehensive analysis of AML survivorship outcomes including psycho-social well-being and somatic health status and conducted a questionnaire-based study collecting data from AML long term survivors (AML-LTS). This report focuses on overall and health-related quality of life. Somatic morbidity in AML-LTS is reported separately (Moret et al.).

Results: 427 former AML patients who had been enrolled in AMLCG trials (AMLCG-1999, AMLCG-2004, AMLCG-2008) or the AMLCG patient registry, participated in this study between 5 and 18.6 years (y) after their initial AML diagnosis (median, 11.3y). Median age of AML-LTS was 61y (range 28y-93y), and 56% were female. Thirty-eight percent of participants had been treated with chemotherapy alone, while 62% received at least one allogeneic stem cell transplant (alloHSCT). A relapse occurred in 24% of the participants.

Unexpectedly, age- and sex-normalized quality of life and general life satisfaction summary scores were significantly higher in AML-LTS (p<.001) compared to adults without the diagnosis of AML. Raw score points of AML-LTS on the FACT-G summary scale also were higher than in age and sex-matched normal adults by a median of 4.7 points (95% CI: 2.82 – 7.2) – a differences that likely is clinically not relevant, considering an established cutoff for clinical relevance of 7 raw score points. No difference between AML-LTS and normal adults was found for health-related life satisfaction (hLS).

Using the cutoff for clinical importance (i.e., 7 points below age- and sex-matched population norm), 26.1% of participants reported relevant impairment of overall QoL. To identify factors potentially associated with poor overall QoL, we constructed a logistic regression model including pre-specified cofactors (age, sex, time since initial diagnosis, relapse and alloHSCT) and additional covariables that associated with QoL in univariate analyses (Figure 1). We found that participants with no children, lower educational level, shorter time since diagnosis and altered financial situation reported significantly lower QoL. No influence was found for disease- and treatment related factors including treatment (alloHSCT vs. no alloHSCT), previous relapse, or de novo vs secondary or therapy-related AML.

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Summary/Conclusion: Unlike previous studies of AML survivorship, our large cohort included a diverse spectrum of patients regarding age, time since diagnosis, and treatment modalities, which allows for new insight into long-term QoL. Our study establishes that overall QoL in AML long-term survivors is comparable to the general population, with further improvement from five years post diagnosis onwards. Importantly, disease- and treatment-related factors, such as prior relapse or alloHSCT, are not associated with overall QoL. However, we were able to identify risk factors for worse QoL, delineating a subgroup of patients that may still have a need for targeted psycho-social interventions five or more years after an AML diagnosis.

S302: COST-EFFECTIVENESS OF KTE-X19 IN PATIENTS WITH RELAPSED/REFRACTORY MANTLE CELL LYMPHOMA

M. Marchetti 1,* , C. Visco 2 3

1 Hematology & TMO Unit, Azienda Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Alessandria; 2 University of Verona; 3 Section of Hematology, Department of Medicine, Azienda Ospedaliera Universitaria Integrata, Verona, Italy

Background: Relapsed/refractory (R/R) mantle-cell lymphoma (MCL) post BTK-i has a poor prognosis when treated with currently available treatments with median overall survival between 6 to 15 months.

In the ZUMA-2 study, KTE-X19, a chimeric antigen receptor (CAR) T-cell therapy, displayed very high response rate (93% overall objective response rate) with 83% of patients in the study alive at 12 months (Wang et al 2020). An indirect comparison of overall survival from ZUMA-2 with patients receiving standard of care (SOC) treatment reported propensity-adjusted HR 0.32-0.49 (Hess et al 2021). Based on such data, international scientific societies (NCCN, ESMO) recommend the use of CAR-T for R/R MCL patients and marketing authorization of KTE-X19 was granted by FDA and EMA.

Aims: The present study aimed to estimate the long-term clinical benefits and overall healthcare costs of KTE-X19 in R/R MCL patients post BTK-i from the perspective of the Italian HealthCare System, compared to current standard therapy for these patients in Italy: rituximab-bendamustine-cytarabine (R-BAC).

Methods: A partitioned-survival model was used to extrapolate overall survival, progression-free survival, quality-adjusted survival and healthcare costs of R/R MCL patients over a lifetime horizon, assuming that patients who had not relapsed within 5 years experienced long-term remission. R-BAC (6 cycles) was used as baseline SOC, however a mix of treatments was tested as sensitivity analysis. The source of safety and survival data for KTE-X19 was the ZUMA-2 trial (median potential follow up 28.8 mo), while data from retrospective real-world studies, including SCHOLAR-2, were used for SOC.

The healthcare costs for patients assigned to the CAR-T strategy included bridging chemotherapy (36.8% of the patients), hospitalization (median length 21.2 days, of which 23% in intensive care), and the price of KTE-X19. The healthcare costs of patients assigned to SOC included in addition to R-BAC treatment costs, the cost for allogeneic hematopoietic stem cell transplant (€179,418) for 31% of patients (McCullogh 2020). Life years (LYs), quality-adjusted life years (QALYs) and costs were estimated by discounting future outcomes (3% per year). Uncertainty analysis included one-way and probabilistic sensitivity

Results: Over a lifetime horizon, mean estimated survival was 8.85 years for KTE-X19 and 1.56 years for R-BAC, while discounted QALYs were 6.40 for KTE-X19 versus 1.20 for R-BAC. Discounted lifetime costs were €411,403 for KTE-X19 versus €74,415 for R-BAC, which corresponds to a cost of €64,798 per QALY gained and €46,264 per LY gained. Detailed results are reported in the table below. The ICERs for KTE-X19 versus SCHOLAR-2 based and meta-analysis based comparisons were €57,915, and €56,010, respectively.

The most influential model parameters were patients age and quality of life values, KTE-X19 acquisition cost, and the proportion of patients receiving allo-SCT in the comparator arm, and model time horizon.

Probabilistic sensitivity analysis showed that at a willingness-to-pay of €70,000 per QALY, the probability of KTE-X19 to be cost-effective was 65%.

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Summary/Conclusion: This analysis shows that KTE-X19 may be a cost-effective alternative to R-BAC for patients with RR-MCL pre-exposed to BTK-i and chemoimmunotherapy, from an Italian third-party payer perspective

S303: EFFECT OF PEGCETACOPLAN ON QUALITY OF LIFE IN COMPLEMENT-INHIBITOR NAÏVE PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: RESULTS FROM THE PHASE 3 PRINCE STUDY

D. Gomez-Almaguer 1,* , R. Wong 2 , T. Dumagay 3 , M. Al-Adhami 4 , J. Savage 4 , Z. Hakimi 5 , A. Bogdanovic 6

1 Hematology Service, Hospital Universitario “Dr José Eleuterio González,” Universidad Autónoma de Nuevo León, Monterrey, Mexico; 2 Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 3 Department of Cellular Therapeutics, Makati Medical Centre, Makati, Philippines; 4 Apellis Pharmaceuticals, Inc., Waltham, United States of America; 5 Swedish Orphan Biovitrum AB, Stockholm, Sweden; 6 Clinic of Hematology, Clinical Center of Serbia, Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening disease characterized by complement-mediated hemolysis and thrombosis. Historically, PNH was treated with C5 inhibitors (eculizumab [ECU)/ravulizumab). Despite their proven efficacy in the control of intravascular hemolysis (IVH), up to 72% of patients experience persistent hemolysis leading to suboptimal hemoglobin (Hb) levels and 36% require transfusions despite ECU treatment which results in a significant impact on the quality of life (QoL), including persistent fatigue. Pegcetacoplan (PEG) is an FDA/EMA-approved C3 complement-inhibitor that provides broad hemolysis control (including IVH and extravascular hemolysis) in patients with PNH.

Aims: This analysis evaluates QoL measures in the Phase 3 PRINCE study ( NCT04085601 ), a multicenter, randomized, open-label, controlled study evaluating the efficacy and safety of PEG compared to control treatment (CTRL; excluding complement-inhibitors) in complement-inhibitor naïve patients with PNH.

Methods: PRINCE has been described previously and was superior for the coprimary endpoints of hemoglobin (Hb) stabilization (avoidance of >1 g/dL decrease in Hb from baseline), change from baseline in lactate dehydrogenase at 26 weeks and PEG-treated patients saw clinically meaningful increases in the FACIT-Fatigue scores (Wong SR, et al. Blood 2021; 138 [Supplement 1]: 606). Secondary endpoints specific to QoL measures included the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 Scale (EORTC QLQ-C30), Linear Analog Scale Assessment (LASA) and The Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue). The EORTC QLQ-C30 contains 30 questions comprising 5 functional, 9 individual symptoms, and one global health status/QoL item(s), where a ≥10-point change in score is considered clinically meaningful. The LASA consists of 3 sections asking to rate the perceived level of functioning (scale 0-100, where a higher score corresponds to a better QoL) and contains specific domains for activity level, ability to carry out daily activities, and overall QoL. The FACIT-Fatigue is a 13-item Likert scaled instrument (scale 0-51), where a ≥3-point increase is considered clinically meaningful.

Results: Relevant baseline characteristics for PRINCE are reported in Table A. Mean baseline scores of the EORTC QLQ-C30 functional and symptom scales were generally similar between groups (except for role and cognitive functioning and insomnia; Table B). After 26 weeks of PEG-treatment, patients displayed clinically meaningful improvements in most of the functional scales and the global health status/QoL at Week 26 as indicated by a ≥10-point score increase from baseline (Table B). Improvements in the symptom scales, including fatigue and dyspnea scales, were also observed in the PEG group at Week 26 as indicated by a reduction in the symptom score close to the general population norms (general population norm: fatigue: 24.1, dyspnea: 10.9). Mean total LASA scores increased in the patients receiving PEG treatment for 26 weeks (Table B). FACIT-Fatigue scores increased for PEG-treated patients to near the general population norm [43.6], supporting the gains shown in the EORTC QLQ-C30-Fatigue scale.

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Summary/Conclusion: Substantial and clinically relevant improvements in QoL were consistently observed with PEG across both EORTC QLQ-C30, total LASA and FACIT-Fatigue scores and clinically significant improvements in fatigue and dyspnea are highly relevant to the symptomology of PNH.

S304: SUTIMLIMAB, A COMPLEMENT C1S INHIBITOR, PROVIDES SUSTAINED IMPROVEMENTS IN PATIENT-REPORTED OUTCOMES IN PATIENTS WITH COLD AGGLUTININ DISEASE (CAD): 2 YEAR FOLLOW-UP FROM THE CARDINAL STUDY

A. Röth 1,* , C. M. Broome 2 , W. Barcellini 3 , T. Henrik Anderson Tvedt 4 , Y. Miyakawa 5 , S. D’Sa 6 , D. Cella 7 , F. Joly 8 , J. Wang 9 , T. Sourdille 9 , F. Shafer 10 , M. Wardęcki 11 , I. C. Weitz 12

1 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2 Division of Hematology, MedStar Georgetown University Hospital, Washington, DC, United States of America; 3 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 4 Section for Hematology, Department of Medicine, Haukeland University Hospital, Bergen, Norway; 5 Thrombosis and Hemostasis Center, Saitama Medical University Hospital, Saitama, Japan; 6 UCLH Centre for Waldenström’s Macroglobulinemia and Related Conditions, University College London Hospitals NHS Foundation Trust, London, United Kingdom; 7 Department of Medical Social Sciences, Center for Patient-Centered Outcomes, Institute for Public Health and Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States of America; 8 Sanofi, Chilly-Mazarin, France; 9 Sanofi, Cambridge, MA; 10 Sanofi, Bridgewater, NJ, United States of America; 11 Sanofi, Warsaw, Poland; 12 Keck School of Medicine of USC, Los Angeles, CA, United States of America

Background: CAD is a rare chronic autoimmune hemolytic anemia characterized by classical complement pathway (CP)-mediated hemolysis, anemia, fatigue, and poor quality of life (QOL). Sutimlimab is a first-in-class humanized monoclonal antibody that selectively inhibits C1s of the C1 complex, preventing CP activation, while leaving the alternative and lectin pathways intact. One-year interim follow-up from the CARDINAL study ( NCT03347396 ) have previously demonstrated continuous classical CP inhibition with sutimlimab resulted in rapid, sustained improvements in all patient-reported outcomes (PROs) measures evaluated.

Aims: To report sutimlimab effect on PROs at 2 years, from the CARDINAL Part B extension.

Methods: CARDINAL was a Phase 3, open-label, single-arm study with a 26-week treatment period (Part A) and a 2-year extension (Part B) after the last patient finishes Part A. Sutimlimab was administered through intravenous infusions on Days 0 and 7, followed by biweekly dosing. PRO data through Week 135, the last data recording within the 2-year Part B time period, are reported here. Efficacy endpoints included hemolytic markers and the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) as a measure of QOL. Exploratory QOL endpoints included mean change from baseline in EuroQol 5-dimension 5-level (EQ-5D-5L) scores, 12-Item Short Form Health Survey (SF-12), Patient Global Impression of Change (PGIC) and Patient Global Impression of Fatigue Severity (PGIS). PRO measures were evaluated every 3 months and conducted in the following order: FACIT-Fatigue, PGIS, PGIC, SF-12 and EQ-5D-5L. Descriptive statistics, frequency, or percentage were used to analyze outcomes.

Results: Overall, 24 patients enrolled and 22 finished Part A and entered Part B, with 19 (86.4%) patients completing Part B. The mean (SD) FACIT-Fatigue score at baseline was 32 (11) and improved by 7 (8) within 1 week of sutimlimab treatment; the mean change score from baseline remained ≥5 from Week 1 to Week 135 (minimum score of 38 (9) at Week 123, maximum score of 44 (5) at Week 7), consistent with a clinically meaningful improvement. Efficacy for the EQ-5D-5L visual analogue scale (VAS) was sustained over 2 years; the mean (SD) change from baseline in EQ-5D-5L visual analogue scale (VAS) score (n=15/22) at Week 135 was 17.1 (21.6) (Figure A). The majority of evaluable patients (n=13/15) indicated an improved disease state compared to baseline on the PGIC at Week 135. No or mild fatigue was reported in patients (80%, n=12/15) on completing PGIS at Week 135, compared with one-third at baseline (n=2/6). The mean increase in SF-12 physical and mental component scores (n=6/22) from baseline to Week 123 were 4.7 (6.9) and 3.8 (14.1) (Figure B), consistent with the clinically important changes of 3.9 and 2.8 respectively.

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Summary/Conclusion: From this CARDINAL follow-up extension study, sutimlimab has shown to produce rapid and sustained improvements in FACIT-Fatigue and other PRO measures evaluated up to 2 years, demonstrating the continued meaningful impact of sutimlimab on patient QOL.

Posters

P305: COMPREHENSIVE TRANSCRIPTIONAL AND CYTOGENETIC PROFILING IMPROVES CLASSIFICATION AND DETECTION OF RISK-STRATIFYING MARKERS IN THE B-OTHER PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA

Ž. Antić 1,* , P. Chouvarine 1 , J. Lentes 1 , C. Schröder 1 , J. Alten 2 , A. Möricke 2 , M. Brüggemann 3 , E. Carrillo-de Santa Pau 4 , T. Illig 1 , T. Laguna 4 , D. M. Schewe 2,5 , M. Stanulla 6 , M. Tang 1 , M. Zimmermann 6 , M. Schrappe 2 , B. Schlegelberger 1 , G. Cario 2 , A. K. Bergmann 1

1 Institute of Human Genetics, Hannover Medical School, Hannover; 2 Berlin-Frankfurt-Münster ALL Study Group Germany (BFM-G), Department of Pediatrics; 3 Department of Hematology, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Germany; 4 Computational Biology Group, Precision Nutrition and Cancer Research Program, IMDEA Food Institute, Madrid, Spain; 5 University Children’s Hospital, Medical Faculty, Otto-von-Guericke-University-Magdeburg, Magdeburg; 6 Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy. Improvements in the genetic-based risk stratification and treatment adaptations have resulted in an increased overall survival, reaching 90% in the contemporary treatment protocols. However, in 25-30% of patients, known as B-other, no recurrent genetic aberrations relevant for the risk stratification and treatment personalization can be detected using conventional cytogenetic methods. Therefore, identification and implementation of new risk-stratifying markers in the current treatment protocols may aid further improvement in the management of children with BCP-ALL.

Aims: Our aim was to investigate the applicability of the integrated use of the whole transcriptome RNA sequencing and conventional cytogenetic methods in the routine diagnostic of the B-other BCP-ALL cases.

Methods: We performed RNA sequencing and analyzed gene fusions, expression profiles, and mutations in diagnostic samples of 174 children with B-other ALL. In order to further refine genetic classification and validate findings obtained with RNA sequencing, we integrated our results with the findings obtained using immunophenotyping, FISH, karyotyping, arrayCGH and Sanger sequencing.

Results: Our analysis unraveled the presence of risk-stratifying fusion transcripts, in the cases in which these alterations were not detectable using conventional cytogenetic methods. These included six cases with cryptic KMT2A rearrangements and one of each with TCF3-PBX1 and TCF3-HLF fusion genes. In addition, we were able to detect 10 cases with recently described fusions involving ZNF384 gene ( ZNF384 r), four cases with NUTM1 rearrangements and three cases with fusions involving MEF2D gene. Gene expression-based clustering unraveled a subset of B-other cases which cluster together with the known subtypes, indicating the presence of previously described ZNF384 r-like, ETV6-RUNX1 -like and BCR-ABL1 -like subtypes. Furthermore, we identified 27 previously unassigned B-other cases co-clustering together with 13 DUX4 -positive ALL cases. This finding suggests that gene expression-based clustering can identify the cases with fusions involving DUX4 gene, known to be cryptic to most of cytogenetic and NGS approaches.

We further assessed the ability of the analysis pipeline to detect fusion transcripts in the samples with <50% of tumor blasts. In five tested cases, with BCR-ABL1 or ETV6-RUNX1 fusions, our analysis pipeline was able to detect the presence of fusion transcripts, with high reliability, even in the samples with down to 7% of tumor blasts. Finally, we assessed the applicability of using commonly available EDTA tubes and RNA stabilizing PAXgene tubes for bone marrow sampling, RNA isolation and whole transcriptome sequencing. We assessed the quality of isolated RNA, library complexity and ability of our analysis pipeline to detect relevant fusion transcripts. PCA analysis of matched samples stored in either EDTA or PAXgene tubes showed high similarity in gene expression, while the correlation between TPM expression values and decay constants indicates that genes over-abundant in EDTA samples are not dominated by slow-degrading transcripts. These findings suggest that for short-term storage EDTA tubes are a viable alternative to the RNA stabilizing PAXgene tubes.

Summary/Conclusion: Taken together, our findings demonstrate the applicability of whole transcriptome sequencing for personalized diagnostics in pediatric ALL, including the tentative classification of the B-other cases that are difficult to diagnose using conventional methods.

P306: PAX5 DEFICIENCY AND GERMLINE SUSCEPTIBILITY TO PEDIATRIC LEUKEMIA

F. Auer 1,* , A. Escudero 2 , M. Sipola 3 , A. Viitasalo 3 , M. Morcos 1 , U. A. Friedrich 4 , A. Pandyra 5 , A. Borkhardt 5 , M. Takagi 6 , J. Hauer 1

1 Department of Pediatrics, Technical University Munich, Munich, Germany; 2 Institute of Medical and Molecular Genetics (INGEMM) La Paz University Hospital, Madrid, Spain; 3 Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland; 4 Pediatric Hematology and Oncology, Department of Pediatrics, University Hospital “Carl Gustav Carus”, TU Dresden, Dresden; 5 Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine University Duesseldorf, Medical Faculty, Duesseldorf, Germany; 6 Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

Background: Somatic mutations affecting the PAX5 gene are common in pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, germline PAX5 mutations are extremely rare. While it is known that PAX5 acts as an indispensable master regulator of proliferation and differentiation in B-cells, the pre-leukemic setting mediated by reduced PAX5 levels is still poorly understood.

Aims: To investigate the genetic and immunological effect of inherited PAX5 germline mutations in humans and to characterize the effect of Pax5 heterozygosity on B-cell development utilizing single-cell RNA Sequencing in mice.

Methods: Aiming to understand the etiology of childhood BCP-ALL in the setting of reduced Pax5 levels, we used 9-color FACS staining and scRNA/bulk RNA-Sequencing, as well as whole exome Sequencing technology.

Results: Here, we describe two new families with inherited PAX5 germline variants affecting amino acid 183 and B-ALL development, including one family with a novel amino acid substitution p.Gly183Arg. Exome analysis of both families together with one family harboring PAX5 p.Gly183Ser previously reported by Auer et al. , ( Leukemia, 2014 ) yielded no additional common germline variants that could explain the incomplete penetrance in the observed leukemia development. A study of the respective B-cell differentiation comparing PBMCs from unrelated non-carriers, healthy carriers, and patients revealed a significant decrease of intermittent B-cells and memory B-cells in carriers and patients compared to non-carriers (Student’s T-test; p<0.05 and p<0.01, respectively). Finally, immunological studies performed using bone marrow samples from a patient and an unrelated control showed a reduction in the number of mature B cells, suggesting a block of the B-cell differentiation at the pre-B stage in the patient.

In line, a comprehensive flow cytometry analysis of the B-cell development in the bone marrow of healthy Pax5 heterozygous ( Pax5 +/- ) mice, showed a robust and age independent enrichment of the pre-BII population (B220 + CD19 + IgM - CD25 + ) compared to their Wildtype (WT) littermates (Student’s t-test; p=0.003). Bulk RNA-Sequencing of sorted preB-II cells (3 WT vs. 3 Pax5 +/- mice) revealed deregulations in major B-cell receptor (BCR) signaling components including downregulation of Cd79a/b, Lyn, MTor and various ITIMs. Subsequent scRNA-Sequencing (4 WT vs. 4 Pax5 +/- mice, pool of 20,000 cells hashtag labeled, with 50,000 reads/cell and VDJ-rearrangement analysis) confirmed a delayed BCR assembly and showed a skewing of light chain rearrangements towards the lambda isotype in Pax5 +/- mice.

Summary/Conclusion: Overall, our data provide evidence for B-cell dysregulations mediated by reduced Pax5 levels in the germline of mice and men. Ultimately these characterizations will help to advance the understanding of molecular mechanisms and susceptible populations associated with B-ALL. In turn, these findings are important to address the far more frequent somatic PAX5 mutations with the goal to prevent or treat a significant proportion of childhood leukemias.

P307: DEL(17)(Q11) IS TYPICAL MARKER OF IMMATURE T-ALL OF ADULTS, WITH NF1, UTP6, AND SUZ12 HAPLOINSUFFICIENCY, GENOME INSTABILITY, AND GENE DOWNREGULATION

V. Bardelli 1,* , V. Pierini 1 , S. Arniani 1 , E. Mvridou 1 , C. Matteucci 1 , A. G. Lema Fernandez 1 , M. Moretti 1 , L. Elia 2 , F. Giglio 3 , F. Forghieri 4 , M. Cerrano 5 , N. Fracchiolla 6 , M. Delia 7 , S. Sica 8 , C. Mecucci 1 , R. La Starza 1

1 Hematology and Bone Marrow Transplantation Unit, Department of Medicine and Surgery, University of Perugia, Perugia; 2 Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome; 3 Hematology and Bone Marrow Transplantation Unit, IRCCS Ospedale San Raffaele, Milan; 4 Section of Hematology, Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, Modena; 5 Department of Molecular Biotechnology and Health Sciences, Division of Hematology, University of Turin, Turin; 6 Hematology, Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan; 7 Hematology and Stem Cell Transplantation Unit, AOUC Policlinico of Bari, Bari; 8 Section of Hematology, Department of Radiological and Hematological Sciences, Catholic University of the Sacred Heart, Rome, Italy

Background: T-ALLs derive from the accumulation of multiple genetic and epigenetic events. Some deregulate T-cell transcription factors and define specific genetic subtypes, while the large majority are involved in pivotal cellular processes, such as signaling, cell cycle, apoptosis, proliferation, ribosome biogenesis, and epigenetic modulation. Genomic imbalances, and in particular deletions, are the most frequent cytogenetic rearrangements in T-ALL. Whether cryptic or large, they often share loss regions - the common deletion region (CDR) - where they map putative suppressor genes that undergo haploinsufficiency or inactivation, thus driving the leukemogenic process.

Aims: Our study focused on T-ALL cases harboring interstitial del (17q) with the aim of assessing the incidence and distribution of this cytogenetic marker, and to detect associated clinical and molecular features.

Methods: The study was carried out on 378 adult (=223) and pediatric (=155) T-ALL, including 279 males and 99 females. The genetic background was investigated by integrated molecular-cytogenetics (FISH and SNPa), 1 RNA microarrays (Affymetrix), targeted sequencing (custom card by Sophia Genetics, Arrow Diagnostics), and RTq-PCR. Data analysis was carried out referring to available databases (UCSC, NCBI, Data of Genomic Variants, Biocarta, KEGG, Reactome).

Results: Cases were classified according to genetic rearrangements as HOXA (=104), TAL/LMO (=74), TLX1/NKX2.1 (=50), TLX3 (=42), BCL11B -a (=8), SPI.1 (=2); 98 were undetermined. Interstitial monoallelic del(17q) was detected 31/378 cases and was mainly found in cases with HOXA related abnormalities (8/104) or cases undetermined (17/98). There were 27 adults and 4 children (p<0.0001). In 19/24 with available flow cytometry the diagnosis was consistent with ETP/near ETP ALL (p<0.001).

All cases shared a common deleted region (CDR) of about 650kb, that invariably involved SUZ12 , UTP6 , and NF1 which were expressed at a significantly lower level than in T-ALL without del(17q). RNA microarray showed that 148 differentially expressed genes (DEG), 67 up- and 81 down- regulated, distinguished del(17q) T-ALL cases from T-ALL without. Among up-regulated there were MEF2C, MN1, and IGFBP7 , which have been all associated with an immature phenotype, and MAF and RUNX2 transcription factors. Instead, among down-regulated, there were putative suppressors involved in genome stability, i.e. BRCA1, FANCI, BRIP1, XRCC2, and CHEK1 . Accordingly, cell cycle, homologous recombination, DNA replication, and Fanconi anemia pathways, were significantly deregulated in T-ALL with del(17q), and a significant association with deletions of RB1 (37%)(p<0.001) and TP53 (20%)(p=0.017) emerged. In addition, del(17q) cases had a higher number (≥5) of copy number abnormalities (p<0.001). Other recurrent alterations affected JAK/STAT members and/or modulators (56% of cases).

Summary/Conclusion: Del(17q) was a recurrent marker in immature T-ALL of adults and characterized 19% of cases undetermined at molecular-cytogenetic level. It caused the monoallelic loss/haploinsufficiency of three genes, i.e. NF1 , UTP6 , and SUZ12, that restricted the CDR. Our study provided evidence that del(17q) identified a major leukemogenic mechanism, through the cooperation of: 17q genes loss of function, JAK/STAT constitutive activation, and altered cell cycle. Interestingly, RUNX2 and IGFBP7 , high-risk markers in T-ALL,2,3 were aberrantly expressed in this subset of T-ALL.

1 La Starza R, et al. J Mol Diagn 2020

2 Matthijssens F, et al. JCI 2021

3 Bartram I, et. al., BMC Cancer 2015

P308: NOVEL COPY NUMBER ABERRATION-BASED CLASSIFICATION METHODS REFINE RISK ASSESSMENT IN PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

G. Bedics 1,* , B. Egyed 2 , L. Kotmayer 1 , A. Benard-Slagter 3 , K. de Groot 3 , A. Bekő 1 , L. L. Hegyi 1 , S. Krizsán 1 , G. Kriván 4 , D. J. Erdélyi 2 , G. Kovács 2 , B. Kajtár 5 , L. Pajor 5 , Á. Vojcek 6 , G. Ottóffy 6 , A. Ujfalusi 7 , C. Kiss 8 , I. Szegedi 8 , K. Bartyik 9 , G. Péter 10 , E. Sebestyén 1 , Z. Jakab 11 , A. Matolcsy 1 , S. Savola 3 , C. Bödör 1 , D. Alpár 1

1 HCEMM-SU Molecular Oncohematology Research Group, Department of Pathology and Experimental Cancer Research; 2 2nd Department of Pediatrics, Semmelweis University, Budapest, Hungary; 3 Department of Oncogenetics, MRC Holland, Amsterdam, Netherlands; 4 Central Hospital of Southern Pest - National Institute of Hematology and Infectious Diseases, Budapest; 5 Department of Pathology; 6 Department of Pediatrics, University of Pécs Clinical Centre, Pécs; 7 Department of Laboratory Medicine; 8 Department of Pediatric Hematology-Oncology, Institute of Pediatrics, Faculty of Medicine, University of Debrecen, Debrecen; 9 Department of Paediatrics and Paediatric Health Care Center, Faculty of Medicine, University of Szeged, Szeged; 10 Hemato-Oncology Unit, Heim Pál Children’s Hospital; 11 Hungarian Childhood Cancer Registry, Hungarian Pediatric Oncology Network, Budapest, Hungary

Background: The genomic landscape of pediatric acute lymphoblastic leukemia (ALL) is heterogeneous with distinct copy number aberrations (CNAs) being detectable in vast majority of the patients. A subset of these alterations provides prognostic and/or predictive information; therefore, various CNA-based patient classifiers were introduced in the past.

Aims: We applied a next-generation sequencing (NGS) based method to comprehensively screen for recurrent, disease-relevant CNAs in a cohort of Hungarian patients, allowing us to establish novel patient risk stratification approaches.

Methods: Diagnostic bone marrow samples from 261 children with B-ALL and 22 matching samples drawn from 21 patients at first or second relapse were investigated by digital multiplex ligation-dependent probe amplification (digitalMLPA) using the ALL-specific D007 probemix. DigitalMLPA libraries were sequenced on an NGS platform. Whole chromosome gains and losses, as well as subchromosomal CNAs were simultaneously profiled. Survival rates were estimated using the Kaplan-Meier method and compared by log-rank tests in R version 4.1.2.

Results: In total, 1,400 CNAs including numerical chromosomal aberrations and subchromosomal CNAs were detected in 93.5% of the diagnostic samples. On average, 5.36 CNAs were observed per patient with a mean of 2.45 subchromosomal alterations. Subtype-defining aberrations were identified in 36.0% of the patients with hyperdiploidy and iAMP21 detected in 32.2% and 3.8% of the cases, respectively. Numerous CNAs in disease-relevant genes responsible for cell cycle control, lymphoid development, signaling, or tumor suppression were identified. Considering all affected exons and an upstream region, 10 distinct patterns of IKZF1 deletion were observed. Unbiased co-segregation analysis revealed 15 positive and 3 negative correlations between the various CNAs, e.g. common co-occurrence of iAMP21 and CDKN2A/B deletion, and negative correlation between ETV6 and BTG1 deletions. Comparative analysis of diagnostic and matching relapse samples revealed characteristic temporal changes of copy number profiles with additional aberrations (65%), as well as both emerging and disappearing CNAs (30%) detected at relapse as compared with diagnosis. One patient did not show CNAs in the analyzed samples (5%). Prognostic subgroups determined based on cytogenetic findings were combined ( i ) with IKZF1del/IKZF1plus status and ( ii ) with CNA subgroups defined by a comprehensive digitalMLPA profiling. The combined genetic classification methods identified 3 and 4 patient subgroups, respectively, with significantly different 5-year progression-free survival rates.

Summary/Conclusion: Comprehensive and highly optimized CNA profiling with digitalMLPA revealed subtype-defining gross-chromosomal changes and additional disease-relevant subchromosomal CNAs in a large cohort of Hungarian patients treated with ALL IC BFM protocols. Comparative scrutiny of matching diagnostic and relapse samples from 21 patients unveiled two different patterns of temporal evolution. Two novel risk stratification approaches have been established by combining cytogenetic data with digitalMLPA-based copy number profiling, with one of those laying more emphasis on CNA data than seen in previously introduced classifications, and the other one combining cytogenetic data with IKAROS status for the first time. DigitalMLPA offers a fast, reliable and standardizable DNA copy number analysis which is easily implementable in the diagnostic workflow of pediatric ALL.

P309: IMAGE-BASED HIGH-CONTENT DRUG SENSITIVITY SCREENING IDENTIFIES CONVENTIONAL, EMERGING, AND POTENTIAL NOVEL THERAPEUTIC TARGETS IN HIGH-RISK ADULT ACUTE LYMPHOBLASTIC LEUKEMIA

H. Bell 1,* , M. Singh 1 , H. Blair 1 , A. Poll 1 , E. Law 1 , O. Heidenreich 2 , F. van Delft 1 , A. Moorman 1 , J. Lunec 3 , J. Irving 1

1 Wolfson Childhood Cancer Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom; 2 Prinses Maxima Centrum, Utrecht, Netherlands; 3 Paul O’Gorman Building, Newcastle University, Newcastle upon Tyne, United Kingdom

Background: Despite concerted efforts to optimize and intensify treatment approaches in adult acute lymphoblastic leukemia (ALL) more than half of patients still succumb to their disease within 5 years of diagnosis, emphasizing the limits of current therapy and the critical requirement for more effective and safer therapeutics. Patient-derived xenograft (PDX) ALL models represent reliably predictive preclinical models and allow in vivo amplification of patient material in quantities sufficient for high-throughput screening applications.

Aims: To identify novel and effective therapies for high-risk ALL using an in vitro co-culture model of PDX ALL blasts and mesenchymal stem cells (MSCs) by high-content screening of >1200 pharmacologically-active small-molecule drug compounds in various stages of clinical development.

Methods: PDX leukemic cells derived from seven patients with high-risk ALL, including relapsed Philadelphia-positive B-ALL, low hypodiploid B-ALL, and ABL -class T-ALL, were used for the drug screen. ALL cells were co-cultured with hTERT-immortalized MSCs to support survival of the ALL blasts during a 96 hour short-term culture with the drug library compounds (final concentration, 1mM) in 384-well format. Drug sensitivity was assessed using an automated, high-throughput, fluorescence image-based microscopy pipeline which identifies and discriminates live ALL cells and MSCs using random forest machine learning algorithms based on cellular nuclear staining.

Results: Drug sensitivity profiles revealed a wide diversity of responses to the library compounds reflected by different “hit” rates (4.5-7.4%), where “hit” is defined as compounds reducing cell survival by more than 50%. Despite this heterogeneity in response, 59 unique compounds were identified with broad anti-leukemic activity; that is, “hits” in 4/7 PDX samples. Concurrent image analysis of MSCs in the co-culture system revealed most hit compounds did not affect MSC numbers relative to vehicle-treated controls ( n =50/59), indicating a possible therapeutic window for targeting leukemic cells specifically. Several hit compounds ( n =14) were drugs or from drug classes which form part of conventional ALL treatment regimens — including anthracyclines, glucocorticoids, and nucleoside analogues — validating the screening approach for successful identification of anti-leukemic compounds. Drug classes with recently recognized and emerging preclinical anti-leukemic activity were also identified including cyclin-dependent kinase (CDK) inhibitors ( n =5), cell cycle-related kinase inhibitors ( n =6), bromodomain and extra-terminal domain (BET) inhibitors ( n =4), histone deacetylase family inhibitors ( n =3), and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors ( n =3). Nine hit compounds elicited significant reductions in cell survival consistently across the panel of PDX samples (7/7), providing some evidence towards anti-leukemic activity independent of cytogenetic background. These included pan-CDK family inhibitors alvocidib, CGP60474 , and roscovitine-derived CR8. Additionally, the selective CDK (-1, -2, -9) inhibitor AZD 5438 exhibited anti-leukemic activity in all samples ( n =6/7), bar the Philadelphia-positive sample.

Summary/Conclusion: We provide evidence that high-content drug screening in a co-culture system is an effective strategy to identify clinically-relevant novel and emerging therapeutic targets in high-risk ALL. Our analyses promote a strong rationale for exploring the targeting of CDKs as a potential pan-effective approach in high-risk adult ALL.

P310: BH3 PROFILING IDENTIFIES SELECTIVE BCL-2 DEPENDENCE OF ADULT EARLY T-CCELL PROGENITOR AND TYPICAL ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS

K. Bhatia 1 , A. Mahesh 1 , E. Olesinski 2 , J. Garcia 2 , N. Jain 3 , W. Y. Jen 4 , M. Ooi Gaik Ming 5 , A. Letai 2 , M. Konopleva 3 , S. Bhatt 1,*

1 Department of Pharmacy, National University of Singapore, Singapore, Singapore; 2 Department of Medical Oncology, Dana-Farber Cancer Institute, MA; 3 Department of Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, Texas, United States of America; 4 Department of Haematology-Oncology, National University of Singapore; 5 Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Background: T-cell acute lymphoblastic leukemia (ALL) results from malignant transformation of immature T-cells, accounting for 10-15% of childhood and 20-25% of adult ALL cases. Although long-term survival rates for standard-risk childhood T-cell ALL (T-ALL) have shown striking improvements to 90%, response outcomes in adults remain much lower. Among T-ALL, a high-risk subtype originating from clonal expansion of recently immigrated thymocytes, defined as early T-cell precursor (ETP) leukemias, has a significantly worse outcome in adults.

Aims: BH3 mimetics, small molecule antagonists of anti-apoptotic BCL-2 family proteins, have shown remarkable clinical success in the treatment of hematological malignancies. We previously showed that pediatric ETP-ALL, is dependent on BCL-2, while typical T-ALL is dependent on BCL-XL. These findings importantly led to the clinical trial of venetoclax in combination with hyperCVAD in relapsed/refractory (R/R) T-ALL patients. However, whether adult patients with T-ALL also show the selective pattern of anti-apoptotic dependence related to the differentiation stage of T-cell has not been studied.

Methods: BH3 profiling, and cell cytotoxicity assay.

Results: We investigated the survival dependencies of adult ETP-ALL and T-ALL on BCL-2 family proteins using 42 primary samples collected from 3 centers (Dana-Farber, MD Anderson, and National Cancer Institute Singapore). All 15 ETP-ALL samples showed robust mitochondrial depolarization response to the BAD peptide as compared to the HRK peptide and DMSO control using BH3 profiling assay, suggesting primary dependence on pro-survival BCL-2 protein. We next performed cell death assays by exposing ETP-ALL primary tumors to short-term treatment with venetoclax and navitoclax and found that consistent with BCL-2 dependence, adult ETP-ALL can be efficiently targeted for apoptosis via venetoclax. We then asked if there is a difference in dependencies between adult ETP-ALL and T-ALL. In ETP-ALL, cytochrome c release was caused by the BAD peptide alone, while in T-ALL, both BAD and HRK peptides induced cytochrome c release. This suggests that ETP-ALL primary tumors are primarily BCL-2 dependent, while T-ALL primary tumors are co-dependent on BCL-2 and BCL-XL. Further, this dependence endorses mechanistic efficacy of venetoclax and navitoclax, where venetoclax serves to antagonize BCL-2, and navitoclax serves to antagonize BCL-XL and to a lesser extent BCL-2, through BIM displacement.

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Summary/Conclusion: We hereby validated that BH3 profiling predicted remarkable on-target cytotoxicity of venetoclax in adult ETP-ALL and T-ALL. In addition, some T-ALL with enriched HRK signaling and BCL-XL dependence revealed that these patients can benefit from navitoclax. These findings provide pre-clinical evidence for venetoclax and navitoclax as a potentially efficacious combination therapy of BH3 mimetics for adults with T-ALL.

P311: IMBALANCES IN HOMOCYSTEINE METABOLISM AND LEUKEMIA

N. Bouayed Abdelmoula 1,*

1 Genomics of Signalopathies at the service of Medicine, Medical University of Sfax, Sfax, Tunisia

Background: The correlation between imbalances in homocysteine metabolism and leukaemia is a matter of debate. While the effects of polymorphisms of key homocysteine metabolism enzymes on homocysteinemia are confirmed, their direct causality in the genesis of leukaemias or their role as disease markers (diagnostic or prognostic) remains controversial. It has been reported in several studies that the C677T and A1298C polymorphisms of the MTHFR may play a role in the pathophysiology of acute leukaemias and various other types of signalopathies such as cancers. Homocysteine could indeed modulate the expression of certain tumour suppressor genes and induce epigenetic alterations.

Aims: In this work, the aim was to identify this correlation using a molecular study of MTHFR polymorphisms in a Tunisian cohort with acute lymphoblastic leukemia.

Methods: The molecular study involved a cohort of 41 Tunisian children with acute lymphoblastic leukemia. Genomic DNA was extracted from blood samples. Amplification of the regions of interest of the MTHFR gene at exons 4 and 7 was performed by PCR using primers designed in silico. Genotyping of the 677C>T and 1298 A>C polymorphisms was performed by PCR-RFLP of the amplicons using two restriction enzymes HinfI and MboII.

Results: MTHFR polymorphisms were detected in 39% of the children. The frequency of polymorphic alleles corresponded to 15.85% for the A1298C polymorphism and 4.87% for the C677T polymorphism. In fact, The C677T and A1298C MTHFR polymorphisms were detected in respectively 4 and 11 patients as heterozygous. Only one patient harbored homozygous A1298C MTHFR polymorphism. Only two cases were heterozygotes for both polymorphisms.

Summary/Conclusion: Imbalances in MTHFR homocysteine metabolism associated with the polymorphisms do not appear to be a risk factor for ALL in the study cohort.

P312: EMPLOYING WHOLE GENOME OPTICAL MAPPING TO CHARACTERIZE THE LANDSCAPE OF STRUCTURAL VARIANTS IN B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

D. Brandes 1,2,* , T. Brozou 1 , S. Soura 1 , A. Bergmann 3 , U. Fischer 1,4 , A. Borkhardt 1,4 , R. Wagener 1,4

1 Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Germany and University Hospital Duesseldorf; 2 Duesseldorf School of Oncology (DSO), Duesseldorf; 3 Institute of Human Genetics, Hannover Medical School (MHH), Hannover; 4 German Cancer Consortium (DKTK), partner site Essen/Duesseldorf, Duesseldorf, Germany

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent pediatric cancer. The most common subtypes are ETV6 :: RUNX1 + BCP-ALL, which harbor a reciprocal translocation between chromosome 12 and 21 leading to a juxtaposition of ETV6 to RUNX1, and high hyperdiploid (HHD) BCP-ALL harboring 51-67 chromosomes. In large-scale genomic studies, the mutational landscape of somatic single nucleotide variants and indels as well as copy number gains and losses based on array CGH have been well described in both subtypes. However, a comprehensive analysis of somatic structural variants (SVs) is still lacking which may reveal significant new insights on tumor development, progression, and treatment options in BCP-ALL.

Aims: We aimed to primarily employ the novel technology whole genome optical mapping (WGOM) to unmask the landscape of structural variants in BCP-ALL. Firstly, we determined the concordance of chromosomal abnormalities identified by WGOM in comparison to standard techniques. Superiorly, we explored the landscape of somatic aberrations in ETV6::RUNX1 + and HHD tumors to identify recurrent alterations and novel SVs affecting genes not yet related to BCP-ALL pathogenesis.

Methods: We extracted ultra-high molecular weight (UHMW) DNA of 13 ETV6::RUNX1 + and 18 HHD BCP-ALL with a tumor cell content of 25-96% at diagnosis. Additionally, we isolated DNA of fibroblasts or remission material of each pediatric patient as matching non-tumor control. Informed consent was obtained from all participants. DNA was fluorescently labeled by a sequence specific enzyme and subsequently linearized and imaged using a Saphyr instrument (Bionano Genomics). Using this approach, we generated comprehensive datasets of 31 matched tumor/non-tumor pairs, allowing the identification of somatically acquired SVs >500bp. In addition, corresponding karyotype, fluorescence in situ hybridization and SNP-array data of the leukemia samples were integrated and compared to WGOM data.

Results: We validated 95% of 233 events observed by standard techniques with WGOM. All hallmark alterations including ETV6::RUNX1 fusion and high hyperdiploidy were identified by WGOM. In addition, we detected a median of 11 (1-36) somatically acquired events in leukemic cells with ETV6::RUNX1 fusion, compared to 3 (1-10) events in HHD cases. WGOM identified recurrent somatic structural aberrations occurring in at least three BCP-ALL cases, including focal deletions affecting ETV6 (9/31), PAX5 (5/31), ARPP21 (3/31), CD200/BTLA (3/31) and RAG2 (3/31). We detected subtype specific recurrent focal deletions disrupting ATF7IP (7/13) and BTG1 (5/13) in ETV6::RUNX1 + subclones whereas deletion of the CDKN2A/B locus (3/18) was only observed in HHD BCP-ALL. Interestingly, we discovered novel recurrently deleted regions on 12q24.11 ( GPN3 , 4/31) and Xq25 ( STAG2 , 3/31) in all BCP-ALL cases as well as focal loss of 19q13.11 ( UBA2 , 3/13) specific for ETV6::RUNX1 + subclones using WGOM. Moreover, complex three-way translocations in ETV6::RUNX1 + tumors were discovered by WGOM, including t(6;8;12)(p12.3;q24.23;q24.21) that leads to the disruption of MED13L and HMGCLL1 .

Summary/Conclusion: Our study showed that WGOM detects SVs as translocations and aneuploidies in BCP-ALL as good as conventional methods with a 95% concordance. However, WGOM allows a higher resolution and more precise localization of chromosomal breakpoints compared to standard techniques. Hence, we identified novel recurrent somatically acquired SVs including a deletion on Xq25 affecting STAG2 , which might play an important role in pediatric BCP-ALL leukemogenesis.

P313: RECURRENT PATHOGENIC GERMLINE CHEK2 VARIANTS IN PEDIATRIC PATIENTS WITH HEMATOLOGICAL MALIGNANCIES

T. Brozou 1,* , R. Wagener 1 , U. Fischer 1 , A. Borkhardt 1

1 Department of Pediatric Hematology, Oncology and Clinical Immunology, Uniklinik Dusseldorf, DUSSELDORF, Germany

Background: CHEK2 is a tumor-suppressor gene encoding CHEK2 kinase that plays a crucial role in DNA repair and cell cycle arrest as part of the ATM-CHEK2-p53 pathway. Predisposing germline variants in CHEK2 are associated with breast cancer and other adult-type malignancies such as prostate, gastric and thyroid cancer. Only few studies have examined the occurrence of germline CHEK2 variants in adults with leukemia, but the association between inherited CHEK2 variants and hematological malignancies in childhood has not been studied.

Aims: Our aim is to understand the frequency and the impact of germline variants in the cancer predisposing CHEK2 gene in pediatric hematological malignancies.

Methods: We analyzed parent-child WES data of 150 children ( ≤ 18yrs) with hematological malignancies (n=107 leukemia, n=43 lymphoma) and mined the data for CHEK2 variants. We performed functional analysis of the identified pathogenic variants to analyze the effect of the alteration on protein function.

Results: We detected in 15/150 children with hematological malignancies a CHEK2 germline variant (Figure 1A). The majority of those patients had a leukemia (n=11 BCP-ALL, n=1 T-ALL, n=1 CML, n=2 Hodgkin Lymphoma). Overall, eight different CHEK2 variants including three truncating variants were detected. Three variants were recurrent (p.Ile157Thr n=5, p.Ile160Met n=3, p.Thr367Metfs*15 n=2). According to the classification of the American College of Medical Genetics and Genomics 7/8 variants were predicted to be pathogenic or likely pathogenic. For two of these variants an impaired function had been already described in the literature. To test the functional consequences of the five remaining variants, we overexpressed these, in comparison to two control CHEK2 variants, in U2OS cells. Four out of seven variants (n=1 truncating, n=1 in-frame deletion, n=2 missense) led to loss of protein expression (Figure 1B) and loss of CHEK2-Thr68 phosphorylation upon irradiation induced DNA damage. Five out of fifteen patients carried a CHEK2 variant impairing its function. Interestingly, 4/5 patients with a pathogenic variant had a BCP-ALL and 1/5 had a CML that rapidly progressed to CD10+, CD19+ lymphoid blast crisis while under imatinib therapy (400 mg/m 2 ).

Figure 1: (A) Schematic representation of the CHEK2 protein indicating the localization of the identified variants. The length of the bars correlates to the number of patients with the respective CHEK2 variant and the color of the bars to the variant type (green: missense, red: frameshift indels, orange: splicing). (B) Western blot analysis of HA-tagged CHEK2 protein variants ectopically expressed in U2OS cells. GAPDH was used as a loading control. CHEK2 variants Arg145Trp and Glu161 del were included as controls in our experiments. (C) Bar blot depicting the quantification of the CHEK2 variant protein expression analyzed by Western blotting of three independent experiments. Each experiment is represent by a dot, the bar indicates the mean of all three experiments.

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Summary/Conclusion: We identified heterozygous CHEK2 variants in the germline of 10% of children with hematological malignancies by WES-based screening of 150 cases. Expression studies revealed that two of the variants identified in our cohort were associated with reduction/loss of CHEK2 expression. We conclude that reduced CHEK2 expression/phosphorylation upon DNA damage may be an important germline-encoded host factor predisposing to the development of BCP-ALL in children.

P314: THERAPEUTICALLY TARGETING THE UNIQUE BARCODE OF MLL/AF4

R. Cameron 1,* , L. Swart 1 , M. Rasouli 1 , O. Heidenreich 1

1 Prinses Maxima Centrum, Utrecht, Netherlands

Background: Patients harbouring MLL ( KMT2A )-rearrangements with AF4 remain a high-risk subgroup in acute lymphoblastic leukaemia cases, with currently <40% event free survival in infants (<1 year). Occurring in >70% of infant cases, improved therapies are essential. The presence of the MLL/AF4 is vital for transformation and maintenance of the leukaemia, making it an ideal therapeutic target. Small interfering RNAs (siRNA) can silence disease driving genes. Fusion genes such as MLL/AF4 present with a unique sequence across the breakpoint that is only present in the cancer cells, potentially allowing for an on-target. We therefore designed an siRNA targeting the fusion site within MLL/AF4 . Since siRNA alone has been shown to have poor pharmacokinetic (PK) and pharmacodynamic (PD) properties, we aimed to improve efficacy chemically modifying siRNA and encapsulating them in lipid nanoparticles.

Aims: The aim of this study is to develop a lipid nanoparticle delivery system to encapsulate a therapeutic siRNA targeting the MLL/AF4 breakpoint and explore the impact of this in t(4;11) cell lines.

Methods: Cells expressing the MLL/AF4 fusion were treated with an MLL/AF4 (siMA6) or control (siMM) siRNA through electroporation or encapsulation by various LNP formulations. Cellular uptake and on target efficiency were investigated in tissue culture using the t(4;11)-positive cell line SEM.

Results:: Electroporations were used to optimise chemically modified siRNA for improving PK and PD of siMA6, this data suggested that >200 nM was required to achieve 60% knockdown (KD) within 24 hours. Following this we tested to reduced concentrations of siRNA to investigate if LNPs improve delivery and efficacy. Cells were given 2ug/ml of siMA6 encapsulated in either cholesterol and PEG rich LNP (LNP-chol) or sphingomyelin rich LNP (LNP-SM) and compared. Uptake of LNP-SM is slower compared to LNP-chol within the first 3 hours, 60% and 90% retrospectively. However, by 6 hours, only a 5% difference with LNP-SM achieving 90% compared to 95% in LNP-chol. Surprisingly, functional studies suggest that consequential effects are enhanced in LNP-SM. Sequentially dosed cells with LNP-chol shows a knockdown ranging from 25%-65% in both MLL/AF4 and downstream target genes when compared to the control over 9 days of sequential dosing. A 10-15% reduction in proliferation and viability was seen at day 9. When treated with LNP-SM these consequences were enhanced, KD of MLL/AF4 is reduced 40-60% and downstream genes such as PROM1 and ANGPT1 to >70% by day 9. Furthermore, proliferation of the cells is impaired, reflected in substantial decrease in viable cells by 1.5-fold and notable increase was observed in the G1 and subg1 populations of the cells cycle. Surprisingly, both LNP-chol and LNP SM exhibited a strong impact on self-renewal with a reduction of ≥50% and ≥60% retrospectively by day 9.

Summary/Conclusion: This study has demonstrated the advantage of targeting fusion genes with a therapeutic siRNA. We further prove the impact of using an LNP delivery system to improve therapeutic potential of siRNA. This allowed use to more clinically relevant concentrations to achieve a 60% of MLL/AF4 and downstream target genes. Notably, using an LNP-SM formulation enhanced activity of siMA6, showing a substantial impact on cell proliferation, viability, and capacity to self-renew. The next important step of this project is to test PK properties in -vivo and validate the promising findings observed in -vitro . These encouraging results could lead to improved treatments for these ALL patients with a clinically poor prognosis

P316: THE ROLE OF TET2 IN (PRE)-LEUKEMIC T-ALL: TET2 TO THE RESCUE?

S. De Coninck 1,2,3,* , P. Van Vlierberghe 1,2,3 , S. Goossens 2,4 , B. Lintermans 1,2,3 , J. Roels 1,2,3

1 Biomolecular Medicine; 2 Cancer Research Institute Ghent (CRIG); 3 Center for Medical Genetics; 4 Diagnostic Sciences, Ghent University, Ghent, Belgium

Background: T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of pediatric and 25% of adult ALL patients. Therapy has been intensified over the last decades, leading to gradual improvements in survival. However, 10-15% of pediatric and 50% of adult T-ALL cases relapse and ultimately die because of refractory disease.

During malignant transformation of T-cells, a clonal expansion of immature thymic cells is selected through the gradual accumulation of advantageous epigenetic changes and genetic mutations. Long-lived preleukemic stem cells (pre-LSCs) were uncovered as an initiating event in various blood-born cancers, including in mouse models of T-ALL.

Advanced next-generation sequencing technology allowed for the identification of these predisposing mutations in preleukemic myelodysplastic syndrome patients before they progress towards acute myeloid leukemia. Substantial enrichment for mutations in regulators of DNA methylation were identified, including loss of TET2. TET2 is an enzyme that catalyzes DNA demethylation and Tet2 knockouts are prone to develop lymphoid malignancies, including T-cell lymphoblastic leukemia/lymphoma. In T-ALL, the role of TET2 remains to be investigated. We hypothesize that TET2 acts as a tumor suppressor in T-ALL, involved in a premalignant clonally expanded pre-LSC population.

Aims: We aimed to evaluate the tumor suppressor role of TET2 in the development of preleukemic stem cells that ultimately give rise to T-ALL, and to gain a better understanding of the molecular mechanisms involved in this process of malignant T cell transformation.

Methods: We evaluated the expression of TET2 in RNAseq data of primary T-ALL samples and compared this with their normal T cell counterparts. Next, we used a spontaneous T-ALL mouse model, CD2-LMO2 tg mice. Already at young age, preleukemic CD2-Lmo2 tg thymocytes gain aberrant self-renewal capacity, which allows clonal expansion of an immature thymocyte precursor subpopulation. This T-ALL mouse model was crossed with a R26-Tet2 knock-in mouse model that allows conditional (CD2-icre) Tet2 expression in all lymphocytes, at an early differentiation stage, before T-cell commitment.

Results: In T-ALL, TET2 mutations are sporadically identified. However, gene expression profiling of primary T-ALL samples identified a large subset of T-ALLs characterized by a profound loss of TET2 expression compared to healthy T cell precursors (Fig. 1A). These low TET2 levels occur in patients from different T-ALL subtypes and were confirmed in a subset of T-ALL cell lines.

To further explore the relevance of TET2 silencing and its potential implications in clonal expansion, preleukemic CD2-Lmo2 tg mice were sacrificed at different time points (8 and 24 weeks). Gene expression levels in premalignant T-cells were compared with age matched wild-type control mice. A gradual decrease of Tet2 expression, with a co-occurrent increase of Tet2 promotor methylation, was detected in preleukemic CD2-Lmo2 tg mice (Fig. 1B-C), which confirms the important role of Tet2 in pre-LSC.

To support the tumor suppressor role of Tet2 in T-ALL, an aging cohort of CD2-Lmo2 tg with and without Tet2 overexpression was built. Survival analysis showed that CD2-LMO2 tg mice with Tet2 overexpression have an increased latency of T-ALL (Fig. 1D, p=0,03).

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Summary/Conclusion: Tet2 plays an important role in pre-LSC and can act as a tumor suppressor in a spontaneous T-ALL mouse model, which supports the possible tumor suppressor role of TET2 in a subgroup of T-ALL patients. Loss of TET2 in these patients might be a good predictor for response to DNA methylation inhibitors.

P317: TARGETING HYPERACTIVE PDGFR-B IN T-ALL AND T-LBL

S. De Coninck 1,2,3,* , P. Van Vlierberghe 1,2,3 , S. Goossens 2,4 , R. De Smedt 1,2,3 , B. Lintermans 1,2,3

1 Biomolecular Medicine; 2 Cancer Research Institute Ghent (CRIG); 3 Center for Medical Genetics; 4 Diagnostic Sciences, Ghent University, Ghent, Belgium

Background: T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. General prognosis of T-ALL has been improving over time. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains dismal.

To this end, we have screened a T-ALL/T-LBL patient cohort for copy number variations and identified a novel MYH9-PDGFR-β fusion in a T-LBL case, driving aberrant activation of platelet derived growth factor β (PDGFR-β). Moreover, RNA sequencing data of an independent T-ALL patient cohort showed overexpression of PDGFR-β in the TLX subgroup and in immature T-ALL patients.

PDGFR-β is a transmembrane glycoprotein dimer molecule that functions as a receptor tyrosine kinase. Ligand binding results in receptor dimerization and subsequent activation via autophosphorylation, causing downstream activation of various signaling pathways such as PI3K/AKT, MAPK/ERK, JAK/STAT and Notch.

As constitutively active PDGFR-β is observed in a variety of malignancies, multi-target kinase inhibitors are already used in the clinic. However, they are not specific and often result in adverse side-effects.

CP-673451 is a novel and selective PDGFR inhibitor. Mechanistically, CP-673451 inhibits autophosphorylation of dimeric PDGFR-β and in this way prevents phosphorylation of downstream PI3K/AKT and GSK-3α/β. Previous studies showed growth inhibition of multiple tumor xenografts (lung and colon carcinomas) mainly due to a direct antitumor effect.

Aims: We aim to assess whether specific PDGFR-β inhibitors can be used as a novel and targeted therapy in T-ALL/T-LBL treatment or if they can be of added value to the current chemotherapy regimen for T-ALL/T-LBL patients. In the future, we would like to investigate the transforming capacity of the MYH9-PDFR-β fusion in an in vitro pro-T cell culture system.

Methods: T-ALL cell lines were screened for PDGFR-β levels and cell lines were selected with both high (CTV-1, SEM) and low (MOLT-16, Jurkat, Loucy) PDGFR-β levels. CP-673451 was tested on the selected cell lines. Cell viability was measured using an ATP assay (CellTiter Glo) and downstream signaling pathways were quantified with flow cytometry and western blot. Additionally, spleen cells from PDGFR-β high and PDGFR-β low T-ALL PDX models were treated ex vivo using CP-673451.

Results: Both PDGFR-β high cell lines and PDGFR-β high PDX models showed sensitivity to CP-673451 treatment in nanomolar range, whereas PDGFR-β low models did not (Fig. 1A-B). Further characterization of CP-673451 treatment showed inhibition of PDGFR-β autophosphorylation both on western blot and flow cytometry. Downstream, phosphorylation levels of STAT3, STAT5 and GSK-3β were decreased upon PDGFR-β inhibition (Fig. 1C). Dephosphorylation of GSK-3β results in activation of this kinase, leading to phosphorylation of MCL1, promoting degradation of this antiapoptotic protein. Interestingly, high MCL1 levels are associated with lowered sensitivity to glucocorticoids, core components of T-ALL/T-LBL treatment. To this end, combination therapy of PDGFR-β inhibition with glucocorticoids can result in synergistic effects. Next, we will conduct in vivo studies to verify if specific PDGFR-β inhibitors could be advantageous in the treatment of PDGFR-β high T-ALL/T-LBL cases.

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Summary/Conclusion: In this study, we have identified MYH9-PDGFRB as a novel rare fusion activating PDGFR-β in a T-LBL patient sample. Moreover, in vitro evaluation in T-ALL cell lines and ex vivo treatment of T-ALL PDX models showed therapeutic benefits from PDGFR-β inhibition by CP-673451.

P318: A DUAL ROLE FOR PSIP1 IN T-ALL

L. Demoen 1,2,* , F. Matthijssens 1,2 , Z. Debyser 3 , S. Goossens 2 , P. Van Vlierberghe 1,2

1 Center for Medical Genetics; 2 Cancer Research Institute Ghent, Ghent University, Gent; 3 Department of Pharmaceutical and Pharmacological Sciences, Katholieke Universiteit (KU) Leuven, Leuven, Belgium

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by the diffuse infiltration of malignant T-cell progenitors in the bone marrow. Currently, the cure rates have augmented to 80% for pediatric and 60% for adult T-ALL cases due to modern intensified chemotherapy treatments. These cure rates sadly come at the cost of severe side effects and current therapy still provides a poor outcome for primary therapy-resistant or relapse cases. Therefore, there is an urgent need for the identification of key drivers and pathways in T-ALL to develop targeted and less toxic therapies. PSIP1, a histone mark reader, was primarily found as a dependency factor in MLL-rearranged AML, which was dispensable for normal hematopoietic development, making it an interesting therapeutic target. Nevertheless, the identification of inactivating mutations and deletions in T-ALL patients and lower expression in all T-ALL subtypes, with exception of the TLX1-subtype, compared to normal T-cell subsets, is rather an indication that PSIP1 can also serve as a tumor suppressor in certain contexts.

Aims: In pursuance of determining the role of PSIP1 in tumor initiation and maintenance in T-ALL, we studied the effect of loss of PSIP1 by utilizing several different T-ALL mice models and human cell lines.

Methods: To identify the role of PSIP1 during tumor initiation, we crossed a conditional Psip fl/fl knock-out mouse model with two different spontaneous T-ALL mouse models. On the other hand, to unravel the consequences of losing PSIP1 expression during tumor maintenance a proliferation competition assay was performed and RNA-seq profiling was used to identify downstream targets of PSIP1. Furthermore, tamoxifen-inducible Psip fl/fl mouse cell lines were created from Lmo2-driven T-ALL mouse tumors to test the loss Psip1 during tumor maintenance both in vitro and in vivo.

Results: A conditional Psip fl/fl knock-out mouse model was crossed with two different spontaneous T-ALL mouse models, namely the Notch1 independent Lck-Cre tg/+ Pten fl/fl model and the Notch1 dependent CD2-Lmo2 tg/+ transgenic, which can also give rise to ETP-like murine T-ALL tumors. The loss of Psip1 was shown to significantly accelerate T-ALL development in both models, with a p-value of 0.0156 and 0.0002 (see figure) respectively.

Interestingly, in contrast with the role of PSIP1 loss in tumor initiation, the loss of PSIP1 expression impaired cell proliferation in five different human T-ALL cell lines. Further, tamoxifen-inducible Psip1 knock-out mouse cell lines were established and are being used to further confirm the dependency role of Psip1 during tumor maintenance. Lastly, COX20, an assembly factor of the cytochrome c oxidase in the mitochondria, was found to be a downstream target of PSIP1 upon knock-down and could explain the impairment of proliferation upon the loss of PSIP1 in these cell lines.

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Summary/Conclusion: Altogether, these data indicate that PSIP1 can exert a dual role in the context of T-ALL, either as a tumor suppressor gene during tumor initiation or as a dependency factor in tumor maintenance. Its dispensability for normal hematopoiesis and its dependency role during tumor maintenance, illustrate the potential of PSIP1 as a therapeutic target in T-ALL.

P319: THE TESTICULAR NICHE OF ACUTE LYMPHOBLASTIC LEUKEMIA – MOLECULAR AND CELLULAR FACTORS FOR THE PREFERENTIAL MIGRATION AND SURVIVAL

T. Skroblyn 1 , J. J. Joedicke 1 , M. Pfau 2 , K. Krüger 1 , J.-P. Bourquin 3 , S. Izraeli 4 , C. Eckert 2,* , U. E. Höpken 1

1 Max-Delbrück-Center for Molecular Medicine; 2 Paediatric Oncology/Haematology, Charité - Universitätsmedizin Berlin, Berlin, Germany; 3 University Children’s Hospital, Zurich, Switzerland; 4 Schneider Children’s Medical Center of Israel, Petach Tiqva, Israel

Background: The testis is the second most frequent (30%) non-hematological extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL) treated according to European protocols. Testicular relapses usually occur late (> 6 months after completion of frontline treatment), and boys with a ETV6::RUNX1 fusion gene positive ALL have a significantly higher risk of a testicular relapse. In adult ALL, only about 1% of patients have a testicular relapse. Event-free survival is between 40%-80% depending on timing of relapse, involvement of the contralateral testis and of the bone marrow. The surgical removal of the clinically-involved testis or high-dose irradiation are the only current treatment options for testicular relapses to achieve a long-term event-free survival, but it impacts long-term quality of life. The molecular mechanisms regulating leukemic cell migration, growth, and survival in the testis have not been sufficiently assessed.

Aims: We aimed at investigating our hypothesis that the testis is a frequent source of extramedullary relapse in pediatric ALL because of the physiologically strong CXCL12 gradient. This allows circulating leukemia cells to migrate into the testis during pre-puberty and to survive for potentially longer periods because of the specific immune suppressive microenvironment at this unusual anatomical site.

Methods: To dissect the pre-pubertal cellular requirements and molecular pathways contributing to testicular leukemic cell dissemination and survival, we combined analysis of primary human leukemias with a patient derived xenograft (PDX) mouse model. We analyzed chemokine receptor expression profiles of pediatric B-ALL samples from patients with different relapse sites, studied the crosstalk of leukemia cell-stroma in co-cultures, and established a pediatric B-ALL PDX mouse model with testicular involvement.

Results: The CXCL12-CXCR4 was identified as the driving force for B-ALL cell migration and survival in the testicular leukemic niche. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B-ALL cells both at the time of diagnosis and relapse. One prerequisite for leukemic cell infiltration in the testis mice was high surface expression of CXCR4 on PDX-ALL cells, and CXCL12 secretion from testicular stroma. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Another requirement for migration and survival of leukemia cells in the testis was pre-pubertal age of the recipient mice. Leukemic cell interactions with specific testis macrophage subpopulations, isolated from affected testes, altered their phenotype towards a pro-tumorigenic M2-like phenotype. The blockage of CXCR4-mediated functions by anti-CXCR4 antibody treatment reduced testicular infiltration of PDX-ALL cells.

Summary/Conclusion: Collectively, a pre-pubertal condition together with high CXCR4 expression are factors affecting the leukemia permissive testicular microenvironment. CXCR4 could be proposed as a promising target for therapeutic prevention of testicular relapses in childhood B-ALL.

P320: TCF3-PBX1 LEUKEMIA INVADES IPSC-DERIVED CEREBRAL ORGANOIDS

P. Gebing 1,* , S. Hänsch 2 , L. Lenk 3 , D. Schewe 3,4 , A. Borkhardt 1,5 , U. Fischer 1,5 , S. Bhatia 1,5

1 Department of Pediatric Oncology, Hematology and Clinical Immonology, Medical Faculty, Heinrich Heine University Düsseldorf; 2 Center for Advanced Imaging - Cai, Heinrich Heine Universität Düsseldorf, Düsseldorf; 3 Department of Pediatrics I, ALL-BFM Group, Christian-Albrechts University Kiel and University Medical Center, Kiel; 4 Department of Pediatrics, Otto-von-Guericke-University, Megdeburg; 5 German Cancer Consortium (DKTK), Düsseldorf, Germany

Background: Infiltration to the central nervous system (CNS) remains a major clinical challenge to this day in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The relevant biological mechanisms underlying the invasion of the CNS, are not yet understood and are mainly investigated using 2D cell culture and in vivo mouse models 1 . Differences between species exist, particularly in cell types and architecture between human and murine CNS, as well as the lack of specificity of infiltration between leukemia subtypes. Human brain organoids potentially address some of these drawbacks and may therefore present a complemental approach to study CNS invasion.

Aims: We aimed to establish human iPSC-derived cerebral organoids 2 as a 3D model for CNS invasion. Applying these organoids in coculture with primary patient-derived xenograft (PDX) BCP-ALL cells (TCF3-PBX1), we aimed to investigate CNS-specific engraftment while using cord-blood-derived CD34-positive hematopoietic stem cells as a healthy control.

Methods: Brain organoids were derived from induced pluripotent cell lines (iPSCs) of different donors and differentiated for 30 and 60 days. Intermediate progenitor, as well as matured neuronal cell types, were controlled by immunofluorescent staining of their respective markers (PAX6/Nestin, TUJ1/MAP2) which assemble in the complex neuroepithelium of cerebral organoids. We cocultured CFDA-SE stained TCF3-PBX1-positive patient-derived leukemia cells and cell lines with cerebral organoids. The initial co-cultivation was designed to investigate when/if infiltration could occur. Furthermore, a limiting dilution was introduced to determine the lowest cell concentration required for invasion. 3D imaging of cleared organoids was performed to analyze invasion.

Results: Patient-derived TCF3-PBX-leukemic xenografted cells and cell lines invade cerebral organoids after 14 days of coculture. We found that this is independent of the iPSCs donors (testing in different batches an iPSC donors). The invasiveness was determined by quantifying single and larger colonies of cells engrafted into the organoids. TCF3-PBX1 revealed high invasiveness (P-value: 0.0006) compared to normal cord-blood-isolated CD34+ hematopoietic stem cells (HSCs). In addition, as few as 50 cells (TCF3-PBX1) per organoid were sufficient to invade the organoid tissue. Interestingly, the shRNA-downregulation of CD79a/Igα in TCF3-PBX1 (697) cells, impeded CNS infiltration to cerebral organoids (P-value: 0.0006), annealing their results in murine models 1 .

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Summary/Conclusion: Human iPSC derived brain organoids display some key specificity with regards to of BCP-ALL CNS infiltration. Although key features (vasculature, leptomeninges etc.) which could strengthen the model, have not yet been successfully developed for brain organoids, they may present a promising 3D culture system to adopt in CNS infiltration studies.

References :

1. Lenk, L. et al . Commun. Biol. 4 , 73 (2021).

2. Lancaster, M. A. et al. Nature 501 , 373–379 (2013).

P321: PERTURBATIONS IN STIMULUS-INDUCED CYTOKINE RESPONSES IN CHILDREN WITH BCP-ALL

N. Ruechel *1, , M. Oldenburg *1 , S. Janssen *2 , A. Pandyra 1 , L. Wei 3 , D. Hein 1 , V. Jepsen 1 , U. Fischer 1 , J. Hauer 4 , F. Auer 4 , A. Beer 5 , O. Adams 6 , C. Mackenzie 7 , M. Jaeger 8 , M. Netea 9 , A. Borkhardt *1 , K. Goessling *1

1 Pediatric Oncology, Hematology and Clinical Immunology, University Hospital Duesseldorf, Duesseldorf; 2 Algorithmic Bioinformatics, Department of Biology and Chemistry, Justus Liebig University, Gießen; 3 Department of Molecular Medicine II, Medical Faculty, Heinrich-Heine University, Duesseldorf; 4 Department of Pediatrics and Children’s Cancer Research Center (CCRC), Technical University (TU), Muenchen; 5 Pediatric Hematology and Oncology, Department of Pediatrics, University Hospital Carl Gustav Carus, Dresden; 6 Institute for Virology, Medical Faculty, University Hospital; 7 Institute of Medical Microbiology and Hospital Hygiene, University Hospital Duesseldorf, Duesseldorf, Germany; 8 Radboudumc, Nijmegen, Department of experimental internal medicine, Radboucumc; 9 Radboudumc, Nijmegen, Department of experimental internal medicine, Radboudumc, Nijmegen, Netherlands

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common subtype of childhood leukemia with a peak during early childhood when children are exposed to viral infections. About 5% of all newborns carry a preleukemic clone, but only about 0,2% of them develop BCP-ALL later in life. It has been assumed that a dysregulated, altered immune response underlies the outgrowth of a pre-leukemic clone in infection-triggered B-cell precursor acute lymphoblastic leukemia (BCP-ALL).

Aims: We investigated whether children with BCP-ALL differ in their cytokine response from healthy, age-matched, non-leukemic children, after immune cells were challenged with viral, fungal or bacterial stimuli.

Methods: We set up a functional experimental platform with 73 stimulus-cytokine pairs to comprehensively characterize the immune profile of pediatric patients with BCP-ALL with the two most common genetic subtypes, either carrying the ETV6/RUNX1 (E/R) gene fusion or the high-hyperdiploid (HD) karyotype in comparison with an age-and gender-matched healthy control cohort without BCP-ALL and their healthy parents (in total n = 101 individuals). Children were in stable first remission, at least two years after the end of therapy and had a fully reconstituted immune system. Blood was taken of patients and controls in parallel and peripheral blood mononuclear cells (PBMCs) were isolated using a density gradient centrifugation. Cells had been stimulated with various fungal, bacterial and viral pathogens, such as Candida albicans , Staphylococcus aureus , Influenza virus , Respiratory Syncytial virus and toll-like receptor ligands (TLR), such as lipopolysaccharide (TLR4), R848 (TLR7/8) or CpG (TLR9); subsequently cytokines (IFNa, IL-1b, IL-1Ra, IL-10, IL-12p70, IL-17, IFNg) had been measured in the cell culture supernatant.

Results: Cytokine base line level did not differ between the groups, while the challenge with various infectious antigens resulted in an overall elevated cytokine production of the BCP-ALL patients with E/R fusion (n=11) (p < 0.01, Mann-Whitney-Wilcoxon test two-sided with Benjamini-Hochberg correction). By deciphering the characteristics of the immune response, a remarkable difference for the anti-viral immune response (p < 0.01) was identified, while the anti-bacterial and anti-fungal response did not differ: The IFNa-response induced by Influenza virus and R848 and the IL-17-response induced by R848 differed most prominently, although not significantly after correction for multiple testing. Further analysis of the cytokine profile of the E/R parents compared with an age- and gender-matched healthy control group revealed an overall significantly elevated cytokine response (p < 0.05) after stimulation, but the different sub-features, such as the fungal, bacterial and viral immune responses were not different. Most strikingly, we could identify this pattern specifically for the E/R cohort. Comparing the cytokine responses of the HD patients (n=8) with the healthy controls (n=22) did not result in any significant differences.

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Summary/Conclusion: Our study suggests that an altered pro-inflammatory anti-viral cytokine response pattern in children with E/R-BCP-ALL is specific to those children, who later developed the BCP-ALL. It is still present years after chemotherapy and possibly contributes to the outgrowth of a pre-leukemic clone, consistently generated in utero during fetal hematopoiesis.

P322: THERAPEUTIC EFFECT OF TARGETING CASEIN KINASE II / WDR5 AXIS IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Q. Han 1 , C. Song 2,3 , Z. Ge 1,*

1 Department of Hematology, Zhongda Hospital，School of Medicine, Southeast University, Institute of Hematology Southeast University, Nanjing, China; 2 Hershey Medical Center, Pennsylvania State University, Hershey; 3 Division of Hematology, The Ohio State University Wexner Medical Center, the James Cancer Hospital, Columbus, United States of America

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignance with limit treatment choices and extremely poor prognosis. WD repeat domain 5 (WDR5) is essential for the methyltransferase activity of MLL1 complex and plays an important role in various biological functions. We reported that targeting Casein Kinase II (CK2)/Ikaros axis by CK2 inhibitor CX-4945 has therapeutic efficacy in B-ALL. However, the effect of WDR5 inhibitor and CX-4945 in T-ALL remains unknown.

Aims: This study is to explore the synergistic effect of WDR5 inhibitor OICR-9429 and CK2 inhibitor CX-4945 and the potential underlying mechanisms of this novel drug combination in T-ALL.

Methods: The expression profile and clinical significance of WDR5 and ATAD2 in T-ALL were explored by analyzing the transcriptome data of T-ALL patients from the GEO database. CCK-8 assay was performed to analyze the synergistic effect of OICR-9429 combined CX-4945 in CEM and MOLT4 cell lines. Cell cycle effect of inhibitors were detected by flow cytometry after 72h cultured. RNA-seq was performed after CEM cells were treated with OICR-9429, CX-4945, and vehicle control for 72 hours. Differential expression genes (DEGs) and pathways were analyzed by R-software. WDR5 and ATAD2 were knockdown by shRNA in CEM and MOLT4 cells. qPCR and Western Bolt were performed to test the DEGs detected by RNA-seq.

Results: WDR5 is significantly over-expressed in T-ALL patients compared with healthy controls in two independent cohorts and is associated with shorter overall survival ( P <0.01). Bioinformatic analysis revealed that the cell cycle was the most significantly altered pathway between WDR5 high and low expression patients ( P <0.001) (Figure 1A). In vitro assay showed that OICR-9429 and CX-4945 alone exhibited an anti-proliferation effect on T-ALL cells after being treated for 72h. In addition, we found that OICR-9429 and CX-4945 had a significant synergistic effect on cell proliferation arrest in CEM (Figure 1B) and MOLT4 cells compared with a single drug ( P <0.05). Cell cycle assay revealed that OICR-9429 (Figure 1C) and CX-4945 induced a G1-phase arrest in CEM and MOLT4 cells and this effect was significantly increased upon OICR-9429 and CX-4945 combination ( P <0.01) (Figure 1D). The anti-proliferation and cell cycle arrest effects were also exhibited in WDR5 knockdown CEM and MOLT4 cells ( P <0.05). Pathway analysis of the DEGs showed that the cell cycle pathway was obviously altered after OICR-9429 and CX-4945 treated ( P <0.001) (Figure 1E). In addition, CX-4945 strongly down-regulated WDR5 (Figure 1F) expression in both transcriptome level and protein level, and knockdown of WDR5 increased the anti-leukemic effect of CX-4945 ( P <0.01) (Figure 1G), suggesting that WDR5 was the downstream target of CK2 and CX-4945 in T-ALL. Moreover, RNA-seq data showed that ATAD2, an important oncogene was suppressed upon treatment of OICR-9429, CX-4945 (Figure 1F), and WDR5 knockdown ( P <0.001). Moreover, the combination of OICR-9429 and CX-4945 strongly induced ATAD2 downregulation compared with either single drug ( P <0.001) (Figure 1H). Likewise, we found that ATAD2 is up-regulated in T-ALL patients and associated with poor prognosis and cell cycle pathway abnormal activation ( P <0.001). ATAD2 knockdown significantly inhibited cell proliferation and induced a G1-phase cell cycle arrest in T-ALL cells ( P <0.01) (Fig 1I).

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Summary/Conclusion: Our data showed that OICR-9429 and CX-4945 have synergistic efficacy in T-ALL. Dual targeting WDR5 and CK2 may be a potential therapeutic approach for T-ALL through the CK2-WDR5-ATAD2 axis.

P323: STREAMLINING PRECLINICAL IN VIVO TREATMENT TRIALS BY MULTIPLEXING GENETICALLY LABELLED PDX MODELS OF SEVERAL PATIENTS IN A SINGLE MOUSE

K. Hunt 1,* , D. Amend 1 , R. Ludwig 1,2 , B. Vick 1,2 , A. K. Wirth 1 , T. Herold 1,3 , I. Jeremias 1,2,4

1 Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Munich; 2 German Cancer Consortium (DKTK), partner site Munich; 3 Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich; 4 Department of Pediatrics, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany

Background: Better treatment options are intensively needed for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Before application in clinical trials, novel therapies require preclinical testing which is resource intensive. In previous work, we had shown that multiplexing several PDX cell populations within a single mouse in competitive in vivo experiments gives identical results to studying each population in separate mice (Liu et al., Biomarker Research 2020).

Aims: Here, we aimed at transferring multiplexing to preclinical in vivo treatment trials. We aimed to increase the efficiency of preclinical drug testing in vivo by simultaneously testing several samples in parallel in a single mouse, using a competitive in vivo approach.

Methods: From primary patient material, we had established patient derived xenograft (PDX) mouse models of ALL and AML which allow serial transplantation in immunodeficient NSG mice. We designed and cloned 5 different lentiviral constructs to label 5 individual samples with both a unique fluorochrome as well as a connected unique genetic barcode, for later detection by flow cytometry or next generation sequencing, respectively. In addition, luciferase was recombinantly expressed in all PDX models to allow repetitive monitoring of leukemia growth and treatment response using bioluminescence in vivo imaging (BLI). Up to 5 PDX samples, either ALL or AML, were multiplexed and aliquots injected into groups of mice (n=4-6). For post mortem analysis, PDX cells were reisolated from murine bone marrow and spleen and absolute tumor burden of PDX cells was quantified by flow cytometry of exactly 1/10 of the bone marrow and spleen. Determining fluorochrome composition allowed quantifying the proportion that each of the 5 samples contributed to the entire tumor load. In case of treatment, a response rate for each individual samples was determined by comparing cell numbers in treated versus control mice.

Results:

From our pool of established PDX samples, we selected 5 ALL or 5 AML samples with different molecular alterations, but similar in vivo growth behaviour. Each PDX model was labelled by its individual fluorochrome and genetic barcode, while all models expressed luciferase. Tumor growth as well as the ratio between the samples was repetitively analysed over the entire course of leukemia outgrowth. The composition of the injection mixture was optimized such that each sample accounted for roughly 20% of the total leukemic burden at the time point of start of treatment.

Mice containing multiplexed PDX cells from 5 different patients with ALL were treated for 2 weeks with either verum treatment or with solvent as control. In a study using the BCL-2 inhibitor Venetoclax in PDX ALL models, we were able to distinguish between sensitive and resistant PDX samples. While two samples showed a drastic decrease in tumor burden, three samples showed no or only a mild response. This effect could be observed both in cells isolated from bone marrow as well as spleen. Variances between mice were rather small, with few exceptions for some treated mice.

With this approach, we were able to define the effect of a single drug on up to five distinct samples in parallel, reducing resources by factor 5.

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Summary/Conclusion:

Taken together, we established a multiplex protocol for in vivo therapy trials that allows simultaneous testing of up to 5 PDX samples in competitive in vivo trials. The approach reduced the required number of experimental mice by a factor 5, in line with the 3R concept. In the future, our approach might rationalize in vivo drug trials.

P324: PHARMACOLOGIC INHIBITION OF DYRK1A RENDERS HIGH-RISK KMT2A-R ALL SENSITIVE TO VENETOCLAX

C. Hurtz 1,* , G. Wertheim 2 , J. Chukinas 3 , R. Bhansali 4 , S. Swaminathan 5 , J. Crispino 6 , J. Shi 7 , S. Tasian 3 , M. Carroll 8

1 Fels Institute for Personalized Medicine, Temple University; 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania; 3 Oncology, CHOP, Philadelphia; 4 Hematology Oncology, Upenn, Pehiladelphia; 5 Systems Biology, City of Hope, Monrovia; 6 Experimental Hematology, St. Jude Children’s Research Hospital, Memphis; 7 Cancer Biology; 8 Medicine, Upenn, Philadelphia, United States of America

Background: KMT2A -rearranged (R) ALL is a high-risk disease with a frequency of 70% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches.

Aims: Determine if pharmacologic DYRK1A inhibition may be a novel treatment strategy for patients with KMT2A-R ALL.

Methods: To identify novel targets in KMT2A -R leukemia, we performed a domain-specific kinome-wide CRISPR screen and identified multiple kinases required for cell growth. We focused on DYRK1A as it met the following three criteria: 1 ) Growth inhibition upon kinase targeting was greater in KMT2A -R leukemic cells than in non- KMT2A -R cells, 2 ) DYRK1A was not found to be common essential gene assessed through the Cancer Dependency Map, 3 ) Small molecule inhibitors are available.

Results: We analyzed multiple ChIP-Seq experiments and identified that KMT2A-fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses demonstrate that KMT2A -R ALL cells treated with a menin inhibitor to disrupt the transcriptional activity of the KMT2A -R complex, downregulate DYRK1A, indicating direct regulation of DYRK1A by the KMT2A-fusion. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced leukemic cell growth inhibition in vitro and in vivo , demonstrating that DYRK1A could be a new therapeutic target in KMT2A -R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A -R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A -R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A -R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt’s Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A -R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells and significantly reduced the leukemia burden in vivo .

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Summary/Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo validation is needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A -R ALL.

P325: A KINASE-DEAD VARIANT OF CDK6 IS ASSOCIATED WITH REDUCED TUMORIGENIC POTENTIAL AND A DISTINCT TRANSCRIPTIONAL REWIRING IN AN ALL MODEL

T. Klampfl 1,* , S. Nebenführ 1 , M. Zojer 1 , F. Bellutti 1 , V. Sexl 1 , K. Kollmann 1

1 Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria

Background: The cell cycle kinase Cdk6 is a major regulator of cell cycle progression from G1 to S phase. Cdk4/6 kinase inhibitors are approved for the treatment of breast cancer and show promising results in pre-clinical studies of other cancers, including hematological malignancies. Cdk6 was also shown to exhibit kinase-independent functions including transcriptional regulation of gene expression.

Aims: A systematic understanding of the kinase independent functions of Cdk6 should allow for a better comprehension of the mechanism of action of Cdk4/6 inhibitors.

Methods: To model kinase-independent functions of Cdk6 in Acute Lymphoblastic Leukemia (ALL), mouse pre/pro B-cell lines were generated. Whole bone marrow from mice carrying a kinase-dead mutant of Cdk6 (Cdk6-K43M), Cdk6 knock-out (Cdk6-KO) or wildtype Cdk6 (Cdk6-WT) were retrovirally transduced with the BCR-ABL1 p185 fusion gene. The resulting cell lines were analyzed in-vivo in transplant settings as well as on the molecular level where RNA-seq and ChIP-seq were performed.

Results: Tail vain injection of Cdk6-K43M, Cdk6-KO and Cdk6-WT pre/pro B-cell lines into NSG mice led to the development of lymphoid malignancies albeit with different latencies depending on genotype. Mice injected with Cdk6-KO lines showed a significantly longer survival than mice injected with Cdk6-WT lines. The injection of Cdk6-K43M cells led to an intermediate phenotype with significantly shorter survival than Cdk6-KO, but significantly longer survival than Cdk6-WT. Subcutaneous injection of the cell lines also allowed for the formation of tumors, where tumor weight was largest in mice injected with Cdk6-WT cells, intermediate in the case of Cdk6-K43M cells and lowest in the case of Cdk6-KO cells. Therefore, while kinase-dead Cdk6 had reduced tumorigenic potential compared to Cdk6-WT, loss of kinase function alone exhibited stronger tumorigenic potential than the complete Cdk6 KO.

Differential gene expression analysis between cell lines of the three genotypes revealed common alterations associated with Cdk6-K43M and Cdk6-KO compared to Cdk6-WT. Some of these changes could directly be attributed to the loss of kinase function, including for example the downregulation of E2F target genes. Additionally, changes specific for either Cdk6-K43M or Cdk6-KO were observed. As Cdk6-K43M retains kinase-independent functionality, which includes transcriptional regulation, we investigated the DNA binding profile of transcriptional complexes containing Cdk6-K43M. ChIP-seq analysis revealed that such complexes bind the DNA at largely similar sites as complexes containing Cdk6-WT. The majority of genes differentially expressed between Cdk6-WT and Cdk6-K43M were associated with transcriptional complexes harboring Cdk6.

Summary/Conclusion: Pre/pro B-cell lines carrying kinase-dead Cdk6 (Cdk6-K43M) showed intermediate tumorigenic potential between lines with Cdk6-WT and Cdk6-KO. While Cdk6-K43M is associated with the DNA at similar sites as Cdk6-WT, there are distinct transcriptional changes associated with this mutation. The further elucidation of these alterations is expected to provide a better understanding of both, kinase independent functions of Cdk6 as well as the effects of its pharmacological inhibition.

P326: PROGNOSTIC UTILITY OF NON-HOMOLOGOUS END JOINING DNA REPAIR PATHWAY GENES IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

S. KUMARI 1,* , J. SINGH 1 , M. Arora 2 , S. Bakhshi 3 , J. K. Palanichamy 2 , A. Sharma 3 , P. Tanwar 1 , A. R. Singh 1 , I. Qamar 4 , A. Chopra 1

1 Lab Oncology Unit; 2 Department of biochemistry; 3 Department of medical oncology, All India Institute of Medical Sciences, New Delhi; 4 School of Biotechnology, Gautam Buddha University, Gerater Noida, India

Background: T-ALL is a molecularly heterogeneous malignancy characterized by transformation and differentiation blockage at different stages of T cell development. Efficient DNA repair has shown to enable leukemic cells to survive the damaging effects of therapeutics, therefore, understanding of DNA repair alterations in leukemia may provide novel druggable targets and prognostic biomarkers. Notably, it is important to determine that whether the variable ability of T cell to repair DNA damage also translates into the corresponding molecular profiles of T-ALL. Several components of different DNA repair pathways have been recently identified to play critical role in ALL pathophysiology and drug resistance. However, the clinical significance of major DNA double strand break repair pathway Non-Homologous End Joining (NHEJ) in T cell acute lymphoblastic leukemia remains to be studied in detail.

Aims:

This study aims to assess the importance of DNA damage response pathways in T-cell acute

lymphoblastic leukemia cancer (ALL).

Methods: We assessed the gene expression pattern of 21 genes involved in NHEJ pathwayincluding six core genes, accessory genes and other regulators such as MRE complex genes in 207 T-ALL patients. In order to determine the changes in expression pattern of NHEJ genes during the course of T-ALL, gene expression was assessed at different time points, including clinical sample at diagnosis, minimal residual diseaseand at relapse. Real time PCR was used to assess the expression of these genes. We further determined the association of NHEJ pathway gene expression with clinical and molecular features.

Results: We observed higher expression of TOX, WRN, NHEJ1, APLF, TDP2 and reduced expression of XRCC4, POLL, PRKDC, APTX, PNKP, XRCC5, DCLRC1C, MRE11, XRCC6, NBN in diagnostic T-ALL samples compared to normal bone marrow. Further, PRKDC, RAD50, and XRCC6 displayed lesser expression in prednisolone resistant group compared to sensitive group. At minimal residual disease, XRCC4, POLL, WRN, POLM, APTX, PNKP, XRCC5, DCLRC1C, XRCC6, NBN, TDP1 exhibited higher expression and TOX, LIG4, MRE11 exhibited reduced expression compared to diagnostic samples. XRCC4, PRKDC, NHEJ1, PNKP, XRCC5, DCLRC1C, MRE11, XRCC6, NBN and TDP1 exhibited higher expression in relapse samples compared to diagnostic samples.

The clinical significance of expression of NHEJ genes was explored in detail. Low expression of XRCC4, PRKDC, RAD50, XRCC5, XRCC6 and TDP1 gene was associated with immature immunophenotype (IPT) as compared to cortical and mature IPT.Patients with ETP-ALL IPT more frequently had lesser expression of XRCC4, PRKDC, RAD50, LIG4, APTX, MRE11, XRCC6 andgene compared to other IPT groups. In diagnostic samples, higher XRCC4 expression was associated with better overall survival (OS) and even free survival (EFS). Additionally, low WRN expression was associated with poor EFS and relapse free survival (RFS). Interestingly, high DCLRC1C expression was associated with favorable OS, EFS and RFS.

Summary/Conclusion: We observed a distinct expression profile of NHEJ genes among molecularly heterogeneous groups of T-ALL patient’spossibly explaining different extent of DNA repair among patient with different immunological groups and in response to therapy. Interestingly, this depicted a clear change in the DNA repair responses in the leukemia patients before and post chemotherapy. Gene expression emerged as the predictor of OS, EFS and RFS in T-ALL patientsthat might be helpful in the improvement of the treatment outcome in T-ALL therapy

P327: QUANTITATIVE EXPRESSION PROFILING OF SURFACE ANTIGENS ON PERIPHERAL BLOOD LEUKOCYTE SUBSETS AND CHILDHOOD T-ALL CELLS USING A STANDARDIZED FLOW CYTOMETRY WORKFLOW: A HCDM CDMAPS INITIATIVE

D. Kužílková 1,* , J. Puñet-Ortiz 2 , P. M. Aui 3 , J. Fernandez 2 , F. Karel 1 , P. Engel 2 , M. C. van Zelm 3,4 , T. Kalina 1

1 CLIP (Childhood Leukemia Investigation Prague), Department of Pediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, Prague, Czech Republic and University Hospital Motol, Prague, Czechia; 2 Department of Biomedical Sciences, University of Barcelona, Barcelona, Spain; 3 Department of Immunology and Pathology, Central Clinical School, Monash University; 4 Department of Allergy, Immunology and Respiratory Medicine, Central Clinical School, Monash University and Alfred Hospital, Melbourne, Australia

Background: Human Leukocyte Differentiation Antigen (HLDA) workshops are organized by the Human Cell Differentiation Molecules (HCDM) consortium to test and validate the reactivity of particular antibody clones to specific targets. Thereby the consortium provides the scientific community validated antibody clones reactive to particular cluster of differentiation (CD) markers. Although this approach has been used since the 1980s, quantitative profiling of CD markers at “single-cell“ level and benchmarking of reagents are currently lacking.

Aims: We aimed to develop a flow cytometric procedure allowing CD marker expression profiling in a standardized way in time and place

Methods: First, we developed and titrated two antibody panels with a free position in the phycoerythrin (PE) channel. The panels enable identification of 27 innate and adaptive leukocyte cell populations present in peripheral blood. The panels were custom dried in 96-well plates, and the Quantibrite™ PE Beads were used for quantification of the PE signal. Subsequently, we developed a high content framework to evaluate the titration of PE conjugated monoclonal antibodies using fluorescently barcoded cell lines and peripheral blood cells. The selected titer and critical antibody information (such as clone, catalogue number, vendor, gene and CDname etc.) were centrally stored in an inventory table, which was expanded into an experiment master table (EMT) following inclusion of experimental details (e.g. the position of individual mAbs in 96-well plate, experiment name, operator etc.). The EMT was used to generate an experimental protocol with automated calculation of reagent amounts and volumes. Post acquisition, the fcs files were annotated using all relevant experimental information from the EMT table.

Results: To validate our approach, we quantified protein expression of four selected CD markers (CD11b, CD31, CD38 and CD40) with well-known expression pattern on peripheral blood leukocytes that showed high reproducibility across centers. We also performed benchmarking of four anti-CD3 clones, of which the titration curves revealed variable performance: from high-performance TB3 clone, through intermediate-performance UCHT1 and SK7 clones to low-performance MEM-57 clone. Three out of four anti-CD3 clones showed similar pattern of staining, whereas the MEM-57 clone showed decreased intensity on CD4 and CD8 T cells while retaining comparable intensity to the other clones for TCRγδ+ T cells. Our pilot results on childhood T-ALL patient samples (n=7) revealed potential targets for minimal residual disease monitoring.

Summary/Conclusion: In summary, we optimized a procedure for quantitative expression profiling of surface antigens on subsets of blood leukocyte and proved its feasibility with inter-laboratory comparison in three different laboratories. The presented workflow enables (i) to map the expression patterns of HLDA-approved antibody clones to CD markers, (ii) to benchmark new antibody clones to established CD markers, (iii) to define new clusters of differentiation in future HLDA workshops and (iv) mapping of childhood T-ALL cells.

Acknowledgement: The reagents were kindly provided by Exbio and BioLegend. The work was financially supported by project NU20-05-00282 of the Czech Republic Ministry of Health

P328: DYNAMIC EVOLUTION OF TCF3-PBX1 LEUKEMIAS AT THE SINGLE-CELL LEVEL UNDER CHEMOTHERAPY PRESSURE

M. Kusterer 1 , M. Lahnalampi 2,* , M. Voutilainen 2 , G. Gentile 1 , S. Pennisi 1 , J. Norona 1 , G. Greve 1 , M. Lübbert 1 , R. Sankowski 3 , M. Prinz 3 , S. Killmer 4 , M. Salvat Lago 4 , B. Bengsch 4 , M. Cleary 5 , V. Zachariadis 6 , M. Enge 6 , O. Lohi 7 , M. Heinäniemi 2 , J. Duque-Afonso 1

1 Department of Hematology/Oncology/Stem Cell Transplantation, University of Freiburg Medical Center, Freiburg, Germany; 2 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland; 3 Department of Neuropathology; 4 Department of Gastroenterology, University of Freiburg Medical Center, Freiburg, Germany; 5 Department of Pathology, Stanford University, Standford, United States of America; 6 Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden; 7 Tampere Center for Child, Adolescent, and Maternal Health Research, Tampere University, Tampere, Finland

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While research has been focused on high-risk patients, the biology of low-to-intermediate-risk patients has so far been inadequately investigated and can lead to overtreatment with severe side effects and under treatment with risk of relapse. The translocation t(1;19) codes for chimeric fusion protein TCF3-PBX1, which is associated with intermediate risk ALL. Using our previous generated TCF3-PBX1 conditional knock-in mice, we established a model to study in vivo chemotherapy resistance.

Aims: We hypothesize that chemotherapy and microenvironment play a crucial role in development of resistance in TCF3-PBX1 leukemias by influencing transcriptional regulation, activation of signaling pathways and the hierarchical structure of leukemic cells. In this project we aim to characterize dynamic changes of TCF3-PBX1 leukemia cells in different tissues under chemotherapy pressure.

Methods: Recipient C57/BL6 healthy mice were sub-lethally irradiated, transplanted with mouse TCF3-PBX1 leukemia cells and treated with vehicle (n=15), prednisolone (n=13) and daunorubicin (n=15) for 20 days. Mice were monitored for signs of disease regularly and circulating GFP+ TCF3-PBX1 leukemia cells were assessed by flow cytometry. Sick mice were sacrificed, leukemia cells were isolated from five different organs (bone marrow, spleen, lymph nodes, spinal cord and brain) and characterized by immunophenotyping, sanger sequencing, bulk RNA sequencing (bulk RNAseq) of GFP+ sorted cells, single cell RNA sequencing and mass cytometry (CYTOF).

Results: All mice transplanted with TCF3-PBX1 leukemia cells and treated with vehicle succumbed to disease with a median survival of about 70 days. We optimized chemotherapy drug concentration and transplanted cell dose, so 60% of mice treated with prednisolone or daunorubicin survived at least 150 days. Leukemic infiltration was showed by histological stainings in analyzed tissues including central nervous system (CNS) (spinal cord, brain) and GFP+ leukemia cells were quantified by flow cytometry. No major differences were observed in immunophenotype of TCF3-PBX1 leukemia and variant allele frequency (VAF) of the known PTPN11 mutation in analyzed tissues or depending on in vivo treatments. Bulk RNAseq of FACS-sorted GFP+ TCF3-PBX1 leukemia cells were clustered and CNS cells separated from other tissues. scRNAseq revealed additional heterogeneity within each tissue based on signaling pathway and cell cycle activity. Interleukin signaling, regulation of apoptosis pathways, and signaling by the B cell receptor (BCR) were regulated based on pathway analysis in CNS compared to other tissues. Hence, we elucidated the hierarchical structure of hematopoietic cells, interaction of leukemic cells with the microenvironment, and changes in signaling response to chemotherapy in vivo treatment by mass spectrometry (CyTOF) to validate the identified altered signaling pathway activities at protein level.

Summary/Conclusion: We have developed a mouse model in order to characterize in vivo chemotherapy resistance depending on niche. Global transcriptomics and phospho-proteomics at the single-cell level might elucidate novel mechanisms of chemotherapy resistance suitable for pharmacological therapies.

P329: UNRAVELING THE ROLE OF GATA3 IN EARLY HUMAN T CELL DEVELOPMENT AND EARLY T CELL PRECURSOR ALL (ETP-ALL)

N. Lambrechts 1,2,* , K. L. Liang 1,2 , T. Putteman 1 , J. Roels 1,3 , J. Van Hulle 1 , T. Taghon 1,2

1 Diagnostic Sciences, Ghent University; 2 Cancer Research Institute Ghent; 3 Biomolecular Medicine, Ghent University, Ghent, Belgium

Background: ETP-ALL is a subtype of T cell acute lymphoblastic leukemia (T-ALL) and can be identified by a characteristic immunophenotype and gene expression profile. Treatment of relapsed ETP-ALL remains challenging using conventional chemotherapy and little targeted therapies are available. A better understanding in the molecular mechanisms involved in the leukemogenesis and maintenance of ETP-ALL could lead to improved and more targeted treatments. An estimated 9% of ETP-ALL patients carry mutations in GATA3 , a transcription factor known to regulate T cell commitment by inducing T lineage genes and inhibiting genes important for other lineages such as NK and B cells. Although these mutations have been described to be loss-of-function mutations, the exact mechanisms through which they contribute to ETP-ALL remain to be elucidated.

Aims: We aim to gain insights into the molecular mechanisms through which GATA3 regulates normal human early T cell development and how disruption of GATA3 function, as a result of mutation, can contribute to the emergence of ETP-ALL.

Methods: We have studied the effect of the most prevalent GATA3 mutation (c.827G>A, p.R276Q) in ETP-ALL on normal human T cell development using in vitro differentiation cultures. We examined the impact of GATA3 p.R276Q on gene expression, DNA binding and chromatin accessibility through RNA-, CUT&Tag- and ATAC-sequencing respectively, starting from primary CD34 + CD1a - early human T cell progenitors isolated from thymus or the ETP-like cell line PER117.

Results: While overexpression of wild-type GATA3 in thymic progenitor T cells induces an acceleration of development towards the CD4 + CD8ab + double positive stage, overexpression of the mutant form results in a delay and an accumulation of CD4 immature single positive cells in OP9-DL1 co-cultures. Remarkably, the inhibition of NK cell development, which is an important function of wild-type GATA3, is maintained by GATA3 R276Q. This indicates that GATA3 R276Q is not merely a loss-of-function mutation. RNA-, CUT&Tag- and ATAC sequencing after overexpression in human CD34 + CD1a - immature thymocytes or the ETP-ALL-like cell line PER117 revealed that most key regulatory effects are maintained by GATA3 R276Q compared to its wild-type counterpart, including induction of T cell genes and inhibition of stem cell and NK cell genes, binding to its target regions by recognizing GATA motifs and exerting a pioneering function by opening and closing chromatin.

Summary/Conclusion: Continuous GATA3-R276Q has a detrimental effect on T cell development in vitro . Since the inhibition on NK cell development is maintained, these findings suggest that this decision is uncoupled from the induction of T cell lineage commitment. Many important effects on RNA expression, DNA binding and chromatin accessibility are maintained by the R276Q mutant compared to wild-type GATA3, pointing out that this mutation not purely acts as a loss-of-function. The exact mechanisms through which GATA3 R276Q contributes to ETP-ALL still remain to be elucidated, but our results show that inhibition of T cell development most likely is one piece of the puzzle that leads to malignant transformation.

P330: TARGETING BTN2A1 BY A UNIQUE ACTIVATING MAB IMPROVES ANTI-TUMOR FUNCTIONS OF VΓ9VΔ2 T CELLS

A.-C. Le Floch 1,* , C. Imbert 1 , A. de Gassart 2 , A. Le Roy 1 , L. Gorvel 1 , N. Vey 3,4 , A. Anastasio 1 , A. Briantais 1 , N. Boucherit 1 , C. E. Cano 2 , D. Olive 3,4

1 Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068; 2 ImCheck Therapeutics; 3 Institut Paoli-Calmettes; 4 Aix-Marseille Université UM105, CNRS UMR 7258, Marseille, France

Background: Vγ9Vδ2 T cells are new promising cytotoxic effectors in cancer immunotherapy. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. But very few data are currently available on susceptibility of acute lymphoblastic leukemia (ALL) cells to Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A targeting agonist antibody ICT01 is being developed in a multicentric Phase 1 and 2 study called EVICTION by Imcheck Therapeutics. Recently the biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 seems to be mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown.

Aims: Here, we show we show that Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts and that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1 called 107G3B5. Mechanistically, anti-BTN2A1 enhances interactions between Vγ9Vδ2 T cells and target cells and improves the binding of Vγ9Vδ2 TCR on target cells.

Methods: 15 hematological cancer cell lines and PBMC from 17 adults ALL patients at diagnosis (7B-ALL, 7T-ALL and 3Ph+ ALL) were tested in functional assays.

We quantified the relative surface expression of BTN2A (using 107G3B5 and 7.48 mAbs) and BTN3A (using 20.1 and 108.5 mAbs) on cell lines and primary ALL blasts. In parallel, allogenic Vγ9Vδ2 T cells functions against cell lines and primary ALL blasts were evaluated. ALL samples were also tested for their expansion capacities after 14 days of PBMC culture in presence of Zoledronate. Interactions of Vγ9Vδ2 T cells with target cells was investigated using a 3D imaging in real-time by holo-tomographic microscopy. Binding assays were realized using a recombinant tetramerized Vγ9Vδ2 TCR.

Results: We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against hematological cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 agonist mAb (107G3B5) significantly enhanced Vγ9Vδ2 T cells mediated apoptosis, in comparison to control condition. This effect was increased over time even for the less spontaneously susceptible cells. We confirmed these observations with autologous Vγ9Vδ2 T cells expanded from 4 ALL patients at diagnosis for which effector functions were increased after treatment with 107G3B5. In live microscopy, 107G3B5 enhanced interactions between target cells cocultured during 24h with Vγ9Vδ2 T cells. Finally, we observed that 107G3B5 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells.

Summary/Conclusion: Here, we demonstrated that targeting BTN2A1 led to improve Vγ9Vδ2 T cell antitumor response at molecular, cellular and functional levels. Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as reported previously as well as with BTN2A1 mAbs.

These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types.

P331: T-CELL RECEPTOR REPERTOIRE DIVERSITY AND L-ASPARAGINASE HYPERSENSITIVITY IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA

S. Lee 1,* , Z. Li 1 , E. Lim 2 , E. Chiew 2 , A. M. Tan 3 , H. Ariffin 4 , J. J. Yang 1 , A. Yeoh 2

1 St. Jude Children’s Research Hospital, MEMPHIS, United States of America; 2 National University of Singapore; 3 KK Women and Children’s Hospital, Singapore, Singapore; 4 University of Malaya Medical Centre, Kuala Lumpur, Malaysia

Background: Asparaginase is an indispensable component of therapy of acute lymphoblastic leukemia (ALL). Treatment with asparaginase is associated with a plethora of adverse effects, amongst which the occurrence of hypersensitivity is one of the most common. Hypersensitivity affects the ability to maintain dose intensity which in turn results in poorer outcomes, especially if there is no suitable replacement.

The basis of L-asparaginase hypersensitivity is undoubtedly immune-mediated. T-cell recognition of antigens is arguably one of the most important facets for establishing an immune response, of which a crucial component is the repertoire and diversity of T-cell receptors (TCR). The exact aspects of T-cell immunity underpinning association with asparaginase allergy has yet to be characterized.

Aims: To evaluate the associations of TCR repertoire and its diversity with asparaginase hypersensitivity in children with ALL.

Methods: We longitudinally profiled the TCR repertoires in 67 children with ALL treated on the frontline Ma-Spore ALL 2010 trial, all of whom receive native L-asparaginase in induction, consolidation, and reinduction. Only patients who developed grade 2 or higher reactions (CTCAE 3.0) were included in this study. These children had bone marrow cells sampled at 3 time-points – diagnosis, week 5 (post-induction), and week 12 (post-consolidation). We performed DNA-based TCR-sequencing on N=180 samples, and evaluated TCR characteristics to determine their associations with asparaginase hypersensitivity.

Results: 12 out of 67 children (17.9%) had allergy in our cohort, representing 16 episodes of varying severity (Figure 1A). First, we evaluated the association of TCR-repertoire diversity early on in treatment with the occurrence of subsequent allergy. We found that a higher TCR diversity at all time-points was significantly associated with late allergy (P=0.03 for Shannon’s entropy, P=0.01 for inverse Simpson’s index, Figure 1B). This TCR diversity was characterized by a higher proportion of infrequent clones occurring <0.5% (P=0.008, Figure 1C). Patients with allergy had significantly lower proportion of shared clonotypes (P=0.003, Figure 1D). Examining the dynamic changes of the TCR repertoire between diagnosis and week 5 for each patient, we found that patients with allergy had a much less similar clonotypic set between timepoints compared to non-allergic patients (P=0.003, Figure 1E). In fact, patients who demonstrated a higher variability in their clonotypes between timepoints (i.e. similarity coefficient of <0.05) had an 8.1-fold risk of allergic event (95% CI 1.7 – 39.1, P=0.001, Figure 1F). Evaluating the TCR-repertoire before and after an allergy, we found that there was convergence of TCR towards a common antigen (Figure 1G), where clonotypes became more closely related after an allergy (Figure 1H). Finally, we found that allergic patients had a lower proportion of public clonotypes (i.e. public clonotypic sequences found on VDJDB database) compared to non-allergic patients (P=0.02, Figure 1I), supporting the “hygiene hypothesis” as a predisposing factor in development of allergy.

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Summary/Conclusion: Higher TCR-repertoire diversity is associated with the risk of L-asparaginase hypersensitivity. This more diverse immunological mileau is characterized by infrequent, dissimilar, and less shared clonotypes, which increases the chance of a reactive clonotype matching to the allergen. Understanding the immunological basis of T-cell response in allergy may help in developing strategies to mediate this toxicity, to further improve outcomes in children with ALL.

P332: MIR-625-5P IS NOVEL CANDIDATE ONCOMIR IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IMPLICATED IN REGULATION OF APOPTOSIS VIA REPRESSION OF HARAKIRI

N. Maćkowska-Maślak 1,* , M. Drobna-Śledzińska 1 , R. Jaksik 2 , M. Kosmalska 1 , M. Witt 1 , M. Dawidowska 1

1 Institute of Human Genetics Polish Academy of Sciences, Poznan; 2 Institute of Automatic Control Silesian University of Technology, Gliwice, Poland

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL, arising from T-cell precursors. miRNAs are non-coding RNAs, contributing to leukemogenesis by involvement in key cellular functions such as proliferation, apoptosis, and signaling. We previously found in miRNA transcriptome analysis that miR-625-5p is overexpressed in T-ALL patients, thus is a potential oncomiR in T-ALL. Here, we confirm the implication of miR-625-5p in the regulation of apoptosis in T-ALL cells in vitro . We show that it acts via repression of HARAKIRI ( HRK ). HRK was originally identified as a proapoptotic gene induced by reduced levels of cytokine in hematopoietic cells and repressed by the expression of death-repressor proteins. Although its proapoptotic function is well described, little is known about the post-transcriptional mechanisms that may participate in HRK inactivation.

Aims: The aim of this study was to investigate the mechanism of miR-625-5p oncogenic action via unraveling its targetome and functional effects in T-ALL in vitro.

Methods: T-ALL JURKAT cells (with high endogenous miR-625-5p level) were transduced for stable inhibition of this miRNA. To evaluate the influence of miR inhibition on growth of T-ALL cells, we used flow cytometry GFP competition assay and CCK8 proliferation assay. To identify the genes mediating the effect of miR-625-5p, we subjected JURKAT cells to Ago2-RIP-seq. We used magnetic beads coated with anti-AGO2 antibody to immunoprecipitate RNA bound to RISC complexes. Ago2-IP and total RNA fractions were sequenced (polyA RNA-seq, 150 bp reads, 60M PE reads/sample, Illumina NovaSeq 6000). Transcripts depleted in Ago2-IP fraction upon miR inhibition were screened for the presence of miR-625-5p binding sites. Luciferase assay was performed to confirm direct interaction between hsa-miR-625-5p and 3’UTR of HRK gene. The changes of HRK protein level upon miR-625-5p inhibition were evaluated via Western Blot. To estimate the apoptosis rate in T-ALL cells we used two flow cytometry assays based on Annexin V and Caspase 3/7 staining.

Results: Inhibition of miR-625-5p decreased the growth of JURKAT cells. In RIP-seq analysis we identified 384 transcripts depleted in RISC by at least 20% upon miR-625-5p inhibition and having at least one putative 3’UTR binding site for this miRNA. Overrepresentation analysis performed for these genes indicated their involvement in the apoptotic process.

Among transcripts depleted in RIP-seq with predicted binding sites for miR-625-5p, we found HRK gene, previously reported to have proapoptotic activity. We postulate that repression of this gene by overexpressed miR-625-5p contributes to decreased apoptosis and to growth advantage of T-ALL cells.

To further confirm that miR-625-5p affects growth of T-ALL cells via negative regulation of apoptosis, we performed apoptotic assays in JURKAT cells and showed that inhibition of this miRNA increased apoptosis rate, in line with our hypothesis on the oncogenic potential of miR-625-5p.

We confirmed the direct interaction between miR-625-5p and HRK 3’UTR by luciferase assay. Additionally, in Western blot a subtle (approaching statistical significance) increase of HRK protein upon miR-625-5p inhibition was observed, supporting our notion that this miRNA is indeed a negative regulator of HRK.

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Summary/Conclusion: miR-625-5p is a novel candidate oncogenic miRNA in T-ALL, implicated in the negative regulation of apoptosis. Post-transcriptional repression of the proapoptotic HRK gene is a putative mechanism contributing to oncogenic properties of this miRNA when overexpressed in T-ALL cells.

P333: MULTI-COHORT GENE EXPRESSION RESOURCE FOR AUTOMATIC SUBTYPING OF B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

V.-P. Mäkinen 1,* , J. Rehn 1 , J. Breen 2 , D. Yeung 1 , D. White 1

1 South Australian Health and Medical Research Institute; 2 University of Adelaide, Adelaide, Australia

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and comprises multiple distinguishable genomic subtypes. RNA sequencing provides a functional snapshot that we propose will provide rapid diagnostic information for most ALL subtypes and deep biological insight to guide diagnostic efforts of atypical cases.

Aims: The aims of this study were to elucidate the predictive associations between mRNA-seq profiles and independently verified genomic lesions, and to develop easy-to-interpret transcriptome-wide biomarkers for ALL subtyping in the clinical setting.

Methods: A total of 1,279 ALL patients were included in the discovery dataset from six publicly available North American cohorts and 767 Australian ALL patients were included as an external validation cohort. Additional publicly available mRNA-seq data (n = 1,160) were used for quality control modelling. A novel batch correction method was introduced and applied to adjust for cohort differences. Three machine learning models (random forest, projections to latent structures and nearest Euclidean neighbor) were trained with the discovery set and validated in the Australian dataset.

Results: Out of 18,503 genes with usable expression, 11,830 (64%) were confounded by cohort effects and excluded. There were no substantial differences between the machine learning methods in the validation set; we chose the nearest neighbor technique as the simplest alternative. Six ALL subtypes (ETV6-RUNX1, KMT2A, DUX4, PAX5 P80R, TCF3-PBX1, ZNF384) that covered 32% of patients were robustly detected by mRNA-seq (PPV ≥ 87%). Five other frequent subtypes (CRLF2, hypodiploid, hyperdiploid, PAX5 alterations and Ph-positive) were distinguishable in 40% of patients, although overlapping transcriptional profiles led to lower accuracy (52% ≤ PPV ≤ 73%). Based on these findings, we developed the Allspice R package that predicts ALL subtypes and driver genes from unadjusted mRNA-seq read counts as encountered in real-world settings.

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Summary/Conclusion: We demonstrated high diagnostic accuracy for a fully automated mRNA-seq method for a third of ALL patients and additional contribution to diagnostic efforts for patients with inconclusive or mixed genomic characteristics.

P334: CHARACTERIZATION OF A DUX4-R INHIBITOR AS A POSSIBLE TREATMENT FOR ACUTE LYMPHOBLASTIC LEUKEMIA

S. Mara 1,* , V. Runfola 1 , M. Pannese 1 , C. Caronni 1 , R. Giambruno 1 , D. Campolungo 1 , C. Ghirardi 1 , D. Gabellini 1

1 Division of Genetics and Cell Biology, IRCCS San Raffaele Hospital, Milan, Italy

Background: Acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer and the major cause of cancer-related death before the age of 20. In up to 10% of cases, the disease is caused by translocation of the Double Homeobox 4 (DUX4) transcription factor to the immunoglobulin heavy chain (IGH) locus, giving rise to rearranged DUX4 (DUX4-r), maintaining the DNA binding domain (dbd) but with a rearranged C-terminus. Contrary to wild type DUX4, which induces apoptosis, DUX4-r acquires transforming ability in cellular and animal models. Through proteomics, we identified a potential DUX4-r inhibitor (iD) for its ability to bind directly to DUX4-r dbd.

Aims: The goal of my project is to test the antileukemic potential of iD in cellular and animal models of DUX4-r leukemia.

Methods: Human and murine cell lines and primary murine bone barrow progenitor cells are lentivirally-transduced to induce the expression of DUX4-r alone or in combination with iD, and the effects on proliferation, transformation, and B lymphoid differentiation potential are being evaluated.

To establish a B-ALL model, DUX4-r PDX cells have been expanded in NSG mice. These cells have been transduced with GFP or iD fused to GFP (GFP-iD) and injected into new NSG recipients for expansion. Serial xenograft transplantation of the purified transduced cells have been performed to test iD-dependent effect on PDX cells expansion and leukemia latency in mice. Human markers and GFP expression were monitored over time in peripheral blood (PB) to assess disease progression.

Results: We confirmed that DUX4-r can transform NIH-3T3 murine fibroblasts and inhibits B-cell differentiation of primary murine bone marrow progenitor cells in vitro . We found that iD decreases DUX4-r dependent transactivation. Moreover, lentiviral expression of iD in B-ALL NALM6 cells (carrying an endogenous DUX4-r translocation) reduces the levels of DUX4-r targets and impairs cell proliferation compared to transduced control cells. Preliminary results show that PDX cells expressing GFP-iD expand less and have a delayed PB engraftment compared to GFP control cells. Moreover, we noticed that in iD cohort only contaminant GFP-ve PDX cells take over in vivo despite purification by FACS sorting of transduced cells.

Summary/Conclusion: So far, iD expression is associated with reduced proliferation of human DUX4-r leukemia cells and reduced DUX4-r targets expression. Pre-clinical validation of iD could identify effective therapeutic approaches for the treatment of DUX4-r B-ALL patients.

P335: GENOMIC DETERMINANTS OF THERAPY RESPONSE IN ETV6-RUNX1 LEUKEMIA

L. Oksa 1,* , S. Moisio 2 , K. Maqbool 3,4 , H. Foroughi 3,4 , R. Kramer 2 , A. Nikkilä 1 , V. Zachariadis 5 , M. Enge 5 , K. Vepsäläinen 6 , J. Duque-Afonso 7 , J. Hauer 8 , V. Wirta 3,4 , O. Lohi 1,9 , M. Heinäniemi 2

1 Tampere Center for Child, Adolescent, and Maternal Health Research, Faculty of Medicine and Health Technology, Tampere University, Tampere; 2 The Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland; 3 Clinical Genomics facility, Science for Life Laboratory; 4 Department of Microbiology, Tumor and Cell biology; 5 Department of Oncology-Pathology, Karolinska Institute, Stockholm; 6 Department of Pediatrics, Kuopio University Hospital, Kuopio, Sweden; 7 Department of Hematology/Oncology/Stem Cell Transplantation, Faculty of Medicine, University of Freiburg Medical Center, Freiburg; 8 School of Medicine; Department of Pediatrics, Technical University of Munich, Munich, Germany; 9 Tays Cancer Centre, Tampere University Hospital, Tampere, Finland

Background: Acute lymphoblastic leukemia (ALL) is the most common cancer in children, with about 3500 children diagnosed yearly in Europe. A quarter of cases harbor the ETV6-RUNX1 (E/R) fusion gene. E/R leukemias are usually classified into low risk group. However, a fraction of patients still encounters disease recurrence. The increased relapse risk has been linked with a non-optimal initial therapy response as measured by minimal residual disease (MRD).

Aims: The aim of this study is to identify genetic differences between E/R cases stratified by MRD and to identify potential mediators of poor therapy response.

Methods: We analyzed the genetic landscape of E/R leukemia in a discovery cohort of 35 patients treated with the NOPHO2008 protocol using whole genome sequencing (WGS). Targeted sequencing was included in validation, including additional cohorts. Patients were classified into three categories (slow, intermediate or fast responder) based on MRD at the end of induction therapy (day 29). Variant callers available in the Balsamic workflow were used to detect DNA alterations and subclonal events analyzed using Battenberg and FastClone tools. Results were compared against published recurrent ALL mutations, bone marrow single cell RNA-sequencing and genome-wide CRISPR-screens in the E/R+ cell line REH, with vincristine, cytarabine, methotrexate, L-asparaginase, maphosamide, daunorubicin and 6-mercaptopurine sensitivity. Nalm-6 screen data was used for evaluating glucocorticoid response.

Results: We found comparable number of single nucleotide variants (SNVs), indels and structural variants (SVs) between the MRD-categories. However, based on mutation signature analysis, a significant negative correlation of MRD-level at day 15 was found with Signature 2 (APOBEC), while day 29 MRD correlated positively with Signature 3 (failure of DNA double-strand repair). Interestingly, the signature related to AID/APOBEC family has been reported with oncogenic role in T-ALLs, where APOBEC3 family genes display high expression during thymocyte development. We used single cell RNA-seq profiles from healthy bone marrow to study the relevance of APOBEC3 expression during precursor B-cell development, and showed that APOBEC3B is highly expressed in the dividing hematopoietic precursor, pro-B and especially pre-B cell stages.

Well-known ALL-related driver genes did not differ between MRD categories, with the exception of KRAS . Instead of activating mutations, we found prevalent deletions in the fast responder category.

To focus specifically on drug response modulating genes, we focused on genes that significantly increased or decreased sensitivity in genome-wide CRISPR screens (top 5% screen hits). Overall, each case harbored multiple DNA alterations in these genes. Moreover, fast responder cases had increased number of hits in sensitizing genes.

Summary/Conclusion: Combining MRD analysis with WGS studies carries the potential to reveal crucial information about sensitizing and resistance mutations and underlying cellular mechanisms. The found DNA variants that associate with MRD-status can be utilized in the design and implementation of personalized therapy in E/R+ ALL.

P336: A CHEMOTRANSCRIPTOMIC SCREENING IDENTIFIES THE REVERSAL OF GLUCOCORTICOID RESISTANCE IN NOTCH1 MUTATED T-ALL

L. Pagliaro 1,* , L. Moron Dalla Tor 1 , V. Federica 2 , P. Andrei 3 , L. Monica 3 , S. Kleissle 4 , M. Neuenschwander 5 , A. Gherli 1 , E. Cerretani 2 , A. D’Antuono 1 , E. Simoncini 1 , A. Montanaro 1 , G. Roti 1

1 Medicine and Surgery, University of Parma, Parma; 2 University of Ferrara, Ferrara; 3 University of Parma, Parma, Italy; 4 Max Delbrueck Center for Moleculare Medicine in the Helmholtz Association (MDC); 5 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

Background: Gain-of-function NOTCH1 mutations are the most common genetic abnormality in T-cell Acute Lymphoblastic Leukemia (T-ALL), accounting for 55-60% of the cases. Consequently, modulators of the Notch pathway, such as γ-secretase inhibitors (GSI), would be expected to have clinical efficacy (Rao, Cancer Res 2009). However, their application was limited by an excess of toxicity due to the suppression of wild-type (WT) NOTCH1 proteins in normal tissue (Deangelo, JCO 2006; Doody, Alzherimer’s Res Ther 2015).

In the past, we identified the Sarco-Endoplasmic Ca 2+ ATPase (SERCA) as a gatekeeper of the oncogenic Notch1 signaling. Thapsigargin (TG), a potent SERCA inhibitor (SI), possesses an anti-NOTCH1-leukemia activity both in vitro and in vivo by preferentially targeting mutated NOTCH1 proteins over WT ones (Roti, Cancer Cell 2013).

Aims: Since SIs display a favorable therapeutic index by targeting mutated NOTCH1 proteins, development of new SIs is under preclinical development (Marchesini, Cell Chem Biol 2020). Thus, is important to establish molecular mechanisms portending resistance in order to develop effective therapeutic strategies to overcome or prevent drug resistance.

Methods: We established drug-resistant clones (R) from parental ALL/SIL T-ALL cell line using a stepwise increase in treatment dose with TG. We quantified the abundance of differentially expressed (DE) genes ( P adj<0.05) in the sensitive or resistant cell line by RNA-seq and identified enriched pathways in the insensitive SI cell line. In parallel, we screened a small molecule library of nearly 2500 bioactive compounds (from the European Chemical Biology Library provided by EU-OPENSCREEN) in ALL/SIL and ALL/SIL R. Compound hits were marked by their ability to inhibit each cell line or both. Confirmatory experiments were completed in multiple T-ALL preclinical models.

Results: ALL/SIL R cells displayed a mutation (c.G770T -> p.Gly257Val) in the ATP2A2 gene occurring between the Asp254-Leu260 in the third SERCA2 transmembrane (TM) helix. This variation avoids the efficient TG binding to the catalytic domain, resulting in a diminished inhibitory effect. These cells showed an increase of TG IC50 >150-fold compared to the parental line while remaining partially sensitive to CAD204520, an inhibitor that binds SERCA in a pocket different from the one occupied by TG (Marchesini, Cell Chem Biol 2020). This result suggests that part of the resistance mechanism is transcriptionally mediated and virtually common to several SI. In SI-R cells, transcriptional analysis of 6241 DE genes revealed enrichment in steroids synthesis and response pathways, including cholesterol and lipid metabolism. Consistently, among small molecules targeting the steroid hormone receptors subfamily 3, glucocorticoids (GC) scored among the top hits in ALL/SIL R. While naïve cells were resistant to GC, R cells showed an enhanced sensitivity at low nanomolar concentrations of several GC including dexamethasone, clobetasol and fluticasone. This effect is at least in part mediated by the GC receptors since the R cells displayed an elevated GC receptors protein level, as showed in western blotting and RT-PCR, and the effect on viability were reversed by RU486 co-treatment.

Consequently, the association of SI plus GC displayed a synergistic effect in multiple preclinical models, including steroid-resistant cell lines, primary leukemia cells, and PDX models.

Summary/Conclusion: These findings suggest that SERCA-Ca 2+ modulation mediates GC and steroid signaling and that innovative SI can pharmacologically modulate glucocorticoid resistance in T-ALL.

P337: LONG TERM INFECTION-DRIVES CHANGES IN THE BONE MARROW MICRO-ENVIRONMENT OF PAX5+/- MICE PREDISPOSED TO B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

W. Liu 1 , O. Stencel 2 , Z. Lu 2 , C. Xu 1 , P. Lang 1 , F. Auer 3 , J. Hauer 3 , U. Fischer 2 , A. Borkhardt 2 , A. Pandyra 2,*

1 Department of Molecular Medicine II; 2 Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine University, Düsseldorf; 3 Department of Pediatrics, Technical University of Munich, Munich, Germany

Background: Pax5 heterozygosity ( Pax5 +/- ) in mice mimics germline or somatic Pax5 dysregulation (resulting in reduced Pax5 levels) observed in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). While a link between general non-specific infectious exposure and BCP-ALL has been demonstrated in the Pax5 +/- mouse model, the effects of a specifically tractable infection are poorly understood. In particular, little is known about potential interactions of pre-leukemic early progenitor B cell populations and immune cells present in the bone marrow microenvironment (BME) during an infection.

Aims: We aimed to characterize the short-term and long-term effects of a viral infection in the BME of the Pax5 +/- mouse model employing the Lymphocytic choriomeningitis virus (LCMV). LCMV is a non-cytopathic virus that has been extensively utilized to investigate virus-induced immunopathology, effector responses and immune tolerance.

Methods: Pax5 +/- mice backcrossed to the C57BL/6J background (N10) were infected with LCMV. Using flow cytometry, ELISA and plaque assay, innate and late adaptive immune responses in infected Pax5 +/- and WT ( Pax5 +/+ ) were assessed. Pre-leukemic early progenitor B cell populations, antigen-specific CD8 + T cells and regulatory T cells (Treg) were evaluated in the BME. This was complemented by short-term ex vivo assays where B cells were stimulated with differing immune stimuli.

Results: When we investigated immune responses in a short-term ex vivo culture system using the bone marrow of Pax5 -/+ and WT mice, we found differences in the induction of the interleukin 7 receptor (IL-7r) on immature Pro-B cells as well as the production of cytokines (IL-6 and TNF-▫▫) in response to the toll-like receptor (TLR7) agonist R848 and the lymphocytic choriomeningitis virus (LCMV) Docile strain. When we infected Pax5 -/+ and WT mice with LCMV-Docile, we found that innate (early interferon production) and adaptive immune responses (tetramer positive CD8 + T cells and early viral titers) were not intrinsically impaired in Pax5 -/+ mice. However, at day 90 post-infection, LCMV-specific T cells were present within the BME and tetramer specific CD8 + T cells raised against the LCMV nucleocapsid protein (np 396) were increased in the BM of Pax5 -/+ infected mice (Figure 1A). Furthermore, when we evaluated Major Histocompatibility Complex Class II (MHC-II) expression on different B cell subsets in the BME of infected and uninfected mice, we found that there was a significant upregulation in all immature B-cell subsets (Pro-B, PreBI-II) in response to infection in the WT but not in the Pax5 -/+ infected mice. Pax5 -/+ mice were susceptible to LCMV infection resulting in shorter long-term survival compared to infected WT mice (Figure 1B).

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Summary/Conclusion: This study demonstrates that the BME environment as well as survival of pre-leukemic Pax5 -/+ mice is differently affected by LCMV infection supporting the observations that immune dysregulation could contribute to the emergence of the leukemic clone 1 .

1 Supported by the Carreras foundation project no. DJCLS07R/2019

P338: BRANCHED CHAIN AMINO ACID TRANSAMINASE 1 ASSOCIATES WITH THE KU70/KU86 HETERODIMER AND MODULATES THE DNA DAMAGE RESPONSE IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS

L. Di Martino 1,* , V. Tosello 2 , S. Dalla Santa 1 , A. Papathanassiu 3 , G. Arrigoni 4 , P. van Vlierberghe 5 , E. Piovan 1,2

1 Surgery, Oncology and Gastroenterology, University of Padova; 2 Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, Padova, Italy; 3 Ergon Pharmaceuticals, LLC, Washington DC, United States of America; 4 Department of Biomedical Sciences, University of Padova, Padova, Italy; 5 Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Branched Chain Amino Acid Transferase 1 (BCAT1), a cytosolic aminotransferase converts BCAAs into their corresponding branched-chain α-keto acids and vice versa. Aberrant expression of BCAT1 has been demonstrated in different tumor models, including acute leukemia. However, its biological role in T-ALL remains to be elucidated.

Aims: The purpose of this study was to dissect the functional role of BCAT1 in T-ALL, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression.

Methods: We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (ALL0434 protocol). BCAT1 transcript and protein levels were determined in T-ALL cell lines (n=13) and patient derived xenografts (PDX; n=12). Metabolomics analysis (using 13 C 6 labeled Leucine) was performed following NOTCH1 inhibition. Further, five PDX samples were treated with a γ secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined by Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Kinetics of the DNA damage response (DDR) was evaluated in BCAT1 knock-down cells. Drug combination experiments using the DNA damaging agent (etoposide) and a specific BCAT inhibitor (ERG245) were performed in vitro and in vivo in PDX T-ALL models.

Results: We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Interestingly, NOTCH1 mutated PDX samples also showed higher BCAT1 expression compared to un-mutated cases. Metabolic studies disclosed that BCAAs oxidation is decreased following NOTCH1 inhibition (reduction in 3-methyl-2 oxovaleric acid). Further, inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. ChIP analysis and luciferase reporter assays suggest that NOTCH1 may directly regulate BCAT1 expression. Functionally, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair and rRNA processing. Indeed, we found Ku70/Ku86 proteins to interact with BCAT1. Evaluation of the DDR following DNA damage, disclosed that BCAT1 deficient cells present an accentuated but defective DDR which translates into marked DNA damage leading to pronounced cell death. Consistently, a marked synergistic therapeutic response was found between the DNA damaging drug etoposide and the BCAT specific inhibitor ERG245 in numerous T-ALL models.

Summary/Conclusion: NOTCH1 may directly regulate BCAT1 expression in T-ALL cells. BCAT1 is involved in sustaining genomic integrity following DNA damage through its interaction with the Ku70/Ku86 heterodimer, known to mediate classical non-homologous end-joining. Our results identify BCAT1 as a novel therapeutic target and suggest that the combination between DNA double stand break inducing agents (such as etoposide) and a BCAT inhibitor could be particularly useful in NOTCH1-mutant T-ALL cases.

P339: BOOSTING THE EFFICACY OF CD20-TARGETING IMMUNOTHERAPY IN B CELL ACUTE LYMPHOBLASTIC LEUKEMIA

M. Poprzeczko 1,* , K. Domka 1,2 , A. Pastorczak 3 , K. Fidyt 2 , L. Komorowski 1 , M. Winiarska 2 , A. Dabkowska 2 , K. Siudakowska 2 , A. Wojciechowska 2 , E. Patkowska 4 , M. Firczuk 1,2

1 Laboratory of Immunology, Mossakowski Medical Research Institute Polish Academy of Sciences; 2 Department of Immunology, Medical University of Warsaw, Warsaw; 3 Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Lodz; 4 Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland

Background: CD20 is a B cell-specific surface protein that appears at the stage of pre-B cells during B cell development. Currently, monoclonal antibodies (mAb) against CD20 are commonly used for immunotherapy of mature B-cell malignancies. B cell precursor acute lymphoblastic leukemia (BCP-ALL) originates from immature B cells in a bone marrow. Overall, the effects of treatment with high-dose multi-agent chemotherapy are good. However, for relapsed/refractory patients, novel treatment options (mainly immunotherapy-based) targeting B cell-specific antigens are already used in clinics and further optimized in preclinical models. Recent clinical trials conducted in Philadelphia (Ph) chromosome-negative BCP-ALL patients with CD20 expression revealed that the outcome of young adults may be improved by a combination of chemotherapy with rituximab, the anti-CD20 mAb. However, in most patients, the expression of CD20 on BCP-ALL blasts is heterogeneous and may be insufficient for effective treatment with CD20 mAb. Also, the regulation of CD20 in BCP-ALL is not well defined.

Aims: The aim of this study is to identify drugs that are able to upregulate CD20 in BCP-ALL, in order to improve the efficacy of anti-CD20 mAbs in preclinical models of the disease.

Methods: We tested the impact of 38 anti-cancer drugs used for the treatment of BCP-ALL as well as tested in clinical trials on the CD20 surface levels after 48hrs of incubation. For the in vitro studies, we employed cell lines and primary cells representing selected high-risk subtypes (Ph-positive, Ph-like, B-other). The levels of CD20 were determined by flow cytometry. Next, the potential of selected drugs to activate the anti-CD20 mAb, rituximab, was tested in a functional assay, Antibody-Dependent Cellular Cytotoxicity (ADCC) with Human Peripheral Blood Mononuclear Cells (PBMC) or primary NK cells, isolated from healthy donors.

Results: Based on the initial screening of cell lines, we selected 10 agents which increased the level of CD20: three histone deacetylases inhibitors, one inhibitor of MDM2 protein (p53 signaling pathway), one apoptosis inducer, three kinase inhibitors, one mitosis inhibitor, and one antimetabolic agent. Importantly, we found that many tyrosine kinases inhibitors and some retinoids significantly decreased CD20 levels in BCP-ALL cell lines. Some of these findings were also confirmed in primograft BCP-ALL blasts co-cultured with OP9 murine stromal cells. Furthemore, we found that five out of ten drugs enhance antibody-dependent cytotoxicity of rituximab, in functional assay. Moreover, two of the tested drugs increased phagocytosis of primograft BCP-ALL cells by primary macrophages differentiated from human peripheral blood monocytes.

Summary/Conclusion: In summary, we identified agents upregulating CD20 levels in BCP-ALL cells in vitro. More importantly, some of the selected drugs improved RTX-mediated effector mechanisms such as ADCC and immunophagocytosis. Further studies aiming to elucidate the mechanisms of CD20 upregulation and to validate our in vitro findings in murine models are ongoing.

P340: IN VITRO AND IN VIVO EFFICACY OF A NOVEL KINASE INHIBITOR TARGETING JAK2 GENE REARRANGEMENTS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA

M. Quadri 1,* , C. Saitta 1 , S. Palamini 1 , C. Palmi 1 , A. Biondi 1 , G. Cazzaniga 1 , G. Fazio 1

1 Centro di Ricerca Fondazione M. Tettamanti, Monza, Italy

Background: Although risk-based treatment is curative for 85% of children with B-cell precursor acute lymphoblasticleukemia (BCP-ALL), relapse remains a leading cause of mortality, urging the need of novel molecular targets. The JAK/STAT alterations represent about 7% of the ‘Philadelphia-like’ cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis and itsmutations have been widely studied, whereas JAK2 fusion genes are still poorly characterized.

Aims: This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients, developing a target strategy in preclinical models.

Methods: We applied RNA Next Generation Sequencing to find JAK2 fusion genes in a cohort of high risk BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. In vivo expansion of patients’ cells has been carried out in NSG mice. After drug treatments with JAK2 inhibitors, phosphoflow and apoptosis assays were done.

Results: We identified 10 pediatric cases carrying a JAK2 fusion with different partners, where PAX5 gene was the only recurrent.After in vivo expansion of cells from 3 cases, carrying PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2, we demonstrated that JAK2 signaling was activeat basal level, through phosphorylation of JAK2 Y1007-1008 compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.03); and also compared to P2RY8::CRLF2 rearrangements and JAK2 mutation(+40%, two-tailed P value 0.16). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated.

We targeted JAK2using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes and till48h, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5::JAK2, -22% in ATF7IP::JAK2 and -35% in ZEB2::JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%) and the significant reduction of phosphorylation on PI3K pathway,downregulating PDPK1, AKT, 4pEBP1 and pS6.

After 48h monotherapy treatment by CHZ868, we detected decrease in cell viability (20-75% at IC50). In combination with dexamethasone, a further decrease of viability was observed.

In the PAX5::JAK2 case, we also performed treatments with BIBF1120/Nintedanib, LCK inhibitor (activated downstream to PAX5 fusions) and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+45%, p<0.01), with reduction of apoptosis. Indeed, active caspase 3 increased after ruxolitinib and chloroquine (autophagy inhibitor) combination(+20% vs ruxolitinib alone, p<0.01). Instead, CHZ868 alone or in combination with chloroquine does not induce autophagy, with no effect on bothLC3-II and active caspase 3 levels.

Finally, we demonstrated the in vivo efficacy of CHZ868 in PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2 patient-derived-xenografts. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in BM(-43-85%), spleen(-72-89%), CNS (-13-62%) and PB (-46-80%).Moreover, CHZ868 in vivo treatment significantly reduced the phosphorylation of pJAK2 (-18-46%), pSTAT5 (-23-71%) and pAKT(Ser473) (-18-34%).

Summary/Conclusion: CHZ868 is a promising drugfor the treatment of JAK2 fusionsBCP-ALL. Further studies will include combination with standard chemotherapy drugs, by reducing the intensity and toxicity of chemotherapy.

P341: A NOVEL BISPECIFIC T CELL ENGAGER (UMG2-CD3) IS EFFECTIVE AGAINST CORTICAL-DERIVED ACUTE LYMPHOBLASTIC LEUKEMIA

C. Riillo 1,* , D. Caracciolo 1 , K. Grillone 1 , N. Polerà 1 , F. M. Tuccillo 2 , P. Bonelli 3 , G. Juli 1 , S. Ascrizzi 1 , F. Scionti 4 , M. Arbitrio 5 , M. Lopreiato 1 , M. A. Siciliano 6 , S. Sestito 6 , G. Talarico 7 , E. Galea 7 , M. C. Galati 7 , M. Rossi 7 , A. Ballerini 8 , M. Gentile 9 , M. T. Di Martino 1 , P. Tagliaferri 1 , P. Tassone 1,10

1 Clinical and Experimental Medicine, Università Magna Graecia, Catanzaro; 2 Istituto Nazionale Tumori “ Fondazione Pascale” - IRCCS; 3 Istituto Nazionale Tumori “ Fondazione Pascale” - IRCCS, Napoli; 4 Institute of Research and Biomedical Innovation (IRIB), Italian National Council (CNR), Messina; 5 Institute of Research and Biomedical Innovation (IRIB), Italian National Council (CNR); 6 Università Magna Graecia; 7 Pugliele-Ciaccio Hospital, Catanzaro; 8 biovelocITA srl, Milano; 9 Annunziata Hospital, Cosenza, Italy; 10 College of Science and Technology,Temple University, Philadelphia, United States of America

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived by T cell precursors and characterized by poor prognosis.The immunotherapy has revolutionized the outcome of B cell acute lymphoblastic leukemia (B-ALL), but the absence of tumor-specific T cell antigen hampers its efficacy in T-ALL. Therefore, the development of novel immune-therapeutic options for the management of this orphan disease is eagerly awaited. CD1a is a glycoprotein expressed on cortical T-ALL and only on healthy thymocytes and Langerhans cells. Taking into account its safe pattern of expression, CD1a might represent a valuable therapeutic target for thetreatment of T-ALL.

Aims: With the aim to provide an effective immune-therapeutic strategy for T-ALL, we developed a bispecific T cell engager (BTCE) derived from a novel UMG2 mAb that recognize a previously uncharacterized CD1a epitope.

Methods: To evaluate the specificity of UMG2 binding to CD1a epitope, HEK293T cells which do not express CD1a endogenously, have been used. In this regards, cells were transfected with a plasmid that encode for CD1a or with an empty vector and UMG2 reactivity has been evaluated by flow cytometry. To assess the unicity of UMG2 mAb binding, a competitive binding assay between UMG2 and commercially available CD1a antibodies has been performed. The UMG2 expression profile on peripheral blood cells from healthy donors and on a panel of cortical T-ALL cells has been evaluated. To develop a UMG2-CD3 construct, an asymmetric 2 + 1 UMG2-CD3 bispecific T cellengager (BTCE) has been generated by using knobs into hole technology. UMG2-CD3 T cell-redirected cytotoxicity has been evaluated on HEK293T wild type, on HEK293T-CD1a+ and on patient-derived T-ALL cells, co-cultured with peripheral blood mononuclear cells (PBMCs), CD4/CD8 depleted and CD56 enriched lymphocytes at different E:T ratio.Moreover, T cell activation has been assesed by flow cytometry. UMG2-CD3 anti-tumor activity against a CD1a+ T-ALL cells has been evaluated in vivo. For this purpose,Hu-PBMCs NSG mouse model has been generated and tumor growth has been assessed by fluorescent imaging probe via IVIS system.

Results: UMG2 mAb recognizes a novel CD1a epitope and does not compete with any of the commercially available anti-CD1a mAbs with the exception of a partial competition with NA1/34-HLK clone. A strong UMG2 reactivity has been observed on T-ALL cells, while no binding has been found on normal blood cells. A concentration-dependent T cell cytotoxicity on CD1a+ T-ALL cells co-cultured with PBMCs in the presence of UMG2-CD3 has been observed. Minimal UMG2-CD3 residual anti-tumor activity has been observed in CD4/CD8 depleted and CD56 enriched lymphocytes. CD56 depleted and Fc-blocked BMCs were able to induce an anti-T-ALL activity comparable to total PBMCs, demonstrating that UMG2-CD3 could not recruit monocytes and NK cells through Fc-FcyR interaction. Moreover, the concentration-dependent increase of i ) T cell proliferation, ii ) cytotoxic degranulation marker (CD107a), iii ) expression of cell surface activation markers (CD25, CD69),and iv ) pro-inflammatory cytokine secretion (IL-2, TNF-α, IFN-γ) has been observed in the presence of UMG2-CD3. Importantly, in an in vivo immune-humanized NSG mice model,UMG2-CD3 was able to significantly inhibit tumor growth and then conferring the survival advantage of treated animals.

Summary/Conclusion: All our findings, demonstrated that UMG2-CD3 BTCE represents a promising immune-therapeutic agent against T-ALL to be further investigated for clinical translation.

P342: BASELINE GENE EXPRESSION ANALYSIS OF RELAPSED ACUTE B-LYMPHOBLASTIC LEUKEMIA PATIENTS TREATED WITH INOTUZUMAB OZOGAMICIN

C. Sartor 1,* , I. Vigliotta 2 , V. Robustelli 1 , G. Cristiano 1 , J. Nanni 1 , L. Zannoni 1 , S. Parisi 2 , S. Paolini 2 , G. Marconi 1,3 , G. Martinelli 3 , A. Curti 2 , M. Cavo 1,2 , C. Terragna 2 , C. Papayannidis 2

1 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Istituto di Ematologia Seràgnoli; 2 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna; 3 Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, 47014, Meldola (FC), Italy

Background: Despite introduction of novel agents, relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) carries dismal outcome. Inotuzumab ozogamicin (InO) is a humanized anti-CD22 monoclonal antibody conjugated to calicheamicin approved for R/R B-ALL able to obtain high CR rates but of short duration. Mechanisms underlaying InO resistance are largely unknown.

Aims: To characterize the baseline differentially expressed genes in a series of R/R B-ALL patients in relation with patient response to InO in order to individuate potential pathways involved in resistance.

Methods: Gene expression profile of 18 R/R B-ALL patient samples was analyzed with RNA-seq, before InO exposure. All patients received at least 1 InO course. Patient population was divided in poor/non-responders (NR) and responders (R), based on InO response. R were defined as patients with duration of response (DoR) ≥3 months after bone marrow (BM) complete remission (CR) achievement. NR were defined either refractory or with a DoR <3 months after CR achievement. The list of significant (p<0.05) differentially expressed genes (DEGs) in NR, as compared to R whose absolute fold change (FC) was ≥2, was analyzed. P values were corrected with the Benjamini-Hochberg algorithm (false discovery rate; FDR). Gene expression results were analyzed with QIAGEN Ingenuity Pathway Analysis (IPA).

Results: Eighteen R/R BM samples of CD22-positive B-ALL were analyzed with RNA-seq. Eight patients were defined as NR and 10 patients as R. Patient disease characteristics in NR and R groups were homogeneous for age, sex, number of Philadelphia (Ph)-positive ALL patients, duration of 1 st remission, number of previous therapy lines including hematopoietic stem cell transplantation (HSCT) and Blinatumomab. At the time of InO therapy all patients had morphological BM relapse and all were CD22-positive. Median CD22 expression percentage on leukemic blasts was 100% (range 100-100) in NR patients and 100% (range 70-100) in R patients (p=0.177). Two patients in R group had a CD22 expression blast percentage of 70% and 76%. Median CD22-fluorescent intensity (CD22-FI) in NR as compared to R was 75.38 (IQR 59.58, 89.51) and 136.51 (IQR 114.38, 151.57) respectively, with significantly lower values in NR group (p = 0.04).

Overall, 370 genes were differentially expressed (p<0.05) in NR, as compared to R patients, of which 32 were significantly differentially expressed (corrected p < 0.05, FC ≥2). Thirty-one were down- and 1 was up-regulated in NR vs R. DEGs were involved in basophil differentiation, carbon dioxide transport, erythrocyte and myeloid cell development, heme metabolic and porphyrin-containing compound biosynthetic processes, cation and cellular ion homeostasis. Both IPA upstream regulator and regulator effect analysis identified the serine/threonine homeodomain-interacting protein kinase-2 (HIPK2) as predicted downregulated. The inhibition of HIPK2 was predicted to be the causal upstream condition of the under-expression of six DEGs from the set ( FECH, ANK1, SCL4A1, EPOR, GATA1, HBZ ) with activation Z score of -2.449 and p value of overlap = 1.02E-09. No difference in terms of HIPK2 expression was appreciated in the two groups, suggesting downregulation at post-transcriptional level.

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Summary/Conclusion: A unique pattern of gene expression signature based on HIPK2 downregulation was identified in poor responders to InO, providing potentially important insights in mechanisms of resistance. HIPK2 downregulation needs to be further validated.

CT and CP contributed equally

P343: GENOMIC CHARACTERISATION OF B-OTHER ALL IN UKALL2003 PATIENTS BY NEXT GENERATION SEQUENCING

C. Schwab 1,* , R. Cranston 1 , S. Ryan 1 , E. Butler 1 , E. Winterman 1 , Z. Hawking 1 , M. Bashton 1 , J. Murray 1 , A. Enshaei 1 , J. Gibson 1 , A. Vora 2 , A. Moorman 1 , C. Harrison 1

1 Leukaemia Research Cytogenetics Group, Newcastle University, Newcastle upon Tyne; 2 Department of Haematology, Great Ormond Street Hospital, London, United Kingdom

Background: Incorporating genetics into risk stratification for the treatment of childhood acute lymphoblastic leukaemia (ALL) has contributed to increased survival rates. About 30% of patients (B-other-ALL) harbour none of the known major chromosomal changes. Recently, we estimated that up to two thirds of B-other-ALL can be classified into novel subtypes using FISH and MLPA alone. We showed that ABL-class fusions and ERG deletions were linked to prognosis (Schwab et al , BJHaem, 2022).

Aims: To characterise B-other-ALL in the UKALL2003 trial using next generation sequencing (NGS), to evaluate the added benefits of these techniques and validate the prognostic significance of B-other-ALL subtypes.

Methods: B-other-ALL patients were tested using whole genome sequencing (WGS) (n=158) and bespoke targeted NGS (t-NGS) for the detection of abnormalities in 64 genes commonly mutated or rearranged in B-ALL (n=180), 42 patients had both WGS and t-NGS. Data were integrated with results from FISH, MLPA and cytogenetics.

Results: A representative cohort of 347 B-other-ALL was classified into subgroups, including six with ≥20 cases (Figure).

Among patients tested by WGS, 92% (n=146/158) were classified compared to 75% (n=104/138) tested by t-NGS. NGS-based approaches allowed for the detection of PAX5 P80R, IKZF1 N169Y and ZEB2 H1038R and fusion partner genes. Identification of DUX4-r was not possible using t-NGS.

PAX5 alt was the most frequently observed (n=90), including dic(9;20) (n=27), dic(9;12) (n=11), PAX5 fusions (n=21), PAX5 mutation (n=11) and PAX5 -ITD (n=12). Additionally, 11 patients were observed with PAX5 P80R mutations, classified as a separate subgroup due to the associated gene expression signature. PAX5 alt had an outcome similar to B-other-ALL overall (overall survival (OS) at 10yrs 82% vs 86%). There was no significant difference in outcome between PAX5 abnormalities, although an association with age was observed: dic(9;20) was more commonly observed in children aged 1-4 ( p < 0.001), while both dic(9;12) and PAX5 P80R were seen in older children aged 10-15 years ( p =0.005).

One subgroup with DUX4 rearrangements and/or ERG deletions ( DUX4 -r/ ERG -d) (n=78) had DUX4 involvement confirmed by WGS in 57 patients. In the remaining 21 patients ERG -d were identified by t-NGS and/or MLPA. ERG -d is exclusive to DUX4 -r patients, thus can be used as a surrogate marker. In line with other studies, patients with DUX4 -r had a lower relapse rate (RR) (5%) and improved OS (96%) compared to other subgroups.

Patients classified as ETV6-RUNX1 -like (n=21) were characterised by multiples abnormalities of ETV6 , including rearrangements (n=17) and/or deletion (n=12) as well as rearrangements (n=7) and/or deletions (n=6) of IKZF1 . An ETV6-RUNX1 -like gene expression signature was confirmed by RNA-Seq (n=6). No relapses or deaths were reported within this subgroup.

As previously reported, ABL-class fusions (n=25) were associated with an inferior outcome compared to other subgroups (RR=63% and OS=50%). Patients with rearrangements of CRLF2 (n=52) and ZNF384 (n=37) had similar outcomes to the cohort overall.

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Summary/Conclusion: A combination of techniques has classified a representative cohort of B-other-ALL patients from UKALL2003 into clinically relevant subgroups. The identification of DUX4 -r, subgroup defining mutations and fusion partner genes demonstrates the value of NGS-based approaches. We have confirmed the good and poor prognostic associations of DUX4 -r and ABL-class fusions, respectively, and identified ETV6-RUNX1 -like subgroup to have a good prognosis.

P344: THE DUAL BCL-2 AND BCL-XL INHIBITOR AZD4320 SHOWS ON-TARGET ACTIVITY IN ALL AND ACTS SYNERGISTICALLY WITH MCL-1 INHIBITION

M. C. Wichert 1 , A. Niedermayer 1 , S. Enzenmüller 1 , K.-M. Debatin 1 , L. H. Meyer 1 , F. Seyfried 1,*

1 Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany

Background: Targeting anti-apoptotic BCL-2 family proteins by BH3-mimetics has become a promising treatment strategy in acute lymphoblastic leukemia (ALL). Heterogeneous activity of the selective BCL-2 inhibitor venetoclax has been observed, but other anti-apoptotic proteins including BCL-XL promote venetoclax insensitivity. In clinical trials, targeting BCL-XL has previously resulted in a dose-limiting decrease of platelets. AZD4320 was developed as a dual inhibitor of BCL-2 and BCL-XL, and its dendrimer conjugate (AZD0466) was recently reported to demonstrate anti-tumor activity in hematological cancer models, while showing only a transient thrombocytopenia.

Aims: In this study, the anti-leukemia activities of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of MCL-1-selective AZD5991 were evaluated and compared to the effects of other BH3-mimetics (BCL-2-selective venetoclax, BCL-XL-selective A-1331852 and MCL-1-selective S63845 ). The on-target activity of the inhibitors was functionally characterized and combination effects were analyzed.

Methods: Cell viability assays were performed in ALL cell lines and patient-derived xenograft (PDX) samples analyzing half maximal effective concentrations (EC 50 ). Protein complexes and expression of apoptosis regulators were analyzed by immunoprecipitation and immunoblotting. Dynamic BH3 profiling using synthetic BH3-peptides was performed to determine the dependency of ALL cells on BCL-2 family proteins. Combination effects were assessed by dose-response matrix analyses.

Results: First, we determined the efficacy of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of the MCL-1 inhibitor AZD5991 for cell death induction in seven B-cell precursor (BCP) ALL cell lines and in a series of 13 PDX samples, showing heterogeneous responses. Interestingly, sensitivities of individual samples to both compounds were not associated with each other. However, we found a significant correlation of sensitivity to the dual inhibitor (AZD4320) with BCL-2 inhibition (venetoclax; N=13; r s =0.56; p=0.049), but no association with BCL-XL inhibition (A-1331852). Analyzing activities of all five BH3-mimetics including venetoclax, we found lowest EC 50 values for AZD4320, indicating particular sensitivity for this dual inhibitor as compared to the single inhibitors (p<0.001).

Investigating dependencies of ALL cells on BCL-2 family proteins by dynamic BH3 profiling, we found a shift towards MCL-1-dependence upon exposure to AZD4320, while AZD5991 induced an increased combined dependence on BCL-2 and BCL-XL, indicating potential synergistic activity of triple inhibition. Using co-immunoprecipitation analyses, we found that the exposure of ALL cells to AZD4320 reduced binding of both BCL-2 and BCL-XL to BIM, confirming on-target activity. Moreover, AZD5991 reduced binding of BIM to MCL-1. Accordingly, combined treatment with both inhibitors results in the release of BIM and downstream apoptosis signaling. Assessing combinatorial treatment in a primary PDX sample using multi-dose matrix analyses of both inhibitors revealed a positive mean Bliss synergy score of +4.8 indicating synergistic activity. Importantly, the highest synergism was found at low concentrations of both inhibitors, suggesting efficacy at moderate concentrations, which could potentially be achieved in vivo .

Summary/Conclusion: In summary, our study demonstrates sensitivity, on-target activity and synergism of the dual BCL-2 and BCL-XL inhibitor AZD4320 with inhibition of MCL-1, thereby providing strong evidence for further clinical evaluation in ALL.

P345: AN IMMUNE SCREEN IDENTIFIES 5-NONLOXYTRYPTAMINE AS A NOVEL ANTI-CANCER AGENT CAPABLE OF IMPROVING ANTI-TUMOR IMMUNITY IN COMBINATION WITH ANTI-PD1 THERAPY

P. Stachura 1,* , W. Liu 1 , A. Wlodarczyk 2 , C. Xu 1 , S. Bhatia 2,3 , P. A. Lang 1 , A. Borkhardt 2,3 , A. A. Pandyra 2,3

1 Department of Molecular Medicine II; 2 Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine-University Dusseldorf; 3 German Cancer Consortium (DKTK), Partner site Essen/Düsseldorf, Dusseldorf, Germany

Background: Exploiting the immune system, particularly CD8 + T cells to eliminate tumors has revolutionized the treatment landscape. However, despite the evident successes of CD8 + T cell-targeting immunotherapies such as anti-PD1 checkpoint inhibitors, many patients fail to respond especially those with poorly immunogenic tumors. Additional obstacles to successful immunotherapy responses include therapy-induced toxicity, lack of bio-markers of response, an immunosuppressive environment leading to T cell dysfunction and exhaustion and poor immune infiltration highlighting a need for novel combinatorial approaches to augment immunotherapeutic activity.

Aims: We aimed to uncover novel therapeutic agents capable of augmenting the anti-tumoral responses of CD8 + T cells in ex vivo systems and in vivo syngenetic poorly immunogenic tumor models.

Methods: To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen, using Lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells that were combined tumor cells expressing the LCMV gp33 (B16) peptide CTL epitope and incubated with an NIH 770 compound library. Hits that increased tumor-cell killing when combined with LCMV-primed splenic T cells were further validated ex vivo. The key candidate was assessed in vivo in C57BL/6J syngeneic tumor models. Immunocompromised NSG mice as well as antibody depletion were used validate CD8 + T cell dependence. In vitro and in vivo immunomodulatory effects were deciphered using flow cytometry, immunoblot, CRISPR/Cas9 and histological analyses.

Results: We identified 5-Nonyloxytryptamine (5-NL), a serotonin receptor (HTR) agonist, as increasing the ability of T cells to target tumor cells. In vitro, 5-NL induced apoptosis in melanoma and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) at low micromolar levels. 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8 + T cells as their depletion abrogated the phenotype (Figure 1A-B). 5-NL’s pro-immune effects were attributed to the upregulation of antigen presenting machinery in melanoma, BCP-ALL and other poorly immunogenic tumors with low MHC-I/HLAA-C expression (Figure 1C). Importantly, the upregulation of MHC-I/HLAA-C occurred without concomitant increases in PD-L1 expression and was linked to upregulation of cAMP Response Element-Binding Protein (CREB) in vitro and in vivo. As demonstrated through CRISPR/Cas9 HTR1D knockout, the immunomodulatory and pro-apoptotic effects of 5-NL occurred independently of receptor target signaling. 5-NL was successfully combined with an anti-PD1 antibody in vivo for maximal tumor growth inhibition.

Figure 1: The serotonin agonist 5-Nonyloxytryptamine (5-NL) was identified as potentiating

T cell mediated anti-tumor immunity. (A) C57BL/6J or (B) CD8 depleted C57BL/6J mice were subcutaneously injected with 5 x 105 gp33 expressing tumor cells. 7 days post-tumor injection, mice were randomized and treated daily 6.25 mg/kg of 5-NL or vehicle for five consecutive days and tumor volume was measured (n = 9-12). H2-Db (MHC-I, murine cell lines) and HLAA-C (human cells lines) were assessed by FACS analysis following treatment with 5-NL for 24 hours (n = 3-9). Error bars in all experiments indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed).

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Summary/Conclusion: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors and increasing their responsiveness to immunotherapies.

P346: THE ROLE OF CYTOKINES, TNF, TGFΒ1 AND NEUTROPHIL-TO-LYMPHOCYTE RATIO IN THE COURSE OF THE ACUTE LEUKEMIA

V. Barilka 1,* , V. Matlan 2 , S. Prymak 3 , O. Shalay 4

1 Laboratory of Immunology and Cytogenetics of Blood Neoplasms, SI “Institute of Blood Pathology and Transfusion Medicine of National Academy of Medical Science of Ukraine”; 2 Associate professor; 3 •Department Oncology and Medical Radiology, Lviv National Medical University named Danylo Galytsky; 4 Head of the •Laboratory of Immunology and Cytogenetics of Blood Neoplasms, SI “Institute of Blood Pathology and Transfusion Medicine of National Academy of Medical Science of Ukraine”, Lviv, Ukraine

Background: Lymphocytes (Lym) and mature segmental neutrophils (Neu) together with blasts are most commonly detected in the peripheral blood (PC) of patients (pta) with acute leukemia (AL). Besides, Neu, Lym and blast cells are an important source of transforming growth factor β1 (TGFβ1), tumor necrosis factor (TNF) production. Accumulation of TNF, TGFβ1 in the plasma of AL pts is considered an unfavorable sign in AL. However, the role of TNF, TGF β1 in the relationship with neutrophil-to-lymphocyte ratio (NLR) in the course of AL is not definitively established.

Aims: Determine the role of cytokines, TNF, TGF β1 in the relationship with NLR to assess the course of AL.

Methods: 42 adult AL pts were examined before treatment, who at the time of the examination had blast cells in the peripheral blood (52.54 ± 5.63 x 10 9 / L). The NLR was calculated from peripheral blood cell counts obtained at the time of diagnosis of AL by dividing the Neu count by the Lym count. The median NLR (0.99) was used to dichotomize patients into high- NLR and low- NLR groups. The concentration of TNF, TGF β1 was determined by biological methods using sensitive cell lines L929 and CCL 64, respectively. Statistical processing of the results was estimated by the value of p ˂ 0, 005. The correlation between the indicators was estimated by the value of r.

Results: There were 25 people in the low-NLR group. Median age 66 years (36 - 81 years). Among the pts were 9 people with ALL and 16 people with AML. Number of blasts 55, 83 ± 6.38 x 109 / L; New count was 11.73 ± 2.95 109 / L, Lym count - 21.27 ± 3.64 109 / L. The NLR index is 0.53 ± 0.09. In this group, 88% of pts died in the short term without achieving remission, the remaining 12% of pts had short-term remission.

The concentration of TNF in plasma was 1.20 ± 0.13 ng / ml, TGF β1 - 4.02 ± 0.48 ng / ml, which in both cases was higher than in healthy individuals (p˂0.001 and p˂0.05, respectively). TNF levels were positively correlated with Lym (r = 0.30) and blasts (r = 0.40), suggesting a possible role for TNF in neoplastic clone survival.

There were 17 people in the high-NLR group, including 10 pts with ALL and 7 people with AML who achieved remission. The median age is 38 years (from 16 to 68 years). The number of blasts was 33.20 ± 9.67 x 109 / L and was significantly lower than in the low-NLR group (p˂0.05); New count was 22.23 ± 0.10 x 109 / L; Lym count - 22.83 ± 3.04 109 / L. The NLR was 1.24 ± 0.16. In this group, 94% of pts had remission. Plasma TNF and TGF concentrations were lower than in the low-NLR group, apparently due to lower blast content. However, no statistical difference in cytokine concentration between the compared groups was found.

Summary/Conclusion: More favorable prognostic features in AL could be NLR, the median of which is equal to or greater than 0.99. Under these conditions, a decrease in the number of blast cells, the concentration of cytokines TNF and TGF β1 in the plasma and the achievement of remission were observed. The data obtained may indicate a prognostic role of Neu and Lym blood subpopulations in the course of AL and in the relationship with cytokines, TNF and TGF β1, may be potential targets for the correction of treatment of AL pts.

P347: COOPERATION OF TLX3 AND FLT3-ITD IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IS ENHANCED BY TLE4 INACTIVATION

Q. Van Thillo 1,2,3,* , L. Lauwereins 1,2,3 , S. Demeyer 1,2,3 , S. Provost 1,2 , N. Mentens 1,2 , C. de Bock 4 , J. Cools 1,2,3

1 Center for Human Genetics, KU Leuven; 2 Center for Cancer Biology, VIB; 3 Leuvens Kankerinstituut, KU Leuven - UZ Leuven, Leuven, Belgium; 4 Children’s Cancer Institute, UNSW, Sydney, Australia

Background: One fifth of T-cell acute lymphoblastic leukemia (T-ALL) cases is defined by the aberrant expression of TLX3 . Being similar to the closely related TLX1, its expression is mainly associated with mutations in the JAK/STAT pathway (Liu et al., Nat Genetics 2017), which have been shown to cooperate with TLX1 in driving T-ALL (Vanden Bempt et al., Cancer Cell 2018). However, it is not known whether TLX3 has the same oncogenic role as TLX1. Additionally, TLX3 expression is also regularly found with FLT3-ITD mutations, which are virtually absent in TLX1-positive cases (Liu et al., Nat Genetics 2017), suggesting there are some important functional differences between TLX3 and TLX1.

Aims: To set up a model of cooperation between TLX3 and FLT3-ITD and to elucidate the oncogenic role of TLX3 in T-ALL.

Methods: We performed in vivo bone marrow transplant (BMT) assays by transducing lineage-negative cells derived from C57BL/6 mice with the respective oncogenes using retroviruses. Pro-T cell growth curves were performed in the absence of interleukin-7 (Il7) as described previously (Van Thillo et al., Nat Communications 2021). Fluorescence activated cell sorting (FACS) staining was performed on the MACSQuant VYB (Miltenyi Biotec) or Fortessa (BD). RNA was prepared at the KU Leuven Genomics Core and 3’ prime end sequencing (QuantSeq) was performed. Differential gene expression analyses were conducted using the DeSeq2 R package (v1.22.0).

Results: The combination of TLX3 and FLT3-ITD in a constitutive BMT assay resulted in a rapid leukemia with an immature myeloid immunophenotype (CD11b+, Gr1-, CD4-, CD8-). The leukemia cells were positive for both FLT3-ITD (GFP) and TLX3 (mCHERRY), indicating cooperation between the two oncogenes in vivo . Similarly, in our in vitro pro-T-cell model, the TLX3 and FLT3-ITD double-positive cells outcompeted the single-positive cells after withdrawal of Il7. Next, we performed RNA-sequencing in CreER (estrogen receptor) pro-T cells 24 hours after activation of TLX3 expression by adding tamoxifen. This showed both up- and downregulation of many genes, including Tle4 among the downregulated genes. Interestingly, TLE4 expression was also lowest in the TLX3-subgroup of patients (Liu. et al. Nat Genetics 2017). TLE proteins are transcriptional co-repressors that can interact with the engrailed-homology 1 (Eh1) domain of transcription factors (Riz et al. Biochem Biophys Res Commun 2009). Therefore, we inserted a point mutation in the Eh1 domain of TLX3, resulting in a phenylalanine to glutamic acid substitution at position 18, to impair this interaction. Strikingly, TLX3(F18E) in combination with FLT3-ITD led to a proliferative advantage in pro-T cells after the omission of Il7 compared to wild-type TLX3. On top of that, cells with TLX3(F18E) and FLT3-ITD were able to grow in the absence of stem cell factor (Scf).

Summary/Conclusion: We demonstrate that TLX3 cooperates with FLT3-ITD in vivo and in vitro in a pro-T-cell context. TLX3 downregulates the co-repressor Tle4 and inhibiting the interaction between TLX3 and Tle4 reinforces the proliferative capacity of pro-T cells in the absence of growth factors.

P348: MRK-560 AND DEXAMETHASONE ARE SYNERGISTIC IN THE TREATMENT OF T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

C. Vandersmissen 1,2,3,* , C. Prieto Fernàndez 1 , O. Gielen 1 , K. Jacobs 1 , I. Govaerts 1 , J. Maertens 4 , H. Segers 5 , J. Cools 1

1 Human Genetics, KU Leuven; 2 Cancer biology, VIB; 3 Leuvens Kanker Institutie (LKI), KU Leuven - UZ Leuven; 4 Microbiology, Immunology and Transplantation, KU Leuven; 5 Oncology, KU Leuven - UZ Leuven, Leuven, Belgium

Background: NOTCH1 activating mutations are found in the majority of T-cell acute lymphoblastic leukemia (T-ALL) cases and inhibition of NOTCH1 activation is therefore a potential therapeutic option in T-ALL. Gamma-secretase inhibitors have been shown to block NOTCH1 activation, but are also associated with dose-limiting toxicity, preventing their use for T-ALL treatment. We have recently shown that a more selective inhibition of the gamma-secretases with PSEN1-selective γ-secretase inhibitors, such as MRK-560, retains a strong anti-leukemia effect with minimal gastrointestinal toxicities.

Aims: We wanted to investigate if MRK-560 would show synergy with currently used chemotherapeutic drugs, such as dexamethasone, vincristine and doxorubicin, and if it could be safely combined with chemotherapy for treating T-ALL patients.

Methods: Dose response curves were performed in DND-41 and SUPT-1 T-ALL cell lines, which were treated with DMSO/MRK-560 alone or in combination with different concentrations of chemotherapy. Apoptosis assay was conducted with annexin-V/PI staining. Human primary T-ALL samples were transduced with a luciferase vector to obtain luciferase positive xenografts in immunodeficient NSG mice that could be used for in vivo treatment. Mice were treated for 3 weeks (5 days on – 2 days off) with vehicle, dexamethasone (5mg/kg, IP), MRK-560 (15mg/kg, IP) or the combination. Leukemia progression was followed by weekly blood sampling and bioluminescent imaging after injection of luciferin.

Results: Treatment with MRK-560 increased the sensitivity to dexamethasone in NOTCH1-dependent T-ALL cell lines (DND-41 and SUPT-1) and we documented a strong synergy between these drugs. The combination between MRK-560 and doxorubicin/vincristine showed only an additive effect. Furthermore, we demonstrated that treatment with MRK-560 and dexamethasone increased apoptosis 2.5 fold compared to single dexamethasone treatment. Dexamethasone acts by activating the glucocorticoid receptor (NR3C1) which activates several target genes including BIM, an inducer of apoptosis. Mechanistically, we found that mRNA and protein levels for NR3C1 and BIM were significantly more upregulated after combination treatment compared to single drug treatment. This was associated with strong downregulation of HES-1 levels. These data indicate that MRK-560 synergizes with dexamethasone via downregulation of HES-1 (downstream of NOTCH1), resulting in enhanced glucocorticoid signaling with higher BIM expression levels and apoptosis. Finally, we also studied the synergy between MRK-560 and dexamethasone in patient-derived xenograft models of T-ALL. Bioluminescent imaging showed significant reduction in leukemia progression in combination treatment compared to single treatments. Furthermore, combination treatment (41 days) significantly prolonged survival compared to single MRK-560 (34 days), dexamethasone (30 days) or placebo (16 days) treatment.

Summary/Conclusion: We have shown that the PSEN1-selective gamma-secretase inhibitor MRK-560 can be safely combined with chemotherapy for the treatment of T-ALL patients thereby demonstrating a synergistic effect between MRK-560 and dexamethasone. Mechanistically, MRK-560 acts in a similar way as broad spectrum gamma-secretase inhibitors, but does not show gastrointestinal toxicity.

P349: CLONAL ARCHITECTURE DISSECTING AT SINGLE-CELL RESOLUTION REVEALS MECHANISMS OF CHEMOTHERAPY RESISTANCE AND RELAPSE IN T CELL ACUTE LYMPHOBLASTIC LEUKEMIA

J. Zhang 1,* , Y. Duan 1 , Y. Zhang 1 , X. Zhu 1

1 Institute of Hematology & Hospital of Blood Diseases,CAMS &PUMC, Tianjin, China

Background: T-lineage acute lymphoblastic leukemia (T-ALL) comprises approximately 10-15% of pediatric ALL cases with distinct feature in biology and largely inferior outcome compared to B-ALL. Growing evidence has reflected pivotal roles of clonal evolution in T-ALL recurrence, but bulk sequencing may not serve as the perfect model to reliably infer clonal heterogeneities and their immunomodulatory milieu during leukemia development. In this study, single-cell sequencing was applied to uncover leukemic clonal relationships with relapse throughout chemotherapy in T-ALL at a more accurate resolution.

Aims: We performed bulk and single cell multi omics analysis for diagnostic and relapse T-ALL, with the aim to dissect mechanisms of chemotherapy resistance and relapse in T cell acute lymphoblastic leukemia.

Methods: We performed bulk whole-exome sequencing for sorted CD7+ BMMCs from 5 pairs of diagnosis-relapse (Dx_Rel) samples, revealing a series of well-reported hotspot mutations in T-ALL.

To dissect clonal diversities within and across the 5 Dx_Rel T-ALL pairs, we carried out high-throughput droplet-based 5’-single-cell RNA-seq (scRNA-seq) and paired T cell receptor sequencing (scTCR-seq).

Results: By performing unsupervised clustering of scRNA- seq profiles encompassing 10 samples, we identified 23 distinct T-lineage clusters (Cluster 0-22) based on the two-dimensional UMAP visualization. In 2 out of 5 patients, diffusion map of T-lineage sub-clusters between diagnostic and relapsed samples appeared to be almost identical, while distinct shifts from diagnosis to relapse in the compositions have been observed in the other 3 out of 5 patients.

We sought to further deconvolute the clonal architecture trajectory for T-ALL Dx_Rel pairs. We observed that dominant diagnostic clones of 4 patients diminished or vanished at relapse, sparing newly emerged subclones predominantly substituted at relapse. We clearly depicted two distinct patterns of evolutionary trajectories in these 4 Dx_Rel pairs by comprehensively mapping hierarchical TCR clonotypes onto leukemic clonotypes at single cell levels. Specifically, in T956 and T723, we observed significant outgrowth of incidental diagnostic sub-clones at relapse, whereby surrogate TCR repertoires correspondingly enumerated, suggestive of dynamic shifts in dominant clone over continuous chemo-exposure. Whereas in T593 and T856, expanding clones at relapse were showed up with completely different gene signatures from the diagnostic ones, but dominant clones at diagnosis and relapse were surprisingly presented with identical TCR repertoires. This was undoubtedly informative of leukemic “clonal drift” within which hypothetical intrinsic transformation happened to the same subclones over persistent chemotherapy.

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Summary/Conclusion: Collectively, our presented study accurately distinguished leukemic cells from normal T cells in T-ALL at a single-cell resolution. By tracking transcriptomic profiles within and across Dx_Rel T-ALL pairs, we further identified distinct clonal evolutionary patterns, which may determine diversified fates of leukemic clones in response to therapeutic pressures, extending significant implications for future precise chemotherapies.

P350: BLINATUMOMAB AND DONOR LYMPHOCYTE INFUSION (DLI) FOR MOLECULAR RELAPSE AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION IN PEDIATRIC PATIENTS

F. Muriano 1,* , F. Cacace 2 , V. Caprioli 2 , M. R. D’amico 2 , G. De Simone 2 , G. Giagnuolo 3 , G. Menna 3 , F. P. Tambaro 2

1 Hematology, AOU Federico II; 2 BMT UNIT; 3 Hematology, AORN Santobono Pausilipon, Naples, Italy

Background: Detection of measurable residual disease (MRD) after Hematopoietic Stem Cell Transplantation (HSCT) is an early predictor of frank relapse. The role of DLI as a pre-emptive treatment to prevent relapse is still debated. Blinatumomab, a CD3/CD19 bispecific T-cell engager molecule, is approved in adult patients with MRD and in children with relapsed or refractory acute lymphoblastic leukemia (ALL).

Aims: We evaluated the safety and feasibility of blinatumomab plus DLI to treat MRD-positive pediatricALLafter HSCT. Here, we report our experience of three patients with post-transplant MRD-positive ALL who received blinatumomab and DLI.

Methods: Three pediatric patients with high-risk B-ALLwere treated according to AEIOP ALL 2009 and AIEOP ALL 2017 protocols and referred for allo-HSCT. Patients became MRD-positive after allo-HSCT and received blinatumomab with DLI.Patients’ characteristics are summarized in the Table.

Results: Patient 1 developed CNS relapse during maintenance, then received salvage therapy according to the INTREALL HR 2010 and 1 course of blinatumomab, achieving MRD-positive CR1.Patient 2, a 16-year old female, had secondary ALL and achieved CR1 after 1 course of chemotherapy and subsequently received 2 courses of blinatumomab, due to chemotherapy-associated toxicity and achieved MRD-undetectable CR1. Patient 3, a 17-year old male was primary refractory to 2 lines of chemotherapy and blinatumomab, received 2 courses of inotuzumab and achieved an MRD-positive complete remission (CR).All patients proceeded to allo-SCT and were MRD-positive at 8, 31 and 14 weeks post-allo-SCT, respectively. All patients discontinued immunosuppressive therapy and received 2, 1 and 4 courses of DLI with blinatumomab, respectively. Blinatumomab was started one week after each DLI, was given at a dose of 15-28cg/kg for a total of 4 weeks each course. Only patient 3 experienced Grade 2 liver GVHD. None of the patients experienced serious adverse events requiring blinatumomab discontinuation. Patient 3 developed isolated CNS relapse after 4 courses of combined DLI plus blinatumomab; patient 2 achieved and remains in MRD-undetectable CR with current follow up and a patient 1 achieved MRD-negative CR after two courses of combined treatment and CNS relapse.

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Summary/Conclusion: The role of cellular therapy with DLI is still debated in ALL. Most post-transplant relapses are associated withloss of surface HLA bythe leukemia cells, hence DLI alone has limited efficacy in patients with post HSCT relapse. Blinatumomab directs T cells to bind CD19 present on malignant B cells and engages CD3 on T cells causing activation and inducing cytotoxicity against ALL blasts. We, therefore, tested the efficacy of blinatumomab plus DLI in 3 MRD-positive patients with ALL after allogeneic HSCT. The use of blinatumomab allowed recruitment of fit donor-derived T lymphocytes (not exposed to immune suppressive agents) against ALL B cells. This hypothesis is supported by the fact thatpatient 3, who received blinatumomab pre-HSCT and had disease progression, probably due to lack of T cells showed by flow cytometry, achieved MRD-undetectablestatus after receiving DLI plus blinatumomab post-transplant. Twopatients reached stable MRD-undetectablestatusand 2 subsequently developed CNS relapse; none received CNS prophylaxis post-HSCT. We hypothesize that blinatumomab plus DLI can clear the hematologic disease, but it is not effective in preventing CNS relapse. Although very interesting, this hypothesis should be confirmed in a larger number of patients.

P351: DOSE-ADJUSTED EPOCH + INOTUZUMAB OZOGAMICIN (DA-EPOCH-INO) IS SAFE AND ACTIVE IN ADULTS WITH RELAPSED/REFRACTORY (R/R) B LYMPHOBLASTIC LEUKEMIA (B-ALL): INITIAL RESULTS OF A PHASE I TRIAL

R. Cassaday 1,2,3,* , K.-L. Garcia 1 , T. Gooley 2 , C. Martino 3 , M.-E. Percival 1,2,3 , A. Halpern 1,2,3 , V. Oehler 1,2,3 , J. Abkowitz 1,2,3 , R. Walter 1,2,3 , C. Ghiuzeli 1,3 , E. Estey 1,2,3

1 University of Washington; 2 Fred Hutchinson Cancer Research Center; 3 Seattle Cancer Care Alliance, Seattle, United States of America

Background: Despite new drugs, survival with R/R B-ALL remains poor. Combination therapies may improve outcomes, but none are proven superior, and toxicities may pose limits. InO has been added to low-intensity chemotherapy, but the risks/benefits of such combinations are poorly defined. As we recently found DA-EPOCH to be effective for untreated ALL, we here tested the addition of InO to DA-EPOCH (# NCT03991884 ).

Aims: The primary objective was to estimate the maximum tolerated dose (MTD) of InO when added to DA-EPOCH.

Methods: Eligible adults had R/R CD22+ B-ALL with ≥5% blasts in blood/marrow or ≥1 site of extramedullary disease [EMD] ≥1.5 cm in diameter. No history of sinusoidal obstructive syndrome (SOS) or chronic liver disease was allowed. Other key eligibility criteria included bilirubin ≤1.5x upper limit of normal (ULN), AST/ALT ≤2.5x ULN, creatinine ≤1.5x ULN, QTc ≤500 msec, and ECOG 0-2. All patients (pts) gave written informed consent.

DA-EPOCH was given on Days 1-5 with G-CSF. Three dose levels (DL) of InO given on Days 8 and 15 were studied in 28-day cycles: DL1, 0.3 and 0.3 mg/m 2 ; DL2, 0.6 and 0.3 mg/m 2 ; and DL3, 0.6 and 0.6 mg/m 2 (respectively). In Cycles 2+, EPOCH was dose-adjusted based on the hematologic nadir from the prior cycle. Up to 4 cycles were permitted. Hepatic prophylaxis with ursodiol and CNS-directed therapy with intrathecal chemotherapy was recommended.

MTD was the dose of InO yielding a true dose-limiting toxicity (DLT) rate of 33%. Initial dose-escalation plan for InO was a 6 + 6 design, but after 5 pts enrolled, this was modified to Bayesian Optimal Interval Design (BOIN) with target accrual of 24 pts. Secondary objectives were descriptions of treatment-related adverse events (TRAEs) per NCI CTCAE v5; efficacy by rate of complete remission without or with incomplete hematologic recovery (CR/CRi) and response at EMD sites per NCCN; measurable residual disease negativity (MRD-) by multiparameter flow cytometry (MFC) and high-throughput sequencing (HTS; clonoSEQ) of bone marrow aspirate; and survival.

Results: To date, 17 pts have been enrolled (Table). All are evaluable for DLT. 5 pts were treated at DL1; then per BOIN, 3 pts at DL2, and 9 pts at DL3. Median number of cycles given was 2 (range, 1-3). There have been 4 DLTs: 1 at DL2 (20%; grade 4 sepsis) and 3 at DL3 (33%; grade 4 hyponatremia, grade 4 SOS, and prolonged pancytopenia). The single case (6%) of SOS occurred after allografting and is ongoing. There have been no treatment-related deaths. Grade 3+ non-heme TRAEs were seen in 13 pts (76%). Those seen in >1 pt were infections (5 pts; 8 events), neutropenic fever (5 pts; 5 events), oral mucositis (3 pts; 4 events), and increased ALT (2 pts; 3 events); all these events resolved. Other hepatic toxicity was generally mild. Of 10 pts who received ≥2 cycles, 5 (50%) escalated the dosing of EPOCH, while 2 (20%) de-escalated.

17 pts are evaluable for response. Of 13 with >5% blasts, 12 (92%; 90% confidence interval 68-100%) achieved CR/CRi, 10 (77%) after 1 cycle. MRD- by MFC was achieved in 9 (75%); 1 had prior InO. In the 5 pts with EMD, 4 (80%) responded including 3 CRs. To date, 6 pts (35%) have undergone allograft post-study. Median follow-up of survivors is 7 mo, with 7 pts in ongoing remission beyond 6 mo.

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Summary/Conclusion: DA-EPOCH-InO has a manageable toxicity profile with infrequent DLTs and only 1 case of SOS in heavily pre-treated adults with B-ALL. Nearly all pts with >5% blasts at enrollment achieved CR, with most being MRD- by MFC. Responses in EMD were common. Enrollment is ongoing. Updated follow-up and responses by HTS will be presented.

P352: SYMPTOMATIC VTE INCIDENCE IN ADULT ALL TREATED WITH PEG-ASPARAGINASE COMPARED TO THE NATIVE L-ASPARAGINASE IN WITH AN ASPARAGINASE-BASED PROTOCOL

R. Chen 1,* , J. Seki 1 , L. Liu 1 , E. Atenafu 1 , K. Yee 1 , A. Schuh 1 , V. Gupta 1 , S. Chan 1 , A. Schimmer 1 , D. Maze 1 , M. Minden 1 , H. Sibai 1

1 University of Toronto, Toronto, Canada

Background: Venous thromboembolism (VTE) is a well-known complication in adults receiving aspraginase (ASNase)-based chemotherapy for adults in acute lymphoblastic leukemia, including the ASNase-intensive Dana-Farber Cancer Institute (DFCI) 91-01 protocol. Previous studies have shown an VTE incidence of up to 41% in patients receiving the DFCI protocol and up to 29% with prophylactic anticoagulations. Since 2019, native L-asparaginase is no longer available in the Canada such that pegylated (PEG)-ASNase has gradually replaced native e.coli ASNase (L-ASNase) as part of the DFCI protocol at our centre.

Aims: The object of this study is to evaluate the incidence of VTE between patients who received PEG-ASNase vs L-ASNase, with emphasis on the highest risk periods of induction and intensification phases.

Methods: A single centred retrospective cohort study of 249 adult patients with Philadelphia chromosome negative (Ph-) between 2011 to 2021 were conducted. There were 178 patients received L-ASNase between 2011 to 2019 and 71 patients received PEG-ASNase between 2018-2021. Of note, all patients received enoxaparin for DVT prophylaxis during the intensification phase.

Results: During the induction phase, 19 out of 178 (10.6%) patients who received L-ASNase during induction developed VTE, whereas 21 out of 71 (29.6%) patients with PEG-ASNase developed VTE (p-value = 0.0002, OR = 3.079, 95 CI 1.43-6.64), with multivariable analysis after adjusting for age, sex, weight, and central line types. Similarly, during the intensification phase, 19 out of 135 (14.1%) patients had VTE on L-ASNase while 10 out of 34 (29.4%) patients on PEG-ASNase developed VTE (p-value =0.014, OR= 2.94, 95% CI 1.21-7.13) after adjusting for age, sex, weight, and central line types. Of note, PICC line was noted to be associated with higher incidence of CVC lines with during induction (p-value= 0.021, OR= 2.52, 95% CI 1.15-5.51).

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Summary/Conclusion: PEG-ASNase is associated with higher incidence of VTE compared to L-ASNase, both during induction and intensification, despite the administration of prophylactic anticoagulation. Further investigations and strategies on reducing VTE risk associated with PEG-ASNase is needed in adult ALL patients who receive an asparaginase-based protocol.

P353: FORTY MONTHS UPDATE OF THE GIMEMA LAL2116 (D-ALBA) PROTOCOL AND ANCILLARY LAL2217 STUDY FOR NEWLY DIAGNOSED ADULT PH+ ALL

S. Chiaretti 1,* , R. Bassan 2 , A. Vitale 1 , L. Elia 1 , A. Piciocchi 3 , P. Viero 2 , F. Ferrara 4 , M. Lunghi 5 , F. Fabbiano 6 , M. Bonifacio 7 , N. Fracchiolla 8 , P. Di Bartolomeo 9 , P. Fazi 3 , M. S. De Propris 1 , M. Vignetti 3 , A. Guarini 10 , A. Rambaldi 11 , R. Foà 1

1 Translational and Precision Medicine, Sapienza University of Rome, Rome; 2 Hematology, Ospedale dell’Angelo, Venice; 3 GIMEMA Data Center, Fondazione GIMEMA – Franco Mandelli Onlus, Rome; 4 Hematology, Ospedale Antonio Cardarelli, Naples; 5 Translational Medicine, University of Eastern Piedmont, Novara; 6 Hematology, Hospital Villa Sofia-cervello, Palermo; 7 Medicine, Verona University, Verona; 8 Fondazione IRCCS, Ca’ Granda-Ospedale Maggiore Policlinico, Milan; 9 Hematology, Transfusion Medicine and Biotechnologies, Ospedale Civile, Pescara; 10 Molecular Medicine, Sapienza University of Rome, Rome; 11 Hematology and Bone Marrow Transplant Unit, ASST Papa Giovanni XXIII, Bergamo, Italy

Background: The outcome of adult Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients greatly improved since the introduction of tyrosine kinase inhibitors (TKIs). A further improvement was achieved with a chemo-free induction/consolidation strategy based on the sequential administration of dasatinib followed by blinatumomab - the D-ALBA GIMEMA LAL2116 trial - with overall survival (OS) and disease-free survival (DFS) rates of 95% and 88% at 18 months (Foa et al, NEJM 2020).

Aims: To provide an updated follow-up of the D-ALBA trial and to document the long-term management of previously enrolled patients.

Methods: In the D-ALBA trial, after the primary endpoint, patients were followed for 12 months. Data on subsequent treatment and survival are being collected in the ancillary study GIMEMA LAL2217.

Results: As reported, 63 patients were enrolled (median age 54 years, 24-82; no upper age limit); the median follow-up is 40 months (0.9-62.5). Since enrollment in the D-ALBA trial, 9 relapses have been documented: 4 hematologic (1 major protocol deviation and 1 in an 82-year old woman who rapidly went off study), 4 at CNS and 1 nodal; median time to relapse was 4.4 months (1.9-25.8). Six deaths were recorded (3 post-allogeneic transplant) in 1 st complete hematologic remission (CHR).

Of the 58 patients who started blinatumomab, 29 continued treatment with TKI: 21 with dasatinib (85% after receiving all 5 cycles of blinatumomab), 2 switched to imatinib due to intolerance (all after the 5 cycles of blinatumomab) and 5 switched to ponatinib for a molecular minimal residual disease increase or medical decision (66% had received all 5 cycles of blinatumomab). Twenty-nine patients were allografted, including 6 patients in 2 nd CHR: 9 from a sibling, 13 from an unrelated donor, 6 from a haploidentical donor and 1 from a cord blood. Six transplants were performed in 2 nd CHR. Before transplant, 8 patients received 2 and 3 cycles of blinatumomab, respectively, 5 4 cycles and 6 5 cycles (2 patients were allografted after 1 cycle). Among grafted patients, 6 deaths occurred, 3 of which in cases transplanted in 2 nd CHR; thus, the transplant-related mortality in 1 st CHR is 10%. When considered in a covariate model, transplant did not impact on both OS and DFS; similar results were obtained also considering allograft only in 1 st CHR. It must, however, be underlined that the allografted population was enriched in cases who did not achieve a molecular response (23 pts). Further investigation is ongoing on this aspect. In the updated follow-up, the estimated 48 months OS is 78% (95% CI: 66-92%) and the DFS is 75% (95% CI: 64-87%). DFS was significantly better in patients achieving a molecular response upon induction than in those who did not (100% vs 67%, p=0.016, Fig. 1A), with no events recorded in the group of complete molecular responders and positive-not-quantifiable cases. Furthermore, we confirmed the inferior DFS for patients carrying an IKZF1 plus genotype compared to that of cases with no IKZF1 deletions/ IKZF1 deletions alone (85% vs 47%, p=0.012, Fig. 1B).

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Summary/Conclusion: In the updated analysis of the D-ALBA trial and the ancillary GIMEMA LAL2217 study, with a median OS of 40 months, we confirm the reported very favorable outcome. Among the few relapsed cases, we observed a rather high incidence of CNS involvements. These results represent the scientific rationale for the ongoing phase 3 GIMEMA LAL2820 trial, where dasatinib has been substituted by ponatinib, CNS prophylaxis has been increased, and transplant allocation is based on genomic features and minimal residual disease monitoring.

P354: REAL-WORLD ANALYSIS OF THE RISKS OF THROMBOTIC AND BLEEDING EVENTS IN PATIENTS RECEIVING PEGYLATED ASPARAGINASE FOR TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA

B. Mautner 1 , B. Brewer 2 , J. Mort 1 , E. Pierce 1 , F. Ghamsari 1 , S. Dibenedetto 1 , E. Morse 1 , L. Wells 1 , A. Morris 1 , C. Allen 3 , I. Patel 4 , D. D’Souza 4 , C. Jackson 5 , M. Perciavalle 6 , B. Yelvington 6 , R. Miller 6 , K. Abernathy 6 , K. Walsh 6 , H. Wolfe 7 , S. Locke 7 , D. Reed 5 , V. Duong 4 , B. Dholaria 6 , T. LeBlanc 7 , R. Shallis 3 , M. Keng 1 , B. Horton 2 , F. El Chaer 1,1,*

1 Hematology and Oncology; 2 Public Health Sciences, Division of Translational Research and Applied Statistics, University of Virginia, Charlottesville; 3 Hematology and Oncology, Yale University, New Haven; 4 Hematology and Oncology, University of Maryland Medical Center, Baltimore; 5 Hematology and Oncology, Wake Forest Baptist Medical Comprehensive Cancer Center, Winston-Salem; 6 Hematology and Oncology, Vanderbilt University Medical Center, Nashville; 7 Hematology and Oncology, Duke Cancer Institute, Durham, United States of America

Background: The prognosis of younger adult patients with acute lymphoblastic leukemia (ALL) has improved with the adoption of pediatric-inspired chemotherapy regimens. PEGylated asparaginase (PEG-Asp) is essential to these regimens but is associated with an increased risk of coagulopathy, venous thromboembolism, and depletion of antithrombin III (AT3). There are limited data evaluating the risk of clinical coagulopathy (thrombosis and bleeding) and the optimal antithrombotic interventions in these patients are unknown.

Aims: We aimed to assess the risk of thrombotic and bleeding events in patients with ALL treated with PEG-Asp and compare the treatment modalities with either AT3 + fibrinogen repletion vs the addition of prophylactic anticoagulation (AC) with either subcutaneous heparin 5000 TID or subcutaneous lovenox 30-40mg/day.

Methods: We performed a multicenter retrospective analysis of patients ≥ 18 yo who received PEG-Asp for B- or T-ALL as part of multiagent chemotherapy from 2015-2020 treated across 6 US academic institutions. Categorical variables were summarized using means and standard deviations. The former was compared using Fisher’s exact test; the latter was compared using either the Wilcoxon text (for two groups) or the Kruskal-Wallis (for more than two groups) as appropriate. One way ANOVA test was used to compare continuous variables.

Results: Our study included 129 treated patients who met eligibility criteria for induction and 73 patients who underwent post-induction therapy. During induction, the majority of the patients (90%) had AT3 and fibrinogen repletion, while only 10% received additional prophylactic anticoagulation. The rates of any thrombotic events were lower in the AT3/cryoprecipitate-only treatment group (18% vs 50%, p=0.015). Venous thromboembolism accounted for the majority of the thrombotic events (91% and 100% in the AT3/cryoprecipitate vs AT3/cryoprecipitate + prophylactic anticoagulation, respectively). Bleeding rates were numerically higher in patients who received AT3/cryoprecipitate + prophylactic anticoagulation (33% vs 14%, p=0.18). The rates of thromboses or bleeding events were not different when either prednisone or dexamethasone were included in the induction treatment regimen. During the post-induction phase, the rates of thrombotic and bleeding events were similar across all groups. The median time to thrombotic event was 11 days after the last PEG-Asp administration during induction, and 123 days during post-induction.

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Summary/Conclusion: Although the increased thrombotic risk in adult patients receiving PEG-Asp is well-characterized, rates of reported thromboses vary widely. Studies examining bleeding risk associated with PEG-Asp regimens are even scarcer and with more variable outcomes. In our study, there was both a statistically significant increase in the rate of any thrombotic event as well as a trend towards increased bleeding in patients receiving AT3/cryoprecipitate + prophylactic anticoagulation versus those receiving AT3/cryoprecipitate alone. More investigation into the relative thrombotic and bleeding risks associated with PEG-Asp is needed to determine optimal antithrombotic interventions in adult patients with ALL.

P355: MINI-HYPER-CVD PLUS INOTUZUMAB OZOGAMICIN, WITH OR WITHOUT BLINATUMOMAB, IN OLDER ADULTS WITH NEWLY DIAGNOSED B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA: UPDATES FROM A PHASE II TRIAL

F. Haddad 1,* , H. Kantarjian 1 , N. Short 1 , F. Ravandi 1 , N. Jain 1 , W. Macaron 1 , T. Kadia 1 , Y. Alvarado 1 , N. Daver 1 , G. Borthakur 1 , C. DiNardo 1 , M. Konopleva 1 , W. Wierda 1 , J. Jacob 1 , E. Roy 1 , C. Loiselle 1 , A. Milton 1 , J. Rivera 1 , R. Garris 1 , E. Jabbour 1

1 Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Blinatumomab (Blina) and inotuzumab ozogamicin (INO) improve the outcomes of patients (pts) with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). These drugs may allow for use of less chemotherapy and improve remission durations and overall survival (OS) in older pts with newly diagnosed B-ALL.

Aims: To evaluate the combination of low-intensity chemotherapy and INO with or without Blina, in older pts with newly diagnosed B-ALL.

Methods: Pts ≥60 years with newly diagnosed Philadelphia chromosome (Ph)-negative B-ALL were eligible to receive mini-hyper-CVD for up to 8 cycles. Initially, INO was given at 1.3-1.8mg/m 2 on day (D) 3 of cycle 1 and 0.8-1.3mg/m 2 on D3 of cycles 2-4. Rituximab (if CD20+) and prophylactic IT chemotherapy were given for the first 4 cycles. POMP maintenance was given for up to 3 years. Starting with pt #50, the protocol was amended and INO was administered in fractionated doses with each of the 4 cycles of hyper-CVD (0.6 mg/m 2 on D2 and 0.3 mg/m 2 on D8 of cycle 1; 0.3 mg/m 2 on D2 and 8 of cycles 2-4) followed by 4 cycles of blina. The cumulative doses of INO given before and after this amendment were 4.3 mg/m 2 and 2.7 mg/m 2 , respectively. Maintenance was also amended to include 12 cycles of POMP and 4 cycles of blina (1 cycle of blina after every 3 cycles of POMP).

Results: 80 pts have been treated with a median age of 68 years (range, 60-87); 30 pts (38%) were ≥70 years. 6 pts were in complete remission (CR) at enrollment and unevaluable for morphological response. Pt characteristics are summarized below (Figure 1 A ).

The overall response rate (ORR) among 74 evaluable pts was 99% (CR, n=66; CRi, n=7). MRD negativity by flow cytometry was achieved in 61/76 pts (80%) after 1 cycle and 74/79 pts (94%) overall. The 30-day and 60-day mortality rates were 0% and 3%, respectively. Among 79 pts who achieved remission, 33 (42%) are still in ongoing remission without stem cell transplant (SCT), 31 (39%) died in remission, 11 (14%) relapsed without SCT, and 4 (5%) proceeded to SCT in first remission (1 of whom subsequently relapsed). Notably, 9 pts (11%) later developed MDS/AML, 7 of whom had a TP53 mutation. Overall, 6 pts (8%) developed VOD, 1 after allogeneic SCT.

With a median follow-up of 55 months (range, 3-119), the 5-year continuous remission and OS rates were 76% and 47%, respectively (Figure 1 B ). Pts aged 60-69 years had better outcomes compared with pts ≥70 years (5-year OS rates: 59% vs 27%, respectively; P=0.04) and as did those without poor cytogenetics (i.e. complex , KMT2A -rearranged, low-hypodiploidy/near-triploidy) compared with poor cytogenetics (5-year OS rates: 54% and 25%, respectively; P=0.02). Deaths in remission were more frequent in pts ≥70 years compared with those 60-69 years (70% vs 35%; P=0.005). The 5-year OS rates for pts age 60-69 without poor-risk cytogenetics (n=37), age 60-69 with poor-risk cytogenetics (n=13), age ≥70 without poor-risk cytogenetics (n=24), and age ≥70 with poor-risk cytogenetics (n=6) were 69%, 39%, 36% and 0% respectively (Figure 1 C ). Presence of a TP53 mutation did not significantly impact OS (P=0.22).

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Summary/Conclusion: Reduced-intensity chemotherapy plus INO, with or without blina, resulted in an ORR of 99% and a 5-year OS rate of 47% in older pts with newly diagnosed Ph-negative B-ALL. Outcomes were particularly favorable in those 60-69 years of age without poor-risk cytogenetics. Pts ≥70 years were at a higher risk of death in remission. A chemotherapy-free approach, with INO plus blina only, is therefore now being explored in this population.

P356: SUBGROUP ANALYSES OF KTE-X19, AN ANTI-CD19 CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELL THERAPY, IN ADULT PATIENTS WITH RELAPSED OR REFRACTORY B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (R/R B-ALL) IN ZUMA-3

B. D. Shah 1,* , R. D. Cassaday 2 , J. H. Park 3 , R. Houot 4 , O. O. Oluwole 5 , A. C. Logan 6 , N. Boissel 7 , T. Leguay 8 , M. R. Bishop 9 , M. S. Topp 10 , D. Tzachanis 11 , K. M. O’Dwyer 12 , M. L. Arellano 13 , Y. Lin 14 , M. R. Baer 15 , G. J. Schiller 16 , M. Subklewe 17 , M. Abedi 18 , M. C. Minnema 19 , W. G. Wierda 20 , D. J. DeAngelo 21 , P. Stiff 22 , D. Jeyakumar 23 , J. Dong 24 , S. Adhikary 24 , L. Zhou 24 , P. C. Schuberth 24 , B. Kharabi Masouleh 24 , A. Ghobadi 25

1 Moffitt Cancer Center, Tampa, FL; 2 University of Washington, Fred Hutchinson, Seattle Cancer Care Alliance, Seattle WA; 3 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 4 CHU Rennes, University Hospital Rennes, Inserm & EFS, Rennes, France; 5 Vanderbilt University Cancer Center, Nashville, TN; 6 UCSF Medical Center, San Francisco, CA, United States of America; 7 Hôpital Saint-Louis, Paris; 8 Service d’hématologie clinique et thérapie cellulaire Hôpital du Haut-Leveque CHU de Bordeaux, Bordeaux, France; 9 The University of Chicago Medicine, Chicago, IL, United States of America; 10 Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany; 11 University of California San Diego, San Deigo, CA; 12 Wilmot Cancer Institute of University of Rochester, Rochester, NY; 13 Winship Cancer Institute of Emory University, Atlanta, GA; 14 Mayo Clinic, Rochester, MN; 15 University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD; 16 David Geffen School of Medicine at UCLA, Los Angeles, CA, United States of America; 17 Ludwig-Maximilians-Universität München, Munich, Germany; 18 University of California Davis Comprehensive Cancer Center, Sacramento, CA, United States of America; 19 University Medical Center Utrecht, on behalf of HOVON/LLPC, Utrecht, Netherlands; 20 The University of Texas MD Anderson Cancer Center, Houston, TX; 21 Dana-Farber Cancer Institute, Boston, MA; 22 Loyola University Chicago Stritch School of Medicine, Maywood, IL; 23 University of California Irvine Medical Center, Irvine, CA; 24 Kite, a Gilead Company, Santa Monica, CA; 25 Washington University School of Medicine, St Louis, MO, United States of America

Background: Brexucabtagene autoleucel (KTE-X19) is approved in the US for the treatment of adult patients with R/R B-ALL based on the positive results of the ZUMA-3 study. In the pivotal Phase 2 portion of ZUMA-3, the overall complete remission (CR) rate (CR + CR with incomplete hematologic recovery [CRi]) was 71% (N=55; median follow-up 16.4 months; Shah et al. Lancet 2021).

Aims: To assess outcomes in ZUMA-3 by prior number of therapy lines, prior blinatumomab, prior allogeneic stem cell transplant (alloSCT), and subsequent alloSCT.

Methods: Eligible adults had R/R B-ALL and received a single infusion of KTE-X19 following leukapheresis and conditioning chemotherapy. Outcomes assessed by independent review are reported in all patients treated at the pivotal dose (1×10 6 CAR T cells/kg).

Results: As of 23 July 2021, the median follow-up among all treated patients in Phase 2 (N=55) was 26.8 months (range, 20.7-32.6). At baseline, 10 patients (18%) had 1 prior line of therapy, and 45 patients (82%) had ≥2 prior lines. The overall CR/CRi rates (95% CI) for patients with 1 or ≥2 prior lines of therapy were 90% (55-100) and 67% (51-80), respectively. In patients with 1 or ≥2 prior lines of therapy, the median (95% CI) duration of remission (DOR) was 4.7 months (1.8-not estimable [NE]) and 14.6 months (9.4-NE), and the median (95% CI) overall survival (OS) was not reached (NR) (2.1-NE) and 25.4 months (14.2-NE), respectively. Grade ≥3 cytokine release syndrome (CRS) occurred in 10% and 27% of patients with 1 or ≥2 prior lines of therapy, and Grade ≥3 neurologic events occurred in 30% and 24%, respectively.

Among the 25 patients (45%) who had prior blinatumomab at baseline, the overall CR/CRi rate (95% CI) was 60% (39-79), and 80% (61-92) in the 30 patients (55%) without prior blinatumomab. The median (95% CI) DOR was 19.1 months (1.3-NE) and 10.3 months (5.2-NE) in patients with or without prior blinatumomab, and the median (95% CI) OS was 14.2 months (3.2-26.0) and NR (18.6-NE), respectively. In patients with or without prior blinatumomab, rates of Grade ≥3 CRS were 24% and 23%, and rates of Grade ≥3 neurologic events were 20% and 30%, respectively.

The overall CR/CRi rate in the 23 patients (42%) who had prior alloSCT vs the 32 patients (58%) who did not was 70% (47-87) vs 72% (53-86), respectively. In patients with vs without prior alloSCT, the medians (95% CI) for DOR were 14.6 months (8.7-23.6) vs NR (4.7-NE), and the medians (95% CI) for OS were 25.4 months (14.2-NE) vs NR (9.0-NE), respectively. Grade ≥3 CRS occurred in 17% and 28% of patients with or without prior alloSCT, and Grade ≥3 neurologic events occurred in 26% and 25%, respectively.

For patients who achieved CR/CRi and did (n=10) or did not (n=29) proceed to subsequent alloSCT post–KTE-X19 infusion, the median (95%CI) DOR was NR (NE-NE) and 14.6 months (8.7-23.6), and the median (95% CI) OS was NR (7.6-NE) and 26.0 months (18.6-NE), respectively.

A pooled analysis of all Phase 1 and 2 patients treated at the pivotal dose (N=78) was newly conducted, with a median follow-up of 29.7 months (range, 20.7-58.3). The similar results observed in this expanded data set as shown in the Table further support the subgroup outcomes described above in Phase 2 patients.

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Summary/Conclusion: Adults with R/R B-ALL benefitted from KTE-X19, with manageable safety, regardless of prior exposure to blinatumomab or prior alloSCT, though survival appeared better in patients without these prior therapies and in earlier lines of therapy, with limited patient numbers in some subgroups.

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P357: MULTICENTER RETROSPECTIVE ANALYSIS OF CLINICAL OUTCOME OF ADULT PATIENS WITH MIXED-PHENOTYPE ACUTE LEUKEMIA (MPAL) DIAGNOSED AND TREATED IN THE LAST TEN YEARS. A CAMPUS-ALL STUDY.

D. Lazzarotto 1,* , I. Tanasi 2 , A. Vitale 3 , M. Piccini 4 , M. Dargenio 5 , F. Giglio 6 , F. Forghieri 7 , N. Fracchiolla 8 , M. Cerrano 9 , E. Todisco 10 , C. Papayannidis 11 , M. Leoncin 12 , M. Defina 13 , F. Guolo 14 , C. Pasciolla 15 , M. Delia 16 , P. Chiusolo 17 , A. Mulè 18 , A. Candoni 1 , M. Bonifacio 2 , G. Pizzolo 2 , R. Foà 3

1 Clinica Ematologica, Azienda Sanitaria Universitaria Friuli Centrale, Udine; 2 Dipartimento di Medicina, Sezione di Ematologia, Università di Verona, Verona; 3 Ematologia, Dipartimento di Medicina Traslazionale e di Precisione, “Sapienza” Università di Roma, Roma; 4 SOD Ematologica, AOU Careggi, Firenze; 5 S.C. Ematologia, Ospedale Vito Fazzi, Lecce; 6 Unità di Ematologia e Trapianto di Midollo Osseo, IRCCS Ospedale San Raffaele, Milano; 7 S.C. Ematologia, Azienda Ospedaliero Universitaria di Modena, Modena; 8 U.O. Ematologia, IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milano; 9 S.C. Ematologia 2, AO Città della Salute e della Scienza, Torino; 10 Divisione di Ematoncologia, Istituto Europeo di Oncologia, Milano; 11 Dipartimento di Oncologia e Ematologia, Policlinico S. Orsola-Malpighi, Bologna; 12 U.O. di Ematologia, Ospedale dell’Angelo, Mestre; 13 UOC Ematologia, Azienda Ospedaliero Universitaria Senese, Siena; 14 Ematologia e Terapie Cellulari, IRCCS Policlinico San Martino, Genova; 15 U.O. di Ematologia, IRCCS Istituto Tumori Giovanni Paolo II; 16 U.O. Ematologia con Trapianto, Azienda Ospedaliero-Universitaria Consorziale, Policlinico di Bari, Bari; 17 Servizio di Ematologia, Policlinico Gemelli, Roma; 18 Divisione di Ematologia ad indirizzo oncologico, A.O. Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy

Background: Mixed phenotype acute leukemia (MPAL) is a very rare disease in adults. Most data derive from small retrospective series, some including also pediatric patients. Generally, treatment is similar to that of acute lymphoblastic leukemia (ALL), but the outcomes in adults and the role of allogeneic stem cell transplantation (AlloSCT) are not well defined.

Aims: Purpose of this retrospective study was to provide data on a large cohort of adult patients diagnosed and treated in 18 Italian hematology centers in the last 10 years.

Methods: Seventy-seven adult patients diagnosed with MPAL according to the EGIL and WHO criteria in the last 10 years (2011-2021) and treated with curative intent were included in the study. Endpoints were the CR rate after induction, overall survival (OS), disease-free survival (DFS) and rate of AlloSCT.

Results: Forty-eight of the 77 (62%) patients had B/myeloid MPAL, 27 (35%) had T/myeloid MPAL and 2 (3%) had B/T/myeloid MPAL. Median age at diagnosis was 49 years (range 17 - 77); it was lower in patients with T/myeloid MPAL than in B/myeloid MPAL cases (39 vs 54 years, P=0.04). Extramedullary involvement was detected in 26/77 (34%) of patients (mainly lymph nodes and spleen), but none had a central nervous system involvement.

Cytogenetic analysis was normal in 40% of patients; 36% of cases had a complex karyotype, 24% had other cytogenetic abnormalities (including 8% with a BCR-ABL1 rearrangement). FLT-ITD mutation was detected in 14% of patients. No differences were found between B/myeloid and T/myeloid MPAL cases.

Thirty/77 (39%) of patients were treated with an acute myeloid leukemia (AML)-like induction regimen, while 47 (61%) were treated with an ALL-like induction. Globally, CR rate after induction was 62% and 42% of these cases were also MRD negative. Patients treated with an AML-like induction had a higher rate of refractory disease after induction (33% vs 10%, P=0.02). The death during induction (DDI) rate was similar in the 2 groups (7% vs 8%). In univariate analysis an ALL-like therapy was the only variable associated with a better CR rate (P=0.0447). Patients refractory to AML-like induction were mainly salvaged with a subsequent AlloSCT or ALL-like therapy followed by an AlloSCT, and 60% of them achieved a CR.

The median OS and DFS of the entire cohort were respectively 41.9 and 37.6 months, with an OS and DFS at 5 years of 43% and 39%, respectively. Age, type of induction therapy, karyotype, presence of FLT3-ITD mutation, extramedullary involvement and type of MPAL (B/myeloid vs T/myeloid) were tested in univariate analysis for OS and DFS. Age below 60 years was the only variable associated with a better OS (median OS of 67 months vs 26 months, P=0.0138), while no variable impacted on DFS.

AlloSCT was performed in 50 patients (65%), 80% of them (40 patients) as part of the frontline treatment (36/40 patients transplanted in CR1). Among transplanted patients, we observed a 5-year OS of 54% (median not reached), that improved to 69% in patients transplanted as part of the frontline treatment.

Summary/Conclusion: This study describes one of the largest cohorts of adult patients with MPAL. These data outline that this disease responds better to ALL-like induction therapy than to AML-like therapy and that consolidation therapy should include, whenever possible, an AlloSCT. Prospective studies are needed to uniform the therapeutic approach and to identify variables capable of predicting outcome and to guide therapy.

P358: COMPREHENSIVE DIAGNOSTICS OF ACUTE LYMPHOBLASTIC LEUKEMIA BY WHOLE TRANSCRIPTOME RNA SEQUENCING

J. Li 1,*

1 SINO-US Diagnostics, Tianjin, China

Background: Acute lymphoblastic leukemia (ALL) has genetic heterogeneity, which is helpful to clinical diagnosis, prognosis stratification and treatment guidance. Gene aberrations are structurally diverse and are currently analyzed by different assays.

Aims: This study aims to establish a single and comprehensive platform for ALL diagnosis by whole transcriptome RNA sequencing (WTRS), which can detect fusion, mutations and expression.

Methods: We analyzed the data of 70 ALL patients by WTRS.

Results: We found that the fusion coincidence rate of WTRS was 100% (19 / 19) comparing with quantitative real-time polymerase chain reaction (qPCR). At the same time, six rare fusion forms were detected, which increased the diagnosis rate of fusion gene from 27% (19 / 70) to 36% (25 / 70). The expression of CRLF2 gene in 5 cases was consistent with that of qPCR. Seven cases with IKZF1 gene skipping identified by qPCR were consistent with the results of WTRS, and a rare IKZF1 deletion subtype was detected. Of the 51 genes associated with ALL, 100% (103/103) gene mutation at the RNA level by WRTS is consistent with the results at the DNA level by targeted DNA sequencing (except 3 splice site mutations and 4 mutations with poor coverage).

Summary/Conclusion: In conclusion, the standardization of experimental operation and improvement of biological information pipline of WRTS are very important factors in its use in standard diagnostic procedures. WRTS has the potential to provide accurate comprehensive diagnostic information for clinic and further improve the ALL diagnostic rate.

P359: IMPROVED OVERALL SURVIVAL AND MRD CLEARANCE WITH BLINATUMOMAB VS CHEMOTHERAPY AS PRE-TRANSPLANT CONSOLIDATION IN PEDIATRIC HIGH-RISK FIRST-RELAPSE B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL)

F. Locatelli 1,* , G. Zugmaier 2 , C. Rizzari 3 , J. Morris 4 , B. Gruhn 5 , T. Klingebiel 6 , R. Parasole 7 , C. Linderkamp 8 , C. Flotho 9 , A. Petit 10 , C. Micalizzi 11 , Y. Zeng 4 , R. Desai 12 , W. Kormany 4 , C. Eckert 13 , A. Möricke 14 , M. Sartor 15 , O. Hrusak 16 , C. Peters 17 , V. Saha 18,19 , L. Vinti 1 , A. von Stackelberg 20

1 IRCCS Ospedale Pediatrico Bambino Gesù, Sapienza, University of Rome, Rome, Italy; 2 Amgen Research (Munich), Munich, Germany; 3 MBBM Foundation, ASST Monza, University of Milano-Bicocca, Monza, Italy; 4 Amgen Inc., Thousand Oaks, United States of America; 5 Universitaetsklinikum Jena, Jena; 6 Universitätsklinikum Frankfurt am Main, Frankfurt, Germany; 7 Azienda Ospedaliera di Rilievo Nazionale Santobono Pausilipon, Napoli, Italy; 8 Medizinische Hochschule Hannover, Hanover; 9 Universitätsklinikum Freiburg, Freiburg, Germany; 10 Hopital Armand Trousseau, APHP.Sorbonne Université, Paris, France; 11 Istituto Pediatrico di Ricerca e Cura a Carattere Scientifico G Gaslini, Genova, Italy; 12 IQVIA Inc., Durham, United States of America; 13 Charité Campus Virchow-Klinikum Pädiatrie m.S. Onkologie/Hämatologie, Berlin; 14 Univ.-Klinikum Schleswig-Holstein, Kiel, Germany; 15 Westmead Hospital, Sydney, Australia; 16 Charles University, Motol University Hospital, Prague, Czechia; 17 St Anna Children’s Hospital, Vienna, Austria; 18 Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom; 19 Tata Translational Cancer Research Centre, Tata Medical Center, Kolkata, India; 20 Charite Universitaetsmedizin CVK Berlin, Berlin, Germany

Background: Children with high-risk first-relapse B-ALL have a poor prognosis. We previously reported that, as compared with chemotherapy, treatment with blinatumomab resulted in prolonged event-free survival (EFS) and overall survival (OS) and higher rates of minimal residual disease (MRD) remission in a phase 3 trial (JAMA 2021;325:843-54; ASH 2021:Abs 1231).

Aims: Here we provide follow-up (FU) data as of Sep 2021, with a focus on survival and MRD response.

Methods: In this phase 3 trial (Amgen NCT02393859 ), children >28 days and <18 years old with high-risk first-relapse B-ALL were randomized 1:1 after induction and 2 cycles of consolidation to receive either a third intensive multidrug chemotherapy consolidation or blinatumomab (15 μg/m 2 /day, 4 weeks, continuous IV infusion). Study enrollment required M1 (<5% blasts) or M2 (≥5% and <25% blasts) bone marrow (BM) at randomization. Patients with complete remission (CR, ie, M1 BM) after treatment could undergo allogeneic hematopoietic stem cell transplant (alloHSCT). EFS, the primary endpoint, was calculated from randomization to whichever occurred first of relapse or M2 BM after CR, failure to achieve a CR at the end of treatment, second malignancy, or death (any cause). Survival outcomes and response rates were analyzed by baseline MRD, ie, MRD at enrollment prior to chemotherapy or blinatumomab. Parents or a legally acceptable representative provided written informed consent.

Results: Enrollment was stopped based on the interim analysis for benefit of blinatumomab (7/17/19 primary analysis). The following results reflect an updated analysis as of Sep 2021. Between Nov 2015 and Aug 2019, 111 patients were randomized: 54 (49%) to blinatumomab and 57 (51%) to chemotherapy at 47 centers in 13 countries. Baseline characteristics were comparable in both arms as previously reported. After a median FU of 44 months, EFS was significantly higher with blinatumomab vs chemotherapy (at 4 years: 59% vs 27%, stratified log-rank p<0.001, hazard ratio [HR]: 0.35, 95% CI: 0.20-0.61). Blinatumomab also showed a strong benefit for the secondary endpoint of OS (at 4 years: 77% vs 49%, stratified log-rank p=0.002, HR: 0.34, 95% CI: 0.17-0.69). EFS, OS, and MRD remission (<10 -4 blasts) were all improved with blinatumomab, both overall and by baseline MRD subgroup (< or ≥10 -3 ) (Table 1a-b, Figure). Further, patients with and without extramedullary disease both had a benefit with blinatumomab. AlloHSCT in second CR was performed in 51/54 patients in the blinatumomab arm and in 39/57 patients in the chemotherapy arm. No new safety signals were identified. Second relapses occurred in 16/54 (30%) patients in the blinatumomab arm and 34/57 (60%) patients in the chemotherapy arm. In the blinatumomab arm, these second relapses were extramedullary in 5/54 patients, CNS in 5 (9.3%), 2 of which were solely CNS. In the chemotherapy arm, these second relapses were extramedullary in 8/57 patients, CNS in 3 (5.3%), 2 of which were solely CNS. CD19-negative relapse was seen in 3 patients in the blinatumomab arm (3/54) and 1 patient in the chemotherapy arm (1/57) prior to any subsequent CD19-directed therapy (ie, after study therapy).

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Summary/Conclusion: In children with high-risk first-relapse B-ALL, treatment with 1 cycle of blinatumomab vs chemotherapy before alloHSCT resulted in superior EFS and improved OS and MRD response rates, all independent of baseline MRD. The incidence of CD19-negative relapse after blinatumomab was low.

P360: EFFICACY AND SAFETY OF DARATUMUMAB IN PEDIATRIC AND YOUNG ADULT PATIENTS WITH RELAPSED/REFRACTORY T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA OR LYMPHOBLASTIC LYMPHOMA: RESULTS FROM PHASE 2 DELPHINUS STUDY

A. Vora 1,* , T. Bhatla 2 , D. Teachey 3 , F. Bautista 4,5 , J. Moppett 6 , P. Velasco Puyó 7 , C. Micalizzi 8 , C. Rossig 9 , N. Shukla 10 , G. Gilad 11,12 , F. Locatelli 13 , A. Baruchel 14 , C. M. Zwaan 4,15 , E. A. Raetz 16 , N. Bandyopadhyay 17 , L. Lopez Solano 18 , R. M. Dennis 18 , R. Carson 18 , L. E. Hogan 19

1 Department of Haematology, Great Ormond Street Hospital for Children, London, United Kingdom; 2 Department of Pediatrics, Children’s Hospital of New Jersey at Newark Beth Israel Medical Center, Newark, NJ; 3 Division of Oncology, Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 4 Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands; 5 Department of Pediatric Oncology and Hematology, Hospital Niño Jesús, Madrid, Spain; 6 Paediatric Haematology, Bristol Royal Hospital for Children, Bristol, United Kingdom; 7 Servicio de Oncología y Hematología Pediátricas, Vall d’Hebron Hospital, Barcelona, Spain; 8 Clinical Experimental Haematology Unit, IRCCS Istituto Giannina Gaslini, Genova, Italy; 9 University Children’s Hospital Münster, Münster, Germany; 10 Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 11 Department of Pediatric Hematology-Oncology, Schneider Children’s Medical Center of Israel, Petach Tikva; 12 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 13 Department of Pediatric Hematology and Oncology, IRCCS Ospedale Pediatrico Bambino Gesù, Sapienza University of Rome, Rome, Italy; 14 Pediatric Hematology and Immunology Department, Robert Debré University Hospital (APHP and Université de Paris), Paris, France; 15 Department of Pediatric Oncology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, Netherlands; 16 New York University Langone Medical Center, New York, NY; 17 Janssen Research & Development, LLC, Raritan, NJ; 18 Janssen Research & Development, LLC, Spring House, PA; 19 Department of Pediatrics, Stony Brook Children’s, Stony Brook, NY, United States of America

Background: Approximately 15-20% of pediatric patients with T-cell acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL) will be refractory to/relapse after frontline treatment; relapsed disease is associated with poor outcomes. In a phase 2 study, 2 of 7 (28.6%) patients with T-cell ALL in first relapse achieved a complete response (CR) using the vincristine, prednisone, PEG-asparaginase, and doxorubicin (VPLD) reinduction backbone. Daratumumab (DARA), a human IgGκ monoclonal antibody targeting CD38 approved for treating multiple myeloma, has shown preclinical efficacy in ALL models.

Aims: Here we report the initial results of DARA plus VPLD in pediatric and young adult patients with relapsed/refractory T-cell ALL or LL enrolled in the phase 2, open-label DELPHINUS study.

Methods: Eligible patients were aged 1-30 y, had T-cell ALL or LL in first relapse or refractory to 1 prior induction/consolidation regimen, and had a performance status ≥70. DARA (16 mg/kg IV QW) was given with VPLD in Cycle 1 and with methotrexate, cyclophosphamide, cytarabine, and 6-mercaptopurine in Cycle 2. Patients received age/risk-adjusted intrathecal therapy. Response was measured at the end of each cycle by local bone marrow morphology. The primary endpoint for ALL patients was CR rate in pediatric patients at the end of Cycle 1. Patients achieving CR after Cycles 1 or 2 could proceed to allogeneic HSCT off study. Overall response rate (ORR) was defined as CR or CR with incomplete hematological recovery (CRi) at any time before start of subsequent therapy or HSCT. Minimal residual disease (MRD) negativity (<0.01%) at any time before disease progression, start of subsequent therapy, or HSCT was centrally reviewed by flow cytometry.

Results: Twenty-four pediatric (age 1-17 y) and 5 young adult (age 18-30 y) ALL patients and 10 LL patients (age 1-30 y) received ≥1 DARA dose. Median (range) age was 10.0 (2-25) y (ALL) and 14.5 (5-22) y (LL); median (range) time from initial diagnosis to first study treatment was 2.0 (0.1-6.1) y (ALL) and 0.8 (0.5-6.0) y (LL). Pediatric ALL patients received a median (range) of 2 (1-3) treatment cycles; young adult ALL patients and LL patients each received 2 (1-2). Among pediatric ALL patients, 10 (41.7%) patients (90% CI, 24.6-60.3) achieved CR at the end of Cycle 1. ORR was 83.3% (CR, 13 [54.2%] patients; CRi, 7 [29.2%] patients) in pediatric and 60.0% (all CR) in young adult ALL patients and 40.0% (all CR) in LL patients. Ten (41.7%) pediatric ALL patients achieved MRD negativity. All pediatric ALL patients had a grade 3/4 TEAE. No pediatric ALL pt discontinued DARA primarily due to AEs and 1 (4.2%) died due to TEAEs (brain edema and hepatic failure) attributed to study treatment but unrelated to DARA.

Summary/Conclusion: The addition of DARA to VPLD in pediatric and young adult patients with relapsed/refractory T-cell ALL or LL showed initial activity, generating improved response rates compared to those achieved with backbone therapy alone, with a manageable safety profile.

P361: EARLY MEASURABLE RESIDUAL DISEASE ANALYSIS IS ONLY PREDICTIVE OF SURVIVAL OUTCOMES AFTER TWO CYCLES OF TREATMENT IN ADULTS WITH B-LYMPHOBLASTIC LEUKEMIA RECEIVING HYPER-CVAD INDUCTION.

R. Nedumannil 1,2,* , D. Ritchie 3,4 , A. Bajel 3,4 , A. P. Ng 3,5 , S. J. Harrison 3,4 , D. Westerman 1,4

1 Department of Pathology, Peter MacCallum Cancer Centre, Melbourne; 2 Department of Diagnostic Haematology, Royal Melbourne Hospital, Parkville; 3 Department of Clinical Haematology, Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, Melbourne; 4 Sir Peter MacCallum Department of Oncology, The University of Melbourne; 5 Immunology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia

Background: Measurable residual disease (MRD) monitoring using multi-parametric flow cytometry (MFC) has a well-established role in B-lymphoblastic leukemia (B-ALL). However, the optimal time-point (TP) for early MRD testing and the associated prognostic impact remain undefined in adult B-ALL patients receiving Hyper-CVAD induction chemotherapy, a dose-intensive regimen comprising alternating A cycles (hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone) and B cycles (methotrexate and cytarabine).

Aims: To evaluate the utility of MRD analysis by MFC after one cycle (TP1) in comparison to that after two cycles (TP2) of induction treatment in adult B-ALL patients receiving frontline Hyper-CVAD chemotherapy.

Methods: A retrospective chart review was performed of consecutive patients older than 18 years with newly diagnosed B-ALL who received frontline Hyper-CVAD induction chemotherapy at two Australian tertiary referral centres from 2010-2020. Patient inclusion required data regarding remission and MRD statuses after each 21-day cycle of induction treatment (defined as two [A and B] cycles of Hyper-CVAD chemotherapy). Two flow cytometry panels (a two tube 8-color panel and a single tube 10-color panel) were used; samples were analyzed using Kaluza Analysis Software (Beckman Coulter) on FACSCanto II (BD Biosciences) and Navios EX (Beckman Coulter) cytometers, respectively. The sensitivity of these assays was 0.001%-0.01%. Clinicopathological and survival data [pertaining to overall survival (OS) and event-free survival (EFS)] were collected from review of patient records.

Results: 49 of 76 (64%) adult B-ALL patients received frontline Hyper-CVAD chemotherapy and had available MFC MRD assessments at each of the two TPs. 19 patients (39%) had Philadelphia chromosome-positive (Ph+) disease and received concomitant tyrosine kinase inhibitor (TKI). Median times to TP1 and TP2 relative to start of treatment were 21 and 45 days, respectively. At TP1, 40 patients (82%) achieved complete remission (CR), amongst whom 12 (30%) were MRD negative. At TP2, the majority of patients (48, 98%) achieved CR, of whom 23 (48%) were MRD negative. Among those who achieved CR after Hyper-CVAD induction, 25 (52%) received Hyper-CVAD consolidation chemotherapy ± TKI while 24 (50%) proceeded to alloSCT in first CR.

After cycle 1A, patients with residual morphological disease compared to those who attained CR had a median OS of 10 months and 44 months, respectively (HR, 6.15; 95% CI, 1.36-27.94; P = 0.019), while the median EFS was 58 months and not reached, respectively (HR, 1.31; 95% CI, 0.33-5.17; P = 0.423). MRD negativity at TP1 was not associated with a significant improvement in either OS ( P = 0.426) or EFS ( P = 0.335) when compared to patients with MRD positivity. In contrast, MRD negativity at TP2 was associated with significantly higher OS ( P = 0·005) and EFS ( P = 0.047) over patients with MRD positivity. Multivariate analysis demonstrated that KMT2A -rearrangement and MRD positivity at TP2 were the only factors (including Ph+ status) that correlated with worse EFS and OS.

Summary/Conclusion: In the absence of residual morphologic disease, MRD analysis after one cycle of Hyper-CVAD induction chemotherapy does not correlate with survival outcomes when compared to MRD testing after two cycles of Hyper-CVAD in adult B-ALL patients. Although validation with larger studies is required, initial MRD testing after two cycles of Hyper-CVAD induction (i.e. after day 42) promotes appropriate resource utilization and ensures no loss in the prognostic power of early MRD monitoring in adult B-ALL.

P362: MINIMAL-LOSS-CYTOMETRY FOR EVALUATION OF CEREBROSPINAL FLUID IN PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA. SINGLE CENTER STUDY.

M. Nováková 1,2,* , I. Janotová 2 , E. Vodičková 3 , A. Houdková 3 , J. Hanzalová 4 , M. Vášková 1,2 , D. Kužílková 1,2 , E. Mejstříková 1,2 , L. Šrámková 2 , J. Starý 2 , O. Hrušák 1,2

1 CLIP-Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University; 2 Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol; 3 Department of Clinical Hematology, University Hospital Motol; 4 Department of Immunology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czechia

Background: Central nervous system (CNS) involvement at diagnosis of acute lymphoblastic leukemia (ALL) represents a risk factor for CNS relapse. Currently, examination of the cerebrospinal fluid (CSF) is performed and evaluated quantitatively using a counting chamber and qualitatively using cytomorphological analysis of cytospin. Recently, flow cytometry (FC) evaluation of CSF was shown to provide more reliable results and to predict relapse risk (de Haas et al., 2021, Thastrup et al., 2020). Independently of these studies, we applied a lyse-no wash technique aiming at minimizing cell loss during preparation and at improved cell concentration estimate.

Aims: Our aim was to compare our findings with morphological criteria and published data and correlate them with clinical outcome in pediatric patients with ALL.

Methods: We included pediatric patients with newly diagnosed ALL (04/2014 – 01/2022), whose CSF examination was performed in CLIP laboratory (n=208). Patients were classified as B cell precursor ALL (BCP ALL; 85%) and T ALL (15%) and treated according to following protocols: AIEOP-BFM ALL 2009 (n=118), AIEOP-BFM ALL 2017 (n=71), EsPhALL(n=8), Interfant06 (n=5), off-protocol (n=6). During follow up, 14 patients relapsed (5 CNS incl. combined, 9 non-CNS). CSF (drawn into tube with no fixative agent and processed within 2 hours) was concentrated by centrifugation and incubated with combination of antibodies (CD20/CD10/CD45/CD34/CD19/CD3/Syto41 in BCP ALL and CD4/CD99/CD5/CD3/CD7/CD16 + 56/CD8/CD45/Syto41 in T ALL). Residual erythrocytes were lysed with NH 4 Cl solution, followed by immediate acquisition on BD FACS Lyric or BD LSRII cytometers. Blasts were quantified to initial CSF volume. Cluster of 20 events was required to assign samples as FC positive (FC+). In parallel, CNS status was concluded according to morphological criteria, being classified as CNS1 (no blasts), CNS2a/b (≤5 WBC/μL with blasts), CNS2c/CNS3 (≥5 WBC/μL with blasts) according to treatment protocol guidelines. Continuous variables were compared with the Mann–Whitney test, survival data were analyzed using Mantel-Cox test. P values<0.05 were considered as significant.

Results: Using FC, we identified atypical blasts in 50 out of the 208 analyzed samples (24%). Median of analyzed CSF volume was 367µL (range 143-1033µL), which enabled median sensitivity 0.054 events/µL (range 0.02-0.14ev/µL). Mean blast concentration was 0.47ev/µL (range 0-25ev/µL) in all samples and 2 ev/µL (range 0.01-25ev/µL, median 0.53ev/µL) in FC+ samples. We observed significantly higher blast concentration in patients with T ALL, in patients with CNS2 and CNS3 and those who subsequently relapsed. Patients with FC+ had significantly lower relapse-free survival than FC- patients (55% vs 94%), which provided better separation than CNS status (68% vs.85% for CNS2/3 vs. CNS1, respectively).

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Summary/Conclusion: Using fresh CSF sample with lyse-no wash protocol enabled us to detect approximately 10x higher blast count (0.24ev/µL in FC+CNS1 and 1.25ev/µL in FC+CNS2/3) than was recently published (0.015ev/µL in FC+CNS1 and 0.073ev/µL in FC+CNS2/3 in Thastrup et al., 2020), and thus it better correlates to cytomorphological data. Interestingly, the proportion of FC+ patients in our cohort is comparable to (24% vs 25%), showing that although in our method we lose fewer cells, we identify the same proportion of FC+ patients. The presented study shows a higher impact of FC positivity on relapse risk than described previously. The disadvantage of our method is a need for a fresh sample, which is investigated locally.

Supported by UNCE/MED/015, NU20J-07-00028.

P363: TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA DURING COVID-19

E. Paramonova 1,* , E. Zhelnova 1 , E. Misyurina 1 , E. Baryakh 1 , E. Karimova 1 , E. Zotina 1 , E. Grishina 1 , D. Gagloeva 1 , M. Lysenko 1

1 hematology, moscow city clinical hospital 52, Moscow, Russia

Background: During the coronavirus pandemic, the risk of severe COVID-19 and mortality are higher in certain groups, in particular in patients with oncohematological diseases. Acute lymphoblastic leukemia (ALL) is a special group of oncohematological diseases in which mortality in the era of COVID-19 has increased 2-3 times. Currently, there is no consensus on the treatment of ALL during coronavirus infection.

Aims: To determine the basic principles and features of the management of patients with ALL during COVID-19.

Methods: 46 patients with ALL and COVID-19 (men 52.2%, women 47.8%) aged 18-74 years (median-44.5) were treated at the Moscow City Clinical Hospital 52 on 01.04.20-01.11.21. B-ALL was 58.7% (27 patients), T-ALL - 34.8% (16 patients), biphenotypic - 4.3% (2 patients), not defined - 2.2% (1 patient), Ph-positive ALL - 17.4% (8 patients). The status of the disease of patients upon admission to the hospital differed: debut of ALL - 20 patients (43.5%), remission - 16 patients (34.8%), relapse and refractory course - 10 patients (21.7%). All patients were treated COVID-19 in accordance with the current guidelines for the prevention, diagnosis and treatment of COVID-19 (interleukin 6 inhibitor, anticoagulant and antibacterial therapy, glucocorticoids (GCs), human immunoglobulin (IG) against COVID-19). According to vital indications and with stabilization of the patient’s condition, 18 patients (39.1%) received chemotherapy (CT).

Results: There were no deaths in the group of patients with remission of ALL. In patients with the debut of ALL, mortality was 45% (9 patients), in relapse and refractory course - 50% (5 patients) (p=0.005). Mortality in the group who did not receive CT was 35.7%, and in the group who received CT - 22.2%. 6 patients with Ph-positive ALL (75.0%) continued therapy with tyrosine kinase inhibitors (TKI). According to the protocol for the treatment of ALL, full doses of GCs (100%) and anthracyclines (ATC) (100%) were used, lumbar punctures (LP) and intrathecal (IT) injections of CT (100%) were continued. Due to the high risk of thrombotic complications in COVID-19 and asparaginase therapy, anticoagulant therapy was performed (100%). Prevention of pneumocystis pneumonia (PCP) (89.1%), antifungal (37.0%) and antibacterial (87.0%) therapy were carried out in the treatment of COVID-19. With the persistence of COVID-19 and the absence of antibodies to COVID-19, 2 patients received repeated transfusion of human IG against COVID-19.

Summary/Conclusion: During the COVID-19 pandemic, patients in remission of ALL coronavirus infection are treated and controlled. Treatment of COVID-19 in patients with ALL is carried out according to general protocols for the treatment of COVID-19, taking into account the peculiarities of nosology (agranulocytosis, high risk of PCP and fungal infection with long-term therapy of GCs, persistence of COVID-19). When the patient’s condition is stabilized, the issue of CT should be decided individually in each case, taking into account all the risks of ALL and COVID-19. During CT, use full doses of GCs, ATC. In patients with mild and moderate COVID-19, continue LP and IT injections of CT, therapy with TKI.

P364: DONOR-DERIVED CD19 CAR-T CELLS AND CHEMOTHERAPY PLUS DONOR LYMPHOCYTE INFUSION IN THE TREATMENT OF RECURRENT CD19+ B-ALL AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

X. Tan 1 , C. Zhang 1 , L. Gao 1 , L. Gao 1 , P. Kong 1 , Q. Wen 1 , S. Lou 2 , X. Zhang 1,*

1 Medical center of hematology, Xinqiao hospital, Army medical university; 2 Department of hematology, the second affiliated hospital, chongqing medical universtiy, Chongqing, China

Background: Donor derived CD19 car T cells plus donor lymphocyte infusion is rarely used in the treatment of recurrent CD19 positive B-ALL after allogeneic hematopoietic stem cell transplantation (allo-HSCT)

Aims: To compare the efficacy of donor derived CD19 car T cells and chemotherapy plus donor lymphocyte infusion in the treatment of recurrent CD19 positive B-ALL after allogeneic hematopoietic stem cell transplantation (allo-HSCT)

Methods: We retrospectively analyzed 43 patients with B-ALL who relapsed after allo-HSCT from March 2016 to November 2019，including 22 patients treated with donor derived CD19-CAR-T cells (CAR-T group) and 21 patients treated with chemotherapy plus donor lymphocyte infusion (Chemo-DLI group).

Results: The rates of complete remission (77.3%) in the CAR-T group were significantly higher than those in the Chemo- DLI group (38.1%); The 1- and 2-year leukemia-free survival(LFS) in the CAR-T group and Chemo-DLI group were 54.5% vs 9.5%, 45.5% vs 4.8%, respectively. The 1- and 2-year overall survival(OS) in the CAR-T group and Chemo-DLI group were 68.2% vs 19%, 63.6%vs 9.5%, respectively. Six patients (28.6%) in the Chemo-DLI group developed grade II-IV acute graft-versus-host disease (aGVHD). Two patients (9.1%) with grade I-II aGVHD were found in the CAR-T group. The incidence of CRS in the CAR-T group was 86.4%, the grade I-II CRS was59.1%, the grade III CRS was 27.3%. There was no grade 4 CRS. Two subjects developed ≤grade-2 immune effector cell-associated neurotoxicity syndrome (ICANS).

Summary/Conclusion: Compared with chemotherapy plus DLI, donor-derived CD19 CAR-T is more effective and safe in the treatment of recurrent B-ALL after allogeneic HSCT.

P365: INCORPORATION OF NELARABINE (NEL), PEGYLATED ASPARAGINASE (PEG) AND VENETOCLAX (VEN) IN THE FRONTLINE THERAPY OF ADULT PATIENTS WITH T-ACUTE LYMPHOBLASTIC LEUKEMIA/T-LYMPHOBLASTIC LYMPHOMA (T-ALL/LBL)

F. Ravandi 1,* , E. Jabbour 1 , N. Jain 1 , T. Kadia 1 , B. Gautam 1 , M. Konopleva 1 , W. Wierda 1 , J. Burger 1 , G. Issa 1 , A. Maiti 1 , H. Balkin 1 , M. Kelly 1 , R. Garris 1 , P. Kebriaei 1 , A. Ferrajoli 1 , G. Garcia-Manero 1 , Y. Alvarado 1 , N. Short 1 , H. Kantarjian 1

1 The University of Texas - MD Anderson Cancer Center, HOUSTON, United States of America

Background: NEL is effective for patients (pts) with relapsed T-ALL/LBL but its incorporation into the front-line therapy has not been investigated. In children, addition of NEL to the induction regimen improved disease-free survival (DFS) but not overall survival (OS) (Dunsmore KP, JCO, 2020). VEN has shown preclinical and clinical activity in relapsed T-ALL/LBL.

Aims: We investigated addition of NEL and PEG into the hyperCVAD (HCVAD) regimen in adult pts with T-ALL/LBL. We then explored the safety and efficacy of addition of VEN to this regimen.

Methods: Pts with previously untreated or minimally pre-treated T-ALL/LBL were eligible if they had an ECOG PS ≤3, creatinine ≤ 2 mg/dL, bilirubin ≤ 2 mg/dL and ALT/AST ≤ 4 x ULN. Pts received 8 cycles of HCVAD (cycles 1,3, 5, 7) alternating with high dose ara-C and methotrexate (MTX) (cycles 2, 4, 6, 8) at approximately 3 weeks intervals. 2 cycles of NEL (650 mg/m% daily x 5) were initially administered after the 8 cycles (cohort 1) but later moved to after cycle 4 (cohort 2) and then PEG (1500 IU/m2 capped at 3750 IU) was added on day 5 of NEL cycles (cohort 3). More recently, VEN 400 mg daily on the first 7 days of each of 8 cycles of therapy was added (cohort 4) and this was later adjusted to be given for 7 days on the induction cycle and reduced to 3 days per cycle in pts with ETP-ALL or with persistent MRD with no VEN after cycle 1 in all other pts (cohort 5) Pts with ETP-ALL were referred for allogeneic stem cell transplant in CR. After the completion of the intensive phase, all pts in all cohorts received 30 cycles of maintenance with monthly POMP and early intensification with NEL/PEG on cycles 6 and 7 and late intensification with MTX/PEG in cycle 18 and HCVAD on cycle 19. All pts received 8 intrathecal doses of MTX alternating with ara-C and mediastinal radiation was considered in pts with balky mediastinal disease.

Results: Between 8/2007 and 12/2021. 120 pts were enrolled in the 5 study cohorts sequentially (cohort 1= 30, 2= 49, 3=17, 4=16, 5=8)(Table). 74 pts had T-ALL, 45 T-LBL and 1 biphenotypic T/myeloid leukemia. Median age was 35 yrs (range 18-78), 93 pts were male (78%) and 106 pts (88%) had PS 0-1. In pts with T-ALL, median bone marrow blast % was 81% (range, 20-97%). 6 pts (5%) had CNS disease. Cytogenetics was diploid in 77 pts (64%), abnormal in 34 (28%), and unavailable in 9 pts (7.5%). Pts were further characterized as thymic (n=53), early T-cell precursor (ETP; n=23), near ETP (N=19) mature (n=11), not otherwise specified (NOS; n=11), and early-non-ETP (n=3). 25 pts had received 1-2 prior cycles of therapy prior to enrollment with 18 having achieved CR. 30-day mortality was 0%. 106 pts (88%) had ≥ one grade 3/4 non-hematological treatment-emergent adverse events (TEAE) with the most frequent grade 3/4 TEAEs being neutropenic infections in 92 pts (77%) and elevated liver enzymes in 25 pts (21%). Overall response rate (CR/CRi/PR) was 97% with CR/CRp in 95/102 pts (93%), PR in 4 (4%), and no response in 3 (3%). The median no. of cycles to response (including negative PET scans) was 1 (range 1-10). CR/CRp rate in T-ALL was 88% and T-LBL was 100%. At a median follow-up of 48 months (mo; 2-168), 79 pts (66%) are alive with a median PFS and OS of 135 and 135 mo (Figure) and 73 pts (61%) remain in CR.

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Summary/Conclusion: Addition of NEL/PEG cycles to the HCVAD regimen is feasible and may be beneficial. Addition of VEN to each cycle is associated with significant myelosuppression. Lower doses of VEN may benefit selected pts.

P366: BLINATUMOMAB ADDED TO PREPHASE AND CONSOLIDATION THERAPY IN NEWLY DIAGNOSED PRECURSOR B-ALL IN ADULTS. A PHASE II HOVON TRIAL

A. Rijneveld 1,* , P. Gradowska 2 , M. Bellido 3 , O. de Weerdt 4 , A. Gadisseur 5 , D. Deeren 6 , L. van der Wagen 7 , Y. Jauw 8 , R. Fijnheer 9 , D. van Lammerren 10 , D. Selleslag 11 , S. Halkes 12 , B. Biemond 13 , D. Breems 14 , I. Moors 15 , G. van Sluis 16 , M. Bakkus 17 , C. Homburg 18 , V. Janda 19 , V. van der Velden 20 , J. Cornelissen 21

1 Hematology; 2 HOVON data Center, Erasmus MC, Rotterdam; 3 hematology, Univerisity Medical Center Groningen, Groningen; 4 Hematology, Sint Antonius Hospital, Nieuwegein, Netherlands; 5 Hematology, Universitair ziekenhuis Antwerpen (UZA), Antwerp; 6 Hematology, AZ Delta, Roeselare, Belgium; 7 Hematology, University Medical Center, Utrecht; 8 Hematology, Amsterdam UMC, VU University Medical Center, Amsterdam; 9 Hematology, Meander Medical Center, Amersfoort; 10 Hematology, Haga Teaching Hospital, The Hague, Netherlands; 11 Hematology, St Jan hospital, Brugge, Belgium; 12 Hematology, Leiden University Medical Center, Leiden; 13 Hematology, Amsterdam UMC, Amsterdam Medical Center, Amsterdam, Netherlands; 14 Hematology, ZNA Stuivenberg, Antwerpen; 15 Hematology, Universiteits ziekenhuis Gent, Gent, Belgium; 16 Hematology, Isala Clinic, Zwolle, Netherlands; 17 Hematology, University Hospital Brussels, Brussels, Belgium; 18 Immunocytology, Sanquin Diagnostic Services, Amsterdam, Netherlands; 19 Immunology, UZ Gent, Gent, Belgium; 20 Immunology; 21 Hematology, Erasmusm University Medical Center, Rotterdam, Netherlands

Background: The bispecific antibody blinatumomab (blina) is approved for patients(pts) with relapsed/refractory precursor B-cell ALL (ALL). It has also shown to be highly effective in MRD+ pts in first line treatment.

Aims: To evaluate whether blina added in upfront therapy to prephase and after consolidation (cons)-1 would increase MRD negativity measured by qPCR or flowcytometry at a cut-off of <10 -4 (primary endpoint). Secondary endpoints included CR, EFS, OS, adverse events and treatment-related mortality.

Methods: Pts, 18-70 years(y) old, with newly diagnosed CD19+ ALL (incl. Ph+), were included. Treatment was based on a pediatric inspired protocol ( HOVON 70, Rijneveld et al., 2011 ) with reduced doses of anthracyclines, MTX, etoposide and PEG-ASP for pts ≥40y old. Prephase consisted of 10 days steroids, from day 5 combined with 14 days blina in the standard step-up dosing schedule. After cons-1 and after intensification 2, two 4-week blina courses were added irrespective of MRD. The protocol was amended twice due to toxicity. First, in 2018 the first PEG-ASP administration was omitted. Second, in 2021 doxorubicin, dexamethasone and PEG-ASP were reduced during intensification 1. Rituximab (if CD20+), prophylactic ITs and imatinib (if Ph+) were standard. AlloHSCT was offered to intermediate and high-risk pts. Trial was registered with ClinicalTrials.gov, identifier NCT03541083 .

Results: Seventy-one pts were enrolled. Pt characteristics are presented (Table). Fifteen pts discontinued treatment before blina cons-1 due to refractory disease (n=8), toxicity (n=7) or death (n=2). In the total study population, 55/71 pts (77%) achieved CR after (blina) cons-1. Among pts still on treatment after cons-1, 55/56 (98%) pts achieved CR, 50/55 (91%) reached MRD negativity. After prephase, CR was already reached in 63% and MRD negativity in 53%. Blina related AEs in prephase were as expected (83% of pts had ≥1 AE and 10% had ≥1 SAE (hepatotoxicity 3 pts, pain lymph node 1, CRS 1, pneumonia 1, renal insufficiency 1)). CRS was observed in 35% of pts, 32% of whom experienced grade 3 and no grade ≥4. During prephase 5 pts discontinued blina; during blina cons-1, 4 pts stopped blina. With a median follow-up of 17,6 months, the estimated 2-y EFS was 64% standard error (SE) ± 7% (≤60y 71% SE ± 9% and >60y 47% SE ± 12%). Overall, 14 (20%) pts died. OS after 2y was 73% SE ± 7% (≤60y 82% SE ± 8% and >60y 52% SE ± 14%) (Figure 1). For pts with Ph+ ALL, 2-y EFS was 88% SE ± 6%and OS also 88% SE ± 7%. For Ph- ALL, 2-y EFS and OS were 53% SE ± 9% and 68% SE ± 9%, resp. Among pts who reached CR on protocol (n=60), 5 (8%) had relapse, 6 (10%) died and 6 (10%) discontinued treatment due to toxicity. Until now, 22 pts proceeded to alloHSCT and 11 with maintenance.

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Summary/Conclusion: Blina can safely be added to prephase of an intensified pediatric schedule for newly diagnosed ALL up to 70y of age, albeit with dose reductions for PEG-ASP, doxorubicin and dexamethasone. The combination increases CR and MRD negativity rate. The early addition of blina resulted in very early achievement of MRD negativity with 53% after prephase and 91% after blina cons-1. Further reductions of chemotherapy should be explored (especially for Ph+) if these results are maintained with longer follow-up.

P367: LONG TERM OUTCOMES OF NEWLY DIAGNOSED CRLF2 REARRANGED B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

J. Senapati 1,* , E. J. Jabbour 1 , N. J. Short 1 , F. Ravandi 1 , P. Kebriaei 1 , T. M. Kadia 1 , G. Borthakur 1 , N. Pemmaraju 1 , R. S. Garris 1 , D. Bansal 1 , S. Konoplev 1 , S. Wang 1 , W. Wang 1 , G. Tang 1 , K. P. Patel 1 , M. Konopleva 1 , N. Jain 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: CRLF2 rearranged B cell acute lymphoblastic leukemia (B-ALL), a subtype of Philadelphia (Ph) -like ALL, constitutes a high-risk subset of B-ALL with poor outcomes with chemotherapy. Targeted therapies such as blinatumomab (blina) or inotuzumab (ino) may improve treatment outcomes for these patients (pts).

Aims: To study the outcomes of newly diagnosed CRLF2 rearranged B-ALL treated with hyper CVAD based regimens and the impact of targeted therapies like inotuzumab and blinatumomab

Methods: We retrospectively analyzed pts with newly diagnosed B-ALL (diagnosed between 01/2001 and 12/2021) at our center who had documented CRLF2 overexpression. Initial therapy, including use of ino and blina in CR1 were noted. Outcomes measures included CR/CRi, MRD response, relapse free survival (RFS) and overall survival (OS). RFS was censored at allogeneic stem cell transplantation (ASCT).

Results: A total of 76 pts with a median age of 38 years (yrs) (range, 18-80) were identified, of which 70% were males and 81% were of Hispanic ethnicity. All pts had overexpression of CRLF2 documented by flow cytometry or gene expression profile. A subset of pts (n=37) had a concomitant CRLF2 FISH performed with all confirming CRLF2 rearrangement. Baseline disease parameters, treatment and outcomes are detailed in Table 1. Sixty-five pts (85%) received Hyper-CVAD based induction therapy [HCVAD-based (n=51); mini-CVD-based (n=14)] and 11 (15%) received augmented BFM. We focus on the outcomes of pts treated with HCVAD/mini-HCVD (n=65); among these pts, 24/65 (37%) received blina in CR1 during consolidation at a median of 3.6 months after starting induction therapy, 22/65 (34%) received ino in CR1 (in C1 as part of mini-HCVD-ino, n=14; in C2 as part of HCVAD, n=8). A total of 14/65 (22%) received both ino and blina in CR1; 32/65 (49%) received ino and/or blina. The median follow-up was 39 months (mos). CR/CRi rate after C1 was 52/65 (80%) with 25/47 (53%) MRD evaluable pts and 25/65 (38%) overall achieving MRD-neg post C1. The median RFS and OS was 17.6 and 26.6 mos, respectively (Fig. 1). Among the 32 pts who received ino and/or blina, the median RFS and OS was 17.6 mos and 38.8 mos respectively. CR/CRp rate after C1 among pts who received mini-HCVD-ino in C1 was 100% (14/14) with 79% MRD-neg. On landmark analysis to the time to blina initiation, blina treated pts had similar RFS and trended for improved OS. A total of 19/65 (29%) had ASCT in CR1; all were MRD-neg prior to ASCT. Landmark analysis for OS based on the time of ASCT (6 mos) in CR1 favored ASCT in CR1 (47.2 vs. 17.6 mos, p=0.04).

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Summary/Conclusion: Despite improvements in treatment options, CRLF2 overexpressed B-ALL continue to have inferior outcomes. Earlier initiation of targeted therapies might improve outcomes.

P368: A PHASE II STUDY OF INOTUZUMAB OZOGAMICIN FOR THE TREATMENT OF MEASURABLE RESIDUAL DISEASE-POSITIVE B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

J. Senapati 1,* , H. Kantarjian 1 , N. Short 1 , Y. Alvarado 1 , J. Burger 1 , N. Jain 1 , M. Konopleva 1 , F. Ravandi 1 , C. DiNardo 1 , L. Masarova 1 , K. Sasaki 1 , P. A. Thompson 1 , A. Ferrajoli 1 , J. O. Jacob 1 , E. D. Mayor 1 , A. M. Milton 1 , C. Loiselle 1 , R. S. Garris 1 , E. J. Jabbour 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: Persistence or re-emergence of measurable residual disease (MRD) in B-cell acute lymphoblastic leukemia (B-ALL) is strongly associated with shorter relapse-free survival. Inotuzumab ozogamicin (INO) is an anti-CD22 antibody-drug conjugate with the potential to eradicate MRD in B-ALL.

Aims: We aimed to evaluate the efficacy of INO in clearing MRD in patients (pts) with persistent or recurrent MRD positivity after treatment with conventional chemotherapy.

Methods: This is a single arm phase II trial of pts with B-ALL in complete remission (CR) who did not achieve MRD negativity (MRD-ve) or had MRD-positive (MRD+ve) relapse after at least 3 months (mos) from the start of frontline therapy (i.e. CR1) or 1 month from the start of any salvage therapy (i.e. ≥ CR2). Eligibility was defined by MRD+ve at ≥ 0.01%. MRD-ve was defined as undetectable MRD by flow cytometry at a minimum sensitivity of 10 -4 for Philadelphia (Ph) negative (Ph-) B-ALL and undetectable MRD by both flow and PCR at 10 -4 for Ph positive (Ph+) B-ALL. INO was given at a dose of 0.6 mg/m 2 on D1 and 0.3 mg/m 2 on D8 of C1 and 0.3 mg/m 2 on D1 and 8 of subsequent cycles (up to 6 total cycles, given every 21-28 days) along with ursodiol prophylaxis. Pts with Ph+ ALL received concomitant TKI, the choice of which was decided by the treating physician.

Results: Between 11/2018 and 1/2022, 20 pts with MRD+ve B-ALL, with a median age of 40 years (range, 19-68) were treated. Twelve pts (60%) had Ph+ B-ALL, 11 of whom received concurrent ponatinib and 1 dasatinib. Eight pts were Ph- (including 3 Ph-like ALL). Fourteen pts (70%) were in CR1, and 6 pts (30%) were in ≥ CR2 (3 in CR2, 2 in CR3 and 1 in CR4). Eleven pts (55%) had received prior blinatumomab (blina) and 4 pts (20%) had prior stem cell transplantation (SCT). The median BCR-ABL1 level in Ph+ B-ALL was 0.58% (range, 0.001-17.5%) and median MRD level by flow cytometry in Ph- B-ALL was 0.2% (range, 0.05-1.24%). Twelve pts (60%) became MRD-ve (responders), 10 after cycle 1 and 2 after cycle 2. In the Ph+ group, 6 pts (50%) responded; another 3 pts attained MMR as best response. Six of 8 (75%) Ph- pts responded. The median number of cycles were 3 (range, 1-6). Seven of 9 pts (78%) without prior blina exposure became MRD-ve, compared with 5/11 (45%) pts with prior blina exposure ( p =0.22). Patient disposition is shown in Fig. 1 A . Amongst the 12 responders, 5 pts were consolidated with ASCT after a median of 3 cycles (range, 1-3) of INO, all in MRD-ve CR pre-ASCT. Three responders (25%) relapsed (including 1 relapse after ASCT). At a median follow-up of 17 mos, 15 pts (75%) are alive, 8 of whom are in continued MRD-ve CR. The estimated 18 mos RFS and OS were 63% and 73% respectively, for the whole group (Fig. 1 B ), and the 18 mos OS was 87% and 55% for responders and non-responders, respectively. Outcomes were similar between pts with Ph+ ALL vs. Ph- ALL, prior blina exposure vs. no prior exposure, and pts in CR1 vs. ≥ CR2.

INO was overall well-tolerated with no grade 4 non-hematological toxicities. Grade 3 non-hematological toxicities included elevated transaminases in 1 pt and veno-occlusive disease in 1 pt without history of prior ASCT. Ten pts (50%) had grade 3-4 hematological toxicities (primarily neutropenia).

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Summary/Conclusion: Low-dose, fractionated INO is a well-tolerated option for MRD eradication in Ph+ and Ph- B-ALL pts, including in those with prior blina exposure.

P369: A PHASE II STUDY OF MINI-HYPER-CVD PLUS VENETOCLAX IN PATIENTS WITH PHILADELPHIA CHROMOSOME-NEGATIVE ACUTE LYMPHOBLASTIC LEUKEMIA

J. Senapati 1,* , H. Kantarjian 1 , N. Short 1 , M. Konopleva 1 , F. Ravandi 1 , N. Jain 1 , P. A. Thompson 1 , N. Pemmaraju 1 , W. G. Wierda 1 , G. Borthakur 1 , T. M. Kadia 1 , G. Garcia-Manero 1 , M. Yilmaz 1 , J. Thankachan 1 , M. Zhao 1 , C. Loiselle 1 , M. T. Talley 1 , M. I. Kwari 1 , R. S. Garris 1 , E. J. Jabbour 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: Venetoclax (VEN), an orally active Bcl-2 antagonist, has shown activity in acute lymphoblastic leukemia (ALL) in preclinical models and in combination with navitoclax (BCL-X L inhibitor) in early clinical studies. Combining VEN with other active agents in ALL might synergize this antileukemic effect.

Aims: We aimed to evaluate the efficacy and tolerability of VEN added to mini-hyper CVD (mHCVD) chemotherapy in patients (pts) with relapsed/refractory (R/R) ALL.

Methods: Pts ≥18 years of age with R/R Philadelphia chromosome negative (Ph-) B- or T-cell ALL/lymphoblastic lymphoma (LBL) were eligible. Pts were required to have a PS of ≤3, bilirubin ≤1.5 mg/dl, AST/ALT ≤3 x ULN and creatinine ≤2 mg/dl. Treatment consisted of mHCVD for up to 8 cycles. VEN was given at a dose of 400 mg daily on D1-14 of C1 and on D1-7 of C2-8. Rituximab (if CD20+) and prophylactic IT chemotherapy x 8 doses were given in C1-C4. Pts with T-cell ALL received an additional 2 cycles of nelarabine (650 mg/m2 daily on D1-5) and peg-asparaginase (1,500 IU/m2 [capped at 3750 IU] on D5), without VEN, during consolidation and another 2 cycles during maintenance. Responding pts received vincristine and prednisone maintenance with VEN daily on D1-14 of each 28-day cycle for up to 2 yrs.

Results: From 6/2019 to 2/2021, 20 pts with R/R ALL were treated, 15 (75%) with B-ALL, 4 (20%) with T-ALL (including 1 ETP-ALL) and 1 (5%) with T-LBL. The median age was 45 yrs (range, 20-70). The median lines of prior therapy was 2 (range 1-5). Of the 15 B-ALL pts, 6 had previously received both blinatumomab (blina) and inotuzumab (INO), 7 had received blina without INO, and 2 had received neither. Overall, 11 pts (55%) had undergone prior stem cell transplantation (SCT).

Pt disposition is shown in Fig. 1 A. One pt was in measurable residual disease (MRD)-positive CR at trial enrollment; all others had active disease and were evaluable for response assessment. Pts received a median of 2 cycles of mHCVD-VEN (range, 1-6). Overall, 12 of 19 evaluable pts (63%) responded (9 CR, 3 CRi), 9 (75%) of whom after 1 cycle. Response in B-ALL was 9/15 (60%) and in T-ALL was 3/4 (75%). Six of the 12 responders (50%) attained MRD negativity (MRD-ve). Of the 12 responders, 4 (33%) underwent SCT, 3 of whom subsequently relapsed and 1 of whom is in continued MRD-ve CR. All of the 8 responders (67%) who did not undergo SCT, subsequently relapsed and died, with a median duration of response of 2.1 mos. Six of the 7 non-responders have died. With a median follow up of 15 months (mos), 5 pts are alive with a median RFS and OS of 6.2 and 7.1 mos respectively (Fig. 1B. The median OS in responders was 8.8 mos (Fig. 1C).

Fig.1: (A) Patient disposition and survival, (B) OS and RFS for the entire cohort, (C) OS of responders and non-responders

The therapy was generally well-tolerated. There were no grade 4-5 adverse events. Four pts had a grade 3 related event (hyperbilirubinemia and mucositis in 1 pt and transaminitis, fatigue and tumor lysis syndrome in 1 pt each). One pt required VEN dose reduction due to cytopenias. The median times to neutrophil and platelet recovery in C1 in responding pts were 18 and 27 days, respectively.

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Summary/Conclusion: Low dose combination chemotherapy with VEN in a population of heavily pretreated R/R ALL was well-tolerated and resulted in a response rate of 63%. This study has now been amended to add navitoclax in an attempt to further improve outcomes.

P370: EARLY RESULTS OF A SAFETY AND EFFICACY STUDY OF ALLOGENEIC TRUUCAR™ GC502 IN PATIENTS WITH RELAPSED/REFRACTORY B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (R/R B-ALL)

S. Li 1,* , Z. Yuan 1 , L. Liu 1 , Y. Li 1 , S. Martina 2 , J. Liu 2 , Z. Li 2 , X. Wang 2 , J. He 2 , W. Zhao 2 , L. Shen 2 , X. Zhang 3 , S. Wang 1

1 920th Hospital, Kunming; 2 Gracell Biotechnologies Ltd, Shanghai; 3 The Second Affiliated Hospital of Army Medical University, Chongqing, China

Background: CD19 targeted autologous CAR-T therapies have been approved for the treatment of r/r B-ALL and greatly improved outcome. However, some patients may not be eligible to receive autologous CAR-T. TruUCAR™ GC502 is an allogeneic, universal CAR-T product with CD19/CD7 dual directed CAR. Preclinical data of GC502 were reported at ASH 2021 (Abstract 148500).

Aims: Here, we report early clinical results from a phase I open-label, non-randomized, prospective investigator initiate trial (IIT) of GC502 in r/r B-ALL patients to evaluate safety and preliminary efficacy

Methods: GC502 is manufactured using leukopaks from HLA-unmatched healthy donors. It contains a 4-1BB based CD19/CD7 dual directing CAR, a T cell enhancer, and genetically disrupted TRAC and CD7 loci to avoid GvHD and fratricide.

Patients (pts) with r/r B-ALL were enrolled and treated with one of two different formulations (A or B) in escalating dose levels ranging from 1.0x10 7 (DL1) to 1.5x10 7 (DL2) cells/kg. Prior to infusion of GC502, pts received a Flu/Cy based lymphodepletion regimen. Adverse events, disease response and expansion kinetics were evaluated in this study.

Results: At date cut off of Feb. 22, 2022, 4 pts (15-34 yrs) had been enrolled into the investigator initiate study of GC502 ( NCT05105867 ). All of patients were heavily pretreated, and had received either autologous or donor derived CD19 or CD19-CD22 targeted CAR-T in prior lines of therapy. Baseline marrow blast levels ranged from 19.5% to 92% (median 48.1%). 1 pt had extramedullary (EM) involvement.

At data cut-off all pts had received a single dose of GC502: 1 pt at DL 1 1.0x10 7 cells/kg in formulation A and 3 patients at DL 2 1.5x10 7 cells/kg – out of which 2 were treated with formulation B. At day 28 post CAR-T infusion, 3 out of 4 response evaluable patients had achieved CR/CRi; 1 pt with EM achieved PR at month 1 and subsequently received allo-HSCT on day 39.

TEAEs presented as Gr 3 febrile neutropenia (4/4), Gr 4 thrombocytopenia (1/4) and Gr 3 anemia (3/4). All TEAE resolved after treatment with SOC. Non-hematological TEAE presented as Gr≥3 γ-GT increase (3/4), Gr≤3 AST increase (2/4) and Gr≤3 ALT increase (3/4). 2 pts received formulation A and experienced Gr 3 CRS with a duration of 7 and 10 days respectively (CRS was graded according ASTCT Consensus Grading). CRS presented as Gr 2 in the 2 patients with formulation B with a duration of 9 and 15 days respectively. CRS in all pts was manageable and resolved after treatment with Ruxolitinib, SOC and supportive care. No ICANS or aGvHD were observed. The pt treated in DL 1 did not show adequate GC502 cellular expansion. Peak expansions of GC502 in peripheral blood were observed between week 1-2 in DL 2. Median peak CAR copies were 149,945 copies/ug DNA (range 10,849-195,400).

Summary/Conclusion: TruUCAR™ GC502 demonstrated promising early results with a manageable safety profile. Robust CAR-T cell expansion was observed in DL2 at 1.5x10 7 cells/kg in heavily pretreated r/r B-ALL patients, including those previously treated with CD19 or CD19-CD22 CAR-T therapies. The study is ongoing and continues accruing patients.

P371: HYPER-CVAD WITH SEQUENTIAL BLINATUMOMAB, WITH OR WITHOUT INOTUZUMAB OZOGAMICIN, IN ADULTS WITH NEWLY DIAGNOSED PHILADELPHIA CHROMOSOME-NEGATIVE B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

N. Short 1,* , H. Kantarjian 1 , F. Ravandi 1 , M. Yilmaz 1 , T. Kadia 1 , P. Thompson 1 , X. Huang 2 , M. Konopleva 1 , A. Ferrajoli 1 , N. Jain 1 , K. Sasaki 1 , Y. Alvarado 1 , G. Borthakur 1 , C. Dinardo 1 , M. Ohanian 1 , W. Macaron 1 , S. Kornblau 1 , M. Zhao 1 , M. Kwari 1 , C. Loiselle 1 , R. Delumpa 1 , A. Milton 1 , J. Rivera 1 , S. Lewis 1 , R. Garris 1 , E. Jabbour 1

1 Department of Leukemia; 2 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Blinatumomab and inotuzumab ozogamicin (INO) are highly effective in relapsed/refractory B-cell acute lymphoblastic leukemia (ALL); blinatumomab is also the only approved therapy for eradication of persistent or recurrent measurable residual disease (MRD) after initial ALL therapy. We hypothesized that early incorporation of blinatumomab and INO in patients (pts) with newly diagnosed Philadelphia chromosome (Ph)-negative B-cell ALL would lead to deeper and more durable responses, reduce relapses, and improve survival.

Aims: We evaluated the efficacy and safety of hyper-CVAD with sequential blinatumomab, with or without INO, in pts with newly diagnosed Ph-negative B-cell ALL.

Methods: Pts 14-59 years of age with newly diagnosed Ph-negative B-cell ALL, including pts who had received no more than 1 prior cycle of chemotherapy, were eligible. Pts were required to have a performance status of ≤3, total bilirubin ≤2 mg/dl, creatinine ≤2 mg/dl, and no significant CNS pathology (with the exception of CNS leukemia). Pts received hyper-CVAD alternating with high-dose methotrexate and cytarabine for up to 4 cycles, followed by 4 cycles of blinatumomab at standard doses. Pts with CD20+ disease (≥1% cells) received 8 doses of ofatumumab (2000 mg) or rituximab (375 mg/m 2 ). Eight doses of prophylactic IT chemotherapy were given. Maintenance was with alternating blocks of POMP (given in maintenance cycles 1-3, 5-7, 9-11, and 13-15) and blinatumomab (given in maintenance cycles 4, 8, and 12). Those with high-risk disease features started blinatumomab after 2 cycles of hyper-CVAD. Beginning with pt #39, INO at a dose of 0.3 mg/m2 on day 1 and 8 was added to the 2 cycles of MTX/Ara-C and to 2 cycles of blinatumomab consolidation (4 total cycles with INO).

Results: As of February 2022, 58 pts have been treated (38 without INO and 20 with INO). Pt characteristics are summarized in Table 1. Median age was 34 years (range, 17-59 years). 13 pts were in CR at enrollment.

Among 45 pts with active disease at study entry, 100% achieved CR, with 80% achieving CR after the first cycle. MRD negativity by 6-color flow cytometry was achieved in 32/42 evaluable pts (76%) after 1 cycle and 55/58 evaluable pts (95%) overall. Two of the 3 pts who did not achieved MRD negativity were later found to have a NUP214 :: ABL1 fusion.

The median duration of follow-up is 26 months (range, 3-61 months). Overall, 5 pts (9%) did not undergo stem cell transplant (SCT) and subsequently relapsed, 18 pts (34%) underwent SCT in first remission (including 2 additional pts who relapsed post-SCT), 2 pts (3%) died in CR, and 33 pts (57%) remain in continuous remission without SCT. All relapses occurred in pts with ≥1 poor-risk feature(s), and no relapses have occurred beyond 2 years from the start of treatment.

For the entire cohort, the 3-year continuous remission and OS rates were 84% and 85%, respectively (Figure 1). No relapses or deaths have occurred in the INO group, and the estimated 1-year OS rate is 100%.

Treatment was overall well-tolerated. One pt discontinued blinatumomab due to a related adverse event (grade 2 encephalopathy and dysphasia). No pts discontinued INO due to toxicity, and no cases of veno-occlusive disease have been observed.

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Summary/Conclusion: Hyper-CVAD with sequential blinatumomab, with or without INO, is highly effective as frontline treatment of Ph-negative B-cell ALL, with an overall MRD negativity rate of 95% and a 3-year OS rate of 85%. Early outcomes with the addition of INO to this regimen are encouraging, with no relapses or deaths observed to date.

P372: DISMAL OUTCOMES OF PATIENTS WITH RELAPSED/REFRACTORY B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA AFTER FAILURE OF BOTH INOTUZUMAB OZOGAMICIN AND BLINATUMOMAB

W. Macaron 1,* , E. Jabbour 1 , M. Konopleva 1 , F. Ravandi 1 , N. Jain 1 , G. Issa 1 , T. Kadia 1 , K. Sasaki 1 , P. Kebriaei 1 , M. Yilmaz 1 , P. Thompson 1 , K. Takahashi 1 , H. Abbas 1 , W. Wierda 1 , R. Garris 1 , H. Kantarjian 1 , N. Short 1

1 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: The development of novel monoclonal antibodies such inotuzumab ozogamicin (INO) and blinatumomab (Blina) results in superior response rates and survival in patients (pts) with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL). However, outcomes of pts after failure of both INO and Blina are not well-established, and effective treatment options for these pts are limited.

Aims: To determine response rates to subsequent therapies and overall survival (OS) in pts with R/R Philadelphia chromosome (Ph)-negative B-cell ALL after failure of both INO/Blina.

Methods: We conducted a retrospective analysis of outcomes in adult pts with Ph-negative B-cell ALL who relapsed or were refractory to both INO/Blina, at least one of which given as salvage therapy.

Results: The baseline characteristics of the 65 pts included are shown in Table 1. Of 29 pts with paired flow cytometry for CD19 and CD22 prior to receipt of either INO/Blina, 4 pts (14%) lost CD19 expression and 3 pts (10%) lost CD22 expression at time of INO/Blina failure.

After INO/Blina failure, 54 pts (83%) received subsequent therapy, with a median of 2 therapies (range, 1-6). Of 53 evaluable pts, 16 pts (30%) achieved CR/CRi with the first salvage therapy received after INO/Blina failure (CR=9%, CRi=21%), and 22 pts (42%) achieved CR/CRi with at least one salvage therapy (CR=17%, CRi=25%). Of the 22 responders, 16 pts (73%) achieved measurable residual disease (MRD) negativity by flow cytometry (sensitivity=0.01%) at best response. Nine responses (41%) were achieved with an investigational drug/regimen and 13 (59%) were achieved with commercial agent(s). Among 54 pts who received at least one subsequent therapy, 12 pts (22%) received CAR T-cells, and 9 pts (17%) proceeded to hematopoietic stem cell transplant (HSCT).

With a median follow-up of 22.3 months, the median OS from the time of INO/Blina failure for the entire population was 3.8 months, and the 1-year OS was 22% (figure 1 A ). Median relapse-free survival (RFS) was 3.5 months, with a 1-year RFS of 12%. Median duration of response (DOR) to subsequent therapy was 5.0 months, with a 1-year DOR of 17%. Median OS from INO/Blina failure in pts who received salvage therapy compared to those who did not were 4.7 months and 1.4 months, respectively (figure 1 B ). In the 9 pts who underwent HSCT, 2 (22%) are still alive and in continuous remission. The median OS from time of HSCT was 13.4 months, and the 1-year post-HSCT OS was 59%. In the 12 pts who received CAR-T cells as a subsequent therapy, 6 (50%) responded and 2 were bridged directly to HSCT. The median OS from time of CAR T-cell therapy was 3.9 months, and the 1-year OS was 36%.

Outcomes of pts who received both INO and Blina as salvage therapy (n=50), excluding those who received 1 of these agents in the frontline or MRD setting, were also analyzed. Median OS from the time of INO/Blina failure was 2.6 months. Thirty-nine (78%) of these 50 pts received subsequent therapy after INO/Blina failure. Of 38 evaluable pts, 15 pts (39%) achieved CR/CRi with at least one subsequent salvage therapy (CR=13%, CRi=26%). Median RFS and OS in pts who underwent subsequent therapy were 5.9 months and 3.9 months, respectively.

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Summary/Conclusion: This study highlights the very poor outcomes of pts with R/R B-cell ALL after failure of both INO/Blina. For pts who respond to subsequent salvage therapy, a consolidative approach with SCT may improve outcomes. These data provide a historical reference for expected outcomes that may serve as a benchmark for the evaluation of novel drugs and combinations in the setting of INO/Blina failure.

P373: CIRCULATING ENDOTHELIAL PROGENITOR CELLS AND METABOLIC FACTORS IN CHILDHOOD CANCER SURVIVORS

E. Athanasopoulos 1,* , G. Martimianaki 1 , P. Iordanis 1 , M. Stratigaki 1 , N. Katzilakis 1 , E. Stiakaki 1

1 Department of Pediatric Hematology-Oncology and Laboratory of Blood Diseases and Childhood Cancer Biology, University Hospital of Heraklion, Medical School, University of Crete, Heraklion Crete, Greece

Background: Circulating Endothelial Progenitor cells (cEPCs) participate in the regulation and maintenance of vascular integrity, balancing the coagulation–anticoagulation mechanisms, as well as the immune response. Obesity and hypertension constitute late effects of chemotherapy in children and numerous studies have shown a decrease in cEPCs number, underlining poor vascular repair capability.

Aims: The determination of cEPCs in children treated for Acute Lymphoblastic Leukemia (ALL), Lymphomas (LYM) and solid tumors (ST) and the study of their levels in correlation with patients’ Body Mass Index (BMI) and blood pressure (BP) in different post-treatment time points.

Methods: Peripheral blood from children with ALL (n=166), LYM (n=37), ST (n=109) and children without malignancies (Control, n=191) were studied. The cEPCs population was determined by Flow cytometry based on CD34, CD45, CD133, VEGFR2 expression. The BMI and the blood pressure (BP) values were registered and the corresponding percentiles calculated. Patients were divided into three groups: <1 year post-treatment, ≤ 1-3 years post-treatment, and ≥ 3 years after completion of treatment. Statistical analysis was performed using t-test (Holm-Sidak) and 2way ANOVA (Tukey’s multiple comparisons test).

Results: In ALL group decreased levels of CD34+VEGFR2+ and CD34+CD133+VEGFR2+ were estimated in contrast with the LYM, ST and Control group (p=0,0144, 0,0262 respectively). Decreased levels of both populations were observed immediately after treatment completion in ALL, while in the post treatment period the levels gradually increased. On the contrary, in the LYM and ST group, at completion of treatment higher level of cEPCs were measured which gradually decreased (p= 0,0405 & 0,0026 respectively). The obesity was correlated with higher levels of cEPCs in the ST and LYM compared to the ALL group. The hypertensive patients had elevated cEPCs.

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Summary/Conclusion: Conditions causing vascular damage such as obesity and hypertension are correlated with increased cEPCs in patients treated for ALL, LYM and ST. The effect of treatment seems to have a different impact in these groups, possibly not only related with the biology of the disease but with the differences of treatment protocols as well. The increase of cEPCs during the 1 st post-treatment year probably reflects an effort to repair vascular damage caused by the treatment. Further investigation is needed to clarify these results.

P374: BLINATUMOMAB AS CONSOLIDATION FOR ADULT B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA. A REAL-WORLD STUDY

I. Urbino 1,* , E. Lengline 2 , M. Cerrano 1 , F. Rabian 2 , R. Kim 3 , M. Sebert 2 , R. Itzykson 2 , E. Audisio 1 , L. Ades 2 , H. Dombret 2 , E. Raffoux 2 , D. Ferrero 4 , E. Clappier 3 , N. Boissel 2

1 Hematology Department, AOU Città della Salute e della Scienza di Torino, Torino, Italy; 2 Hematology Department; 3 Hematology Laboratory, Saint-Louis Hospital - Université de Paris, Paris, France; 4 Department of Molecular Biotechnology and Health Sciences, Division of Hematology, University of Torino, Torino, Italy

Background: Blinatumomab is a CD3/CD19 bispecific T-cell engager approved for the treatment of relapsed/refractory (R/R) or minimal residual disease (MRD)-positive B-cell precursor acute lymphoblastic leukemia (B-ALL). Recent phase 3 studies in children and young adults with B-ALL in first relapse suggested that blinatumomab used as consolidation after chemotherapy salvage could be more beneficial than given as single agent in overt relapse. Whereas current strategies aim to demonstrate the benefit of blinatumomab in first line, the optimal use of blinatumomab in adult patients with first B-ALL relapse still deserves to be further explored

Aims: To evaluate the efficacy of blinatumomab given as consolidation in adults with B-ALL in a real-world setting

Methods: We retrospectively included 115 consecutive patients with B-ALL treated with blinatumomab from April 2012 until June 2021 at Saint-Louis Hospital, Paris, France (n=100), and at AOU Città della Salute e della Scienza, Turin, Italy (n=15). Patients included in clinical trials were excluded. Patients were divided in three subgroups: 68 patients treated in 1st complete remission (CR1), 31 patients treated in 2 nd CR after chemotherapy-base salvage therapy (CR2), and 16 patients treated in overt relapse (R/R). Patients in CR1 received blinatumomab for MRD persistence (n=59/68, 87%) or due to inability to receive standard consolidation (n=9/68, 13%, off-label use). The number of blinatumomab cycles along with the use of chemotherapy and/or of allogeneic hematopoietic stem cell transplant (alloHSCT) after blinatumomab was up to physician choice.

Results: The median age of patients was 37 years (range, 15-84). Among the 115 patients, 24% (n=28) were Philadelphia (Ph)-positive. Age, sex, baseline disease features and genetic risk categories did not differ between subgroups. After blinatumomab a complete MRD-response was achieved in 83% of CR1 and 86% of CR2 patients (p=.99). A complete remission was reached by 9/15 R/R patients (60%). In the 3 subgroups, the median number of blinatumomab cycles given was 2 (range, 1-6). Forty-six patients (42%) treated in CR (41% CR1, 45% CR2) and 4 R/R patients (25%) were bridged to allo-HSCT in continuous CR after blinatumomab. With a median follow up of 3.1 years, 3-year DFS was 68% in CR1 and 67% in CR2 patients (p=0.41); 3-year OS was 80% in CR1 and 71% in CR2 patients (p=0.32). In R/R patients, 3-year DFS and OS were 13% and 20% respectively. Considering patients who received blinatumomab in CR (CR1+CR2), univariate analysis showed that higher MRD levels both before and after blinatumomab were associated with shorter DFS, while only MRD response to blinatumomab was associated with OS. Both pre- and post-blinatumomab MRD levels retained significance in bivariate analysis for DFS

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Summary/Conclusion: The present study underlines the efficacy of blinatumomab in consolidation after chemotherapy-based salvage, showing comparable outcomes between patients treated in CR2 and CR1. In CR2 patients, promising DFS and OS were observed as compared to historical cohorts of patients treated with chemotherapy alone or with blinatumomab in overt relapse. Our data suggest that blinatumomab should be preferably used in consolidation rather than as salvage therapy in patients with B-ALL in first relapse

P375: CHALLENGES IN MANAGEMENT OF ACUTE LYMPHOBLASTIC LEUKAEMIA IN A RESOURCE CONSTRAINED SETTING IN LOWER-MIDDLE-INCOME COUNTRIES (LMICS)

T. Vaid 1,* , R. Dhawan 1 , M. Aggarwal 1 , P. Kumar 1 , J. Dass 1 , G. VIswanathan 1 , T. Seth 1 , S. Tyagi 1 , M. Mahapatra 1

1 Hematology, All India Institute of Medical Sciences, Delhi, India

Background: Improvement in supportive care, introduction of more intensive chemotherapy regimens and strategic use of allogenic stem cell transplant in some patients has resulted in significant improvement in outcomes of patients with acute lymphoblastic leukemia (ALL). However, this improvement is largely restricted to the developed world. We explore these challenges encountered in treating ALL in one of the largest government hospitals in India.

Aims: This study aims to assess the baseline characteristics, outcomes and challenges in management of acute lymphoblastic leukaemia in a resource constrained setting in lower-middle-income countries (LMICs)

Methods: Consecutive patients diagnosed with acute lymphoblastic leukaemia in the department of Hematology from 1 January 2017 to 31 December 2019 were included. Pediatric patients were treated with standard or augmented BFM protocols depending on risk stratification. Adolescent and yound adults (AYA) were treated with pediatric inspired protocols or GMALL like protocols. Adult patients were treated with GMALL like protocol.

Results: Of the 273 patients diagnosed with ALL, only 197 (72%) were able to get admitted for treatment at our institute. Median time from diagnosis to admission was 12.5 days (range: 0 -190 days) and the median distance of the patient’s permanent residence and our institute was 217 km (range 3-2400km). Baseline characteristics of these 197 patients are mentioned in Table 1.

Of the 197 patients treated at our centre, only 165 (83.7%) were able to receive induction chemotherapy. Of the remaining 32 patients, 26 (13.2%) died before treatment could be initiated and 6 (3%) were lost to follow up. Patients who died prior to starting chemotherapy had significantly higher incidence of baseline infection and organ dysfunction compared to those receiving induction.

Neutropenic fever was present in 50.56% patients at the time of admission. Focus of infection was lungs in 68.5%, sinusitis in 6.8%, skin & soft tissues in 5.6%, neutropenic enterocolitis in 2.2%, pyelonephritis in 1.1%, splenic abscess in 1.1% & bacteraemia with no obvious focus in 14.7% patients. 58.6% of the patients developed a new episode of neutropenic fever during induction chemotherapy. Post induction, a cumulative of 70 episodes of neutropenic fever and other infections were noted in 57 patients, of these 19 patients needed hospitalization.

At the end of induction, 64.5% (n=127) patients achieved complete remission. Induction mortality was 27.9% (n=55) and 4.1% (n=8) patients were lost to follow up. Primary refractory disease was noted in 3.5% (n=7) patients.

After a follow up period of 4 years, only 22.8% (n=45) patients were alive and on regular therapy, 32% (n=63) patients died & 25.4% (n=50) patients were lost to follow up. CNS relapse & medullary relapse was noted in 7.1% (n=14) & 9.1% (n=18) patients respectively.

4-year event free survival was 57% for both standard risk and high risk paediatric ALL patients respectively, 44% & 35% for standard risk and high risk AYA patients respectively & 20% and 27% for standard risk and high-risk adult ALL patients.

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Summary/Conclusion: ALL patients in our study had a high incidence of infection and organ dysfunction at baseline that complicated therapy. With limited access to healthcare, poor socio-economic status and high incidence of infection, developing countries face a unique set of challenges. Outcomes of ALL patients in our cohort are at odds with those reported from the developed world.

P376: CD38: A FUNCTIONING TARGET IN RELAPSED/REFRACTORY ACUTE LYMPHOBLASTIC LEUKEMIA. LIMITATIONS IN TREATMENT AND DIAGNOSTICS.

B. Vakrmanova 1,* , M. Novakova 1 , P. Riha 2 , M. Zaliova 1 , E. Fronkova 1 , E. Mejstrikova 1 , L. Reznickova Rezkova 1 , J. Stary 2 , O. Hrusak 1 , L. Sramkova 2

1 Department of Paediatric Haematology and Oncology, CLIP, Second Faculty of Medicine and Motol University Hospital; 2 Department of Paediatric Haematology and Oncology, Second Faculty of Medicine and Motol University Hospital, Prague, Czechia

Background: The prognosis of relapsed T-acute lymphoblastic leukemia (ALL) is dismal and there is a need for new treatment options. Daratumumab, a monoclonal kappa chain antibody against CD38 is routinely used in multiple myeloma treatment. CD38 is also expressed in malignant cells of most cases with pediatric ALL. Accordingly, daratumumab can be used experimentally in treatment of relapsed ALL but data about efficacy of such a treatment is limited. The loss of CD38 described in myeloma patients can be one of the reasons of treatment failure.

Aims: Does daratumumab provide a benefit in relapsed ALL?

Can we observe the inability of anti-CD38 mAbs to bind to leukemia cells after daratumumab treatment as described in myeloma patients?

Methods: We treated five patients with relapsed ALL with daratumumab between 10/2019 – 10/2021 (four of them for a first relapse of T-ALL and one for a second CD19 neg relapse of B cell precursor (BCP) ALL). In three patients, daratumumab was used in combination with chemotherapy, in one patient, chemotherapy was early discontinued for toxicity and the remaining patient received it in monotherapy due to clinical condition. Blast positivity of CD38 by a diagnostic monoclonal antibody (mAb) at relapse was confirmed in all patients before treatment started.

Results: Three of five patients responded to daratumumab plus chemotherapy and were in second complete remission (CR2) and underwent stem cell transplantation (SCT). CD38 expression on blasts after daratumumab was not evaluated due to blast elimination in these cases. Two of these patients relapsed with CD38 pos ALL 5 and 7 months after SCT, respectively. Third patient is in CR2 one year after SCT. In two of five patients (one with T-ALL and one with a CD19 neg BCP ALL) blasts were still detected by flow cytometry after daratumumab. In patient 4 disease progressed under daratumumab treatment. Under the progressing disease patient died 52 days after the relapse. In the other patient, the amount of blasts decreased by one log after the first course of treatment. Nevertheless, the blasts remained on the same log level. Therefore, daratumumab treatment was stopped and replaced by inotuzumab and subsequent SCT, which led to a molecular CR3. We could not detect binding of a diagnostic anti-CD38 (clone T16, HIT2) in neither of the two cases early after the start of daratumumab treatment. We then tested whether the lack of anti-CD38 binding could be caused by steric hindrance with daratumumab molecules as had been described in myeloma patients. We proved our patients had blasts CD38 positive on mRNA level and intracellularly (patient 4). We also detected daratumumab directly on the blasts by detecting its kappa chain on the blasts (patients 4). Moreover, adding daratumumab to blasts of a patient freshly diagnosed with CD38 pos BCP ALL blocked anti-CD38 antibody (clone T16) binding. Collectively, the data showed that like in myeloma, daratumumab may block binding of some diagnostic antibodies. We are currently testing the feasibility of multiepitope CYT-38F2 antibody in children with ALL on daratumumab treatment.

Summary/Conclusion: In conclusion, daratumumab can lead to CR2 in relapse/refractory ALL, however, the effect is often temporary. Daratumumab administration may result in weeks lasting inability of anti-CD38 mAbs to bind to leukemia cells.

Supported by Ministry of Health of the Czech Republic, grants nr. NU20-03-00284 and NU20J-07-00028.

P377: GENETIC CHARACTERISTICS AND CD7-CAR-T THERAPY OF TRAD::MYC TRANSLOCATION POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS

T. Wang 1,2 , J. Ni 3 , H. Zhang 3 , S. Hui 1 , T. Liu 3 , X. Zhang 4 , G. Zhang 4 , X. Ma 3 , X. Wang 1 , Y. Zhang 1 , Q. Zhang 1 , H. Liu 1,2,*

1 Division of Laboratory Medicine, Hebei Yanda Lu Daopei Hospital, Langfang; 2 Beijing Lu Daopei Institute of Hematology, Beijing; 3 Division of Laboratory Medicine, Hebei Yanda Lu Daopei Hospital, Langfang; 4 department of chemotherapy, Hebei Yanda Lu Daopei Hospital, Langfang, China

Background: TRAD::MYC is a recurrent but rare translocation in leukemia and lacks relevantreports.

Aims: To investigate the genetic characteristics of acute lymphoblastic leukemia (ALL) with TRAD::MYC translocation and the clinical outcome of chimeric antigen receptor T cells (CAR-T) therapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Methods: A retrospective analysis was performed of 5,450 patients with ALL admitted to our hospital from Apr. 1, 2016, to Dec. 31, 2021. Cases positive for t(8;14)(q24.1;q11.2) in karyotype analysis were selected, TRAD::MYC translocation and CDKN2A/B gene deletion were confirmed by fluorescence in situ hybridization (FISH). The TRAD::MYC positive cases were further screened for a panel of 36 common leukemia fusion genes (FGs), RNA-seq for comprehensive analysis of FGs, and mutation screening of a panel of 86 leukemia driver genes.

Results: A total of 12 cases were TRAD::MYC positive, 9 males and 3 females, aged 3-28 years (median 15 years), 9 children (<18 years old) and 3 adults. The diagnosis included 11 T-ALL and 1 B-ALL, and 7 cases (63.6%) manifested high white blood cells (180-486.18×109/L) at the onset. Nine cases had karyotype results at the initial onset, of which three were positive for t(8;14)(q24.1;q11.2), and the rest 6 acquired this translocation at relapse. In the 3 cases with no karyotype results at the initial onset, t(8;14)(q24.1;q11.2) was detected in relapse specimens. Of the 12 cases, 11 showed additional karyotype abnormalities in t(8;14)(q24.1;q11.2) positive specimens. FISH confirmed the involvement of TRAD and MYC genes corresponding to t(8;14)(q24.1;q11.2) in all 12 cases, and CDKN2A/B gene loss was detected in 4 (33%) cases. All cases were screened for fusion genes, and RNA-seq was performed in five cases. Six cases were positive for driver FGs, including 3 STIL::TAL1, 1 KMT2A::AF9 in the B-ALL case, 1 CTCF::NFATC3, and 1 ZC3HAV1::ABL2. Six cases (50%) were positive for PTEN mutation and 3 positive for FBXW7 mutation, other mutated genes including NOTCH1, TP53, CCND3, etc. Eleven cases were followed up for 6-67 months (median 11 months), and 8 died during the follow-up period. Eight cases (72.7%) experienced central nervous system (CNS) leukemia, and 3 had mediastinal invasion. All patients relapsed after chemotherapy, and the leukemia cells proliferated rapidly. Six cases received CD7-CAR-T therapy, and all achieved disease remission after CAR-T cell infusion, of which 1 died from secondary severe lung infection and 2 died from relapse. Four cases underwent allo-HSCT, including 2 salvage transplantation and 2 bridging transplantation after CD7-CAR-T regimen. All 3 cases that remained alive received CD7- CAR-T therapy, 2 of which were bridged to allo-HSCT.

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Summary/Conclusion: Recurrent t(8;14)(q24.1;q11.2)/TRAD::MYC have a low incidence, mainly in T-ALL cases, but can also be seen in B-ALL. More than half of cases acquired the translocation at relapse and in some cases accompanied with other driver FGs in T-ALL such as STIL::TAL1, KMT2A fusions, and ABL family gene fusions. TRAD::MYC translocation confers aberrant MYC expression and hyperproliferative of tumor cells. Half cases were also accompanied by PTEN mutation, which further adversely affected the prognosis. In these cases, the response to conventional chemotherapy is extremely poor and prone to early relapse, and CNS infiltration is common. CD7-CAR-T regimen bridging to allo-HSCT might be a promising therapeutic strategy for better survival in this subgroup, and the long-term outcomes remain to be further investigated.

P378: THE PROGNOSIS FACTORS OF CAR-T THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY B-ALL

Y. Wang 1 , X. Hu 1,* , D. Zhang 1 , Q. Gao 1 , X. Zhai 1 , H. Wang 1 , Y. Gao 1 , Y. Miao 1 , Y. Guo 1 , W. Zhang 1 , X. Ru 1 , X. Li 2 , F. Guan 2

1 Department of Hematology, Shaanxi Provincial People’s Hospital, Xi’An; 2 School of Medicine, Northwest University, Xi’An, China

Background: CAR-T therapy showed good clinical efficacy on relapsed/refractory B-ALL (R/R B-ALL) patients. However, some latest studies revealed that most patients tend to relapse during long-term follow-up after CAR-T infusion. Few studies have examined the factors associated with the prognosis of R/R B-ALL patients.

Aims: The aim of the study is to investigate the factors associated with the long-term survival of R/R B-ALL patients after CAR-T therapy.

Methods: Thirty-eight R/R B-ALL patients were included in the study. Patients received CAR-T cells infusion from May 2015 to August 2018 in Shaanxi Provincial People’s Hospital. Peripheral blood mononuclear cells were collected, and CD3+ cells were sorted. CAR-T cells were prepared by lentivirus. These cells were transfused 3 to 5 days after lymphocyte removal. Bone marrow smears, flow cytometry, and QT-PCR were used to assess the development of disease between 2 and 4 weeks after infusion. The endpoint of follow-up was the time of death or Feb 15, 2022. Cox regression models were used to analyse prognosis factors.

Results: The median age of the 38 patients was 25 years (6-59 years), including 21 males and 17 females. 26.3% (10/38) of patients had leukemic cells higher than 30% in their bone marrow smears. There were 34.21% (13/38) of patients with Ph(+), 34.21% (13/38) with extramedullary disease (EMD), 10.52% (4/38) with MLL-AF4 fusion gene (+), and 5.26% (2/38) had received HSCT prior to CAR-T therapy. Within 4 weeks of CAR-T cells infusion, 86.84% (33/38) of patients achieved MRD (-). 13 patients were disease-free by Feb 15, 2022. For all the 38 patients, the median OS and DFS were 19 months and 16.5 months. We found that a higher proportion of leukemic cells in bone marrow, MRD (+) after CAR-T infusion and the presence of the MLL-AF4 fusion gene could predict a worse prognosis. We found that sex, Ph (+), and HSCT after CAR-T infusion is not associated with the long-term survival of patients. Patients who had received maintenance therapy after CAR-T therapy showed better OS (49 months vs 9 months, P<0.001) and DFS (49 months vs 6 months, P<0.001) compared to those who received no maintenance therapy. In addition, we unexpectedly found that patients with EMD showed better OS (49 months vs 15 months, P=0.019) and DFS (49 months vs 12 months, P=0.045) than those without EMD.

Summary/Conclusion: R/R B-ALL patients could achieve long-term survival with CAR-T cell therapy. The proportion of leukemic cells in bone marrow, MRD(+), and the presence of the MLL-AF4 fusion gene could predict a worse prognosis of R/R B-ALL patients. We found that maintenance treatment after CAR-T therapy could significantly improve patients’ long-term survival and disease-free time, while HSCT showed no significant benefit to long-term prognosis.

P379: MUTATIONAL LANDSCAPE IN THERAPY-RELATED ACUTE LYMPHOBLASTIC LEUKEMIA

K. D. Hofer 1,* , E. Haralambieva 2 , C. Fritz 2 , M. Roncador 1 , C. Ruetsche 3 , M. Buehler 2 , J. Tchinda 4 , U. Schanz 1 , C. C. Widmer 1,5

1 Department of Medical Oncology and Hematology; 2 Institute of Pathology; 3 Hematology, University Hospital Zurich; 4 Oncology Laboratory, University Children’s Hospital Zurich, Zurich; 5 Hematology, University Hospital Basel, Basel, Switzerland

Background: There is increasing evidence that therapy-related acute lymphoblastic leukemia (t-ALL) with a reported incidence of 3% to 9% is a distinct entity, which is associated with inferior survival compared with de novo ALL (dn-ALL). It appears that this results from a poor cytogenetic predisposition, but data are still very limited due to the rarity of the disease and the mutational landscape has not been adequately explored, as molecular analyses are sparse in this unique patient cohort.

Aims: This study attempted to investigate the molecular landscape of patients with an initial diagnosis of ALL and a prior history of malignancy with consecutive genotoxic therapy and to evaluate the cytogenomic results and survival.

Methods: Data were collected from 131 adult patients with an initial diagnosis of ALL treated at the Department of Medical Oncology and Hematology, University Hospital Zurich, Switzerland. Patients with prior treatment of another malignancy with alkylating agents, topoisomerase inhibitors, radioactive iodine ablation (for thyroid cancer) and/or irradiation were classified as t-ALL.

Results: Within our ALL cohort, 14% of the patients met the criteria for t-ALL. The median latency between genotoxic therapy and diagnosis of t-ALL was 6.5 years, with breast cancer being the most common neoplasm. KTM2A rearrangement was not significantly more common in t-ALL than in dn-ALL (15.7% vs 11%, p = 0.15), a finding which was not as expected from the literature. In therapy-related myeloid neoplasms large chromosomal deletions, such as del(5q) and del(7q) are known to be associated with alkylating agents and topoisomerase II inhibitors. In 12% of our t-ALL patients deletions in chromosome 7 were found, but none in chromosome 5. On the other hand, a high proportion of Philadelphia chromosome (Ph) 36.8% was present in the t-ALL group. On a molecular level, the most frequently observed mutation was KMT2D , followed by myeloid neoplasm-associated mutations of CDKN2A , KRAS and DNTM3A , but no TP53 mutation was found. KRAS mutation was never present in combination with Ph+, but was associated with KMT2A rearrangement. Outcome was particularly poor in Ph+ t-ALL patients compared to Ph+ dn-ALL, an effect that was mitigated by allogeneic stem cell transplantation.

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Summary/Conclusion: In addition to KMT2A rearrangement as a high-risk genomic subtype, a high proportion of Ph+ was observed in the t-ALL group. Ph+ t-ALL showed a different mutational landscape than KTM2A -mutated patients, suggesting that the development of t-ALL is driven by multiple pathways with particularly bad outcome for patients with Ph+. Although there is overlap in the molecular profile of t-ALL and myeloid neoplasms, no TP53 mutation was found in our t-ALL cohort. The heterogeneity of the genetic aberrations in ALL render molecular genome sequencing of somatic variants an important step to advance our understanding of the disease, especially in those patients without KTM2A rearrangement but Ph positivity. Our findings help further define therapy-associated ALL as a distinct entity, but also highlight the need for a better molecular understanding of its specific disease evolution.

P380: EX VIVO IMMUNE ACTIVATION WITH THE MACROPHAGE-TARGETING IMMUNOTHERAPY, ANTI-CLEVER-1 ANTIBODY BEXMARILIMAB, IN ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME

S. Aakko 1,2,* , A. Ylitalo 3 , H. Kuusanmäki 2 , M. Björkman 1 , J. Mandelin 1 , J. Jalkanen 1 , M.-L. Fjällskog 1 , C. Heckman 2 , M. Hollmén 3 , M. Kontro 2,4

1 Faron Pharmaceuticals Ltd, Turku; 2 Institute for Molecular Medicine Finland, HiLIFE, University of Helsinki, Helsinki; 3 MediCity Research Laboratory, University of Turku, Turku; 4 Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland

Background: Despite the recent approvals of targeted therapeutic agents for acute myeloid leukemia (AML), the treatment options remain few and for myelodysplastic syndrome (MDS), even fewer. The potential of immunotherapies in these myeloid malignancies remains under investigation. To this end, bexmarilimab, an anti-Clever-1 antibody, aims to harness the therapeutic potential of macrophages. Clever-1 ( STAB1 ) is a scavenger receptor, expressed on the surface of a subpopulation of immunosuppressive macrophages. By inhibiting Clever-1, bexmarilimab demonstrates potential to turn the anti-inflammatory macrophages to pro-inflammatory and activate CD8+ T cells (Virtakoivu R. et al. 2021. Clin. Cancer Res ) along with promising anti-tumor activity against solid tumors in patients with multiple lines of previous treatment (Bono P. et al. 2019. Ann. Oncol. ). As Clever-1 is expressed in myeloid cells and high STAB1 levels associate with poor survival in AML (Lin S.Y. et al. 2019. Mol. Ther. Nucleic Acids ), bexmarilimab may possess therapeutic potential in AML and MDS.

Aims: The aim of this study was to profile Clever-1 expression in AML and MDS and to test in preclinical models bexmarilimab’s growth inhibitory and immunomodulatory potential, as a single agent and in combination with azaciditine and venetoclax.

Methods: AML cell lines (n=11) and frozen mononuclear cells, extracted from AML (n=27) and very high risk MDS (n=4) patient bone marrow (BM) aspirates provided by the Finnish Hematology Registry and Biobank, were used in this study. Samples were treated for 48h with bexmarilimab alone, or in combination with azacytidine and/or venetoclax. Flow cytometry was used to detect different myeloid and TBNK cell populations along with Clever-1, HLA-DR, PD-(L)1 and Granzyme B protein expression.

Results: Our results confirm Clever-1 protein expression in AML cell lines with high STAB1 level and most importantly, in AML and MDS patient-derived BM monocytes and blasts. AML samples from FAB M4/M5 subtypes exhibited highest Clever-1 levels, along with a FAB M2 sample collected prior to allogeneic stem cell transplantation and consequent rapid relapse. Clever-1 expression correlated negatively with monocyte MHC class II molecule, HLA-DR, expression, and BM T cell frequency, in line with the immunosuppressed state associated with high Clever-1.

Ex vivo treatment of the primary AML cells with bexmarilimab resulted in a notable, 5-10x fold increase of monocyte HLA-DR in samples with low basal HLA-DR and high Clever-1. The combination of azacitidine with bexmarilimab augmented HLA-DR induction by 20-110% (mean 44%). On the other hand, combination with bexmarilimab overcame the HLA-DR suppressing effect of venetoclax.

The BM TBNK cell populations and thus bexmarilimab’s effect on the effector cells, showed great inter-sample variability. FAB M1/M2 AML showed increase of activation markers, such as Ki67, CXCR3 and Granzyme B after ex vivo bexmarilimab treatment in CD8+ T cells. Furthermore, bexmarilimab reduced PD-1 expression in NK- and CD8+CXCR3+ T cell populations of FAB M0-M2 AML and MDS-EB2.

Summary/Conclusion: Our results confirm that Clever-1 is expressed in AML and MDS patient BM blasts and monocytes. Ex vivo treatment with bexmarilimab, alone or in combination with azacytidine or venetoclax, demonstrated increased HLA-DR expression, indicating enhanced antigen presentation capability. Furthermore, NK and CD8+ T cells showed decreased PD-1 and increase of activation markers. These results confirm the therapeutic potential of bexmarilimab in myeloid malignancies and await further validation in clinical trials.

P381: DNA METHYLATION PROFILING OF MESENCHYMAL STROMAL CELLS ISOLATED FROM FEMURAL HEAD BONE MARROW VERSUS BONE MARROW ASPIRATES: RELEVANCE FOR AML STUDY BASED CONTROLS

A. Abdul-Aziz 1,* , C. Weigel 2 , A. Kovacs 2 , Y.-Z. Wu 2 , J. Byrd 1 , E. Hertlein 1 , C. Oakes 2

1 Department of Internal Medicine, University of Cincinnati, Cincinnati; 2 Department of Internal Medicine, The Ohio State University, Columbus, United States of America

Background: Bone marrow mesenchymal stromal cells (BMSCs) are important precursors for multiple differentiated cell types. Alterations in BMSCs contribute to several disease states, including acute myeloid leukemia (AML) and other hematologic cancers. As BMSCs are used as controls for research and obtained from various bone tissue locations with active hematopoiesis, it is important to understand the developmental capacity and potential differential phenotypes of BMSCs derived from various anatomical sites. Epigenetic regulation plays an essential role in cell lineage specification and development; carefully controlled transcription factor (TF) activity and other chromatin-associated proteins modify the genomic landscape to generate stable, cell type-specific global patterns of gene expression.

Aims: We examined genome-wide DNA methylation patterns in BMSCs derived from either femoral head bone marrow or bone marrow aspirates of the iliac crest of healthy donors to identify the optimal control cell population for hematological malignancy research.

Methods: Hip-derived BMSCs were obtained from femur heads obtained during hip replacement surgeries (n=19). All patients provided informed consent in accordance with an IRB-approved protocol and the Declaration of Helsinki. Aspirate-derived BMSCs (n=7) were expanded from bone marrow mononuclear cells obtained from a commercial source (AllCells). For genome-wide DNA methylation analyses, the EPIC/850K array platform was used following manufacturer’s protocols (Illumina). Analyses included data obtained from the Gene Expression Omnibus (GEO, GSE79695 and GSE124390 ) generated from 450K (n= 12) and 850K array data (n=10), respectively. Overlapping probes between the arrays that passed QC were used for downstream analysis. Sex chromosomes and non-CpG probes were removed. The data was reduced to 5,000 most-variable CpGs followed by clustering analysis as described in results.

Results: Unsupervised clustering using principal component analysis (PCA) and hierarchical clustering revealed two groups that segregated perfectly between hip-derived versus aspirate-derived BMSCs. There were no observable differences in aspirate-BMSCs generated by our group and those obtained from GEO. Two group supervised analysis (hip versus aspirate) revealed 4,568 CpGs (probes) hypomethylated in hip-derived samples (q<0.05, difference>20%) and 1,433 probes hypermethylated in hip-derived samples (q<0.05, difference>20%). DNA sequence motif enrichment analysis using the HOMER software revealed that hypomethylation in hip-derived BMSCs involved activation of CRE, HOX and AP-1 family TFs. We also observed parallel hypomethylation of the promoters of CREB5, genes from the HOXB and HOXC clusters, and several genes of the MAPK pathway. Hypermethylation in hip-derived BMSCs conversely involved RUNX motifs, corresponding with hypermethylation of the RUNX3 gene. We also observed robust DNA methylation changes at the promoters of other lineage-specifying genes including TBX3, TBX5, PITX2, NR2F2, and EN1. Pathway analysis of genes demonstrating promoter hypomethylation in hip-derived samples revealed strong enrichment for adipocyte progenitors and development.

Summary/Conclusion: These findings demonstrate that hip-derived BMSCs are phenotypically distinct from aspirate-derived BMSCs, which may affect their potential use in both therapeutic and research settings where stem-like non-primed BMSCs are required. Our study highlights the importance of using proper selection of control cells to study the role of BMSCs in hematologic malignancies.

P382: PTK2B MEDIATES TYROSINE KINASE INHIBITOR DRUG RESISTANCE IN AML ASSOCIATED WITH ALTERED MIGRATION AND ADHESION PROPERTIES

C. Allert 1,2,* , S. Zimmermann 2,3 , S. Göllner 1 , D. Heid 1,4,5 , M. Bruckmann 1 , M. Janssen 1 , B. Besenbeck 1 , J. Krijgsveld 2,3 , C. Müller-Tidow 1,2,4 , M. F. Blank 1,3,4

1 Medical Department V Hematology, Oncology and Rheumatology, Heidelberg University Hospital; 2 University of Heidelberg Medical Faculty; 3 Division Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ); 4 Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 5 European Molecular Biology Laboratory EMBL, Heidelberg, Germany

Background: Tyrosine kinase inhibitor (TKI) therapy is a well-established therapy approach in FLT3-mutated AML. However, TKI resistance occurs frequently while the underlying mechanisms remain incompletely understood. One concept of chemotherapy-resistance in leukemia is cell adhesion mediated drug resistance (CAM-DR).

Aims: In this study we aimed to investigate the role of CAM-DR at an early stage of therapy resistance in FLT3-ITD mutated AML.

Methods: We characterized FLT3-ITD mutated AML cell lines upon shorter and longer TKI exposure. Emerging TKI resistance was assessed by transcriptome and total proteome analyses, as well as nascent proteomics and MS-based protein stability/degradation assays. Further, we investigated the impact of emerging TKI-resistance on cell adhesion and migration. After identification of a highly expressed protein in TKI resistant cells with implications in cell adhesion, i.e. PTK2B, we investigated the effects of available inhibitors of this protein on AML cell lines in the presence or absence of other TKIs or chemotherapeutic agents and confirmed the effects in primary AML patient samples (n=6).

Results: We discovered that protein tyrosine kinase 2b (PTK2B) was induced during early TKI resistance development. Interestingly, PTK2B, a member of the focal adhesion kinase superfamily, was upregulated at the transcriptional and translational level. PTK2B was also post-translationally stabilized in TKI resistant cells. Of note, PTK2B was highly expressed in leukemic stem cells (LSCs) from FLT3-ITD mutated patients.

The upregulation of PTK2B was accompanied by increased migration of TKI-resistant cells with concomitant loss of cell adhesion, suggesting altered niche interactions. Notably, treatment with a PTK2B inhibitor, such as PF-431396, reverted the resistance-associated phenotypes. Enhanced migration was abolished upon PF-431396 treatment. Significantly altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon treatment of resistant cells with a PTK2B inhibitor.

The FAK/PTK2B inhibitors PF-431396 and Defactinib synergized with several TKIs, such as midostaurin or gilteritinib, and commonly used chemotherapeutic agents, such as daunorubicin, in FLT3-ITD mutated AML cells. Synergism was restricted to FLT3 mutated cells. This synergism was significantly more pronounced in TKI-resistant cells and the addition of a PTK2B inhibitor could overcome TKI resistance to various extent. Also in primary AML patient samples, Defactinib showed lower IC50s specifically for FLT3-ITD mutated patients.

Summary/Conclusion: These data establish that FLT3-ITD AML resistance towards TKI depends on PTK2B which alters leukemia cell adhesion and migration. PTK2B inhibitors might be synergistic with TKI in FLT3-ITD AML therapy. PTK2B inhibition could thus prevent the outgrow of drug-resistant clones at an early stage and may improve the outcome of FLT3-ITD mutated AML.

P383: USING CF-DNA AS STARTING MATERIAL FOR MRD STUDIES BY NGS IMPROVES THE SENSITIVITY OF THE METHOD AGAINST CTCS.

N. Álvarez Sánchez-Redondo 1,* , S. Dorado 2 , A. Martín 3 , L. Rufián 1 , V. Garrido 1 , A. Giménez 1 , L. Moreno 1 , Y. Heredia 4 , J. Martínez-López 5 , S. Barrio 6 , R. Ayala 7

1 1. Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC; 2 3.Computer Science and 4.Altum Sequencing Co., Madrid, Spain. Engineering Department. Carlos III University, Madrid and; 3 4.Altum Sequencing Co., Madrid, Spain.; 4 4. Altum Sequencing Co., Madrid, Spain.; 5 1.Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC, and 2.Complutense University of Madrid; 6 1.Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC, and 4.Altum Sequencing Co., Madrid, Spain.; 7 1.Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC, and 2.Complutense University of Madrid., Madrid, Spain

Background: The quantification of Minimal Residual Disease (MRD) has become a relevant marker as it can detect acute myeloid leukemia (AML) patients with a high risk of relapse. Although the reference MRD method in AML is to quantify tumor cells present in bone marrow (BM), the study of circulating tumor cells (CTC) in peripheral blood (PB) is also being incorporated into clinical protocols. However, cfDNA as an MRD biomarker in AML has not yet been validated.

Keywords: AML, MRD, Liquid Biopsy, NGS.

Aims: The purpose of the study is to evaluate the use of cfDNA by NGS test as a new method for MRD quantification in AML and compare it application with the use of PB CTCs in the follow-up of the disease.

Methods: Three AML patients treated at the Hospital Universitario 12 de Octubre and with cfDNA available have been included. The mutational profile was defined by NGS (Ion Torrent System) at diagnosis using a panel of 42 genes involved in myeloid pathologies. Then, somatic mutations were selected, and MRD quantification was performed in liquid biopsy combining whole blood cells (WBC) and cfDNA. Both fractions were informatically differentiated by including molecular tags in the first PCR amplification. A total of 18 follow-up samples were analyzed with a threshold for EMR positivity set at 1x10^ (-4) as we previously described (Onecha E et al. Haematologica 2019). An average of 66ng of cfDNA and 660ng of gDNA from WBC were used. The results were compared with those obtained by multiparametric flow cytometry (MFC) in BM.

Results: In the first patient, four variants affecting the genes CBL (VAF =7.4%), MPL (50%), JAK2 (51.5%) and ETV6 (10,6%) were detected at diagnosis in BM. The Leukemia-associated variant affecting ETV6 was selected as an MRD biomarker and detected in cfDNA (0.86%) post-consolidation 1. On this time-point (TP), the result of MRD by MFC on BM was also positive (0.002%), being negative in CTC-PB by NGS. The Non-leukemia-associated variants ( JAK2 , MPL , CBL ) were also followed-up. Of interest, those alterations showed constant VAF levels in pre-transplant remission. After transplant, the VAF of JAK2 and MPL correlated with the donor chimerism studied by conventional methods after transplantation. On the other hand, CBL VAF was below the threshold sensitivity limit (0.01%) during all follow-ups. This result that CBL was also associated with leukemia, likely affecting a treatment-sensitive subclone.

In patient 2, variants affecting IDH2 (92%), RUNX1 (43%), and DNMT3A (49%) genes were detected by at diagnosis. IDH2 mutation was selected as MRD biomarker, showed a VAF of 9.4% at relapse, correlating with the blast count of 10% defined by the morphology study of BM. Of note, in this TP, MRD-BM by MFC was 0.011%.

In patient 3, the SF3B1 (43%) and EPAS1 (49%) mutations were detected by NGS in BM at diagnosis. The leukemia-associated variant ( SF3B1 ) was observed in the cfDNA sample (0,0003) at the first follow-up sample but was negative at the following TPs. MRD by CTCs and MFC-BM were also negative. This patient has not relapsed to date (follow-up time six months after diagnosis).

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Summary/Conclusion: MRD quantification based on the use of cfDNA by NGS offers promising results and, in the future, could be a good option for disease monitoring or early detection of relapse. The results of the whole series will be presented at the meeting.

P384: THE ROLE OF THE MANNOSE RECEPTOR C-TYPE 2 (MRC2) IN LEUKEMIC STEM CELL MAINTENANCE IN AML

M. Arnone 1,* , E. Görsch 1 , S. Pöschel 1 , P. Godavarthy 1 , A. Stanger 1 , S. Rudat 1 , C. Lengerke 1

1 Innere Medizin 2, University Hospital Tuebingen, Tuebingen, Germany

Background: Therapy-resistant leukemia-initiating cells, so called leukemic stem cells (LSC), are responsible for disease relapse and a major cause of death in patients with acute myeloid leukemia (AML). Effective identification and targeting of LSCs are thus important goals. Our previous RNA sequencing analyses revealed that the MRC2 gene, which encodes a transmembrane receptor known for its ability to bind and degrade collagen as well as for its pro-migratory and -invasion functions, is enriched in LSCs.

Aims: We aim to gain a better understanding of the function(s) of MRC2 in AML in general and LSCs in particular.

Methods: We used flow cytometry to characterize MRC2 expression in primary AML samples and healthy cells from the hematopoietic system. Imaging flow cytometry experiments were performed to study the role of MRC2 in gelatin uptake.

Results: We found that mRNA expression of the MRC2 gene was increased in the LSC compartment of 5 AML patients compared to their non-LSC counterparts. Analyzing MRC2 cell surface presentation by flow cytometry, we found that AML cell lines (n=10) displayed the highest levels of MRC2 expression followed by healthy CD34 + cord blood (CB) cells (n=7), while primary AML patient samples (n=21) were characterized by an intermediary MRC2 cell surface presentation with the lowest levels of MRC2 presentation in differentiated healthy hematopoietic cells. Interestingly, high MRC2 expression correlated with increased clonogenic activity as only sorted MRC2 + but not MRC2 - primary AML cells from the same sample were able to form colonies in a methylcellulose assay; and siRNA-mediated knockdown of MRC2 in the bulk of primary AML cells led to a reduction in colony formation when compared to control siRNA transfected cells. Furthermore, using CRISPR/Cas9 we generated a MRC2 knock-out AML cell line and observed in in vitro assays that MRC2 expression is required for migration, invasion and adhesion to collagen and laminin 421. Finally, we could show by imaging flow cytometry (ImageStream) that AML cells lacking MRC2 expression were unable to internalize fluorescently-labelled gelatin.

Summary/Conclusion: Together, our results suggest that AML cells benefit from MRC2 expression due to its multiple functions involving migration, invasion and the interaction with extracellular matrix components. Additionally, MRC2 has a potential role in LSC biology such as the maintenance of stemness-associated properties. Further in vivo studies will improve our understanding of MRC2, which could then inform us on novel therapeutic approaches.

P385: DETECTION OF KMT2A PARTIAL TANDEM DUPLICATIONS IN ACUTE MYELOID LEUKEMIA PATIENTS BY NGS

A. Artigas-Baleri 1,* , G. Oñate 2 , A. Brell 2 , J. Esteve 3 , S. Vives 4 , M. Tormo 5 , M. Arnan 6 , A. Garcia 7 , R. Coll 8 , A. Sampol 9 , J. Bargay 10 , F. Vall-llovera 11 , O. Salamero 12 , J. Nomdedéu 2 , M. Pratcorona 2

1 Hematology, Insitut de Recerca de Hospital de la Santa Creu i Sant Pau; 2 Hematology, Hospital de la Santa Creu i Sant Pau; 3 Hematology, Hospital Clínic, Barcelona; 4 Hematology, Institut Català d’Oncologia, Hospital Germans Trias i Pujol, José Carreras Leukemia Research Institute, Badalona; 5 Hematology, Hospital Clínico Universitario, Biomedical Research Institute INCLIVA, Valencia; 6 Hematology, Insitut Català d’Oncologia, Hospital Duran i Reynals, Institut Biomèdica de Bellvitge (IDIBELL), L’Hospitalet de Llobregat; 7 Hematology, Hospital Universitari Arnau de Vilanova, Lleida; 8 Hematology, Institut Català d’Oncologia, Hospital Josep Trueta, Girona; 9 Hematology, Hospital Son Espases; 10 Hematology, Hospital Son Llàtzer, Palma de Mallorca; 11 Hematology, Mutua Terrassa, Terrassa; 12 Hematology, Hospital Vall d’Hebron, Barcelona, Spain

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic disease consisting of an aberrant proliferation and differentiation of hematopoietic stem cells in the bone marrow. In the last years the identification of cytogenetic and molecular biomarkers has improved the AML classification considering prognosis, and allowing a better risk-adapted treatment strategy. Regarding molecular biomarkers, NPM1, RUNX1 and ASXL1 mutations, FLT3 -ITD (internal tandem duplications), CEBPA bi or KMT2A -PTD (partial tandem duplications) have been shown to have clinically significant prognostic value and they define specific AML disease subtype. Our study focuses on the detection of PTD in KMT2A which is involved in the regulation of gene expression during hematopoiesis. KMT2A -PTDs are found in 5-10% of normal karyotype patients and have been associated with poorer disease-free and overall survival rates. Mainly, PTDs in this gene span exons 2 to 8-10 and their detection is based on long-distance genomic PCR with extracted DNA from bone marrow. This is a time-consuming technique, and that is probably the main reason to explain why this marker is not widely performed at the time of diagnosis.

Aims: We aimed to analyze the potentiality of Next Generation Sequencing (NGS) in the identification of KMT2A -PTD, a technique that is being implemented in many laboratories and could be a good strategy to detect this marker.

Methods: This study included patients from CETLAM group (Grupo Cooperativo de Estudio y Tratamiento de las Leucemias Agudas y Mielodisplásicas) with a diagnosis of AML in 2021 (from January to October) and age≤70. The study of KMT2A -PTDs was carried out with DNA samples through two different techniques: PCR and NGS. DNA was extracted from bone marrow with the AllPrep DNA/RNA Mini Kit (Qiagen®). For the PCR amplification we used both custom 8-exon forward primer and 2-exon reverse primer. NGS was performed by hybridization capture technology (Nonacus®) using a custom panel which includes 43 genes. MiSeq (Illumina®) system was used for sequencing and Datagenomics for analysing the results. Hybridization capture allows the comparison of the number of reads per position between samples to identify structural variants as PTDs. This approach permits to know exactly which exons have been duplicated.

Results: We included a total of 72 AML patients who were genotyped for KMT2A -PTD with both PCR and Hybridization capture NGS. We identified 4 and 7 patients with a partial tandem duplication in KMT2A gene by PCR and NGS, respectively. The four KMT2A -PTDs identified with PCR were also detected by NGS. In contrast, three PTDs were observed only with NGS. In two of them the PTD spanned exons 2 to 8 (Figure 1), while in the other it spanned exons 4 to 8. The first two PTDs were confirmed with the classic long-distance genomic PCR but performed with cDNA, skipping the presence of a large non-coding DNA in the rearrangement which could not be amplified by conventional PCR. To confirm the third PTD a new reverse primer in exon 4 was designed. All three NGS observed KMT2A -PTDs could be verified.

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Summary/Conclusion: Our results show that Hybridization capture NGS is a good technique to detect KMT2A -PTDs, with a better sensibility than PCR. NGS is a cost-effective method as it is becoming an essential tool for AML classification at diagnosis.

P386: CELL FATE DETERMINING TRANSCRIPTION FACTORS AS VULNERABILITIES IN CHILDHOOD AND ADULT MLL-REARRANGED AND T(8;16) ACUTE MYELOID LEUKEMIA

S. Arza Apalategi 1,* , B. Heuts 2 , S. Bergevoet 1 , S. van Heeringen 3 , J. Jansen 1 , J. Martens 2 , B. van der Reijden 1

1 Laboratory Medicine, Laboratory of hematology, Radboudumc; 2 Molecular Biology; 3 Molecular Developmental Biology, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands

Background: The MLL-AF9 protein contributes to acute myeloid leukemia (AML) development through transcriptional activation of oncogenic gene programs. Remarkably, MLL-AF9 AML consists of two subgroups, one with high oncogenic MECOM expression and dismal outcome; the other with a unique gene expression profile lacking MECOM expression and a favourable outcome. How the differences between these two clinically and biologically different subgroups are brought about in the context of the same MLL-AF9 genetic background is currently unknown.

Aims: To identify vulnerabilities in AML beyond the MLL-AF9 oncofusion gene we determined cell fate determining transcription factors pertinent to the two clinically and biologically different subgroups.

Methods: Transcription factors key to cell fate were determined by first analysing differential transcriptional start sites of genes and gene enhancers by genome-wide “Cap Analysis Gene Expression (CAGE)”-sequencing of three primary MECOM positive and negative MLL-AF9 AML samples. Sequencing data were subsequently analysed using a new bioinformatic tool that builds gene regulatory networks based on differential gene expression and enhancer activities to predict key transcription factors in cell fate determination. For predicted factors AML cohort-wide gene expression patterns, transforming potential and downstream gene programs were determined.

Results: Bioinformatic analyses of CAGE-seq data identified the transcription factor MECOM as top hit for cell fate determination in MECOM+ MLL-AF9, confirming the power of this approach. In the group lacking MECOM expression we identified HMX3 as most influential transcription factor. HMX3 is essential for neuronal development and not expressed in any healthy blood cell type. In a large adult and childhood AML cohort (totalling 630 cases), high HMX3 expression was observed in 4% and 11% cases, respectively. HMX3 positive cases clustered tightly together in unsupervised clustering analyses, and 80% of cases were positive for MLL-rearranged (MLL-AF9, AF10, ENL) and t(8;16) AML. None were positive for other known recurrent AML mutations except for RAS mutations. In line with these observations, MLL-AF9 positive human cell lines expressed HMX3 (eg. THP1 and MOLM13). HMX3 silencing in THP1 and MOLM13 cells caused pronounced cell cycle arrest and apoptosis evidenced by PI, Annexin and 7AAD staining in flow cytometry. Forced lentiviral HMX3 expression in primary human GCSF-mobilized CD34+ cells resulted in an over 70% clonogenic growth inhibition compared to control transduced cells. To identify relevant gene programs we silenced HMX3 in MOLM13 cells followed by RNA-sequencing. Gene ontology analyses of over 3000 differentially expressed genes (p<0.01) suggested a role for HMX3 in cell cycle progression, sister chromatid separation, and immune response suppression (p<0.05).

Summary/Conclusion: Bioinformatic analyses of CAGE-sequencing of primary leukemia samples predicted the transcription factors MECOM and HMX3 key to poor and good outcome MLL-AF9 AML, respectively. High HMX3 expression associated with a unique gene expression profile and associated strongly with MLL-rearranged and t(8;16) AML. HMX3 only was unable to transform healthy human immature blood cells but appeared essential for survival of MLL-AF9 leukemia cell lines, potentially by positively regulating cell cycle progression and immune evasion. Insight into the factors that activate HMX3 expression in AML could provide potential targets for therapies in MLL-rearranged and t(8;16) AML.

P387: UNRAVELING LEUKEMIC STEM CELLS MITOCHONDRIAL DEPENDENCY IN PEDIATRIC ACUTE MYELOID LEUKEMIA

M. Benetton 1,* , A. Da Ros 1 , G. Borella 1 , G. Longo 1 , C. Tregnago 1 , F. Locatelli 2 , M. Pigazzi 1

1 Department of Women’s and Children’s Health, Haematology-Oncology Clinic and Laboratory, University of Padova, Padova; 2 Department of Pediatric Hematology and Oncology, IRCCS Ospedale Pediatrico Bambino Gesù and Sapienza University of Rome, Roma, Italy

Background: Relapse still represents an unsolved problem for children with acute myeloid leukemia (AML), therefore more effective therapeutic strategies are needed for a complete blasts eradication. Leukemic stem cells (LSCs) have been proposed as the therapy-resistant reservoir of cells responsible for failure of antiproliferative chemotherapy and disease recurrence in AML due to their self-renewal capacity and quiescence allowing evasion from chemotherapy. Thus, the identification of LSCs-specific vulnerabilities is needed for more efficacious treatments.

Aims: We aim to dissect novel LSCs characteristics to unravel mechanisms of chemoresistance and disease recurrence, and characterize biological properties of LSCs that can be targeted to completely eradicate AML.

Methods: To study LSCs derived from primary pediatric AML samples, we based on reactive oxygen species (ROS) content since it was shown that functionally defined LSCs have relatively low levels of ROS. We isolated the lowest 20% of cells fluorescing with CellROX probe (ROS low) and the highest 20% (ROS high) by cell sorting, and analyzed cell cycle, engraftment in mice and RNA sequencing to identify deregulated pathways.

Results: After isolating ROS low and ROS high cells, we first assessed classical stemness features. By colony-forming assay, we observed that ROS low and ROS high cells have similar colony-forming capacity, but only ROS low cells preserve this capacity after a second replating whereas the ROS high population does not (p=0.002). By BrdU assay, we found that ROS low cells are relatively dormant, being more in G0/G1 cell cycle phase (41.9%) than the ROS high counterpart (7.9%, p=0.001), also confirmed by CFSE staining, revealing that ROS low cells proliferate more slowly (at day 4, 31% undivided cells in ROS low vs 8% in ROS high). According to our previously published data, we confirmed that ROS low cells express higher levels of CDK6-AS1 (RQ=1.38) and CDK6 genes (RQ=2.69; RQ=1 for ROS high) and that, being more quiescent, are characterized by lower levels of mitochondrial membrane potential by TMRE and lower content of mitochondrial ROS, byproducts of mitochondrial activity, by MitoSOX staining. We also verified that ROS low cells have a lower mitochondrial mass by MitoTracker and TOMM20 staining (mitochondria to nucleus ratio 1.18 vs 1.35 for ROS high, p=0.0005), suggesting that mitochondrial mass and functions might be involved in quiescent cells properties. However, engraftment experiments did not reveal different capacity of ROS low or ROS high cells in recreating leukemia in NSG mice, neither in terms of human CD45+ cells in mice peripheral blood (23.3% vs 41.9% respectively, p=0.12) nor bone marrow infiltration (73% vs 84.8%, p=0.44) at 8 weeks post-engraftment, opening for further dissection of the presence of LSCs within ROS high cells. By RNA sequencing, we found that the top 150 differentially expressed genes between ROS low and ROS high cells are involved in focal adhesion (logP=-13.51), regulation of hemopoiesis (logP=-12.31), cell response to stress (logP=-8.73) and cytokine production (logP=-8.67). Further analyses will confirm the role of these pathways in maintaining ROS low LSCs. Moreover, we identified 5 differentially expressed surface antigens that are under investigation as putative ROS low LSCs markers.

Summary/Conclusion: In conclusion, the ROS low strategy permits to identify a quiescent AML subpopulation with peculiar mitochondrial features and transcriptome allowing to pinpoint novel molecular pathways that might be targeted to enhance LSCs clearance.

P388: PREFERENTIAL SYNERGISTIC EFFECT OF INDISULAM IN COMBINATION WITH VENETOCLAX AND AZACYTIDINE IN AML CELLS WITH SF3B1 MUTATIONS.

P. Bernardo 1,2,* , M. Pereira 1 , B. Galvão 1 , J. Desterro 1,3 , A. Nunes 3 , M. D. J. Frade 3 , J. Lobato 3 , M. G. da Silva 3 , M. Lemos 4 , M. Silva 4 , P. Ribeiro 4 , S. Matos 5 , N. Custódio 1 , M. Carmo-Fonseca 1

1 Instituto Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa; 2 Serviço de Hematologia Clínica, Hospital da Luz Lisboa; 3 Serviço de Hematologia Clínica, Instituto Português de Oncologia de Lisboa Francisco Gentil; 4 Serviço de Hematologia Clínica, Centro Hospitalar Lisboa Central - Hospital de St. António dos Capuchos; 5 GenoMed - Diagnósticos de Medicina Molecular SA, Lisbon, Portugal

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis characterized by a high rate of relapse after conventional combination chemotherapy, highlighting the need for improved therapeutic interventions. Genes involved in pre-mRNA splicing, namely SF3B1, U2AF1 and SRSF2, are frequently mutated in AML.Recent studies suggest that pharmacological targeting of the splicing machinery results in preferential lethality of splicing mutant AML cells, potentially providing a novel strategy for treatment of this disease. Existing anti- cancer sulfonamides have been shown to interfere with splicing and induce preferential killing of splicing mutant AML cell lines. A particular sulfonamide drug, indisulam, was previously studied in cancer clinical trials where it was found to be safe but demonstrated limited efficacy, most likely because neither the mechanism of action nor potential biomarkers of response were known.

Aims: We investigated, in this study, the efficacy of indisulam, either alone or in combination with ABT-199 (venetoclax) and azacitidine, in cultured primary human AML cells.

Methods: Bone marrow cells were collected from adults newly diagnosed with AML, non-treated. Cells were cultured and incubated for 48 and 72 hours with indisulam at a range of nanomolar concentrations. Additionally, cells were incubated with ABT-199 (10nM) and azacitidine (1μM), alone or in combination with indisulam. Cell viability and cell death were measured. For mutation profiling, a targeted myeloid gene panel was used.

Results: We found that treatment with indisulam resulted in lethality of primary AML cells harboring mutations in SF3B1, but had minor or no effect on AML cells with other mutations including mutations in splicing factors U2AF1 and SRSF2. In SF3B1-mutated cells, a clear synergistic effect was further observed when indisulam was combined with ABT-199 and azacitidine. The correlation between efficacy of indisulam and synergy with ABT-199 and azacitidine in AML cells with different mutations will be shown and discussed.

Summary/Conclusion: Our results indicate that indisulam is preferentially active and synergistic with venetoclax and azacitidine in SF3B1-mutated cells from newly diagnosed, non-treated AML patients.

P389: BISPECIFIC ANTIBODIES ACTIVATE AUTOLOGOUS LEUKEMIA-REACTIVE T CELLS IN ACUTE MYELOID LEUKEMIA

D. Böhm 1,* , D. Primo 2 , J. Ballesteros 2 , K. Wennerberg 1

1 BRIC - Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, Denmark; 2 Vivia Biotech, Tres Cantos, Madrid, Spain

Background: Acute myeloid leukemia (AML) is a highly immunosuppressive disease with a high risk of relapse. This makes the finding of effective immunotherapy-based treatments important, but also difficult. T cell-engaging bispecific antibodies (TCEs) are designed to bind to both T cells and target cells, activate the T cells and thus induce an immune response against the leukemia cells without employing the MHC-T cell receptor (TCR) interaction. Several therapies of this kind are under development for treatment of AML. Surprisingly, both in vitro and in vivo , a subset of these TCE immunocoached T cells (ICTs) can subsequently target autologous AML cells and mediate efficient TCR-MHC-dependent AML cell killing in the absence of the TCE. This points to that a) a portion of the T cells present in an AML are leukemia-reactive but have been immunosuppressed - a suppression that can be reversed by the addition of a TCE, b) that this anti-AML T cell reactivation may play an important part of the mechanism of action of AML-targeted TCEs and c) that by understanding this reactivation mechanism, we will be able to design improved AML immunotherapies in the future.

Aims: To understand the mechanisms by which a TCE induces reactivation of leukemia-reactive T cells (LRTs) in AML.

Methods: T cells in biobanked AML patient bone marrow samples were activated with either an anti-CD3 x anti-CD123 TCE or a conventional anti-CD3 antibody for 5 days before isolating the ICTs. ICT assay: ICTs were washed to remove the TCE, and isolated ICTs were co-cultured with additional autologous bone marrow cells at different E:T ratios. Killing efficiency and T cell expansion were determined over several days using flow cytometry. Trogocytosis assay: ICTs were co-cultured with stained additional autologous AML cells for 1 h and T cell populations of interest were isolated using FACS. T cell repertoires were analyzed by targeted RNAseq of the TCR.

Results: ICTs generated with a TCE kill autologous AML cells efficiently without further addition of the TCE. Adding additional TCE at this stage also does not increase the AML killing efficiency of the ICTs. ICTs generated with an activating anti-CD3 antibody show a similar AML killing efficiency and a similar trogocytotic behavior as TCE ICTs. Most of the TCR clones present in the AML bone marrow pre-stimulation were also represented among the ICTs and trogocytotic ICTs (ICTs that are directly targeting the AML cells after either TCE or anti-CD3 stimulation; Figure 1), suggesting that the bulk of T cells in an AML bone marrow represent heterogeneous immunosuppressed LRTs which can be equally well reactivated by a TCE or an anti-CD3 antibody. Trogocytotic ICTs generated from both types of T cell activation have a similar TCR distribution. ICTs derived from the bone marrow target autologous AML cells more efficiently than ICTs from peripheral blood.

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Summary/Conclusion: Similarities between TCE ICTs and CD3 ICTs in regards to TCR distributions and killing efficiencies suggest that the TCE reactivates leukemia-reactive T cells via a CD3-dependent mechanism rather than a more complex proximity mechanism. Our data further suggests that T cells in the AML bone marrow are highly heterogeneous and leukemia targeted to a very high degree. In summary, AML bone marrow ICTs are targeting and killing leukemic cells efficiently in a polyclonal fashion and our results highlight that boosting this mechanism can leverage the clinical impact of AML TCEs and be utilized in other novel AML immunotherapy approaches.

P390: ACUTE MYELOID LEUKEMIA SUPPRESSION BY PALBOCICLIB AND PONATINIB IN PATIENT-DERIVED XENOGRAFT

D. Busa 1,* , M. Culen 1,2 , T. Loja 3 , I. Jeziskova 1,2 , A. Folta 1,2 , J. Mayer 1,2,3

1 Department of Internal Medicine - Hematology and Oncology, Masaryk University; 2 Department of Internal Medicine - Hematology and Oncology, University Hospital Brno; 3 Central European Institute of Technology, Brno, Czechia

Background: Palbociclib, a CDK4/6 inhibitor approved for breast cancer treatment, and ponatinib , a BCR/ABL1 inhibitor with a multi-kinase activity approved for chronic myeloid and acute lymphoid leukemia, were previously shown to be effective in vitro against different cancer types including acute myeloid leukemia (AML).

Aims: To test palbociclib and ponatinib, along with reference drugs – venetoclax, azacitidine, cytarabine+doxorubicine (chemotherapy) on 2 primary AML samples in a patient-derived xenograft model.

Methods: Two newly diagnosed AMLs (AML #1: myelomonocytic AML, intermediate cytogenetic risk; AML #2: AML with myelodysplasia-related changes, poor cytogenetic risk) were xenotransplanted into NOD SCID gamma mice. Treatment was initiated at detection of approximately 5-20% hCD45+ cells in mouse peripheral blood (PB). Chemotherapy (cytarabine+doxorubicine, AraC/Dox) was administered as a 5 + 3 regimen. Azacitidine was administered subcutaneously, five days per week in 3 cycles – 1 week on, 1 week off. Palbociclib, ponatinib, venetoclax, and vehicle were administered orally for 3 weeks, 5 days per week. Gene sequencing was performed using targeted 37 gene Archer Variantplex Core Myeloid Kit panel.

Results: From the reference treatments, significant AML reduction and prolonged overall survival (OS) was achieved with venetoclax in both AMLs, and with chemotherapy in AML #1. No statistically significant effect was observed with azacitidine, but interestingly in AML #2 the azacitidine treatment induced the longest remission in the whole study, almost 10 weeks (<1% hCD45+ cells in PB) in 2/4 mice.

Of the tested drugs, palbociclib showed the same effect as venetoclax, with significantly reduced AML and prolonged OS in both AMLs. Ponatinib led to prolonged OS only in AML #1 (Fig. 1A-D).

Overall, none of the treatments led to complete AML eradication and all PDX groups gradually relapsed.

No phenotype changes were observed in the relapsed cells in palbociclib and ponatinib groups compared to vehicle (SSClowCD45dim blasts, SSCdimCD45high monocytes, CD34+ and CD34+CD38- cells). Azacitidine group showed altered monocyte, CD34+, and CD34+CD38- fractions in AML #1. Azacitidine, chemotherapy, and venetoclax showed altered blast and monocyte fraction in AML #2 and venetoclax also altered CD34+ fraction in AML #2.

Mutational status in AML #1 remained unchanged ( FLT3 -TKD and NRAS mutated) in all PDX groups at relapse. In AML #2, KRAS mutation from the primary tumor remained conserved, whereas all PDXs lost an IDH1 mutation and gained a WT1 mutation. This indicates that mutation selection was induced by the xenotransplantation rather than treatment. In addition, a second WT1 mutation was gained only in the palbociclib group.

Treatment toxicity manifested by weight decrease after the end of treatment was only seen with chemotherapy and was accompanied by early mouse mortality in 3/4 mice for AML #2.

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Summary/Conclusion: Palbociclib demonstrated in vivo AML suppression that was comparable to venetoclax and superior to chemotherapy. Effect of ponatinib was only limited. This encourages further investigation of treatment efficacy in different AML subtypes and in combination with other drugs.

Funding: MUNI/A/1330/2021

P391: FLT3-ITD MEASUREMENT AT DIAGNOSIS AND FOR THE ASSESSMENT OF MINIMAL RESIDUAL DISEASE AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA: CDNA VS. DNA

D. Carbonell 1,2,* , M. Chicano 1,2 , A. Cardero-Illán 1 , G. Rodriguez-Macías 1 , P. Muñiz 1,2 , R. Bailén 1,2 , G. Oarbeascoa 1,2 , I. Gómez-Centurión 1,2 , J. Anguita 1,2 , M. Kwon 1,2 , J. L. Díez-Martín 1,2,3 , I. Buño 1,2,4,5 , C. Martínez-Laperche 1,2

1 Hospital General Universitario Gregorio Marañón; 2 Instituto de Investigación Sanitaria Gregorio Marañón; 3 Department of Medicine; 4 Department of Cell Biology, Complutense University of Madrid; 5 Genomics Unit, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain

Background: DNA is standardly preferred to cDNA for the analysis of FLT3 - internal tandem duplication (ITD). Although its screening is mandatory at diagnosis in clinical routine, its use as minimal residual disease (MRD) marker after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains controversial due to the poor sensitivity of its detection method and its apparent instability during the disease.

Aims: To compare FLT3 -ITD mutation analysis in DNA and cDNA samples at diagnosis and to prove the usefulness of its expression measurement as MRD marker after allo-HSCT.

Methods: Forty-six diagnosis samples from 46 patients and 80 samples from 34 patients who underwent an allo-HSCT (34 pre-HSCT samples, 34 at day 30 after infusion and 12 before relapse) were analyzed. DNA was purified using a Maxwell RSC Blood DNA Kit (Promega), RNA was isolated using TRIzol reagent (Invitrogen) and cDNA synthesis was performed using the First Strand cDNA Synthesis kit (Roche). FLT3 -ITD was analyzed in both DNA and cDNA samples by fragment analysis using an ABI3130xl DNA sequencer (Applied Biosystems). Fragment analysis was analyzed through Peak Scanner Software (Thermo Fisher). Allelic ratio (AR) quantification was performed by dividing the area under the curve of the mutant allele by the area under the curve of the wild type allele. Every clone was analyzed as an event, comparing its DNA ratio with its cDNA ratio.

Results: Median AR of the 58 FLT3 -ITD clones at diagnosis were 0.54 [0-9.47] in DNA samples and 0.63 [0.012-13] in cDNA samples, (Wilcoxon test, p<0.001, Figure A). In six patients, FLT3 -ITD AR was <0.5 in DNA and ≥0.5 in cDNA, changing their prognosis based on ELN algorithm. One of them was not candidate for chemotherapy and of the five remaining, four were refractory or relapsed after intensive treatment. In terms of sensitivity, cDNA was better than DNA, quantifying higher AR values in most cases, optimizing the detection of minor clones and the prognostic classification. In respect of HSCT samples, seven patients were positive for FLT3 -ITD mutation pre-HSCT in cDNA and two in DNA. Of the seven patients which were positive by cDNA, four relapsed after HSCT. The three patients who did not relapse had FLT3 -ITD AR lower than 0.05. As for the two patients who tested positive in the DNA samples, only the one who also tested positive for cDNA relapsed. Despite the small sample size, these results reveal that the analysis of pre-HSCT cDNA samples could have a predictive value for relapse. No patient was positive on day 30 after HSCT, suggesting that it may not be useful, whether the DNA or cDNA sample is studied. However, the analysis at this time point could be crucial in those patients who may relapse early. On respect of the last sample before relapse (a median of 22 days before relapse, range 7-85), of the 12 cases, three were positive for FLT3 -ITD mutation in DNA samples and nine were positive in cDNA samples. Great differences were observed between AR of both type of samples (Wilcoxon test, p<0.001, median 0 [0-0.04] vs . 0.15 [0-0.57], Figure B). In three patients FLT3 -ITD cDNA measurement did not anticipate the relapse, due to the loss of the mutation or because the day on which the sample was analyzed was too early to detect the mutation, issues that can be solved by complementing with other MRD markers.

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Summary/Conclusion: In conclusion, cDNA fragment analysis of FLT3 -ITD by capillary electrophoresis is an easy-to-implement technique that could be a great alternative approach in AML patients at diagnosis and in allo-HSCT monitoring.

P392: PHARMACOLOGICAL INHIBITION OF SYK CONFERS ANTI-PROLIFERATIVE AND NOVEL ANTI-TUMOR IMMUNE RESPONSES IN AML

L. A. Carvajal 1,* , B. Robinson 2 , Y. Kosaka 2 , T. Jacob 2 , J. Lee 2 , T. Hood 1 , K. Baker 2 , A. Kaempf 2 , S. N.-A. Amara 1 , J. Pucilowska 2 , E. Lind 2 , C. Tognon 2 , J. Tyner 2 , P. Kumar 1 , T. Vu 2 , J. DiMartino 1

1 Kronos Bio, Inc., San Mateo; 2 Oregon Health & Science University (OHSU), Portland, United States of America

Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1.

The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1 -driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY ( NCT05020665 ), in combination with intensive induction/consolidation chemotherapy in patients (pts) with treatment-naive NPM1 -mutated ( NPM1 m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in pts with relapsed or refractory FLT3 -mutated AML ( NCT05028751 ).

Aims: To investigate the effects of ENTO and LANRA in T-cell responses in AML.

Methods: All pt-derived bone marrow (BM) and peripheral blood samples were obtained in accordance with IRB (OHSU#004422) approval. Results from gene expression profiling studies (RNA sequence [RNA-seq]) in bone marrow/peripheral blood mononuclear cells (MNCs) derived from 152 pts with AML, for which matching NPM1 status and ex vivo sensitivity to ENTO was available, previously published by Tyner et al (2018), were reanalyzed using trimmed mean of M values normalization and differential expression using edgeR. ENTO/LANRA sensitivity was assessed ex vivo after 72 hours in culture at 37°C by MTS assay. T-cell functional assays were performed by culturing MNCs with anti-CD3 and treating with ENTO or LANRA, singly and in combination with anti-PD1. CD3+ T-cell proliferation as measured by Ki67 expression and pSYK activation were assessed by quantitative single-cell immunofluorescence microscopy. Six formalin-fixed, paraffin-embedded BM biopsies from newly diagnosed AML pts were assessed for spatial expression of mRNA using a digital spatial profiling method.

Results: In AML, mutations in NPM1 with co-expression of HOXA9/MEIS1 at baseline predicted anti-proliferative activity to ENTO in ex vivo drug sensitivity studies. Accordingly, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. Pathway analysis of archival RNA-seq data from pts enrolled in the Leukemia and Lymphoma Society BEAT AML Master Protocol ( NCT03013998 [BAML-16-001-S6]) revealed gene expression signatures at baseline associated with the observed sensitivity to ENTO, including significant enrichment in the expression of genes associated with leukemogenesis, myeloid differentiation and immune regulation. This was supported by T-cell functional studies, which demonstrated that ENTO and LANRA, singly as well as in combination with anti-PD1, could restore T-cell proliferation in primary AML pt samples that exhibited suppression. Lastly, Cancer Transcriptome Atlas analysis on archival BM biopsies obtained from these AML pts showed a strong correlation between immune checkpoint response and gene expression associated with immune pathway signaling.

Summary/Conclusion: Inhibition of SYK is a promising therapeutic approach in NPM1 m AML. Our studies illustrate that ENTO and LANRA may restore T-cell proliferation in a subset of AML pts with dysfunctional T-cell responses, suggesting a novel mechanism of action. Additional studies are required to fully understand the mechanism of SYK-mediated antitumor immune responses in AML.

P393: SYNERGISTIC EFFECT OF EZH2 INHIBITOR WITH BCL2 INHIBITOR BY ENHANCING PIK3IP1 EXPRESSION IN ACUTE MYELOID LEUKEMIA

C. Yang 1 , C. Song 2,3 , Z. Ge 1,*

1 Department of Hematology, Zhongda Hospital，Medical School of Southeast University, Institute of Hematology Southeast University, Nanjing, China; 2 Hershey Medical Center, Pennsylvania State University Medical College, Hershey; 3 Division of Hematology, The Ohio State University Wexner Medical Center, the James Cancer Hospital, Columbus, United States of America

Background: Acute Myeloid Leukemia(AML) is a highly heterogeneous blood cancer with poor outcomes. PI3K Interacting Protein 1(PIK3IP1) binds to the p110 catalytic submit and downregulate PI3K activity. EZH2 is the catalytic subunit of polycomb repressive complex 2(PRC2). Inhibition of EZH2 activity is a therapeutic vulnerability in cancer cells. Combination of the BCL2 inhibitor Venetoclax(Ven) with hypomethylating agents has been approved for treatment in AML with substantial benefits, however, most patients develop resistance. Thus, a more efficacious treatment by targeting the drug resistance paradigm based on BCL2 inhibitor is required.

Aims: To investigate the anti-leukemic activity of the combination of the EZH2 inhibitor DZNeP with BCL2 inhibitor Ven and to evaluate the association of PIK3IP1 with clinical characteristics of patients with AML.

Methods: EZH2 and PIK3IP1 mRNA level was examined by qPCR in newly-diagnosed AML patients and healthy control from Feb. 1, 2016, to Jan. 30, 2022, at our institute with approval of the Ethics Committee. Cell Counting Kit-8 assay(CCK8) was used for cell proliferation and cytotoxicity in U937 and MV-4-11 AML cells treated with vehicle control, EZH2 inhibitor(DZNeP), Ven, and Combination (Ven+DZNeP). Synergistic effect was analyzed with Calcusyn. RNA-seq was performed with total RNA isolated from U937 cells treated with 2μM DZNeP for 48 hours. Apoptosis was measured by cell staining with Annexin V+propidium iodide (PI) following flow cytometry analysis. Lentiviral PIK3IP1 shRNA knockdown was performed in AML cells. Gene expression was analyzed by qPCR and Western blot.

Results: EZH2 mRNA level was significantly increased in 53 newly diagnosed patients with AML compared to 20 healthy bone marrow control(p<0.0001)(Fig.1A). Both DZNeP and Ven had a dose-dependent and time-dependent effect on cell proliferation arrest in AML cells. DZNeP significantly sensitized the effect of Ven on cell proliferation arrest compared to single drug-only control. CalcuSyn analysis showed the synergistic effect of the Ven+DZNeP(Fig.1B). The total apoptosis rate increased significantly in the Ven+DZNeP group(Fig.1C). RNAseq analyses showed that PIK3IP1 is dramatically upregulated(Fig.1D). GSEA enrichment analyses showed that the Differentially Expressed Genes mainly upregulated apoptosis and downregulated cell cycle pathway(Fig.1E). Moreover, the PIK3IP1 mRNA level was significantly decreased in the aforementioned AML cohort compared to the control(P<0.005)(Fig.1F). The AML patients were divided into two PIK3IP1 High expression groups(n=29) and PIK3IP1 Low expression groups(n=24). PIK3IP1 low expression is significantly associated with a lower overall response rate (96.55% vs 79.17%, p=0.047), higher relapse or death rate(58.33% vs 24.14%, p=0.029). A moderately negative correlation between EZH2 and PIK3IP1 was observed(r=-0.376, p=0.006)(Fig.1G). In addition, qPCR data showed that PIK3IP1 is significantly upregulated by DZNeP or Ven+DZNeP(Fig. 1H). PIK3IP1 is efficiently knocked down in U937 and MV-4-11(Fig.1I), and PIK3IP1 knockdown significantly blocked the combination of DZNeP and Ven mediated cell proliferation arrest and apoptosis(Fig.1J-K).

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Summary/Conclusion: The combination of EZH2 inhibitor DZNeP and BCL2 inhibitor Venetoclax exhibited a strong synergistic cell proliferation arrest and apoptosis of AML cells in PIK3IP1 dependent manner. PIK3IP1 low expression is associated with oncogenic markers and poor outcomes in AML. Our results highlight the likelihood of further in vivo pre-clinical study for the potential clinical trial of the novel combination in AML patients.

P394: INSIGHTS INTO PATIENT-DERIVED XENOGRAFT MODELS FOR DEK-NUP214 LEUKEMIA

F. Charles Cano 1,* , A. Kloos 1 , T. Fangmann 1 , N. Kattre 1 , R. Schottmann 1 , K. Döhner 2 , A. Ganser 1 , M. Heuser 1

1 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Medizinische Hochschule Hannover, Hannover; 2 Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany

Background: Acute myeloid leukaemia (AML) with the translocation DEK-NUP214 (t(6;9)(p23;q34)) is a rare subtype present in 1% of adult and 2% of paediatric patients and is recognized by the world health organization (WHO) as a unique subgroup of AML. These patients, typically, are diagnosed with de novo AML and have poor clinical outcome and resistance to chemotherapy. DEK-NUP214 is the sole cytogenetic abnormality in the majority of these patients at the time of diagnosis, suggesting its central role in disease development. This subgroup of leukaemia remains poorly characterized. There is only one cell line (FKH-1) and only very few reported DEK-NUP214 patient derived xenograft (PDX) models. The major limitation is the lack of serially transplantable PDX models, which are required for pharmacologic studies. The development of DEK-NUP214 PDX models will aid in the understanding of the molecular pathogenesis of this disease.

Aims: To establish patient-derived xenograft models that recapitulate DEK-NUP214 leukaemia.

Methods: Five patient samples were selected based on their cytogenetic profile and 1x10 6 cells were injected intravenously into irradiated NSG-SGM3 (NSGS) mice (n=3) per sample. Engraftment of human cells in peripheral blood by flow cytometry and blood counts was monitored monthly. Engrafted cells were used for serial transplantations. Cytospin preparations of peripheral blood, spleen and bone marrow of the PDX samples as well as the patient samples were stained with Wright-Giemsa stain. The DEK-NUP214 fusion transcript was confirmed by Sanger sequencing. Mutational analysis was performed with a custom myeloid sequencing panel.

Results: Three patient samples showed a mean of 12.5±6.0 (PDX1), 0.68±0.23 (PDX2) and 4.9±2.5% (PDX3) engraftment of human CD45+CD33+ cells at 20-24 weeks in peripheral blood and were transplanted into secondary mice. The characteristics of the patients at diagnosis, whose samples engrafted, were 19, 53 and 51 years old, respectively, with a median of 52.8 x10 9 /L (range 34.1-116 x10 9 /L) white blood cells (WBC), 7.4 g/dl (range 6-9.4 g/dl) of haemoglobin (Hb), and 38 x10 9 /L (range 26-61 x10 9 /L) platelets (Plt). The average circulating blasts were 56.7±18% in peripheral blood and blasts in bone marrow were 84.7±4.7%. PDX models at the 1 st transplantation (Tx) had an increased WBC and decreased Hb at the time of sacrifice. Average spleen weights were 196±24.1, 59.7±3.18, and 103±35.4 g, respectively. The morphological analysis revealed large blasts with partially granulated cytoplasm and few vacuoles similar to the patients’ cells. The engrafted cells showed a myeloid phenotype with expression of CD38, CD33 and CD14, with higher engraftment in bone marrow than spleen. Secondary transplants from PDX1 showed 12.5% of hCD45+ cells at 16 weeks in peripheral blood, with average spleen weight of 168 g, with a similar immunephenotype as primary mice, and were transplanted into tertiary mice. Mutational analysis showed that the PDX samples retained the same mutations as observed in the patient sample (FLT3-ITD for PDX1 and PDX2, and WT1 for PDX1) and the fusion transcript as well.

Summary/Conclusion: We have successfully established AML PDX models for DEK-NUP214 leukaemia that reflect the morphological, immunophenotypic and molecular genetics of this disease subtype. This will enable pharmacologic studies in this rare disease and will allow a better understanding of the involved pathways.

P395: PHARMACEUTICAL TARGETING OF RAS IN ACUTE MYELOID LEUKAEMIA

D. Coleman 1,* , L. Strate 1 , J. Griffin 1 , T. Rabbitts 2 , P. Cockerill 1 , C. Bonifer 1

1 University of Birmingham, Birmingham; 2 The Institute of Cancer Research, London, United Kingdom

Background: Acute Myeloid Leukaemia is characterised by a small number of co-occurring driver mutations which combine to make a diverse collection of disease subtypes. Mutations in genes associated with cell signalling are a key group of driver mutations and lead to the constitutive activation of these proteins and their associated signalling cascades. RAS mutations occur in 6% of AML and confer a proliferative advantage. However, 36% of AMLs include mutations which lead to activated RAS such as FLT3 mutations and are associated with a worse outcome after induction chemotherapy 1 .

Inhibitors which bind to mutated and activated members of the RAS protein family to block the interaction between the protein and its downstream targets have been developed 2 which allow us to investigate the effects of pan-RAS inhibition in Acute Myeloid Leukaemia. These Pan-RAS inhibitors inhibit RAS-RAF and RAS-PI3K signalling leading to reduced phosphorylation of ERK and AKT.

Aims: 1. We have used novel specific and panRAS RAS inhibitors designed in the Rabbitts lab followed by system-wide analyses to identify the specific gene regulatory network of mutant RAS AML and identify RAS-responsive genes

2. We have interrorgated key transcription factor nodes with RNAi

Methods: Primary cells were co-cultured on primary hMSCs for perturbation experiments. Fresh blood or bone marrow samples from patients with AML were purified and sorted for CD34 or CD117, these samples were profiled by RNA-seq an ATAC-seq. Data from multiple samples with RAS mutations was integrated and compared to data from healthy CD34+ PBSCs to establish the gene regulagtory network.

Results: In vitro panRAS inhibitors cause cytotoxicity in primary AML samples with RAS activating mutations whilst cells with WT RAS exhibit reduced sensitivity. An exception to this finding are cells with FLT3-ITD mutations, which were also sensitive to RAS inhibition. This is of particular interest as patients treated with FLT3 inhibitors can relapse with a RAS mutant subclone.

To understand the role of RAS mutations in AML we have constructed a gene regulatory network from the genomic and transcriptomic profiling of primary cell samples from patients which has allowed us to identify a core network of transcription factors. The direct association of aberrant RAS signalling with these transcription factors has been determined through perturbation experiments using RAS inhibitors, and by targeting key transcription factor nodes with RNAi. These include transcription factors ETV5 and KANK1, both of which have been linked to RAS signalling in solid tumour models 3,4 .

Summary/Conclusion: These experiments will identify targets of the RAS pathway essential for the survival of AML cells.

1. Ball, B.J., et al. (2019), “RAS Mutations Are Independently Associated with Decreased Overall Survival and Event-Free Survival in Patients with AML Receiving Induction Chemotherapy. ” Blood , 134 (Supplement_1): 18-18.

2. Cruz-Migoni, A., et al. (2019), “Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds.” Proceedings of the National Academy of Sciences 116 (7): 2545-2550.

3. Mus, L.M., et al. (2020), “The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness.” Sci Rep 10 (218)

4. Jonathan R. Dry, et al. (2010), “Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)”. Cancer Res ; 70 (6): 2264–2273

P396: DYSREGULATED DNAM-1 AND KIR2DL1 EXPRESSION IN NATURAL KILLER CELLS CORRELATES WITH POOR OUTCOME IN ACUTE MYELOID LEUKEMIA

A. F O Costa 1,2,* , L. O Marani 1 , I. A Lopes 1 , L. S Binelli 1 , P. S Scheucher 1 , J. L S Schiavinato 1 , M. I. A Madeira 1 , K. B B Pagnano 3 , B. K Duarte 3 , A. B. F Glória 4 , E. M Rego 5 , F. Traina 1 , R. Welner 2 , L. L Figueiredo-Pontes 1

1 Hematology Division, Department of Medical Images, Hematology, and Clinical Oncology, Ribeirao Preto Medical School at University of Sao Paulo, Ribeirao Preto, Brazil; 2 Division of Hematology and Oncology, Department of Medicine, UAB Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, United States of America; 3 Hemocentro, University of Campinas, Campinas; 4 Hematology Division, Hospital das Clinicas da Universidade Federal de Minas Gerais, Belo Horizonte; 5 Internal Medicine, Medical School of the University of Sao Paulo, Sao Paulo, Brazil

Background: Although improvements have been made in understanding the physiopathology of Acute Myeloid Leukemia (AML), most patients still relapse leading to poor long-term prognosis and cure rates. Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms underlying the control of hematopoietic neoplasms by these cells remain unclear.

Aims: We hypothesized that FLT3-ITD mutation, a molecular marker of adverse prognosis, is associated with phenotypically and functionally impaired NK cells due to imbalanced expression of inhibitory and activating receptors during AML.

Methods: We analyzed the expression of DNAM-1 and KNG2D activating and NKG2A, KIR2DL1 inhibitory receptors on CD56 + , CD56 bright CD16 - and CD56 dim CD16 + NK subsets and its correlation with FLT3-ITD mutation in 70 AML patients and 12 healthy bone marrows (NBM) by flow cytometry. Seven marrows from AML patients in complete remission after induction chemotherapy (AML-CR) were used to evaluate NK cell and receptor recovery. Mann Whitney or Kruskal-Wallis tests (P < 0.05) were performed using GraphPad Prism (V8.0.2).

Results: A decreased frequency of all NK cell subsets was found in AML (CD56 + , P=0.03; CD56 bright , P=0.04, CD56 dim , P<0.01). Decreased expression of NKG2D (P<0.01) and a tendency of decreasing DNAM-1 (P=0.38) was found in CD56 + NK in AML, recovering to values closer to normal in AML-CR. Expression of NKG2D was decreased in both CD56 bright and CD56 dim NK subsets (P=0.03 and P<0.01), while DNAM-1 was found to be considerably decreased in CD56 dim cytotoxic NK (P=0.02). FLT3 mutated patients showed even lower expression of DNAM-1 (P=0.02) and NKG2D (P=0.13) in CD56 + NK. Even though the latter was not statistically significant, we observed a decreasing pattern in these patients, that was confirmed by an important decrease of both activating receptors uniquely in the CD56dim subset (DNAM-1, P=0.03; NKG2D, P=0.05), which are known to contain the most cytotoxic activity. As for the inhibitory receptors, our cohort demonstrated a trending increase in NKG2A expression on all NK subsets, although not significant, and no difference was found in FLT3 mutated patients. Meanwhile, KIR2DL1 expression was higher in CD56 + , CD56 bright and CD56 dim NK cells in AML (P<0.01; P=0.06 and P<0.01), while AML-CR showed frequencies comparable to healthy In FLT3-ITD mutated patients, CD56 dim NK had increased expression of KIR2DL1 (P=0.03), strongly correlating with an imbalance between activation and inhibition of NK cell activity.

Summary/Conclusion: Our data indicate that CD56 dim NK show an exhaustion immunophenotype in FLT3-ITD mutated AML, with high expression of KIR2DL1 inhibitory receptor and low expression of both NKG2D and DNAM-1 activating receptors and that this imbalance likely leads to impaired cytotoxicity, compromised anti-leukemic activity and could be contributing to worst disease outcome, which will be confirmed through functional assays. Closing these gaps in knowledge informs the fundamental characteristics of NK dysfunction during leukemia and is of significant interest for targeting, therapeutic interventions and NK cell-mediated immunotherapy.

P397: PROLACTIN RECEPTOR CONFERS CHEMORESISTANCE DUE TO SENESCENCE ENTRANCE IN ACUTE MYELOID LEUKEMIA

L. Cuesta-Casanovas 1,2,* , J. M. Cornet-Masana 1 , J. M. Carbó 1 , J. Delgado-Martínez 1,3 , L. Clément-Demange 4 , A. Banús-Mulet 5 , F. Guijarro 6,7 , J. Esteve 5,6,7,8 , R. M. Risueño 1

1 Leukemia Stem Cell, Josep Carreras Leukemia Research Institute, Badalona; 2 Faculty of Bioscience, Autonomous University of Barcelona; 3 Faculty of Pharmacy, University of Barcelona; 4 Leukos Biotech, Barcelona; 5 Josep Carreras Leukemia Research Institute, Badalona; 6 Faculty of Medicine, University of Barcelona; 7 Department of Hematology, Hospital Clínic; 8 Institut d’Investigacions Biomèdiques August Pi I Sunyer, Barcelona, Spain

Background: Acute myeloid leukaemia (AML) is a hematopoietic neoplasm characterized by frequent relapse because of chemotherapy resistance, which confers poor outcome. For this reason, the study of new therapeutic approaches that completely eradicate the disease is an urgent need. A previous in silico analysis performed in our laboratory demonstrated that PRLR signalling pathway is overactivated in leukemic stem cells (LSCs) in comparison to hematopoietic stem cells (HSCs), suggesting a relationship between PRLR and AML resistance and progression.

Aims: The main objective was to decipher the role of PRLR and its signalling pathway in the transformation events associated with the initiation and maintenance of AML.

Methods: mRNA expression of PRLR and Stat5a was analysed from public repositories. Surface PRLR expression was assessed by flow cytometry and total protein expression by western blot. Stat5 activation (phosphorylation status) and induced gene regulation were determined in AML cells ectopically expressing the wildtype PRLR (PRLRwt) and the dominant negative isoform (PRLRsh) and treated with its natural ligand (PRL) or an antagonist (G129R-PRL), by western blot and luciferase reporters. Senescence status was analysed by SA-β-galactosidase staining by microscopy and quantified using ImageJ software. Cytarabine resistant cells (AraC-R) were obtained by treating them during several weeks with increasing doses of cytarabine. PRLR expression was silenced using the CRISPR-Cas9 technology. Clonogenicity was studied in AML samples and healthy linage-depleted cord blood cells treated with PRL and hPRL-G129R and seeded in a semisolid medium in presence of instructive cytokines.

Results: AML patient cells express higher levels of PRLR in terms of mRNA, protein surface expression and total protein in comparison to healthy donors. Stat5 mRNA was overexpressed in AML, and PRL induced its phosphorylation and activation in presence of PRLR wt isoform, while the dominant negative did not respond to PRL. The antagonist of the receptor did not induce any effect. Stat5 mRNA expression was increased at relapse, and so it was the PRLR surface and total protein when AML cells acquire chemoresistance. Moreover, PRLR wildtype cells showed greater levels of SA-β-galactosidase, an indicative of the entrance in senescence. This effect was reverted when PRLR was knocked-down using CRISPR-Cas9 technology. The inhibition of PRLR with an antagonist or its downregulation with the CRISPR-Cas9 technology decreased clonogenicity capacity of AML patient cells, without affecting healthy donor cells.

Summary/Conclusion: PRLR expression is higher in AML patient samples, and correlates with chemoresistance and refractoriness, making the receptor an adequate biomarker for AML diagnosis and follow-up. PRLR-PRL signalling promotes the activation of Stat5, which is increased in AML samples, especially at relapse, suggesting an important role in AML progression and chemoresistance, a process related with increasing levels of PRLR surface expression. Moreover, the suppression of PRLR expression sensitizes the cells to chemotherapy.

Those results demonstrate for the first time that PRLR suppression increases the sensitivity to chemotherapy and decreases the clonogenicity capacity of AML cells, suggesting the use of antagonists of the receptor as new therapeutic strategies against AML.

P398: DEVELOPMENT AND CHARACTERIZATION OF PRECLINICAL IN VIVO MODELS FOR THE IDENTIFICATION AND TESTING OF NEW THERAPEUTIC APPROACHES FOR PEDIATRIC ACUTE MYELOID LEUKEMIA

A. Da Ros 1,* , V. Indio 2 , E. Porcù 1 , G. Borella 1 , M. Benetton 1 , C. Tregnago 1 , G. Longo 1 , S. Cairo 3 , B. Michielotto 1 , S. Bresolin 1 , B. Buldini 1 , A. Pession 4 , F. Locatelli 5 , M. Pigazzi 1

1 Department of Women’s and Children’s Health, Haematology-Oncology Clinic and Laboratory, University of Padova, Padova; 2 Department of veterinary medical science, University of Bolgna, Bologna, Italy; 3 XenTech, Evry, France; 4 Pediatric Unit, IRCCS Azienda Ospedaliera-University of Bologna, Bologna; 5 Department of Pediatric Hematology and Oncology, IRCCS Bambino Gesù Children’s Hospital, Sapienza University of Rome, Rome, Italy

Background: In pediatric acute myeloid leukemia (AML) chemotherapy is the standard of care, but >25% of patients still relapse and after a disease recurrence the survival probability is extremely low (<50%). To ameliorate patients’ outcome there is an urgent need to discover new treatments. Nevertheless, pediatric drug development is extremely reduced by the need of a better understanding of the adverse event profile of adult cancer indications in children, by the lack of pediatric-specific formulations, and by the reduced number of pediatric AML patients that can be included in clinical trials. Thus, robust preclinical AML models to faithfully predict new drug efficacy is urgently needed to advance new drugs in clinical setting.

Aims: This study aims to generate and characterize AML patient derived xenografts (PDXs) and accelerate the evaluation of innovative medicines for AML.

Methods: We generated PDXs from primary AML samples by inoculating blasts in NSG mice and, when engrafted, in 3 consequent mice recipients (namely P0, P1 and P2-PDX). We characterized AML by immunophenotipic profile and by RNA and whole-exome sequencing (WES). According to somatic mutations and copy number alterations we determined AML clonal compositions. We selected drugs and performed drug testing in vivo , after the expansion of P3-PDXs.

Results: We generated 22 AML-PDXs representing high-risk AML subtypes for genetic characterization harboring NUP98-NSD1 , or NPM1-MLF1 , or CBFA2T3-GLIS2 , or FUS-ERG or KMT2A somatic translocations, or FLT3 -ITD mutation. We monitored the AML associated immunophenotype in PDXs finding it was similar to that of the original AML. By WES we detected a consistent number of variants in each patients’ AML (ranging from 28 to 69), confirming an high AML intra-tumoral heterogeneity. Furthermore, we did not find any mutation recurrence among models, underlining an high inter-tumoral heterogeneity. In all models we tracked clonal evolution from patients’ AML to P2-PDX highlighting that most of the variants were maintained, with very few variants acquired during model development. Monitoring clonal dynamics we recognize a specific “founder” clone characterized by an average of 30 variants which are maintained up to P2 at the same allelic frequency, other small clones with average of 10 variants increasing the allelic frequencies in P2 and, in a restricted number of models, we observed that some clones were lost. By WES and transcriptome analysis we highlighted druggable mutations and pathways allowing the selection of novel targeted drugs. We screened their efficacy in vitro alone or combined with chemotherapic (Arabinoside) and biological agents (Venetoclax) by using AML ex vivo cells and mesenchymal stromal cells in a 3D co-culture system for exploring their synergy in reducing AML proliferation. Four selected drugs are under evaluation in AML-PDX models.

Summary/Conclusion: We have created a series of paired AML and xenograft models for advancing pediatric AML therapeutics. Our models represent a concrete perspective for both, the identification of new variants and pathways involved in AML progression, and the possibility to perform novel drug screenings useful to increase AML drug portfolio.

P399: UNRAVELING THE TUMOR SUPPRESSIVE ROLE OF STAT3Β IN ACUTE MYELOID LEUKEMIA

S. Edtmayer 1,* , A. Witalisz-Siepracka 1 , B. Zdársky 1 , T. Eder 2 , V. Poli 3 , F. Grebien 2 , D. Stoiber 1

1 Department of Pharmacology, Physiology and Microbiology, Karl Landsteiner University of Health Sciences, Krems an der Donau; 2 Institute of Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria; 3 Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin, Italy

Background: Signal transducer and activator of transcription 3 (STAT3) is a mediator of cytokine signaling existing in two alternatively spliced isoforms known as the full-length isoform STAT3α (770aa) and the C-terminally truncated isoform STAT3β (722aa). Initially STAT3β was described as the dominant negative form of STAT3α. It gained more attention as it turned out to initiate transcription independent of STAT3α and being capable to rescue embryonic lethality in absence of STAT3α. In acute myeloid leukemia (AML) patients, STAT3 has been reported to be constitutively activated thereby promoting proliferation and blast survival. However, most of those studies did not consider the two distinct isoforms of STAT3. Recently, our group identified STAT3β as a novel tumor suppressor and favorable prognostic marker in AML, however the underlying mechanism in leukemic cells remained elusive.

Aims: With this study we aim to provide in-depth knowledge on the role of STAT3 isoforms in AML development and disease outcome.

Methods: By using murine AML cell lines lacking either STAT3α or STAT3β, we aim to elucidate the STAT3 isoform-specific impact on cellular processes shaping AML development and progression. To gain better understanding we use AML in vivo models and in vitro assays in combination with next-generation sequencing (NGS) approaches to identify molecular mechanisms explaining the tumor suppressive function of STAT3β in AML. In brief, fetal liver-derived stem cells, isolated from STAT3α and STAT3β deficient mice on C57BL/6J background, were transduced with a retrovirus introducing the human fusion-oncogene MLL-AF9, and intravenously transplanted into immunocompromised mice.

Results: Animals transplanted with leukemic cells lacking STAT3β showed accelerated disease progression and poorer overall survival confirming its tumor suppressive properties. Furthermore, absence of STAT3β favored leukemic infiltration and migration. Additionally, RNA sequencing revealed that lack of STAT3β in leukemic cells leads to a dysregulation of crucial genes driving leukemogenesis.

Summary/Conclusion: Understanding the mechanisms behind the tumor suppressive property of STAT3β has potential translational impact and is crucial to identify novel therapies and preventative strategies. Further validation and ChIP sequencing will provide novel direct target genes of STAT3β which could serve as prognostic or therapeutic molecules.

P400: PROTEOGENOMIC CHARACTERIZATION OF 5-AZACYTIDINE EFFECTS ON ACUTE MYELOID LEUKEMIA IMMUNOPEPTIDOME

G. Ehx 1,* , N. Nandita 2 , C. Durette 2 , J. Courtois 1 , M.-P. Hardy 2 , K. Vincent 2 , J.-P. Laverdure 2 , J. Lanoix 2 , F. Baron 1 , P. Thibault 2 , C. Perreault 2

1 GIGA-I3: Hematology, University of Liege, Liege, Belgium; 2 IRIC, University of Montreal, Montreal, Canada

Background: Hypomethylating agents like 5-azacytidine (AZA) are licensed for the treatment of acute myeloid leukemia (AML) patients ineligible for allogeneic hematopoietic cell transplantation. While previous reports suggested that AZA promotes the recognition of AML blasts by cytotoxic T cells, the mechanism behind this improved recognition is not fully understood. Specifically, AZA is assumed to promote the expression of transcripts repressed by genomic methylation, namely cancer-testis antigens (CTA) and endogenous retroelements (ERE), resulting in the presentation of immunogenic MHC-I-associated peptides (MAPs, collectively referred to as the immunopeptidome) derived from the translation of these transcripts. However, the presentation of such MAPs after AZA treatment has not been firmly demonstrated thus far.

Aims: Our study aims to characterize how AZA treatment shapes the identity of MAPs presented by AML cells.

Methods: Four different AML cell lines, THP-1, OCI-AML3, SKM-1, and MOLM-13 were treated with a non-cytotoxic dose of AZA for 3 days. Their transcriptome has been characterized by high-coverage RNA sequencing (RNA-seq) and their immunopeptidome by shotgun mass spectrometry (MS). To identify MAPs deriving from unannotated genomic regions, we have designed a cutting-edge proteogenomic pipeline using the RNA-seq data to build personalized MS databases that enabled the identification of non-canonical MAPs such as ERE-derived MAPs.

Results: Paired transcriptomic comparisons between treated and untreated cells showed that AZA induces a large-scale gene upregulation (87% differentially-expressed transcripts were upregulated). Among them, 38% were EREs and 6% were CTAs, suggesting that AZA-induced MAPs have greater chances of deriving from EREs than from CTAs. However, we could not identify a single ERE-derived upregulated MAP among the immunopeptidome of AZA-treated AML cells while multiple CTA-derived MAPs (0.4 - 0.7% of the upregulated MAPs) were presented at greater levels by AZA. Because a GO-term analysis of the upregulated protein-coding genes evidenced a robust innate immune response in AZA-treated cells, we conclude that AZA-induced enhanced CTL recognition is more dependent on CTA- than on ERE-derived MAP presentation and that ERE enhanced expression could rather trigger the observed innate immune response. An in-depth analysis of the immunopeptidome showed that MAPs having an altered presentation following AZA treatment derived only partially from transcripts whose expression was affected by AZA. The analysis of the other AZA-induced MAPs showed that they resulted preferentially from the activity of the constitutive proteasome (vs the immunoproteasome) and derived preferentially from proteins having less aromatic residues (and therefore more prone to misfolding). Because the degradation of misfolded proteins by the constitutive proteasome can indicate that AZA induces an unfolded protein response, we have examined whether AZA stimulates autophagy, a process frequently triggered in association with such stress. Accordingly, flow cytometry assays on AML cells treated with AZA for 24h evidenced a robust autophagy induction.

Summary/Conclusion: Altogether our results show that AZA promotes the presentation of CTA-derived rather than ERE-derived MAPs and that autophagy induction could enable the survival of AML cells to AZA-induced proteotoxic stress. Our findings suggest that autophagy inhibitors could synergize with AZA in AML therapy.

P401: NEXT-GENERATION FLOW CYTOMETRY TO DETECT MEASURABLE RESIDUAL DISEASE IN ACUTE MYELOID LEUKEMIA USING A 19-COLOR FULL SPECTRUM FLOW CYTOMETRY PANEL

H. Fokken 1,* , N. Kattre 1 , A. Kloos 1 , M. Heuser 1 , T. Kacprowski 2,3 , M. Tobias 4 , A. Schwarzer 1,5

1 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover; 2 Division Data Science in Biomedicine, Peter L. Reichertz Institute for Medical Informatics of TU Braunschweig and Hannover Medical School; 3 Braunschweig Integrated Centre of Systems Biology (BRICS), TU Braunschweig, Braunschweig; 4 Department of Pediatric Hematology; 5 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany

Background: The detection of residual leukemic cells (“measurable residual disease”/MRD) has dramatic prognostic impact on relapse incidence and patient survival in AML and has become key to guide therapeutic decision making. MRD can be measured by PCR, next-generation sequencing, and conventional multicolor flow cytometry (Flow-MRD, 8-10 colors), respectively. Flow-MRD relies on the aberrant expression of surface marker combinations on leukemic blasts (leukemia-associated immunophenotypes - LAIPs), which are not found on normal hematopoietic cells. Hence, the challenge of Flow-MRD is to detect and track small populations of leukemic cells against the complex and heterogenous background of human bone marrow cells. With the development of next-generation Spectral Flow Cytometers, it is now possible to stain cells with 20-30 colors at once which would greatly increase the number of LAIPs that can be tracked and increase sensitivity of Flow-MRD when the number of available cells is limited. We hypothesized that spectral Flow-MRD allows for monitoring leukemic disease burden with unprecedented resolution.

Aims: To establish a one-tube full spectrum MRD detection assay and a semi-automated computational workflow for improved detection of minimal residual disease in AML.

Methods: We established a 19-color full spectrum flow cytometry panel with one tube containing CD45, CD34, CD117, CD13, CD33, HLA-DR, CD2, CD4, CD5, CD7, CD11b, CD14, CD15, CD19, CD22, CD38, CD56, CD64 and CD133. Limit of detection and quantification, linearity, intra-/inter-assay precision, and robustness were determined via spike-in assays. We measured normal bone marrow of healthy donors and MRD samples from AML patients. Data were analyzed with GemStone using its unique highly automated approach to characterizing normal cells in bone marrow based on a template model designed specifically for data generated with this panel. Results were then explored with the Cen-Se dimensionality reduction algorithm (Bagwell et al. J Biom Biostat (2019)) and subsequently compared to expert gating of data generated on the same samples using a well-established 5-Tube conventional flow cytometry panel.

Results: We defined reference values for known leukemia-associated immunophenotypes using normal bone marrow. Expert analysis of data simultaneously analyzed with the full-spectrum panel and the 5-tube conventional flow cytometry panel yielded highly correlating results for the proportions of granulocytes, lymphocytes, monocytes, and immature blasts in measured bone marrow with r 2 values greater than 0.90. In addition, quantitative and qualitative MRD results correlated very well when comparing semi-automated analysis of our full-spectrum panel using GemStone and expert analysis of the 5-tube conventional flow cytometry panel.

Summary/Conclusion: We have developed an integrated pipeline for full-spectrum flow cytometry that improves the applicability and performance of flow-cytometry-based MRD detection in AML.

P402: MUTATIONS IN BONA FIDE ONCOGENES KRAS AND DNMT3A ARE NOT RELATED TO DEPENDENCY IN ESTABLISHED TUMORS, IN PDX MODELS OF ACUTE LEUKEMIA IN VIVO

Y. Gao 1,* , M. Ghalandary 1 , M. Becker 1 , D. Amend 1 , M. Rothenberg-Thurley 2 , K. Metzeler 2,3 , I. Jeremias 1,4,5

1 Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU); 2 Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, Ludwig Maximilians University (LMU), Munich; 3 Department of Hematology and Cell Therapy, University Hospital Leipzig, Leipzig; 4 German Cancer Consortium (DKTK), Partner Site; 5 Department of Pediatrics, University Hospital, Ludwig Maximilians University (LMU), Munich, Germany

Background: The role of oncogenic mutations during the process of tumor formation was intensively studied in genetically engineered mouse models, while much less is known about their function in established tumors of patients.

Aims: To bridge the gap, we studied frequently mutated genes for their dependency in established tumors in vivo, using genetic engineering in patient-derived xenograft (PDX) models.

Methods: Primary tumor cells from seven patients with acute myeloid leukemia (AML) were transplanted into immunocompromised NSG mice. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA. The customized sgRNA library was designed and cloned using our CLUE platform ( www.crispr-clue.de; Becker et al., Nucleic Acids Res. 2020). PDX cells transgenic for Cas9 and the CRISPR/Cas9 sgRNA library were transplanted into NSG mice, grown until advanced AML disease and sgRNA distribution measured by next-generation sequencing. From dropout hits in PDX in vivo screens, single molecules were validated by fluorochrome-guided competitive in vivo experiments, comparing cells with and without knockout in the same mouse and determining cell distributions by flow cytometry gating on the different recombinant fluorochromes. Human AML cell lines were studied in vitro for comparison.

Results: We cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo . In hit validation experiments, knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1 . KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS , suggesting that patients suffering tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS . DNMT3A belongs to the genes most frequently mutated in AML, but most AML cell lines tested in vitro did not depend on DNMT3A and knockout of DNMT3A did not reduce, but rather increased AML cell line growth in vitro . In contrast, in PDX models in vivo , we found a clear essential function for DNMT3A in certain AML samples and PDX in vivo results were discordant to cell line in vitro data. These data highlight the complementary use of PDX models to study gene dependencies. To our surprise, the dependency of AML cells on DNMT3A was unrelated to the presence or absence of a hot spot mutation in DNMT3A .

Summary/Conclusion: We conclude that both KRAS and DNMT3A harbor an essential function in certain patients established AML tumors in vivo , although independently from the presence of absence of a mutation in the gene. Warranting verification in additional patient samples, oncogenes and tumor entities, our data might indicate re-consider basic principles of decision making in Molecular Tumor Boards.

P403: PROGNOSTIC SIGNIFICANCE OF LONG NON-CODING RNA GAS5 AND MICRORNA-222 EXPRESSION PROFILES IN YOUNGER AML PATIENTS

Ð. Pavlović 1 , N. Tošić 1 , B. Zukić 1 , I. Marjanović 1 , Z. Pravdić 2 , N. Suvajdžić Vuković 2,3 , S. Pavlović 1 , V. Gašić 1,*

1 Institute of Molecular Genetics and Genetic Engineering; 2 Clinic of Hematology; 3 Faculty of Medicine, Belgrade, Serbia

Background: Acute myeloid leukemia (AML) is a heterogeneous malignant disease, that accounts for 80% of all acute leukemias in adults. The treatment protocol and prognosis for AML are based on the classification of the patients into risk groups based on the pretreatment karyotype analysis. However, nearly half of the patients don’t have any detectable cytogenetic changes. Therefore, there is a constant need to find new prognostic markers, as well as markers that can be used as targets for therapeutics that could improve the treatment. Recently, using high-throughput technologies, the search for new biomarkers has pointed towards non-coding RNAs, especially long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). Numerous studies have shown lncRNA GAS5 expression level dysregulation in multiple cancers, but it was poorly investigated in AML. Additionally, miR-222 expression levels have shown significant influence on cancers in general, including AML. Since GAS5 acts like a molecular sponge for miR-222, co-expression profiles of these non-coding RNAs could be novel prognostic markers in AML.

Aims: We have investigated the expression level pattern of lncRNA GAS5 in younger AML patients and examined its potential influence on disease outcome. GAS5 expression levels were also investigated in AML patients with normal karyotype, while taking into account miR-222 expression levels, in order to examine their potential role as dual biomarkers.

Methods:: 94 bone marrow samples of younger (<65 years) AML patients have been collected. Diagnostics and stratification were based on cytomorphology, immunophenotyping and cytogenetic analysis. All patients received the same induction chemotherapy. Along with them, 14 healthy controls were analysed. The AML patient group with normal karyotype (AML-NK) had 39 patients. GAS5 and miR-222 expression levels were analysed using Real-Time PCR. Relative quantification was performed using the ddCt method, respective healthy controls were used as the calibrator for each non-coding RNA. The ROC curve analyses found the optimal cut-off value to discriminate between Gas5high and GAS5low. While the cut-off value for dividing miR-222 expression levels into miR-222high and miR-222low was performed using the value of the median level of expression among healthy controls.

Results: Our results showed that GAS5 expression level in AML patients was lower compared to healthy controls (p < 0.01). Lower GAS5 expression on diagnosis was related to an adverse prognosis, since higher levels of lactate dehydrogenase was detected in the GAS5low group compared to the GAS5high group (p < 0.01). The disease-free survival (DFS) and the overall survival (OS) were significantly lower in the GAS5low group, compared to the GAS5high group, but survival analysis failed to confirm this finding. In the AML-NK group patients had higher expression of miR-222 compared to healthy controls (p < 0.01). A synergistic effect of GAS5low/miR-222high status on disease prognosis was not established.

Summary/Conclusion: This is the first study focused on examining the lncRNA GAS5 and miR-222 expression pattern in AML patients. Its initial findings indicate potential prognostic significance of GAS5 expression and the need for further investigation of these two non-coding RNAs, their potential roles in leukemogenesis, and the prognosis of AML patients.

P404: PROGNOSTIC IMPACT OF CEBPA-MUTATIONAL SUBGROUPS IN ADULT AML – RESULTS OF A LARGE METAANALYSIS IN MORE THAN 1000 CEBPA-MUTANT PATIENTS

J.-A. Georgi 1,* , S. Stasik 1 , M. Kramer 2 , C. Röllig 1 , T. Haferlach 3 , P. Valk 4 , D. Linch 5 , T. Herold 6 , N. Duployez 7 , F. Taube 1 , U. Platzbecker 8 , H. Serve 9 , C. Baldus 10 , C. Müller-Tidow 11 , C. Haferlach 3 , M. Meggendorfer 3 , S. Koch 3 , E. L. Boertjes 4 , R. K. Hills 12 , A. Burnett 13 , G. Ehninger 2 , K. Metzeler 8 , M. Rothenberg-Thurley 6 , A. Dufour 6 , H. Dombret 14 , C. Pautas 15 , L. Fenwarth 7 , M. Bornhäuser 1 , R. Gale 5 , C. Thiede 1,16

1 Medizinische Klinik und Poliklinik I, Universitätsklinikum Dresden; 2 AvenCell Europe GmbH, Dresden; 3 MLL Münchner Leukämielabor GmbH, München, Germany; 4 Erasmus University Medical Center, Rotterdam, Netherlands; 5 Department of Haematology, UCL Cancer Institute, London, United Kingdom; 6 Labor für Leukämiediagnostik, Medizinische Klinik und Poliklinik III, Universitätsklinikum München, München, Germany; 7 Institut de Recherche contre le Cancer de Lille, Centre Hospitalier Universitaire de Lille, Lille, France; 8 Klinik und Poliklinik für Hämatologie, Zelltherapie und Hämostaseologie, Universitätsklinikum Leipzig, Leipzig; 9 Medizinische Klinik 2, Universitätsklinikum Frankfurt, Frankfurt am Main; 10 Klinik für Innere Medizin II, Universitätsklinikum Schleswig-Holstein, Kiel; 11 Klinik für Hämatologie, Onkologie und Rheumatologie, Universitätsklinikum Heidelberg, Heidelberg, Germany; 12 Nuffield Department of Population Health, Oxford University, Oxford; 13 Department of Haematology, Cardiff University, University Hospital of Wales, Cardiff, United Kingdom; 14 Institut de Recherche Saint-Louis, Université de Paris, Hôpital Saint-Louis, Paris; 15 Service d’Hématologie Clinique, Hôpital Henri Mondor, Créteil, France; 16 AgenDix GmbH, Dresden, Germany

Background: CEBPA double mutations (dm) are included in the current AML WHO-classification as a favorable-risk independent entity, but recent data suggest this might be limited to those approximately 90% of patients (pts) with in-frame mutations in the basic leucine zipper (bZIP) domain, and that bZIP single allele mutations (sm) might also be favorable.

Aims: To investigate further, we performed a meta-analysis involving detailed sequencing data from 1010 CEBPA -mutant pts.

Methods: Primary pt data was obtained from six study groups: 98 from ALFA, 104 AMLCG, 191 HOVON, 200 MLL, 240 SAL, 177 UK(MRC/NCRI). All pts were treated with curative intention using age and risk-adapted strategies. CEBPA sequences were classified by localization (bZIP vs transactivation domains [TAD] 1 + 2), allelic status (sm vs dm), and type of mutation (in-frame [if] or frameshift [fs] insertions/deletions [InDel] or missense [ms]). Data for GATA2 , WT1 , FLT3, NPM1 co-mutations were available for most pts.

Results: Overall, 546 (54%) had dm and 464 (46%) sm CEBPA (289 TAD1/2; 175 bZIP). To study the impact of the different mutations, we generated 8 groups: dm CEBPA pts with 1/more bZIP InDel-if (Gr1), bZIP InDel-fs (Gr2) or bZIP ms (Gr3) mutation or 2 TAD mutations (Gr4), as well as the corresponding sm groups (Gr5-8). Of note, dm/sm pts with bZIP InDel-if mutations (Gr1 + 5) were significantly younger and predominantly had de novo AML (Tab.1) whereas Gr2-4 and Gr6-8 pts were older with higher incidence of sAML. GATA2 and WT1 mutations were more common in Gr1 + 5 pts; mutations in NPM1 , spliceosome ( SF3B1, U2AF1, SRSF2 and ZRSR2 ) and DNA-methylation ( ASXL1, DNMT3A, TET2, IDH1/2 ) associated genes were confined to the other groups. Pts in Gr1 showed the highest rate of CR1 (Tab.1), better RFS and OS (Fig. 1), whereas dm CEBPA pts in Gr2-4 did significantly worse. Outcome of Gr5 pts was less favorable than Gr1, but still better than for the other subgroups (Gr2-4 and 6-8). In a combined analysis of Gr2-4 and Gr6-8, prognostic factors according to ELN2017 identified three clearly separated subgroups, with the favorable subgroup predominantly associated with mut. NPM1 ; Fig. 1B). The prognostic impact of Gr1 for CR, OS and RFS was confirmed in multivariable analyses taking into account the individual study groups.

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Summary/Conclusion: The results of this large metaanalysis confirm our previous results that only dmCEBPA with InDel-if mutations show a specific biology and very favorable prognostic implications, and further supports the notion that the subgroups with other dm pts differ substantially and have a significantly worse outcome. Sm CEBPA bZIP InDel-if mutant pts and those with other CEBPA mutations and favorable ELN2017 genotype also show an improved outcome.

P405: INHIBITION OF CKS1-DEPENDENT PROTEOSTASIS REVEALS VULNERABILITIES IN LEUKAEMIC STEM CELLS WITH CONCOMITANT PROTECTION OF HEALTHY HAEMATOPOIETIC STEM CELLS

W. Grey 1,2,* , A. Rio-Machin 3 , P. Casado-Izquierdo 3 , E. Gronroos 2 , S. Ali 2 , J. Miettinen 4 , F. Bewicke-Copley 3 , A. Parsons 4 , C. Heckman 4 , C. Swanton 2 , P. Cutillas 3 , J. Gribben 3 , J. Fitzgibbon 3 , D. Bonnet 2

1 York Biomedical Research Institute, The University of York, York; 2 The Francis Crick Institute; 3 Bart’s Cancer Institute, London, United Kingdom; 4 HiLIFE, Helsinki, Finland

Background: Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low two-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs, and the significant toxicity towards healthy hematopoietic cells. Whilst much work has been done to identify genetic and epigenetic vulnerabilities in AML LSCs, little is known about protein homeostasis – so called “Proteostasis” – in drug resistance and relapse.

Aims:

- Characterisation of proteostatic targeting in AML and haematopoiesis.

- Identify proteostatic vulnerabilities in LSCs.

Methods: This study used a range of primary patient AML samples, highthroughput drug screening, in vivo transplantation models and proteomics to demonstrate mechanism of action and functionality of a small molecule inhibitor of CKS1-dependent protein degradation.

Results: We tested a range of proteostatic targeting agents against primary, poor risk, AML patient samples and demonstrate a selective vulnerability of both AML blasts and LSCs to inhibition of CKS1-dependent protein degradation in vitro and in vivo using a small molecule inhibitor. Mechanistically, inhibition of CKS1 leads to hyperactivation of RAC1, increased NADPH production and critical levels of intracellular ROS, resulting in death of LSCs.

Conversely, CKS1 inhibition has the opposite effect on healthy haematopoiesis. Healthy haematopoietic stem and progenitor cells (HSPCs) display increased quiescence upon inhibition of CKS1, a phenotype which confers chemoprotection of healthy HSPCs during clinical chemotherapy protocols (Cytarabine + Doxorubicin, 5 + 3). A phenotype conserved by intestinal stem and progenitor cells.

Summary/Conclusion: Together these findings demonstrate a key vulnerability of AML-LSCs to CKS1-inhibition, while it preserves healthy stem cells. This offers the prospect of both depleting LSCs in vivo and bringing back clinically unfit patients into the pool for intensive chemotherapeutic treatment.

P406: ENHANCED SIGNIFICANCE OF FLT3-ITD RESIDUAL DISEASE DETECTION ON TREATMENT OUTCOME IN ACUTE MYELOID LEUKEMIA

T. Grob 1,* , M. Sanders 1 , C. Vonk 1 , F. Kavelaars 1 , M. Rijken 1 , D. Hanekamp 1 , P. Gradowska 1 , J. Cloos 2 , Y. Fløisand 3 , M. V. Marwijk Kooy 4 , M. Manz 5 , G. Ossenkoppele 2 , L. Tick 6 , V. Havelange 7 , B. Löwenberg 1 , M. Jongen-Lavrencic 1 , P. Valk 1

1 Hematology, Erasmus Medical Center, Rotterdam; 2 Hematology, Amsterdam University Medical Center, Amsterdam, Netherlands; 3 Hematology, Oslo University Hospital, Oslo, Norway; 4 Hematology, Isala Hospital, Zwolle, Netherlands; 5 Hematology, University Hospital Zurich, Zurich, Switzerland; 6 Hematology, Maxima Medisch Centrum, Eindhoven, Netherlands; 7 Hematology, Cliniques Universitaires Saint-Luc, Brussels, Belgium

Background: The prognostic significance of FLT3 -internal tandem duplications ( FLT3 -ITD) in acute myeloid leukemia (AML) in relation to the allelic mutational burden and other concurrent gene mutations at diagnosis remains subject of scientific controversy. Increasing evidence indicates that treatment outcome prediction can be improved by assessing the kinetics and depth of response during therapy by detection of minimal residual disease (MRD). Until now, FLT3 -ITD MRD detection by RQ-PCR has been hampered by the variety in patient specific FLT3 -ITD (i.e. sequence, position and length) and is currently not recommended because of potential instability of FLT3 -ITD clones during relapse. However, systematic studies evaluating the applicability of FLT3 -ITD MRD detection with next-generation sequencing (NGS) in AML are currently lacking.

Aims: Here, we set out to compressively investigate the impact of NGS-based FLT3 -ITD MRD detection on treatment outcome in a cohort of newly diagnosed patients with AML in the context of current prognostic factors at diagnosis and other MRD measurements during therapy, including mutant NPM1 and multiparameter flow cytometry (MFC).

Methods: In 176 de novo FLT3 -ITD AML patients who were treated in HOVON-SAKK multicenter prospective phase III clinical trials, NGS was performed at diagnosis and in complete remission (CR) following two cycles of standard induction chemotherapy. The NGS libraries were paired-end sequenced (2×221-bp) with custom primers on an Illumina MiSeq according to manufacturer’s recommendation (Illumina, San Diego, CA). We used our in-house data analysis pipeline for variant calling. The primary endpoints of the study were relapse and overall survival. The Cumulative Incidence of Relapse (CIR) was estimated with competing-risks regression analyses according to the method of Fine & Gray. The Cox proportional hazard model was used to calculate overall survival estimates.

Results: NGS-based FLT3 -ITD MRD was present in 43 of 176 (24%) AML patients with a median variant allele frequency of 0.01% (range 3.1x10 -4 % to 3.10%). Presence of FLT3 -ITD MRD was associated with increased risk of relapse (4-year risk of relapse, 79% FLT3 -ITD MRD vs. 34% no FLT3 -ITD MRD; P<0.001) and inferior overall survival (4-year rate of overall survival, 27% FLT3 -ITD MRD vs. 57% no FLT3 -ITD MRD; P<0.001). In multivariate analysis, detection of FLT3 -ITD MRD in CR confers independent prognostic significance for relapse (hazard ratio, 4.00; P<0.001) and overall survival (hazard ratio 2.78; P<0.001). Strikingly, FLT3 -ITD MRD exceeds the prognostic value of most generally accepted clinical and molecular prognostic factors, including FLT3 -ITD allelic ratio at diagnosis and MRD assessment by NGS-based mutant NPM1 detection or MFC.

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Summary/Conclusion: NGS-based detection of FLT3 -ITD MRD in CR identifies AML patients with a profound risk of relapse and death that outcompetes the significance of most accepted prognostic factors at diagnosis and during therapy, and furnishes support for FLT3 -ITD as a clinically relevant biomarker for dynamic disease risk assessment in AML.

P407: SYNERGISTIC EFFECT OF A NOVEL COMBINATION OF HDACI CHIDAMIDE WITH CLADRIBINE ON CELL PROLIFERATION ARREST AND APOPTOSIS IN ACUTE MYELOID LEUKEMIA BY TARGETING MYC/HDAC2 SIGNALING

S. Gu 1 , C. Song 2,3 , Z. Ge 1,*

1 Department of Hematology, Zhongda Hospital, School of Medicine, Southeast University, Institute of Hematology Southeast University, Nanjing, China; 2 Hershey Medical Center, Pennsylvania State University Medical College, Hershey; 3 Division of Hematology, The Ohio State University Wexner Medical Center, the James Cancer Hospital, Columbus, American Samoa

Background: Chidamide (CHI), a novel Histone deacetylase inhibitor (HDACi), which is now involved in the salvage treatment of relapsed/refractory acute myeloid leukemia in a clinical trial. Cladribine (2-chloro-2’-deoxyadenosine (2-CdA)), is a purine nucleoside antimetabolite analog, which resists degradation by adenosine deaminase and therefore accumulates to cytotoxic levels. The anti-leukemia effects of either single drug have been reported by inducing cell apoptosis through suppressing multi-oncogenic pathways in human tumor cells. However, whether and how the combination of the two drugs exerts the synergistic anti-tumor effects are still undetermined in AML.

Aims: This study is to investigate the synergistic therapeutic effects of 2-CdA in combination with CHI in AML.

Methods: Cell Counting Kit-8 (CCK-8) cell proliferation assay, Annexin V apoptosis assay, Propidium Iodide cell cycle assay, and Western Blot (WB) were performed in U937 AML cells in presence of CHI only, 2-CdA only, CHI+ 2-CdA, and vehicle control for 48h. RNA-seq was performed in U937 cells treated with CHI and 2-CdA; the differentially expressed genes (DEGs) were identified by R studio, and pathway enrichment of the DEGs was analyzed with Metascape online tool. Co-Immunoprecipitation (co-IP) was used to examine the interaction of cMYC with HDAC1/2. Data were expressed as the mean ± standard deviation. T-tests statistical analysis was employed for determining the significant difference between groups. A synergistic effect was determined by the combination indices (CIs) with CompuSyn software.

Results: The 50% inhibiting concentration (IC50) of CHI and 2-CdA on U937 cell proliferation is 9.358±1.448μmol/L and 0.024±3.071μmol/L, respectively. The combination of various doses of CHI with either IC50 or 1/2 IC50 of 2-CdA significantly decreases the survival rate of U937 cells compared to single drug control (Fig.1a). The CI analysis showed the high synergistic therapeutic effects of CHI with ether dose of 2-CdA (Fig. 1b). The combination of (8mM) CHI with (0.02mM) 2-CdA ignificantly elevated the apoptosis rate and induced the G0/G1 cell phase arrest of AML cells compared with either single drug alone ( p <0.0001, Fig. 1c, d). Moreover, the expression of apoptotic markers, caspase-3, PARP-1, and caspase-9 is elevated, and the cell cycle marker p21 proteins were significantly upregulated, but Cyclin-dependent kinase CDK2 and Cyclin E2 down-regulated in the combination group compared to either single drug control ( p <0.05, Fig. 1e). To further understand the underlying mechanism of the synergy, RNA-seq analysis was performed in U937 cells treated with either CHI or 2-CdA, respectively. The overlapped altered DEGs were identified, and cMYC is dramatically down-regulated. The pathway analysis of the altered DEGs showed the enrichment of MYC pathways further indicated the critical role of MYC (Fig. 1f). It is reported that cMYC is associated with the histone deacetylase complex. Indeed, the co-IP assay showed an interaction of MYC with HDAC2, not HDAC1 in U937 (Fig. 1g). These data suggested the role of cMYC/HDAC2 signaling in the synergistic effect of the combination of CHI with 2-CdA in the disease.

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Summary/Conclusion: The combination of Chidamide and Cladribine has a synergistic effect on cell proliferation arrest, apoptosis, and cell cycle arrest in AML cells through MYC/HDAC2 pathway. Our data provide experimental evidence for the novel potential combination of AML therapy. More in vivo pre-clinical studies will be explored for future clinical trials.

P408: NRAS MUTATIONS ARE INVOLVED IN ACUTE MYELOID LEUKAEMIA DRUG RESISTANCE

F. Healy 1,* , V. Marensi 1 , J. Woolley 1 , D. MacEwan 1

1 Department of Pharmacology and Therapeutics, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom

Background: Acute Myeloid Leukaemia is the most common adult leukaemia, typically associated with poor prognosis. While rates of response to initial cytarabine-based induction chemotherapy are high, the majority of patients will relapse even with haematopoietic stem cell transplant. Thus, the addition of targeted therapies to induction regimens are becoming more widespread, particularly the use of midostaurin and gilteritinib. Even so, resistance is emerging to FLT3 inhibitors, and can often be attributed to NRAS mutations.

Aims: Here, we use over-expression and CRISPR-Cas9 to assess the involvement of NRAS mutations in FLT3-inhibitor resistance cell line models (MOLM-13, FLT3-ITD heterozygous and MV4-11, FLT3-ITD homozygous) that had been previously generated in our lab.

Methods: Drug sensitivity was determined by quantification of apoptosis 72 hours post-treatment (Annexin-V/Propidium Iodide staining). NRAS mutational hotspots associated with increased activation and drug resistance were analysed by Sanger Sequencing. NRAS over-expression and knock-out cell lines were generated using either transfection (HEK293T) or lentiviral transduction (MOLM-13 and MV4-11). Signalling dynamics driving resistance were determined by immunoblotting.

Results: Mutations arose in the MOLM-13-resistant and MV4-11 resistant cell lines at common NRAS mutational hotspots (Q61 and G12, respectively). NRAS Q61K over-expression in the parental MOLM-13 cell line decreased sensitivity to the FLT3 inhibitors gilteritinib, quizartinib, as well as cytarabine, relative to the parental cell line. However, they remained more sensitive to FLT3 inhibition and cytarabine than the resistant cells. In contrast, NRAS-Q61K over-expression caused ~3-fold decrease in sensitivity to trametinib, the MEK inhibitor that acts downstream of NRAS, relative to both the MOLM-13 and MOLM-13-resistant cell lines (Table 1). MOLM-13-NRAS-Q61K over-expression cells showed a growth advantage compared to parental cells in normal culture. In MV4-11-NRAS-Q61K cells, preliminary mechanistic analysis has shown a decrease in ERK phosphorylation. Conversely, CRISPR-Cas9-mediated NRAS knockout in HEK293T appeared to increase ERK activity.

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Summary/Conclusion: NRAS mutations contribute to a FLT3-inhibitor-resistant phenotype, confer a growth advantage in FLT3-ITD+ AML and may act independently of ERK. We have engineered the MOLM-13-resistant and MV4-11-resistant cell lines to express Cas9 under a doxycycline-inducible promoter, to permit stable reversion of the respective mutations. Future work will use these cells to create a CRISPR-mediated reversion of the NRAS mutations which have arisen in the resistant cell lines, to better understand the role of NRAS mutations in FLT3-ITD+ AML. We will better characterise the impacts of a host of NRAS mutations within AML using over-expression and knockout cell lines, and assess drug sensitivity and subsequent pathway re-wiring.

P409: NPM1 MUTATED SAMPLES ARE ESPECIALLY VULNERABLE WHEN TARGETING RAC1 IN PATIENT-DERIVED AML CELLS IN VITRO

A. Hemsing 1,2,* , K. P. Rye 2 , K. J. Hatfield 3 , H. Reikvam 1,2

1 Department of Medicine, Haukeland University Hospital; 2 Department of Clinical Science, University of Bergen; 3 Department of Immunology and Transfusion Medicine, Haukeland University Hospital, Bergen, Norway

Background: Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase signaling molecule known to be overexpressed in many solid cancers and leukemia. Rac1 plays a part in several pathways regulating, e.g., cell migration, adhesion, and cell proliferation. Recent studies have revealed activation and widespread implication of the Rac1 molecule in acute myeloid leukemia (AML), and in vitro Rac1 inhibition in AML cell lines induces apoptosis and improves chemosensitivity.

Aims: We studied the in vitro effect on proliferation, apoptosis, and cytokine release of five RAC1 inhibitors in a cohort of heterogeneous, consecutive patient-derived AML cells at diagnosis. We aimed to map the antileukemic effect in a relatively large AML cohort to identify potential subgroups of AML patients that could be particularly vulnerable to RAC1 inhibition.

Methods: 79 patient-derived AML cell samples from diagnosis were thawed after preservation in liquid nitrogen and cultured at standard in vitro methods with the Rac1-inhibitors ZINC69391, ITX3, EHOP-16, 1A-116, and NSC23766. Cell proliferation was measured by the 3H-thymidine incorporation assay, and apoptosis was measured by the Annexin V/Propidium Iodine apoptosis assay for flow cytometry. Assessment of cytokine profiles in culture media was done with Luminex multiplex immunoassay.

Results: Measured IC 50 values for each compound were as follows; ZINC69391 23 µM (95% CI 15 -29 µM), ITX3 24 µM (15 – 40µM), EHOP-16 3 µM (1,8 – 3,9 µM), 1A-116 10 µM. (8-11 µM) and NSC23766 12 µM (9-15 µM). All five Rac1 inhibitors resulted in an overall antiproliferative effect, tested at dosage around IC 80 . Using bioinformatical classification, we observed that high versus intermediate/low antiproliferative effect was more common in NPM1 mutated (Fischer’s exact test, p=0.002) and CD34 negative (p=0.008) patient samples. The differences were insignificant regarding the presence of FLT3 mutation or according to cytogenetics, FAB classification, gender, and age.

All five compounds did induce significant apoptosis and necrosis compared to untreated control by Wilcoxon signed-rank test (p< 0.0001) at dosages around IC 40 and IC 80 . For NSC23766 only, the presence of NPM1 mutation was associated with more severe apoptosis and necrosis (p=0.01). The presence of FLT3 mutation or CD34 negativity or positivity did not influence the depth of apoptosis and necrosis.

Presence of NPM1 mutation was associated with reduced viability after treatment with EHOP-16 (p=0.025), ITX3 (p=0.047) and NSC23766 (p=0.005). CD34 negativity was significantly associated with reduced viability after treatment with NSC23766 (p=0.006). Finally, Rac1 inhibition significantly reduced the cytokine release of several cytokines crucial for leukemogenesis, with the strongest effects observed for Rac1 inhibitors 1A-116 and NSC23766 (Figure 1, Euclidean clustering).

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Summary/Conclusion: Our results suggest potent and significant antiproliferative and antiapoptotic effects of Rac1 inhibition in primary AML samples. Interestingly, patients harboring the NPM1 mutation seem significantly more vulnerable to such inhibition. Our findings support further research regarding the role of Rac1 and its pharmacological inhibition in AML.

P410: LPIN1 REGULATES LIPID HOMEOSTASIS AND MAINTAINS STEM CELL ENRICHED POPULATIONS IN AML

K. Huber 1,2,* , S. Garg 3 , L. He 1,4 , A. Pouya 1 , C. Rohde 1,4 , C. Lüchtenborg 5 , C. Arnold 4,6 , J. Zaugg 4,6 , S. Raffel 1 , C. Müller-Tidow 1,4 , B. Brügger 5 , C. Pabst 1,4

1 Medizinische Klinik V für Hämatologie, Onkologie und Rheumatologie, Universitätsklinikum Heidelberg, Heidelberg; 2 Medizinische Klinik II für Hämatologie und Onkologie, Universitätsklinikum Schleswig-Holstein, Kiel, Germany; 3 Dana-Farber Cancer Institute, Boston, United States of America; 4 Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 5 Heidelberg University Biochemistry Center (BZH); 6 European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Background: Leukemic stem cells (LSCs) often escape chemotherapy treatment due to the acquisition of stem cell like properties such as slow cell cycle progression, which makes them main drivers of cytologic relapse in acute myeloid leukemia (AML). Identification of novel therapeutic targets to specifically eradicate residual chemoresistant AML cells therefore represents a key challenge in AML therapy. Since LSCs show altered metabolic features, which include dependency on low ROS levels and oxidative phosphorylation of amino acids, targeting LSC metabolism has evolved as a promising approach. Recently the phospholipid phosphatidylserine was shown to be required for LSC stemness and differentiation. However, the role of the main phospholipid synthesis pathway in which the phosphatidic acid phosphatase (PAP) lipin1 catalyzes a rate limiting step remains largely unexplored.

Aims: We investigated the role of the phosphatidic acid phosphatase LPIN1 in AML and healthy hematopoietic cells.

Methods: We applied a gene depletion approach using shRNAs and CRISPR/Cas9 on AML cell lines, primary AML samples and cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) to monitor the effects of LPIN1 depletion on proliferation, differentiation, colony forming capacity and bone marrow engraftment in mice. To gain insights into the mechanisms of action, we combined RNA-sequencing (RNA-seq) with lipidomics assays.

Results: Here we show that regulation of lipid homeostasis by lipin1 is required for HSPC and LSC function. RNA-seq data analysis of healthy hematopoietic cells revealed highest LPIN1 expression in the immature CD34 positive compartment, which steadily decreases during myeloid differentiation. In normal karyotype (NK) AML LPIN1 was utmost expressed in an AML subpopulation characterized by high LSC frequency and co-positivity for the LSC markers CD34 and GPR56. Knock down (KD) of LPIN1 in CD34 positive HSPCs and patient derived xenograft (PDX) leukemic cells hampered proliferation and reduced the CD34 positive LSC compartment in vitro . In xenotransplantation assays LPIN1 KD reduced bone marrow engraftment of healthy HSPC and human AML cells. Furthermore, in vivo differentiation of CD34 positive cells into B-cells was almost completely abrogated upon LPIN1 KD. In the leukemia setting, LPIN1 KD particularly reduced the LSC-enriched CD34+GPR56+ co-positive fraction. While RNA-seq of the AML cell line OCI-AML3 and CD34 positive cells showed only subtle gene expression changes in phospholipid metabolism upon LPIN1 KD, lipidomics analyses revealed significant differences in the composition of phospholipids upon LPIN1 KD.

Summary/Conclusion: Our data establish LPIN1 as an essential enzyme maintaining lipid homeostasis and stem cell functions in the hematopoietic system. Identification of phospholipid entities with differential impact on healthy versus leukemic cells might help refine LSC targeted therapies.

P411: SF3B1 MUTATIONS IN AML ARE STRONGLY ASSOCIATED WITH MECOM REARRANGEMENTS AND MAY BE INDICATIVE OF AN MDS PRE-PHASE

S. Huber 1,* , S. Hutter 1 , M. Meggendorfer 1 , G. Hoermann 1 , W. Walter 1 , C. Baer 1 , W. Kern 1 , T. Haferlach 1 , C. Haferlach 1

1 MLL Münchner Leukämie Labor GmbH, München, Germany

Background: Mutations in the splicing factor gene SF3B1 are most frequent in myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) but are also recurrently found in acute myeloid leukemia (AML). Splicing factor mutations have been suggested to have additive value if incorporated into the current risk classification of AML (van der Werf et al., Blood Advances 2021).

Aims: Explore the genetic landscape of SF3B1 mutated AML and determine the prognostic impact of SF3B1 mutations.

Methods: We analyzed 735 patients diagnosed with AML based on cytomorphology, cytogenetics, and molecular genetics according to WHO classification 2017. Reads from amplification-free whole genome sequencing (WGS, median coverage 103x, Illumina, San Diego, CA) were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7.

Results: We identified SF3B1 mutations in 41 of 735 (6%) AML patients. SF3B1 mutations were found in AML with recurrent genetic abnormalities (24/471; 5%), AML with myelodysplasia-related changes (MRC; 11/158; 7%) and AML not otherwise specified (NOS; 6/106; 6%). When focussing on SF3B1 mut cases, AML-MRC (11/41; 27%) and AML with GATA2::MECOM (10/41; 24%) were most frequent. 9 additional cases showed another MECOM rearrangement (-rear) resulting in 46% (19/41) of patients with SF3B1 mutations accompanied by a MECOM -rear. A prior history of MDS or MDS/MPN was documented in 20% (8/41) of SF3B1 mut patients. Thereof, 63% (5/8) harbored a MECOM rearrangement and 25% RUNX1 mut . On average, SF3B1 mut patients harbored 2.85 mutations (including SF3B1 ). The most frequent additional mutations of SF3B1 mut patients were RUNX1 (9/41; 22%) and NRAS (8/41; 20%), and NPM1, TET2, or DNMT3A mutations or FLT3 -ITD were detected in 15% (6/41) each. SF3B1 showed an average variant allelic frequency (VAF) of 41%. Notably, the VAF of SF3B1 in AML with NPM1 or RUNX1 was higher than the VAFs of NPM1 or RUNX1 in 9/11 (82%) cases, suggesting that SF3B1 preceded these mutations. In 16/41 (39%) cases molecular follow-up data was available. The SF3B1 mutation persisted during the entire disease course in 8 cases (including 1 with relapse). In particular, in two of these AML patients with mutated NPM1 , the SF3B1 mutation remained detectable by NGS, despite complete hematologic remission and undetectable NPM1 mutation by high-sensitive qPCR. In all 5 cases with relapse the SF3B1 mutation re-occurred at relapse. In 4 patients, the VAF of SF3B1 decreased, similar to accompanying mutations. In contrast to MDS, SF3B1 mutations did not affect the overall survival (median OS: 16.9 months in SF3B1 mut ; 5.7 months in unmutated group; p=0.822) in the total AML cohort. When stratified for AML sub-entities, the prognosis of the SF3B1 mut AML seems to be dominated by the sub-entity.

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Summary/Conclusion: Within AML, SF3B1 mutations are enriched in AML patients with GATA2::MECOM and AML-MRC. This association with poor risk subtypes dominates the prognosis of SF3B1 mut AML. The high VAF of SF3B1 mutations and the persistence of SF3B1 mutations in AML patients in complete remission suggest the early acquisition of SF3B1 mutations in a pre-leukemic clone in a number of patients. While an antecedent MDS or MDS/MPN has been documented in some cases of SF3B1 mut AML, it might be unidentifiable in others. Alternatively, SF3B1 mut clonal hematopoiesis of indeterminate potential (CHIP) may represent a relevant pre-phase of SF3B1 mut AML.

P412: LOW EXPOSURE, EXTENDED DOSING MIMICKING CLINICAL EXPOSURES OF ORAL AZACITIDINE SYNERGIZES WITH VENETOCLAX IN PRECLINICAL AML MODELS

D. Jeyaraju 1,* , M. Alapa 1 , A. Polonskaia 1 , A. Risueno Perez 2 , R. Hurren 3 , X. Wang 3 , M. Gronda 3 , A. Ahsan 1 , C. Wang 1 , P. Subramanyam 4 , J. Sriganesh 4 , A. Anand 4 , M. Bysani Reddy 4 , K. Ghosh 5 , C. Kyriakopoulos 2 , N. Lailler 6 , C. Hartl 6 , D. Lopes de Menezes 7 , A. Schimmer 3 , P. Hagner 1 , A. Gandhi 1 , A. Thakurta 1

1 Translational Medicine, Bristol Myers Squibb, Summit, United States of America; 2 BMS Center for Innovation and Translational Research Europe, Bristol Myers Squibb, Seville, Spain; 3 Princess Margaret Cancer Center, Toronto, Canada; 4 BBRC; 5 Bristol Myers Squibb, Bangalore, India; 6 Rancho Biosciences, San Diego; 7 Bristol Myers Squibb, San Francisco, United States of America

Background: Acute myeloid leukemia (AML) has a high incidence of relapse despite the advancement of novel therapies. AML patients that are ineligible for intensive chemotherapy are typically administered injectable azacitidine (AZA) at a dose of 75 mg/m 2 for 7 days in combination with BCL-2 inhibitor venetoclax (VEN) as induction therapy. Injectable-AZA dose was determined primarily through safety, tolerability, and bioavailability studies based on the CALGB and AZA-001 trials and not through a dose escalation study. Thus, it is not known if similar efficacy and combinability of AZA with VEN could be achieved at lower exposures and extended duration with oral-AZA dosing. The approval of oral-AZA in AML maintenance therapy, albeit at a lower cumulative exposure conferred at doses of 300 mg for an extended duration of 14 days of a 28-day dosing cycle, creates opportunities to answer these questions.

Aims: To investigate the mechanism of action and combinability of oral-AZA with VEN in pre-clinical models.

Methods: For in vitro cell-line based work, 1 day x 1mM AZA and 5days x 0.2mM AZA were used to mimic injectable-AZA like and oral-AZA like regimens respectively. For in vivo studies we modeled injectable-AZA and oral-AZA doses with 3mg/kg, qd x 5 days and 1 mg/kg, qd x 15 days respectively (intraperitonially in both cases, modified from Vu et al., Nat. Commun. 2020 ). VEN was administered orally at 100 mg/kg, qd x21 days. CRISPR screen was conducted with OCI-AML2-Cas9 cells using the Cellecta sgRNA library in the presence of AZA (121nm (IC 50 ) x 5 days).

Results: To identify synthetic lethal targets of oral-AZA, we conducted a genome-wide CRISPR screen. Among others, BCL-2 emerged as a synergistic hit. During in vitro validation of the CRISPR screen data, VEN synergized (anti-proliferative effect) with oral-AZA like exposure only when added after 5 days (0.2mM /day) of repeated AZA dosing. On the other hand, injectable-AZA like exposure synergized with VEN in 24 hours suggesting that the Oral-AZA/VEN synergy was driven more through epigenetic changes and not by stress response as has been reported for injectable-AZA. To further validate the CRISPR screen findings, we utilized in vivo AML xenograft mouse (injected with human AML cell lines) treated with an oral-AZA like or injectable-AZA like regimen with/without VEN. Mouse survival data demonstrated that both AZA regimens combined with VEN with equivalent efficacy.

The persistence of leukemic stem cells (LSCs) is one of the reasons for relapse in AML. In vitro , oral-AZA like exposure resulted in decreased numbers of CD34+ CD38- LSCs. In an in vivo primary AML xenograft model using intrafemoral engraftment, oral-AZA like exposure was effective in reducing engraftment of leukemic cells from the right to the left femur (10-fold reduction). Analysis of cells at the site of injection (right femur) revealed induction of differentiation from a granulocyte-monocyte progenitor (GMP) to a megakaryocyte-erythrocyte progenitor (MEP) or common myeloid progenitor (CMP) state in response to oral-AZA.

Summary/Conclusion: Collectively, our pre-clinical in vitro and in vivo data in AML models indicates that oral-AZA can combine effectively with VEN. In addition, oral-AZA can effectively eliminate LSCs and induce differentiation of immature cells. Through the CRISPR screen, our work identifies potential novel combination partners for oral-AZA.

P413: THE POTENTIAL USE OF AN ORAL HYPOMETHYLATING AGENT, OR-2100, AS A COMBINATION THERAPY FOR MYELOID MALIGNANCIES

K. Kamachi 1,2,* , H. Ureshino 1,2 , K. Kawasoe 1,2 , N. Yoshida-Sakai 1,2 , Y. Yamamoto 1 , Y. Fukuda-Kurahashi 2,3 , Y. Kurahashi 1,2,3 , T. Watanabe 2 , S. Kimura 1,2

1 Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine; 2 Department of Drug Discovery and Biomedical Sciences, Saga university, Saga; 3 OHARA Pharmaceutical Co.,Ltd., Shiga, Japan

Background: The combination therapy of hypomethylating agents (HMAs), azacytidine (AZA) and decitabine (DAC), with venetoclax (VEN), a selective BCL-2 inhibitor, have revolutionized the outcome of elderly patients with acute myeloid leukemia (AML), which have demonstrated the potential effect of HMAs as a combination therapy with molecular targeting agents for myeloid malignancies. However, there are a few studies on the combined effect of HMAs with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and the mechanism by which HMAs enhance the efficacy of molecular targeting agents remain poorly understood. We have developed OR-2100 (OR21), an oral HMA, a 5’-O-trialkylsilylated DAC. OR21 is as effective as DAC, but less myelosuppressive, in xenograft mouse models of solid and hematological malignancies ( Hattori et al. Clin Epigenetics 2019, Watanabe et al. Blood 2020, Ureshino et al. Mol Cancer Ther 2021, Kamachi et al. Cancer Letters 2022 ). We investigated the efficacy and mechanism of OR21 in combination with VEN or TKIs for myeloid malignancies, AML, and CML.

Aims: To investigate the efficacy and mechanism of the combination therapy of OR21 for myeloid malignancies.

Methods: We investigated the efficacy and mechanism of OR21 in combination with VEN against AML and with TKIs against CML, in vitro and in vivo .

Results: In AML, OR21 plus VEN synergistically inhibited cell growth, increased apoptosis in AML cell lines as similar to AZA and DAC, and significantly prolonged survival in a xenograft mouse model using HL60 cell line ( p<0.05 ). Gene expression analysis using AML cell lines (HL60, KG1a) showed that OR21 plus VEN significantly downregulated VAMP7 which regulates mitophagy/autophagy to maintain mitochondrial homeostasis compared to VEN monotherapy. Consistently, OR21 plus VEN enhanced apoptosis by increasing ROS accumulation and attenuated mitophagy/autophagy response triggered by decreased mitochondrial membrane potential. The addition of mitophagy inducers, such as rapamycin and p62-mediated mitophagy inducer, to OR21 plus VEN decreased ROS accumulation and apoptosis, indicating OR21 enhanced ROS accumulation via attenuating VEN-induced mitophagy/autophagy pathway.

In CML, OR21 plus TKIs (imatinib, dasatinib, ponatinib) synergistically inhibited cell growth, increased apoptosis in CML cell lines as similar to AZA and DAC, and significantly suppressed the colony-forming capacity of bone marrow CD34 + stem/progenitor cells in CML patients. OR21 plus imatinib significantly suppressed tumor growth compared to imatinib monotherapy in a xenograft mouse model using K562 cell line ( p<0.05 ). In phosphoproteomic, transcriptome and methylome analysis (K562, KBM5), OR21 augmented imatinib-induced apoptosis by dephosphorylating of STAT and Src family proteins by upregulating several tumor suppressor genes, including PTPN6 , through demethylation of their promoter CpG regions.

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Summary/Conclusion: OR21 enhanced the anti-tumor effect in combination with the molecular agents for myeloid malignancies with different mechanisms: OR21 increased ROS accumulation in combination with VEN, and dephosphorylated BCR-ABL1 downstream protein kinases by demethylating effect in combination with TKIs. With its advantage as an oral HMA, OR21 is expected to have future clinical trials as a combination therapy for myeloid malignancies.

P414: RNA MODIFICATION-FOCUSED CRISPR-CAS9 SCREEN REVEALS POTENTIAL THERAPEUTIC TARGETS MEDIATING CHEMORESISTANCE IN AML

M. Kienhöfer 1,2,3,* , C. Pauli 1,2 , S. Delaunay 2 , C. Rohde 1,3,4 , M. F. Blank 1,2,4 , D. Heid 1,4 , M. Frye 2 , C. Müller-Tidow 1,3,4,5

1 Department of Medicine V, Hematology, Oncology and Rheumatology, University Hospital Heidelberg; 2 German Cancer Research Center (DKFZ); 3 University of Heidelberg Medical Faculty; 4 Molecular Medicine Partnership Unit, European Molecular Biology Laboratory–Heidelberg University Hospital; 5 National Center for Tumor Diseases (NCT), Heidelberg, Germany

Background: Acquired therapy resistance is one of the main causes of death in Acute Myeloid Leukemia (AML). RNA-modifying proteins (RMP) are a diverse group of enzymes responsible for the deposition and the removal of over 170 chemical modifications, impacting key features of RNA biology. The RMPs represent a new focus for target therapies.

Aims: The objective of the current study is to uncover how RNA-modifying proteins affects resistance to treatment in AML.

Methods: A CRISPR-Cas9-based screening approach was utilized to study RNA-modifying proteins in an AML cell line and a derived cell line resistant to cytarabine. The library pool targeted 150 RNA modifying enzymes, the 250 most abundant expressed snoRNAs in AML, as well as a group of control genes and non-targeting sgRNAs. A lentiviral system was used to introduce the CRISPR system. Multiplicity of infection (MOI) was held under 0.3 to guarantee one gene knockout per cell. Post-infection, the cell lines were divided into a control and a treatment group. The treatment group was stimulated with increasing doses of cytarabine over 22 days. Samples were analyzed via NGS.

Results: In the CRISPR screen, we observed that many of the gene knockouts led to cell depletion. Overall, 37 genes knockouts were depleted in the control group of both cell lines without additional drug treatment, suggesting that the related genes are required for survival and/ or proliferation. Further, 28 RNA-modifying enzymes and 15 snoRNAs were altered upon drug treatment in at least the parental or the resistant cell line. Interestingly, 15 of those 28 enzymes show additional effects on proliferation. Based on this data, we chose a methyltransferase responsible for specific modification on tRNAs for further analysis. Single knockout of the enzyme sensitized different AML cell lines for cytarabine treatment and showed impairment of proliferation as well as the ability to form colonies.

Summary/Conclusion: RNA modifications promote several hallmarks of cancer. This study reports the involvement of specific RNA-modifying enzymes for therapy resistance and proliferation in AML. However, it remains unclear whether these enzymes are dysregulated in leukemic cells and therefore could display a targetable vulnerability. In addition, further studies of the affected cellular pathway could contribute to a better understanding of the underlying resistance mechanisms.

P415: TARGETED 3RD-GENERATION (NANOPORE) SEQUENCING REVEALS MANY NOVEL CIRCULAR RNAS OF THE APOPTOSIS-RELATED BCL2L12 GENE, EXPRESSED IN HUMAN CELLS FROM ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES

K. Xenou 1 , C. Sotiropoulou 1 , P. Karousi 1 , N. Machairas 1 , A. Scorilas 1 , V. Pappa 2 , S. Papageorgiou 2 , C. Kontos 1,*

1 Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens; 2 Second Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, Athens, Greece

Background: Circular RNAs (circRNAs) are non-coding RNAs generated via back-splicing. They are indirect – yet pivotal – regulators of gene expression, as many of them bind microRNAs (miRNAs) and render them unavailable to exert their direct regulatory functions. They also interact with chromatin, altering its structure and affecting transcription of several genes. Lastly, though considered as non-coding, few of them have also been shown to encode short, functional polypeptides. Due to their multifaceted role and the deregulation of their expression in cancer, circRNAs are related to the development and progression of malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). BCL2L12 is a prominent member of the apoptosis-related BCL2 family, with an already established role in AML. Moreover, BCL2L12 mRNA expression has been associated with chemoresistance of malignant bone marrow blasts. However, the identity and role of the circRNAs produced by alternative back-splicing of BCL2L12 primary transcripts in MDS and AML has not been investigated, so far.

Aims: We sought to determine the identity of BCL2L12 circRNAs expressed in MDS and AML cell lines and to explore their putative interactions with miRNAs having an established role in these disease entities.

Methods: MDS-L cells [MDS with del(5q) and complex karyotype], NB-4 and HL-60 cells (acute promyelocytic leukemia with and without PML-RARA, respectively), U-937 and THP-1 cells (acute monocytic leukemia), KASUMI-1 cells (AML with t(8;21)(q22;q22.1); RUNX1 - RUNX1T1 ) and MOLM-13 cells (AML with t(9;11)(p21.3;q23.3); KMT2A - MLLT3 ) were propagated. Total RNA was extracted from these seven cell lines; 2μg of each RNA extract were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of the BCL2L12 gene; thus, only cDNAs from BCL2L12 circRNAs were amplified. After having mixed and purified all nested-PCR products of each cell line cDNA, nanopore sequencing libraries were built. Following nanopore sequencing and basecalling in a MinION Mk1C 3 rd -generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA identification was performed using an in-house –built, PERL-based algorithm; miRDB was used for prediction of their interactions with miRNAs.

Results: We discovered 83 novel BCL2L12 circRNAs, 9 of which were common between MDS-L cells and at least one AML cell line. The exon structure of these novel circRNAs showed a remarkable diversity, comprising exons also encountered in messenger RNAs (mRNAs) of BCL2L12 as intronic regions. This phenomenon was mostly observed in MDS-L cells. Several BCL2L12 circRNAs in NB-4 and HL-60 cells exhibited complete intron retention; additionally, a novel exon was present at a remarkable frequency in NB-4 cells. Moreover, several BCL2L12 circRNAs are predicted to sponge miRNAs with a key role in AML (miR-7-5p, miR-125b-5p and miR-182-5p) and/or MDS (miR-150-5p).

Summary/Conclusion: Numerous circRNAs are produced through alternative back-splicing of the primary BCL2L12 transcripts. The circRNA expression profile of this gene differs among the MDS and AML cell lines, as well as among the six AML cell lines. Intronic regions of this apoptosis-related gene are included in several circRNAs, augmenting the repertoire of miRNAs predicted to be bound by BCL2L12 transcripts. This phenomenon was more common in the MDS cell line, followed by both acute promyelocytic leukemia cell lines. Our data suggest an important regulatory role for this gene in MDS and AML subtypes.

P416: CLONAL HEMATOPOIESIS IS COMMON IN AML LONG-TERM SURVIVORS AND MAY ASSOCIATE WITH DIABETES AND SECONDARY NEOPLASIAS, BUT NOT OTHER HEALTH OUTCOMES

S. M. Krauß 1,* , E. Telzerow 2 , A. S. Moret 2 , D. Richter 3 , M. Rothenberg-Thurley 2 , D. Görlich 4 , M. C. Sauerland 4 , W. E. Berdel 5 , B. Wörmann 6 , U. Krug 7 , J. Braess 8 , P. Heussner 9 , W. Enard 3 , W. Hiddemann 2 , K. Spiekermann 2 , U. Platzbecker 1 , K. H. Metzeler 1

1 Dept. Hematology and Cell Therapy, University Hospital Leipzig, Leipzig; 2 Dept. of Medicine III, University Hospital, Ludwig-Maximilians University; 3 Dept. Biology II, Ludwig-Maximilians University, Munich; 4 Institute of Biostatistics and Clinical Research; 5 Dept. of Medicine, Hematology and Oncology, University of Münster, Münster; 6 Deutsche Gesellschaft für Hämatologie und medizinische Onkologie, Berlin; 7 Dept. of Medicine 3, University Hospital Leverkusen, Leverkusen; 8 Dept. of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg; 9 Psycho-Oncology, Hospital Garmisch-Partenkirchen, Garmisch-Partenkirchen, Germany

Background: Clonal hematopoiesis (CH), the outgrowth of hematopoietic stem cells and their progeny with somatically acquired gene mutations, is a common phenomenon in elderly individuals without known hematological disorders. CH has been linked to an increased risk of developing myeloid neoplasia and cardiovascular diseases, resulting in higher all-cause mortality. More specifically, clonal hematopoiesis of indeterminate potential (CHIP) has been defined by hematopoietic clones with a variant allele frequency (VAF) ≥2% in the absence of hematologic disease. CH is also known to be common in patients with acute myeloid leukemia (AML) briefly after therapy, but prevalence and relevance of CH in AML long-term survivors (AML-LTS) has not been investigated so far.

Aims: We aimed to study CH in a cohort of 380 AML-LTS using a targeted next generation sequencing (NGS) assay utilizing single-molecule Molecular Inversion Probes (smMIPs), and to analyze determinants of CH and its effects on somatic health outcomes.

Methods: We employed smMIP-technology, which follows a hybridization-capture protocol for NGS library preparation to selectively enrich and amplify hundreds of genomic target loci covering 82 regions in 24 CH and AML-related genes in a single reaction. Unique molecular identifiers (UMIs) within probes enabled computational correction of sequencing errors to significantly increase precision of variant calls.

Results: We successfully established a UMI-based smMIP NGS assay as well as a custom computational analysis pipeline to enable the reliable detection of variants ≥0.5% VAF. In whole blood specimens from 380 AML-LTS we detected CH in 61.4% of individuals, including CHIP (VAF ≥2%) in 35.8%. Stratification by treatment group, i.e. chemotherapy/autologous hematopoietic stem cell transplantation only (chemo) vs. allogeneic hematopoietic stem cell transplantation (alloSCT), revealed a significantly lower prevalence of CHIP in the alloSCT group (26.0% vs. 52.1%; p<.001). Concordantly, median VAF of CH mutations after alloSCT was significantly lower than in the chemo group (1.2% vs. 1.8%; p=.005). CHIP prevalence increased with age in the chemo group, from 21.7% (<50 years) to 70.0% (≥70 years), whereas it was independent of patient age in the alloSCT group (Figure A). Conversely, CHIP prevalence was independent from time since start of therapy in the chemo group, but increased with time after transplantation in the alloSCT group, from 18.4% (0-5 years) to 46.2% (16-18 years; Figure B). In both treatment groups, the known CHIP driver genes DNMT3A and TET2 were most frequently mutated. Notably, PPM1D (p<.001) and TP53 (p<.001) were more frequently mutated in the chemo group (Figure C). We did not detect significant differences in complete blood counts of AML survivors according to CH status. While most somatic comorbidities showed no clear association with CH status, prevalence of diabetes and development of secondary cancer after leukemia therapy were increased in patients with CH (VAF <2%), and more so in those with CHIP (Figure D).

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Summary/Conclusion: We report a high prevalence of CH in AML long-term survivors, particular in those treated with chemotherapy without alloSCT. CHIP prevalence increases with age in patients treated with chemotherapy, and with time elapsed after transplantation in patients who underwent alloSCT. We reveal distinct mutation spectra for both groups. Although associations between CH and somatic morbidities seem to be weak overall, we found associations with diabetes and the development of secondary cancer after leukemia treatment, which warrant further exploration.

P417: A SINGLE AMINO ACID AT THE PNT DOMAIN OF ERG MEDIATES ITS LEUKEMOGENIC ACTIVITY THROUGH INTERACTION WITH THE NCOR-HDAC3 CO-REPRESSOR COMPLEX.

E. Kugler 1,2,* , S. Madiwale 2 , D. Yong 3 , Y. Birger 2,4 , J. A. I. Thoms 5,6 , D. B. Sykes 7,8 , M. Yassin 9 , N. Aqaqe 9 , A. Rein 2,4 , H. Fishman 2 , I. Geron 2 , C.-W. Chen 10,11,12 , B. Raught 13 , Q. Liu 10 , M. Milyavsky 9 , J. Pimanda 5,6 , G. G. Privé 3,13 , S. Izraeli 2,4

1 Institute of Hematology, Davidoff Cancer Center, Rabin medical center, Petah Tikva; 2 Department of Human Molecular Genetics and Biochemistry, Sackler Medical School, Tel Aviv University, Tel Aviv, Israel; 3 Department of Biochemistry, University of Toronto, Toronto, Canada; 4 The Rina Zaizov Pediatric Hematology and Oncology Division, Schneider Children’s Medical Center, Petach Tikva, Israel; 5 Adult Cancer Program, Lowy Cancer Research Centre; 6 Faculty of Medicine, UNSW Sydney, Sydney, Australia; 7 Center for Regenerative Medicine, Massachusetts General Hospital, Boston; 8 Harvard Stem Cell Institute, Cambridge, United States of America; 9 Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 10 Department of Systems Biology, Beckman Research Institute, City of Hope, Duarte; 11 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston; 12 City of Hope Comprehensive Cancer Center, Duarte, United States of America; 13 Princess Margaret Cancer Centre, University Health Network, Toronto, Canada

Background: The ETS transcription factor ERG is essential for the maintenance of hematopoietic stem cells. However, it has also been implicated as an oncogene in the development of acute leukemia. Our studies and those of others have demonstrated that ERG directly contributes to the initiation and maintenance of lymphoid and myeloid acute leukemia subtypes. Nevertheless, ERG co-factors critically involved in leukemogenesis remain largely uncharacterized.

Aims: Here we report a critical role for the conserved amino-acid proline at position 199, at the 3’ end of the PNT domain, for ERG’s leukemogenic activity.

Methods: A genomic and proteomic analysis has been used to study the role of P199 in the leukemogenic pathway of ERG.

Results: We specifically demonstrate in functional and gene expression assays that P199 is required for ERG-induced myeloid differentiation restriction and for self-renewal and maintenance of murine hematopoietic stem and progenitor cells (HSPC).

As P199 is located within the PNT domain (protein interaction domain), we attempted to identify key protein interactions associated with leukemia progression induced by ERG. To this end, we used proximity ligation-mass spectrometry (BioID). In HEK293 cells, proximity maps of WT-ERG and a mutated form of ERG at position 199 (P199L-ERG) were compared. A total of 240 putative protein interactors with a significance analysis of interactome were identified. The hits were highly enriched with chromatin modifiers. Most significantly, the mutation severely impaired ERG’s interaction with components of the NCoR-HDAC3 complex, resulting in a 40% reduction in the number of spectral counts in comparison with wild-type ERG.

ChIP sequencing for histone markers conducted on ER-Hoxb8 cells (transformed murine GMPs) demonstrated a decrease in H3K27ac signature at over 1500 unique sites in cells transduced with WT-ERG compared to those transduced with the mutant. Interestingly, these sites were predominantly associated with enhancers of genes expressed in mature myeloid cells.

Next, we addressed the role of HDAC3 in ERG’s transcriptional activity. RNA sequencing of human ER-Hoxb8 cells treated with BRD3308 (an HDAC3 inhibitor) for 48 hours was performed. Interestingly, leukemia-associated pathways (Hoxa9-Meis1, CBFA2T3 and repression of myeloid differentiation genes) were ranked at the leading edge of gene sets that were significantly perturbed after HDAC3 inhibition.

Moreover, we tested the significance of the ERG/NCoR-HDAC3 interaction in models of human leukemia. We demonstrated that ERG-dependent human AML cells as SKNO1 (AML1-ETO) and TF1 (CBFA2T3-ABDH12) were more sensitive to HDAC3 inhibition (RGFP966) as compared to ERG independent cells.

Furthermore, RGFP966 exhibited a significant in-vivo antileukemic effect as measured by the burden of disease in the bone marrow of NSG mice transplanted with SKNO1 cells.

By using the CRISPR-dCAS9 system we were able to show that leukemia development is abrogated in mice transplanted with SKNO1 cells expressing a reduced level of HDAC3. Remarkably, this effect was translated into a significant survival advantage.

Summary/Conclusion: Taken together, our findings indicate that the aberrant overexpression of ERG maintains HSPCs in an undifferentiated state and promotes AML development. We suggest that the interaction between ERG and the NCoR-HDAC3 complex has an important role in the leukemogenic process, and that HDAC3 inhibition could be beneficial in AML characterized by high ERG expression.

P418: MODELLING AND TARGETING ACUTE MYELOID LEUKAEMIA CELLS IN THE BONE MARROW PROTECTIVE NICHE

E. E. Ladikou 1,2,* , K. Sharp 1 , T. A. Burley 1 , E. Kennedy 1 , T. Chevassut 2 , C. Pepper 1 , A. G. Pepper 1

1 Clinical and Experimental Medicine, Brighton and Sussex Medical School; 2 Department of Haematology, Brighton and Sussex University Hospital Trust, Brighton, United Kingdom

Background: AML is a therapeutic challenge due to its aggressiveness and biological heterogeneity. Although 80% of patients initially achieve a complete remission, the long-term disease-free survival is poor. One issue contributing to disease relapse, is persistence of disease in the protective niche of the Bone Marrow microenvironment (BMME). Here, AML cells are surrounded by other cell types that promote their survival, which enables them to evade therapeutic destruction and promotes the emergence of drug resistance. Clinical trials of single agent mobilising drugs, like Plerixafor, have yielded promising results but they are not curative. So, the development of combinatorial therapies that simultaneously target different components of AML cell adhesion may release more AML cells into the circulation, where they can be targeted by standard therapies.

Aims: a) To develop a multi-cellular, co-culture system to recapitulate the adhesive BMME, b) To test rational drug combinations and identify targets with the potential to mobilise anchored AML cells and c) To perform paired transcriptomic and phenotypic analysis of persistently adhered versus mobilised AML cells to detect novel targets.

Methods: We have developed a novel 96-well plate multi-cellular, co-culture system using stromal cells (HS5), endothelial cells (HUVEC), osteoblasts (hFOB 1.19) and AML cells (OCI-AML3 and KG1a). Multiple mobilisation drugs, alone and in combination, were tested and AML cell release was quantified by flow cytometry. Drugs tested to date include: Plerixafor (CXCR4 inhibitor), Natalizumab (anti-α4-integrin antibody), ONO-7161 (novel CXCR4 inhibitor), purified anti-CD44 and anti-E-Selectin. Phenotypic and transcriptional comparisons were made between the persistently adherent and mobilised AML cells.

Results: The most adhesive physiological “BMME mix” was identified as HS5, HUVEC and hFOB 1.19 in equal 1/3 proportions (p=0.0001). Co-culture with AML cells resulted in 74% and 68% adherence of KG1a and OCI-AML3, respectively. Phenotypic analysis of adhered vs non-adhered AML cells identified CD44, CXCR4, CD49d and CD38 as important markers of optimal BMME adhesion. Subsequently, our novel co-culture system was used as a drug testing platform to assess clinically available agents. Plerixafor and Natalizumab increased detachment by 1.3-fold (p=0.0132) and 1.2-fold (p=0.015) respectively. In contrast, ONO7161 and anti-E-selectin had little effect. The most promising candidate, anti-CD44, resulted in a 2-fold and 1.6-fold detachment of KG1a (p=0.004) and OCI-AML3 (p=0.027), respectively. However, even in the presence of the maximum dose of anti-CD44 (5µg/mL)), 46% of KG1a and 48% of OCI-AML3 cells remained persistently adhered. Synergy experiments with anti-CD44 in combination with either Plerixafor or Natalizumab yielded no better release than anti-CD44 alone.

Summary/Conclusion: We have developed a novel, multi-cellular co-culture system that recapitulates the adhesive BMME. We are using this as a drug testing platform for AML to identify the most potent release agents and test the effects of cytotoxic drugs on those most persistently adhered. To-date, anti-CD44 is the most effective release agent in our model, but it still failed to release all AML cells. Transcriptomic analysis of the persistently adherent AML cells should enable us to delineate the critical biological interactions required for BMME adhesion. This will enable us to identify rational new targets to effectively remove chemo-resistant AML cells from the protective BM niche.

P419: IDENTIFICATION OF NOVEL THERAPEUTIC OPTIONS FOR VENETOCLAX-RESISTANT AML CELLS THROUGH DRUG REPURPOSING

A. Ladungova 1,2,* , D. Busa 3 , Y. Lodhi 1,4 , J. Hyl 3 , M. Culen 3,5 , M. Smida 1,3,5

1 Central European Institute of Technology, Masaryk University; 2 National Centre for Biomolecular Research, Faculty of Science, Masaryk University; 3 Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine, Masaryk University; 4 Department of Biology, Faculty of Medicine, Masaryk University; 5 Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czechia

Background: Acute myeloid leukemia (AML) is a malignant disease derived from the bone marrow precursors of myeloid lineage. Treatment options are rather limited, primarily based on chemotherapy, and often result in disease progression. Recently, venetoclax-based therapies have transformed the frontline regimens of elderly patients and patients unfit for intensive chemotherapy. Despite its promising outcomes in clinical studies, multiple resistant subclones evolved during the treatment acting as a barrier in disease regression. Understanding the venetoclax-resistance mechanisms and detecting the major determinants could reveal previously unrecognized novel perspectives for therapeutic strategies to improve patients’ outcomes. Moreover, performing high-throughput screenings with clinically approved drugs could reveal novel treatment options for resistant subclones of AML.

Aims: Our project aims 1) to identify novel FDA-approved drugs effective for treating venetoclax-resistant AML cells through a high throughput screening, 2) to validate the most effective compounds , prove their specificity, and 3) to investigate the molecular background mechanisms underlying the observed sensitivity.

Methods: Two different models mimicking the microenvironment of venetoclax-resistance formation have been established: 1.) Patient-derived mouse xenograft (PDX) model. AML PDX model was generated by using AML patient sample transplanted into an immunocompromised murine host and treated with different regimens of venetoclax; 2.) Venetoclax-resistant cell lines (MOLM-13, OCI-AML3, HL-60). Generation of the venetoclax-resistant AML cell lines was achieved through the chronic administration of the compound, inhibiting the cell viability to 80-90% in multiple rounds with gradually increasing concentrations until the cells become fully resistant. Both models are next used for the high-throughput screening of 859 approved drugs, covering broad chemical space. The standard concentration (1 μM) of drugs in the library is automatically added to cells on 384-well-plates with a programmed liquid handling system epMotion (Eppendorf). Cell viability is determined after 72 hours by a Cell-titer Glo assay. Compounds with the strongest effect upon resistant AML cell viability are validated through 10-point dose-response curves with 2-fold-dilutions. Moreover, the investigation of molecular mechanisms responsible for the sensitization of resistant AML cells is planned by using various cell biology and molecular biology techniques.

Results: Treating the PDX mouse model with venetoclax for 6 weeks resulted in the generation of resistant cells. Importantly, these cells also acquired additional resistance to topoisomerase inhibitors, DNA-damaging agents and many other compounds. On the contrary, both 6-week as well as 3-week venetoclax treatments generated cells exquisitely sensitive to the iron chelator deferoxamine mesylate and a purine nucleoside analogue clofarabine. In addition, we generated a venetoclax-resistant MOLM-13 cell line with 1500-fold decreased sensitivity and have subjected it to the drug screening to correlate its response with the PDX model.

Summary/Conclusion: The outcomes of this research project point out the importance of novel treatment options to overcome venetoclax-resistance in AML cells. Further validations and investigation of the molecular mechanisms are planned for the top-performing drugs to strengthen the translational potential for AML therapy.

This project was supported by the grants number MUNI/A/1330/2021, MUNI/A/1419/2021 and by OPRDE (No.CZ.02.2.69/0.0/0.0/19_073/0016943).

P420: FORCING LEUKAEMIC STEM CELL TO LEAVE QUIESCENT STATE BY INHIBITING HEDGEHOG, NOTCH AND WNT/BETA-CATENIN SIGNALLING PATHWAYS

D. Láinez-González 1,* , J. Serrano-López 1 , P. Llamas-Sillero 2 , J. M. Alonso-Dominguez 2

1 Experimental Hematology Lab, Instituto de Investigación Sanitaria Fundación Jiménez Díaz; 2 Department of Hematology, Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain

Background: Acute myeloid leukaemia (AML) is a clonal neoplasm with a high relapse rate. It is thought that this relapse is caused by chemoresistance of leukaemic stem cells (LSC) due to their quiescent state. Numerous signalling pathways have been described that regulate quiescence. Key players in this setting are the Hedgehog (Hh), Notch and WNT/β-Catenin pathways. Current chemotherapeutic agents target the cell population in the proliferative phase of the cell cycle. Therefore, cells in quiescence are not affected by drugs such as cytarabine (AraC). Theoretically, forcing quiescent cells into the proliferative phase would help to eradicate the LSC population and avoid relapses.

Aims: To force quiescent LSC to enter cell cycle by inhibiting Hedgehog, Notch or/and Wnt/Beta-Catenin.

Methods: Three acute myeloid leukemia cell lines, HL60, OCI-AML3 and KASUMI1, and 13 primary samples were used to study cell cycle and cytotoxicity. They were treated during 24-48 hours with Glasdegib, Nirogacestat and PRI-724 which target Smoothened (Hedgehog), Gamma-Secretase (Notch) and Beta-Catenin (Wnt) proteins respectively. Studies of drug combination were also performed to inhibit different signaling pathways at the same time. Cytarabine was employed during 48-72 hours in cell lines or during 24-48 hours in primary samples. Cytotoxicity was analyzed by WST8 assay in cell lines and flow cytometry in primary samples by using KI67 and Annexin V. Cell cycle was study by flow cytometry using KI67 and 7AAD, and in primary samples CD45, CD34 and CD38 antibodies were used to detect LSC. Cell lines experiments were triplicated.

Results: In HL60, double inhibition during 48h of Hedgehog and Notch with 15 nM Glasdegib + 5 nM Nirogacestat reduced quiescent state in 67.27% compared with DMSO. Furthermore, this proposed treatment in combination with 109.1 nM AraC decreased 27-70% viability compared with AraC monotherapy at 72h. Similar results were obtained in OCI-AML3, in which the proposed treatments reduced quiescent phase compared with control, but no changes were observed in viability. On the other hand, in KASUMI1 there were no differences either in cell cycle or viability assay. In primary CD34+ samples, when Hedgehog and Notch were inhibited during 48h, quiescent state decreased in 33.06%, but no effects in viability were observed.

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Summary/Conclusion: The number of primary samples should be increased to shed light on the efficacy of this interesting therapeutic strategy. Nevertheless, inhibition of Hh, Notch and WNT pathways reduce the quiescent population either measuring the disease as a whole or looking at the LSC population. Differences observed between different AML cell lines regarding increase of the citotoxicity might be explained by AML genetic heterogeneity.

P421: LOWER VΓ9VΔ2 T CELLS RELATIVE FREQUENCIES PREDICT POORER SURVIVAL IN NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA

A.-C. Le Floch 1,2,* , F. Orlanducci 1,2 , A. Le Roy 1,2 , J.-F. Hamel 3 , N. Ifrah 4 , P. Cornillet-Lefebvre 5 , J. Delaunay 6 , C. Récher 7 , E. Delabesse 8 , A. Pigneux 9 , M.-C. Béné 10 , N. Vey 11 , A.-S. Chretien 1,2 , D. Olive 1,2

1 Equipe Immunité et Cancer, Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR7258, Institut Paoli-Calmettes, Aix-Marseille Université, UM105; 2 Plateforme d’immunomonitoring, Institut Paoli-Calmettes, Marseille; 3 Département de Biostatistiques, CHU d’Angers, Université d’Angers; 4 Département d’Hématologie, CHU d’Angers, Université d’Angers, Inserm, CRCINA, Angers; 5 Laboratoire d’Hématologie, CHU de Reims, Reims; 6 Département d’Hématologie, CHU de Nantes, Nantes; 7 Département d’Hématologie; 8 Laboratoire d’Hématologie, CHU de Toulouse, Institut Universitaire du Cancer de Toulouse Oncopôle, Université Toulouse III Paul Sabatier, Toulouse; 9 Département d’Hématologie et Thérapie Cellulaire, CHU de Bordeaux, Bordeaux; 10 Laboratoire d’Hématologie, CHU de Nantes, Nantes; 11 Département d’hématologie, CRCM, INSERM U1068, CNRS UMR7258, Institut Paoli-Calmettes, Aix-Marseille Université UM105, Marseille, France

Background: Major role of γδ T cells has recently emerged in anti-tumor immunity. Across several tumor subtypes, increased relative frequencies of γδ T cells had the best prognostic value in comparison to other immune subsets. In acute leukemias, patients with increased γδ T cells reconstitution after allogenic stem cell transplantation had better outcomes. The less studied putative prognostic impact of Vγ9Vδ2 T cells abundance in newly diagnosed (ND) AML relies almost on inference of transcriptomic data or post induction frequencies and has not been assessed with respect to other confounding factors.

Aims: The aim of this study was to determine the predictive and prognostic impact of blood Vγ9Vδ2 T cells frequencies at diagnosis, as measured using phenotypic analysis, taking into account biological and clinical parameters in patients treated for AML with induction.

Methods: Proportion of Vγ9Vδ2 T cells among lymphocytes has been assessed by flow or mass cytometry on PBMCS from AML patients at diagnosis, obtained from the Department of Hematology of Institut Paoli Calmettes or from the tumor bank of the FILO group. Correlations between clinical outcome, patient or disease characteristics, and Vγ9Vδ2 T cells frequencies have been analyzed. For some of the samples, we characterized immune alterations displayed by Vγ9Vδ2 T cells, or performed Vγ9Vδ2 T cell expansion and degranulation assays of autologous Vγ9Vδ2 T cells.

Results: 198 patients were included in this study. Mean age was 56 years [18.9-81.5], 79 (39.9%) and 119 (60.1%) patients had respectively a favorable or an intermediate ELN classification. Mean CR rate was 81.3%. Analysis of overall survival (OS) and relapse free survival (RFS) based on Vγ9Vδ2 T cell frequencies among lymphocytes, highlighted that the 0.8% threshold was the most significant. Vγ9Vδ2 T cells relative frequencies were considered low for values below this threshold.

By univariate analysis, patients with lower Vγ9Vδ2 T cells at diagnosis had significantly lower 5-year OS and RFS (HR = 1.54, 95%CI = 1.03-2.31, p = 0.028 and HR = 1.69, 95%CI = 1.09-2.62, p = 0.015). These results were confirmed in multivariate analysis (HR = 1.55 [1.04-2.30], p = 0.030 and HR = 1.64 [1.06, 2.53], p = 0.025). Phenotypic alterations found in patients with lower Vδ2 included a loss of some cytotoxic Vγ9Vδ2 T cell subsets and a decrease in BTN3A expression on the surface of blasts. Finally, in vitro, samples irrespective of their Vγ9Vδ2 T cells numbers did expand and displayed similar functional effects against target cells after stimulation with zoledronate or anti-BTN3A 20.1 agonist mAb.

Summary/Conclusion: This study confirmed the prognostic value of Vγ9Vδ2 T cells predominance among lymphocytes, in ND AML patients treated with induction. Vγ9Vδ2 T cells frequencies could be easily included in algorithm of treatment decision. Our findings contribute also to a strong rationale to elaborate consolidation protocols targeting enhancement of Vγ9Vδ2 T cells response, such as anti-BTN3A agonist antibodies that are under development (Marabelle et al. ESMO 2021).

P422: OMIPALISIB, A DUAL PI3K/MTOR INHIBITOR, TARGETS MITOCHONDRIA AND IMPAIRS OXIDATIVE PHOSPHORYLATION IN ACUTE MYELOID LEUKEMIA

C.-Y. Tseng 1,* , C.-Y. Kuo 1 , Y.-H. Fu 1,2 , H.-A. Hou 3 , H.-F. Tien 3 , L.-I. Lin 1

1 Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan; 2 Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, United States of America; 3 Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan

Background: Mitochondria play a central role in cell metabolism. Recent studies explored mitochondrial alterations contributing to metabolic vulnerability in myeloid leukemia cells may considered as therapeutic targets. We previously reported that omipalisib, a PI3K/mTOR dual inhibitor, could significantly inhibit the growth of myeloid leukemia cells and impair their mitochondrial respiration. However, the precise mechanism of the mitochondria dysfunction in response to omipalisib is still not fully studied.

Aims: To explore the mechanism of the mitochondria dysfunction in response to omipalisib.

Methods: OCI-AML3 and THP-1 myeloid leukemia cell lines were used in this study. Omipalisib (GSK2126458) and another PI3K/mTOR dual inhibitor gedatolisib (PF-05212384) were used. Flow cytometry was used for mitochondrial analysis. RNA-seq was performed by using an Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) between control and omipalisib (or gedatolisib) were identified by EBseq with a threshold of p ≤ 0.05. The mRNA, nuclear and mitochondrial DNA quantification were measured by using QuantStudio 3 Real-Time PCR Systems. The parameters of mitochondrial respiration were analyzed by using the XFe 24 extracellular flux analyzer.

Results: We demonstrated the anti-proliferative effect of omipalisib and gedatolisib on a panel of AML cell lines with different genetic backgrounds. OCI-AML3 cell lines had significant response to omipalisib and gedatolisib with IC 50 of 17.45 nM and 153.38 nM, respectively. Subsequently, the transcriptome analysis of 50 nM omipalisib or 200 nM gedatolisib-treated OCI-AML3 cells were analyzed. Compared to DMSO-treated, a total of 1199 (595 upregulated and 604 downregulated) and 326 (218 upregulated and 108 downregulated) DEGs were identified in the omipalisib-treated cells and the gedatolisib-treated cells, respectively. Gene set enrichment analysis (GSEA) indicated that both omipalisib and gedatolisib could suppress “E2F targets”, “Myc targets”, and “G2M checkpoint”, and trigger “TNFα signaling via NFκB”, and “Inflammatory response”. Of them, we found that omipalisib had more significant effect to inhibit “Oxidative phosphorylation” (NES: -2.01) than gedatolisib did (NES: -1.40), especially mitochondrial biogenesis and amino acid metabolism-related pathways. Omipalisib had an inhibitory effect on the mitochondrial basal respiration rate and maximal respiration. Further analysis revealed quantitative reverse-transcription PCR analysis revealed that both omipalisib and gedatolisib could potently suppress serine synthesis ( PHGDH , PSAT1 , PSPH ), glycine synthesis ( SHMT1/2 ), and tetrahydrofolate cycle-related genes ( MTHFD1/2 ) at the transcriptional level. On the other hand, rather than gedatolisib, omipalisib could significantly inhibit expression of glycolysis-related genes ( GLUT1 , HK2 , PKM2 , and LDHA ) as well as mitochondrial biogenesis-related genes ( PPARGC1B , TFAM , and NDUFS6 ). In addition, omipalisib could strongly decrease mtDNA and mitochondrial mass. These results suggest that omipalisib could suppress the mitochondrial biogenesis through alternative mechanisms, including but not limited to PI3K-AKT-mTOR signaling.

Summary/Conclusion: Rather than gedatolisib, omipalisib could significantly inhibit mitochondrial biogenesis and suppress the respiration of mitochondria in myeloid leukemia cells. Our results indicate that omipalisib could suppress cell proliferation not only through PI3K-AKT-mTOR signaling but also via impairing mitochondrial biogenesis. This information may be potentially suitable for future clinical applications.

P423: ADAPTING CRISPR CAS9 DROPOUT SCREENS TO IN VIVO PDX MODELS OF ACUTE LEUKEMIAS

R. Ludwig 1,2,* , D. Amend 1 , E. Bahrami 1 , I. Jeremias 1,2,3

1 Apoptosis in Hematopoietic Stem Cells, German Research Center for Environmental Health (HMGU), Munich; 2 German Cancer Consortium (DKTK), partner site Munich; 3 Department of Pediatrics, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany

Background: Acute leukemias require better treatment and targeted therapies represent interesting future therapeutic options. Such therapies precisely inhibit molecules with essential function, also called vulnerabilities or dependencies, so that leukemia cells die once the target is inhibited.

Aims: Here, we aimed at identifying therapeutic targets on a patient individual level and in the surrounding of a living organism. Towards this aim, we established CRISPR Cas9 dropout screens in PDX models of acute leukemias in vivo .

Methods: Primary patient acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples were transplanted into immunocompromised NSG mice and re-passaged to develop serially transplantable PDX models. To overcome limited transduction efficiency inherent to PDX leukemia cells, lentiviruses were used.

Results: We first elaborated on the maximum library size to be used in PDX models in vivo . As only a limited number of PDX cells home into mice, library size is limited to ensure the coverage of the library. We used, screening sgRNAs as genetic barcodes in Cas9 negative samples to determine the maximum library size upon next generation sequencing (NGS); a regular distribution of all sgRNAs recovered from mice at the end of the experiments indicated a suitable library size. We found that PDX ALL models tolerated a larger library compared to PDX AML models that might reflect lower intra-sample heterogeneity and higher leukemia stem cell frequency in ALL compared to AML.

To perform knockout screens, a split-construct for Cas9 was used to reduce plasmid size and optimize lentiviral transduction efficiency into PDX cells; concomitantly split-GFP was used to enrich cells expressing both Cas9 split plasmids by flow cytometry.

A customized library consisting of 146 target genes combined with positive and negative control genes was designed at 5 sgRNAs per gene using the CLUE platform ( www.crispr-clue.de ) (Becker et al., Nucleic Acids Res. 2020) and cloned into a lentiviral vector backbone. The sgRNA library vectors include either a puromycin-resistance cassette or an H-2Kk surface marker, each combined with a BFP fluorescent marker. This allows to determine transduction efficiency by FACS-based detection of BFP and to enrich the transduced PDX cells either by puromycin selection or by H-2Kk-MACS selection. Input samples were collected after library transduction and enrichment to >90% CRISPR/Cas9 sgRNA library positive cells, while remaining cells were injected into immunocompromised NSG mice, grown in vivo and re-isolated at advanced disease stage. Analysis of input and endpoint samples using NGS enabled to determine the frequency of sgRNAs and bioinformatically compare the abundance of different sgRNAs between the control and the samples of interest using MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout). A significant sgRNA dropout was characterized by a p-value below 0.05 and a FDR below 0.1.

The screening experiments led to the top 10 depleted dropout genes for ALL and AML PDX samples.

Besides individual dropouts of the different samples, also shared dropouts were detected. Especially sample overlapping dropouts might represent interesting starting points for new therapeutic options.

Summary/Conclusion: In summary, we have established a CRISPR Cas9 screening pipeline, which allows investigating therapeutic targets on a patient-individual level in ALL PDX models and, for the first time, in AML PDX models in vivo .

P424: INVESTIGATING GPR56 FUNCTION IN HUMAN IPSC-DERIVED HEMATOPOIETIC CELLS USING A TET-ON MODEL OF CONSTITUTIVE ACTIVATION

L. Mackintosh 1,* , A. Maglitto 1 , C. Rodriguez Seoane 1 , S. A. Mariani 1 , A. G. Rossi 1 , E. Dzierzak 1

1 Centre For Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom

Background: GPR56 (ADGRG1) is a widely expressed adhesion G-coupled protein receptor in humans and mice which is implicated to play a role in both haematopoietic development and Acute Myeloid Leukaemia (AML). In AML, there exists a low frequency subpopulation of leukaemic stem cells (LSCs) which, similar to their benign Hematopoietic Stem Cell (HSC) counterparts, exhibit stem cell behaviours. High expression of GPR56 forms part of the molecular signature of these LSCs, and AML cases with high levels of GPR56 at diagnosis have poorer outcomes. Our hypothesis is that GPR56 activity underlies some of the stem cell behaviours seen in both HSCs and LSCs.

Aims: To characterise the effects of GPR56 receptor activation so as to better understand its role in AML and hematopoietic development.

Methods: We developed a Tet-On human inducible stem cell line (M1) transfected with a construct encoding a truncated GPR56 protein. This constitutively active GPR56 receptor form is overexpressed following doxycycline administration. Flow cytometry-based cell cycle and apoptosis studies were performed on the M1 line and wild type Sci55 (WT) iPSCs +/- doxycycline exposure. IncuCyte® transmigration studies utilising an SDF-α gradient were performed to examine cell migration. Finally, the M1 cells were subjected to a hematopoietic differentiation protocol and assessed for CD34 + CD38 - compartment size at day 12 of differentiation.

Results: GPR56 activation alters cell cycle progression with significant accumulation of M1 cells in the G0/1 and G2/M phases of the cell cycle compared to WT (p <0.005). GPR56 activation also causes a significant reduction in apoptotic cells in dox-treated M1 cells (p <0.005), despite doxycycline being pro-apoptotic in WT cells (p <0.05). GPR56 activation reduces the number of transmigrating M1 cells in response to an SDF-α gradient (p <0.05). Importantly, GPR56 activation during differentiation increases the CD34 + CD38 - compartment size in M1 cells treated with doxycycline at day 12 (p<0.05) as compared to untreated cells

Summary/Conclusion: GPR56 activation results in accumulation of undifferentiated M1 iPSCs in the G0/1 and G2/M phases, suggestive of cell cycle checkpoint arrest, and exerts a powerful anti-apoptotic effect. GPR56 activation also reduces cell transmigration in response to SDF-α, suggesting receptor involvement in cell adhesion and migration. Finally, GPR56 activation during hematopoietic differentiation results in an increased yield of CD34 + CD38 - cells, suggesting that GPR56 activity may influence the proportion of immature cells arising during haematopoietic differentiation. These results are of interest in relation to normal HSC functions and may indicate the mechanisms through which overexpression of GPR56 (with increased activation) in LSCs contributes to poorer outcomes in AML. Future work will include identification and characterisation of the signalling pathways downstream of the GPR56 receptor to better understand this receptor’s role in the haematopoietic system in health and disease.

P425: ROLE OF LYSOSOMAL-ASSOCIATED GENE LAPMT4B IN LEUKEMIA

J. Delgado-Martínez 1,2,* , J. M. Cornet-Masana 1 , L. Cuesta-Casanovas 1,3 , J. M. Carbó 1,4 , L. Clément-Démange 4 , R. M. Risueño 1

1 Josep Carreras Leukaemia Research Institute; 2 Faculty of Pharmacy, University of Barcelona; 3 Faculty of Bioscience, Autonomous University of Barcelona; 4 Leukos Biotech, Leukos Biotech, BARCELONA, Spain

Background: Despite the efforts to find druggable targets in acute myeloid leukemia (AML), successful therapeutic approaches are needed in this disease. Due to the particular characteristics of lysosomes in cancer, the clinical relevance of key lysosomal genes’ expression was searched in public repositories. LAPTM4B gene expression was found to be directly correlated with a worse clinical outcome in AML.

Aims: To decipher the role of the lysosome-associated gene LAPTM4B in AML will be the main objective of this study.

Methods: Generation of AML cells overexpressing both LAPTM4B isoforms (35kDa -long- and 24kDa -short) were obtained by lentiviral transduction. Sensitivity to chemoresistance was measured by flow cytometry. To determine the role of LAPTM4B in the leukemia regeneration capacity, conditioned NSG mice were transplanted with both LAPTM4B isoforms-expressing AML cells and the engraftment was analysed by flow cytometry. Activation of key secondary messengers was measured by luciferase response elements and Western Blot.

Results: LAPTM4B overexpression directly correlated with a higher resistance to cytarabine. LAPTM4B short isoform presented an increased clonogenic potential versus the long isoform. Interestingly, a competitive bone marrow transplantation assay confirmed this effect in terms of in vivo engraftment capacity. Moreover, LAPTM4B-mediated activation was able to induce MAPK signalling in the leukemic cell context.

Summary/Conclusion: LAPTM4B overexpression not only increases the regeneration potential but also confers resistance to cytarabine, suggesting an effect in the maintenance of leukemia. In summary, this study will increase the knowledge of the role of lysosomes in leukemogenesis, and additionally, it will help to identify new lysosome-related biomarkers.

P426: A SINGLE-CENTER RETROSPECTIVE ANALYISIS OF THE MUTATIONAL PROFILE IN ACUTE MYELOID LEUKEMIA PATIENTS

Y. Martínez Díez 1,* , A. Franganillo Suárez 1 , J. Cornago 1 , L. Solan 2 , R. Ayala 3 , J. Martinez 3 , P. Llamas 1 , J. L. Lopez Lorenzo 1

1 Hematology; 2 Hospital Fundación Jiménez Díaz; 3 Hematology, Hospital 12 de Octubre, Madrid, Spain

Background: Acute myeloid leukemia (AML) is a clonal disorder originating from specific genetic aberrations in myeloid precursor cells. Next generation sequencing (NGS) analyses provide clinicians with a deeper knowledge of AML pathogenesis and is being implemented as a routine test at diagnosis due to its relevance in management and prognosis, following the characterization of molecular subgroups.

Here we provide a summary of our experience in a single centre (Fundación Jiménez Díaz University Hospital (FJDH), Madrid, Spain), describing the molecular profile of our AML patients, studied as part of a national Spanish project lead by the PETHEMA group, and an analysis on which alterations could be significant to overall survival (OS).

Aims: Analyse prognosis associated to molecular profile of new cases of AML

Methods: AML new diagnoses and relapses in FJDH from 2018 to 2020 were retrospectively evaluated. The samples were referred to a central laboratory (12 de Octubre University Hospital, Madrid, Spain) where NGS custom panels were run. Other significant variables were analysed.

Results: 66 AML patients were evaluated with a mean age of 60yo (22-93). 191 mutations were detected and most NGS were run at diagnosis 57 (86.4%). Most of the sample received an intensive treatment 42 (63.6%), however only 25 (37.9%) patients achieved CR, 2 (3%) CRi, 6 (9.1%) +MRD, 4 (6.1%) PR and 26 (39.4%) were refractory. 11 (16.7%) patients relapsed. Median OS was 13 months (CI 95% 7.9 – 18.1). Survival analysis confirmed significant differences between the 3 ELN prognostic groups and some of the genetic alteration (Figure 1).

In this series there was high incidence of secondary and adverse prognosis AML, as expected from the high expression of ASXL1 (12.1%) and TP53 (15.2%) mutations. DNMT3A mutations were most frequent (25.8%), followed by NPM1 (22.8%). We also found IDH mutations (28.8%), which though less common could also be expected in adverse prognosis AML. Our sample size was big enough that prognostic differences within each ELN classification group were observed, as well as related to treatment. There was statistical significance in OS between patients with wildtype vs mutated TP53 (8.9 vs 22.8; p=0.037) or JAK2 (3.8 vs 22.5, p=0.001), as supported by literature. Interestingly, we also found DNMT3A mutated AML had a worse OS (24.1 vs 12.7; p=0.023). This may support recommending HSCT in DNMT3A -mutated AML, as argued for TP53 - and JAK2 -mutated AML.

Although our OS graphs inclined towards favourable prognosis in the presence of NPM1 mutations (27.9 vs 19.9; p=0.12) and adverse prognosis with ASXL1 (11.1 vs 22.5; p=0.055), the statistical analysis did not show significance in either case, probably due to small sample size. RUNX1 did not seem to play a role in prognosis (19.1 vs 20.4; p= 0.741) which might be because most patients underwent HSCT, although current studies seem to suggest RUNX1 mutations are independent to prognosis. Similarly, FLT3-ITD mutations did not confer worse outcomes (25.5 vs 20.1; p=0.464). This may be due to the mutation often being absent in disease relapse or progression, sometimes in relation to targeted therapies. In our sample IDH mutations did not confer changes to OS (18.2 vs 21.9; p=0.707), though this could be explained by the limited availability of targeted therapies in our country.

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Summary/Conclusion: We found high incidence of high risk AML. Most of our data agree with the published literature. Of note, RUNX1 doesn’t seem to play an important role in prognosis. However, DNMT3A mutations alone and mainly as a group with TET2 and ASXL1 were related to a bad outcome.

P427: THE SINGLE CELL T CELL LANDSCAPE OF AML PATIENTS POST ALLOGENEIC STEM CELL TRANSPLANTATION

A. Mathioudaki 1,2,* , X. Wang 3 , R. Huth 3 , J. Zaugg 4 , C. Pabst 3,4

1 Molecular Medicine Partnership Unit, European Molecular Biology Laboratory (EMBL); 2 Faculty of Biosciences, Heidelberg University; 3 Department of Medicine V, Hematology, Oncology and Rheumatology, University Hospital Heidelberg; 4 Molecular Medicine Partnership Unit, EMBL Heidelberg, Heidelberg, Germany

Background: In Acute myeloid leukemia (AML), chemotherapy may lead to long-term remission, but allogeneic stem cell transplantation (alloSCT) often remains the only curative therapeutic approach, particularly in AML with high-risk genetics. However, not every patient responds to alloSCT. Several hypotheses have been associated with therapy failure, such as the incapability of T cells to recognize and eliminate residual leukemia stem cells (LSCs), which thus escape the graft-versus-leukemia (GVL) effect. However, the role of the T cells’ repertoire in therapy outcome still remains unclear.

Aims: Here, we investigated the impact of the bone marrow (BM) T cell composition on the therapy outcome of AML patients after alloSCT.

Methods: We performed single-cell RNA sequencing (scRNA-seq) of T cells and hematopoietic stem and progenitor cells (HSPCs) isolated from BM aspirates 100 days post alloSCT of three patients in complete remission (CR) versus three suffering relapse (REL). We investigated the prognostic impact of putative T-cell biomarkers in therapy outcome, using flow cytometry analysis on an independent cohort.

Results: Using scRNA-seq, we identified 21 T cell and 9 HSPC populations and observed differences in T cell population abundances between REL and CR. In particular, we observed an enrichment of specific CD8+ T cell subsets in the CR group, while certain CD4+ T cell subsets, such as regulatory T cells were enriched in REL patients. Furthermore, we observed an increased T cell cytotoxicity signature in the CR group, that potentially contributes to better clinical outcome. Within the genes upregulated in the CR condition, we identified surface molecules linked to T cell cytotoxicity. Subsequent flow cytometry analysis corroborated a link between these markers and better clinical outcome. Our data suggest that the early bone marrow T cell signature post alloSCT might reflect GVL-effects required to achieve long-term survival.

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Summary/Conclusion: Understanding the role of T cell composition on therapy response may offer novel approaches for both predicting prognosis as well as developing new therapeutic strategies.

P428: SYK INHIBITION DRIVES DEEP RESPONSES IN A BIOMARKER GUIDED SUBSET OF AML ALONE AND IN RATIONAL COMBINATIONS

M. McKeown 1,* , L. A. Carvajal 1 , M. A. Day 1 , C. Noe 1 , P. Kumar 1 , J. DiMartino 1 , D. Saffran 1

1 Kronos Bio, Inc., San Mateo, United States of America

Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1.

The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1 -driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY ( NCT05020665 ), in combination with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1 -mutated ( NPM1 m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3 -mutated AML ( NCT05028751 ).

Aims: To assess the potential of biomarker-guided responses to ENTO and LANRA in mutationally defined subsets of AML and its activity in combination with other targeted agents in translationally relevant models.

Methods: Full kinome profiling was run at fixed 1uM concentration followed by dose response IC50 determination for top hits. AML and lymphoma cell lines were tested for antiproliferative effects using CellTiter Glo (CTG) at 5 days. AML patient-derived ex vivo models were tested in microtiter plate viability assays with CTG or in methylcellulose colony viability assays for the NPM1 m/ FLT3 -ITD model used in combination studies.

Results: Kinase selectivity profiling found >10-fold higher selectivity of ENTO and LANRA for SYK vs other kinases with improved potency and selectivity compared with the approved first-generation agent, fostamatinib. SYK inhibition with either agent showed robust anti-proliferative activity in a panel of AML and lymphoma cell lines with varying mutational backgrounds. In addition, synergy was observed when ENTO or LANRA was combined with a Menin inhibitor in MLL r, FLT3 -ITD cell lines. LANRA and ENTO showed strong anti-proliferative activity in NPM1 m AML patient samples validating NPM1 m as a patient selection biomarker for ENTO and LANRA. In an analysis of a large public dataset of ex vivo patient samples, the presence of NPM1 m and/or FLT3 -ITD was significantly predictive of response to ENTO. In an ex vivo model of NPM1 m/ FLT3 -ITD AML, consistent sub-micromolar response to LANRA was observed. Combinations with AML standard-of-care and investigational agents, including azacytidine, showed at least additive effects. Strong synergy with the JAK inhibitor ruxolitinib was consistent mechanistically with a reporter model that demonstrated the ability of LANRA to block activation of STAT5 in response to proleukemic paracrine signaling. Finally, robust synergy across a range of LANRA concentrations was found when paired with venetoclax and gilteritinib. These results prompted further testing with an in vivo, patient-derived xenograft.

Summary/Conclusion: SYK is a promising therapeutic target for subsets of AML patients defined by specific mutations. The highly predictive response for SYK inhibition with concurrent NPM1 m/ FLT3 -ITD mutations and synergy with gilteritinib observed in patient derived models is supportive of its clinical evaluation in combination with a FLT3 inhibitor. Our work supports the rationale for an ongoing Phase 1b/2 study of LANRA in combination with gilteritinib in patients with R/R FLT3 -mutated AML.

P429: CLONALLY RESOLVED SINGLE-CELL MULTI-OMICS IDENTIFIES LEUKEMIA SURFACE ANTIGENS

A. K. Merbach 1,* , S. Beneyto-Calabuig 2 , J.-A. Kniffka 1 , C. Szu-Tu 2 , C. Rohde 1 , M. Antes 1 , M. Janssen 1 , A. Waclawiczek 3 , J. J. M. Landry 4 , V. Benes 4 , J. Anna 5 , M. Brough 5 , B. Besenbeck 1 , J. Felden 1 , S. Bäumer 6 , M. Hundemer 1 , T. Sauer 1 , C. Pabst 1 , M. Scherer 2 , S. Raffel 1 , L. Velten 2 , C. Müller-Tidow 1

1 Department of Medicine, Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany; 2 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain; 3 Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ) and DKFZ-ZMBH Alliance; 4 Genomics Core Facility, European Molecular Biology Laboratory (EMBL); 5 Institute of Human Genetics, University Hospital Heidelberg, Heidelberg; 6 Department of Medicine A, Hematology and Oncology, University Hospital Muenster, Münster, Germany

Background: At the time of diagnosis, AML is a highly heterogeneous disease, usually consisting of various epigenetically and genetically distinct pre-leukemic, as well as leukemic subclones of various maturation stages that coexist in a competitive, or even cooperative manner. This clonal heterogeneity and, in particular, the strong resemblance of leukemic cells with their healthy counterparts presents a significant challenge in the identification of cancer specific gene expression and surface markers, both prerequisites for the development of targeted therapies. Existing approaches to add clonal resolution to droplet-based single-cell RNAseq data use qualitative information available from calls of individual mitochondrial or genomic single nucleotide variants as well as copy number variants. These single cell mutational calls, however, cannot be taken at face value, but need to be analysed through appropriate statistical methods, which are currently missing.

Aims: Here we aimed at developing a novel method, CloneTracer, to add clonal resolution to new or existing human single-cell transcriptomic or multiomic datasets to ultimately allow identification of leukemia surface antigens through intra-sample comparison of cancerous and healthy cells of matched differentiation state.

Methods: CloneTracer amplifies mitochondrial and selected nuclear SNVs, infers copy number variation, and estimates clonal hierarchies and identities through a Bayesian model that accounts for the noise properties of single-cell RNA-seq data.

Results: Through combination of clonal identities with information on surface markers at single cell resolution, CloneTracer identified novel leukemia markers that enabled quantification of the leukemic burden in clinical specimens and enrichment for leukemic or healthy cells by FACS.

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Summary/Conclusion: In summary, our data illustrate the benefits of clonally resolved single cell multi-omics for identifying cancer specific surface antigens and potential druggable targets by intra-patient comparisons.

P430: MOLECULAR CHARACTERIZATION OF CLINICAL RESPONSE IN NEWLY-DIAGNOSED ACUTE MYELOID LEUKEMIA PATIENTS TREATED WITH IVOSIDENIB + AZACITIDINE COMPARED TO PLACEBO + AZACITIDINE

S. de Botton 1,* , S. Choe 2 , D. Marchione 2 , P. Montesinos 3 , C. Recher 4 , S. Vives Polo 5 , E. Zarzycka 6 , J. Wang 7 , G. Bertani 8 , M. Heuser 9 , R. Calado 10 , A. Schuh 11 , S.-P. Yeh 12 , J. Hui 2 , S. Pandya 2 , D. Gianolio 2 , S. Daigle 2 , C. DiNardo 13 , H. Dohner 14

1 Institut Gustave Roussy, Villejuif, France; 2 Servier Pharmaceuticals LLC, Boston, United States of America; 3 Hospital Universitari i Politècnic La Fe, València, Spain; 4 Institut Universitaire du Cancer de Toulouse Oncopole, Toulouse, France; 5 Hospital Universitario Germans Trias i Pujol-ICO Badalona, Josep Carreras Research Institute, Badalona, Spain; 6 Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland; 7 Institute of Hematology & Hospital of Blood Disease, Tianjin, China; 8 ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 9 Hannover Medical School, Hannover, Germany; 10 Ribeirão Preto School of Medicine, University of São Paulo, São Paulo, Brazil; 11 Princess Margaret Cancer Centre, Toronto, Canada; 12 China Medical University, Taichung, Taiwan; 13 The University of Texas M.D. Anderson Cancer Center, Houston, United States of America; 14 Ulm University Hospital, Ulm, Germany

Background: Acute myeloid leukemia (AML) is a disease with a dynamic mutational landscape; 6–10% of patients (pts) have somatic mutations in isocitrate dehydrogenase 1 ( IDH1 ), which can drive oncogenesis. Ivosidenib (IVO) is a potent oral targeted inhibitor of mutant IDH1 (mIDH1). IVO 500 mg QD + azacitidine (AZA) 75 mg/m 2 SC or IV for 7 days in 28-day cycles was shown to significantly improve event-free survival (HR=0.33 [95% CI 0.16, 0.69], p=0.0011), median overall survival (24.0 vs 7.9 months), and complete remission + partial hematologic recovery rates (CR/CRh; 52.8% vs 17.6%) vs placebo + AZA in the double-blind phase 3 AGILE study ( NCT03173248 ) in pts with newly diagnosed IDH1 -mutated AML (ND-AML).

Aims: To assess the impact of IVO+AZA on IDH1 -mutation clearance ( IDH1 -MC) and baseline co-mutation analysis from AGILE.

Methods: Genomic DNA from bone marrow mononuclear cells (BMMCs) or peripheral blood mononuclear cells (PBMCs), and/or bone marrow aspirate (BMA) were used for molecular studies. IDH1 -MC analysis on BMMCs was performed by BEAMing digital PCR (limit of detection 0.02%-0.04%). BMA, BMMCs and PBMCs were utilized for co-mutational analysis by next-generation sequencing, ACE Extended Cancer Panel (detection limit 2%). All patients gave written informed consent.

Results: 146 pts were randomized: 72 to IVO+AZA; 74 to placebo+AZA. Median (range) baseline m IDH1 variant allele frequency in BMMCs was 36.7% (3.1–50.5%) in the IVO+AZA arm and 35.5% (3.0–48.6%) in the placebo+AZA arm. Updated IDH1-MC data (October 2021) from 47 IVO+AZA and 32 placebo+AZA treated pts with at least 1 on-treatment sample demonstrated IDH1 -MC in 21/35 (60%) IVO+AZA pts achieving CR/CRh vs 4/11 (36%) placebo+AZA pts. In CR/CRh pts with time points available after IDH1 -MC, suppression of the m IDH1 was durable and IDH1 -MC maintained in all subsequent samples in 17/17 (100%) IVO+AZA treated pts and 1/3 (33%) placebo+AZA pts. Further analysis of baseline co-mutations on 120 pts (IVO+AZA: n=58; placebo+AZA: n=62) showed that DNMT3A , SRSF2 , and RUNX1 were the most frequent in both treatment arms. Importantly, comparison of CR/CRh and non CR/CRh responses by cohort did not identify any single gene or pathway associated with an inferior outcome in IVO+AZA pts compared to placebo+AZA pts (p<0.05, Fisher’s Exact test). Several genes ( DNMT3A , RUNX1 , SRSF2 , STAG2 ) and pathways (Differentiation, Epigenetics, Splicing) were associated with improved outcomes with IVO+AZA, including the RTK pathway, which was previously reported to be associated with primary resistance to IVO monotherapy. Further analysis of patient subgroups, including R132 variants (i.e., R132C vs R132S), will be presented.

Summary/Conclusion: These data suggest that improved clinical outcomes with IVO+AZA are associated with sustained clearance of the m IDH1 clone including pts with disease that harbor mutations implicated in resistance to IVO monotherapy (e.g., with RTK pathway mutations).

P431: THE ROLE OF SMARCA4 IN HEMATOPOIESIS AND ACUTE MYELOID LEUKEMIA

F. Modemann 1,2,* , L. Ramke 1 , J. Muschhammer 1 , L. Behrmann 1 , V. Thaden 3 , M. Schoof 3,4 , C. Göbel 3 , S. Neyazi 3,4 , C. Bokemeyer 1 , J. Wellbrock 1 , U. Schüller 3,4,5 , W. Fiedler 1

1 Department of Oncology and Hematology, University Medical Center Hamburg-Eppendorf; 2 Mildred Scheel Cancer Career Center, University Medical Center Hamburg-Eppendorf; 3 Research Institute Children’s Cancer Center Hamburg; 4 Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf; 5 Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Background: SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a) is the central ATPase containing enzyme in the SWI/SNF- (switch/sucrose non-fermentable) complex, which influences gene transcription by regulating chromatin accessibility. In solid tumors, SMARCA4 mainly possesses tumor suppressive features. However, in the setting of acute myeloid leukemia (AML), SMARCA4 seems to play a different role, acting as a proliferation stimulus for myeloid blasts.

Aims: The aim was to investigate the role of SMARCA4 in normal hematopoiesis and in pathogenesis and maintenance of AML to evaluate the prognostic and potential therapeutic relevance of this protein.

Methods: We screened 152 bone marrow samples of newly diagnosed adult AML patients compared to 29 healthy bone marrow samples for SMARCA4 expression by using RT-qPCR. SMARCA4 expression was correlated to overall and relapse free survival. We further analyzed proliferation and colony-formation capacity of AML cell lines with lentivirally transduced shRNA-mediated SMARCA4 knockdown or SMARCA4 overexpression induced by lentiviral transduction. Two mouse models were generated: Firstly, to evaluate the impact of SMARCA4 knockout on normal hematopoiesis, a transgenic mouse model with Cre-recombinase mediated conditional knockout of SMARCA4 under the control of an Mx-1 promoter was developed. Blood counts, bone marrow smear, colony formation assays, and RNA expression profiles were analyzed. Secondly, a xenograft mouse model was generated by transplantating the AML cell line MV4-11 with SMARCA4 knockdown or overexpression in NSG mice to analyze overall survival.

Results: Expression of SMARCA4 was significantly higher in adult AML samples compared to healthy CD34+ cells (p<0.0001). High SMARCA4 expression was associated with worse overall (p=0.001) and relapse free survival (p=0.0073). SMARCA4 knockdown in AML cell lines resulted in significantly lower proliferation rate in three different AML cell lines compared to controls, whereas SMARCA4 overexpression led to higher proliferation rates. Overall survival of mice transplanted with SMARCA4 overexpressing MV4-11 cells was significantly worse than in the control group, whereas mice transplanted with SMARCA4 knockdown MV4-11 cells lived significantly longer.

Transgenic SMARCA4 knockout mice showed significantly lower leukocyte, reticulocyte, and thrombocyte counts and showed lower colony forming capacity compared to Cre-negative control mice. Bone marrow smears showed aplasia of all cell lines in SMARCA4 knockout mice. RNA-sequencing revealed 749 upregulated and 520 downregulated genes with a log fold change ≥ +/- 1 in the bone marrow of SMARCA4 knockout mice compared to Cre-negative control mice. Relevant pathways according to REACTOME data base performing over representation analysis included genes of generation of second messenger molecules, cell surface interactions at the vascular wall, and immunoregulatory interactions between lymphoid and non-lymphoid cells.

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Summary/Conclusion: SMARCA4 is upregulated in primary AML cells and is an unfavorable prognostic marker in AML patients. SMARCA4 overexpression in AML cell lines leads to higher proliferation rate and to a worse overall survival in transplanted mice. In normal hematopoiesis, SMARCA4 knockout leads to decreased cell growth in bone marrow, highlighting the dependence on SMARCA4 for proliferation of hematopoietic (stem) cells.

P432: RISK STRATIFICATION IN ACUTE MYELOID LEUKEMIA: EXTERNAL VALIDATION OF THE STELLAE-123 GENE EXPRESSION MODEL AND COMPARISON WITH CURRENT PROGNOSTIC SCORES

A. Mosquera Orgueira 1 , A. Peleteiro Raindo 1,* , J. A. Diaz Arias 1 , M. Cid Lopez 1 , R. Abal Garcia 1 , B. Antelo Rodriguez 1 , M. S. Gonzalez Perez 1 , J. C. Vallejo Llamas 1 , M. M. Perez Encinas 1

1 Hematology, CHUS, Santiago de Compostela, Spain

Background: The identification of recurrently mutated genes and cytogenetic anomalies has provided high prognostic and therapeutic significance in patients diagnosed with AML. Currently, some of this information has been incorporated into risk stratification guidelines, such as those of the European Leukemia Net (ELN). However, these guidelines are based on cytogenetic analyses and a limited number of mutations, and they don’t consider the genomic complexity of AML. Recently, we presented a new prognostic score based on gene expression analysis (Stellae-123) which achieved high discriminative power in survival prediction of AML patients, particularly among those bearing high-risk mutations. It is necessary to evaluate this signature in new AML cohorts and to test its performance compared with standard risk stratification scores.

Aims: To validate the prognostic precision and discriminative power of the Stellae-123 gene expression signature in external cohorts and to compare its performance with standard prognostic scores: ELN-2017 in adult patients and clinical risk score in pediatric patients.

Methods: RNA-seq data from 2 adult cohorts and one pediatric AML cohort was retrieved. The BeatAML (N=334) and the AMLCG-2008 (N=199) cohorts consisted of adult AML patients, whereas the TARGET AML cohort (N=144) was composed solely of pediatric patients. Gene expression estimates (FPKM) were normalized between cohorts using ComBat. Common genes were retrieved, and we selected those included in the Stellae-123 predictor, reaching a total of 69 genes. Random forests were built to predict survival in the largest cohort (BeatAML). The precision of the predictors was evaluated using cross-validated time-dependent AUCs derived from cox models. In the particular case of BeatAML, gene expression survival models were based on out-of-bag predictions in order to reduce the risk of overfitting during the training phase of the model.

Results: We initially built a survival predictor using random forests based on the expression of the 69 genes incorporated in Stellae-123. This predictor was trained in the BeatAML cohort, and external validation was performed on the remaining two cohorts. The model achieved c-indexes of 0.635, 0.645 and 0.598 in the BeatAML, AMLCG-2008 and TARGEL AML cohorts, respectively. We then evaluated the precision of this signature to predict survival at 6 months, 1 year and 2 years after diagnosis using cross-validated cox models. The results indicated that this model achieved greater precision than the ELN-2017 and the pediatric clinical risk score in the majority of evaluated time points (Figure 1).

Since age is a variable deeply associated with survival in AML, we evaluated the performance of the models including this covariate. We observed clear improvements in AUCs for the BeatAML and TargetAML, but not for the AMLCG-2008 cohort, a finding which is probably related to the fact that these were fit adult patients recruited in a clinical trial. Notably, we also observed an improved performance of the gene expression signature plus age model compared with the ELN-2017 and pediatric clinical risk scores plus age models, particularly in the prediction of survival within the first year after diagnosis.

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Summary/Conclusion: The Stellae-123 gene expression signature can predict overall survival in AML with greater precision than the ELN-2017 and the pediatric clinical risk score. Therefore, gene expression profiling emerges as a powerful tool to optimize patient risk stratification. There is a growing need to standardize these tests for clinical use.

P433: ADAPTER HOLIDAYS MAINTAIN IN-VITRO ADCAR-T CELL FUNCTIONALITY AGAINST AML

D. Nixdorf 1,2,* , M. Sponheimer 1,2 , D. Berghammer 2 , N. Zieger 1,2 , L. Rohrbacher 1,2 , S. Dapa 3 , C. M. Seitz 4 , K. Brandstetter 5 , M. von Bergwelt-Baildon 1 , H. Leonhardt 5 , J. Mittelstaet 6 , A. Kaiser 3 , V. Bücklein 1,2 , M. Subklewe 1,2

1 Department of Medicine III; 2 Laboratory for Translational Cancer Immunology, LMU, Munich; 3 R&D Department, Miltenyi Biotec GmbH, Bergisch Gladbach; 4 Department of General Pediatrics, University Children’s Hospital Tuebingen, Tuebingen; 5 Department of Biology II, LMU, Munich; 6 R&D Department, Miltenyi Biotech GmbH, Bergisch Gladbach, Germany

Background: The rise of immunotherapies has sparked hope for improving the treatment options of acute myeloid leukemia (AML). However, bispecific antibodies (AB) and CAR-T cells demonstrated only moderate efficacy in clinical trials. The heterogeneous nature of the disease requires more flexible, potent and safer approaches to make T-cell based treatments available for AML patients. We have recently developed a novel, highly modular adapter CAR-T cell (AdCAR) platform capable of targeting multiple antigens via biotin-labeled adapter molecules (AMs) in the context of a specific linker (Seitz et al., 2021).

Aims: Utilizing an AdCAR-T cell approach to overcome well described obstacles in AML we evaluated several key parameters of our platform, including cytotoxicity, AM internalization and serial targeting capability. Furthermore, we highlight the possibility of intermittent (int.) AM dosing regimens (AM holidays) to delay AdCAR-T cell dysfunction due to continuous (cont.) stimulation.

Methods: The cytotoxic capacity of AdCAR-T against AML cell lines and primary AML (pAML) samples was evaluated in co-culture assays utilizing Fab-based AMs. Specific lysis was assessed by FC after 48-72 h. Internalization kinetics of AMs were assessed via surface staining of AML cells with AMs and secondary staining with anti-Biotin-PE AB. The % surface bound AMs over time at 37 °C vs 4 °C was analyzed. For serial targeting experiments the AMs were either added once, replenished every 3 days, or changed to AMs with different target specificity. To mimic AM holidays in vitro , cont. vs int. AM-exposed AdCAR-T were co-cultured with OCI-AML-3 (21 d vs 7 d AM exposure, followed by 7 d AM absence). Cytotoxic capacity, T cell proliferation and cytokine secretion were assessed.

Results: AdCAR-T cells elicited high cytotoxicity against several AML cell lines utilizing AMs of different specificity (AMs for CD33, CD123, CLL-1; IC 50 = 2.1, 0.6, 1.9 ng/ml). Against pAML similar data were obtained in an E:T and AM-concentration dependent manner. We demonstrated feasibility of serial AM usage by performing long-term pAML/AdCAR-T co-cultures. In comparison to one-time or low-dose cont. CD33 AM exposure, which led to an outgrowth of pAML cells after 12 d, increasing AM concentrations and/or switching the target antigen improved AdCAR-T cell efficacy. We further observed receptor-mediated endocytosis of AMs (reduction of MV4-11 surface bound CD33 AM after 6 h = 81%) and postulate that AM internalization is a common form of antigen sink in AM-based T-cell recruiting therapies. Internalization of AMs reduces AM half-life and decreases cytotoxic capacity of AdCAR-T cells. While limited AM levels can be overcome by repetitive dosing, AM internalization potentially also contributes to faster off-switch dynamics. Importantly, we observed that cont. AdCAR-T cell stimulation leads to T cell dysfunction (Fig 1) in in vitro long-term efficacy studies. Strikingly, these effects can be mitigated by implementing int. AM dosing regimens, shown by significantly increased T cell proliferation (fold change d14: 1.9 vs 3.5), cytotoxic capacity (% specific lysis d14: 43 vs 64) and IL-2 production (202 vs 823 pg/ml; all mean+/-SEM).

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Summary/Conclusion: Taken together, AdCAR-T cells provide flexibility in target antigen choice, safety through short half-life of the AMs and long-term functionality by mitigating AdCAR-T cell dysfunction through AM holidays. Future trials need to consider the high risk of CAR-T cell exhaustion upon cont. stimulation and AdCAR-T cells might pave the way for rational design of more sophisticated treatment schedules.

P434: MITOCONDRIAL MCL1 REGULATES LEUKEMIC CELLS METABOLISM VIA DIRECT INTERACTION WITH HEXOKINASE II. METABOLIC SIGNATURE AT ONSET PREDICTS OVERALL SURVIVAL IN AMLS’ PATIENTS

N. I. Noguera 1,2,* , G. Catalano 1,2 , A. Zaza 1,2 , C. Banella 3 , T. Ottone 2,4 , E. Pelosi 5 , S. Travaglini 1,2 , M. Divona 6 , M. I. Del Principe 1 , F. Buccisano 1 , L. Maurillo 1 , E. Amatuna 7 , U. Testa 5 , A. Venditti 1 , M. T. Voso 1,2

1 Tor Vergata University; 2 Fondazione Santa Lucia, Rome; 3 Meyer Children’s University Hospital, Florence; 4 Tor Vergata University, Roma; 5 Istituto Superiore di Sanità; 6 Policlinico Tor Vergata, Rome, Italy; 7 University Medical Center Groningen, Groningen, Netherlands

Background: Metabolic reprogramming is a basic feature in cancer. Leukemia associated cellular reprogramming, metabolic heterogeneity at onset and metabolic clonal evolution, driven by therapy, are essential insights that still remain unclear

Aims: We compared Acute Myeloid Leukemias’ (AMLs) cells metabolism to hematopoietic progenitors’’ and normal maturing bone marrow cells’, to acquire useful prognostic information and to uncover actionable therapeutic targets.

Methods: Methods We analyzed fresh primary blast from 19 AML patients, hematopoietic progenitors obtained from normal CD34+ cells differentiated to promyelocytes and granulocyte and the MV4-11, OCI-AML2 and OCI-AML3 cell lines, using a Seahorse Bioscience XFe96 analyzer. We silence MCL1 by siRNA and evaluated the interaction between HK2, MCL-1 and VDAC by co-immunoprecipitation and confocal microscopy. We assessed expression of MCL-1 and HK2 by western blot; HK2 by q-RTPCR.

Results: Primary AML blast cells feature a lower spare respiratory capacity (SRC) (p=0.02) and lower glycolytic capacity (p=0.02) as compared to early progenitors/precursors (EP/P) from cultured CB CD34+ cells at day 7 of culture (N7, mostly promyelocytes) (Figure 1 a and b). Primary AML blast depend principally on fatty acids (p<0.05); they display a great flexibility, switching to glucose or glutamine to meet their energetic needs (Figure 1c). Consistent with the high adaptability of AML cells, and the emergence of resistance to therapy. We could define two populations (cut off value 10 pmol/min/x105 cells): one with higher (22±12 pmol/min/x105 cells) and on with lower (3±2 pmol/min/x105 cells) p<0.0001 levels of proton leak. The cases with higher proton leaks levels presented a reduced, extremely short, overall survival (p=0.048). We defined two SRC populations (cut off value 80 pmol/min/x105 cells): higher (124±47 pmol/min/x105 cells) and lower (52±25 pmol/min/x105cells) levels (p=0.0001). Interestingly the cases with higher SRC showed a trend of reduced overall survival. Associating high proton leak levels with high SRC the significance increases to p= 0.007. Considering the Basal OXPHOS of the AML patient’s cells, we observed two populations higher (54±12 pmol/min/x105 cells) and lower (16±6 pmol/min/x105cells) levels (p=0.0001). In patients with both high SRC plus high basal OXPHOS, the overall survival shortage is significant (p=0.002), indicating that an high SRC associated to higher basal respiration in AML blast cells confers greater aggressiveness and resistance to the therapy (Figure 1d). Patients with high mitochondrial respiration had a significantly higher myeloid cell leukemia 1 protein (MCL1) expression. We ascertained that MCL1 directly binds to Hexokinase II (HK2) on the outer mitochondrial membrane (OMM) affecting its stability.

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Summary/Conclusion: We demonstrate that high proton leak and high mitochondrial respiration at onset, arguably with the concourse of MCL1/HK2 action, is significantly linked with a shorter overall survival in AMLs’ patients. Our data describe a new function of MCL1 protein in AMLs’ cells, forming a complex with HK2 co-localized to the voltage dependent anion channel (VDAC) on the OMM, thus promoting glycolysis and OXPHOS, ultimately conferring metabolic plasticity and promoting resistance to therapy. The lower spare respiratory capacity and lower glycolytic capacity of AML patients’ blast respect to normal early hematopoietic precursors suggest a therapeutic window to use glycolytic and mitochondrial inhibitors in resistant AML patients.

P435: ANALYSIS OF MYELOBLAST IMMUNOPHENOTYPE PROFILE IN ACUTE MYELOID LEUKAEMIA AS PREDICTOR FOR CLINICAL OUTCOME

M. Sobas 1 , M. Nowak 1,* , D. Szymczak 1 , I. Andrasiak 2 , T. Wróbel 1

1 Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical Univeristy; 2 Independent Researcher, -, Wrocław, Poland

Background: The European LeukemiaNet (ELN) classification, is currently widely accepted risk stratification of acute myeloid leukaemia (AML). ELN stratification is based on genetic abnormalities and divides AML in low, intermediate and high risk groups. However, the outcome of AML patients, even within one classified group may be different. Flow cytometry analysis of blast cells play an essential role in diagnosis of AML, but the prognostic value of particular antigen expression is still unknown.

Aims: The aim of this study was to investigate the prognostic impact of the immunophenotype of blast cells.

Methods: We performed a retrospective analysis on 146 AML patients (aged 21-65) who were diagnosed and treated with intensive chemotherapy between 2017-2020. Allo-HSCT recipients were labelled TPL1, the others - TPL0. Patients with acute promyelocytic leukaemia were excluded from the study. Quantitative value (%) of particular antigen (CD2, CD4, CD7, CD9, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD25, CD33, CD34, CD36, CD38, CD41, CD45, CD56, CD61, CD64, CD65, CD71, CD73, CD96 CD117, CD123, HLA-DR) expression was collected. ELN classification at diagnosis, cytogenetic, mutational status (FLT3-ITD, FLT3-TKD, NPM1, CEBPA, CBFB-MYH11, RUNX1-RUNX1T1) and outcome (complete remission (CR), primary resistance, relapse after achieving CR were recorded). Categorical variables were presented as frequencies with percentages, median and interquartile range (IQR) were used to describe continuous variables. Non-normally distributed variables were analysed using Mann Whitney test and Kruskal-Wallis test. The Kaplan-Meier method and Cox proportional-hazards model were used to estimate time-to-event variables and determine hazard ratio (HR). All statistical tests were two-tailed with the significance level set at α=0,05. The study was performed in accordance with the Declaration of Helsinki.

Results: The baseline characteristics of the patients are presented in a table. The median age at diagnosis was 54 (aged 21-65). CR rate was 73%. The median follow up of 146 AML was 443 days. The median value of HLA-DR of relapsed (n=31, 21%) vs non-relapsed AML (n=115, 79%) was higher: 100 (90-100) vs 90 (67-99); p=0.006. There was no relationship between the HLA-DR expression (%) and ELN risk (p=0.705) nor mutational status (p>0.05). The primary resistance was more common in patients with higher CD34 and lower CD38 levels. The median value of CD34 in resistant AML vs those who achieved CR was 93% (22-99) vs 47% (0-91); p=0.017 and the median value of CD38 was 61% (31-93) vs 93% (83-98); p=0.011. The median CD34 value was significantly higher in the adverse vs favourable ELN-group: 79% (18-96) vs 2% (0-69), respectively (p=0.001) while the median CD38 value was significantly higher in the favourable vs adverse ELN-group, 96% (88-99) vs 86% (58-96), respectively, (p=0.005). Improved EFS was reported for decrease of HLA-DR (HR=1.01; p=0.049) and increase of CD38 (HR=0.99; p=0.046) among TPL0 patients. These variables are borderline during OS prediction for TPL0 - HLA-DR (HR=1.01; p=0.081) and CD38 (HR=0.99; p=0.051).

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Summary/Conclusion: Together with genetic mutations, immunophenotyping could be an equivalent component of risk assessment. The levels of CD34 and CD38 turned out to modify the first line treatment response and correlate with the ELN stratification. Higher percentage of CD34 and lower of CD38 may characterise refractory to standard intensive therapy AML, while higher HLA-DR may proceed relapse. Further, analyses, on prospective trials, should be conducted to confirm these correlations.

P436: TARGETED DELIVERY OF T22-PE24-H6 TO CXCR4 POSITIVE CELLS FOR ACUTE MYELOID LEUKEMIA TREATMENT

Y. Núñez Amela 1,2,* , A. Garcia-León 1,2 , A. Falgàs 1,2,3 , N. Serna 3,4,5 , L. Sánchez-García 3,4,5 , A. Garrido 2,6 , J. Sierra 2,6 , A. Gallardo 1,7 , U. Unzueta 1,2,3,5 , E. Vázquez 3,4,5 , A. Villaverde 3,4,5 , R. Mangues 1,2,3 , I. Casanova 1,2,3

1 Biomedical Research Institute Sant Pau (IIB-Sant Pau), Barcelona; 2 Josep Carreras Leukaemia Research Institute (IJC), Badalona; 3 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid; 4 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona; 5 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallès; 6 Department of Hematology, Hospital de la Santa Creu i Sant Pau; 7 Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

Background: Acute myeloid leukemia (AML) is a malignant heterogeneous group of hematological diseases that originates from a hematopoietic progenitor with altered maturation capacity. Recently, considerable advances have been made in the development of targeted therapies for distinct molecularly defined subtypes. Although many AML patients initially respond to current treatment, the majority of them relapse. Therefore, new targeted therapies are needed to overcome drug resistance and improve treatment effectiveness. CXCR4 is a chemokine receptor that is expressed in over 20 cancer types, both hematologic cancers and solid tumors. CXCR4 receptor and its ligand CXCL12 are key mediators of the interactions between AML cells and the bone marrow (BM) microenvironment. Moreover, CXCR4 is highly expressed in around 50% of AML patients. Thus, we have generated a nanoparticle (T22-PE24-H6) that selectively delivers the exotoxin A from Pseudomonas aeruginosa to CXCR4 + leukemic cells.

Aims: To assess the selectivity of T22-PE24-H6 cytotoxicity in CXCR4 + AML cell lines as well as in BM samples from AML patients. In addition, we evaluated the antineoplastic effect of the nanoparticle in a CXCR4 + AML disseminated mouse model.

Methods: OCI-AML-3, MONO-MAC-6 and HEL cell lines were characterized for CXCR4 expression using flow cytometry and immunohistochemistry (IHC). The in vitro antineoplastic effect of T22-PE24-H6 was determined by cell viability assays. Competition assays with the CXCR4 antagonist AMD3100 were performed to demonstrate the CXCR4-dependent cytotoxicity of the nanoparticle. In vivo effect was assessed in NSG mice intravenously injected with 1x10 6 luminescent MONO-MAC-6 cells. Animals were treated with 5 μg T22-PE24-H6 or Buffer (166 mM NaCO3H pH 8) daily for 10 doses. Bioluminescence signal (BLI) was monitored to quantitatively measure and evaluate in vivo disease progression. Animal tissue samples were collected after mice euthanasia and used to analyze AML dissemination and toxicity. CXCR4 expression levels and T22-PE24-H6 effect were also determined in BM samples from 10 newly-diagnosed AML patients.

Results: T22-PE24-H6 nanoparticle demonstrated a potent in vitro anticancer effect in MONO-MAC-6 cell line. The specific cytotoxic activity of the nanoparticle was CXCR4 dependent as demonstrated by performing competition assays with AMD3100. Furthermore, T22-PE24-H6 intravenous administrations showed significant BLI reduction in a CXCR4 + AML disseminated mouse model compared to buffer-treated mice. In addition, no differences between groups in mouse body weight, biochemical parameters or histopathological changes in normal tissues were detected. Finally, T22-PE24-H6 causes a significant cell viability reduction in AML patient samples with high CXCR4 expression. In contrast, the nanoparticle has no effect in AML patient samples with low expression of CXCR4.

Summary/Conclusion: T22-PE24-H6 selectively kills AML cell lines and BM samples from AML patients with CXCR4 overexpression. The nanoparticle also induces a high antineoplastic effect in a CXCR4 + AML disseminated mouse model without detectable systemic toxicity. These data strongly support that T22-PE24-H6 may obtain therapeutic benefit for AML patients with high CXCR4 expression.

P437: SURVIVAL-BASED 4-GENE PROGNOSTIC INDEX IMPROVES THE ELN-RISK CLASSIFICATION IN ACUTE MYELOID LEUKEMIA

C. A. Ortiz Rojas 1,2,3,* , D. A. Pereira-Martins 1,2,3,4 , C. C. Bellido More 5 , J. R. Hilberink 4 , J. L. Coelho-Silva 1,3 , I. Weinhäuser 1,2,3,4 , L. Yamamoto de Almeida 1,3 , V. M. de Deus Wagatsuma 1,3 , C. Hassibe Thomé 1 , G. Aguiar Ferreira 1 , E. Ammatuna 4 , G. Huls 4 , J. J. Schuringa 4 , E. Magalhães Rego 1,2,3

1 Center for Cell-Based Therapy, University of Sao Paulo, Ribeirão Preto; 2 Hematology Division, LIM31, Faculdade de Medicina, University of Sao Paulo, Sao Paulo; 3 Department of Medical Imaging, Hematology, and Oncology, Ribeirão Preto Medical School, University of Sao Paulo, Ribeirão Preto, Brazil; 4 Department of Hematology, Cancer Research Centre Groningen, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands; 5 Department of Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirão Preto, Brazil

Background: The European Leukemia-Net (ELN) criteria have been routinely used for risk categorization in acute myeloid leukemia (AML), classifying patients into favorable, intermediate and adverse-risk groups based on the presence of specific chromosomal and molecular alterations (Döhner et al., 2017). Despite this classification, high heterogeneity in the disease course is still a clinical barrier since leukemia can progress differently than expected. In this sense, the identification of markers that could improve the risk prediction is an unmet medical need.

Aims: We decide to perform a multicohort analysis of transcriptomic data to identify a gene signature consistently associated with prognosis in AML patients.

Methods: Publicly available data of five AML cohorts treated with intensive chemotherapy were included in this study: TCGA, n=122; BeatAML, n=230; HOVON ( GSE6891 ), n=392; NTUH ( GSE71014 ), n=104; German ( GSE37642 ), n=136. Also, we retrospectively included 38 patients diagnosed with AML, treated with intensive chemotherapy at the University Medical Center Groningen, in order to validate our gene signature by qRT-PCR. Univariate and multivariate Cox regression and area under the ROC curve (AUC) was applied separately to each gene in each cohort, in order to filter genes associated with prognosis. Prognostic indexes (PIs) models were generated by weighted sum using the pooled β coefficient. Then, the best PI was evaluated in its ability of improving ELN-risk classification.

Results: We found seven genes whose expression levels were associated with overall survival. After modeling possible PIs, we selected the PI that generates a better model in combination with ELN classification by using the Akaike’s Criterion Information (AIC) estimator. Thus, we selected a 4-gene prognostic index (4-PI) composed of CYP2E1, DHCR7, IL2RA, and SQLE genes. Survival stratification by 4-PI resulted in 5-year survival rates of 44.7 vs. 7.8% in the TCGA cohort, 28.5 vs. 0% in the BeatAML cohort, 47.7 vs. 26.6% in the HOVON cohort, 41.5 vs. 9.1% in the German cohort, and 88.7 vs. 44.1% in the NTUH cohort. Next, after correction for confounders variables, the 4-PI showed to be independently associated with prognosis, with an HR=3.94, 2.1, and 1.78 for TCGA, BeatAML, and HOVON cohorts, respectively. Among the patient molecular features, we found that inv(16) and dmCEBPA cases were more frequent in the low 4-PI, while TP53 alterations were frequent in patients with a higher 4-PI. Next, we evaluated the utility of the 4-PI in recategorizing patients from the ELN-risk subgroups in a best-fitted risk category. In the BeatAML cohort, ELN2017-adverse patients represented 36.9% (n=85) of patients, with a median OS of 12.2 months, but in the integrated risk, the adverse group increased to 38.7% (n=89) with a median OS of 10.3 months. We found similar results after integration of the ELN2010 with our 4-PI in the TCGA and HOVON cohorts. This power of stratification was maintained after applying the revised ELN proposed by Herold et al., 2020. Next, in our validation cohort, when we integrated the ELN2017 with the 4-PI, 14 patients were recategorized from the intermediate to the adverse group, improving the difference between them (median OS=33.8 vs. 7.6 months). Finally, we found a consistent enrichment of cholesterol metabolism related-pathways in cases with high 4- PI in all the cohorts.

Summary/Conclusion: We conclude that our 4-PI outperforms currently used prognosis predictors in AML and its incorporation into ELN categorization can improve risk stratification.

P438: ARYL HYDROCARBON RECEPTOR SIGNALLING IN NORMAL HAEMATOPOIETIC (HSC) AND LEUKEMIA STEM CELLS (LSC): DIFFERENTIAL EFFECT OF AHR ANTAGONIST ON LSC COMPARED TO HSC

A. Oryshchuk 1,* , R. Desai 1 , P. Kakadiya 1 , S. Bohlander 1

1 Molecular medicine and pathology, University of Auckland, Auckland, New Zealand

Background: Acute myeloid leukaemia (AML) is a devastating disease with poor prognosis. It is driven by leukemia stem cells (LSCs), which are able to survive treatment and drive relapse. This highlights the importance of characterizing LSCs. LSC-targeted treatment strategies are limited because there are no known surface markers that are specific for LSCs in every leukaemia. Targeting a signalling pathway that is altered in all LSCs could be a novel, promising treatment strategy. The aryl hydrocarbon receptor (AHR) pathway was shown to be critical for the self-renewal properties of normal haematopoietic stem cells (HSCs).

Aims: We aimed to study the consequences of manipulating the AHR pathway in vivo in a murine leukaemia model.

Methods: We used the following methods: murine bone marrow transplantation leukemia model (MBMTLM) - CALM-AF10 with limiting dilution assay (LDA); competitive repopulation assay (CRA) with LDA (CD45.2 donors into CD45.1 recipient mice), transcriptome analysis (sequencing with the Illumina platform and analisys with the DESeq2 ). Treatment of the cells was performed at various experiments with the AHR agonist, ITE, and an AHR antagonist, CH-223191 (a vehicle control is DMSO).

Results: We used a murine retroviral transduction bone marrow transplantation leukemia model (MBMTLM)) with the CALM-AF10 minimal fusion gene (CAMF) as the oncogenic driver, to study the effect of AHR agonist (ITE) and an AHR antagonist (CH-223191) treatment on LSCs. Interestingly, both AHR agonist and antagonist treatment affected LSC frequency and survival of mice transplanted with treated cells. The LSC frequency as measured by limiting dilution transplantation assays (LDA) was 3.7 times lower after agonist treatment (1:163, blue survival curve in fig. 1, A) compared to a vehicle control (1:44, DMSO, black survival curve)). Very interestingly, after antagonist treatment the LSC frequency was so low that could not be measured as none of the mice transplanted with antagonist-treated cells in the LDAs developed leukaemia (n=9, CH-223191 (red curve in fig. 1, A)). Of note, our CAMF MBMTLM has a very high LSC frequency of about 1:4 in cells that were not cultured after thawing. We performed RNA-Seq on 90 samples to determine changes in gene expression patterns after treatment of healthy bone marrow cells and CAMF leukemia cells with the AHR agonist and antagonist. Preliminary differential expression analysis with DESeq2 revealed differential expression of genes involved in apoptosis and proliferation in leukaemia cells treated with CH-223191. As leukemia did not develop in recipients of cells treated with the antagonist CH-223191, genes differentially regulated in these cells might be critical and specific to LSC function and will be candidate targets for LSC-specific treatments. To confirm that AHR antagonist treatment affects LSCs differently than normal HCS, we performed competitive repopulation assay (CRA). Our preliminary data (n = 12) show that treatment of HSCs with CH-223191 results in a steady increase in the engraftment over a 47-week period following transplantation (coral bars, fig. 1, B), as opposed to the grafts from the ITE-treated arm (blue bars), vehicle or untreated HSCs (black and green bars, respectively).

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Summary/Conclusion: In summary, our data show that the LSCs react differently than normal HSCs to changes in AHR signalling. These differences suggest it might be possible to specifically inhibit LSCs while sparing normal HSCs, thus paving the way to develop strategies to treat leukemia based on manipulating AHR signalling.

P439: ASSOCIATING EX VIVO DRUG SENSITIVITY WITH METABOLIC STATUS IDENTIFIES EFFECTIVE COMBINATION STRATEGIES IN ACUTE MYELOID LEUKEMIA

D. A. Pereira-Martins 1,2,3,* , E. Griessinger 1 , I. Weinhauser 1,2 , J. L. Coelho-Silva 2,3 , D. R. Silveira 4 , L. Quek 4 , A. Erdem 1 , J. R. Hilberink 1 , E. V. de Paula 5 , S. T. Olalla Saad 5 , E. Ammatuna 1 , G. Huls 1 , E. M. Rego 2 , A. R. Lucena-Araujo 3 , J. J. Schuringa 1

1 Department of Experimental Hematology, Cancer Research Centre Groningen, University Medical Center Groningen, Groningen, Netherlands; 2 Center for Cell Based Therapy, University of Sao Paulo, Ribeirao Preto; 3 Department of Genetics, Federal University of Pernambuco, Recife, Brazil; 4 Myeloid Leukaemia Genomics and Biology Group, School of Cancer and Pharmaceutical Sciences, King’s College London, London, United Kingdom; 5 Hematology and BloodTransfusion Center, University of Campinas, Campinas, Brazil

Background: Metabolic reprogramming is a hallmark of cancer, and acute myeloid leukemia (AML) is no exception. Yet, we and others have shown that clear heterogeneity exists in metabolic programming between genetically distinct AML subtypes and altered function of mitochondria (mt) might be linked to chemoresistance in some patients.

Aims: Here, we evaluated the mitochondrial function of AML cells in more detail.

Methods: Retrospective analysis of the mtDNA content (mtDNAc) in a large cohort of Brazilian patients with de novo AML (03 centers, n=482, age:18-65y) treated with the 3 + 7 scheme, and sex- and age-paired healthy donors (HD, n=308), revealed higher mtDNAc in 34% of the AML samples when compared to the highest value of mtDNAc in the HD group.

Results: High mtDNAc was independently associated with increased relapse risk. These findings were confirmed in a validation cohort of AML patients treated with the 3 + 7 scheme at the UMCG in the Netherlands (n=92, age:18-65y). Clustering analysis using label-free quantified proteome of sorted CD34 + cells from AML samples (n=30) associated high mtDNAc with oxidative phosphorylation (OxPhos) metabolism and L-GMP leukemia. Flow cytometry analysis for Hematopoietic progenitors (defined by CD34, CD38, CD123, and CD45RA gated inside the CD45 + cells) and mitochondrial markers (Mitotracker Green/DeepRed for mitochondrial mass/potential, respectively) in a set of primary AML samples (n=32), showed an enhanced proportion of L-GMP cells and a positive correlation with the mitochondrial potential (r=0.51) in AMLs with high mtDNAc. Consistently, oxygen consumption rate (OCR) and lactate production measured by Seahorse was increased in primary AML blasts (n=11) and AML cell lines (n=14) with high mtDNAc, suggesting increased energetic metabolism. Multiomics analysis of AML cell lines (n=19) revealed a positive correlation between mtDNAc and mitochondrial metabolism and apoptosis regulation. To evaluate the role of mtDNAc during drug-induced apoptosis, we treated primary AML blasts (n=67) with cytarabine (AraC, 100 nM) for 48h. We observed a negative correlation between mtDNAc and apoptosis rate (r=-0.75). Moreover, the reduction of mtDNAc in AML blasts (n=16) using shRNA targeting the POLG gene resulted in mixed phenotypes. POLG silencing impaired cell proliferation and induced monocytic differentiation for AMLs with high mtDNAc, while promoted cell proliferation in mtDNAc low AMLs, suggesting a completely distinct dependency on OxPhos for these 2 groups of AMLs. Pharmacological treatment of 9 AML cell lines with ETC complex I inhibitors (CIi; rotenone, 50 nM and metformin, 1 mM) in combination with AraC (10-500 nM) and Venetoclax (VEN, 10-1000 nM), resulted in a reduction of mtDNAc and functional respiration in AML cells. Combination therapy of CIi with AraC resulted in increased cell death in OxPhos - but not in glycolysis-driven AML cells. In contrast, the combination of CIi+VEN resulted in enhanced cell death in all AML cells, regardless of their metabolic state. Since metformin is an FDA-approved drug, we assessed the metformin-induced apoptosis, in combination with AraC or VEN in 64 primary AML samples ex vivo . Metformin treatment resulted in a reduction of basal OCR of AML blasts (n=7). The metformin-induced apoptosis rate positively correlated with mtDNAc (r=0.89), displaying additive effects with AraC- and VEN-induced apoptosis in AML samples.

Summary/Conclusion: Overall, our findings suggest that mtDNA content can be used to identify patients with a higher risk of chemoresistance and supports the idea of repurposing metformin into AML therapy.

P440: THE COMBINATION VENETOCLAX PLUS MCL1 OVERCOMES DRUG RESISTANCE IN ACUTE PROMYELOCYTIC LEUKEMIA

D. A. Pereira-Martins 1,2,* , N. I. Noguera 3 , T. Ottone 3,4 , A. Diepstra 5 , C. Ortiz 1 , I. Weinhauser 1,2 , G. Huls 2 , J. J. Schuringa 2 , E. M. Rego 1 , E. Ammatuna 2

1 Center for Cell Based Therapy, University of Sao Paulo, Ribeirao Preto, Brazil; 2 Department of Experimental Hematology, Cancer Research Centre Groningen, University Medical Center Groningen, Groningen, Netherlands; 3 Department of Biomedicine and Prevention, University of Tor Vergata; 4 Neuro-Oncohematology, Santa Lucia Foundation, I.R.C.C.S., Rome, Italy; 5 Department of Pathology and Medical Biology, Cancer Research Centre Groningen, University Medical Center Groningen, Groningen, Netherlands

Background: Although acute promyelocytic leukemia (APL) is the most favorable subtype of acute myeloid leukemia, it remains a life-threating disease for high-risk patients. Despite progress in APL with arsenic (ATO) plus retinoic acid (RA) as front-line treatment, approximately 10-15% of patients will acquire resistance to ATO and/or RA and relapse. Relapse after ATO/RA based therapy remains very challenging, and novel drug schemes to overcome resistance are urgently needed. Analysis of bone marrow (BM) biopsies of 15 consecutive APL patients at diagnosis, revealed a homogeneous staining for BCL2 in all cases. These results suggest BCL2 inhibition could be a therapeutic option for resistant APL. In vitro treatment with the BCL2 inhibitor Venetoclax (VEN), resulted in increased MCL1 expression, suggesting a resistance mechanism to this therapy.

Aims: Here, we investigated the effects of VEN+MCL1 inhibitor S64315 in APL cells resistant to ATO/RA, and evaluated the effect of the combination therapy in vivo , using a cell line derived xenograft (CLDX) model for APL with the ATO-resistant associated mutation PML A216V.

Methods: Primary leukemic blasts from APL mice (n=4) and patients (age, 25-52y; n=10) were treated with VEN (250-500nM), ATO, ATRA (1µM), and S64315 (10-50nM) monotherapies and combinations and evaluated for cell survival. In addition, we evaluated the transcriptome of 16 APL patients (age, 25-74y; 7 males) enrolled in the TCGA study regarding the BCL2 expression. NB4/NB4R2 (RA-resistant) cell lines were transduced with PML A216V. Additionally, U937 cells were transduced with the inducible PML - RARA system. After induction of PML - RARA expression with Zinc for 0-24h, BCL2 protein levels were evaluated by western blot. Additionally, RA/ATO treated (1µM) NB4 cells for 6-72h were evaluated for BCL2/MCL1 levels. For the CLDX model, NB4 PML A216V cells were transplanted into NSG mice, and after chimerism detection (hCD45 + ) in the peripheral blood, mice were equally distributed into 4 groups of treatment: vehicle, VEN (25mg/kg/day), S64315 (20mg/Kg/day) and combination (n=5/group) for 21 consecutive days.

Results: Gene set enrichment analysis using the transcriptome of APL patients with BCL2 high associated these patients with the terms “EPPERT_HSC_LSC” and “17_LSC”. These results suggest that APL patients with high BCL2 expression differ in their metabolic and proliferative state compared to patients with BCL2 low . Functionally, the induction of PML - RARA expression in U937 cells resulted in increased BCL2 expression in a time-dependent manner (peak at 6h). Contrarily, treatment with ATO/RA resulted in decreased BCL2 levels with upregulation of the MCL1 protein. BH3 profile revealed that ATO/RA treated NB4 cells displayed an increase in the MCL1 anti-apoptotic dependency. Additionally, in vitro and ex vivo treatment of APL cells (cell lines and primary blasts) revealed synergic activity of S64315 , but not the BCL2 inhibitor (VEN), in combination with ATO. Furthermore, treatment of ATO resistant NB4 cells (by gradual exposure to ATO or by expression of the PML A216V) revealed sensitivity of those cells to the combination VEN+ S64315 . Finally, in vivo CLDX model revealed limited effect of VEN and S64315 monotherapy, while the combination was able to effectively reduce the leukemic burden, resulting in increased overall survival (vehicle:15 vs Combo:42 days).

Summary/Conclusion: The combination of BCL2 and MCL1 inhibition is a potential effective treatment in ATO/RA resistant APL patients and could be clinically explored in this category of patients with very poor prognosis.

P441: DIFFERENCES IN GENE EXPRESSION PATTERN AND IMMUNOPHENOTYPE OF BONE MARROW MULTIPOTENT MESENCHYMAL STROMAL CELLS IN PATIENTS AT THE ONSET OF ACUTE LEUKEMIA AND AFTER ACHIEVING REMISSION

A. Sadovskaya 1,* , N. Petinati 2 , N. Kapranov 2 , N. Drize 2 , A. Vasilieva 2 , O. Gavrilina 2 , E. Parovichnikova 2

1 Faculty of biology, Department of Immunology, Lomonosov Moscow State University; 2 NATIONAL MEDICAL RESEARCH CENTER FOR HEMATOLOGY, Moscow, Russia

Background: The stromal microenvironment plays a key role in both maintaining normal hematopoiesis and assisting malignant cells during leukemia development. In patients with acute leukemia, the properties of multipotent mesenchymal stromal cells (MSCs) are changed both at the onset of the disease due to interaction with tumor cells and upon reaching remission due to chemotherapy. As a result, the ability of MSCs to maintain normal hematopoiesis is reduced. A detailed study of these changes may allow the development of rehabilitation measures for restoring the stromal microenvironment of patients.

Aims: The aim of this work was to study general changes in gene expression pattern and immunophenotype of MSCs in patients with acute leukemia at the onset of the disease and in remission.

Methods: The study included MSCs obtained from the bone marrow (BM) of 15 patients: 4 with ALL (1 male, 3 female, median age 26.5) and 11 AML (4 male, 7 female median age 33), BM of 30 donors of appropriate age was used as control. All donors and patients signed informed consent. MSCs were cultured by standard methods. Immunophenotypes were studied by flow cytometry. Relative expression levels (REL) of several genes were investigated by real time PCR.

Results: The study of the mean fluorescence intensity (MFI) of MSC markers revealed a significant decrease in MFI of CD274 (PD-L1) in primary patients compared to healthy donors and its absence on the cells of patients in remission. At the onset both MFI of CD54 (ICAM-1) and the proportion of CD54+ MSCs were decreased. In remission these parameters decreased even more, so the treatment reduced the adhesive capacity of MSCs. The expression of CD105 was reduced at the onset of the disease and increased after the end of treatment, but did not normalize.

In patients at the onset of the disease and in remission, compared with healthy donors, the expression of IL1R1, PDGFRβ, ANG1 was reduced; expression of IL6, PPARγ, JAG1, was increased (see Figure). Decreased level of IL8 and SOX9 normalized in remission.

The decrease in growth factor (GF) receptors PDGFRβ and IL1R1 (IL1β may act as a GF for BM cells) in patients may be a result of negative feedback loops: GFs typically act as auto- and paracrine regulators, and malignant cells often overexpress various GF. The fact that these genes’ expression does not return to normal in remission may be a result of damage from treatment. IL1β expression in patients was not different from normal, but its increase in remission compared to onset implies stroma restoration processes.

Another important effect is the alteration in MSCs’ interactions with stem cells: JAG1 product helps to ensure stem cells’ quiescent state and bond with stroma. In contrast, there is an increase in IGF1 expression at the onset, which can lead to stem cell activation.

Alterations in the RELs of differentiation markers (SOX9, SPP1, PPARγ) indicate changes in the predisposition of MSCs to differentiation. PPARγ is a multifunctional nuclear receptor, and can inhibit proliferation. PPARγ upregulation may once again be a result of negative feedback networks preventing MSCs’ uncontrolled proliferation in GF-rich microenvironment.

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Summary/Conclusion: The data suggest dramatic alterations in hematopoietic and stromal cells interaction at the onset of acute leukemia that were not undone by achieving remission. This is supported by decreased levels of endoglin and ICAM-1 and alteration of gene expression pattern.

The materials supported by grants from the Russian Foundation for Basic Research project 19-29-04023 were used.

P442: SPINK2 PROTEIN EXPRESSION QUANTIFIED BY IMMUNOHISTOCHEMISTRY REFINES RISK STRATIFICATION AND PREDICTS THERAPY OUTCOMES IN AML

H. A. Pitts 1,* , C. K. Cheng 1 , J. S. Cheung 1 , M. K. H. Sun 1 , M. H. Ng 1

1 Blood Cancer Cytogenetics and Genomics Laboratory, Department of Anatomical & Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, SAR, China

Background: Suboptimal prognostication, therapy refractoriness and relapse contribute to the poor outcome of Acute Myeloid Leukemia (AML) patients. Leukemic stem cells (LSC) are considered crucial drivers of relapse and therapy resistance. Clinicopathological characterization of LSC-associated genes with convenient assessment tools such as immunohistochemistry (IHC) is needed. Our initial screening of AML datasets for LSC-associated genes identified Serine Protease Inhibitor Kazal type 2 ( SPINK2 ) with high expression in AML, particularly in LSC fractions. However, in-depth analysis of its clinicopathological associations and prognostic utility in AML are lacking.

Aims: To assess SPINK2 expression in adult AML with IHC, and determine its clinicopathological associations, prognostic impact and association with therapy response

Methods: We studied the expression of SPINK2 by IHC in 172 non-M3 adult AML patients (median age:52yrs, range: 18-86yrs) treated at the Prince of Wales Hospital. The majority (90.8%) were de novo type and 72.3% had intermediate-risk(IR) cytogenetics, with 88.5% receiving Daunorubicin + Ara-C (DA 7 + 3) induction. SPINK2 expression was assessed by 3 hematopathologists in a double-blinded manner and quantified by a composite score (range: 0-16). Clinical data was collected for all cases.

Results: SPINK2 staining in leukemic blasts was consistently cytoplasmic, and its protein expression strongly correlated with mRNA levels by qPCR in a subset of 128 patients (r=0.716, P <0.0001). The cohort was dichotomized at the median score 3, since this cut-off showed strongest association with poor event-free survival (EFS) and overall survival (OS). High SPINK2 ( SPINK2 hi ) patients accounted for 77/172 (44.8%) cases, and low SPINK2 ( SPINK2 lo ) patients for 95/172(55.2%).

SPINK2 hi associated with IR cytogenetics ( P =0.014), normal karyotype ( P =0.019) and NPM1 mutation ( P <0.001), while SPINK2 lo with t(8;21) and CEBPA double-mutation (both P <0.001). Survival and treatment-response analyses were performed on a subgroup of 137 patients who received DA 7 + 3 based induction. SPINK2 hi patients had lower CR rates (73.8% vs 88.3%, P =0.028), higher 6-month relapse rates (31.8% vs 9.1%, P =0.004), lower 5-yr RFS (25.8% vs 46.8%, P= 0.004), EFS (16.6% vs 37.2%, P <0.001) and OS (25.3% vs 51.2%, P <0.001). SPINK2 status also identified high risk patients of the IR cytogenetic subgroup. Of these, SPINK2 hi patients had lower CR rates (68.6% vs 90.0%, P =0.013), higher 6-month relapse rates (31.4% vs 6.9%, P =0.018), lower 5-yr RFS(27.0% vs 44.6%, P= 0.018), EFS(15.4% vs 38.3%, P <0.001) and OS(22.9% vs 51.5%, P =0.002). In this subgroup, median SPINK2 score was higher in patients requiring ≥2 inductions to achieve CR vs. patients requiring 1 course (7 vs 2, P =0.009). Multivariate analyses in whole and IR cytogenetics cohorts showed the poor prognostic effect of SPINK2 hi on EFS and OS independent of age, cytogenetic risk, mutations and achievement of CR1, including stem cell transplantation given in CR (Table 1). In our NPM1 mut subgroup (N=41), SPINK2 hi status associated with poor EFS (HR:3.3, P =0.04) and OS (HR: 4.0, P =0.007) independent of age and FLT3 -ITD with high allelic ratio. Incorporation of SPINK2 with FLT3 status refined ELN2017 risk definition, whereby 15/22 favorable-risk patients could be reclassified as intermediate-risk.

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Summary/Conclusion: High SPINK2 protein expression by IHC predicted increased chemoresistance and early relapse risk in AML, and was an independent adverse prognostic biomarker, particularly in patients with intermediate-risk cytogenetics and NPM1 mutations.

P443: IMPACT OF PHARMACOGENETIC VARIANTS ON CYTARABINE AND ANTHRACYCLINE EFFECT ON OUTCOMES IN ADULT AML PATIENTS

Z. Pravdic 1,* , M. Virijevic 1,2 , M. Mitrovic 1,2 , N. Pantic 1 , V. Gasic 3 , S. Pavlovic 3 , N. Sabljic 1 , D. Pavlovic 3 , I. Marjanovic 3 , N. Suvajdzic Vukovic 1,2

1 Clinic of Hematology, UCCS; 2 Faculty of Medicine, University of Belgrade; 3 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia

Background: Acute myeloid leukaemia (AML) is primarily treated with combination of cytarabine and anthracyclines. Despite high remission rates, the 5-year overall survival (OS) is <50% and <20% for<60-year-old a ≥60-year-old patients, respectively. This grim clinical outcome could be partly explained by the patients’ genetic variability of proteins involved in metabolic paths of cytarabine and anthracyclines. Pharmacogenetic variants of the main cytarabine membrane transporter SLC29A1 (solute carrier family 29 member 1), DCK (Deoxycytidine kinase) the first enzyme in cytarabine stepwise activation process, genes coding main anthracycline efflux pump ABCB1 (ATP binding cassette) and genes coding glutathione S-transferases GSTM1 and GSTT1, main cytosolic detoxifiers od anthracycline-induced oxidative stress, are shown to influence clinical outcome in AML patients.

Aims: to evaluate: 1) the effects of variants in pharmacogenes SLC29A1, DCK , ABCB1, GSTM1 and GSTT1 , on complete remission (CR) and relapse rate (RR), disease free survival (DFS) and overall survival (OS), 2) the influence of demographic, laboratory and AML-related parameters on CR, RR, DFS and OS.

Methods: 100 newly-diagnosed consenting adults (18-62 years old) diagnosed with AML, except acute promyelocytic leukaemia in Clinic of Haematology UCCS from January 2015 to January 2018, were included in retrospective cohort. Patients received one or two inductions ‘’3 + 7’’ cycles. In patients achieving CR either three consolidation cycles with high/intermediate doses of cytarabine or allogenic SCT in selected patients were performed. Demographic, standard laboratory and AML-related parameters (blood/bone marrow blast percentage, cytologic, flow cytometry and genetic markers enabling ELN risk stratification were collected from patients’ health records. Variants SLC29A1 rs9394992, DCK rs12648166, ABCB1 rs2032582 and GSTM1 and GSTT1 gene deletions were detected by methodology based on PCR, fragment analysis and direct sequencing. The study was approved by the Ethics Committee of the UCCS. The methods of descriptive and analytic statistics were used, while survival analysis was done by the Kaplan-Meier method using the Log-Rank test.

Results: 100 patients (53 males) were included in the study, with the median age of 51 (range 18-62). Median laboratory parameters were: WBC 15.5x10 9 /L (range 1-348.8), Hgb 97g/L (range 20-166), Plt 53.5 (range 1-422), LDH 249U/L (1-4169). CD34 was positive in 56% patients. According to ELN 15, 55 and 30 patients were classified in favorable, intermediate and adverse risk group, respectively. CR was achieved in 57 patients (41 after the first and 16 after the second induction cycle). A total of 34% of patients relapsed. Median DFS was 5.3 months (range 0.25-61.5), while median OS was 6 months (range 0.3-75). There was a significant difference in median DFS between CC, CT and TT genotype of SLC29A1 : 13, 8 and 1.75 months, respectively (p=0.00037). Median DFS was significantly decreased in null genotype of GSTM1 (9.5 (null) vs 18 months (non-null); p=0.028). Significant difference in OS within ABCB1 genotypes TT, GG, GT and AG+AT was registered: 13, 7, 6.5 and 3 months, respectively (p=0.0036). Other parameters did not differ significantly.

Summary/Conclusion: Our results, regarding impact of variants of SLC29A1, GSTM1 on DFS and impact of variant ABCB1 on OS in AML patients, are in line with previous studies. Although further studies, with larger cohorts, are needed, these pharmacogenetic variants show a potential to become prognostic markers in AML.

P444: IDENTIFICATION OF FUNCTIONAL GENETIC DEPENDENCIES IN CEBPA-MUTATED ACUTE MYELOID LEUKEMIA

L. Proietti 1,* , E. Heyes 1 , T. Eder 1 , T. Brandstoetter 2 , G. Manhart 1 , V. Sexl 2 , F. Grebien 1

1 Institute for Medical Biochemestry; 2 Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria

Background: C/EBPα is a key myeloid transcription factor that regulates the switch between proliferating uncommitted cells and terminally differentiated cells. The CEBPA gene is mutated in 10-15% of AML patients. CEBPA lesions are divided into C-terminal mutations that abrogate DNA binding and N-terminal frameshift mutations, which cause expression of an N-terminally truncated isoform, termed p30. A large fraction of AML patients carries biallelic CEBPA mutations. In these patients, the shorter C/EBPα p30 variant is the only functional isoform of C/EBPα. While we and others have identified molecular mechanisms underlying the development and maintenance of CEBPA -mutated AML, a comprehensive analysis of vulnerabilities that is associated with this disease subtype is lacking.

Aims: We aimed to characterize the functional genetic dependencies of AML with biallelic CEBPA mutations.

Methods: We generated an immortalized AML cell line by serial transplantation of fetal liver cells carrying biallelic CEBPA mutations (CNC, C ebpa N -terminal/ C -terminal) followed by prolonged culture of leukemic cells. After stable expression of Cas9, we performed a genome-scale CRISPR/Cas9 loss-of-function screen in CNC cells and compared the results to genome-wide screening results from the HPC7 hematopoietic multipotent progenitor cell line, which were used as a representative of the healthy, untransformed state.

Results: Transplantation of CNC cells into C57BL/6 mice induced an aggressive Mac1 + /Gr1 + /c-Kit + leukemia within 7 weeks, similar to the initial model (Bereshchenko et al , 2009). This immuno-phenotype was preserved upon prolonged in vitro culture. Analysis of the CRISPR/Cas9 screen in CNC cells identified 2488 genes whose mutational inactivation was associated with reduced fitness - 438 of these genes are “core” essential, as they have been annotated to be required for the survival of both cancer and normal cells. In HPC7 cells, the loss of 1674 genes caused cell depletion, including 388 core essential genes. Intersection of screening results from CNC and HPC7 cells revealed 1290 commonly depleted genes, of which 380 were core essential. We identified 308 genes that were exclusively required for the proliferation and survival of CNC cells. This list of high-confidence candidates is enriched for genes involved in mRNA metabolism, DNA repair and signaling pathways. We are currently in the process of validating prioritized target genes using a variety of approaches.

Summary/Conclusion: This approach allows the acquisition of deeper insights into the molecular mechanisms underlying the development and maintenance of leukemia with CEBPA mutations. Multi-layered validation of high confidence candidates will be performed in vivo and in vitro to identify leukemia-specific vulnerabilities that can serve as starting points for rational targeting strategies.

P445: CRYPTIC TRANSLOCATION T(5;11)(Q35;P15) RESULTING IN NUP98::NSD1 GENE FUSION IN ADULTS WITH DE NOVO ACUTE MYELOID LEUKEMIA (AML)

S. Ransdorfova 1,* , J. Markova 1 , M. Valerianova 1 , J. Brezinova 1 , M. Onderkova 1 , I. Mendlikova 1 , L. Lizcova 2 , L. Pavlistova 2 , K. Svobodova 2 , S. Izakova 2 , A. Jonasova 3 , C. Salek 1 , Z. Racil 1 , Z. Zemanova 2

1 Institute of Hematology and Blood Transfusion; 2 Center of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and First Faculty of Medicine, Charles University; 3 1st Medical Department, General University Hospital and First Faculty of Medicine, Charles University, Prague, Czechia

Background: The NUP98::NSD1 fusion, a product of the cryptic translocation t(5;11)(q35;p15.5), is a recurrent genetic change in cytogenetically normal patients with AML. It occurs most frequently in children (16%) and young (2%) AML patients, very rarely in adult patients. The coexistence of internal tandem duplication of FLT3 gene ( FLT3::ITD ) found in more than 70% of NUP98::NSD1 positive patients is always associated with a poor prognosis and results in high frequency of induction failure.

Aims: The aim of this study was to determine the incidence of the NUP98::NSD1 fusion gene in adults with AML and NUP98 rearrangement.

Methods: We examined the bone marrow cells of 268 newly diagnosed AML patients using conventional karyotyping in combination with FISH (Abbott, MetaSystems) and mFISH/mBAND (MetaSystems). We used RT-PCR followed by direct sequencing to detect fusion genes. We processed the PCR product by ExoSAP-IT and directly sequenced on ABI Prism 310 genetic analyzer using the Big Dye Terminator kit v. 3.1.

Results: In nine out of 268 cases we identified rearrangement of the 11p13-15 region. We confirmed t(5;11)(q35;p15.5) with NUP98::NSD1 gene fusion in four of them (4/268; 1.5%). Sequence analyses proved the NUP98-NSD1 transcript, arising from fusion of NUP98 exon 12 with exon 6 in NSD1 gene, in all four patients (2M/2F; FAB M4/M5b; age 42, 54, 64 and 64 years). FLT3::ITD mutation was detected in all of them. Specific primers and a probe have been designed to monitor minimal residual disease during therapeutic treatment of the patients. Out of 4 patients, two died (median OS 9 months) and two patients are alive (4 and 1 year after allogenic bone marrow transplantation).

Summary/Conclusion: Our study demonstrated the occurrence of t(5;11)(q35;p15.5) with NUP98::NSD1 gene fusion also in adults over 60 years of age. We confirmed the NUP98::NSD1 fusion gene in 1.5% adults AML patients. With respect to the poor prognosis of the patients with NUP98::NSD1 fusion gene, we suggest pre-screening of NUP98 gene rearrangement using FISH in cytogenetically normal AML patients with confirmed FLT3::ITD mutation.

Supported by MH CZ-DRO-VFN64165, DRO-UHKT00023736.

P446: USE OF MIRNA EXPRESSION PATTERNS REVEALS ACTIVIN RECEPTOR 2 PATHWAY IN MEDIATING CHEMO-RESISTANT AML

P. Reichelt 1,* , S. Bernhart 2 , F. Wilke 1 , U. Platzbecker 1 , M. Cross 1 , G. Behre 3

1 Department of Hematology and Cell Therapy, University Hospital Leipzig; 2 Interdisciplinary center for bioinformatics Leipzig, Leipzig; 3 Clinic for Internal Medicine I, Municipal Hospital Dessau, Dessau, Germany

Background: Acute myeloid leukemia (AML) is a highly aggressive and heterogeneous disease and the most prevalent type of acute leukemia in adults. Standard chemotherapy using cytarabine and idarubicin/daunorubicin usually achieves remission, but this is commonly followed by chemoresistance and relapse. The treatment of chemoresistant AML remains a major challenge that will require personalized approaches.

Aims: With this study, we aim to establish NGS microRNA expression profiling and pathway analysis to identify pathways that are regulated differentially between chemo-sensitive and -resistant AML as potential targets for targeted therapy.

Methods: MicroRNA expression profiles were analyzed by NGS (next generation sequencing) of chemo-resistant and -sensitive subclones of AML cell line HL60. Bioinformatical identification of candidate deregulated pathways was performed using miRTarbase and stringdb. Pathway activity in cell sublines was assessed by protein expression, proliferation and flow cytometric analysis, using western blotting, phosphlow and MTS assays. Paired samples of AML pre- and post- relapse were used to validate gene expression changes by qPCR and analyses were extended to the publically available dataset of the AML patient cohort of the cancer genome atlas (TCGA).

Results: Our microRNA based bioinformatic approach predicted 27 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways to be represented differentially between resistant and sensitive cells (FDR<0.05). Prominent among these were significant changes in signaling pathways of the TGFβ ligand family. Cell culture experiments and protein expression analysis confirmed both a higher sensitivity to TGFβ induced growth arrest and increased abundance of TGFβ signaling proteins in chemo-resistant HL60 cells. Consistent differences in TGFß pathway gene expression were also detected in AML patients of the TCGA dataset. Furthermore, our data suggest a shift of the predominantly expressed isoform of activin receptor 2B to activin receptor 2A in chemo-resistance. Analysis of paired bone marrow samples from AML patients at diagnosis and first relapse showed the latter to be associated with higher expression of TGFβ receptors and the TGFβ target protein CDKN2B as well as significant upregulation of activin receptor 2A.

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Summary/Conclusion: Here we show how miRNA screening approaches can be used to predict deregulated cellular signaling pathways as potential targets for the therapy of chemoresistant AML. MicroRNA based prediction of targetable pathways combined with database screening identified the activin receptor 2A/B as a potential modulator of chemoresistance.

P447: BCL2-INHIBITION TARGETS LEUKEMIC STEM CELLS IN ACUTE MYELOID LEUKEMIA INDEPENDENT OF MONOCYTIC BLAST POPULATIONS

S. Renders 1,2,* , A.-M. Leppä 1 , A. Waclawiczek 1 , C. Reyneri 1 , M. Janssen 2 , E. Donato 1 , J. Unglaub 2 , C. Pabst 2 , R. Schlenk 2 , M. Hundemer 2,3 , S. Raffel 2 , T. Sauer 2 , C. Müller-Tidow 2 , A. Trumpp 1

1 DKFZ/ Hi-Stem; 2 Department of Hematology, University Hospital Heidelberg, Heidelberg; 3 MVZ Hämatologische Diagnostik, Heppenheim, Germany

Background: Treatment with Hypomethylating agents (HMA) in combination with the BCL-2 inhibitor Venetoclax (VEN) has recently become the standard of care for AML patients unsuitable for intensive induction chemotherapy and shows superior results to treatment with Azacitidine (AZA) alone (DiNardo et al., 2020, NEJM). However, upfront resistance and relapse remain major obstacles.

It has recently been proposed that monocytic differentiation associated with increased expression of antiapoptotic-protein MCL-1 predicts resistance to AZA/VEN treatment in AML (Pei et al., 2020 Cancer Discovery). However, other studies did not find impaired outcome in patients with monocytic AMLs treated with HMA/VEN (DiNardo et al., 2021, Lancet Oncology; Stein et al., 2021 Blood Advances).

Aims: We aimed to understand apoptotic dependencies linked to differentiation states in AML subpopulations in combined with clinical data to understand and treatment effects and resistance upon HMA/VEN therapy.

Methods: We performed retrospective analysis of >50 patients at Heidelberg University hospital to identify risk factors of treatment failure. For functional investigation, we studied 24 AML cell lines and generated Xenografts in NSG mice of 12 AML patients to study mature and leukemic stem cell (LSC)-like primary AML subpopulations. We characterized these samples further by RNA sequencing and BCL-2 family level assessment. To functionally validate our findings, we performed ex vivo drug treatment and subpopulation specific BH3 profiling.

Results: In our clinical cohort the only factors associated with HMA/VEN resistance were previous myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) and complex karyotype, but not monocytic differentiation. The CD64+CD11b+, mature blast population making up >50% of leukemic cells in monocytic and <20% in primitive samples showed high levels of resistance to AZA/VEN therapy in both primitive and monocytic leukemias but did not engraft when transplanted into NSG mice, arguing they do not contain LSCs. In contrast, we found immature GPR56+ LSCs to be sensitive to AZA/VEN treatment irrespective whether they were derived from monocytic or primitive primary AMLs. LSCs from monocytic and primitive AMLs initiated disease in NSG mice, highlighting that targeting LSCs is essential to treat AML. Next, we investigated expression of BCL2, MCL1 and BCL-xL levels in the same primary patient samples and observed high MCL1 expression in monocytic AML samples. However, MCL1 expression predominantly derived from the mature population whereas LSCs expressed robust levels of BCL2 but comparatively low levels of MCL1, independent of whether monocytic or primitive AMLs were analyzed. Additionally, transcriptomes of LSCs from either monocytic or primitive samples did not display significant differences. We next performed BH3 profiling and found LSC to be dependent on BCL2 and mature blasts on MCL1 in all samples studied. In line, ex vivo HMA/VEN treatment specifically eliminated LSCs. We further monitored blast clearance in longitudinal patient samples and found robust loss of LSC within hours in patients, while mature blasts persisted for days.

Summary/Conclusion: We find that LSCs, independent of the overall mature blast content, are sensitive to HMA/VEN treatment and highlight that genetic risk factors and disease history are relevant predictors of treatment failure.

P448: PROGNOSTIC IMPACT OF SOMATIC CEBPA BZIP DOMAIN MUTATIONS IN ACUTE MYELOID LEUKEMIA

F. G. Ruecker 1,* , A. Corbacioglu 1 , F. Theis 1 , M. Christopeit 2 , U. Germing 3 , G. Wulf 4 , M. Abu Samra 5 , L. Teichmann 6 , M. Lübbert 7 , M. W. Kühn 8 , M. Bentz 9 , J. Westermann 10 , L. Bullinger 10 , V. I. Gaidzik 1 , E. Jahn 1 , M. Gröger 1 , S. Kapp-Schwoerer 1 , D. Weber 1 , F. Thol 11 , M. Heuser 11 , A. Ganser 11 , H. Döhner 1 , K. Döhner 1

1 Department of Internal Medicine III, University Hospital of Ulm, Ulm; 2 Medical Clinic, Department of Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, Tübingen; 3 Department of Hematology, Oncology and Clinical Immunology, Heinrich Heine University, Düsseldorf; 4 University Medical Center Göttingen, Göttingen; 5 Department of Internal Medicine IV, University Hospital of Gießen, Gießen; 6 Department of Medicine and Polyclinic III, Bonn University Hospital, Bonn; 7 Department of Hematology, Oncology and Stem Cell Transplantation, University of Freiburg Medical Center, Freiburg; 8 Department of Hematology, Medical Oncology and Pneumology, University Medical Center Mainz, Mainz; 9 Department of Internal Medicine III, Hospital of Karlsruhe, Karlsruhe; 10 Department of Hematology, Oncology and Tumor Immunology, Charité University Medicine, Berlin; 11 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Hannover, Germany

Background: Mutations of CEBPA ( CEBPA mut ) are present in ~5-10% of newly diagnosed adult acute myeloid leukemia (AML), and approximately half of the patients (pts) exhibit biallelic mutations ( CEBPA bi ). CEBPA bi defines a distinct entity within the 2016 WHO classification and is categorized as favorable in the 2017 risk stratification by the European LeukemiaNet. CEBPA mut can be divided into basic leucine zipper domain (bZIP) or transcription activation domains (TAD) mutations, respectively. Recent studies have demonstrated CEBPA bZIP mutations, in particular in-frame mutations ( CEBPA bZIP-inf ), to be associated with favorable outcome, regardless of mono- or biallelic status.

Aims: To evaluate the prognostic impact of CEBPA bZIP , in particular CEBPA bZIP-inf mutations in AML.

Methods: Investigating a cohort of 454 intensively treated CEBPA mutated AML pts entered into the AMLSG BiO Registry study ( NCT01252485 ).

Results: Of the 454 pts, 223 had CEBPA bi and 231 monoallelic CEBPA mutations ( CEBPA sm ) affecting bZIP in 78 pts or TAD in 153 pts. Genotypes differed significantly with regard to clinical and genetic features: CEBPA bi pts were younger than CEBPA smbZIP and CEBPA smTAD (median age in yrs: 52 vs 59 vs 60; P <.001), had a higher rate of de novo AML (97% vs 81% vs 84%; P <.001), lower platelet counts (median G/l 36 vs 57 vs 52; P <.001), higher peripheral blood (PB) blast counts (median 71% vs 60% vs 44%; P <.001), and showed an inverse correlation with FLT3 internal tandem duplication ( FLT3 -ITD) and NPM1 mutation ( NPM1 mut ) (6% vs 21% vs 35% and 0% vs 26% vs 50%; P <.001 each).

Outcome analysis revealed a significant improved overall (OS) and event-free survival (EFS) for CEBPA bi with no difference between CEBPA smbZIP and CEBPA smTAD (5-year OS: 62% vs 42% vs 51%; P <.001, 5-year EFS: 47% vs 27% vs 43%; P <.022). Subgroup specific analysis within CEBPA smbZIP revealed an improved outcome for CEBPA bZIP-inf pts (n=46) (5-year OS: 52% vs 29%; P =.001, and 5-year EFS: 34% vs 18%; P= .018).

To further address the impact of CEBPA bZIP-inf , pts were categorized as CEBPA bZIP-inf (n=250), irrespective of the allelic status, vs all others ( CEBPA other ) (n=204). CEBPA bZIP-inf pts were younger (median age in yrs: 52 vs 62; P <.001), had a higher rate of de novo AML (97% vs 81%; P <.001), lower platelet counts (median G/l 36 vs 53; P <.001), higher white blood cell (WBC) (median G/l 25.4 vs 16.1; P =.029), and higher PB blast counts (median 72% vs 46%; P <.001); FLT3 -ITD and NPM1 mut were less common in CEBPA bZIP-inf pts (9% vs 30% and 3% vs 44%; P <.001 each). CEBPA bZIP-inf exhibited a significant improved OS (5-year OS: 60% vs 48%; P <.001) and EFS (5-year EFS: 47% vs 37%; P =.007); in multivariate Cox models for OS and EFS, including allogeneic hematopoietic cell transplantation (HCT) in first complete remission as time-dependent covariate, age (HR: 1.50; P <.001), WBC (HR: 1.40; P =.018), and adverse cytogenetics (HR: 2.33; P =.010) were unfavorable factors for OS, whereas NPM1 mut (HR: 0.63; P =.040), HCT (HR: 0.46; P =.010), and CEBPA bZIP-inf (HR: 0.59; P =.007) revealed as favorable. For EFS, age (HR: 1.19; P =.002), WBC (HR: 1.30; P =.028), and FLT3 -ITD (HR: 1.83; P =.003) were unfavorable, whereas NPM1 mut (HR: 0.65; P =.028) and HCT (HR: 0.33; P <.001) were favorable.

Summary/Conclusion: In this cohort of 454 CEBPA mutated adult AML pts, CEBPA bZIP-inf was associated with specific clinical and genetic characteristics. Furthermore, CEBPA bZIP-inf pts had a significantly superior outcome irrespective of the allelic status. This study confirms recent findings suggesting a prognostic role of this mutation type.

FGR and AC contributed equally

P449: NOVEL, FIRST-IN-CLASS, SOMATIC IDH2 DELETION-INSERTION VARIANT C.516_518DELINSTGC CONFERS BORDERLINE PHENOTYPE AND DOES NOT SHOW SUSCEPTIBILITY TO ENASIDENIB IN-VITRO

L. Ruhnke 1,* , D. M. Poitz 2 , S. Herold 3 , S. Dressler 4 , C. Dill 1 , M. Peitzsch 2 , U. Oelschlägel 1 , J. Frimmel 1 , D. Kunadt 1 , T. Kretschmann 1 , H. Altmann 1 , F. Stölzel 1 , C. Röllig 1 , T. Chavakis 2 , G. Baretton 3 , K. Schäfer-Eckart 4 , M. Bornhäuser 1

1 Department of Internal Medicine I; 2 Institute of Clinical Chemistry and Laboratory Medicine; 3 Institute of Pathology, University Hospital Dresden, Dresden; 4 Department of Internal Medicine V, Nuremberg Hospital North, Paracelsus Medical University, Nuremberg, Germany

Background: Mutations in IDH2 are among the most common genetic alterations in patients with acute myeloid leukemia (AML). So far, only substitution variants (also known as single nucleotide variants (SNVs)) - in particular affecting the hotspots at codon Arg140 and Arg172 - have been identified.

.

Aims: Here we report and functionally characterize a novel, first-in-class, somatic IDH2 deletion-insertion (delins) variant affecting codons Arg172/His173 identified in a patient diagnosed with AML with myelodysplasia related changes (AML-MRC).

Methods: The novel delins variant was identified performing amplicon-based targeted next generation sequencing (NGS), Sanger sequencing was used for verification. For functional evaluation high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS), global DNA methylation and DNA hydroxymethylation assays and in-vitro drug testing was performed; AML patients without evidence of IDH1 or IDH2 mutations and patients with IDH2 Arg172 mutation served as negative and positive controls, respectively.

Results: A 63-year-old male patient (unique patient number (UPN) 1) was diagnosed with AML-MRC. Further work-up revealed an aberrant karyotype (47,XY,+11/ 46,XY,der(7)t(7;11)(q31;q12)/ 46,XY), a partial tandem duplication of KMT2A ( KMT2A -PTD) as well as variants in IDH2 , DNMT3A and NRAS (Figure 1.) (no CBFB::MYH11 , PML::RARA or RUNX1::RUNX1T1 fusion, no ASXL1 , CEBPA , FLT3 , NPM1 , RUNX1 or TP53 mutation) (Figure 1A). He received induction therapy with liposomal daunorubicin/cytarabine, achieved CR and underwent allogeneic hematopoietic cell transplantation.

Of note, the IDH2 variant identified was a novel three base-pair deletion-insertion variant (c.516_518delinsTGC) leading to the replacement of amino acids arginine and histidine at codon 172 and 173 for serin and alanine (p.Arg172_His173delinsSerAla), variant allel frequency was 44%. The novel delins variant could be validated performing Sanger sequencing; parallel Sanger sequencing of buccal saliva DNA ruled out germline origin (Figure 1B). Since there are no valid in-silico prediction tools for delins variants, we decided to assess the potential pathogenicity of the novel alteration. First, we applied LC-MS/MS to evaluate 2-hydroxyglutarate (2-HG) levels as compared to BM-MNCs obtained from IDH1/IDH2 WT AML patients (UNP2-4) and IDH2 MUT AML patients (UPN5-10). In line with previous reports, 2-HG levels were increased in patients with IDH2 mutations as compared to their IDH1 / IDH2 WT counterparts (1000-fold). However, in UPN1 only slightly elevated 2-HG levels were found (10-fold) (Figure 1C); no differences in isocitrate or ketoglutarate levels were seen (data not shown). Similar results were obtained when performing DNA methylation and DNA hydroxymethylation studies. While the percentage of methylated DNA in UPN1 was comparable to the amount of 5-methylcytosine (5-mC) seen in the IDH2 mutated group (UNP5-10), 5-hydroxymethylcytosine (5-hmC) levels in UPN1 tended towards those observed in the IDH2 WT cohort (UPN2-4) (data not shown). Finally, we performed in-vitro drug testing to evaluate response to the IDH2 inhibitor (IDH2i) enasidenib. Interestingly, enasidenib did not affect granulocytic differentiation of BM-MNCs as assessed by CD11b, CD15 and CD16 flow cytometry (Figure 1D).

Image:

Иллюстрация — в первоисточнике ↗

Summary/Conclusion: Here, we report a novel, somatic delins variant in IDH2 , which, albeit affecting the known Arg172 hotspot, does not show susceptibility to the IDH2i enasidenib in-vitro . Our report argues for functional characterisation of novel variants to ensure a valid biomarker-driven therapy in AML patients.

P450: PRECLINICAL AND CLINICAL SIGNS OF RVU120 EFFICACY, A SPECIFIC CDK8/19 INHIBITOR IN DNMT3A MUTATION POSITIVE AML AND HR-MDS

U. Pakulska 1 , N. Angelosanto 2 , M. Mikula 3 , H. Nogai 2 , R. Dudziak 2 , C. Abboud 4 , M. Obacz 1 , K. Goller 1 , M. Cybulska 3 , M. Mazan 1 , M. Kozakowska 1 , T. Rzymski 1,*

1 R&D; 2 Clinical Department, Ryvu Therapeutics, Kraków; 3 Maria Skłodowska-Curie Institute - Cancer Center, Warsaw, Poland; 4 Washington University School of Medicine in St. Louis, St Louis, United States of America

Background: CDK8 and its paralog CDK19 regulate transcription as a part of mediator complex, that links enhancers with core promoters. AML likewise many other cancers highjack CDK8 and CDK19 to maintain undifferentiated state and prevent apoptotic cell death. First CDK8/CDK19 inhibitor RVU120 has reached clinical development phase Ib ( NCT04021368 ) in AML and HR-MDS patients.

Aims: It is now critical to establish relationship between preclinical and clinical efficacy results, and molecular characteristics in order to identify actionable markers predicting response to CDK8/CDK19 inhibitors.

Methods: Association of molecular profiles with responses to RVU120 has been performed on genetically annotated AML PDCs, followed by flow cytometry and bioinformatic analysis. PDCs were implanted intravenously into NSG-SGM3 mice and after disease onset animals were treated orally with RVU120. Profiling of transcriptional response to RVU120 in DMNT3A mutant cells has been performed by RNA-seq. The First in Human study of RVU120 is currently active enrolling R/R AML or HR-MDS patients, in a dose escalation design aimed at exploring safety/tolerability and identify the RPD2. RVU120 is administered orally, each other day for 7 total doses per 21 days cycle, until disease progression/unacceptable toxicity. Response to the study drug is assessed according to Dohner 2017 criteria.

Results: Screening of AML PDC against RVU120 indicated high anti-cancer efficacy in >40% of tested samples. Correlation of efficacy with genetic profile of samples indicated specific enrichment of DNMT3A or NPM1 mutants in responder group. These results were further corroborated in disseminated PDX AML model, showing complete clearance of DNMT3A and NPM1 mutation- positive blasts and recovery of murine BM in animals treated with RVU120. Transcriptomic analysis of DNMT3A and NPM1 positive AML cells indicated distinct transcriptomic profiles, characterized by high expression of homeobox genes, involved in determination of cell faith. Immunophenotyping of responder cells indicated elimination of lineage committed blast cells.

At the date of this abstract submission, 13 patients have been enrolled into CLI120-001 trial, including 2 patients with DNMT3A mutations. Notably, first R/R AML patient that achieved CR was positive for DNMT3A and NPM1 mutations. At study entry this patient was progressing with pancytopenia, 65% BM blasts and extramedullary localization of leukemia. BM showed a complete clearance of blasts at the end of 1 st cycle of 75mg dose, followed by hematological recovery and signs of monocytic differentiation. Importantly, monocytic differentiation is observed as a pharmacodynamic effect of RVU120 in preclinical studies. Skin leukemia lesions improved gradually up to a CR in cycle 7. Second DNMT3A mutation patient with HR-MDS, escalated from 50 to 75 mg dose from cycle 7, continues treatment at the cycle 24 with SD and erythroid hematological improvement.

Summary/Conclusion: AML PDCs with DNMT3and NPM1 mutations show differential sensitivity to RVU120 treatment both in vitro and in vivo. Anti-cancer efficacy of RVU120 was associated with transcriptomic reprogramming and lineage commitment. Preliminary evidence of clinical response to RVU120 has been also shown in R/R AML and HR-MDS patients positive for DNMT3A mutations. Further molecular studies in greater number of patients under RVU120 treatment are ongoing and will provide evidence for predictive markers of response to RVU120 in AML.

P451: PRMT2: AN ANTI-INFLAMMATORY EPIGENETIC FACTOR INVOLVED IN ACUTE MYELOID LEUKEMIA AGGRESSIVENESS

C. Sauter 1,* , J. Simonet 1 , C. Fournier 1,2 , M. Mounier 1,3 , M. Rebourgeon 1 , L. Brignoli 1 , A. Aznague 1,2 , A. Largeot 1 , Y. Hérault 4 , G. Sauvageau 5 , F. Guidez 1 , M. Callanan 1,2 , J.-N. Bastie 1,6 , L. Delva 1 , R. Aucagne 1,2

1 UMR 1231 Inserm, Équipe Epi2THM, Équipe Labex LipSTIC, Université de Bourgogne Franche-Comté; 2 Unité d’Innovation en Génétique et Épigénétique en Oncologie (IGEO), Plateforme CRISPR Genomics (CRIGEN), CHU François-Mitterrand; 3 Registre des hémopathies malignes de Côte d’Or, Université de Bourgogne Franche-Comté, Dijon; 4 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département of de Génomique Fonctionnelle, UMR 7104 CNRS, UMR 964 Inserm, Université Louis Pasteur, Collège de France et Institut Clinique de la Souris (ICS), Illkirch, France; 5 Laboratoire de Génétique Moléculaire des Cellules Souches, Institut de Recherche en Immunologie et Cancer, Université de Montréal, Montréal, Canada; 6 Service d’Hématologie Clinique, CHU François-Mitterrand, Dijon, France

Background: Protein Arginine Methyltransferases (PRMTs) are epigenetic factors involved in several cellular processes including regulation of gene expression through methylation of histone tails. Overexpression or overactivity of the two major PRMTs (1 and 5) have been previously identified in Acute Myeloid Leukemia (AML) patients and their biological or pharmacological inhibition leads to a decreased leukemia progression in mice (PRMT1, 4, 5). Among the nine members of this protein family, PRMT2 has been less well characterized. It has been shown to be overexpressed in glioblastoma and is responsible for the H3R8me2a epigenetic mark leading to transcription activation of target genes. In addition, PRMT2 has also been demonstrated to have a role in the inflammatory pathway but its effect is not fully understood yet. Moreover, it is well known that severe chronic inflammation can contribute to the development of certain cancers. Exacerbated inflammatory responses and aberrant myeloproliferation are thus interconnected.

Aims: We are investigating the role of PRMT2 in the development of AML and we aim to discover its precise role in the control of inflammatory processes.

Methods: We used a PRMT2 knockout mouse model (Prmt2 KO ), AML patient cohorts of Dijon (Dijon university hospital, France) and Montréal (IRIC, QC, Canada). We also generated a knockout human AML HL-60 cell line using the CRISPR-Cas9 system and a HL-60 cell line overexpressing PRMT2 through the use of a Citrine-tagged fusion protein.

Results: Prmt2 KO mice do not exhibit difference in bone marrow progenitor or mature cell populations compared to wild type mice, indicating that PRMT2 has little or no influence in the hematopoietic stem cell maintenance. In contrast, we observed that Prmt2 KO bone marrow-derived macrophages (BMDM) are more sensitive to lipopolysaccharides (LPS) stimulation and express higher levels of pro-inflammatory cytokine mRNA, suggesting a role of PRMT2 in the negative regulation of the inflammatory processes. Analysis of datasets from a cohort of 371 AML patients (Guy Sauvageau, Leucegene project, IRIC, Montréal, QC, Canada) reveals that patients with a lower PRMT2 expression display a worse survival rate compared to patients with a higher PRMT2 expression. Gene Set Enrichment Analysis of this cohort show that PRMT2 is involved in the inflammatory response signaling pathway, thus confirming our hypothesis. In a cell model we show that PRMT2 knockout cells exhibit higher levels of phosphorylated STAT3 protein compared to wild type cells after LPS stimulation. PRMT2 could therefore be involved directly or indirectly in the STAT3 pathway by preventing its phosphorylation and activation. Ongoing transcriptomic and epigenetic studies could provide new clues for better understanding the role of PRMT2 in an inflammatory context in AML and the association between inflammation and leukemogenesis.

Summary/Conclusion: Taken together, our data suggest that PRMT2 has an anti-inflammatory effect in mice and AML patients with a low PRMT2 expression inducing a higher inflammatory phenotype, thus resulting to worse overall survival. PRMT2 could be involved in the STAT3 signaling pathway by preventing its activation leading to increased inflammation.

P452: THE INTERACTOME OF CDK6 IN DIFFERENT ONCOGENIC CONTEXTS

L. Scheiblecker 1,* , S. Nebenfuehr 1 , M. Zojer 1 , E. Doma 1 , V. Sexl 1 , K. Kollmann 1

1 Institute for Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria

Background: The mammalian cell cycle is controlled by a complex signaling network that is frequently deregulated in cancer. Cyclin-dependent kinases (CDKs) are the key players in governing cell cycle progression.

CDK6 and its close homologue CDK4 are responsible for driving the cell cycle from G1 to S-Phase. In complex with their regulatory subunits, the D-type cyclins, CDK4/6 phosphorylate the retinoblastoma protein family members (Rb) to induce G1 to S-Phase progression and DNA replication.

CDK6, and not CDK4, plays an important role in hematopoiesis and is frequently found overexpressed in hematologic malignancies. In the recent years, CDK6 has been shown to regulate transcription by interacting with chromatin in a kinase dependent and independent manner. This function is crucial in leukemia as well as hematopoietic stem cells.

Aims: To better understand the transcriptional role of CDK6 in transformed and untransformed cells we will analyze the interactome of CDK6. We hypothesize that different oncogenes lead to the expression of different CDK6-dependent transcriptional programs and to different CDK6-containing protein complexes on DNA level. We aim to identify common and leukemia subtype-specific interaction partners of CDK6 that will represent novel therapeutic vulnerabilities in combination with CDK6 inhibition.

Methods: To characterize the interactome of CDK6 we transformed the murine hematopoietic progenitor cell line HPC7 with the BCR-ABL or the MLL-AF9 oncogene that give rise to myeloid leukemia. We isolated CDK6-containing protein complexes by immunoprecipitation followed by mass spectrometry. To extend our data and to get a specific set of interaction partners involved in the transcriptional role of CDK6, we performed a nuclear fractionation prior to immunoprecipitation and mass spectrometry. This enabled us to focus on interaction partners solely in the nucleus.

Results: We obtained proteins interacting with CDK6 on a global cell level as well as specific for the nucleus that represent co-factors of CDK6-mediated transcriptional regulation. By intersecting the proteomics data from transformed and untransformed HPC7 cells, we detected interaction partners of CDK6 that are common for all tested leukemia entities, specific for leukemic subtypes and interaction partners which just occur in untransformed cells. As a next step, the determined proteins will be subjected to pathway analysis to reveal CDK6 dependent signaling networks. These proteins might serve as valuable targets for potential combinatorial treatments together with CDK6 inhibition.

Summary/Conclusion: Combining our results with next generation sequencing data will allow us deciphering the distinct transcriptional signatures regulated by CDK6 in different leukemia entities. Our insights will provide a better understanding of malignancies with enhanced CDK6 expression and may help to develop novel treatment strategies by interfering with CDK6 interaction partners.

P453: CDK6 DEGRADATION IS IMPEDED BY P16INK4A AND P18INK4C IN AML

B. Schmalzbauer 1,* , T. Thondanpallil 1 , G. Heller 2 , C.-M. Sperl 1 , I. M. Mayer 1 , V. M. Knab 1 , S. Nebenfuehr 1 , M. Zojer 1 , A. C. Mueller 3 , F. Fontaine 3 , T. Klampfl 1 , V. Sexl 1 , K. Kollmann 1

1 Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna; 2 Department of Medicine I, Division of Oncology, Medical University of Vienna; 3 CeMM – Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

Background: Acute myeloid leukemia (AML) is a highly aggressive disease that comprises a heterogeneous group of genetically distinct subtypes. AML is initiated by leukemic cells, which are able to self-renew and give rise to malignant myeloid blasts. A key regulator and prognostic biomarker for AML is the cell cycle regulator Cyclin-dependent kinase 6 (CDK6) which represents a novel therapeutic target for the treatment of certain subtypes of AML. CDK4/6 kinase inhibitors are extensively studied in several cancer types but the beneficial effects may be limited by primary and secondary resistance mechanisms. Pharmacological CDK6 degraders, which eliminate kinase -dependent and -independent effects, represent an alternative therapeutic option. The exact mechanism and efficacy of CDK6 degraders in AML subtypes remain unknown. The involvement of p16 INK4A in Palbociclib resistance and the role of INK4 proteins in AML disease progression require a systematic investigation of INK4 proteins in the context of CDK6 degradation in distinct AML subtypes.

Aims: We aim to elucidate CDK6 degrader efficacy in two CDK6-dependent AML subtypes harboring either MLL-AF9 or AML1-ETO. We hypothesize an involvement of INK4 proteins on CDK6 degrader efficacy. Understanding the exact mechanisms of the CDK6 degrader will help to determine AML subtypes that would benefit from CDK6-targeted therapy.

Methods: We took advantage of a novel hematopoietic progenitor cell model (HPC LSK s) to study CDK6 degrader efficacy in a genetically defined setting of AML subtypes. We analyzed INK4 RNA expression of human AML patients and the HPC LSK cells using RNA-Seq and Microarray data sets. We analyzed cell proliferation and cell cycle changes using flow cytometry. CDK6 Co-immunoprecipitation followed by Mass Spectrometry was performed to screen for CDK6 interaction partners that are enriched upon CDK6 Degrader treatment. These findings were validated via immunoblotting. Human AML cell lines were used to further validate the results in a different in vitro system and to translate them to human settings.

Results: We show that efficacy of the CDK6 specific protein degrader varies among AML subtypes and depends on low expression of the INK4 proteins p16 INK4A and p18 INK4C . INK4 protein levels are significantly elevated in MLL-AF9+ compared to AML1-ETO+ cells, contributing to the different CDK6 degradation efficacy. We demonstrate that CDK6 complexes containing p16 INK4A or p18 INK4C are protected from pharmacological degradation and that INK4 levels define the proliferative response to CDK6 degradation. These findings define INK4 proteins as predictive marker for CDK6 degradation – targeted therapies in AML.

Summary/Conclusion: We here identify p16 INK4A and p18 INK4C as dominant CDK6 binding partners that counteract pharmacological protein degradation in AML. Our data highlight the need for novel CDK6 specific therapies targeting CDK6 sites not competing with INK4 or CIP/KIP binding to overcome therapeutic limitations.

P454: VENETOCLAX AND GILTERITINIB SYNERGIZE IN FLT3 WILDTYPE ACUTE MYELOID LEUKEMIA BY SUPPRESSION OF MCL-1 VIA COMBINED AXL AND FLT3 TARGETING

C. Schmidt 1,* , M. Janssen 1,2 , P.-M. Bruch 1,2 , M. F. Blank 1,2,3 , C. Rohde 1,2 , A. Waclawiczek 4 , S. Renders 1,4 , D. Heid 1,2 , S. Göllner 1 , L. Vierbaum 1 , K. Weidenauer 1 , S. Herbst 1 , M. Knoll 1 , C. Kolb 1 , B. Besenbeck 1 , M. Fabre 5,6 , M. Gu 5,6 , R. Schlenk 1 , F. Stölzel 7 , M. Bornhäuser 7 , C. Röllig 7 , U. Platzbecker 8 , C. Baldus 9 , H. Serve 10 , T. Sauer 1 , S. Raffel 1 , C. Pabst 1,2 , G. Vassiliou 5,6,11 , B. Vick 12,13,14 , I. Jeremias 12,13,14,15 , A. Trumpp 4 , J. Krijgsveld 3,16 , C. Müller-Tidow 1,2 , S. Dietrich 1,2

1 Department of Medicine V, Hematology, Oncology and Rheumatology, University hospital Heidelberg; 2 Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 3 Division Proteomics of Stem Cells and Cancer; 4 Division of Stem Cells and Cancer, German Cancer Research Centre (DKFZ), Heidelberg, Germany; 5 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton; 6 Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom; 7 Department of Medicine I, University hospital Carl Gustav Carus, Dresden; 8 Medical Clinic and Policlinic I, Hematology and Cellular Therapy, Leipzig University Hospital, Leipzig; 9 Department of Hematology and Oncology, University hospital Schleswig-Holstein, Campus Kiel, Kiel; 10 Department of Medicine II, Hematology-Oncology, Goethe University Hospital Frankfurt, Frankfurt am Main, Germany; 11 Department of Hematology, University of Cambridge, Cambridge, United Kingdom; 12 Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München; 13 German Research Center for Environmental Health (HMGU); 14 German Consortium for Translational Cancer Research (DKTK); 15 Department of Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig Maximilians University München, Munich; 16 Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany

Background: BCL-2 inhibition has been shown to be effective in older patients with acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, treatment resistance and relapse represent major clinical challenges. Thus, there is an unmet need to overcome resistance to current venetoclax-based regimens.

Aims: In order to establish an alternative treatment approach for AML in elderly patients, we aimed at identifying a highly synergistic drug combination partner for venetoclax.

Methods: We performed a high-throughput drug screening to identify the most effective combination partner for venetoclax in AML. Overall, 64 anti-leukemic drugs were screened in 31 primary high-risk AML samples in the presence or absence of venetoclax. We calculated drug synergies, validated top venetoclax combination partners in AML cell lines and primary patient samples and performed mechanistic analyses.

Results: Established anti-leukemic drugs exhibited only weak synergism with venetoclax in high-risk AML samples. In contrast, when targeting distinct pathways such as FLT3 signaling, we observed increased synergistic activity. Gilteritinib exhibited highest synergy with venetoclax in FLT3 wildtype AML, with especially high synergy values in TP53 mutated samples. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability and abolished colony-formation potential in FLT3 wildtype as well as venetoclax-azacitidine resistant cell lines and primary patient samples. Proteomics revealed increased levels for proteins involved in FLT3 wildtype signaling in specimens with low in-vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159 and proteasomal degradation. Compared to other FLT3 inhibiting drugs, gilteritinib showed highest synergism with venetoclax due to the additional inhibition of AXL that reinforced the downregulation of MCL-1.

Gilteritinib and venetoclax proved efficacious in an FLT3 wildtype PDX model with TP53 mutation. Furthermore, the drug combination reduced leukemic burden in four patients with venetoclax-azacitidine refractory AML, who received gilteritinib and venetoclax as an individual treatment approach.

Summary/Conclusion: In summary, our results identified gilteritinib as a potential therapy combination partner for venetoclax in patients with FLT3 wildtype AML.

P455: BREAKING THE PUMP: TARGETING THE SODIUM-POTASSIUM PUMP AS A THERAPEUTIC STRATEGY IN ACUTE MYELOID LEUKEMIA

C. Schneider 1,2,* , H. Spaink 1 , G. Alexe 1,2 , N. V. Dharia 1,2 , D. Khalid 1 , S. Scheich 3,4 , B. Haeupl 4,5 , T. Oellerich 4,5 , K. Stegmaier 1,2

1 Pediatric Oncology, Dana-Farber Cancer Institute, Boston; 2 The Broad Institute of MIT and Harvard, Cambridge; 3 Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, United States of America; 4 Department of Medicine II, Department for Hematology/Oncology, Goethe University, Frankfurt; 5 German Cancer Research Center and German Cancer Consortium, Heidelberg, Germany

Background: Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy with poor overall survival despite intensive cytotoxic chemotherapy and newer targeted agents. With a 5-year survival rate of only 29% in adults with AML, new therapeutic strategies are needed.

Aims: In order to identify novel, selective targets for AML, we used the Cancer Dependency Map, which provides genome-scale CRISPR/Cas9 depletion screen data in hundreds of cancer cell lines. Here, we identified ATP1B3 as a context-specific dependency that could be therapeutically exploited.

ATP1B3 is the smaller glycoprotein subunit (beta) of the sodium-potassium pump (Na/K-ATP pump). Together with the catalytic alpha subunit ATP1A1, it forms a heterodimer located in the plasma membrane, regulating the electrochemical gradient through the transport of Na and K ions across the membrane. The Na/K-ATP pump is of vital importance in the maintenance of cellular homeostasis and membrane potential and because of its function as a receptor and signal transducer. While the ATP1A1 carries out ion transport and enzymatic activity, making it a common essential gene (a gene which ranks among the most depleted genes in at least 90% of screened cell lines), ATP1B3 is not essential in all cancer cells. This beta subunit seems to be crucial for intracellular transport and stabilization of the alpha subunit in the membrane. The beta subunit has 4 paralogs, showing similar expression patterns among tissues, with one exception, ATP1B1.

Methods: We used a diverse panel of functional genomic based assays, validating our finding using CRISPR/Cas9 knockout and overexpression constructs for the Na/K-ATP pump beta subunit paralogs. In an approach to analyze protein-protein interactions we generated BioID constructs for a mass spectrometry-based analysis. For an in vivo study we used a bioluminescent imaging (BLI)-based orthotopic mouse model of AML, carrying non-targeting, ATP1B1 or ATP1B3 knockout guides.

Results: By using CRISPR/Cas9 knockout we could validate ATP1B3 as a selective dependency and performing competitive growth assays we showed that loss of ATP1B3 in ATP1B1 low expressing AML cells leads to synthetic lethality. The absence of both paralogs of the beta subunit results in the loss of their common essential binding partner ATP1A1 in hematologic malignancies, while higher expression of ATP1B1 can stabilize ATP1A1 under the loss of ATP1B3 in solid tumors or in ATP1B1 overexpressing AML cells.

Next, we validated our findings in vivo . In a BLI-based orthotopic mouse model of AML, we found that the AML model with ATP1B3 knockout showed lower leukemia burden and normal spleen weight in comparison to a non-targeting or ATP1B1 knockout control. To understand the specific role of ATP1B3 we produced BioID (proximity-dependent biotin identification) constructs for ATP1B1 and ATP1B3. In ongoing studies, through this mass spectrometry-based analysis of protein-protein interactions, we will gain a greater insight into the protein partners of the Na/K-ATP pump.

Summary/Conclusion: Taken together, we identified ATP1B3 as a selective dependency in AML. We propose that the elimination of ATP1B3 leads to the destabilization of the sodium-potassium pump when ATP1B1 levels are low, making it a potential tumor-selective therapeutic target for AML and other hematologic malignancies with low expression of ATP1B1.

P456: A LONG READ SEQUENCING AND CRISPR-CAS9 BASED APPROACH FOR RAPID COPY-NUMBER ALTERATION AND STRUCTURAL VARIATION DETECTION IN HEMATOLOGIC MALIGNANCIES

J. Schrezenmeier 1,2,* , J. E. Straeng 1 , O. Blau 1 , A. Elashy 1 , A. Lazarides 1 , S. Skambraks 3 , E. Jahn 3 , B. Gillißen 1 , C. Eckert 4 , A. Nogai 1 , I.-W. Blau 1 , K. Giannopoulos 5 , M. Heuser 6 , K. Döhner 3 , L. Bullinger 1 , A. Dolnik 1

1 Hematology, Oncology and Tumorimmunology, Charité-Universitätsmedizin Berlin; 2 Berlin Institute of Health at Charité – Universitätsmedizin Berlin, BIH Biomedical Innovation Academy, Berlin; 3 Department of Internal Medicine III, University of Ulm, Ulm; 4 Pediatric Hematology and Oncology, Charité-Universitätsmedizin Berlin, Berlin, Germany; 5 Department of Experimental Hematooncology, Medical University of Lublin, Lublin, Poland; 6 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany

Background: Current standard karyotyping methods are labor-intensive and have severe limitations in terms of resolution and duration to results. For hematologic malignancies, but not restricted to, this represents a relevant obstacle in translating molecular findings into time critical treatment decisions.

Aims: To develop a sequencing based approach for rapid and scalable cytogenetics that can be applied to a wide spectrum of hematologic malignancies to detect clinically relevant molecular markers within 48hrs

Methods: For copy number alteration detection, whole genome sequencing was performed on a GridION sequencer (Oxford Nanopore Technologies, ONT, using the SQK-LSK109 kit) in a cohort of 53 hematologic neoplasms, including acute myeloid leukemia (AML, n=18), chronic lymphocytic leukemia (CLL, n=8), multiple myeloma (MM, n=17) and pediatric acute lymphocytic leukemia (ALL, n=10) samples. For the identification of balanced alterations, transcriptome sequencing data were generated using ONT (SQK-DCS109 kit), and we applied an in-house developed analysis pipeline based on minimap2 alignment followed by Blast with an ensuing filtering algorithm based on orientation, inter-read distance and length filtering. For targeted sequencing of structural aberrations, we developed a CRISPR-Cas9 based tiling approach using sgRNA pools (Integrated DNA Technologies, IDT). This approach allows to cover AML relevant targets such as t(8;21), the KMT2A (MLL) breakpoint area, and the FLT3-ITD region as well as lymphoma relevant regions such as the immunoglobulin heavy chain locus (library preparation was performed using SQK-CS9109 kit).

Results: For copy number profiling a median whole-genome coverage of 2.6 was reached (range 0.27-7 fold). ONT sequencing and conventional karyotyping approaches showed a high concordance with Pearson correlation coefficients >0.95 for copy number alteration comparisons between conventional cytogenetics and ONT sequencing results for all investigated disease entities. Regarding the detection of structural aberrations transcriptome sequencing and fusion gene analysis using the above described analysis and filtering pipeline we could e.g. reliably detect the t(9;22) translocation from the K562 cell line and the t(8;21) translocation from Kasumi-1. Additionally, using this method we were able to correctly identify the primary AML sample harboring a t(8;21) translocation that was among the n=12 AML patient samples. In addition to an RNA-based work-flow, we established a CRISPR-Cas9 based DNA enrichment approach for the detection of recurrent structural aberrations from specified genomic DNA loci. The enrichment of genomic regions of interest using sgRNA libraries led e.g. to a median coverage of 44 reads (range 5-136-fold) of the t(8;21) region-of-interest in primary AML samples, which allow the detection of the exact coordinates of the breakpoints.

Summary/Conclusion: Long read based sequencing based copy number alteration and structural variation detection based on combination of different long read sequencing approaches like low-coverage whole genome sequencing, transcriptome sequencing and CRISPR-Cas9 based sequencing of genomic loci of interest represents a highly promising tool for high resolution and high speed cytogenetics that has the potential to overcome many limitations of conventional cytogenetics.

P458: TRANSCRIPTIONAL AND TRANSLATIONAL SIGNATURES OF TRIPLE MUTATED DNMT3AKO/FLT3ITD/NPM1C MICE SHOW ALTERED RNAS PROCESSING AND CELL CYCLE PROGRESSION

A. Scialdone 1,* , D. Sorcini 1 , A. Stella 1 , V. Tini 1 , A. Marra 1 , C. Rompietti 1 , F. De Falco 1 , E. Dorillo 1 , F. M. Adamo 1 , E. C. Barcelos 1 , R. Arcaleni 1 , M. P. Martelli 1 , P. Sportoletti° 1 , B. Falini°;° co-last author 1

1 Medicine and Surgery, University of Perugia, Perugia, Italy

Background: The top ranking recurrently mutated genes in AML are NPM1 , FLT3 and DNMT3A 1,2 . Co-occurrence of these three mutations is associated to a unique AML subset characterized by high peripheral blood and bone marrow blast counts, normal cytogenetic and very poor EFS and OS 3 . Nevertheless, the cooperative leukemogenic mechanisms of these three mutations have not been deeply explored and a successful targeting treatment of AML patients DNMT3Amut/ FLT3 -ITD/ NPM1 c still represents an unmet clinical need.

Aims: To dissect how DNMT3A loss of function, FLT3 Internal Tandem Duplication (ITD) and NPM1 mutations ( NPM1 c ) cooperate in driving AML onset and progression through a picture of the transcriptome and the translatome of triple mutated AML cells.

Methods: Mx1-Cre- NPM1 c and/or Mx1-Cre- DNMT3A KO and FLT3 ITD mutant mice were crossed to obtain the following lines: DNMT3A KO / FLT3 ITD / NPM1 c , FLT3 ITD / DNMT3A KO and FLT3 ITD / NPM1 c . Peripheral blood (PB) was periodically taken and analysed, previous anesthesia, while bone marrow (BM) and spleen were harvested two months after pIpC induction or at conclamate disease (n=7 each group). To investigate the mechanisms underlying the triple mutated mice phenotype, we looked BM leukemic samples from all groups both at transcriptional level by RNA sequencing (RNAseq) and at translational level (protein synthesis capacity of AML blasts) by Ribosome footprinting (Riboseq) 4 . Unique Differentially Expressed Genes (DEGs) (filtered by p -value<0.05 and FC >1.5) belonging to FLT3 ITD / DNMT3A KO / NPM1 c vs FLT3 ITD / DNMT3A KO were identified by Venn diagram and used in enrichment analysis on Reactome database and MSigDB (C6-oncogenic signature).

Results: The FLT3 ITD/ DNMT3A KO / NPM1 c, DNMT3A KO / FLT3 ITD and FLT3 ITD / NPM1 c displayed a fully penetrant leukemic phenotype. As expected, FLT3 ITD/ DNMT3A KO / NPM1 c showed a more aggressive disease characterized by higher WBC counts, more pronounced blasts infiltration of the spleen and shorter survival (>2 fold vs double mutants). The FLT3 ITD/ DNMT3A KO / NPM1 c mice displayed 1429 (842 UP and 587 DOWN) DEGs compared to FLT3 ITD / DNMT3A KO mice. The enrichment analysis suggested that DNMT3A and NPM1 mutations, in a constitutive FLT3 ITD expression context, cooperate to activate transcriptional programs sustaining the proliferation and maintenance of blasts through concerted upregulation of a number of pathways, including the Anaphase-Promoting Complex (APC), which regulates cell cycle progression, the m-TOR-mediated autophagy inhibition, the Rho-GTPases effectors (with Rac1 overexpression), the Hippo-mediated self renewal, the HOXA9 signature and TP53 -regulated apoptosis. Interestingly, genes involved in transport of mature transcripts to cytoplasm and nuclear RNAs metabolism were also upregulated. In keeping with these RNAseq findings, the Riboseq data showed that both the quote of processed transcripts (15 vs 9%) and the percentage of processed pseudogenes were more abundant in triple vs double mutated mice. On the other hand, the long intergenic noncoding RNAs (lincRNA) fraction was reduced in triple mutants. This finding may reflect dysfunctional remodeling of tumor microenvironment, contributing to AML cells immune escape in the BM5.

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Summary/Conclusion: The leukemic DNMT3A KO / FLT3 ITD / NPM1 c genotype is characterized by unique transcriptional and translational signatures. The information about deregulated pathways and altered protein synthesis establishes the rational basis for designing new therapeutic approaches in this poor prognosis AML genotype.

P459: NUC-7738 REGULATES BETA-CATENIN SIGNALLING RESULTING IN REDUCED PROLIFERATION AND SELF-RENEWAL OF AML CELLS

A. M. Shahid 1,* , M. Elshani 1 , D. J. Harrison 1

1 School of Medicine, University of St Andrews, St Andrews, United Kingdom

Background: NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine triphosphate (3’-dATP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I/II clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. We have recently shown in cell lines a potential therapeutic role for NUC-7738 in patients with acute myeloid leukemia (AML), whereby NUC-7738 induced myeloid cell differentiation and mitochondrial-mediated apoptosis. AML leukemia stem cells (LSCs) are required for the initiation and maintenance of the disease. Activation of the Wnt/β-catenin pathway is required for the survival and development of LSCs and therefore, targeting β-catenin is a potential therapeutic strategy.

Aims: The aim of this study was to determine whether NUC-7738 regulates β-catenin and the expression of its target genes in AML cells

Methods: AML cell lines KG1a, OCI-AML3, HL-60 and U937 were treated with NUC-7738 for 48 or 72 hours. LSCs were determined by the expression of surface markers CD34, CD38 and CD123 via flow cytometry. Self-renewal was assessed by performing the colony forming assay using MethoCult Enriched media. Cell proliferation was determined by the Alamar Blue assay. Immunoblotting was performed for β-catenin, PI3K-p110, phosphorylated AktSer 473 and phosphorylated GSK3β Ser9

Results: NUC-7738 reduced PI3K-p110, phosphorylated AktSer 473 and phosphorylated GSK3β Ser9 resulting in reduced β-catenin expression. NUC-7738 decreased the percentage of CD34 + CD38 - CD123 + (LSCs) from 15% to 7% and significantly reduced the total number and size of leukemic colonies. NUC-7738 inhibited cell proliferation in U937, OCI-AML3 and HL-60 cells, with IC 50’s of 1.55 µM, 7 µM and 16 µM, respectively.

Summary/Conclusion: Self-renewal pathways, including Wnt/β-catenin, are key for the initiation and long-term maintenance of LSCs. Through the ability to reduce PI3K-p110, phosphorylated AktSer 473 , and phosphorylated GSK3β Ser9 and suppress β-catenin signalling, NUC-7738 exploits a key developmental process of AML, supressing the expansion and survival of LSCs. These results indicate that targeting LSCs with NUC-7738 may offer a successful therapeutic strategy for treating patients with leukemia.

P460: RECOMBINANT SLIT2 INHIBITS ACUTE MYELOID LEUKEMIA CELL PROLIFERATION IN VITRO AND IN VIVO

L. Araujo de Albuquerque Simoes 1,2,3,4,* , I. Weinhäuser 2,5 , D. A Pereira-Martins 2,5 , C. A. Ortiz Rojas 2 , T. Mantello Bianco 2 , R. D. C. Cavaglieri 6 , E. Magalhães Rego 2,3,4,7

1 Center of Translational Research in Oncology, ICESP - Faculty of Medicine, USP, Sao Paulo; 2 Center for Cell Based Therapy, Fundação Hemocentro de Ribeirão Preto, Ribeirão Preto; 3 Medical Research Laboratory on Molecular Hematology (LIM31), University of São Paulo; 4 Hematology, Faculty of Medicine, University of Sao Paulo, Sao Paulo, Brazil; 5 Department of Experimental Hematology, University Medical Center Groningen, Groningen, Netherlands; 6 Medical Research Laboratory on Molecular Hematology (LIM31), Faculty of Medicine, University of Sao Paulo; 7 Center of Translational Research in Oncology, ICESP - Faculty of Medicine, USP, Sao Paulo, Brazil

Background: The SLIT/ROBO axis has been shown to play a key role in many physiological processes such as organogenesis, axon guidance and angiogenesis. Additionally, several studies conducted in solid tumors observed frequent hypermethylation of either Slit or Robo at their promoter site. In the context of leukemia, Golos et al., 2019 showed that the low expression of SLIT2 was associated with lower overall survival in adults with acute myeloid leukemia (AML). Furthermore, it was demonstrated by our group that the knockdown of SLIT2 in Acute Promyelocytic Leukemia (APL) cells leads to an increase of cell proliferation in vitro and a more aggressive course of the disease in vivo (Weinhäuser et al., 2020).

Aims: Given the evidence of the relevance of SLIT2 in APL, we opted to transfer our gained knowledge to a more challenging leukemia subset and decided to study the role of SLIT2 in AML.

Methods: We first evaluated the methylation pattern of SLIT2 in AML patients compared to healthy donors by analyzing publicly available datasets ( GSE58477 , normal karyotype blasts: 62, healthy CD34+: 10; GSE63409 , LSC: 14, HSC: 5), followed by the assessment of the level of SLIT2 in the bone marrow (BM) plasma of AML patients and healthy donors by ELISA. Additionally, to functionally assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV4-11, and MOLM13) with recombinant SLIT2 (50ng/mL) in vitro. We performed the knockdown of SLIT2 in AML cell lines (THP-1 and OCI-AML3) and evaluated their proliferation capacity as well. Moreover, AML cells were treated with decitabine and recombinant SLIT2. Finally, we evaluated the anti-leukemic effects of SLIT2 in vivo. To do so NSGS mice were transplanted with luciferase-transduced MV4-11 cells and the animals were either treated with vehicle (control group) or recombinant SLIT2 (25 ng/g of body weight) two days after transplant for a period of one week.

Results: Our analysis indicated increased methylation at the SLIT2 promoter site in AML patients compared to healthy CD34 + . In accordance with our analysis, we detected decreased level of SLIT2 protein in the bone marrow (BM) plasma of AML patients (1.43 ng/mL) when compared to healthy donors (3.51 ng/mL) (p<0.05). The functional role of SLIT2 was determined with the treatment of KASUMI1, MV4-11 and MOLM13 cells with recombinant SLIT2 in vitro. Our results showed that SLIT2 treatment reduced the cell proliferation and colony formation capacity and induced cell cycle retention in the G1 phase for all AML cell lines. Contrarily, the knockdown of SLIT2 promoted increased cell proliferation in THP-1 and OCI-AML3 cell lines. Moreover, we observed increased induction of decitabine-induced apoptosis when MV4-11 and MOLM13 cells were treated in the presence of recombinant SLIT2. Finally, the engraftment and progression of the disease of NSGS mice transplanted with luciferase-transduced MV4-11 cells were monitored by the detection of luciferase bioluminescent signals. Our results showed, that SLIT2 treatment was able to significantly delay the progression of AML in vivo . Mice treated with SLIT2 presented improved overall survival (vehicle 15d: CI 13-16d; SLIT2 19d: 95% CI: 16-22d. P value = 0.0320), decrease leukemic infiltration in the BM and spleen, reduced spleen size (vehicle: 125 mg; SLIT2: 100 mg P value= 0.04), indicating lower disease burden when compared to the control group.

Summary/Conclusion: In conclusion, our results suggest that SLIT2 has tumor suppressive functions in AML in vitro and in vivo highlighting the therapeutic potential with low cytotoxicity of SLIT2 in AML.

P461: MYELOID KINOME INHIBITOR HM43239 OVERCOMES ACQUIRED RESISTANCE IN ACUTE MYELOID LEUKEMIA MODELS

R. Bejar 1,* , S. J. Baek 2 , N. Abbasi 1 , A. Krasny 1 , R. Sinha 1 , H. Zhang 1 , S. Howell 3 , W. Rice 1

1 Aptose Biosciences Inc., San Diego, United States of America; 2 Hanmi Pharmaceutical Company, Seoul, South Korea; 3 Moores Cancer Center, University of San Diego Health, San Diego, United States of America

Background: Oral HM43239 is in development for the treatment of acute myeloid leukemia (AML) because of its capacity to potently inhibit kinases that drive myeloid malignancies, including diverse forms of the FLT3, SYK, JAK and c-KIT kinases. Wildtype FLT3 is overexpressed in most AML patients, and approximately 30% of newly diagnosed adult AML patients harbor internal tandem duplications (ITDs) or point mutations in the tyrosine kinase domain (TKD). These mutations drive aberrant activation of downstream proliferation pathways and are associated with a high risk of relapse. Likewise, the c-KIT alternative receptor kinase, as well as the SYK and JAK1/2 intracellular kinases, mediate oncogenic signaling in AML that can promote drug resistance to certain FLT3 inhibitors. HM43239 was developed to overcome shortcomings of other FLT3 inhibitors. It inhibits a broad set of mutant and wildtype forms of FLT3, while simultaneously disrupting downstream SYK, JAK/STAT5, ERK, and other rescue signaling pathways. This rationale supports the development of HM43239.

Aims: Evaluate the activity of HM43239, an orally active drug, as a FLT3-focused myeloid kinome inhibitor in human AML models.

Methods: Biochemical kinase assays were performed by RBC and Carna. The effects of HM43239 on cell proliferation (IC 50 ), growth rate (GR 50 ) and concentration at half-maximal effect (Growth Effective Concentration; GEC 50 ) were determined using the MTS assay with vehicle controls. Cell-based inhibition of target phosphorylation was assessed by Western blot and flow cytometric analyses. The AML cell lines tested included MV-4-11, MOLM-13, MOLM-14 and BAF3/ITD. In vivo efficacy was assessed using the MOLM-14 FLT3-Mutated xenograft model.

Results: HM43239 inhibited the enzymatic activities of FLT3-WT, -ITD, and -D835Y variants with IC 50 values of 1.1, 1.8 and 1.0 nM, respectively. Binding K d values for FLT3-WT, -ITD, -D835Y, -D835H, -ITD/D835V and -ITD/F691L were 0.58, 0.37, 0.29, 0.4, 0.48, and 1.3 nM, respectively. HM43239 killed AML cells that harbor the FLT3-ITD mutation (MV-4-11, MOLM-13 and MOLM 14) with IC 50 , GR 50 and GEC 50 values ranging from 4 to 10 nM. In cell lines expressing wild type FLT3 (KG1, HEL92.1, SKM-1, and THP-1) the values for these parameters ranged from 0.05 nM to 3 µM. Western blot and flow cytometric analyses of MV-4-11, MOLM-14 cells and Ba/F cells transfected with target proteins revealed that the phosphorylation of FLT3 and SYK was reduced by 50-90% at HM43239 concentrations of 10 – 100 nM, and that these concentrations produced >80% reduction in phosphorylation of ERK and JAK/STAT5 that function downstream of FLT3. HM43239 inhibited FcγR-induced SYK and JAK/STAT5 activation in KG-1a (FLT3-WT) cells that upregulate RAS signaling which is a mechanism of acquired resistance to gilteritinib. In murine xenograft studies, HM43239 was more effective than gilteritinib in both SC and orthotopic FLT3-F691L and FLT3-ITD/F691L mutant MOLM-14 models.

Summary/Conclusion: HM43239 inhibits wild type and mutant forms of FLT3 at low nM concentrations and demonstrates in vivo efficacy at doses that are well tolerated. Its ability to also inhibit SYK and, by reducing the activity of these upstream kinases, to also impair the activity of EKR1/2 and JAK/STAT5 that participate in rescue pathways, makes this a particularly interesting molecule with the potential of offsetting the development of resistance that is common with other FLT3 inhibitors. A Phase 1/2 trial of HM43239 in patients with AML is open and accruing patients ( NCT03850574 ).

P462: IDENTIFICATION OF TARGETED THERAPIES DIRECTED TO ACUTE MYELOID LEUKEMIA MINIMAL RESIDUAL DISEASE

N. van Gils 1,* , F. Kessler 1 , M. Broux 2 , F. Denkers 1 , S. Demeyer 2 , J. Cools 2 , D. C. De Leeuw 1 , J. Janssen 1 , L. Smit 1

1 Hematology, UMC Amsterdam, location VUmc, Cancer Center Amsterdam, Amsterdam, Netherlands; 2 Center for Human Genetics, KU Leuven, Leuven, Belgium

Background: Despite good responses to intensive polychemotherapy in the initial treatment phase, the biggest challenge in the treatment of acute myeloid leukemia (AML) is persistence of residual therapy-resistant cancer cells (measurable residual disease, MRD) that can develop into recurrence. Leukemia cells with stem cell features (“leukemic stem cells”, LSCs) residing within MRD are thought to be at the origin of relapse. Current knowledge gaps on residual leukemic cell persistence and ways to target MRD are hampering progress in development of treatments successfully preventing relapse and increasing AML cure rates.

Aims: To identify transcriptional and epigenetic characteristics and vulnerabilities of AML MRD, guiding the design of “targeted” combination treatment strategies that successfully eliminate relapse-initiating cells in individual AML patients.

Methods: We developed in vivo patient-derived xenograft (PDX) NOD/SCID/IL2Rγc-deficient (NSG) mouse models mimicking MRD by 1) intravenous (IV) injection of primary AML diagnosis samples and, as soon as leukemic cells were detectable in the peripheral blood, treatment with combination chemotherapy, and 2) IV injection of AML patient samples at the stage of MRD in NSG mice. Moreover, we selected samples from individual AML patients at different time points, diagnosis, MRD and relapse, and purified by flow cytometry the leukemic cells using the leukemia-associated immunophenotype (LAIP) expressed on the AML cells. To elucidate the mutational landscape, transcriptional program and chromatin accessibility involved in development and persistence of MRD, we performed DNA-, RNA- and the assay for transposase-accessible chromatin (ATAC)-sequencing on the purified leukemic cells.

Results: We revealed that in patient AML samples at the stage of MRD there is clonogenic capacity of leukemic stem/progenitors (Figure 1A). Treatment with combination chemotherapy significantly reduced leukemic burden in the bone marrow of NSG mice (Figure 1B top). Interestingly, the number of CD34 + CD38 - leukemic cells in AML1 was hardly affected by chemotherapy, whereas treatment significantly reduced the number of these cells in AML2 (Figure 1B bottom), indicating that leukemic CD34 + CD38 - cells residing within different patients have a heterogeneous response to polychemotherapy. Injection of AML MRD from several patients resulted in leukemia engraftment (example Figure 1C). For one case, marked as MRD-negative by flow cytometry, we even observed that MRD could initiate high leukemia load in the mice (Figure 1D), while after injection of diagnosis and relapsed AML from the same patient no engraftment was observed. Together, our results implicated that AML cells after therapy, even if the patient is marked as MRD-negative, could have high leukemia-initiating potential, which could have been induced by chemotherapy.

Using RNA-sequencing we identified and compared the gene expression profile and chromatin accessibility of leukemic cells at diagnosis, MRD and relapse from patients samples. We identified several genes upregulated in AML MRD cells as compared to diagnosis AML. Moreover, we showed increased chromatin accessibility, indicating open chromatin structure and potential transcriptional activity, at several loci in MRD compared to diagnosis AML, giving opportunities to therapeutically target MRD.

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Summary/Conclusion: We generated AML MRD PDX mouse models, and identified transcriptional and epigenetic characteristics of AML MRD that might guide the design of “targeted” combination treatment strategies potentially eliminating relapse-initiating cells.

P463: RE-PURPOSING OF GENE SIGNATURES IN AML UNCOVERS NOVEL ENERGETICS-ASSOCIATED MOLECULAR SUBTYPES

P. Strain 1,* , E. E. Scanlon 1 , G. Jellema 2 , R. D. Kennedy 1,2 , K. I. Mills 1 , J. K. Blayney 1

1 Patrick G Johnston Centre for Cancer Research, Queens University Belfast, Belfast; 2 Almac Diagnostics, Almac, Craigavon, United Kingdom

Background: AML is a highly heterogeneous disease with great diversity in clinical features and patient response to treatments. Despite recent improvements in disease understanding, treatments have remained unchanged for 30 years. This presents the need to characterise the disease more fully and characterise its underpinning molecular subtypes. The re-use of published and validated prognostic and predictive gene signatures e.g. Cancer Hallmarks presents an invaluable in silico opportunity to uncover the biological mechanisms underpinning treatment response in AML.

Aims: To develop an automated statistical analysis pipeline combined with machine learning techniques to derive robust patient clusters representative of novel molecular AML subtypes significantly associated with clinical variables and survival outcomes.

Methods: Analysis was carried out using a primary dataset (AML-OHSU: 451 AML patient samples) and a validation dataset (TCGA-LAML: 200 AML patient samples). Both datasets had been processed using Almac’s claraT platform, a software-driven solution which provides a comprehensive overview of tumour profiles using gene expression signatures.

An automated analytical pipeline was developed using Consensus Clustering (CC), a method that determines the number and membership of potential clusters, using different combinations of 8 distances and 7 linkages within a dataset. Robustness was tested via bootstrapping. This pipeline was used to stratify patients via a dimension reduction approach whereby clustering was performed on 210 gene signatures categorised by 10 different hallmarks of cancer. Analysis was performed to identify clinical associations within robust clusters that were linked to differences in survival.

Results: The automated clustering pipeline analysed a total of 1,314 stable clusters across 10 cancer hallmarks in the AML-OHSU dataset. Stable clusters were subsequently processed via log rank analysis (OS right-censored at 60 months) identifying 134 stable clusters with significant differences (p-value <0.05) in survival outcome.

Stable clusters with significant survival differences were tested against 32 clinical categorical variables present in the AML-OHSU dataset. The results were filtered for a significant threshold (chi square p-value <0.05 and BH p-value <0.2). Here we found gene signatures representative of the Energetics hallmark, incorporating 22 signatures, to be one of the most frequently clustered throughout our results, ranking highest where K =3.

A significant difference in overall survival probability ( Log rank p-value: 0.033 ) was found between clusters (Energetics, K=3 ). Patients in the poorest survival cluster were characterised by a refectory induction response to treatment, having the lowest number of fusions, a low frequency of NPM1 mutations and a high proportion of patients above the age of 65.

To validate energetics results from the AML-OHSU dataset, CC was again performed using gene signatures from the TCGA dataset that were representative of the energetics hallmark. A significant difference between overall survival probability ( Log rank p-value: 0.019 ) was again found between stable clusters of the energetics hallmark ( K = 3).

Summary/Conclusion: We have demonstrated that a novel analytical pipeline developed here to analyse Hallmark-related gene signatures can aid in the discovery of new molecular subtypes in AML associated with prognosis. We have subsequently validated these in an independent dataset. The Energetics hallmark has not, to our knowledge, been linked with AML prognosis before, and may suggest novel biology linked to treatment response.

P464: COMPARED TO CLASSICAL CYTOGENETICS OPTICAL GENOME MAPPING (OGM) DETECTS MULTIPLE ADDITIONAL STRUCTURAL CHROMOSOMAL ABERRATIONS IN PEDIATRIC ACUTE MYELOID LEUKEMIA (AML)

J. Suttorp 1,* , J. L. Lühmann 2 , D. Steinemann 2 , D. Reinhardt 1 , N. von Neuhoff 1 , M. Schneider 1

1 Clinic of Pediatrics III, University Hospital Essen, Essen; 2 Department of Human Genetics, Hannover Medical School, Hannover, Germany

Background: AML is the 2nd most frequently diagnosed blood cancer in children affecting approximately 130 patients annually in Germany. Pediatric AML is a heterogeneous malignancy that has multiple genetic aberrations. This is relevant for classification, prognosis and selection of optimal therapy. To detect these alterations established procedures (karyotyping, FISH, RNA-based techniques) are used with well-known limitations (resolution of karyotyping and FISH, respectively; cell cultivation is necessary to generate metaphases; cost-intensiveness; necessity of experienced and intensively trained staff). OGM is an emerging technique addressing these limitations. Based on extraction of ultra-high weight DNA followed by fluorescent labelling at the sequence CTTAAG a barcode-like pattern of the genomic DNA is created. The DNA strands labelled in this way are stretched and drawn as single ultra-long fragments through nanochannels. The results are translated into molecule maps which are compared to the reference genome (resolution down to 500 bp) allowing the identification of genetic alterations. This method combines high resolution with picturing almost whole chromosomes without cell cultivation.

Aims: The aim of this study was to test to what extend OGM might supplement classical cytogenetics (CCG) to detect risk defining genetic aberrations at initial diagnosis in pediatric patients with AML.

Methods: We analyzed 24 specimen of pediatric AML, MPAL and bilineage leukemia patients by OGM starting from stored frozen material (bone marrow, peripheral blood) obtained at initial diagnosis. Results of OGM were compared with karyotyping and FISH. Primer walking and breakpoint spanning PCR were used to validate newly detected aberrations by OGM in selected cases. Applying breakpoint-spanning PCR, validated aberrations were used as markers for assessment of minimal residual disease (MRD) in selected cases during AML treatment.

Results: Overall, we detected discrepant results between CCG and OGM in 17/24 (70%) cases including 9 cases in which karyotyping detected aberrations which were not found by OGM. However, these aberrations were not relevant for risk stratification. In general, focusing on genetic aberrations important for risk stratification the results between CCG and OGM were concordant in 23/24 (96%) cases. In a single case OGM was able to detect a high-risk aberration which was missed by CCG. In total, 33 previously unknown genetic alterations were identified by OGM. Out of these four newly detected variants (2 deletions, 2 translocations) were validated by PCR. The translocations t(2;12) and t(8,12) both affected gene ETV6 on chr. 12 with different fusion partners (chr. 2: AC064875.1 ; chr. 8: NSMCE2 ). In both translocations sequences at the breakpoints were determined. Two deletions affecting genes RNF157 on chr. 17 and MEF2B on chr. 19 were validated. These aberrations had not been described before in pediatric AML. In 2 cases - both without a previously identified MRD marker – the newly detected translocations by OGM were used for MRD monitoring.

Summary/Conclusion: OGM considerably expands the range of techniques to optimize the diagnosis of pediatric AML and has much potential to omit limitations of classical cytogenetics. Furthermore, the results will contribute to a better understanding of the leukemogenesis of pediatric AML. In addition, OGM offers the possibility to identify new aberrations which can serve as patient specific MRD markers especially in cases without a previously known MRD marker.

P465: A LIPID NANOPARTICLE DELIVERY SYSTEM FOR TARGETING THE LEUKAEMIC FUSION GENE RUNX1/ETO BY SIRNA

L. Swart 1,* , P. Derevyanko 1 , A. van Oort 1 , M. Ashtiani 1 , A. Krippner-Heidenreich 1 , A. Koekman 2 , C. Seinen 2 , H. Issa 3 , H. Blair 4 , R. Schiffelers 2 , O. Heidenreich 1,4

1 Princess Maxima Center for Pediatric Oncology; 2 Department of Central Diagnostic Laboratory Research, University Medical Center Utrecht, Utrecht University, Utrecht, Utrecht, Netherlands; 3 Department of Pediatrics, University Hospital Frankfurt, Frankfurt (Main), Germany; 4 Wolfson Childhood Cancer Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

Background: The leukaemic fusion gene RUNX1/ETO initiates and drives leukaemogenesis and, thus, constitutes an ideal cancer-specific target. Direct and exclusive targeting of leukaemic fusion proteins using RNA interference is an attractive concept, but has proven challenging because of the unfavourable pharmacokinetic properties of siRNA. To address this challenge, we have developed lipid nanoparticle (LNP) formulations that interfere with the expression in leukaemic cell lines and in primary cells. To further harness their use in patient-derived AML cells, we are exploring and optimizing targeted LNPs for effective siRNA delivery to primary leukaemic cells.

Aims: To develop and validate a delivery system for the therapeutic application of fusion gene-specific siRNAs.

Methods: LNPs encapsulating siRE (active siRNA targeting the RUNX1/ETO fusion transcript) and siMM (mismatch siRNA control with two nucleotides swapped in the sense strand) were prepared using microfluidic mixing. Ligand or fluorescent dyes were conjugated to polyethylene glycol (PEG) following a copper-free click chemistry approach. LNPs were characterized by dynamic light scattering (DLS) and cryo electron microscopy (EM). Functional experiments with patient-derived t(8;21) cells were performed in co-culture with mesenchymal stromal cells under conditions that preserved and expanded immature VLA-4 expressing leukaemic populations as determined by an 11 antibody panel and flow cytometry analysis. Transcript and protein levels of RUNX1/ETO and target genes were determined by bulk and single cell (sc) RNA-seq, real-time polymerase chain reaction and western blotting.

Results: To monitor uptake of 50 – 60 nanometers sized LNPsiRNA we labelled the surface with Cy3 and a ligand by a PEG post-insertion approach. To improve uptake and tissue retention, we chose a modified short peptide ligand harbouring an LDV motif with high affinity towards the very late antigen-4 (VLA-4) that is widely expressed across haematopoietic cells. Decoration of the LNPs with the LDV resulted in an enhanced uptake in t(8;21) cell lines within the first 8 hours. While undecorated LNPs were hardly taken up by patient-derived material, the decoration with LDV resulted in an efficient uptake and a concomitant knockdown of RUNX1/ETO transcript and protein after a single dose. Sequential treatment leads to longer-lasting reduction of RUNX1/ETO transcript and associated target genes ( LAPTM5 , CCND2 , C/EBPA and ANGPT1 ) and loss of RUNX1/ETO protein. Combined multiparameter flow analysis and scRNAseq of patient cells show that RUNX1/ETO depletion causes massive shifts in cell populations and an almost complete loss of immature populations (CD34+ CD38- CD117+ CD45RA+) associated with a gain in mature (CD15+) populations, indicating a release from the differentiation block caused by the fusion protein. Gene Set Enrichment Analysis (GSEA) of scRNAseq data confirm the role of RUNX1/ETO in promoting stemness and cell cycle progression and highlight a key function of this fusion protein in coordinating metabolism in support of leukaemic propagation. Currently, we are performing in vivo biodistribution and efficacy studies using orthotopic mouse models.

Summary/Conclusion: Decoration of LNPs with an LDV motif enhances uptake of fusion transcript-specific siRNAs and enables efficacious knockdown of RUNX1/ETO in patient-derived cells. The biologic consequences validate RUNX1/ETO as a highly promising and leukaemia-specific target and highlight the LNP-mediated delivery of siRNAs as a promising new approach for the treatment of fusion gene-driven leukaemias.

P466: AMPLIFIED EPOR/JAK2 GENES DEFINE A UNIQUE SUBTYPE OF ACUTE ERYTHROID LEUKEMIA

J. Takeda 1,* , K. Yoshida 1 , M. Nakagawa 1 , Y. Nannya 1 , A. Yoda 1 , R. Saiki 1 , Y. Ochi 1 , L. Zhao 2 , R. Okuda 1 , X. Qi 1 , T. Mori 1 , A. Kon 1 , K. Chiba 3 , H. Tanaka 4 , Y. Shiraishi 3 , M.-C. Kuo 5 , C. Kerr 6 , Y. Nagata 6 , D. Morishita 7 , N. Hiramoto 8 , A. Hangaishi 9 , H. Nakazawa 10 , K. Ishiyama 11 , S. Miyano 4 , S. Chiba 12 , Y. Miyazaki 13 , T. Kitano 14 , K. Usuki 9 , N. Sezaki 15 , H. Tsurumi 16 , S. Miyawaki 17 , J. Maciejewski 6 , T. Ishikawa 8 , K. Ohyashiki 18 , A. Ganser 19 , M. Heuser 19 , F. Thol 19 , L.-Y. Shih 5 , A. Takaori−Kondo 20 , H. Makishima 1 , S. Ogawa 1

1 Department of Pathology and Tumor Biology; 2 Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto; 3 Division of Genome Analysis Platform Development, National Cancer Center Research Institute; 4 M&D Data Science Center, Tokyo Medical and Dental University, Tokyo, Japan; 5 Division of Hematology−Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan; 6 Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 7 Chordia Therapeutics Inc., Kanagawa; 8 Department of Hematology, Kobe City Medical Center General Hospital, Kobe; 9 Department of Hematology, NTT Medical Centre Tokyo, Tokyo; 10 Department of Hematology, Shinshu University Hospital, Matsumoto; 11 Department of Hematology, Kanazawa University, Kanazawa; 12 Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba; 13 Department of Hematology, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki; 14 Department of Hematology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka; 15 Department of Hematology, Chugoku Central Hospital, Hiroshima; 16 Department of Hematology, Gifu University, Gifu; 17 Division of Hematology, Tokyo Metropolitan Ohtsuka Hospital; 18 Department of Hematology, Tokyo Medical University, Tokyo, Japan; 19 Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; 20 Department of Hematology / Oncology, Kyoto University, Kyoto, Japan

Background: Acute erythroid leukemia (AEL) is a rare subtype of acute myeloid leukemia (AML) characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and erythroid/myeloid (EML) phenotypes based on the degree of erythroid hyperplasia. Despite an intensive mutational analysis, the mechanism of erythroid hyperplasia in AEL is still poorly understood and so are feasible therapeutic targets.

Aims: To understand the mechanism of erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL.

Methods: We analyzed a total of 124 adult AEL cases, where whole genome/exome sequencing of 35 cases were followed by targeted-capture sequencing in all cases. RNA sequencing was also performed in 23 cases. The mutational profile of AEL cases was compared to that of 409 cases with non-erythroid AML (non-AEL). Patient-derived xenograft (PDX) mouse models of AEL with JAK2 and/or EPOR amplification were established from 6 AEL patients with these abnormalities. These models were tested for their response to JAK1/2 inhibitor.

Results: In accordance with a recent report, AEL cases were classified into 4 genomic groups (A-D), which are characterized by biallelic TP53 mutations and complex karyotype (Group-A), mutated NPM1 (Group-B) and STAG2 (Group-C), and other mutations in histone modifiers and transcription factors (Group-D). In particular, all but one PEL cases belonged to Group-A. Also found in non-AEL cases, these group-defining lesions in AEL were uniquely associated with focal gains/amplifications of EPOR , JAK2 , and/or ERG/ETS2 loci (Group-A), PTPN11 mutations (Group-B), and KMT2A -PTD (Group-C), which might be responsible for the AEL phenotype. Highly enriched in PEL cases (7/13), EPOR / JAK2 focal gains/amplifications were implicated in their extreme erythroid hyperplasia and associated with particularly poor prognosis, even compared to other Group-A cases, who had shorter survival than those in other groups. As expected, JAK2/EPOR -amplified cases showed upregulated STAT5 signaling compared to non-AEL cases, which however, was also observed in other AEL cases, suggesting that upregulated STAT5 signaling is a hallmark of AEL. Based on these findings, we tested the effect of JAK2 inhibition on cell growth of EPOR/JAK2 -amplified AEL cells in in vitro culture and xenograft model. Of interest, ruxolitinib-mediated JAK2 inhibition resulted in significantly suppressed in vitro cell growth of EPOR/JAK2 -amplified AEL cells and prolonged overall survival in 4 PDX models with phospho-STAT5 downregulation, although other 2 models were resistant to JAK2 inhibition with persistent STAT5 activation.

Summary/Conclusion: AEL is a heterogenous subgroups of AML characterized in common by upregulated JAK/STAT5 signaling. Gains/amplifications of JAK2 / EPOR are frequent in TP53 -mutated cases, particularly those with the PEL phenotype, and could be exploited as potential therapeutic targets using JAK2 inhibitors.

P467: FUNCTIONAL CHARACTERIZATION OF ABERRANT GATA1 PROTEIN COMPLEXES IN NORMAL AND MALIGNANT HUMAN ERYTHROBLASTS

S. Tauchmann 1,* , F. Otzen Bagger 2 , T. Bock 3 , R. Sivalingam 1 , T. Eder 4 , E. Heyes 4 , A. Fagnan 5 , M. von Lindern 6 , T. Mercher 5 , F. Grebien 4 , J. Schwaller 1

1 Childhood Leukemia, University of Basel, University Children`s Hospital Basel, Department Biomedicine, Basel, Switzerland; 2 University of Copenhagen, Center of Genomic Medicine, Copenhagen, Denmark; 3 Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland; 4 Institute for Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria; 5 INSERM U1170, Equipe Labellisée Ligue Contre le Cancer, Institut Gustave Roussy, Université Paris-Saclay, Université de Paris, Paris, France; 6 Department Hematopoiesis, Sanquin Research, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands

Background: Terminal differentiation of erythroid progenitor cells is controlled by the master transcription factor GATA1 acting in activating and repressive protein complexes. In acute erythroid leukemia (AEL), cell differentiation is impaired leading to accumulation of immature erythroblasts. Previous work in mouse models and primary human cells suggested impaired function of abundantly expressed GATA1 protein in AEL cells. Interestingly, GATA1 overexpression induced or enhanced terminal erythroid differentiation of the human AEL cell line K562 or immortalised HUDEP2 erythroblasts, respectively.

Aims: We hypothesize that impaired terminal erythroid differentiation in AEL might be the consequence of aberrant GATA1 protein interactions.

Methods: We compared the proteomes of three human AEL cell lines (F36P, K562, KMOE2) and primary AEL patient cells with HUDEP2 cells and primary human erythroblasts (hEBST) that were capable of in vitro terminal erythroid differentiation, using a tandem mass tag(TMT)-based approach (n=3/type). In parallel, we compared the GATA1 interactomes by immunoprecipitation (IP) followed by mass spectrometry (MS) (n=3/type). In addition, we performed a targeted CRISPR screen in Cas9 -expressing K562 cells in which we monitored growth kinetics upon mutational disruption of candidate genes and simultaneously assessed changes in erythroid differentiation by measuring CD71 and CD235a surface markers.

Results: Unsupervised hierarchical clustering displayed a clear separation of AEL proteomes from those of normal erythroblasts with 386 proteins higher expressed in the first and 623 proteins more abundant in the latter group (logFC≥2;q<0.05). GATA1-IP-MS analysis of nuclear lysates from all six cell types identified 1616 proteins, of which 126 were differentially associated with GATA1 depending on cell state. 54 proteins preferably interacted with GATA1 in the AEL group, whereas 72 proteins were more enriched in “normal erythroblasts” (q<0.5). GATA1 interactomes in hEBST and HUDEP2 cells highly overlapped and were more similar to GATA1 partners identified in F36P and KMOE, than those of K562 and primary AEL cells. Integrative analysis revealed 118 proteins that were differentially expressed and/or precipitated by GATA1-IP, of which 49 were enriched in malignant and 69 proteins in normal erythroblasts (q<0.5). To identify if GATA1-associated proteins are involved in erythroid differentiation we functionally characterized them in a CRISPR screen in K562 cells. 4 patterns were observed: inactivation of cluster 1 genes such as ZEB2 or IKZF1 did not change cell differentiation or growth, loss of cluster 2 genes like RPS21 or RANGAP1 did not change differentiation, but impaired cell growth/survival. Disruption of cluster 3 genes like SND1 , or EIF5A resulted in an increase in CD71 and/or CD235a expression without significant changes in cell growth, and deletion of cluster 4 genes including SMC1A or ATP1A1 caused significant signs of differentiation together with cell depletion over time. Overall, knockout of 41 genes caused significantly increased CD71 and/or CD235a surface expression, indicating that these GATA1-interacting proteins affect erythroid differentiation.

Summary/Conclusion: Our work suggests that impaired erythroid differentiation of AEL cells involves aberrant GATA1 protein complexes. Ongoing gain- and loss of function validation experiments of selected candidate genes and chromatin assays will identify novel regulators of terminal differentiation in normal and malignant erythropoiesis.

P468: MRD AS A BIOMARKER FOR RESPONSE TO DONOR LYMPHOCYTE INFUSION AFTER AL-LOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

K. Teich 1,* , M. Stadler 1 , R. Gabdoulline 1 , C. Wienecke 1 , B. Heida 1 , P. Klement 1 , K. Büttner 1 , L. Venturini 1 , M. Wichmann 1 , W. Puppe 2 , C. Schultze-Florey 1 , G. Beutel 1 , M. Eder 1 , A. Ganser 1 , M. Heuser 1 , F. Thol 1

1 Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation; 2 Department of Virology, Hannover Medical School, Hannover, Germany

Background: Donor lymphocyte infusions (DLIs) can be employed for acute myeloid leukemia (AML) patients as therapeutic or prophylactic treatment after allogeneic hematopoietic cell transplantation (alloHCT). Application of DLI after alloHCT may induce graft-versus-host disease (GVHD). Therefore, biomarkers that predict the effect of DLIs may be useful to guide DLI use. Molecular measurable residual disease (MRD) is prognostic for the response to standard chemotherapy as well as for targeted therapies.

Aims: To evaluate MRD as a prognostic marker for DLIs after alloHCT.

Methods: Patients with AML or myelodysplastic syndrome (MDS) aged ≥18 years undergoing DLI at Hannover Medical School between 1998 and 2018 with available PB and/or BM DNA samples were included. Patients were excluded if they had no detectable mutations at diagnosis, had a second alloHCT in the observed time or if MDS patients had <10% bone marrow blasts at diagnosis.

DNA libraries were prepared using the TruSight Myeloid Panel or Nextera Flex for enrichment (Illumina) and sequenced on a MiSeq device. Error-corrected sequencing of patient specific mutations was performed 30 days (Follow Up 30 days, FU30) and 90 days (FU90) after the first DLI. All mutations detected at diagnosis were used for MRD monitoring. Marker positivity was defined as variant allele frequency above the limit of detection as not all patients were in complete remission (CR), in CR with incomplete hematological recovery (CRi) or in morphologic leukemia free state (MLFS).

Results: The median time from alloHCT to DLI was 11.3 months (range 3.1 to 74.4 months). Of 78 patients, 24 patients were marker negative and 54 patients were marker positive before DLI (of the marker positive patients 23 were in CR/CRi/MLFS). As expected, marker negative patients before DLI had a significantly better overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) than marker positive patients. From the 54 marker positive patients before DLI, 27 patients were in CR/CRi at FU90, 22 patients did not reach CR/CRi and five patients died before FU90. OS, EFS and RFS were significantly improved in patients that were in CR/CRi at FU90. CR/CRi status at FU90 correlated strongly with remission status before DLI (p<0.001).

Next, the prognostic effect of MRD in CR/CRi patients at FU90 (n=27) was analyzed. 18 patients reached MRD negativity at FU90 and nine patients stayed MRD positive. Baseline characteristics such as patient sex, cytogenetic risk group, age at diagnosis, the median time between alloHCT and DLI or use of prophylactic vs. therapeutic DLIs were similar between MRD negative and positive patients. Treatment intensity until FU90 was similar between the two groups. Remission status before DLI was not associated with MRD negativity at FU90 (p=0.782). There was no significant difference in OS (p=0.593), EFS (p=0.229) and RFS (p=0.226) between FU90 MRD negative and positive patients. Exclusion of DTA ( DNMT3A, TET2, ASXL1 ) mutations as MRD markers affected the number of patients in CR/CRi at FU90 (20 patients MRD negative and five patients MRD positive), while patient characteristics and outcome did not differ between the two groups.

Summary/Conclusion: Patients that achieve CR/CRi until FU90 after DLIs have a significantly better outcome than the remaining patients. However, the MRD status in patients in CR/CRi at FU90 had no prognostic effect in the patient cohort investigated here.

P469: CHEMORESISTANCE IN ACUTE MYELOID LEUKEMIA: A SINGLE-CELL RNA SEQUENCING APPROACH

C.-L. Teng 1,* , P.-L. Cheng 1 , T.-H. Hsiao 2 , C.-H. Chen 3

1 Division of Hematology/Medical Oncology; 2 Department of Medical Research, Taichung Veterans General Hospital, Taichung; 3 National Institute of Cancer Research, National Health Research Institutes, Zhunan, Taiwan

Background: Our previous study demonstrated that myc, mitochondrial oxidative phosphorylation, mTOR, and stemness were independently responsible for chemoresistance in AML.

Aims: The current study aimed to further identify the potential mechanisms of chemoresistance of the “7 + 3” induction in AML using a single-cell RNA sequencing (scRNA-seq) approach.

Methods: Thirteen untreated de novo AML patients were enrolled and stratified into the complete remission (CR) (n=8) and the non-CR (n=5) groups. We used scRNA-seq to analyze the genetic profiles of 28950 AML cells from these 13 patients. A previously published bulk RNA-seq dataset validated our results.

Results: Chemoresistant AML cells had more premature accumulation in early hematopoiesis. The hematopoietic stem cell-like cells from the non-CR group expressed more leukemic stem cell markers (CD9, CD82, IL3RA and, IL1RAP) than those from the CR group. Besides, chemoresistant progenitor cells had impaired myeloid differentiation due to early hematopoiesis arrest. Importantly, the AML cells analyzed by the scRNA-seq and bulk RNA-seq harbored comparable myeloid lineage cell fraction, which internally validated our results. Using the TCGA database, our analysis demonstrated that AML patients with a higher expression of chemoresistant genetic markers (IL3RA and IL1RAP) had a worse overall survival (p < 0.01 for IL3RA; p < 0.05 for IL1RAP).

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Summary/Conclusion: The AML cells responsive and resistant to the “7 + 3” induction had diverse cell origins according to specific genetic biomarkers using a scRNA-seq approach. Besides, hematopoiesis arrest occurred earlier in chemoresistant AML cells. This result provided an insight into a more understanding of chemoresistance in AML.

P470: NGS-BASED MINIMAL RESIDUAL DISEASE DETECTION IN PERIPHERAL BLOOD SHOWS GOOD PROGNOSTIC VALUE FOR OS AND EFS IN PATIENTS WITH ACUTE MYELOID LEUKEMIA RECRUITED IN THE UNIFY TRIAL

C. Thiede 1,2,* , C. Schuster 2 , C. Krippendorf 2 , G. Koenen 3 , T. Medts 3 , P. Marques Ramos 3 , L. Gou 4 , P. Montesinos 5 , W. Fiedler 6 , R. Müller 7 , J. Krauter 8 , S. Sica 9 , J. Westermann 10 , M. Levis 11 , R. Stone 12 , J. Sierra 13 , H. Döhner 14

1 Medizinische Klinik und Poliklinik, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden; 2 AgenDix, Gesellschaft für angewandte molekulare Diagnostik mbH, Dresden, Germany; 3 Novartis Pharma AG, Basel, Switzerland; 4 Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States of America; 5 Hospital Universitario i Politecnico La Fe Valencia, Valencia, Spain; 6 Med. Klinik, Universitätsklinikum Hamburg Eppendorf, Hamburg, Germany; 7 Klinik für Medizinische Onkologie und Hämatologie, Universitäts Spital Zürich, Zürich, Switzerland; 8 Städtisches Klinikum Braunschweig, Braunschweig, Germany; 9 Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma, Italy; 10 Charité University Medical Center, Berlin, Germany; 11 The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD; 12 Dana-Farber Cancer Institute, Boston, United States of America; 13 Hospital Santa Creu i Sant Pau, Universitat Autonoma of Barcelona, Jose Carreras Leukemia Research Institute, Barcelona, Spain; 14 Universitätsklinikum Ulm, Ulm, Germany

Background: Assessment of measurable residual disease (MRD) to evaluate the depth of remission at the time of achieving morphological complete remission (CR) or CR with incomplete blood count recovery (CRi) by multiparameter flow cytometry (MFC) or quantitative polymerase chain reaction (qPCR) has been shown to be predictive of outcome in patients (pts) with acute myeloid leukemia (AML), and is recommended according to European LeukemiaNet guidelines. However, accurate MRD assessment is still limited by lack of suitable markers in all pts (qPCR) and/or limited specificity/sensitivity (MFC). Next generation sequencing (NGS) holds promise to overcome some of these limitations and allows versatile and sensitive MRD assessment in almost all AML pts. However, data on NGS-MRD in this setting are still limited.

Aims: To further extend the experience with NGS-based MRD assessment, we prospectively collected samples to explore prognostic implications of MRD as detected by NGS at CR/CRi in the context of a randomized, placebo-controlled phase 3 study (UNIFY; NCT03512197 ), investigating midostaurin added to conventional 7 + 3 based chemotherapy in FLT3 wt pts. Following the recommendation by an independent DMC, the UNIFY study was stopped in Sep 2019 due to futility. The current subset analysis was performed to investigate the impact of NGS-MRD results on long-term outcomes.

Methods: Peripheral blood mononuclear cell (PBMC) or bone marrow mononuclear cell (BMMC) samples were collected from pts treated in the UNIFY study. NGS-MRD was performed on genomic DNA using targeted NGS-error-controlled sequencing method (Ion-Torrent S5XL) with a sensitivity between 0.1 and 0.001%. Targets for MRD detection were identified by NGS of BM samples taken at diagnosis (Archer Myeloid Panel) covering 74 genes frequently mutated in myeloid disease. MRD positivity was defined as any mutation call >0.1%, excluding mutations in genes associated with clonal hematopoiesis, ie., DNMT3A, TET2 and ASXL1.

Results: Total 731 BMMC (from 305 out of the total 501 pts) and 564 PBMC samples (from 204 pts) were collected and analyzed, of which 467 were paired (from 175 pts). The 10 most frequently used genes for MRD monitoring were NRAS, IDH2, RUNX1, SRSF2, TP53, PTPN11, IDH1, BCOR, CEBPA and GATA2 . Analyses in paired samples showed numerically higher MRD + rates in BMMC vs. PBMC (57% vs 46% MRD + at the end of induction, respectively). No differences in MRD rates were seen between treatment arms, and data was pooled for analysis. A high correlation of variant allele frequency (Pearson’s r≥0.84) and a high level of concordance in MRD calls were seen between BMMC and PBMC (91% overall percent agreement). For pts who fulfilled the criteria of a successful induction treatment, detection of MRD in either BMMC or PBMC in CR/CRi pts at the end of induction was associated with significantly lower event free survival (EFS) and overall survival (OS) compared to MRD − status (1-year EFS of 26% vs 72% in BMMC and 34% vs 71% in PBMC; 1-year OS of 73% vs 93% in BMMC and 67% vs 94% in PBMC) (Figure). Preliminary multivariate analysis, controlling for age and sex, confirmed the independent prognostic value of NGS-MRD for OS. Additional analysis is ongoing.

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Summary/Conclusion: Taken together, these data indicate that MRD by NGS at the time of CR/CRi in both BMMC and PBMC, is prognostic for EFS and OS. Although the results should be interpreted with caution due to the premature termination of the study and limited follow-up, these further support the use of molecular MRD assessment to predict outcome in pts with AML.

P471: ERYTHROID/MEGAKARYOCYTIC DIFFERENTIATION BIAS IN BONE MARROW OF AML/MDS PATIENTS AFTER DECITABINE TREATMENT

F. Tiso 1,* , A. O. de Graaf 1 , L. I. Kroeze 2 , B. A. van der Reijden 1 , S. M. C. Langemeijer 3 , K. M. Hebeda 2 , J. H. Jansen 1

1 Department of Laboratory Medicine, Laboratory of Hematology; 2 Department of Pathology; 3 Department of Hematology, Radboudumc, Nijmegen, Netherlands

Background: The hypomethylating agent Decitabine is used mainly in AML patients who are not eligible for standard induction chemotherapy or as a bridge therapy before a stem cell transplantation. Decitabine is an analogue of cytidine deoxynucleoside and it is incorporated into the DNA as decitabine triphosphate. In the DNA decitabine triphosphate inhibits the methylation of the DNA, leading to the reactivation of tumour suppressor genes which have been epigenetically silenced, induces cell differentiation and also apoptosis.

Aims: We studied early signs of morphological changes in bone marrow biopsies (BMB) of AML/MDS patients after treatment with Decitabine and correlated these with the mutational background.

Methods: We investigated 45 patients with AML/MDS who were treated with Decitabine. Bone marrow biopsies were morphologically examined, and we assessed the mutational status of the BMB. Sequencing was performed using a panel of 27 commonly mutated genes in myeloid malignancies, using a targeted error corrected approach.

Results: We observed a striking erythroid/megakaryocytic differentiation bias after treatment in 16/45 patients, whereas the other patient group (n=29) showed predominant myeloid differentiation. Patients developing this erythroid/megakaryocytic bias showed a trend towards a higher frequency of mutations in genes encoding for spliceosome components (U2AF1, SF3B1 and SRSF2), in particular U2AF1 was found significantly (p-value=0.05) more mutated. Furthermore, this group carried a significantly (p-value=0.03) lower number of mutations in genes encoding for proteins involved in signalling and kinase pathways (ETNK1, FLT3, CBL, JAK2, KIT, KRAS, MPL, BRAF, NRAS) compared to the group with predominant myeloid differentiation.

Summary/Conclusion: A bias towards erythroid/megakaryocytic differentiation was observed in the bone marrow of one third of the patients after treatment with Decitabine. This bias was over-represented in patients with mutations in spliceosome component encoding genes, particularly U2AF1. Patients with myeloid predominant Decitabine response harboured an increased number of mutations in signalling and kinase proteins encoding genes.

P472: THE FLT3-ITD RECEPTOR CAN PROMOTE PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM THROUGH THE OXIDOREDUCTASE ERO1

M. Turos Cabal 1,* , A. M. Sánchez Sánchez 1 , N. Puente Moncada 1 , C. Rodriguez 1 , F. Herrera 2 , V. Martin 1

1 Morphology and Cellular Biology, University of Oviedo, Oviedo, Spain; 2 Department of Chemistry and Biochemistry, University of Lisbon, Lisbon, Portugal

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and the most common myeloid tumour in adults. Internal tandem duplications (ITD) in FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This receptor is synthesized and processed in the endoplasmic reticulum (ER) before being transported to the plasma membrane, where it is activated by ligand binding. Upon activation, it regulates cell differentiation, proliferation, and survival. Mutations in FLT3, such as ITD mutations, not only constitutively activate this receptor, but also prevent its correct processing in the endoplasmic reticulum. Therefore, it is mostly retained in this organelle.

Aims: We decided to study if the aberrant accumulation of FLT3-ITD affects the functioning of the ER.

Methods: Experiments were performed on wild-type and FLT3-ITD AML cell lines, as well as on stably transfected FLT3 and FLT3-ITD Ba/F3 cell lines. Protein aggregation, reactive oxygen species (ROS) and glutathione (GSH) levels were determined by Thioflavin T, DCFH-DA and Thioltracker Violet fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion.

Results: Protein folding in the ER generates reactive oxygen species (ROS). Antioxidant molecules such as GSH are needed to maintain redox homeostasis. We found that FLT3-ITD cell lines have lower levels of protein aggregates, as well as ROS and GSH. This data could suggest that mutant cell lines have more efficient protein folding mechanisms. In fact, among the ER proteins that are involved in the protein folding process, we found the oxidoreductase ERO1 to be increased in FLT3-ITD cell lines. Supporting our findings, we saw that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol. This was reflected by a slight decrease in cell proliferation compared to wild-type cell lines. We confirmed that the decrease in cell viability in FLT3-ITD cell lines was not accompanied by cell death, as opposed to wild-type cell lines. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We decided to study if there were differences in the basal levels of UPR by western blot. We found FLT3-ITD cell lines to have lower levels of UPR, reflected by lower protein expression of the active forms of PERK and IRE (p-PERK and p-IRE respectively), together with the protein BiP, the major chaperone found in the ER and binds to misfolded proteins. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, reflected by lower levels of chaperones and UPR proteins, possibly because they rely on more efficient protein folding mechanisms. To confirm the importance of protein folding process in acute myeloid leukemia, we tested the effect of ERO1 inhibitor (EN460), and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the ITD mutation in the FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell lines carrying the normal or mutated FLT3 receptor. We found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cell lines.

Summary/Conclusion: Current AML treatments are mainly based on the administration of FLT3 inhibitors, with limited success. Our data suggest that FLT3-ITD cell lines, with greater resistance to treatments, have more efficient protein folding mechanisms. This process should be studied in more detail to determine its potential as a therapeutic target for AML treatment.

P473: DEVELOPMENT OF POTENTIAL BIOMARKERS FOR IRAK4 INHIBITOR EMAVUSERTIB IN HUMAN ACUTE MYELOID LEUKEMIA

A. Ugolkov 1,* , M. Pilichowska 2 , C.-C. Li 1 , R. C. Hok 1 , M. Samson 1 , M. Lane 1 , R. Von Roemeling 1 , R. Martell 1

1 Curis, Inc., Lexington, MA; 2 Tufts Medical Center, Boston, MA, United States of America

Background: Acute myeloid leukemia (AML), the second most common form of leukemia in adults, remains a highly fatal disease. Interleukin-1 receptor-associated kinase 4 (IRAK4) has been demonstrated as a potential therapeutic target in human AML. IRAK4-mediated activation of NF-kappaB signaling pathway could play a critical role in NF-kappaB-regulated survival and chemoresistance of cancer cells. The results of ongoing Phase 1 study demonstrated clinical activity of IRAK4 inhibitor emavusertib (CA-4948) in patients with relapsed/refractory AML and high-risk MDS. To support the development of companion diagnostic for emavusertib, we developed an immunohistochemical (IHC) assay and explored expression of potential biomarkers in bone marrow (BM) samples obtained from AML patients.

Aims: The objective of the present study was to analyze expression of IRAK4, NF-kappaB p-p50 and NF-kappaB p-p65 by IHC staining in human AML with further evaluation of these molecules as potential biomarkers for emavusertib therapy in AML patients.

Methods: We used IHC staining and immunoblotting to determine the expression of IRAK4, NF-kappaB p-p50 S337 and NF-kappaB p-p65 S536 proteins in human leukemia cell lines and clinical AML samples. Exploratory biomarker evaluations were performed using serial sections of formalin-fixed paraffin-embedded BM clot samples obtained from 19 AML patients. Eight BM AML samples were purchased from Analytical Biological Services and 11 BM samples were obtained at the screening from evaluable patients with relapsed/refractory AML enrolled in CA-4948-102 clinical trial.

Results: Using IHC staining, we found IRAK4 nuclear expression in blasts in 9/19 AML cases. To the best of our knowledge, this is the first report showing nuclear accumulation of IRAK4 in cancer cells. In support of our findings in clinical AML samples, we detected IRAK4 protein expression in nuclear lysates prepared from leukemia cell lines THP-1, HL-60 and K562. Using AML BM samples, we found that IRAK4 nuclear expression in blasts was significantly correlated with activation of NF-kappaB as determined by nuclear accumulation of NF-kappaB p-p50 and p-p65 in 9/19 cases. Cytoplasmic expression of IRAK4 was detected in 2/19 cases whereas IRAK4, NF-kappaB p-p50 and p-p65 expression was not detectable in 8/19 cases of AML. Clinical response data will be presented in the context of these novel findings.

Summary/Conclusion: Our findings uncovered a previously unknown nuclear expression of IRAK4 in leukemia cells. Our results demonstrated co-expression of nuclear IRAK4, NF-kappaB p-p50 and p-p65 in blasts suggesting a potential novel mode of interaction between IRAK4 and NF-kappaB in human AML. Although the role of nuclear IRAK4 in leukemia cells remains to be investigated, our preliminary findings revealed new perspectives into emavusertib treatment stratification and demonstrate the possibility of discovering novel biomarkers through IHC analysis of clinical samples. Additional accrual is ongoing to better define emavusertib biomarkers in AML patients.

P474: PIEZO1 AND THE THERAPEUTIC POTENTIAL OF MECHANORECEPTORS IN ACUTE MYELOID LEUKAEMIA

M. Velasco Estevez 1,* , A. Fernandez 1 , A. Otero-Sobrino 1 , P. Aguilar-Garrido 1 , M. A. Navarro-Aguadero 1 , R. Sanchez 2 , A. Gimenez Sanchez 2 , V. Garrido Garcia 2 , L. Carneros Blanco 2 , J. Martinez-Lopez 2 , M. Gallardo 1

1 H12O-CNIO Haematological Malignancies Group, Clinical Research Unit, Centro Nacional de Investigaciones Oncologicas (CNIO); 2 Grupo de Hematología Traslacional, Hospital 12 de Octubre de Madrid, Madrid, Spain

Background: Mechanotransduction is the process through which cells sense the mechanical input in and out the cell, and translate it into biochemical signals adapt and respond. In the past few years, it has been observed the important contribution of mechanical input in cell differentiation, fate, stemness, senescence and cancer, amongst others.

Piezo1 is a mechanoreceptor discovered in 2010 and constitutes a cation channel activated through stretch and shear flux leading to a calcium influx in the cell that activates a myriad of different pathways. Interestingly, despite being a mechanoreceptor, Piezo1 can also be activated through a small molecule, Yoda-1, and blocked by a peptide, GsMTx4, allowing the pharmacological study of this receptor. Mutations in PIEZO1 lead to pathologies as Xerocitosis hereditaria and it has been linked to erythroid formation. However, the expression and role of Piezo1 in haematological malignancies is yet to be explored

Aims: We aim to analyse the expression of Piezo1 in Acute Myeloid Leukaemia (AML) and healthy donors, and investigate the putative role of Piezo1 in cell differentiation and fate as well as its therapeutic potential for AML.

Methods: Mouse embryonic stem cells (mESC) were cultured and treated with Yoda-1 and GsMTx4. PIEZO1 expression in AML and healthy donors was analysed through RNAseq databases (Bloodspot, GEPIA2 and MILE study) and corroborated by ICC staining of human samples. Lastly, mononuclear cells in viability from AML or healthy donors were cultured in vitro for dose-response curves to Yoda-1 and GsMTx4 and in methylcellulose to analyse the phenotype and proliferation under those treatments. Both imaging and immunophenotyping with flow cytometry were performed in those samples.

Results: When analysing the expression of PIEZO1 in AML, it was seen that Piezo1 was higher in AML compared to healthy donors, and interestingly, there was a much higher expression in AML inv(16), same chromosome as PIEZO1 is (Figure 1A). We observed that activation of Piezo1 in mESCs led to a higher expression of stemness marker Oct3/4 (Figure 1B), suggesting that it is involved in the stemness state of haematopoietic stem cells. Furthermore, we observed a dramatic effect of both Yoda-1 and GsMTx4 in leukaemia haematopoietic cells (LSCs) from AML patients grown in methylcellulose, where GsMTx4 caused an increment in the cell number and shifted the cell fate towards the erythroid lineage, while Yoda-1 decreased the number of cells and ablated the erythroid differentiation. Interestingly, such effects were barely significant in cells from healthy donors, showing a wide window for treatment (Figure 1C-D).

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Summary/Conclusion: Our study shows that Piezo1 changes with cell differentiation and it could be a mechanism through which stem cells direct their fate. Furthermore, Piezo1 is highly expressed in AML and its blockage leads to a shift towards the erythroid differentiation while its activation blocks cell proliferation and differentiation, which such effects being much more significant in AML samples than healthy donors, suggesting a potential as therapeutic target.

This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140) and IF-Marie Sklodowska Curie Actions grant (MAtChinG – 101027864).

P475: CONTROL LEUKEMIA BY INDUCING ANTI-CANCER IMMUNE REACTIVITY IN VIVO? POTENTIAL OF A DC-TRIGGERED MECHANISM

G. Filippini Velazquez 1,* , D. Amberger 2 , L. Klauer 2 , E. Rackel 2 , M. Atzler 2 , S. Ugur 2 , C. Plett 2 , A. Rabe 3 , C. Kugler 2 , A. Rank 1 , M. Inngjerdingen 3 , T. Baudrexler 2 , B. Eiz-Vesper 4 , C. Schmid 1 , H. Schmetzer 2

1 Department of Hematology and Oncology, Section for Stem Cell Transplantation, Augsburg University Hospital, Augsburg; 2 Medical Department III, Department for Hematopoietic Cell Transplantation, Munich University Hospital, Munich, Germany; 3 Institute of Clinical Medicine, Department of Immunology, University of Oslo, Oslo, Norway; 4 Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Hannover, Germany

Background: There are virtually no treatment options for therapy-refractory or relapsed AML/MDS and high rates of relapse in successfully treated patients. The combination of the (clinically approved) immune-modulatory compounds GM-CSF+ Prostaglandine (PGE)1, the combination referred to as KIT-M converts myeloid blasts into dendritic cells of leukemic origin (DC leu ). After stimulation with DC leu , antileukemic (T)cells are activated.

Aims: Kit-M treatment may be an attractive tool for immunotherapy in myeloid leukemia.

Methods: Generation of DC from leukemic whole blood (WB) samples. Mixed lymphocyte culture (MLC) followed by functional antileukemic assays.

Results: 1. ex vivo : Treatment of 65 leukemic WB samples with KIT-M does not induce blast proliferation, but triggers generation of mature DC/DC leu and reduces tolerogenic DC. Kit treated WB activates the adaptive and innate immune system after MLC (Tcell proliferation, antitumor-supportive Tcells (TCRgd,Tb7), memory cells (Tcm,Tb7cm) and downregulates immunosuppressive Tcells (Treg4 and 8). Moreover leukemia-specific (interferon g (g) and/or degranulating ( deg )) adaptive (g-degT4,T8,TCRgd,Tb7,Tcm) and innate cells (g-degNK,NKb7,CIKb7) are increased and regulatory cells (g-degTreg4) downregulated. In addition, blast lysis is increased vs control. Ex vivo achieved blast lysis correlates positively with frequencies of mature DC/DC leu , leukemia-specific T3,T4,T8,TCRgd,Tb7 and NK cells and negatively with Treg4 and 8. Blast lysis does not correlate with age, sex, ELN risktype, blast counts, or response to chemotherapy.

2. In vivo - rats: Kit-M treatment of 3 leukemically diseased (vs 3 control) rats (followed by sacrification after treatment) leads to reduced blasts and Tregs in blood and spleen and increased DC leu and memory-like Tcells.

3 . In vivo - human: Kit-M therapy was offered to a 72 year old pancytopenic male as an individual salvage attempt (applied as continuous infusion), after discussion with the ethical commitee, the patient’s information about the experimental nature of the treatment and his written consent. The treatment was well tolerated and the patient improved clinically. Neutrophils in WBC increased from 10% to 50%, thrombocytes reached 100 G/l after 24 days. Immune monitoring showed a continuous increase of proliferating and non-naïve Tcells, NK, CIK- and NKT-, TH17 cells, B mem -cells and DC in PB. The production of IFNg producing T-, CIK and NKT-cells was demonstrated, suggesting an in vivo production/activation of (potentially leukemia-specific) cells. Immune stimulatory effects decreased after discontinuation of therapy. After 4 weeks of treatment, the patient was discharged in good clinical condition. Unfortunately, at two weeks from discharge, AML progressed and the patient died few days later.

Summary/Conclusion: Treatment of WB ex vivo with Kit-M leads to activation of adaptive and innate (leukemia-specific) immune reactive cells (and downregulated suppressive mechanisms) via a DC/DC leu triggered mechanism – resulting in significantly improved blast lysis compared to controls (independent of patients‘ risk classification, MHC, age or sex). In vivo treatment of leukemically diseased rats or humans was well tolerated, led to an increase of platelets and granulocytes and stable (low) blast counts in PB – probably mediated by a (leukemia specifically) DC/DC leu activated immune system. A dose defining clinical trial in carefully selected patients to confirm clinical safety and underscore clinical efficacy is being prepared.

P476: LOSS OF CHD4 IN ACUTE MYELOID LEUKAEMIA GIVES RISE TO INCREASED DNA DAMAGE REPAIR DEFECTS AND ALTERED CELL CYCLE PROGRESSION

D. Venney 1,* , Y. Atlasi 2 , A. Mohd-Sarip 2 , K. Mills 1

1 Haematology; 2 Queens University Belfast, Belfast, United Kingdom

Background: Acute Myeloid Leukaemias (AMLs) are an array of blood cell disorders arising from alterations within myeloid precursors. Cancer genome studies highlighted alterations within the nucleosome remodelling and deacetylation (NuRD) complex occur in over 20% of all cancers. The NuRD complex controls nucleosome assembly and chromatin accessibility; CHD4 is a core subunit of the NuRD which is involved in a multitude of functions including epigenetic regulation, cell cycle progression and DNA replication and repair. CHD4 is linked to PARP dependent DNA damage response (DDR) pathway by rapid but transient accumulation at sites of damage, preventing transcription which enables homologous recombination (HR).

Aims: We aim to explore the function of CHD4-NuRD in AML using a CHD4 knockout (CHD4-/-) isogenic cell line models created using CRISPR/Cas9 to assess the mechanism of function of CHD4 within AML disease progression and evaluate if there is targetable therapeutic potential.

Methods: CRISPR-Cas9 was used to generate isogenic CHD4-/- models in two representative AML cell lines. CHD4-/- models underwent functional studies; proliferation, cell cycle and protein expression assays were used to phenotype models before inducing DNA damage at a clinically relevant dose and reassessing cell response. A high throughput drug screen was performed to identify DNA damage compounds that showed significant sensitivity in CHD-/- models and validation was assessed using single compound treatments and co-culture assays.

Results: Analysis of publicly available TCGA AML transcriptomic data set (N=161), found that AML patients (N=82) who express below median CHD4 levels have a poorer prognostic outcome suggesting a link between stalled transcription enabling HR-based DDR and patient survival (P=0.047). Analysis of the intermediate cytogenetic risk group showed patients with a low CHD4 expression had a worse outcome; which was comparable to the adverse risk group patients (P=0.032), indicating that loss of CHD4 may contribute to a more aggressive phenotype AML in this group.

Phenotype analysis of CHD4-/- models showed DNA damage markers 53BP1 and PARP expression increases significantly, highlighting the role of CHD4 within the DDR. This increased DNA damage response was accompanied by reduced cellular proliferation in CHD4-/- cells. Accordingly, cell cycle analysis showed a decrease in cells entering the G2/M phase alongside an increase in cells in G1 and S phase suggesting a potential cell cycle checkpoint deficiency causing stunted proliferation in CHD4-/- cells. Of note, we did not observe a significant compensatory increase in CHD3-NuRD components expression in the absence of CHD4 suggesting there is no compensation for the loss of CHD4.

We measured behaviour of CHD4-/- cells to stress-inducing conditions; in response to irradiation and chemotherapy drugs commonly used in AML. CHD4-/- cells display much stronger reduction in cellular proliferation post-2Gy irradiation compared to control while cell cycle analysis showed further cellular arrest in G2/M. Furthermore, a drug screen (160 DNA damaging compounds) showed an increased sensitivity of CHD4-/- cells to compounds targeting DNA/RNA synthesis pathways, including Tubercidin and Cytarabine, indicating that loss of CHD4 results in hyper-increase in damage.

Summary/Conclusion: Our data has identified a role of CHD4 in AML progression mediated through loss of cell cycle progression control and an elevated response to DNA damage. Therefore, targeting cells with lower CHD4 expression with compounds targeting DNA/RNA synthesis may have an impact on patient outcome.

P477: PHENOTYPICALLY-DEFINED STAGES OF LEUKEMIA ARREST PREDICT MAIN DRIVER MUTATIONS SUBGROUPS, AND OUTCOME IN ACUTE MYELOID LEUKEMIA

F. Vergez 1,* , L. Largeaud 1 , S. Bertoli 2 , M.-L. Nicolau-Travers 1 , J.-B. Rieu 1 , I. Vergnolle 1 , E. Saland 1 , A. Sarry 2 , S. Tavitian 2 , F. Huguet 2 , M. Picard 2 , J.-P. Vial 3 , N. Lechevalier 3 , A. Bidet 3 , P.-Y. Dumas 4 , A. Pigneux 4 , I. Luquet 1 , V. Mansat-De Mas 1 , E. Delabesse 1 , M. Carroll 5 , G. Danet-Desnoyers 5 , J.-E. Sarry 6 , C. Recher 2

1 Laboratory of Hematology; 2 Department of Hematology, CHU Toulouse, Toulouse; 3 Laboratory of Hematology; 4 Department of Hematology, CHU Bordeaux, Bordeaux, France; 5 Stem Cell and Xenograft Core, UPENN, Philadelphia, United States of America; 6 UMR1037, INSERM, Toulouse, France

Background: Normal blood cell maturation is organized according to a functional hierarchy at the top of which are multipotent hematopoietic stem cells (HSC). Immunophenotypically, human HSC are enriched in a population of Lin - , CD34 + , CD38 - , CD90 + , CD45RA - cells. Upon differentiation, HSC give rise to multipotent progenitors (MPP), which retain the ability to produce all blood lineages but have lost their self-renewal capacity. The classical model of hematopoiesis postulates that MPP are then orientated toward either the lymphoid or myeloid lineage, developing into common myeloid progenitors (CMP) or lymphoid-primed multipotent progenitors (LMPP) which can still produce defined myeloid cell types. Along the myeloid pathway, CMP can differentiate into either granulocyte-monocyte progenitors (GMP) or megakaryocyte-erythroid progenitors (MEP). GMPs finally differentiate into granulocyte progenitors (GP) or monocyte progenitors (MP).

Aims: In this study, we hypothesized that the stages of leukemic arrest (SLA) in myeloid maturation can be defined by flow cytometry by analogy with the stages of hematopoiesis and they correlate with the oncogenic events implicated in the mechanism of differentiation block.

Methods: Six stages of leukemia differentiation-arrest categories based on CD34, CD117, CD13, CD33, MPO and HLA-DR expression by flow cytometry were identified in 2 independent fully annoted cohorts of 2087 and 1209 AML patients. Next-generation sequencing was performed in 409 AML patients.

Results: HSC/MPP-like AMLs display low proliferation rate, inv(3) or RUNX1 mutations, and high leukemic stem cell frequency as well as poor outcome, whereas GMP-like AMLs have CEBPA mutations, RUNX1-RUNX1T1 or CBFB-MYH11 translocations, lower leukemic stem cell frequency, higher chemosensitivity and better outcome. NPM1 mutations correlate with most mature stages of leukemia arrest together with TET2 or IDH mutations in GP-like AML or with DNMT3A mutations in MP-like AML.

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Summary/Conclusion: AML immunophenotyping can establish a new SLA classification that strongly correlates with cellular behaviour of the leukemic bulk, predicts main genetic subgroups early at diagnosis and outcome after intensive chemotherapy. Each SLA is defined by specific oncogenic events whose penetrance may be dependent on the differentiation stages of hematopoiesis and their gene expression. Identifying disrupted gene pathways specific for each SLA should therefore form the basis for targeted therapies aimed at inducing AML differentiation.

P478: THE GERMLINE VARIANT GFI1-36N PROMOTES GENETIC INSTABILITY IN LEUKEMIC CELLS AND OPENS NEW THERAPEUTIC APPROACHES FOR ABOUT 15% OF ALL AML PATIENTS

J. Vorwerk 1,* , D. Frank 1 , K. Sun 1 , F. Neumannn 2 , M. Heuser 3 , D. Kunadt 4 , W. E. Berdel 1 , J.-H. Mikesch 1 , C. Schliemann 1 , G. Lenz 1 , A. K. Jayavelu 5,6,7,8,9 , C. Khandanpour 1

1 Department of Medicine A, University Hospital Münster; 2 Fluorescence Microscopy Facility Münster, Institute of Medical Physics and Biophysics, University of Münster, Münster; 3 Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover; 4 Department of Internal Medicine I, University Hospital Dresden, Dresden; 5 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Munich; 6 Clinical Cooperation Unit Pediatric Leukemia, German Cancer Research Center (DKFZ); 7 Department of Pediatric Oncology, Hematology, and Immunology; 8 Hopp Children’s Cancer Center Heidelberg (KiTZ); 9 Molecular Medicine Partnership Unit, European Molecular Biology Laboratory (EMBL), Heidelberg University, Heidelberg, Germany

Background: The zinc finger protein Growth Factor Independence 1 (GFI1) acts as a transcriptional repressor regulating differentiation of myeloid and lymphoid cells. In a single-nucleotide polymorphism (SNP) of GFI1, GFI1-36N , the amino acid serine (S) is replaced by asparagine (N) at position 36. GFI1-36N has a prevalence of 7% in healthy Caucasians and 15% in acute myeloid leukemia (AML) patients, hence predisposing to AML. We have shown previously that this is particularly since the GFI1-36N protein differs from the GFI1-36S variant regarding its ability to induce epigenetic changes leading to a derepression of oncogenes. Now we have evidence that GFI1 is implicated in DNA repair as AML blast cells of patients carrying the GFI1-36N SNP feature more frequently complex-aberrant karyotypes and more mutations leading to an inferior prognosis.

Aims: Therefore, in this work, we explored whether presence of GFI1-36N alters DNA repair activity and whether this can be targeted therapeutically.

Methods: We analyzed the association between GFI1-36N and chromosomal aberrations in human AML samples. As a murine model of human AML, we knocked in the human GFI1-36S or GFI1-36N variant into the murine Gfi1 gene locus and retrovirally expressed MLL-AF9 to induce AML. We then performed γ-H2AX immunofluorescence and flow cytometry to quantify the number of DNA double-strand breaks (DSBs), confocal immunofluorescence microscopy to detect DNA repair foci of key enzymes of non-homologous end joining (NHEJ) and homologous recombination (HR) as well as proteomics and immunoblotting to determine the level of DNA damage response enzymes and cell cycle kinases. Cell viability assay and colony-forming unit assay were used to examine the response of GFI1-36S and GFI1-36N leukemic cells to palbociclib treatment.

Results: A complex aberrant karyotype was found twice as often in patients carrying the GFI1-36N SNP compared to GFI1-36S controls (38% vs. 20%, p ** = 0.0080). Murine GFI1-36N-MLL-AF9 cells showed 43 times more genetic and cytogenetic aberrations than GFI1-36S-MLL-AF9 controls ( p *** = 0.0003). Upon exposure to irradiation, murine GFI1-36N leukemic cells featured more DSBs and delayed repair. Regarding the two main DSB repair pathways, we did not see a significant difference in the ability of GFI1-36N or GFI1-36S leukemic cells to perform NHEJ, but a reduced HR capacity in presence of GFI1-36N . On a molecular level, GFI1-36N was associated with less foci of the HR key enzyme RAD51, reduced protein levels of the DNA damage response enzymes CHK1 and CDK2, higher levels of the cell cycle propagating CDKs, and reduced levels of the CDK inhibiting CDKNs. As especially CDK4 and CDK6 protein levels were significantly increased in GFI1-36N leukemic cells, we subsequently treated malignant cells with the established CDK4/6 inhibitor palbociclib. We observed that GFI1-36N leukemic cells were more susceptible to this treatment, indicated by 49% less CFUs compared to GFI1-36S leukemic controls ( p ** = 0.0063).

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Summary/Conclusion: The findings suggest that the GFI1-36N SNP increases cell cycling and reduces DNA repair activity. This could promote emergence of chromosomal aberrations and hence contribute to genomic instability of leukemic cells (Figure 1). On a therapeutic level, GFI1-36N could possibly be a marker for a specific subset of AML patients that is sensitive to CDK4/6 inhibitors.

P479: DNA METHYLATION PROFILING REFINES THE PROGNOSTIC CLASSIFICATION OF ACUTE MYELOID LEUKEMIA PATIENTS TREATED WITH INTENSIVE CHEMOTHERAPY

S. Vosberg 1,* , A. Ohnmacht 2 , C. Moser 3 , A. Arneth 2 , V. Jurinovic 3 , K. H. Metzeler 4 , M. C. Sauerland 5 , D. Görlich 5 , W. E. Berdel 6 , J. Braess 7 , S. Amler 8 , U. Krug 9 , W. Hiddemann 3 , K. Spiekermann 3 , C. C. Oakes 10 , M. P. Menden 2 , T. Herold 3 , P. A. Greif 3

1 Department of Internal Medicine, Medical University of Graz, Graz, Austria; 2 Institute of Computational Biology, Helmholtz Zentrum Munich, Neuherberg; 3 Department of Medicine III, University Hospital LMU Munich, Munich; 4 Department of Hematology, Cellular Therapy, and Hemostaseology, University of Leipzig, Leipzig; 5 Institute of Biostatistics and Clinical Research; 6 Department of Medicine, Hematology and Oncology, University of Münster, Münster; 7 Department of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg; 8 Friedrich-Loeffler-Institut, Greifswald-Insel Riems; 9 Department of Medicine III, Hospital Leverkusen, Leverkusen, Germany; 10 Department of Internal Medicine, The Ohio State University, Columbus, United States of America

Background: Multiple studies have shown that DNA methylation is frequently altered in acute myeloid leukemia (AML).

Aims: Here we propose a prognostic classifier for the survival of AML patients that describes DNA Methylation-based Risk Assessment (“DMRA”), trained and validated using a total of 538 adult AML patients from two independent cohorts, all treated with intensive cytarabine-based chemotherapy with curative intent, and compared it to the current ELN 2017 classification.

Methods: Patients from the first cohort were either treated within one of two consecutive clinical trials of the German AMLCG study group or documented within the AMLCG registry. DNA methylation profiles of 377 diagnostic AML samples were generated using Illumina Infinium MethylationEPIC BeadChip arrays, selected based on their chemotherapy regimen (i.e. 7 + 3, S-HAM, HAM(-HAM), or TAD-HAM). The clinical annotation includes overall survival (OS) and the ELN risk assessment. Our DMRA classifier was trained on the most variable CpG sites genome wide, weighted upon their relevance to OS based on ridge regression. Three prognostic subgroups (“low”, “medium” and “high” risk) were identified using cutoffs by optimizing the prediction error to avoid over- and underfitting. We validated our approach by 10-fold cross-validation within AMLCG data, ensuring that samples are either used for training or validation at the same time, as well as using an independent cohort of diagnostic AML samples, selected for cytarabine-based “standard chemotherapy” (n=161, BeatAML).

Results: Overall, the three risk groups defined by DMRA reveal more distinct survival characteristics between each other as compared to between ELN risk groups (Figure 1A,B), with 3-year OS of 64% / 44% / 31% by ELN vs. 64% / 39% / 15% by DMRA. Of note, nearly half of patients were assigned to a different risk group using DMRA as compared to ELN (n=240/538, 45%, Figure 1C).

Importantly, we were able to define more precise risk groups within ELN subgroups, with significant differences in OS. Within ELN favorable AML, 22% of patients (n=44/203) were classified with increased risk (3-year OS 70% vs. 44%, p=0.003 “low” vs. “medium”, log-rank test, Figure 1D). Within ELN intermediate AML, 33% of patients (n=44/135) were classified as low risk AML (3-year OS 58% vs. 38%, p=0.03 “low” vs. “medium”, Figure 1E). Within ELN adverse AML, 71% of patients (n=142/200) were classified with reduced risk (n=27 “low risk”, n=115 “medium risk”, 3-year OS 38% vs. 37% vs. 13%, p=0.0003 “low” vs. “high”, p=0.0002 “medium” vs. “high”, Figure 1F).

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Summary/Conclusion: Based on this approach, we were able to distinguish clinically relevant prognostic subgroups of AML patients within well described (cyto-) genetic risk groups. Patients with favorable (cyto-) genetics but high-risk epigenetics might benefit from alternative consolidation treatment strategies, such as allogeneic stem cell transplantation. On the other hand, patients with adverse (cyto-) genetics but low-risk epigenetics might be considered for consolidation chemotherapy instead of stem cell transplantation in first complete remission. Finally, patients with both, adverse (cyto-) genetics and high-risk epigenetics, must be considered as very poor risk subgroup with very short OS (median 228 days).

Our results demonstrate that DNA methylation profiling is a feasible and robust approach for risk stratification in AML, able to refine current risk assessment based on (cyto-) genetics alone. We propose that DNA methylation-based risk assessment may serve as a promising complement for current standards in prognostic classification of AML.

P480: NKG2D-MEDIATED ANTI-TUMOR IMMUNITY CONTRIBUTES TO THE FAVORABLE PROGNOSIS IN APL

H. Wang 1,* , X. Zhang 1 , S. Gong 1 , H. Du 1 , N. Mei 1

1 First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a poor prognosis. Acute promyelocytic leukemia (APL) is a unique subtype of AML, with PML-RARA fusion caused by t(15;17)(q22;q12) translocation, and is now considered a highly curable disease. Exploring the difference between APL and non-APL AML, may shed new insight on the treatment of AML. NKG2D is an activating receptor on NK cells that recognizes ligands on the surface of tumor cells and plays a crucial role in tumor cell killing. Prior studies have demonstrated that one mechanism of AML immune evasion is downregulation of NKG2D ligands (NKG2DL) or secretion of receptor-blocking soluble ligands. Whereas, reports on NKG2DL expression in APL are still lacking. In this study, we investigate the differences of NKG2DL expression on APL and non-APL cells.

Aims: Exploring the difference between APL and non-APL AML, may shed new insight on the treatment of AML.

Methods: Bone marrow samples were derived from de novo APL and non-APL AML patients, diagnosed according to French-American-British (FAB) criteria in The First Affiliated Hospital of Xi’an Jiao Tong University. Flow cytometry was performed with the anti-NKG2DL mAbs (MIC-A/B-PE, ULBP-1-APC, ULBP-2/5/6-PerCP). Leukemic blasts were identified using the CD45/SSC gating procedure.

Non-APL AML patients were divided into high or low group based on the median expression levels of NKG2DL for survival analysis.

Results: Totally 46 patients comprising of 23 APL and 23 non-APL AML were enrolled in this study. There was no significant difference in the baseline characteristics between the two groups. All non‐APL AML cells had low or no NKG2DL surface expression, while most APL cells expressed high NKG2DL levels. In particular, just two APL patients was negative for the NKG2DL but positive for CD34. The median percentage of NKG2DL among non-APL AML cells and APL cells were 1.2% vs 20.3% in MIC-A/B (P<0.01), 1.1% vs 54.8% in ULBP-1 (P=0.017), and 3.8% vs 36.35% in ULBP-2/5/6 (P<0.01). (Fig.1)

23 non-APL AML patients were enrolled for study inclusion; one patient dropped out due to cerebral infarction. The median follow-up time was 17.2 weeks. The high ULBP-2/5/6 group showed a superior overall survival rate compared to the low ULBP-2/5/6 group (P < 0.01), but there was no significant difference between high and low MIC-A/B groups (P = 0.31) or between the high and low ULBP-1 groups (P = 0.52).

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Summary/Conclusion: NKG2DL was decreased significantly on the surface of non-APL AML cells compared with APL cells. These results may indicate that NKG2D-mediated anti-tumor immunity contributes to the favorable prognosis in APL, while immune escape is an underlying mechanism for unfavorable prognosis in non-APL AML. NKG2DL may be used as a potential biomarker to predict prognosis of patients with non-APL AML, and upregulation of its expression could improve the clinical efficacy. Further studies with larger sample size and longer follow-up are required to confirm our conclusion.

P481: MP0533, A NEW MULTISPECIFIC DARPIN CD3 ENGAGER TARGETING THREE TUMOR ASSOCIATED ANTIGENS, INDUCES SPECIFIC T-CELL ACTIVATION AND AML TUMOR KILLING IN VIVO

A. Croset 1,* , M. Bianchi 1 , M. Franchini 1 , T. Lekishvili 1 , Y. Kaufmann 1 , A. Auge 2 , M. Hänggi 1 , W. Ali 1 , C. Zitt 1 , S. Wullschleger 1 , M. Matzner 1 , C. Reichen 1 , S. Fischer 1 , Y. Grübler 1 , A. Eggenschwiler 1 , T. Looser 1 , R. Watson 1 , P. Spitzli 1 , V. Kirkin 1 , D. Steiner 1 , A. Goubier 1

1 Molecular Partners AG, Schlieren, Switzerland; 2 Molecular Partners AG, Molecular Partners AG, Schlieren, Switzerland

Background: AML treatment options are generally focused on chemotherapy followed by allogeneic hematopoietic stem cell transplantation. However, many patients are ineligible for these options and often receive palliative treatments with poor long-term survival and there is an urgent need for improved therapeutic solutions. Newer therapies, including T cell engagers (TCE) and chimeric antigen receptor (CAR) T cells that target specific molecules overexpressed on leukemic blasts and stem cells are promising alternative treatment options for AML. However, achieving efficacy and low toxicity remains challenging.

MP0533 is a multi-specific, half-life extended, T cell engaging DARPin in development for treatment of patients with AML and higher risk myelodysplastic syndrome (HR-MDS). MP0533 consists of a chain of 6 covalently linked DARPin domains. Three of these domains engage CD33, CD123 and CD70 on the target cells, utilizing an avidity driven approach to ensure that binding occurs preferentially when 2 or more TAAs are present. A fourth DARPin domain targets CD3 for T-cell engagement. Lastly 2 additional HSA binding DARPin domains prolong half-life in circulation. In vitro , MP0533 has demonstrated, in both allogeneic and autologous settings, single to double digit pM potency against AML cell lines and AML primary cells expressing any combination of at least 2 of the 3 targeted TAAs and low cytokine release.

Aims: To evaluate and confirm that the previously established i n vitro and ex vivo efficacy and safety profiles of MP0533 are also observed in an in vivo human PBMC xenograft mouse model.

Methods: NXG mice were injected intraperitoneally with hPBMC from healthy donors and xenografted with MOLM-13 cells subcutaneously two days after hPBMC injection. Therapeutic treatment was initiated eight days after tumor implantation once tumors were established. DARPins were injected intravenously three times per week for two weeks. Several readouts such as T cell activation and human immune cell infiltration in tumors were performed by flow cytometry three days after first treatment. Additionally, cytokine and chemokine release were analyzed in serum four hours after the first treatment injection and in tumor supernatant 3 days after the first treatment injection.

Results: MP0533 induced AML tumor killing in vivo (P value <0.0001 at day 18 compared to PBS). Three days after the first injection, MP0533 recruited human immune cells in the tumor (around 10% of hCD45+ cells detected by FACS, P value = 0.0044 compared to PBS) and induced T cell activation (around 46% of CD4+/CD25+/CD69+ cells and 38% of CD8+/CD25+/CD69+ cells detected by flow cytometry, P value <0.0001 compared to PBS) which triggered cytokines and chemokines release in the tumor such as IL-6, INFγ and TNFα. However, MP0533 didn’t induce significant cytokine release, including at the highest tested dose, in mice serum four hours after the first injection.

Summary/Conclusion: We were able to generate a multi-specific CD3 engaging DARPin molecule with tailored affinities towards different TAAs showing significant efficacy in vivo that induced T cell activation without significant cytokine/chemokine release.

P482: NOVEL BISPECIFIC INNATE CELL ENGAGER AFM28 FOR THE TREATMENT OF CD123 POSITIVE ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME

J.-J. Siegler 1,* , N. Schmitt 2 , J. Pahl 1 , T. Haneke 1 , I. Kozlowska 1 , S. Sarlang 1 , A. Beck 2 , S. Knackmuss 1 , P. Ravenstijn 1 , U. Reusch 1 , J. Medina-Echeverz 1 , J. Endell 1 , T. Ross 1 , D. Nowak 2 , C. Merz 1

1 Affimed GmbH, Im Neuenheimer Feld 582, Heidelberg; 2 Department of Hematology and Oncology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

Background: Novel treatments for patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS) with relapsed or refractory (R/R), or measurable residual disease are urgently needed. Targeting of the surface antigen CD123 expressed on both leukemic blasts and stem cells (LSCs) of most patients allows for depletion of both compartments, which is considered a prerequisite for deep anti-tumor responses and induction of prolonged remission. AFM28 is a novel bispecific Innate Cell Engager (ICE ® ) designed for bivalent high-affinity binding of CD16A on natural killer (NK) cells, redirecting effector cell cytotoxicity to CD123 + tumor cells. Allogeneic NK cell therapy is emerging as a next-generation treatment with demonstrated activity in R/R AML and a very benign safety profile. Retargeting of NK cells with AFM28 may be a particularly effective treatment strategy in patients with CD123 + AML and HR-MDS.

Aims: 1. Preclinical characterization of AFM28 pharmacology and toxicology in vitro and in vivo to support clinical development.

2. Assessment of binding and activation of NK cells by AFM28 to test suitability for combination development with allogeneic NK cell therapy.

Methods: Flow cytometry (FC) was used to assess binding and retention of AFM28 on NK cells, NK cell activation (degranulation, IFNγ expression) and depletion of leukemic cells from AML and MDS patient specimens. Quantification of secreted cytokines in leukocyte cultures employed bead-based luminex methodology. In vivo efficacy data were acquired by BLI measurement from a murine AML model. Cynomolgus data were acquired from non-GLP dose range finding and a GLP toxicology study using standard methods (ELISA, IHC, FC).

Results: AFM28 induced NK cell activation at low picomolar concentrations and mediated efficacious depletion of CD123 + tumor cell lines and primary leukemic cells, including cells with low CD123 expression. NK cell activation was induced more potently than by an effector-enhanced IgG1 and was accompanied by more pronounced degranulation and IFNγ production. Similarly, AFM28 induced depletion of leukemic cells in patient bone marrow samples, without lysis of CD34 + /CD123 − cells, suggesting sparing of healthy hematopoietic progenitors. Potent anti-tumor activity was confirmed in vivo in a murine tumor model. In preclinical toxicology models, AFM28-induced target cell depletion was associated with minimal release of inflammatory cytokines, including IL-6, TNFα and IFNγ from leukocytes in vitro , and only low-level release of IL-6 in cynomolgus monkeys, despite efficacious depletion of CD123 + basophils and plasmacytoid dendritic cells (pDCs). Characterization of NK cell binding revealed high-avidity interaction, long cell surface retention, and potent induction of NK cell-mediated tumor cell lysis with no impact on NK cell viability.

Summary/Conclusion: These data demonstrate that AFM28 exhibits potent pharmacological activity in vitro and in vivo and efficaciously induces NK cell-mediated depletion of primary leukemic cells. Further, toxicology data suggest good tolerability with a low risk of cytokine release syndrome. Together, these findings support the clinical investigation of AFM28 as a potential novel treatment of CD123 + AML and HR-MDS. A first-in-human trial investigating safety and activity of single agent AFM28 in patients with R/R AML is currently in preparation. In addition, these data support the combination of AFM28 with allogeneic NK cell therapy, which holds potential as an effective, novel treatment strategy.

P483: STAT3 DRIVES IMMUNE EVASION OF MLL-AF9-DRIVEN ACUTE MYELOID LEUKEMIA

B. Zdarsky 1 , A. Witalisz-Siepracka 1,* , S. Boigenzahn 1 , K. Fiedler 1 , D. Stoiber 1

1 Department of Pharmacology, Physiology and Microbiology, Division Pharmacology, Karl Landsteiner University of Health Sciences, Krems, Austria

Background: Acute myeloid leukemia (AML) is a heterogenous disease with poor prognosis. Although the treatment opportunities in AML expanded in recent years, relapse still remains a major issue and novel therapies aiming to eradicate minimal residual disease are needed. Natural killer (NK) cell immunotherapy is put forward as a potential strategy fulfilling these unmet clinical needs. Importantly, NK cell function of AML patients is largely diminished due to different mechanisms of tumor-mediated immune suppression and escape. Signal transducer and activator of transcription 3 (STAT3) is a member of the JAK-STAT signaling pathway driving transcriptional responses downstream of many cytokines. To escape the immune surveillance, tumor cells tend to downregulate NK cell responses and STAT3 was proposed to drive this process in solid cancers.

Aims: We aim to investigate the function of STAT3 in AML evasion from NK cell surveillance.

Methods: Using CRISPR/Cas9 we generated STAT3-deficient human AML cell lines carrying the MLL-AF9 oncogene. The cell lines were characterized via flow cytometry, Western blot and qPCR and used as target cells for a human NK cell line in cytotoxicity assays.

Results: Using publicly available databases, we show that STAT3 expression negatively correlates with several NK cell-activating ligands in AML patients. Moreover, when studying a panel of human AML cell lines with a range of STAT3 expression levels, we observed a trend for the most efficient NK-cell mediated killing in cell lines expressing the lowest levels of STAT3. AML cells lacking STAT3 showed no major impairment in survival and proliferation under homeostatic conditions but were less resistant to different types of cellular stress. Interestingly, loss of STAT3 significantly enhanced killing by NK cells. The effect was also supported by elevated expression of NK-activating ligands in STAT3-deficient cells. We are currently testing if STAT3 inhibition recapitulates the observed effect.

Summary/Conclusion: Our in vitro data indicate that targeting STAT3 in AML might be a strategy to enhance NK cell-mediated killing. We plan to extrapolate our findings into in vivo xenograft systems with adoptive transplants of primary human NK cells. The study will provide important insights into AML evasion from NK cell-mediated surveillance and potentially give a novel rationale for STAT3 inhibition in AML.

P484: WHOLE EXOME SEQUENCING (WES) CHARACTERIZES THE MUTATIONAL LANDSCAPE OF BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM (BPDCN)

H. Witte 1,* , A. Kuenstner 2 , J. Schwarting 3 , V. Bernard 4 , H. Merz 4 , N. von Bubnoff 3 , H. Busch 2 , A. Feller 4 , N. Gebauer 3

1 Hematology and Oncology, German Armed Forces Hospital Ulm, Ulm; 2 Medical Systems Biology Group, University of Luebeck; 3 Hematology and Oncology, UKSH Campus Lübeck; 4 Haematopathology, Haematopathology Luebeck, Luebeck, Germany

Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and clinically aggressive hematologic malignancy. Due to novel technologies pathogenetic concepts of BPDCN underlie a dynamic process. However, its genomic features including novel therapeutic vulnerabilities remain sparsely characterized.

Aims: The present study aimed to illustrate the mutational landscape of BPDCN.

Methods: To delineate the mutational landscape of BPDCN, whole-exome sequencing (WES) of 47 cases was performed. Significantly mutated genes, oncogenic drivers and perturbed pathways were compared with data from acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML).

Results: Median age was 73 years (range 26 – 91) and 72.3% were male. Primary involvement of the skin and the bone marrow was present in 28 (59.6%) and 15 (34.9%) cases, respectively. A median of 98 mutations per sample was detected resulting in a tumor mutational burden (TMB) of 2.57 mutations/Mb/sample. WES and consecutive gene set enrichment analysis revealed an accumulation of oncogenic mutations in epigenetic regulators of gene-expression (95.7% of cases; TET2, DNMT3A, KMT2D, SETD2, IDH2 ), RTK-RAS (93.6%; NRAS, MET, EGFR ), NOTCH (76.6%; NOTCH2, CREBBP, EP300 ) and WNT signaling pathways (59.6%; CTNNB1, MED12 ). Moreover, WES identified a subset of shared myeloid drivers across the spectrum of BPDCN, AML and CMML. In the era of precision oncology, exome profiling revealed several potential therapeutic targets such as NOTCH2, NRAS, EGFR or EZH2 .

Summary/Conclusion: To the best of our knowledge, we provide genomic profiling in the largest WES cohort in BPDCN to date. Current results confirmed previous genomic features associated with myeloid pathogenesis, identified additional significantly mutated drivers and novel potential therapeutic vulnerabilities.

P485: TARGETING HISTONE LYSINE-SPECIFIC DEMETHYLASES BY JIB-04 IMPROVES RESPONSE OF AML CELLS TO VENETOCLAX TREATMENT

K. Wohlan 1,* , D. Fan 2,3 , J. Su 2,3 , A. G. Guzman 1 , M. A. Goodell 1,4,5,6,7 , R. E. Rau 8

1 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, United States of America; 2 Institute of Biomedical Big Data; 3 School of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, China; 4 Program in Developmental Biology; 5 Stem Cells and Regenerative Medicine Center, and Center for Cell and Gene Therapy; 6 Interdepartmental Program in Integrative Molecular and Biomedical Sciences; 7 Department of Molecular and Human Genetics, Baylor College of Medicine; 8 Department of Pediatrics, Baylor College of Medicine, Texas Children’s Hospital, Houston, United States of America

Background: Acute myeloid leukemia (AML) is a heterogeneous, hematologic malignancy that is associated with a poor prognosis. Available treatment options are limited, especially for older patients and others who are ineligible for intensive chemotherapy. The BCL2-specific inhibitor venetoclax has proven efficacy in AML treatment particularly in combination with hypomethylating agents (HMA) or low-dose cytarabine. However, data from a clinical phase II study indicated a 30% failure rate after venetoclax+HMA treatment of newly diagnosed patients (PMID: 30361262), suggesting additional options are needed for patients who do not respond to available treatments or develop resistance. Compared to normal bone marrow histone lysine-specific demethylases are more highly expressed in AML, suggesting they are potential targets for therapeutic intervention. JIB-04 is a potent small molecule inhibitor for multiple members of the KDM 4, 5 and 6 families with KDM5A being the most sensitive. Combined pharmacological inhibition of KDM6 and KDM4 family members has been shown to reduce proliferation and increase apoptosis of primary AML cells of various subtypes (PMID: 29111428). Knockdown of KDM4 and KDM5 proteins leads to proliferation inhibition, cell cycle arrest, and apoptosis in AML cells (PMID: 34017391; PMID: 29602065). Together, these data highlight that KDM4, KDM5, and KDM6 proteins play an important role in the survival of AML cells.

Aims: Describe the efficacy of JIB-04 alone and in combination with venetoclax for the treatment of AML. Identify the molecular changes due to JIB-04 in AML cells of varying genetic subtypes.

Methods: We treated AML cell lines, patient AML blasts, and CD34+ cells from healthy donors with JIB-04, venetoclax, or the combination for 72 hours. Apoptosis was quantified by annexinV/propidium iodide staining. Cell proliferation of AML cell lines treated with JIB-04 was evaluated using MTT assay and transcriptome profiling was done by RNA-Seq.

Results: Treatment of AML cell lines with low nanomolar doses (IC 50 range from 8-60nM) of JIB-04 resulted in inhibition of proliferation and apoptosis. The combination of JIB-04 with venetoclax showed a strong synergistic effect in all AML cell lines including AML cell lines with relative resistance to venetoclax (Figure 1A). Moreover, patient AML blasts also showed a synergistic response to the drug combination compared to single-drug treatments (Figure 1B). In contrast, viability of normal CD34+ hematopoietic cells was not affected by JIB-04 treatment at a dose of 100nM that leads to apoptosis in AML cells, indicating a therapeutic window.

Changes in the transcriptome after JIB-04 treatment were analyzed in AML-OCI3 and MOLM-13 cells. In both cell lines, genes related to the mTOR pathway and stress response were downregulated, indicating possible mechanisms reducing proliferation and increasing apoptosis. Further experiments will be performed to confirm the identified regulated genes as druggable targets in AML that improve venetoclax response.

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Summary/Conclusion: While JIB-04 alone shows some anti-leukemic effect, this study highlights the potential therapeutic benefit of combining JIB-04 and venetoclax in AML. This novel drug combination synergistically inhibits cell growth and induces apoptosis in AML cells with various subtypes. In vivo studies are currently underway to show the efficacy of this drug combination in patient derived xenograft models.

P486: CD9 MARKS DIFFERENTIATION AND ANTI-TUMOR IMMUNITY IN PEDIATRIC ACUTE MYELOID LEUKEMIA

Y. Xu 1,* , K. Y. Y. Chan 1 , S. P. Fok 1 , C. K. Li 1 , K. T. Leung 1

1 Department of Paediatrics, The Chinese University of Hong Kong, Shatin, Hong Kong

Background: Despite intensive treatment regimes, the clinical outcomes for children with acute myeloid leukemia (AML) remain suboptimal, underscoring the need to decipher the underlying pathology and translate into therapeutic modalities. Emerging evidence suggest myeloblasts could evolve multiple machineries to evade immune patrol and hinder immunotherapies.

Aims: We recently reported the importance of CD9 in pediatric ALL but its role in AML remains unknown. In this disease context, we pursued to characterize its prognostic significance, elucidate its regulation and function, and identify its role in leukemia immunosurveillance.

Methods: Patients were stratified based on CD9 status for comparison of long-term survival. Epigenetic control of CD9 was investigated by ChIP-sequencing and confirmed by HDACi treatment. Impact of CD9 on leukemia aggressiveness was measured by competition and colony formation assays. Influence of CD9 on leukemia progression was evaluated in xenograft models, coupled with global transcriptome profiling of AML samples. Immunomodulatory effect of CD9 was dissected by single-cell transcriptomics and validated by flow cytometry. Immune-related CD9 interactors were identified by IP-MS, followed by proof-of-function experiments in an immune-reconstituted mouse model.

Results: The expression of CD9 on blasts of AML patients (12.2%, n=82) was significantly lower than those of ALL patients (90.4%, n=219, P <0.001) or stem cells from normal bone marrow donors (48.4%, n=22, P =0.014). Among AML cases, the blasts of 32 patients (39%) were CD9+. The 5-year relapse-free survival rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P =0.018).

A marked decrease of H3K9/27Ac occupancy in the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P <0.05), and strongly correlated with CD9 repression ( r =0.585-0.719, P <0.01). Exposure of CD9- AML cell lines (n=8) or samples (n=9) to panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P <0.05), and restored activating histone acetylation marks (4.1-41.6-fold, P <0.05).

Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation ( P <0.01) and colony formation ( P =0.002). NOD/SCID mice receiving CD9+ AML exhibited a drastic reduction of leukemic load by 70.7-91.8%. Molecular expression analysis revealed decreased stemness (NES: -1.7, P =0.01) and increased monocyte (NES: 1.8, P =0.034) gene signatures in CD9+ patient samples. Concordantly, a profound up-regulation of CD9 (9.4-51.1-fold, P <0.01) was observed in PMA-mediated monocyte/macrophage differentiation but not in ATRA-mediated neutrophil differentiation of myeloblasts.

Mechanistically, CD9 promoted basal and IFNγ-induced MHC-I/II expression ( P <0.01) through the JAK2/STAT5 axis. Inter-patient comparisons of bone marrow samples (n=31) revealed a higher MHC-I expression in CD9+ AML ( P <0.001). Interestingly, CD9 physically bound to MHC-I/II, formed an immune complex and regulated intracellular trafficking. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13), concomitant with a robust bone marrow infiltration of cytotoxic T cells.

Summary/Conclusion: Our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining differentiation and immune recognition in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy.

P487: SMALL NON-CODING RNAS ASSOCIATE WITH CHROMATIN TO MAINTEIN PROPOGATION OF ACUTE MEYLOID LEUKEMIA

H. Yun 1,2,* , F. Zhou 1,2 , C. Rohde 1,2 , J. Zoller 1 , X. Yu 1,2 , S. Göllner 1 , C. Müller-Tidow 1,2,3

1 Department of Medicine V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital; 2 Molecular Medicine Partnership Unit, European Molecular Biology Laboratory (EMBL); 3 National Center for Tumor Diseases (NCT), Heidelberg, Germany

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the clonal expansion of myeloid progenitors without terminal differentiation. The interplay between genome and epigenome alterations drives aberrant gene network linking to leukemia transformation. Altered transcription may occur at three-dimensional chromatin level and be orchestrated by a combination of multiple regulatory factors. Although some RNA molecules, especially non-coding RNAs, were reported to modulate transcription in leukemia cells, the regulatory role of genome-wide RNAs on transcription through chromatin association remains largely unexplored.

Aims: To identify novel RNA molecules as chromatin-association factors critical for leukemogenesis through modulating transcription

Methods: We performed in situ Mapping of RNA-Genome Interactome (iMARGI) in a human acute leukemia cell line – MV4-11 cells. Chromatin association of candidate RNAs was verified using Chromatin Isolation by RNA Purification (ChIRP) by means of biotinylated oligonucleotides for target capture. A series of ChIP-seq assays were performed in multiple AML cell lines to profile chromatin occupancy by Fibrillarin (FBL), a catalytic component of C/D box ribonucleoproteins (snoRNPs), as well as to observe the remodelling of chromatin states marked by H3K27ac and H3K9me3 upon shRNA-mediated knockdown of FBL. Loss-of-function experiments using antisense oligonucleotides were performed on candidate chromatin-associated RNAs in AML cell lines, AML primary samples, and CD34 + healthy bone marrow cells.

Results: In total, about 52.1 million RNA-chromatin interactions were identified out of deep sequencing of four iMARGI libraries. Whereas the vast majority (50.5 million, 97%) of RNA-chromatin interactome were intra-chromosome, a small fraction (1.6 million, 3%) occurred inter chromosome. Though protein-coding mRNAs predominate the chromatin-associated RNA species, other non-coding RNAs were also captured. Intriguingly, different from protein-coding mRNAs and long intergenic non-coding RNAs (lincRNAs) which mainly form intra-chromosome interactions, two small non-coding RNA classes, small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs), were found with prevalent frequency (62% and 92%, respectively) of inter-chromosome associations.

SNORD3A (U3) and SNORD118 (U8) were C/D box snoRNAs ranked top by the number of their associated interactions in MV4-11 cells as well as in other cell types. Chromatin association of U3 and U8 was verified using ChIRP, with the observation of unique chromatin binding for both U3 and U8, and of specific binding motifs of transcription factors at their binding sites. ChIP-seq revealed that the snoRNP FBL occupied only a small set of genes (n=118), and shRNA-mediated knockdown of FBL had a minimal impact on H3K27ac and H3K9me3, suggesting that snoRNA-chromatin interactions may function independently on FBL-associated snoRNPs.

ASO-mediated loss of U3 or U8 significantly impairs leukemia cells proliferation and colony forming capacity in multiple AML cell lines and primary AML blast samples, whereas only mildly affects CD34 + healthy cells.

Summary/Conclusion: We identified chromatin-associated snoRNAs U3 and U8 which were essential for leukemia maintenance and may serve as therapeutic targets for leukemia eradication.

P488: THZ1, A COVALENT CDK7 INHIBITOR, INDUCES APOPTOSIS IN ACUTE MYELOID LEUKEMIA CELLS AND EXERTS SYNERGISTIC ANTILEUKEMIA ACTIVITY WITH AZACITIDINE

S. Zhang 1,* , H. Liu 1

1 Beijing Hospital, Beijing, China

Background: Acute myeloid leukemia (AML) is a malignant blood cancer that develops mainly in elderly adults and has dismal clinical outcomes. Azacitidine (AZA) is still the preferred treatment for elderly patients unfit for intensive chemotherapy. However, the remission rate of azacitidine monotherapy is dismal, which necessitates the multidrug combination therapy. THZ1, a CDK7 inhibitor, has been proven to be effective in various cancers by regulating transcription and the cell cycle and inducing cell death. Here, we investigated the antitumor effect of THZ1 monotherapy and its combination treatment with AZA.

Aims: To investigate the antitumor activity of the CDK7 inhibitor - THZ1 in acute myeloid leukemia (AML).

Methods: We first examined the cell viability of THZ1 against THP-1, MOLM-13, OCI-AML3 cell lines using CCK-8 assays. We used flow cytometry to detect apoptosis after cells were stained with Annexin V-FITC/Propidium Iodide (PI), and western blot to detect the expression levels of proteins. We detect cell cycle by flow cytometry after cells were stained with 50 µg/ml PI and then analyzed with ModFit LT 5.0. RNA-sequencing analysis was performed to explore the potential mechanism of THZ1 in AML cells. In the double-drug combination experiment, we used CCK-8 assays to detect the effect of drugs on the viability of the three cell lines, used CompuSyn software to calculate the combination index (CI), which indicates additive effects (CI = 1.0), synergism (CI < 1.0), and antagonism (CI > 1.0).

Results: THZ1 decreased viability and induced apoptosis in THP1, MOLM-13, and OCI-AML3 cells in a dose- and time-dependent manner. Besides, THZ1 inhibited phosphorylation of Ser2, Ser5, and Ser7 residues of RNA Pol II CTD and induced cell cycle arrest at G0/G1 phase in AML cells. RNA-sequencing analysis revealed that THZ1 induced changes in the expression of genes involved in apoptosis, the cell cycle, and DNA repair. Combined treatment with AZA and THZ1 showed a synergetic effect (CI < 1.0). Western blot analysis of apoptosis markers, including cleaved caspase3 and PARP1, demonstrated that THZ1 increased the expression of these markers and potentiated AZA-related apoptosis. Compared with monotherapy, combined treatment with AZA and THZ1 resulted in more Annexin V positive cells detected by flow cytometry, which further verified the synergistic effect of the two drugs. Western blot results indicate that combined treatment with AZA and THZ1 downregulates MCL1 at the protein level without an apparent decrease in BCL2 protein expression.

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Summary/Conclusion: Our data demonstrate that the CDK7 inhibitor THZ1 induces the apoptosis of AML cells and exerts synergistic antileukemia activity with azacytidine, which provide the rationale for combination treatment with AZA and THZ1 for AML.

P489: THE LONG NON-CODING RNA LINC01547 PROMOTES PROLIFERATION, CLONOGENICITY, AND CELL CYCLE PROGRESSION IN ACUTE MYELOID LEUKEMIA

S. M. N. Zimmermann 1,2,3,* , M. F. Blank 1,2,4 , D. Heid 1,4,5 , M. Bruckmann 1 , C. Müller-Tidow 1,3,4 , J. Krijgsveld 2,3

1 Department of Medicine V, Hematology, Oncology and Rheumatology, University Hospital Heidelberg; 2 Division Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ); 3 University of Heidelberg Medical Faculty; 4 Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 5 European Molecular Biology Laboratory EMBL, Heidelberg, Germany

Background: Non-coding sequences account for about 98% of the human genome, and it has become increasingly apparent that non-protein coding transcripts drive numerous physiological and pathological cellular processes. Long non-coding RNAs play important roles in cancer, but functions in leukemogenesis and leukemic maintenance, as well as underlying mechanisms, remain to be elucidated.

Aims: In this study, we characterize the functional and mechanistic implications of the long non-coding RNA LINC01547 in acute myeloid leukemia (AML).

Methods: We generated single-cell derived, biallelic LINC01547 knock-out clones in Kasumi-1 cells using the CRISPR/Cas9 system with paired sgRNAs. Knock-out and control cells were assessed for differences in proliferation and clonogenicity by cell growth and methylcellulose assays. Alterations in proliferation, cell cycle progression, apoptosis, and global protein synthesis were subsequently evaluated in flow cytometry-based assays. Finally, we characterized the impact of LINC01547 loss on RNA and protein homeostasis by RNA sequencing, as well as nascent and steady-state proteomics.

Results: Bioinformatic analyses of expression data suggested a role for LINC01547 in AML. LINC01547 was highly expressed in AML (TCGA data), and its expression is associated with worse overall survival in AML.

Biallelic deletion of LINC01547 suppressed AML cell growth and colony formation capacity. Similarly, EdU-based proliferation assays showed significantly reduced proliferation upon loss of LINC01547. Overall protein synthesis, assessed by O-propargyl-puromycin (OPP) assays, and apoptosis, analyzed by annexin V assays, were not significantly affected.

Transcriptome and steady-state proteome analyses revealed that depletion of LINC01547 impacted the expression of multiple mRNAs and proteins. An overall weak correlation between differential expression on the transcript and the protein level indicated that LINC01547 might mediate its effects through both, transcriptional and post-transcriptional mechanisms. In line, mass-spectrometric analysis of nascent and steady-state proteomes upon LINC01547 depletion showed numerous alterations on the translational and total protein level, whereas the respective mRNAs often remained unaffected. Especially proteins involved in cell cycle regulation exhibited reduced translation rates upon loss of LINC01547. Consistently, cell cycle assays showed that depletion of LINC01547 slowed cell cycle progression and led to accumulation of cells in G0/G1 phase. Overlapping our transcriptome, total proteome, and nascent proteome data sets, we identify potential downstream targets through which LINC01547 presumably exerts its newly uncovered pro-oncogenic function.

Summary/Conclusion: We demonstrate that the previously uncharacterized long non-coding RNA LINC01547 has wide-ranging functions in AML. Its overexpression in AML and negative correlation with overall survival are substantiated by its uncovered implications in cell proliferation, clonogenicity, and cell cycle progression. In-depth characterization and overlap of its impact on transcriptome, nascent proteome and total proteome reveal numerous genes and pathways affected by LINC01547 depletion. Further assessment of LINC01547 and its downstream targets will improve our understanding of the mechanisms underlying leukemogenesis and potentially reveal novel leverage points in AML therapy.

P490: SNORNA-DERIVED RNAS (SDRNAS) ARE IMPORTANT DRIVERS OF LEUKEMOGENESIS AND LEUKEMIC MAINTENANCE IN AML

R. Zinz 1,2,* , C. Rohde 1,3 , D. Heid 1,3,4 , M. Bruckmann 1 , M. Bornhäuser 5 , C. Röllig 5 , U. Platzbecker 6 , C. Baldus 7 , H. Serve 8 , F. Zhou 1 , C. Pabst 1,3 , C. Müller-Tidow 1,2,3 , M. F. Blank 1,3,9

1 Medical Department V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital; 2 University of Heidelberg Medical Faculty; 3 Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory (EMBL); 4 European Molecular Biology Laboratory (EMBL), Heidelberg; 5 Medical Clinic and Policlinic I, University Hospital Carl Gustav Carus and Medical Faculty of the TU Dresden, Dresden; 6 Department of Hematology, Cellular Therapy and Hemostaseology, Leipzig University Hospital, Leipzig; 7 Medical Department II, Hematology/Oncology, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel; 8 Department of Medicine II, Hematology/Oncology, University Hospital Frankfurt, Goethe-University Frankfurt, Frankfurt; 9 Division Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany

Background: Small non-coding RNAs play important roles in leukemogenesis. The canonical function of small nucleolar RNAs (snoRNAs) is to facilitate 2’-O-methylation of rRNA via a target site-specific sequence and thus promote ribosomal biogenesis and function. Recently, we have shown that a subset of small non-coding RNAs, i.e. C/D box snoRNAs are overexpressed in acute myeloid leukemia (AML) and required for AML1/ETO-driven leukemia initiation and maintenance. Depletion of snoRNP protein components or even single snoRNAs can potently inhibit AML cell growth or decrease clonogenicity, and the levels of many snoRNAs positively correlate with leukemic stem cell frequency in AML patients. Over the past years, it has been shown that snoRNAs can be further processed into shorter snoRNA-derived RNA fragments (sdRNAs), but the expression patterns of sdRNAs in AML, as well as mechanistic and functional implications, remain widely elusive.

Aims: In this study, we investigated sdRNA expression patterns, functions and mechanisms in AML and healthy hematopoiesis.

Methods: We prospectively characterized snoRNA and sdRNA expression in 96 AML patient samples, as well as in healthy hematopoietic stem and progenitor cells and differentiated white blood cells by small RNA sequencing. mRNA sequencing was performed to investigate connections between snoRNAs, sdRNAs and the transcriptome. Further, we depleted AML cells of different snoRNA loci by CRISPR/Cas9-based knock-out and performed lentiviral rescue overexpression of the respective sdRNA or full-length snoRNA.

Results: We discovered that more than 120 snoRNAs are further processed into sdRNA fragments in AML. Single snoRNAs often evolve into multiple sdRNA isoforms. Interestingly, sdRNA expression varied significantly between different AML patients. Correlation of sdRNA and full-length snoRNA expression with patient characteristics, clinical outcome and mutational status revealed numerous implications. Those include e.g. a distinct pattern of enriched sdRNAs in NPM1-mutated patients. Expression of several sdRNAs was associated with poor overall, event-free and relapse-free survival in AML. We identified sdRNAs that were overexpressed in AML as compared to HSCs and found characteristic expression patterns among differentiated white blood cells. Notably, ratios of sdRNAs and their host snoRNA were not stable across different samples, suggesting an active mechanism regulating sdRNA processing and thus their downstream pathways. Gene set enrichment analyses based on mRNA and sdRNA correlations suggested distinct cell type specific properties.

Forced expression of several sdRNAs rescued the reduced clonogenic potential observed upon depletion of the respective host snoRNA locus. This effect was often identical or even more pronounced than the rescue with the respective snoRNA precursor. Of note, overexpression of distinct sdRNAs in AML cell lines enhanced clonogenic potential.

Summary/Conclusion: SnoRNA-derived sdRNAs are a common feature of AML with characteristic expression patterns determined by mutation status and clinical features. Targeting certain sdRNAs or factors involved in sdRNA processing or function might constitute promising novel therapeutic leverage points in AML.

P491: PREDICTIVE FACTORS OF DIFFERENTIATION SYNDROME IN PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA

M. Ben Salah 1,* , M. Bchir 1 , R. Berred 1 , R. Kharrat 1 , L. Aissaoui 1 , Y. Ben Abdennebi 1 , R. Ben Lakhal 1 , H. Ben neji 1 , B. Meddeb 1

1 Hematology department, Tunis El-Manar University, Faculty of medicine of Tunis, Aziza Othmana Hospital, Tunis, Tunisia

Background: Differentiation syndrome (DS) may be a life-threatening complication in patients with acute promyelocytic leukemia (APL) treated with all-trans retinoic acid (ATRA) and anthracycline chemotherapy. Identifying high risk patients enables a more efficient follow up and an optimal prophylaxis strategy.

Aims: The aim of this study was to analyze the incidence, characteristics, and the risk factors of DS occurring during induction treatment in APL patients treated with ATRA and anthracycline.

Methods: We conducted a retrospective single-center study, including patients diagnosed with APL between 2010 and 2019 in our department and treated with ATRA and chemotherapy according to PETHEMA protocols LPA99 and LPA2005. Obesity, renal failure and a high risk (according to the Sanz score) represented an indication for a DS prophylaxis prescription, using whether prednisone (LPA99) or dexamethasone (LPA2005). A univariate and a multivariate analysis were conducted, in order to identify predictive factors of DS.

Results: Ninety patients were included in our study, with an average age of 34 years old. According to Frankel’s diagnostic criteria, 16 patients (18%) experienced a DS, with a mean age of 39 years [13-71 years], and a sex ratio of 0.6. Half of this group was classified as high risk according to the Sanz score, 44% as intermediate risk, and 6% as low risk. A severe differentiation syndrome was described in 7 patients (44%). The syndrome occurred at a median of 4 days after the start of induction therapy [2-26 days]. A Prophylaxis had been prescribed for 10 patients (62.5%): 8 were classified as high risk, and 2 had obesity.

In order to identify the risk factors of DS, the following parameters were analyzed: age, sex, performance status, Body Mass Index, clinical presentation at diagnosis, treatment protocol, Sanz score, creatinine level, LDH level, hemoglobin level, white blood cell (WBC) count, platelets, cytological type, PML-RARa variant, CD2, CD15, CD34, CD56 expression, fibrinogen level, prothrombin time, presence of disseminated intravascular coagulopathy (DIC), occurrence of bleeding and thromboembolic complications, and corticosteroid prophylaxis.

The univariate analysis identified a statistically significant difference between the group of patients with DS and the one without, for the following factors: WBC greater than 50G/L (31,3% versus 8,1%; p=0,02), the presence of DIC at diagnosis (81.3% versus 18.7%; p=0.01), the expression of CD34 (p=0.017), the LDH level (a median of 307 versus 590; p=0.026), and the treatment protocol LPA 99(11 patients (68.8%) treated according to LPA99 versus 5 (31,3%) treated according to LPA2005; p=0.003). Upon multivariate analysis, WBC count greater than 50 G/L (p=0,007), the presence of DIC at diagnosis (p=0,03), and the treatment protocol LPA99 (p=0,02), remained independent risk factors of DS (Table1).

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Summary/Conclusion: Differentiation syndrome is a significant complication secondary to the ground-breaking treatment of APL. It is necessary to accurately identify high risk patients in order to elaborate effective risk-adapted prophylaxis and treatment.

P492: TAMIBAROTENE IN COMBINATION WITH VENETOCLAX AND AZACITIDINE IN PREVIOUSLY UNTREATED ADULT PATIENTS SELECTED FOR RARA-POSITIVE AML WHO ARE INELIGIBLE FOR STANDARD INDUCTION THERAPY (SELECT AML-1)

E. Stein 1,* , S. de Botton 2 , A. Pigneux 3 , C. McMahon 4 , B. Ball 5 , G. Borthakur 6 , A. Eghtedar 7 , S. Kambhampati 8 , J. Tache 9 , E. Wang 10 , H. Kelley 11 , A. Volkert 11 , K. Baker 11 , Q. Kang-Fortner 11 , G. Hodgson 11 , C. Madigan 11 , E. Warlick 11 , D. Roth 11 , M. Kelly 11 , D. Pollyea 4

1 Department of Medicine, Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, United States of America; 2 Institut Gustave Roussy, Paris; 3 CHU de Bordeaux - Hôpital Haut-Lévèque, Bordeaux, France; 4 University of Colorado, Aurora; 5 City of Hope, Duarte; 6 Department of Leukemia, Division of Cancer Medicine, MD Anderson Cancer Center, Houston; 7 Colorado Blood Cancer Institute, Sarah Cannon Research Institution, Denver; 8 HCA Midwest Research Medical Center, Sarah Cannon Research Institution, Kansas City; 9 BRCR Global, Plantation; 10 Roswell Park, Buffalo; 11 Syros, Cambridge, United States of America

Background: RARA-positive (RARA+) AML is a novel genomically defined patient subset with an actionable biological target for treatment with tamibarotene, an oral and selective RARα agonist (McKeown 2017). RARA+ patients can be selected by a blood-based biomarker test, with approximately 30% of newly diagnosed (ND) AML patients being RARA+ (Vigil 2017). As a biologically targeted agent for patients with RARA overexpression, tamibarotene has the potential to provide benefit irrespective of mutation or cytogenetic risk classification. In RARA+ ND AML patients ineligible for standard induction therapy, tamibarotene plus azacitidine (aza) led to a CR/CRi rate of 61% and a rapid onset of response (de Botton 2020).

Approximately one-third of patients with ND unfit AML do not respond to front-line standard of care venetoclax (ven)/aza (DiNardo 2020). Translational data suggest RARA positivity enriches for monocytic features reported to be associated with ven resistance (Fiore 2020, Pei 2020). This data suggests the RARA biomarker selects for patients who may respond to tamibarotene and may be less likely to respond to ven/aza. Given that tamibarotene plus aza has been generally well tolerated, with no increase in myelosuppression compared to single agent aza (de Botton 2020), it is anticipated that tamibarotene can be administered safely in combination with ven/aza.

Aims: This is a Phase 2, open-label, multi-center study in the U.S. and France comparing the clinical activity of tamibarotene/ven/aza to ven/aza in treatment-naive RARA+ AML patients ineligible for standard induction chemotherapy. The primary objectives are to characterize the safety of the combination and to compare the CR/CRi rate of tamibarotene/ven/aza vs. ven/aza, with secondary objectives to compare CR rate, CR/CRh rate, duration of response, and time to response. The overall response rate using tamibarotene/ven/aza following ven/aza treatment failure will be explored. Clinical activity will be characterized by ELN criteria (Dohner 2017).

Methods: This 3-part trial includes a safety lead-in, randomized efficacy study, and salvage arm. Following the safety lead-in, approximately 80 patients will be randomized 1:1 to receive tamibarotene/ven/aza or ven/aza. In the salvage arm, tamibarotene will be added for study patients randomized to ven/aza who experience progressive disease, relapse, or treatment failure. Patients will be treated with aza at 75 mg/m 2 IV/SC daily on days 1-7, ven on days 1-28 per VENCLEXA USPI, followed by tamibarotene at 6 mg twice per day by mouth on days 8-28 of each 28-day cycle.

Results: Response rates and 95% exact binomial confidence intervals will be calculated by treatment group.

Summary/Conclusion: The SELECT AML-1 trial ( NCT04905407 ) opened in July 2021 with ongoing enrollment.

P493: SINGLE CENTER EXPERIENCE OF VENETOCLAX (VEN) IN COMBINATION WITH FLAG-IDA IN PATIENTS (PTS) WITH NEWLY DIAGNOSED (ND) AND RELAPSED/REFRACTORY (R/R) ACUTE MYELOID LEUKEMIA (AML)

Y. Abaza 1,* , T. Khan 1 , S. Dinner 1 , O. Frankfurt 1 , J. K. Altman 1

1 Northwestern University, Chicago, United States of America

Background: Despite the high complete remission (CR) rates achieved with intensive chemotherapy in AML, relapse rates remain high. This underscores the need for novel regimens capable of inducing deeper remissions via eradicating measurable residual disease (MRD). Recently, FLAG-IDA + VEN was shown to be an effective regimen in AML inducing high rates of MRD negative CR in both ND-AML and R/R AML allowing patients to be successfully bridged to allogenic stem cell transplantation (allo-SCT) (DiNardo 2021).

Aims: Assess the clinical activity of FLAG-IDA + VEN in pts with high-risk AML

Methods: We conducted a single-center retrospective study to assess the clinical activity of FLAG-IDA + VEN in a high-risk pt population. Standard cytogenetic (CN) testing was conducted and molecular data was obtained using a 40-gene next generation sequencing platform. Responses were based on the modified International Working Group criteria and included CR, CR with incomplete platelet recovery (CRp), morphologic leukemia-free state (MLFS), and partial remission (PR).

Results: From March 2020 to January 2022, 17 pts with AML (10 ND and 7 R/R) were treated using FLAG-IDA + VEN (Table 1). Median age was 48 years (range, 21-68) with male predominance (76%). Among the 10 pts with ND-AML (5 de novo, 3 AML-MRC, 2 treated-secondary), 3 had extramedullary disease (EMD). The most common ELN cytogenetic risk groups were adverse (N=2), complex (N=4), and KMT2A-rearranged (N=2). Five pts (50%) harbored a RAS mutation. Median time to initiation of therapy was 5 days (range, 2-14). Five pts [KMT2A-r: 2; complex CN: 2; diploid: 1] achieved CR/CRp including 1 pt with EMD. Median time to count recovery (ANC ≥ 500 and platelets ≥ 50,000) was 21 days (range, 21- 42). Number of cycles of consolidation given to date were 1, 1, 1, 2, and 5 cycles, respectively, and none of these pts have received allo-SCT yet. MRD status was available for 3 pts of which 2 achieved MRD negativity using flow cytometry (<10 -3 ) and one remained MRD positive (NPM1 PCR) after induction. With median follow-up of 4.7 months (range, 1.6-7.6), 4 pts remain alive in CR/CRp and 1 pt [complex CN, del 17p, TP53 mutant (VAF: 90%)] relapsed after 2 cycles of therapy with no response to salvage decitabine (DAC) plus VEN and died due to progressive disease (PD). Of the remaining 5 nonresponders, 2 had treated-secondary AML, 2 with adverse CN [inv (3), -(7)], and 2 pts had EMD. All 5 pts died due to PD including 3 pts who had no response to salvage therapy.

Among the 7 pts with R/R AML, 6 were in salvage 1 and 1 pt received prior allo-SCT. Responses were observed in all patients: 4 CR, 1 CRp, 1 MLFS, and 1 PR. Median time to count recovery in R/R AML pts was 33 days (range, 24-78). Of the 5 pts who achieved CR/CRp, 4 were bridged to allo-SCT and 1 pt died in CR after 3 cycles of consolidation due to sepsis. Of the 4 SCT recipients, 2 pts relapsed 2 months post-SCT (1 died due to PD, 1 alive receiving salvage therapy) and 2 remain alive in CR for 4+ and 9+ months, respectively. The 2 pts who achieved MLFS and PR died due to PD and fungal pneumonia after 5 and 1.5 months of starting therapy, respectively. After median follow-up of 5.3 months (range, 0.7-13.2), median overall survival for the entire population was 6.2 months and the median duration of response for all pts who achieved CR/CRp was not reached.

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Summary/Conclusion: FLAG-IDA plus VEN is a feasible and active regimen in AML. Longer follow-up and larger multicenter studies are needed to confirm the efficacy of this regimen.

P494: MYELOID LEUKEMIA IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES: A RETROSPECTIVE COHORT STUDY

A. Abomhya 1,* , A. Razzaq 2 , S. Lukose 1

1 Internal Medicine, The Brooklyn Hospital Center, Brooklyn, United States of America; 2 St. George’s University School of Medicine, West Indies, Grenada

Background: Inflammatory bowel diseases include ulcerative colitis and Crohn’s disease and affect around 3.1 million adults in the United States (US). The data on the risk of myeloid leukemia among patients with IBD are limited and there has been only a couple of studies that evaluated the incidence of myeloid leukemia in this patient population.

Aims: This study aimed to estimate the prevalence of myeloid leukemia in patients with IBD. Secondary outcomes included mortality, length of stay, all-cause 30-day non-elective readmission rate, and total cost of hospitalization.

Methods: This is a retrospective cohort study for patients with IBD in the US. We queried the Nationwide Readmission Databases 2016-2018 using ICD-10-CM codes to identify all adult patients admitted for IBD. Patients with a comorbid diagnosis of myeloid leukemia including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), Myeloid sarcoma, and Unspecified Myeloid leukemia were identified. Figure 1 summarizes the case selection process. Median and IQR were used to describe Continuous variables, and proportions were used with categorical variables. Comparison between groups was performed by Mann Whitney test for continuous variables and Chi-Square test for Categorical variables. Multivariate regression analysis was performed to study the impact of comorbid myeloid leukemia on inpatient mortality and non-elective readmissions. Statistical analyses were performed using SPSS Version 25 (IBM Corporation, Armonk, NY, USA).

Results: We extracted 365,152 index hospitalization records for IBD, 1052 (0.3%) had myeloid leukemia. Six hundred sixteen patients had acute myeloid leukemia (AML), 341 patients had chronic myeloid leukemia, 10 patients had myeloid sarcoma and 104 had unspecified myeloid leukemia. IBD patients with myeloid leukemia were older (64; Interquartile Range (IQR): 52-73 vs. 56; IQR: 38-70, P <0.001), more common to be males (50.8% vs. 49.2%, P <0.001) compared to IBD patients without myeloid leukemia. Having myeloid leukemia was associated with increased length of stays in days (7; Interquartile Range (IQR): 3-23 vs. 3; IQR: 2-6, P <0.001), increased median total charges ($74,413; IQR: $31,700 - $259,676 vs. $32,586; IQR: $17,827 - $62,260, P <0.001). On multivariate analysis; having myeloid leukemia was associated with increased mortality (Odds ratio (OR): 6.544; 95% confidence interval (CI): 5.318-8.052, P <0.001) and higher odds of all-cause 30-day non-elective readmission (OR: 1.4; 95% CI: 1.155-1.697, P= 0.001).

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Summary/Conclusion: In our nationwide cohort of IBD patients, 0.3% had myeloid leukemia. Given the associated mortality and morbidity, we recommend considering a hematological consult for IBD patients with leukocytosis or leukopenia for further evaluation and appropriate management. More research studies are needed to investigate the pathogenesis of myeloid leukemia in IBD patients.

P495: PHASE 2 STUDY OF ASTX727 (DECITABINE/CEDAZURIDINE) PLUS VENETOCLAX IN PATIENTS WITH RELAPSED/REFRACTORY ACUTE MYELOID LEUKEMIA (AML) OR PREVIOUSLY UNTREATED, ELDERLY PATIENTS UNFIT FOR CHEMOTHERAPY

T. Abuasab 1,* , Y. Alvarado 1 , G. Issa 1 , R. Islam 1 , N. Short 1 , M. Yilmaz 1 , N. jain 1 , L. Masarova 1 , S. Kornblau 1 , E. Jabbour 1 , N. Pemmaraju 1 , G. Montalban-Bravo 1 , S. Pierce 1 , C. DiNardo 1 , T. Kadia 1 , N. Daver 1 , M. Konopleva 1 , G. Garcia-Manero 1 , F. Ravandi 1

1 Department of Leukemia, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Houston, United States of America

Background: ASTX727, is an oral formulation of the fixed dose combination of decitabine and cytidine deaminase inhibitor cedazuridine (35 mg/100 mg).

Aims: To investigate whether a total oral therapy regimen of ASTX727+venetoclax (ven) is feasible and safe.

Methods: Pts aged ≥18 years (yrs) with relapsed/refractory AML (R/R) or pts with AML aged ≥ 75 or 18 -74 with comorbid conditions prohibiting intensive chemotherapy were eligible to participate (frontline-FL). Other eligibility criteria included adequate renal and hepatic function and an ECOG performance status (PS) of≤2. ASTX727 is administered daily on days 1‐5 of each treatment cycle and ven on days 1‐28 of the 1 st cycle after a dose ramp up of 100-200-400 mg over 3 days (with tumor lysis prophylaxis precautions and with ven dose adjustments as needed. A bone marrow exam is performed on day 21±3 of 1 st cycle and ven is held if blasts <5% to allow count recovery. Cycles are repeated every 4-8 weeks depending on count recovery and Ven is administered for 21 days in subsequent cycles, with dose adjustments as necessary depending on count recovery.

Results: Between March 2021 and January 2022, 28 pts (15 FL and 13 R/R) have been treated on the study. The median age is 75 yrs (range, 47-90) with FL cohort 81 and R/R cohort 72. 9 FL pts (60%) were ≥80 and 5 (30%) 70-80 years. In R/R cohort 9 pts (69%) were 70-80 yrs. The median PS is 2 (range 0-3) and in the R/R cohort, the median number of prior treatments is 2 (range, 1-4). In the FL cohort 5 (33%) had normal and 6 (40%) a complex karyotype; 3 had other. In the R/R cohort, 15% had normal, 46% complex karyotype and 31% other. Mutations of note in the FL cohort were RUNX1 (33%), ASXL1 (33%), DNMT3A (7%), TET2 (40%) and TP53 (20%).

The overall response (ORR) including complete response (CR), CR with incomplete count recovery (CRi) and morphological leukemia free state (MLFS) in the FL cohort is 61% (4 CR, 4 CRi, 1 MLFS and 3 non-responders). 3 pts received only one day of therapy for severe adverse events unrelated to therapy (1 due to ischemic stroke, 1 septic shock and 1 debilitation) and were not evaluable for response. In the R/R cohort, the ORR rate was 45% (2 CR, 2 CRi, 2 MLFS with 5 non-responders and 2 not evaluable). The median number of cycles received is 2 (range, 1-5) for both cohorts. With a median follow-up of 5 months, the median survival for the FL cohort has not been reached (range, 0.6 – 7.3) and is 7.2 (range, 0.8-7.3) months for the R/R cohort. Grade 3 or higher adverse events directly attributable to therapy were mainly myelosuppression-related and included neutropenic infections in 3 (11%) and elevation of liver enzymes in 1 (4%) pt.

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Summary/Conclusion: The combination of ASTX727+ven is feasible, particularly in the advanced elderly population, and demonstrates significant efficacy in pts unfit for chemotherapy both in the FL and R/R settings.

P496: CLINICAL CHARACTERISTICS OF SECONDARY MYELOID NEOPLASMS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

T. Abuasab 1,* , S. F. Mohadam 1 , H. Hwang 2 , X. Wang 2 , K. Sasaki 1 , M. Yilmaz 1 , T. Kadia 1 , C. DiNardo 1 , N. Daver 1 , N. Pemmaraju 1 , G. Borthakur 1 , F. Ravandi 1 , G. Garcia-Manero 1 , K. Takahashi 1

1 Department of Leukemia; 2 Department of Biostatistics, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Houston, United States of America

Background: Inflammatory Bowel Disease (IBD) comprises two major disorders: ulcerative colitis (UC) and Crohn’s disease (CD). Patients (pts) with IBD are at an increased risk of cancer secondary to long-standing intestinal inflammation and secondary to immunosuppressive therapies, including but not limited to colorectal cancer, hepatobiliary tract cancer, Hodgkin, and non-Hodgkin lymphomas. However, very little data is available regarding the risk of secondary myeloid neoplasm (MNs) in pts with IBD except for the well-known association between azathioprine with therapy related MNs.

Aims: To describe the clinical characteristics of secondary MNs in patients with IBDs.

Methods: Retrospective chart review of patients with MNs who were previously treated for IBDs was performed. A descriptive statistic was performed to define the demographics, clinical and biological characteristics of the pts included in the study.

Results: Between 2012 and 2020, 43 pts were identified to have developed a secondary MN during or after the treatment of IBDs. Sixty-three percent (27/43) were female, and 37% (16/43) were male, with most of the pts being of white ethnicity (35/43, 81%). Seventy percent (30/43) of the secondary MNs arose after the therapy for CD, whereas 30% (13/43) arose after UC therapy. The median age at the time of MN diagnosis was 59 years (yrs) (range, 23-83) and latency from IBD diagnosis was 16 years (range, 0-56). Twenty-five pts were on active treatment for IBD at the time of MN diagnosis (9 on biological agent, 11 on mesalamine, and 2 on azathioprine), whereas 18 were in remission. In addition, 8 pts (19%) had secondary cancer before the MN diagnosis (2 pts lymphoma, 2 pts skin cancer, ovary Ca, uterine Ca, schwannoma, and adrenal tumor) and 3 of them received therapy (chemotherapy and/or radiation therapy).

Seventy four percent (32/43) of MN diagnoses were Acute myeloid leukemia (AML), 9 (21%) pts had myelodysplastic syndrome (MDS), and one each for MDS/myeloproliferative neoplasm (MPN) and chronic myelomonocytic (CMML). Seventy percent of the patients had cytogenetic abnormalities:13 pts (30%) with complex karyotype and 6 pts (14%) with core-binding factor (CBF) abnormalities (5 with inv16 and 1 with t [8;21]). The most common somatic mutations included: TP53 (N = 10, 23%), FLT3 (N = 7, 16%), RAS (N = 8, 19%), TET2 (N = 7, 16%), and DNMT3A mutations (N = 6, 14%). Interestingly, all the pts with CBF-AML had prior history of CD, two of them treated with anti TNF-α treatment.

The median overall survival (OS) was 2.17 yrs for the whole cohort, and there was no difference in OS between MNs arose from CD and UC (2.35 and 2.08 yrs for CD and UC, respectively). Seventeen patients underwent allogenic stem cell transplantation, which resulted in remission of IBDs in 13 pts (76%).

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Summary/Conclusion: We described the clinical characteristics of secondary MNs after IBD therapy. Secondary MNs after IBD therapy included higher than expected prevalence of core-binding factor AML, which was strongly associated with prior history of CD. This observation raises an important question about the association between CD and CBF-AML. Of interest, allogeneic stem cell transplantation led to remission of both MNs and IBDs, highlighting the role of allo SCT in autoimmune diseases.

P497: PROGNOSTIC IMPACT OF RAS AND C-KIT MUTATIONS (SINGLE VS. MULTIPLE) IN CORE-BINDING FACTOR ACUTE MYELOID LEUKEMIA TREATED WITH FLUDARABINE, CYTARABINE, G-CSF (FLAG) BASED REGIMEN

T. Abuasab 1,* , J. Senapati 1 , T. Kadia 1 , F. Ravandi 1 , C. DiNardo 1 , N. Pemmaraju 1 , M. Ohanion 1 , Y. Alvarado 1 , H. Kantarjian 1 , G. Borthakur 1

1 Department of Leukemia, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Houston, United States of America

Background: Core-binding factor acute myeloid leukemia (CBF-AML), including inv (16) and t (8;21) AML, represent 15%-20% of all adult AML patients (pts) and is generally recognized as favorable AML subgroups. Despite being favorable, up to 30-50% of patients with CBF-AML eventually relapse. Both RAS (particularly multiclonal) and KIT mutations in CBF-AML have been associated with poor outcomes, but our initial analysis for patients treated with a fludarabine-based regimen did not support a negative impact of these mutations. In addition, we showed that the optimal reduction of disease defining transcripts (RUNX 1-RUNX1T1 and CBFB-MYH11 ) by PCR at end of induction and end of consolidations, has the most impact on the relapse free survival of these patients.

Aims: We studied the prognostic impact, in a consecutive cohort of CBF-AML patients treated uniformly, of kinase mutations ( RAS and KIT ) and the number (Single vs. multiple) of mutations in addition to the impact of mutation status on attainment of optimal PCR reductions at most informative pre-defined time points; end of induction and end of treatment.

Methods: Pts aged ≥ 18 years (yrs) with de novo CBF-AML, treated with FLAG based regimen with idarubicin or gemtuzumab ozogamicin between April 2013 and December 2019 at our center were included in this analysis. RAS and KIT mutations were tested by next generation sequencing. PCR for disease-specific transcripts was monitored at the end of C1 and end of therapy (EOT). Optimal response was considered as PCR transcripts < 0.1% post C1 and <0.01% at EOT.

Results: A total of 91 pts with CBF AML were evaluated with a median age of 52 yrs (range, 22-79), 41 of whom were females (45%). Fifty-three pts (58%) had Inv 16 and the rest had t (8;21). Twenty-six pts (29%) had FLT3 mutation (ITD and/or TKD), 32 pts (35%) had RAS mutation (19 pts with NRAS, 3 pts with KRAS, and 10 pts with both mutation), and 30 pts (33%) had KIT mutation. Out of 32 pts with RAS mutation, 16 (50%) had single mutation and16 (50%) had multiple RAS mutations, while of 30 pts with KIT mutation, 25 pts (83%) had single KIT mutation, and 5 pts (17%) had multiple KIT mutations.

On regression analysis of presence of KIT or RAS mutation and their impact on end of induction and end of treatment PCR, only KIT mutation was associated with lower odds (0.33, 95% CI 0.11-0.9) of optimal EOT response. Amongst mutated pts, the presence of single versus multiple KIT or RAS mutation did not affect optimal PCR responses.

At a median follow up of 53.1 months (mos), the median relapse free survival (RFS) for the whole cohort was 27.7 mos (range,1.7-93.6).

RAS mutated pts had longer RFS compared to non-mutated pts (not reached vs. 43.78 mos, p=0.02); however, KIT mutation did not affect RFS. In addition, the number of the RAS or KIT mutation (One mutation vs. multiple mutations) also did not impact RFS.

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Summary/Conclusion: KIT mutation might negatively affect attainment of optimal PCR responses at end of FLAG based therapy, but it lacked significant impact on relapse free survival. Single versus multiple KIT or RAS mutations also do not seem to have an impact on therapy response.

P498: CLINICAL AND BIOLOGICAL MARKERS ASSOCIATED WITH LONG-TERM SURVIVAL FOR PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) IN REMISSION AFTER CHEMOTHERAPY IN THE QUAZAR AML-001 TRIAL OF ORAL AZACITIDINE

A. H. Wei 1,* , H. Döhner 2 , H. Sayar 3 , F. Ravandi 4 , P. Montesinos 5 , H. Dombret 6 , D. Selleslag 7 , K. Porkka 8 , J.-H. Jang 9 , B. Skikne 10 , C. Beach 10 , T. Prebet 10 , G. Zhang 10 , A. Risueño 11 , M. Ugidos Guerrero 11 , W. L. See 10 , D. Menezes 10 , G. J. Roboz 12

1 Department of Clinical Haematology, Alfred Hospital, and the Australian Centre for Blood Diseases, Monash University, Melbourne, Australia; 2 Department of Internal Medicine III, Ulm University Hospital, Ulm, Germany; 3 Indiana University Cancer Center, Indianapolis; 4 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America; 5 Hospital Universitario y Politécnico La Fe, Valencia, Spain; 6 Hematology, Hôpital Saint-Louis, Assistance Publique – Hôpitaux de Paris (AP-HP), and Institut de Recherche Saint-Louis, Université de Paris, Paris, France; 7 AZ Sint-Jan Brugge-Oostende AV, Bruges, Belgium; 8 HUS Comprehensive Cancer Center, Hematology Research Unit Helsinki and iCAN Digital Precision Cancer Center Medicine Flagship, University of Helsinki, Helsinki, Finland; 9 Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 10 Bristol Myers Squibb, Princeton, United States of America; 11 BMS Center for Innovation and Translational Research Europe (CITRE, a Bristol-Myers Squibb Company), Seville, Spain; 12 Weill Cornell Medicine and New York Presbyterian Hospital, New York, United States of America

Background: In the randomized, phase 3 QUAZAR AML-001 trial, oral azacitidine (Oral-AZA) significantly prolonged overall survival (OS) compared with placebo (PBO) (median OS 24.7 vs 14.8 months [mo], respectively) in older patients (pts) with AML in first remission after intensive chemotherapy (IC). At an updated data cutoff performed in Sep 2020, 34.9% of pts in the Oral-AZA arm and 24.4% of pts in the PBO arm remained alive at ≥3 y from randomization.

Aims: Assess clinical and biological variables associated with long-term survival (LTS) in QUAZAR AML-001.

Methods: In all, 472 pts were randomized 1:1 to receive Oral-AZA 300 mg or PBO QD ×14d/28d within 4 mo of achieving first complete remission (CR) or CR with incomplete blood count recovery (CRi) after IC. The primary endpoint was OS, time from randomization until death, withdrawal of consent, or loss to follow-up. The LTS cohort comprised pts who were alive ≥3 y from randomization as of Sep 2020 and the non-LTS cohort included pts who died or were censored before 3 y. Variables assessed for association with LTS were diagnostic (Dx [pre-IC]) features (AML subtype, cytogenetic risk, NPM1 and FLT3 mutations [ mut ]); pre-study treatment (Tx) variables (response to IC [CR/CRi], receipt of consolidation, number of consolidation cycles); baseline (BL) demographic and disease characteristics, hematologic parameters (red blood cells [RBCs], hemoglobin, platelets, and leukocyte subsets), and measurable residual disease (MRD) status; and post-BL variables (MRD response [conversion from MRD+ at BL to MRD– on study], timing of MRD– [MRD– response on-study vs BL MRD–], and receipt of transplant after Tx discontinuation [D/C]). Associations of LTS with bone marrow immune parameters (CD3, CD4, and CD8 T-cell counts, and expression of PD-1/TIM-3 T-cell exhaustion markers) were investigated in a subset of pts (n=108). Variables were compared within Tx arms (LTS vs non-LTS) in univariate analyses with P values corrected for multiple testing. A logistic multivariable regression analysis of the effects of prognostic BL covariates on LTS was performed.

Results: The LTS cohort included 83/238 pts (34.9%) in the Oral-AZA arm and 57/234 pts (24.4%) in the PBO arm. Within both arms, factors significantly associated with LTS were intermediate (Int)-risk cytogenetics and NPM1 mut at Dx, and MRD response (MRD+ to MRD–) on study ( Figure ). MRD response rate was 2-fold higher with Oral-AZA vs PBO (37% vs 19%, respectively), and while early attrition was more common in the PBO arm, most MRD responses occurred within 6 mo. Factors significantly associated with LTS only in the PBO arm were BL (post-IC) MRD– status and receipt of transplant after Tx D/C. No significant associations were observed between BL hematological or immune parameters and LTS.

The multivariable analysis (MVA) confirmed Oral-AZA Tx as independently significantly predictive of LTS vs PBO. Other covariates significantly associated with LTS in MVA were Int-risk cytogenetics and NPM1 mut at Dx, and MRD– status at BL.

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Summary/Conclusion: Oral-AZA Tx was significantly associated with LTS vs PBO. In univariate analysis, Int-risk cytogenetics and NPM1 mut at Dx, and MRD response on-study, were significantly prognostic of LTS in both arms, whereas MRD– status at BL (post-IC) was associated with LTS only in the PBO arm.

P499: THE PHASE 1B OMNIVERSE TRIAL OF ORAL AZACITIDINE IN COMBINATION WITH VENETOCLAX FOR TREATMENT OF RELAPSED/REFRACTORY OR NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA (TRIAL IN PROGRESS)

A. H. Wei 1,* , H. E. Carraway 2 , L. Taningco 3 , E. Laille 3 , J. Gong 3 , T. Prebet 3 , D. Lopes de Menezes 3 , F. Ravandi 4

1 Department of Clinical Haematology, Alfred Hospital, and the Australian Centre for Blood Diseases, Monash University, Melbourne, Australia; 2 Leukemia Program, Taussig Cancer Institute, Cleveland Clinic, Cleveland; 3 Bristol Myers Squibb, Princeton; 4 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: For older patients (pts) with acute myeloid leukemia (AML) who are ineligible for standard intensive chemotherapy (IC) regimens, traditional lower-intensity AML treatment (Tx) options include low-dose cytarabine (LDAC) or hypomethylating agents (HMAs; azacitidine [AZA] and decitabine), which are generally well tolerated but associated with suboptimal outcomes vs IC [Vey 2020]. Therefore, there remains a need for less toxic and more efficacious lower-intensity AML Tx regimens, especially agents that can be administered in the outpatient setting, which may increase convenience and reduce resource utilization.

Venetoclax (VEN) is an oral, selective, small-molecule BCL2 inhibitor that has demonstrated considerable activity when combined with LDAC or HMAs in older pts with AML. In a recent phase 3 trial, VEN + injectable AZA significantly improved response and survival vs AZA alone in IC-ineligible pts with newly diagnosed (ND) AML [DiNardo 2020]. In the USA, VEN is approved in combination with an HMA or LDAC for Tx of pts with ND AML ≥ 75 years (y) of age or who cannot use IC due to comorbidities.

Oral-AZA (CC-486) is approved for pts with AML in first remission (CR1) after IC who are ineligible for transplant or other curative therapies. In the randomized, phase 3 QUAZAR AML-001 trial of Oral-AZA in pts with AML in CR1 after IC, Oral-AZA 300 mg QD for 14 days (d)/28d Tx cycle was generally well tolerated and associated with significantly improved overall survival vs placebo. Gastrointestinal symptoms were the most common adverse events reported with Oral-AZA [Wei 2020]. Oral-AZA has also shown clinical activity in pts with active AML [Savona 2015].

As incorporation of AZA into DNA is S-phase-restricted, extended Oral-AZA dosing regimens (> 7d/28d Tx cycle) increase drug incorporation into cycling tumor cells to prolong epigenetic activity throughout the Tx cycle [Laille 2015]. An all-oral VEN + Oral-AZA combination regimen allows for outpatient administration and thus improves pt adherence [Eek 2016].

Aims: Describe the study design and objectives of the OMNIVERSE trial of Oral-AZA + VEN in older pts with ND AML or pts with AML relapsed/refractory (R/R) to prior Tx.

Methods: OMNIVERSE ( NCT04887857 ) is a multicenter, open-label, 2-part phase 1b trial ( Figure ). The key objectives of the trial are to assess safety and determine the maximum tolerated dose of Oral-AZA + VEN in pts with R/R AML (WHO 2016 criteria) ineligible to receive further IC ( part I ), and then in pts with ND AML ≥ 75 y of age, or those ≥ 18–74 y of age ineligible for IC or HSCT due to comorbidities ( part 2 ). Main eligibility criteria include an ECOG performance status of 0–2 (ECOG 3 is allowed for pts ≥ 18–74 y of age with comorbidities) and an unfavorable cytogenetic risk profile for pts with ND AML. The initial dose of Oral-AZA is 300 mg QD × 14d/28d cycle, with de-escalation to 200 mg × 14d/28d cycle allowed for dose-limiting toxicities. VEN 400 mg is taken orally QD in continuous 28d cycles (or for 21d/cycle for dose level –2). A modified toxicity probability interval-2 design is used to evaluate the planned dose levels. The sample size is dependent on the dose levels included in the study (≤ 18 pts/part).

Results: N/A

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Summary/Conclusion: Study enrollment began in 2021. The trial is ongoing at clinical sites in the United States and Australia.

P500: A REAL WORLD MULTI CENTRE STUDY OF CPX-351 REVEALS NO DIFFERENCE IN OVERALL SURVIVAL WHEN COMPARED WITH FLAG-IDA AND 3 + 7 IN HIGH RISK AML.

C. Andrews 1,* , I. AlNabhani 1 , T. Young 1 , E. G. Atenafu 1 , S. E. Assouline 2 , J. M. Brandwein 3 , S. M. Chan 1 , S. Chow 4 , D. Khalaf 5 , V. Gupta 1 , D. D. H. Kim 6 , D. Maze 1 , M. M. Minden 1 , T. Murphy 1 , D. Sanford 7 , A. D. Schimmer 1 , A. C. Schuh 1 , J. Sibai 1 , K. Yee 1 , H. Sibai 1

1 Division of Medical Haematology and Oncology, Princess Margaret Cancer Centre, Toronto; 2 Division of Hematology, Sir Mortimer B. Davis-Jewish General Hospital, Montreal; 3 Division of Hematology, University of Alberta, Edmonton; 4 Odette Cancer Centre, Sunnybrook Health Science Centre, Toronto; 5 Division of Hematology, Juravinski Cancer Centre, McMaster University, Hamilton; 6 Hans Messner Allogeneic Blood and Marrow Transplant Program, Princess Margaret Cancer Centre, Toronto; 7 Leukemia/Bone Marrow Transplant Program of British Columbia, University of British Columbia, Vancouver, Canada

Background: CPX-351 is a liposomal formulation of daunorubicin and cytarabine in a fixed synergistic ratio of 5:1. It has been approved by both the FDA and EMA for use in high-risk AML including therapy related AML (t-AML) and AML with myelodysplastic related change (AML-MRC). However, this is little data of CPX-351 in a real-world setting. There is also no data comparing CPX-351 to FLAG-IDA which is used in our institution front line for high-risk AML

Aims: The objective of our multi-centre study was to assess outcomes of CPX-351 and then compare these outcomes in patients who received FLAG-IDA and 3 + 7 for high risk AML(tAML and AML-MRC).

Methods: Patients aged 18 and over who were treated with induction chemotherapy and who met the WHO criteria for t-AML and AML-MRC were included in the study. Retrospective data was collected from 10 centres throughout Canada for CPX-351. The Princess Margaret Cancer Centre database was used to collect data for patients who received induction with FLAG-IDA and 3 + 7. Targeted sequencing was performed on DNA samples using the TruSight Myeloid Sequencing Panel. Overall survival (OS) and progression free survival (PFS) rates were calculated using the Kaplan-Meier method.

Results: 76 patients treated with CPX-351 were identified with baseline characteristics seen on Table 1. Targeted sequencing was performed on 72% of patients (55/76) and the average number of mutations was 2(0-7). RUNX1 was the most commonly mutated gene found in 22% (12/55), followed by ASXL1 mutated in 19% (10/55) and SRSF2 in 15%. Assessing treatment responses, 53% (38/76) of patients achieved a complete remission (CR) or a complete remission with incomplete recovery (CRi). The median CR duration was short at 7.3 months. Median follow up was 7.78 months (range 0.2 to 20 months). OS was 57% at 12 months and 38% at 18 months. PFS was 40% at 12 months and 23% at 18 months. There were no differences in OS when stratified by ELN risk (p=0.46), adverse risk cytogenetics (p=0.1485), or poor risk mutations such as RUNX1 (p=0.73), ASXL1 (p=0.47) or TP53(p=0.53).Patients who received an ASCT had significant improvement of OS of 82% at 18 months compared to those who did not receive a transplant of 15% at 18 months (Fig 2; p=0.0001).

Patients who received 3 + 7 and FLAG-IDA for the same indications as CPX-351 (tAML and AML-MRC) were identified. Baseline characteristics are shown in Table 1. 69% of patients receiving FLAG-IDA and 67% of those receiving 3 + 7 had adverse ELN risk stratification. CR rates were highest with FLAG IDA with 75% achieving a CR or CRi. 68% of patients who received CPX-351 proceeded to ASCT compared with 59% with FLAG-IDA and 57% with 3 + 7. OS at 18 months was 42% for FLAG-IDA and 39% for 3 + 7. There was no statistical difference in OS when comparing CPX-351 to FLAG-IDA and 3 + 7 (p=0.855) (Figure 1). Similarly, there was no difference in PFS when comparing all 3 inductions (p=0.26). Patients who received a ASCT had significant improvement of OS of 59% at 18 months compared to those who did not receive a transplant of 24% at 18 months (p=<0.0001)irrespective of what induction was used.

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Summary/Conclusion: High risk AML remains an unmet clinical need which is supported by our study which reveals no difference in OS or PFS when treated with either CPX-351, FLAG-IDA or 3 + 7. Unsurprisingly, ASCT improved OS across all patients irrespective of induction type. Further study with a prospective randomised control trial is required.

P501: CLI120-001 PHASE1B DOSE ESCALATION STUDY OF RVU120 IN PATIENTS WITH AML OR HIGH RISK MDS SAFETY AND EFFICACY DATA UPDATE

C. Abboud 1 , Z. Jan Maciej 2 , G. Borthakur 3 , S. Solomon 4 , B. Howard 5 , T. Bradley 6 , E. Mouhayar 7 , N. Angelosanto 8,* , H. Nogai 8 , A. Glasmacher 9 , R. Dudziak 8 , K. Brzozka 10 , T. Rzymski 10 , P. Littlewood 11 , E.-L. Maranda 12

1 Division of Oncology, Washington University In Saint Louis, Saint Louis, United States of America; 2 Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland; 3 Department of Leukemia, MD Anderson Cancer Center, Houston; 4 Blood and Marrow Transplant Leukemia and Immunotherapy Unit, Northside Hospital, Atlanta; 5 Sarah Cannon Research Institute, Sarah Cannon, Nashville; 6 Division of Hematology, University of Miami Health System, Miami; 7 Department of Cardiology, MD Anderson Cancer Center, Houston, United States of America; 8 CLINICAL DEVELOPMENT, Ryvu therapeutics, Krakow, Poland; 9 Dep. of Internal Medicine III, University Clinic Bonn, Bonn, Germany; 10 Research & Preclinical Development; 11 DMPK Department, Ryvu Therapeutics, Krakow; 12 Deaprtment of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland

Background: CDK8 and its paralog CDK19 have central roles in maintenance of cancer cell viability and undifferentiated state for a variety of tumor types. (Dannappel et al. 2019; Rzymski et al. 2015; Philip et al. 2018). RVU120 (SEL120), a novel CDK8/CDK19 kinase inhibitor with significant efficacy in preclinical AML models, has shown clinical efficacy in a currently ongoing phase Ib trial in patients with relapsed/refractory (R/R) AML or HR-MDS ( NCT04021368 ). This paper provides update with new available data on disease evaluation from ongoing patients and further enrolment into next cohort level 85 mg.

Aims: The primary objective of the study is to determine preliminary safety profile, dose limiting toxicities (DLTs), maximum tolerated dose (MTD) and the recommended phase 2 dose of RVU120 as a single agent. Secondary objectives include PK, antileukemic activity and exploratory PD characterization.

Methods: The study comprises at least 7 dose escalating cohorts. The first 3 cohorts followed an accelerated scheme, 1 patient enrolled/cohort from 10 to 50 mg dose levels, from cohort 4 (75 mg) to 7 (100 mg) doses onwards a 3 + 3 design is followed. Data from each cohort is evaluated by a data review committee (DRC). RVU120 is administered orally every other day, for a total of 7 doses, in a 3-week treatment cycle until disease progression/unacceptable toxicity. Adverse events are graded according to NCI-CTCAE v.5.0. DLTs are assessed at completion of C1. Disease evaluation is performed according to Dohner 2017 and Cheson 2006 response criteria for AML and MDS respectively. PK parameters are calculated by non-compartmental analysis. Pharmacodynamic (PD) activity is assessed by flow cytometry measure of pSTAT5 Ser725 levels, that are highly dependent on the activity of CDK8 and CDK19 in AML/MDS cells.

Results: At data cut off of 23 rd Feb22, 13 pts have been enrolled, median age 73 years and median 2 previous lines of therapy, ECOG PS 2 in 4 pts, 1 in 7, 0 in 2. No DLTs were observed, all 14 Serious Adverse Events, including 1 COVID19 death and 1 pancreatitis, were not related to study drug (G1 fever, G2 Upper Respiratory Infection, G3: pseudomonas sepsis; urinary tract infection; febrile neutropenia; lung infection, pain, hemoptysis, pleural effusion, G5 pneumonitis, death NOS, pancreatitis). Cohort 1 pt, 10 mg dose level, and cohort 2 pt, 25 mg, showed stable (SD) and progressive disease (PD) respectively at the end of C1. Cohort 3 pt, an 81 YO male HR-MDS, escalated from 50 to 75 mg dose from C7, is SD at C24D13 with Erythroid Hematological Improvement on C5, C7, C10, C18. Cohort 4, 75 mg dose pt, a 62 YO male with AML DNMT3A pos, relapsing after Ven/Dec, achieved CRi at the end of C1 and CR in C7, and progressed at the end of C8. Two out of the remaining 4 pts treated at 75 mg reached SD (1 still ongoing at C3D15 and another died on C3D20 while on SD), 1 pt died of COVID-19 pneumonitis on C1D18, 1 pt with AML secondary to MPN was SD at C2 and progressed on C4. Two pt were treated at 110 mg (cohort 5), 1 not evaluable died for pancreatitis and 1 was SD at the end of C1. 2 pt entered cohort 6, 85 mg, and will be evaluable at the end of March 2022.

Summary/Conclusion: Preliminary results from the first 6 cohorts have shown a favorable safety and a predictable PK profile of RVU120. Meaningful PD activity and clinical efficacy were observed at 50 and 75 mg doses. Enrollment is currently ongoing at 85 mg cohort

P502: CLINICAL IMPLEMENTATION OF GERMLINE GENETIC TESTING FOR HEMATOLOGIC DISORDERS

S. Ansar 1,* , J. Malcolmson 2,3 , K. M. Farncombe 4 , K. Yee 1 , R. H. Kim 1,5,6 , H. Sibai 1

1 Division of Medical Oncology and Hematology; 2 Familial Cancer Clinic, Princess Margaret Cancer Centre, University Health Network; 3 Department of Molecular Genetics, University of Toronto; 4 Toronto General Hospital Research Institute, University Health Network; 5 Division of Clinical and Metabolic Genetics, The Hospital for Sick Children; 6 Department of Medicine, University of Toronto, Toronto, Canada

Background: Up to 18% of adult hematology patients (pts) suspected of having an inherited predisposition are found to have a germline mutation leading to a hereditary hematologic disorder (HHD). Timely identification of these disorders leads to heightened surveillance for malignancies, specific treatment regimens, donor selection, transplant conditioning regimens, cascade testing in family members and supportive care. Unlike solid tumour hereditary cancer syndromes, the investigations in HHD are more complex and there is no formal consensus of referral criteria or genetic testing criteria. We describe our initial experience in this patient population at our centre, the Princess Margaret Cancer Centre (PM). We have established a workflow to initiate germline genetic testing in pts under suspicion for a HHD. This involves a collaboration with a genetics clinic to 1) capture pts based on their personal/family history of cancer and suggestive genetic findings identified on bone marrow/blood, 2) procure a skin biopsy for fibroblast culture and germline DNA extraction and 3) provide follow-up counselling and management for positive results.

Aims: Our goal is to evaluate the positivity rate of germline mutations in adult hematology pts who were referred for genetics assessment.

Methods: The PM is the largest leukemia Centre in Canada, and has seen over 3000 adult pts with a malignant disorder from 2015-2021 (AML:1597, MDS:472, ALL:286, MPN:664 and marrow failure:22). We performed a retrospective chart review of all adult hematology pts referred to cancer genetics service for HHD workup during this time period.

Results: 116 pts (66 male, 50 female) were suspected for a HHD and referred for germline genetic testing on fibroblast DNA. 52 pts (45%) were ≤40y old and 64 pts (55%) >40y old (age range 18-86y, median 53y). 71 pts (61%) were referred with a positive family history of hematologic disorders (HD). In addition, 61 pts presented with a myeloid malignancy (46 with MDS, AML, or CML, and 15 with a MPN), 42 presented with a lymphoid malignancy (ALL, CLL, or lymphoma), 8 presented with bone marrow failure, and 5 presented with other HD. 40 pts were found to have a germline genetic mutation, 7 of which were associated with carrier status. In total, 33 (28.4%) referred pts were found to have at least one actionable germline mutation. This corresponds to a positive genetic result found in 30% of pts referred with MDS/AML, 33% of pts referred with a lymphoid malignancy, and 18% of pts referred with bone marrow failure, MPN, or other HD. Additionally, 18 (55%) of these pts had a positive family history of HD, while 15 (45%) presented with no family history of HD. Of these 33 positive cases, 13 (11.2% of all referrals) occurred in a gene associated with a hematologic malignancy and resulted in a HHD diagnosis. 20 pts were found to have a mutation in a gene associated with a non-hematologic hereditary cancer syndrome. After the implementation of a HHD genetics workflow in 2018, we saw an increase in the total number of referrals to a genetics clinic over the past seven years, with 25 pts (6 positive cases) referred between 2015-2018, and 91 pts (27 positive cases) referred between 2019-2021.

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Summary/Conclusion: Our overall positivity rate was 28.4% which includes mutations in any hereditary cancer gene, and 11.2% for HHDs. Our high pickup rate across all age groups and the increase in the number of referrals to genetics service suggests that more pts with HD, including older pts, would benefit from consultation with specialized centres that are experienced in the evaluation and treatment of these disorders.

P503: MRD MONITORING DURING INTENSIVE CHEMOTHERAPY IN PEDIATRIC AML- DATA OF THE AML-BFM GROUP

E. Antoniou 1,2,* , S. Hahn 1,2 , S. Sendker 1,2 , N. von Neuhoff 1,2 , D. Reinhardt 1,2 , M. Schneider 1,2

1 Clinic of pediatrics III, University hospital of Essen; 2 AML-BFM study group, Essen, Germany

Background: During the past decades, minimal residual disease (MRD) has been established as a diagnostic tool, providing critical prognostic information in pediatric acute myeloid leukemia (AML). To measure initial treatment response, immunophenotyping was used by most cooperative study groups. However, known limitation of sensitivity and specificity supported the continuous evaluation of qPCR-based MRD detection in pediatric AML.

Aims: To determine the prognostic role of MRD monitoring by RT-qPCR in the pediatric AML.

Methods: A total of 238 pediatric AML patients were monitored for MRD by reverse-transcriptase quantitative PCR (RT-qPCR) assay from September 2012 to December 2020. Since the inclusion to this study is based on the presence of quantifiable genetic markers, the patient group is strongly biased. The treatment was conducted according to the AML-BFM study 2004 and 2012 as well as the register 12 and 17. All measurements were performed in bone marrow samples or in peripheral blood at initial diagnosis. MRD monitoring included the following aberrations: t(8;21), inv(16), inv(11), NPM1, FLT3-ITD, t(15;17), t(5;11), t(9;11), t(6;9), WT1, t(1;11), t(11;11), t(1;22), t(11;12), t(17;19), t(3;5), t(4;10), t(11;19), t(2;11), t(6;11), t(X;11), t(6;8), t(8;16) and t(8;22).

Results: The patients had a mean age at diagnosis of 9.2 years (range 10 days to 18.6 years) and 51 % were females. They were distributed to the following stratification groups: standard risk (56 %), intermediate risk (27 %) and high risk (17 %). The event free survival was 71 % (±3 %) and the OS reached 90 %(±2 %).

In core binding factor acute myeloid leukemia (CBF-AML) (n=91) a positive MRD level persists in the majority of cases. Following the 3 rd treatment block 64% of cases remained MRD-positive (MRD threshold ≥1x10 -5 ). However, MRD monitoring showed no prognostic relevance as relapses were detected in 18 % (6/33) and 17 % (10/58) of MRD-positive and negative cases, respectively.

For patients with NPM1 mutations (n=26) MRD-positivity and -negativity (threshold of ≥1x10 -6 ) showed a similar relapse rate of 10 %. This data are in contrast to reports in adults (Ivey e t al. NEJM 2016).

In patients with an AML and t(9;11) MRD-positivity after first and second induction indicated a significant higher risk of relapse. After first induction MRD-positive cases (n=19) had a relapse rate of 32 % compared to 13 % for negative cases (n=24). After second induction the relapse rates were 63 % and 22 % for MRD-positive (n=8) and negative (n=27) cases, respectively.

Summary/Conclusion: MRD monitoring is a powerful tool to improve risk group stratification in pediatric AML. Despite its high sensitivity and specificity, the prognostic informativeness of MRD monitoring through RT-qPCR varies widely among different genetic subgroups. In CBL-AML or NPM1-positive AML, which are prognostically more favorable, persisting MRD-positivity alone was not associated with an increase in the risk of relapse. In contrast, MRD-positivity in AML with t(9;11) appears to be highly informative.

Further studies with larger number of patients are needed to further define AML subgroups and thresholds for MRD-positivity in the pediatric AML.

P504: CLINICAL MUTATIONAL ANALYSIS BY NEXT-GENERATION SEQUENCING AND REAL-LIFE VALIDATION OF THE REVISED 2017 EUROPEAN LEUKEMIANET GENETIC RISK STRATIFICATION IN AML PATIENTS

C. Aparicio Pérez 1,* , F. Salas Hernandez 1 , A. C. Gonzalez Teomiro 1 , F. Jimenez Najar 1 , I. Fernandez Camacho 1 , C. Martin Calvo 1 , J. Sánchez García 1,2 , J. Serrano López 1,2

1 hematology, Reina Sofia University Hospital; 2 IMIBIC, Córdoba, Spain

Background: Recently, a census of mutated genes in AML has been described but their value in clinical practice is not fully elucidated. The revised 2017 European LeukemiaNet (ELN) recommendations for genetic risk stratification of AML have been widely adopted, but have not yet been validated in large cohorts of AML patients in real life.

Aims: The objective of this work is to analyze the mutations detected by Next Generation Sequencing (NGS) and to study whether the application of ELN 2017 scale improves prognostic risk stratification with respect to ELN 2010 and Medical Research Council (MRC).

Methods: We included 112 adult AML patients diagnosed in our center between Jun2017 and Dec2021 and who were studied at diagnostic using the spanish PLATFOLMA PETHEMA. The prognostic risk was established according to MRC, ELN2010 and ELN2017 classification. Baseline demographic, disease characteristics, treatment procedures and mutations by functional groupsare summarized in Table 1

NPM1 and FLT3-ITD were determined by melting curve analysis and standard PCR-EC technique according to Thiede et al (Blood 2002) in ABI 3130 Analyzer (Thermofisher). For NGS, the commercial panel Myeloid SolutionTM (Sophia Genetics) KAPA Kit amplification libraries and sequencing on ILUMINA Myseq platform were used. Variant analysis was performed using DDM software (Sophia Genetics).

Results: The majority of the patients (97.1%) had at least one mutation at diagnosis detected by NGS. The median number of mutations was 2.36 (0-6). Grouped by functional groups, the most frequent were those related to DNA methylation (44.6 %) and signaling/kinase pathways (37.5%). The most prevalent were FLT3 (29 %), IDH1/IDH2 (27%) and TP 53 (19.6%) followed by NPM1 and RUNX-1 (17%).

Patients older than 60 years, presented higher percentage of unfavorable ASXL1 (18.6%), RUNX1 (20%) and TP53 (25.7%) mutations with respect to younger (4.8%, 11.9% and 9.5 %) being these differences statistically significant in ASXL 1 and TP 53 (χ 2 p=0.044 and p=0.047 respectively). NPM1 (28.6%) and FLT3 (40.5%) mutations were significantly more frequent in younger patients (χ 2 p=0.017 and p=0.013 respectively), than in >60 years (10% and 17.1%, respectively).

Re-stratifying according to ELN 2017, the 53% of patients categorized within the intermediate risk group according to MRC change prognostic group: 28.9% became redefined as unfavorable risk and 24.1% as favorable.

In the overall series the overall survival analysis shows statistically significant differences taking into account either ELN 2017 or ELN 2010 and MRC (T.logrank p=0.003, p=0.018 and p=0.012, respectively). However, according to ELN 2017 there are greater differences between intermediate and unfavorable groups than in the other classifications.

Statistically significant differences in overall survival according to ELN 2017 (T.logrank p=0.028) are also found in patients receiving intensive treatment. The estimated overall survival at two years is 72.%, 52.1% and 46.8% in the favorable, intermediate and unfavorable group, respectively.

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Summary/Conclusion: - NGS proves its usefulness by detecting more clinically relevant alterations than conventional cytogenetic techniques and PCR, and stratifies a larger group of patients as favorable and unfavorable.

- Our results validate the prognostic significance of the ELN2017 classification in real life, both in the overall series and in candidates for intensive QT.

- ELN 2017 establishes greater survival differences between the intermediate and unfavorable group than ELN 2010 and MRC redefining the intermediate group.

P505: SAFETY AND EFFICACY OF CASEIN KINASE 1Α AND CYCLIN DEPENDENT KINASE 7/9 INHIBITION IN PATIENTS WITH RELAPSED OR REFRACTORY AML: A PHASE 1, FIRST-IN-HUMAN STUDY OF BTX-A51

B. J. Ball 1,* , G. Borthakur 2 , A. Stein 1 , K. W. Chan 3 , D. Thai 3 , E. M. Stein 4

1 Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte; 2 Leukemia, MD Anderson Cancer Center, Houston; 3 BioTheryx, San Diego; 4 Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Inactivation of p53 and overexpression of Mcl1 are common mechanisms that cancer cells use to evade apoptosis. BTX-A51 is a novel, oral, direct inhibitor of casein kinase 1α (CK1α), cyclin dependent kinase 7 (CDK7), and CDK9. CDK7 and CDK9 phosphorylate RNA polymerase II (Pol II) to enable transcriptional initiation and elongation, particularly at large clusters of transcriptional enhancers termed super-enhancers (SE). Preclinical studies have demonstrated that BTX-A51 robustly increased p53 protein levels via CK1α inhibition and Mdm2 downregulation while preferentially decreasing SE transcription of key oncogenes such as Myc and Mcl1, enabling selective apoptosis of leukemia cells. Here, we report the interim results of the phase 1, first-in-human (FIH) study of BTX-A51 in patients (pts) with R/R AML.

Aims: 1. To evaluate the safety and preliminary efficacy of BTX-A51 in patients with R/R AML

2. To determine the pharmacokinetics and pharmacodynamics of BTX-A51

Methods: This is an open-label, multi-center, FIH Phase 1 study. The study utilizes a hybrid accelerated titration with single pt cohorts and a Bayesian optimal interval design to assess 9 potential dosing cohorts. Key eligibility criteria include age ≥ 18 years, R/R AML or R/R high-risk MDS, ECOG ≤ 2, adequate kidney and liver function, WBC ≤ 25K/uL. PD studies include digital droplet PCR from PB to quantify target genes and measurement of macrophage inhibitory cytokine level by ELISA from serum at serial timepoints.

Results: As of 25 January 2022, 30 pts (28 with AML; 2 with HR-MDS) enrolled at dose levels between 1 and 42mg; 2 pts remain on treatment. Monotherapy doses between 1 and 42 mg were administered orally 3 days/week (wk) (3 wk in a 28-day cycle) and at 21 mg (4 wk in a 28-day cycle). Baseline characteristics include median age 75 years, median number of prior therapies 3, 97% received prior treatment with venetoclax, 97% had prior HMA, and 43% had prior induction failure.

The most common treatment-emergent AEs (TEAEs) were nausea (60%), vomiting (52%), hypokalemia (48%), diarrhea (36%), and hypotension (36%). The most common Grade 3 or higher TEAEs were anemia (28%), febrile neutropenia (28%), platelet count decreased (24%), and hypokalemia (20%). The only DLTs were grade 3 hepatic failure at the 42 mg dose and grade 3 alkaline phosphatase elevation at the 21 mg dose. All events resolved after holding study drug.

Plasma PK of BTX-A51 was roughly dose-proportional between 1 and 42 mg with accumulation based on AUC between Day 1 and Day 5. Estimated half-life was between 18 and 55 hours.

Among the 30 pts with R/R AML and MDS, CR/CRi rate was 10% (3/30) with 1 pt at the 11 mg and 2 pts at the 21 mg dose levels attaining CRi. Bone marrow (BM) blast reduction >50% occurred in 4 patients including the 3 responders, all at the 11 and 21mg dose levels. All 4 pts with >50% BM blast reduction had RUNX1 mutations; 9 pt with RUNX1 enrolled in the trial. The median duration of response for pts achieving CR/CRi was approximately 1.5 month. Responses were not observed in MDS pts. PD data will be provided in the full presentation. Based on the clinical data from dose escalation, the RP2D is 21 mg administered 3 days/wk for 4 wk of a 28-day cycle.

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Summary/Conclusion: In this FIH study, BTX-A51 demonstrated an acceptable safety profile and promising antileukemic activity in pts with heavily pretreated R/R AML. The 21 mg dose administered 3x/wk for 4 wk was identified as the RP2D. RUNX1 mutations were enriched among responders and pts attaining >50% BM blast reduction.

P506: ACHIEVEMENT OF MEASURABLE RESIDUAL DISEASE CLEARANCE IS A STRONGER PREDICTOR OF PATIENT OUTCOME THAN TREATMENT INTENSITY IN NEWLY DIAGNOSED PATIENTS WITH ACUTE MYELOID LEUKEMIA

A. Bazinet 1,* , T. Kadia 1 , N. Short 1 , G. Borthakur 1 , S. Wang 2 , S. Loghavi 2 , J. Jorgensen 2 , K. Patel 2 , C. DiNardo 1 , N. Daver 1 , Y. Alvarado 1 , F. Haddad 1 , S. Pierce 1 , M. Andreeff 1 , E. Jabbour 1 , M. Konopleva 1 , H. Kantarjian 1 , F. Ravandi 1

1 Leukemia; 2 Hematopathology, University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Modern therapy for acute myeloid leukemia (AML) can consist of either intensive or low-intensity regimens selected based on patient (pt) age/comorbidities. Measurable residual disease (MRD) has emerged as a strong independent prognostic factor in AML. It is unknown whether pts who achieve similar levels of MRD clearance experience comparable outcomes irrespective of treatment intensity.

Aims: To establish the relative prognostic contribution of treatment intensity and MRD status on overall survival (OS) and relapse-free survival (RFS) in newly diagnosed AML pts who have achieved a first response.

Methods: We conducted a retrospective chart review to identify non-CBF, non-APL AML pts treated at our institution between 2010 and 2021 with intensive (IA; int/high-dose cytarabine + anthracycline-based, without venetoclax) or low-intensity (Lo + VEN; low-dose cytarabine/hypomethylating agent-based, with venetoclax) regimens who had achieved CR/CRi/MLFS and undergone MRD testing by multiparameter flow cytometry at time of first response. Differences in baseline pt characteristics were evaluated using the Chi-square or Wilcoxon-Mann-Whitney tests for categorical and continuous variables, respectively. The Kaplan-Meier method was used to estimate median OS and RFS, with pts censored at the time of stem cell transplantation (SCT). Multivariate analysis was performed using a Cox proportional hazards model.

Results: We identified 635 pts meeting inclusion criteria. Baseline characteristics are shown in Table 1. Compared to the IA-treated pts (n=385), pts treated with Lo + VEN (n=250) were significantly older, more likely to be male, less likely to achieve MRD clearance, and more likely to be classified as adverse risk by ELN 2017. The SCT rate in the Lo + VEN cohort was half that of the IA cohort (25% vs 50%). Median OS was 51m, 24.6m, 15m, and 9.9m in the IA MRD(-), Lo + VEN MRD(-), IA MRD(+), and Lo + VEN MRD(+) groups, respectively. Median RFS was 27.8m, 15.4m, 7.3m, and 5.2m in the IA MRD(-), Lo + VEN MRD(-), IA MRD(+), and Lo + VEN MRD(+) groups, respectively. Pts within the same MRD category (+ or -) had significantly higher OS and numerically higher RFS if treated with IA versus Lo + VEN. When the analysis was restricted to pts aged ≥ 60 years (n=56 in IA, n=239 in Lo + VEN; Fig. A-B), the differences within MRD categories did not reach statistical significance (median OS NR in IA MRD(-) vs 24.6m in Lo + VEN MRD(-), p=0.08; median OS 13m in IA MRD(+) vs 10.6m in Lo + VEN MRD(+), p=0.50; median RFS NR in IA MRD (-) vs 15.4m in Lo + VEN MRD(-), p=0.07; median RFS 25.1m in IA MRD(+) vs 5.3m in Lo + VEN MRD(+), p=0.24). The SCT rate was 29% (87/295) in the age ≥ 60 cohort. Given the confounding effect of unbalanced pt characteristics between the IA and Lo + VEN cohorts, we performed a multivariate analysis on the full population (n=635) taking into consideration the effects of age, treatment intensity, MRD status, and ELN category on OS and RFS (all significant by univariate analysis). In the multivariate analysis for OS, ELN adverse risk was the strongest predictor (HR 2.09, p<0.001), followed by MRD(+) status (HR 1.68, p<0.001), while treatment intensity and age were not significantly predictive. For RFS, ELN adverse risk (HR 2.31, p<0.001) and MRD(+) status (HR 1.98, p<0.001) were also the only two significant predictors.

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Summary/Conclusion: In newly diagnosed AML patients, MRD status at time of first response and ELN risk are stronger predictors of pt outcome than intensity of therapy received.

P507: AGREEMENT BETWEEN REAL-WORLD PHYSICIAN RESPONSE ASSESSMENT AND THE 2017 EUROPEAN LEUKEMIANET (ELN) CRITERIA IN ACUTE MYELOID LEUKEMIA (AML): A COMPARATIVE ANALYSIS

P. A. Patel 1,* , F. Hoff 1 , A. J. Belli 2 , E. Hansen 2 , C. Anderson 2 , A. Barcellos 2 , L. L. Fernandes 2 , H. Foss 2 , M. He 2 , M. Schulte 2 , C.-K. Wang 2

1 University of Texas Southwestern Medical Center, Dallas, TX; 2 COTA, Inc., New York, NY, United States of America

Background: Real-world data (RWD) is a valuable resource to understand the experience of oncology patients treated outside a clinical trial. Increasingly, RWD is being used in support of regulatory decision-making to evaluate outcomes compared to what is observed in clinical trials. In the real-world setting, physicians often do not utilize the specific inclusion/exclusion criteria and objective response criteria that are implemented in clinical trials. Rather, a descriptive assessment of response and clinical benefit is often used. Additionally, response assessment frequency may not adhere to the requirements from clinical trials. Subsequently, it is critical that real-world, physician-assessed responses are investigated to understand the concordance with objective criteria in order to contextualize endpoints reported using RWD.

Aims: To evaluate the concordance and performance of physician-assessed response in the COTA real-world database to derived response using the 2017 ELN criteria as the established standard for response assessment in clinical trials.

Methods: A total of 879 patients meeting the following criteria were identified in the COTA database: diagnosed with AML on or after April 1st, 2017, age ≥18 years at diagnosis, received systemic treatment after diagnosis, and had at least 28 days of follow up or a post-therapy bone marrow assessment. The COTA database is a USA-based dataset composed of longitudinal, de-identified data on the diagnosis, clinical management, and outcomes of patients with cancer. Physician-assessed response was manually captured as documented in electronic health records (EHRs). Derived response was defined as 2017 ELN response based on bone marrow findings and complete blood count (CBC) results available in the EHR. Rates of agreement and disagreement between physician-assessed and derived response to first line treatment were calculated. Overall response rate (ORR) was estimated by both response definitions.

Results: The study population (n=879) had a median age of 67 years and were predominately white (78%), treated in the community setting (65%), and adverse risk per ELN criteria (42%). Overall agreement between response categories was 65.1% with the highest agreement among CR (61.7%). Agreement by response category is shown in Table 1. In a selected group of patients with all required bone marrow and lab values to derive ELN response (n=435), the ORR (95% confidence interval) was 80.5% (76.4, 84.1) for derived responders and 79.1% (75, 82.8) for physician-assessed responders.

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Summary/Conclusion: In our real-world cohort of patients with AML, agreement between derived response per 2017 ELN criteria and physician-assessed response was 65.1%. Among patients with derivable ELN response, the ORR calculations were similar by both response definitions. These findings are significant in showing that responses assessed by physicians treating patients with AML in the real-world setting are comparable to those derived via 2017 ELN criteria. Of interest, discordance in derived response vs. physician-assessed response was most common in the setting of CRi and MLFS. These two response types require the incorporation of CBC results and bone marrow cellularity. Future research will investigate the outcomes of patients with discordant response assessments and incorporate additional response categories including CR with partial hematologic recovery (CRh) and CR without minimal residual disease (CR MRD-).

P508: REAL LIFE EXPERIENCE USING FRONT-LINE CPX-351 FOR THERAPY-RELATED AND AML-MRC: RESULTS FROM THE SPANISH PETHEMA REGISTRY.

T. Bernal 1,* , G. Rad 2 , A. de Laiglesia 3 , C. Benavente 4 , A. Garcia Noblejas 5 , D. Garcia Belmonte 6 , R. Riaza 7 , O. Salamero 8 , A. Foncillas 9 , A. Roldán 10 , V. Noriega Concepcion 11 , J. Perez de Oteyza 12 , J. M. Bergua Burgues 13 , S. Lorente de Uña 14 , A. de la Fuente Burguera 15 , M. J. Garcia Perez 16 , J. L. Lopez Lorenzo 17 , P. Martinez 18 , C. Alaez 19 , M. Callejas 20 , C. Martinez Chamorro 21 , J. Rifon Roca 22 , L. Amador Barciela 23 , M. Lopez 24 , K. Gomez Correcha 25 , E. Lavilla Rubiera 26 , M. L. Amigo 27 , F. Vall-Llovera 28 , A. Garrido 29 , M. Garcia Fortes 30 , D. de Miguel Llorente 31 , A. Aules Leonardo 32 , C. Cervero 33 , R. Coll Jorda 34 , M. Perez Encinas 35 , M. Polo Zarzuela 4 , D. Martinez Cuadron 36 , P. Montesinos 36

1 Hematology, Hospital Universitario Central Asturias, ISPA, IUOPA; 2 Hematology, Hospital Universitario Central Asturias, ISPA, Oviedo; 3 Hematology, Hospital Puerta de Hierro; 4 Hematology, Hospital Clinico San Carlos; 5 Hematology, Hospital La Princesa; 6 Hematology, Hospital Universitario Sanitas La Zarzuela; 7 Hematology, Hospital Universitario Severo Ochoa, Madrid; 8 Hematology, Hospital Vall d´Hebron, Barcelona; 9 Hematology, Hospital Infanta Leonor; 10 Hematology, Hospital Infanta Sofia San Sebastian de los Reyes, Madrid; 11 Hematology, Complejo Hospitalario de A Coruña, A Coruña; 12 Hematology, Hospital Madrid Norte San Chinarro, Madrid; 13 Hematology, Hospital San Pedro Alcantara, Caceres; 14 Hematology, Hospital Vithas Xanit Internacional, Malaga; 15 Hematology, M.D. Anderson Cancer Center, Madrid; 16 Hematology, Complejo Hospitalario Torre Cardenas, Almeria; 17 Hematology, Fundacion Jimenez Diaz; 18 Hematology, Hospital Doce de Octubre; 19 Hematology, Hospital Universitario La Moncloa; 20 Hematology, Hospital Universitario Principe de Asturias; 21 Hematology, Hospital Universitario Quiron Pozuelo, Madrid; 22 Hematology, Clinica Universitaria de Navarra, Pamplona; 23 Hematology, Complejo Hospitalario Pontevedra, Pontevedra; 24 Hematology, Hospital General de Valencia, Valencia; 25 Hematology, Hospital Juan Ramon Jimenez, Huelva; 26 Hematology, Hospital Lucus Augusti, Lugo; 27 Hematology, Hospital Universitario Morales Messeguer, Murcia; 28 Hematology, Hospital Mutua Tarrasa; 29 Hematology, Hospital de la santa Creu i San Pau, Barcelona; 30 Hematology, Hospital Universitario Virgen de la Victoria, Malaga; 31 Hematology, Hospital Universitario de Guadalajara, Guadalajara; 32 Hematology, Hospital Miguel Servet, zZragoza; 33 Hematology, Hospital Virgen de la Luz, Cuenca; 34 Hematology, ICO Girona, Hospital Universitario Dr Josep Trueta, Girona; 35 Hematology, Hospital Universitario Santiago de Compostela, Santiago de Compostela; 36 Hematology, Hospital Universitari i Politecnic La Fe, Valencia, Spain

Background: Acute Myeloid Leukemias arising after cytotoxic therapy (t-AML) or with myelodysplasia-related changes (AML-MRC) share adverse risk features and poor outcomes after standard 3 + 7 chemotherapy. In a randomized clinical trial, CPX-351 has shown superior overall survival (OS) compared to standard anthracycline-cytarabine schedule in these AML subtypes.

Aims: to evaluate the effectiveness of CPX-351 treatment in a real-world setting.

Methods: adult t-AML and AML-MRC patients who have been treated upfront with CPX-351 in 35 Spanish centers between 2018 and 2021. All patients were included in the PETHEMA registry ( NCT02606825 ). Primary end-point was OS. Secondary end points were complete remission with or without hematological recovery (CR/CRi), proportion of minimal residual negativity (MRD), rate of allogeneic hematopoietic stem cell transplant (HSCT) and safety.

Results: CPX-351 was administered to 74 patients as first induction chemotherapy. Median age was 67 (63-71) years, with 40% (30/74) of female patients. ECOG performance status score was 0-1 in 85% (53/62) patients. A diagnosis of t-AML was present in 30% (22/74) patients. Hypomethylating agents were used for MDS or CMML phase in 15%. Hematopoietic Transplant Comorbidity Index was low, intermediate and high in 14% (10/74), 43% (32/74) and 43% (32/74) patients. ELN17 risk classification was favourable, intermediate, adverse and indeterminate in 9% (7/74), 38% (28/74), 46% (34/74) and 5% (4/74).

CR/CRi was obtained in 43% (32/74) patients after first cycle with CPX-351. Three additional patients achieved Morphological Leukemia Free Status. A second induction with CPX-351 was administered in 5 patients with 100% CR (CR/CRi after 2 cycles 37/74, 50%). MRD was evaluated in 97% (36/37) of CR/CRi patients, being low (<0.1%) in 47% (17/36) of them. In univariate analysis, no factor was associated with response. However, 71% (5/7) response rate was observed in the favourable ELN17 genetic risk group compared to 48% (32/67) in the non-favourable.

HSCT was performed in 27% (20/74) patients, in 1 st CR/CRi in 70% (14/20), MLFS in 15% (3/20) and active disease in 10% (2/20). With median follow up of 11.9 months (7.2-23.6) from HSCT, median OS was not reached (95% CI 9.5-NR months (Figure). In univariate analysis no factors were significantly associated with OS after HSCT.

Median time to absolute neutrophil count ≥0.5 x10 9 /L and platelets ≥50 x10 9 /L were 30.5 (25-35) and 30 (26-39) days, respectively. Deaths within the first 30 days after induction occurred in 12% (9/74) patients. Causes of early death: infection in 5 patients, haemorrhage in 3 and respiratory failure in 1. With 6.1 (1.8-14) months of median follow up, median OS was 10.5 (6.9-NR) months, with significant differences between HSCT and non-HSCT patients [NR, (95% CI: 12.7-NR), vs. 6 months, (95% CI: 4-NR), P<0.001]. In univariate analysis, factors associated with lower OS were non-favourable ELN17 [8.9 vs 23.5, P=0.04]; ECOG≥2 [0.7 vs 12., P=0.002]; age ≥65 [7.9 vs NR, P=0.007] and male [6.6 vs NR, P= 0.007]. In multivariate analysis, age above 65 years [HR 1.6, P<0.001], male [HR -0.9, P=0.02] and ECOG≥2 [HR 1.9, P<0.001] were associated with OS.

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Summary/Conclusion: Our real-life cohort was comparable to the target population of the pivotal trial including patients diagnosed with sAML (t-AML, AML-MRC and ELN17 high risk AML), obtaining similar outcomes than the CPX-351 phase 3 trial arm. To optimize treatment outcomes CPX-351 should be preferably offered to t-AML and AML-MRC patients who are suitable for HSCT.

P509: CLONAL HEMATOPOIESIS-ASSOCIATED MUTATIONS AS MEASURABLE RESIDUAL DISEASE MARKERS IN ACUTE MYELOID LEUKEMIA PATIENTS FOLLOWING ALLOGENEIC STEM CELL TRANSPLANTATION

L. Bischof 1,* , J. Ussmann 1 , D. Brauer 1 , D. Backhaus 1 , L. Herrmann 1 , G.-N. Franke 1 , V. Vucinic 1 , K. H. Metzeler 1 , U. Platzbecker 1 , S. Schwind 1 , M. Jentzsch 1

1 Hematology, Leipzig University Hospital, Leipzig, Germany

Background: Clonal hematopoiesis (CH)-associated mutations (mut) are frequent in acute myeloid leukemia (AML), appear early in leukemogenesis, and often persist in remission after chemotherapy. Following allogeneic hematopoietic stem cell transplantation (HSCT), which replaces the patients’ hematopoiesis, CH mut may present useful measurable residual disease (MRD) markers.

Aims: To evaluate patient specific CH-associated mut as MRD markers in AML patients (pts) after allogeneic HSCT.

Methods: We analyzed 31 AML pts with CH-associated mut present at diagnosis ( DNMT3A : n=17; SRSF2 : n=9; IDH2 : n=7 ; ASXL1 : n=4; TET2 : n=2, and JAK2 : n=1), 23 pts had 1, 7 had 2, and 1 patient had 3 CH mut. All received a myeloablative (36%), reduced-intensity (48%), or non-myeloablative (16%) HSCT (median age 58, range 32-72 years). 84% were transplanted in complete remission (CR) or CR with incomplete recovery (CRi). European Leukemia Net (ELN) risk was 23% favorable, 37% intermediate, and 40% adverse. Mut-specific digital droplet PCR assays were developed using a competitive probe-approach. MRD pos was defined as variant allele frequency (VAF) ≥2% for ASXL1 & ≥0.05% for all other analyzed mut. 209 samples with a median of 6 (range 1-13) blood or bone marrow samples per patient were available after HSCT with a median of 1.2 years follow-up time for pts alive.

Results: Prior to HSCT, 89% of pts remained CH-associated mut positive in CR/CRi. 11 pts relapsed after HSCT (35%), all with the same CH-associated mut present at diagnosis (median VAF at relapse 15.6, range 0.2-41.2%). Of those, 6 pts had at least 1 MRD sample available prior to relapse. In 5 pts, impeding relapse was predicted by a CH-associated mut MRD conversion with a median VAF of 0.27 (range 0.06-1.0)% at a median of 103 (range 14-199) days prior to relapse. The lead time was longest for a DNMT3A mut patient (199 days) & shortest for a SRSF2 mut patient (14 days). For the patient relapsing without prior CH MRD positivity, the last available sample was taken 169 days before relapse & was CH MRD neg . 20 pts retained remission during follow-up. Of those, 17 pts remained CH MRD neg in all samples (median samples per analyzed mutation 6, range 1-13). 2 pts had 2 MRD pos samples directly after HSCT but became MRD neg in subsequent samples. 1 patient received Enasidenib maintenance after HSCT with fluctuating CH-MRD values after HSCT ( SRSF2 & IDH2, VAFs <0.05-0.34% & <0.05-0.14%, respectively).

Of the pts with MRD samples within the first year after HSCT available, pts with at least 1 MRD pos sample after HSCT had a significantly higher cumulative incidence of relapse (CIR, P =.008, Figure 1A) & shorter relapse-free survival (RFS, P =.02, Figure 1B). 8 pts had concurrent NPM1 mut (n=4) or RUNX1 mut (n=4), which showed concordant MRD results in 7 pts (4 remained MRD neg , 3 concurrently converted MRD pos ). 1 patient suffered a NPM1 MRD neg relapse which was predicted by 2 re-detected CH-associated mut ( DNMT3A mut & IDH2 mut, 112 & 199 days prior to relapse, respectively, Figure 1C).

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Summary/Conclusion: Prior to HSCT most CH-associated mut remain detectable in pts in CR/CRi. However, pts with at least one CH MRD pos sample following allogeneic HSCT had higher CIR & shorter RFS. All relapsing pts were positive for the same CH-associated mut present at diagnosis. 5 of 6 showed increasing VAFs prior to hematologic relapse & the majority of pts retaining remission remained CH MRD neg . While CH-associated mut in chemotherapy treated AML pts have limited MRD value, these mut are feasible MRD markers after HSCT & may inform relapse preventing therapy decisions.

P510: THE IMPACT OF POST-REMISSION GRANULOCYTE COLONY-STIMULATING FACTOR USE IN THE PHASE 3 STUDIES OF VENETOCLAX COMBINATION TREATMENTS IN PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA

C. DiNardo 1,* , K. Pratz 2 , P. Panayiotidis 3 , X. Wei 4 , V. Vorobyev 5 , Á. Illés 6 , I. Kim 7 , V. Ivanov 8 , G. Ku 9 , C. L. Miller 10 , M. Zhang 10 , F. Tatsch 10 , J. Potluri 10 , X. Schmidt 10 , C. Recher 11

1 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 2 Abramson Cancer Center, University of Pennsylvania, Philadelphia, United States of America; 3 Haematology Clinic and BMT Unit, NKUA, Laiko General Hospital, Athens, Greece; 4 The Affiliated Cancer Hospital of Zhengzhou University/Henan Cancer Hospital, Zhengzhou, China; 5 Department of Hematology, S. P. Botkin City Clinical Hospital, Moscow, Russia; 6 Department of Hematology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary; 7 Seoul National University Hospital, Seoul, South Korea; 8 Almazov National Medical Research Centre, Saint Petersburg, Russia; 9 Genentech, South San Francisco; 10 AbbVie, Inc., North Chicago, United States of America; 11 Centre Hospitalier Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse Oncopole, Université de Toulouse 3 Paul Sabatier, Toulouse, France

Background: In the randomized Phase 3 VIALE-A ( NCT02993523 ) and VIALE-C ( NCT03069352 ) trials, venetoclax (Ven) + azacitidine (Aza) and Ven + low-dose cytarabine (LDAC), respectively, improved outcomes vs low intensity chemotherapy in patients (pts) with newly diagnosed acute myeloid leukemia (AML) ineligible for intensive chemotherapy (IC). Evidence is limited on the use of granulocyte colony-stimulating factor (G-CSF) and impact on outcomes in this pt population.

Aims: To explore outcomes in pts with post-remission G-CSF use in the VIALE-A and VIALE-C studies.

Methods: In VIALE-A, pts received Ven 400 mg or placebo (PBO) daily plus Aza 75 mg/m 2 on d 1−7 (28-d cycles). In VIALE-C, pts received Ven 600 mg or PBO daily plus LDAC 20 mg/m 2 on d 1−10 (28-d cycles). G-CSF use was given per institutional practice. Here, pts who achieved a best response of complete remission (CR) or CR with incomplete hematologic recovery (CRi) were analyzed by post-remission G-CSF use.

Results: In VIALE-A, 190/286 pts (66%) treated with Ven+Aza and 41/145 (28%) pts treated with PBO+Aza achieved CR/CRi; 93 (49%) and 10 (24%) pts with CR/CRi received G-CSF post-remission (median time to first post-remission G-CSF use [range], 36 d [2−483] and 35 d [4−127]), respectively. In VIALE-C, 69/143 pts (48%) treated with Ven+LDAC and 9/68 (13%) pts treated with PBO+LDAC achieved CR/CRi; 30 (43%) and 2 (22%) received G-CSF post-remission (median time to first post-remission G-CSF use [range], 30 d [2−459] and 229 d [169−289]), respectively. In VIALE-A and VIALE-C, baseline demographics were generally similar in Ven-treated pts regardless of post-remission G-CSF use ( Table ).

In VIALE-A, median duration of response (mDOR) for CR/CRi (95% CI) was not reached (NR; 17.5−NR) and was 12.9 mo (7.9−17.3) in the Ven+Aza+G-CSF and Ven+Aza+non-G-CSF groups, respectively ( Table ); DOR at 12 mo was 67% and 53%. In VIALE-C, mDOR was 10.8 (4.9−17.8) and 11.8 mo (5.9−NR) in the Ven+LDAC+G-CSF and Ven+LDAC+non-G-CSF groups, respectively; DOR at 12 mo was 45% and 49%. In VIALE-A, median overall survival (mOS [95% CI]) was NR (NR−NR) with Ven+Aza+G-CSF and was 21.1 mo (15.2−NR) with Ven+Aza+non-G-CSF; 12-mo OS rates were 83% and 71%. In VIALE-C, mOS was 20.8 (11.9−NR) with Ven+LDAC+G-CSF and 13.7 mo (10.8−NR) with Ven+LDAC+non-G-CSF; 12-mo OS estimates were 68% and 57%. Among 164 evaluable pts in VIALE-A, measurable residual disease response (MRD<10 −3 ) was achieved by 67, of whom 38 (57%) had post-remission G-CSF. In VIALE-C, MRD<10 −3 was achieved by 9 of 64 evaluable pts, of whom 6 (67%) had post-remission G-CSF.

In VIALE-A, post-remission Gr ≥3 neutropenia and febrile neutropenia (FN) rates were 33% (n=31) and 39% (n=36) with Ven+Aza+G-CSF, and 29% (n=28) and 20% (n=19) with Ven+Aza+non-G-CSF, respectively. Median durations of post-remission Gr ≥3 neutropenia and FN in VIALE-A were 12.5 d and 8 d (Ven+Aza+G-CSF), and 16 d and 10.5 d (Ven+Aza+non-G-CSF). In VIALE-C, post-remission Gr ≥3 neutropenia and FN rates were 53% (n=16) and 23% (n=7) with Ven+LDAC+G-CSF, and 51% (n=20) and 8% (n=3) with Ven+LDAC+non-G-CSF, respectively. Median durations of post-remission Gr ≥3 neutropenia and FN in VIALE-C were 15 d and 6 d (Ven+LDAC+G-CSF), and 12.5 d and 29 d (Ven+LDAC+non-G-CSF).

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Summary/Conclusion: In VIALE-A and VIALE-C, G-CSF was frequently used per institutional practice post-remission in responding patients to manage neutropenia. Overall, there was a trend towards shorter durations of Gr ≥3 neutropenia and FN with post-remission G-CSF use, without evidence of negative impact on DOR or OS.

P511: RISK FACTORS AND INCIDENCE OF CARDIAC EVENTS IN A LARGE COHORT OF 525 ADULT PATIENTS WITH NEWLY DIAGNOSED NON-M3 ACUTE MYELOID LEUKEMIA

B. Boluda 1,2,* , A. Solana-Altabella 2,3 , I. Cano 1,2 , D. Martínez-Cuadrón 1,2 , E. Acuña-Cruz 1,2 , L. Torres-Miñana 1,2 , R. Rodríguez-Veiga 1,2 , D. Martínez-Campuzano 1 , R. García-Ruiz 1 , P. Lloret 1 , P. Asensi 1 , A. Serrano 1 , A. Osa-Sáez 4 , J. Agüero-Ramón-Llín 4 , M. Rodríguez-Serrano 4 , F. Buendía-Fuentes 4 , J. E. Megías-Vericat 3 , B. Martín-Herreros 1,2 , E. Barragán 1 , C. Sargas 1,2 , M. Salas 5 , M. Wooddell 5 , C. Dharmani 5 , M. A. Sanz 1,2 , J. De La Rubia 1 , P. Montesinos 1

1 Hematology, Hematology Department, Hospital Universitari i Politècnic La Fe, València, Spain; 2 Instituto de Investigación Sanitaria La Fe; 3 Pharmacy; 4 Cardiology, Hospital Universitari i Politècnic La Fe, Valencia, Spain; 5 Daiichi Sankyo, Inc., Basking Ridge, United States of America

Background: The incidence of cardiac morbidity and mortality in acute myeloid leukemia (AML) population is not well known. AML patients (pts) receive potentially cardiotoxic drugs such as anthracyclines, QTc prolonging drugs such as FLT3 inhibitors, azoles, among others. Cardiac events can emerge in pts with previous cardiac diseases, or due to the toxicity of chemotherapy and other concomitant medications. So far, no previous studies have provided a holistic view of cardiac issues in a real-world series of AML pts.

Aims: To estimate the cumulative incidence (CI) of cardiac events in a large series of unselected pts with AML in a single Spanish institution, and to identify risk factors associated with their development.

Methods: Between January 2011 and June 2020, 571 pts were consecutively diagnosed with AML in Hospital La Fe, Valencia. The median follow-up of alive patients was 33 months. Clinical records were reviewed from diagnosis to death/last follow-up. Cardiac events were coded according to CTCAE. There were 525 treated pts, 285 (54%) with intensive chemotherapy and 240 (46%) with non-intensive therapy.

Results: Among 46 untreated pts, 7 (15%) died due to cardiac events. Median age of 525 treated pts was 65 years, 331 (58%) male, and 73 (13.9%) had prior relevant cardiac comorbidities; 77 (14.7%) had FLT3-ITD mutation and 38 (7.2%) received front-line FLT3 inhibitors. Overall, 488 cardiac events were recorded, 19 (3.6%) pts had fatal cardiac events and 288 (54.9%) had any grade of non-fatal cardiac events. The 9-years CI of grade 1-2 QTcF prolongation was 11.2%, 2.7% pts had grade 3 QTcF prolongation >500ms, and no patient had grade 4 o 5. The 9-years CI of grade 1-2 cardiac failure was 1.3%, grade 3-4 15%, and grade 5 2.1%. The 9-years CI of grade 1-2 arrhythmia was 1.9%, grade 3-4 9.1%, and no pts had grade 5. Among 307 pts with cardiac events, 38 (12%) pts developed the first event before starting treatment, 132 (43%) during first cycle, 31 (10%) during consolidation, 55 (18%) during non-intensive further cycles, 14 (5%) during hematopoietic cell transplantation and 37 (12%) during follow-up.

In the univariate analysis, pts with prior cardiac antecedents had increased incidence of fatal cardiac events compared with pts with no prior cardiac disease (CI at 9 years 20.1% vs 4.9%, p<0.001). Pts older than 65 years of age (64.4% vs 49.9%, p<0.001), prior cardiac disease (73% vs 54.6%, p=0.004) or included in clinical trial (65.2% vs 50.8%, p<0.001) had increased CI of non-fatal cardiac events. Multivariate analysis showed that prior cardiac disease was associated with increased incidence of fatal cardiac events [Hazard Ratio (HR) 1.9, p<0.001]. Age >65 (HR 2.2, p<0.001), prior cardiac disease (HR 1.4, p=0.02) and non-intensive chemotherapy (HR 1.8, p=0.004) were associated with increased CI of non-fatal cardiac event.

Median overall survival (OS) in the overall cohort was 11.4 months (9.6-13.4 months, IC 95%). Among 285 intensive therapy pts, median OS was 22 months, 34 months in pts without cardiac events (n=125), 43 months with grade 1-2 (n=52), 15 months with grade 3-4 (n=98), and 5.2 months with grade 5 (n=10) (p<0.001).

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Summary/Conclusion: We observed a high CI of cardiac events (58.5%) in a real-world series of patients undergoing AML therapy, associated with significant mortality due to cardiotoxicity (3.6%), and decreased OS. Prior history of cardiac comorbidity was associated with an increased risk of fatal cardiac events. Older age, cardiac comorbidities, and non-intensive therapies were related to non-fatal events.

P512: PRELIMINARY RESULTS OF VEN-A-QUI STUDY: A PHASE 1-2 TRIAL TO ASSESS THE SAFETY AND EFFICACY OF THE COMBINATION OF AZACITIDINE OR LOW-DOSE CYTARABINE WITH VENETOCLAX AND QUIZARTINIB IN NEWLY DIAGNOSED

J. M. Bergua-Burgues 1,* , R. Rodríguez-Veiga 2 , I. Cano 2 , F. Vall-llovera 3 , A. García-Guiñon 4 , J. Gómez-Estruch 5 , M. Colorado 6 , I. Casas-Avilés 1 , J. Esteve-Reyner 7 , M. V. Verdugo 8 , F. Ramos 9 , M. Valero 10 , E. Acuña-Cruz 2 , B. Boluda 2 , L. Torres-Miñana 2 , J. Martínez-López 11 , E. Barragán 2 , R. Ayala 11 , D. Martínez-Cuadrón 2 , P. Montesinos 2

1 Hospital San Pedro de Alcántara, Cáceres; 2 Hospital Universitari i Politècnic La Fe, Valencia; 3 Hospital Universitari Mútua Terrassa, Barcelona; 4 Hospital Arnau de Vilanova, Lleida; 5 Hospital Clinico Universitario Virgen de la Arrixaca, Murcia; 6 Hospital Marqués de Valdecilla, Santander; 7 Hospital Clinic Barcelona, Barcelona; 8 Hospital de Jerez, Jerez de la Frontera; 9 Hospital Clinico de León, León; 10 Hospital Arnau de Vilanova, Valencia; 11 Hospital 12 de Octubre, Madrid, Spain

Background: Venetoclax (VEN) combined with Azacitidine (AZA) or Low Dose Cytarabine (LDAC) has emerged as new therapeutic option for unfit acute myeloid leukemia (AML) patients (pts), but primary resistance is observed in roughly 40% of them, while relapses occur in the vast majority. We speculate that adding a FLT3-ITD inhibitor could improve the complete remission (CR) and overall survival (OS) rates in this setting.

Aims: To explore the safety and efficacy of VEN-AZA or VEN-LDAC regimens in combination with Quizartinib (QUI) (VEN-A-QUI trial; EUDRACT2020-000406-28).

Methods: The target population comprised newly diagnosed pts aged ≥ 60 years old unfit for intensive treatment, including those with secondary AML, with or without prior exposure to AZA. The Phase I consisted in two arms, one with AZA (Arm A) and the other with LDAC (Arm B) plus VEN combined with QUI to establish the recommended phase 2 dose (RP2D) of both triplets. Phase 1 scheme was based in 3 + 3 cohorts of patients observing cycle 1 dose limiting toxicities. Once established the RP2D the phase 2 comprised randomized 1:1 assignment of 60 patients (48 FLT3 wild type and 12 FLT3-ITD mut) to VEN-AZA-QUI vs. VEN-LDAC-QUI, comparing the CR/CRi rate of both arms. Secondary objectives were to evaluate the CR/CRi after cycle 1 and 4, compare OS and RFS between both triplets, quality of life, medical resources, exploration of biomarkers, and immune recovery.

Results: Data cut-off for preplanned interim analysis included 57 pts screened and 45 enrolled, 16 in phase 1 and 29 in phase 2. Median age was 76,5 years (range 67-87), males/females (28/23). Previous MDS or MPN was present in 28 pts (59%); and 22 (48%) had previous treatment with AZA for MDS or MPN phase.

We included 16 pts in phase 1, 9 with AZA and 7 with LDAC. RP2D of QUI was 60 mg in AZA arm and 40 mg in LDAC arm. No DLT was observed in arm B, and in arm A a brain hemorrhage after more than 40 days of thrombocytopenia at dose of 60 mg. The safety committee recommended performing an early (day 14-21) bone marrow assessment in cycle 1, leading to VEN interruption in case of aplastic morphology with grade 4 neutropenia or thrombocytopenia. No grade ≥3 related non-hematological adverse events (AEs) were noted during phase 1. The most frequent non-hematological serious AEs during phase 1 were infections (n=23), and gastrointestinal (n=20). No grade 3 QTc prolongation was observed. Objective responses were CR+CRh+CRi 7 pts (44%), PR 1 (6%), death 4 (25%), and resistance/progression 4 (25%).

Twenty-nine pts (4 with FLT3-ITD mut) were enrolled in the phase 2 (15 in AZA and 14 in LDAC Arm). A median of 1 cycle (range 1-4) was administered at data cut-off, with best response among 24 evaluable pts: CR+CRh+CRi 10 (42%), MLFS in 3 (12%), PR 5 (21%), death 4 (17%), and resistance/progression 2 (8%). The overall response (CR+CRh+CRi+MLFS) was 54%. The more frequent non-hematological AEs were infections (n=35) and gastrointestinal (n=31). Two cardiac failures, 1 chest pain and 1 atrial fibrillation were noted in phase 2 (all of them unrelated to VEN or QUIZ). No grade 3 QTc prolongation was observed.

Summary/Conclusion: This interim report shows an overall response rate of 54% using triplets (VEN-AZA-QUI or VEN-LDAC-QUI) for newly diagnosed unfit AML pts. However, substantial toxicity and early death cases were observed. Of note, 59% of enrolled pts had secondary AML, and 48% was exposed to AZA before inclusion. Final analyses with more pts and follow-up will clarify the efficacy and tolerability of these triplets.

P513: CPX-351 TREATMENT FOR ACUTE MYELOID LEUKEMIA IN ENGLAND: REAL-WORLD OUTCOMES IN ADULTS AGED <60 YEARS VERSUS ≥60 YEARS

A. Legg 1,* , R. Muzwidzwa 2 , A. Lambova 2 , K. Styles 2 , P. Doubleday 2 , G. Medalla 1

1 Jazz Pharmaceuticals, Oxford; 2 IQVIA Inc., London, United Kingdom

Background: CPX-351 (Vyxeos ® liposomal) is a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio. Since 2018, NICE and the EMA have recommended the use of CPX-351 for adults with newly diagnosed, therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (AML-MRC) due to prior myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) or de novo AML with myelodysplasia-related cytogenetic changes.

Aims: To compare the characteristics and overall survival (OS) of younger (18–60 years) versus older (≥60 years) adults with AML who were treated with CPX-351 in England.

Methods: This retrospective study utilized the Cancer Analysis System database available through England’s National Cancer Registration and Analysis Service. A diagnosis of t-AML or AML-MRC between 01/01/2013 and 31/03/2021 was determined either directly using International Classification of Diseases for Oncology, Third Edition (ICD-O-3) codes or indirectly using nonspecific ICD-O-2, ICD-O-3, or ICD-10 AML codes in combination with prior systemic anticancer therapy or radiotherapy (t-AML) or a prior diagnosis of MDS/CMML (AML-MRC). OS was estimated from the diagnosis date or landmarked from the hematopoietic cell transplantation (HCT) date.

Results: Overall, 211 patients with AML received CPX-351; their median (IQR) age was 65 (59, 70) years, 133 (63%) were male, 187 (89%) were White, 87 (41%) had secondary AML (t-AML or prior MDS/CMML), 33 (16%) had other AML-MRC, and 91 (43%) had unspecified de novo AML. Of the 60 (28%) patients aged <60 years, 12 were aged 18–44 years and 48 were aged 45–59 years. Of the 151 (72%) patients aged ≥60 years, 97 were aged 60–69 years and 54 were aged ≥70 years.

The cutoff date for OS was 31/08/2021, with a median (IQR) follow-up of 11.3 (4.7, 20.1) months. The estimated median OS (95% CI) was 12.9 (10.5, 17.5) months overall, 18.5 (11.0, not estimable) months for adults aged <60 years, and 11.2 (8.5, 15.9) months for adults aged ≥60 years, with 2-year survival of 35%, 44%, and 32%, respectively ( Figure ). Early mortality at 30 days was 6% overall, 3% for adults aged <60 years, and 7% for adults aged ≥60 years. Among adults aged <60 years, HCT was reported for 29/60 (48%) patients, including 29/50 (58%) with ≥3 months of follow-up. Among adults aged ≥60 years, HCT was reported for 55/151 (36%) patients, including 55/115 (48%) with ≥3 months of follow-up. When landmarked from the HCT date, median OS was not reached in either age group ( Figure ).

In a treatment patterns analysis, after receiving CPX-351, 14/60 (23%) adults aged <60 years and 63/151 (42%) aged ≥60 years died without salvage therapy, and 19/60 (32%) adults aged <60 years and 37/151 (25%) aged ≥60 years were alive without receiving subsequent therapy by the end of the study period. The most common salvage therapy after CPX-351 was FLAG-based chemotherapy (aged <60 years: n=12/60; aged ≥60 years: n=17/151).

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Summary/Conclusion: This study provides real-world survival outcomes in younger and older adults with AML who were treated with CPX-351 in England. These results suggest CPX-351 is an effective treatment for patients with AML aged <60 and ≥60 years in a real-world setting that bridged many patients to HCT, with promising post-HCT outcomes.

P514: V-FAST MASTER TRIAL: PRELIMINARY RESULTS OF TREATMENT WITH CPX-351 PLUS MIDOSTAURIN IN ADULTS WITH NEWLY DIAGNOSED FLT3-MUTATED ACUTE MYELOID LEUKEMIA

J. McCloskey 1,* , V. Pullarkat 2 , G. Mannis 3 , T. L. Lin 4 , S. A. Strickland 5 , A. T. Fathi 6 , H. P. Erba 7 , S. Faderl 8 , D. Chakravarthy 8 , Y. Lutska 9 , V. Chandrasekaran 9 , R. S. Cheung 8 , M. Levis 10

1 John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ; 2 City of Hope Comprehensive Cancer Center, Duarte, CA; 3 Stanford University Medical Center, Palo Alto, CA; 4 University of Kansas Medical Center, Kansas City, KS; 5 Vanderbilt-Ingram Cancer Center, Nashville, TN; 6 Massachusetts General Hospital Cancer Center/Harvard Medical School, Boston, MA; 7 Duke University School of Medicine, Durham, NC; 8 Jazz Pharmaceuticals, Palo Alto, CA; 9 Jazz Pharmaceuticals, Philadelphia, PA; 10 Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, United States of America

Background:: CPX-351 (Europe: Vyxeos ® liposomal; United States: Vyxeos ® ), a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio, is approved for newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in adults in Europe and patients aged ≥1 year in the United States. In a phase 3 study in older adults with newly diagnosed, high-risk/secondary AML, CPX-351 significantly improved overall survival and remission rates versus conventional 7 + 3, with a comparable safety profile. Preclinical data suggest CPX-351 may have synergistic activity with targeted agents, including the FLT3 inhibitor midostaurin.

Aims: Herein, we report preliminary results for the cohort of adults treated with CPX-351 + midostaurin in the V-FAST ( V yxeos – F irst Phase As sessment with T argeted Agents) trial.

Methods: V-FAST is an open-label, multicenter, multi-arm, nonrandomized, phase 1b master trial ( NCT04075747 ) to evaluate the safety and preliminary efficacy of CPX-351 combined with targeted agents (midostaurin, venetoclax, enasidenib). Eligible adults in the CPX-351 + midostaurin cohort were aged 18 to 75 years, had newly diagnosed AML with a FLT3 internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutation, were fit for intensive chemotherapy, and had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2. The dose-exploration phase (3 + 3 design) determined a recommended phase 2 dose of CPX-351 100 units/m 2 (daunorubicin 44 mg/m 2 + cytarabine 100 mg/m 2 ) on Days 1, 3, and 5 + midostaurin 50 mg twice daily on Days 8 to 21. There were no dose-limiting toxicities, and additional patients were enrolled in the expansion phase at this dose.

Results: A total of 23 patients received CPX-351 + midostaurin and had sufficient data to be included in the analysis (cutoff date: 20 January 2022). Patient baseline characteristics are shown in the Table . Treatment-emergent adverse events (TEAEs) in ≥40% of patients included febrile neutropenia (78%), nausea (65%), increased alanine aminotransferase (57%), leukopenia (57%), thrombocytopenia (57%), headache (43%), and hyponatremia (43%). All patients experienced a grade 3/4 TEAE, primarily hematologic events. Nonhematologic grade 3/4 TEAEs in ≥2 patients included pneumonia (17%), lung infection (13%), and hyperglycemia (9%). There were no grade 5 TEAEs and no deaths on or before Day 60. Complete remission was achieved by 18/22 (82%) patients with an evaluable remission assessment after the first induction cycle.

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Summary/Conclusion: Preliminary results from the V-FAST trial suggest the combination of CPX-351 + midostaurin is feasible, with a manageable safety profile and promising remission rates in adults with newly diagnosed AML who have a FLT3 mutation.

P515: LOWER-INTENSITY CPX-351 + VENETOCLAX FOR PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA WHO ARE UNFIT FOR INTENSIVE CHEMOTHERAPY

G. L. Uy 1 , V. A. Pullarkat 2,* , P. Baratam 3 , R. K. Stuart 3 , R. B. Walter 4 , E. S. Winer 5 , S. Faderl 6 , V. Chandrasekaran 6 , Q. Wang 6 , D. Chakravarthy 6 , R. S. Cheung 6 , T. L. Lin 7

1 Washington University School of Medicine, St. Louis, MO; 2 City of Hope Comprehensive Cancer Center, Duarte, CA; 3 Medical University of South Carolina, Charleston, SC; 4 Fred Hutchinson Cancer Research Center, Seattle, WA; 5 Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; 6 Jazz Pharmaceuticals, Palo Alto, CA; 7 University of Kansas Medical Center, Kansas City, KS, United States of America

Background: CPX-351 (Europe: Vyxeos ® liposomal; United States: Vyxeos ® ) is a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio. CPX-351 is approved for newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in adults in Europe and patients aged ≥1 year in the United States who are candidates for intensive chemotherapy (IC). However, the appropriate dosage of CPX-351 in patients unfit for IC may be different from the label dosage. Venetoclax (VEN; BCL-2 inhibitor) + low-dose cytarabine has demonstrated efficacy in unfit patients with AML, and drug synergism/additivity in preclinical studies provided a rationale for combining CPX-351 + VEN clinically.

Aims: Our study evaluates the safety and efficacy of lower-intensity CPX-351 + VEN in adults with newly diagnosed AML who are unfit for IC.

Methods: This is an ongoing, open-label, phase 1b study ( NCT04038437 ). Patients who achieve at least partial remission after 1 or 2 cycles may receive up to 4 similar cycles in the dose-exploration phase (DEP) or up to 8 similar cycles in the expansion phase (EP). Patients are assessed for response (morphology, measurable residual disease [MRD]) and monitored for safety and survival.

Results: The data include 31 patients enrolled by 15 September 2021, with a data cutoff of 2 December 2021: 4 patients in the DEP at dose level 1 (CPX-351 20 units/m 2 on Days 1 and 3 + VEN 400 mg on Days 2 to 21 of each cycle), 7 patients in the DEP at dose level 2 (CPX-351 40 units/m 2 + VEN 400 mg), and a total of 20 patients in the DEP and EP at dose level 1b (CPX-351 30 units/m 2 + VEN 400 mg), which was established as the recommended phase 2 dose. Patients were considered unfit for IC based on age ≥75 years (n=15) or health (Eastern Cooperative Oncology Group performance status of 2 to 3 and/or comorbidities [n=16]). Median age was 74 years (range: 60, 90); 65% were male; 77% had de novo AML; 58% had poor-risk disease; and 23% had a TP53 mutation.

Nonhematologic treatment-emergent adverse events (TEAEs) in ≥20% of patients were diarrhea (26%), cough (23%), dyspnea (23%), and nausea (23%). Hematologic grade ≥3 TEAEs were reported in 17 (55%) patients; no nonhematologic grade ≥3 TEAE was reported in >10% of patients. There were no deaths by Day 30; mortality at Day 60 was 13%, with deaths due to myocardial infarction unrelated to therapy (n=1), worsening lung infection (n=1), and disease progression (n=2). Median (interquartile range) recovery times were 30 days (22, 34.5) to neutrophils ≥500/μL and 21 days (21, 27) to platelets ≥50,000/μL.

Complete remission (CR) or CR with incomplete neutrophil or platelet recovery (CRi) was achieved by 16/28 (57%) patients with an evaluable remission assessment. All 16 of these patients achieved remission (CR or CRi) after the first treatment cycle. MRD negativity was achieved by 12/16 (75%) patients with CR or CRi, primarily after Cycle 1 (Cycle 1: n=8; Cycle 2: n=2; Cycle 3: n=1; Cycle 4: n=1). Survival data are not yet mature.

Summary/Conclusion: Lower-intensity CPX-351 + VEN was generally well tolerated in adults with newly diagnosed AML who are unfit for IC and showed promising initial efficacy, with CR or CRi in the majority of patients.

P516: ADULT LANGERHANS CELL HISTIOCYTOSIS WITH THYROID GLAND INVOLVEMENT: CLINICAL PRESENTATION, GENOMIC ANALYSIS AND OUTCOME

H. Cai 1,2,* , T. Liu 1 , H. Cai 1,2 , M. Duan 1,2 , J. Li 1,2 , D. Zhou 1,2 , X. Cao 1,2

1 Department of Hematology; 2 State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China

Background: Langerhans cell histiocytosis (LCH) is a rare, clonal disorder derived from CD1a-positive and CD207-positive immature myeloid dendritic cells. The clinical presentations and outcomes of LCH are extremely variable. Thyroid gland involvement is rare, and solitary thyroid gland involvement with LCH is extremely rare, with most cases presenting as part of multisystem disease.Until now, little information exists on the genomic analysis of LCH with thyroid gland involvement with or without coexisting papillary thyroid carcinoma.

Aims: To evaluate the characteristics and treatment outcomes of adult Langerhans cell histiocytosis (LCH) patients with thyroid involvement.

Methods: We retrospectively described the clinical, biological, and genomic characteristics of a series of 36 LCH patients with thyroid involvement in our centre between January 2001 and December 2021.

Results: We retrospectively described the clinical, biological, and genomic characteristics of a series of 36 LCH patients with thyroid involvement in our centre between January 2001 and December 2021. At the time of diagnosis, only 1 patient was classified as having single-system LCH, and 35 patients were classified as having multisystem (MS) LCH. Three patients had coexisting papillary thyroid carcinoma. Patients with thyroid gland involvement had higher frequencies of pituitary (88.6% vs. 53.4%, P<0.001), liver (45.7% vs. 20.7%, P=0.003) and lymph node (54.3% vs. 31.6%, P=0.012) involvement and a lower frequency of bone (45.7% vs. 72.0%, P=0.003) involvement than patients without thyroid gland involvement. Sixteen patients had abnormal thyroid function, including 9 patients with primary hypothyroidism, 1 patient with central hypothyroidism, and 6 patients with subclinical hypothyroidism. BRAF V600E , BRAF N486_P490 and MAP2K1 mutations were detected in 14.3%, 57.1% and 7.1% of patients, respectively. After a 43-month median follow-up, none of the patients died, and 15 patients experienced reactivation. The median event free survival was 37.5 months. Two of 6 patients with subclinical hypothyroidism had normal thyroid function, and 12 patients still had hypothyroidism after treatment.

Summary/Conclusion: As the largest adult LCH cohort with thyroid gland involvement to date, we found that patients with thyroid gland involvement had different clinical characteristics, genetic profiles and outcomes than patients without thyroid gland involvement.

P517: PROSPECTIVE MULTICENTRIC STUDY ON INFECTIOUS COMPLICATIONS AND CLINICAL OUTCOME IN 230 UNFIT AML PATIENTS TREATED IN FIRST-LINE WITH HYPOMETHYLATING AGENTS ALONE OR IN COMBINATION WITH VENETOCLAX.

A. Candoni 1,* , M. Piccini 2 , D. Lazzarotto 1 , V. Bonuomo 3 , M. Dargenio 4 , M. Riva 5 , L. Mellillo 6 , C. Papayannidis 7 , M. Stulle 8 , G. Dragonetti 9 , M. I. Del Principe 10 , C. Cattaneo 11 , C. Pasciolla 12 , R. De Marchi 13 , M. Delia 14 , M. C. Tisi 15 , M. E. Zannier 1 , G. Nadali 3 , M. Sciumè 16 , A. Spadea 17 , C. Sartor 7 , D. Griguolo 8 , E. Buzzatti 10 , C. M. Basilico 18 , C. Sarlo 19 , A. L. Piccioni 20 , R. Cairoli 5 , A. Olivieri 21 , L. Pagano 9

1 Division of Hematology and Stem Cell Transplantation, ASUFC, University of Udine, Udine; 2 Division of Hematology, Azienda Ospedaliero-Universitaria Careggi, Florence; 3 Division of Hematology, AOUI, Policlinico GB Rossi, Verona; 4 Division of Hematology, Ospedale Vito Fazzi, Lecce; 5 Dipartimento di Ematologia ed Oncologia, Niguarda Cancer Center ASST Grande Ospedale Metropolitano, Milano; 6 Division of Hematology, Lecce and IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Lecce; 7 Institute of Hematology and Medical Oncology “L. and A. Seragnoli”, University of Bologna, Bologna; 8 Division of Hematology, ASUGI, Trieste; 9 Division of Hematology, Polo Onco-Ematologico, Fondazione Policlinico A. Gemelli-IRCCS, Università Cattolica del Sacro Cuore; 10 Hematology, Fondazione Policlinico Tor Vergata, Roma; 11 Section of Hematology, Spedali Civili, Brescia; 12 Haematology Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari; 13 Onco Hematology, Department of Oncology, Veneto Institute of Oncology IOV, IRCCS, Padova; 14 Hematology and Bone Marrow Transplantation Unit, Azienda Ospedaliero-Universitaria Consorziale Policlinico-University of Bari, Bari; 15 Cell Therapy and Hematology, San Bortolo Hospital, Vicenza; 16 U.O. Oncoematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milano; 17 Hematology and Stem Cell Transplant Unit, IRCCS Regina Elena National Cancer Institute, Roma; 18 Division of Hematology, ASST Sette Laghi, Ospedale Circolo e Fondazione Macchi, Varese; 19 Hematology and Stem Cell Transplantation Unit, University Campus Bio-Medico; 20 Dipartimento di Ematologia, Azienda Ospedaliera San Giovanni Addolorata, Roma; 21 Division of Hematology, Azienda Ospedaliero-Universitaria Ospedali Riuniti di Ancona, Ancona, Italy

Background: The hypomethylating agents (HMAs) are an important therapeutic option for older patients (pts) with AML and have become the backbone for combination regimens (eg, with Venetoclax). However, there are very limited real-life prospective studies regarding clinical outcome of these pts, including infectious complications and infection related mortality (IRM) during treatment.

Aims: To investigate the infectious complications and clinical outcome in AML patients treated with HMAs± Venetoclax (V) outside of clinical trials.

Methods: The recruitment of this prospective multicentric study (CE-Id-study:2908) has been completed on December 31, 2020. We enrolled 230 AML pts with a median age of 75 years (range 25-94); 157 pts (68%) had >2 relevant comorbidities. Of the 230 cases, 132 (57%) received a first-line therapy with a combination of HMAs+V while 98 (43%) were treated with HMAs monotherapy (azacitidine or decitabine). A total of 1550 cycles of HMAs have been administered (680/1550 with HMAs+V).

Results: The best response achieved, with HMAs treatment, was: CR in 44% of cases (57,6% with HMAs+V and 25,5% with HMAs alone, P=0,0001), PR in 17% and SD in 14% of cases (ORR 61%; 72% in HMAS+V and 46% in HMAs alone, P=0,0007). The microbiological or radiological proven infectious complications (almost one) occurred in 160/230 (70%) of pts, mainly pneumonia (in 42% of pts) and/or bacteremia/sepsis (one or more events in 29% of pts). Febrile neutropenia (one or more episodes) occurred in 38% of pts and 14 cases of Covid-19 (6%) were reported. After a median follow-up of 9 months (1-24) from the start of HMAs therapy, 144 (63%) pts died and 86 (37%) were alive. The 1 yr OS probability was 46% with a median OS of 10,3 months (11 months in HMAs+V and 9 months in HMAs alone; P=ns). The primary causes of death were: progression of AML (42%), Infection (26%-37/144), Infection+AML (24%), other causes (8%). The IRM was 26% and 19/144 (13%) pts died of infectious complication while in CR/PR (16 in HMAs+V group and only 3 in HMAs group; P=0,005). Data on antibiotic prophylaxis, hospitalization, drug-doses modulation, are available and analyzed in this study.

Summary/Conclusion: The results of this real-life, multicentric, prospective study, confirm a higher CR rate in pts treated with HMAs+V compared to HMAs alone (P=0,0001). However, we found a high rate of infectious complications and IRM (26%) with a higher infection related deaths in patients in CR/PR who were treated with HMAs+V (P=0,005). Findings from this study highlight the critical relevance of infection prevention in reducing infectious mortality, which adversely impacts the OS of this frail AML population.

P518: PEDAL/EUPAL INTERNATIONAL COLLABORATION TO IMPROVE THE OUTCOME OF CHILDREN WITH RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA

V. Ceolin 1,2,3,* , S. Ishimaru 1 , F. Bautista 1,4 , B. Goemans 1 , E. A. Kolb 5 , L. Di Laurenzio 6 , T. M. Cooper 7 , M. Bakker 1 , J. Wahlstrom 8 , G. Sunkersett 8 , G. Ku 9 , C. Zwaan 1,3,4 , D. Reinhardt 10 , G. Nichols 6

1 Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands; 2 Regina Margherita Children’s Hospital, University of Turin, Turin, Italy; 3 Sophia Children’s Hospital, Erasmus University MC, Rotterdam, Netherlands; 4 ITCC consortium, Paris, France; 5 Nemours Centers for Childhood Cancer Research & Cancer and Blood Disorders, Wilmington; 6 The Leukemia & Lymphoma Society, New York; 7 Seattle Children’s Hospital, Washington; 8 AbbVie One Oncology; 9 Genentech Inc, San Francisco, United States of America; 10 University Hospital of Essen, Essen, Germany

Background: The prognosis of children with acute myeloid leukemia (AML) has increased stepwise over the last decades, and the overall survival (OS) is currently in the 75-80% range. However, at relapse, OS is approximately 40-50%. Over the last decade, many novel drugs have been developed specifically for use in adults with AML and few have been authorized for children. Given the medical need, effective and less toxic therapies are urgently also needed for children to overcome the limitations of current intensive chemotherapy.

Aims: The Leukemia & Lymphoma Society (LLS) Pediatric Acute Leukemia (PedAL) program is a strategic initiative that aims to overcome the obstacles in treating children with acute leukemia via a Transatlantic collaboration. The aim is to run a program of early and late phase clinical trials evaluating the safety and the efficacy of new agents in pediatric leukemia, eventually setting a new standard of treatment for relapsed AML. In Europe this will be implemented as a master clinical trial with sub-trials. The European Pediatric Acute Leukemia (EuPAL, including United Kingdom) consortium was set up to coordinate the European efforts for the PedAL initiative.

Methods: In Europe, the current project consists of a registry protocol (EuPAL2021 Registry) and a Master protocol with sub-trials (ITCC-101). In North America patients will be enrolled in the PedAL Screening Protocol - APAL2020SC. Any patient with known or suspected relapsed or refractory AML, who is less than 22 years of age will be eligible for inclusion in the registry in Europe or in the screening protocol in North America, to provide confirmation of relapse and target expression using standardized methodologies. In Europe, the EuPAL 2021 Registry will lead to harmonization of diagnostics in country/collaborative group-specific centralized laboratories where the analysis will be carried out as part of standard of care. In North America, Australia and New Zealand APAL2020SC will utilize commercial laboratories (Hematologics, Foundation Medicine). The ITCC-101 PedAL/EuPAL Master protocol is a complex clinical trial with a stratification approach to allocate these patients to sub-trials, according to relevant biomarkers and disease related information. After confirmation of relapse, patients may be enrolled in one of the open sub-trials, provided the detailed eligibility criteria are met. Each sub-trial will effectively run as one study performed on a global scale, and data will be entered in one database. Sub-trials may not start at the same time and may be added by a substantial amendment to the master. In North America each sub-trial will be added as an amendment to the LLS Investigational New Drug. For the first wave of sub-trials, Investigational Medicinal Product prioritization was mainly achieved through international collaboration at the Pediatric Forum for AML organized by ACCELERATE Strategy Forum in April 2020.

Results: The first sub-trial will be APAL2020D, an open-label Phase III randomized multicenter trial to assess if venetoclax combined with FLA+GO (fludarabine, high-dose cytarabine, and gemtuzumab ozogamicin) will improve overall survival compared to FLA+GO, with the aim to establish the standard treatment for the 2 nd relapsed AML patients ( NCT05183035 ) and will open in Q2/Q3 2022.

Summary/Conclusion: The PedAL/EuPAL international collaboration will determine, using biology-based selection markers for treatment stratification, the standards of care for AML in 1 st and 2 nd relapse and define the safety and efficacy parameters necessary for the efficient evaluation of novel targeted therapies.

P519: TRANSFUSION INDEPENDENCE AMONG NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA PATIENTS RECEIVING VENETOCLAX-BASED COMBINATIONS VS OTHER THERAPIES: RESULTS FROM THE AML REAL WORLD EVIDENCE (ARC) INITIATIVE

O. Wolach 1 , J. S. Garcia 2 , P. Vachhani 3 , J. Zeidner 4 , C. Lai 5 , Y. Moshe 6 , M. Xavier 7 , S. Lee 8 , T. Zuckerman 9 , D. A. Pollyea 10 , S. Abedin 11 , E. C. Chen 2 , S. Bathini 3 , W. Edwards 7 , C. N. Bui 12 , W. W.-H. Cheng 12 , A. Svensson 12 , A. Guérin 13 , J. Maitland 13 , E. Ma 14 , M. Montez 14 , M. Grunspan 15 , A. D. Goldberg 16,*

1 Rabin Medical Center, Petah Tikya, Israel; 2 Dana-Farber Cancer Institute, Boston; 3 O’Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham; 4 University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill; 5 University of Pennsylvania Perelman Center for Advanced Medicine, Philadelphia, United States of America; 6 Tel Aviv (Sourasky) Medical Center, Tel Aviv, Israel; 7 Scripps-MD Anderson, San Diego; 8 Janssen Research and Development, Springhouse, United States of America; 9 Rambam Health Care Campus, Haifa, Israel; 10 University of Colorado School of Medicine, Aurora; 11 Medical College of Wisconsin, Milwaukee; 12 AbbVie Inc, North Chicago, United States of America; 13 Analysis Group Inc, Montreal, Canada; 14 Genentech, South San Francisco, United States of America; 15 AbbVie Inc, Hod-Hasharon, Israel; 16 Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Venetoclax (VEN), a novel BCL-2 inhibitor, is FDA approved in combination with hypomethylating agents (azacitidine or decitabine) or low-dose cytarabine for the treatment of newly-diagnosed (ND) acute myeloid leukemia (AML) in adults ≥75 years or those who have comorbidities that preclude use of intensive induction chemotherapy. This study is part of the ongoing AML Real world evidenCe (ARC) Initiative which aims to provide insight into the use and relative efficacy of VEN-based combinations among ND patients (pts) with AML.

Aims: To describe transfusion independence (TI) among ND AML pts treated with VEN-based combinations vs non-VEN regimens in clinical practice.

Methods: The ARC Initiative is a multicenter chart review study of adult pts with AML who received VEN ≥April 2016 (VEN cohort) or non-VEN regimens ≥May 2015 (control cohort). Pts in the VEN cohort were matched 1:1 to pts in the control cohort based on age category (<60; 60-74; ≥75) and European Leukemia Net (ELN) risk classification. Outcomes, including TI, hematopoietic cell transplantation (HCT), and best response, were assessed during first-line therapy over a transfusion observation period defined as time from initiation of therapy to the earliest amongst progression, initiation of second-line therapy, admission to hospice, death, or last visit at study site. TI was defined as no red blood cell (RBC) or platelet (PLT) transfusions during any consecutive ≥56-day period during the transfusion observation period. Kaplan-Meier (KM) analyses were conducted to assess the TI rate at 3 months after treatment initiation. Interim descriptive results are presented from data cutoff of Sept 2021; data will be updated for the meeting.

Results: A total of 119 VEN and 119 matched control pts with ND AML who had available information on transfusions were included in this analysis (Table 1). Among pts in the control cohort, 65.5% received a high intensity regimen (e.g., cytarabine+daunorubicin [37 pts, 31.1%], CPX-351 [16 pts, 13.4%]) and 34.5% received a low intensity regimen (decitabine [24 pts, 20.2%], azacitidine [15 pts, 12.6%]). Of pts tested for genetic mutations, common mutations were TP53 (VEN: 23.1%; control: 15.8%), RUNX1 (VEN: 17.1%; control: 17.5%), and IDH1/IDH2 (VEN: 12.8%; control: 14.0%). During a median transfusion observation period of 4.0 months in the VEN cohort and 3.6 months in the control cohort, 87.4% of VEN and 93.3% of control pts received ≥1 RBC or PLT transfusion. Pts in the VEN cohort received a median of 2.0 RBC and/or PLT transfusions per month and 5.0% received HCT; pts in the control cohort received a median of 3.5 RBC and/or PLT transfusions per month and 13.4% received HCT (all p <0.05). Among the 103 pts in the VEN cohort and 99 pts in the control cohort who had ≥56 days of follow-up, 61.2% of VEN pts and 50.5% of control pts achieved TI during first-line therapy (p=0.17), and 60.6% of VEN pts and 55.8% of control pts with response data achieved response (p=0.59). The KM rate of achieving TI at 3 months was 45.0% in the VEN cohort and 35.9% in the control cohort (log-rank p=0.12). Pts in the VEN and control cohort achieved TI after a median of 2.3 and 2.8 months, respectively (p=0.08).

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Summary/Conclusion: Interim ARC initiative results show that pts receiving VEN-based combinations receive statistically fewer transfusions and are trending towards achieving TI more frequently and reaching TI more quickly after initiation of treatment compared to matched control pts receiving low and high intensity therapies. Further analyses are planned to understand the real-world outcomes of ND pts with AML.

P520: PHASE 1/2 STUDY OF SEL24/MEN1703, A FIRST-IN-CLASS DUAL PIM/FLT3 KINASE INHIBITOR, IN PATIENTS WITH IDH1/2-MUTATED ACUTE MYELOID LEUKEMIA: THE DIAMOND-01 TRIAL.

G. Martinelli 1,* , A. Santoro 2 , C. Gambacorti-Passerini 3 , S. Vives Polo 4 , S. R. Solomon 5 , S. Mukherjee 6 , E. Lech-Maranda 7 , M. Yair Levy 8 , A. Wierzbowska 9 , M. Calbacho-Robles 10 , G. Marconi 11 , M. Benedetta Giannini 11 , I. Cano 12 , L. Torres Miñana 13 , E. Acuña-Cruz 12 , N. Angelosanto 14 , A. Galleu 15 , S. Baldini 15 , S. Blotta 15 , F. Ravandi 16 , P. Montesinos 17

1 IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori”-IRST S.r.l., Meldola; 2 Department of Oncology, IRCCS, Humanitas Research Hospital; 3 Department of Hematology, University of Milano-Bicocca, Milan, Italy; 4 Institut Català d’Oncologia-Hospital Germans Trias i Pujol, Badalona, Spain; 5 Northside Hospital Cancer Institute, Atlanta; 6 Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 7 Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland; 8 Texas Oncology-Baylor Charles A. Sammons Cancer Center, Dallas, United States of America; 9 Department of Hematology, Medical University of Lodz, Lodz, Poland; 10 Department of Hematology, Ramón y Cajal University Hospital, Madrid, Spain; 11 IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST S.r.l., Meldola, Italy; 12 Department of Hematology; 13 Department of Hematology, La Fe University Hospital, Valencia, Spain; 14 13Clinical Development-Hematology, Ryvu Therapeutics, Krakow, Poland; 15 Hematologic Malignancies - Global Oncology TA, Menarini Group, Florence, Italy; 16 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America; 17 La Fe University Hospital, Valencia, Spain

Background: Mutations in the FLT3 tyrosine kinase are the most frequent mutations that occur in adults with acute myeloid leukemia (AML) and can co-occur with mutations in IDH1 or IDH2 (collectively IDH m) in up to 30% of cases. SEL24/MEN1703 is an orally available, first-in-class, dual PIM/FLT3 kinase inhibitor. Preliminary results from the phase 1/2 first-in-human DIAMOND-01 trial ( NCT03008187 ) evaluating single-agent SEL24/MEN1703 showed activity in adults with relapsed/refractory (R/R) IDH m AML, where 3 of 8 IDH m patients responded.

Aims: Here we report the first safety and efficacy results from an additional expansion cohort of the DIAMOND-01 trial in 20 patients with R/R IDH m AML.

Methods: Patients with IDH m R/R AML and no standard therapeutic options were eligible. The recommended dose of 125 mg SEL24/MEN1703 was given orally, once daily for 14 days over a 21-day cycle until disease progression or unacceptable toxicity. The primary endpoint was safety, and adverse events (AEs) were graded according to NCI CTCAE v4.03. The secondary endpoint was antileukemic activity including overall response rate (ORR).

Results: As of 10 January 2022, 14 patients were enrolled in the IDH m cohort. Median age was 68 years (range 37–79). Four patients had AML secondary to myelodysplastic syndrome and 7 patients had intermediate cytogenetic risk. Median number of prior lines of treatment was 2 (range 1–3). Seven patients had IDH2 , 1 had IDH1/2 , and 4 had IDH1 mutations. Concomitant mutations in FLT3 -ITD were detected in 2 patients. Median duration of treatment was 2 cycles (range 1–8). Safety data (N=12) showed that serious treatment-emergent AEs (TEAEs; reported in ≥5% patients) were pneumonia (33%), skin infection, and clostridial gastroenteritis (8% each). These events were all unrelated to study drug. Drug-related TEAEs were liver injury and hyponatremia (8% each). The drug-related liver injury occurred in a patient who was concomitantly receiving other drugs with known hepatotoxic potential. Grade ≥3 TEAEs (≥10% patients) were pneumonia (33%) and asthenia (17%), both unrelated to study drug. No differentiation syndrome was observed. Of the 7 patients who completed ≥1 treatment cycle and had ≥1 postbaseline assessment or clear disease progression, ORR was 28.6%; 1 patient achieved a complete response with incomplete blood count recovery (CRi) at cycle 3 and underwent hematopoietic stem cell transplant, 1 patient had a partial response at cycle 4 (confirmed at cycle 7 and still on treatment), 4 had disease progression, and 1 discontinued for an AE (not drug related). Among the 7 remaining patients, 3 discontinued before completion of cycle 1 without progression or response, while 4 patients were ongoing and have not yet had any postbaseline assessment.

Summary/Conclusion: Preliminary results in the IDH m cohort confirm that SEL24/MEN1703, a first-in-class, orally available, dual PIM/FLT3 inhibitor, has a manageable safety profile and single-agent activity in patients with R/R IDH m AML. Updated results will be presented at the congress.

P521: TREATMENT OF BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM IN PEDIATRIC PATIENTS WITH TAGRAXOFUSP, A CD123-TARGETED THERAPY

N. Pemmaraju 1,* , B. Cuglievan 1 , J. Lasky 2 , A. Kheradpour 3 , N. Hijiya 4 , A. S. Stein 5 , S. Meshinchi 6 , C. Mullen 7 , E. Angelucci 8 , L. Vinti 9 , T. I. Mughal 10,11 , A. Pawlowska 12

1 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 2 Cure 4 The Kids Foundation, Las Vegas; 3 Department of Pediatric Hematology and Oncology, Loma Linda University Children Hospital, Loma Linda; 4 Division of Pediatric Oncology, Hematology, and Stem Cell Transplantation, Columbia University Irving Medical Center, New York; 5 Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte; 6 Department of Pediatrics, University of Washington School of Medicine, Seattle; 7 Division of Pediatric Hematology/Oncology, Department of Pediatrics, Golisano Children’s Hospital, University of Rochester, Rochester, United States of America; 8 Hematology and cellular therapy unit, IRCCS Ospedale Policlinico San Martino, Genova; 9 Department of Paediatric Haematology/Oncology, Cell and Gene Therapy, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy; 10 Division of Hematology-Oncology, Tufts University Medical School, Boston; 11 Stemline Therapeutics Inc, New York; 12 Department of Pediatrics, City of Hope National Medical Center, Duarte, United States of America

Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy derived from plasmacytoid dendritic cell precursors that overexpress CD123, the interleukin-3 receptor alpha. Though primarily a disease of the elderly, BPDCN has been documented in infants and children in 10–20% of all cases. The rarity of BPDCN in the pediatric population makes collecting robust safety and efficacy data challenging and there is a paucity of data published for pediatric patients (pts). A first-in-class, CD123-directed therapy, tagraxofusp (TAG, SL-401) is the only approved treatment for patients with BPDCN. A multicohort prospective study ( NCT02113982 ) demonstrated a tolerable safety profile and a durable overall response rate of 75% (median duration 24.9 months) in first-line (1L) pts. TAG is approved by the US FDA for the treatment of BPDCN in pts over the age of 2 years with newly diagnosed and relapsed or refractory (R/R) BPDCN; it is also approved by the EMA for 1L treatment of adult pts. Herein, we present a case series of pediatric pts with BPDCN treated with TAG, to provide further insight into the efficacy and safety of this therapy in this understudied patient population.

Aims: To expand on the body of data regarding TAG therapy in pediatric pts with BPDCN.

Methods: Pediatric case reports of BPDCN were collected from centers across the US and Europe. TAG was administered according to local institutional guidelines 1L, or R/R. Data was collated and summarized descriptively. Analyses included tumor response, survival, and safety (adverse events [AEs] and laboratory values).

Results: Eight pediatric pts with BPDCN were treated with TAG and included in this analysis. Median age was 15.5 years (range 2 – 21 years), and 7 of the 8 pts were female. Five pts were treatment naïve, and 3 pts were R/R. Four pts had skin only disease. Two pts had skin, bone marrow (BM), and lymph node (LN) involvement; 1 of these also had central nervous system (CNS) involvement. One patient had BM and LN, but no skin involvement, and 1 patient had BM only disease. A 2-year-old patient received 7 mcg/kg TAG in combination with venetoclax and azacitidine at second relapse; all other pts received 12 mcg/kg throughout all treatment cycles. The number of TAG cycles administered ranged from 1 to 4 at the time of data cutoff. A decrease in albumin occurred in 2 pts, and 2 pts had a rise in transaminases, all of which were manageable. One patient experienced headaches, hot flashes, fatigue, and mouth sores. One patient experienced grade 2 capillary leak syndrome in cycle 1, which was treated and resolved, and the patient went on to receive subsequent cycles. No AEs were seen in 3 pts. Three 1L pts achieved a complete response, including 1 patient with extensive disease (skin, bone marrow, and CNS) who also received intrathecal therapy. One 1L and 1 R/R patient achieved stable disease (no progression after 4 TAG cycles each), and 1 patient with R/R BPDCN progressed while on TAG. Five pts treated with TAG were bridged to a stem cell transplant (n=3 1L, n=2 R/R; Table 1). Median survival data will be presented (6 of 8 pts remain alive).

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Summary/Conclusion: These cases expand the knowledge base of BPDCN treatment in pediatric pts. TAG was well tolerated in all pts, with a manageable safety profile that was similar to that reported for adults in the pivotal study. Treatment with TAG demonstrated promising efficacy, with responses that enabled 5 of 8 pediatric pts, including 3 1L and 2 R/R, to be bridged to stem cell transplant.

P522: ABSOLUTE LYMPHOCYTE COUNT IS AN INDEPENDENT SURVIVAL PREDICTOR IN PATIENTS WITH ACUTE MYELOID LEUKEMIA TREATED WITH INTENSIVE CHEMOTHERAPY

G. Cristiano 1,* , J. Nanni 1 , L. Zannoni 1 , C. Sartor 1 , S. Parisi 2 , S. Paolini 2 , C. Papayannidis 2 , M. Cavo 1,2 , A. Curti 2

1 Hematology, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna; 2 Hematology, IRCCS Azienda ospedaliero-universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy

Background: Absolute lymphocyte count (ALC) is known to be an independent prognostic factor for overall survival (OS) in patients receiving autologous transplantation for Hodgkin’s and non-Hodgkin’s lymphomas, multiple myeloma and breast cancer. In acute myeloid leukemia (AML) patients, enhanced ALC recovery after intensive chemotherapy (IC) has been associated with superior OS and leukemia-free survival (LFS). However, few if any data correlated ALC recovery with novel therapies as well as with updated prognostic factors, such as European Leukemia Net (ELN) risk-classification.

Aims: Our study aims at evaluating the predictive value of ALC recovery in AML patients treated with IC by analysing its impact through different patient subgroups according to therapy response, type of chemotherapy regimen and the use of allogeneic stem cell transplantations (HSCT).

Methods: We evaluated 148 newly diagnosed adult AML patients treated with IC at Bologna Seràgnoli Hematology Institute since 2007. We defined 4 ALC time-points (TPs): at 15, 21 and 28 days from the start of induction chemotherapy and before consolidation therapy (CC). ALC cut-off was established at 500/mm 3 . In addition to the assessment of the single TP, patients were also grouped in those who obtained ≥500/mm 3 ALC in all 4 TPs and those who had <500/mm 3 ALC in at least one TP. Median follow-up was 51,4 months.

Results: When ALC recovery was assessed at the single TP, only ALC-CC recovery showed a statistically significant association with better OS and LFS. However, significant outcome differences were observed among CR patients (76,3%), subdivided in those who achieved ALC in all TPs vs others. A trend toward better correlation of ALC with OS was observed in patients who received “3 + 7-based” regimen (30,8%;) versus those who were treated with “fludarabine-based” regimen (69,1%;). Better outcome was observed in “3 + 7” patients who had ALC recovery in all TPs and at day 15 post-chemotherapy. Of note, in the “3 + 7” group, FLT3-ITD positive patients, who achieved early ALC recovery at day 21 had better outcomes. We, then, analysed the impact of post-chemotherapy ALC recovery in patients who received HSCT (66,3%) vs those who did not (33,6%) (Fig.1). As expected, transplanted patients had globally better outcome than patients who received only chemotherapy. In patients who received HSCT the impact of ALC recovery after chemotherapy was minimal. In contrast, patients who had ALC <500/mm 3 ALC in at least one TP and did not undergo HSCT had the worst outcome. Interestingly, patients who were not transplanted but had ≥500/mm 3 ALC in all 4 TPs had a similar outcome as patients who received HSCT.

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Summary/Conclusion: Our study indicates ALC recovery after IC as a promising survival predictor in AML patients, especially in those who do not undergo HSCT. In the path that aims to investigate the complex interaction between leukemia and the immune system, our study may provide the clinical rationale for the construction of an immunological score to be integrated in the current disease-centered risk-classification system. Future studies in larger cohorts addressing the impact of ALC recovery in association with the use of novel agents and minimal residual disease status are highly warranted.

P523: MIDOSTAURIN PLUS INTENSIVE CHEMOTHERAPY IN FLT3 MUTATED AML. “REAL LIFE” DATA VERSUS THE RATIFY STUDY

A. De La Fuente 1,1,* , M. Diaz Beya 2 , P. Beneit 3 , C. Botella 4 , A. Fernandez Moreno 5 , A. Sampol 6 , M. Arnan Sangerman 7 , A. Yeguas Bermejo 8 , M. D. L. L. Amigo 9 , J. Labrador 10 , A. Garcia Guinon 11 , A. Garrido 12 , J. Serrano 13 , S. Vives Polo 14 , M. Garcia Fortes 15 , M. J. Sayas 16 , J. M. Bergua 17 , M. T. Olave 18 , F. Vall LLovera 19 , J. Bargay 20 , M. Pereiro Sanchez 21 , R. Garcia Boyero 22 , A. Diaz Lopez 23 , M. Tormo 24

1 Hematology, MD Anderson CC Madrid, Madrid; 2 Hematology, H. U. Clinic IDIBAPS, Barcelona; 3 Hematology, H. U. San Juan de Alicante; 4 Hematology, H. General de Alicante, Alicante; 5 Hematology, H. U. Central de Asturias, Oviedo; 6 Hematology, H. U. Son Espases, Palma de Mallorca; 7 Hematology, ICO Hospitalet, L’Hospitalet de Llobregat, Barcelona; 8 Hematology, H. U. Salamanca, Salamanca; 9 Hematology, H. U. Morales Meseguer, Murcia; 10 Hematology, H. U. de Burgos, Burgos; 11 Hematology, H. U. Arnau de Vilanova, Lleida, Lleida; 12 Hematology, Hospital de la Santa Creu i Sant Pau. Instituto de investigación Jose Carreras, Barcelona; 13 Hematology, H. U. Reina Sofia, Cordoba; 14 Hematology, ICO H. Germans Trias i Pujol, Barcelona; 15 Hematology, H. Virgen de la Victoria, Malaga; 16 Hematology, H. U. Doctor Peset, Valencia; 17 Hematology, H. U. san Pedro de Alcantara, Caceres; 18 Hematology, H. U. Lozano Blesa, Zaragoza; 19 Hematology, H. U. Mutua Terrassa, Barcelona; 20 Hematology, H. U. Son LLatser, Palma de Mallorca; 21 Hematology, C. H. U. Orense, Orense; 22 Hematology, H. G. U. de Castellon, Castellon de la Plana; 23 Hematology, Fundacion MD Anderson Madrid, Madrid; 24 Hematology, 20Clinico Universitario Instituto de Investigación Sanitaria INCLIVA, Valencia, Spain

Background: FLT3 mutation is associated with adverse prognosis in AML (ELN2017, Dohner et al, Blood 2017) and Intensive chemotherapy (IC) has been the standard of treatment for FLT3 mutated AML for decades. Midostaurin (Midos) has been approved in combination with IC for FLT3-mutated AML based on an improvement in overall survival noted in the RATIFY phase 3 trial (Stone et al, N Engl J Med 2017)

Aims: The aims of this study are to analyze safety and effectiveness of Midos plus IC in FLT3 AML, to validate RATIFY results in a “real-world” setting and to identify risk factors.

Methods: We carried out a retrospective multicenter study (MDA-AML-2018-06) in 27 Spanish centers. Inclusion criteria: age >18 years, FLT3-mutated AML diagnosis according to WHO criteria and start of treatment with midostaurin in combination with IC between June 2016 and June 2021. We evaluated the response according to 2017 ELN criteria, toxicity according to CTCAE v4.0 and overall survival (OS) by Kaplan-Meier. Statistical analysis was performed using SPSS program version 20.0.

Results: A total of 175patients (pt) were included (93 female), median age 53 (18-76), of them 111were younger than 60yrs and 110pts had ECOG<2. ELN2017 stratification was favorable in 53pt; Intermediate: 72pt; adverse in 26pt. Median WBC count 44.500/µL (0.4-395.000). A total of 133pts had FLF3-ITD mutation and of them 74pt with ratio ≥0.5. Safety: Total Midos cycles 548 (median 2), 20 pt (11.5%) suffered QT prolongation, 26pt (14,8%) Midos interruption and 10pt (5.7%) Midos reduction. There were no deaths related to Midos. Effectiveness: 144 (81.4%) pt achieved CR after Induction1or2, of them 76pt were consolidated with alloSCT, 24pt received maintenance and 41pt proceed to W&W. Median OS for the whole population was not reached and the 24months OS was 68%. In our experience ELN2917 classification and ECOG<2 identify groups with differences in OS (p0.019 and p0.04 respectively). Age <60vs≥60 resulted in no differences for OS. We observed significant differences for maintenance versus W&W (p=0.001) in favorable and intermediate risk patients. Comparing maintenance vs alloSCT we observe no differences in the Intermediate ELN2017 group.

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Summary/Conclusion: Our experience confirms safety and effectiveness of Midos plus IC as first line in FLT3 AML patients with similar 2yOS as the previous reported in the RATIFY trial. Interesting that in our experience age 60 and above resulted in no differences for OS.

P524: PHASE 1B/2 STUDY ON SAFETY, PK, PD, AND PRELIMINARY EFFICACY OF THE SELECTIVE SYK INHIBITOR LANRAPLENIB IN COMBINATION WITH THE FLT3 INHIBITOR GILTERITINIB, IN FLT3-MUTATED R/R AML (KB-LANRA 1001)

E. M. Stein 1,* , A. Patel 2 , L. C. Michaelis 3 , G. Schiller 4 , R. Swords 5 , L. A. Carvajal 6 , G. Bray 6 , J. DiMartino 6 , M. Y. Levy 7

1 Memorial Sloan Kettering Cancer Center, New York; 2 The University of Chicago Medical Center, Chicago; 3 Medical College of Wisconsin, Milwaukee; 4 University of California Los Angeles (UCLA), Los Angeles; 5 Oregon Health and Science University, Portland; 6 Kronos Bio, Inc., San Mateo; 7 Texas Oncology - Baylor Charles A. Sammons Cancer Center, Dallas, United States of America

Background: Spleen tyrosine kinase (SYK) is a key driver of lymphoid and myeloid cell signaling pathways and has been implicated in the pathogenesis of acute myeloid leukemias (AML) defined by dysregulated expression of HOXA9 and MEIS1 transcription factors. SYK also cooperates with internal tandem duplication (ITD)-mutated FMS-like tyrosine kinase 3 ( FLT3 ) to drive leukemogenesis. Combined pharmacologic inhibition of SYK and FLT3 results in robust antileukemic effects in preclinical models of FLT3 ITD-driven AML. Lanraplenib (LANRA) is an oral, selective SYK inhibitor. Studies demonstrate dose-dependent reductions in viability of leukemic cells ex vivo from newly diagnosed FLT3 ITD-mutated AML patients (pts) as well as additive reductions in viability when combined with the selective FLT3 inhibitor, gilteritinib. Once daily (QD) LANRA was well-tolerated at doses up to 50mg for 7 days and 30mg for up to 12 weeks in healthy volunteers and pts with autoimmune disorders.

Aims: The KB-LANRA 1001 trial will investigate the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of LANRA when combined with gilteritinib in relapsed or refractory pts with FLT3 -mutated AML. We will also evaluate the rate of complete responses (CR) and CR with partial hematologic recovery (CRh), duration of response (DoR), event-free (EFS) and overall survival (OS).

Methods: KB-LANRA 1001 is a global, multicenter trial of LANRA in combination with standard dose gilteritinib (120mg QD) in FLT3 -mutated AML pts aged ≥18 with at least 1 prior therapy. Those who have been treated with midostaurin or other multikinase inhibitors are eligible, though pts who failed to achieve at least a partial response or who relapsed following prior exposure to gilteritinib or other next-generation FLT3 inhibitor monotherapy are excluded. Both medications will be administered daily in sequential, 28-day cycles until progression/relapse, intolerance, or failure to achieve at least a partial remission after 6 cycles. The Phase 1b component will define the maximum tolerated and/or recommended Phase 2 dose (MTD/RP2D) of LANRA when added to gilteritinib in accordance with a 3 + 3 dose escalation design. PK and PD parameters will be characterized for LANRA monotherapy and when co-administered with gilteritinib. Four LANRA doses are planned for evaluation (20, 40, 60 and 90mg). All decisions regarding dose escalation including declaration of the MTD will be made by a dose-escalation committee based on the prospectively defined definition of dose-limiting toxicities and other safety metrics. The Phase 2 component will further evaluate safety, PK, PD and preliminary efficacy of the combination at the LANRA RP2D. For both trial components, response assessments will be conducted on Day 1 of Cycles 2 and 3 and every 3 cycles thereafter until 2 consecutive assessments indicate CR or CRh. A 2-stage design will be employed to test the null hypothesis that the rate of CR/CRh for the combination is ≤23%, as reported for gilteritinib monotherapy, versus the alternative hypothesis of CR/CRh ≥46%, among pts treated at the LANRA RP2D. All pts will be followed for relapse, EFS and OS. Key exploratory endpoints will be assessments of measurable residual disease among pts who achieve CR/CRh, correlations with selected baseline biomarkers that may predict efficacy outcomes and assessment of LANRA PD properties (including target engagement) when administered alone and in combination with gilteritinib.

Results: Trial in progress.

Summary/Conclusion: Accrual is ongoing.

P525: PHASE 3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY OF THE EFFICACY AND SAFETY OF ENTOSPLETINIB ADDED TO INTENSIVE INDUCTION AND CONSOLIDATION CHEMOTHERAPY IN NEWLY DIAGNOSED NPM1-MUTATED AML

J. C. Byrd 1,* , J. E. Cortes 2 , M. D. Minden 3 , T. Oellerich 4 , E. M. Stein 5 , J. Elder 6 , P. Kumar 7 , G. Bray 7 , J. DiMartino 7 , W. Stock 8

1 University of Cincinnati, Cincinnati; 2 Georgia Cancer Center at Augusta University, Augusta, United States of America; 3 Princess Margaret Cancer Centre, Toronto, Canada; 4 Frankfurt Cancer Institute, Frankfurt am Main, Germany; 5 Memorial Sloan Kettering Cancer Center, New York; 6 PharPoint Research, Inc., Durham; 7 Kronos Bio, Inc., San Mateo; 8 University of Chicago Medicine, Chicago, United States of America

Background: Spleen tyrosine kinase (SYK) is a component of both lymphoid and myeloid cell signaling pathways and is implicated in the pathogenesis of a subset of acute myeloid leukemia (AML) defined by dysregulated expression of HOXA9 and MEIS1 transcription factors. Entospletinib (ENTO) is an oral, selective SYK inhibitor that is acceptably tolerated when administered with intensive induction and consolidation in newly diagnosed AML patients (pts). In a Phase 2 study, following induction with cytarabine and daunorubicin (7 + 3) plus ENTO, higher rates of complete response (CR) or CR with incomplete hematologic recovery (CRi) occurred in pts with rearrangements of the KMT2A gene ( MLL -r) and mutations of the nucleophosmin 1 ( NPM1 ) gene, both of which are associated with aberrant expression of HOXA9 and MEIS1 , compared to pts without these mutations. In an exploratory analysis, pts with HOXA9 / MEIS1 expression levels above the median experienced superior overall survival (OS) compared to pts with expression levels below the median.

Aims: We hypothesize that the addition of ENTO to intensive induction/consolidation in newly diagnosed pts with NPM1 -mutated AML will improve the CR rate without evidence of measurable residual disease (MRD-negative CR) post-induction and duration of event-free survival (EFS).

Methods: AGILITY is a global, multi-center, double-blind, placebo-controlled trial of ENTO in combination with cytarabine plus daunorubicin or idarubicin induction (7 + 3) and age-adjusted high-dose cytarabine (HiDAC) consolidation in newly diagnosed AML pts aged 18–74 who are candidates for intensive induction and harbor a documented NPM1 mutation. Pts with co-mutated FLT3 are excluded. Pts will be stratified based on age (<60 vs ≥60 years) and anthracycline administered during induction (daunorubicin vs idarubicin). Approximately 180 pts will be randomized to receive 7 + 3 induction and HiDAC consolidation with ENTO (400mg orally twice daily) or with placebo. Pts with <5% leukemic blasts after induction cycle 1 will proceed to the first cycle of consolidation; pts with ≥5% residual blasts will undergo a second induction cycle. Pts who do not achieve CR after the last cycle of induction plus ENTO or placebo will be designated as induction treatment failures (ITF) for purposes of EFS estimation. Pts who achieve or remain in CR after 2 chemotherapy cycles (either 2 induction cycles or 1 induction and 1 consolidation cycle) will be evaluated for MRD in bone marrow based on enumeration of mutant NPM1 alleles using a molecular assay. Pts may receive ≤3 cycles of consolidation with HiDAC and ENTO or placebo per their original randomized treatment assignment beyond chemotherapy cycle 2. All pts will be followed for relapse and survival. The primary endpoint is the rate of MRD-negative CR (<0.01% mutant NPM1 alleles). A key secondary endpoint is EFS, defined as time from randomization to earliest occurrence of ITF, relapse from CR, or death from any cause. OS will be evaluated. Key exploratory endpoints will be the correlation between recurring genomic mutations and response or progression and longitudinal assessment of peripheral blood for detection of NPM1 -m alleles among pts who achieve MRD-negative CR post-induction.

Results: Trial in progress.

Summary/Conclusion: Accrual is ongoing.

P526: REAL-WORLD COMPARISON OF DIFFERENT TREATMENT MODALITIES FOR FAVORABLE RISK ACUTE MYELOID LEUKEMIA: STANDARD DOSE ANTHRACYCLINE VS HIGH DOSE ANTHRACYCLINE VS ADDITION OF GEMTUZUMAB OZOGAMICIN

J. Mort 1 , B. Brewer 2 , B. Mautner 1 , S. Dibenedetto 1 , E. Pierce 1 , E. Morse 1 , L. Wells 1 , F. Ghamsari 1 , A. Morris 1 , S. Clark 3 , J. Reid 3 , I. Patel 4 , C. Jackson 5 , M. Perciavalle 6 , B. Yelvington 6 , R. Miller 6 , K. Abernathy 6 , H. Wolfe 7 , S. Locke 7 , J. Zeidner 3 , D. Reed 5 , V. Duong 4 , B. Dholaria 6 , T. LeBlanc 7 , M. Keng 1 , B. Horton 2 , F. El Chaer 1,*

1 Hematology and Oncology; 2 Public Health Sciences, Division of Translational Research and Applied Statistics, University of Virginia, Charlottesville; 3 Hematology and Oncology, University of North Carolina, Chapel Hill; 4 Hematology and Oncology, University of Maryland Medical Center, Baltimore; 5 Hematology and Oncology, Wake Forest Baptist Medical Comprehensive Cancer Center, Winston-Salem; 6 Hematology and Oncology, Vanderbilt University Medical Center, Nashville; 7 Hematology and Oncology, Duke Cancer Institute, Durham, United States of America

Background: The standard remission induction regimen for medically fit patients with acute myeloid leukemia (AML) consists of a backbone of cytarabine & an anthracycline (“7 + 3” therapy). However, the choice & dose of the anthracycline varies between institutions & practitioner preference, particularly in AML with favorable risk cytogenetics. Gemtuzumab ozogamicin (GO) may improve outcomes in this patient population but is associated with increased toxicities.

Aims: We aimed to compare the outcomes of 3 cohorts of newly diagnosed AML with favorable cytogenetics who received induction 7 + 3 induction therapy: 1. Standard dose (45 or 60 mg/m2 daunorubicin) (SD); 2. Higher dose (90 mg/m2 daunorubicin or 12 mg/m2 idarubicin) (HD) of anthracycline, 3. 7 + 3 with fractionated GO.

Methods: We performed a multicenter retrospective analysis of medically fit patients ≥ 18 yo with favorable risk AML determined using ELN 2017 guidelines from 2015-2020 treated across 6 US academic institutions. Categorical variables were summarized using means and standard deviations. The former was compared using Fisher’s exact test; the latter was compared using either the Wilcoxon text or the Kruskal-Wallis as appropriate. One way ANOVA test was used to compare continuous variables. Kaplan-Meier curves were generated to provide visual summaries of survival and time-to-event data.

Results: Our study included 189 treated patients who met eligibility criteria with a median follow up of 2 years. Patients’ characteristics and outcomes are summarized in Table 1. Sixty-nine (37%) received induction with SD, 100 (53%) received HD, and 20 (11%) received GO. Patients in the SD and HD group were younger (median age 50 and 52 yo, respectively) and the majority (>94%) had de novo AML. The rates of composite complete remission in the SD, HD, and GO groups were 66.7%, 70% and 75%, respectively (p=0.761). RFS and OS were similar between all groups (p=0.4 and 0.3, respectively). The rate and duration of cytopenias at day 40 after induction, major bleeding, infections, admission to the intensive care unit, new onset cardiomyopathy (ejection fraction<50%), liver dysfunction and acute renal injury were similar among all groups. No patients developed veno-occlusive disease.

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Summary/Conclusion: Our analysis demonstrated no differences in RFS and OS in patients with AML treated with either GO, HD or SD. In addition, the rates of complications secondary to chemotherapy were similar among all groups. However, a measurable residual disease analysis is ongoing for further characterization of disease response.

P527: PHASE 1 TRIAL OF PEGCRISANTASPASE IN COMBINATION WITH VENETOCLAX IN ADULTS WITH RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA (R/R AML) - SAFETY, EFFICACY AND PK/PD IN THE FIRST TWO COHORTS

Y. Liu 1 , O. M. Bah 1 , D. Bollino 1 , K. Caprinolo 1 , J. Zarrabi 1 , S. Philip 1 , R. G. Lapidus 1 , E. T. Strovel 2 , Z. N. Singh 3 , M. E. Kallen 3 , Y. Ning 3 , R. Koka 3 , S. Niyongere 1 , V. H. Duong 1 , M. R. Baer 1 , M. Graveno 1 , A. Emadi 1,*

1 University of Maryland Greenebaum Comprehensive Cancer Center; 2 Pediatrics; 3 Pathology, University of Maryland School of Medicine, Baltimore, United States of America

Background: Glutamine depletion induced by Erwinia asparaginases including pegcrisantaspase (PegC) downregulates the mTOR/p70S6K pathway and inhibits 4EBP1 phosphorylation. We recently found that the combination of PegC with the Bcl-2 inhibitor venetoclax (Ven) has strong in vitro and in vivo activity in poor-prognosis AML, mediated by enhancement of eIF4E-4EBP1 interaction on the cap-binding complex and decreasing protein synthesis. We hypothesized that Ven-PegC treatment will be safe and effective in the treatment of R/R AML. Here we report the results from the first two cohorts of a phase 1 trial of Ven-PegC in adults with R/R AML.

Aims: The primary objective is to estimate the maximum tolerated dose and to determine the regimen-limiting toxicities (RLT) of Ven-PegC. The exploratory endpoints include plasma amino acids levels 1- and 2-weeks post-PegC administration.

Methods: This is an open-label phase 1b clinical trial ( NCT04666649 ) of daily oral Ven in combination with biweekly out-patient IV PegC in 28-day cycles that will be administered to 4 cohorts (Table 1A) based on a standard 3 + 3 design.

Results: Eight patients (pts) were enrolled into the first two cohorts; 7 received ≥ 1 dose of PegC and one dropped out before receiving study drugs. Pts’ characteristics are shown in Table 1B. Median age was 66 (59-76) years; two (29%) were female; six (86%) had adverse-risk AML by ELN classification; and four (57%) had complex karyotypes.

Pharmacokinetic (PK) and pharmacodynamic (PD) data are available for 5 pts; all pts achieved a nadir plasma asparaginase activity > 0.1 IU/mL during cycle 1 (3/3 in cohort 1 within 3 weeks and 2/2 in cohort 2 within 10 days). Mean plasma amino acid levels (µmol/L) at baseline were: asparagine (Asn) 29 (17-38), glutamine (Gln) 421 (384-465), glutamate (Glu) 59 (22-103), serine (Ser) 66 (49-80) and glycine (Gly) 203 (166-207). All pts achieved an Asn level of 0 by day 7; Asn remained undetectable throughout the study. Plasma Gln (µmol/L) reduction from baseline occurred in 4/5 (80%) pts within cycle 1: pt4 423→273 (Day15), pt5 446→86 (Day28), pt6 467→216 (Day22), pt7 388→156 (Day8), pt10 420→395 (Day10). Plasma Glu levels increased in all pts, indicating conversion of Gln to Glu by PegC. Interestingly, plasma levels of Ser increased 1.5-2-fold at the nadir of plasma Gln (Figure 1A-C, maximum amino acid changes after Ven-PegC).

Seven pts from cohorts 1 (4 pts) and 2 (3 pts) who received at least one dose of Ven-PegC were included in analysis of toxicity and efficacy, with data cutoff February 26, 2022 (Table 1B). No RLT has been observed. In cohort 1, pt2 died before completing cycle 1 due to septic shock and delay in seeking medical care, pt4 died of AML progression. In cohort 2, pt7 had progressive disease after cycle 1, pt9 opted for comfort care before evaluation of response, and pt10 died of AML progression,

In cohort 1, pt6 achieved measurable residual disease (MRD)-negative complete remission with partial hematologic recovery (CRh) and proceeded to allogeneic hematopoietic stem cell transplant (allo-HSCT) after 3 cycles of Ven-PegC, and bone marrow blasts in pt5 decreased from 78% to 9% after cycle 1. These two pts had the lowest plasma Gln levels (103 [pt6, cycle2 day29] and 86 [pt5, cycle1 day28]).

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Summary/Conclusion: No RLT has been observed following Ven-PegC in cohorts 1 or 2. The Ven-PegC combination at the lowest dose of PegC administered, produced an MRD negative CRh in one patient and a transient substantial reduction in bone marrow blast count in another, both associated with decreases in plasma Gln levels. The study is ongoing.

P528: A MIRNA SIGNATURE RELATED TO STEMNESS IDENTIFIES HIGH-RISK PATIENTS IN PEDIATRIC ACUTE MYELOID LEUKEMIA

E. Esperanza Cebollada 1,2,* , S. Gomez Gonzalez 3 , N. Vega García 1,2 , C. Vicente Garcés 1,2 , M. Torrebadell 1,2,4 , S. Rives 2,4,5 , J. L. Dapena 2,5 , A. Català 2,4,5 , M. Camós 1,2,4

1 Hematology Laboratory, Hospital Sant Joan de Déu; 2 Leukaemia and other Pediatric Hemopathies. Developmental Tumors Biology Group; 3 Developmental Tumor Biology Laboratory, Institut de Recerca Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona; 4 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid; 5 Pediatric Hematology and Oncology Department, Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain

Background: MicroRNAs (miRNAs) regulate gene expression and may function as tumor suppressors or cancer promoters. MicroRNAs regulating hematopoietic stem cells (HSC) could be prognostic biomarkers in leukemia, particularly in KMT2A -rearranged ( KMT2A+ ) patients, in whom few mutations have been described.

Aims: To analyze the role of miRNAs involved in HSC self-renewal and stemness pathways (NOTCH, WNT/beta-catenin, HOX, and FLT3) in a pediatric acute lymphoblastic leukemia (ALL) and myeloid acute leukemia (AML) cohort of patients.

Methods: We analyzed by quantitative PCR the expression of 89 selected miRNAs. To calculate miRNAs ∆∆CT expression differences between paired subgroups we used the lmFit function of the Limma R package, and the false discovery rate method to adjust the p-values with a 0.05 cut-off.

Results: We studied 110 patients (38% female, median age 6.1 years (range 0-17.4)), including 70 ALL, 39 AML, and one undifferentiated leukemia. Our series was primed with KMT2A + cases of all lineages (n=28). Globally, in the unsupervised hierarchical cluster analysis (HCA), patients were separated by lineage. Based on the sub-branching, we tried to correlate the clusters with clinic-biological variables. We found no association with age, sex, central nervous system infiltration, or leukocyte count. However, Kaplan-Meier analysis revealed that some clusters had significantly different event-free survival (EFS) (p=0.014): poor (clusters P1 and P4), good (P2), and intermediate (P3). Of note, clusters with good and poor survival branched off again according to lineage (P1 included most of AML cases and P4 most of ALL patients). We then studied survival according to lineage. Within ALL patients, we saw a group with a non-significant worse outcome (P4ALL), as compared with the remaining patients (P2ALL and P3ALL). In contrast, in AML patients, the HCA distinguished a group of 14 patients (36%, P1AML) with significantly worse outcome: OS at 5 years of 64.3%±12.8 vs. the remaining AML patients, all showing excellent survival (grouped in P2AML, 90.5%±6.4), p=0.028. Notably, differences in EFS at 5 years were even higher: 43%±13.2 (P1AML) vs. 86.5%±7.2 (P2AML), p=0.003. P1AML included cases from all molecular categories ( RUNX1::RUNX1T1, CBFB::MYH11, KMT2A+, and cases without recurrent genetic alterations). Of note, no PML::RARA cases fell in this cluster. We identified 24 miRNAs (23 underexpressed and 1 overexpressed) differentially expressed in P1AML patients. The 24 miRNAs signature included underexpressed miRNAs known to act as tumor suppressors regulating cell proliferation or apoptosis (hsa-miR-223-3p, hsa-miR-193a-3p, hsa-miR-181a-5p, hsa-miR-181b-5p, hsa-miR-181c-5p, hsa-miR-708-5p, hsa-miR-34b-5p, and hsa-miR-22-3p). The only miRNA overexpressed, hsa-miR-9-5p, promotes proliferation and inhibits apoptosis, functioning as oncomiR in KMT2A -rearranged AML cases. The remaining miRNAs (hsa-miR-199b-5p, hsa-miR-222-5p, hsa-miR-373-3p, hsa-miR-195-5p, hsa-miR-151a-5p and hsa-miR-1290, hsa-miR-20b-5p and hsa-miR-17-5p, hsa-miR-24-3p, hsa-miR-128-3p, hsa-miR-21-5p, hsa-miR-331-5p, hsa-miR-30b-5p, hsa-let-7g, and hsa-let-7i-5p), among their pleiotropic functions, regulate cellular stemness and proliferation. Our results suggests that within each molecular category, a miRNA signature could distinguish those patients with activated stemness and proliferation pathways and a more aggressive leukemia.

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Summary/Conclusion: We obtained a distinctive signature of 24 miRNAs capable of distinguishing pediatric AML patients with excellent or poor outcome.

P529: FIRST EUROPEAN REAL-WORLD EVIDENCE PROSPECTIVE REGISTRY OF FIRST-LINE ADULT PATIENTS WITH BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM TREATED WITH FIRST-IN-CLASS CD123-TARGETED THERAPY TAGRAXOFUSP

U. Platzbecker 1,* , E. Angelucci 2 , P. Montesinos 3 , R. M. Lemoli 4 , A. Spyridonidis 5 , J. Casariego 6 , T. I. Mughal 7,8 , M. Mohty 9

1 Medical Clinic and Policlinic of Hematology, Cell Therapy and Hemostaseology, University Hospital Leipzig, Leipzig, Germany; 2 Hematology and Cellular Therapy Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 3 Department of Hematology, Hospital Universitari i Politècnic La Fe, València, Spain; 4 Clinic of Hematology, Department of Internal Medicine, IRCCS San Martino Hospital, University of Genoa, Genoa, Italy; 5 Department of Internal Medicine, BMT Unit, University of Patras, Patras, Greece; 6 Aldebaran Research & Development SL, Madrid, Spain; 7 Division of Hematology-Oncology, Tufts University School of Medicine, Boston; 8 Stemline Therapeutics, New York, United States of America; 9 Department of Hematology and Cellular Therapy, Hôpital Saint-Antoine, Sorbonne University, Paris, France

Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy derived from plasmacytoid dendritic cells that overexpress interleukin-3 (IL-3) receptor alpha (CD123). It is a highly aggressive disease characterized by skin and bone marrow involvement and is associated with a poor prognosis (median overall survival 8–14 months). Tagraxofusp (TAG, SL-401) is a first-in-class CD123-targeted therapy comprising human IL-3 fused to a truncated diphtheria toxin payload. It is currently the only approved first-line (1L) treatment for adult patients (pts) with BPDCN in Europe. As of July 2021, 65 pts with BPDCN have received TAG in a clinical setting outside the US as part of an expanded access program. Collecting real-world clinical data from TAG is important to increase understanding of clinical outcomes, especially in pts commonly underrepresented in clinical trials. We describe the first real-world evidence prospective registry in pts with BPDCN receiving TAG. It has been designed to meet requirements outlined in the marketing authorization of the European Commission for further evaluation of the efficacy and safety of TAG in pts with 1L or relapsed/refractory BPDCN.

Aims: To investigate effectiveness and safety of TAG in pts with 1L BPDCN.

Methods: This is a noninterventional, single-arm, post-authorization study (EudraCT: 2021-001684-24) in adult pts with BPDCN prescribed TAG under real-world routine clinical practice conditions (12 mcg/kg intravenously [IV] once daily on days 1–5 of a 21-day cycle, per the summary of product characteristics recommendation). A minimum of 80 pts will be included in approximately 40–55 sites in Europe, estimated over 4 years. Eligible pts have a diagnosis of BPDCN and are to be treated (or have recently started treatment) with TAG monotherapy as 1L treatment, per physician’s decision.

Primary endpoints are rate of complete response (CR), defined as CR + clinical CR (CR with residual skin abnormality not indicative of active disease) after 3 months of treatment, and safety of TAG including the incidence and severity of capillary leak syndrome (CLS). Secondary endpoints include number of pts bridging to SCT, progression-free survival, overall survival, best overall response, duration of response, TAG dose interruptions/administration of IV albumin supplementation in pts with CLS or CLS symptoms, as well as safety and incidence and severity of adverse events of special interest (which include CLS and hepatic, renal, and cardiac events).

Quarterly data collection is anticipated, as well as collection at screening, enrollment, early discontinuation, and study end ( Figure ). Safety data collection will end 18 months after the last enrolled pt’s first visit (LPFV). Interim analyses will be performed annually; an effectiveness interim analysis is scheduled at 20 months post-LPFV. Analyses will be performed using descriptive statistics. Survival data will be summarized using the Kaplan-Meier method. Subgroup analyses for effectiveness and safety may be performed, on the basis of gender, age, or baseline Eastern Cooperative Oncology Group performance status. Enrollment is planned to start by June 2022.

Taking the registry as the basis, upon study finalization a complementary comparative analysis of effectiveness and safety data from the TAG registry with a retrospective clinical cohort will be undertaken using appropriate methodology, such as propensity score matching or inverse probability treatment weighting.

Results: This is a TiP and there are no results at this time.

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Summary/Conclusion: This is a TiP and there are no results at this time.

P530: TAGRAXOFUSP IN BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM WITH/WITHOUT CENTRAL NERVOUS SYSTEM INVOLVEMENT AND INTRATHECAL CHEMOTHERAPY AS PRIMARY TREATMENT OR PROPHYLAXIS: AN ITALIAN EXPERIENCE.

G. Rivoli 1,* , G. Beltrami 1 , A. Raiola 1 , A. Dominietto 1 , M. Riggi 2 , E. Angelucci 1

1 Hematology and cellular therapy unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy; 2 Stemline Therapeutics Switzerland GmbH, Zug, Switzerland

Background: The precise frequency of central nervous system involvement (CNS+) in patients (pts) with blastic plasmacytoid dendritic cell neoplasm (BPDCN) is currently unknown. Recent small series of clinical experience suggest the incidence to be about 10% at baseline and 30% at first relapse. CNS is a ”sanctuary” for any systemic treatment and can lead to disease recurrence even after the achievement of a complete response (CR) at the medullary and skin level. Thus, it is essential to ensure clearance (or prophylaxis) of the disease including the meningeal compartment. Intrathecal (IT) chemotherapy is a valid complementary tool aimed at eradication of the disease, with modest systemic side effects; it is currently indicated as both prophylaxis and primary treatment. Combining IT chemotherapy with systemic treatment has made it possible to obtain long remissions in pts with BPDCN even in the presence of CNS+. Tagraxofusp (TAG), a first-in-class CD123-targeted therapy, was approved by the European Medicines Agency in 2021 for treatment of BPDCN in first-line adult pts.

Aims: Data from 5 pts with BPDCN with/without CNS+ treated with TAG were retrospectively collected in our center in Geneva. The aim was to evaluate if CNS treatment/prophylaxis impacts prognosis and efficacy of systemic treatment with TAG.

Methods: A diagnosis of BPDCN was confirmed by hematopathology with biomarkers, including CD123, CD4, and CD56, and presence of CNS involvement by the characteristic morphology and “8-color flow cytometry” for CD123. Pts received TAG intravenous infusions at 12 mcg/kg once daily on days 1–5 of a 21-day cycle. TAG was repeated for 1–4 cycles, depending on the level of response and potential bridge to hematopoietic stem cell transplant (HSCT). Hospitalization was mandatory for the first cycle; the following cycles were administered in an outpatient setting if no severe complications occurred during the first cycle. IT chemotherapy (methotrexate 12.5 mg, dexamethasone 4 mg, and cytarabine 50 mg) was administered at each cycle at the same doses until negative cerebrospinal fluid (CSF) was observed in CNS+ pts. After 4 treatment cycles, response was evaluated, including CSF examination.

Results: TAG was administered in 5 first-line pts with BPDCN; 3 were CNS+ (1 clinically evident case and 2 with cytofluorometric positivity only). All pts received IT chemotherapy, both as a primary-intention treatment in the 3 CNS+ pts, and as prophylaxis in the 2 CNS-negative pts. Table 1 reports the main pt characteristics. Pts 2, 3, and 5 with CNS+ achieved clearance of the disease and none reported CNS relapse. In pts receiving CNS prophylaxis, pt 1 received TAG plus HSCT and is in CR after 29 months; pt 4 received high-dose chemotherapy, achieved a good partial remission after TAG, and remains alive after 4 months with HSCT shortly planned. No pts developed CNS adverse events following IT chemotherapy.

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Summary/Conclusion: These preliminary results confirm the feasibility of IT chemotherapy with systemic TAG. Baseline disease and CNS involvement did not appear to predispose pts to different efficacy results or treatment-related adverse events. IT chemotherapy effectively cleared and controlled CNS involvement. IT prophylaxis should be considered in pts with BPDCN who receive CD123-targeted therapies.

P531: AN OBSERVATIONAL, MULTICENTER, RETROSPECTIVE ANALYSIS OF PATIENTS WITH BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM TREATED WITH TAGRAXOFUSP IN THE EUROPEAN EXPANDED ACCESS PROGRAM

M. Herling 1,* , E. Deconinck 2 , M. Anant 3 , D. Manteigas 4 , M. Riggi 3 , E. Angelucci 5

1 Department of Hematology, Cell Therapy and Hemostaseology, University Hospital Leipzig, Leipzig, Germany; 2 Regional University Hospital of Besançon, Besançon, France; 3 Stemline Therapeutics Switzerland GmbH, Zug, Switzerland; 4 Cmed, West Sussex, United Kingdom; 5 Hematology and Cellular Therapy Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy

Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare aggressive hematologic malignancy, with poor prognosis (median age 67 yr). It is characterized by clonal expansion of plasmacytoid dendritic tumor cells expressing specific markers including the interleukin-3 receptor alpha (CD123). Primary sites are skin and bone marrow, followed by peripheral blood, lymph nodes, viscera, and central nervous system. Tagraxofusp (TAG), a CD123-targeted therapy, was approved by the European Medicines Agency on 07.01.2021. In 08.2019, a Global Expanded Access Program (EAP) was implemented to provide access to patients (pts) prior to regulatory authorization of TAG in real-world practice.

Aims: We conducted a European multicenter non-interventional, retrospective analysis of BPDCN pts treated with TAG. Main objectives were rates of complete response, and incidence and severity of capillary leak syndrome (CLS). Secondary outcomes included rate of pts bridged to stem cell transplantation, progression-free survival, and overall survival, safety of TAG measured by the incidence and severity of adverse events (AEs), and number of TAG doses administered in each cycle.

Methods: The main inclusion criterion was diagnosis of BPDCN, confirmed by hematopathology with established marker panels (including CD123). The physician who signed the supply form of TAG in each participating center informed the pts on their treatment. Training of the physicians, nurses, and pharmacists was mandatory before delivering the treatment. The analysis included all pts enrolled in the European EAP from 08.2019 to 12.2021. Pts received TAG intravenous infusions, at 12 mcg/kg once daily on days 1–5 (up to day 10 allowed) of a 21-day cycle. Hospitalization was required only for the first cycle (subsequent cycles were allowed to be administered in an outpatient setting).

Results: Overall, 76 adult (median age 64 yr, range 21–85 yr) and 4 pediatric pts (1, 4, 14, and 16 yr) were included across 57 European centers: Germany 18 centers, France 17, Italy 9, Switzerland 5, United Kingdom 4, Spain 3, and Austria 1. Most pts were male (78%), representing real-world distribution. Sixty-three pts received first-line TAG and 17 pts as second or further line of treatment. The median number of cycles (based on the number of treatments delivered for the TAG EAP supply of each 21-day cycle) was 2.5 (range 1–8) in first line and 2.6 (range 1–13) cycles in second-line and further pts, respectively. No deaths due to CLS were reported. The analysis is still ongoing at the time of abstract finalization; complementary data on safety, efficacy, number of pts transplanted, and time-related parameters will be reported at the meeting.

Summary/Conclusion: The is the largest retrospective analysis of real-world clinical practice outside of a clinical trial in BPDCN pts treated with TAG. The EAP was carefully followed in all 57 centers with initial training provided to the multidisciplinary teams before treatment initiation. This is thought to have positively affected prevention and management of CLS and other grade 3–4 AEs; no death related to CLS occurred. The preliminary results confirm the feasibility and safety of TAG, allowing the administration also in elderly pts, with manageable safety. In fact, AEs mainly occurred in cycle 1 in an inpatient setting, with similar rates and severity in older vs younger pts. Baseline characteristics did not appear to predispose pts to different treatment-related AEs.

P532: ACUTE MYELOID LEUKEMIA (AML): UNICENTRIC REPORT ON 1029 PATIENTS DIAGNOSED IN TERTIAL REFERAL CENTER

A. Fricke 1,* , K. Nachtkamp 1 , K. Döhner 2 , B. Hildebrandt 3 , B. Betz 3 , M. Rudelius 4 , M. Seidl 5 , C. Zahner 1 , G. Kobbe 1 , A. Kündgen 1 , P. S. Jäger 1 , B.-N. Baermann 1 , U. Germing 1

1 Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Düsseldorf; 2 Department of Internal Medicine III, University Hospital Ulm, Ulm; 3 Institute of Human Genetics, University Düsseldorf, Düsseldorf; 4 Pathological Institute, Ludwig-Maximilians-University Hospital Munich, Munich; 5 Pathological Institute, University Hospital Düsseldorf, Düsseldorf, Germany

Background: Since 2016 patients with AML can be diagnosed according to the WHO classification proposals. The ELN genetic risk classification is a tool for treatment planning.

Aims: In order to validate the WHO and the ELN classification, we collected data from 1029 patients diagnosed and / or treated in our institution from 2005 to 2021.

Methods: Patients were followed up until December 2021. Diagnoses were based on cytomorphology, histomorphology, flow cytometry, karyotyping, and molecular genetics performed at the University of Düsseldorf and partly at the University of Ulm. Treatment intensity was defined as follows: no treatment at all, best supportive care (BSC) (including low dose Ara-C and Hydroxyurea), Hypomethylating agents (HMA), induction and consolidation, or allogeneic hematopoietic stem cell transplantation (HSCT).

Results: Median age at diagnosis was 64 years (ys) (19 – 94). 46% were females. There were 270 patients with recurrent genetic abnormalities (26.5%), 346 with AML-MRC (34%), 121 with tAML (11.9%), 243 with AML-NOS (23.9%), 3 with myeloid sarcoma (0.3%), 4 with blastic plasmacytoid dendritic neoplasm (0.4%), 20 with biphenotypic AML (2%), and in 22 patients the classification was not possible.

17.7% of patients belong to the good risk category of ELN classification, 21.6% to the intermediate risk category and 30% to the adverse risk group. In 30.7%, the classification was not possible due to missing data.

274 patients were treated with HSCT as most intensive therapy (28%), 348 patients with induction and consolidation (35.6%). 209 patients received HMA (21.4%). 127 patients were treated with BSC (13%) and in 20 patients (2%) no therapy was administered. In 51 patients, AML was diagnosed at our laboratory, but treatment took place outside of our department.

The median survival time of the entire group was 18 months with 39% of the patients alive by the end of 2021. Median survival was 21 months in females as compared to 16 months in males (p = 0.04). Patients aged over 65 ys had a median survival of 7 months as compared to 134 months in patients aged less than 65 ys (p < 0.0005). There was no difference in overall survival in patients younger than 50 ys of age.

Patients with recurrent genetic abnormalities did not reach median survival whereas median survival of patients with AML-MRC, tAML, and NOS was 11, 12, and 14 month (p < 0.0005). The latter three categories did not differ in terms of median survival (p = 0.23). Within the group of AML with recurrent genetic abnormalities, only the patients with t(6;9) and inv(3) reached median survival whereas all other categories had a favorable outcome.

Patients of the good risk ELN group did not reach the median survival time whereas median survival of the intermediate and adverse risk groups was 27 and 12 months (p < 0.0005). Patients who underwent HSCT did not reach median survival. Patients treated with induction and consolidation had a median survival time of 123 months compared to 8 months median survival in patients who got HMA (p < 0.0005).

In a multivariate analysis including intensity of therapy, age at diagnosis, and ELN group, only intensity of treatment and ELN group were independently influencing overall survival.

Summary/Conclusion: The WHO and the ELN classification could be assessed correctly in about 70% of patients only, primarily due to the missing examination of somatic mutations. The ELN classification together with the treatment intensity have major impact on outcome. Patients who neither receive induction and consolidation nor HSCT are facing an unfavorable outcome, even if they are treated with HMA.

P533: SINGLE-CENTER PHASE 2 STUDY OF PD-1 INHIBITOR COMBINED WITH DNA DEMETHYLATION AGENT + CAG REGIMEN IN PATIENTS WITH HIGH-RISK ACUTE MYELOID LEUKEMIA: INTERIM ANALYSIS

X.-N. Gao 1,* , Y.-F. Su 1 , Y. Jing 2 , J. Wang 1 , L. Xu 1 , L.-L. Zhang 2 , A. Wang 1 , Y.-Z. Wang 1 , Y.-F. Li 2 , D.-H. Liu 1

1 Senior Department of Hematology, the Fifth Medical Center; 2 Department of Hematology, the First Medical Center, Chinese PLA General Hospital, Beijing, China

Background: The expression of PD-L1 is increased in acute myeloid leukemia (AML) cells. However, blocking the immune checkpoint alone has limited efficacy as a single agent in highly proliferative leukemia cells. Here we report the results of an interim analysis of an ongoing single-arm phase 2 trial (no. NCT04541277 ) of PD-1 inhibitor combined with DNA demethylation agent + CAG regimen in patients with high-risk AML.

Aims: To assess safety and response to Tislelizumab + Azacytidine/Decitabine + CAG regimen after minimum 1 cycle of therapy in patients who failed at least one prior induction course of therapy or developed relapse (Cohort 1), and patients with persistent minimal residual disease (MRD)-positivity after consolidation treatment (Cohort 2).

Methods: Patients must have failed at least one prior induction course of therapy or have a relapse or have a persistent MRD-positive disease after consolidation treatment. Other eligibility criteria included in ECOG performance status ≤ 2, normal organ function, and no autoimmune diseases requiring systemic immunosuppression. The patients received Azacytidine 75 mg/m 2 subcutaneously daily, day 1-7 or Decitabine 20 mg/m 2 intravenously daily, day 1-5 plus CAG regimen (cytarabine 100 mg intravenously every 12 hours, day 1-5; aclarubicin 20 mg intravenously daily, day 1-5; and concurrent use of G-CSF 5 mg/kg/day subcutaneously) with Tislelizumab 200 mg beginning on the next day after chemotherapy was stopped and every 4 weeks thereafter. This study was approved by the Ethics Committee of the Chinese PLA General Hospital, and signed informed consents were obtained from all patients.

Results: Efficacy: Fifteen patients has been enrolled (Table 1). Fourteen (93.3%) are evaluable for response, 7 (46.7%) completed 2 cycles and 8 (53.3%) completed 1 cycle: Of the 12 patients in the previous treatment failure cohort, 7 achieved complete remission (CR)/CR with incomplete hematologic recovery (CRi) (1/7) (Table 1B), 3 partial remission (PR), and 2 non-remission. Of the 2 patients in the persistent MRD-positivity after consolidation treatment cohort, 1 achieved MRD negativity and 1 had no response. The 8-week mortality were 18.8%: 2 patients died of rapidly progressive disease concomitant with lung infection at 34 days and 56 days after treatment, respectively. One died of relapse of disease at +4 months after received HLA-haploidentical peripheral blood stem cell transplantation (PBSCT). One died of acute graft-versus-host disease of the gastrointestinal tract at +35 days after received a HLA-haploidentical PBSCT in NR status at transplant. With a median follow-up of 5.4 months (1.1 -12.7), the median overall survival (OS) is not reach with an estimated 1-yr OS of 59.6% (95%CI, 17.8%-85.6%). The median OS for responders (CR/CRi+PR) is not reach vs . 1.9 months for non-responders ( p =0.083). The median event-free survival (EFS) is 6.5 months with an estimated 1-yr EFS of 58.3% (95%CI, 7.7%-89.3%).

Toxicity: Grade 2 immune-related adverse events (IRAEs) were observed in 2 (13.3%) patients, included in 1 patient with grade 2 skin rash which resolved with steroids, and the other with grade 2 peripheral sensory neuropathy. All patients developed treatment-related grade 3 or 4 hematologic toxicities. Two patients died of lung infection at 34 days and 56 days after treatment in the setting of PD.

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Summary/Conclusion: Tislelizumab + Azacytidine/Decitabine + CAG regimen is safe and effective for high-risk AML patients. IRAEs are mild, low-grade. Special attention needs to be paid to patients who have an increased risk of developing graft-versus-host disease.

P534: AZACITIDINE PLUS VENETOCLAX FOR THE TREATMENT OF RELAPSED AND FIRST-LINE AML PATIENTS

S. Garciaz 1,* , M.-A. Hospital 1 , A.-S. Alary 2 , C. Saillard 1 , Y. Hicheri 1 , B. Mohty 1 , J. Rey 1 , E. D’incan 1 , A. Charbonnier 1 , V. Ferdinand 1 , V. Maisano 1 , L. Lombardi 1 , A. Ittel 3 , M.-J. Mozziconacci 3 , V. Gelsi-Boyer 3 , N. Vey 1

1 Hematology; 2 Molecular biology; 3 Biopathology, Institut Paoli-Calmettes, MARSEILLE, France

Background: Venetoclax (VEN) belongs to a novel BH3-mimetic class of small molecules that selectively targets BCL-2, activating the apoptosis effectors BAX and BAK to drive mitochondrial outer membrane permeabilization, cytochrome c release and cell death. Combination of VEN and the hypomethylating agent azacitidine (AZA) has deeply changed the paradigm of treatment of newly diagnosed (ND) AML patients ineligible for high dose chemotherapy because of older age or comorbidities. There is scarce evidence for the utilization of VEN-AZA for relapsed or refractory (R/R) AML, a category of patients classically associated with an extremely poor outcome.

Aims: The objective of our study was to describe a R/R AML cohorts of AML patients treated with VEN-AZA in our institution and to compare the clinical and molecular characteristics predicting response in R/R AML versus ND AML

Methods: This retrospective study included consecutive patients treated with VEN-AZA for R/R AML and ND AML. Patients received AZA at standard dose of 75 mg/m 2 QD for seven days and VEN was administrated either at 400 mg or at 100 mg when associated with strong CYP3P450 A3 inhibitors after three days ramp up. Response was determined using the ELN 2017 criteria. The ORR was defined as the combination of complete response (CR), CR with incomplete hematologic recovery (CRi), and morphologic leukemia-free state (MLFS).

Results: We compared the outcome of 39 R/R AML and 38 concomitant ND AML patients treated in our institution between Jan. 2020 and Dec. 2021. The median age was 69 (22-86) and 73 (61-81) in the R/R and ND groups, respectively. Thirty-five percent of patients had MRC-AML. Adverse cytogenetics was found in 36% of patients in the R/R group and 59% of patients in the ND group. Most frequent mutations were ASXL1, RUNX1, TET2, IDH1/2 and TP53 found in 33%, 33%, 28%, 24% and 22% of patients, respectively. Overall response rate was lower in R/R AML (37% versus 56%) including 13% CR, 8% CRi, 3% PR and 13% MLFS in the R/R AML group and 32% CR, 13% CRi and 13% MLFS in the ND AML group. Adverse cytogenetics was associated with treatment failure only in the R/R group (Relative Risk=0.10, p=0.005). ASXL1, IDH1/2 and SFSR2 mutations were associated with a trend in a higher response rate in the R/R group. Median overall survival (OS) were 5.9 months in the R/R group and 9.4 months in the ND group. In the R/R group, median OS were 2.2 months in the adverse cytogenetics group versus 8.7 months in the intermediate cytogenetics group (p=0.02). Median leukemia-free survival of responding patient was not different between the two groups (9 months), indicating that VEN-AZA can be efficient as a salvage treatment for selected R/R AML patients.

Summary/Conclusion: We described one of the largest series of R/R AML patients treated wiht VEN-AZA. By a doing a direct comparison between R/R AML and ND AML treated concomitantly, we found that adverse cytogenetics was associated with treatment failure only in the R/R group suggesting that this subgroup of patients should not be treated with VEN-AZA. Further analyses including more patients are needed to determine which subgroup may benefit from the VEN-AZA as a salvage treatment.

P535: UPDATES FROM ITALIAN MULTICENTER REAL-LIFE EXPERIENCE ON CPX-351 THERAPY IN YOUNG PATIENTS (<60 YEARS OLD).

B. Garibaldi 1,* , M. Franciosa 2 , F. Pilo 3 , D. Menotti 4 , V. Cardinali 5 , L. Brunetti 4 , E. A. Martino 6 , E. Vigna 6 , I. Tanasi 7 , A. Duminuco 1 , C. Maugeri 8 , M. S. Parisi 8 , P. F. Fiumara 8 , E. Mauro 8 , M. Gentile 6 , M. P. Martelli 5 , D. Capelli 4 , C. Romani 3 , S. Galimberti 2 , G. A. Palumbo 1,8 , F. Di Raimondo 8 , C. Vetro 8

1 Postgraduate School of Hematology, University of Catania, Catania; 2 Division of Haematology, A.O.U. Pisana, U.O. Ematologia, Pisa; 3 U.O.C. Ematologia e Trapianto di cellule staminali emopoietiche, A.O. Brotzu, Cagliari; 4 SOD clinica ematologica, Ospedale Umberto I di Ancona, Ancona; 5 Division of Haematology, A.O. S. Maria della Misericordia, Perugia; 6 Division of Haematology, A.O. di Cosenza, Cosenza; 7 Division of Haematology, A.O.U. Policlinico ‘G.B. Rossi’, Verona; 8 Division of Haematology, A.O.U. Policlinico “G.Rodolico” - S.Marco, Catania, Italy

Background: CPX-351 has been indicated as a valid treatment approach in acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) and therapy-related AML (t-AML). Data on young patients are limited.

Aims: Explore CPX-351 efficacy in younger patients (pts) (<60 years old) in real-life.

Methods: Since September 2019 we treated 32 pts from 7 Italian centres with CPX-351. Median age was 55 (range 32-59) and ECOG range 0-2. Our cohort consisted in 6 pts with t-AML and 26 pts with MRC-AML (10 morphologic, 11 secondary to MDS, 3 secondary to MDS/MPN, 6 with MRC cytogenetics features including 2 secondary to MDS and 2 morphologic AML-MRC). 2 pts (both with AML-MRC) harboured FLT3 -ITD and 1 patient FLT3 -TKD, 4 pts IDH1 and 3 pts TP53 mutations. NPM1 was negative in all pts. 10 pts had complex karyotype (CK) and 5 pts received prior treatment with hypomethylating agents for MDS.

Results: All pts underwent induction therapy with intravenous administration of CPX-351 at day 1, 3 and 5 except 1 patient who received two doses due to pneumonia. 2 pts (6%) died during induction. Most frequent complication was febrile neutropenia (46%). At disease re-evaluation, we obtained 16 (50%) Complete Remission (CR), 5 (16%) CR with incomplete hematological recovery (CRi), 1 (3%) Morphological Leukemia Free State (MLFS), 4 (12,5%) Partial Remission (after reinduction converted to 2 CR, 1 CRi, while 1 patient was refractory) and 4 (12,5%) refractory pts who switched to salvage therapies except for one who died due to disease progression. Overall response rate (ORR) was 68% after induction and 78% after reinduction. All 25 responding pts underwent consolidation, except one that proceeded directly to allogenic stem cell transplantation (HSCT). 16 out 25 responding pts (64%) underwent HSCT. CR rate in patient with CK was 60%. At induction, median days of severe neutropenia (defined as neutrophils ≤ 500/uL) and thrombocytopenia (defined as platelets ​​≤20.000/uL) were respectively 26 and 22. Regarding subgroup analysis by driving mutations, the 4 IDH1 pts obtained a CR and 3 of them received HSCT. 2 out of 3 TP53 pts were refractory and the other one obtained a CR followed by HSCT. About the 2 FLT3 -ITD pts, one died during induction and the other one obtained CR after reinduction and then underwent HSCT. After a median follow-up of 19 months (mo), 6 pts out of the 25 who reached CR/CRi (24%) relapsed and 20 out of 32 (63%) pts are alive. Median relapse-free survival (RFS) for responding pts was not reached. RFS was inferior for t-AML compared to AML-MRC (7 mo vs not reached median, p=0.032). RFS was negatively influenced by CK (median RFS 7 mo for CK vs not reached if no CK, p=0.009) and performance status (PS) (median RFS not reached for PS 0 vs 15 months for PS 1 vs 7 mo for PS 2, p=0.017). Median overall survival (OS) was 20 mo. Pts responding to induction did not reach median OS, while refractory pts had a median OS of 9 months. Noteworthy, OS was not influenced by cytogenetic risk, subtype of AML (if MRC or t-AML) or mutational status. Pts undergoing HSCT at first CR, did not reach median OS, while not transplanted pts had a median OS of 17 mo. COX multivariate analysis showed that reaching CR was the only significative parameter for OS (HR 8, 95% CI 1.18-54).

Summary/Conclusion: CPX-351 is active in young pts with t-AML and AML-MRC with an ORR higher than that reported in the pivotal study (78% vs 47%) and high rate of bridge to transplant at first remission (56%) also considering the different age range. CK and PS 1-2 negatively impacted RFS. OS was influenced by response to induction and HSCT.

P536: VALUE OF MEASURABLE RESIDUAL DISEASE BY MULTIPARAMETRIC FLOW CYTOMETRY IN NON-PROMYELOCYTIC ACUTE MYELOID LEUKEMIA. REAL WORLD EVIDENCE

A. D. Gimenez Conca 1,* , J. Gonzalez 2 , M. M. Rivas 3 , A. Navickas 4 , I. Fernandez 5 , I. Rey 6 , H. Dick 7 , S. Cranco 8 , M. Moirano 9 , L. Ferrari 5 , M. Clavijo 10 , A. Suero 11 , R. Ramirez 12 , A. L. Basquiera 13 , N. Carnelutto 14 , M. L. Rapan 15 , A. Jorge Alberto 1 , C. Belli 16

1 Hospital Italiano de Buenos Aires; 2 Hospital Durand, Buenos Aires; 3 Hospital Austral, Pilar; 4 Hospital El Cruce, Florencio Varela; 5 Fundaleu; 6 Hospital Ramos Mejia, Buenos Aires; 7 Hospital Italiano de La Plata, La Plata; 8 Instituto Alexander Fleming, Buenos Aires; 9 Hospital San Martin, La Plata; 10 Hospital Aleman; 11 Hospital Cesar Milstein, Buenos Aires; 12 Instituto Conciencia, Neuquen; 13 Hospital Privado, Cordoba; 14 Hospital de Clínicas; 15 Sanatorio Sagrado Corazon; 16 Academia Nacional de Medicina, Buenos Aires, Argentina

Background: Measurable residual disease (MRD) allows recognition of a group of patients with acute myeloid leukemia (AML) with a higher risk of relapse. There are, however, remaining open questions, since clinical studies applied variable techniques, cutoff values and time points. Multiparameter flow cytometry (MFC) is the most accessible method in Argentina, since real time PCR assessment is not available for the majority of the institutions. We designed, therefore, this real-life study using decentralized assessment of MRD by MFC to solve the role of positive MRD at different times during treatment with standard chemotherapy.

Aims: Our aim was to assess the impact of positive MRD at the end of induction and after first consolidation (C1) as a prognostic factor for relapse-free survival (RFS) and overall survival (OS) in patients with non-promyelocytic AML.

Methods: The registry of the Argentinian Group of Acute Leukemia (GALA) from the Argentine Society of Hematology (SAH) includes data from 20 centers. We retrospectively selected an analytical cohort with all patients with AML, treated with an intensive regimen and in complete remission (CR) after induction, from January 2010 to March 2021. The OS and RFS were estimated with Kaplan-Meier method and its comparison was evaluated by log-rank, censoring patients at the time of bone marrow transplantation (BMT). The cut-off point for MRD analysis was 0.1% evaluated at the end of induction and after C1. A subanalysis of patients with unknown and intermediate cytogenetic risk (CRM modified according to available molecular data of FLT3, NPM1 and CEBPA) was performed.

Results: Of the 1,160 patients enrolled in the registry, 493 met the inclusion criteria and 20 were excluded due to lack of MRD data at the end of induction. Among the 473 patients analyzed: 238 were women, the median age was 49.5 years [IQR 36.6-59.7]), 184 (38.9%) were intermediate risk and 51 (10.8%) with not evaluable cytogenetic and molecular data. With a median follow-up of 18.9 months, 158 (33.4%) relapsed and 130 (27.5%) died.

The median (Me) OS for patients with post induction negative MRD (MRDneg) was 61.2 (CI95% not evaluable) versus 25.9 months (CI95% 2.5-49.2) with positive MRDpos, p =0.03. The RFS were 20.4 (CI95 8-32.8) and 12.4 (CI95% 6.5-18.3), p=0.06, for MRDneg and MRDpos respectively.

Regarding the evaluation of MRD post C1, the Me OS was not reached for patients with MRDneg, while it was 22.1 months (CI95% 9.3-34.8) among patients with MRDpos, p=0.03. Their respective RFS were 30.3 (CI95% not evaluable) and 14.4 months (CI95% 0-30.7), p=0.03, respectively.

In the subanalysis carried out in the group with intermediate or unknown cytogenetic risk post C1, the Me OS was not reached in MRDneg versus 17.5 months (CI95% 9.3-25.7) in MRDpos, p=0.007. In addition, a median RFS of 20.4 months (CI95% 0.7-40.1) vs 7.4 (CI95% 3.9-10.9), p=0.003, among those with MRDneg and MRDpos, respectively.

Summary/Conclusion: The assessment of MRD by MFC showed predictive power at the different moments of follow-up evaluated. It was more evident after consolidation, with statistically significant impact in OS and RFS. The post-consolidation MRD subanalysis in the group with IR or non-evaluable cytogenetics showed promising results. This real-practice data is consistent with those published recently. Post-C1 MRD could be a useful tool in clinical practice deciding the best post-induction strategy in intermediate-risk patients. A further detailed study is required to confirm our preliminary results.

P537: SURVIVAL AFTER ALLOGENEIC TRANSPLANTATION FOR ACUTE MYELOID LEUKEMIA IN ADULTS IN DENMARK FROM 2000 TO 2020: A POPULATION-BASED COHORT STUDY

L. K. Gjærde 1,2,* , L. H. Jakobsen 3,4 , C. Juhl-Christensen 5 , G. Olesen 5 , I. Petruskevicius 5 , M. T. Severinsen 3 , C. W. Marcher 6 , K. Theilgaard-Mönch 1 , N. S. Andersen 1 , L. S. Friis 1 , B. Kornblit 1 , S. L. Petersen 1 , I. Schjødt 1 , H. Sengeløv 1,2

1 Department of Hematology, Rigshospitalet; 2 Department of Clinical Medicine, University of Copenhagen, Copenhagen; 3 Department of Hematology, Aalborg University Hospital; 4 Department of Mathematical Sciences, Aalborg University, Aalborg; 5 Department of Hematology, Aarhus University Hospital, Aarhus; 6 Department of Hematology, Odense University Hospital, Odense, Denmark

Background: Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment of acute myeloid leukemia (AML), but survival is challenged by the risk of relapse and treatment-related/non-relapse mortality (NRM). In the recent two decades, older patients (>50 years of age) and patients with comorbidities have been able to receive HCT due to the introduction of non-myeloablative conditioning in Denmark in 2001.

Aims: We aimed to investigate trends in overall survival (OS) and other HCT-related outcomes in a population-based cohort study of all adults (≥18 years) who received a first HCT for AML in Denmark between 2000–2020.

Methods: Indications for HCT comprised intermediate- or adverse-risk (including secondary/treatment-related) AML in 1 st complete remission (CR) and all patients in ≥2 nd CR. The total effect of calendar year of HCT on transplant outcomes was tested in a Cox model for OS and Fine-Gray competing risks models for NRM, relapse, grade II–IV acute graft-versus-host disease (GvHD) and chronic GvHD. Time-specific OS estimates and cumulative incidences of HCT outcomes were derived using the pseudo-observation framework. Cure fractions (the fraction of patients who experienced no excess mortality compared to the Danish general population matched on calendar year, age and sex) were estimated in a mixture cure model.

Results: From 2000 to 2020, 659 adults received a first HCT for AML (95% de novo AML; 5% secondary/therapy-related AML) in Denmark. Median (min–max) age at HCT was 56 (19–74) years, going from 44 years in 2000–2005 to 59 years in 2016–2020. 63% of patients were transplanted in 1 st CR, 35% in ≥2 nd CR, and 2% after primary induction failure. Non-myeloablative conditioning was performed in 60% of patients, going from 26% in 2000–2005 to 65% in 2016–2020. Median follow-up was 7.5 years. Main causes of death during follow-up were relapse (56%), infection (11%), and organ failure (10%). OS at 2- and 5-years for all patients was 66% (95% CI: 63–70%) and 57% (95% CI: 53–61%), respectively. OS differed over time (p = 0.02), decreasing from 2000 to 2010 from when it increased until 2020 (Figure Panel A). This change was mainly driven by corresponding changes in NRM over time (p < 0.001, Figure Panel B) rather than significant increases in the relapse rate over time (p = 0.16, Figure Panel C). Adjusting for age did not influence the trends notably. For acute GvHD, the risk decreased over time (p = 0.004), ranging from a 1-year cumulative incidence of 42% (95% CI: 26–62%) in 2000 to 26% (95% CI: 19–34%) in 2020, whereas the risk of chronic GvHD remained stable (p = 0.49), with a 2-year cumulative incidence of 49% (95% CI: 45–53%) in the full cohort. Cure fractions differed over time (p = 0.05) decreasing from 63% (95% CI: 41–78%) in 2000 to 46% (95% CI: 38–54%) in 2010, and increasing again to 68% (95% CI: 52–80%) in 2020.

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Summary/Conclusion: Survival after HCT for AML in adults in Denmark decreased in the first decade after introducing non-myeloablative conditioning regimens for older or comorbid patients, mainly because of higher NRM, but survival improved again in the later decade. The risk of acute, but not chronic, GvHD decreased over time. The latest improvements in peri-HCT supportive care and post-HCT maintenance treatments may further improve survival in the coming decade.

P538: TREATMENT PATTERNS AND CLINICAL OUTCOMES IN ACUTE PROMYELOCYTIC LEUKEMIA: REAL-WORLD DATA

V. B. Goli 1,* , H. Jain 1 , L. Nayak 1 , J. Thorat 1 , B. Bagal 1 , A. Rajendra 1 , S. P G 2 , D. Shetty 3 , H. Jain 3 , P. Tembhare 2 , N. Patkar 2 , M. Sengar 1

1 Medical Oncology; 2 Pathology; 3 Cytogenetics, TATA MEMORIAL HOSPITAL, MUMBAI, India

Background: All-trans retinoid acid (ATRA) and arsenic trioxide (ATO) have changed the treatment paradigm of acute promyelocytic leukemia (APML). Current therapeutic strategies are based on the risk stratification at diagnosis and molecular response at the end of consolidation.We report our experience of treatment patterns and clinical outcomes of newly diagnosed APML patients.

Aims: The primary objective of our study was to evaluate event-free survival in all risk categories of APML.Secondary objectives include disease-free survival, overall survival, rates of complete remission (CR) at the end of induction, rates of molecular CR at the end of consolidation.

Methods: We included treatment-naïve APML patients age>14 years who were treated between May 2014 to May 2018.The diagnosis of APML was established by peripheral blood/bone marrow morphology and flow cytometry (FCM) and confirmed by fluorescent in situ hybridization (FISH) and reverse-transcriptase polymerase chain reaction (RT-PCR) for PML-RARA.The details of patients were retrieved from electronic medical records.Patients with low/intermediate risk were treated with ATO-ATRA based induction and consolidation,where as patients with high risk were treated with varying combination of ATO/ATRA/chemotherapy with or without maintenance.

Results: We registered 149 patients during the study period.The median age was 37 years(range 15-72 years, male 56.6%, female-37%).93 patients (62.4%) were stratified as low risk and 56 patients (37.5%) as high risk based on Sanz’s score.The induction therapy for high risk APML was single agent ATO (42.8%),ATO/ATRA/anthracycline (26.7%),ATO/anthracycline (25%),and ATO/ATRA (5%).The consolidation for high risk APML includes ATRA/daunorubicin (50%),ATO/ATRA (26.8%),HIDAC (4%) and APL 2000 in one patient.All low-risk APML patients received ATO-ATRA in induction and consolidation.Anthracycline was added to three patients in induction to decease white cell count.The proportion of deaths that occurred in first week include 6.5% in low risk and 9% in high risk group.The median follow up was 42 months.The hematological CR at the end of induction was 85%.(128/149 patients,expired during induction-14,lost to follow up-7).The rate of end of consolidation PCR negativity was 83%.Differentiation syndrome occurred in 75% of patients.For the overall patient population, the 3-year probability for OS, EFS, DFS was 88.1% (95% C.I, 82.9 93.6), 75.9% (95% C.I, 68.9-83.5), 81.4% (95% C.I, 75-88.4) respectively. The 3-year survival probability for OS, EFS, and DFS for low risk APML was 90% (95% C.I, 84.1-96.4), 80.1% (95% C.I, 72-89.1), 84.5% (95% C.I, 76.9-92.7) respectively whereas for high risk APML was 84.7% (95% C.I, 75.3-95.2), 68.6% (95% C.I, 56.9-82.9), 76.1% (95% C.I-65-89) respectively.The total number of relapses were 11.The median time to relapse for low risk and high risk was 23months(18-32months) and 20months(range 5-39months) respectively.

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Summary/Conclusion: Treatment with ATO and ATRA based regimen are effective in real world setting.The emphasis should be to reduce the early death rate due to bleeding and infectious complications.

P539: PROGNOSTIC RELEVANCE OF MINIMAL RESIDUAL DISEASE IN THERAPY RELATED AND SECONDARY ACUTE MYELOID LEUKEMIA RECEIVING CPX-351 OR FLUDARABINE-BASED INDUCTION.

F. Guolo 1,2,* , P. Minetto 1 , C. Riva 1,2 , F. Parodi 2 , M. Miglino 1,2 , E. Tedone 3 , N. Colombo 3 , E. Carminati 4 , C. Nurra 4 , A. Cagnetta 1 , M. Cea 1,2 , R. M. Lemoli 1,2

1 Clinic of Hematology, Department of Internal Medicine (DiMI), IRCCS Ospedale Policlinico San Martino; 2 University of Genoa; 3 Flow Cytometry Unit, Department of Pathological Anatomy; 4 Molecular Biology Unit, Department of Pathological Anatomy, IRCCS Ospedale Policlinico San Martino, GENOVA, Italy

Background: Minimal residual disease (MRD) assessment retains high prognostic value in Acute Myeloid Leukemia patients (AML) undergoing intensive induction therapy. Widely available MRD techniques include multicolor flow cytometry (MFC), RT-PCR for recurrent genetic lesion and, for patients lacking specific markers, RT-PCR for the pan- leukemic marker WT1. However, most of the data on the prognostic value of MRD come from trials including younger patients treated with conventional 3 + 7 regimen. Furthermore, AML arising from a previous myelodisplastic syndrome (s-AML) and therapy-related AML (t-AML) are usually under-represented in trial involving younger patients. Few data are, therefore, available on the kinetics and the prognostic value of MRD in elderly s- AML and t-AML patients; especially in the context of more modern frontline treatment such as CPX-351.

Aims: We evaluated MRD in a cohort of elderly s-AML or t-AML patients receiving induction therapy either with an age-adjusted fludarabine-containing regimen (FLAI3) or CPX-351, in order to compare the probability of achieving MRD negativity, to assess the prognostic value of MRD and to define the best time-points for MRD assessments.

Methods: A total of 151 elderly (>60 year, median age 68, range 60-77) patients were analyzed in this study. Patients were treated between January 2005 and January 2020 in our Center, either with CPX-351 (n=50) or fludarabine- high dose cytarabine-idarubicin (FLAI), with (n=72) or without (n =29) gemtuzumab-ozogamicin (GO). MRD was analyzed in all patients achieving hematological complete remission (CR) with both MFC and WT1 expression levels. All patients were affected by s-AML or t-AML, defined according to the WHO 2016 criteria.

Results: After induction, CR was achieved in 95 patients (59%). CR rate was 40/50 in patients treated with CPX-351 (80)% significantly higher when compared to patients receiving FLAI (55/101, 54.5%, p<0.05). The addition of GO to FLAI did not increase CR rate. Among CR patients, a total of 51 (53.7%) and 53 patients (55.8%) achieved MRD negativity, with MFC or WT1-based methods, respectively. MFC MRD negativity probability was higher among patients receiving CPX-351 as induction therapy (MFC MRD negativity rate of 26/40, 65% and 25/55, 45% in CR patients who received CPX-351 or FLAI, respectively, p<0.05). Adding GO to FLAI did not improve MRD negativity probability. The most informative timepoint was after first cycle. WT1-based MRD assessment led to similar results. In multivariate analysis, MRD showed significant prognostic value for Overall Survival (OS) in all treatment group (2-year OS of 34% and 77% in patients with or without residual MFC MRD after induction, respectively, p<0.05). Similarly, consolidation with allogeneic stem cell transplantation (HSCT) was correlated with higher OS. Notably, 12/40 (30%) CR patients treated with CPX 351 underwent HSCT.

Summary/Conclusion: In conclusion MRD assessment retains a strong prognostic value also in s-AML and t-AML patients. The evaluation of MRD with both methods lead to similar conclusions and allowed us to obtain MRD data from virtually all AML patients treated in the selected time period. CPX-351 treatment resulted in higher CR rate with deeper MRD responses, if compared to FLAI3 and allowed a significant number of elderly AML patients to proceed to HSCT.

P540: PEVONEDISTAT, AZACITIDINE AND VENETOCLAX FOR PATIENTS WITH RELAPSED/REFRACTORY ACUTE MYELOID LEUKEMIA– A PHASE I STUDY

G. S. Guru Murthy 1,* , S. Kaufmann 2 , A. Saliba 2 , A. Szabo 3 , L. Michaelis 1 , S. Abedin 1 , L. Runaas 1 , K. Carlson 1 , S. Maldonado-Schmidt 4 , A. Hinman 4 , A. Thomas 4 , A. Baim 4 , M. Litzow 2 , E. Atallah 1

1 Hematology-Oncology, Medical College of Wisconsin, Milwaukee; 2 Hematology-Oncology, Mayo Clinic, Rochester; 3 Biostatistics; 4 Clinical trials, Hematology-Oncology, Medical College of Wisconsin, Milwaukee, United States of America

Background: Outcomes of patients with relapsed/refractory acute myeloid leukemia (RR-AML) have remained poor. Venetoclax therapy in RR-AML is associated with lower complete remission (CR) as compared to newly diagnosed AML. Preclinical studies suggest overexpression of anti-apoptotic MCL-1 as a mechanism of BCL-2 inhibitor resistance (Konopleva et al. Cancer Cell 2006). Pevonedistat is a first in class inhibitor of Nedd8 activating enzyme that induces pro-apoptotic NOXA leading to neutralization of MCL-1 and apoptosis. In preclinical AML models, combination of pevonedistat and venetoclax showed synergistic effect (Knorr KL et al. Cell Death Differ. 2015).

Aims: To assess the safety and outcomes of adding pevonedistat to azacitidine and venetoclax in patients with RR-AML.

Methods: This is a phase I multicenter study ( NCT04172844 ) with 3 + 3 design to determine the safety and recommended phase 2 dose (RP2D) of pevonedistat, venetoclax and azacitidine in RR-AML. Patients aged 18 years or above with morphologically documented RR-AML, ECOG performance status 0-2 and adequate organ function were eligible. Exclusion criteria were isolated extramedullary relapse, hematopoietic stem cell transplantation (HSCT) within 100 days of enrollment, and active acute graft versus host disease. Previous therapy with hypomethylating agent (HMA) or venetoclax was not an exclusion. Treatment included azacitidine (75 mg/m 2 daily x 7 days), venetoclax (400 mg daily x 28 days), and pevonedistat in escalating doses (10-20 mg/m 2 IV days 1,3,5 of each cycle) in 28-day cycles. Pevonedistat was given at 10 mg/m 2 dose in cohort 1, 15 mg/m 2 in cohort 2 and 20 mg/m 2 in cohort 3.

Results: Sixteen patients with RR-AML participated in the study (15 evaluable for response). Median age was 73 years (61-91), 37.6% had secondary/therapy related AML, 56.3% received prior venetoclax/HMA and 18.1% had relapse after prior allogeneic HSCT. Most common grade 3 or higher adverse events included neutropenia (44%), thrombocytopenia (38%), febrile neutropenia (25%), anemia (25%), and sepsis (19%). There was 1 dose limiting toxicity (DLT) in cohort 1 (atrial fibrillation), but no subsequent DLT despite planned dose escalation. Pevonedistat 20 mg/m 2 was established as the RP2D. The rate of CR/CRi/morphological leukemia free state (MLFS) was 40% (CR/CRi 33%) for the overall cohort and 85.7% in venetoclax/HMA naïve RR-AML with a median time to response of 1 cycle. Among patients achieving CR/CRi, 60% attained minimal residual disease negativity by flow cytometry.

Correlative studies with BH3 mimetic profiling showed variable baseline sensitivity to BH3 mimetics and sequential western blot assays showed upregulation of PUMA (for two patients in CR) and NOXA (for one patient with MLFS). Higher levels of DNMT1 were seen in patients with CR or MLFS. One patient with CR also had evidence of leukemic stem cell/progenitor cell sensitivity to pevonedistat at 50-100 nM.

Summary/Conclusion: Combining pevonedistat with venetoclax and azacitidine is safe and tolerable in patients with RR-AML. Dose escalation yielded encouraging efficacy with pevonedistat 20 mg/m 2 as the RP2D.

P541: OUTCOMES AND MANAGEMENT OF PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA PRESENTING WITH HYPERLEUKOCYTOSIS

F. Haddad 1,* , K. Sasaki 1 , T. Abuasab 1 , S. Venugopal 1 , D. Rivera Delgado 1 , A. Bazinet 1 , R. Babakhanlou 1 , K. Kim 1 , J. Senapati 1 , F. Ong 1 , S. Desikan 1 , N. Short 1 , N. Pemmaraju 1 , G. Borthakur 1 , C. DiNardo 1 , N. Daver 1 , E. Jabbour 1 , G. Garcia-Manero 1 , F. Ravandi 1 , T. Kadia 1

1 Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Patients with acute myeloid leukemia (AML) presenting with hyperleukocytosis have higher mortality rates and inferior outcomes. Analyzing the factors associated with mortality and survival in AML patients with a white blood cell count (WBC) ≥100 x 10 9 /L can guide management and improve early mortality.

Aims: To analyze the outcomes of patients with AML presenting with hyperleukocytosis and establish a treatment algorithm for the management of those patients.

Methods: In a retrospective analysis, we screened patients who presented to our institution for newly diagnosed AML over the period of 10 years and identified those with WBC ≥100 x 10 9 /L. Logistic regression models estimated odds ratios (OR) for 4-week mortality and Cox proportional hazard models estimated hazard ratios (HR) for overall survival (OS).

Results: We identified 129 patients with newly diagnosed AML and hyperleukocytosis. Median age was 65 years (range, 23-86 years) and 66% of patients had ECOG performance status (PS) <2. Median WBC was 146 x 10 9 /L (range, 100-687) and 78 patients had clinical leukostasis (CL) including renal failure in 31 patients (24%), new onset hypoxia in 29 patients (23%), headache in 19 patients (15%), chest pain in 9 patients (7%) and neurological symptoms in 3 patients (2%). FLT3 and RAS pathway mutations were found in 63% and 27% of patients, respectively. 29 of 129 patients (22%) had poor-risk cytogenetics.

Compared with patients without evidence of CL, those with CL were less likely to have good PS (ECOG PS <2, 58% vs 82%; P = 0.006), had higher 4-week mortality (16% vs 2%; P = 0.015) and 8-week mortality (19% vs 6%; P = 0.038).

Cytoreduction consisted of hydroxyurea in 124 patients (96%), cytarabine in 69 patients (54%) and leukapheresis in 31 patients (24%). Patients who underwent leukapheresis were less likely to receive cytarabine compared with those who did not (35% vs 59%; P = 0.024); and tended to have more CL compared with patients who did not (74% vs 56%; P = 0.093). 30 patients had tumor lysis syndrome (TLS). TLS risk did not increase with WBC and was not associated with the cytoreductive modality used. 11 patients had intracranial hemorrhage (ICH): 9 patients (82%) with WBC ≥150 x 10 9 /L and 18% with WBC <150 x 10 9 /L (P = 0.048). No association was observed between the incidence of ICH and the cytoreductive therapy.

4-week and 8-week mortality rates were 10% and 14%, respectively. After a median follow-up of 49.4 months (95% CI, 26.2-72.6), median OS was 14.3 months (95% CI, 7-21.6), with 2-year OS of 40%. Median OS was 12.3 months (95% CI, 7.4-17.2) compared with 29 months (95% CI, 2.1-55.6) in patients with or without CL, respectively (P = 0.007). Median OS was 9.9 months (95% CI, 7.5-12.2) and 21.3 months (95% CI, 10.7-31.8) among those who did or did not undergo leukapheresis, respectively (P=0.003). Median OS was 42 months (95% CI, 14.2-69.8) in patients younger than 65 years compared with 8 months (95% CI, 6-10) in those 65 years and older.

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Summary/Conclusion: Older age, poor-risk cytogenetics, TLS and disseminated intravascular coagulation (DIC) were associated with early mortality and inferior OS in patients with hyperleukocytosis. Careful monitoring of those patients with prompt cytoreduction and management of complications may help improve their outcomes.

P542: REAL WORLD OUTCOMES FOR PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA IN BRITISH COLUMBIA (BC): EXCELLENT OUTCOMES WITH LOW EARLY DEATH RATE AND HIGH OVERALL SURVIVAL IN A POPULATION BASED STUDY

R. Henderson 1,* , Y. Eissa 1 , R. Stubbins 1 , Y. Abou Mourad 1 , S. Chung 1 , D. Forrest 1 , K. Hay 1 , F. Kuchenbauer 1 , S. Nantel 1 , T. Nevill 1 , M. Power 1 , J. Rodrigo 1 , C. Roy 1 , D. Sanford 1 , K. Song 1 , H. Sutherland 1 , C. Toze 1 , J. White 1 , S. Narayanan 1

1 Leukaemia/Bone Marrow Transplant Programme, Vancouver General Hospital, Vancouver, Canada

Background: Acute promyelocytic leukaemia (APL) therapy has significantly improved with the use of All trans retinoic acid (ATRA) and Arsenic Trioxide (ATO) for low risk disease (Lo-Coco 2013). Treatment of high risk disease remains more challenging with a higher risk of early mortality & greater concern of relapse. All patients (pts) with APL in British Columbia, Canada are treated by the Leukemia/BMT Program at Vancouver General Hospital, Vancouver. Since 2014, all pts have received ATRA/ATO based therapy based on the Le-Coco study. Pts with high risk disease (WCC > 10 x 10 9 /L at presentation) also receive a 3 day course of doxorubicin (60mg/m2) for induction only.

Aims: We sought to review treatment outcomes since initiating these treatment protocols with emphasis on drug toxicities, dose modifications & management of high risk & elderly patients in order to describe real world outcomes.

Methods: Ethics was approved by the board of the BCCA and UBC. Data was collected by chart reviews, hospital databases & collated on a central file. Demographics, risk stratification, treatment received, adverse events & survival was collected & analysis performed on Microsoft excel v16.57 & SPSS Statistics v28.0.1.1. Kaplan Meier curves were performed for PFS & OS, student t and chi squared tests were performed to assess differences between subgroups.

Results: We identified 67 pts with APL during the study period. 40% were male 60% female, the median age was 61 yrs. (21-83yo). 36(54%) pts were > 60 yrs at diagnosis. The median duration of follow up was 2.9 yrs. All had molecular confirmation of disease. 16(24%) pts had high risk disease at diagnosis. At time of censor overall survival was 97% and PFS 94%. 55(82%) pts had a documented toxicity. Common toxicities included differentiation syndrome (DS) in 27(40%) pts, hepatotoxicity 17 (25%), QTc prolongation 13(19%) & neurological effects 9 (13%). Infections were documented in 40(59%) pts including bacterial infections in 25 patients (37%), culture negative febrile neutropenia in 12 (20%), viral infections in 5 (7%) & fungal infections in 1 (1.5%). Clinically significant bleeding was documented in 10 (15%) of patients, 5 of which were CNS/retinal bleeds.

DS was more common in high risk disease though this difference was not statistically significant (p=0.365). 8(30%) pts with DS needed ICU admission. There were 2 early deaths, in the cohort, both in pts > 75 yo with high risk disease due to multi-organ failure and bleeding within 5 days of presentation.

Dose modifications were assessed in 64 pts (2 deaths & one lost to follow up). 37 (58%) were described as having had a dose modification. 13 (20%) had a reduction in treatment dose, 16(25%) had doses/days of drug omitted. 6(9%) had reductions in dose and omissions. The most common reasons for dose modifications were DS 12(19%), QTc prolongation 10 (15%) & hepatotoxicity 8 (12%). Pts over 60yo were more likely to have a dose modification than pts below 60yo (73% vs 43% p = 0.014).

65 pts diagnosed achieved morphological remission & 64 a molecular remission at the end of planned therapy. 2 relapses occurred in our cohort, 1 had a concomitant cytogenetic abnormality incorporating a Tp53 deletion. The second initially received ATRA only induction due to age & comorbidity.

Summary/Conclusion: This real world, population based data for patients with APL in BC confirms high response rates, survival and relatively low early death rate and acceptable toxicity even in older patients with high risk disease and comorbidities. In our cohort appropriate dose modifications did not impact remission or relapse rates.

P543: TARGETING SAMHD1 WITH HYDROXYUREA IN FIRST-LINE CYTARABINE-BASED THERAPY OF NEWLY DIAGNOSED ACUTE MYELOID LEUKAEMIA: RESULTS FROM THE HEAT-AML TRIAL

M. Jädersten 1,* , I. Lilienthal 2 , N. Tsesmetzis 2 , M. Lourda 2 , S. Bengtzén 3 , A. Bohlin 3 , C. Arnroth 4 , T. Erkers 4 , B. Seashore-Ludlow 4 , G. Giraud 5 , G. S. Barkhordar 6 , S. Tao 7 , L. Fogelstrand 8 , L. Saft 4 , P. Östling 4 , R. Schinazi 7 , B. Kim 7 , T. Schaller 9 , G. Juliusson 10 , S. Deneberg 1 , S. Lehmann 11 , G. Rassidakis 4 , M. Höglund 11 , J.-I. Henter 2 , N. Herold 2

1 Department of Hematology, Karolinska University Hospital; 2 Childhood Cancer Research Unit; 3 Centre for hematology and regenerative medicine; 4 Department of Oncology-Pathology, Karolinska Institutet, Stockholm; 5 Department of Immunology, Uppsala University, Uppsala; 6 Department of Clinical Genetics and Genomics, Sahlgrenska University Hospital, Gothenburg, Sweden; 7 Department of Pediatrics, Emory University, Atlanta, United States of America; 8 Department of Laboratory Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; 9 Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, Germany; 10 Department of Hematology, Skåne University Hospital, Lund; 11 Department of Medical Sciences, Uppsala University, Uppsala, Sweden

Background: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-CTP levels through targeted inhibition of SAMHD1.

Aims: To evaluate feasibility, safety, and efficacy of adding hydroxyurea to standard AML-directed therapy according to national guidelines.

To perform translational studies including SAMHD1-staining on bone marrow sections, pharmacokinetics and drug-sensitivity analysis on leukemic cells ex vivo.

Methods: This phase-1 trial (EudraCT-number: 2018-004050-16) was run at two sites (Karolinska University Hospital and Uppsala University Hospital, Sweden). Eligibility criteria included age >18 years, newly diagnosed non-promyelocytic AML, and fitness for intensive chemotherapy. Patients with CBF-AML eligible for treatment with gemtuzumab-ozogamicin were excluded. Treatment comprised 2 to 4 cycles of ara-C 1000 mg/m 2 i.v. b.i.d. on day 1-5 during all 4 cycles and daunorubicin 60 mg/m 2 i.v. q.d. on day 1-3 during cycles 1 and 2, and on day 1-2 during cycle 3. Patients with FLT3 -mutated AML received midostaurin 50 mg b.i.d. on day 8-21 of each cycle. Risk-adapted allo-HSCT was performed at the discretion of the treating haematologist. The dose of hydroxyurea was escalated in a 3 + 3 design: 500 + 500 mg (level 1), 1000 + 500 mg (level 2), and 1000 + 1000 mg (level 3), each dose being given 1 hour prior to start of the ara-C infusion b.i.d. on day 1-5.

Here we report the results of the first 9 patients in the run-in phase 1 part of the study. The phase 2 part will include an additional 60 patients. Recruitment is ongoing, utilizing the highest dose of hydroxyurea.

Expression of SAMHD1 was assessed using a double-immunostaining method (SAMHD1/CD68), an autostainer system (BenchMark Ultra, Ventana, Rotkreuz, Switzerland) and previously validated protocols 20 . CD68 + /SAMHD1 + histiocytes (macrophages) served as internal controls in all bone marrow biopsies assessed.

Drug sensitivity analysis was performed on AML mononuclear cells utilized a high-throughput system evaluating >500 cytotoxic agents including combinations of ara-C and hydroxyurea in different doses.

Results: All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow-cytometry or RT-qPCR) had an MRD level <0.1% after two cycles of chemotherapy. Six of nine patients underwent hematopoietic stem cell transplantation. With a median follow-up of 13.2 months, no relapse has been observed.

No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in ara-CTP levels (1.5-fold; P =0.04) in the 6 patients receiving single doses of 1000 mg hydroxyurea. Drug-sensitivity analysis indicated an additive effect of ara-C and hydroxyurea on leukemic cells ex vivo . There was no apparent correlation between expression of SAMHD1-expression and efficacy; all patients had deep responses.

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Summary/Conclusion: The high rate of complete remission and MRD negativity together with the pharmacokinetic and ex vivo evidence suggest that the efficacy of cytarabine-based AML treatment can be enhanced by addition of hydroxyurea as a targeted inhibitor of SAMHD1. Importantly, orally administered hydroxyurea may provide a safe, inexpensive, and broadly accessible strategy to improve outcome in AML. These results will have to be validated in a larger patient cohort.

P544: MOLECULAR PREDICTORS OF RESPONSE AND SURVIVAL IN TREATMENT-NAÏVE PATIENTS WITH ACUTE MYELOID LEUKEMIA FOLLOWING VENETOCLAX AND HYPOMETHYLATING AGENTS

I. Johnson 1,* , K. McCullough 2 , F. Farrukh 3 , A. Al-Kali 3 , H. Alkhateeb 3 , K. Begna 3 , A. Mangaonkar 3 , M. Litzow 3 , W. Hogan 3 , M. Shah 3 , M. Patnaik 3 , A. Pardanani 3 , A. Tefferi 3 , N. Gangat 3

1 Department of Medicine; 2 Divison of Hematology; 3 Mayo Clinic, Rochester, United States of America

Background: Venetoclax (Ven) in combination with hypomethylating agents (HMA) is FDA-approved for elderly/unfit AML patients. Limited data exists on molecular predictors of response and survival. In a prior study, response was superior (CR/CRi >80%) with NPM1 , IDH1/2 , DNMT3A mutations, inferior with TP53 , RUNX1 , FLT3/ITD , RAS and prolonged survival with NPM1 and IDH2 mutations (2 yr OS 71.8%/79.5%) ( DiNardo, Blood, 2020 ).

Aims: Our objective was to determine the impact of mutations on response and survival in treatment-naïve AML patients receiving Ven+HMA.

Methods: Treatment-naïve AML patients receiving Ven+HMA outside a clinical trial at the Mayo Clinic were included. Molecular studies were performed by next-generation sequencing. Response was assessed according to the 2017 European LeukemiaNet (ELN) criteria. Standard statistical analyses were performed using JMP Pro (Version 16.0.0).

Results: i) Patient characteristics

103 AML patients (median age 74 years, 67% male, 62% de novo ) received upfront Ven+HMA. ELN cytogenetic risk included favorable (6%, n=6 ), intermediate (50%, n=52 ) or adverse (44%, n=45 ). Mutations involved TP53 in 25 patients (25%), TET2 in 24 (23%), IDH1/IDH2 in 20 (19%), RUNX1 in 19 (19%), ASXL1 in 18 (18%),), SRSF2 in 18 (18%), K/NRAS in 15 (15%), NPM1 in 13 (13%), DNMT3A in 13 (13%), FLT3-ITD in 10 (10%) patients.

ii) Predictors of response

40 (39%) patients achieved complete remission (CR), 20 (19%) CR with incomplete hematological recovery (CRi), resulting in CR/CRi in 60 (58%) patients.

In univariate analysis, presence of ASXL1 mutation was associated with favorable response (CR/CRi 83% vs 53%, p=0.01), secondary AML (CR/CRi 49% vs 65%, p=0.09), adverse karyotype (49% vs 67%, p=0.11), presence of TP53 (32% vs 67%, p=0.002) and FLT3-ITD mutations (30% vs 61%; p=0.06) predicted inferior response. In multivariable analysis, including the aforementioned variables, presence of ASXL1 mutation (83% vs 53%; OR 4.5) and absence of TP53 (67% vs 32%; OR 3.3) and FLT3-ITD mutations predicted favorable response (61% vs 30%; OR 6.4). Moreover, in ASXL1 mutated patients, CR/CRi was not impacted by presence of TP53 mutation (100% vs 81%) whereas in ASXL1 unmutated patients, presence of TP53 mutation predicted inferior response (26% vs 63%; p=0.001). Presence of NPM1 (CR/CRi; 69% vs 57%, p=0.41), IDH1/2 (70% vs 55%; p=0.23), DNMT3A (54% vs 59%; p=0.73), RUNX1 (58% vs 58%; p=0.1), RAS (60% vs 58%, p=0.88) did not impact response.

iii) Predictors of survival

After a median follow up of 6.6 months, 68 patients died and 9 underwent allogeneic stem cell transplant. Median overall survival (mOS) was 8.5 months and longer in transplanted patients (not reached vs 8.4 months, p=0.08).

Age-adjusted analysis in 94 patients not transplanted, identified CR/CRi (p<0.0001), NPM1 (p=0.009), IDH1/2 mutations (p=0.02) as favorable, and TP53 (p=0.01), ASXL1 mutations (p=0.17) adverse karyotype (p=0.05) unfavorable risk factors for survival. Despite higher CR/CRi, ASXL1 mutated patients had shortened mOS due to higher relapse.

Multivariable analysis confirmed the negative survival impact of not achieving CR/CRi, ASXL1 mutation and adverse karyotype. Accordingly, a three-tiered model was generated by using the three variables (Figure 1).

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Summary/Conclusion: Our observations differ from those of DiNardo, et al. ( Blood, 2020 ). The current study identifies presence of ASXL1 mutation and absence of TP53 and FLT3-ITD mutations as predictors of favorable response. Additionally, we propose a novel three-tiered survival model based on response, ASXL1 mutation and karyotype.

P545: CHARACTERISTICS AND OUTCOME OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND TRISOMY 19

S. Kayser 1,2,* , D. Martínez-Cuadrón 3,4 , R. Rodriguez-Veiga 5 , M. Hänel 6 , M. Tormo 7 , K. Schäfer-Eckart 8 , C. Botella 9 , F. Stölzel 10 , T. Bernal del Castillo 11 , U. Keller 12 , C. Rodriguez-Medina 13 , G. Held 14 , M.-L. Amigo 15 , C. Schliemann 16 , M. Colorado 17 , M. Kaufmann 18 , M. Barrios Garcia 19 , S. W. Krause 20 , M. Görner 21 , E. Jost 22 , B. Steffen 23 , A. D. Ho 24 , C. Baldus 25 , H. Serve 26 , U. Platzbecker 1 , C. Müller-Tidow 24 , C. Thiede 27 , M. Bornhäuser 28 , P. Montesinos 4,5 , C. Röllig 10 , R. F. Schlenk 24,29,30

1 Medical Clinic and Policlinic I, Hematology and Cellular Therapy, University Hospital Leipzig, Leipzig; 2 National Center of Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg, Germany; 3 Hematology Department, 3HemaHospital Universitari i Politècnic, La Fe, Valencia; 4 CIBERONC, Instituto Carlos III, Madrid; 5 Hematology Department, Hospital Universitari i Politècnic, La Fe, Valencia, Spain; 6 Klinikum Chemnitz, Chemnitz, Germany; 7 Hematology Department, Hospital Clínico Universitario, INCLIVA Research Institute, University of Valencia, Valencia, Spain; 8 Hospital Nord, Nürnberg, Germany; 9 Hospital General, Alicante, Spain; 10 Department of Medicine I, University Hospital Carl-Gustav-Carus, Dresden, Germany; 11 Hospital Central de Asturias, Asturias, Spain; 12 Department of Hematology, Oncology and Tumor Immunology, Charité-University Medical Center, Campus Benjamin Franklin, Berlin, Germany; 13 Hematology Department, Hospital Universitario de Gran Canaria Doctor Negrín, Las Palmas de Gran Canaria, Spain; 14 Westpfalz Klinikum, Kaiserslautern, Germany; 15 Hospital General Universitario Morales Meseguer, Murcia, Spain; 16 University Hospital Muenster, Münster, Germany; 17 16Hospital Universitario Marqués de Valdecilla, Santander, Spain; 18 Robert Bosch Hospital Stuttgart, Stuttgart, Germany; 19 Department of Hematology, 18DepartmentHospital Regional Universitario de Málaga, Malaga, Spain; 20 Department of Internal Medicine 5 – Hematology/Oncology, 19Department of Internal Medicine 5 – HeUniversity Hospital of Erlangen, Erlangen; 21 Klinik für Hämatologie, Onkologie und Palliativmedizin, Klinikum Bielefeld Mitte, Bielefeld; 22 University Hospital Aachen, Aachen; 23 Department of Internal Medicine II, Department oUniversity Hospital of Frankfurt Main, Frankfurt am Main; 24 Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg; 25 Department of Internal Medicine II, University Hospital of, Kiel; 26 Department of Internal Medicine II, 22Department of InterUniversity Hospital of Frankfurt Main, Frankfurt am Main; 27 Department of Medicine I, 9DepartmUniversity Hospital Carl-Gustav-Carus; 28 Department of Medicine I, 9DepUniversity Hospital Carl-Gustav-Carus, Dresden; 29 NCT Trial Center, National Center of Tumor Diseases, German Cancer Research Center (DKFZ); 30 Department of Medical Oncology, National Center for Tumor Diseases (NCT), Heidelberg University Hospital, Heidelberg, Germany

Background: Trisomy 19 is a recurrent but rare cytogenetic abnormality reported in patients with acute myeloid leukemia (AML). The prognostic significance of this abnormality in AML patients is not clear. Prognosis of AML patients with trisomy 19 seems to be poor as compared to that of patients with intermediate-risk cytogenetics. Allogeneic hematopoietic stem cell transplantation (allo-HCT) may improve survival if applied early in first complete remission (CR).

Aims: To characterize AML patients with trisomy 19 and compare outcomes according to different treatment strategies.

Methods: We retrospectively studied 97 AML patients with trisomy 19 (median age at diagnosis, 57 years; range, 17-83 years) treated between 2001 and 2019 within 2 study groups (SAL & PETHEMA). Standard statistical methods were applied.

Results: Median white blood cell count at diagnosis was 6.7/nl (range, 0.1-151/nl) and platelets 48.5/nl (range, 4-307/nl). Type of AML was de novo in 66 (68%), secondary after myelodysplastic syndrome/ myeloproliferative neoplasm in 16 (16%), and therapy-related in 9 (9%) patients (missing, n=6; 6%). Thirty-five (36%) patients were female. Cytogenetic analysis revealed trisomy 19 as the sole abnormality in 10 (12.5%), additional abnormalities in a non-complex karyotype in 8 (8%) and a complex karyotype in 79 (81.5%) patients. Most frequent additional cytogenetic abnormality was trisomy 8 (n=46); in 17 patients karyotypes were characterized by trisomies only. A total of 65 patients (67%) had NPM1 and FLT3 -ITD mutation testing. Of those, only 3 (5%) and 1 (2%) harbored NPM1 and FLT3 -ITD mutations, respectively. Only 4 (8%) of 51 patients were CEBPA mutated. Ninety-two patients (95%) were treated intensively and 4 (4%) received non-intensive therapy (missing, n=1, 1%). In intensively treated patients early death rate was 10% (n=9); CR was achieved in 52% (n=48) and 35% (n=35) patients were refractory. Factors associated with response to intensive induction therapy were trisomy 19 as sole abnormality or within a karyotype characterized by trisomies only (OR, 5.64; 95%-CI, 1.71-18.63; P=0.005) and age at diagnosis (10 years difference OR, 0.57; 95%-CI, 0.40-0.80; P=0.001). One of 4 patients treated non-intensively achieved a CR. An allo-HCT was performed in 34 (35%) patients, of whom 19 patients were transplanted in first CR after induction therapy. Type of donor was matched-related in 12 and matched-unrelated in 22 patients. Median follow-up of the whole cohort was 6.4 years (95%-CI, 2.91-8.97 years). Five-year overall (OS) and relapse-free survival rates were 20% (95%-CI, 13-31%) and 26% (95%-CI, 16-43%). OS rates were significantly higher in intensively treated patients with trisomy 19 as sole abnormality or within a karyotype characterized by trisomies only (P=0.05). An Andersen-Gill model including allo-HCT as a time dependent covariable on OS revealed as significant parameters trisomy 19 as sole abnormality or within a karyotype characterized by trisomies only (HR, 0.47; 95%-CI, 0.25-0.89; P=0.021) and age at diagnosis (10 years difference; HR, 1.29; 95%-CI, 1.10-1.52; P=0.002), whereas allo-HCT had no beneficial impact (HR, 1.45; 95%-CI, 0.81-2.59; P=0.21).

Summary/Conclusion: Patients with trisomy 19 are very heterogeneous in particular with respect to cytogenetic and molecular abnormalities. In our cohort, patients with trisomy 19 as sole abnormality or within a karyotype characterized by trisomies only had a high CR rate and better clinical outcome. In the cohort of intensively treated patients, allo-HCT did not improve OS.

P546: EFFECTS OF CHEMOTHERAPY DOSE REDUCTIONS IN OVERWEIGHT AND OBESE PATIENTS WITH ACUTE MYELOID LEUKEMIA – A DANISH NATIONWIDE COHORT STUDY

D. Kristensen 1,2,* , L. B. Nielsen 1,2 , L. H. Jakobsen 1,3 , T.-C. C. Kristensen 1 , T. C. El-Galaly 1,2 , A. S. Roug 1,2,4 , M. T. Severinsen 1,2

1 Department of Hematology, Clinical Cancer Research Center, Aalborg University Hospital; 2 Department of Clinical Medicine; 3 Department of Mathematical Sciences, Aalborg University, Aalborg; 4 Department of Hematology, Aarhus University Hospital, Aarhus, Denmark

Background: The majority of chemotherapeutic agents are dosed according to a body weight derived variable. Studies in solid cancers have shown that overweight patients frequently receive dose reduction (DR) of chemotherapy, despite no evidence corroborates increased toxicity of full dosing. Rather, DR to ≤95% of actual weight-based dose, has been shown to result in shortened overall survival (OS). Consequently, the American Society of Clinical Oncology does not recommend up-front dose reduction based on body mass index (BMI) or body surface area (BSA) in overweight patients. Current evidence regarding DR and outcome among overweight patients with acute myeloid leukemia (AML) receiving induction chemotherapy (IC) is limited.

Aims: The purpose of this study was to investigate the association between DR and outcome in overweight patients with AML.

Methods: We utilized the Danish National Acute Leukemia Registry to conduct a retrospective cohort study. Overweight (BMI≥25) AML patients aged 18-75 years and treated with IC between 2000-2012 were included. We defined DR as ≤95% of actual BSA-based chemotherapy dose. Relative risks (RR) for DR, complete remission (CR) rates, and 30- and 90-day mortality were modeled, and OS and relapse-free-survival (RFS) were calculated and compared using the 5-year restricted mean survival time difference (Δ5y-RMST).

Results: The study population included 536 overweight AML-patients of whom 54 patients (10.1%) were categorized as DR (mean reduction 11.2%). Risk factors for DR in univariate analysis were increasing BMI (30-34.9: RR, 2.52 [95% CI, 1.32-4.71]; ≥35: RR, 4.66 [95% CI, 2.37-8.91]), increasing BSA (2.0-2.2: RR 4.61 [95% CI, 1.96-12.6]; ≥2.2: RR 15.21 [95% CI, 6.75-40.67]), therapy-related AML (RR 2.85 [95% CI, 1.12-7.24]) and favorable risk cytogenetics (RR 2.20 [95% CI, 1.02-4.33]). No significant differences were observed for rates of CR, 30- and 90-day mortality between patients receiving DR and non-DR IC. Dose reduction did not affect median RFS (DR, 14.5 [95% CI, 9.0 to 41.7] months; non-DR, 15.0 [12.3 to 19.3]) with an adjusted Δ5y-RMST of 0.2 (-8.4 to 8.8) months nor median OS (DR, 17.0 [11.9-45.5] months; non-DR, 17.5 [14.8-20.5]) with an adjusted Δ5y-RMST of 0.8 (-5.7 to 7.3) months (figure panel A+C). We constructed a case-matched cohort matched on age, sex, AML subtype and BMI (figure panel B+D) and performed a sensitivity analysis using ≤90% cut-off to define DR which led to the same conclusions.

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Summary/Conclusion: This study demonstrates that ~10% of Danish overweight AML-patients treated with IC are dose reduced ≥5% compared to full BSA-based doses. Risk factors were increasing BMI and BSA in addition to therapy-related AML and favorable cytogenetic risk. Importantly, our results suggest that IC dose reduction does not adversely impact AML outcomes including 30- and 90-day mortality, rates of CR, RFS and OS. However, we encourage future prospective clinical studies to address this question with specific and uniform standards or protocol specifications for dose reduction or dose capping in overweight and obese patients with AML.

P547: CHARACTERISTICS AND OUTCOMES OF PATIENTS WITH ACUTE MYELOID LEUKAEMIA TREATED WITH VENETOCLAX COMBINATION THERAPY: REAL-WORLD EXPERIENCE IN BOTH FRONTLINE AND RELAPSED/REFRACTORY SETTINGS

H. P. J. Lam 1,2,* , S. Leong 1,2 , Z. Kirkham 1,2 , A. Wilson 1 , R. Burt 1 , R. Sellar 1 , A. Fielding 1 , R. Gupta 1 , E. Payne 1 , M. Mansour 1 , P. Kottaridis 1 , A. Khwaja 1 , J. O’Nions 1,2

1 Department of Haematology; 2 NIHR UCL Clinical Research Facility, University College London Hospitals NHS Foundation Trust, London, United Kingdom

Background: Venetoclax (Ven) in combination with hypomethylating agents, such as azacitidine (Aza) and low dose cytarabine (LDAC) has been shown to be effective therapy in acute myeloid leukaemia (AML) and has become standard of care for newly-diagnosed patients unfit for intensive chemotherapy (DiNardo et al., 2020; Wei et al., 2019; Pollyea et al., 2020). Efficacy has also been shown in the relapsed/refractory (R/R) setting in more limited data sets (Báez-Gutiérrez et al., 2021; Pollyea et al., 2020, Stahl et al., 2020; DiNardo et al., 2019). Ven combination therapy has become widely used in newly-diagnosed patients in the UK since its approval during the COVID-19 pandemic as an alternative to intensive chemotherapy and subsequently for patients unfit for intensive therapy.

Aims: We describe the characteristics and outcomes of patients with AML or high risk myelodysplastic syndrome (HR-MDS) receiving Ven combinations in frontline and R/R settings to provide real-world insight into their use in UK clinical practice.

Methods: A retrospective analysis was performed of all patients with AML or HR-MDS who received Ven combination therapy at University College London Hospital between April 2020 and September 2021. Patient demographics, treatment history and bone marrow results were obtained from electronic health care and laboratory records. Disease stratification and response assessments were made as per European LeukemiaNet (ELN) criteria (Döhner et al., 2017).

Results: At the time of analysis, 95 patients received Ven combinations (61 as frontline treatment and 34 for R/R AML), with a median follow up of 14 months. The majority of patients in both groups had adverse risk ELN classification (70.5% of frontline patients, 64.7% of R/R) and received Ven-Aza (100% frontline and 91.1% R/R) (Table 1). The median ages were 72 and 59 years respectively. The incidence of composite CR/CRi was 70.5% in the frontline setting, with median duration of response (DoR) of 8.3 months and overall survival (OS) of 7.1 months. In R/R AML, the CR/CRi rate was 64.7%, median DoR 10.5 months and median OS 9.8 months. Four out of the 43 patients who achieved CR/CRi (9.3%) following frontline treatment and 9 of the 22 R/R (40.9%) patients proceeded to allogeneic stem cell transplant (alloSCT) post induction. The median survival for all patients who underwent alloSCT is not reached in this analysis. The highest CR/CRi rates were observed in intermediate risk patients (90.9% in frontline treatment, 71.4% in R/R), with lower rates in both favourable (80% and 66.7%) and adverse risk patients (65.1% and 59.1% respectively). The presence of NPM1 and IDH1/2 mutations were associated with high CR/CRi rates in both the frontline (85.7% and 84.6% respectively) and R/R groups (100% and 81.8%), with below average response rates seen in TP53 mutated AML (62% in frontline, 40% in R/R). Notable responses were seen in patients with RUNX1 mutations in both settings (77.8% frontline, 66.6% R/R).

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Summary/Conclusion: Our data describes real world effectiveness for venetoclax combinations as both frontline and salvage therapy in UK clinical practice, similar to that seen in clinical trials. This further contributes to our understanding of these therapies, in particular their use as a viable treatment option in R/R patients and as a bridge to alloSCT, and highlights the importance of further characterisation of genetic predictors of response to inform treatment decisions in real-world practice.

P548: BEMCENTINIB COMBINED WITH LOW-DOSE CYTARABINE IS EFFICACIOUS AND WELL TOLERATED IN RELAPSED AML PATIENTS UNFIT FOR INTENSIVE CHEMOTHERAPY. UPDATES FROM THE ONGOING PHASE II TRIAL ( NCT02488408 )

S. Loges 1,2,3,* , M. Heuser 4 , J. Chromik 5 , G. Sutamtewagul 6 , S. Kapp-Schwoerer 7 , M. Crugnola 8 , N. Di Renzo 9 , R. Lemoli 10 , D. Mattei 11 , I. Ben-Batalla 1,2,3 , J. Waizenegger 1,2,3 , L.-M. Rieckmann 1,2,3 , M. Janning 1,2,3 , C. D. Imbusch 2 , N. Beumer 1,2,12 , D. Micklem 13 , C. Gorcea-Carson 14 , G. Lawson 14 , J. Nautiyal 14 , S. Deharo 14 , W. Fiedler 15 , Y. Alvarado-Valero 16 , B. Gjertsen 17

1 DKFZ-Hector Cancer Institute, University Medical Center Mannheim, Mannheim; 2 Division of Personalized Medical Oncology (A420), German Cancer Research Center (DKFZ), Heidelberg; 3 Department of Personalized Oncology, University Hospital Mannheim and Medical Faculty Mannheim, University of Heidelberg, Mannheim; 4 Hematology, Haemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover; 5 University Hospital Frankfurt, Frankfurt, Germany; 6 University of Iowa Hospitals and Clinics, Iowa City, United States of America; 7 University Hospital of Ulm, Ulm, Germany; 8 University of Parma, Parma; 9 Haematology and SCT Unit, Vito Fazzi Hospital, Lecce; 10 University of Genoa, Genoa; 11 ASOS. Croce e Carle, Cuneo, Italy; 12 Faculty of Biosciences, Heidelberg University, Heidelberg, Germany; 13 BerGenBio ASA, Bergen, Norway; 14 BerGenBio Ltd, Oxford, United Kingdom; 15 University Medical Center Hamburg, Hamburg, Germany; 16 The University of Texas M.D. Anderson Cancer Center, Houston, United States of America; 17 Haukeland University Hospital, Bergen, Norway

Background: The new standard of care (SOC) in newly-diagnosed AML patients (pts) unfit for intensive chemotherapy (IC) due to age or co-morbidities yields favourable efficacy. However, beyond 1st line, these pts have limited treatment options, with a dismal mOS of 2.4 months at relapse, highlighting a significant unmet need for new treatments in this pt population. Bemcentinib (BEM) is a first-in-class, orally bioavailable, highly selective AXL-inhibitor. AXL (a receptor tyrosine kinase) represents an important target in AML, as its expression on AML cells and stem cell compartment is associated with poor prognosis, resistance to chemotherapy and decreased antitumor immune response.

Aims: The ongoing BGBC003 PhII trial studied the safety and efficacy of the BEM+LDAC combination in relapsed (REL) AML pts unfit for IC; in addition, a comprehensive translational biomarker analysis was pursued. Here, we present preliminary efficacy and safety data and include initial translational multiomics data from bone marrow mononuclear cells (BMMNC).

Methods: Pts received BEM at 200 mg/OD with 3 loading doses at 400 mg/OD and LDAC at SOC schedule. Study endpoints included overall survival (OS), objective response (OR), clinical benefit rate (CBR) (OR+unchanged [UC]) and exploratory biomarker analyses.

Longitudinal BMMNC samples (n=32) from 13 pts underwent scRNA-seq and CITEseq (Chromium 10x genomics; TotalSeq, Biolegend). Pts were stratified by best response: CR, CRi, PR for Responders; UC, PD for Non-Responders. Cell type annotations were inferred from expression of known markers at RNA and protein level and for identification of malignant cells copy number variation was included.

Results: As of 13 Dec 2021, 22 REL AML pts (10/22 in 1st relapse) were treated with BEM+LDAC. Median age was 75.5yrs [66-86], median prior lines of therapy 1 [1-8] and adverse cytogenetic risk in 7/22 (32%). Only 19/22 pts were evaluable for efficacy. The overall REL population (n=19) demonstrated a composite CR (CRc=CR+CRi) of 26% (5/19) and CBR of 79% (14/19); AML pts in 1st relapse (10/19) showed a CRc of 30% (3/10) and CBR of 70% (7/10) whereas REL pts with time on treatment (ToT) > 3 months (11/19) achieved CRc of 45% (5/11) and CBR 91% (9/11); median ToT of 12.9 months for CR/CRi pts; 1 pt remains on treatment. Median OS was 6.2 months in the overall REL pts (n=19), 7.4 months in the AML pts in 1st relapse (10/19) and 11.3 months in REL pts with ToT >3 months. Late onset responses suggest an immunological mechanism of action (iMOA) and may contribute to a longer ToT and survival.

CITEseq analysis identified differences in the immune compartment, underscoring an iMOA associated with response to BEM+LDAC. Furthermore, increased gene set enrichment of TNFα signalling via NfkB at screening in malignant blasts of responders emphasizes the influence of the TNFα signalling pathway as an interesting field of further research.

The safety profile of BEM+LDAC in the REL AML pts (n=19) is comparable with the known safety profile of LDAC. TRAEs of ≥G3 observed in ≥10% of pts were anaemia (33% BEM+LDAC; 22% BEM), and ECG QT prolonged (11% BEM+LDAC; 11% Bem). No G5 TRAEs reported.

Summary/Conclusion: BEM+LDAC is efficacious and well tolerated in REL AML pts unfit for IC, with promising survival benefit compared to historical data. Translational research showed activation of CD8+T cells and B/Plasma cells in response to treatment, indicating that BEM elicits activation of two major adaptive immune cell populations responsible for anti-AML immune responses. BEM+LDAC warrants further evaluation in this population.

P549: ANALYSIS OF THE CLINICAL SIGNIFICANCE AND PROGNOSTIC IMPACT OF TET2 SINGLE NUCLEOTIDE POLYMORPHISM I1762V IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

Y. Li 1 , X. Lyu 1,*

1 Central Lab, Henan Cancer Hospital, Zhengzhou, China

Background: Acute myeloid leukemia (AML) is a malignancy that originates from myeloid hematopoietic stem/progenitor cells in the bone marrow. The currently recognized cause of AML is chromosomal abnormalities or genetic mutations in myeloid hematopoietic stem/progenitor cells induced by various physicochemical factors such as benzene, pesticides, ionizing radiation, and chemotherapeutic drugs. With the development of high-throughput sequencing technology, a variety of acquired somatic mutations are used for the diagnosis, treatment and prognostic assessment of AML. Also, genetic susceptibility due to many single nucleotide polymorphisms (SNPs) is thought to be important in the development of AML. The TET2 gene encodes a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine and contributes to the epigenetic regulation of myelopoiesis. Mutations in the TET2 gene lead to dysmethylation and myeloid transformation. The TET2 SNP I1762V (rs2454206AG/GG, c.5284A>G, p.Ile1762Val) has an allele frequency of approximately 21% in the Chinese population. Some studies have shown that pediatric AML patients carrying mutations at this locus have a better prognosis. In this study, we investigated the clinical significance of the TET2 SNP locus I1762V in AML patients and its impact on prognosis through a comprehensive analysis of high-throughput sequencing results from 413 AML patients.

Aims: This study aimed to explore the clinical significance and effect on the prognosis of TET2 Single nucleotide polymorphism I1762V in patients with acute myeloid leukemia (AML).

Methods: Target sequencing on 58 hematological tumor-related genes in bone marrow samples of 413 AML patients was performed using the high-throughput sequencing method. TET2 I1762V and other somatic mutations were annotated and compared with patients’ clinical information and prognosis.

Results: I1762V was detected in 154 patients with AML, which was significantly different from the normal population in NyuWa Chinese Population Variant Database (χ2=72.4, P<0.001). I1762V was not related to sex, age, and karyotype of AML patients (P>0.05). The proportion of NPM1 gene mutations and KIT gene mutations in patients with I1762V was significantly higher than the others(P < 0.001). NPM1 mutations and KIT mutations were mutually exclusive. The survival analysis results showed that the OS and PFS of AML patients with I1762V were significantly higher than those of wild type patients (HR=0.55, P < 0.05), while the OS and PFS in AML patients with DNMT3A mutation (with or without I1762V mutation) were lower than those of wild type patients (HR=1.79, P < 0.05).

Summary/Conclusion: TET2 SNP I1762V is an factor affecting the prognosis of AML patients, which can be used to guide the treatment and evaluate the prognosis of AML. TET2 SNP I1762V influenced the concomitant or reciprocal mutations in AML patients; I1762V was closely associated with the prognosis of AML patients. The findings demonstrate that the TET2 SNP locus I1762V is an important guide for the diagnosis, treatment and prognostic assessment of AML patients.

P550: A PHASE 3, RANDOMIZED TRIAL OF MAGROLIMAB IN COMBINATION WITH VENETOCLAX AND AZACITIDINE IN PREVIOUSLY UNTREATED PATIENTS WITH ACUTE MYELOID LEUKEMIA INELIGIBLE FOR INTENSIVE CHEMOTHERAPY (ENHANCE-3)

N. G. Daver 1,* , K. Liu 2 , S. Werneke 2 , E. Rustia 2 , G. Ramsingh 2 , P. Vyas 3

1 The University of Texas MD Anderson Cancer Center, Houston; 2 Gilead Sciences, Inc., Foster City, United States of America; 3 Department of Medicine, University of Oxford Radcliff, Oxford, United Kingdom

Background: Acute myeloid leukemia (AML) is an aggressive clonal hematopoietic malignancy of myeloid cells associated with limited outcomes for patients who are ineligible for intensive chemotherapy due to age or comorbidities. Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a “don’t eat me” signal on cancer cells, resulting in tumor phagocytosis by macrophages. A triplet regimen of magrolimab + azacitidine + venetoclax has shown promising activity in patients with AML.

Aims: To evaluate the efficacy, safety, and tolerability of magrolimab + azacitidine + venetoclax in previously untreated patients with AML who are ineligible for intensive chemotherapy.

Methods: This is a phase 3, randomized, double-blind, placebo-controlled, multicenter study. Approximately 432 patients will be randomized 1:1 to receive magrolimab + azacitidine + venetoclax (experimental arm) or placebo + azacitidine + venetoclax (control arm) (Figure). Randomization will be stratified by age, cytogenetic risk group, and geographic region. Patients are eligible if they are ≥75 years of age or 18 to 74 years of age with specific comorbidities. Receipt of prior antileukemic therapy for AML is not allowed. Magrolimab will be administered intravenously with an initial 1 mg/kg priming dose on days 1 and 4 to mitigate on target anemia. The dose will then be escalated to 15 mg/kg on day 8, then to 30 mg/kg on days 11 and 15, then weekly for 5 doses, followed by every other week beginning 1 week after the fifth weekly 30 mg/kg dose. Placebo will be dosed at the same frequency as magrolimab. Venetoclax will be administered orally at 100 mg on day 1, 200 mg on day 2, and 400 mg daily starting on day 3 and thereafter. Azacitidine will be given intravenously or subcutaneously at 75 mg/m 2 on days 1 to 7, or days 1 to 5 and 8 to 9 of 28-day cycles. Patients will remain on study until disease progression, relapse, loss of clinical benefit, or unacceptable toxicities, or until other discontinuation criteria are met. The dual primary endpoints are complete remission (CR) rate within 6 treatment cycles and overall survival. Secondary endpoints include duration of CR, transfusion independence, and event-free survival.

Results: Trial in progress.

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Summary/Conclusion: Patient enrollment is ongoing. Clinical trial information: NCT05079230 .

P551: A PHASE 3, RANDOMIZED, OPEN-LABEL STUDY EVALUATING THE SAFETY AND EFFICACY OF MAGROLIMAB IN COMBINATION WITH AZACITIDINE IN PREVIOUSLY UNTREATED PATIENTS WITH TP53-MUTANT ACUTE MYELOID LEUKEMIA

N. G. Daver 1,* , P. Vyas 2 , M. P. Chao 3 , G. Xing 3 , C. Renard 3 , G. Ramsingh 3 , D. A. Sallman 4 , A. H. Wei 5

1 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2 Weatherall Institute of Molecular Medicine, MRC Molecular Hematology Unit, University of Oxford, Oxford, United Kingdom; 3 Gilead Sciences, Inc., Foster City; 4 Moffitt Cancer Center, Tampa, United States of America; 5 Department of Haematology, Alfred Hospital and Monash University, Melbourne, Australia

Background: Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a macrophage immune checkpoint and “don’t eat me” signal on cancer cells, that eliminates leukemia stem cells by inducing tumor phagocytosis. Hypomethylating agents synergize with magrolimab by inducing “eat me” signals on leukemic blasts, thereby enhancing phagocytosis. Magrolimab with azacitidine (AZA) has shown encouraging activity in frontline acute myeloid leukemia (AML) unfit for intensive chemotherapy and myelodysplastic syndrome. In TP53 -mutant ( TP53 m) AML, it demonstrated an objective response rate of 69%, complete response (CR) rate of 45%, and median overall survival (OS) of 12.9 months. The TP53 gene mutation is observed in approximately 10-15% of newly diagnosed AML and is associated with poor survival. The published first-line median OS in TP 53m AML is 4-7 months, regardless of whether patients are treated with intensive chemotherapy or non-intensive approaches, such as a hypomethylating agent with venetoclax (VEN). AML patients harboring the TP53 gene mutation represent a significant unmet medical need.

Aims: To evaluate the efficacy, safety, and tolerability of magrolimab+AZA vs physician’s choice of VEN+AZA or “7 + 3” chemotherapy in patients with previously untreated TP53 m AML.

Methods: This is a phase 3, randomized, open-label, multicenter study. Approximately 346 patients will be randomized (1:1) to magrolimab+AZA (experimental arm) or physician’s choice of VEN+AZA or 7 + 3 chemotherapy, based on patient fitness. Randomization will be stratified by appropriateness for non-intensive vs intensive therapy, geographic region (US vs non-US sites), and age (<75 vs ≥75 years). Patients are eligible if they are ≥18 years old with histologically confirmed AML with no prior antileukemic therapy, with at least one TP53 gene mutation that is not benign or likely benign (confirmed by central laboratory) or biallelic 17p deletions (based on a locally evaluated karyotype/fluorescence in situ hybridization report). During the first 28-day cycle, patients randomized to magrolimab+AZA will receive magrolimab intravenously (IV) at a priming dose of 1 mg/kg on days 1 and 4, 15 mg/kg on day 8, and 30 mg/kg on days 11, 15, and then weekly for 5 doses, followed by every other week beginning 1 week after the fifth weekly 30 mg/kg dose. For patients randomized to the VEN+AZA arm, VEN and AZA will be administered according to labeled indications. Patients treated with 7 + 3 chemotherapy will receive 1-2 induction cycle(s) with IV daunorubicin or idarubicin on days 1-3 and cytarabine 100 mg/m 2 or 200 mg/m 2 on days 1-7 followed by up to 4 consolidation cycles with high-dose cytarabine (3000 mg/m 2 ). Patients receiving magrolimab+AZA or VEN+AZA will remain on treatment until disease progression, relapse, loss of clinical benefit, unacceptable toxicities, or stem cell transplant. Patients can undergo stem cell transplantation per investigator decision. The primary endpoint is OS in patients appropriate for non-intensive therapy, and the key secondary endpoint is OS in all patients.

Results: Trial in progress.

Summary/Conclusion: Patient enrollment is ongoing. Clinical trial information: NCT04778397 .

P552: TREATMENT OUTCOMES IN NEWLY DIAGNOSED, UNTREATED PATIENTS WITH TP53-MUTATED ACUTE MYELOID LEUKEMIA: A SYSTEMATIC LITERATURE REVIEW AND META-ANALYSIS

N. G. Daver 1,* , S. Iqbal 2 , C. Renard 2 , R. J. Chan 2 , K. Hasegawa 2 , H. Hu 2 , P. Tse 3 , J. Yan 3 , M. J. Zoratti 3 , F. Xie 3 , G. Ramsingh 2

1 The University of Texas MD Anderson Cancer Center, Houston; 2 Gilead Sciences, Inc., Foster City, United States of America; 3 McMaster University, Hamilton, Canada

Background: TP53 mutations are present in 10% to 15% of patients with acute myeloid leukemia (AML) and are associated with resistance to therapy and poor outcomes. Currently available frontline therapies for TP53 -mutated AML include intensive chemotherapy (IC), hypomethylating agents (HMAs), and venetoclax combined with HMA (VEN+HMA).

Aims: To describe response and survival outcomes associated with IC, HMAs, and VEN+HMA in newly diagnosed, untreated patients with TP53 -mutated AML.

Methods: EMBASE and MEDLINE were systematically searched to identify prospective and retrospective studies that reported complete remission (CR), CR with incomplete hematologic recovery (CRi), median overall survival (OS), event-free survival (EFS), or duration of response (DOR) outcomes in patients with TP53 -mutated AML treated with IC, HMAs, or VEN + HMA. Screening and data extraction were conducted independently and in duplicate by 2 reviewers. Response outcomes (CR and CRi) were pooled using random-effects models, and the median of medians method was used for time-related outcomes (OS, EFS, DOR).

Results: From the 3006 abstracts identified (May 20, 2021), 17 publications (12 studies: 6 randomized controlled trials, 2 single-arm trials, and 4 retrospective studies) satisfied the inclusion criteria (Table). The percentage of patients achieving CR was highest with IC (43%; 95% CI, 30%-56%; N=133), followed by VEN+HMA (33%; 95% CI, 22%-47%; N=54;) and HMA (21%; 95% CI, 7%-49%; N=14). The CRi rate was highest with VEN+HMA (20%; 95% CI, 12%-33%; N=54), followed by IC (6%; 95% CI, 1%-20%; N=35). No studies reported CRi outcomes for HMA alone. The rates of CR/CRi were 49% (95% CI, 37%-60%; N=121) for VEN+HMA, 46% (95% CI, 39%-53%; N=200) for IC, and 13% (95% CI, 2%-48%; N=28) for HMA alone. The median OS was similarly low across the 3 treatments: 6.5 months for IC (95% CI, 5.1-8.5 months; N=155), 6.2 months for VEN+HMA (95% CI, 5.2-7.2 months; N=73), and 6.1 months for HMA (95% CI, 4.9-7.2 months; N=34). The median EFS was 3.7 months (95% CI, 1.6-5.7 months; N=133) with IC. The median DOR was 3.5 months (N=35) with IC and 5.0 months (95% CI, 3.5-6.5 months; N=54) with VEN+HMA. No studies reported EFS for HMA alone or VEN+HMA or DOR for HMA alone.

Summary/Conclusion: CR, CR/CRi, DOR, and median OS were modest across all treatments for newly diagnosed, untreated patients with TP53 -mutated AML. Limitations of this analysis include low study and patient numbers for some interventions, the lack of direct trial comparisons, and that CR/CRi outcomes were not reported separately for DOR. The findings of this review highlight the significant unmet need for this very difficult-to-treat population.

P553: A PHASE 2, OPEN-LABEL, MULTIARM, MULTICENTER STUDY TO EVALUATE MAGROLIMAB COMBINED WITH ANTILEUKEMIA THERAPIES FOR FIRST-LINE, RELAPSED/REFRACTORY, OR MAINTENANCE TREATMENT OF ACUTE MYELOID LEUKEMIA

P. Vyas 1,* , N. G. Daver 2 , M. P. Chao 3 , G. Xing 3 , C. Renard 3 , G. Ramsingh 3 , A. H. Wei 4 , D. A. Sallman 5

1 Weatherall Institute of Molecular Medicine, MRC Molecular Hematology Unit, University of Oxford, MRC Molecular Haematology Unit and Oxford Biomedical Research Centre, University of Oxford and Oxford University Hospitals, Oxford, United Kingdom; 2 The University of Texas MD Anderson Cancer Center, Houston; 3 Gilead Sciences, Inc., Foster City, United States of America; 4 Department of Haematology, Alfred Hospital and Monash University, Melbourne, Australia; 5 Moffitt Cancer Center, Tampa, United States of America

Background: Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a macrophage immune checkpoint and “don’t eat me” signal on cancer cells. Magrolimab induces tumor phagocytosis and eliminates leukemia stem cells. Chemotherapy and hypomethylating agents synergize with magrolimab by inducing “eat me” signals on leukemic blasts, thereby enhancing phagocytosis. Magrolimab+azacitidine (AZA) has shown encouraging clinical efficacy with an objective response rate (ORR) of 63% and a complete remission (CR) or CR with incomplete hematologic recovery (CRi) rate of 54% in first-line acute myeloid leukemia (AML). Newly diagnosed AML patients (pts) who are ineligible for intensive chemotherapy (IC) are incurable despite the progress made with AZA+venetoclax (VEN; median overall survival [OS] 14-18 months), while AML pts with relapsed/refractory (R/R) disease after intensive regimens have a dismal prognosis. Furthermore, for pts in remission, maintenance therapy with oral AZA has improved OS and prevented relapse, although relapse rates remain high. Magrolimab combinations are being evaluated in each of these settings to improve the standard of care.

Aims: To evaluate the safety, efficacy, and tolerability of magrolimab combined with antileukemia therapies in first-line pts ineligible for IC (cohort 1), R/R pts after IC (cohort 2), and pts in CR/CRi with presence of minimal residual disease (MRD) after IC (cohort 3).

Methods: This is an open-label, multiarm, multicenter study that includes 3 safety run-ins with corresponding phase 2 cohorts. Pts in cohort 1 must be ≥75 years or 18-74 years with comorbidities that preclude IC. Pts in cohort 2 must have R/R AML after initial IC. Pts in cohort 3 must have achieved a CR/CRi with MRD positivity, as assessed by flow cytometry, after IC, and not be candidates for hematopoietic stem cell transplant. Each cohort will initially enroll 6 pts for 1 cycle (28 days) to assess dose-limiting toxicities and determine the recommended phase 2 dose (RP2D). Pts in cohort 1 will receive a combination of magrolimab, VEN, and AZA. Pts in cohort 2 will receive magrolimab in combination with mitoxantrone, etoposide, and cytarabine (MEC). Pts in cohort 3 will receive magrolimab with oral CC-486. All will receive magrolimab on the same schedule. In cycle 1, magrolimab will be administered intravenously (IV) as priming and ramp-up doses at 1 mg/kg on days 1 and 4, 15 mg/kg on day 8, 30 mg/kg on days 11, 15, and then weekly for 5 doses, followed by every other week beginning 1 week after the fifth weekly 30 mg/kg dose. For cohort 1, AZA (75 mg/m 2 ) will be administered IV/subcutaneously on days 1-7 or 1-5, 8, and 9 of each cycle. VEN, MEC, and CC-486 will be administered according to labeled indications. After completion of the safety run-in, additional pts will be enrolled in cohort 1 (n=40), cohort 2 (n=30), and cohort 3 (n=40) for phase 2. In phase 2, pts in cohorts 1 and 3 will receive study treatment at RP2D until disease progression, unacceptable toxicity, or loss of clinical benefit. Pts in cohort 2 will receive magrolimab with MEC for up to 3 cycles followed by magrolimab alone for a total of 12 magrolimab cycles. In cohorts 1 and 2, the primary efficacy endpoint is the CR rate. Secondary endpoints of interest include OS, ORR, and MRD-negative CR/CRi rates. The primary efficacy endpoint in cohort 3 is the MRD-negative CR rate.

Results: Trial in progress.

Summary/Conclusion: Patient enrollment is ongoing. Clinical trial information: NCT04778410 .

P554: GILTERITINIB VERSUS SALVAGE CHEMOTHERAPY FOR RELAPSED/REFRACTORY FLT3-MUTATED ACUTE MYELOID LEUKEMIA: A PHASE 3, RANDOMIZED, MULTICENTER, OPEN-LABEL TRIAL IN ASIA

J. Wang 1,* , B. Jiang 2 , J. Li 3 , L. Liu 4 , X. Du 5 , H. Jiang 6 , J. Hu 7 , M. Yuan 8 , T. Sakatani 9 , T. Kadokura 9 , M. Takeuchi 9 , S. Izuka 9 , L. Girshova 10 , J. Tan 11 , S. Bondarenko 12 , L. L. L. Wong 13 , A. Khuhapinant 14 , E. Martynova 15 , N. Hasabou 16 , R. Tiu 16

1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin; 2 Department of Hematology, Peking University International Hospital; 3 Department of Hematology, Peking Union Medical College Hospital, Beijing; 4 Department of Hematology, Shanghai Tongren Hospital, Shanghai; 5 Department of Hematology, Guangdong Provincial People’s Hospital, Guangzhou; 6 Department of Hematology, Peking University Peoples Hospital, Beijing; 7 Fujian Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian; 8 Astellas Pharma, Inc., Beijing, China; 9 Astellas Pharma, Inc., Tokyo, Japan; 10 Almazov National Medical Research Centre, St. Petersburg, Russia; 11 Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, Sarawak, Malaysia; Department of Hematology, Ampang Hospital, Selangor, Malaysia; 12 Clinical Research Institution of Pediatric Hematology and Transplantation under the Name of Raisa Gorbacheva, State Medical University, St. Petersburg, Russia; 13 Hematology Unit, Queen Elizabeth Hospital, Kota Kinabalu, Sabah, Malaysia; 14 Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; 15 Krasnoyarsk Regional Clinical Hospital, Krai, Russia; 16 Astellas Pharma, Inc., Northbrook, United States of America

Background: Due to poor prognosis, treatment options for patients (pts) with relapsed/refractory to therapy (R/R) FLT3 -mutated ( FLT3 mut+ ) acute myeloid leukemia (AML) are needed globally. The phase 3 ADMIRAL trial showed superior survival benefit and favorable safety for pts with R/R FLT3 mut+ AML receiving gilteritinib vs salvage chemotherapy (SC); randomized, controlled trials of treatments in a mostly Asian population are lacking.

Aims: We evaluated the efficacy and safety/tolerability of gilteritinib vs SC in Asian pts with R/R FLT3 mut+ AML after first-line therapy.

Methods: In this phase 3, open-label COMMODORE ( NCT03182244 ) trial, adult pts in China, Russia, Singapore, Thailand, and Malaysia with R/R FLT3 mut+ AML were randomized 1:1 to gilteritinib 120 mg orally per day or SC (low-dose cytarabine; mitoxantrone/etoposide/intermediate-dose cytarabine; or fludarabine/high-dose cytarabine/granulocyte colony-stimulating factor) over continuous 28-day cycles. Pts had ECOG score ≤2; pts with acute promyelocytic leukemia, BCR-ABL–positive leukemia, active central nervous system disease, or secondary AML were excluded. Primary endpoint was overall survival (OS); key secondary efficacy endpoints were event-free survival (EFS) and complete remission (CR). Other endpoints were duration of remission, composite CR (CRc), and safety/tolerability. OS and EFS were analyzed with stratified Cox proportional hazard models; response rates were analyzed with the Cochran-Mantel-Haenszel test. Subgroup analyses were planned. Interim analysis results are presented.

Results: As of June 30, 2020, 234 pts were randomized (gilteritinib, n=116; SC, n=118). Median age was 51.5 and 49.5 years in the gilteritinib and SC groups, respectively; most pts had not received prior FLT3 inhibitors (87.9% and 93.2%). Baseline FLT3 mutations in the gilteritinib vs SC groups were: FLT3 -ITD (91.4% vs 83.1%), FLT3 -TKD (6.0% vs 11.9%), and both FLT3 -ITD and FLT3 -TKD (2.6% vs 5.1%). Median OS follow-up duration was 11.1 mo for gilteritinib and 6.9 mo for SC. Median OS was longer with gilteritinib (9.0 mo) vs SC (4.7 mo; HR 0.549 [95% CI: 0.379, 0.795]; P =0.00126); 1-year survival rate was 33.3% and 23.2%, respectively. OS benefit was seen with gilteritinib vs SC across most subgroups ( Figure ). Pts on gilteritinib had longer EFS than pts on SC (median EFS 2.8 vs 0.6 mo; HR 0.551 [95% CI: 0.395, 0.769]; P =0.00004). More pts had CR on gilteritinib (16.4%) vs SC (10.2%; P =0.17690); CRc rates were 50.0% and 20.3% ( P <0.00001). Grade ≥3 adverse events (AEs) with gilteritinib (97.3%) vs SC (94.2%) were comparable; serious AE rates were higher for gilteritinib (73.5%) vs SC (61.5%). Adjusted for treatment exposure, AE rates were lower with gilteritinib (grade ≥3, 55.56 events/pt-year [E/PY]; serious, 6.19 E/PY) than SC (grade ≥3, 164.00 E/PY; serious, 12.40 E/PY). Most common AEs for gilteritinib were anemia (76.1%), thrombocytopenia (46.9%), pyrexia (41.6%), and increased blood lactate dehydrogenase (41.6%); for SC, most common AEs were anemia (64.4%), decreased white blood cell count (41.3%), and thrombocytopenia (38.5%). AEs leading to death occurred in 22 (19.5%) and 15 (14.4%) pts receiving gilteritinib or SC, respectively.

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Summary/Conclusion: Gilteritinib significantly prolonged OS and EFS vs SC in pts with R/R FLT3 mut+ AML in Asia. Safety/tolerability adjusted for treatment exposure was favorable for gilteritinib vs SC. COMMODORE results further validate/affirm the clinical efficacy/safety data from ADMIRAL, reinforcing the significant benefit of gilteritinib in R/R FLT3 mut+ AML.

P555: A PHASE 1B/2 STUDY OF THE CD123-TARGETING ANTIBODY-DRUG CONJUGATE PIVEKIMAB SUNIRINE (IMGN632) IN COMBINATION WITH VENETOCLAX (VEN) AND AZACITIDINE (AZA) FOR PATIENTS WITH CD123-POSITIVE AML

N. G. Daver 1,* , P. Montesinos 2 , A. Aribi 3 , G. Martinelli 4 , J. Altman 5 , G. Roboz 6 , E. S. Wang 7 , P. W. Burke 8 , D. Jeyakumar 9 , R. B. Walter 10 , D. J. DeAngelo 11 , H. P. Erba 12 , A. Advani 13 , L. Gastaud 14 , X. Thomas 15 , E. Todisco 16 , N. Pemmaraju 1 , L. Mendez 17 , A. de la Fuente 18 , G. Gaidano 19 , A. Curti 20 , N. Boissel 21 , C. Recher 22 , C. Schliemann 23 , P. Vyas 24 , C. M. Sloss 25 , J. Wang 25 , K. A. Malcolm 25 , P. A. Zweidler-McKay 25 , K. L. Sweet 26

1 Department of Leukemia, The University of Texas MD Anderson, Houston, United States of America; 2 Hospital Universitari i Politècnic La Fe, Valencia, Spain; 3 Gehr Family Center for Leukemia Research, City of Hope, Duarte, United States of America; 4 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. e A. Seràgnoli”, Bologna, Italy; 5 Division of Hematology and Oncology, Northwestern University Feinberg School of Medicine, Chicago; 6 Division of Hematology & Medical Oncology, Weill Cornell Medicine/New York Presbyterian Hospital, New York; 7 Roswell Park Comprehensive Cancer Center, Buffalo; 8 Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan, Ann Arbor; 9 University of California Irvine, Chao Family Comprehensive Cancer Center, Orange; 10 Fred Hutchinson Cancer Research Center, Seattle; 11 Department of Medical Oncology, Harvard Medical School, Dana-Farber Cancer Institute, Boston; 12 Division of Hematologic Malignancies and Cellular Therapy, Duke University, Durham; 13 Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 14 Medical Oncology Department, Antoine Lacassagne Hospital, Nice; 15 Department of Hematology, Hospices Civils de Lyon, Lyon-Sud Hospital, Lyon, France; 16 Division of Onco-Hematology, European Institute of Oncology IRCCS, Milano, Italy; 17 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States of America; 18 MD Anderson Cancer Center Madrid, Madrid, Spain; 19 Division of Hematology, Department of Translational Medicine, Università del Piemonte Orientale, Novara; 20 IRCCS Azienda ospedaliero-universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy; 21 Université de Paris, Service Hématologie, Hôspital Saint-Louis, Paris; 22 Service d’Hématologie, CHU de Toulouse - Institut Universitaire du Cancer Toulouse Oncopole, Toulouse, France; 23 Department of Medicine A, Hematology, Oncology and Pneumology, University Hospital Muenster, Muenster, Germany; 24 MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford NIHR Biomedical Research Centre, and Oxford University Hospitals NHS Trust, Oxford, United Kingdom; 25 ImmunoGen, Inc., Waltham; 26 Department of Malignant Hematology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States of America

Background: Overexpression of CD123 occurs in multiple hematological malignancies, including acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute lymphoblastic leukemia (ALL) and others. With limited expression on normal hematopoietic progenitor cells, the CD123 antigen is an attractive target for new therapeutics. IMGN632 is a CD123-targeting antibody-drug conjugate (ADC) comprising a novel anti-CD123 antibody coupled, via a peptide linker, to a unique DNA-alkylating cytotoxic payload of the IGN (indolinobenzodiazepine pseudodimer) class. In preclinical models of AML, IMGN632 exhibited potent anti-leukemia activity, with a wide therapeutic index. Confirming preclinical expectations, encouraging single-agent activity has emerged for IMGN632 in the ongoing Phase I trial in patients with CD123-positive BPDCN and AML (Daver. Blood (2019) 134 (Supplement_1):734.).

Preclinical data have demonstrated increased activity with the addition of IMGN632 to AZA alone and to AZA+VEN in multiple AML xenograft and PDX models, leading to improved survival in these models (Kuruvilla. Blood (2019) 134 (Supplement_1):1375.). We have reported compelling activity (ORR 59% and CCR rate 38%) of the IMGN632 triplet in relapsed or refractory AML patients (Daver. ASH 2021 Abstract #372). The safety profile of the triplet included rates of cytopenias similar to those observed with AZA+VEN; additional low-grade adverse events included infusion-related reactions, dyspnea, fatigue, gastrointestinal toxicities, electrolyte imbalances, and pneumonia.

Aims: This Phase 1b/2 study is designed to determine the safety, tolerability, and preliminary anti-leukemia activity of IMGN632 when administered in combination with AZA and VEN to patients with relapsed and frontline CD123-positive AML.

Methods: Dose escalation for the IMGN632+AZA+VEN triplet is complete. The Recommended Phase 2 Dose levels are: IMGN632 45 mcg/kg given on day 7 with 14 days of VEN, and either AZA 50 OR 75 mg/m2 for 7 days of a 28-day cycle.

Results: N/A

Summary/Conclusion: Phase 2 expansion cohorts for patients with untreated/frontline and relapsed AML are enrolling to further characterize the safety profile and assess the antileukemic activity. NCT04086264

P556: CHANGES IN HEALTH-RELATED QUALITY OF LIFE IN PATIENTS WITH NEWLY-DIAGNOSED ACUTE MYELOID LEUKEMIA RECEIVING IVOSIDENIB + AZACITIDINE OR PLACEBO + AZACITIDINE

A. Schuh 1,* , S. de Botton 2 , C. Recher 3 , S. Vives Polo 4 , E. Zarzycka 5 , J. Wang 6 , G. Bertani 7 , M. Heuser 8 , R. Calado 9 , S.-P. Yeh 10 , J. Hui 11 , S. Pandya 11 , D. Gianolio 11 , C. Chamberlain 11 , H. Dohner 12 , P. Montesinos 13

1 Princess Margaret Cancer Centre, Toronto, Canada; 2 Institut Gustave Roussy, Villejuif; 3 Institut Universitaire du Cancer de Toulouse Oncopole, Toulouse, France; 4 Hospital Universitario Germans Trias i Pujol-ICO Badalona, Josep Carreras Research Institute, Badalona, Spain; 5 Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland; 6 Institute of Hematology & Hospital of Blood Disease, Tianjin, China; 7 ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 8 Hannover Medical School, Hannover, Germany; 9 Ribeirão Preto School of Medicine, University of São Paulo, São Paulo, Brazil; 10 China Medical University, Taichung, Taiwan; 11 Servier Pharmaceuticals LLC, Boston, United States of America; 12 Ulm University Hospital, Ulm, Germany; 13 Hospital Universitari i Politècnic La Fe, València, Spain

Background: Ivosidenib (IVO) is a potent, targeted inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1) that is approved for acute myeloid leukemia (AML). IVO plus azacitidine (AZA) demonstrated clinical benefit compared with placebo and AZA in the AGILE study ( NCT03173248 ).

Aims: To assess the impact of IVO+AZA versus placebo+AZA on health-related quality of life (HRQoL) using data from the AGILE study.

Methods: In the double-blind, placebo-controlled phase 3 AGILE study, patients (pts) were randomized 1:1 to IVO 500 mg QD + AZA 75 mg/m 2 SC or IV for 7 days in 28-day cycles, or placebo+AZA. HRQoL was a secondary endpoint assessed using two validated questionnaires: the European Organisation of Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30) and the EuroQol 5-dimension 5-level questionnaire (EQ-5D-5L). Questionnaires were administered pre-dose on cycle (C) 1 Day (D) 1, on C1D15, C2D1, C2D15, and on D1 of every odd cycle thereafter until the end of treatment. Score change from baseline across visits for all subscales of EORTC QLQ-C30 was analyzed with mixed models. A 10-point threshold in EORTC QLQ-C30 subscale score was used to evaluate clinically meaningful changes from baseline or differences between arms. Two-sided nominal p-values are reported. All patients gave written informed consent.

Results: At baseline, 69 and 68 pts out of 72 receiving IVO+AZA completed the EORTC QLQ-C30 and EQ-5D-5L, respectively, and 66 pts out of 74 receiving placebo+AZA completed both. Mean baseline HRQoL scores were similar between treatment arms (EORTC QLQ-C30 global health status [GHS/QoL] score was 56.3 and 53.2 in the IVO+AZA and placebo+AZA arms, respectively). There was an initial decline in HRQoL (EORTC QLQ-C30 GHS/QoL) in both arms for ~4 months, consistent with time to response, and which was generally not clinically meaningful (Table). IVO+AZA was associated with preserved or improved HRQoL compared to baseline for most subscales of the EORTC QLQ-C30 from C5 to C19 (after which no placebo+AZA HRQoL data were available), and at most timepoints for EQ-5D-5L VAS scores and index values. EORTC QLQ-C30 subscales with clinically meaningful improvements from baseline at most timepoints from C5 to C19 in the IVO+AZA arm included GHS/QoL (Table), fatigue, pain and appetite loss. In contrast, there were few clinically meaningful improvements from baseline in placebo+AZA pts. GHS/QoL scores were significantly improved (p≤0.05) for IVO+AZA versus placebo+AZA at C2D1, C2D15, C7 and C9 (Table), and differences between treatment arms were clinically meaningful at C2D1, C2D15, C7, C9, C13, C15 and C19 (Table). Likewise, improvements in EORTC QLQ-C30 fatigue, appetite loss, nausea and vomiting, diarrhea, cognitive functioning and social functioning favored IVO+AZA over placebo+AZA at multiple timepoints.

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Summary/Conclusion: Data from the AGILE study show that patients with mIDH1 AML receiving treatment with IVO+AZA tended to report maintenance or improved HRQoL from cycle 5 through to cycle 19 compared with placebo+AZA.

P557: HEMATOLOGIC IMPROVEMENTS WITH IVOSIDENIB + AZACITIDINE COMPARED WITH PLACEBO + AZACITIDINE IN PATIENTS WITH NEWLY-DIAGNOSED ACUTE MYELOID LEUKEMIA

H. Dohner 1,* , P. Montesinos 2 , S. Vives Polo 3 , E. Zarzycka 4 , J. Wang 5 , G. Bertani 6 , M. Heuser 7 , R. Calado 8 , A. Schuh 9 , S.-P. Yeh 10 , A. de la Fuente 11 , C. Cerchione 12 , S. Daigle 13 , J. Hui 13 , S. Pandya 13 , D. Gianolio 13 , C. Recher 14 , S. de Botton 15

1 Ulm University Hospital, Ulm, Germany; 2 Hospital Universitari i Politècnic La Fe, València; 3 Hospital Universitario Germans Trias i Pujol-ICO Badalona, Josep Carreras Research Institute, Badalona, Spain; 4 Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland; 5 Institute of Hematology & Hospital of Blood Disease, Tianjin, China; 6 ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 7 Hannover Medical School, Hannover, Germany; 8 Ribeirão Preto School of Medicine, University of São Paulo, São Paulo, Brazil; 9 Princess Margaret Cancer Centre, Toronto, Canada; 10 China Medical University, Taichung, Taiwan; 11 MD Anderson Cancer Center Madrid, Madrid, Spain; 12 Hematology Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy; 13 Servier Pharmaceuticals, Boston, United States of America; 14 Institut Universitaire du Cancer de Toulouse Oncopole, Toulouse; 15 Institut Gustave Roussy, Villejuif, France

Background: Ivosidenib (IVO) is a potent oral targeted inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1). IVO plus azacitidine (AZA) significantly improved event-free survival (EFS), overall survival and complete remission + partial hematologic recovery rates compared with placebo + AZA, in patients (pts) with newly diagnosed IDH1 -mutant acute myeloid leukemia (AML) in the Phase 3 AGILE trial ( NCT03173248 ).

Aims: To report blood count recovery results from the AGILE trial.

Methods: Pts were randomized 1:1 to IVO 500 mg QD + AZA 75 mg/m 2 SC or IV for 7 days in 28-day cycles (n=72), or placebo+AZA (n=74). Red blood cell (RBC)/platelet transfusion history were assessed at screening and follow-up. Bone marrow (BM) and peripheral blood samples were obtained at screening, and during weeks 9, 17, 25, 33, 41, 53, and every 24 weeks thereafter, and at end of treatment and during EFS follow up. Samples were analyzed at each local site according to ICSH guidelines. All patients gave written informed consent.

Results: In the IVO+AZA and placebo+AZA arms, 4.2% and 5.5% of pts, respectively, received concomitant granulocyte colony-stimulating factor. Hemoglobin levels steadily increased from baseline at a similar rate in both treatment arms. Mean platelet count recovered from baseline values in the IVO+AZA and placebo+AZA arms (71.0 and 92.6 x 10 9 /L, respectively) as early as week 9 of treatment (171.1 and 155.1 x 10 9 /L, respectively) and continued to steadily increase thereafter in the treated population. In pts receiving IVO+AZA, mean neutrophil counts rapidly increased from baseline (0.99 x 10 9 /L) to week 2 (2.05 x 10 9 /L) and week 5 (4.07 x 10 9 /L), and then generally stabilized to within the normal range to study end (last available cycle value; ~2.0 x 10 9 /L). Mean neutrophil counts initially declined with placebo+AZA before slowly recovering to near-normal levels after 36-40 weeks. The increased blood counts were accompanied by a rapid decrease in the mean BM blast percentage from 54.8% at baseline to 12.0% and 7.2% at week 9 and 17, respectively, in IVO+AZA treated patients and were maintained for 149 weeks. The decline in BM blasts was slower in the placebo+AZA arm (53.7%, 34.6% and 19.6% at baseline, week 9 and week 17, respectively). Among patients who were RBC/platelet transfusion-dependent at baseline (~54.0% in both groups), 46.2% in the IVO+AZA group achieved RBC/platelet transfusion independence compared with 17.5% in the placebo+AZA arm (1-sided p=0.0032). Additionally, fewer adverse events of febrile neutropenia (28.2% vs 34.2%) and infections (28.2% vs 49.3%) were reported in the IVO+AZA arm compared to the placebo+AZA arm.

Summary/Conclusion: IVO+AZA demonstrated a significant clinical benefit compared with placebo+AZA and this sub-analysis demonstrated a rapidly improved recovery of blood counts and a reduced dependence on RBC and/or platelet transfusion. Moreover, rates of febrile neutropenia and infections were reduced with IVO+AZA.

P558: GERMAN AMLCG-SURVIVORSHIP STUDY – SOMATIC LONG-TERM CONSEQUENCE OF AML AND ITS THERAPY: FROM HEART TO KIDNEY.

A. S. Moret 1,* , E. Telzerow 1 , C. Sauerland 2 , M. Rothenberg-Thurley 1 , F. H. A. Mumm 1 , J. Braess 3 , B. Wörmann 4 , U. Krug 5 , W. E. Berdel 6 , W. Hiddemann 1 , K. Spiekermann 1 , P. Heußner 7 , K. Kraywinkel 8 , D. Görlich 2 , K. H. Metzeler 9

1 Department of Medicine III and Comprehensive Cancer Center (CCC Munich LMU), University Hospital, LMU Munich, Munich; 2 Institute of Biostatistics and Clinical Research, University of Münster, Münster; 3 Department of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg; 4 Charité University Hospital Berlin, Berlin; 5 Department of Medicine 3, Hospital Leverkusen, Leverkusen; 6 Department of Medicine A, University of Münster, Münster; 7 Hospital Garmisch-Partenkirchen, Garmisch-Partenkirchen; 8 Department of Epidemiology and Health Monitoring, Robert Koch Institute, Berlin; 9 Department of Hematology, Cell Therapy and Hemostaseology, University Hospital Leipzig, Leipzig, Germany

Background: As outcomes of patients with acute myeloid leukemia (AML) have improved, the fraction of patients surviving long-term are increasing. Information on somatic and psycho-social health consequences of AML and its treatment is sparse.

Aims: Our aim was to perform a multi-dimensional analysis of health outcomes in AML long-term survivors (AML-LTS). This report focuses on somatic morbidity; overall and health-related quality of life are reported separately (Telzerow et al.).

Methods: We conducted a cross-sectional study including AML survivors who had been enrolled in clinical trials or the patient registry of the AML-CG study group and were alive ≥5 years after initial diagnosis. Data concerning somatic health status were collected through patient questionnaires, assessment by the patients’ physicians, and medical and laboratory reports. An age- and sex-matched control cohort was derived from German population-based health surveys (Robert Koch Institute, DEGS1 survey; n=6013).

Results: Data on somatic health status was available for 355 survivors, aged 28-93 years, who participated 5 to 19 years after their AML diagnosis. Thirty-eight percent of survivors were treated with chemotherapy with or without an autologous transplant, whereas 62% had undergone allogeneic transplantation (alloSCT).

We compared the prevalence of somatic diseases between AML-LTS and the general German population (DEGS1 sample), focusing on cardiovascular diseases and risk factors due to their relevance for overall morbidity and mortality, and their known association with survivorship in other cancers. Using age- and sex-adjusted multivariate models (Figure A), we found that AML survivors had a 2-fold higher risk of type 1/2 diabetes (DM 1/2), and a 3.5-fold increased risk of congestive heart failure (CHF) compared to the general population. Prevalence of hypertension, coronary artery disease (CAD) and myocardial infarction were similar between the groups. To identify factors associated with development of CHF among AML-LTS, we constructed multivariate models incorporating patient- and treatment-related covariables. We found an increased risk of CHF for AML-LTS who have had AML relapse (OR, 3.16; 95% CI: 1.46 – 6.83; P=0.004) and, in trend, for patients with sAML or tAML (OR 2.19; 95%CI: 0.92 – 5.22, P=0.076). Disease- or treatment-related factors did not significantly associate with any of the other comorbidities we studied.

Furthermore, AML-LTS had significantly impaired kidney function, as assessed by the glomerular filtration rate (eGFR) estimated using the CKD-EPI-formula. Figure B displays the median eGFR according to age groups for AML-LTS and DEGS1. For each age group AML-LTS had significantly lower eGFR in comparison to DEGS1. We did not find an association between kidney function and time since diagnosis (p=.5) and no differences between AML-LTS treated by chemotherapy or alloSCT (p=.5), survived a relapse (p=.9) or were diagnosed with de novo-AML vs. sAML/tAML (P=.6).

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Summary/Conclusion: Compared to the German general population, AML-LTS had increased risks of CHF and diabetes, but not hypertension or CAD. We identified AML relapse as a risk factor for developing of CHF, suggesting that cumulative chemotherapy exposure might be causally involved. Notably, AML-LTS who had undergone alloSCT did not have increased risks of CHF, CAD, hypertension or diabetes, compared to survivors treated with chemotherapy only. In addition, AML-LTS had a higher prevalence of impaired renal function. We did not identify treatment- or disease-related factors that might cause the lower kidney function in AML-LTS.

P559: HUMORAL RESPONSE TO MRNA-BASED COVID-19 VACCINE IN PATIENTS WITH MYELOID MALIGNANCIES

A. Mori 1,* , M. Onozawa 2 , S. Tsukamoto 1 , T. Ishio 1 , E. Yokoyama 1 , K. Izumiyama 1 , M. Saito 1 , H. Muraki 3,4 , M. Morioka 1 , T. Teshima 2 , T. Kondo 1

1 Blood Disorders Center, Aiiku Hospital; 2 Department of Hematology, Hokkaido University Faculty of Medicine; 3 Sapporo Clinical Laboratory Inc.; 4 Division of Laboratory, Aiiku Hospital, Sapporo, Japan

Background: The end of the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease-19 (COVID-19), is not foreseen. Vaccination using two subtypes of mRNA-based vaccines, BNT162b2 or mRNA-1273, is an effective public health measure to reduce the risk of infection and severe complications from COVID-19. However, COVID-19 vaccine response data for patients with myeloid malignancy, who are at severe risk in case of infection, has not emerged.

Aims: We investigated the antibody titers of COVID-19 in patients with myeloid malignancies who received two doses of mRNA-based COVID-19 vaccine.

Methods: Previously treated, currently treated, and newly diagnosed 46 patients with acute myeloid leukemia (AML) and 23 patients with myelodysplastic syndrome (MDS) were included in this study. Anti-spike SARS-CoV-2 antibody titers were measured at 3 months after the second vaccination and compared them to those in healthy controls.

Results: Seroconversion rates for AML and MDS were 94.7% and 100%, without significant difference from healthy controls (100%). In AML patients, the median antibody titers of patients in complete remission (CR) (816.5 [interquartile range (IQR): 250.0-2063.5] U/ml vs 1023.0 [640.0-1535.0] U/ml, P =0.668), especially those who were under treatment-free observation in CR (1630.0 [806.0-2454.0] U/ml vs 1023.0 [640.0-1535.0] U/ml, P =0.1220), were comparable to those in healthy controls. On the other hand, even in CR, the antibody titer in AML patients under maintenance therapy was significantly lower than that in patients under treatment-free observation (154.0 [126.0-289.0] U/ml vs 1630.0 [806.0-2454.0] U/ml, P =0.0003). Among the AML patients in CR, patients receiving maintenance treatment had a significantly lower median absolute lymphocyte count (0.81 [0.71-1.46] x 10 9 /l vs 1.58 [1.29-1.93] x 10 9 /l, P =0.0094) and a significantly lower median absolute neutrophil count (1.45 [1.15-1.64] x 10 9 /l vs 3.45 [2.68-4.28] x 10 9 /l, P <0.0001) than that in patients under treatment-free observation.

Significantly lower antibody titers were associated with current active treatment (92.2 [37.5-216.3] U/ml vs 1630.0 [806.0-2454.0] U/ml, P <0.0001), AML with myelodysplasia-related changes (50.8 U/ml [39.9-109.1] vs 816.5 [283.0-1935.3] U/ml, P =0.0022), advanced age more than the median age of 68 years (195.0 [43.3-743.0] U/ml vs 1630.0 [806.0-3391.0] U/ml, P =0.0002), and vaccine subtypes of BNT162b2 (285.0 [127.8-1045.3] U/ml vs 3037.0 [2198.50-4537.0] U/ml, P =0.0002) in AML patients and with current active treatment (41.0 [10.7-227.5] U/ml vs 623.5 [173.8-1613.3] U/ml, P =0.0233), subtypes of excess blasts (11.1 [4.8-34.1] U/ml vs 212.0 [81.7-600.0] U/ml, P =0.0293), and high and very high risk of the revised international prognostic scoring system in MDS patients (9.0 [3.4-41.0] U/ml vs 169.0 [48.5-327.0] U/ml, P =0.0380).

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Summary/Conclusion: This is one of the first studies on the effect of COVID-19 vaccines focusing on patients with AML and MDS, and there are many new findings. The response to COVID-19 vaccine appears to be related to disease and treatment status. Myeloid malignancies may have less impact than lymphoid malignancies on the vaccine response. AML patients under treatment-free observation in CR could be expected to have a vaccine effect that is comparable to that in healthy individuals. In contrast, since the response to vaccination might be insufficient in AML patients undergoing maintenance therapy, maintenance therapy should be continued with strict measures for prevention of infection even after vaccination.

P560: ANALYSIS OF GENE MUTATION SPECTRUM AND CORRELATION BETWEEN GENETIC ABNORMALITIES IN 207 PATIANTS WITH T(8; 21) AML

Y. Nie 1,* , Y. Li 1 , H. Zhang 1 , H. Li 1 , J. Zhang 1 , C. Wang 1 , Z. Chen 1 , S. Yang 1 , Y. Lin 1 , K. Ru 1

1 Sino-us diagnostics, Tianjin, China

Background: The core binding factor (CBF) involves a subgroup of AML which is composed of a and b subunits encoded by RUNX1(AML1) and CBFβ genes respectively, and CBF is responsible for regulating multiple hematopoietic genes of myeloid differentiation. AML with abnormal CBF accounts for 12%-15% of adult AML, including t(8;21)(q22;q22) and inv (16)(p13q22), which produce RUNX1-RUNX1T1 (also known as AML1-ETO) and CBFβ-MYH11 fusion gene respectively.

Next generation sequencing(NGS) revealed that AML with t (8; 21) had other somatic gene mutations at the same time. In addition to the known genetic abnormalities of RAS and tyrosine kinase signaling pathways, the genes such as epigenetic regulation showed recurrent genetic abnormalities. Some of these gene abnormalities, such as KIT, JAK2 and FLT3-ITD mutations, have been reported in many studies and result in poor prognosis. However, due to the relatively small sample or detected genes size, there are some differences in the research conclusions. This study analyzed the molecular correlation test results of 207 patients with t (8; 21) AML, and conducted a comprehensive retrospective analysis by 56 gene targeted sequencing, so as to clarify the gene distribution and related pathways involved in t (8; 21) AML patients in Chinese population, as well as the correlation between different gene mutations and chromosome abnormalities.

Aims: To evaluate the gene mutational profile and its correlation with chromosomal abnormalities in acute myeloid leukemia (AML) patients with t (8; 21).

Methods: The data of 207 patients with t(8;21) AML was retrieved from Sino-us diagnostics lab from June 2015 to October 2021. We analyzed the gene mutational spectrum and characterized the pathways of 56 AML-associated genes by NGS technology.

Results: Among these 207 patients with t(8;21) AML, the incidence of KIT(53%) gene mutation was the highest, followed by NRAS (18%), ASXL2 (17%), FLT3 (12%), and EZH2 (10%). Most mutations were enriched in signal transduction, chromatin modification, and transcriptional regulation pathways. According to their median VAF, we found that DNA methylation genes, such as IDH1(50.8%), IDH2(49.0%), TET2 (43.8%) and DNMT3A (37.2%) or Chromatin remodeling genes, like ASXL1 (42.1%), ASXL2 (37.3%) are present in the majority of the cells. Meanwhile, alterations in RAS/RTK signaling genes like FLT3 (23.1%), KIT (32.5%) and the RAS GTPases NRAS (8.6%) and KRAS (13.3%) have the lower VAF. In addition, most KIT mutations (121/154) were located in exon 17, mainly on the SNV with D816 or N822. According to the correlation analysis, we found that IDH1/2 and complex karyotype (CK) or ZBTB7A mutation, ASXL2 and CCND2 mutation, t (x; 8; 21) abnormality and JAK2 or FLT3 mutation may co-mutate. On the contrary, KIT and NRAS mutation were mutually exclusive.

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Summary/Conclusion: From the patients with t(8;21) AML, most gene mutations occurred in pathways involving signal transduction, chromatin modification, or transcriptional regulation. DNA methylation or Chromatin remodeling genes may occur in the early stage of leukemogenesis while RAS/RTK signaling genes may indicated later events. The coexistence or mutual exclusion between genes and chromosomes may provide a basis for further definitive diagnosis and therapeutic regimen.

P561: VIALE-T: A RANDOMIZED, OPEN-LABEL, PHASE 3 STUDY OF VENETOCLAX IN COMBINATION WITH AZACITIDINE AFTER ALLOGENEIC STEM CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

C. Craddock 1,* , U. Platzbecker 2 , M. Heuser 3 , V. Pullarkat 4 , S. Chaudhury 5 , D. Wu 6 , S. Addo 7 , B. Chyla 7 , Q. Jiang 7 , P. Lee 7 , J. E. Wolff 7

1 University of Birmingham, Birmingham, United Kingdom; 2 University Hospital in Leipzig, Leipzig; 3 Hannover Medical School, Hannover, Germany; 4 City of Hope, Duarte, CA; 5 Ann & Robert H. Lurie Children’s Hospital of Chicago, Northwestern Feinberg School of Medicine, Chicago, United States of America; 6 The First Affiliated Hospital of Soochow University, Soochow, China; 7 AbbVie Inc, North Chicago, United States of America

Background: Acute myeloid leukemia (AML) is an aggressive malignancy and is the most common and second most common form of acute leukemia in adults and children, respectively. The combination of the highly selective BCL-2 inhibitor venetoclax and the hypomethylating agent azacitidine was shown to be safe and effective in clinical trials (DiNardo et al. Blood . 2019;133:7-17; DiNardo et al. N Engl J Med . 2020;383:617-629) and is approved by the United States Food and Drug Administration and European Medicines Agency for the treatment of patients with AML who are not eligible to receive intensive chemotherapy. Following allogeneic stem cell transplantation (alloSCT), most patients do not receive antileukemic therapy; however, an unmet need remains as disease relapse and graft-versus-host disease (GvHD) commonly occur posttransplant. In addition to the antileukemic effect of venetoclax shown in clinical studies, preclinical studies suggest venetoclax may mitigate the risk of GvHD. VIALE-T is a Phase 3, randomized, open-label trial in progress ( NCT04161885 ) evaluating the safety and efficacy of venetoclax in combination with azacitidine versus best supportive care (BSC) as maintenance therapy following alloSCT in patients with AML.

Aims: N/A

Methods: This Phase 3 study consists of 2 parts (Figure). Key inclusion criteria include diagnosis of AML; plans to receive alloSCT or have received alloSCT within the past 30 days; bone marrow blasts <10% before pretransplant conditioning and <5% posttransplant; have received myeloablative, or reduced intensity, or nonmyeloablative pretransplant conditioning protocols. Grafts are allowed from various sources (bone marrow, peripheral blood stem cells, cord blood cells). Patients must be ≥18 years old for Part 1 and ≥12 years old for Part 2. Additionally, patients must meet key laboratory values for absolute neutrophil count (Part 1, ≥1500/µL; Part 2, ≥1000/µL), platelet count (Part 1, ≥80,000/µL; Part 2, ≥50,000/µL), bilirubin ≤3 times the upper limit of normal, and creatinine clearance >30 mL/min. Patients who have received venetoclax and had no history of disease progression while receiving venetoclax are eligible.

Part 1 evaluates dose levels of venetoclax combined with azacitidine to determine the recommended Phase 3 dose (RP3D), which will be confirmed in approximately 12 additional patients enrolled in the Safety Expansion Cohort. Part 2 will be a randomized, open-label evaluation of the RP3D of venetoclax combined with azacitidine and BSC versus BSC only in adults and children aged 12 years or older. All venetoclax-treated patients will receive antibiotic prophylaxis during Cycle 1.

The primary endpoint for Part 1 is the frequency of dose-limiting toxicities. The primary endpoint for Part 2 is relapse-free survival as assessed by an independent review committee. Key secondary endpoints for Part 2 include overall survival, GvHD-free relapse-free survival, and the rate of patients without higher grade GvHD at 90 days after randomization. Enrollment into the Safety Expansion Cohort will be completed in 2021. Part 2 will enroll approximately 400 patients across approximately 175 participating study sites in 17 countries, with recruitment beginning in 2022.

Results: N/A

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Summary/Conclusion: N/A

P562: LEMZOPARLIMAB (LEMZO) WITH VENETOCLAX (VEN) AND/OR AZACITIDINE (AZA) IN PATIENTS (PTS) WITH ACUTE MYELOID LEUKEMIA (AML) OR MYELODYSPLASTIC SYNDROMES (MDS): A PHASE 1B DOSE ESCALATION STUDY

J.-Z. Hou 1,* , N. G. Daver 2 , D. A. Stevens 3 , T. Yamauchi 4 , Y. Moshe 5 , C. Y. Fong 6 , A. Marzocchetti 7 , R. Adamec 8 , M. Patel 8 , S. Lambert 9 , K. Y. Wu 8 , C. Röllig 10

1 UPMC Hillman Cancer Center, Pittsburgh, PA; 2 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX; 3 Norton Cancer Institute, Louisville, KY, United States of America; 4 University of Fukui Hospital, Fukui, Japan; 5 Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 6 Department of Clinical Haematology, Austin Health, Heidelberg, Victoria, Australia; 7 AbbVie S.r.l., Rome, Italy; 8 AbbVie Inc., North Chicago, IL; 9 AbbVie Inc., South San Francisco, CA, United States of America; 10 Universitätsklinikum Carl Gustav Carus an der TU Dresden, Dresden, Germany

Background: Despite treatment advances, pts with AML or higher-risk MDS who are ineligible for standard intensive treatments still have poor survival, highlighting the need for novel treatments.

Overexpression of CD47 is common in leukemic stem cells and AML blasts and correlates with poor clinical outcomes. Lemzo is an anti-CD47 antibody with red blood cell–sparing properties. Treatment with ven plus aza has shown favorable safety and efficacy in older/unfit pts with AML and higher-risk MDS. Blocking CD47 is hypothesized to hypersensitize AML cells to the antitumor activity of ven and aza.

This study will evaluate the safety and dose-limiting toxicities (DLTs) of lemzo with ven + aza for pts with treatment-naïve AML, as well as lemzo with aza ± ven for treatment-naïve higher-risk MDS.

Aims: N/A

Methods: This phase 1b, open-label, dose-escalation study ( NCT04912063 ) is enrolling adults with: (1 ) treatment-naïve AML with adverse cytogenetic/molecular risk not suitable for induction therapy, with an Eastern Cooperative Oncology Group Performance Status (ECOG-PS) 0–2 (aged ≥75 years) or 0–3 (aged ≥18–74 years) excluding acute promyelocytic leukemia; or (2 ) treatment-naïve higher-risk MDS (Revised International Prognostic Scoring Score >3) with <20% bone marrow blasts, ECOG-PS 0–2, and no immediately planned stem cell transplant.

For each 28-day cycle, aza is administered subcutaneously or intravenously (IV) daily for 7 days within the first 9 days (7-0-0 or 5-2-2 schedule); ven is administered orally daily on days 1–28 (AML, following dose ramp-up) or days 1–14 (ven-containing MDS cohorts). Lemzo is administered IV at a schedule that is to be determined in this study.

Dose escalation has Bayesian optimal interval design and may be expanded to investigate alternate dosing for lemzo. Dose expansion will initiate at recommended phase 2 dose. Treatment discontinuation criteria are unacceptable toxicity, progressive disease, lack of partial/complete remission or clinical benefit within 6 cycles, or at physician’s discretion. Pts who discontinue study treatment without progression will continue with posttreatment follow-up; pts who progress will enter survival follow-up.

The primary endpoints are DLTs of lemzo. Secondary endpoints for both cohorts include best overall responses of complete remission, duration of response, event-free survival, and overall survival. Exploratory biomarker endpoints are included. Safety assessments include adverse event (AE, graded per National Cancer Institute Common Terminology Criteria for AEs v5.0) monitoring, physical examinations, vital signs, electrocardiograms, and laboratory tests.

Response rates will be analyzed with estimates and 95% confidence intervals based on exact binomial distribution. Time-to-event endpoints will be analyzed using Kaplan–Meier methodology.

Results: N/A

Summary/Conclusion: N/A

P563: A RANDOMIZED, DOUBLE-BLIND, 2-ARM, MULTICENTER, PH3 STUDY OF VENETOCLAX & ORAL AZACITIDINE VS. ORAL AZACITIDINE AS MAINTENANCE THERAPY FOR PTS WITH AML IN FIRST REMISSION AFTER INTENSIVE CHEMOTHERAPHY

V. Ivanov 1,* , S.-P. Yeh 2 , J. Mayer 3 , L. Saini 4 , A. Unal 5 , M. Boyiadzis 6 , D. M. Hoffman 7 , K. Kang 7 , S. N. Addo 7 , W. L. Mendes 7 , A. T. Fathi 8

1 Almazov National Medical Research Centre, Saint Petersburg, Russia; 2 China Medical University Hospital, Taichung City, Taiwan; 3 Fakultni Nemocnice Brno, Lískovec, Czechia; 4 London Health Sciences Center, Ontario, Canada; 5 Erciyes University Medical School, Kayseri, Turkey; 6 Genentech Inc., South San Francisco, CA; 7 AbbVie Inc, North Chicago, IL; 8 Massachusetts General Hospital, Boston, MA, United States of America

Background: Acute myeloid leukemia (AML) is an aggressive, heterogenous hematologic malignancy with poor prognosis. For eligible patients, standard treatment includes intensive chemotherapy during induction, followed by consolidative chemotherapy or stem cell transplantation. Despite current therapies, the prevention of relapse is still a major therapeutic challenge and an unmet need for patients in remission.

Venetoclax (Ven) is a selective, potent, oral BCL-2 inhibitor that induces apoptosis in AML cells. Ven in combination with Azacitidine (Aza) leads to prolonged overall survival (OS) and rapid, durable remissions in treatment-naïve AML patients ineligible for intensive chemotherapy. The QUAZAR AML-001 ( NCT01757535 ) trial showed that the median OS in patients receiving maintenance oral Aza was superior to patients receiving placebo following upfront induction and consolidation; this benefit was also observed for patients who achieved either complete remission (CR) or CR with incomplete blood count recovery (CRi).

VIALE-M is a randomized, double-blind, two-part study ( NCT04102020 ) to determine the recommended Phase 3 dose (RPTD) of Ven in combination with oral Aza (CC-486) that can be safely administered as maintenance therapy, and to evaluate the safety and efficacy of Ven in combination with oral Aza + BSC compared to placebo and oral Aza + BSC in patients with AML who have achieved CR or CRi after intensive induction and consolidation.

Aims: N/A

Methods: The study is enrolling patients aged ≥18 years with newly diagnosed AML per WHO 2016 classification, with confirmed CR or CRi following intensive induction and consolidation therapies, from > 200 sites worldwide. Patients must have achieved first CR or CRi (after induction) ≤120 days of first dose of study drug or ≤75 days past last dose of last consolidation cycle, have intermediate or poor risk cytogenetics per NCCN 2016 categorization, ECOG ≤2, and no history of acute promyelocytic leukemia or active central nervous system involvement with AML. For the dose finding part of the study, patients will receive Ven QD for up to 24 cycles and oral Aza QD on D1-14 of each 28-day cycle for up to 24 cycles to determine the RPTD. For safety expansion, patients will receive Ven QD for up to 24 cycles and oral Aza QD on D1-14 of each cycle for up to 24 cycles at the RPTD. For the randomization part, patients will be randomized 1:1 to receive placebo or Ven QD at the RTPD and oral Aza QD on D1-14 of each cycle for 24 cycles. In all parts, treatment may discontinue due to relapse/unacceptable toxicity, or continue beyond 24 cycles under investigator’s discretion.

For the randomization portion, the primary endpoint is relapse-free survival; secondary outcomes include OS, minimal residual disease conversion, and improvement in quality of life.

Results: N/A

Summary/Conclusion: N/A

P564: FINAL RESULTS OF THE QOLESS AZA-AMLE RANDOMIZED TRIAL TO EVALUATE THE EFFICACY OF 5-AZA FOR POST-REMISSION THERAPY OF ACUTE MYELOID LEUKEMIA IN ELDERLY PATIENTS

E. N. Oliva 1,* , P. Salutari 2 , F. Di Raimondo 3 , G. Reda 4 , D. Capelli 5 , G. Iannì 6 , G. Tripepi 7 , C. Alati 1 , C. Mammì 8 , M. G. D’Errigo 8 , P. Niscola 9 , C. Selleri 10 , P. Musto 11 , E. Vigna 12 , A. Volpe 13 , N. Cascavilla 14 , M. C. Cannatà 8 , D. Mannina 15 , A. Candoni 16

1 U.O.C. di Ematologia, Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio di Calabria; 2 U.O. di Ematologia, Ospedale Civile di Pescara, Pescara; 3 U.O.C. di Ematologia, Policlinico Catania, Catania; 4 U.O.C. di Ematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano; 5 Clinica di Ematologia, Ospedali Riuniti di Ancona, Ancona; 6 Dielnet SRL; 7 Ifc-CNR; 8 U.O.S.D. Genetica Medica, Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio di Calabria; 9 U.O. di Ematologia, Ospedale Sant’Eugenio, Roma; 10 Dipartimento di Oncoematologia, AOU San Giovanni di Dio e Ruggi d’Aragona, Salerno; 11 Department of Emergency and Organ Transplantation, “Aldo Moro” University School of Medicine, and Unit of Hematology and Stem Cell Transplantation, AOUC Policlinico, Bari; 12 U.O. di Ematologia, Ospedale L’Annunziata, Cosenza; 13 U.O. di Ematologia, Azienda Ospedaliera San Giuseppe Moscato, Avellino; 14 U.O. Ematologia, Ospedale Casa Sollievo della Sofferenza IRCCS, San Giovanni Rotondo; 15 U.O.C. di Ematologia, Azienda Ospedaliera Papardo, Messina; 16 Clinica Ematologica, Centro Trapianti e Terapie Cellulari, Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy

Background: Elderly patients in complete remission (CR) after first-line therapy for acute myeloid leukemia (AML) relapse within months unless additional therapy is given. In elderly patients with AML in CR after intensive chemotherapy, the HOVON 97 study reported on the efficacy and safety of azacitidine (5-Aza) post-remission therapy and the randomized QUAZAR trial showed that CC-486 as maintenance therapy was associated with significantly longer overall and relapse-free survival than placebo. We report final results of the QoLESS AZA-AMLE Phase III, randomized, open label, Italian multicentre, trial to evaluate the efficacy of 5-Aza for post-remission therapy of elderly AML patients compared to best supportive care (BSC).

Aims: The primary endpoint is disease-free survival (DFS, from CR to relapse/death at 2 and 5 years. Secondary endpoints are post-remission hospitalizations, overall survival (OS) at 2 and 5 years and changes in quality of life (QoL).

Methods: Patients aged ≥ 61 years with untreated AML, “de novo” or evolving from MDS, fit for intensive chemotherapy but ineligible for stem cell transplantation received induction chemotherapy consisting of 2 courses of 3 + 7 (Daunorubicin 40 mg/m2 daily days 1-3 and cytarabine 100 mg/m2 intravenous infusion days 1-7. Cases in CR received cytarabine 800 mg/m2 3-hour infusion twice daily for 3 days. Patients in CR were randomized to receive BSC or 5-Aza 50 mg/m2 for 7 days every 28 days and dose increase after 1st cycle to 75 mg/m2 for further 5 cycles, followed by cycles every 56 days for 4.5 years. QoL was assessed with QOL-E version 3 and EORTC QLQ-C30 version 3.0.

Results: 149 patients were enrolled to finally randomize 54 patients alive and in CR (Table 1). After randomization, no patients died before relapse, 7 cases completed the study while 43 subjects relapsed: at 2 years, 22 cases in BSC (median DFS: 6.0 months, 95% CI: 0.2–11.7) versus 18 in the 5-Aza arm (median DFS: 10.8 months, 95% CI: 1.9-19.6; p=0.20); at 5 years, 1 subject on BSC withdrew consent and 23 subjects relapsed (median DFS: 6.0 months, 95% CI: 0.2–11.7) while 2 subjects on 5-Aza withdrew consent, 1 discontinued for bladder cancer and 20 relapsed (median DFS: 10.8 months, 95% CI: 1.9-19.6; p=0.23). Age modified the effect of the allocation arm on DFS. Data adjustment for cytogenetic risk further amplified the efficacy of 5-Aza in patients aged >68 years, both at 2 (HR: 0.24, 95% CI: 0.08-0.69, P=0.008) and 5 years (HR: 0.28, 95% CI: 0.10-0.76, P=0.012). The effect of 5-Aza versus BSC tended to be statistically significant on DFS at 2 (P=0.053) and 5 years (P=0.068) in Cox models including cytogenetic risk and MRD as covariates. Kaplan-Meier analyses of the effect of 5-Aza on DFS (at 2 and 5 years) showed that patients aged >68 years and positive MRD tended to have a benefit in terms of DFS at both 2 and 5 years (P=0.056). In the 5-Aza arm, 5 subjects out of 27 (19%) were hospitalised whereas no patient was hospitalised in the BSC arm (P=0.023). QoL was similar in both arms after randomization. Twenty grade 3-4 adverse events, mainly neutropenia, occurred in 5-Aza versus 1 in BSC arm (p=0.002).

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Summary/Conclusion: 5-Aza is well tolerated in elderly patients with AML who have achieved CR following standard chemotherapy. MRD remains an important predictor of DFS in the long-term, independent of 5-Aza maintenance. No statistically significant differences were observed between the two arms in terms of DFS, but after data adjustment it is suggested that patients aged >68 years and positive MRD may benefit from 5-Aza maintenance.

P565: IMPROVED OUTCOME OF PATIENS WITH ACUTE MYELOID LEUKEMIA HARBORING FLT3 MUTATION IN THE ERA OF TARGETED THERAPY

G. Oñate 1,2,* , M. Pratcorona 1 , A. Garrido 1 , A. Artigas 1 , A. Bataller 3 , M. Tormo 4 , M. Arnan 5 , S. Vives 6 , R. Coll 7 , O. Salamero 8 , F. Vall-Llovera 9 , A. Sampol 10 , A. Garcia 11 , M. Cervera 12 , S. Garcia Avila 13 , J. Bargay 14 , X. Ortin 15 , J. Esteve 3 , J. Sierra 1

1 Hematology, Hospital de la Santa Creu i Sant Pau; 2 Universitat Autònoma de Barcelona; 3 Hematology, Hospital Clínic, Barcelona; 4 Hematology, Hospital Clínico Universitario, Biomedical Research Institute INCLIVA, Valencia; 5 Hematology, Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, Hospitalet de Llobregat; 6 Hematology, ICO-Hospital Germans Trias i Pujol, Badalona; 7 Hematology, ICO-Hospital Josep Trueta, Girona; 8 Hematology, Hospital Universitari Vall d’Hebron, VHIO; 9 Hematology, Hospital Universitari Mútua Terrassa, Barcelona; 10 Hematology, Hospital Son Espases, Palma de Mallorca; 11 Hospital Universitari Arnau de Vilanova, Lleida; 12 ICO-Hospital Joan XXIII, Tarragona; 13 Hematology, Hospital del Mar, Barcelona; 14 Hospital Son Llàtzer, Palma de Mallorca; 15 Hematology, Hospital Verge de la Cinta, Tortosa, Spain

Background: In recent years, the treatment of AML is rapidly evolving due to the advances in targeted therapy, risk-adapted protocols and MRD-guided decisions. The prognosis of patients harbouring FLT3 -ITD differs according to its allelic ratio and the presence of NPM1 co-mutation ( NPM1 mut). Nonetheless, FLT3 -ITD prognostic impact and allogeneic hematopoietic cell transplantation (alloHCT) indication in this setting might be redefined by the widespread use of FLT3 inhibitors. Since 2012, chemotherapy eligible patients are treated following a risk-adapted and MRD-guided protocol in the CETLAM group (AML-12). Since 2016, targeted therapy with midostaurin was progressively incorporated in the protocol for FLT3 -mutated ( FLT3 mut) patients.

Aims: To analyse the outcome of FLT3 mut AML treated with a risk-adapted protocol in two different time periods, mainly differentiated by the advent of midostaurin.

Methods: All adult patients with de novo AML and available FLT3 status treated with the intensive protocol AML-12 (clinicaltrials.gov # NCT04687098 ) in the Spanish CETLAM centres from 2012 to 2020 were retrospectively analysed. Molecular testing of NPM1 , and FLT3 -ITD and its allelic ratio were studied as previously described. Patients were divided into two study cohorts: the early (2012-2015) and the late cohort (2016-2020). All patients received the same chemotherapy protocol except for the addition of midostaurin since 2016 in most FLT3 mut patients. Complete remission (CR), overall survival (OS), event-free survival (EFS) and risk of relapse (RR) followed ELN criteria. OS and EFS were studied with the Kaplan-Meier method and log-rank test, cumulative incidence and Grey test were used to estimate relapse risk (CIR) and non-relapse mortality (NRM).

Results: A total of 906 patients were selected, 390 from the early and 516 from the late cohort. Median follow-up was 38 months; FLT3 mut was present in 227 cases (25%) and constitute the focus of this study. There were not differences on main variables between cohorts (age, cytogenetic risk, ELN category, frequency of NPM1 mutation or FLT3 -ITD allelic ratio). TKD mutations were only available since 2015. Midostaurin was administered to 62% of FLT3 mut patients from the late cohort: 63 with ITDs (20 low and 43 high ratio) and 20 TKDs. CR rates were similar between cohorts. AlloHCT in first CR (CR1) was performed in 60% (early) and 67% (late) patients (p=ns). A higher RR was observed in the early group in comparison to the late cohort (2-year RR 42±11% vs 28±10%; p=0.014), without differences in NRM (Figure 1B), translating into an improved EFS (median EFS 9.4 vs. 26 months, p=0.014) and OS in the late cohort (median OS: 15 months vs. non-reached, p=ns, Figure 1A). Among NPM1 mut AML patients, FLT3 -ITD and ELN 2017 categories retained its prognostic value in the early cohort, with a worse OS, EFS, and higher RR among FLT3 -ITD vs wt patients (HR for OS 1.9 95% CI 1.15-3.2, p=.011; figure 1C) and FLT3 mut ELN-intermediate vs favourable patients (OS p<0.001; figure 1D). On the contrary, in the late cohort, FLT3 -ITD and FLT3 high allelic ratio lost their adverse prognostic impact, with comparable RR, EFS, and OS regardless of FLT3 -ITD status (Figure 1E and F).

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Summary/Conclusion: This real-life study demonstrated an improved outcome among FLT3 mut AML patients treated in the most recent period, probably reflecting the beneficial effect of midostaurin on relapse prevention in this context. Future analyses should revise the role of alloHCT in CR1 for non-favourable FLT3 mut AML patients treated with FLT3 inhibitors who achieve MRD eradication.

P566: IMPACT OF FRONTLINE INDUCTION APPROACH ON POST-STEM CELL TRANSPLANT (SCT) OUTCOMES IN OLDER ADULTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA (AML)

F. Ong 1,* , F. Ravandi 1 , U. Popat 2 , T. M. Kadia 1 , N. Daver 1 , C. DiNardo 1 , M. Konopleva 1 , G. Borthakur 1 , E. J. Shpall 2 , B. Oran 2 , G. Al-Atrash 2 , R. Mehta 2 , E. J. Jabbour 1 , M. Yilmaz 1 , G. C. Issa 1 , G. Garcia-Manero 1 , A. Maiti 1 , H. A. Abbas 1 , R. E. Champlin 1 , N. J. Short 1

1 Leukemia; 2 Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, HOUSTON, United States of America

Background: A hypomethylating agent plus venetoclax (VEN) is standard of care for older, unfit patients (pts) with newly diagnosed AML. However, the optimal induction regimen for older pts with AML who are adequately fit for SCT is not well-established.

Aims: We investigated the post-SCT outcomes of older adult pts with newly diagnosed AML based on the intensity of the induction regimen received.

Methods: This is a retrospective analysis of pts ≥60 years (yr) of age with newly diagnosed AML who received allogeneic SCT after achieving first remission (CR, CRi or MLFS) between 9/2012 and 7/2021 at our cancer center. Out of 127 pts, 44 pts received intensive chemotherapy (IC) without VEN as induction therapy, 36 pts received low-intensity therapy (LIT) without VEN, and 47 pts received LIT with VEN. Overall survival (OS), relapse-free survival (RFS), cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) after SCT were compared among the groups.

Results: The baseline characteristics of the groups are shown in Table 1. Pts in the IC cohort was significantly younger (median age of 63 yrs) than pts who received LIT with or without VEN (median age of 68 yrs, p<0.01). Pts in the IC group were more likely to have ECOG PS of 0 (34%) as compared to those who received LIT (14%, p=0.07). Most pts had adverse ELN cytomolecular risk on presentation (43% in IC, 50% in LIT without VEN, and 55% in LIT with VEN). The median numbers of cycles prior to SCT were 3 in all groups. Pts in LIT without VEN group were more likely to receive matched unrelated donor (75%) than those in the LIT with VEN (49%) and IC groups (50%) and were less likely to receive a haploidentical SCT (3% vs. 17% and 14%, respectively). Pts in LIT with VEN group were more likely to receive reduced-intensity conditioning (92%) prior to SCT, compared with 53% and 58% in the IC and LIT without VEN groups, respectively (p<0.01). HLA matching was well-balanced in 3 groups. The majority of pts were in CR/CRi prior to SCT (100% in the IC group, 95% in the LIT without VEN group, and 92% in the LIT with VEN group), while the rest of pts achieved MLFS. MRD negativity prior to SCT was achieved in more pts treated with IC (77%) or LIT with VEN (66%), compared with LIT without VEN (49%). For the entire cohort, the median follow-up was 37 months (mos) post-SCT. Post-SCT outcomes are shown in Figure 1. The 2-yr OS was highest in the LIT with VEN group (73%) when compared with the IC or LIT without VEN groups (58% and 44%, respectively); OS was statistically superior with LIT with VEN compared with LIT without Ven (p=0.02) and there was a trend towards superior OS compared with IC (p=0.17). 2-yr RFS was also superior in the LIT with VEN group (62%) as compared with the IC or LIT without VEN groups (54% and 42%, respectively). 2-yr CIR was highest in LIT without VEN group (36%), and was similar in the LIT with VEN (19%) and IC groups (18%). 2-yr NRM was highest in IC group (27%) as compared with the LIT with VEN (11%) and LIT without VEN groups (22%) (p=0.02 for IC vs. LIT with VEN).

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Summary/Conclusion: LIT with VEN resulted in comparable—and possibly superior—outcomes compared with IC in SCT eligible patients ≥60 yrs with newly diagnosed AML. Despite the older age and worse PS of the LIT with VEN group, their post-SCT NRM was lower than those who received IC, which translated to numerically superior OS. These results support LIT with VEN as an effective and tolerable induction strategy in older SCT-eligible pts with newly diagnosed AML.

P567: CENTRAL NERVOUS SYSTEM INVOLVEMENT IN ADULT PATIENTS WITH ACUTE MYELOID LEUKEMIA – INCIDENCE AND OUTCOME

M. Virijevic 1,2,* , I. Djunic 1,2 , M. Mitrovic 1,2 , N. Pantic 1 , Z. Pravdic 1 , N. Sabljic 1 , A. Novkovic 3 , M. Cvetovic 2 , J. Rajic 1 , A. Vidovic 1,2 , M. Todorovic-Balint 1,2 , N. Suvajdzic-Vukovic 1,2

1 Clinic of Hematology, University Clinical Center of Serbia; 2 Faculty of Medicine, University of Belgrade; 3 Department of Hematology, Clinical Hospital Center “Zemun”, Belgrade, Serbia

Background: Examination of central nervous system (CNS) involvement is not routine diagnostic in adult patients with acute myeloid leukemia (AML). Therefore, its impact on the disease course is not well defined. There are studies showing that CNS involvement in AML is associated with a worse outcome, whereas others did not confirm such impact on long-term survival.

Aims: To determine the incidence of CNS involvement, its impact on the disease free and overall survival, and to define risk factors for CNS involvement.

Methods: This single center, retrospective study involved 645 adult patients with nonpromyelocytic AML, diagnosed in period of 2013. and 2021. In patients with the presence of CNS symptoms, with increased leukocyte count (≥30x10 9 /L), FLT3 mutation, monocyte phenotype, CD56 positivity, a lumbar puncture was performed. All cerebrospinal fluid (CSF) samples were examined by flow cytometry (FCM). Treatment of CNS+ include intrathecal CT: cytarabine 100 mg. Assessment of remission of CNS+ was performed after 8 i.t. CT. The following parameters were estimated as risk factors for CNS+ at diagnosis of AML: age, WBC (<30x10 9 /L vs. ≥30x10 9 /L), ECOG PS, HCT CI score, elevated lactate dehydrogenase (LDH), leukemia-related parameters (cytogenetics (including ELN risk stratification), flow cytometry). The methods of descriptive and analytical statistics were used. Univariate and multivariate Cox Proportional Hazards models were used to identify risk factors.

Results: CNS involvement examination was performed in 272 patietns. A total of 55/272 (20.2%) patients had proven CNS involvement, while 217/272 (79.8%) were without neuroleukemia. Complete remission was achieved by 168/272 (61.7%) of patients, but there was no statistically significant difference between CNS positive and CNS negative patients (p=0.619). Patients with CNS involvement at diagnosis had a significantly higher frequency of hyperleukocytosis (≥30x10 9 /L), age ≤50 years, French–American–British M5 subtype, expression of CD56 and CD15 antigen. Multivariate analysis identified CD56+ as the most important risk factor for CNS+ at diagnosis in AML patients: p=0.003, HR 2.271; 95% confidental interval (CI)= 1.320-3.908. There were no statistically significant differences in 5-year overall survival and disease free survival between CNS positive and CNS negative patients.

Summary/Conclusion: Our study showed a high incidence of CNS involvement in AML patients, compared to studies in which the incidence was significantly less (3.3%). Our study confirmed the accuracy of the factor we used to assess CNS involvement. There was no difference in the outcome between patients with CNS involvement and those without.

P568: REAL-LIFE EXPERIENCE OF TREATMENT REFRACTORY/RELAPSED ACUTE MYELOID LEUKEMIA PATIENTS WITH VENETOCLAX COMBINATION THERAPY

H. Pomares Marin 1,* , J. Villarreal Hernández 2 , M. Condom Esteve 2 , S. Vives Polo 3 , M. Cervera Calvo 4 , R. Coll Jorda 5 , C. Maluquer Artigal 1 , G. Ibarra Fernández 3 , A. Torrent Catarineu 3 , M. Galiano Barajas 1 , A. M. Sureda Balari 1 , M. Arnan Sangerman 1

1 Servei d’Hematologia, Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat; 2 Servei d’Hematologia, Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat; 3 Servei d’Hematologia, Institut Català d’Oncologia, Hospital Germans Trias i Pujol, Institut de Recerca contra la Leucèmia Josep Carreras, Universitat Autònoma de Barcelona, Badalona; 4 Servei d’Hematologia, Institut Català d’Oncologia, Hospital Universitari Joan XXIII, Tarragona; 5 Servei d’Hematologia, Institut Català d’Oncologia, Institut d’Investigació Biomèdica de Girona (IDIBGI), Universitat de Girona, Girona, Spain

Background: Venetoclax, a BCL-2 specific inhibitor, used in combination with azacitidine in patients with newly-diagnosed acute myeloid leukemia (AML) who were ineligible for intensive chemotherapy treatment, has shown high response rate and overall survival compared with azacitidine monotherapy ( Di Nardo et al. N Engl J Med 2020; 383:617-629 ). Moreover, Venetoclax in combination with hypomethylating agents or with low-dose cytarabine is being explored in other settings being frequently used in relapsed/refractory (R/R) AML.

Aims: We performed a retrospective study of patients with R/R AML receiving venetoclax combinations in the Catalan Institute of Oncology (ICO) in order to determine the efficacy and safety of the combination.

Methods: We analyze 60 patients diagnosed with R/R AML at 4 hospitals belonging to ICO in Spain, treated with venetoclax (400mg/24h; initial daily dose of 100mg with a 3-day ramp-up to target dose of 400mg) in combination with hypomethylating agents (Azacitidine 75mg/m 2 7/28 days or Decitabine 20mg/m 2 5/28 days) or low-dose cytarabine (20mg/m 2 10/28 days) from May 2019 until December 2021. Event was defined as death, refractoriness to treatment or progressive disease.

Results: Characteristics of our cohort are described in Table 1. Notably, 31 (52%) patients had high-risk AML according to ELN 2017 classification. Fourteen (23%) patients received venetoclax in combination with decitabine, 34 (57%) patients with azacitidine, and 12 (20%) patients with low-dose cytarabine. The median number of cycles received was 3 (range 1-28), with a median of one cycle to achieve the best response (range: 1-4). Early mortality in the first 30 days was 15% (9 patients), 5 due to progression disease, 2 due to infection and 2 due to clinical worsening. Overall response rate after first cycle (Complete response (CR) + complete response without haematological recovery (CRi) + partial response (PR)) was 58%. Three patients were treated in a molecular-MRD positive status, and all of them achieved molecular response. Six of 19 patients (32%) were transitioned to allogeneic stem cell transplantation (alloSCT). The median event-free survival and overall survival was 5.03 months and 7.2 months, respectively. Response to treatment after 3-4 cycles, discriminate two groups of patients with an OS of 12.66 months in those patients who achieved CR or PR vs 2.4 months in non-responders (p0.000). Patients relapsed after alloSCT (7 patients) presented a poor outcome (median OS was 1.6 months).

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Summary/Conclusion: Our study showed that real-world experience of treating patients with R/R AML with venetoclax in combination with hypomethylating agents or low-dose cytarabine is feasible as salvage treatment in patients relapsed to hypomethylating agent and as bridge therapy to alloSCT with a rapid response rate. Moreover, rapid responses shown with the combination, allow us to identificate those patients who may benefit from this approach.

P569: GILTERITINIB AND QUIZARTINIB IN RELAPSED/REFRACTORY (R/R) ACUTE MYELOBLASTIC LEUKEMIA (AML) WITH FLT3 MUTATIONS: A REAL-LIFE EFFECTIVENESS AND SAFETY STUDY

D. Quintela 1,* , M. Morgades 1 , A. Serrano 2 , M. Cervera 3 , A. Balerdi 4 , M. Díaz-Beyá 5 , M. Arnan 6 , A. Garrido 7 , R. Coll 8 , M. Tormo 9 , J. López-Marin 10 , B. Merchan 11 , S. Garcia 11 , M. Casado 12 , A. Sampol 13 , J. Esteve 5 , D. Martínez-Cuadrón 2 , J. Sierra 7 , M. Á. Sanz 2 , J. M. Ribera 1 , P. Montesinos 2 , S. Vives 1

1 Clinical Hematology, ICO-Hospital Universitari Germans Trias i Pujol, Institut de Recerca Josep Carreras, Badalona; 2 Clinical Hematology, Hospital Universitari i Politècnic La Fe, Valencia; 3 Clinical Hematology, ICO-Hospital Joan XXIII, Tarragona; 4 Clinical Hematology, Hospital Universitario Cruces, Barakaldo; 5 Clinical Hematology, Hospital Clínic de Barcelona, Barcelona; 6 Clinical Hematology, ICO-Hospital Duran i Reynals, L’Hospitalet del Llobregat; 7 Clinical Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona; 8 Clinical Hematology, ICO-Hospital Josep Trueta, Girona; 9 Clinical Hematology, Hospital Clínico Universitario de Valencia, Valencia; 10 Clinical Hematology, Hospital General Universitario de Alicante, Alicante; 11 Clinical Hematology, Hospital del Mar, Barcelona; 12 Clinical Hematology, Hospital Universitario de Badajoz, Badajoz; 13 Clinical Hematology, Hospital Universitari Son Espases, Palma, Spain

Background: Patients with R/R AML have poor prognosis. FLT3 mutations worsened the prognosis but offer a targeted therapy. FLT3 inhibitors have demonstrated efficacy as monotherapy in FLT3-positive R/R AML.

Aims: The objective of the present study was to analyze the efficacy and safety of Gilteritinib and Quizartinib in R/R FLT3-mutated AML in real-life context.

Methods: Between December 2016 and April 2021, 22 patients were treated with Gilteritinib and 5 patients with Quizartinib in 13 centers of the Spanish PETHEMA and CETLAM groups.

Results: The median age was 62 years (range 25; 81) and 56% were women. Most patients presented ITD FLT3 (80%), 56% were refractory and 44% were relapsed (CR1 duration ≥6 months in 60%). The 1 st line therapy was intensive chemotherapy (78%, 38% with midostaurin) or HMA (22%). A total of 13/27 patients (48%) had previously received a FLT3 inhibitor (midostaurin=6, sorafenib=2, quizartinib=2, midostaurin + crenolanib vs placebo=2 and midostaurin + quizartinib=1). Seventy percent of the patients received >1 line of treatment prior to Gilteritinib/Quizartinib (the most frequent was FLAG-IDA). Compared with diagnosis, patients in R/R had poorer functional status (ECOG<2 88% vs. 73%) but lower leukocyte counts, LDH level and FLT3 ITD ratio (21.5 x10 9 /L vs 4.7x10 9 /L, 563 U/L vs 330 U/L and 0.60 vs 0.42, respectively). Three patients displayed clonal evolution in R/R with acquisition of cytogenetic alterations in patients with altered karyotype at diagnosis and appearance of alterations in patients with normal karyotype. The ORR was 63% (21% CR, 21% CRi and 21% PR) and the median OS was 5.8 (95%CI, 3.8; 7.9) months. Time to achieve better response was 1.8 months (range 0.9;4.1). Eight patients received the FLT3 inhibitor as a bridge to SCT, and six of them were finally transplanted. Gilteritinib dose was 120mg/day, increasing to 200mg/d in 41% of patients (due to lack of response after 28 days). Toxicity was observed in 69% of patients (g3/4 in 30%). Febrile neutropenia was the most frequent toxicity (35%) followed by hepatotoxicity (28%) and QTc prolongation (16%). One patient died of toxicity attributed to Gilteritinib (febrile neutropenia).

Summary/Conclusion: Treatment with Gilteritinib and Quizartinib as monotherapy is an effective and tolerable option for patients with R/R FLT3-mutated AML in real-life, with similar response rates and toxicity to those reported in Phase 3 trials, despite the fact that the study population from our series was more heterogeneous.

P570: REAL-WORLD EFFICACY OUTCOMES OF VENETOCLAX PLUS AZACITIDINE VS INTENSIVE CHEMOTHERAPY FOR INDUCTION THERAPY IN ADULT PATIENTS WITH ACUTE MYELOID LEUKEMIA

A. M. Zeidan 1,* , D. A. Pollyea 2 , U. Borate 3 , A. Vasconcelos 4 , R. Potluri 5 , D. Rotter 5 , Z. Kiendrebeogo 5 , L. Gaugler 4 , G. Bonifacio 4 , T. Prebet 4 , C. Chen 4

1 Yale University School of Medicine, New Haven, CT; 2 University of Colorado School of Medicine, Aurora, CO; 3 Oregon Health & Science University, Portland, OR; 4 Bristol Myers Squibb, Princeton, NJ; 5 SmartAnalyst Inc., New York, NY, United States of America

Background: Intensive chemotherapy (IC) is commonly used to achieve remission in patients with newly diagnosed acute myeloid leukemia (AML), although the relapse rate remains high. The combination of venetoclax, a BCL2 inhibitor, plus intravenous or subcutaneous azacitidine (ie, VEN-AZA), is FDA- and EMA-approved for the treatment of patients with newly diagnosed AML aged ≥ 75 years or with comorbidities that preclude the use of IC. Because of the challenging induction and maintenance decisions for some patients, it is important to understand the real-world patterns of use and outcomes of VEN-AZA vs IC.

Aims: To compare US real-world outcomes for the use of VEN-AZA vs IC, particularly in older populations.

Methods: This retrospective analysis was performed on a cohort of patients from the US-based Flatiron Health electronic health record-derived de-identified database, who had newly diagnosed AML and received induction therapy with VEN-AZA or IC between 21 Nov 2018 and 01 Jun 2021. Using an intention-to-treat analysis (including patients with less than 2 months of follow-up), patient groups were propensity score-matched in a 1:1 ratio using nearest neighbor matching with calipers set to 0.01 to reduce bias. Matched-patient assessments included morphological complete remission, overall survival (OS; defined as from first day of therapy to death), and relapse-free survival (RFS; from first day of therapy to relapse or death). Univariate analyses were used to investigate possible treatment-associated OS and RFS benefit in patient subgroups, including those with high-risk mutations.

Results: Of 7484 patients in the Flatiron database with newly diagnosed AML, 1188 met the selection criteria and 436 were propensity score-matched resulting in 218 per treatment group. In the matched VEN-AZA vs IC cohorts, patients had a mean age of 69.9 vs 69.0 y and Charlson Comorbidity Index of 0.5 vs 0.7, respectively; 39% vs 38% were female. The rates of patients with complete remission (CR) and those with stem cell transplant (SCT) were significantly lower with VEN-AZA vs IC at 46% vs 62% ( P =0.001) and 16% vs 31% ( P <0.001), respectively. Fewer VEN-AZA– vs IC-treated patients received an early posttreatment bone marrow biopsy, with respective rates of 31% vs 69% (1 mo) and 57% vs 86% (2 mo). Median OS for VEN-AZA vs IC in the matched population was 13.0 vs 17.2 mo ( P =0.263), respectively; it was 11.0 vs 15.6 mo ( P =0.410) with censoring for SCT. Median RFS was numerically shorter for VEN-AZA than for IC (9.5 vs 11.0 mo; P =0.689); when including censoring for SCT, RFS was longer for VEN-AZA (7.5 vs 5.7 mo; P =0.027). Among subgroups, including those with high-risk mutations, no significant factor was found to be associated with OS in the matched patients (Figure). The frequency of known patients with high-risk mutations in RUNX1 , ASXL1 , and TP53 was similar for both treatment groups. Similar subgroup results were observed for RFS.

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Summary/Conclusion: Rates of CR and SCT were significantly higher with IC. OS was nominally longer with IC vs VEN-AZA, with and without censoring for SCT; differences were not statistically significant. None of the subgroups tested, including patients with high-risk mutations, showed a significant OS association. Longer follow-up time may contribute to better characterized outcomes achieved with IC vs VEN-AZA in routine clinical practice. These data support more investigation on strategies to improve survival outcomes, including the impact of maintenance therapy.

P571: ORAL AZACITIDINE VS MIDOSTAURIN AS MAINTENANCE TREATMENT FOR FLT3 MUTANT ACUTE MYELOID LEUKEMIA IN COMPLETE REMISSION: AN INDIRECT TREATMENT COMPARISON

E. N. Oliva 1,* , C. Chen 2 , S. N. Rahim 2 , B. Seuro 3 , A. Vasconcelos 2 , L. Gaugler 2 , B. Skikne 2 , T. Prebet 2 , H. Cameron 3 , C. Beach 2 , G. Thompson 2

1 Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio Calabria, Italy; 2 Bristol Myers Squibb, Princeton, NJ; 3 Eversana, San Diego, CA, United States of America

Background: Patients (pts) with FLT3 -mutant (mut) acute myeloid leukemia (AML) have a high risk of relapse, poor prognosis, and limited treatment (tx) options. A trend toward improved survival with oral azacitidine (Oral-AZA) vs placebo (PBO) as maintenance treatment (MT) was observed in pts in first complete remission (CR) after intensive chemotherapy (IC) induction ± consolidation in an exploratory analysis of the phase 3 QUAZAR AML-001 study (QUAZAR; NCT01757535 ). Midostaurin (MIDO) plus chemotherapy was FDA-approved as induction and consolidation tx for pts with FLT3 -mut AML based on results of the phase 3 RATIFY study ( NCT00651261 ); absence of randomization prior to MT makes inferences about the effectiveness of MIDO as MT difficult, although MIDO was EMA-approved for MT. The relative efficacy of MT with Oral-AZA vs MIDO has not been compared directly in clinical trials.

Aims: To compare the survival outcomes of MT with Oral-AZA vs MIDO in pts with FLT3 -mut AML who achieved first CR after IC (± consolidation tx) by performing an indirect tx comparison (ITC) of data from QUAZAR and RATIFY.

Methods: An ITC of overall survival (OS) and relapse-free survival (RFS) comparing Oral-AZA vs MIDO was performed, in which data from QUAZAR pts with FLT3 -mut AML who had achieved a CR with full blood count recovery (matching RATIFY eligibility criteria) were compared with efficacy outcomes from the RATIFY MT phase (Larson RA, et al. Leukemia 2021). Primary analyses utilized the anchored Bucher method, which assumes the relative tx effect is comparable across studies with similar effect modifiers. Time-varying methods (parametric and spline models) were also explored to account for violation of the proportional hazards (PH) assumption.

Results: Of 472 pts in the intent-to-treat population of QUAZAR, 56 pts with FLT3 -mut AML achieving a CR (Oral-AZA, 24; PBO, 32) were included in the primary analysis population, as were all 205 RATIFY MT-phase pts (MIDO, 120; PBO, 85). In the QUAZAR and RATIFY populations, respectively, mean age was 66.4 and 42.7 y, 55% and 50% were female, 36% and 29% had point mutations in the FLT3 tyrosine kinase domain, 0% and 57% had favorable cytogenetic risk, and 0% and 5% underwent stem cell transplantation during first CR. Bucher ITCs of both OS and RFS appeared to favor Oral-AZA over MIDO, but were not statistically significant (Table). To assess age imbalance (QUAZAR pts were ≥ 55 y; RATIFY pts, 18–59 y), an exploratory analysis with QUAZAR pts (n = 9; age-matched to RATIFY pts) showed OS and RFS results with Oral-AZA vs PBO were similar to those of all QUAZAR pts. Evidence suggests the PH assumption had been violated in some of these analyses, so best fitting parametric models were explored. Landmark OS and RFS rates (generalized gamma model) were found to be higher with Oral-AZA than MIDO (Table). Similar results were obtained using a spline model.

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Summary/Conclusion: Despite more favorable prognostic factors among pts in RATIFY (eg, younger age), the Bucher ITC analysis showed a trend toward longer OS and RFS with Oral-AZA vs MIDO as MT for pts with FLT3 -mut AML. Analyses that accounted for the PH assumption violation also indicated a survival benefit for Oral-AZA vs MIDO. Limitations included trial differences and small sample size. More research is needed to support this observation.

P572: VENETOCLAX AND HYPOMETHYLATING AGENT COMBINATIONS FOR THE TREATMENT OF ADVANCED MYELOPROLIFERATIVE NEOPLASMS AND ACUTE MYELOID LEUKEMIA WITH EXTRAMEDULLARY DISEASE

K. Sanber 1,* , K. Ye 2 , H.-L. Tsai 1 , M. Newman 3 , A. Ambinder 1 , A. DeZern 1 , T. Jain 1

1 Department of Oncology; 2 School of Medicine; 3 Department of Pharmacy, Johns Hopkins University, Baltimore, United States of America

Background: Progression of myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), into accelerated phase (AP) or blast phase (BP) disease is associated with poor prognosis and decreased responsiveness to intensive chemotherapy regimens that are typically used in young and fit patients with acute myeloid leukemia (AML). Although the combination of a hypomethylating agent (HMA) and venetoclax (HMA/venetoclax) is being increasingly utilized to treat patients with advanced MPN or MDS/MPN as well as AML with extramedullary disease (EMD), outcomes in these subgroups of patients is not well characterized as they were largely excluded from the pivotal VIALE-A trial.

Aims: We sought to evaluate the outcomes associated with HMA/venetoclax in disease groups that were not well-represented in the pivotal phase III VIALE-A trial.

Methods: We performed an IRB-approved, retrospective chart review of patients who received HMA and venetoclax (for at least 14 days) between 1/1/2016 and 5/1/2021 at Johns Hopkins University. Composite complete response included patients who attained a complete molecular response (CMR), complete cytogenetic response (CCR) or acute leukemia response-complete (ALR-C) for patients with advanced MPN or MDS/MPN based on consensus guidelines (Mascarenhas et al., 2012), as well as complete response (CR) and CR with incomplete hematologic recovery (CRi) for patients with AML and EMD based on the ELN 2017 criteria (Dohner et al., 2017).

Results: The composite CR rate and median overall survival (OS) were: 42.9% and 9.7 months for the overall population (n=35), 42.9% and 10.2 months for the BP cohort (n=21), 60% and 9.0 months for the AP cohort (n=5), 33.3% and 8.1 months for the AML with EMD (n=9). A summary of patient outcomes and their mutational landscape is represented in Figure 1. Five out of 15 patients who achieved a composite CR went on to receive an allogeneic bone marrow transplant. Patients with advanced MDS/MPN had numerically higher composite CR rate (70.0% versus 31.3%; P = 0.1054) and median OS (11.9 months versus 5.1 months; P = 0.1585) compared to patients with advanced MPN. Patients with a mutation in the SRSF2 gene had a higher composite CR rate (80% versus 20%; p= 0.0082) and median OS (10.9 months versus 8.0 months; P = 0.2614) compared to those with a wild-type SRSF2 gene. On the other hand, there was a trend towards lower composite CR in AML/blast phase patients who had EMD compared to those without EMD (33% vs 67%; P =0.1414).

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Summary/Conclusion: The composite CR rate (42.9%) and median OS (9.7 months) for the overall patient population in our study are lower than those reported in the VIALE-A trial (42.9% versus 66.4% and 9.7 months versus 14.7 months, respectively). This is not unexpected since advanced MPN and extramedullary AML are known to have a worse prognosis than de novo AML. While limited by a small sample size, our study suggests that HMA/venetoclax may be associated with more favorable outcomes in patients with advanced MDS/MPN compared to those with advanced MPN. Mutations in the SRSF2 gene may also be associated with more favorable outcomes. These findings will need to be confirmed in larger studies and their biological basis will need to be further investigated.

P573: PHARMACOKINETIC EXPOSURE EQUIVALENCE AND PRELIMINARY EFFICACY AND SAFETY FROM A RANDOMIZED CROSS OVER PHASE 3 STUDY OF AN ORAL HYPOMETHYLATING AGENT DEC-C COMPARED TO IV DECITABINE IN AML PATIENTS

K. Geissler 1 , Z. Koristek 2 , T. Bernal del Castillo 3 , J. Novák 4 , G. Rodriguez Macias 5 , S. K. Metzelder 6 , A. Illes 7 , A. Nagy 8 , J. Mayer 9 , M. Arnan 10 , M.-M. Keating 11 , J. Krauter 12 , M. Lunghi 13 , N. Stefano Fracchiolla 14 , U. Platzbecker 15 , V. Santini 16 , Y. Sano 17,* , A. Oganesian 17 , H. Keer 17 , M. Lübbert 18

1 Clinic Hietzing, Vienna, Austria; 2 University Hospital Ostrava, Ostrava, Czechia; 3 Hospital Universitario Central de Asturias, Oviedo, Spain; 4 Department of Haematology, 3rd Faculty of Medicine, Charles University and Faculty Hospital Kralovske Vinohrady, Prague, Czechia; 5 Hospital General Universitario Gregorio Marañón, Madrid, Spain; 6 Philipps-Universität Marburg, Marburg, Germany; 7 Division of Haematologoy, Department of Internal Medicine, Faculty of Medicine, University of Debrecen, Debreccen; 8 1st Department of Internal Medicine, University of Pécs, Pécs, Hungary; 9 Fakultní Nemocnice, Brno, Czechia; 10 Hematology Department, Servei d’Hematologia, Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain; 11 Queen Elizabeth II (QEII) Health Sciences Centre, Halifax, Nova Scotia, Canada; 12 Städtisches Klinikum Braunschweig, Braunschweig, Germany; 13 Azienda Ospedaliero-Universitaria Maggiore della Carità Novara, Novara; 14 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milano, Italy; 15 University Hospital Leipzig, Leipzig, Germany; 16 MDS Unit, AOU Careggi, DMSC, University of Florence, Firenze, Italy; 17 Astex Pharmaceuticals, Inc., Pleasanton, United States of America; 18 Universitaetsklinikum Freiburg, Freiburg, Germany

Background: Parenterally administered hypomethylating agents (HMAs), decitabine (DEC) and azacitidine (AZA), are approved in Europe for adult patients with acute myeloid leukemia (AML) who are not candidates for standard induction chemotherapy as single agent or in combination with venetoclax. ASTX727 (DEC-C) is a fixed dose combination (FDC) tablet of 35 mg DEC and 100 mg cedazuridine, a novel cytidine deaminase inhibitor (CDAi). In clinical trials with myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemia (CMML) patients, DEC-C provides DEC exposures that are equivalent to IV DEC at the approved dose of 20 mg/m 2 daily×5 and is approved as INQOVI® in the US, Canada, and Australia.

Aims: To demonstrate DEC exposure bioequivalence of oral DEC-C and IV-DEC and generate clinical data using DEC-C in AML patients.

Methods: The ASCERTAIN study was a randomized cross over design. Patients were randomized 1:1 to either Sequence A: DEC-C (35 mg DEC/100 mg cedazuridine) in Cycle 1 followed by IV-DEC at 20 mg/m 2 in Cycle 2, or Sequence B: receiving IV-DEC in Cycle 1 followed by DEC-C on Cycle 2 to compare PK [primary endpoint Area Under the Curve (AUC) equivalence over 5 days of dosing]. All patients received DEC-C from Cycle 3 onwards until treatment discontinuation to assess safety and clinical efficacy. Patients were eligible as per the EMA-approved decitabine label (newly diagnosed AML who are not candidates for standard induction chemotherapy). Clinical responses were assessed according to modified International Working Group (IWG) 2003 response criteria.

Results: 89 patients were randomized, of whom 87 were treated. The median age of patients was 78.0 years (range, 61 to 92) with 31 (35.6%) males and 56 (64.4%) females. Cytogenetic risk classification was poor-risk in 33 (37.9%) and intermediate-risk in 45 (51.7%) patients. For the primary endpoint, preliminary PK data was available from 69 patients who successfully completed PK assessments for both IV DEC and DEC-C cycles, and the DEC AUC 0-24 (h*ng/mL) 5-Day geometric mean estimate was 904 for DEC-C and 907 for IV-DEC resulting in an oral/IV geometric LSM AUC ratio of 99.64% (90% CI of 91.23-108.8%). Safety findings were consistent with those anticipated for IV-DEC (related Grade ≥ 3 AEs in more than 10% were thrombocytopenia, anemia, febrile neutropenia, neutropenia, and pneumonia). As of the data cutoff date (10 SEP 2021), median follow up was 7.95 months (IQR 6.11-11.86). Of the 77 patients who had ≥6 months of follow up or discontinued treatment, the best response was complete response (CR) in 17 (22.1%, 95% CI: 13.4, 33.0%). In addition, 4 patients (5.2%) had CR with incomplete blood cell count recovery (CRi), with 1 patient (1.3%) who had CR with incomplete platelet recovery (CRp), resulting in composite response rate [CR + CRp] of 23.4% [18/77 patients, 95% CI: 14.5, 34.4%]. These results obtained with DEC-C are consistent with those observed for IV DEC. Based on preliminary and limited study follow-up with ~46% censored observations, the median survival was approximately 7.9 months (95% CI: 5.9, 13.0).

Summary/Conclusion: This randomized phase 3 study in AML patients not candidates for standard induction chemotherapy demonstrates that the oral FDC of DEC-C (35mg/100 mg) resulted in an equivalent DEC AUC exposure to IV-DEC at 20 mg/m 2 over 5 days. In addition, safety findings and preliminary clinical activity is also consistent with published data from IV-DEC, suggesting that DEC-C has the potential to be an oral alternative to the standard IV decitabine Daily×5 regimen.

P574: CPX-351 COMBINED WITH HEMATOPOIETIC CELL TRANSPLANTATION WITH REGULATORY AND CONVENTIONAL T CELL IMMUNOTHERAPY FOR HIGH-RISK ACUTE MYELOID LEUKEMIA

S. Sciabolacci 1,* , L. Ruggeri 1 , V. Cardinali 1 , L. Brunetti 1 , S. Tricarico 1 , S. Saldi 2 , F. Marzuttini 1 , M. Griselli 1 , G. Perta 1 , V. Viglione 1 , G. Cimino 1 , A. Osmani 1 , M. Caridi 1 , R. Sembenico 1 , A. Terenzi 1 , T. Zei 1 , R. Iacucci Ostini 1 , M. F. Martelli 1 , B. Falini 1 , A. Velardi 1 , C. Aristei 2 , C. Mecucci 1 , A. Carotti 1 , M. P. Martelli 1 , A. Pierini 1

1 Division of Hematology and Clinical Immunology; 2 Department of Surgical and Biomedical Science, Perugia General Hospital, Perugia, Italy

Background: Patients with newly diagnosed acute myeloid leukemia with myelodysplasia-related changes (MRC-AML) or therapy-related AML (t-AML) have an extremely poor prognosis. CPX-351, a dual-drug liposomal encapsulation of cytarabine and daunorubicin, improved significantly rates of remission and overall survival (Lancet JE et al, JCO 2018). Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative strategy for such patients, but incidence of post-transplant relapse is still unacceptably high. We developed a novel HSCT strategy that combines an age-adapted myeloablative irradiation-based conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy (Treg/Tcon HSCT). HLA-haploidentical Treg/Tcon HSCT ensured low relapse rate (4%) and resulted in an unprecedented chronic graft versus host disease/relapse free survival (CRFS, 75%) in AML patients (Pierini et al., Blood Adv. 2021).

Aims: We report preliminary outcomes of the combination therapy with CPX-351 and Treg/Tcon HSCT for MRC-AML and t-AML at our center.

Methods: We included in the study all patients with MRC-AML or t-AML who were referred at our center from September 2019 to January 2022 and could be treated with CPX-351. Treg/Tcon HSCT consisted of a myeloablative conditioning regimen with total marrow/lymphoid irradiation, thiotepa, fludarabine and cyclophosphamide followed by an infusion of 2x10 6 /Kg donor Tregs on day -4, 1x10 6 /Kg Tcons on day -1 and a megadose (>6x10 6 /Kg) of purified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GvHD prophylaxis was given post-transplant (clinicaltrials.gov: # NCT03977103 ).

Results: Seventeen patients with a median age of 57.5 years (46-72) were treated with CPX-351: 13 with newly diagnosed AML (11 MRC-AML and 2 t-AML) and 4 patients who relapsed after a previous HSCT and who acquired novel cytogenetic abnormalities (2 complex karyotypes, CK). 9/13 patients with newly diagnosed AML had karyotype abnormalities: 4 monosomy 7, 1 CK, 1 t(3;3), 1 isolated del(20q) and 2 (both t-AML) t(11;20) which resulted in NUP98-TOP1 fusion. Nine patients achieved complete remission (CR), 2 partial response (PR) and 2 had persistent disease. No patient experienced grade>3 extrahematologic toxicity.

Ten/13 patients underwent a first HSCT (8 in CR, 2 in PR). Eight underwent Treg/Tcon HSCT (3 from HLA-matched, 5 from HLA-haploidentical donor), while 2 unmanipulated haploidentical HSCT followed by post-transplant cyclophosphamide (PT-Cy). All patients achieved full-donor engraftment. No patients experienced non-relapse mortality (NRM). Three/10 patients had a grade ≥ III acute GvHD; all of them are alive and off immunosuppressive therapy. At a median follow up of 16.2 months (range 9-29 months), 9/10 patients are alive and in CR with no chronic GvHD. No relapse occurred in the 8 patients who underwent Treg/Tcon HSCT, while one patient who underwent HSCT followed by PT-CY relapsed.

The 4 patients who were treated after one previous HSCT received one single induction cycle with CPX-351, achieved CR and experienced no grade>3 extra-hematologic toxicity. All of them could undergo a second HSCT. NRM occurred in 1 patient (septic shock), while 3 patients are alive to date.

Summary/Conclusion: CPX-351 allowed most of MRC-AML and t-AML patients to reach HSCT in CR despite high risk genetics and even when used in patients who relapsed after a 1 st HSCT. Combination of CPX-351 with Treg-Tcon HSCT is safe and exert powerful antileukemic activity. Thus, it appears to be a promising strategy to ensure high rate of CRFS in very high-risk AML.

P575: IMPACT OF MOLECULAR RESPONSE AND CHEMOTHERAPY REGIMEN ON OUTCOMES IN CORE BINDING FACTOR AML

J. Senapati 1,* , F. Haddad 1 , F. Ravandi 1 , T. Kadia 1 , C. DiNardo 1 , N. Daver 1 , N. Pemmaraju 1 , Y. Alvarado 1 , M. A. Brandt 1 , H. Kantarjian 1 , G. Borthakur 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: Fludarabine based induction/consolidation regimens result in excellent outcomes in core binding factor acute myeloid leukemia (CBF-AML) Attainment of early polymerase chain reaction (PCR) based optimal minimal residual disease (MRD) responses positively impact long term outcomes and provide early decision timepoints for alternative therapies including stem cell transplantation (SCT).

Aims: We studied factors that affected early and late optimal PCR responses (OPR) and survival in patients (pts) of CBF AML who were treated with fludarabine based regiemns (fludarabine, cytarabine and GCSF) with idarubicin (FLAG-IDA) or gemtuzumab (FLAG-GO).

Methods: This is a prospective study of pts with N/D CBF-AML treated at our center on FLAG based regimens. Pts received up to 7 cycles of therapy with appropriate dose reductions based on age or toxicity. PCR for the disease defining transcripts ( CBFB::MYH11 or RUNX1::RUNX1T1 ) were monitored after C1, C3 and end of therapy (EOT). Based on our earlier report (Boddu et. al., Leukemia 2018 ) OPR was defined as PCR transcript <0.1% after C1 and <0.01% for later time points.

Results: Between 4/2007 to 12/2019, 174 pts, with a median age of 51 years (range, 19-78) were included. A total of 109 pts (63%) received FLAG-IDA, while the rest got FLAG-GO therapy. This was mostly due to withdrawal of approval of GO in the US for a period of time. Median number of cycles received was 6 (range, 1-7) Baseline parameters are described in Table 1.

PCR data is available for 94% pts after C1, 90% after C3 and 92% at EOT. EOT was considered the timepoint at which pt stopped further FLAG based therapy beyond C1. The percent of patients achieving OPR at each time point by regimen are: 68% vs. 41% (p=0.002) after C1, 75% vs. 39% (P< 0.0001) after C3 and 92% vs. 55% (p<0.0001) at EOT for FLAG-GO and FLAG-IDA respectively. On univariate analysis, FLAG-GO regimen favored attainment of post C1 OPR over FLAG-IDA (OR, odds ratio=3.1, 95%CI 1.6-6.2) while age, baseline BM blast, cytogenetic subtype of CBF, ACA and kinase mutations did not. The benefit of FLAG-GO in attaining OPR after C1 was maintained even on a multivariate (MV) model. For EOT OPR, FLAG-GO (OR= 8.5, 95% CI 3.4-26) fared better over FLAG-IDA. Absence of KIT mutation (OR=2.4) and attainment of post C1 OPR (OR= 11.3) were the other relevant factors. On MV analysis, only chemo arm and post C1 OPR remained significant.

At a median follow up of 92 months (mos), the RFS was 124 mos and OS not reached (NR) for the whole cohort (RFS 68 mos and OS 102 mos for FLAG-IDA treated pts vs. both NR for FLAG-GO treated pts, p=0.004 and 0.04 respectively). Across the 2 groups there was no difference in RFS or OS between pts who attained early or late OPR. However, on stratifying serial PCR responses with therapy arms, even in pts with early OPR (after C1), RFS was better (NR) in FLAG-GO arm vs 101 mos for FLAG-IDA arm (p= 0.03), though OS was similar, likely reflecting efficacy of salvage therapies in this subset of AML. In a Cox proportional hazards model that included age, therapy arm, kinase mutations, cumulative therapy cycles, post C1 and EOT PCR responses; only younger age and EOT OPR favorably affected RFS; however FLAG-GO treated pts had lower hazards for RFS at any level of PCR response compared to FLAG-IDA treated pts (Fig. 1 ).

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Summary/Conclusion: In CBF AML, FLAG-GO regimen leads to higher rate of earlier and end of therapy OPR than FLAG-Ida and substantially better RFS. At any combination of PCR response, FLAG-GO treated patients had lower hazards for relapse than FLAG-IDA treated pts.

P576: A PHASE I/II STUDY OF MILADEMETAN (DS3032B) IN COMBINATION WITH LOW DOSE CYTARABINE WITH OR WITHOUT VENETOCLAX IN ACUTE MYELOID LEUKEMIA

J. Senapati 1,* , J. Ishizawa 1 , H. A. Abbas 1 , S. Loghavi 1 , G. Borthakur 1 , G. C. Issa 1 , S. I. Dara 1 , R. Pourebrahim 1 , N. Daver 1 , E. J. Jabbour 1 , S. M. Kornblau 1 , N. Pemmaraju 1 , G. Garcia-Manero 1 , F. Ravandi 1 , M. Muftuoglu 1 , J. Khoury 1 , C. DiNardo 1 , M. Andreeff 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: In TP53 wild type (WT) acute myeloid leukemia (AML), inhibition of MDM2 increases p53 protein expression and mediates antileukemic effects. MDM2i monotherapy in AML has shown only modest responses; combining MDM2i with anti-leukemic therapies with additive or synergistic pre-clinical activity and non-overlapping toxicities might potentiate therapeutic benefit.

Aims: To evaluate safety and efficacy of milademetan (DS-3032b), an MDM2i, with low dose cytarabine (LDAC) ± venetoclax (VEN) in patients (pts) with AML.

Methods: This open label, single arm Phase I/II trial ( NCT03634228 ) included pts with AML and WT TP53. The phase I utilized a 3 + 3 design to identify safety and tolerability of the combination and determine the recommended combination dose to proceed to the expansion phase (phase II). Pts included were adult (≥18 years), ECOG PS ≤ 3, with adequate renal and hepatic function. Key exclusion criteria included acute promyelocytic leukemia, prior therapy with MDM2i, or stem cell transplantation (SCT) in the preceding 60 days.

Doses of milademetan and combination therapy are provided in Table 1. Toxicity was graded according to the NCI-CTCAE v.5 and responses according to the ELN 2017 AML response criteria.

Results: A total of 21 pts were screened; 16 pts were enrolled and treated in phase I. Median age was 70 years (range, 23-80) and 11 (69%) were women. Median number of prior therapies was 3 (range, 1-7), with 6 pts (37%) who had undergone prior SCT. Two pts (12.5%) were newly diagnosed with treated secondary AML; having received 3 and 2 lines of therapy for prior MDS. Milademetan combination therapy was reasonably well tolerated at all dose levels, the most common adverse events being gastrointestinal. One pt had an attributable grade 3-4 toxicity (diarrhea), which was considered a dose limiting toxicity. Pts received a median of 1 cycle (range, 1-4). Two pts (12.5%) achieved an overall response (CRp, 12.5%, 1 each at dose level 1 and 2). At a median follow up of 10.5 mos (range, 0.6-17), 5 pts are alive (31%), all on subsequent lines of therapy. Ten of 11 deaths occurred in non-responding pts and were attributed to disease progression or infection. One responding pt died of infection in cycle 3. Given the minimal response rates at all dose levels, the phase II expansion portion of the trial was not conducted.

p53 immunohistochemistry was performed on baseline bone marrow (BM) samples from 11 pts, all of whom had WT expression pattern. BM samples of 1 amongst 7 pts evaluated post C1 demonstrated acquisition of mutant p53. We performed CyTOF analysis of sequentially collected samples to interrogate signalling pathways, the p53-MDM2 axis, and the abundance of pro/anti-apoptotic molecules. We observed that CD34+ leukemia blasts expressed high levels of Bcl-2 and were eliminated after therapy in a patient with CRp while Bcl-2 low non-malignant monocytic cells preferentially enriched. Comparative single-cell proteomic analysis in responders and non-responders are underway to identify proteomic correlates of favourable response vs. therapy resistance.

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Summary/Conclusion: The combination of milademetan with LDAC ± venetoclax was reasonably well tolerated but showed only minimal clinical responses at all 3 dose levels in a heavily pre-treated AML population. Further studies are needed to decipher the most appropriate combination regimen and pt population to benefit from milademetan.

P577: 20% BLAST CRITERIA FOR DIAGNOSING AML/MDS: IS IT TIME TO MOVE BEYOND?

V. Sheth 1,* , R. Walter 2,3 , C. Shaw 3 , E. Estey 2,3 , M.-B. Percival 2,3

1 Deprtment of hematology and Bone marrow transplant, UAB, Birmingham; 2 Division of hematology, University Washington; 3 Clinical research division, Fred Hutch, SEATTLE, United States of America

Background: Classification of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) is driven by an arbitrary blast percentage cut off (20%, using the 2016 WHO criteria), which subsequently affects treatment options (including enrollment in clinical trials) without taking genomic and biological characteristics into consideration. It has recently been posited that AML and MDS with excess blasts (EB) exist on a continuum based on biological features rather than blast percentages, and their outcomes post treatment are very similar.

Aims: We therefore hypothesized that the IPSS-R (MDS prognostication using hematologic parameters and cytogenetic features) should predict outcomes for WHO-defined AML, and similarly, ELN2017 (AML prognostication using molecular and cytogenetic features) should predict outcomes for WHO-defined MDS-EB (5-19% blasts).

Methods: We retrospectively analyzed 1034 adult patients from the UW/FHCRC registry database, who received treatment for AML or MDS-EB between the years 2005-2018. WHO-defined AML occurred in 777 patients (67%) and WHO-defined MDS-EB in 257 (23%). Median follow up was 5 years. Outcomes were relapse-free survival (RFS) and overall survival (OS).

Results: Median age of patients in AML group was 63 years (range 18-91), and in MDS group was 60 years (range 22-85). The majority of the patients in AML cohort had intermediate (37%) or high-risk disease (37%) using ELN2017 classification; many were reclassified to very high risk by IPSS-R (54%). Most of the patients in MDS cohort were very high risk (54%) using IPSS-R; many were reclassified to ELN high risk (46%). For AML patients, classification using the IPSS-R was highly predictive of OS for AML in univariate (p=0.001) and multivariate analysis (reference low risk/intermediate risk) (p=0.001, HR-2.1, 95% confidence interval (CI) 1.1-2.9 for very high risk and p=0.2, HR-1.4, 95%CI 0.76- 1.9 for high risk in AML), as well as RFS in univariate (p=0.001) and multivariate analysis (reference low risk/intermediate risk) (p=0.001, HR-2.08, 95%CI 1.2-2.8 for very high risk and 0.2, HR-1.4, 95%, CI 0.2-1.8 for high risk in AML). Similarly, ELN 2017 was predictive of OS in MDS cohort in univariate (p=0.025), and multivariate analysis (reference ELN adverse (p=0.005, HR-0.1, 95%CI 0.02-0.5, ELN favorable risk), as well as RFS in univariate (p=0.043) and multivariate analysis (reference ELN adverse) (p=0.019, HR-0.25 for ELN favorable risk, 95%CI 0.04- 0.8).

In the subgroup of AML patients who achieved CR/CRi, IPSS-R was predictive of RFS in univariate analysis (p=<0.001), and multivariate analysis (ref low/int) (p=0.001, HR=2.08 for very high risk). Similarly, in the subgroup of MDS patients who achieved CR/CRi, ELN 2017 was predictive of RFS (p=<0.001), but this effect was not sustained in multivariable analysis.

Summary/Conclusion: Data from our cohort suggest that IPSS-R can be predictive of survival outcomes in WHO-defined AML patients and ELN 2017 can similarly be predictive in WHO-defined MDS patients. Therefore, we suggest that diagnostic criteria for AML and MDS should include more biological and genomic features rather than a blast percentage cut-off, using tools such as the recently presented IPSS-molecular for MDS. Clinical trials of new therapies should include all patients having 3 5% blasts (MDS-EB and AML). This study, however, carries a potential drawback of retrospective classification and lack of complete molecular data through comprehensive targeted next generation sequencing panel in patients treated at earlier time periods

P578: GILTERITINIB FOR RELAPSED/REFRACTORY FLT3 MUTATED ACUTE MYELOID LEUKEMIA A REAL-WORLD, MULTI-CENTER, MATCHED ANALYSIS

S. Shimony 1,2,3 , J. Canaani 3,4 , E. Kugler 2,3,* , B. Nachmias 5 , R. Ram 6,7 , A. Frisch 8 , C. Ganzel 9 , V. Vainstein 5 , Y. Moshe 3,10 , S. Aumann 5 , M. Yeshurun 2,3 , Y. Ofran 9 , P. Raanani 2,3 , O. Wolach 2,3

1 Leukemia, Dana Farber Cancer Institute, Boston, United States of America; 2 hematology, Rabin medical center, Petah Tikva; 3 Sackler Medical School, Tel Aviv University, Tel Aviv; 4 Leukemia, Chaim Sheba Medical Center, Ramat Gan; 5 hematology, Hadassah Medical Center and Hebrew university Faculty, Jerusalem; 6 BMT Unit; 7 Sackler Medical School, Tel Aviv Sourasky Medical Center, Tel Aviv; 8 Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, and Rappaport Faculty of Medicine - Technion, Haifa; 9 hematology, Shaare Zedek Medical Center, and Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem; 10 hematology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

Background: Patients with FLT3-mutated relapsed or refractory (R/R) acute myeloid leukemia (AML) have a dismal prognosis. Gilteritinib is a FLT3 tyrosine kinase inhibitor (TKI) recently approved for patients with R/R AML.

Aims: We aimed to characterize real-world data regarding gilteritinib treatment in FLT3-mutated R/R AML and to compare outcomes with matched FLT3-mutated R/R AML patients treated with chemotherapy-based salvage regimens.

Methods: Survival analysis was performed by Kaplan Meyer with log rank test to compare between subgroups. Cox proportional hazards regression models were fitted to predict effect of covariates on overall survival (OS) and event free survival (EFS). All statistical analyses used 2-sided p value with a threshold of 0.05.

Results: Twenty-five patients from six academic centers were treated with gilteritinib for FLT3-mutated R/R AML. The median age was 58 (IQR1-3 47-73) years, 80% (n=20) were treated with a prior intensive induction regimen and 40% of them received prior TKI therapy. After a median time of seven months post gilteritinib initiation (range 1-34), 12 patients (48%) achieved complete response (CR) with gilteritinib (figure 1A). The estimated median overall survival (OS) of the entire cohort was eight (CI 95% 0-16.2) months and was significantly higher in patients who achieved CR compared to those who did not (16.3 months, CI 95% 0-36.2 vs. 2.6 months, CI 95% 1.47-3.7; p value=0.046, figure 1B). In a multivariate cox regression analysis, achievement of CR was the only predictor for longer OS (HR 0.33 95% CI 0.11-0.97, p=0.044). Prior TKI exposure did not affect OS but was associated with better event-free survival (HR 0.15 95% CI 0.03-0.71, p=0.016). An age and ELN-risk matched comparison between patients treated with gilteritinib and intensive salvage revealed similar response rates (50% in both groups); median OS was 9.6 months (CI 95% 2.3-16,8) vs. 7 months (CI 95% 5.1-8.9) in gilteritinib and matched controls, respectively (p = 0.869, figure 1C).

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Summary/Conclusion: In the real-world setting gilteritinib is effective, including in heavily pre-treated, TKI exposed patients.

P579: A PHASE 4 STUDY OF FRACTIONATED GEMTUZUMAB OZOGAMICIN ON QT INTERVAL AND SAFETY IN PATIENTS WITH RELAPSED/REFRACTORY CD33-POSITIVE ACUTE MYELOID LEUKEMIA

P. Montesinos 1,* , V. Kota 2 , J. Brandwein 3 , P. Bousset 4 , R. J. Benner 5 , E. Vandendries 6 , M. F. McMullin 7

1 Department of Hematology, Hospital Universitario i Politècnico la Fe, Valencia, Spain; 2 Department of Medicine: Hematology and Oncology, Medical College of Georgia, Augusta University, Augusta, United States of America; 3 Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada; 4 Pfizer Inc., Paris, France; 5 Pfizer Inc., Groton; 6 Pfizer Inc., Cambridge, United States of America; 7 School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, United Kingdom

Background: Gemtuzumab ozogamicin (GO) is a CD33-directed antibody-drug conjugate indicated for treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML). Fractionated dosing of GO has demonstrated an enhanced safety profile without hampering efficacy.

Aims: To assess the effect of fractionated GO as monotherapy on corrected QT (QTc) interval, safety, pharmacokinetics (PK), and immunogenicity of GO in patients (pts) with R/R AML.

Methods: Pts aged ≥12 y with R/R AML were enrolled in a single-arm, open-label, phase 4 study ( NCT03727750 ) and received a fractionated dose of GO 3 mg/m 2 on days 1, 4, and 7 of each cycle, up to 2 cycles. Pts receiving ≥1 dose of GO were evaluable for QTc, safety, and PK. The primary endpoint was mean change from baseline in QTc. Secondary endpoints included PK parameters, adverse events (AEs), including veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS), incidence of anti-drug antibodies (ADAs)/neutralizing antibodies (NAb), overall survival (OS), and response rate of complete remission (CR)/CR with incomplete hematologic recovery (CRi). This analysis includes data from pts aged ≥18 y.

Results: Of 51 adult pts enrolled (median age 67 y), 50 (98%) pts received ≥1 dose of GO during cycle 1; 9 (18%) pts were treated with GO during cycle 2. The upper limit of the 2-sided 90% CI for least squares mean differences in QTc according to Fridericia’s formula (QTcF) was <10 msec for all timepoints during cycle 1 (Table). Most pts had a QTcF ≤450 msec (90%) and a maximum increase from baseline QTcF ≤30 msec (94%). No pts had a post-baseline QTcF >480 msec or a change from baseline >60 msec. Treatment-emergent AEs (TEAEs) occurred in 98% of pts; 54% were grade 3/4. The most common (>10%) grade 3/4 TEAEs were febrile neutropenia (36%) and thrombocytopenia (18%). One pt experienced grade 3 atrial fibrillation and supraventricular tachycardia, unrelated to GO. No pts experienced grade 4 or higher cardiac conduction TEAEs. VOD/SOS was not reported; however, one pt experienced treatment-related grade 5 capillary leak syndrome associated with pleural effusion, ascites, hyperbilirubinemia, and endothelial syndrome. The overall incidence of ADAs and NAbs were 12% and 2%, respectively. ADAs were persistent in 5/6 pts (83%). Of the 6 pts with treatment-induced ADAs, 2 experienced pyrexia (grade 1 and grade 3) within 24 h of the infusion. Among ADA-negative pts (n=44), infusion-related reactions were observed in 7 (16%) pts. These were grade 1/2, except for 1 instance of grade 3 urticaria. Median (95% CI) OS was 2.8 (1.7–4.2) mo. Disease progression was the most common cause of death in 35/45 (78%) pts. A CR/CRi was observed in 5/51 (10%) pts.

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Summary/Conclusion: Fractionated dosing of GO (3 mg/m 2 per cycle) is not predicted to pose a clinically significant safety risk for QT prolongation in pts with R/R AML, with the upper limit of the 2-sided 90% CI for the least squares mean change from baseline falling below the 20 msec threshold of clinical concern for oncology drugs. TEAEs reported in this study are consistent with the known safety profile of GO, and the presence of ADAs does not appear to be associated with any potential safety concerns. Response rates were lower than previously reported in the MyloFrance 1 study, which included less heavily pretreated patients (i.e., patients only in first relapse; Taksin et al, Leukemia, 2007).

P580: THE USE OF LEUKAEMIA Q-FUSION GENE SCREENING ASSAY (Q30) IN THE DIAGNOSTIC EVALUATION OF ACUTE MYELOID LEUKAEMIA (AML)

S. Tayabali 1,* , R. Baker 1,2 , E. Nacheva 1,2 , J. O’Nions 1,2 , R. Gupta 1,2 , A. J. Wilson 1,2 , K. Xu 1,2

1 University College London Hospital; 2 UCLH Specialist Integrated Haematology Malignancy Diagnostic Service, Health Services Laboratories, London, United Kingdom

Background: AML is a clinically heterogenous disease characterised by chromosomal and genetic abnormalities which guide risk stratification and management. Hence timely cytogenetic and molecular profiling is essential to initiate appropriate treatment. Approximately 30% of AML cases have fusion genes. We use a combination of fluorescence in situ hybridization (FISH) to rapidly screen for common chromosomal abnormalities and fusion genes, and chromosomal microarray (CMA) to assess copy number variations across the whole genome. However, cytogenetic analysis is resource intensive and may take several days which can delay patient management.

The QuanDx© Leukaemia Q-Fusion Genes Screening Kit (Q30) uses multiplex reverse transcription real-time PCR (RT-qPCR) to allow simultaneous detection of 30 fusion genes with 140+ breakpoints within hours. The use of Q30 may allow earlier risk stratification and treatment before FISH and CMA results are available.

Aims:

To evaluate the Q30 Kit to detect rearrangements on bone marrow and peripheral blood specimens from newly diagnosed AML patients; and compare concordance of Q30 and FISH in identifying leukaemia fusion genes.

Methods:

We retrospectively reviewed FISH, CMA and Q30 data for new AML cases diagnosed in the specialist integrated haematological malignancy diagnostic service (SIHMDS) at University College London Hospital over a 2 year period: 01 Jan 19 to 31 Dec 20.

Results:

114 cases were included. The median age was 59 [range 19 to 91]. Male 59 (52%), Female 55 (48 %).

30 (26%) of 114 patients had a detectable fusion gene by Q30 (Figure 1). 25/30 were confirmed by FISH either on our standard panel or subsequently using targeted probes.

The 5/30 fusion genes detected by Q30, but not by FISH were:

- t(3;5)(q25;q34) NPM1-MLF1 (n=1)

- t(9;22)(q34;q11) BCR-ABL1 with a high cycle threshold (Ct) value (n=3)

- t(16;21)(p11;q22) TLS-ERG (n=1)

The discordant results were due to high Ct values (detecting low level gene fusions below the limit of FISH sensitivity), or a lack of commercially available fusion probes.

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Summary/Conclusion: The ability to rapidly risk-stratify newly diagnosed AML patients allows earlier initiation of targeted therapies. We found that Q30 is highly sensitive and showed 100% concordance in identifying fusions associated with good cytogenetic risk AML in our cohort. Q30 can also identify high risk fusion genes including DEK-CAN, MLL-AF6 and other high risk MLL translocations included in the panel which are rare and for which FISH fusion probes are not routinely used. These may have been otherwise undetected, but can subsequently be confirmed by FISH, and importantly allow the generation of molecular measurable residual disease (MRD) assays to assess treatment response.

We conclude that Q30 is a rapid, simple and sensitive adjunct to conventional FISH in the diagnosis of AML and, in combination with CMA, may preclude the requirement for conventional and time consuming G-banding analysis.

P581: PHASE II STUDY OF LOWER-INTENSITY FRONTLINE THERAPY FOR NEWLY DIAGNOSED PATIENTS WITH AML WHO ARE UNFIT OR OTHERWISE NOT ELIGIBLE FOR FRONTLINE CLINICAL TRIALS

S. Venugopal 1,* , E. Jabbour 1 , N. Pemmaraju 1 , G. Montalban-Bravo 1 , K. S. Chien 1 , N. Daver 1 , N. Jain 1 , J. Burger 1 , Y. Alvarado 1 , A. Maiti 1 , C. D. DiNardo 1 , G. Borthakur 1 , R. Malla 1 , G. Garcia-Manero 1 , F. Ravandi 1 , H. Kantarjian 1 , T. Kadia 1

1 Leukemia, The University of Texas, MD Anderson Cancer Center, Houston, United States of America

Background: Pts with newly diagnosed (ND) AML frequently present with abnormal organ function, poor performance status (PS), concurrent active malignancies, and active infections. These factors often preclude these pts from enrollment on frontline clinical trials, since standard eligibility routinely exclude them.

Aims: Therefore, we designed a lower-intensity regimen of cladribine plus low-dose cytarabine (LDAC) alternating with decitabine (DAC) with less stringent inclusion criteria in ND pts unfit or otherwise ineligible for existing clinical trials.

Methods: Pts >18 years with untreated AML and ineligible for other frontline AML clinical trials were enrolled ( NCT01515527 ). Eligibility criteria included either creatinine ≥2mg/dL; or total bilirubin ≥2 mg/dL; or ECOG PS of 3 or 4; or ineligible for participation in a higher priority protocol. Pts with active concurrent malignancies and ongoing infection related to their AML could be enrolled. Induction was cladribine 5 mg/m2 IV on D1-5, Cytarabine 20 mg SQ twice daily on D 1-10, followed by consolidation with cladribine 5 mg/m2 IV on D1-3, Cytarabine 20 mg SQ twice daily on D 1-10 alternating with decitabine 20 mg/m2 IV, daily on D 1-5. The primary objective was 60-d survival rate.

Results: A total of 25 pts have been enrolled. The median age was 73 years (range, 52-82) with 76% of the cohort older than 70 years. Six pts (24%) had concurrent active malignancy, 4 (16%) had baseline creatinine >2mg/dL, and 9 (36%) had PS ≥3. 76% of them were adverse risk per ELN 2017. Baseline characteristics are shown in figure.

Among 25 evaluable pts, 17 (68%) achieved a composite complete response (CRc) including 10 (40%) CR and 7 (28%) CR with incomplete count recovery (CRi). Among responders, 6 pts (35%) achieved MRD neg by flow. Of 7 pts with no response, all were ELN adverse risk. Median cycles to response was 1 (range: 1 - 4). 30- and 60 d mortality was 8% and 16%, respectively, including 2 pts (8%) who died due to pseudomonal sepsis on D8, and the other due to pneumonia on D11.

At a median follow up of 9.4 months (range, 0.4- 19.9 m),median OS (6-mo OS% - 61 %), and EFS was 8.3 mo each (6-mo RFS% - 56 %) with a 60-d OS and EFS rate of 83% each, with a median RFS of 5.8 mo (2-mo RFS%-66%; 6-mo RFS% - 49 %). In this challenging patient population, this lower-intensity regimen was well tolerated, with an acceptable toxicity profile.

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Summary/Conclusion: In an unfit patient population with a high comorbidity burden, that were ineligible for other clinical trials, induction therapy with Cladribine plus LDAC was feasible and effective in newly diagnosed pts with AML. The regimen produced high rates of response, encouraging EFS, OS, and low early mortality in a cohort predicted to have a high risk of early death. Treating this pt population on a clinical trial is feasible and can allow pts to achieve remission and move on to effective post-remission therapy.

P582: FIRST RESULTS OF A PHASE II STUDY (STIMULUS-AML1) INVESTIGATING SABATOLIMAB + AZACITIDINE + VENETOCLAX IN PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA

A. M. Zeidan 1,* , J. Westermann 2 , T. Kovacsovics 3 , S. Assouline 4 , A. C. Schuh 5 , H.-J. Kim 6 , G. Rodriguez Macias 7 , D. Sanford 8 , M. R. Luskin 9 , E. M. Stein 10 , K. Malek 11 , J. Lyu 12 , M. Stegert 11 , J. Esteve 13

1 Yale University and Yale Cancer Center, New Haven, United States of America; 2 Charité-University Medical Center Berlin, Campus Virchow Clinic, Berlin, Germany; 3 University of Utah and Huntsman Cancer Institute, Salt Lake City, United States of America; 4 Jewish General Hospital, McGill University, Montreal; 5 Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada; 6 Catholic Hematology Hospital, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea; 7 Hospital General Universitario Gregorio Marañón, Madrid, Spain; 8 BC Cancer, University of British Columbia, Vancouver, Canada; 9 Dana-Farber Cancer Institute, Harvard Medical School, Boston; 10 Memorial Sloan Kettering Cancer Center, New York, United States of America; 11 Novartis Pharma AG, Basel, Switzerland; 12 Novartis Institutes for BioMedical Research, Shanghai, China; 13 Hospital Clínic, Barcelona, Spain

Background: The BCL-2 inhibitor venetoclax (VEN) in combination with a hypomethylating agent (HMA) has improved outcomes for patients with newly diagnosed (ND) acute myeloid leukemia (AML) who are unfit for intensive chemotherapy (IC; DiNardo CD, N Engl J Med, 2020); however, responses are often transient. Sabatolimab is a novel immuno-myeloid therapy targeting TIM-3, an immune regulator expressed on immune cells and myeloid leukemic progenitors but not on normal hematopoietic stem cells. Sabatolimab + HMA has shown promising durable responses in a Phase Ib study in patients with ND-AML and myelodysplastic syndrome (Wei AH, ASH 2021; NCT03066648 ). Treatment with sabatolimab + VEN + azacitidine (AZA) may also induce different pathways of cancer cell elimination.

Aims: In this first presentation of the study, we report findings from the dose-escalation part of STIMULUS-AML1 ( NCT04150029 ), a Phase II, single-arm study of sabatolimab + AZA + VEN in adult patients with AML.

Methods: Adult patients with ND-AML ineligible for IC were enrolled. In dose-escalation part 1 (safety run-in), patients received either 400 mg (Cohort 1) or 800 mg (Cohort 2) sabatolimab every 4 weeks (Q4W) plus 400 mg VEN once a day every day, plus 75 mg/m 2 AZA on Days (D) 1-7, or D1-5+D8-9, or D1-6+D8 of a 28-day cycle (Zeidan A, ESH-AML 2022). For the safety run-in part, the primary endpoint was assessment of the incidence of dose-limiting toxicities (DLTs) between Cycle 1 D8 and the end of Cycle 2.

Results: As of the Sep 6, 2021 data cutoff, 18 patients have been treated in part 1 of the study with sabatolimab + AZA + VEN (Cohort 1, n=5; Cohort 2, n=13). Baseline demographics are shown in Table 1. Treatment was ongoing for 9 (50%) patients (Cohort 1, n=1; Cohort 2, n=8). Reasons for study treatment discontinuation included adverse events (AEs; n=3), progressive disease (n=3, including a case of chloroma), planned stem cell transplant (n=2), and physician decision (n=1). Only 1 DLT (elevated troponin T/asymptomatic myocarditis) was reported in Cohort 2 and no DLTs were reported in Cohort 1. Table 2 summarizes AEs observed in >20% of patients in the study. Consistent with AEs often observed during AZA + VEN therapy (DiNardo CD, N Engl J Med, 2020), the 5 most common AEs regardless of relationship to study treatment were constipation (all grades [gr]: 39%; gr ≥3: 0%) and hematologic events, including anemia (33%; 28%), decreased platelets (33%; 33%), neutropenia (39%; 39%), and febrile neutropenia (gr: 50%). Treatment-related AEs gr ≥3 in >25% of all patients included neutropenia (39%), decreased platelet count (33%), and decreased neutrophil count (28%). Treatment-related AEs led to study treatment discontinuation in 3 patients (platelet count decreased [n=2], troponin level increased/asymptomatic myocarditis [n=1]). AEs led to sabatolimab dose interruption in 6 patients, all in Cohort 2. No sabatolimab dose reduction was observed. Fourteen patients had dose interruption of VEN due to AEs. Five patients had dose reduction of VEN (none due to AEs). Serious AEs were reported in 14 (78%) patients; the most common was febrile neutropenia (44%). No other serious AEs were reported in >1 patient. Efficacy data will be presented at the EHA meeting.

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Summary/Conclusion: Safety and tolerability of sabatolimab + AZA + VEN were comparable at 2 dose levels (400 and 800 mg) of sabatolimab and were overall comparable to the reported safety profile of VEN + AZA doublet therapy. These findings supported initiation of the expansion cohort of STIMULUS-AML1 at a sabatolimab 800 mg dose.

P583: PATIENTS AT HIGH RISK OF RELAPSE POST-TRANSPLANT: A PHASE 1 STUDY DESIGN WITH A NOVEL TREATMENT STRATEGY USING THE ESTIMAND FRAMEWORK

R. Zeiser 1,* , C. Schmid 2 , G. Al-Atrash 3 , Y. Xu 4 , H.-J. Weber 5 , L. Eldjerou 6 , S. Weber 7 , L. Widmer 7 , C. Craddock 8

1 Department of Medicine I, Medical Center, University Freiburg, Freiburg; 2 Department of Hematology and Oncology, Augsburg University Hospital and Medical Faculty, Augsburg, Germany; 3 Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston; 4 Oncology Analytics, Novartis Pharmaceutical Corporation, East Hanover, United States of America; 5 Oncology Analytics, Novartis Pharma AG, Basel, Switzerland; 6 Clinical Development, Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 7 Advanced Exploratory Analytics, Novartis Pharma AG, Basel, Switzerland; 8 Centre for Clinical Haematology, University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom

Background: The risk of disease relapse in patients (pts) allografted for acute myeloid leukemia (AML) is high and remains the main cause of transplant failure. The great majority of pts destined to relapse post-allogeneic stem cell transplant (SCT) will do so within the first 12 months and survival after early relapse is dismal. Therefore, strategies to prevent relapse are urgently required. A potent graft-versus-leukemia (GvL) effect is exerted after SCT, and strategies that may augment a GVL effect have the potential to reduce relapse risk post-SCT. Siremadlin (HDM201) is a novel investigational MDM2 inhibitor with single-agent (SA) anti-AML activity. MDM2 inhibitors, including siremadlin, possess potent immunomodulatory effects (IME) in murine solid tumor and AML models. The post-allogeneic SCT setting is ideal for investigating the IME of MDM2 inhibition on enhancing GvL effect. Here we present a phase Ib/II proof-of-concept study designed to assess safety and preliminary efficacy of a treatment strategy with siremadlin as monotherapy and, subsequently, in combination with donor lymphocyte infusions (DLIs) for AML in post-SCT setting using the estimand framework (ICH E9 (R1) addendum).

Aims: The objective of the planned treatment strategy is to enhance GvL post-SCT by siremadlin as monotherapy and in combination with DLIs. Due to the risk of Graft-vs-Host Disease (GvHD) with DLI early after SCT, siremadlin will be given as priming monotherapy to prevent early relapse prior to starting siremadlin/DLI combination therapy. SA siremadlin maintenance after the combination therapy is intended to prolong and enhance the GvL reaction to eradicate residual leukemic blasts.

Methods: The estimand framework mandates a careful definition of the experimental intervention according to the clinical question of interest. In this trial, the experimental intervention follows a treatment strategy: Patients (pts) with AML in complete remission (CR) post-SCT but at high risk for relapse based on pre-SCT risk factors will start with siremadlin priming monotherapy. Pts who tolerate the siremadlin dose with no evidence of GvHD are eligible to start siremadlin/DLI combination therapy. After combination therapy, pts may continue with SA siremadlin maintenance. Depending on the treatment journey, pts may receive only a part of the intervention’s sequence (e.g., pts in siremadlin priming monotherapy who are not eligible for DLI combination will continue the priming part). The clinical questions addressed in this trial are to determine the recommended dose (RD) of siremadlin across all parts of the treatment strategy and to assess preliminary efficacy regardless of the parts of the treatment strategy pts received. The doses of interest were preselected based on SA studies and in pts who received siremadlin after SCT.

Results: The proposed approach is tailored to investigate safety and efficacy of the treatment strategy. The RD of siremadlin will be determined over 2 parts: A Bayesian logistic regression model will guide dose finding of siremadlin during priming (Cycle 1) and a Bayesian time-to-DLT model will assess the siremadlin dose for combination. Efficacy will be assessed by proportion of pts remaining in CR for at least 6 months.

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Summary/Conclusion: In clinical practice, treatment decisions follow a strategy. The estimand framework guided the design of a clinical study in AML post SCT to conceptualize the assessment of a treatment strategy. Accordingly, safety and efficacy of the treatment strategy as a whole will be assessed taking into account different patient journeys.

P584: OVERALL SURVIVAL WITH INTENSIVE CHEMOTHERAPY (IC) VS NON-IC IN PATIENTS (PTS) WITH NEWLY DIAGNOSED (ND) AML FROM THE CONNECT® MYELOID DISEASE REGISTRY INELIGIBLE FOR RANDOMIZED CLINICAL TRIALS (RCT)

H. Erba 1,* , D. Pollyea 2 , M. Sekeres 3 , G. Garcia-Manero 4,5 , K. Seiter 5 , I. DeGutis 6 , P. Kiselev 6 , A. McBride 6 , E. Yu 6 , G. Roboz 7,8

1 Duke University, Durham; 2 University of Colorado, Aurora; 3 Sylvester Comprehensive Cancer Center, University of Miami Health System, Miami; 4 The University of Texas MD Anderson Cancer Center, Houston; 5 New York Medical College, Valhalla; 6 Bristol Myers Squibb, Princeton; 7 Weill Cornell Medicine; 8 New York-Presbyterian Hospital, New York, United States of America

Background: Pts with AML in RCTs often do not reflect the population seen in clinical practice due to strict eligibility criteria.

Aims: To evaluate patient outcomes of IC vs venetoclax (VEN)-containing regimens based on eligibility criteria from a recent RCT (the VIALE-A trial) in a broad cohort of real-world pts with AML from the Connect ® Myeloid Disease Registry ( NCT01688011 ).

Methods: Pts were stratified into 3 groups based on the non-IC phase 3 VIALE-A trial eligibility criteria: 1) “eligible” pts who would have met all VIALE-A inclusion criteria; 2) “unfit” pts who would have been ineligible for VIALE-A due to ≥ 1 of the following: abnormal liver/kidney function, high ECOG, recent prior malignancy, comorbidities score ≥ 2 by ACE-27, hepatic grade ≥ 1, AIDS grade ≥ 1; 3) “fit” pts who would have been ineligible for a VEN-based regimen in VIALE-A because they would have qualified for IC (defined as: ≤ 74 y of age, low ECOG, no apparent cardiovascular/renal comorbidities, and did not meet any criteria in #2). Baseline characteristics were summarized by eligibility group. Overall survival by group was estimated using the Kaplan–Meier method. Induction regimens were categorized as IC (any regimen containing 7 + 3, MEC, CLAG, FLAG) or VEN-based. Hazard ratios (HR) for induction regimens among each group were estimated using Cox models adjusted for age, ELN risk, ECOG, frailty score, and comorbidity index.

Results: Of 734 enrolled pts with AML (Dec 2021), most were male (61%) and white (84%); median age 71 y (range, 55–97). Only 26% of pts (n = 192) were eligible for a non-IC RCT, 45% (n = 327) were ineligible due to unfitness, and 29% (n = 215) were ineligible due to overall fitness. The main reason for non-IC RCT ineligibility was high overall comorbidity grade (n = 265 [36%]). At baseline, fit pts intended to undergo transplant more often compared with unfit pts. Median duration of overall survival for eligible, unfit, and fit pts was 14, 10, and 22 months, respectively (HR [95% CI], eligible vs unfit, 0.02 [−0.19 to 0.22], P = 0.8735; eligible vs fit, 0.57 [0.33–0.81], P < 0.0001; unfit vs fit, 0.55 [0.33–0.77], P < 0.0001). Among unfit pts, those receiving IC had significantly longer overall survival compared with pts receiving a VEN-based regimen (median overall survival, 14 vs 6 months, respectively; HR, 0.51 [95% CI, 0.27–0.98]; P = 0.042; Figure). Unfit patients who received IC went on to transplant more frequently than those who received VEN-based therapies (16% [n = 17] vs 1% [n = 1], respectively). Eligible pts who received IC (n = 31) tended to have shorter median overall survival (13 months) vs pts who received VEN-based therapies (n = 27; 23 months; HR, 1.45, 95% CI, 0.66–3.17; not sig.). Among fit pts, median overall survival was 19 months for those receiving IC (n = 69) but could not be estimated for pts receiving VEN-based therapies due to small sample size (n = 10).

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Summary/Conclusion: The majority of pts with ND AML in the Connect ® Myeloid Disease Registry would have been ineligible for a non-IC RCT due to being too fit or unfit. Among pts ineligible for an RCT due to unfitness, there was an association with increased overall survival in pts receiving IC vs those receiving VEN-based therapies, and pts in the IC group were more likely to receive a transplant. This analysis suggests that RCTs may be excluding pts who appear unfit but can potentially tolerate IC and experience improved survival outcomes.

P585: VENETOCLAX IN COMBINATION WITH INTENSIVE TREATMENT PROTOCOLS FOR PATIENTS WITH HIGH-RISK ACUTE MYELOID LEUKEMIA – A MULTICENTER REAL-WORLD ANALYSIS

O. Wolach 1,* , A. Frisch 2 , L. Shargian 1 , M. Yeshurun 1 , A. Apel 3 , V. Vainstein 4 , Y. Moshe 5 , S. Shimony 1 , O. Amit 5 , Y. Bar On 5 , Y. Ofran 6 , P. Raanani 1 , B. Nachmias 4 , R. Ram 5

1 Hematology, Rabin Medical Center, Petah Tikva; 2 Hematology, Rambam Health Care Campus, Haifa; 3 Hematology, Shamir Medical Center, Zriffin; 4 Hematology, Hadassah Medical Center, Jerusalem; 5 Hematology, Tel Aviv Sourasky Medical Center, Tel Aviv; 6 Hematology, Shaare Zedek Medical Center, Jerusalem, Israel

Background: The addition of venetoclax, a selective BCL2-inhibitor, to low intensity therapies was shown to improve the survival of patients with acute myeloid leukemia (AML) that are ineligible for intensive induction chemotherapy. Based on the encouraging anti-leukaemic properties of venetoclax and safety profile, several studies are currently exploring venetoclax in combination with high-intensity chemotherapy in high-risk AML scenarios such as ELN adverse-risk AML and in relapsed and refractoy (R/R) AML. The addition of venetoclax to established intensive chemotherapy protocols in these trials are generally reported to be associated with enhanced efficacy, frequently at the cost of significant hematological and infectious toxicities that requires dose modifications of both chemotherapy and venetoclax dosing

Aims: To assess the safety and efficacy of venetoclax in combination with intensive treatment protocols in a ‘real-world’ setting.

Methods: A multicenter retrospective cohort study in six academic centers in Israel. The decision to treat a patient with venetoclax in addition to an intensive treatment protocol as well as the choice of protocol and dosing were at discretion of the treating physician.

Results: Twenty-nine patients were included in analysis (median age 53.4 years). Most patients were treated for R/R AML (n=27, 93%) with a median of one (0-5) previous lines of therapy and 48% of patients (n=14) having prior allogeneic hematopoietic-cell transplantation (HCT).

Most patients were treated with venetoclax in combination with FLAG-IDA protocol (n=25, 86%). Median follow-up was 6.3 (0.7-23.7) months. Platelet and neutrophil recovery were observed at a median of 31 (95%CI 17.6-38.3) and 24 (95%CI 20-28) days, respectively. Infectious complications were common (blood stream infections, 41% and invasive fungal infections, 28%) and 30-day mortality was 14%.

Composite complete remission (CRc) was 72% for the entire cohort and 91% in patients treated for post-HCT relapse. Incidences of relapse-free and overall survival at 12 months were 90% (95% CI 76%-98%) and 55% (95% CI 21%-63%), respectively.

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Summary/Conclusion: The addition of venetoclax to intensive therapy is effective in high-risk AML, especially in post-HCT relapse setting. Prophylaxis and surveillance for infections are crucial.

P586: IADADEMSTAT COMBINATION WITH AZACITIDINE SHOWS ENCOURAGING SAFETY AND EFFICACY DATA IN ELDERLY AND UNFIT AML PATIENTS

O. Salamero 1,* , T. Somervaille 2 , A. Molero 1 , E. Acuña-Cruz 3 , J. A. Perez-Simon 4 , R. Coll 5 , M. Arnan Sangerman 6 , B. Merchan 7 , A. Perez 8 , I. Cano 3 , R. Rodriguez-Veiga 3 , M. Arevalo 9 , S. Gutierrez 9 , C. Buesa 9 , F. Bosch 8 , P. Montesinos 3

1 Dep. of Hematology, Vall d’Hebron University Hospital. Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain; 2 The Christie Hospital NHS Foundation Trust, Cancer Research Manchester Institute, Manchester, United Kingdom; 3 Hospital Universitario y Politécnico La Fe, Valencia; 4 Hospital Universitario Virgen del Rocio; Instituto de Biomedicina de Sevilla (IBIS)/CSIC/U. de Sevilla, Sevilla; 5 ICO Hospital Dr Josep Trueta, Girona; 6 Institut Català d’Oncología (ICO), Hospital de Bellvitge, Hospitalet de Llobregat; 7 Hospital del Mar; 8 Vall d’Hebron University Hospital. Vall d’Hebron Institute of Oncology (VHIO), Barcelona; 9 Oryzon Genomics SA, Cornella de Llobregat, Spain

Background: Acute Myeloid Leukemia (AML) is an hematological malignancy with highest incidence and lower survival rates in elderly. Previously reported ORR with hypomethylating agents such as azacitidine alone is less than 30%. Recently, combinations of hypomethylating agents with venetoclax have shown improved response ratios. However, refractoriness or relapse is still a challenge for most patients (pts), particularly in the elderly and the high-risk subpopulations. It has been shown that LSD1 is involved in malignant transformation in AML. Iadademstat (iada) is a potent and selective LSD1 inhibitor that has been administered to more than 100 oncology pts in Ph1 and Ph2 trials, showing manageable toxicity and signs of preliminary activity.

Aims: ALICE (EudraCT 2018-000482-36) is a Phase IIa study to assess the safety, tolerability and the recommended Phase II dose (RP2D) of iada (PO 5d ON, 2d OFF every week in 28d cycles) in combination with azacitidine (sc., d1-5 and 8-9 every cycle) for the treatment of adult patients diagnosed with AML, as per WHO 2016 classification, who have not received prior treatment and are ineligible for intensive chemotherapy. We present an update of the ongoing study six months after completion of recruitment.

Methods: Two doses of iada are studied in the trial: 60 and 90µg/m 2 /d. Besides the safety and tolerability primary endpoints, secondary endpoints investigate anti-leukemic activity including ORR according to ELN recommendations, time to response (TTR) and duration of responses (DoR). Additional assessments include residual detectable disease status, overall survival and PK/PD determinations.

Results: At EHA 2021, ALICE data from 27 pts were reported. In October 2021, recruitment of the targeted 36 pts was completed. Patient baseline characteristics are shown in Table 1. Main safety events by the end of 2021 included 348 adverse reactions (AR). The most frequently reported AR was platelet reduction, observed in 44% of pts, however, Grade ≥3 thrombocytopenia was already present at baseline in 61% of pts (Table 1). Only three Grade 3-4 nonhematological treatment-related ARs were observed in two pts (asthenia, dysgeusia and weight decrease). Among the 71 serious AR reported, only 2 were considered as potentially related to iada, corresponding to a differentiation syndrome and a fatal intracranial hemorrhage, previously presented at EHA 2021.

By the time this abstract is written, among the pts intended to treat (34), 27 have had at least 1 bone marrow evaluation after cycle 1 and are therefore evaluable for efficacy. 78% of them (21 of 27) achieved an objective response; of which, 62% were CR/CRi. Moreover, 80% of the CRi pts also achieved partial hematology recovery (CRh). Among CR/CRi pts, 5 out of the 8 already assessed by MRD presented no residual detectable disease by flow cytometry. DoR is encouraging with 77% of the CR/CRi lasting more than 6 months, with a mean DoR increasing as the study progresses, with the longest CR to date above 1,100 days. Iada at 90 µg/m 2 /d is the confirmed RP2D for the combination, showing deep responses with manageable toxicity.

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Summary/Conclusion: ALICE data confirms that the combination of iada with azacitidine gives robust, fast and durable responses in unfit, previously untreated, AML patients. Considering iada’s efficacy, pharmacologic properties, its manageable toxicity and low anticipated drug-drug interactions, iada combinations may offer additional therapeutic options for AML patients in first line or in the R/R setting.

P587: COVALENT-101: A PHASE 1 STUDY OF BMF-219, A NOVEL ORAL IRREVERSIBLE MENIN INHIBITOR, IN PATIENTS WITH RELAPSED/REFRACTORY ACUTE LEUKEMIA, DIFFUSE LARGE B-CELL LYMPHOMA, AND MULTIPLE MYELOMA

F. Ravandi-Kashani 1,* , A. Kishtagari 2 , H. Carraway 3 , G. Schiller 4 , E. Curran 5 , B. Yadav 6 , A. Cacovean 6 , S. Morris 6 , T. Butler 6 , J. Lancet 7

1 Department of Leukemia, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston; 2 Vanderbilt-Ingram Cancer Center, Nashville; 3 Cleveland Clinic Foundation, Cleveland; 4 University of California Los Angeles, Los Angeles; 5 University of Cincinnati Medical Center, Cincinnati; 6 Biomea Fusion, Inc, Redwood City; 7 Department of Malignant Hematology, Moffitt Cancer Center, Tampa, United States of America

Background: Menin, a protein involved in transcriptional regulation, impacting cell cycle control, apoptosis, and DNA damage repair, plays a direct role in oncogenic signaling in multiple cancers. Inhibition of menin is therefore a novel approach to cancer treatment. Preclinical data of BMF-219, a highly selective, orally bioavailable, small-molecule irreversible inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling in vitro and in vivo. BMF-219 exhibited a strong anti-proliferative effect on various menin-dependent acute myeloid leukemia (AML) cell lines, diffuse large B-cell lymphoma (DLBCL) cell lines representing Double/Triple Hit Lymphoma (DHL/THL) & Double Expressor Lymphoma (DEL), and multiple myeloma (MM) cell lines with diverse mutational backgrounds. BMF-219 also showed high potency ex vivo in patient samples from MLL-rearranged and NPM1-mutant AML, THL and MYC-amplified DLBCL, and bone marrow mononuclear cells from treatment-naive and relapsed/refractory (R/R) MM.

Aims: COVALENT-101 (BF-MNN-101: NCT05153330 ) is a prospective, open-label, multi-cohort, non-randomized, multicenter Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of once daily oral BMF-219 in patients with R/R acute leukemia (AL), DLBCL and MM who have received standard therapy.

The primary objective of the study is to determine independently for each cohort/indication, the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-219 oral monotherapy. Key secondary objectives include further evaluation of safety and tolerability, characterization of the pharmacodynamics and pharmacokinetics of BMF-219, and assessment of its antitumor activity based on best overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), and time to progression (TTP) per disease-specific response criteria as assessed by the investigator. Food-effect studies will be performed in DLBCL and MM patients at certain dose levels.

Methods: Utilizing an accelerated titration design, doses of BMF-219 will be escalated in single-subject cohorts independently for each indication until 1 subject experiences either a ≥ Grade 2 related adverse event or dose limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 + 3” design. Treatment will continue in 28-day cycles until progression or intolerability. Expansion cohorts for each indication will enroll patients to obtain further safety and efficacy data.

Patients with R/R AL, R/R DLBLC who received ≥ 2 but ≤ 5 therapies, and R/R MM who received ≥ 3 therapies and have either failed or are ineligible for any standard therapies are eligible. Patients must have ECOG PS ≤ 2, and adequate organ function. Key exclusion criteria include known CNS disease involvement, prior menin inhibitor therapy, and clinically significant cardiovascular disease.

Results: The study is ongoing.

Summary/Conclusion: The enrollment commenced in January 2022.

P588: IMPROVED OUTCOME OF RELAPSED/REFRACTORY ACUTE MYELOID LEUKEMIA BY ADDITION OF LIPOSOMAL ADRIAMYCIN TO CLAG CHEMOTHERAPY

H. Yao 1,* , C. Zhang 1 , J. Li 2 , X. Yin 3 , J. Rao 1 , X. Tan 1 , T. Chen 1 , L. Gao 1 , P. Kong 1 , X. Zhang 1

1 Medical Center of Hematology, Xin Qiao Hospital, Chong Qing; 2 Hematology, Changsha Central Hospital Affiliated to Nanhua University, Hu Nan; 3 Department of Hematology,the 923rd Hospital of the Joint Logistics Support Force of the People′Liberation Army of China, Guang Xi, China

Background: Prognosis of refractory and relapsed acute myeloid leukemia (r/r AML) is challenge and no standard of treatment exists. Cladribine based regimen in treatment of r/r AML proved to be effective,while the total rate of response is limited.

Aims: To explore whether the addition of liposomal adriamycin can improve the response.

Methods: We registered a clinical trial, which is CLAG plus liposomal adriamycin (CLAG+PLD) regimen for re-induction of r/r AML (ChiCTR1800017569), to observe the response, overall survival (OS) and disease free suivival (DFS) of the patients. In addition to that, CLAG alone as salvage therapy (no PLD group) for RR-AML in our medical center were chosen as historical comparison

Results: To observe the response, overall survival (OS) and disease free suivival (DFS) of the patients. In addition to that, CLAG alone as salvage therapy (no PLD group) for RR-AML in our medical center were chosen as historical comparison.Of the 55 patients who were evaluable for remission after CLAG +PLD treatment(PLD group), 27（49.1%）achieved CR and negative of MRD, 12（21.8%）achieved CR and positive of MRD, 5（9.1%）achieved partial response, and 11（20%）no response. In Contrast, no PLD group has inferior response rate,with the corresponding data is 24（46.6%）, 6（10.9%）,10（18.2%）,and 13（23.6%）respectively (p= 0.008).As for the 2-year OS and DFS, the median OS and DFS for PLD group has not reached and 10 months for noPLD group (p= 0.023 and p= 0.045 respectively). The safety observation did not find increasing toxicity in PLD group.In subgroup analysis of PLD group, the response between FLT3-ITD mutated group and wild group showed significant difference ( p =0.006). Moreover, hematopoietic stem cell transplantation (HSCT) group exert superiority in both OS and DFS compared to no HSCT group with 2-year of follow-up（p＜0.0001 and p＜0.00001 respectively）.

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Summary/Conclusion: In conclusion, CLAG + PLD is a prospective salvage regimen for r/r AML which brings improved response rates, 2-year OS and DFS and similar toxicities compared to CLAG.

P589: AGE AND AGE-ADAPTED RISK FACTORS HIGHLY INFLUENCE SURVIVAL PROBABILITY IN AML PATIENTS – A RETROSPECTIVE ANALYSIS

K. Nachtkamp 1 , C. Zahner 1,* , A. Fricke 1 , T. Ulrych 1 , F. I. Schulz 1 , A. Kündgen 1 , P. S. Jäger 1 , G. Kobbe 1 , U. Germing 1

1 Klinik für Hämatologie, Onkologie und Klinische Immunologie, Uniklinik Düsseldorf, Düsseldorf, Germany

Background: Advanced age is strongly associated with inferior overall survival in patients with acute myeloid leukemia (AML). Despite advances in modified treatment regimen and genetic analyses of underlying mutations the prognosis for patients above the age of 65 remains poor.

Aims: The aim was to determine patient-related risk factors and disease-associated variables.

Methods: 915 AML patients (498 < 65 years, 417 ≥ 65 years) treated at the University Hospital of Düsseldorf were analyzed. Patients were diagnosed between 2008 and 2020 comprising 484 males and 431 females. Median age was 63 years. Patients’ survival was calculated using Kaplan-Meier-plots and multivariate regression analyses were performed to identify prognostic factors for survival.

Results: Age itself proved to be an independent risk factor for survival: 6.9 months within the group of patients ≥ 65 years, 166 months in patients < 65 years (p < 0.001). In patients ≥ 65 years aberrant cytogenetic findings and an inferior ECOG status were more frequent at the time of diagnosis. Comparison of survival within the same cytogenetic risk group yielded significant differences between the two age-stratified cohorts: Bearing cytogenetic aberrations associated with a favorable prognosis in the group with patients aged over 65 was associated with a median survival of 18 months whereas patients younger than 65 did not reach estimated median survival time (p < 0.001). When comparing patients with the same ECOG-status, advanced age was the most decisive prognostic factor for survival. This was highly significant in patients with ECOG status 0. In the group with higher ECOG status an influence of age was notable but of lower significance. In patients with pathologic lung function tests age turned out to be significant for survival: 26 months (≥ 65 years) vs. not reached (< 65 years) (p = 0.008). In patients with pathologic cardiac ultrasound findings results were similar with 26 months (≥ 65 years) vs. not reached (< 65 years) (p = 0.001). Whether AML was de novo or secondary affected survival probability only in the group aged below 65 (166 months de novo AML, 69 months secondary AML).

Summary/Conclusion: Age has significant influence on patient- and leukemia-related factors, thus modulating the impact of other parameters on the prognosis. We were able to confirm the following independent negative predictive parameters on survival in AML patients: increasing age ≥ 65 years, high-risk genetic aberrations, comorbidities (lung, heart) and higher ECOG-status.

P590: PRELIMINARY SAFETY AND EFFICACY OF BGB-11417, A POTENT AND SELECTIVE B-CELL LYMPHOMA 2 (BCL2) INHIBITOR, IN PATIENTS (PTS) WITH ACUTE MYELOID LEUKEMIA (AML)

J. Shortt 1,* , S. Y. Tan 2 , P. Cannell 3 , T. F. Ng 4 , C. Y. Fong 5 , S. Ramanathan 6 , R. Rajagopal 7 , S. Leitch 8 , R. Gasiorowski 9 , C. Grove 10 , D. Lenton 11 , P. Tan 12 , C. DiNardo 13 , M. T. Ling 14 , S. Cheng 14 , Y. Liu 14 , M. Co 14 , W. Y. Chan 14 , D. Simpson 14 , A. H. Wei 12,15

1 School of Clinical Sciences, Monash University and Monash Health, Clayton; 2 St Vincent’s Hospital Melbourne, Fitzroy, Victoria; 3 Fiona Stanley Hospital, Murdoch, Western Australia; 4 Gold Coast University Hospital, Southport, Queensland; 5 Austin Health, Heidelberg, Victoria; 6 The Saint George Hospital-Kogarah, Kogarah, New South Wales, Australia; 7 Middlemore Hospital; 8 North Shore Hospital, Auckland, New Zealand; 9 Concord Repatriation General Hospital, Concord West, New South Wales; 10 Linear Clinical Research & Sir Charles Gairdner Hospital, Nedlands, Western Australia; 11 Orange Health Service (Central West Cancer Care Centre), Orange, New South Wales; 12 One Clinical Research, Nedlands, Western Australia, Australia; 13 University of Texas MD Anderson Cancer Center, Houston, TX; 14 BeiGene (Shanghai) Co., Ltd., Shanghai, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 15 Department of Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Victoria, Australia

Background: BCL2, a key regulator of apoptosis promoting cancer cell survival, is aberrantly expressed in many hematologic malignancies. The BCL2 inhibitor, venetoclax, is standard of care for the treatment of newly diagnosed AML in adults unfit for induction chemotherapy. Although venetoclax-based treatments have improved outcomes for pts with AML, there are concerns surrounding disease resistance after continued use and adverse events (AEs) such as gastrointestinal toxicities and neutropenia. Clinical findings have shown that 20-30% of pts using venetoclax are refractory to treatment, and a majority of pts who initially achieve remission ultimately relapse ( N Engl J Med. 2020;383(7):617-629). The highly selective investigational BCL2 inhibitor, BGB-11417, demonstrated more potent antitumor activity than venetoclax in preclinical studies ( Cancer Res . 2020;80[suppl, abstr]:3077).

Aims: To present preliminary safety and efficacy results of BGB-11417 in combination with azacitidine in pts with AML enrolled in BGB-11417-103 ( NCT04771130 ).

Methods: BGB-11417-103 is an ongoing phase 1b/2, global, multicenter, dose-escalation and expansion study evaluating the combination of BGB-11417 and azacitidine in pts with AML (either treatment-naïve [TN] unfit for intensive induction chemotherapy or relapsed/refractory [R/R]), myelodysplastic syndrome, or myelodysplastic syndrome/myeloproliferative neoplasm. Pts who received prior azacitidine or BCL2 inhibitors were excluded. For pts with AML, the 10-day regimen consisted of BGB-11417 at 40 mg (Cohort 1), 80 mg (Cohort 2), or 160 mg (Cohort 3) in combination with azacitidine (75 mg/m 2 x 7 days). In Cycle 1, a 4-day ramp-up of BGB-11417 was utilized starting at 1/8 of the target dose. The window to assess dose-limiting toxicity (DLT) was up to Day 28 for nonhematologic toxicities and Day 42 or initiation of Cycle 2 for hematologic toxicities. Treatment-emergent AEs were graded per Common Terminology Criteria for AEs v5.0. Responses were assessed using the 2017 European LeukemiaNet criteria ( Blood. 2017;129(4):424-447). All pts gave informed consent.

Results: As of 10 January 2022, 27 pts with AML were treated with the combination therapy (6 in Cohort 1; 15 in Cohort 2; 6 in Cohort 3; Table). Median age was 80 yrs (TN; 18 pts) and 70 yrs (R/R AML; 9 pts); 44% of pts had adverse karyotype. Median prior line of therapy for R/R AML was 1 (range 1-2). At a median study follow-up of 2.1 months and median duration of treatment of 1.84 months (range 0.3-7.6), 2 of 23 evaluable pts experienced DLTs (Grade 4 neutropenia and Grade 4 thrombocytopenia at the 80 mg x 10 day dose level), which did not meet the safety stopping protocol criteria. Laboratory tumor lysis syndrome (TLS) was observed in 1 pt with a known history of chronic kidney disease treated with 160 mg x 10 day; pt was asymptomatic and TLS resolved within 4 days. Most common nonhematologic AEs were constipation (37%) and azacitidine injection-site reaction (33%). Most common Grade ≥3 hematologic AEs were neutropenia (44%), thrombocytopenia (41%), and anemia (37%). No pts required dose reductions of BGB-11417. Majority (n=17; 63%) of pts are continuing study treatment. Ten pts discontinued due to AEs (n=3), proceeding to transplant (n=3), consent withdrawal (n=2), or disease progression (n=2). Preliminary CR/CRh rates for TN and R/R were 56% and 44%, respectively. Seven of the 9 CRs were achieved by end of Cycle 1.

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Summary/Conclusion: Preliminary results show that a 10-day regimen of BGB-11417 plus azacitidine resulted in a majority of CR observed by the end of Cycle 1 and was well tolerated in pts with AML.

P591: PRELIMINARY RESULTS OF A PHASE I STUDY OF CLIFUTINIB, A HIGHLY SELECTIVE, POTENT ORAL FLT-3 INHIBITOR, IN PATINETS WITH FLT3-MUTATED RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA

W. Yu 1 , X. Wei 2 , Z. Ge 3 , Y. Li 4 , Z. Jiang 5 , L. Yang 6 , L. Lin 7 , Y. Yao 8 , X. Deng 9 , X. Du 10 , Y. Li 11 , T. Chen 12 , X. Feng 13 , J. Zhou 14 , M. Hou 15 , R. Fu 16 , J. Lan 17 , X. Hu 18 , S. Huang 19 , J. Wang 20 , X. Du 21 , H. Yang 22 , H. Yang 23 , H. Wang 1 , L. Zheng 24 , Z. Wang 24 , B. Liu 24 , N. Kang 24 , Y. Zhuang 24 , Y. Zhang 24 , J. Jin 1,*

1 The First Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou; 2 Henan Cancer Hospital, Zhengzhou; 3 Zhongda Hospital, Southeast University, Nanjing; 4 Zhujiang Hospital of Southern Medical University, Guangzhou; 5 The First Affiliated Hospital of Zhengzhou University, Zhengzhou; 6 The Second Hospital of Shanxi Medical University, Taiyuan; 7 ​Hainan General Hospital, Haikou; 8 Baoji Central Hospital, Baoji; 9 Weihai Municipal Hospital, Weihai; 10 Guangdong General Hospital; 11 Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou; 12 Huashan Hospital, Shanghai; 13 The Affiliated Hospital of Qingdao University, Qingdao; 14 Wuhan Tongji Hospital, Wuhan; 15 Qilu Hospital of Shandong University, Jinan; 16 Tianjin Medical University General Hospital, Tianjin; 17 Zhejiang Provincial People’s Hospital, Hangzhou; 18 Beida Medical Treatment Luzhong Hospital, Zibo; 19 Xi’an Gaoxin Hospital, Xian; 20 Aerospace Center Hospital, Beijing; 21 The Second People’s Hospital of Shenzhen, Shenzhen; 22 Shunde Hospital of Southern Medical University, Foshan; 23 The 1st Affiliated Hospital of He’nan University of Science and Technology, Luoyang; 24 Sunshine Lake Pharma Co., Ltd, Dongguan, China

Background: An internal tandem duplication (ITD) in the juxtamembrane domain of FLT3 on chromosome 13q12, which accounts for 30% of patients with Acute Myeloid Leukemia (AML), is associated with poor prognosis, including a lower complete remission (CR) rate, a reduced duration of remission (DOR), a higher relapse rate, and a decreased overall survival. Clifutinib, a highly selective oral FLT-3 inhibitor, demonstrated potent activity against FLT3-ITD mutated human cell lines, both in vitro and in vivo .

Aims: This present study ( NCT04827069 ) is a first-in-human study of Clifutinib, including dose-escalation and dose-expansion phases, with a purpose to evaluate the safety, tolerability, pharmacokinetics (PK) and antitumor activity in Chinese patients with relapsed/ refractory (R/R) FLT3-mutated AML. Here we report the preliminary results of the study.

Methods: AML patients aged≥18 years with refractory to ≥ 2 cycles of standard induction chemotherapy, or relapsed after achieved remission from prior treatments were enrolled. Previous use of FLT3 inhibitors was allowed. The primary endpoints included safety and tolerability, including dose limiting toxicity (DLT), adverse event (AE). Secondary endpoints were PK and anti-leukemic effect [CR/ CRh (Complete Remission with Partial Hematologic Recovery) rate, Composite Complete Remission (CRc) rate, DOR, and overall survival (OS)]. Clifutinib was administered orally on an empty stomach at 10~70 mg daily, 28 consecutive days as a treatment cycle. Resumption of Clifutinib was not allowed after hematopoietic stem cell transplantation (HSCT). Modified 3 + 3 and accelerated titration design was utilized.

Results: As of December, 2021, fifty-seven patients were enrolled, including one at 10 mg, one at 20 mg, 6 at 55 mg, 34 at 40 mg, 15 at 70mg dose groups. The median age was 52 years, and one third of the subjects received prior FLT-3 inhibitors treatments. Two subjects discontinued from the study due to AEs. Eighteen subjects were evaluable for DLT determination. Only one subject in the 55 mg group experienced DLT (Grade 3 QT prolongation), and MTD had not been reached. Fifty-three subjects were evaluable for safety analysis. The most common treatment-related AEs of any grade included platelet count decreased (69.8%), decreased white cell count (69.8%), neutrophil count decreased (60.4%), anemia (43.3%), lymphocyte count decreased (32.1%). Plasma concentrations of Clifutinib increased with increasing dose, with t max between 2 and 6 hours. Accumulation was observed following repeat dosing, with estimated t 1/2 values of 68.3 to 114.2 hours based on accumulation index. Approximately dose-linear PK parameters were observed over the dose range for both single and repeat dosing. Of 53 FLT3-ITD positive, evaluable subjects, CR/CRh rate was 17.0% (9/53), and CRc rate was 45.3% (24/53). Among subjects administered with Clifutinib 40 mg daily, the CR/CRh rate was 18.2% (6/33) with 3 patients achieved CR, median DOR of CR/CRh was 5.7 months, the CRc rate was 48.5% (16/33), and median OS was 7.4 months. Among patients experienced only one prior regimen, the CR/CRh rate of 40 mg dose group was 25% (4/16), the CRc rate was 50% (8/16), and the median OS was 13.0 months.

Summary/Conclusion: Preliminary results of this phase 1 study demonstrated that Clifutinib has an acceptable safety profile and promising antitumor activity, especially at the dose of 40 mg daily. A confirmatory phase 3 study is planned to further evaluate the efficacy and safety of Clifutinib at 40 mg daily in FLT3-mutated R/R AML.

P592: GILTERITINIB IN COMBINATION WITH VENETOCLAX, LOW DOSE CYTARABINE AND ACTINOMYCIN D FOR FLT3 MUTATED RELAPSED OR REFRACTORY ACUTE MYELOID LEUKEMIA

A. Žučenka 1,* , R. Pileckytė 1 , K. Maneikis 1 , V. Vaitekėnaitė 1 , L. Kevličius 1 , L. Griškevičius 1

1 Hematology, Oncology, Transfusion Medicine Center, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania

Background: A second generation FLT3 inhibitor Gilteritinib has become a standard of care for FLT3 mutated relapsed or refractory acute myeloid leukemia ( FLT3 m R/R AML). However, remission duration and overall survival remain unsatisfactory. Preliminary results of doublet Gilteritinib + Venetoclax and triplet Gilteritinib + Venetoclax + Hypomethylator regimens are encouraging. Herein, we report the quadruplet regimen consisting of Gilteritinib, Venetoclax, Low Dose Cytarabine and Actinomycin D (ACTIVE + G) for the treatment of FLT3 m R/R AML in the clinical practice setting.

Aims: To evaluate the efficacy and safety of the ACTIVE + G regimen.

Methods: This was an observational, retrospective study. The patients were at least 18 years of age and had FLT3 m R/R AML. All patients provided informed consent for treatment and data collection. The ACTIVE + G regimen consisted of Venetoclax 600mg/d p/o from day 1 up to day 28, Cytarabine 20mg/m2 s/c on days 1-10, Actinomycin D 12.5 µg/kg i/v on days 1-3 (on days 1-2 for patients ≥65 years) and Gilteritinib 120mg/d p/o starting from either day 4 or day 10 and continued up to day 28. Indications for stopping Venetoclax and Gilteritinib before day 28 were life-threatening infections or faster hematological recovery in responding patients. A second ACTIVE + G cycle was administered in non-responders without evidence of progressive disease after Cycle 1 or in responders with positive measurable residual disease (MRD). Responders after ACTIVE + G could proceed to either allogeneic stem cell transplantation (alloSCT) or maintenance therapy with Venetoclax, Low Dose Cytarabine and Gilteritinib. We evaluated baseline characteristics, composite CR (CRc = CR + CRi + CRp), overall response (ORR = CRc + MLFS), MRD negativity rates (<0.1% by multiparameter flow cytometry), overall survival (OS), relapse-free survival (RFS), grade 3-5 non-hematological toxicities and day 30 and day 60 mortality rates.

Results: Fifteen patients had been treated with ACTIVE + G, of whom 8 (53%) were female. The median age was 66 years (34-87), median ECOG was 2 (0-3). FLT3-ITD mutation was confirmed in 80% (12/15) of cases and 20% (3/15) had FLT3-TKD . The most common co-mutations were NPM1 (53%, 8/15), DNMT3A (27%, 4/15), IDH1 (20%, 3/15) and IDH2 (20%, 3/15). Three patients (20%) had adverse cytogenetics. The median number of previous treatment lines was 2 (1-5). Twelve patients (80%) had received prior intensive chemotherapy, 3 patients (20%) had prior Venetoclax exposure and 9 patients (60%) had been previously treated with FLT3 inhibitors (Midostaurin – 7, Sorafenib – 1, Gilteritinib – 1). Three patients (20%) had relapsed after alloSCT. The majority of patients (80%, 12/15) had received 1 cycle of ACTIVE + G, 3 patients (20%) had been treated with 2 cycles. The CRc and the ORR were 67% (10/15) and 93% (14/15), respectively. MRD negativity was confirmed in 50% (5/10) of CRc cases. The median OS and RFS were 8.6 and 12.9 months, respectively. The most common non-hematological grade 3-5 adverse events were febrile neutropenia (73%, 11/15), sepsis/bacteremia (53%, 8/15), pneumonia (33%, 5/15) and secondary hemophagocytosis (13%, 2/15). Day 30 and day 60 mortality rates were 13% (2/15) and 20% (3/15), respectively.

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Summary/Conclusion: A quadruplet regimen ACTIVE + G demonstrated high efficacy in this small group of R/R FLT3 m AML patients irrespective of their prior exposure to FLT3 inhibitors or Venetoclax. The main toxicities were infectious complications attributable to prolonged myelosuppression. Prospective clinical trials are needed to verify our results.

P593: MULTIDIMENSIONAL ANALYSIS OF THE B CELL RECEPTOR OFFERS INSIGHT INTO THE ONTOGENETIC RELATIONSHIP OF MONOCLONAL B-CELL LYMPHOCYTOSIS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

A. Agathangelidis 1,* , C. Galigalidou 2 , A. Iatrou 2 , L. Zaragoza-Infante 2 , L. Scarfò 3 , M. C. Maniou 2 , P. Ranghetti 3 , N. Pechlivanis 2 , V. Junet 4 , A. Skaftason 5 , M. Tsagiopoulou 2 , F. Psomopoulos 2 , R. Rosenquist 5 , P. Ghia 6 , A. Chatzidimitriou 2 , K. Stamatopoulos 2

1 Department of Biology, Section of Genetics and Biotechnology, National and Kapodistrian University of Athens, Athens; 2 Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 3 Division of Experimental Oncology, Università Vita-Salute San Raffaele/Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale San Raffaele, Milan, Italy; 4 Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Barcelona, Spain; 5 Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 6 Division of Experimental Oncology, Università Vita-Salute San Raffaele/Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale San Raffaele, Milan, Greece

Background: The clonotypic B-cell receptor immunoglobulin (BcR IG) is key to the pathogenesis of CLL. Against that, however, scant information exists regarding the role of BcR IG in early stages of CLL ontogenesis, i.e. monoclonal B cell lymphocytosis (MBL).

Aims: Here, we aimed to address this issue through the integrative analysis of: (i) the BcR IG gene repertoire, (ii) the transcriptome, and, (iii) the BcR IG reactivity profile, of both the high and the low-count MBL subtypes (HC-MBL and LC-MBL, respectively), complemented by comparisons to CLL.

Methods: The study group comprised 17 individuals with LC-MBL and 14 with HC-MBL. “CLL-like” cells were flow-sorted. BcR IG NGS data was annotated with IMGT/HighV-QUEST and TRIPR. Clonotypes were defined as IG gene rearrangements with identical IG variable gene and CDR3 amino acid sequence. Stereotypy analysis in MBL cases focused on abundant clonotypes, which were compared against data from >39,000 CLL cases. Intraclonal diversification was assessed through graph networks of all distinct nucleotide variants of each clonotype. Transcriptome profiling was performed on polyA-selected RNA from 16 LC-MBL and 8 HC-MBL cases; comparisons were undertaken against RNAseq data from 74 CLL patients. Count normalization, differential gene expression and splice event analysis were performed with DESeq2, Limma and SGSseq, respectively; gene enrichment analysis was performed with Enrichr (p<0.05). BcR IG reactivity profiling was performed for 6 LC-MBL cases, 10 HC-MBL cases and 9 CLL cases against 123 autoantigens using a protein microarray; validation tests were performed with ELISA.

Results: Clonality assessment revealed a monoclonal profile for most HC-MBL cases (86%), whereas LC-MBL cases were mainly oligoclonal (53%). Stereotypy analysis revealed a stronger immunogenetic connection of HC-MBL to CLL, since 19/47 abundant clonotypes (40%) from HC-MBL were assigned to CLL stereotyped subsets as opposed to only 19/167 (11%) from LC-MBL. Intraclonal diversification analysis was undertaken for: (i) 26 monoclonal cases (from HC-MBL or LC-MBL), (ii) 10 oligoclonal LC-MBL cases and 73 monoclonal CLL cases. Significant differences (p<0.05) were evident between oligoclonal MBL vs monoclonal MBL vs CLL; the former showed wide branching of variants due to distinct somatic hypermutations (SHM). In contrast, monoclonal MBL and CLL displayed progressively higher levels of variant connectivity due to the presence of shared SHMs, likely reflecting stronger antigenic pressure. Comparative transcriptomic analysis of either type of MBL vs CLL disclosed significant (p<0.05) down-regulation of genes in the Notch, AMPK and BcR signaling pathways. Moreover, LC-MBL showed overexpression of genes implicated in antigen processing and presentation and OXPHOS compared to either HC-MBL or CLL. In addition, both MBL types were characterized by virtual absence of alternative splicing, contrasting CLL where splicing events were identified in 14 genes of the BcR signaling pathway. Finally, BcR IG reactivity analysis revealed different patterns among individual cases, however all examined cases from MBL and CLL consistently bound a set of autoantigens, including myelin, centromere protein B, β 2 -microglobulin, C-reactive protein and the nucleosome.

Summary/Conclusion: In conclusion, we highlight important differences between MBL and CLL stemming from the attributes of the BcR IG and the related signaling pathway. Overall, the reported findings allude to different maturation processes that likely contribute to shaping a distinct BcR IG signaling capacity in MBL, particularly LC-MBL, vs CLL.

P594: INCREASED FREQUENCIES OF IGH LOCUS SUICIDE RECOMBINATION POINTS ON CHRONIC LYMPHOCYTIC LEUKEMIA WITH LOW RATE OF AID RELATED SOMATIC MUTATIONS, MYC OVEREXPRESSION AND SHORT TELOMERES

I. Al Jamal 1,2,* , M. Parquet 1 , H. Boutouil 1 , K. Guiyedi 1 , D. Rizzo 1,3 , M. Dupont 1,3 , M. Boulin 1,3 , S. Aoufouchi 4 , S. Al Hamaoui 2 , N. Makdissy 2 , J. Feuillard 1,3 , N. Gachard 1,3 , S. Peron 1

1 Centre National de la Recherche Scientifique (CNRS), UMR 7276/INSERM U1262,Limoges University, Limoges, France; 2 Faculty of Sciences, GSBT Genomic Surveillance and Biotherapy Team, Lebanese University, Tripoli, Lebanon; 3 Limoges University Hospital Center, Biological Hematology Laboratory, Limoges; 4 UMR 9019 Genome Integrity and Cancers, Villejuif, France

Background: In normal B-cells, Activation Induced-cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and IGHV somatic hypermutation (SHM). AID is also implicated in another IgH rearrangement, the Locus Suicide Recombination (LSR). LSR occurs in activated B-cells and recombines the IgH locus between the switch µ (Sµ) region and one 3’a2 regulatory region (3’a2RR) of the IgH locus. LSR results in the complete deletion of the cluster of IgH constant genes. When LSR hits the active IgH locus, it induces the loss of BCR expression and the death of the concerned B cell. Chronic lymphocytic leukemia (CLL) is an indolent non-Hodgkin B-cell lymphoma. Tumor CLL B-cells weakly express a B cell receptor (BCR) on the surface which is composed, in the vast majority of cases, of immunoglobulins (Ig) of the mu (µ) and delta (δ) isotypes and Ig class-switched CLL are rare, raising the question of abnormalities in the Ig gene recombination machinery in this B-cell cancer.

Aims: Searching for abnormalities of IgH locus recombination in CLL, we investigated CSR and LSR in CLL.

Methods: In this study, we used different molecular biology technics as high throughput Sequencing (HTS) to analyze CSR and LSR junctions, IgHV, and PIM-1 mutation. Quantitative reel time PCR (quantification of AID, cMYC and IgH locus transcripts). Samples are from CLL patients (N=47) with more than 98% blood tumor cell infiltration and controls consisted in healthy volunteers (HV) (N=9). Bioinformatics uses CSReport tool. Statistics are from graph pad or R.

Results: CSR levels were lower in CLL than in HV samples contrariwise to LSR that was found at comparable levels in both HV and CLL groups (Fig. A, B). Moreover, some patients exhibited increased LSR counts. Because distribution of LSR counts was bimodal with a valley at 27, that value being also the mean of LSR counts in HVs, we separated CLL patients in two groups so called LSR-High and LSR-Low with that threshold of 27 LSR count per sample. We analyzed the diversity of the LSR junctions using Shannon Index. LSR junction diversity was significantly higher in LSR-High than in LSR-Low CLLs and HVs. This result is in opposition with the IgHV mutation rate since LSR-High CLLs exhibited a stronger homology to IgHV reference sequences (unmutated CLLs). We also found that LSR-High CLLs exhibited a marked lowest rate of AID off-target PIM1 mutations (Fig. C). AID expression was low and at comparable levels in both LSR-Low and LSR-High CLLs. Because diversity of LSR junctions in LSR-High CLLs is evocative of an on-going process, we analyzed the expression of productive and non-productive transcripts of the constant part of IgH locus, which we found increased in LSR-High when compared to LSR-Low CLLs. We also observed shorter telomere lengths and c-Myc overexpression in the LSR-High group. Both shorter telomeres and c-Myc overexpression are very likely to reflect an increased number of DNA replication cycles in the history of the CLL tumor cell (Fig. D, E). Consistently, Kaplan Meyer curves of Treatment Free Survival (TFS) show that TFS of LSR-High CLLs was significantly shorter than the one of LSR-Low counterpart, an indication of a more rapidly evolving CLLs (Fig. F).

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Summary/Conclusion: Altogether, these results indicate the accessibility of IgH locus and the proliferation in CLL patients with high rate and increased diversity of LSR junctions could be increased in Myc dependent manner resulting in shorter survival and pointing on an AID independent mechanism of IgH recombination.

P595: DISCOVERY OF NOVEL CIRCULAR RNAS (CIRCRNAS) OF THE APOPTOSIS-RELATED BAX AND BCL2L12 GENES WITH A MULTIFACETED ROLE IN CHRONIC LYMPHOCYTIC LEUKEMIA, USING CUTTING-EDGE NANOPORE SEQUENCING TECHNOLOGY

P. Artemaki 1,* , N. Machairas 1 , P. Karousi 1 , V. Pappa 2 , A. Scorilas 1 , S. Papageorgiou 2 , M. Batish 3 , C. Kontos 1

1 Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens; 2 Second Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, Athens, Greece; 3 Department of Medical and Molecular Sciences, University of Delaware, Newark, DE, United States of America

Background: Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key indirect regulators of gene expression, with deregulated expression and established involvement in several human malignancies. Many circRNAs have been shown to bind microRNAs (miRNAs), thus making miRNAs unavailable to regulate protein-coding gene expression. Moreover, some of them have been described to interact with chromatin and affecting its structure. Despite their pivotal roles in the development and progression of cancer, only three studies have investigated their expression and role(s) in chronic lymphocytic leukemia (CLL), so far. The products of BCL2 and its homologues, including BAX and BCL2-like 12 ( BCL2L12 ), are implicated in CLL, as apoptosis regulators. Moreover, expression levels of both BAX and BCL2L12 mRNAs are deregulated in CLL and are associated with patients’ response to therapy and overall survival. However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their regulatory potential.

Aims: We sought to further elucidate the contribution of both BAX and BCL2L12 in CLL by unraveling the identity of their circRNAs in a B-cell leukemic cell line (EHEB) and by studying in silico their potential interactions with miRNAs having an already established role in CLL.

Methods: EHEB cells were propagated according to ATCC instructions. After total RNA extraction, 2μg of total RNA were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of BAX and BCL2L12 genes; thus, only cDNAs from BAX and BCL2L12 circRNAs were amplified, respectively. After having mixed and purified all nested-PCR products, nanopore sequencing libraries were built according to an optimized protocol. Following nanopore sequencing and basecalling in a MinION Mk1C 3 rd -generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA sequences were identified using an in-house –built, PERL-based algorithm. A single-molecule resolution fluorescent in situ hybridization (FISH) method called circFISH was used to visualize the circRNA distribution in the cells. Lastly, miRDB was used to predict interactions of the most abundant BAX and BCL2L12 circRNAs with miRNAs.

Results: We discovered 18 and 21 novel circRNAs produced by BAX and BCL2L12 genes, respectively, in this leukemic cell line. The exon structure of these novel circRNAs showed remarkable diversity, also comprising genomic regions that have previously been considered as intronic, as they are not included in messenger RNAs (mRNAs). Interestingly, one of the two most abundant BAX circRNAs is composed of a single exon, also spanning two introns. Similar intron retentions have been observed in a few BCL2L12 circRNAs. The gamut of miRNAs predicted to be bound by novel circRNAs includes miR-484, miR-181a-2-3p and miR-214-3p. Particularly, the inhibition of miR-214-3p is associated with elevated apoptosis in leukemic B-cells.

Summary/Conclusion: Alternative splicing and back-splicing mechanisms of the primary BAX and BCL2L12 transcripts produce multiple distinct circRNAs in this B-cell CLL cell line. Interestingly, one of the two most abundant circular transcripts of BAX is a circRNA spanning three exons and two intervening introns of this gene. Lastly, several miRNAs are predicted to be sequestered by circRNAs of either of these apoptosis-related genes, including miR-181 family members that have been associated with chemosensitivity. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

P596: CLINICAL IMPACT OF TP53 DISRUPTION IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS TREATED WITH A BCR INHIBITOR. A CAMPUS CLL EXPERIENCE

R. Bomben 1,* , F. M. Rossi 1 , F. Vit 1 , T. Bittolo 1 , A. Zucchetto 1 , R. Papotti 2 , E. Tissino 1 , F. Pozzo 1 , M. Degan 1 , E. Zaina 1 , I. Cattarossi 1 , P. Nanni 1 , R. Marasca 3 , G. Reda 4 , L. Laurenti 5 , J. Olivieri 6 , A. Chiarenza 7 , L. Ballotta 8 , A. Cuneo 9 , M. Gentile 10 , F. Morabito 11 , A. Tafuri 12 , F. Zaja 13 , R. Foà 14 , F. Di Raimondo 15 , G. Del Poeta 16 , V. Gattei 1

1 Clinical and Experimental Onco-Heamtology Unit, Centro di Riferimento Oncologico IRCCS, Aviano; 2 International PhD School in Clinical and Experimental Medicine, University of Modena and Reggio Emilia; 3 Hematology Unit, Department of Oncology and Hematology, Azienda-Ospedaliero Universitaria (AOU) of Modena, Policlinico, Modena; 4 Division of Ematologia, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico di Milano, Milano; 5 Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Universitaria Policlinico A Gemelli di Roma, Roma; 6 Clinica Ematologica, Centro Trapianti e Terapie Cellulari “Carlo Melzi” DISM, Azienda Ospedaliera Universitaria S. Maria Misericordia, Udine; 7 Division of Hematology, Policlinico, Department of Surgery and Medical Specialties, University of Catania, Catania; 8 Dipartimento Clinico di Scienze Mediche, Chirurgiche e della Salute, University of Trieste, Trieste; 9 Hematology Section, Department of Medical Sciences, University of Ferrara, Ferrara, Ferrara; 10 Hematology, Unit AO of Cosenza; 11 Biothecnology Research Unit, AO of Cosenza, Cosenza; 12 Department of Clinical and Molecular Medicine and Hematology, Sant’Andrea - University Hospital - Sapienza, University of Rome, Rome; 13 Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste; 14 Hematology, Department of Translational and Precision Medicine, ‘Sapienza’ University, Roma; 15 Division of Hematology, Policlinico, Department of Surgery and Medical Specialties, University of Catania, Catania; 16 Division of Haematology, University of Tor Vergata, Roma, Italy

Background: In chronic lymphocytic leukemia (CLL), the presence of TP53 aberration (mutations and/or deletion) predicts an increased risk of relapse and death after chemo-immunotherapy (CIT). Although TP53 deletion and mutations mostly co-occur and are considered equal prognosticators, the clinical impact of isolated or concomitant mutations and deletions remains unclear in the context of targeted agents.

Aims: To investigate the clinical relevance of isolated or concomitant TP53 aberrations in a large cohort of CLL patients treated with BCR inhibitors.

Methods: In the framework of an institutional Italian multicenter working group on CLL (Campus CLL), a retrospective analysis of 229 CLL patients (51 treatment naïve, and 178 relapsed/refractory) treated with ibrutinib was carried out. All patients referred to a single institution for del17p analyses by FISH (167-kb 17p13 orange probe, MetaSystems) and TP53 mutations by NGS, both analyses carried out on CD19-purified (>85% pure) CLL samples within 6 months prior the start of ibrutinib treatment. The median follow-up from ibrutinib treatment was 36.3 months (95% CI 29.5-41.5 months). Overall survival (OS) and progression free survival (PFS) were computed from the start of ibrutinib treatment.

Results: In the CLL cohort, 76 patients with del17p showed a trend for inferior OS respect to wt cases (P=0.0662, Fig.1A), while a significant correlation was found with PFS ( P=0.0162 ). With regard to TP53 mutations, 296 TP53 mutations were found in 126 patients (range of mutations/patients 1-11). As with CIT, TP53 -mutated patients, irrespective of VAF (Fig.1B), experienced a significantly worse OS and PFS than wt cases also by univariate analyses ( P=0.0160 , and P=0.0378 , respectively). The combination of del17p with TP53 mutation data identified 95 cases with no TP53 aberrations, 8 cases del17p only, 58 cases TP53 -mutated only, and 68 cases bearing both del17p deletion and TP53 mutation (Fig.1CD). Only patients with concomitant TP53 mutations and del17p experienced significantly shorter OS and PFS compared to TP53 wt cases ( P=0.0122 , and P=0.0076 , respectively; Fig.1CD). Conversely, patients presenting a single aberration showed no significant differences compared to wt patients (Fig.1CD). The simultaneous presence of TP53 mutations and del17p remained an independent predictor factor both for OS and PFS by multivariate analysis, together with the previous therapy lines (0-1 vs. >1), and anemia. The evolution of TP53 -mutated clones was assessed by longitudinal NGS analysis of sequential PB samples collected from 38 patients (16 relapsed, and 22 on treatment) corresponding to 127 TP53 mutations. Among relapsed cases, 7 showed a prominent expansion of the TP53 -mutated clone, 8 remained stable, and the remaining case displayed an evident decrease (Fig.1E). In non-relapsed patients, 3 cases presented an increase of TP53 mutations, 13 remained stable, and 6 showed a reduction (Fig.1F), with no significant difference compared to relapsed cases (P=0.0623, c 2 test). BTK and PLCG2 mutations were found in 9/16 (56%) relapsed cases and in 3/22 (14%) patients still on ibrutinib ( P=0.0492 , c 2 test). Of note, only 3/7 relapsed cases that presented a positive selection for TP53 mutations showed the presence of BTK mutations at the time of relapse.

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Summary/Conclusion: This retrospective study indicates that only the concomitant presence of TP53 mutations and deletion is an independent negative prognostic factor for OS and PFS in patients with CLL on ibrutinib treatment; the simultaneous investigation of del17p and TP53 mutations may lead to a more precise risk assessment.

P597: HIGH DOSE IRON IMPAIRS MALIGNANT B-CELL VIABILITY IN CHRONIC LYMPHOCYTIC LEUKEMIA

J. Bordini 1,* , C. Lenzi 2 , L. Toscani 1 , P. Ranghetti 1 , E. Perotta 1 , L. Scarfò 2 , P. Ghia 2 , A. Campanella 2

1 Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute; 2 Division of Experimental Oncology, Vita-Salute San Raffaele University, Milan, Italy

Background: Identification of new strategies to improve Chronic Lymphocytic Leukemia (CLL) outcome is fundamental since the disease remains incurable. Malignant B-cells show high levels of reactive oxygen species (ROS), which favors adaptation and survival but also enhances sensitivity to pro-oxidant therapeutics. Redox homeostasis is affected by iron balance since iron excess works as pro-oxidant, and cancer cells rearrange iron trafficking proteins to promote iron uptake. This adaptation might be exploited by exposing malignant cells to iron excess to cause ROS generation, lipid peroxidation, and eventually ferroptosis. We previously demonstrated the feasibility of this strategy in multiple myeloma and prostate cancer pre-clinical models.

Aims: We aim at exploiting high dose iron to negatively affect malignant cell survival and increase therapeutic efficacy of current target therapies (BTK and BCL2 inhibitors). We aim at dissecting the molecular mechanisms underlying iron toxicity and at testing whether iron can improve immune dysfunctions.

Methods: CLL cell lines (MEC-1, MEC-2 and PCL-12), patients peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) and B lymphocytes purified from patients PB were treated with 300 µM ferric ammonium citrate (FeAC) alone or in combination with the BTK inhibitor ibrutinib (IBR, 5-10 µM) or the BCL2 inhibitor venetoclax (VNTX, 1.25-2.5 nM). We evaluated cell viability by annexin PI staining, lipid peroxidation and ferroptosis by measuring malondialdehyde (MDA) levels and antioxidant gene expression levels by qRT-PCR. We additionally evaluated T cell activation by measuring CD25 expression. MEC-1 xenografts generated in immunodeficient RAG2 -/- yc -/- mice were treated with 20 mg/Kg ferric carboximaltose.

Results: In vitro , iron treatment impaired cell proliferation in all CLL cell lines analyzed, inducing accumulation of MDA and cell death (p<0.01). In vivo , iron supplementation reduced the amount of MEC-1 cells in bone marrow and spleen of xenograft mice as compared to control saline-treated mice. In patients primary samples, iron induced cell death of leukemic lymphocytes (CD19 + CD5 + ) either as purified cells or within bulk PBMC (p<0.01). Moreover, combination of iron with IBR or VNTX induced cell death at a higher extent compared to each single drug or to iron alone (p<0.01). Leukemic cells in BMMC samples were also iron sensitive and the effect became more evident upon combination with IBR or VNTX (p<0.05). Neither iron nor iron-drug combinations induced cell death in non-malignant cells. Iron also increased CD25 expression in CD8 + T cells and we are currently exploring whether this might indicate that iron can also improve cytotoxic mediated immune response against leukemic cells.

As single patient analysis revealed heterogeneity of response, we investigated whether we might predict iron sensitivity by analyzing the antioxidant gene expression profile. All CLL cell lines analyzed showed a lower basal expression of NFE2L2, HMOX1, and GPX4 antioxidant genes than typical iron-resistant cell lines, such as prostate cancer PC-3 cells, suggesting that differences in antioxidant gene expression levels may mediate iron response and that this association is worth to be explored in CLL primary samples to explain heterogeneous iron response.

Summary/Conclusion: Our pre-clinical studies suggests that exploiting iron toxicity might be a valuable strategy to improve current target therapies in CLL. Additional studies aimed at distinguishing iron sensitive patients from poor responders will improve the translational potential of this therapeutic approach.

P598: EBI3/IL-27 DEPLETED MICROENVIRONMENT FAVOURS TUMOUR PROGRESSION IN CHRONIC LYMPHOCYTIC LEUKAEMIA

I. C. Botana 1,* , G. Pagano 1 , W. Marina 1 , P. M. Roessner 2 , B. Qu 3 , A. Ramsay 4 , B. Stamatopoulos 5 , M. Seiffert 2 , J. Paggetti 1 , E. Moussay 1

1 Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg; 2 Molecular Genetics, German Cancer Research Centre (DKFZ), Heidelberg; 3 Biophysik, Medizinische Fakultät der Universität des Saarlandes, Homburg, Germany; 4 School of Cancer and Pharmaceutical Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom; 5 Clinical Cellular Therapy Research Laboratory (LTCC), Institut Jules Bordet, Brussels, Belgium

Background: Chronic Lymphocytic Leukaemia (CLL) is the most common adult leukaemia in western countries. Unfortunately, CLL remains an incurable disease, evidencing the urgency to develop novel effective treatments for this malignancy. CLL cells are known to be highly dependent on interactions with non-malignant cells in their tumour microenvironment (TME) for survival and proliferation. Such dependency highlights the potential of immunotherapy as a therapeutic approach in CLL. Recently, several pro-inflammatory cytokines have emerged as a powerful tool to reactivate the immune system in a wide range of malignancies. IL-27 is a member of the IL-12 family of heterodimeric cytokines reported to have pleiotropic functions during cancer development in different malignancies. Based on the existing literature, we wondered whether IL-27 affects the development and progression of CLL.

Aims: The goal of this study is to elucidate the role of IL-27 in CLL development and progression.

Methods: We used a constitutive knockout mouse model of the EBI3 subunit of IL-27 to abrogate IL-27 expression in vivo. We performed adoptive transfer (AT) of TCL1 derived CLL cells into Ebi3 -/- and WT mice, as well as generated the transgenic mouse model Eµ-TCL1-Ebi3 -/- .The splenic TME of these mice was characterized using flow cytometry. In addition, in vivo neutralization of IL-27 using a blocking antibody was conducted. Immunodeficient Rag2 -/- mice were also injected with TCL1-derived CLL cells and either WT or Ebi3 -/- CD3+ T cells to assess the role of T-cells in leukaemia control in the presence and absence of IL-27. Finally, the transcriptional differences among WT and Ebi3 -/- T cells were assessed using bulk-RNA sequencing, and their impact on T cell cytotoxicity was investigated both in humans and mice using in vitro killing assays. The serum levels of IL-27 in CLL patients and sick mice were quantified via ELISA and compared to those of healthy controls.

Results: A strikingly enhanced CLL development and survival reduction was observed when CLL was AT into Ebi3 -/- mice, as well as in the transgenic Eµ-TCL1-Ebi3-/- mice when compared to controls (A ). Consistently, flow cytometry analysis revealed an increasingly immunosuppressive splenic TME in the absence of EBI3, characterized by an enrichment of highly activated and immunosuppressive Tregs and a terminally exhausted CD8+ T cell subset. The aforementioned results were recapitulated in an IL-27 neutralization experiment (B ). Using Rag2-/- mice, we demonstrated that T cells from EBI3 -/- mice were less efficient in controlling CLL development (C ). RNA sequencing revealed major changes in CD8+ T cells transcriptional program, in particular a decreased expression of crucial transporters (D ). Finally, in vitro killing assays showed an increased cytotoxicity of both human and murine T cells in the presence of IL-27, while serum protein quantification showed a decrease in the levels of IL-27 in both CLL patients (E ) and sick mice (F ) when compared to their healthy counterparts.

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Summary/Conclusion: Overall, our results demonstrate the antitumor role of IL-27 in CLL development and progression by promoting T-cell-mediated anti-tumor immunity, as well as establish this cytokine as a potential immunotherapeutic agent in CLL.

P599: RARE GERMLINE VARIANTS IN ATM INFLUENCE THE PATHOGENESIS OF CLL

B. L. Lampson 1 , A. Gupta 1 , S. Tyekucheva 2 , Z. Wang 2 , N. Wojciechowska 3 , C. J. Shaughnessy 1 , P. O. Baker 1 , S. M. Fernandes 1 , A. S. Kim 3 , J. R. Brown 1,*

1 Medical Oncology; 2 Data Science, Dana-Farber Cancer Institute; 3 Pathology, Brigham and Women’s Hospital, Boston, United States of America

Background: Germline missense variants of unknown significance (VUS) are increasingly identified in cancer-related genes by next generation sequencing. The ATM gene on chromosome 11 is a tumor suppressor gene that is frequently deleted or mutated in CLL, and also carries over 1,000 germline missense VUS.

Aims: We sought to evaluate whether germline ATM variants are more frequent in chronic lymphocytic leukemia (CLL) compared to other hematologic malignancies and whether they influence the clinical characteristics of CLL. We sought to determine whether one of the most common VUS has an impact on ATM function.

Methods: In our hematologic malignancy clinic, we identified 3,128 patients (including 825 CLL patients), who underwent clinical-grade sequencing of the entire coding region of ATM between 2014 and 2019. We evaluated the frequencies of germline ATM variants in different hematologic neoplasms. In patients with CLL, we determined whether these variants affect CLL-associated characteristics such as somatic 11q deletion. Finally we generated an ATM L2307F knock-in cell line to characterize the functional impact of the L2307F VUS on response to etoposide treatment or irradiation.

Results: Germline ATM variants are present in 24% of patients with CLL, significantly higher than in patients with other lymphoid malignancies (16% prevalence), myeloid disease (15%), or no hematologic neoplasm (14%). CLL patients with germline ATM variants are younger at diagnosis and twice as likely to have 11q deletion. The ATM variant p.L2307F is present in 3% of CLL patients and is associated with a three-fold increase in rates of somatic 11q-deletion. CLL in patients with two other common ATM VUS, p.S707P and p.F858L, was also significantly enriched for ATM -aberrant disease. Cell-based assays evaluating response to etoposide treatment or irradiation demonstrated that ATM p.L2307F shows hypomorphic function.

Summary/Conclusion: Germline ATM variants cluster within CLL and affect the phenotype of the CLL that develops, implying that some of these variants (such as ATM L2307F ) have functional significance and should not be ignored. Further larger studies are needed to determine whether these variants affect the response to therapy or account for some of the inherited risk of CLL.

P600: HIGH-DEPTH RNA-SEQUENCING IDENTIFIES CD8+ STAT3 MUTATED T-LGLL PATIENTS AS A DISTINCT BIOLOGICAL ENTITY WITHIN THE DISEASE HETEROGENEITY

G. Calabretto 1,* , A. Binatti 2 , A. Teramo 1 , G. Barilà 3 , A. Buratin 4 , V. R. Gasparini 1 , C. Vicenzetto 1 , E. Gaffo 5 , V. Trimarco 6 , L. Trentin 1 , M. Facco 1 , F. Vianello 1 , G. Semenzato 1 , R. Zambello 1 , S. Bortoluzzi 2

1 Department of Medicine - University of Padua, Veneto Institute of Molecular Medicine; 2 Department of Molecular Medicine, University of Padua, Padua; 3 Hematology Unit, Ospedale dell’Angelo, Mestre-Venezia; 4 Department of Molecular Medicine and Department of Biology; 5 CRIBI; 6 Department of Medicine, University of Padua, Padua, Italy

Background: T-Large granular lymphocyte leukemia (T-LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-LGL. We recognized a most common CD8+ subtype (CD8+ T-LGLL) and a CD4+ variant (CD4+ T-LGLL). STAT3 and STAT5B activating mutations, the main genetic lesions, are linked to the immunophenotype of the leukemic clone, T-LGLL clinical manifestations and disease prognosis. To schematize the biological heterogeneity of the disease, 4 main T-LGLL subsets can be identified: CD8+ STAT3M, CD8+ WT, CD4+ STAT5BM and CD4+ WT (M, mutated; WT, wild-type). Although STAT3 and STAT5b may regulate the transcription of genes involved in disease severity, the relationship between T-LGL immunophenotype (CD8+ or CD4+), STATs mutational status and gene expression profiles (GEP), including non-coding-RNAs, has never been investigated.

Aims: The aim of the study was to disclose T-LGLL transcriptome unbalances accounting for disease pathogenesis and clinical manifestations, by investigating GEP of leukemic T-LGL from each disease subtype and healthy controls (CTR).

Methods: High-depth RNA-seq was performed with a HiSeq3000 (Illumina) on RNA extracted from immuno-magnetically purified T-LGL of 20 T-LGLL patients (stratified in the 4 groups) and 5 CTR; 150 paired-end reads were generated (315 million reads/sample, on average). RNA-seq data were analyzed for reads alignment and transcripts quantification by CircComPara pipeline. Differentially expressed genes (DEGs) were assessed using DESeq (adj. p<0.01) and validated by RT-qPCR.

Results: Unsupervised multi-dimensional scaling analysis of GEP clearly separated T-LGLL from CTR samples. Of note, CD8+ STAT3M patients, characterized by a symptomatic disease and reduced survival, resulted as a distinct biological entity with respect to the other T-LGLL subgroups, that showed similar transcriptomic features. In detail, we found 1612 DEGs in CD8+ STAT3M and 1707 DEGs in the other T-LGLL cases, with the majority being down-regulated as compared to CTR. Of these, 641 genes were commonly dysregulated in all T-LGLL subgroups, although a more marked dysregulation was observed in presence of STAT3 mutations. 971 genes were differentially expressed uniquely in CD8+ STAT3M cases, with 571 being differentially expressed also versus other T-LGLL subgroups. Overall, dysregulated genes included tumor suppressors, protein kinases and phosphatases, cytokines and receptors. Of note, two non coding-RNAs with a known oncogenic role in other malignancies were up-regulated in LGLL samples.

Next, Gene Set Enrichment Analyses (GSEA) disclosed pathway dysregulation. In CD8+ STAT3M cases proteasome and Interferon signaling emerged among the most aberrantly activated signaling pathways with respect to CTR, whereas the cell cycle checkpoints pathway resulted to be activated as compared to the other T-LGLL subgroups.

Considering CD4+ STAT5BM T-LGLL, we observed the upregulation of a JAK/STAT axis negative regulator, reported to be down-expressed in other STAT5B-related disorders characterized by poor prognosis. This transcriptional signature might account for the peculiar indolent clinical course of STAT5BM T-LGLL patients.

Summary/Conclusion: We identified new genetic players involved in T-LGLL pathogenesis and disease severity, showing a peculiar GEP of CD8+ STAT3M cases, which are the most symptomatic and treatment-requiring patients. Most importantly, the identified DEGs are disclosing novel potential therapeutic targets to design innovative RNA-based therapies for T-LGLL patients.

P601: TP53 ANALYSIS AND REPORTING IN CHRONIC LYMPHOCYTIC LEUKAEMIA: ARE LABORATORIES IN COMPLIANCE WITH THE EUROPEAN RESEARCH INITIATIVE ON CLL (ERIC) RECOMMENDATIONS?

A. Cartwright 1,* , S. Scott 1 , L. Whitby 1

1 UK NEQAS for Leucocyte Immunophenotyping, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom

Background: The presence of TP53 aberrations, specifically del(17p) and/or TP53 nucleotide variants, in CLL are associated with adverse clinical outcomes and resistance to immunochemotherapy. As such, analysis of TP53 has become part of routine clinical diagnostics prior to initial and subsequent treatments, ensuring appropriate risk stratification and therapeutic intervention. Deletions of TP53 are identified by fluorescent in situ hybridisation, however, TP53 variants can be identified through various molecular techniques, including Sanger sequencing and next-generation sequencing (NGS). ERIC recommendations outline the requirements for testing, analysis and reporting of TP53 variants in CLL.

Aims: The aim of this study was to evaluate laboratory approaches to analysis and reporting of TP53 variants in CLL in line with ERIC recommendations.

Methods: The UK National External Quality Assessment Service for Leucocyte Immunophenotyping has provided external quality assessment for use of NGS gene panels in CLL since 2018. Samples are manufactured from cell line material and one sample per annum is issued for participating laboratories to evaluate using their in-house methodology. Between 2018-2021, three samples were formulated to contain TP53 variants and distributed. Laboratories were requested to report methodology and variants of clinical significance, in line with HGVS nomenclature at both the DNA and protein level. Additionally, details of assay limit of detection (LOD), reference sequences and interpretation databases utilised were also requested. Data returns were then evaluated relating to TP53 variant analysis and reporting.

Results: In total, 34 (100%) participating laboratories reported the use of either amplicon or capture based NGS methods, with assay LOD ranging from 1-15%, with a median LOD of 5%. All participating laboratories sequenced the ‘minimum’ exons indicated in the guidelines (exons 4-10), with 91.2% (31) laboratories sequencing ‘optimal’ exons 2-11. Furthermore, 97.1% (33) laboratories utilised the recommended Locus Reference Genomic sequence LRG_321t1 ( NM_000546 ), with one laboratory reporting use of LRG_321t3 ( NM_001126114 ) variant transcript. The IARC TP53 locus-specific database for variant interpretation was utilised by 70.6% (24) laboratories and despite recommendations, use of dbSNP for filtering of polymorphic/neutral variants was undertaken by 76.5% (26) laboratories. Across the 3-sample period, 97.1% (33) laboratories reported correct DNA HGVS nomenclature and 58.8% (20) laboratories reported correct protein HGVS nomenclature. Of the 14 laboratories reporting inaccurate protein HGVS nomenclature, 11 (78.6%) reported minor nomenclature issues precluding full compliance.

Summary/Conclusion: In this study, there is a high level of consensus amongst laboratories in the reporting and analysis of TP53 variants, except for protein nomenclature usage. Guidelines serve to promote harmonisation and standardisation of processes within laboratories and ERIC recommendations have achieved this in relation to TP53 variants in CLL, ensuring that patients are appropriately stratified for correct therapeutic interventions. Whilst there is currently a high level of consensus seen in analysis and reporting, the recommendations are likely to evolve as evidence and understanding of the genetic landscape in CLL therapeutics increases and further technological advances are made. As such, laboratories will need to ensure continued adoption to reflect new data reporting methods, treatments and standards of care for CLL patients.

P602: DISTINCT IMMUNE-RESPONSE PROFILE OF RICHTER TRANSFORMATION: HIGH EXPRESSION OF IL-10, LAG-3 AND OTHER IMMUNE CHECKPOINT MOLECULES

Q. Chen 1,* , A. Behdad 2 , S. Ma 2 , M. Schipma 2 , Y.-H. Chen 2

1 Pathology; 2 Northwestern University, Chicago, United States of America

Background: Richter transformation (RT) is an aggressive progression of chronic lymphocytic leukemia (CLL), often to diffuse large B cell lymphoma (DLBCL), with poor prognosis and limited response to standard therapy. Our prior studies have shown differences in immune checkpoint molecule expression between RT and CLL; in particular, expression of programmed cell death 1 (PD1) in lymphoma cells and its ligands (PDL1) in background immune cells. Significantly, we have shown high PD1 expression in RT predicts its clonal-relatedness to CLL.

Aims: To investigate immune-response pathway dysregulation in RT by gene expression profiling. To discover immune pathways and molecules that may serve as targets for immune therapy. To discover biomarkers that may help identifying patients that will benefit from checkpoint inhibitor therapy.

Methods: We studied 16 patients diagnosed with RT and 18 with CLL. We performed a 400-gene targeted NGS gene expression profiling on RT and CLL cases using the Oncomine immune-response assay from ThermoFisher. Data analysis (RNA-seq with STAR and DESeq2) was done by aligning quality-sufficient reads to the human genome and counting reads for each gene. Gene normalization and differential expression were then calculated, with adjusted p-values <0.05 determining statistically significant differentially expressed genes. Next, a pathway analysis was performed (Metascape) to identify significant pathways among the differentially expressed genes. We also performed PD1 and LAG3 immunohistochemical stains in all cases.

Results: Gene profiling analysis revealed significant differences in expression levels of 89 immune-response genes between RT and CLL cases. The top upregulated genes in RT comparing to CLL included several checkpoint pathway genes: LAG-3, HAVCR2/TIM3, and CD274/PD-L1. Several immune modulation/immune suppressive genes were significantly upregulated, in particular IL-10 and TGFb1. Other top upregulated genes included those involved in innate immunity (S100A9, S100A8, IGSF6), and in cytokine signaling (CCR1, IL-1B, IL-18). In addition to immune-response genes, MYC, TP63 and several cell proliferation pathway genes were upregulated in RT. The top down-regulated genes in RT compared to CLL included CD79B (B-cell receptor signaling), CD160 (checkpoint pathway), KLF2 and CD40LG (T-cell regulation), CIITA (IFNg signaling) and SELL (cell adhesion). To further confirm these findings we performed LAG3 IHC. LAG3 was highly expressed by large neoplastic B-cell in PD1-positive RT and only in scattered large cells in proliferation centers of CLL, and largely negative in PD1-negative RT cases. Average positive neoplastic cells were 67% in PD1+ RT (n=12), 2.8% in PD1- RT (n=4), and 10.8% in CLL (n=18) (P<0.05).

Summary/Conclusion: Using a targeted NGS gene profiling analysis, we demonstrate a significant difference in expression levels of multiple immune-response genes in RT vs CLL. In particular, we found upregulation in immune modulation genes and checkpoint pathway genes PD1, LAG3 and TIM3, and immunosuppressive cytokine IL-10. Overexpression of LAG3 in large neoplastic B-cells of RT, specifically in cases clonally-related to CLL is an important finding that may serve as a diagnostic marker. Checkpoint molecules LAG3 and TIM3 can serve as new immune-therapy targets for the treatment of RT. IL-10 blockade has been shown to enhance T-cell immunity, and may be a potential therapeutic target.

P603: GUT MICROBIOME IN CHRONIC LYMPHOCYTIC LEUKEMIA SHOWS DEPLETION OF SHORT-CHAIN FATTY ACIDS PRODUCING BACTERIA

T. Faitova 1,* , M. Jørgensen 2 , R. Svanberg 1 , C. da Cunha-Bang 1 , E. E. Ilett 2 , C. MacPherson 2 , C. Niemann 1,3

1 Department of Hematology; 2 PERSIMUNE Centre of Excellence, Rigshospitalet, Copenhagen University Hospital; 3 Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark

Background: Recent studies have shown extensive crosstalk between our immune system and gut microbiome (GM). The host immune system plays a vital role in the maintenance of GM homeostasis by 1) establishing a balance between eliminating invading pathogens and promoting the growth of beneficial microbes, 2) producing short-chain fatty acids (SCFA), the main source of nutrition for the colon cells, and 3) modulating the immune system by cytokine production. Mounting evidence shows that the GM of patients with high rates of infection are characterized by an imbalance of bacteria, inducing proinflammatory states and reduced capacity for SCFA synthesis. As chronic lymphocytic leukemia (CLL) is, among others, also characterized by high rate of infectious complications and an altered immune system, it is warranted to explore composition of the CLL microbiome.

Aims: We aim to investigate the hypothesis that deviation of the GM from homeostasis, i.e. loss of ‘health promoting’ gut microbes and/or overgrowth of pathogenic bacteria, distinguishes patients with CLL from the background population.

Methods: Feces samples of patients with CLL were collected, immediately fixated and frozen within 72 h; total genomic DNA was sequenced. Feces samples of healthy controls were chosen to match the CLL population with respect to age, demographic data, sample collection method, and sequencing platform. Taxonomical profiling was done using an in-house bioinformatics pipeline.

Results: A total of 61 CLL patients and 30 healthy individuals were included in the study. We observed reduced GM alpha diversity, and depletion of bacterial members of Lachnospiraceae and Ruminococcaceae families among the CLL patients when compared to healthy individuals. Our data show that members of the Lachnospiraceae family ( Anaerostipes hadrus , Coprococcus comes , Blautia spp., Dorea spp.), and 3 members of Ruminococcaeae family ( Ruminococcus torques , Ruminococcus bromii , Faecalibacterium prausnitzii ) were among the most differentially abundant bacterial species between the microbiomes of healthy individuals and CLL. Their mean proportions were shown to be significantly higher in healthy microbiome samples. As further differentially abundant species we observed Bacteroides sp. and Alistipes finegoldii , which both demonstrated significantly higher mean proportions in CLL microbiomes (Fig 1).

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Summary/Conclusion: To sum up, the CLL microbiomes in comparison to healthy controls demonstrated lower enrichment of Lachnospiraceae and Ruminococcaceae families, the major SCFAs-producing bacterial taxa reported to have a protective effect against inflammation. This supports the notion that proinflammatory risk factors identified in other cohorts with GM dysbiosis are also present within CLL patients. As CLL represents an antigen driven malignancy with immune dysfunction, we hypothesize that GM dysbiosis could both be implicated in the pathogenesis of CLL, through antigenic drive, and contribute to the distortion of the immune system in CLL.

Notably, both immune dysfunction and treatment (e.g. antimicrobials) may influence the CLL microbiome itself and therefore confound cross-sectional clinical studies. We therefore plan to investigate the interaction between microbiome and CLL development in the TCL1 mouse model of CLL. Also, identification of potential mechanistic links between the GM and the microenvironment in CLL should be investigated, e.g. extravesicular vesicles or cytokines released from or impacted by the GM. In addition, modulation of the microbiome in animal models may help to establish causal connections between the GM and CLL development.

P604: CATALASE EXPRESSION IN LEUKEMIA CELLS IS CONTROLLED BY GENETIC AND EPIGENETIC MECHANISMS

M. Galasso 1,* , E. Dalla Pozza 1 , R. Chignola 2 , S. Gambino 1 , C. Cavallini 3 , A. Pilatone 1 , F. M. Quaglia 4 , O. Lovato 3 , I. Dando 1 , G. Malpeli 5 , M. Krampera 4 , M. Donadelli 1 , M. G. Romanelli 1 , M. T. Scupoli 1,3

1 Department of Neurosciences, Biomedicine and Movement Sciences; 2 Department of Biotechnology; 3 Research Center LURM, Interdepartmental Laboratory of Medical Research; 4 Department of Medicine, Section of Hematology; 5 Department of Surgery, Dentistry, Pediatrics, and Gynecology, University of Verona, Verona, Italy

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in Western countries. It is an incurable disease characterized by an extremely variable clinical course and response to treatment. We have recently shown that high catalase ( CAT ) expression identifies a more aggressive clinical behavior in CLL. However, molecular mechanisms controlling catalase expression in leukemia cells are still poorly characterized. The rs1001179 single nucleotide polymorphism (SNP) as well as DNA methylation in the CAT promoter have been shown to regulate CAT expression in various cell types.

Aims: With the aim to characterize regulatory mechanisms underlying differential expression of catalase in CLL, in this study we investigated the role of rs1001179 SNP and CpG Island II methylation encompassing this SNP in the regulation of CAT expression.

Methods: Peripheral blood mononuclear cells (PBMC) from 55 CLL patients and 50 healthy donors (HD) were used for the study. CAT mRNA levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). DNA samples were genotyped for the rs1001179 SNP using Restriction Fragment Length Polymorphism (RFLP)-PCR. Bioinformatic analysis was used to predict transcription factor (TF) binding to SNP sequences. Chromatin Immunoprecipitation (ChIP)-qPCR was performed to validate predicted TF binding sites. Methylation levels of CpG sites within the CAT promoter was determined by pyrosequencing of bisulfite-converted DNA.

Results: The rs1001179 SNP genotyping showed that CLL cells harboring the T allele exhibited a significantly higher catalase expression compared with cells bearing the CC genotype. Interestingly, bioinformatic analysis predicted that the SNP provide distinct binding sites for various TF, including ETS-1 and GR-β. ChIP assay confirmed that CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β, but not to the other analyzed TFs. In addition, CLL cells exhibited lower methylation levels within the CAT promoter compared with HD B cells, in line with the higher catalase mRNA and protein levels expressed by CLL versus HD B cells. Moreover, methylation levels negatively correlated with CAT expression in CLL cells. Remarkably, inhibition of methyltransferase activity in leukemic cells induced a significant increase of CAT mRNA and protein levels, thus showing that DNA methylation could control catalase expression in CLL. Moreover, expression of DNA methyl transferase 1 ( DNMT1 ) resulted significantly reduced in CLL cells compared with HD B cells and inversely correlated with CAT expression in CLL, thus suggesting that differences in methylation levels underlying catalase expression could be driven by the DNMT1 enzyme. Finally, modeling analysis showed that the CT/TT genotypes exhibited a lower methylation and a higher CAT expression level, suggesting that the rs1001179 T allele and methylation cooperate in inducing CAT gene expression.

Summary/Conclusion: Our data show that SNP as well as methylation of the catalase promoter are involved in controlling catalase expression in CLL. The key advance of this study is to provide new insights into the regulatory mechanisms underlying differential expression of catalase in leukemic cells, which is of clinical relevance in CLL. Moreover, our study may form the basis for future challenges aimed at developing combinatorial therapies targeting catalase regulatory pathways.

P605: T-CELL PROLYMPHOCYTIC LEUKEMIA: MOLECULAR CHARACTERIZATION OF A COHORT OF 18 PATIENTS AND EVALUATION OF BORTEZOMIB AS A POSSIBLE THERAPEUTIC STRATEGY

V. R. Gasparini 1,* , A. Martines 2 , G. Barilà 3 , A. Teramo 1 , G. Calabretto 1 , C. Vicenzetto 1 , S. Carraro 4 , V. Trimarco 4 , L. Trentin 4 , M. Facco 4 , G. Semenzato 1 , L. Bonaldi 2 , R. Zambello 1

1 Department of Medicine, University of Padova and Veneto Institute of Molecular Medicine; 2 Immunology and Molecular Oncology Unit, Veneto Institute of Oncology, IOV-IRCCS, Padova; 3 Hematology Unit, Ospedale dell’Angelo, Mestre-Venezia; 4 Department of Medicine, University of Padova, Padova, Italy

Background: T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive mature T-cell malignancy characterized by a progressive clinical course and resistance to chemotherapy, whose pathogenesis is still largely unknown. Recurrent somatic mutations were detected within the JAK/STAT axis (i.e., JAK3 and STAT5b genes). T-PLL patients present with progressive lymphocytosis, splenomegaly and lymphadenopathy. New therapeutic strategies are needed to overcome the overall survival of two years observed in T-PLL patients even after treatment with alemtuzumab and consolidation with allogeneic stem cell transplantation. Ex-vivo drug sensitivity and resistance tests suggested proteasome inhibitors as novel candidates for the treatment of T-PLL.

Aims: The investigation of the molecular features of T-PLL aimed at understanding whether JAK/STAT genetic lesions might have a role in the clinical management of patients. Ex-vivo treatment with Bortezomib (Bz), a proteasome inhibitor, was performed to test the affordability of this compound as a new option for T-PLL therapy.

Methods: Sanger sequencing was performed on peripheral blood mononuclear cells (PBMC) of 18 T-PLL patients to detect the presence of somatic mutation in the hotspot regions of JAK3 and STAT5b genes. Patients were recruited at the Padova University Hospital. Immunophenotypic characterization of the leukemic clone was performed in all cases, while 11 patients underwent cytogenetic analysis upon mitogen stimulation. PBMC of 10 patients were cultured for 48h with different concentrations (1.3, 2.6, 5.2 and 7.8nM) of Bz according to literature. Annexin staining and western blot analyses were performed at both 24h and 48h to assess the efficacy of the treatment.

Results: At least one mutation within the JAK/STAT pathway was detected in 39% (7/18) of patients. Consistent with the literature’s data, N642H variant was the most recurrent STAT5b mutation. Conversely, the most frequent variant in JAK3 gene was N564S, which has never been detected in T-PLL and not listed in the COSMIC database. Cytogenetic analysis revealed a complex karyotype in 9/11 (82%) patients with specific primary T-PLL aberrations: inv(14)(q11q32) or variant rearrangements in 78% (7/9) and t(X;14)(q28;q11) in 2 cases. Known secondary alterations such as those involving chromosome 8 and deletion of 11q22 were found in 89% (8/9) and 67% (6/9) of cases, respectively. Analysis of the clinical data did not reveal a correlation with the patient’s mutational status. Ex-vivo treatment with Bz induced apoptosis of the leukemic clone in a dose and time dependent manner. Compared to the untreated condition (16%), Bz concentration of 5.2nM increased the mortality of 1.7 (27%) and 4 (63.5%) times at 24h and 48h, respectively. The apoptosis was confirmed also at protein levels by a trend of caspase 9 and PARP cleavages upregulation at 48h. No difference in the sensitivity to treatment with Bz was observed between mutated and wild-type patients.

Summary/Conclusion: No correlations between recurrent lesions within the JAK/STAT axis and clinical outcome were found. This points against a pathogenetic role of these mutations in T-PLL, conversely from other leukemias where STAT mutations are regarded as prognostic markers. This hypothesis is further sustained by the same response to the treatment with Bz. Since the treatment of T-PLL mostly remains an unmet clinical need, the finding of an increased apoptosis in Bz-treated cells paves the way for a deeper study on molecular mechanisms involved in the apoptosis of the leukemic clone.

P606: META-ANALYSIS OF LOW ALLELIC BURDEN C481 BTK-MUTATIONS CALLS FOR CAUTION IN IBRUTINIB RESISTANCE EVALUATION

M. H. Hansen 1,2,* , S. R. Veyhe 1,2 , N. Abildgaard 1,2 , C. G. Nyvold 1,2 , H. Frederiksen 2

1 Hematology-Pathology Research Laboratory, Research Unit for Hematology and Research Unit for Pathology; 2 Department of Hematology, Odense University Hospital, Odense, Denmark

Background: Introduction of the orally administered tyrosine kinase inhibitor ibrutinib, a drug approved for relapsed or refractory mantle cell lymphoma in late 2013 and chronic lymphocytic leukemia in 2014, has changed treatment algoritms for B-cell malignancies and improved progression-free survival. Ibrutinib irreversibly targets Bruton’s tyrosine kinase ( BTK ), thus interrupting B-cell receptor signaling. However, it is known that mutations in the BTK gene, located on the X chromosome, can mediate acquired resistance, specifically modifying cysteine residue 481 to serine (C481S, X:101356176, GRCh38).

Aims: Despite this knowledge, it is still unknown how low mutational burden can contribute to general resistance towards ibrutinib treatment. Thus, we performed a meta-analysis based on previous cardinal studies to investigate the collective and gender-specific distribution of variant allele frequencies.

Methods: We implemented meta-analysis of single-nucleotide variant calls from seven studies involving whole-exome sequencing (Woyach et al. 2014 and 2017, Maddocks et al. 2015, Ahn et al. 2017, Kanagal-Shamanna et al. 2019, Gángó et al. 2020, Bödör et al. 2021) on mutational profiling concerning ibrutinib resistance and CLL. Four of these studies contained gender specific data. Computational analyses, statistics, and visualization were performed in Wolfram Language, R, and GraphPad Prism.

Results: From the available data included in these studies, we were able to base our analyses on 226 non- BTK mutations and 170 BTK mutations within a variant allele frequency (VAF) range of 0.0001–0.9, of which 93 mutation candidates from males (VAF < 0.9) and 77 from females (VAF < 0.44) were observed. Extrapolative analysis of allele frequency distribution from all other variants (excl. BTK) in the studies revealed an exponential increase in the number of low burden variants below 10% VAF (R2>0.99). These variants were not significantly different between genders (P Mann-Whitney =0.24, n male =130, n female =73). In contrast, a significant difference was observed between male variant allele frequencies (P Mann-Whitney =0.005) and females owing to BTK hemizygosity in males. However, this significance persisted when female BTK mutations were transformed to reflect a single copy (VAF/2, P Mann-Whitney =0.015, Δ VAF =0.05) for gender comparison and even when observations for males were alternatively transformed to reflect two copies (2*VAF, P Mann-Whitney <0.001, Δ VAF =0.19). The previously discovered threshold of 10% was set as the detection limit.

Summary/Conclusion: Defining specific molecular contributions in drug resistance is imperative for the correct use of targeted therapy. It has been questioned how the low allelic burden of BTK mutations plays a role. Therefore, it is crucial to investigate the lower limit of detection. The meta-analysis suggests that variants below a certain threshold, here empirically 10%, may in many cases be false-positive variants, in agreement with other reports involving WES in general, introduced by erroneous base calling, PCR errors, etc. Although intriguing, it remains to elaborate whether the haploinsufficiency of BTK in males skew in the degree of ibrutinib resistance.

P607: A NOVEL LYMPH NODE-MIMICKING 3D CULTURE SYSTEM DISPLAYS LONG-TERM T CELL-DEPENDENT CLL PROLIFERATION AND SURVIVAL

M. Haselager 1,2,3,4,* , E. Perelaer 1 , A. Kater 2,3,4,5 , E. Eldering 1,2,3,4

1 Experimental Immunology, Amsterdam University Medical Center; 2 Lymhoma and Myeloma Center Amsterdam, LYMMCARE; 3 Cancer Center Amsterdam; 4 Amsterdam Infection & Immunity Institute; 5 Hematology, Amsterdam University Medical Center, Amsterdam, Netherlands

Background: Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in the absence of microenvironmental survival signals 1 . No current system permits long-term expansion of CLL cells in vitro due to difficulties of mimicking a physiological microenvironment. The lymph node (LN) is the critical site of in vivo CLL proliferation and is also thought to play a key role in the development of resistance to targeted agents 2,3 . Developing an in vitro culture system for CLL with pathophysiological relevance will greatly benefit studies of CLL proliferation, microenvironment, clonal outgrowth and relevant drug screening.

Aims: To design an in vitro CLL culture system that incorporates key aspects of the CLL LN; contribution of non-CLL cells and cytokines, and induction of drug resistance and prolonged proliferation of CLL cells.

Methods: Primary CLL cells were cultured in ultra-low attachment (ULA) plates in parallel to standard 2D cultures. PBMCs were cultured with or without T cells (in a specific CLL:T cell ratio to mimic the LN composition), or PBMCs were co-cultured with primary lymph node fibroblasts. CLL cells were either stimulated directly with a B cell cocktail (BCC) consisting of IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28.

Results: Compared to 2D cultures, 3D cultures showed several important CLL LN features. First, significantly increased CLL proliferation independent of IGHV mutation status, which was abrogated in the absence of T cells, or by JAK inhibitors. Notably, treatment with BTK inhibitors significantly inhibited CLL proliferation and resulted in disintegration of the 3D spheroid architecture. Second, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations. Third, 3D cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the 3D model. Fourth, when PBMCs were stimulated with BCC, spheroids developed proliferation center-like structures after 4 weeks of culture where T cells localized together with enrichment of Ki-67+ CLL cells. Finally, either B or T cell stimulation resulted in an induction of venetoclax resistance, showing that T cells are able to confer drug resistance to CLL cells in this model.

Summary/Conclusion: We present a 3D culture system that underlines the role of T cells in sustained CLL proliferation, permitting investigation of CLL cells in the context of a protective niche consisting of multiple cell types, thereby opening up new avenues for clinically useful applications.

P608: DISTINCT P53 PHOSPHORYLATION PATTERNS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS ARE REFLECTED IN CIRCUMJACENT PATHWAYS’ ACTIVATION UPON DNA DAMAGE

M. Pesova 1,2,* , V. Mancikova 1,2 , R. Helma 1,2 , S. Pavlova 1,2 , V. Hejret 1 , P. Taus 1 , J. Hynst 1 , K. Plevova 1,2,3 , J. Kotaskova 1,2,3 , J. Malcikova 1,2 , S. Pospisilova 1,2,3

1 Central European Institute of Technology (CEITEC), Masaryk University; 2 Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine of Masaryk University and University Hospital; 3 Institute of Medical Genetics and Genomics, Faculty of Medicine, Masaryk University, Brno, Czechia

Background: Presence of defects in the TP53 gene represents a clinically relevant biomarker in chronic lymphocytic leukemia (CLL). Besides gene mutations and deletions, the functionality of p53 protein can be disrupted by other mechanisms, such as aberrant phosphorylation.

Aims: To uncover how p53 phosphorylations affect its functions in CLL cells.

Methods: P53 phospho-patterns were analyzed in 71 TP53 -intact primary CLL samples using Zn2+-Phos-tag™ SDS-PAGE after induction of p53 protein by DNA damage (fludarabine and doxorubicin treatments). Differences in gene expression were studied using RNA sequencing and compared to CLL cells carrying the mutated TP53 gene. The ability of p53 to activate transcription of its target genes ( BAX , BBC3 , CDKN1A , GADD45A ) was assessed by qPCR. The presence of gene mutations was analyzed using a targeted NGS panel.

Results: While fludarabine induced a consistent phospho-pattern, two distinct p53 phosphorylation profiles were identified upon doxorubicin treatment. Profile I featured heavily phosphorylated p53 protein, while only low phosphorylation occurred in profile II. On the transcriptomic level, samples from the profile II were less capable of activating p53 target genes upon doxorubicin treatment, thus resembling TP53 -mutant cells, whereas proper p53 signaling was triggered in profile I. Consistently, induction of the studied downstream effector genes CDKN1A , BAX , BBC3 and GADD45 after doxorubicin treatment was lower in profile II than in profile I samples but still higher than in TP53 mutants. The differences between the two phoshoprofiles were also observed in untreated cells: the expression of miR-34 in profile II showed an intermediate pattern between profile I and TP53 -mutant cells. Furthermore, profile II samples had significantly higher basal p53 protein levels than profile I samples, where p53 was generally detectable only upon DNA damage. The PROGENy analysis of the basal activity of selected cancer-related pathways in untreated cells pointed to the different regulation of the hypoxic pathway: the activity of hypoxic pathway in profile II samples was in between high levels found in TP53 mutants and low levels in profile I. Finally, we have found ATM locus/gene defects more frequently in profile II.

Summary/Conclusion: Our study suggests that wt- TP53 CLL cells with less phosphorylated p53 show TP53 -mutant-like behavior after DNA damage. Hypophosphorylation of p53 protein and the corresponding lower ability to respond to DNA damage are linked to ATM locus defects and to the higher basal activity of the hypoxia pathway.

The project was supported by GACR 19-15737S, MZCR-RVO 65269705, MUNI/A/1330/2021 and AZV NU21-08-00237.

P609: CLINICOBIOLOGICAL CHARACTERISTICS AND TREATMENT EFFICACY OF NOVEL AGENTS IN CHRONIC LYMPHOCYTIC LEUKEMIA WITH IGLV3-21R110

P. Hengeveld 1,2,* , Y. E. Ertem 1 , J. Dubois 3 , C. Mellink 4 , A.-M. van der Kevie-Kersemaekers 4 , L. Evers 3 , K. Heezen 1 , P. M. Kolijn 1 , O. Mook 4 , M. M. Motazacker 4 , K. Nasserinejad 5 , S. Kersting 6 , P. Westerweel 2 , C. Niemann 7 , A. Kater 3 , A. Langerak 1 , M.-D. Levin 2

1 Department of Immunology, Erasmus MC, Rotterdam; 2 Department of Internal Medicine, Albert Schweizer Ziekenhuis, Dordrecht; 3 Department of Hematology; 4 Department of Human Genetics, Amsterdam UMC, Amsterdam; 5 Department of Hematology, Erasmus MC, Rotterdam; 6 Department of Hematology, Haga Ziekenhuis, Den Haag, Netherlands; 7 Department of Hematology, Rigshospitalet, Copenhagen, Denmark

Background: The composition of the clonotypic B cell receptor (BCR) is of key importance in chronic lymphocytic leukemia (CLL). Recently, a novel immunogenetically defined CLL subset was described, characterized by a clonotypic BCR using the immunoglobulin lambda (IGL) IGLV3-21*01/IGLV3-21*04 gene with a distinctive G110R somatic hypermutation (IGLV3-21 R110 ). IGLV3-21 R110 CLL accounts for approximately 20% of all CLL and is associated with an adverse prognosis. However, the clinicobiological profile and predictive impact of IGLV3-21 R110 CLL in the context of novel therapies remains incompletely characterized.

Aims: To characterize the cytogenetic, immunogenetic and mutational landscape of IGLV3-21 R110 CLL and to assess the predictive impact of this genotype in the context of novel therapies.

Methods: We characterized the light chain genotype, clinicobiological features and response to therapy of patients enrolled in the HOVON-139/GIVE trial and the Dutch sub-cohort of the HOVON-141/VIsion trial. The HOVON-139/GIVE phase-II trial evaluated first-line minimal residual disease (MRD)-guided duration of treatment with obinutuzumab and venetoclax in CLL patients unfit for treatment with chemoimmunotherapy. The HOVON-141/VIsion phase-II trial evaluated MRD-guided ibrutinib and venetoclax combination treatment in relapsed or refractory (R/R) CLL patients.

Results: The IGLV3-21 R110 genotype was present in 16/65 patients (25%) in the first-line cohort and in 32/129 patients (25%) in the R/R cohort. Loss of 13q14 and 11q22 were enriched in IGLV3-21 R110 patients, compared to other patients (del13q14: 79% vs. 57%, P =0.009; del11q22: 34% vs. 18%, P =0.03). In contrast, the IGLV3-21 R110 genotype and trisomy 12 or loss of 17p13 were mutually exclusive (trisomy 12: 0% vs. 12%, P =0.008; del17p13: 0% vs. 12%, P =0.01). Mutations in the SF3B1 and ATM genes were significantly more common in IGLV3-21 R110 patients, compared to other patients ( SF3B1 : 49% vs. 21%, P =0.0008; ATM : 36% vs. 18%, P =0.02). IGHV and IGHD gene usage of IGLV3-21 R110 patients was markedly skewed. The IG heavy-chain complementarity determining region 3 (HCDR3) was shorter in IGLV3-21 R110 patients, compared to other patients (median HCDR3 aa length 13 [range: 9-17] vs. 19 [range 8-30], P <0.0001). Whereas the IGHV SHM imprint of IGLV3-21 R110 patients was centered around the 98% cutoff between unmutated and mutated IGHV, the range was wider in other patients (range 95.1%-99.7% vs. 85%-100%, P =0.01). When comparing patients with IGLV3-21 R110 CLL versus all other light chains, were no differences regarding MRD (%uMRD<10 -4 in peripheral blood, HOVON-139/GIVE after 12 cycles: 87% vs. 98%, P =0.15; HOVON-141/VIsion after 15 cycles: 52% vs. 61%, P =0.11) and progression-free survival (24-month PFS, HOVON-139/GIVE: 100% vs. 94%, P =0.52; HOVON-141/VIsion: 91% vs. 92%, P =0.74) in either trial.

Summary/Conclusion: We have characterized the clinicobiological features of the largest cohort of IGLV3-21 R110 patients reported thus far. We demonstrate that CLL with IGLV3-21 R110 is typified by a distinct cytogenetic, mutational and immunogenetic profile. There was no evidence for a predictive impact of the IGLV3-21 R110 genotype on the efficacy of the novel therapies with venetoclax and ibrutinib employed in the HOVON-139/GIVE and HOVON-141/VIsion trials. Our results suggest that novel targeted therapies may mitigate the adverse risk profile of IGLV3-21 R110 CLL. To determine whether these patients should preferentially receive novel therapies, characterization of the predictive impact of IGLV3-21 R110 in the setting of chemoimmunotherapy is warranted.

P610: SYNERGISTIC ACTIVITY OF FTO INHIBITOR FB23-2 WITH IBRUTINIB IN XENOGRAFT MURINE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA

X. Hu 1,* , H. Wang 2 , Y. Han 1 , X. Zhang 1 , Z. Tian 1 , Y. Zhang 2 , X. Wang 1

1 Department of Hematology, Shandong Provincial Hospital，Cheeloo College of Medicine, Shandong University; 2 Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China

Background: Several studies have documented that FB23-2, as the selective inhibitor of RNA N6-methyladenosine (m 6 A) demethylase FTO, exerts crucial role in dampening tumorigenesis. Indeed, FTO deactivation was proved to render resistant leukemia cells sensitive to targeted agents, which suggests a potential combination therapy strategy of FB23-2.

Aims: The present study was aimed to identify the synergistic utility of FB23-2 combination with ibrutinib in CLL.

Methods: A disseminated leukemia xenograft murine model was established to explore the efficacy of FB23-2 in CLL. Leukemia burden of CLL mice was quantified by luciferase intensity using bioluminescence imaging. Bone marrow and spleen infiltration of leukemic cells was assessed by flow cytometry. And cell survival was determined by cell counting kit-8.

Results: To investigate the combinatory effect of FB23-2 and ibrutinib, cell viability assay was performed firstly. FB23-2 displayed potent synergistic activity with ibrutinib in promoting cytotoxicity in CLL cells (Figure 1A). Consistently, FTO silencing mediated by lentivirus exhibited exquisite sensitivity to ibrutinib in CLL cells (Figure 1B). Besides, CLL primary cells and MEC-1 cells with treatment of FB23-2 and ibrutinib simultaneously triggered increased cell apoptosis compared to single agent (Figure 1C). Further underlying cooperative mechanism investigation demonstrated an aberrant activation of DNA damage pathway after serial dilution of FB23-2 treatment. Hence, enhanced expression of p-ATM, p-chk2 and p-H2AX was detected in FTO knockdown and ibrutinib co-treatment group, indicating FTO inhibition inducing ibrutinib-sensitivity via regulating DNA damage (Figure 1D).

In addition, we investigated the efficacy of FB23-2 alone and corporation in CLL xenograft murine model. Compared to ibrutinib alone, combination treatment with FB23-2 significantly prolonged the lifespan of CLL mice (median survival time 20 versus 22.5 days, respectively, p =0.02, Figure 1E). FB23-2+ibrutinib group showed an obviously declined proportion of CLL cells compared to ibrutinib-treated alone both in bone marrow(20.93%±2.95% versus 37.17%±4.22%, p =0.02) and spleen (23.58%±6.03% and 54.58%±9.01%, p =0.03, Figure 1F-G). Similar results were obtained with three treated group relative to control group (all p <0.05). Moreover, FB23-2 combined with ibrutinib diminished leukemia burden and splenomegaly in CLL mice (Figure 1H-I). As shown in Figure 1J, leukemia involvement and malignant proliferation were more evident in control group, confirming the potent anti-leukemic activity of FB23-2.

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Summary/Conclusion: Taken together, our investigation provides pre-clinical evidence for the utility of FB23-2 in CLL, especially combinatory benefit with ibrutinib. FB23-2 represents a promising strategy to novel treatment optimum in CLL.

P611: NEXT-CLL: A NEW NGS-BASED METHOD FOR RAPID ASSESSMENT OF IGHV MUTATIONAL STATUS IN CHRONIC LYMPHOID LEUKEMIA

E. Bourbon 1 , K. Chabane 2 , A.-S. Michallet 3 , E. Ferrant 1 , I. Mosnier 2 , S. Poulain 4 , M. Giraud 5 , A. Bouvard 2 , H. Ghesquieres 6 , S. Hayette 2 , P. Sujobert 2 , S. Huet 2,*

1 Hematology clinics; 2 Laboratory of hematology, Hospices Civils de Lyon, pierre-benite; 3 Hematology clinics, Centre Leon Berard, Lyon; 4 Laboratory of hematology; 5 Bio-informatics, CHRU de Lille, Lille; 6 Hematology clinics, Hospices Civils de Lyon, Lyon, France

Background: Chronic Lymphocytic Leukemia (CLL) is a highly heterogeneous disease both in terms of biological landscape and clinical course. Current guidelines recommend IGHV mutation status (MS) determination prior to treatment initiation in order to guide the first-line therapeutic choice between chemoimmunotherapy and novel agents. Currently, the IGHV MS is determined in most routine laboratories by low-throughput Sanger sequencing that allows for the detection of the dominant clone in the majority of CLL cases. However, this technic remains technically challenging despite best practice guidelines, does not provide any insight into subclonal architecture and intraclonal diversity, and has a general failure rate ranging from 10 to 20%. Besides, commercially available NGS solutions have drawbacks (costs) and constraints (as they necessitate mandatorily a MiSeq sequencing machine and are not feasible on other devices) that restrict their use by any laboratory in routine practice.

Aims: In this study, we aimed at providing a ready-to-use and rapid strategy to evaluate the IGHV gene MS using NGS in routine practice. We present a new method called Next-CLL, allowing rapid assessment on any NGS device available in routine diagnostic laboratories.

Methods: Peripheral blood samples were obtained from 80 patients with typical CLL at diagnosis. Genomic DNA (gDNA) and complementary DNA (cDNA) were used. In order to fully cover the IGHV rearranged gene sequence, 4 mutliplex PCR reactions were designed: [LEADER-FR2R/JHc], [FR1c-JHc], [FR1-JHc] and [FR2-JHc], producing overlapping amplicons. Final products from the 4 PCR reactions were mixed and purified, subjected to A-tailing and ligated to Illumina-single indexed adapters. The library was then sequenced (2x150 bp) on a NextSeq 500 (Illumina). A reassembly of IGHV overlapping amplicons was performed using the BBTools Suite. The outputs were then uploaded on the Vidjil plateform for IGHV clonal analysis. The MS was calculate using the IMGT web interactive tool following ERIC recommendations. IgHV MS was assessed in parallel by Sanger sequencing or the commercial NGS assay IdentiClone® Assay (Invivoscribe) as reference. Gene usage, % identity to the germline and stereotype subsets were compared with the results obtained from Next-CLL.

Results: Next-CLL identified a productive clone in 100% of cases (n=80), compared to PCR with Sanger sequencing (15% of failure). All cases that failed using the Sanger sequencing were subjected to a second NGS technique (Invivoscribe) that confirmed the results from Next-CLL, ruling out any possibility of false positive cases. Next-CLL showed 100% concordance with the reference techniques for IGHV gene identification and allowed assessment of the MS from the leader sequence, as recommended by the ERIC consortium. Only 2 cases showed discordant IGHV MS, close to the 98% identity cut-off, between Next-CLL and Sanger sequencing (the latter being unable to capture the leader sequences), thus providing 97% of concordance between the 2 techniques (Figure 1). Moreover, 100% concordant results were obtained between gDNA and cDNA (n=32 cases analysed for both) using Next-CLL.

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Summary/Conclusion: Our new method Next-CLL allows assessment of the IGHV MS by NGS with an easy and rapid workflow, providing clinically useful information from the IGHV leader sequence and allowing analysis of intra-clonal diversity (not possible with the standard Sanger sequencing). Importantly, it might be used both on gDNA and cDNA and on any sequencing machine, on the contrary to existing commercially available kits.

P612: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)-DERIVED T-CELLS HAVE DISTURBED FATTY ACID METABOLISM, POSSIBLY CONTRIBUTING TO T-CELL DYSFUNCTION

C. F. Jacobs 1,2,3,4,* , H. Simon-Molas 1,2,3,4 , N. Zelcer 5 , A. P. Kater 2,3,4,6 , F. S. Peters 1,2,3,4

1 Experimental Immuno-Hematology; 2 Hematology, Amsterdam University Medical Center; 3 Cancer Center Amsterdam; 4 Amsterdam institute for Infection & Immunity; 5 Medical Biochemistry, Amsterdam University Medical Center; 6 Lymphoma and Myeloma Center Amsterdam, Amsterdam, Netherlands

Background: T-cells from CLL patients are dysfunctional and display reduced activation, proliferation and cytotoxicity upon in-vitro activation. Whilst the role of glucose metabolism in activated T cells is extensively studied, the contribution of other energy sources is less clear. Fatty acid (FA) and lipid uptake in T cells involves the scavenger membrane transporter CD36. CD36-mediated lipid transport has been identified to (metabolically) modulate intratumoral T cells (both T reg and cytotoxic CD8 + T cells). However, lipid metabolism and a potential role for CD36 has not been studied in the context of T cell dysfunction in CLL

Aims: To examine the role of FA metabolism and the fatty acid transporter CD36 in acquired T-cell dysfunction in CLL.

Methods: Untreated CLL patients with a white blood cell count (WBC) greater than 20×10 9 cells/L and, as control, age-matched healthy donors (HD) were studied. T cells were analyzed by flow cytometry, confocal microscopy or extracellular flux analysis (Seahorse), either directly after thawing or after a 2 day culture with or without αCD3/αCD28 antibodies.

Results: Analysis of CD36+ vs CD36- CD4 and CD8 T-cells from HD revealed that CD36+ cells had higher long-chain fatty acid (LCFA) and glucose uptake, and higher expression of the glucose transporter GLUT-1 and the rate-limiting mitochondrial FA transporter CPT1α (Fig. 1A). The cells expressing CD36 were also more likely to increase their CD25 expression and mitochondrial mass upon stimulation, indicating a metabolic and functional advantage potentially conferred by the surface expression of CD36. The absolute amount and percentage of T cells (CD4 and CD8) expressing FA transporter CD36 was lower in CLL T-cells as compared to HD (Fig. 1B). The levels of LCFA uptake and expression of CPT1α were comparable, indicating that the lower amount of CD36 might successfully cater to the FA demands of resting CLL T cells(Fig. 1C). However, while stimulation of HD T-cells markedly upregulated CD36 abundance, LCFA uptake and CPT1α, concomitantly with the activation marker CD25, CLL-derived T-cells failed to mount a similar response (Fig. 1D). This lack of induction of CD36 expression was also observed in CLL-derived T cells that increased CD25, which implies that this response is independent of T-cell activation. The transcription factors peroxisome proliferator-activated receptors (PPAR) α and γ are important regulators of FA metabolism. We observed reduced expression of these transcription factors in resting CLL T cells. However, distinct from CD36, CLL T cells were indistinguishable from HD T-cells in their ability to increase levels of both PPARα and PPARγ upon stimulation, indicating that alternative mechanisms might be implicated in the regulation of CD36 and FA metabolism in CLL T cells.

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Summary/Conclusion: Collectively, our results show that disturbances in FA metabolism are present on multiple levels in CLL T cells and that lack of LCFA uptake into CLL T-cells could represent a limiting step in FA metabolism. We also highlight CD36+ cells as a metabolically distinct T-cell subtype that is largely absent in CLL T-cells, independently of their activation status. We are currently investigating strategies to increase CD36 expression and FA metabolism in general in CLL T cells, which could ameliorate CLL T cell dysfunction and provide targets to improve autologous T cell therapies.

P613: TLR9 SIGNALLING IS A POTENTIAL TUMOUR ESCAPE MECHANISM FOLLOWING BTKI THERAPY FOR THE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKAEMIA.

E. Kennedy 1,* , S. Mitchell 1 , T. A. Burley 1 , T. Liloglou 2 , R. Johnston 3 , I. Ashworth 1 , D. Bak-Blaz 3 , K. Chamberlain 3 , E. Ladikou 1 , S. Mackay 4 , C. Pepper 1 , A. G. Pepper 1

1 Department of Clinical and Experimental Medicine, Brighton and Sussex Medical School, Falmer; 2 Molecular and Clinical Cancer Medicine, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool; 3 Brighton and Sussex University Hospitals NHS Trust, Brighton; 4 Cell Biology and Drug Discovery and Design Groups, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom

Background: CLL cell trafficking is fundamental to CLL progression and an important focus when identifying novel therapeutic targets. Toll-like receptor 9 (TLR9) is an intracellular pattern recognition receptor, and a potential contributor to CLL cell chemotaxis and tumour maintenance. TLR9 recognises unmethylated CpG motifs within bacterial/viral/mitochondrial DNA, and TLR9 activation promotes an NFkB and STAT3-driven activation/migratory phenotype in primary CLL cells. We have previously shown cell-free unmethylated DNA to be up to 28-fold higher in CLL patient plasma, relative to healthy controls.

Aims: To investigate TLR9 activation as a potential mechanism of resistance to current B-cell receptor (BCR)-targeted therapeutics.

Methods: Migration assays were performed using primary CLL cells stimulated -/+ 1μM ODN 2006 (TLR9 agonist), -/+ 1μM ibrutinib (BTK-inhibitor), 2μM ODN INH-18 (TLR9 antagonist) or -/+ 2.5-10μM CW15337 (NIK inhibitor). CLL cells migrated through 5μM pore membranes towards a CXCL12-gradient.

Results: TLR9 activation induced a dichotomous migratory response in CLL patient samples. Following stimulation with ODN 2006, 25/42 (60%) patient samples showed an increase in CLL cell migration (‘Responders’) and 17/42 (40%) showed either no change or a decrease in CLL cell migration (‘Non/Reverse Responders). Interestingly, IGHV-mutated (M-CLL) samples were almost exclusively ‘Responders’ (i.e., 14/17 [82%]) and IGHV-unmutated (U-CLL) samples were equally likely to be ‘Responders’ or ‘Non/Reverse Responders’ (i.e., 11/25 [44%] vs 14/25 [56%] respectively).

Whilst there was no difference in the expression levels of TLR9 in M-CLL vs U-CLL cells, U-CLL samples expressed significantly higher basal levels of CD69 (B-cell activation marker), and their TLR9-induced migratory response negatively correlated with their basal migration. We therefore hypothesised that U-CLL ‘Non/Reverse Responders’ may have reached maximal stimulatory capacity through BCR-signalling alone, rendering them unresponsive to further activation.

To test this hypothesis, we simulated BCR and TLR9 stimulation in M-CLL and U-CLL cells using mathematical modelling. Our simulations showed M-CLL cells (with low basal BCR-activation) to induce NFkB signalling in response to TLR9 activation and U-CLL cells (with high basal BCR-activation) to be unresponsive. Importantly, when simulating treatment with a Bruton’s Tyrosine Kinase inhibitor (BTKi), U-CLL cells gained sensitivity to TLR9 activation. These results have since been verified in vitro using BTKi-treated patient samples, where we found a subset of U-CLL ‘Non/Reverse Responders’ to become ‘Sensitised’ to TLR9 activation in the presence of ibrutinib. In the ‘Responder’ subgroup, ODN INH-18 (TLR9 antagonist) and ibrutinib inhibited CLL cell migration synergistically. Together, these data implicate TLR9 signalling as a tumour escape mechanism following BTKi therapy. Since both the BCR and TLR9 signalling pathways culminate in NFkB activation, we are currently investigating components of NFκB signalling as potential novel therapeutic targets. Preliminary data using the NIK selective inhibitor ‘CW15337’ shows CLL cell migration to be inhibited with an IC50 of <1μM.

Summary/Conclusion: CLL patient plasma contains high levels of TLR9 ligand (unmethylated DNA), which may promote CLL cell trafficking and BTKi resistance in select subgroups. We believe that targeting downstream NFκB signalling has the potential to inhibit both BCR and TLR9 signalling, and to increase treatment efficacy in TLR9 ‘Responder’ and BTKi ‘Sensitised’ patients.

P614: SUBCLONAL ARCHITECTURE OF CHROMOSOMES REVEALED BY SINGLE-CELL ANALYSIS OF GENE EXPRESSION IN A PATIENT WITH CLONAL EVOLUTION OF RELAPSING/REFRACTORY CLL

J. Kotaskova 1,2,3,* , T. Kurucova 2 , K. Reblova 1,2,3 , M. Valisova 1,2 , K. Zavacka 1,2 , A. Mareckova 1 , M. Bohunova 1 , S. Pavlova 1,2 , J. Malcikova 1,2 , V. Navrkalova 1,2 , M. Jarosova 1,3 , M. Doubek 1,2,3 , K. Plevova 1,2,3 , S. Pospisilova 1,2,3

1 Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University; 2 Central European Institute of Technology, Masaryk University; 3 Institute of Medical Genetics and Genomics, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czechia

Background: Chronic lymphocytic leukemia (CLL) manifests by remarkable intraclonal heterogeneity of genomic defects, especially in patients with relapsed/refractory disease, often connected with complex karyotype. In cancer cells, chromosomal changes are often multiple and complex. Aberrations can be present only in subclones and often underlie disease refractoriness. Using the bulk analysis such as WGS, WES, or genomic array to precisely determine the co-occurrence of aberrations in the individual cell provides limited information.

Aims: To perform an in-depth, single-cell RNA sequencing (scRNAseq) study of subclonal changes associated with relapsed/refractory CLL.

Methods: A bulk analysis of DNA from separated malignant cells using comprehensive NGS panel LYNX (PMID 34082072) was performed on a representative CLL case at three time-points (TPs): at dg (TP1, month 0, Rai II), before first therapy (TP2, month 19) and during relapse (TP3, month 59). Simultaneously, the transcriptome was analyzed using scRNAseq (Chromium system, 10x Genomics) in 2330 cells (TP1), 2397 cells (TP2), and 1902 cells (TP3). A detailed data analysis was carried out with the Seurat R package. In addition, we used the InferCNV tool to detect the chromosomal aberrations in every tested cell. We identified multiple chromosomal changes and compared the findings with the results of bulk DNA analysis using a genomic array (ThermoFisher Scientific) and FISH.

Results: We detected a number of aberrations that were already present at TP1, including del13q, del11q, mutations in NOTCH1 , RPS15 , ZMYM3 , and minor mutations in TP53 and ATM . At TP2, additional changes comprised mutations in PIM1 , XPO1, and del(3q). Between TP2 and TP3, the patient underwent several treatment lines (FCR, BR, COP) without an apparent therapeutic effect. The cells from TP3 carried complex changes, including additional mutations in BIRC3 and RB1 . Unsupervised clustering of scRNAseq gene expression data defined major clones bearing aberrations (losses on chromosomes 1, 9, 13, 17, 18). Moreover, these clones were composed of cells with additional aberrations defining a number of separate subclones. The proportion of subclones was typically under the detection limit of the genomic array and showed extreme intraclonal heterogeneity of the relapsed/refractory CLL case.

Summary/Conclusion: We showcase how genomic alterations influence the expression of affected genes. Such fact can be exploited to reconstruct chromosomal disruptions in individual cells using scRNAseq and to uncover the subclonal architecture of the disease.

Grants: MH-CZ_AZV_NU20-08-00314, MH-CZ_AZV_NV19-03-00091, MEYS-CZ_MUNI/A/1330/2021, MH-CZ_RVO_65269705.

P615: BCL2 RESISTANCE MUTATIONS IN A REAL-WORLD COHORT OF PATIENTS WITH VENETOCLAX-TREATED CHRONIC LYMPHOCYTIC LEUKAEMIA

L. Kotmayer 1,* , T. László 1 , R. Kiss 1 , L. L. Hegyi 1 , G. Mikala 2 , P. Farkas 3 , A. Balogh 3 , T. Masszi 3 , J. Demeter 4 , J. Weisinger 3 , H. Alizadeh 5 , L. Gergely 6 , A. Sulák 7 , M. Egyed 8 , M. Plander 9 , P. Pettendi 10 , D. Lévai 11 , T. Schneider 11 , Z. Pauker 12 , A. Masszi 11 , R. Szász 6 , C. Bödör 1 , D. Alpár 1

1 Department of Pathology and Experimental Cancer Research, Semmelweis University; 2 South-Pest Central Hospital – National Institute of Hematology and Infectious Diseases; 3 Department of Internal Medicine and Hematology; 4 Department of Internal Medicine and Oncology, Semmelweis University, Budapest; 5 1st Department of Internal Medicine, Clinical Centre, University of Pécs, Pécs; 6 Division of Hematology, Department of Internal Medicine, University of Debrecen, Debrecen; 7 2nd Department of Internal Medicine and Cardiology Center, University of Szeged, Szeged; 8 Kaposi Mór University Teaching Hospital of County Somogy, Kaposvár; 9 Markusovszky University Teaching Hospital, Szombathely; 10 Hetényi Géza Hospital and Clinic of County Jász-Nagykun-Szolnok, Szolnok; 11 National Institute of Oncology, Budapest; 12 Borsod-Abaúj-Zemplén County Hospital and University Teaching Hospital, Miskolc, Hungary

Background: The Bcl-2 inhibitor venetoclax has transformed the therapeutic landscape of therapy refractory and treatment naïve chronic lymphocytic leukaemia (CLL). Despite the remarkable response rates in both patient groups, a subset of CLL patients receiving venetoclax treatment experience disease progression, frequently associated with pathogenic variants of the BCL2 gene. Apart from the most common hotspot G101V and D103Y mutations, pathogenic variants affecting several different loci of the coding region of BCL2 have been described, however data and international recommendations on the sensitive detection and monitoring of these mutations are still absent to date.

Aims: Our aim was to develop a droplet digital PCR (ddPCR) based assay for the detection of BCL2 G101V and D103Y mutations and to reveal further resistance mutations emerging at relapse by performing targeted ultra-deep next-generation sequencing (NGS).

Methods: Peripheral blood samples from 71 patients treated with venetoclax-rituximab or first line venetoclax-obinutuzumab combination were collected from 11 Hungarian oncohematological centres. Genomic DNA was extracted from peripheral blood mononuclear cells and leukemic cell purity was assessed by flow cytometry. Fractional abundance of the most common hotspot BCL2 mutations G101V and D103Y was assessed using the QX200 digital droplet PCR system. Following ddPCR analyses, targeted ultradeep NGS was performed on samples obtained at relapse using a custom-designed panel covering the whole coding regions of BCL2 , BTK , PLCG2 and TP53 genes. Fractional abundance of BCL2 mutations identified by NGS was assessed retrospectively in serial peripheral blood samples by ddPCR using custom-designed, allele-specific assays.

Results: Lower quantitative limit of detection of the BCL2 ddPCR assays was tested and could ubiquitously be established as 0.01%. With a median follow up of 14 months, BCL2 G101V and D103Y mutations were detected in 8.5% (6/71) and 9.9% (7/71) of the patients, respectively, with 90% (9/10) of them experiencing relapse during the follow-up period. Three patients harbored both resistance mutations as detected by ddPCR. BCL2 G101V and/or D103Y were observed in 43.5% (10/23) of the cases showing signs of disease progression. In four cases, emergence of the mutations predated the first clinical signs of relapse with a median 4 months. In samples obtained at relapse, targeted ultradeep NGS uncovered two additional BCL2 variants (V156D and A113G), which were successfully backtracked and detected by ddPCR. Both V156D and A113G were detected in patients previously found to harbor G101V or D103Y, with no further BCL2 resistance mutations identified in G101V and D103Y wild type cases.

Summary/Conclusion: In a real-world cohort of CLL patients receiving venetoclax in combination therapy, comprehensive and sensitive screening of BCL2 mutations can identify molecular mechanisms of venetoclax resistance in nearly half of the patients experiencing relapse. In patients harboring multiple BCL2 mutations, convergent evolution of the CLL subclones may contribute to the driver mechanisms of resistance, justifying the comprehensive approach for the detection of these variants. In secondary venetoclax resistant cases displaying wild type BCL2 , further molecular screening methods are required to reveal alternative genetic or non-genetic reasons for disease progression.

P616: THE LOSS OF IΚBΕ INHIBITOR ACCELERATES DISEASE DEVELOPMENT IN CHRONIC LYMPHOCYTIC LEUKEMIA

C. Lenzi 1,* , J. Bordini 2 , A. Pseftogkas 1 , A. Morabito 2 , G. Tsiolas 3 , F. E. Psomopoulos 3 , A. Campanella 1 , M. Frenquelli 2 , P. Ghia 1

1 Division of Experimental Oncology, Università Vita-Salute San Raffaele; 2 Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy; 3 Center for Research and Technology Hellas-CERTH, Thessaloniki, Greece

Background: Chronic lymphocytic leukemia (CLL) is an indolent B cell malignancy in which B lymphocytes accumulate in peripheral blood (PB), bone marrow (BM) and spleen (SP), as a result of a complex intertwining between microenvironmental stimuli and genetic defects. Molecular studies revealed recurrent mutations in a large number of genes, each present with distinct frequency in different subgroups of patients. Among these genes, NFKBIE, a negative regulator of the NF-κB pathway, has been found mutated in a relevant proportion of patients with CLL, mainly those belonging to the clinically aggressive subset #1, suggesting that a reduced expression of NFKBIE may contribute to CLL aggressiveness through constitutive NF-κB activation independent of BcR signaling.

Aims: We aimed at studying the effects of NFKBIE in the pathogenesis of CLL using functional inactivation of the gene both in vitro and in vivo .

Methods: We engineered the MEC-1 CLL cell line using CRISPR/Cas9 technology to generate NFKBIE- knock-out (KO) cell lines. We analyzed the effect of NFKBIE ablation studying in vitro cell proliferation and migration, gene expression, BcR signalling pathway, and response to treatment with the BTK inhibitor Ibrutinib. We also evaluated the relevance of NFKBIE manipulation in vivo by injecting the engineered cells into RAG2 -/- γc -/- immunodeficient mice. In addition, we generated NFKBIE -/wt mice and crossed them with Em-TCL1 mice, a validated CLL mouse model, to generate immunocompetent Em-TCL1-NFKBIE -/wt . We analyzed disease expansion by flow cytometry, measuring the amount of malignant B cells in the PB, SP and BM.

Results: MEC-1 cells carrying NFKBIE ablation showed faster proliferation in vitro as compared to controls (p<0.05). RNAseq analyses indicated de-regulation of migratory pathway that was functionally confirmed by migration experiments with the specific chemokines (i.e. CCL19, CCL21 and SDF1). In particular, the percentage of migrated KO cells was significantly higher than that of control cells, either in the presence or the absence of each of the three chemokines. As predicted by the function of the gene, biochemical analyses in KO cells revealed the increase of cRel and p50 protein levels and the increase of p65 phosphorylation, all NF-κB transcription factors indicating unabated cellular activation. Interestingly, in vitro culture of the cells in the presence of Ibrutinib showed that NFKBIE KO cells are more resistant to apoptosis induction than control cells (p<0.05). The effect of NFKBIE manipulation was also more evident in vivo . Xenografts generated with MEC-1 NFKBIE KO cells in RAG2 -/- γc -/- immunodeficient mice showed an increased disease expansion in the SP and BM (p<0.05) and a shorter survival (p<0.05) than mice injected with control cells. In immunocompetent Em-TCL1 mice, which develop the CLL-like disease with aging, preliminary analysis at 3, 6 and 9 months of age showed higher malignant cells expansion in PB of Em-TCL1-NFKBIE -/wt compared with control Em-TCL1 mice.

Summary/Conclusion: The loss of NFKBIE function appears to be associated with a more aggressive disease in all CLL models tested, both in vitro and in vivo , accelerating disease development under CLL pro-tumorigenic conditions. In addition, it appears to be potentially involved in the onset of resistance to the BTK inhibitor Ibrutinib, thus providing an alternative mechanism that may explain drug failure in the resistant patients who do not carry BTK/PLCG2 mutations.

P617: FACING LIMITED DATASET IN HEMATOLOGICAL MALIGNANCIES: OPTIMIZATION OF MACHINE LEARNING MODELS WITH THE GENERATION OF NEW TRAINING VARIABLES AND HYPER-PARAMETERS SELECTION.

R. BIZOÏ 1,* , L. Mauvieux 2,3

1 DBSYS; 2 Laboratoire d’hematologie, Hopital Hautepierre; 3 IRFAC, INSERM U1113, Strasbourg, France

Background: There is a growing interest in the application of Machine-Learning (ML) techniques in medical research. However, the datasets in this field concern a limited number of individuals. Our goal was to implement technologies and measures for estimating learning performance in a small dataset of B-cell chronic lymphoid malignancies with extreme individuals (outliers) and a modality imbalance for the target variable.

Aims: We postulated that a limited set of antigen expression could decipher the different diagnosis of chronic B-cell malignancies (CBCM). In this aim we retrospectively studied the flow cytometry results obtained at the diagnosis for 673 patients explored in our center: 212 CLL/lymphocytic lymphoma, 160 lymphoplasmacytic lymphoma (LPL, including Waldenstrom Macroglobulinemia), 76 mantle cell lymphoma (MCL), 169 marginal zone lymphoma (MZL), 35 Hairy cell lymphoma (HCL) and 27 follicular lymphoma. We focused on median fluorescence intensity (MFI) values of CD148, CD180 and CD200 and three categorical “positive or negative” markers (CD5, CD23, FMC7).

Methods: We used the Scikit-Learn python library and LightGBM to test several ML models and develop techniques for generating new variables and selecting hyper-parameters. The treatments on the dataset were the following:

1. The three quantitative dimensions were normalized using Box-Cox transformation for LightGBM, LogisticRegression, RandomForest, KNN and GaussianNaiveBayes algorithms.

2. 70% of the data for training and 30% for testing were randomly separated.

3. Outlier removal and diagnostic dimension, modality balancing treatments were performed only on the learning sample using different unsupervised methods: localOutlierFactor, IsolationForest, OneClassSVM and EllipticEnvelope.

4. In order to avoid overfitting, the number of individuals of the least frequent modalities were balanced using (Synthetic Minority Over-sampling Technique) SMOTE algorithm.

5. For a better accuracy of classification algorithms, a discretization was set on the quantitative dimensions (CD148, CD180 and CD200 MFI). Thus all the values of the three dimensions were merged into one and this dimension was discretized into 96 quantiles.

6. New variables were created, that allowed a better learning for the LightGBM and RandomForest classification algorithms. Polynomial transformation and a principal component analysis were used for Logistic Regression.

7. Algorithms hyperparameters of ML were optimized in order to improve performance using GridSearchCV. The selection of the best ML models was done using cross-validation.

Results: The models were used for the prediction of CLL, LPL, MCL and MZL diagnosis. The results for each of the classifiers are summarized in Table 1. The global accuracy of the prediction using only 6 antigenic markers was very stable, ranging from 80 to 85% for the different algorithms. The F1-score, that represents the harmonic mean of the precision and recall of prediction (TP/(TP+ ½* (FP + FN)) was the best for CLL (f1 = 91,4%) and the lowest values were obtained for MCL and LPL.

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Summary/Conclusion: In conclusion, the methodology presented here allows an optimization of machine learning algorithms for limited set of data. Using only the results of six flow cytometry stainings, that can be analyzed in a single tube, high diagnosis prediction rates were obtained. These results, that need to be confirmed in different series, open the way to innovating diagnosis assistance tools in the field of hematological malignancies. These methods can also be applied to other diagnosis issues.

P618: SECOND-GENERATION CD19 TARGETING TRI-SPECIFIC KILLER ENGAGER DRIVES ROBUST NK CELL FUNCTION AGAINST B CELL MALIGNANCIES

J. Miller 1,* , B. Kodal 1 , P. Hinderlie 1 , D. Vallera 2 , G. Berk 3 , V. Bachanova 1 , M. Felices 1

1 Medicine; 2 Radiation Oncology, University of Minnesota, Minneapolis; 3 GT Biopharma, Brisbane, United States of America

Background: Chronic Lymphocytic Leukemia (CLL) involves uncontrollable clonal expansion of CD5+/CD19+ B lymphocytes. While patients can sometimes coexist with CLL for years, eventually there is progression to bulky adenopathy and pancytopenia from marrow suppression requiring therapy. In recent years, targeted therapies, including utilization of the inhibitors for B cell receptor (BCR) signaling and for B cell lymphoma 2 (Bcl-2) protein, have been effective treatment options. However, these treatments are associated with limitations such as development of drug resistance and the lower treatment efficacy ratio in high-risk patients. Meanwhile, CAR-T cell immunotherapy failed to produce effective treatment outcomes against CLL, mostly due to defects in the effector T cells leading to product failures, as well as being associated with high levels of off-target cytotoxicity.

Aims: Thus, approaches to CLL treatment are necessary and NK cell immunotherapy presents a viable and non-toxic option to this problem.

Methods: Our group previously demonstrated the immunotherapeutic potential of utilizing first-generation CD19-targeting 161519 Tri-Specific Killer Engager (TriKE ® ) to drive natural killer (NK) cell cytotoxicity against B-cell malignancies. We have now produced a novel second-generation CD19 TriKE, ‘CAM161519’, utilizing a humanized camelid anti-CD16 VHH single domain antibody (sdAb) ‘CAM16’, a wild-type IL-15 component, and anti-CD19 tumor antigen scFv all linked via short peptide linkers. The TriKE primarily works by generating a cytolytic bridge between NK cells and CD19 expressing B cell malignancies while also providing an expansion and survival signal to the NK cells. Using flow-based assays and imaging based cytolytic assays we explored the capability of CAM161519 to target a variety of B cell lines (Raji, Daudi, and Nalm6) and primary B cell malignancies from patients with CLL and B cell acute lymphoblastic leukemia (B-ALL). Expanded NK cells were incubated with primary CLL targets from 6 different patients and no treatment (NT), rhIL-15 (30 nM), Rituximab (10 ug/mL), or CAM16159 (30 nM). NK cell degranulation and IFNg production was evaluated after 5 hours of co-culture (Figure). CLL killing was evaluated in a similar co-culture, but with a staining mix containing CD5 and CD19, to identify CLL tumor cells in order to determine the percent CLL targets killed normalized to incubation of CLL cells with NK cells alone (Figure Image).

Results: The CAM161519 robustly activated NK cell degranulation (CD107a) and inflammatory cytokine production (IFNg) against B cell lines. The activation was specific as it was strongly reduced against CD19 knockout Nalm6 cells. The TriKE also induced specific NK cell proliferation. IncuCyte imaging based cytolytic assays demonstrated strong killing activity against Raji targets. When evaluating activity against primary targets, CAM161519 induced enhanced NK cell degranulation, cytokine production, and cytotoxicity using flow cytometry-based assays against primary CLL (Figure Image) and B-ALL targets when compared to IL-15 treatment. CAM161519 also rescued functionality of NK cells from CLL patients against their autologous CLL cells.

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Summary/Conclusion: Taken together, these data are indicative of the potential for clinical translation of the CAM161519 TriKE in the CLL and B-ALL settings alone or in combination with cellular products.

P619: TARGETING TRANSLATION BY DISRUPTING THE NEWLY IDENTIFIED PROHIBITIN-EIF4F COMPLEX AS A NOVEL THERAPEUTIC STRATEGY IN CHRONIC LYMPHOCYTIC LEUKEMIA

A. Largeot 1 , V. Klapp 1 , S. Gonder 1,* , E. Viry 1 , M. Szpakowska 2 , D. Perez Hernandez 2 , G. Dittmar 2 , A. Chevigné 2 , L. Ysebaert 3 , B. Stamatopoulos 4 , L. Desaubry 5 , J. Paggetti 1 , E. Moussay 1

1 Department of Cancer Research, Luxembourg Institute of Health, Luxembourg; 2 Department of Infection and Immunity, Luxembourg Institute of Health, Esch sur Alzette, Luxembourg; 3 Service d’Hématologie, IUCT-Oncopole, Toulouse, France; 4 Laboratory of Clinical Cell Therapy, ULB-Research Cancer Center (U-CRC), Jules Bordet Institute Université Libre de Bruxelles, Brussels, Belgium; 5 Regenerative Nanomedicine Laboratory (UMR1260), Faculty of Medicine, FMTS, INSERM-University of Strasbourg, Strasbourg, France

Background: CLL is the most common type of leukemia in adult, and despite great advance in the standard of care in the last decades, there is still no cure available. CLL cells are dependent on their microenvironment for proliferation and survival. Microenvironmental stimuli are associated with an increase in translation globally but also at the level of specific transcripts, including Myc (Yeomans et al ., 2016, Blood).

Aims: Here, we tested the targeting of translation initiation in CLL as a novel therapeutic strategy and identified prohibitin as partner of the translation initiation machinery.

Methods: In order to target translation, we used the FL3 molecule, a synthetic flavagline, which is a known inhibitor of translation initiation in other types of cancers (Boussemart et al. , 2014, Nature). This drug binds to the scaffold proteins prohibitins (PHBs), but the mechanism for translation inhibition is still unclear. PHBs are crucial partners for the activation of the Ras-Raf-MEK-ERK pathway, ultimately leading to eIF4E (a factor of the eIF4F translation initiation machinery) phosphorylation and to activation of translation.

Results: We confirmed the increase in translation in human primary CLL cells upon stimulation ( A ). In addition, we showed that in the Eµ-TCL1 murine model, CLL cells have a higher translation rate compared to normal B cells and T cells. In vitro treatment of human CLL cells (either cell lines or primary patient samples) with FL3 leads to a decrease in translation rate. It is associated with a strong decrease of cell viability ( B ) and apoptosis induction, even at a low nanomolar dose. By western blot, we showed that in CLL, contrary to other cancer types, FL3 does not prevent RAF1 and ERK phosphorylation. However, eIF4e phosphorylation is impaired. This observation strongly suggests a direct impact of the drug through its molecular target PHBs on the translation initiation machinery. By co-immunoprecipitation, proximity-ligation assay ( C ) and Nanoluciferase experiments, we demonstrated the interaction between PHBs and the members of the eIF4F complex. In addition, gene silencing of PHB by shRNA leads to a decrease in translation. This indicates for the first time a direct role of PHBs in translation, and allows a better understanding of FL3’s mechanism of action. We thus propose that PHB is necessary for the correct assembly of the eIF4F complex. Interestingly, we showed that the loss of eIF4e phosphorylation is neither responsible for the impairment of translation nor for the decrease in cell proliferation. By pulse SILAC experiments, we determined the proteins that are subjected to increased translation upon TLR stimulation, and to decreased translation upon FL3 treatment. Among others, oncogenes such as c-MYC or ETS-1 ( D ) have been identified. In vivo treatment of mice with FL3 after TCL1 adoptive transfer leads to a significant delay in CLL progression and an increased survival ( E-F ), demonstrating the relevance and possible impact of this molecule. Finally, high expression of translation initiation-related genes correlated with poor survival and unfavorable clinical parameters in CLL patients (G ).

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Summary/Conclusion: To conclude, we identified translation initiation as a potential therapeutic target in CLL. The use of the inhibitor of translation FL3, efficiently impairs proliferation of CLL cells in vitro and controls CLL development in vivo . Moreover, we start to unveil the mechanism of action of this molecule, by the identification of a novel interactor of the translation initiation machinery, namely prohibitins.

P620: DYNAMICS OF GENOMIC ABERRATIONS IN RELATION TO DISEASE ACTIVITY IN UNTREATED PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

V. Navrkalova 1,2,* , J. Kotaskova 1,2 , K. Plevova 1,2 , T. Reigl 2 , M. Valisova 1 , M. Bohunova 1 , M. Zenatova 1 , L. Radova 2 , A. Panovska 1 , S. Pospisilova 1,2

1 Internal Medicine - Hematology and Oncology, University Hospital Brno and Masaryk University; 2 Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czechia

Background: Chronic lymphocytic leukemia (CLL) manifests remarkable clinical variability linked with extensive heterogeneity of genomic defects. Despite the progress with novel therapeutic agents, some patients still face relapse and disease persistence due to subclonal populations, which harbor various somatic defects, growth dynamics, and therapy sensitivity. Clonal evolution of genomic defects was observed mainly in treated CLL patients, whereas progression and therapy need occur at some point during the disease course suggesting clonal dynamics even in untreated patients.

Aims: To describe impact of clonal dynamics in gene mutations and chromosomal defects on clinical course of the disease in paired samples of untreated CLL patients.

Methods: 200 samples from 100 CLL patients were analyzed by well-established comprehensive NGS panel LYNX (PMID 34082072) in two time points; at the diagnosis and before first therapy. Variants in 70 genes associated with B-cell malignancies, copy-number alterations (CNAs) and copy-neutral loss of heterozygosity (CN-LOH) across the whole genome were evaluated by a dedicated in-house bioinformatic pipeline. Clinical data were correlated with obtained results.

Results: The surveillance period median between two sampling points was 36 months (range 7-174). One third of patients was still untreated during the follow-up (median 86 months, range 21-271), and the time to first treatment among 68 patients was 38 months (range 11-141). At the baseline, the most frequent defects were mutations in NOTCH1 (15 %), SF3B1 (15 %), ATM (12 %) genes, and 13q- (56 %), 11q- (19 %) and tri12 (15 %). We observed common clonal and subclonal co-occurrence of genomic defects in the pretreatment period. In sequential samples, we detected a change in the composition of genomic aberrations. Most alterations were stable (59 % mutations, 70 % chromosomal defects), but we observed dynamic changes in the rest with the predominance of growing allelic frequency and occurrence of new defects. Unsupervised clustering according to the presence of individual genomic defects with the emphasis on their dynamic changes and CLL driver attribute enabled to distinguish three clusters. The correlation with clinical data showed significant differences among clusters in IGHV status, CLL activity (categorized according to therapy need), and an increase of absolute lymphocyte count (ALC) per month. Obviously, the most adverse was cluster 1 with unmutated IGHV, rapid increase in ALC, and dynamics in CLL activity. Cluster 2 seemed to be intermediate and cluster 3 indolent. Groups also significantly differed in the presence and evolution of 11q-, 13q-, ATM mutations, tri12, chr13 LOH, 3p-, 2p+, chr9 LOH. Importantly, stable defects of chr13 were the most abundant in cluster 3, stable tri12 and expansions of 13q- in cluster 2, dynamics defects including 11q-, ATM mutations, 3p-, 2p+ and LOH 9 in cluster 1.

Summary/Conclusion: In untreated CLL patients, clonal stable genomic defects prevail. However, common subclonal alterations and dynamic changes were observed in a considerable proportion of patients. After unsupervised clustering, a group of patients showing the most adverse clinical disease course harbored ATM defects, 3p-, 2p+, and chr9 LOH, possibly standing behind fast progression and therapy need. Hence, detailed monitoring of clonal evolution can help understand the heterogeneity in the CLL activity in the pretreatment period.

Supported by AZV NV19-03-00091, MZ-CR RVO 65269705, MUNI/A/1330/2021.

P621: SUBCLONAL EVOLUTIONARY TRAJECTORIES IN IGLV3-21R110 CHRONIC LYMPHOCYTIC LEUKEMIA

L. Paschold 1,* , D. Simnica 1 , R. Benitez Brito 2 , C. Schultheiss 1 , T. Zhang 1 , C. Dierks 1 , M. Binder 1

1 Department of Internal Medicine IV, Oncology/Hematology, Martin-Luther-University Halle-Wittenberg, Halle, Germany; 2 Department of Hematology, Inselspital, University of Bern, Bern, Switzerland

Background: There is a large body of evidence supporting the importance of antigenic interactions of the B cell receptor (BCR) with self or non-self antigens that make this receptor and its signaling machinery one of the most important drivers in many types of non-Hodgkin lymphoma. This has led to a new pathobiological understanding of these diseases as well as novel therapeutic strategies.

Chronic lymphocytic leukemia (CLL) is a prototype disease in which BCR-BCR self-interactions induce autonomous signaling. While virtually all CLL BCR are prone to autonomous signaling induced by such contacts, the IGLV3-21 R110 molecular interaction found in 10% of CLL patients defines a distinct clinical subset of patients that show a very aggressive clinical course despite novel treatments. In these cases, a single amino acid replacement from G to R located at the boundary of variable and constant region of the BCR is an important tumor promotor driving neoplastic B cell growth.

Aims: It has been unclear how the R110 point mutation is generated. We set out to investigate the trajectory of the malignant CLL B cell clone and subclonal heterogeneity in IGLV3-21 R110 cases in order to gain insight into the molecular pathogenesis of this unique amino acid exchange.

Methods: We screened an initial CLL cohort of 127 cases by flow cytometry using an anti-IGLV3-21 R110 antibody. Positive cases were subjected to immunosequencing of the light chain locus via amplicon based next-generation sequencing and characterized for typical chromosomal deletions and driver gene mutations.

Results: We identified twelve CLL cases with IGLV3-21 R110 . Of these, we found a higher frequency of mutations in ATM (42%) and SF3B1 (25%) than in unselected CLL cohorts, which is in line with findings reported by others. As expected, the repertoire of healthy B cells in the blood of IGLV3-21 R110 CLL patients contained IGLV3-21 light chains with the wild-type G110 residue, indicating that the R110 exchange is of acquired nature. Interestingly, in half of the IGLV3-21 R110 CLL cases we found additional clones with IGLV3-21 and R110, including cases with divergent J gene cassettes originating from different pre-B cells. This indicates that the R110 exchange may occur multiple times within independent B cell clones in the same patient. Due to the unique location at the variable-constant boundary and the high correlation of the IGLV3-21 R110 patient subset with mutations of genes involved in DNA repair (ATM/SF3B1), R110 is likely generated as a result of incorrect V-J recombination facilitated by the error-prone-environment.

Summary/Conclusion: Our work indicates that the G110R point mutation results from incorrect V-J rearrangements in the error-prone environment that may be induced by SF3B1 and ATM mutations associated with this CLL subset. We found evidence for a branching light chain evolution that leads to a striking convergence of separate clones on the identical light chain amino acid exchange in the same patient. This strongly emphasizes the role of this mutation as a tumor promotor involved in malignant transformation of this subset of CLL.

P622: DOUBLE-HIT TRAF3 DELETION AND MUTATION IDENTIFIED BY HIGH-THROUGHPUT SEQUENCING DEFINE A NEW INDEPENDENT PROGNOSTIC FACTOR IN CHRONIC LYMPHOCYTIC LEUKEMIA

C. Perez-Carretero 1,2,* , M. Hernández-Sánchez 1,2 , M. Quijada-Álamo 1,2 , T. González 1,2 , A. Rodríguez-Sánchez 1,2 , M. Martín-Izquierdo 1,2 , J. Matías-Martín 1 , A. Rubio 3 , J. Dávila 4 , A. García de Coca 5 , J. Galende 6 , P. Jimenez-Montero 7 , J.-A. Queizán 7 , I. González-Gascón y Marín 8 , J.-Á. Hernández-Rivas 8 , R. Benito 1,2 , J.-M. Hernández-Rivas 1,2 , A.-E. Rodríguez-Vicente 1,2

1 Department of hematology, Hospital Universitario de Salamanca; 2 Universidad de Salamanca, IBSAL, Centro de Investigación del Cáncer, IBMCC-CSIC, Salamanca; 3 Department of hematology, Hospital Miguel Servet, Zaragoza; 4 Department of hematology, Hospital Nuestra Señora de Sonsoles, Ávila; 5 Department of hematology, Hospital Clínico, Valladolid; 6 Department of hematology, Hospital El Bierzo, Ponferrada; 7 Department of hematology, Hospital General, Segovia; 8 Department of hematology, Hospital Universitario Infanta Leonor, Madrid, Spain

Background: Chromosome 14q abnormalities involving the IGH gene are present in 5-15% of chronic lymphocytic leukemia (CLL) patients. Besides IGH translocations, interstitial 14q32.33/IGH deletions have been reported at lower frequencies (2%), and their clinical implications and underlying molecular mechanisms remain unknown. In other B-cell malignancies such as multiple myeloma, TRAF3 gene, proximal to the IGH locus, may be encompassed in the deletion, with mutations in the remaining allele. However, the molecular status of TRAF3 in CLL and their impact in prognosis have not been studied in depth.

Aims: To analyze the genetic landscape of CLL patients with IGH deletion and evaluate the molecular status of TRAF3 to elucidate their impact in CLL pathogenesis and prognosis.

Methods: A total of 897 CLLs were analyzed by FISH with the IGH break-apart probe and the 4-probe CLL panel (ATM, CEP12, D13S319 and TP53) (Vysis). 324 untreated CLLs (54 with IGH deletion (del-3’IGH) and 270 as the control group) were sequenced by targeted-NGS, with a custom panel including 54 CLL-related genes (Nextseq platform, Illumina). Copy number variations (CNVs) of TRAF3 were assessed by a NGS approach and validated by SNP Array 6.0.

Results: FISH analyses identified a total of 54 out of 897 CLLs (6%) with a 300 kb deletion of the centromeric side of IGH (del-3’IGH CLLs). Moreover, our results showed that 76% of del-3’IGH CLLs had additional FISH abnormalities, being the most frequent the loss of 13q (42%), +12 (22%), del(11q) (11%) and 17p- (9%). NGS analyses showed that the most recurrently mutated genes in del-3’IGH CLL were NOTCH1 (30%), ATM (20%) and TRAF3 (13%). Notably, the mutational frequency of TRAF3 mutations was significantly higher in del-3’IGH CLLs than in the control group (13% vs. 0.5%, p<0.001).

Given the surprisingly incidence of TRAF3 mutations in this subgroup, we further assessed its CNV status, noticing that TRAF3 loss was enriched in del-3’IGH CLLs when compared to the control group (11/54, 20% vs . 4/270, 1.5%, p<0.001). By integrating mutational and CNV information, we observed that 7 out of 11 patients with a TRAF3 loss harbored mutations in this gene. The deletion was present in a higher percentage of cells than the mutation (mean: 70.5% vs . 15.6%), which may suggest that TRAF3 mutation appears as a subclonal event, leading to a biallelic inactivation of this gene. Our results showed a novel biallelic alteration in CLL, reminding the double null event that occurs in other gene drivers such as TP53 and ATM.

Of note, this double-hit on TRAF3 contributed to a shorter time to first treatment (TFT) in del-3’IGH CLL patients (5 vs. 54 months, p<0.001). Furthermore, TRAF3 biallelic inactivation had a similar TFT than TP53 or ATM -altered patients with an adverse prognosis (median TRAF3 : 5 vs. 59 months, p<0.001, ATM : 2 vs 59, p<0.001 and TP53 : 12 vs 59, p=0.002), being also an independent risk factor in the multivariate analyses in the CLL cohort (n=324) (HR=0.21, 95% CI=0.05-0.85, p=0.029) (Table 1).

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Summary/Conclusion: Our work demonstrates that TRAF3 mutations and deletions are highly enriched in CLL patients with del-3’IGH, resulting in the biallelic inactivation of this gene. The presence of this double-hit on TRAF3 worsen the prognosis of CLL patients, being and independent prognostic factor with similar impact on TFT than the biallelic loss of TP53 .

Funding: PI21/00983; FI19/00191 (CPC); CD19/00222 (MHS); FEHH (MQÁ); USAL (RBS)

P623: IBRUTINIB REGULATES MIR-181A/B VIA C-FOS IN CHRONIC LYMPHOCYTIC LEUKEMIA

A. Ramassone 1,* , F. Palmiero 1 , A. Tomasso 2 , I. Innocenti 2 , L. Laurenti 2 , R. Visone 3

1 Center for Advanced Studies and Technology, “G. d’Annunzio” University, Chieti; 2 Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma; 3 Department of Medicine, Dentistry and Biotechnology, “G. d’Annunzio” University, Chieti, Italy

Background: B-cell receptor (BCR) signalling controls B-cell survival and proliferation, and its deregulation has a pathogenic role in Chronic Lymphocytic Leukemia (CLL), a B-cell malignancy. Currently, several BCR signalling inhibitors are used in therapy, among them the Ibrutinib. We previously reported that the BCR-signalling inhibition mediated by Ibrutinib leads to miR-181a/b activation; those miRNAs are significant biomarkers of CLL progression, and their downregulation is associated to a poor prognosis; furthermore, low levels of miR-181a/b impair several anti-apoptotic proteins, contributing to cell survival. BCR signalling also controls c-Fos/AP-1 pathway in pre-B cells. Recently, we suggested the involvement of c-Fos in miR-181b expression, however the mechanism by which this regulation is exerted is still not explored. Moreover, the involvement of c-Fos in miR-181a/b transcription during Ibrutinib is still not clear.

Aims: Assess the impact of Ibrutinib on c-Fos expression; assess the involvement of the transcription factor c-Fos in miR-181a/b transcription; assess the impact of miR-181a/b in CLL cells survival during Ibrutinib treatment.

Methods: To assess the impact of Ibrutinb on c-Fos transcription, we evaluated its expression in CLL cells treated with Ibrutinib or vehicle. To assess the involvement of c-Fos in miR-181a/b transcription, we evaluated i) the expression of the miRs and of their primary transcripts in CLL cells overexpressing c-Fos, and ii) the ability of c-Fos to bind miR-181a/b host gene promoter by luciferase, ChIP and EMSA assays. To assess the impact of the miRs on CLL cells survival during Ibrutinib treatment, we performed MTT assay in CLL cells after miR-181a/b silencing and Ibrutinib treatment.

Results: Considering that c-Fos was found to be controlled by BCR signalling in pre-B cells, we evaluated whether BCR signalling affect c-Fos also in CLL cells: we found that c-Fos expression increases in primary CLL cells after Ibrutinib treatment. Since miR-181a/b was up-regulated after Ibrutinib treatment in vitro and in vivo in CLL cells, and considering that a possible involvement of c-Fos in miR-181b expression was already suggested, we sought to identify if c-Fos was a direct regulator of miR-181a/b expression. We confirmed that c-Fos overexpression affects miR-181b levels in CLL patients, but not miR-181a; however, the dysregulation of the miR shows heterogeneity among patients. Indeed miR-181b expression increases in 3 and decreases in 2 of 7 patients. To better understand how c-Fos impact on miRs expression, we analysed both miR-181a and miR-181b at the transcriptional level in CLL and lymphoblastoid cell lines, finding that c-Fos negatively regulates the pri-miR-181a/b. To clarify the mechanism by which c-Fos regulates miR-181a/b transcription, we analysed the miRs host gene (MIR181A2HG) promoter. Since MIR181A2HG promoter shows two consensus sequences for the transcription factor c-Fos, we studied whether c-Fos binds these regions. We found that c-Fos binds at MIR181A2HG promoter by recognizing the two consensus sequences. Accordingly, after their deletion, the ability of c-Fos to recognize the promoter was overcome. Finally, since miR-181a and miR-181b are involved in cell death, we assessed whether those miRNAs participate in CLL cells death induced by Ibrutinib: we found that the silencing of both miRNAs improves CLL cell survival during Ibrutinib treatment.

Summary/Conclusion: c-Fos is a downstream target of BCR signalling and a direct regulator of miR-181a/b transcription in CLL. MiR-181a/b are effectors of Ibrutinib-induced CLL cells death.

P624: CHROMOTHRIPSIS IN PATIENTS WITH CLL AND COMPLEX KARYOTYPE: PATTERNS OF ABERRATIONS AND PROGNOSTIC VALUE

S. Ramos-Campoy 1,2,* , A. Puiggros 1,2 , J. Kamaso 1,2 , S. Beà 3 , S. Bougeon 4 , M. J. Larráyoz 5 , D. Costa 3 , H. Parker 6 , G. M. Rigolin 7 , M. L. Blanco 8 , R. Collado 9 , I. Ancín 10 , R. Salgado 11 , M. Moro 12 , T. Baumann 3 , E. Gimeno 1,13 , C. Moreno 8,14 , M. J. Calasanz 5 , A. Cuneo 7 , J. C. Strefford 6 , F. Nguyen-Khac 15 , D. Oscier 16 , C. Haferlach 17 , J. Schoumans 4 , B. Espinet 1,2

1 Molecular Cytogenetics Laboratory, Pathology Department, Hospital del Mar; 2 Translational Research on Hematological Neoplasms Group, Cancer Research Program, Institut Hospital del Mar d’Investigacions Mèdiques (IMIM); 3 Hematopathology Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBERONC, Barcelona, Spain; 4 Oncogenomic Laboratory, Hematology Service, Lausanne University Hospital, Lausanne, Switzerland; 5 Department of Genetics, Universidad de Navarra, Pamplona, Spain; 6 Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; 7 Hematology Section, St. Anna University Hospital, Ferrara, Italy; 8 Department of Hematology, Hospital Universitari de la Santa Creu i Sant Pau, Barcelona; 9 Department of Hematology, Consorcio Hospital General Universitario, Valencia; 10 Department of Hematology and Hemotherapy, Hospital Universitario Cruces, Bilbao; 11 Department of Hematology, Fundación Jiménez Díaz, Madrid; 12 Department of Hematology, Hospital Universitario Central de Asturias, Oviedo; 13 Applied Clinical Research in Hematological Malignances, Cancer Research Program, Institut Hospital del Mar d’Investigacions Mèdiques (IMIM), Barcelona; 14 Josep Carreras Leukemia Research Institute, Badalona, Spain; 15 Hematology Department and Sorbonne University, Hôpital Pitié-Salpêtrière, APHP, INSERM U1138, Paris, France; 16 Department of Molecular Pathology, Royal Bournemouth Hospital, Bournemouth, United Kingdom; 17 MLL Munich Leukemia Laboratory, Munich, Germany

Background: Chromothripsis is a unique catastrophic event found in 1-5% of chronic lymphocytic leukemia (CLL) patients, associated with poor prognostic factors and short survival (Edelmann et al, 2012; Puente et al, 2015; Leeksma et al, 2021). It is characterized by the presence of multiple genomic rearrangements in one chromosome, resulting in oscillating switches between 2-3 copy number states by chromosomal microarrays (CMA) or next-generation sequencing (Stephens et al, 2011). Optical genome mapping (OGM) is a new method based on the imaging of long DNA molecules (>250kb) labelled at specific motifs, generating a unique pattern. This allows mapping of the genomic location of each molecule and detection of structural and numerical chromosomal abnormalities with high resolution and sensitivity.

Aims: 1. To analyze the genome complexity associated with chromothripsis and the patterns of rearrangements detected by OGM; 2. To determine the prognostic impact of chromothripsis in a cohort of CLL patients enriched in complex karyotypes (CK).

Methods: A total of 33 patients with chromothripsis detected by CMA were included: 11 with chromothripsis-like patterns (≥7 switches), 22 with classical chromothripsis (≥10 switches). Nine of the cases were studied with OGM (Bionano Genomics). Clinico-biological features and time to first treatment (TTFT) were analyzed. Results were compared with a cohort of 129 patients showing CK without chromothripsis (control group) (Ramos-Campoy et al, 2021).

Results: All patients with chromothripsis showed a high genomic complexity (30/33 with CK by chromosome banding analysis and 3/33 classified as complex by CMA [range: 10-24 CNV, being at least three of them >5Mb (range: 3-6)]. Forty-six chromothriptic events were identified. These affected 1-4 chr/patient, but a single chromosome was involved in 25/33 patients (76%). Chromothripsis was distributed throughout all chromosomes, most frequently affecting 3 (n=5), 6 (n=5) and 13 (n=5). The events were mainly multiple losses (25/46) or alternated gains and losses (19/46). Size and coordinates of CNVs detected by OGM and CMA were highly concordant. OGM detected multiple rearrangements cryptic by CMA (median: 9/chromothriptic event). Two patterns of chromothripsis-related rearrangements were identified: one clustered in the chromothriptic region (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (range: 1-6 chr) (6/9). One patient showed 36 rearrangements throughout the genome, suggesting the coexistence of chromothripsis and chromoplexy, characterized by the presence of multiple chained translocations (Figure). Patients with chromothripsis showed a higher frequency of TP53 del/mut (70% vs 39%; p =0.001) and a shorter TTFT (15m vs 2m; p =0.013) than the control group. No differences were observed for IGHV and del(11q) status. When stratifying patients based on TP53 status, the presence of chromothripsis was not associated with shorter TTFT. In the multivariate analysis including TP53 , genomic complexity by CMA and chromothripsis, only TP53 was statistically significant (HR=1.6; p =0.029).

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Summary/Conclusion: 1. Chromothriptic events involve multiple loci in CLL patients; 2. OGM allows not only the detection of CNV but also the identification of two rearrangement patterns associated with chromothripsis (clustered or involving non-chromothriptic chromosomes) whose clinical impact should be further explored. 3. In CLL patients with CK, the adverse prognosis of chromothripsis is associated with a higher frequency of TP53 del/mut .

P625: SINGLE-CELL RNA SEQUENCING REVEALS THE HETEROGENEITY OF TUMOR CELLS AND IMMUNE MICROENVIRONMENT IN RICHTER SYNDROME AND ACCELERATED CLL

Y. Sha 1,2,* , Y. Miao 1,2 , S. Qin 1,2 , R. Jiang 1,2 , W. Wu 1,2 , Y. Xia 1,2 , L. Wang 1,2 , L. Fan 1,2 , W. Xu 1,2 , H. Jin 1,2 , H. Zhu 1,2 , J. Li 1,2

1 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University; 2 Department of Hematology, Pukou CLL Center, Pukou division of Jiangsu Province Hospital, Nanjing, China

Background: Richter Syndrome (RS) is defined as development of aggressive lymphoma arising from chronic lymphocytic leukemia/Small lymphocytic leukemia (CLL/SLL). Accelerated CLL/SLL (aCLL/SLL) is pathologically defined as a highly proliferative form of CLL/SLL (either expanded proliferation center or Ki-67>40%). The underlying mechanism behind transformation from indolent CLL/SLL to aggressive DLBCL remains largely unknown. The biological difference between RS DLBCL and aCLL/SLL has not been explored yet.

Aims: To explore intra- and inter-tumor and immune microenvironment (TME) heterogeneity of RS and aCLL/SLL. To disclose the molecular mechanism underlying transformation from CLL/SLL to DLBCL.

Methods: Single-cell RNA sequencing (scRNA-seq) of paired lymph node (LN) and peripheral blood (PB) was done among 2 RS (P1, P2) and 2 aCLL/SLL (P3, P4) patients (pathologically diagnosed in LN, Fig. A) and bioinformatics analysis was conducted.

Results: Identification of cell type in 8 samples showed great heterogeneity (Fig. B). Single-cell trajectory analysis was done using “monocle” inferred tumor development and differentiation of each patient. Monoclonal B cells with high Ki-67 expression and high proportion of G2M phase cells were enriched in the end of pseudotime trajectories (Fig. C). Further analysis of CD5, CD20 and CD23 expression alongside pseudotime trajectories were also conducted, indicating that our trajectories could depict the transformation from indolent CLL/SLL to aggressive DLBCL. Therefore, cell states were clustered according to the trajectories. P1 and P2 were clustered as seven groups respectively (P1A-G, P2A-G) and P3 were clustered as three groups (P3A-G). Notably, although minor proportion of proliferating cells were enriched in the end of P4 pseudotime trajectories, clusters could not be split in P4 patients. All cell states groups were mapped to UMAP analysis and the proportion of G2M phase was highest in state G of RS patients. Single-sample GSEA (ssGSEA) analysis was done to disclose the pathway activation in transformation process and found that pathways including cell cycle, TCA cycle, spliceosome, MYC targets, unfolded protein response, epigenetics, DNA repair, glycolysis and oxidative phosphorylation were gradually activated alongside trajectories from state A-G in RS. Moreover, pathways including NOTCH, P53, PI3K-AKT, Antigen processing and presentation, apoptosis, hypoxia and etc. were found significantly upregulated in RS (Fig. E). Further analysis of immune microenvironment of RS and aCLL/SLL showed that exhaustion T cells were significantly enriched in RS lymph node compared with aCLL/SLL, indicating the immune surveillance escape mediated by exhaustion T cells might prompt DLBCL transformation. PDCD1 (PD-1), CTLA4, LAG3, HAVCR2 (TIM3), TIGIT expression were significantly upregulated in exhaustion T cells of RS patients, suggesting potential treatment target of RS for immune checkpoint inhibitors (Fig. F).

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Summary/Conclusion: Our study was the first one using scRNA-seq to disclose the tumor heterogeneity and immune microenvironment of RS and aCLL/SLL in single-cell dimension. Evolution trajectories from indolent CLL/SLL to aggressive DLBCL were constructed and pathway activation in the process of transformation were further explored. Moreover, immune microenvironment analysis of RS and aCLL/SLL showed that exhaustion T cells were significantly enriched in RS lymph node and immune checkpoints including PDCD1 (PD-1), CTLA4, LAG3, HAVCR2 (TIM3), TIGIT were significantly upregulated in exhaustion T cells of RS patients, suggesting potential treatment target of RS.

P626: FUNCTIONAL SCREENING OF PI3K INHIBITORS STRATIFIES RESPONDERS TO IDELALISIB AND INDICATES DRUG CLASS ACTIVITY IN IDELALISIB-REFRACTORY CLL

S. Skånland 1,* , Y. Yanping 1 , P. Athanasiadis 1 , L. Karlsen 1 , A. Urban 1 , I. Murali 2 , S. Fernandes 2 , A. Hilli 3 , K. Taskén 1 , F. Bertoni 4 , A. Mato 5 , E. Normant 6 , J. Brown 2 , G. Tjønnfjord 1 , T. Aittokallio 1

1 Oslo University Hospital, Oslo, Norway; 2 Dana-Farber Cancer Institute, Boston, United States of America; 3 Diakonhjemmet Hospital, Oslo, Norway; 4 Università della svizzera italiana, Bellinzona, Switzerland; 5 Memorial Sloan Kettering Cancer Center; 6 TG Therapeutics, New York, United States of America

Background: The phosphatidylinositol 3-kinase inhibitors (PI3Ki) idelalisib and duvelisib are approved for relapsed chronic lymphocytic leukemia (CLL). While patients may show an initial response to these therapies, development of treatment resistance or intolerance remains clinical challenges. Prediction of individual treatment responses based on clinically actionable biomarkers is needed to overcome these challenges.

Aims: 1. To characterize functional responses to 10 PI3Ki in CLL

2. To study PI3Ki drug class activity in idelalisib-refractory CLL

3. To investigate whether ex vivo drug sensitivity can predict in vivo treatment responses

Methods: CLL cells from patients that were treatment naïve (n=7), idelalisib refractory (n=9), or on idelalisib treatment (longitudinal samples from n=6 patients) were screened against 10 PI3Ki (buparlisib, compound 7n, copanlisib, duvelisib, idelalisib, nemiralisib, pictilisib, pilaralisib, umbralisib, ZSTK474), both alone and in combination with the B-cell lymphoma 2 (Bcl-2) antagonist venetoclax. Two idelalisib-resistant lymphoma cell lines were used as reference. Treatment effects on cell signaling were profiled using phospho flow and effects on cell viability were monitored with detection of cleaved caspase-3 and using the CellTiter-Glo assay.

Results: Pan-PI3Ki were most effective in inhibiting PI3K signaling both after 30 min and 24h exposure. Inhibition of cell signaling correlated with induction of apoptosis. Treatment with a PI3Ki induced cell death in 72h CellTiter-Glo assays, although to varying extent. The pan-PI3Ki were overall most potent in inhibiting CLL cell viability. Interestingly, pan-PI3Ki exhibited sustained activity both in idelalisib-resistant lymphoma cell lines and in CLL cells from idelalisib-refractory patients. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Notably, CLL cells from idelalisib-refractory patients remained sensitive to idelalisib+venetoclax combination. Ex vivo drug testing on CLL cells from a patient who presented with relapsed disease after sequential treatment with FCR, ibrutinib, idelalisib and venetoclax revealed sensitivity to PI3Ki+venetoclax treatment. The patient consequently started treatment with idelalisib+venetoclax. She obtained an initial partial response, but then suffered intolerable adverse effects and the therapy was stopped. Protein profiling of the patient’s CLL cells collected at 5 time-points before, during and after the combination regimen showed that B-cell signaling was reduced by the treatment, but again significantly increased when the therapy was stopped. Increased phosphorylation of Bruton’s tyrosine kinase (BTK) and extracellular signal-regulated kinase (ERK) at the latest time-point was mirrored by increased sensitivity to venetoclax combined either with a BTK or MEK inhibitor. Unfortunately, the patient passed away and the efficacy of these alternative therapies could not be tested. To more systematically assess the predictive value of ex vivo drug sensitivity data, we analyzed drug responses to 73 combinations on CLL cells from patients treated with idelalisib. CLL cells collected at baseline from patients who obtained a long-term response to idelalisib showed significantly higher drug sensitivity scores than CLL cells from patients who obtained a short-term response.

Summary/Conclusion: Our findings indicate PI3Ki drug class activity in idelalisib-refractory CLL, and suggest that ex vivo drug sensitivity may guide precision medicine and predict treatment responses. These results warrant further testing in larger cohorts and in clinical trials.

P627: COMBINED DRUG AND CRISPR/CAS9 SCREENING REVEALS SPECIFIC TARGETS FOR SF3B1- AND NOTCH1-MUTATED CLL CELLS

L. Dostálová 1,2 , A. Ladungová 1,3 , D. Škrnová 1 , N. Varma Gottumukkala 1,2 , Y. Lodhi 1,2 , M. Smida 1,4,*

1 CEITEC Masaryk University; 2 Department of Biology, Faculty of medicine, Masaryk university; 3 National Centre for Biomolecular Research, Faculty of Science, Masaryk university; 4 Department of internal medicine - Oncology and hematology, University hospital Brno and Medical faculty of Masaryk university, Brno, Czechia

Background: Chronic lymphocytic leukemia (CLL) is genetically heterogeneous disease with a plethora of mutations occurring in relatively low fraction of patients. Mutations in SF3B1 and NOTCH1 genes are among the most frequent recurrent mutations found in 10-20% CLL cases. The presence of these mutations is considered as a negative prognostic factor, yet, there is no specific treatment available to achieve more prominent response.

Aims: The aim of this study was to use the combination of CRISPR/Cas9 screening and high-throughput drug screening on cells with introduced SF3B1 or NOTCH1 mutations in order to reveal unique vulnerabilities specific to the cells carrying these frequent CLL mutations.

Methods: Using CRIPSR/Cas9-based homology directed repair, we have introduced specific mutations most recurrent within the hotspots of SF3B1 (K700E) and NOTCH1 (P2514fs) into CLL-derived cell line HG3. These two knock-in cell line variants were then subjected to a drug screening with a library of 859 approved drugs, enriched with oncology-indicated drugs. Cell viability in response to 10 uM library concentration upon 72h was assessed by Cell-Titer Glo and evaluated against the wild-type counterparts. Simultaneously, genome-wide CRISPR/Cas9 knockout screen was performed in these cell lines to identify genes whose deletion is selectively lethal to SF3B1 or NOTCH1-mutated cells.

Results: The CRISPR/Cas9 dropout screen has revealed several interesting genes that are in synthetic lethal relationship with the introduced point mutations. In particular, SPDYE1, a gene involved in cell cycle regulation, and LUC7L3, involved in splicing and cAMP regulatory element binding, were found to be synthetically lethal with the NOTCH1 mutation. On contrary, SNUPN, responsible for transport of spliceosomal snRNPs, and UQCRC1, involved in cytochrome c regulation and oxidative phosphorylation, were found to be essential for SF3B1-mutated cells. Drug screening using library of approved drugs demonstrated general hypersensitivity of both the mutant cell lines and the wild-type controls to the inhibitors of proteasome and HDAC enzymes. In addition, exquisite sensitivity of NOTCH1-mutant and SF3B1-mutant cell lines, contrary to their wild-type counterparts, was revealed towards the inhibitors of various hormone receptors or inhibitors of 20S proteasome.

Summary/Conclusion: The combination of diverse screening approaches may reveal novel vulnerabilities specific for individual mutations frequently recurring in CLL patients. Thorough validations of the identified hits is ongoing, together with the analyses into the possible mechanisms behind these unique targets.

This research was partly financed by the grant MUNI/A/1330/2021, MUNI/IGA/1516/2020 and MUNI/A/1419/2021.

P628: HIGH THROUGHPUT IMMUNOGENETIC EVIDENCE FOR EXTENSIVE IN VIVO CLASS SWITCH RECOMBINATION EVENTS IN CLL

E. Sofou 1,2,* , A. Agathangelidis 3 , A.-M. Antoniadou 1 , E. Georgiou 2 , M. Papaioannou 4 , N. Pechlivanis 1 , F. Psomopoulos 1 , K. Stamatopoulos 1 , A. Chatzidimitriou 1

1 Institute of Applied Biosciences, Centre for Research and Technology Hellas; 2 Laboratory of Biological Chemistry, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki; 3 Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens; 4 1st Department of Internal Medicine, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

Background: In vivo Class Switch recombination (CSR) has previously been reported for a fraction of patients with chronic lymphocytic leukemia (CLL) expressing unmutated IGHV genes (U-CLL) and high mRNA levels of activation-induced cytidine deaminase compared to patients expressing mutated IGHV genes (M-CLL). This observation is certainly intriguing, however inherently limited due to the low-sensitive, Sanger-based approach used for the immunogenetic characterization.

Aims: Here, we sought to overcome the aforementioned limitation and obtain more insight into in vivo CSR in CLL using a highly-sensitive, next generation sequencing (NGS) methodology.

Methods: We studied 73 patients (48 U-CLL, 25 M-CLL) sampled at diagnosis. Case selection was based on the dominant expression of the IgM isotype, as determined by flow cytometry. IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified for the μ, γ, and α transcripts utilizing IGHV Leader and IGHC-specific primers. All obtained PCR products (n=215) were subjected to paired-end NGS. High-quality, productive IGHV-IGHD-IGHJ gene rearrangements (n=23,217,561; median= 97,932/sample) were annotated with IMGT/HighV-QUEST. Clonotypes were defined as rearrangement sequences expressing the same IGHV gene and identical complementarity-determining region 3 (VH CDR3) amino-acid (AA) sequence. Clonotype variants were defined as different nucleotide sequences clustered in the same clonotype.

Results: The IGH gene repertoire of μ transcripts was clearly monoclonal in all cases, showing a dominant CLL clonotype (DCC) with a median frequency of 93.5% for either U-CLL or M-CLL. Subsequently, the IGH gene repertoires of γ and α transcripts were scanned for the presence of the DCC. In U-CLL, 46/48 cases expressed switched transcripts with median frequencies of 14% (range 0.01-97.2%) and 21.3% (range 0.1-95.3%) for γ and α transcripts, respectively. In M-CLL, 24/25 patients expressed switched transcripts albeit at significantly lower levels than U-CLL (p=0.01) (median frequencies: 0.2% and 13.6%; ranges 0.03-94% and 0.04-91.2% for the γ and α transcripts, respectively). Interestingly, within U-CLL, cases belonging to stereotyped subset #1 (n=21) and its satellite subset #99 (n=2) exhibited significantly higher levels of switched transcripts (median frequencies: 37.9% and 37.6% for the γ and α transcripts, respectively) than all other cases (p=0.04 for γ transcripts, p=0.002 for α transcripts). Analysis of somatic hypermutation (SHM) in the DCC within the repertoires of μ, γ and α transcripts of U-CLL cases revealed the presence of shared SHMs (even among cases with 100% IGHV gene identity) but also distinct SHMs shared by the γ and α clonotypic transcripts but not the IgM, indicative of common selection pressure post-CSR. Interestingly, M-CLL patients displayed less intraclonal diversity in the γ and α clonotypic transcripts, despite the significant burden of SHM within the IgM DCC.

Summary/Conclusion: In conclusion, our data reveal that the B cell receptor IGH gene repertoire is highly dynamic and interconnected in both U-CLL and M-CLL, albeit considerably more pronounced in the former. This is especially the case for patients assigned to the clinically aggressive stereotyped subsets #1 and #99, prompting speculations regarding the precise nature of the respective affinity maturation process. Shared SHMs between clonotype variants only among clonotypic γ and α transcripts rather than the μ transcripts indicate that CSR likely precedes SHM, particularly in U-CLL.

P630: TARGETING NEGATIVE REGULATION OF MAPK SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA

M. Stumpf 1,* , V. Ecker 1 , P. Giansanti 2 , J. Lu 3 , T. Zenz 4 , I. Ringshausen 5 , B. Küster 6 , J. Ruland 1 , M. Buchner 1

1 TranslaTUM - Central Institute for Translational Cancer Research, Technische Universität München, München; 2 Bavarian Biomolecular Mass Spectrometry Center (BayBioMS), Technical University of Munich, Freising; 3 European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 4 Department of Medical Oncology and Hematology, University Hospital and University of Zurich, Zurich, Switzerland; 5 Wellcome Trust/MRC Cambridge Stem Cell Institute and Department of Haematology, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, United Kingdom; 6 Chair of Proteomics and Bioanalytics, Technical University of Munich (TUM), Freising, Germany

Background: The MAPK-ERK pathway is a key signaling cascade for cellular homeostasis. Following MAPK activation, feedback loops increase the production of pathway inhibitors, such as dual specificity phosphatases (DUSPs), to restrict MAPK signal. DUSPs can inhibit tumor growth by preventing ERK activation and may act as oncogenes by helping tumor cells in adjusting to elevated MAPK levels. Despite mounting evidence that MAPK signaling is critical for CLL formation and progression, little is known about the role of MAPK negative feedback via the DUSP family in CLL.

Aims: In this study, we investigated the significance of DUSP1 and DUSP6’s negative regulation of MAPK in CLL and evaluated their usage as possible therapeutic targets in drug resistant disease.

Methods: We examined the expression levels of DUSP1 and DUSP6 in primary CLL patients using RNAseq and immunoblot analysis. To determine their functional relevance, we employed the small molecule inhibitor BCI on primary murine and human (drug resistant) CLL cells in vitro and in vivo and generated genetic knockout MEC-1 CLL cells, to validate the inhibitor effects. In addition, we investigated the downstream effects of DUSP1/6 inhibition on CLL signaling events using global phosphoproteomics in primary CLL and MEC-1 cells. Finally, using particular inhibitors in in vitro experiments, we confirmed the importance of discovered pathway activations.

Results: To evaluate a potential role of negative feedback regulation of MAPK signaling in CLL, we first analyzed mRNA expression levels of DUSP1 and DUSP6 in CLL samples (n=210). Both negative regulators were expressed in all of the CLL samples studied, with DUSP6 levels being upregulated in cases with activating mutations in the KRAS or BRAF genes (12.1±17 RNAseq counts for BRAF/KRASwt cases vs. 46.7±19 RNAseq counts for BRAF/KRAS mutated cases). Furthermore, after microenvironmental stimulation, DUSP levels increased in CLL lymph nodes compared to peripheral blood (DUSP1: log2-fold change of 2.6, p=0.0094; DUSP6: log2-fold change of 1.8, p=2.2*10e-7; Herishanu et al, 2011) and stromal coculture. Notably, increased DUSP6 mRNA expression was linked to a poor clinical outcome, as evidenced by a shorter time to treatment (p=0.0109; Log-rank Test) and overall survival (p=0.0075; Log-rank Test). We next used the dual-specific DUSP1/6 inhibitor BCI to block the phosphatase activity and found that this causes cell death in CLL cells, but not or only mildly affected the survival of healthy donor B cells or other T- and B-cell lines. In the TCL1-driven CLL mouse model, administration of the DUSP1/6 inhibitor is effective in lowering CLL cell progression (>50% reduction of total CLL cell count in the spleen; n=5; p=0.0029). We generated genetic knockout lines of DUSP1 or DUSP6, which inhibited CLL cell expansion, to confirm the inhibitor’s specificity. We next used phosphoproteomics to explore the signaling consequences of DUSP1/6 inhibition in CLL, and discovered that DUSP1/6 inhibition caused acute stimulation of BCR/MAPK signaling. Subsequently, the DNA damage response (DDR) pathway is activated, resulting in the induction of apoptotic cell death. Finally, we tested whether DUSP1/6 inhibition is efficacious in drug-resistant CLL by using BCI to treat therapy-naive and ibrutinib refractory CLL samples, and we discovered that DUSP1/6 inhibition is extremely successful in targeting treatment-resistant CLL.

Summary/Conclusion: Conclusion: Based on these results, we propose transient DUSP1/6 inhibition as a novel and unexpected concept for eliminating drug-resistant CLL cells.

P631: A PCR-BASED ASSAY FOR IGLV3-21R110 SCREENING CONFIRMS ITS PROGNOSTIC VALUE IN AN INDEPENDENT COHORT OF 613 PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA.

C. Syrykh 1,2,* , N. Russiñol 1 , T. Baumann 3,4 , M. Kulis 1,5 , M. Alcoceba 5,6 , M. González 5,6 , E. Colado 7 , Á. R. Payer 7 , M. Aymerich 1,3,5 , M. J. Terol 8 , S. Ruiz-Gaspà 1 , A. López-Guillermo 1,3,5,9 , J. I. Martín-Subero 1,5,9,10 , D. Colomer 1,3,5,9 , J. Delgado 1,3,5,9 , E. Campo 1,3,5,9 , F. Nadeu 1,5

1 Institute d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 2 Department of Pathology, Institut Universitaire du Cancer, CHU de Toulouse, Toulouse, France; 3 Hospital Clinic of Barcelona, Barcelona; 4 Hospital Universitario 12 de Octubre; 5 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid; 6 Biología Molecular e Histocompatibilidad, IBSAL-Hospital Universitario, Centro de Investigación del Cáncer-IBMCC (USAL-CSIC), Salamanca; 7 Servicio de Hematología y Hemoterapia, Hospital Universitario Central de Asturias, Oviedo; 8 Servicio de Hematología, Hospital Clínico Universitario, INCLIVA, Universidad de Valencia, Valencia; 9 Universitat de Barcelona; 10 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Background: Recent studies have shown that expression of the immunoglobulin lambda light chain IGLV3-21 gene carrying a point mutation in the amino acid 110 (named IGLV3-21 R110 ) defines a subset of chronic lymphocytic leukemia (CLL) with an intermediate epigenetic subtype and poor prognosis (Maity et al. PNAS, 2020; Nadeu et al. Blood, 2021). This mutation is found in up to 6.5% of patients with CLL at diagnosis and up to 18% of cases enrolled in clinical trials. In addition, IGLV3-21 R110 seems to have prognostic value independently of the IGHV gene somatic hypermutation (SHM) status, stereotype subset #2, and disease stage, which makes it a promising prognostic biomarker. However, further studies on independent cohorts are needed to support its application in clinical practice.

Aims: To develop a rapid detection method of the IGLV3-21 R110 mutation and to assess its prognostic significance in a cohort of 613 previously unpublished CLL patients.

Methods: A multiplex IGLV3-21 R110 mutation-specific polymerase chain reaction (msPCR) assay was established in a cohort of 12 patients (including 6 IGLV3-21 R110 mutated) and validated in 159 cases (including 7 mutated; Nadeu et al. Blood, 2021). This msPCR assay was applied on an independent cohort of 613 CLL patients (575 CLL, 38 monoclonal B-cell lymphocytosis). Clinical analyses were performed for time to first treatment (TTFT) and overall survival (OS) from time of diagnosis considering only cases diagnosed as CLL.

Results: A msPCR approach was designed integrating two forward primers aligning to distinct regions of the IGLV3-21 gene and two R110-specific reverse primers matching the IGLJ1 and IGLJ2/3 genes, respectively. A third pair of primers targeting exon 9 of FBXW7 was used as an internal control. PCR conditions were set up on a cohort of 12 cases and subsequently validated on 159 previously published cases with 100% concordance. We then applied this msPCR assay to a cohort of 613 previously unpublished CLL patients and identified the R110 mutation in 22 (3.6%) cases, including 75% mutated IGHV (M-CLL) and 25% unmutated IGHV (U-CLL). Moreover, 84.6% of IGLV3-21 R110 cases carried non-stereotyped immunoglobulin genes while the remaining stereotyped IGLV3-21 R110 cases were subset #2. Ten of the IGLV3-21 R110 cases were classified according to the epigenetic subtypes and all belonged to the intermediate CLL (i-CLL) subgroup. Clinically, M-CLL patients carrying the IGLV3-21 R110 as well as i-CLL with IGLV3-21 R110 had a shorter TTFT compared to M-CLL and i-CLL lacking IGLV3-21 R110 , respectively, and similar to U-CLL/naïve-like CLL (p<0.005). Multivariable analyses including IGLV3-21 R110 , disease stage, and IGHV gene SHM status or epigenetic subtypes confirmed the independent prognostic value of IGLV3-21 R110 on TTFT (p<0.005). IGLV3-21 R110 had also a prognostic impact on OS since M-CLL carrying the IGLV3-21 R110 had shorter OS similar to that of U-CLL (Figure). Finally, we combined these novel data with those of our previously published 489 CLL cohort (Nadeu et al. Blood, 2021) (N total=1102). Clinical analyses of the whole cohort confirmed the independent prognostic value of IGLV3-21 R110 .

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Summary/Conclusion: We have developed a reliable, easy-to-use msPCR assay suitable for IGLV3-21 R110 screening in large cohorts. By applying this msPCR on a cohort of 613 CLL patients, our results corroborate the relevance of IGLV3-21 R110 testing to improve the risk stratification of CLL patients in the clinical practice, especially within M-CLL and i-CLL subtypes.

P632: ORAL AND GUT MICROBIAL DIVERSITY CORRELATES WITH PROGNOSTIC FEATURES IN CHRONIC LYMPHOCYTIC LEUKEMIA

M. Szelest 1,* , M. Paziewska 1 , M. Kiełbus 1 , M. Masternak 1,2 , J. Zaleska 1 , E. Wawrzyniak 3 , E. Kalicińska 4 , P. Jabłonowska 4 , T. Wróbel 4 , A. Wolska-Washer 5,6 , J. Z. Błoński 3,6 , K. Giannopoulos 1,2

1 Department of Experimental Hematooncology, Medical University of Lublin; 2 Department of Hematology, St John’s Cancer Centre, Lublin; 3 Department of Hematology, Medical University of Lodz, Lodz; 4 Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Wroclaw; 5 Department of Experimental Hematology, Medical University of Lodz; 6 Copernicus Memorial Hospital, Lodz, Poland

Background: For the last years it has been speculated that immunologic and inflammatory factors, including antigen stimulation, could be involved in the pathogenesis of chronic lymphocytic leukemia (CLL). With increasing evidence that the activation of cellular proinflammatory signaling pathways driven by somatic mutations and an increased release of proinflammatory cytokines is associated with CLL development, it seems relevant to investigate the role of chronic inflammation in the pathogenesis of this disease. Recent studies have revealed that the intestinal microbiota could modulate the activity of immune system through creating an imbalance between cell proliferation and apoptosis. Thus, the relationship of microbial dysbiosis and specific taxon abundances with CLL biology and clinical outcome should be examined.

Aims: As it was reported that the activity of human microbiome is involved in cancer development and host response against anticancer therapy, the aim of this study was to investigate the stool and oral microbiome alterations in CLL patients with respect to selected prognostic and predictive markers.

Methods: The study included 92 patients with untreated CLL. Microbiota composition of 85 stool samples and 76 oral samples from these cases were determined by 16S rRNA next-generation sequencing and analyzed with respect to the Binet stage, IGHV and TP53 mutation status, CD38 and ZAP-70 expression, and cytogenetic aberrations. The species richness and evenness were analyzed using Shannon and Chao1 indexes, respectively. The DADA2 tool was used to classify sequences into appropriate taxonomic groups. The differences in taxonomic profiles and species diversity in oral and stool samples with distinct prognostic features were investigated using the Wilcoxon or Kruskall-Wallis tests.

Results: Alpha-diversity analysis indicates that there were no significant differences in richness and evenness between probes with distinct CLL features in both fecal and oral samples. However, at phylum level, an increase in Proteobacteria , a common feature of gut dysbiosis, was observed in stool samples from patients with stage Binet C compared to stage Binet A (p=0.042), and in oral samples with del11q compared to samples with no del11q (p=0.021). The abundance of Bacteroidota was higher in fecal samples collected from patients with stage Binet A in comparison to stage Binet B (p=0.051) and C (p=0.009). Moreover, Bacteroidota was relatively more abundant in oral samples from patients with stage Binet A in comparison to stage Binet C (p=0.008). Bacteroidota was significantly less abundant in stool samples from patients with unmutated IGHV compared to samples with mutated IGHV (p=0.029). Stool samples from patients with CD38 + exhibited decreased abundance of Bacteroidota and increased abundance of Firmicutes in comparison to CD38 - cases (p=0.031 and p=0.022, respectively). Furthermore, fecal samples from patients with del6q showed a higher abundance of Firmicutes and lower abundance of Bacteroidota compared to samples with no del6q (p=0.006 and p=0.018, respectively). Additionally, a relative increase in Actinobacteriota and Firmicutes was found in oral samples with tri12 in comparison to samples with no tri12 (p=0.039 and p=0.016, respectively).

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Summary/Conclusion: Our findings provide new insights into oral and gut microbial diversity which is related to distinct prognostic features in CLL and might point to the inflammatory-related modulation of the clinical course of disease.

P633: ACTIVE CHROMATIN REGULATORY LANDSCAPE OF STEREOTYPED SUBSETS IN CHRONIC LYMPHOCYTIC LEUKEMIA REVEALS A DISTINCTIVE SIGNATURE IN SUBSET #8

M. Tsagiopoulou 1,2,* , V. Chapaprieta 3 , N. Russiñol 3 , N. Pechlivanis 1 , N. Papakonstantinou 1 , N. Stavroyianni 4 , F. Psomopoulos 1 , E. Campo 3 , K. Stamatopoulos 1 , J. I. Martin-Subero 3

1 Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 2 CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain; 3 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain; 4 Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece

Background: Chronic lymphocytic leukemia (CLL) patients with stereotyped B cell receptors have been characterized from the perspective of genetic and DNA methylation alterations. Recent studies have characterized the chromatin landscape of non-stereotyped CLLs and normal B cells (Beekman, et al. Nat Med. 2018).

Aims: To characterize the active chromatin regulatory landscape of 4 major stereotyped subsets as compared to IGHV status-matched non-stereotyped CLLs.

Methods: We used chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) with an antibody for the H3K27ac (a bona fide histone mark for regulatory element activation) in sorted CLL cells from 15 normal B cell subpopulations and 44 CLL, including 18 cases from stereotyped subsets #1, #2, #4, and #8. To understand their functional impact, we integrated our data with 16 RNA-seq data considering the 3D chromatin configuration (Hi-C data: GM12878 and a representative U-CLL case) (Figure 1)

Results: Unsupervised principal component analysis (PCA) of H3K27ac data revealed different profiles in CLL as a whole, regardless of stereotypy status, vs normal B cells (PC1, 25%). Of note, PC2 (14%) revealed a clear separation between subset #8, a highly aggressive CLL subtype with the highest risk of Richter’s transformation amongst all CLL, vs other CLLs or normal B cells, suggesting that subset #8 may have a specific acetylation pattern. Indeed, supervised differential acetylation analysis (FDR<0.01) uncovered a signature of 903 Differentially Acetylated Regions (DAR) in subset #8 vs U-CLL (Figure 1) and no differences for the other subsets. This signature was unrelated to +12, highly prevalent (~60%) in subset #8, or IgG expression, a hallmark of subset #8. Hierarchical clustering analysis of this signature revealed 6 clusters of regions with different chromatin dynamics highlighting the de novo active cluster1 (n=197 regions) in subset #8 vs other CLLs or normal B cells. These differentially acetylated regions (DARs) were enriched (FDR<0.05) in binding sites of several differentially expressed TFs in #8 vs U-CLL, e.g. members of SOX, GATA, and CEBP families as well as MEF2A and HNF1A, which showed the highest enrichment. Next, we used the topologically associating domains (TAD) resulting from Hi-C data and RNAseq data to identify the potential target genes of the 197 de novo DARs in subset #8. We identified the target genes in 78/197 (39.6%) DARs that were linked to 112 genes, as some active regulatory regions were associated with more than one gene. Among them, the DAR located in chr9:137085994-137086793 bp was related to the expression levels of 8 genes ( LCNL1, CLIC3, FBXW5, AJM1, MAMDC4, CCDC183, TRAF2 and an un-annotated transcript) within the same TAD. This region also contained 6 additional H3K27ac peaks with higher signal in subset #8, though not reaching significance. This finding suggests deregulation of a broad region in chromosome 9 leading to the coordinated upregulation of 8 transcripts. Concerning the activated genes, TRAF2 is a remarkable example since TRAF family members (TRAF6, -4, -1) are found overexpressed in U-CLL and here we report an active region in subset #8 leading to TRAF2 overexpression.

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Summary/Conclusion: We report a subset #8-specific chromatin acetylation signature containing de novo activated regions, linked to the upregulation of target genes, in some cases including blocks of adjacent genes. This study further supports the distinct biological nature of subset #8 and may help to understand the aggressive clinical behavior of this distrinctive CLL variant.

P634: MHC-BASED LARGE-SCALE SCREENING FOR ANTI-TUMOR T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA REVEALS CD8+ T CELLS WITH SPECIFICITY AGAINST THE CLONOTYPIC B-CELL RECEPTOR IMMUNOGLOBULIN

A. Vardi 1,2,* , Y. Basavaraju 3 , A. Agathangelidis 2,4 , N. Wulff Pedersen 3 , M. Karipidoy 2 , A.-L. Schaap-Johansen 3 , A. Fylaktou 5 , N. Stavroyianni 1 , M. Iskas 1 , A. Anagnostopoulos 1 , A. Chatzidimitriou 2 , P. Marcatili 3 , S. R. Hadrup 3 , K. Stamatopoulos 2

1 Hematology Department & HCT Unit, G.Papanikolaou Hospital; 2 Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 3 Department of Health Technology, Technical University of Denmark, Lyngby, Denmark; 4 Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens; 5 Department of Immunology, National Peripheral Histocompatibility Center, Thessaloniki, Greece

Background: Chronic lymphocytic leukemia (CLL) is currently incurable, indicating a need for novel strategies towards disease eradication, including reinvigoration of anti-tumor immune responses. T cells in CLL appear selected by restricted antigens, with evidence suggesting that the selecting epitopes may lie within the clonotypic B-cell receptor immunoglobulins (BcR IG).

Aims: We previously performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. Here, we performed major histocompatibility complex (MHC)-based large-scale screening to identify autologous CD8 + T cells recognizing the predicted neoepitopes.

Methods: We evaluated 653 peptides derived from the clonotypic BcR IG of 25 CLL patients (median 21 predicted neoepitopes/patient, across 13 MHC-I alleles). Considering the MHC-I typing of each patient, we constructed patient-specific peptide-MHC multimers labeled with a unique DNA barcode plus a fluorochrome (PE). We generated MHC-specific multimer libraries which we then mixed with respective autologous T-cell enriched peripheral blood mononuclear cells (PBMCs). Duplicate samples/patient were analyzed. In addition, known viral peptide-MHC multimers labeled with a different fluorochrome (APC) as well as three MHC-matched healthy donor PBMCs were used as controls. PE- and APC-positive multimer-binding CD8 + T cells were sorted for each cell sample; subsequently, DNA barcode amplification and sequencing was performed. Sequencing data were processed (Barracoda software) to obtain the number of clonally reduced barcode reads assigned to a given sample and peptide-MHC specificity. The number of clonally reduced reads for a given pMHC specificity was used to estimate the frequency of T cells specific for a given epitope, based on the average number of T cell receptor–MHC multimer interactions detected in the total MHC multimer-binding T cell pool in a given cell sample. Peptides with a log fold change >2 were considered as significant [log fold change: (number of DNA barcode reads associated with a specific pMHC)/3x (number of total barcode reads derived from the same sample)].

Results: Overall, 3 peptide-MHC multimers were recognized by CD8 + T cells: (i) VTVADTAVYY (peptide A) and (ii) INLNPSLKRR (peptide B), both within the context of the A03*01 allele, and (iii) YSFTSYWINW (peptide C) within the context of the A24*02 allele. Peptide A derived from the IGHV4-34 FR3 region of a somatically hypermutated clonotypic BcR IG, containing a single A to V somatic hypermutation (SHM) at position 96. Peptide B derived from the IGHV4-39 CDR2-FR3 junction of a somatically hypermutated clonotypic BcR IG, containing 3 SHMs (T to I at position 65, Y to L at position 67 and S to R at position 74). Peptide C derived from the IGHV5-10-1 CDR1-FR2 junction of a clonotypic BcR IG assigned to stereotyped subset #1, containing the sole SHM of the IG (S to N at position 40). The immunogenicity of these peptides was further corroborated by the fact that they were recognized not only by the autologous T cells of the patient from whom the peptide derived, but also by T cells of other patients as well as a healthy donor sharing the respective MHC allele.

Summary/Conclusion: In conclusion, we offer proof of concept that the targeted SHM which shapes the CLL BcR IG repertoire may produce idiotypic targets for T cell-based therapy or peptide vaccine design. Characterization of the epitope-binding T cells is currently underway by our group, aiming to provide further insight on how these cells could be recruited into effective anti-tumor responses.

P635: TARGETING THE ONE-CARBON METABOLISM AS NOVEL THERAPEUTIC STRATEGY IN CHRONIC LYMPHOCYTIC LEUKEMIA

E. Viry 1,* , A. Largeot 1 , S. Gonder 1 , S. Chemlal 1 , N. Kiweler 2 , J. Meiser 2 , T. Helleday 3 , E. Moussay 1 , J. Paggetti 1

1 Tumor Stroma Interactions, Department of Cancer Research; 2 Cancer Metabolism Group, Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg; 3 Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institute, Solna, Sweden

Background: Chronic Lymphocytic Leukemia (CLL), the most common type of leukemia in adults, is characterized by the clonal expansion of CD5 + CD19 + B cells. CLL progression is highly dependent on complex interactions with non-malignant cells of the tumor microenvironment. Despite great advance in the standard of care in the last decades, there are still unmet medical needs (long-life treatment, resistance…). Altered cellular metabolism has emerged as a hallmark of cancer by sustaining the uncontrolled growth of cancer cells. The one-carbon pathway is a major driver for tumor proliferation, providing building blocks for biosynthesis of nucleotides through pyrimidines (dTTP) and purines synthesis, redox metabolism (GSH), and energy balance by suppling ATP and NADPH to cells (A ). Understanding the metabolic reprogramming occurring in cancer, notably in CLL, may provide insights to support the development of novel therapies.

Aims: We aim to get insights into one-carbon metabolism regulation in CLL, identify new therapeutic targets and find new treatment options for CLL patients.

Methods: Through a collaboration with a biotech company that develops inhibitors of the one-carbon metabolism, we tested new MTHFD1/2 inhibitors, which we believe, are promising in the context of CLL. The cytotoxic activity of the inhibitors was evaluated on a panel of murine and human CLL cell lines and primary cells, and also on others B cell malignancies such as Mantle Cell Lymphoma (MCL), Diffuse Large B-Cell Lymphoma (DLBCL), and Multiple Myeloma (MM). The molecular mechanisms sustaining the cytotoxic activity were evaluated by performing metabolic tracing, rescue experiments and CRISPR/Cas9 KO.

Results: We showed a strong expression of both MTHFD1 and 2 in all the cancer cell lines tested and in stimulated primary human CLL cells. A higher expression of both enzymes was also observed in primary CLL cells, isolated from the Eµ-TCL1 murine model of CLL compared to normal B cells. In vitro treatment with the inhibitors efficiently reduced cell viability of CLL cell lines and primary cells, as for MCL and DLBCL cell lines, at low nanomolar dose while no effect was observed on MM cell lines ( B ). This effect is associated with a blockade of cell proliferation. Using 13 C-serine isotope tracing, we could showed that the inhibitors significantly reduced ATP production from serine in CLL cells, probably resulting in altered nucleotides biosynthesis. By performing rescue experiments with thymidine or hypoxanthine, we confirmed that the cytotoxic effect of the inhibitors on CLL cells is mediated through a defect in pyrimidine synthesis ( C ). Interestingly, we demonstrated that the resistant MM cell lines overexpressed the SHMT1 enzyme, and could be sensitized to MTHFD1/2 inhibition by combination therapy with SHMT1/2 inhibitor ( D ). Taken together these results suggest that SHMT1 may be a key player in the response to MTHFD1/2 inhibition. Furthermore, we have previously reported that CLL induces a dramatic remodeling of tumor microenvironment, notably leading to the accumulation of highly immunosuppressive Tregs (Wierz et al. , Blood, 2018). We showed that both enzymes are overexpressed in those highly immunosuppressive Tregs, and that MTHD1/2 inhibitors are able to reduce the viability of ex vivo polarized Tregs.

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Summary/Conclusion: MTHFD1/2 inhibitors display a high cytotoxic activity in CLL and other B-cell malignancies by impairing cell proliferation through a defect in pyrimidine synthesis. These inhibitors also exhibit cytotoxic effect on activated Treg cells ex vivo , reinforcing its therapeutic potential for CLL treatment.

P636: REAL WORLD OUTCOME WITH IBRUTINIB IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA FROM THE GERMAN REALITY STUDY

M. Welslau 1,* , R. Schlag 2 , B. Heinrich 3 , L. Vornholz 4 , S. Buchholz 4 , B. Marquard 4 , S. Dörfel 5 , G. Anke 6

1 Oncology and Hematology, MVZ at Medical Clinic Aschaffenburg-Alzenau, Aschaffenburg; 2 Oncology and Hematology, Oncological Practice, Würzburg; 3 Oncology and Hematology, Oncological Practice, Augsburg; 4 Medical & Scientific Affairs, Janssen-Cilag GmbH, Neuss; 5 Oncology and Hematology, Onco Center Dresden, Dresden; 6 Hematological and Oncological Diseases, MVZ Potsdam, Potsdam, Germany

Background: Chronic lymphocytic leukemia (CLL) is one of the most common forms of leukemia, characterized by abnormal survival and proliferation of mature B cells in peripheral blood, bone marrow, and lymph nodes. Ibrutinib is a once-daily Bruton’s tyrosine kinase inhibitor (BTKi) with a significant benefit in both progression free survival (PFS) and overall survival (OS) demonstrated in multiple phase 3 trials for patients (pts) with previously untreated (1L) or relapsed/refractory (R/R) CLL. In 2014, ibrutinib was approved by the European Medicines Agency and made available to CLL pts in Germany. Prior to the REALITY study, limited data were available on the effectiveness of ibrutinib-treated pts with CLL in routine clinical practice in Germany.

Aims: REALITY was a prospective, multicenter, non-interventional study to evaluate the effectiveness of ibrutinib as the 1 st , 2 nd , or ≥3 rd line of treatment (cohorts C1, C2, and C3 respectively) in pts with CLL during the first 2 years of observation period in a real-world setting.

Methods: 302 pts were enrolled in the three cohorts (C1: 104 pts; C2: 90 pts; C3: 108 pts) and treated in 57 study sites in Germany from January 2017 to July 2021. Written informed consent was obtained from all pts. Key study endpoints include retention rate (defined as ratio of pts on ibrutinib treatment to the number of pts at risk), start of next therapy, PFS, OS, and safety. The results of this study are descriptive, and no formal hypotheses was pre-specified.

Results: Median age at time of ibrutinib treatment initiation was 74.0 years for the 1L pts in C1, and 72.5 and 73.0 years in the R/R cohorts of C2 and C3, respectively.

In cohort C1 (1L), pts with genetic high risk features del(17p) and TP53 mutation were more frequent compared to C2 and C3. In C1 del(17p) was reported in 34/51 (66.7%) pts and TP53 mutations in 31/49 (63.3%) pts. In the R/R cohorts C2 and C3 del(17p) was reported in 11/57 (19.3%) pts and 13/65 (20.0%) pts, respectively. TP53 mutations were reported in 14/48 (29.2%) pts in C2 and 21/40 (52.5%) pts in C3.

The primary study endpoint was retention rate, 77.9% pts continued to be on treatment after 1 year in C1 (1L). In C2 and C3 (R/R), the 1-year retention rates were 74.4% and 58.3% respectively.

In C1, with median follow-up of 30.6 months, median PFS was not reached (NR). The 1-year PFS and OS in the C1 cohort (1L) were 91.3% and 96.2%, respectively. Only 8 pts initiated next line treatment in C1 after 1 year.

In C2, with a median follow-up of 31.5 months, median PFS was NR. The 1-year PFS and OS were 85.6% and 93.3%, respectively. 11 pts initiated next line treatment in C2 after 1 year.

In C3 median follow-up was 30.9 months and median PFS was NR. The 1-year PFS and OS were 83.3% and 93.5%, respectively. 26 pts initiated next line treatment in C3 after 1 year.

Detailed results after 1 and 2 years of observation are summarized in Table 1.

No new safety signals for ibrutinib were observed.

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Summary/Conclusion: This first, comprehensive data from clinical practice of ibrutinib-treated pts with CLL in Germany showed that ibrutinib is a highly effective treatment option, especially as a 1 st line treatment. The effectiveness and safety profile were consistent with clinical trials.

P637: FRONTLINE THERAPY CLL WITHOUT 17P DEL/P53 ABERRATIONS: SYSTEMATIC REVIEW AND BAYESIAN NETWORK META-ANALYSIS

G. Caridà 1 , D. Ciliberto 1 , G. Pelaia 2 , N. Staropoli 1 , M. A. Siciliano 3 , N. D. Calandruccio 1 , R. Toscano 4 , M. Rossi 5,6,*

1 Oncology Unit; 2 Pediatric Unit, “Magna Graecia” University, Catanzaro; 3 Oncology Unit, “S. Giovanni di Dio” Hospital, Crotone; 4 Oncology Unit, AO “S. Francesco di Paola”, Paola (CS); 5 Department of Experimental and Clinical Medicine, Magna Græcia University, Catanzaro, Italy, “Magna Graecia” University; 6 Hematology Unit, AO “Pugliese Ciaccio”, Catanzaro, Italy

Background: Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia, with a median age at diagnosis of 72 years. IgVh unmutated CLL without 17p del/p53 aberrations (UN-only CLL) still carry a poorer prognosis as compared to IgVh mutated (MU-only) cases. Although many treatment options are currently available for the front-line therapy, the best choice and treatment sequencing stratified on efficacy and tolerability for UN/MU-only CLL remains to be defined.

Aims: We performed a systematic review and Bayesian network meta-analysis to define best frontline therapy and treatment sequence for UN/MU-only CLL.

Methods: Analysis of the literature was performed through the main databases. Due to different therapeutic approaches to front-line therapy of CLL, we chose to conduct a Bayesian Network Meta-Analysis (NMA), allowing to rank (from best to worst) multiple treatments in a single analysis. We generated a rank for Progression Free Survival (PFS), Overall Response Rate (ORR), Minimal Residual Disease (MRD), assessed by flow cytometry on peripheral blood, and tolerability. PFS was evaluated in unselected population for IgVh, and in UN/MU-only CLL patients.

Results: Fifteen clinical trials, with a total of 7958 patients, have been included, while 16 different treatments were considered to build four different network meta-analyses to identify the best treatment in PFS, ORR, MRD, and tolerability: Acalabrutinib (ACA); ACA + Obinutuzumab (ACA-O); Alemtuzumab; Bendamustine + Rituximab; Chlorambucil; Chlorambucil + O; Chlorambucil + Ofatumumab; Chlorambucil + Rituximab; Fludarabine + Cyclophosphamide + Rituximab; Fludarabine + Cyclophosphamide; Ibrutinib (IBR); IBR + O; IBR + Rituximab; Lenalidomide, Venetoclax + IBR (VEN-IBR), Venetoclax + Obinutuzumab + Ibrutinib (VEN-O-IBR), Venetoclax + O (VEN-O). ACA-O scored the best in PFS in an unselected population (SUCRA 100%, probable the best 99.9%), UN- and MU- only CLL patients. Our analysis showed that VEN-O ranked the first for ORR (SUCRA value 100%, probably the best 100%). VEN-O ranked the first also for MRD (SUCRA 90.4%, probably best 36.2%) versus the second VEN-O-IBR (SUCRA 84.0%, probably best 35.9); in the direct comparison, the difference between VEN-O and VEN-O-IBR is not statistically significant (Odds Ratio 1.44, CrI 0.00,563.57). Finally, ACA monotherapy is the most tolerable therapy (SUCRA value 100%, probably the best 100%), whereas IBR-O turned to be the most toxic treatment (SUCRA value 0%, probably the best 1%). Interestingly, IBR monotherapy ranked the 9th in the network, whereas all combination regimens including Obinutuzumab carried a high risk of toxicity.

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Summary/Conclusion: To our knowledge, this is the first network meta-analysis that compares at the same time PFS, tolerability, ORR, and MRD. Our PFS analysis showed that ACA-O was the best treatment in both MU/UN-only patients. Furthermore, ACA turned out to be the most tolerated regimen, whereas the BTKis-O based regimens significantly carried a high toxicity profile as compared to BTKis alone, counterbalancing the advantage of O-addition in terms of ORRs, where O-based regimens achieved the higher rank, when combined either with VEN or BTKis. ACA-O was the best treatment for PFS, although with a worse toxicity profile as compared to ACA monotherapy. VEN-O scores as the best in ORR and MRD. The combination ACA-O has the highest probability to be the best treatment for prolonging the PFS in untreated, UN/MU-only CLL patients. ACA monotherapy is a valuable alternative considering both the safety and efficacy profile.

P638: THE RISK OF SECOND PRIMARY MALIGNANCIES AND CAUSE-SPECIFIC MORTALITY AMONG PATIENTS WITH T-CELL PROLYMPHOCYTIC LEUKEMIA IN THE UNITED STATES: A POPULATION-BASED STUDY.

K. Chamarti 1,* , M. Mannem 1 , B. Bakhati 1

1 Department of Internal Medicine, Texas Tech University Health Sciences Center at Permian Basin, Odessa, United States of America

Background: T cell prolymphocytic leukemia (T-PLL) is a rare lymphoid malignancy, compromising 2% of mature lymphocytic leukemias. The T-PLL follows an aggressive clinical course with median survival of 7.5 months with conventional chemotherpay. In the era of targeted therapy with anti-CD52 antibody for patients with T-PLL, there is data lacking on the spectrum of second primary malignancies (SPMs) and cause-specific mortality.

Aims: In this current study we aim to identify the risk of SPM and cause specific mortality in patients with T-PLL in the era of targeted therapies with anti-CD52 antibodies using a national registry from the United States.

Methods: The incidence of SPMs and cause-specific mortality among two-month survivors of T-PLL, was estimated using the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER)-13 registries. We included patients from 1992-2018 with ICD-O-3 code 9834/3 from the registry. We estimated the risk of SPMs using standardized incidence ratios (SIRs) and cause-specific mortality by standardized mortality ratios (SMRs) and absolute excess risk (AER)/10,000 population.

Results: We included a total of 314 patients with T-PLL in this study. The median age at diagnosis was 68.5 years (range 1-85 years). The median follow-up duration was 12 months (range 0-196 months). A total of 13 (4%) patients developed 14 SPMs at the last follow-up. The SPM risk for the cohort was significantly elevated (SIR 2.54, 95% confidence interval [CI] 1.39-4.27) compared with general population. The median latency period for SPM development was 28 months (range 8-108 months). The SIR was significant among males (SIR 2.96, 95% CI 1.42-5.44), but not in females (SIR 1.88, 95% CI 0.51-4.82). The SIR according to age groups was significant in patients with age < 60 years (SIR 3.84, 95% CI 1.25-8.97), but not in age > 60 years (SIR 2.14, 95% CI 0.98-4.06). Specific SPMs with the highest risk included nasopharyngeal cancers (SIR 200.87, 95% CI 5.09-1,119.20) and multiple myeloma (SIR 33.39, 95% CI 6.89-97.58). At last follow-up, 155 (49%) patients had died, of which 89% were due to malignant neoplasms. There was statistically significant risk of mortality due to all malignant cancers combined (SMR 58.35, 95% CI 49.03-68.94), largely due to mortality from lymphocytic leukemia (SMR 2587, 95% CI 2021-3264, AER 1757), non-Hodgkin lymphoma (SMR 100, 95% CI 45.88-190.45, AER 220), stomach and duodenal ulcers (SMR 73.74, 95% CI 1.87-410.84, AER 24.43), uterine cancer (SMR 66.55, 95% CI 1.68-370.78, AER 24.39), and pneumonia and influenza (SMR 11.88, 95% CI 2.45-34.71, AER 68.04). The risk of death from lymphocytic leukemia was highest within the first year of diagnosis of T-PLL and continued to decline slowly thereafter, although it remained statistically significantly elevated more than other causes of death (SMR 1yr=3405, AER 2666; SMR 1-5 yrs=2623, AER 1715; SMR 5-10 yrs=1015, AER 516).

Summary/Conclusion: Our study reveals that the risk of developing SPMs remains elevated in patients with T-PLL compared to the general population. Additionally, men are at an increased risk for SPMs compared to women. Despite therapeutic advances in T-PLL, hematologic neoplasms (including lymphocytic leukemia) have remained the leading cause of death in the last three decades. Further studies are needed to better define the healthcare needs of T-PLL patients beyond initial treatment to reduce the burden of mortality from T-PLL and other SPMs.

P639: OUTCOMES FOR PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA PREVIOUSLY TREATED WITH A COVALENT BTK AND BCL2 INHIBITOR IN THE UNITED STATES: A REAL-WORLD DATABASE STUDY

A. R. Mato 1,* , Y. Chen 2 , A. C. Girvan 2 , L. M. Hess 2 , L. Bowman 2 , P. Abada 2 , H. Konig 2 , R. A. Walgren 2

1 Memorial Sloan Kettering Cancer Center, New York; 2 Eli Lilly and Company, Indianapolis, United States of America

Background: Covalent BTK inhibitors (cBTK) and B-cell lymphoma 2 inhibitors (BCL2i) have transformed outcomes in patients with chronic lymphocytic leukemia (CLL). Although these agents are highly effective, they are not curative, and a proportion of patients will require additional therapy. No prospective randomized clinical trials have evaluated the efficacy of therapies following treatment with these two modern drug classes.

Aims: We therefore evaluated treatment outcomes in patients with CLL following treatment with at least these two drug classes using a real-world dataset from the United States (US).

Methods: De-identified patient-level electronic medical record data linked to claims data in the US from ConcertAI were utilized for this study. Eligible patients were ≥18 years old, diagnosed with CLL between December 2011 and October 2020. To ensure a minimum of 1 year follow-up for all patients, data were available through July 2021. Patients were required to have completed treatment with at least one cBTKi and a BCL2i during the 1st-3rd lines of therapy. Time-to-event analyses utilized the Kaplan-Meier method.

Results: 382 patients (median age 73.4 years, male 64.4%) met eligibility criteria. Of these, 228 (59.7%) received subsequent therapy following BTK inhibitor and BCL2 inhibitor regimens, while the remaining 154 (40.3%) did not receive further treatment. Among those who received further treatment, the most common therapies included retreatment with a venetoclax-containing regimen (n=112, 49.1%). Of note, only 2.6% (n=6) of patients received immediate post-dual failure therapy with a PI3K inhibitor in this setting. Among those who received subsequent therapy (n=228), the median time from the start of the immediate post-cBTKi/BCL2i therapy to discontinuation or death was 5.5 months (95% CI: 3.5-6.9). For the entire cohort of patients with CLL who discontinued BTKi and BCL2i regimens (n=382), the median time from the end of the last of BTKi/BCL2i therapy to discontinuation of subsequent therapy or death was 5.6 months (95% CI: 4.3-6.0).

Summary/Conclusion: Patients with CLL who have been previously treated with both a cBTKi and BCL2i experience poor outcomes as observed by duration of treatment and time to treatment discontinuation of subsequent therapy. This population represents a growing area of unmet medical need for patients with CLL.

P640: PIRTOBRUTINIB, A HIGHLY SELECTIVE, NON-COVALENT (REVERSIBLE) BTK INHIBITOR IN COMBINATION WITH VENETOCLAX±RITUXIMAB IN RELAPSED/REFRACTORY CLL: RESULTS FROM THE BRUIN PHASE 1B STUDY

L. E. Roeker 1,* , A. R. Mato 1 , J. R. Brown 2 , C. C. Coombs 3 , N. N. Shah 4 , W. G. Wierda 5 , M. R. Patel 6 , K. L. Lewis 7 , M. Balbas 8 , J. Zhao 8 , N. C. Ku 8 , J. F. Kherani 8 , D. E. Tsai 8 , B. Nair 8 , C. Y. Cheah 7

1 Memorial Sloan Kettering Cancer Center, New York; 2 Dana-Farber Cancer Institute and Harvard Medical School, Boston; 3 University of North Carolina at Chapel Hill, Chapel Hill; 4 Medical College of Wisconsin, Milwaukee; 5 MD Anderson Cancer Center, Houston; 6 Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota, United States of America; 7 Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 8 Loxo Oncology at Lilly, Stamford, United States of America

Background: Covalent Bruton tyrosine kinase inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia (CLL), but patients (pts) discontinue these agents due to resistance or intolerance. Pirtobrutinib is an oral, highly selective, non-covalent (reversible) BTKi with promising efficacy and safety in heavily pretreated relapsed/refractory (R/R) CLL pts, regardless of BTK C481 mutation status. Recent clinical studies reported on the safety and efficacy of time-limited venetoclax and covalent BTKi combination regimens.

Aims: We evaluated the safety and efficacy of pirtobrutinib combined with venetoclax ± rituximab in pts with R/R CLL.

Methods: BRUIN is a phase 1/2 global, multicenter study ( NCT03740529 ) of pirtobrutinib in pts with advanced B-cell malignancies. The phase 1b portion evaluated the safety of pirtobrutinib at a continuous dose of 200 mg QD from Cycle 1, Day 1 plus venetoclax starting on Cycle 2, Day 1 with a standard 5-week dose ramp to 400 mg QD (PV) and PV plus rituximab at 375 mg/m2 on Cycle 1, Day 1, then 500 mg/m2 on Day 1 of Cycles 2-6 (PVR). Prior BTKi was allowed; prior venetoclax was not permitted. Objectives included safety and overall response rate (ORR) of each combination.

Results: As of 27 SEP 2021, 15 pts received PV and 10 pts received PVR. Median age was 66 years (range, 39-78). Median prior lines of therapy was 2 (range, 1-4). The majority of pts in both cohorts had received prior chemotherapy (56%, n=14), CD20 monoclonal antibody (72%, n=18), and/or covalent BTKi (68%, n=17). No dose-limiting toxicities were reported. Safety profiles were generally similar across both cohorts. The most common treatment-emergent adverse events (TEAE) of any grade, regardless of attribution, were neutrophil count decrease (36%), nausea (32%), fatigue (32%), diarrhea (28%), and constipation (24%). The only Grade ≥3 TEAE to occur in more than 2 pts was neutrophil count decrease (36%, n=9). One pt experienced Grade 4 clinical tumor lysis syndrome with resultant acute kidney injury during venetoclax dose escalation, which resolved with supportive measures. No pts discontinued treatment due to AEs. For the 22 pts with efficacy data available as of 03 NOV 2021, median duration of follow-up was 9 months (range, 3.9-15) and the ORR was 95.5% (95% CI, 77-100). All responding pts except 1 remain on therapy (PVR responder discontinued due to death unrelated to study treatment). As all responses were ongoing and early, and MRD analysis was not yet performed.

Summary/Conclusion: Pirtobrutinib combined with venetoclax ± rituximab was well tolerated and had a safety profile consistent with known drug class findings and no clear additive toxicities in pts with R/R CLL. Early results demonstrate promising efficacy with combination therapy.

P641: RETREATMENT WITH VENETOCLAX AFTER VENETOCLAX, OBINUTUZUMAB +/- IBRUTINIB: POOLED ANALYSIS OF 13 PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) TREATED IN GCLLSG TRIALS

P. Cramer 1,* , M. Fürstenau 1 , A. Giza 1 , S. Robrecht 1 , E. Tausch 2 , C. Schneider 2 , C.-M. Wendtner 3 , M. Hoechstetter 3 , J. Schetelig 4 , S. Böttcher 5 , P. Dreger 6 , A.-M. Fink 1 , P. Langerbeins 1 , O. Al-Sawaf 1 , K. Fischer 1 , S. Stilgenbauer 2 , B. Eichhorst 1 , M. Hallek 1

1 Department I of Internal Medicine and German CLL Study Group; Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), University of Cologne, Cologne; 2 Department III of Internal Medicine, University Hospital Ulm, Ulm; 3 Department of Hematology, Oncology, Immunology, Palliative Care, Infectious Diseases and Tropical Medicine, Klinikum Schwabing, Munich; 4 Department I of Internal Medicine, University Hospital Carl Gustav Carus, Dresden; 5 Department III of Internal Medicine, University Hospital Rostock, Rostock; 6 Department V of Internal Medicine, University Hospital Heidelberg, Heidelberg, Germany

Background: Fixed duration venetoclax (V)-containing therapies are commonly used in CLL, either combined with obinutuzumab (Ven-G) for treatment-naïve or with rituximab for relapsed/refractory disease. Current clinical trials also combine Ven-G with ibrutinib (GIVe) or acalabrutinib (GAVe) to increase efficacy and possibly shorten treatment duration with minimal residual disease (MRD)-guided discontinuation strategies.

Aims: There is only little evidence regarding the best sequence of currently available therapies and if time-limited therapies can be repeated at progression. Therefore, we pooled the patients (pts) with two consecutive V-containing treatment lines from GCLLSG trials, to study efficacy and safety of a re-treatment with V-based regimens.

Methods: Thirteen pts with two sequential V-containing lines (V1 and V2) were identified within the following 5 multicenter phase-II or -III trials: 3 pts each received V1 in CLL2-BAG and CLL2-GIVe, 2 Ven-G in CLL13 (excluded from time-to event analyses due to unavailable data) and 1 in CLL14; all were subsequently treated in CLL2-BAAG (V2). Four additional pts received V1 and V2 in CLL2-BAG.

Results: At the start of V1, the median age was 58 (range 49-65) years and 11 pts (92%) had a high/very high CLL-IPI, all 13 pts had an unmutated IGHV, 8 (62%) a del(17p) and/or TP53mut and 6 (46%) a complex karyotype. Four pts had already received a median of 2.5 (range 1-4) prior treatment lines (chemo(immuno)therapies only).

V1 was Ven-G in 10 pts, including 4 pts with a bendamustine debulking, and GIVe in 3 pts. Median duration of V1 was 13 (range 11-29) months for all pts and 14 (range 13-26) months in the 9 pts with a MRD-guided discontinuation strategy who achieved uMRD, one pt failed to achieve uMRD in bone marrow and had to stop after approximately 2.5 years as per protocol. Three pts received V1 with a fixed duration of approximately one year.

V2 was GAVe in 9 pts, including 2 with a bendamustine debulking, and Ven-G or Ven in 2 each, all with a MRD-guided discontinuation. Thus far, 5 pts have stopped V2: two pts due to uMRD and one each due to adverse events (allergic skin reactions), planned allogeneic stem cell transplantation and end of study treatment after approximately 2.5 years. Median treatment duration was 16 (range 8-30) months for V2, which is already longer than with V1 (see above) but 8 pts still continue V2.

All pts experienced adverse events (AEs) during V1 and V2, AEs CTC °III/IV occurred in 11 and 8 pts during V1 and V2, respectively and serious AEs in 6 and 4, respectively. Hematological AEs °III/IV occurred in 6 and 2 pts during V1 and V2, tumor lysis syndromes °III in 2 and 3 pts and infections °III/IV in 2 each.

All pts responded to V1 and V2, uMRD in PB was achieved at the end of V1 in 12/13 pts (92%) and thus far with V2 in 9/13 pts (69%). Median time between end of V1 and start of V2 was 29 (range 15-55) months. Median progression-free survival from start of V1 was 42 months and after V2 no progressions occurred so far at a median follow up time of 19 (range 8-33) months. All pts are alive, see also Fig. 1.

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Summary/Conclusion: In this pooled analysis of 13 pts with two consecutive time-limited V-containing therapies, which includes mainly pts with adverse risk factors and a short remission duration, V-based re-treatment appeared to be safe and efficacious: no increased rate of AEs was seen so far and all pts responded with at least 2/3 even achieving uMRD again.

P642: IMPACT OF TYPE 2 DIABETES ON SURVIVAL AND TREATMENT IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

E. Curovic Rotbain 1,2,3,4,* , K. Rostgaard 2,4 , M. A. Andersen 1,4,5 , N. Vainer 1 , C. Da Cunha-Bang 1 , H. Hjalgrim 1,2,4,6 , H. Frederiksen 3,7,8 , S. Slager 9 , C. Utoft Niemann 1,6

1 Department of Hematology, Rigshospitalet; 2 Hematology Group, Danish Cancer Society Research Center, Copenhagen; 3 Department of Hematology, Odense University Hospital, Odense; 4 Department of Epidemiology, Statens Seruminstitut; 5 Department of Clinical pharmacology, Bispebjerg and Frederiksberg Hospital; 6 Department of Clinical Medicine, Copenhagen University, Copenhagen; 7 Departemnt of Clinical Research, University of Southern Denmark; 8 Academy of Geriatric Cancer Research (AgeCare), Odense University Hospital, Odense, Denmark; 9 Department of Health Sciences Research, Mayo Clinic, Rochester, United States of America

Background: Because of a generally advanced age at diagnosis of chronic lymphocytic leukemia (CLL), this group of patients is often burdened by comorbid conditions. Comorbid conditions are associated with increased mortality in CLL, due to causes both related and unrelated to CLL. Diabetes is one of the most common comorbid conditions with a prevalence of 8-21% at time of CLL-diagnosis, and the prevalence of diabetes is projected to double in the general population within the next two decades. While many comorbid conditions may have limited potential for improvement of management, treatment for type 2 diabetes (T2D) is rapidly evolving with new treatment combinations displaying survival benefits.

Aims: To assess the impact of T2D on time to treatment and mortality for patients with CLL from time of diagnosis and from time of first-line treatment.

Methods: In Denmark, all citizens have access to free health care services. Contacts with the health care system are registered by the unique personal identification number, enabling linkage of data on person level across national registers. We followed all patients registered with newly diagnosed CLL in the Danish CLL Register or the Danish Cancer Register in the period 2002-2018 from 1 month after CLL-diagnosis until death or end of follow-up. Data on causes of death were obtained through the Danish National Register of Causes of Death. T2D was defined as having an International Classification of Diseases-10 diagnosis of T2D (E11) in the Danish National Patient Register or ≥1 prescription for non-insulin anti-diabetic drugs (ATC code A10B) in the Danish Prescription Register. Fine-Gray and Cox proportional hazards regressions were fitted, and corresponding Kaplan-Meier curves computed.

Results: In total, 7 446 patients with CLL were identified, of whom 802 (11%) had T2D at CLL diagnosis. T2D was associated with longer time to first treatment (hazard ratio (HR) 0.81, 95% confidence interval (CI) [0.67-0.99]) and higher mortality (HR 1.58, 95% CI [1.42-1.76]) from time of CLL diagnosis. Patients with T2D had an increased mortality due to causes both related (HR 1.15, 95% CI [0.99-1.34]) and unrelated (HR 2.07, 95% CI [1.67-2.55]) to CLL. Among 1 487 patients receiving first-line treatment, 164 (11%) had T2D. Upon first-line treatment, patients with T2D had shorter event-free survival (HR 1.40, 95% CI [1.07-1.79]) and higher mortality (HR 1.22, 95% CI [0.95-1.56]) when compared with patients with CLL without T2D. Treatment with fludarabine, cyclophosphamide, and rituximab (FCR) was not associated with a superior survival compared with bendamustine and rituximab (BR) in patients with T2D (HR 2.58, 95% CI [0.61-10.86]).

Summary/Conclusion: T2D is associated with an inferior prognosis in CLL. CLL patients with T2D had a lower probability of receiving CLL-treatment yet higher mortality. While the latter may be related to surveillance bias, further investigation in to causes is required. Though not statistically significant, analyses suggest that among patients with CLL and T2D, BCR treatment might be superior to FCR.

P643: RISK FACTORS FOR UNFAVORABLE OUTCOME OF HOSPITALIZED PANTIENTS WITH CONCURENT CHRONIC LYMPHOCYTIC LEUKEMIA AND COVID-19 – EXPERIENCE OF THREE SERBIAN UNIVERSITY CENTERS

Z. Cvetkovic 1,2,* , O. Markovic 2,3 , K. Markovic 4 , A. Novkovic 1 , A. Divac 3 , N. Vukosavljevic 4 , M. Tanasijevic 1 , T. Bibic 1

1 Department of Hematology, Clinical Hospital Center Zemun; 2 University of Belgrade, Medical Faculty; 3 Department of Hematology, Clinica Hospital Center Bezanijska kosa; 4 Department of Hematology, Clinical Hospital Center Zvezdara, Belgrade, Serbia

Background: Vulnerability of patients (pts) with chronic lymphocytic leukemia (CLL) and their susceptibility to Covid-19 infection is documented in several studies with reported case fatality rates (CFRs) up to 40%, but there is still paucity of data on identifying risk factors of their adverse outcome.

Aims: To evaluate demographic, patient-related, CLL-related and Covid-19 related risk factors in hospitalized pts with concurrent CLL and Covid-19.

Methods: Total of 81 CLL pts were identified in medical records of three University centers in Belgrade: Clinical Hospital Center (CHC) Zemun, CHC Bezanijska kosa and CHC Zvezdara dedicated to treatment of Covid-19 pts during pandemic (from 15 March 2020 to 31 December 2021).

Results: For all 81 pts CFR was 32.1%. Age (median age 68 yrs;range 45-90 yrs) and sex (apparent male prevalence: 61 male and 20 female; M:F=3.05) had no influence on outcome. Pts with Charlson comorbidity index >4 (29/81;35.8%) had significantly higher CFR (38% vs 9.5%, p=0,025). Concerning CLL-directed treatment: 26/81(32.1%) pts were on active treatment (5 pts were on Bruton tyrosine kinase inhibitor, 21pts receiving imunochemotherapy), 11/81(13.6%) pts were in remission on previous lines of therapy, while 44/81(54.3%) pts were treatment naive. CLL treatment history had no impact on CFR, as well as anemia (Hb<100g/l) that was present in 29/81(35.8%)pts, hipogammaglobulinemia (21/81;26%pts) and hiperferritinemia>450ng/mL (50/81;61.7%pts). Of evaluated laboratory parameters, high levels of lactate-dehydrogenase (LDH>2xUNL:6/81;7.4%pts), D-dimer (>1000ng/mL:36/81;44.4%pts), and C-reactive protein (CRP>100mg/L: 31/81;38.3%pts) proved to be associated with adverse outcome; p-values 0.002, 0.039 and <0.001, respectively. According to Covid-19 clinical course, the severe Covid-19 score had 35(43,2%)pts, and critical 19(23.5%)pts. Covid-19 infection was treated according to current National guidelines. Corticosteroids were administrated to 81.5% of pts, antiviral agents to 38.3%, IL-6 receptor inhibitor to 11.1%, antiviral monoclonal antibodies to 7.4% and intravenous immunoglobulin to 19.8% of pts. None of listed therapeutic approaches had impact on CFRs. Antibiotics were administrated to 43/81 (53.1%) of pts with documented or highly suspected concomitant bacterial infection (procaltitonin level>0.5ng/mL and/or chest X-Ray image corresponding to bacterial pneumonia), and the bacterial coinfection had adverse impact on CFR (51.2% vs.10.2%; p<0.001). Significantly higher mortality was documented in pts who needed supplemental oxygen (58/81;71%) (CFR 43.1 vs.4.3%; p<0.001), and intensive care unit (ICU) admission (25/81-30.9%; 19/25 needed mechanical ventilation) (CFR 88% vs.7.1%;p<0.001). In multivariate analysis, bacterial coinfection and ICU admission proved to be the most significant adverse parameters influencing outcome (p=0.012).

Summary/Conclusion: Our study proved the dismal outcome of CLL pts with concurrent Covid-19. That could be mainly attributed to the high proportion of bacterial coinfections reflecting their frailty and sucessibility to both viral and bacterial infections.

P644: THE LIFE EXPECTANCY OF PATIENTS WITH HAIRY CELL LEUKEMIA VERGES UPON THE LIFE EXPECTANCY OF THE GENERAL POPULATION: A POPULATION-BASED STUDY IN THE NETHERLANDS

A. Dinmohamed 1,2,3,* , C. Maas 1,2 , O. Visser 4 , J. Doorduijn 5 , R. Mous 6 , R. Raymakers 6 , A. Kater 3 , W. Posthuma 1,7,8

1 Research and Development, Netherlands Comprehensive Cancer Organisation (IKNL), Utrecht; 2 Public Health, Erasmus MC, Rotterdam; 3 Hematology, Amsterdam UMC, Amsterdam; 4 Registration, Netherlands Comprehensive Cancer Organisation (IKNL), Utrecht; 5 Hematology, Erasmus MC Cancer Institute, Rotterdam; 6 Hematology, UMC Utrecht, Utrecht; 7 Internal Medicine, Reinier de Graaf Gasthuis, Delft; 8 Hematology, Leiden University Medical Center, Leiden, Netherlands

Background: At present, no other therapeutic strategy has substantially outperformed purine nucleoside analogues (PNAs)—introduced around the late 1980s and early 1990s—to manage hairy cell leukemia (HCL). The most recent population-based study in HCL showed that 10-year relative survival of HCL patients diagnosed in the Netherlands during 2001-2015 was 97%, 95%, and 83% in the age groups <60, 60-69, and ≥70 years, respectively (Dinmohamed AG et al. Blood ; 2018). Given the improved longevity of HCL patients, relative survival rates fall short to inform on longevity across the entire patients’ life span.

Aims: Studies estimating the life expectancy of HCL patients are hitherto lacking. Therefore, we assessed trends in the life expectancy of HCL patients from a historical and contemporary perspective.

Methods: We selected all HCL patients diagnosed between 1989 and 2019—with follow-up for survival up to January 1, 2021—from the Netherlands Cancer Registry (N=1,828; median age, 60 years; interquartile range, 50-70 years; 78% males). We estimated the loss in expectation of life (LEL)—i.e., the difference between the life expectancy of patients and an age-, sex-, and period-matched group from the general population—using flexible parametric relative survival models. The LEL is interpreted as the average number of life years lost due to an HCL diagnosis. The LEL can vary markedly across ages because life expectancy is age-dependent. Therefore, the proportional LEL (PLEL) was estimated. These survival measures were presented by year of diagnosis for four ages at diagnosis (i.e., 40, 50, 60, and 70 years), stratified by sex.

Results: The life expectancy of HCL patients increased gradually across all ages between 1989-2019, irrespective of sex (Fig A). It is noteworthy that HCL patients diagnosed in 1990 lost comparatively few life-years due to their diagnosis, ranging from a LEL of 1.7 to 3.9 life-years lost depending on age and sex (Fig B). Estimates for patients diagnosed in 2019 ranged from 0.8 to 1.9 life-years lost (Fig B).

Over time, the decrease in LEL was most pronounced for younger patients (Fig B). For example, a 50-year-old male diagnosed with HCL in 1990, on average, has a LEL of 3.8 years (95% confidence interval [CI]: 2.1-5.5), whereas a male with a HCL diagnosis in 2019, on average, has a LEL of 1.0 years (95% CI, 0.3-1.7). The corresponding estimates for a 70-year-old male HCL patient were 2.3 (95% CI, 1.4-3.1) and 1.1 (95% CI, 0.6-1.7), respectively.

The PLEL estimates also portrays that outcomes in HCL patients improved over time across all ages (Fig C). Nevertheless, there was a persistent age differential in the PLEL over time. More specifically, younger patients consistently have more remaining life-years than older patients, reflected in lower PLEL estimates in younger patients.

Of note, the life expectancy estimates of female patients should be interpreted with caution by considering the wideness of the 95% CIs due to the comparative rarity of HCL in females to estimate the life expectancy accurately.

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Summary/Conclusion: The life expectancy of HCL patients verges upon the life expectancy of the general population. This encouraging finding was already objectified around the early-1990s when PNA therapy was introduced for HCL management. Thereafter, the life expectancy gradually increased over time. Novel therapeutic strategies may reduce the minimal excess mortality encountered in contemporary diagnosed patients, particularly among the elderly.

P645: THE INFLUENCE OF ACTIVE CARDIAC MONITORING WITH REMOTE CONTROL ON THE SURVIVAL OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA, RECEIVING IBRUTINIB.

E. Emelina 1,* , G. Gendlin 1 , I. Nikitin 1

1 Hospital Therapy №2, Pirogov Russian National Research Medical University, Moscow, Russia

Background: Survival of chronic lymphocytic leukemia (CLL) patients receiving ibrutinib (Ib) is influenced by many factors, among which cardiovascular disease, both in history and during treatment with Ib, is of great importance.

Aims: To assess the impact on 5-year overall survival (5-OS) of patients CLL treated with Ib two options for dynamic monitoring by a cardiologist: standard cardiac monitoring (SC) and active cardiac monitoring with remote control (ACM).

Methods: We observed in dynamics for 5 years’ period 217 patients with CLL, constantly receiving therapy with Ib 420 mg/day. In the ACM group (n=89), in addition to the standard examination, every week or more often using messengers, we did active medical monitoring of the patient’s symptoms and well-being, assessment of blood pressure and heart rate, control of cardioprotective medicines taken, correction of the therapy, calling patients for examination and additional examination. The remaining 128 patients were examined in dynamics, but did not support remote control, constituting the SC group. The age of patients in the ACM group and in the SC group did not differ and was 66.0 (60.0–70.0) years and 66.0 (59.0–74.0) years, respectively (p = 0,39). There were slightly more men in the SC group (68.8%) than in the active cardiac monitoring group (53.9%), p = 0.026. The proportion of patients with CLL with the number of pretreatment lines from 0 to 2 (median -1) in the ACM group was 52.7%, in the SC group it was 58.1%; with the number of lines from 3 to 12 (median - 4) in the AKM group - 47.3%, in the SC group - 41.9% (p = 0.53). The AKM and SC groups did not differ in the results of the Geriatric 8 scale, Charlson index, and echocardiography parameters at visit 1. In the ACM group, there were more patients with cardiac problems: with arterial hypertension (AH) (p < 0.0001) and atrial fibrillation (p < 0.0001), receiving anticoagulants (p < 0.0001), a comparable number of patients with coronary artery disease.

Results: In the ACM group, 70 out of 77 (90.9%) patients with CLL and AH achieved a stable level of target blood pressure values, in contrast to the SC group - 26 out of 66 (39.9%), p < 0.0001. Significantly more events requiring cardiac surgery (stenting of the coronary arteries, installation of a pacemaker) were detected in the ACM group – 12, versus 0 in the SC group (p = 0.0004). In the ACM group, despite a more pronounced cardiac comorbidity, 5-OS was significantly better, than in the SC group in both men (p < 0.0001) and women (p < 0.0001) with CLL and in patients older than 70 years old (p = 0.0004). 5-OS was also better in the ACM group than in the SC group in patients with CLL with a median number of previous lines of therapy equal to 1 (p<0.0001) and in patients with a median number of chemotherapy lines equal to 4 (p<0.0001), in patients with genetic abnormalities (p=0.004) and pretreated with fludarabine and/or anthracyclines (p < 0.0001).

Summary/Conclusion: Early detection and correction of cardiovascular complications/events, achievement of stable target blood pressure values, constant monitoring of cardioprotective treatment in the ACM group explain the statistically highly significant differences in 5-OS in patients with CLL who are on constant Ib therapy. Conducting active cardiomonitoring with remote control makes it possible to achieve higher rates of total 5-OS in patients with CLL receiving Ib.

P646: EFFECTIVENESS AND SAFETY OF VENETOCLAX IN COMBINATION WITH RITUXIMAB (VENR) IN R/R CLL PATIENTS WITH OR WITHOUT RISK-ASSOCIATED GENETIC MARKERS – DATA FROM THE OBSERVATIONAL VERVE STUDY

I. Schwaner 1,* , H. Hebart 2 , C. Losem 3 , T. Wolff 4 , K. Famulla 5 , J. Huelsenbeck 5 , B. Schmidt 6 , T. Nösslinger 7 , D. Rossi 8

1 Onkologie Kurfürstendamm, Berlin; 2 Kliniken Ostalb, Mutlangen; 3 MVZ Onkologie und Hämatologie, Neuss; 4 Onkologie Lerchenfeld, Hamburg; 5 AbbVie Deutschland GmbH & Co KG, Wiesbaden; 6 Hämatologisch-Onkologische Gemeinschaftspraxis, München, Germany; 7 Medizinische Abteilung für Hämatologie und Onkologie, Hanusch Krankenhaus, Wien, Austria; 8 Institute of Oncology Research, Bellinzona, Switzerland

Background: Treatment with Venetoclax in combination with Rituximab (VenR) has shown promising efficacy and good tolerability in clinical trials 1 However, there is limited real-world data on VenR in CLL patients with or without genetic markers, known to be associated with unfavorable outcomes.

Aims: The non-interventional observational study VeRVe aims to investigate safety and effectiveness of VenR for treatment of CLL under real-world conditions in Germany, Austria, and Switzerland. In this analysis we focus on safety and effectiveness of VenR in CLL patients with or without TP53 mut and/or del(17p) as well as IGHV status.

Methods: CLL patients included in this study required therapy and were eligible for VenR treatment according to local label 2 . Study documentation is possible at baseline, weekly during ramp-up, monthly until the end of month 6 and quarterly afterwards up to a maximum of 3 years. Response assessment can be documented at end of ramp-up, after 3, 12, 24 and 36 months.

Results: At the time of analysis (November 4 th , 2021), 100 patients were enrolled in the VenR arm of the VeRVe study and received at least one dose of Ven. 86 of them did also receive at least one dose of rituximab. 28 patients were female, 32 had aberrant TP53 ( TP53 mut and/or del(17p)) and 40 were IGHV unmutated. TP53, del(17p) and IGHV status was unknown for 20, 18 and 42 patients, respectively. The median age was 72 y in the population with TP53 aberration and 71 y in the population with wild-type (wt) TP53. Within the TP53 aberrant vs the TP53 wt group 48% vs 67% had received 1, 48% vs 33% had received 2 or more prior lines of therapy. At least 1 comorbidity was reported for 85% of TP53 aberrant and 81% of TP53 wt patients.

After 12 months of therapy, the best ORR was 88% in the total, 90% in the TP53 aberrant and 91% in the TP53 wt population. Thereby, CR/CRi was achieved in 54% of TP53 aberrant and 44% of TP53 wt patients. When subdivided according to IGHV status, the best ORR was 88% in IGHV mutated and 91% in IGHV unmutated patients. 29% of IGHV mutated and 62% of IGHV unmutated patients reached CR/CRi as best ORR within 12 months. However IGHV status was not documented for 42 patients.

Estimated PFS-rates after 1 year were 88% in the total population and 86% and 90% in the TP53 aberrant and TP53 wt population, respectively (figure). Estimated OS-rates after 1 year were 90% in the total population, 93% in the TP53 aberrant population and 90% in the TP53 wt population.

After a median observation time of 451 days (range 1-1016), CTCAE grade ≥3 AEs were reported in 57% (n=57) of all patients, 61% (n=20) in TP53 aberrant patients and 64% (n=27) in the TP53 wt patients. SAEs were reported in 36% (n=36) of all, 42% (n=14) in TP53 aberrant and 40% (n=17) in the TP53 wt patients. TLS occurred in 10 patients (10%) of the overall population. 2 of them where clinical TLS (both in the group TP53 wt).

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Summary/Conclusion: In conclusion, patients with and without TP53 aberrations were comparable regarding age and burden of comorbidities, but in the group with TP53 aberrations slightly more patients had a high number of prior therapy lines. However, both populations responded to VenR treatment with comparably high best ORR, estimated PFS-rates and estimated OS-rates. This was also the case for IGHV mutated vs unmutated patients, however patient number was low for this analysis, because the IGHV status was often not documented in this real-world study. VenR treatment was well tolerated in the overall population and all subgroups and no new safety signals were detected.

P647: SAFETY AND EFFECTIVENESS OF VENETOCLAX MONOTHERAPY IN R/R CLL PATIENTS WITH OR WITHOUT RISK-ASSOCIATED GENETIC MARKERS – DATA FROM THE OBSERVATIONAL VERVE STUDY

I. Schwaner 1,* , H. Hebart 2 , C. Losem 3 , T. Wolff 4 , K. Famulla 5 , J. Huelsenbeck 5 , B. Schmidt 6 , T. Nösslinger 7 , D. Rossi 8

1 Onkologie Kurfürstendamm, Berlin; 2 Kliniken Ostalb, Mutlangen; 3 MVZ Onkologie und Hämatologie, Neuss; 4 Onkologie Lerchenfeld, Hamburg; 5 AbbVie Deutschland GmbH & Co KG, Wiesbaden; 6 Hämatologisch-Onkologische Gemeinschaftspraxis, München, Germany; 7 Medizinische Abteilung für Hämatologie und Onkologie, Hanusch Krankenhaus, Wien, Austria; 8 Institute of Oncology Research, Bellinzona, Switzerland

Background: In chronic lymphocytic leukemia (CLL), the association of genetic markers such as del(17p), TP53 and IGHV status with disease prognosis is well established. In clinical trials, treatment with Venetoclax (Ven) monotherapy has shown promising efficacy and good tolerability in all subgroups 1,2 . However, there is limited real-world data on Ven usage in specific genetic subgroups.

Aims: The non-interventional observational study VeRVe aims to investigate safety and effectiveness of Ven for treatment of CLL in a real world setting in Germany, Austria, and Switzerland. Here, we present VeRVe data focusing on Ven monotherapy in CLL patients with or without the genetic risk factors TP53 mut and/or del(17p) as well as IGHV status.

Methods: CLL patients included in this study required therapy and were eligible for Ven monotherapy treatment according to local label 3 . Study documentation is possible at baseline, weekly during ramp-up, monthly until the end of month 6 and quarterly afterwards up to a maximum of 2 years. Response assessment can be documented at end of ramp-up, after 3, 12 and 24 months.

Results: On Nov 4 th , 2021, 77 patients were enrolled in the VeRVe study and received at least one dose of Ven monotherapy, 29 of which were female. 40 of them had TP53 aberrations ( TP53 mut and/or del(17p)) and 27 were IGHV unmutated. For 14 patients TP53 mut status, for 10 patients del(17p) and for 40 patients IGHV status was not documented. At start of therapy, the median age of patients with TP53 aberrations was 73 years and 71 years for patients with wildtype (wt) TP53 . In the population with TP53 aberrations, 40% had received 1, 28% 2 and 32% 3 or more prior therapies. In the population with wild-type (wt) TP53 , 12% had received 1, 40% 2 and 48% 3 or more prior therapies. 80% of patients with TP53 aberration and 72% of patients with wt TP53 had at least 1 comorbidity.

After 12 months of therapy, the best ORR (CR/CRi) was 74% (34%) in the total population. Thereby, the best ORR (CR/CRi) was 76% (43%) in patients with TP53 aberrations and 69% (27%) in patients without TP53 aberrations. When subdivided according to IGHV status, the best ORR (CR/CRi) was 83% (33%) in IGHV mutated and 85% (43%) in IGHV unmutated patients (see figure).

Moreover, estimated PFS-rate after 1 year was at 80% in the total population. Thereby, estimated PFS-rate was at 78% in patients with TP53 aberrations and at 83% in patients with wt TP53 . Estimated OS-rate after 1 year was 82% in the total population, 77% in patients with TP53 aberration and 84% in patients with wt TP53 .

With a median observation time of 294 days (range 0-1130), CTCAE grade AEs ≥3 were reported in 56%(n=43) of the total population, 53% (n=21) in the TP53 aberrant and 64% (n=16) in the TP53 wt population. SAEs were reported in 37% (n=39) of the total population, 45% (n=18) in the TP53 aberrant and 32% (n=8) in the TP53 wt population. Overall, 5 patients had a TLS with 3 of them being clinical TLS. No new safety signals were detected.

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Summary/Conclusion: In summary, patients with TP53 aberration were slightly more pre-treated and suffered from co-morbidities more frequently than patients with wt TP53 . Nevertheless, best ORR, estimated PFS-rates and OS-rates were comparably high in both populations. IGHV status was often not determined in this real-world study. For the available data, best ORR in IGHV mutated and IGHV unmutated patients were comparable. Surprisingly, CR/CRi rates were highest in patients with TP53 aberration or IGHV unmutated patients. Treatment was well tolerated in the overall population and all subgroups.

P648: T CELL PHENOTYPIC CHANGES IN PARTICIPANTS OF A MULTIPHASE OPTIMIZATION STRATEGY (MOST) BEHAVIORAL INTERVENTION OF HEALTHY DIET AND EXERCISE IN PATIENTS (PTS) WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)

M. Gordon 1,* , J. Crane 2 , C. Y. Lee 3 , S. Fares 3 , K. Basen-Engquist 3 , M. Markofski 4 , A. Ferrajoli 5 , E. LaVoy 4

1 Cancer Medicine, University of Texas MD Anderson Cancer Center; 2 Biology, University of Houston; 3 Behavioral Science, University of Texas MD Anderson Cancer Center; 4 Health and Human Performance, University of Houston; 5 Leukemia, University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Clinical outcomes in pts with CLL are influenced by disease biology, host immune response and comorbidities (Roessner et al, Leukemia, 2020 and Rotbain et al, Leukemia, 2021). Intensive physical exercise can modulate T cell function and slow leukemic progression (Sitlinger et al, Blood Adv., 2020). Here we report baseline and 4 month follow up of T cell phenotype in CLL pts after participation in a MOST exercise program.

Aims: Assess the association of comorbidities and T cell subsets in pts with CLL participating in an organized exercise program.

Methods: Pts with CLL treated at MD Anderson were randomized to one of 16 combinations of 5 evidence-based behavioral interventions. Pts with baseline and follow up data were included. T cell phenotype was identified by multicolor flow cytometry assessing CD3, CD4, CD8, HLADR and PD1. Difference in baseline and pre/post intervention T cell subset was assessed by t-test and Mann-Whitney. A p-value <.1 was considered significant.

Results: Median age was 63 years in the 24 pts with CLL. TP53 aberrancy was present in 8%, unmutated IGHV in 46% and 63% had moderate-severe comorbidities (CLL comorbidity index [CI] score, 1-2). Median prior lines of therapy were 1, 85% were receiving a BTK inhibitor (ibrutinib n=7, acalabrutinib n=2 and zanubrutinib n=2); 50% were untreated.

Baseline T cell phenotype showed that a CLL-CI score of 1-2, compared to 0 was associated with lower HLADR+CD4+ T cells (p=.07). CLL-CI score of 1 vs 0 was associated with high PD1+CD8+ T cells (p=.03) and PD1+CD4+ T cells (p=0.06; Fig 1). By individual comorbidity category only upper gastrointestinal comorbidities (n=6) were associated with T cell phenotype, lower HLADR+CD8+ T cells (p=.04). Pts ≥65 years had lower HLADR+CD4+ (p=.04) and HLADR+PD1+CD4+ (p=.09) T cell populations. Advanced Rai stage (2-4) was associated with higher CD8+ T cells (p=.03). Treatment, IGHV and Beta 2 microglobulin were not associated with T cell phenotype.

T cell phenotype at 4 months showed that a CLL-CI score of 1-2 vs 0 was associated with lower HLADR+CD4+ T cells (p=.06). Both PD1+CD4+ and PD1+CD8+ T cells were higher in pts with a CLL-CI score of 1 vs 0 (p=.09 for both). Pts with vascular comorbidities had lower CD8+ (p=.09) and higher HLADR+PD1+CD4+ (p=.09) T cells. Endocrine comorbidities were associated with lower HLADR+CD4+ (p=.08) and lower HLADR+PD1+CD4+ (p=.09) T cells. Advanced Rai stage was associate with increased HLADR+CDC8+ T cells (p=.02). Treatment and age did not significantly impact T cell phenotype.

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Summary/Conclusion: Comorbidities are associated with shorter survival and more rapid progression of CLL (Gordon et al, CCR, 2021 and Rotbain et al, Blood adv., 2022). But the mechanism(s) underlying these observations are not well described. High HLADR+PD1+CD4+ T cells are associated with CLL progression (Elston et al, BJH, 2020). We hypothesized that comorbidities, measured by the CLL-CI, could influence T cell function and thus lead to CLL progression. CLL-CI score was associated T cell phenotype in this study. Pts with comorbidities had higher PD1 expression at baseline. Interestingly, after participation in the exercise intervention, pts with vascular disease had higher HLADR+PD1+CD4+ T cells while those with endocrine comorbidities had lower HLADR+PD1+CD4+ T cells. This suggest that pts with obesity and diabetes may particularly benefit from participation in behavioral interventions by lowering the % of HLADR+PD1+CD4+ T cells.

P649: A FIRST-IN-HUMAN PHASE 1 TRIAL OF NX-2127, A FIRST-IN-CLASS ORAL BTK DEGRADER WITH IMID-LIKE ACTIVITY, IN PATIENTS WITH RELAPSED AND REFRACTORY B-CELL MALIGNANCIES

A. Mato 1,* , A. Danilov 2 , M. R. Patel 3 , M. Tees 4 , I. Flinn 5 , W. Ai 6 , K. Patel 7 , M. Wang 8 , S. M. O’Brien 9 , S. Nandakumar 10 , M. Tan 10 , E. Meredith 10 , M. Gessner 10 , S. Y. Kim 10 , A. Wiestner 11 , W. G. Wierda 8

1 Memorial Sloan Kettering Cancer Center, New York; 2 City of Hope National Medical Center, Duarte; 3 Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota; 4 Colorado Blood Cancer Institute, Denver; 5 Sarah Cannon Research Institute and Tennessee Oncology, Nashville; 6 University of California San Francisco Medical Center, San Francisco; 7 Swedish Cancer Institute, Seattle; 8 MD Anderson Cancer Center, Houston; 9 Chao Family Comprehensive Cancer Center, University of California, Irvine; 10 Nurix Therapeutics, Inc., San Francisco; 11 National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, United States of America

Background: Bruton’s tyrosine kinase inhibitors (BTKi) have received regulatory approvals and are standard of care for patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and WaldenstrÖm macroglobulinemia (WM). However, BTKi-resistant disease remains a clinical challenge with limited options for subsequent therapy.

Immunomodulatory drugs (IMiDs, e.g., lenalidomide) are approved as monotherapy for follicular lymphoma (FL), MZL, and MCL, in combination with other therapies for diffuse large B-cell lymphoma (DLBCL) and have shown synergy with BTK-targeted therapy. Dual activity of BTK protein degradation with IMiD-like activity offers a unique approach to overcome known resistance to BTKi.

NX-2127 is an oral small molecule that induces BTK degradation via recruitment of cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NX-2127 has shown preclinical activity similar to IMiDs by catalyzing the ubiquitination of Ikaros (IKZF1) and Aiolos (IKZF3), ultimately leading to increased T-cell activation. NX-2127 was shown to degrade both wild-type (WT) and C481-mutated (ibrutinib-resistant) BTK protein in vitro. Robust BTK degradation was also shown in non-human primate studies. Further, NX-2127 demonstrates potent tumor growth inhibition in BTK-dependent mouse xenograft tumor models expressing either WT or ibrutinib-resistant C481S BTK-mutant protein. This dual activity of BTK degradation and IMiD-like activity offers a promising treatment for patients who have failed prior therapy.

Aims: The primary objectives are to evaluate safety and tolerability and to determine the maximum tolerated dose (Phase 1a), and to evaluate the early clinical activity of NX-2127 in expansion cohorts (Phase 1b).

Methods: NX-2127-001 is a first-in-human, dose escalation (Phase 1a) and cohort expansion (Phase 1b) study designed to evaluate the safety, tolerability, and preliminary efficacy of NX-2127 in adult patients with relapsed/refractory B-cell malignancies with once daily oral dosing. Dose escalation will proceed using a modified Fibonacci design with 1 patient per cohort, proceeding to a standard 3 + 3 design based on protocol specified criteria. There will be up to 5 expansion cohorts in Phase 1b enrolling patients with CLL/SLL, DLBCL, FL, MCL, MZL, and WM. Key eligibility criteria include ≥ 2 two prior lines of therapy ( ≥ 1 prior for WM); measurable disease; and an Eastern Cooperative Oncology Group performance status of 0 or 1. Approximately 130 patients (30 in Phase 1a, 100 in Phase 1b) will be enrolled and treated until disease progression or unacceptable toxicity.

The primary endpoint are dose-limiting toxicities and maximum tolerated dose (Phase 1a), objective response (Phase 1b), and safety (Phase 1a and Phase 1b) of NX-1607. Secondary endpoints (Phase 1a and Phase 1b, unless otherwise indicated) include pharmacokinetics, pharmacodynamics, duration of response, progression-free survival, overall survival (Phase 1b), safety (Phase 1b), and complete response rate/complete response rate with incomplete marrow recovery.

Results: The Phase 1a part of this study is currently enrolling in the United States.

Summary/Conclusion: Accrual is ongoing. Clinical trial information: NCT04830137 .

P650: A FIRST-IN-HUMAN PHASE 1 TRIAL OF NX-5948, AN ORAL BTK DEGRADER, IN PATIENTS WITH RELAPSED AND REFRACTORY B-CELL MALIGNANCIES

K. Linton 1,* , G. P. Collins 2 , D. El-Sharkawi 3 , R. Mous 4 , F. Forconi 5 , M. Tan 6 , S. Nandakumar 6 , E. Meredith 6 , K. L. Jameson 6 , S. G. Injac 6 , J. Doorduijn 7

1 The University of Manchester, Manschester; 2 Oxford University Hospitals NHS Trust, Oxford; 3 Royal Marsden NHS Foundation Trust, Sutton, United Kingdom; 4 UMC Utrecht Cancer Center, University Medical Center, Utrecht, Netherlands; 5 University Hospital Southampton NHS Trust, Southhampton, United Kingdom; 6 Nurix Therapeutics, Inc., San Francisco, United States of America; 7 Erasmus MC Cancer Institute, Rotterdam, Netherlands

Background: Bruton’s tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) signaling pathway and chronic activation of BTK-mediated BCR signaling is a hallmark of many B-cell malignancies. BTK-targeted therapy has been shown to be safe and effective in a variety of B-cell lymphomas, including chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM). However, BTK inhibitor (BTKi) resistant disease remains a clinical challenge with limited options for salvage therapy and overall poor patient outcomes. BTK degradation may be effective in patients who have developed resistance to BTKi or in B-cell indications where treatment with BTKi has been less effective.

NX-5948 is an oral, small molecule that induces BTK degradation through the recruitment of cereblon, an adaptor protein of the E3 ubiquitin ligase complex, and does not induce degradation of other cereblon neosubstrates. NX-5948 degrades both wild type BTK and BTKi resistant mutants (C481) and inhibits ibrutinib-resistant tumor cell line growth at concentrations where ibrutinib and acalabrutinib are inactive. NX-5948 potently inhibits tumor growth in xenograft models that contain either wild type BTK or BTKi-resistant mutations. Further, NX-5948 crosses the blood-brain barrier, thus positioning it as a therapeutic agent in patients with primary central nervous system lymphoma (PCNSL) and other lymphomas that have CNS involvement.

Aims: The primary objectives are to evaluate safety and tolerability, determine the maximum tolerated dose (MTD; Phase 1a only), and the early clinical activity in each expansion cohort (Phase 1b only) of NX-5948 in patients with relapsed and refractory B-cell malignancies.

Methods: NX-5948-301 is a first-in-human, dose escalation (Phase 1a) and cohort expansion (Phase 1b) study designed to evaluate the safety, tolerability, and preliminary efficacy of NX-5948 in adult patients with relapsed and refractory B-cell malignancies. NX-5948 will be given orally daily and dose escalation will proceed using a standard 3 + 3 design. There will be up to 5 expansion cohorts in Phase 1b composed of patients with the following tumor types: (a) CLL/SLL with a C481 mutation; (b) CLL/SLL without a C481 mutation; (c) aggressive subtypes diffuse large B-cell lymphoma (DLBCL) and MCL; (d) indolent subtypes FL, MZL, and WM; and (e) primary/secondary CNS lymphoma. Other key eligibility criteria include 2 or more prior lines of therapy (only 1 prior for WM and PCNSL); measurable disease; and an Eastern Cooperative Oncology Group performance status of 0 or 1. Up to 130 patients (30 in Phase 1a, 100 in Phase 1b) will be enrolled and treated until disease progression or unacceptable toxicity.

Results: Enrollment in this study has begun in Europe.

Summary/Conclusion: Accrual is ongoing. Clinical trial information: NCT05131022 .

P651: VENETOCLAX-BASED TREATMENT OF PATIENTS WITH RICHTER SYNDROME: OUTCOMES FROM A MULTICENTER RETROSPECTIVE STUDY

P. Hampel 1,* , S. Parikh 1 , W. Wierda 2 , A. Ferrajoli 2 , N. Jain 2 , J. Burger 2 , T. Call 1 , Y. Wang 1 , S. Kenderian 1 , W. Ding 1 , P. Thompson 2

1 Hematology, Mayo Clinic, Rochester; 2 Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, United States of America

Background: Patients (pts) with chronic lymphocytic leukemia (CLL) who develop Richter Syndrome (RS) have a poor prognosis with median overall survival (OS) often reported as less than 12 months (Tsimberidou JCO 2006; Rogers BJH 2018), using chemoimmunotherapy (CIT) regimens typically administered to de novo large cell lymphoma. Venetoclax (ven) has demonstrated single-agent activity in RS with an overall response rate of 43% (Davids JCO 2017). Ven combined with DA-EPOCH-R achieved a median OS of 19.6 months and the highest complete response (CR) rate (50%) reported in pts with RS (Davids Blood 2022). These results support a synergistic effect when compared to CR rates of 20% and 0% with EPOCH-R and ven monotherapy, respectively.

Aims: To evaluate the outcomes of pts with RS treated with ven-based treatment, outside clinical trials, including novel-novel combinations and CIT combinations.

Methods: We analyzed pts with RS treated with a ven-based regimen at MDACC (n=37) or Mayo Clinic (n=18) between 12/2013 and 8/2021. Patient and disease characteristics from the time of ven-based treatment start were ascertained. Retrospective response assessment was as per Lugano 2014 guidelines. Toxicity was recorded per iwCLL 2018 guidelines (hematologic toxicity) or CTCAE v5.0 (non-hematologic toxicity). OS and progression-free survival (PFS) were analyzed using the Kaplan-Meier method, with and without censoring for allogeneic hematopoietic stem cell transplantation (alloSCT). No formal statistical comparisons were made between different treatment groups.

Results: Fifty-five pts were identified with a median age of 66 years (range 43-83 years); 31% were female. High-risk CLL disease characteristics were frequently identified: 92% unmutated IGHV , 42% del(17p), 57% TP53 mutation, and 62% complex karyotype. The median number of prior CLL-directed therapies was 2 (range 0-7), including prior chemotherapy (42%), prior Bruton tyrosine kinase inhibitor (BTKi; 38%), and prior ven (22%); 62% of pts were previously untreated for RS.

Median follow-up from the start of ven-based RS treatment was 9 months (mo). The most common ven-based combination regimens achieved overall response and CR rates as follows: 50%/40% with intensive CIT + ven (n=20), 40%/30% with BTKi + ven +/- CD20 antibody (n=20), 60%/50% with R-CHOP + ven (n=10). Among the remaining 5 patients, 2 received ven monotherapy and 3 received varied ven-based combination regimens. Nine pts proceeded to subsequent alloSCT.

The median PFS for the total cohort when censored at alloSCT was 4.0 mo; PFS uncensored for alloSCT was 4.4 mo (Figure 1 A ). Median PFS by treatment group (Figure 1 B ) was as follows: 3.7 mo for intensive CIT + ven, 3.9 mo with BTKi + ven +/- CD20 antibody, not reached with R-CHOP + ven. The median OS for the total cohort was 9 mo. The estimated median OS by treatment group (Figure 1 C ) was 8.4 mo with intensive CIT + ven, 10.6 mo with BTKi + ven +/- CD20 antibody and not reached with R-CHOP + ven.

Grade 3-4 neutropenia and thrombocytopenia were more common with intensive CIT + ven (71%; 69%) and R-CHOP + ven (77%; 67%) compared to BTKi + ven +/- CD20 antibody (35%; 25%). Febrile neutropenia occurred in 42%, 33%, and 25% of pts in these three groups, respectively, with 37%, 33%, and 30% experiencing a grade 3-4 infection.

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Summary/Conclusion: In the notoriously difficult-to-treat RS patient population, ven-based combination regimens achieve high response rates, including some with prolonged duration, particularly with the use of R-CHOP + ven. A prospective clinical trial evaluating this combination is ongoing ( NCT03054896 ).

P652: MEASURING MINIMAL RESIDUAL DISEASE BEYOND 10-4 THROUGH IGHV LEADER-BASED NEXT GENERATION SEQUENCING IMPROVES PROGNOSTIC STRATIFICATION IN CHRONIC LYMPHOCYTIC LEUKEMIA

P. Hengeveld 1,2,* , M. van der Klift 1 , P. M. Kolijn 1 , F. Davi 3 , F. Kavelaars 4 , E. de Jonge 5 , S. Robrecht 6 , J. Assmann 1 , L. van der Straten 1,2 , M. Ritgen 7 , P. Westerweel 2 , K. Fischer 6 , V. Goede 8 , M. Hallek 6 , M.-D. Levin 2 , A. Langerak 1

1 Department of Immunology, Erasmus MC, Rotterdam; 2 Department of Internal Medicine, Albert Schweizer Ziekenhuis, Dordrecht, Netherlands; 3 Department of Hematology, Pitié-Salpêtrière Hospital, Paris, France; 4 Department of Hematology; 5 Department of Clinical Chemistry, Erasmus MC, Rotterdam, Netherlands; 6 Department I. of Internal Medicine, Center for Intergrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne; 7 Department of Medicine II, University Hospital of Schleswig Holstein, Kiel; 8 Division of Oncogeriatrics, St Marien Hospital, Cologne, Germany

Background: The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10 -4 . Measuring MRD <10 -4 could help to further distinguish between CLL patients with durable remission and those at risk of early relapse.

Aims: To develop an academically sourced IGHV leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL.

Methods: The IGHV leader primer set developed by the EuroClonality-NGS working group was used to amplify all IGH rearrangements present in an end of treatment (EOT) DNA pool. The IGH clonal target was amplified in a single-step PCR and subsequent sequencing was performed on the Illumina MiSeq platform. To calculate MRD depth, NGS output was annotated through in the ARResT/Interrogate immunoprofiler. Technical validation of the IGHV leader-based assay was performed on contrived MRD samples, created by serial dilution of pretreatment CLL DNA pooled PBMC DNA from healthy donors. Clinical validation of the IGHV leader-based assay was performed on EOT samples from the CLL11 trial ( NCT01010061 ).

Results: In 176/183 (96%) measurements on contrived samples, the CLL-specific rearrangement was detected. The limit of detection and limit of quantitation were estimated at 3.4 [95% CI 1.9-16.0] and 3.8 [95% CI 1.7-8.2] malignant cells per assay, respectivelly. The limit of blank was found to be 0. Linearity was established in the MRD 10 -2 -10 -5 range ( r =0.94 [95%CI 0.91-0.96]). Of note, when provided with sufficient DNA input, MRD could even be detected down to MRD 10 -6 . There was high inter-assay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative PCR ( r =0.92, [95%CI 0.86-0.96]) and droplet digital PCR ( r =0.93, [95%CI 0.88-0.96]).

In a cohort of 67 patients from the CLL11 trial, using 5μg DNA input and MRD 10 -5 as a cut-off, undetectable MRD (uMRD) was associated with superior progression-free survival (PFS) and time to next treatment. Importantly, deeper MRD measurement allowed for additional stratification of patients with MRD <10 -4 but ≥10 -5 . Patients with MRD in this range had a significantly shorter PFS, compared to patients with uMRD <10 -5 , but significantly longer, compared to patients with MRD ≥10 -4 (median PFS: ≥10 -4 , 10.4 months; <10 -4 but ≥10 -5 , 27.5 months; uMRD <10 -5 , NR, 4-year PFS rate: ≥10 -4 , 66.7%; <10 -4 but ≥10 -5 , 25.0%; uMRD <10 -5 , 7.2%, P <0.001) (Figure 1).

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Summary/Conclusion: We here present an academically-developed, IGHV leader-based NGS assay for the detection and quantification of MRD in CLL beyond MRD 10 -4 . The assay has high sensitivity and is quantitative and linear up to MRD 10 -5 when using 5μg DNA input. Measurement to MRD 10 -6 is feasible, conditional on sufficient DNA input. The deeper MRD measurements enabled by the IGHV leader-based NGS assay resulted in improved stratification of CLL patients following treatment.

P653: OUTCOMES FOLLOWING TREATMENT WITH A COVALENT BTK AND BCL2 INHIBITOR AMONG PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)/SMALL LYMPHOCYTIC LYMPHOMA (SLL): A REAL-WORLD STUDY OF A LARGE U.S. DATABASE

T. A. Eyre 1,* , L. Hess 2 , T. Sugihara 3 , R. A. Walgren 2 , P. B. Abada 2 , H. Konig 2 , J. Pagel 4 , L. E. Roeker 5 , A. Mato 5

1 Oxford University Hospitals, NHS Foundation Trust, Oxford, United Kingdom; 2 Eli Lilly and Company, Indianapolis; 3 Syneos Health, Austin; 4 Loxo Oncology, Stamford; 5 Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Covalent Bruton tyrosine kinase inhibitors (cBTKi) and B-cell lymphoma 2 inhibitors (BCL2i) have improved the outcomes for patients with CLL/SLL. Although these agents are highly effective, they are not curative, and over time patients will require additional therapy. Real-world data of patient outcomes post cBTKi and BCL2i exposure in CLL/SLL are limited to small retrospective studies, and the optimal treatment in this setting is unknown.

Aims: This study was designed to address this data gap by evaluating the characteristics and clinical outcomes about patients with CLL/SLL after treatment with both cBTKi and BCL2i.

Methods: Eligible patients were adult patients in the Flatiron Health Electronic Health Record (EHR)-derived de-identified database diagnosed with CLL/SLL between December 2011 and October[LMH1] 2020. Follow-up data were available through October 2021 at the time of analysis. Patients were required to have at least one record of receiving both a cBTKi and a BCL2i. Time-to-event analyses using Kaplan-Meier method included duration of therapy, time to next treatment discontinuation, transformation or death, and overall survival (OS) from the time of cBTKi/BCL2i discontinuation.

Results: 339 patients (median age 66 years, interquartile range 59, 73; male 69.6%) met eligibility criteria. Most patients received cBTKi and BCL2i as early lines of therapy: 40.4% received cBTKi in first line and BCL2i in second line; an additional 21.8% received cBTKi in the second line and BCL2i in third line. Transformation was recorded among 24 (7.1%) patients during the study period. Of all eligible patients, 215 had discontinued both cBTKi and BCL2i and could be evaluated for post-cBTKi/BCL2i outcomes. A total of 47 patients (21.9% of the 215 who discontinued therapy) died before receiving subsequent therapy, 116/215 (54%) received subsequent therapy, and the remaining 52/215 (24.2%) were alive without additional treatment after cBTKi/BCL2i exposure. The median time from discontinuation of cBTKi/BCL2i (whichever agent was last) to the immediate next treatment discontinuation, transformation, or death was 4.6 months (95% confidence interaval [CI]: 3.0-6.9). Among those who received subsequent therapy, the most common immediate next line of therapy included additional BCL2i-based therapy (venetoclax re-treatment with or without other agents, n=71/116; 61.2%) or anti-CD20 antibody-based therapy (with or without other agents, 73/116 (62.9%). Fewer patients received PI3K inhibitor-based treatment (n=14/116; 12.1%) or additional cBTKi therapy (n=19/116; 16.4%) at any time after discontinuation of the initial cBTKi/BCL2i. As shown in the Figure, for those who received subsequent therapy (n=116), the median duration of the immediate post-cBTKi/BCL2i therapy was 4.8 months (95% CI: 3.0-7.3). Among the 215 who discontinued cBTKi/BCL2i therapy, median OS from discontinuation was 14.1 months (95% CI: 11.7-21.0).

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Summary/Conclusion: Patients with CLL/SLL who have been previously treated with both a cBTKi and BCL2i experience poor outcomes as observed by time to next treatment discontinuation, duration of subsequent therapy and overall survival. There remains a need for more effective therapies for patients with CLL/SLL after progression on cBTKi/BCL2i.

P654: IBRUTINIB PLUS VENETOCLAX IN PATIENTS WITH COMPLEX KARYOTYPE AND CHRONIC LYMPHOCYTIC LEUKEMIA

A. Petrenko 1,2,* , M. Kislova 1 , E. Dmitrieva 1 , T. Novikova 3 , M. Kislitsyna 3 , T. Obukhova 3 , E. Nikitin 1,2 , V. Ptushkin 1,2

1 Moscow City Center of Hematology, Botkin hospital; 2 Russian Medical Academy of Continuous Medical Education; 3 Karyology laboratory, National Medical Research Center for Hematology, Moscow, Russia

Background: In the largest study of Baliakas et al. (2019) the presence of at least 5 abnormalities, was associated with dismal clinical outcome, independently of the somatic hypermutation status and TP53 status. The presence of 3 or 4 aberrations is defined as clinically relevant in the absence of TP53. Studies by Kittai (2021) and Al-Sawaf (2020) showed the impact of karyotypic complexity on survival in patients with chronic lymphocytic leukemia (CLL) treated with ibrutinib or venetoclax. The complex karyotype (CK) is a topic that is being intensively researched, both in the aspect of increasing karyotypic complexity stratification and clonal evolution.

Optimal therapy for patients with CLL has not yet been developed. The combination therapy of ibrutinib and venetoclax was superior to chlorambucil and obinutuzumab in terms of undetectable minimal residual disease (MRD) responses according to data from the GLOW trial (Tunir, 2021). The importance of achieving a complete response with undetectable MRD as the goal of therapy in CLL was proposed (Montserrat, 2005).

Aims: The aim of our study is to evaluate the effectiveness of therapy with ibrutinib and venetoclax in combination for the patients with CLL and CK.

Methods: This ambilinear observational study included patients with CLL with high genetic complexity (high-CK), defined as >=5 aberrations or CK (>=3 aberrations) in combination with a 17p deletion (CK+del17p). The first retrospective cohort included patients treated with ibrutinib monotherapy (Imono) to progression or intolerable toxicity since May 2015. The second prospective cohort included patients receiving ibrutinib in combination with venetoclax (IVen) since July 2019. Venetoclax therapy was started at the 3rd month of ibrutinib (from the escalation phase). Combination therapy was continued until a complete response, defined as three consecutive PET-CT-negative and MRD-negative results 3 months apart. If this criterion was not achieved at 24th month of therapy, venetoclax was discontinued and ibrutinib continued indefinitely.

Results: Seventy-nine patients are included in the study. Twenty-nine patients in the first cohort and 50 patients in the second cohort. The characteristic is presented in Table. At the current follow-up periods, there were no significant differences in PFS and OS regarding a follow-up period <= 24 months (with the exception of death from COVID-19, since patients were not observed at parallel time intervals). In the group of patients treated with Imono, the majority of patients achieved partial remission or partial remission with lymphocytosis by 12 months. In 21 patients from Iven group, with a median follow-up of 7.4 months, a complete remission was achieved (72.4%); of these, 8 had unmeasurable MRD. Four patients did not complete the escalation period. There was a significant difference in the median MRD response achieved between 3 (log10>10) and 12 (log10<0,1) months in IVen group (p=0,03). In 2 patient from the IVen group progression of the disease was noted.

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Summary/Conclusion: Combination therapy with ibrutinib and venetoclax is an effective oral regimen for high-risk patients with complex karyotype disorders. PFS in both groups is currently not significantly different, which is obviously due to the short follow-up period. Patients receiving the IVen regimen achieve a significantly better response, which paves the way for allogeneic transplantation in these patients.

P655: RACIAL AND SOCIOECONOMIC DISPARITIES AMONG PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA IN THE US: ANALYSIS OF SURVEILLANCE, EPIDEMIOLOGY, AND END RESULTS PROGRAM DATA

A. Kittai 1,* , Y. Huang 1 , J. Fisher 1 , S. Bhat 1 , M. Grever 1 , E. Paskett 1 , K. Rogers 1 , J. Woyach 1

1 The Ohio State University, Columbus, United States of America

Background: Therapy for chronic lymphocytic leukemia (CLL) has changed dramatically over the past 20 years. With the cost of new therapies and rapid practice changes, it is unclear if patients are benefiting equally from this progress. We assessed Surveillance, Epidemiology, and End Results (SEER) program data to determine how race and socioeconomic status (SES) affect survival for patients with CLL.

Aims: Aim 1 - Determine the prognostic significance of race on overall survival for patients with CLL.

Aim 2 - Determine the prognostic significance of socioeconomic status on overall survival for patients with CLL.

Methods: CLL cases reported to 18 SEER Program registries from 2006 – 2018 were included. Patient characteristics such as age at diagnosis, sex, year of diagnosis, race, and SES as determined by rural/urban census tract residence (RUCA), and neighborhood (as represented by the Yost Index, a composite measure of 7 variables assessing different aspects of the SES of a census tract) were collected and analyzed. Multivariable cox regression (MVA) was used to determine adjusted odds of survival. Two separate databases were utilized, one which included data to 2018, and another which contained SES data but only had data available to 2016.

Results: 46,605 cases from 2009 – 2018 were identified without SES data. The median age was 70 years, 60% were male, and there was an even distribution of patients diagnosed with CLL annually from 2009 – 2018. Of the cases with race reported, 89.9% were white, 7.3% Black, 2.4% Asian/Pacific islander, and 0.3% American Indian/Alaska Native. After a median follow up of 47 months, the median 3-, 5-, and 10-year overall survival (OS) was 79.5%, 69.5%, and 48.8%, respectively. MVA showed Black race (HR 1.5, 95% CI 1.4 – 1.6) as the strongest independent prognostic variable for worse OS controlling for year of diagnosis, suggesting race was a significant factor in OS in the era of modern therapies.

Using the linked RUCA and Yost tertiles for SES, 47,867 cases of CLL from 2006 – 2016 were analyzed. Like the prior analysis, median age was 70 years, 60% were male, and there was an even distribution of patients diagnosed with CLL annually from 2006 – 2016. Of the cases with race reported, 90.4% were white, 7.1% Black, 2.3% Asian/Pacific islander, and 0.3% American Indian/Alaska Native. Per the Yost index, 24.8%, 33.8%, and 41.4% of patients were in Groups 1, 2, and 3, respectively. Per RUCA categories, 8.9% and 91.1% of patients were residents of rural and urban areas, respectively. MVA showed American Indian/Alaska Native, and Black race as independent prognostic variables for worse OS, and Yost group 2 and 3, representing higher SES, were found to be significant independent prognostic variables for improved OS (Table 1). In this analysis, race remained an independent variable for worse OS after controlling for SES.

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Summary/Conclusion: Black race and low SES are prognostic of OS in CLL. Further research is needed to determine whether this is due to access to therapy, quality of care, social determinants of heath, or disease biology.

P656: INCIDENT VENOUS THROMBOEMBOLISM (VTE) IN PATIENTS WITH MONOCLONAL B-CELL LYMPHOCYTOSIS (MBL) AND CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) – A POPULATION-BASED STUDY

A. Koehler 1,* , M. Moise 1 , T. Call 1 , K. Rabe 2 , S. Achenbach 2 , D. Crusan 2 , K. Bailey 2 , T. Petterson 2 , W. Ding 1 , S. Kenderian 1 , J. Leis 3 , Y. Wang 1 , E. Muchtar 1 , P. Hampel 1 , S. Schwager 1 , S. Slager 2 , N. Kay 1 , A. Ashrani 1 , S. Parikh 1

1 Hematology; 2 Biostatistics, Mayo Clinic, Rochester; 3 Hematology, Mayo Clinic, Scottsdale, United States of America

Background: Data on risk of VTE after MBL/CLL diagnosis is limited;it is unclear if MBL/CLL truly confers increased risk of VTE as seen in other malignancies.

Aims: To determine incidence of VTE after MBL/CLL diagnosis and compare it to age- and sex-matched VTE incidence in the general population.

Methods: We identified all individuals with newly diagnosed, untreated MBL or CLL between 1998-2021 within a 27-county region surrounding Rochester, MN. Patient demographics and CLL-specific characteristics were extracted from the Mayo Clinic CLL Database and the electronic health record (EHR). Incident VTE after diagnosis of MBL/CLL was identified by querying a) the Mayo Clinic CLL Database; b) EHR for VTE-specific ICD-9 and ICD-10 diagnosis codes; and c) all radiographic studies performed during longitudinal care of these patients. Risk of VTE was estimated using Cox proportional hazards model with CLL/SLL status as a time-dependent variable. The unadjusted cumulative risk of VTE was estimated using Kaplan-Meier methods in SAS 9.4. Rate of incident VTE among age- and sex-matched population residing in Olmsted County, MN from 2001-2015 was pulled from the Rochester Epidemiology Project. Age- and sex-specific VTE risk associated with MBL/CLL (standardized incidence rate ratio; SIR) was estimated by dividing observed post-MBL/CLL diagnosis VTE incidence rate by expected VTE incidence rate in the general population. We performed an exact binomial test in the statistical package R (version 4.1.2) to evaluate whether the proportion of VTE cases after MBL/CLL diagnosis (versus those occurring in the general population) was consistent with their respective proportion of person-years at-risk for VTE.

Results: We identified 946 patients with newly diagnosed MBL/CLL, of whom 42 (4%) reported a prior history of VTE and were excluded from this analysis. Of the 904 evaluable subjects, 293 had MBL and 611 had CLL. The median age was 69 years (range, 28 - 96 years) and 587 (65%) were male.

After a median follow-up of 6 yrs (range: 1 day-23 yrs), 70 of 904 patients developed VTE after MBL/CLL diagnosis; 43 (61%) with deep vein thrombosis (DVT), 24 (34%) with pulmonary embolism (PE), and 3 (4%) with both DVT and PE as the first event. Risk of VTE was similar in CLL compared to MBL [HR (95% CI) = 0.90 (0.49-1.65)]. The 5-year and 10-year cumulative risk of VTE was 4.9% and 11.5%, respectively. Forty-seven (68%) patients had an identifiable provoking factor apart from MBL/CLL including second active malignancy (n=21), surgery (n=9), recent hospitalization (n=6), travel (n=4), trauma (n=3), line-associated VTE (n=3), and immobility (n=1).

The age-adjusted VTE incidence rates for females and males with newly diagnosed MBL/CLL were 1275 and 1228 per 100,000 person-years, respectively. In contrast, the age-adjusted VTE incidence rates for females and males residing in Olmsted County, MN were 193 and 218 per 100,000 person-years, respectively. The overall VTE incidence rate in females and males after MBL/CLL diagnosis was 6.0 (95% CI: 4.0-8.8) and 5.7 (95% CI: 4.1-7.7) higher, respectively, compared to the general population; the risk of VTE was highest in 18–49-year-old MBL/CLL patients (SIR: 16.2, 95% CI: 3.3-47.6) (Figure 1).

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Summary/Conclusion: In this large population-based cohort study, 1 in 12 patients with MBL/CLL developed VTE after a median follow-up of 6 yrs. Patients with MBL/CLL demonstrated a 6-fold increased risk of VTE compared to the age- and sex-matched general population. Importantly, nearly 3 in 4 MBL/CLL patients had additional provoking factors at time of VTE, most notably second active malignancy.

P657: REAL-WORLD TREATMENT PATTERNS OF PATIENTS DIAGNOSED WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IN THE UNITED STATES (US)

X. Yang 1,* , E. Zanardo 2 , D. Lejeune 3 , E. De Nigris 1 , E. Sarpong 1 , N. Lema 2 , M. Farooqui 1 , F. Laliberté 3

1 Merck & Co., Inc., Kenilworth; 2 Analysis Group, Inc., Boston, United States of America; 3 Groupe d’analyse, Ltée, Montréal, Canada

Background: CLL represents the most common type of leukemia among adults in the US, but current data on the treatment patterns of patients in clinical practice is limited. Standard of care frontline therapy consists of various types of targeted therapy or chemoimmunotherapy (CIT), but it is reserved for patients with advanced-stage disease.

Aims: This study aims to describe real-world treatment patterns among patients diagnosed with CLL in the US, with a focus on ibrutinib therapy.

Methods: A retrospective database analysis was conducted using the Optum Clinformatics DataMart TM database (01/2007–07/2020). Patients with ≥2 medical encounters with a diagnosis code for CLL or small lymphocytic lymphoma (SLL) on different dates were selected (earliest date was defined as the index date). At least 12 months of continuous enrollment pre-index date (baseline period) and ≥18 years of age as of the index date were required. Patients with a diagnosis for mantle cell lymphoma or with evidence of anticancer therapy (antineoplastic, radiation, or cell therapy) during baseline were excluded. A subset of patients with ≥1 pharmacy claim for ibrutinib was identified. An adapted algorithm developed from previously published studies was used to identify lines of therapy (LOTs). Treatment patterns, including duration of therapy (DOT) and regimens were reported. DOT spanned from LOT initiation up to discontinuation of all agents in the LOT, a switch to another LOT, or the addition of a new agent. Median time to the first line (1L) and from 1L to second line (2L) were evaluated using a Kaplan-Meier analysis (KM) to account for censoring.

Results: Among 23,087 patients with CLL, the median age was 74 years and 43% were female. Of these, 1,387 were treated with ibrutinib (median age: 75; female: 38%). Patients had a mean Charlson comorbidity index score of 1.9. Analysis of treatment patterns (Table 1) showed that 7,192 patients (31%) were treated with ≥1 LOT (mean ± standard deviation [SD] DOT of 1L: 1.6 ± 2.0 years), and 10% of patients were treated with ≥2 LOT (mean ± SD DOT of 2L: 1.2 ± 1.7 years). Ibrutinib was used by 907 patients in 1L (DOT in 1L: 1.0 ± 1.1 years) and 617 in subsequent LOTs (DOT in 2L+: 0.9 ± 1.1 years). Median time from index date to 1L initiation was 8.1 years. Of the patients who used antineoplastics therapies in 1L, a majority used CLL-related ones (79%). Most treated patients received targeted therapy (37%; rituximab: 19% and ibrutinib: 12%) and CIT (32%; bendamustine + rituximab: 13% and cyclophosphamide + fludarabine + rituximab: 5%). In 2L, 16% of patients used ibrutinib, 12% a bendamustine + rituximab regimen, and 4% chlorambucil. Median time from 1L initiation to 2L initiation was 4.4 years. Proportion of patients treated with CIT tended to diminish with subsequent LOT, whereas the proportion treated with ibrutinib tended to increase.

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Summary/Conclusion: This real-world long-term data shows that patients receive their 1L many years after a first CLL diagnosis, with a median time to treatment of ~8 years. Among US patients with CLL, rituximab, bendamustine + rituximab, and ibrutinib were identified as the most used 1L regimens, and the latter two as the most used 2L regimens. As novel therapies are increasingly used, and genetic testing becomes more available, further research will be needed to evaluate the changes in the way CLL is treated and its effects on clinical outcomes.

P658: COMPARISON OF CHARACTERISTICS AND OUTCOME OF COVID-19 INFECTION IN PATIENTS WITH LYMPHOPROLIFERATIVE DISEASE AND IN PATIENTS FROM THE GENERAL POPULATION - EXPERIENCE FROM THREE UNIVERSITY CENTERS

O. Markovic 1,2,* , A. Divac 2 , Z. Cvetkovic 1,3 , D. Stanisavljevic 4 , K. Markovic 5 , I. Bukurecki 2 , T. Bibic 3 , N. Stanisavljevic 1,2 , J. Bila 1,6 , M. Zdravkovic 1,7

1 University of Belgrade, Medical Faculty; 2 Department of Hematology, CHC Bezanijska kosa; 3 Department of Hematology, CHC Zemun; 4 Institut of Statistics, University of Belgrade, Medical Faculty; 5 Department of Hematology, CHC Zvezdara; 6 Clinic of Hematology, Clinical Center of Serbia; 7 Department of Cardiology, CHC Bezanijska kosa, Belgrade, Serbia

Background: Patients with lymphopproliferative diseases (LPD) and covid-19 have poor outcome as consequence of inadequate humoral and cellular immunity due to the hematological disease itself but also due to the administered chemotherapy which further increases the risk of complications and mortality.

Aims: The aim of this study is to analyze demographic and clinical characteristics, laboratory parameters, the presence of comorbidities, laboratory parameters, disease status, as well as outcome of the patients with COVID-19 and lymphoproliferative disease and compare them with characteristics of covid-19 infection in patients from general population (GP).

Methods: This is a prospective multicenter observational study conducted in the following 3 University centers in period from 15 March 2020 to 31 October 2021. The study included hospitalized patients diagnosed with COVID-19 infection: 161 patients with LPD and 162 patients from the GP. Statistical analysis included demographic statistics, the χ 2 test, the Mann-Whitney test, Kaplan-Meier method for analysis of survival and multivariate logistic regression model for analysis of risk factors for mortality.

Results: In the LPD group, there were 54 patients (33.54%) with chronic lymphocytic leukemia (CLL), 72 patients (44.72%) with Non-Hodgkin lymphoma/Hodgkin lymphoma (NHL/HL) and 35 patients (21.74%) with multiple myeloma (MM). Ninety-six (59,63%) patients were on active treatment and 14(8.7%) patients were newly diagnosed. The LPD and GP group differed significantly in relation to age (66 vs. 54 years), gender (male: 60.2% vs. 75.3%), presence of comorbidities (109, 67.7% vs. 81, 50%) patients, covid score (mild 22.5% vs. 1.9%, moderate 80, 50.3% vs. 121, 74.7%), and severe/critical 44(27.1%) vs. 38(23.4%) patients. Group of patients with LPD had also significantly lower level of hemoglobin, lowest value of lymphocytes, platelets, higher level of CRP, ferritin, D-dimer (on admission and maximal values) and LDH with respect to group of patients from GP. Mortality rate was higher in LPD group of patients than in GP group (45, 28% vs. 26, 16%) patients. Among the LPD group, the highest mortality rate was observed in patients with MM (16, 45.71%) patients, followed by CLL (15, 27.9%) patients and NHL/HL group (14, 19.4%) patients. Independent factors related to survival are high value of D dimer, anemia (hemoglobin <100g/l) and moderate/critical COVID score in LPD group, while maximal value of CRP, anemia, leucocytosis and age (>60 years) in GP group.

Summary/Conclusion: Our study showed significant difference in the characteristics and outcome in covid-19 between patients with LPD and patients from GP. Patients with LPD are older, they have significantly higher inflammatory parameters and more frequent presence of comorbidities compared to patients from GP. Independent factors related to survival in the LPD group are high values of D dimer, moderate/critical COVID score and anemia, while maximal values of CRP, anemia and older age are identified in the GP group.

P659: IBRUTINIB IN OVER-EIGHTIES PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: A MULTICENTER ITALIAN COHORT

G. Reda 1 , V. Mattiello 1,* , A. M. Frustaci 2 , A. Visentin 3 , F. R. Mauro 4 , I. Innocenti 5 , M. Gentile 6 , D. Giannarelli 7 , A. Noto 1 , R. Cassin 1 , L. Laurenti 5 , A. Tedeschi 2

1 Hematology Unit, IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 2 Department of Hematology, Niguarda Cancer Center, ASST Grande Ospedale Metropolitano Niguarda, Milano; 3 Hematology and Clinical Immunology unit, Department of Medicine University of Padua, Padova; 4 Hematology, Department of Translational and Precision Medicine, ‘Sapienza’ University, Rome; 5 Dipartimento Scienze Radiologiche ed Ematologiche, Divisione di Ematologia Fondazione Policlinico universitario A Gemelli, Roma; 6 Azienda Ospedaliera of Cosenza, Cosenza; 7 Biostatistic Unit, Scientific Directorate, Fondazione Policlinico Universitario A. Gemelli, IRCCS, Roma, Italy

Background: Ibrutinib is a first-in-class covalent inhibitor of Bruton’s tyrosine kinase that has changed the treatment paradigm of chronic lymphocytic leukemia (CLL) in both treatment-naive (TN) and relapsed/refractory (R/R) setting. Clinical trials, in selected populations showed that the BTK inhibitor is manageble even in the elderly. Nevertheless, few studies are focused on the role of ibrutinib in unselected patients aged ≥80 years.

Aims: We aimed to assess the activity and safety of ibrutinib in a cohort of patients with CLL aged ≥80 years at therapy start. The primary endpoint was safety evaluation. Key secondary endpoints were overall (ORR), complete (CR) and partial (PR) response rates according to iwCLL 2018, median progression free survival (PFS) and overall survival (OS).

Methods: Sixty consecutive patients (age ≥80 years) diagnosed with TN (20 patients) or R/R CLL (40 patients) who started ibrutinib were enrolled in this multicenter study from six Italian sites; data were retrospectively analyzed. Pre-existing cardiovascular risk factors were present in 66.7% of patients; 23.3% had experienced a cardiovascular event prior ibrutinib initiation. (Table 1)

Results: After a median follow up of 27 months, at least one adverse event (AE) occurred in 68.3% patients, leading to treatment discontinuation in 23.3%. The most common grade ≥3 events were infections (23%) and neutropenia (6%). Cardiovascular events occurred in 31.6% of patients, with an incidence of atrial fibrillation (AF) and arterial hypertension both increasing over time and reaching 16% at 24 months. Although no significant increase in incidence of cardiovascular events was noted among patients with concomitant cardiovascular risk factors or previous events, baseline higher left atrial diameter at echocardiography was predictive of AF occurrence. OS did not differ between patients experiencing AF or not. During treatment, two patients experienced sudden cardiac death. Bleeding was the most frequent AE, occurring in 36.6% of patients, with a median time to event of 24 months and predominantly in patients assuming concomitant antiplatelet or anticoagulant drugs. A total of 23 infective events was registered, mostly in the first 12 months, leading to drug permanent discontinuation in 5 patients. Of note, the infectious rate was higher in del(17p) CLL (p=0.05). The obtained ORR was 88.3%, with 21.7% of patients achieving CR and 66.7% PR; median PFS was 51.8 months (95% CI: 47.4-56.2). No significant difference in PFS was observed comparing TN to R/R patients (p=0.83), or IGHV mutated and unmutated patients (p=0.45). Furthermore, no difference emerged comparing PFS data of patients with TP53 dysfunction (del17 and/or TP53 mutation) to patients without TP53 dysfunction (p=0.13). Patients achieving a response during ibrutinib experienced a prolonged PFS compared to patients achieving SD (p<0.0001). Drug withholding for more than 7 days due to ibrutinib-related toxicities affected patients’ outcome, translating in a shorter PFS with a trend to statistical significance (p=0.07). Median overall survival was 53.2 months (95% CI: 43.3-63.0).

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Summary/Conclusion: A high proportion of patients had an overall response to ibrutinib and the risk-benefit profile was favourable, providing further evidence for use of ibrutinib in this subset of elderly and unselected patients. Safety profile remains consistent with literature data with no emergent adverse events, thus making ibrutinib an attractive therapeutic possibility even in patients with advanced age and multiple comorbidities.

P660: SEROLOGIC RESPONSE TO THE SECOND AND THIRD DOSE OF THE SARS-COV-2 VACCINE IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: RESULTS OF A PROSPECTIVE, CENTRALIZED, MULTICENTER STUDY.

F. R. Mauro 1,* , D. Giannarelli 2 , C. Galluzzo 3 , A. Visentin 4 , A. M. Frustaci 5 , P. Sportoletti 6 , C. Vitale 7 , G. Reda 8 , M. Gentile 9 , L. Levato 10 , R. Murru 11 , D. Armiento 12 , C. Ielo 13 , R. Maglione 13 , E. Crisanti 13 , A. Cipiciani 14 , V. Mattiello 15 , V. Gianfelici 10 , L. Barabino 16 , R. Amici 17 , M. Coscia 18 , A. Tedeschi 19 , L. Trentin 20 , S. Baroncelli 17

1 Department of Translational and Precision Medicine-Sapienza University of Rome, Italy, Sapienza University; 2 Design and Analysis of Clinical Trials Unit, Scientific Directorate, Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy; 3 National Center for Global Health, Istituto Superiore di Sanità, Rome; 4 Hematology and Clinical Immunology Unit, Department of Medicine University of Padua, Padova, University of Padua, Padova; 5 Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milan; 6 Institute of Hematology-Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, Perugia; 7 Department of Molecular Biotechnology and Health Sciences, University of Torino and Division of Hematology, A.O.U. Città della Salute e della Scienza di Torino, Rome; 8 Hematology Unit, Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore, University of Milan, Milan, Italy., Milan; 9 Hematology and Oncology Department, Biotechnology Research Unit, Cosenza, Italy., Cosenza; 10 Department Hematology-Oncology, Azienda Ospedaliera Pugliese-Ciaccio, Catanzaro; 11 Hematology and Stem Cell Transplantation Unit, Ospedale Oncologico A. Businco, ARNAS “G. Brotzu”, Cagliari; 12 Hematology, Stem Cell Transplantation Unit, University Campus Bio-Medico; 13 Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome; 14 Institute of Hematology-Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, Perugia; 15 Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore, University of Milan, Milan; 16 Hematology and Stem Cell Transplantation Unit, Ospedale Oncologico A. Businco, ARNAS “G. Brotzu”, Cagliari; 17 National Center for Global Health, Istituto Superiore di Sanità, Rome; 18 Department of Molecular Biotechnology and Health Sciences, University of Torino and Division of Hematology, A.O.U. Città della Salute e della Scienza di Torino, Torino; 19 ASST Grande Ospedale Metropolitano Niguarda, Milan; 20 livio.trentin@unipd.it, Hematology and Clinical Immunology Unit, Department of Medicine, University of Padua, Padova, Italy

Background: Patients with chronic lymphocytic leukemia (CLL) show high infection-related morbidity and mortality due to variable degree of humoral and cellular immune deficiency. High Covid-related mortality and reduced response to the SARS-Cov-2 vaccine have been reported in this patient population.

Aims: We carried out a prospective multicenter study to define the rate of CLL patients with an appropriate immune response after the mRNA SARS-CoV2 vaccine (Pfizer-BioNTech; Moderna).

Methods: Two-hundred patients with CLL received the first dose of the SARS-CoV-2 vaccine between February and August 2021. Centralized assessment of the anti-SARS-Cov-2 IgG levels (Sero Index, Kantaro Quantitative SARS-CoV-2 IgG Antibody, RUO-R&D System) was performed at the Istituto Superiore di Sanità of Rome, Italy. The median follow-up of this study is 10.7 months (range 1-12.9).

Results: The median age of patients was 70 years, the median IgG level was 635 mg/dl, 61% of patients were IGHV unmutated, and 34% showed TP 53 disruption. The majority of patients, 83.5%, were previously treated. Prior treatment included chemoimmunotherapy in 20 (10%) patients, ibrutinib–based therapy in 72 (36%; front-line, 21%; advanced line, 15%), venetoclax-based therapy in 75 (37.5%; front-line, 13.5%; advanced line, 24%). Overall, 135 (77.5%) patients had been previously treated with rituximab, 33 (16.5%) of them within 12 months before vaccination. We assessed the serologic response after the second dose of the SARS-CoV2 vaccine in 195 patients while five were excluded from the analysis (positive test before vaccination, 3 patients; lost to the follow-up, 1; Richter syndrome, 1). Adequate levels of anti-SARS-Cov-2 IgG were detected in 76/195 (39%) patients. Age (<70 vs.≥ 70 years; p <0.0001), CIRS value (<6 vs. ≥6; p=0.005), beta-2 microglobulin (<3.5 vs. ≥ 3.5mg/dl; p=0.04), IgG levels (<550 vs. ≤ 550 mg/dl; p <0.0001), prior treatment (p=0.0001), number of prior treatments (0 + 1 vs. ≥ 2; p=0.002) and the time between prior rituximab and vaccination (>12 vs. ≤12 month; p=0.001) showed a significant impact on the humoral response. In multivariate analysis only age (OR: 0.92 [95% CI: 0.92-0.97] p=0.0001), IgG levels (OR: 0.28 [95% CI: 0.13-0.58] p<0.001), and the time between prior rituximab and vaccination (OR: 0.10 [95% CI: 0.03-0.37] p=0.001), revealed a significant and independent impact on response. When the analysis was restricted to patients who received targeted therapy, in addition to the younger age (OR: 0.96 [95% CI: 0.92-0.99] p=0.04), higher IgG levels at baseline (OR: 0.31 [95% CI: 0.12-0.79] p=0.014), longer time between the start of ibrutinib or venetoclax-based therapy and vaccination (<18 vs.≥18 months; OR: 0.17 [95% CI: 0.06-0.44], p <0.0001) showed a favorable and independent impact on response. Ninety-three% (182/195) of patients received a third dose of the vaccine. A significant increase in the rate of serologic responses, 51.5% (85/165 evaluated patients, p=0.019), was observed after the booster dose. Moreover, a response was detected in 25% (26/103 evaluated patients) of previously seronegative patients.

Summary/Conclusion: In this prospective, multicenter, centralized study, we recorded an effective immune response to the SARS-CoV-2 vaccine in about a third of patients with CLL. Younger age, higher IgG levels, no prior treatment, or stable disease after targeted therapy that suggest preserved immunocompetence were associated with a greater likelihood of achieving an effective immune response. A booster dose of the SARS-CoV-2 vaccine proved beneficial also in previously seronegative patients.

P661: POPULATION-WIDE PATTERNS OF CARE IN CHRONIC LYMPHOCYTIC LEUKEMIA IN AUSTRALIA: AN ANALYSIS OF THE PHARMACEUTICAL BENEFITS SCHEME DATASET

C. Tam 1,* , F.-L. Zhao 2 , R. Gauba 2 , K. Yang 3 , S. Azmi 3 , S. C. Li 4

1 Peter MacCallum Cancer Centre, St. Vincent’s Hospital, and University of Melbourne, Melbourne; 2 BeiGene AUS PTY Ltd., Sydney, Australia; 3 BeiGene USA, Inc., San Mateo, United States of America; 4 University of Newcastle, Callaghan, Australia

Background: The treatment landscape in patients with chronic lymphocytic leukemia (CLL) is changing with the approvals of Bruton’s tyrosine kinase inhibitors (BTKis) in Australia.

Aims: We sought to understand the practice impact of the introduction of publicly funded novel agents for the treatment of CLL. The objective of this study was to describe the evolving treatment patterns of Australian patients with CLL over the last 10 years using population-wide prescription records.

Methods: Patients who initiated a treatment for CLL between 01/01/2011 and 07/31/2021 were extracted from the Services Australia 10% Pharmaceuticals Benefits Scheme (PBS) dataset. This dataset includes the dispensing records for 10% of the Australian population and captures all publicly funded treatments in Australia. The index date was defined as the commencement of any drug for the treatment of CLL. First-line (1L) therapy was defined as the first treatments prescribed for CLL. A patient was defined as relapsed/refractory (R/R) if they had commenced a drug which was in a different therapeutic category, or if they re-started the same regimen after a gap of more than 180 days. Descriptive analyses were conducted to examine the use of treatment regimens for the overall 10-year population by line of therapy. Analyses by calendar year were also performed to assess changes in treatment patterns.

Results: Overall, 803 patients with CLL were identified. The majority of patients were male (65%) and age > 60 years (77%), with most being aged 70-79 years (33% of total). Many patients were receiving comedications at baseline, including antihypertensives (47%), antipsychotics or antidepressants (17%), and/or anticoagulants (13%). In the overall population (2011-2021), the majority of patients had received 1L treatment with fludarabine-cyclophosphamide-rituximab (FCR, 49%), chlorambucil ± CD20 (27%), or CD20 monotherapy (17%). The most commonly used regimens in R/R patients at any subsequent episode of treatment included CD20 monotherapy (56%), BTKi (41%) or FCR (33%). A trend in adoption of novel agents was observed throughout the years following their PBS listing. Analysis by calendar year showed that from 2011 to 2020 use of FCR in 1L decreased from 78% to 10%; and use of BTKis in R/R increased from 0% to 62%.

Summary/Conclusion: CLL treatment patterns have significantly changed in Australia since the introduction of the BTKis (e.g., ibrutinib, acalabrutinib). The use of FCR in 1L CLL has decreased and use of BTKis in R/R patients has increased.

P662: PATIENT-REPORTED OUTCOMES FROM A PHASE 3 RANDOMIZED STUDY OF ZANUBRUTINIB VERSUS BENDAMUSTINE PLUS RITUXIMAB (BR) IN PATIENTS WITH TREATMENT-NAÏVE (TN) CLL/SLL

P. Ghia 1,* , G. Barnes 2 , K. Yang 2 , C. Tam 3,4,5,6 , P. Hillmen 7 , T. Robak 8 , J. Brown 9 , B. Kahl 10 , T. Tian 2 , A. Szeto 2 , J. Paik 2 , M. Shadman 11,12

1 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 2 BeiGene USA, Inc., San Mateo, United States of America; 3 Peter MacCallum Cancer Centre, Melbourne; 4 University of Melbourne, Parkville; 5 St Vincent’s Hospital Melbourne, Fitzroy; 6 Royal Melbourne Hospital, Parkville, Australia; 7 St James’s University Hospital, Leeds, United Kingdom; 8 Medical University of Lodz, Lodz, Poland; 9 Dana-Farber Cancer Institute, Boston; 10 Washington University School of Medicine, St. Louis; 11 Fred Hutchinson Cancer Research Center; 12 Medicine, University of Washington, Seattle, United States of America

Background: Zanubrutinib is an oral, highly selective, next-generation Bruton tyrosine kinase (BTK) inhibitor. In cohort 1 of the phase 3 SEQUOIA trial (BGB-3111-304; NCT03336333 ), efficacy and safety of zanubrutinib and BR were compared in adult patients (pts) with TN chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) without del(17p).

Aims: Here, we report the health-related quality of life (HRQoL) outcomes from an interim analysis of the SEQUOIA trial.

Methods: Patient-reported outcomes (PROs) were secondary endpoints and assessed using the EORTC QLQ-C30 and EQ-5D-5L VAS. Patients completed these questionnaires at baseline, every 12 weeks for 96 weeks, and then every 24 weeks until disease progression, death, or withdrawal from study. The PRO endpoints included global health status (GHS), physical and role functions, and symptoms of fatigue, pain, diarrhea, and nausea/vomiting, measured by QLQ-C30, with critical clinical cycles of Weeks 12 and 24. Descriptive analyses were performed on all questionnaire responses, and a mixed model for repeated measures was performed on the PRO endpoints at Weeks 12 and 24.

Results: Baseline demographics and disease characteristics between the zanubrutinib (n=241) and BR (n=238) arms were similar. Across all pts, adjusted completion rates for PROs (# of pts who completed the questionnaire at each cycle divided by # of pts expected to complete the questionnaires) were high (~80%) at Weeks 12 and 24. Compared with pts who received BR, pts treated with zanubrutinib experienced greater improvements in HRQoL at Weeks 12 and 24 as reported on the QLQ-C30 ( Table ). By Week 24, significant improvements were observed with zanubrutinib vs BR in GHS, physical functioning, role functioning as well as greater reductions in diarrhea, fatigue, and nausea/vomiting. Per EQ-5D-5L VAS, comparable improvements from baseline between zanubrutinib and BR in the health status were observed at Weeks 12 (4.3 vs 3.5) and 24 (4.5 vs 4.9), respectively.

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Summary/Conclusion: In the SEQUOIA trial, zanubrutinib was associated with significant improvements in HRQoL in pts with TN CLL/SLL without del(17p), as indicated by the PRO endpoints of the GHS, physical and role functions, and greater reductions in symptoms of fatigue, diarrhea and nausea/vomiting compared with BR.

P663: HEALTH-RELATED QUALITY OF LIFE OUTCOMES ASSOCIATED WITH ZANUBRUTINIB VS IBRUTINIB MONOTHERAPY IN PATIENTS WITH RELAPSED/REFRACTORY (RR) CLL/SLL: RESULTS FROM THE RANDOMIZED PHASE 3 ALPINE TRIAL

P. Hillmen 1,* , J. Brown 2 , N. Lamanna 3 , S. O’Brien 4 , C. Tam 5,6,7,8 , L. Qiu 9 , K. Yang 10 , G. Barnes 10 , K. Wu 10 , T. Salmi 11 , B. Eichhorst 12

1 St James’s University Hospital, Leeds, United Kingdom; 2 Medical Oncology, Dana-Farber Cancer Institute, Boston; 3 Herbert Irving Comprehensive Cancer Center, Columbia University, New York; 4 Chao Family Comprehensive Cancer Center, University of California, Irvine, United States of America; 5 Peter MacCallum Cancer Centre, Melbourne; 6 University of Melbourne, Parkville; 7 St Vincent’s Hospital Melbourne, Fitzroy; 8 Royal Melbourne Hospital, Parkville, Australia; 9 Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; 10 BeiGene USA, Inc., San Mateo, United States of America; 11 BeiGene Switzerland GmbH, Basel, Switzerland; 12 Department of Internal Medicine, University of Cologne, Center for Integrated Oncology Aachen, Cologne, Germany

Background: In the phase 3 ALPINE trial (BGB-3111-305; NCT03734016 ), efficacy and safety of zanubrutinib, a highly selective, next-generation Bruton tyrosine kinase inhibitor (BTKi), were compared with the first-generation BTKi, ibrutinib, in adult patients with RR chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).

Aims: This abstract, based on the interim analysis of the ALPINE trial, describes the effects of zanubrutinib monotherapy and ibrutinib monotherapy on the health-related quality of life (HRQoL).

Methods: Health-related quality of life was examined by patient-reported outcomes (PROs) measures assessed by EORTC QLQ-C30 and EQ-5D-5L at baseline, Cycle 1, and then every 3rd cycle until end of treatment. Key PRO endpoints included global health status (GHS), physical and role functions, and fatigue, pain, diarrhea, and nausea/vomiting. Descriptive analysis on all the scales was conducted as was a mixed model repeated-measure (MMRM) analysis of the longitudinal QLQ-C30 data. Data presented are from key cycles (7 and 13), corresponding to 6 and 12 months of treatment, respectively.

Results: In the intent-to-treat population (N=652; zanubrutinib, n=327; ibrutinib, n=325), adjusted completion rates were high (>85%) in both arms at Cycles 7 and 13. On the QLQ-C30, estimated mean treatment differences and 95% CI in key PRO endpoints demonstrated treatment differences, in favor of zanubrutinib, in GHS, physical functioning, and fatigue in Cycle 7, and diarrhea in Cycle 13 ( Table ). Mean change from baseline (SD) in EQ-5D-5L VAS showed consistently more improvement with zanubrutinib compared with ibrutinib at both Cycle 7 (8.4 [18.2] vs 4.0 [16.6]) and Cycle 13 (6.8 [18.8] vs 5.2 [17.5]).

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Summary/Conclusion: In the ALPINE trial, patients with RR CLL/SLL who received zanubrutinib monotherapy reported improvements in key HRQoL endpoints compared with patients who received ibrutinib monotherapy.

P664: GRADING QUALITY OF EVIDENCE AND STRENGTH OF RECOMMENDATIONS FOR THE TREATMENT OF RELAPSED/REFRACTORY CLL.

S. Molica 1,* , C. Patti 2 , P. Sportoletti 3 , A. Chiarenza 4 , A. M. Giordano 5 , F. Chiurazzi 6 , N. Di Renzo 7 , P. Musto 8 , F. Pane 9 , F. Di Raimondo 4 , L. Trentin 10 , F. R. Mauro 11 , D. Giannarelli 12

1 Department Hematology, Hull University Teaching Hospitals NHS Trust, Hull, UK, Hall, United Kingdom; 2 hematology, Ospedale Cervello, Palermo; 3 Hematology Unit, University Perugia, Perugia; 4 Hematology Unit, Ferrarotto Hospital, Catania; 5 5Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, University of Bari, Bari; 6 Department of Clinical Medicine and Surgery, Hematology Unit, Federco II Medical School Napoli, Napoli; 7 Hematology Unit, Presidio Ospedaliero Vito Fazi, Lecce; 8 Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, University of Bari, Bari; 9 Department of Clinical Medicine and Surgery, Hematology Unit, Federico II Medical School Napoli, Napoli; 10 Hematology and Clinical Immunology Unit, Department of Medicine, University of Padua, Padua; 11 Hematology, Department of Translational and Precision Medicine, Sapienza University Rome; 12 Biostatistic Unit, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Rome, Italy

Background: Changes in the treatment landscape of CLL have complicated the decision-making processes, especially in the context of relapsed/refractory (R/R) disease. In this setting current guidelines do not provide clear information on the sequencing of targeted agents (TAs).

Aims: With this in mind a series of evidence-based recommendations were produced to support pts, clinicians, and other health care professionals in their decisions about the management of R/R CLL.

Methods: A multidisciplinary panel that included specialists in methodology, CLL management, and hematology, performed a systematic review of the literature using the databases MEDLINE, EMBASE, and the Cochrane Library, then participated in a virtual consensus conference held at the end of December 2021. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to formulate recommendations. Specifically, the strength of recommendations was determined by the balance between desirable and undesirable consequences of alternative management strategies, quality of evidence, variability in values, preferences, and resource used.

Results: Firstly, the panelists prioritized 10 scientific questions in the context of 4 specific scenarios of R/R CLL: (1) relapse after frontline chemo-immunotherapy (CIT), (2) progression of disease (PD) while receiving ibrutinib (IBR), (3) progression after treatment with venetoclax (VEN), and (4) impact of adherence to IBR or VEN on the clinical outcome.

The following recommendations were produced:

1- A TA should be offered to all pts relapsing after first-line CIT (strong recommendation based on high-quality evidence). In absence of head-to-head trials comparing a BTKi vs an anti-BCL2 inhibitor, the panel suggests a patient-oriented choice consisting of either continuous BTKi (i.e., IBR or acalabrutinib) or time-limited (TL) VEN-based therapy in both standard or high genomic risk (conditional recommendation based on moderate-quality evidence).

Remarks : TL VEN-based regimen is more cost-effective than continuous IBR.

2- The panel recommends switching to VEN for those pts who experience PD whilst on a BTKi (in absence of a valid alternative the recommendation becomes strong even if the quality evidence is moderate). There is no evidence to recommend the combination of VEN plus rituximab or an alternative covalent BTKi over VEN single agent (conditional recommendation based on low certainty in the evidence of effects). Noncovalent BTKi pirtobrutinib needs validation in phase 3 studies.

3- The panel recommends utilizing a BTKi monotherapy instead of re-challenging VEN for pts who have progressed after completing VEN-based therapy (conditional recommendation based on low certainty in the evidence of effects).

4- Finally, panelists agreed that the impact of adherence to IBR or VEN on PFS in R/R pts is supported by a low quality of evidence (conditional recommendation based on low certainty in the evidence of effects).

Summary/Conclusion: These evidence-based consensus recommendations strongly support the use of a TA in R/R CLL patients. The choice between TL and continuous TA is patient-oriented with the remark that a TL approach is cost-effective within current pricing structures. A cross-over approach to an alternative TA is suggested in pts progressing on BTKi or BCL2i.

However, these recommendations are guided by the fundamental principle that optimal patient care involves ongoing discussions between clinicians and pts, continuously addressing goals of care and the relative risk-benefit balance of treatment.

P665: CARDIAC ADVERSE EVENTS AND SECOND PRIMARY MALIGNANCIES WITH ACALABRUTINIB IN CHRONIC LYMPHOCYTIC LEUKEMIA -A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS

K. Parmar 1,* , D. Pawar 1 , K. Bharathidasan 1 , G. DelRioPertuz 1 , L. Tijani 1

1 Texas Tech University Health Sciences Center, Lubbock, United States of America

Background: Cardiac adverse events were the most common cause of treatment discontinuation in patients with chronic lymphocytic leukemia (CLL) on ibrutinib. Acalabrutinib is a new, selective, irreversible Bruton’s tyrosine kinase inhibitor (BTKi) that is approved for the treatment of CLL.

Patients with CLL are inherently immunodeficient and are at increased risk of second primary malignancies (SPM). Additionally, BTKi have been studied in association with risk for secondary malignancies. The risk of cardiac adverse events and SPM with acalabrutinib in patients with CLL.

Aims: We conducted this systematic review and meta-analysis to estimate risk of cardiac adverse events and SPM with acalabrutinib in patients with CLL.

Methods: A comprehensive literature search was performed in PubMed, EMBASE, Clinical trials.gov and meeting abstracts till January 31,2021. All randomized controlled trials comparing acalabrutinib-based regimens with a control group in patients with CLL, with cardiac adverse events and SPM reported as adverse effects were included. Pooled risk ratios (RR) with 95% confidence intervals (CI) were calculated using the Mantel-Haenszel method of the fixed-effect model. Heterogeneity of effect size was quantified using the I 2 statistic. Statistical analysis was performed using the Review Manager, version 5.3.

Results: Three phase III RCT with total of 1362 patients were included for our analysis (Figure). Byrd et al., compared acalabrutinib with ibrutinib. The ASCEND trial compared acalabrutinib with investigator’s choice chemotherapy, either idelalisib plus rituximab or bendamustine plus rituximab. In the ELEVATE -TN study, the comparator arm included the combination of chlorambucil and Obinutuzumab. Acalabrutinib was used at a dose of 100 mg twice daily in all 3 studies. All but ELEVATE -TN study included patients previously treated with CLL. Atrial fibrillation was the most common cardiac adverse event across all trials. Non melanoma skin cancer was the most common SPM across all trials.

There was no increase in any grade cardiac adverse events with acalabrutinib when compared to control arm (pooled RR 1.06; 95%CI 0.84-1.34). There was no increased risk of atrial fibrillation (pooled RR 0.85; 95%CI 0.57-1.26), ventricular arrythmias and cardiac arrest (Figure). There was significant increase in hypertension (HTN) in the patients on the acalabrutinib containing regimens (pooled RR 1.78; 95%CI 1.24-2.56; P= 0.002); however, there was no increase in grade 3+ HTN.

There was a decrease in the SPM with acalabrutinib as compared to the control group (pooled RR 0.67; 95%CI 0.46-0.96; P= 0.03) (Figure). There was also significant decrease in non-melanoma skin cancer in the acalabrutinib as compared to the control group (pooled RR 0.59; 95%CI 0.37-0.93; P 0.02). There was no significant difference in the genitourinary cancer, lung cancer and hematologic cancer.

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Summary/Conclusion: There is no increase in risk of atrial fibrillation with the acalabrutinib based regimens as compared to the non-acalabrutinib – based therapies; however, there was a significantly increased risk of HTN. There is lower risk of SPM and non-melanoma SPM in the acalabrutinib based regimens than the control arm.

P666: ACALABRUTINIB ± OBINUTUZUMAB VS OBINUTUZUMAB + CHLORAMBUCIL IN TREATMENT-NAIVE CHRONIC LYMPHOCYTIC LEUKEMIA: 5-YEAR FOLLOW-UP OF ELEVATE-TN

J. P. Sharman 1,* , M. Egyed 2 , W. Jurczak 3 , A. Skarbnik 4 , K. Patel 5 , I. W. Flinn 6 , M. Kamdar 7 , T. Munir 8 , R. Walewska 9 , L. M. Fogliatto 10 , Y. Herishanu 11 , V. Banerji 12 , G. Follows 13 , P. Walker 14 , K. Karlsson 15 , P. Ghia 16 , A. Janssens 17 , F. Cymbalista 18 , E. Ferrant 19 , W. G. Wierda 20 , V. Munugalavadla 21 , T. Yu 21 , M. H. Wang 21 , J. A. Woyach 22

1 Willamette Valley Cancer Institute and Research Center, Eugene, OR, United States of America; 2 Somogy County Mór Kaposi General Hospital, Kaposvár, Hungary; 3 Maria Skłodowska-Curie National Research Institute of Oncology, Krakow, Poland; 4 Novant Health Cancer Institute, Charlotte, NC; 5 Swedish Cancer Institute, Center for Blood Disorders and Stem Cell Transplantation, Seattle, WA; 6 Sarah Cannon Research Institute and Tennessee Oncology, Nashville, Tennessee; 7 University of Colorado Cancer Center, Aurora, CO, United States of America; 8 Haematology, Haematological Malignancy Diagnostic Service (HMDS), St. James’s Institute of Oncology, Leeds; 9 Cancer Care, University Hospitals Dorset, Bournemouth, United Kingdom; 10 Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil; 11 Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 12 Departments of Internal Medicine, Biochemistry & Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba and CancerCare Manitoba, Winnipeg, Canada; 13 Department of Haematology, Addenbrooke’s Hospital NHS Trust, Cambridge, United Kingdom; 14 Peninsula Health and Peninsula Private Hospital, Frankston, Melbourne, Australia; 15 Skåne University Hospital, Lund, Sweden; 16 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 17 University Hospitals Leuven, Leuven, Belgium; 18 Bobigny: Hématologie biologique, Hopital Avicenne, Université Sorbonne Paris Nord, Bobigny; 19 Hospices Civils de Lyon, Centre Hospitalier Lyon Sud, Service d’Hématologie Clinique, Pierre-Bénite, France; 20 Department of Leukemia, Division of Cancer Medicine, MD Anderson Cancer Center, Houston, Texas; 21 AstraZeneca, South San Francisco, CA; 22 The Ohio State University Comprehensive Cancer Center, Columbus, OH, United States of America

Background: For ELEVATE-TN ( NCT02475681 ), we previously reported superior efficacy of acalabrutinib (A) ± obinutuzumab (O) vs O + chlorambucil (Clb) in patients with treatment-naive (TN) chronic lymphocytic leukemia (CLL) at 28.3 and 46.9 months median follow-up.

Aims: To report the updated efficacy and safety results from the ELEVATE-TN study after a median follow-up of 5 years.

Methods: Patients were randomized to A+O, A, or O+Clb after informed consent was obtained. Patients who progressed on O+Clb could cross over to A monotherapy. Investigator-assessed (INV) progression-free survival (PFS), INV overall response rate (ORR), overall survival (OS), and safety were evaluated.

Results: A total of 535 patients (A+O, n=179; A, n=179; O+Clb, n=177) had a median age of 70 years; 63% had unmutated IGHV and 9% had del(17p). At a median follow-up of 58.2 months (range, 0.0–72.0; data cutoff Oct 1, 2021), median PFS was not reached (NR) (hazard ratio [HR]: 0.11) for A+O and A (HR: 0.21) vs 27.8 months for O+Clb (both P <0.0001). The estimated 60-month PFS rates were 84% (A+O), 72% (A), and 21% (O+Clb). In patients with unmutated IGHV, the median PFS was NR for A+O and A vs 22.2 months among patients in the O+Clb arm (HR: 0.06 [A+O] and 0.12 [A]; both P <0.0001); estimated 60-month PFS rates were 82% (A+O), 72% (A), and 6% (O+Clb). In patients with del(17p), the median PFS was NR and 64.1 months for A+O and A vs 17.7 months for O+Clb (HR: 0.21, P =0.0031 [A+O]; HR: 0.29, P =0.0130 [A]); estimated 60-month PFS rates were 75% (A+O), 71% (A), and 27% (O+Clb). Median OS was NR in any treatment arm, and significantly longer in the A+O vs O+Clb arms (HR: 0.55; P =0.0474); estimated 60-month OS rates were 90% (A+O), 84% (A), and 82% (O+Clb). ORR was significantly higher with A+O (96%; 95% CI 92–98) and A (90%; 85–94) vs O+Clb (83%; 77–88; P <0.0001 [A+O], P =0.0499 [A]). Complete response (CR)/CR with incomplete hematologic recovery (CRi) rates were higher with A+O (29%/3%) vs O+Clb (13%/1%); 13%/1% had CR/CRi with A; CR increased since the interim analysis (previously 21% [A+O] and 7% [A]). Adverse events (AEs) and treatment exposure are shown in the Table. Treatment is ongoing in 65% (A+O) and 60% (A) of patients; the most common reasons for treatment discontinuation were AEs (17% [A+O], 16% [A], 14% [O+Clb]) and progressive disease (6%, 10%, 2%, respectively). Crossover from O+Clb to A occurred in 72 (41%) patients; 25% of these patients discontinued A (10% due to AEs and 11% due to progressive disease).

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Summary/Conclusion: After 5 years of follow-up, efficacy and safety of A+O and A monotherapy were maintained, with significantly longer OS in the A+O arm compared with O+Clb.

P667: LONG-TERM EFFICACY OF ACALABRUTINIB-BASED REGIMENS IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA AND HIGHER-RISK GENOMIC FEATURES: POOLED ANALYSIS OF CLINICAL TRIAL DATA

M. Davids 1,* , J. Sharman 2 , P. Ghia 3 , J. Woyach 4 , W. Jurczak 5 , T. Siddiqi 6 , P. Miranda 7 , M. Shahkarami 8 , T. Yu 8 , U. Emeribe 7 , J. Byrd 9

1 Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts; 2 Willamette Valley Cancer Institute and Research Center, Eugene, Oregon, United States of America; 3 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 4 The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America; 5 Maria Skłodowska-Curie National Research Institute of Oncology, Krakow, Poland; 6 City of Hope Comprehensive Cancer Center, Duarte, California; 7 AstraZeneca, Gaithersburg, Maryland; 8 AstraZeneca, South San Francisco, California; 9 Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America

Background: Chronic lymphocytic leukemia (CLL) has a highly variable disease course; patients (pts) with higher-risk genomic features typically have poor response to chemoimmunotherapy, and pts with del(17p) also have short, suboptimal response to venetoclax + obinutuzumab. Thus, novel agents such as Bruton tyrosine kinase inhibitors (BTKi), including the highly selective covalent BTKi acalabrutinib (A), are preferred treatments in this higher-risk population based on demonstrated effectiveness in higher-risk subgroups in individual studies.

Aims: To assess long-term efficacy of A-based regimens in pts with treatment-naive (TN) or relapsed/refractory (R/R) CLL and higher-risk genomic features.

Methods: Data were pooled from CLL pts with higher-risk genomic features treated with A ± obinutuzumab (O) in 3 clinical studies in TN CLL (ELEVATE-TN, CL-001 [TN cohort], CL-003) and 3 in R/R CLL (ASCEND, ELEVATE-RR, CL-001 [R/R cohort]). Higher risk was defined as del(17p) and/or TP53 mutation [del(17p)/ TP53 m], unmutated IGHV (uIGHV), or complex karyotype (CK, ≥3 chromosomal abnormalities). Efficacy analyses (progression-free survival [PFS], overall survival [OS], response rates) focus on del(17p)/ TP53 m and uIGHV; safety analyses also include pts with CK.

Results: 801 pts (TN 313; R/R 488, median 2 prior therapy lines [range 1–10]) were included; 64 (20%) TN and 219 (45%) R/R pts had del(17p)/ TP53 m, among whom 44 (69%) and 170 (78%) also had uIGHV. Overall, 288 (92%) TN and 425 (87%) R/R pts had uIGHV and 47 (15%) and 160 (33%) had CK. Median pt age was 68 (TN) and 66 (R/R) y. For A-based regimens combined (A and A+O), overall response rate (ORR) was 91% (n=58/64; complete response [CR]: 20% [n=13/64]) in TN pts with del(17p)/ TP53 m and 96% (n=276/288; CR 16% [n=47/288) in TN pts with uIGHV. At 47.3 mo median follow-up (range 1.0–82.0), median PFS was not reached (NR) in TN pts with del(17p)/ TP53 m with A-based regimens; PFS rates at 48 mo suggest similar efficacy with A and A+O in TN pts with del(17p)/ TP53 m (76% and 77%, respectively) ( Fig 1A ). Median PFS was NR among TN pts with uIGHV with both A and A+O; PFS rates at 48 mo with A and A+O were 84% and 86%, respectively ( Fig 1B ). Median OS was NR with A and A+O in TN pts with del(17p)/ TP53 m or uIGHV; OS rates at 48 mo were similar for A and A+O in pts with del(17p)/ TP53 m (89% for both) and in pts with uIGHV (92% and 95%).

In the R/R cohort (A monotherapy only), ORR was 86% (n=184/214; CR 5% [n=11/214]) in pts with del(17p)/ TP53 m and 87% (n=356/408; CR 7% [n=29/408]) in pts with uIGHV. At 44.0 mo median follow-up (range 0–88.0), median PFS was 38.6 mo and 46.9 mo and PFS rates at 36 mo were 54% and 65% in pts with del(17p)/ TP53 m and uIGHV, respectively ( Fig 1C ). Median OS was 60.6 mo and NR in pts with del(17p)/ TP53 m and uIGHV, respectively; OS rates at 36 mo were 73% and 82%.

Among all higher-risk pts, incidences of grade ≥3 treatment-related AEs were 30% (TN) and 35% (R/R). Discontinuations due to treatment-related AEs occurred in 4% (TN) and 6% (R/R) of pts; 60% and 27%, respectively, remained on treatment at data cutoff.

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Summary/Conclusion: In this pooled analysis of clinical trial data in 801 CLL pts with higher-risk genomic features, efficacy of A-based regimens led to high PFS and OS rates at a median follow-up of nearly 4 y. The safety profile in this analysis was similar to the overall safety profile of acalabrutinib. Our results demonstrate the long-term benefit of A-based regimens in CLL pts with higher-risk genomic features, regardless of line of therapy.

P668: ACALABRUTINIB VS RITUXIMAB PLUS IDELALISIB OR BENDAMUSTINE IN RELAPSED/REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA: ASCEND RESULTS AT ~4 YEARS OF FOLLOW-UP

P. Ghia 1,* , A. Pluta 2 , M. Wach 3 , D. Lysak 4 , M. Simkovic 5 , I. Kriachok 6 , A. Illes 7 , J. de la Serna 8 , S. Dolan 9 , P. Campbell 10 , G. Musuraca 11 , A. Jacob 12 , E. Avery 13 , J. H. Lee 14 , G. Usenko 15 , M. H. Wang 16 , T. Yu 17 , W. Jurczak 18

1 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 2 Department of Hematological Oncology, Oncology Specialist Hospital, Brzozow; 3 Department of Hemato-Oncology and Bone Marrow Transplantation, Medical University of Lublin, Lublin, Poland; 4 Fakultní Nemocnice Plzen, Pilsen; 5 University Hospital Hradec Kralove, Charles University, Hradec Kralove, Czechia; 6 National Cancer Institute, Kiev, Ukraine; 7 University of Debrecen, Faculty of Medicine, Department of Hematology, Debrecen, Hungary; 8 Hospital Universitario 12 de Octubre, Madrid, Spain; 9 Saint John Regional Hospital, University of New Brunswick, New Brunswick, Canada; 10 Barwon Health, University Hospital Geelong, Geelong, Victoria, Australia; 11 Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, Italy; 12 The Royal Wolverhampton NHS Trust, Wolverhampton, United Kingdom; 13 Nebraska Hematology Oncology, Lincoln, Nebraska, United States of America; 14 Gachon University Gil Medical Center, Incheon, South Korea; 15 City Clinical Hospital No. 4 DCC, Dnipro, Ukraine; 16 AstraZeneca, South San Francisco, California; 17 AstraZeneca, South San Francisco, California, United States of America; 18 Maria Sklodowska-Curie National Institute of Oncology, Krakow, Poland

Background: Acalabrutinib (acala) is a next-generation, highly selective, covalent Bruton tyrosine kinase (BTK) inhibitor approved for patients (pts) with chronic lymphocytic leukemia (CLL). In the primary analysis of ASCEND (median follow-up 16.1 mo), acala showed superior efficacy with an acceptable tolerability profile vs idelalisib (Id) plus rituximab (R) (IdR) or bendamustine (B) plus R (BR) in pts with relapsed/refractory (R/R) CLL (Ghia et al. J Clin Oncol . 2020;38:2849-2861).

Aims: We report the results of the ASCEND study at ~4 years of follow-up.

Methods: In this multicenter, randomized, open-label, phase 3 study ( NCT02970318 ), pts with R/R CLL received oral (PO) acala 100 mg BID until progression or unacceptable toxicity or investigator’s (INV) choice of IdR (Id: 150 mg PO BID until progression or unacceptable toxicity; R: 375 mg/m 2 x1 then 500 mg/m 2 IV [8 total infusions]) or BR (B: 70 mg/m 2 IV; R: 375 mg/m 2 x1 then 500 mg/m 2 IV [6 cycles]). Progression-free survival (PFS), overall survival (OS), overall response rate (ORR), and safety were assessed.

Results: A total of 310 pts (acala, n=155; IdR, n=119; BR, n=36) were randomized (median age 67 y; del(17p) 15%, unmutated IGHV 74%, Rai stage 3/4 42%). At median follow-up of 46.5 mo (acala) and 45.3 mo (IdR/BR), acala significantly prolonged INV-assessed PFS vs IdR/BR (median not reached [NR] vs 16.8 mo; P <0.0001); 42-mo PFS rates were 62% for acala vs 19% for IdR/BR. In pts with del(17p), median PFS was NR (acala) vs 13.8 mo (IdR/BR; P <0.0001). In pts with unmutated IGHV, median PFS was NR (acala) vs 16.2 mo (IdR/BR; P <0.0001). Median OS was NR in both arms; 42-mo OS rates were 78% (acala) vs 65% (IdR/BR). ORR was 83% (acala) vs 84% (IdR/BR) (ORR + partial response with lymphocytosis: 92% [acala] vs 88% [IdR/BR]). AEs led to drug discontinuation in 23% of acala, 67% of IdR, and 17% of BR pts. Events of clinical interest (acala vs IdR/BR) included all-grade atrial fibrillation/flutter (8% vs 3%), all-grade hypertension (8% vs 5%), all-grade major hemorrhage (3% vs 3%), and grade ≥3 infections (29% vs 29%).

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Summary/Conclusion: At ~4 years of follow-up, acala maintained efficacy compared with standard-of-care regimens and a consistent tolerability profile in R/R CLL.

P669: FIXED-DURATION (FD) IBRUTINIB + VENETOCLAX FOR FIRST-LINE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)/SMALL LYMPHOCYTIC LYMPHOMA (SLL): 3-YEAR FOLLOW-UP FROM THE PHASE 2 CAPTIVATE STUDY FD COHORT

C. Moreno 1,2,* , W. G. Wierda 3 , P. M. Barr 4 , T. Siddiqi 5 , J. N. Allan 6 , T. J. Kipps 7 , L. Trentin 8 , R. Jacobs 9 , S. Jackson 10 , A. Tedeschi 11 , S. Opat 12 , R. Bannerji 13 , B. J. Kuss 14 , L. J. Croner 15,16 , E. Szafer-Glusman 15,16 , C. Zhou 16 , A. Szoke 16 , J. P. Dean 16 , P. Ghia 17 , C. S. Tam 18

1 Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona; 2 Josep Carreras Leukemia Research Institute, Barcelona, Spain; 3 Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX; 4 Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY; 5 City of Hope National Medical Center, Duarte, CA; 6 Weill Cornell Medicine, New York, NY; 7 UCSD Moores Cancer Center, La Jolla, CA, United States of America; 8 University of Padova, Padova, Italy; 9 Levine Cancer Institute, Charlotte, NC, United States of America; 10 Middlemore Hospital, Auckland, New Zealand; 11 ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 12 Monash University, Clayton, VIC, Australia; 13 Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United States of America; 14 Flinders University and Medical Center, Bedford Park, SA, Australia; 15 AbbVie, North Chicago, IL; 16 Pharmacyclics LLC, an AbbVie Company, South San Francisco, CA, United States of America; 17 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 18 Peter MacCallum Cancer Center & St. Vincent’s Hospital and the University of Melbourne, Melbourne, VIC, Australia

Background: CAPTIVATE (PCYC-1142) is a multicenter phase 2 study of first-line ibrutinib + venetoclax in CLL. The primary analysis evaluating FD treatment with ibrutinib + venetoclax was previously presented (Ghia et al., ASCO 2021).

Aims: To present 3-year follow-up results from the FD cohort of CAPTIVATE.

Methods: Patients aged ≤70 years with previously untreated CLL/SLL received 3 cycles of ibrutinib then 12 cycles of ibrutinib + venetoclax (ibrutinib 420 mg/day orally; venetoclax ramp-up to 400 mg/day orally). Responses were investigator assessed per iwCLL 2008 criteria. Undetectable minimal residual disease (uMRD; <10 -4 ) was measured by 8-color flow cytometry. Serious AEs (SAEs) deemed related to ibrutinib reported >30 days after last dose of study drug were collected.

Results: 159 patients were enrolled (median age 60 years), including patients with high-risk features of del(17p)/ TP53 mutation (17%), unmutated IGHV (uIGHV; 56%), and complex karyotype (19%). 147 (92%) and 149 (94%) patients completed treatment with ibrutinib and venetoclax, respectively. With 1 year of additional follow-up since primary analysis, median time on study was 39 months (range 1-41). ORR was 96% and was consistent (96%-97%) in patients with high-risk features (Table). The primary endpoint of complete response (CR) including CR with incomplete bone marrow recovery (CRi) rate in patients without del(17p) (n=136) increased nominally from 56% (95% CI, 48-64) to 58% (95% CI 50-66); in all patients, CR rate increased from 55% (95% CI 48-63) to 57% (95% CI 50-65). In patients achieving CR, 93% had durable responses lasting ≥12 months post-treatment. Of patients with uMRD in peripheral blood at 3 months post-treatment, 66/85 (78%) evaluable patients maintained uMRD through 12 months post-treatment. At 36 months, PFS was 88% (95% CI 82‒92) and OS was 98% (95% CI 94-99); similar rates were seen in patients with high-risk features (Table). All patients are off treatment; no new SAEs of any kind have occurred since the primary analysis. Available data on relevant mutations in BTK , PLCɣ2 , or BCL-2 at time of PD will be presented. As of January 2022, 12 patients were retreated with single-agent ibrutinib after progressive disease (treatment duration range 3-29 months); of evaluated patients, 7/9 had partial responses and 2/9 had stable disease.

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Summary/Conclusion: Fixed duration ibrutinib + venetoclax continues to provide deep, durable responses and clinically meaningful PFS, including in patients with high-risk disease features, representing an all-oral, once-daily, chemotherapy-free FD regimen for previously untreated patients with CLL/SLL. With an additional 1 year of follow-up, no OS events or SAEs occurred. Manageable safety profile is unchanged as previously reported. To date, successful single-agent ibrutinib retreatment responses are observed.

P670: ABSENCE OF BTK, BCL2, AND PLCG2 MUTATIONS IN RELAPSING CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AFTER FIRST-LINE TREATMENT WITH FIXED-DURATION IBRUTINIB (I) PLUS VENETOCLAX (V)

L. J. Croner 1,2,* , J. N. Allan 3 , N. Jain 4 , J. F. Seymour 5 , K. Eckert 2 , L. W. K. Cheung 1,2 , E. Szafer-Glusman 1,2

1 AbbVie, North Chicago, IL; 2 Pharmacyclics LLC, an AbbVie Company, South San Francisco, CA; 3 Weill Cornell Medicine, New York, NY; 4 MD Anderson Cancer Center, Houston, TX, United States of America; 5 Peter MacCallum Cancer Center & Royal Melbourne Hospital, Melbourne, VIC, Australia

Background: CAPTIVATE (PCYC-1142; NCT02910583 ) is a multicenter phase 2 study of first-line I+V in patients (pts) with CLL in 2 cohorts: Minimal Residual Disease (MRD) and Fixed Duration (FD). We previously reported that fixed-duration I+V provides deep, durable responses (Ghia et al, ASCO 2021; Wierda et al, J Clin Oncol 2021). Clinically-relevant mutations in BTK , PLCG2 , and BCL2 have previously been reported in pts with progressive disease (PD) following single agent BTK or BCL2 inhibitor therapies. Such resistance-associated mutations may occur with increasing frequency after prolonged drug exposure. Fixed duration I+V is hypothesized to decrease the likelihood of developing resistance mutations and facilitate extended clinical benefit.

Aims: We sought to ascertain presence or absence of resistance mutations at the time of PD in samples from pts treated with fixed-duration I+V in CAPTIVATE. Baseline genomic risk features and potentially prognostic mutations in CLL-related genes of interest were also evaluated in pts with and without subsequent PD.

Methods: The analysis was conducted in 191 pts aged ≤70 y with previously untreated CLL who completed fixed-duration I+V, defined as 3 x 28d cycles of I then 12 cycles of I+V (I 420 mg/d orally; V ramp-up to 400 mg/d orally); this included pts from the FD cohort, and pts from the MRD cohort with confirmed undetectable MRD who received an additional cycle of I+V and were randomized to placebo. Samples from pts at the time of PD were evaluated for resistance mutations by targeted next-generation sequencing (NGS) using a custom-designed high-read-depth 100-gene panel (limit of detection 1% VAF, negative predictive value ≥95% assuming population prevalences <20%, for BTK , PLCG2 , or BCL2 variants) in CD19-enriched peripheral blood (PB) or bone marrow. Mutations in additional genes of interest ( Table ) were evaluated by NGS using the Personalis ACE panel in baseline PB samples. Genomic risk features ( Table ) were also assessed at baseline. Mutations and genomic risk features evident at baseline were compared across PD and non-PD pts.

Results: Baseline mutations in genes of interest and genomic risk features in 190 pts who completed fixed-duration I+V and with available baseline data are shown in the Table . Rates of mutations were similar between pts with (n=30) and without (n=160) subsequent PD. Of 30 pts with PD events (occurring between 16 and 53 mos after start of treatment), 22 (73%) had baseline mutations in genes of interest and 27 (90%) had ≥1 baseline genomic risk feature. Pts with PD generally had more baseline genomic risk features than pts without PD (median 3 vs 2; Wilcoxon P =0.02; analysis restricted to FD cohort pts to avoid bias). To date, 26 of 30 PD patient samples have been evaluated for mutations; no resistance-associated variants in BTK , PLCG2 , or BCL2 were detected.

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Summary/Conclusion: Earlier work has demonstrated a promising safety profile and efficacy of fixed-duration I+V in first-line CLL. Importantly, the current results show that no resistance-associated mutations in BTK , PLCG2 , or BCL2 were identified in the pts with PD evaluated to date after first-line, fixed-duration I+V. The absence of such mutations at PD suggests that fixed-duration I+V treatment may offer the possibility of effective subsequent retreatment options with I and/or V and extend clinical benefit.

P671: TREATMENT SEQUENCES AND OUTCOMES OF PATIENTS WITH CLL TREATED WITH TARGETED AGENTS IN REAL-WORLD SETTINGS

A. R. Mato 1,* , B. S. Manzoor 2 , C. C. Coombs 3 , N. Lamanna 4 , H. H. Tuncer 5 , J. R. Brown 6 , L. E. Roeker 1 , M. Shadman 7 , J. N. Allan 8 , C. Ujjani 7 , B. Eichhorst 9 , L. Leslie 10 , I. Fleury 11 , H. Alhasani 2 , J. Rhodes 12 , B. T. Hill 13 , P. M. Barr 14 , A. Skarbnik 15 , M. S. Davids 6 , R. Bannerji 16 , M. Fuldeore 2 , F. Lansigan 17 , A. Schuh 18 , L. Pearson 19 , C. P. Fox 20 , I. Pivneva 21 , L. Popadic 22 , A. Guerin 21 , T. A. Eyre 18

1 Memorial Sloan Kettering Cancer Center, New York; 2 AbbVie, Inc., North Chicago; 3 Lineberger Comprehensive Cancer Center, Chapel Hill; 4 Columbia University, New York; 5 The Cancer Center at Lowell General Hospital, Lowell; 6 Dana-Farber Cancer Institute, Boston; 7 Fred Hutchinson Cancer Research Center, Seattle; 8 Weill Cornell Medicine University Medical Center, New York, United States of America; 9 University of Cologne, Cologne, Germany; 10 John Theurer Cancer Center, Hackensack, United States of America; 11 Hôpital Maisonneuve-Rosemont, Montreal, Canada; 12 Northwell Health Cancer Institute, Lake Success; 13 Cleveland Clinic, Cleveland; 14 Wilmot Cancer Institute, Rochester; 15 Novant Health, Charlotte; 16 Rutgers Cancer Institute of New Jersey, New Brunswick; 17 Dartmouth-Hitchcock Medical Center, Lebanon, United States of America; 18 Churchill Hospital, Oxford, United Kingdom; 19 Tufts Medical Center, Boston, United States of America; 20 Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; 21 Analysis Group, Inc., Montreal, Canada; 22 Analysis Group, Inc., New York, United States of America

Background: Treatment (tx) options for patients (pts) with chronic/small lymphocytic leukemia (CLL/SLL) have expanded following regulatory approval of targeted agents. However, real-world evidence regarding pt outcomes and tx sequences is evolving.

Aims: We assessed tx trends, sequences and outcomes following the introduction of targeted agents.

Methods: The CLL Collaborative Study of Real-World Evidence (CORE), a retrospective, international, observational study (23 centers) for CLL/SLL pts provided data for this analysis. Pts were diagnosed with CLL/SLL, treated in the relapsed/refractory (R/R) setting after 2/12/2014. Tx sequences following targeted agents (ie, BTKi: acalabrutinib, ibrutinib; BCL2i: venetoclax), clinical response (physician-assessed in medical charts and reported as overall response rate [ORR]), and progression-free survival (PFS) were assessed.

Results: Of the 1,624 study pts, in 1L 48.8% initiated CT/CIT, 40.6% targeted agents (BTKi: 32.6%; BCL2i: 4.5%), and 10.6% other therapies. We observed 1L CT/CIT use decreased starting 2014 (pre-2014: 58.1%; 2014–2016: 46.0%; 2017–2019: 24.6%; 2020–2021: 0%), while 1L targeted agent use increased over time (pre-2014: 30.5%; 2014–2016: 43.1%; 2017–2019: 67.0%; 2020–2021: 94.7%). Post-targeted agent introduction in 2014, the most frequent tx sequence has changed from 1L CT/CIT→BCRi in 2014–2016 to 1L BCRi→BCL2i in 2017–2021 (Figure 1).

Across lines and years of tx, among 650 R/R CLL/SLL pts who initiated therapy >2/12/2014, 536 pts (82.5%) received BTKi-based regimens and 214 pts (32.9%) received BCL2i-based regimens. Of the 536 pts receiving BTKi-based regimens (ibrutinib alone: 454 [85.5%]; acalabrutinib alone: 21 [4.0%]), majority was in 2L and 3L+ (291 pts (54.3%); 167 pts (31.2%), respectively). Of the 457 pts with response assessed, 343 pts (75.1%) achieved ORR in the line of therapy (LOT) with BTKi-based regimen. The median PFS for pts receiving BTKi-based regimen was 36.4 months (95% confidence interval [CI]: 33.1, 41.9). 236 pts (44.0%) receiving a BTKi-based regimen had a subsequent LOT; of the 177 pts with response assessed, 120 pts (67.8%) achieved ORR. The most common subsequent tx were BCL2i-based (N=113; ORR: 76.2%) or another BTKi-based (N=41; ORR: 54.8%) regimens.

Of the 214 pts receiving BCL2i (venetoclax alone: 129 [60.3%]), majority was in 2L and 3L+ (77 pts [36.0%] and 132 pts [61.7%], respectively). Of the 164 pts with response assessed, 127 pts (77.4%) achieved ORR in the LOT with BCL2i-based regimen. The median PFS for pts receiving BCL2i was 26.2 months (95% CI: 20.0, 38.7). 47 pts (22.0%) receiving BCL2i had a subsequent LOT; of the 37 pts with response assessed, 27 pts (73.0%) achieved ORR. The most common subsequent tx were BTKi-based (ibrutinib-based regimen: 14 [29.8%]; acalabrutinib-based regimen: 5 [10.6%]; ORR: 76.9%) or another BCL2i-based (venetoclax-based re-tx: 8 [17.8%]; ORR: 80.0%) regimens.

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Summary/Conclusion: 1L CT/CIT use is declining since 2014 and 1L targeted agent use is now the standard of care in real-world practice. Of currently available targeted therapies, ibrutinib was the most commonly used. These results demonstrate sequences of BTKi use following venetoclax (venetoclax→BTKi) are effective, as well rechallenging with venetoclax (venetoclax→venetoclax), providing support for either strategy in the R/R CLL/SLL management. These results provide clinicians with valuable insights regarding pt outcomes associated with different tx sequences currently considered in modern clinical practice. Updated pt level data and survival analyses are underway.

P672: REAL-LIFE EFFICACY AND SAFETY OF VENETOCLAX MONOTHERAPY IN RELAPSED/ REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA - INTERIM ANALYSIS OF MULTICENTRIC STUDY VERONE

L. Ysebaert 1,* , X. Troussard 2 , V. Levy 3 , R. Le Calloch 4 , R. Guieze 5 , S. Leprêtre 6 , A.-S. Michallet 7 , V. Leblond 8 , P. Feugier 9 , J. Ramier 10 , A. Delmer 11

1 Hematology Unit, IUCT Oncopole, Toulouse; 2 Hematology Unit, CHU Caen, Caen; 3 Hematology Unit, Hopital Avicenne, Bobigny; 4 Hematology Unit, CH Cornouaille, Quimper; 5 Hematology Unit, CHU Clermont-Ferrand, Clermont-Ferrand; 6 Hematology Unit, Henri Becquerel Cancer Research Center, Rouen; 7 Hematology Unit, Leon Berard Cancer Reseach Center, Lyon; 8 Hematology Unit, Hôpital Pitié-Salpêtrière, Paris; 9 Hematology Unit, CHRU Nancy, Nancy; 10 AbbVie France, Rungis; 11 Hematology Unit, CHU Reims, Reims, France

Background: The efficacy and safety of venetoclax (VEN), the first oral inhibitor of BCL-2, has been demonstrated in clinical trials in patients with chronic lymphocytic leukemia (CLL). However, few real-life data in France is available today on this treatment.

Aims: VERONE is an observational ongoing study, it aims to describe the efficacy and tolerance of VEN in a real-life situation, as well as its use in current medical practice.

Methods: This longitudinal non-interventional study is conducted in adults starting treatment with VEN for CLL, with a follow-up over 48 months. This second interim analysis was performed in patients treated with monotherapy until progression, 43 months after the first inclusion. The overall response rate (ORR, assessed by investigator) and progression-free survival (PFS) were estimated 1 and 2 years after the first intake of VEN in patients with at least one follow-up visit in the study (evaluable population).

Results: From March 2018 to September 2021, 196 patients included in 64 centers started VEN as monotherapy for CLL (tolerance population), 195 met the selection criteria (analysis population) and 113 could be evaluated (evaluable population). In the analysis population (n = 195), patients and CLL characteristics at baseline were: median age 74.0 years (33.0-91.0), male 68.2%, ECOG ≥2 18.5%, ≥1 comorbidities 87.7%, median time since diagnosis 8.3 years (0.0-29.4), 37.3% of 169 genetic examinations was TP53 and/ or del (17)p, median number of previous treatments 2 (1-9) (chemoimmunotherapy 87.2%, BCR inhibitors (ibrutinib, idelalisib) 70.8%). Before the first intake of VEN, preventive measures against tumor lysis syndrome (TLS) were taken in the majority of cases (hospitalization 85.2%, hydration 85.2% and anti-hyperuricemia prophylaxis 66.3%). The median duration of the titration period was 5.0 weeks, and 77.0% of patients reached the recommended dose of 400mg at the time of analysis.

In the 113 evaluable patients, the overall response rate was 91.2% [95%CI 84.5%-95.1%] at 1 year and 92.9% [95%CI 86.7%-96.4%] at 2 years. After a median follow-up of 23.2 months, the PFS rate was 81.8% [95%CI 72.5%-88.2%] at 1 year and 71.1% [95%CI 58.0% 80.7%] at 2 years. The median duration of treatment was 15.5 months.

In terms of safety, 85.2% of the 196 patients presented adverse events [AEs, neutropenia (46.4%) and diarrhea (24.5%)], including 64.3% of AEs of grade ≥3 (including 32.7% neutropenia). Permanent discontinuation of treatment due to AEs related to VEN was observed in 20.4% of patients. 4 AEs grade 5 related to VEN was observed in 3 patients (deterioration of general condition, ascites, hepatic failure, death).

At inclusion, the risk level of developing TLS, assessed in 149 patients, was low (28.2%) moderate (69.8%) or high (2.0%). During the dose escalation phase, 19 patients (9.7%) developed TLS and 4 (2.0%) stopped VEN treatment.

Summary/Conclusion: This real-life study confirms the efficacy of VEN in heavily treated patients with a safety profile little different from that observed in selected populations in clinical trials.

P673: DEPLETION AND RECOVERY OF NORMAL B-CELLS DURING AND AFTER TREATMENT WITH CHEMOIMMUNOTHERAPY, IBRUTINIB OR VENETOCLAX.

A. Rawstron 1,* , N. Webster 2 , A. Pitchford 3 , S. Dalal 2 , A. Bloor 4 , R. de Tute 1 , A. Hockaday 3 , S. Jackson 3 , D. Cairns 3 , N. Greatorex 3 , D. Allsup 5 , T. Munir 6 , P. Hillmen 2

1 HMDS, Leeds Cancer Centre; 2 Experimental Haematology, University of Leeds; 3 Leeds Cancer Research UK Clinical Trials Unit, Leeds Institute of Clinical Trials Research, Leeds; 4 Haematology, The Christie NHS Foundation Trust, Manchester; 5 Haematology, Hull York Medical School, Hull; 6 Haematology, Leeds Cancer Centre, Leeds, United Kingdom

Background: Infectious complications are a major cause of morbidity and mortality in Chronic Lymphocytic Leukaemia (CLL). Therapeutic approaches that deplete CLL cells also affect normal B-cells. Optimal treatment would result in eradication of CLL cells and recovery of normal immune function. FLAIR (ISRCTN01844152) is a phase III trial for previously untreated CLL comparing ibrutinib plus rituximab (IR) with fludarabine, cyclophosphamide and rituximab (FCR) and subsequently amended to also compare ibrutinib plus venetoclax (I+V) and ibrutinib alone (I) with FCR. Measurable residual disease (MRD) and normal B-cell levels were assessed at multiple timepoints.

Aims: To assess the depletion of normal B-cells during treatment and recovery after end of treatment.

Methods: Participants aged under 75 years with <20% TP53-deleted cells were initially randomised to FCR or IR and subsequently to FCR, IR, I+V or I with the IR arm closed after randomisation of 771 participants to FCR/IR. FCR was given for 6 cycles, while treatment in the IR, I and I+V arms continued for up to 6 years except in participants attaining <0.01% MRD who continued treatment for the time taken to achieved MRD <0.01% and then stopped if MRD remained <0.01%. Month (M) 24 was earliest permitted stopping point. MRD flow cytometry was performed according to ERIC guidelines (panel: CD19/5/20/43/79/81+ROR1, acquisition of 0.5–2.2 million cells, BD Biosciences Lyric). Additional analysis of normal B-cell subsets was performed in a cohort of >500 patients (panel: CD19 to identify B-cells, CD20/5/79b+ROR1 and CD3 to exclude CLL & contaminating cells, with CD27/ 38/IgD/IgM to characterise normal B-cell subsets using a Coulter Cytoflex LX).

Results: Normal B-cells were undetectable during FCR treatment and only rarely detectable until 12 months after last FCR cycle. Circulating normal B-cells were reduced in number or undetectable in participants receiving ibrutinib-containing regimens with greater depletion in the I+V and IR arms relative to I monotherapy. B-progenitors persist through FCR treatment but were depleted during I, I+R or I+V treatment. Normal B-cell levels at 24 and 36 months after randomisation, with time off-treatment if applicable, are shown in Figure 1.

In the ibrutinib-containing arms (IR, I, and I+V), there was a trend towards fewer COVID-associated SAE at any time point for participants with detectable B-cells at 24M (4/181, 2.2%) compared to those with no detectable B-cells (14/344, 4.1%) and COVID-associated SAEs were not observed in FCR-treated participants who had recovered any level of normal B-cells by 24M (0/215). However, the data on COVID infections are limited and there was no apparent association between normal B-cell levels at 24M with the proportion of participants experiencing an infectious SAE overall. Assessment of normal B-cell subsets during ibrutinib-based treatment demonstrated a mix of naïve and memory B-cells. Serological response to COVID infection/vaccination in this cohort is currently being performed.

Participants stopping I+V treatment at 24-30 months post-randomisation due to MRD eradication showed rapid recovery of normal naive B-cells within 6-12 months after end of treatment in the vast majority (>95%) of evaluable cases.

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Summary/Conclusion: Normal circulating B-cells are depleted during treatment with rituximab but can persist at a low level during I, IR or I+V treatment. Most patients in remission after treatment with FCR or I+V show recovery of normal B-cells at 12 months of stopping treatment.

P674: UPDATED RESULTS OF A PHASE 1B STUDY OF IBRUTINIB (IBR) PLUS OBINUTUZUMAB (OBIN) IN PATIENTS WITH RELAPSED OR REFRACTORY (R/R) CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)

C. Ryan 1,* , D. Brander 2 , P. Barr 3 , S. Tyekucheva 4 , Y. Ren 4 , L. Hackett 1 , M. McDonough 1 , K. Pena Del Aguila 1 , M. McEwan 1 , M. Collins 1 , J. Abramson 1 , E. Jacobsen 1 , A. LaCasce 1 , D. Fisher 1 , J. Brown 1 , M. Davids 1

1 Medical Oncology, Dana-Farber Cancer Institute, Boston; 2 Duke University Cancer Center, Durham; 3 University of Rochester Wilmot Cancer Institute, Rochester; 4 Data Science, Dana-Farber Cancer Institute, Boston, United States of America

Background: Combination ibr + obin was approved in frontline CLL based on results of iLLUMINATE; however, less data are available on this regimen in R/R CLL. Though active in R/R CLL, ibr monotherapy in RESONATE led to 4-year (yr) PFS/OS of ~45%/65%, leaving room for improvement. We previously reported initial safety and efficacy results of this phase 1b study investigating ibr + obin in R/R CLL (Davids et al., EHA 2020). Here, we report updated results, with median follow-up now of 41.5 months (mos).

Aims: To assess longer-term safety and efficacy of ibr + obin in patients (pts) with R/R CLL.

Methods: We conducted a phase 1b, investigator-initiated study of ibr + obin in R/R CLL ( NCT02537613 ). The primary objective was to assess safety of 3 different drug sequencing regimens in cycle 1; the main secondary objective was to assess efficacy. Pts were randomized 1:1:1 to either receive obin 1 mo before ibr, ibr 1 mo before obin, or start concomitantly. Both drugs were given at the approved doses and schedule, with 6 total cycles of combination followed by ibr monotherapy until progression or unacceptable toxicity. Notable eligibility criteria: requiring treatment (tx) for R/R CLL/SLL by 2008 iwCLL criteria, ECOG PS ≤2, no prior ibr or obin. Assessments: toxicities by CTCAE v4, efficacy by 2008 iwCLL criteria, MRD by 4-8 color flow (10 -4 sensitivity). Serum concentrations of 21 cytokines were assessed pre-tx and after 1 week of combination tx in a representative subset of 27 pts (n=9 per arm) by bead-based multiplex immunoassay (Eve Technologies, Calgary).

Results: 52 pts were accrued, and all received ≥1 cycle of combination tx. Median age: 67 yrs (range 33-84); 77% male; median # prior tx: 1 (range 1-6); 25% del(17p) or TP53 mut; 27% del(11q); 50% unmutated IGHV; 27% bulky LAD (nodes ≥5 cm).

As of 14 Feb 2022: median follow-up 41.5 mos (range 2.3-73.3). 27 pts (52%) remain on tx. All-grade heme toxicities: thrombocytopenia (88%; 23% Gr3/4), neutropenia (71%; 37% Gr3/4), febrile neutropenia (8%, all Gr3/4), anemia (73%; 8% Gr3). Notable non-heme toxicities: bruising (58%, all Gr1/2), arthralgia (38%, all Gr1/2), diarrhea (37%, all Gr1/2), transaminitis (35%; 4% Gr3), ≥Gr3 infection (17%, all Gr3), and cardiotoxicities: hypertension (46%; 10% Gr3), atrial fibrillation (21%; 10% Gr3). 1 pt had sudden cardiac death after 11 mos on study. 7 pts required ibr dose-reduction due to toxicity.

The best ORR is 96%, including 50% CR and 46% PR (Fig. 1A). All 3 pts who previously progressed on venetoclax responded (1 CR, 2 PR). By ITT, 10/52 (19%) achieved a best response of undetectable MRD in bone marrow (BM, 44 pts tested), and 14/52 (27%) in peripheral blood (PB, 34 pts tested). 4-yr PFS and OS are 74% and 93% (Fig. 1B-C), with 3 deaths (n=1 each: sudden death, MDS, Richter’s syndrome). Pts who achieved BM and PB undetectable MRD had a significantly larger decrease in circulating CCL4 (p=0.02) and CXCL13 levels (p=0.01), respectively, comparing baseline to 1 week of combination tx (Fig. 1D).

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Summary/Conclusion: Ibr + obin is a highly active combination in R/R CLL, achieving 50% CR and 4-yr PFS/OS of 74%/93%, suggesting a potential positive impact of obin (though possibly also reflecting an earlier relapse population than RESONATE). Responses were observed in all 3 pts who had progressed after venetoclax, including 1 CR. With ~3.5 yrs median follow-up, and some pts on tx over 6 yrs, cardiotoxicity was consistent with prior ibr studies, and no new safety concerns have emerged. Our data support continued exploration of combination BTK inhibitor plus obin in R/R CLL.

P675: TREATMENT OF HYPERTENSION IN PATIENTS RECEIVING BTK INHIBITORS: A MULTICENTER RETROSPECTIVE STUDY

M. Shadman 1,2,* , J. Voutsinas 3 , B. Fakhri 4 , S. Khajavian 3 , S. Spurgeon 5 , D. Stephens 6 , A. Skarnbik 7 , A. Mato 8 , C. Broome 9 , A. Gopal 1,2 , S. Smith 1 , R. Lynch 1 , M. Rainey 10 , S. Kim 5 , O. Barrett-Campbell 11 , E. Hemond 11 , M. Tsang 12 , D. Ermann 6 , N. Malakhov 13 , D. Rao 8 , M. Shakib Azar 3 , B. Morrigan 3 , A. Chauhan 14 , T. Plate 15 , T. Gooley 1 , K. Ryan 16 , F. Lansigan 11 , B. Hill 10 , G. Pongas 15 , S. Parikh 17 , L. Roeker 8 , J. Allan 18 , R. Cheng 2 , C. Ujjani 1

1 Fred Hutchinson Cancer Research Center / University of Washington; 2 University of Washington; 3 Fred Hutchinson Cancer Research Center, Seattle; 4 University of California San Francisco (UCSF), San Francisco; 5 Oregon Health and Science University (OHSU), Portland; 6 University of Utah, Salt Lake City; 7 Novant Health, Charlotte; 8 Memorial Sloan Kettering Cancer Center, New York; 9 Georgetown University, Washington DC; 10 Cleveland Clinic, Cleveland; 11 Dartmouth-Hitchcock Medical Center, Lebanon; 12 UCSF, San Francisco; 13 New York-Presbyterian Hospital, Weill Cornell Medicine, New York; 14 Georgia Cancer Center at Augusta University, Agusta; 15 Sylvester Comprehensive Cancer Center, University of Miami, Miami; 16 Astra Zeneca, Gaithersburg; 17 Mayo Clinic, Rochester; 18 Weill Cornell Medicine, New York, United States of America

Background: Hypertension (HTN) is an adverse event (AE) associated with the use of ibrutinib and less commonly with second generation BTK inhibitors (BTKis) as shown by a significant lower rate of HTN with acalabrutinib compared to ibrutinib in the ELEVATE-RR trial. Nevertheless, HTN is considered an AE of BTKis as a class.

Aims: We aimed to identify a class or combination of antihypertensive drugs (anti-HTNs) that may be most effective in pts with HTN while taking a BTKi.

Methods: In this multicenter retrospective real-world study, we included consecutive pts with lymphoid malignancies taking BTKis and anti-HTNs with at least 3 months of follow-up. Eligible pts were stratified into 2 groups: 1) Pts with pre-existing HTN before starting BTKi (pre-HTN) and 2) Pts who developed incident HTN after starting BTKis ( de novo HTN). The anti-HTN drugs were categorized as follows: 1) Angiotensin-converting-enzyme inhibitors and Angiotensin receptor blockers (ACEi/ARB); 2) Beta-blockers (BB); 3) Calcium channel blockers (CCB); 4) Hydrochlorothiazide (HCTZ ) and 5) non-HCTZ diuretics (other-DU). Timepoints for mean arterial pressure (MAP) measurements and start/end time of each anti-HTN were documented in relation to the index date (first date of concurrent use of BTKi and anti-HTN drug). Generalized estimating equations were used to assess the association between time varying MAPs and the individual drug categories, as well as the number of medication categories.

Results: We included 196 pts (118 pre-HTN and 78 de novo HTN) from 12 centers. The majority of pts had CLL (94%) on ibrutinib (90%) for first (49%), second (25.5%) or later (25.5%) lines of therapy. The median age was 67 (IQR 37-88), most pts were male (72%), and caucasian (94.4%). The median duration of follow-up was 38.9 months (IQR 3.5-110). Pre-BTKi comorbidities included diabetes (17.3%), coronary disease (11.7%), cerebrovascular accidents (2.6%) and chronic kidney disease (12.8%). BTKi dose was reduced in 53 pts (27%) with 9 (4.6%) having dose reduction because of HTN. Twelve pts (6.2%) switched to other BTKis and 5 pts (2.6%) did so for HTN. In the pre-HTN cohort, a trend for statistically significant BP reduction was only achieved when 4+ anti-HTN drugs were used [Est -4.81; 95% CI (-9.93, 0.307); p = 0.07]. In this cohort, concurrent use of HCTZ and BB showed a significant trend for a lower BP [Est -5.05; 95% (-10.0, -0.06); p= 0.04]. In de novo HTN cohort, treatment with 3+ anti-HTN drugs was associated with a statistically significant reduction in the BP [Est -5.70; 95% CI (-9.94, -1.46); p =0.008]. In de novo cohort combination of HCTZ and ACEi/ARB was associated with a significant MAP reduction [Est -5.47; 95% CI (-10.9, -0.001); p= 0.05]. When the 2 cohorts were combined, treatment with 4+ drugs [Est -6.27; 95% CI (-11.8, -0.69); p = 0.02] and treatment with HCTZ and ACEi/ARB combination [Est -5.05; 95% CI (-9.61, -0.48); p = 0.03] or HCTZ and BB [Est -5.05; 95% CI(-9.61,-0.48); p= 0.03] were associated with MAP reduction. Gender or race did not modify these associations.

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Summary/Conclusion: Treatment of HTN in pts taking Ibrutinib is challenging and requires utilization of multiple anti-hypertensive drugs both in pre-HTN and de novo HTN pts. Among different anti-HTN classes, we observed a statistically significant MAP reduction in pts with pre-HTN on HCTZ and BB combination and in pts with de novo HTN on HCTZ and ACEi/ARB combination. These findings need to be further assessed in pts taking newer generation BTKi and confirmed in large prospective studies.

P676: LOW-DOSE FCR COMPARED TO BR IN PREVIOUSLY UNTREATED PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA WITHOUT TP53 ABERRATIONS: A REAL – WORLD RETROSPECTIVE ANALYSIS BY THE CZECH CLL STUDY GROUP.

L. Smolej 1,* , Y. Brychtová 2 , E. Cmunt 3 , M. Oršulová 2 , P. Vodárek 1 , M. Dostál 1 , P. Turcsányi 4 , R. Urbanová 4 , A. Panovská 2 , J. Zuchnická 5 , J. Mihályová 5 , D. Lysák 6 , M. Brejcha 7 , H. Móciková 8 , K. Klásková 8 , M. Šimkovič 9 , M. Špaček 3 , M. Doubek 10

1 4th Department of Medicine - Hematology, University Hospital and Faculty of Medicine, Hradec Králové; 2 Department of Internal Medicine – Hematology and Oncology, University Hospital, Brno; 3 First Department of Internal Medicine - Hematology, General University Hospital, Prague; 4 Department of Hemato - oncology, University Hospital, Olomouc; 5 Department of Hematooncology, University Hospital, Ostrava; 6 Department of Hematology - Oncology, University Hospital, Pilsen; 7 Department of Hematology, Hospital Agel, Nový Jičín; 8 Department of Internal Medicine - Hematology, University Hospital Královské Vinohrady, Prague; 9 4th Department of Medicine - Hematology, University Hospital, Hradec Králové; 10 Department of Internal Medicine - Hematology, University Hospital, Brno, Czechia

Background: Low – dose fludarabine, cyclophosphamide, and rituximab (LDFCR) regimen showed promising activity in treatment-naïve elderly / comorbid patients (pts) with chronic lymphocytic leukemia (CLL) (Smolej et al., Br J Haematol. 2021). However, no data are available regarding comparison to bendamustine and rituximab (BR), which has been one of the predominant first - line options for CLL patients ineligible for full – dose FCR.

Aims: To perform a historical comparison of the efficacy and safety of LDFCR and BR regimens used as the first - line therapy of CLL within routine practice. Patients with 17p deletion and/or TP53 mutation were not included in this analysis because chemoimmunotherapy is no longer indicated in this extremely unfavourable subgroup due to very low activity.

Methods: The analysis included 237 pts treated with LDFCR and 320 pts treated with BR (median age, 69 vs 70 years; males, 70 vs 61%; median CIRS score, 6 vs 7; Rai stage III/IV, 57 vs 59%; bulky lymphadenopathy > 5cm, 35 vs 35%; unmutated IGHV, 74 vs 70%, deletion 11q, 28 vs 26%) treated between March 2009 and December 2019 at 17 centers cooperating within the Czech CLL Study Group. LDFCR consisted of fludarabine 20 mg/m 2 orally or 12 mg/m 2 on days 1-3, cyclophosphamide 150mg/m 2 orally or iv on days 1-3, and rituximab 500 mg/m 2 D1 (375 mg/m 2 in Cycle 1). BR included bendamustine 90 mg/m 2 iv on days 1-2 and rituximab 500 mg/m 2 D1 (375 mg/m 2 in Cycle 1). Median follow – up was 98 months for LDFCR and 57 months for BR.

Results: Distribution of demographic data and prognostic factors was comparable between LDFCR and BR groups. The overall response rate / complete remissions were similar for LDFCR vs BR (84/46% vs. 88/51%, p=n.s.). The median progression – free survival (PFS) was 30 vs 34 months (hazard radio [HR] 0.90; p=n.s.); PFS was markedly longer in pts with mutated IGHV and absence of del 11q (LDFCR vs BR, median 58 vs 59 months, HR 0.84; p=n.s.; Fig. 1a). Median overall survival (OS) was 73 vs 77 months for LDFCR vs BR (HR 1.09; p=n.s.) but markedly longer (median 116 months vs not reached) in patients with mutated IGHV without del 11q (HR 0.90; p=n.s.; Fig. 1b). Grade ≥ 3 neutropenia occurred in 55% (LDFCR) vs 51% (BR) pts but serious infections developed in 15% vs 20% pts only.

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Summary/Conclusion: Our data indicate that both low-dose FCR and BR are well – tolerated regimens with similar efficacy and safety and represent a suitable first - line treatment alternative for elderly / comorbid CLL patients with favourable biological prognosis. Detailed results including multivariate analysis will be presented.

P677: CLINICAL EFFICACY AND TOLERABILITY OF VENETOCLAX PLUS RITUXIMAB IN RELAPSED OR REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA – REAL WORLD ANALYSIS OF POLISH ADULT LEUKEMIA STUDY GROUP

A. Soboń 1,* , J. Drozd-Sokołowska 2 , E. Paszkiewicz-Kozik 3 , L. Popławska 3 , M. Morawska 4,5 , J. Tryc-Szponder 6 , L. Bołkun 7 , J. Rybka 8 , K. Pruszczyk 1 , A. Juda 9 , A. Majeranowski 10 , E. Iskierka-Jażdżewska 11 , P. Steckiewicz 12 , K. Wdowiak 13 , B. Budziszewska 1 , K. Jamroziak 2 , I. Hus 1 , E. Lech-Marańda 1 , B. Puła 1

1 Department of Hematology, Institute of Hematology and Transfusion Medicine; 2 Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw; 3 Department of Lymphoid Malignancies, Maria Sklodowska-Curie National Research Institute of Oncology, Warszawa; 4 Experimental Hematooncology Department, Medical University of Lublin & Hematology Department; 5 St. John’s Cancer Center, Lublin; 6 Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznań; 7 Department of Hematology, Medical University of Białystok, Białystok; 8 Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Wrocław; 9 Department of Hematology and Bone Marrow Transplantation, Medical University of Lublin, Lublin; 10 Department of Hematology and Transplantology, Medical University of Gdańsk, Gdańsk; 11 Department of Hematology, Medical University of Łódź, Copernicus Memorial Hospital, Łódź; 12 Department of Hematology, Holy Cross Cancer Center, Kielce; 13 Department of Internal Diseases and Oncological Chemotherapy Faculty of Medical Sciences, Medical University of Silesia, Katowice, Poland

Background: The introduction of venetoclax into clinical practice has improved the outcome of patients with relapsed/refractory chronic lymphocytic leukemia (RR-CLL). The results of the MURANO trial published in March 2018 showed significantly longer progression-free survival (PFS) and overall survival (OS) in RR-CLL patients treated with venetoclax and rituximab (VEN-R) comparing to bendamustine and rituximab (BR) and resulted in the approval of VEN-R in the therapy of RR-CLL in the European Union and the United States. It should be noted that the results of registration studies often do not correspond with the data from real-life observations.

Aims: To study the clinical efficacy and safety profile of VEN-R treatment in RR-CLL patients outside clinical trials.

Methods: We performed retrospective analysis of RR-CLL patients treated with VEN-R in hematology centers of the Polish Adult Leukemia Study Group (PALG) from 2019 to 2021.

Results: Clinical data of 117 RR-CLL patients treated with VEN-R were collected. Median patient age upon initiation of VEN-R therapy was 67 years (range 33 – 84 years). Seventy-two patients (61.5%) were men. Median Cumulative Illness Rating Scale (CIRS) was 6 (range 2 - 16). Patients were treated with a median of 2 (range 1–9) previous lines of therapy, whereas 32 patients (27.4%) had relapsed following the first line of treatment. Overall, 25 patients (21.4%) had 17p deletion, whereas TP53 mutation was identified in 13 patients (11.1%). The median follow-up was 9.96 months (range 0.27 - 29.13). The overall response rate (ORR) was 95.2%. Seventeen patients (14.5%) achieved complete remission (CR), 83 (70.9%) partial remission (PR), while in 5 patients (4.3%) disease progression was noted. In the patients with 17p deletion (n=22) or TP53 mutation (n=11), CR and PR were observed in 4 (12.1%) and 29 (87.9%) patients, respectively. The median PFS in the whole cohort was 20.8 (95% CI 18.43 - not reached) months and the median OS was not reached. In our study none of the analyzed clinico-pathological factors had significant impact on ORR, PFS and OS. During the follow-up time four (3.4%) cases of Richter transformation were diagnosed. There were 18 deaths recorded during the course of observation; 3 (16.7%) due to disease progression and 7 (38.9%) due to COVID-19 infection. The others were due to infections other than SARS-CoV-2 (n=3, 16.7%) and the cause of death could not be specified in five cases (27.8%). Eighty-three patients (70.9%) remain on treatment, while treatment was discontinued in thirty-four cases (29.1%). Reasons for therapy discontinuation included patient’s death (52.9%), treatment-related cytopenias (17.6%), disease progression (14.7%), Richter’s transformation (11.8%), autoimmune hemolytic anemia (5.9%), diarrhea (2.9%) and infections (8.8%). In one case treatment discontinuation was due to consent withdrawal and one patient was lost to follow-up. The following adverse events of VEN-R treatment were reported during the study: all grade neutropenia (71.8% with grade 3/4 in 55.6%), anemia (51.3%), thrombocytopenia (47%), pneumonia (9.4%), neutropenic fever (6.8%), autoimmune hemolytic anemia (4.3%), immune thrombocytopenic purpura (1.7%), diarrhea (4.3%) and in one case exacerbation of heart failure was observed.

Summary/Conclusion: In this retrospective analysis the outcomes of treatment with the VEN-R regimen in real-life setting were worse than those reported in the MURANO trial.

P680: SARCOPENIA EVALUATED BY CT SCAN IS ASSOCIATED WITH SHORTER SURVIVAL IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA TREATED WITH TARGETED THERAPIES. A PROSPECTIVE STUDY.

A. Visentin 1,* , F. Crimì 2 , A. Corso 2 , R. Angelone 2 , C. A. Cavarretta 1 , A. Cellini 1 , F. Angotzi 1 , V. Ruocco 1 , S. Pravato 1 , E. Quaia 2 , L. Trentin 1

1 Hematology and Clinical Immunology unit, Department of Medicine, University of Padova; 2 Radiology Institute, Department of Medicine, University of Padova, Padova, Italy

Background: Chronic lymphocytic leukemia (CLL) usually affects elderly patients with comorbidities. Although drugs targeting BTK, BCL2 and PI3K have been proved to be better tolerated than chemoimmunotherapy and to improve the survival of CLL patients, comorbidities, assessed by Cumulative Illness Rating Scale (CIRS) score, are still associated with a worse outcome. Indexes of sarcopenia, subcutaneous or visceral adipose tissues assessed by CT scan, are emerging as survival markers in cancer patients.

However, CT scan is not recommended by iwCLL guideline nor we known the clinical impact of body composition in CLL.

Aims: The aim of this study was to assess the impact of muscle and adipose tissue indexes in patients with CLL treated with targeted agents.

Methods: We performed a single center prospective study of CLL patients treated with BTK, BCL2 and PI3K inhibitors out of clinical trials. Inclusion criteria were diagnosis of CLL and need of treatment according to iwCLL guideline, creatinine clearance >30ml/min and CT scan performed at baseline. For each patient, with NIH ImageJ software, we extracted the areas of subcutaneous fat, visceral fat and the skeletal muscle from an unenhanced axial CT image at vertebra L3. The values obtained (cm 2 ) were divided by the square of patient’s height (m 2 ), obtaining the subcutaneous adipose tissue index (SATI), the visceral adipose tissue index (VATI) and the skeletal muscle index (SMI). Overall survival (OS) was calculated as months from starting targeted therapies to death (event) or last available follow-up (censored). Survival curves with Log-rank test.

Results: We included 118 patients, 70 (59%) were males, mean age was 71.6±8.6 years, 74 (63%) were at Rai stage III-IV, 50 (42%) had a CIRS≥6, 96 (81%) unmutated IGHV, 41 (35%) 17p13 deletion, 39 (33%) TP53 mutation. The median number of therapies was 3 (range 1-9). Sixty-six patients (56%) received ibrutinib, 27 (23%) idelalisib+rituximab and 25 (21%) venetoclax±rituximab, including 17 (14%) front line therapies (13 ibrutinib, 4 venetoclax).

We observed that SMI values correlated with SATI (p=0.035) and VATI (p<0.0001). SMI, SATI and VATI were higher in males vs females, median 48.4 vs 42.2 (p=0.007), 61.0 vs 67.9 (p=0.0087) and 70.0 vs 50.3 (p=0.0113)(Fig 1A). Using these thresholds, low-SMI, suggestive of sarcopenia, and low SATI correlated with a more advance number of previous therapies (p=0.027 and p=0,037). Low-SMI was more common in patients with CIRS≥6 (p=0.039). SMI, SATI and VATI did not correlated with age, stage and biological markers.

After a median follow-up of 32 months the median OS for the whole cohort was 46.7 months. We confirmed that CIRS≥6 correlated with a worse OS in our study (p<0.0001, Fig. 1B). The 3-year OS was 53% and 66% for patients with low-SMI vs high-SMI, and the median OS was 40.3 months for low-SMI but not reached for high-SMI patients (p=0.0199, Fig. 1C). SATI and VATI did not correlate with OS.

Combining data of CIRS≥6 and low-SMI, 40 (34%) patients, 47 (40%) and 31 (26%) displayed 0, 1 and 2 markers (Fig. 1D). The 3-year OS was 80%, 56% and 42% for patients at score 0, 1 and 2. The median OS was not reached for score 0 patients, but decreased from 42 to 23 months for patients at score 1 and 2 (p=0.0006, Fig. 1E).

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Summary/Conclusion: In this prospective study we found that that basal CT scan identifies sarcopenic CLL patients with low SMI featured by an adverse prognosis even if treated with targeted oral drugs, in particular when combined with the CIRS score. The prognostic and predictive impact of basal CT scan in CLL deserves further investigation.

P681: EUROFLOW STANDARDIZATION TECHNIQUE AND NORMALIZATION PROCEDURES IN LONGITUDINAL FLOW CYTOMETRIC EXPRESSION ANALYSIS OF CD20 IN CLL PATIENTS RECEIVING ANTI-CD20 DIRECTED THERAPY

P. J. Walter 1,* , A. Schilhabel 1 , P. Cramer 2 , J. von Tresckow 3 , S. Kohlscheen 1 , K. Fischer 2 , B. Eichhorst 2 , S. Böttcher 4 , M. Brüggemann 1 , M. Kneba 1 , M. Hallek 2 , M. Ritgen 1

1 Laboratory for Specialized Hematological Diagnostics, Medical Department II, University Hospital Schleswig-Holstein, Kiel; 2 Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, German CLL Study Group, University Hospital Cologne, Cologne; 3 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen; 4 Medical Department III Hematology, Oncology, Palliative Care, Center for Inner Medicine, University Hospital Rostock, Rostock, Germany

Background: CD20 expression is still a controversial issue regarding its prognostic value for therapy outcome. While up to 50% of patients relapse within four of years after anti-CD20 directed treatment and constitute a need for a salvage or relapse/refractory treatment, only few publications address the longterm influence on CD20 expression assessed by flow cytometry. The technical complexity of the method and factors affecting the variability of measurements of samples at different time points and from different sources/patients are well recognized by the field and addressed by different approaches as standardization of instruments, automated gating and alignment of population or normalization.

Aims: In order to evaluate long term influence of anti-CD20 directed therapy on CD20 expression in patients with chronic lymphocytic leukemia who showed only weak MRD responsiveness to therapy in different clinical trials, we aimed to establish a normalization approach based on the instrument standardization to reduce the proportion of technical variance within data.

Methods: Mean fluorescence intensities (MFI) of peak seven of standardized fluorescence beads from daily quality control and instrument standardization of flow cytometers according to the EuroFlow protocol were used to establish a normalization approach. Proof of prinicple was performed using the residual peaks covering the MFI range provided by the beads. To assess different sources of variance ANOVA of a 3x3x3 experiment evaluating marker expressions on different cell populations in the blood of healthy donors using Quantibrite-PE beads was performed addressing technical variation (day, instrument) and biological variation. CD20 and CD19 MFI on CLL cells were retrospectively assessed from MRD measurements of peripheral blood samples of patients enrolled in the phase II GCLLSG trials CLL2-BIG (n=57), -BAG (n=58), -BIO (n=58), -BCG (n=22), combining facultative Bendamustin (B) debulking with either Obinutuzumab (G), Ofatumumab (O), Venetoclax (A), Ibrutinib (I) or Idelalisib (C).

Results: Long term shifts of fluorescence intensities, and coefficients of variation were reduced 2 to 10 times across the MFI range covered by the fluorescence beads without distorting the longitudinal course of instrument performance. Most of the variance in assessments of molecules equivalent soluble fluorochrome from MFIs of NK cell and B cell markers in peripheral blood of healthy donors were related to inter-donor variations (p<0.002). Normalized MFIs for CD19 and CD20 on CLL cells do not significantly differ from the measured values in the Mann-Whitney-U test (p>0.85). Higher CD20 expression at therapy start seems to be correlated to strong MRD response solely in the CLL2-BIO trial (p=0.036, Fig. 1). Prognostic factors TP53-, IGHV-, and NOTCH1- mutation did not seem to correlate with the observed CD20 expression differences. Therapy strata showed differences (statistically not significant) in the proportion of firstline and r/r treated patients.

Figure 1 Pre-treatment CD20 MFI on CLL cells of patients categorized according to treatment and variable (strong, medium, weak, and no) MRD response.

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Summary/Conclusion: Following standardized staining and instrument monitoring, it should be possible to robustly assess longitudinal biological variations of marker expression based on MFI values. In a cross trial comparison Obinutzumab showed highest proportion of patients with strong MRD response independent from initial CD20 expression, whereas strong response to Ofatumumab seems to correlate with higher CD20 expression levels.

P682: NEMTABRUTINIB (MK-1026), A NON-COVALENT INHIBITOR OF WILD-TYPE AND C481S MUTATED BRUTON TYROSINE KINASE FOR B-CELL MALIGNANCIES: EFFICACY AND SAFETY OF THE PHASE 2 DOSE-EXPANSION BELLWAVE-001 STUDY

J. Woyach 1,* , I. W. Flinn 2 , F. T. Awan 3 , H. Eradat 4 , D. Brander 5 , M. Tees 6 , S. A. Parikh 7 , T. Phillips 8 , W. Wang 9 , N. M. Reddy 10 , M. Z. Farooqui 10 , J. C. Byrd 11 , D. M. Stephens 12

1 Division of Hematology, The Ohio State University, Columbus, Columbus; 2 Sarah Cannon Center Research Institute, Nashville, Nashville; 3 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas; 4 Department of Hematology-Oncology, David Geffen School of Medicine at UCLA, Los Angeles; 5 Duke Cancer Institute, Duke University Medical Center, Durham; 6 Colorado Blood Cancer Institute, Denver; 7 Division of Hematology, Mayo Clinic, Rochester; 8 Division of Hematology and Oncology, University of Michigan Rogel Cancer Center, Ann Arbor; 9 Veristat, LLC, Southborough; 10 Merck & Co., Inc., Kenilworth; 11 Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati; 12 Division of Hematology and Hematologic Malignancies, University of Utah Huntsman Cancer Institute, Salt Lake City, United States of America

Background: For patients (pts) with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and certain B-cell neoplasms, resistance to Bruton tyrosine kinase inhibitors (BTKi) develops primarily through mutations at the cysteine binding site (C481) or PLCγ2 mutations. Nemtabrutinib (MK-1026, formerly ARQ-531) is a non-covalent, potent inhibitor of both wild type and C481-mutated BTK.

Aims: In the phase 1/2 dose-escalation and dose-expansion BELLWAVE-001 study ( NCT03162536 ), the preliminary recommended phase 2 dose (RP2D) of nemtabrutinib was determined to be 65 mg once daily. The efficacy and safety of nemtabrutinib in patients (pts) with CLL/SLL and B-cell non-Hodgkin lymphoma (NHL) were also evaluated at a higher dose during the dose-expansion phase.

Methods: In this open-label, single-arm phase 2 study, 9 expansion cohorts were initiated following determination of preliminary nemtabrutinib RP2D. Approximately 10-25 eligible pts were enrolled into Cohort A (relapsed/refractory (r/r) CLL/SLL, with ≥2 prior therapies including covalent BTKi, with documented C481 mutation), Cohort B (r/r CLL/SLL progressed on/intolerant to a BTKi, with ≥2 prior therapies without C481 mutation), Cohort C (Richter transformation with ≥1 prior therapy), Cohort D-H (follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, high-grade B cell lymphoma with known MYC, BCL-2 or BCL-6 translocations, and Waldenström macroglobulinemia [WM], respectively, who received ≥2 prior therapies), Cohort I (food-effect cohort including pts with B-cell NHL, CLL/SLL, and WM). Primary end point was ORR (per iwCLL criteria, by investigator) for participants with CLL/SLL. Secondary end points included DOR and safety and tolerability.

Results: Among 118 pts enrolled, 44 had B-cell NHL, 68 CLL/SLL, and 4 WM. Of these, 94 (79.6%) were treated at the preliminary RP2D, including 51 (54.3%) participants with CLL/SLL. Pts with CLL/SLL had a median (range) number of prior therapies of 4 (1-18), 43 (84%) had prior BTKi therapy, 12 (23%) had del17p, 30 (59%) had IGHV unmutated status, and 32 (63%) had C481S BTK mutation. At data cut-off (April 7, 2021), median (range) follow-up was 4.6 mo (0.1-26.7) for all treated pts. ORR was 57.9% (22/38; 1 CR, 21 PR/PRL) per iwCLL criteria in the efficacy-evaluable population of pts with CLL/SLL treated at preliminary RP2D. Median duration of response was not estimable [NE] (range, 8.3 mo-NE). Among all treated participants, 114 (97%) had a treatment-emergent adverse event (TEAE), with 78 (66%) having a drug-related TEAE, and 9 (8%) having a drug-related TEAE that led to discontinuation. Common TEAEs (≥ 20%) included fatigue (33%), constipation (31%), dysgeusia (28%), cough (25%), nausea (25%), pyrexia (25%), dizziness (23%), hypertension (23%), peripheral edema (22%), diarrhea (21%), and arthralgia (20%). Grade ≥3 TEAEs occurred in 80 (68%) participants. Grade 5 TEAEs occurred in 7 (6%) participants and included death following disease progression 3 (3%), sepsis 1 (1%), dyspnea 1 (1%), and respiratory failure 2 (2%). Common drug-related TEAEs (≥10%) included dysgeusia (15%), nausea (11%), fatigue (11%), and decreased neutrophil count (10%). Grade 3-4 drug-related TEAEs occurred in 31 (26%) participants. No drug-related TEAEs led to death.

Summary/Conclusion: Nemtabrutinib has promising antitumor activity with a manageable safety profile in pts with CLL/SLL exposed to multiple lines of therapy, including in those who had progression of disease on prior covalent BTKi. Further evaluation of nemtabrutinib in B-cell malignancies is ongoing.

P683: A FIRST-IN-HUMAN PHASE 1 STUDY OF ORAL LOXO-338, A SELECTIVE BCL2 INHIBITOR, IN PATIENTS WITH ADVANCED HEMATOLOGIC MALIGNANCIES (TRIAL IN PROGRESS)

W. Jurczak 1,* , A. J. Alencar 2 , G. S. Guru Murthy 3 , M. Hoffmann 4 , J. M. Pagel 5 , V. Patel 5 , J. M. Pauff 5 , P. L. Zinzani 6 , S. Le Gouill 7 , A. R. Mato 8 , L. E. Roeker 8

1 Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 2 Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, FL; 3 Division of Hematology/Oncology, Medical College of Wisconsin, Milwaukee, WI; 4 Hematologic Malignancies and Cellular Therapeutics, University of Kansas Cancer Center, Overland Park, KS; 5 Loxo Oncology at Lilly, Stamford, CT, United States of America; 6 Institute of Hematology Seràgnoli, University of Bologna, Bologna, Italy; 7 Institut Curie Hospital, Paris, France; 8 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America

Background: B-cell lymphoma 2 (BCL2) is a key regulator of apoptosis and provides protection from cell death in many hematological malignancies. LOXO-338 is a novel, orally bioavailable small molecule inhibitor of BCL2, designed to achieve selectivity over BCL-xL and thus avoid dose-limiting thrombocytopenia associated with BCL-xL inhibition. In preclinical studies, LOXO-338 showed a favorable pharmacological profile, selectively inhibited BCL2, and was well-tolerated in vivo . LOXO-338 also demonstrated dose-dependent tumor growth inhibition in various murine xenograft models, and showed improved efficacy in combination with pirtobrutinib, a highly selective, non-covalent (reversible) BTK inhibitor (Brandhuber et al. Cancer Res 2021;81,13 Suppl:1258).

Aims: To determine the recommended phase 2 dose of LOXO-338 and evaluate the safety profile of LOXO-338 as monotherapy and in combination.

Methods: LOXO-BCL-20001 is a global, open-label, multi-center, first-in-human phase 1 study of oral LOXO-338 in patients with advanced hematologic malignancies who have received standard therapy ( NCT05024045 ). The study will be conducted in 2 parts. Part 1 will evaluate LOXO-338 as monotherapy, and will explore different dosing strategies. Part 2 will evaluate LOXO-338 in combination with pirtobrutinib. Part 1 dose escalation portion of the study will follow an i3 + 3 design. Each cycle will be 28 days (4 weeks).

Eligible patients include those with CLL/SLL, mantle cell lymphoma, and Waldenstrӧm macroglobulinemia (WM) who received standard therapy. Patients with other B-cell non-Hodgkin lymphomas (NHL) who failed standard therapy or, in the opinion of the investigator, have no known available options to provide benefit for the patient’s condition, are also eligible. Patients must have recovered from prior treatment-related adverse events. Patients with active or suspected Richter transformation, transformed low grade lymphoma, Burkitt or Burkitt-like lymphoma, and multiple myeloma are also eligible. Patients with AL amyloidosis are eligible in monotherapy dose expansion; all other patients are eligible in both monotherapy and combination dose expansion. Patients must not have progressed while receiving prior BCL2 inhibitor; those with WM or AL amyloidosis must not have received a prior BCL2 inhibitor. Key exclusion criteria include history of CNS involvement, stem cell transplant or CAR-T therapy <60 days, concurrent anticancer therapy, and clinically significant cardiovascular disease.

The primary objective of part 1 is to determine the recommended phase 2 dose of oral LOXO-338 in patients who were previously treated for CLL/SLL and other B-cell NHL, administered alone or in combination with pirtobrutinib. Determining antitumor activity in patients with WM and AL amyloidosis is an additional primary objective of part 1 monotherapy. Secondary objectives include determining the safety profile and tolerability, PK properties and antitumor activity of LOXO-338 in combination with pirtobrutinib. Antitumor activity will be evaluated based on overall response rate (ORR), progression-free survival (PFS), time to progression (TTP) and duration of response (DOR) based on disease-specific response criteria per investigator assessment.

Results: N/A

Summary/Conclusion: N/A

P684: PHASE 1 STUDY OF LP-108, A SELECTIVE BCL-2 INHIBITOR, IN PATIENTS WITH RELAPSED OR REFRACTORY B-CELL NON-HODGKIN LYMPHOMA

W. Xu 1,* , R. Feng 2 , L. Wang 1 , Y. Song 3 , H. Zhu 1 , L. Fan 1 , X. Guo 2 , X. Wei 2 , N. Lin 3 , C. Xu 4 , Z. Wang 5 , X. Xiao 5 , Y. Shen 5 , Y. Chen 6 , Y. Chen 6 , F. Tan 5 , S. P. Anthony 6 , J. Li 1

1 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing; 2 Department of Hematology, Nanfang Hospital of Southern Medical University, Guangzhou; 3 Department of Lymphoma, Beijing Cancer Hospital, Beijing; 4 Phase I Unit, Nanfang Hospital of Southern Medical University; 5 Department of Clinical Development, Guangzhou Lupeng Pharmaceutical Co., Ltd., Guangzhou, China; 6 Department of Clinical Development, Newave Pharmaceutical Inc., Pleasanton, United States of America

Background: Targeting BCL-2 family proteins is a well-recognized therapeutic approach. Globally approved BCL-2 inhibitor venetoclax has shown efficacies in certain hematologic malignancies (HMs) but requires weekly ramp-up to the target dose to mitigate the risk of tumor lysis syndrome (TLS). LP-108 is a novel, highly potent, orally bioavailable, and selective BCL-2 inhibitor that has shown promising preclinical antitumor activity in various HMs.

Aims: To present preliminary results from an ongoing phase 1 study of LP-108 under daily dose ramp-up in patients with relapsed or refractory (r/r) B-cell non-Hodgkin lymphoma (NHL) ( NCT04356846 ).

Methods: This is an open-label, multicenter, phase I study to assess the safety, pharmacokinetics (PK) and preliminary efficacy of LP-108 in Chinese patients with r/r B-cell NHL, consisting of a dose-escalation phase and a dose-expansion phase. Adult r/r B-cell NHL patients requiring therapy are eligible if they had received at least one prior line of treatment (except BCL-2i). LP-108 is orally administered once daily in 28-day cycles until disease progression, unacceptable toxicity, or per investigator discretion. All patients provided and signed informed consent.

Results: As of February 18, 2022, 19 patients have been enrolled and were evaluable for safety, with disease types of MCL (n = 7), CLL/SLL (n = 7), DLBCL (n = 2), FL, WM, and MALT (n = 1 each), and median age of 61 years (range, 39 to 83). Patients had received a median of 3 prior lines of therapy (range, 1~6), of which 13 (68%) patients were previously BTKi exposed. No dose-limiting toxicity (DLT) has been observed in patients who were treated with LP-108 up to 600 mg, thus the maximum tolerated dose (MTD) was not identified. With median duration of treatment of 55 days (range, 4~472), treatment-related adverse events (TRAEs) were reported in 17 patients (89.4%), most of which were grade 1 to 2 in severity. TRAEs of any grade occurred ≥20% of patients were neutropenia (36.8%), leukopenia (31.5%), ALT elevation (31.5%), AST elevation (26.3%), hyperphosphatemia (21%), hyperuricemia (21%), and diarrhea (21%). Grade 3 or 4 TRAEs were reported in 7 patients (36.8%), including neutropenia, thrombocytopenia, lymphopenia, laboratory TLS, and rash. Serious TRAEs included rash, thrombocytopenia, and laboratory TLS, each occurring in 1 patient. No patient discontinued treatment due to toxicity, and no clinical TLS was observed. No deaths occurred during treatment or within 28 days of the end of treatment. At doses ≥200 mg, 3 of 10 efficacy evaluable patients achieved an objective response (1 CR and 2 PR), 4 patients had stable disease (2 with substantially reduced lymphomegaly), and 3 patients had progressive disease. Rapid reduction in ALC was observed in CLL patients (pending imaging assessment) during ramp-up and the first cycle of treatment at doses as low as 20mg. At data cutoff, 11 patients remain on treatment and 8 patients discontinued treatment due to disease progression or patient decision. The PK profile showed that plasma exposure proportionally increased with doses ranging from 20 to 600 mg (mean AUC 24h range, 1.26~46.5 h*μg/ml, mean C max range, 0.087~2.67 μg/ml). Plasma concentrations peaked approximately 4 to 10 hours after dosing, and the average terminal half-life of LP-108 was approximately 11.8 hours (range, 6.9~16.6).

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Summary/Conclusion: LP-108 showed favorable safety profile up to 600 mg with daily dose ramp-up schedule, and antitumor activity in patients with r/r B-cell NHL. Dose escalation beyond 600 mg and further dose expansion are ongoing.

P685: REAL WORLD EVIDENCE OF IMPACT OF ATRIAL FIBRILLATION ON CLINICAL AND ECONOMIC OUTCOMES IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

K. Yang 1 , T. Liu 2 , B. Tang 1 , A. Chanan-Khan 3,*

1 Beigene USA, San Mateo; 2 Beigene USA, Emeryville; 3 Mayo Clinic, Jacksonville, United States of America

Background: While the incidence of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) has been increasing, the implications of AF in real-world CLL patients remain understudied.

Aims: This study aimed to assess the impact of AF on clinical and economic outcomes in CLL patients.

Methods: This retrospective observational study used the US IBM MarketScan® commercial claims/Medicare supplement database (2017-2020) to identify newly diagnosed CLL patients (≥18 years) and incidence of AF after index date, defined as the first CLL diagnosis date. Patients were followed for ≥3-months pre-index, and from index to last follow-up or death. In addition to patient characteristics, clinical outcomes (incidence of heart failure [HF], bleeding and stroke) and economic outcomes (costs and healthcare resource utilization [HRU]) were compared between CLL patients with AF (CLL+AF) and CLL patients without AF (CLL-AF). HRU was evaluated by inpatient, outpatient, ER, and pharmacy visits. Multivariable regression analyses were conducted to examine the association between AF and clinical outcomes.

Results: Among a total of 16,800 newly diagnosed CLL patients included in the study, 20% developed AF. CLL+AF were significantly older than CLL-AF (median: 77 vs 62 years; P < .001). Compared to CLL-AF, CLL+AF also had significantly more comorbidities at baseline, as shown by higher Charlson comorbidity index (CCI) (median: 1.0 vs 3.0; P < .001) and more patients with previous AF history (0.3% vs 8.4%; P < .001). Further assessing clinical outcomes in CLL+AF vs CLL-AF, significantly higher incidence of HF (26.3% vs 3.0%; P < .001), bleeding (12.2% vs 4.8%; P < .001) and stroke (6.6% vs 1.2%; P < .001) were observed. For HRU, CLL+AF were reported to have significantly higher rates of ER visits (29.4% vs 12.9%; P < .001) and hospitalizations (42.2% vs 14.5%; P < .001) than CLL-AF. In CLL+AF, the average total AF-related costs were $13,520.21 within 30 days after AF diagnosis, and $22,304.82 within 60 days after AF diagnosis. Controlling for demographics and comorbidities, multivariable regressions reported statistically significant associations between AF and HF, as well as AF and stroke (Table).

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Summary/Conclusion: This real-world study reported significantly higher incidence of HF, bleeding and stroke incurred by CLL patients who developed AF compared with those who did not. The presence of HF, bleeding and stroke further increased HRU and costs. These findings highlight the importance of better disease management and treatment selection to prevent AF in CLL patients.

P686: A PHASE 1 FIRST IN-HUMAN STUDY OF BGB-16673, A BRUTON TYROSINE KINASE PROTEIN DEGRADER, IN PATIENTS (PTS) WITH B-CELL MALIGNANCIES (TRIAL IN PROGRESS)

C. S. Tam 1,2,3,* , C. Cheah 4,5,6 , D. A. Stevens 7 , K. By 8 , X. Chen 8 , B. Tariq 8 , G. S. Vosganian 8 , J. Huang 8 , M. Alwan 9

1 Peter MacCallum Cancer Centre, Melbourne, Victoria; 2 University of Melbourne; 3 Royal Melbourne Hospital, Parkville, Victoria; 4 Department of Haematology, Sir Charles Gairdner Hospital and Pathwest Laboratory Medicine, Nedlands, Western Australia; 5 Medical School, University of Western Australia, Crawley, Western Australia; 6 Linear Clinical Research, Nedlands, Western Australia, Australia; 7 Norton Cancer Institute, Louiseville, KY; 8 BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 9 Perth Blood Institute, West Perth, Western Australia, Australia

Background: Bruton tyrosine kinase (BTK) functions downstream of the B-cell antigen receptor (BCR) and plays a critical role within the BCR signaling pathway and the pathogenesis of several B-cell malignancies. BTK inhibitors (BTKi) have revolutionized management of B-cell malignancies. However, resistance caused by BTK mutations, which can abrogate BTKi binding capacity or BTK scaffold function, or cause kinase hyper activation, may limit therapeutic options in subsequent lines of therapy. Re-challenging patients with agents that can overcome the resistance due to BTK mutations may provide a novel treatment option. BGB-16673 is an investigational, orally available agent with preclinically demonstrated BTK degradation activity against both wild type and mutant forms commonly identified in pts who have progressed on BTKi.

Aims: In the dose-escalation part of the study, we aim to assess the safety and tolerability of BGB-16673 in selected relapsed or refractory (R/R) B-cell malignancies, to characterize its pharmacokinetic (PK) and pharmacodynamic (PD) profiles, to determine a recommended phase 2 dose (RP2D) and to evaluate anti-tumor activity. In the dose-expansion part of the study, we aim to evaluate the safety, tolerability, PK, PD, and anti-tumor activity under the RP2D in pts with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL).

Methods: BGB-16673-101 ( NCT05006716 ) is a phase 1 open-label, dose-escalation and -expansion study evaluating BGB-16673 in adult pts with R/R B-cell malignancies. Key inclusion criteria include confirmed diagnosis of R/R B-cell malignancy (including marginal zone lymphoma, follicular lymphoma, MCL, CLL/SLL, and Waldenström macroglobulinemia [WM]), Eastern Cooperative Oncology Group performance status score of 0 to 2, and adequate organ function. Key exclusion criteria include current or history of central nervous system involvement, autologous stem cell transplant (ASCT) within 3 months or chimeric antigen receptor T cell therapy or allogeneic SCT within 6 months of the first dose of BGB-16673 or requiring treatment with strong inhibitors or inducers of CYP3A. All pts will be followed for safety and tolerability, including treatment-emergent adverse events that occur during treatment and up to 30 days after treatment discontinuation, or until the initiation of another anti-cancer therapy, whichever occurs first. The totality of the available safety, efficacy, PK, and PD data from the dose-escalation part will be used by the Safety Monitoring Committee to determine the RP2D. The dose-expansion part will commence subsequent to RP2D determination. Responses will be evaluated per the 2014 Lugano Classification, the 2018 International Workshop on CLL guidelines response assessment with modification for treatment-related lymphocytosis, or the 6 th International Workshop on WM consensus criteria. Additional efficacy analyses will include progression-free survival and overall survival. All pts will give informed consent.

Results: This is a trial in progress; safety and tolerability results of BGB-16673 are expected.

Summary/Conclusion: BGB-16673-101 is the first in-human study of the BTK degrader BGB-16673.

P687: A PHASE 1 STUDY WITH THE NOVEL B-CELL LYMPHOMA 2 (BCL2) INHIBITOR BGB-11417 AS MONOTHERAPY OR IN COMBINATION WITH ZANUBRUTINIB (ZANU) IN PATIENTS (PTS) WITH B-CELL MALIGNANCIES: PRELIMINARY DATA

S. Opat 1,2,* , C. Y. Cheah 3,4,5 , M. Lasica 6 , E. Verner 7,8 , P. J. Browett 9 , H. Chan 10 , J. D. Soumerai 11 , E. González Barca 12 , J. Hilger 13 , Y. Fang 13 , J. Huang 13 , D. Simpson 13 , C. S. Tam 14,15,16

1 Monash Health, Clayton; 2 Monash University, Clayton, Victoria; 3 Sir Charles Gairdner Hospital and Pathwest Laboratory Medicine, Nedlands; 4 Medical School, University of Western Australia, Crawley; 5 Linear Clinical Research, Nedlands, Western Australia; 6 St. Vincent’s Hospital Melbourne, Fitzroy, Victoria; 7 Concord Repatriation General Hospital, Concord; 8 University of Sydney, Sydney, New South Wales, Australia; 9 Auckland City Hospital; 10 North Shore Hospital, Auckland, New Zealand; 11 Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, United States of America; 12 Institut Català d’Oncologia-Hospitalet, IDIBELL, Universitat de-Barcelona, Barcelona, Spain; 13 BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 14 Peter MacCallum Cancer Centre, Melbourne; 15 University of Melbourne, Parkville; 16 Royal Melbourne Hospital, Parkville, Victoria, Australia

Background: BCL2 is aberrantly expressed in many hematologic malignancies and promotes tumorigenesis by enabling cells to evade apoptosis. BCL2 inhibitors have an established role in the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and acute myeloid leukemia. The currently approved BCL2 inhibitor, venetoclax, is associated with mild gastrointestinal toxicities, neutropenia, and the development of BCL2 mutations leading to resistance. BGB-11417 is a highly selective inhibitor of BCL2 with superior potency to venetoclax in human acute lymphoblastic leukemia, mantle cell lymphoma (MCL) cell lines, and xenograft model of diffuse large B-cell lymphoma (DLBCL). BGB-11417 has favorable pharmacokinetics with excellent bioavailability and selectivity for BCL2. Toxicology studies have shown a broad therapeutic index and an improved safety profile.

Aims: BGB-11417-101 is an ongoing first-in-human phase 1/1b dose-escalation and expansion study ( NCT04277637 ) evaluating the safety, tolerability, maximum tolerated dose (MTD), and recommended phase 2 dose of oral BGB-11417 as monotherapy or in combination with the BTK inhibitor ZANU, in pts with B-cell malignancies.

Methods: In separate monotherapy and combination therapy dose-escalation cohorts, pts with relapsed/refractory (R/R) B-cell malignancies received escalating doses of BGB-11417 (40, 80, 160, 320, or 640 mg once daily [QD]) with a weekly or daily ramp-up to intended target dose. Pts in the combination therapy cohorts received ZANU (320 mg QD or 160 mg twice daily) 8-12 weeks before BGB-11417. Dose-limiting toxicity for each dose cohort was evaluated by a Bayesian logistic regression model during dose ramp-up through day 21 at intended dose. Adverse events (AEs) were reported per Common Terminology Criteria for AEs v5.0.

Results: As of 17 Dec 2021, 58 pts received BGB-11417 (32 monotherapy; 26 combination). Of the pts receiving monotherapy, 26 with R/R non-Hodgkin lymphoma (NHL; 17 DLBCL, 6 follicular lymphoma, and 3 marginal zone lymphoma) received BGB-11417 ≤640 mg and 6 with R/R CLL/SLL received ≤160 mg. Of the pts receiving combination treatment, 19 with R/R CLL/SLL received BGB-11417 ≤160 mg and 7 with R/R MCL received ≤80 mg. MTD has not yet been reached. Median follow-up was 3.9 months (range, 0.1-20.4). AEs across all dose levels are listed in the table. Only 2 grade ≥3 AEs (1 neutropenia, 1 autoimmune hemolytic anemia) were reported in combination cohorts. Twenty pts discontinued treatment (17 disease progression; 1 AE; 2 other reasons). One high-risk pt with CLL on monotherapy had laboratory tumor lysis syndrome (TLS) that resolved with no intervention (laboratory TLS <2%). Efficacy data are early: most pts had reduction in sum of product of perpendicular diameters; 2 pts with NHL (monotherapy) achieved responses (1 complete response). Pts with CLL/SLL had notable reductions in absolute lymphocyte count at doses as low as 1 mg; 2 responses (partial response or better) were seen with monotherapy and 12 responses with combination (partial response with lymphocytosis or better).

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Summary/Conclusion: These preliminary data show promising efficacy for BGB-11417 and an improved safety profile, particularly in combination cohorts. Grade ≥3 neutropenia was uncommon. BGB-11417 is tolerable up to doses of 640 mg as monotherapy and up to 160 mg in combination with ZANU. Dose escalation continues as an MTD has not yet been reached in any dose-escalation cohort. Enrollment continues, with data for Waldenström macroglobulinemia and treatment-naïve CLL/SLL cohorts forthcoming.

P688: VIRTUAL SCREENING PROTOCOL TO IDENTIFY TYROSINE KINASE INHIBITORS WITH A POTENTIAL TO INTERACT WITH P-GLYCOPROTEIN AND TO ACT AS MDR CHEMOSENSITIZERS: A VALIDATION STUDY

E. Beleva 1,2,3,* , I. Tsakovska 1 , P. Alov 1 , T. Pencheva 1 , I. Lessigiarska 1 , I. Pajeva 1

1 QSAR and Molecular Modeling, Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia; 2 Clinical Oncology, Medical University - Plovdiv, Plovdiv; 3 Faculty of Mathematics and Informatics, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria

Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) has been attributed to both BCR-ABL-dependent pathways - acquisition of point mutations and BCR-ABL-independent ones – drug efflux mediated by ATP-binding cassette proteins. Primary multidrug resistance (MDR) has been mostly attributed to aberrant expression of P-glycoprotein (Pgp). Therefore research efforts have been directed to improving anticancer drug efficacy by co-administration of TKIs as Pgp modulators. Recently novel SRC family kinase (SFK) inhibitors based on the pyrazolol[3,4-d]-pyramidine scaffold were shown to inhibit Pgp in two MDR cancer cell lines with the proforms outlining a stronger potential to modulate Pgp [1].

Aims: In this study we aimed: (i) to validate the in-house developed virtual screening (VS) protocol for TKIs with a potential to interact with Pgp; (ii) to compare the Pgp binding potential of the novel SFK inhibitors (SI306, SI221, pro-SI306, pro-SI221) with that of widely explored TKIs (core group), including those used for treatment of CML - imatinib, nilotinib and dasatinib.

Methods: Molecular modelling was performed in MOE software [2]. Two VS protocols were explored in the study: (A) docking without pharmacophore filter and with triangle matching placement; (B) an in-house developed VS protocol based on pharmacophore filter and docking with pharmacophore placement. The following docking settings were applied: (i) rigid receptor / flexible ligand mode; (ii) London dG scoring function applied at both the placement stage and the rescoring stage. VS protocol B is based on an in-house library of 43 TKIs collected from the scientific literature and DrugBank database. The library includes structural identificators of the compounds and experimental data proving their P-gp binding. It is freely available at http://biomed.bas.bg/qsarmm/Tyrosine_kinase_inhibitors.The VS protocols were applied to predict the Pgp binding affinity of the SFK inhibitors SI306, SI221, pro-SI306, and pro-SI221 as well as the core group drugs.

Results: The results are presented in Table 1. The pro-forms showed higher interaction energies in the docking simulations compared to their parent ones in agreement with the experimental results pointing to the pro-forms having a stronger potential to modulate Pgp (Table 1) [1]. Further the comparison with the reference compound Dasatinib shows that except for SI221, the novel SFK inhibitors have a similar or even higher potential to interact with P-gp. The comparison with the core group of drugs outlines the following order of predicted P-gp affinity: (i) for VS protocol A: pro-SI306>pro-SI221>Nilotinib; (ii) for VS protocol B: pro-SI221>Lapatinib>pro-SI306>Tepotinib>Nilotinib. In both cases pro-SI306 and pro-SI221 are among the top-ranked P-gp ligands.

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Summary/Conclusion: In summary, the previously developed VS screening protocol based on pharmacophore filtering and docking was validated on novel SFK inhibitors that were predicted as Pgp ligands in agreement with the experimental results. Further, the VS results outlined their similar or even higher affinity to Pgp compared to Dasatinib, which was used as reference compound due to its dual BCR-ABL and SRC inhibitory activity. Therefore they are suitable lead structures in the design of Pgp modulators able to restore sensitivity to TKIs in resistant tumour cell lines. The presented protocols as well as the investigated compounds might be useful for virtual screening/drug design of TKIs that act as MDR chemosensitizers in a combined anti-cancer therapy.

P690: GENE MUTATIONAL PROFILE ASSOCIATED WITH TREATMENT FAILURE OR PROGRESSION IN CHRONIC MYELOID LEUKEMIA

N. Estrada 1,2,* , B. Xicoy 3 , M. Cabezón 3 , S. Marcé 3 , A. Senin 4 , A. Angona 5 , E. Alonso 4 , M. Ratia 4 , M. E. Plensa 6 , J. Buch 7 , X. Ortín 8 , L. Zamora 3

1 Myeloid Neoplasms, Josep Carreras Leukaemia Research Institute; 2 Germans Trias i Pujol Health Science Research Institute (IGTP); 3 Institut Català d’Oncologia (ICO)-Hospital Germans Trias i Pujol, Josep Carreras Leukaemia Research Institute, Badalona, Barcelona; 4 Institut Català d’Oncologia - Hospital Duran i Reynals, Hospitalet de Llobregat; 5 Institut Català d’Oncologia - Hospital Universitari Doctor Josep Trueta, Girona; 6 Hospital de Mataró, Mataró; 7 Hospital de Calella and Institut Català d’Oncologia-Girona, Girona; 8 Hospital de Tortosa Verge de la Cinta, Tortosa, Spain

Background: Approximately one third of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKI) fail to achieve optimal response due to resistance. It is known that BCR-ABL1 kinase domain (KD) mutations are associated with TKI resistance. However, only half on non-responsive patients carry BCR-ABL1 KD mutations. Other resistance mechanisms independent of BCR-ABL1 KD mutations, such as mutations in myeloid-related genes, may participate in the lack of response. Moreover, resistance to TKI therapy confers a much higher risk of progression to advanced disease stages of CML, such as accelerated phase (AP) and blast crisis (BC), and consequently, a worse prognosis.

Aims: The main objective of this study was to explore the impact of mutations in myeloid-related genes in TKI resistance and progression to AP/BC by targeted deep-sequencing (TDS).

Methods: The mutation profile study was done by TDS using a 32 myeloid-related gene panel (Myeloid Solution Capture Kit, Sophia Genetics, Switzerland). The platform Sophia DDM (Sophia Genetics) was used for data analysis. Regarding variant filtering, sequencing and mapping errors were eliminated and intronic and synonymous variants were filtered out. Variants present with a minor allele frequency (MAF) >1%, according to population databases (ExAc, 1000 genomes), were considered polymorphic changes without clinic relevance. COSMIC, VARSOME and Franklin databases as well as in silico functional predictors (SIFT, PolyPhen2 and MutationTaster) were used for variant interpretation. Variants were also filtered according to the variant allele frequency (VAF): all variants with VAF ≥5% were reported, as well as hot-spot variants with VAF 2-5% and at least 25 reads. Only variants described as pathogenic or potentially pathogenic were reported. Finally, BCR-ABL1 mutations were analyzed by Sanger in resistant patients.

Results: TDS was performed in a total of 78 samples from 67 patients. Across patients with available sample at diagnosis, 25% (16/64) of patients harbored at least one mutation. Overall, 11 (17.2%) patients had 1 mutation, 3 (4.6%) had 2 mutations, 1 (1.6%) patient had 3, and 1 (1.6%) patient had 4 mutations (Figure 1A). Most frequently mutated genes at diagnosis were ASXL1 (14.1%), DNMT3A (6.3%), JAK2 (3.1%) and TET2 (3.1%), followed by ETV6 , EZH2 , IDH2 , SETBP1 , SRSF2 , TP53 and WT1 , all of them at a frequency of 1.6% (Figure 1B). Of the 67 CML patients included, 15 (22.4%) had resistance to first-line TKI and 5 of them (33%) progressed to AP/BC. From the 15 resistant patients, TDS was performed in 12 cases at diagnosis, in 5 at time of resistance, and at progression in 4/5 patients. At diagnosis, 6 (50%) resistant patients harbored 1 mutation in ASXL1 (3/12), DNMT3A (2/12) or TET2 (1/12) and 1 patient harbored 2 mutations ( ASXL1 and WT1 ). Only 9 of 52 (17.3%) non-resistant patients harbored ≥1 mutation at diagnosis; most frequently mutated gene was ASXL1 (5/9). At time of resistance, 5 patients had BCR-ABL1 mutations by Sanger technique and 3 patients harbored cancer-related gene mutations by TDS ( ASXL1 in 2 patients). At progression, all patients harbored at least 1 mutation by TDS, except one patient in which only 2 mutations in BCR-ABL1 KD were detected.

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Summary/Conclusion: ASXL1 mutation was the most frequent mutation in CP at diagnosis and ASXL1 and ABL1 were the most common variants at resistance and progression. Acquisition of mutations was detected during CML progression.

Acknowledgments: this work was supported by a grant from Incyte (EU-ES-H-18021)

P691: MODELLING OF IMMUNE RESPONSE IN CHRONIC MYELOID LEUKEMIA PATIENTS SUGGESTS POTENTIAL FOR TREATMENT REDUCTION PRIOR TO CESSATION

E. Karg 1 , C. Baldow 1 , T. Zerjatke 1 , R. E. Clark 2 , I. Roeder 1,3 , A. C. Fassoni 4 , I. Glauche 1,*

1 Institute for Medical Informatics and Biometry (IMB), TU Dresden, Dresden, Germany; 2 Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom; 3 National Center for Tumor Diseases (NCT), Partner Site Dresden, Dresden, Germany; 4 Instituto de Matemática e Computação, Universidade Federal de Itajubá, Itajubá, Brazil

Background: Chronic myeloid leukemia (CML) is a malignancy driven by the characteristic BCR-ABL1 -fusion oncogene, while the BCR-ABL1 transcript is used as a marker for diagnosis and monitoring. The introduction of tyrosine kinase inhibitors (TKI) marked a substantial progress in CML therapy. Current efforts focus on the discontinuation of TKI therapy for well-responding patients as the long-term drug administration is associated with side effects and high economical costs. Many studies reported that about 50% of patients can achieve sustained treatment free remission (TFR) while 50% present with molecular disease recurrence, typically within two years of stopping. It has been speculated that the immune system (IS) plays a major role in the control of residual disease levels and influences the individual recurrence behaviour. The recently published DESTINY trial (Clark et al., Lancet Haematol, 2019; NCT 01804985 ) showed that TKI dose reduction prior to cessation can proactively increase the fraction of patients to remain in sustained TFR. However, there has been no systematic investigation to evaluate how dose reduction regimens can further improve the success of TKI stop trials.

We have previously shown that mathematical models of CML can correctly describe patient time courses after TKI stop (Hahnel et al., Cancer Res, 2020). We classified patients into different groups according to their predicted immune system configuration and showed that one class of patients required complete CML eradication to achieve TFR, while other patients were able to control residual leukemia levels after treatment cessation. While a prospective stratification of the patients is difficult to achievable, we aimed to investigate whether a further optimized dose reduction scheme prior to stopping could further improve TFR.

Aims: We aim to use a mathematical description of patient time courses to investigate different TKI dose reductions schemes prior to therapy cessation and evaluate them with respect to the total amount of drug used and the expected TFR success.

Methods: Our analysis is based on a cohort of 72 patients from the DESTINY trial (Clark et al., Lancet Hematol, 2019; NCT 01804985 ). We applied an established mathematical model (Hahnel et al., Cancer Res, 2020) and obtain individual fits for all patients using a genetic algorithm. Applying a re-sampling approach we derive estimates of recurrence times and fractions for different dose reduction schemes in which the timing and the amount of dose reduction are varied in a systematic manner.

Results: We use computer simulations to show that TKI dose reduction prior to treatment cessation appears as an equally efficient approach to achieve high levels of CML patients remaining in TFR compared to full dose treatment for the same duration. Our model simulations confirm clinical findings that the overall time of TKI treatment is a major determinant of TFR success, while at the same time indicating that lower dose TKI treatment is sufficient for many patients. Our model results indicate that a stepwise dose reduction prior to TKI cessation (e.g. 12 month post MR4 at 100% TKI, 12 more months at 50% and 12 more months at 25%) does not limit the overall success rate of TFR, while it substantially reduces the amount of total administered TKI and thereby decreases side effects as well as overall treatment costs.

Summary/Conclusion: Our findings illustrate the potential of dose reduction schemes prior to treatment cessation and suggest corresponding and clinically verifiable strategies that are applicable to many CML patients.

P692: IMMUNE FACTORS MAINTAINING TREATMENT-FREE REMISSION IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA AFTER DISCONTINUATION OF IMATINIB

P. Kwaśnik 1,* , J. Zaleska 1 , D. Link-Lenczowska 2 , M. Zawada 2 , B. Ochrem 2 , G. Bober 3 , E. Wasilewska 4 , E. Mędraś 5 , I. Hus 6,7 , M. Szarejko 8 , W. Prejzner 8 , O. Grzybowska-Izydorczyk 9 , A. Klonowska-Szymczyk 9 , T. Sacha 2 , K. Giannopoulos 1,10

1 Department of Experimental Hematooncology, Medical University of Lublin, Lublin; 2 Department of Haematology, Jagiellonian University Hospital in Krakow, Krakow; 3 Department of Hematooncology and Bone Marrow Transplantation, Medical University of Silesia, School of Medicine in Katowice, Katowice; 4 Department of Hematology, Medical University of Bialystok, Bialystok; 5 Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Medical University of Wroclaw, Wroclaw; 6 Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw; 7 Department of Clinical Transplantology, Medical University of Lublin, Lublin; 8 Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk; 9 Department of Hematology, Medical University of Lodz, Copernicus Memorial Hospital, Lodz; 10 Department of Hematology, St John’s Cancer Center, Lublin, Poland

Background: CML treatment aims to achieve long-term remission without treatment (TFR). The mechanisms that are responsible for CML relapses remain elusive. It is suggested that maintaining TFR is not directly related to the total disposing of the gene transcript BCR-ABL1 , but it might be a result of the immune surveillance restoration in CML.

Aims: To determine immune factors responsible for elimination or control over residual CML cells, which may be crucial for achieving long-term relapse-free survival (RFS) and TFR after imatinib discontinuation.

Methods: Peripheral blood mononuclear cells from 63 CML patients were analyzed using flow cytometer analysis at two-time points: at discontinuation of imatinib and 3 months after withdrawal. The major populations of the immune system that have a potential effect on the maintenance of the patient in remission were assessed. BCR-ABL1 transcript were analyzed using two molecular biology methods: standard real-time quantitative polymerase chain reaction (RQ-PCR) and droplet digital PCR (ddPCR).

Results: We found a significant increase in the percentage of HLA-DR+ cells (median 1.25 vs 1.90%, p<0.0001), mDC (median 0.20 vs 0.28%, p<0.01), pDC (median 0.10 vs 0.15%, p<0.01) and a significant decrease in PD-1 expression on mDC cells (median 35.81 vs 32.23%, p=0.03) in 3 months after treatment discontinuation.

There was a significantly reduced level of NKT cells (median 14.45 vs 12.80%, p=0.02), a significantly increased PD-1 expression on CD161+ cells (median 21.02 vs 21.28%, p=0.02) and a significantly decrease of PD-1 expression on CD56brightCD16- cells (median 2.31 vs 2.08%, p=0.04).

Analyses of T cells showed a significant decrease in frequencies of CD4+CD25+ T cells (median 18.01% vs 13.42%, p<0.0001), CD4+CD25+FOXP3+ (median 3.00 vs 2.89%, p=0.02) and a significantly lower expression levels of PD-1 on CD4+ cells (median 21.59 vs 19.34%, p=0.02) after 3 months compared to moment of withdrawal.

We found a weak negative correlation between age and CD19+ cell depletion (p<0.01, R=-0.39) and a moderate positive correlation between age and PD-1 expression on CD19+ cells (p<0.001, R=0.43) at the moment of withdrawal. In addition, we found a weak positive correlation between age and HLA-DR+ cell count (p=0.01, R=0.32) and a weak negative correlation with CD19+ cell count (p=0.04, R=-0.26) in 3 months after stopping treatment.

Significantly higher levels of NK cells: CD56dimCD16+ (median 8.93 vs 14.23%, p<0.01) and CD56brightCD16- (median 0.33 vs 0.52%, p=0.02) were found in men compared to women 3 months after withdrawal.

Analysis of the BCR-ABL1 transcript by both RQ-PCR and ddPCR methods in relation to the immune subpopulations showed significant differences between the percentage of HLA-DR+ cells and the depth of the molecular response: in RQ-PCR greater than or equal to MR4.0 at the moment of withdrawal vs loss of MR4.0 after 3 months (median 1.24 vs 1.87, p=0.03); greater than or equal to MR4.0 at the moment of withdrawal vs greater than or equal to MR4.0 after 3 months (median 1.24 vs 1.92, p<0.001).

Summary/Conclusion: The demonstrated changes in the percentage of populations of immune cells show the importance of immunocompetent cells’ involvement in maintaining the patient in TFR. The characterization of the immune system, which probably plays the most important role in achieving long-term relapse-free response and maintaining remission without treatment, may help define the group of patients who could safely discontinue imatinib.

Funding: NCN 2018/31/B/NZ6/03361

P693: DIAGNOSTIC ROLE OF CD26+ LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA

S. Tiwari 1,* , J. Dass 1 , G. Vishwanathan 1 , R. Dhawan 1 , M. Agarwal 1 , P. Kumar 1 , T. Seth 1 , S. Tyagi 1 , M. Mahapatra 1

1 Hematology, All India Institute of Medical Sciences, New Delhi, India

Background: Diagnosis of CML consists of persistent leucocytosis with the demonstration of the Philadelphia (Ph) chromosome abnormality, the t(9;22)(q34;q11), by routine cytogenetics, fluorescence in situ hybridization (FISH) or by molecular studies such as RT-PCR.These conventional methods suffer from a longer turn around time, higher costs and requirement of labs with adequate expertise. CD26 is a highly specific marker expressed in CML stem cells, which is not present on normal hematopoietic stem cells or on Leukemia stem cells (LSCs) of other myeloid neoplasms. A flow-cytometry based assessment of CD26+LSCs may prove to be an optimal biomarker for the diagnosis and monitoring of CML patients.

Aims: 1)Assessment of CD 26 expression in suspected cases of CML-chronic phase

2)Correlation of CD 26+ stem cells with clinico-pathological parameters at baseline and its kinetics on tyrosine kinase inhibitor (TKI) treatment on further follow-up at 12 months.

Methods: Suspected patients of CML-CP were included in the study. Peripheral blood were utilized for flowcytometric assessment of CD26+ LSCs. The results were correlated with conventional diagnostic tests of CML. Patients with myeloid neoplasms other than CML who proved negative for BCR-ABL1 by RT-PCR and normal HSCs donors treated with granulocyte-colony stimulating factors (G-CSF), were included as negative controls. All samples were processed using standard stain -lyse-wash method within 24 hr of collection. To reach a sensitivity of 10−5, acquisition of at least 1.0 × 106 cells was performed on FACS Canto II flow cytometer data a FACS DIVA 8.0.3 software (BD, Biosciences).CD26 expression was evaluated by a sequential gating strategy. After exclusion of doublets and debris CD45 dim to intermediate/CD34+/CD38- population was gated sequentially and then the expression of CD 26+ was studied on this population. All the samples were simultaneously assessed for BCR-ABL1 transcript by standard RT-PCR analysis. Patients on treatment were also followed up at 12 months to detect levels of CD26+ LSCs and correlated with BCR -ABL1 levels by qPCR.

Results: A total of 86 patients of suspected cases of CML were included in the study from June 2020 to Dec 2021.The On flowcytometry analysis, CD 26 expression was present in 75 cases. All the cases with positive CD26+ LSCs were found to be positive on RT-PCR for BCR-ABL transcript. Eleven cases which were scored negative for CD26+ LSCs were also found to be negative for any BCR-ABL transcript. These negative cases included 8 cases of primary myelofibrosis, 2 cases of chronic myelomonocytic leukemia and 1 case of Polycythaemia Vera. The median percentage of CD26+ LSCs was 39.8% (Range-0.5-100). There was no significant correlation of the CD26 levels with age (p value=0.96), sex (p value=0.7), percentage of blasts (p value = 0.88), total leucocyte counts (p value=0.32), basophils (p value = 0.19), Sokal score (p value = 0.99) and ELTS score (p value = 0.99). Follow-up data was available for 7 patients and at 12 months all of these patients had achieved major molecular remission on TKIs. Two patients scored negative for CD26+LSCs while five patients showed significant fall in the level of CD 26+ LSCs however it was still detectable by flowcytometry (p value= 0.04).

Summary/Conclusion: Flowcytometry assessment of CD26 + LSCs is a rapid and cheap diagnostic tool for diagnosis of CML with high diagnostic efficacy. Falling levels on TKIs warrants further research to prove it as a surrogate for monitoring response by conventional methods.

P694: INVESTIGATING THE ROLE OF CELL ADHESION MOLECULES IN TUNNELING NANOTUBES FORMATION IN CHRONIC MYELOID LEUKEMIA MICROENVIRONMENT

L. Turos-Korgul 1,* , A. Zieminska 1 , L. Parker 2 , K. Piwocka 1

1 Laboratory of Cytometry, Nencki Institute of Experimental Biology PAS, Warsaw, Poland; 2 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, United States of America

Background: The cross-talk and interactions between leukemia cells and their microenvironment play an important role in the development of drug resistance. Tunneling nanotubes (TNTs) represent a novel type of direct cell-cell communication way allowing for direct transfer of different types of cargo between distant cells. Our previous studies (Kolba et al., 2019) have shown that these structures are formed between bone marrow stromal HS-5 cells and chronic myeloid leukemia (CML) K562 cells. Significantly, TNT-mediated transfer of membrane vesicles from stromal to leukemic cells resulted in the stroma-mediated protection of CML cells from imatinib-induced apoptosis. Despite the efforts made to understand the molecular mechanisms regulating TNTs formation, they still remain unclear - especially in the leukemia microenvironment. We have shown that TNTs are formed between stromal and leukemic cells, following direct cell-cell interaction and cell dislodgement. Thus, we have focused our attention on cell adhesion molecules, as potential key players in tunneling nanotubes formation and TNT-mediated vesicles transfer between leukemic and stromal cells.

Aims: The aim of presented studies was to elucidate the role of CD44 and ITGβ1 - one of the most important cell adhesion molecules, in TNTs formation between leukemic and stromal cells and TNT-mediated exchange of vesicles.

Methods: To examine this, we established the HS-5 stromal cell line with silenced CD44 protein expression. Then, we co-cultured control (wt) or CD44-silenced HS-5 cells with leukemic K562 cells. For ITGβ1 studies, we used blocking anti-ITGβ1 antibody. Using confocal microscopy, three types of TNTs in co-cultures were counted: homotypic TNTs formed between stromal cells, homotypic TNTs formed between CML cells and TNTs formed between stromal and CML cells (heterotypic). Moreover, the TNT-mediated transfer of cellular vesicles from stromal to leukemic cells was assessed in these co-cultures using flow cytometry. Since CD44 expression increases in CML cells after co-culture with stroma, we examined its potential role in the drug resistance in CML acceptor cells. To do this, rhodamine-123 efflux assay was used.

Results: We found that both, tunneling nanotubes number and uptake of cellular vesicles by leukemia, decreased after co-culture with CD44-silenced stromal cells and after blocking with anti-ITGβ1 antibody. Flow cytometry analyses of rhodamine-123 efflux showed significantly increased efflux pump activity in leukemic acceptor cells.

Summary/Conclusion: Our data suggest that CD44 and ITGβ1 can play an important role in formation of tunneling nanotubes between leukemic and stromal cells and TNT-mediated cellular vesicles exchange. Moreover, CD44 presented in acceptor cells is involved in the development of drug resistance in leukemic cells, by promoting activity of efflux transporters.

P695: PROGNOSTIC IMPACT OF ASXL1 MUTATIONS IN CHRONIC PHASE CML

A. Bidikian 1,* , H. Kantarjian 1 , E. Jabbour 1 , N. Short 1 , F. Ravandi 1 , G. Issa 1 , K. Sasaki 1

1 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America

Background: While the clinical impact of mutations in the ABL1 gene on response to therapy in chronic phase chronic myeloid leukemia (CP-CML) is well established, less is known about mutations in genes frequently involved in myeloid malignancies.

Aims: To characterize the clinical impact of mutations in non- ABL1 genes in CP-CML.

Methods: We identified 66 patients (pts) with CP-CML where targeted next generation sequencing was performed at our institution. This panel included 81 genes recurrently mutated in myeloid malignancies. Event-free survival (EFS) was measured from treatment initiation to the time of an event defined as loss of hematologic response, loss of major cytogenetic response (MCyR), transformation to accelerated or blast phase and lack of MCyR after 12 months of treatment. Failure-free survival (FFS) was measured similarly with the additional events of drug discontinuation due to lack of response or adverse effect. We subsequently performed univariate and multivariate analyses for survival.

Results: The median age of pts was 61 years (18 – 80 years) and 48% were female. The most common first line therapy in these patients was Dasatinib (52%), followed by Imatinib (19%), Dasatinib + Venetoclax (16%), Nilotinib (8%), Bosutinib (3%) and Ponatinib (2%). The distribution of evaluable patients to low, intermediate and high Sokal risk groups was 22%, 62% and 16%, respectively. Non- ABL1 mutations were found in 22/66 patients (33%), with 5 (8%) of pts with at least 2 mutations. Most common mutations involved ASXL1 (8/66 pts; 12%), DNMT3A (4/66 pts; 6%) and RUNX1 (3/66 patients; 5%). Mutations in ASXL2, CALR, EZH2, GNAS, IDH2, SF3B1, SMC3, SRSF2, STAG2 and SUZ12 were detected at least once. Unlike mutations in other genes, mutated ASXL1 was associated with worse EFS compared with wild-type with a median of 33 vs 88 months ( P = 0.009) (Figure 1A) and worse FFS with medians of 32 vs 58 months, respectively ( P = 0.05) (Figure 1B). There was no difference in overall survival by mutational status (Figure 1C). In a multivariate analysis including all baseline prognostic variables, mutated ASXL1 was independently associated with worse EFS with a hazard ratio of 5.94 (95% CI 1.25 – 28.21; P = 0.03). There was no difference in rates of cytogenetic and molecular responses by mutational status. However, time to achievement of response was longer in patients with mutated ASXL1 albeit not statistically significant. (Figure 1D)

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Summary/Conclusion: Mutations in ASXL1 are associated with delayed responses and worse outcomes when detected in chronic phase CML.

P696: SUBTHERAPEUTIC TKI DOSES FOR THE MAINTENANCE OF CML PATIENTS WITH INTOLERANCE OR REFUSAL TO DISCONTINUE: A SINGLE CENTER FEASIBILITY STUDY.

A. Borrero 1,* , A. Segura 1 , S. Sanchez 1 , R. Stuckey 1 , J. F. López 1 , C. Bilbao 1 , M. T. Gomez casares 1

1 HOSPITAL UNIVERSITARIO DE GRAN CANARIA DOCTOR NEGRIN, LAS PALMAS DE GRAN CANARIA, Spain

Background: Discontinuation has become a common practice in patients with CML treated with TKI who have maintained a deep molecular response. 50-60% of patients successfully discontinue treatment without relapsing (treatment-free remission, TFR), which implies that 40-50% will have to remain with TKI treatment for life. Many studies have been carried out on TKI treatment with reduced doses, often used to manage adverse events and TKI intolerance. Such studies have shown that most patients maintain MMR and many even achieve it when reduced doses are administered from diagnosis. There are fewer cases reported of patients who receive subtherapeutic doses, i.e. doses lower than those considered in the TKI data sheet. Reasons why subtherapeutic doses may be employed in clinical practice include the patient’s refusal to discontinue or a failed discontinuation attempt (loss of TFR), as a maintenance strategy, or in those patients with intolerance. Although its use is likely, information on patient outcome is scarce.

Aims: We investigated the feasibility of the use of subtherapeutic doses in CML patients. We studied the reason for the dose reduction and the impact of the dose reduction on molecular response.

Methods: In this observational, retrospective, single-center study, we searched the medical records of all patients diagnosed with CML in chronic phase at our hospital between 2003 and 2020. Patients were identified who were treated with TKIs at subtherapeutic doses. Subtherapeutic doses were defined as doses lower than those considered in the TKI data sheet: dasatinib 20 mg, imatinib 100 mg or 200 mg, nilotinib 150 mg, bosutinib 100 mg.

Results: Medical records were searched and 13 CML patients who received infratherapeutic TKI doses were identified, 7 men (53.9%) and 6 women (46.2%) with a mean age of 75.5 years. Of these patients, 7 (53.8%) received imatinib (6 with 200 mg doses and 1 with 100 mg), 1 (7.7%) received bosutinib 100 mg and 5 (38.5%) received dasatinib 20 mg. The mean follow-up time from the start of the dose reduction was 60.5 months and 40.7 months from the last reduction. None of the patients presented TKI resistance or progression during the follow-up period. 3 patients reduced the dose as a practical alternative for fear of discontinuation; 2 patients discontinued, but after relapse they remained with the minimum dose to maintain MMR; the rest of the patient reduced doses because of intolerance. All patients presented MMR at last follow-up and 92.3% deep MMR (≥MR4).

Summary/Conclusion: Our results show that TKI doses can be optimized on an individual basis to infratherapeutic levels and MMR or better can be maintained. Although only a small series of patients was studied, no patients developed TKI resistance or progression while receiving infratherapeutic doses during an average period of 5 years. For intolerant patients who received infratherapeutic doses, adverse events were resolved and quality of life improved. The administration of infratherapeutic TKI doses could also be a good option for patients who relapse after a discontinuation attempt or for those who do not want to discontinue, as a form of “maintenance” therapy. The use of infratherapeutic doses, rather than a full TKI discontinuation, would still provide economic savings. Moreover, it would be interesting to evaluate if long-term use of infratherapeutic doses can prevent the development of serious off-target TKI toxicities, such as vascular events. Studies with larger patient cohorts are needed to validate the feasibility of this clinical practice.

P697: PONATINIB IN A REAL-LIFE SETTING: A RETROSPECTIVE ANALYSIS FROM THE MONITORING REGISTRIES OF THE ITALIAN MEDICINES AGENCY (AIFA)

M. Breccia 1,* , P. P. Olimpieri 2 , S. Celant 2 , O. M. Olimpieri 2 , F. Pane 3 , A. Iurlo 4 , V. Summa 2 , P. Corradini 5 , P. Russo 2

1 Department of Translational and Precision Medicine, Sapienza University; 2 Italian Medicines Agency, AIFA, Roma; 3 Federico II University, Napoli; 4 Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 5 Università degli Studi di Milano & Divisione Ematologia, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milano, Italy

Background: The efficacy and safety of ponatinib, a third-generation tyrosine kinase inhibitor (TKI), are mainly reported in sponsored clinical trials.

Aims: The aim of this study, based on a retrospective analysis from the monitoring Registries of the Italian Medicines Agency (AIFA wMRs), is to provide real-world data on daily practice management, treatment modifications and outcome of a large cohort of chronic myeloid leukemia (CML) patients treated with ponatinib.

Methods: AIFA wMRs is an administrative database whose main scope is monitoring the appropriateness of drug prescription in Italy. Information collected through the wMRS included demographic (place of birth, age, and sex) and clinical data (BCR-ABL levels, mutations and chromosomal abnormalities if evaluated), drug prescription and administration data (date of prescription and administration, dose administered, dose changes, occurrence of adverse events as yes/no dichotomic variable, reasons for treatment interruption and/or discontinuation) and response to treatment (response assessment was requested every 90 days). Subjects who had not been administered ponatinib for ≥ 120 days were adjudicated as “discontinued” even in the absence of the “end of treatment form”. Time to treatment discontinuation (TTD) was defined as the time occurring between the date of first administration and the date of treatment discontinuation for any cause, including death.

Results: Overall, 666 CML subjects were eligible for analysis: 515 patients had chronic phase (CP) CML, 50 accelerated phase (AP) and 101 blast phase (BP). Median age at baseline was 58.7 years, male prevalence (57.1%). Median time from diagnosis to start of ponatinib was 2.35 years: 259 (38.9%) subjects had received 2 lines of treatment, 260 (39.0%) 3 lines and 147 (22.1%) 4 or more lines. Mutational status was available for 58.3% of patients (n=388), with a T315I mutation reported in 46 (6.9%) patients and other mutations (the most represented being E255K, F317L, Y253H and V299L) in 99 (14.9%) subjects. At baseline, 181 patients (27.2%) had arterial hypertension, 33 (5.0%) a history of arterial and/or venous thromboses, 32 (4.8%) a pre-existing ischemic heart disease, and 13 (2.0%) a history of congestive heart failure. Overall, 593 patients (89.0%) were evaluable for best response. Ten cases (1.7%, 3 AP/BP and 7 CP) did not achieve molecular response, whereas 58 patients (8.7%, 26 AP/BP patients and 32 CP) reached a BCR/ABL1 ratio between 1% and 10% IS. A MR2 (less than 1%IS) was obtained by 82 subjects (12.3%, 20 AP/BP and 62 CP patients); 128 patients (19.2%, 12 AP/BP and 116 CP patients) achieved a molecular response ranged between MR3 (0.1%) and MR4 (0.01% IS) and 266 (39.9%, 44 AP/BP and 222 CP patients) a deeper molecular response (< 0.01% IS). With a median follow-up of 14.4 months, 136 subjects (20.4%) required at least one dose reduction due to adverse events; 309 patients (46.4%) had their dose decreased in the absence of any evidence of side effects. Treatment discontinuation occurred in 144 patients (21.6%): intolerance (7.4%), primary resistance (3.5%) and acquired resistance (5.6%). Kaplan-Meier estimated TTD was 47.2 months (CI 95% 39.3 – NA) and 7.3 months (CI 95% 4.6 – 11.8) for CP and AP/BP patients, respectively. The probability of treatment discontinuation did not significantly differ for ponatinib in second, third or subsequent line of therapy (p=0.58).

Summary/Conclusion: This real-life investigation shows that ponatinib dose reductions were mainly performed in the absence of reported toxicity rather than owing to the occurrence of adverse reactions.

P698: BOSUTINIB DOSE OPTIMIZATION IN THE SECOND-LINE TREATMENT OF ELDERLY CML PATIENTS: EXTENDED 3-YEAR FOLLOW-UP AND FINAL RESULTS OF THE BEST STUDY

F. Castagnetti 1,2,* , M. Bocchia 3 , E. Abruzzese 4 , I. Capodanno 5 , M. Bonifacio 6 , G. Rege Cambrin 7 , M. Crugnola 8 , G. Binotto 9 , C. Elena 10 , A. Lucchesi 11 , M. Bergamaschi 12 , F. Albano 13 , L. Luciano 14 , F. Sorà 15 , F. Lunghi 16 , F. Stagno 17 , M. Cerrano 18 , A. Iurlo 19 , A. R. Scortechini 20 , S. Leonetti Crescenzi 21 , R. Spadano 22 , E. Trabacchi 23 , M. Lunghi 24 , G. Spinosa 25 , D. Ferrero 26 , D. Rapezzi 27 , M. Ladetto 28 , L. Nocilli 29 , G. Gugliotta 1 , M. Iezza 1 , M. Cavo 1,2 , G. Saglio 30 , F. Pane 14 , G. Rosti 11

1 Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna; 2 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, BOLOGNA; 3 Hematology Unit, University of Siena, Siena; 4 Hematology Unit, S. Eugenio Hospital, Tor Vergata University, Roma; 5 Hematology Unit, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Reggio Emilia; 6 Department of Medicine, Section of Hematology, University of Verona, Verona; 7 Internal Medicine Unite, San Luigi Gonzaga Hospital, Orbassano (TO); 8 Hematology Unit, Azienda Ospedaliero-Universitaria di Parma, Parma; 9 Department of Medicine, Hematology and Clinical Immunology, University Hospital of Padova, Padova; 10 Department of Hematology Oncology, Foundation IRCCS Policlinico San Matteo, University of Pavia, Pavia; 11 Hematology Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola (FC); 12 Hematology Unit, Policlinico S.Martino-IRCCS, Genova; 13 Hematology Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari; 14 Hematology Unit, Federico II University, Napoli; 15 Hematology Unit, Policlinico A. Gemelli, Università Cattolica del Sacro Cuore, Roma; 16 Hematology and Bone Marrow Transplant Unit, San Raffaele Scientific Institute IRCCS, Milano; 17 Hematology Section and BMT Unit, AOU Policlinico V. Emanuele, Catania; 18 Hematology Unit, AOU Città’ della Salute e della Scienza, Torino; 19 Hematology Unit, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano; 20 Hematology Unit, A.O.U. Ospedali Riuniti, Ancona; 21 Hematology Unit, San Giovanni - Addolorata Hospital, Roma; 22 Hematology Unit, Casa Sollievo Della Sofferenza, San Giovanni Rotondo (FG); 23 Hematology and BMT Unit, Department of Hematology and Oncology, Guglielmo da Saliceto Hospital, Piacenza; 24 Hematology Unit, AOU Maggiore della Carità, Novara; 25 Hematology Unit, Azienda Ospedaliero Universitaria Ospedali Riuniti, Foggia; 26 Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino; 27 Hematology Unit, AO S. Croce e Carle, Cuneo; 28 Hematology Unit, Ospedale SSAntonio e Biagio e Cesare Arrigo, Alessandria; 29 Hematology Unit, Azienda Ospedaliera Papardo, Messina; 30 Divisione Universitaria di Ematologia e Terapie Cellulari, A.O. Ordine Mauriziano, Torino, Italy

Background: The median age of CML patients requiring a second-line treatment is older than 60 years and many of these patients have relevant comorbidities. Bosutinib (BOS) is a second-generation TKI, with similar efficacy to dasatinib and nilotinib, but more favorable toxicity profile in the elderly, because of lower incidence of cardiovascular (CV) adverse events (AEs). BOS safety profile may be an added value in this setting, but a fixed initial dose of 500 mg OAD may be higher than necessary.

Aims: All TKIs have been approved in CML at a specific initial dose, with dose reductions only in case of toxicity. The aim of our study was to evaluate the efficacy of low-dose BOS in the second line treatment of elderly CML patients, with subsequent dose increase only in patients without optimal molecular response at given timepoints (dose optimization).

Methods: A prospective phase 2 single-arm study has been designed by the GIMEMA CML Working Party ( NCT02810990 ). Study design: all patients, ≥ 60 yrs old, started BOS 200 mg for 2 weeks (“run-in” period), then the dose was increased to 300 mg; after 3 months, pts with BCR-ABL IS transcript ≤ 1% continued 300 mg, while in pts with transcript > 1% the dose is furtherly increased to 400 mg, in absence of relevant toxicity. The primary endpoint was the rate of MR3 at 12 months (core phase), but all pts were followed for additional 2 years (extension phase) to describe the long-term efficacy and outcome.

Results: Sixty-three pts have been enrolled. Median age: 73 yrs (range 60-90). Reasons for switching to BOS: intolerance 65%, resistance 35%. First-line TKI: imatinib 83%, DAS 11%, NIL 6%. The rate of pts in MR3 at 12 mos was 59% (already reported). Median follow-up 38 mos (range 35-59). Maximum BOS dose: 400 mg, 21%; 300 mg, 73%; 200 mg, 6%. The probabilities of achieving or maintaining MR3, MR4 and MR4.5 by 36 mos were 78%, 54% and 46%, respectively (excluding pts with response at baseline, 73%, 50% and 44%, respectively); the probabilities were lower in the resistant cohort (64%, 36% and 18%, respectively). Overall, 24%, 33% and 11% of pts had 1 log, 2 logs or ≥ 3 BCR-ABL IS transcript logs reduction from baseline (68% of pts had a molecular improvement from baseline). At 36 mos, 36 pts (57%) were still on BOS (33 pts in optimal response, 3 pts in MR2), while 27 pts (43%) discontinued the study drug. Events leading to permanent treatment discontinuation: 7 CML unrelated deaths, 9 AEs (transaminase increase in 5 pts, skin rash, myalgia, GI toxicity and renal failure in 1 patient each), 9 unsatisfactory responses (without progression to advanced phases), 1 treatment-free remission attempt, 1 other reason. The overall survival probability was 81%. Pts with CV AEs: acute coronary syndromes, 6 pts; pericarditis, 2 pts; peripheral arterial thrombosis, 1 pt (all pts had CV risk factors). No pleural effusions were observed. The incidence of GI toxicity was lower than reported elsewhere. Thirty-six pts were still on BOS at the last contact: 6% on 400 mg, 50% on 300 mg, 44% on 200 mg.

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Summary/Conclusion: A progressive dose increase, based on molecular response, in elderly CML patients treated with second-line bosutinib produced high response rates; few patients required a dose increase to 400 mg, while most pts remained on 300 mg or less. Not unexpectedly, given the old age, unrelated CV AEs and deaths occurred, but the study drug was generally well tolerated. An initial use of low dose TKIs, with dose optimization in case of absence of optimal response, is a promising strategy in elderly patients.

P699: REAL-LIFE OUTCOMES OF PONATINIB TREATMENT IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) OR PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (PH+ALL): 5-YEAR-DATA FROM A BELGIAN REGISTRY

T. Devos 1,2,* , V. Havelange 3 , K. Theunissen 4 , S. Meers 5 , F. S. Benghiat 6 , A. Gadisseur 7 , G. Vanstraelen 8 , H. Vellemans 9 , B. Bailly 10 , N. Granacher 11 , P. Lewalle 12 , A. De Becker 13 , K. Van Eygen 14 , M. Lejeune 15 , M. Janssen 16 , A. Triffet 17 , I. Vrelust 18 , D. Deeren 19 , D. Mazure 20 , M. Beck 21 , H. Sebti 21 , D. Selleslag 22

1 Department of Hematology, University Hospitals Leuven; 2 Department of Microbiology and Immunology, Laboratory of Molecular Immunology (Rega Institute), Leuven; 3 UCL Saint-Luc, Woluwe-Saint-Lambert; 4 Jessa Ziekenhuis, Hasselt; 5 Algemeen Ziekenhuis Klina, Brasschaat; 6 Hôpital Erasme, Bruxelles; 7 Universitair Ziekenhuis Antwerpen, Edegem; 8 CHR Verviers, Verviers; 9 CHU UCL Namur, Site Godinne, Yvoir; 10 Hôpital de Jolimont, Haine-Saint-Paul; 11 Ziekenhuis Netwerk Antwerpen Stuivenberg, Antwerpen; 12 Institut Jules Bordet, Université Libre de Bruxelles, Bruxelles; 13 Universitair Ziekenhuis Brussel, Jette; 14 Algemeen Ziekenhuis Groeninge, Kortrijk; 15 Centre Hospitalier Universitaire de Liège (Sart-Tilman), Liège; 16 Ziekenhuis Oost-Limburg, Genk; 17 Centre Hospitalier Universitaire Charleroi Vésale, Charleroi; 18 Algemeen Ziekenhuis Sint-Elisabeth, Turnhout; 19 Algemeen Ziekenhuis Delta, Roeselare; 20 Universitair Ziekenhuis Gent, Gent, Belgium; 21 Incyte Biosciences Benelux B.V., Amsterdam, Netherlands; 22 Algemeen Ziekenhuis Sint-Jan Brugge, Brugge, Belgium

Background: Ponatinib is a third-generation tyrosine kinase inhibitor (TKI) indicated for adult patients with chronic, accelerated or blast phase CML resistant or intolerant to nilotinib or dasatinib or with Ph+ALL resistant or intolerant to dasatinib or for patients with the T315I mutation. Real-world data on ponatinib treatment are limited but are important to evaluate the effectiveness and safety of ponatinib and optimize its use in daily practice.

Aims: To report 5-year-Belgian registry data on ponatinib use in CML and Ph+ALL patients in routine clinical practice.

Methods: This ongoing prospective multi-center registry ( NCT03678454 ) includes ≥18-year-old patients with CML or Ph+ALL eligible for ponatinib treatment per product label. Data on demographics, effectiveness (major molecular response [MMR] rate) and safety were collected for patients enrolled from 01-March-2016 onwards and are presented here up to 17-May-2021.

Results: In this interim analysis, 77 patients from 21 hospitals were enrolled (50 CML, 27 Ph+ALL). Of the 28 patients (39%; 14 CML, 14 Ph+ALL) with mutations in the BCR-ABL1 kinase domain, 17 (8 CML, 9 Ph+ALL) had the T315I mutation at entry. Median age at ponatinib start was 61 years for CML and 56 years for Ph+ALL patients. 88% of CML and 96% of Ph+ALL patients had received ≥2 prior TKIs. Potential pre-existing risk factors for TKI cardiovascular toxicity were observed: history of cardiovascular disease (27 patients), hypertension (23), smoking (15), diabetes (13), hypercholesterolemia (8), hyperlipidemia (6). The most frequently reported risk factor was history of cardiovascular disease (36% CML, 33% Ph+ALL). Median follow-up was 545 (14-3190) days for CML and 210 (26-2933) days for Ph+ALL patients. Reasons for starting ponatinib therapy were intolerance to previous TKIs (26 CML, 11 Ph+ALL), relapse on/refractoriness to previous TKIs (11 CML, 8 Ph+ALL), T315I mutation (6 CML, 6 Ph+ALL) and disease progression (7 CML, 2 Ph+ALL). Ponatinib starting doses were: 45 mg/day (66%), 30 mg/day (10%), 15 mg/day (22%) in CML patients (1 patient started with 15 mg every 2 days) and 45 mg/day (67%), 30 mg/day (15%), 15 mg/day (19%) in Ph+ALL patients. MMR was achieved in 58% of CML and 52% of Ph+ALL patients. The median time-to-MMR was 170 days in CML and 86 days in Ph+ALL patients. Of the 37 patients who started ponatinib due to intolerance to previous TKIs, 59% (15 CML, 7 Ph+ALL) achieved MMR. Adverse reactions (ARs) were reported in 63 patients (82%); the most common were rash (23% of all), constipation (18% of CML) and headache (11% of Ph+ALL patients). Seventeen patients developed 26 serious AR. Serious cardiovascular ARs were reported in 7 patients (coeliac artery stenosis [in 2 patients], ischemic stroke [1], worsening hypertension [1], hypertension [1], deep venous thrombosis [1], possible transient ischemic attack [1]). Eight deaths were recorded, none related to ponatinib treatment. 56% of CML and 24% of Ph+ALL patients discontinued ponatinib treatment due to an AR.

Summary/Conclusion: This real-world Belgian registry over 5 years supports the use of ponatinib in CML and Ph+ALL patients resistant or intolerant to previous TKIs or carrying the T315I mutation. Results obtained in routine clinical practice are in concordance with clinical trials, including PACE, in which CML patients had durable and clinically meaningful responses to ponatinib.

Funding: Incyte Biosciences Benelux BV

Acknowledgements: Medical writing support was provided by Adina Truta (Modis)

P700: TREATMENT-FREE REMISSION (TFR) AFTER DASATINIB IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE (CML-CP) AND DEEP MOLECULAR RESPONSE (DMR): FINAL 5-YEAR RESULTS OF DASFREE

N. P. Shah 1,* , V. García-Gutiérrez 2 , A. Jiménez-Velasco 3 , S. Saussele 4 , D. Rea 5 , F.-X. Mahon 6 , M. Y. Levy 7 , M. T. Gómez-Casares 8 , M. J. Mauro 9 , O. Sy 10 , P. Martin-Regueira 10 , J. H. Lipton 11

1 UCSF School of Medicine, San Francisco, United States of America; 2 Servicio Hematología y Hemoterapia, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid; 3 Hospital Universitario Carlos Haya, Malaga, Spain; 4 Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany; 5 Hôpital Saint-Louis, Paris; 6 Institut Bergonié Cancer Center, Université Bordeaux, Bordeaux, France; 7 Baylor Charles A. Sammons Cancer Center, Dallas, United States of America; 8 Hospital Universitario de Gran Canaria Dr. Negrín, Las Palmas de Gran Canaria, Spain; 9 Memorial Sloan Kettering Cancer Center, New York; 10 Bristol Myers Squibb, Princeton, United States of America; 11 Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada

Background: Patients (pts) with CML-CP who have a DMR are eligible to discontinue treatment (Tx) to attempt TFR. DASFREE ( NCT01850004 ) is a single-arm, open-label, phase 2 trial evaluating dasatinib discontinuation in pts with a stable DMR. At the 2-year (y) follow-up, 46% of pts remained in TFR; in a multivariate analysis, longer duration of prior dasatinib, first-line (1L) dasatinib, and older age were significantly associated with TFR maintenance (Shah et al. Leuk Lymphoma 2019).

Aims: To report the final 5-y results assessing TFR maintenance after dasatinib discontinuation.

Methods: Eligible pts were aged ≥18 y, received dasatinib Tx as 1L or subsequent therapy for ≥2 y, and had a DMR (MR 4.5 or BCR-ABL ≥0.0032% on the international scale, confirmed during pre-screening and with 2 central lab assessments 3 months [mo] apart) for ≥1 y prior to study entry. Pts discontinued dasatinib and were monitored for up to 5 y. All pts provided written informed consent. If loss of major molecular response (MMR) occurred, pts restarted dasatinib at the same dose as at enrollment. A univariate analysis was conducted to identify baseline correlates of maintaining TFR. Rate of TFR maintenance (event-free survival) in the extended follow-up, rate of MMR recapture after relapse, identification of predictive factors for TFR maintenance, and safety were all key secondary or exploratory endpoints.

Results: A total of 84 pts discontinued dasatinib. At a minimum follow-up of 60 mo (database lock, Dec 2021), 44% (n=37) of pts remained in TFR and the remaining 56% (n=47) had lost MMR and restarted Tx. No relapses occurred later than 39 mo after discontinuation. Baseline characteristics were balanced between pts who remained in TFR versus those who restarted Tx, except for age (65% of pts who remained in TFR vs 85% who restarted Tx were aged <65 y). Among enrolled pts, 24 discontinued the study early (4 due to drug-related adverse events [AEs]; none due to death); 60 discontinued as planned per protocol (end of study). Patients who discontinued from the study were evaluated for response regained before discontinuation. All evaluable pts (n=46; one patient withdrew from the study 1 month after restarting Tx and did not undergo molecular assessment) who restarted Tx regained MMR and MR 4.5 in a median (range) time of 1.9 mo (0.9–3.7) and 3.3 mo (1.5–29.6), respectively. The 5-y TFR rate was 43.8% (95% CI, 33.1–54.4; Figure ). A univariate analysis of baseline correlates identified older age and 1L dasatinib as significantly associated with maintaining TFR. Musculoskeletal and connective tissue disorders (any grade) were experienced by 39% of pts (n=33). The most common any-grade AEs were arthralgia (18%) and hypertension (13%). There were no deaths due to CML. Withdrawal events (n=15) were experienced by 9 pts (11%) after a median (range) of 3.7 (<1–18) mo from dasatinib discontinuation, with no new events beyond 18 months of study follow-up; 10 events resolved in a median (range) of 5.98 (<1–70) mo.

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Summary/Conclusion: Discontinuation of dasatinib after 1L therapy and beyond is a viable option for pts with CML-CP in sustained DMR. About half of the pts who discontinued dasatinib maintained TFR at 5 y. Re-treatment after relapse was successful; all evaluable pts who lost MMR regained MMR and MR 4.5 after therapy was reinitiated. The overall safety profile was consistent with the known safety profile of dasatinib and with the 2-y results.

P701: COVID-19 IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN TREATMENT-FREE REMISSION: DISEASE SEVERITY AND IMPACT ON TFR STATUS

S. Saußele 1 , N. Evans 2 , F. E. Nicolini 3 , E. Chelysheva 4 , D. Réa 5 , F. Castagnetti 6 , S. Claudiani 7 , R. Rodrigues 8 , C. Kok 9 , T. Hughes 2,9,*

1 III. Med. Klinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany; 2 International CML Foundation, London, United Kingdom; 3 Hematology department, Centre Léon Bérard and INSERM1052, Lyon, France; 4 National Research Center for Hematology, Moscow, Russia; 5 Hematology department, Saint-Louis Hospital, Paris, France; 6 Istituto di Ematologia “Seràgnoli”, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 7 Department of Haematology, Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom; 8 Departamento de Hematologia, Instituto Português de Oncologia de Lisboa - IPO, Lisbon, Portugal; 9 Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, Australia

Background: Severe SARS-CoV-2 infections associated with high mortality rates are reported in a higher percentage of patients (pts) with hematologic malignancies compared to general population. In chronic myeloid leukemia (CML), pts with uncontrolled disease have a higher mortality risk. The impact of SARS-CoV-2 infection on CML pts in treatment-free remission (TFR) has not been studied so far. In particular, as immune control of residual disease may be important for TFR, the concern is that the infection could induce loss of TFR.

Aims: To evaluate the outcome of SARS-CoV-2 infection in CML pts in TFR and assess any impact on maintenance of TFR.

Methods: From March 2020 to December 2021, the CANDID study organized by the international CML Foundation has collected data on COVID-19 positive CML pts worldwide. Details on the registry were presented recently (Pagano ASH 2021). For this sub-analysis on pts in TFR additional information were collected including; molecular remission status (BCR::ABL1 ratios) before, during and after SARS-CoV-2 infection covering at least 6 months.

For molecular analyses, BCR::ABL1 ratios were classified according to Cross et al (Leukemia 2015). In addition, ratios of 0% without indication of sensitivity were allocated as MR4 i.e. 0.01% IS . PCR outlier results were identified using the ROUT method by nonlinear regression with a maximum false discovery rate (FDR) of 1% (Motulsky et al 2006). Time to molecular relapse (MR) was measured from the date of COVID-19 diagnosis to the date of MR defined as loss of major molecular remission (MMR, BCR::ABL1 >0.1% IS ) or the date of last molecular test. Molecular relapse-free survival (MRFS) and overall survival (OS) were estimated with the Kaplan-Meier method. The statistical difference between groups was performed using log-rank test.

Results: By December 2021, 1050 COVID-19 positive CML pts were registered. 95 pts were in TFR at the time point of SARS-CoV-2 infection of which 89 (93.68%) recovered and 6 deceased (6.32%). Median age of TFR pts was 57 years, male were 51 (53.68%). Median time from CML diagnosis to reporting date was 13 years (range 3.7-27.0 years). TFR duration was 2.83 years in median (range 0.5 months – 10.1 years) including 19 pts with a duration < 1 year.

From the 89 recovered TFR pts, 74 pts completed the 6-month follow up (83%), a further 6 pts with molecular follow-up of 3-5 months after COVID-19 diagnosis were still in TFR, 9 pts were lost to follow-up.

Of 74 pts with complete reports, 69 pts remained in TFR (93%) and 5 pts lost TFR. For 71 pts, PCR results were obtained before, during and after infection. With the ROUT method 10 pts demonstrated outlier PCR tests, 61 pts demonstrated stable PCR results. There was no statistically significant difference in PCR results before and during/after infection (p>0.2). MRFS for these 71 pts 15 months after COVID-19 diagnosis was 86%. Probability of TFR loss was higher in pts with a TFR duration < 6 months compared to pts with TFR duration >6 months (27% vs 10%, Fig 1A). Additionally, there were no statistically differences in hospitalization rate (16% vs 23%, p=0.12) and severity of COVID-19 symptoms (12.6% vs 12%, p=0.87) comparing TFR and TKI treated pts. OS of COVID-19 positive TFR pts did not differ from COVID-19 positive pts on TKI therapy (HR 1.1, CI 0.47-2.54) (Fig 1B).

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Summary/Conclusion: In this sub-analysis of the CANDID study, CML pts in TFR had similar severity and survival to CML pts who were on TKI therapy and there was no evidence of an increased risk of TFR loss after SARS-CoV-2 infection.

P702: FIRST INTERIM RESULTS OF THE PROSPECTIVE STUDY OF DOSE REDUCTION AND DISCONTINUATION OF TYROSINE KINASE INHIBITORS (READIT-2020) IN CHRONIC MYELOID LEUKEMIA PATIENTS WITH DEEP MOLECULAR RESPONSE

M. Gurianova 1,* , O. Shukhov 1 , E. Chelysheva 1 , A. Petrova 1 , A. Bykova 1 , I. Nemchenko 1 , E. Kuzmina 1 , N. Tsyba 1 , L. Gavrilova 2 , E. Stepanova 1 , A. Kohno 1 , A. Turkina 1

1 National Research Centre for Hematology, Moscow; 2 Republican Clinical Hospital №4, Saransk, Russia

Background: The feasibility of the one-step tyrosine kinase inhibitors (TKI) dose reduction before treatment-free remission (TFR) phase in chronic myeloid leukemia (CML) patients (pts) has been confirmed in several trials.There are no data of the prospective two-step dose reduction trials of different TKIs followed up with TFR observation.The experience of resuming TKIs at reduced doses after the molecular relapse is limited as well.

Aims: To analyze the interim data of the survival without loss of major molecular response (MMR,BCR::ABL1≤0,1%) after TKI stop in CML pts taking reduced-dose TKI therapy and the probability of MMR and deep MR (DMR, BCR::ABL1≤0.01%) recovery after resuming TKIs at reduced doses (Clinicaltrials.gov NCT 04578847 ).

Methods: The prospective trial had 2 phase:1)TKI dose reduction phase for at least 12 months (mo),2)TFR phase for at least 24 mo.TKI dose reduction consisted of 2 steps each lasting for 6 mo.The inclusion of pts was possible at any step if they met the key inclusion criteria:CML in chronic phase,age ≥18 years (y),TKI therapy duration ≥ 3 y,MMR and DMR duration ≥ 2 y and ≥ 1 y.The inclusion into TFR was done after the dose reduction phase in pts with a DMR duration≥2 y and ≥MR4.5 at the time of TKI stop.In case of MMR loss the TKI dose was increased by +1 level from the dose at which the MMR loss developed.

Results: A total of 103 CML pts were included from Dec.2019 till Dec.2021 at different trial phases ( Tab 1 ). Female 60%, Median (Me) age at diagnosis and at inclusion 45 y (23-74 y) and 51 y (23-74 y),respectively;ELTS score 61%:17%:1%, 21% for low,intermediate,high and unknown risk group.A history of at least one TKI stop was in 29 pts.

Me TKI duration was 7 y (3-19,7 y);Me duration of MMR and DMR was 3,3 y (2-126 y) and 2,5 y (1,2-10,5 y), respectively.

At baseline 69 (67%) pts received imatinib (IM) and 34 (33%) pts-second-generation (2G)TKI (Tab 1). The reasons for 2GTKI switch were treatment failure (n=12),drug toxicity (n=25),others (n=1).

Sixty-four pts completed the 1 st dose reduction step lasting for 6 mo. There was no MMR loss after the 1 st dose reduction step. Four pts lost DMR on IM 300 mg,but three pts restored DMR in 3 mo at the same dose thereafter.

Fifty-seven pts completed the 6 mo 2 nd step of dose reduction.Two pts lost MMR on IM 200 mg.Nine pts lost DMR (without MMR loss):6 pts on IM 200 mg,1 pt on dasatinib (DAS) 25 mg and 2 pts on nilotinib (NIL) 200 mg.

Forty-nine pts were included in TFR phase,16 pts (32,6%) had a history of at least one TKI stop.Eight pts had a history of resistance to TKI therapy.Me follow-up after TKI discontinuation was 10 mo (1-24 mo).

The survival without MMR loss was 61% and 41% in pts with 1st and 2nd TKI stop,respectively (Fig 1). The survival without MMR loss was 54% after 12 mo in total group.

TKI therapy was resumed at reduced doses after MMR loss:IM 200 mg(n=12),NIL 200 mg (n=4),DAS 25 mg (n=2);BOS 200 mg (n=3).The probability of recovery of MMR and DMR was 86% and 83% after 6 mo resumption of TKIs at reduced doses (Fig 2).

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Summary/Conclusion: The 2-step dose reduction of TKIs before TFR phase is a promising approach for CML pts.Use of the reduced TKI doses in pts with DMR lasting for≥1 y is a safe treatment option under the strict molecular monitoring. The 61% survival rate without MMR loss in CML pts having the 1 st TKI discontinuation after the dose reduction phase is encouraging.The high probability of MMR and DMR recovery after resuming TKIs at reduced doses allows to consider this possibility in case of the molecular relapse.

P703: ASSOCIATION BETWEEN BARIATRIC SURGERY AND OUTCOMES IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA TREATED WITH ORAL TYROSINE KINASE INHIBITORS

F. Haddad 1,* , H. Kantarjian 1 , E. Jabbour 1 , N. Short 1 , A. Bidikian 1 , J. Ning 2 , L. Xiao 2 , N. Pemmaraju 1 , K. Marx 1 , F. Ravandi 1 , K. Sasaki 1 , G. Issa 1

1 Leukemia; 2 Statistics, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Bariatric surgery is widely used in patients (pts) with morbid obesity with great success in reduction of associated metabolic complications. However, it can also alter the pharmacokinetics of oral medications. Oral tyrosine kinase inhibitors (TKIs) represent the mainstay of chronic myeloid leukemia (CML) therapy. The impact of bariatric surgery on CML treatment outcomes is largely unknown.

Aims: To evaluate the clinical impact of bariatric surgery on outcomes of CML pts treated with TKIs.

Methods: In a retrospective analysis, we screened pts with CML treated at our institution and identified those who had any type of bariatric surgery including gastric bypass, gastric sleeve or gastric banding. We then compared their responses and outcomes to a control cohort of pts without history of bariatric surgery, using propensity score matching for Sokal Risk and body mass index (BMI) at a 2:1 ratio. In addition to cytogenetic and molecular responses, we assessed times to achieving responses and BCR::ABL1 halving times to investigate differences in response dynamics. We assessed comorbidities using the Charlson Comorbidity Index. Event-free survival (EFS) was measured from treatment start to loss of response, progression, or death, whereas failure-free survival (FFS) additionally accounted for therapy discontinuation for other reasons such as intolerance. Overall survival (OS) was measured from treatment start date to death or censored at last follow-up. Univariate and multivariate analyses (MVA) were used to assess the association between characteristics and survival outcomes.

Results: We identified 28 pts with CML and a history of bariatric surgery (22 pts had their surgery before the diagnosis of CML) and 56 pts as a matched control. Their baseline characteristics are summarized below.

Among pts with bariatric surgery, 61% required >1 TKI throughout the course of their disease due to intolerance or resistance, compared with 25% of the control group (P=0.002). BCR::ABL1 halving time was higher in the bariatric surgery group vs control (26 days vs 13 days; P<0.0001), suggesting slower response dynamics. The median time to achieve complete cytogenetic response (CCyR) and major molecular response (MMR) was significantly longer in pts with history of bariatric surgery: 6 months vs 3 months (P<0.0001) and 12 months vs 6 months (P=0.001), respectively. Bariatric surgery was associated with inferior EFS (10-year: 31% vs 69%; P=0.009) and FFS (10-year: 16% vs 54%; P<0.0001) compared with control, with a trend for worse OS (10-year: 65% vs 85%; P=0.09) (Figure 1).

We subsequently conducted univariate and MVA and assessed the impact of bariatric surgery and covariates such as sex, BMI, Sokal risk, TKI used, age and use of proton pump inhibitors. Bariatric surgery was the only independent predictor for the risk of treatment failure (hazard ratio [HR] 5.95, 95% CI 2.38-14.89; P=0.0001) or EFS (HR 3.6, 95% CI 1.38-7.91; P=0.007) in the MVA but did not independently affect the risk of death. The Charlson Comorbidity Index was the only independent predictor for the risk of death (HR 1.65, 95% CI 1.10-2.48; P=0.02).

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Summary/Conclusion: Bariatric surgery is associated with slower responses to TKIs and higher rates of treatment failure. There is an unmet need to design treatment strategies for these patients. Although not readily available in the clinical setting, studies measuring drug level in these patients are needed to assess which TKI has a better bioavailability, which in turn could translate to improved outcomes.

P704: ASCIMINIB PROVIDES DURABLE MOLECULAR RESPONSES IN PATIENTS (PTS) WITH CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE (CML-CP) WITH THE T315I MUTATION: UPDATED EFFICACY AND SAFETY DATA FROM A PHASE I TRIAL

T. Hughes 1,* , J. E. Cortes 2 , D. Réa 3 , M. J. Mauro 4 , A. Hochhaus 5 , D.-W. Kim 6 , K. Sasaki 7 , F. Lang 8 , M. C. Heinrich 9 , M. Breccia 10 , M. Deininger 11 , Y.-T. Goh 12 , J. J. Janssen 13 , M. Talpaz 14 , V. Gómez García de Soria 15 , P. le Coutre 16 , S. Kapoor 17 , S. Cacciatore 18 , F. Polydoros 18 , N. Agrawal 18 , F.-X. Mahon 19

1 SA Pathology and South Australian Health and Medical Research Institute, University of Adelaide, Adelaide, Australia; 2 Georgia Cancer Center, Augusta, United States of America; 3 Hôpital Saint-Louis, Paris, France; 4 Memorial Sloan Kettering Cancer Center, New York, United States of America; 5 Universitätsklinikum Jena, Jena, Germany; 6 Uijeongbu Eulji Medical Center, Geumo-dong, Uijeongbu-si, South Korea; 7 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 8 Goethe University Hospital, Frankfurt, Germany; 9 Portland VA Health Care System and OHSU Department of Medicine, Division of Hematology and Oncology, Knight Cancer Institute, Portland, United States of America; 10 Policlinico Umberto I-Sapienza University, Rome, Italy; 11 Versiti Blood Research Institute, Milwaukee, United States of America; 12 Singapore General Hospital, Bukit Merah, Singapore; 13 Amersterdam University Medical Centers, Amersterdam, Netherlands; 14 University of Michigan Rogel Cancer Center, Ann Arbor, United States of America; 15 Hospital Universitario La Princesa, Madrid, Spain; 16 Charité - Universitätsmedizin Berlin, Berlin, Germany; 17 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 18 Novartis Pharma AG, Basel, Switzerland; 19 Institut Bergonié, Bordeaux, France

Background: In pts with CML, the BCR::ABL1 T315I mutation is associated with poor clinical outcomes and confers resistance to previously approved ATP-competitive tyrosine kinase inhibitors (TKIs). Until recently, ponatinib (PON) was the only TKI available for these pts, but its use may be limited by associated cardiovascular events. In the primary analysis of the phase I trial X2101, asciminib—the 1st BCR::ABL1 inhibitor to S pecifically T arget the A BL M yristoyl P ocket (STAMP)—demonstrated efficacy and a favorable safety profile in heavily pretreated pts with CML with T315I. These results supported the FDA approval of asciminib as a new treatment option for pts with CML-CP with T315I (NCCN 2021). We report updated efficacy and safety data in these pts (data cutoff: January 6, 2021).

Aims: Provide updated safety and efficacy data for pts with CML-CP with T315I treated with asciminib monotherapy 200 mg twice daily (BID) after added exposure.

Methods: Pts with CML-CP with T315I were enrolled if treated with ≥1 prior TKI and no other effective therapy was available, provided informed consent, and received asciminib 200 mg BID.

Results: 48 pts with T315I were included; 2 (4.2%) pts had additional BCR::ABL1 mutations at baseline. Eight (16.7%), 15 (31.3%) and 25 (52.1%) pts received 1, 2, and ≥3 prior TKIs, respectively. At data cutoff, treatment was ongoing in more than half (27 [56.3%]) of pts; the predominant reason for treatment discontinuation was physician’s decision (11 [22.9%]), mainly due to lack of efficacy.

Of the 48 pts, 45 were evaluable ( BCR::ABL1 IS >0.1% at baseline) for major molecular response (MMR); 3 were excluded for BCR::ABL1 atypical transcripts. Among evaluable pts, 19 (42.2%) achieved MMR by wk 24 and 22 (48.9%) by wk 96; 19 were still in MMR at the cutoff date. Evaluable pts included 26 PON-pretreated and 19 PON-naive pts; 34.6% and 68.4%, respectively, achieved MMR by the cutoff date (Table). The probability of pts maintaining MMR for ≥96 wks was 84% (95% CI, 68.1-100.0). Thirteen (28.9%) and 11 (24.4%) pts achieved MR 4 and MR 4.5 , respectively. Twenty (54.1%) and 23 (62.2%) of 37 pts with BCR::ABL1 IS >1% at baseline achieved BCR::ABL1 IS ≤1% by wk 48 and 96, respectively.

The median duration of exposure was 2.08 (range, 0.04-4.13) yrs with more than half (27 [56.3%]) of pts receiving treatment for ≥96 wks; the median daily dose intensity was 398.3 (range, 179-400) mg/day. The safety/tolerability profile of asciminib remained favorable after ≈9 months of added follow-up (Table). The most common (≥5%) grade ≥3 adverse events (AEs) were lipase increase (18.8%, all asymptomatic elevations), thrombocytopenia (14.6%), and vomiting, ALT increase, abdominal pain, hypertension, anemia, neutropenia, and neutrophil count decrease (6.3% each). Arterial occlusive events occurred in 4 (8.3%) pts; none led to dose adjustment/interruption/discontinuation. AEs leading to discontinuation were reported in 2 new pts since the previous data cutoff; both pts discontinued and died due to COVID-19. These were the only study deaths reported in this pt population.

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Summary/Conclusion: Asciminib monotherapy 200 mg BID exhibited a sustained, favorable safety profile after added exposure with no new safety signals in pts with CML-CP with T315I—a population with high unmet medical need. The clinical efficacy of asciminib is demonstrated by the high proportion of pts achieving durable MMR and BCR::ABL1 IS ≤ 1%. The updated analysis confirms asciminib as a treatment option for pts with CML-CP with T315I, including those for whom treatment with PON has failed.

P705: MULTI-CENTER CHART REVIEW STUDY EXAMINING TREATMENT PATTERNS AND CLINICAL OUTCOMES AMONG PATIENTS WITH CHRONIC PHASE (CP) CHRONIC MYELOID LEUKEMIA (CML) TREATED IN THIRD-LINE (3L) OR LATER IN FRANCE

F. E. Nicolini 1 , G. Etienne 2 , F. Huguet 3 , M. Gu 4 , C. Bouvier 1 , A. Yocolly 5 , R. Favier 6 , M. Trancart 6 , L. Huynh 4,*

1 Centre Leon Berard, Lyon; 2 Institut Bergonié, Bordeaux; 3 Institut Universitaire de Cancérologie de Toulouse, Toulouse, France; 4 Analysis Group, Inc., Boston, MA; 5 Novartis Services, Inc., East Hanover, NJ, United States of America; 6 Novartis Oncologie, Rueil-Malmaison, France

Background: The standard of care for CML has substantially evolved over a relatively short period of time; pharmacologic inhibition of ABL1 with adenosine triphosphate (ATP)-competitive tyrosine kinase inhibitors (TKIs) remains the cornerstone of modern treatment. Clinical guidelines for CP-CML recommend that patients who have failed ≥2 prior TKIs switch to an alternative 2 nd or 3 rd generation TKI. However, up to 65% of these patients have prior exposure to ATP-competitive TKIs and history of either TKI intolerance or resistance.

Aims: This retrospective, multi-center, chart review study aimed to characterize the disease burden, treatment patterns, and clinical outcomes of CP-CML patients who have failed ≥2 TKIs.

Methods: De-identified demographic and clinical data for adult patients diagnosed with CP-CML treated in 3L or later at three reference centers in France were abstracted from medical charts using electronic case report forms. Descriptive statistics were summarized for patient characteristics, clinical outcomes during 3L treatment, and adverse events. Molecular data were standardized and expressed in the international scale (IS). The cumulative incidence of patients achieving molecular response (major molecular response [MMR, 0.01% IS ≤0.1%] or deep molecular response [MR 4.0 , 0.0032% < BCR–ABL1 IS ≤0.01% or MR 4.5 , BCR–ABL1 IS ≤0.0032%]) were summarized at specific time points. Progression-free survival (PFS) and overall survival (OS) from 3L initiation were examined using the Kaplan–Meier (KM) method.

Results: Medical data for 157 CP-CML patients were assessed; the mean follow-up was 66.9 months, the median age at 3L initiation was 62.1 years, 56% were male, and 90% had major BCR-ABL1 rearrangement (among the 89 patients with mutation status assessed, 7 [8%] had BCR-ABL1 T315I mutation, 3 [3%] had M244V, and 3 [3%] had F359I). According to EUTOS long-term survival score, 40% of patients had low risk, 25% had intermediate risk, and 11% had high risk of death due to CML. Cardiovascular diseases were present in 55% of patients at 3L; mean systolic and diastolic blood pressure were 140.9 and 78.9 mmHg, respectively. Mean ± SD low-density lipoprotein cholesterol was 141.0 ± 54.0 mg/dl. Median duration of 3L therapy was 17.0 months. TKIs received in 3L were dasatinib (32%), nilotinib (19%), imatinib (18%), ponatinib (17%), and bosutinib (14%). Treatment-free remission was initiated by 16 (10%) patients. In patients with documented responses, 42% patients achieved MMR, 27% achieved MR 4.0 , and 14% achieved MR 4.5 at 12 months. Although median PFS and OS were not reached as of data collection, 19 (12%) patients died due to disease progression (n=7), toxicity (n=1), or other reasons (n=10) and 1 patient had an unknown cause of death. Approximately 50% of patients discontinued treatment; 37% had ≥4 lines of treatment and 16% had ≥5 lines. The primary reasons for discontinuation (not mutually exclusive) were intolerance (54/78 [69%]), resistance (18/78 [23%]), and signs of ineffectiveness (14/78 [18%]). AEs were documented for 139/157 patients (89%). The mean number of AEs per patient was 2.7; infections (18%) and asthenia (13%) were the more commonly documented AEs.

Summary/Conclusion: CP-CML patients continue to experience a substantial disease burden and poor prognosis after 3L treatment with available TKIs, underscoring the need for novel therapies that are well tolerated and can achieve durable responses.

P706: ASCIMINIB USE IN CML: THE UK EXPERIENCE

A. Innes 1,2,* , V. Orovboni 3 , S. Claudiani 1 , F. Fernando 1 , A. Khan 1 , J. Byrne 4 , P. Gallipoli 5 , M. Copland 6 , G. Horne 6 , C. Arnold 7 , A. Collins 8 , N. Cunningham 7 , A. Danga 9 , R. Frewin 10 , P. Garland 11 , G. Hannah 12 , S. Hassan 13 , S. Makkuni 14 , K. Rothwell 15 , L. Foroni 2 , C. Hayden 3 , J. Apperley 2 , D. Milojkovic 1

1 Department of Haematology, Hammersmith Hospital, Imperial College Healthcare NHS Trust; 2 Centre for Haematology, Faculty of Medicine, Department of Immunology and Inflammation, Imperial College London; 3 SIHMDS (Molecular Laboratory), Hammersmith Hospital, Imperial College Healthcare NHS Trust, London; 4 Nottingham University Hospitals NHS Trust, Nottingham; 5 Cancer Research UK Barts Centre, London; 6 Institute of Cancer Sciences, Paul O’Gorman Research Centre, Gartnavel General Hospital, Glasgow; 7 Belfast City Hospital, Belfast; 8 Norfolk and Norwich University Hospital NHS Foundation Trust, Norwick; 9 The Hillingdon Hospital, London; 10 Gloucestershire Hospital NHS Trust, Gloucester; 11 Princess Royal University Hospital; 12 King’s College Hospital NHS Foundation Trust, London; 13 Queen’s Hospital, Romford, London; 14 Mid and South Essex NHS Foundation Trust, Essex, London; 15 Calderdale and Huddersfield NHS Foundation Trust, Huddersfield, United Kingdom

Background: Asciminb is an allosteric BCR-ABL1 inhibitor that has been available for patients with chronic myeloid leukaemia (CML) in the United Kingdom under a managed access program (MAP) from Novartis since 2016.

Aims: The aim of this project was to share real-world experience of asciminib use in the UK.

Methods: Using a national survey proforma (REC reference: 21/HRA/2157), we collated baseline, outcome and toxicity data on 44 patients treated with asciminib in the UK. Responses were assess using BCR-ABL1 IS values and categorised by ELN criteria. Intolerances were reported using CTCAEv5.0 criteria.

Results: The median age at treatment was 58 [22-88] years. Vascular comorbidities were seen in 22 (50%) patients, the most frequent being hypertension (n=12, 27%), peripheral vascular disease (n=6, 14%), AF (n=6, 14%) and ischaemic heart disease (n=6, 14%). Chronic kidney disease was present in 7 (16%) patients. All but one patient was in chronic phase at the time of treatment, but tyrosine kinase domain mutations (TKDM) were common prior to treatment initiation (T315I in 11/44 (25%), other TKDM in 8/44 (18%)). The median number of prior TKIs were 4 [2-5], with 44% of prior lines stopped for resistance, and 56% for intolerance. The most recent TKI was discontinued for resistance in 14 (32%) and intolerance in 30 (68%). Eight (18%) patients had developed pleural effusions on a previous TKIs. Most patients had previously received ponatinib (n=30, 68%) which has been discontinued for resistance in 8 (27%) and intolerance in 22 (73%), with 7 (16%) vascular events on treatment. Five (11%) patients had received prior allogeneic stem cell transplant.

At the time of data cut-off, 25 (57%) patients remained on treatment with a median treatment duration of 20 [3-51] months. Reason for treatment discontinuation was resistance (n=7, 16%), intolerance (n= 7, 16%), pregnancy (n=1, 2%), treatment-free remission (n=1, 2%) and non-compliance (n=1, 2%). Two patients died whilst on treatment of unrelated causes. The median daily dose delivered was 240mg [20-400] in patients with T315I-TKDM and 80mg [20-80] in others.

While 11 patients had no significant response (including those with early treatment discontinuation for intolerance), 23 (53%) patients achieved MR3 or better, with 14 (32%) achieving MR4 or better. Of 9 patients in MR3 or better at asciminib initiation, 3 (33%) did not tolerate treatment, 2 (22%) maintained their response, and 4 (44%) deepened their response by one category or more. The presence of a non-T315I TKDM was associated with a lower incidence of achieving MR3, with 1 of 6 (16%) evaluable patients in this group achieving MR3 or better, compared with 22 of 31 (71%) evaluable patients with no TKDM or a T315I-only TKDM (p=0.04) (Table1). Haematological toxicity was seen in 17 (38%) patients, and was grade 3-4 in 6 (14%) (predominantly anaemia and thrombocytopaenia). Non-haematological toxicity was reported in 19 (43%) patients (most commonly fatigue (n=4), insomnia (n=3), bone pain (n=2), back pain (n=2), nausea (n=2), fluid retention (n=2)), with only 2 (4%) patients experiencing grade 3-4 toxicity (fatigue in both cases). No worsening of baseline vascular co-morbidity or renal dysfunction was noted. One patient developed a new pleural effusion on the 200mg bd dose, which did not recur on dose reduction.

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Summary/Conclusion: These data show that asciminib is a well-tolerated, effective treatment for patients who are resistant to or intolerant of multiple TKIs, many of whom often have a number of co-morbidities including vascular disease.

P707: DOSE MODIFICATION DYNAMICS OF PONATINIB IN PATIENTS WITH CHRONIC-PHASE CHRONIC MYELOID LEUKEMIA (CP-CML) FROM THE PACE AND OPTIC TRIALS

J. Apperley 1,* , H. Kantarjian 2 , M. Deininger 3 , E. Abruzzese 4 , J. Cortes 5 , C. Chuah 6 , D. J. DeAngelo 7 , J. DiPersio 8 , A. Hochhaus 9 , J. Lipton 10 , F. Nicolini 11 , J. Pinilla-Ibarz 12 , D. Rea 13 , G. Rosti 14 , P. Rousselot 15 , M. Mauro 16 , N. Shah 17 , M. Talpaz 18 , A. Vorog 19 , X. Ren 19 , E. Jabbour 2

1 Imperial College London, London, United Kingdom; 2 The University of Texas MD Anderson Cancer Center, Houston, TX; 3 University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, United States of America; 4 S. Eugenio Hospital, Tor Vergata University, Rome, Italy; 5 Georgia Cancer Center at Augusta University, Augusta, GA, United States of America; 6 Singapore General Hospital, Duke-NUS Medical School, Singapore, Singapore; 7 Dana-Farber Cancer Institute, Boston, MA; 8 Washington University School of Medicine, St. Louis, MO, United States of America; 9 Universitätsklinikum Jena, Jena, Germany; 10 Princess Margaret Cancer Centre, Toronto, Ontario, Canada; 11 Centre Léon Bérard, Lyon, France; 12 H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, United States of America; 13 Hôpital Saint-Louis, Paris, France; 14 IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola (FC), Italy; 15 Hospital Mignot University de Versailles Saint-Quentin-en-Yvelines, Paris, France; 16 Memorial Sloan Kettering, New York, NY; 17 University of California San Francisco, San Francisco, CA; 18 Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI; 19 Takeda Development Center Americas, Inc., Lexington, MA, United States of America

Background: Ponatinib, a potent oral, third-generation tyrosine kinase inhibitor (TKI), is FDA approved for treatment of patients with relapsed/refractory CML. In the pivotal Phase 2 PACE (Ponatinib Ph+ ALL and CML Evaluation) trial ( NCT01207440 ), patients with resistant/intolerant CP-CML demonstrated deep, lasting responses to ponatinib 45 mg once daily. The Phase 2 OPTIC (Optimizing Ponatinib Treatment in CP-CML) trial ( NCT02467270 ) prospectively evaluated a response-based dose-reduction strategy in an attempt to optimize the dose schedule of ponatinib in patients with CP-CML resistant to second-generation (2G) BCR::ABL1 TKI therapy or with a T315I mutation.

Aims: Since the unique dosing strategies in the PACE and OPTIC trials provide the opportunity to closely evaluate the dose and schedule of ponatinib, we conducted an in-depth analysis of dosing dynamics between the 2 trials and compare efficacy and safety outcomes.

Methods: Adults with resistant/intolerant CP-CML from the PACE and OPTIC trials were enrolled. In PACE, patients received an initial dose of ponatinib 45 mg once daily, in OPTIC, patients were randomly assigned (1:1:1) to an initial oral dose of ponatinib 45 mg, 30 mg, or 15 mg once daily. In PACE, proactive dose reductions were mandated in ≈2 years from initiation of first patient (in 2013) as arterial occlusive events (AOEs) emerged as notable adverse events (AEs). OPTIC was designed to incorporate a mandatory response-based dose-reduction strategy; patients in the 45-mg and 30-mg cohorts in OPTIC achieving ≤1% BCR::ABL1 IS reduced their dose to 15 mg once daily; doses also were reduced to manage AEs. This analysis includes data from patients with CP-CML in PACE and from the 45-mg starting dose cohort in OPTIC. Efficacy outcomes include ≤1% BCR::ABL1 IS , progression-free survival (PFS), and overall survival (OS). Safety outcomes include treatment-emergent adverse events (TEAEs) and treatment-emergent AOE (TE-AOE) rates.

Results: Together, 364 patients with CP-CML had ≥1 prior 2G TKI or had a T315I mutation and received a starting dose of ponatinib 45 mg (PACE, n=270; OPTIC, n=94). Percentages of patients receiving ≥2 or ≥3 prior TKIs were 93% and 60% in PACE and 99% and 53% in OPTIC, respectively. Percentages of patients with vascular disorders were 44% in PACE and 32% in OPTIC. Median follow-up was 57 months (PACE) and 32 months (OPTIC). ≤1% BCR::ABL1 IS response by 24 months was 52% in PACE and 56% in OPTIC, 2-year PFS was 68% in PACE and 80% in OPTIC, and 2-year OS was 86% in PACE and 91% in OPTIC. Median time to ≤1% BCR::ABL1 IS response was 5.6 months in PACE and 6 months in OPTIC. Median duration of response was not reached in either trial. Median time to dose reduction for AEs was 2.85 months in PACE and 3.64 months in OPTIC and dose reductions due to AEs occurred in 82% of patients in PACE and 46% in OPTIC. Median time on therapy was 12.6 months in PACE and 19.5 months in OPTIC. Per 100-patient years, exposure-adjusted TE-AOEs were 15.8 events at 0 to <1 year in PACE and 7.6 events at 0 to <1 year in OPTIC. Individual dosing dynamics by safety and efficacy will be presented.

Summary/Conclusion: The response-based dose-reduction strategy in OPTIC resulted in comparable or better efficacy outcomes, fewer dose reductions related to AEs, fewer exposure-adjusted TE-AOEs, and longer median time on therapy compared with PACE. These results further demonstrate the benefit of the response-based dosing regimen used in OPTIC.

This abstract is an encore from the American Society of Hematology 2021 Annual Meeting.

P708: CANADIAN AND RUSSIAN EXPERIENCES OF ASCIMINIB IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS WHO FAILED MULTIPLE LINES OF TYROSINE KINASE INHIBITOR (TKI) THERAPY.

F. Khadadah 1,* , A. G. Turkina 2 , E. Lomaia 3 , E. V. Morozova 4 , O. A. Shukhov 2 , A. Petrova 2 , T. Chitanava 3 , J. J. Vlasova 5 , E. Kuzmina 2 , I. Nemchenko 2 , E. Y. Chelysheva 2 , A. Bykova 2 , M. Gurianova 2 , A. Xenocostas 6 , L. Busque 7 , K. Jamani 8 , S. Cerquozzi 8 , P. Kuruvilla 9 , R. Kaedbey 10 , B. Leber 10 , S. Assouline 11 , D. D. H. Kim 1

1 Princess Margaret Cancer Centre, Toronto, Canada; 2 National Medical Research Center for Hematology, Moscow; 3 Almazov National Medical Research Centre; 4 . Raisa Gorbacheva Memorial Research Institute of Children’s Oncology, Hematology and Transplantation; 5 Raisa Gorbacheva Memorial Research Institute of Children’s Oncology, Hematology and Transplantation, Saint-Petersburg, Russia; 6 London Health Sciences Centre, Victoria Hospital, London; 7 Maisonneuve-Rosemont Hospital, Montreal; 8 Tom Baker Cancer Centre, Calgary; 9 William Osler Health System, Brampton; 10 Juravinski Cancer Center, Hamilton; 11 Jewish General Hospital, Montreal, Canada

Background: Asciminib (ASC) is a novel, first in class ‘specifically targeting the ABL myristate pocket (STAMP) inhibitor. Recent update of the ASCEMBL phase 3 data showed a superior cumulative incidence of major molecular rate (MMR) of 33.2% with ASC over bosutinib (BOS) at 18.6% by 48 weeks and higher MR4 rate ( BCR-ABL1 ≤0.01%) of 14.0% with ASC over 6.6% with BOS at 48 weeks in CML patients (pts) in chronic phase (CP) who have failed at least two lines of TKI therapy.

Aims: Canadian and Russian groups individually reported their real-world experiences of ASC therapy under the Managed Access Program in heavily pre-treated CML patients.

Methods: Data was collected, updated and merged from 80 CML pts treated with ASC between Nov 2018 - Dec 2021 (n=57 in Russia; n=23 in Canada). Median age was 57 years (range 20-92). The median number of previous TKIs was 4 (range 2-6); Imatinib (n=74, 93%), dasatinib (n=64, 80%), nilotinib (n=58, 73%), bosutinib (n=51, 64%), ponatinib (n=35, 44%), and others (n=13, 16%). Median duration from diagnosis to ASC treatment was 92 mo (months) (range 11-310). 27 (34%) pts had a past history of clinically significant cardiovascular disease or high risk profile for cardiovascular disease. Of the 80 pts, 27 (34%) had a preexisting T315I mutation and 18 (23%) had a non-T315I mutation. Pts failed previous TKI therapy due to A)resistance or suboptimal response (n=59; 74%) and B)intolerance (n=21; 26%). BCR-ABL qPCRs were monitored at each institution. Achievement of MMR and molecular response of 4 log reduction (MR4) was assessed at 6 and 12 mo.

Results: With a median of 9 mo of follow-up (range 1-38), MMR was noted in 15/68 (22%) and 14/36 pts (39%) evaluated at 6 and 12 mo, respectively. MR4 was noted in 11/68 (16%) and 9/36 pts (25%) at 6/12 mo. The cumulative incidence of MMR and MR4 was 32.6% (20.9-44.8%) and 15.9% (7.9-26.3%) at 12 mo. The probability of freedom from treatment failure (FTF) at 12 mo was 42.4% (27.6-56.4%), based on the ELN 2013 failure criteria for 2 nd line therapy or beyond.

In the overall population (n=80), FTF was significantly associated with reason for switch to ASC (p=0.006), and there was a trend toward association of FTF with disease phase (p=0.08) and ponatinib therapy (p=0.12), but not with ABL1 kinase domain mutation (p=0.402), additional cytogenetic abnormality (p=0.908), nor cardiovascular risk profile (p=0.345).

When analysis was restricted to the patients in CML-CP (n=65), a median duration of FTF was calculated as 11.6 mo, while the 12 mo FTF rate was 46.8% (28.9-62.8%). The patients previously treated with ponatinib (n=25) showed a 27.7% (8.4-51.4%) FTF rate at 12 mo, which was lower than that in those naïve to ponatinib (n=40), 61.7% (34.9-80.2%; p=0.023). Those switched to ASC for intolerance (n=21) showed higher 12 mo FTF rate of 81.2% (41.5 -95.2%) compared to 29.4% (11.3-50.2%) in those with resistance (n=44; p=0.008).

Fifteen pts discontinued ASC due to treatment failure (n=11), progression of disease to blast phase (n=3) or grade 4 thrombocytopenia (n=1). No cardiovascular event was noted.

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Summary/Conclusion: This combined Canadian and Russian real-world experience of ASC study includes heavily pretreated CML pts including 15 pts in advanced phase. The MMR and MR4 rates were comparable in those in CP, while it was lower in non-CP pts. The 12 mo FTF rate of 42.4% was comparable to the one reported from the ASCEMBL study, 57.7%, considering that this group includes more patients who had heavily pre-treated with advanced disease.

*AGT and FK contributed equally as co-first author.

P709: CLINICAL OUTCOME OF ASCIMINIB TREATMENT IN A REAL-WORLD MULTI-RESISTANT CML PATIENT POPULATION.

C. C. Kockerols 1,* , J. J. Janssen 2 , N. M. Blijlevens 3 , S. K. Klein 4 , L. G. van Hussen-Daenen 5 , G. N. van Gorkom 6 , W. M. Smit 7 , P. van Balen 8 , B. J. Biemond 9 , M. J. Cruijsen 10 , M. F. Corsten 11 , P. A. te Boekhorst 12 , H. R. Koene 13 , G. L. van Sluis 14 , J. J. Cornelissen 12 , P. E. Westerweel 1

1 Internal Medicine, Albert Schweitzer Hospital, Dordrecht; 2 Hematology, Amsterdam University Medical Center, location VUMC, Amsterdam; 3 Hematology, Radboud University Medical Center, Nijmegen; 4 Hematology, University Medical Center Groningen, Groningen; 5 Hematology, University Medical Center Utrecht, Utrecht; 6 Hematology, Maastricht University Medical Center, Maastricht; 7 Hematology, Medisch Spectrum Twente, Enschede; 8 Hematology, Leiden University Medical Center, Leiden; 9 Hematology, Amsterdam University Medical Center, location AMC, Amsterdam; 10 Hematology, Catharina Hospital, Eindhoven; 11 Hematology, Meander Medical Center, Amersfoort; 12 Hematology, Erasmus University Medical Center, Rotterdam; 13 Hematology, St.-Antonius Hospital, Nieuwegein; 14 Hematology, Isala, Zwolle, Netherlands

Background: Asciminib is a first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) tyrosine kinase inhibitor (TKI). Asciminib was found to have superior efficacy compared to bosutinib in patients (pts) intolerant or refractory to ≥2 currently registered TKI with good tolerability. While awaiting its EMA approval, asciminib is made available by Novartis to pts with high clinical need in an early access programme (EAP) in The Netherlands.

Aims: We aimed to assess treatment outcomes in this nationwide patient cohort.

Methods: Dutch hematologists who treated CML pts in the asciminib EAP were asked to provide clinical data of consenting pts. Pts were included if ≥18 years and treated in this asciminib EAP, thus outside clinical trials. Treatment failure was defined as progression to advanced stage disease (AP/BC), not reaching a complete cytogenetic response or BCR-ABL1 <1%IS (CCYR/MR2.0) (=primary) or losing previously achieved responses (=secondary). Event-free survival (EFS) was defined as time from asciminib start until allogeneic stem cell transplantation (SCT), progression to AP/BC or death; whatever came first. Responses were assessed with the cumulative incidence competing risk (CICR) method considering the competing event of TKI discontinuation for any cause.

Results: All hematologists agreed to participate and 53 out of 56 pts from 14 medical centers consented and were included. Most pts (72%) had been exposed to ≥3 TKIs prior to asciminib. The majority (79%) started with asciminib because of multi-resistance (with or without intolerance) including 15 pts with primary ponatinib-failure, 15 pts harboring the T315I mutation and 3 pts with compound mutations. Two pts were treated with asciminib post-SCT and were excluded from response analyses.

Median asciminib exposure was 7 months (IQR 3-16). Frequently reported adverse events (AE) were cytopenia and folliculitis, classified as probably related to asciminib and manageable generally without dose modification. Only one pt definitely discontinued asciminib due to persistent anemia, but in the context of also a failing response. Two vascular events were reported, one recurrent TIA therefore classified as unrelated and one NSTEMI classified as possibly related to asciminib.

Sixteen pts discontinued asciminib, mostly because of treatment failure (primary n=8, secondary n=2) or proceeding to SCT (n=5). Six out of 7 pts with AP/BC discontinued asciminib within 6 months because of treatment failure/SCT.

The CI of maintaining or reaching CCYR/MR2.0 and MMR by 6 months for CP pts were 60% and 44%, respectively. Of note, only one out of 10 pts in CP with primary ponatinib-failure reached MR2.0 and none of these patients reached MMR. However, CP pts with secondary ponatinib-failure responded well with CCYR/MR2.0 or better in 78% (7/9).

During asciminib treatment 5 pts progressed to advanced stage disease, 9 pts eventually underwent SCT and 7 pts died of whom 6 with a CML-related cause. Overall survival and EFS were 87% and 72%, with a median follow-up time of 10 and 8 months, respectively.

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Summary/Conclusion: Our results show that asciminib is a well-tolerated novel treatment option for CML with adequate response rates, even in a heavily pretreated multi-resistant patient cohort. Most frequently reported AEs were manageable cytopenia and folliculitis. No patient discontinued asciminib solely due to intolerance. The CI of maintaining or reaching MR2.0/CCYR at 12 months was 57%. In our cohort, prior primary ponatinib-failure generally heralded subsequent asciminib treatment failure, both in the presence and absence of a T315I-mutation.

P710: CLINICAL STUDY ON THE RELATIONSHIP BETWEEN NON-ABL1 KINASE REGION MUTATION AND TKI DRUG RESISTANCE AND DISEASE PROGRESSION IN CHRONIC MYELOID LEUKEMIA

W. xianwei 1 , L. xiaodong 1,*

1 Central Lab, Henan Cancer Hospital, Zhengzhou, China

Background: The emergence of small molecule tyrosine kinase inhibitors (The Tyrosine Kinase Inhibitors, TKI) has gradually transformed chronic myelocytic leukemia (CML) from a fatal malignant disease into a chronic disease, while ABL1 kinase Kinase Domain (KD) mutation is the main reason for poor efficacy or resistance to TKIs. In recent years, with the clinical application of Next Generation Sequencing (NGS) technology, it has been found that CML patients are often accompanied by non-ABL1 kinase region gene mutations, but whether these accompanying mutations are related to the efficacy of TKIs and disease progression in CML? still uncertain. This study retrospectively analyzed the correlation between CML with non-ABL1 KD mutation and TKI efficacy and disease progression, in order to provide basis and scientific guidance for further accurate diagnosis and treatment of CML patients.

Aims: To investigate the relationship between CML associated with non ABL1 Kinase Domain (KD) mutation and TKI efficacy and disease progression.

Methods: 108 blood tumor related genes in 120 patients with CML were detected by Next-generation sequencing (NGS). The non ABL1 KD mutations and their relationship with relevant clinical information and ABL1 KD mutation were analyzed.

Results: Non ABL1KD mutations were detected in 58 patients (48%), and the mutation proportion of patients with additional chromosome abnormalities (63%) was significantly higher than that of patients without additional chromosome abnormalities (34%) ( P =0.003). In the analysis of mutation proportion in patients with different stages of CML, the mutation proportion in blastic phase(73%), accelerated phase (67%) and chronic phase (42%) decreased gradually ( P =0.031). The proportion of WT1 mutation and transcriptional regulation related mutations in patients with AP CML (WT1 + : 17%, P =0.027; Transcriptional regulatory mutations 42%, P =0.001) were higher than those in CP. In the analysis of TKI treatment response, the proportion of mutations detected in the best response group (30%), warning group (52%) and drug resistance group (61%) increased gradually ( P =0.018). The proportion of RUNX1 mutation in TKI treatment failure group (17%) was higher than that in warning group (2%) ( P =0.029). The proportion of ABL1 KD mutation in CML patients with non ABL1 KD mutation (60%) was significantly higher than that in patients without non ABL1 KD mutation (36%) ( P =0.022).

Summary/Conclusion: Non ABL1 KD mutations are closely related to TKI efficacy and disease progression, while WT1 and RUNX1 mutations may be new molecular targets for accurate diagnosis of CML.

P711: KINETICS OF CITED2 GENE EXPRESSION IN CHRONIC MYELOID LEUKEMIA PATIENTS

B. Atef 1,* , S. El-Ashwah 1 , L. M. Saleh 2 , H. Gawish 3 , M. Nasr 1

1 Hematology Unit, Internal Medicine Department; 2 Hematology Unit, Clinical Pathology Department; 3 Diabetes & Endocrinology Unit, Internal Medicine Department, Faculty of Medicine Mansoura University, Mansoura, Egypt

Background: In chronic myeloid leukemia (CML), BCR-ABL1 oncoprotein induces overexpression of STAT5 and its target genes. Some of these genes are involved in regulating the leukemic stem cells (LSCs) cell cycle. CITED2 gene controls the balance between the proliferation and the quiescence states of the LSCs that resist tyrosine kinase inhibitors (TKIs). Recently, studies reported that adding peroxisome proliferator‐activated receptor γ agonists (approved for type 2 Diabetes mellitus treatment) to TKI therapy may decrease the transcription of STAT5 and its target genes, eliminate the quiescent LSCs and ameliorate the patients’ response.

Aims: The aim of the study is to clarify the value of combining the glitazones with the TKIs in the treatment of denovo CML CP patients as regard the patients’ response and effect of this combination therapy on the expression of CITED2 gene.

Methods: In a context of a clinical trial (approved by the ethical Committee of the Faculty of Medicine Mansoura University), eligible denovo patients of CML chronic phase (N=26) were treated with imatinib 400 mg combined and pioglitazone 15 mg (off label use) daily for 6 months after having their consent. Responders continued on same TKI and their sequential BCR/ABL Q-PCR was monitored. The non-responder patients were switched to another line and excluded. Pre and post-treatment samples were collected to assess the expression levels of CITED2 gene.

Results: The study group included 15 males (57.7%) and 11 females (42.3%) with a mean age of 43.2 years. Their median BCR-ABL gene expression before treatment was 79% (range = 0.63-380) that decreased to 3.65% (range = 0.05-20) and 0.750 % (range = 0.002-46) on 3 rd and 6 th months post-treatment. Among the study group 100% of the patients achieved hematological response (HR) at 3 months. At 6 months 65.4% of the patients had response more than CCyR and 23% of them had MMR at 6 months respectively. The most frequent complains among patients were increase body weight and edema (p value < 0.001). The median of CITED2 gene pre-treatment and post-treatment expression levels were: 276.3 (range= 1-241221.7) and 2.6 (range= 0.006-86475.3) with a significant decrease in expression (p=0.005) after the combined treatment.

Summary/Conclusion: Pioglitazone in combination with imatinib improved the patients’ response and downregulate the expression of the CITED2 overexpressed gene. Longer follow up is planned and needed to confirm whether it can effectively induce more deep molecular response among the patients or not.

P712: ASCIMINIB ITALIAN MANAGED ACCESS PROGRAM: EFFICACY PROFILE IN HEAVILY PRE-TREATED CML PATIENTS

M. Breccia 1,* , A. V. Russo Rossi 2 , B. Martino 3 , E. Abruzzese 4 , M. Annunziata 5 , G. Binotto 6 , A. Ermacora 7 , C. Fava 8 , L. Giaccone 9 , V. Giai 10 , A. P. Nardozza 11 , P. Coco 11 , A. Gozzini 12 , L. Levato 13 , A. Lucchesi 14 , L. Luciano 15 , M. Maria Cristina 16 , G. Rege-Cambrin 17 , M. Santoro 18 , B. Scappini 19 , A. R. Scortechini 20 , P. Sportoletti 21 , E. Trabacchi 22 , F. Castagnetti 23

1 Department of Translational and precision medicine-Az, Policlinico Umberto I-Sapienza University, Rome; 2 Hematology and Transplantation Unit, University of Bari, Bari; 3 Azienda Ospedaliera “Bianchi Melacrino Morelli”, Reggio Calabria; 4 Hematology, Sant Eugenio Hospital, Rome; 5 Division of Hematology, AORN Cardarelli, Naples; 6 Hematology Unit, University of Padova, Padova; 7 Department of Internal Medicine, Pordenone General Hospital, Pordenone; 8 Clinical and Biological Sciences, University of Turin; 9 Department of Molecular Biotechnology and Health Sciences, University of Turin, Struttura Semplice Dipartimentale Allogeneic Stem Cell Transplant, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino; 10 Division of Haematology, Città della Salute e della Scienza, Turin; 11 Novartis Oncology, Medical Department, Novartis Farma SpA, Origgio; 12 Department of Cellular Therapies and Transfusion Medicine, AOU Careggi, Florence; 13 Department Hematology-Oncology, Azienda Ospedaliera Pugliese-Ciaccio, Catanzaro; 14 Hematology Unit, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola; 15 Hematology Unit, AUOP Federico II, Naples; 16 Hematology Department, San Bortolo Hospital, Vicenza; 17 Medicina Interna e Ematologia, Ospedale San Luigi, Università di Torino, Orbassano; 18 Hematology, University Hospital Paolo Giaccone, Palermo; 19 Hematology Unit, Azienda Ospedaliero-Universitaria Careggi, Florence; 20 Division of Hematology, Azienda Ospedaliero Universitaria Ospedali Riuniti Ancona, Ancona; 21 Institute of Hematology, Centro Ricerche Emato-Oncologiche, Ospedale S. Maria della Misericordia, University of Perugia, Perugia; 22 Hematology Unit and BMT Center, Ospedale G. Saliceto, Piacenza; 23 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy

Background: Chronic myeloid leukemia (CML) patients who have experienced failure and/or intolerance to multiple lines of treatment have limited therapeutic possibilities. Novel treatment options are needed to achieve sustained responses and increased tolerability in this subset of patients. Asciminib, unlike all approved tyrosine kinase inhibitors (TKIs) that bind to the ATP site of the BCR-ABL1 oncoprotein, is an allosteric, first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor with a new mechanism of action that has shown efficacy in heavily pre-treated patients after two TKIs or more.

Aims: We share our experience on asciminib use in CML patients outside of clinical trials.

Methods: We describe retrospective data from 34 chronic phase CML patients in their 3 rd or later line, treated with asciminib between April 2019 and October 2021 in 22 Italian institutions. The drug was granted by Novartis under a Managed Access Program (MAP). BCR::ABL1/ABL ratio was expressed as % IS in all centers. Efficacy was analyzed by comparing the response registered at the latest time point (34 pts evaluable) or at 3 months (27 pts evaluable – we excluded those patients for whom molecular analysis was not assessed) vs the response at baseline. Patient recruitment, dosing regimen and safety were recorded according to the MAP recommendations. The safety information was extracted from Argus database, a Novartis database where all adverse events related to Novartis compounds are stored.

Results: Median time of asciminib treatment was 8.3 months (3.3–33.2) for the whole cohort. Patients’ characteristics are displayed on Fig 1. Twenty-five patients (73.5%) were pretreated with at least 3 or more TKIs and 50% have reported to have ≥3 comorbidities. Approximately 59% of patients received prior ponatinib. Switch to asciminib occurred for resistance in 21 pts (61.8%) and intolerance in 10 pts (29.4%). Ten patients (29.4%) harbored mutations in ABL kinase domain, and 5 (14.7%) have a T315I mutation. All the T315I patients had a previous ponatinib exposure.

Nineteen out of 27 (70.4%), 12/27 (44.4%) and 6/27 (22.2%) achieved or maintained respectively Molecular Response of 2 log reduction (MR2), Major Molecular Response (MMR) and Deep Molecular Response (DMR) at three months’ time point. Twenty-four out of 34 (70.5%), 14/34 (41.7%) and 8/34 (23.5%) achieved or maintained respectively MR2, MMR, DMR response at the last follow up. After 3 months and at the last follow up, 18/27 (66.7%) and 20/34 (58.8%) of patients, respectively, showed an improvement of previous baseline response. Approximately 37% and 35%, without MMR response at baseline, reached at least an MMR at three months and last follow up, respectively. We also observed that ponatinib treated patients showed a reduced probability of reaching MR2, MMR and DMR responses compared to ponatinib naive patients at both 3 months and last follow up time points. Seventy-five percent of patients with a T315I mutation showed a response improvement, respect to baseline, already after 3 months on asciminib. The safety profile of asciminib, based on the information spontaneously reported by physicians, remained consistent with that already reported, with no new safety findings.

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Summary/Conclusion: Asciminib has shown a promising efficacy profile and tolerability in a setting of CML patients resistant and/or intolerant to 2 or more TKIs and with a high comorbidity burden. Our results confirmed what observed in sponsored trials and in the other world-wide programs, strongly suggesting a possible role for this new agent in the future therapeutic scenario.

P713: REAL-WORLD MANAGEMENT OF PATIENTS WITH CHRONIC MYELOID LEUKAEMIA (CML): FIRST ANALYSIS FROM THE UK NATIONAL REGISTRY FOR CML

G. Nesr 1,* , R. Szydlo 1 , B. Braithwaite 2 , S. Frackelton 2 , P. Mathew 2 , M. Hashmi 2 , F. Fernando 1 , A. Innes 3 , S. Claudiani 1 , A. Khan 1 , A. Rao 4 , L. Foroni 1 , D. Milojkovic 3 , J. F. Apperley 1 , R. E. Clark 5

1 Centre for Haematology, Imperial College London, London; 2 The Royal Liverpool University Hospitals, Liverpool; 3 Department of Clinical Haematology, Imperial College Healthcare NHS Trust; 4 Department of Clinical Haematology, Great Ormond Street Hospital for Children, London; 5 Department of Molecular & Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom

Background: Analysing data emerging from population based cancer registries (PBCR) informs current practice. The UK National Registry for chronic myeloid leukaemia (CML) patients (ptn) is a web-based PBCR, initially established as a regional registry in 2010, then expanded nationally in 2015.

Aims: To understand real world management of UK CML ptn.

Methods: The registry utilizes an online interface to capture baseline and yearly follow up data from contributing centres (CC). Data are securely transferred using the N3 backbone of the NHS network. Ptn are consented for enrolment at their respective centres. A “treatment episode” is defined as the duration during which a ptn received a specific tyrosine kinase inhibitor (TKI). The 2013 version of the ELN guidelines was used to define treatment milestones. For each milestone timepoint, treatment switches were counted if they occurred before the next milestone.

Results: Demographic and baseline disease characteristics data were available for 748 ptn from 43 CC (Figure 1). Of those, 79%, 75% and 94% had available karyotypes, FISH for BCR-ABL1, and RT-PCR for BCR-ABL1, respectively, at diagnosis and/or follow up. Follow up and treatment data were available for 570 and 534 ptn, respectively. Median follow up duration was 4.7 years (range: 0.1-23.7). Median age at diagnosis was 53.3 years (range: 14.6-95.1) and 53% were males. Karyotype and RT- PCR for BCR ABL1 at diagnosis were available for >75% of ptn in 40% of CC. Molecular monitoring data were available for 540 ptn: 342 (63%) had a median testing frequency of ≤ 3 months, 167 (31%) between 3-6 months, 20 (4%) between 6-12 months, and 11 (2%) of > 12 months. Imatinib (IM), nilotinib (NIL), dasatinib (DAS), bosutinib and ponatinib were prescribed as 1st or subsequent line therapy in 46%, 23%, 21%, 14%, and 7% of the treatment episodes, respectively. IM remained the most frequent 1st-line treatment after NICE approval of 2GTKI in newly diagnosed ptn. The choice of 1st line TKI (IM vs 2G) was not influenced by the Sokal, Euro, EUTOS or ELTS scores. Of ptn started on 1st line IM, NIL, and DAS; 94%, 63% and 100% received the licensed doses (400 mg OD, 300 mg BD, and 100 mg OD), respectively. Optimal responses at 3,6 and 12 months were achieved in 73%, 63%, and 51% of pts. Failure at 3 months was not evaluable as the registry did not capture sequential FBCs. Of those with a suboptimal response at 3 months, 15% changed treatment. At 6 months 28% and 50% of ptn with suboptimal and failure responses, respectively, switched their TKI. At 12 months, 10% and 28% of ptn in the suboptimal and failure categories changed TKI. Between 1-6 treatment switches, for failure and/or intolerance, occurred in 46% of ptn. Treatment switches on IM were more common for resistance than intolerance and vice versa for 2G and 3G TKI. Of all ptn who switched TKI, 40% underwent a further TKI switch. Kinase domain mutation analysis was requested in 36% and 33% of ptn resistant to their TKI at 6 and 12 months, respectively. Five-year overall survival (OS) probability was 93% for CP-CML ptn. Ptn achieving CCyR had a significantly higher OS probability than those who did not (p<0.001 by log rank test). However, differences in OS probability were not statistically significant for ptn attaining deeper responses (p<0.5 for MR3, p<0.076 for MR4, p<0.141 for MR4.5, and p<0.084 for MR5).

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Summary/Conclusion: Our results highlight the lack of conformance to the current guidelines in various aspects of CML management, the excellent OS survival achievable with currently available TKIs and the importance of achieving CCyR for better OS.

P714: EFFECT OF TYROSINE KINASE INHIBITORS ON MALE FERTILITY IN PATIENTS WITH CHRONIC PHASE CHRONIC MYELOID LEUKAEMIA

G. Nesr 1,* , S. Claudiani 1 , D. Milojkovic 2 , I. Caballes 2 , S. Lovato 1 , J. Channa 3 , J. F. Apperley 1

1 Centre for Haematology, Imperial College London; 2 Department of Clinical Haematology, Imperial College Healthcare NHS Trust; 3 Section of Investigation Medicine, Imperial College London Faculty of Medicine, London, United Kingdom

Background: With the current availability of 3+ generations of tyrosine kinase inhibitors (TKIs) and the resultant survival benefit, chronic phase chronic myeloid leukaemia (CP-CML) is considered a chronic disease for most patients. Consequently, haematologists are faced more with questions regarding the effects of treatment on fertility and pregnancy outcomes. Whilst the teratogenic effect of TKIs is well established, data on their effect on fertility, and especially male fertility, are scarce.

Semen analysis parameters (sperm number, motility and morphology) remain the cornerstone in assessing male fertility, despite its limitations in comprehensively assessing sperm functions.

Aims: To compare semen analysis parameters before and after TKI therapy in CP-CML patients treated in the haematology department of Imperial College Healthcare NHS Trust (ICHNT).

Methods: In collaboration with the Andrology Department, newly diagnosed men with CP-CML are offered sperm banking before starting cytoreductive or TKI therapy, and sperm analyses are routinely undertaken.

Patients treated with TKIs who had sperm banking at diagnosis were invited for repeat semen analysis.

Treatment history and semen analysis parameters were collected from patients’ paper/electronic medical records.

Median and range were used to summarize continuous non-parametric data. Paired sample t-test was used to compare continuous semen analysis parameters before and after TKI therapy. Double-sided p value of < 0.05 was considered as statistically significant. SPSS version 25 software was used for statistical analysis.

Results: Twenty-five patients were identified to have a semen analysis performed around the time of starting TKI therapy and were approached for repeat testing. Of those, 13 refused to participate, 1 was an international patient no longer residing in the UK and 1 stopped TKI therapy for treatment free remission trial. Therefore, 10 patients were finally included.

Median age at diagnosis was 35.6 years (range: 22.9-45.4) and patients were diagnosed between 2001 to 2014.

Six and 2 patients received hydroxyurea and interferon α, respectively, before TKI therapy. Six patients received 1 TKI (imatinib (IM), n = 4; nilotinib (NIL), n = 1; bosutinib (BOS), n = 1), 2 received 2 TKIs (IM followed by NIL), and 2 received 3 TKIs (IM, DAS, NIL and IM, NIL, DAS). Switches were for failure and/or intolerance.

Median duration of TKI therapy at the time of repeat semen analysis was 5.9 years (range: 3.3-16.5). Patients 6 and 10 had the initial semen analysis after 736 and 475 days, respectively, from commencing TKI therapy and all remaining patients had it before starting. All patients had a deep molecular response at the time of the repeat semen analysis.

The differences between sperm concentration, % progressive motility and % total motility were all non-statistically significant (p = 0.457, 0.535, and 0.574, respectively). In 2 patients the semen parameters were below the reference ranges in both occasions. Morphology (% normal forms) was less than the reference 4% in the repeat samples of 5 patients (Table 1).

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Summary/Conclusion: In our cohort, no significant differences were found in sperm concentration or motility before and after treatment with different TKIs for a prolonged period. The relevance of the finding of a low % of normal forms, in the presence of normal sperm concentration and motility, in some of the patients in the semen sample after TKI therapy is not clear. Studies with larger sample size would be needed to confirm or refute our findings.

P715: MULTICENTER OBSERVATIONAL STUDY OF PONATINIB IN CML PATIENTS IN ARGENTINA. REAL-WORLD EXPERIENCE.

M. J. Mela Osorio 1 , M. V. Osycka 2 , B. Moiraghi 2 , M. A. Pavlovsky 1 , A. I. Varela 2 , I. Fernandez 1 , F. Sackmann Massa 1 , L. Ferrari 1 , I. Giere 1 , C. Sighel 1 , M. Riddick 3 , C. Pavlovsky 1,*

1 HEMATOLOGY, FUNDALEU; 2 HEMATOLOGY, Hospital General de Agudos Jose Maria Ramos Mejia; 3 HEMATOLOGY, Instituto de Investigaciones Fisicoquímica Teóricas y Aplicadas (INIFTA, Facultad de Ciencias Exactas, Universidad Nacional de La Plata- CONICET). Departamento de Matemática, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Buenos Aires, Argentina

Background: Ponatinib is a 3 rd generation tyrosine kinase inhibitor (TKI) indicated for adults with resistant or intolerant chronic phase (CP), accelerated phase (AP) or blast phase (BP) chronic myeloid leukemia (CML) as well as in patients carrying the T315I mutation. There is a lack of data on its use in the real-world setting.

Aims: We aimed to evaluate treatment patterns, outcomes and cardiovascular toxicity in CML patients treated with ponatinib in Argentina.

Methods: This retrospective study included all patients aged ≥ 18 years with CP, AP and BP CML who initiated ponatinib in routine clinical practice across 2 medical centers in Argentina from 2013 to 2021. Demographic, efficacy and safety data were collected from patient medical charts. Continuous variables were dichotomized. Association was evaluated through Fisher exact test.

Results: A total of 56 patients were analyzed, 53 in CP, 2 in AP and 1 in BP. One patient (1.8%) received ponatinib in first-line, 19 (34%) in second-line (2L), 35 (62.4%) in 3L, and 1 (1.8%) in ≥4L. Prior cardiovascular (CV) risk factors were recorded: hypertension 8/56 14%, body mass index ≥25kg/m2 32/56 57%, hyperlipidemia (>220mg/dl) 13/56 23.2%, smokers 10/56 18% and diabetes mellitus 4/56 7%.

Median age at ponatinib start was 43.5 years (range, 18–73). Of 38 evaluated patients, 23 (60.5%) had a confirmed ABL1 mutation, including 19 (50%) with the T315I mutation. Starting doses of ponatinib were 45 mg (34%), 30 mg (53.5%), or 15 mg (12.5%). The median follow-up was 130 months (range, 13-324) and median treatment duration was 38 months (range, 1–105). At baseline, 51 patients (91%) with CP CML had less than CCyR and 2 (3.5%) were in CCyR. For 3 patients (5.3%), assessment was not available.

At 3 months, 15/51 (29%) evaluable patients with CP CML were in CCyR; at 6 months, 17/44 (38.6%) evaluable patients with CP CML were in CCyR and at 12 months, 19/34 (56%) evaluable patients with CP CML were in CCyR.

Additionally, 21 (37.5%) and 9 (16%) patients with CP CML achieved a major molecular response (MMR) or a deep molecular response (MR4.0–MR5.0) at least once during follow-up, respectively.

Progression-free survival rates estimated for patients with CP CML at 12 and 24 months were 90.3% (95% CI, 82.6–98.8%) and 86.2% (95% CI, 77.3–96.3%), respectively. Corresponding overall survival rates were 100%, and 96.1% (95% CI, 90.9–100%).

Serious arterial thrombotic adverse events (AT-AEs) occurred in 8 patients (14%), 60% of these were coronary artery disease, 30% cerebrovascular and 10% peripheral arterial disease. AT-AEs events occurred after a median time on ponatinib of 5 months (range, 2-48), the majority of the events were severe but resolved, however two were fatal.

Patients who developed an AT-AE after starting ponatinib were older than 60 years (p=0.0174), and had higher cholesterol levels (p=0.03979). No associations were found regarding obesity (p=0.403), previous TKI lines (p>0.5), smoking history (p>0.5) or ponatinib starting dose ≤30mg vs >30mg (p=0.4248).

Summary/Conclusion: This is the first analysis of ponatinib treated patients in real-world in Argentina and demonstrates that ponatinib has a favorable efficacy and safety profile in patients with CML treated in standard clinical practice. Cardiovascular toxicity of ponatinib in routine clinical practice is increased. Older (>60y) and dyslipemic patients were at higher-risk of occlusive events. Prompting aggressive control of CV risk factors as well as reducing doses at optimal time-points may help optimize the use of ponatinib during daily practice.

P716: OUTCOMES AND PATTERNS OF TREATMENT IN CHRONIC MYELOID LEUKEMIA, A GLOBAL PERSPECTIVE BASED ON A REAL-WORLD DATA GLOBAL NETWORK

A. Sanz 1,* , R. Ayala 1 , G. Hernández 2 , N. López 1 , D. Gil 1 , R. Gil 1 , R. Colmenares 1 , G. Carreño-Tarragona 1 , J. M. Sánchez Pina 1 , R. A. Alonso 1 , N. García Barrio 3 , D. Pérez-Rey 2 , L. Meloni 4 , M. Calbacho 1 , M. Pedrera-Jiménez 3 , P. Serrano-Balazote 3 , J. de la Cruz 5 , J. Martínez-López 1

1 Hematology, Hospital 12 de Octubre; 2 Biomedical Informatics Group, Universidad Politécnica de Madrid; 3 Data Science Group, Research Institute imas12, Madrid, Spain; 4 Trinetx, LLC, Cambridge, United States of America; 5 Research Institute imas12, Hospital 12 de Octubre, Madrid, Spain

Background: The natural history of chronic myeloid leukemia (CML) experienced a major change in the early 2000s with the development of the first BCR-ABL1 specific tyrosine kinase inhibitors (TKIs); however, there is limited supporting evidence from electronic health records (i.e., real-world data [RWD]). TriNetx is a global health research platform that provides researchers access to electronic medical records from healthcare organizations (HCOs) for conducting research studies. It allowed us to analyze trends in survival in CML patients throughout the years from a global perspective, as well as examine relevant comorbidities and long-term toxicities.

Aims: The objective of this study is to better define the current survival landscape of CML and the long-term toxicities, so that we can have a better and updated understanding of the disease and the actual clinical practice in local, regional and global settings.

Methods: We analyzed data from more than 7500 patients diagnosed with CML from four research networks (Hospital 12 de Octubre (H12O), the EMEA network, the US network, the Global Network) covering more than 16 million subjects. Propensity score matching for age and gender was applied, comparing samples equal in size (CML and controls). This study was conducted with anonymized data accessed via the TriNetX platform, which provided diagnoses, procedures, medications, laboratory values, and genomic information from approximately 6,000 CML patients treated with TKIs from 57 health care organizations.

Results: Survival at 20 years was not significantly different between patients with CML treated with TKIs and non-cancer patients (63·4% vs 69·05%, HR 1.095 (0·924 - 1·298) p = 0·295, Figure 1). No significant change in survival probability was seen comparing patients treated 2001-2010 and 2011-2020 (HR 1·113 (0·858 - 1·443), p = 0.42) or comparing imatinib with second-generation TKIs (imatinib vs dasatinib HR 1·017 (0·895 - 1·157), log-rank p = 0·80; imatinib vs nilotinib HR 1·101 (0·925 - 1·310).

When comparing the occurrence of second malignancies, an increased incidence was seen in CML patients (64·4% vs 17·2%, OR 8·72 (7·955, 9·549)). In the case of cardiovascular diseases, CML patients again showed a higher incidence than non-CML patients (74·2% vs 60·5%, p<0·001, OR 1·879 (1·729, 2·041)). No specific TKI was associated with the increased risk of any of these conditions.

Regarding TKI usage and switching patterns, dasatinib was the most common second-line treatment for both H12O and the US, but not in the EMEA where it was nilotonib. Ponatinib assumed a larger role in third-line and beyond.

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Summary/Conclusion: From this large real-world analysis, we conclude that in the TKI era, the CML population has the same survival as a non-cancer population. However, the main TKI toxicities and comorbidities remained high in the CML population, and despite them not seeming to impact survival, it could affect the quality of life. Therefore, strategies to stop lifelong TKI use should be prioritized to reduce adverse events.

P717: BOSUTINIB IN NEWLY DIAGNOSED CHRONIC MYELOID LEUKEMIA: GASTROINTESTINAL, LIVER, EFFUSION AND RENAL SAFETY CHARACTERIZATION IN THE BFORE TRIAL

J. E. Cortes 1,* , D. Milojkovic 2 , C. Gambacorti-Passerini 3 , V. García-Gutiérrez 4 , M. J. Mauro 5 , E. Leip 6 , S. Purcell 7 , A. Viqueira 8 , T. H. Brümmendorf 9

1 Georgia Cancer Center, Augusta, United States of America; 2 Hammersmith Hospital, London, United Kingdom; 3 University of Milano-Bicocca, Monza, Italy; 4 Universitario Ramón y Cajal, Ramón y Cajal Health Research Institute, Madrid, Spain; 5 Memorial Sloan Kettering Cancer Center, New York; 6 Pfizer Inc, Cambridge, United States of America; 7 Pfizer Ltd, London, United Kingdom; 8 Pfizer SLU, Madrid, Spain; 9 Universitätsklinikum RWTH Aachen, Aachen, Germany

Background: Bosutinib is approved for the treatment of Philadelphia chromosome–positive (Ph+) chronic myeloid leukemia (CML) resistant/intolerant to prior therapy and newly diagnosed Ph+ chronic phase (CP) CML. Efficacy and safety of bosutinib vs imatinib in patients with newly diagnosed CP CML was assessed in the phase 3 BFORE trial ( NCT02130557 ).

Aims: The safety profile of bosutinib after 5 years follow-up, with a focus on gastrointestinal, liver, effusion and renal treatment-emergent adverse events (TEAEs), was characterized.

Methods: Patients who received ≥1 dose of bosutinib (n=268) or imatinib (n=265) 400 mg/day in BFORE were included. Adverse events (AEs) of special interest were analyzed by selecting prespecified MedDRA terms to generate TEAE clusters. This analysis is based on the final database lock: June 12, 2020.

Results: Median duration of treatment was 55 months for patients receiving bosutinib or imatinib; respective median (range) dose intensity was 393.6 (39–583) vs 400.0 (189–765) mg/d. Any grade TEAEs occurred in 98.9% and 98.9% of bosutinib- vs imatinib-treated patients. The most common newly occurring TEAEs (any grade) after 12 months were increased lipase (9.0%) with bosutinib, and diarrhea (8.3%) with imatinib. In bosutinib- vs imatinib-treated patients, 25.4% vs 14.3% had AEs leading to permanent treatment discontinuation; the majority discontinued in year 1 (14.2% vs 10.6%). The most frequent AEs leading to discontinuation were increased ALT (overall, 4.9%; year 1, 4.5%) with bosutinib vs thrombocytopenia (overall, 1.5%; year 1, 1.5%) with imatinib.

Gastrointestinal, liver, effusion and renal TEAEs, respectively, occurred in 79.9%, 44.0%, 6.0% and 10.4% (maximum grade 3/4 [G3/4]: 9.0%, 26.9%, 1.1% and 2.2%) of bosutinib- vs 61.5%, 15.5%, 2.3% and 9.8% (G3/4: 1.1%, 4.2%, 0.4% and 0.8%) imatinib-treated patients. One grade 5 renal TEAE occurred in the bosutinib arm and was not considered related to treatment. Cumulative rates per treatment year are shown in the Table. The most common gastrointestinal TEAEs were diarrhea (bosutinib vs imatinib: 75.0% vs 40.4% [G3/4: 9.0% vs 1.1%]) with bosutinib, and nausea (37.3% vs 42.3% [G3/4: 0% vs 0%]) with imatinib. In both arms, the most common liver, effusion and renal TEAEs, respectively, were increased ALT and/or AST (34.0% vs 8.3% [G3/4: 22.0% vs 2.3%]), pleural effusion (5.2% vs 1.9% [G3/4: 0.7% vs 0.4%]) and increased blood creatinine (6.7% vs 8.3% [G3/4: 0.4% vs 0.4%]). Gastrointestinal, liver, effusion and renal TEAEs infrequently led to treatment discontinuation (1.9%, 7.8%, 0.7% and 0.7% vs 1.1%, 0.8%, 0% and 0.4%).

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Summary/Conclusion: The safety profiles of bosutinib and imatinib in BFORE were distinct, with no new safety signals identified after 5 years of follow-up. The onset of TEAEs occurred primarily during year 1 (e.g., gastrointestinal and liver), with an increased incidence of some TEAEs (e.g., effusion and renal) in later years. Discontinuations due to AEs generally occurred early into treatment, with low rates of discontinuation due to gastrointestinal, liver, effusion and renal AEs. These long-term safety results further support the use of first-line bosutinib as a standard of care in patients with CP CML.

P718: ASCIMINIB MANAGED-ACCESS PROGRAM (MAP) IN RUSSIA

O. Shukhov 1,* , A. Turkina 1 , E. Lomaia 2 , E. Morozova 3 , A. Petrova 1 , T. Chitanava 2 , Y. Vlasova 3 , E. Chelysheva 1 , E. Kuzmina 1 , I. Nemchenko 1 , A. Bykova 1 , M. Gurianova 1 , A. Kohno 1 , E. Parovichnikova 1

1 National Medical Research Center for Hematology, Moscow; 2 Almazov National Medical Research Centre; 3 Raisa Gorbacheva Memorial Research Institute of Children’s Oncology, Saint-Petersburg, Russia

Background: Asciminib is aa ABL kinase inhibitor that works by Specifically Targeting the ABL Myristoyl Pocket (STAMP) that has shown efficacy and a good safety profile in phase I and III studies in patients with Ph-positive leukemia failing prior TKIs. In Russia, asciminib is available under the Novartis approved Managed Access Program (MAP).

Aims: to present interim results of the use of aciminib in clinical practice under the MAP program in Russia.

Methods: From September 2019 to January 2022, 68 patients (pts) with CML were enrolled in the MAP program from three Russian clinics. We analyzed therapy results from 50 pts. Eleven pts who received asciminib for less than 3 months and 7 pts who underwent bone marrow transplantation were excluded from the analysis. Pts recruitment, dosing regimen, response monitoring, and toxicity control were performed according to the MAP treatment plan. Complete cytogenetic response (CCyR), major molecular response (MMR) and deep molecular response (MR4) were assessed by cumulative incident function (CIF) with Gray’s test for comparison responses in subgroups. Multivariate analysis was performed to find independent factors for MMR. Differences were considered significant if the p ≤ 0.05

Results: Baseline characteristics: female 66%; Ме age 53 years (range 26-81); median duration of CML before asciminib 8 years (range 1-24); 42 pts were in chronic phase (CP) CML, 7 and 1 pts had a history of accelerated phase (AP) and myeloid blast crisis (MBC), respectively. Twenty nine (58%) pts had BCR::ABL1 mutations, 20 pts (40%) had BCR::ABL1 T315I , 8 (16%) pts had at least two mutations. Eight (16%) pts had additional chromosomal abnormalities (ACAs). Among the thirty three (66%) pts who received ≥4 TKIs, 20 (40%) had a history of ponatinib treatment. Me follow-up period was 11 months (range 4-30), 7(14%) pts discontinued asciminib (5 due to resistance, 2 due to progression). One-year survival rate without treatment discontinuation was 92%. In 30 (60%) pts, The initial dose was 40 BID in 30 (60%) pts and 200 mg BID in 20 (40%) pts

The 2-year overall survival was 96%, two (4%) pts progressed to advanced phase and died. CIF of CCyR, MMR and MR4 at 12 month was 37.5% (CI:24-58), 32% (CI:20-52) and 14% (CI:7-31), respectively. Univariate analysis was performed for the following factors: history of advanced phases, initial dose of asciminib, presence of BCR::ABL1 mutations and ACAs, best molecular response on previous TKIs, molecular response at the time of asciminib starting, history of ponatinib treatment, number of prior TKIs and duration of TKI therapy before asciminib. Best molecular response to previous TKIs, molecular response at the time of asciminib starting, number of TKIs prior to asciminib, and history of previous ponatinib treatment were identified as predictors of MMR at 12 months (Table 1). Best molecular response on previous TKIs (≤1% vs 1-10% vs ≥10%) was found to be independently significant factor due to multivariate analysis (p=0,0072, hazard ratio 7,6 (1,7-33))

Twenty two (44%) patients experienced adverse events (AEs) of any grade and 8 (16%) had AEs of grade 3-4 (Table 2), reported during treatment, regardless of the connection with the drug. No one stopped treatment because of toxicity. No differences in toxicity were found between doses of 80 and 400 mg/day.

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Summary/Conclusion: Asciminib shows promising results for the treatment of highly pre-treated CML patients and should be considered as an therapeutic options for pts with failure to previous TKIs

P719: EFFICACY AND SAFETY OF BOSUTINIB IN LATER-LINE PATIENTS WITH CHRONIC MYELOID LEUKEMIA: A SUB-ANALYSIS FROM THE PHASE 4 BYOND TRIAL

C. Gambacorti-Passerini 1,* , T. H. Brümmendorf 2 , T. Ernst 3 , E. Leip 4 , S. Purcell 5 , A. Viqueira 6 , F. J. Giles 7 , G. Rosti 8 , A. Hochhaus 3

1 University of Milano-Bicocca, Monza, Italy; 2 Universitätsklinikum RWTH Aachen, Aachen; 3 Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany; 4 Pfizer Inc, Cambridge, United States of America; 5 Pfizer Ltd, London, United Kingdom; 6 Pfizer SLU, Madrid, Spain; 7 Developmental Therapeutics Consortium, Chicago, United States of America; 8 IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola, Italy

Background: Bosutinib is approved for patients with Philadelphia chromosome-positive chronic myeloid leukemia (CML) resistant or intolerant to prior therapy and newly diagnosed patients in chronic phase. The efficacy and safety of bosutinib in patients with CML resistant or intolerant to prior tyrosine kinase inhibitors (TKIs) was evaluated in the phase 4 BYOND trial ( NCT02228382 ; Gambacorti-Passerini et al, Blood, 2021).

Aims: The efficacy and safety of bosutinib in resistant and intolerant patients with CML treated with 2 (3L) and 3 (4L) prior TKIs was characterized.

Methods: The BYOND trial included 163 patients with CML resistant or intolerant to prior TKIs. A sub-analysis of 48 patients treated with 2 (3L) and 3 (4L) prior TKIs, categorized by resistance or intolerance to the last received TKI, was performed. This sub-analysis is based on the final November 23, 2020 database lock.

Results: There were 18 and 30 patients resistant or intolerant to the last received TKI who entered the study without complete cytogenetic response (CCyR) or major molecular response (MMR), respectively. Median (range) treatment duration was 10.6 months (1.6–48.5) vs 28.3 months (0.2–48.6) and median (range) dose intensity was 447.1 mg/d (131.3–520.4) vs 288.8 mg/d (79.7–500.0) for resistant vs intolerant patients. Prior TKIs included imatinib (88.9% vs 100.0%), dasatinib (88.9% vs 83.3%), and nilotinib (66.7% vs 63.3%) for resistant vs intolerant patients. Overall, 61.1% vs 66.7% of resistant vs intolerant patients discontinued bosutinib, mostly commonly due to adverse events (AEs) in 27.8% vs 16.7% patients; 16.7% vs 6.7% discontinued bosutinib due to insufficient clinical response. Rates of CCyR and MMR are shown in the Table. Among responders (resistant vs intolerant patients), median (range) time to CCyR was 5.1 months (2.8–8.8) vs 3.0 months (2.7–6.1); median (range) time to MMR was 5.8 months (2.8–9.4) vs 3.2 months (2.8–9.3). In resistant vs intolerant patients, any grade treatment-emergent AEs (TEAEs) were reported by 100.0% vs 96.7% patients; grade 3/4 TEAEs were reported by 72.2% vs 83.3% patients. Grade 3/4 TEAEs >10% in resistant patients were thrombocytopenia (22.2%) and neutropenia (11.1%), and in intolerant patients were increased alanine aminotransferase (26.7%), diarrhea (23.3%), pleural effusion (13.3%), and rash (13.3%).

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Summary/Conclusion: This sub-analysis of resistant and intolerant patients without baseline CCyR or MMR shows bosutinib was active in heavily pretreated patients with resistance or intolerance to the last received TKI. Despite a difference between resistant and intolerant patients with CML, efficacy outcomes, though lower than the overall BYOND population, are encouraging, and safety was generally consistent with previous reports.

P720: PROACTIVE DASATINIB DOSE REDUCTION IN THE ALLG CML 12 DIRECT STUDY BASED ON TROUGH LEVELS MINIMISE TOXICITY AND PRESERVE EFFICACY

D. Yeung 1,2,* , A. Grigg 3 , N. Shanmuganathan 1 , A. Soltenbeck 4 , D. White 2 , S. Branford 5 , N. Viiala 6 , P. Rowlings 7 , A. Mills 8 , J. Shortt 9 , C. Tiley 10 , D. Ross 11 , D. Kipp 12 , R. Harrup 13 , I. Cunningham 14 , J. Kwan 15 , R. Eek 16 , H. Mutsando 17 , K.-S. Tan 18 , K. Burbury 19 , M. Wright 20 , T. Hughes 1,2 , On Behalf of the ALLG 21

1 Haematology, Royal Adelaide Hospital; 2 Precision Medicine, SAHMRI, Adelaide; 3 Haematology, Austin Hospital; 4 Statistical Revelations, Melbourne; 5 Molecular Pathology, SA Pathology, Adelaide; 6 Haematology, Liverpool Hospital, Sydney; 7 Haematology, Calvary Mater Hospital, Newcastle; 8 Haematology, Princess Alexandra Hospital, Brisbane; 9 Haematology, Monash Medical Centre, Melbourne; 10 Haematology, Gosford Hospital, Gosford; 11 Haematology, Flinders Medical Centre, Adelaide; 12 Haematology, Barwon Health, Geelong; 13 Haematology, Royal Hobart Hospital, Hobart; 14 Haematology, Concord Hospital; 15 Haematology, Westmead Hospital, Sydney; 16 Haematology, Border Medical Oncology, Albury; 17 Haematology, Toowoomba Hospital, Toowoomba; 18 Nephrology, Princess Alexandra Hospital, Brisbane; 19 Haematology, Peter MaCallum Cancer Centre, Melbourne; 20 Haematology, Finoa Stanley Hospital, Perth; 21 ALLG, Melbourne, Australia

Background: Dasatinib treatment leads to excellent molecular responses in chronic phase chronic myeloid leukemia (CP-CML) but at 100mg leads to a substantial risk of pleural effusions, which may be higher in the elderly. Rousselot et al (BJH 2021) suggested this risk may be mitigated through dose modifications as guided by trough levels (Cmin).

Aims: The CML12 (DIRECT) study, run by the ALLG, is a single arm phase II study, aiming to minimise dasatinib related toxicity whilst preserving efficacy, through dose adaptation as guided by dasatinib trough levels. We report here the primary end point - cumulative incidence of pleural effusion (PEf) at 24 months (mos) and key efficacy secondary end points.

Methods: DIRECT enrolled newly diagnosed CP-CML pts. The first 34 patients (pts) were aged >60 years (yrs), after which the protocol was amended to include pts aged >18 yrs at the recommendation of the trial management committee. All pts started on dasatinib 100mg QD, with Cmin taken at 7, 28, 56 & 90 days, then every 3 mos hence. Pts sequentially dose reduced to 70mg, then to 50mg QD, for Cmin >3nM, within the first 2 mos, prior to assessment of early molecular response at 3 mos. Doses <50mg QD were permitted temporarily only for toxicity management. Chest x-ray and transthoracic echocardiograms were performed at baseline & 24 mos.

Results: Eighty pts were enrolled from 14 centres, with a median follow up of 36 mos (range 24-60). Median age was 64 yrs (range 21-86) and 46% were female. The ELTS score was low and intermediate in 54% and 34% respectively, high in 5% and missing in 8%. Cmin and treatment assigned at various timepoints are detailed in Table 1. Older patients had higher Cmin, particularly prior to dose adjustments. The majority of pts were dose reduced to 50mg QD. Fifteen cases of pleural effusion (PEf) occurred, 5 within the first mo, and 12 in total by 24 mos; 3 were asymptomatically detected on chest x-ray. The cumulative incidence of PEf by 24 mos was 15% overall (95% CI 8-25%, Fig 1); with values of 7.6%, 14.3% and 45.5% respectively in patients <60 yrs, 60-75 yrs and ≥75 yrs. The Cmin prior to the PEf was >3nM in 8/15 pts.

At 24 mos, 59 (74%) of pts remained on dasatinib. Reasons for discontinuation were intolerance / adverse events (AE) (n=15, 19%); treatment failure (n=4, 5%); death (n=1, 1%) and consent withdrawn (n=1, 1%). AEs leading to discontinuation were PEf (n=6, 7.5%) pulmonary hypertension (n=4, 5%; 2 reported as SAEs), pericardial effusion (n=2, 2.5%) and cardiac failure (n=2, 2.5%). There were no cases of peripheral vascular disease or strokes.

An early molecular response (BCR::ABL1 <10% IS at 3 mos) was achieved in 77 pts (96%). Cumulative incidence of MMR (<0.1%) by 12 and 24 mos were 75% (95% CI 66-84%) and 92% (95% CI 86-97%), respectively; MR4.5 by 24 and 48 mos were 48% (95% CI 38-60%) and 76% (95% CI 64-90%), respectively. No patient progressed to AP/BC on or off study. Three pts have died of non CML-related causes (infection n=2; biliary carcinoma n=1).

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Summary/Conclusion: High rates of MMR/MR4.5 were achieved, despite significant dose reductions, according to target drug levels over the first 2 months. Toxicity profile was also broadly favourable. However, such adaptive approaches had limited capacity to influence early development of PEf. A strategy using lower starting doses, with escalation for failure to achieve molecular targets, may further minimise toxicity, especially in the elderly.

P721: PREDICTIVE SCORING SYSTEMS FOR MOLECULAR RESPONSES IN PERSONS WITH CHRONIC MYELOID LEUKEMIA RECEIVING INITIAL IMATINIB THERAPY

X. Zhang 1,* , Z. Li 1 , M. Zhang 1 , P. G. Robert 2 , X. Huang 1 , Q. Jiang 1

1 Peking University People’s Hospital, Beijing, China; 2 Centre for Hematology Research, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

Background: Nowadays more patients have benefited from tyrosine kinase inhibitors (TKIs) therapy to achieve a stable major molecular response (MMR) and even deep molecular responses (DMR, at least MR 4 ), which prevented disease progression and allows for pursuing treatment-free remission (TFR). However, there were still lacking of robust prediction models for molecular responses including MMR or DMR to TKI-therapy.

Aims: Develop and validate predictive scoring systems for MMR and MR 4 in persons with newly-diagnosed chronic phase chronic myeloid leukemia (CML) receiving imatinib.

Methods: Data from 1364 consecutive subjects were randomly-assigned 2:1 to training (n=909) and validation (n=455) datasets. Co-variates significantly associated with MMR and MR 4 in multi-variable analyses in the training dataset were used to develop a predictive scoring system based on standardized regression coefficients (beta [β]) and the model tested in the validation dataset. Time-dependent area under the receiver-operator characteristic curves (AUROC), calibration plots and decision curve analyses (DCAs) were used to evaluate predictive accuracy.

Results: In the training dataset, male sex (β=-0.350, HR=0.7 [95%CI: 0.6, 0.8]), WBC ≥ 130 ×10E+9/L (β=-0.538, HR=0.6 [0.5, 0.7]), hemoglobin < 110 g/L (β=-0.490, HR=0.6 [0.5, 0.8]) and EUTOS long-term survival (ELTS) risk score (intermediate-risk: β=-0.464, HR=0.6 [0.5, 0.8]; high-risk: β=-0.743, HR =0.5 [0.3, 0.7]) were significantly-associated with lower cumulative incidence of MMR. Each co-variate was assigned 1 point in the predictive scoring system for MMR except WBC ≥ 130 ×10E+9/L and ELTS high-risk assigned 2 points. Using the predictive scoring system, subjects were classified into (1) low- (score=0-1; n=429, 47%), (2) intermediate- (score=2-4; n=340, 38%); and (3) high- (score=5-6; n=140, 15%) risk subgroups. 5-year cumulative incidences of MMR were 86% (84, 89%), 61% (55, 65%) and 34% (27, 43%) ( p for trend <0.001; Figure A ). Comparable 5-year cumulative incidences in validation dataset were 86% (82, 90%), 69% (64, 74%) and 31% (22, 40%) ( p for trend<0.001; Figure B ). AUROC values were 0.77 and 0.74 in the training and validation datasets. Similar analyses for MR 4 in training dataset identified male sex (β=-0.282, HR=0.8 [0.6, 0.9]), WBC ≥ 120 ×10E+9/L (β=-0.747, HR=0.5 [0.4, 0.6]), hemoglobin<110 g/L (β=-0.496, HR=0.6 [0.5, 0.8]) and ELTS risk score (intermediate-risk: β=-0.509, HR=0.6 [0.4, 0.8]; high-risk: β=-1.212, HR=0.3 [0.1, 0.6]) were significantly-associated with lower cumulative incidence of MR 4 . Male sex was assigned 1 point; ELTS intermediate-risk and hemoglobin < 110 g/L, 2 points; WBC ≥ 120 ×10E+9/L, 3 points; and ELTS high-risk, 4 points in the predictive scoring system for MR 4 . Subjects in the training dataset were classified into (1) low- (score=0-2; n=416, 46%), (2) intermediate- (score=3-7; n=348, 38%); and (3) high- (score=8-10; n=145, 16%) risk cohorts. 5-year cumulative incidences of MR 4 were 71% (70, 73%), 35% (31, 39%) and 16% (10, 22%) ( p for trend<0.001, Figure C ). Similar 5-year cumulative incidences in validation dataset were 66% (61, 71%), 45% (38, 52%) and 21% (12, 30%) ( p for trend<0.001, Figure D ). AUROC values were 0.76 and 0.72. Calibration plots and DCAs showed good performance for predicting MMR and MR 4 .

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Summary/Conclusion: We developed the predictive scoring systems for MMR and MR 4 in persons with chronic phase CML receiving imatinib. These data may help physicians make an appropriate therapy decision.

P722: IS THE SECOND-GENERATION TYROSINE KINASE INHIBITOR A BETTER THERAPY THAN IMATINIB FOR PERSONS WITH CHRONIC MYELOID LEUKEMIA PRESENTING IN ACCELERATED-PHASE?

X. Zhang 1,* , S. Yang 1 , Y. Liu 1 , Q. Jiang 1

1 Peking University People’s Hospital, Beijing, China

Background: Although most patients with chronic myeloid leukemia (CML) were presenting in the chronic phase at diagnosis, approximately 5% presented with features of accelerated phase. The features of accelerated phase were variably defined by different criteria, such as MD Anderson and European LeukemiaNet (ELN) criteria most often used. Regardless of these criteria used to defined accelerated phase, only scant data were available regarding the prognostic co-variates for outcome. Moreover, evidence that patients could benefit more from the second generation (2G-) tyrosine kinase inhibitor (TKI) than imatinib as initial therapy was still extremely limited.

Aims: Explore whether patients with chronic myeloid leukemia (CML) presenting in accelerated-phase could benefit from initial second-generation tyrosine kinase inhibitor (2G-TKI) therapy.

Methods: We interrogated data from consecutive patients with CML presenting in accelerated phase at diagnosis by the MD Anderson criteria (n = 325) or ELN criteria (n = 275). Propensity score matching (PSM) analysis was used to compare cytogenetic and molecular responses and outcomes in those receiving initial imatinib versus a 2G-TKI. COX multi-variable regression model was performed to identify the prognostic co-variates.

Results: In the cohort defined by MD Anderson criteria, PSM analysis showed that patients receiving initial 2G-TKI (n = 89) had higher probability of achieving a CCyR ( p = 0.01), MMR ( p = 0.04) and MR 4.5 ( p = 0.004) than those receiving imatinib (n = 156), however, probabilities of FFS, PFS and survival were similar. Multi-variable analyses showed that lower platelets and higher blasts in blood or bone marrow were significantly-associated with poor FFS, PFS and/or survival (Figure A ). In an attempt to determine whether choice of therapy contributed to the differences in responses and outcomes, platelets < 600 × 10E+9/L and blood / bone marrow blasts ≥ 5% were identified as the prognostic categorical co-variates by ROC analyses. The entire cohort were classified into low- (possessing none of these co-variates, n = 57), intermediate (possessing any 1 of these co-variates, n = 127) or high-risk (possessing all of these co-variates, n = 61) subgroups. There were significant differences in probabilities of outcomes among these risk subgroups (all p values < 0.001, Figure B ). In each risk subgroup, patients receiving initial 2G-TKI had the similar outcomes to those receiving imatinib except higher incidences of responses including CCyR, MMR and MR 4.5 . The same results were observed in the cohort defined by ELN criteria.

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Summary/Conclusion: Initial 2G-TKI lead to similar outcomes as imatinib in patients with CML presenting in accelerated-phase except higher therapy responses.

P723: LOSS OF THE NK MOTIF AND ANKYRIN REPEAT DOMAIN 1 (KANK1) LEADS TO LYMPHOID COMPARTMENT DYSREGUATION IN MICE

M. Almosailleakh 1,* , S. Narcisi 1 , C. Roger Michel Côme 1 , H. Luche 2 , K. Grønbæk 1,3

1 Biotechnology Research and innovation Center, University of Copenhagen, Copenhagen, Denmark; 2 Centre d’Immunophénomique, Parc Scientifique et Technologique de Luminy, Marseille, France; 3 Department of Hematology, Regishospitalet, Copenhagen, Denmark

Background: We identified a novel germline loss of heterozygosity (LOH) mutation encompassing the NK Motif And AnKyrin Repeat Domain 1 ( KANK1 ) gene in a young Myelodysplastic syndrome (MDS) patient and his healthy father, with no additional MDS-related mutations or chromosomal abnormalities. Clinical investigation suggested development of MDS following severe infection, likely triggered by an autoimmune mechanism. KANK1 protein plays a role in the control of cytoskeleton formation by regulating actin polymerization. A t(5;9) translocation resulting in a fusion of the platelet-derived growth factor receptor beta gene ( PDGFRB ) and KANK1 was detected in a patient with myeloid neoplasm (MN) characterized with severe thrombocythemia. The protein is thought to have a tumour suppressor function, as its expression is downregulated or missing in several tumor tissues, while over-expressing the protein was reported to inhibit the proliferation of tumor cells in solid cancer models. In addition, defects in cytoskeletal proteins are reported to contribute to immunological dysfunction and disruption of normal immune processes.

Aims: To investigate the role of KANK1 in the regulation of normal hematopoiesis and the development of myeloid disorders.

Methods: We generated a new genetically modified KANK1 ( Kank1 -/- ) knockout mouse model and analysed the phenotypic and developmental changes in hematopoiesis in young and aged mice.

Results: We confirmed the loss of Kank1 mRNA and protein expression by q-PCR and targeted protein mass-spectrometry respectively. The mice are viable and fertile, and reproduced according to mendelian ratio. Over the two years observation period, Kank1 -/- mice did not exhibit any signs of disease, and their peripheral blood parameters remained within the normal range. Flow cytometry analysis of bone marrow (BM) cells from 17-20 weeks kank1 -/- old mice showed minor increase in CD3 + T-cell population, while the expression of the double positive B220/CD19 B-cells population was reduced in Kank1 -/- compared to Kank1 +/+ . Comprehensive flow cytometry analysis of spleen and thymus cells from young (n=8 each, 6-7 weeks old) and aged (n=8, 68-71 weeks old) Kank1 -/- mice showed further dysregulation of the lymphoid compartment when compared to Kank1 +/+ . In young mice, a slight decrease of Treg cells is noticed in the spleen, coupled with a slight decrease of activated CD4 in the peripheral blood. In the spleen of aged Kank1 -/- mice, the proportions of macrophages and Treg cells were decreased, while the central memory CD4 + T-cells were increased. Total protein analysis from BM cells of Kank1 -/- (n=3) compared to Kank1 +/+ mice (n=3) showed a significant reduction (FC >0.5, adj.p. <0.05) in proteins associated with cytoskeleton formation such as Myosin1 (MYH1), Tropomysin-1 (TDM1) and Annexin (ANXA2), and extracellular matrix components such as Collagena(CO1A1) and Matrix metalloproteinase9 (MMP9). We also detected a significant increase in expression of the DNA licensing factor minichromosome maintenance complex component 7 (MCM7), and the mitochondrial membrane associated transporter ATP binding Cassette Subfamily B Member 10 (ABCBA).

Summary/Conclusion: Dysregulation of the immune system has been widely recognized to play a role in the development of MDS. Based on our results, we propose a role for KANK1 in regulating the development of immune cells in the mouse through it invovlement in cytoskeleton regulation. In vivo infectious challenge experiments using our model will futher elucidate the importance of KANK1 in the development of MDS.

P724: THE BONE MARROW MESENCHYMAL CELL-DERIVED EXTRACELLULAR MATRIX IS SIGNIFICANTLY ALTERED IN PATIENTS WITH MELODYSPLASTIC SYNDROMES

A. Bains 1,* , L. Behrens Wu 2 , M. Richter 1 , V. Magno 3 , S. Rother 4 , M. Cross 1 , C. Werner 3 , M. Wobus 2 , U. Platzbecker 1

1 Medical Clinic and Policlinic I, Hematology and Cellular Therapy, University Hospital Leipzig, Leipzig; 2 Department of Medicine I, University Hospital Carl Gustav Carus; 3 Leibniz Institute of Polymer Research, Technische Universität, Dresden, Dresden; 4 Center for Molecular Signaling, Saarland University School of Medicine, Homburg (Saar), Germany

Background: Myelodysplastic syndromes (MDS) are characterised by ineffective hematopoiesis and peripheral cytopenia in one or more hematopoietic lineages. MDS is driven by genetic and epigenetic lesions in the hematopoietic clone itself, but also by changes in the bone marrow microenvironment. Here, the mesenchymal stromal cells (MSCs) regulate hematopoietic cell behaviour through cell-cell contact, paracrine signalling and metabolic support. The MSC-derived Extracellular matrix (ECM) plays a key role in coordinating both physical and biochemical interactions but the involvement of ECM in MDS has yet to be determined.

Aims: We aimed to characterise the relevant parameters of the mesenchymal cell-derived ECM generated by MSCs from healthy donor and MDS patient bonemarrow.

Methods: MSC from low risk (LR) and high risk (HR) MDS patients and age-matched healthy donors were cultured for 10 days in DMEM Glutamax medium supplemented with 10% FBS on glass slides pre-coated sequentially with poly-octadecene-alt-maleic anhydride (POMA) and human fibronectin. The confluent monolayers were washed in PBS, decellularized by washing with 20mM ammonia and were subject to DNase treatment. The resulting ECM were stored for up to 4 weeks at 4oC. Matrix structure was analysed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), glycosaminoglycan (GAG) content by staining with peanut and wheat germ agglutinin and collagen composition by immunostaining with antibodies to collagens 1 and 4. Total collagen content was quantified by Sircol assay. In parallel, the expression of matrix-related genes was measured by real time PCR of RNA extracted from the MSCs.

Results: In SEM, the ECM network generated by MSCs derived from both low risk (LR) and high risk (HR) MDS was thicker and of a higher density than that produced by MSCs from healthy donors. AFM analysis showed ECM of MDS-derived MSCs also had a significantly reduced rigidity. An increase in overall collagen content in MDS-derived ECM was detected by sircol assay and confirmed by immunostaining for the most prevalent collagen subtypes 1 and 4. Lectin staining revealed disease stage-specific differences in GAG composition: The levels of both GAGs carrying N-acetyl glucosamine and those carrying N-acetyl galactosamine sugars were increased in ECM from LR-MDS, while ECM from HR-MDS retained high levels of N-acetyl glucosamine but carried only low levels of N-acetyl galactosamine GAGs. Finally, the pattern of hyaluronan synthase (HAS) gene expression differed markedly between MSCs from healthy donor, LR- and HR-MDS. Specifically, HAS1 was strongly increased in LR-MDS and remained significantly above control levels in HR-MDS, while HAS3 was down-regulated specifically in HR-MDS. Consistent with this, increased levels of low molecular weight hyaluronic acid was observed in ECM from LR-MDS

Summary/Conclusion: We report both structural and compositional differences between the MSC-derived ECM from healthy donors, LR-MDS and HR-MDS. The thicker and softer structure of the MDS matrix may be both a consequence and a driver of changes in the MSC phenotype. Increase in the major collagens 1 and 4 are consistent with an increase in overall ECM, but it remains unclear whether the decreased rigidity of ECM in MDS is due to differences in protein cross-linking or in the levels of hydrated glycosaminoglycans. MDS-associated changes in hyaluronic acid expression suggest that MSC-derived HA may contribute to the inflammatory bone marrow state characteristics of LR-MDS.

P725: METABOLIC PROPERTIES OF MESENCHYMAL STROMAL CELLS IN MYELODYSPLASTIC SYNDROMES

E. Balaian 1,2 , K. Sockel 1,2 , M. Cross 3 , A. Funk 4 , M. Bornhäuser 1,5 , T. Chavakis 4,5 , M. Wobus 1,2,*

1 Medical Department 1, University Hospital Carl Gustav Carus Dresden, Dresden; 2 German Cancer Consortium (DKTK), Partner Site Dresden and German Cancer Research Center (DKFZ), Heidelberg; 3 Medical Department I - Hematology and Cell Therapy, University of Leipzig Medical Center, Leipzig; 4 Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus Dresden, Dresden; 5 National Center for Tumor Diseases, Partner Site Dresden and German Cancer Research Center (DKFZ), Heidelberg, Germany

Background: The crosstalk between the mutant hematopoietic stem and progenitor cells and the bone marrow microenvironment plays an important role in the pathogenesis of myelodysplastic syndromes (MDS). Mesenchymal stromal cells (MSC) represent one of the key cellular elements in this context. The functional properties of MDS-derived MSCs including impaired hematopoietic support, as well as increased senescence and defective osteogenic differentiation have been extensively studied. Additionally, the detrimental effect of high erythropoietin (Epo) concentration on MDS-MSCs has been demonstrated by our group. However, whether these deficiencies are associated with cellular metabolic changes has not been hitherto addressed.

Aims: We aimed to assess the basal metabolic characteristics in MDS-derived MSCs and the effect of Epo on their energetic profile in vitro .

Methods: MSC from bone marrow aspirates of patients with MDS (low risk n=3, MDS del5q n=1, chronic myelomonocytic leukemia n=2, high risk n=3) or age-adjusted healthy donors (n=3) were isolated by Ficoll density centrifugation and plastic adherent culture. Metabolic measurements were performed before and after incubation with Epo (50 IU/ml) for 2 days. Seahorse X96 Flux Analyzer (Seahorse Biosciences, Billerica, MA, USA) was used to analyse glucose metabolism by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Metabolite concentrations were measured in the culture supernatants using NMR spectroscopy.

Results: MSC from healthy donors demonstrated a higher OCR/ECAR ratio compared to MDS counterparts (6.19±4,71 vs 1.14±0.28, p=0.005), suggesting enhanced glycolysis engagement of MDS-MSCs. This was confirmed by increased levels of lactate in the culture medium from MDS-MSCs after 48 hours of cultivation (healthy: 3.78±0.28 mM vs MDS: 6.75±2.35 mM, p=0.049), whereas the concentrations of pyruvate, alanine and acetate did not differ. Treatment with Epo resulted in a significant increase of the OCR/ECAR ratio (from 1.14±0.28 to 3.25±1.75, p=0.003) in MDS-MSCs, whereas changes in healthy MSCs were not significant (from 6.19±4,71 to 3.81±1.86, p=0.46).

Summary/Conclusion: In contrast to healthy MSCs which rely mainly on mitochondrial respiration, MDS-MSCs preferentially utilize the glycolytic pathway under normoxic conditions in vitro . Epo treatment leads to increased mitochondrial respiration in MDS-MSCs, but not in healthy counterparts. Whether this effect has a causative link to the disturbed function of MDS-MSCs remains to be clarified.

P726: MYELOID-DERIVED SUPPRESSOR CELLS AS POTENTIAL TARGETS FOR IMMUNE THERAPY IN CCUS AND LOW RISK MDS

S. Bentivegna 1,* , M. O. Holmström 2 , M. H. Andersen 2 , K. Grønbæk 1,3

1 Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen; 2 National Center for Cancer Immune Therapy (CCIT-DK), Department of Oncology, Copenhagen University Hospital, Herlev; 3 Department of Hematology, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark

Background: Myeloid-derived suppressor cells (MDSC) are powerful immune suppressing cells that infiltrate the bone marrow (BM) microenvironment in myelodysplastic syndrome (MDS) and suppress both a potential immune response against the malignant HSPC and the normal hematopoiesis (Chen et al. , 2013). Different groups reported an expansion of MDSC in MDS, and an increased number of MDSC is associated with disease progression (Chen et al. , 2013; Kittang et al. , 2016). MDSC mediate their immunoregulatory effects releasing immunosuppressive cytokines, such as TGFβ, and enzymes such as indoleamine 2,3-dioxygenase (IDO) and arginase (ARG) (Mondanelli et al. , 2017). The presence of self-reactive T cells that specifically target immunosuppressive cells in both healthy donors and cancer patients has previously been described (Andersen, 2017). These cells, termed anti-regulatory T cells (anti-Tregs), recognize epitopes derived from immunosuppressive proteins such as IDO, ARG, which are expressed by MDSC (Andersen, 2018). We speculate that the stimulation of anti-Tregs cells using peptide-based vaccines can lead to the depletion of the immunosuppressive cells in the BM, and it will help to prevent disease progression in patients with clonal cytopenia of undetermined significance (CCUS) or MDS.

Aims: The aim of this work was to characterize the immune cell composition of blood and BM samples from CCUS/MDS patients and investigate the presence of circulating anti-Treg cells in blood samples and their reactivity towards immunogenic peptides.

Methods: Using flow cytometry we analyzed blood and BM samples from 20 patients (pts) with CCUS/MDS (10 CCUS and 10 low risk-MDS) and 6 elderly healthy controls (EHC). The presence of anti-Tregs was investigated in 7 pts, by ELISPOT assay on patient T cells that had been stimulated with epitopes derived from the immunosuppressive proteins ARG, IDO or TGFβ.

Results: Although there were no significant differences in the percentage of MDSC in BM samples between controls and pts, the proportion of IDO+ and ARG+ MDSC in pts was significantly higher compared to EHC. In addition, there was a significantly higher percentage of CTLA-4+ and PD-1+ T cells present in the BM samples from pts compared to EHC. Interestingly, in the ELISPOT assay, 4 out the 7 pts showed a significant response towards the ARG peptide, 2 showed a response towards IDO peptide and in 4 of the patients there was a significant response towards the TGFβ peptide. Overall, of the 7 pts analyzed, 6 harbored T cells specific to one or more of the immunosuppressive proteins ARG, IDO and TGFβ (figure 1).

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Summary/Conclusion: There is an increased proportion of activated MDSC in BM in pts with CCUS/MDS compared to EHC. As the immune checkpoint molecules are expressed after T cell activation, the increased percentage of CTLA-4+ and PD-1+ cells in pts BM suggests that there is a T cell immune response in the BM of pts with CCUS/MDS. The ELISPOTs showed that circulating anti-Tregs are present in CCUS/MDS patients. These results suggest, that the tumor specific immune response in patients with CCUS/MDS is present, albeit suppressed by (amongst others) MDSC. We speculate that therapeutic cancer vaccination targeting immunosuppressive cells such as MDSC by vaccination using e.g. IDO, ARG or TGFβ derived epitopes will incite an anti-regulatory immune response, which may break the local immune suppression in the BM and allow effector immune cells to exert killing of transformed cells.

P727: NK-CELL IMMUNOEDITING BY MESENCHYMAL STROMAL CELLS IN MDS

V. Bisio 1,2,* , B. Schell 1,2 , L.-P. Zhao 1,2 , E. Lereclus 1,2 , M. Boy 1,2 , A. Bonaud 1,2 , A. Caignard 1,2 , A. Toubert 1,2,3 , P. Fenaux 4 , M. Espéli 1,2 , K. Balabanian 1,2 , L. Adès 4 , N. Dulphy 1,2,3

1 Université de Paris, Institut de Recherche Saint Louis, EMiLy, Inserm U1160; 2 OPALE Carnot Institute, The Organization for Partnerships in Leukemia, Hôpital Saint-Louis; 3 Laboratoire d’Immunologie et d‘Histocompatibilité, Assistance Publique des Hôpitaux de Paris (AP-HP), Hôpital Saint-Louis; 4 Université de Paris, Department of Hematology, AP-HP, Paris, France

Background: Disorders of the immune system contribute to the pathophysiology and progression of myelodysplastic syndrome (MDS). Cytotoxic Natural Killer (NK) lymphocytes have been extensively studied for their anti-leukemic activity. However, whether the dysplastic bone marrow (BM) in MDS can directly imprint NK cells and hence alter immunosurveillance is still controversial. Extensive characterizations of MDS patients uncovered deep perturbations in the phenotype and function of cytotoxic lymphocyte subsets and of the stromal compartment, in particular mesenchymal stromal cells (MSC), linked with disease predisposition, initiation and progression. However, few studies have been undertaken to characterize the interactions established within the MDS immune microenvironment between immune and stromal cells.

Aims: Recent studies on MDS BM ecosystem suggest a possible symbiotic relationship between cancer, immune and stromal cells. Our project thus aims to underpin the influence of the pathological MSC from MDS BM over NK cells to create a leukemia-permissive microenvironment allowing disease development and progression.

Methods: We performed an extensive in vitro characterization of the NK and MSC interactions by co-culture system. Healthy donor (HD) NK cells were co-cultured either with MSC isolated from HD or MDS patient BM samples and were analyzed by high-parametric flow-cytometry, qRT-PCR, secretome and imaging assays.

Results: Analyses of supernatants from MSC/NK co-cultures unveiled altered secretion of soluble factors related to inflammation and BM homing such as IL6, CCL2 and CXCL10 in the pathologic condition compared to the healthy one. MDS MSC did not support efficiently NK-cell viability. Moreover, NK cells cultured with MDS MSC were less efficient at eliminating cancer cells compared to NK cells cultured with HD MSC. Finally, metabolic analyses uncovered an altered NK cell energetic profile dictated by the different MSC that could contribute to their poor survival and reduced cytolytic function.

Summary/Conclusion: Altogether, our results suggest that MSC from MDS BM edit NK cells to promote an immunosuppressive environment facilitating MDS escape and progression. Further analyses are ongoing to underpin the pivotal role of immune-microenvironment interactions in the treatment response. We expect this study to identify new biomarkers to forecast disease progression and open an avenue for new combination therapies targeting the tumour microenvironment.

P728: GENE EXPRESSION PROFILE REVELS T-LYMPHOCYTES ACTIVATION THROUGH CELL CYCLE PROGRESSION AND MITOCHONDRIAL METABOLISM REGULATION IN MYELODYSPLASTIC SYNDROMES WITH DEL(5Q) AFTER LENALIDOMIDE TREATMENT

M. del Rey 1,* , L. A. Corchete 2 , T. González 1 , F. López-Cadenas 3 , E. Lumbreras 1 , S. M. Toribio 1 , B. Xicoy 4 , J. Sánchez-García 5 , T. Bernal 6 , G. F. Sanz 7 , P. Fenaux 8 , J. M. Hernández-Rivas 9 , M. Diez-Campelo 10

1 Cancer Research Center - University of Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL); 2 Instituto de Investigación Biomédica de Salamanca (IBSAL); Hematology Department, Hospital Clínico Universitario de Salamanca; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC); 3 Hematology Department, Hospital Clínico Universitario de Salamanca, Salamanca; 4 Hematology Department, Institut Català d’Oncologia-Hospital Germans Trias i Pujol; Josep Carreras Leukemia Research Institute, Universitat Autònoma de Barcelona, Badalona; 5 Hematology Department, Hospital Reina Sofía, Córdoba; 6 Hematology Department, Hospital Universitario Central de Asturias, Oviedo; 7 Hematology Department, Hospital Universitario y Politécnico La Fe, Valencia, Spain; 8 Hôpital Saint-Louis, Paris, France; 9 Hematology Department, Hospital Clínico Universitario de Salamanca; Cancer Research Center - University of Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL); 10 Hematology Department, Hospital Clínico Universitario de Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain

Background: Lenalidomide is a potent drug whose mechanisms of action in del(5q) clone from Myelodysplastic Syndromes (MDS) have been extensively described. Lenalidomide also induces immune modulation independent of del(5q) and has multiple effects on T-cell signaling; however, the molecular and cellular determinants that mediate this immunomodulatory function remain elusive.

Aims: To better understand the immunomodulatory activity of lenalidomide in MDS patients with del(5q) by in vivo sequential gene expression profile studies of T lymphocytes in response to the treatment.

Methods: Our study was conducted in patients included in the Sintra-REV Clinical Trial (phase III). Sequential study was carried out in 26 samples from 13 paired patients. Seven out 13 were treated with lenalidomide and achieved a major erythroid and cytogenetic response. Peripheral blood (PB) samples were collected before and one month after treatment in treated patients and at the same time points for non-treated patients. CD3+ cells were collected from PB samples and total RNA was isolated. SureSelect Strand Specific RNA library (Agilent Technologies) was applied to study changes in RNA levels. Raw reads were aligned against the Human genome GRCh37 using the STAR aligner. Counts were assigned to Ensembl gene IDs through HTseq using its UNION version. Differential gene expression was determined with DESeq2, considering as statistically significant those genes with FDR < 0.05. Pathway over-representation analysis (ORA) was conducted in the Webgestalt suite.

Results: 332 genes were differentially expressed in CD3+ lymphocytes one month after lenalidomide treatment in our cohort of patients; of note, none of them were observed after one month in non-treated patients. The ORA revealed significant differences in the gene expression profile of sixteen cytokines (e.g. IL10, TNFSF10, IFNG, IL6 and MBP6 ). These genes could contribute to activation of an antileukemic immune response, help to correct the anemia as well as attenuate inflammation signaling in MDS patients with del(5q). The ORA also showed an enrichment of genes involved in cell cycle (35 genes). The most represented up-regulated genes related to this pathway were: cyclines ( CCNB1, CCNB2, CDK1 ), centromere genes ( CENPE, CENPM, CENPU ), kinesin family members ( KIF18A, KIF23, KIF2C ) and genes involved in cell division ( CDC6, CDC7, CDC25A ). It has been described that lenalidomide inhibits CDC25A selectively in the del(5q) clone resulting in G2/M arrest and apoptosis. By contrast, our study showed that this gene was upregulated in T-lymphocytes promoting cell cycle and proliferation of these cells. In addition, 456 genes were differentially expressed between non-treated and lenalidomide treated samples; 30 of them were related to mitochondrial metabolism (e.g HIBCH, ECHS1, DECR1, ACSM1, HADH, COQ3, COQ5, SDHAF3, SDHAF4 ) and mitochondrial translation (e.g. MRPL15, MRPL24, MRPL48, MRPL51, MRPS33 ). Thereby, T lymphocytes adaptive responses as well as sustained killing by cytotoxic T cells could be mediated by mitochondria in treated patients.

Summary/Conclusion: To our knowledge, this is the first report describing RNA expression profiles in PB CD3+ lymphocytes collected from lenalidomide-treated del(5q) patients, contributing to overall understanding of lenalidomide action. A better understanding of the molecular targets that mediate the immunomodulatory activity is needed to harness the full potential of this agent. Functional experiments trying to test the new biological mechanisms described above are currently conducted.

P729: DEREGULATION OF NONCODING RNAS DERIVED FROM KDM GENES IS ASSOCIATED WITH AZACITIDINE RESPONSE IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES AND ACUTE MYELOID LEUKEMIA

M. Dostalova Merkerova 1,* , A. Hrustincova 1 , I. Trsova 1 , Z. Krejcik 1 , D. Kundrat 1 , J. Klema 2 , K. Szikszai 1 , A. V. Le 2 , J. Cermak 3 , A. Jonasova 4 , M. Belickova 1

1 Department of Genomics, Institute of Hematology and Blood Transfusion; 2 Department of Computer Sciences, Czech Technical University; 3 Laboratory of Anemias, Institute of Hematology and Blood Transfusion; 4 First Department of Medicine, General University Hospital, Prague, Czechia

Background: Prediction of response to azacitidine (AZA) treatment is an important challenge in the management of higher-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To date, no clinical, cytogenetic, or molecular markers of AZA treatment outcome have been validated to support clinical decisions. Moreover, little is known about how AZA efficiency can be affected by various noncoding RNAs (ncRNAs), such as long ncRNAs (lncRNAs) and circular RNAs (circRNAs). KDM genes encode demethylases of histone lysine residues (e.g., KDM1 mediates demethylation of H3K4 and H3K9, and KDM4 acts on H3K9, H3K36, and H1K26). KDMs are often differentially expressed in leukemia and they cooperate with transcription factors to activate or repress gene expression.

Aims: Our aims were to provide biological insight into the contribution of ncRNAs to AZA mechanisms of action and to propose novel disease biomarkers that would be able to predict future response to AZA treatment. Based on our results, we particularly focused on expression and function of KDM -related ncRNAs.

Methods: Whole transcriptome RNA sequencing (RNA-seq) was performed in CD34+ bone marrow cells from 26 patients with MDS or AML with myelodysplasia-related changes (AML-MRC) before AZA treatment (11 responders and 16 nonresponders), and 9 healthy controls. Expression of protein coding genes (PCGs), lncRNAs and circRNAs was bioinformatically processed.

Results: Overall, differential expression analysis between AZA responders and nonresponders identified significant deregulation of 202 PCGs, 34 lncRNAs, and 21 circRNAs. Surprisingly, machine learning data showed that predictive potential of ncRNAs was stronger than that of PCGs. Within the data, deregulation of several KDM -related ncRNAs was observed, e.g., CTC-482H14.5 (antisense RNA to KDM4B gene, downregulated in AZA responders), hsa_circ_0003889 (circRNA processed from KDM1A , downregulated in AZA responders), and hsa_circ_0001580 (circRNA processed from KDM1B , upregulated in AZA responders). By contrast with these noncoding transcripts, the levels of corresponding PCGs (namely KDM1A/1B/4B ) were not altered between AZA responders and nonresponders, suggesting that the deregulated ncRNAs play specific roles independent of their host genes. Further, the best predictor genes were defined by machine learning using RNA-seq data and pathway analysis linked these markers to cellular processes related to MDS/AML. Interestingly, CTC-482H14.5 was identified as one of the strongest predictors of AZA treatment response. Expression of this KDM -related ncRNA was coregulated with expression of DNMT1, EZH2, and multiple MCM genes, associating this transcript with epigenetic regulation and recombinational repair pathways.

Summary/Conclusion: Several ncRNAs processed from KDM genes seem to be closely related to the responsiveness of MDS/AML-MRC patients to AZA treatment. In this study, novel functions were predicted for these ncRNAs, pointing to possible mechanisms by which their deregulation could affect AZA treatment response.

Acknowledgements: Supported by GA CR (No. 20-19162S) and MH CZ-DRO (UHKT, 00023736).

P730: GENETIC LANDSCAPE OF SOMATIC MYELOID MUTATIONS IN THE PRESENCE OF RARE TERT VARIANTS AND THEIR RELATION WITH MYELOID NEOPLASIA

A. Ferrer 1,2,* , A. Mangaonkar 1 , T. Lasho 1 , M. Wylam 3 , C. Finke 1 , J. Fernandez 1 , R. He 4 , D. Viswanatha 4 , M. Patnaik 1,2

1 Division of Hematology; 2 Center for Individualized Medicine; 3 Division of Pulmonary and Critical Care Medicine; 4 Department of Hematopathology, Mayo Clinic, Rochester, United States of America

Background: Genetic variants in TERT , the gene that encodes telomerase reverse transcriptase, can result in accelerated telomere attrition. Impaired telomere maintenance is implicated in the pathogenesis of myelodysplastic syndromes (MDS), with mechanisms remaining to be elucidated.

Aims: To explore the clinical and genetic associations of TERT variants in an unselected cohort of patients screened with a myeloid mutation next generation sequencing panel due to clinical suspicion of a myeloid neoplasia.

Methods: Using a research protocol approved by the Mayo Clinic Institutional Review Board, we performed a retrospective chart review to identify a cohort of unselected individuals within all historical data at our institution that had undergone somatic mutational analysis for suspected myeloid neoplasia using our insitutional amplicon based next generation sequencing panel (42 genes, including exons 2 to 16 of TERT , with a read depth of at least 250X). TERT variants were classified into those with CADD score >20 and <20 in line with previous literature to identify variants within the top 1% according to their predicted deleteriousness. Additionally, we identified within our cohort individuals that received a diagnosis of MDS, clinical and survival information.

Results: We identified 55 different TERT variants (46 missense, 3 in canonical splice sites, 4 synonymous and 2 small inframe deletions) in 148 individuals tested from April of 2016 to November of 2021 (Fig. 1A&B). The five most recurrent variants were c.1234C>T (48 patients), c.1323_1325del (29 patients), c.3164C>T (5 patients), c.604G>A (4 patients) and c.969G>A (4 patients), all with CADD<20. None of these patients were diagnosed with a bona fide telomere biology disorder and no clinical information about telomere length status was available. All variants were considered germline since their allele frequency was near 50%, although no germline confirmation was performed. All variants were clinically classified as uncertain significance according to the American College of Medical Genetics guidelines. Of the 148 total patients, 17 (12%) carried TERT variants with CADD>20 that were distributed thorough all the gene domains with no clustering or presence of hotspots (Fig.1A&B). 59% (10 patients) of CADD>20 variants carriers were diagnosed with MDS: 5 patients with <5% bone marrow blasts (4 with normal karyotype) and 5 patients with 5-19% blasts, all with complex karyotype. This percentage was significantly higher compared to CADD<20 variant carriers (34%, 49 patients, p=0.048 by Chi-square test, Fig. 1C). However, no major differences were found between the two groups in terms of age at diagnosis (median 70 vs 73 years old, p = 0.2305) or AML-free survival (median 10 vs 15.5 months since diagnosis, p=0.4674). Similarly, the percentage of patients presenting additional somatic variants in other myeloid-related genes was similar between CADD<20 and CADD>20 TERT variant carriers (60% vs 64%). The two most frequenly mutated genes in CADD>20 carriers were TP53 (22%), TET2 (18%), RUNX1 (9%), U2AF1 (9%) and BCOR (9%) while in the CADD<20 carriers were TET2 (14%), ASXL1 (11%), SRSF2 (9%), TP53 (7%) and JAK2 (6%). Our data showed that TP53 pathogenic variants were significantly enriched in CADD>20 TERT variant carriers compared to CADD<20 carriers. (22.7% vs 7.4%, p=0.0169, Fig. 1D).

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Summary/Conclusion: Carriers of TERT variants with CADD score of >20 were more likely to be associated with a diagnosis of MDS and with somatic TP53 mutations in comparison to those with CADD score <20, findings that we plan to validate functionally and prospectively.

P731: INTERPLAY BETWEEN INFLAMMATION AND EPIGENETICS IN TUMOR REPROGRAMMING OF MESENCHYMAL STROMAL CELLS (MSCS) IN MYELODYSPLASTIC SYNDROMES (MDS)

C. Giallongo 1,* , I. Dulcamare 2 , D. Tibullo 3 , D. Pieragostino 4 , M. C. Cufaro 4 , M. A. Amorini 5 , G. Lazzarino 6 , A. Romano 7 , G. Scandura 2 , T. Zuppelli 2 , M. Di Rosa 8 , A. Duminuco 2 , G. Broggi 9 , R. Caltabiano 9 , R. Floresti 10 , R. Motterlini 11 , F. Di Raimondo 7 , G. A. Palumbo 1

1 Department of Medical, Surgical Sciences and Advanced Technologies G.F. Ingrassia; 2 Division of Hematology, AOU Policlinico; 3 Department of Biomedical and Biotechnological Sciences, Section of Biochemistry, University of Catania, Catania; 4 Department of Innovative Technologies in Medicine and Dentistry, ‘‘G. d’Annunzio’’, University of Chieti-Pescara, Chieti; 5 Department of Biomedical and Biotechnological Sciences, Section of Biochemistr, University of Catania, Catania; 6 UniCamillus - Saint Camillus International University of Health Sciences, University of Rome, Rome; 7 Department of General Surgery and Medical-Surgical Specialties; 8 7Department of Biomedical and Biotechnological Sciences, Human, Histology and Movement Science Section; 9 Department of Medical and Surgical Sciences and Advanced Technologies “G.F. Ingrassia”, Anatomic Pathology, University of Catania, Catania, Italy; 10 University Paris-Est Créteil, INSERM, IMRB, University Paris-Est; 11 University Paris-Est Créteil, INSERM, IMRB, University Paris Est, Paris, France

Background: The alteration of hematopoiesis in MDS patients is deeply associated with microenvironment alterations, in particular in MSCs. Stromal cells are epigenetically reprogrammed to function in cooperation with leukemic cells and propagate the disease as a “tumor unit”. Notably, dysfunctions of MDS-MSCs persist following expansion ex vivo suggesting a hereditable epigenetic dysregulation which endures despite removal of disease-associated microenvironment factors.

Aims: Here, we investigated the role of histone H2A variant MacroH2A1 (mH2A1) in promoting pro-tumorigenic inflammation and metabolic reprogramming of MSCs.

Methods: MSCs were collected from BM of MDS patients (n=18) and matched healthy controls (n=8; HC). HS-5 cells were used as model of healthy MSCs. Real time, western blot, immunohistochemistry and immunofluorescence were used. Proteomic and metabolic analysis were performed.

Results: MDS patients had significant higher signature of mH2A1 in bone marrow slides compared to age-matched control. Investigating mH2A1 expression in stromal cells, MDS-MSCs had increased levels of expression in respect of HC-MSCs. In accordance with the aberrant inflammation described in MDS microenvironment, MDS-MSCs showed higher levels of the sum of NO2 + and NO3 + associated to increased GSH and NADP + /NAPH. TLR4 also was upregulated and positively correlated to mH2A1 expression. To better investigate the relationship between mH2A1 and TLR4 in MDS-MSCs, we induced mH2A1 overexpression in HS-5 cells (mH2A1-OE) by mH2A1-CT-MYC plasmid. Our data showed that mH2A1-OE upregulated TLR4 and increased NFkB nuclear translocation compared to cells transfected with empty vector (CTL). Proteomic analysis confirmed upregulation of intracellular serine protease inhibitors (SerpinB2, B8, B6) strongly induced during inflammation and important for the maintenance of TLR4 activation. Moreover, proteomic approach identified upregulation of several proteins associated to hypermethylation of DNA and histones in mH2A1-OE. In particular, S-adenosylhomocysteine hydrolase which regulates the concentration of S-adenosylhomocysteine (SAH), a strong inhibitor of methyltransferase reactions and of the methyl donor S-adenosyl-methionine (SAM), resulted overexpressed. HPLC analysis showed higher SAM/SAH ratio associated to a significant reduction of SAH in mH2A1-OE, confirming the increase of the methylation index. In addition, the higher levels of CBX3, a suppressive epigenetic mark which recognizes histone H3K9me3, contributes to the maintenance of the heterochromatin. The higher levels of H3K9me3 in mh2A1-OE were confirmed by western blot analysis. Overexpression of mH2A1 also induced metabolic reprogramming with the acquisition of a more glycolytic metabolism characterized by decreased levels of NAD + /NADH, upregulation of LDHA and MCT4. As LDHA could translocate into the nucleus, we evaluated if it used a noncanonical enzymatic activity in mh2A1-OE. Data showed increased nuclear localization of LDHA producing histone H3K79 hypermethylation. Nuclear LDHA was also observed in MDS-MSC. Finally, we treated ex-vivo HC- and MDS-MSCs with azacytidine founding a significant reduction both of mH2A1 and TLR4 associated to lower levels of nuclear NFKB.

Summary/Conclusion: Our data provide a key role of mH2A1in driving the crosstalk between inflammation, metabolism and epigenetic signatures in MDS-MSC. Future experiments will define the contribution of mH2A to ineffective hematopoiesis and leukemic evolution.

P732: TH17/TREG IMBALANCE IN LOW RISK MDS PATIENTS

M. Boada 1 , N. Trias 2 , A. Brugnini 2 , C. Guillermo 1 , S. Grille 1,2,*

1 Catedra de Hematologia; 2 Laboratorio de Citometria y Biologia Molecular. Depto. Básico de Medicina, Hospital de Clinicas, Montevideo, Uruguay

Background: Myelodysplastic Syndromes (MDS) are a heterogeneous group of clonal haematopoietic stem/progenitor cells (HSPC) disorders characterized by ineffective haematopoiesis, peripheral cytopenia, and risk of acute myeloid leukemia (AML). MDS pathogenesis is complex and depends on the interaction between HSPC and microenvironment. Many studies have highlighted the role of different immune cells in immune dysregulation leading to pathogenesis of MDS and progression of disease to AML. Immune response differs between risk groups favouring a pro-inflammatory profile in low risk, whereas in high-risk patients there is a trend towards immunosuppressive environment. Regulatory T cells (TRegs) and T helper 17 cells (Th17) are CD4 T cell functional subsets and their balance (TH17/TReg) is essential for maintaining immune haemostasis

Aims: To study T cell subsets and cytokines plasma levels in low risk MDS patients and healthy controls.

Methods: This is a case control study conducted in Hospital de Clinicas Montevideo, Uruguay. Patient inclusion criteria were Low Risk MDS (R-IPSS <4.5) and control group were healthy blood donors. We assessed peripheral blood T cell subsets by multiparametric flow cytometry using the following monoclonal antibodies: CD45, CD3, CD4, CD8, CXCR3, CCR6, CCR4, CD25 and CD127. Subpopulations were defined as reported by Maecker et al (1). Cytokine’s plasma levels were determined by Cytometric Bead Array (BD). We studied pro-inflammatory, TH1, TH2 and TH17 cytokines (IL-2, IL-4, IL-6, IL-10, INFg, TNFa, and IL-17). The protocol was authorized by the Ethics Committee of the institution and all subjects signed an informed consent.

Results: We included 24 MDS patients and 19 controls with a median age of 68.5 (47-78) and 48 (32-55) years respectively. Male:Female ratio was 1.3:1 in controls and 0.9:1 in patients (p˃0.05). According to R-IPSS we included 13 (30.2%) Very Low Risk, 11 (25.2%) Low risk and 19 (44.2%) Intermediate patients. MDS patients showed lower proportion of Treg cells (p<0.001) and higher TH17/Treg ratio (p<0.001) than controls (Figure 1). There were no statistical differences in total T CD4+ cells and TH1/TH2 ratio. IL-2, IL-6 and IFN-γ levels were higher in patients than controls. Median levels of IL-2 were 0.2 (0.1-4.5) pg/mL in patients and 0.1 (0.0-2.6) pg/mL in controls, p=0.002. Median levels of IL-6 were 9.4 (0.5-10.5) pg/mL in patients and 0.5 (0.2-2.9) pg/mL in controls, p=0.001. Median levels of IFN-γ were 2.2 (1.1-3.3) pg/mL in patients and 0.1 (0.0-2.6) pg/mL in controls, p=0.003. There was no statistical difference in IL-4, IL-10, TNFa, and IL-17A between groups.

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Summary/Conclusion: Our results showed that TH17/Treg balance is impaired, revealing a predominant proinflammatory state in in MDS Low risk patients. This occurs in concordance with elevated pro-inflammatory cytokines (IL-2, IL-6 and IFN-γ). These findings agree with previous reports that evidenced TH17/Treg ratio was higher in low risk MDS patients when compared with high risk. However, those studies highlighted the importance of TReg expansion and its prognostic impact in high risk. Evidence comparing TH17/TReg balance between low risk MDS patients and controls is scant. We believe that these results support the importance of a pro-inflammatory response in low-risk patients.

1. Maecker HT et al. Standardizing immunophenotyping for the Human Immunology Project. Nature Reviews Immunology . 2012;12(3):191-200. doi:10.1038/nri3158

P733: DHX9 HELICASE DYSFUNCTION CONTRIBUTES TO DISEASE PROGRESSION IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES

N.-F. Huang 1 , C.-K. Chang 1 , Q. He 1 , L.-Y. Wu 1 , Z. Zhang 1 , X. Li 1 , F. Xu 1,*

1 Department of Hematology, ShangHai Jiaotong University Affiliated Sixth People’s Hospital, ShangHai, China

Background: DHX9 is a member of the DEAH (Asp-Glu-Ala-His) helicase family, and participates in regulating DNA replication and RNA processing. DHX9 dysfunction promotes tumorigenesis in several solid cancers. However, the role of DHX9 in myelodysplastic syndromes (MDS) is still unknown.

Aims: to investigate the role of DHX9 in the pathogenesis of myelodysplastic syndromes.

Methods: DHX9 expression was analyzed in 120 MDS patients and 42 non-MDS benign controls by quantitative PCR. Clinical significance of changes in DHX9 expression was investigated in disease progression and survival. Lentivirus-mediated DHX9 knockdown experiments were performed to investigate its biological function. The mechanistic involvement of DHX9 in MDS development was explored in cell functional assays, gene microarray and pharmacological intervention. Considering the importance of DHX9 in regulation of R-loop, we also investigated the role of DHX9 and R-loop in MDS.

Results: We found that overexpression of DHX9 is frequent in MDS patients, and associated with poor survival and high risk of AML transformation. Cell functional assays showed that DHX9 is essential for the maintenance of malignant proliferation of leukemia cells, and knockdown of DHX9 results in increased cell apoptosis and growth inhibition. Gene expression profile and bioinformatics analysis reveal that DHX9 may be involved in PI3K-AKT signaling pathways. Validating experiments show that DHX9 is indispensable for the activation of PI3K-AKT signaling as knockdown of DHX9 inactivates the PI3K-AKT signaling, reduces the expression of anti-apoptosis genes such as CCND2 , MYC and BCL2. In addition, we found that DHX9 knockdown leads to R-loop accumulation in MDS cells, and induces R-Loop-dependent DNA damage through ATR-Chk1 activation. It suggests that overexpressed DHX9 could counteract cell growth defect caused by R-Loop which is quite common in MDS patients especially in those with splicing genes mutations.

Summary/Conclusion: Our data suggest that DHX9 contributes to disease progression by activation of PI3K-AKT signaling and correcting R-loop-mediated growth defect in MDS, and may serve as a novel prognostic marker for AML transformation and therapeutic target in MDS.

P734: DOWN-REGULATION OF RING1 AND YY1 BINDING (RYBP) LEADS TO EPIGENETIC OVEREXPRESSION OF ANTI-APOPTOTIC PROTEIN BCL2 IN MYELODYSPLASTIC SYNDROMES

F. Xu 1,* , X. Li 1 , Q. He 1 , L.-Y. Wu 1 , Z. Zhang 1 , C.-K. Chang 1

1 Department of Hematology, ShangHai Jiaotong University Affiliated Sixth People’s Hospital, ShangHai, China

Background: Excessive activation of oncogenes plays a crucial role in apoptosis resistance and disease progression to leukaemia in myelodysplastic syndromes (MDS). RYBP is a member of BCOR complex for epigenetic transcription repression, and also considered as an apoptosis-associated protein. However, the role of RYBP is still unclear in the myelodysplastic syndromes.

Aims: To investigate the role of RING1 and YY1 binding (RYBP) in the myelodysplastic syndromes.

Methods: The expression of RYBP mRNA and its clinical significance was analyzed in MDS patients. Lentivirus-mediated RYBP knockdown and overexpression were performed to investigate its biological function. The mechanism of RYBP in MDS development was investigated by cell functional assays, gene microarray and chromatin immunoprecipitation.

Results: In this study, we found that reduced RYBP expression is observed in MDS patients and associated with high AML transformation. Overexpression of RYBP inhibits cell proliferation, and results in an increased cell apoptosis along with p53 activation in myeloid cell lines. However, knockdown of RYBP promotes cell proliferation, induces apoptosis resistance to etoposide and decitabine, and simultaneously reduces H2AK119ub1 level. Gene expression profile analysis identifies BCL2 as a possible target of RYBP. Further functional analysis shows that depletion of RYBP leads to transcription activation of BCL2 through removal of H2AK119ub1 at the promoter region of BCL2 gene. BCL-2 specific inhibitor ABT-199 significantly induces cell apoptosis in RYBP-depleted cells, and restores the apoptosis sensitivity to etoposide and decitabine.

Summary/Conclusion: Reduced RYBP expression is frequent in MDS patients and associated with high AML transformation. RYBP inhibition leads to epigenetic overexpression of anti-apoptotic protein BCL2 in MDS, which contributes to apoptotic resistance of clonal cells and disease progression. BCL2 inhibitor may be considered as a candidate agent in advanced MDS.

P735: THE ROLE OF SF3B1 MUTATIONS IN MYELODYSPLASTIC SYNDROMES

S. Huber 1,* , S. Hutter 1 , M. Meggendorfer 1 , G. Hoermann 1 , W. Walter 1 , C. Baer 1 , W. Kern 1 , T. Haferlach 1 , C. Haferlach 1

1 MLL Münchner Leukämie Labor GmbH, München, Germany

Background: SF3B1 is one of the most commonly mutated genes in patients with myelodyplastic syndromes (MDS), associated with ring sideroblasts (RS) and an indolent disease course. Malcovati et al. proposed MDS with mutated SF3B1 fulfilling certain criteria as distinct MDS entity (Malcovati et al., Blood, 2020).

Aims: Study the SF3B1 mutation in MDS with respect to the genomic landscape, AML transformation rate and clinical outcome.

Methods: We analyzed 734 patients diagnosed with MDS based on cytomorphology, cytogenetics and molecular genetics and classified according to WHO classification 2017. Amplification-free WGS was performed with a median coverage of 103x (Illumina, San Diego, CA). Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7.

Results: SF3B1 mutations were identified in 231 of 734 (31%) MDS patients and were mainly found in MDS-RS (171/200; 86%; MDS-RS-SLD: 43/51, 84%; MDS-RS-MLD: 128/149; 86%). In addition, 13% (37/300) of MDS with excess blasts (MDS-EB1/2) and 20% of MDS with isolated del(5q) harbored SF3B1 mutations. In the total cohort SF3B1 mutations were associated with better outcome (median OS: 79 vs. 53 month; p<0.0001). Within the different MDS entities SF3B1 mutations were favorable in MDS-RS-SLD (median OS: 106 vs. 25 month; p=0.009), MDS-RS-MLD (median OS: 82 vs. 64 month; p=0.049) and MDS-EB-2 (median OS: 129 vs. 25 month; p=0.011), but were associated with shorter survival in MDS with isolated del(5q) (median OS: 69 vs. 79 month; p=0.044). 144/231 (62%) SF3B1 mutated samples fulfilled the criteria proposed by Malcovati et al. ( SF3B1 ent). These cases were associated with longer survival compared to SF3B1 mutated samples not falling into the proposed SF3B1 entity ( SF3B1 nent). Within SF3B1 ent 47% (67/144) did not harbor any additional mutation resulting in an average of 1.8 mutations (including SF3B1 ) in this group. SF3B1 nent patients showed on average more mutations (MDS with isolated del(5q): 1.9; MDS-EB: 2.7; MDS-RS: 3.1). The most frequent additional mutations in all SF3B1 mutated patients were TET2 (29%), DNMT3A (16%) and ASXL1 (9%). Regarding cytogenetic abnormalities, 69/231 (30%) SF3B1 mutated samples showed aberrant karyotypes ( SF3B1 ent: 27/144, 19%; SF3B1 nent: 42/87, 48%). Of SF3B1 mutated patients 7% (15/231) progressed to AML compared to 15% (75/503) of SF3B1 wildtype patients (median follow-up: 112 months). Notably, 80% of SF3B1 nent showed AML transformations in comparison to 20% of SF3B1 ent (median follow-up: 122 and 112 months). Within the 15 SF3B1 mutated patients progressing to AML 47% showed additional RUNX1 and 27% DNMT3A or TET2 mutations at MDS diagnosis. In AML-transforming patients with a low SF3B1 variant allelic frequency (VAF, n=2) additional spliceosome mutations ( SRSF2 or ZRSF2 ) were identified at the time of diagnosis. Two other SF3B1 mutated patients showing disease progression to AML harbored MECOM rearrangements when MDS was diagnosed. During progression one patient gained a MECOM rearrangement and two patients other chromosomal aberrations present in the AML state.

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Summary/Conclusion: SF3B1 mutations occur in several MDS entities, are associated with good clinical outcome and mainly co-occurred with TET2 mutations. Patients showed a better prognosis (longer OS, lower AML transformation rate) when categorized into the proposed SF3B1 entity. AML transformation of SF3B1 mutated MDS patients is determined by the entire genomic landscape indicating RUNX1 as potential driver.

P736: BCOR MUTATION REGULATES MYELODYSPLASTIC SYNDROMES PROGRESSION THROUGH REPRESSING AUTOPHAGY FLUX MEDIATED BY HDAC6 ACTIVATION

C. Jianan 1 , Y. dongqin 2 , J. jiacheng 1 , W. LINGYUN 1,*

1 xueye, shanghai no.6 hospital; 2 xueye, shanghai no.8 hospital, shanghai, China

Background: Past studies have revealed that mutations in bcor are involved in various human cancers including MDS and AML. Autophagy plays a critical role in cancer progression. However, the role of bcor mutations in autophagy and MDS progression remains subject to debate.

Aims: This study investigated the role of bcor mutations in autophagy and MDS progression.

Methods: Cell lines with bcor p483L stable overexpression were established with a recombinant lentiviral vector. The effects of bcor mutations on cell proliferation, cell cycle progression, and cell apoptosis were detected by CCK8 assay, colony formation assay, and flow cytometry. We detected changes in autophagy levels by Western blot (WB), quantitative polymerase chain reaction (qPCR), and most importantly, transmission electron microscopy. The function of histone deacetylase 6 (HDAC6) was inhibited by Tubasatin A （TBA）, and We detected the consequent changes in the functional phenotype of the cells by WB and qPCR. The interactions between related proteins were analyzed by a co-immunoprecipitation assay. To test cell lines for mitochondrial dysfunction, bcor p483L mutant cells, negative control cells, and wild-type cells were double-stained with MitoTracker Green and MitoTracker Red, or single-stained with MitoSOX followed by fluorescence-activated cell sorting (FACS) analysis. The changes in pyroptosis level caused by bcor p483L mutation were detected by WB and qPCR.

Results: The cell proliferative ability of bcor p483L mutant cells was repressed compared to the negative control cells. Besides, bcor p483L mutation could promote cell apoptosis and block the cell cycle progression to the G2 phase. Notably, bcor p483L mutation repressed autophagic flux in cultured cells, as corroborated by decreased expression of autophagy marker genes (e.g., ATG7, ATG10) and reduced numbers of autophagosomes or autolysosomes, which were accompanied by upregulation of HDAC6, downregulation of FoxO1 and ac-FOXO1. HDAC6 was required for inhibiting the deacetylation of FoxO1. Enhanced HDAC6 expression and repression of FOXO1 were required for bcor p483L -mutation-deduced autophagy. Depression of HDAC6 by TBA was sufficient for increasing the expression of FOXO1, ac-FOXO1, and inducing autophagic flux. Notably, we examined the direct interaction between HDAC6 and FOXO1, FOXO1 and ATG7. Bcor p483L mutated cells accumulated dysfunctional, non-respiring mitochondria and increased mitochondrial ROS production compared to negative control cells. And we found upward adjustment of pyroptosis level and DNA damage.

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Summary/Conclusion: Bcor p.P483L mutation could disrupt BCOR protein structure and function, therefore reducing the transcriptional repressive effect of BCOR on HDAC6. HDAC6 was upregulated and decreases the acetylation of FOXO1, further repressing autophagy. We further verified the protein: protein interaction between HDAC6 and FOXO1, FOXO1 and ATG7. Defects in the clearance of damaged mitochondria by bcor p483L -mutation-regulated autophagy contribute to the accumulation of cellular reactive oxygen species（ROS）, thus promoting cell apoptosis and repressing cell proliferation. The escalation in pyroptosis levels was found after bcor mutations and may be partly due to increased ROS levels. We also found increased gene damage and increased likelihood of gene mutations in bcor p483L mutant cells, which would promote MDS transformation into AML. These findings indicate a completely novel BCOR-HDAC6-FOXO1-ATG7 axis in the regulation of autophagy and MDS progression, providing potential possibilities for MDS treatment.

P737: FUNCTIONAL ROLES OF DDX41 MUTATIONS IN THE DEVELOPMENT OF MYELOID MALIGNANCIES

A. Kon 1,* , M. Nakagawa 1 , K. Kataoka 2 , H. Makishima 1 , M. Nakayama 3 , H. Koseki 4 , Y. Nannya 1 , S. Ogawa 1

1 Dept. Pathology and Tumor Biology, Kyoto University, Kyoto; 2 Division of Molecular Oncology, National Cancer Center Research Institute, Tokyo; 3 Department of Technology Development, Kazusa DNA Research Institute, Chiba; 4 Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

Background: DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~3-7 % of sporadic cases with MDS/AML, in which a germline protein-truncating allele is compounded by a somatic missense mutation affecting the helicase domain (p.R525H) in the remaining allele in typical cases. However, little is known about the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms.

Aims: This study aimed to clarify the molecular pathogenesis of DDX41 mutated myeloid neoplasms.

Methods: We generated mice models carrying conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles in various combinations. By crossing these mice with Rosa26-CreERT2 transgenic mice, we obtained heterozygous Ddx41 KO ( Ddx41 +/- ), homozygous Ddx41 KO ( Ddx41 -/- ), heterozygous Ddx41 R525H mutation ( Ddx41 R525H/+ ), and Ddx41 R525H/KO ( Ddx41 R525H/- ) mice, in which expression of the mutant allele was induced by tamoxifen administration. We then investigated the effect of these alleles on hematopoietic cells.

Results: First, we assessed cell intrinsic effects of these Ddx41 alleles, in noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were reconstituted with bone marrow (BM) from Ddx41 -/- or Ddx41 R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF). By contrast, the mice transplanted with BM from Ddx41 +/- mice showed significantly reduced WBC counts and myeloid skewing in long-term observation.

We also assessed the reconstitution capacity of BM cells from different Ddx41 mutant mice using peripheral blood (PB) chimerism after competitive transplantation with Ddx41 +/+ cells. The PB donor chimerism of Ddx41 -/- and Ddx41 R525H/- mice-derived cells was markedly reduced compared to that of Ddx41 +/+ cells. In contrast, Ddx41 +/- and Ddx41 R525H/+ mice-derived cells showed a slightly reduced PB chimerism compared to Ddx41 +/+ mice-derived cells. Of interest, when Ddx41 R525H/- -derived BM cells were co-transplanted with Ddx41 +/- -derived BM cells, some of the recipient mice showed impaired hematopoiesis, suggesting the presence of a non-cell autonomous effect of Ddx41 R525H/- cells on Ddx41 +/- cells.

Transcriptome analysis of stem cells (Kit + Sca-1 - Lin low cells) from different Ddx41 mutant mice revealed significant changes in gene expression and splicing patterns in many genes in all mutant mice, with larger changes for Ddx41 R525H/- than Ddx41 +/- and Ddx41 R525H/+ cells. Notably, Ddx41 R525H/- -derived stem cells exhibited a significant upregulation of genes involved in innate immunity, including an upregulation of cGAS-STING innate immunity pathways, as well as an enhanced Trp53 pathway, whereas there was a downregulation of genes related to RNA metabolism and ribosome biogenesis.

Summary/Conclusion: Our results suggest that monoallelic Ddx41 loss leads to age-dependent impaired hematopoiesis, while compound biallelic loss-of-function and R525 alleles showed early development of severe BM failure, where activated innate immunity and impaired ribosome functions may play important roles. The role of DDX41 mutations in myeloid neoplasms, however, need further evaluation.

P738: EXPRESSION OF TRANSPOSABLE ELEMENTS IN CD34+ CELLS IN MYELODYSPLASTIC SYNDROMES

Z. Krejcik 1,* , A. Hrustincova 1 , I. Trsova 1 , D. Kundrat 1 , K. Szikszai 1 , M. Belickova 1 , J. Cernak 2 , A. Jonasova 3 , M. Dostalova Merkerova 1

1 Department of Genomics; 2 Laboratory of Anemias, Institute of Hematology and Blood Transfusion; 3 First Department of Medicine, General University Hospital, Prague, Czechia

Background: Myelodysplastic syndromes (MDS) are a heterogenous group of malignant hematopoietic stem cell (HSC) disorders characterized by genomic instability resulting in aberrant differentiation of HSCs, peripheral blood cytopenia, and a tendency toward leukemic transformation. Transposable elements (TEs) are common in the human genome and, with regard to their ability to change the position in the genome, may represent one of the sources of this instability. Although TE dysregulation has previously been reported in different hematological malignancies, e.g., in acute myeloid leukemia (AML), information about TEs in MDS is rare. In the past, Colombo et al. demonstrated suppressed TE expression in higher risk MDS in a small cohort of patients. They proposed this suppression as a mechanism for immune escape in MDS/AML because the higher TE expression can potentially trigger immune-mediated cell clearance of cancer cells via “viral mimicry” pathways.

Aims: Our aims were to identify, quantify and compare TE expression in different stages of MDS on a larger cohort of patients to provide a deeper insight into their occurrence and possible roles in MDS.

Methods: We prepared, sequenced, and analyzed rRNA-depleted RNA libraries of CD34 + HSCs in a cohort of 75 MDS patients, and 13 healthy controls, using the SalmonTE tool for the TE detection.

Results: Comparison between healthy controls and MDS patients revealed significantly upregulated class I LTR (Long Terminal Repeat) retrotransposons (p = 0.0006, logFC = 0.040), especially then ERV1 (Endogenous Retrovirus) class (p = 0.0002, logFC = 0.052), in MDS patients. Detailed analysis revealed 11 significantly (FDR < 0.05) differentially expressed TEs, nine upregulated (ERV1: HERV-Fc1, MER51E, LTR27E, MER65C, HUERS-P1, PABL_A, ERV3: MER54B, CR1: X5A_LINE, SINE (Short Interspersed Element): MIR3) and only two downregulated (ERV1: LTR71A, LTR26E) in the group of MDS patients. After stratification of patients into lower (IPSS-R score ≤ 3.5, n = 48) and higher risk (IPSS-R score ≥ 4, n = 27) categories, we observed significant dysregulation of non-LTR retrotransposons in general, and in particular of SINE (p = 6.2x10 -14 , logFC = -0.21) and LINE1 (Long Interspersed Element) (p = 3x10 -7 , logFC = 0.06) clades. Detailed differential expression analysis revealed that 18 out of 21 most significantly (FDR < 0.001) deregulated TEs were suppressed in higher risk MDS, namely AluY, AluYb8, AluYd8, AluYe2, AluSq4, AluYa1, AluYa5, AluYb3a2, AluYc1, AluYbc3a, AluYe5, AluYf2, SVA_B and SVA_F (all SINE), L1HS (LINE1), LTR12E and LTR1A2 (both ERV1); and only three TEs were upregulated (HARLEQUIN, LTR24C, PABL_AI (all ERV1)).

Summary/Conclusion: To our knowledge, this is the first report on TE expression and MDS-risk specific dysregulation in a larger cohort of MDS patients, revealing their importance in the pathogenesis of MDS. Significant suppression of TEs observed in higher risk MDS patients supports the previously formulated potential mechanism of immune escape in these MDS patients.

Acknowledgements: Supported by AZV CR (NU20-03-00412) and MH CZ-DRO (UHKT 00023736).

Reference: Colombo et al. Suppression of transposable elements in leukemic stem cells. Sci Rep. 2017. 1;7(1):7029. doi: 10.1038/s41598-017-07356-9.

P739: THE EPI-GENOMIC LANDSCAPE OF MONOSOMY 7 IN ADULT MDS/AML

A. G. Lema Fernandez 1,* , C. Nardelli 1 , V. Di Battista 1 , M. Quintini 1 , F. Pellanera 1 , C. Matteucci 1 , V. Pierini 1 , B. Crescenzi 1 , M. Moretti 1 , V. Bardelli 1 , P. Gorello 1 , C. Mecucci 1

1 Department of Medicine, University of Perugia, Perugia, Italy

Background: Monosomy 7 is one of the most frequent aneuploidies in myeloid malignancies often occurring in the context of high-risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) and associated to poor prognosis. Despite several studies have focused on the pathogenetic role of genes mapping at chromosome 7q, its molecular landscape remains undetermined.

Aims: The aim of this study was to characterize biological features of adult MDS/AML with isolated monosomy 7 by investigating the epi-genomic landscape.

Methods: Epi-genomic characterization included karyotype, Fluorescent In Situ Hybridization (FISH), Single Nucleotide Polymorphism Array (SNPa), Targeted Genomic Sequencing, RNA sequencing and multiplex Enhanced Reduced Representation Bisulfite Sequencing (mERRBS). This comprehensive approach was used in analyzing a cohort of three adult MDS/AML cases with isolated monosomy 7 against a cohort of three non-neoplastic cytopenias and against dic(1;7)(q10;p10) cases previously characterized by our group (Leukemia, 2019).

Results: Karyotype identified the presence of isolated monosomy 7 in all cases, interphase-FISH showed clonality as accounting for 65-85% of the total bone marrow population. Targeted sequencing showed multiple variants affecting myeloid genes (mean 5.3 events; from 2 to 9 per case) but did not identify a common mutational marker. Gene expression profile (FDR<0.1, log 2 FC>I1I) provided us with a specific signature differentiating monosomy 7 from both controls [632 differentially expressed genes (DEGs): 405 up and 227 down] and dic(1;7) [1.706 DEGs: 832 up and 874 down]. In keeping with monosomy, a strong gene dosage effect at chromosome 7 genes emerged. Functional pathways analysis specifically identified the presence of active stemness programs in monosomy 7, through deregulation of mTOR pathway, Rho-GTPases and hematopoietic stem cell markers. Deregulation of genes involved in apoptosis escape, included in TP53 and heat-shock proteins signaling, supported the undifferentiated phenotype. Interestingly, PTCH1 tumor suppressor downregulation was a differential feature of monosomy 7 with respect to the other groups and it was validated in an independent cohort of MDS/AML with isolated monosomy 7. Global methylation further delineated specific boundaries between monosomy 7 and dic(1;7) cases showing a methylation gradient that defined monosomy 7 as the most hypermethylated group. Private localization of differentially methylated regions in monosomy 7 emerged at intergenic enhancers, enriched for homeo-domain transcription factors binding sites, and at CTCF binding sites linked to promoters. A subset of hypermethylated enhancers in monosomy 7 was in accordance with repression of their respective target genes, almost totally located on chromosome 7 ( ARPC1A , CCZ1 , IKZF1 , PMS2 , RBAK , RBM33 , RHEB , RNF216P1 , ZNF12 and ZNF853 ). Their downregulation was related not only to the chromosome loss but also to the effect of enhancer hypermethylation in the retained chromosome, supporting a combined cytogenetic and epigenetic mechanism in determining biallelic deregulation. Among these deregulated genes, IKZF1 and its target genes accounted for > 6% of the whole typical expression profile.

Summary/Conclusion: In conclusion our results first identified a specific signature of DNA methylation and an active epi-genomic program reminiscent of stem cells in MDS/AML with monosomy 7, providing new insights to the knowledge of biological targets in this aggressive aberration.

P740: IMPACT OF MAGROLIMAB IN COMBINATION WITH AZACITIDINE ON RED BLOOD CELLS IN PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME (HR MDS)

J. Y. Chen 1,* , L. Johnson 2 , K. M. McKenna 2 , T. S. Choi 2 , J. Duan 2 , D. Feng 2 , J. M. Tsai 3 , N. Garcia-Martin 4 , K. Sompalli 2 , R. Maute 2 , P. Vyas 4 , R. Majeti 5 , C. H. M. Takimoto 2 , J. Liu 1 , G. Ramsingh 2 , M. P. Chao 2 , J.-P. Volkmer 2 , I. L. Weissman 5

1 Stanford University School of Medicine, Stanford; 2 Gilead Sciences, Inc., Foster City; 3 Brigham and Women’s Hospital, Boston, United States of America; 4 Molecular Hematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 5 Ludwig Cancer Center and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, United States of America

Background: Magrolimab is a monoclonal antibody that blocks CD47, a “don’t eat me” signal expressed on cancer cells, to escape immune surveillance and macrophage-mediated clearance. Prior preclinical studies have shown that CD47 is critical to red blood cell (RBC) homeostasis, with CD47 deficiency decreasing RBC half-life. Fc-mediated opsonization also depletes RBCs, raising concerns that potential on-target anemia could result from the use of anti-CD47 agents via multiple mechanisms. Nonetheless, several clinical trials have demonstrated that magrolimab can be safely administered as monotherapy, with an initial lower “priming” dose yielding transient anemia with compensatory reticulocytosis and no anemia observed at subsequent higher maintenance doses. However, the mechanism underlying this observed protection has not been fully defined.

Aims: To describe manageable anemia in patients with HR MDS treated with magrolimab in combination with azacitidine (AZA) in a phase 1 clinical trial ( NCT03248479 ) and further investigate the underlying mechanisms in preclinical models.

Methods: In a multicenter prospective study, complete blood counts (CBCs), peripheral blood, and bone marrow (BM) were collected at prespecified time points from patients with HR MDS (n=57) treated with magrolimab in combination with AZA. CBCs were measured, and blood and BM samples were analyzed by flow cytometry for expression of CD47 on RBCs and white blood cells (WBCs). Magrolimab was initially administered as a priming dose (1 mg/kg) followed by an initial weekly maintenance dose (30 mg/kg) before transitioning to maintenance dosing every 2 weeks. AZA 75 mg/m 2 was administered on days 1-7 of each 28-day cycle. Preclinical modeling studies were conducted with intact and Fc-deficient anti-mouse CD47 (MIAP410) and anti-human CD47 (magrolimab) antibodies in murine models, including C57BL/6J B-hSIRPA/hCD47 mice.

Results: Combination treatment with magrolimab and AZA resulted in tolerable anemia that correlated with rapid, near-complete loss of CD47 in RBCs but not WBCs. The initial 1-mg/kg priming dose was sufficient for this CD47 loss, which persisted with subsequent 30-mg/kg maintenance doses. Both findings are consistent with prior clinical observations of magrolimab monotherapy in patients with solid tumors and magrolimab in combination with rituximab in patients with lymphoma. Our preclinical studies with mouse models revealed that CD47 removal is mechanistically independent of previously described RBC antigen modulation mechanisms and cellular compartments. Instead, this CD47 loss requires anti-CD47 cross-linking between RBCs and non-RBCs.

Summary/Conclusion: Overall, these results support the idea that on-target magrolimab-mediated anemia is mitigated by a near-complete loss of RBC CD47. Patients with HR MDS treated with magrolimab in combination with AZA had tolerable anemia with the use of priming and maintenance doses.

P741: DDX41 MUTATIONS DEFINE A UNIQUE SUBTYPE OF MYELOID NEOPLASMS.

H. Makishima 1,* , R. Saiki 1 , Y. Nannya 2 , S. Korotev 3 , C. Gurnari 4 , J. Takeda 1 , Y. Momozawa 5 , S. Best 6 , P. Krishnamurthy 6 , T. Yoshizato 1 , Y. Atsuta 7 , Y. Shiozawa 1 , Y. Iijima-Yamashita 8 , K. Yoshida 1 , Y. Shiraishi 9 , Y. Nagata 1 , N. Kakiuchi 1 , M. Onizuka 10 , K. Chiba 9 , H. Tanaka 9 , A. Kon 1 , Y. Ochi 1 , M. Nakagawa 1 , R. Okuda 1 , T. Mori 1 , H. Itonaga 11 , Y. Miyazaki 11 , M. Sanada 8 , H. Tsurumi 12 , S. Kasahara 13 , C. Müller-Tidow 14 , A. Takaori-Kondo 15 , K. Ohyashiki 16 , T. Kiguchi 17 , F. Matsuda 18 , J. Jansen 19 , C. Polprasert 20 , S. Miyano 9 , L. Malcovati 21 , T. Haferlach 22 , M. Kubo 5 , M. Cazzola 21 , A. Kulasekararaj 6 , L. Godley 3 , J. Maciejewski 4 , S. Ogawa 1

1 Department of Pathology and Tumor Biology; 2 Kyoto University, Kyoto, Japan; 3 Departments of Medicine and Human Genetics, Section of Hematology/Oncology, The University of Chicago, Chicago; 4 Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 5 Laboratory for Genotyping Development, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan; 6 King’s College Hospital NHS Foundation Trust, London, United Kingdom; 7 Department of Healthcare Administration, Nagoya University Graduate School of Medicine; 8 Department of Advanced Diagnosis, Clinical Research Center, Nagoya Medical Center, Nagoya; 9 Division of Genome Analysis Platform Development, National Cancer Center Research Institute, Tokyo; 10 Department of Hematology and Oncology, Tokai University School of Medicine, Isehara; 11 Department of Hematology, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki; 12 Department of Hematology, Gifu University; 13 Department of Hematology, Gifu Municipal Hospital, Gifu, Japan; 14 Heidelberg University Hospital, Heidelberg, Germany; 15 Department of Hematology, Kyoto University, Kyoto; 16 Department of Hematology, Tokyo Medical University, Tokyo; 17 Chugoku Central Hospital, Fukuyama; 18 Center for Genomic Medicine, Kyoto University, Kyoto, Japan; 19 Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Nijmegen, Netherlands; 20 Department of Medicine, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Bangkok, Thailand; 21 Department of Molecular Medicine, University of Pavia, Pavia, Italy; 22 MLL, Munich Leukemia Laboratory, Munich, Germany

Background: DDX41 was identified as a causative gene for late-onset familial myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While DDX41 is thought to be one of the most frequent targets of germline mutations responsible for sporadic cases with AML/MDS and other myeloid neoplasms (MN), the entire spectrum of pathogenic DDX41 variants and their effect size therein are still to be elucidated. The clinical and genetic pictures of DDX41 -mutated myeloid neoplasms have not fully been investigated in a large cohort of patients, either.

Aims: In this study, we enrolled a total of 9,081 sporadic and familial cases with different MNs from different ethnicities, in which germline and somatic mutations in DDX41 and other major driver genes in AML/MDS were analyzed using next generation sequencing.

Methods: To estimate the size of the risk of MN development associated with pathogenic germline DDX41 variants, we calculated their enrichment in 3,672 MN cases as compared with a control cohort ( n =20,238). We also estimated the penetrance of pathogenic DDX41 germline variants for the development of MNs.

Results: We identified 292 cases with pathogenic or likely pathogenic DDX41 mutations with conspicuous ethnic diversity, of which 159 (54%) accompanied somatic mutations as well, while the remaining 53 cases had somatic mutations alone. Among 292 germline variants, 197 were truncating variants, whereas only 2.6% of somatic mutations were truncating ( P <0.0001). Pathogenic germline mutations were significantly more frequent in Asian than in Caucasian cases (odds ratio (OR)=1.64), consistent with a significantly higher frequency of pathogenic germline DDX41 variants in the former cases. We observed a significant enrichment of 5 DDX41 variants in myeloid neoplasms, including p.A500fs, p.E7*, p.Y259C, p.E256K, and p.S363del, compared with 20,238 control cases, with a mean odds ratio of 10.6. To estimate the penetrance of NMs associated with pathogenic germline variants, we calculated cumulative incidence of MN in a cohort of 384 first-degree relatives (kin-cohort) of DDX41 -mutated NM cases. Penetrance of pathogenic DDX41 germline variants was only negligible under 40 years of age, but rapidly elevated thereafter, reaching as high as 38.5% at the age of 85, confirming late onset of DDX41 -mutated MNs.

DDX41 variants were significantly more common in higher risk MDS (7.1%) and secondary AML (9.7%), compared with other myeloid neoplasms (1.9%). Patients with MDS having both germline and somatic mutations were more likely to classified in refractory anemia with excess blasts (RAEB), compared with those with germline or somatic alone ( P =0.029). DDX41 variants were significantly associated with lower WBC and hypocellular bone marrow. Most frequently co-occurring mutations included those in ASXL1 , SRSF2 , TET2 , CUX1 , and DNMT3A , of which only CUX1 mutations were statistically significant. Among MDS cases, DDX41 mutations were associated with a significantly higher incidence of leukemic evolution ( P =0.006) than DDX41 -unmutated cases, which was virtually confined to those with truncating DDX41 variants. However, despite this, DDX41 -mutated cases had a better OS than un-mutated cases, particularly when treated with hypomethylating agents (HMAs) ( P< 0.001).

Summary/Conclusion: In summary, DDX41 -mutated myeloid neoplasms define a distinct entity of myelodysplasias associated with higher incidence of leukemic evolution and better responsiveness to hypomethylating agents.

P742: TRANSCRIPTOMIC ANALYSIS OF ISOLATED CD4+ AND CD8+ T CELLS REVEALS DIFFERENCES IN TCR REPERTOIRE IN PATIENTS WITH HIGH RISK MDS COMPARED TO AML.

K. Katsiki 1 , A. Tasis 1 , A. Filia 1 , E. Lamprianidou 1 , K. Liapis 1 , I. Kotsianidis 1 , I. Mitroulis 1,*

1 Department of HEmatology, Democritus University of Thrace, Alexandroupolis, Greece

Background: Myelodysplastic syndrome (MDS) is a group of clonal disorders of hematopoietic progenitor cells, which often progresses towards acute myeloid leukemia (AML). Even though MDS is a clonal hematopoietic disease, bone marrow microenvironment and more specifically inflammation has been linked to its progression. Among other cell types, CD4+ and CD8+ T cells are major players in the regulation of the microenvironment in malignancies.

Aims: In this study, we aimed to investigate the molecular signature of bone marrow CD4+ and CD8+ T cells from patients with high-risk MDS, AML and chronic myelomonocytic leukemia (CMML).

Methods: RNA sequencing was performed on isolated CD4+ and CD8+ T cells from the bone marrow of six patients with high risk MDS (RAEB-II), six patients with CMML and four patients with AML, using cell sorting. Except from the analysis of gene expression, we performed TCR repertoire analysis using VDJtools, an open-source Java/Groovy-based framework that can analyze immune repertoire sequencing (RepSeq) data and allows applying a diverse set of post-analysis strategies. To do so, RNA sequencing data were used as input for the V-(D)-J junction mapping software and analysis was performed on the MiXCR platform. The output of MiXCR wereused as input for the VDJtools after running a convert routine with -S mixcr argument to prepare datasets in a format for VDJtools analysis.

Results: Analysis of CD8+ T cells from patients with high risk MDS and AML identified 38 differentially expressed genes. We observed that the expression of the TCR variants TRAV20 and TRAV13-1 was increased in patients with AML compared to high risk MDS (Figure 1A). Further analysis of the TCR repertoire revealed that the diversity of TCR was higher in CD8+ T cells from patients with AML (Figure 1B), whereas multidimentional scaling analysis revealed a clear separation of the two groups (Figure 1C). To the same direction, clustering of the usage of TRAV-TRAJ further confirmed this finding (Figure 1D). On the other hand, we did not observe any difference in the TCR diversity of the variant usage when we compared CD8+ T cells from patients with CMML and AML (Figure 1E-F), or in the comparison between CD4+ T cells from patients with high risk MDS and AML (Figure 1G-I).

Image:

Иллюстрация — в первоисточнике ↗

Summary/Conclusion: CD8+ T cells are a major cell population that exerts anti-tumour properties in cancer. Herein, we compared the TCR repertoire of CD8+ and CD4+ T cells in the bone marrow of patients with high risk MDS and AML and observed that despite the common features of these two disorders, there are significant differences in the TCR repertoire specifically in CD8+ T cells, especially concerning TCR diversity. These findings imply that there is more active T cell response in the bone marrow in AML compared to MDS. However, it is not clear to what degree this T cell response targets AML neo-antigens.

P743: THE PIVOTAL ROLE OF GLUTAMINOLYSIS IN MYELODYSPLASTIC SYNDROME (MDS): A NOVEL STRATEGY FOR THE TARGETED THERAPY OF MDS

S. Okabe 1,* , Y. Tanaka 1 , A. Gotoh 1

1 Department of Hematology, TOKYO MEDICAL UNIVERSITY, Tokyo, Japan

Background: Myelodysplastic syndromes (MDS) is indicated by bone marrow failure and increased risk of transformation to acute myelogenous leukemia (AML). MDS is more common with advancing age and a combination of comorbidities is associated with significantly worse overall survival. Moreover, iron overload (IOL) starts to develop in MDS patients by transfusion in many cases. Because the outcome of MDS is poor, alternative therapeutic strategies are required to improve the survival of MDS and AML patients.

Aims: Glutamine is the most abundant amino acid in blood. Glutaminase (GLS) is the initial enzyme in glutamine metabolism and glutaminolysis contributes to tumor growth. Because GLS is frequently activated in various types of cancer, GLS inhibitor could suppress MDS and AML cells in combination with BCL2 inhibitor.

Methods: In this study, we investigated whether GLS was involved in MDS progress. We also investigated the efficacy of GLS inhibitor (CB-839 or IPN-60090) and BCL2 inhibitor, venetoclax by using MDS and AML cell line, SKM-1, MDS-L, MOLM-14, THP-1, MV4;11 and osteoblastic cell line, MC3T3-E1.

Results: We first investigated the relationship between GLS expression and MDS patients by microarray gene expression data from the online Gene Expression Omnibus (GEO). In mammalian cells, there are two paralogous GLS genes, GLS1 and GLS2. GLS1 expression was increased in refractory anemia with excess of blasts (RAEB)-2 cells compared to normal control cells ( GSE19429 ). In contrast, GLS2 expression was not changed. High GLS1 expression is associated with poor prognosis in AML patients. Deprivation of glutamine in culture medium revealed that cellular growth inhibition. We next evaluated the effect of GLS inhibitor (CB-839 or IPN-60090) or venetoclax on proliferation of MDS and AML cell lines. 72 h treatment of MDS and AML cells were inhibited by GLS inhibitor or venetoclax in a dose dependent manner. Cellular cytotoxicity and caspase 3/7 activity was also increased. Glutamine is converted by GLS into glutamate and related nicotinamide adenine dinucleotide phosphate (NADP) production. Intracellular NADPH and NADH were reduced by GLS inhibitor. Co-treatment with GLS inhibitor and venetoclax was superior effect than single drug alone. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased. The bone marrow is a relatively hypoxic microenvironment. Gene expression of GLS1 is increased under hypoxia condition. The proteasome 20S activity and phosphorylation of nuclear factor-kappa B (NF-κB) were increased. Efficacy of venetoclax is reduced under hypoxia condition. Co-treatment with GLS inhibitor and venetoclax overcome in hypoxia mediated drug resistance. GLS1 shRNA transfected cells reduced cellular proliferation. Cell cycle analysis showed G1 arrest and increased sensitivity of venetoclax. Because MDS patients with IOL have reduced overall survival and poorer outcomes, we next examined IOL by using osteoblastic cell line, MC3T3-E1. The cellular proliferation was reduced by ferric ammonium citrate (FAC). Intracellular ROS was increased by FAC. GLS inhibitor treatment protected FAC mediated cell death.

Summary/Conclusion: Targeting of glutaminolysis and BCL2 inhibition combine to enhance therapeutic efficacy and has been proposed as a novel strategy high-risk MDS and AML. We also provide the promising clinical relevance as a candidate drug for treatment of MDS and AML patients.

P744: UNBALANCED TRANSLOCATION DER(1;7)(Q10;P10) AS A DISTINCT SUBTYPE IN MYELODYSPLASTIC SYNDROMES

R. Okuda 1,* , Y. Ochi 1 , K. Chonabayashi 2,3 , N. Hiramoto 4 , M. Sanada 1,5 , H. Handa 6 , S. Kasahara 7 , S. Sato 8 , N. Kanemura 9 , T. Kitano 10 , M. Watanabe 3 , W. Kern 11 , M. Creignou 12 , Y. Shiraishi 13 , M. Watanabe 14 , K. Usuki 15 , S. Imashuku 16 , E. Hellstrom-Lindberg 12 , T. Haferlach 11 , S. Chiba 17 , N. Sezaki 18 , L.-Y. Shih 19 , Y. Miyazaki 8 , Y. Yoshida 2 , T. Ishikawa 14 , K. Ohyashiki 20 , Y. Atsuta 21 , Y. Shiozawa 1 , S. Miyano 13,22 , H. Makishima 1 , Y. Nannya 1 , S. Ogawa 1,12,23

1 Department of Pathology and Tumor Biology; 2 Center for iPS Research and Application; 3 Department of Hematology and Oncology, Kyoto University, Kyoto City; 4 Department of Hematology, Kobe City Medical Center General Hospital, Kobe City; 5 Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya City; 6 Department of Hematology, Gunma University Graduate School of Medicine, Gunma; 7 Department of Hematology, Gifu Municipal Hospital, Gifu; 8 Japan Adult Leukemia Study Group, Japan Adult Leukemia Study Group, Japan; 9 Department of Internal Medicine, Gifu University, Gifu; 10 Department of Hematology, Kitano Hospital, Osaka, Japan; 11 MLL Munich Leukemia Laboratory, MLL Munich Leukemia Laboratory, Munich, Germany; 12 Department of Medicine, Karolinska Institute, Stockholm, Sweden; 13 Laboratory of Sequence Data Analysis, The University of Tokyo, Tokyo; 14 Department of Hematology, Hyogo Prefectural Amagasaki General Medical Center, Amagasaki; 15 Department of Hematology, NTT Medical Center Tokyo, Tokyo; 16 Department of Laboratory Medicine, Uji-Tokushukai Medical Center, Uji; 17 Department of Hematology, University of Tsukuba, Tsukuba; 18 Department of Hematology, Chugoku Central Hospital, Hiroshima, Japan; 19 Division of Hematology-Oncology, Chang Gung University, Taoyuan, Taiwan; 20 Department of Hematology, Tokyo Medical University, Tokyo; 21 The Japanese Data Center for Hematopoietic Cell Transplantation, The Japanese Data Center for Hematopoietic Cell Transplantation, Japan; 22 Laboratory of DNA Information Analysis, The University of Tokyo, Tokyo; 23 Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto City, Japan

Background: der(1;7)(q10;p10) (der(1;7)) is an unbalanced translocation between chromosomes 1 and 7 found in 1.5-6% of patients with myelodysplastic syndromes (MDS), depending on different ethnicity, where the common consequence is +1q and -7q. Previous studies have noted a higher incidence of RUNX1 mutation and better prognosis for der(1;7) cases compared to -7/del(7q) MDS cases. While previous studies have described several features of der(1;7), the understanding of der(1;7)+ MDS/AML is largely limited due to their small study size.

Aims: To elucidate the clinical and genetic characteristics of der(1;7) with integrated omics analysis in a large cohort of myeloid neoplasms.

Methods: We enrolled 148 cases with der(1;7) and analyzed their mutation profiles and clinical features using whole exome and/or targeted-capture sequencing of major driver genes of myeloid neoplasms and RNA sequencing. Distinct features of der(1;7) were investigated by comparing the results with those from an additional 3,238 non-der(1;7) cases with different myeloid neoplasms comprising -7/del(7q), +1q, and other cases.

Results: 148 der(1;7) cases comprised 72.3% MDS, 23.7% AML, and 3.4% MDS/MPN cases. Compared to -7/del(7q) AML cases, der(1;7) AML cases were more likely to be AML with dysplasia or MDS-derived AML and therapy-related AML (84.4% vs 53.2% and 9.1% vs 1.9%, respectively) (p<0.001). Targeted-capture sequencing of der(1;7) cases revealed that 91.9% of cases harbored at least one copy number change or mutation. The frequency of genetic mutation and copy number alteration for der(1;7) cases substantially differed compared to -7/del(7q), +1q, and other cases. der(1;7) cases had frequent mutations affecting RUNX1 (37.8%), EZH2 (18.9%) and ETNK1 (18.2%), which were less common in -7/del(7q) cases (OR=5.58, OR=6.52, and OR=6.23, respectively). By contrast, TP53 mutations were less frequent in der(1;7) cases (OR=0.021). Copy number analysis revealed a frequent co-occurrence of +8 and del(20q) with der(1;7) cases (18.9% and 37.4%), while del(5q), which frequently co-occurred with del(7q), was not found in der(1;7) cases. Prognosis of der(1;7) MDS cases tended to be better than -7/del(7q) cases (HR=0.71, p=0.098), but poorer than +1q (HR=1.36, p=0.11) and others cases (HR=1.8, p<0.001). The most frequent cause of death in der(1;7) cases was infection (45.5%), followed by disease progression (36.4%). This high frequency of infection as a cause of death was unique in der(1;7) as compared to -7/del(7q) (infection 13.9% and disease progression 72.3%) and others (infection 10.8% and disease progression 76.9%). Finally, RNA-sequencing analysis between der(1;7) MDS cases and non-der(1;7) MDS cases showed unique expression profiles in der(1;7). 1,463 differentially expressed genes were identified, of which 895 were down-regulated and 573 were up-regulated. GSEA analysis of major pathways showed down-regulation of those related to cell cycles (including E2F targets), innate immunity, and TNFα signaling via NFκB. Gene ontology analysis also revealed down-regulation of cell cycle-related pathways, further supporting the significance of the down-regulated cell cycles in der(1;7) MDS cases, compared to non-der(1;7) cases.

Summary/Conclusion: der(1;7)-positive MDS and AML represent a distinct subtype of myeloid neoplasms, characterized by unique patterns of co-mutations/copy-number lesions and gene expression profiles showing down-regulation of the cell-cycle pathway.

P745: ASSESSMENT OF MIRNA BIOGENESIS GENES VARIANTS IN MDS DEVELOPMENT AND PROGRESSION

D. Bug 1,* , A. Tishkov 1 , I. Moiseev 2 , Y. Porozov 3,4 , N. Petukhova 1

1 Bioinformatics Research Center; 2 R.M. Gorbacheva Scientific Research Institute of Pediatric Hematology and Transplantation, Pavlov University, St Petersburg; 3 World-Class Research Center “Digital Biodesign and Personalized Healthcare”, I.M. Sechenov First Moscow State Medical University, Moscow; 4 Department of Computational Biology, Sirius University of Science and Technology, Sochi, Russia

Background: Experimental studies demonstrated that knockout of miRNA processing enzymes Dicer and Drosha is associated with the development of phenotype similar to myelodysplastic syndrome (MDS). DICER1 inherited syndrome is associated with cancer predisposition. There is a limited number of clinical studies evaluating the spectrum of mutations in the microRNA processing genes in patients with hematological malignancies and the pathogenic significance of these mutations is unknown. Thus, we conducted a bioinformatics analysis of sequencing data from our data set of MDS patients and available results in the public databases.

Aims: We aim to identify DICER1 and DROSHA variants in hematological malignancies that lead to the protein function alteration based on bioinformatics analysis.

Methods: Data on 35 previously sequenced high-risk MDS patients (Moiseev IS et al ., PLoS One. 2021 Mar 17;16(3):e0248430) was analyzed, and the list of DICER1 and DROSHA variants was enriched with disease-specific coding variants retrieved from COSMIC database, and 3’-UTR-variants - from dbSNP. All collected variants were subjected to bioinformatics analysis. We used 3D-structures of Drosha and Dicer for molecular dynamics (MD) simulations for 200 and 300 ns, respectively (Schrödinger Suite 2020-4, Schrödinger, LLC, New York, NY, USA, 2020). The IntaRNA and MicroSNiPer algorithms were used to calculate interaction energies of 3’-UTRs hybridizing with the most abundant miRNAs in MSCs to evaluate noncoding variants.

Results: The prevalence of mutations in DICER1 and DROSHA was 54% and 17%, respectively, predominantly located in 5’-UTR and 3’-UTR regions. Detected allele frequencies and prevalence suggested their association with minor clones within the bone marrow niche.

MD simulation demonstrated apparent structural alterations with consequent protein dysfunction. RMSD results and calculated total energy of studied proteins were higher for all mutants compared to wild-type Drosha (at least 30% RMSD shift). All mutations were shown to be located in close proximity of the miRNA binding site, all were destabilizing for protein structure alone with consequent alterations in bonds and protein-miRNA binding. The most significant structural shift and protein destabilization was detected for DICER1 variants F508C and T993R characterized by local transformation of bonds interplay. The conformational changes in the protein due to Dicer variants were studied in 300 ns: differences in average RMSD values were within ~ 1Å between WT protein and mutants. The most significant changes were observed at the local area for I445S, F508C, and T993R characterized by shift of molecular bonds and alteration of protein stability parameters.

A list of SNPs that exhibit the highest repression of DICER1 and DROSHA was obtained (Table 1). The most prominent SNPs (rs1479981622, rs536092006, rs1296755923, rs1207839989) had less than 0.03% population allele frequency which is in concordance with their possible pathogenicity.

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Summary/Conclusion: We identified potential coding and non-coding “hot spots” for future analysis. Also the study shows that 3’-UTR SNPs of DICER1 , DROSHA have more potential to impact MDS progression than coding variants. Simultaneously, we found low frequency of reports for these mutations in the available databases, indicating bias and absence of these genes in the targeted sequencing panels. This builds the foundation for future research of DICER1 and DROSHA clinical role in hematological malignancies.

P746: COMBINATION OF RAS/MAPK MODULATOR AND AZACITIDINE AFFECTS HISTONE MARKS AND IMPACTS THE INNATE IMMUNE SIGNALING PATHWAY IN THE MDS-L CELL LINE

R. Rai 1,* , F. Patel 1 , J. Feld 1 , S. Melana 1 , S. Navada 1 , R. Odchimar-Reissig 1 , E. Demakos 1 , E. P. Reddy 2 , A. Horowitz 2 , L. Silverman 1

1 Division of Hematology and Medical Oncology; 2 Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, United States of America

Background: Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis, and a propensity to transform into acute myeloid leukemia. MDS patients exhibit chronic activation of the innate immune response and a hyperinflammatory microenvironment (Barryero L, et al. Blood, 2018). Azacitidine (Aza), as a single agent has an overall response rate (ORR) of only 50% (Silverman LR, et al., JCO 2002; Fenaux P et al., Lancet Oncol. 2009). The response to Aza is not durable and all patients relapse with worsening bone marrow failure. Aza combined with a multi-kinase inhibitor of RAS and MAPK pathways, rigosertib (RAS/MAPK), reverses the bone marrow failure state and demonstrates an ORR of 54% in patients who failed a prior HMA based therapy. (Navada SC, et al. EHA Library 2019). This represents a critical observation in overcoming the epigenetic clinical resistance phenotype, though the mechanism is still elusive. Aberrant RAS/MAPK signaling has also contributed to MDS pathophysiology.

Aims: To investigated the impact of RAS modulation combined with Aza on the innate immune signaling pathway.

Methods: We used the MDS-L cell line as a model to limit the heterogeneity observed in MDS patients. The cells were treated with, Aza, RAS/MAPK and RAS/MAPK-Aza for 48 hrs and further analyzed by qPCR and western blot. We studied the expression of several histone proteins and various pattern recognition receptors (TLRs, RIG-I, MDA5, STING), their intermediate adaptor molecules myeloid differentiation factor 88 (MYD88), mitochondrial antiviral signaling (MAVS) gene; and interferon regulatory factor (IRF)-3 and -7 by qPCR.

Results: We found an increase in H3K9ac protein expression with RAS/MAPK and RAS/MAPK-Aza; Aza and RAS/MAPK alone each had similar effects on H3K4me3, however, its expression was markedly upregulated with RAS/MAPK-Aza. Effects on H3K36me3 were comparable in all treated cells. We observed marked effects on the repression marks H3K9me3 and H3K27me3 by RAS/MAPK and RAS/MAPK-Aza combination. Furthermore, we observed that Aza, RAS/MAPK and RAS/MAPK-Aza significantly inhibit the expression of TLR1, 2 and 6. However, TLR-3, 9, RIG-I, MDA5, STING, MAVS, MYD88, and IRF-3, 7 were significantly inhibited by RAS/MAPK and RAS/MAPK-Aza.

Summary/Conclusion: We find that the RAS/MAPK modulator rigosertib has histone modifying effects alone and when combined with Aza. The combination has effects on histone modification of both activator and repressor marks and is associated with down-regulation of the innate immune signaling pathway. In the clinic RAS/MAPK modulation combined with Aza reverses bone marrow failure in MDS. Further studies are underway to determine the correlation of the histone modification and innate immune signaling changes, as well as the role of RAS/MAPK modulation, to determine how these mechanisms contribute to the improvement in hematopoiesis in MDS patients.

P747: LR-MDS IS CHARACTERIZED BY DIFFERENTIAL EXPRESSION OF INFLAMMASOME-RELATED GENES IN SPECIFIC CELL POPULATIONS

C. Rolfs 1,* , M. Schneider 1 , M. Trumpp 1 , L. Fischer 2 , S. Winter 2 , S. Uxa 1 , V. Menger 1 , K. Nenoff 1 , A. S. Kubasch 1 , K. Metzeler 1 , K. Sockel 2 , C. Thiede 2 , L. Hofbauer 3 , A. Brüderle 4 , J. Woo 5 , M. Cross 1 , U. Platzbecker 1

1 Department of Hematology and Cell Therapy, University Hospital Leipzig, Germany, Leipzig; 2 Medical Clinic and Policlinic I; 3 Department of Medicine III, University Hospital Carl Gustav Carus, Dresden, Germany; 4 Novartis Oncology; 5 Translational Clinical Oncology, Novartis Institutes for BioMedical Research, Basel, Switzerland

Background: Myelodysplastic Syndromes (MDS) are a group of hematopoietic neoplasms characterized by clonal expansion of hematopoietic stem and progenitor cells (HSPC) and peripheral blood cytopenia. MDS can develop from clonal hematopoiesis of indeterminate potential (CHIP) and progress through low-risk (LR) to high-risk (HR)-MDS and further to acute myeloid leukemia (AML). Recent findings emphasize the role of sterile inflammation in the bone marrow (BM) as a driver of neoplastic progression. Our previous analysis of whole BM mononuclear cells (BM-MNC) has revealed distinct expression patterns of inflammasome-related genes, including IL1B and IL18, to be associated with LR-MDS subgroups and clinical features. This raises the prospect of interrupting disease evolution at an early stage by targeted, personalized anti-inflammatory therapy. The development and stratification of such therapies will ultimately require more detailed knowledge of the variety and consequences of inflammation networks in MDS bone marrow.

Aims: Our objective was to assess inflammasome-related gene expression levels in individual cell types in the BM of CHIP, LR-MDS and HR-MDS individuals as well as non-CHIP controls in order to determine a) which cell types contribute to the expression of inflammasome-related genes and b) whether specific disease states are associated with consistent patterns of cell-specific gene expression.

Methods: Cryopreserved BM-MNC from 3 non-CHIP controls, 3 CHIP individuals, 14 LR-MDS and 5 HR-MDS patients were obtained from the MDS registry and the BoHemE Study ( NCT02867085 ) at the University Hospitals in Dresden and Leipzig. Hematopoietic stem and progenitor cells (HSPC), monocytes, monocytic myeloid-derived suppressor cells (M-MDSC), polymorphonuclear MDSC (PMN-MDSC), B lymphocytes, T lymphocytes and CD45 - cells (control) were sorted from thawed BM-MNC on a BD FACS Jazz. RNA was isolated and the gene expression of IL1B, IL18, S100A9, IRAK4, NLRP3, PYCARD, CASP1 and NLRC4 was assessed by RT-qPCR.

Results: The majority of the inflammasome-related genes, including IL1B , NLRP3 , NLRC4 and S100A9 were expressed in all disease states predominantly by monocytes and M-MDSC (p<0.001). However, IL18 was expressed at the highest level in HSPC. Consistent to our previous analysis of whole BM-MNC, we found an association between cell-specific gene expression and LR-MDS genetics, with HSPC-derived IL18 mRNA being highest in SF3B1 -mutated LR-MDS, while monocytic IL1B mRNA was highest in del(5q) LR-MDS. Furthermore, an apparent progression in expression from non-CHIP through to LR-MDS was detected, with mRNA levels of IL1B and NLRP3 in monocytic cells and PYCARD and CASP1 in HSPC increasing from non-CHIP to CHIP between 1.5- and 7.9-fold. From CHIP to LR-MDS, mRNA levels of CASP1 and NLRC4 in monocytic cells and PYCARD both in HPSC and in monocytic cells increased by 1.8- to 2.1-fold.

Summary/Conclusion: We identify features of inflammasome-related gene expression in individual cell populations and disease states reflecting the progression from non-CHIP through CHIP to MDS. Our analysis suggests strongly that high resolution studies of gene expression in specific populations or single cells will resolve a spectrum of MDS-related inflammation states relevant to prognosis and personalized therapy. Therefore, we are currently performing RNA-Seq from sorted cell populations in order to extend our analysis beyond the core inflammasome reported here.

P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML

A. Schnegg-Kaufmann 1,2,* , J. Thoms 2 , G. S. Bhuyan 3 , H. Henry 2 , L. Vaughan 2 , K. Rutherford 4 , P. Kakadia 5 , E. Johansson 6 , T. Failes 7 , A. Greg 7 , J. Koval 8 , R. Lindeman 9 , P. Warburton 10 , A. Rodriguez-Meira 11 , A. Mead 11 , A. Unnikrishnan 3 , S. Bohlander 12 , E. Pappaemmanuil 13 , O. Faridani 2 , C. Jolly 2 , F. Zanini 3 , J. Pimanda 14

1 Department of Haematology and Central Haematology Laboratory, Inselspital, Bern, Switzerland; 2 School of Medical Sciences, Lowy Cancer Research Centre; 3 Prince of Wales Clinical School, Lowy Cancer Research Centre, UNSW Sydney, Sydney, Australia; 4 Computational Oncology Service, Department of Epidemiology & Biostatistics, Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York, United States of America; 5 University of Auckland, Auckland, New Zealand; 6 Flow Cytometry Facility, Mark Wainwright Analytical Centre; 7 Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney; 8 Ramaciotti Centre for Genomics, UNSW Syndey, UNSW; 9 Department of Haematology, Prince of Wales Hospital, Sydney; 10 Department of Haematology, Wollongong Hospital, Wollongong, Australia; 11 Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 12 Leukaemia and Blood Cancer Research Unit, Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; 13 Computational Oncology Service, Department of Epidemiology & Biostatistics, Center for Computational Oncology, Memorial Sloan Kettering Cancer Center, New York, United States of America; 14 School of Medical Sciences, Lowy Cancer Research Centre, UNSW, Sydney, Sydney, Australia

Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear.

Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML.

Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC).

Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ~10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells.

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Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

P749: PROGNOSTIC SIGNIFICANCE OF SERIAL CIRCULATING TUMOR DNA STATUS POST TREATMENT IN MYELODYSPLASTIC SYNDROMES AND ACUTE MYELOID LEUKEMIA

H. Tong 1,* , X. Zhou 1 , W. Lang 1 , C. Mei 1 , Y. Ren 1 , L. Ma 1 , G. Xu 1 , L. Xu 1 , Y. Li 1

1 department of hematology, The first affiliated hospital Zhejiang university school of medicine, Hangzhou, China

Background: A major advance in our understanding of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) biology has been the implementation of next generation sequencing (NGS) which influenced diagnostic, prognostic, and therapeutic decisions in myeloid malignancies. Circulating tumor DNA (ctDNA) sequencing, as as a novel and minimally invasive measure, was reported to exhibit excellent correlations with matched bone marrow NGS in MDS and AML. However, the clinical relevance of dynamic ctDNA monitor during active treatment is unknown.

Aims: In this study, we assessed the role of ctDNA as a biomarker to monitor therapeutic response and clonal evolution in MDS and AML.

Methods: Thirty-one MDS and AML patients who had both bone marrow NGS and ctDNA at baseline were included for concordance analysis. Twenty-seven patients who had at least two serial ctDNA assessment with at least one month interval were included for dynamic ctDNA analysis. Targeted deep sequencing was performed on bone marrow and ctDNA using a customized panel of 165 genes known to be recurrently mutated in MDS and acute myeloid leukemia.

Results: Thirty out thirty-one patients were identified with ctDNA mutation at baseline. Diagnostic ctDNA and matched bone marrow DNA exhibited excellent correlations with variant allele frequencies (VAFs) (R 2 =0.721, p<0.001). For 27 patients who had ctDNA mutation in baseline and treated with HMA or chemotherapy, the average mutation VAFs at the end of treatment was lower in patients achieving a CR/CRi versus those with mCR, mLFS or SD (2.7% vs 24.1%, respectively, p<0.001). As expected, mutation VAFs from all patients with treatment failure did not decrease during treatment. (Figure 1). Similarly, the average decrease in mutation VAFs from pretreatment to end of treatment was greater in patients achieving a CR versus those who did not (87.7% vs 35.5%, respectively, p= 0.001). ctDNA negative post treatment was associated with longer PFS (median PFS not reached (NR) vs. 5.6 (95% CI 4.08-7.13) months; P=0.009)(Fig 2A) and OS (median OS NR vs. 12.0 (95% CI 9.08-15.0) months; P=0.023) (Fig 2B).

Seven of ten MDS patients who transformed to AML were identified with lately acquired subclones harboring FLT3 or NF1, mutations involving in signaling pathway. Moreover, new subclones were detected 0.5 to 4 months prior to AML transformation in 4 cases, indicating that genetic progression can predate morphological progression.

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Summary/Conclusion: ctDNA status post treatment was relevant to clinical response and was of prognostic significance for disease progress and relapse. Dynamic ctDNA changes revealed complex patterns of clonal structure of MDS and AML in response to treatment. ctDNA sequencing could be an attractive, prospective measure for disease monitoring.

P750: CLONAL HEMATOPOIESIS AND EPIGENETIC AGE ACCELERATION IN ELDERLY DANISH TWINS

M. Tulstrup 1,2,3,4,* , M. Soerensen 2,5 , J. W. Hansen 1,3,4 , J. Weischenfeldt 3,4,6 , K. Grønbæk 1,3,4 , K. Christensen 2,5,7

1 Department of Hematology, Rigshospitalet, Copenhagen; 2 The Danish Twin Registry, University of Southern Denmark, Odense; 3 Biotech Research and Innovation Centre; 4 The Danish Stem Cell Centre (DanStem), University of Copenhagen, Copenhagen; 5 Department of Clinical Genetics, Odense University Hospital, Odense; 6 Finsen Laboratory, Rigshospitalet, Copenhagen; 7 Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is commonly ocurring in the elderly and associated with increased morbidity and mortality. CHIP is associated with accelerated epigenetic age (Robertson et al. Curr Biol 29 , R786–R787, 2019; Nachun et al., Aging Cell, 20 , e13366, 2021) and especially with intrinsic measures of epigenetic age, i.e. measures which reflect cell-type independent effects of aging on DNA methylation, as opposed to extrinsic measures that reflect age-related changes to the immune-cell composition of the peripheral blood. Furthermore, Nachun et al. reported that the combined measure of Hannum and GrimAge acceleration modifies the effect of CHIP mutation status, resulting in strongly increased risk of all-cause mortality in individuals with both CHIP and accelerated Hannum age and GrimAge age.

Aims: To replicate and extend these findings in elderly Danish twins, especially investigate gene-specific effects on extrinsic vs. intrinsic estimators and the effect of shared environmental and genetic factors in intra-twin pair analyses.

Methods: Blood samples collected from elderly Danish twins (mean age 79 years, range 73–90) in 1997 were analyzed for CHIP mutations using a custom Illumina TruSeq next-generation sequencing panel targeting 21 og the most commonly mutated genes in CHIP. DNA methylation profiling was performed on the Illumina HumanMethylation450K BeadChip. Associations between CHIP and epigenetic age were investigated using linear regression (individual-level analyses) and conditional logistic regression (intra-pair analyses). The interaction between CHIP and epigenetic age acceleration was analysed using Cox proportional hazards regression.

Results: Of 308 individuals (154 twin pairs), 116 carried a CHIP mutation. The strongest association with CHIP in individual-level analyses was seen for the Intrinsic Epigenetic Age Acceleration (IEAA) estimator (CHIP carriers 1.40 years older [95% CI: -0.01–2.81]), and in intra-pair analyses Hannum age showed the strongest association (OR 1.1 [1.01–1.19]). In mutation-specific analyses, TET2 mutations were associated with the extrinsic Hannum age estimator in both individual-level (3.12 years [1.07–5.16]) and intra-pair analyses (OR 1.11 [0.99–1.24]). DNMT3A mutations were associated with IEAA in individual-level (1.82 years, [0.07–3.57]) but not intra-pair analysis (OR 1.03, 0.92–1.14). Analyses of logit-transformed VAF were consistent with these results. Individuals with both CHIP and accelerated Hannum and GrimAge estimates did not have an increased mortality risk in our cohort (HR 1.02, [0.70–1.46]).

Summary/Conclusion: TET2 mutations were predominantly associated with extrinsic epigenetic age acceleration and the effect remained when controlling for shared factors between co-twins. Conversely, DNMT3A mutations were associated with intrinsic epigenetic age acceleration, and the intra-pair analysis indicated that this association may be due to a shared genetic and/or environmental background. The association between TET2 mutations and extrinsic epigenetic aging indicates that TET2 mutated clones may be drivers of immunosenescence, while the association between DNMT3A mutations and intrinsic epigenetic aging may be due to increased stem cell proliferation rates in individuals with DNMT3A mutations. The CHIP-age acceleration interaction reported by Nachun et al was not replicated, indicating that this mortality model should be used with caution, especially in this age group.

P751: CHARACTERIZING CIRCULAR RNA EXPRESSION IN MYELODYSPLASTIC SYNDROME

E. Wedge 1,2,3,* , C. R. M. Côme 2,3 , J. W. Hansen 1,2,3 , J. S. Jespersen 2,3,4 , M. Dahl 1,2,3 , C. Schöllkopf 1 , K. Raaschou-Jensen 5 , B. Porse 2,3,4 , J. Weischenfeldt 2,3,4 , L. S. Kristensen 6 , K. Grønbæk 1,2,3

1 Department of Hematology, Rigshospitalet; 2 Biotech Research and Innovation Center (BRIC); 3 Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), Faculty of Health and Medical Sciences, University of Copenhagen; 4 The Finsen Laboratory, Rigshospitalet, Copenhagen; 5 Department of Hematology, Odense University Hospital, Odense; 6 Department of Biomedicine, Aarhus University, Aarhus, Denmark

Background: Circular RNA (circRNA) research is an expanding field, reflecting a recognition that circRNAs may have various roles in cancer pathology. CircRNAs are formed when back-splicing events create covalently closed loop structures from pre-mRNA molecules (Kristensen LS, Nat Rev Genet, 2019 ). CircRNA expression is cell-type specific, also during hematopoietic differentiation (Nicolet BP, Nucleic Acids Res, 2018 ), but their biological significance in myeloid cancers is still largely unknown.

Aims: We aimed to profile circRNA expression in myelodysplastic syndrome (MDS), along with the related diseases chronic myelomonocytic leukemia (CMML) and clonal cytopenia of undetermined significance (CCUS), in order to identify disease-specific expression patterns and relevant candidates for biological or biomarker exploration.

Methods: Seventy-one patients and eight healthy age-matched controls were included. Bone marrow aspirate underwent FACS sorting to obtain CD34+ stem and progenitor cells. RNA was extracted and total RNA libraries were created using enzymatic ribosomal RNA removal. The RNA was sequenced on a NovaSeq 6000 (mean 270 million paired-end reads per sample, read length 150 bp). Bioinformatic pipelines find_circ and CIRI2 were used to identify and quantify reads originating from the back-splicing junctions of circRNAs. Detection by both pipelines was required, plus presence in at least 10% of the samples. DESeq2 was used for normalization and comparison of groups. CircRNAs with a mean expression of >10 normalized reads were considered highly abundant and this subset was the focus of analysis.

Results: In total, 8,651 unique circRNAs passed the filtering criteria, and the top 826 were highly abundant. Global circRNA expression showed upregulation in CCUS relative to healthy controls (p<0.001) and MDS relative to CCUS (p<0.001) (Figure A). We found that 110 unique circRNAs were significantly upregulated in MDS relative to healthy controls, whilst none were significantly downregulated (Figure B). In addition, high or very high IPSS-R score was associated with significantly higher circRNA expression than lower scores (p<0.001). We used LASSO regression to identify 14 circRNAs which may be related to clinical outcomes, and used these to calculate a Myeloid Circ Score (MCS). Patients were designated ‘high MCS’ or ‘low MCS’ with a cut-off at the median. A high MCS was associated with poorer progression-free survival (PFS) with a hazard ratio (HR) of 33.5 (95%CI 7.8-143.5, p<0.001) and poorer overall survival (OS) with a HR of 16.5 (95%CI 3.8-71.5, p<0.001) in the whole cohort. The same could be seen in analysis of MDS patients only (PFS HR 14.2 (95%CI 3.2-62.7, p<0.001), OS HR 8.5 (95%CI 1.9-38.3, p=0.005)). This a greater hazard ratio than that of the IPSS-R in this cohort (Very high/high/Intermediate IPSS-R relative to low/very low IPSS-R; HR for PFS of 4.27 (95%CI 1.7-11.1, p=0.003), and HR for OS of 2.5 (95%CI 0.9-7.3, p=0.09)).

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Summary/Conclusion: We have observed associations between overall circRNA abundance and disease severity in myeloid cancer, including a global upregulation along the spectrum of disease from healthy to CCUS to MDS, and upregulation in high risk MDS compared to lower risk MDS. CircRNAs may be directly implicated in disease biology or be reflective of other changes at the cellular level. The Myeloid Circ Score has potential in risk stratification but requires validation in independent cohorts and further investigation in more readily available tissues such as bulk bone marrow or peripheral blood.

P752: TARGETING S100A9 IN THE MYELODYSPLASTIC INFLAMMATORY BONE MARROW MICROENVIRONMENT BY TASQUINIMOD IMPROVES THE SUPPORTIVE FUNCTION OF MESENCHYMAL STROMAL CELLS

M. Wobus 1,2,* , E. Balaian 1,2 , M. Lissner 1 , R. Towers 1 , U. Oelschlägel 1 , R. Wehner 2,3 , T. Chavakis 4 , M. Törngren 5 , H. Eriksson 5 , E. Bondesson 5 , U. Platzbecker 6 , M. Bornhäuser 1,2 , K. Sockel 1

1 Department of Medicine 1, University Hospital TU Dresden, Dresden; 2 German Cancer Consortium (DKTK), Partner Site Dresden, and German Cancer Research Center (DKFZ), Heidelberg; 3 Institute of Immunology; 4 Institute of Clinical Chemistry and Laboratory Medicine, University Hospital TU Dresden, Dresden, Germany; 5 Active Biotech AB, Lund, Sweden; 6 Medical Clinic and Policlinic 1, Hematology and Cellular Therapy, Leipzig University Hospital, Leipzig, Germany

Background: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. An aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species (ROS). Tasquinimod (TASQ, Active Biotech) is a novel, small molecule inhibitor of S100A9 which has been investigated in several solid tumor entities and is currently under investigation in a phase Ib/IIa trial of patients with relapsed/refractory multiple myeloma. So far, little is known about its effects on myeloid malignancies.

Aims: We aimed to investigate the role of S100A9 in cellular models of MDS and the in vitro potential of TASQ to target S100A9 within the MDS microenvironment.

Methods: Mesenchymal stromal cells (MSCs) from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) ± TASQ (10µM). Subsequently, TLR4 downstream and proinflammatory signaling was analyzed by Western blot and real-time PCR. ROS levels were determined with a CellROX Flow Cytometry Kit (Thermo Fisher). Moreover, MSC differentiation and colony-forming unit-fibroblast (CFU-F) capacity were tested. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium.

Results: Exposure of MDS and healthy MSCs to S100A9 induced TLR4 downstream signaling as demonstrated by increased expression of IRAK1 and NF-kB-p65 as well as gasdermin, an inductor of pyroptosis. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. In line with this, OXPHOS and ROS levels were decreased after addition of TASQ. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1β and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50% both at the mRNA and protein level. Moreover, addition of TASQ to MDS MSC cultures resulted in a significantly higher CFU-F number as well as abolished blocked the excessed adipogenic differentiation.

In co-cultures with HSPCs, we observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs, which could be restored by TASQ pre-treatment.

Summary/Conclusion: We provide evidence that TASQ mitigates the pathological inflammasome activation in the myelodysplastic BM niche in vitro by inhibition of the S100A9-mediated TLR4 signalling as well as its effects on NF-kB-p65 transcription and PD-L1 expression, resulting in improved hematopoietic support by MSCs, which suggests a beneficial effect in cytopenic MDS patients.

P753: DYSFUNCTIONAL BONE MARROW ENDOTHELIAL PROGENITOR CELLS ARE INVOLVED IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES

T. Xing 1,2,* , Z.-S. Lyu 1,2 , C.-W. Duan 3 , H.-Y. Zhao 1 , S.-Q. Tang 1 , Q. Wen 1 , Y.-Y. Zhang 1 , M. Lv 1 , Y. Wang 1 , L.-P. Xu 1 , X.-H. Zhang 1 , X.-J. Huang 1,2 , Y. Kong 1

1 Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University People’s Hospital; 2 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing; 3 Key Laboratory of Pediatric Hematology and Oncology Ministry of Health and Pediatric Translational Medicine Institute, Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of medicine, Shanghai, China

Background: Myelodysplastic syndromes(MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective haematopoiesis and immune deregulation. Emerging evidence has shown important roles of the bone marrow(BM) microenvironment in regulating haematopoiesis and immune balance. As an important component of BM microenvironment, murine studies and our previous studies( Blood 2016, EbioMedicine 2020, CEI 2021) reported endothelial progenitor cells(EPCs) modulate the physiology and regeneration of haematopoietic stem cells(HSCs) and differentiation of effector T cell subsets. Some evidences suggest that EPCs demonstrate inferior supporting ability to normal HSCs in MDS. However, the dual supporting abilities of BM EPCs to normal HSCs and malignant cells in patients with different stages of MDS remain unclear. In addition, the immunomodulatory ability of BM EPCs in MDS needs to be elucidated.

Aims: To determine the number, the functions and the abilities of haematopoiesis and immune regulation of BM EPCs in patients with different stages of MDS. Moreover, to explore the correlation between dysfunctional BM EPCs and different stages of MDS and underlying mechanisms of dysfunction.

Methods: In this study, patients with lower-risk MDS(N=15), higher-risk MDS(N=15) and de novo acute myeloid leukaemia(AML)(N=15) and healthy donors(HDs)(N=15) were enrolled. The number of primary BM EPCs were detected by flow cytometry. The functions of BM EPCs were evaluated by DiI-Acetylated low-density lipoprotein uptake and FITC-UEA-1 binding assay, tube formation and migration assays. In order to assess the gene expression profiles of BM EPCs, RNA sequencing and gene set enrichment analysis were performed. The supporting abilities of BM EPCs from MDS patients to normal HSCs, leukaemia cells and T cells were assessed by in vitro coculture experiments(coculture with HSCs and T cells from HDs and HL-60 cells, respectively).

Results: Increased but dysfunctional BM EPCs were found in MDS patients compared with HDs, especially in patients with higher-risk MDS. RNA-seq indicated the changes of haematopoiesis- and immune-related pathways in MDS BM EPCs. In vitro coculture experiments verified that BM EPCs from HD, lower-risk MDS, and higher-risk MDS to AML exhibited a progressively decreased ability to support normal HSCs, manifested as elevated apoptosis rates and intracellular reactive oxygen species(ROS) levels and decreased colony-forming unit plating efficiencies of HSCs. Moreover, BM EPCs from higher-risk MDS patients demonstrated an increased ability to support leukaemia cells, characterized by increased proliferation, leukaemia colony-forming unit plating efficiencies, whereas decreased apoptosis rates and apoptosis-related genes of HL-60 cells. Furthermore, BM EPCs induced normal T cell differentiation towards more immune-tolerant cells, like regulatory T cells, in higher-risk MDS patients in vitro. In addition, the levels of intracellular ROS and the apoptosis ratio were increased in BM EPCs from MDS patients than HDs, especially in higher-risk MDS patients, which may be underlying mechanisms of dysfunctional BM EPCs.

Summary/Conclusion: The current study demonstrated that increased but dysfunctional BM EPCs in MDS patients. Moreover, the dysfunctions of BM EPCs were more severe in higher-risk than lower-risk MDS patients, manifested as more supportive ability to leukaemia cells but worse ability to normal HSCs. Our data indicated that repair of dysfunctional BM EPCs may be a promising therapeutic approach for MDS patients.

P754: INITIAL RESULTS OF PHASE I/II STUDY OF AZACITIDINE IN COMBINATION WITH QUIZARTINIB FOR PATIENTS WITH MYELODYSPLASTIC SYNDROME AND MYELODYSPLASTIC/MYELOPROLIFERATIVE NEOPLASM WITH FLT3 OR CBL MUTATION

T. Abuasab 1,1,* , E. Jabbour 1 , N. Short 1 , M. Konopleva 1 , K. S. Chien 1 , S. Fareed Mohamed 1 , N. Daver 1 , R. Kanagal-shamanna 2 , G. Garcia-Manero 1 , G. Montalban-Bravo 1

1 Department of Leukemia; 2 Department of Hematopathology, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Houston, United States of America

Background: FMS-like tyrosine kinase 3 ( FLT3 ) mutations occur in about 1% of patients (pts) with newly diagnosed myelodysplastic syndrome (MDS) and up to 19% at time of failure of hypomethylating agent (HMA). In addition, Casitas B-lineage Lymphoma mutations ( CBL ) are observed in 10% of pts with MDS/MPN and up to 13-15% of pts with CMML (mainly proliferative type). Preclinical studies suggest that CBL -mutant cells are dependent on FLT3 signaling.

Aims: The primary objective is to determine the safety, tolerability, and maximum tolerable dose (MTD) of quizartinib. Secondary objectives include evaluation of overall response per IWG 2006 criteria, in addition to overall survival (OS), duration of response, leukemia-free survival (LFS), relapse-free survival (RFS).

Methods: We designed a phase I/II study of azacitidine in combination with quizartinib for pts with newly diagnosed or previously treated MDS or MDS/MPN with detectable FLT3 and/or CBL mutations. The study included an initial phase I dose escalation portion followed by a phase II dose expansion. Dose escalation was planned to evaluate 3 dose levels of quizartinib (30, 40 and 60mg, respectively) administered on days 1-28 of every cycle in combination with azacitidine (75 mg/m2/day) on days 1-5 each 28-day cycle.

Results: As of February 25, 2022, a total of 10 pts have been enrolled (9 in dose escalation, 1 in dose expansion): 7 pts with CMML, two with MDS with excess blast and one with atypical chronic myeloid leukemia (aCML). A total of 8 pts (80%) had normal karyotype, one ptn (10%) had complex karyotype and one (10%) had monosomy 7. Six pts had FLT3 mutations and 4 pts CBL mutations. Pts characteristics are summarized in Table 1. Nine out of 10 pts were evaluable at the time of the analysis. Median number of cycles administered was 5 (range 2-10). All pts have achieved response so far with 8 pts (89%) having achieved marrow CR (mCR), including hematological improvement (HI) in 1 patient out of 3 pts with baseline cytopenias evaluable for HI. One patient had hematologic improvement-erythropoietic (HI-E) with control of leukocytosis and thrombocytosis to normal levels. During cycle 1, responses were associated with recovery of platelets to above 50 and 100x10 9 /L by day 28 of therapy in 67% and 56% of pts, respectively. In addition, absolute neutrophils count (ANC) was recovered to 1 x10 9 /L by day 28 in all pts except of one patient at day 52.

With a median follow up of 9.87 months (range 2.5-18), the median response duration is 6.3 months (range 1.6-14.3) with a median OS of 17.34 months (range 2.5 – 18). A total of 4 pts (44%) have stopped the treatment: 1 patient due to disease relapse and one due to disease progression after both received 6 cycles of treatment and two proceeded to allogenic stem cell transplantation after achieving response. No dose-limiting toxicities have been observed to date up to the maximum dose of 60 mg of quizartinib. Two pts required dose reductions of Quizartinib, one due to cytopenias and the other one due to concomitant posaconazole administration. Regarding the grade 3 or more adverse events (AEs): 3 pts (33%) had severe anemia/thrombocytopenia, 2 pts (22%) had skin infection, one patient (11%) had cardiac arrythmia with Mobitz II AV block and one patient (11%).

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Summary/Conclusion: Preliminary data suggest azacitidine in combination with quizartinib for pts with MDS and MDS/MPN with FLT3 or CBL mutations have an acceptable toxicity profile and associated with promising responses. Further follow-up with a larger patient cohort are required to emphasize the safety and efficacy.

P755: HYPERFERRITINEMIA IS A PREDICTIVE BIOMARKER OF POOR CLINICAL OUTCOMES IN CMML

L. E. Aguirre 1,* , S. Ball 1 , A. Jain 1 , N. Al Ali 1 , D. Sallman 1 , A. Kuykendall 1 , K. Sweet 1 , J. Lancet 1 , E. Padron 1 , R. Komrokji 1

1 Malignant Hematology, Moffitt Cancer Center, Tampa, United States of America

Background: CMML is a heterogenous disease exhibiting features innate to MPN and MDS. Increasing evidence supports a close interplay between systemic inflammation and risk of myeloid malignancies, notably for those with history of infection or autoimmune disease. CMML has been associated with inflammation and end-organ damage related to CKD and CVD. Analysis of gene signatures from CMML-derived monocytes has shown them to be highly proinflammatory. High ferritin may serve as a practical biomarker of disease activity to help identify pts at higher risk of poor outcomes.

Aims: We aimed to identify whether hyperferritinemia correlated with worse outcomes in CMML and to characterize relevant baseline clinical and molecular features associated with this clinical phenotype.

Methods: Retrospective data was collected from a database of pts with CMML treated at Moffitt Cancer Center. Pts were stratified in 2 cohorts based on ferritin levels (<1000 or ≥1000 ng/mL). Hyperferritinemia was defined as ferritin >1000 as seen at diagnosis or during follow-up. Kaplan–Meier was used to estimate OS. Cox regression was used for multivariate analysis.

Results: Between August 1995 and October 2020 729 pts with CMML were identified. Median age at diagnosis was 71 (17-95). Out of 571 pts with available ferritin levels 29% (n=168) developed hyperferritinemia vs 71% (n=403) who did not. mOS was 32.4 mos (95%CI 30-35 mos).

Pts with higher ferritin tended to present with CMML-2 (p=0.001) and harbor a proliferative phenotype (p=0.01). They presented with higher marrow cellularity (mean 83%, p =0.08), PLT (mean 177k, p= 0.038), and lower Hb (mean 9.5, p<0.05). There was no association with % circulating IMC, monocytes, WBC or ANC at baseline. Hyperferritinemia was associated with more profound fibrosis (p=0.007), cytopenias (p<0.05), % peripheral blasts (p<0.05), RBC and PLT transfusion dependence (p<0.05).

Pts with hyperferritinemia had higher risk disease per IPSS-R, CPSS and all CMML models (p<0.05); and had higher rates of AML transformation (p<0.05). Pts were also more likely to require treatment earlier (within 3 yrs of diagnosis) (p<0.05).

ASXL1 (p=0.002), EZH2 (p=0.003), and SETBP1 (p=0.019) mutations were more common among pts who developed hyperferritinemia. Conversely, TET2 (p=0.001), CBL (p=0.028) and SRSF2 (p=0.003) mutations were less common.

mOS for pts with hyperferritinemia was 23.9 mos (95%CI 19.9-27.9 mos), much lower than for those with ferritin <1000 (mOS 40.5 mos, 95%CI 35.4-45.5 mos) (p<0.05). In multivariate analysis, hyperferritinemia was a significant independent covariate for OS after adjusting for CPSS, transfusion dependence and disease phenotype (dysplastic vs proliferative) (HR= 0.69; 95%CI 0.53-0.89; p=0.005).

Summary/Conclusion: Almost 1/3 of pts with CMML will develop hyperferritinemia. This is associated with more aggressive disease and rates of AML transformation leading to dismal outcomes. ASXL1, EZH2, and SETBP1 MTs confer a higher risk of hyperferritinemia. Our findings indicate that hyperferritinemia is an independent prognostic biomarker that may serve as a surrogate representative of disease biology and comorbidities in CMML.

P756: ALLOGENEIC STEM CELL TRANSPLANTATION IN MYELODYSPLASTIC SYNDROME/MYELOPROLIFERATIVE NEOPLASM (MDS/MPN) OVERLAP SYNDROMES. A SINGLE CENTER EXPERIENCE AND DISSECTION OF MUTATIONAL PROFILE

A. Avendaño Pita 1,* , M. Martín Izquierdo 1 , S. Muntión Olave 1 , T. Jímenez Solas 1 , M. García Antúnez 1 , L. Eva 1 , M. Del Rey 1 , S. Toribio Castelló 1 , A. Yeguas Bermejo 1 , T. González 1 , J. M. Hernández-Rivas 1 , A. Á. Martín López 1 , M. Cabrero 1 , E. Pérez López 1 , A. Cabero 1 , M. Baile 1 , L. Vázquez 1 , L. López Corral 1 , F. Sánchez-Guijo 1 , D. Caballero Barrigon 1 , M. Cortés Rodríguez 1 , M. Díez Campelo 1

1 Hematology, University Hospital of Salamanca / IBSAL, Salamanca, Spain

Background: MDS/MPN (myelodysplastic syndrome/myeloproliferative neoplasm) overlap syndromes are myeloid malignancies for which allogeneic hematopoietic stem cell transplant (allo-HSCT) is potentially curative in the absence of disease-modifying therapies.

Aims: To describe clinical, biological and molecular characteristics at diagnosis and transplant outcomes of our series trying to detect witch parameters may have impact on survival.

Methods: We retrospectively reviewed 35 consecutive patients who underwent to allo-HSCT from 1999 to 2021 as MDS/MPNS overlap syndrome. Descriptive statistics and survival analysis were performed trough jamovi (Version 2.2.).

Results: Clinical, biological and molecular baseline characteristics are summarized in image. Median age at allo-HSCT were 58 (27-70) with a median time from diagnosis to allo-HSCT of 8 months (1-128). Twenty patients (57%) were referred in complete remission (CR). Preferred graft source were peripheral blood in 34 patients (97.1%). Regarding to conditioning regimen, 26 patients (74.3%) received reduced intensity, 7 patients (20%) received myeloablative and 2 patients (5.7%) sequential conditioning. Eighteen patients (51.4%) had matched sibling donor, 8 patients (22.8%) had matched unrelated donor, 3 patients (8.6%) mismatched unrelated donor and 6 (17.1%) haploidentical donor. All patients reached neutrophil graft and 31 patients (88.6%) reach platelet graft with a median time of 19 (12-29) and 14 (10-36) days respectively. On day +100, 29 patients (82.9%) were in CR, 4 patients (11.4%) did not reach response and 2 (5.7%) were not evaluable. Twenty three patients (65.7%) developed acute graft versus host disease (GVHD), in 19 cases (54.3%) grades II-IV, and 15 patients (42.9%) developed chronic GVHD, in 8 cases (22.9%) moderate-severe grade.

Median follow-up were 13 months (2-153) with 17 months (2-153) for chronic myelomonocitic leukemia (CMML) and 11.5 months (2-133) for atypical chronic. myeloid leukemia (aCML). Overall survival (OS) at 1, 3, and 5 years were 69.7%, 54.9% and 42.5% respectively.

Sixteen patients (45.7%) relapsed with a median time of 7.5 months (0-85). Among relapsed patients, 5 patients (31.3%) were not treated, 3 patients (19.8%) received donor lymphocyte infusion, in 5 patients (31.3%) we administered hypometilating agents and salvage chemotherapy in 2 (12.5%) remaining patients reaching second allo-HSCT in two cases. Non-relapse mortality were 31.4%, in most cases due to infection. Transplant related mortality (TRM) at day +100 and global TRM were 5.7% and 14.3% respectively. At last follow-up, 13 patients (37.1%) remain alive, 11 (31.4%) in CR and 2 (5.7%) in relapsed situation.

On the univariate analysis, harboring signal transduction mutation (p = 0.008), mutational global burden by variant allelic frequency (VAF) at diagnosis greater than 30% (p = 0.045) were asociated with worse outcomes. On the other hand, reaching CR at day +100 (p <0.0001) and developing chronic GVHD (p = 0.0001) seems to improve survival. If we look at the CMML subgroup, we found that new CMML transplant score significantly stratifies outcomes (p = 0.006).

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Summary/Conclusion: Once again, poor results are confirmed for this MDS/MPN overlap syndromes with only a third of the patients being long term survivors. Molecular data offered by NGS may help to refine prognostic scores and to stablish transplant referral. Mutations in signaling pathways seems to confer poor prognosis as a late event in disease course and provide more aggressive clinical behavior.

P757: RESULTS OF A PHASE 1 STUDY OF AZACITIDINE COMBINED WITH VENETOCLAX FOR TREATMENT-NAIVE AND RELAPSED HIGH-RISK MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA

A. Bazinet 1,* , E. Jabbour 1 , H. Kantarjian 1 , K. Chien 1 , C. DiNardo 1 , M. Ohanian 1 , N. Daver 1 , R. Kanagal-Shamanna 2 , T. Kadia 1 , K. Takahashi 1 , L. Masarova 1 , N. Short 1 , Y. Alvarado 1 , P. Thompson 1 , G. Montalban-Bravo 1 , M. Yilmaz 1 , F. Ravandi 1 , M. Konopleva 1 , M. Andreeff 1 , S. Kornblau 1 , N. Pemmaraju 1 , D. Hammond 1 , H. Schneider 1 , B. Mirabella 1 , G. Garcia-Manero 1

1 Leukemia; 2 Hematopathology, University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Patients (pts) with higher-risk myelodysplastic syndromes (HR-MDS) who are ineligible for allogeneic stem cell transplant (SCT) have limited treatment options beyond the hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DAC). Responses to HMA monotherapy are time-limited and prognosis after HMA-failure is dismal. Venetoclax (VEN) is an oral small molecule BCL-2 inhibitor with the potential to prolong/deepen responses when combined with an HMA.

Aims: To evaluate the safety/tolerability, recommended phase 2 dose (RP2D), and preliminary efficacy of VEN in combination with AZA for the treatment of HR-MDS/CMML.

Methods: This was a single-center dose-escalation phase 1 study with “3 + 3” design. Key inclusion criteria were a diagnosis of MDS or CMML by WHO 2016, age ≥ 18 yrs, int-2 or high risk by IPSS, and bone marrow (BM) blasts of 5-19%. Pts could be treatment-naïve or relapsed/refractory, defined as failure after 4+ cycles of HMA. Prior VEN exposure was not permitted. Treatment consisted of AZA 75 mg/m2 on D1-5 and escalating doses of VEN on D1-7 or 1-14 (Table 1) every 28D. VEN dosing was adjusted for pts on concomitant CYP3A inhibitors. The primary objective was safety/tolerability and determination of the RP2D. Secondary objectives were response rates, overall survival (OS), and progression-free survival (PFS). Adverse events (AEs) were graded as per the CTCAE v5.0 and responses assessed using the modified IWG 2006 criteria. All pts provided informed consent. The study was registered on ClinicalTrials.gov ( NCT04160052 ).

Results: Data cutoff was February 19th, 2022. 23 pts (13 HMA-naïve MDS, 4 HMA-naïve CMML, 4 HMA-failure MDS, 2 HMA-failure CMML) were enrolled. The median age was 68 years (range 58-84) and median BM blasts 11% (range 6-19%). IPSS was int-2 in 78% of pts and high in 22% of pts. 8/23 (35%) had complex cytogenetics, 6/23 (26%) had TP53 mutations, and 5/23 (22%) had therapy-related MDS/CMML. A single dose-limiting toxicity (DLT) occurred at dose level +2 (BM aplasia and delayed count recovery). The maximum tolerated dose (MTD) was not reached. The most common grade 3/4 AEs were neutropenia (39%), thrombocytopenia (39%), anemia (13%), and infections: pneumonia (30%), neutropenic fever (17%), diverticulitis (9%), sepsis (9%), cellulitis (4%), and splenic abscess (4%). The RP2D was established at AZA 75 mg/m2 on D1-5 plus VEN 400 mg on D1-14. 3 deaths occurred on study, all from sepsis. 30 and 60-day mortality were 4% and 9%, respectively. Other reasons for discontinuing therapy included progression of MDS/CMML (n=6), transformation to AML (n=4), SCT (n=4), and social reasons (n=1). The ORR by intention-to-treat (ITT) analysis was 82% (14/17; 3 CR, 5 mCR+HI, 6 mCR) in the HMA-naïve cohort and 100% (6/6; 6 mCR) in the HMA-failure cohort. A median of 3 cycles (range 1-11) were given. Responses occurred after a median of 1 cycle (range 1-2) and lasted a median of 4.5m (range 1.2m to 21.2m). At a median follow-up of 13.2m, median OS was not reached (1-year OS 68.6%) in the HMA-naïve cohort and 8.3m (1-year OS 22%) in the HMA-failure cohort. Median PFS was 13.1m (1-year PFS 51.5%) in the HMA-naïve cohort and 6.8m (1-year PFS 0%) in the HMA-failure cohort.

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Summary/Conclusion: AZA combined with VEN is tolerable and yields a high ORR in HR-MDS/CMML, with or without prior HMA. Rates of myelosuppression and infection are high and careful monitoring is required. The RP2D is AZA 75 mg/m2 on D1-5 plus VEN 400 mg on D1-14, every 28D. The phase 2 dose expansion component of this study is ongoing.

P758: INHIBITION OF ATR WITH CERALASERTIB (AZD6738) FOR THE TREATMENT OF PROGRESSIVE OR RELAPSED MYELODYSPLASTIC SYNDROMES AND CHRONIC MYELOMONOCYTIC LEUKEMIA: A PHASE IB/II STUDY

A. Brunner 1,* , Y. Liu 2 , L. Mendez 3 , J. Garcia 2 , P. Amrein 1 , D. Neuberg 2 , E. Dean 4 , S. Smith 4 , R. Stone 2 , A. Fathi 1 , M. Walter 5 , T. Graubert 1 , M. Jacoby 5

1 Massachusetts General Hospital; 2 Dana-Farber Cancer Institute; 3 Beth-Israel Deaconess Medical Center, Boston, United States of America; 4 AstraZeneca, Cambridge, United Kingdom; 5 Washington University School of Medicine, St. Louis, United States of America

Background: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are enriched for mutations in the spliceosome complex, including the genes SF3B1, SRSF2, ZRSR2, and U2AF1 . Ceralasertib (AZD6738) is a selective and potent inhibitor of Ataxia Telangiectasia and Rad3 Related (ATR) kinase. We have previously shown that cells with splicing factor (SF) mutations have increased R loops, which require ATR for resolution and preferentially undergo apoptosis after ATR inhibition.

Aims: We hypothesized that inhibition of ATR may be useful in patients with MDS or CMML, particularly those who harbor SF gene mutations.

Methods: This 2-part study evaluated ceralasertib monotherapy in adult patients with MDS or CMML progressing on or not responsive to front-line therapy (DNMTI for HR MDS, ESA for LR MDS). Enrollment cohorts included SF mutant ( SF3B1 , SRSF2 , U2AF1 , or ZRSR2 ) and SF wildtype (wt). Part 1 enrolled 3-6 patients at de-escalating dose levels: 160mg BID days 1-14 of 28 d cycles, 120mg BID d1-14, and 80mg BID d1-14. DLTs were assessed the first 30d and de-escalation would occur for DLTs in >1/6 patients. Higher-risk MDS (HR-MDS, IPSS-R > 3.5) and CMML patients were enrolled first; after a dose was deemed safe, lower-risk MDS (LR-MDS, IPSS-R 3.5, transfusion-dependent post/ineligible for ESA or severe neutropenia/thrombocytopenia) was evaluated. Part 2 enrolled up to 20 SF mut patients and 20 SF wt patients for efficacy. A Simon 2-stage design mandated that at least 1/10 patients in each group needed to respond (by IWG 2006 criteria) to enroll the final 10 patients.

Results: At the data cut-off (12/15/21) 32 patients have been enrolled with 30 evaluable: 22 with HR-MDS or CMML, and 8 with LR-MDS. The median age was 73 (range 43-88) and predominantly male (26/30). 21 patients harbored SF mut disease while 9 had SF wt MDS/CMML. The most frequently seen ≥grade 3 adverse events (AEs) for higher-risk patients (n=20) included anemia (n=6), febrile neutropenia (n=4), neutropenia (n=5), thrombocytopenia (n=6). In LR-MDS (n=8), ≥grade 3 AEs included thrombocytopenia (n=2), and neutropenia (n=2). 5 deaths occurred within 30d of study treatment; 1 patient with active CMML had an intracranial bleed with thrombocytopenia, possibly related to study treatment, while 4 deaths were attributed to underlying disease (3 with progression to AML, 1 MDS patient with sepsis). No DLTs were observed in the HR or LR groups and 160mg BID days 1-14 of 28 days was deemed the RP2D.

Responses were assessed across the cohort as well as according to SF mutant status (Table). A total of 8 patients had an IWG response (BOR: CR, n=1; mCR, n=3; HI, n=4), including 6/21 SF mut patients (ORR 29%), and 2/9 SF wt patients (ORR 22%). The responses in SF wt patients included one mCR (5% to 1% marrow blasts in a patient with DNMT3A and ETV6 mut and del(chr7)), and one HI-P (82 BL to 234 max in a patient with DNMT3A, IDH2, ETV6 mut and del(chr7)). Responses in SF mut included CR (n=1), mCR (n=2, one with associated HI-N), SD with HI (n=3). An additional 3 SF mut patients had improvements in blood counts but not lasting 8 weeks (ANC n=2, platelets n=1) and one with platelet improvement but a baseline not meeting IWG criteria (plt avg=108k prior to C1D1).

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Summary/Conclusion: Ceralasertib is an oral ATR inhibitor with preliminary activity in patients with progressive/refractory MDS and CMML at doses of 160mg BID on days 1-14 of a 28 day cycle. The study is ongoing and will evaluate additional dosing schedules.

P759: CORRELATION ANALYSIS OF DNA METHYLATION LEVEL AND CLINICAL CHARACTERISTICS IN JMML PATIENTS

Y. Cai 1,* , J. Zhang 1 , M. Yi 1 , X. Liu 1 , X. Zhang 1 , Y. Wan 1 , L. Chang 1 , L. Zhang 1 , X. Chen 1 , Y. Guo 1 , Y. Zou 1 , Y. Chen 1 , Y. Zhang 1 , W. Yang 1 , X. Zhu 1

1 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: As a rare, aggressive pediatric myeloproliferative disease, juvenile myelomonocytic leukemia (JMML) encompassed both biological features of myelodysplastic syndrome and myeloproliferative neoplasm. Studies have shown that the methylation level in JMML patients is closely related to prognosis, and patients with high methylation level have poor prognosis.

Aims: This study aimed to find clinical indicators that were associated with different methylation levels and prognosis.

Methods: The clinical information of 24 JMML patients with DNA samples admitted to our center from December 2013 to May 2020 was retrospectively analyzed, and the DNA methylation level of their whole genome was detected.

Results: The median age of onset was 14.5 months (0.1-153 months) among the 24 cases, including 17 males and 7 females. At diagnosis, the median WBC count was 27.1×10 9 /L (6.2-98.1×10 9 /L), and the median platelet count was 38×10 9 /L (10-277×10 9 /L). Chromosome karyotype abnormalities were found in 12.5% (3/24) of patients. Next-generation sequencing results showed that 79.2% (19/24) patients had at least one Ras pathway-related classical gene mutation, and 41.7% (10/24) patients had two or more somatic mutations. Genomic DNA methylation levels were divided into three groups: 11 cases in the hypomethylation group, 2 cases in the moderate methylation group, and 11 cases in the hypermethylation group. The concordance rate of our methylation subgroup compared with the international consensus classification was 91.7%. There were significant differences in age, platelets, PTPN11 gene mutation and the number of somatic mutations ≥2 in different methylation groups (P<0.05). Correlation analysis showed that hypermethylation level was significantly correlated with PTPN11 gene mutation and ≥2 somatic mutations (P<0.001).

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Summary/Conclusion: JMML patients with high methylation level in the DNA genome at diagnosis were older and with lower platelet levels, and hypermethylation were significantly correlated with high-risk prognostic factors such as PTPN11 gene mutation and ≥2 somatic mutations.

P760: PATTERNS OF HYPOMETHYLATING AGENT FAILURE IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES

K. Chien 1,* , K. Kim 1 , Z. Li 2 , R. Kanagal Shamanna 3 , F. Ong 1 , G. Montalban Bravo 1 , T. Kadia 1 , E. Jabbour 1 , N. Pemmaraju 1 , D. Hammond 1 , N. Short 1 , F. Ravandi 1 , Y. Alvarado 1 , S. Pierce 1 , X. Q. Dong 1 , H. Kantarjian 1 , G. Garcia-Manero 1

1 Leukemia; 2 Biostatistics; 3 Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Hypomethylating agents (HMA), such as azacitidine and decitabine, are the current standard-of-care for patients (pts) with myelodysplastic syndrome (MDS). Though HMA improve cytopenias and delay progression of disease, most pts eventually response to these agents with poor survival outcomes afterward. This poses significant treatment challenges after HMA failure (HMA-F) and an increased risk of transformation to acute myeloid leukemia (AML).

Aims: Here, we assess the clinicopathologic characteristics and outcomes of pts with HMA-F MDS.

Methods: We retrospectively evaluated all pts with MDS seen at a single tertiary cancer center from July 2017 to July 2021 and identified those who were previously untreated and later developed HMA-F. Pt characteristics, laboratory values, and bone marrow (BM) data, including cytogenetics and next generation sequencing (NGS), were assessed at both diagnosis and the time of HMA-F. Genomic DNA was extracted from whole BM aspirate samples and subject to 28- (n=12) or 81-gene (n=131) target PCR-based sequencing using a NGS platform at diagnosis and an 81-gene NGS panel (n=85) at the time of HMA-F. Survival data was updated in January 2022.

Results: Out of 799 untreated MDS pts, 147 pts (32%) developed HMA-F with a median follow-up time of 44.0 months (mo) from diagnosis. Baseline pt characteristics and subsequent treatment are summarized in Table 1. The median age was 71 with therapy-related MDS (t-MDS) in 37%. At diagnosis, most pts had higher-risk MDS with IPSS-R high (29%) or very high (39%) disease, and complex karyotype was seen in 44%. The median number of mutations identified was 2 with TP53 (44%) and ASXL1 (21%) the most frequently discovered mutations. Regarding HMA therapy, 67% received HMA monotherapy and 33% received HMA in combination with various agents. The median number of cycles of HMAs received was 7 (range: 1-37).

At the time of HMA-F, pts presented with higher BM blasts (6% vs 10%, p<0.001) and lower platelet counts (63 K/µL vs 36 K/µL, p<0.001) than at diagnosis. The median number of cytogenetic abnormalities increased from 1 to 2 with no change in the median number of mutations from diagnosis to HMA-F, but both had significant increases by paired t-test (p<0.001). The median time from diagnosis to HMA-F was 10.8 mo, and the median overall survival (mOS) from HMA-F was 6.8 mo (95% CI: 5.4, 9.0). Survival was well-stratified by both IPSS-R at diagnosis (14.6 mo in lower-risk MDS vs 5.4 mo in higher-risk MDS, p=0.001) and at HMA-F (11.4 mo in lower-risk MDS vs 3.5 mo in higher-risk MDS, p<0.001). TP53 mutations at diagnosis (HR 3.02, p<0.001) and t-MDS (HR 2.50, p<0.001) were associated with shorter mOS. In pts with higher-risk MDS, initial response to HMA therapy (7.2 mo in responders vs 2.7 mo in non-responders, p=0.011) and stem cell transplantation at HMA-F (4.7 mo in non-transplanted vs not reached in transplanted, p<0.001) were associated with improved survival. Progression to acute myeloid leukemia (AML) was seen in 72 pts (49%), and the mOS from the time of AML diagnosis was 3.4 mo.

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Summary/Conclusion: Pts with HMA-F MDS have high-risk features, including poor cytogenetics and adverse mutations, and are often therapy-related. At the time of HMA-F, patients present with higher BM blasts, worsening thrombocytopenia, and more cytogenetic abnormalities and mutations. Unfortunately, after failure of HMA, pts have frequent transformation to AML and dismal overall survival. Further understanding of the underlying biology of HMA-F MDS is warranted with an urgent need for therapeutic interventions after failure of HMA therapy.

P761: MONOCYTOSIS IN PRIMARY CARE AND HEMATOLOGICAL MALIGNANCIES

M. Christensen 1,2,* , V. Siersma 2 , M. Kriegbaum 2 , B. Lind 3 , J. Samuelsson 4 , K. Grønbæk 1 , L. Granfeldt 5 , C. L. Andersen 1

1 Department of Hematology, Rigshospitalet; 2 Institute for Public Health, University of Copenhagen, Copenhagen; 3 Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark; 4 Department of Hematology, Universitetssjükhuset, Lindköping, Sweden; 5 Department of Hematology, Odense University Hospital, Odense, Denmark

Background: Monocytosis (blood monocyte count > 0.8 x10 9 /L) is relatively often observed in routine blood work from primary care, however, the significance is not always apparent as monocytosis may be present in a spectrum of diseases spanning from mild infections to chronic myelomonocytic leukemia.

Aims: We aimed to examine the predictive value of monocytosis in primary care by relating monocyte count to subsequent 3-year incidence of hematological malignancy.

Methods: We included 663.184 adult patients from primary care in the greater Copenhagen Area who had a complete blood cell count (CBC) performed at any time between 2000-2016. For patients with multiple samples registered, a random sample was selected and samples predating the index CBC by 3 months were assessed to account for sustained monocytosis. Using the extensive Danish health data registers, we collected data on incident hematological malignancy for 3 years following the CBC. We modelled the association between monocyte count and hematological malignancies as well as all-cause mortality using multiple logistic regression analysis.

Results: Monocytosis was observed in 4,6 % of CBCs. We saw an increased risk of myeloid malignancies with the most noticeable being an odds ratio (OR) for CMML > 100 with monocytosis > 1.0 x 10 9 /L. Sustained monocytosis increased this risk to OR >140. The incidence was, however, still low with only 0.1% of patients with monocytosis developing CMML within 3 years. Risk of lymphoproliferative and M-component related diseases were also increased, however, other relevant cell line were affected as well in these cases.

Summary/Conclusion: Monocytosis is a common finding in primary care, but even with monocytosis > 1 x 10 9 /L hematological malignancies are rare. Optimized referral to secondary care remains a challenge and if the clinical presentation leaves doubt of the diagnosis, repeated measurements help demask if malignancy is the cause of the monocytosis.

P762: NONLINIEAR IMPACT OF CLINICAL FEATURES ON THE SURVIVAL OUTCOME OF MYELODYSPLASTIC SYNDROMES PATIENTS

F. Darbaniyan 1,* , G. Montalban Bravo 2 , K. S. Chien 2 , R. Kanagal Shamanna 3 , K. sasaki 2 , H. Yang 2 , K. A. Soltysiak 2 , Z. Li 1 , K.-A. Do 1 , G. Garcia-Manero 2

1 Biostatistics; 2 Leukemia; 3 hematopathology, MD Anderson Cancer Center, Houston, United States of America

Background: Myelodysplastic syndromes (MDS) are a group of hematopoietic stem-cell disorders with heterogenous prognosis. The revised international prognostic scoring system (IPSS-R) (Greenberg et al., Blood, 2012) is the most widely used score for prognostication. However, the allocation of IPSS-R risk categories remains imprecise for some patients leading to unsatisfactory therapy decisions. Newer molecular prognostic models (Nazha et al., JCO, 2021 and Bernard et al., ASH, 2021) have added mutation data to improve survival predictions for MDS patients.

Aims: Our aims were to investigate the nonlinear association of normalized clinical data in combination with molecular and cytogenetic data and to improve the prognostication potential of IPSS-R for patients with MDS.

Methods: A multivariable fractional polynomials (MFP) algorithm was applied on clinical data of 943 MDS patients and nonlinear associations between continuous covariates and survival outcomes were efficiently modeled within Cox regression. Selected transformed variables were picked and the potential overfit was avoided by a closed test procedure for function selection (Royston et al., John Wiley & Sons, 2008). The performance of the model was evaluated in an independent set of 436 MDS patients and the accuracy was assessed using a concordance (c)index and the area under the Receiver Operating Characteristic curve (AUC) at each time point. Mutation data was obtained in a subset of 695 patients using next-generation sequencing, evaluating a panel of 81 genes at the time of diagnosis.

Results: An MFP-model consisting of cytogenetics per IPSS-R and WHO subtypes, continuous forms of age and hemoglobin, and non-linear transformations of platelets and bone marrow blast percentage yielded a c-index performance of 0.82 vs 0.78 for IPSS-R and a significant AUC upgrade at all time points in the training set (Figure a). In comparison with the IPSS-R, absolute neutrophil count was not identified as an independent prognostic covariate and common features were treated in a non-linear fashion. Validation of the model in an independent set of 438 patients yielded a higher c-index (0.72 vs 0.68) with an improved AUC over time compared to the IPSS-R (Figure b). Based on MFP risk score, patients were then clustered into 5 different risk categories and compared with IPSS-R classifications. Our data showed no significant difference in time to death between IPSS-R groups for very low risk and low risk or between low risk and intermediate risk (Figure c); however, MFP classification showed 5 significantly different subgroups in predicting time to death for MDS patients (Figure d).

We further investigated the effect of molecular data on disease prognostics in a subset of 695 patients sequenced using an 81 gene panel. In addition to the selected clinical covariates, occurrence of TP53 , SRSF2 , and ZRSR2 mutations were independently associated with lower survival, while the presence of TET2 mutation showed a depression effect on TP53 mutation towards a better outcome. As SF3B1 was not selected in the final model, we further evaluated its potential interactions and identified its correlation with cytogenetics and platelets, which predominated in the MFP model. This might be due to heterogeneity within the data set (e.g., 25% co-occurrence of SF3B1 with TP53 mutation) and needs to be investigated in larger cohorts.

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Summary/Conclusion: We have established a nonlinear model that yields improved risk assessment compared to IPSS-R criteria.

P763: VENETOCLAX WITH AZACITIDINE IN RELAPSE/REFRACTORY HIGHER RISK MYELODYSPLASTIC SYNDROME: UPDATED PHASE 1 RESULTS

S. P. Desikan 1,* , G. Montalban-Bravo 1 , M. Ohanian 1 , N. Daver 1 , T. Kadia 1 , S. Venugopal 1 , K. Chien 1 , H. Kantarjian 1 , G. Garcia-Manero 1

1 Leukemia, MD Anderson, houston, United States of America

Background: Patients with higher risk (HR) MDS status post HMA failure have poor survival, 4-6 months. [Lancet Oncology Apr 2016] The combination of azacitidine(Aza) and venetoclax(Ven) has produced encouraging results in the frontline settings for patients with HR disease.(Garcia et.al ASH 2019)

Aims: The primary objective is to establish the safety of azacitidine and venetoclax in HR MDS after HMA failure. The secondary objectives include rates of CR, marrow response, transfusion independence, duration of response, and overall survival.

Methods: In this phase 1 study, patients 18 years and older with HMA failure (relapse or progression after 4 cycles), adequate organ function and performance status are being enrolled. Patients who had received prior Ven were excluded. All patients received Aza 75mg/m2 on D1-5. A 3 + 3 design was utilized with Ven. A 3 + 3 design was utilized with Ven with dose levels 0, 1, 2 receiving 100mg, 200mg, and 400mg respectively. Patients are currently being enrolled in the expansion cohort(400mg). Response was determined based on IWG06 criteria.

Results: Baseline characteristics are in Table 1. Fifteen patients have been enrolled with a median age of 78[67 – 81]. The median blast% on enrollment was 9%[6 – 17] with a median ANC, Hgb, and PLT of 1.67 K/µL, 7.7mg/dL, and 39 K/µL. When stratified based on cytogenetics, 1 (7%) had good risk, 7 (47%) patients had intermediate risk, 2 (13%) had poor risk, and 5(33%) had very poor risk. When stratified based on IPSS-R criteria: 2(13%) had intermediate risk, 5(33%) had high risk, and 8(54%) had very high risk disease. ASXL1 and TP53 mutations were enriched, occurring in 46% and 53% of patients.

Four(33%) patients had Grade ¾ cytopenias (either thrombocytopenia or neutropenia) relating to this combination. Three patients required dose reductions. No tumor lysis syndrome has occurred to date.

Two patients recently started treatment and could not be included for response assessment. Among the 13 patients evaluable, the ORR is 62%(n=8). The median number of cycles to response was 1[1-4]. Responses consisted of 1 CR, 4 marrow responses with either platelet or neutrophil recovery, and 3 marrow responses without count recovery. Three patients achieved transfusion independence. Five patients had NR with 4 having TP53 mutations.

The median OS was 8.5 months with a 30 day and 60 day mortality of 8% and 23% respectively. Early mortality was related to infection in 2 patients and progression in another.

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Summary/Conclusion: With a response rate of 62% and an OS of 8.5 months, this combination may have benefit in HR RR MDS. TP53 mutations and higher cytogenetic risk confer poor prognosis.

P764: RESPONSE AND SURVIVAL OUTCOMES WITH HYPOMETHYLATING AGENTS IN AN ARGENTINEAN COHORT OF 113 PATIENTS WITH CHRONIC MYELOMONOCITIC LEUKEMIA

J. Gonzalez 1,* , A. Perusini 2 , F. Russo 3 , L. Fuentes 4 , T. Deluca 5 , A. L. Basquiera 6 , A. Navickas 7 , L. Kornblihtt 8 , N. Pintos 9 , E. Nucifora 2 , M. Iastrebner 10 , A. Enrico 5 , J. Arbelbide 2 , C. Belli 11

1 Hematology, Hospital Agudos Carlos G Durand; 2 Hematology, Hospital Italiano de Buenos Aires; 3 Hematology, Hospital Paroissien; 4 Hematology, Instituto Alexander Femming, CABA; 5 Hematology, Hospital Italiano de La Plata, La Plata; 6 Hematology, Hospital Privado de Cordoba, Cordoba; 7 Hematology, Hospital El Cruce, Florencio Varela; 8 Hematology, Hospital de Clinicas Jose de San Martin; 9 Hematology, Sanatorio Mendez; 10 Hematology, Sanatorio Sagrado Corazon; 11 Genetic, IMEX-CONICET/ANM, CABA, Argentina

Background: Hypomethylating agents (HMA) are the first line option for high-risk patient and lower-risk patient with transfusion dependence, or according to worsening clinical features. Data regarding HMA efficacy in CMML has been largely retrospective, or from MDS studies that included CMML patients, with overall response rates (ORR) ranging from 40% to 50% and true CR rates being <20%. Local data is scarce.

Aims: To examine the influence of prognostic factors at diagnosis and during the follow-up in the outcome and response to HMA therapy in a CMML cohort from Argentine.

Methods: We performed a retrospective analysis of 113 CMML patients from the Argentine Registry of MDS promoted by the Argentine Society of Hematology who were treated between January-07/February-22. Statistical analysis included Kaplan-Meier survival analysis, Cox proportional hazard and Chi2/Fisher’s exact test.

Results: The median age at diagnosis was 67 years (IQR 58-74) being 71.7% >60 years old, 18.8% a Charlson’s Index (CCI)>2, 81 (72%) were males, and 59 (52%) CMML-0, 25 (22%) CMML-1 and 29 (26%) CMML-2. At treatment initiation, 70.5% showed hemoglobin <10g/dL, 22.1% platelet counts <30,000/µL, 13.1% poor karyotypes and 91.2% at high risk according to the CPSS, IPSS, IPSS-R or Bournemouth scoring systems. During the follow-up, median 20 months (IQR 8.6-42.7), 45.1% evolve to AML and 75.2% died. Regarding HMA therapy, the median time to treat was 2.4 months, 78.8% patients received AZA and 22.2% DAC, the median number of cycles of 7 (IQR 4-12) during a median period of 8.1 months (m). The median overall survival (OS) of the cohort was 25.5m (95%CI 18.9-32.1), since treatment initiation 17.1m (95%CI 13.3-20.8), and after cessation 5.3m (95%CI 4.3-6.3).

Most of parameters and scoring systems analyzed were useful to predict outcome from diagnosis or from treatment initiation to last follow up. Cox regression analysis revealed that CCI>2 (HR 2.7, 95%CI 1.5-5.0, p=0.001), WHO classification (ref. CMML-0, CMML-1, HR 2.1, 95%CI 1.1-4.1, p=0.026; CMML-2, HR 3.4, 95%CI 1.6-6.8, p=0.001), lower hemoglobin level (<10g/dL, HR 1.9, 95%CI 1.1-3.5, p=0.023), platelet count <30.000/µL (HR 3.5, 95%CI 2.0-6.0, p<0.001), time to treat <6m (HR 4.7, 95%CI 2.4-9.1, p<0.001) and the presence of blast in PB (HR 1.7, 95%CI 1.0-2.8, p=0.05) and were independently associated with a reduced OS. Almost all parameters, with the exception of lower Hb levels, sustained their independency since treatment initiation.

A total of 99 patients were evaluated for response to treatment with an overall response rate (at 4-6 cycles) of 59.6% (CR/mCR/PR: 36.4%, HI: 23.2%, SD: 14.1%). The median overall survival of responders was 44.8m, similar to those with SD (33.2m, p=0.246), afterward grouped, with 8.3m in non-responders (p<0.001). We also evaluated whether prognostic factors were useful to predict response. Only the WHO 2016 proposal (CMML-0 61.6% vs 34.6%, CMML-1 21.9% vs 30.8%, CMML-2 16.4 vs 34.6%, p=0.046) and the absence of peripheral blasts (61.1% vs 36.0%, p=0.037) were associated with statistically different rates of response, with a tendency to treat later ≥6m (37.0% vs 15.4%, p=0.05).

Summary/Conclusion: The present series represents the first experience in Latin America evaluating HMA agents in CMML patients. Our results highlight the adverse impact of several parameters, including the severity of the thrombocytopenia <30,000/µL on the outcome of CMML patients under HMA. However, clinical parameters are limited to predict the response rate.

P765: UPDATED ENROLLMENT AND RESULTS FROM THE PHASE 1 SUBSTUDY OF IVOSIDENIB IN PATIENTS WITH IDH1-MUTANT RELAPSED/REFRACTORY MYELODYSPLASTIC SYNDROME (R/R MDS)

C. D. DiNardo 1,* , J. M. Foran 2 , J. M. Watts 3 , E. M. Stein 4 , S. de Botton 5 , A. T. Fathi 6 , G. T. Prince 7 , R. M. Stone 8 , P. A. Patel 9 , G. J. Roboz 10 , M. L. Arellano 11 , H. P. Erba 12 , A. Pigneux 13 , P. Baratam 14 , R. K. Stuart 14 , X. Thomas 15 , I. R. Lemieux 16 , X. Bai 16 , S. M. Kapsalis 16 , G. Garcia-Manero 1 , D. A. Sallman 17

1 MD Anderson Cancer Center, University of Texas, Houston; 2 Mayo Clinic, Jacksonville; 3 Sylvester Comprehensive Cancer Center, University of Miami, Miami; 4 Memorial Sloan Kettering Cancer Center, New York, United States of America; 5 Institut Gustave Roussy, Villejuif, France; 6 Massachusetts General Hospital, Harvard Medical School, Boston; 7 Johns Hopkins Hospital, Baltimore; 8 Dana-Farber Cancer Institute, Boston; 9 University of Texas Southwestern Medical Center, Dallas; 10 Weill Cornell Medicine and The New York Presbyterian Hospital, New York; 11 Winship Cancer Institute, Emory University School of Medicine, Atlanta; 12 Duke University, Durham, United States of America; 13 Centre Hospitalier Universitaire de Bordeaux Haut-Lévêque, Pessac, France; 14 Medical University of South Carolina, Charleston, United States of America; 15 Centre Hospitalier Universitaire de Lyon-Sud, Lyon, France; 16 Servier Pharmaceuticals LLC, Boston; 17 Moffitt Cancer Center, Tampa, United States of America

Background: Isocitrate dehydrogenase 1 ( IDH1 ) is mutated in ~3% of patients with MDS, increasing the risk of transformation to acute myeloid leukemia (AML). Ivosidenib (IVO), an oral, potent, targeted inhibitor of the mutant IDH1 (mIDH1) enzyme, is FDA approved for m IDH1 R/R AML and m IDH1 newly diagnosed AML in patients ≥75 years old or with comorbidities precluding the use of intensive induction chemotherapy. Promising safety and efficacy findings in the first-in-human, open-label, dose escalation and expansion study of IVO in patients with m IDH1 advanced hematologic malignancies ( NCT02074839 ) led to the FDA granting Breakthrough Therapy designation to IVO in m IDH1 MDS. 12 patients with R/R MDS received IVO 500 mg once daily (QD). Median age was 72.5 (range 52–78) years and all had received prior MDS treatment. The investigator-assessed overall response rate (ORR) was 75%, with median response duration of 21.4 months. This substudy enrolled additional patients with m IDH1 R/R MDS; therefore, we report updated results.

Aims: To further evaluate safety, tolerability, clinical activity, and pharmacokinetics/pharmacodynamics of IVO in patients with m IDH1 R/R MDS.

Methods: This substudy of the single-arm study of IVO evaluated patients with m IDH1 R/R MDS after documented failure or relapse following prior standard therapy including intensive chemotherapy and hypomethylating agents. Patients must have given informed consent, have high disease burden based on IPSS or IPSS-R risk at baseline, and an Eastern Cooperative Oncology Group performance status score of 0–2. IVO 500 mg QD was given orally on days 1–28 of 28-day cycles.

Results: As of 08May2021, 16 patients with R/R MDS were enrolled: 3 of the 16 who received 500 mg IVO QD were women; median age was 73.5 (range 52–78) years and 44% were ≥75 years of age. As of the data cut, 5 (31%) patients remained on treatment and free from leukemic transformation and 11 (69%) had discontinued including 6 for disease progression, 1 for allogeneic stem cell transplantation, and 1 owing to an adverse event (AE) of sepsis (the only fatal AE; reported by investigator as not related to IVO). Differentiation syndrome was observed in 2 patients (grade 2). Electrocardiogram QT prolonged was reported in 2 patients (grade 1 and 2). The Table outlines AEs. Complete response (CR) was achieved by 7 of 16 patients (44%; 95% CI, 20%, 70%), partial response (PR) by 1 (6%), and marrow CR by 5 (31%), giving an ORR of 81% (95% CI, 54%, 96%). At 12 months, the Kaplan-Meier estimate of duration of CR+PR was 60.0%. 3 patients experienced CRs lasting 24.0, 63.7, and 65.4 months, which remain ongoing. 11 of 16 (69%) patients achieved hematologic improvement in ≥1 lineage. Among 7 patients who were transfusion dependent at baseline, 5 (71%) became independent of red blood cell or platelet transfusions for 56 or more consecutive days on treatment. Further translational data are being processed.

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Summary/Conclusion: IVO monotherapy induced durable remissions and transfusion independence in patients with m IDH1 R/R MDS, with a manageable safety profile. These results contribute to IVO’s potential as an effective, oral, targeted treatment for patients with m IDH1 R/R MDS.

P766: CLINICAL OUTCOMES IN PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROMES RECEIVING HYPOMETHYLATING AGENTS: A LARGE POPULATION-BASED ANALYSIS

A. M. Zeidan 1,* , E. S. Mearns 2 , C. Ng 2 , A. Shah 2 , N. Lamarre 3 , A. Yellow-Duke 2 , N. Alrawashdh 2 , B. Yang 4 , W. Cheng 5 , C. N. Bui 5 , A. Svensson 5

1 Yale Cancer Center, Yale University School of Medicine, New Haven; 2 Genentech, Inc., South San Francisco; 3 Real World Data Analytics, Genesis Research, Hoboken; 4 Roche Diagnostics, Santa Clara; 5 AbbVie, North Chicago, United States of America

Background: Myelodysplastic syndromes (MDS) are a spectrum of hematopoietic neoplasms characterized by variable degrees of cytopenias and risk of progression to acute myeloid leukemia (AML). Although the hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DEC) are widely used to treat higher-risk (HR)-MDS in the United States (US), questions remain about their longer-term clinical benefits.

Aims: To understand the survival outcomes and progression-related events of patients (pts) treated with AZA or DEC for HR-MDS in the US.

Methods: Older adults (≥66 years old) diagnosed with refractory anemia with excess blasts (RAEB), an established proxy for HR-MDS, between January 2009 and December 2017, were included from the Surveillance, Epidemiology, and End Results (SEER)-Medicare linked database. The index date was date of HMA initiation. Pts who received AZA or DEC, had ≥12 months (mo) of continuous enrollment prior to the index date, and who did not receive a stem cell transplant (SCT) prior to the index date were included. Overall survival (OS) from the index date to death was estimated using Kaplan–Meier methodology. Baseline platelet and red blood cell (RBC) transfusion dependence (TD) were defined as ≥2 transfusion episodes in the 8 weeks prior to the index date; baseline transfusion use (TU) was defined as one transfusion episode in the 8 weeks prior to the index date. A competing risk analysis was used to assess the incidence of progression to AML, with death as the competing event.

Results: Of 973 pts with HR-MDS treated with HMA, 75.8% received AZA and 24.2% received DEC. Median time from diagnosis to HMA initiation was 1.2 mo (interquartile range [IQR]: 0.6–2.6). Median treatment duration was 5.7 mo (IQR: 2.3–11.5) overall (5.8 mo [IQR: 2.6–11.8] for AZA and 5.0 mo [IQR: 1.9–11.0] for DEC). At baseline, 13.8% of pts had platelet TD/TU, 29.9% had RBC-TD and 27.0% had RBC-TU. Only 6.4% of pts received SCT during follow-up.

Median follow-up from the index date until death or censoring was 13.8 mo (IQR: 6.2–25.3). Median OS (mOS) was 13.9 mo (95% confidence interval: 12.9–15.0). There was no significant difference in mOS for pts treated with AZA vs DEC (13.4 vs 15.2 mo, respectively; p=0.22). Pts with RBC-TD/TU had shorter mOS vs those with RBC-transfusion independence (TI) (10.7 vs 18.6 mo, respectively; p<0.0001). Similarly, pts with platelet TD/TU had shorter mOS vs those with platelet TI (8.4 vs 14.9 mo, respectively; p<0.0001). There were no statistically significant differences in OS by the route of AZA administration, insurance category, county metropolitan status, or county poverty level. Overall, 38.0% of HMA-treated pts progressed to AML; median time from HMA initiation to AML progression was 8.2 mo (IQR: 3.5–15.1). Cumulative incidences of AML and death were 25.7% and 30.7%, respectively, at Year 1 and 34.3% and 45.8%, respectively, at Year 2, with the rates of progression slowing over time.

Summary/Conclusion: Compared with previously published literature from the SEER-Medicare database (Zeidan Br J Haematol 2016; mOS: 11 mo [AZA] and 12 mo [DEC] in pts diagnosed with HR-MDS between 2004 and 2011), pts with HR-MDS treated with HMA have improved mOS, but OS remains substantially worse vs the landmark AZA-001 trial (Fenaux Lancet Oncol 2009; mOS: 24.5 mo). The incidence of death and AML progression was highest in the first year after initiation of therapy. SCT rates remain very low among pts with HR-MDS, further emphasizing the urgent need for novel treatment options for pts with HR-MDS.

P767: CLINICAL SIGNIFICANCE OF ROUTINE HIGH-RESOLUTION STRUCURAL VARIANT PROFILING IN MYELODYSPLASTIC SYNDROMES

H. Yang 1 , G. Garcia-Manero 1 , K. Sasaki 1 , G. Montalban-Bravo 1 , Z. Tang 1 , Y. Wei 1 , T. Kadia 1 , K. Chien 1 , D. Rush 1 , H. Nguyen 1 , A. Kalia 1 , M. Nimmakayalu 1 , C. Bueso-Ramos 1 , H. Kantarjian 1 , L. J. Medeiros 1 , R. Luthra 1 , R. Kanagal-Shamanna 1,*

1 THE UNIVERSITY OF TEXAS M.D. ANDERSON CANCER CENTER, Houston, United States of America

Background: Structural Variant Profiling (SVP) using high-resolution technologies, in combination with NGS-based mutation analysis, can inform critical cytogenomic data needed for improving the prognostication and outcomes in patients (pts) with MDS. Optical Genome Mapping (OGM) is a novel non-sequencing-based technique for genome-wide high-resolution SVP for all types of SVs: copy number alterations, structural rearrangements, inversions and partial tandem duplications (PTD).

Aims: The goal was to evaluate the clinical implications of SVP on prognosis and risk-stratification in a large series of consecutive treatment naïve MDS pts.

Methods: All patients (pts) underwent conventional chromosomal banding analysis (CBA), OGM and targeted 81-gene panel NGS. We compared the cytogenetic [based on comprehensive cytogenetic scoring system (CCSS)] and R-IPSS risk groups between data generated by CBA and OGM respectively and correlated with outcomes. For OGM, CCSS was calculated from the predicted karyotype generated by consolidatied alignment of multiple individual SVs.

Results: There were 101 newly diagnosed MDS pts (71 men; 29 women). The median age was 72 (25-92). The distribution of CCSS groups [Very Good (3%), Good (43%), Intermediate (22%), Poor (10%), Very Poor (21%)] and R-IPSS risk categories [Very low (5%), low (30%), Intermediate (27%), High (17%), Very high (20%)]. Two patients had incomplete karyotype.

By generating predicted karyotypes from OGM data, there were 224 clinically significant unique aberrations. Of these, 94 (40%) aberrations (seen across 34 patients) were not detected by CBA. These included SVs affecting KMT2A, MECOM, TP53, NUP98 among others. Comparison of CCSS cytogenetic groups between OGM and CBA showed that OGM data upgraded the CCSS cytogenetic-risk in 12 pts (resulting in R-IPSS upgrade in 9 pts) and downgraded in 7 pts (R-IPSS downgraded in 6 pts). Together with the results for 2 patients with non-informative karyotype, OGM modified the CCSS for 21% pts, and R-IPSS category for 17 (17%) pts. Since CCSS is influenced by the number of alterations, there were patients where OGM data, though clinically important, did not change the CCSS/R-IPSS category. To capture this, we defined an “actionable SV” as (1) prognostic, not recognized in the CCSS criteria ( KMT2A -PTD, TP53 allele status) or (2) potential eligibility for a clinical trial, such as rearrangements in KMT2A , MECOM, or NUP98 . Well-established SVs defined in CCSS were ignored. Based on this, 13 (13%) pts had an “actionable alteration” (9 with a positive finding). When both the CCSS change and actionable alterations were combined, OGM results provided informative data in 28 (28%) patients.

Next, we evaluated the impact of CCSS-CBA and CCSS-OGM groups using outcome data. Over a median follow-up duration of 34 months, the median OS was 86 months. Using Harrell’s concordance index, OGM had a slightly improved prediction of prognosis for both CCSS [0.7 vs. 0.665] and R-IPSS categories [0.705 vs. 0.69] compared to CBA. By UVA, the following parameters associated with outcome: SF3B1 mutation [p=0.030; HR 0.343; CI 0.130-0.901], TP53 mutation [p=0.001; HR 3.232; CI 1.582-6.601], CCSS score by CBA [p=0.001; HR 1.690; CI 1.250-2.286] and CCSS by OGM [p<0.001; HR 2.051; CI 1.488-2.827]. By MVA analysis, CCSS by OGM [p<0.001; HR 1.956; CI, 1.367-2.798], but not CCSS by CBA), BM blast percentage [p=0.026; HR 1.079; CI, 1.009-1.153] and TP53 mutation [p=0.040; HR 2.335; CI, 1.038-5.250] associated with prognosis.

Summary/Conclusion: High-resolution SVP by OGM improved the prognostic risk stratification in MDS.

P768: GUADECITABINE (SGI-110) VS. TREATMENT CHOICE (TC) IN RELAPSED/REFRACTORY(R/R) MYELODYSPLASTIC SYNDROME (MDS), RESULTS OF A GLOBAL, RANDOMIZED, PHASE 3 STUDY.

G. Garcia-Manero 1,* , S. Bart 2 , J. K. McCloskey 3 , P. Fenaux 4 , D. Selleslag 5 , G. Reda 6 , D. Valcárcel 7 , V. Santini 8 , J. Mayer 9 , B. Xicoy 10 , H. Yamaguchi 11 , M. Lübbert 12 , Y. Miyazaki 13 , H. Keer 14 , Y. Hao 14 , M. Azab 14 , H. Döhner 15

1 The University of Texas MD Anderson Cancer Center, Houston; 2 Fred Hutchinson Cancer Research Center, Seattle; 3 John Theurer Cancer Center at Hackensack University Medical Center, Hackensack, United States of America; 4 Hôpital Saint Louis, Paris, France; 5 Algemeen Ziekenhuis Sint-Jan Brugge-Oostende, Brugge, Belgium; 6 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy; 7 Hospital Universitari Vall d’Hebrón, Barcelona, Spain; 8 MDS Unit AOU Careggi, DMSC University of Florence, Florence, Italy; 9 Fakultní Nemocnice Brno, Brno, Czechia; 10 Institut Català d’Oncologia-Hospital Universitari Germans Trias i Pujol, Badalona, Spain; 11 Nippon Medical School Hospital, Tokyo, Japan; 12 Universitaetsklinikum Freiburg, Freiburg, Germany; 13 Nagasaki University Hospital, Nagasaki, Japan; 14 Astex Pharmaceuticals, Inc., Pleasanton, United States of America; 15 Ulm University Hospital, Ulm, Germany

Background: Guadecitabine (G) is a next-generation hypomethylating agent (HMA), administered as a small-volume subcutaneous (SC) injection, designed with the potential to overcome pharmacokinetic resistance to first-generation HMAs (decitabine and azacitidine). Preliminary guadecitabine phase 2 data in r/r MDS showed an overall survival of almost 12 months (Garcia-Manero,G, et al; Lancet Haematol http://dx.doi.org/10.1016/S2352-3026 [19]30029-8) leading to the ASTRAL-3 study.

Aims: Compare overall survival of guadecitabine to that of TC consisting of low-dose cytarabine (LDAC), intensive chemotherapy (IC), or best supportive care (BSC). Secondary objectives included multiple standard assessments of response and safety.

Methods: Subjects with r/r MDS or Chronic Myelomonocytic Leukemia (CMML) who had progressed or failed to respond after 6 full cycles of standard HMA therapy were randomized 2:1 between guadecitabine (60 mg/m2) SC Days 1-5 every 28 days vs. a pre-selected TC (BSC, IC, or LDAC). Stratification was by disease, disease burden (>10% bone marrow blasts or not, geography, and pre-selected TC option). Primary endpoint was overall survival (OS) and the study was designed with ~90% power to detect a hazard ratio of 0.68 (approximately 2.8 month difference in median survival).

Results: 417 MDS/CMML subjects were randomized to G or TC (G:277, TC: 140). Demographics and disease status were reasonably balanced across the groups (see Table 1 below) as was percentage of commonly associated genetic mutations (TET2, DNMT3A, SF3Baa, RUNX1, and TP53). Median guadecitabine exposure was 4 cycles and 3 cycles for TC. Neither overall survival (G: median 9.1 months, TC: median 8.3 months, p=0.61, HR:0.94 with 95% CI: [0.74-1.19]) nor leukemia-free survival (LFS) (G: median 5.7 months, TC: median 5.9 months; p=0.38) demonstrated a significant difference between the two groups and subgroup analyses did not suggest a difference between guadecitabine and any of the different TC options or different genetic mutations. Transfusion dependence for 8 weeks was similar with 32.1% and 22.4% of the G group and 37.9% and 20.0% of the TC group being platelet transfusion or red blood cell transfusion independent, respectively. Safety was consistent with known profiles of the respective agents with the G group having a slightly higher overall incidence of adverse events (AE) (99.3% vs 92.6% for TC) and grade 3 or higher AE (92.2% vs 70.5% for TC). The AEs with the highest incidence in subjects who received guadecitabine were febrile neutropenia (38.5 vs 18.9% for TC), pneumonia (34.4% vs 18.9% for TC), neutropenia (34.1% vs 15.6% for TC), and thrombocytopenia (32.2% vs 21.3% for TC).

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Summary/Conclusion: This large, global, randomized phase 3 study did not demonstrate superiority of guadecitabine over Standard TC in MDS/CMML patients who were refractory or relapsed following full course of prior HMA treatment.

P769: MUTATION PROFILES AND RISK STRATIFICATION IN HYPOCELLULAR MYELODYSPLASTIC SYNDROME

K. Kim 1,* , K. Chien 1 , O. Faustine 1 , G. Montalban-Bravo 1 , R. Kanagal-Shamanna 2 , T. Kadia 1 , E. Jabbour 1 , Y. Alvarado 1 , K. Sasaki 1 , X. Q. Dong 1 , S. Pierce 1 , C. Bueso-Ramos 2 , H. Kantarjian 1 , G. Garcia-Manero 1

1 Department of Leukemia; 2 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Hypocellular myelodysplastic syndrome (hMDS) is a subset of MDS, defined by marrow cellularity less than 30% in age under 70 and less than 20% in age 70 or more. The clinicopathologic features of hMDS have been previously studied, but genetic data and clinical impact of hMDS remain unclear.

Aims: We conducted a comparative study of hMDS to normo-/hypercellular MDS (non-hMDS) with clinical characteristics and mutations and further stratified the risk by mutation profile.

Methods: We conducted a retrospective review using a single tertiary cancer center registry of untreated MDS patients from 2020 to 2021. Mutation data was obtained by 28- or 81-gene next-generation sequencing (NGS) panels. We analyzed clinicopathological and mutation data comparing hMDS to non-hMDS pts and conducted risk stratification in hMDS using mutation profiles.

Results: In a total of 1899 patients, 174 (9%) patients had hMDS. 1352 (71%) pts had mutation profiles that were further analyzed; 56% and 44% patients had 81-gene and 28-gene NGS panels performed, respectively. The median follow-up time was 19.5 months. Thrombocytopenia (p=0.016) and neutropenia (p<0.0001) were more common in hMDS than in non-hMDS. By IPSS-R cytogenetic scores, hMDS had scores of 3 than non-hMDS (20% vs. 9%, p<0.001), but non-hMDS had more cytogenetic scores of 4 (27% vs. 19%, p=0.018). The distribution of other IPSS-R cytogenetic scores or IPSS-R risk classifications was similar between the 2 groups. hMDS patients were more likely to be age<65 (68% vs. 47%, p<0.001) and have therapy-related MDS (t-MDS) (44% vs. 32%, p=.001). hMDS patients also had a median overall survival (mOS) of 29.2 months, which was similar to that observed in non-hMDS pts (HR 1.04, p=0.702). This finding was maintained after multivariate adjustment including age, IPSS-R, t-MDS, and certain high-risk mutations ( TP53, ASXL1, RUNX1, RAS pathway). Hypocellularity did not affect survival in either de novo or t-MDS. AML transformation rates (20% in both groups) were similar. Interestingly, pts with hMDS had lower rates of high-risk mutations (38% vs. 51%, p=0.009) with less RUNX1 (p=0.079) and ASXL1 (p=0.010) mutations while the frequency of TP53 and RAS pathway mutations were comparable. In t-MDS, hMDS pts had less frequent TP53 (p=0.005) or U2AF1 (p=0.087) mutations. In de novo MDS, hMDS patients had less RUNX1 (p=0.036), SRSF2 (p=0.014), and TET2 (p=0.019) mutations. In hMDS, mutations in the RAS pathway (HR 3.64, p=0.007) or in TP53 (HR 2.47, p=0.002) were associated inferior mOS, while TET2 mutations (HR 0.29, p=0.036) correlated with superior mOS. hMDS also had worse mOS in RAS pathway-mutated MDS (HR 2.62, p=0.043).

Based on mutation data and multivariate selection models, patients were stratified into 3 groups: (1) pts with TET2 or SF3B1 (only available 81-gene panels) mutations, (2) pts with TP53 mutations or RAS pathway mutations, and (3) pts without TET2/SF3B1/TP53/RAS mutations. The TET2/SF3B1 group showed better mOS (HR 0.34, p=0.041) compared to the TP53/RAS group showing inferior mOS (HR 4.64, p<0.001) (shown in figure). The mOS difference observed with the TET2 / SF3B1 group did not carry over into non-hMDS pts (p=0.889).

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Summary/Conclusion: Our study showed that hMDS patients have similar outcomes with non-hMDS pts with unique molecular profiles. hMDS pts have less frequent high-risk mutations with distinct risk stratification. Improving risk stratification tools in hMDS by incorporating genomic data, such as TP53/RAS pathway and TET2/SF3B1 mutations, would be a study of interest.

P770: A COMPARATIVE STUDY OF LEUKEMIC TRANSFORMATION IN THERAPY-RELATED AND DE NOVO MYELODYSPLASTIC SYNDROME AFTER HYPOMETHYLATING AGENT FAILURE

K. Kim 1,* , F. Ong 1 , Z. Li 2 , R. Kanagal-Shamanna 3 , G. Montalban-Bravo 1 , T. Kadia 1 , E. Jabbour 1 , N. Pemmaraju 1 , D. Hammond 1 , N. Short 1 , F. Ravandi 1 , Y. Alvarado 1 , S. Pierce 1 , X. Q. Dong 1 , H. Kantarjian 1 , G. Garcia-Manero 1 , K. Chien 1

1 Department of Leukemia; 2 Department of Biostatistics; 3 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Therapy-related myelodysplastic syndrome (t-MDS) is subset of myelodysplastic syndrome (MDS), known for having dismal survival outcomes. The majority of patients (pts) with t-MDS have similar clinical courses with de novo MDS patients after hypomethylating agent failure (HMA-F) that often results in transformation to acute myeloid leukemia (AML).

Aims: We conducted a study to identify unique clinicopathologic features of t-AML from t-MDS compared to secondary AML (sAML) from de novo MDS after HMA-F.

Methods: We identified sAML patients after HMA-F from all untreated MDS pts in a tertiary cancer center registry from 2017-2021. Clinical characteristics and pathologic data including cytogenetic (CG) and next generation sequencing (NGS) information were collected at the time of MDS diagnosis (dx), HMA-F, and AML dx.

Results: 71 (48%) MDS pts with AML transformation after HMA-F were included in the study with a median follow-up time of 15.4 months (mo). At the time of MDS dx, 31 (43%) pts had t-MDS: 97%, 58% and 29% had a history of chemotherapy, radiation, and prior stem cell transplant (SCT), respectively. t-MDS patients were more likely to present with age<65 (48% vs. 28%, p=0.070) and thrombocytopenia (33,000 vs. 75,500, p=0.004) when compared to de novo MDS pts. The majority of t-MDS pts had very high risk disease by IPSS-R (67% vs. 39%, p=0.031), while de novo MDS pts frequently had high risk disease (50% vs. 20%, p=0.013). 90% of t-MDS pts had adverse CG (IPSS-R CG score 3-4) at MDS dx, compared to 46% in de novo MDS pts (p=0.004). NGS at MDS dx showed more frequent TP53 mutations in t-MDS than in de novo MDS (73% vs 36%, p=0.003). Interestingly, ASXL1 (36% vs. 1%, p=0.001), RUNX1 (9% vs. 0%, p=0.004), STAG2 (21% vs. 0%, p=0.013), TET2 (13% vs. 0%, p<0.001), and SRSF2 (18% vs. 0%, p=0.027) were more frequently found in de novo MDS (Table). t-MDS had worse overall survival (OS) from MDS dx (HR 1.82, p=0.026). The median OS from MDS dx was 19.4 mo and 14.3 mo in de novo MDS and t-MDS, respectively.

The number of HMA cycles received before HMA-F was 6 (range: 1-13) in t-MDS and 5 (range: 1-23) in de novo MDS. At the time of HMA-F, t-MDS pts presented with more anemia (p=0.005) and thrombocytopenia (p=0.064). The number of CG clones were similar, but the number of CG abnormalities were higher in t-MDS pts (p=0.025). Among pts with subsequent AML transformation after HMA-F, the median time from HMA-F to AML was 1.5 mo in t-MDS to AML, compared to 4.6 mo in de novo MDS to AML.

At time of AML dx, 48 (68%) pts had mutation panels available. All TP53 mutation status stayed the same in t-MDS pts at the time of MDS and AML dx. Other changes in mutations were shown in Table. Though the median OS from AML transformation was short in all pts, t-MDS pts had inferior outcomes (2.4 mo vs. 5.0 mo, HR 1.70, p=0.043). The initial response rate to salvage therapy was comparable between t-MDS and de novo MDS after AML transformation (50% and 56%, p=0.761), but more pts underwent SCT with de novo MDS compared to t-MDS (10% vs. 3%, p=0.383).

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Summary/Conclusion: Transformation to AML after HMA-F results in poor outcomes, but pts with t-MDS have worse survival with distinct CG and mutation profiles. Further understanding of the pathogenesis of AML transformation in both t-MDS and de novo MDS after HMA-F is warranted with improved therapy options an area of unmet need.

P771: PROGNOSTIC CHARACTERISTICS OF PATIENTS WITH MDS WITH ABERRATIONS OF CHROMOSOME 5. DATA FROM THE DÜSSELDORF MDS REGISTRY

F. Schulz 1 , J. Kostova 1,* , B. Hildebrandt 2 , K. Nachtkamp 1 , C. Strupp 1 , N. Gattermann 1 , M. Lübbert 3 , S. Blum 4 , S. Parmentier 5 , K. Götze 6 , J. Lipke 7 , F. Beier 8 , W.-K. Hofmann 9 , U. Germing 1

1 Department of Hematology, Oncology and Clinical Immunology; 2 Institute of Human Genetics, University Hospital Düsseldorf, Düsseldorf; 3 Department of Medicine I: Hematology, Oncology, and Stem-Cell Transplantation, University Medical Center Freiburg, Freiburg, Germany; 4 Department of Hematology and Oncology, Lausanne University Hospital, Lausanne; 5 Oncology and Hematology, Tumor Center, St. Claraspital, Basel, Switzerland; 6 Department of Internal Medicine III, University Hospital Klinikum Rechts der Isar, Munich; 7 Gefos Dortmund, Dortmund; 8 Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, University Hospital Aachen, Aachen; 9 Department of Hematology and Oncology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

Background: Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with a wide spectrum of biological and clinical features. Up to 20% of MDS patients present with aberrations of chromosome 5, either as isolated del(5q) or combined with other aberrations. Patients with a medullary blast count < 5% and isolated del(5q), or with one additional aberration except anomalies of chromosome 7, are diagnosed as MDS del(5q), a defined entity according to the WHO 2016 classification. They have a favorable prognosis compared to other types of MDS, although profound anemia, chronic transfusion dependency and the risk of disease progression into AML present clinical challenges.

Aims: We analyzed the prognostic impact of karyotype complexity in MDS del(5q).

Methods: From the Düsseldorf MDS registry we retrieved 499 patients with MDS or CMML according to WHO 2016, who showed any kind of del(5q). Biological and clinical parameters such as karyotype, IPSS-R and type of treatment were evaluated. Survival and progression to AML was calculated using Kaplan-Meier-plots. Cox regression analysis was performed to identify disease-related prognostic factors. Patients were followed up until Dec 31th 2021.

Results: 58% of the patients were female, median age was 67 years. Median medullary blast count was 4% (0-19%), and median survival in the entire group was 28 months. For assessing the natural course of disease, we screened for patients with non-intensive treatment only (best supportive care, lenalidomide, low-dose Ara-C, hydroxyurea). Separation by medullary blast count (< 5%, 5-9% or ≥ 10%) was associated with significantly different median survival times, ranging from 63 months in the first group to 3 months in patients with ≥ 10% medullary blasts (p < 0.001). Survival was also affected by the cytogenetic risk group according to IPSS-R. Patients in the low-risk group (median survival 72 months) differed significantly from patients in the very high risk group (median survival 5 months) (p < 0.001). A similar trend was observed according to karyotype complexity represented by number of aberrations apart from del(5q). Median survival of patients with an isolated del(5q) was 86 months, while patients with a complex karyotype (del(5q) and ≥3 additional aberrations) had a median survival of 5 months. Biological risk factors were assessed using Cox regression analyses. Considering overall survival as endpoint, medullary blast percentage, cytogenetic risk group, and complexity of the karyotype had a significant impact on survival (p < 0.001). In the entire patient cohort, as well as among patients receiving non-intensive treatment, the rate of AML progression was significantly influenced by karyotype complexity (p < 0.001). While the median time to AML progression in both isolated del(5q) and del(5q) combined with one aberration had not been reached at the end of the observation period, the median time to AML progression for patients with del(5q) as part of a complex karyotype was 31 months in the entire cohort and 14 months in the low-intensity treatment group.

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Summary/Conclusion: Both the IPSS-R cytogenetic risk group and the complexity of the 5q- karyotype showed prognostic impact in patients with del(5q). This was true for overall survival as well as risk of AML progression. Regardless of therapy or medullary blast count in patients with del(5q), the risk of AML progression increased dramatically with increasing complexity of the 5q- karyotype.

P772: PROGNOSTIC IMPACT OF DISEASE-RELATED RISK FACTORS AND TREATMENT APPROACHES IN PATIENTS WITH ADVANCED MYELODYSPLASTIC SYNDROMES (MDS)

C. Lesch 1,* , A. Kündgen 1 , K. Nachtkamp 1 , A. Kasprzak 1 , C. Strupp 1 , M. Rudelius 2 , N. Gattermann 1 , G. Kobbe 1 , B. Hildebrandt 3 , U. Germing 1

1 Department of Haematology, Oncology and Clinical Immunology, Heinrich-Heine University Hospital, Düsseldorf, Duesseldorf; 2 Institute of Pathology, Ludwig-Maximilians-University of Munich, Munich; 3 Institute of Human Genetics, Heinrich- Heine University Hospital, Düsseldorf, Duesseldorf, Germany

Background: Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell disorders. Treatment approaches depend on the type of MDS according to the WHO classification and on risk assessment according to the international prognostic scoring system (IPSS-R). For patients with advanced MDS, as defined by an excess of blasts, therapeutic options include hypomethylating agents and allogeneic stem cell transplantation.

Aims: To determine the prognostic impact of disease-related risk factors and treatment approaches in 432 patients with MDS EB1 and EB2.

Methods: Patients included in this analysis were classified as having MDS EB1 (5-9% marrow blasts) or MDS EB2 (10-19% marrow blasts) at diagnosis. All therapeutic procedures prior to the development of acute myeloid leukemia (AML) were documented. Patients were followed up until 31 st December 2021, or until death. Therapeutic approaches were categorized as best supportive care (BSC) only, low-dose chemotherapy, immunomodulatory treatment, hypomethylating agents (HMA), intensive chemotherapy (IC) and allogeneic stem cell transplantation (alloSCT). The mentioned therapy regimes were weighted in different categories according to their intensity, with BSC defined as the therapy with the lowest intensity and alloSCT as the one with the highest (BSC < low-dose-chemotherapy < immunomodulatory treatment < HMA < IC < allo SCT).

Results: MDS EB1 was diagnosed in 182 patients (42,1%), and MDS EB2 in 250 patients (57,9%). Distribution among IPSS-R risk groups was as follows: 2% low risk, 26.3% intermediate risk, 38% high risk, and 33,7% very high risk. Median life expectancy in the entire cohort including patients with all types of treatment, was 22 months. Progression to AML occurred in 186 (43%) of the patients.

Patients receiving supportive care only (42.6%) had a median survival of 13 months. Patients treated with a hypomethylating agent (18%) showed an overall survival of 26 months, which was significantly better compared with best supportive care (p=0.002). Patients undergoing induction chemotherapy (6%) had a median survival of 21 months, comparable with HMA treatment. Patients receiving low-dose chemotherapy (4.6%) had a median survival of 9 months. The longest median survival (131 months) was seen in patients undergoing alloSCT.

Multivariate Cox regression analysis showed that treatment category (p<0.00005), together with cytogenetic risk category according to IPSS-R (very low, low and intermediate versus high versus very high) (p<0.0005), and hemoglobin (< ≥ 10g/dl) (p=0.003) were the only parameters influencing survival independently, whereas WHO type, gender, ANC, and platelets did not show independent influence on survival.

Summary/Conclusion: Stratification according to cytogenetic risk groups is very important for the prognostic assessment of MDS patients. Assignment to the cytogenetic group high risk and very high risk has greater prognostic impact than the medullary blast count. Even though hypomethylating agents can improve overall survival compared to best supportive care, long-term survival can only be achieved through allogeneic stem cell transplantation. Therefore, all patients eligible for intensive treatment should be evaluated for this treatment option at the time of diagnosis. Furthermore, in recognition of the fact that clonal evolution may occur in MDS patients, consideration should be given to repeating the chromosome analysis as the most important prognostic factor at regular intervals.

P773: THE ELEVATION OF RED BLOOD CELL DISTRIBUTION IS AN INDEPENDENT PROGNOSTIC FACTOR FOR JUVENILE MYELOMONOCYTIC LEUKEMIA

W. Liang 1,* , C. Liu 2 , J. Zhang 2 , M. Yi 2 , Y. Cai 2 , A. Zhang 2 , L. Liu 2 , L. Zhang 2 , X. Chen 2 , Y. Zou 2 , Y. Chen 2 , Y. Guo 2 , Y. Zhang 2 , X. Zhu 2 , W. Yang 2

1 Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; 2 Department of Pediatrics, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Juvenile myelomonocytic leukemia (JMML) is an overlapping myeloproliferative and myelodysplastic disorder of infants and early children. The existing clinical and laboratory prognostic features are inadequate to predict a worse outcome alone. New prognostic indicators are urgently needed since the great clinical heterogeneity. Red cell distribution width (RDW) is a parameter of the complete blood count (CBC) measured automatically by haematology analyzers to reflect the heterogeneity of erythrocyte size. In recent years, RDW has been suggested to have a high negative value for outcomes in various cancers. However, the role of RDW at diagnosis in patients with JMML is still unclear.

Aims: Our aim is to investigate the prognostic role of RDW in children with JMML.

Methods: We conducted a single center retrospective study to investigate the prognostic role of RDW in children with JMML. Seventy-seven patients were enrolled from January 1, 2008 to November 31, 2019. Survival rates were calculated and compared using Kaplan-Meier methods and log-rank tests. Univariate and Multivariate analysis was carried out according to the Cox proportional hazard regression model.

Results: The mean red cell distribution width standard deviation (RDW-SD) for patients is 53.3(39.3-73.7) fl. Cox proportional hazard models showed that patients with RDW-SD >50.50fl at diagnosis were significantly associated with poor overall survival (OS) (HR = 5.22, CI = 1.50-18.21, P=0.010). The multivariate analysis demonstrated that the RDW-SD count was the independent risk factor for OS. Patients with PTPN11 mutation and RDW elevation displayed the worst survival rate, in comparison with other subgroups (P=0.00013).

Summary/Conclusion: The results suggested that the elevated level of RDW is an independent prognostic factor for JMML. Furthermore, the PTPN11 mutation and a high RDW level may involve a synergy during the natural course of JMML.

P774: CLINICAL OUTCOMES ASSOCIATED WITH AZACITIDINE MONOTHERAPY FOR TREATMENT-NAIVE PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME (HR MDS): A SYSTEMATIC LITERATURE REVIEW AND META-ANALYSIS

K. Hasegawa 1,* , A. H. Wei 2 , G. Garcia-Manero 3 , N. G. Daver 3 , N. Rajakumaraswamy 1 , S. Iqbal 1 , R. J. Chan 1 , H. Hu 1 , P. Tse 4 , J. Yan 4 , M. J. Zoratti 4 , F. Xie 4 , D. A. Sallman 5

1 Gilead Sciences, Inc., Foster City, United States of America; 2 Department of Clinical Haematology, The Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Australia; 3 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 4 McMaster University, Hamilton, Canada; 5 Moffitt Cancer Center, Tampa, United States of America

Background: Azacitidine (AZA) is a standard of care for patients with HR MDS; however, the number of large data sets describing outcomes of AZA monotherapy in HR MDS is limited.

Aims: To aggregate clinical outcomes data associated with AZA monotherapy for treatment-naive patients with HR MDS.

Methods: CENTRAL, EMBASE, and MEDLINE were searched to identify interventional, prospective, and retrospective observational studies using AZA monotherapy in treatment-naive HR MDS (defined as intermediate-2 or high risk by International Prognostic Scoring System [IPSS] or intermediate to very high risk by Revised IPSS). Inclusion of observational studies was limited to those with >20 patients in the AZA monotherapy arm. Responses according to International Working Group (IWG) 2000 or IWG 2006 criteria—including complete remission (CR) rate, overall response rate (ORR; defined as CR, marrow CR [mCR], partial remission, and hematologic improvement), overall survival (OS), duration of response (DOR), and time to response (TTR)—were extracted. All articles were reviewed by 2 independent abstractors. Response rates were synthesized using random-effects models; median of medians was used for OS, DOR, and TTR.

Results: Of 3250 abstracts identified, 34 publications describing 16 studies met inclusion criteria: 5 randomized controlled trials (RCTs), 3 prospective studies, and 8 retrospective studies. None of the response endpoints were reported in all studies (Table). The pooled CR rates, reported in 2 RCTs (N=55), 1 prospective study (N=27), and 3 retrospective studies (N=509), were 14%, 11%, and 16%, respectively. The pooled CR rate across all studies (N=591) was 16% (95% CI, 13%-19%). The mCR rate from 1 RCT was 19% (95% CI, 10%-34%; N=42), and the pooled mCR rate from 2 retrospective studies was 18% (95% CI, 13%-26%; N=126). The pooled ORRs ranged from 44% to 55% across RCTs, prospective studies, and retrospective studies.

The pooled median OS was 16.7 months in 3 RCTs (N=161), 16.5 months in a single prospective study (N=34), and 14.4 months in 5 retrospective studies (N=1472). The pooled median OS across all studies (N=1667) was 16.4 months (95% CI, 12.0-17.3 months).

The pooled median DOR was 10.1 months (95% CI, 9.1-11.0 months), and the median TTR was 4.6 months (95% CI, 3.0-9.0 months).

Summary/Conclusion: These findings provide evidence that benefit with AZA monotherapy in treatment-naïve patients with HR MDS is limited. Opportunity exists for novel therapies to increase CR rates and prolong survival.

P775: CLINICAL OUTCOMES AND SOCIOECONOMIC FACTORS IN CHRONIC MYELOMONOCYTIC LEUKEMIA IN THE UNITED STATES USING THE NATIONAL CANCER DATABASE: 2004-2017

N. Mclaughlin 1,* , G. Ruan 2 , K. Wudhikarn 3 , A. Al-Kali 2 , A. Tefferi 2 , N. Gangat 2 , R. Go 2 , M. Patnaik 2 , M. Shah 2

1 Division of Internal Medicine; 2 Division of Hematology; 3 Mayo Clinic, Rochester, United States of America

Background: Chronic myelomonocytic leukemia (CMML) is a rare myeloid malignancy characterized by proliferation of dysplastic monocytic cells and carries a risk for leukemic transformation. Previously, we described the incidence and outcomes of CMML at the population level (2004-2015) using the Surveillance, Epidemiology, and End Results Program (SEER) and National Cancer Database (NCDB) (Ruan et al., ASH 2020). We studied population-based outcomes in CMML using the NCDB from 2004-2017.

Aims: Our aim was to understand the impact of social determinants of health on outcomes in CMML. Secondly, we studied if the outcomes of CMML have improved.

Methods: The NCDB is a nationwide oncology outcomes database for >1500 cancer programs in the US and Puerto Rico, capturing 70% of all newly diagnosed cases of cancer. We queried the NCDB using the ICD-O-3 diagnosis code 9945/3 from 2004-2017. Only patients (pts) with diagnostic confirmation via histopathologic examination were included. Percent without high school degree (HSD) reflects the education level within the pt’s zip code, which was stratified into 4 quartiles. Median household income was estimated by matching the pt’s zip code at time of diagnosis against US Census 2000 data, which was stratified into 4 quartiles. Hazard ratios (HR) with confidence intervals (CI) were calculated using a Cox proportional hazards model. Overall survival (OS) was estimated using the Kaplan-Meier method. Variables significant in univariable analysis were included in a multivariable analysis.

Results: We identified 7143 CMML pts in the NCDB. The median age at diagnosis was 72 years (interquartile range [IQR] 65-81) and 4386 (61%) were males. With a median follow up of 6.1 years (IQR 3.6-9.4), the median OS was 1.4 years (IQR 0.4-3.8). 1600 (23%) had private insurance, 4862 (70%) had Medicare, 298 (4%) had Medicaid, and 129 (2%) were uninsured. In univariate analysis, private insurance had an improved OS compared to patients with Medicaid (HR 1.2, 95% CI 1.06-1.4; p value 0.007), Medicare (HR 1.5, 95% CI 1.4-1.6; p value < 0.001), and other government insurance (HR 1.5, 95% CI 1.1-2.0; p value 0.004). Pts living in a zip code with percent of adults without an HSD of < 14% had an improved OS compared to zip codes with > 29% (HR 1.2, 95% CI 1.1-1.3; p value < 0.001). Median household income by zip code of ≥ $46,000 had an improved OS compared to < $30,000 (HR 1.2, 95% CI 1.1-1.4; p value < 0.001). Receiving treatment at an academic program had an improved OS compared to community cancer programs (HR 1.2, 95% CI 1.1-1.3; p value < 0.001), comprehensive community cancer programs (HR 1.3, 95% CI 1.3-1.4; p value < 0.001), and integrated network cancer programs (HR 1.3, 95% CI 1.2-1.4; p value < 0.001).

Finally, pts diagnosed in 2012-2017 had an improved OS compared to pts diagnosed in 2004-2011 (HR 0.8, 95% CI 0.8-0.9; p value < 0.001). Variables significant in univariate analysis were included in multivariate analysis (Table 1). When education level and median household income per zip code were included in multivariate analysis, education level remained statistically significant and income did not.

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Summary/Conclusion: CMML continues to have a poor prognosis, but OS has improved in recent years. Socioeconomic factors including insurance status, level of education and median household income per zip code, and facility type appear to be associated with outcomes. On multivariable analysis, age < 65, private insurance (vs government insurance), living in a zip code with increased percent HSD, and treatment at an academic program had improved outcomes.

P776: A PHASE 2, OPEN-LABEL, ASCENDING DOSE STUDY OF KER-050 FOR THE TREATMENT OF ANEMIA IN PATIENTS WITH VERY LOW, LOW, OR INTERMEDIATE RISK MYELODYSPLASTIC SYNDROMES

S. Tan 1,* , A. Arbelaez 2 , L. Chee 3 , C. Y. Fong 4 , D. Hiwase 5 , G. Kannourakis 6 , J. Kwan 7 , J. Liang 8 , A. Puliyayil 9 , H. Rose 10 , D. Ross 11 , T.-C. Teh 12 , D. Westerman 13 , J. Wight 14 , W. Feng 15 , J. Lachey 15 , A. McGinty 15 , H. Natarajan 15 , C. Rovaldi 15 , S. Cooper 15

1 St Vincent’s Hospital, Melbourne; 2 Tweed Hospital, Tweed Heads; 3 Royal Melbourne Hospital and Peter MacCallum Cancer Centre, Melbourne; 4 Austin Health, Hiedleberg; 5 Royal Adelaide Hospital, Adelaide; 6 Ballarat Oncology, Ballarat; 7 Westmead Hospital, Westmead, Australia; 8 Middlemore Hospital, Auckland, New Zealand; 9 Albury Wodonga Health, Albury-Wodonga Regional Cancer Centre, Auckland; 10 Barwon Health, Geelong; 11 Flinders Medical Centre, Adelaide; 12 Box Hill Hospital, Eastern Health, Victoria; 13 1. Peter MacCallum Cancer Centre. 2. University of Melbourne, Melbourne; 14 Townsville University Hospital, James Cook University, Douglas, Australia; 15 Keros Therapeutics, Lexington, United States of America

Background: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis; many patients develop high transfusion burden (HTB) requiring ≥4 units RBCs every 8 weeks and have high unmet medical need. KER-050, an investigational modified activin receptor type IIA inhibitor, is designed to target TGF-β ligands to promote differentiation of erythroid and megakaryocyte lineages to improve anemia and thrombocytopenia.

Aims: Evaluate safety, tolerability, pharmacodynamics (PD), efficacy of KER-050 in MDS in an open-label Phase 2 study.

Methods: IPSS-R lower-risk anemic MDS patients are enrolled, including non-transfused, low-transfusion burden (LTB) and HTB patients. In Part 1 base study, ascending dose cohorts receive KER-050 subcutaneously q4w for 4 doses until a recommended Part 2 dose is determined. Safety endpoints include incidence of adverse events (AEs); erythroid efficacy endpoints (≥8 weeks duration) include rates of transfusion independence (TI) and reduction in RBC transfusions by ≥4 units (IWG 2006 HI-E) in HTB patients. Safety-set includes patients receiving ≥1 dose. PD/efficacy results are reported in efficacy evaluable (EE) patients who contributed ≥8 weeks of HGB and transfusion data. New analyses presented here describe response and markers of erythropoiesis and thrombopoiesis in HTB patients.

Results: At data cut-off (25-Oct-2021), median follow-up was 124 days (range 10-217). 24 patients received ≥1 dose of KER-050 across 4 dose levels (0.75mg/kg to 3.75mg/kg q4W). Of these, 16 were EE (8 not EE due to: duration of treatment (n=6), death (n=1), study discontinuation (n=1)). Median time from diagnosis was 2.2 years (range 0.2 – 8.6), 14 (58.3%) were HTB. Of HTB patients, 7 were RS+, 6 were on iron chelators.

No DLTs were reported in the safety-set. 5 (20.8%) reported grade ≥3 treatment-emergent AEs (TEAEs), 5 (20.8%) reported SAEs (none related), 4 (16.7%) developed grade 1 or 2 treatment-related AEs. Most frequent TEAEs (≥10%) were diarrhea, dyspnea, fatigue, nausea, anemia, headache. 2 discontinued study drug: 1 participant decision, 1 death. 1 required dose modification due to unrelated TEAE; none required dose modification for increased HGB or thrombocytosis.

Results from 16 EE cohort 1-3 patients were presented at ASH 2021 (Poster #3675), 8 (50%) achieved HI-E or TI. New analyses focused on HTB patients found that 6 of 9 HTB, EE patients achieved HI-E and 4 achieved TI for ≥8 weeks (Panel A). Increases in reticulocytes (RETs) (Panel B) and sTfR and decreases in serum ferritin (Panel C) in these HTB patients were observed. Mean maximum RETs increase was 56.87 x10 9 /L, range 15.7-142.5x10 9 /L with mean maximum ferritin reduction of 29.7%, range -17 to 65% and mean maximum sTfR increases of 51%, range 29-90%. Increases in platelets were observed in HTB patients achieving HI-E or TI (Panel D). Maximum increase from baseline was 98 x10 9 /L (mean), range 33-179 x10 9 /L.

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Summary/Conclusion: KER-050 was generally well-tolerated as of data cut-off date. HI-E and TI ≥8 weeks have been observed in HTB patients treated with KER-050. Observed PD effects in RETs, sTfR and ferritin support the proposed mechanism of action of increasing erythropoiesis. Increases in platelets have been observed in HTB patients achieving HI-E or TI which supports the potential of KER-050 as a treatment for multilineage cytopenias in difficult-to-treat HTB patients. Dose escalation is ongoing in this Phase 2 study of anemic patients with MDS; updated data from Part 1 with safety, PD and efficacy data from cohorts 4 and 5 will be presented for the first time at the meeting.

P777: RUNX1 VARIANTS WITH HIGH VARIANT ALLELE FREQUENCY IN MYELOID NEOPLASMS. GERMLINE OR NOT?

N. J. Nitschke 1,2,* , M. Krogh 3 , K. Raaschou-Jensen 4 , M. T. Severinsen 5 , A. S. Roug 6 , J. S. Jespersen 2 , J. W. Hansen 1,2 , J. Weischenfeldt 2 , M. K. Andersen 7 , K. Grønbæk 1,2

1 Department of Hematology, Rigshospitalet; 2 Biotech Research and Innovation Center (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen; 3 Department of Hematology, Sjælland University Hospital, Roskilde; 4 Department of Hematology, Odense University Hospital, Odense; 5 Department of Hematology and Clinical Cancer Research, Aalborg University Hospita, Aalborg; 6 Department of Clinical Medicine, Aarhus University Hospital, Aarhus; 7 Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark

Background: RUNX1 is a transcription factor frequently mutated in myeloid neoplasms e.g. 10% of acute myeloid leukemia (AML) patients, but RUNX1 variants detected in tumor tissue at a variant allele frequency (VAF) close to 50% can potentially be germline. As pathogenic germline RUNX1 variants are causative for RUNX1 Familial Platelet Disorder with Associated Myeloid Malignancies it is crucial to distinguish between somatic and germline variants. Previous studies in AML patients estimate the frequency of germline RUNX1 variants to be approximately 3%. The frequency of RUNX1 germline variants in lower risk myeloid neoplasms (MN) such as myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), clonal cytopenia of undetermined significance (CCUS) and idiopathic cytopenia of undetermined significance (ICUS) remains unknown.

Aims: To estimate the frequency of RUNX1 germline variants in MN.

Methods: Patients referred to a hematological department in Denmark with suspicion of MN (excluding myeloproliferative disorders) were included in the study. At first visit, a skin biopsy was drawn and frozen dry at -80 °C. RUNX1 variants were identified in either blood or bone marrow using 4 different amplicon based next generation sequencing panels. Variants detected with a VAF > 40 % were further classified as somatic or germline by bi-directional sanger sequencing of germline DNA purified from skin biopsies.

Results: Among 595 patients, 85 (14%) had variants in RUNX1 and 28 patients (5%) had variants with a VAF above 40%. High VAF RUNX1 variants were either missense, frameshift, nonsense, or splice-site variants. A majority of the variants were located in the runt homology domain (RHD). We observed a higher proportion of patients with AML and a lower proportion of patients with CCUS in patients with a RUNX1 variant compared to patients without a RUNX1 variant, and this tendency was enhanced if the RUNX1 variants were present at VAF > 40%.

Among 22 patients with a high VAF RUNX1 variant and available germline DNA two variants (9%) were classified as germline ( NM_001754.5 ; c.668A>G, c.649G>A, minimal allel frequency reported in gnomAD; 0.012%, 0.003%). The c.668A>G variant was detected in a patient with a low platelet count and CCUS diagnosed at age 24. The c.649G>A variant was detected in a patient with a normal platelet count and MDS diagnosed at age 64. Both germline variants were missense variants located in the region between the RHD and the transactivating domain (TAD) and both were predicted to be pathogenic in-silico by several prediction tools. Unfortunately, family history wasn’t available for any of the two patients with germline variants.

Pathogenic variants co-occurring at similar VAF as RUNX1 were detected in 17 out of 20 patients with a somatic RUNX1 variant, but not in any of the patients with germline variants (Figure 1). Pathogenic variants in SRSF2 , TET2 and ASXL1 were frequently observed to co-occur with high VAF RUNX1 variants.

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Summary/Conclusion: RUNX1 variants with a VAF above 40% were classified as germline in two out of 22 MN patients (9%). The variants were detected in patients with MDS and CCUS indicating that germline testing should be considered not only in AML, but also in other MN with high VAF of a RUNX1 variant. Furthermore, a high VAF RUNX1 variant without co-occurring variants with similar VAF in other genes should increase the consideration for germline testing.

P778: LONG-TERM UTILIZATION AND BENEFIT OF LUSPATERCEPT IN PATIENTS (PTS) WITH LOWER-RISK MYELODYSPLASTIC SYNDROMES (LR-MDS) FROM THE MEDALIST TRIAL

P. Fenaux 1,* , V. Santini 2 , R. S. Komrokji 3 , A. Zeidan 4 , G. Garcia-Manero 5 , R. Buckstein 6 , D. Miteva 7 , K. Keeperman 8 , N. Holot 8 , J. Zhang 8 , J. A. Nadal 7 , B. Rosettani 7 , A. Yucel 8 , U. Platzbecker 9

1 Service d’Hématologie Séniors, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris and Université Paris 7, Paris, France; 2 MDS Unit, AOU Careggi, University of Florence, Florence, Italy; 3 Moffitt Cancer Center, Tampa; 4 Department of Internal Medicine, Yale School of Medicine and Yale Cancer Center, New Haven; 5 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America; 6 Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Canada; 7 Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland; 8 Bristol Myers Squibb, Princeton, United States of America; 9 Medical Clinic and Policlinic 1, Hematology and Cellular Therapy, University Hospital Leipzig, Leipzig, Germany

Background: Luspatercept has been shown to improve anemia in the phase 3 MEDALIST trial of pts with LR-MDS ineligible/intolerant or refractory to erythropoiesis-stimulating agents (ESAs).

Aims: To report the long-term clinical value of luspatercept treatment (Tx) in pts from the MEDALIST study including dosing, duration of Tx (DOT) and response, baseline characteristics of pts receiving luspatercept in a long-term follow-up (LTFU) study, and rates of progression to acute myeloid leukemia (AML) and high-risk MDS (HR-MDS).

Methods: Eligible pts were ≥18 y of age, had LR-MDS requiring regular red blood cell (RBC) transfusions, and were ineligible/intolerant or refractory to ESAs. Pts were randomized 2:1 to subcutaneous luspatercept or placebo every 3 wk for 24 wk. The primary endpoint was RBC transfusion independence (RBC-TI) ≥8 wk during wk 1–24. MEDALIST pts who continued to receive luspatercept due to ongoing clinical benefit were eligible for enrollment into the LTFU study, a phase 3b rollover study to evaluate the long-term safety of luspatercept in pts who participated in other luspatercept studies. DOT was calculated as (Tx end date − date of first dose) + 1) / 7. Duration of response was determined by Kaplan–Meier analysis. Total person-years for pts at risk of HR-MDS/AML progression was calculated from LR-MDS diagnosis to HR-MDS/AML diagnosis, or to last HR-MDS/AML follow-up date for pts who did not progress.

Results: As of Jan 15, 2021, the median (95% confidence interval [CI]) DOT was 11.70 (8.97–16.33) mo for luspatercept pts and 5.52 (5.52–5.59) mo for placebo pts. Median (interquartile range [IQR]) DOT for placebo pts who transitioned to LTFU was 24.0 (24.0–25.3) wk and 24.0 (24.0–39.0) wk for placebo pts who discontinued prior to LTFU. The median (IQR) DOT for luspatercept pts who transitioned to LTFU was longer than for those pts who discontinued (190.0 [86.0–211.0] wk vs 31.0 [24.0–64.1] wk). Baseline characteristics more frequently observed in luspatercept pts who transitioned to LTFU than in pts who discontinued include younger age, female sex, and lower Eastern Cooperative Oncology Group performance status score, transfusion burden, erythropoietin, and serum ferritin levels (Table A). In MEDALIST, 106/153 (69.3%) pts receiving luspatercept and 64/76 (84.2%) receiving placebo escalated to the maximum dose of 1.75 mg/kg. During the entire Tx phase, RBC-TI ≥8 wk was observed in 74/153 (48.4%) and 12/76 (15.8%) luspatercept and placebo pts, respectively, with a median (95% CI) cumulative duration of response of 80.7 (53.71–154.14) wk and 21.0 (10.86–NE) wk, respectively. During the entire Tx period, RBC-TI ≥16 wk was observed in 48/153 (31.4%) and 6/76 (7.9%) luspatercept and placebo pts, respectively (Table B). Among luspatercept pts, 13/153 (8.5%) progressed to HR-MDS/AML during the entire Tx period, compared with 5/76 (6.6%) placebo pts. The total person-years for pts randomized to luspatercept at risk of progressing to HR-MDS/AML was 401.7 y vs 190.9 y for placebo.

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Summary/Conclusion: Pts receiving luspatercept had an extended period of clinical benefit and >50% of pts continued to receive luspatercept for >1 y, the majority of whom underwent dose escalations to achieve an optimal response. Pts experienced durable responses with luspatercept, with a median cumulative duration of RBC-TI response of approximately 20 mo. Pts receiving luspatercept also remained on Tx longer than placebo regardless of participation in the LTFU study, suggestive of luspatercept’s clinical benefit with significant durability.

P779: OPTIMIZING TREATMENT OUTCOMES FOR CHILDREN WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME UNDERGOING ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

Y. Ren 1,* , F. Liu 1 , X. Chen 1 , X. Li 1 , L. Liu 1 , W. Yang 1 , Y. Guo 1 , X. Zhu 1

1 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Myelodysplastic syndrome (MDS) are rare clonal hematopoietic disorders in children. Risk stratification system for adults is unfit for children. Refractory anemia with excess blasts(RAEB), acute myeloid leukemia with myelodysplasia-related changes(AML-MRC), monosomy 7 and complex karyotype always indicate poor prognosis. Hypomethylating agent (HMA) improved survivals of high-risk MDS in adults, and decitabine as part of conditioning regimen for allogenetic hematopoietic stem cell transplantation(allo-HSCT) was associated with favorable outcomes. However, these findings have not been tested in pediatric cohorts broadly.

Aims: To evalute the efficacy and safety of hypomethylating agents as a part of pre-transplant treatments and contidioning regimens in children with higher-risk MDS.

Methods: Patients from 1 to 14 years of age who were diagnosed with higher-risk MDS and underwent allo-HSCT consecutively between February 2019 and August 2020 in Pediatric Blood Diseases Center were enrolled. All identified patients were retrospectively reviewed with a collection of clinical data, laboratory test results, and outcome.

Results: We reviewed 18 pediatric patients with higher-risk MDS, including 7 with RAEB, 8 with AML-MRC, and 3 with refractory cytopenia of childhood with monosomy 7. Regardless of initial diagnosis, monosomy 7 is the most common cytogenetic aberration (8/18, 44.4%). Next-generation sequencing (NGS) was performed on 15 specimens and the most frequent detection of mutation gene was SETBP1, followed by ETV6. Before transplantation, ten patients received monotherapies or combined chemotherapies with HMA, two patients received AML-like chemotherapies, and nine of those achieved bone marrow complete remission. The stem cell sources came from matched sibling donor (n=1), haploidentical donor (n=11), and unrelated cord blood (n=6), respectively. Low-dose decitabine-containing myeloablative conditioning regimens were administered in all cases. About 50% of patients (n=9) experienced Grade II- IV acute graft versus host diseases post-transplant. The cumulative incidence of transplantation-related mortality (TRM) was 11.1%. During a median follow-up of 10.5 (range, 2–36) months after transplantation, 15 of 18 patients were alive. At time of last follow-up, all survivors had resolution of hematologic disorder without relapse.

Summary/Conclusion: In summary, administration of hypomethylating agents to allogeneic SCT resulted in excellent outcomes in children with higher-risk MDS.

P780: COMBINATION THERAPY OF ELTROMBOPAG AND CYCLOSPORINE A CAN IMPROVE HEMATOPOIESIS IN PATIENTS WITH LOWER RISK MYELODYSPLASTIC SYNDROME

L. Song 1,* , C. Chang 1

1 Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid disorders characterized by ineffective hematopoiesis and cytopenias, with variable risks of progression to acute myelogenous leukemia (AML). Peripheral blood cytopenias (thrombocytopenia, leucopenia, and anemia) are the major sources of morbidity and mortality for lower-risk MDS (LR-MDS) patients. Among the cytopenias, thrombocytopenia is a severe complication of ineffective hematopoiesis and is associated with poor overall survival.Eltrombopag (EPAG) is an orally administered small-molecule, nonpeptide thrombopoietin (TPO) receptor agonist, C-mpl is a TPO receptor expressed on megakaryocytes, HSCs, and progenitor cells. The interaction between EPAG and megakaryocytes regulates platelet production, maturation, and release through the binding of C-mpl on megakaryocytes.

Aims: The above studies provided a rationale for the use of the combination of EPAG and CysA in the treatment of LR-MDS. In this study, we investigated the efficacy and safety of the combination of EPAG and CysA in LR-MDS in a non-randomized phase II investigator-initiated trial (IIT).

Methods: Herein, a non-randomized phase II investigator-initiated trial was conducted to investigate the efficacy and safety of the combination of EPAG and cyclosporine A (CysA) in LR-MDS. EPAG dose was escalated from 50 mg/day to a maximum of 100 mg/day. The dose of CysA is 3–5 mg/kg/d to maintain serum trough concentration at 200 mg/mL.

Results: All the 31 patients were evaluated for response at the primary endpoint. Among them, 21 (67.7%) patients achieved treatment response in at least one eligible lineage with an objective response rate (ORR), 4 (12.9%) patients achieved a CR, 5 (16.1%) patients achieved bi-lineage responses (HI-PE R), 11 (24.3%) patients achieved platelet lineage responses (HI-P R), and 1 (3.2%) patient achieved erythroid lineage response (HI-E R). The median time to response was 8 (range, 2–24) weeks. Of the 21 patients eligible for an erythroid lineage response (RBC transfusion-dependent or Hb <90 g/L),10 patients (47.6%) had responded by the endpoint and the platelet response rate was 64.5% (20/31). The median increase in Hb levels and platelets was from 60.4±18.2 g/L to 68.0±21.2 g/L (P=0.015) and from 12.3±11.2×109/L to 32.5±37.3×109/L (P=0.003). The median DORs of HI-E and HI-P were not achieved, but the response rates of erythroid and platelet lineages in 20 months were 80.0% and 70.6%, respectively. The RBC and platelet transfusion-dependent rate decreased from 42.9% (9/21) to 28.6% (6/21) (X2=0.933，P=0.334) and from 58.0% (18/31) to 25.8% (8/31) (X2=6.624, P=0.010). respectively. The median time to response is 8 weeks.

All 31 patients were evaluated for toxicity, and the treatment was found to be well-tolerated. The most commonly reported adverse events (AEs) of any grade were nausea (6/31,19.4%), elevated liver transaminases (4/31,12.9%), and headache (3/31,9.7%); no severe AEs had been reported until the cutoff date.

Summary/Conclusion: In conclusion, our results indicated that the addition of Epag to CysA is well-tolerated and is an effective treatment for patients with low to intermediate-1 risk MDS. These response rates are better than CysA monotherapy, especially HI-P. Also, the hematological responses are durable, although continued Epag administration might be necessary. Further large prospective and controlled studies are warranted to define the role of this combination treatment in LR-MDS in comparison to CysA

P781: CLINICAL OUTCOMES AND HEALTHCARE RESOURCE UTILIZATION (HCRU) IN PATIENTS WITH LR-MDS REINITIATING ESAS FOLLOWING PREVIOUS ESA TREATMENT

G. Garcia-Manero 1,* , R. K. Matsuno 2 , A. McBride 3 , T. D. Brown 2 , D. Idryo 2 , R. Broome 2 , A. Herriman 2 , T. Johnson 2 , K. Wilkinson 2 , S. Walters 2 , A. Schrag 2 , C. Johanson 2 , M. Izano 2 , S. Mukherjee 4

1 MD Anderson Cancer Center, Houston; 2 Syapse, San Francisco; 3 Bristol Myers Squibb, Princeton; 4 Cleveland Clinic, Cleveland, United States of America

Background: The median survival for patients with lower-risk myelodysplastic syndromes (LR-MDS) is estimated to be 5–10 years. Erythropoiesis-stimulating agents (ESAs) are utilized as first-line treatment for anemia in LR-MDS; however, real-world treatment patterns, clinical outcomes, and healthcare resource utilization (HCRU) for patients with LR-MDS who reinitiated ESAs following previous discontinuation are not well-established.

Aims: To examine treatment patterns, clinical outcomes, and HCRU in patients with LR-MDS who reinitiated treatment with ESAs.

Methods: This was a retrospective study of patients with LR-MDS in a network of US community health systems who initiated ESAs as first-line therapy between January 1, 2016 and June 30, 2019 and were followed through June 30, 2021. Patients were required to have discontinued (had a ≥ 3-week gap between treatment with epoetin alfa, or a ≥ 6-week gap between treatment with darbepoetin alfa) and subsequently reinitiated ESA therapy at least once during follow-up. Outcomes included failure to achieve transfusion independence (TI), progression to acute myeloid leukemia (AML), overall survival (OS), number and type of health system visits, and medication use. TI was defined per IWG 2006 criteria in the subset of patients who had received ≥ 1 transfusion in the 8 weeks prior to ESA initiation.

Results: There were 108 patients with confirmed LR-MDS who initiated ESA-based therapy. Patients had a median age of 79 years at diagnosis (interquartile range [IQR] 73–85), were predominantly male (58%), White (97%), and overweight or obese (52%). The median follow-up period was 17.0 months (IQR 7.2–34.7). The most used ESA was darbepoetin alfa (60%), followed by epoetin alfa (38%) and epoetin alfa-epbx (2%). Of the 33 patients who received a baseline transfusion, 52% did not achieve TI; 53% and 43% failed to achieve TI at 6 and 12 months, respectively. The majority of patients were repeatedly treated with ESAs (n = 61, 56%). Among these patients, 2% progressed to AML and 48% died during follow-up; median OS was 45 months. The median number of health system visits per patient-month was 3.2 (IQR 0.6–4.9); 89%, 62%, and 72% of pts had ≥ 1 outpatient, emergency department, or inpatient visit respectively, with median length of hospitalization of 12 days (IQR 4.8–27.8). Most patients (54%) received an antibiotic at least once during follow-up; overall, use of immunosuppressive therapy (2%) or iron chelation (3%) was rare.

Summary/Conclusion: In this real-world study of community practice in the USA, reinitiation of ESA treatment after prior discontinuation in LR-MDS was prevalent. In these patients, poor clinical outcomes and frequent health system visits were observed. This study highlights the need to consider alternative treatment options for patients with LR-MDS.

P782: IMMUNOPHENOTYPIC AND MOLECULAR GENETIC MARKERS OF UNFAVORABLE PROGNOSIS IN PATIENTS WITH MYELODYSPLASTIC SYNDROME

H. Trubkina 1,* , I. Iskrou 2 , V. Smolnikova 3 , T. Lebedeva 3 , I. Lendzina 1

1 hematology department №3, State Institution “Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology”; 2 Department of Clinical Hematology and Transfusiology, Belarusian Medical Academy of Postgraduate Education; 3 clinical laboratory STCM, State Institution “Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology”, Minsk, Belarus

Background: Therapy is selected based on risk, need for transfusion of blood components, percentage of bone marrow blasts, and cytogenetic profile. In low-risk groups, the goal of treatment is to reduce the need for replacement therapy and prevent transformation to higher-risk or AML. In higher risk groups, the goal is to prolong survival.

Currently, there are no approved criteria for selecting therapy for patients with MDS based on the classifications and prognostic scales used, treatment regimens for progression of MDS or its refractory forms.

Aims: The aim of the study was to identify unfavorable prognostic immunophenotic and molecular genetic markers of blast cells in patients with high-risk myelodysplastic syndrome

Methods: The prospective cohort study included 68 patients with newly diagnosed MDS in the period from January 2017 to December 2021. The patients underwent a primary diagnostic complex of MDS, including immunophenotyping and molecular genetic research of bone marrow aspirate. The age of patients is 25-82 years, the median age is 64 years.

According to the IPSS classification included: high risk (10 pats), intermediate-2 (26 pats), intermediate-1 (26 pats), low risk (6 pats). According to the IPSS-R classification included: very high risk (23 pats), high risk (17 pats), intermediate (14 pats), low risk (12 pats), very low risk (2 pats). The following molecular genetic markers were found: complex aberrations >3 (10 pats), trisomy 8 chromosome (8 pats), absence of molecular genetic disorders (27 pats), isolated 5q chromosome (13 pats), any changes of the 7 chromosome (8 pats), deletion of the 20 chromosome (3 pats), deletion of the 11 chromosome (1 pats), TP53 (2 pats).

To explore immunophenotic and molecular genetic markers we use three state “illness-death” model. We consider the following states: diagnosis, transformation into leukemia; death.

Results: We identified high-risk immunophenotypic and molecular genetic markers

- · for transition “diagnosis-death”:

- CD38 <50% HR 3.7 (1.2-11.5 CI; p = 0.022),

- CD13> 50% HR 8.7 (1.1-67.8 CI; p = 0.04),

- complex aberrations >3 HR 5.8 (1.4-24.6 CI; p=0,015);

- · for transition “diagnosis-transformation”:

- CD71 ≥65% HR 4.1 (1.35-12.4 CI; p = 0.013);

- CD13> 75% HR 2.8 (1.1-7.1 CI; p = 0.034),

- complex aberrations >3 HR 2.8 (0,86-9,0 CI; p=0,087);

- · for transition “transformation-death“:

- CD25 <5% HR 6.3 (1.4-28 / 4 CI; p = 0.017),

- CD 33 <50% HR 6.6 (1.3-34.7 CI; p = 0.026)),

- complex aberrations >3 HR 0,22 (0,06-0,84 CI; p=0,027),

- trisomy 8 chromosome HR 7.9 (0.71-88 CI; р=0,093).

Figure 1 – The probability of various conditions depending on the time since the diagnosis.

State 1 - estimation of the probability of being in the state «diagnosis-transformation»

State 2 - estimation of the probability of being in the state «transformation-death»

State 3 - estimation of the probability of being in the state «diagnosis-death»

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Summary/Conclusion: The selection of groups of patients with identified immunophenotypic and molecular genetic markers of a high risk of transformation into acute leukemia and a high risk of death (“diagnosis-death”: CD38<50%, CD13>50%, complex aberrations>3; “diagnosis-transformation”: CD71≥65%, CD13>75%, complex aberrations>3; “transformation-death“: CD25<5%, CD33<50%, complex aberrations>3, trisomy 8 chromosomes) requires consideration of a new approach to therapy.

P783: SYSTEMIC THERAPY UTILIZATION AND HEMATOLOGIC OUTCOMES IN LOWER-RISK MYELODYSPLASTIC SYNDROMES (LR-MDS): FINDINGS FROM A REAL-WORLD MEDICAL RECORD REVIEW STUDY IN THE US, UK, AND EUROPE (EU)

M. Diez-Campelo 1,* , A. Yucel 2 , R. Goyal 3 , R. C. Parikh 3 , S. Dhuliawala 3 , M. Jimenez 3 , M. Sluga-O’Callaghan 4 , C. Hughes 5 , D. Tang 2 , U. Germing 6

1 Hematology Department, Institute of Biomedical Research of Salamanca, University Hospital of Salamanca, Salamanca, Spain; 2 Bristol Myers Squibb, Princeton, NJ; 3 RTI Health Solutions, Research Triangle Park, NC, United States of America; 4 RTI Health Solutions, Lyon, France; 5 Bristol Myers Squibb, Summit, NJ, United States of America; 6 University Hospital of Dusseldorf, Dusseldorf, Germany

Background: Patients (pts) with LR-MDS experience a mean life-year loss of 6 years, and the treatment focus is improvement of quality of life and prolongation of life expectancy.

Aims: This retrospective medical record review assessed real-world data to determine prevailing LR-MDS treatment patterns and hematologic outcomes.

Methods: Eligible pts (≥18 years of age) with LR-MDS (Jul 1, 2013–Sep 30, 2018) were identified by participating hematologist-oncologists in the US, Canada (CAN), UK, and EU (France, Germany, Spain). Study measures and hematologic outcomes were descriptively assessed (data abstraction Jun–Nov 2021). Aggregated analyses used pooled US/CAN and UK/EU data sets for the overall population and pt subgroups treated with clinician-defined first-line (1L) and second-line (2L) erythropoiesis-stimulating agents (ESAs).

Results: Medical record data were abstracted by 114 US/CAN (n=174 pts; median age 60.6 years; 68.4% male) and 166 UK/EU clinicians (n=319 pts; median age 67.1 years; 72.4% male). Median follow-up duration from MDS diagnosis was 60.6 months (US/CAN) and 67.6 months (UK/EU). Approximately half of the pts had “low” risk status (49.4% US/CAN; 53.9% UK/EU) categorized by the Revised International Prognostic Scoring System at initial diagnosis. The most common karyotype abnormality was del(5q) (27.6% US/CAN; 20.4% UK/EU). SF3B1 mutation was recorded for 7.5% US/CAN and 8.2% UK/EU pts. Almost all pts had received ≥1 line of systemic treatment for managing MDS-associated anemia (US/CAN: 165 [94.8%] 1L, 77 [44.3%] 2L; UK/EU: 294 [92.2%] 1L, 133 [41.7%] 2L). In 1L, ESA-based regimens were the most common treatment with median duration of therapy of ≥2 years (US/CAN: 142 [86.1%], of which the majority (80.3%) was ESA monotherapy; UK/EU: 264 [89.8%], comprising 82.2% ESA monotherapy). Treatment characteristics and outcomes are shown in the Table. Among pts who were transfusion dependent (TD) before 1L ESA, a very small proportion achieved transfusion independence (TI) or red blood cell transfusion reduction ≥50%, which was maintained for a median of <1.5 years. Among the 1L ESA-treated pts who were TD, TI was achieved by 5.7% (US/CAN) and 12.9% (UK/EU). In 2L ESA-treated pts (n=23 US/CAN; n=46 UK/EU), almost all were previously treated with a 1L ESA-containing regimen (95.7% US/CAN; 84.8% UK/EU). The most common reasons for reinitiating ESA in 2L (n=22, 15.5% US/CAN; n=39, 14.8% UK/EU) were overall health status (40.9% US/CAN; 33.3% UK/EU), treatment efficacy (40.9% US/CAN; 48.7% UK/EU), and compliance with national guidelines (13.6% US/CAN; 46.2% UK/EU). Among the pts who reinitiated ESA in 2L, the most common reasons for discontinuation of 1L ESA were completion of planned course of therapy (45.5% US/CAN; 28.2% UK/EU), pt decision (40.9% US/CAN; 20.5% UK/EU), progressive disease (36.4% US/CAN; 51.3% UK/EU), and adverse events (13.4% US/CAN; 15.4% UK/EU). Lenalidomide was used in 1L therapy among 8.5% pts in US/CAN and 5.8% in UK/EU. Among those with del(5q) mutation, ESA was used in 1L therapy among 83.3% pts in US/CAN and 87.7% in UK/EU.

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Summary/Conclusion: ESA-based regimens were the most common treatment for anemia management in pts with LR-MDS; >1/3 of pts were TD before initiating 1L ESA therapy. A considerable proportion of 1L ESA-treated pts reinitiated ESA in 2L despite the majority having discontinued 1L treatment due to planned therapy completion, pt decision, or progressive disease. This real-world study suggests that there is an unmet need in addressing anemia burden with ESAs, and novel and effective treatments are needed.

P784: A PHASE I/II STUDY OF VENETOCLAX IN COMBINATION WITH ASTX727 (DECITABINE/CEDAZURIDINE) IN TREATMENT‐NAÏVE HIGH‐RISK MYELODYSPLASTIC SYNDROME (MDS) OR CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML)

S. Venugopal 1,* , H. kantarjian 1 , A. Maiti 1 , N. Short 1 , G. Montalban-Bravo 1 , Y. Alvarado 1 , K. S. Chien 1 , R. Kanagal-Shamanna 2 , N. Pemmaraju 1 , N. Daver 1 , T. Kadia 1 , G. Borthakur 1 , E. Jabbour 1 , G. Garcia-Manero 1

1 Leukemia; 2 Hematopathology, The University of Texas, MD Anderson Cancer Center, Houston, United States of America

Background: In MDS, hypomethylating agents (HMA) remain the standard of care. ASTX727, an oral fixed dose combination of HMA decitabine (35mg) and cytidine deaminase inhibitor cedazuridine (100mg), was recently approved in the US for the treatment of MDS and CMML. Venetoclax (Ven), an orally bioavailable BCL-2 inhibitor in combination with azacitidine has shown preliminary clinical activity in treatment-naïve, higher risk MDS

Aims: Based on this data, we designed a study to evaluate a total-oral regimen of Ven+ASTX727 combination in pts with higher risk MDS or CMML

Methods: This single arm Phase I/II study of orally administered ASTX727 in combination with Ven ( NCT04655755 ) is enrolling patients ≥18 years with treatment‐naïve, higher risk MDS (intermediate-2- or high-risk categories) per IPSS or CMML with excess blasts ≥5%. The primary objective is to determine the safety and tolerability (phase 1) and overall response rate (ORR, defined as CR+mCR) (phase 2) of Ven+ASTX727 combination. ASTX727 is administered orally daily on D1‐5 and Ven is administered orally daily on D1‐14 of 28-d cycles. 3 dose levels of venetoclax in combination with ASTX727 will be tested. To mitigate tumor lysis syndrome, pretreatment white blood cell count should be less than 10 × 10 9 /L. Cytoreduction is allowed. The safety population includes all patients who received any dose of Ven+ ASTX727, and the efficacy population includes patients who have a valid baseline and post-baseline disease assessment and had received at least one dose of the study drug

Results: 15 pts have been enrolled to date (Figure). The median age is 72 years (range 54-94) with 12 pts aged ≥65 years. These patients had a median bone marrow blast count of 12% (range 6-15%) and harbored a median number of 4 (range 1-9) mutations. Most pts had adverse risk mutations such as ASXL1 (67%) and RUNX1 (47%).

No DLTs were observed in the initial 6 patient safety lead-in. There were no deaths during the 30-day and 60-day window. No tumor lysis syndrome was observed. The ORR was 87% with 3 pts achieving CR (20%) and 10 pts achieving marrow CR (67%). All pts achieved a response within 1 cycle among which 4 pts, including one with TP53 mut ,proceeded to hematopoietic stem cell transplant. 2 pts had stable disease at the end of 1 cycle. At a median follow up of 8.8 months, the median duration of response was not reached (range 0.9-11.1 months), and the median overall survival was not reached (range 1.0-12.1 months).

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Summary/Conclusion: Ven+ASTX727 combination appears safe and demonstrates preliminary efficacy in pts with higher risk MDS or CMML with excess blasts. Total-oral regimen of Ven+ASTX727 combination appears to be a promising strategy for high-risk MDS or CMML pts and may alleviate the burden of chronic, long-term parenteral HMA treatment

P785: CURRENT CARDIOVASCULAR DISEASE RISK PREDICTION MODELS ARE NOT APPLICABLE IN MDS PATIENTS: PRELIMINARY RESULTS OF A PROSPECTIVE OBSERVATIONAL SINGLE-CENTRE COHORT STUDY

C. Misidou 1 , E. Gkolia 2 , C. Kymparidou 1 , V. Papadopoulos 1 , M. Papoutselis 1 , K. Liapis 1 , I. Mitroulis 1 , G. Vrachiolias 1 , S. P. Deftereos 2 , D. Stakos 3 , I. Kotsianidis 1,*

1 Department of Hematology, Democritus University of Thrace Medical School; 2 Department of Radiology, University Hospital Of Alexandroupolis; 3 Cardiology Department, Democritus University of Thrace Medical School, Alexandroupolis, Greece

Background: Cardiovascular disease (CVD) is a leading cause of death in lower-risk Myelodysplastic syndrome (LR-MDS) patients and a shared pathobiology between MDS and CVD has been postulated. However, current evidence emanates only from retrospective cohorts suffering from crucial limitations.

Aims: Prospective annotation can overcome these limitations and delineate the role of MDS as an independent CVD risk factor. For this reason we conducted a prospective observational single-centre cohort study in LR-MDS patients.

Methods: Patients underwent thorough evaluation for CVD every 6 months. Cerebrovascular, peripheral and coronary vascular beds were assessed for atherosclerosis by ultrasound and coronary artery calcium (CAC), respectively. Cardiac structure and function was assessed by echocardiography. Multi-Ethnic Study of Atherosclerosis (MESA), Framingham (FRS) risk scores and serum markers of myocardial injury (Serum high-sensitivity troponin T, hsTnT), stress (N-terminal pro–B-type natriuretic peptide, NT-proBNP), and systemic inflammation (high-sensitivity C-reactive protein, hsCRP) were also estimated at each visit. Chi-square, Wilcoxon signed-ranks tests and Pearson’s correlation coefficient were used as appropriate. Multiple linear regression was applied for modeling the relationship between a dependent and one or more independent variables. The level of statistical significance was set to P=0.05.

Results: Table 1 presents baseline characteristics of 24 patients included so far. There were no correlations of CAC, MESA and FRS with MDS subtype and IPSS-R risk category when adjusting for MDS-CI, preexisting CVD, age, sex, and transfusion dependence. In sharp contrast to the general population ( Okwuosa, TM et al. JACC 2011 ), CAC score was not associated with FRS and was disproportionately increased in low and very low FRS risk patients (Figure 1). Likewise, the number of affected vascular beds (AVB) did not correlate with FRS (Figure 2), further suggesting that the current CVD risk prediction tools are inaccurate in MDS and arguing for an intrinsic tendency of MDS towards CVD development.

In 9 out of 24 patients a second evaluation at 6 months was completed. All 9 patients increased significantly CAC (p=0.004) and MESA (p=0.003) scores at 6 months, while 5/9 patients also increased the number of AVB. The average increase of CAC score (189.77 +/-212.89) was markedly higher than expected in the age-matched general population ( Budoff, MJ et al. JACC 2013 ) and was not associated with FRS at baseline, while it was also independent of MDS subtype and IPSS-R risk category after adjusting for MDS-CI, preexisting CVD, age, sex, and transfusion dependence. Finally, baseline NT-proBNP levels correlated strongly with CAC score (Figure 3) in line with numerous evidence showing that NT-proBNP can serve as a predictor of CVD risk in both MDS and non-MDS individuals.

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Summary/Conclusion: To our knowledge this is the first study in LR-MDS patients assessing longitudinally all critical parameters and providing objective measurements of subclinical atherosclerosis in all vascular beds. Our preliminary results indicate that the established CVD prediction models do not operate in LR-MDS patients and reinforce the notion that MDS represents an independent factor for CVD development. Accurate assessment of CVD risk and prompt detection of subclinical cardiovascular disease in MDS patients will assist clinical decisions on the introduction of intensive monitoring and preventive use of CVD-specific interventions in these individuals.

P786: ANTI-PD-1 ANTIBODY (SINTILIMAB) PLUS DECITABINE AS FIRST LINE TREATMENT FOR PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME（MDS）: PRELIMINARY RESULTS FROM A SINGLE-ARM, OPEN-LABEL, PHASE II STUDY

J. wang 1,* , S. li 1 , H. Jiang 1 , Y. Chang 1 , X. Zhao 1 , J. Jia 1 , X. Zhu 1 , L. Gong 1 , X. Liu 1 , W. Yu 1 , X. Huang 1,2

1 Peking University People’s Hospital, Peking University Institute of Hematology. National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation; 2 XiaoJun Huang, Correspondence.Peking University, People’s Hospital, Peking University Institute of Hematology, 11 Xizhimen South Street, Beijing, China

Background: Hypomethylating agents (HMAs) are the preferred treatment for untreated patients (pts) with higher-risk MDS, but the survival of pts after HMAs treatment is poor. It has been demonstrated that the PD-1/PD-L1 expression was upregulated by HMAs in MDS pts, providing a strong rationale for combining HMAs with PD-1 antibody for MDS treatment.

Aims: To evaluate safety and efficacy of Sintilimab plus decitabine for pts with higher-risk MDS in a single-arm, open-label,phase 2 study(ChiCTR2100044393).

Methods: Adult pts with higher-risk MDS by the IPSS-R were enrolled. Patients received decitabine 20mg/m 2 intravenously daily for 5 days and sintilimab 200mg IV starting on the first and 22nd day of a cycle every 42 days until unacceptable toxicity, relapse, or progression, for a maximum of 8 cycles. The primary endpoint was overall response rate (CR+PR+mCR). Simon’s optimal two-stage design was employed.If five or more pts in stage I (13 pts) achieved ORR, the study would enroll 34 additional pts in stage II (47 pts).Secondary endpoints included safety, and survival outcomes. The relationship between expression levels of immune-checkpoint and efficacy of the combination therapy, as well as other potential biomarkers were also explored via genomic profiling.

Results: At data cut-off (February 22, 2022), 21 pts were enrolled with a median follow-up time of 6.5 months. The median age of pts was 64 years (range 30-83), and the other characteristics are summarised in Table 1.The ORR was 62%, with six pts reaching complete response (CR),four pts reaching marrow CR and three pts achieved marrow CR+ hematologic improvement (HI). In addition,four pts reaching HI,so the overall improvement rate was 81%. The most common grade 3 treatment-emergent adverse events(TEAEs)(>10%) were febrile neutropenia (76.2%) and pulmonary infection (38.1%). No grade 4 TEAEs and treatment-related deaths occurred. A total of 12 pts （57.1%）experienced immune-related adverse event, including rash (28.6%), pneumonia (9.5%), hypothyroidism (9.5%), elevated serum bilirubin (4.8%) and transpeptidase (4.8%), which were all resolved by glucocorticoid. In these 21 pts, the most frequently mutated genes are ASXL1（28.6%）， RUNX1 (14.3%) and TET2(14.3%). Updated biomarker data will be presented during the EHA meeting.

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Summary/Conclusion: To the best of our knowledge, this is the first study to evaluate the efficacy and safety of sintilimab plus decitabine in pts with untreated higher-risk MDS. The preliminary results demonstrate that the combination therapy is relatively safe with anti-tumor activity.

P787: SABATOLIMAB (MBG453) COMBINATION TREATMENT REGIMENS FOR PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROMES: THE MYELODYSPLASTIC SYNDROMES STUDIES IN THE STIMULUS IMMUNO-MYELOID CLINICAL TRIAL PROGRAM

A. M. Zeidan 1,* , A. Al-Kali 2 , U. Borate 3 , T. Cluzeau 4 , A. E. DeZern 5 , J. Esteve 6 , A. Giagounidis 7 , K. Kobata 8 , R. Lyons 9 , U. Platzbecker 10 , D. A. Sallman 11 , V. Santini 12 , G. F. Sanz 13 , M. A. Sekeres 14 , A. H. Wei 15 , Z. Xiao 16 , M. Van Hoef 17 , C. Nourry-Boulot 17 , I. Sadek 18 , F. Ma 18 , A. Iordan 17 , J. Sabo 18 , G. Garcia-Manero 19

1 Yale University and Yale Cancer Center, New Haven; 2 Mayo Clinic, Rochester; 3 Oregon Health & Science University, Portland, United States of America; 4 Centre Hospitalier Universitaire de Nice, Nice, France; 5 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins, Baltimore, United States of America; 6 Hospital Clinic, Barcelona, Spain; 7 Marien Hospital, Düsseldorf, Germany; 8 None, Los Angeles; 9 The US Oncology Network, San Antonio, United States of America; 10 University Hospital Leipzig, Leipzig, Germany; 11 H. Lee Moffitt Cancer Center & Research Institute, Tampa, United States of America; 12 University of Florence, Florence, Italy; 13 Hospital Universitari i Politécnic La Fe; and CIBERONC, Instituto de Salud Carlos III, Valencia; Madrid, Spain; 14 Sylvester Comprehensive Cancer Center, University of Miami, Miami, United States of America; 15 Department of Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Australia; 16 Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin, China; 17 Novartis Pharma AG, Basel, Switzerland; 18 Novartis Pharmaceuticals Corporation, East Hanover; 19 MD Anderson Cancer Center, Houston, United States of America

Background: Patients (pts) with higher-risk myelodysplastic syndromes (HR-MDS) unfit for hematopoietic stem cell transplant (HSCT) have poor outcomes with single-agent hypomethylating agent (HMA) therapy. Novel treatments with durable responses and survival benefit are needed. TIM-3 regulates innate and adaptive immunity and is expressed on leukemic stem cells (LSCs) and blasts but not normal hematopoietic stem cells. Sabatolimab is a novel immuno-myeloid therapy that binds to TIM-3 on immune cells, facilitating immune activation and phagocytosis of leukemic cells. Sabatolimab also binds to TIM-3 on leukemic cells, potentially impeding self-renewal of LSCs. Sabatolimab + HMAs had promising efficacy and response durability in HR-MDS in early-phase (ph) trials, with few significant immunologic adverse events (AEs; Brunner AM. ASH 2021. Oral 244).

Aims: The STIMULUS immuno-myeloid clinical trial program investigates the safety, efficacy, and durability of sabatolimab-based combination therapies in patients with myeloid malignancies. Exploration of sabatolimab’s mechanism of action and identification of potential biomarkers predictive of response are planned. This abstract summarizes the design of 4 ongoing STIMULUS trials in previously untreated adult pts with HR-MDS ineligible for intensive chemotherapy or HSCT.

Methods: STIMULUS-MDS1 ( NCT03946670 ) is an international, randomized, double-blind, placebo-controlled, ph II trial evaluating sabatolimab + HMA in pts with very-high, high-, or intermediate-risk (vH/H/IR)-MDS who have completed enrollment (N=127). Primary endpoints are complete remission (CR) and progression-free survival (PFS).

STIMULUS-MDS2 ( NCT04266301 ) is an international, randomized, double-blind, placebo-controlled, ph III trial of sabatolimab + azacitidine (AZA) in pts with vH/H/IR-MDS or chronic myelomonocytic leukemia-2; the study has completed recruiting (N=530). Primary endpoint is overall survival (OS). Key secondary endpoints are time to definitive deterioration of fatigue; red blood cell transfusion-free interval; and improvement of fatigue, physical, and emotional functioning.

STIMULUS MDS-US ( NCT04878432 ) is a US-based, open-label, single-arm, ph II trial of sabatolimab + HMA of investigator’s choice (AZA intravenous [IV] or subcutaneous, decitabine IV, or oral decitabine/cedazuridine) in pts with vH/H/IR-MDS. Target enrollment is 90 pts. Primary endpoint is safety. Secondary endpoints include CR, PFS, leukemia-free survival (LFS), and OS.

STIMULUS-MDS3 ( NCT04812548 ) is an international, open-label, single-arm, ph II trial that explores triplet therapy of sabatolimab + AZA + BCL-2 inhibitor venetoclax (VEN) in pts with vH/HR-MDS. Part 1 is a safety run-in comprising 2 safety cohorts with ~6 pts receiving sabatolimab 400 mg + AZA + VEN and ~12 pts receiving sabatolimab 800 mg + AZA + VEN. Primary endpoint of part 1 is safety. If (both 400- and 800-mg doses) sabatolimab + AZA + VEN are safe, the trial will move to an expansion cohort (~58 pts) of sabatolimab 800 mg Q4W + AZA + VEN. Primary endpoint is CR. Secondary endpoints include CR + mCR rate, overall response rate (CR + mCR + PR + hematologic improvement), OS, PFS, LFS, and event-free survival.

The STIMULUS immuno-myeloid clinical trial program is investigating the efficacy and safety profiles and the quality-of-life improvements for sabatolimab-based combination therapies in pts with myeloid malignancies. The STIMULUS-MDS trials aim to gain insight into the durable benefit of sabatolimab combination therapies in pts with HR-MDS.

Results: None.

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Summary/Conclusion: None.

P788: FACTORS DRIVING TREATMENT DECISION IN PATIENTS WITH INTERMEDIATE-RISK MYELODYSPLASTIC SYNDROME (MDS): A RETROSPECTIVE ANALYSIS FROM THE GRUPO ESPAÑOL DE SÍNDROMES MIELODISPLÁSICOS SPANISH MDS REGISTRY

M. Díez-Campelo 1,2,* , L. Hernandez-Donoso 3 , E. Sasse 3 , S. Colicino 3 , J. Curto 4 , A. Molero Yordi 5 , M. Tormo Díaz 2,6,7 , M. Arnan 8 , G. Sanz 9,10 , M. Díaz-Beyá 11 , M. T. Cedena Romero 12 , A. Jerez 13 , D. Valcárcel Ferreiras 2,5

1 Hospital Universitario de Salamanca, Salamanca; 2 Grupo Español de Síndromes Mielodisplásicos (GESMD), Valencia, Spain; 3 Novartis Pharma AG, Basel, Switzerland; 4 MFAR Clinical Research SL, Madrid; 5 Vall d’Hebron Institute of Oncology (VHIO), University Hospital Vall d’Hebron, Barcelona; 6 Hospital Clínico Universitario Valencia; 7 INCLIVA Biomedical Research Institute, Valencia; 8 Institut Català d’Oncologia, Hospital Duran i Reynals, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona; 9 Hospital Universitario y Politécnico La Fe, Valencia; 10 CIBERONC, Instituto de Salud Carlos III, Madrid; 11 Hospital Clinic Barcelona, Barcelona; 12 Hospital Universitario 12 de Octubre, Madrid; 13 Hospital Universitario Morales Meseguer, Murcia, Spain

Background: Current treatment (tx) options for patients (pts) with MDS are limited, although multiple potential txs are in development. The use of the Revised International Prognostic Scoring System (IPSS-R) for diagnosis of MDS supports tx decisions and prognostic assessment. Pts with very low/low-risk (vL/LR) MDS usually receive supportive care (SC) to manage symptoms, whereas those with very high/high-risk (vHR/HR) MDS commonly receive active txs such as hypomethylating agents (HMAs), allogeneic hematopoietic stem cell transplantation (alloSCT), or chemotherapy (CT). There is limited agreement on whether active tx or standard SC should be used for pts with intermediate-risk (IR) MDS.

Aims: We aimed to identify factors possibly associated with the real-world use of tx in pts with IR-MDS in the Spanish MDS registry.

Methods: This was a retrospective, observational study of data from pts registered in the Grupo Español de Síndromes Mielodisplásicos (GESMD) from January 2008-June 2020. Adult pts with MDS (WHO 2008 classification) who signed an informed consent and had a minimum follow-up of 6 months in the registry were included. Pts with unavailable data on IPSS-R risk or HMA tx were excluded. We collected demographic, clinical, and laboratory data, and tx used, including azacitidine (AZA), CT, alloSCT, SC (eg, transfusions, growth factors) and other tx (eg, immunosuppressors, androgens). Univariate analysis was performed to evaluate the association of potential risk factors, using categorical and continuous variables, with the tx use. We focused our analysis on AZA and SC in pts with IR-MDS ineligible for alloSCT. Odds ratio, 95% confidence interval, and exploratory P value were reported for comparison between tx use.

Results: Overall, 4604 pts (median age: 76 y; male: 61%; IPSS-R score vHR: 432, 9.4%; HR: 487, 10.6%; IR: 761, 16.5%; LR: 1795, 39%; vLR: 1129, 24.5%) were included. Pts with IR-MDS were 63% male and similar in number by score and median age at diagnosis compared with the total study population: 3.5 (n=268, 73 y), 4.0 (n=270, 75 y) and 4.5 (n=223, 75 y). Most common treatments received were AZA (n= 241, 31.7%) and SC (n=250, 32.9%); a minority received CT and/or alloSCT (n=84, 11%), while 186 pts (24.4%) did not receive any. AZA was the only HMA used in this population. The median time from diagnosis to start of AZA in this group was 3 months. When comparing AZA with SC use in pts with IR-MDS, univariate analysis (Table 1) showed that gender, age at diagnosis, year of diagnosis, bone marrow and peripheral blood blasts, transfusion dependency, and hematologic parameters at diagnosis (eg, hemoglobin, platelet, and monocyte counts) were associated with tx selection. A multivariate analysis is ongoing for deeper understanding of these data.

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Summary/Conclusion: These preliminary data show several factors significantly associated with tx use, suggesting that in some pts with IR-MDS, AZA is more likely to be used versus SC when these factors are considered. It is possible that selection bias, misclassification and confounding may have occurred as the registry only collects routine data according to local practices, with variability in the quality and completeness of data across participating centers. Other factors not captured, such as pt characteristics, comorbidities, access to health care, and social/familial support, could have also influenced the tx decision. Further analyses are ongoing to identify the relationship of multiple factors at once and assert the strength of these associations while removing the effect of confounders.

P789: ANALYSIS OF THE CLINICAL FEATURES, PROGNOSIS AND GENE MUTATIONS IN PRIMARY MYELODYSPLASTIC SYNDROME PATIENTS WITH MYELOFIBROSIS

G. Xu 1,* , S. Zhu 1 , H. Tong 1

1 Hematology, Hospital, Hangzhou, China

Background: Bone marrow(BM) fibrosis in myelodysplastic syndrome (MDS-MF) is observed approximately in 10–50% of primary MDS patients in the previous reports. BM biopsy is essential for the diagnosis and evaluation of MDS-MF patients and they differ from MDS without MF in terms of clinical performance, prognosis and survival. Although MDS-MF is associated with an adverse prognosis in the view of most hematologists, MDS-MF is not independently listed as a subtype of MDS according to the 2016 World Health Organization classification, indicating the need for further investigation. In this study, we retrospectively analyzed 417 newly diagnosed primary MDS cases in our hospital and to compare the clinical characteristics, prognosis and gene mutations of the two group patients with or without myelofibrosis.

Aims: This study aims to analyze the clinical features, prognosis and characteristics of the gene mutations of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients, ultimately to improve the cognition of MDS-MF.

Methods: From January 1, 2013, to February 1, 2020, 417 newly diagnosed primary MDS patients in the first affiliated hospital of Zhejiang University with bone marrow (BM) biopsy examination were included. They were divided into two groups according to whether BM associated with fibrosis (2005 European Myelofibrosis Network criteria), the MDS-MF group(MF grades 1–3）and the MDS without MF group (MF grade 0）.The clinical features, prognosis and gene alterations were analyzed between the two groups retrospectively.

Results: ① MF was confirmed in 46.3％ cases of all the MDS patients（193/417）, of which 66.3％（128/193）were MF-1, 25.9％（50/193）were MF-2 and 7.8% were MF-3 respectively. There is no statistically significant difference in the proportion of subtypes patients according to the 2016 who classification (P=0.342).

②Compared with those without MF, MDS-MF were significantly associated with worse OS(P=0.035) and worse PFS(p<0.001). Subgroup analysis showed that both OS and PFS of either the MDS-MF1-2 group or the MDS-MF 3 group were also significantly worse than those of the MDS without MF group（P＝0.013 and <0.001 respectively）.

③The clinical features of the two groups were statistically significant differences in the higher number of BM blast cell(P=0.006), higher prevalence of death (P=0.031), higher rate of Leukemic transformation (P<0.001), higher level of serum lactate dehydrogenase (LDH) (p<0.001), higher level of β2-microgram (p=0.003) and higher level of CRP in the peripheral blood (P=0.004) in the MDS-MF group than in the MF0 group.

④Among all the patients,337 were finished Next-generation-sequencing (NGS) test. Using the same panel analysis, U2AF1 gene mutation was more frequently in the MDS-MF compared with those patients without MF (P=0.027). There was totally 14.2% (48/417) patients with the mutation of U2AF1, of which 19.7%（29/147) in MDS-MF group and 10.0%（19/190) in MDS without MF group. There was significant difference concomitant with U2AF1 mutation between the two groups(P=0.038).

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Summary/Conclusion: MDS-MF has unique laboratory and clinical characteristics. MF is an independent risk factor for shorter overall survival (OS) and worse progression-free survival (PFS) in primary MDS. Evaluation of MF is very significant for prognosis judgment in primary MDS. The U2AF1 mutation may be a biomarker related to the poor prognosis of MDS-MF. However, the mechanism of U2AF1 in MDS-MF patients needs to be further explored.

P790: THE EXPRESSION LEVEL OF WT1 MRNA IN PATIENTS WITH IDIOPATHIC CYTOPENIA OF UNDETERMINED SIGNIFICANCE (ICUS) PROGRESSING TO MYELODYSPLASTIC SYNDROME (MDS)

X. Ye 1,* , J. Huang 1

1 Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China

Background: Idiopathic cytopenia of undetermined significance (ICUS) is defined as patients with one or more unexplained cytopenia who do not meet diagnostic criteria for myelodysplastic syndrome (MDS) or another hematologic disorder. In the clinical course of ICUS, some patients eventually have typical MDS changes. Until now, there is no effective method to predict the development of the disease. Recent studies have shown that the expression level of WT1 mRNA in peripheral blood has important clinical significance in the diagnosis, occurrence, development and prognosis of MDS. At the same time, some studies have proved that there is a significant difference in the expression level of WT1 mRNA between MDS and hemocytopenia caused by other reasons.

Aims: In order to clarify the clinical significance of WT1 mRNA expression in the progression of ICUS to MDS.

Methods: Between April 2017 and January 2020, untreated adult patients with ICUS or lower-risk MDS were enrolled in the study. All patients detected the WT1 mRNA expression of bone marrow and peripheral blood at diagnosis. Then patients with ICUS were followed up. Every three months, the patients with ICUS detected the WT1 mRNA expression of peripheral blood, until the patient progressed to MDS. The results of WT1 transcript levels are depicted as WT1 (copies/mL)/c-ABL (copies/mL) as a percentage.

Results: Total of 80 patients with ICUS and 40 patients with lower-risk MDS were enrolled. At diagnosis, the median expression level of WT1 mRNA in MDS group was 5.99 (0.08-79.27), and that in ICUS group was 0.50 (0.16-22.48). There was significant difference between them (P = 0.000). Then 80 patients with ICUS were followed up to January 2022. The median follow-up time was 30 months (11-54 months). 8 patients developed MDS, the progression rate was 10%, and the median progression time was 6.5 months (2-19 months). The median expression level of WT1 mRNA at the initial diagnosis in ICUS group without progressing to MDS was 0.44 (0.16-7.58), and that in progressing to MDS group was 9.44 (0.26-22.48). There was significant difference between the two groups (P = 0.031). The median expression level of WT1 mRNA was 19.185 (0.73-42.30) when MDS was diagnosed. The median maximum expression level of WT1 mRNA in peripheral blood of ICUS group without MDS was 0.98 (0.22-7.58). There was significant difference between the two groups (P = 0.007).

Summary/Conclusion: The study clarified the WT1 mRNA expression status of ICUS patients. There were significant differences in WT1 mRNA expression level between patients with ICUS and lower-risk MDS, and the WT1 mRNA expression level of patients with lower-risk MDS was relatively high. The study confirmed that patients with high WT1 mRNA expression level in the patients with ICUS (WT1 mRNA expression level > 9.44) are very likely to progressing to MDS; Dynamic monitoring of WT1 mRNA expression in peripheral blood is an effective method for following-up after ICUS diagnosis. The progressive increase of WT1 mRNA expression is one of the indicators for researchers to judge the progress of ICUS to MDS.

P791: COMMON VARIANTS IN COMPLEMENT PROTEINS, C3 AND CR1, ENHANCE COMPLEMENT ATTACK ON PNH ERYTHROCYTES AND INCREASE RISK FOR EXTRAVASCULAR HAEMOLYSIS

A. J. Baral 1,* , C. Mckinley 2 , R. Fellows 1 , T. Hallam 1 , T. Cox 1 , D. Payne 3 , S. Richards 2 , A. Pike 4 , R. Kelly 4 , M. Griffin 4 , P. Hillmen 2 , K. Marchbank 1 , D. Kavanagh 5 , D. Newton 6 , C. Harris 1

1 Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne; 2 Division of Haematology and Immunology, Leeds Institute of Medical Research, University of Leeds; 3 Haematological Malignancy Diagnostic Service, St. James’s University Hospital; 4 Department of Haematology, St James University Hospital, Leeds; 5 Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne; 6 Division of Haematology and Immunology, Leeds Institute of Medical Research, University of Leeds, Leeds, United Kingdom

Background: In paroxysmal nocturnal haemoglobinuria (PNH), absence of glycosylphosphatidylinositol (GPI) anchors leads to loss of the complement inhibitor, CD59, on PNH erythrocytes (E) causing complement-mediated intravascular haemolysis. Treatment with anti-C5 anitbody (eculizumab or ravulizumab) rescues PNH-E but loss of another GPI-anchored regulator, CD55, leads to accumulation of C3 fragments on E and extravascular haemolysis (EVH) due to C3-driven erythrophagocytosis. Therefore, approximately 30% of patients still require blood transfusion. Deposition of C3b and generation of downstream inactivation fragments such as iC3b and C3dg is controlled by binding of complement receptor type 1 (CR1) to C3b with factor I (FI), forming a trimolecular complex (TMC). We hypothesise that polymorphisms in C3 and CR1 influence the efficacy of this inactivation process and dictate propensity for EVH.

Aims: To determine the combined effect of C3-S/F and CR1 density polymorphisms in susceptibility to EVH by the inactivation of C3b/iC3b on PNH-E.

Methods: Forty-two eculizumab-treated PNH patients were genotyped for their single nucleotide polymorphism in C3 (rs2230199,S/F) and CR1 (rs11118133,H/L). C3 loading on PNH-E of patients were measured by flow cytometry as part of the routine analysis at the Leeds Haematological Malignancy Diagnostic Service. Lactate dehydrogenase, haemoglobin levels and reticulocyte count were also recorded. Soluble CR1 (sCR1) constructs were generated using recombinant technology and C3 variants were purified from human plasma using classical chromatography techniques. Interactions between C3b and CR1 and formation of the alternative pathway regulatory TMC (C3b:CR1:FI) were analysed using surface plasmon resonance (SPR).

Healthy donors were genotyped for their CR1 density polymorphism and relative CR1 expression on E was quantitated by flow cytometry. Impact of CR1 density polymorphism on C3b inactivation was studied using C3b-coated streptavidin beads and solubilised E from CR1 H/H or H/L donors and C3b breakdown to iC3b/C3dg was measured using flow cytometry.

Results: There was a trend for higher mean percentage of C3 loading in patients who have the C3-S/S allele (n=28, 30%) compared to patients with the C3-S/F allele (n=12, 18%) and a patient who had the C3-F/F allele (7%). Patients with high (H/H) CR1 expression (n=25, 20%) showed a trend for lower mean percentage of C3 loading on PNH-E than patients with intermediate (H/L) CR1 expression (n=17, 32%). In patients with the C3-S/S polymorphism, haemoglobin levels were significantly lower (p=0.0132) and higher numbers of patients (20 of 29) had at least one transfusion event.

Using SPR, we demonstrated that CR1 bound C3b-F with a higher affinity than C3b-S. This led to higher levels of TMC formation with FI and more effective decay of C3-F convertase (C3b-F:Bb) by CR1. Solubilised E from a CR1-H/H donor converted iC3b more effectively to C3dg compared to E from CR1-H/L donors.

Summary/Conclusion: These data indicate that both C3-S/F and CR1 density polymorphisms may influence C3b loading on PNH-E, with C3 fragments influencing EVH risk. Weaker binding of C3b-S to CR1 led to a decreased regulatory TMC formation with FI and slower decay of C3-S convertase (C3b-S:Bb). These data also indicate that E from individuals with lower CR1 expression convert the iC3b to C3dg more slowly. Overall, these data demonstrate mechanisms that enhance erythrophagocytosis in susceptible PNH patients by increased C3 loading on PNH-E and a decreased in removal of phagocytic opsonin, iC3b.

P792: CRISPR/CAS9-BASED MODEL OF HETEROZYGOUS CXCR4 WT/R334X MUTATION TO STUDY CELLULAR PHENOTYPES IN WHIM SYNDROME

K. Zmajkovicova 1,* , S. Pawar 1 , S. Maier-Munsa 1 , A. Badarau 2 , A. G. Taveras 3

1 X4 Pharmaceuticals (Austria) GmbH; 2 Former Employee at X4 Pharmaceuticals (Austria) GmbH, Vienna, Austria; 3 X4 Pharmaceuticals, Boston, United States of America

Background: WHIM syndrome is a phenotypically heterogenous primary immunodeficiency characterized by Warts, Hypogammaglobulinemia, Infections, Myelokathexis, neutropenia and lymphopenia. WHIM syndrome pathogenesis is causally linked to heterozygous gain-of-function (GOF) mutations in the C-terminus of the chemokine receptor CXCR4, a master regulator of immune cell trafficking and homeostasis, causing desensitization defects and hyperactivation of downstream signaling. The most frequently reported mutation in patients with WHIM syndrome is c.1000C>T, which results in a C-terminal truncation of the receptor at position R334 (R334X).

Aims: In vitro assays using cell lines with exogeneous overexpression of CXCR4 WHIM variants can model the GOF cellular phenotypes of WHIM syndrome but may not entirely mimic the condition in patients, who typically have one wildtype (WT) CXCR4 allele. We aimed to characterize a cellular model of homozygous and heterozygous CXCR4 R334X in the endogenous locus, to better understand the pathogenic impact of harboring mutations in one or both alleles.

Methods: CRISPR-Cas9 platform was used to establish a model of heterozygous mutations found in patients with WHIM syndrome. Jurkat cell line (with endogenous expression of WT CXCR4 ) was edited to harbor the c.1000C>T/R334X mutation in a single allele (WT/RX) or in both alleles (RX/RX). Unedited parental Jurkat cell line and Jurkat cells with edited silent mutations (WT/WT) were used as controls.

Results: Upon stimulation with C-X-C chemokine ligand 12 (CXCL12), RX/RX cell lines displayed an internalization defect (65% of CXCR4 receptors remaining on the cell surface at 100 nM CXCL12) compared to the parental (24%) and WT lines (20%). The RX/WT-expressing cells had an intermediate phenotype (43%). We analyzed the signaling responses downstream of activated CXCR4, for which GOF phenotypes had been reported in R334X patient cells. Calcium mobilization in response to CXCL12 was enhanced in cells harboring the R334X mutation, reaching a 2.5- to 3-fold higher maximum effect (Emax) compared to cells with WT CXCR4. ERK activation downstream of CXCR4 reached a higher amplitude (8-fold increase over baseline) and duration after stimulation with CXCL12 in all lines expressing R334X compared to the parental and WT/WT cell lines (6-fold increase). Presence of a single mutant allele seemed to confer the full GOF phenotype in both signaling readouts in this cell line. Migration of cells toward CXCL12 was significantly enhanced in R334X-expressing cells. Mavorixafor, a CXCR4 antagonist currently in clinical trials for the treatment of patients with WHIM syndrome, was active in an unbiased manner on a spectrum of CXCR4-related functions (ligand binding inhibition, calcium mobilization, ERK activation and chemotaxis) with comparable biological activity and potency in cells expressing the WT and R334X CXCR4 receptor.

Summary/Conclusion: We established the first model recapitulating the heterozygous CXCR4 WT/R334X mutations found in patients with WHIM syndrome using the CRISPR/Cas9 platform. This cellular model recapitulates the functional defects found in immune cells from patients with WHIM syndrome. When stimulated with CXCL12, WT/RX -expressing cells appeared to display full GOF phenotype in downstream signaling assays compared to RX/RX-expressing cells. This observation is consistent with the dominant inheritance pattern of WHIM syndrome. The present study brings several novel insights in CXCR4 WHIM biology and enriches the toolbox of models available for studying WHIM syndrome.

P793: SCREENING OF NATURALLY OCCURRING CXCR4 VARIANTS FOR IDENTIFICATION OF NOVEL PATHOGENIC MUTATIONS FOR WHIM SYNDROME

S. Pawar 1,* , I. Wiest 1 , S. Maier-Munsa 1 , B. Maierhofer 1 , N. Sondheimer 2 , A. G. Taveras 3 , A. Badarau 4 , K. Zmajkovicova 1

1 X4 Pharmaceuticals (Austria) GmbH, Vienna, Austria; 2 Division of Clinical and Biochemical Genetics, The Hospital for Sick Children, Toronto, Ontario, Canada; 3 X4 Pharmaceuticals, Boston, United States of America; 4 Former Employee at X4 Pharmaceuticals (Austria) GmbH, Vienna, Austria

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare, autosomal-dominant primary immunodeficiency marked by neutropenia and lymphopenia. Classically, WHIM syndrome pathogenesis is causally linked to a variety of heterozygous gain-of-function mutations in the C-terminus of chemokine receptor CXCR4 , a master regulator of immune cell trafficking and homeostasis. As of February 2022, 18 variants in CXCR4 have been implicated in WHIM syndrome, while a substantial number remain unexplored for their association with the disease. Using in vitro functional assays, we previously found that impaired receptor internalization and enhanced chemotaxis are conserved in all CXCR4 WHIM variants and that defective internalization correlates with neutropenia, the most penetrant phenotype of patients with WHIM syndrome. We used this approach to confirm functional defects in primary cells isolated from patients harboring a novel variant, suggesting that the in vitro profiling approach can potentially drive identification of pathogenic variants.

Aims: We aimed to functionally characterize multiple previously uncharacterized variants of CXCR4 using in vitro assays. We also examined allele frequencies of these novel variants to estimate the potential number of individuals harboring these variants with a long-term goal of determining the actual prevalence of WHIM syndrome.

Methods: The CXCR4 -negative K562 cell line was used as a model system to express 53 novel CXCR4 variants identified in patient and population databases (ClinVar, Ensembl, CentoMD, GnomAD) and genetic screening initiatives (Invitae PATH4WARD) found in the C-terminus (hotspot of CXCR4 WHIM mutations) as well as throughout the protein. The effects of these mutations on CXCR4 internalization and chemotaxis were studied in cells stimulated with the C-X-C chemokine ligand 12 (CXCL12). The in vitro functional parameters were compared with known pathogenic CXCR4 variants and were used to assign a cumulative score (impaired internalization + enhanced chemotaxis) of functional defect severity.

Results: Out of 53 selected variants, 42 were missense (ms), 6 were frameshift (fs), 3 were nonsense (ns), and 2 were transcript variants (tv). Eighteen of the screened variants led to a decreased internalization of CXCR4 in comparison to the wild-type (WT) receptor upon stimulation with CXCL12 while 20 variants demonstrated enhanced chemotaxis of cells toward CXCL12. Overall, 34 variants showed defects in at least one CXCR4-dependent marker. Correlation analysis and scoring of the functional parameters in cells expressing mutated or CXCR4 WT revealed 17 variants (9 ms, 5 fs, 2 ns, 1 tv) that co-segregated with the known pathogenic WHIM variants. Finally, white blood cell counts in 3 patients harboring novel variants showed that CXCR4 internalization defects in vitro correlated with the severity of neutropenia.

Summary/Conclusion: This is the first study characterizing functional impairments in naturally occurring CXCR4 variants via a pipeline of assays intended to predict pathogenicity. Seventeen CXCR4 variants were identified (several outside the C-terminus of CXCR4 ) that caused in vitro functional defects resembling those exhibited by known WHIM variants. Many (10/17) of these newly identified variants are present at high frequencies (4.4 × 10 -4 –8.0 × 10 -6 ) in genomic databases. Potential association of these variants with WHIM syndrome is under investigation and is likely to expand current estimates of WHIM syndrome prevalence.

P794: DIFFERENTIATION POTENTIAL AND RELATIVE GENE EXPRESSION LEVELS ARE CHANGED IN CFU-F FROM BONE MARROW OF APLASTIC ANEMIA PATIENTS AT THE ONSET OF THE DISEASE

A. Dorofeeva 1,* , I. Shipounova 1 , A. Luchkin 1 , Z. Fidarova 1 , E. Mikhailova 1

1 National Research Center for Hematology, Moscow, Russia

Background: Aplastic anemia (AA) is a heterogeneous blood system disease characterized by the bone marrow (BM) aplasia in and pancytopenia. There are non-severe (NAA), severe (SAA) and very severe (VSAA) forms of AA. The main cause of AA is believed to be the damage to the hematopoietic stem cells (HSC) by autoreactive cytotoxic T-lymphocytes. Another reason for the AA development may be a dysfunction of the BM stromal microenvironment. One of the ways of study the BM stroma is the analysis of colony-forming units of fibroblasts (CFU-F).

Aims: To determine the CFU-F concentration in BM of AA patients, to assess the differentiation potential of individual colonies and the expression level of genes associated with proliferation, differentiation, immune response, and maintenance of HSC in CFU-F from BM of untreated AA patients.

Methods: The study included 38 AA (15 with SAA, 4 with VSAA and 19 with NAA) patients in the debut. VSAA was combined with SAA for the analysis. The control group included 22 healthy donors. BM was aspirated at diagnostic punctures in patients and planned exfusion from donors after informed consent. CFU-F cultures were obtained according to the standard method. The colonies number was counted, and total RNA was isolated. Individual CFU-F colonies were obtained by plating 15000 BM mononuclear cells per well of a 96-well plate in aMEM with 20% FBS. Wells with individual clones had been analyzed further. The differentiation potential of CFU-F colonies was determined by the level of marker gene expression after incubation in a medium with inducers of osteogenic or adipogenic differentiation. Gene expression was determined by TaqMan RT-PCR. Data are presented as mean±standard error of the mean. Differences were considered significant at p<0.05 using an unpaired Student’s t-test.

Results: CFU-F concentration in the BM of AA patients was not altered (33.58±9.12 per 10 6 BM mononuclear cells versus 34.43±8.39 in donors). The differentiation potential of CFU-F clones from SAA patients was skewed to osteogenesis when compared with donors and NAA patients. The proportion of CFU-F clones that failed to differentiate was decreased in SAA patients, while in NAA patients the proportion of such clones increased in comparison with healthy donors. These data may indicate a change in the ratio of stromal precursors of different maturity in the BM of patients with NAA and SAA compared with healthy donors (Table 1).

CFU-F cultures of SAA patients significantly differed from those of healthy donors in the expression level of 10 genes (Table 2). The similar changes in 5 of these genes ( PDGFRA, PDGFRB, ANG1, CFH, NES ) were also found in NAA, but, with the exception of NES , the changes were not significant. Most of these genes are associated with the HSC maintenance, thus, the identified changes could be the result of BM aplasia. The expression level of the other 5 genes in CFU-F of NAA patients did not differ from that in donors ( FGFR1, IL1B, PPARG, HLA-DR ) or tended to change oppositely than in SAA ( IL10 ). These genes are functionally associated with immune response and differentiation. Possibly, the activity of these genes prevents the development of more pronounced aplasia in NAA patients.

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Summary/Conclusion: CFU-F of NAA and SAA patients differ from CFU-F of healthy donors by their differentiation potential and gene expression. Changes in CFU-F of SAA patients are more pronounced, which may be associated with deeper aplasia. This work was supported by the Russian Foundation for Basic Research, grant no. 19-015-00280.

P795: CHARACTERIZATION OF ERCC6L2 SYNDROME PATIENTS’ TRANSCRIPTOME

S. P. Douglas 1,2,* , I. Kaaja 1,2 , T. H. Räisänen 1,2 , E. Pitkänen 1,3 , O. Kilpivaara 1,2,4 , U. Wartiovaara-Kautto 1,5

1 Applied Tumor Genomics Research Program, Faculty of Medicine; 2 Department of Clinical and Medical Genetics, Medicum; 3 Institute for Molecular Medicine Finland (FIMM), University of Helsinki; 4 HUSLAB Laboratory of Genetics, HUS Diagnostic Center, Helsinki University Hospital; 5 Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, University of Helsinki, Helsinki, Finland

Background: Biallelic germline mutations in ERCC6L2 cause a bone marrow failure syndrome with a tendency for acquiring somatic TP53 mutations and possible progression to acute myeloid leukemia with erythroid characteristics. The disease and some functions of ERCC6L2 have only recently been characterized but a lot remains uncovered. ERCC6L2 affects DNA repair, but also plays a role in immunoglobulin class-switching and mitochondrial function. Although the biallelic ERCC6L2 mutation is in all the patient’s cells, it seems to affect the hematopoietic cells most drastically. However, we have seen differences also in the metabolism and growth of the ERCC6L2-deficient patients’ skin fibroblasts. Constitutional changes in RNA expression levels and altered metabolism due to ERCC6L2 deficiency may also affect the bone marrow niche and help us understand the disease more profoundly.

Aims: We aimed to characterize ERCC6L2 syndrome on the transcriptome level using patient-derived samples (blood, skin fibroblasts, lymphoblastoid cell lines [LCLs]).

Methods: 32 blood samples were collected from 15 ERCC6L2 patients (some in multiple time points), and 6 healthy controls.

Skin fibroblast cell lines from 4 ERCC6L2 patients, and 3 healthy controls were grown in duplicates. In total 14 samples were grown in high-glucose (25mM) media until about 80% confluence and cells were harvested after six hours of changing the media.

LCLs derived from EBV-transformed PBMCs from 6 ERCC6L2 patients and 4 healthy controls were grown in duplicates in two different media compositions (in total 20 samples).

3’ RNA sequencing was performed on RNA extracted from all patient samples. Differential expression analysis was done with DESeq2 and pathway enrichment analysis with PathfindR.

Results: ERCC6L2-patients and controls differ significantly in their mRNA expression levels in multiple cell types and enriched pathways differ between cell types.

In the patient blood samples, global genome nucleotide excision repair and transcription-coupled nucleotide excision repair (TC-NER) pathways are overrepresented compared to healthy controls. ERCC6L2 has been suggested to be involved both in TC-NER and non-homologous end joining of DNA double strand breaks. Also neddylation, which is a ubiquitin-like posttranslational modification affecting gene expression, is highly overrepresented. Cellular response to chemical stress is also upregulated, which demonstrates the reported sensitivity of ERCC6L2-deficient cells to some chemicals.

The patients’ skin fibroblasts have different expression patterns compared to healthy controls in pathways related to cell differentiation, response to stimuli and extracellular matrix composition. These results support our previous observations about impaired fibroblast growth and metabolism. Expression changes in fibroblasts can tell us about effects that the germline ERCC6L2 mutation can have on extra-hematopoietic cells and tissues in the absence of somatic TP53 mutations.

LCLs from ERCC6L2 patients’ cells differ from healthy controls by various immune system pathways: the enrichment of complement activation, phagocytosis, B-cell receptor regulation, but also in their response to metal ions.

Summary/Conclusion: Multiple cell types of ERCC6L2 patients’ cells differ significantly from healthy controls’ cells in pathways related to DNA repair, stress response, cell morphology and immune function. These enriched pathways demonstrate the diverse effects the germline ERCC6L2 mutation has on the patients’ cells.

P796: ENRICHED CD16+ MONOCYTES DRIVE T LYMPHOCYTES CELL RESPONSES IN APLASTIC ANEMIA

M. Ge 1,* , H. Wu 1 , J. Huo 1 , Y. Shao 1 , Y. Zheng 1

1 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China

Background: Monocytes, an important component of innate and acquired immunity, encompass three subsets, the classical monocytes (CM, CD14 ++ CD16 - ), intermediate monocytes (IM, CD14 ++ CD16+) and nonclassical monocytes (NCM, CD14 + CD16 ++ ). IM and NCM are collectively referred to CD16 + monocytes, which has been studied in many inflammatory diseases. Whereas, the distribution and function of monocytes in acquired aplastic anemia (AA) remain unclear.

Aims: This study aims to determine the distribution of peripheral blood (PB) and bone marrow (B M) monocyte subpopulations in AA and expound the role of CD16 + monocytes on T lymphocytes function.

Methods: The proportions of monocyte subsets in the PB and BM of AA patients and healthy donors (HD) were analyzed by flow cytometry. CD16 + monocytes were isolated by magnetic beads and co-cultured with CD3 + T cells, afterwards, the basic biological characteristics of T cells including proliferation, activation and differentiation etc. were evaluated.

Results: Results showed that the percentage of CD16 + monocytes in PB and BM was significantly increased in AA patients compared with that in HDs, and the proportion of CM was decreased. However, there was no difference between patients with SAA and NSAA. CD16 + monocytes were further divided into two subgroups, which named IM and NCM. In contrast with HD, the two subgroups were both elevated in AA, mainly the IM. After treatment, NCM and IM were reduced and CM was increased in the patients of complete and partial remission. Then the effect of CD16 + monocytes on T cells function were elaborated. Using a co-culture model, we discovered that CD16 + monocytes in AA patients had stronger ability to promote the proliferation of CD3 + T, CD3 + CD4 + T and CD3 + CD8 + T cells than that in HDs (Fig 1A, b-d). Furthermore we found that AA-CD16 + monocytes played an important role in the over activation of CD3 + CD4 + T and CD3 + CD8 + T cells (Fig 1B, b-d). As expected, AA-CD16 + monocytes were extremely potent inducers of Th1、Th17 and cytotoxic T cells (Tc1) differentiation in vitro (Fig 1C, b-d), nevertheless, there was no difference on the effect of monocytes on Treg between AA and HD.

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Summary/Conclusion: This study provided that the expanded CD16 + monocytes, including NCM and IM，had a major role in driving T cell responses in AA patients.

P797: METABOLIC PROFILING IN ERCC6L2 AND SHWACHMAN DIAMOND SYNDROME

I. Kaaja 1,2,* , S. P. Douglas 1,2 , T. H. Räisänen 1,2 , K. Haimilahti 3,4 , A. I. Nieminen 5 , E. Pirinen 3,6 , U. Wartiovaara-Kautto 1,7 , O. Kilpivaara 1,8

1 Applied Tumor Genomics Research Program, Faculty of Medicine; 2 Department of Medical and Clinical Genetics, Medicum, Faculty of Medicine; 3 Clinical and Molecular Metabolism Research Program, Faculty of Medicine; 4 Stem Cells and Metabolism Research Program, Faculty of Medicine; 5 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki; 6 Research Unit for Internal Medicine, Faculty of Medicine, University of Oulu, Oulu; 7 Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, University of Helsinki; 8 HUSLAB Laboratory of Genetics, HUS Diagnostic Center, Helsinki University Hospital, Helsinki, Finland

Background: Germline mutations in genes involved in DNA repair, telomere maintenance, and ribosome biogenesis are established causes of bone marrow failure (BMF) syndromes. Biallelic ERCC6L2 germline mutations cause BMF and accumulation of somatic TP53 mutations in bone marrow, contributing to the disease progressing into acute myeloid leukemia. ERCC6L2 is tentatively indicated in DNA repair and mitochondrial function, however, understanding its function is still in its infancy.

Aims: To particularly understand the role of mitochondrial function in ERCC6L2 syndrome, we compared cellular metabolism in ERCC6L2 syndrome and Shwachman Diamond syndrome (SDS), a ribosomopathy also prone to somatic TP53 mutagenesis. Our aim was to discover any underlying alterations in mitochondrial function and to perceive any differences between the two syndromes that are similar in phenotype but different in genotype.

Methods: To study the effect of the ERCC6L2 and SDBS germline mutations on cellular metabolism in isolation from the effect of accumulating somatic mutations in the bone marrow, we examined patient-derived skin fibroblasts (ERCC6L2 syndrome, n=2; SDS, n=2) and healthy controls (n=2). Mitochondrial function was studied with Seahorse XFe96 Analyzer where mitochondrial oxygen consumption rate (OCR) was measured under different glucose and glutamine concentrations. Furthermore, we inhibited mitochondrial pyruvate carrier (MPC) to examine the metabolic compensation for mitochondrial pyruvate deficiency via Seahorse Substrate Oxidation Stress Test. Mitochondrial DNA (mtDNA) amount was quantified in patient samples using qPCR. To further investigate the metabolism in each BMF syndrome, untargeted metabolomic screening of ERCC6L2 (n=4), SDS (n=3) and healthy control (n=3) fibroblasts in normal and low-glucose conditions was conducted by liquid chromatography mass spectrometer. We cross-referenced differentially expressed (DE) metabolites with DE genes (3’ RNAseq) in a matched set of samples to identify important biological pathways.

Results: Our results indicate impaired mitochondrial respiration under low energy availability in both ERCC6L2 syndrome and SDS, as both BMF syndromes showed decreased maximal respiratory and reserve capacity in low-glucose conditions. Equal mtDNA amount was confirmed in patient and control cells suggesting that the decline was not related to changes in mitochondrial content. In ERCC6L2, but not in SDS cells, maximal respiratory and reserve capacity improved upon the addition of L-glutamine. In addition, we observed a lower glutamine concentration in both ERCC6L2 and SDS in low-glucose conditions. MPC inhibition lowered the respiration in SDS cells, but not in ERCC6L2 cells, indicating that pyruvate may be compensated by other substrates, such as glutamine oxidation, in ERCC6L2 but not in SDS cells. Together, these findings imply increased consumption of glutamine in ERCC6L2 cells and possibly impaired glutamine utilization in SDS cells. Metabolomic screening and integration of metabolomics data with transcriptomics detected subtle trends indicating changes in TCA metabolite and gene expression as well as in nicotinate/nicotinamide metabolism.

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Summary/Conclusion: We show altered mitochondrial function in low-glucose conditions in two BMF syndromes, and propose glutamine-dependency as a factor for challenged mitochondrial function under low energy substrate availability in ERCC6L2 syndrome. Further studies on the metabolic behavior of the hematopoietic stem cell and its niche in BMF syndromes are needed to understand the role of metabolism in the disease progression.

P799: SPATIAL ANALYSIS IDENTIFIES A SPECTRUM OF IMMUNE DYSREGULATION IN ACQUIRED BONE MARROW FAILURE CONDITIONS

R. Koldej 1,2,* , A. Prabahran 1,2,3 , C. W. Tan 2,4 , M. Davis 2,4 , D. Ritchie 1,2,3

1 ACRF Translational Research Laboratory, Royal Melbourne Hospital; 2 Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne; 3 Clinical Haematology, Royal Melbourne Hospital; 4 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

Background: Poor Graft Function (PGF), manifested by multilineage cytopenias with complete donor chimerism post allogeneic stem cell transplantation, and acquired Aplastic Anaemia (AA) are acquired bone marrow failure syndromes. Both are thought to result from T cell activation resulting in excessive interferon-γ (IFNγ) production, which in turn suppresses haematopoiesis. In AA, this can promote clonal stem cell selection and potentially progression to myeloid malignancy. Despite similarities in the clinical presentation and proposed mechanism, no studies to date have compared the immunobiology of the bone marrow (BM) microenvironment in these conditions.

Aims: To examine the immune microenvironment of the BM in archival AA and PGF BM trephines and identify common immunopathologies.

Methods: Archival BM trephines were sourced from patients with PGF (n=20), AA at Diagnosis (AA_DX) (n=15), AA at Progression (AA_PROG) to MDS/AML (n=15) and normal controls (NC) (n=20). 6 x 300µm regions identified by dual CD3/CD45 immunofluorescent staining were analysed per BM trephine using NanoString GeoMX™ digital spatial profiling for the expression of 57 proteins with an immunology focused panel. Data was analysed using a Limma-Voom bioinformatics pipeline.

Results: Significant dysregulation was identified across multiple proteins. Overall, 22 proteins had significantly different expression in AA_DX samples (13 up, 9 down) compared to NC with AA_PROG and PGF having 8 (4 up, 4 down) and 14 (7 up, 7 down) respectively. As would be expected given the hypocellular BM, expression of CD45 was significantly reduced compared to NC across AA_DX (adj P = 5.413e-10), AA_PROG (adj P = 8.299e-8) and PGF (adj P = 8.634e-9) groups.

When AA_DX patients were stratified into those who progressed (n=6) vs those who did not (n=9), or when AA_DX vs AA_PROG was compared in patients with matched samples (n=6) there were no significantly differentially expressed proteins identified. This suggests that while AA provides an immune microenvironment that is permissive to MDS/AML pathogenesis, it may not directly impact on clonal evolution.

Total monocytes were unchanged by CD11c expression. However, AA and PGF samples exhibited upregulation of CD163 compared to NC (AA_DX adj P = 4.5e-6; AA_PROG adj P = 1.2e-5; PGF adj P = 1.58e-8) suggesting an increase in monocyte/macrophage lineage cells. Expression of CD66b was also downregulated in AA_DX (adj P = 8.459e-24), AA_PROG (adj P = 1.193e-10) and PGF (adj P = 2.244e-14) suggesting a reduction in non-classical monocytes, neutrophils or granulocytic MDSCs.

Importantly, VISTA was downregulated across AA_DX (adj P = 6.84e-27), AA_PROG (adj P = 1.1e-5) and PGF (adj P = 1.58e-8) and STING was downregulated in AA_DX compared to NC (adj P = 6.139e-13) with a trend towards reduced expression in PGF. Decreased expression of these key immunoregulatory proteins may have wide ranging effects on BM resident macrophages, dendritic cells, MDSCs and T cells leading to reduced immunoregulation and increased T cell activation/IFNγ production, ultimately resulting in stem cell depletion. Data on cell specific expression is currently being obtained to refine this model.

Summary/Conclusion: Spatial analysis demonstrated that patients with AA and PGF exhibit similar patterns of protein expression likely resulting in a BM immune microenvironment of decreased immunoregulation. AA_DX samples exhibited a greater degree of dysregulation of multiple markers compared to PGF suggesting that these diseases represent a spectrum of immune dysregulation.

P800: BLOCKADE OF COMMON GAMMA CHAIN CYTOKINE SIGNALING WITH REGN7257, AN INTERLEUKIN 2 RECEPTOR GAMMA (IL2RG) MONOCLONAL ANTIBODY, PROTECTED MICE FROM GRAFT-VERSUS-HOST DISEASE AND IMMUNE APLASTIC ANEMIA

A. Le Floch 1,* , K. Nagashima 2 , T. Norton 2 , L. Perlee 2 , A. Murphy 2 , M. Sleeman 2 , J. Orengo 2

1 Inflammation and Immune Diseases; 2 Regeneron, Tarrytown, United States of America

Background: Pathogenic T-cell responses in T cell-mediated diseases can be driven by cytokines of the common gamma chain (γc) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γc cytokines signal through their corresponding receptors, expressed primarily on immune cells (including T cells), that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling.

Aims: To understand the roles of γc cytokines in driving graft-versus-host disease (GVHD) and bone marrow failure disorders such as immune aplastic anemia (AA), we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and we tested its ability to suppress pathogenic T-cell responses in mouse models of T cell-mediated disease.

Methods: We evaluated the efficacy of REGN7257 in a xenogeneic mouse model of GVHD, where immunodeficient NOD- scid -IL2RG null mice were engrafted with human peripheral blood mononuclear cells (huPBMC). Mice were treated with REGN7257 either prophylactically or therapeutically, and monitored for weight loss and survival, peripheral human T-cell engraftment as well as serum pro-inflammatory cytokine levels over time. In a separate experiment, mice were sacrificed at day 49 post-huPBMC injection (after 4 weeks of antibody treatment) for tissue analysis (liver, lung, skin and bone marrow) of immune cell infiltration (T cells and macrophages), inflammation and/or fibrosis.

Results: In a xenogeneic model of GVHD, both prophylactic and therapeutic γc cytokine signaling blockade with REGN7257 effectively protected mice from weight loss and resulted in improved survival, by reducing T-cell expansion in blood and production of pro-inflammatory cytokines in serum. Consistent with the classic pathology of GVHD, lungs, liver and skin of control mice were highly infiltrated by T cells, while γc cytokine signaling blockade strongly reduced T-cell infiltration, with reduced tissue levels of pro-inflammatory cytokines. Importantly, therapeutic γc cytokine signaling blockade in established GVHD (i.e. when mice already showed weight loss) provided similar benefit. Blockade of γc cytokine signaling also led to a reduction in the severity of chronic GVHD, with decreased macrophage infiltration in liver and associated hepatic fibrosis. In this xenogeneic model of GVHD, hemoglobin levels and platelet numbers in blood were both reduced, indicating anemia and thrombocytopenia, respectively, which are two complications associated with aplastic anemia. In addition to peripheral pancytopenia, recipient mice that were engrafted with huPBMC also showed severe marrow aplasia. Importantly, this phenotype of aplastic anemia was prevented by blockade of γc cytokine signaling.

Summary/Conclusion: Blockade of γc cytokine signaling with REGN7257 protected against T cell-mediated pathology in a xenogeneic GVHD mouse model that uniquely presents hallmarks of both acute and chronic GVHD, with T-cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. These data provide evidence of γc cytokines as key drivers of T cell-mediated responses, offering a potentially novel strategy for the management of T cell-mediated diseases, such as GVHD and immune AA.

P801: EVALUATION OF COMMON GAMMA CHAIN CYTOKINE SIGNALING BLOCKADE WITH REGN7257, AN INTERLEUKIN 2 RECEPTOR GAMMA (IL2RG) MONOCLONAL ANTIBODY, ON IMMUNE CELL POPULATIONS ACROSS SPECIES

A. Le Floch 1,* , K. Nagashima 2 , D. Birchard 2 , H. Pan 2 , C. Korgaonkar 2 , T. Norton 2 , L. Perlee 2 , A. Murphy 2 , M. Sleeman 2 , J. Orengo 2

1 Inflammation and Immune Diseases; 2 Regeneron, Tarrytown, United States of America

Background: The common γ chain cytokine receptor (γc; IL2RG) family of cytokines includes interleukin 2 (IL2), IL4, IL7, IL9, IL15, and IL21. This set of cytokines exhibits broad pleiotropic actions on both the innate and adaptive immune system with each cytokine sharing the IL2RG chain as part of its signaling receptor complex. Mutations in the IL2RG gene result in X-linked severe combined immunodeficiency (XSCID) in humans, whereby patients present with dramatically diminished numbers of T cells and NK cells, and dysfunctional B cells.

Aims: Given the crucial role for γc cytokines in the development and function of lymphocytes, modulating their activities may offer therapeutic potential in a range of immune-mediated diseases. To understand the effects of γc cytokine blockade on immune cells including T-cell subsets, we utilized REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and tested its ability to suppress immune cell populations and their functions across species (mouse, cynomolgus monkey [CM] and human).

Methods: We evaluated the effects of γc cytokine signaling blockade with REGN7257 on immune cell populations in Il2rg hu/hu mice and CM by flow cytometry. Mixed lymphocyte reaction assays were performed to look at the impact of γc cytokine signaling blockade on activation/proliferation of human T cells. To further analyze the contribution of γc cytokines to T-cell differentiation and function, we performed in vitro transcriptomic studies on human peripheral blood mononuclear cells stimulated with anti-CD3/CD28 beads.

Results: γc cytokine signaling blockade with REGN7257 in Il2rg hu/hu mice efficiently reduced circulating B, NK and T cell populations, with no changes in blood neutrophil counts. In addition, the impact of γc cytokine signaling blockade on lymphocyte populations in vivo were investigated in CM in a single dose pharmacokinetic/pharmacodynamic study and a repeat-dose toxicology study. Inhibition of γc cytokine signaling with REGN7257 in CM led to similar phenotypic changes to that observed in Il2rg hu/hu mice, with decreases in peripheral T cells and NK cells, without impacting B cells, granulocytes, platelets or red blood cells. γc cytokine signaling blockade led to a reduction in both CD4 + and CD8 + T cell counts with effector memory T cells being the most impacted T-cell population studied. Furthermore, blockade of γc cytokine signaling led to reduction in activated and proliferating T cells in CM and demonstrated potent inhibition of allogeneic responses in mixed human lymphocyte reaction assays, by preventing T-cell activation and proliferation. Two-tailed gene set enrichment analysis identified 4 distinct patterns of significantly enriched gene sets that were impacted by γc cytokine signaling upon CD3/CD28-induced activation of human T cells: gene sets involved in inflammatory responses, differentiation and proliferation, activation and immune responses, as well as those related to adhesion and migration. These signatures were blocked with REGN7257 treatment.

Summary/Conclusion: Taken together, these pharmacological observations highlight the major role for γc cytokine signaling in maintenance of lymphocyte populations (i.e. NK cells and T cells) in mouse and CM, but not other immune cell populations (i.e. granulocytes, platelets and red blood cells). Furthermore, our data highlight the importance of γc cytokines in driving functions of CM and human T cells, opening a potential new route for the management of T cell-mediated diseases.

P802: PATTERNS OF T-CELL AUTOREACTIVITY DIFFER BETWEEN PEDIATRIC APLASTIC ANEMIA AND HEPATITIS-ASSOCIATED BONE MARROW FAILURE

M. Nováková 1,2,* , M. Svatoň 1,2 , A. Skotnicová 1 , M. Suková 2 , L. Řezníčková 1,2 , T. Valová 1,2 , D. Pospíšilová 3 , O. Fabri 4 , P. Švec 4 , J. Trka 1,2 , O. Hrušák 1,2 , J. Starý 2 , E. Mejstříková 1,2 , E. Froňková 1,2

1 CLIP-Paediatric Haematology and Oncology; 2 Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague; 3 Department of Pediatrics, Palacky University and University Hospital Olomouc, Olomouc, Czechia; 4 Department of Pediatric Hematology and Oncology, National Institute of Children’s Diseases and Medical Faculty, Comenius University, Bratislava, Slovakia

Background: Both idiopathic aplastic anemia (AA) and hepatitis-associated bone marrow failure (HABMF) are considered to be autoreactive T cell mediated diseases. This is based on indirect evidence, most often the success of immunosuppression or oligoclonality with T cell repertoire restriction.

Aims: Our questions were: Does the composition of T cells and T-cell receptor beta (TRB) repertoire differ between HABMF, BMF without preceding hepatitis (nonHABMF) and normal bone marrow (BM)? Are there any shared clonotypes suggesting common antigenic stimulus? How does immunosuppressive therapy affect T-cells and does this correlate with outcome?

Methods: In total 22 pediatric patients diagnosed in 2004-2021 with HABMF were included in the study. Sixteen HABMF patients were treated with immunosuppressive therapy (IST) as a frontline treatment, of which 8 patients did not respond. As a control group, we included 10 nonHABMF patients with AA (n=6) and refractory cytopenia of childhood (n=4), all treated with IST with poor response in 6 patients, as well as 10 samples of healthy donor BM grafts and 8 newborn peripheral blood (NBPB) samples. We performed flow cytometry immunophenotyping at diagnosis (D0) and on day 120 (D120) after initiation of IST in HABMF and nonHABMF patients. We performed sequencing of the TRB gene rearrangements according to the EuroClonality-NGS working group protocols using DNA isolated from these samples and normalized DNA input for TRB library preparation to the equivalent of 20 000 CD3+ cells per sample based on flow cytometry data, if possible. Whole exome sequencing (WES) was performed from diagnostic samples of HABMF patients.

Results: Patients with HABMF had a significantly lower proportion of CD3+ T cells in their BM compared to the nonHABMF with the predominance of CD8+ T cells and their activation by expression of HLA-DR observed with flow cytometry. The analysis of TRB repertoire was performed in 22 D0 and 10 corresponding D120 samples of HABMF patients that had undergone IST and we compared the diversity and clonotype composition with the nonHABMF patients as well as the BM graft samples and NBPB. Expansion of individual TRB clonotypes (>5% of all reads) at D0 was observed more frequently in HABMF patients (9 out of 22) compared to the nonHABMF patients (1 out of 10). None of these expanded clonotypes were shared among more patients based on their nucleotide or amino acid sequence. There was no correlation of expanded T cell clone dynamics and response to IST. The diversity of TRB repertoire was reduced in the nonHABMF group after IST, however we did not observe any correlation between the clinical response to the IST and initial TRB diversity in either group of patients. WES did not reveal any pathogenic variants in genes associated with immune dysregulation.

Summary/Conclusion: Both the proportion of T cells and T-cell composition in the BM differed between HABMF and nonHABMF patients at diagnosis. Although we observed an expected reduction in the CD3+ cells after IST both in the HABMF and nonHABMF group, there was no significant difference in the TRB repertoire diversity in the HABMF patients between D0 and D120. We did not observe any correlation between the repertoire diversity and clonotype evolution and the clinical response to IST. Our data further support the hypothesis that T-cells play a significant role in both AA and HABMF, but suggest that the pathogenetic mechanism of both entities is different.

Supported by NV20-03-00284, NV19-05-00332, UNCE/MED/015, NU20J-07-00028 and GAUK 534120.

P803: TELOMERE SHORTENING IN BONE MARROW MESENCHYMAL STEM CELLS OF ACQUIRED APLASTIC ANEMIA PATIENTS ASSOCIATE WITH ALTERED EXPRESSION OF GENES INVOLVED IN TELOMERE MAINTENANCE, DNA DAMAGE, AND SENESCENCE

J. Srivastava 1,* , P. Saxena 1 , N. Tripathy 1 , S. Nityanand 1 , C. P. Chaturvedi 1

1 Stem Cell Research Centre, Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

Background: Telomeres are repetitive nucleotide sequences that protect the chromosome ends from DNA damage. Compelling data suggest that telomere attrition is associated with alteration in the DNA damage pathways, thereby inducing cell senescence. Idiopathic acquired aplastic anemia (AA) is a bone marrow (BM) failure disease characterized by pancytopenia and a hypoplastic fatty marrow. Several studies have reported shortening of telomere length (TL) in hematopoietic stem cells (HSCs) and lymphocytes in AA patients. The bone marrow mesenchymal stem cells (BM-MSC), which are the key cells of the BM niche, have garnered lots of interest as they are found to be functionally impaired in AA patients. However, a study highlighting telomere dysfunction in the BM-MSC of AA patients remains obscure. Therefore, we aims to study the telomere length shortening and its correlation with the alteration of genes involved in the telomere maintenance, DNA damage, and cellular senescence in AA patients.

Aims: The study aims to evaluate the telomere length in BM-MSC of AA patients in comparison to that of controls. Further, it correlates the telomere length shortening with the alteration of genes involved in telomere maintenance, DNA damage, and cellular senescence in Bm-MSC of AA patients.

Methods: BM-MSC were harvested from the BM of newly diagnosed idiopathic acquired AA patients (n=20) and healthy control (n=12) after informed consent. The telomere length and the expression of genes associated with telomere maintenance, DNA damage responses, and cell senescence of the BM-MSC of AA patients were analyzed by real-time quantitative-PCR (RT-qPCR). The correlation between telomere shortening and expression of telomere maintenance, DNA damage, and cellular senescence associated genes was done using Pearson’s correlation. Student’s t-test and Mann Whitney test were used to compare differences between groups. All the results were analyzed using GraphPad Prism software. The data was represented as mean ± standard deviation, and p-value <0.05 was considered significant.

Results: Twenty patients with idiopathic acquired AA (11 non-severe aplastic anemia (NSAA) patients and 9 severe aplastic anemia (SAA)) patients and 12 healthy controls were included in the study. The TL was significantly shorter in the BM-MSC of AA patients [0.77 (Range: 0.4 – 1.56) as compared to that of healthy controls [1.40 (range: 0.71 – 3.22); p=0.002 ] (Figure 1). The TL in BM-MSC was not influenced by age ( p=0.360 ), disease severity, and other hematological parameters. A significant alteration was observed in the expression of genes involved in telomere maintenance, DNA damage, and cell senescence with a positive correlation between the telomere length and telomere maintenance genes TRF2 (r=0.788; p=0.007 ), TPP1 (r=0.636; p=0.04 ) and TOP1 (r=0,676; p=0.03 ) as well as with gene involved in DNA damage responses CDKN1A (r= 0.667; p= 0.03 ) and ATM (r=0.783; p=0.007 ).

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Summary/Conclusion: This is the first study to highlight telomere shortening in the BM-MSC of AA patients. Furthermore, our study demonstrates that telomere shortening is in BM-MSC of AA patients is accompanied by altered expression of genes involved in telomere maintenance, DNA damage, and cellular senescence. These results shed new insight into that telomere attrition in the BM-MSC of AA patients could be attributable to DNA damage induced cell senescence or vice-versa. Overall, the findings of this study reveal that BM-MSC of AA patients potentially contributes towards the disease pathobiology.

P804: EX VIVO TREATMENT WITH ELTROMBOPAG RESCUES HEMATOPOIETIC STEM CELLS FROM ANTI-THYMOCYTE GLOBULIN-RELATED DAMAGE DURING IMMUNOSUPPRESSIVE THERAPY IN PATIENTS WITH APLASTIC ANEMIA

M. Vieri 1,2,* , B. Rolles 1,2 , M. Crocioni 1,2 , S. Isfort 1,2 , J. Panse 1,2 , T. H. Brümmendorf 1,2 , F. Beier 1,2

1 Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation; 2 Center for Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), Uniklinik RWTH Aachen, Aachen, Germany

Background: Immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG) and cyclosporine A is one first-line treatment option for patients (pts) with aplastic anemia (AA). One side effect of ATG is the release of interferon-gamma (IFN-γ), which is considered a major factor in the pathogenic autoimmune depletion of hematopoietic stem cells (HSC). Recently, eltrombopag (EPAG) was introduced for therapy of refractory AA pts due to its ability to improve HSC function. Clinical trials have evidenced that EPAG used alongside with IST leads to a higher response rate as compared to its later administration e.g. 2 weeks after ATG start. However, the potential underlying cause for this difference has not been clarified to date.

Aims: We hypothesize that EPAG might protect HSC from negative effects of ATG-induced IFN-γ release.

Methods: Sera of six pts with moderate to severe AA were collected pre-and post-ATG administration on days +1 and +2. Bone marrow (BM) of AA pts was collected and cryopreserved before ATG administration. In addition, we collected CD34 + cells isolated from five healthy donors (HD) undergoing HSC mobilization. HD-derived CD34 + or AA-derived BM cells were cultured in presence of pre as well as post-ATG serum from pts with AA, in combination with 5 µM EPAG and/or an IFN-γ neutralizing antibody (NAB; 2.5 µg/mL). After 48 h, colony forming unit (CFU) assay with cultured cells and intracellular staining of phospho-SMAD2/3 (known to be down-regulated in response of IFN-γ) were performed, while IFN-γ was measured via flow cytometry in collected sera.

Results: Serum IFN-γ levels were 0.56 ± 0.6 pg/mL (mean ± standard deviation) post-ATG, whereas they were 0.015 ± 0.04 pg/mL pre-ATG (n= 6; p=0.08). When we tested effects of ATG sera on the clonogenic potential (CP) of HD HSC (n=5), a significant decrease of the number of CFU colonies (NoC) treated with post-ATG (148 ± 75) sera was found compared to pre-ATG treatment (201 ± 75, p= 0.032). This negative effect was mitigated by adding EPAG to the post-ATG sera (223 ± 100; p=0.036). To specifically analyze the potential suppressive role of IFN-γ, we added a IFN-γ NAB to the post-ATG serum (n=4), which also significantly increased colony formation (NoC 225.7 ± 33.4) compared to 140.1 ± 9.3 post-ATG without AB (p=0.014). Furthermore, phosphorylation of phospho-SMAD2/3 was found to be significantly lower in post-ATG (2052 ± 428, mean fluorescence intensity, n=4) compared to pre-ATG (3323 ± 32; p=0.03) treated cells. This effect was partially rescued by adding EPAG to the post ATG sera (2247 ± 627, p=0.239). Finally, we analyzed whether EPAG could rescue AA BM in presence of their matching serum, pre- and post- ATG treatment (n=6). As expected for AA BM, the NoC obtained was substantially reduced. Adding post-ATG serum resulted in further impairment of their CP (NoC: 2.0 ± 1.6) compared to the pre-ATG serum (6.0 ± 6.7; p=0.04). Again, addition of EPAG to the post-ATG serum was able to rescue AA BM cells’ CP from such effect (NoC: 5.6 ± 4.6; p=0.04).

Summary/Conclusion: We provide evidence that treatment with ATG has an initial negative impact on the CP of HSC from HD and AA pts, likely mediated through acute release of IFN-γ. The addition of both IFN-y-NAB as well as of EPAG protects HSC from this effect. These results indicate a potential explanation for the superior response rates of EPAG given concurrently with ATG-based IST compared to later application and suggest that administration of EPAG should be started simultaneously with IST in order to maximize its beneficial effect in AA pts.

P805: ALEMTUZUMAB IN RELAPSED SEVERE APLASTIC ANEMIA: LONG-TERM RESULTS OF A PHASE II STUDY

N. Aggarwal 1,* , A. L. Manley 1 , J. Durrani 1 , R. Shalhoub 2 , O. Rios 1 , J. Lotter 1 , B. Patel 1 , C. Wu 2 , N. Young 1 , E. Groarke 1

1 Hematology Branch; 2 Office of Biostatistics Research, National Heart, Lung, and Blood Institute, Bethesda, United States of America

Background: Aplastic anemia (AA) is characterized by pancytopenia and a hypocellular bone marrow from immune-mediated bone marrow destruction. Immunosuppressive therapy (IST) is a good alternative to hematopoietic stem cell transplantation (HSCT), but relapse occurs in about 30% of cases, necessitating further therapy. We investigated alemtuzumab, a humanized IgG1 monoclonal antibody targeting CD52. We initially reported on 25 relapsed patients with 56% response. Here, we present long-term results of a total 42 patients.

Aims: Assess the efficacy and long-term outcomes in patients with relapsed severe AA (SAA) who received alemtuzumab.

Methods: Participants met Camitta criteria for SAA, had received at least one course of antithymocyte globulin (ATG)-based IST, and had relapsed. Alemtuzumab was administered intravenously (IV) (n=28) or subcutaneously (SC) (n=14). The primary endpoint was hematologic response at 6 months, with blood counts no longer meeting SAA criteria. Secondary endpoints included robust response (platelet or absolute reticulocyte count >50x10 9 /L at 6 months), relapse, clonal evolution to myelodysplasia and leukemia, response to therapy after relapse, and survival. Patients were assessed annually, and additional AA therapy and transfusion dependence was also recorded. Cumulative incidence curves were used to estimate the probability of relapse among responders and clonal evolution, with death as a competing risk. Overall survival probabilities were evaluated using Kaplan-Meier curves. This trial is registered at clinicaltrials.gov ( NCT00195624 ).

Results: Patients were enrolled over 9 years with a median follow-up of 6 years. Median age was 32 years and 57% were female. Five patients (12%) had neutrophil count <0.2x10 9 /L prior to receiving alemtuzumab. At 6 months, 18 (42%) patients had achieved a response, and 12 (29%) attained a robust response. Response was achieved in 15 (54%) of those who received alemtuzumab IV, versus 3 (21%) who received SC. Of non-responders or those off study at 6 months (n=24), 2 achieved a late response by 1 year.

Another relapse occurred in 8 patients, and cumulative incidence at median follow-up was 39%. Of those who were refractory to or relapsed (n=28) after alemtuzumab, 4 (15%) proceeded to HSCT, 12 (43%) to other medical AA therapies, and 10 (37%) to both. Subsequent treatments included eltrombopag (n=13), r-ATG (n=8) CSA (n=6), androgen (n=3) or other (n=4); response to further treatment occurred in 2 (29%) of relapsed and 5 (38%) of refractory patients, with response unknown in 7.

Clonal evolution occurred in 9 patients; cumulative incidence at median follow-up was 23%. Of these, 4 were non-responders. Evolution was high risk (morphological MDS, AML, or chromosome 7 abnormality) in 7 patients.

Overall survival was 67% at median follow-up (Figure 1). At last follow-up, 10 of the 18 responders (56%) had durable response and were both transfusion- and AA therapy-independent. Patients had prolonged immunosuppression after alemtuzumab, with CD4+ T cell counts <200 in 81% of assessed patients at 6 months, 67% at 1 year, and 42% at 2 years.

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Summary/Conclusion: Alemtuzumab is effective for relapsed SAA. The rate of hematologic response was greater with IV rather than SC administration. Most responders achieved durable long-term hematologic improvement, and the rate of clonal evolution was similar to that seen in our SAA patients treated with rabbit ATG. Immunosuppression can persist for years following alemtuzumab therapy, requiring long-term monitoring and antimicrobial prophylaxis.

P806: IMPACT OF MARROW HEMOPHAGOCYTOSIS IN DIAGNOSTIC DEFINITION AND PROGNOSTIC SIGNIFICANCE IN ADULT POPULATION

C. Almeida 1,* , T. Cardoso 2 , A. Roque 1 , S. Moreira 2 , J. Cascais Costa 2 , N. Silva 2 , L. Santos 2 , C. Geraldes 1

1 Hematology Department; 2 Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal

Background: Hemophagocytic lymphohistiocytosis (HLH) is associated with high mortality and is an entity difficult to diagnose. Bone marrow hemophagocytosis (BMHF) is the most easily accessible histomorphological criterion, but per si is neither specific nor sensitive for the diagnosis of HLH and its clinical significance is still not clear, mainly in adult population

Aims: To evaluate the clinical and prognostic significance of BMHF.

Methods: Retrospective analysis of adult patients with documentation of BMHF between 2007 and 2020. The identification of the patients was carried out by searching the database by the following keywords: “phagocytosis”, “phagocyte” and “histiocyte”. For the definition of HLH, the HLH-2004 and the revised HLH-2009 criteria were applied. HScore values ​​>169 were considered highly suggestive of HLH. Stratified models for each of the risk scores (HLH-2004 and HLH-2009) were compared using Akaike’s information criterion (AIC) and the Harrell’s concordance index (C-index).

Results: Ninety-five patients (pts) were included, 63.2% males with median age of 64 (20-95) years old. Twenty-two pts (23.2%) presented ≥5 HLH-2004 criteria and 24 (25.3%) HLH-2009 criteria, with only 6 patients (27.3%) with simultaneous diagnosis by both criteria (Cohen’s kappa 0.0253). Forty-three (45.3%) patients presented ≥3 HLH-2004 criteria and 34 pts (35.8%) had Hscore>169.

After a median follow-up of 24.3 months, the median overall survival (OS) was 39.4 months. OS was significantly lower in patients with ≥5 HLH-2004 (0.9 vs 86.5 M; HR 3.56; p<0.001), with ≥3 HLH-2004 criteria (3.17 vs NR; HR 2.95; p<0.001), with HLH-2009 criteria (5.5 vs 78.0 M; HR 1.89; p=0.034), and HScore>169 (1.45 vs 86.5; HR 3.08; p<0.001), compared with those that do not have.

Excluding variables included in the above scores, the only clinical/analytical variable that was statistically significant for OS in univariate analysis was LDH>UNL (HR 1.02; p<0.001).

After multivariate analysis for age, aetiology and LDH>ULN, ≥5 HLH-2004 (HR 4. p<0.001), HLH-2009 2009 (HR 1.87 p=0.045), ≥3 HLH-2004 (HR 3.06; p<0.001) and Hscore>169 (HR 3.39; p=0.001), the four scores retain significance for OS.

In patients with BMHF, HLH-2004 criteria discriminated best between patients with poor and favourable OS than HLH-2009 (C-index 0.6364 vs 0.5601; AIC 400.24 vs 411.53).

Concerning the aetiology, 51.6% was neoplastic (24.5% [n=12] with ≥5 HLH-2004 criteria), 13.7% infectious, 28.4% autoimmune and in 28,4% the trigger was not identified.

Summary/Conclusion: In our cohort, HLH-2004 criteria had the best predictive value for OS, compared with HLH-2009

However, in the absence of the data need to calculate these scores, both ≥3 HLH-2004 criteria and Hscore>169 are independent predictors of OS, which, due to the deleterious prognosis of HLH, support their utility to start treatment earlier

P807: APLASTIC ANEMIA FOLLOWING THE SARS-COV-2 VACCINE

R. Babakhanlou 1,* , T. Kadia 1 , K. Chien 1 , P. Thompson 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: There have been several reports describing the possible relationship between the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine and the development of hematological diseases, such as autoimmune hemolytic anemias (AIHA), paroxysmal nocturnal hemoglobinuria or immune thrombocytopenic purpura. Limited data is available on the relationship between the SARS-CoV-2 vaccine and the development of aplastic anemia (AA). We report a series of four cases of acquired AA developing in patients, who had received an mRNA-based SARS-CoV-2 vaccine.

Aims: To study the temporal association of COVID-19 vaccination and acquired aplastic anemia

Methods: We reviewed the cases of all patients who presented to MD Anderson Cancer Center between January 2021 and December 2021 for pancytopenia. We identified patients who were diagnosed with aplastic anemia after receiving two doses of the SARS-CoV-2 vaccine and summarized baseline characteristics and clinical course.

Results: Four patients with newly diagnosed AA were identified. All patients were male with a median age of 56 years (range, 19-69). Two patients (50%) had received the Pfizer-BioNTech vaccine and two (50%) the Moderna mRNA-1273 vaccine. One of the patients had contracted COVID 8 weeks prior to his vaccination. Three patients suffered from chronic medical problems including type 1 diabetes, hyperlipidemia, and hypertension. None of those patients developed any symptoms after the first dose of the vaccine. Symptoms occurred on average three weeks after the second vaccine and have been outlined in Table 1. Most common symptoms were fatigue, bruising and bleeding, development of petechiae, dizziness and shortness of breath. Specific autoimmune workup was done and all patients received baseline bone marrow aspiration and biopsy with cytogenetics and next-generation sequencing. In all four patients cytogenetics showed a diploid karyotype with no chromosomal abnormalities. Flow cytometry and molecular diagnostics did not show any abnormalities.

In two cases the bone marrow was hypocellular and in two cases the bone marrow was acellular. All patients received combination immunosuppressive therapy with steroids, horse anti-thymocyte globulin, cyclosporine and eltrombopag, three on a clinical trial and one off protocol.

Patients were on therapy for a median of 26 weeks (16-48). One patient achieved complete remission at 12 weeks. One patient has remained transfusion dependent, while the remaining two patients became transfusion independent at six months.

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Summary/Conclusion: Although we cannot prove a causative relationship, in rare circumstances, the SARS-CoV-2 vaccine could be associated with the development of AA. Additional epidemiologic and laboratory studies are planned to further evaluate any potential causative relationship.

P808: CONGENITAL NEUTROPENIA PREVALENCE AMONG POLISH CHILDREN – SUMMARY OF A NATIONWIDE GENETIC SCREENING CAMPAIGN

K. Bąbol-Pokora 1,* , W. Dobrewa 1 , M. Bielska 1 , J. Madzio 1 , S. Janczar 1 , W. Młynarski 1

1 Department of Pediatrics Oncology and Hematology, Medical University of Lodz, Lodz, Poland

Background: Severe congenital neutropenia (SCN) is a heterogenous group of rare primary immunodeficiency disorders, characterized by impaired maturation of neutrophil granulocytes. Patients with severe congenital neutropenia are prone to recurrent gingivitis, mouth and rectal ulcerations, and often life-threatening bacterial infections. They are also predisposed to myelodysplastic syndromes or acute myeloid leukemias. Nine types of severe congenital neutropenia have been distinguished so far with the most frequent pathogenetic defects in the ELANE gene. This study explores the distribution and mutation spectrum of disease-causing genetic variants associated with congenital neutropenia in the Polish pediatric cohort and the observed ten-fold increase in the referral rate following a nationwide information campaign.

Aims: The aim of this study was to estimate prevalence of congenital and autoimmune neutropenia among Polish children and to improve the efficiency of recruiting patients with suspected SCN by launching the FIX-NET project with a nationwide advertising campaign.

Methods: Molecular analyses were performed either by direct Sanger sequencing (78 patients recruited in 2008-2015) or by Targeted NGS (201 patients recruited in 2016-2021) using Illumina platform, focused on the 54 genes related to SCN and neutropenia associated syndromes. Overall 279 neutropenic patients were enrolled in the studies and written informed consent has been given. The following results describe the largest cohort of genetic variation associated with SCN in Poland.

Results: We identified pathogenic changes in genes associated to severe congenital neutropenia or related syndromes among 66 patients, most of which were variants in the ELANE gene (44%). There were a number of patients with pathogenic changes in CXCR4 (12%) and SBDS (11%) genes, and also in the recently described dominant SCN-related genes – SRP54 (8%) and CLPB (5%). Novel mutations accounted for 30% of all reported variants. In addition among 32 patients we found changes in genes that cause neutrophil dysfunctions or may lead to autoimmune neutropenia, such as AIRE , FAS , or CTLA4 .

Summary/Conclusion: Our study shows the prevalence of congenital neutropenia in Poland in the largest pediatric neutropenic cohort described so far. It indicates that since the introduction of NGS to the diagnosis of primary and secondary neutropenia, more genetic changes have been detected, however, the greatest impact on increasing awareness among doctors, and thus improving the effectiveness of recruitment had the nationwide information campaign.

P809: MODULATION OF ARACHIDONIC ACID PATHWAY BY IBUPROFEN RESULTS IN COMPLETE HEMATOLOGICAL RESPONSE IN GHOSAL HEMATODIAPHYSEAL DYSPLASIA WITHOUT NEED FOR CORTICOSTEROID

N. Barrett 1,* , C. McDonnell 2,3 , M. Cotter 4

1 Paediatric Haematology, Royal Hospital for Children, Glasgow, United Kingdom; 2 Paediatric Endocrinology, CHI Temple Street; 3 Discipline of Paediatrics, Univerisity of Dublin Trinity College Dublin; 4 Paediatric Haematology, CHI Temple Street, Dublin, Ireland

Background: Ghosal hematodiaphyseal dysplasia (GHDD) is a rare autosomal recessive disorder characterized by pancytopenia and increased bone density of the long bones. First described in families of Indian and Middle Eastern descent, subsequent studies identified the genetic lesion in this disease as due to loss of function mutations in the TBXAS1 gene encoding thromboxane synthase, a key component of the arachidonic acid pathway responsible for the formation of thromboxane A2 from prostaglandin H2.

As well as pancytopenia, laboratory features include platelet aggregation defects and increase in osteoblastic activity, the latter being the basis of increased bone density in patients with Ghosal syndrome. Simultaneously, loss of thromboxane synthase activity may be associated with shunting of prostaglandin precursors away from production of thromboxane into production of pro-inflammatory prostaglandins. This may be the mechanism responsible for the observed marrow suppression seen in these patients.

We describe a 3 year old female of non-consanguineous parents of Filipino origin who presented with marked pancytopenia. Next Generation Sequencing bone marrow failure gene panel revealed a novel homozygous mutation in TBXAS1 . Subsequent DXA scanning revealed increased bone mineral density consistent with the diagnosis (Z -score +2.4 for lumbar spine (L1-4)) although clinical sequelae of bony disease were not apparent.

Aims: While multiple case reports document that these patients have hematological response to long term corticosteroid therapy, we hypothesized that inhibition of cyclooxygenase (COX) upstream of thromboxane synthase in the arachidonic acid pathway may be effective in this condition.

Methods: Patient was treated with ibuprofen at regular therapeutic doses (approximately 30 mg/kg/24 hours daily initially) and serial full blood counts were monitored.

Results: The patient exhibited a rapid and complete response to ibuprofen with normalization of full blood counts. The dose of ibuprofen was reduced to 10 mg/kg/day, and hematologic response is maintained on low dose after one year.The treatment is well tolerated with no side effects.

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Summary/Conclusion: Treatment of GHDD with ibuprofen is a novel approach that offers clear potential benefits in comparison to the known adverse effects profile of long term corticosteroids. Bone density scan and markers are in process to ascertain the impact of this treatment on bone density, although this may take longer to show beneficial effects. The treatment was easily tolerated and well managed by the patient and family at home with minimum disruption to her quality of life.

P810: BONE MARROW FAILURE IN PATIENTS CARRYING VARIANTS ON CARD11 GENE.

A. Beccaria 1,* , A. Grossi 2 , F. Fioredda 3 , M. Lanciotti 3 , E. Palmisani 3 , P. Terranova 3 , M. Lupia 3 , G. Dell’Orso 3 , I. Ceccherini 2 , C. Dufour 3 , M. Miano 3

1 DOPO Clinic and Hematology Unit, Division of Pediatric Hematology and Oncology; 2 Laboratory of Genetics and Genomics of Rare Diseases; 3 Hematology Unit, Division of Pediatric Hematology and Oncology, IRCCS Istituto Giannina Gaslini, Genova, Italy

Background: An accurate differential diagnosis in children with Marrow Failure (MF) is crucial for the clinical management of the disease. In addition to the classical congenital MF (cMF), the role of Inborn Errors of Immunity (IEI) have been recently highlighted as potential cause of the disease. CARD11 is a membrane protein acting as a key signaling scaffold which controls the antigen-induced lymphocyte activation during the adaptive immune response (NF-kB, JNK and mTOR pathway). Germline CARD11 mutations can result in gain/loss of function of the protein leading to different phenotypes (SCID, BENTA disease, atopy, CVID). Several heterozygous hypomorphic/dominant negative (DN) variants of CARD11 have demonstrated a loss of protein function, exhibiting high penetrance and variable expressivity. In particular, mutations in the GUK (Guanylate Kinase) domain seem to be involved in modulating the self-inactivating capacity of CARD11. No CARD11 variants have ever been described to be associated with MF. Herein, we report 4 patients with MF carrying variants on the CARD11 gene.

Aims: To evaluate the clinical/immunological features and genetic profile of patients with MF carrying CARD11 variants and followed in our Centre.

Methods: A restrospective review of clinical, immunological and genetic data was performed from patients’ records. Molecular analysis was conducted using Next-Generation Sequencing (NGS), targeted panels including genes involved in both cMF and IEI, or by Whole Exome Sequencing (WES). Filtering of variants was performed according to Mendelian disease segregation and zigosity (OMIM), in silico prediction of pathogenicity (ACMG criteria in “Varsome”), and minor allele frequency (reported in GnomAD).

Results: Four children carrying variants in the CARD11 gene presented with MF as initial sign of the disease (n=2) or with immune cytopenias further evolving into MF (n=2). Their clinical/immunological features, genetic profile, treatment and follow-up are reported in Table1. Lymphocytes subsets analysis revealed a deficiency of B memory, Tregs and an increased of HLADR+ T cells in all patients. Two cases showed elevated CD4-CD8- TCR αβ+ T-cells (“double negatives T-cells”). All patients showed autoimmune stigmata and 2 of them also presented autoimmune hepatitis. No patients responded to first-line treatment. One responded to second-line treatment with Mychofenolate-mofetil (MMF) and periodic infusion of immunoglobulins. The remaining 3 patients underwent hematopoietic stem cell transplantation (SCT) from haploidentical-αβCD19 depleted transplant (n=2) or HLA-identical cousin (n=1) (Table1).

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Summary/Conclusion: This report highlights novel phenotypic characteristics of patients carrying variants in CARD11 such as MF and autoimmune hepatitis, thus widening the clinical spectrum of the disease. This underlines the need for an enlarged molecular analysis in pediatric cases of MF and suggests that, in some patients, immune-mediated destruction of blood and marrow cells cooperate in generating the cytopenia. Three variants detected in our cohort are located in the GUK domain of the gene, close to other reported pathogenic dominant negative mutations leading to haploinsufficiency, suggesting interference with the CARD11’s ability to self-inactivate. Functional study will be necessary to confirm the pathogenic role of such variants. Although treatment with MMF may be considered, SCT represents the only curative option for such patients.

P811: A SINGLE CENTER HISTORICAL CONTROL STUDY OF ELTROMBOPAG ADDED TO IMMUNOSUPPRESSIVE THERAPY FOR SEVERE APLASTIC ANEMIA IN CHILDREN

B. yang 1,* , L. fu 1 , H. li 1 , H. chen 1 , J. ma 1

1 Hematology Oncology Center, Beijing Children Hospital, Beijing, China

Background: Severe aplastic anemia is caused by immune-mediated destruction of bone marrow. Standard immunosuppressive therapy (rabbit anti-thymocyte globulin combined with cyclosporine) is effective, but the effect needs to be improved. A thrombopoietin-receptor agonist, eltrombopag, added to standard immunosuppressive therapy led to clinically significant increases in blood counts. We combined eltrombopag and standard immunosuppressive therapy in pediatric severe aplastic anemia patients.

Aims: To evaluate the efficacy and safety of eltrombopag added to immunosuppressive therapy for severe aplastic anemia in children.

Methods: The clinical data of patients with severe aplastic anemia and received immunosuppressive therapy from March 2013 to July 2020 were collected and analyzed retrospectively. We conducted a historical control study, in which the patients treated alone with immunosuppressive therapy were used as the control group. We compared the cases between control group and eltrombopag group, included hematological response, effective time, relapse, clonal evolution, adverse reactions, event free survival (EFS) and overall survival (OS).

Results: 1. A total of 115patients (60 males), median aged 5.77 years, median follow-up time was 45 months, were enrolled in this study. All patients were diagnosed with severe or very severe aplastic anemia, including 49 patients in the historical control group and 66 patients in the eltrombopag group. 2. The complete response rate (CRR) at 3 months was 49.0% in eltrombopag cohort and 8.2% in historical control. The overall response rate (ORR) at 3months was 63.6% and 30.3% respectively. The CRR at 6 months was 50.0% in eltrombopag cohort and 10.2% in control cohort. The ORR at 6months were 71.2% and 57.1% respectively. There were significant differences of the CRR between two groups, there was no significant difference between two groups.3. The median effective time of the control group and eltrombopag group was 90 days and 71 days respectively (P = 0.007), the median complete remission time was 360 days and 98 days respectively (P < 0.001). The median time of separated from granulocyte colony stimulating factor (G-CSF), Red Blood Cell transfusion and Platelet transfusion in the control group were 105 days, 46 days and 54 days respectively, and 68 days, 45 days and 45 days in the eltrombopag group. There were significant differences in the time of separated from G-CSF (P = 0.003), Red Blood Cell transfusion (P = 0.001) and Platelet transfusion (P < 0.001) in the two groups. 4.The relapse rate was 4.1% (n = 2) in the control group and 10.2% (n = 5) in the eltrombopag group. One patient (2.0%) in the control group developed clonal evolution and progressed to myelodysplastic syndrome.5. The common adverse reaction were transient and reversible hyperbilirubinemia (13.6%, n = 9), elevated liver enzymes (4.5%, n = 3) and hyperuricemia (1.5%, n = 1).6. The 1-year OS and 2-year or of the control group was 100% and 98.5% respectively, comparing to 95.9% and 97.0% in eltrombopag group. The 2-year EFS of the control group and the eltrombopag group was 79.6% and 74.2%, respectively, and the 3-year EFS were 73.5% and 72.7%, respectively.

Summary/Conclusion: The addition of eltrombopag to standard immunosuppressive therapy was associated with markedly higher rates and effected more quickly of hematologic response among pediatric severe aplastic anemia patients than in a historical cohort and showed a reliable security.

P812: LONG-TERM COMPLEMENT INHIBITION AND SURVIVAL OUTCOMES IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: AN INTERIM ANALYSIS OF THE RAVULIZUMAB CLINICAL TRIALS

A. Kulasekararaj 1,* , R. Brodsky 2 , M. Griffin 3 , A. Kulagin 4 , M. Ogawa 5 , J. Wang 5 , A. Mujeebuddin 5 , J.-I. Nishimura 6 , R. Peffault de Latour 7 , J. Szer 8 , J. W. Lee 9

1 King’s College Hospital, National Institute of Health Research/Wellcome King’s Clinical Research Facility and King’s College London, London, United Kingdom; 2 Division of Hematology, Johns Hopkins Medicine, Baltimore, United States of America; 3 St James Hospital, NHS Teaching Hospitals, Leeds, United Kingdom; 4 RM Gorbacheva Research Institute, Pavlov University, St Petersburg, Russia; 5 Alexion, AstraZeneca Rare Disease, Boston, MA, United States of America; 6 Osaka University Graduate School of Medicine, Suita, Japan; 7 Hôpital Saint-Louis AP-HP, Paris, France; 8 Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, Melbourne, VIC, Australia; 9 Department of Hematology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, chronic, hematologic disorder characterized by uncontrolled terminal complement activation, intravascular hemolysis, thrombotic events and significant morbidity and mortality. Complement component 5 (C5) inhibitors, eculizumab and ravulizumab, are the current standard of care in patients with PNH. Eculizumab has been the mainstay of treating patients with PNH since approval by both US Food and Drug Administration (FDA) and European Medicines Agency (EMA) in 2007. Ravulizumab (approved in 2018 [FDA] and 2019 [EMA]) is a new treatment for patients with PNH, offering complete complement C5 inhibition throughout the 8-week (q8w) dosing interval. The ravulizumab clinical trial program has allowed for analysis into the long-term management of patients with PNH treated with C5 inhibitors and their associated clinical outcomes.

Aims: To analyze patient survival using long-term data from the ravulizumab clinical trial program.

Methods: This analysis utilized pooled long-term survival data across the ravulizumab phase 1b, 2 and 3 trials (103 [ NCT02598583 ], 201 [ NCT02605993 ], 301 [ NCT02946463 ] and 302 [ NCT03056040 ]) with up to 4 years of open-label extension. Complement C5 inhibitor (eculizumab/ravulizumab) and dose received varied during the initial treatment period. During open-label extension, patients received weight-based dosing of intravenous ravulizumab q8w. Deaths reported, patient survival over time and adverse events resulting in patient death were analyzed. Instances of patients that died owing to infection or sepsis were specifically analyzed to provide insight into the events leading to death.

Results: This analysis reported 1479.0 patient-years of follow-up in 475 patients with PNH treated with ravulizumab. Of the 475 patients who received ravulizumab, 12 (2.5%) died during the initial treatment period and 4-year open-label extension with an overall incidence of 0.8 per 100 patient-years. Nine of these deaths occurred within the first 3 years of treatment. The remaining three patients died during the fourth (n = 2) and fifth (n = 1) years of treatment. Of the 12 deaths reported, six were attributed to infection or sepsis (Table 1). These patients were predominantly male (83.3%), and white or Asian (50.0%, respectively), and median (range) age was 61 (43–75) years. In addition, of these six patients, two had aplastic anemia and one patient had pancytopenia. Most (83.3%) of these patients were hospitalized owing to onset of infection; however, one patient (white male; 43 years old; ravulizumab to ravulizumab) was hospitalized owing to worsening of aplastic anemia. During hospitalization, this patient was diagnosed with acute respiratory infection and died owing to sepsis. One patient (Asian male; 62 years old; ravulizumab to ravulizumab) died owing to meningococcal sepsis (strain unknown). This patient was vaccinated against meningococcal groups A, C, Y and W-135, but had not received prophylactic antibiotics. Other causes of death included cardiac disorders (n = 1), neoplasms (n = 4; acute myeloid leukemia, metastatic urothelial carcinoma of the kidney, metastatic malignant neoplasm of the lung and non-small cell lung carcinoma) and respiratory disorders (n = 1).

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Summary/Conclusion: This analysis reports the longest period of follow-up in 475 patients with PNH treated with ravulizumab. Overall, long-term ravulizumab treatment was associated with a low incidence of death and few patients died owing to infection. This analysis supports the long-term use of ravulizumab for PNH.

P813: EFFICACY, TREATMENT ADMINISTRATION SATISFACTION AND SAFETY OF SUBCUTANEOUS RAVULIZUMAB THROUGH 1 YEAR IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA WHO RECEIVED PRIOR INTRAVENOUS ECULIZUMAB

M. N. Yenerel 1,* , F. Sicre de Fontbrune 2 , C. Piatek 3 , F. Sahin 4 , W. Füreder 5 , M. Ogawa 6 , I. Tomazos 6 , H. Doll 7 , A. Ozol-Godfrey 6 , J. R. Sierra 6 , J. Szer 8

1 Division of Hematology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; 2 Hematology and Bone Marrow Transplant Unit, Assistance Publique Hôpitaux des Paris, Saint Louis Hospital, Paris, France; 3 Jane Anne Nohl Division of Hematology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States of America; 4 Department of Hematology, Ege University Bornova, Izmir, Turkey; 5 Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria; 6 Alexion, AstraZeneca Rare Disease, Boston, MA, United States of America; 7 Clinical Outcomes Solutions, Folkstone, United Kingdom; 8 Haematology at Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, Melbourne, VIC, Australia

Background: The efficacy of ravulizumab (intravenous [IV] formulation; administered every 8 weeks) for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH) has been demonstrated in several randomized trials ( NCT02946463 , NCT03056040 , NCT03406507 ). In study 303 ( NCT03748823 ), subcutaneous (SC) ravulizumab, administered weekly via an on-body delivery system, showed pharmacokinetic non-inferiority to IV ravulizumab in adult patients with PNH who were clinically stable on prior IV eculizumab treatment. Here, we report results from the first 1 year of SC treatment, starting at day 15 for patients who continued SC ravulizumab during the extension period (SC/SC) and day 71 for patients who switched from IV ravulizumab to SC ravulizumab (IV/SC).

Aims: To evaluate the efficacy, treatment administration satisfaction and safety of SC ravulizumab through the first 1 year (day 351) of treatment in adult patients with PNH previously treated with eculizumab.

Methods: Patients (≥ 18 years) with clinically stable PNH (lactate dehydrogenase [LDH] levels ≤ 1.5 × upper limit of normal [246 U/L]) and ≥ 3 months prior eculizumab treatment were enrolled in the study, which consisted of a screening period (day -1 to day -30), a 10-week randomized treatment period and an extension period of up to 172 weeks. During the randomized treatment period, patients were assigned (2:1 ratio) to receive either SC ravulizumab or IV ravulizumab; all patients received SC ravulizumab during the extension period. Efficacy endpoints included: change in LDH from baseline; incidence of breakthrough hemolysis; transfusion avoidance; and stabilized hemoglobin (avoidance of a ≥ 2 g/dL decrease in hemoglobin in the absence of transfusion). Treatment administration satisfaction was assessed via the Treatment Administration Satisfaction Questionnaire (TASQ), which has been validated in a PNH population. Safety, including adverse events (AEs), serious AEs (SAEs) and adverse device effects (ADEs), were also assessed up to the 1-year data cut-off.

Results: In total, 128 patients received SC ravulizumab (SC/SC: n = 84; IV/SC: n = 44; mean [range] duration of SC treatment: 486.4 [37–709] days). Efficacy endpoints (SC/SC and IV/SC) remained stable over time through 1 year of SC ravulizumab treatment. Mean (standard deviation [SD]) percentage change in LDH from baseline to SC day 351 was 0.9% (20.5%). Breakthrough hemolysis events were infrequent: 5/128 patients (3.9%); no event was considered free C5-related. Transfusion avoidance was maintained in 83.6% of patients during SC treatment, and 79.7% achieved stabilized hemoglobin. Improvement in total TASQ score with SC ravulizumab (compared with baseline IV eculizumab) was apparent at the first post-SC treatment assessment (SC day 29) and maintained until data cut-off ( Figure ). The most common AEs (reported by ≥ 10% of patients, excluding ADEs related to device product issues) during SC treatment were headache (14.1%, all grade ≤ 2), COVID-19 (14.1%, one death) and pyrexia (10.9%); injection site reaction (4.7%) was the most common non-device related ADE. Treatment-emergent SAEs were experienced by 21.1% of patients through to data cut-off. Although many patients had ≥ 1 device issue ADE, full SC dose administration was achieved in 99.9% of attempts. ADE incidence decreased over time.

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Summary/Conclusion: The SC method of administration provides an additional treatment option for patients with PNH receiving ravulizumab therapy. Patients may be switched from IV eculizumab or IV ravulizumab to SC ravulizumab without loss of efficacy.

P814: CLINICAL CHARACTERISTICS AND GENE MUTATION ANALYSIS OF 148 CHILDREN WITH FANCONI ANEMIA IN CHINA

L. Chang 1,* , L. zhang 1 , W. An 1 , Y. wan 1 , Y. cai 1 , Y. Lan 1 , M. Ruan 1 , X. liu 1 , Y. Zou 1 , X. Zhu 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical Colleg, Tian jin, China

Background: Fanconi anemia (FA) is a rare autosomal recessive, X-linked (FANCB) or autosomal dominant (FANCR/RAD51) disease of bone marrow failure. Pathogenesis is mainly caused by gene mutation related to the FA pathway. At present, at least 23 genes have been discovered to play a role in the FA pathway. The gene mutations of FA have been reported in more and more countries. In China, there no large-scale cases have been reported.

Aims: In this study, we analyzed the clinical characteristics of 148 FA children from 20 provinces in China from October 2003 to October 2021. We explored the relationship between genotype and phenotype in 105 children with FA genotyping results. This is the largest series of subtyped Chinese Fanconi anemia patients to date, and the results will be helpful for future clinical management.

Methods: A total of 148 Fanconi anemia patients from 140 families were diagnosed by clinical phenotype, family history, chromosome breakage test induced by mitomycin C, Single cell gel electrophoresis experiment, targeted - sequence, whole exon sequencing, and MLPA method from October 2003 to October 2021. Since there were no genetic test results for the children before 2010, only the 105 patients after 2010 had gene sequencing results, which allowed genotyping. DNA damage repair defects were detected by MMC-induced Single cell gel electrophoresis and chromosome breakage experiments. Targeted-seq was used to identify the FA mutations of the patients. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large fragment deletions in patients with negative results in the targeted-seq but conforming to the FA by MMC-induced chromosome breakage experiments test and clinical manifestations. We performed WES of DNA samples from five patients and their parents.

Results: 1) The most common subtype of FA in China is FA-A, followed by FA-D2 and FA-P。2) The most common deformities in FA-A patients are finger deformities and skin pigmentation, while heart deformities are more common in FA-D2/B/G/I/P subtypes. 3) The common mutations of FANCA are exon23c.2101A>G, exon28c.2778 + 1G>A, exon34c.3348 + 1G>A, exon21c.1844delC, and exon30 C2941T>G, which are different from Korea and Japan. 4) Homozygous nonsense mutation of exon32 c.3188G>A and exon29 c.2851C>T in FA-A likely benign, but the homozygous splicing mutation in exon29c.2852 + 1G>T is harmful. 4) Children with splicing and Del mutations of FANCD1 gene had poor clinical prognosis, while children with homozygous mutations of exon10c. 1792A>G is likely benign. 5) Cases of initial diagnosis of MDS or poor disease progression tend to occur in FA-B/C/E/G/J/L/M and FA-S subtypes. 6)Chromosome 1, 3, 7, and 8 abnormalities and mutations of SF3B1 P53 are related to disease progression.

Summary/Conclusion: We made a comprehensive description and analysis of the clinical characteristics and mutant genes of Children with FA in China. We proposed the mutation sites related to clinical prognosis and the cytogenetic abnormalities of disease progression, providing new data for our comprehensive understanding of FA. Our data will be useful for FA future menagement.

P815: PHARMACOKINETIC AND PHARMACODYNAMIC SIMILARITY OF ABP 959 AND ECULIZUMAB REFERENCE PRODUCT: UNBOUND ECULIZUMAB AND CH50 FROM THE RANDOMIZED, DOUBLE-BLIND, SINGLE-DOSE STUDY IN HEALTHY VOLUNTEERS

V. Hanes 1 , J. Pan 1 , D. Mytych 1 , V. Chow 1,*

1 Amgen Inc, Thousand Oaks, United States of America

Background: ABP 959 is being developed as a biosimilar to eculizumab reference product (RP). ABP 959 and the RP are humanized recombinant IgG2/4κ mAbs which bind with specificity to the human complement C5, blocking its cleavage to C5a and C5b and preventing the generation of the terminal complement complex (TCC), thereby blocking cell lysis. Excessive activation or insufficient control of C5 is believed to play a role in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), and neuromyelitis optical spectrum disorder (NMOSD). Drug binding to C5 blocks this biological function and contributes to efficacy across these indications for which the RP is approved.

Aims: The goal of this analysis is to further support the previously demonstrated pharmacokinetic (PK)/pharmacodynamic (PD) similarity of ABP 959 with eculizumab RP by providing additional data on similarity in PK exposure with respect to unbound eculizumab RP and 50% total hemolytic complement activity (CH50).

Methods: This was a randomized, double-blind, single-dose, 3-arm, parallel-group trial in healthy adult male subjects, aged between 18 and 45 years, with a body mass index of 18 to 30.0 kg/m 2 , who were randomized following informed consent, to receive a 300-mg IV infusion of ABP 959, or FDA-licensed eculizumab RP (eculizumab US), or EU-authorized eculizumab (eculizumab EU). Serum samples for PK and PD evaluations were collected over 56 days. Primary objectives were to demonstrate PK and PD equivalence of ABP 959 versus eculizumab US and ABP 959 versus eculizumab EU, as assessed by area under the total serum concentration versus −time curve (AUC) from time 0 extrapolated to infinity (AUCinf) and an area between the effect curve (ABEC) of 50% total hemolytic complement activity (CH50). Secondary PK endpoints included AUC from time 0 to the time of the last observed quantifiable concentration (AUC last ) and maximum observed concentration (C max ). Pre-specified equivalence criterion for the primary PK and PD parameters was 90% confidence interval (CI) for geometric means (GM) ratio within 0.80 to 1.25. Other secondary endpoints included the safety, tolerability, and immunogenicity of the investigational products (IPs).

Results: A total of 219 subjects were randomized (ABP 959, n=71; eculizumab US, n=74; eculizumab EU, n=74). As with PK and PD parameters of total eculizumab following a single 300 mg IV infusion of IP, 1 PK/PD parameters for unbound eculizumab were similar between ABP 959 versus eculizumab US and ABP 959 versus eculizumab EU.

The GM and GM coefficient of variation (%CV) of AUC inf (h.µg/mL) for unbound eculizumab was: 5072.1 (25) for ABP 959, 5527.6 (31) for eculizumab US, and 5070.3 (31) for eculizumab EU.

The GM and %CV of ABEC of CH50 (%*h) was 17724.5 (38.75) for APB 959, 16549.4 (42.23) for eculizumab US, and 16361.1 (36.94) for eculizumab EU. The resulting 90 and 95% CI of GM ratios were within the bioequivalence criteria of 0.80 to 1.25.

1 Eur J Haematol. 2020;105:66–74.

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Summary/Conclusion: These additional clinical pharmacology results demonstrate bioequivalence of ABP 959 to eculizumab RP with regard to unbound eculizumab and CH50. These results further support the PK/PD similarity of ABP 959 with eculizumab RP.

P816: TRANSIENT MONOSOMY 7 IN SAMD9/9L SYNDROMES: IS IT SAFE TO WATCH AND WAIT?

M. Erlacher 1,* , F. Andresen 1 , A. Yoshimi 1 , M. Sukova 2 , J. Stary 2 , B. de Moerloose 3 , M. Dworzak 4 , S. Polychronopoulou 5 , G. Göhring 6 , C. Kratz 7 , J. van der Werff ten Bosch 8 , M. Seidel 9 , B. Strahm 10 , M. Wlodarski 10 , C. Niemeyer 10

1 Division of Pediatric Hematology and Oncology, University Medical Center Freiburg, Freiburg, Germany; 2 Department of Pediatric Hematology and Oncology, University Hospital Motol, Prague, Czechia; 3 Department of Pediatric Hematology-Oncology, Ghent University Hospital, Ghent, Belgium; 4 Department of Pediatrics, St. Anna Children’s Hospital and Children’s Cancer Research Institute, Vienna, Austria; 5 Department of Pediatric Hematology/Oncology, Aghia Sophia Children’s Hospital, Athens, Greece; 6 Department of Human Genetics; 7 Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany; 8 Department of Pediatric Onco-Hematology, Universitair Ziekenhuis Brussels, Brussels, Belgium; 9 Division of Pediatric Hemato-Oncology, Medical University Graz, Graz, Austria; 10 Division of Pediatric Hematology and Oncology, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Background: Monosomy 7 is a non-random but poorly understood somatic event in myelodysplastic syndrome (MDS). Most enigmatic is the fact that monosomy 7 can disappear spontaneously. Such transient monosomy 7 was recently associated with germline mutations in sterile alpha-motif domain-containing protein 9 ( SAMD9 ) and its paralog SAMD9-like ( SAMD9L ), both located on chromosome 7q. Mechanistically, gain-of-function (GOF) SAMD9/9L mutations inhibit proliferation and reduce survival in different cell types thus leading to bone marrow failure, immunodeficiency and non-hematological phenotypes. Hematopoietic cells are under a high selection pressure to inactivate this toxic gene and achieve this by multiple mechanisms: uniparental disomy, monosomy 7, del(7q) or somatic loss-of-function (LOF) mutations of the mutated allele. Multiple “rescued” clones exist in parallel within the bone marrow of patients, and dominance of an UPD or somatically mutated clone can displace the monosomy 7 clone and result in hematological remission.

Aims: We recently identified SAMD9/9L mutations in 8% of children with MDS, and a strong association with monosomy 7 (EWOG-MDS; Sahoo et al, Nat Med, 2021). This retrospective analysis revealed transient monosomy 7 and spontaneous hematological remission in some infants. To prospectively study the propensity of patients to lose their monosomy 7 clone, EWOG-MDS elected to delay immediate hematopoietic stem cell transplantation (HSCT) in children <5 years of age diagnosed of refractory cytopenia (RCC) with germline SAMD9/9L mutation and monosomy 7.

Methods: Patients with SAMD9 or SAMD9L mutation and monosomy 7, younger than 5 years of age, were closely monitored with bone marrow morphology, SAMD9/9L mutational analysis and cytogenetic studies every 3-4 month. HSCT was recommended in case of severe infections, persistent severe neutropenia, progression with additional cytogenetic aberrations or increase in blasts (MDS-EB), or persistence of monosomy 7 beyond the age of 5 years.

Results: Eight patients diagnosed at a median age of 11 months (range 4-36 months) with SAMD9L (n=7) or SAMD9 (n=1) germline mutation were studied. Two (P5 and P8) were born small for gestational age, P5 presented with global developmental delay and cerebellar atrophy. With a median follow-up of 27 months, 6 of the 8 patients are alive. One patient succumbed to infection prior to HSCT (P4), another to transplantation-related toxicity (P8). HSCT was performed or is currently scheduled in 4 of the 8 patients; indications were severe neutropenia/infection (P5, P6), immunodeficiency (P7) or MDS-EB (P8). The monosomy 7 clone regressed in 3 patients (P1-P3) and was no longer detectable at 3 months (P1) or 1 year (P2) from diagnosis (P3 was lost to follow-up at 12 months). Somatic SAMD9L mutations were noted in P1 and P2, both of which experienced a complete hematological and cytogenetic recovery.

In sum, we followed 8 patients with SAMD9/9L syndrome and monosomy 7. Transient monosomy 7 with hematological recovery was observed in 2 patients and accompanied by gain of somatic rescuing mutations.

Summary/Conclusion: Transient monosomy 7 in young children with SAMD9/SAMD9L germline disorder and RCC with monosomy 7 is a rare event. Initiation of a watch and wait strategy instead of timely HSCT will require a stringent surveillance strategy including repetitive BM analyses for SAMD9/9L sequencing, cytogenetics and search for somatic oncogenic events.

P817: SYNDROMES PREDISPOSING TO LEUKEMIA ARE A MAJOR CAUSE OF INHERITED CYTOPENIAS IN CHILDREN

O. Gilad 1,2,* , O. Dgany 3 , S. Noy -Lotan 3 , T. Krasnov 3 , J. Yacobovich 4 , R. Rabinowicz 4 , T. Goldberg 5 , A. Kuperman 6 , A. Abu-Quider 7 , H. Miskin 8 , N. Kapelushnik 9 , N. Mandel-Shorer 10 , S. Shimony 11 , D. Harlev 12 , T. Ben-Ami 13 , E. Adam 14 , C. Levin 15 , S. Aviner 16 , R. Elhasid 17 , S. Berger-Achituv 17 , L. Chaitman-Yerushalmi 18 , Y. Kodman 4 , N. Oniashvilli 19 , M. Hameiri- Grosman 19 , S. Izraeli 4 , H. Tamary 1,3,20 , O. Steinberg-Shemer 1,3,20

1 Sackler faculty of Medicine, Tel Aviv University, Tel Aviv; 2 Department of Hematology-Oncology, Scneider Children’s medical center of Israe; 3 Pediatric Hematology Laboratory, Felsenstein Medical Research Center; 4 Department of Hematology-Oncology, Schneider Children’s Medical Center of Israel; 5 Department of Hematology-Oncology, Scneider Children’s medical center of Israel, Petach Tikva; 6 Blood Coagulation Service and Pediatric Hematology Clinic, Galilee Medical Center, Nahariya; 7 Pediatric Hematology, Soroka University Medical Center, Ben-Gurion University, Beer Sheva; 8 Pediatric Hematology Unit, Shaare Zedek Medical Center, Jerusalem; 9 Goldschleger Eye Institute, Sheba Medical Center, Tel Hashomer; 10 Department of Pediatric Hematology-Oncology, Rambam Healthcare Campus, Haifa; 11 Institute of Hematology, Davidoff Cancer Centre, Rabin Medical center, Petach-Tikva; 12 Pediatric Hematology-Oncology Department, Hadassah University Medical Center, Jerusalem; 13 Pediatric Hematology Unit, Kaplan Medical Center, Rehovot; 14 Pediatric Hematology-Oncology Department, Sheba Medical Center, Tel Hashomer; 15 Pediatric Hematology Unit and Research Laboratory, Emek Medical Center, Afula; 16 Department of Pediatrics, Barzilai University Medical Center, Ashkelon; 17 Department of Pediatric Hemato-Oncology, Tel Aviv Medical Center; 18 Genoox, Health Care Technology, Tel Aviv; 19 Department of Hematology-Oncology, Schneider Children’s Medical Center of Israel; 20 Department of Hematology-Oncology, Schneider Children’s Medical Center of Israel, Petach Tikva, Israel

Background: Prolonged cytopenias are a non-specific sign with a wide differential diagnosis. Among inherited disorders, cytopenias predisposing to leukemia require a timely and accurate diagnosis to ensure appropriate medical management, including adequate monitoring and stem-cell transplantation prior to the development of leukemia.

Aims: We aimed to define the types and prevalences of the genetic causes leading to persistent cytopenias in children.

Methods: The study comprises children with persistent cytopenias, myelodysplastic syndrome (MDS), aplastic anemia, or suspected inherited bone marrow failure syndromes, who were referred for genetic evaluation from all pediatric hematology centers in Israel during 2016-2019. For variant detection, we used a custom-made targeted next-generation sequencing panel covering 226 genes known to be mutated in inherited cytopenias followed, if needed, by whole exome sequencing. Sanger sequencing was used for validation.

Results: In total, 189 children with persistent cytopenias underwent a genetic evaluation. Pathogenic and likely pathogenic variants were identified in 59 patients (31.2%), including 47 with leukemia predisposing syndromes. Most of the latter (32, 68.1%) had inherited bone marrow failure syndromes, 9 (19.1%) had inherited thrombocytopenia predisposing to leukemia, and 3 (6.4%) had congenital neutropenia. Twelve patients had cytopenias with no known leukemia predisposition, including nine children with inherited thrombocytopenia and three with congenital neutropenia (not yet known to predispose to leukemia). Importantly only 3 had myelodysplastic syndrome. Thus, the majority of leukemia predisposition could not have been deduced from the morphological presence of MDS

Summary/Conclusion: almost one-third of 189 children referred with persistent cytopenias had an underlying inherited disorder; 79.7% of whom had a germline predisposition to leukemia. Precise diagnosis of children with cytopenias should direct follow-up and management programs and may positively impact disease outcome.

P818: NEXT GENERATION TARGETED SEQUENCING FOR ENHANCED GENOTYPING OF DIAMOND BLACKFAN ANEMIA IN ISRAEL

T. Goldberg 1,* , O. Steinberg-Shemer 1,2,3 , O. Dgany 3 , S. Noy-Lotan 3 , T. Krasnov 3 , O. Gilad 1,2 , J. Yacobovich 1,2 , H. Tamary 1,2,3

1 Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah Tikva; 2 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; 3 Pediatric Hematology Laboratory, Felsenstein Medical Research Center, Petah Tikva, Israel

Background: Diamond Blackfan anemia (DBA), an erythroid-specific congenital bone marrow failure syndrome, is predominantly driven by mutations in one of several genes encoding ribosomal proteins (RP), leading to protein haploinsufficiency. In recent years, a number of patients with “DBA-like” syndrome were found to have mutations in non-RP genes including CECR1 . Next generation sequencing (NGS) is rapidly replacing conventional Sanger sequencing approaches in the molecular diagnosis of patients with DBA and other inherited bone marrow failure syndromes. Targeted NGS panels enable high-resolution deep sequencing of all known genes associated with a specific clinical presentation such as congenital anemias.

Aims: This study aims to use a targeted NGS panel specific for inherited anemias to genotype previously undiagnosed patients with clinical DBA in Israel. Enhanced genotyping efficiency provided by NGS may provide an opportunity to evaluate for genotype-phenotype correlations.

Methods: A targeted NGS panel including over 260 genes was used as an adjunct to Sanger sequencing and multiplex ligation-dependent probe amplification for genotyping patients from the Israeli inherited bone marrow failure registry (I-IBMFR) with a clinical diagnosis of DBA. Over 90% of 48 patients with DBA from the Israeli registry had available DNA for genotyping.

Results: Forty-eight patients with clinical DBA, registered to the I-IBMFR, have been followed for a median of 12.1 years. The majority of patients presented with anemia in the first year of life, with a median age at presentation of 3 months. Forty-four of these patients underwent genotyping. Pathogenic RP gene alterations were detected in 31 patients in 9 different RP genes; homozygous CECR1 mutations were found in 8 separate patients. Forty percent of these molecular diagnoses were detected by targeted NGS panel technology. More than half of the patients exhibited at least one congenital malformation. Cardiac anomalies were observed broadly in patients with small and large subunit RP gene mutations. Thumb anomalies were found exclusively in patients with RPL5 mutations; cleft lip and palate were reported only in patients with RPS10 and RPL11 mutations. Fewer than half of the patients given a steroid trial were responsive; patients with RPS19 mutations responded significantly better to steroid treatment than patients with RPL5 mutations. Just under half of the patients with RP gene mutations became red blood cell (RBC) transfusion dependent, while almost all of the patients with CECR1 mutations required RBC transfusions. Three patients, including two genetically undiagnosed patients, went into spontaneous remission after receiving chronic treatment.

Summary/Conclusion: Use of targeted NGS has dramatically enhanced the efficiency of genotyping for patients with DBA and other inherited bone marrow failure syndromes, allowing for focused management of patients and discovery of novel genotype-phenotype correlations. Ongoing investment in registries and comprehensive genotyping are crucial in increasing our understanding of this rare disease and optimizing patient care.

P819: TRANSPLANT, TREATMENT AND TRANSFUSION FREE (TTT-FREE) SURVIVAL AS RELEVANT CLINICAL ENDPOINT AFTER IMMUNOSUPPRESSIVE TREATMENT FOR ACQUIRED APLASTIC ANEMIA IN ADULTS

E. Koster 1 , C. Halkes 1,* , E. Bogers 1 , C. Hazenberg 2 , F. Heubel-Moenen 3 , S. Langemeier 4 , E. Meijer 5 , E. Nur 5 , M. Raaijmakers 6 , T. Snijders 7 , M. de Witte 8 , J. Tjon 1 , L. de Wreede 9

1 Hematology, Leiden University Center, Leiden; 2 Hematology, University Medical Center Groningen, Groningen; 3 Hematology, Maastricht University Medical Center, Maastricht; 4 Hematology, Radboud Medical Center, Nijmegen; 5 Hematology, Amsterdam Univeristy Medical Center, Amsterdam; 6 Hematology, Erasmus Medical Center, Rotterdam; 7 Hematology, Medical Center Twente, Enschede; 8 Hematology, University Medical Center Utrecht, Utrecht; 9 Department of Biomedical Data Sciences, Leiden University Center, Leiden, Netherlands

Background: Acquired aplastic anemia (AA) is characterized by an aplastic bone marrow and pancytopenia. Adult patients with AA can be treated with immunosuppressive treatment (IST) consisting of ATGAM in combination with cyclosporin (CsA) or with an allogeneic hematopoietic stem cell transplantation (alloSCT). Patients <40 years with an HLA-identical sibling preferentially receive an alloSCT. Other patients are treated with IST as 1 st line treatment. IST leads to transfusion independency in the majority of patients but it can take up to 6 months before this response occurs. Some patients need long-term treatment with CsA to maintain this response and durable efficacy is hampered by relapsing aplasia or the development of MDS, AML or PNH. While graft versus host disease and relapse-free survival (GRFS) is used as a favorable composite outcome after alloSCT, a widely accepted measurement for treatment success after IST is missing.

Aims: In this study we examined Transplant, Treatment and Transfusion-free survival (TTT-free survival) as a relevant clinical outcome after standard IST with ATGAM and CsA in AA adult patients. We determined the 5-year TTT-free survival after standard IST in patients ≤40 years and >40 years.

Methods: The Dutch Adult Aplastic Anemia Registry started in 2014 and contains detailed data of all consecutive AA patients who have been treated with ATGAM-based therapy in the participating hospitals, offering a unique possibility to evaluate real-life long-term efficacy and safety of 1 st line IST treatment in AA. To determine TTT-free survival, a multistate model (MSM) was developed, to take into account that patients can have transient periods of treatment success and failure. Patients started in the dynamic state treatment & transfusion at time of the start of ATGAM. This state also included all other (2 nd line) non-alloSCT treatments (for example Eltrombopag, Rabbit-ATG or Danazol). Other dynamic states patients could enter and leave during follow-up were treatment & no transfusion, transfusion & no treatment and no treatment & no transfusion (considered as TTT-free survival). Absorbing states, accessible from all dynamic states, were death, alloSCT for AA, treatment for MDS/AML and treatment for PNH.

Results: ATGAM with CsA as 1 st line treatment was started in 117 patients (median age 54 years, range 18-79). Overall survival after 5 year is 77% (95% Confidence Interval (95%CI) 67-86%) Figure 1 shows the results of the MSM. After 5 years, the TTT-free survival was 42% (95%CI: 33-55%) for the total cohort. 19% (95%CI: 12-31%) of the patients was transfusion independent but still needed medication at this time (mainly CsA or Eltrombopag). 15% (95%CI: 10-23%) had received an alloSCT as 2 nd line treatment for AA, 5% (95%CI: 2-13%) had started treatment for MDS/AML and 2% (95%CI: 0-11%) had started treatment with complement inhibition for PNH. 5-year TTT-free survival was 60% (95%CI:44-82%) in patients aged ≤40 years (n=36), but only 33% (95%CI: 23-49%) in patients aged >40 years (n=81).

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Summary/Conclusion: TTT-free survival can be used to evaluate treatment success after IST in AA patients, allowing achievement, loss and recovery of response. We showed that 5-year TTT-free survival is superior in patients ≤40 years compared to patients >40 years. This MSM can be used to predict outcomes in AA patients receiving IST and can help in the decision whether and when to offer a patient an alloSCT.

P820: PREDICTING RESPONSE TO RABBIT ATG-BASED INTENSIVE IMMUNOSUPPRESSIVE THERAPY COMBINED WITH ELTROMBOPAG IN CHINESE ADULT PATIENTS WITH SEVERE APLASTIC ANEMIA

R. Li 1 , Z. Liu 1 , X. Chen 2 , Q. Long 2 , Y. Yang 3 , S. Lin 4 , J. Jia 5 , G. He 1,* , J. Li 1

1 Hematology, the First Affiliated Hospital of Nanjing Medical University; 2 Hematology, the Second People’s Hospital of Nanjing, Nanjing; 3 Hematology, the First Bethune Hospital of Jilin University, Changchun; 4 Hematology, Zhejiang Province Hospital of Traditional Chinese Medicine, Hangzhou; 5 Hematology, Peking University People’s Hospital, Beijing, China

Background: The intensive immunosuppressive therapy (IST) consisted by antithymocyte immunoglobulin (ATG) and cyclosporin (CsA) combining with eltrombopag (E-PAG) is the initial therapy for adult patients with severe aplastic anemia (SAA). There is rabbit ATG (r-ATG) rather than horse ATG in the mainland of China and E-PAG is recommended with 75mg/d for East Asian population.

Aims: To explore factors predicting the efficacy of E-PAG (75mg/d) plus r-ATG based IST for SAA patients in East Asia.

Methods: From February 2018 to December 2020, we conducted a retrospective, multicenter study to analyze 58 adult SAA patients with rabbit ATG-based IST and E-PAG in the China Eastern Cooperation Group for Anemia (CECGA). The dosage of r-ATG was 3.5mg·kg -1 ·d -1 for 5 days with intravenous infusion and CsA was 3-5mg·kg -1 ·d -1 early in the regimen maintaining concentration at 150-200ng/ml, whose dosage could be modulated on the basis of drug concentration and severe adverse events (SAEs). E-PAG was recommended with 75mg/d at least 6 months. If or SAEs occurred or the count of platelet was higher than 200×10 9 /L, E-PAG would be discontinued.

Results: 58 patients with median age of 42.5 years (18 years to 74 years) were treated with r-ATG based IST plus E-PAG. Cohort 1 and cohort 2 included patients with or without a response at 6 months, whist the median age were 50.5 years (24 years to 74 years) and 38 years (18 years to 71 years), separately. The median time between diagnosis and treatment was 3 weeks (1 week to 106 weeks) and 3 weeks (1 week to 166 weeks), respectively. The response rates of E-PAG combining with IST at 3, 6 and 12 months after IST were 64%, 76% and 85% (44 of 52), while the complete response rates at 3, 6 and 12 months were 19%, 21% and 29% (15 of 52), separately. The baseline absolute lymphocyte count (ALC) (area under a curve (AUC)=0.706, 95%CI 0.522-0.890, P =0.057), red cell distribution width – coefficient of variation (RDW-CV) (AUC=0.722, 95%CI 0.494-0.950, P =0.040) and reticulocyte (Ret) percentage (AUC=0.798, 95%CI 0.640-0.956, P =0.006) were highly predictive of response at 6 months (Figure 1). The tipping values of Ret percentage, ALC and RDW-CV were 0.45%, 1.06×10 9 /L and 11.75%, respectively. The sensitivity and specificity of Ret percentage were 81.6% and 66.7%; RDW-CV were 94.7% and 55.6%; ALC were 55.3% and 88.9%. The 2-year overall survival was 93%.

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Summary/Conclusion: The baseline Ret percentage, RDW-CV and ALC were potential factors to predict a favorable effect in SAA patients of East Aisan.

P821: LATE ONSET AND/OR LONG LASTING NEUTROPENIA IN CHILDHOOD: CLINICAL AND HEMATOLOGICAL CHARACTERISTICS AND OUTCOMES

C. Kelaidi 1,* , K. Antoniadi 1 , V. Tzotzola 1 , V. Papadakis 1 , M. Ampatzidou 1 , C. Pontikoglou 2 , I. Mavroudi 2 , M. Tzanoudaki 3 , G. Paterakis 4 , S. I. Papadhimitriou 5 , K. Tsitsikas 1 , A. Bountali 1 , K. Manola 6 , H. A. Papadaki 2 , S. Polychronopoulou 1

1 Pediatric Hematology-Oncology, Aghia Sophia Children’s Hospital, Athens; 2 Haemopoiesis Research Laboratory, University of Crete, School of Medicine, Heraklion; 3 Flow cytometric cell analysis, Laboratory of Immunology, Aghia Sophia Children’s Hospital; 4 Flow Cytometry Analysis, Laboratory of Immunology; 5 Laboratory of Hematology, Georgios Gennimatas General Hospital; 6 Laboratory of Health Physics, Radiobiology & Cytogenetics, National Centre for Research Demokritos, Athens, Greece

Background: In contrast with primary autoimmune neutropenia of infants and pre-school children, characterized by the presence of anti-polymorphonuclear antibodies (anti-PNN), benign course and spontaneous resolution at age 3-5 years, late onset (LO) and/or long lasting (LL) neutropenia of childhood is vaguely described.

Aims: To address characteristics and outcomes in a cohort of children with LO/LL diagnosed and followed by our Department.

Methods: Cross-sectional study of patients aged 0-16 years with LO (age >5 years, in accordance with Fioredda et al, 2020) or LL (duration >3 years) neutropenia (1.5 G/L) +/- leukopenia (Z-score ≤2 according to age and gender); with a first visit or continued follow-up in our Department of Pediatric Hematology-Oncology (T.A.O.) during the period 2015-2020. Patients with severe congenital neutropenia (SCN) were excluded. Detection of anti-PNN using GAT and GIFT was centralized.

Results: From 1972 to 2021, 534 cases of neutropenia/leukopenia were recorded (5, 40, 82, 134, 273 during 1970-1979, 1980-1989, 1990-1999, 2000-2009, 2010-2021, respectively); 152 were included in the cross-sectional study. Sixty-one (40%) had no LO/LL (age<5 years, resolution within≤3 years), 77 (51%0 had LO and 14 (9%) had LL. Male/female ratio was 47% vs 52% vs 28% in no LO/LL vs LO vs LL, respectively (P=0.27), median age 1 vs 11.1 vs 3.1 years (P<10-4), median neutrophil count 0.5 vs 1.2 vs 0.6 G/L (P<10-4), and with concomitant leukopenia in 27% vs 53% vs 36% (P=0.009) or lymphopenia in 3% vs 10% vs 7% of cases (P=0.27). Anti-PNN were detected in 83% vs 42% vs 60% of patients no LO/LL vs LO vs LL, respectively (P=0.12). Familial neutropenia was observed in three pairs of siblings/cousins with LL, possible ethnic neutropenia in 7 patients, probable genetic predisposition to cancer in 4 patients LO/LL and a congenital metabolic syndrome in one patient LL. Infection, mostly cutaneous, occurred in 17% vs 24% vs 66% no LO/LL vs. LO vs. LL (P=0.02). Bone marrow examination was performed in 8 patients (4 LO, 4 LL) and showed mild hypoplasia in 2 (LO). Mild peripheral blood lymphocytic subsets abnormalities were observed in ¼ of the patients, particularly in LO, and mild reduction of serum immunoglobulin class levels in ¼ of the patients, without difference between no LO/LL and LO. Genetic typing performed in 9% of patients LO/LL (targeted pediatric MDS panel N=2, primary immunodeficiency N=2, SCN N=1, whole-exome sequencing N=2) showed a heterozygous TACI mutation in one patient with LL and frequent infections of moderate severity. In total, MDS, immunodeficiency and autoimmunity was noted in 1%, 5% and 4% of patients LO and immunodeficiency in 14% of patients LL. One patient LL developed B-cell precursor ALL. One patient LO with Evans syndrome received eltrombopag, without response, and MMF with response. All patients are alive. Neutropenia resolved spontaneously in 54% of patients. Two-year probability of unresolved neutropenia was 26% vs 78% vs 100% in patients no LO/LL, LO and LL, respectively (P<10-4).

Summary/Conclusion: LO/LL neutropenia might be associated with bone marrow failure syndromes, immunodeficiency or autoimmunity. Long-term follow-up and investigations are needed in children with LO/LL neutropenia. Genetic typing in specialized reference networks, like EuNet-INNOCHRON or EWOG, might identify diagnostic subgroups of clinical relevance.

P822: A PHASE 1 SINGLE ASCENDING DOSE STUDY OF CAN106, A LONG-ACTING ANTI-C5 COMPLEMENT MONOCLONAL ANTIBODY IN CLINICAL DEVELOPMENT FOR PNH AND OTHER COMPLEMENT-MEDIATED DISEASES

C. M. KHOO 1,* , X. SONG 2 , Q. WU 2 , G. F. COX 3

1 Department of Medicine, National University Hospital, Singapore, Singapore; 2 Clinical Development and Medical Affair, CANbridge Pharmaceuticals Inc., Shanghai, China; 3 Clinical Development and Medical Affair, CANbridge Pharmaceuticals Inc., Cambridge, MA, United States of America

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired genetic disorder caused by clonal mutations in the PIGA gene. Deficiency of PIGA protein renders erythrocytes susceptible to complement-mediated destruction, causing intravascular and extravascular hemolysis, which can lead to anemia, fatigue, thromboembolism, major organ damage, and death. Despite the approval of one anti-C3 and two anti-C5 therapies for PNH, high treatment costs limit market access in many countries. CAN106 is a novel anti-C5 monoclonal antibody designed for optimal pH-dependent binding to C5 and enhanced binding to FcRN to increase intracellular recycling and prolong its half-life.

Aims: This first-in-human Phase 1 study aimed to evaluate the single-dose safety and tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of CAN106.

Methods: This was a randomized, double-blind, placebo-controlled, single-ascending-dose study. We enrolled 31 healthy adult subjects at a single site in Singapore who received CAN106 (mg/kg) or placebo at one of the following 6 doses (mg/kg) and ratios: (0.25, n=1:0; 0.75, n=1:0; 2, n=3:2; 4, n=6:2; 8, n=6:2 or 12, n=6:2). The primary endpoint was the incidence of adverse events overall and by intensity, seriousness, type, and relatedness to study drug. Secondary endpoints were the pharmacokinetic characterization of CAN106, the pharmacodynamic effects on free C5 (target engagement) and CH50 (serum hemolytic activity), and the incidence of anti-drug antibodies. The original study duration of 196 days was shortened to 112 days based upon the observed half-life of CAN106 and the return of CH50 to normal values.

Results: The mean age of subjects was 34 years, and 97% were male. All 31 subjects completed the study. Seven CAN106 subjects experienced 20 treatment-emergent adverse events (TEAEs), and three placebo subjects experienced five TEAEs. Most TEAEs were mild and not related to CAN106, and all resolved without any sequelae. Three CAN106 subjects experienced seven drug-related TEAEs at the two highest doses. Most were mild, none was serious, and all resolved without any sequelae. These events included a moderate infusion-related reaction that resolved upon discontinuing treatment; mild dizziness; and mild elevations in ALT, AST, hemoglobin, hematocrit, and RBC. CAN106 exposure (Cmax and AUC) was dose-proportional, linear, and had low inter-subject variability (<20% CV). The terminal elimination half-life was approximately 32 days. CAN106 led to dose-dependent reductions in free C5 and CH50 within 24 hours. At the 8 and 12 mg/kg doses, all subjects showed >99% reduction in free C5 and >90% inhibition of CH50, with the latter sustained for 2 to 4 weeks. Two subjects in the 4 mg/kg cohort tested positive for anti-drug antibodies at single time points (pre-dose and Day 28).

Summary/Conclusion: CAN106 was safe and well-tolerated at single doses up to 12 mg/kg in healthy adult subjects. The dose-exposure relationship was linear, and the 32-day half-life in healthy subjects was similar to that of ravulizumab, which is dosed every 8 weeks in patients, suggesting the potential for an extended dosing interval. CAN106 led to rapid, dose-dependent reductions in free C5 and CH50, and at the two highest doses, achieved complete and sustained complement blockade (>90% inhibition of CH50) for up to 4 weeks. These promising results support further clinical development of CAN106 in PNH and other complement-mediated diseases.

P823: THE INCIDENCE, OUTCOMES, AND RISK FACTORS OF SECONDARY POOR GRAFT FUNCTION IN HAPLOIDENTICAL HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR ACQUIRED APLASTIC ANEMIA

F. lin 1,* , T. han 1 , Y. zhang 1 , Y. cheng 1 , Z. xu 1 , X. mo 1 , F. wang 1 , C. yan 1 , Y. sun 1 , J. wang 1 , F. tang 1 , W. han 1 , Y. chen 1 , Y. wang 1 , X. zhang 1 , K. liu 1 , X. huang 1 , L. xu 1

1 Peking university institute of hematology, Beijing, China

Background: Secondary poor graft function (sPGF) increases the risk of life-threatening complications after transplantation and is an obstacle to achieving better outcomes after hematopoietic stem cell transplantation (HSCT).

Aims: We aimed to determine the incidence, clinical outcomes, and risk factors of sPGF in haploidentical HSCT (haplo-HSCT) for acquired aplastic anemia (AA) patients.

Methods: We retrospectively reviewed 430 consecutive AA patients receiving haplo-HSCT in Peking University People’s Hospital between January 2006 and December 2020.

Results: We reported the estimated 3-year cumulative incidence of sPGF was 4.64%, while no primary PGF occurred. The median time to sPGF was 121 days (range 30-626 days) after transplantation. To clarify the risk factors for sPGF, 17 sPGF cases and 382 without PGF were further analyzed. Compared to patients without PGF, the 2-year overall survival was significantly poorer for sPGF patients (67.7% vs 90.8%, p =.002). Twelve sPGF patients were alive until the last follow-up, and 7 achieved transfusion independency. The multivariable analyses revealed that a history of refractory cytomegalovirus viremia (CMViremia) (OR=7.038, p=.002) post-transplantation independently increased the risk of sPGF. There was weak evidence that a history of grade 3-4 acute graft-versus-host disease (aGvHD) increased the risk of sPGF (p=.092).

Summary/Conclusion: In conclusion, sPGF can develop in 4.64% of AA patients after haplo-HSCT and significantly decreases survival. The independent hazard elements for sPGF were a history of refractory CMViremia and grade 3-4 aGvHD. We advocated better post-transplantation strategies to balance the risk of immunosuppression and viral reactivation for haplo-HSCT in AA patients.

P824: HEMATOLOGIC FEATURES IN 102 FRENCH PATIENTS DIAGNOSED WITH GERMLINE MUTATIONS OF TELOMERES RELATED GENES AGED 15 YEARS OLD OR MORE

F. Maillet 1,* , J.-E. Galimard 2 , R. Borie 3 , C. Kannengiesser 4 , E. Lainey 5 , R. Peffault De Latour 1 , F. Sicre de Fontbrune 1

1 Hematology Department, Saint Louis Hospital; 2 Statistical Unit, EBMT; 3 Pulmonology Department; 4 Genetics Department, Bichat Hospital; 5 Hematology Department, Robert Debré Hospital, Paris, France

Background: Short telomere syndrome (STS) is a group of genetic disorders characterized by germline mutations in telomeres related genes (TRG) leading to premature telomere shortening. In the recent years, the spectrum of STS diseases has widened and now includes heterogeneous associations of hematologic, pneumologic, hepatic and osteoarticular features, and cancer predisposition. STS clinical landscape remain unknown. We took advantage of a large cohort of patients to describe hematologic and extra-hematologic features of this rare disease.

Aims: The aim of this study was to describe hematologic and extrahematologic features in patients with STS diagnosed at age 15 or more and followed in adult centers.

Methods: All data were obtained from the French National reference Center for Aplastic Anemia. Between 2003 and 2021, we retrospectively included all patients 15 years old or older at diagnosis of STS, with pathogenic germline variant(s) in TRG, and at least one bone marrow assessment (bone marrow aspiration or biopsy). Overall survival (OS) was calculated from date of diagnosis to death or last follow-up using the Kaplan-Meier estimator.

Results: One hundred and two patients were included: 89 (87%) were propositus and 13 were diagnosed after genetic counseling. The median follow-up after diagnosis was 3.6 years. The 4 years OS of the index cases was 63% (95% CI: 49 – 74).

62 (70%) and 6 (46%) patients were male, and median age at diagnosis was 42 years (IQR 28-56) and 27 years (IQR 21-48) for index cases and non-index cases, respectively. Median age at onset of first symptoms was 28 years (IQR 20-49) and 29 years (IQR 18-38), with a median duration from first symptoms to STS diagnosis of 5 years (IQR 2-11) and 6 years (IQR 0-7) for index cases and non-index cases, respectively. In index cases, TRG mutations involved TERT in 43 patients (49%), TERC in 24 patients (28%), RTEL1 in 11 patients (13%), DKC1 in 3 patients (3%), and in 1 patient each for NHP2, CTC1, PARN, TINF2, TERT plus TERC (0.9%). In index cases, mutations were heterozygous in 92%, homozygous in 6%, and X-linked in 2%. Among 68 patients with available telomere evaluation, the median telomere length was 6.2% (IQR 4.8-7.4%), whereas the median telomere length at first percentile was 7.6%. Telomere length was shorter than first percentile in 81.5% of patients. 69% of index cases had at least 1 relative with STS.

Among index cases, 73 (82%) presented an hematological involvement. At diagnosis, the hematological presentation was BMF in 49 patients (70%), MDS in 17 (24%) and AML in 2 (3%), 1 CVID (1.5%), 1 lymphoma (1.5%) and not specified for 3 patients. During follow-up, evolution toward MDS was observed in 2 patients, and toward AML in 4. 14 patients received allo-HSCT (13.7%): 9 for BMF and 5 for MDS.

Extra hematologic features among index cases: 49 patients (55%) had at least one clinical feature of dyskeratosis congenita . Pulmonary involvement was present in 55 patients (62%), of those 93% had diffuse interstitial pneumonia. 12 patients underwent lung transplantation. Hepatic involvement was present in 42 patients (47%), including nodular regenerative hyperplasia (29%), cirrhosis (33%) and portal hypertension (43%). 3 patients underwent liver transplantation. Cancer occurred in 7 patients: 4 solid cancers and 3 lymphomas.

Summary/Conclusion: This study in STS patients allows a better understanding of the spectrum of this disease. Hematological presentation was the main feature, followed by lung and hepatic involvement. A long-term multidisciplinary close follow-up is mandatory in this population of patients.

P825: FACTOR D INHIBITION WITH ORAL BCX9930 MONOTHERAPY LEADS TO SUSTAINED CONTROL OF HEMOLYSIS AND SYMPTOMS OVER 48 WEEKS IN SUBJECTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA NAÏVE TO C5 INHIBITORS

A. McDonald 1,* , J. L. R. Malherbe 2 , A. Kulasekararaj 3 , W. Füreder 4 , M. Griffin 5 , M. Cornpropst 6 , M. K. Farmer 6 , D. Kargl 6 , D. Collins 6 , W. Sheridan 6 , A. Risitano 7

1 Netcare Pretoria East Hospital, Moreleta Park; 2 University of the Free State, Bloemfontein, South Africa; 3 Kings College Hospital NHS Foundation Trust, London, United Kingdom; 4 Medical University of Vienna, Vienna, Austria; 5 Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom; 6 BioCryst Pharmaceuticals, Durham, United States of America; 7 AORN Moscati, Avellino, Italy

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis due to uncontrolled activity of the alternative pathway (AP) of complement. BCX9930 is an oral Factor D inhibitor that targets the rate-limiting enzyme of AP with the potential to prevent both intravascular hemolysis and extravascular hemolysis. We previously reported safety and effectiveness of BCX9930 at 22 weeks in subjects naïve to complement inhibitors (CI; McDonald et al. EHA 2021). Here, we report further long-term safety, tolerability, and effectiveness of BCX9930.

Aims: This analysis evaluated effectiveness of BCX9930 in subjects completing at least 48 weeks of treatment at target dose of 400 mg BID or 500 mg BID. Additionally, safety data are presented for all subjects (n=16, including 6 subjects with an inadequate response to C5 inhibitors) enrolled in an open-label, dose-ranging study ( NCT04330534 ) and extension study ( NCT04702568 ).

Methods: Study BCX9930-101 enrolled subjects with PNH with either no history of C5 inhibitor use or those with an inadequate response to C5 inhibitors (transfusion within 3 months of screening or Hb <10 g/dL). Subjects in each of three multiple dose cohorts received BCX9930 at 50 to 400 mg orally twice daily (BID) titrated to target dose of 400 or 500 mg BID. CI-naïve subjects deriving clinical benefit after 28 days of treatment could receive up to 48 additional weeks of BCX9930 before continuing into an extension study.

Safety and effectiveness outcomes were evaluated monthly. Effectiveness endpoints were change from baseline (CFB) to 48 weeks in hemoglobin (Hb), red blood cell (RBC) transfusions, lactate dehydrogenase (LDH), absolute reticulocyte count (ARC), and % PNH Type II+III RBC clone size relative to PNH white blood cell (WBC) clone size. CFB in fatigue was assessed by Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue Scale (clinically important difference ≥3-point change, higher scores indicate less fatigue).

Results: Ten CI-naïve adult subjects enrolled in BCX9930-101 and 9 completed at least 48 weeks of treatment. Mean (SD) age and time since PNH diagnosis were 29.2 (8.09) years and 2.6 (1.70), mean (SD) number of RBC units transfused in 12 months prior to study entry was 7.6 (8.2), and median (range) treatment exposure at 400 mg or 500 mg BID was 470 (350, 533) days. Mean (SEM) Hb CFB to Week 48 was 3.75 (0.89) g/dL (8.25 [0.60] g/dL to 12.0 [0.85] g/dL), and all 9 subjects were transfusion-free versus 2 of 9 in 12 months prior to study. At Week 48, mean (SEM) ARC CFB was – 57 (21.8) 10 3 /µL (178 [20.7] vs. 121 [15.5] 10 3 /µL), mean (SEM) % PNH RBC/WBC clone size increased from 51.9% (4.4%) to 93% (2.7%), and mean (SEM) LDH CFB was –70.5% (6.18%). Clinically meaningful improvements in FACIT-Fatigue scores were observed over 48 weeks, with mean (SEM) CFB of 5.3 (1.82) points (mean score 40.7 vs 46.0). The most common adverse events in all subjects (n=16) were headache (38%), self-limited rash (25%), and abdominal pain (19%).

Summary/Conclusion: BCX9930 at doses up to 500 mg BID was generally well-tolerated. Notable improvements from baseline to 48 weeks were observed in Hb, LDH, ARC, and fatigue scores, with concomitant increases in PNH RBC clone size. Proximal AP inhibition of Factor D by BCX9930 demonstrated sustained relief of anemia and fatigue, and freedom from transfusions, supporting ongoing pivotal trials of BCX9930 monotherapy for treatment of PNH.

P826: FACTOR D INHIBITION WITH ORAL BCX9930 LEADS TO SUSTAINED CONTROL OF HEMOLYSIS AND SYMPTOMS OVER 48 WEEKS IN SUBJECTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA INADEQUATELY CONTROLLED ON C5 INHIBITORS

A. Kulasekararaj 1,* , W. Füreder 2 , A. McDonald 3 , J. L. R. Malherbe 4 , S. Gandhi 1 , D. Collins 5 , M. Cornpropst 5 , S. Dobo 5 , M. K. Farmer 5 , D. Kargl 5 , W. Sheridan 5 , M. Griffin 6 , A. Risitano 7

1 Kings College Hospital NHS Foundation Trust, London, United Kingdom; 2 Medical University of Vienna, Vienna, Austria; 3 Netcare Pretoria East Hospital, Moreleta Park; 4 University of the Free State, Bloemfontein, South Africa; 5 BioCryst Pharmaceuticals, Durham, United States of America; 6 Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom; 7 AORN Moscati, Avellino, Italy

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis due to uncontrolled activity of the alternative pathway (AP) of complement. BCX9930 is an oral Factor D inhibitor that targets the AP rate-limiting enzyme with the potential to prevent both intravascular hemolysis and extravascular hemolysis. We previously presented safety and effectiveness data for BCX9930 at 11 weeks in subjects with an inadequate response (IR) to C5 inhibitors (C5 INH; Kulasekararaj et al. EHA 2021). Here, we report further long-term safety, tolerability, and effectiveness of BCX9930.

Aims: This analysis evaluated effectiveness of BCX9930 in subjects completing at least 48 weeks of treatment at target dose of 400 mg BID or 500 mg BID. Additionally, safety data are presented for all subjects enrolled (n=16, including 10 subjects naïve to C5 INH) in an open-label, dose-ranging study ( NCT04330534 ) and extension study ( NCT04702568 ).

Methods: Study BCX9930-101 enrolled subjects with PNH with either no history of C5 INH use or those with an IR to C5 INH (defined as having a transfusion within 3 months of screening or Hb <10 g/dL). For subjects with an IR to C5 INH treatment, BCX9930 was added to C5 INH and subjects were treated with 200 mg orally twice daily (BID) escalated to 400 mg BID, or 400 mg BID escalated to 500 mg BID. Subjects deriving clinical benefit could rollover into an extension study after 28 days, and C5 INH could be withdrawn.

Safety and effectiveness outcomes were evaluated monthly. Effectiveness endpoints were change from baseline (CFB) to 48 weeks in hemoglobin (Hb), red blood cell (RBC) transfusions, lactate dehydrogenase (LDH), absolute reticulocyte count (ARC), and % PNH RBCs relative to PNH white blood cells (WBC). CFB in fatigue was assessed by Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue Scale (clinically important difference ≥3-point change, higher scores indicate less fatigue).

Results: Six adult subjects with an IR to C5 INH enrolled in Study BCX9930-101 and rolled over into the open-label extension study, and 4 completed 48 weeks of treatment (3 of 4 on BCX9930 monotherapy at Week 48, mean [range] duration on monotherapy 107 [85, 129] days). Mean (SD) age and time since PNH diagnosis were 31 (9.42) and 7.7 (5.80) years, mean (SD) number of RBC units transfused in 12 months prior to study entry was 5.5 (4.4), and median (range) treatment exposure at 400 mg or 500 mg BID was 413.5 (337, 420) days. Mean (SEM) Hb CFB to Week 48 was 2.69 (0.97) g/dL (8.91 [0.56] g/dL to 11.60 [1.27] g/dL). 3 of 4 subjects were transfusion-free at Week 48 versus 1 of 4 in 12 months prior to study. At Week 48, Mean (SEM) ARC CFB was –63 (17.1) 10 3 /µL (204 [15.5] vs. 141 [19.6] 10 3 /µL), and mean (SEM) % PNH RBC/WBC clone size increased from 59% (4.4%) to 92% (3.8%,). Clinically meaningful improvements in FACIT-Fatigue scores were observed over 48 weeks, with mean (SEM) CFB of 10.7 (6.0) points (mean score 37.3 vs. 47.9). The most common adverse events for all subjects (n=16) were headache (38%), self-limited rash (25%), and abdominal pain (19%).

Summary/Conclusion: BCX9930 at doses up to 500 mg BID was generally well-tolerated. Notable improvements were observed from baseline to 48 weeks in Hb, ARC, and fatigue scores, with concomitant increases in PNH RBC clone size. Proximal AP inhibition of Factor D by BCX9930 in combination with C5 INH (with 3 of 4 subjects transitioning to monotherapy) led to sustained relief of anemia, fatigue, and reduction of transfusion burden, supporting ongoing pivotal trials of oral BCX9930 monotherapy for treatment of PNH.

P827: EFFICACY AND SAFETY OF ROMIPLOSTIM ADDED TO IMMUNOSUPPRESSIVE THERAPY AS A FIRST-LINE TREATMENT IN PATIENTS WITH APLASTIC ANEMIA: A PHASE 2/3 CLINICAL TRIAL

H. Yamazaki 1,* , J. W. Lee 2 , J. H. Jang 3 , M. Sawa 4 , M. Kizaki 5 , Y. Tomiyama 6 , K. Nagafuji 7 , K. Usuki 8 , J.-P. Gau 9 , Y. Morita 10 , J.-L. Tang 11 , H. Chang 12 , M. Noshiro 13 , A. Matsuda 14 , K. Ozawa 15 , K. Mitani 16 , Y. Kanda 15 , S. Nakao 1

1 Kanazawa University Hospital, Ishikawa, Japan; 2 Seoul St. Mary’s Hospital​; 3 Samsung Medical Center​, Seoul, South Korea; 4 Anjo Kosei Hospital, Aichi; 5 Saitama Medical Center, Saitama; 6 Osaka University Hospital, Osaka; 7 Kurume University Hopital, Fukuoka; 8 NTT Medical Center Tokyo, Tokyo, Japan; 9 Taipei Veterans General Hospital​, Taipei, Taiwan; 10 Kindai University Hospital, Osaka, Japan; 11 National Taiwan University Hospital​, Taipei; 12 Chang Gung Medical Foundation- Linkou Branch​, Taoyuan, Taiwan; 13 Kyowa Kirin Co.,Ltd., Tokyo; 14 Saitama Medical University International Medical Center, Saitama; 15 Jichi Medical University Hospital; 16 Dokkyo Medical University Hospital, Tochigi, Japan

Background: Romiplostim, a thrombopoietin (TPO) mimetic protein, has been shown to promote tri-lineage hematopoiesis in patients with acquired aplastic anemia (AA) refractory to immunosuppressive therapy (IST) or eltrombopag; however, its effectiveness in the combination therapy with antithymocyte globulin (ATG) plus cyclosporine (CsA) as a first-line treatment remains unknown.

Aims: To clarify this issue, we conducted a phase 2/3 clinical trial to evaluate the efficacy and safety of romiplostim combined with rabbit ATG plus CsA in patients with AA requiring transfusions who had not been exposed to IST.

Methods: This study was a multi-national, open-label study with romiplostim in IST-naïve adult patients with AA ( NCT03957694 ). 10 µg/kg of romiplostim was started on day 1 of ATG therapy (2.5mg/kg/day for 5 days) and was weekly given for the first 4 weeks. The dose was adjusted from 5 to 20 µg/kg according to the prespecified dose adjustment procedure. Treatment lasted until Week 26. For those who intend to continue treatment, romiplostim administration was extended to Week 52. The primary endpoint was the hematological response rate (HRR) at Week 27 based on the response assessment criteria (Table 1). The secondary endpoints included the HRR at Week 14; the time to the hematological response; the proportion of patients who achieved transfusion independence or who showed a reduction of transfusion requirement among patients who had received a transfusion within 8 weeks prior to the first romiplostim administration. The bone marrow and cytogenetic analyses were performed prior to enrollment and at Week 27 and 53.

Results: Twenty-six patients were screened, of which 17 (9 Japan, 7 Korea, and 1 Taiwan; 5 transfusion dependent non-severe AA, 6 severe AA (SAA) and 6 very SAA) were enrolled in the study. The median age was 44.0 years (range: 25-70). Two patients discontinued romiplostim before Week 27. The HRR (CR or PR) at Week 27 was 76.5% (95% CI: 50.10%, 93.13%), of which 6 (35.3%) achieved CR. The HRR at Week 14 was 41.2% (95% CI: 18.44%, 67.03%). The median day to achieve the first hematological response was 93.0 (range: 63-181). Of the 16 patients who depended on platelet transfusion before romiplostim administration, 14 (87.5%) achieved transfusion independence or showed a reduction of transfusion requirement at Week 27. In addition, of 16 who required erythrocyte transfusion at baseline, 13 (81.3%) achieved transfusion independence or showed a reduction of transfusion requirement at Week 27.

The frequently reported adverse events (AEs) were constipation (41.2%) followed by headache (35.6%). The frequently reported drug-related AEs were headache (12.9%) followed by muscle spasms (9.7%), alanine aminotransferase increased, fibrin D dimer increased, malaise, and pain in extremity (each 6.5%). In bone marrow examination, increases of reticuline grade (grade 1 to 2) were observed in 3 patients at Week 27 and 1 patient at Week 53. No chromosomal abnormality or transformation into MDS and/or AML was observed.

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Summary/Conclusion: This is the first clinical trial of romiplostim combined with rabbit ATG plus CsA in patients with acquired AA requiring transfusions who were previously untreated with IST. This regimen produced higher HRR at Week 27 (76.5%) than those of historical control received rabbit ATG plus CsA (approximately 50% at 6 months), with manageable AEs, and could therefore serve as a new first-line treatment option in patients with AA.

P828: NORMALIZATION OF HEMATOLOGIC AND HEALTH-RELATED QUALITY OF LIFE MARKERS IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA TREATED WITH PEGCETACOPLAN AND BASELINE HEMOGLOBIN AT OR ABOVE 10 G/DL

J. Panse 1,* , N. Daguindau 2 , S. Okuyama 3 , R. Peffault de Latour 4 , P. Schafhausen 5 , N. Straetmans 6 , M. Al-Adhami 7 , T. Ajayi 7 , E. Persson 8 , M. Yeh 7 , R. Wong 9

1 Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, University Hospital RWTH Aachen, Aachen, Germany; 2 Hematology Department, CH Annecy Genevois, Annecy, France; 3 Hematology/Oncology Division, Denver Health Medical Center, Denver, United States of America; 4 French Reference Center for Aplastic Anemia and Paroxysmal Noctural Hemoglobinuria, Assistance Publique - Hôpitaux de Paris; Université de Paris, Paris, France; 5 Medical Clinic and Polyclinic II - Hematology, Oncology, University Medical Center of Hamburg-Eppendorf, Hamburg, Germany; 6 Department of Hematology, Cliniques Universitaires Saint-Luc, Woluwe-Saint-Lambert, Belgium; 7 Apellis Pharmaceuticals, Inc., Waltham, United States of America; 8 Swedish Orphan Biovitrum AB, Stockholm, Sweden; 9 Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare life-threatening disease characterized by chronic complement-mediated hemolysis and thrombosis. Patients living with PNH can experience anemia with associated fatigue, dyspnea, and require blood cell transfusions which can negatively impact quality of life (QoL). Pegcetacoplan (PEG), is a C3 complement-inhibitor approved by the FDA/EMA for adults with PNH.

Aims: This post hoc analysis evaluated hemoglobin (Hb), lactate dehydrogenase (LDH), absolute reticulocyte count (ARC), and Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue normalization rates and safety after PEG treatment in a subgroup of patients with PNH and baseline Hb levels ≥10.0g/dL from the PADDOCK ( NCT02588833 ), PEGASUS ( NCT03500549 ), and PRINCE ( NCT04085601 ) studies.

Methods: PADDOCK evaluated PEG treatment (270-360mg/day subcutaneously [sc]) in complement-inhibitor naïve patients. PEGASUS enrolled patients with Hb levels <10.5g/dL at screening despite stable eculizumab (ECU) treatment (≥3 months). PEGASUS patients were randomized 1:1 to ECU or PEG (1080mg sc 2x weekly) during the randomized controlled period (RCP) through Week 16. Patients who received PEG during the RCP continued with PEG monotherapy (PEG-to-PEG) and ECU patients switched to PEG monotherapy (ECU-to-PEG) during the open-label period (OLP) through Week 48. PRINCE compared PEG treatment (1080mg sc 2x weekly) in complement-inhibitor naïve patients with PNH and Hb levels below the lower limits of normal (males: ≤13.6g/dL; females: ≤12.0g/dL) to control treatment (CTRL; excluding complement-inhibitors i.e., ECU/ravulizumab). The post hoc analysis included adult patients with PNH with baseline Hb levels ≥10.0g/dL and no transfusions within 14 days of the baseline measurement. Hb, LDH, and ARC normalization, as well as FACIT-Fatigue normalization (defined as increase to population norm) was evaluated after 16 weeks of PEG monotherapy for all three studies. Patients who withdrew or were lost to follow-up without providing efficacy data at the specified timepoints were classified as non-responders. Safety endpoints included incidences of adverse events (AEs).

Results: Overall, 22 patients were included in the post hoc analysis: 5 PADDOCK, 9 PEGASUS, and 8 PRINCE patients. After 16 weeks of pegcetacoplan treatment, patients in all three studies showed improvements in Hb normalization, LDH normalization, and ARC normalization (Table 1). Additionally, improvements in FACIT-Fatigue normalization were demonstrated in PADDOCK, PRINCE, and PEGASUS (PEG-to-PEG) patients (Table 1). No incidences of serious AEs were reported in PADDOCK and PRINCE subgroups. One PEGASUS patient had a serious AE of breakthrough hemolysis (severe, unrelated to PEG), following an upper respiratory infection which resolved and did not lead to study discontinuation. Incidences of injection site reactions and infections and infestations occurred at similar rates between subgroups in all three studies (Table 1). No thrombotic events occurred in this patient population.

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Summary/Conclusion: While this patient population had near normal Hb at baseline (median: 10.4g/dL), they also had elevated ARC and LDH prior to PEG therapy, suggesting ongoing hemolysis which was normalized in most patients after PEG treatment initiation. Overall, these results suggest PEG can be efficacious long-term in patients with less severe anemia, regardless of prior complement-inhibitor treatment, resulting in further clinical improvements in markers of hemolysis and QoL. The safety profile of PEG was consistent with results from previous studies.

P829: A PHASE III RANDOMIZED CLINICAL TRIAL COMPARING SB12 (PROPOSED ECULIZUMAB BIOSIMILAR) WITH REFERENCE ECULIZUMAB IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

J. H. Jang 1,* , R. Demichelis Gomez 2 , H. Bumbea 3 , L. Nogaieva 4 , L. L. L. Wong 5 , S. M. Lim 6 , J. Park 7 , Y. Kim 8 , S. Cho 7

1 Hematology-Oncology, Samsung Medical Center, Seoul, South Korea; 2 Hematology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico; 3 Hematology, Bucharest Emergency University Hospital, Bucharest, Romania; 4 Regional Treatment-Diagnostic Hematology Center, Communal Nonprofit Enterprise Cherkasy Regional Oncology Dispensary of Cherkasy Oblast Council, Cherkasy, Ukraine; 5 Hematology Unit, Hospital Queen Elizabeth, Kota Kinabalu; 6 Department of Medicine, Hospital Sultanah Aminah, Johor Bahru, Malaysia; 7 Clinical Development; 8 Biometrics, Samsung Bioepis, Incheon, South Korea

Background: SB12, a proposed eculizumab biosimilar, is a humanized monoclonal antibody that blocks complement C5 cleavage, thereby inhibits terminal complement-mediated intravascular hemolysis.

Aims: The randomized, double-blind, multicenter, cross-over study was aimed to demonstrate comparable clinical efficacy by evaluating the lactate dehydrogenase (LDH), safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of SB12 and reference eculizumab (ECU) in paroxysmal nocturnal hemoglobinuria (PNH) patients ( NCT04058158 ).

Methods: A total of 50 patients aged ≥ 18 years with a confirmed diagnosis of PNH and ≥ 1.5 upper limit of normal range (ULN) of LDH without previous exposure to a complement inhibitor were included. All patients provided written informed consents and were randomized (1:1) to treatment sequence I (TS1: SB12 to ECU, n=25) or II (TS2: ECU to SB12, n=25). Patient received 600 mg of SB12 (TS1) or ECU (TS2) intravenously every week for first 4 weeks (initial phase) and 900 mg for the fifth week, followed by 900 mg every 2 weeks thereafter (maintenance phase). The treatment was switched to ECU (TS1) or SB12 (TS2) at Week 26, and switched treatment was provided until Week 50.

Primary endpoints were LDH level at Week 26 and time-adjusted area of under the effect curve (AUEC) of LDH from Week 14 to Week 26 and Week 40 to Week 52. Equivalence was declared for LDH level at Week 26 if the two-sided 95% confidence interval (CI) of the mean difference in between SB12 and ECU lied within the pre-defined equivalence margin of [−1.2 × ULN, 1.2 × ULN] = [−337.2, 337.2], where ULN = 281 U/L. Equivalence was declared for time-adjusted AUEC of LDH if the two-sided 90% CI of the ratio of geometric means between SB12 and ECU lied within the pre-defined equivalence margin of [0.77, 1.29]. Secondary endpoints were LDH profile over time and number of units of packed red blood cells (pRBCs) transfused throughout the study period, safety, PK, PD, and immunogenicity.

Results: Of the 50 randomized patients, 49 patients received the study treatment and 46 patients completed the study. Baseline demographic and disease characteristic were comparable between the two treatment sequences. The 95% CI of mean difference in LDH level at Week 26 between SB12 and ECU (SB12 − ECU: 34.48, 95% CI [−47.66, 116.62]) lied within the pre-defined equivalence margin. The 90% CI of ratio of time-adjusted AUEC of LDH between SB12 and ECU (SB12/ECU: 1.08, 90% CI [0.95, 1.23]) lied within the pre-defined equivalence margin. The overall LDH profile during the study period was also comparable. The mean number of units of pRBCs transfused during Period 1 (TS1: 1.1 U; TS2: 0.9 U, respectively) and Period 2 (TS1: 1.1 U; TS2: 1.0 U, respectively) was comparable.

Treatment-emergent adverse events (TEAEs) was reported in 72.3% for SB12 and 68.1% in ECU. Three patients in SB12 and 2 patients in ECU had serious TEAEs. None of three serious TEAEs reported in SB12 were related to the treatment. Two of three TEAEs (cellulitis; infusion site hypersensitivity) reported in ECU were treatment-related, and 1 death due to major adverse vascular event (portal vein thrombosis) not related to the treatment was reported in ECU. The mean eculizumab serum trough concentrations and the mean terminal complement activities in both treatment sequences were comparable. No patient developed anti-drug antibodies during the study period.

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Summary/Conclusion: SB12 showed clinical equivalence to ECU measured by LDH. SB12 and ECU were comparable in terms of safety, PK, PD, and immunogenicity.

P830: CROVALIMAB MAINTAINS CLINICAL BENEFIT OVER LONG-TERM TREATMENT IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: RESULTS FROM THE COMPOSER TRIAL OPEN-LABEL EXTENSION

A. Röth 1,* , M. Egyed 2 , S. Ichikawa 3 , Y. Ito 4 , J. S. Kim 5 , Z. Nagy 6 , N. Obara 7 , J. Panse 8 , H. Schrezenmeier 9 , S. Sica 10 , J. Soret 11 , K. Usuki 12 , S.-S. Yoon 13 , K. Benkali 14 , M. Buri 15 , P. Lundberg 15 , H. Patel 16 , K. Shinomiya 17 , S. Sreckovic 15 , J.-I. Nishimura 18

1 Department of Hematology and Stem Cell Transplantation, West German Cancer Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2 Kaposi Mor Oktato Korhaz, Kaposvar, Hungary; 3 Tohoku University Hospital, Miyagi; 4 First Department of Internal Medicine, Hematology Division, Tokyo Medical University, Tokyo, Japan; 5 Yonsei University College of Medicine, Severance Hospital, Seoul, South Korea; 6 Semmelweis University, Budapest, Hungary; 7 Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan; 8 University Hospital RWTH Aachen, Aachen; 9 Institute of Transfusion Medicine, University Hospital Ulm, Ulm, Germany; 10 Fondazione Policlinico Universitario A. Gemelli-Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy; 11 Centre d’Investigation Clinique, Hôpital Saint-Louis, Paris, France; 12 NTT Medical Center Tokyo, Tokyo, Japan; 13 Department of Internal Medicine, Seoul National University Hospital, Seoul, South Korea; 14 Certara, Inc., Paris, France; 15 F. Hoffmann-La Roche, Ltd., Basel, Switzerland; 16 Genentech, Inc., South San Francisco, CA, United States of America; 17 Chugai Pharmaceutical Co., Tokyo; 18 Graduate School of Medicine, Osaka University, Osaka, Japan

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a potentially life-threatening acquired hematopoietic stem-cell disorder, resulting in hematuria, anemia, and acquired thrombophilia. In the Phase I/II COMPOSER trial ( NCT03157635 ), patients (pts) with PNH who were treatment-naive or switched from eculizumab were treated with crovalimab, a novel anti-C5 monoclonal antibody designed with Sequential Monoclonal Antibody Recycling Technology (Röth et al. Blood 2020).

Aims: To report the long-term efficacy, safety, pharmacokinetic (PK) and pharmacodynamic (PD) outcomes in pts with PNH treated with crovalimab in the open-label extension (OLE) period of the COMPOSER trial.

Methods: COMPOSER consists of four sequential parts followed by an OLE period. Pts with PNH who were treatment-naive or switched from eculizumab in Parts 2, 3, and 4, who completed the primary treatment period through Week 20 and derived benefit from crovalimab treatment, were eligible to enter the OLE (Röth et al. EHA 2019). Long-term efficacy, safety, PK and PD data are presented for the OLE. Efficacy endpoints include change in lactate dehydrogenase (LDH), transfusion avoidance and hemoglobin stabilization (both assessed in 24-week intervals), and breakthrough hemolysis (BTH). PK and PD endpoints include assessment of serum concentrations of crovalimab, complement activity by liposome immunoassay (LIA), and free C5 over time.

Results: Overall, 43 of 44 pts entered the OLE and at the clinical cutoff (Nov 1, 2021), 38 pts were ongoing.

Mean normalized LDH was maintained at ≤1.5 × upper limit of normal (ULN) during the OLE, and 80% to 100% of evaluable pts achieved LDH ≤1.5 × ULN at each visit (Figure). Transfusion avoidance was achieved in 83% to 92% of pts over each 24-week interval in the OLE. Similarly, hemoglobin stabilization remained relatively stable during the OLE, with 79% to 88% of pts achieving hemoglobin stabilization across 24-week intervals. There was a total of five BTH events during the OLE across all pts (none leading to withdrawal) for an overall BTH rate of 0.05 events per pt-year (95% confidence interval: 0.01, 0.11).

The incidence of adverse events (AEs) was reported cumulatively from baseline. Median treatment duration (range) for treatment-naive and switched pts on the study was 3.4 years (0.9-4.4) and 2.9 years (0.4-3.9), respectively. Any grade AEs occurred in 95% (42/44) of pts; none led to withdrawal from treatment. Severe AEs occurred in 17% (3/18) of treatment-naive pts and 19% (5/26) of switched pts. Serious AEs occurred in 32% (14/44) of pts (33% [6/18] in treatment-naive and 31% [8/26] in switched pts), of which 5% (2/44) were assessed as related to study treatment by the investigator. No meningococcal infections or deaths were reported.

Crovalimab concentrations were stable and within the expected range over time. Terminal complement activity by LIA was near the lower limit of quantification (10 U/mL) and free C5 levels were maintained at low levels (<0.001 g/L) in most pts. PK/PD relationships between free C5 or LIA with crovalimab confirmed that crovalimab levels of ≈100 μg/mL resulted in complete inhibition of terminal complement activity.

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Summary/Conclusion: Treatment-naive and switched pts with PNH treated with crovalimab in the COMPOSER trial maintained disease control over long-term follow-up. No new safety signals were identified, and complete terminal complement inhibition was maintained during the OLE. These data support the continued clinical development of crovalimab in PNH.

P831: PATIENT-REPORTED OUTCOMES IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA TREATED WITH CROVALIMAB: RESULTS FROM THE COMPOSER TRIAL

A. Röth 1,* , J. Panse 2 , B. Gentile 3 , M. Buri 4 , H. Patel 3 , Z. Nagy 5

1 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen; 2 University Hospital RWTH Aachen, Aachen, Germany; 3 Genentech, Inc., South San Francisco, CA, United States of America; 4 F. Hoffmann-La Roche Ltd., Basel, Switzerland; 5 Semmelweis University, Budapest, Hungary

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a potentially life-threatening acquired hematopoietic stem cell disorder, resulting in anemia, hemoglobinuria, and acquired thrombophilia. Patients with PNH can experience a variety of disease-related symptoms, particularly fatigue, that can negatively impact their day-to-day functioning and health-related quality of life. In the Phase I/II COMPOSER trial ( NCT03157635 ), patients with PNH who were treatment-naive or switched from eculizumab were treated with crovalimab, a novel anti-C5 monoclonal antibody designed with Sequential Monoclonal Antibody Recycling Technology (Röth et al. Blood 2020).

Aims: To explore the efficacy of crovalimab from the perspective of patients with PNH using patient-reported outcome (PRO) tools to assess fatigue, functioning, and global health status (GHS)/quality of life (QoL).

Methods: COMPOSER included treatment-naive (Parts 2 and 4 Arm A [4A]) and switched (Parts 3 and 4 Arm B [4B]) patients who received different crovalimab dosing regimens for a 20-week treatment period (Figure). Paper PROs were completed at baseline and at scheduled visits during the treatment period (up to Week 10 for Part 2 and Week 20 for Parts 3 and 4). Fatigue was assessed with the Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue scale. Additional symptoms, functioning, and GHS/QoL were assessed with the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30).

Results: The PRO completion rate at baseline was 100% of evaluable patients for Parts 2 (n=10), 3 (n=19), and 4A (n=8), and 86% for 4B (n=7). Completion rates remained ≥86% for all follow-up visits.

Mean FACIT-Fatigue scores at baseline among treatment-naive patients in Parts 2 (29.7, standard deviation [SD]=13.7) and 4A (33.9, SD=8.7) were below or within the range for severe fatigue (30-34; Eek et al. J Patient Rep Outcomes 2021). Mean scores improved while on treatment with crovalimab, from baseline to Week 10 in Part 2 patients (8.8, SD=10.2) and to Week 20 in Part 4A patients (6.0, SD=6.1), exceeding the threshold for clinically meaningful improvement (5 points; Cella et al. ASH 2021). Switched patients in Parts 3 and 4B had less fatigue on average at baseline (37.3, SD=12.6; 37.0, SD=9.9, respectively) compared with treatment-naive patients, and showed largely minor changes at each follow-up. Similar results were observed for the EORTC QLQ-C30 Fatigue scale. Part 2 and Part 4A patients by Week 10 and 20, respectively, had mean fatigue severity levels comparable to (FACIT-Fatigue) or lower than (EORTC QLQ-C30 Fatigue) mean baseline values in switched patients.

Mean EORTC QLQ-C30 scores in all functioning domains (i.e., physical, role, social, emotional, and cognitive) improved from baseline to Week 10 in Part 2 patients. Improvements in physical, role, and social functioning only were observed in Part 4A patients at all scheduled follow-up visits. Patients in both treatment-naive cohorts exhibited improvements in GHS/QoL. Switched patients, on average, exhibited limited change from baseline in EORTC QLQ-C30 scores, with mean improvements only observed for emotional functioning in Part 4B patients.

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Summary/Conclusion: Treatment-naive patients experienced improvements from baseline in fatigue, functioning, and GHS/QoL whereas switched patients maintained baseline levels. These data provide supportive evidence for the treatment efficacy of crovalimab from the patient perspective.

P832: CLINICAL OUTCOMES AND IMMUNE RESPONSES TO SARS-COV-2 VACCINATION IN PATIENTS WITH SEVERE APLASTIC ANEMIA

R. Rajput 1,* , X. Ma 2 , K. L. Boswell 3 , M. Gaudinski 3 , E. M. Groarke 1 , J. Lotter 1 , O. Rios 1 , I. Darden 1 , C. O. Wu 2 , R. A. Koup 3 , N. S. Young 1 , B. Patel 1

1 Hematology Branch, National Heart Lung and Blood Institute; 2 Office of Biostatistics Research; 3 Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland, United States of America

Background: Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 has led to significant morbidity and mortality in patients with hematological disease. Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder that presents with pancytopenia and a hypocellular marrow due to immune-mediated destruction of hematopoietic stem cells (HSC). Patients are at high risk for infection due to neutropenia and treatment with immunosuppressive therapy (IST). Acute COVID-19 infection has been reported to cause worsening underlying marrow failure and death in unvaccinated SAA patients. Patients with SAA are often not vaccinated to protect against routine viruses due to concerns of ineffective protective specific antibody response to viral antigens and potential pathogenic global immune system activation. To date, the clinical impact and efficacy of COVID-19 vaccination in SAA has not been described.

Aims: Assess the impact of SARS-CoV-2 vaccination on SAA disease status and to determine the humoral and cellular response to vaccination.

Methods: SAA treated with IST including horse antithymocyte globulin (h-ATG), cyclosporine (CSA), and eltrombopag (EPAG) at the National Institute of Health with confirmation of SARS-CoV-2 vaccination between January and November 2021 were included. Current SAA disease status at the time of vaccination was recorded as complete response (CR), partial response (PR) and non-response (NR). Univariate analysis was performed using the Student paired t-test to compare available blood counts. Peripheral mononuclear blood cells (n=27) and serology (n=12) after completion of vaccination series were analyzed to assess cellular and humoral response, respectively.

Results: Blood samples from fifty patients with SAA with median age of 42 years (9-78), 29 females, and 21 males, were studied. At time of vaccination, 15 patients (30%) were receiving CSA. Forty-seven of 50 (94%) of patients did not have any changes in disease status after vaccination. Among 40 patients with available data 3 months prior to and after completion of vaccination series, there was no significant difference in HGB (p=0.52) PLT (p=0.67), ANC (p=0.98), and ALC (p=0.42; Figure 1A-C). Relapse after vaccination, defined as progressive and substantial decline in blood counts, was noted in 3 (6%) cases. All 3 relapsed patients were deemed weak PR at time of vaccination (PLT < 50 K/μL) and were 6 months, 3 years, and 4 years from initial IST. Two of the 3 patients received an initial Pfizer inoculation and did not receive the second dose because of declining blood counts. Relapse occurred 4 weeks after completion of a full Moderna series in the third patient, who had demonstrated a decline in counts 6 months prior to vaccination. Vaccinated SAA patients all had adequate antibody production to the SARS-CoV-2 spike protein. They also exhibited a CD4+ Th1 dominant response, similar to healthy controls, even when receiving CSA at time of vaccination (Figure D). CD8+ responses to vaccination were observed, but patients with SAA appear to have qualitatively blunted responses compared to controls.

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Summary/Conclusion: SARS-CoV-2 vaccination in SAA may result in transient decline in blood counts but true relapse is rare. Relapse may be more likely in patients whose blood counts are declining prior to vaccination. Appropriate humoral and cellular responses are seen in patients with SAA. A risk versus benefit assessment to vaccinate against SARS-CoV-2 in SAA may be warranted in patients with weak partial response to initial therapy or whom have clinical or laboratory evidence suggestive of active disease.

P833: CATEGORIZING HEMATOLOGICAL RESPONSE TO PEGCETACOPLAN IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA: A POST HOC ANALYSIS OF THE PHASE 3 PRINCE STUDY DATA

A. Risitano 1,* , R. Wong 2 , M. Al-Adhami 3 , J. Savage 3 , R. Horneff 4 , R. Peffault de Latour 5

1 Hematology and BMT Unit, AORN, San Giuseppe Moscati, Avellino, Italy; 2 Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 3 Apellis Pharmaceuticals, Inc., Waltham, United States of America; 4 Swedish Orphan Biovitrum AB, Stockholm, Sweden; 5 French Reference Center for Aplastic Anemia and Paroxysmal Noctural Hemoglobinuria, Assistance Publique - Hôpitaux de Paris; Université de Paris, Paris, France

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare complement-mediated hematological disorder characterized by anemia, hemolysis, and bone marrow failure. Pegcetacoplan (PEG), an FDA/EMA-approved targeted C3 therapy for the treatment of PNH, can control intravascular and prevent extravascular hemolysis. A prior post hoc analysis of PEGASUS reported a higher proportion of patients presenting good or better hematologic responses to PEG versus eculizumab (Risitano A, et al., Blood , 2021;138 [Supplement 1]: 1104). The PRINCE study demonstrated efficacy/safety of PEG compared to control treatment (CTRL; supportive treatment of PNH without complement inhibitors) in complement-inhibitor naïve patients with PNH.

Aims: This post hoc analysis categorized the hematologic response to PEG and CTRL at baseline and Week 26 in the PRINCE Trial (phase 3, multicenter, randomized, open-label, controlled trial [ NCT04085601 ]).

Methods: Adult complement-inhibitor naïve (no complement-inhibitor treatment within 3 months prior to screening) patients with PNH who had low hemoglobin (Hb) levels and high lactate dehydrogenase levels were included. Patients were randomized 2:1 to receive PEG or CTRL through Week 26. Hematologic response to PEG and CTRL was categorized as complete, good, partial, or minor at baseline and at Week 26 using the number of packed red blood cells transfusions needed in the last 6 months and Hb level at the specified timepoints (revised categorization criteria per Debureaux PE, et al. Bone Marrow Transplant . 2021; 56(10):2600-2602). Hematologic response categorization was performed according to the following criteria: Complete response: no transfusions, no anemia (≥12 g/dL); Good response: no transfusions, but with chronic mild anemia (10-12 g/dL); Partial response: no/occasional transfusions (≤2 units/6 months) and chronic moderate anemia (8-10 g/dL); Minor response: regular transfusions (≥3 units/6 months) and chronic moderate/severe anemia (<10 g/dL), or no/occasional transfusions (≤2 units/6 months) and chronic severe anemia (<8 g/dL). Any patients with missing data at Week 26 were evaluated using the most recent value within the 6 weeks prior. Statistical tests assessed significance for responses of ‘Good’ or ‘Complete’, collectively (Fisher’s Exact Test between PEG and CTRL groups at baseline; McNemar’s test within PEG group at baseline vs. Week 26).

Results: In this study, 53 patients were randomized to PEG (n=35) or CTRL (n=18). At baseline, most patients had a partial or minor response to supportive therapy prior to randomization, with only 5.7% of patients in the PEG group and 0.0% in the CTRL group presenting a Good or Complete hematologic response (p= 0.5428). Hematologic response categorization for patients in the PEG group are shown for baseline and Week 26 (Figure). At Week 26, 80.0% of patients in the PEG group exhibited a Good or Complete response. The proportion of patients in the PEG group who demonstrated a Good or Complete response was significantly higher at Week 26 as compared to baseline (p<0.0001). One patient treated with PEG had missing data at Week 26 due to being lost to follow-up and was not evaluated.

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Summary/Conclusion: This post hoc analysis of PRINCE trial data revealed that a substantial proportion of patients achieved good or complete hematologic response to PEG after 26 weeks. These findings further support the positive efficacy of PEG on hematologic parameters and provide evidence for the role of proximal complement inhibition on hematological and clinical improvements for patients with PNH.

P834: REAL-WORLD EVIDENCE OF SAFETY AND EFFECTIVENESS OF ECULIZUMAB AND SWITCH TO RAVULIZUMAB IN A SWISS PATIENT POPULATION WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

A. Rovó 1,* , L. Simeon 2 , M. Gavillet 3 , K. Samii 4 , N. Cantoni 5 , D. Heim 6 , G. Stüssi 7 , M. Bissig 8

1 Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, Bern; 2 Department of Hematology and Central Hematology Laboratory, Cantonal Hospital Lucerne, Luzern; 3 Service and Central Laboratory of Hematology, Department of Oncology and Department of Laboratory Medicine and Pathology, Lausanne University Hospital (CHUV), Lausanne; 4 Hematology Division, Department of Oncology, Geneva University Hospitals, Geneva; 5 Division of Oncology, Hematology and Transfusion Medicine, Kantonsspital Aarau, Aarau; 6 Department of Hematology, University Hospital Basel, Basel; 7 Clinic of Hematology, Oncology Institute of Southern Switzerland, Bellinzona; 8 Department of Medical Oncology and Hematology, University Hospital of Zurich, Zurich, Switzerland

Background: Eculizumab (ECU) a complement component 5 (C5) inhibitor, is the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH). Ravulizumab (RAV) is engineered from ECU with the main advantage of having an extended 8-week (vs 2-week with ECU) dosing interval, which decreases treatment burden markedly. Hence, RAV is expected to become the new standard of care to treat PNH. In Switzerland, RAV was approved for PNH treatment in 2020.

Aims: The objective of this study was to evaluate data from the PNH Swiss Soliris and Ultomiris Reimbursement Registry (SSURR) to assess key parameters for safety and effectiveness of ECU and to assess the adoption of RAV for PNH treatment by physicians under real-world conditions.

Methods: The SSURR was designed in 2012 as a prospective, longitudinal, multi-center registry study in Switzerland. Patients were recruited and followed in 9 centers. Health-related data were collected at the first consultation (baseline) and during follow-up visits. In this report, results are based on data collected from 23 February 2012 to 30 September 2021. Data were summarized using descriptive analysis. Ethics approval was obtained. All participants provided written informed consent.

Results: In total, 56 patients with a median age of 51 (range 20-84) years were enrolled in the Registry, 52% were female. For 42 patients, the 12-month follow-up (FU) was available: 39 patients on ECU and 3 on RAV (switched from ECU). Median FU was 27 (range 12-108) months. Hemoglobin (Hb) mean was 63 ± 47 g/L at baseline (n=41), and Hb mean was 105 ± 25 g/L (n=26) at the last visit. The lactate dehydrogenase (LDH) decreased from a mean 1457 ± 706 U/L (baseline, n=42) to 403 ± 212 U/L after 12 months FU (n=40; 65% of patients < 1.5 × upper limit of normal [ULN]), and was for 88% of the patients < 1.5 × ULN at their last visit (n=24). Thrombosis events (TE) were reported in 33% of patients prior to treatment start (n=42) and 3 (7%) had thrombosis during FU of which 2 patients had no prior history of TE. Within 12 months prior to treatment start, 21 patients (50%) received one or more red blood cell (RBC) transfusions. At 12 months of FU, 91% (n=42) were RBC transfusion-free. RBC transfusions remained reduced (82%; n=27 at last visit). After 12 months FU, 19% (n=8) of the patients had withdrawn from the registry due to allogenic bone marrow transplantation, low PNH clone size, transformation to hypoplastic MDS, and 5 non-disease related reasons.

Eight patients from the 56 enrolled received RAV as initial treatment. They were all C5-inhibitor treatment-naïve with a mean LDH level of 1481 ± 1432 U/L at inclusion, which decreased to 230 ± 62 U/L at the 6-month visit (n=4; all below 1.5 × ULN). Of the 56 enrolled patients with PNH, 43% were switched from ECU to RAV. These patients (58% female) had a median age of 55 (range 26-85) years and 75% of them had LDH < 1.5 × ULN at their last LDH measurement on ECU before RAV administration. There were 7 patients with a 6-month FU after switching at which 6 of them had a LDH below 1.5 × ULN.

Summary/Conclusion: The current analysis provides real world evidence for the safety and effectiveness of ECU in the Swiss PNH patient population, demonstrated by pronounced reduction in LDH, low need for RBC transfusion and stable hemoglobin as well as reduced occurrence of thrombosis. The fast adoption of RAV demonstrates RAV may likely become the new standard of care for patients with PNH. In time, more data will become available for safety and effectiveness of RAV under real-world conditions.

P835: GENETIC BACKGROUND AND CLINICAL CHARACTERISTICS OF CONGENITAL NEUTROPENIAS IN ISRAEL

O. Steinberg-Shemer 1,2,3,* , L. Yeshareem 4 , S. Noy-Lotan 3 , O. Dgany 3 , T. Krasnov 3 , G. Berger Pinto 1 , B. Bielorai 2,5 , A. A. Kuperman 6,7 , R. Laor 8 , N. Mandel-Shorer 9,10 , A. Ben Barak 9 , C. Levin 10,11 , A. Mahdi 12 , H. Miskin 12 , S. Revel-Vilk 13,14 , D. Levin 15 , M. Benish 15 , T. Zuckerman 10,16 , O. Wolach 2,17 , I. Pazgal 2,18 , O. Gilad 1,2 , T. Goldberg 1 , J. Yacobovich 1,2 , S. Izraeli 1,2 , H. Tamary 1,2,3

1 Hematology-Oncology, Schneider Children’s Medical Center of Israel, Petach Tikva; 2 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; 3 Pediatric Hematology Laboratory, Felsenstein Medical Research Center; 4 Pediatrics A, Schneider Children’s Medical Center of Israel, Petach Tikva; 5 Pediatric Hematology-Oncology, Sheba Medical Center, Tel Hashomer; 6 Blood Coagulation Service and Pediatric Hematology Clinic, Galilee Medical Center, Nahariya; 7 Azrieli Faculty of Medicine, Bar-Ilan University, Safed; 8 Hematology Service, Bnei Zion Medical Center; 9 Department of Pediatric Hematology-Oncology, Ruth Rappaport Children’s Hospital, Rambam Healthcare Campus; 10 Rappaport Faculty of Medicine, Technion-Institute of Technology, Haifa; 11 Pediatric Hematology Unit and Research Laboratory, Emek Medical Center, Afula; 12 Pediatric Hematology, Soroka University Medical Center, Ben-Gurion University, Beer Sheva; 13 Pediatric Hematology/Oncology Unit, Shaare Zedek Medical Center; 14 Faculty of Medicine, Hebrew University, Jerusalem; 15 Pediatric Hemato-Oncology, Tel Aviv Medical Center, Tel Aviv; 16 Hematology and Bone Marrow Transplantation Institute, Rambam Healthcare Campus, Haifa; 17 Institute of Hematology, Davidoff Cancer Center, Rabin Medical Center; 18 Comprehensive Center of Thalassemia, Hemoglobinopathies & Rare Anemias, Institute of Hematology, Beilinson Hospital, Rabin Medical Center, Petach Tikva, Israel

Background: Congenital neutropenias comprise a heterogenous group of disorders causing low neutrophil counts with a variable clinical presentation. Patients may suffer from severe infections and have a high risk of myeloid transformation. To date, over 30 genes are known to cause congenital neutropenias, with a diverse relative frequency in different ethnicities. Understanding the spectrum of genetic causes of neutropenia in ethnically diverse populations and the related clinical presentation may direct the molecular work-up and clinical care. Israel is enriched with multiple ethnicities and high consanguinity.

Aims: To evaluate the clinical and genetic spectrum of patients with congenital neutropenias in Israel.

Methods: All patients diagnosed with congenital neutropenia from the Israeli Inherited Bone Marrow Failure registry were included. Data were extracted from the registry and patients’ charts. Sanger sequencing was first performed for ELANE or G6PC3, followed by less commonly mutated genes. In recent years, patients with wild-type ELANE/G6PC3 were referred for targeted next generation sequencing, followed by whole exome sequencing.

Results: The cohort encompasses 66 patients, from 50 families, including 7 with cyclic neutropenia. Fifty patients (76%) were genetically diagnosed; 22 (33% of the cohort) had mutations in ELANE , 9 (13.6%) had homozygous G6PC3 mutations, 7 (10.6%) SBDS mutations, 6 (9%) patients had SRP54 mutations, two each had heterozygous GFI1 and homozygous JAGN1 mutations and one each had TAZ and SLC37A1 mutations. All patients with G6PC3 and GFI1 mutations were of consanguineous Muslim Arab origin. Extra-hematopoietic manifestations were common among patients with mutations in G6PC3 . Most patients (82%) presented with severe infections and this was also the most common cause of death. Eight patients (12%) developed myeloid transformation; in 6 of those a somatic molecular work-up was performed, and truncating mutations in the granulocyte-colony stimulating factor were detected in 4. Eighteen (27%) patients underwent hematopoietic stem cell transplantation, most often due to insufficient response to treatment with granulocyte-colony stimulating factor.

Summary/Conclusion: The prevalence of genes causing congenital neutropenias in Israel is unique, as it includes a relatively high frequency of patients with mutations in G6PC3 and an absence of patients with HAX1 mutations, thus directing the genetic work-up and the clinical care. Similar to other registries, a molecular diagnosis was not achieved for 24% of the patients, suggesting yet unknown genetic causes.

P836: LONG-TERM DYNAMICS OF CLONAL HEMATOPOIESIS IN CHRONIC IDIOPATHIC NEUTROPENIA (CIN)

G. Tsaknakis 1,2,* , S. Papadakis 1,2 , P. Kanellou 3 , E. Boutakoglou 1,2 , I. Mavroudi 1,2 , C. Pontikoglou 1,2 , E. Papadaki 1,2

1 Haemopoiesis Research Laboratory, University of Crete School of Medicine; 2 Department of Hematology, University Hospital of Heraklion; 3 Department of Hematology, Venizeleion General Hospital, Heraklion, Greece

Background: We have previously performed next generation sequencing (NGS) analysis of genes recurrently mutated in myeloid malignancies in a cohort of CIN patients. We have estimated for the first time the frequency of clonal hematopoiesis in these patients and found that clonal CIN patients have significantly higher risk to develop MDS/AML compared to non-clonal CIN patients.

Aims: To conduct longitudinal follow-up NGS analyses of myeloid genes in CIN patients in order to assess clonal dynamics and evolution of clonal hematopoiesis in these patients.

Methods: We have performed longitudinal NGS analyses in 53 patients with CIN diagnosis (mean absolute neutrophil counts -ANCs- 1320±460/μL; median 1500, range 200-1700/μL) according to previously published criteria i.e no evidence of any underlying condition related to neutropenia after an extended clinical/laboratory investigation including bone marrow biopsy, karyotype, immunophenotype. Genomic DNA was extracted from bone marrow or peripheral blood samples at baseline and follow-up timepoints, sequencing libraries were prepared and subjected to targeted NGS on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 myeloid genes.

Results: Longitudinal NGS analysis in the patients was performed over a period of 4-173 months (median 31 months). The incidence of clonal hematopoiesis was similar at baseline (16/53 i.e. 30.2%) and follow-up (16/53 i.e. 30.2%) (Figure 1A). The mutation spectrum (i.e. absence of mutations, multiple mutated genes or presence of a single mutated gene) at baseline (Figure 1B) was comparable to that at follow-up (Figure 1C). In total, 28 mutations were detected in 17 CIN patients at one or both time-points. The variant allele frequencies (VAF) did not show a significant difference at baseline (9.68%) and follow-up (13.23%) and correlation analysis showed that VAFs at both points strongly correlated (r=0,59; P<0.01), with the majority of CIN patients showing a stable clone size (Figure 1D). We performed the same analysis for the most frequently mutated genes in our cohort of patients, namely DNMT3A , TET2, SRSF2 (Figure 1E-G). Overall, DNMT3A mutations did not show any positive selection over time, with a median increase in VAF of 1.78% (P=0.67) during the follow-up period (median period of 47.5 months, range 12-164). Similarly non-significant increases in VAF was seen for TET2 (0.49%) and SRSF2 (0.04%), within a median period of 26 months (range 22-111) and 55 months (range 12-98), respectively. One patient with newly developing clonal CIN and one patient with disappearance of clonal hematopoiesis were found at follow-up after 4 and 98 months, respectively. Two patients acquired a second mutation at follow-up. At baseline they carried mutations in SRSF2 and ZRSR2 , respectively and at follow-up they both developed TET2 truncating mutations. No significant changes at baseline and follow-up in ANCs were observed in patients with clonal disease. Overall, four patients (7.55%) transformed to AML/MDS.

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Summary/Conclusion: In the majority of CIN patients tested for clonal evolution over time, most mutant hematopoietic clones appeared to be remarkably stable, with limited VAF expansion over the follow-up period and no acquisition of new molecular alterations. Only two patients acquired an additional mutation over time and this propensity was associated with mutations in spliceosome genes. None of the individuals bearing DNMT3A or TET2 mutations acquired additional mutations. This study contributes to the better understanding of clonal hematopoiesis in CIN.

P837: ELTROMBOPAG (EPAG) INDUCES HEMATOLOGIC RESPONSES FOR PATIENTS WITH POST-ALLOGENIC HEMATOPOIETIC STEM CELL TRANSPLANT POOR GRAFT FUNCTION (POST-ALLO HSCT PGF): RESULTS OF THE ELTION STUDY IN SPAIN

C. Vallejo 1,* , I. García 2 , F. Sanchez Guijo 3 , M. Cuesta 4 , C. Solano 5 , A. Sampol 6 , T. Torrado 7 , M. Vendranas 8 , M. Marigil 8

1 University Hospital Donostia, San Sebastian, Spain, University Hospital Donostia, San Sebastian, Spain, Santiago de Compostela; 2 Department of Hematology, University Hospital Santa Cruz y San Pau, Barcelona; 3 Department of Hematology, University Hospital of Salamanca, Salamanca; 4 Department of Hematology, Carlos Haya University Hospital, Malaga; 5 Department of Hematology, Hospital Clinico de Valencia, Valencia; 6 Department of Hematology, University Hospital Son Espases, Palma de Mallorca; 7 Department of Hematology, University Hospital Alvaro Cunqueiro, Vigo; 8 Medical Affairs Department, Novartis, Barcelona, Spain

Background: Persistent cytopenia due to poor graft function (PGF) is a life-threatening complication in patients undergoing allogeneic HSCT (allo-HSCT). Several therapeutic approaches have been tested in this subset of patients with poor clinical results.

Aims: The objective of this multicenter open-label interventional prospective phase II Novartis study (ELTION, ClinicalTrials.gov id: NCT03718533 ), was to analyze efficacy and safety of EPAG in patients with post-allo-HSCT poor graft function.

Methods: Adult patients diagnosed with PGF (defined as severe cytopenia after day +30 post-transplant, with two or more of the following: platelets <20000/µL -mandatory-, ANC <1000/µL, hemoglobin< 10 g/dL), and full donor chimerism, were eligible to enter the trial. Study treatment consisted of EPAG. at 150 mg/day administered up to 36 weeks; dose adjustments were contemplated as per protocol on an individual basis. The primary efficacy endpoint was the overall hematologic response (partial and complete), as determined by platelet, hemoglobin and neutrophil counts by 16 weeks after the initiation of EPAG.

Results: Although the aim of the study was to include 33 patients, recruitment stopped prematurely due to the difficulties for hospital visits posed during COVID-19 pandemic, and eventually only 10 patients were included. The decision for this premature termination is not related to any safety concern related to the drug. Patient characteristics are shown in the Table 1 attached below. At EPAG. initiation, all 10 patients showed thrombocytopenia (<20000/µcL), 5 presented with anemia (Hgb <10 g/dL), and 4 had neutropenia (ANC <1000/µcL). Four patients discontinued EPAG before week 12 due to: disease progression/relapse (2 patients), protocol deviation (1 patient), and CMV infection (1 patient). In none of the cases, the event was related to study drug. At week 16, 4 patients (4/10, 40%) and at week 24, 5 patients, showed improvement in at least one of the 3 hematologic cell lines (partial response), respectively. Counts pre- and post-EPAG and global response in patients who stayed on treatment > 12 weeks are displayed below:

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Summary/Conclusion: In our experience, EPAG worked well in subjects with PGF, an otherwise life-threatening condition for patients, and its use at 150 mg/day is safe and well tolerated in this setting. Our data suggest that eltrombopag might improve hematologic cell counts in patients with PGF, especially in those patients who remained on treatment at week 24, however further research is warranted to extend its applicability for larger cohorts

P838: PEGCETACOPLAN RAPIDLY STABILIZES COMPLEMENT INHIBITOR NAÏVE PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA EXPERIENCING HEMOLYSIS WITH ACUTE HEMOGLOBIN DECREASES; PRINCE TRIAL POST HOC ANALYSIS

R. Wong 1,* , M. Al-Adhami 2 , J. Savage 2 , R. Horneff 3 , M. Yeh 4 , T. Dumagay 5 , D. Gomez-Almaguer 6

1 Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 2 Apellis Pharmaceuticals, Inc., Waltham, United States of America; 3 Swedish Orphan Biovitrum AB, Stockholm, Sweden; 4 Apellis Pharmaceuticals, Waltham, Waltham, United States of America; 5 Department of Cellular Therapeutics, Makati Medical Centre, Makati, Philippines; 6 Hematology Service, Hospital Universitario “Dr José Eleuterio González”, Universidad Autónoma de Nuevo León, Monterrey, Mexico

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and life-threatening disease characterized by complement-mediated hemolysis and thrombosis. Pegcetacoplan (PEG) is the first FDA/EMA-approved C3 complement-inhibitor that provides broad hemolysis control in patients with PNH.

Aims: This is the first report from the PRINCE trial on the efficacy of PEG in control arm rescue patients using a post hoc time-aligned analysis.

Methods: PRINCE is a phase 3, randomized, open-label study, that evaluates the efficacy of PEG in improving lactate dehydrogenase (LDH) and hemoglobin (Hb) levels in patients with PNH. 53 adult (≥18 years old), complement-inhibitor naïve (no prior eculizumab/ravulizumab) patients with LDH ≥1.5x the upper limit of normal (ULN) (≥339 U/L) and Hb below the lower limits of normal (LLN) (males: ≤13.6 g/dL; females: ≤12.0 g/dL), were enrolled. Patients were randomized 2:1 to receive PEG (1080 mg subcutaneously twice weekly, n=35; PEG arm) or to the Control arm (trials were in regions where C5 inhibitors were not available, n=18) for 26 weeks. Patients in the Control arm had the option to escape to PEG treatment (Rescue arm) should they exhibit signs of hemolysis and acute Hb decline by ≥2 g/dL from their baseline. Key endpoints included both changes from baseline in LDH and Hb levels. Because patients could be rescued at any point, a post hoc analysis was performed to time-align the Rescue patients (n=11), based on the start of PEG dosing, with patients in the PEG arm, to compare changes in LDH and Hb levels over time by PEG exposure.

Results: 61% (11/18) of Control arm patients experienced acute Hb decreases allowing rescue by PEG treatment. All Rescue patients had a pre-rescue LDH ≥2X ULN (mean 2424.0 U/L) and all had a rapid response to PEG treatment with a significant decrease in LDH level by Week 1 (mean 422.1 U/L) with 5/11 patients achieving ≤1.5X ULN; by Week 2 mean LDH levels were in the normal range (186.6 U/L) with 8 patients below ULN and 2 patients below 1.5X ULN, suggesting complete control of intravascular hemolysis. These results are similar to PEG arm patients (150.4 U/L) (Figure A).

Seven of the eleven patients showed a significant increase in Hb level (Week 2 mean Hb for Rescue patients: 10.9 g/dL; PEG patients: 11.19 g/dL); four patients showed no change, potentially due to underlying bone marrow dysfunction (3 had a history of aplastic anemia). Three patients had a transfusion within 5 days of starting PEG treatment; 1 of them remained transfusion dependent after rescue and did not have a Hb change.

The time-align plots (Figures A and B) show Rescue patients had rapid improvements in LDH to normal levels and Hb improvements similar to PEG arm patients.

PEG demonstrated a favorable risk-benefit profile. No deaths or serious adverse events deemed related to PEG occurred and no drug discontinuations due to PEG-related adverse events were reported. The most common AEs were injection site reaction (PEG [including Rescue patients for all AEs], 30.4%, n=14; Control arm, 0.0%), hypokalemia (PEG, 13.0%, n=6; Control arm, 11.1%, n=2), dizziness (PEG, 10.9%, n=5; Control arm, 0.0%), and fever (PEG, 8.7%, n=4; Control arm, 0.0%).

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Summary/Conclusion: PEG treatment was able to rapidly stabilize complement-inhibitor naïve patients experiencing acute hemolysis with substantial Hb decrease, and normalize LDH and raise Hb levels. The treatment effect of PEG on both LDH and Hb levels over time was largely similar between the PEG arm patients and Rescue patients. PEG represents a new effective therapeutic option with a favorable safety profile for PNH patients.

P839: PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA TREATED WITH PEGCETACOPLAN SHOW IMPROVEMENTS IN D-DIMER NORMALIZATION AND DECREASE IN INCIDENCE OF THROMBOSIS

I. Weitz 1,* , M. Al-Adhami 2 , J. Min 2 , E. Persson 3 , M. Yeh 2 , J. Savage 2 , D. Dingli 4

1 Jane Anne Nohl Division of Hematology, Keck-USC School of Medicine, Los Angeles; 2 Apellis Pharmaceuticals, Waltham, United States of America; 3 Swedish Orphan Biovitrum AB, Stockholm, Sweden; 4 Mayo Clinic, Rochester, United States of America

Background: Thrombosis is the main life-threatening complication of paroxysmal nocturnal hemoglobinuria (PNH), a rare acquired hematologic disease. Pegcetacoplan (PEG) is a C3 complement-inhibitor approved by the FDA/EMA for treatment of adults with PNH.

Aims: This post hoc analysis examined incidence of thrombosis, anti-thrombotic therapy (ATT), and D-dimer normalization in adult patients with PNH treated with PEG in the Phase 3 PEGASUS ( NCT03500549 ) and PRINCE ( NCT04085601 ) studies.

Methods: PEGASUS enrolled adult patients with PNH with prior suboptimal response to eculizumab (ECU) despite stable ECU treatment (≥3 months) and hemoglobin (Hb) levels <10.5 g/dL at screening. For the randomized controlled period (RCP), patients were randomized 1:1 to ECU (n=39) or PEG (n=41; 1080 mg subcutaneously [sc] 2x weekly) for 16 weeks. Thereafter, ECU patients were switched to PEG monotherapy and PEG RCP patients remained on PEG during the open-label period (OLP, n=77) through Week 48. PRINCE compared PEG treatment (n=35; 1080 mg sc 2x weekly) in complement-inhibitor-naïve patients to patients receiving control treatment (CTRL; excluding complement-inhibitors i.e., ECU/ravulizumab; n=18). CTRL patients had the option to escape to the PEG group if Hb levels decreased ≥2 g/dL from baseline (n=11). Safety analyses in all trials included monitoring of thrombotic events. D-dimer normalization, defined as D-dimer level less than the upper limit of normal (0.5 µg/mL) was examined at baseline, Week 8, and the end of the study period (PEGASUS: 16 weeks [RCP] and 48 weeks [OLP], PRINCE: 26 weeks).

Results: Prior to study entry, 31% (n=25) of PEGASUS patients (6 of these while on ECU therapy) and 9% (n=5) of PRINCE patients experienced at least one type of thrombosis. During the PEGASUS OLP, 2 of 77 patients experienced a thrombotic event, one in the setting of non-Hodgkin’s lymphoma and one during sepsis; neither event was deemed related to PEG. No patients in the PEGASUS RCP or PRINCE trial experienced a thrombotic event.

Pooled analysis of completed clinical trials of PEG including PEGASUS and PRINCE indicated there were 2 cases of thrombosis in 164 patients comprising 130 patient-years (1.54 events per 100 patient-years), compared to 1.77 events per 100 patient-years for patients on ECU treatment before entry into PEGASUS.

In PEGASUS, 34% (n=27) of patients were on ATT prior to study entry. During the PEGASUS RCP, concomitant ATT was observed in 37% (PEG, n=15) and 33% (ECU, n=13) of patients, and during the PEGASUS-OLP in 23% (n=18) of patients (PEG monotherapy). In PRINCE, concomitant ATT decreased from 21% (n=11) prior to the study to 7% (PEG, n=3) and 0% (CTRL, n=0) during the study.

In PEGASUS, D-dimer normalization in the PEG arm increased from 73% (baseline, n=30) to 89% (Week 8, n=33) and was largely sustained at Week 16 and Week 48 (Table 1). D-dimer normalization in the ECU arm increased once ECU patients switched to PEG monotherapy, from 76% (Week 16, n=29) to 89% (Week 48, n=17) (Table 1). In PRINCE, D-dimer normalization in the PEG arm increased from 51% (baseline, n=18) to 67% (Week 8, n=18) and increased further to 68% (n=19) at Week 26 (Table 1).

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Summary/Conclusion: Overall, these results demonstrate that PEG treatment can increase D-dimer normalization and reduces incidence of thrombotic events in patients with PNH who are complement-inhibitor naïve or remained anemic after ECU treatment, suggesting that PEG treatment is as effective as ECU in these outcomes.

P840: A MATCHING-ADJUSTED INDIRECT COMPARISON OF THE EFFICACY OF PEGCETACOPLAN USING PRINCE TRIAL DATA VERSUS RAVULIZUMAB AND ECULIZUMAB IN COMPLEMENT-NAÏVE PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

R. Wong 1 , J. Fishman 2,* , K. Wilson 3 , M. Yeh 2 , Z. Hakimi 3 , C. Yee 4 , L. Huynh 4 , M. S. Duh 4

1 Sir Y.K. Pao Centre for Cancer & Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 2 Apellis Pharmaceuticals, Waltham, United States of America; 3 Swedish Orphan Biovitrum AB, Stockholm, Sweden; 4 Analysis Group, Boston, United States of America

Background: Pegcetacoplan (PEG) is a PEGylated peptide targeting proximal complement protein C3 that has been shown to control both intravascular and extravascular hemolysis and recently approved by the FDA and EMA for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). PRINCE ( NCT04085601 ), was a phase-3, randomized, open-label trial assessing the efficacy and safety of PEG compared to control (CTL) treatment, excluding complement inhibitors among treatment naïve patients with PNH. PEG demonstrated superior efficacy to CTL by improving hemoglobin (Hb) levels and reductions in lactate dehydrogenase (LDH) levels in patients with PNH. To date, no head-to-head clinical trials exist comparing PEG to complement inhibitors in treatment naïve patients.

Aims: To assess the comparative effectiveness of PEG for complement-naïve patients with PNH against ravulizumab (RAV) and eculizumab (ECU) using matching-adjusted indirect comparison (MAIC) methodology to conduct cross-trial comparisons.

Methods: Individual patient data from PRINCE as well as aggregated published results of RAV and ECU in the ALXN1210-PNH-301 (“301”) study (Lee et al. 2019) were used. To adjust for cross-study differences, propensity score weighting was developed using logistic regression analyses and used to balance demographic and clinical characteristics between the PEG arm and RAV and ECU arms from 301. Outcomes were compared at 26 weeks using unanchored MAIC between PEG versus RAV and PEG versus ECU and included mean and percent change in LDH levels from baseline, LDH normalization (defined as LDH levels dropping <1xULN [246 U/L] in the absence of transfusions during the randomized controlled period), Hb stabilization (defined as the avoidance of a ≥2 g/dL decrease in hemoglobin level in the absence of transfusions during the randomized controlled period), and transfusion avoidance. Weighted Wald tests and 95% confidence intervals were computed for comparisons of categorical and continuous outcomes.

Results: A total of 34 patients were included in the PEG arm, 125 patients in the RAV arm, and 121 patients in the ECU arm. Prior to matching, significant differences existed between arms in the proportion of patients with White and American Indian or Alaska Native race, and mean LDH (all p<0.001). After matching the PEG to the RAV and ECU arms, separately, most patient characteristics were well balanced with a mean age of 43.8 years, 44.8 years, 46.2 years, respectively, 43.0-50.0% females, and a mean Hb of 9.4-9.6g/dL across arms; however, there were significant differences in Asian race and baseline LDH (all p<0.01). At week 26, patients in the PEG arm showed a significantly greater improvement in their LDH levels from baseline, with a mean of -523 U/L (-11.2%) compared to RAV and -578 U/L (-12.0%) compared to ECU (all p<0.001; Figure 1). Patients receiving PEG also showed significant improvements in LDH normalization (35.6% compared to RAV, p<0.001; 39.6% compared to ECU, p<0.001), Hb stabilization (28.2% compared to RAV, p<0.001; 31.7% compared to ECU, p<0.001), and transfusion avoidance (22.6% compared to RAV, p<0.001; 30.1% compared to ECU, p<0.001; Figure 1).

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Summary/Conclusion: In the absence of head-to-head comparisons, this unanchored MAIC study shows that PEG is more efficacious versus RAV and ECU in complement inhibitors treatment naïve PNH patients in terms of significant improvements across all evaluated endpoints, Hb stabilization, LDH improvement, LDH normalization and transfusion avoidance.

P841: EXTRACELLULAR VESICLE PROTEINS AS BIOMARKER OF OUTCOME IN MULTIPLE MYELOMA - A REAL-WORLD STUDY

E. Arnault Carneiro 1,* , B. Ferreira 1,2,3 , C. Pestana 1,4 , F. Barahona 1,3 , J. Caetano 1,3,5 , R. Lopes 1,6 , P. Lúcio 5 , M. Neves 5 , H. C. Beck 7 , A. S. Carvalho 8 , R. Matthiesen 8 , B. Costa-Silva 9 , C. João 1,3,5

1 Myeloma Lymphoma Research Group, Champalimaud Foundation, Lisbon; 2 Hemato-Oncology Unit, Champalimaud Foundation, Odense; 3 NOVA Medical School, NOVA Medical School; 4 Centre of Statistics and its Applications, Faculty of Sciences, University of Lisbon; 5 Hemato-Oncology Unit, Champalimaud Foundation; 6 Faculty of Medicine, University of Lisbon, Lisbon, Portugal; 7 Centre for Clinical Proteomics, Odense University Hospital, Odense, Denmark; 8 Computational and Experimental Biology, CEDOC, NOVA Medical School; 9 Systems Oncology Group, Champalimaud Foundation, Lisbon, Portugal

Background: Multiple myeloma (MM) is a hematological malignancy characterized by clonal plasma cells accumulation within the bone marrow. MM diagnosis and prognosis rely on bone marrow biopsy. Given clonal plasma cell patchy distribution, bone marrow biopsies may not reflect disease heterogeneity whilst submitting patients to invasive procedures. Liquid biopsies, such as extracellular vesicles (EV) from peripheral blood, may overcome these limitations. In MM, there is evidence that EV intervene in key processes such as tumor progression and drug resistance. Most studies analyzing EV from MM patients focus on genome, and real-world studies on EV proteome are scarce.

Aims: We characterized blood and bone marrow samples from the precursor stage, MGUS and from MM patients to determine whether EV content can discriminate between diagnosis and predict patient prognosis. These results were compared to healthy donor samples.

Methods: After informed consent signed, a cohort of 102 patients and 19 healthy donors were followed in median time of 25 months. Blood and BM samples were collected at diagnosis, response and relapse of patients. EV were isolated by ultracentrifugation. Protein and EV particle concentrations were quantified. Proteomic analysis was performed by mass spectrometry (LC-MS/MS). Diagnostic and prognostic impacts of EV characteristics were investigated. A multivariable longitudinal logistic regression model was built to determine the association with common myeloma-related blood parameters.

Results: We report a set of peripheral blood EV-proteins (PDIA3, C4BPA, BTN1A1, APRIL, PSMB8 and PDE8B) that have the potential to be used as new diagnosis biomarker for myeloma patients. Functional enrichment analysis revealed that proteins differentially expressed in patient vs healthy donors are strongly related to immune response, supporting increased immune dysfunctions in patients with active multiple myeloma.

We identified that the level of EVcargo (protein/particle ratio) is significantly associated to patient overall survival. High EVcargo patients (>0.6 µg/10 8 particles) had a shorter overall survival compared to low EVcargo patients (≤0.6 µg/10 8 particles). High EVcargo is associated with high sFLC lambda, IgA immunoparesis and shorter time in response. The downregulation of IGHA1/IGHA confirmed patients immunoparesis. Upregulation of Ig lambda production proteins confirmed the increased presence of sFLC in the same patients. Also, we report an association between the progression free survival time and expression level at diagnosis of SERPINA2, RPS26 and UBBP4, suggesting their potential as prognosis biomarkers.

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Summary/Conclusion: To our knowledge, this is the first report of EV protein content from a real-world MGUS and MM patient cohort followed for more than 2 years. Our findings show that PDIA3, BTN1A1, APRIL and complement proteins should be further explored in myeloma, as peripheral blood biomarkers of diagnosis. Circulating EVcargo is a promising prognostic biomarker for MM patients. Patients with high EVcargo had 12 times increased risk of dying and high EVcargo is associated with important prognostic features such as immunoparesis, sFLC or duration of treatment response. EVcargo is an indirect and more affordable measure to infer EV protein load compared to mass-spectrometry. Our results show that EV have the potential to be used as diagnosis biomarker in MGUS and MM patients and as an added approach to discriminate patients with poor survival. Our results substantiate the interest of EV as liquid biopsies in myeloma and future validation in independent clinical settings is urged.

P842: NOVEL MULTIFUNCTIONAL TETRAVALENT CD38 NKP46 FLEX-NK ENGAGERS ACTIVELY TARGET AND KILL MULTIPLE MYELOMA CELLS

L. Lin 1 , H.-M. Chang 1 , C. Nakid 2 , S. Frankel 1 , D. Wu 3 , J. Kadouche 4 , D. Teper 4 , O. Mandelboim 5 , J.-C. Bories 2 , A. Arulanandam 1,* , W. Li 1

1 Cytovia Therapeutics, Natick, United States of America; 2 INSERM U976 and Université de Paris, Paris, France; 3 Lynx Biosciences, San Diego; 4 Cytovia Therapeutics, Aventura, United States of America; 5 Hebrew, University of Jerusalem, Jerusalem, Israel

Background: Given that CD38 is a clinically validated target for natural killer (NK) cell mediated cytotoxicity in multiple myeloma (MM), we sought to leverage our FLEX-NK TM platform to create a NK engager antibody targeting CD38. FLEX-NK TM is a proprietary platform for production of tetravalent IgG1-like multifunctional NK engager antibodies with a novel FLEX-linker to allow for simultaneous binding of both the targeted cancer cells and NK cells via the activation receptor NKp46.

Aims: Derive preclinical proof of concept with CD38 targeted NK-cell engager bispecific antibody in Mulitple Myeloma

Methods: CYT-338 was expressed in CHOZEN cells and purified by step column chromatography. PB-NK cells were purified from healthy donor blood. PB-NK cytotoxicity against MM tumors was evaluated in the presence of CYT-338 or isotype control antibodies at a fixed E/T ratio. CYT-338 pharmacokinetics in mice was evaluated following a single intravenous injection. Anti-tumor efficacy of CYT-338 was evaluated in NSG-mice injected with PB-NK cells and MM cell line MM1S expressing luciferase. CYT-338 mediated fratricide was evaluated in purified PB-NK’s. CYT-338 cytokine release assessments and hemato-cytotoxicity was evaluated in-vitro in human PBMC assays.

Results: CYT-338 showed dose dependent binding to CD38 expressing MM cell lines MM1S and KMS11 and no binding to a CD38 knock out MM.1S cell line. CYT-338 bound MM cell lines with ~ 2-fold higher mean fluorescence intensity than anti-CD38 monoclonal antibody (mAb) or daratumumab alone. Epitope mapping studies for our CD38 mAb binder using alanine scanning mutagenesis showed that amino acid S274 on CD38, critical for binding to daratumumab, is not important for CD38 binding, suggesting a distinct epitope detected by our CD38 binder. CYT-338 showed greater dose dependent NK cell redirected cytolysis, degranulation and cytokine production against MM1S cells compared to daratumumab. The CYT-338 pharmacokinetics study in mice showed a terminal half-life of 41 hrs. Preliminary in-vivo tumor model studies in NSG mice using PB-NK and CYT-338 injections showed tumor growth inhibition of MM1S tumor cells. Low NK cell fratricide was observed with CYT-338 while daratumumab showed significantly higher NK cell fratricide. In peripheral blood mononuclear cell hemato-toxicity studies, depletion of monocytes and NK cells were observed with daratumumab but no significant depletion was observed with CYT-338. Cytokine release assessment of CYT-338 in the human PBMC assay showed no evidence of cytokine release while high levels of cytokine release was observed with daratumumab, anti-CD28 (TGN1412) and CD3 antibody controls.

Summary/Conclusion: These results suggest that the CYT-338 engager has a favorable NK cell engager profile for targeting CD38 expressing multiple myeloma distinct from daratumumab.

P843: METABOLOMIC CHARACTERIZATION OF HUMAN MULTIPLE MYELOMA CELL LINE TO STUDY TUMOR RESISTANCE TO DIFFERENT CLASSES OF THERAPEUTIC AGENTS

A. Steer 1,* , D. Chemlal 1 , E. Varlet 2 , A. Machura 1 , A. Kassambara 1 , E. Alaterre 2 , G. Requirand 3 , N. Robert 3 , C. Hirtz 4 , H. De Boussac 1 , A. Bruyer 1 , J. Moreaux 2

1 Diag2Tec; 2 Institute of Human Genetics, UMR 9002 CNRS-UM; 3 Laboratory for Monitoring Innovative Therapies, Department of Biological Hematology, CHU Montpellier; 4 Clinical Proteomics Platform, LBPC, IRMB, CHU, UM Montpellier, Montpellier, France

Background: Multiple myeloma (MM) is the second most common hematological malignancy, characterized by the abnormal accumulation of plasma cells in the bone marrow. Although the latest treatments, including proteasome inhibitors, immunomodulating agents, or immunotherapy, have greatly improved patient survival, a residual subset of cells remains resistant to therapies and usually causes relapses.

Aims: The drug resistance could be explained, among others, by the interactions with the microenvironment, MM’s high molecular heterogeneity, or the appearance of adaptive survival mechanisms after treatment exposure. However, a better understanding of the mechanisms involved in drug resistance remains of significant interest. Among the factors influencing the resistance of cancer cells, the “metabolic plasticity” of the tumor and, therefore, its ability to adapt to stress conditions is a mechanism increasingly studied in recent years in cancer. Although measuring mitochondrial metabolism has been identified as a major factor influencing response to treatments in several cancers, few studies have been documented in MM.

Methods: Here, we characterized the metabolic profiles of a panel of 20 human MM cell lines (HMCL) representative of the molecular heterogeneity found in MM patients. This panel included 10 commercial HMCL and 10 HMCL derived in our laboratory. First, oxygen consumption rates (OCR), extracellular acidification rate (ECAR), and spare respiratory capacity (SRC) of our HMCL panel were assessed in a Mito Stress Assay using a Seahorse XFe96 analyzer.

Results: Interestingly, the metabolic activities were shown very heterogeneous in HMCLs with a part of cell lines more dependent of the glycolysis activity and another part more dependent of the mitochondrial respiration. By integrating the HMCL’s metabolic profiles with their respective transcriptomic data (RNAseq), we defined a metabolomic score to classify the HMCL into different groups and represent their glycolysis level or mitochondrial activity. The score was calculated from the expression of 112 genes involved in the electron transport chain (Oxphos) or in glycolysis. For a given gene, the expression values were first log2-transformed and then normalized by subtracting the average. The score is computed as the difference between the Oxphos and glycolytic genes’ average expression. Interestingly, high significant correlations between the HMCL’s functional metabolic profiles and their calculated metabolomic score were identified. Furthermore, we investigated the potential prognostic value of this gene-based metabolomic score in the MMRF CoMMpass cohort (newly diagnosed MM patients, n=674) using the maxstat algorithm, which segregated the cohort into two groups with a significantly different outcome. We identified that 32% of patients with a high gene-based metabolomic score, were associated with poor overall survival (P = 3.1x10 -6 ). We then validated this prognostic value in a second cohort of MM’s patients: the cohort of 206 patients (ArrayExpress public database under accession number E-MTAB-362). Moreover, we performed correlation analyses between the metabolic profiles of those HMCL and their respective response to the conventional treatments (IC50). We found a significant correlation between a high mitochondrial ATP production and the resistance to proteasome inhibitor (P = 0.023, n= 12).

Summary/Conclusion: Altogether, we demonstrated that metabolomic deregulation could participate in drug resistance in MM. Inhibitors targeting metabolic activities may be of therapeutic interest to overcome drug resistance in MM.

P844: CHARACTERIZATION OF MULTIPLE MYELOMA CELL LINES WITH ACQUIRED-RESISTANCE TO PROTEASOME INHIBITORS HIGHLIGHTS A LINK BETWEEN RESISTANCE AND METABOLIC DEREGULATION

H. De Boussac 1,* , A. Machura 1 , A. Steer 1 , A. Kassambara 1 , M. Gely 1 , D. Chemlal 1 , C. Gourzones 2 , G. Requirand 3 , N. Robert 3 , L. Vincent 4 , C. Herbaux 4 , A. Bruyer 1 , J. Moreaux 5

1 Diag2Tec; 2 UMR 9002 CNRS-UM; 3 Laboratory for Monitoring Innovative Therapies, Department of Biological Hematology; 4 CHU Montpellier; 5 Institute of Human Genetics, UMR 9002 CNRS-UM, Montpellier, France

Background: Characterized by an abnormal clonal proliferation of malignant plasma cells, Multiple myeloma (MM) is the second most common hematological malignancy. Novels agents have significantly improved clinical outcomes, but most of the MM patients eventually relapse. A better understanding of the drug resistance mechanisms remains of significant interest to improve the treatment of patients

Aims: We aimed to investigate the mechanisms involved in the resistance to proteasome inhibitors (PI).

Methods: For that purpose, we have derived and characterized 6 Bortezomib (Btz)-resistant human myeloma cells lines (BR-HMCLs) from different molecular subgroups and still dependent on the addition of IL-6 including XG1BR t(11;14), XG2BR t(12;14), XG7BR, XG20BR, XG24BR t(4;14) and XG19BR t(14;16). These cell lines were cultured continuously with escalating concentrations of Btz during 12 months.

Results: Interestingly, BR-HMCLs demonstrated significant resistance to Btz compared to their parental counterparts (CTR-HMCLs) (mean IC50: BR-HMCLs =5nM vs CTR-HMCL=2.3nM, p<0.05). In addition, these BR-HMCLs also showed significant cross-resistance to Carfilzomib (Cfz) and Ixazomib (Ixa) PIs (p<0.05 for Cfz or Ixa). Finally, no significant cross-resistance was observed with other therapeutic agents (melphalan, dexamethasone or IMIDs), indicating that the observed drug resistance mechanisms are especially related to PIs.

We then used genomic approaches (whole genome sequencing and transcriptomic analyses) to understand the PIs resistance mechanisms acquired by MM cells. Remarkably, as observed in relapsed MM patients, among the 40 mutations identified in BR-HMCLs compared to the CTR-HMCLs, we identified a mutation residing in the Btz-binding pocket of the proteasome beta5-subunit (PSMB5), that reduces the PI binding capacity, thus preventing inactivation of the catalytic activity of the 20S proteasome.

Further transcriptomic analyses on BR-HMCLs underlined significant deregulation of genes involved in cell metabolism and drug clearance that could allow the BR-cells to maintain metabolic homeostasis and survival in stringent redox conditions. Thus, in the BR-HMCL we identified an upregulation of enzymes directly involved in glycolytic and energy metabolism (ALDOC, ENO3, HK1, PDK1/3, PFKB3/4, PFKL, SLC2A1 (FC>1.5 p-value <0.05), but also a significant downregulation of 8 solute carrier protein (SLC) intake transporters together with a significant upregulation of xenobiotic receptors (FC> or < 1.5; p-value <0.05).

We then tested the metabolomic of BR-HMCL using the Seahorse assay. This technology allows to analyze the glycolysis via the extracellular acidification rate (ECAR) and cell respiration via the mitochondrial oxidative phosphorylation based on the oxygen consumption rate (OCR). Following the increased gene expression of several glycolytic enzymes found in the BR-HMCLs, we observed that while CTR and BR displayed equivalent respiration and glycolytic activities, their treatment with Btz induces major metabolic modifications, including a significant increase of the glycolytic and mitochondrial activity of the BR-HMCL while it decreases it in the CTR-HMCL, suggesting a higher capacity of the BR-HMCL to respond to cell stress.

Summary/Conclusion: Altogether, we developed acquired PIs resistant HMCLs that exhibit PSMB5 mutation as observed in patients, and we identified pathways linked to metabolism deregulation in these cell lines. These results make our PI-resistant models, an attractive preclinical model to test new therapeutic strategies to overcome PI resistance in MM.

P845: IDENTIFICATION OF MULTIPLE MYELOMA BIOMARKERS VIA LIQUID BIOPSY BASED ON CELL-FREE DNA USING A CAPTURE-HYBRIDIZATION PANEL

N. Buenache Cuenda 1,* , A. Sánchez-delaCruz 1 , I. Cuenca Navarro 1 , L. Rufián Vázquez 1 , V. Garrido García 1 , A. Giménez Sánchez 1 , R. Alonso Fernández 2 , J. M. Sánchez Pina 2 , Y. Ruiz Heredia 1 , S. Barrio García 1 , I. Rapado Martínez 1,2 , R. Ayala Díaz 1,2,3,4 , J. Martínez-López 1,2,3,4 , J. M. Rosa-Rosa 1

1 Department of Translational Haematology, Research Institute Hospital 12 de Octubre (i+12) Haematological Tumors Clinical Research Unit H12O-CNIO; 2 Department of Translational Haematology, Haematology Service, Hospital 12 de Octubre; 3 Department of Medicine, Faculty of Medicine, Complutense University; 4 CIBERONC, Madrid, Spain

Background: Multiple myeloma (MM) is a haematological malignancy characterized by clonal proliferation of pathogenic CD138+ plasma cells (PPCs) in bone marrow (BM). In recent years, the treatment of MM has shown great evolution. However, most patients who achieve a complete response eventually relapse. For that, an early detection of PPCs could be very beneficial for MM patients, and therefore, a sensitive detection of this stage could allow early therapeutic interventions. Liquid biopsy using cell-free DNA (cfDNA) as a minimally invasive approach could be an alternative not only for the diagnosis but also for the detection of recurrences.

Aims: The main objective of this study was the quantification of MM patient-specific biomarkers in cfDNA compared to PPCs and BM samples.

Methods: Patients with active MM were treated with VRD, autologous stem cell transplantation and maintenance. We studied gDNA from 23 PPCs from BM aspirates, 16 gDNA from whole BM and 12 cfDNA from peripheral blood samples obtained at diagnosis. Capture panel was aimed to detect most common and relevant aberrations known in MM such as coding regions of 37 genes involved in MM progression and drug resistance, canonical IGH and IGK to capture IGH rearrangements and regions to cover traditional translocations. Capture libraries were generated with SureSelect Reagent kits (Agilent Technologies). We used 50 ng of genomic DNA for both PCCs and BM and for cfDNA a median of 10-200 ng input. Final libraries were run on an Illumina NextSeq 500 platform. A specific bioinformatics pipeline was applied: alignment to the hg38 genome, SNVs and Indels identification by combination of variant callers, and IGH / K rearrangements with MiXCR.

Results: A total of 36 different mutations were identified in the PPCs with frequencies ranging from 0.011-0.570. Comparison between PPCs and cfDNA showed that 20 out of 23 (87%) mutations identified in the PPCs were observed in the cfDNA, whereas 32 out of 35 (91%) mutations were observed in BM. Moreover, 6 out of 11 (55%) mutations identified in the PPCs were observed in cfDNA and BM (Figure 1A). In 5 out of 5 patients at least 1 mutation of those evaluated in PPCs was detected in cfDNA. Interestingly, for the classical oncogenes ( NRAS , KRAS and BRAF ), 5 of 8 (63%) hotspot mutations were observed in cfDNA and 7 of 8 (88%) in BM. Using the ratio between the observed frequency in cfDNA/BM and that observed in PPCs, we estimated an average tumour burden of 6% in cfDNA and a mean clonality of ~30% in BM. In addition, 5 out of 5 translocations identified in PPCs were observed in BM and unexpectedly 1 out of 2 was observed in cfDNA. Regarding IGH / K rearrangements, 50% of the patients presented at least one rearrangement in the cfDNA fraction that was identified in the PPCs, and 93% in the BM fraction. Furthermore, the average reduction in frequency observed in cfDNA and BM was ~12% compared to the frequency seen in PPCs. Finally, 2 out of 5 patients showed the same IG rearrangement in the three fractions studied[jm1] (Figure 1B).

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Summary/Conclusion: Most of molecular alterations identified in PPCs seemed to be present in peripheral blood of patients with MM, surprisingly, even translocation breakpoints could be seen in cfDNA. However, liquid biopsy monitoring is limited by signal dilution, as an approximately 1-log reduction in cfDNA was observed compared to PPCs, which should be considered in bioinformatic analyses. On the other hand, IGH / K rearrangements could be identified in fewer patients, but the signal reduction was only ~12%, confirming IG rearrangements as strong biomarkers for MM in cfDNA.

P846: EXPRESSION SIGNATURE OF TP53 BIALLELIC INACTIVATION IDENTIFIES A GROUP OF MULTIPLE MYELOMA PATIENTS WITHOUT THIS GENETIC CONDITION BUT WITH DISMAL OUTCOME.

C. De Ramón 1,* , E. A. Rojas 1 , I. J. Cardona-Benavides 1 , M. V. Mateos 1 , L. A. Corchete 1 , N. C. Gutiérrez 1

1 Hematology, University Hospital of Salamanca, IBSAL, Cancer Research Center-IBMCC (USAL-CSIC), Salamanca, Spain

Background: Cytogenetic abnormalities remain the most relevant prognostic factors, especially those related to TP53 gene. Biallelic inactivation of TP53 , included in the definition of double-hit (DH) MM, entails an ominous prognosis, although is present in less than 5% of newly diagnosed MM (NDMM). However, ultra-high-risk MM, defined as those patients with a median survival less than 24 months, represents 15-20% of the MM population. While other high-risk cytogenetic abnormalities may account for this adverse prognosis, these kind of cytogenetic alterations are not present in all patients with such an unfavorable outcome. On the other hand, p53 can be deregulated by other mechanisms different from changes in DNA gene sequence, such as epigenetic regulation or altered expression of its regulators.

Aims: To define the transcriptional signature of DH -TP53 and to find out if it was present in other patients who did not have biallelic inactivation of TP53 .

Methods: We analyzed RNA-seq, whole-genome and whole-exome sequencing data from 660 newly diagnosed MM (NDMM) patients from the MMRF (Multiple Myeloma Research Foundation) CoMMpass study to characterize the transcriptional signature of DH- TP53 MM. This gene signature was used to build a score based on a Spearman correlation coefficient and a scaled GINI index. Survival data were retrieved from the IA16 release and the analysis was performed using the Kaplan-Meier estimator and log-rank test. Multivariable Cox models were fitted in R. GSE4581 and GSE136400 gene expression series from GEO were used as validation cohorts.

Results: TP53 biallelic inactivation, defined in this work as DH- TP53 group, was found in 23 out of 660 patients (3.5%) and was associated with an exclusive gene expression signature consisted of 78 genes. Based on these genes, we calculated the DH- TP53 score, which identified a subgroup of 50 patients that shared the same transcriptional profile (DH- TP53 -like group). The prognosis of this group was particularly unfavorable with a median overall survival (OS) of 23 months; and a progression-free survival (PFS) of 15 months, even worse than that described for DH- TP53 patients (HR = 1.83 [95% CI, 1.00-3.35], p = 0.046). The prognostic value of the DH- TP53 was confirmed as an independent prognostic factor for PFS (HR 3.84 [95% CI 2.51-5.88], p < 0.001) and OS (HR 3.32 [95% CI, 2.31-4.77], p < 0.001) in the multivariable analysis. We also observed that survival for any of the cytogenetic abnormalities was significantly shortened in the DH- TP53 -like group ( p < 0.05). Furthermore, the prognostic value of the DH- TP53 score was externally validated using gene expression data obtained by microarray analysis

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Summary/Conclusion: The expression signature of DH- TP53 MM was shared by other MM patients without TP53 biallelic inactivation (DH- TP53 -like group). The DH- TP53 score identified an ultra-high-risk group of MM patients with a median overall survival below 24 months. In addition, this score refined the prognostic stratification of cytogenetic abnormalities, and was externally validated using expression data analyzed by microarrays.

Funding: This study has been funded by ISCIII (PI16/01074 and PI19/00674) (co-funded by FEDER), Castilla y Leon (GRS 2058/A/19 and GRS 2331/A/21) and AECC (PROYE20047GUTI). CDR, EAR and IJCB were supported by the AECC (CLJUN18010DERA), the “Consejería de Educación de Castilla y León” and the ISCIII (FI20/00226), respectively.

P847: MRD BY MASS SPECTROMETRY IN PERIPHERAL BLOOD AND NEXT GENERATION SEQUENCING IN BONE MARROW IN A PHASE 2 STUDY OF DARATUMUMAB, CARFILZOMIB, LENALIDOMIDE, AND DEXAMETHASONE FOR MULTIPLE MYELOMA

B. Derman 1,* , J. Rosenblatt 2 , D. Avigan 2 , A. Major 1 , M. Rampurwala 1 , D. Barnidge 3 , A. Stefka 1 , K. Jiang 1 , A. Jakubowiak 1

1 Section of Hematology/Oncology, University of Chicago, Chicago; 2 Beth Israel Deaconess Medical Center, Boston; 3 The Binding Site Group, Rochester, MN, United States of America

Background: Measurable residual disease (MRD) as assessed by next generation sequencing (NGS) using bone marrow (BM) aspirate carries prognostic significance in multiple myeloma (MM). Though its analytical sensitivity reaches 10 -6 , MRD assessed from BM may be vulnerable to false negatives due to patchy or extramedullary disease, or due to inadequate aspirate sample. Mass spectrometry (MS) is a promising peripheral blood (PB) assay that may approximate current methods of MRD detection.

Aims: In this phase 2 study evaluating the safety and efficacy of extended daratumumab, carfilzomib, lenalidomide, and dexamethasone (Dara-KRd) without autologous stem cell transplant (ASCT) in newly diagnosed MM, we are evaluating the concordance of MRD by NGS in BM and by MS in PB.

Methods: Forty-one patients have been enrolled from two MM Research Consortium sites into this phase 2 study (planned enrollment 45 patients). All patients receive 24 cycles of Dara-KRd in 28-day cycles without ASCT. With optional stem cell collection for ASCT-eligible candidates after cycle 4. MRD by NGS was assessed from BM aspirates by the clonoSEQ® assay (Adaptive Biotechnologies) with a limit of detection <10 -6 . MRD by MS was evaluated using both an automated MALDI-TOF (EXENT) and liquid-chromatography-MS (LC-MS) by the Binding Site Group (assays under development). Paired PB MS and BM NGS samples were available for 13 patients at early (post-cycle 4) and 18 patients at later (cycles 8-24) timepoints. There were 44 paired NGS/EXENT samples (14 post-cycle 4) and 42 paired NGS/LC-MS samples (13 post-cycle 4). Cohen’s kappa test was used to assess concordance between MS and NGS samples.

Results: For the early post-cycle 4 timepoint, there was low agreement between NGS (sensitivity threshold 10 -6 ) and MS; there was 46% agreement (Cohen’s kappa -0.18) between NGS and EXENT and 54% agreement (Cohen’s kappa -0.15) between NGS and LC-MS. Of the discordant cases for NGS and EXENT, 4/7 (57%) were NGS - /EXENT + . Two of these 4 patients converted to EXENT - at C8. For discordant NGS/LC-MS cases, 5/6 (83%) were NGS - /LC-MS + . None converted to LC-MS - . The one NGS + /LC-MS - case was from a patient with kappa light chain MM.

For the later timepoints (cycles 8-24), there was higher concordance between NGS and MS. There was 63% agreement (Cohen’s kappa 0.27) between NGS and EXENT and 59% agreement between NGS and LC-MS (Cohen’s kappa 0.13). Of the discordant cases for NGS and EXENT, 4/11 (36%) were NGS - /EXENT + and 1 of these cases was followed by conversion to EXENT - . For discordant NGS/LC-MS cases, 9/12 (75%) were NGS - /LC-MS + . None converted to LC-MS - . Two of the three NGS - /LC-MS + cases were from a patient with kappa light chain MM.

With median follow-up of 10 months, there have been no progressions or deaths among these patients. The prognostic significance of persistent NGS + or LC-MS + patients cannot be determined at this time and requires longer follow-up.

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Summary/Conclusion: MRD assessment by MS (EXENT and LC-MS) in PB and NGS in BM serve complementary roles. Early in treatment, MS positivity may represent false positives due to immunoglobulin recycling. EXENT from PB appears to more closely approximate the sensitivity of MRD by NGS at a sensitivity threshold of 10 -5 , while LC-MS from PB appears to reach and possibly exceed the sensitivity of MRD by NGS in BM at a sensitivity threshold of 10 -6 . The prognostic significance of persistent LC-MS positivity is unclear and requires longer follow-up.

P848: TINOSTAMUSTINE (EDO-S101), AN ALKYLATOR AND HISTONE DEACETYLASE INHIBITOR, ENHANCES THE EFFICACY OF DARATUMUMAB IN PRECLINICAL MODELS OF MULTIPLE MYELOMA

A. Diaz-Tejedor 1,* , L. San-Segundo 1 , L. A. Corchete 1 , L. González-Méndez 1 , M. Martín-Sánchez 1 , M. Lorenzo-Mohamed 1 , M. González-Rodríguez 1 , M. Cruz-Hernández 1 , M. Sánchez-Blázquez 1 , N. C. Gutiérrez 1 , M.-V. Mateos 1 , M. Garayoa 1 , E. M. Ocio 2 , T. Paíno 1

1 Department of Hematology, Cancer Research Center-IBMCC; University Hospital of Salamanca-IBSAL, Salamanca; 2 Department of Hematology, University Hospital Marques de Valdecilla (IDIVAL); University of Cantabria, Santander, Spain

Background: Tinostamustine (EDO-S101) is a novel alkylating and deacetylase inhibiting molecule designed to improve drug access to DNA strands, induce DNA damage and counteract its repair in cancer cells. It has shown anti-myeloma (MM) activity in different preclinical models both in monotherapy and in combination with bortezomib.

Aims: To evaluate whether tinostamustine enhances the anti-myeloma effect of daratumumab.

Methods: Tinostamustine was provided by Mundipharma and daratumumab was obtained from pharmacy surplus of the University Hospital of Salamanca. Mechanisms of action of daratumumab were studied by flow cytometry (FCM) in MM cell lines pretreated with tinostamustine and cultured as specified: 1) with F(ab) 2 fragments (direct apoptosis via crosslinking); 2) with 10% human serum (CDC assays); 3) co-cultures of MM and NK cells (ADCC assays). Expression of CD38 and NKG2D ligands (MICA and MICB) after tinostamustine treatment was evaluated by FCM, WB and qPCR. The effects of tinostamustine + daratumumab combination were also studied ex vivo in bone marrow (BM) samples from MM patients, and in vivo in two plasmacytoma models developed in CB17-SCID and NK-cell-humanized NSG mice.

Results: Pretreatment of U266, JJN3, MM.1S, NCI-H929, RPMI-8226 and MOLP-8 cell lines with tinostamustine (1-2.5 μM) for 48 h increased CD38 expression. Tinostamustine-pretreated myeloma cells showed a higher level of daratumumab binding, as demonstrated by anti-human-IgG1-AF488 staining. Also, tinostamustine (1-2.5 μM) increased MICA and MICB expression in MM cell lines. In ex vivo cultures, tinostamustine increased expression of CD38 in 4 out of 10 patients and MICA in 3 out of 5 patients.

Next, the influence of tinostamustine pretreatment (2.5 μM) on several daratumumab mechanisms was evaluated. In apoptosis via crosslinking, the percentage of apoptotic cells in presence of daratumumab was higher in tinostamustine-pretreated MOLP-8 cells vs DMSO-pretreated cells (90.57%±7.35 vs 60.36%±2.86; p<0.001). Likewise, pretreatment with tinostamustine increased the percentage of Annexin-V+/7AAD+ MOLP-8 cells in CDC assays with daratumumab, although not reaching statistical significance (93.22%±1.05 vs 73.39%±11.93). In MM-NK co-cultures in absence of daratumumab (NK cell-mediated direct cytotoxicity), the percentages of apoptotic cells in tinostamustine-pretreated vs DMSO-pretreated cells were: 83.64%±5.28 vs 61.58%±6.81 in MOLP-8 cells (p<0.05); 54.27%±5.99 vs 34.24%±7.37 for RPMI-8226 (p<0.05); and 26.15%±4 vs 12.32%±1.97 in MM.1S (p<0.05). However, in the presence of daratumumab (ADCC) these percentages showed a similar tendency although not significant: 88.57%±3.77 vs 78.89%±5.57 in MOLP-8; 70.75%±5.69 vs 58.28%±7.52 for RPMI-8226; and 56.74%±7.25 vs 45.68%±7.1 in MM.1S. Ex vivo experiments with patients’ BM samples (n = 10) showed that simultaneous combination of daratumumab + tinostamustine significantly increased the percentage of eliminated MM cells compared to individual treatments, with an acceptable toxicity on healthy lymphocytes (Figure 1a).

Finally, in the CB17-SCID model, tinostamustine treatment (24 h) followed by daratumumab administration controlled tumor growth significantly better than daratumumab alone (Figure 1b). The combination prolonged the median survival as compared to vehicle, and monotherapy with tinostamustine or daratumumab (Figure 1b). Similar results were obtained in the NSG model.

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Summary/Conclusion: Our preclinical data demonstrate that tinostamustine increases the anti-myeloma effect of daratumumab presumably due to enhanced expression of CD38 and MICA.

P849: DEPLETION OF DIS3 IN MULTIPLE MYELOMA LEADS TO PERTURBATION IN RNA METABOLISM, CELL CYCLE PROGRESSION AND MITOTIC CHECKPOINT.

V. Favasuli 1,* , I. Silvestris 1 , K. Todoerti 2 , D. Ronchetti 1 , S. Erratico 3 , D. giannandrea 4 , Y. Torrente 5 , R. Chiaramonte 4 , S. Fabbris 1 , N. Bolli 1 , L. Baldini 1 , A. Neri 1 , E. Taiana 1

1 Oncology and Hemato-oncology, University of Milano; 2 Phatology and Molecular Oncology, IRCCS Istituto Nazionale dei Tumori; 3 Novystem Spa, Novystem Spa; 4 Department of Health Sciences; 5 Stem Cell Laboratory, Unit of Neurology, University of Milano, Milan, Italy

Background: MM is a genetically heterogeneous neoplasm in which the co-occurrency of multiple genomic events play a crucial role in tumor development, progression and drug resistance. DIS3 gene maps at 13q22 and encodes for a highly conserved ribonuclease involved in the RNA processing, quality control and degradation. Deletion of DIS3 gene is a common characteristic of MM patients (approximately 50% of cases); moreover, DIS3 mutations occur in about 11% of MM cases and are frequently (70%) associated with loss of the gene. DIS3 mutations along with del(13q) have been recently described by us as associated with a strong negative effect on clinical outcome.

Aims: The purpose of this study is to shed light on the biological role and impact of DIS3 transcript in MM.

Methods: LNA gapmeRs were used to silence DIS3 in HMCLs by gymnotic delivery in four human myeloma cell lines, two of which showing a monoallelic del(13q). qRT-PCR has been used to evaluate DIS3 silencing efficiency. Cell viability and cellular growth was evaluated by viable counts and by Cell Titer Glo assay. Modulation of cell cycle phases distribution was assessed by flow cytometry. Cell cycle synchronization was obtained using SynchroSet kit. Transcriptional analysis was performed using Affymetrix microarray. Protein expression was evaluated by Western Blot. Mitotic spindle organization was analyzed by immunofluorescence (IF).

Results: DIS3 silenced HMCLs show a significant decrease of cellular growth right after 4 days from LNA gapmeR delivery. DIS3 depleted cells showed a significant perturbation of cell cycle in all the HMCLs tested. Analysis of synchronized HMCLs revealed a more pronounced modulation of all cell cycle phases distribution confirming the involvement of DIS3 in cell cycle regulation. Accordingly, DIS3 silencing lead to an important modulation of cell cycle checkpoint proteins and mitotic spindle checkpoints. Confocal microscopy analysis performed in DIS3 silenced HMCLs revealed the presence of mitotic defects and microtubule organization abnormalities. Transcriptional profile analysis of DIS3 -KD samples is associate to a strong modulation of cell cycle regulation and mitotic spindle related pathways, together with the expected, strong modulation of RNA degradation pathway.

Summary/Conclusion: Our data indicate that DIS3 silencing in HMCLs leads to a cell cycle perturbation accompanied to an increase of mitotic defects. To the best of our knowledge, this is the first evidence in the context of the pathological role of DIS3 in MM. These results should be considered of interest considering that DIS3 abnormalities are a common feature of MM patients. Moreover, the transcriptional analyses may contribute to identify putative DIS3 -related pathways and genes (with coding or non-coding potential) involved in tumorigenic mechanisms in MM and possibly implicated in the therapeutic settings.

P850: THE HUMORAL AND CELLULAR RESPONSE TO SARS-COV-2 MRNA VACCINATION IN PATIENTS WITH MULTIPLE MYELOMA AND PRE-MALIGNANT MONOCLONAL GAMMOPATHIES: IMPACT OF OMICRON VARIANT

P. Storti 1,* , V. Marchica 1 , R. Vescovini 1 , V. Franceschi 1 , L. Russo 1 , V. Raimondi 1 , D. Toscani 1 , J. Burroughs Garcia 1 , N. T. Iannozzi 1 , B. Dalla Palma 2 , M. T. Giaimo 1,2 , L. Notarfranchi 1 , G. Donofrio 1 , N. Giuliani 1,2

1 Department of Medicine and Surgery, University of Parma; 2 Hematology, “Azienda Ospedaliero-Universitaria”, Parma, Italy

Background: Multiple myeloma (MM) patients have a high risk of infections with a possible reduced response to vaccines including anti-SARS-CoV-2 one. Currently, the impact the emerging new variants in MM patients underwent to anti-SARS-CoV-2 vaccination is still unknown.

Aims: The humoral and cellular response to SARS-CoV-2 mRNA full vaccination and booster dose and the impact of new spike-mutated variants, including Omicron have been investigated in a cohort of MM patients and those with pre-malignant monoclonal gammopathies.

Methods: We enrolled 38 COVID-19-naïve patients: 6 monoclonal gammopathies of undetermined significance (MGUS), 10 smoldering myeloma (SMM), 9 newly diagnosed MM (NDMM) and 13 relapsed MM (MMR). Peripheral blood (PB) samples were collected before the first dose and 14±2 days after the second dose (POST) of the mRNA BNT162b2 vaccine. In a subset of 16 patients with MM (MMD and MMR), PB samples were also collected after 14±2 days of a heterologous booster dose (BOOSTER) with mRNA-1273 vaccine. SARS-CoV-2 spike IgG antibodies (Abs) were measured by the COVID-SeroIndex Kantaro SARS-CoV-2 IgG test. The fraction of neutralizing Abs (NAbs) against SARS-CoV-2 spike of Wuhan-Hu-1 strain and five variants (alpha, beta, gamma, delta and Omicron) was assessed in the collected sera by a SARS-CoV-2 pseudovirus neutralization assay. Finally, the collected PB-mononuclear cells were stimulated, with overlapping peptides pools spanning SARS-CoV-2 spike glycoprotein and SARS-CoV-2 spike-specific CD4+ and CD8+ T cells were identified by intracellular cytokine staining for Interferon-gamma (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), and CD107a+ using flow cytometry assay.

Results: The seropositivity rate for SARS-CoV-2 spike IgG Abs in the total cohort was 86.8% with 13.2% patients exhibiting no detectable Abs. We found that MMR patients had significantly lower SARS-CoV-2 spike IgG Abs and NAbs compared with MGUS, SMM and NDMM patients after full vaccination. All the analyzed variants, remarkably Omicron, had a significant negative impact on the neutralizing ability of the vaccine-induced Abs in all patients with MM and also with SMM. We also observed a variable spike-specific CD4+ and CD8+ T cell responses. We found that both SMM and NDMM patients had significantly reduced spike-specific IL-2+CD4+ T cell responses compared to MGUS patients. MM patients showed reduced IFN-γ+ and TNF-α+CD8+ T cells, compared to MGUS. In BOOSTER samples, MMR patients retained significantly lower anti-spike-IgG-Abs levels compared to MMD patients. After the booster dose, MMD patients lost the negative impact of the spike variants seen after full vaccination, due to a significant increase of the NAbs titers. On the other hand, MMR patients retained NAbs titers against Beta, Delta and Omicron variants significantly lower than against Whuan-Hu-1 spike. The booster dose increased SARS-CoV-2 spike-specific T cells in both MMD and MMR reaching the 100% of responder patients with an increased percentage of IL-2+CD4+ T cells compared to full vaccination.

Summary/Conclusion: Our study underlines the negative impact of Omicron variants on the neutralizing ability of the vaccine-induced Abs in MM patients as well as in SMM after a full vaccination. Booster dose immunization improved spike humoral and cellular responses in MMD patients and in most, but not all, MMR patients suggesting these patients need to be considered still at risk of Omicron SARS-CoV-2 infection with a clinically relevant disease.

P851: TARGETING GENE DEPENDENCIES IN MYC OVEREXPRESSING MULTIPLE MYELOMA

L. Hasan Bou Issa 1,* , L. Fléchon 1 , W. Laine 1 , A. Ouelkdite 1 , G. Escure 2 , S. Gaggero 1 , T. Ingegnere 1 , R. Sklavenitis Pistofidis 3 , I. M. Ghobrial 3 , T. Facon 2 , S. Mitra 1 , J. Kluza 1 , B. Quesnel 1,2 , S. Manier 1,2

1 Univ Lille, Canther, INSERM UMR-S1277 - CNRS UMR9020; 2 CHU Lille, Department of Hematology, Lille, France; 3 Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States of America

Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by a proliferation of clonal plasma cells in bone marrow. MM progresses from precursor stages, named monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM), to the symptomatic myeloma, MM. MYC abnormalities play a critical role in the disease progression. However, MYC is not therapeutically targetable due to its nuclear localization and the short half-life of the protein.

Aims: To overcome this, we hypothesized that the proliferative advantage promoted by MYC overexpression induces differential genomic dependencies on other signaling pathways thus creates vulnerabilities that can be exploited therapeutically.

Methods: We searched for genetic vulnerabilities associated with MYC overexpression by leveraging genome-scale pooled short hairpin RNA screening data for 236 cancer cell lines from Project Achilles. We generated an isogenic model of MYC overexpression (OE) in U266 cell line using EF1A-C-MYC lentiviral vector. Then, we performed RNA-seq, quantitative proteomics by Tandem Mass Tag mass spectrometry (TMT-MS) and a drug screening with ~2000 compounds. For validation, we performed pharmacological inhibition of glutamine catabolism as well as shRNA-mediated GLS1 knockdown. To determine the functional mechanisms, we used capillary electrophoresis-mass spectrometry (CE-MS) and Agilent Seahorse XF analyzer.

Results: Achilles analysis revealed main dependencies associated with MYC overexpression on glutamine metabolism and specifically GLS1 (glutaminase) and SLC1A1 (glutamine transporter). Using a screening of 2000 small molecules, we further observed that inhibitors of NAD synthesis and mTORC1, which rely on the intracellular glutamine pool, had preferential effect on the proliferation of U266/MYC. Both RNA-seq and quantitative proteomics showed no significant upregulation of glutaminolysis related genes, suggesting a non-oncogenic dependency. Our validation tests confirmed this metabolic dependency, MYC OE cell lines failed to proliferate in the context of glutamine starvation and showed higher sensitivity to CB-839 and V-9302 inhibiting GLS1 and SLC1A5, respectively. Using the Seahorse analyzer, we measured the oxygen consumption rate (OCR) induced by glutamine. U266/MYC cells possess the ability to oxidize glutamine at a higher rate compared to U266/Ctrl. Additionally, higher sensitivity of U266/MYC to CB-839 was observed on both baseline and FCCP-induced OCR highlighting the role of glutamine in controlling mitochondrial OXPHOS in MYC OE cells. To understand the differential metabolic rewiring in MYC OE cells, we performed metabolomic analysis and observed higher GLS1 activity in U266/MYC presented by elevated glutamine to glutamate flux. We also identified the enriched metabolic pathways under GLS1 inhibition. Notably, CB-839 in U266/MYC results in more pronounced changes in TCA cycle and energy debt. This effect was blunted by the co-incubation with a-ketoglutarate, in a rescue experiment. Interestingly GLS1 inhibition was not limited to this effect, but extended to redox balance. CB839 significantly decreased glutathione level in U266/MYC. Furthermore, the intracellular concentrations of the glutamate-dependent amino acids were more depleted under GLS1 inhibition in U266/MYC compared to U266/Ctrl.

Summary/Conclusion: Combining these observations, we were able to identify vulnerabilities to glutamine metabolism in MYC overexpressing cells. These results may lead to developing new therapeutic strategies to target MYC in clinical practice.

P852: NEOANTIGENS PREDICTED BY THE GNE NEOANTIGEN PIPELINE IN PATIENTS DIAGNOSED WITH DE NOVO MULTIPLE MYELOMA

Y. Jiang 1 , C. Henneges 2,* , S. Ruppert 3 , P. Kimes 2 , A. Amitai 2 , A. Qamra 2 , C. Dos Santos 3 , J. N. Paulson 2 , A. Bazeos 1

1 Product Development, Oncology, Roche Products Limited, Welwyn, United Kingdom; 2 Product Development Data Sciences; 3 Oncology Biomarker Development, gRED, Genentech, Inc., South San Francisco, United States of America

Background: Neoantigens are tryptic peptides expressed and presented on MHC-I characteristic for specific tumor mutations. They enable immune surveillance against cancer and may be potential targets for individualized cancer immuno-therapy.

Aims: Here we present neoantigen evaluations from reprocessing de novo Multiple Myeloma patients recruited into the CoMMpass study.

Methods: Whole Exome/Genome Sequencing data as well as RNA-sequencing data was provided by the Multiple Myeloma Research Foundation CoMMpass (SM) Longitudinal Study (research.themmrf.org) [ NCT01454297 ]. Data was processed with the GNE/Roche neoantigen pipeline applying Mutect/Strelka for mutation calling, RNAseq and hlahd for HLA haplotyping prior to predicting peptide binding affinity and rank using NetMHCpan 4.0. Only strong binding peptides (rank score <0.5) are displayed here and analyzed using the nested-model approach: the baseline model of primary therapy (PI, IMiD, both) was extended by a term for presence of individual neoantigens in a gene (prevalence >=5%) for modeling Progression-free (PFS) and Overall Survival (OS) and Number of Progression Events during Observation period. Neoantigens are reported if the Likelihood-Ratio-Test was statistically significant for the Cox PH model or a Poisson regression at the 5% level, or if the AIC was better than the reference model. We also report the c-Index and calibration slope, estimated on a predefined training:validation split.

Results: In N=730 patients, we identified nine proteins with high affinity neoantigens in total (LRT p-value<0.05): MT-CYB, IGHJ5, MAGEC1, IGKV1_5, MT_ND1, IGLV3_1, KRAS, AC245369.1 , and NRAS. All, except NRAS (p=0.055), had a statistically significant Risk-Ratio when included in the Poisson regression model. Of these, IGHJ5 and IGKV1-5 correlated with PFS; MT-ND1 and KRAS correlated with OS. HR and Risk-Ratios with 95% CI in Table. Neoantigens occur at different frequencies ranging from 4.5% to 31.1% in the study population. The most commonly shared neoantigens are from IGLV3-1 (31.1%). Mapping neoantigens to protein domains using the PFAM database, they are present in domains related to energy turn-over (PF00032, PF00033), RAS signaling (PF00071) or in the Ig-like domain (PF07686).

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Summary/Conclusion: We identified nine neoantigens, including two pairs each affecting PFS and OS, that had an effect on the number of progression event rate. Most were associated with immuno-globulin chain mutations, four were found as mitochondrial mutations and or being part of signaling. More data is needed to determine the correlations of these neoantigens with clinical and cytogenetic risk factors in patients with multiple myeloma. In addition, further studies are needed to determine factors that affect the commonality of neoantigens in this population.

P854: HEXABODY-CD38 INDUCES TROGOCYTOSIS AND EFFECTIVELY INDUCES COMPLEMENT-MEDIATED TUMOR CELL LYSIS AFTER TREATMENT WITH DARATUMUMAB OR ISATUXIMAB IN VITRO

I. H. Hiemstra 1,* , K. Santegoets 1 , M. L. Janmaat 1 , W. Ten Hagen 1 , S. Van Dooremalen 1 , B. E. De Goeij 1 , S. Bosgra 1 , A. K. Sasser 2 , E. C. Breij 1

1 Genmab BV, Utrecht, Netherlands; 2 Genmab US, Inc, Plainsboro, NJ, United States of America

Background: HexaBody-CD38 (GEN3014) is a next-generation CD38-specific IgG1 antibody with a hexamerization-enhancing mutation. HexaBody-CD38 is designed to induce strong anti-tumor activity in patients with CD38-expressing hematological malignancies through potent complement-dependent cytotoxicity (CDC) and other Fc-mediated effector functions. The safety and preliminary efficacy of HexaBody-CD38 are currently being evaluated in a first-in-human trial in relapsed/refractory multiple myeloma (MM) patients ( NCT04824794 ). Trogocytosis has been suggested as effector mechanism of daratumumab: reduction of CD38 expression on tumor and immune cells is thought to reduce local immunosuppression and contribute to improved adaptive immune responses against MM cells (Krejcik, 2018, Oncotarget 9 , 33621).

Aims: The present study aimed to increase our understanding of the MoA of HexaBody-CD38, by studying its capacity to induce trogocytosis as well as its capacity to induce CDC in CD38 + tumor cells in the presence of daratumumab or isatuximab in vitro .

Methods: Trogocytosis was evaluated as the amount of membrane transfer in absence of transfer of cytoplasm in a flow cytometry-based assay using Wien-133 cells as target cells and healthy donor monocytes as effector cells. Binding of HexaBody-CD38 to CD38 + cell lines SU-DHL-4, NCI-H929, and Wien-133 that were pre-treated with daratumumab or isatuximab was allowed for 15 min, 1 h, 4 h or 24 h, while daratumumab or isatuximab remained present at saturating concentrations. HexaBody-CD38 mediated CDC of daratumumab or isatuximab-opsonized Wien-133 cells, which were insensitive to CDC induction by daratumumab or isatuximab, was assessed by flow cytometry at 45 min, 4 h, or 24 h after adding human complement in the presence of daratumumab or isatuximab.

Results: HexaBody-CD38 induced dose-dependent transfer of plasma membrane from CD38 + tumor cells to human monocytes (n=7). The mean EC 50 for trogocytosis activity of HexaBody-CD38 was 8.79 ± 2.49 ng/mL (0.018 ± 0.014 nM). This was in the same range as EC 50 s of daratumumab and the HexaBody-CD38 parental antibody without E430G mutation, suggesting that the hexamerization mutation has limited impact on trogocytosis induction.

HexaBody-CD38 was found to compete with daratumumab and isatuximab for binding to CD38. Binding of HexaBody-CD38 to CD38 + cells increased in time and with increasing concentration, generally faster and more extensive in the presence of isatuximab compared to daratumumab. At equimolar concentrations, near-complete displacement of isatuximab and daratumumab was observed after incubations ≥1 h and ≥4 h, respectively. Accordingly, after 24 h HexaBody-CD38 induced comparable maximum CDC of CD38 + cells in presence or absence of daratumumab. Comparable CDC activity in the presence or absence of isatuximab was already observed after 4 h.

Summary/Conclusion: HexaBody-CD38 was shown to induce efficient monocyte-mediated trogocytosis of CD38 + tumor cells in vitro , suggesting HexaBody-CD38 may reduce CD38-associated immunosuppression in the tumor microenvironment. In addition, it has previously been reported that the main differentiating effector mechanism activity of HexaBody-CD38 compared to daratumumab and isatuximab is its increased capacity to induce CDC. Here we confirmed the CDC potency of HexaBody-CD38 even in the presence of saturating concentrations of daratumumab and isatuximab. This suggests that HexaBody-CD38 is capable of inducing CDC of CD38 + myeloma cells in patients who have received prior anti-CD38 mAb treatment with residual daratumumab or isatuximab present in their circulation.

P855: SYNERGISTIC EFFICACY OF COMBINED SUMOYLATION AND PROTEASOME INHIBITION IN MULTIPLE MYELOMA

U. Keller 1,* , G. Heynen 1 , F. Baumgartner 1 , M. Heider 2 , U. Patra 3 , J. Braune 1 , P. Mertins 4 , S. Müller 3 , F. Bassermann 2 , J. Krönke 1 , M. Wirth 1

1 Hematology, Oncology and Cancer Immunology, Charite - Universitätsmedizin Berlin, Berlin; 2 Medicine III, Technische Universität München, Munich; 3 Biochemistry II, Goethe Universität Frankfurt, Frankfurt; 4 Proteomics Core, Max-Delbrück Center for Molecular Medicine, Berlin, Germany

Background: Multiple myeloma (MM) is a genetically and clinically heterogeneous neoplastic plasma cell disorder. Treatment regimens for MM include proteasome inhibitors (PIs) which are routinely combined with dexamethasone and chemotherapeutics or immunomodulatory drugs (IMiDs). While such regimens have resulted in significant improvement of disease control and survival, the development of drug resistance remains a major clinical problem. Relapsed/refractory MM (r/r MM) is associated with particularly poor prognosis. New therapeutic strategies to further improve the management of MM and to overcome drug resistance are therefore urgently needed. SUMOylation is a post-translational protein modification pathway closely related to ubiquitylation. Hyperactive SUMOylation (SUMO) signalling is involved in both cancer pathogenesis and cancer progression. A state of increased SUMOylation has been associated with aggressive cancer biology.

Aims: We sought to identify stress response mechanisms associated with adverse prognosis and PI resistance. The overall goal was to develop novel therapy combinations targeting molecular vulnerabilities associated with advanced and/or PI-resistant MM.

Methods: RNA-sequencing, unbiased mass spectrometry-based proteomics and bioinformatics analyses were used to interrogate the biology and in particular stress response pathways in MM. Cell culture finding were informed with primary MM patient data from gene expression studies and and survival data from clinical trials. Novel drug combination treatments were tested in MM cell-based models in vitro, in MM xenograft models in vivo, and in primary MM patient samples.

Results: Here, we found that r/r MM is characterized by a state of activated SUMOylation, a ubiquitin-related post-translational protein modification pathway. High expression of the SUMO E1 activating enzyme ( SAE1 / UBA2 ) was associated with poor overall survival. Induced resistance to the second generation PI carfilzomib (CFZ) enhanced SUMO pathway activity. Accordingly, CFZ-pretreated patients showed enhanced SUMO pathway activity in the MM compartment. Treatment of MM cell lines with subasumstat (also TAK-981), a novel small-molecule inhibitor targeting the SUMO E1 activating enzyme, showed synergistic treatment efficacy with CFZ in both PI-sensitive and PI-resistant MM cell lines irrespective of the TP53 state. Combination therapy was effective in two murine MM xenograft models, where in vivo growth was significantly inhibited, and in patient-derived primary MM cells in vitro . Mechanistically, combinatorial treatment of subasumstat and CFZ enhanced genotoxic and proteotoxic stress and apoptosis.

Summary/Conclusion: Our findings reveal activated SUMOylation as a therapeutic target in MM and point to combined SUMO/proteasome inhibition as a novel and potent strategy for the treatment of patients with MM.

P856: A SINGLE-CELL FUNCTIONAL PRECISION MEDICINE LANDSCAPE OF MULTIPLE MYELOMA

K. Kropivsek 1,* , P. Kachel 2 , S. Goetze 3,4,5 , R. Wegmann 1 , Y. Severin 1 , B. D. Hale 1 , Y. Festl 1 , J. Mena 1 , A. van Drogen 3,4,5 , N. Dietliker 2 , J. Tchinda 6 , B. Wollscheid 3,4,5 , M. G. Manz 2 , B. Snijder 1

1 Department of Biology, Institute of Molecular Systems Biology, ETH Zurich; 2 Department of Medical Oncology and Hematology, University Hospital Zurich; 3 Department of Health Sciences and Technology, Institute of Translational Medicine; 4 Swiss Multi-Omics Center, PHRT-CPAC, ETH Zurich, Zürich; 5 Swiss Institute of Bioinformatics, Lausanne; 6 Pediatric Oncology, Children’s Research Centre, University Children’s Hospital Zurich, Zürich, Switzerland

Background: Multiple myeloma (MM) is a cancer of plasma cells, defined by complex genetics and extensive intra- and inter-patient heterogeneity. Despite improved patient survival driven by a plethora of treatment options, the disease remains incurable.

Molecularly-guided precision medicine to individualize treatment strategies in MM has had limited success, in part due to the genetic and molecular complexity of the disease. Functional precision medicine, a complementary approach in which patient treatment is guided by the ex vivo drug response of patient cells, has not yet been evaluated for MM systematically.

Aims: Our observational study has two aims: 1) To evaluate single-cell resolved functional precision medicine (also called Pharmacoscopy) as a way to tailor treatment for individual MM patients; 2) To identify new treatment options for, and improve our molecular understanding of, MM.

Methods: In our observational clinical study, image-based ex vivo drug screening (Pharmacoscopy; PCY) measured the single-cell level responses to drug and immunotherapy combinations for 138 patient samples from 98 patients covering all stages of the disease. Single-cell morphologies are analyzed by deep convolutional neural networks, quantifying the cellular composition and morphological myeloma heterogeneity at time of sampling. PCY data is integrated with 1) sample-matched proteomics of purified cell subpopulations, 2) bone marrow serum cytokine profiling, as well as with 3) patient-matched genetics and clinical data. Lastly, ex vivo drug sensitivities are matched to clinical responses.

Results: Our large-scale single-cell morphological characterization of Multiple Myeloma samples reveals three reoccurring cell community modes, which we call PhenoGroups (PGs). These PGs represent different disease stages, with both genetic and inflammatory associations, independently confirmed by orthogonal data. The ex vivo responses to 61 existing and novel therapeutic strategies (ranging from single drugs to quadruple drug combinations) reveal considerable differences in patients’ ex vivo responses to the same treatments. Integration with proteotype data discovers the comprehensive molecular network underlying variable drug sensitivities, recovering known regulators of drug response in myeloma and identifying new possible mechanisms of action. Furthermore, integration with genetics and clinical characteristics across patients recovers known biomarkers, e.g. proteasome inhibitor resistance for TP53 mutant patient samples, and proposes novel biomarkers for individualized patient treatments. The strongest predictor of ex vivo drug sensitivity is the sample composition as captured by the PhenoGroups, which we find is also reflected in the clinical course. Finally, longitudinal follow-up and Kaplan-Meier analyses confirm that Pharmacoscopy-measured drug responses are significantly predictive of the clinical response of patients (Figure 1).

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Summary/Conclusion: Our single-cell functional precision medicine platform (Pharmacoscopy) combined with patient-matched OMICs provides molecular and clinical insights into existing and novel treatment strategies for MM patients. Furthermore, Pharmacoscopy is significantly predictive of clinical response in MM, warranting further clinical investigation.

P857: BORTEZOMIB- AND CARFILZOMIB-RESISTANT MYELOMA CELLS SHOW INCREASED ACTIVITY OF ALL THREE ARMS OF UNFOLDED PROTEIN RESPONSE AND ENRICHMENT OF SIRTUIN SIGNALING PATHWAY

T. Kubicki 1,* , K. Bednarek 2 , M. Kostrzewska-Poczekaj 2 , M. Łuczak 3 , Z. Kanduła 1 , K. Lewandowski 1 , L. Gil 1 , M. Jarmuż-Szymczak 2 , D. Dytfeld 1

1 Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences; 2 Institute of Human Genetics, Polish Academy of Science; 3 Institute of Bioorganic Chemistry, Polish Academy of Science, Poznan, Poland

Background: Proteasome inhibitors (PIs) are among most potent and widely-used class of drugs in multiple myeloma (MM) treatment. One of the main challenges in MM therapy is acquired resistance to drugs, PIs are no exemption from this phenomenon. Several theories were proposed to describe mechanisms responsible for resistance to most commonly used PIs – bortezomib and carfilzomib. One of the most promising explanations involves modulation of unfolded protein response (UPR) pathway. The pathway is initiated by conformational changes in three sensor proteins (PERK, IRE1α, ATF6) in response to overaccumulation of misfolded proteins. Recent study (Sarin et al, Leukemia 2020) suggests that MM cell lines used for in vitro experiments differ substantially in resemblance to patients’ tumors, partially explaining difficulties in translation of results from laboratory models to MM treatment. Here we evaluated MM1S cells, that are among best fitted to resemble actual MM patients’ biology.

Aims: The study aimed at describing functional differences between PI-sensitive MM1S cells (MM1S WT) and their daughter cells, resistant to either bortezomib (MM1S/R BTZ) or carfilzomib (MM1S/R CFZ), as well as between both resistant cell lines.

Methods: Resistant cell lines were generated by continuous culture with increasing concentrations of drugs. Subsequently proteomic profiling of sensitive and resistant cells was performed. To functionally analyze proteomic results proteasome activity and generation of reactive oxygen species (ROS) were measured. Finally, changes in UPR activation were assessed by Western blot analysis of key proteins involved in this pathway.

Results: BTZ- and CFZ-resistant cell lines were successfully generated. IC 50 values were 3-fold higher for the resistant cells (MM1S WT IC 50 =15.2 nM for BTZ, IC 50 =8.3 nM for CFZ; MM1S/R BTZ IC 50 =44.5 nM; MM1S/R CFZ IC 50 =23.0 nM). When exposed to different PI than during resistance generation period MM1S/R BTZ cells were resistant to CFZ (IC 50 =43.5 nM) whereas MM1S/R CFZ cells were similarly sensitive to BTZ as MM1S WT (IC 50 =24.0 nM). Unsupervised principal component analysis revealed that MM1S/R BTZ and MM1S/R CFZ differ significantly from MM1S WT cells and also from each other. Canonical pathway analysis showed similar pathways enriched in both comparisons – MM1S WT vs MM1S/R CFZ and MM1S WT vs MM1S/R BTZ, however important differences were present in terms of statistical significances of particular pathways (Figure). Sirtuin signaling was identified as the most enriched pathway in BTZ-resistant cells (top-4 in CFZ-resistant) and EIF2 signaling in CFZ-resistant cells. In functional studies, both PIs continued to block chymotrypsin-like proteasome activity in resistant cells. The relative reduction in the activity was similar for resistant and sensitive cells – 65% for MM1S/WT in BTZ, 45% for MM1S/R BTZ, 96% for MM1S/WT in CFZ, 77% for MM1S/R CFZ. Baseline activity of all 3 catalytical domains of proteasome was significantly higher in the resistant cells. MM1S/R BTZ cells generated lower amount of ROS in comparison to MM1S WT (64%), while there were no differences in similar comparison for MM1S/R CFZ cells. Both baseline and drug-induced activity of UPR was higher in PI-resistant cells then in MM1S WT and included all three arms of UPR - IRE1α/XBP1s, ATF6 and EIF2α/ATF4 (downstream effectors of PERK).

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Summary/Conclusion: Contrary to some previous reports, performed mainly on other MM cell lines, PI-resistant MM1S cells show upregulation of UPR. This reflects heterogeneity of MM and prompts further studies on UPR role and its interplay with sirtuin signaling.

P858: EPIGENETIC REGULATION OF CD155 ON MULTIPLE MYELOMA CELLS INFLUENCES ANTI-TUMOR T-CELL CYTOTOXICITY

L. Martinez-Verbo 1,* , P. Llinàs-Arias 1 , L. Villanueva 1 , C. A. García-Prieto 1,2 , D. Piñeyro 1 , G. Ferrer 1,3,4 , M. Esteller 1,5,6

1 Cancer Epigenetics, Josep Carreras Leukaemia Research Institute, Badalona; 2 Barcelona Supercomputing Center, Barcelona, Spain; 3 Feinstein Institutes for Medical Research, Northwell Health, New York, United States of America; 4 Centro de investigación Biomédica en Red Cancer, Madrid; 5 Institució Catalana de Recerca i Estudis Avançats; 6 Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain

Background: DNA methylation remains as one of the key molecular players regulating gene expression. In multiple myeloma (MM) and thanks to data available, we identified CD155 as a putative epigenetically regulated gene. Cytotoxic T-cells (CD8+) are crucial in the clearance of malignant cells and their activation is tightly regulated by immune checkpoint events (IC). Tumor cells take advantage of this system and escape immune recognition, avoiding T-cell attacks by inducing exhaustion. CD155 is involved in the CD8+ T-cell activation IC and it is of interest because could act as an inhibitory or stimulatory signal. We investigated the role of CD155 in MM and its importance in T-cell inhibition in-vitro , in relation to its epigenetic state and expression.

Aims: To evaluate the role of CD155 methylation in MM cells and how it affects T-cell activation and tumor elimination.

Methods: The methylation status of the promoter region of CD155 was studied in several MM cell lines from COSMIC database and experimentally validated in four (RPMI-8226, MM.1S, AMO-1 and KMS-12-BM). RPMI-8226 was selected as an unmethylated cell line for CD155 knock down model (depleted expression) and scramble model by lentiviral transduction. We analyzed if CD155 has an impact in cell proliferation and cell cycle with MTT assay and flow cytometry. Co-culture experiments were performed with the CD155 models and T-cells isolated from healthy donors for 48 hours and studied T-cell cytotoxicity by analyzing the remaining MM cells with flow cytometry. Finally, we analyzed CD155 expression and survival in MM from CoMMpass project public data.

Results: We validated the in-silico methylation data by bisulfite sequencing and negative regulation by Western Blot and quantitative PCR in four different cell lines. Depletion of CD155 did not have a significant impact on cell growth, apoptosis or cell cycle. For instance, when we co-cultured the CD155 models with pre-activated T-cells, we detected more cytotoxicity towards CD155 depleted cells, whereas CD155 expressing cells resisted T-cell activity (p=0.02). In order to determine if the inhibition was mediated by interaction CD155-TIGIT, we added 10ug/ml of neutralizing αTIGIT and/or αPD1 antibody to the co-culture systems. While T-cells co-cultured with CD155 depleted cells in the presence of αTIGIT did not change their levels of cytotoxicity, expressing cells were eliminated more successfully. In the presence of αPD1, we saw a significant restoration of T-cell cytotoxicity against both models and antibody combination showed a synergic effect in expressing models’ co-culture whereas CD155 depleted levels remained independent of αTIGIT presence. Supporting th ese results , the data obtained from the CoMMpass project showed the MM patients (N= 793) with higher expression of CD155 had shorter overall survival than lower expressing ones (p<0.001).

Summary/Conclusion: In MM cells, the expression of CD155 is regulated by the methylation status of its promoter region. Unmethylated MM cells expressing CD155 promote T-cell inhibition and its depletion resulted in an improvement of T-cell cytotoxicity activity against malignant cells. The addition of anti-TIGIT and anti-PD1 validated that the CD155 T-cell inhibition is mediated by the interaction with TIGIT, independently of PD1. These findings indicate that CD155 unmethylated MM cases are at higher risk warranting further investigation.

P859: ANTI-CD38 NANOBODY JK36 ALLOWS RELIABLE MRD DETECTION IN DARATUMUMAB TREATED MULTIPLE MYELOMA PATIENTS

E. Meseguer Martinez 1,* , J. Marco Buades 1 , A. García Feria 1 , P. Ribas García 1 , M. J. Fernandez Llavador 1 , A. López Gabaldon 1 , S. Broseta Tormos 1 , E. Francés Aracil 1 , O. Cortés Ortega 1 , E. Donato Martínez 1 , M. Fernandez Zarzoso 1 , M. Panero Ruiz 1 , M. J. Cejalvo Andújar 1 , A. Tolosa Muñoz 1 , M. L. Juan Marco 1 , D. Ivars Santacreu 1 , E. Gómez Beltrán 1 , M. J. Sayas Lloris 1

1 Hematología y Hemoterapia, Hospital Universitario Doctor Peset, Valencia, Spain

Background: The introduction of immunotherapies such as daratumumab, a CD38 monoclonal antibody has improved survival in multiple myeloma (MM), however a few considerations on minimal residual disease (MRD) assessment by flow cytometry have been raised and deserve our attention. In flow cytometry CD38 is frequently used as a gating marker of plasma cells (PCs), but daratumumab can hampering myeloma cell detection. A strategy to overcome this interference is to staining PCs with anti-CD38 nanobody JK36. Nanobodies are single variable domain antibody fragments (VHH) that often expose a long complementarity-determining region 3 (CDR3), feature that allows anti-CD38 nanobody JK36 recognizing a cryptic epitope not masked by anti-CD38 therapies.

Aims: To compare the median fluorescence intensity (MFI) of CD38 multiepitope (ME) antibody and anti-CD38 nanobody in bone marrow samples from MM patients not treated with daratumumab. Secondly, to compare flow cytometric MRD data obtained using CD38-ME and anti-CD38 nanobody from daratumumab treated MM patients.

Methods: Samples were obtained from 10 MM patients not treated with daratumumab to compare the MFI of CD38-ME vs CD38-nanobody at baseline and after incubation with daratumumab at a concentration of 10 nM for 60 min at room temperature. Marrow samples were stained using panel CD38/CD56/CD19/CD138/CD45 with variable CD38, CD38-ME vs anti-CD38 nanobody. Data were analyzed using Kaluza 2.1.1 software (Beckman Coulter). Moreover, 17 bone marrow samples were processed using bulk lysis and subsequently stained using the MM-MRD EuroFlow 8-color antibody combination panel and tubes 1 and 2 with variable CD38, CD38-ME vs anti-CD38 nanobody. Samples were acquired on CytoFlex flow cytometer (Beckman Coulter) and data were analyzed using Infinicyt 2.0 software (Cytognos). Statistical analysis was performed using SPSS version 24, verifying the assumption of normality of the variable, pairwise comparisons were carried out using the T-tests for related samples.

Results: The MFI values of CD38 ME on samples from daratumumab not treated patients was higher than samples after incubation with daratumumab (p=0.003). For CD38 nanobody, no significant differences were observed for the MFI values of samples, independent of daratumumab incubation (p=0,139) (figure 1A). The percentage of loss of MFI were higher in the CD38 ME group (-36,32%) than in the CD38 nanobody group (-0,35%). Regarding 17 patients treated with anti-CD38 therapies, 14 were treated with daratumumab and 3 with isatuximab. Sixteen samples were MRD positive with a sensitivity of 10 -5 or 10 -6 by both approaches. As shown in figure 1B, CD38-ME and CD38 nanobody both showed high and comparable MFI on PCs from patients treated with anti-CD38 therapies.

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Summary/Conclusion: The anti-CD38 nanobody JK36 construct has lower coefficient of variation than CD38 ME antibody. It is a viable alternative to CD38 ME antibody to gate PCs by flow cytometry allowing reliable MRD detection to identify PCs in the presence of anti CD38 biologics. Nanobody technology open new avenues in the detection capability of MRD flow cytometry studies in the era of immunotherapy.

P860: THE DE NOVO DNA METHYLTRANSFERASE DNMT3B PLAYS AN IMPORTANT ROLE IN MM CELL GROWTH, CLONOGENICITY AND DRUG RESPONSE

C. Muylaert 1,* , L. Van Hemelrijck 1 , P. Vlummens 1,2 , A. Maes 1 , K. De Veirman 1 , E. Menu 1 , K. Vanderkerken 1 , E. De Bruyne 1

1 Department of Hematology and Immunology-Myeloma Center Brussels, VUB, Brussels; 2 Department of Internal Medicine, Ghent University Hospital, Ghent, Belgium

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy due to the development of drug resistance (DR). In about half of the MM patients, modifications are observed in epigenetic modifiers (epiplayers) at the time of diagnosis and this frequency is further increased at relapse, indicating an important role for epiplayers in MM cell DR. However, so far, only for two epiplayers, MMSET and EZH2, a clear role in MM cell DR development has been established. With the aim to identify new, clinically relevant epiplayers involved in MM progression and relapse, we recently compared the RNASeq data from matched newly diagnosed and relapsed patients from the MMRF CoMMpass study. We found that the epiplayer DNMT3B is significantly increased in relapsed patients, suggesting a possible role for DNMT3B in MM relapse.

Aims: The aim of this study is to explore the role of DNMT3B in MM cell biology and drug response.

Methods: Publicly available gene expression profiling data of three independent cohorts of newly diagnosed MM patients ( GSE4581 , E-MTAB-372, MMRF-COMMPASS), one cohort of relapsed patients ( GSE9782 ), BM plasma cells (E-MTAB-372), primary MM cells (E- MTAB-372), and human MM cell lines (HMCLs; E-TABM-1088 and E-TABM-937) was used. DNMT3B specific targeting was achieved by using the DNMT3B specific inhibitor Nanaomycin A and genetic inhibition using doxycycline inducible shRNA against DNMT3B. Viability and apoptosis were assessed using a CellTiter-Glo assay and an AnnexinV/7AAD staining respectively. In addition, BrdU incorporation and cell cycle analysis was evaluated to assess cell proliferation. Clonogenic capacity was evaluated by a colony formation assay.

Results: We found that DNMT3B mRNA levels increase during disease progression and high DNMT3B mRNA expression correlates with a worse disease outcome in both newly diagnosed and relapsed patients, indicating a role for DNMT3B in MM progression and DR. In line, Nanaomycin A treatment (up to 72h) led to a significant and dose-dependent decrease in cell viability and proliferation and increase in apoptosis in the XG-2, XG-7 and AMO-1 HMCLs. We also validated the anti-MM activity of Nanaomycin A on human primary MM cells. Since DNMT3B is thought to play a significant role in cancer cell stemness, we next evaluated the effect on clonogenicity. A significant and dose-dependent decrease in the number of colonies was observed when low doses of Nanaomycin A were added to AMO-1 (100 and 200 nM) and XG-2 (30 and 50 nM) cells on the day of plating (day 0), but not when added 7 days after plating (day 7). In contrast, treatment with a high Nanaomycin A concentration (800 nM) significantly reduced the number of colonies at both timepoints, indicating that high Nanaomycin A doses are cytotoxic whereas low doses impair the proliferation and clonogenicity of the MM cells. The anti-clonogenic effect of DNMT3B targeting was confirmed by DNMT3B knockdown in AMO-1 cells. Finally, combining Nanaomycin A (100 nM) with bortezomib (Bz, 4 nM) resulted in a significant decrease in colony formation compared to either Nanaomycin A or Bz treatment alone.

Summary/Conclusion: Taken together, our findings indicate that DNMT3B is a novel promising target to overcome or delay relapse in MM. DNMT3B targeting impairs MM cell proliferation and clonogenicity and sensitizes the cells to the proteasome inhibitor Bz. In the near future, the anti-myeloma activity of DNMT3B targeting will be validated in vivo using the 5TMM murine MM models and the underlying mechanisms will be further determined.

P861: SIALOFUCOSYLATED STRUCTURES ENABLE PLATELET BINDING TO MYELOMA CELLS CONFERRING PROTECTION FROM NK-MEDIATED CYTOTOXICITY

A. Natoni 1,* , M. Cerreto 1 , M. S. De Propris 1 , M. T. Petrucci 1 , I. Del Giudice 1 , S. Intoppa 1 , M. L. Milani 1 , L. Kirkham-McCarthy 2 , R. Henderson 3 , D. Swan 3 , M. O’Dwyer 2 , A. Guarini 4 , R. Foà 1

1 Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy; 2 Biomedical Sciences, School of Medicine, National University of Ireland Galway; 3 Department of Haematology, Galway University Hospital, Galway, Ireland; 4 Department of Molecular Medicine, Sapienza University, Rome, Italy

Background: Multiple myeloma (MM) is a plasma cell disorder that develops in the bone marrow (BM), characterized by an unchecked proliferation and by the ability to disseminate in different parts of the skeleton. Several studies have highlighted a prominent role of platelets in the metastatic dissemination of cancer cells. Indeed, by binding to tumor cells, platelets promote adhesion to target organs and evasion from natural killer (NK)-mediated cytotoxicity. Importantly, tumor/platelet interactions are mostly mediated by P-selectin. We have previously shown that MM cells enriched for Sialyl Lewis X (SLe x ), a sialofucosylated structure recognized by selectins, efficiently home into the BM and become resistant to bortezomib in vivo (Natoni et al , 2017).

Aims: We herein hypothesized that sialofucosylation participates in platelet recruitment on the surface of MM cells via P-selectin, forming a “cloak” that protects tumor cells from NK-mediated cytotoxicity.

Methods: Platelets were purified from peripheral blood samples obtained from healthy individuals. MM cell lines and their derivative that have been enriched for SLex, which strongly bind to selectins, were co-cultured with platelets for 30 min and platelet binding was assessed by flow cytometry using the CD41/CD61 antibody. In some experiments, cells were incubated with platelets in the presence of an anti-P-selectin blocker antibody. To examine the NK-mediated cytotoxicity towards tumor cells, parental and SLex MM cells were co-cultured with platelets for 24 h and then incubated with autologous NK cells expanded in vitro for 7 days. NK-mediated cytotoxicity was assessed after 4h by flow cytometry using Annexin V/7-AAD staining. To examine platelet/MM binding in patients, BM samples from MM patients at different disease stages were analyzed by flow cytometry using a panel of antibodies to identify platelet binding to malignant plasma cells.

Results: In MM cell lines, platelets bound exclusively to SLe x enriched cells, suggesting that efficient binding requires sialofucosylation. This binding could be blocked by a P-selectin antibody, indicating that the interactions between MM cells and platelets are partially dependent on P-selectin. Platelets significantly decreased NK-mediated cytotoxicity in the SLe x enriched (P=0.0025) but not in the parental MM cells, confirming the important role of sialofucosylation in platelet binding. Platelets also bound to malignant cells from primary BM aspirates. Interestingly, the binding to platelets occurred in SLe x positive and negative cells, suggesting that in vivo non-sialofucosylated ligands may also be involved in platelet binding. Importantly, the SLe x CD41/CD61 double positive population seems to accumulate in MM patients at relapse (median diagnosis vs relapse: 0.86 vs 3.03; P=0.0034).

Summary/Conclusion: We herein report that MM cell lines enriched in SLe x interact with platelets in vitro , and that this interaction partially protects MM cells from NK-mediated cytotoxicity. These results suggest that in vivo , platelets may promote immune evasion facilitating the dissemination of MM cells. Moreover, SLe x positive primary malignant BM plasma cells bind platelets, particularly from patients at relapse, suggesting that in vivo these cells may accumulate as the disease progresses and becomes resistant to treatment. Finally, the interactions between platelets and MM cells could be disrupted by a P-selectin antibody, indicating a possible therapeutic target to restrict metastasis and re-sensitize MM cells to NK cells.

P862: SERUM MASS SPECTROMETRY TO ANALYZE DISEASE RESPONSE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA RECEIVING ARI0002H, AN ACADEMIC BCMA-DIRECTED CAR T-CELL THERAPY

I. Ortiz De Landazuri 1,* , A. Oliver-Caldés 2 , M. Español-Rego 1 , M. T. Contreras 3 , A. Zabaleta 4 , C. Agulló 3 , N. Puig 5 , V. Cabañas 6 , V. González-Calle 5 , R. Jiménez 2 , S. Inogés 4 , P. Rodríguez-Otero 4 , B. Martin-Antonio 7 , J. L. Reguera 8 , A. López-Diaz de Cerio 4 , D. Benítez-Ribas 1 , L. G. Rodríguez-Lobato 2 , E. A. González 1 , L. Rosiñol 2 , J. Yagüe 1 , J. M. Moraleda 6 , Á. Urbano-Ispizua 2 , M. V. Mateos 5 , M. Juan 1 , B. Paiva 4 , M. Pascal 1 , C. Fernández de Larrea 2

1 Immunology Department; 2 Hematology Department, Hospital Clínic de Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona; 3 Clinical Biochemistry Department, Hospital Universitario de Salamanca, Salamanca; 4 Clínica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA), IDISNA, CIBERONC, Pamplona; 5 Hematology Department, Hospital Universitario de Salamanca, Salamanca; 6 Hospital Clinico Universitario Virgen de la Arrixaca., Murcia; 7 Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Madrid; 8 Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS/CSIC/CIBERONC), University of Sevilla, Sevilla, Spain

Background: ARI0002h is an academic BCMA-directed CAR T-cell that has been reported as effective in patients (pts) with relapsed/refractory multiple myeloma (RRMM). In these pts, next generation flow cytometry (NGF) in bone marrow (BM) allows the identification of deeper responses than serum protein immunofixation (IFE) after treatment.

Aims: Here, we explore EXENT Quantitative Immunoprecipitation Mass Spectrometry (EXENT QIP-MS) as a highly-sensitive, serum-based, alternative monitoring technique and able to identify therapeutic monoclonal antibodies (t-mAb) interferences.

Methods: Thirty-three RRMM pts with measurable disease in serum by IFE or free light chains were included. All of them received ≥2 prior regimens including a proteasome inhibitor, an immunomodulatory drug and daratumumab, and were refractory to the last one. These pts were treated with ARI0002h, 30 in the setting of the CARTBCMA-HCB-01 clinical trial ( NCT04309981 ) and 3 as compassionate use. The M-protein (MP) was analyzed in serum by EXENT QIP-MS using IgG/A/M/κ/λ isotypic beads at three time points: before ARI0002h infusion, and 28 and 100 days post-infusion. Finally, 98 samples were analyzed by EXENT QIP-MS. Every patient had received daratumumab previously, but the last infusion date varied widely. The presence of disease in BM was also investigated by NGF (sensitivity ≥ 2x10 -6 ) at 28 and 100 days post-infusion. The median follow-up of the pts was 15 months (range 5 to 20).

Results: Before ARI0002h infusion, MP could be identified in 33 (100%) pts by EXENT QIP-MS. After 28 and 100 days post-infusion, MP was also detected by this method in serum in 24 (72.7%) and 18 (56.3%) pts, respectively.

Investigating the ability of EXENT QIP-MS to identify the patient´s MP, interferences due to daratumumab and tocilizumab were successfully detected in serum with a IgG-kappa peak in the mass spectrum of 11693 and 11750 m/z, respectively. Before ARI0002h, daratumumab was identified in 10 pts (30.3%) and persisted even at day +100 in 5 of them (15.2%). Tocilizumab was detected at day +28 in 15 (71.4%) pts out of the 21 receiving it, persisting at day +100 in 3 of them (14.3%).

Then, we compared the results obtained by EXENT QIP-MS and IFE at the two time-points analyzed (n=63). Results were concordant in 81% of samples and discordant in 19% (p<0.0001). All discordances were due to EXENT QIP-MS(+)/IFE(-) results, except in one case. In this patient, the serum was mistakenly considered as IFE(+) (IgG-kappa) owing to the interference of daratumumab. Discordant results were equally noted at day +28 and +100 (Fig.1).

From 25 and 26 MRD-evaluable pts at day +28 and +100, 96% and 92% were MRD(-) in BM by NGF, respectively. NGF was discordant in all cases (EXENT QIP-MS(+)/NGF(-)), except in 1 patient at +28 days and 3 pts at +100 days, reflecting the MP persistence in the absence of BM disease. After 16 months, 75% of pts with EXENT QIP-MS(-)/NGF(-)/IFE(-) at day +28 were alive and without progression, and only 2 pts have relapsed from the disease, both after 12 months (14 and 16 months, respectively).

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Summary/Conclusion: In this study including pts with RRMM and measurable disease, serum EXENT QIP-MS allowed the identification of the MP in all cases with high sensitivity. EXENT QIP-MS was also able to differentiate between the MP and t-mAb, which translated in a correct labeling of treatment response. As compared to both IFE in serum and NGF in BM, EXENT QIP-MS was able to identify residual disease in a higher proportion of cases.

P863: GENERATION OF A FIRST-IN-CLASS INHIBITOR FOR THE MASTER ONCOREGULATOR HNRNP K IN MULTIPLE MYELOMA

Á. Otero Sobrino 1,* , J. Le Coq 2 , P. Aguilar-Garrido 1 , M. Á. Navarro Aguadero 1 , M. I. Albarrán 3 , J. Klett 3 , C. Blanco 3 , R. Fernández-Leiro 4 , J. Martínez-López 5 , M. Velasco 1

1 H12O-CNIO Haematological Malignancies Clinical Research Unit; 2 Electron Microscopy Unit; 3 Biology Section; 4 Genome Integrity and Structural Biology Group, Spanish National Cancer Research Centre (CNIO); 5 Grupo de Hematología Traslacional, Hospital 12 de Octubre de Madrid, Madrid, Spain

Background: Haematological malignancies constitute a plethora of different neoplasms including leukaemia, lymphomas and myeloma. Despite all of them having robust first-line treatments and a myriad of therapeutic alternatives, some remain incurable due to the resistance and refractoriness of tumour cells. The heterogeneous nuclear ribonucleoprotein K, hnRNP K, is a master oncoregulator that binds C-rich tracks of nucleic acids and is implicated in multiple biological functions such as splicing, polyadenylation and translation. Indeed, hnRNP K contributes to treatment resistance and poor outcomes in haematological malignancies. Compared to other hnRNPs, hnRNP K has the unique structural characteristic of containing three K-homology (KH) domains that allows it to bind both ssDNA and RNA sequences, as well as a K-interactive (KI) region that binds to multiple critical proteins such as Src, Fyn or Lyn amongst others. Interestingly, hnRNP K regulates the p53/p21 and c-Myc pathways, and our group has characterised hnRNP K as a driver of leukaemia so far, thus proving its role in haematological neoplasms.

Aims: We aim to develop a first-in-class inhibitor for hnRNP K and test its efficacy in circumventing resistance of haematological malignancies.

Methods: We have produced full-length (FL) hnRNP K protein in-house and verified that it was properly folded and could bind RUNX1 RNA and mRNA sequence-based ssDNA, using Tycho and fluorescence anisotropy technologies. We then carried out a high-throughput screen of over 7,000 small molecules (both FDA and non-FDA approved) using AlphaLISA technology and validated both chemically and biologically the inhibition of hnRNP K with those identified compounds. Lastly, we have performed phenotypical analysis of the effects of the hnRNP K inhibitors in Multiple Myeloma cell line AMO1 and L363 overexpressing hnRNP K through CRISPR synergistic activation mediator (SAM).

Results: We have successfully produced and purified the FL hnRNP K protein, with stable inflexion temperatures (Ti) as measured by Tycho. Furthermore, we corroborated that it maintained its ssDNA and RNA binding abilities through fluorescence anisotropy. Lastly, we set up the AlphaLISA system to screen a library of over 7,000 small molecules and identified circa 10 interesting hits, from which we validated the 5 best based on their inhibition capacity and specificity of hnRNP K. We verified the chemical properties of these small molecules. Lastly, we confirmed that they had a biological effect of decreasing the levels of hnRNP K downstream pathways as well as inducing changes in viability dose-response curves, using Multiple Myeloma cells (AMO1 and L363) genetically modified with CRISPR/SAM technology to overexpress hnRNP K. Likewise, we investigated the absence of such biological effect in hnRNP K-Knock Out (KO) AMO1 and L363 cells, obtained by CRISPR-Cas9.

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Summary/Conclusion: We have identified a first-in-class hnRNP K inhibitor that specifically binds and blocks hnRNP K in modified Multiple Myeloma cell lines. This constitutes a novel and promising therapeutic approach that could help overcome the current drug resistance in haematological neoplasms currently found in the clinic.

This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140) and IF-Marie Sklodowska Curie Actions grant (MAtChinG – 101027864).

P864: CTPS1 IS A NOVEL THERAPEUTIC TARGET IN MYELOMA - SELECTIVE SMALL MOLECULE INHIBITION DELIVERS SINGLE AGENT ACTIVITY AND SYNERGISES WITH ATR INHIBITION

C. Pfeiffer 1,* , P. Beer 2 , H. Asnagli 2 , A. Bolomsky 3 , A. Grandits 4,5 , A. Schneller 1 , J. Huber 1 , N. Zojer 1 , M. Schreder 1 , A. Parker 2 , H. Ludwig 1

1 Department of Medicine I, Klinik Ottakring, Wilhelminen Cancer Research Institute, Vienna, Austria; 2 Step Pharma, Saint-Genis-Pouilly, France; 3 National Cancer Institute, Center for Cancer Research, Lymphoid Malignancies Branch, National Institutes of Health, Bethesda, United States of America; 4 Department of Medicine I, Division of Oncology, Medical University of Vienna; 5 Comprehensive Cancer Center, Vienna, Austria

Background: Recent years have seen major improvements in the treatment of myeloma; however, relapse occurs in the majority of patients. Novel treatment approaches are moving away from cytotoxic chemotherapy towards targeted therapies with defined mechanisms of action. Malignant cells have an increased demand for key cellular components and a reliance on de novo synthesis pathways. CTP synthetase 1 (CTPS1) plays a pivotal role in pyrimidine production, by catalysing the rate limiting step in the de novo synthesis of CTP, which is required for DNA, RNA and phospholipids. Human genetic studies have identified an essential and non-redundant role for CTPS1 in lymphoid cell proliferation which is complemented by the homologous CTPS2 isoform outside the haemopoietic system.

Aims: To evaluate the role of CTPS1 as a novel target in myeloma, elucidate the molecular consequences of CTPS1 inhibition and test rationally designed combination therapy.

Methods: STP938 is a potent small molecule CTPS1 inhibitor with >1,300-fold selectivity for CTPS1 over CTPS2. The effects of STP938 on cell proliferation (metabolic activity, tetrazolium indicator), apoptosis (annexin V) and cell cycle (propidium iodide) were assessed in vitro using 12 myeloma cell lines. The role of CTPS1 was further assessed in CRISPR knock-out (KO) experiments. Activation of proteins in the replication stress and DNA damage response pathways was analysed by western blotting. Additive/synergistic effects of combining STP938 with two different ATR inhibitors were assessed on cell line proliferation and enumerated by Bliss score (Synergyfinder 2.0).

Results: STP938 showed single agent activity against 6 of 12 cell lines tested, with IC 50 values for sensitive lines ranging from 19 to 128 nM (Figure A). STP938 showed cytotoxic activity against sensitive lines, evidenced by induction of apoptosis. KO experiments confirmed that MM cell lines depend on CTPS1 for proliferation. Exposure to STP938 was associated with accumulation of cells in S phase, induction of a replication stress response as evidenced by activation (phosphorylation) of CHEK1, and induction of the DNA damage response pathway as evidenced by activation of CHEK2 (albeit to a lesser extent than activation of CHEK1). Importantly, S phase accumulation, induction of replication stress and induction of DNA damage response was observed in cell lines where STP938 did not produce significant anti-proliferative activity (Figure B). Given the ability of STP938 to activate CHEK1, STP938 was tested in combination with an ATR inhibitor, as ATR is the main upstream activator of CHEK1. The ATR inhibitor ceralasertib demonstrated additive or synergistic activity when combined with STP938 (Figure C). Notably, synergy between STP938 and ATR inhibition, along with induction of apoptosis, was observed in cell lines resistant to single agent STP938. Results were confirmed with a second ATR inhibitor (VE-821, Figure C).

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Summary/Conclusion: Inhibition of CTPS1 by single agent STP938 showed anti-proliferative activity against 6 of 12 myeloma cell lines, and induced replication stress in all cell lines. Combined STP938 and ATR inhibition showed anti-proliferative activity in all cell lines, including those resistant to single agent STP938. These data suggest a model whereby myeloma cells with high background replication stress are sensitive to killing by single agent STP938, whereas cells with lower background replication stress are sensitised by STP938 to cell death induced by ATR inhibition (Figure D). STP938 will shortly enter clinical development for patients with late stage lymphoid neoplasms.

P865: BIOMARKER ANALYSIS TO SUPPORT DOSE OPTIMIZATION OF IBERDOMIDE AS MONOTHERAPY AND IN COMBINATION WITH STANDARD OF CARE TREATMENTS FOR MULTIPLE MYELOMA FROM A PHASE 1/2 TRIAL

M. Amatangelo 1,* , Y. Cheng 1 , W. Pierceall 1 , N. W. van de Donk 2 , S. Lonial 3 , M. Wang 1 , J. Emerson 1 , K. Hong 1 , P. Maciag 1 , T. Peluso 4 , A. Gandhi 1 , A. Thakurta 1

1 Bristol Myers Squibb, Princeton, NJ, United States of America; 2 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Department of Hematology, Cancer Center Amsterdam, Amsterdam, Netherlands; 3 Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 4 Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland

Background: Iberdomide (IBER), a novel cereblon E3 ligase modulator (CELMoD ® ) compound, induces ubiquitination and proteasome-dependent degradation of Ikaros and Aiolos proteins, resulting in tumoricidal and immunomodulatory activity in multiple myeloma (MM). IBER is being investigated for the treatment of relapsed/refractory MM (RRMM) in a phase 1/2 study ( NCT02773030 ). Due to the recent focus by health authorities on dose selection for oncology products other than the maximum tolerated dose (MTD), a comprehensive analysis of pharmacodynamics (PD) and pharmacokinetics (PK) was implemented to support dose optimization of IBER.

Aims: To inform dose selection of IBER as monotherapy, and in combination with dexamethasone (DEX; Iber+d) and with DEX and daratumumab (DARA; IberDd).

Methods: PK samples were collected on treatment of cycles (C) 1–4 to estimate IBER exposure using population PK (area under the concentration curve over 24-h dosing interval [AUC τ ]). Biomarkers included analysis of peripheral blood samples on C1 day (D)1 and mid-cycle through C4 for immunophenotyping, absolute neutrophil counts (ANC), and assessment of serum free light chain (sFLC), as a biomarker of tumor burden. Bone marrow samples were collected at screening and C2D15 for immunohistochemistry.

Results: IBER AUC τ increased in a dose-proportional manner between 0.3 and 1.0 mg as monotherapy and 0.3 and 1.6 mg in combination with DEX, with moderate variability. Median time to maximum serum concentration of IBER was 2–4 h post dose with an elimination half-life of ~12 h. Comparable IBER exposure was observed between monotherapy and Iber+d. Reductions in Ikaros/Aiolos protein levels in tumor cells were observed at all dose levels in both cohorts with a >90% reduction at the 0.45-mg dose. However, a >50% decrease in sFLCs was observed only at doses ≥0.9 mg, and doses of 1.6 mg induced faster and deeper decreases vs lower doses. In the immune compartment, IBER treatment induced similar dose-/exposure-dependent PD changes as monotherapy and Iber+d, which appeared to saturate at higher doses. A >80% reduction in mature B cells and ~2-fold increase in proliferating/activated T and NK cells were observed at doses ≥1.0 mg. PD activity of IBER was not attenuated by prior refractoriness to immunomodulatory drugs (IMiD ® agents) or anti-CD38 therapy. Based on these results, doses between 1.0 and 1.6 mg were tested in the IberDd cohort. PK and PD of IBER were similar with concomitant administration of DARA. In this cohort, a reduction in mature B cells and sFLCs was more consistently observed at 1.6 mg vs lower doses; however, higher IBER exposures were associated with more pronounced decreases in ANC when compared with Iber+d. This was clinically manageable and the MTD of IBER was not determined in any cohort.

Summary/Conclusion: Similar PK and PD results were observed in patients treated with IBER monotherapy, Iber+d, and IberDd. Across all cohorts, a >20% difference in dose was needed for meaningful change in IBER exposure, immune PD began to saturate at doses ≥1.0 mg, and decreases in tumor burden were greatest at the 1.6-mg dose. Based on these results, IBER doses of 1.0, 1.3, and 1.6 mg were chosen for dose optimization of IberDd for RRMM. Taken together with data suggesting immune surveillance is a driver of maintenance therapy efficacy and a desire to increase IBER tolerability with long-term treatment, doses of 0.75, 1.0, and 1.3 mg were selected for dose optimization of IBER monotherapy in the newly diagnosed MM maintenance setting.

P866: CORRELATIVE ANALYSIS TO DEFINE PATIENT PROFILES ASSOCIATED WITH MANUFACTURING AND CLINICAL ENDPOINTS IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS TREATED WITH IDECABTAGENE VICLEUCEL (BB2121)

J. Rytlewski 1,* , J. Fuller 1 , D. R. Mertz 1 , C. Freeman 2 , S. Manier 3 , N. Shah 4 , T. B. Campbell 1

1 Bristol Myers Squibb, Princeton, NJ; 2 Moffitt Cancer Center, Tampa, FL, United States of America; 3 CHU de Lille, University of Lille, Lille, France; 4 University of California San Francisco, San Francisco, CA, United States of America

Background: The anti-B-cell maturation antigen (BCMA) autologous chimeric antigen receptor (CAR) T cell therapy idecabtagene vicleucel (ide-cel; bb2121) has achieved deep and durable responses in relapsed/refractory multiple myeloma (RRMM) patients (Munshi, N Engl J Med 2021). Prior studies have described the correlation between patient characteristics and clinical outcomes from CD19 CAR T cell therapy (Finney, J Clin Invest 2019; Fraietta, Nat Med 2018).

Aims: We sought to define patient profiles correlated with manufacturing and clinical endpoints in RRMM patients treated with ide-cel in clinical trials using unsupervised clustering and multivariate machine learning across multiple key variable domains.

Methods: Clinical and manufacturing data were harmonized across 164 RRMM patients treated with ide-cel in KarMMa ( NCT03361748 ) and KarMMa-2 cohort 1 ( NCT03601078 ). Based on individual multivariate importance, 10 selected peripheral blood mononuclear cell (PBMC), drug product (DP), and in-process cell growth variables were used to define patient clusters. Random forest classifiers were generated to identify patient characteristics associated with manufacturing and clinical endpoints. Wilcoxon rank sum and Kruskal-Wallis tests were used to compare 2 and 3+ categorical groups; Cox proportional hazards were used to compare groups with time-to-event data.

Results: Using an unsupervised method, 4 patient clusters were identified that represented 10%, 57%, 15%, and 18%, respectively, of the total 164 patients. Cluster 4 was the most favorable and was characterized by a higher frequency of T cells in PBMCs; increased T-cell size during manufacturing; higher DP T-cell transduction, potency, and vector copy number; and ultimately a > 3-fold higher CAR T cell yield compared with the least favorable cluster 1. Cluster 2 contained most patients and was associated with intermediate manufacturing endpoints. Patients in cluster 4 had a higher complete response rate, longer progression-free survival, and were defined by lower tumor burden, higher absolute lymphocyte count (ALC), and longer washout period after alkylator treatment, among others (Table).

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Summary/Conclusion: The current study identified patient profiles in RRMM using accessible laboratory or medical history data that correlated with longitudinal outcomes. These findings may inform patients likely to achieve improved outcomes with CAR T cell therapy.

P867: TUMOR PROFILING OF IDECABTAGENE VICLEUCEL (IDE-CEL; BB2121) PATIENTS IN KARMMA SHOWED COMPARABLE RESPONSES IN EXISTING MOLECULAR HIGH-RISK SUBSETS AND PRELIMINARY GENE SIGNATURE OF DURABLE RESPONSE

N. Martin 1,* , A. Xu **1 , N. Stong **1 , J. Rytlewski 1 , O. Finney 2 , T. Campbell 1 , W. Pierceall 1 , E. Flynt 1 , E. Thompson 1 , S. Kaiser 1

1 Bristol Myers Squibb, Princeton, NJ; 2 2seventy bio, Boston, MA, United States of America

Background: Multiple myeloma (MM) tumors exhibit increasing prevalence of high-risk/resistance (HR) features with each successive relapse, leading to poorer outcomes in late-line patients (pts). This may arise primarily as malignancy heterogeneity increases, and with selective pressure from successive treatment regimens. Idecabtagene vicleucel (ide-cel; bb2121) was the first approved anti-BCMA CAR T therapy in late-line relapsed/refractory MM (RRMM), and CAR Ts represent a novel mechanism of action (MOA) in the MM treatment landscape. In the KarMMa study ( NCT03361748 ), high incidences of overall responses (OR) to ide-cel were observed, including in the pt subgroup with HR cytogenetic features.

Aims: Assess baseline multi-omics molecular profiles in KarMMa pt tumors to characterize known and novel molecular features and ide-cel outcomes. Evaluate post–ide-cel tumor composition by analyzing paired pt specimens (pretreatment and at relapse).

Methods: Bone marrow aspirates were collected from KarMMa pts at pretreatment (n=97 RNA, n=68 DNA) and progression (n=64 RNA, n=33 DNA). RNA sequencing and whole genome sequencing was performed on CD138+ cells. Known molecular HR genomic (biallelic p53 inactivation, high cancer clonal fraction del17p, HR t(4,14), and cereblon mutation) and transcriptomic (MDMS8 gene signature) features were analyzed, and correlations with OR and progression-free survival (PFS) evaluated. BCMA mutation and copy number variation were evaluated. Differential gene expression and principal component analyses explored novel transcriptomic signatures and response.

Results: Molecular HR features were identified in 44% (43/97) of pts at pretreatment and 48% (31/64) at progression. Some tumors had multiple HR features consistent with heterogeneity in late-line RRMM. Paired pre/post–CAR T samples did not show dominant enrichment for ≥ 1 HR features at progression. OR and PFS were similar in those with vs. without each molecular HR feature analyzed. One principal component (PC4) was correlated with PFS (p=0.002). The top weighted genes in PC4 may be a novel signature associated with durable ide-cel responses, and exploratory analyses of this signature are ongoing. Loss of one copy of BCMA was observed in 4% (3/68) and 12% (4/33) of pts at pretreatment and progression, respectively. Biallelic loss of BCMA at progression was observed in 6% (2/33) of pts, one of whom had single copy number loss pretreatment.

Summary/Conclusion: Baseline multi-omic–based tumor HR features were not associated with OR. This finding suggests ide-cel activation and expansion remains a critical axis for OR that may not be substantially influenced by tumor intrinsic features. Biallelic loss of BCMA was not observed pretreatment and infrequently at progression, consistent with previous reports. Baseline HR features did not correlate with PFS, and a dominant selection for any one HR feature at progression was not noted. These observations were consistent with previous reports in non-molecularly defined HR subgroups from KarMMa and the hypothesis that CAR T MOA may have a broader spectrum of clinical activity across more diverse molecular subtypes of pts, especially existing HR subsets; this could be a consideration as CAR Ts are developed in earlier lines of therapy. A transcriptional signature was associated with more durable ide-cel responses, which we postulate may outline a distinct suboptimal pretreatment feature in an ide-cel context; further exploration of this preliminary signal is ongoing.

**Authors contributed equally to this abstract.

P868: A CIRCULATING SERUM MIRNAS-BASED MODEL TO PREDICT EARLY MORTALITY IN MULTIPLE MYELOMA PATIENTS TREATED WITH BORTEZOMIB-BASED REGIMENS.

A. Puła 1,2,* , P. Robak 2,3 , D. Jarych 4 , D. Mikulski 2,5 , I. Dróżdż 6 , J. Szemraj 7 , T. Robak 1,2

1 Department of Hematolgy, Medical University of Lodz; 2 Copernicus Memorial Hospital; 3 Department of Experimental Hematology, Medical University of Lodz; 4 Laboratory of Virology, Institute of Medical Biology, Polish Academy of Sciences; 5 Department of Biostatistics and Translational Medicine; 6 Department of Clinical Genetics; 7 Department of Medical Biochemistry, Medical University of Lodz, Lodz, Poland

Background: Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow. Despite the progress made in the treatment of MM, some patients die within the first year of diagnosis. Numerous studies underline the role of miRNAs in the pathogenesis of MM and their potential role in prognosis.

Aims: The aim of this study was to analyze the expression of selected miRNAs in the serum of MM patients treated with bortezomib-based regimens and determine their potential to predict early mortality.

Methods: The study was conducted in prospectively-recruited patients with newly-diagnosed MM who were qualified for bortezomib-based treatment regimens. All were admitted to the Department of Hematology, Medical University of Lodz between 2017 and 2021. Venous blood samples were collected from the patients before treatment initiation, processed, and stored for further use. Total RNA, including miRNA, was isolated from serum, and reverse-transcribed to cDNA. The expression of 31 selected miRNAs was determined using a miRCURY LNA miRNA Custom PCR Panel. The miRNA selection was based on the results of our previous study (Robak et al., Cancers Cancers (Basel) 2020; 12(9): 2569). Three miRNAs were used for expression normalization: hsa-miR-23b-3p, hsa-miR-151a-5p and hsa-miR-152-3p. Clinical data, including patient characteristics on diagnosis, treatment regimen, response to treatment and follow-up were obtained from hospital records. Differential expression analysis, univariate and multivariate logistic regression models were performed using R version 3.6.3.

Results: A total of 69 MM patients were included in the study with a mean age at diagnosis of 64.9 ± 11.0 years. Among them, 17 patients experienced death within 12 months of diagnosis. Patients with early mortality were significantly older (72.6 vs. 62.3 years, p=0.0005), and more frequently men (70.6% vs. 42.3%, p=0.0429). No differences were observed in R-ISS distribution or CRAB symptoms between the groups, nor were any significant differences between percentage myeloma infiltration in the bone marrow and various laboratory findings, including LDH, serum M protein, albumin, CRP, and uric acid. In the differential expression analysis, two miRNAs were significantly downregulated in early mortality group- hsa-miR-328-3p (fold change- FC: 0.72, p=0.0342) and hsa-miR-409-3p (FC: 0.49, p=0.0357). Similarly, hsa-miR-328-3p (OR 0.44, 95%CI: 0.20-0.97, p=0.0415) and hsa-miR-409-3p (OR 0.69, 95%CI: 0.48-0.98, p=0.0400) were found to have a protective effect against early mortality in univariate analyses. The multivariate logistic regression analysis found that miRNAs retained their significance when established clinical prognostic factors were included. The final model consisted of hsa-miR-409-3p (OR 0.61, 95%CI: 0.37-0.99, p=0.0480), hsa-miR-328-3p (OR 0.33, 95%CI: 0.13-0.87, p=0.0254), age (OR 1.13, 95%CI: 1.03-1.23, p=0.0096) and R-ISS 3 (OR 2.91, 95%CI: 0.63-13.47, p=0.1723). A receiver operating characteristics (ROC) analysis for the model yielded an area under the curve (AUC) of 0.863 (95%CI: 0.761-0.965). At the optimal cut-off value of 0.28, the sensitivity and specificity reached 88.2% and 77.5%, respectively.

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Summary/Conclusion: hsa-miR-409-3p and hsa-miR-328-3p are independent factors related to early mortality in MM patients. Our results were used to generate a multiple regression model that may have the potential to predict early mortality. Further external validation of our model is necessary to confirm its clinical value.

P869: PRODUCTION BY GUT MICROBIOTA AS PROGNOSTIC BIOMARKER IN MM

A. Rodríguez-García 1,* , R. Garcia-Vicente 1 , M. L. Morales 1 , R. Gómez-Gordo 2 , P. Justo 1 , C. Cuéllar 1 , J. Sánchez-Pina 1 , D. Gómez-Garre 1 , J. Martínez-López 1 , M. Linares 3

1 Department of Hematology, Hospital Universitario 12 de Octubre, Hematological Malignancies Clinical Research Unit H120-CNIO; 2 Microbiota and Vascular Biology Laboratory, Hospital Clínico San Carlos-Instituto de Investigación Sanitaria San Carlos (IdISSC); 3 Department of Biochemistry and Molecular Biology, Universidad Complutense de Madrid, Madrid, Spain

Background: There is increasing evidence compelling that the microbiota has a strong impact on cancer. The presence of these microorganisms and their activity could have an impact on immune cells and the bone marrow, playing an important role in the progression of multiple myeloma (MM). The short-chain fatty acids (SCFAs) metabolites from gut microbiota have been linked to beneficial effect in the response of MM to treatment.

Aims: In this work we explore whether microorganisms and metabolites of the gut microbiota are imbalanced in monoclonal gammopathies and their possible role in the development of MM.

Methods: We collected 51 patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 12), smoldering (SMO) (n=7) and MM at diagnosis (MMdx) (n = 11), at relapse (MMrf) (n=7) and at complete remission (CR) (n = 7, 3 of them paired with their diagnostic stage). Furthermore, healthy patients (n = 8) were included as controls (C). After the extraction of the microbial DNA from fecal samples, the ribosomal subunit of the bacterial 16S gene was sequenced on the Illumina® MiSeq ™ and bioinformatic analysis and quantification of absolute abundance were carried out. The analysis of metabolites released by the gut bacteria was performed by the quantification of SCFAs acetate, butyrate and propionate on serum samples by MRM (LC-QQQ-MS). The effect of SCFAs on the proliferation of human MM tumor cell lines was evaluated using standard MTT assays.

Results: Patients with MMrf showed the lowest estimated richness with significant differences in Alpha diversity measured by Pielou’s index (Fig 1A). Beta diversity clustered separately and was significant in patients with MM at complete remission compared to MMdx in paired samples (Fig. 1B). Then, we compare the taxonomic composition of the gut microbiota of patients at different stages of the disease, and we found significant differences in the relative abundance of some microorganisms. On one hand, the genus Butyricimonas decreased in MM and its preclinical stages compared to controls and the genus Blautia was lower at MMdx compared to MMcr patients. Both microorganisms are butyrate producers which suggests a possible beneficial role of these microorganisms. In contrast, some genus such as Lachnoclostridium were increased in MMdx (Fig. 1C). To delve into the functional profiles of the microbial communities, we predicted metabolic pathways based on taxonomic profiles and we observed an increased in pathways involving the SCFA propionate in MMcr compared to MMrf (Fig. 1D). The SCFA butyrate was found decreased in MMdx compared with MGUS patients in serum levels (Fig. 1E). The in vitro assays with MM cell lines showed that the SCFAs acetate, butyrate and propionate had an antiproliferative effect (Fig. 1E).

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Summary/Conclusion: Our study showed differences in the abundance levels of gut microbiota both on the progression and the response of the MM disease. The presence of some microorganisms and their metabolites, such as SCFAs, suggests a beneficial role which could be exploited as biomarkers of prognosis.

P870: DEFINING A NOVEL FUNCTION FOR THE POST-TRANSLATIONAL MODIFICATION UFMYLATION IN THE ADAPTIVE RESPONSE TO ARGININE DEPRIVATION IN MULTIPLE MYELOMA

A. Romano 1,* , G. Scandura 1 , C. Giallongo 2 , E. La Spina 1 , A. Barbato 1 , D. Tibullo 3 , K. Todoerti 4 , A. Neri 4 , G. A. Palumbo 2 , F. Di Raimondo 2

1 Dipartimento di Chirurgia e Specialità Medico Chirurgiche; 2 DIPARTIMENTO DI SCIENZE MEDICHE, CHIRURGICHE E TECNOLOGIE AVANZATE; 3 DIPARTIMENTO DI SCIENZE BIOMEDICHE E BIOTECNOLOGICHE, Università degli Studi di Catania, CATANIA; 4 Department of Oncology and Hemato-oncology, University of Milan, Milano, Italy

Background: Our previous work showed that arginine deprivation is associated to multiple myeloma (MM) bortezomib refractoriness, but the molecular basis of this adaptive response is still largely unknown.

Aims: We designed an integrative transcriptional and metabolic study to explore the contemporary transcriptional and metabolic changes occurring upon arginine deprivation in four human myeloma cell lines (HMCLs, U266, NCI-H929, OPM2 and MM1.s).

Methods: HMCLs were individually cultured with customized complete with different Arg concentrations, correspondent respectively to 100%, 25% and 10% of the arginine concentration in MM bone marrow. After 24-48 hours of culture, the cells were collected for global metabolomic analysis (Metabolon Inc) and transcriptome profiling by Illumina platform. Apoptosis and mitochondrial depolarization were measured by FACS. Protein expression was evaluated by western blot analysis.

Results: In all tested HMCLs the progressive arginine deprivation (range 1124-0 µM) induced delay in cell cycle until proliferation arrest (in total lack of arginine in culture media), with increase of G0-G1 length, at specific timepoints for each cell line tested. However global protein synthesis was not affected and surprisingly, in all tested cell lines, the monoclonal component secretion in extracellular medium was preserved after 96 hours of arginine starvation.

At 48 hours of arginine deprivation (range 1124-100 µM) GCN2 pathway was induced in HMCLs with consequent increase of basal levels of ATF4, p62, GABARAP and autophagy flux. After 48 hours of arginine starvation, coupled metabolomics and RNA-seq, disclosed an impairment in the pool of nucleotides and activation of salvage purine pathway, that could explain changes in cell cycle and proliferation. Arginine-deprived MM cells had higher levels of choline and phospho-ethanolamine with lower levels of glycerophosphorylcholine (GPC) compared to controls suggesting an effect of arginine-deprivation on membrane dynamics, associated to increased production of fatty acids, palmitate (16:0) and stearate (18:0), required for the generation of new membranes. Culture in arginine deficient media resulted in complete depletion of reduced glutathione (GSH) and oxidized glutathione (GSSG), with depletion of gamma-glutamylcysteine, indicating increased levels of oxidative stress. Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements, involving upregulation of genes required for the unfolded protein response, NF-kB response to TNF, p53 pathway and networks and proteosome degradation, with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation. In all cell lines, transcripts involved in the UFMylation pathway were affected, with reduction of UFSP2 and increase of UFMYlation targets RPL26, PKM2, ENO-1, PGK-1 and SQSTM1/p62. In a large series of primary samples (9N - 20MGUS - 33SMM - 170MM - 24PPCL - 12SPCL) and 18 MM cell lines, through bioinformatic analysis of previously published datasets (GENE1.0_BAv20_Neri-Gutierrez), we found that myeloma progression was associated to increased expression of UFMYlation targets ENO-1, PGK1 and DLD.

Summary/Conclusion: Taken together, our data clearly indicate how arginine starvation can induce an heterogeneous adaptation processes occur in individual tumour clones. The ER stress adaptive response triggered by arginine deprivation promotes UFMylation activation, due to downregulation of UFSP2 and accumulation of RPN1/RPL26 targets.

P871: ISB 1442, A FIRST-IN-CLASS CD38 AND CD47 BISPECIFIC ANTIBODY INNATE CELL MODULATOR FOR THE TREATMENT OF CD38 POSITIVE HEMATOLOGICAL MALIGNANCIES

C. Grandclement 1 , C. Estoppey 1 , E. Dheilly 1 , M. Panagopoulou 1 , E. Martini 1 , V. Labanca 1 , S. De Angelis 1 , J. Frei 1 , A. Drake 1 , A. Rubod 1 , G. Gudi 2 , V. Udupa 3 , J. Koch Olsen 4 , R. Giovannini 4 , M. A. Doucey 1 , E. Feldman 2 , C. Konto 2 , A. Srivastava 1 , M. Perro 1 , S. Sammicheli 1,*

1 Ichnos Sciences, Epalinges, Switzerland; 2 Ichnos Sciences, New York, United States of America; 3 Glenmark Pharmaceuticals Limited, Mahape, India; 4 Ichnos Sciences, La Chaux-de-Fonds, Switzerland

Background: ISB 1442 is a first in class 2 + 1 biparatopic bispecific antibody targeting CD38 x CD47 using the BEAT ® 2.0 (Bispecific Engagement by Antibodies based on the TCR) platform. The CD38 binding arm consists of bi-paratopic Fabs that strongly bind to CD38 in two different epitopes on tumor cells and do not functionally compete with daratumumab. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the high affinity anti-CD38 Fab arms preferentially drive binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

Aims: ISB 1442 was developed for the treatment of hematologic malignancies that express CD38, including multiple myeloma (MM), acute myeloid leukemia (AML), and T-acute lymphoblastic leukemia (T-ALL), with the intention of overcoming mechanisms of resistance to CD38-targeted therapies such as daratumumab and isatuximab, and minimizing hemagglutination/hemolysis on red blood cell (RBC) observed with anti-CD47 monoclonal antibodies (mAb) such as magrolimab.

Methods: ISB 1442 was tested for its capacity in vitro to induce ADCP, ADCC and CDC relative to daratumumab and magrolimab across a broad range of MM, AML and T-ALL cell lines expressing different levels of CD38 and CD47. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells (ADCC), autologous macrophages (ADCP), and complement from human serum (CDC). In vivo, ISB 1442 was assessed in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice compared to daratumumab and magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays.

Results: In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing MM, AML and T-ALL tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. In the CDC, ADCC and MMoAK assays, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab in MM cell lines.

In vivo, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab.

ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. In monkeys, ISB 1442 showed more pronounced binding on RBC than magrolimab due to higher expression of CD38, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies.

Summary/Conclusion: In summary, we report a novel approach for the treatment for CD38 positive hematologic malignancies by co-targeting CD38 and CD47 using a first in class multispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance.

P872: DEL(1P32) REMAINS A POWERFUL PROGNOSTIC FACTOR IN A LARGE COHORT OF NDMM PATIENTS: AN UPDATE

A. Schavgoulidze 1,2,* , A. Perrot 2,3 , T. Cazaubiel 4 , X. Leleu 5 , S. Manier 6 , L. Buisson 2,7 , S. Maheo 2,7 , L. Do Souto Ferreira 2,8 , R. Lannes 7,9 , L. Pavageau 2,7 , C. Hulin 4 , J.-P. Marolleau 10 , L. Voillat 11 , K. Belhadj 12 , M. Divoux 13 , B. Slama 14 , S. Brechignac 15 , M. Macro 16 , A.-M. Stoppa 17 , L. Sanhes 18 , F. Orsini-Piocelle 19 , J. Fontan 20 , M.-L. Chretien 21 , H. Demarquette 22 , M. Mohty 23 , H. Avet-Loiseau 2,7 , J. Corre 2,7

1 Unit for Genomics in Myeloma, IUCT-Oncopole; 2 CRCT INSERM U1037 and Paul Sabatier University; 3 Hematology Department, University Hospital IUC-Oncopole, Toulouse; 4 Hematology Department, University Hospital, Bordeaux; 5 Hematology Department, University Hospital, Poitiers; 6 Hematology Department, University Hospital, Lille; 7 Unit for Genomics in Myeloma, University Hospital IUC-Oncopole; 8 Unit for Genomics in Myeloma, University Hospital, Toulouse, France; 9 Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States of America; 10 Hematology Department, University Hospital, Amiens; 11 Hematology Department, Hospital, Chalon-sur-Saône; 12 Hematology Department, University Hospital, Créteil; 13 Hematology Department, University Hospital, Nancy; 14 Hematology Department, Hospital, Avignon; 15 Hematology Department, University Hospital, Bobigny; 16 Hematology Department, University Hospital, Caen; 17 Hematology Department, Institut Paoli Calmettes, Marseille; 18 Hematology Department, Hospital, Perpignan; 19 Hematology Department, Hospital, Annecy; 20 Hematology Department, University Hospital, Besançon; 21 Hematology Department, University Hospital, Dijon; 22 Hematology Department, Hospital, Dunkerque; 23 Hematology Department, Saint-Antoine University Hospital, Paris, France

Background: Multiple myeloma (MM) is the second hematological malignancy in the Western countries. Currently, the Revised International Staging System (R-ISS) is widely used to assess patients’ prognosis. Cytogenetic abnormalities (CA) affecting the chromosome 1, gain 1q and del(1p32), were not included in the new criteria of HR CA despite their relatively high frequencies (respectively 35% and 11%), due to insufficient data in the study. However, we have recently confirmed the significant impact on del(1p32) on myeloma‘s prognosis, being the second most pejorative abnormality, just after del(17p).

Aims: The aim of this study is to update our data about the prognostic impact of del(1p32) on a large cohort of NDMM patients.

Methods: Clinical data were obtained from 2551 NDMM patients, followed up for ≥ 36 months or having died or progressed within 36 months post treatment. Informed consent was obtained for all included patients. 1258 patients were treated by an intensive therapy. Follow-up duration was estimated using reverse Kaplan-Meier method. Overall survival (OS) and progression free survival (PFS) curves were estimated using the Kaplan-Meier method and were compared using the log-rank test. Tests were two-sided, and P < .05 was considered significant. All analyses were performed using R version 4.1.1.

Results: We observed 12.4% of patients displaying del(1p32), which was the expected proportion. Median follow-up was 67.4 months. The OS of patients harboring del(1p32) was twice as short as the OS of patients without del(1p32) (60.4 and 123.9 months, respectively, P < 0.0001) (Figure 1). Likewise, PFS was significantly shorter in del(1p32) patients (18.1 and 29.1 months, respectively, P < 0.0001). These poorer outcomes were observed even in patients treated with an intensive therapy (del(1p32) vs. no del(1p32); PFS 26.5 vs. 37.0 months, P < 0.0001; OS 72.0 vs. 127.4 months, P < 0.0001).

We observed higher frequencies of del(17p) and gain 1q in del(1p32) patients (21.3% and 53.2% respectively), compared to general MM population. To check if the poor survival is not only due to these higher levels of association, we have decided to focus on patients harboring del(1p32) without the main high-risk (HR) CA. HR CA were defined by the presence of del(17p), t(4;14) and/or gain 1q. Patients without HR CA had a lower PFS and OS when they carried del(1p32) (del(1p32) vs. no del(1p32); PFS 25.6 vs. 34.8 months, P = 0.0004; OS 83.0 vs. 136.1 months, P = 0.0002).

It is now widely admitted that cumulating HR CA worsen the prognosis. Thus, we have assessed the effect of additional CA on the prognosis of del(1p32) patients. Additional CA were CA defined as HR CA above. As expected, the overall survival of del(1p32) patients significantly decreases when this abnormality was associated with other CA (OS: del(1p32) alone 83.0 months, del(1p32) with 1 HR CA 45.8 months, del(1p32) with 2 HR CA or more 36.5 months, P < 0.0001).

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Summary/Conclusion: Here we have confirmed the pejorative impact of del(1p32) in multiple myeloma on the largest cohort of NDMM patients ever evaluated to our knowledge (316 del(1p32) patients). Our results demonstrate the importance of the detection of del(1p32) at diagnosis because of its huge impact on the prognosis, especially in the era of risk-adapted treatment strategy. The multivariate analysis is in progress.

P873: CARFILZOMIB RESISTANCE IS ASSOCIATED WITH SIGNIFICANT DEREGULATION OF THE BH3 FAMILY PROTEINS

A. Schneller 1,* , A. Bolomsky 1 , N. Zojer 1 , M. Schreder 1 , H. Ludwig 1

1 Clinic Ottakring, Department of Medcin I, Wilhelminen Krebsforschung, Wien, Austria

Background: Despite the remarkable clinical activity of PIs (Proteasome inhibitors) in MM (multiple myeloma), most patients ultimately relapse and become PI-resistant. Deregulation of the apoptosis pathway has been identified as a possible cause of drug resistance in tumor cells. The intrinsic apoptosis pathway is regulated by the BCL2 protein (BH3) family. Previous studies indicated a substantial role of the BH3 family in PI resistance and in response to PI. For instance, the sensitivity to the BCL-2 inhibitor venetoclax is increased in PI resistant AMO-1 cells and a deregulation of BH3 proteins Noxa and MCL1 was noted in MM cell lines exposed to bortezomib. These data suggest that mechanisms involved in PI resistance are closely interconnected with the intrinsic apoptosis pathway orchestrated via the BH3 family, revealing novel opportunities for therapeutic intervention.

Aims: Here, we analyze the interplay between BH3 protein family members in PI resistant vs. sensitive cell lines aiming to decipher survival strategies of PI resistance.

Methods: MM cell lines were exposed to serially increasing doses (max. 25nM) of carfilzomib for > 1 year, leading to the outgrowth of clones with acquired carfilzomib resistance. Analysis of BH3 protein expression and sensitivity to BH3 mimetics in PI resistant cell lines were performed via western blot and viability assays, respectively. Analysis of BH3 protein dependency was done using the flow cytometry-based BH3 profiling method. Thereby, cytochrome c release was measured upon exposure to peptides specifically binding anti-apoptotic BH3 proteins, allowing to identify the relevance of distinct anti-apoptotic BH3 proteins in the cell line of interest. Apoptosis induction of the cells was analyzed by flow cytometry.

Results: Viability assays indicate that all 7 tested carfilzomib resistant MM cell lines developed a cross resistance to the MCL-1 inhibitors S63845 and AZD-5991. Decreased sensitivity to the BCL-2 inhibitor venetoclax as well as to the BCL-xL inhibitor A1331852 was observed in resistant KMS12PE cells. As previously shown, these effects are linked to MDR1 upregulation in carfilzomib resistant cells and reversible by MDR-1 inhibition. BH3 profiling indicates that under naïve conditions the changes in BH3 protein dependency could be observed in 3/7 carfilzomib resistant cell lines. Specifically, resistant KMS12PE cells exhibit a decreased sensitivity to the MCL-1 binding peptide, carfilzomib resistant OPM-2 cells show an increased sensitivity towards peptides binding to BCL-xL and A1 and resistant AMO-1 cells demonstrate a reduced sensitivity a peptide binding to BCL-xL, Bcl-2 and BCL-w. Protein expression analysis of carfilzomib sensitive vs. resistant cell line variants indicate alterations of BCL2 family members (MCL-1, BCL-xL, BCL-2, BAD, BID, BAX, BAK, BIM, NOXA and PUMA) in untreated conditions as well as under carfilzomib treatment (Figure 1). Notably, carfilzomib treatment upregulated BCL-xL in 5 and BAK in 7 out of 7 carfilzomib resistant cell line variants.

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Summary/Conclusion: Our data show significant deregulation of the BH3 protein family in carfilzomib resistant cell lines highlighting the importance of the apoptosis signaling pathway in PI resistance. This is further emphasized by the coherent deregulation of BCL-xL and BAK throughout carfilzomib exposed carfilzomib resistant cell lines when compared to their sensitive cell line variants. Moreover, BH3 profiling indicates a change in dependency on anti-apoptotic BH3 protein in carfilzomib resistant vs. sensitive MM cell lines.

P874: DECIPHERING PLASMA CELL HETEROGENEITY AND TUMOR MICROENVIRONMENT IN LIGHT-CHAIN AMYLOIDOSIS USING SINGLE CELL RNA-SEQUENCING

A. Srivastava 1,* , R. Kumari 1 , S. Adhikari 1 , P. Sergeev 1 , J. J. Miettinen 1 , M. H. Suvela 1 , K. Flanagan 2 , A. Slipicevic 2 , N. N. Nupponen 2 , F. Lehmann 2 , C. Heckman 1

1 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland; 2 Oncopeptides AB, Stockholm, Sweden

Background: Immunoglobulin light-chain amyloidosis (AL) is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils, which deposit on vital organs causing organ dysfunction. Similar to AL, multiple myeloma (MM) is also caused by atypical plasma cell (PC) expansion in the bone marrow. Although, much research has been done on MM to understand disease mechanisms and to develop effective treatments, knowledge related to AL is still limited. Notably, there is little information available regarding the associated tumor microenvironment (TME) of AL. As the TME plays an important role in disease development and treatment response, comprehensive investigation of the AL TME could improve disease diagnosis and treatment. Furthermore, information distinguishing PCs from AL and MM patients is relatively sparse, but could help determine if treatments developed for MM would also be applicable for the treatment of AL.

Aims: The main objective of our study was to decipher the underlying disease mechanisms of the AL at cellular and molecular level by analyzing AL patient samples using single cell RNA sequencing (scRNAseq). To achieve this, we aimed to understand heterogeneity within AL vs MM plasma cells and the TME at the cellular and gene expression level.

Methods: The Finnish Hematology Research Biobank provided 23 viably frozen bone marrow mononuclear cell (BM-MNC) samples from 22 AL patients and 24 samples from 23 MM patients. The BM-MNCs were thawed and sorted based on their CD138 expression and cell viability (7AAD dead cell marker). The viable CD138+ and CD138- cells were mixed in a ratio where CD138+ were enriched, but not exceeding 50%. The Chromium Single Cell 3’RNAseq run and library preparation were done using Chromium™ Single Cell 3’ Reagent version 3 chemistry. 10x Genomics Cell Ranger v3.0.1 pipelines were used for the initial data processing. Further, the quality control and data preprocessing was performed using Seurat 4.1.0. For cell type annotations to identify the clusters, specific cell type markers for the immune cells were obtained from sctype database, GSEA-MSigDB and manual curation.

Results: From the integrated analysis of scRNAseq data from 22 AL patient samples, we identified a total of 27 distinct clusters of cells belonging to 10 unique cell types. Out of the 27 clusters, 10 clusters were identified as PC sub-populations. The proportions of PCs in the samples negatively correlated to the other major cell types such as NK cells, T cells, gamma delta T cells and monocytes. High transcriptional variability was observed between the PC clusters of the AL patients indicating a high degree of heterogeneity among the AL PCs. Analysis of the MM scRNAseq data resulted in 14 PC clusters, however variability between these clusters was not much distinct as compared to AL. A comparison between AL and MM PC clusters resulted in overlapping transcriptional profiles of 2 AL PC clusters with one MM PC cluster. 5 out of 10 clusters of PCs in AL patients were found to be associated with protein processing in the endoplasmic reticulum and apoptosis pathway.

Summary/Conclusion: Within the AL TME, we observed negative correlation of major immune cell types with PCs, suggesting that immune cells may contribute to controlling tumor cell burden in AL. In addition, PC sub-populations in amyloidosis patients are very heterogeneous at the inter-individual level compared to MM patients further indicating that AL patients may benefit from more tailored treatment approaches.

P875: ACTIVATION OF LNCRNA NEAT1 LEADS TO SURVIVAL ADVANTAGE OF MULTIPLE MYELOMA CELLS BY SUPPORTING A REGULATORY LOOP WITH DNA REPAIR PROTEINS: RATIONAL BASES FOR NEAT1 THERAPEUTIC TARGETING IN THE DISEASE

E. Taiana 1,2,* , D. Ronchetti 1,2 , V. K. Favasuli 1,2 , I. Silvestris 1,2 , N. Puccio 1,2 , K. Todoerti 3,4 , C. Bandini 5,6 , I. Craparotta 7 , L. Di Rito 7 , S. Erratico 8,9 , D. Giannandrea 10 , R. Piva 5,6 , M. Bolis 7 , Y. Torrente 9 , R. Chiaramonte 10 , N. Bolli 1,2 , L. Baldini 1,2 , A. Neri 1,2

1 Oncology and Hemato-oncology Department, University of Milan; 2 Hematology, Fondazione Cà Granda IRCCS Policlinico; 3 Pathology Department; 4 Molecular Oncology 1 Department, IRCCS Istituto Nazionale dei Tumori, Milan; 5 Molecular Biotechnology and Health Sciences Department, University of Turin; 6 Città Della Salute e della Scienza Hospital, Turin; 7 Oncology Department, Mario Negri IRCCS; 8 Novystem Spa; 9 Unit of Neurology, Fondazione Cà Granda IRCCS Policlinico; 10 Health Sciences Department, University of Milan, Milan, Italy

Background: Multiple myeloma (MM) is a fatal malignant proliferation of antibody-secreting bone marrow plasma cells characterized by marked genomic instability.

Long non-coding RNA (lncRNA) represents the largest class of non-protein coding genes in the human genome. Their crucial role in solid tumor and hematological malignances, including MM, is well documented.

NEAT1 is a highly expressed nuclear lncRNA, representing the core structural component of paraspeckle organelles. We previously demonstrated that NEAT1 silencing negatively regulates proliferation and viability of MM cells, both in vitro and in vivo , highlighting its pivotal role in DNA integrity maintenance, by regulating the homologous recombination. Despite the increasing information obtained by loss-of-function approaches, there is still a lack of information regarding possible oncogenic benefits acquired by MM cells upon NEAT1 overexpression.

Aims: Taking advantage of the use of the CRISPR/Cas9 SAM genome editing approach, the present study aimed to shed light on the biological and molecular implication of NEAT1 overexpression in MM.

Methods: We adopted the CRISPR/Cas9 Synergistic Activation Mediator editing system to transactivate NEAT1 in AMO-1 MM cell line. LNA-gapmeR antisense oligonucleotide technology was used to silence NEAT1 in MM cells by gymnotic delivery. Hypoxia was induced by culturing the cells into a modular incubator chamber flushed with a mixture of 1% O 2 , 5% CO 2 and 94%N 2 at 37 °C. NEAT1 expression was assessed by qRT-PCR and RNA-FISH. Cell cycle phases distribution and apoptotic cells percentage were assessed by flow cytometry. Clonogenic potential was evaluated by methylcellulose assay. Protein expression was investigated by WB and IF. Cell morphological analysis was performed after May-Grunwald Giemsa staining. Transcriptomic analysis was performed by RNA sequencing, following the TruSeq Stranded Total RNA protocol. Libraries with optimal quality and quantity were run on NextSeq 500. Statistical significance was determined by Student t test analysis; differences were considered significant when P-value was <0.05.

Results: We performed a transcriptomic analysis of AMO-1 cells either transactivated or silenced for NEAT1. Our data revealed a NEAT1 pivotal role in the maintenance of DNA integrity, showing a significant deregulation of almost all the crucial cellular DNA repair mechanisms for both single and double strand breaks. In particular, we found that NEAT1 transactivation affects DNA repair mechanisms through the activation of a molecular axis including two fundamental kinase proteins, i.e. ATM and DNA-PKcs, and the direct target pRPA32. Furthermore, our data strongly confirmed the activation of this NEAT1-orchestrated molecular axis whether MM cells are exposed to nutrient starvation and hypoxia, suggesting its implication in conferring oncogenic and pro-survival properties to MM cells. The disruption of this oncogenic loop leads to MM cell death and the loss of pro survival advantages, thus suggesting that NEAT1 should be considered a crucial factor for MM cells survival.

Summary/Conclusion: Overall, we provided novel important insights into the role of NEAT1 in MM pathobiology, demonstrating that its deregulation strongly correlates with adaptation to stress condition, often associated with late stages of the disease. Taken together, our results suggest that NEAT1 could represent Achilles’ heel for MM cells and should be considered a potential therapeutic target for MM treatment.

P876: INCIDENTAL DISCOVERY OF PROBABLE PATHOGENIC GERMLINE VARIANTS IN MULTIPLE MYELOMA PATIENTS UNDERGOING SOMATIC GENOMIC PROFILING

S. Thibaud 1,* , T. Brander 2 , M. Balwani 2 , T. Mouhieddine 1 , D. Melnekoff 2 , O. Van Oekelen 2 , A. Lagana 2 , J. Houldsworth 3 , A. Chari 1 , J. Richter 1 , L. Sanchez 1 , S. Richard 1 , C. Rodriguez 1 , A. Rossi 1 , H. J. Cho 1 , S. Jagannath 1 , K. Onel 4 , S. Parekh 1

1 Hematology & Medical Oncology; 2 Genetics and Genomic Sciences; 3 Molecular Pathology, Icahn School of Medicine at Mount Sinai, New York; 4 Sema4, Stamford, United States of America

Background: Next-generation sequencing (NGS) is increasingly used to characterize the somatic mutational landscape of patients with multiple myeloma (MM) and guide precision medicine approaches. Somatic genomic profiling (SGP) in cancer patients can lead to the incidental discovery of pathogenic or likely-pathogenic germline variants (PGVs) in ~4% of cases (PMID 26787237). Our previous analysis of whole exome germline data from 895 MM patients in the MMRF CoMMpass dataset showed ~8% of MM patients carry a PGV in a hereditary cancer gene (HCG), and identified shared characteristics among PGV carriers including younger age at diagnosis, higher likelihood of having family history of hematologic malignancy, higher likelihood of t(11;14) MM, and favorable outcomes (Thibaud et al, ASH 2021).

Aims: The present study aims to assess the rate of detection of probable PGVs (PPGVs) in MM patients undergoing targeted SGP.

Methods: We retrospectively reviewed results of 1,614 commercial NGS panels performed by a single genetic testing laboratory on bone marrow aspirates of 1,095 MM patients treated at our institution since 2015 who were not enrolled in the MMRF CoMMpass study. We considered individual variants to be PPGVs when all of the following criteria were met: 1) variant found in a panel of 106 well-established HCGs (same panel used in our prior study, but excluding TP53 given high rate of somatic mutations in MM); 2) variant unambiguously listed as pathogenic or likely pathogenic (P/LP) in ClinVar, or variant classified as “conflicting interpretations of pathogenicity” but reported by ≥2 CLIA-certified labs as P/LP; 3) variant allele frequency (VAF) >30% (per PMID 33236764); 4) variant detected in all available test reports for a given patient (and VAF >30% in all available test reports). Well known founder variants (FV) in specific populations were labeled as PPGV-FVs.

Results: 95 MM patients (8.7%) had a genetic variant meeting criteria to be classified as a PPGV. Two patients had 2 distinct PPGV. PPGV were most commonly detected in the following HCGs (Table 1): APC (n=22), CHEK2 (n=20), ATM (n=7), BRCA1 (n=6), MUTYH (n=6). Of all PPGV carriers, 47 (49%) carried a PPGV-FV. Median VAF for PPGVs was 48.2% (range 30-85%). 34 PPGV carriers had serial NGS tests available (range 2-5 reports); in these patients, VAF standard deviation across serial measurements was <5% in 89% of cases. Taken together, these findings indicate a high likelihood that PPGV detected in these MM patients while undergoing tumor sequencing are indeed incidentally-discovered PGV warranting referral to a genetic counselor for consideration of confirmatory testing.

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Summary/Conclusion: SGP of bone marrow aspirate in MM patients incidentally detected PPGV in well-established HCG in 8.7% of cases. These findings are consistent with those of our prior study, which found an 8.6% prevalence of PGV in newly-diagnosed MM patients. In MM patients undergoing SGP, detection of a mutation involving a HCG and with a VAF >30% should prompt referral to genetic counseling for confirmatory testing, as PGV detection can have clear implications for patients and their families.

P878: HIGH MUTATIONAL BURDEN OF CHROMATIN MODIFYING GENES DEFINES A RELATIVELY INDOLENT SUBGROUP OF PLASMA CELL DYSCRASIAS

X. Wang 1,* , B. Qiao 2 , Y. Yu 1 , J. Xu 2 , L. Zuo 2 , J. Chen 1 , H. Cai 1 , H. Han 1 , X.-X. Cao 1 , C. Sun 2 , J. Li 1

1 Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing; 2 Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Background: Plasma cell dyscrasias (PCDs), including multiple myeloma (MM), light chain amyloidosis (AL), POEMS syndrome, and MGUS, present different clinical features. But as malignancies of plasma cells, they potentially have common mutational patterns associated with chemotherapy and prognosis, which haven’t been elucidated.

Aims: We aimed to reveal the clinically relevant subgroups of MM, AL, POEMS, and MGUS defined by mutational patterns of bone marrow plasma cells (BMPCs).

Methods: We conducted targeted gene sequencing including 370 genes of BMPCs from patients with MM (n=163), AL (n=121), POEMS (n=67) and MGUS (n=13). All the subjects provided informed consent.

Results: Principal component analysis of the top 100 variable genes revealed 2 major subgroups among patients with PCDs (Fig. 1a). Approximately 25% of MM, AL, POEMS and MGUS, subgroup 1 was featured by the heavily mutated genes enriched in histone modifications and chromatin organization, such as KMT2D, EP400, RUNX1, SOX9, KDM5A, NCOR2, and ARID1B. Subgroup2 showed lower total mutational burden and was featured by mutations of proto-oncogene and B cell differentiation-associated genes, such as NCOR1, APOBEC4, KRAS, TP53, FGFR3, FAM46C, and MYC (Fig. 1b). The high mutational burden of chromatin-modifying genes was exclusively observed in subgroup 1 and was comparable among MM, AL, POEMS, and MGUS (Fig. 1c). Patients in subgroup 1 showed worse responses to the first-line CyBorD for MM and AL (Fig. 1d), but better responses to the lenalidomide-based VRD for MM and Rd for POEMS. Overall, patients in subgroup 1 demonstrated longer progression-free survival (PFS), which was observed in MM, AL (Mayo12, Stage I, II), and POEMS, respectively (Fig. 1e).

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Summary/Conclusion: These results provide mechanistic insights into a relatively indolent subgroup among patients with MM, AL, POEMS, and MGUS, which is featured by a high mutational burden of chromatin-modifying genes. This subgroup can be identified by detecting the mutations of chromatin-modifying genes in clinical management. Overall longer PFS and better response to lenalidomide-based first-line therapy can be expected.

P879: EXENT AND FLC MASS SPECTROMETRY FOR SEROLOGICAL IDENTIFICATION AND QUANTIFICATION OF MONOCLONAL IMMUNOGLOBULINS IN MULTIPLE MYELOMA

S. Huhn 1,* , A. M. Poos 1 , E. K. Mai 1 , M. Hänel 2 , H. J. Salwender 3 , J. Dürig 4 , I. W. Blau 5 , C. Scheid 6 , K. C. Weisel 7 , M. Munder 8 , O. Berlanga 9 , U. Bertsch 1 , H. Goldschmidt 1 , N. Weinhold 1

1 Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg; 2 Department of Internal Medicine III, Clinic Chemnitz, Chemnitz; 3 Asklepios Tumorzentrum Hamburg, AK Altona and AK St. Georg, Hamburg; 4 Department for Hematology and Stem Cell Transplantation, University Hospital Essen, Essen; 5 Medical Clinic, Charité University Medicine Berlin, Berlin; 6 Department of Internal Medicine I, University of Cologne, Cologne; 7 Department of Oncology, Hematology and BMT, University Medical Center Hamburg-Eppendorf, Hamburg; 8 Third Department of Medicine, University Medicine Mainz of Johannes Gutenberg, Mainz, Germany; 9 The Binding Site Group, Ltd, Birmingham, United Kingdom

Background: Mass spectrometry has broad potential for screening and monitoring monoclonal gammopathies. The technology overcomes some of the limitations inherent to standard electrophoretic methods for identifying monoclonal immunoglobulins and is amenable to automation.

Aims: Here we assess the performance of the EXENT assays for the quantification of M-proteins and whether free light chain evaluation by mass spectrometry (FLC-MS) offers added sensitivity during multiple myeloma monitoring.

Methods: The MM5 trial compared two regimens of bortezomib-based induction therapy and of lenalidomide consolidation followed by lenalidomide maintenance in newly diagnosed multiple myeloma patients. We used mass spectrometry for identifying serum M-proteins in samples at baseline (n=434), post-induction (n=340), and post-consolidation (n=294). Briefly, polyclonal antibodies (anti-IgG,-IgA,-IgM,-total κ and -total λ [EXENT, The Binding Site, UK] and anti-free κ and -free λ [FLC-MS]) covalently attached to paramagnetic microparticles were separately incubated with serum, washed, treated to elute and reduce patient immunoglobulins, and spotted onto MALDI plates with HCCA matrix. Spectra were generated using the MALDI-TOF-MS system. Light chain mass spectra were obtained using EXENT software to yield immunoglobulin isotype, mass-to-charge ratio ( m/z ) and quantity (g/L). Results were compared to serum protein electrophoresis, immunofixation, and FLC ratios (Freelite®, The Binding Site, UK).

Results: EXENT demonstrated improved sensitivity over immunofixation for identifying the M-protein at each time-point of the MM5 trial: 99% vs. 95% at baseline; 91% vs 80% post-induction; and 63% vs. 46% post-consolidation. Agreement between methods was 87%.

The addition of FLC-MS to EXENT further improved sensitivity to 100%, 95.0%, and 72% at each respective time-point, whereas the combination of immunofixation and FLC ratios resulted in sensitivities of 100%, 90%, and 54%. Overall concordance between EXENT+FLC-MS and the combination of immunofixation and FLC ratios was 100% at baseline, 93% post-induction, and 73% post-consolidation. In all, 158 (11%) samples had an M-protein by EXENT+FLC-MS only, and 28 (2%) by the combination of immunofixation and FLC ratios.

18 and 92 patients achieved an IMWG complete response after induction and consolidation, respectively. EXENT identified an M-protein in 5 (28%), and 23 (25%) of these patients, whereas EXENT+FLC-MS was more sensitive and detected monoclonal proteins in 11 (61%), and 38 (41%) patients, respectively.

There was acceptable quantitative agreement for M-protein measurements between EXENT and serum protein electrophoresis (n=631: Passing-Bablok fit: y=0.85x-0.58; coefficient of determination R 2 = 0.89; Bland-Altman relative bias (95%CI): -32.62% (-123.79-58.55%)). However, there was a substantial discordance in 285 samples <10g/L by serum protein electrophoresis (y=0.58x+0.54; R 2 =0.28; -44.84% (-179.29 – 84.60%)) compared to 346 samples >10g/L (y=0.85x-0.25; R 2 =0.82; -19.91% (95%CI: -97.32 – 23.56%)); suggesting overestimation by serum protein electrophoresis at lower levels.

Summary/Conclusion: EXENT provides superior performance for the identification of M-proteins throughout monitoring. Sensitivity can be improved by incorporating FLC-MS assessments, thus providing a means for determining serological residual disease in patients at complete response by IMWG criteria. Quantification of the M-protein by mass spectrometry compares well to standard methods and offers accurate results at levels below the analytical threshold of confidence for electrophoretic approaches.

P880: OVEREXPRESSION OF PFKFB4 PROMOTES ADAPTATION TO HYPOXIC MICROENVIRONMENT IN MULTIPLE MYELOMA

J. Yang 1,* , F. Wang 1 , B. Chen 1

1 Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China

Background: Multiple myeloma (MM) is a hematological malignancy with monoclonal proliferation of plasma cells, which is closely related to hypoxic bone marrow microenvironment. Previous studies have found that 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) is involved in glycolysis, cell cycle progression, autophagy, and metastasis in various tumor diseases and overexpression of PFKFB4 is associated with poor prognosis. However, the underlying mechanism of PFKFB4 in hypoxia adaptation in MM has not been reported.

Aims: The aim of this study was to explore the role of PFKFB4 in MM cells adaptation to the hypoxic BM microenvironment and provide potential therapeutic target for clinical treatment.

Methods: We analyzed expression profile GSE80140 and GSE80545 downloaded from National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) database, and conducted cell transcriptome sequencing. 5MPN, a specific inhibitor of PFKFB4, was employed to evaluate the effect of PFKFB4 in hypoxia adaptation in MM by cell proliferation, apoptosis, and cycle assays, F-2,6-BP measurement, and Western blot.

Results: MM cell lines (RPMI-8226 and U266) was found to express higher level of PFKFB4 under hypoxia (1% O 2 ) compared to normoxia (20% O 2 ). 5MPN inhibited MM cells proliferation, suppressed intracellular F-2,6-BP concentration, and induced cell cycle arrest in G1 phase. Inhibition of PFKFB4 function had no significant effect on inducing cell apoptosis. Western blot detected the expression of proliferation, apoptosis and cell cycle related proteins, and the results were consistent.

Summary/Conclusion: Overexpression of PFKFB4 promotes MM proliferation and cell cycle progression. 5MPN effectively reserves the process, suggesting that PFKFB4 is a potential therapeutic target in MM.

P881: CDK7 CONTRIBUTES TO METABOLIC REPROGRAMMING IN MM CELLS THROUGH C-MYC MEDIATED TRANSCRIPTIONAL CONTROL OF GLYCOLYTIC GENES

Y. Yao 1,2 , J. Fong Ng 1 , W. D. Park 3 , D. Gramegna 1 , A. Samur 1 , M. Samur 1 , E. Morelli 1 , M. Chesi 4 , C. Mitsiades 1 , K. C. Anderson 1 , C. Lin 3 , N. Munshi 1 , M. Fulciniti 1,*

1 Dana Farber Cancer Institute, Boston, United States of America; 2 Xuzhou Medical University, Xuzhou, China; 3 Baylor College of Medicine, Houston; 4 Mayo Clinic, Scottsdale, United States of America

Background: We have recently elucidated the biological role of CDK7 and explore the functional consequence of its inhibition in MM using chemical and genetic approaches. We reported a specific vulnerability in MM cells upon disruption of the CDK7-driven molecular programs, supporting a model where cell cycle and transcriptional deregulation are counteracted by inhibition of CDK7 activity leading to cell cycle arrest and subsequent apoptosis in MM. Importantly, we established that CDK7 positively correlates with c-MYC and E2F1 transcriptional outputs in primary cells from MM patients, suggesting its regulatory role for both transcription factors.

Aims: Bioinformatics analyses of RNA-seq data revealed that CDK7 kinase activity is required for the expression of many components of the glycolytic cascade; in contrast, most genes for OXPHOS were only weakly modulated by YKL-5-124. Interestingly, recent studies have identified CDK7 as an activator of glucose consumption in lung cancer cells, and autosomal dominant polycystic kidney disease. These observations prompted us to delve into the potential role of CDK7 in regulating glucose metabolism in MM cells.

Methods: CDK7 inhibition was achieved with 1. selective covalent inhibitor YKL-5-124; 2. protein degradation dTAG system; 3. inducible KD/KO systems. The transcriptional networks were evaluated by integrated ChIP-seq and RNA-seq data analysis. Metabolic changes were evaluated by ECAR and OCR assessment using Seahorse analyzer.

Results: To evaluate if CDK7 inhibition suppresses glycolysis we assessed control and treated cells during a glycolysis stress test with Seahorse analyzer using extracellular acidification rate (ECAR) as a measure of glycolytic activity. Compared to control, treated cells displayed a significant defect in glycolysis, glycolytic capacity, and glycolytic reserve in a c-MYC dependent manner. These differences in glycolytic activity upon YKL-5-124 treatment were not accompanied by changes in basal oxygen consumption rate (OCR). Similarly to the drug compound, CDK7 protein degradation using dTAG system diminished glycolytic activity, suggesting that CDK7 regulates glycolysis in MM model systems. Evaluation of tumors retrieved from mice after treatment revealed significant reduction in glycolysis rate. Cancer cells catabolize glucose to lactate in aerobic conditions: a sharp time dependent increase in extracellular lactate release was observed in MM cells over time which was suppressed by YKL-5-124 treatment via regulation of lactate dehydrogenase A (LDHA) expression and enzymatic activity. Together with impairment of aerobic glycolysis, YKL-5-124 treatment resulted in increased ROS levels and induction of apoptotic cell death. The cellular level of ROS is an important oxidative molecule causing endogenous DNA damage. We indeed observed induction of DNA damage as measured by investigation of γH2AX levels after YKL-5-124 treatment. To explore whether YKL-5-124 induced cell death by ROS-mediated mechanisms, we examined the effects of the thiol antioxidant N-acetyl-L-cysteine (NAC) on MM cells upon YKL-5-124 treatment. NAC treatment prevented ROS production, cell death, apoptosis and DNA damage accumulation induced by CDK7i, indicating a causative link between increased ROS levels upon CDK7i and tumor cell death.

Summary/Conclusion: Our data demonstrate that CDK7 through its ability to link cell cycle, transcription and cellular metabolism affects MM cells at several levels, representing an attractive and therapeutically actionable molecular vulnerability in MM.

P882: IDENTIFICATION OF M7G RNA METHYLATION REGULATORS AND M7G-RELATED GENES IN MULTIPLE MYELOMA

M. Yu 1,* , C. Li 2 , J. Xu 3 , M. Liu 2 , X. Cui 2

1 Shandong University of traditional Chinese Medicine; 2 The Second Affiliated Hospital of Shandong University of traditional Chinese medicine; 3 Affiliated Hospital of Shandong University of traditional Chinese medicine, Jinan, China

Background: N7-methylguanosine (m7G) is a positively charged, essential modification at the 5’ cap of eukaryotic mRNA, impacting RNA processing and function, especially mRNA sublocation, translation, and splicing, which is crucial for the regulation of tumor progression. However, the m7G modification pattern of multiple myeloma (MM) is still unclear.

Aims: To explore the impact of m7G regulators on MM prognosis and further conducted a comprehensive investigation on the differences in m7G-related genes among different m7G modification patterns.

Methods: We analyzed the mRNA expression of m7G regulators in healthy donors (n = 9) and MM (n = 174) bone marrow plasma cells, investigated their prognostic values, and comprehensively evaluated the m7G modification patterns of 559 MM bone marrow samples. These data are from Gene Expression Omnibus (GEO) datasets. ConsensusClusterPlus R package (1.56.0) was used to group patients.

Results: Compared with healthy donors, the expression of three m7G writers (METTL1, RNMT, and WBSCR22) was significantly upregulated in MM patients (Fig. 1A). The Kaplan-Meier (K-M) survival curve showed that three m7G regulators (METTL1, WDR4 and RNMT) were significantly correlated with survival, and their high expression was associated with shorter overall survival (OS) (Fig. 1B). Based on the expression levels of m7G regulators, MM patients with qualitatively distinct m7G modification patterns were classified into three different groups. The K-M curve analysis showed a significant survival difference among the subgroups, of which cluster A showed a worse outcome than clusters B and C (Fig. 1C), and the expression of m7G regulators was higher in cluster A (Fig. 1D). To further investigate the biological behaviors of these distinct m7G modification patterns, gene set variation analysis (GSVA) was performed. Compared with clusters B and C, cluster A was markedly enriched in cell proliferation-related pathways. Recent studies have found that the m7G cap in eukaryotic mRNAs is necessary to regulate sublocation, translation, and splicing. Therefore, we used the Pearson correlation coefficient to determine the potential m7G-related mRNAs. As a result, 88 interactions and 88 m7G-related mRNAs (absolute correlation coefficient >0.3, P <0.05) were identified. Further univariate Cox analysis revealed that 27 mRNAs were independent prognostic factors for MM patients, 20 of which were high-risk genes, with varying expression levels in each group (Fig. 1E and 1F). Among them, CDK4, a high-risk gene highly expressed in cluster A (Fig. 1G), is closely associated with cell proliferation by regulating the cell cycle, which is consistent with the GSVA results. As with other cancers, cell cycle dysregulation leads to the progression of MM, where CDK4/6 disorders are common.

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Summary/Conclusion: This work showed that the high expression of m7G regulators was correlated with unfavorable prognostic outcomes in MM. The difference in m7G modification patterns may be an important factor in determining the prognosis of MM patients via causing disorders of the cell cycle.

P883: TARGETING CALCINEURIN/NFATC1 SIGNALING SENSITIZES PROTEASOME INHIBITION TREATMENT IN MULTIPLE MYELOMA

J. Zhang 1,* , X. Luo 1 , T. Xia 1 , K. C. Cheng 1 , M. H. Ng 1

1 Blood Cancer Cytogenetics and Genomics Laboratory, Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China

Background: Proteasome inhibitors (PIs) (e.g., Bortezomib) are key treatments to improve the clinical outcome of multiple myeloma (MM). However, disease relapse from the emergence of PI-resistant clones is the main problem. Insights into the mechanisms underlying PI-resistance will provide novel druggable targets to prevent relapse. It was well-recognized that PIs induce endoplasmic reticulum (ER) stress in MM cells. ER stress is associated with the Calcineurin/nuclear-factor-of-activated-T-cells (NFAT) signaling. Accumulating studies indicated the importance of Calcineurin/NFAT signaling in tumorigenesis and drug resistance. However, the involvement of Calcineurin/NFAT signaling in PI-resistance in MM has not yet been investigated.

Aims: To dissect the role of the Calcineurin/NFAT axis in Bortezomib (BTZ) treatment response and explore the therapeutic potential by combining Bortezomib and Calcineurin/NFAT inhibitors in MM.

Methods: We studied the expression pattern of PPP3CA and NFATC1 from MM datasets and MM cell lines and their association with BTZ responses in MM. The expression of the four principal NFAT members was detected by expression microarray and their activation status by western blotting in MM cell lines. We identified the most prominent NFAT member and assessed its activation under BTZ treatment. A lentivirus-transduction system was used for overexpressing genes, and siRNA transfection was applied to induce NFATC1 knockdown in a BTZ-resistant cell line ARH77. Flow cytometry and western blotting assessed the combinational effect of Calcineurin/NFAT inhibitors and BTZ on MM cells.

Results: The expression level of PPP3CA , which encodes Calcineurin Catalytic subunit A (CnA), was correlated to the poor clinical outcome of recurrent MM patients (MMRF cohort). Calcineurin was highly expressed in ARH77, which is less sensitive to BTZ treatment than other MM cell lines. Among the four NFAT members, we found that NFATC1 and NFATC3 were the most highly expressed members in MM cell lines. However, NFATc1 was the only member suppressed by CsA but activated by the calcium channel agonist Ionomycin in ARH77. Consistent with Calcineurin, NFATc1 was highly expressed in ARH77, with reference to a confirmed NFATc1-positive cell line Raji and the nuclear-NFATc1 overexpressing HEK-293T. Additionally, BTZ activated CnA and NFATc1 time-dependently in ARH77 but failed to activate CnA and NFATc1 in the BTZ-sensitive cell line NCI-H929. Immunofluorescence showed that BTZ triggered the nuclear translocation of NFATc1. From GEO datasets, NFATC1 was overexpressed in patients less responsive to BTZ, but was unchanged in patients subjected to conventional therapies. The Calcineurin/NFATc1 was thus a potential mechanism of BTZ-resistance in MM. Depleting NFATc1 enhanced BTZ-induced DNA damage-related apoptosis in ARH77. The combinational treatment of Calcineurin/NFAT inhibitors (CsA and FK506) and BTZ decreased the nuclear/cytosolic ratio of NFATc1 and promoted apoptosis in two MM cell lines compared to the treatment of BTZ alone.

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Summary/Conclusion: The activation of the Calcineurin/NFATc1 axis is an important mechanism that contributes to BTZ-resistance in MM. Targeting this axis confers translational potential for improving BTZ-based therapies.

P884: POTENTIAL BENEFIT OF POST-TRANSPLANT DOXYCYCLINE IN AL ACHIEVING HEMATOLOGICAL RESPONSE AFTER AUTOLOGOUS STEM CELL TRANSPLANT – LONG TERM FOLLOW UP

N. Abdallah 1,* , A. Dispenzieri 1 , E. Muchtar 1 , F. Buadi 1 , P. Kapoor 1 , M. Lacy 1 , Y. Hwa 1 , A. Fonder 1 , M. Hobbs 1 , S. Hayman 1 , N. Leung 1 , D. Dingli 1 , R. Go 1 , Y. Lin 1 , W. Gonsalves 1 , M. Binder 1 , T. Kourelis 1 , R. Warsame 1 , R. Kyle 1 , S. Rajkumar 1 , M. Gertz 1 , S. Kumar 1

1 Hematology, Mayo Clinic, Rochester, United States of America

Background: Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by multiorgan deposition of fibrils composed of misfolded immunoglobulin light chains. Current treatment strategies targeting the plasma cell clone do not result in immediate organ recovery, and thus effective fibril-directed therapies are needed. Doxycycline has been shown to disrupt amyloid fibril formation in preclinical models. In 2012, we reported initial results on the impact of posttransplant prophylaxis with doxycycline using data from 445 AL amyloidosis patients; prophylaxis with doxycycline was associated with improved survival compared to penicillin in those achieving hematologic response. Since then, few studies have evaluated doxycycline use in AL amyloidosis, mainly in combination with induction chemotherapy, with mixed results. We provide here updated results on outcomes of AL amyloidosis patients who received doxycycline prophylaxis post autologous stem cell transplantation (ASCT) in our institution.

Aims: To evaluate the impact of doxycycline use for post-ASCT antimicrobial prophylaxis on survival in AL amyloidosis patients.

Methods: We included 553 patients who underwent ASCT from July 1996 to June 2014. We excluded patients who died within 100 days of ASCT. Doxycycline 100 mg daily was substituted for penicillin prophylaxis in patients with penicillin allergy; since 2013, doxycycline has become the standard for prophylaxis in AL amyloidosis at our institution. Prophylaxis was typically continued for a year after ASCT. Antibiotic type was determined by review of medical records. The Kaplan-Meier method was used to estimate time to next treatment (TTNT) and overall survival (OS), both measured from the time of transplant; curves were compared using the log-rank test.

Results: Median age was 59 and 60% were male. The median time from diagnosis to ASCT was 4 months; 90% had early ASCT (within 1 year of diagnosis) and 57% received upfront ASCT without prior induction. Penicillin was used for prophylaxis in 67% (372 patients); the rest received doxycycline. Hematologic and organ response rates were similar in the 2 groups. Median follow up from ASCT was 10.5 vs. 13.6 years in the doxycycline and penicillin groups, respectively. Among patients with early ASCT, TTNT was 5.5 (95%CI: 4.2-8.1) vs. 5.8 (95%CI: 4.7-6.8) years, respectively (P=0.95). Median OS was 12.0 (95%CI: 11.0-19.6) vs. 11.0 (95%CI: 9.6-12.7) years, respectively (P=0.17); 5- and 10-year OS rates were 81% vs. 75% and 62% vs. 54% in the doxycycline and penicillin groups, respectively. Among patients with day 100 hematologic response, there was a non-statistically significant trend towards improved OS with doxycycline, but no difference in those without hematologic response: median OS with doxycycline vs. penicillin was 21.1 vs. 14.6 years in patients with day 100 dFLC <4 mg/dL (P=0.18) Figure 1 a . In patients with normal day 100 FLCr, median OS was NR vs. 12.8 years, respectively (P=0.10) Figure 1 b . There was no difference between the 2 groups when OS was stratified by Mayo 2012 stage (1-2 vs 3-4), number of involved organs (1 vs. >1), ASCT period (before or after 2012), or pre-ASCT induction (Yes vs. No).

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Summary/Conclusion: After 13 years of follow up we observed a trend towards improved OS with doxycycline as post-ASCT prophylaxis in patients who achieve a hematological response. Prospective studies are needed to elucidate whether doxycycline improves outcomes in AL amyloidosis and identify the optimal dose, duration, timing, and the subset of patients likely to benefit.

P885: PROSPECTIVE ASSESSMENT OF MYELOMA TUMOUR BURDEN AND BONE DISEASE USING DW-MRI AND EXPLORATORY BONE BIOMARKERS

G. Agarwal 1,* , G. Nador 1 , S. Varghese 1 , H. Getu 1 , C. Palmer 2 , C. Rodgers 3 , C. Pereira 2 , G. Sallemi 2 , K. Partington 4 , N. Patel 4 , R. Soundarajan 5 , R. Mills 5 , R. Brouwer 5 , A. Shah 6 , D. Peppercorn 6 , C. Edwards 2 , M. Javaid 2 , S. Gooding 1 , K. Ramasamy 1

1 Department of Clinical Haematology, Oxford University Hospitals NHS Foundation Trust; 2 The Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford; 3 Department of Clinical Neurosciences, University of Cambridge, Cambridge; 4 Department of Radiology, Oxford University Hospitals NHS Foundation Trust; 5 University of Oxford, Oxford; 6 Department of Radiology, Hampshire Hospitals NHS Foundation Trust, Hampshire, United Kingdom

Background: Key clinical priorities for multiple myeloma (MM) are to reduce tumour burden and complications, of which lytic bone disease is the major cause of morbidity. To guide treatment, there is a pressing need for biomarkers that can accurately quantify MM tumour burden and associated bone disease. To this end, Diffusion-Weighted Magnetic Resonance Imaging (DW-MRI) and bone turnover markers are promising radiological and serum metrics, respectively, although their clinical value in prospective assessment is unclear.

Aims: LOOMIS was a single-centre observational cohort study that evaluated the relative merits of DW-MRI and bone turnover markers in prospectively assessing tumour burden and bone disease when added to standard clinical assessment, in patients with MM and monoclonal gammopathy of undetermined significance (MGUS).

Methods: A total of 67 patients were enrolled (14 newly diagnosed MM, 12 relapsed MM, 15 smouldering MM, 14 MGUS and 12 healthy volunteers) between March 2018 and March 2020. At baseline (and 6-month follow-up for MM/MGUS), participants had a DW-MRI scan and serum measurements of established (P1NP, CTX-1, ALP) and exploratory (DKK1, sclerostin, RANKL:OPG ratio) bone turnover markers. MM/MGUS patients additionally had standard myeloma bloods and Dual-Energy X-ray Absorptiometry (DXA) at each visit, as correlates of tumour burden and bone loss. DW-MRI scans were double reported by expert Radiologists for Apparent Diffusion Coefficient (ADC) measurements of lytic bone lesion(s), and Myeloma Response Assessment and Diagnosis System (MY-RADS) Response Assessment Category (RAC) scoring. Patients were classified by International Myeloma Working Group (IMWG) response criteria as a clinical correlate of therapy response.

Results: On assessment of baseline tumour burden, there was no correlation between single lesion DW-MRI ADC and serum paraprotein [ p>0.05 ]; however, there was moderate positive correlation between serum DKK1 and serum paraprotein [ r=0.39, p=0.04 ]. At follow-up, radiological MY-RADS RAC scoring correlated with conventional IMWG response criteria [ p=0.015 ]; additionally, longitudinal relative change in serum DKK1 differed between IMWG-defined therapy responders (37% decrease) and non-responders (15% increase) [ p<0.01 ]. On baseline assessment of bone loss, there was moderate positive correlation between serum sclerostin and DXA bone mineral density at femoral neck [ r=0.40, p<0.01 ] and lumbar spine [ r=0.54, p<0.001 ]. At follow-up, there was a moderate negative correlation between longitudinal % change in the RANKL:OPG ratio and % change in DXA bone mineral density at femoral neck [ r= -0.45, p<0.01 ].

Summary/Conclusion: Our prospective trial validates DW-MRI-based MY-RADS RAC scoring as a qualitative radiological tool to assess therapy response. Whilst previous work has supported single lytic lesion ADC measurements as a correlate of tumour volume, we did not find this; small sample size and prior chemotherapy in our study may have limited our ability to detect this. In comparison, serum bone turnover markers such as DKK1 may provide a more global measure to quantify myeloma burden both at baseline diagnosis and longitudinally with therapy. Additionally, our data highlight serum sclerostin and RANKL:OPG ratio as potential biomarkers to assess and monitor bone loss. Overall, our study highlights emerging radiological and serum biomarkers of tumour burden and associated bone loss in MM and MGUS, which merit further exploration alongside uniformly treated cohorts, to better understand their clinical utility.

P886: AFRICAN AMERICAN PATIENTS WITH SMOLDERING MULTIPLE MYELOMA MAY HAVE A LOWER RISK OF PROGRESSION COMPARED TO WHITE PATIENTS

T. Akhlaghi 1,* , K. Maclachlan 2 , N. Korde 2 , S. Mailankody 2 , A. Lesokhin 2 , H. Hassoun 2 , S. X. Lu 2 , D. Patel 2 , U. Shah 2 , C. Tan 2 , A. Derkach 3 , O. Lahoud 4 , H. J. Landau 4 , G. L. Shah 4 , M. Scordo 4 , D. J. Chung 4 , S. A. Giralt 4 , S. Z. Usmani 2 , O. Landgren 5 , M. Hultcrantz 2

1 Department of Internal Medicine, Icahn School of Medicine, Mount Sinai Morningside and West; 2 Myeloma Service, Department of Medicine; 3 Department of Epidemiology and Biostatistics; 4 Adult Bone Marrow Transplant Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York; 5 Myeloma Service, Sylvester Comprehensive Cancer Center, University of Miami, Miami, United States of America

Background: The incidence of multiple myeloma (MM) and its precursor stages is two to threefold higher in African Americans (AAs) compared to whites when adjusted for socioeconomic status, age, and sex. However, there is limited information on whether racial background affects the risk of progression from smoldering MM (SMM) to MM.

Aims: To assess the effect of race on the progression from SMM to MM.

Methods: Patients with SMM presenting to Memorial Sloan Kettering Cancer Center between the years 2000 and 2019 and who identified as either AA or white were included in this retrospective study. Baseline patient and disease characteristics were collected at the time of diagnosis including laboratory, imaging, and pathology reports. Differences in distributions of continuous and discrete characteristics were assessed by Kruskal-Wallis and chi-square tests. Time to progression (TTP) was assessed using the Kaplan-Meier method with log-rank test for comparisons. Univariate and multivariate Cox proportional hazard models were used to estimate effects of risk factors on TTP with hazard ratios (HR) and 95% confidence intervals (CI).

Results: A total of 576 patients were included (70 were AA, 12%). Median follow-up time was 3 years in AAs and 4 years in whites. Differences in baseline characteristics between AAs and whites included median age (60 years in AAs [IQR 51-67] vs 64 years in whites [IQR 56-72], p = 0.01), median hemoglobin level (12.3g/dL in AA [IQR 11.8-13] vs 12.8g/dL in whites [IQR 11.8-13.9], p = 0.02), and immunoparesis including 1 or 2 uninvolved immunoglobulins (31% and 10% in AAs vs 56% and 27% in whites, p = 0.002). There was no difference in bone marrow plasma cell percentage (BMPC), M-spike, free light chain ratio, or Mayo-2018 SMM risk score. AA race was associated with a significantly decreased risk of progression in the univariate model (HR 0.57, CI 0.34-0.94). In the multivariate model adjusting for age, sex, and variables associated with an increased risk of progression in the univariate model (BMPC, M-spike, free light chain ratio, immunoparesis and low albumin), AA race remained associated with a decreased risk of progression (HR 0.39, CI 0.16-0.95). Overall, AA patients with SMM had a significantly (p = 0.027) longer median TTP (9.7 vs 6.2 years), and a lower 2-year (12.6% vs 20.1%) and 5-year (34% vs 44.6%) progression rate than whites. Because AA patients were younger at diagnosis, we stratified patients by age group, < 65 vs ≥65 years. In patients < 65 years, there was no difference in progression rate. In patients aged ≥65 years, AA patients continued to have a longer TTP than whites (9.8 vs 5.2 years, p = 0.02).

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Summary/Conclusion: In our retrospective single institution experience, AA patients with SMM had a lower risk of progression to MM compared to whites. Both groups had similar Mayo-2018 risk scores, however, AA patients had a lower degree of immunoparesis at baseline. Future studies are needed to better understand if these differences are explained by differences in disease biology including genomic mechanisms, immune microenvironment, and systemic immune response.

P887: IN-HOUSE ANTI BCMA CAR-T THERAPY FOR THE TREATMENT OF RELAPSED REFRACTORY MULTIPLE MYELOMA: PHASE 1 RESULTS.

N. Asherie 1,* , S. Kfir-Erenfeld 1 , B. Avni 1 , S. Grisariu 1 , M. Assayag 1 , T. Sharon 1 , E. Lebel 2 , C. J. Cohen 3 , M. E. Gatt 2 , P. Stepensky 1

1 Bone Marrow Transplantation; 2 Hematology, Hadassah Medical Center, Jerusalem; 3 The Laboratory of Tumor Immunology and Immunotherapy, Bar-Ilan University, Ramat Gan, Israel

Background: B-Cell Maturation Antigen (BCMA) targeted CAR-T cell therapy is a treatment strategy showing great promise. Recently, anti-BCMA CAR-T cell therapy has been approved by the FDA for treatment of relapsed/refractory multiple myeloma. However, the major drawbacks of this treatment are the length of the manufacturing process, limited access and the excessive cost. Thus, there is an urgent need for development of in-house manufactured CAR-T cell treatments. Based on favorable pre-clinical results using our novel CAR-T-anti BCMA construct, in February 2021 we have launched a BCMA-targeted CAR-T cell based clinical trial treating relapsed refractory (R/R) multipe myeloma (MM) patients ( NCT04720313 ).

Aims: This phase I-II clinical trial is a dose escalation study. The first goal was to explore the safety of our product. The phase I first part of the trial consisted of three escalating cell doses ranging between 150- and 800x10^6 CAR+ cells.

Methods: We enrolled 20 R/R MM patients having failed more than three lines of previous therapies, to receive our CAR-T-anti BCMA product at escalating doses of 150 x 106 (cohort 1, 6 patients), 450 x 106 (cohort 2, 7 patients) and 800 x 106 (cohort 3, 7 patients) cells. The median age was 62 (range: 44-75) years, with a median time of 55 months from initial disease diagnosis (range, 8-241) and a median number of 5.5 previous treatment lines (range, 3-13). Eighty percent had bone marrow (BM) involvement and the majority were categorized as ISS/R-ISS II. The vast majority of patients underwent previous autologous BM transplantation and were refractory to proteasome inhibitors, immunomodulatory agents (IMiD) and anti-CD38 antibody, while only 10 patients (50%) were previously exposed to an anti-BCMA agent (belantamab). Patients in cohort 1 had a higher rate of extramedullary disease, high LDH and massive BM involvement (>50% plasma cells in BM biopsy), while patients in cohort 3 had a higher ECOG performance score and a lower rate of cytogenetic high-risk disease.

Results: At a median follow up of 156 days, overall response was 75% for the entire cohort with 2/6,1/7 and 1/7 patients not responding in cohorts 1, 2 and 3, respectively. Six patients achieved MRD negative CR (4 of them in cohort 3), while 3 achieved MRD positive (or MRD not evaluated) CR and 6 had achieved VGPR (4 of them in cohort 2). The median PFS for the entire study group was 166 days (range, 24-289); 75 days (range, 24-289), 181 days (range, 61-209) and not reached (range, 20-124) for cohort 1,2 and 3, respectively. The median duration of response (DOR) for the entire study group was 151 days (range, 32-259). The progression rate in patients exposed to belantamab, was higher (7/9, 78%) compared to the non-exposed patients (4/11, 36%). On the day of last follow up, 75% of patients were still alive. Eleven (55%) patients progressed. Four (25%) patients died due to disease progression. Ninety percent of patients developed cytokine release syndrome (CRS), mostly on the day of cell infusion (median day 0, range 0-21), while none suffered from grade 3-4 CRS. None of the patients developed immune effector cell-associated neurotoxicity syndrome (ICANS).

Summary/Conclusion: The anti-BCMA CART - cell therapy developed at Hadassah Medical Center proves safe at the three cohorts, with manageable toxicities and evidence of initial short term favorable responses attesting to its efficacy in heavily pretreated R/R MM patients. These data are very encouraging and demonstrate the capability of single center local medical institution to develop, manufacture and deliver in-house CART.

P888: REPLACING STEROIDS IN TRANSPLANT-INELIGIBLE MULTIPLE MYELOMA: THE PHASE 2 ISATUXIMAB-BORTEZOMIB-LENALIDOMIDE-DEXAMETHASONE REST STUDY

F. B. Askeland 1,2,* , E. Haukås 3 , T. S. Slørdahl 4 , D. Schjøll 5 , A. Lysen 1 , E. Hermansen 6 , F. Schjesvold 1,2

1 Oslo Myeloma Center, Department of Hematology; 2 KB Jebsen Center for B Cell Malignancies, Oslo University Hospital, Oslo; 3 Department of Cancer and Blood Diseases, Stavanger University Hospital, Stavanger; 4 Department of Hematology, St. Olavs Hospital, Trondheim, Norway; 5 Oslo Myeloma Center, Department of Hematology, Oslo University Hospital, Oslo; 6 Department of Hematology, Zealand Iniversity Hospital, Roskilde, Denmark

Background: Standard therapies for newly diagnosed multiple myeloma (NDMM) patient’s ineligible for autologous stem-cell transplantation (ASCT) have been lenalidomide-dexamethasone (Rd), bortezomib-lenalidomide-dexamethasone (VRd) or bortezomib-melphalan-prednisone (VMP). The benefit of adding an anti-CD 38 monoclonal antibody (mAb) to the standard treatments of VMP and Rd has been demonstrated in two large phase 3 studies (Facon, 2019. Mateos, 2018). Isatuximab is an anti-CD38 mAb approved in combination with pomalidomide-dexamethasone and carfilzomib-dexamethasone to treat relapsed/refractory MM, and has also shown clinical responses as combination therapy to treat NDMM (Trudel, 2019).

Corticosteroids have been the backbone of most myeloma targeted therapies since the discovery of their effectiveness. Due to the continuous treatment paradigm, the cumulative dose of corticosteroids in patients is substantial. Steroids inflict reduction in both short- and long-term quality of life and increase patients’ receptiveness for infections. Infections are one of the main reasons for complications and death during active first line treatment, and the rate rises with age. Both steroid-free regimens and regimens with reduced corticosteroids have demonstrated improved safety and similar efficacy as regimens containing steroids.

Aims: The study will evaluate isatuximab (Isa) in combination with bortezomib (V) and lenalidomide (R) with minimal dexamethasone (d) as first-line treatment in transplant-ineligible patients. The primary endpoint is the number of patients who achieve measurable residual disease negative (Euroflow NGF 10 -5 ) complete response during and/or after 18 cycles of study treatment. Secondary endpoints include progression free survival, overall survival, overall response rate (ORR), safety evaluations and patient-reported outcome.

Methods: The REST study is an academic, single arm, open-label, phase 2 study of NDMM patients ineligible for ASCT. 50 patients will be included and receive Isa-VRd (Isa:10 mg/kg IV Days 1, 8, 15, 22 during cycle 1, QW cycle 2-18; V: 1,3 mg/m 2 SC Days 1,8,15 during cycle 1-8; R: 25 mg PO Days 1-21 during all cycles; d: 20 mg PO Days 1,8,15,22 only for the first 2 cycles ), all 28-day cycles.

Results: As of January 31, 2022, 17 patients have been enrolled. Baseline characteristics can be found in Table 1. At data-cutoff, the median number of cycles started by patients was 3.6 (range 1-8) and the median duration of exposure was 16 (range 3-28) weeks. 8 patients developed 14 grade ≥ 3 non-hematological adverse eventd (AE), including infections (n=7), fever (n=1), skeletal pain (n=2), acute renal failure (n=1), peripheral sensory neuropathy (n=1), diarrhea (n=1) edema (n=1). There were no AEs leading to definitive treatment discontinuation. At a follow up of 6 months, the ORR was 100% (15/15) and the ≥ very good partial response rate was 53.3% (8/15 patients).

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Summary/Conclusion: Isa-VRd in the transplant ineligible population has a tolerable safety profile. Isa-VRd is showing encouraging preliminary efficacy in NDMM ineligible for transplant. Enrollment and follow-up is ongoing.

Acknowledgements: Sanofi sponsors the REST study.

P889: WILL OUTCOME IMPROVE BY TREATING MULTIPLE MYELOMA PATIENTS AT MRD RELAPSE? THE REMNANT STUDY (RELAPSE FROM MRD NEGATIVITY AS INDICATION FOR TREATMENT)

F. B. Askeland 1,2,* , A.-M. Rasmussen 1,2 , A. Lysen 1 , E. Haukås 3 , A. Eilertsen 4 , M. Moksnes 5 , G. Tsykunova 6 , A. Vik 7,8 , B. D. Eiken 9 , J. H. Sørbø 10 , J. Rolke 11 , K. O. Sand 12,13,14 , R. F. Hallstensen 15 , L. Myrseth 1 , T. S. Slørdahl 16,17 , F. Schjesvold 1,2

1 Oslo Myeloma Center, Department of Hematology; 2 KG Jebsen Center for B Cell Malignancies, Oslo University Hospital, Oslo; 3 Department of Hematology and Oncology, Stavanger University Hospital, Stavanger; 4 Department of Hematology, Akershus University Hospital, Oslo; 5 Cancer and Hematology Center, Vestfold Hospital, Tønsberg; 6 Department of Medicine, Haukeland University Hospital, Bergen; 7 Department of Hematology, University Hospital of North Norway; 8 Institute of Clinical Medicine, UiT, The Artic University of Norway, Tromsø; 9 Department of Oncology, Hospital Østfold, Kalnes; 10 Medical Department, Levanger Hospital, Levanger; 11 Department of Hematology, Sørlandet Hospital, Kristiansand; 12 Department of Medicine, Ålesund Hospital; 13 Department of Reasearch and Innovation, Møre og Romsdal Hospital Trust; 14 Department of Health Sciences in Ålesund, Norwegian University of Science and Technology, Ålesund; 15 Department of Hematology, Nordland Hospital, Bodø; 16 Department of Hematology, St. Olavs Hospital; 17 Norwegian University of Science and Technology (NTNU), Trondheim, Norway

Background: Although many new treatment modalities have become available, multiple myeloma (MM) is nevertheless considered an incurable disease.

In recent years, the importance of achieving measurable residual disease (MRD) negativity is becoming more clearly defined in patients with MM. There is solid evidence that bone marrow MRD negativity is one of the strongest prognostic factors, and deeper responses correlate with increasingly favorable outcomes. The deepest and most durable responses are achieved in the first line (1.L) of treatment.

Aims: The REMNANT study will evaluate if treating MRD relapse after 1.L therapy prolongs progression free survival (PFS) and overall survival (OS) compared to starting treatment at biochemical relapse/progressive disease (PD) in MM patients.

Methods: The REMNANT study is an academic, multicenter, open-label, randomized phase II/III study of newly diagnosed (ND) MM patients eligible for autologous stem cell transplant (ASCT). Few exclusion criteria apply and patients with kidney failure and amyloidosis can be enrolled.

391 NDMM patients (age 18-75 years) eligible for ASCT will be enrolled in the phase II part of the study and receive standard of care (SOC) 1.L treatment according to Norwegian national guidelines; four pre-transplant induction and four post-transplant consolidation cycles of bortezomib, lenalidomide and dexamethasone (VRd). After induction, patients will undergo tandem or single transplant, depending on toxicity and response to first transplant.

Following 1.L treatment 176 patients who are ≥ complete response (CR) and MRD negative (NGF Euroflow) will be enrolled in part 2 of the study. Patients will be randomized in a 1:1 ratio to receive second line treatment at MRD relapse in arm A or at PD in arm B. Randomization will be stratified by R-ISS stage at diagnosis and single versus tandem transplant. At loss of MRD negativity in arm A or at PD in arm B, second line treatment will be carfilzomib, dexamethasone and daratumumab until progressive disease.

In part 2 we will compare different methods for measuring MRD; NGF Euroflow, Next Generation Sequencing (NGS), mass spectrometry and PET-CT. We will evaluate how the different methods compare when it comes to sensitivity and how they correlate to outcome (PFS and OS). The microbiota composition has shown to impact outcome in myeloma patients. Therefore, we will investigate how the microbiota at diagnosis differs in patients who achieve MRD negativity and those who still are MRD positive after treatment.

Results: We will present updated results from part 1 of the study. As of February 16 th 2022, 136 patients have been enrolled in part 1.

Twenty-six patients have been evaluated for MRD status post consolidation. Thirteen patients (50%) were ≥ CR and MRD negative and have been enrolled in part 2 of the study. In Table 1 baseline characteristics and results are presented.

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Summary/Conclusion: Part 1 of the REMNANT study will clarify the overall response rate and depth of response measured by MRD, after SOC Norwegian 1.L treatment

Part 2 will determine if treating patients at MRD relapse, when tumor burden is lower, will improve outcome.

P890: THE OUTCOME OF SECOND PRIMARY MALIGNANCIES DEVELOPING IN MM PATIENTS

I. Avivi 1,* , D. H vesole 2 , J. Dávila Valls 3 , L. Usnarska-Zubkiewicz 4 , V. Milunovic 5 , B. B. Bogumiła Osękowska 6 , A. Kopińska 7 , M. Gentile 8 , B. P. MARTÍNEZ 9 , P. Robak 10 , E. crusoe 11 , R. LUIS GERARDO 12 , M. Gajewska 13 , V. Gergely 14 , M. Delforge 15 , Y. Cohen 16 , G. Alessandro 17 , C. peña 18 , C. Shustik 19 , G. Mikala 20 , K. Žalac 21 , A. H. Denis 22 , B. Peter 23 , K. Weisel 24 , J. martinez lopez 25 , A. Waszczuk-Gajda 26 , M. Krzystanski 27 , A. Jurczyszyn 28

1 Head of Hematology Division, Tel Aviv Medical Center, Tel AVIV, Israel; 2 John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, United States of America; 3 Complejo Asistencial de Avila, Complejo Asistencial de Avila, Avila, Spain; 4 Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw, Poland; 5 Division of Hematology, Clinical Hospital Merkur, Zagreb, Croatia; 6 Pomeranian Medical University in Szczecin, Pomeranian Medical University in Szczecin, Szczecin; 7 Department of Haematology, Oncology and Internal Diseases, University Clinical Centre, Medical University of Warsaw, Warsaw, Poland; 8 Hematology Unit, AO Cosenza, Cosenza, Italy; 9 (University Hospital of Salamanca, Cancer Research Center-IBMCC, Salamanca, Spain; 10 Medical University of Lódź, Medical University of Lódź, Lódź, Poland; 11 : Federal University Of Bahia, University Hospital and Rede D’or Oncologia, Bahina, Brazil; 12 Clinic Barcelona Hospital Universitari, Clinic Barcelona Hospital Universitari, Barcelona, Spain; 13 Department of Hematology and Bone Marrow Transplantation, Silesian Medical University, Katowice, Poland, Warsaw, Poland; 14 Department of Internal Medicine and Haematology, Semmelweis University, Budapest, Hungary; 15 Department of Hematology, UZ Leuven, Leuven, Belgium; 16 Hematology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; 17 Hematology, Department of Medical Science, Surgery and Neuroscience, University of Siena, Siena, Italy; 18 Sección Hematología, Hospital del Salvador, Santiago, Chile; 19 Hematology Department, MUHC - McGill University Health Centre, montreal, Canada; 20 Department of Hematology and Stem Cell Transplantation, South-Pest Central Hospital, Budapest, Hungary; 21 Department of hematology of Clinics for Internal Medicine, University Hospital Center ‘‘Sestre Milosrdnice’’, Zagreb, Croatia; 22 N Ireland Centre for Stratified Medicine, Ulster University, Londonderry, Ireland; 23 Department of Medicine, Warren Alpert Medical School, Brown University, Rhode Island, United States of America; 24 : University Medical-Center Hamburg-Eppendorf, Hamburg, Germany; 25 Nieves Lopez-Muñoz Hospital, Madrid, Spain; 26 Department of Hematology, Oncology and Internal Diseases, Warsaw Medical University, Warsaw; 27 Department of Hematology; 28 Department of Hematology, Jagiellonian University Medical College, crakow, Poland

Background: The continuing improvement in response rates and overall survival (OS) of multiple myeloma (MM) patients exposed to the cumulative effect of multiple anti-MM agents including IMiDs, PIs, mAbs and cytotoxic chemotherapy, has resulted in an increased risk for second primary malignancies (SMPs).

There is minimal detailed information regarding the clinical characteristics of patients who develop SPMs, risk factors predicting development, time to onset, SPM’s management and outcomes in patients treated with novel agents outside clinical trials.

Aims: To investigate the characteristics and outcome of SPMs reported in MM patients and define impact of SPM diagnosis on MM management and outcome.

Methods: A multicenter international ‘real-world’ retrospective analysis, reviewing MM databases of 25 participating centers and analyzing the characteristics and outcome of SPM reported in patients that were treated with novel-based induction. In addition, the impact of SPM diagnosis on MM management and outcome are reported.

Results: Two hundred and one consecutive MM patients, experiencing SPM ≥ 6 months since initiation of first line therapy were included in the analysis. Clinical characteristics included the following: 64% were male, median age at 66±10.57 years, 10% with a prior history of cancer. 36% and 25% of evaluable cases presented with high ISS-3 and high-risk cytogenetics. 87(43%) underwent an autologous hematopoietic cell transplantation and 51(27%) received maintenance therapy. Median number of lines of therapy prior to SPM defection was 2 (1-7).

With a median follow-up of 50± 45 months since MM diagnosis, 115 patients (57%) were diagnosed with solid cancers: colorectal cancer (n=20, 17.4%), lung (n=18, 15.6%), breast (n=14, 12%), prostate (n=8, 7%), melanoma (n=10, 9%), pancreas (n=10, 9%), bladder (n=7, 6%) and gastric (n=7, 6%), skin cancer (n= 34, 16.9%) and cancer of unknown origin (n=9, 4.5%). 46 patients (22.9%) developed hematological cancers, including lymphoma and MDS/AML; reported in 7(6%) and 27 (13.4%) cases respectively. 79% (n=158) were actively treated for SPM and 39.5% responded to therapy.

Forty Eight percent (n=98) of the patients were concurrently receiving an anti-MM therapy at the time of SPM diagnosis. MM treatment remained unchanged in 27 patients, discontinued and not substituted with any other anti-MM therapy until MM progression in 59 patients and changed to a new anti-MM therapy in 12 patients.

Within a median follow-up period of 112 months since MM diagnosis and 31 months since SPM detection, 50.5% (n-101) have died; 32 due to solid cancer (32/101, 28%), 11 due to hematological cancer (11/46, 24%) and 13 (6%) due to MM progression. Cause of death was not available for 45 cases. Median OS since MM diagnosis and since SPM detection were 63 months and 8.5 months, respectively (Figure 1). (Detailed analysis regarding causes of death and risk factors to be presented at the meeting).

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Summary/Conclusion: With the continuing improvement in OS, a higher proportion of MM patients are likely to develop SPM. OS since SPM diagnosis is shortened, approaching 8.5 months only, suggesting that the SPMs, originating in this scenario, are generally aggressive. Surveillance for early detection and treatment of SPMs are warranted. Further studies are necessary to determine the factors associated with the development of SPM to avoid precipitating agents especially in patients with lower risk disease since these individuals should have extended survival that may be substantially shortened by the development of treatment-related SPM.

P891: IXAZOMIB, POMALIDOMIDE AND DEXAMETHASONE (IXPD) IN RELAPSED OR REFRACTORY MULTIPLE MYELOMA (RRMM) CHARACTERIZED WITH HIGH-RISK CYTOGENETICS. IFM 2014-01

A. Bobin 1 , S. Manier 2 , J. De Keizer 1 , L. Karlin 3 , A. Perrot 4 , C. Hulin 5 , D. Caillot 6 , C. Mariette 7 , P. Lenain 8 , V. Richez 9 , M. Tiab 10 , C. Touzeau 11 , A. Jaccard 12 , O. Decaux 13 , C. Araujo 14 , K. Belhadj 15 , L. Benboubker 16 , C. Déal 17 , M. Macro 18 , L. Vincent 19 , B. Arnulf 20 , B. Bareau 21 , T. Braun 22 , C. Calmettes 23 , D. mamoun 24 , H. Zerazhi 25 , H. Demarquette 26 , P. Feugier 27 , C. Fohrer-sonntag 28 , S. Godet 29 , M.-O. Petillon 2 , H. Avet-Loiseau 4 , X. Leleu 1,*

1 CHU de Poitiers, Poitiers; 2 chu de lille, lille; 3 CHU de Lyon, Lyon; 4 CHU de Toulouse, Toulouse; 5 CHU de Bordeaux, Bordeaux; 6 CHU de Dijon, Dijon; 7 CHU de Grenoble, Grenoble; 8 CHU de Rouen, Rouen; 9 CHU de Nice, Nice; 10 CH la Roche sur Yon, La Roche Sur Yon; 11 CHU de Nantes, Nantes; 12 CHU de Limoges, Limoges; 13 CHU de Rennes, Rennes; 14 CH de Bayonne, Bayonne; 15 CHU de Créteil-APHP, Créteil; 16 CHU de Tours, Tours; 17 Intergroupe Francophone du Myélome, Paris; 18 CHU de Caen, Caen; 19 CHU de Montpellier, Montpellier; 20 Hopital Saint Louis, Paris; 21 Hopital Privé Sevigne, Cesson Sevigne; 22 Hopital Avicenne, Bobigny; 23 CH de Périgueux, Périgueux; 24 CHU de Angers, Angers; 25 CH de Avignon, Avignon; 26 CH de Dunkerque, Dunkerque; 27 CHRU de Nancy, Nancy; 28 CHU de Strasbourg, Strasbourg; 29 CHU de Reims, Reims, France

Background: High risk (HR) cytogenetics remains of poor prognosis, particularly in the RRMM setting. IFM 2010-02, Pd in advanced HR RRMM, had demonstrated limited activity with no addition of a proteasome inhibitor (PI), and a good safety profile. We hypothesized that addition of Ixazomib (oral PI at increased dose density) to Pd (IxPd) in HR RRMM would improve convenience, thus adherence to treatment, and in parallel improve efficacy with no increased toxicity.

Aims: We aimed to determine the efficacy and safety profile of the association ixazomib, pomalidomide and dexamethasone HR RRMM.

Methods: Eligible patients had a RRMM in L2 refractory to lenalidomide but not to pomalidomide and ixazomib. HR was defined by presence of either del(17p) and/or t(4;14) at diagnosis or study entry. Patients received 17 induction cycles, 21-days long, consisting of ixazomib 3mg/day (d 1, 4, 8 and 11), pomalidomide 4mg/day (d1 to 14) and weekly dexamethasone, followed by a maintenance phase of 28-day cycles with ixazomib 4mg/day (d 1, 8 and 15) and pomalidomide 4mg/day (d 1 to 21), until progression. The primary endpoint was time to progression (TTP). The number of patients to be recruited was initially calculated based on an expected doubling of the median TTP obtained in IFM 2010-02 with Pd in either 2 HR RRMM population, but the number was recalculated due to difficulties in recruitment to demonstrate an expected doubling of the median TTP from 3 months, as a whole. No statistical comparison can be done across HR groups given that the study was powered to analyze the 2 groups as a whole and not separately with 26 patients.

Results: Twenty-six patients were enrolled in the study. Median age was 72 years (Interquartile range: 67-78). Ten patients presented with del(17p) and twelve patients with t(4;14). All patients were refractory to lenalidomide.

With a median follow-up time of 9.8 months, the median TTP was 9.7 months (95% confidence interval (CI) 4.4;-). The median OS was 12.2 months (CI95% 11.1;-). The ORR and ≥VGPR rate was 15 (58%) pts and 7 pts (27%) in the study as a whole. The ≥VGPR rate was 30% and 33% in del(17p) and t(4;14), respectively. No CR was observed with IxPd in our series.

The most common adverse events (≥10% occurrence) were neutropenia (n=13), thrombocytopenia (n=12), asthenia (n=9), anemia (n=7), rash (n=4), diarrhea (n=3), dizziness (n=3), general physical health deterioration (n=3), muscle spasms (n=3), peripheral oedema (n=3), pyrexia (n=3). Ten SUSARs were declared with no modification to the safety profile of ixazomib and pomalidomide according to the independent data monitoring committee.

Summary/Conclusion: The study IFM 2014-01/IxPd in HR L2 RRMM met its primary end point objective since the median TTP was superior to 3 months, the median TTP reported in IFM2010-02 as a whole, with the addition of Ixazomib to Pomalidomide and dexamethasone in this population characterized with a very poor outcome. This data confirms the importance of proteasome inhibitors in HR RRMM. This phase 2 study needs confirmation in a larger cohort.

P893: CARFILZOMIB IN COMBINATION WITH LENALIDOMIDE AND DEXAMETHASONE (KRD) AS SALVAGE THERAPY FOR MULTIPLE MYELOMA PATIENTS: ITALIAN, MULTICENTER, RETROSPECTIVE EXPERIENCE OUTSIDE OF CLINICAL TRIALS

E. A. Martino 1,* , C. Conticello 2 , E. Zamagni 3 , V. Pavone 4 , S. Palmieri 5 , M. Musso 6 , P. Tacchetti 7 , A. Mele 4 , L. Catlano 8 , E. Vigna 1 , A. Bruzzese 9 , F. Mendicino 1 , C. Botta 10 , D. Vincelli 11 , G. Farina 12 , M. Barone 13 , C. Cangialosi 14 , K. Mancuso 15 , I. Rizziello 15 , S. Rocchi 15 , A. P. Falcone 16 , G. Mele 17 , G. Reddiconto 18 , B. Garibaldi 19 , E. Iaccino 20 , G. Tripepi 21 , F. Di Raimondo 22 , P. Musto 23 , A. Neri 24 , M. Cavo 3 , F. Morabito 25 , M. Gentile 1

1 Azienda Ospedaliera di Cosenza, COSENZA; 2 University of Catania, Catania; 3 Seràgnoli Institute of Hematology and Medical Oncology, University of Bologna, Bologna; 4 Hospital Card. G. Panico, Tricase; 5 Ospedale Cardarelli, Napoli; 6 Dipartimento Oncologico, La Maddalena, Palermo; 7 Istituto di Ematologia “Seràgnoli”, Bologna; 8 AUOP “Federico II”, Napoli; 9 Azienda Ospedaliera of Cosenza, Cosenza; 10 Internal Medicine and Medical Specialties, University of Palermo, Palermo; 11 Great Metropolitan Hospital “Bianchi-Melacrino-Morelli”, Reggio Calabria; 12 AORN “Sant’Anna e San Sebastiano”, Caserta; 13 ”Tortora” Hospital, Pagani; 14 A. O. Ospedali Riuniti Villa Sofia-Cervello, Palermo; 15 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna; 16 IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo; 17 Hematology Unit, Brindisi; 18 Hospital Vito Fazzi, Lecce; 19 Azienda Policlinico-OVE, University of Catania, Catania; 20 University “Magna Graecia” of Catanzaro, Catanzaro; 21 National Research Institute of Biomedicine and Molecular Immunology, Reggio Calabria; 22 Hematology Section, University of Catania, Catania; 23 ”Aldo Moro” University School of Medicine, Bari; 24 Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano; 25 Biotechnology Research Unit, AO di Cosenza, Cosenza, Italy

Background: Carfilzomib in combination with lenalidomide, and dexamethasone (KRd) have been approved for the treatment of relapsed and refractory multiple myeloma (RRMM) based on ASPIRE clinical trial. Efficacy and safety of the triplet outside clinical trial are still the object of investigation by many groups to confirm ASPIRE results in the setting of RRMM treated in real-life who don’t meet clinical trial restrictive inclusion criteria.

Aims: The aim of the present study is to investigate the efficacy and safety of KRd regimen in the setting of RRMM treated in real-life.

Methods: Here we report a retrospective multicenter analysis of 600 RRMM patients treated with KRd between December 2015 and December 2018

Results: The median age at KRd start was 64 years (range 33-85) and the median number of previous therapies was 2 (range 1-11). After a median number of 11 KRd cycles, the ORR was 79.9%. The median PFS was 22 months and the 2-year probability of PFS was 47.6%. At multivariate analysis advanced ISS (III), and high-risk cytogenetic were significantly associated with shorter PFS. The median OS was 34.8 months; the 2-year probability of OS was 63.5% At multivariate analysis creatinine clearance<30 ml/min, more than 1 line of previous therapy, and high-risk FISH were significantly associated with poor prognosis. After a median follow-up of 16 months (range 1-50), 259 withdrew from therapy. The main reason for discontinuation was progressive disease (81.8%). Seventy-four patients (12.3%) discontinued therapy for toxicity. The most frequent side effects were hematological (anemia 49.3%, neutropenia 42.7%, thrombocytopenia 42.5%) and cardiovascular (hypertension 14.5%, heart failure 2.5%, arrhythmias 3.6%).

Summary/Conclusion: Our study confirms the safety and efficacy of KRd in the real-life setting of RRMM patients and encourages its use in clinical practice.

P894: A PHASE II TRIAL TO EVALUATE THE EFFICACY OF DARATUMUMAB WITH DCEP IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS WITH EXTRAMEDULLARY DISEASE AFTER BORTEZOMIB BASED TREATMENT

J. M. Byun 1,* , C.-K. Min 2 , K. Kim 3 , S.-M. Bang 4 , J.-J. Lee 5 , J. S. Kim 6 , S.-S. Yoon 1 , Y. Koh 1

1 Seoul National University Hospital; 2 Seoul St Mary’s Hematology Hospital; 3 Samsung Medical Center, Seoul; 4 Seoul National University Bundang Hospital, Seongnam; 5 Chonnam National University Hwasun Hospital, Hwasun; 6 Yonsei University College of Medicine, Seoul, South Korea

Background: Extramedullary multiple myeloma (EMM) is an aggressive subentity of multiple myeloma (MM), characterized by the ability of a subclone to thrive and grow independent of the bone marrow microenvironment, resulting in a high-risk state associated with increased proliferation, evasion of apoptosis and treatment resistance. Despite improvement in survival for most patients with MM over recent decades, outcomes are generally poor when EMM develops. Thus, better understanding of the disease and more innovative therapeutic approaches are needed.

Aims: To study the efficacy and safety of daratumumab in combination with dexamethasone, cyclophosphamide, etoposide and cisplatin (DCEP)

Methods: This was a multi-center, prospective phase II study (ClinicalTrials.gov identifier: NCT04065308 ). A total of 33 patients older than 19 years with multiple myeloma according to IMWG, relapsed/refractory to bortezomib, and with EMM (short-axis ≥1cm by CT or PET-CT) were enrolled. As shown in the Figure, patients received 3 cycles of daratumumab in combination with DCEP, followed by daratumumab maintenance. The primary objective was complete response rate after 3 cycles of daratumumab with DCEP. The secondary objectives included: 1) overall response rates; 2) progression free survival (PFS); 3) overall survival; and 4) safety and toxicity profiles. Here we present the interim data of 24 patients.

Results: The median age at MM diagnosis was 57 years (range 34-71) and 79.2% patients had EMM at diagnosis. There were 10 patients with t(4;14), 4 patients with del(17p) and 6 patients with t(14;16). There were 6 patients (25%) with ISS I disease, 10 (41.7%) with ISS II disease, and 8 (33.3%) with ISS III disease. The median number of prior lines of treatment was 3 (range 1-5), with all patients being exposed to bortezomib prior to enrollment. Also, 70.8% patients had prior exposure to carfilzomib, 87.5% to lenalidomide, and 50% to pomalidomide.

There were 17/24 (70.8%) patients who completed 3 cycles: 3 showed CR; 2 showed VGPR; 11 showed PR and 7 with progressive disease as shown in the Figure. Six patients were able to complete the study protocol. For all patients the median PFS was 4 months, but for those who completed the study protocol the median PFS was not reached and all but 1 remained in remission until the data cut-off in 2022-Jan-31. Patients achieving PR or better response after cycle 3 showed significantly better PFS (6 vs 2 months, p<0.001) compared to those who didn’t.

Most patients (22/24) underwent cyclophosphamide/etoposide/cisplatin dose reduction by 30% at cycle 1. One patient required further dose reduction at cycle 2. There was 1 patient who went off trial at D15 of cycle 1 according to attending physician’s decision and 2 patients who had to skip D22 daratumumab during cycle 1 due to thrombocytopenia. No patients required dexamethasone dose adjustments. During cycle 1, 12.5% showed anemia ≥grade 3, 25% showed thrombocytopenia ≥grade 3, 4.2% showed lymphopenia ≥grade 3, and 4 (16.7%) events of febrile neutropenia. During cycle 3, 5.3% showed anemia ≥grade 3, 10.5% showed thrombocytopenia ≥grade 3, while 15.8% showed lymphopenia ≥grade 3. The most common non-hematological adverse events was nausea (20.8%).

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Summary/Conclusion: In conclusion, daratumumab in combination with DCEP showed overall response of 66.7% (CR 25%), and durable remission in 20.8% of the enrolled patients.

P895: DARATUMUMAB, BORTEZOMIB, AND DEXAMETHASONE (D-VD) VERSUS BORTEZOMIB AND DEXAMETHASONE (VD) ALONE IN CHINESE PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA (RRMM): UPDATED ANALYSIS OF LEPUS

W. Fu 1,* , W. Li 2 , J. Hu 3 , G. An 4 , Y. Wang 5 , C. Fu 6 , L. Chen 7 , J. Jin 8 , X. Cen 9 , Z. Ge 10 , Z. Cai 8 , T. Niu 11 , M. Qi 12 , X. Gai 13 , Q. Li 13 , W. Liu 14 , W. Liu 13 , X. Yang 14 , X. Chen 14 , J. Lu 15,16

1 Shanghai Changzheng Hospital, Shanghai; 2 The First Hospital of Jilin University, Jilin; 3 Fujian Medical University Union Hospital, Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian; 4 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College; 5 Department of Hematology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin; 6 The First Affiliated Hospital of Soochow University; 7 The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Jiangsu; 8 The First Affiliated Hospital of Zhejiang University, College of Medicine, Zhejiang; 9 Peking University First Hospital, Beijing; 10 Zhongda Hospital Southeast University, Jiangsu; 11 Department of Hematology, West China Hospital Sichuan University, Sichuan, China; 12 Janssen Research & Development, LLC, Spring House, PA, United States of America; 13 Janssen Research & Development, LLC, Beijing; 14 Janssen Research & Development, LLC, Shanghai; 15 Peking University People’s Hospital, National Clinical Research Center for Hematologic Disease, Beijing; 16 Collaborative Innovation Center of Hematology, Soochow, China

Background: D-Vd prolonged progression-free survival (PFS) and induced deeper and more durable responses versus Vd alone in patients with RRMM in the global phase 3 CASTOR study (Weisel K, et al. ASH 2019. Abstract 3192). Results from a prespecified interim analysis of the phase 3 LEPUS study (median follow-up, 8.2 months) showed significant clinical benefit with D-Vd versus Vd in Chinese patients with RRMM and a safety profile that was generally consistent with that of CASTOR (Lu J, et al. Clin Lymphoma Myeloma Leuk 2021. 21[9]:e699-e709).

Aims: To examine updated efficacy of D-Vd versus Vd in LEPUS at a median follow-up of 25.1 months.

Methods: In the randomized, multicenter, phase 3 LEPUS study ( NCT03234972 ), Chinese patients with RRMM and ≥1 prior line of therapy were randomized 2:1 to 8 cycles (21 days/cycle) of Vd (bortezomib 1.3 mg/m 2 SC on Days 1, 4, 8, and 11; dexamethasone 20 mg PO/IV on Days 1, 2, 4, 5, 8, 9, 11, and 12) ± daratumumab (16 mg/kg IV once weekly in Cycles 1-3, every 3 weeks in Cycles 4-8, and every 4 weeks thereafter until disease progression). PFS was the primary endpoint.

Results: A total of 211 patients were randomized (D-Vd, n = 141; Vd, n = 70), with a median age of 61 (range, 28-82) years. Patients received a median of 2 (range, 1-11) prior lines of therapy; 79.1% of patients previously received bortezomib, 26.5% were refractory to lenalidomide, and 64.0% were refractory to their last prior line of therapy. At a median follow-up of 25.1 months, D-Vd prolonged PFS versus Vd alone (median: 14.8 vs 6.3 months; hazard ratio [HR], 0.35; 95% confidence interval [CI], 0.24-0.51; P <0.00001). The PFS benefit of D-Vd versus Vd was maintained across subgroups, including patients with prior bortezomib (HR, 0.36; 95% CI, 0.25-0.53), patients with high-risk cytogenetics (HR, 0.41; 95% CI, 0.23-0.71), and patients who were refractory to last prior line of therapy (HR, 0.42; 95% CI, 0.27-0.65). Overall response rate (ORR; 84.7% vs 66.7%; P = 0.00314) and rates of very good partial response or better (71.5% vs 34.9%; P <0.00001) and complete response or better (40.1% vs 14.3%; P = 0.00016) were higher with D-Vd versus Vd. Median time to first response was numerically shorter (0.79 months vs 1.23 months) and median duration of response was numerically longer (16.8 months vs 9.0 months) with D-Vd versus Vd. The estimated 18-month overall survival rate was 78.8% in the D-Vd group and 65.8% in the Vd group.

Summary/Conclusion: In this updated analysis of LEPUS, D-Vd continued to demonstrate substantial efficacy benefits versus Vd in terms of PFS, ORR, and depth of response. These data further support the use of D-Vd as a standard of care in Chinese patients with RRMM.

P896: DEVELOPMENT AND VALIDATION OF A FRAILTY PREDICTION MODEL MORE SUITABLE FOR MULTIPLE MYELOMA IN CHINESE PATIENTS: THE TM FRAILTY SCORE.

Y. Chen 1 , J. Gu 1 , B. Huang 1 , J. Liu 1 , X. Li 1 , J. Li 1,*

1 Department of Hematology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

Background: Frailty is associated with adverse clinical outcomes in patients with multiple myeloma (MM). Accurate evaluation of frailty is critical when choosing appropriate chemotherapy regimens for elderly patients. Currently, the IMWG geriatric assessment (GA) is widely used to assess tolerance to chemotherapy for elderly patients. However, the applicability of the IMWG GA in the Chinese MM population is limited.

Aims: To developed a new frailty model that is more suitable for Chinese patients with MM.

Methods: The applicability of the IMWG GA was evaluated in 167 consecutive patients diagnosed with MM from June 2019 to September 2021 at the First Affiliated Hospital of Sun Yat-sen University. Eight comprehensive geriatric assessment (CGA) domains were analyzed in 135 of these patients to screen items most closely associated with the outcomes, including grade ≥ 3 adverse events (AEs), treatment discontinuation, TTP, and OS. A new frailty prediction model was developed, and its fitness and predictive performance were compared to those of the IMWG GA.

Results: The patients in the IMWG GA Frail group had a higher rate of grade ≥3 AEs, a higher rate of treatment discontinuation, and a greater risk of death than those in the Fit group (P<0.05). No significant differences were observed in patients in the Int-fit group who experienced chemotherapy AEs, treatment discontinuation, and TTP compared with the Fit group. By screening items in all 8 CGA domains, multivariate analysis confirmed that the Timed Up and Go test （TUG） and the Mini Nutritional Assessment Short-Form（MNA-SF） were independent prognostic factors for grade ≥3 AEs and OS. We found that the TUG and MNA-SF scores could stratify the risk of grade ≥ 3 AEs in the IMWG GA Int-fit group (P < 0.05). After MNA-SF was added to construct the IMWG GA Plus model, Harrell’s concordance index (C-index) of the IMWG GA Plus model for the prediction of grade ≥3 AEs increased from 0.662 to 0.701. The C-index for treatment discontinuation rose from 0.636 to 0.669. We further combined the TUG test and the MNA-SF to construct the TM frailty scoring system. The KM curve analysis showed that the TM frailty score could well distinguish the risk of AEs between the Fit and Frail groups (P<0.05). The C-index of the TM frailty score was 0.741, 0.690, and 0.702 for grade ≥ 3 AEs, treatment discontinuation, and OS, respectively, suggesting good predictive ability, which was higher than that of the IWMG GA (C-index=0.662, 0.636 and 0.631, respectively) and the IMWG GA Plus model (C-index=0.701, 0.656 and 0.618, respectively).

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Summary/Conclusion: A novel scoring system—the TM frailty score—was developed by combining the TUG (T) and MNA-SF (M). The TM frailty score is more appropriate than the IMWG GA for evaluating frailty in the Chinese population.

P897: UPDATED RESULTS FROM THE ONGOING PHASE 1 STUDY OF ELRANATAMAB, A BCMA TARGETED T-CELL REDIRECTING IMMUNOTHERAPY, FOR PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA

A. Dalovisio 1,* , N. Bahlis 2 , N. Raje 3 , C. Costello 4 , B. Dholaria 5 , M. Solh 6 , M. Levy 7 , M. Tomasson 8 , H. Dube 9 , M. Damore 9 , S. Jiang 10 , C. Basu 10 , A. Skoura 11 , E. Chan 12 , S. Trudel 13 , A. Jakubowiak 14 , M. Chu 15 , C. Gasparetto 16 , M. Sebag 17 , A. Lesokhin 18

1 Department of Hematology and Oncology, Ochsner Health, Jefferson, United States of America; 2 Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada; 3 Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston; 4 Moores Cancer Center, University of California San Diego, La Jolla; 5 Vanderbilt University Medical Center, Vanderbilt-Ingram Cancer Center, Nashville; 6 Blood and Marrow Transplant Group of Georgia, Northside Hospital, Atlanta; 7 Department of Medical Oncology, Baylor Scott and White Health, Dallas; 8 Holden Comprehensive Cancer Center, University of Iowa, Iowa City; 9 Oncology Research and Development; 10 Early Clinical Development, Pfizer, San Diego; 11 Early Clinical Development, Pfizer, Collegeville; 12 Oncology Research and Development, Pfizer, South San Francisco, United States of America; 13 Princess Margaret Cancer Centre, University Health Network, Toronto, Canada; 14 Department of Medicine, University of Chicago Medical Center, Chicago, United States of America; 15 Cross Cancer Institute, Edmonton, Canada; 16 Department of Medicine, Duke University Cancer Institute, Durham, United States of America; 17 Cedars Cancer Center, McGill University Health Center, Montreal, Canada; 18 Division of Hematology and Oncology, Memorial Sloan Kettering Cancer Center/Weill Cornell Medical College, New York, United States of America

Background: Elranatamab (PF-06863135), a bispecific molecule that engages B-cell maturation antigen (BCMA) on multiple myeloma (MM) and CD3 on T-cells, induces targeted proliferation and activation of T cells to redirect the immune response against MM.

Aims: The Phase 1 study, MagnetisMM-1 ( NCT03269136 ), aims to characterize the safety, pharmacokinetics (PK), pharmacodynamics and efficacy of elranatamab as a single agent or in combination with immunomodulatory agents for patients (pts) with relapsed or refractory MM.

Methods: After informed consent, elranatamab was given subcutaneously weekly or every 2 weeks (Q2W) at doses from 80 to 1000µg/kg. A subset of pts received a single priming dose (600µg/kg or 44mg equivalent) followed 1 week later by the recommended Phase 2 dose (RP2D; 1000µg/kg or 76mg equivalent) thereafter. Treatment-emergent adverse events (TEAEs) were graded by Common Terminology Criteria for Adverse Events (v4.03) and cytokine release syndrome (CRS) by American Society for Transplantation and Cellular Therapy criteria. PK, cytokine and soluble BCMA profiling, and lymphocyte subset analyses were performed. Response was evaluated by International Myeloma Working Group (IMWG) criteria. Minimal residual disease (MRD) was assessed by next generation sequencing at a sensitivity of 1×10 -5 in accordance with IMWG criteria.

Results: A total of 55 pts received elranatamab monotherapy at doses ≥215µg/kg as of 1-Nov-2021. Median age was 64 years (range 42-80), 27% of pts were Black/African American or Asian, and 27% had high risk cytogenetics at baseline. Median number of prior lines of therapy was 6 (range 2-15), 91% were triple-class refractory, 22% received prior BCMA-targeted therapy, and 56% had prior stem cell transplant. The most common TEAEs (all causality) were CRS, neutropenia, anemia, injection site reaction, and lymphopenia. With a single priming dose and premedication, the incidence of CRS at the RP2D was 67% and divided equally between Grade 1 and 2, with no events greater than Grade 2. PK exposure was dose dependent, and elranatamab 1000µg/kg Q2W achieved exposure associated with anti-myeloma activity. Elranatamab therapy induced peripheral T-cell proliferation with a median time to response of 36 days (range 7-73), and the level of soluble BCMA decreased with disease response. With a median follow up of 8.1 months (range 0.3-21) and including only IMWG confirmed responses, overall response rate (ORR) was 64% (95% CI 50-75%), and 31% of pts achieved complete response or better. For responders (n=35), the probability of being event free at 6 months was 91% (95% CI 73-97%). Single-agent elranatamab induced durable clinical and molecular responses, and updated results including serial MRD assessment will be presented.

Summary/Conclusion: Elranatamab demonstrates a manageable safety profile and achieves durable clinical and molecular responses for pts with relapsed or refractory MM.

P898: COMPARISON OF TECLISTAMAB WITH BELANTAMAB MAFODOTIN IN PATIENTS WITH TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA USING MATCHING-ADJUSTED INDIRECT TREATMENT COMPARISON

M. Delforge 1,* , S. Z. Usmani 2 , N. W. van de Donk 3 , A. L. Garfall 4 , P. Moreau 5 , A. Oriol 6 , A. K. Nooka 7 , L. Rosinol 8 , N. Bahlis 9 , P. Rodriguez-Otero 10 , T. Martin 11 , J. Diels 12 , S. Van Sanden 12 , L. Pei 13 , E. Ammann 14 , R. Kobos 13 , M. Slavcev 14 , J. Smit 15 , A. Londhe 16 , A. Krishnan 17

1 University of Leuven, Leuven, Belgium; 2 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 3 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 4 Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 5 University Hospital Hôtel-Dieu, Nantes, France; 6 Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 7 Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 8 Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain; 9 Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, AB, Canada; 10 Clínica Universidad de Navarra, CIMA, CIBERONC, IDISNA, Pamplona, Spain; 11 University of California, San Francisco, San Francisco, CA, United States of America; 12 Janssen Pharmaceutica NV, Beerse, Belgium; 13 Janssen Research & Development; 14 Janssen Global Services, Raritan, NJ; 15 Janssen Research & Development, Spring House, PA; 16 Janssen Research & Development, Titusville, NJ; 17 City of Hope Comprehensive Cancer Center, Duarte, CA, United States of America

Background: Treatment options are limited for patients with relapsed/refractory multiple myeloma (RRMM) who are triple-class exposed (TCE) to immunomodulatory drugs, proteasome inhibitors, and anti-CD38 antibodies. While there is no standard of care for treatment of patients with TCE RRMM, belantamab mafodotin (belamaf) is a recently approved, novel therapeutic option. Teclistamab (tec; JNJ-64007957) is a B-cell maturation antigen × CD3 bispecific antibody currently being evaluated in the single-arm, phase 1/2 MajesTEC-1 study ( NCT04557098 ) in patients with TCE RRMM who received ≥3 prior lines of therapy.

Aims: Given the absence of a control arm in MajesTEC-1, the efficacy outcomes of patients treated with tec at the recommended phase 2 dose in MajesTEC-1 were compared with those treated with belamaf in the phase 2 DREAMM-2 study ( NCT03525678 ).

Methods: An unanchored matching-adjusted indirect comparison was conducted using individual patient-level data (IPD) from 150 patients treated with tec 1.5 mg/kg weekly in MajesTEC-1 (clinical cutoff of Sep 7, 2021), and published summary-level data from 97 patients treated with the approved dose of belamaf (2.5 mg/kg every 3 weeks) in DREAMM-2. After applying the DREAMM-2 eligibility criteria to patients from the intent-to-treat population of MajesTEC-1, IPD from MajesTEC-1 were weighted to match the aggregated DREAMM-2 baseline patient characteristics. Baseline characteristics of prognostic significance (such as cytogenetic profile, International Staging System stage, presence of extramedullary disease, number of prior lines of therapy, and refractory status) were adjusted for the analysis. Comparative efficacy of tec vs belamaf was estimated for overall response rate (ORR), complete response or better (≥CR) rate, duration of response (DOR), overall survival (OS), and progression-free survival (PFS). An odds ratio (OR) and 95% confidence interval (CI) derived from a weighted logistic regression analysis was used to compare the relative effects of tec vs belamaf for binary outcomes (ORR and ≥CR rate). A weighted Cox proportional hazards model was used to estimate time-to-event endpoints (DOR, OS, and PFS).

Results: After adjustment, the effective sample size of the MajesTEC-1 cohort was 33. Baseline characteristics were balanced between MajesTEC-1 and DREAMM-2 after reweighting the MajesTEC-1 cohort. Tec-treated patients had improved outcomes compared with patients treated with belamaf: ORR (OR 2.05; 95% CI 0.92–4.57; P =0.0786), ≥CR rate (OR 2.13; 95% CI 0.80–5.65; P =0.1283), DOR (hazard ratio [HR] 0.19; 95% CI 0.05–0.73; P =0.0149), OS (HR 0.95; 95% CI 0.47–1.92; P =0.8897), and PFS (HR 0.63; 95% CI 0.34–1.15; P =0.1338). For most outcomes, the lack of statistical significance may be due to the reduced effective sample size following adjustment.

Summary/Conclusion: In this comparative analysis, tec showed statistically improved DOR when compared with belamaf and numerically favorable results for other outcomes. This highlights the potential of tec as a treatment option for patients with TCE RRMM who received ≥3 prior lines of therapy.

P899: REAL-WORLD ASSESSMENT OF TREATMENT PATTERNS AND OUTCOMES IN PATIENTS WITH LENALIDOMIDE-REFRACTORY RELAPSED/REFRACTORY MULTIPLE MYELOMA FROM THE OPTUM DATABASE

B. Dhakal 1,* , H. Einsele 2 , R. Potluri 3 , J. Schecter 4 , W. Deraedt 5 , N. Lendvai 4 , A. Slaughter 6 , C. Lonardi 7 , S. Nair 5 , J. He 8 , N. Joseph 9 , P. Cost 8 , S. Valluri 8 , F. Yalniz 10 , L. Pacaud 10 , K. Yong 11

1 Medical College of Wisconsin, Milwaukee, United States of America; 2 Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Wuerzburg, Germany; 3 SmartAnalyst Inc, New York; 4 Janssen R&D, Raritan, United States of America; 5 Janssen Pharmaceutica NV, Beerse, Belgium; 6 Cilag GmbH International, Zug, Switzerland; 7 Janssen, Buenos Aires, Argentina; 8 Janssen Global Services, LLC, Raritan; 9 Janssen Scientific Affairs, LLC, Horsham; 10 Legend Biotech USA, Piscataway, United States of America; 11 University College London Cancer Institute, London, United Kingdom

Background: New treatment combinations using proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and anti-CD38 monoclonal antibodies have significantly improved survival outcomes in patients with multiple myeloma (MM). However, for patients who relapse and/or become refractory after exposure to PIs and IMiDs, selecting the next regimen remains a challenge. There are limited data characterizing treatments and outcomes in this difficult-to-treat population.

Aims: To characterize treatment patterns and outcomes in difficult-to-treat patients with MM, stratified by their number of prior lines of therapy (PL).

Methods: Data were derived from the Optum deidentified US claims and electronic health record (EHR) database. Patients with MM (index diagnosis in or after Jan 2011) were selected if they received 1-3 PL, including with a PI and an IMiD, and were refractory to lenalidomide. Subsequent treatments beginning in or after Jan 2016 were included to focus on contemporary therapies. Time zero (T0) was the date when the first subsequent therapy started after patient met inclusion criteria. Patient characteristics were assessed at T0. Time-to-event analyses were estimated using the Kaplan-Meier method (starting at T0) for overall survival (OS) and time to next treatment (TTNT). The log-rank test for trend was applied to compare Kaplan-Meier curves for OS by line of therapy.

Results: The database included a total of 13,615 (claims) and 22,626 (EHR) patients with an index diagnosis of MM and ≥1 line of therapy. 1,028 (claims) and 1,416 (EHR) patients met inclusion criteria. Median age was 72 (claims) and 68 (EHR) years; 53.4% (claims) and 52.1% (EHR) were male. 12.2% (claims) and 4.9% (EHR) of patients received stem cell transplant prior to T0. Patients in both cohorts had significant comorbidities with mean Charlson Comorbidity Index 3.9 (claims) and 3.2 (EHR). Hypertension and renal failure were among the most prominent comorbidities. The most common subsequent treatment regimens based on hierarchy were daratumumab (22.1% claims; 17.4% EHR), carfilzomib (10.6% claims; 18.4% EHR), and pomalidomide based (12.3% claims; 8.7% EHR). Daratumumab/pomalidomide/dexamethasone was the most frequently used regimen (5.4% claims; 3.8% EHR). For claims data, median OS (months) was 36.7 (95% CI: 31.7-41.2). Median OS was not reached (42.3-NE) for patients with 1 PL (n=546), was 26.2 (21.7-35.7) for patients with 2 PL (n=380) and was 12.1 (7.6-25.0) for patients with 3 PL (n=102). Median TTNT (months) was 5.3 (4.9-5.9) overall, including 4.6 (4.3-5.1) for patients with 1 PL, 6.4 (5.3-7.6) for patients with 2 PL, and 6.0 (4.9-9.4) for patients with 3 PL. For EHR data, median OS was 34.0 (28.2-42.8), with median OS of 46.9 (42.8-NE) for patients with 1 PL (n=587), 28.2 (23.8-41.7) for patients with 2 PL (n=584), and 20.8 (14.9-29.8) for patients with 3 PL (n=245). Median TTNT was 5.8 (5.1-6.5) overall, with values of 5.1 (4.7-6.5) for patients with 1 PL, 6.2 (5.3-7.6) for patients with 2 PL, and 5.8 (4.4-8.2) for patients with 3 PL.

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Summary/Conclusion: PI-exposed, lenalidomide-refractory patients with 1-3 PL were treated with various regimens. These patients have poor OS and progress quickly through current therapies, suggesting poor progression-free survival. OS is reduced with each successive line of therapy. This analysis highlights the need for new effective regimens for this patient population.

P900: SALVAGE AUTOLOGOUS STEM CELL TRANSPLANT IN RELAPSED MULTIPLE MYELOMA: A SINGLE CENTRE EXPERIENCE

S. Duarte 1,* , A. Roque 1,2 , D. Mota 1 , J. Carda 1,2 , C. Geraldes 1,2

1 Clinical Hematology Department, University Hospital Center of Coimbra; 2 Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Background: Multiple myeloma (MM) is an incurable disease and most patients (pts) will relapse during their lifetime. Although salvage therapy has improved with the advent of new drugs, salvage autologous stem cell (ASC) transplant (SAT) remains appropriate for pts relapsing after primary therapy that includes ASCT with initial remission duration >18 months (mo).

Aims: Evaluate the outcome of SAT in MM in the new drugs era and efficacy and toxicity associated to this treatment.

Methods: Single centre retrospective analysis of MM pts submitted to SAT between 2007-2021. Tandem ASCT excluded. Engraftment defined according to EBMT criteria.

Results: Fifty-three pts submitted to SAT, 58.5% male, median age of 60 yo (37-71), 27.8% with international staging system score III. Median number of prior lines of therapy before SAT was 2 (2-4), 51 (96.2%) including proteasome inhibitors (PI), 38 (71.7%) immunomodulators (IMiD) and 13 (24.5%) daratumumab. Twenty-five (50%) pts submitted to maintenance therapy with thalidomide (Thal) after ASCT and 15 (30.6%) with lenalidomide (Len) after SAT.

Pre-SAT response to therapy was complete remission (CR) in 9.6%, very-good partial response (VGPR) in 50% and PR in 40.4%. Most pts received conditioning with melphalan 200mg/m 2 for both transplants (1 st : 100%, 2 nd : 96.2%).

After SAT, median time to neutrophil engraftment was 12 days (6-21) and for platelet recovery was 13 days (3-26). Grade 3 oral and gastrointestinal mucositis observed in 14% and 6%, respectively, and febrile neutropenia in 91% of pts. SAT response evaluation showed CR in 28.3%, VGPR in 32.1% and PR in 37.7%. One patient showed progressive disease. No treatment related mortality (TRM) at 100 days post-SAT.

Median PFS after 1 st ASCT was 32 mo. Median time between 1 st and 2 nd ASCT was 46 mo (22-140). Median PFS and OS after SAT was 28 and 78 mo, respectively. Comparison between PFS post-ASCT and post-SAT was not significant (P=0.09).

ISS stage with no impact on PFS (HR 1.99; p=0.263) and OS (HR 2.03; p=0.398), after SAT. No statistically significant difference in post-SAT PFS (HR 1.23 p=0.592) and OS (HR 1.84, p=0.312) between patients submitted to transplant before and after 2016, despite more intensive therapeutic protocols (100% triplets and 24.5% anti-CD38-based regimens in former period compared with duplets in the first). PFS of pts who reached CR post-SAT was significantly higher compared to pts in VGPR/PR (NR vs 22 mo, HR 2.97, p=0.044). However, CR post-SAT with no impact on OS (NR vs 118 mo, HR 1.27 p=0.71). Maintenance therapy with (Thal) post-ASCT with no impact on PFS (33 vs 32 mo, HR 1.24, p=0.47). Similarly, the impact of post-SAT maintenance with Len on PFS (51 vs 28 mo, HR 1.35, p=0.49) and OS (NR vs 73 mo, HR 4.40, p=0.157) was not significant.

Pts with remission time duration after 1 st ASCT ≥36 mo had median PFS and OS after SAT significantly longer (51 vs 22 mo, HR 2.41, p=0.024, and NR vs 71 mo, HR 6.24, p=0.015, respectively), compared to pts with post-ASCT remissions <36 mo.

Univariate analysis failed to show other clinical and analytical variables with impact on PFS and OS.

Summary/Conclusion: In our cohort, median PFS and OS after SAT was 78 mo and 28 mo, respectively. Pts with remissions after 1 st ASCT ≥36 mo showed significantly longer PFS and OS. PFS was significantly improved in pts reaching CR after SAT. Post-SAT Len maintenance improved PFS and OS, however with no statistical significance. Severe toxicities were rare and never resulted in TRM. Overall, our centre experience shows that SAT is an effective and safe treatment in MM in the new drugs era.

P901: POMALIDOMIDE, BORTEZOMIB, AND DEXAMETHASONE IN LENALIDOMIDE–PRETREATED MULTIPLE MYELOMA: A SUBANALYSIS OF OPTIMISMM BY FRAILTY

A. Oriol 1,* , M. Dimopoulos 2 , F. Schjesvold 3,4 , M. Beksac 5 , M. Yagci 6 , A. Larocca 7 , S. Guo 8 , Y. Mu 8 , K. Hong 9 , S. Dhanasiri 10 , P. Richardson 11 , K. Weisel 12

1 Hematology Department, Institut Català d’Oncologia, Barcelona, Spain; 2 National and Kapodistrian University, Athens, Greece; 3 Oslo Myeloma Center, Department of Hematology, Oslo University Hospital; 4 KG Jebsen Center for B Cell Malignancies, University of Oslo, Oslo, Norway; 5 Ankara Üniversitesi Tip Fakültes; 6 Gazi University Medical Faculty, Ankara, Turkey; 7 SSD Clinical trials in onco-ematologia e mieloma multiplo, Division of Hematology, University of Torino, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, Torino, Italy; 8 Evidera, Waltham; 9 Bristol Myers Squibb, Princeton, United States of America; 10 Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland; 11 Dana-Farber Cancer Institute, Boston, United States of America; 12 University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Background: Patients (pts) with multiple myeloma (MM) are likely to be older adults, and advanced age is associated with lower survival rates, in part due to comorbidities and frailty. Results from the phase 3 OPTIMISMM trial ( NCT01734928 ) demonstrated that pomalidomide (P) in combination with bortezomib + dexamethasone (Vd) significantly improved progression-free survival (PFS) in lenalidomide (LEN)–pretreated pts with relapsed/refractory MM (RRMM) vs Vd, irrespective of age and Eastern Cooperative Oncology Group performance status (ECOG PS).

Aims: Here, we report results from a post hoc analysis assessing outcomes of PVd vs Vd in pts with RRMM by frailty status.

Methods: Pts with MM and 1–3 prior lines of therapy (including a LEN-containing regimen) who had been randomized 1:1 to PVd or Vd were assessed for frailty. Frailty scores were calculated using age (≤ 75 yr = 0; 76–80 yr = 1; > 80 yr = 2), the Charlson Comorbidity Index (≤ 1 = 0; > 1 = 1), and ECOG PS (0 = 0; 1 = 1; ≥ 2 = 2). Pts were classified as non-frail (NF; combined score: 0 or 1) or frail (F; combined score: ≥ 2). PFS, overall response rate (ORR), and safety outcomes were assessed by treatment group and frailty status.

Results: In the intent-to-treat population (N = 559) (data cut-off Oct 26, 2017), 93/281 (33.1%) pts who received PVd and 93/278 (33.5%) who received Vd were frail. Baseline characteristics were similar between treatment groups within each frailty subgroup. Median PFS was longer with PVd vs Vd in NF ( P = 0.001) and F ( P = 0.006) subgroups ( Table ). ORR was higher with PVd vs Vd in NF ( P < 0.001) and F ( P < 0.001) subgroups. Incidence of grade ≥ 3 (G3) treatment-emergent adverse events (TEAEs) was higher with PVd vs Vd in NF (88.1 vs 61.3%) and F (96.8 vs 87.9%) subgroups; peripheral neuropathy, acute renal failure, and hypertension were the most common. Treatment discontinuation due to G3 TEAEs was greater with PVd vs Vd in NF (19.2 vs 18.5%) and F (30.1 vs 20.9%) subgroups. PVd had a longer median treatment duration vs Vd in NF (8.8 vs 5.7 mo) and F (8.9 vs 4.3 mo) subgroups.

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Summary/Conclusion: Frail pts with RRMM who received PVd had longer PFS and higher ORR than pts who received Vd, consistent with the overall OPTIMISMM results. Frail pts experienced more G3 TEAEs and treatment discontinuations with PVd vs Vd, but treatment duration was longer with PVd vs Vd.

P902: RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS. A MULTICENTER RETROSPECTIVE ANALYSIS OF ELIGIBILITY CRITERIA FOR CAR-T CELL THERAPY

F. Fazio 1,* , A. Di Rocco 1 , M. T. Giaimo 2 , T. Za 3 , N. Ciccone 4 , M. Sessa 4 , B. Gamberi 5 , V. Tomarchio 6 , L. De Padua 7 , V. Bongarzoni 8 , S. Mariani 9 , A. Rago 10 , A. Piciocchi 11 , F. Merli 5 , A. Cuneo 4 , V. De Stefano 3 , R. Foà 1 , M. Martelli 1 , M. T. Petrucci 1

1 Hematology - Azienda Policlinico Umberto I, Department of Traslational and Precision Medicine Sapienza University of Rome, Roma; 2 Hematology - Azienda Ospedaliera Parma, University of Parma, Parma; 3 Hematology - Fondazione Policlinico A. Gemelli IRCCS, Department of Radiological and Hematological Sciences, Catholic University, Roma; 4 Hematology - Ospedale Sant’Anna, University of Ferrara, Ferrara; 5 Hematology, AUSLL/IRCCS Santa Maria Nuova Hospital, Reggio Emilia; 6 Hematology - Stem Cell Transplantation, University Campus Bio Medico, Roma; 7 Hematology - Ospedale Fabrizio Spaziani, Frosinone; 8 Hematology - Azienda Ospedaliera San Giovanni; 9 Hematology - Ospedale Sant’Andrea, Sapienza University of Rome; 10 UOSD Hematology ASLRoma 1; 11 Italian Group For Adult Hematologic Diseases (GIMEMA), Roma, Italy

Background: The overall survival (OS) of multiple myeloma (MM) patients (pts) has improved over the years due to the introduction of novel drugs, such as proteosome inhibitors (PI), immunomodulatory drugs (IMiDs) and anti- CD38 monoclonal antibodies (moAb). Nevertheless, the majority of pts continues to relapse and MM remains an incurable disease. No standard of care has been established for relapsed/refractory (RR) MM pts who have been exposed to the main anti-myeloma drugs. The outcome of pts failing standard of care regimens, which is now defined as triple-refractory, is poor, with a median progression-free survival (PFS) of 3-4 months and OS of 8-9 months. Novel therapeutic strategies are warranted to overcome the natural occurrence of relapse or therapy resistance in RRMM pts. Chimeric antigen receptor (CAR)-modified T cells are a promising new therapy approach for triple refractory RRMM. Specific CAR-T targets are being studied, but BCMA-directed CAR-T cells have so far provided the most convincing evidence of activity, with one product (idecabtagene vicleucel) recently approved by FDA and EMA.

Aims: The primary endpoint of this observational and retrospective study was to define the clinical characteristics and outcome of a cohort of RRMM pts potentially eligible to CAR-T cell treatment according to the KarMMa trial criteria. Secondary endpoints were aimed at defining specific factors influencing CAR-T cell therapy eligibility and at identifying a real-life estimate of RRMM pts truly eligible for CAR-T cells.

Methods: This is a cohort analysis on RRMM pts managed between January 2018 and July 2021 at 10 Italian hematology centers. At the time of data collection, 108 RRMM pts had underwent at least 3 prior therapy regimens; they had received a previous PI, IMiDs and a moAb, and were considered refractory to the last regimen.

Results: Median age was 68 years (38-86); 63 (59%) pts were >65 years; 57 (53%) were male. Of 108 pts, 87 (80%) were ECOG 0-1 and 33 (35%) were ISS III. The majority of pts, 72 (67%), had undergone an autologous stem cell transplantation; 93 pts received >3 prior lines of therapy. Sixty-seven (62%) were triple-refractory and 41 (38%) were penta-refractory. Based on the KarMMa trial criteria, 49/108 pts (45%) would be defined as eligible and 59 (55%) not eligible for CAR-T cell therapy. Organ dysfunction such as impaired renal function, anemia, thrombocytopenia, neutropenia and a FEV <45% was the most common criteria for ineligibility (86%). Twenty-one pts (19%) were not eligible because of an ECOG >2. Of the 59 pts considered ineligible for CAR-T cell therapy, 46 (62%) presented ≥2 ineligibility criteria.

After a median follow-up of 27 months (mo) (8-40.5), the median OS for the entire cohort was 21.1 mo (Fig. 1A). The median OS was 33 mo in eligible pts vs 8.3 mo in non-eligible pts (p=0.002) (Fig. 1B). The median PFS of the entire cohort was 8.7 mo (Fig. 1C) and the median PFS was 19.4 mo in eligible pts vs 6 mo (p=0.001) in non-eligible pts (Fig. 1D).

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Summary/Conclusion: Despite the limits of a retrospective study and a limited cohort, our real-life analysis shows that heavily treated pts with RRMM are less likely to be eligible for CAR-T cell therapy. Considering the emergent role of quadruplet combined approaches for first- line therapy and given the therapeutic relevance of CAR-T cells for the management of RRMM pts previously exposed to PI, IMiDs and moAb, our data could help to better define pts who could benefit from CAR-T cells under the current indications, while waiting for an extension of this approach to earlier disease stages.

P903: DARATUMUMAB PLUS BORTEZOMIB AND DEXAMETHASONE IN NEWLY DIAGNOSED PATIENTS WITH MAYO 2004 STAGE 3 LIGHT-CHAIN AMYLOIDOSIS: A PROSPECTIVE PHASE 2 STUDY

Y.-J. Gao 1,* , K.-N. Shen 1 , L. Chang 1 , J. Feng 1 , L. Zhang 1 , Y.-Y. Mao 1 , X.-X. Cao 1 , D.-B. Zhou 1 , J. Li 1

1 Department of Hematology, Peking Union Medical College Hospital, Beijing, China

Background: Patients with systemic light-chain (AL) amyloidosis at the advanced cardiac stage exhibit extremely poor survival. Although daratumumab has shown superior outcome in treatment of AL amyloidosis according to the result of ANDROMEDA trial, whether these feeble patients can benefit from daratumumab therapy need further investigation.

Aims: To prospectively explore the value of daratumumab plus bortezomib and dexamethasone in patients with AL amyloidosis at the advanced heart-stage.

Methods: This is a phase 2, open-label, single center clinical trial planning to include 40 newly diagnosed patients with Mayo 2004 stage 3a and 3b AL amyloidosis at Peking Union Medical College Hospital (Beijing, China). Eligible patients should have measurable hematological disease (baseline dFLC >50mg/L). Initiation treatment includes daratumumab (intravenously at 16 mg/kg weekly during cycles 1-2, once every two weeks during cycles 3-6 and once every 4 weeks thereafter for up to 12 cycles), bortezomib (at 1.3mg/m 2 subcutaneously weekly during cycles 1-6) and dexamethasone (20mg weekly during cycles 1-6). Each cycle consists of 4 weeks. Treatment responses are evaluated every week during the first cycle and at the end of each cycle since cycle 2. Termination of the therapy is considered when patients have progressed disease, serious side effects related to treatment or initiation of the second-line treatment. (ClinicalTrials.gov identifier: NCT04474938 ）

Results: From 28 th May, 2021 to 28 th January, 2022, 38 patients were enrolled. Twenty-nine (76.3%) patients were male and the median age was 59 years (range 41-77). The median NT-proBNP was 10665 pg/ml (range 803 ~ >35000) and the median cTnI was 0.17ug/L (range 0.07-3.07). Twenty-one patients (55.3%) were stage 3b. Median dFLC was 265 mg/L (range 72-2966). Twenty-four patients (63.2%) had NYHA class III or IV heart function. The median number of organs involved was 2 (range 0-4). Fourteen (36.8%) patients had kidney involvement and nine (23.7%) patients had liver involvement.

The median number of treatment cycles was 3.25 (range 0.25-9). For the best hematologic response, 34 of 38 patients (89.5%) reached ≥PR, including 18 patients (47.4%) with CR and 8 (21.1%) patients with VGPR. At the end of the first cycle, 8 patients (22.9%) achieved CR; 12 patients (34.3%) reached VGPR; 9 patients (25.7%) had PR and 6 patients (17.1%) were NR. The hematological ORR was 74.1% at 3 months (40.7% with CR and 18.5% with VGPR). The median time to the first hematologic remission was 7 days (range 7-21) and the median time to ≥VGPR was 14 days (range 7 days – 3 months). The cardiac response rate was 18.5% at 3 months and 23.5% at 6 months. After the median follow-up time of 4.5 months (range 0.7-8.6), the median OS was not reached. The 6-month survival rate of all patients was 76.6%, with 84.7% for stage 3a and 69.0% for stage 3b.

The most common grade 3 or 4 adverse events were infection (n=8, 21.1%). Infusion reaction was recorded in 5 patients (13.2%) and only 1 patient was categorized as grade 3 reaction. All of them occurred during the first time of infusion. Other serious adverse effects included grade 3 diarrhea (n=3), pneumothorax (n=1), bone fracture (n=1), deep venous thrombosis (n=1), hematuresis (n=1), gastrointestinal bleeding (n=1), ischemic stroke (n=1) and intestinal obstruction (n=1). No patients withdrew due to adverse events.

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Summary/Conclusion: Our results showed that daratumumab plus bortezomib and dexamethasone had favorable safety and potential advantage among patients with AL amyloidosis presenting severe cardiac involvement.

P904: CILTACABTAGENE AUTOLEUCEL VS TREATMENTS FROM REAL-WORLD CLINICAL PRACTICE FOR TRIPLE CLASS EXPOSED PATIENTS WITH MULTIPLE MYELOMA: ADJUSTED COMPARISONS BASED ON CARTITUDE-1 AND THE EMMY FRENCH COHORT

O. Decaux 1,* , C. Hulin 2 , A. Perrot 3 , M. Macro 4 , L. Frenzel 5 , J. Diels 6 , N. J. Perualila 6 , F. Ghilotti 7 , B. Haefliger 8 , E. Goldsztajn 9 , S. Vernet 10 , J. Thevenon 10 , M. Willaime 11 , N. Texier 11 , J. M. Schecter 12 , D. Madduri 12 , C. Jackson 12 , S. Valluri 13 , P. Moreau 14

1 Internal medicine and clinical immunology, CHU Rennes, Rennes; 2 Hematology and cell therapy, CHU Bordeaux, Bordeaux; 3 Hematology, Oncopole, Toulouse; 4 Hematology, CHU Caen, Caen; 5 Hematology, Necker-enfants malades hospital, Paris, France; 6 Health Economics, Market Access & Reimbursement, Janssen Pharmaceutica NV, Beerse, Belgium; 7 Health Economics, Market Access & Reimbursement, Janssen Pharmaceutica NV, Cologno Monzese, Italy; 8 Health Economics, Market Access & Reimbursement, Cilag GmbH International, Zug, Switzerland; 9 Market Access & Reimbursement; 10 Medical affairs, Janssen, Issy-les-Moulineaux; 11 Biometry, Kappa Sante, Paris, France; 12 Research and Development, Janssen R&D; 13 Health Economics, Market Access & Reimbursement, Janssen, Raritan, United States of America; 14 Clinical hematology, University Hospital Hotel-Dieu, Nantes, France

Background: The prognosis of patients with triple-class (TC) exposed, relapsed and refractory multiple myeloma (RRMM) is poor. CARTITUDE-1 is an open-label, single arm, phase 1b/2 clinical trial performed to assess the safety and efficacy of ciltacabtagene autoleucel (cilta-cel), a novel chimeric antigen receptor T-cell therapy. Adjusted comparisons to other therapies used in real-world clinical practice (RWCP) help inform the relative efficacy of cilta-cel in TC exposed patients. The E pidemiology of the therapeutic management of M ultiple MY eloma in France (EMMY) study is a retrospective study capturing information on the therapeutic management of patients from 71 centers in France, and can serve as an external control arm for CARTITUDE-1.

Aims: To compare cilta-cel with treatments from RWCP in terms of overall response rate (ORR), very good partial response or better rate (≥VGPR), progression-free survival (PFS), and overall survival (OS).

Methods: Adjusted comparisons were estimated using individual patient data (IPD) from CARTITUDE-1 (cut-off date July 2021) and reconstructed IPD from EMMY (cut-off date March 2021). Within EMMY, all treatment lines initiated after a patient met eligibility criteria (TC exposed patients in L4+ and ECOG<2) were used for analysis, so long as eligibility remained intact. Inverse probability weighting (IPW) was used to balance the cilta-cel and RWCP groups on prognostic baseline variables including refractory status, time to progression on prior line of treatment (LOT), number of prior LOTs, ECOG performance status, age, sex, years since MM diagnosis, average duration of prior LOTs, MM type, and prior transplant. Weights derived from propensity scores estimated with a multivariable logistic regression and were assigned to EMMY patients, such that the weighted EMMY cohort reflected the CARTITUDE-1 population. Balance was assessed using standardized mean differences (SMD). The relative efficacy of cilta-cel versus RWCP was assessed using relative response rates (RR) for binary outcomes and hazard ratios (HR) for time-to-event outcomes, using weighted logistic regression and proportional hazards modeling, respectively. Main analyses were performed in the population of infused patients in the cilta-cel group and an aligned population of EMMY RWCP patients that included those still progression-free after 52 days (mean time between apheresis and cilta-cel infusion). Similar analyses were performed for all enrolled patients.

Results: 113 patients were enrolled in CARTITUDE-1 and 97 received cilta-cel infusions. 437 LOTs (238 patients) from EMMY were included in the RWCP external control arm and 309 LOTs (227 patients) were alive and progression-free after 52 days. More than 100 treatment regimens within the RWCP group were used. After reweighting, baseline traits were balanced between groups, with all SMD <0.20. ORR (RR 5.1, 95% CI: 3.7;7.1) and ≥VGPR (RR 14.5 [8.0; 26.1] were superior with cilta-cel vs. RWCP, as were both PFS (HR 0.15 [0.10; 0.22]) and OS (HR 0.21 [0.12; 0.37]). Results for the enrolled populations were consistent (see Table ).

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Summary/Conclusion: Adjusted comparisons between cilta-cel and RWCP in France showed superior efficacy on all outcomes for cilta-cel vs. RWCP, with 5.1 and 14.5-fold higher response rates for ORR and ≥VGPR, respectively, and reduced risks of disease progression/death and death by 85% and 79% with cilta-cel versus RWCP. These findings align with results from similar comparisons in other countries. Cilta-cel offers substantial clinical benefits for patients with triple class exposed RRMM.

P905: IXAZOMIB-THALIDOMIDE-DEXAMETHASONE INDUCTION FOLLOWED BY IXAZOMIB OR PLACEBO MAINTENANCE IN NON-TRANSPLANT ELIGIBLE NEWLY DIAGNOSED MULTIPLE MYELOMA PATIENTS; LONG-TERM RESULTS OF HOVON-126/NMSG 21.13

K. Groen 1,* , M. R. Seefat 1 , B. van der Holt 2 , F. H. Schjesvold 3,4 , C. A. Stege 1 , M.-D. Levin 5 , M. Hansson 6,7 , R. B. Leys 8 , J. Regelink 9 , A. Waage 10 , D. Szatkowski 11 , P. Axelsson 12 , T. H. Do 13 , A. Svirskaite 14 , E. van der Spek 15 , E. Haukas 16 , D. Knut-Bojanowska 17 , P. F. Ypma 18 , C. Blimark 19 , U.-H. Mellqvist 20 , N. W. van de Donk 1 , P. Sonneveld 21 , A. Klostergaard 22 , A. J. Vangsted 23 , N. Abdilgaard 24,25 , S. Zweegman 1

1 Hematology, Amsterdam UMC, Amsterdam; 2 HOVON Data Center, Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 3 Hematology, Oslo Myeloma Center; 4 KG Jebsen center for B Cell malignancies, University of Oslo, Oslo, Norway; 5 Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, Netherlands; 6 Skane University Hospital, Lund; 7 Sahlgrenska Academy, Göteborg, Sweden; 8 Department of Internal Medicine, Maasstad Ziekenhuis, Rotterdam; 9 Department of Internal Medicine, Meander Medisch Centrum, Amersfoort, Netherlands; 10 IKOM, Norwegian University of Science and Technology, Trondheim; 11 Førde Central Hospital, Førde, Norway; 12 Skanes University Hospital, Lund, Sweden; 13 Herlev Hospital, Herlev; 14 Aalborg Hospital, Aalborg, Denmark; 15 Department of Internal Medicine, Rijnstate, Arnhem, Netherlands; 16 Stavanger University Hospital-Rogaland Hospital, Stavanger, Norway; 17 NU-Hospital - Uddevalla Hospital, Uddevalla, Sweden; 18 Department of Hematology, Haga Ziekenhuis, Den Haag, Netherlands; 19 Sahlgrenska University Hospital, Gothenburg; 20 Sodra Alvsborgs Sjukhus Boras, Boras, Sweden; 21 Erasmus Medical Center Cancer Institute, Rotterdam, Netherlands; 22 Aarhus University Hospital, Aarhus; 23 Department of Hematology, Rigshospitalet, Copenhagen; 24 Department of Hematology; 25 Academy or Geriatric Cancer Research, Odense University Hospital, Odense, Denmark

Background: In the HOVON 126/NMSG 21.13 trial non-transplant eligible newly diagnosed multiple myeloma (NTE-NDMM) patients were treated with 9 induction cycles of ixazomib, thalidomide and dexamethasone (ITd), followed by randomization between either ixazomib or placebo until progression or unacceptable toxicity. The overall response rate and PFS data have been previously published.

Aims: We here present the long-term PFS2 and overall survival data.

Methods: Patients were treated with 9 induction cycles (28 days) of ixazomib (4mg on day 1, 8 and 15), thalidomide (100mg on day 1-28) and dexamethasone (40mg on day 1, 8, 15 and 22), followed by maintenance with either ixazomib or placebo (4mg, both on day 1, 8 and 15, every 28 days). Patients were classified as fit, intermediate fit or frail, based on a modified IMWG frailty index which incorporated age, the Charlson Comorbidity Index (CCI) and the WHO performance as a proxy for (instrumental) Activities of Daily Living (iADL) (scoring WHO 0 as 0 points, WHO 1 as 1 point, and WHO 2-3 as 2 points).

Results: From registration: 143 eligible patients were included in the study. After a median follow-up (FU) of 67.4 months (m), the median PFS was 14.3m (95% CI 11.5-16.8), median PFS2 was 34.6m (30.7-41.5) and median OS was 58.3m (50.5-65.0). There was no difference in PFS between frailty subgroups. In contrast, median PFS2 and OS were longer in fit patients (PFS2: 49.1m (34.6-74.1), OS: NR (66.6-NR)) versus intermediate-fit (30.1m (25.1-39.0); 51.2m (32.3-63.9) resp.) and frail patients (30.9m (24.0-42.3); 50.5m (32.9-59.4) resp.).

From randomization: 78 (55%) patients were randomized, 39 patients in each arm. After a median FU of 60 months from randomization, there was no difference in PFS between the ixazomib-arm (median 9.5m; 95% CI 5.5-14.8) and the placebo-arm (8.4m; 3.0-13.8). Median PFS2 was 39.8m (28.8-60.0) for patients on ixazomib, as compared to 28.7m (22.8-43.2) for patients in the placebo arm, although this difference was not statistically significant. Median OS was not reached for the ixazomib arm and was 50.7m (41.3-58.1) for the placebo arm (HR 0.39; 95% CI 0.19-0.78, p=0.008).

In both arms 32 (82%) patients received 2 nd line treatment. With the caveat of low numbers and heterogeneous treatment regimens, more patients in the ixazomib arm received daratumumab-lenalidomide-dexamethasone (4 patients, 13%) and panobinostat-bortezomib-dexamethasone (6 patients, 19%), compared to the placebo arm (2 patients (6%) and 3 patients (9%) respectively).

In order to explain the difference in OS, subsequent lines of therapy are currently being investigated.

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Summary/Conclusion: With longer FU, we here confirm that ixazomib maintenance therapy did not improve PFS, compared to placebo. However, PFS2 tends to be longer and OS was superior in patients treated with ITd followed by maintenance with ixazomib versus placebo.

P906: IXAZOMIB, DARATUMUMAB AND LOW DOSE DEXAMETHASONE IN FRAIL PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA (NDMM): RESULTS OF THE MAINTENANCE TREATMENT OF THE PHASE II HOVON 143 STUDY

K. Groen 1,* , M. Seefat 1 , K. Nasserinejad 2 , C. A. Stege 1 , E. van der Spek 3 , Y. M. Bilgin 4 , A. Kentos 5 , M. Sohne 6 , R. J. van Kampen 7 , I. Ludwig 8 , N. Thielen 9 , N. Durdu-Rayman 10 , N. C. de Graauw 11 , N. W. van de Donk 1 , E. G. de Waal 12 , M.-C. Vekemans 13 , G. J. Timmers 14 , M. van der Klift 15 , S. Soechit 16 , P. A. Geerts 17,18 , M. H. Silbermann 19 , M. Oosterveld 20 , I. Nijhof 1,21 , P. Sonneveld 22 , S. K. Klein 23 , M.-D. Levin 24 , S. Zweegman 1

1 Hematology, Amsterdam UMC, Amsterdam; 2 HOVON Data Center, Department of Hematology, Erasmus MC Cancer Institute, Rotterdam; 3 Department of Internal Medicine, Rijnstate Hospital, Arnhem; 4 Department of Internal Medicine, Admiraal de Ruijter Hospital, Goes, Netherlands; 5 Department of Hematology, Centre Hospitalier Jolimont, Haine-Saint-Paul, Belgium; 6 Department of Internal Medicine/Hematology, St Antonius Hospital, Nieuwegein; 7 Department of Internal Medicine, Zuyderland Medical Center, Sittard-Geleen; 8 Department of Hematology, Bernhoven Hospital, Uden; 9 Department of Internal Medicine, Diakonessenhuis, Utrecht; 10 Department of Internal Medicine, Franciscus Hospital, location Vlietland, Schiedam; 11 Department of Internal Medicine, Bravis ziekenhuis, Roosendaal; 12 Department of Internal Medicine, Medisch Centrum Leeuwarden, Leeuwarden, Netherlands; 13 Department of Hematology, St Luc Hospital, Bruxelles, Belgium; 14 Department of Internal Medicine, Amstelland Hospital, Amstelveen; 15 Department of Internal Medicine, Amphia Hospital, Breda; 16 Department of Hematology, Reinier de Graaf Groep, Delft; 17 Department of Internal Medicine, Deventer Hospital, Deventer; 18 currently Isala, Zwolle; 19 Department of Internal Medicine, Tergooi Hospital, Hilversum; 20 Department of Internal Medicine, Canisius-Wilhelmina Hospital, Nijmegen; 21 currently St Antonius Hospital, Nieuwegein; 22 Department of Hematology, Erasmus MC Cancer Institute, Rotterdam; 23 Department of Internal Medicine, Meander Medical Center, Amersfoort; 24 Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, Netherlands

Background: Frail patients with newly diagnosed multiple myeloma (NDMM) have an inferior PFS and OS, a higher treatment discontinuation rate and more grade ≥3 non-hematologic toxicity, compared to fit patients. In order to improve their outcome we investigated a three-drug regimen with a presumed low-toxicity profile; ixazomib, daratumumab and low-dose-dexamethasone (Ixa-Dara-dex).

This trial is registered at www.trialregister.nl as NTR6297.

Aims: To present the long term follow up outcome, with an emphasis on the maintenance phase.

Methods: NDDM patients, who were frail according to the IMWG-Frailty Index, were included. Patients defined frail based on age only (frail-age) were compared to frail patients based on other reasons (impairments (i)ADL and/or CCI≥2; frail-other) and patients frail based on age as well as other reasons (frail-both).

After nine cycles of Ixazomib (4mg; days 1, 8, 15), daratumumab (16mg/kg iv; cycles 1-2: days 1, 8, 15, 22; cycles 3-6: days 1, 15; cycles 7-9: day 1) and low dose dexamethasone (on the days daratumumab was administered; cycle 1-2: 20mg; subsequent cycles 10mg), patients without progressive disease or excessive toxicity continued with maintenance treatment, consisting of 8-week cycles with ixazomib (4mg orally on days 1, 8, 15, 29, 36, 43), daratumumab (16mg/kg iv or 1800mg subcutaneously on day 1) with dexamethasone (10mg intravenously on day 1), until progression, for a maximum of two years.

Results: Sixty-five frail patients were included. After a median follow-up of 39 months, the median progression free survival was 13.8m (95%CI: 9.2-17.7). The median PFS2 for all patients was 30.7m (22.2-39.1), 39.1 months in patients classified as frail-age, 24.5 months in frail-other patients and 26.6 months in frail-both patient (log-rank p=0.30). Median OS for all patients was 34.0m (24.0-41.2), not reached (frail age), 28.1m (frail-other) and 30.7 months (frail-both) (log-rank p=0.29) (Table 1).

Thirty-two patients (49%) proceeded to the maintenance phase. During maintenance treatment, 6 patients (19%) had improvement of response: 1 SD to MR, 3 PR to VGPR, 1 VGPR to CR and 1 VGPR to sCR. The rate of VGPR or better improved from 41% to 50% during maintenance.

During maintenance, 21/32 (66%) patients discontinued therapy, because of progressive disease (14/21; 67%), toxicity (2/21; 10%; infection and intracranial hemorrhage), non-compliance (1/21; 5%), intercurrent death (1/21; 5%) and other reasons (3/21; 14%; physician’s choice, dementia and patient condition).

Hematologic ≥3 grade adverse events (AEs) during maintenance were limited: neutropenia 0%, anemia 3% and thrombocytopenia 9%. Non-hematologic ≥3 grade AEs occurred in 18 (56%) patients. Most common AEs were infections (9%), nervous system disorders (9%; cognitive disturbance, stroke and syncope) and gastro-intestinal complaints (6%). There were 2 (6%) second primary malignancies and one patient (3%) experienced grade 3 neuropathy.

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Summary/Conclusion: Ixa-dara-dex maintenance treatment in frail patients was safe, and resulted in an improvement in response rate in 19% of patients. Patients frail based on age, had higher PFS, PFS2 and OS, as compared to other frail subgroups.

P907: CARFILZOMIB, DEXAMETHASONE, AND DARATUMUMAB (KDD) VS CARFILZOMIB AND DEXAMETHASONE (KD) IN RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): FRAILTY SUBGROUP ANALYSIS OF THE CANDOR STUDY

H. Quach 1,* , X. Leleu 2 , M.-V. Mateos 3 , S. Z. Usmani 4 , A. K. Nooka 5 , A. Goldrick 6 , R. Najdi 6 , N. Shu 7 , T. Facon 8

1 University of Melbourne, St Vincent’s Hospital, Melbourne, VIC, Australia; 2 CHU de Poitiers, La Miletrie/INSERM CIC 1402, Poitiers, France; 3 University Hospital Salamanca/ISAL, Salamanca, Spain; 4 Memorial Sloan Kettering Cancer Center, New York; 5 Winship Cancer Institute, Emory University, Atlanta; 6 Amgen, Thousand Oaks, United States of America; 7 Parexel, Chengdu, China; 8 Hôpital Claude Huriez, Lille, France

Background: Frailty scores based on age, comorbidities, and functional status can help identify frail patients (pts) at risk of higher rates of treatment-related toxicity or poor outcomes. An analysis of carfilzomib-treated pts in ASPIRE, ENDEAVOR, and ARROW found that efficacy and safety of carfilzomib regimens is consistent regardless of frailty status (Facon Blood Adv 2020).

Aims: To compare efficacy and safety across frailty subgroups in pts with RRMM treated with KdD vs Kd in the phase 3 CANDOR study.

Methods: This post hoc analysis used a planned interim readout (June 15, 2020) of CANDOR ( NCT03158688 ). Pts were categorized as fit, intermediate (int), or frail based on a frailty score of 0, 1, or ≥2. Scoring used an algorithm based on the International Myeloma Working Group (IMWG) frailty index: the final score is based on age (0 if ≤75 years, 1 if 76-80 years, 2 if >80 years), modified Charlson Comorbidity Index (CCI [Facon Blood Adv 2020]) (0 if CCI ≤1, 1 if CCI >1), and Eastern Cooperative Oncology Group performance status (ECOG PS) (0 if ECOG PS=0, 1 if ECOG PS=1, and 2 if ECOG PS=2). Progression-free survival (PFS) was summarized descriptively, with hazard ratio (HR) and 95% CI estimated by stratified Cox proportional hazards model. Overall response rate (ORR) was summarized descriptively, with odds ratio (OR) and 95% CI estimated by Mantel-Haenszel method. Response and progression were assessed by the Onyx Response Computer Algorithm using IMWG criteria.

Results: Frailty status was generally proportional between arms, with 27% and 35% classified as fit, 43% and 36% int, 25% and 27% frail, and 5% and 2% unknown for KdD and Kd, respectively. Although baseline characteristics of the overall population were generally balanced, more pts had prior transplant in the KdD vs Kd arm for int (65% vs 36%) and frail (41% vs 27%) subgroups, and in the frail subgroup fewer pts were lenalidomide exposed (37% vs 61%)/refractory (25% vs 46%) in the KdD vs Kd arm. Median duration of treatment for KdD vs Kd was 99 weeks (wks) vs 68 wks for fit pts, 82 wks vs 31 wks for int pts, and 51 wks vs 21 wks for frail pts. Median PFS in KdD vs Kd arms was not reached (NR) vs 17.6 months (HR 0.64, 95% CI 0.38–1.07) for fit, NR vs 11.1 months (HR 0.44, 95% CI 0.28–0.69) for int, and 18.5 vs 9.3 months (HR 0.66, 95% CI 0.38–1.14) for frail pts. ORR for KdD vs Kd arms was 89% vs 89% (OR 1.09, 95% CI 0.35–3.38) for fit pts, 87% vs 70% (OR 2.95, 95% CI 1.31–6.62) for int pts, and 75% vs 54% (OR 2.39, 95% CI 1.09–5.22) for frail pts ( Figure ). Any grade treatment-emergent adverse events (TEAEs) occurred in >95% of pts across arms and subgroups. Grade ≥3 TEAEs occurred in 87% (KdD) and 70% (Kd) of fit pts, 84% and 71% of int pts, and 91% and 90% of frail pts. Fatal TEAEs occurred in 4% and 2% of fit pts, 11% and 9% of int pts, and 16% and 8% of frail pts in the KdD and Kd arms. Carfilzomib was discontinued due to TEAEs in 25% vs 17% of fit pts, 22% vs 29% of int pts, and 35% vs 23% of frail pts. Among TEAEs of interest, acute renal failure occurred in 0 fit pts, 3% vs 14% of int pts, and 8% vs 5% of frail pts, and infusion reactions occurred in 16% vs 2% of fit pts, 13% vs 4% of int pts, and 14% vs 13% of frail pts in the KdD vs Kd arms (Table ).

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Summary/Conclusion: Consistent with previous findings of the efficacy and safety benefits of KdD, a PFS benefit with KdD vs Kd was observed across frailty subgroups, without increased toxicity. The clinically meaningful ORR benefit in the frail subgroup may help physicians evaluate treatment options in this challenging group.

P908: FACTORS ASSOCIATED WITH THE CLINICAL PROGNOSTIC PERFORMANCE OF MINIMAL RESIDUAL DISEASE ASSESSMENT BY NEXT GENERATION FLOW CYTOMETRY IN MULTIPLE MYELOMA

B. Nandakumar 1 , A. Baranwal 1 , M. Ebraheem 1 , H. Olteanu 2 , M. Shi 2 , S. Kumar 1 , W. Gonsalves 1 , D. Jevremovic 2,*

1 Hematology; 2 Laboratory Medicine and Pathology, Mayo Clinic, Rochester, United States of America

Background: Minimal Residual Disease (MRD) negativity (-) using next generation flow (NGF) cytometry with a minimum sensitivity of 10 -5 is strongly associated with improved progression-free survival (PFS) and overall survival (OS). However, clinicopathologic factors affecting the prognostic performance of this assay are not clear at this time, and despite the achievement of MRD (-) status, disease progression is still readily observed in multiple myeloma (MM).

Aims: We investigated quantitative non-clonal plasma cell factors measurable in the bone marrow sample that could predict disease progression in MRD (-) MM patients.

Methods: We retrospectively reviewed all patients with MM who underwent a bone marrow biopsy at the Mayo Clinic, Rochester, USA from July 2017 to December 2020. MRD testing was done on bone marrow using the established Euroflow protocol with analytic sensitivity between 10 -5 and 2x10 -6 . In addition to quantitative measurements of the number of clonal plasma cells, the number of hematogones, polyclonal plasma cells and mast cells were collected.

The time to next therapy (TTNT) was defined as the time from the date of MRD testing to the date of starting a new treatment regimen due to disease progression. Patients who did not require change in treatment for MM were censored at the last known follow-up. Kaplan-Meier curves and log-rank method were used to compare TTNT.

Results: A total of 1,142 NGF assessments, obtained in 783 different patients with MM, were found to have an MRD (-) result. The median age of patients at the time of collection was 63 years. The median absolute number of non-aggregate events captured was 8,586,360 (range: 840,904 - 9,975,075). For the entire sample cohort, the median number of polyclonal plasma cells were 1,874 (range: 0 - 121,221), the median number of hematogones were 70,307 (range: 0 - 6,486,739) and the median number of mast cells were 734 (range: 0 - 116,834).

Of the 1,142 NGF assessments, follow-up data was available on 675 samples, of which 204 (30%) were associated with disease progression requiring a change in therapy.

Number of polyclonal plasma cells and mast cells greater than their respective medians were considered elevated and were associated with disease progression (Χ 2 p < 0.001 for polyclonal plasma cells and Χ 2 p = 0.046 for mast cells).

Patients with higher polyclonal plasma cell events were found to have significantly higher TTNT compared to those with lower plasma cell events (median NA vs. 31 months, p < 0.001). The median TTNT for patients with higher mast cell events was also significantly higher compared to those with lower mast cell events (median NA vs. 36 months, p = 0.002).

Summary/Conclusion: The number of polyclonal plasma cells and mast cells in the bone marrow sample could be useful in predicting myeloma progression in patients with a MRD (-) bone marrow assessment by NGF. This observation may be related to the quality of the specimen (hemodilution), true biologic activity of polytypic plasma cells and mast cells, or both.

P909: RENAL RESPONSE OF POMALIDOMIDE WITH BORTEZOMIB AND DEXAMETHASONE IN NEWLY DIAGNOSED MULTIPLE MYELOMA PATIENTS WITH RENAL IMPAIRMENT

Y. Jian 1 , L. Chang 2 , M. Shi 3 , Y. Sun 4 , X. Chu 5 , H. Xue 6 , X. Shen 7 , J. Ma 8 , G. Jia 9 , Y. Feng 10 , Z. Xi 11 , Y. Zhao 12 , Y. Ma 13 , J. Xiao 14 , G. Ma 15 , Q. Wang 16 , W. Huang 17 , L. Bao 18 , Y. Dong 19 , H. Zhou 20 , C. Sun 21 , G. Su 22 , Y. Yan 23 , Q. Saiyin 24 , L. Su 25 , S. Gao 26 , W. Tian 27 , X. Sun 28 , H. Jing 29 , D. Gao 30 , W. Chen 1 , J. Li 2 , W. Gao 1,*

1 Beijing Chaoyang Hospital, Capital Medical University; 2 Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing; 3 The First Affiliated Hospital of Kunming Medical University, Hematology Research Center of Yunnan Province, Kunming; 4 Chifeng Municipal Hospital, Chifeng; 5 The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Qingdao; 6 Affiliated Hospital of Hebei University, Baoding; 7 Heping Hospital Affiliated To Changzhi Medical College, Changzhi; 8 The First Affiliated Hospital of Zhengzhou University, Zhengzhou; 9 The First Affiliated Hospital of Baotou Medical College, Inner Mongolia University of Secience and Technology, Baotou; 10 The Third People’s Hospital Of Datong, Datong; 11 Linfen People’s Hospital, Linfen; 12 The First Affiliated Hospital, Harbin Medical University, Harbin; 13 Second hospital of Shanxi Medical University, Taiyuan; 14 Yantaishan Hospital Affiliated to Binzhou Medical University, Yantai; 15 The Forth Hospital of Hebei Medical University, Shijiazhuang; 16 The Second Affiliated Hospital of Nanchang University, Nanchang; 17 The Fifth Medical Center of People’s Liberation Army (PLA) General Hospital; 18 Beijing Jishuitan Hospital; 19 Peking University First Hospital; 20 Beijing Luhe Hospital, Capital Medical University, Beijing; 21 Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan; 22 Cangzhou Central Hospital, Cangzhou; 23 Bayannur Hospital, Bayannur; 24 Ordos Central Hospital, Ordos; 25 Shanxi Cancer Hospital, Taiyuan; 26 The First Hospital of Jilin University, Changchun; 27 Shanxi Bethune Hospital, Taiyuan; 28 The First Affiliated Hospital of Dalian Medical University, Dalian; 29 Peking University Third Hospital, Beijing; 30 The Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China

Background: Renal impairment (RI) is a common complication of multiple myeloma (MM), usually related to poor outcomes. Bortezomib-based regimens have been recommended as first-line therapy of MM with RI. Recently, pomalidomide also has shown significant efficacy and an acceptable safety profile in MM patients with RI. However, few prospective studies evaluated the value of combination of pomalidomide with bortezomib and dexamethasone (PVD) in these special patients.

Aims: To evaluate the renal response, hematological response and safety of pomalidomide in combination with bortezomib and dexamethasone (PVD) as first-line therapy in newly diagnosed MM (NDMM) with RI.

Methods: This study is an ongoing, prospective, open-label, multicenter, phase 2 study (ChiCTR2100043748). Eligible adult NDMM patients with MM-related RI (defined as estimated glomerular filtration rate (eGFR) < 40ml/min) who had measurable disease are enrolled in the study. Exclusion criteria included RI caused by other reasons than tubular nephropathy, such as renal amyloidosis, light chain deposition disease, etc. Patients received a maximum of 9 cycles of PVD therapy. For transplant-eligible patients, autologous stem cell transplantation (ASCT) was administrated after 3 to 6 PVD cycles. Primary endpoint was renal overall response rate (ORR) at 3 months. Secondary endpoints included best renal response, hematological response, minimal residual disease (MRD), progression-free survival (PFS), overall survival (OS) and safety. Both hematological response and renal response were assessed by International Myeloma Working Group (IMWG) criteria.

Results: Between Feb 28, 2021 and Jan 21, 2022, sixty-one patients were enrolled across 28 Chinese hospitals, with a median age of 61 years (range: 38-79); 60.7% were male. Fifty-eight (95.1%) patients had cytogenetic information by FISH at diagnosis, fourteen (24.1%) of them had high-risk cytogenetic abnormalities (defined as t(4;14), t(14;16), and/or del(17p)). Median serum creatine level and eGFR were 349 μmol/L (range: 155-1718) and 12.9 mL/min (range: 2.5-36.3), respectively. Twelve (19.7%) patients received dialysis. Median number of PVD treatment cycles was 4 (range: 1-9). Six patients had received ASCT. Among 56 patients with evaluable hematological response, ORR was 91.1% (69.6% ≥VGPR, 41.1% CR). Forty-eight (78.7%) patients had got the primary endpoint with renal ORR at 3 months, which was 89.4% (48.9% ≥ renal-PR, 35.4% renal-CR). Median time to best renal response was 1.5 months (range: 0.3-11.0). Dialysis independence rate was 58.3% (7/12). Median time from first dose of PVD therapy to dialysis independence was 1 month (range: 0.5-2.0). Rapid reduction of serum free light chain (FLC) was seen at the first cycle of PVD therapy. Median reduction proportion of involved FLC at C1D8 was 77.5% (range: 0-98.5%), while at C1D22 was 91.7% (range: 0-99.9%). The most common adverse effects (incidence >10%) were infection (29.5%), myelosuppression (18.0%), peripheral neuritis (16.4%) and skin rash (11.5%). Median follow-up time was 6 months. Twelve patients (19.7%) discontinued PVD treatment, mainly due to toxicity and patients’ intention. Median PFS and OS has not been reached.

Summary/Conclusion: PVD regimen is an effective and well-tolerated regimen for NDMM patients with RI, which offered high renal response (renal ORR at 3 months of 89.4%).

P910: DOES MINIMAL RESIDUAL DISEASE OF STEM CELL COLLECTION HAVE PROGNOSTIC IMPACT ON PATIENTS WITH MULTIPLE MYELOMA?

X. Jingyu 1,* , Y. Wenqiang 1 , F. Huishou 1 , L. Jiahui 1 , L. Lingna 1 , D. Chenxing 1 , D. Shuhui 1 , S. Weiwei 1 , X. Yan 1 , Q. Lugui 1 , A. Gang 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Blood Diseases Hospital & Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Multiple myeloma (MM) is a kind of hematological malignancy involved in monoclonal plasma cells. In most clinical guidelines autologous stem cell transplantation (ASCT) is considered as the standard of care for transplant-eligible patients (TEMM) as long as patients achieving partial response (PR). However, it is unclear that under the circumstance of not achieving complete response (CR) weather it would lead to the presence of neoplastic plasma cells in the stem cell collection (SCC) and then result in negative impact on survival prognosis.

Aims: Here, we evaluated the effect of the minimal residual disease (MRD) of SCC in the TEMM.

Methods: We analyzed retrospectively clinical data of 90 patients with MM undergoing ASCT between January 1, 2013 to June 1, 2021 and MRD evaluation of both bone marrow (BM) and SCC were carried out at the same time. MRD was evaluated by multiparameter flow cytometry (MFC) with 10 -4 -10 -5 sensitivity. The best response was defined as the deepest response during the follow-up. Here we defined the time from ASCT to disease progression or death as modified progression-free survival (mPFS) and the time from ASCT to death as modified overall survival (mOS).

Results: A total of 90 patients met the inclusion criteria. The median age is 54 (37-69) and 62.2% were males. There were 25 (27.8%) patients presenting high-risk cytogenetic abnormalities by FISH, defined as the presence of at least one of t (4;14), t (14;16) or del (17p). Before ASCT 36.7% of patients achieved MRD negativity in BM and 76.7% in SCC. After the comparison among MRD-positivity status with different sensitivity and numbers of detectable MRD neoplastic plasma cells, we found that the percentage of patients with MRD positivity in SCC was much less than that in BM no matter the sensitivity ( P <0.001). Neither mPFS ( P =0.861, median mPFS, 40.83m vs. 34.17m for negativity vs. positivity) nor mOS ( P =0.747, median mOS, 67.02m vs. 58.86m for negativity vs. positivity) was affected by MRD status in SCC. According to MRD status they were divided into 4 groups, namely MRD negativity in BM and SCC (Group A, 34.4%), MRD positivity in BM but negativity in SCC (Group B, 22.2%), MRD positivity in BM and SCC (Group C, 41.1%) as well as MRD negativity in BM but positivity in SCC (Group D, 2.3%). Having taken the inaccuracy of biopsy into consideration we excluded Group D. The median follow-up of the cohort was 26.8 months (15.1-105.1m). Patients among the three groups experienced similar mPFS ( P =0.403, median mPFS, 41.07m vs. 34.17m vs . 40.83m, for Group A, B and C, respectively) and similar mOS ( P =0.933, median mOS, 67.02m vs . 58.86m vs. 58.61m for Group A, B and C, respectively). Achievement of CR with the presence of MRD negativity was associated with prolonged mPFS and mOS compared with MRD-positive CR or ≤VGPR ( P ＜0.001, median mPFS, 55.88m vs. 23.03m vs. 27.10m, respectively; P =0.026, median mOS, 67.02 vs . 46.16m vs. 41.65m, respectively).

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Summary/Conclusion: Our results demonstrated that neoplastic plasma cells in SCC have little impact on the survival prognosis in MM patients and it is sound to carry out ASCT when TEMM patients achieving PR. Also, MRD-negativity status can be considered more valuable on prognosis than CR.

P911: SYMPTOM BURDEN AND ITS IMPACT ON DAILY LIFE AMONG PATIENTS WITH IDIOPATHIC MULTICENTRIC CASTLEMAN DISEASE (IMCD) – FINDINGS FROM AN INTERNATIONAL IMCD PATIENT SURVEY

F. Shupo 1 , N. Mason 2 , E. Jones 2 , G. Wayi-Wayi 1 , M. Repasky 3 , M. Franklin 4 , J. Brazier 4 , N. Zibelnik 1 , S. Mukherjee 5,*

1 EUSA Pharma, Hemel Hempstead; 2 BresMed Health Solutions, Sheffield, United Kingdom; 3 Castleman Disease Collaborative Network, Paso Robles, United States of America; 4 School of Health and Related Research, Sheffield, United Kingdom; 5 Hematology and Medical Oncology, Cleveland Clinic Main Campus, Cleveland, United States of America

Background: Idiopathic Multicentric Castleman disease (iMCD) is a rare lymphoproliferative disorder driven by proinflammatory hypercytokinemia. The presentation of iMCD is heterogeneous and can range from mild constitutional symptoms to chronic burdensome symptoms and in extreme cases life-threatening multiorgan failure. Consequently, disease-related symptoms in iMCD patients are likely to adversely impact daily life. To date, characterization of symptom burden and their impact on daily living in iMCD patients has not been systematically studied.

Aims: We aimed to investigate, characterize, and map the symptoms and associated burden on daily life experienced by patients with various subtypes of iMCD.

Methods: We developed an international patient-based online survey informed by clinical practice and published literature to elicit the burden of disease-related symptoms and effects of symptoms on daily life from a patient perspective. Eligible patients were > 18 years old with physician-confirmed diagnosis of iMCD-NOS (not otherwise specified), TAFRO (thrombocytopenia, anasarca, reticulin fibrosis of the bone marrow, renal dysfunction, and organomegaly) and POEMS—associated MCD (multicentric Castleman disease with polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes). This survey was shared with iMCD communities in Australia, Canada, the UK, and the US via the Castleman Disease Collaborative Network (CDCN). Burden of Illness (BOI) was quantitatively measured using a 5-point frequency Likert scale (from 0 ‘Does not affect my daily life’ to 4 ‘Very severely affects my daily life’), and mean impact scores (MIS) were calculated. Ethics approvals/waivers were attained for this one-time, cross-sectional, bespoke 47-question survey.

Results: A total of 57 patient responses were collected during April–November 2021. On average, patients experienced 7.0 symptoms (range: 0─22) in the week prior to survey completion. Tiredness was the most frequently reported symptom (77%), followed by physical weakness (44%) and night sweats (40%). Individual symptoms were clustered into clinically relevant categories and frequency of symptom groupings reported by iMCD subtype (Fig 1). Constitutional (82%) and neuropsychiatric (68%) symptoms were most frequently experienced across all respondents in the week prior to survey completion. The average number of symptoms by iMCD subtypes were 7.1 by iMCD-NOS, 5.5 by TAFRO and 8.5 by POEMS-associated MCD. 91% of all patients with iMCD reported experiencing at least one symptom in the week prior to survey completion. When rating their most impacted aspects of daily life due to their symptoms (Table 1), patients with iMCD-NOS reported pain and discomfort (MIS 2.09) and personal relationships (MIS 2.08), patients with MCD-POEMS reported sexual functioning (MIS 3.40) and pain and discomfort (MIS 2.67), while patients with TAFRO reported sexual functioning (MIS 2.44) and ability to travel (MIS 2.22).

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Summary/Conclusion: To our knowledge, this is the first study of its kind to characterize and map the BOI in iMCD patients assessed by symptom frequency, symptom burden (multiplicity of symptoms) and its adverse effects on different aspects of daily living. Through our ongoing work we hope to develop a symptom burden score/scale that captures the symptom severity and its impact on daily living which can then be incorporated as a patient reported outcome measure for shared treatment decision-making and response assessment in addition to the laboratory and radiologic parameters.

P912: COVID19 SEVERITY AND THERAPEUTIC OPTIONS IN PATIENTS WITH MULTIPLE MYELOMA

E. Karimova 1,* , E. Zhelnova 1 , E. Baryakh 1 , K. Yatskov 1 , E. Zotina 1 , E. Grishina 1 , E. Paramonova 1 , D. Gagloeva 1 , E. Misyurina 1

1 hematology, Moscow city hospital №52, Moscow, Russia

Background: Patients with multiple myeloma have higher risk of SARS-CoV-2 infection and higher risk of death than patients without MM

Aims: Analysis of features of the course of COVID-19 in patients with MM.

Methods: 89 pts with MM and COVID-19 were treated in the hospital from March 2020 to October 2021, 53 (60%) female, 36 (40%) male. The median age was 64 years (35-88 years). 32 pts (36%) were presented with active myeloma, 19 (21%) – R/R myeloma, 21 (24%) – CR, 17 (19%) - PR or VGPR. The majority of patients - 56 (63%) had III st. (Durie-Salmon). Kidney damage was presented in 33 pts (37%). The majority of patients had severe lung damage: CT 2 st. - 26 pts (30%), III st. - 35 (39%), IV st.-17 (19%).

Patients received standard therapy for COVID19: etiotropic (antiviral, viral neutralizing MAB, convalescent plasma, immunoglobulin), pathogenetic (GCS, interleukin inhibitors, anticoagulants), therapy for secondary infectious complications.

Results: IL inhibitor therapy was given 76 patients (85%), 35 (39%) required repeated administration of IL inhibitors. 58 pts (65%) received GCS therapy in various doses.

Anticoagulant therapy was conducted in all patients, 51 pts (57%) received LMWH, 37 (42%) received continuous infusion of heparin, 1 patient received NOAC. The frequency of hemorrhagic complications on LMWH and heparin therapy was comparable (5.9% and 5.5%), mortality and the frequency of TEC in patients on heparin therapy were higher (49% and 13% versus 14% and 2% in patients on LMWH therapy, p<0.001 and p=0.034, respectively). It can be explained by the initial severity of patient’s condition, which required heparin therapy (30 pts - 81% had an initially high degree of lung damage - CT 3-4 st.)

Secondary infectious complications were reported in 80% of patients: bacterial pneumonia in 71 pts (80%), sepsis in 24 (27%). Infectious endocarditis (2 pts), meningitis (2 pts), colitis (9 pts), colon gangrene complicated by peritonitis (1 patient) were less common. Fungal infections were verified in 19 pts (21%): 6 (7%) – mucosal candidiasis, 13 (14%) – invasive aspergillosis. Infection caused by herpes viruses was detected in 5 patients (6%). There was no significant connection between severity of COVID19, frequency of secondary infections and mortality and level of normal immunoglobulins.

The median duration of hospitalization was 19 days versus 9 among patients without MM. 25 patients (28%) were hospitalized in the ICU. The overall mortality was 27% (24 pts), among this patients 71% (17 pts) had active or R/R myeloma and stage III of the disease. The mortality in the ICU was 88% (22 pts). The most frequent causes of fatality were secondary infectious complications in 92% (22 pts), progression of MM in 8% (2 pts). 27 patients (30%) required chemotherapy during COVID-19.

Summary/Conclusion: Patient with R/R MM have the highest risk of mortality from COVID19. For patients with multiple myeloma the same COVID19 therapeutic options are effective as for non-immunodeficient patients. Patients with MM and COVID19 also have a high risk of secondary infectious complications.

P914: EFFICACY AND SAFETY OF BELANTAMAB MAFODOTIN MONOTHERAPY IN PATIENTS WITH RELAPSED OR REFRACTORY LIGHT CHAIN AMYLOIDOSIS: A PHASE 2 STUDY BY THE EUROPEAN MYELOMA NETWORK

E. Kastritis 1,* , G. Palladini 2 , M. A. Dimopoulos 1 , A. Jaccard 3 , G. Merlini 2 , F. Theodorakakou 1 , D. Fotiou 1 , M. C. Minnema 4 , A. Wechalekar 5 , S. Gkolfinopoulos 6 , K. Manousou 6 , P. Sonneveld 7 , S. Schönland 8

1 Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 2 Amyloidosis Research and Treatment Center, University of Pavia, Pavia, Italy; 3 Referral Center for AL amyloidosis, Limoges, France; 4 Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands; 5 Clinical Haematology, Cancer Division, University College London Hospital, London, United Kingdom; 6 Health Data Specialists, Dublin, Ireland; 7 Erasmus MC Cancer Institute, Rotterdam, Netherlands; 8 University of Heidelberg, Heidelberg, Germany

Background: Managing patients (pts) with relapsed/refractory (RR) light chain (AL) amyloidosis is challenging, as there is no current standard treatment, many available options are associated with low efficacy and toxicity, and options for daratumumab (DARA)- and bortezomib-exposed patients are limited. Belantamab mafodotin (belamaf), a multi-modal antibody-drug conjugate targeting BCMA, has shown efficacy and tolerability in heavily pretreated pts with RR multiple myeloma, including those refractory to DARA. Since clonal plasma cells in AL amyloidosis and MM are phenotypically similar, belamaf could be a novel treatment option in AL amyloidosis.

Aims: To evaluate the efficacy and safety of belamaf monotherapy off-label in pts with RR AL amyloidosis.

Methods: The ongoing prospective, open-label, multinational, phase 2, EMN27 study ( NCT04617925 ) aims to enroll 36 adult pretreated pts with AL amyloidosis who require therapy. Pts at Mayo cardiac stage 3b are excluded. Belamaf monotherapy at 2.5mg/kg is administered by intravenous infusion every 6 weeks for a maximum of 8 cycles; dosing can be reduced to 1.92mg/kg for toxicity. Per study design, a safety analysis (after 6 pts received ≥1 treatment cycle) and an efficacy analysis (after 13 pts are enrolled) were planned. The safety analysis revealed no new safety signals, and pt accrual continued to 13 pts. The efficacy analysis is currently conducted; however, already 3 pts achieved complete response, or very good partial response (VGPR), or low difference of involved to uninvolved serum free light chains (dFLC) response and enrollment is continuing to include all planned pts. This descriptive analysis included pts initiating study treatment ≥3 months before the cut-off date (15/01/2022).

Results: Of 11 pts included in the analysis, 4 (36.4%) continued treatment by the cut-off date, and 7 (63.6%) discontinued (disease progression: 5 [45.5%], death: 2 [18.2%]). The pts median age was 69.0 years (range 46.0–80.0), and most were males (7, 63.6%). At baseline, 3 (27.3%) and 8 (72.7%) pts had New York Heart Association class I and II symptoms, respectively; the median N-terminal pro-brain natriuretic peptide, high-sensitivity troponin T, and dFLC were 1,979 pg/mL (range 190.0–4,135.0), 41.6 pg/mL (range 11.0–80.8), and 34.2mg/dl (range 4.4–279.1), respectively. Except for the heart, commonly involved organs were the nervous system (4 pts, 36.4%) and the soft tissue (2 pts, 18.2%). The median number of previous AL amyloidosis treatments was 3.0 (range 1.0–7.0), including DARA. The median duration of belamaf therapy was 3.1 months (range 1.4–5.7). At a median follow up of 9.4 months (range 3.1–10.0), the overall response rate was 72.7% (8 pts; VGPR: 27.3% [3 pts] and partial response: 45.5% [5 pts]). Median time to first hematological response was 8.5 days (range 1.0–28.0) and to VGPR or better 15.0 days (range 8.0–15.0). The 3-month organ (heart, kidney, or liver) response rate was 36.4% (4 pts). All pts had ≥1 non serious adverse event (SAE). Four (36.4%) pts had ≥1 SAE, including 2 (18.2%) pts with a belamaf-related grade 2 and 4 visual impairment (1 [9.1%] pt each). Eight (72.7%) pts had ≥1 adverse event of special interest. Two (18.2%) pts had a fatal SAE (pneumonia and intestinal perforation, 1 [9.1%] pt each), both unrelated to belamaf.

Summary/Conclusion: In this prospective study, belamaf monotherapy induced rapid, clinically meaningful responses with a manageable safety profile in heavily pretreated pts with RR AL amyloidosis. As the study progresses, additional data will be generated.

P915: EFFICACY AND SAFETY OF DARATUMUMAB MONOTHERAPY IN NEWLY DIAGNOSED PATIENTS WITH STAGE 3B LIGHT CHAIN AMYLOIDOSIS: A PHASE 2 STUDY BY THE EUROPEAN MYELOMA NETWORK

E. Kastritis 1,* , M. C. Minnema 2 , M. A. Dimopoulos 1 , G. Merlini 3 , F. Theodorakakou 1 , D. Fotiou 1 , A. Huart 4 , K. Belhadj 5 , S. Gkolfinopoulos 6 , K. Manousou 6 , P. Sonneveld 7 , G. Palladini 3

1 Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 2 Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands; 3 Amyloidosis Research and Treatment Center, University of Pavia, Pavia, Italy; 4 Department of Nephrology and transplantation, Rangueil University Hospital, Toulouse; 5 Lymphoid Malignancies Unit, Henri Mondor Hospital, Créteil, France; 6 Health Data Specialists, Dublin, Ireland; 7 Erasmus MC Cancer Institute, Rotterdam, Netherlands

Background: Cardiac involvement and severity of cardiac dysfunction in light chain (AL) amyloidosis is a critical prognostic factor. Patients (pts) at Mayo cardiac stage 3b have a poor prognosis with a median overall survival (OS) of just 4 months and high rates of early death with current therapies; thus, there is a need for novel, non-toxic, effective treatments for these pts. Daratumumab (DARA), a human anti-CD38 antibody, has shown efficacy and tolerability in pts with AL amyloidosis.

Aims: To evaluate the efficacy and safety of DARA monotherapy used off-label in newly diagnosed pts with stage 3b AL amyloidosis.

Methods: The ongoing EMN22 phase 2, multinational, open-label study ( NCT04131309 ) aims to enroll 40 newly diagnosed pts with stage 3b AL amyloidosis. Eligible adult pts have high-sensitivity troponin T (hsTnT) >54 pg/mL and N-terminal pro-brain natriuretic peptide (NT-proBNP) ≥8,500 pg/mL. DARA monotherapy, 16 mg/mL by intravenous infusion (09/2019–01/2020) and 1,800 mg by subcutaneous injection (01/2020 and thereafter), is administered weekly during cycles (C)1 and 2, every 2 weeks for C3–6, and every 4 weeks thereafter. Pts not achieving a hematological very good partial response (VGPR) or better by the end of C3 can receive additional weekly bortezomib and low dose dexamethasone (Vd). Treatment continues up to 2 years from initiation or until disease progression or initiation of a new therapy. Primary endpoint is OS rate at 6 months. This descriptive analysis included pts initiating treatment ≥6 months before the cut-off date (14/01/2022); the median (95% confidence interval [CI]) OS was obtained by Kaplan-Meier analysis.

Results: Of 27 pts included, 8 (30%) continued study treatment by the cut-off date and 19 (70%) had discontinued. The pts median age was 68 (range 45–84) years, and most were male (16, 59%). At screening, 10 (37%) and 17 (63%) pts had New York Heart Association class II and IIIA symptoms, respectively; the median NT-proBNP was 15,512 pg/mL (range 8,816–72,522), hsTnT was 133 pg/mL (range 60–692), and the difference of involved to uninvolved free light chains was 406 mg/l (range 24–3,377). Beyond the heart, the median number of other organs involved was 2 (range 0–5), most commonly kidneys (14 pts, 52%) and peripheral nerves (11 pts, 41%). The median duration of DARA therapy was 7 months (range <1–24); seven (26%) pts received additional Vd. At a median observation time of 8 months (range <1–11), the overall response rate (ORR) was 67% (18 pts; complete response [CR]:19.0% [5 pts], VGPR:37% [10 pts], partial response:11% [3 pts]). The ORRs at 1, 2, and 3 months were 59% (16 pts), 63% (17 pts), and 63% (17 pts), respectively. Median time to first response was 7 days (range 6–114), and to VGPR or better 54 days (range 6–219). Median OS was 9 months (95% CI, 3–not reached). The 6- and 12-month median (95% CI) OS rates were 63% (42–78) and 49% (28–67), respectively. Twenty-five (93%) pts had ≥1 non-serious adverse event. Twenty (74%) pts had ≥1 serious adverse event (SAE), comprising 15 (56%) pts with ≥1 cardiac-related SAE and 11 (41%) pts with a fatal SAE. Six SAEs were treatment-related: 2 with DARA (grade 3 pneumonia, grade 5 sepsis); 3 with bortezomib (grade 2 fatigue, grade 2 fall, and grade 3 troponin I increase); and 1 with dexamethasone (grade 3 cardiac failure).

Summary/Conclusion: Among pts with Mayo stage 3b AL amyloidosis, a subgroup with poor prognosis, DARA monotherapy induced rapid and deep hematological responses and no new safety signals; the median OS surpassed that reported previously.

P916: COEXISTENCE OF ≥2 HIGH-RISK MOLECULAR ABNORMALITIES SUPERVENES THE PROGNOSTIC VALUE OF THE REVISED INTERNATIONAL STAGING SYSTEM FOR MYELOMA: REAL-WORLD DATA ANALYSIS FROM THE GREEK MYELOMA STUDY GROUP

E. Katodritou 1,* , D. Dalampira 1 , T. Triantafyllou 1 , M. Gavriatopoulou 2 , S. Delimpasi 3 , A. Pouli 4 , T. Papadopoulou 1 , E. Verrou 1 , A. Sevastoudi 1 , K. Tsirou 1 , L. Katsika 1 , G. Douganiotis 1 , N. Karampatzakis 1 , T.-E. Metallinou 1 , V. Palaska 1 , M. Kotsopoulou 5 , C. Lalayianni 6 , M.-C. Kyrtsonis 7 , E. Spanoudakis 8 , D. Maltezas 5 , A. Chatzivasili 9 , M.-A. Dimopoulos 2 , E. Terpos 2 , E. Kastritis 2

1 Hematology, Theagenio Cancer Hospital, Thessaloniki; 2 Clinical Therapeutics, National and Kapodistrian University of Athens; 3 Hematology and Bone Marrow Unit, Evangelismos General Hospital; 4 Hematology, Agios Savvas Cancer Hospital; 5 Hematology, Metaxa Cancer Hospital, Athens; 6 Hematology and Bone Marrow Unit, George Papanicolaou General Hospital, Thessaloniki; 7 First Department of Propaedeutic Internal Medicine, National and Kapodistrian University of Athens, Athens; 8 Hematology, University Hospital of Alexandroupolis, Alexandroupolis; 9 Hematology, Venizelion General Hospital, Heraclion, Greece

Background: Revised International Staging System (R-ISS) has improved the prognostic value of ISS in multiple myeloma (MM). Recently, the Mayo Additive Staging System incorporated +1q21 to determine a 5-factor 3-tier system, providing an add-on value on R-ISS. The prognostic impact of the coexistence of ≥2 high-risk molecular abnormalities, including +1q21, compared to R-ISS, has not been validated adequately.

Aims: The aim of this study was to evaluate the prognostic impact on survival of the coexistence of ≥2 high-risk molecular abnormalities, defined as Ultra High Risk (UHR) MM, in comparison with R-ISS and other established prognostic markers, in the real-world setting.

Methods: We analyzed the data of 1352 consecutive newly diagnosed MM patients (M/F: 655/697, median age: 66, range: 29-87, IgG: 817, IgA: 363, light chain: 144, IgD: 9, IgM: 3, non-secretory: 16), treated between 2002-2021 and which had been tested for molecular abnormalities i.e. del17p, t(14;16), t(4;14) and +1q21 using fluorescence in situ hybridization. Patients with ≥2 high-risk features were classified as UHR. We compared the two groups for age, performance status, ISS, R-ISS, lactate dehydrogenase (LDH), albumin, hemoglobin (Hb), β2-microglobulin, estimated glomerular filtration rate (eGFR), 1st and 2 nd line therapies and response rates. A Cox regression model was used to determine independent prognostic factors for overall survival (OS). Progression-free survival (PFS) and OS were plotted with Kaplan-Meier; a p<0.05 was considered as statistically significant.

Results: One hundred sixteen patients (9%) were classified in the UHR group vs. 1236 (91%) in the non-UHR group; 106 patients had 2, and 10 patients had 3 molecular abnormalities. The most common combination of high-risk features was +1q21 plus t(4;14) (40%). Median age, sex, performance status, LDH and serum albumin, did not differ, whereas the UHR group had lower eGFR, higher β2-microglobulin and lower Hb (p<0.05). Early stage (ISS1/R-ISS1) was more frequent in the non-UHR group (p<0.05); 1 st line treatment, including autologous transplantation (ASCT), and second line treatment were well balanced between groups (p<0.05); 66% of patients overall, received triplet/quadruplet combinations and 29% underwent ASCT upfront. Overall response rate after induction therapy was 86% and did not differ between groups (p<0.05). Complete response was lower in the UHR group (11% vs. 19%; p=0.03). After a median follow up of 49 months (95% CI: 44-54), 59% of patients were alive. Median PFS was significantly shorter for the UHR group (15.8 vs. 31.9 months, p<0.001; HR: 0.45, 95% CI: 0.36-0.58). Median OS was 28 months (95% CI:18-38) for patients in the UHR group vs. 69 months (95% CI: 61-77) for others (p<0.001). In the univariate analysis, age, anemia, eGFR, upfront ASCT, R-ISS and UHR myeloma were independent predictors for OS (p<0.05). In the multivariate analysis UHR myeloma was the strongest independent predictor for OS (p<0.001; HR: 0.42), supervening the prognostic value of R-ISS (R-ISS1 vs. R-ISS2 HR=0.58, R-ISS2 vs. R-ISS3 HR: 0.75). Additionally, UHR status singled out a distinct group within R-ISS2 patients with significantly worse OS (38 months, 95% CI: 28-48 vs. 64mo, 95% CI: 55-72) (p<0.001).

Summary/Conclusion: According to our analysis of a large cohort of newly diagnosed MM patients UHR myeloma, was the strongest independent predictor for OS, supervening the prognostic value of R-ISS. Moreover, UHR status could serve as an additional prognostic marker for R-ISS2 patients, helping thus to optimize therapeutic approach of MM patients.

P917: HOW COMORBID ARE OUR MYELOMA PATIENTS AND HOW MANY MAKE IT TO THE SECOND-LINE TREATMENT: REAL-WORLD DATA OF 251 MYELOMA PATIENTS TREATED IN THE HEMATOLOGICAL NETWORK OF THE OEGK

F. Keil 1,* , P. Attalla 1

1 3rd Medical Department for Hematology and Oncology, Hanusch hospital, Vienna, Austria

Background: Comorbid conditions have a negative impact on overall survival (OS) in multiple myeloma (MM) patients. Furthermore, a European multi-center study reported that only 61% of patients proceed to second-line treatment.

Aims: The objective of this study was to determine the prevalence of common comorbidities and their impact on OS. Furthermore, we examined the mortality during first-line treatment and the percentage of patients proceeding to second-line treatment.

Methods: This retrospective study was conducted in the hematology care network “Hämatologieverbund der Österreichischen Gesundheitskasse (OEGK)” , consisting of the Hanusch hospital and three outpatient centers, in Vienna, Austria. We included all patients who were observed and/or treated for multiple myeloma between 2012 and 2018 within the hematology care network. Follow-up was conducted until March 2021.

Results: We analyzed 251 patients (median age 69 years). Prevalent concomitant conditions at diagnosis were hypertension (67.4%), further cardiovascular diseases excluding hypertension (23.1%), obesity (15.2%), diabetes (13.8%) and chronic lung disease (9.2%). For the evaluation of renal impairment, we used the CCI, which defines kidney disease as a creatinine >3mg/dl or the need for dialysis, as well as the chronic kidney disease (CKD) classification system. While 7.8% of patients fulfilled the CCI criteria for kidney disease, 39.5% had a CKD stage ≥3A.

Comorbidity in general, as well as specific comorbid conditions had a negative effect on overall survival. Patients with a CCI score of 0 points at diagnosis showed a median OS of 8.7 years compared to 3.8 years and 2.6 years for patients with a score of 1 point and ≥2 points, respectively (p <0.001). We also observed a reduced OS of 4.9 years in patients with at least one creatinine clearance (CrCl) value <60 ml/min throughout the observation period compared to 16.4 years in patients with a CrCl consistently ≥60 ml/min (p <0.001).

In total, 232 patients received a first-line treatment. During the observation period after first-line therapy, 146 patients proceeded to second-line treatment due to progress, 30 showed no sign of progress so far and 12 had an ongoing first-line maintenance therapy. 21 patients were referred from other centers for a second opinion and/or ASCT. These patients were still alive at the end of the observation period, but data about further treatment lines were missing. One patient eligible for second-line treatment refused further therapeutic measures. Furthermore, 22 patients died during first-line treatment or the subsequent treatment-free interval. Of these 22 patients, 11 died during active first-line treatment due to progression of myeloma or treatment-related toxicity. The other 11 patients died after completion of first-line treatment during the observation period due to secondary neoplasms (4) and infection (2). For the remaining five patients the cause of death was unknown. In total, 90.5% of patients who had received a first-line treatment were or still are eligible for second-line treatment.

Summary/Conclusion: Comorbid conditions were prevalent in our population and exerted a major impact on mortality. In comparison to the CCI, the chronic kidney disease classification system showed superior sensitivity for detection of renal impairment, which is a good predictor of survival. In contrast to previously published data, a high proportion of patients were eligible for second-line treatment. Disease-specific mortality prevented only a small number of patients from receiving second-line treatment.

P918: HIGH RESPONSES RATES WITH SINGLE AGENT BELANTAMAB MAFODOTIN IN RELAPSED SYSTEMIC AL AMYLOIDOSIS

J. Khwaja 1,* , J. Bomsztyk 2 , S. Mahmood 2 , B. Wisniowski 2 , R. Shah 1 , A. Tailor 1 , K. Yong 1 , R. Popat 1 , N. Rabin 1 , C. Kyriakou 1 , J. Sive 1 , S. Worthington 1 , A. Hart 1 , E. Dowling 1 , N. Correia 1 , C. Bygrave 3 , A. Rydzewski 4 , K. Jamroziak 5 , A. Wechalekar 2

1 Department of Haematology, University College London Hospital; 2 National Amyloidosis Centre, University College London (Royal Free Campus), London; 3 Department of Haematology, University Hospital of Wales, Cardiff, United Kingdom; 4 Department of Internal Medicine, Nephrology and Transplantation Medicine, Central Clinical Hospital of the Ministry of Internal Affairs; 5 Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw, Warsaw, Poland

Background: Systemic AL amyloidosis is an incurable relapsing plasma cell disorder. Despite therapeutic advances, there are no approved treatments for relapse disease. Treatment is often challenging due to underlying organ dysfunction. Belantamab mafodotin is an antibody-drug conjugate targeting B-cell maturation antigen with approval for relapsed refractory myeloma. In multiply pre-treated myeloma, the DREAMM-2 phase II trial showed an overall response rate of 32% for those with 2.5 mg/kg dose administered every three weeks with 2/3 rd patients reporting keratopathy. A small case series of 6 patients with relapsed AL amyloidosis (Zhang et al , ASH 2021) was recently reported and a phase 2 trial is recruiting for patients with refractory amyloidosis ( NCT04617925 ).

Aims: We report our initial results using Belantamab monotherapy for the treatment of patients with AL amyloidosis with relapsed disease.

Methods: Data for consecutive patients who were administered Belantamab at a specialist referral centre, National Amyloidosis Centre, University College London, was analysed.

Results: Eleven patients were included 8 male, 3 female. Median age at Belantamab initiation was 65 (range 42-74) years. Eight patients had λ AL-type and three κ AL-type. At diagnosis, median involved free light-chain concentration was 534 (range 73-7181) mg/l. A median of two organs involved at baseline (range 1-3): 4 had cardiac involvement (half Mayo stage 2; half Mayo stage 3a) and 8 had renal involvement. The median prior lines of therapy was 3 (range 2-5) with all exposed to prior immunomodulatory drugs, proteasome inhibitors and 73% to anti-CD38 antibody treatments. Thirty-six percent had relapsed after melphalan-conditioned autologous stem cell transplantation.

A median of 3 cycles of belantamab were delivered (range 1-8). The most frequent adverse event was ocular toxicity which was experienced in 8 patients (grade 1-3), necessitating dose modification of the three-weekly schedule. One patient developed transient grade 1 dyspnoea and liver dysfunction. No patients developed cytopenias, unlike previous reports (Zhang et al , 2021), nor infections beyond COVID (2 patients mild with no hospital admissions). The majority of the cohort required dose reduction either at initiation (patient 4, due to end stage renal failure; patient 11, post-renal transplant) or during therapy (n=5; three to 1.9mg/kg, two to 1.25mg/kg) due to ocular toxicity. Only one patient remained on the standard dose of 2.5mg/kg for ≥ 3 cycles. Ocular toxicity improved after treatment interruption (drug intervals 4-6 weeks) and no patients required complete treatment cessation. One patient is too early to assess response. Haematological responses (PR or better) were seen in 7 patients with 3 complete responses and two very good partial responses (VGPR) which are ongoing. Both renal patients (patients 4 and 11) commenced a dose of 1.25mg/kg and sustained a VGPR with no additional toxicity. Patient 3 had a 42% reduction in sFLC after two doses but then a prolonged gap due to keratopathy and has lost the response. There were no cardiac or renal toxicities observed.

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Summary/Conclusion: Belantamab mafodotin demonstrates significant activity in patients with heavily pre-treated AL amyloidosis with 70% achieving a ≥PR. Apart from keratopathy requiring dose modification, no other substantial toxicity was observed. Two patients with renal impairment (stage V CKD and ESRD) and one patient post-renal transplant tolerated treatment with no additional toxicity. Belantamab mafodotin shows promise in treatment of relapsed AL and needs further prospective trials.

P919: SIGNIFICANCE OF IHC AND BIOCHEMICAL MARKERS OF BONE METABOLISM FOR PREDICTING OSTEODESTRUCTIVE SYNDROME IN PLASMA CELL PROLIFERATION

Z. Kozich 1,* , V. Martinkov 1 , M. Zhandarov 1 , J. PUGACHEVA 1 , S. Mihno 1 , N. Klimkovich 2

1 The Republican Research Center for Radiation Medicine and Human Ecology, Gomel; 2 Belarusian Medical Academy for Postgraduate Education, Minsk, Belarus

Background: Multiple myeloma (MM) is a malignant disease of a lymphoid nature accompanied by the proliferation of tumor plasma cells, in its development passing through the stage of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SM). One of the main manifestations of MM is the lesion of the skeleton bones, which may already manifest at the stage of MGUS and SM and further it can lead to a decrease in the quality of life of patients. Our work is devoted to the study of the role of markers that contribute to the detection of the progression of the destructive syndrome at the stage of MGUS and SM.

Aims: To study significance of IHC and biochemical markers of bone metabolism for predicting osteodestructive syndrome in MGUS and SM.

Methods: All patients underwent aspiration and BM biopsy for cytological and histopathological assessment of PC infiltration. An immunological study of blood serum and the determination of biochemical markers of bone metabolism of serum were also performed. All patients underwent CT and MRI of the whole body. The diagnosis of MGUS was based on international criteria: the presence of less than 10% of clonal plasma cells in the bone marrow aspirate, the concentration of M-protein in the blood serum <30 g/l. Among patients with MM, a group without osteodestructive lesions of the skeleton bones was identified. Statistical processing of the results was carried out using the Statistica 6.1 software package. Differences were considered statistically significant at p<0.05.

Results: The study included 132 patients (63 MM patients and 68 MGUS patients), who did not differ in age at the time of diagnosis, p = 0.089, the median age was 64.0 years (25% and 75% - 56.0 to 69, 0) and 61.0 years (25% and 75% - 53.0 and 66.0), respectively. In the MG group, female patients predominated (70.1%), they were significantly more common than in the MM group (53.1%), p=0.045.

Damage to the skeleton bones during primary diagnosis (including SP) was detected in 37.4% (68) of cases. Bone tissue destruction was more common in males (p=0.029). Changes in bone tissue for patients with MGUS and SM detected by MRI in most cases are presented as a diffuse lesion, foci of bone tissue rarefaction without obvious foci of destruction or SP. The presence of destructive lesions was more frequently detected in MGUS patients with subsequent progression to MM (p<0.002). Time to progression was about 14 months on average (range from 3 to 25 months).

When analyzing the obtained results, in MGUS patients, an excess of the β CrossLaps level in serum occurred in 7.8% of cases. At the stage of MGUS, 25.3% of patients (according to the level of osteocalcin) and 16.1% (according to the level of VAR) had disorders in the processes of bone tissue formation. Bone tissue destruction was detected more often in patients with IgM secretion (p=0.014), the ratio of immunoglobulin light chains κ/λ <0.1 and >10 and more than 10% of CD 138+ plasma cells in immunohistochemical studies.

Summary/Conclusion: At the stage of MGUS, disturbances in the processes of bone tissue remodeling occur, which is accompanied by the appearance of deviations in the level of biochemical markers (osteocalcin, β CrossLaps). Therefore, these markers have the potential to be used to identify individuals at increased risk of developing a destructive syndrome, and in conjunction with other risk factors (IgM secretion of more than 10% of CD 138+ plasma cells on immunohistochemistry) and to identify patients at increased risk of progression during MM.

P920: EFFECTIVENESS AND SAFETY OF SELINEXOR-BASED REGIMEN IN THE TREATMENT OF RELAPSED AND REFRACTORY MULTIPLE MYELOMA: A MULTICENTER REAL-WORLD STUDY FROM CHINA

L. Kuang 1 , B. Fang 2 , W. Chen 3 , A. Liu 3 , C. Li 4 , L. Bao 5 , C. Fu 6 , J. Chen 7 , H. Li 7 , Y. Pang 8 , A. Liao 9 , Y. Liang 10 , Y. Wei 11 , J. Li 1,*

1 First Affiliated Hospital of Sun Yat-sen University, Guangzhou; 2 Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Zhengzhou; 3 Beijing Chao-yang Hospital, Capital Medical University, Beijing; 4 Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan; 5 Beijing Jishuitan Hospital, Beijing; 6 The First Affiliated Hospital of Soochow University, Suzhou; 7 Sichuan Provincial Hospital of University of Electronic Science and Technology of China, Chengdu; 8 The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou; 9 Shengjing Hospital of China Medical University, Shenyang; 10 Sun Yat-sen University Cancer Center; 11 Nanfang Hospital, Southern Medical University, Guangzhou, China

Background: Selinexor is the first approved oral selective nuclear exportin inhibitor. Clinical trials have shown that selinexor-based regimens improved the survival of RRMM patients. However, there are limited reports on the effectiveness and safety of selinexor in the real world.

Aims: To evaluate the effectiveness and safety of selinexor-based regimens in the treatment of patients with relapsed and refractory multiple myeloma (RRMM) in real world settings.

Methods: The clinical data of 53 patients with RRMM who were treated with selinexor-based regimens in 11 centers in China from July 2020 to December 2021 were retrospectively analyzed, and the response, survival and side effects were evaluated.

Results: The median age of the 53 patients with RRMM at the time of treatment with the selinexor-based regimens was 60 years (range: 41-79). The median time since initial diagnosis was 30 months (range: 1-156). The median number of prior lines of therapy was 4 (range: 1-11), including 22 (41.5%) cases who had received autologous hematopoietic stem cell transplantation in the past, 8 cases (15.0%) who had been exposed to CART, 34 cases (64.1%) who were double-class refractory (proteasome inhibitors and immunomodulators), and 20 cases (33.7%) who were triple-class refractory(proteasome inhibitors, immunomodulators and daratumumab). The number of patients with high-risk cytogenetics [17p-/t (14;16)/t (4;14)/1q21] at onset was 24 (66.7%), and 20 (55.6%) at the time of the lastest relapse. The responses were evaluable in 47 patients, and the overall response rate (ORR, ≥PR) was 44.7%, including 3 cases with CR (6.4%), 4 cases with VGPR (8.5%), and 14 cases with PR (29.8%). The median follow-up time was 2.75 months (range: 0.14-17.5). In the responders, the median time to first response was 1.07 months (range: 0.25-2.71), and the median duration of response (DOR) was 7.75 months (95% CI: 4.705-10.795). The median progression-free survival (PFS) was 6.107 months (95%CI: 2.856, 9.358), and the median overall survival (OS) had not been reached. The major grade 3-4 adverse effets were hematological toxicity, and the incidences of grade 3-4 neutropenia, lymphopenia, and thrombocytopenia were 41.2%, 37.9%, and 53.0%, respectively. The incidence of dose reductions of selinexor due to myelosuppression was 7.1%. Among the non-hematological adverse effects, the most common grade 3-4 adverse effects were nausea and vomiting (38.7%), fatigue (18.6%), and infection (18.6%).

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Summary/Conclusion: In the real world, the selinesor-based regimens have good effectiveness and safety profile in the treatment of RRMM.

P921: UPDATED EFFICACY AND SAFETY RESULTS OF TECLISTAMAB, A B-CELL MATURATION ANTIGEN X CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA FROM MAJESTEC-1

J. Martínez-López 1,* , P. Moreau 2 , S. Z. Usmani 3 , A. Garfall 4 , N. W. van de Donk 5 , J. F. San-Miguel 6 , A. Oriol 7 , A. Chari 8 , L. Karlin 9 , M.-V. Mateos 10 , R. Popat 11 , A. K. Nooka 12 , S. Sidana 13 , D. Trancucci 14 , R. Verona 15 , S. Girgis 15 , C. Uhlar 15 , T. Stephenson 15 , A. Banerjee 15 , A. Krishnan 16

1 Haematological Malignancies Clinical Research Unit, Hospital 12 de Octubre Universidad Complutense, CNIO, CIBERONC, Madrid, Spain; 2 Hematology Clinic, University Hospital Hôtel-Dieu, Nantes, France; 3 Levine Cancer Institute/Atrium Health, Charlotte, NC; 4 Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 5 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 6 Clínica Universidad de Navarra, CIMA, CIBERONC, IDISNA, Pamplona; 7 Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Barcelona, Spain; 8 Mount Sinai School of Medicine, New York, NY, United States of America; 9 Service d’Hématologie Clinique, Centre Hospitalier Lyon Sud, Pierre-Bénite, France; 10 University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain; 11 University College London Hospitals, NHS Foundation Trust, London, United Kingdom; 12 Winship Cancer Institute, Emory University, Atlanta, GA; 13 Stanford University School of Medicine, Stanford, CA; 14 Janssen Research & Development, Raritan, NJ; 15 Janssen Research & Development, Spring House, PA; 16 City of Hope Comprehensive Cancer Center, Duarte, CA, United States of America

Background: Teclistamab (JNJ-64007957), a bispecific antibody targeting both B-cell maturation antigen (BCMA) and CD3 receptors, mediates T cell activation and subsequent lysis of BCMA-expressing myeloma cells. In the multi-cohort, open-label, phase 1/2 MajesTEC-1 ( NCT03145181 ) study, the safety and efficacy of teclistamab in patients with relapsed/refractory multiple myeloma (RRMM) who previously received ≥3 lines of therapy (LOT) are being investigated. In phase 1, weekly subcutaneous dose of teclistamab 1.5 mg/kg, preceded by step-up doses of 0.06 and 0.3 mg/kg, was identified as the recommended phase 2 dose (RP2D). Initial results from phase 1/2 showed that teclistamab at the RP2D was well tolerated and provided encouraging efficacy in patients with no prior exposure to an anti–BCMA-targeted treatment.

Aims: We report updated efficacy and safety results from MajesTEC-1 in patients treated at the RP2D, including additional patients and longer follow-up.

Methods: MajesTEC-1 included patients aged ≥18 years with documented MM (as per the International Myeloma Working Group [IMWG] criteria) who had received ≥3 prior LOT including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody. Patients previously exposed to BCMA-targeted therapy were not eligible in Phase 1. Patients received teclistamab at the RP2D. Overall response rate (ORR, assessed per the IMWG 2016 criteria) was the primary endpoint. CTCAE v4.03 (cytokine release syndrome [CRS] and ICANS graded per ASTCT guidelines) was used for grading adverse events (AEs). Results are based on a Sep 7, 2021 data cutoff for safety and a Nov 9, 2021 data cutoff for efficacy (N=165).

Results: The median age was 64 y (range 33–84), 58% were male, and patients had received 5 (range 2–14) median prior LOT; 100% of patients were triple-class exposed, 78% were triple-class refractory, 70% were penta-drug exposed, and 30% were penta-drug refractory. ORR was 64% (95% CI 56–72), with 30% of patients achieving a complete response or better. Durable responses were observed, which deepened over time. The 12-month duration of response (DOR) rate was 66% (95% CI 49–79); median DOR was not reached. A reduction in soluble BCMA was observed in the first cycle of treatment in a majority of patients who responded to teclistamab. Neutropenia (65%; grade 3/4: 57%), anemia (50%; grade 3/4: 35%), thrombocytopenia (38%; grade 3/4: 21%), and lymphopenia (34%; grade 3/4: 32%) were the most common hematologic AEs. Infections occurred in 104 patients (63%; grade 3/4: 35%). The most common nonhematologic AE was CRS in 72% patients (grade 3, 0.6%; no grade 4/5). The median (range) time to CRS onset was 2 days (1–6) and median duration was 2 days (1–9). A total of 9 ICANS events (all grade 1/2; all resolved) were reported in 5 (3%) patients, of which 7 ICANS events were concurrent with CRS (all resolved). No dose reductions due to AEs were required, and no treatment-related deaths were reported.

Summary/Conclusion: The deep and durable responses achieved with teclistamab in patients with highly refractory MM were reaffirmed with data from ~9 months of follow-up. No new safety signals were identified. Additional data with longer follow-up, including subgroup analyses and progression-free survival, will be presented.

P922: HEALTH-RELATED QUALITY OF LIFE WITH TECLISTAMAB, A B-CELL MATURATION ANTIGEN X CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA FROM MAJESTEC-1

R. Popat 1,* , P. Moreau 2 , S. Z. Usmani 3 , A. Garfall 4 , M.-V. Mateos 5 , J. F. San-Miguel 6 , A. Oriol 7 , A. K. Nooka 8 , L. Rosinol 9 , A. Chari 10 , L. Karlin 11 , A. Krishnan 12 , N. Bahlis 13 , T. Martin 14 , B. Besemer 15 , J. Martínez-López 16 , M. Delforge 17 , J. Fastenau 18 , K. S. Gries 18 , N. W. van de Donk 19

1 University College London Hospitals, NHS Foundation Trust, London, United Kingdom; 2 Hematology Clinic, University Hospital Hôtel-Dieu, Nantes, France; 3 Levine Cancer Institute/Atrium Health, Charlotte, NC; 4 Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 5 University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca; 6 Clínica Universidad de Navarra, CIMA, CIBERONC, IDISNA, Pamplona; 7 Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Barcelona, Spain; 8 Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 9 Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain; 10 Mount Sinai School of Medicine, New York, NY, United States of America; 11 Service d’Hématologie Clinique, Centre Hospitalier Lyon Sud, Pierre-Bénite, France; 12 City of Hope Comprehensive Cancer Center, Duarte, CA; 13 Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, CA; 14 University of California San Francisco, San Francisco, CA, United States of America; 15 University of Tuebingen, Tuebingen, Germany; 16 Haematological Malignancies Clinical Research Unit, Hospital 12 de Octubre Universidad Complutense, CNIO, CIBERONC, Madrid, Spain; 17 Universitaire Ziekenhuizen Leuven, Leuven, Belgium; 18 Janssen Research & Development, Raritan, NJ, United States of America; 19 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands

Background: Because patients with multiple myeloma (MM) have impaired health-related quality of life (HRQoL), it is important that patient-reported outcomes (PROs) are assessed in addition to clinical outcomes. Teclistamab (JNJ-64007957) is a B-cell maturation antigen (BCMA) x CD3 bispecific antibody designed to mediate T cell-induced lysis of BCMA-expressing MM cells. In the phase 1/2 MajesTEC-1 trial, initial results from the pivotal cohort (patients who received ≥3 prior lines of therapy [LOT], including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody) showed that teclistamab was well tolerated with encouraging efficacy.

Aims: We report PROs of the pivotal cohort from MajesTEC-1.

Methods: MajesTEC-1 included patients aged ≥18 years with documented relapsed/refractory multiple myeloma (RRMM) as per the International Myeloma Working Group diagnostic criteria, progressive/measurable disease, and previous exposure to ≥3 prior LOT. Patients who had received prior anti-BCMA treatment were ineligible. Patients were given weekly subcutaneous teclistamab at the recommended phase 2 dose (1.5 mg/kg with step-up doses of 0.06 and 0.3 mg/kg). PRO assessments were performed at screening and at every even treatment cycle; results for cycles 2-8 are reported here. HRQoL was assessed by EORTC QLQ-C30 (range: 0–100; with higher scores indicating better global health status [GHS] but greater symptom severity [symptom scales]) and EuroQol 5-dimensional descriptive system (using the visual analog scale [VAS] with “0” indicating worst imaginable health state and “100” indicating best imaginable health state). Mixed-effects model with repeated measures was used to determine treatment effect. Meaningful improvement was defined as the proportion of patients with a change of ≥10 points. Kaplan-Meier estimate was used to determine time to worsening.

Results: The analysis included 110 patients, with median follow-up duration of 7.8 months. The PRO compliance rates were high at baseline (85–90%) and through treatment cycles 2–8 (80–94%). Overall HRQoL was improved with teclistamab, as shown by improved GHS scores (cycle 2–8) and reduced pain (-4.2 [cycle 2] to -15.1 [cycle 8]; Table), with no overall changes in physical functioning and fatigue. Meaningful improvements from baseline to cycle 8 were observed in GHS (50% of patients), physical function (35%), pain (65%), and fatigue (73%). Meaningful improvement in overall health (VAS) was also observed in 50% of patients. Median time to improvement from baseline was ~1.5 months with longer time to improvement seen for nausea/vomiting and fatigue. Across all symptoms, median time to worsening ranged from 2 months to not estimable.

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Summary/Conclusion: Teclistamab provided rapid, clinically meaningful improvements in HRQoL in patients with RRMM, which are consistent with clinical outcomes in MajesTEC-1.

P923: TANDEM AUTOLOGOUS STEM CELL TRANSPLANTATIONS STILL BENEFIT FOR MYELOMA PATIENTS: A POOLING META-ANALYSIS

C.-H. Lee 1,* , C.-L. Ho 1

1 Hematology and Oncology, Tri-Service General Hospital/National Defense Medical Center, Neihu Dist, Taiwan

Background: Although high-dose therapy and autologous stem cell transplant combined with novel agents continues to be the hallmark of first-line treatment in newly diagnosed transplant-eligible multiple myeloma patients, disease progression remains an issue. To deepen the treatment response and prolong disease free survival, tandem autologous stem cell transplant is one of treatment choice. Compared to toxicity of sub-sequent pharmacotherapy or allogeneic transplant, tandem ASCT is well-tolerated. However, the efficacy of tandem ASCT is not fully defined yet. Thus, we aimed to assess effectiveness of tandem ASCT.

Aims: To evaluate the true effect of tandem autologous/autologous stem cell transplantation for myeloma patients in current era.

Methods: Eligible randomized controlled trials published before Dec 2021 were retrieved from databases. Myeloma patients received tandem autologous stem cell transplantation was included. Systematic review and pairwise meta-analysis were performed. We calculated the hazard ratio (HR) with 95% confidence intervals (CIs).

Results: Ten articles, involving 3,123 patients, met the selection criteria. All studies demonstrated acceptable quality. The mean age was 58.2 year-old with mean 36.2% high risk cytogenetics feature. Eight trials were international multi-centers phase III RCTs. Tandem autologous stem cell transplantation compared to single ASCT was associated with a significantly superior progression free survival (random-effect HR 0.89; 95% CI 0.82-0.97; I2 = 0.0%), but no significance difference regard to overall survival (random-effect HR 0.98; 95% CI 0.94–1.03; I2 = 0%). For high risk myeloma subgroup, 4 trials compared tandem ASCT versus lenalidomide, tandem ASCT demonstrated superior progression free survival (random-effect HR 0.87; 95% CI 0.82–0.91; I2 = 0%)

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Summary/Conclusion: In conclusion, tandem ASCT are still to be considered as an acceptable treatment options in general myeloma patients. High-risk cytogenetics is frequently observed in newly diagnosed myeloma with worsens outcome after single autologous, whereas a tandem autologous transplant strategy may overcome onset poor prognosis.

P924: TREATMENT EFFICACY OF PROGRESSION FREE SURVIVAL FOR REFRACTORY/RELAPSED MULTIPLE MYELOMA IN GERIATRIC PATIENTS: A NETWORK META-ANALYSIS

C.-H. Lee 1,* , C.-L. Ho 1

1 Hematology and Oncology, Tri-Service General Hospital, Neihu Dist, Taiwan

Background: In recent 10 years, there were many studies explored the treatment efficacy of refractory/relapse multiple myeloma (R/R MM). Emerging evidences proved combination of new treatment modalities is significant better than traditional therapy. However, very few studies focus on geriatric patient who are more fragile and may not be able to tolerate treatment.

Aims: Our aim was to synthesize all efficacy evidence, enabling an integrated comparison of all current treatment options in geriatric patients with R/R MM.

Methods: We performed a systematic literature review to identify all publicly available randomized controlled trials (RCT) evidence. We searched Embase, PubMed, Cochrane Central Register of Controlled Clinical Trials, and the Web site www.ClinicalTrials.gov . Geriatric Patients with a diagnosis of R/R MM (including patients in second-line or more than second-line treatments) received subsequent active treatment were included. Geriatric patients should be set at least age 65 or above as a cut-off level. Data was extracted from subgroup analysis of each study. The evidence was synthesized using a Bayesian network meta-analysis. Hazard ratio (HR) was adopted.

Results: In total, 4,966 citations were retrieved from the databases; 60 full texts were screened, of which 47 were excluded. In total, 13 RCTs were identified for quantitive analysis, including 14 treatment options (figure). Total 4337 geriatric patient were enrolled for network meta-analysis, twelve trials set cut-off level of age about 65, one set age 75 as a cut-off level. All trials had good quality. The triple combination therapy of daratumumab, lenalidomide and dexamethasone (DaraLenDex) was identified as the best treatment option in patients with R/R MM (figure). It was most favorable in terms of (1) HR for progression free survival (0.15; 95% credible interval (CI): 0.09 to 0.25) with significance and (2) probability of being best (96% of the cumulative ranking).

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Summary/Conclusion: To the best of our knowledge, this is the first NMA on R/R MM that includes all regimens currently evaluated in randomized trials in elderly R/R MM. Our findings suggest that a 3-drug regimen containing the lenalidomide-dexamethasone backbone, preferentially combined with anti-MM mAbs daratumumab or elotuzumab, has the highest probability of being ranked as the best treatment in this setting, underlying the role of immunotherapy in MM. Prospective randomized trials are eagerly awaited to clarify the optimal sequencing of treatments for MM. We emphasize that it remains essential to conduct phase III RCTs to obtain more direct head-to-head evidence. Until such evidence becomes available, our results are highly important for informed decision making in everyday clinical practice

P925: IMPACT OF PRIOR TREATMENT EXPOSURE ON THE EFFECTIVENESS OF IXAZOMIB-LENALIDOMIDE-DEXAMETHASONE IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS TREATED IN ROUTINE CLINICAL PRACTICE (THE INSURE STUDY)

H. C. Lee 1,* , K. Ramasamy 2 , M. Macro 3 , F. E. Davies 4 , R. Abonour 5 , F. van Rhee 6 , V. T. Hungria 7 , N. Puig 8 , K. Ren 9 , J. Silar 10 , V. Enwemadu 11 , D. Cherepanov 9 , D. M. Stull 11 , X. Leleu 12

1 MD Anderson Cancer Center, Houston, United States of America; 2 Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 3 CHU de Caen, Caen, France; 4 Perlmutter Cancer Center, NYU Langone, New York; 5 Indiana University School of Medicine, Indianapolis; 6 University of Arkansas for Medical Sciences, Little Rock, United States of America; 7 Clinica São Germano and Santa Casa Medical School, São Paulo, Brazil; 8 Hospital Universitario de Salamanca Hematología, Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain; 9 Takeda Development Center Americas, Inc. (TDCA), Lexington, United States of America; 10 Institute of Biostatistics & Analyses, Ltd, Brno, Czechia; 11 Takeda Pharmaceuticals U.S.A., Inc., Lexington, United States of America; 12 Pôle Régional de Cancérologie, Department of Hematology, CHU La Milétrie-Poitiers, Poitiers, France

Background: Results from INSURE, a pooled, global analysis of 3 observational studies, show that the effectiveness of ixazomib-lenalidomide-dexamethasone (IRd) used to treat relapsed/refractory multiple myeloma (RRMM) in routine clinical practice is comparable to its efficacy seen in the TOURMALINE-MM1 trial (median progression-free survival [PFS], 19.9 vs 20.6 months [mos]), with no new safety concerns (Leleu ASH 2021 #2701). Data on effectiveness outcomes following retreatment with agents used in earlier lines of therapy (LoTs) are limited, but may be of particular value for MM pts previously treated with lenalidomide (LEN) or proteosome inhibitors (PIs).

Aims: To characterize the impact of prior exposure & refractoriness to LEN or PIs on the effectiveness & safety of IRd in RRMM.

Methods: INSURE is a pooled analysis of data from 3 studies: INS IGHT MM, U VEA-IXA, & RE MIX. INSIGHT MM is a prospective, global study of 4307 MM patients (pts) from 15 countries. UVEA-IXA is a multicenter, longitudinal, retrospective cohort study of 309 RRMM pts receiving ixazomib (IXA)-based therapy via an early-access program in Europe. REMIX is a retrospective/prospective study of 198 RRMM pts treated with IRd via a compassionate-use program in France. INSURE included adult RRMM pts who had received IRd in ≥2nd LoT. Primary outcomes were PFS & time-to-next therapy (TTNT). Secondary outcomes included: duration of treatment (DOT), overall survival (OS), overall response rate (ORR), & safety (discontinuations due to adverse events [AEs] were reported separately for each study). In this prespecified analysis pts were grouped by prior LEN or PI exposure (naïve, exposed, or refractory).

Results: 562 pts were included: 391/100/71 were LEN-naïve/exposed/refractory & 37/408/117 were PI-naïve/exposed/refractory. In LEN-naïve/exposed/refractory pts, median age was 69/68/68 years (yrs; 24/14/18% >75 yrs) & 18/13/22% had an Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2 (missing pts excluded from %); pts had received a median of 1/2/3 LoT(s) prior to IRd. In PI-naïve/exposed/refractory pts, median age was 71/68/69 yrs (38/22/15% >75 yrs) & 6/16/27% had an ECOG PS ≥2 (missing pts excluded from %); pts in all 3 subgroups had received a median of 2 LoTs prior to IRd. The Table shows effectiveness outcomes. Median DOT was 15.3/15.6/4.7 mos & median PFS was 21.6/25.8/5.8 mos in LEN-naïve/exposed/refractory pts. Median DOT & PFS in PI-naïve/exposed/refractory pts were 20.4/15.2/7.6 mos & not reached/19.7/12.9 mos, respectively. OS data were immature. The proportions of LEN-naïve/exposed/refractory pts in INSIGHT (n=114/39/28) & UVEA-IXA (n=161/15/19) who discontinued a study drug due to AEs were: IXA, 32/28/25% & 19/7/11%; LEN, 22/28/18% & 16/7/11%; dexamethasone (DEX), 18/21/14% & 11/0/11%, respectively. The proportions of PI-naïve/exposed/refractory pts in INSIGHT (n=9/130/42) & UVEA-IXA (n=18/125/52) who discontinued a study drug due to AEs were: IXA, 44/28/31% & 22/17/15%; LEN, 33/22/21% & 17/16/12%; DEX, 33/18/17% & 17/10/8%, respectively. Data for discontinuations due to AEs were not available for REMIX. Additional safety data will be presented.

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Summary/Conclusion: IRd appeared to be effective in RRMM pts in routine clinical practice regardless of prior LEN or PI exposure, with better outcomes seen in LEN- &/or PI-non-refractory vs -refractory pts. PFS outcomes in the naïve/exposed populations are comparable to those reported in TOURMALINE-MM1. Pts with prior LEN or PI exposure can achieve clinical benefit when retreated suggesting that prior exposure should not preclude use of these agents in later LoTs.

P926: DARATUMUMAB, BORTEZOMIB AND DEXAMETHASONE FOR TREATMENT OF PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA AND SEVERE RENAL IMPAIRMENT: RESULTS FROM THE PHASE 2 GMMG-DANTE TRIAL

L. Leypoldt 1,* , M. Gavriatopoulou 2 , B. Besemer 3 , H. Salwender 4 , M.-S. Raab 5 , A. Nogai 6 , C. Khandanpour 7 , V. Runde 8 , M. Zago 9 , P. Martus 10 , H. Goldschmidt 11 , C. Bokemeyer 1 , M. Dimopoulos 2 , K. Weisel 1

1 Department of Hematology, Oncology and Bone Marrow Transplantation with Section of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2 Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens, Alexandra General Hospital, Athens, Greece; 3 Department of Hematology, Oncology, Immunology, Rheumatology and Pulmonology, University Hospital of Tuebingen, Tuebingen; 4 Asklepios Tumorzentrum Hamburg, AK Altona and AK St. Georg, Hamburg; 5 Department of Internal Medicine, Heidelberg University Hospital, Heidelberg; 6 Department of Hematology, Oncology and Tumor Immunology, Charite Medical School, Berlin; 7 Department of Medicine A, Hematology, Oncology and Pneumology, University Hospital Münster, Münster; 8 Department of Hematology/Oncology, Wilhelm-Anton Hospital Goch, Goch; 9 Center for Clinical Trials, University Hospital of Tuebingen; 10 Department of Clinical Epidemiology and Applied Biostatistics, Eberhard Karls University of Tuebingen, Tuebingen; 11 Internal Medicine V and National Center for Tumor Diseases (NCT), University Hospital Heidelberg, Heidelberg, Germany

Background: Renal function impairment is one of the main characteristics of multiple myeloma (MM) leading to diagnosis and defining treatment requirement in first-line and subsequent relapse. However, limited data exist on safety and efficacy of anti-MM regimens in patients (pts) with severe renal impairment and those requiring hemodialysis as pts are mostly excluded from clinical trials and specific trials in this indication are rare. Proteasome inhibitors as bortezomib (BTZ) are known to represent one of the preferable drug classes in patients with renal impairment due to rapid response, lack of dose adjustment and renoprotective effects. Daratumumab (DARA) is an anti-CD38 monoclonal antibody which enhances response rates and efficacy when added to standard of care regimens, both in newly diagnosed and relapsed and refractory (RR)MM. The combination of BTZ, dexamethasone (DEX), and DARA (DVd) has been shown to be efficacious and safe in RRMM in the phase 3 study CASTOR (Palumbo et al., NEJM 2016). In the CASTOR trial, pts with a GFR < 20 ml/min were excluded.

Aims: Here, we present results from the primary analysis of the investigator-initiated multicenter GMMG-DANTE ( NCT02977494 ) trial investigating DVd in RRMM pts with severe renal impairment including pts on hemodialysis.

Methods: RRMM pts with measurable disease who had received at least 1 prior line of therapy and a GFR < 30 ml/min or undergoing hemodialysis were eligible. Pts received DVd in the approved schedule with 8 cycles (21 d/cycle) of BTZ (1.3 mg/m 2 , SC) on Days 1, 4, 8, and 11 and DEX (20 mg, PO or IV) on days 1, 2, 4, 5, 8, 9, 11, and 12 + DARA (16 mg/kg, IV) given weekly for cycles 1-3, Q3W for cycles 4-8, and Q4W thereafter. 36 patients were planned to be included into the trial. The trial was closed prematurely after 22 patients due to inferior recruitment. The primary endpoint was overall response rate (ORR). Key secondary endpoints were progression-free survival (PFS), overall survival (OS) and safety. Response assessment was performed using the IMWG criteria.

Results: 22 pts from 6 German and one Greek site were included from 2017-2020. Analyzed population included 21 patients. Median age was 70 years. Median GFR was 21.0 ml/min, 8 patients were under hemodialysis. Median number of prior lines was 2 (range 1-10). All patients started DVd treatment. For one patient it was not possible to obtain a response. ORR was 67% (14/20) with 6 pts (29%) showing a partial response (PR), 6 pts (29%) a very good partial response (VGPR) and 2 pts (10%) a complete response (CR). Median treatment duration was 24 weeks (range 2-106). After a median follow-up of 28 months, median PFS was 10.4 months, median OS was not reached (OS after 24 months until 39 months plateau 0.504). The most frequent toxicity ≥ grade 3 was hematologic, with anemia in 23.8%, thrombocytopenia in 23.8% and neutropenia in 9.5%. Main non-hematologic adverse events (AEs) ≥ grade 3 were infections (23.8%) with mainly pneumonias. Polyneuropathy all grade was described in 52.4%.

Summary/Conclusion: In this phase II prospective trial we demonstrated that DVd shows relevant efficacy in RRMM pts with severe renal impairment and can be safely administered. Efficacy is comparable to the one reported in patients with no severe renal impairment. Toxicity does not differ from previously reported data on DVd. Acknowledging the generally reported impaired outcome of MM pts showing severe renal impairment, PFS and OS underline the importance of effective MM regimens which can be safely administered in a difficult to treat population.

P927: CLINICAL CHARACTERISTICS, SURVIVAL OUTCOMES AND PROGNOSIS IN 123 IMMUNOGLOBULIN D MULTIPLE MYELOMA PATIENTS: A RETROSPECTIVE SINGLE-CENTER

J. Liu 1,* , H. Fan 1 , W. Yan 1 , J. Xu 1 , L. Li 1 , L. Qiu 1 , G. An 1

1 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Immunoglobulin D (IgD) myeloma is a rare subtype of multiple myeloma (MM), and has been considered to have a more aggressive course and poor outcome. With the rapid progress of multiple myeloma treatment, including the application of proteasome inhibitors and immunomodulatory drugs and the promotion of autologous stem cell transplantation (ASCT), the prognosis of MM patients has been greatly improved. Considering the development of MM treatment, the survival outcome and prognostic factors of IgD MM patients are now controversial.

Aims: To evaluate the clinical characteristics, survival outcomes and prognosis of IgD myeloma

patients in the era of novel therapy.

Methods: We retrospectively analyzed the clinical characteristics of 123 IgD MM patients and compared them with other MM subtypes. At the same time, we also discussed the survival outcomes and prognostic factors of IgD myeloma patients under different treatment modes.

Results: Male to female ratio was 2.2, median age was 55 years (29-79 years)， with preponderance of lambda light chains (89.4%). IgD myeloma more likely presented advanced stage when diagnosed: higher frequencies of ISS stage Ⅲ, R-ISS stage Ⅲ, renal dysfunction, high lactate dehydrogenase (LDH) level, high β2MG level and high bone marrow plasmacytosis. Additionally, the genetics of IgD myeloma patients were also different from other subtypes myeloma. Complex karyotypes were more common in IgD myeloma patients (23.9% vs. 14.8%, p=0.017). Cytogenetic abnormalities demonstrated by FISH could be found in 86% (80/93) of the IgD myeloma patients. The frequencies of t (11;14) (38.6% vs. 13.6%, p=0.000) and 1q21 amplification (72.0% vs. 53.9%, p=0.001) were much higher in IgD MM patients than other subtypes myeloma, while deletion of 13q (34.4% vs. 45.7%, p=0.032) and high risk cytogenetic abnormalities (HRCA) (23.0% vs. 37.9%, p=0.009) were less. 71 IgD MM patients received induction therapy based on ‘novel therapies’, 26 on ‘traditional therapies’. Compared with ‘traditional therapies’, ‘novel therapies’ significantly improved the prognosis of IgD patients (PFS 16.9 vs. 29.1 months, P=0.008; OS 37.6 vs. 70.8 months, P=0.002). Further analysis of the ‘novel therapies’ group found that, in non-transplant patients, the prognosis of the IgD and non-IgD patients was similar, but in transplant patients, IgD MM patients showed worse outcomes compared to non-IgD MM patients (figure). Multivariate analysis of 71 IgD patients who received ‘novel therapies’ identified that R-ISS stage Ⅲ, complex karyotypes and deletion of 13q were independent adverse factors for OS, whereas complex karyotypes and thrombocytopenia were independent adverse factors for PFS.

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Summary/Conclusion: In summary, our study compared the clinical characteristics of IgD and non-IgD MM patients, IgD myeloma patients are younger, have higher tumor burden and advanced disease stage. In addition, their genetics are significantly different from non-IgD MM: IgD MM patients have more complex karyotypes, amplification of 1q21 and t (11;14), while less deletion of 13q and HRCA. Meanwhile, we found that although new drugs combined with ASCT significantly improved the prognosis of MM patients, the improvement of IgD MM is less obvious than that of non-IgD MM patients, suggesting more targeted drugs need to be developed for patients with IgD MM. Finally, we identified that R-ISS stage Ⅲ, complex karyotypes and deletion of 13q were independent adverse factors for OS, indicating that R-ISS is still useful way to predict the outcomes of patients with IgD myeloma.

P928: REAL-LIFE ANALYSIS OF THE MULTIPLE MYELOMA PATIENT’S SURVIVAL IN A LARGE COHORT OF PATIENTS

N. Lopez-Muñoz 1,* , G. Hernandez-Ibarburu 2 , J. M. Sánchez-Pina 1 , R. A. Alonso 1 , C. Cuellar 1 , M. Calbacho 1 , R. Ayala 1 , R. Iñiguez 1 , N. García Barrio 3 , E. Vera 1 , D. Pérez-Rey 2 , L. Meloni 4 , M. Pedrera-Jiménez 3 , P. Serrano-Balazote 3 , J. De La Cruz 5 , J. Martínez-López 1

1 Hospital 12 de Octubre; 2 Biomedical Informatics Group, Universidad Politécnica de Madrid; 3 Data Science Group, Research Institute imas12, MADRID, Spain; 4 Trinetx, LLC, Cambridge, United States of America; 5 Research Institute imas12, MADRID, Spain

Background: Multiple myeloma (MM) constitutes approximately 10% of hematological malignancies, with a median age at diagnosis of 65 years. Patient survival has improved considerably over the last 20 years with the introduction of new drugs. In 1999, the first immunomodulatory drug, thalidomide, was approved, followed by lenalidomide in 2005. In 2003, proteosome inhibitors such as bortezomib were introduced and, in 2015, anti-CD38 monoclonal antibodies such as daratumumab.

Aims: By relying on the TriNetx platform, a global health research platform, we have analyzed the impact on the outcome of the introduction new drugs for MM in the last 20 years. We also analyzed the difference patterns of treatments between networks.

Methods: First, we retrospectively selected patients diagnosed with symptomatic multiple myeloma between 1999 and 2019 in a tertiary care hospital in Spain, Hospital 12 de Octubre (H12O) in Madrid. Then, we analyzed data from more than 7500 patients diagnosed with CML from research networks: EMEA and the US; covering more than 16 million subjects. We compared the time of survival since the MM diagnosis in three groups depending of the age at MM diagnosis (less than 65, between 65 and 75, and more than 75 years old) over three periods of time: 1999-2009, 2010-2014 and 2015-2019 (2020 was excluded because of pandemic impact). Kaplan-Meier analysis was used for analyzing overall survival (OS) and differences between groups were tested for statistical significance using the log-rank test. Also, we analyzed the patterns of treatment along this time.

Results: Approximately, a total 760 patients were included in the study. In the H12O, median OS was 45.1, 45.2, 65.1 months and not reached for the 1999-2009, 2010-2014 and 2015-2019 periods, respectively (p=0.001). Among the group of patients younger than 65 years old, the median OS was 64 months (47.5-81.4; 95%) among patients diagnosed in the period 1999-2009; 80 months (36.1-124; 95%) in 2010-2014; and median not reached for the 2014-2019 (p<.001).

Similar results were found in a US cohort, including approximately 7000 patients, between the years 2010-2014 vs 2014-2019 (59.71% vs 64.20%, p<0.0001, HR 1.154 (1.074- 1.241) (figure 1 ).

The same results were observed for the 65-75 years cohort, median OS was 62.6 months (50.9-74.3; 95%) for the 1999-2009 period, 70.6 months (33.1-108.1; 95%) between 2010 and 2014, and not reached for the 2014-2019 period (p<.001).

The same occurs among more than 3000 patients from the US base between the years 2010-2014 vs 2014-2019 (53.96% vs 57.73%, p=0.016, HR 1.099 (1.018 - 1.187) (figure 2 ).

Otherwise, patients older than 75 years have a median OS of 31.1 months and do not show a statistically significant difference regardless on the year of MM diagnosis (p=.18). Nor were clinically significant results obtained between the EMEA and US cohorts.

On the other hand, the first-line treatment used in patients was analysed, and in both the EMEA and US cohorts, the use of bortezomib (46% vs 47% in the EMEA cohort; 62% vs 70% in US cohort), lenalidomide (43% vs 57% in EMEA cohort, 15% vs 18% in US cohort) and daratumumab (0% vs 4% in EMEA cohort, 2% vs 4% in US cohort) increases over the years comparing the 2010-2014 vs 2015-2019 time cohorts. This could explain the improved survival in more recent times.

Summary/Conclusion: The introduction of new agents for the treatment of MM has transformed the natural history of the disease, achieving long survival times in younger patients. Thus, it is essential to continue to advance and develop new therapies.

P929: IXAZOMIB AND DARATUMUMAB WITHOUT DEXAMETHASONE (I-DARA) IN ELDERLY FRAIL RELAPSING MYELOMA (RRMM) PATIENTS: RESULTS OF THE PHASE 2 STUDY IFM 2018-02 OF THE INTERGROUPE FRANCOPHONE DU MYELOME (IFM).

M. Macro 1,* , C. Touzeau 2 , C. Mariette 3 , S. Manier 4 , S. Brechignac 5 , L. Vincent 6 , B. Hebraud 7 , O. Decaux 8 , S. Schulmann 9 , C. Lenoir 10 , P. Godmer 11 , A. Farge 12 , L. Peyro Saint Paul 13 , J.-J. Parienti 13 , X. Leleu 14

1 HEMATOLOGY IHBN, University Hospital, CAEN; 2 HEMATOLOGY, University Hospital, Nantes; 3 HEMATOLOGY, University Hospital, Grenoble; 4 Hematology, University Hospital, Lille; 5 Hematology, University Hospital, Paris; 6 Hematology, University Hospital, Montpellier; 7 Hematology, University Hospital, Toulouse; 8 Hematology, University Hospital, Rennes; 9 Hematology, University Hospital, Nancy; 10 Hematology, Private Hospital, Bordeaux; 11 Hematology, Community Hospital, Vannes; 12 Hematology; 13 Clinical Research, University Hospital, CAEN; 14 Hematology, University Hospital, Poitiers, France

Background: Frail patients with multiple myeloma have an inferior outcome, especially in the relapse setting. This adverse prognosis is mainly related to a high discontinuation rate due to treatment (Tx) related adverse events.

Aims: The aim of this phase 2 study is to evaluate efficacy and tolerability of Ixazomib-Daratumumab (I-Dara) without Dexamethasone in elderly frail patients with relapsed myeloma (RRMM) ( NCT03757221 ).

Methods: Ixa-Dara naïve RRMM patients received oral Ixazomib (4 mg: days 1, 8, 15), IV Daratumumab (16 mg/kg; days 1, 8, 15, 22, cycles 1-2; days 1, 15, cycles 3-6; days 1, cycles 7+) and IV Methylprednisolone before Daratumumab (100 mg at day 1, 8, cycle 1 and then 60 mg). They were enrolled after 1 or 2 prior therapy if their frailty score was ≥ 2 by IMWG score. The primary endpoint was ≥ very good partial response rate (VGPR) at one year. Secondary endpoints included overall response rate (ORR), progression free survival (PFS), overall survival (OS) & toxicity according to NCI-CTCAE version 5

Results: Sixty-three patients were screened and 55 enrolled between 03/2018 and 09/2021. Patient were at first (n = 36) or second relapse (n = 19). Thirty-three patients (60%) were previously exposed to bortezomib, 37 (67%) were previously exposed to lenalidomide (Len) and 20 (36 %) were refractory to Len. Median age was 82 (72-93). All patients had a frailty score ≥2 and 13 (24 %) had a 3 or 4 frailty score. In 41 patients ISS at diagnosis was stage I (n = 11), II (n = 18) or III (n = 12). Seventeen (36%) patients harbored high-risk (HR) cytogenetic, including t(4;14) (n = 8) or del17p (n = 10). The median duration of Tx among 28 pts with ongoing Tx was 10 months [5-32] at data cutoff (February, 2). The median duration of Tx among 27 pts who stopped Tx was 6 months [0-18]: 18 had progressive disease. Nine patients died during the study: Daratumumab-related bronchospasm (D1C1); Ixazomib-related overdose (C2); sepsis (n = 4), progressive disease (n = 3). Regarding toxicity, 27 pts had a ≥grade 3 AE (49%). The most common grade 3-4 toxicities were thrombocytopenia (n = 9), other cytopenias (n = 4), infection (n = 8), hypertension (n = 3) and gastrointestinal disorders (n = 3). Fourteen out of 28 were SAE including 5 infections, 1 bronchospasm, 1 acute respiratory failure and 2 ixazomib overdoses. Overall response rate, including minimal response, was 86 % with a ≥VGPR rate of 32 % in the whole group. In Len refractory patients the ORR was 82 % and ≥VGPR 41%, in HR cytogenetic patients ORR was 85 % and ≥VGPR 46%. With a median follow-up of 11.6 months median PFS is 16 months and median OS NR (76% estimated at one year).

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Summary/Conclusion: In this elderly frail population Ixa-Dara is a feasible combination with favorable efficacy profile even in Len refractory and HR cytogenetic patients. Early toxicity remains a concern in this population eventhough more manageable with Dara SC.

P930: ISATUXIMAB, LENALIDOMIDE, BORTEZOMIB AND DEXAMETHASONE AS INDUCTION THERAPY FOR NEWLY-DIAGNOSED MULTIPLE MYELOMA PATIENTS WITH HIGH-RISK CYTOGENETICS: A SUBGROUP ANALYSIS FROM THE GMMG-HD7 TRIAL

E. K. Mai 1,* , U. Bertsch 1,2 , R. Fenk 3 , D. Tichy 4 , B. Besemer 5 , J. Dürig 6 , R. Schroers 7 , I. von Metzler 8 , M. Hänel 9 , C. Mann 10 , A. M. Asemissen 11 , B. Heilmeier 12 , E. Nievergall 1 , S. Huhn 1 , K. Kriegsmann 1 , N. Weinhold 1 , S. Luntz 13 , T. A. W. Holderrried 14 , K. Trautmann-Grill 15 , D. Gezer 16 , M. Klaiber-Hakimi 17 , M. Müller 18 , C. Khandanpour 19 , W. Knauf 20 , C. Scheid 21 , M. Munder 22 , T. Geer 23 , H. Riesenberg 24 , J. Thomalla 25 , M. Hoffmann 26 , M. S. Raab 1 , H. J. Salwender 27 , K. C. Weisel 11 , H. Goldschmidt 1,2

1 Department of Internal Medicine V, University Hospital Heidelberg; 2 National Center for Tumor Diseases, Heidelberg; 3 Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Düsseldorf; 4 Division of Biostatistics, erman Cancer Research Center (DKFZ) Heidelberg, Heidelberg; 5 Department of Internal Medicine II, University Hospital Tübingen, Tübingen; 6 Department for Hematology and Stem Cell Transplantation, University Hospital Essen, Essen; 7 Medical Clinic, University Hospital Bochum, Bochum; 8 Department of Medicine, Hematology/Oncology, University Hospital Frankfurt, Goethe University, Frankfurt am Main; 9 Department of Internal Medicine III, Clinic Chemnitz, Chemnitz; 10 Department for Hematology, Oncology and Immunology, University Hospital Gießen and Marburg, Marburg; 11 Department of Oncology, Hematology and BMT, University Medical Center Hamburg-Eppendorf, Hamburg; 12 Clinic for Oncology and Hematology, Hospital Barmherzige Brueder Regensburg, Regensburg; 13 Coordination Centre for Clinical Trails (KKS) Heidelberg, Heidelberg; 14 Department of Oncology, Hematology, Immuno-Oncology and Rheumatology, University Hospital Bonn, Bonn; 15 Department of Internal Medicine I, University Hospital Dresden, Dresden; 16 Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen; 17 Clinic for Hematology, Oncology and Palliative Care, Marien Hospital Düsseldorf, Düsseldorf; 18 Clinic for Hematology, Oncology and Immunology, Klinikum Siloah Hannover, Hannover; 19 Medical Clinic A, niversity Hospital Münster, Münster; 20 Center for Hematology and Oncology Bethanien, Frankfurt am Main; 21 Department of Internal Medicine I, University Hospital Cologne, Cologne; 22 Department of Internal Medicine III, University Hospital Mainz, Mainz; 23 Department of Internal Medicine III, Diakoneo Clinic Schwäbisch-Hall, Schwäbisch-Hall; 24 Hematology / Oncology Center, Bielefeld; 25 Hematology / Oncology Center, Koblenz; 26 Medical Clinic A, Clinic Ludwigshafen, Ludwigshafen; 27 Asklepios Tumorzentrum Hamburg, AK Altona and AK St. Georg, Hamburg, Germany

Background: The multicenter phase III trial GMMG-HD7 ( NCT03617731 ) demonstrated superior minimal residual disease (MRD) negativity rate after induction therapy in patients with transplant-eligible newly-diagnosed multiple myeloma (NDMM) by addition of the anti-CD38 monoclonal antibody isatuximab (Isa) to lenalidomide / bortezomib / dexamethasone (Isa-RVd), as compared to RVd alone (Goldschmidt H et al., 2021, ASH Annual Meeting).

Aims: Here we present a subgroup analysis on patients with high-risk cytogenetics.

Methods: Patients with transplant-eligible NDMM were equally randomized to receive three 42-day cycles of RVd (lenalidomide 25 mg/d p.o., d1–14 and d22-35; bortezomib 1.3 mg/m 2 s.c. d1, 4, 8, 11, 22, 25, 29, 32; dexamethasone 20 mg/d d1-2, 4-5, 8-9, 11-12, 15, 22-23, 25-26, 29-30, 32-33) in both study arms. Isa was added to Isa-RVd as follows: 10 mg/kg i.v., cycle 1: d 1, 8, 15, 22, 29; cycles 2-3: d 1, 15, 29. Randomization for induction was stratified by Revised International Staging System. Primary endpoint of the trial was MRD negativity rate assessed by next-generation flow (NGF, cut-off 1x10 -5 ) after induction therapy. Fluorescence in-situ hybridization (FISH) analysis was performed centrally on CD138-purified plasma cells. High-risk and ultra high-risk cytogenetics were defined as at least one or two of the following aberrations, respectively: del17p, t(4;14), t(14;16), gain1q21 (≥ 3 copies). Data cut-off for the present analysis was December 2021.

Results: 660 patients (Isa-RVd: 331 and RVd: 329) were eligible for intention-to-treat analysis. The study met its primary endpoint, demonstrating superiority of NGF-MRD negativity rates with Isa-RVd compared to RVd (50.1% vs. 35.6%; odds ratio [OR]=1.82, 95% confidence interval [95% CI]: 1.33-2.48, p<0.001). High-risk cytogenetics were well balanced between the treatment arms. 264 of 584 (45.2%) and 82 of 580 (14.1%) evaluable patients had high-risk and ultra high-risk cytogenetics, respectively. Del17p, t(4;14), t(14;16) and gain 1q21 were present in 59 of 615 (9.6%), 67 of 613 (10.9%), 17 of 609 (2.8%) and 218 of 583 (37.4%) evaluable patients, respectively. Among patients with high-risk cytogenetics, MRD negativity rates were 50.4% (63/125) vs. 37.4% (52/139; OR=1.70, 95% CI: 1.04-2.79, p=0.03) with Isa-RVd vs. RVd. MRD negativity rates for ultra high-risk patients were 56.3% (27/48) vs. 44.1% (15/34; OR=1.63, 95% CI: 0.67-3.99, p=0.28) with Isa-RVd vs. RVd. Similar results were observed for Isa-RVd vs. RVd among the common major single high-risk cytogenetic features: del17p: 56.0% (14/25) vs. 35.3% (12/34), OR=2.33, 95% CI: 0.82-6.88; t(4;14): 57.6% (19/33) vs. 47.1% (16/34), OR=1.53, 95% CI: 0.58-4.06; t(14;16): 66.7% (6/9) vs. 50.0% (4/8), OR=2.00, 95% CI: 0.28-15.67; gain1q21: 48.2% (55/114) vs. 35.6% (37/104), OR=1.69, 95% CI: 0.98-2.92. Dividing evaluable patients in either standard risk (absence of any high-risk aberration; 320/578, 55.4%) vs. high-risk (exactly one high-risk aberration; 176/578, 30.4%) vs. ultra high-risk (≥2 high-risk aberrations; 82/578, 14.2%) yielded similar efficacy results. MRD negativity rates for Isa-RVd vs. RVd were 49.7% (86/173) vs. 36.7% (54/147; OR=1.70, 95% CI: 1.09-2.68) in standard risk patients, 46.7% (35/75) vs. 34.7% (35/101; OR=1.65, 95% CI: 0.90-3.05) in high-risk patients and 56.3% (27/48) vs. 44.1% (15/34; OR=1.63, 95% CI: 0.67-3.99) in ultra high-risk patients.

Summary/Conclusion: Isa-RVd induction therapy is superior to RVd in patients with transplant-eligible NDMM and high-risk or ultra high-risk cytogenetics, consistent with the benefit observed in the overall trial population.

P931: PROSPECTIVE OBSERVATIONAL MULTICENTER STUDY OF GAUCHER DISEASE PREVALENCE IN PATIENTS AFFECTED BY MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE

G. Giuffrida 1,* , U. Markovic 1,2,3 , C. Conticello 1 , D. Nicolosi 1 , V. Calafiore 1 , A. Romano 1,4 , A. Condorelli 1 , S. Grasso 1 , A. Duminuco 1 , M. Calagna 1 , A. Nardo 1 , C. Riccobene 1 , B. Esposito 1 , U. Consoli 5 , V. Di Giacomo 6 , S. Neri 6 , M. R. Cingari 7 , G. Iaria 8 , V. Innao 5,9 , M. Spina 10 , J. Gentile 11 , C. Zizzo 12 , G. Duro 12 , F. Di Raimondo 1,4

1 Division of Hematology, AOU “Policlinico G. Rodolico-San Marco”, Catania; 2 Division of Oncohematology and BMT, Istituto Oncologico del Mediterraneo, Viagrande; 3 Department of Biomedical, Dental, Morphological and Functional Imaging Sciences, University of Messina, Messina; 4 Postgraduate School of Hematology, University of Catania; 5 Unità Operativa Complessa (UOC) di Ematologia, Azienda Ospedaliera di Rilievo Nazionale e di Alta Specializzazione (ARNAS), Catania; 6 UOC di Ematologia, Azienda Ospedaliera Papardo, Messina; 7 Unitá Operativa Semplice Dipartimentale Ematologia, Ospedale San Vincenzo, Taormina; 8 Hemato-Oncology and Radiotherapy Department, Azienda Ospedaliera “Bianchi Melacrino Morelli”, Reggio Calabria; 9 Division of Hematology, Department of Human Pathology in Adulthood and Childhood, University of Messina, Messina; 10 UO Medicina Generale, Ospedale Nuovo di Gragnano, Gragnano; 11 Sanofi Genzyme, Milano; 12 Institute for Biomedical Research and Innovation (IRIB-CNR), National Research Council of Italy, Palermo, Italy

Background: Gaucher disease (GD) is a rare, autosomal recessive genetic disorder, caused by deficiency of glucocerebrosidase, leading to glucosylceramide accumulation in tissue macrophages of hematological, visceral, and skeletal organ systems. Type 1 GD accounts for more than 90% all patients.

Monoclonal gammopathy of undetermined significance is the most common plasma cell disorder, occurring in 3% of the population older than 50 years and is typically detected as an incidental finding. Type 1 GD It is frequently associated with polyclonal and monoclonal gammopathy (MGUS) and is hypothetically caused by long-term immune activation due to accumulated lysolipids, particularly glucosylsphingosine, although the cause remains unknown.

Aims: We evaluated the prevalence of GD in MGUS patients, particularly when other signs and symptoms are present, in order to determine its potential usefulness as screening tool and inclusion in diagnostic framework of GD.

Methods: Between January 2018 and February 2022, dried blood spots (DBS) samples were collected and tested for the acid β-glucosidase (glucocerebrosidase) enzyme activity from MGUS patients, both adult and pediatric, followed in seven hematology units of Sicily, Calabria and Naples. The glucocerebrosidase activity was measured with multiplexed tandem mass spectrometry (MS/MS) using the NeoLSD® assay system, and pathological range was within 0.2 and 2.5 nmol/h/ml. In case of DBS positive result, a confirmatory test was carried over in order to confirm the diagnosis of GD. The study was approved by the local institutional review board and supported by Sanofi Genzyme. All patients provided informed consent for the prospective collection of their data.

Results: A total of 600 patients with MGUS was enrolled at last study update. Around half of the study population was male, with median age of 65 years. Immunoglobulin G was most frequently isolated with serum immunofixation, median level of monoclonal protein was 0.66 g/dL, while median kappa and lambda values based on monoclonal serum free light chain type were 24.8 mg/L and 32.7 mg/L, respectively. Out of 161 patients with available complete blood count median hemoglobin value was 13.5 g/dL with 15 patients having less than 11 g/dL, median platelet count was 224.000/mmc with 10 patients having less than 100.000/mmc platelets and median neutrophil count of 4.280/mmc and seven patients with less than 2.000/mmc neutrophils. Median ferritin level was 77 ng/mL, with more than 400 ng/mL in 10 patients.

The median glucocerebrosidase activity in the entire cohort was 6.9 nmol/h/ml (range 0.3-58.8), with thirteen patients having pathological enzyme activity (median value 1.8 nmol/h/ml, range 0.2-2.5). Sequence analysis of GBA gene evidenced compound heterozygous mutation of the GBA1 gene in three patients (c.1226A>G - N370S and c.1448T>C -L444P in two and c.1226A>G -N370S and c.355G>A -G119R in one respectively), and homozygous mutation (c.1226A>G -N370S) in one patient associated with glucocerebrosidase activity below normal limits. All three patients with compound heterozygous mutational status had signs of GD (hepatosplenomegaly and mild thrombocytopenia). As for the rest of the MGUS patients, in a total of 114 patients sequence analysis evidenced single heterozygous mutation in 16 patients.

Summary/Conclusion: Although type 1 GD remains a rare lysosomal storage disorder, patients with the diagnosis of MGUS could be considered for GD screening with DBS, especially when other symptoms (thrombocytopenia, splenomegaly, hyperferritinemia) are present.

P932: ON-DEMAND PLERIXAFOR WITH CYCLOPHOSPHAMIDE AND G-CSF FOR HEMATOPOIETIC STEM-CELL MOBILIZATION IN MULTIPLE MYELOMA PATIENTS: FINAL RESULTS OF THE MOZOBL06877 STUDY

R. Mina 1,* , F. Bonello 1 , F. Fazio 1 , R. Saccardi 1 , V. Bongarzoni 1 , F. Marchesi 1 , G. Bertuglia 1 , P. Curci 1 , R. M. Lemoli 1 , S. Ballanti 1 , T. Dentamaro 1 , G. Benevolo 1 , A. Capra 1 , R. Floris 1 , P. Tosi 1 , A. Olivieri 1 , D. Rota-Scalabrini 1 , C. Cangialosi 1 , M. Cavo 1 , P. Corradini 1 , G. Milone 1 , M. Boccadoro 1 , A. Larocca 1

1 European Myeloma Network, (EMN), Italy

Background: High-dose melphalan and autologous stem-cell transplant (ASCT) are pillars for the upfront treatment of ASCT-eligible newly diagnosed multiple myeloma (ND)MM patients (pts) or as salvage strategy at relapse. Failure to collect an adequate number of hematopoietic stem cells (HSC) to receive ASCT (<2×106 CD34+ cells/kg) occurs in 5%-15% of MM pts undergoing HSC mobilization with granulocyte colony-stimulating factor (G-CSF) or G-CSF+cyclophosphamide (G-CSF/CY).

Aims: We report the final results of the observational MOZOBL06877 study ( NCT03406091 ; partially supported by Sanofi investigation funds) to prospectively assess the performance of HSC mobilization with G-CSF/CY plus on-demand CXCR4 inhibitor plerixafor (PLX) in NDMM pts treated with novel agents.

Methods: NDMM pts undergoing HSC mobilization with CY (2-4 g/m2) and G-CSF (5-10 mcg/kg/day) were enrolled and observed up to 30 days after mobilization. According to its label, “on-demand” PLX was administered in pts with <20 CD34 + cells/ul after ≥4 days of G-CSF or in case <1×106 CD34+ cells/kg were collected on the first apheresis day. Pts gave written informed consent. The primary endpoint was the poor mobilizer rate, defined as the rate of pts collecting <2×106 CD34+ cells/kg or requiring PLX. Secondary endpoints were the identification of predictive factors for PLX use and safety during mobilization.

Results: 301 NDMM pts were enrolled and analyzed; 72% received induction with bortezomib-thalidomide-dexamethasone, 9% a lenalidomide (Len)-based and 3% a daratumumab (Dara)-based regimen. Overall, 48 pts (16%) were poor mobilizers: 14 (5%) failed to yield ≥2×10 6 /Kg CD34 + , while 34 (11%) required rescue with PLX. Among pts yielding <2×10 6 /Kg CD34+ cells, 4 received PLX, while 10 did not. Among pts who successfully collected ≥2×10 6 /Kg CD34 + (n=287, 95%), 34 (12%) required PLX: 23 (68%) due to a CD34 + /uL count ≤20 after ≥4 days of G-CSF and 11 (32%) to a collection <1×10 6 /Kg after the first apheresis day. In pts mobilized with PLX, median number of CD34 + ×10 6 /L cells increased from 17.5 (IQR 10.8-25.6) before PLX to 58.3 (IQR 34.2-100.2) after PLX. Median number of CD34 + /Kg collected in pts who did not require PLX was 10.2×10 6 (IQR 8.3-13.2). In pts who required PLX, a median of 6.5×10 6 CD34 + /Kg (IQR 4.6-9.6) was collected. An adequate HSC yield for ≥2 ASCTs (≥4×10 6 /Kg) was obtained in 96% and 85% of pts not receiving or receiving PLX, respectively. Median number of apheresis days was 1 (IQR 1-2) in pts not requiring PLX and 2 (IQR 1-2) in pts requiring PLX. In a multivariate analysis ( Table ), factors predicting PLX use were advanced disease stage (R-ISS 3 vs 1-2, OR 5.5, P =0.01), bone marrow (BM) plasma cell (PC) infiltration at diagnosis (PC>60% vs ≤60%, OR 4.2, P <0.001), pre-mobilization absolute neutrophil count (ANC, < vs >2500/mmc, OR 2.8, P =0.01), and the use of Len-based (Yes vs No, OR 3.4, P =0.02) or Dara-based induction regimens (Yes vs No, OR 5.3; P =0.04). Grade 3 infections occurred in 3 pts (1%), with no grade 4-5 reported.

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Summary/Conclusion: Among NDMM pts treated with novel-agent-based induction and undergoing HSC mobilization with CY/GCSF, “on-demand” PLX in case of low CD34 + cell count or insufficient HSC yield was a safe and effective rescue strategy, reducing mobilization failure from 16% to 5%. Predictive factors for PLX use were advanced disease stage, high BM plasmacytosis, low ANC values before mobilization and the use of Len/Dara during induction. Preemptive PLX in pts with ≥1 risk factors should be investigated to further reduce the risk of mobilization failure.

P933: DARATUMUMAB (D) IN COMBINATION WITH VD OR D-RD IN RELAPSED OR REFRACTORY MULTIPLE MYELOMA: SUBGROUP ANALYSIS OF CASTOR AND POLLUX STUDIES IN PATIENTS WITH EARLY OR LATE RELAPSE AFTER INITIAL THERAPY

A. Spencer 1,* , P. Moreau 2 , M.-V. Mateos 3 , H. Goldschmidt 4 , K. Suzuki 5 , M.-D. Levin 6 , P. Sonneveld 7 , S.-S. Yoon 8 , S. Z. Usmani 9 , K. Weisel 10 , D. Reece 11 , T. Ahmadi 12 , H. Pei 13 , W. Garvin Mayo 14 , X. Gai 15 , J. Carey 16 , R. Carson 16 , M. A. Dimopoulos 17

1 Malignant Haematology and Stem Cell Transplantation Service, Alfred Health-Monash University, Melbourne, Australia; 2 Hematology Department, University Hospital Hôtel-Dieu, Nantes, France; 3 University Hospital of Salamanca/IBSAL/Cancer Research Center-IBMCC (USAL-CSIC), Salamanca, Spain; 4 University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 5 Department of Hematology, Japanese Red Cross Medical Center, Tokyo, Japan; 6 Albert Schweitzer Hospital, Dordrecht; 7 Erasmus MC Cancer Institute, Rotterdam, Netherlands; 8 Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea; 9 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 10 Department of Oncology, Hematology and Bone Marrow Transplantation With Section of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 11 Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada; 12 Genmab US, Inc., Plainsboro, NJ; 13 Janssen Research & Development, LLC, Titusville, NJ; 14 Janssen Research & Development, LLC, Raritan, NJ, United States of America; 15 Janssen Research & Development, LLC, Beijing, China; 16 Janssen Research & Development, LLC, Spring House, PA, United States of America; 17 National and Kapodistrian University of Athens, Athens, Greece

Background: High-risk multiple myeloma (MM) is often defined based on cytogenetic abnormalities (ie, t[4;14], t[14;16], and/or del17p); however, patients who relapse early (12-18 months) after initial therapy are considered a functional high-risk group that is also associated with poor prognosis. Daratumumab (DARA), a human IgGk monoclonal antibody targeting CD38, is approved in combination with standard-of-care regimens for MM. In the phase 3 CASTOR and POLLUX studies, DARA in combination with bortezomib plus dexamethasone (D-Vd) and lenalidomide plus dexamethasone (D-Rd) significantly improved progression-free survival (PFS), regardless of cytogenetic risk, and achieved higher rates of complete response or better (≥CR) and minimal residual disease (MRD)–negativity vs Vd or Rd alone in patients with RRMM.

Aims: In this post-hoc analyses of CASTOR and POLLUX we evaluated D-Vd vs Vd and D-Rd vs Rd in patient subgroups with 1 prior line of therapy based on timing of relapse (early or late) after initiation of the first line of therapy.

Methods: In CASTOR and POLLUX, patients with RRMM and ≥1 prior line of therapy were randomized to D-Vd/Vd or D-Rd/Rd, respectively. The primary endpoint was PFS. In this analysis, the early relapse subgroup included patients with 1 prior line of therapy who relapsed <18 months after initiating their first line of therapy; patients with 1 prior line of therapy who relapsed ≥18 months after initiating their first line of therapy were included in the late relapse subgroup.

Results: 49 and 186 patients from CASTOR and 99 and 196 patients from POLLUX were included in the early relapse and late relapse subgroups, respectively. Median follow-up was 72.6 months (CASTOR) and 79.7 months (POLLUX). PFS consistently favored the DARA-containing regimens across subgroups ( Table ). In CASTOR, ≥CR rates were higher with D-Vd vs Vd in the early relapse (21% vs 17%; P = 0.7360) and late relapse (51% vs 14%; P <0.0001) subgroups. In POLLUX, ≥CR rates were higher with D-Rd vs Rd in the early relapse (53% vs 12%; P <0.0001) and late relapse (62% vs 38%; P = 0.0012) subgroups. MRD-negativity rates (10 –5 ) were higher with D-Vd/D-Rd vs Vd/Rd regardless of relapse timing (CASTOR: early, 13% vs 0%; P = 0.1476; late, 23% vs 3%; P <0.0001; POLLUX: early, 30% vs 4%; P = 0.0006; late, 34% vs 14%; P = 0.0009).

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Summary/Conclusion: These post hoc analyses of CASTOR and POLLUX showed PFS and depth of response benefits of DARA-containing regimens in patients with 1 prior line of therapy, regardless of relapse timing (early or late). Our results support the use of D-Vd and D-Rd in RRMM, including in patients who are considered functional high risk.

P934: DARATUMUMAB + LENALIDOMIDE, BORTEZOMIB, AND DEXAMETHASONE IN TRANSPLANT-ELIGIBLE NEWLY DIAGNOSED MULTIPLE MYELOMA: A POST HOC ANALYSIS OF SUSTAINED MINIMAL RESIDUAL DISEASE NEGATIVITY FROM GRIFFIN

C. Rodriguez 1,* , J. L. Kaufman 2 , J. Laubach 3 , D. W. Sborov 4 , B. Reeves 5 , A. Chari 6 , R. Silbermann 7 , L. J. Costa 8 , L. D. Anderson Jr 9 , N. Nathwani 10 , N. Shah 11 , N. Bumma 12 , A. Jakubowiak 13 , R. Z. Orlowski 14 , H. Pei 15 , A. Cortoos 16 , S. Patel 16 , T. S. Lin 16 , P. G. Richardson 3 , P. M. Voorhees 17

1 Wake Forest University School of Medicine, Winston-Salem; 2 Winship Cancer Institute, Emory University, Atlanta; 3 Dana-Farber Cancer Institute, Boston; 4 Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City; 5 University of North Carolina – Chapel Hill, Chapel Hill; 6 Tisch Cancer Institute, Mount Sinai School of Medicine, New York; 7 Knight Cancer Institute, Oregon Health & Science University, Portland; 8 University of Alabama at Birmingham, Birmingham; 9 Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas; 10 Judy and Bernard Briskin Center for Multiple Myeloma Research, City of Hope Comprehensive Cancer Center, Duarte; 11 Department of Medicine, University of California San Francisco, San Francisco; 12 Division of Hematology, The Ohio State University Comprehensive Cancer Center, Columbus; 13 University of Chicago Medical Center, Chicago; 14 Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston; 15 Janssen Research & Development, LLC, Titusville; 16 Janssen Scientific Affairs, LLC, Horsham; 17 Levine Cancer Institute, Atrium Health, Charlotte, United States of America

Background: In the primary analysis of the phase 2 randomized GRIFFIN study, Daratumumab (DARA) + lenalidomide, bortezomib, and dexamethasone (D-RVd) improved the stringent complete response (sCR) rate by end of consolidation for transplant-eligible newly diagnosed multiple myeloma (NDMM) (42.4% vs 32.0%; 1-sided P = 0.068). With longer follow-up (median, 38.6 mo), D-RVd vs RVd improved minimal residual disease (MRD)-negativity (10 –5 ) rates in clinically relevant subgroups (ISS stage III, 71% vs 36%; high cytogenetic risk, 44% vs 29% [del17p, t(4;14), or t(14;16)]; revised high cytogenetic risk, 55% vs 32% [del17p, t(4;14), t(14;16), t(14;20), or gain 1q]).

Aims: In this post-hoc analysis we present results of sustained MRD negativity (median follow-up, 38.6 mo) in the same subgroups and in patients (pts) with ≥CR.

Methods: Transplant-eligible NDMM pts were randomized 1:1 to 4 D-RVd/RVd induction cycles, ASCT, 2 D-RVd/RVd consolidation cycles, and 2 years of maintenance therapy with lenalidomide (R) ± DARA. For induction/consolidation (21-day cycles), pts received R (25 mg PO Days [D] 1-14), V (1.3 mg/m 2 SC D1, 4, 8, 11), and d (40 mg PO weekly) ± DARA (16 mg/kg IV D1, 8, 15 of Cycles 1-4 and D1 of Cycles 5-6). In maintenance (28-day cycles), pts received R (10 mg PO D1-21; if tolerated, 15 mg in Cycles 10+) ± DARA (16 mg/kg IV Q8W/Q4W or 1800 mg SC per protocol amendments). The primary endpoint was sCR rate by end of consolidation.

Results: The following features were balanced among randomized pts (D-RVd, n = 104; RVd, n = 103): high cytogenetic risk (16; 14), revised high cytogenetic risk (42; 37), gain 1q (34; 28), and ISS stage III (14; 14). Sustained MRD-negativity rates at 10 –5 lasting ≥6 and ≥12 months were higher for D-RVd vs RVd among all high-risk subgroups ( Table ). D-RVd was superior to RVd for rates of sustained MRD negativity lasting ≥12 months for pts with ≥CR (53.7% vs 20.3%) and sCR (59.1% vs 17.4%; Table ). Among all pts with sustained MRD negativity, only 1 D-RVd pt subsequently had disease progression, and 1 RVd pt died. Additional data on MRD at 10 –6 and PFS will be presented.

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Summary/Conclusion: MRD data in GRIFFIN show that the addition of DARA to RVd induction/consolidation and R maintenance may lead to durable MRD-negativity (10 –5 ) rates in pts with transplant-eligible NDMM with high cytogenetic risk, ISS stage III, and those who achieve ≥CR or sCR, however larger studies are needed.

P935: REAL WORLD COMPARATIVE ANALYSIS OF THE EFFICACY OF TECLISTAMAB VERSUS CURRENT TREATMENTS IN PATIENTS WITH TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA FROM THE LOCOMOTION STUDY

H. Einsele 1,* , P. Moreau 2 , M. Delforge 3 , N. W. van de Donk 4 , F. Ghilotti 5 , J. Diels 6 , A. Elsada 7 , V. Strulev 6 , L. Pei 8 , R. Kobos 8 , J. Smit 9 , M. Slavcev 10 , K. Weisel 11 , M.-V. Mateos 12

1 Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Wuerzburg, Germany; 2 Hematology Clinic, University Hospital Hôtel-Dieu, Nantes, France; 3 University of Leuven, Leuven, Belgium; 4 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 5 Janssen-Cilag SpA, Cologno Monzese, United States of America; 6 Janssen Pharmaceutica NV, Beerse, Belgium; 7 Janssen-Cilag, High Wycombe, Buckinghamshire, United Kingdom; 8 Janssen Research & Development, Raritan; 9 Janssen Research & Development, Spring House; 10 Janssen Global Services, LLC, Raritan, United States of America; 11 University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 12 University Hospital of Salamanca/IBSAL, CIC, Salamanca, Spain

Background: Patients with triple-class exposed relapsed/refractory multiple myeloma (TCE RRMM) have poor prognosis and limited treatment options after being treated with ≥3 lines of therapy (LOT). Teclistamab (tec), a B-cell maturation antigen × CD3 bispecific antibody, is currently being evaluated in the MajesTEC-1 ( NCT04557098 ) study. MajesTEC-1 is a single-arm, phase 1/2 study in patients with TCE RRMM, previously exposed to an immunomodulatory drug, a proteasome inhibitor, and an anti-CD38 antibody and received ≥3 LOT.

Aims: Since MajesTEC-1 lacks a control arm, we performed a real-world comparative analysis of the efficacy of tec vs currently used treatments in clinical practice by creating an external real-world control arm from LocoMMotion ( NCT04035226 ), a prospective study of real-world clinical practice (RWCP) efficacy and safety outcomes in patients with TCE RRMM who received ≥3 LOT.

Methods: Patients from LocoMMotion (248 patients, clinical cutoff May 21, 2021) who met MajesTEC-1 eligibility criteria were used to create the external control arm for MajesTEC-1. Individual patient-level data were included for 150 patients from MajesTEC-1 treated with tec (1.5 mg/kg weekly) at a clinical cutoff of Sep 7, 2021. Inverse probability of treatment weighting with average treatment effect on the treated was used to adjust for imbalances in prognostically significant baseline covariates: Eastern Cooperative Oncology Group performance status, gender, type of MM, prior transplant, refractory status, International Staging System stage, time to progression on prior LOT, extramedullary disease, number of prior LOT, time since diagnosis, average duration of prior LOT, age, hemoglobin, lactate dehydrogenase and creatinine clearance. Efficacy of tec vs RWCP was measured for overall response rate (ORR), very good partial response (VGPR) rate, complete response or better (≥CR) rate, duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Odds ratio, transformed into a response-rate ratio (RR) (along with 95% confidence interval [CI]), derived from weighted logistic regression was used for binary endpoints (ORR, VGPR rate, and ≥CR rate). Hazard ratios (HRs) and 95% CIs for time-to-event endpoints (DOR, PFS, and OS) were computed using a weighted Cox proportional hazards model.

Results: After reweighting the external RWCP cohort, the 2 cohorts had well balanced baseline characteristics. Patients treated with tec had improved outcomes vs current treatments used in RWCP: ORR (RR: 2.31; 95% CI 1.75–2.87; P <0.0001), VGPR rate (RR: 5.54; 95% CI 3.38–7.70; P <0.0001), ≥CR rate (RR 91.50; 95% CI 12.66–661.43; P <0.0001), DOR (HR 0.17; 95% CI 0.08–0.36; P <0.0001), PFS (HR 0.47; 95% CI 0.34–0.67; P <0.0001), and OS (HR 0.69; 95% CI 0.46–1.05; P =0.08).

Summary/Conclusion: In this analysis, significantly improved efficacy for almost all outcomes was observed with tec when compared with RWCP. This highlights the potential of tec as a highly effective treatment option for patients with TCE RRMM who have been exposed to ≥3 LOT.

P936: TIME TO RESPONSE, DURATION OF RESPONSE, AND PATIENT-REPORTED OUTCOMES WITH DARATUMUMAB PLUS RD VS RD ALONE IN TRANSPLANT-INELIGIBLE PATIENTS WITH NDMM: SUBGROUP ANALYSIS OF THE PHASE 3 MAIA STUDY

T. Facon 1,* , S. K. Kumar 2 , T. Plesner 3 , P. Moreau 4 , N. Bahlis 5 , H. Goldschmidt 6 , M. O’Dwyer 7 , A. Perrot 8 , C. P. Venner 9 , K. Weisel 10 , J. R. Mace 11 , N. Raje 12 , M. Tiab 13 , M. Macro 14 , L. Frenzel 15 , X. Leleu 16 , H. Pei 17 , F. Borgsten 18 , S. Z. Usmani 19

1 University of Lille, CHU Lille, Service des Maladies du Sang, Lille, France; 2 Department of Hematology, Mayo Clinic Rochester, Rochester, MN, United States of America; 3 Vejle Hospital and University of Southern Denmark, Vejle, Denmark; 4 Hematology Department, University Hospital Hôtel-Dieu, Nantes, France; 5 Arnie Charbonneau Cancer Research Institute, University of Calgary, Calgary, AB, Canada; 6 University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 7 Department of Medicine/Haematology, NUI, Galway, Ireland; 8 CHU de Toulouse, IUCT-O, Université de Toulouse, UPS, Service d’Hématologie, Toulouse, France; 9 Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada; 10 Department of Oncology, Hematology and Bone Marrow Transplantation with Section of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 11 Florida Cancer Specialists, St. Petersburg, FL; 12 Center for Multiple Myeloma, Massachusetts General Hospital Cancer Center, Boston, MA, United States of America; 13 CHD Vendée, La Roche sur Yon; 14 Centre Hospitalier Universitaire (CHU) de Caen, Caen; 15 Department of Clinical Haematology, Hopital Necker-Enfants Malades, Paris; 16 CHU Poitiers, Hôpital la Milétrie, Poitiers, France; 17 Janssen Research & Development, LLC, Titusville, NJ, United States of America; 18 Janssen-Cilag, Birkerød, Denmark; 19 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America

Background: In the phase 3 MAIA study, adding daratumumab (DARA) to lenalidomide and dexamethasone (Rd) improved progression-free survival (primary endpoint), overall survival, duration of response, and patient-reported outcomes (PROs) in transplant-ineligible patients with newly diagnosed multiple myeloma (NDMM).

Aims: Here we report a MAIA subgroup analysis of time to response, duration of response, and PROs.

Methods: Transplant-ineligible patients with NDMM received 28-day cycles of Rd (R 25 mg PO on Days 1-21; d 40 mg PO QW) ± DARA (16 mg/kg IV QW in Cycles 1-2, Q2W in Cycles 3-6, and Q4W thereafter) until disease progression or unacceptable toxicity. Secondary endpoints included time to response and duration of response. PROs were measured using the EORTC QLQ-C30, with treatment effects assessed via mixed-effects model with repeated measures.

Results: In total, 368 patients were assigned to the DARA plus lenalidomide and dexamethasone (D-Rd) group and 369 patients to the Rd group; 162 (44%) D-Rd patients and 142 (38%) Rd patients had renal impairment (defined as baseline CrCl ≤60 mL/min). At a 56.2-mo median follow-up, median times to very good partial response or better (≥VGPR) and complete response or better (≥CR) were shorter with D-Rd vs Rd in the overall study population and in the subgroups of patients with and without renal impairment ( Table ). Among patients who achieved ≥CR or partial response or better (≥PR), higher proportions of D-Rd vs Rd patients had not experienced disease progression at 48 mo ( Table ). Among patients with renal impairment, greater improvements from baseline in patient-reported pain, fatigue, and nausea and vomiting symptom scores were observed with D-Rd vs Rd across most timepoints; a notably greater meaningful reduction in pain symptom score was seen with D-Rd vs Rd as early as Cycle 6 Day 1 (least squares mean change from baseline, −14.9 vs −7.0; P =0.0241). Analyses for additional patient subgroups will be presented.

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Summary/Conclusion: In transplant-ineligible patients with NDMM, D-Rd showed more rapid deep responses as well as more durable responses vs Rd, regardless of renal function. Improvements in patient-reported symptoms were generally greater with D-Rd vs Rd in patients with renal impairment. Our results support the use of D-Rd in transplant-ineligible patients with NDMM.

P937: PRECLINICAL DISCOVERY AND EARLY FINDINGS FROM THE PHASE 1, DOSE-ESCALATION STUDY OF WVT078, A BCMA-CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH R/R MULTIPLE MYELOMA

M. S. Raab 1,* , Y. C. Cohen 2,3 , F. Schjesvold 4,5 , K. Aardalen 6 , A. Oka 6 , A. Spencer 7 , M. Wermke 8 , P. Hari 9 , J. L. Kaufman 10 , A. M. Cafro 11 , E. M. Ocio 12 , N. Doki 13 , K. Henson 6 , G. Trabucco 6 , P.-E. Juif 14 , R. Chawla 14 , J. Cotton 6 , A. Fessehatsion 6 , L. Fan 6 , J. Blankenship 6 , B. Granda 6 , H. Lu 6 , S. De Vita 6

1 Department of Internal Medicine, Heidelberg University Hospital, Heidelberg, Germany; 2 Tel-Aviv Sourasky (Ichilov) Medical Center; 3 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 4 Oslo Myeloma Center, Department of Hematology, Oslo University Hospital; 5 KG Jebsen Center for B cell malignancies, University of Oslo, Oslo, Norway; 6 Novartis Institutes for BioMedical Research, Cambridge, MA, United States of America; 7 The Alfred Hospital, Melbourne, VIC, Australia; 8 NCT/UCC Early Clinical Trial Unit, Universitätsklinikum Carl Gustav Carus an der Technische Universität, Dresden, Germany; 9 Division of Hematology & Oncology, Medical College of Wisconsin, Milwaukee, WI; 10 Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 11 Department of Hematology, ASST Grande Ospedale Metropolitano, Niguarda, Milan, Italy; 12 Hospital Universitario Marqués de Valdecilla (IDIVAL), Universidad de Cantabria, Santander, Spain; 13 Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Tokyo, Japan; 14 Novartis Institutes for BioMedical Research, Basel, Switzerland

Background: B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily with selective expression on benign and malignant plasma cells. Signaling through BCMA mediates survival and proliferation of multiple myeloma (MM) cells. BCMA is a validated clinical target with multiple BCMA-directed therapies approved or in development for patients with relapsed and/or refractory (r/r) MM. Bispecific antibodies (BsAbs) offer a convenient alternative to chimeric antigen receptor–modified T-cell (CAR-T) therapies targeting BCMA and are demonstrating high response rates in patients with MM. Here we describe the discovery of WVT078, a novel BCMA-CD3 BsAb and report initial clinical data from the ongoing phase 1 study ( NCT04123418 ) evaluating ascending doses of WVT078 in patients with r/r MM.

Aims: To evaluate the safety and tolerability of the novel BCMA-CD3 BsAb WVT078 in patients with r/r MM.

Methods: Multiple BCMA-CD3 BsAb candidates were characterized in vitro and in vivo for T cell activation and anti-MM activity. The activity and tolerability of prioritized candidates were further assessed in an integrated pharmacology study in cynomolgus monkeys. WVT078 was selected for the phase 1 clinical study and is being tested in patients with MM who are r/r to ≥2 standard of care regimens including an immunomodulatory drug, a proteasome inhibitor, and an anti-CD38 agent (if available). Patients who have received a prior BCMA CAR-T or BCMA-antibody-drug conjugates therapy are included, whereas those with prior treatment with BCMA-CD3 bispecifics are excluded.

Results: WVT078, a BCMA-CD3 BsAb with high affinity binding to BCMA (with subnanomolar K D ), was selected to effectively target MM cells with a low BCMA expression, an emerging mechanism of resistance to BCMA-targeted therapies. WVT078 demonstrated potent anti-MM activity and efficient T-cell activation in preclinical models, and was well tolerated in cynomolgus monkeys. As of December 08, 2021, a total of 33 patients with r/r MM were treated with intravenous WVT078 once weekly at the following dose levels: 3, 6, 12, 24, 48, 64, 96, 192, and 250 µg/kg of body weight. Twenty-five patients (75.8%) discontinued treatment due to progressive disease (n=22), physician decision (n=2) or adverse event (AE, n=1). The most frequent (≥20%) treatment-related AEs (TRAEs) of all grades across all doses included cytokine release syndrome (CRS, 60.6%), pyrexia (39.4%), increased alanine aminotransferase (ALT, 30.3%), increased aspartate aminotransferase (AST) and anemia (24.2% each), and the most common grade ≥3 TRAEs (≥10%) included increased AST, lymphopenia (18.2% each), increased ALT (15.2%), CRS and neutropenia (12.1% each). Clinical activity was observed starting at the dose of 48 µg/kg. At the active doses of 48-250 µg/kg (n=26), the overall response rate (partial response or better) was 34.6% (n=9; 90% CI, 19.4-52.6) and the complete response rate (stringent complete response+complete response) was 11.5% (n=3; 90% CI, 3.2-27.2). The maximum tolerated dose has not been reached. Pharmacokinetic data showed a trend towards dose-proportional relationship and no evidence of accumulation.

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Summary/Conclusion: WVT078, a novel BCMA-CD3 BsAb, demonstrated potent anti-MM activity and favorable tolerability in preclinical models. In the phase 1 study, WVT078 exhibited an acceptable safety profile and preliminary evidence of clinical activity. Clinical evaluation of WVT078 as a single agent and in combination with a gamma secretase inhibitor is ongoing.

P938: CARFILZOMIB-LENALIDOMIDE-DEXAMETHASONE CONSOLIDATION IN MYELOMA PATIENTS WITH A POSITIVE FDG PET/CT AFTER UPFRONT AUTOLOGOUS STEM CELL TRANSPLANTATION: A PHASE II STUDY (CONPET)

J. N. Nørgaard 1,2,3,* , N. Abildgaard 4,5 , A. Lysén 1,3 , G. Tsykunova 6 , A. J. Vangsted 7 , C. Joao 8 , N. Remen 1 , L. Osnes 9 , J. P. Connelly 10 , M.-E. R. Revheim 2,10 , F. Schjesvold 1,3

1 Oslo Myeloma Center, Department of Hematology, Oslo University Hospital; 2 Institute of Clinical Medicine; 3 KG Jebsen Center for B-cell Malignancies, University of Oslo, Oslo, Norway; 4 Department of Hematology, Odense University Hospital; 5 Department of Clinical Research, University of Southern Denmark, Odense, Denmark; 6 Division of Hematology, Haukeland University Hostpital, Bergen, Norway; 7 Departmen of Hematology, Rigshospitalet, Copenhagen, Denmark; 8 Department of Hematology, Champalimaud Centre for the Unknown, Lisboa, Portugal; 9 Department of Immunology; 10 Division for Radiology and Nuclear Medicine, Oslo University Hospital, Oslo, Norway

Background: [18F]-Fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) positivity after first line treatment with autologous stem cell transplantation (ASCT), is strongly correlated with reduced progression free survival and overall survival (Moreau et al. , JCO, 2017). However, FDG PET/CT positive patients who obtain FDG PET/CT negativity after treatment seem to have comparable outcomes to patients who were FDG PET/CT negative at baseline (Davies et al., Haematologica 2018).

Aims: Aiming for FDG PET/CT negativity may be an important goal in myeloma treatment. The use of FDG PET/CT positivity as an indication for consolidation therapy after ASCT has not been studied before. We here present the complete data of the primary endpoint, PET/CT negativity after KRd consolidation.

Methods: Patients with multiple myeloma who had received standard first line treatment including ASCT and achieved very good partial response (VGPR) or better, were examined by FDG PET/CT. Patients who were FDG PET/CT positive defined by the Italian Myeloma criteria for PET USe (IMPETUS) (Nanni C et al ., EJNMMI 2016 and 2018) were included in the treatment phase of the study and were assessed for minimal residual disease (MRD) by Euroflow (sensitivity: 10 -5 ) before treatment. FDG PET/CT examinations were centrally read by two experienced nuclear medicine radiologists. The treatment consisted of four 28-day cycles of KRd (carfilzomib 36 mg/m 2 day 1,2,8,9,15 and 16 (except 20 mg/m 2 day 1 and 2 first cycle), lenalidomide 25 mg day 1-21 all cycles and dexamethasone 40 mg day 1,8,15 and 22 all cycles). After 4 cycles, FDG PET/CT and Euroflow for MRD were repeated for response evaluation. Both patients with FDG PET/CT negativity and patients with FDG PET/CT positivity at baseline are followed for progression free survival and overall survival.

Results: 159 patients were screened with FDG PET/CT. Fifty-three patients of 159 (33%) had a positive FDG PET/CT result. Among FDG PET/CT positive patients, a higher proportion had ISS score III, high-risk cytogenetics and VCd induction was more common (Table 1). Forty-eight patients completed KRd treatment. Sixteen patients (33%) converted into FDG PET/CT negativity. More patients with previous VRd induction than VCd induction converted from FDG PET/CT positive to FDG PET/CT negative (38% vs 20%). No patients stopped treatment due to adverse events. Twenty-seven of 49 (55%) patients with a FDG PET/CT positive result were MRD negative before KRd consolidation. A higher proportion of MRD negative patients vs MRD positive patients before KRd treatment, converted into FDG PET/CT negativity, with 37% vs 28%, respectively. For the proportion of patients that after KRd treatment was both FDG PET/CT negative and MRD negative by EuroFlow, the difference was even higher (37% vs 5% in MRD negative vs MRD positive patients, respectively). In total, the MRD negativity rate increased from 55% to 76%

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Summary/Conclusion: Before KRd consolidation, 33% of patients in VGPR or better after first line treatment including ASCT were FDG PET/CT positive. High-risk cytogenetics, ISS score III and VCd induction were more common in FDG PET/CT positive patients. After four cycles of KRd, 33% of patients converted from FDG PET/CT positivity to negativity. A higher proportion converted into FDG PET/CT negativity in patients who were MRD negative before KRd treatment, and in patients who had received VRd. KRd consolidation is feasible and converts a clinically significant proportion of FDG PET/CT positive patients to negativity. Patients are followed for PFS and OS.

P939: SYNERGISTIC EFFECTS OF LOW DOSE BELANTAMAB MAFODOTIN IN COMBINATION WITH A GAMMA-SECRETASE INHIBITOR (NIROGACESTAT) IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): DREAMM-5 STUDY

A. Nooka 1,* , S. Lonial 1 , S. Grosicki 2 , M. Hus 3 , K. Song 4 , T. Facon 5 , N. S. Callander 6 , V. Ribrag 7 , K. Uttervall 8 , H. Quach 9 , V. Vorobyev 10 , C.-K. Min 11 , S. Cheng 12 , L. M. Smith 12 , J. Yu 13 , T. Collingwood 13 , B. Holkova 13 , B. E. Kremer 13 , I. V. Gupta 13 , P. G. Richardson 14 , M. C. Minnema 15

1 Emory University, Winship Cancer Institute, Atlanta, GA, United States of America; 2 Department of Hematology and Cancer Prevention, Medical University of Silesia, Katowice; 3 Katedra i Klinika Hematoonkologii i Transplantacji Szpiku, Lublin, Poland; 4 Vancouver General Hospital, Vancouver, BC, Canada; 5 Department of Haematology, Lille University Hospital, Lille, France; 6 University of Wisconsin, Carbone Cancer Center, Madison, WI, United States of America; 7 Institut Gustave Roussy, Villejuif, France; 8 Karolinska University Hospital, Stockholm, Sweden; 9 University of Melbourne, St. Vincent’s Hospital Melbourne, Melbourne, VIC, Australia; 10 S P Botkin City Clinical Hospital, Moscow, Russia; 11 Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, South Korea; 12 SpringWorks Therapeutics, Stamford, CT; 13 GlaxoSmithKline, Upper Providence, PA; 14 Dana-Farber Cancer Institute, Boston, MA, United States of America; 15 University Medical Center Utrecht, Utrecht, Netherlands

Background: Preclinical data demonstrate that nirogacestat, a gamma-secretase inhibitor, may increase cell-surface levels of a B-cell maturation antigen (BCMA) and reduce soluble BCMA levels, which could enhance anti-BCMA agent activity in multiple myeloma. In the DREAMM-5 ( NCT04126200 ) Phase I/II platform trial belantamab mafodotin (belamaf; BLENREP), a BCMA-targeting antibody-drug conjugate, is being evaluated in combination with nirogacestat.

Aims: The aim of this study is to determine if the combination can result in similar efficacy and an improved ocular safety profile compared to the currently approved belamaf schedule (single agent dose 2.5 mg/kg Q3W) in patients with RRMM which showed a 31% overall response rate (ORR) and 44.5% Gr3/4 keratopathy (BLENREP US prescribing information).

Methods: This cohort within the DREAMM-5 nirogacestat combination sub-study has a sequential dose-exploration (DE) phase evaluating 0.95 mg/kg Q3W belamaf with 100 mg BID nirogacestat continuously, followed by a randomized cohort expansion (CE) comparing the combination to a belamaf 2.5 mg/kg Q3W arm.

Results: Preliminary results from the 10 patients in the DE cohort with low-dose belamaf + nirogacestat, are presented in this abstract. Patients had a median (range) of 4.5 (3–10) prior lines of therapy. At time of data cut-off (Nov 15, 2021), patients received a median (range) of 7 cycles (1–26). The ORR was 60% (n=6/10) and 20% (n=2) achieved a very good partial response (Table). The key emergent adverse events (AEs) included ocular events (n=7 [70%]; ≥Grade [Gr] 3, n=2 [20%] of which Gr 3 keratopathy was reported in 1 patient [10%]), diarrhea (n=7 [70%]; Gr3, n=1 [10%]) and hypophosphatemia (n=7 [70%]; Gr3, n=1, [10%]). There were 2 Grade 5 AEs, neither of which were related to study treatment. No patient permanently discontinued study due to treatment related AEs.

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Summary/Conclusion: Encouraging clinical activity and a manageable safety profile is observed with low dose belamaf (0.95 mg/kg Q3W) + nirogacestat (100 mg BID continuously) in patients with RRMM. This ongoing sub-study is actively recruiting patients and will continue to evaluate belamaf + nirogacestat efficacy and safety. Updated results will be reported at the congress.

Funding Statement: GSK (208887); drug linker technology licensed from Seagen Inc.; mAb produced using POTELLIGENT Technology licensed from BioWa.

This abstract was previously submitted to the American Society of Clinical Oncology (ASCO) Annual Meeting, June 3–7, 2022, and is submitted on behalf of the original authors with their permission. ©2022 American Society of Clinical Oncology, Inc. Reused with permission. All rights reserved.

P940: SAFETY AND CLINICAL ACTIVITY OF BELANTAMAB MAFODOTIN WITH PEMBROLIZUMAB IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): DREAMM-4 STUDY

A. Suvannasankha 1,* , N. Bahlis 2 , S. Trudel 3 , K. Weisel 4 , C. Koenecke 5 , A. Oriol 6 , P. M. Voorhees 7 , A. A. Alonso 8 , N. S. Callander 9 , M. V. Mateos Manteca 10 , N. Reddy 11 , S. Hakim 12 , N. Patel 13 , D. Williams 12 , R. C. Jewell 13 , X. Zhou 12 , I. Gupta 14 , A. K. Nooka 15

1 Indiana University Simon Cancer Center and Roudebush VAMC, Indianapolis, IN, United States of America; 2 Arnie Charbonneau Cancer Research Institute, University of Calgary, Calgary, AB; 3 Princess Margaret Cancer Centre, Toronto, ON, Canada; 4 University Medical Center Hamburg-Eppendorf, Hamburg; 5 Hannover Medical School, Clinic for Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany; 6 Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 7 Levine Cancer Institute, Atrium Health, Charlotte, NC, United States of America; 8 Hospital Universitario Quirónsalud Madrid, Madrid, Spain; 9 Carbone Cancer Center, University of Wisconsin, Madison, WI, United States of America; 10 Instituto de Investigación Biomédica de Salamanca and Centro de Investigación del Cáncer, Hospital Universitario de Salamanca, Salamanca, Spain; 11 Merck & Co., Inc., Kenilworth, NJ; 12 GlaxoSmithKline, Upper Providence, PA; 13 GlaxoSmithKline, Research Triangle Park, NC; 14 GlaxoSmithKline, Philadelphia, PA; 15 Winship Cancer Institute, Emory University Hospital, Atlanta, GA, United States of America

Background: Belantamab mafodotin (belamaf; BLENREP), a B-cell maturation antigen (BCMA) targeted antibody–drug conjugate approved for adult patients with RRMM, has a multimodal mechanism that eliminates myeloma cells via direct cytotoxicity and a systemic anti-tumor immune response, which may be augmented by an immune checkpoint inhibitor.

Aims: DREAMM-4 ( NCT03848845 ) assessed safety and clinical activity of belamaf with pembrolizumab (pembro) in RRMM.

Methods: This was a Phase I/II, single-arm, open-label study of adults with RRMM after ≥3 lines of therapy (LOT, including anti-CD38 monoclonal antibody, proteasome inhibitor, and immunomodulator). Part 1 established the dose of belamaf 2.5 mg/kg with pembro 200 mg, both IV Q3W up to 35 cycles, for the Part 2 expansion. Primary efficacy endpoint was investigator-assessed overall response rate (ORR, ≥partial response [PR] per IMWG criteria by investigator). Secondary endpoints included duration of response (DoR), progression-free survival (PFS), adverse events (AEs) per NCI-CTCAE v4.03, and pharmacokinetics (PK).

Results: This primary analysis of all treated pts (as of Oct 18, 2021) included 34 pts (6 in Part 1 and 28 in Part 2). In both parts, median prior LOT was 5 (range 3–13); 10 pts (29%) had high-risk cytogenetics and 9 (26%) had extramedullary disease. ORR was 47%, with most responses (10/16 pts) ≥ very good PR (VGPR, Table). Median follow-up was 14.7 months (mo); median (95% CI) DoR was 8.0 (2.1–not reached) mo; median PFS was 3.4 (1.4–5.6) mo. Most pts had ≥1 AE (any grade [Gr]: 97%; Gr ≥3: 74%) and treatment-related AE (TRAE, any Gr: 97%; Gr ≥3: 65%). Most common (≥35%) AEs were keratopathy (any Gr: 76%; Gr ≥3: 38%), vision blurred (any Gr: 38%; Gr ≥3: 0%), and thrombocytopenia (any Gr: 35%; Gr ≥3: 29%). AEs led to dose delays (65%) and dose reductions (32%), but not discontinuation. Nine pts had a serious AE (SAE); 4 pts had ≥1 SAE related to study treatment. Two pts had immune-related AEs of Gr 1 (gout and autoimmune hypothyroidism). Preliminary PK and soluble BCMA data were consistent with single-agent belamaf therapy.

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Summary/Conclusion: Belamaf + pembro demonstrated a favorable ORR compared with single-agent belamaf in heavily pre-treated RRMM. No new TRAEs were identified; AE frequency and severity were similar to single-agent belamaf. Correlative biomarker studies are ongoing.

Funding Statement: GSK (205207) in collaboration with Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. Drug linker technology licensed from Seagen Inc. mAb produced using POTELLIGENT technology licensed from BioWa.

This abstract was previously submitted to the American Society of Clinical Oncology (ASCO) Annual Meeting, June 3–7, 2022, and is submitted on behalf of the original authors with their permission. ©2022 American Society of Clinical Oncology, Inc. Reused with permission. All rights reserved.

P941: SAFETY AND CLINICAL ACTIVITY OF BELANTAMAB MAFODOTIN WITH LENALIDOMIDE PLUS DEXAMETHASONE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): DREAMM-6 ARM-A INTERIM ANALYSIS

H. Quach 1,* , M. Gironella 2 , C. Lee 3 , R. Popat 4 , P. Cannell 5 , R. S. Kasinathan 6 , B. Chopra 7 , R. Rogers 6 , G. Ferron-Brady 6 , S. Shafi-Harji 8 , N. Patel 7 , J. Opalinska 6 , I. Gupta 6 , B. Augustson 9

1 University of Melbourne, St. Vincent’s Hospital Melbourne, Melbourne, Australia; 2 Department of Hematology, University Hospital Vall d’Hebron, Barcelona, Spain; 3 Royal Adelaide Hospital, Adelaide, Australia; 4 NIHR UCLH Clinical Research Facility, University College London Hospitals, NHS Foundation Trust, London, United Kingdom; 5 Department of Medicine, Royal Perth Hospital, Perth, Australia; 6 GlaxoSmithKline, Upper Providence, PA, United States of America; 7 GlaxoSmithKline, London; 8 GlaxoSmithKline, Stevenage, United Kingdom; 9 Department of Haematology, Sir Charles Gairdner Hospital, Perth, Australia

Background: Belantamab mafodotin (belamaf; BLENREP) is a B-cell maturation antigen-targeting antibody–drug conjugate approved for patients with RRMM as monotherapy at 2.5 mg/kg Q3W. Preclinical data demonstrate synergy between belamaf and lenalidomide (Len), suggesting added benefit when combined with standard of care such as Len + dexamethasone (Dex).

Aims: DREAMM-6 ( NCT03544281 ) Arm A is evaluating belamaf in combination with LenDex in patients with RRMM.

Methods: This ongoing, two-part, two-arm, open-label study included patients with RMMM previously treated with ≥1 line of therapy (LOT). Patients received 4 belamaf doses/schedules (1.9 mg/kg Q8W or Q4W; 2.5 mg/kg Q4W or Q4W SPLIT dose [50% on Days (D)1, 8] IV) in combination with Len (20 mg PO D1–21) and Dex (20 mg PO/IV D1, 8, 15, 22). Primary objectives were safety (including treatment-related [TR]AEs related to the combination belamaf and LenDex), tolerability, and efficacy (including overall response rate [ORR] defined as ≥partial response).

Results: As of this interim analysis (data cut: July 23, 2021), 45 patients received ≥1 dose (12 at 1.9 mg/kg Q8W; 4 at 1.9 mg/kg Q4W; 16 at 2.5 mg/kg Q4W; 13 at 2.5 mg/kg Q4W SPLIT). Across cohorts, the median age was 68 years (range: 36–80). Thirteen patients (29%) had high-risk cytogenetics and 6 (13%) had extramedullary disease. Median prior LOT was 3 (range: 1–11) and 26 (58%) had prior Len treatment.

The median duration of follow-up and ORR ranged across cohorts (Table). The median duration of response was only reached in the 1.9 mg/kg Q4W cohort (11.1 mo [95% CI: 3.7–not reached [NR]). At the time of data cut, median progression-free survival was not reached in the 1.9 mg/kg Q8W or 2.5 mg/kg Q4W cohorts. Gr ≥3 TRAEs occurred in 42–85%. Gr ≥3 keratopathy occurred in 0 patients in 1.9 mg/kg Q8W, 1 patient (25%) in 1.9 mg/kg Q4W, 8 patients (50%) in 2.5 mg/kg Q4W, and 6 patients (46%) in 2.5 mg/kg Q4W SPLIT cohorts.

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Summary/Conclusion: Belamaf + LenDex had a tolerable safety profile, with no new safety signals identified in patients with RRMM. AEs, including keratopathy, were common but manageable with dose modifications. Encouraging clinical activity is observed with this combination in patients with RRMM. Follow-up/correlative studies are ongoing.

P942: DREAMM-9: PHASE I STUDY OF BELANTAMAB MAFODOTIN PLUS STANDARD OF CARE IN PATIENTS WITH TRANSPLANT-INELIGIBLE NEWLY DIAGNOSED MULTIPLE MYELOMA

S. Z. Usmani 1,* , M. Mielnik 2 , Y. Koh 3 , A. Alonso Alonso 4 , X. Leleu 5 , H. Quach 6 , C.-K. Min 7 , W. Janowski 8 , A.-O. Abdallah 9 , M. Garg 10 , I. Sandhu 11 , E. M. Ocio San Miguel 12 , A. Oriol 13 , P. Rodriquez-Otero 14 , K. Ramasamy 15 , K. Weisel 16 , B. Besemer 17 , M. Cavo 18 , X. L. Zhou 19 , M. C. Kaisermann 20 , C. M. Bego Marques 21 , D. Williams 20 , F. Carreno 20 , B. E. Kremer 20 , I. V. Gupta 20 , M. Hus 2

1 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 2 Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin, Lublin, Poland; 3 Seoul National University Hospital, Seoul, South Korea; 4 University Hospital Quirón Madrid, Madrid, Spain; 5 CHU de Poitiers, Poitiers, France; 6 St.Vincent’s Hospital Melbourne, Melbourne, Australia; 7 Catholic University of Korea Seoul St. Mary’s Hospital, Seoul, South Korea; 8 Calvary Mater Newcastle, Newcastle, Australia; 9 University of Kansas, Kansas City, KS, United States of America; 10 Leicester Royal Infirmary, Leicester, United Kingdom; 11 University of Alberta, Edmonton, Canada; 12 Hospital Universitario Marqués de Valdecilla (IDIVAL), Universidad de Cantabria, Santander; 13 Institut Català d’Oncologia and Institut Josep Carreras - Hospital Universitari Germans Trias i Pujol (HUGTP), Badalona; 14 Department of Hematology, Clínica Universidad de Navarra, Pamplona, Spain; 15 Churchill Hospital, Headington, Oxford, United Kingdom; 16 University Medical Center Hamburg-Eppendorf, Hamburg; 17 University of Tübingen, Tübingen, Germany; 18 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istitutodi Ematologia “Seràgnoli”, Universitàdegli Studi di Bologna, Bolonga, Italy; 19 GlaxoSmithkline, Waltham, MA; 20 GlaxoSmithKline, Upper Providence, PA, United States of America; 21 GlaxoSmithKline, Barcelona, Spain

Background: The bortezomib, lenalidomide, and dexamethasone (VRd) regimen is a standard of care for newly diagnosed multiple myeloma (NDMM). Belantamab mafodotin (belamaf) is a B-cell maturation antigen-binding antibody-drug conjugate that eliminates myeloma cells by a multimodal mechanism: direct cell kill and anti-myeloma tumor immune response. Belamaf has demonstrated deep and durable responses as a monotherapy in the DREAMM-2 study of patients (pts) with relapsed/refractory multiple myeloma (RRMM). Preclinical evidence of belamaf in combination with bortezomib or lenalidomide suggests enhanced anti-myeloma activity, providing rationale for this treatment combination.

Aims: To evaluate the safety and tolerability of this combination in adult pts with transplant-ineligible (TI) NDMM and establish the recommended Phase III dose.

Methods: DREAMM-9 ( NCT04091126 ) is an ongoing Phase I, open-label, randomized study of belamaf + VRd. The belamaf dose cohorts currently being evaluated are Cohort 1 (1.9 mg/kg Q3/4W), Cohort 2 (1.4 mg/kg Q6/8W), Cohort 3 (1.9 mg/kg Q6/8W), Cohort 4 (1.0 mg/kg Q3/4W), and Cohort 5 (1.4 mg/kg Q3/4W). Belamaf is given with VRd Q3W until Cycle 8, and with Rd Q4W thereafter. After evaluation of safety data for Cohort 1, Cohorts 2–5 were opened in parallel and enrolled pts were randomized 1:1:1:1. Primary endpoint is safety. Secondary endpoints include efficacy, tolerability, and pharmacokinetics (PK).

Results: As of data cutoff (07 Dec 2021), 64 pts were analyzed across all cohorts. Median age (range) was 73.0 (51–88) years, 55% were male, 80% were white, 8% had extramedullary disease, 59% were International Staging System stage II or III, 20% had amp1q, and 17% had high-risk cytogenetics (≥1 of: t(4;14), t(14;16), del17p). The median duration of follow-up varied: Cohort 1 (17.4 months [mo]), Cohort 2 (5.9 mo), Cohort 3 (6.1 mo), Cohort 4 (4.7 mo), Cohort 5 (5.8 mo). Median number of belamaf cycles were: Cohort 1 (6), Cohort 2 (3), Cohort 3 (3.5), Cohort 4 (4.5), and Cohort 5 (5). Most common adverse events (AEs) across cohorts included thrombocytopenia (49%), constipation (43%), diarrhea (32%), and peripheral sensory neuropathy (30%). AEs related to study treatment were experienced by 61 (97%) pts. Belamaf-related grade 3/4 AEs occurred in 24 (38%) pts. Belamaf dose reductions occurred in 11 (18%) pts, with dose delays in 10 (16%) pts. Three pts experienced a fatal severe AE (unrelated to study treatment); 2 due to COVID-19 infection, 1 due to pancreatic adenocarcinoma. Early deep responses were observed; 67–92% pts achieved ≥very good partial response (VGPR) ( Table ), with median time to VGPR of 2.1–2.9 months across cohorts. Of pts with ≥VGPR, 17 were minimal residual disease (MRD) negative, 10 in Cohort 1. As of data cutoff, 8–75% of pts achieved best response of complete response (CR) or stringent CR (sCR). Grade 3 corneal exam findings were reported in 25–58% of pts; grade 3 visual acuity changes were reported in 21–75% of pts. No grade 4 corneal exam findings or visual acuity changes were reported in pts receiving belamaf Q6/8W, compared with 0–17% and 0–8%, respectively, in the Q3/4W cohorts. Belamaf PK profile was similar to that in pts with RRMM, accounting for baseline characteristics.

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Summary/Conclusion: Belamaf + VRd demonstrated high response rates in pts with TI NDMM, with a high rate of MRD negativity indicating deep responses. No new safety signals were observed relative to DREAMM-2. Study is ongoing to evaluate the safety and efficacy of variable dose intensities of belamaf in combination with VRd.

P943: SURVIVAL OUTCOMES OF PATIENTS WITH MULTIPLE MYELOMA IN FRANCE: A COHORT STUDY USING THE FRENCH NATIONAL HEALTHCARE DATABASE (SNDS)

X. Leleu 1,* , B. Gorsh 2 , A. Bessou 3 , P. Paka 2 , J. De Nascimento 3 , X. Colin 4 , S. Landi 5 , P. F. Wang 2

1 Department of Hematology, Centre Hospitalier Universitaire, Université de Poitiers, Poitiers, France; 2 GlaxoSmithKline, Upper Providence, PA, United States of America; 3 IQVIA, Paris; 4 GlaxoSmithKline, Rueil-Malmaison, France; 5 GlaxoSmithKline, Research Triangle Park, NC, United States of America

Background: While treatment options for multiple myeloma (MM) have expanded with the introduction of novel therapies, it remains unclear how this has translated into better patient outcomes. Although some patients can achieve long-term remission, MM remains incurable. In oncology, overall survival (OS) is the gold standard for measuring the clinical benefit of an agent. Real-world studies in several countries have shown increases in OS in recent years for patients with MM; up-to-date real-world data on MM survival in France are limited.

Aims: To assess the OS of patients with MM in France from diagnosis and by line of therapy (LOT) and post triple-class exposure (TCE). Secondary aims were survival outcomes by different subpopulations based on standard of care (SoC) treatment regimens and transplant status.

Methods: This longitudinal retrospective cohort study used data from the French National Healthcare database (Système National des Données de Santé – SNDS) from Jan 2013‒Dec 2019, which included records of outpatient claims, hospital discharges and death registry, demographics, and reimbursement data for hospital and outpatient services. Eligible patients (≥18 years) had ≥2 records of an MM diagnosis (ICD-10 codes C90/C90.0), and/or long-term disease during the study period; ≥1 dispensation/administration of MM drug treatment and date of death available.

LOT was algorithmically defined based on drug regimen, time since administration, and gap between regimens. OS was analyzed from five time points: 1) from time of diagnosis; 2) from the start of line of LOT1 to LOT4; 3) from the start of first TCE (defined as prior exposure to a proteasome inhibitor, an immunomodulatory agent and an anti-CD38 monoclonal antibody); 4) from the subsequent treatment following TCE, and 5) from the subsequent treatment/no treatment following TCE and LOT5+ (indexed at the date of the last observed treatment). Patient OS was assessed by Kaplan–Meier method and death rates were calculated.

Results: A total of 14,309 patients were included in this analysis. Median age was 71 years at diagnosis and 51% of patients were male. Patient survival outcomes deteriorated as their LOT advanced ( Figure ). Median OS from initial diagnosis was >5 years with a slight increase for patients diagnosed more recently (63.8 months for patients diagnosed in 2017 or later vs 60.8 months in those diagnosed in 2014 or later). From LOT1 to LOT4 OS decreased from 61 to 14.8 months or about 30% to 50% with each additional LOT. At the time of first TCE, daratumumab monotherapy/combination therapy were the most common regimens. Median OS following TCE was 3.8 months. Retreatment with previous agents were the SoC following TCE. Among TCE patients who had 4 prior LOTs, 43% of them received a subsequent LOT with a median OS estimate of 8.2 months. For those who discontinued treatment, median OS was only 1 month, indexing at the date of the last observed treatment.

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Summary/Conclusion: Patients with MM experience worsening survival outcomes with increasing time from diagnosis and with subsequent LOT. Prognosis was especially poor among TCE patients who received subsequent therapy and worse for those who did not receive additional treatment. Despite therapeutic advances and slight increase in OS for patients diagnosed more recently, an unmet need for improved access to novel therapies remains, especially in those TCE patients.

P944: A PHASE I STUDY OF C-CAR088, A NOVEL HUMANIZED ANTI-BCMA CAR T CELL THERAPY IN RELAPSED/REFRACTORY MULTIPLE MYELOMA

X. Qu 1,* , G. An 2 , W. Sui 2 , T. Wang 2 , X. Zhang 3 , J. Yang 3 , Y. Zhang 4 , L. Zhang 4 , D. Zhu 5 , J. Huang 5 , S. Zhu 5 , X. Yao 5 , J. Li 5 , C. Zheng 5 , S. Chen 5 , K. Zhu 6 , Y. Wei 5 , X. Lv 5 , L. Lan 5 , Y. Yao 5 , D. Zhou 4 , P. Lu 3 , L. Qiu 2 , J. Li 1

1 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Nanjing; 2 Department of Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin; 3 Department of Hematology, Hebei Yanda Lu Daopei Hospital, Langfang; 4 Department of Hematology, Peking Union Medical College Hospital, Beijing; 5 Cellular Biomedicine Group Inc, Shanghai; 6 University of Maryland School of Medicine, Maryland, China

Background: Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor-T cell (CAR T) therapy shows remarkable efficacy in patients with relapsed or refractory multiple myeloma (RRMM).

Aims: C-CAR088, a novel second-generation humanized anti-BCMA CAR T cell therapy, was developed.

Methods: This phase I dose-escalation and expansion study assessed the safety and efficacy of three dosages of C-CAR088 in patients with RRMM. As of July 2, 2021, 31 patients had been infused with C-CAR088.

Results: Any grade cytokine release syndrome (CRS) developed in 29 patients (93.5%), and grade 3 CRS occurred in three patients (9.7%). One patient from the high-dose group (4.5-6.0 × 10 6 CAR T cells/kg) developed grade 1 neurotoxicity. No dose-limiting toxicities were observed in any dose group, and all adverse events were reversible after proper management. The overall response, stringent complete response, complete response (CR), and very good partial response rates were 96.4%, 46.4%, 10.7%, and 32.1%, respectively. The CR rate in the medium-dose (3.0 × 10 6 CAR T cells/kg) and high-dose (4.5-6.0 × 10 6 CAR T cells/kg) groups was 54.5% and 71.4%, respectively. In the CR group, 15 (93.7%) patients achieved minimal residual disease (MRD) negativity (test sensitivity >1/10 -5 ). All seven patients with double-hit or triple-hit multiple myeloma achieved MRD-negative CR.

Summary/Conclusion: Our findings demonstrate the manageable safety profile and potential efficacy of C-CAR088.

P945: SUBCUTANEOUS ISATUXIMAB ADMINISTRATION BY AN ON-BODY DELIVERY SYSTEM IN COMBINATION WITH POMALIDOMIDE-DEXAMETHASONE IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS: INTERIM PHASE 1B STUDY RESULTS

H. Quach 1,* , G. Parmar 2 , E. M. Ocio 3 , H. M. Prince 4 , A. Oriol 5 , N. Tsukada 6 , K. Sunami 7 , P. Bories 8 , C. Karanes 9 , S. Madan 10 , D. Semiond 11 , M. Inchauspe 12 , S. Macé 13 , P. B. Musholt 14 , F. Suzan 13 , P. Moreau 15

1 Clinical Haematology Service, St Vincent’s Hospital, University of Melbourne, Vic; 2 Illawarra Cancer Care Centre, Wollongong, NSW, Australia; 3 Hospital Universitario Marqués de Valdecilla (IDIVAL), Universidad de Cantabria, Santander, Spain; 4 Molecular Oncology and Cancer Immunology, Epworth Healthcare and University of Melbourne, Melbourne, Vic, Australia; 5 Institut Català d’Oncologia and Institut Josep Carreras, Hospital Germans Trias i Pujol, Barcelona, Spain; 6 Department of Hematology, Japanese Red Cross Medical Center, Tokyo; 7 National Hospital Organization Okayama Medical Center, Okayama, Japan; 8 Early Phase Unit, Institut Claudius Regaud, Institut Universitaire du Cancer Toulouse, Toulouse, France; 9 Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, CA; 10 Banner MD Anderson Cancer Center, Gilbert, AZ; 11 Sanofi, Cambridge, MA, United States of America; 12 IT&M Stats for Sanofi, Neuilly sur-Seine; 13 Sanofi Research & Development, Chilly-Mazarin, France; 14 Sanofi Research & Development, Frankfurt, Germany; 15 Department of Hematology, University Hospital of Nantes, Nantes, France

Background: Intravenous (IV) isatuximab (Isa) + pomalidomide-dexamethasone (Pd) is approved for the treatment of relapsed/refractory multiple myeloma (RRMM) patients (pts). Subcutaneous (SC) delivery would optimize convenience of administration. Prior results showed that SC Isa administered by syringe pump has efficacy and safety profiles comparable to IV Isa; the recommended Phase 2 dose (RP2D) was 1400 mg (IMW21 P-207).

Aims: This multicenter Phase 1b study evaluated safety, pharmacokinetics (PK), and efficacy of SC vs IV Isa + Pd in RRMM pts after ≥2 prior treatment lines.

Methods: Pts were randomized 2:1 to SC1000 mg or IV 10 mg/kg and to SC1400 mg or IV. An expansion cohort was later implemented with SC Isa administered at the RP2D via on-body delivery system (OBDS), a wearable bolus injector applied to the abdomen by a healthcare professional. Primary endpoints (EPs) were safety, including injection site reactions (ISRs), and PK. Main secondary EPs were overall response rate (ORR) and progression-free survival (PFS).

Results: Fifty-six pts were randomized and treated: 12 Isa IV, 12 Isa SC1000, 10 Isa SC1400, and 22 OBDS pts. At study entry, ISS stage II–III was 67% in IV, 33% in SC1000, 60% in SC1400, and 50% in OBDS pts. On Jan 20, 2022, 33% IV, 25% SC1000, 50% SC1400, and 86% OBDS pts remained on treatment. Due to sequential accrual, median follow-up (FU) was longer in IV (20.6 mo) and SC1000 (23.8 mo) than SC1400 (18.1 mo) and OBDS (6.5 mo) pts. Infusion reactions (IRs) were infrequent (≤10% in each cohort, all Grade [G] 2), only at first IV or SC infusion/injection, with no IRs in OBDS. Local tolerability of OBDS was very good, with 5 (22.7%) pts experiencing 7 ISR episodes, all G1, out of 305 administrations (2.3%): 5 injection site erythemas, 1 injection site hemorrhage, and 1 injection site induration. Median duration of OBDS injection was 10 min. Incidence of G4 neutropenia was lower in SC1000 (42%) vs the other cohorts (55–60%). The lower percentage of ≥G3 treatment-related adverse events in OBDS (77%) vs the other cohorts (≥80%) may be due to shorter FU. Best overall responses are shown in table; longer FU is needed for OBDS pts. Median PFS was 22 mo in IV, 12.5 mo in SC1000, and not reached in SC1400 and OBDS pts. PK and CD38 receptor occupancy results in OBDS pts are consistent with those observed in SC1400 with pump.

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Summary/Conclusion: SC Isa administered by OBDS shows a safety profile consistent with IV administration with no IRs and excellent local tolerability. Efficacy in the SC cohorts was comparable to the Phase 3 ICARIA results. Isa SC administration by OBDS is well tolerated, requires a short duration of injection, and provides a convenient hands-free option.

Clinical trial information: NCT04045795 . Research Sponsor: Sanofi.

P946: A PHASE I/II SINGLE ARM STUDY OF BELANTAMAB MAFODOTIN, CARFILZOMIB AND DEXAMETHASONE IN PATIENTS WITH RELAPSED MULTIPLE MYELOMA: AMARC 19-02 BELACARD STUDY.

M. Lasica 1,2,* , A. Spencer 2,3,4 , P. Campbell 2,5,6 , C. Wallington-Gates 2,7,8 , N. Wong Doo 2,9,10 , W. Janowski 2,11 , G. McCaughan 2,12,13 , A. Puliyayil 2,14 , F. Yuen 2,3 , K. Le 2,3 , J. Reynolds 2,3,4 , H. Quach 1,2,15

1 Department of Haematology, St Vincent’s Hospital Melbourne, Fitzroy; 2 Australasian Myeloma Research Consortium; 3 Department of Haematology, Alfred Hospital, Melbourne; 4 Faculty of Medicine, Monash University, Clayton; 5 Department of Haematology, Barwon Health, Geelong; 6 Deakin University, Burwood; 7 Flinders University; 8 Department of Haematology, Flinders Medical Centre, Bedford Park; 9 Department of Haematology, Concord Hospital, Concord; 10 University of Sydney, Camperdown; 11 Department of Haematology, Calvary Mater Newcastle, Newcastle; 12 Department of Haematology, St Vincent’s Hospital Sydney; 13 St Vincent’s Clinical School, University of New South Wales, Sydney; 14 Department of Haematology, Border Cancer Hospital, Albury; 15 University of Melbourne, Parkville, Australia

Background: Belantamab Mafodotin (Belamaf), a first in class anti- B-cell maturation antigen (BCMA) antibody-drug conjugate is efficacious in patients with triple-class exposed/refractory multiple myeloma (RRMM). Combining Belamaf (B) with carfilzomib and dexamethasone (Kd) is potentially synergistic through direct myeloma-cell kill and immune response against myeloma.

Aims: To characterize the safety, tolerability, and preliminary efficacy of BelaMaf in combination with carfilzomib and dexamethasone (Kd) in patients with early relapsed MM.

Methods: BelaCarD is an ongoing, two-part, single-arm, multicentre phase I/II study evaluating an extended schedule of B every 8 weeks in combination with Kd in patients with RRMM after 1-3 prior lines of treatment. Prior refractoriness to proteasome inhibitors was allowed. Here we report a pre-planned analysis of the safety run-in phase of the first 10 patients who have completed at least 1 treatment-cycle. Belamaf (2.5mg/kg) was administered on day (D) 1 of every 2 nd 28-day cycle, K 70mg/m2 iv D1 (20mg/m2 on C1D1), D8 and D15 of every cycle and dexamethasone 40mg weekly (20mg for patients >75 years). Treatment was continued until disease progression. Adverse events (AEs) were graded per CTCAEv4, except for corneal AEs which were graded by the pre-specified keratopathy and visual acuity (KVA) scale. Response was assessed by the International Myeloma Working Group (IMWG) criteria.

Results: At cut-off (Feb 3rd 2022), 19 patients had received B-Kd. The median age of the 10 safety run-in patients was 65 years (range, 48-77); One, five and four patients had 3, 2 and 1 prior lines of therapy respectively including (exposed/refractory %) bortezomib (100/30%), carfilzomib (10/0%) lenalidomide (60/50%), pomalidomide (10/10%), ASCT (70/0%), anti-CD38 monoclonal Ab (mAb) (40/40%). The median number of treatment-cycles was 7 (5-11). The median number of cycles commenced was 9 (range, 2-13). The most frequent AE during cycle 1 was thrombocytopenia (all grade 30%, Gr 3/4 20%) and blurred vision (all grade 20%, Gr 3/4 0%) One patient experienced Gr 4 neutropenia. From cycle 2 onwards, the most frequent AEs included blurred vision (all grade 40%, Gr 3/4 20%), peripheral neuropathy (all grade 30%, Gr 3/4 10%), upper respiratory tract infection (all grade 30%, Gr 3/4 20%), dry eyes (all grade 20%, Gr 3/4 0%), neutropenia (all grade 20%, Gr 3/4 10%) and nausea (all grade 20%, Gr 3/4 0%). Six patients had an SAE, one was related to Belamaf (Gr 1 infusion reaction). Keratopathy occurred in 8 patients; grade 1, 20%; grade 2, 0%; grade 3, 60%. Decline in best corrected visual acuity (BCVA) by at least 2 lines occurred in 8 patients (Gr 3 n=6, Gr 2 n=2). One patient discontinued therapy due to corneal toxicity. Two patients died (progressive disease n=1, unrelated cause n=1).

Of the 10 patients in the safety run-in, 9 achieved a PR or better (CR=3, VGPR=3, PR=3) and 2 are known to have subsequently progressed (Figure 1). At estimated median potential follow-up of 9.95 months, median PFS had not been reached (95%CI: 1.08 –NR).

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Summary/Conclusion: B-Kd with an extended B schedule, has a safety profile that is in keeping with that expected for each individual drug. Deep responses were seen. Recruitment is ongoing in an expansion phase based on the preliminary safety and efficacy.

P947: COMPARATIVE EFFECTIVENESS OF ORAL IXAZOMIB-LENALIDOMIDE-DEXAMETHASONE (IRD) AFTER INITIAL BORTEZOMIB (V)-BASED INDUCTION VS PARENTERAL V-BASED THERAPY IN NEWLY DIAGNOSED MULTIPLE MYELOMA (NDMM)

R. M. Rifkin 1,* , C. L. Costello 2 , R. E. Birhiray 3 , S. Kambhampati 4 , J. Richter 5 , R. Abonour 6 , H. C. Lee 7 , Y. J. Kim 8 , K. Ren 9 , D. M. Stull 10 , D. Cherepanov 9 , K. Bogard 10 , S. J. Noga 9 , S. Girnius 11

1 Rocky Mountain Cancer Centers/US Oncology Research, Denver; 2 Moores Cancer Center, University of California San Diego, La Jolla; 3 Hematology Oncology of Indiana/American Oncology Network, Indianapolis; 4 Kansas City Veterans Affairs Medical Center, Kansas City; 5 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York; 6 Indiana University School of Medicine, Indianapolis; 7 MD Anderson Cancer Center, Houston, United States of America; 8 Data Analytics, Evidera, St-Laurent, Canada; 9 Takeda Development Center Americas, Inc. (TDCA); 10 Takeda Pharmaceuticals U.S.A., Inc., Lexington; 11 TriHealth Cancer Institute, Cincinnati, United States of America

Background: Long-term proteasome inhibitor (PI)-based treatment can improve outcomes for patients (pts) with multiple myeloma (MM). However, prolonged parenteral PI therapy (e.g. with V) can be challenging to achieve in routine clinical practice, and outcomes for pts are often poorer in this setting compared with clinical trials. The phase IV, community-based, single-arm US MM-6 study ( NCT03173092 ) is assessing in -class transition ( i CT) from V-based induction to all-oral IRd in transplant ineligible NDMM pts treated in routine clinical practice, with the objective of increasing the duration of PI-based treatment while maintaining quality of life. INSIGHT MM is the largest global, prospective, observational study of MM pts (>4,200), and provided a subset of patients as the comparator cohort. This enabled assessment of i CT vs V-based therapy in NDMM pts in routine clinical practice in the US.

Aims: To examine the comparative effectiveness of IRd following initial V-based induction (3 cycles; US MM-6 pts; ‘IRd’ cohort) vs continued V-based therapy (INSIGHT MM pts; ‘V-based’ cohort) in NDMM pts.

Methods: A secondary analysis of non-transplant eligible US NDMM pts with ≥stable disease after 3 cycles of V-based induction and baseline Eastern Cooperative Oncology Group performance status (ECOG PS) of 0, 1 or 2 from the US MM-6 (Manda CLML 2020) and INSIGHT MM (Costello Future Onc 2019) studies was performed. Study outcomes included first-line duration of treatment (DOT), overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and reasons for treatment discontinuation. All analyses were weighted using the inverse probability of treatment weighting (IPTW) approach to reduce the imbalance of potential confounding factors between the two cohorts (adjusted analyses). Kaplan–Meier methods were used to examine DOT, PFS, OS, and associated 95% confidence intervals (CIs); the log-rank test was used to compare distribution of time to events. The Clopper-Pearson method was applied to estimate 95% CIs for ORR. Statistical significance was evaluated at alpha=0.05.

Results: 100 pts from the IRd cohort (MM-6) and 111 pts from the V-based cohort (INSIGHT) were included. After IPTW, in the IRd vs V-based cohorts: median age was 75.0 vs 74.8 yrs; 56.7 vs 51.3% of pts were male; 37.4 vs 29.1% had an ECOG PS of ≥2; 48.8 vs 41.4% had International Staging System stage III at initial diagnosis, and 79.5/17.7/2.8 vs 77.3/19.5/3.1% pts had received VRd/ V-cyclophosphamide-d (VCd)/ VRCd as initial induction therapy.

Adjusted ORRs in the IRd vs V-based cohorts were 74.1 (95% CI 66.0–82.2) vs 57.5% (95% CI 47.9–67.1; p<0.0001). After a median follow-up of 20.3 and 15.8 months in the IRd and V-based cohorts, respectively, DOT was 10.8 (95% CI 6.5–24.4) vs 5.3 months (95% CI 4.3–7.0; p<0.0001) (see Figure). Median PFS was not estimable (NE) in either cohort; 24-month PFS rates were 85.7 (95% CI 68.1–94.0; IRd cohort) vs 76.5% (95% CI 62.6–85.8; V-based cohort). Median OS was NE in either cohort; 24-month OS rates were 94.0 (95% CI 77.7–98.5; IRd cohort) vs 84.9% (95% CI 70.6–92.6; V-based cohort). In the IRd and V-based cohorts, 16.8 and 16.9% of pts discontinued IRd and V, respectively, due to an adverse event.

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Summary/Conclusion: US MM-6 NDMM pts who transitioned to IRd after 3 initial cycles of V-based induction had a significantly higher ORR and longer DOT compared with pts who received continued V-based therapy in INSIGHT MM. The results suggest that i CT from continued V-based therapy to all-oral IRd may improve outcomes in pts treated at community oncology clinics.

P948: INCIDENCE AND CLINICAL OUTCOME OF SARS-COV-2 INFECTION AFTER VACCINATION IN PATIENTS WITH MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS)

N. Sgherza 1,* , P. Curci 1 , R. Rizzi 1,2 , D. Roccotelli 2 , M. Croce 2 , M. Avantaggiato 2 , L. Ruga 2 , A. Vitucci 1 , A. Palma 1 , D. Di Gennaro 2 , P. Musto 1,2

1 Hematology and Bone Marrow Transplantation Unit, AOUC Policlinico, Bari, Italy; 2 Department of Emergency and Organ Transplantation, “Aldo Moro” University School of Medicine, Bari, Italy

Background: Our group recently reported that MGUS patients investigated during the early waves of pandemic, when vaccines were still not available, neither have an increased risk of contracting SARS-CoV-2, infection, nor show poorer COVID-19 outcomes with respect to healthy controls (Sgherza N. et al. Haematologica. 2022).

Aims: Aiming to specifically address the clinical effects of vaccines, we compared incidence and outcome of SARS-CoV-2 infection of 1,454 previously described, not vaccinated MGUS patients (91 of whom were SARS-CoV-2 positive), with those observed in a similar population during the national vaccination campaign.

Methods: We obtained retrospective information from 86 individuals found to be SARS-CoV-2 positive among 1,265 MGUS patients analyzed after at least two doses of anti-SARS-CoV-2 vaccine received between April 2021, and January, 2022. Seventy-one subiects positive before vaccination or after only one dose were escluded from analysis.

Results: The mean age of this group was 65.7 +/- 13.3 years (range 31-90); 32 patients were female (37%), and 54 were male (63%). About MGUS-subtypes, the most frequent one was IgG-kappa (n=35; 40.7%), followed by IgG-lambda (n=28; 32.5%), IgM-kappa (n=11; 12.8%) and others (n=12; 14%). Most of patients (81/46, 94.2%) were at low or low-intermediate risk, according to Mayo Clinic prognostic model. Thirty-seven (43%) patients developed SARS-CoV-2 infection after two doses (9, 26 and 2 patients receiving ChAdOx1-S, BNT162b2 mRNA and mRNA-1273 vaccines, respectively), fourty-nine (57%) after three doses (BNT162b2 mRNA or mRNA-1273 as “booster” dose, repectively). The mean number of days between last dose of vaccine and SARS-CoV-2 infection was 103.04 +/-80.28 (range 2-285). The two populations of SARS-CoV2 positive MGUS patients (before and after vaccination) were comparable for age, sex and presence of co-morbidities (data not shown). Overall, rates of symptoms (59.3% vs 26.7%), hospitalization (20.9% vs 2.3%), hospitalization in Intensive Care Unit (11% vs 1.2%) and rate of deaths (8.8% vs 1.2%) were significantly higher in still not vaccinated MGUS patients than in those who had received vaccines (Table 1).

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Summary/Conclusion: Our data indicate that the incidence of SARS-CoV-2 infection is not significantly reduced in fully vaccinated MGUS patients (even after three doses), likely also because of the higher diffusion capacity of the recently recognized Omicron viral variants. However, as observed in normal population and in other hematological contexts, the clinical outcome of COVID-19 may be significantly improved after vaccination in these patients, with symptoms present in less of one third of subjects and no case of hospitalization or death in our series. These observations support extensive vaccination programs in patients with MGUS.

P949: A PHASE 2 TRIAL OF ELRANATAMAB, A B-CELL MATURATION ANTIGEN (BCMA)-CD3 BISPECIFIC ANTIBODY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA: INITIAL SAFETY RESULTS FOR MAGNETISMM-3

A. M. Lesokhin 1,* , B. Arnulf 2 , R. Niesvizky 3 , M. Mohty 4 , N. J. Bahlis 5 , M. H. Tomasson 6 , P. Rodrίguez-Otero 7 , H. Quach 8 , N. S. Raje 9 , S. Iida 10 , M.-S. Raab 11 , A. Czibere 12 , S. Sullivan 12 , E. Leip 12 , A. Viqueira 13 , X. Leleu 14

1 Division of Hematology and Oncology, Memorial Sloan Kettering Cancer Center/Weill Cornell Medical College, New York, United States of America; 2 Hôpital Saint-Louis, Paris, France; 3 Weill Cornell Medical College - New York Presbyterian Hospital, New York, United States of America; 4 Sorbonne University, Hôpital Saint-Antoine, and INSERM UMRs938, Paris, France; 5 Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada; 6 Holden Comprehensive Cancer Center, University of Iowa, Iowa City, United States of America; 7 Clinica Universidad de Navarra, Madrid, Spain; 8 University of Melbourne, Melbourne, Australia; 9 Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, United States of America; 10 Department of Hematology & Oncology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 11 Heidelberg Myeloma Center, Department of Hematology/Oncology, Heidelberg University Hospital, Heidelberg, Germany; 12 Pfizer Inc, Cambridge, United States of America; 13 Pfizer SLU, Madrid, Spain; 14 Centre Hospitalier Universitaire de Poitiers, Poitiers, France

Background: Elranatamab (PF-06863135) is a humanized bispecific antibody that targets both BCMA-expressing multiple myeloma (MM) cells and CD3-expressing T cells. MagnetisMM-3 ( NCT04649359 ) is an open-label, multicenter, non-randomized, phase 2 study to evaluate the safety and efficacy of elranatamab monotherapy in patients with relapsed/refractory (R/R) MM.

Aims: Initial safety results from the MagnetisMM-3 trial in patients with R/R MM and no prior BCMA-targeted treatment are presented.

Methods: MagnetisMM-3 enrolled patients who are refractory to at least 1 proteasome inhibitor, 1 immunomodulatory drug, and 1 anti-CD38 antibody. Patients were assigned to 1 of 2 independent, parallel cohorts: those naïve to BCMA-directed therapies (Cohort A) and those with previous exposure to BCMA-directed antibody-drug conjugates or CAR-T cells (Cohort B). Patients received subcutaneous elranatamab 76 mg QW on a 28-day cycle with a 2-step-up priming dose regimen administered during the first week. Dose modifications were permitted for toxicity. Treatment-emergent adverse events (TEAEs) were graded by CTCAE (v5.0), and cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) by ASTCT criteria.

Results: As of the data cutoff on Dec 31, 2021, 60 patients in Cohort A had received ≥1 dose of elranatamab; the last patient’s first dose was ~2 months prior to the cutoff. Median age was 69.0 years (range, 44−89), 48.3% were male, 63.3% were white, 18.3% were Asian and 11.7% were Black/African American. At baseline, 60.0% of patients had an ECOG performance status 1−2 and patients had received a median of 5 (range, 2−12) prior therapies. Median duration of elranatamab treatment was 9.57 weeks (range, 0.1−46.1); median relative dose intensity was 87.4% (range, 23.1−101.4).

TEAEs were reported in 100% (Grade [G] 3/4, 75.0%) of patients. Most common (≥30%) hematologic TEAEs were neutropenia (36.7% [G3/4, 35.0%]), anemia (36.7% [G3/4, 30.0%]) and thrombocytopenia (30.0% [G3/4, 21.7%]). Among patients who received the 2-step-up priming regimen (n=56), CRS and ICANS, respectively, were reported in 58.9% (G3/4, 0%) and 3.6% (G3/4, 0%); of those patients, 57.6% (n=19/33) and 100% (n=2/2) received tocilizumab and/or steroids. Most common (≥30%) non-hematologic TEAE, other than CRS/ICANS, was fatigue (31.7% [G3/4, 3.3%]). Infections were reported in 46.7% (G3/4, 18.3%) of patients; most frequently reported were upper respiratory tract infections (11.7% [G3/4, 0%]). Discontinuations due to adverse events were reported in 5.0% of patients. No patients permanently discontinued treatment due to CRS or ICANS. There were 10 deaths; causes were MM progression (n=8), septic shock (n=1) and unknown (n=1). Data will be updated at the time of presentation to include ~90 patients.

Summary/Conclusion: Preliminary results of MagnetisMM-3 in patients with R/R MM and no prior BCMA-targeted treatment suggest that 76 mg QW elranatamab with a 2-step-up priming regimen is well tolerated, with no G ≥3 CRS or ICANS observed.

P950: COMBINED PROTEASOME AND AUTOPHAGY INHIBITION IN RELAPSED/REFRACTORY MULTIPLE MYELOMA – A PHASE I TRIAL OF HYDROXYCHLOROQUINE, CARFILZOMIB AND DEXAMETHASONE

T. S. Slørdahl 1,2,* , F. B. Askeland 3,4 , M. S. S. Hanssen 5 , S. M. Sundt-Hansen 1 , N. T. Vethe 6 , H. Hjorth-Hansen 1,2 , M. H. Fenstad 7 , A. Waage 2 , Ø. Hjertner 1 , F. Schjesvold 3,4 , A. Sundan 2

1 Department of Hematology, St. Olavs Hospital; 2 Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim; 3 Oslo Myeloma Center, Department of Hematology, Oslo University Hospital; 4 KG Jebsen Center for B Cell Malignancies, University of Oslo, Oslo; 5 Department of Ophthalmology, St. Olavs Hospital, Trondheim; 6 Department of Pharmacology, Oslo University Hospital, Oslo; 7 Department of Immunology and Transfusion Medicine, St. Olavs Hospital, Trondheim, Norway

Background: Multiple myeloma (MM) is characterized by malignant cells which produce large amounts of monoclonal immunoglobulin. MM cells are highly sensitive to drugs that target protein degradation. There are two important intracellular pathways for protein degradation (Lamark, 2010). The proteasome is responsible for the degradation of poly-ubiquitinated damaged, modified, misfolded and redundant proteins (Jung, 2009). This pathway of degradation is the target of proteasome inhibitors. The other main pathway is that of autophagy and lysosomal degradation, eliminating protein aggregates and damaged organelles. Hydroxychloroquine (HCQ) is an inhibitor of autophagy. In vitro studies have shown that HCQ potentiates carfilzomib (K) toxicity towards myeloma cells and could therefore be an attractive treatment option in myeloma (Baranowska, 2016). Here we present safety and efficacy data from a phase 1 study on the combination of HCQ, K and dexamethasone (d).

Aims: This study aimed to answer the question whether combined inhibition of the proteasome and the autophagosome is safe and tolerable. The primary endpoint was to establish a maximum tolerated dose (MTD) of the combination HCQ-Kd.

Methods: This phase I, single arm, open label dose escalation study (3 + 3 design), included patients with relapsed and/or refractory MM with at least two prior lines of therapy including bortezomib and an immunomodulatory agent. All patients started a 14-day run-in of HCQ at their intended HCQ dose level (DL) before the first cycle of HCQ-Kd. The HCQ DL were 200 mg (DL1), 400 mg (DL2), 600 mg (DL3), and 800 mg (DL4&5) daily. Intravenous carfilzomib was given in a dose of 20 mg/m 2 in cycle 1 day 1, and thereafter 56 mg/m 2 (DL1-4) and 70 mg/m 2 (DL5) on day 8, 15, and in all subsequent cycles and doses. Oral dexamethasone 40 mg (20 mg pts >75y) was administered on day 1, 8, 15 and 22. Patients were treated with HCQ-Kd for a maximum of six 28-day cycles and continued Kd if still responding thereafter. The dose limiting toxicity (DLT) observation period was 28 days. Adverse events (AE) grade (G) 1-5 were registered from day 1 of run-in cycle and until 90 days post last dose of HCQ.

Results: The clinical trial is completed, and we here report the data for MTD, safety and efficacy. 19 patients were included. One patient withdrew consent prior to first treatment cycle and was therefore not included in MTD and efficacy analyses. Median previous lines of therapy were 4 (2-9), 50% had high-risk cytogenetics. Only one DLT occurred at DL2 (G3 pneumonia), and MTD was not reached. 104 AEs were recorded, of which G1 45%, G2 32%, G3 22% and G4 1%. No deaths occurred. Most common AEs were anemia (74%), neutropenia (42%), thrombocytopenia (19%), skeletal pain (32%), respiratory tract infections (26%), insomnia (26%) and vomiting/nausea (21%). A total of 13 serious adverse events (SAEs) occurred during the study of which 2 led to treatment discontinuation. Responses were seen in 8 of 18 (ORR 44 %) including 3 PR (18%), 3 VGPR (18%), 1 CR (6%), 1 sCR (6%). Clinical benefit rate was 78 % when including 6 MR (33%) (figure 1). One patient reached MRD negativity. 7 patients did not complete all 6 cycles of HCQ-Kd, 5 due to PD and 2 due to SAEs.

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Summary/Conclusion: HCQ given up to 800 mg daily in combination with weekly carfilzomib and dexamethasone is well tolerated in patients with relapsed/refractory multiple myeloma. Adverse events were mostly grade 1 and 2. The study results indicate a meaningful clinical efficacy of the combination.

P951: CLINICAL AND LABORATORY CHARACTERISTICS, COURSE AND OUTCOME OF COVID19 INFECTION IN PATIENTS WITH MULTIPLE MYELOMA

A. Sretenovic 1,* , M. Mitrovic 1 , T. Adzic Vukicevic 2 , Z. Bukumiric 3 , O. Markovic 4 , M. Zdravkovic 4 , D. Antic 5 , J. Bila 1

1 Myeloma department, Clinic of hematology University Clinical centre of Serbia; 2 Pulmology, COVID Hospital “Batajnica”, University Clinical Center of Serbia; 3 Medical Faculty, University of Belgrade; 4 Hematology, Clinical-Hospital Center “Bezanijska Kosa”; 5 Lymphoma department, Clinic of hematology University Clinical centre of Serbia, Belgrade, Serbia

Background: During the Covid19 pandemic, patients with multiple myeloma (MM) are particularly vulnerable due to the characteristics of their disease.

Aims: The aim of study was to analyze clinical and laboratory characteristics, course and outcome of Covid19 infection in patients with multiple myeloma.

Methods: The study included 53 patients with MM and Covid 19 infection, diagnosed during period March 2020 - November 2021 (27 male; 26 female, mean age 62 yrs, range 37- 87 yrs). IgG MM was present in 28pts (53%), IgA in 8 (15%), IgM in 3pts (6%), and BJ in 14 (26%). According to the clinical stage (CS, Durie-Salmon), distribution was as follows: III 44pts (83%); II 4pts (8%); I CS 5pts (9%). Renal impairement existed in 18pts (34%). Regarding ISS score, the group included: ISS1 had 18pts (34%), ISS2 12pts (23%) and 23pts (43%) had ISS3. According to the Revised ISS (R-ISS) score, R-ISS1 was found in 9pts (17%), R-ISS2 in 20pts (58%) and R-ISS3 was present in 13pts (25%). All pts were treated according to National Protocol for the Treatment of Covid19 infection, including: antibiotics in 53pts (100%), corticosteroids in 50pts (94%), low molecular weight heparin in 52pts (98%), and intravenous immunoglobulins in 26pts (49%). During period January 2021 - November 2021, 9 of 24pts (37.5%) were vaccinated against Covid19. Variables of importance were analyzed using descriptive and analytical statistics. All calculations were made in SPSS program version 26.0.

Results: At the moment of detected Covid19 infection, 37pts (70%) had active MM, and 16pts (30%) were in the state of follow-up during remission of disease. Immunoparesis was present in all of 37pts (74%) with active MM. Elevated interleukin-6 (IL-6) was found in 19pts (36%), of which 15pts (78.9%) had active MM. Similarly, elevated d-dimer was found in 46pts (87%), of which 35pts had active disease. During the course of Covid19 infection, pneumonia was registered in 52pts (98%), bleeding in 2pts (4%) and thrombosis in 4pts (8%). Bleeding and thrombosis were registered in patients with active MM. In the Intensive Care Units were treated 7pts (13%), with lethal outcome of 6pts. In the Semi-Intensive Care were treated 36pts (64%). A total of 36pts (64%) were cured and 17pts (36%) died. The average time to recovery of our pts was 14 days (max up to 45 days). After recovery 20pts (55%) had active myeloma, reinfection was found in 5pts (13.5%) and they were in active phase of disease. All of 5pts (10%), who were previously vaccinated against Covid19 during MM remission, completely recovered from Covid19 infection. Additionally, 4pts (8%) were vaccinated after recovery from Covid19 infection.

Patients with active MM had signficantly worse outcome of Covid19 infection, (Chi-Square, p=0.003). Immunoparesis was highly frequent in pts with acitive disease (Fisher Exact Test, p=0.000), as well as elevated d-dimer (Fisher Exact Test, p=0.002). Increased IL-6 was observed in pts with active MM, but statistical significance was not established (Chi-Square p=0.088). The poor treatment outcome of pts with MM and Covid 19 was influenced by: Age (T-Test, p=0.022), Renal impairement (Chi-Square, p=0.032), and high R-ISS score 3 (Mann Whitney, p=0.042).

Summary/Conclusion: A significantly worse outcome of Covid19 infection may be expected in patients with active MM and renal impairement, accompanied with findings of elevated d-dimer and IL-6, as well as immunoparesis and R-ISS score 3. All vaccinated patients recovered from Covid 19 infection.

P952: THE ROLE OF AGE IN THE LONG-TERM TREATMENT OF MULTIPLE MYELOMA - A LONGITUDINAL ANALYSIS OF REGULAR CARE DATA

H. T. Steinmetz 1,* , M. Heinz 1 , U. Totzke 2

1 Onkologie Köln, Outpatient Clinic for Hematology and Oncology, Cologne, Cologne, Germany; 2 TOTZKE & DREHER SCIENTIFIC SA, Basel, Switzerland

Background: Recently, we demonstrated in a longitudinal approach that the percentage of multiple myeloma (MM) patients reaching consecutive treatment lines in regular care is much higher than presumed, i.e. 88%, 66%, 44%, and 30% in 2 nd , 3 rd , 4 th , and 5 th line, respectively (Oncol Res Treat 44, 662-71).

Aims: As the annual risk of death generally increases with age, we investigated here the impact of age on the course of MM treatment.

Methods: MM patients treated in our outpatient clinic in Germany over 8 years were assigned to three groups of equal size according to age at diagnosis. Resulting subgroups were analyzed regarding their outcome and the probabilities of reaching consecutive therapy lines using a linear statistical model, as previously described for the analysis of the whole cohort.

Results: Age groups were <65, 65-74, and ≥75 years. As compared to the two remaining groups, the proportion of male patients and of those with ISS 1 was considerably higher in the youngest age group while the median observation time was on average 7 months shorter in the oldest age group (Tab.).

The probability of reaching consecutive treatment lines decreased in all age groups, but most strikingly in that of the oldest patients (Fig.). Age does not appear to play a major role for reaching subsequent lines at ages up to 75 years though. Up to this age, patients were thus even more likely to reach subsequent lines than previously shown for the total cohort, increasing to about a 50% chance for the 5 th line of therapy. The mean age at death was 83 (SD 4.8) years in the oldest age group, but in none of the age groups, the leading cause of death was MM.

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Summary/Conclusion: Only age beyond 75 years at baseline significantly reduces the probability of reaching 2 nd or higher lines of MM therapy.

P953: PHASE I STUDY OF TQB3602 CAPSULE, AN ORAL PROTEASOME INHIBITOR, IN RELAPSED/REFRACTORY MULTIPLE MYELOMA

W. Tang 1 , Y. Li 1 , X. Zhong 1 , Q. Liang 1 , Y. Liu 2 , Y. Zeng 3 , B. Fang 2 , L. Zheng 4 , T. Niu 1,*

1 Department of Hematology, West China Hospital, Sichuan University, Chengdu; 2 Department of Hematology, Henan Cancer Hospital, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou; 3 Department of Hematology, First Affiliated Hospital of Kunming Medical University, Hematology Research Center of Yunnan Province, Kunming; 4 GCP Center/National Institute of Drug Clinical Trial, West China Hospital, Sichuan University, Chengdu, China

Background: The proteasome inhibitor is an effective treatment strategy for multiple myeloma (MM). TQB3602 is a novel oral proteasome inhibitor derived from multiple rounds of optimization based on ixazomib. It has an addition of an acridine ring to the structure of ixazomib, which maintains high activity and selectivity, resulting in better metabolic stability. In preclinical models of MM, TQB3602 displayed potent kinase inhibiting activity for 20S proteasome enzyme with IC50 15.3 nM. It also inhibited cell proliferation in the MM.1S cell line with IC50 10.1 nM. In the MM.1S MM CB-17 CDX model, TQB3602 also showed antitumor efficacy. TQB3602 represents a promising clinical candidate for treating MM.

Aims: This study presents the first-in-human trial designed to evaluate the safety, tolerance, pharmacokinetics, dose-limiting toxicity and maximum tolerated dose of TQB3602 in relapsed/refractory MM (RRMM).

Methods: This study is a multicenter, open-label, 3 + 3 dose-escalation phase I trial ( NCT04275583 ). Adult patients with RRMM who had received at least two kinds of therapy, including bortezomib, lenalidomide or thalidomide, are enrolled. TQB3602 is orally administered at a dose of 0.5mg, 1mg, 2mg, 3mg, 4mg, 5mg, 5.5mg, 6mg or 7mg on days 1, 8, 15 in 28-day cycle until limiting toxicity or disease progression.

Results: Twenty-five patients with RRMM (16 males, 9 females, median age 65, range 37-73) were enrolled between May 2020 and October 2021, with all patients exposed to bortezomib and three patients exposed to ixazomib. Patients received a median of three cycles of TQB3602 (range 1-11). Grade ≥3 drug-related adverse events included anemia (28%), thrombocytopenia (8%), neutropenia (8%) and diarrhea (4%). No grade ≥3 drug-related peripheral neuropathy occurred. Two dose-limiting toxicities occurred at 7mg, including diarrhea and grade 2 peripheral neuropathy. The maximum tolerated dose was determined as 6mg. TQB3602 was rapidly absorbed, resulting in a time to plasma peak concentration from 0.8 to 1.5 hours. After multiple dosing, the terminal half-life was 23 to 152 hours. Following administration of 6 mg, the mean half-life was approximately 82 hours. TQB3602 slightly accumulated in the human body. At a dose of 6 mg, the AUC last and Cmax were approximately 1.9 times higher on day 15 than day 1. The mean plasma concentration-time profiles of day 1 and day 15 are shown in Figures A and B, and the geometric mean plasma Cmax and AUC last on day 1 and day 15 are shown in Figures C and D. Among 25 response-evaluable patients, 65% of patients achieved stable disease or better.

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Summary/Conclusion: TQB3602 is well tolerated with no severe neuropathy adverse events and has shown preliminary efficacy in patients with RRMM. Based on the efficacy, safety, and pharmacokinetic data, 6mg was recommended as the anticipated therapeutic dose and adopted in an ongoing dose-expansion phase.

P954: ESTABLISHING THE NATIONAL INSTITUTE FOR HEALTH RESEARCH (NIHR) HEALTH INFORMATICS COLLABORATIVE (HIC) MULTIPLE MYELOMA (MM) REGISTRY

S. Tayabali 1,* , S. Moore 2 , M. Jenner 3 , F. Djebbari 2 , L. Romao 1,4 , L. English 1,4 , R. Rodriguez 1,4 , B. Briot Ribeyre 1,4 , G. Roadknight 2,5 , K. Varnai 2,5 , S. Little 2,5 , J. Davies 2,5 , K. Woods 2,5 , C. Davis 6 , F. Borca 6 , J. Olza Meneses 6 , H. Phan 7 , W. K. Wong 1,4 , R. Popat 1

1 University College London Hospitals NHS Foundation Trust, London; 2 Oxford University Hospitals NHS Foundation Trust, Oxford; 3 University Hospital Southampton NHS Foundation Trust, Southampton; 4 NIHR University College London Hospitals Biomedical Research Centre, London; 5 NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford; 6 NIHR Southampton Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust; 7 NIHR Southampton Biomedical Research Centre, University of Southampton, Southampton, United Kingdom

Background: Real world data provides unique insights into the natural history of MM, resource utilisation, adherence to standards and outcomes of treatments outside clinical trials. However, the quality of such initiatives can be poor due to the need for manual data entry, inability to integrate multiple data sources, lack of harmonisation of data across centres and limited generalisability due to centre bias. Exemplar registries collate and transform multidisciplinary routinely collected data from Electronic Health Records (EHRs) to a common data language allowing integration, analysis and expansion.

Aims:

To develop a secure national MM registry beginning with a three site pilot.

Methods:

Potential registry providers were evaluated against the following criteria: previous experience, infrastructure and technical capability, data governance, compliance with legal standards and cost effectiveness. The platform required ethical and data sharing approvals. A common data model was investigated and a myeloma focused multidisciplinary dataset was designed and mapped to source data. Data extraction, transformation and analysis is planned.

Results:

8 providers were assessed for suitability. The NIHR HIC was chosen due to its established data sharing network across 30 UK sites and overarching governance framework. This platform permitted the establishment of harmonised data flow, comparable across sites and retaining clinical interpretability. A minimal myeloma dataset of routinely collected information was agreed by clinicians and data scientists to contain the following fields: baseline and disease characteristics including imaging findings eg: extramedullary/paramedullary disease, skeletal/spinal disease, comorbidities, mode of presentation, cytogenetics, treatments including chemotherapy, stem cell transplants/CAR-T cells, spinal interventions, anti-infectives, bisphosphonates and other supportive care. Each variable was mapped to identify the location of raw data in the EHR, its format and suitability for extraction. For unstructured data, eg: cytogenetics and imaging reports, natural language processing is being developed to extract key fields.

To enable harmonisation of data from different systems, data is transformed into a common data model. The Observational Medical Outcomes Partnership common data model (OMOP CDM) was chosen as it provided a framework for systematic analysis of data from disparate data sources, typical of those held within health care information systems. This model allows for international benchmarking and collaboration for research. OMOP CDM maps commonly used coding systems and vocabularies, eg ICD-10, SNOMED-CT into standard concepts permitting raw data entering the registry to be standardised into a research ready format that is person-centric, disease agnostic and syntactically and semantically defined.

Anonymised data from multiple sites will be electronically transferred to a ISO27001 certified, GDPR compliant UCL data safe haven for analysis. Oversight is provided by the Steering Committee of clinicians, data scientists, myeloma patients and Myeloma UK with patient involvement at every step. Data will be analysed to answer 3 initial themes (Image 1), and extended further to include control data for Health Technology Appraisals. To date we have successfully identified an initial cohort of over 4000 myeloma cases since 2015.

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Summary/Conclusion: The NIHR HIC Myeloma registry represents a research ready platform to integrate and analyse UK wide real word data. Detailed data analysis and progress in answering the initial 3 themes is ongoing.

Funding: Myeloma UK & NIHR

P955: CARFILZOMIB IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS: THE REAL-LIFE EXPERIENCE OF EMMY

C. Hulin 1,* , M. Macro 2 , A. Perrot 3 , B. Royer 4 , D. Caillot 5 , K. Belhadj 6 , L. Frenzel 7 , R. Benramdane 8 , H. Demarquette 9 , B. Bareau 10 , S. Darre 11 , C. Calmettes 12 , R. Le Calloch 13 , M. Bouketouche 14 , K. Laribi 15 , N. Texier 16 , M. Willaime 16 , C. Deal 17 , P. Moreau 18 , O. Decaux 19

1 CHU Bordeaux, Bordeaux; 2 CHU Caen, Caen; 3 CHU de Toulouse, IUC T-O, Toulouse; 4 bruno.royer@aphp.fr, paris; 5 CHU Dijon, Dijon; 6 Hôpital Henri Mondor, Creteil; 7 Hôpital Necker, paris; 8 CH Cergy Pontoise, Cergy Pontoise; 9 CH. Dunkerque, Dunkerque; 10 CH Cesson Sévigné, Cesson Sévigné; 11 CH. Arras, Arras; 12 CH. Périgueux, Périgueux; 13 CH Cornouaille, Quimper; 14 CH. St Quentin, St. Quentin; 15 CH. Le Mans, Le Mans; 16 kappa santé; 17 IFM, paris; 18 CHU Nantes, Nantes; 19 CHU Rennes, Rennes, France

Background: EMMY is a large-scale epidemiological study to assess the epidemiology and real-life management of multiple myeloma (MM). Proteasome inhibitors (PI), immunomodulators (IMID) and anti-CD38 (aCD38) provide broad solutions to treat patients. Following pivotal clinical trials, carfilzomib, a second-generation PI, is approved in relapsed/refractory (RR) MM. Its use in real-life setting can be assessed in EMMY.

Aims: To assess the use of carfilzomib in RRMM patients in real-life conditions.

Methods: EMMY is a descriptive, multicenter, national, non-interventional study conducted in 72 IFM (Intergroupe Francophone du Myélome, sponsor) sites in France. Any patient initiating treatment for MM over a 3-month annual observation period, is included, since 2017 This dynamic cohort has included approximately 900 patients each year (2765 patients included in 2019). Data are updated annually from hospital records. Patients receiving carfilzomib (K) for RRMM at inclusion or follow up were identified and classified by the regimens received. The median progression-free survival (mPFS), and median overall survival (mOS) were estimated per regimen and per treatment line.

Results: 512 patients (18.5%) (median age 68.4 years (y), 20.5% ≥75y) received K for RRMM, 111 (21.7%) for a second (L2), 97 (18.9%) a third (L3) and 304 (59.4%) a fourth or more (L4+) line. K was used in a double regimen with dexamethasone (Kd) in 232 patients (45.3%) and in a triple regimen either with an IMID (K-IMID) in 198 (38.7%) patients or with other agents in 82 patients (16%) (K-aCD38 for 35 (6.8%), K-alkylant for 27 (5.3%) and K-venetoclax for 19 (3.7%) patients). 25.4% patients (62/244) were at high cytogenetic risk.

K-IMID was used in patients of 66,8y, with an ECOG ≥ 2 for 20.8%, and comorbidities (≥ 1) for 24.3% including 3% of cardiovascular diseases. K-IMID was used in L2 (42.9%), L3 (21.7%) and L4+ (35.4%). 63.1% were priorly exposed to lenalidomide (len) of which 35,9% were refractory. K was combined with len in 144 (28,1%) and with pomalidomide (pom) in 54 (10,5%) patients. The use of K-pom increased over years: 14,4% in 2018, 28,2% in 2019, 73,9% in 2020. mPFS was overall 10.4 months (m) 95%CI [7.3; 14.7]; it was 23m 95%CI [14.7; -] in L2, 6.2m 95%CI [1.3; 12.2] in L3 and 7.1m 95%CI [4.6; 9.2] in L4+. mOS was not reached in L2, L3 or L4+.

Kd was used in patients of 70.1y, with an ECOG ≥ 2 for 31.3% and comorbidities for 29.3%. 93.5% were priorly exposed to len of which 66.4% were refractory. Kd was used in L2(6%), L3 (17.2%) and L4+ (76.8%). mPFS and mOS were 4.4m [3.7; 5.8] and 19.5m [13.7; 22.3] in L4+.

K-aCD38 was used in patients of 63.5y, with an ECOG ≥ 2 for 10%, and comorbidities (≥ 1) for 37.1%. 91.4% were priorly exposed to len including 45.7% len-refractory. K-aCD38 (33/35 with daratumumab) was used in L2 (31.4%), L3/L4 (35.7%) and L5+ (22.9%). mPFS and mOS (all lines included) were 6m [3.8; 15.1] and 21.3m [16.9; -].

K-alkylant (5/27 with bendamustine, 22/27 cyclophosphamide) and K-venetoclax were used in older patients (71.8y and 70.4y) and in L4 or L5 for >80% of them. More than 90% were pre-exposed to len including 65% of len-refractory patients.

K discontinuation was recorded for 63.4% of patients for progression in 44% and adverse event in 11.7% of them.

Summary/Conclusion: The EMMY study shows that carfilzomib is used in many combinations in extended real-life settings compared to pivotal clinical trials including older patients, largely-exposed to lenalidomide and at advanced disease. The use of lenalidomide in early stages has led to a shift in the management of RRMM patients with carfilzomib.

P956: EMMY COHORT: MANAGEMENT OF NEWLY DIAGNOSED OR RELAPSED/ REFRACTORY MULTIPLE MYELOMA IN PATIENTS AGED OF 80 YEARS AND OVER

M. Macro 1,* , O. Decaux 2 , A. Perrot 3 , B. Royer 4 , M. Chretien 5 , K. Belhadj 6 , M. Mohty 7 , L. Frenzel 8 , X. Leleu 9 , M. Dib 10 , O. Allangba 11 , P. Zunic 12 , I. Botoc 13 , J. Malfuson 14 , P. Moreau 15 , R. Garlantazec 16 , N. Texier 17 , R. Germain 17 , C. Deal 18 , C. Hulin 19

1 CHU Caen, Caen; 2 C.H.U de Rennes, Rennes; 3 Institut Universitaire du Cancer Toulouse Oncopole, Toulouse; 4 hôpital st louis, aphp, Paris; 5 CHU Dijon, Dijon; 6 Hôpital Henri Mondor, Creteil; 7 Hôpital Saint-Antoine; 8 Hôpital Necker, paris; 9 CHU de Poitiers, Potiers; 10 CHU Angers, Angers; 11 C.H. Yves le Foll, ST Brieuc; 12 CHU Réunion Sud, St Pierre; 13 CH Saint-Malo, Saint Malo; 14 Hôpital d’Instruction des Armées Percy, Clamart; 15 CHU nantes, Nantes; 16 CHU Rennes, Rennes; 17 kappa santé; 18 IFM, Paris; 19 CHU Bordeaux, Bordeaux, France

Background: Multiple myeloma (MM) patients aged 80 years and older are a population more prone to comorbidities, frailty, cognitive impairment or physical decline and require appropriate management of their myeloma. They are often underrepresented in pivotal clinical trials, with little data available on their management. The EMMY study is a large-scale epidemiological study to assess the epidemiology and real-life management of MM and can focus on the use and real-life efficacy of treatments for newly diagnosed or relapsed/ refractory elderly MM patients.

Aims: To describe the management of MM in newly diagnosed or relapsed patients aged 80 years and over and to assess the real-life effectiveness of the treatments received.

Methods: EMMY is a descriptive, multicenter, national, non-interventional study conducted in 72 IFM (Intergroupe Francophone du Myélome, sponsor) centers in France. Any patient initiating treatment for MM over a 3-month observation period, from October to December, is included, since 2017. It is a dynamic cohort with the inclusion of approximately 900 patients each year: 2765 patients included at the end of 2019 of which 561 were aged 80 and over (20.3%). Data are updated annually from hospital records up to 2020 at the time of the analysis.

Four cohorts of patients aged 80 years and older were analysed according to the line of treatment initiated during their follow-up: L1, L2, L3 or L4+. Median time to next treatment (mTNT), median progression-free survival (mPFS) and median overall survival (mOS) were assessed in months(m).

Results: Patients ≥80 years initiated 19.8% of L1s (237/1199) 22.7% (232/1022) of L2s, 22.2% (167/751) of L3s and 18.7% (288/1540) of L4+. They are 7.5% patients ≥85 years old to initiate L1, 8.7% L2, 7.4% L3 and 5.1% L4+. When the line is started, an ECOG ≥2 is reported for 39.4% of L1, 27.7% of L2, 35.1% of L3 and 38.5% of L4 patients and comorbidities recorded in 45.1% of L1, 39.6% of L2, 35.4% of L3 and 38.6% of L4+. In L1, the ISS is level I, II, III for 20.2%, 27.4% and 52.4% of patients.

In L1, over the 2017-2020 study period, patients received treatment with PI (58.6%), IMID (32.1%), PI/IMID (4.2%), anti-CD38 (1.3%) or other molecules (3.8%). In L2, they received PI (22.8%), IMID (48.7%), PI/IMID (11.7%), anti-CD38 (14.2%) or other (2.6%), in L3 PI (12.6%), IMID (38, 9%), PI/IMID (9.6%), anti-CD38 (29.3%) or other (9.6%) and in L4+ a PI (23.3%), IMID (20.1%), PI/IMID (5.6%), anti-CD38 (29.2%) or other (21.9%). More than half received a double combination in L1 (50.2%), L2 (59.9%), L3 (54.4%) and L4+ (53.8%).

Regarding efficacy, mTNT is 20.3m [15.2; 24.6] in L1, 14.7m [12.3; 20.4] in L2, 10.8m [9; 13.5] in L3 and 8.2m [6.5; 11.2] in L4+. The mPFS is 19.3m [15.1; 23.2] in L1, 12.6m [10.1; 17.5] in L2, 8.9m [10.1; 17.5] in L3 and 5.9m [4.5;7.4] in L4+. The mOS is not reached in L1 (rate at 30m: 76.7% [70; 83.3]) and L2 (rate at 30m: 58.1% [48.1; 68]; it is 25.7m [17.1; NE] in L3 and 18.3m [13.4; 22.4] in L4+.

Summary/Conclusion: Patients ≥80 years of age account for more than 20% of de novo and relapsed myeloma patients treated in EMMY. Double combinations are used in half of the situations. Over the study period, PI-based treatments were preferred in L1, IMID-based in L2 and antiCD38 for 30% of patients in L3 and L4+. The benefits in terms of survival are real and this specific population is likely to increase significantly in the near future, requiring human and logistical resources.

P957: CARFILZOMIB, LENALIDOMIDE, DEXAMETHASONE FOLLOWED BY A SECOND AUTO-HCT IS AN EFFECTIVE STRATEGY IN FIRST RELAPSE MULTIPLE MYELOMA: A STUDY OF THE CHRONIC MALIGNANCIES WORKING PARTY OF EBMT

R. Tilmont 1,* , I. Yakoub-Agha 1,2 , D.-J. Eikema 3 , N. Zinger 4 , M. Haenel 5 , N. Schaap 6 , C. Herrera Arroyo 7 , C. Schuermans 8 , W. Bethge 9 , M. Engelhardt 10 , J. Kuball 11 , M. Michieli 12 , N. Schub 13 , K. M. O. Wilson 14 , J. H. Bourhis 15 , M. V. Mateos 16 , N. Robin 17 , E. Jost 18 , N. Kröger 19 , J. M. Moraleda 20 , S. Sica 21 , P. J. Hayden 22 , M. Beksac 23 , S. Schönland 24 , S. Manier 1,25

1 Hematologie Clinique, CHU de Lille; 2 CHU de Lille, Univ Lille, INSERM U1286, Infinite, Lille, France; 3 EBMT Statistical Unit; 4 EBMT Leiden Study Unit, Leiden, Netherlands; 5 Klinikum Chemnitz gGmbH, Chemnitz, Germany; 6 Nijmegen Medical Centre, Nijmegen, Netherlands; 7 Hosp. Reina Sofia, Cordoba, Spain; 8 GZA Hospitals, Antwerp, Belgium; 9 Universitaet Tuebingen, Tuebingen; 10 University of Freiburg, Freiburg, Germany; 11 University Medical Centre, Utrecht, Netherlands; 12 Centro di Riferimento Oncologico, Aviano, Italy; 13 University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Germany; 14 Department of Haematology, Cardiff, United Kingdom; 15 Gustave Roussy Cancer Campus, Villejuif, France; 16 Hospital Clínico, Salamanca, Spain; 17 University College London Hospital, London, United Kingdom; 18 University Hospital Aachen, Aachen; 19 University Hospital Eppendorf, Hamburg, Germany; 20 Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; 21 Universita Cattolica S. Cuore, Rome, Italy; 22 Department of Haematology, Trinity College Dublin, St. James’s Hospital, Dublin, Ireland; 23 Ankara University Faculty of Medicine, Ankara, Turkey; 24 Medizinische Klinik u. Poliklinik V, University of Heidelberg, Heidelberg, Germany; 25 Univ Lille, Canther, INSERM UMR-S1277 CNRS UMR9020, Lille, France

Background: Multiple myeloma (MM) is an incurable hematologic malignancy despite recent therapeutic advances. In the context of lenalidomide-sensitive relapse, one of the preferred options from EHA/ESMO and IMWG recommendations is the combination of carfilzomib-lenalidomide-dexamethasone (KRd). This regimen has been approved based on the ASPIRE trial with a PFS of 26.3 months vs 17.6 months for Rd alone (HR = 0.69, p=0.0001). In younger patients, a second autologous hematopoietic cell transplantation (auto-HCT) remains an option in case of a prolonged remission after frontline auto-HCT. Only few data are available on the combination of KRd followed by a second auto-HCT in first relapse.

Aims: Primary objective was to estimate progression-free survival (PFS) and overall survival (OS) in patients who received a second line of treatment with KRd followed by a second auto-HCT. Secondary objectives were to assess the response rates and identify statistically significant co-variables on PFS and OS in this population.

Methods: This international retrospective study was performed in 22 centers affiliated to the European Society for Blood and Marrow Transplant Society (EBMT). Patients with MM were included if they received a second line of treatment with KRd induction followed by a second auto-HCT between January 2016 and December 2018.

Results: A total of 51 patients were included, with a median age of 62 years (range 35 – 69). ISS at diagnosis was stage I for 18 patients (35.3%), stage II for 11 (21.6%),stage III for 14 (27.5%) and missing for 8 patients (15.7%). Twenty-seven patients (52.9%) were of standard cytogenetic risk and 11 patients (21.6%) of high cytogenetic risk according to IMWG criteria, data was missing for 13 patients (25.5%). The median time between the 1 st to the 2 nd transplant was 40.4 months (range 17.9–87.9). Regarding the number of cycles of KRd received in induction, 25 patients received 3 or 4 cycles (49.0%), 17 patients 5 or 6 cycles (33.3%) and 9 patients 7 to 12 cycles (17.7%). The conditioning chemotherapy was melphalan alone in 46 patients (90.2%), a combination of melphalan and another drug in 4 patients (7.8%) and cyclophosphamide alone in 1 patient (2.0%). The median follow-up was 36.7 months (range 5.3-58.0). The median PFS was 32.6 months (95%CI: 30–39.9) and the median OS was not reached, while 36- and 48-months OS rates were 87.5% (95%CI: 78.5–97.4) and 72.8% (95%CI: 57.0–92.8) respectively. In univariate analysis, 2 co-variates were found to be associated with a longer median PFS: an interval between the first and the 2 nd auto-HCT of more than 4 years (mPFS of 36.1 vs . 30.6 months, p = 0.02) and the achievement of a very good partial response (VGPR) or better at the 2 nd transplant (mPFS of 33.5 vs . 27.8 months, p = 0.01). These results were also observed in multivariate analysis, with HR of 0.41 (95%CI: 0.17-0.96, p = 0.04) and HR of 0.45 (95%CI: 0.21-0.98, p = 0.04), respectively. No statistical association was found between the duration of PFS and cytogenetic risk profile, with the limitation of a small number of patients with high-risk cytogenetics. Regarding OS, no significantly relevant co-variables were found in uni- or multivariate analysis.

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Summary/Conclusion: A second auto-HCT after KRd induction is an effective treatment for patients with a first lenalidomide-sensitive relapse of MM. Our study suggests that patients with at least 4 years of remission after a frontline auto-HCT and who achieved at least a VGPR after KRd induction, benefit the most from this treatment strategy.

P958: REAL-LIFE CURRENT STANDARD OF CARE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA: SUBGROUP ANALYSES FROM THE LOCOMMOTION STUDY

H. Einsele 1,* , P. Moreau 2 , V. De Stefano 3 , D. Dytfeld 4 , E. Angelucci 5 , R. Benjamin 6 , H. Goldschmidt 7 , N. W. van de Donk 8 , B. Besemer 9 , C. Scheid 10 , R. Vij 11 , E. I. ’. Groen-Damen 12 , M. Semerjian 13 , V. Strulev 14 , J. M. Schecter 15 , T. Roccia 16 , T. Nesheiwat 17 , R. Wapenaar 12 , K. Weisel 18 , M.-V. Mateos 19

1 Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Würzburg, Germany; 2 University Hospital Hôtel-Dieu, Nantes, France; 3 Section of Hematology, Catholic University, Fondazione Policlinico A Gemelli, IRCCS, Rome, Italy; 4 Poznań University of Medical Sciences, Poznań, Poland; 5 Hematology and Transplant Center, IRCCS Ospedale Policlinico San Martino, Genova, Italy; 6 Department of Haematology, King’s College Hospital, London and School of Cancer and Pharmaceutical Sciences, King’s College, London, United Kingdom; 7 University Hospital Heidelberg, Heidelberg, Germany; 8 Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 9 University Hospital Tubingen, Tubingen, Germany; 10 University of Cologne, Cologne, Germany; 11 Washington University School of Medicine, St. Louis, MO, United States of America; 12 Janssen-Cilag, Breda, Netherlands; 13 Janssen-Cilag, Issy-Les-Moulineaux, France; 14 Janssen Pharmaceutica NV, Beerse, Belgium; 15 Janssen R&D, Raritan, NJ, United States of America; 16 Janssen R&D, High Wycombe, United Kingdom; 17 Legend Biotech USA, Piscataway, NJ, United States of America; 18 University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 19 University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain

Background: Patients (pts) with relapsed/refractory multiple myeloma (RRMM) who have had previous triple-class exposure to a proteasome inhibitor (PI), immunomodulatory drug (IMiD), and anti-CD38 monoclonal antibody have limited treatment options and represent an urgent and unmet clinical need. LocoMMotion ( NCT04035226 ) is the first prospective multinational study to assess real-life standard of care (SOC) treatment in pts with triple-class exposed RRMM.

Aims: To assess efficacy in subgroups of pts treated with SOC therapies in the LocoMMotion study.

Methods: All pts provided informed consent. This noninterventional study was conducted across 76 sites (63 in Europe, 13 in the United States). Pts were included if they had received ≥3 prior lines of therapy (LOT) or were refractory to a PI and an IMiD (double-refractory); received a PI, an IMiD, and anti-CD38 monoclonal antibody and had documented progressive disease during/after their last LOT. Real-life SOC therapies were defined as those used in local clinical practice. Responses and disease progression were based on International Myeloma Working Group criteria and assessed by response review committee. Patient subgroups were categorized according to the following baseline characteristics: age, Eastern Cooperative Oncology Group performance status (ECOG PS), renal function, International Staging System stage, extramedullary plasmacytoma, lactate dehydrogenase, bone marrow plasma cells percentage, number of prior LOT, triple-class or penta-drug exposure, and refractoriness.

Results: 248 pts were enrolled as of May 21, 2021 (median follow-up: 11.0 months). Pts received a median of 4.0 (range: 1-20) cycles of SOC treatment. Efficacy outcomes were generally worse in pts with refractoriness to 3 classes of antimyeloma therapy, presence of extramedullary plasmacytomas, high LDH, and ECOG PS ≥1 compared with pts who did not have these characteristics (Table). Across all subgroups, overall response rate ranged from 20.0% to 43.1%. Efficacy outcomes were not affected by age and number of prior LOT.

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Summary/Conclusion: In this first prospective study of real-life SOC treatment in pts with triple-class exposed RRMM, subgroup analyses indicate that specific pt and disease characteristics were associated with poor outcomes. Outcomes were poor in triple-class refractory and non-triple-class refractory pts; however, the latter subgroup had longer median progression-free survival. These data may help inform bridging strategies for chimeric antigen receptor T-cell therapy.

P959: CILTACABTAGENE AUTOLEUCEL IN LENALIDOMIDE-REFRACTORY PATIENTS WITH PROGRESSIVE MULTIPLE MYELOMA AFTER 1-3 PRIOR LINES OF THERAPY: CARTITUDE-2 BIOLOGICAL CORRELATIVE ANALYSES AND UPDATED CLINICAL DATA

J. Hillengass 1,* , A. D. Cohen 2 , M. Delforge 3 , H. Einsele 4 , H. Goldschmidt 5 , K. Weisel 6 , M.-S. Raab 7 , C. Scheid 8 , J. M. Schecter 9 , K. C. De Braganca 9 , H. Varsos 9 , T.-M. Yeh 9 , P. Mistry 10 , T. Roccia 10 , C. Corsale 9 , M. Akram 11 , L. Pacaud 11 , T. Nesheiwat 11 , M. Agha 12 , Y. C. Cohen 13

1 Roswell Park Comprehensive Cancer Center, Buffalo, NY; 2 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, United States of America; 3 University Hospitals (UZ) Leuven, Leuven, Belgium; 4 Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Würzburg; 5 University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg; 6 University Medical Center Hamburg-Eppendorf, Hamburg; 7 University Hospital Heidelberg, and Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center, Heidelberg; 8 University of Cologne, Cologne, Germany; 9 Janssen R&D, Raritan, NJ, United States of America; 10 Janssen R&D, High Wycombe, United Kingdom; 11 Legend Biotech USA, Piscataway, NJ; 12 UPMC Hillman Cancer Center, Pittsburgh, PA, United States of America; 13 Tel-Aviv Sourasky (Ichilov) Medical Center and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Background: Cohort A of the multicohort phase 2 CARTITUDE-2 ( NCT04133636 ) study is assessing ciltacabtagene autoleucel (cilta-cel), a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor T-cell (CAR-T) therapy, in patients with multiple myeloma (MM) who received 1-3 prior lines of therapy (LOT) and were refractory to lenalidomide (len). This population is difficult to treat and has poor prognosis.

Aims: To present updated results from CARTITUDE-2 Cohort A.

Methods: All patients provided informed consent. Eligible patients had progressive MM after 1-3 prior LOT that included a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD). Patients were len-refractory and had no prior exposure to BCMA-targeting agents. Patients received a single cilta-cel infusion (target dose: 0.75×10 6 CAR+ viable T cells/kg) after lymphodepletion. Cilta-cel safety and efficacy were assessed. The primary endpoint was minimal residual disease (MRD) negativity at 10 -5 by next generation sequencing. Patient management strategies were used to reduce the risk of movement and neurocognitive adverse events (MNTs). Other assessments included pharmacokinetic (PK) analyses (C max and T max of CAR+ T-cell transgene levels in blood), levels of cytokine release syndrome (CRS)-related cytokines (e.g., IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with immune effector cell-associated neurotoxicity syndrome (ICANS), and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS.

Results: As of January 2022 (median follow-up: 17.1 months [range: 3.3-23.1]), cilta-cel was administered to 20 patients (male: 65%; median age: 60 years [range: 38-75]). Median number of prior LOT was 2 (range: 1-3); median time since MM diagnosis was 3.5 years (range: 0.7-8.0). 95% of patients were refractory to their last LOT; 40% were triple-class refractory. Overall response rate was 95%, with 90% of patients achieving ≥complete response and 95% achieving ≥very good partial response. Median time to first response was 1.0 month (range: 0.7-3.3); median time to best response was 2.6 months (range: 0.9-13.6). All MRD-evaluable patients (n=16) achieved MRD negativity at 10 -5 . Median duration of response was not reached. The 12-month progression-free survival rate was 75% and the 12-month event-free rate was 79%. CRS occurred in 95% of patients (grade 3/4: 10%), with a median time to onset of 7 days (range: 5-9) and median duration of 3 days (range: 2-12). 30% of patients had neurotoxicity (5 grade 1/2 and 1 grade 3/4). ICANS occurred in 3 patients (15%; all grade 1/2); 1 patient had facial paralysis (grade 2). No MNTs were observed. 1 death due to COVID-19 occurred and was assessed as treatment-related by the investigator; 2 deaths due to progressive disease and 1 due to sepsis (not related to treatment) also occurred. Based on preliminary PK analyses of CAR transgene by qPCR, peak expansion of CAR-T cells occurred at day 10.5 (range: 8.7-42.9); median persistence was 153.5 days (range: 57.1-336.8).

Summary/Conclusion: A single cilta-cel infusion led to deepening and durable responses at this longer follow-up (median 17.1 months) in patients with MM who had 1-3 prior LOT and were len-refractory. Follow-up is ongoing. We will present updated and detailed PK, cytokine, and CAR-T subset analyses as well as clinical correlation to provide novel insights into biological correlates of efficacy and safety in this difficult-to-treat patient population, which is being further evaluated in the CARTITUDE-4 study ( NCT04181827 ; enrollment concluded).

P960: HEALTH-RELATED QUALITY OF LIFE IN THE LOCOMMOTION STUDY OF REAL-LIFE CURRENT STANDARD OF CARE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

M. Delforge 1,* , P. Moreau 2 , H. Einsele 3 , V. De Stefano 4 , J. Lindsey-Hill 5 , L. Vincent 6 , S. Mangiacavalli 7 , A. Perrot 8 , E. Ocio 9 , S. ten Seldam 10 , E. I. ’. Groen-Damen 11 , M. Semerjian 12 , V. Strulev 13 , J. M. Schecter 14 , T. Roccia 15 , K. S. Gries 14 , T. Nesheiwat 16 , R. Wapenaar 11 , M.-V. Mateos 17 , K. Weisel 18

1 University Hospitals (UZ) Leuven, Leuven, Belgium; 2 University Hospital Hotel-Dieu, Nantes, France; 3 Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, Wuerzburg, Germany; 4 Section of Hematology, Catholic University, Fondazione Policlinico A Gemelli, IRCCS, Rome, Italy; 5 Nottinghamshire University Hospitals NHS Trust, Nottingham, United Kingdom; 6 Département d’hématologie Clinique, Centre Hospitalier Universitaire de Montpellier, Montpellier, France; 7 Fondazione IRCCS Policlinico San Matteo, University of Pavia, Pavia, Italy; 8 Centre Hospitalier Universitaire de Toulouse, Service d’Hématologie, Toulouse, France; 9 Hospital Universitario Marqués de Valdecilla (IDIVAL), Universidad de Cantabria, Santander, Spain; 10 Myeloma Patients Europe, Brussels, Belgium; 11 Janssen-Cilag, Breda, Netherlands; 12 Janssen-Cilag, Issy-les-Moulineaux, France; 13 Janssen Pharmaceutica NV, Beerse, Belgium; 14 Janssen R&D, Raritan, NJ, United States of America; 15 Janssen R&D, High Wycombe, United Kingdom; 16 Legend Biotech USA Inc, Piscataway, NJ, United States of America; 17 University Hospital of Salamanca/IBSAL/CIC, Salamanca, Spain; 18 University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Background: Assessment of patient-reported outcomes (PRO) can inform how real-life standard of care (SOC) treatments affect health-related quality of life (HRQoL) for patients with relapsed/refractory multiple myeloma (RRMM). We present measures of symptoms, functioning, and overall HRQoL from LocoMMotion ( NCT04035226 ), the first prospective, multinational study of real-life SOC in triple-class exposed patients with RRMM.

Aims: To assess HRQoL in patients with RRMM receiving real-life current SOC in the LocoMMotion study.

Methods: All patients provided informed consent. LocoMMotion is a noninterventional study across 76 sites (63 Europe, 13 United States) in patients who had received ≥3 prior lines of therapy (LOT) or were refractory to proteasome inhibitor (PI) and immunomodulatory drug (IMiD). Patients received PI, IMiD, and anti-CD38 monoclonal antibody and had disease progression during/after their last LOT. Real-life SOC treatments were defined as those used in local clinical practice. The following questionnaires were used: European Organisation for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30), 4 single items from EORTC QLQ-myeloma-specific module (EORTC QLQ-MY20), and EuroQol 5-Dimension 5-Level (EQ 5D-5L). HRQoL data were collected at baseline (BL), day 1 of each treatment cycle, end of treatment visit, and during follow-up (every 4 weeks). Established thresholds were used to evaluate improvement compared with BL health status. Mixed models for repeated measures were used to assess within-group change.

Results: The questionnaire completion rate was 75.6% during SOC treatment in the LocoMMotion study (N=248; male: 54.4%; median age: 68 years; median cycles of SOC: 4.0 [range: 1-20]). Most patients did not achieve meaningful improvement (defined by a literature-based minimally important difference of 10 points in mean score) in PRO scores. This was most pronounced in pain symptoms, with 62% of patients showing no meaningful improvement during the first 3 months of treatment and 55% showing no improvement during the full treatment duration. Least square (LS) mean changes from BL during SOC treatment and subsequent LOT for the overall population are described (Table). Patients with ≥very good partial response during SOC treatment showed greater improvement in PRO scores, including LS mean change for pain score (-14.9 [95% confidence interval: -22.9, -7.0]).

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Summary/Conclusion: This first prospective study of real-life current SOC in triple-class exposed patients with RRMM reported limited gains in HRQoL, most notably in pain symptoms. There is an urgent and unmet need for therapies that lead to deep responses and delayed disease progression, as these are associated with improvements in HRQoL.

P961: CILTACABTAGENE AUTOLEUCEL, A BCMA-DIRECTED CAR-T CELL THERAPY, IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA: 2-YEAR POST LPI RESULTS FROM THE PHASE 1B/2 CARTITUDE-1 STUDY

Y. Lin 1,* , T. Martin 2 , J. G. Berdeja 3 , A. Jakubowiak 4 , M. Agha 5 , A. D. Cohen 6 , A. Deol 7 , M. Htut 8 , A. Lesokhin 9 , N. C. Munshi 10 , E. O’Donnell 11 , C. C. Jackson 12 , T.-M. Yeh 12 , A. Banerjee 13 , E. Zudaire 13 , D. Madduri 12 , C. Zhou 14 , L. Pacaud 14 , S. Z. Usmani 9 , S. Jagannath 15

1 Mayo Clinic, Rochester, MN; 2 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA; 3 Sarah Cannon Research Institute, Nashville, TN; 4 University of Chicago, Chicago, IL; 5 UPMC Hillman Cancer Center, Pittsburgh, PA; 6 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA; 7 Karmanos Cancer Institute, Wayne State University, Detroit, MI; 8 City of Hope Comprehensive Cancer Center, Duarte, CA; 9 Memorial Sloan Kettering Cancer Center, New York, NY; 10 Dana-Farber Cancer Institute, Harvard Medical School; 11 Massachusetts General Hospital, Harvard Medical School, Boston, MA; 12 Janssen R&D, Raritan, NJ; 13 Janssen R&D, Spring House, PA; 14 Legend Biotech USA, Piscataway, NJ; 15 Mount Sinai Medical Center, New York, NY, United States of America

Background: The phase 1b/2 CARTITUDE-1 study ( NCT03548207 ) reported early, deep, and durable responses with ciltacabtagene autoleucel (cilta-cel), a chimeric antigen receptor T (CAR-T) cell therapy with 2 B-cell maturation antigen (BCMA)–targeting single-domain antibodies, in heavily pretreated patients with relapsed/refractory multiple myeloma (RRMM). At a median follow-up of ~1 year, the overall response rate (ORR) was 97%, with 67% of patients achieving stringent complete response (sCR). Progression-free survival (PFS) and overall survival (OS) rates at 1 year were 77% and 89%, respectively.

Aims: To report updated 2-year post last patient in (LPI) results (total median follow-up of ~30 months). CARTITUDE-1 results at a median follow-up of 21.7 months are reported here.

Methods: All patients provided informed consent. Patients with RRMM had received ≥3 prior lines of therapy (LOT) or were refractory to a proteasome inhibitor (PI) and immunomodulatory drug (IMiD). Patients had received a PI, IMiD, and anti-CD38 antibody. After apheresis, bridging therapy was allowed. A single cilta-cel infusion was administered at a target dose of 0.75×10 6 CAR+ viable T cells/kg 5-7 days after lymphodepletion. The primary objectives were to evaluate the safety and efficacy of cilta-cel. Response assessments were based on International Myeloma Working Group criteria by independent review committee. Minimal residual disease (MRD) negativity at 10 -5 was assessed by next-generation sequencing.

Results: 97 patients (male: 59%; median age: 61 years) received cilta-cel as of July 22, 2021. The median number of prior LOT was 6 (range: 3-18). 84% of patients were penta-drug exposed, 88% were triple-class refractory, 42% were penta-drug refractory, and 99% were refractory to their last LOT. The ORR was 97.9% (95% CI: 92.7-99.7), with 94.9% of patients achieving very good partial response and 82.5% achieving sCR. Median time to first response was 1.0 month, median time to best response was 2.6 months, and median time to ≥CR was 2.9 months. Median duration of response was not reached. Among 61 MRD-evaluable patients, 92% were MRD negative at 10 -5 , which was sustained in 44% of patients (27/61) for ≥6 months and in 18% of patients (11/61) for ≥12 months. Median PFS and OS were not reached. The 2-year PFS rate was 60.5% (95% CI: 48.5-70.4) overall. The 2-year PFS rate was 91% for patients with sustained MRD negativity ≥6 months and 100% for those with sustained MRD negativity ≥12 months. No new safety signals or new cases of CAR-T cell neurotoxicity, movement/neurocognitive treatment-emergent adverse events, or treatment-related deaths were reported since 1-year median follow-up. Over a median follow-up of ~2-years, 15 second primary malignancies occurred in 11 patients.

Summary/Conclusion: A single cilta-cel infusion resulted in deepening and durable responses in heavily pretreated patients with RRMM at a median follow-up of ~2 years. The safety profile was manageable. Follow-up in CARTITUDE-1 is ongoing. We will present landmark 2-year post LPI data, with ~8 months of additional follow-up (~30 months total median follow-up). Cilta-cel is also being evaluated in earlier LOT and outpatient settings across the CARTITUDE program, including NCT04133636 , NCT04181827 , NCT04923893 .

P962: CHANGE IN SOLUBLE BCMA LEVEL MAY BE A SURROGATE MARKER OF EARLY RESPONSE TO THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM): PRELIMINARY RESULTS FROM A PHASE I STUDY OF CEVOSTAMAB

R. Nakamura 1,* , R. Hendricks 1 , R. Dokhaei 1 , M. Susilo 1 , D. Wilson 1 , S. Trudel 2 , A. Cohen 3 , S. J. Harrison 4 , A. Krishnan 5 , R. Fonseca 6 , J. I. Adamkewicz 1 , M. Li 1 , C. Wong 1 , J. Cooper 1 , T. Sumiyoshi 1

1 Genentech, Inc., South San Francisco, CA, United States of America; 2 Princess Margaret Cancer Centre and University of Toronto, Toronto, ON, Canada; 3 Abramson Cancer Center and University of Pennsylvania, Philadelphia, PA, United States of America; 4 Peter MacCallum Cancer Centre, Sir Peter MacCallum Department of Oncology, Melbourne University, and The Royal Melbourne Hospital, Melbourne, VIC, Australia; 5 City of Hope, Duarte, CA; 6 Mayo Clinic in Arizona, Phoenix, AZ, United States of America

Background: Cevostamab is an FcRH5xCD3 bispecific antibody that facilitates T-cell directed killing of myeloma cells, and has shown promising activity and manageable safety in a Phase I study in patients (pts) with RRMM ( NCT03275103 ; Trudel et al. ASH 2021). B-cell maturation antigen (BCMA) is a membrane-bound protein that is expressed preferentially by malignant plasma cells and has become an important therapeutic target in MM. Shedding of membrane-bound BCMA, mediated by γ-secretase, gives rise to the soluble form of BCMA (sBCMA) which is often present at elevated levels in pts with MM. Normalization of sBCMA may be a predictor of response to therapy, which may be independent of treatment and target.

Aims: To evaluate sBCMA as a biomarker of early response in pts enrolled in the single step-up dosing cohorts of the cevostamab Phase I study.

Methods: Plasma samples were collected at baseline and at Cycle (C) 1 Day (D) 1 (pre-infusion and end of infusion [EOI]), C1D2, C1D4, C1D8 (pre-infusion and EOI), C1D9, C1D11, and C2D1 (pre-infusion and EOI). sBCMA levels were quantified using hybrid immunoaffinity capture with LC-MS/MS. Pts were stratified by refractory status, prior therapy, prior transplant, cytogenetic risk and response (responder [≥PR] or non-responder [<PR]), and samples from each category were grouped for analysis. Associations between baseline sBCMA levels and baseline patient characteristics or response to treatment were evaluated using an unpaired 2-sample Wilcoxon test (significance level: p≤0.05). The relationship between sBCMA dynamics (percent change in sBCMA level from baseline to C2D1 EOI) and best response was evaluated using logistic linear regression. All pts provided informed consent.

Results: At cut-off (January 7, 2022), 97/103 pts in the single step-up cohorts were biomarker evaluable. No clear differences in baseline sBCMA levels were observed in pts stratified by refractory status, prior transplant, or prior anti-CD38 or anti-BCMA therapy. Analysis of a subset of pts with cytogenetic data showed a trend towards lower baseline sBCMA levels in the standard-risk group (n=18) vs the high-risk group (n=42; p=0.032). In the active dose cohorts (3.6/20mg+; n=77), baseline sBCMA levels were comparable between responders and non-responders. However, baseline sBCMA levels were lower in pts achieving a VGPR or better vs those achieving a PR or no response (median: 47.7ng/mL vs 120.5ng/mL; p=0.037). At C2D1 EOI, most responders had a reduction in sBCMA relative to baseline, with the pts who achieved sCR having the greatest decrease (median: –92.10%). In comparison, an increase in sBCMA was observed in non-responders at C2D1 EOI, with the pts who had PD having the greatest increase (median: 93.14%). The extent of reduction in sBCMA from baseline to C2D1 EOI was significantly correlated with best overall response rate (PR or better) (p=1.8x10 –5 ).

Summary/Conclusion: In this study, baseline sBCMA was not associated with refractory status, prior transplant or treatment in pts with RRMM. Patients with standard-risk cytogenetics had lower baseline sBCMA levels vs those with high-risk cytogenetics. The kinetics of sBCMA change from baseline to C2D1 EOI corresponded with response to cevostamab, with greater reductions increasing the probability of best response. These early data suggest that sBCMA dynamics may be a potential tool for early disease monitoring and identification of response in RRMM. Updated data will be presented.

P963: EVOLUTION OF TREATMENT PATTERNS AND OVERALL SURVIVAL IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA RECEIVING A SECOND LINE OF THERAPY BETWEEN 2012 AND 2020: ANALYSIS OF THE PREAMBLE COHORT

D. Kuter 1,* , H. Goldschmidt 2 , D. Cella 3 , M. T. Petrucci 4 , P. Moreau 5 , B. Durie 6 , A. Juarez-Garcia 7 , L. Trong 7 , J. Gu 7 , G. Hernandez Rivera 8 , S. Dhanasiri 8 , T. Marshall 7 , J. Wang 7 , L. Lacoin 9 , K. Ramasamy 10 , R. Vij 11

1 Hematology Division, Massachusetts General Hospital, Boston, MA, United States of America; 2 University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 3 Department of Medical Social Sciences and Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, United States of America; 4 Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy; 5 Service d’hématologie clinique, CHU Hôtel Dieu, Nantes, France; 6 Cedars Sinai Cancer Center, Los Angeles, CA; 7 Bristol Myers Squibb, Princeton, NJ, United States of America; 8 Bristol-Myers Squibb, Boudry, Switzerland; 9 Epi-Fit, Bordeaux, France; 10 Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 11 Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, United States of America

Background: Treatment (Tx) options such as immunomodulatory agents (IMiDs), proteasome inhibitors (PIs), and more recently anti-CD38 monoclonal antibodies (mAbs) have improved clinical outcomes for patients (pts) with multiple myeloma (MM); yet many experience disease relapse or become refractory to Tx. PREAMBLE is a prospective, observational cohort study of pts with MM in North America and Europe.

Aims: To assess Tx patterns and overall survival (OS) evolution over time in pts with relapsed/refractory MM (RRMM) who received a second-line therapy (2L) between 2012 and 2020 in the PREAMBLE study.

Methods: This analysis focused on a subset of pts enrolled in PREAMBLE at the time of initiating 2L Tx for MM. The index date was defined as the enrollment date. Pts were followed until death, completion of 3 years of follow-up (FU), date of loss to FU, or date of last visit prior to database extraction date. Pt characteristics, previous Tx since diagnosis and 2L regimens received were described at index date. Pts were grouped by date of enrollment in 3 cohorts (2012-15, 2016-18, and 2019-20). Duration of 2L Tx and OS were analyzed using the Kaplan Meier method.

Results: 611 pts were included with a median age of 71 years at the start of 2L Tx, 56.8% were male, and 72.7% and 27.3% were from Europe and North America, respectively. 352 pts received 2L in 2012-2015, 199 in 2016-2018 and 60 in 2019-2020. Median FU was 22.8, 32.3 and 18.4 months in the 3 cohorts. The median time from diagnosis to the start of 2L Tx was 33.3 months, increasing from 31.8 months in the 2012-15 cohort to 40.2 months in the 2019-20 cohort. Prior to the study entry, during first line (1L), 31.6% of pts received a stem cell transplant (SCT) (34.9%, 27.6% and 25.0% respectively for the 3 cohorts). Overall, the main 1L regimens received were PI-based regimens (48.0%), IMiD-based regimens (24.9%), and combinations of IMiD and PI (22.7%). Fewer than 5 pts received an anti-CD38 mAb in 1L. The proportion of pts who received a combination of IMiD and PI in 1L increased from 19.3% in 2012-15 to 25.1% in 2016-18 and 35.0% in 2019-20.

Regarding 2L Tx received, single class regimens (IMiD only and PI only) use dropped from 88.0% in 2012-15 to 62.8% in 2016-18 and 18.4% in 2019-20. It was replaced by various combinations of Tx as shown in the figure. The combinations of Tx containing anti-CD38 mAb had increased from 9.0% to 60.1% since the first launch of anti-CD38 in 2016. 9.0% of pts received an SCT during 2L (10.5%, 8.0% and 3.3% for the 3 cohorts). The unadjusted median duration of 2L for the 3 cohorts was of 6.0, 8.1 and 9.9 months (log rank test, p=0.01). A significant improvement in unadjusted OS was observed (log rank test, p<0.001). One year-OS (95% confidence interval [CI]) was 78.4% (73.9-82.9), 84.8% (79.7-89.9) and 93.0% (86.3-99.7); 2 year-OS (95%CI) was 61.3% (55.8-68.5), 74.5% (68.2-80.6) and 77.7% (65.0-90.4) in 2015-16, 2016-18 and 2019-20 cohorts, respectively. Median OS was not reached.

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Summary/Conclusion: Major changes in 2L Tx patterns were observed in pts with RRMM between 2012 and 2020 with the decreased use of single class regimens and the increased use of combination Tx. Pts were likely to be exposed to all 3 main classes of drugs (IMiD, PIs and anti-CD38) earlier in the Tx pathway. A trend towards improvement in OS from start of 2L was also observed over time, likely driven by the changes in Tx patterns. Future analyses of this on-going study will allow to evaluate the long-term impact of those new Tx and the remaining unmet needs.

P964: IMMUNOPHENOTYPED-SUSPENSION-MULTIPLEX FISH BY IMAGING FLOW CYTOMETRY FOR THE SIMULTANEOUS DIAGNOSIS OF THREE PIVOTAL IGH TRANSLOCATIONS IN MULTIPLE MYELOMA

T. Tsukamoto 1,* , M. Kinoshita 2 , K. Yamada 2 , T. Yamaguchi 3 , Y. Chinen 1 , S. Mizutani 1 , T. Fujino 1 , Y. Shimura 1 , T. Kobayashi 1 , J. Inazawa 4 , J. Kuroda 1

1 Division of Hematology and Oncology, Kyoto Prefectural University of Medicine, Kyoto; 2 Sysmex Corporation, Hyogo; 3 General Laboratory, Bio Medical Laboratories, Inc.; 4 Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

Background: The cytogenetic abnormalities including t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32) are associated with the molecular profiles, clinical features, and treatment outcome in multiple myeloma (MM). Although the double-color interphase fluorescence in situ hybridization (DC-FISH) is well established to detect those translocations, the conventional method can analyze only one abnormality on each slide.

Aims: To analyze multiple chromosomal abnormalities simultaneously with high sensitivity and specificity, we aimed to develop a new diagnostic modality of four-color FISH for multiple translocations in immunophenotyped cells in suspension using imaging flow cytometry (immunophenotyped-suspension-multiplex (ISM)-FISH).

Methods: For the ISM-FISH, we first subjected Carnoy-fixed cells in suspension to immunostaining using a brilliant violet 421-conjugated anti-CD138 antibody. Then, we hybridized with four different FISH probes, i.e., Texas Red (TxRed)-conjugated probe for IGH, fluorescein isothiocyanate (FITC)-conjugated probe for FGFR3, Gold-conjugated probe for cyclin D1 (CCND1), and Cy5-conjugated probe for c-MAF, in suspension. Then, more than 2.5 × 10 4 nucleated cells for each sample were analyzed with imaging flow cytometer MI-1000 (Sysmex) by six channels: one for immunophenotyping, four for FISH and one for bright field image. In the process of imaging flowcytometric analysis, we sorted CD138-positive cell fraction, computed the distance between FISH probes using originally developed spot counting tool, and determined to be positive for translocation when the distance is under the threshold.

Results: By this ISM-FISH system, we simultaneously examined three chromosomal translocations, including t(4;14), t(14;16), and t(11;14), in three MM cell lines, KMS-11, KMS-21-BM, KMS-26, and successfully detect the abnormalities. Using different proportions of transformation-positive MM cells spiked with transformation-negative leukemic cells, HL-60, ISM-FISH showed a sensitivity of less than 0.1%. Next, ISM-FISH and conventional DC-FISH were performed for bone marrow nucleated cells from 70 patients with MM or monoclonal gammopathy of undetermined significance (MGUS) and the experiments showed a promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive than standard DC-FISH, examining 200 interphase cells with its optimal sensitivity of up to 1.0%. Furthermore, the ISM-FISH showed a positive concordance of 83.3% and negative concordance of 97.4% using standard DC-FISH, examining 200 interphase cells. Furthermore, in three patients (4%), t(4;14) or t(11;14) were positive by ISM-FISH but negative by DC-FISH examining 200 cells. Extensive DC-FISH study examining additional 1,000 cells showed positive in these ISM-FISH-positive/DC-FISH (200cells)-negative samples, suggesting higher sensitivity of ISM-FISH than that of the standard DC-FISH.

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Summary/Conclusion: This study developed the new diagnostic system of ISM-FISH, which enables the simultaneous diagnosis of three clinically pivotal chromosomal translocations, t(4;14), t(14;16), and t(11;14), in MGUS and MM. This system may facilitate rapid and reliable cytogenetic diagnosis and promote patient-oriented therapy according to the type of chromosomal translocation in the clinical practice setting.

P965: FOLLOW-UP ANALYSIS OF THE RANDOMIZED PHASE II TRIAL OF BORTEZOMIB, LENALIDOMIDE, DEXAMTHASONE WITH/WITHOUT ELOTUZUMAB FOR NEWLY DIAGNOSED, HIGH RISK MULTIPLE MYELOMA (SWOG-1211)

S. Usmani 1,* , A. Hoering 2 , S. Ailawadhi 3 , R. Sexton 2 , B. Lipe 4 , J. Valent 5 , M. Rosenzweig 6 , J. Zonder 7 , M. Dhodapkar 8 , N. Callander 9 , T. Zimmerman 10 , P. Voorhees 11 , B. Durie 12 , S. V. Rajkumar 13 , P. Richardson 14 , R. Orlowski 15

1 Memorial Sloan Kettering Cancer Center, New York; 2 Cancer Research And Biostatistics, Seattle; 3 Mayo Clinic, Jacksonville; 4 University of Rochester Medical Center, Rochester, NY; 5 Cleveland Clinic, Cleveland; 6 City of Hope, Los Angeles; 7 Karmanos Cancer Institute, Detroit, MI; 8 Emory Winship Cancer Institute, Atlanta GA; 9 University of Wisconsin, Madison; 10 Beigene, Chicao; 11 Levine Cancer Institute, Charlotte; 12 Cedar Sinai Medical Center, Los Angeles; 13 Mayo Clinic, Rochester MN; 14 Dana Farber Cancer Institute, Boston MA; 15 MD Anderson Cancer Center, Houston TX, United States of America

Background: The introduction of immunomodulatory agents, proteasome inhibitors, and autologous stem cell transplantation (ASCT) has improved outcomes for patients with multiple myeloma (MM), but those with high risk MM (HRMM) have a poor long-term prognosis. Herein we provide survival outcomes on the first randomized trial in newly diagnosed HRMM, S1211, to follow-up on the previously reported progression-free survival (PFS) ( NCT01668719 , Usmani SZ et al, Lancet Haem 2021).

Aims: S1211 is a randomized phase II trial comparing 8 cycles of lenalidomide, bortezomib and dexamethasone (RVd) induction followed by dose-attenuated RVd maintenance until disease progression with or without elotuzumab (RVd-Elo). Stem cell collection was allowed, but ASCT was deferred until progression.

Methods: HRMM was defined by one of the following: gene expression profiling high-risk (GEP hi ), t(14;16), t(14;20), del(17p), amplification 1q21, primary plasma cell leukemia (pPCL), or elevated serum LDH ( ≥ 2X ULN). Median PFS was the primary endpoint, using a one-sided stratified log-rank test at a one-sided significance level of 0.1. Secondary endpoints included overall response rate (ORR), adverse events (AE), serious adverse events (SAE) and OS. Response was assessed using the IMWG 2009 criteria.

Results: S1211 enrolled 103 evaluable patients, RVd n=54, RVd-Elo n=49. 74% had ISS II/III, 48% amp1q21, 38% del(17p), 11% t(14;16), 9% GEP hi , 7% pPCL, 5% t(14;20) and 4% elevated LDH (17% ≥ 1 feature). With median follow-up of 72 months (mos.), no difference in median PFS was observed [RVd-Elo=29 mos., RVd= 34 mos., HR = 1.11 (80% CI=0.82, 1.49, p=0.66]. No difference in OS was observed [RVd-Elo = median not reached (NR), RVd= 68 mos., HR = 0.85 (80% CI: 0.59, 1.23), p-value = 0.58]. 76% pts had ≥ Grade 3 AEs, no differences in the safety profile were observed. Amongst patients with gain/amp 1q21, median PFS was [RVd-Elo=31 mos., RVd= 37 mos., HR = 1.48 (80% CI= 0.95, 2.31), p=0.25], median OS was [RVd-Elo = 61 mos., RVd= 68 mos., HR = 1.23 (80% CI: 0.72, 2.10), p-value = 0.63]. In patients with del(17p), median PFS was observed [RVd-Elo=41 mos., RVd= 30 mos., HR = 0.98 (80% CI= 0.60, 1.58), p=0.95], median OS was [RVd-Elo = NR, RVd= 72 mos., HR = 0.77 (80% CI: 0.40, 1.48), p-value = 0.61].

Summary/Conclusion: In the first randomized HRMM study reported to date, the addition of Elo to RVd induction and maintenance did not improve PFS and OS with a median follow-up of 6 years. Although the median PFS for Del17p subgroup on RVd-Elo arm is higher than RVd, it did not achieve statistical significance. The PFS and OS observed for gain/amp 1q21 and del17p in the RVd control arm may serve as important benchmarks for future enrichment design HRMM clinical trials. The PFS and OS in both arms of the study exceeded the original statistical assumptions and support the role for PI/IMiD combination induction/maintenance therapy for this population.

P966: DARATUMUMAB, CARFILZOMIB, POMALIDOMIDE AND ELOTUZUMAB FOR THE TREATMENT OF POEMS SYNDROME- THE MAYO CLINIC EXPERIENCE

I. Vaxman 1,2,* , S. Kumar 1 , F. buadi 1 , M. Lacy 1 , D. Dingli 1 , A. Fonder 1 , M. Hobbs 1 , S. Hayman 1 , L. Y. hwa 1 , T. Kouralis 1 , R. Warsame 1 , E. Muchtar 1 , L. Nelson 1 , P. Kapoor 1 , M. Grogan 1 , R. Go 1 , W. Gonsalves 1 , M. Siddiqi 1 , K. Robert 1 , V. Rajkumar 1 , M. Gertz 1 , A. Dispenzieri 1

1 hematology, Mayo Clinic, Rochester, United States of America; 2 hematology, Beilinson, Hod Hasharon, Israel

Background: POEMS (Polyneuropathy, Organomegaly, Endocrinopathies, Monoclonal protein, Skin changes) syndrome is a rare paraneoplastic syndrome and therapies are directed against plasma cells that produce the proteins that cause this syndrome. Novel therapies are widely used in multiple myeloma aiming for plasma cell eradication. However, data on their use in POEMS syndrome are lacking.

Aims: To provide the Mayo Clinic experience in treating 16 patients with relapsed POEMS syndrome with novel agents (daratumumab, carfilzomib, pomalidomide, and elotuzumab).

Methods: We identified all POEMS patients seen at Mayo Clinic Rochester, Minnesota using a prospectively maintained database of patients seen at our center between June 1979 and May 2021. Of these patients, we identified all the patients that were treated with a “novel” agent, defined as daratumumab, carfilzomib, pomalidomide or elotuzumab.

The primary endpoints were response to therapy (hematological, PET, VEGF and clinical, which will be referred to as response H, P, V, C , respectively), and time to next therapy (TTNT), defined as time from institution of regimen of interest to next therapy. The secondary outcome was safety.

Results: The median age at POEMS diagnosis was 57 years (range 39-79) and 15 patients (93%) were men. Among all patients at diagnosis, 10 (63%) had skin changes, 15 (94%) had signs of extravascular volume overload, and 15 (94%) patients had endocrine disorders. Ten and six patients had IgA and IgG isotypes, respectively, and all the patients had lambda light chain isotype. Radiological evidence for sclerotic lesions were found in 13 (81%) patients.

The median time from diagnosis to novel agent’s first dose administration was 50 months (IQR 23-122) and the median lines of therapy prior to novel agent was 2 (range 1-4). Twelve patients (75%) underwent prior autologous stem cell transplantation (ASCT), and 5 patients had prior lenalidomide. The median age at novel agent administration was 63 years (IQR 51-70) and 4 patients (24%) were 70 years or older.

The patients were treated with a a doublet including dexamethasone (N=5) (31%) or in various combinations with other agents: DRd (N=6), DC(V)d (N=3), KRd (N=3), KPd (N=1), DP(V)d (N=5), and EloRd (N=1).

The outcomes with novel agent therapies were favorable (Table 1). Among patients treated with daratumumab based therapies (N=17), 9 patients achieved CR/VGPR H , 7 patients achieved CR V , and 5 patients achieved CR P . Among patients treated with carfilzomib-based therapies (N=6), 3 patients achieved CR/VGPR H and one achieved PR H . Only one patient treated with carfilzomib-based therapies achieved a clinical response.

Neither patient who received pomalidomide and dexamethasone or elotuzumab with lenalidomide and dexamethasone responded. At a median follow-up of 38 months since starting of the novel agent (IQR 24-57), 15 of the patients (94%) are still alive, and the median TTNT was not reached.

None of the patients discontinued therapy due to adverse events and no deaths occurred on therapy. Novel therapies were safe with 7 events of hospitalization due to pneumonia (4 in daratumumab-based therapies and 3 on carfilzomib-based therapies), and 4 patients were hospitalized due to volume overload (all received dexamethasone with therapy). Three patients experienced infusion-related reactions (IRR) to the first dose of IV daratumumab.

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Summary/Conclusion: Response rate was high and the responses were deep. Novel agent therapies were safe, and no death case occurred on therapy. Future studies are needed to clarify the optimal sequence of novel agents and the best combination.

P967: TUMOR BURDEN AS A CRITICAL PROGNOSTIC FACTOR OF PRIMARY EXTRAMEDULLARY DISEASE

M. Vlachová 1,* , M. Štork 2 , S. Ševčíková 1 , T. Jelínek 3 , J. Minařík 4 , J. Radocha 5 , P. Krhovská 4 , L. Pospíšilová 6 , I. Špička 7 , J. Straub 7 , P. Pavlíček 8 , A. Jungová 9 , V. Sandecká 2 , V. Maisnar 5 , R. Hájek 3 , L. Pour 2

1 Babak Myeloma Group, Department of Pathophysiology, Faculty of Medicine, Masaryk University; 2 Clinic of Internal Medicine, Hematology and Oncology, University Hospital Brno, Brno; 3 Department of Hematooncology, University Hospital Ostrava and Faculty of Medicine University of Ostrava, Ostrava; 4 Department of Hemato-Oncology, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc; 5 4th Department of Internal Medicine – Hematology, Faculty Hospital and Charles University in Hradec Kralove, Hradec Kralove; 6 Institute of Biostatistics and Analyses, Ltd., Brno; 7 1st Medical Department - Clinical Department of Haematology, the First Faculty of Medicine and General Teaching Hospital Charles University; 8 Department of Internal Medicine and Hematology, University Hospital Kralovske Vinohrady, Prague; 9 Hematology and Oncology Department, Charles University Hospital, Pilsen, Czechia

Background: Multiple myeloma (MM) is the second most common hematological malignancy characterized by malignant plasma cell (PC) infiltration of the bone marrow (BM). In the case of extramedullary MM (EMD), plasma cells survive and proliferate outside of the bone marrow microenvironment. Primary EMD is found in newly diagnosed MM (NDMM) patients.

Aims: The aim of this study was to describe clinical features and treatment outcomes of primary EMD patients and to define possible risk factors.

Methods: In total, 724 primary EMD patients were diagnosed in the Czech Republic between 2004 and 2021 by using modern imaging methods. As a reference group, 2440 MM patients without any evidence of EMD were used. All patients enrolled into the analysis were treated by novel agents. Patients’ data were analyzed from the Registry of Monoclonal Gammopathies of the Czech Myeloma Group (RMG).

Results: Primary EMD patients had less frequently advanced ISS (ISS 3) (OR 0.51 [95% CI: 0.42–0.63], p<0.001), high paraprotein levels (>20g/l) (OR 0.64 [95% CI: 0.54–0.76], p<0.001) and high PC infiltration of bone marrow (>10%) (OR 0.35 [95% CI: 0.30–0.42], p<0.001) when compared to reference MM patients. Median PFS in newly diagnosed primary EMD patients was comparable to reference MM patients (23.2 months [95% CI: 21.2–26.2] vs. 23.3 months [95% CI: 22.5–24.8], p=0.800). Median OS was comparable to reference patients (52.6 months [95% CI: 46.3–62.0] vs. 55.0 months [95% CI: 51.6–58.9], p=0.504). By multivariate analysis, adjusted for ISS, we found high levels of BM PCs (>10%) together with 3 and more EMD lesions as an independent risk factor for inferior survival in primary EMD patients (PFS: HR 1.92 [95 % CI: 1.28–2.87]; OS: HR 2.06 [95 % CI: 1.35–3.14], both p=0.001).

Summary/Conclusion: We found primary EMD patients distinct from reference MM patients. Survival intervals of both groups of patients were comparable. We found that the intra- and extramedullary tumor burden is an independent key factor influencing primary EMD prognosis.

This work was supported by grant AZV NU21-03-00076.

P968: ADDITION OF IXAZOMIB TO POMALIDOMIDE AND DEXAMETHASONE IMPROVES PROGRESSION-FREE SURVIVAL FOR MULTIPLE MYELOMA PATIENTS PROGRESSING ON LENALIDOMIDE AS PART OF 1ST LINE THERAPY: ALLIANCE A061202

P. Voorhees 1,* , V. Suman 2 , Y. Efebera 3 , N. Raje 4 , S. Tuchman 5 , C. Rodriguez 6 , K. Santo 2 , M. Bova-Solem 7 , D. Carlisle 7 , U. Saad 8 , P. McCarthy 9 , P. Richardson 10

1 Department of Hematologic Oncology and Blood Disorders, Levine Cancer Institute, Atrium Health, Charlotte; 2 Alliance Statistics and Data Management Center, Mayo Clinic, Rochester; 3 Department of Hematology and Oncology, Ohio Health, Columbus; 4 Division of Hematology and Oncology, Massachusetts General Hospital, HArvard Medical School, Boston; 5 Division of Hematology, Lineberger Comprehensive Cancer Center, the University of North Carolina, Chapel Hill; 6 Tisch Cancer Institute, Mount Sinai School of Medicine, New York City; 7 Protocol Operations Program, Alliance for Clinical Trials in Oncology, Chicago; 8 Division of Hematologic Oncology, Memorial Sloan Kettering Cancer Center, New York City; 9 Transplant and Cellular Therapy Program, Roswell Park Comprehensive Cancer Center, Buffalo; 10 Department of Hematology and Oncology, Dana Farber Cancer Institute / Partners Cancer Care, Harvard Medical School, Boston, United States of America

Background: Lenalidomide (LEN) maintenance and continuous LEN-based induction therapy until disease progression have become standard of care for frontline therapy of multiple myeloma (MM). As such, an increasing number of patients (pts) in need of 2nd line therapy have LEN-refractory disease. Optimal treatment in this setting has not been rigorously assessed in randomized studies. The phase I portion of Alliance A061202 demonstrated the safety of the ixazomib-pomalidomide-dexamethasone (IXA-POM-DEX) combination for the treatment of pts with LEN and proteasome inhibitor (PI)-refractory MM.

Aims: In the randomized phase II portion, we evaluated the addition of IXA to POM-DEX for PI naïve / sensitive pts progressing on LEN as part of 1 st line therapy. The primary endpoint was progression-free survival (PFS). Key secondary endpoints included overall response rate (ORR), depth of response, survival and safety.

Methods: Pts were randomized 1:1 to IXA-POM-DEX or POM-DEX and stratified by prior bortezomib exposure, International Staging System stage (1 and 2 vs 3) and the presence of high-risk cytogenetics. POM was administered at 4 mg on days 1 – 21; IXA 4 mg on days 1, 8 and 15; and DEX 20 mg (>75 years (yrs)) or 40 mg (≤75 yrs) on days 1, 8, 15 and 22 of a 28-day cycle. Treatment was continued until disease progression, the emergence of unacceptable side effects or withdrawal of treatment consent.

Results: 38 and 39 eligible pts were assigned to IXA-POM-DEX and POM-DEX, respectively. The median age was 66 yrs (range 41 – 83) and 64 yrs (range 52 – 85). A planned first interim analysis was conducted after 43 out of 57 required events had occurred. PFS favored the IXA-POM-DEX arm (one-sided log rank test value = 4.6345, p=0.03134 [< p-value boundary of 0.058]), yielding a hazard ratio of 0.528 (upper 90% bound = 0.777). A stratified log-rank test found that PFS was superior for the triplet after adjusting for stratification factors (one-sided stratified log rank test value = 5.8371; p=0.0157), adjusted hazard ratio 0.451 (upper 90% bound = 0.694). The ORR favored IXA-POM-DEX (63.2% vs 43.6%, p=0.0853), and the ≥very good partial response was 26.3% vs 5.1%, respectively (p=0.01). The clinical benefit rate (ORR + minimal response rate) was 73.7% and 56.4%. The most common grade 3/4 adverse events included lymphopenia, neutropenia, anemia, and fatigue in 40%, 37%, 16% and 16% of IXA-POM-DEX-treated pts and 26%, 21%, 13%, and 15% of POM-DEX-treated pts. Therapy was discontinued for disease progression in 47.4% of pts on IXA-POM-DEX and 76.9% of pts on POM-DEX and for adverse events in 7.9% and 7.7% of pts, respectively.

Summary/Conclusion: The addition of IXA to the POM-DEX backbone improved the depth of response and PFS for pts relapsing on LEN as part of 1 st line therapy. Hematologic toxicity was increased with the addition of IXA, but side effects were manageable. The ease of administration of this all-oral combination allowed for safer, uninterrupted treatment during the COVID pandemic. Our results should be confirmed in phase III trials but lend support for this regimen as part of 2 nd line therapy for this patient population.

P969: COMPARISON OF VENOUS THROMBOEMBOLISM INCIDENCE IN MULTIPLE MYELOMA PATIENTS RECEIVING LENALIDOMIDE-BASED REGIMENS WITH RIVAROXABAN OR ASPIRIN THROMBOPROPHYLAXIS

Y. Wang 1 , X. Tang 2 , W. Zheng 1 , Z. Wang 1 , J. Xu 1 , X. Tan 2 , Y. Tian 2 , R. Xu 1 , S. Cui 1,*

1 Affiliated Hospital of Shandong University of Traditional Chinese Medicine; 2 Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China

Background: Thromboprophylaxis is routinely used with lenalidomide-based regimens in multiple myeloma because of a substantial risk of venous thromboembolism (VTE). Aspirin is one of the most common thromboprophylaxis agent but also increases bleeding complications.Rivaroxaban represents a selective direct inhibitor of activated coagulation factor X (FXa) having peroral bioavailability and prompt onset of action which can mitigate the risk of higher incidence of VTE without an observable increase in bleeding rates.However, little is known about the incidence of VTE in lenalidomide-based regimens with rivaroxaban thromboprophylaxis.

Aims: The purpose of our study was to compare of venous thromboembolism incidence in multiple myeloma patients receiving lenalidomide-based regimen with rivaroxaban or aspirin thromboprophylaxis.

Methods: We conducted a single-centre retrospective study designed to assess the rates of VTE, including pulmonary embolism (PE) and deep vein thrombosis (DVT), in MM patients receiving lenalidomide-based regimen with aspirin or rivaroxaban thromboprophylaxis.A total of 130 patients who were not anticoagulated for other clinical indications and received at least 1 cycle of lenalidomide-based regimen between January 2015 and December 2021 were included. Clinical bleeding events after treatment were also important indicators in our study.

Results: The incidence of venous thromboembolism in the rivaroxaban group was significantly lower than that in the aspirin prevention group (P<0.05). The incidence of bleeding in the rivaroxaban group was also lower than that in the aspirin prevention group,but there was no statistical significance(P﹥0.05).

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Summary/Conclusion: In this pilot study, we found that rivaroxaban was safe and well-tolerated as the primary preventive measure for venous thromboembolism in patients with multiple myeloma receiving lenalidomide-based regimen. However, these findings warrant further investigation in a larger randomized study to be validated.

P970: SAFETY AND EFFICACY OF BCMA-CAR-T IMMUNE CELLS DERIVED FROM CORD BLOOD IN THE TREATMENT OF RELAPSED AND REFRACTORY MULTIPLE MYELOMA

Y. Wang 1 , X. Hu 1 , Q. Gao 1 , H. Wang 1 , Y. Gao 1,* , W. Zhang 1 , X. Ru 1 , L. Hou 1 , W. Zhou 1 , H. Zhang 1 , Y. Zhang 1 , F. Wang 2 , X. Li 3 , F. Guan 3

1 Department of Hematology, Shaanxi Provincial People’s Hospital, Xi’An; 2 School of Medicine, Shanghai Jiao Tong University, Shanghai; 3 School of Medicine, Northwest University, Xi’An, China

Background: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. The prognosis of MM patients varies, with low-risk patients surviving more than 10 years with standard care and high-risk patients surviving less than 1 year. CAR-T therapy shows promising clinical results on relapsed or refractory MM (R/RMM) patients. However, generating clinically significant doses of CAR-T cells from heavily pre-treated patients for autologous CAR-T therapy is not always possible. Although allogeneic CAR-T cells therapy could potentially overcome such limitations, it poses a considerable risk of graft-versus-host disease (GVHD). Cord blood (CB) is a convenient source of T cells with clear advantages. T cells in CB have low immunogenicity and a relatively low risk of GVHD. Here we present a novel approach to CAR-T cells production from CB, which we hope could overcome the constraints mentioned above.

Aims: The study aims to evaluate the safety and efficacy of the CB-generated BCMA-CAR-T infusion in R/RMM patients.

Methods: This study is based on a clinical investigation during Jan 2021 and Dec 2021 in Shaanxi Provincial People’s Hospital. In this study, CD3+ T cells from the CB samples were selected, activated, and modified by lentivirus to produce anti-BCMA CAR-T cells. The cells were administrated intravenously to patients with R/RMM after expanding for 5-10 days in vitro. 2-3 days after being administered the lymphodepleting chemotherapy regimen of cyclophosphamide and fludarabine, patients received CAR-T cells infusion. We followed the occurrence of adverse events, the level of inflammatory cytokine concentration, CAR-T cells expansion in the peripheral blood, serum monoclonal protein levels, and the proportion of plasma cells in bone marrow smears after CAR-T cells infusion.

Results: This study included 11 patients with R/RMM (7 males and 4 females). The median age was 58 years (ranging from 44 to 65). 18.2% (2/11) of the patients had received other CAR-T therapies and relapsed before the enrolment. 72.73% (8/11) of the patients had the extramedullary disease (EMD). 63.64% (7/11) of the patients had received autologous hematopoietic stem cell transplantation (Auto-HSCT). All patients received CAR-T infusion at the average dose of 7.11 (4.4-15) ×10 6 /kg. After infusion, 18.2% (2/11) of the patients had a fever lasting for 48 hours, and 18.2% (2/11) of them had an increase in heart rate. Nausea and diarrhea occurred in 18.2% (2/11) of the patients. No patients had transient consciousness disorder. No patients received dexamethasone to relieve symptoms. CAR-T cells had expanded in all patients, with no increase in the levels of IL-6, IL-8 and IFN-r in peripheral blood in 2 weeks after infusion. Grade 1 cytokine release syndrome (CRS) was found in 2 patients. No patients had GVHD after infusion. The ORR was 54.5% (6/11), with 18.2% (2/11) of the patients achieving CR, and 36.4% (4/11) achieving PR. Except 1 female died from severe pulmonary infection, other 10 patients were followed up for more than 1 month. Unfortunately, 1 patient who reached CR relapsed during 3 months of follow-up. Those 4 patients who didn’t achieve PR received autologous CAR-T therapy afterwards and had responses better than PR (1 CR, 1 VGPR, 2 PR).

Summary/Conclusion: BCMA-CAR-T cells manufactured from CB show a promising safety profile and certain clinical efficacy for R/RMM patients who are not eligible for autologous CAR-T cells therapy. We also found allogeneic CAR-T cells manufactured from CB could prepare patients for later subsequent autologous CAR-T therapy.

P971: ADJUSTED COMPARISON OF PATIENT REPORTED OUTCOMES FROM CARTITUDE-1 AND LOCOMMOTION COMPARING CILTACABTAGENE AUTOLEUCEL VERSUS REAL WORLD CLINICAL PRACTICE IN TRIPLE-CLASS EXPOSED MULTIPLE MYELOMA

K. Weisel 1,* , M.-V. Mateos 2 , L. Vincent 3 , T. Martin 4 , J. G. Berdeja 5 , A. Jakubowiak 6 , S. Jagannath 7 , Y. Lin 8 , P. Thilakarathne 9 , F. Ghilotti 10 , J. Diels 9 , B. Haefliger 11 , C. Hague 12 , A. Gonzalez 11 , J. M. Schecter 13 , K. S. Gries 14 , V. Strulev 15 , T. Nesheiwat 16 , L. Pacaud 16 , H. Einsele 17 , P. Moreau 18

1 Oncology, Hematology and BMT, University Medical-Center Hamburg-Eppendorf, Hamburg, Germany; 2 CIC, University Hospital of Salamanca/IBSAL, Salamanca, Spain; 3 Clinical Haematology Department, Montpellier University Hospital, Montpellier, France; 4 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco; 5 Sarah Cannon Research Institute, Nashville; 6 University of Chicago, Chicago; 7 Mount Sinai Medical Center, New York; 8 Mayo Clinic, Rochester, United States of America; 9 Health Economics, Market Access & Reimbursement, Janssen Pharmaceutica NV, Beerse, Belgium; 10 Health Economics, Market Access & Reimbursement, Janssen-Cilag SpA, Cologno Monzese, Italy; 11 Health Economics, Market Access & Reimbursement, Cilag GmbH International, Zug, Switzerland; 12 Health Economics, Market Access & Reimbursement, Janssen, High Wycomb, United Kingdom; 13 Research and Development, Janssen R&D, Raritan; 14 Research and Development, Janssen R&D, Los Angeles, United States of America; 15 EMEA Medical Affairs, Janssen Pharmaceutica NV, Beerse, Belgium; 16 Legend Biotech USA, Piscataway, United States of America; 17 Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany; 18 Clinical Hematology, University Hospital Hotel-Dieu, Nantes, France

Background: Adjusted comparisons of the single-arm CARTITUDE-1 clinical trial of ciltacabtagene autoleucel (cilta-cel) and the prospective LocoMMotion study of therapies used in real-world clinical practice (RWCP) have shown the clinical benefits of cilta-cel on response and survival outcomes in patients with triple-class exposed multiple myeloma (TCE-MM). Yet, patients with TCE-MM have a reduced health-related quality of life (HRQoL) compared with age- and sex-matched populations.

Aims: To present adjusted comparisons of patient-reported outcomes (PROs) from TCE-MM patients who received cilta-cel in CARTITUDE-1 vs. RWCP in LocoMMotion.

Methods: EQ-5D-5L, EORTC QLQ-C30 and EORTC QLQ-MY20 questionnaires were administered to all patients in the CARTITUDE-1 Phase 2 and LocoMMotion studies at baseline, day 7, day 28 and every 4 weeks up to 52 weeks. Mixed model repeated measures (MMRM) analyses were performed to analyze changes from baseline (CFB) for each patient cohort and the difference in CFB between cilta-cel and RWCP over time. MMRM models included the baseline PRO score and prognostic characteristics as covariates to balance patient cohorts and to adjust for confounding bias. A sensitivity analysis was implemented assigning worst PRO values to patients who dropped out of the analyses due to death.

Results: A total of 61 patients in CARTITUDE-1 and 202 patients in LocoMMotion had PRO assessments at baseline and during follow-up. At day 7, PRO scores worsened versus baseline in both cohorts, and worsening was more pronounced for cilta-cel in physical, role and social functioning, fatigue and lack of appetite and constipation, coinciding with short term adverse events associated with the cilta-cel infusion and lymphodepleting chemotherapy. From the next assessment at week 4 onwards, PRO values significantly improved over time for cilta-cel patients versus baseline, while improvement from baseline was lower for RWCP for most domains and symptoms. The average improvement versus baseline from week 4 onwards, represented by the absolute difference in CFB between both patient cohorts, was significantly in favor of cilta-cel for Visual Analogue Scale (8.0), Global health status (8.5), pain (-11.4), dyspnea (-8.9), constipation (-8.3), future perspective (16.5) (all p<0.01), emotional functioning (7.4) and feeling restless or agitated (-7.2) (p<0.05) ( Table ). All other PROs were numerically in favor of cilta-cel across the follow-up period, except for nausea and vomiting and lack of appetite, for which small differences in improvement are numerically in favor of RWCP. The sensitivity analyses assigning worst PRO values to patients who dropped out of the main analyses due to death illustrate that these results are inherently biased against the more effective treatment on survival and underestimate the PRO benefit for cilta-cel.

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Summary/Conclusion: Patients with TCE-MM treated with cilta-cel demonstrated significant improvements vs. RWCP increasing over time in multiple PRO endpoints. These findings indicate that cilta-cel can significantly improve patients’ HRQoL in addition to significant efficacy benefits on response, progression free and overall survival and can help address unmet patient needs.

P972: INDIRECT COMPARISON OF TECLISTIMAB IN MAJESTEC-1 VERSUS PHYSICIAN’S CHOICE OF THERAPY IN LONG-TERM FOLLOW-UP OF TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA IN DARATUMUMAB TRIALS

K. Weisel 1,* , A. Chari 2 , S. Z. Usmani 3 , H. Goldschmidt 4 , M.-V. Mateos 5 , K. Qi 6 , A. Londhe 6 , S. Nair 7 , X. Lin 8 , L. Pei 9 , E. Ammann 10 , R. Kobos 9 , J. Smit 11 , T. Parekh 12 , M. Slavcev 10 , P. Moreau 13

1 University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2 Mount Sinai School of Medicine; 3 Memorial Sloan Kettering Cancer Center, New York, United States of America; 4 University Hospital Heidelberg and National Center of Tumor Diseases, Heidelberg, Germany; 5 University Hospital of Salamanca/IBSAL/CIC/CIBERONC, Salamanca, Spain; 6 Janssen Research & Development, Titusville, United States of America; 7 Janssen Pharmaceutica NV, Beerse, Belgium; 8 Janssen Global Services, Horsham; 9 Janssen Research & Development; 10 Janssen Global Services, Raritan; 11 Janssen Research & Development, Spring House; 12 Janssen Research & Development, Bridgewater, United States of America; 13 Hematology Clinic, University Hospital Hotel-Dieu, Nantes, France

Background: Teclistamab (tec), a B-cell maturation antigen × CD3 bispecific antibody, is currently being investigated in MajesTEC-1, a phase 1/2, single-arm trial ( NCT04557098 ) in patients with triple-class exposed relapsed/refractory multiple myeloma (TCE RRMM) who had received ≥3 prior lines of therapy (LOT) and previously exposed to an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 monoclonal antibody. Currently, there are no head-to-head trials comparing the efficacy of tec versus physician’s choice of therapy.

Aims: To evaluate the comparative efficacy of tec versus physician’s choice of therapy.

Methods: Patients in the long-term follow-up of 4 clinical trials of daratumumab (CASTOR, POLLUX, EQUULEUS, and APOLLO) who met the eligibility criteria for MajesTEC-1 were used to create an external control arm for MajesTEC-1. After discontinuation of trial treatments, patients (N=427) subsequently received physician’s choice of therapy, with disease progression and best treatment response assessed by the investigator. The MajesTEC-1 cohort included individual patient-level data from patients who received tec (1.5 mg/kg weekly) at a clinical cutoff of Sep 7, 2021. Inverse probability of treatment weighting with average treatment effect on the treated population was used to adjust for imbalances in baseline covariates of prognostic significance such as International Staging System stage, number of prior LOT, extramedullary plasmacytoma, time since diagnosis, age, hemoglobin, cytogenetic risk, refractory status and progression on last LOT. Efficacy assessments included overall response rate (ORR), rate of complete response or better (≥CR), rate of very good partial response or better (≥VGPR), progression-free survival (PFS), time to next treatment (TTNT), and overall survival (OS) were assessed. For binary endpoints (ORR, ≥CR rate, ≥VGPR rate), odds ratio (OR) along with 95% confidence interval (CI) derived from a weighted logistic regression analysis was used to estimate the relative effect of tec vs physician’s choice of therapy. Hazard ratios (HRs) and 95% CIs were computed using a weighted Cox proportional hazards model for time-to-event endpoints (PFS, OS, and TTNT). Multiple sensitivity analyses were also conducted.

Results: Baseline characteristics were comparable between the 2 cohorts after applying inverse probability of treatment weighting. Patients treated with tec had improved outcomes vs physician’s choice of therapy: ORR (OR 4.58; 95% CI 2.83–7.53; P <0.0001); ≥CR rate (OR 12.62; 95% CI 5.20–38.55; P <0.0001); ≥VGPR rate (OR 11.64; 95% CI 6.49–21.98; P <0.0001); PFS (HR 0.62; 95%CI 0.45–0.84; P =0.0024); TTNT (HR 0.38; 95% CI 0.27–0.52; P <0.0001); and OS (HR 0.47; 95% CI 0.32–0.69; P =0.0001). All sensitivity analyses showed similar results.

Summary/Conclusion: In the present analysis, improved efficacy in all clinical outcomes was observed with tec versus physician’s choice of therapy, highlighting the therapeutic potential of tec to address unmet needs in patients with TCE RRMM who received ≥3 prior LOT.

P973: REAL-WORLD STUDY ON ADOPTION OF STANDARD OF CARE (SOC) FOR FRONTLINE TRANSPLANT-ELIGIBLE MULTIPLE MYELOMA (FLTEMM) PATIENTS BETWEEN 2017 AND 2020/2021 ACROSS FRANCE, GERMANY, SPAIN, AND ITALY

K. Weisel 1,* , A. Wadlund 2 , G. Gungor 3 , E. Dergarabetian 4 , C. Pacheco 5 , N. Masurkar 5 , P. Rodriguez-Otero 6

1 Oncology, Hematology and BMT, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2 Janssen, Janssen-Cilag AB, Kolonnvägen 45, Solna, Sweden; 3 Janssen, Kavacik, Keçeli Plaza, Ertürk Sk. No:13, 34810 Beykoz, Istanbul, Turkey; 4 Janssen-Cilag Limited, 50-100 Holmers Farm Way, High Wycombe, Buckinghamshire, United Kingdom; 5 Cerner Enviza, France SAS, 198 avenue de France, Paris, France; 6 Department of Hematology, Clinica Universidad de Navarra, Pamplona, Spain

Background: Newer therapies for frontline transplant-eligible multiple myeloma (FLTEMM) are underway, but an accurate and updated overview of standard MM treatment management is lacking. Repeated analyses and real-world data are warranted to describe current MM standard of care (SoC), treatment lines, and patient clinical outcomes in daily practice.

Aims: This non-interventional, cross-sectional, retrospective observational database study described the current SoC for FLTEMM patients in France, Germany, Spain, and Italy, and recorded the evolution in regimen adoption in distinct elements of frontline treatment (induction, consolidation, and maintenance) during 2017-2020/2021.

Methods: Clinical information on ongoing (population I) or previous (population II) FLTEMM patients was extracted from the Cancerology database. The primary objective was to describe the treatment regimen considered current SoC for FLTEMM management in all four countries using study population I, and to study the evolution in the use of different regimens in induction phase using study population II. The secondary objective was to describe clinical outcomes, including best tumor response and minimal residual disease (MRD) status, associated with each induction regimen in study population II.

Results: In study populations I and II, the median (min, max) age of the patients at induction initiation was 59.0 (35.0, 86.0) and 60.0 (23.0, 80.0) years and 62.9% (227/361) and 64.8% (287/443) patients were male, respectively. In study population I, the most common induction regimens were bortezomib/lenalidomide/dexamethasone (VRd) in France (75.3%, 33/44) and Spain (44.1%, 53/120), bortezomib/thalidomide/dexamethasone (VTd) in Italy (65.2%, 76/116), and others in Germany (58.9%, 48/81) (including daratumumab-based [19.8%, 16/81] or not). Maintenance was ongoing or planned for 78.3% (34/44), 62.3% (51/81), 65.2% (78/120), and 61.4% (71/116) patients in France, Germany, Spain, and Italy, respectively. Among study population I patients on an ongoing maintenance regimen, lenalidomide was received by 99.6% (20/20) patients in France, 86.8% (34/40) in Germany, 80.2% (45/56) in Spain, and 90.7% (30/33) in Italy. In study population II, VRd use as 1L induction increased from 27.0% (10/36) in 2017 to 65.7% (12/18) in 2019 in France and was relatively low in Germany, Spain, and Italy. The most common 1L induction regimen in Germany was VCd, but its use decreased from 85.2% (31/37) in 2017 to 64.1% (24/38) in 2019. VTd was the most common 1L induction in Spain and Italy, but its use declined from 58.3% (16/27) and 72.4% (34/47) in 2017 to 17.3% (4/22) and 52.8% (19/36) in 2019, respectively. In total, 37.5% (166/443) study population II patients achieved at least a complete response, which was the highest for those on VRd (59.2% versus 34.3% for VTd, 33.4% for VCd, and 33.7% for other non-daratumumab-based regimens, respectively). In total, 42.5% (188/443) study population II patients were tested for MRD status and 18.2% (81/443) were found negative. MRD testing rate varied from 26.2% in France to 14.8% in Germany, 84.4% in Spain, and 47.7% in Italy.

Summary/Conclusion: The use of bortezomib triplets in induction varied markedly over time and between selected countries. Despite not being approved by the European Medicines Agency specifically for FLTEMM patients, VRd use tended to increase with time in France and to a lesser extent in Spain and Italy in this population.

P974: THE CLINICAL CHARACTERISTICS AND PROGNOSIS OF PATIENTS WITH PRIMARY PLASMA CELL LEUKEMIA (PPCL) UNDER THE NEW IWMG DEFINITION CRITERIA

W. Yan 1,* , J. Xu 1 , H. Fan 1 , J. Liu 1 , L. Li 1 , L. Qiu 1 , G. An 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Primary plasma cell leukemia has been described as a distinct entity of plasma cell disorders with special features and poor prognosis. In 2021, IMWG proposed that revised the diagnostic criteria of PCL as CPCs ≥ 5% by peripheral blood (PB) smear. As the new diagnostic standard has been established recently, it is of great significance that illustrate the clinical characteristics and outcomes of the new pPCL cohort.

Aims: Given the limited data for the rare entity, we performed a large retrospective analysis about the clinical characteristics, survival outcomes, and risk factors for the new-defined pPCL patients treated in our hospital.

Methods: We conducted a retrospective analysis of 158 pPCL patients diagnosed from 2000 to 2019 in our hospital. This pPCL cohort was redefined from NDMM patients who appear CPCs ≥ 5% by morphologic evaluation of their PB smear. And we compared them to a control group with 485 NDMM patients. Statistical analyses were performed using the R version 4.1.2.

Results: The characteristics and comparisons between pPCL patients and NDMM group are depicted in Table 1.In general, bone marrow suppression and adverse prognostic biomarkers (ie, anemia, thrombocytopenia, elevated LDH, hypodiploidy, and high-risk cytogenetics) were more common in pPCL patients compared with NDMM patients (P<0.05).

For 130 pPCL patients whose treatment and prognosis data were available, with a median follow-up time of 54.7 months, the median PFS and OS were 16.9 months and 30.0 months respectively, both significantly longer than the control NDMM patients (P<0.001). Interestingly, we find the cohort who attended after 2007 got obvious longer PFS than those who received treatment in 2000-2006(20.6 vs 10.0 months, P=0.017), which showed a similar result in OS (31.6 vs 16.0 months, P=0.034).

A Cox-regression multivariate analysis was performed among the baseline variables. The presence of hypodiploidy and elevated serum LDH were found to be prognostic for worse PFS, whereas age＞60 and elevated LDH were the independent predictors for worse OS. As for the cytogenetic aberrations, did not play an important role in the outcome of pPCL patients other than the presence of del(17p) which could impact OS in the univariate analysis.

There are 98 patients who had response data known, with 80(81.6%) patients achieving objective response in the first-line treatment, 56.4%≥VGPR, and 38.9% CR. When stratifying the cohort by the best response, the median PFS for patients achieved: NR, PR, and ≥VGPR were 2.4, 11.2, and 31.0 months, respectively (P＜0.001). The OS for patients who had ever achieved NR, PR, and≥VGPR were 2.4, 24.9, and 62.1 months, respectively (P＜0.001). Then, we divided the cohort who had achieved≥PR into early response group (≤2 courses), intermediate response group (3-4 courses), and late response group (＞4 courses). Whereas, there is no statistical difference in the prognosis of 3 groups with diverse remission speed rates (PFS: P=0.35; OS: P=0.77). We also found that achieving deep remission was the best independent favorable predictor for long survival in the multivariate analysis.

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Summary/Conclusion: In conclusion, primary plasma cell leukemia (pPCL) defined by the new revised diagnostic criteria (CPCs≥5%), remains an aggressive disease characterized with poor prognosis despite the advancement of treatment regimens. And achieving deep remission (≥VGPR) in the first-line therapy predicts the best prognosis regardless of response rate.

P975: INTERIM ANALYSIS OF PHASE II STUDY OF DARATUMUMAB IN COMBINATION WITH BORTEZOMIB AND DEXAMETHASONE IN PATIENTS WITH MULTIPLE MYELOMA WHO RECEIVED 1 PRIOR LINE OF THERAPY (KMM1906)

K. H. Yoo 1,* , G. W. Gang 2 , J. H. Yi 2 , M. K. Kim 3 , H. J. Kim 4 , S.-H. Kim 5 , J. S. Park 6 , J.-J. Lee 7 , C.-K. Min 8 , J. H. Lee 1 , D. Cho 9 , K. Kim 10

1 Division of Hematology, Department of Internal Medicine, Gachon University Gil Medical Center, Gachon University College of Medicine, Incheon; 2 Division of Oncology and Hematology, Department of Internal Medicine, Korea University Medical Center, Seoul; 3 Department of Internal Medicine, Yeungnam University College of Medicine, Daegu; 4 Division of Hematology/Oncology, Department of Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang; 5 Department of Internal Medicine, Dong-A University College of Medicine, Busan; 6 Department of Hematology-Oncology, Ajou University School of Medicine, Suwon; 7 Department of Hematology-Oncology, Chonnam University Hwasun Hospital, Hwasun; 8 Department of Internal Medicine, Seoul St. Mary’s Hospital, The Catholic University of Korea; 9 Department of Laboratory Medicine and Genetics; 10 Division of Hematology and Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea

Background: Daratumumab in combination with bortezomib and dexamethasone (DVd) demonstrated efficacy in heavily pretreated patients with relapsed or refractory multiple myeloma (MM). In a previous pivotal phase 3 study, DVd showed better efficacy in patients receiving only first-line treatment, while the limited administration of bortezomib and dexamethasone for 8 cycles was criticized.

Aims: We conducted a phase 2 study with DVd regimen in patients with MM who received 1 prior line of therapy.

Methods: We evaluated daratumumab (16 mg/kg IV), bortezomib (1.3 mg/m 2 SQ), and dexamethasone (20 mg IV or PO) with maintaining all three drugs after 9 cycles until disease progression or unacceptable toxicity. The primary endpoint was objective response rate ≥ very good partial response (VGPR). Secondary objectives included progression-free survival (PFS), overall survival (OS), safety and tolerability, and minimal residual disease (MRD)-negativity. MRD was assessed in patients who achieved in stringent complete response (sCR) or CR, and who maintained VGPR over 6 months, with EuroFlow-based next-generation flow (NGF).

Results: From June 2020 to June 2021, 26 MM patients who received 1 prior line of therapy and disease progressed were enrolled at 10 centers in Korea. The median age of all patients was 72 years (range, 47-85), and 8 patients were male. Five patients (19%) had high-risk cytogenetic abnormalities (t(4;14), t(14;16), or del17p), and 6 patients (23%) had extramedullary disease. All patients were treated with 1 prior line, including VTD (N = 11, 42%), VMP (N = 8, 31%), Rd (N = 6, 23%), and CMP (N = 1, 4%). Nineteen patients (73%) and 18 patients (69%) were exposed to bortezomib and immunomodulatory drugs, respectively, from previous treatment (Table 1). During the median follow-up period of 11.7 months, 15 patients (58%) maintained treatment, and 11 patients (42%) discontinued the study due to disease progression (N = 6), death from other causes (N = 2), or withdrawal of consent (N =3). 16 patients (62%) achieved ≥ VGPR (2 sCR, 7 CR, and 7 VGPR). The MRD-negativity (10 -5 ) was 5/12 (42%) (Table 2). The treatment response and PFS of each patient were given in the swimmer plot (Figure 1). The most common adverse event (AEs) ≥ grade 3 was thrombocytopenia (N = 6, 23%). Serious AEs were reported in 8 patients (31%). Dose delay or dose reduction have occurred in 17 patients (65%).

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Summary/Conclusion: Among patients with MM who received 1 prior line of therapy, DVd regimen with maintenance strategy showed an acceptable clinical response and MRD-negativity with manageable toxicity profile.

P976: BENDAMUSTINE-POMALIDOMIDE-DEXAMETHASONE (BPD) FOR RELAPSED AND/OR REFRACTOR MULTIPLE MYELOMA WITH EXTRAMEDULLARY DISEASE

Z. Yuping 1,* , H. Y. Wu 1 , X. Chu 2 , X. Deng 3 , X. Feng 4 , C. Yuan 5 , X. Ran 6 , G. Liu 7 , C. Fan 8 , H. Hao 5 , X. Zhou 1

1 Qingdao Municipal Hospital, Qingdao City, Shandong Province; 2 The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai City, Shandong Province; 3 Weihai municipal hospital, Weihai City, Shandong Province; 4 The Affiliated Hospital of Qingdao University (West Coast Hospital area), Dongying City, Shandong Province; 5 QILU HOSPITAL OF SHANDONG UNIVERSITY(Qingdao), Qingdao City, Shandong Province; 6 Weifang people’s Hospital, Weifang City, Shandong Province; 7 Shengli Oilfield Central Hospital, Dongying City, Shandong Province; 8 Department of Hematology, Qingdao Hospital of Traditional Chinese Medicine (Qingdao Hiser Hospital), Qingdao City, Shandong Province, China

Background: Extramedullary disease (EMD) is a rare manifestation of multiple myeloma (MM). The incidence is about 6-20% in the relapsed MM patients. Although novel agents prolong the survival of patients with MM, patients with EMD are prone to relapse or progress again easily,EMD may be associated with decreased overall survival in MM. No standard therapy has been established for this high-unmet need population. Bendamustine is an old bi-functional alkylating agent which has proved to be effective in Multiple Myeloma (MM). Pomalidomide is a potent immunomodulatory drug agent with anti-angiogenic, anti-proliferative, and immunomodulatory activity against MM. Preclinical studies showed that bendamustine and pomalidomide had higher penetration rate of cerebrospinal fluid. In China, the price of two drugs is relatively economical. In a phase I/II study,the combination of BPd demonstrates deep Response(ORR:61%) and has a favorable tolerance profile in patients with RRMM. To summarize,BPD is an safe,effective, and economical treatment, and there are no studies of BPD in the regimen of RRMM with EMD,so it is worth exploring in domestic clinical trials.

Aims: To evaluate the efficacy and safety of bendamustine, Pomalidomide, and dexamethasone (BPd) for relapsed / refractor multiple myeloma with EMD.

Methods: This is an open-label, multicenter, prospective study（ChiCTR2100048829）.Eligible patients were aged≥18 years, and had a diagnosis of RRMM with EMD, and an Eastern Cooperative Oncology Group performance status of 0-2,Patients received bendamustine 75 mg/m 2 on days 1 and 2(If there is no severe side effects in the first cycle, it can be increased to 80mg/m 2 ), 6 cycles. Pomalidomide 4 mg on days 1-21,until progression or intolerable toxicity. Dexamethasone 20mg on days1-4, 15-18, until progression or unacceptable toxicity. Each cycle is 28days. The primary endpoint is overall response rates (ORR).

Results: A total of 21 patients were enrolled in the study,12(57.14%) patients with first relapse multiple myeloma. The median age is 57.4years(47-72). According to IMWG criteria, all first relapse patients responded(ORR100%)and CR:3pts(25%),VGPR:7pts(58.33%),PR:2pts(16.67%).Patients with ≥2 nd relapese, the ORR was44.44%,≥VGPR:0pts(0%), PR:4pts(44.44%),SD:3pts (33.33%),PD:2pts(22.22%).This results established that BPD has a deep Response in first relapse MM with EMD.The 1-year PFS was79.16%of all patients.The most common grade 3 or 4 hematologic toxicities were neutropenia7(33.33%),thrombocytopenia4(19.05%),anemia 2(9.52%). Overall,the severe toxicities are manageable.

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Summary/Conclusion: Safety data from the study demonstrates that BPD regimen has a favorable tolerance profile in patients for RRMM with EMD. Early efficacy is encouraging, Longer-term results still require more patients have been enrolled and further follow-up. The combination of BPD will be an effective treatment strategy in RRMM with EMD.

P977: BISPECIFIC CS1-BCMA CAR-T CELLS ARE CLINICALLY ACTIVE IN RELAPSED OR REFRACTORY MULTIPLE MYELOMA

C. Li 1,2 , X. Wang 1,2 , Z. Wu 1,2 , W. Luo 1,2 , Y. Zhang 1,2 , M. Du 1,2 , C. Lu 1,2 , H. Kou 1,2 , Y. Kang 1,2 , J. Xu 1,2 , P. Huang 1,2 , W. Xiong 3 , J. Zheng 1 , J. Deng 1 , Y. Hu 1,2 , H. Mei 1,2,*

1 Institute of Hematology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology; 2 Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease; 3 Wuhan Sian Medical Technology Co., Ltd, Wuhan, China

Background: BCMA-targeted CAR-T cell therapy has showed high response rates in multiple myeloma (MM); however, remission is transient in most of patients (pts). Variable and even absent BCMA expression on MM cells have been documented after single BCMA-targeted CAR-T cell treatment. CS1 (CD319, SLAMF7) plays a vital role in myeloma pathogenesis, including promoting MM cells growth, survival and adhesion. CS1 is highly expressed on tumor cells in almost all MM pts, and is also retained at significant levels at relapse. Therefore, we propose to augment BCMA targeting with CS1. Bispecific CS1-BCMA CAR-T cells are effective in targeting MM cells in preclinical studies ( Biomedicines 2021, 9 , 1422).

Aims: Here we report the outcomes of 13 pts with refractory or relapsed (RR) MM in our phase I clinical trial ( NCT 04662099 ).

Methods: CS1-BCMA bispecific CAR contained a murine anti-CS1 scFv (clone 7A8D5) and a murine anti-BCMA scFv (clone 4C8) in tandem, and a 4-1BB costimulatory domain (Figure 1 A ). The enrolled pts must have received at least 2 prior lines of therapy, and previous BCMA- or CS1-targeted immunotherapies were allowed. Pts were subjected to lymphodepleting regimens with cyclophosphamide (250 mg/m 2 , d-5 to d-3) and fludarabine (30 mg/m 2 , d-5 to d-3) daily prior to the CAR-T infusion (d0). Planned dose levels were 0.75, 1.5, and 3.0x10 6 CAR+T cells/kg, and repeated infusions were allowed. Primary objectives were incidence of adverse events. Cytokine release syndrome (CRS) and neurotoxicity were graded using the ASTCT criteria, and other AEs using CTCAE v5.0. Secondary objectives were overall response rate(ORR), overall survival(OS), duration of response (DOR), and progress-free survival (PFS). Response was assessed per the IMWG criteria (2016). Other objectives included in vivo kinetics of CAR-T cells.

Results: As of February 10, 2022, 13 pts received the infusion of CS1-BCMA CAR-T cells and were included in the final analysis. Six pts (46%) carried cytogenetic abnormalities, and 3 pts had only extramedullary diseases (EMD) without detectable MM cells in bone marrow (BM). BCMA and CS1 were highly expressed on MM cells in BM (median BCMA+ 77.2% and CS1 + 96.5%) and EMD (Figure 1 B ). Eight pts (62%) were refractory, and median prior lines of treatment was 5.5 (range 2-10), and 7 pts (54%) received autologous stem cell transplantation.

Four pts (31%) experienced CRS, and grade 3 CRS occurred once and lasted for 5 days. Neurotoxicity was not observed. The ORR is 76.9% (10/13), including 4 stringent complete, 2 very good partial, and 4 partial response (Figure 1 C ). Although CAR-T cells infiltrated into the tumor tissue, response was not observed in the 3 pts with only EMD (Figure 1 D ). Soluble BCMA decreased remarkably in peripheral blood (PB) and BM (Figure 1 E ). The median follow-up and DOR were 290 days. The median OS and PFS weren’t reached, and OS and PFS at 9-month were 77.1%.

On day 14 after infusion, CAR-T cells peaked at 464842 copies/ug DNA in PB (n=13) and 282920 copies/ug DNA in BM (n=10) by droplet digital PCR; CAR-T cell expanded at 400 CAR+T cells/ul PB (n=13) and 271 CAR+T cells/ul BM (n=10) by flow cytometry. CAR was detected in vivo at a median of 5 months. Moreover, we systematically evaluated the immune characteristics of infused products (Figure 1 F ). Limited by small sample size, correlation analysis wasn’t done

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Summary/Conclusion: Our study demonstrates bispecific CS1-BCMA CAR-T cells are clinical active with a good safety profile in heavily pretreated patients with MM.

P978: IXAZOMIB VERSUS LENALIDOMIDE OR IXAZOMIB AND LENALIDOMIDE COMBINATION AS MAINTENANCE REGIMEN FOR PATIENTS WITH MULTIPLE MYELOMA: INTERIM ANALYSIS OF A MULTI-CENTER PROSPECTIVE STUDY IN CHINA

Z. Zhuang 1 , Y. Tian 2 , H. Yu 3 , L. Shi 4 , W.-W. Tian 5 , Q. Liu 6 , D. Zou 7 , F. Dong 8 , Y. Ma 9 , R. Feng 10 , S. Liu 1 , H. Liu 10 , H. Jing 8 , W. Sun 7 , L.-M. Ma 5 , L. Bao 4 , R. Fu 3 , Y. Wu 2 , W. Chen 2 , J. Zhuang 1,*

1 Department of Hematology, Peking Union Medical College Hospital; 2 Department of Hematology, Beijing Chao-Yang Hospital of Capital Medical University, Beijing; 3 Department of Hematology, Tianjin Medical University General Hospital, Tianjin; 4 Department of Hematology, Beijing JishuitanHospital, Beijing; 5 Department of Hematology, Shanxi Bethune Hospital of Shanxi Medical University, Taiyuan; 6 Department of Hematology, The First Affiliated Hospital of Anhui Medical University, Hefei; 7 Department of Hematology, Xuanwu Hospital of Capital Medical University; 8 Department of Hematology, Peking University Third Hospital, Beijing; 9 Department of Hematology, The second Hospital of Shanxi Medical University, Taiyuan; 10 Department of Hematology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science, Beijing, China

Background: Maintenance therapy (MT) deepens response and prolongs progression free survival (PFS) in patients with newly diagnosed multiple myeloma (NDMM) after frontline regimens. Ixazomib, a 2 nd generation oral proteasome inhibitor (PI), has been approved for MT because of its convenience and tolerability.

Aims: We conducted this prospective multi-center study to compare the efficacy and safety of Ixazomib (I-MT) or Ixazomib plus Lenalidomide (IL-MT) to Lenalidomide (L-MT) as maintenance regimen in NDMM patients.

Methods: This study was approved by the Institutional Review Board of Peking Union Medical College Hospital and registered ( NCT04217967 ). NDMM patients were enrolled from 10 centers of North China MM Registry since September 2019. After 4 cycles of front-line induction therapy, patients reached partial response (PR) would receive autologous stem cell transplantation (ASCT) if eligible, or keep up to 5 cycles of front regimens if ineligible, then start maintenance therapy. Patients did not reach PR would switch to 2nd-line induction for 2-5 cycles and start MT once PR was achieved. For MT, 4mg of Ixazomib was given on day 1,8,15, and 25mg of Lenalidomide every other day on days 1–21 of 28day cycles. Patients in dual drug group were administrated with both Ixazomib and Lenalidomide, dose as listed above. The primary endpoint was PFS from MT.

Results: A total of 176 patients were enrolled, including 58 in I-MT, 72 in L-MT and 46 in IL-MT. The demographic and clinical characteristics, including gender ratio, age, paraprotein isotype, ISS, R-ISS, were comparable among different MT regimen groups either receiving ASCT or not at baseline (Table 1). The proportions of patients with high-risk cytogenetic abnormalities (HRCAs), defined as amplification 1q21, deletion 17p, t(4,14) and t(14,16), were lower in L-MT without ASCT and I-MT with ASCT.

The median follow-up duration since MT was 10.6, 12.6 and 11.1 months in I-MT, L-MT and IL-MT groups in non-ASCT patients, while 9.3, 14.0 and 12.2 months in ASCT patients, respectively. In non-ASCT groups, the disease progression rates were 13.5%, 10.2% and 10.8%, whereas 0, 21.7% and 0 in ASCT groups. The median PFS and OS were not reached (NR) in all groups. There were 73.1%, 73.4% and 78.3% of the patients reached VGPR or better before MT in non-ASCT patients, while the rates improved to 80.8%, 81.6% and 86.5% during follow-up. The depth of response did not change in ASCT patients.

In the whole cohort, the prevalence of peripheral neuropathy (PN) was 18.9% on I-MT, 8.3% on L-MT and 21.7% on IL-MT. Grade 2 PN occurred in 3, 0 and 2 patients, respectively. The incidence of gastrointestinal (GI) events was 12.1%, 1.4% and 15.2%, respectively. Grade 3 GI events occurred in 3, 0 and 1 patients. The incidence of grade 3-4 hematologic toxicities was 1.7%, 4.2%, and 2.2%. Infections developed in 10.3%, 2.8% and 4.3% patients. No drug withdrawal was related to adverse events.

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Summary/Conclusion: Due to inadequate access to melphalan and low rate of ASCT, the PFS of NDMM in Chinese patients was relatively inferior. We design this multi-centered prospective study to evaluate if dual drug MT will further strengthen response and make up the gap. Though the primary endpoint--PFS has not been reached in all treatment groups, dual MT improves response most and is quite tolerable.

P979: EXOSOMES IN POLYCYTHEMIA VERA: “MINI PLATELETS“ WITH THROMBOGENIC POTENTIAL

A. Abulafia 1,2,* , G. Spectre 1,2 , E. Ziv 3 , K. R. Geiger 1 , Z. Sarsor 3 , N. Ron 2,3 , E. Beery 3 , S. Revel-Vilk 4,5 , M. Naamad 6 , P. Raanani 1,2,3 , O. Uziel 2,3 , U. Rozovski 1,2

1 Institute of Hematology, Rabin Medical Center, Petah Tikva; 2 Sackler School of Medicine, Tel Aviv University, Tel Aviv; 3 Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva; 4 Gaucher Unit, Pediatric Hematology/Oncology Unit, Shaare Zedek Medical Center; 5 Faculty of Medicine, Hebrew University of Jerusalem; 6 Flow Cytometry Unit, Shaare Zedek Medical Center, Jerusalem, Israel

Background: Polycythemia vera (PV) is characterized by clonal proliferation of myeloid progenitor cells that is caused by activating mutation in the Janus Kinase (JAK)2 gene. Patients with PV tend to develop thrombotic complications across the vasculature tree. Yet, how PV-clonal cells induce pro-thrombotic environment is still under investigation. Secreted by all types of cells, exosomes are nano-scaled particles which carry a molecular cargo that reflects their cell of origin. Exosomes travel in blood, may reach distant sites and affect various physiological and pathological processes. These features led us to think that PV-derived exosomes (hereafter PV-exosomes) carry the mutated JAK2 oncogene and contribute to the thrombotic manifestations seen in PV.

Aims: Determine whether PV-exosomes carry mutated JAK2 transcripts.

Determine the prothrombotic potential of PV-exosomes and their effect on endothelium.

Methods: Exosomes were isolated from eythroleukemia (HEL 92.1.7) cell line (positive for mutated JAK2) and from patients with PV by serial ultracentrifugation. To confirm the presence of exosomes, electron microscopy images were obtained and to quantify their concentration nanoscale tracking analysis was applied (Malvern).

The exosomes were stained with the FM-4-11 lipophilic dye and their uptake by HUVECs and HaCaT cells was followed by flow cytometry. To determine the presence of JAK2 transcript, a 465 bp long DNA segment within exon14 of JAK2 was amplified by qRT-PCR and by Sanger sequencing JAK2 mutational status was determined. Thrombin generation assay (TGA) (Ceveron®, Alpha, Technoclone) was used to assess the thrombotic potential of PV-exosomes. Platelet activation status was assessed by the expression of PAC-1, CD-62 and CD-63 in platelets and Trans Epithelial Electric Resistance (TEER) (Electric cell substrate impedance sensing device, Applied Biophysics) was used to assay endothelial dysfunction in HUVECs.

Results: We isolated exosomes from the sera of 24 healthy donors and 31 patients with PV. We found the presence of the mutated JAK2 mRNA in all samples confirming that the mutated JAK2 transcript is packaged into sera-exosomes. Yet, while healthy donors’ derived exosomes carry only the wild type JAK2 mRNA, we detected both the wild type and the mutated JAK2 isoforms in patients with PV.

We showed that PV-exosomes induced thrombin generation. Then we compared PV-exosomes and healthy-individuals’ derived exosomes and found that thrombin generation potential of PV-exosomes is higher in all measured parameters (p< 0.05 in all parameters). Furthermore, using various platelet markers (CD62, CD63, PAC1) we confirmed that PV-exosomes induced platelet activation. Together, these findings suggest that PV-exosomes may promote thrombosis directly.

Under physiological conditions the integrity of the vascular wall prevents uncontrolled thrombi. Therefore, we wondered whether PV-exosomes induce endothelial dysfunction, and in this way contribute indirectly to the pro-thrombotic phenotype. PV-exosomes were taken up by HUVEC endothelial cells in a dose- and time- dependent manner and TEER assay revealed that exosomal uptake by HUVECs induced endothelial dysfunction by breaking the tight junction between adjacent endothelial cells.

Summary/Conclusion: Exosomes may act as “mini platelets” that promote thrombosis in patients with PV. PV-exosomes may promote thrombosis either directly by activating platelets and inducing thrombin generation or indirectly by inducing endothelial dysfunction.

P980: JAK2V617F-DEPENDENT DOWN REGULATION OF SHP1 EXPRESSION PARTICIPATES IN THE SELECTION OF MPN CELLS IN THE PRESENCE OF TGFΒ

C. Aoun 1,* , N. Salomao 1 , N. Maslah 1 , S. Awan Toor 1 , G. Letort 1 , P. Gou 1 , J.-J. Kiladjian 2 , S. Giraudier 1 , B. Cassinat 3

1 INSERM U1131-hopital saint louis, Paris, France; 2 Centre d’investigations cliniques; 3 Laboratoire de biologie cellulaire, INSERM U1131-Hopital Saint Louis, Paris, France

Background: Myeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells, due to acquisition in HSC of mutations in the JAK2, CALR or MPL genes. The JAK2V617F mutation, leading to a constitutively active form of JAK2, is the most frequent mutation. During disease evolution the clonal selection of mutated cells is dependent on cell-intrinsic and extrinsic factors present in the niche microenvironment such as cytokines. TGFβ, whose local secretion is increased in MPNs, and more particularly in myelofibrosis is known to negatively regulate normal hematopoietic stem cells (HSC) proliferation. However, its mode of action is not clearly established. We hypothesized that JAK2V617F mutant HSC are resistant to TGFβ antiproliferative effect explaining the pathological stem cell invasion in myelofibrosis. Using multiomics approaches to study the intracellular signaling pathways in MPNs we recently identified the down regulation of SHP-1 expression in JAK2V617F cells compared to wild type cells. Of note, the phosphatase SHP-1 has recently been shown to be required for the TGFβ-induced quiescence of HSCs.

Aims: We hypothesized a relationship between SHP-1 down regulation and the resistance of JAK2V617F mutated cells to the antiproliferative effect of TGFβ. The work presented herein aimed at verifying this hypothesis.

Methods: We used the HEL erythroleukemia cell line which carries several copies of the JAK2V617F mutated gene. We also used an isogenic model obtained by transducing either the JAK2 wild type (JAK2wt) of JAK2V617F allele in the UT-7 megakaryoblastic cell line. Proliferation was measured using the CCK8. Protein expression was measured using western blot while gene expression was measured using RT-qPCR.

Results: The expression of SHP-1 was reduced by 30% at the RNA level and 50% at the protein level in UT-7 cells transduced with JAK2V617F compared to JAK2wt -transduced cells. In bone marrow cells taken from JAK2V617F knock-in mice the protein level of SHP1 was reduced by 70% compared to wild-type mice. EPO treatment of UT-7 cells (which express the EPO-R) showed a down regulation of SHP-1 expression which was abrogated in the presence of the JAK2 inhibitor ruxolitinib. These results suggest that SHP-1 expression is modulated by JAK2 signaling leading to its down regulation in JAK2V617F cells. Although TGFβ receptor and SMAD2/3 expression was similar in both cell type, JAK2V617F UT-7 cells were less responsive to TGFβ signaling compared to JAK2wt cells (reduced phosphorylation of SMAD2/3). The proliferation of JAK2wt UT-7 cells was significantly reduced in the presence of TGFβ 10ng/mL while JAK2V617F UT-7 cells were not affected. To confirm the involvement of SHP-1 we transduced HEL cells with the cDNA coding for SHP-1 in order to restore higher levels of the protein in this JAK2V617F mutated cell line and observed a significant reduction in the proliferation when treated with TGFβ compared to cells transduced with the empty vector. Finally, we performed an in vitro competitive assay where JAK2V617F UT-7 cells transduced by an m-cherry fluorescent vector were mixed 50/50 with JAK2wt UT-7 and cultured with TGFβ. After 14 days of culture with TGFβ 10ng/mL we observed a significant positive selection of the JAK2 mutant cells over JAK2wt cells.

Summary/Conclusion: In conclusion, we found a JAK2-dependent down-regulation of SHP1 expression which is related to the resistance of JAK2V617F cells to the antiproliferative effect of TGFβ explaining clonal selection of mutant stem cells in myelofibrosis

P981: NEXT GENERATION SEQUENCING (NGS): AN IMPORTANT TOOL TO CHARACTERIZE MYELOPROLIFERATIVE DISEASES IN CHILDREN

S. Bianchi 1,* , G. Palumbo 1 , V. Filipponi 1 , N. Monaco 1 , G. Pileggi 1 , R. Maglione 1 , M. Rousseau 1 , M. L. Moleti 1 , F. Giona 1

1 Translational and Precision Medicine, Sapienza, University of Rome, Rome, Italy

Background: Mutations in the JAK2, MPL and CALR driver genes are reported in over 90% of adults with BCR-ABL1 -negative chronic myeloproliferative neoplasms (MPNs) and in 22-40% of children, where inherited forms, such as familial erythrocytosis (FE) and hereditary thrombocytosis (HT), are common. Next Generation Sequencing (NGS) is useful to identify clonal markers, other than those found using standardized methods.

Aims: This study was carried out in order to: a) confirm the results obtained in our preliminary experience, using NGS in a larger number of children and adolescents with myeloproliferative diseases (MPDs); b) identify non-canonical driver and/or non-driver mutations in patients (pts) with MPDs; c) evaluate long-term outcome of disease in different subgroups of pts.

Methods: 86 pts (46 male, 41 female; median age at diagnosis: 15 years) with a BM evaluation, molecular analysis for driver mutations for MPN (JAK2V617F, JAK2 exon 12, CALR, MPL), and genes involved in the FE (HIF2α) and HT (MPL), were studied in NGS.

Results: Driver mutations were found in 52/87 pts (60%) (16 JAK2V617F, 10 CALR, 21 MPLS505N, 2 MPLV501A, and 3 HIF2α). A 44-NGS gene panel providing diagnostic information in myeloid malignancies and in rare inherited erythrocytosis/thrombocytosis were performed in 71/86 pts (82.5%), 28 of them (39.5%) without any mutations. The 44-NGS gene panel detected non canonical driver mutations in 5/28 (18%) triple negative pts, and additional non canonical driver mutations in 2 JAK2V617F and CALR type 2 pts. Overall, driver mutations were found in 61/86 (71%) of pts. The NGS panel revealed 28 additional functional non-driver mutations (12 mutations in 5 genes of the signaling group, 5 mutations in 2 genes of the mRNA splicing group, 6 mutations involving the ASXL1 gene of the histone modification group, and 5 mutations involving 2 genes of the DNA methylation group) in 29 pts, 16 of them with no driver mutations.

Pts with acquired MPNs were retrospectively classified according to the WHO 2016 criteria; pts with HIF mutations and/or anamnestic criteria of familial erythrocytosis were considered as FE, pts with MPLS505N, or MPLV501A mutations were defined as HT (Table1). One or more non-driver mutations were found in 17/37 (46%) TE, 4/15 (26.5%) PV and mPV, 6/9 (66.6%) FE pts, and in 0/23 HT pts. During follow-up, 20 pts (12 ET, 5 HT, 2 PV and 1 pre-PMF) presented progressive splenomegaly, after a median of 120 months; in addition, 10 (6 ET, 3 HT, and 1 pre-PMF) of them developed a ≥2 BM fibrosis, after a median of 188 months. Considering driver mutations, 8/20 pts were JAK2V617Fmutated, 5 had CALR mutations, and 5 were MPLS505N mutated; 2 pts were wild type. One or more additional non-driver mutations were present in 3/5 CALR, and in 3/8 JAK2V617Fmutated pts. Overt MFI developed in 3 pts, 2 of them needed hematopoietic stem cell transplantation. Two pts developed a malignant neoplasia. Six (7%) pts, 3 MPLS505N, 2 JAK2V617F, and one wild type, without thrombophilic abnormalities, experienced a thrombotic event. All pts are alive after a median time of 15 years from initial diagnosis.

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Summary/Conclusion: In our experience, the use of a 44-gene NGS panel enabled us to identify non canonical driver, and functional non-driver mutations, in addition to those detected by conventional methods, in about 90% of pediatric pts with acquired and familial MPDs. Additional clonal mutations have been identified in two thirds of FE pts, but none in HT pts. Interestingly, the development of BM fibrosis was observed in pts with HT, but not in FE group.

P982: MYELOID-DERIVED SUPPRESSOR CELLS: CHARACTERIZATION IN PERIPHERAL BLOOD AND SPLEEN TISSUE OF PATIENTS WITH MYELOFIBROSIS

R. Campanelli 1,* , A. Bonometti 2,3 , M. Massa 4 , C. Abbà 1 , L. Villani 1 , A. Carolei 1 , P. Catarsi 1 , M. Paulli 3 , G. Barosi 1 , E. Boveri 3 , V. Rosti 1

1 Center for the Study of Myelofibrosis, General Medicine 2—Center for Systemic Amyloidosis and High-Complexity Diseases, IRCCS Policlinico San Matteo Foundation, Pavia; 2 IRCCS Humanitas Research Hospital, Milan; 3 Department of Diagnostic Medicine, Anatomic Pathology Unit; 4 General Medicine 2—Center for Systemic Amyloidosis and High-Complexity Diseases, IRCCS Policlinico San Matteo Foundation, Pavia, Italy

Background: Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that accumulate in patients with malignancies, sepsis or chronic inflammation. Features of MDSCs are: myeloid origin, immature state, strong ability to reduce cytotoxic functions of T/NK cells, potential to differentiate into endothelial cells favoring neoangiogenesis. MDSCs are conventionally divided into polymorphonuclear (PMN)- and monocytic (M)-MDSCs. PMN-MDSCs are characterized by the production of reactive oxygen species and arginase-1 (Arg-1), that directly inhibits T-cell proliferation and activation, while M-MDSCs predominantly express inducible nitric oxide synthase and produce nitric oxide.

Myelofibrosis (MF) is characterized by clonal expansion of a hematopoietic stem cell, extramedullary hematopoiesis, bone marrow (BM) fibrosis, splenomegaly, BM and splenic neoangiogenesis and an acquired mutation of JAK2 , CALR or MPL gene. Inflammation plays a relevant role in MF pathogenesis, as proven by high levels of inflammatory cytokines with prognostic significance and by a state of chronic oxidative stress.

Aims: To characterize circulating MDSCs and assess their localization and quantification in spleen samples from MF patients.

Methods: Peripheral blood mononuclear cells (PBMCs) from MF patients (n=15) and healthy subjects (HDs; n=10) were stained with surface antibodies (CD11b, CD14, HLA-DR, CD15, CD33) and intracellular Arg-1 and analysed by flow cytometry. Formalin-fixed paraffin-embedded samples of spleen specimens (MF n=41; HDs n=20) were immunostained with anti-Arg-1 antibody using the automated platform Dako Omnis Envision Flex with the EnVision FLEX, High pH, HRP Rabbit/Mouse High pH [GV800] revelation kit.

Results: In PBMCs, the percentage of PMN-MDSCs (identified as CD11b + HLA-DR low/- CD33 dim CD14 - CD15 + ) was higher (p=0) in patients (median 9.2, range 1-59) than HDs (median 1.7, range 0.1-4.5) and directly correlated with the allelic burden (R=0.86, p=0.006) in JAK2-mutated patients. Patients in pre-fibrotic phase (n=7) had levels of PMN-MDSCs (median 9.2, range 1-31.4) comparable to HDs; on the contrary, the percentage of PMN-MDSCs in patients with BM fibrosis (n=8; median 13.3, range 3.5-59) was higher (p=0) than in HDs. All PMN-MDSCs were Arg-1 + , both in MF and in HDs. Gene expression studies are ongoing. The expression of the CXCR4 receptor on PMN-MDSCs was lower, although not statistically significant, in MF (median 1.1, range 0-9.3) than in HDs (median 2.5, range 0-11.9). PMN-MDSCs in MF inversely correlated (R=-0.89 p=0.007) with CXCR4 expressed on CD34 + cells.

The percentage of M-MDSCs (identified as CD11b + HLA-DR low/- CD33 hi CD14 + CD15 - ) was higher (p=0.015) in HDs (median 0.49, range 0.25-1.5) than in MF (median 0.29, range 0.02-0.76).

Immunohistochemical staining of spleen sections showed a higher (p=0) percentage of Arg-1 + cells (calculated on total spleen cells) in MF (median 20, range 5-60) than in HDs (median 7, range 2-20); Arg-1 + cells were present in all patients and predominantly located in the red pulp in MF patients, while in HDs were detected in the white pulp.

Summary/Conclusion: The identification of MDSCs and their relation with the mutational status suggest a new mechanism of MF pathogenesis, either related to the chronic inflammatory status of patients or as players of the neoangiogenesis that characterizes the disease. Moreover, the low expression of CXCR4 on PMN-MDSCs could be, on one hand, related to the low expression of this receptor on CD34 + cells or suggest a MF-specific recruitment mechanism, other than that observed in solid tumors.

P983: SINGLE CELL ANALYSIS ALLOWS THE EARLY DETECTION OF LEUKEMIC CLONES IN MPN PATIENTS

C. Carretta 1,* , S. Parenti 1 , S. Mallia 1 , S. Rontauroli 1 , C. Chiereghin 2 , S. Castellano 1 , E. Bianchi 1 , E. Genovese 1 , S. Sartini 1 , L. Tavernari 1 , M. Mirabile 1 , M. G. Della Porta 2,3 , R. Manfredini 1

1 Center for Regenerative Medicine “S. Ferrari”, University of Modena and Reggio Emilia, Modena; 2 Humanitas Clinical and Research Center -IRCCS, Rozzano; 3 Department of Biomedical Sciences, Humanitas Unviersity, Milan, Italy

Background: Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders resulting in the overproduction of myeloid differentiated cells. Primary Myelofibrosis (PMF) is characterized by the worst prognosis and 15-20% of cases develop secondary Acute Myeloid Leukemia (AML). MPNs driver mutations affect JAK2, CALR or MPL genes. Moreover, mutations in epigenetic regulators can exacerbate the disease and alter response to treatment. Our group recently demonstrated through single cell analysis that MPNs progression is due to increased genetic heterogeneity, loss of heterozygosity and parallel AML evolution.

Aims: In this work we sought to define the genomic architecture of MPN patients during disease evolution, and gain insight into the chromatin and transcriptional perturbations induced by epigenetic modifiers’ mutations.

Methods: In order to describe MPNs clonal hierarchy at the single cell level, we employed Mission Bio Tapestri platform. We analyzed the CD34+ compartment of 3 patients during the chronic phase of the disease and after leukemic transformation through a custom panel comprising 29 genes frequently mutated in myeloid neoplasms. CNV assessment was performed on the same data through Mosaic algorithm.

On one patient, single nucleus RNA (snRNA-seq) and ATAC-seq were then conducted through 10x genomics instrument on CD34+ cells from the chronic and blast phase. Data were analyzed through Seurat R package.

Results: Genomic analysis was performed on 20.652 single cells coming from 7 samples. The analyzed patients suffered from Essential Thrombocythemia (n=2) or PMF (n=1); 2 patients harbored CALR type 1 driver mutation, while one patient carried JAK2V617F variant. In all patients, the first mutational hit occurred on epigenetic remodeler genes (i.e. TET2, ASXL1). Driver mutations’ allele frequency remained stable during disease progression and did not seem to drive leukemic transformation; in one case, JAK2V617F variant was lost in blast phase. In one patient, single cell analysis revealed the acquisition of 3 mutually exclusive pathogenic variants in RAS pathway (two NRAS mutations and a KRAS mutation). Notably, leukemic driver mutations, affecting genes such as IDH2, TP53 and KRAS, were already traceable at very low allele frequency in the chronic phase of the disease of all patients, despite being undetectable by bulk diagnostic NGS analysis. For all patients, CNV analysis highlighted a higher gene dosage imbalance in the leukemic clones when compared with those in the chronic phase.

Preliminary analysis of single cell ATAC+RNAseq data showed that the leukemic sample is enriched in more primitive cell types (e.g. multipotent progenitors). Moreover, genes found to be upregulated and more accessible in blast phase erythroid progenitors and HSC clusters are associated with AML poor prognosis.

Summary/Conclusion: Altogether this analysis suggests that MPNs’ first mutational hit frequently occurs in chromatin remodeler genes and affects a large fraction of neoplastic cells, confirming their impact on MPNs pathogenesis. Single cell multiomic analysis suggests that epigenetic alterations contribute to alter CD34+ cells differentiation state and activate the expression of pro-leukemic genes. Moreover, genomic analysis highlighted that clones carrying driver mutations remain stable during time and do not seem to drive leukemic transformation. On the other hand, genetic alterations, such as SNVs and CNVs, driving AML evolution are early identified by single cell analysis despite being undetectable by bulk sequencing.

P984: MUTATIONAL PATTERNS IN PH-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS (MPN)

I. C. Casetti 1,* , D. Pietra 2 , V. V. Ferretti 2 , A. De Silvestri 2 , O. Borsani 1 , D. Vanni 1 , C. Trotti 1 , S. Catricalà 2 , C. Favaron 1 , M. Cazzola 1 , L. Arcaini 1,2 , E. Rumi 1,2

1 University of Pavia; 2 Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

Background: According to the 2016 WHO classification, Ph-negative MPN include essential thrombocythemia (ET), polycythemia vera (PV), prefibrotic myelofibrosis (prePMF) and overt fibrotic myelofibrosis (PMF). The molecular basis of MPN was clarified with the discovery of driver mutations in JAK2 , MPL and CALR genes, but the biological landscape is more complex than initially assumed. Subclonal somatic mutations, also found in other myeloid neoplasms (MN), have been described and are associated with disease progression and poor prognosis. Those mutations target epigenetic regulators of DNA ( DNMT3A , TET2 , IDH1 / IDH2 ), genes involved in the chromatin structure ( ASXL1 , EZH2 ), spliceosome genes ( SF3B1 , SRSF2 ), transcription factors and tumor suppressor genes ( RUNX1 , TP53 ).

Aims: To correlate driver mutations, subclonal variants, clinical data and diagnosis in 509 MPN patients (236 ET, 91 prePMF, 182 PMF) and to define a diagnostic and prognostic role of these variants.

Methods: We studied DNA variants with the Illumina Nextera Rapid Capture Custom Enrichment Kit and HiSeq2500 platform. We selected a panel of 81 genes based on prior implication in the pathogenesis of MN. Inclusion criteria were (i) a diagnosis of ET, prePMF or overt PMF according to the 2016 WHO criteria, (ii) a peripheral blood sampling at diagnosis or before therapy administration.

Results: Overall, we detected 598 additional somatic variants. PMF showed a larger proportion of patients (pts) with at least one additional variant compared with prePMF and ET (86.3% vs 42.9% vs 34.8%, p<0.001) and a higher average number of variants per pts. These findings suggest that PMF is not an advanced stage of prePMF, but a different clinical entity with high tendency in variants accumulation.

Considering the driver mutation, the number of pts with at least one additional variant was significantly different among the molecular subgroups ( CALR 46.5%, JAK2 58.9%, MPL 82.1% TN 31.1%, p<0.001).

Somatic mutations were grouped based on their role in the cell cycle. In ET the most commonly mutated genes belong to the DNA methylation regulation and the most frequently involved gene was TET2 . In PMF, chromatin structure, RNA splicing and DNA methylation were the most recurrently involved pathway and ASXL1 and TET2 were the most frequently mutated genes. In prePMF most of the variants impaired DNA methylation and RNA splicing and TET2 and SF3B1 were the most frequently mutated genes. RNA splicing and DNA methylation are often involved in myelodisplastic syndromes (MDS). Thus, PMF and prePMF share molecular signatures with MDS, suggesting that MN are part of a continuum spectrum of diseases.

There was no association between driver mutations and additional variants, except for the spliceosome genes. The rate of splicing factors alterations in CALR mutants was significantly lower compared with JAK2 or MPL mutants (3.9% vs 22.3% vs 35.7%, p<0.001).

We finally correlated additional mutations and clinical data (blood count, splenomegaly, LDH, CD34+ circulating cells), overall survival (OS) and progression to blast phase (BP). We focused on the most commonly mutated genes, defined as mutations detected in at least 10 pts (>5%): ASXL1 , DNMT3A , TET2 , EZH2 , SRSF2 . ASXL1 was associated with anemia and splenomegaly, a higher risk of leukemic evolution (sHR=4.5, 95%CI:1.7-11.8, p=0.003) and shorter OS (HR=6.2 (4.3-9.0) p<0.001); SRSF2 had an impact on BP evolution and OS, while EZH2 showed an impact on OS.

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Summary/Conclusion: We suggest that not only histological and clinical criteria, but also distinct mutational patterns might differentiate ET, prePMF and overt PMF.

P985: THE ALLELIC RATIO OF DRIVER AND ASXL1 MUTATIONS IS PROGNOSTICALLY RELEVANT IN PMF

G. Coltro 1,2,* , G. Rotunno 1,2 , F. Mannelli 1,2 , G. G. Loscocco 1,2 , N. Bartalucci 1,2 , C. Mannarelli 1,2 , C. Maccari 1,2 , P. Guglielmelli 1,2 , A. M. Vannucchi 1,2

1 Department of Experimental and Clinical Medicine, University of Florence; 2 CRIMM, Center for Research and Innovation of Myeloproliferative Neoplasms, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy

Background: Primary myelofibrosis (PMF) is a clonal disorder driven by mutations in JAK2 , CALR , and MPL . Somatic mutations in myeloid-associated genes were shown to impact prognosis of PMF patients (pts). Among these, ASXL1 mutations ( ASXL1 mt ) are by far the most frequent and prognostically relevant, in fact they are included in the category of “high molecular risk” (HMR), along with EZH2 , IDH1/2 , SRSF2 , and U2AF1 Q157 mutations.

Aims: To investigate the phenotypic and prognostic implications of ASXL1 mt and variant allele frequency (VAF) in PMF.

Methods: After IRB approval, consecutive pts with WHO-defined PMF were included in the study. Mutational analysis by targeted NGS was performed by previously described methods (Guglielmelli P et al. , JCO 2017).

Results: The study enrolled a total of 384 pts, including 190 (49%) prefibrotic and 194 (51%) overt PMF. Median age was 60 (18-90) years, 236 (61%) were male. JAK2 , CALR and MPL mutations were found in 255 (66%), 83 (22%), and 17 (4%) pts, respectively; 36 (9%) were triple negative (TN). Among HMR mutations, ASXL1 was mutated in 88 (23%) pts, SRSF2 in 35 (9%), EZH2 in 32 (8%), U2AF1 in 15 (5%), and IDH1/2 in 11 (3%). VAF distribution of driver and HMR mutations was as reported in Fig.1A.

We did not find any significant correlation of ASXL1 mt VAF with other clinical and molecular characteristics, including PMF subtype, gender, age, hemoglobin, leukocyte, platelet and blast counts, splenomegaly, BM fibrosis grade, cytogenetics, mutational status for driver and other HMR genes and their VAFs. The only exception was the association of CALR -mutated PMF with lower ASXL1 VAF (median 28% vs 40%, p =.0332).

Median overall survival (OS) was 120 (102-151) months. In univariate Cox analysis, ASXL1 mt VAF did not correlate with OS, nor did other single driver and HMR mutations. When considered as a whole (for the purpose of this study, TN pts were considered has having driver VAF equal to 0), the VAF of driver mutations inversely correlated with survival (HR 0.4 [0.2-0.9], p =.0219). We next investigated the allelic ratio between driver and ASXL1 mutations ( d/A ratio) and its prognostic correlates. Median (range) of d/A ratio was 1.24 (0-13), and ROC analysis with death as an endpoint identified 1.31 as the optimal cut-off value. In univariate analysis, pts with a d/A ratio <1.31 had a significantly worse OS compared to those with a ratio ≥1.31 (median 45 vs 104 months, p =.0014; HR 2.4 [1.4-4.1]) (Fig.1B). When TN pts with ASXL1 mt were considered apart, having a d/A ratio <1.31 retained its inferior prognostic impact compared to the d/A ratio ≥1.31 group (median OS 57 vs 104 months, p <0.0453; HR 1.8 [1-3.2]), with TN/ ASXL1 mt pts being associated with the worst outcome (median OS 24 months) (Fig.1C).

In a multivariate Cox model including VAF-adjusted ASXL1 mutant status ( ASXL1 wt vs ASXL1 mt with d/A ratio ≥1.3 vs ASXL1 mt with d/A ratio <1.3) and other HMR mutations, the former and mutated SRSF2 were confirmed to be independent predictors of inferior OS. Both d/A ratio <1.31 and ≥1.31 remained significant compared to ASXL1 wt with respective HRs of 3.4 (2.3-5.1; p <.0001) and 1.6 (1-2.6; p =.0342). Notably, d/A ratio <1.31 still retained its inferior prognostic impact compared to d/A ratio ≥1.31 (HR 2 [1.2-3.5]; p =.0095).

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Summary/Conclusion: This study explores the implications of ASXL1 mt VAF and its interplay with driver mutant burden. The adverse prognosis of pts with a d/A ratio <1.31 suggests that this disease entity is predominantly driven by ASXL1 mt -clones characterized by a more aggressive biology. Further research is needed.

P986: CYTOGENETIC DIAGNOSIS WITH NEXT-GENERATION CYTOGENETICS BY OPTICAL GENOME MAPPING IN PATIENTS WITH MYELOFIBROSIS.

Á. Díaz González 1,* , G. Avetisyán 1 , E. Mora 1 , M. Santiago 2 , A. Liquori 1 , S. Furió 1 , S. García 1 , A. Villalba 1 , Á. López 1 , J. V. Gil 1 , C. García 1 , E. González 1 , C. Martínez 1 , B. Fernández 1 , M. Guaita 1 , B. Martín 1 , L. Cordon 1 , E. Barragán 1 , J. de la Rubia 1 , J. Cervera 2 , E. Such 1

1 Hematology; 2 Genetics Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain

Background: The prognosis of myelofibrosis patients is highly variable. Due to this heterogeneity, it is important to have prognostic tools that correctly stratify the patients. The most current prognostic scales such as the MIPSS70+ v2.0 or the GIPSS require cytogenetic data. The main limitation in classifying these patients is the lack of information on cytogenetic alterations because of bone marrow fibrosis and absence of metaphases.

Recently, a new strategy has emerged that characterize all cytogenetic alterations using Optical Genome Mapping considered Next Generation Cytogenetics which overcomes the limitations of conventional cytogenetics by using ultra high molecular weight DNA instead of metaphases. This DNA can be obtained from peripheral blood or bone marrow samples.

Aims: The main objective of this project is to perform a complete genomic characterization of 10 patients with myelofibrosis by karyotype, FISH, Next Generation Sequencing (NGS) and Optical Genome Mapping (OGM).

Methods: We have performed the karyotype staining the chromosomes by G-banding and the results were reported according to the International System for Human Cytogenetic Nomenclature (ISCN, 2020). FISH was performed to discard rearrangements of PDGFRA, PDGFRB, FGFR1 and JAK2. NGS libraries were prepared using a panel SOPHiA Myeloid Solution™ kit which includes SNV and CNV of 30 genes related to myeloproliferative neoplasms and leukemia. For study by OGM we obtained ultra-high molecular weight (UHMW) DNA from peripheral blood samples which are labeled with DLGreen fluorophores using Enzyme 1 (DLE-1) reactions following the manufacturer’s protocols (Bionano Genomics). Labeled DNA was loaded on a Saphyr chip and run on a Saphyr instrument (Bionano Genomics). The novo genome map assembly will be performed using BionanoSolve™ and structural variants will be called against the human reference hg38 assembly. Data was analyzed with Bionano Access™ (Bionano Genomics).

Results: Preliminary results were obtained:

- Patient 1 had unsuccessful karyotype and JAK2 (p.Val617Phe, VAF 13%) mutation by NGS.

- Karyotype result of patient 2 suggest that is normal: 46,XY[6]. NGS detected pathogenic mutations in JAK2 (p.Val617Phe, VAF 41%), SRSF2 (p.Pro95His, VAF 42%) and ETV6 (p.Arg105*, VAF 41%) and likely pathogenic mutation in ASXL1 (p.?; c.1720-2A>G, VAF 45%).

- Patient 3 had a normal karyotype: 46,XX[18] with CALR type 1 mutation (p.Leu367Thrfs*?, VAF 37%) by NGS.

We have not detected rearrangements by FISH in none of the 3 patients. 1500 Gbp of DNA were collected to perform the OGM for each patient and we selected structural variants that were present in less than or equal to 0,5 % of the control samples with the same enzyme. Patients 1 and 2 who had unsuccessful karyotype could be characterized by OGM. We have found by OGM a median of 1 insertion (range 0-4), 8 deletions (4-10) and 2 duplications (0-3). No inversion or translocation were detected. No cytogenetic alterations classified as very high-risk karyotype in MIPSS70+ v2.0 scale were detected. After an exhaustive analysis of these structural alterations initially detected, all alterations were ruled out as they are present in general population and therefore correspond to a normal karyotype.

Summary/Conclusion: Optical Genome Mapping suggest to be very promising diagnostic tool that can complete the study of patients with myelofibrosis, especially in those with non-assessable karyotype. The extension to a larger number of patients will confirm the results obtained and detect variables that remain cryptic to the karyotype.

P987: A DIFFERENT BALANCE IN OXIDATIVE STRESS RESPONSE IN CALR AND JAK2 MUTATED MYELOFIBROSIS PATIENTS CORRELATES WITH CLINICAL OUTCOME

E. Genovese 1,* , M. Mirabile 1 , S. Rontauroli 1 , S. Sartini 1 , S. Fantini 1 , L. Tavernari 1 , M. Maccaferri 2 , P. Guglielmelli 3 , E. Bianchi 1 , S. Parenti 1 , C. Carretta 1 , S. Mallia 1 , S. Castellano 4,5,6 , C. Colasante 5 , M. Balliu 3 , N. Bartalucci 3 , R. Palmieri 7 , T. Ottone 7,8 , B. Mora 9 , L. Potenza 5 , F. Passamonti 9 , M. T. Voso 7,8 , M. Luppi 5 , A. M. Vannucchi 3 , E. Tagliafico 4,5 , R. Manfredini 1

1 Life Sciences Department, Centre for Regenerative Medicine, University of Modena and Reggio Emilia; 2 Department of Laboratory Medicine and Pathology, Diagnostic Hematology and Clinical Genomics, AUSL/AOU Policlinico, Modena; 3 Department of Experimental and Clinical Medicine, Center of Research and Innovation of Myeloproliferative Neoplasms (CRIMM), University of Florence, Careggi University Hospital, Florence; 4 Center for Genome Research, University of Modena and Reggio Emilia; 5 Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, AUSL/AOU Policlinico; 6 PhD Program in Clinical and Experimental Medicine, University of Modena and Reggio Emilia, Modena; 7 Department of Biomedicine and Prevention, University of Tor Vergata; 8 Santa Lucia Foundation, I.R.C.C.S., Neuro-Oncohematology, Rome; 9 Division of Hematology, Ospedale ASST Sette Laghi, University of Insubria, Varese, Italy

Background: Myelofibrosis (MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and poor response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, that leads to greater genomic instability and promotes cell transformation.

Aims: In order to unveil the role of driver mutations in oxidative stress response, we evaluated oxidative stress markers levels in CD34+ hematopoietic stem/progenitor cells and plasma from MF patients.

Methods: Firstly, we have measured the levels of ROS, 8-hydroxy-2-deoxy-guanosine (8-OHdG) and activity of superoxide dismutase (SOD) in CD34+ cells from 20 JAK2 and 14 CALR myelofibrosis (MF) patients compared with 17 healthy donors (HD). In particular, the redox-sensitive CM-H2DCFDA was used to measure the intracellular ROS. The levels of 8-OHdG were detected by enzyme-linked immunosorbent assay (ELISA). Moreover, SOD activity was measured by means of colorimetric assay. Subsequently, we evaluated whether a different oxidative status could be detected in 129 plasma samples of MF patients (n=86 JAK2 , n=43 CALR ), as the level of total antioxidant capacity (TAC), an analyte frequently used to assess the antioxidant status of biological samples. Finally, we evaluated the association between TAC plasma levels and overall survival (OS) by means of multivariate analysis.

Results: Our results demonstrated that ROS production in CD34+ cells from CALR -mutated MF patients is strongly increased compared with patients harboring JAK2 mutation, leading to increased oxidative DNA damage. Moreover, CALR -mutated cells show less SOD antioxidant activity than JAK2 -mutated ones. Subsequently, we found that high TAC plasma levels correlate with detrimental clinical features, such as high levels of lactate dehydrogenase (LDH) and circulating CD34+ cells. Moreover, high TAC plasma levels are also associated with a poor OS in JAK2 -mutated patients. Multivariate analysis demonstrated that high plasmatic TAC is an independent prognostic factor for OS that allows the identification of patients with inferior OS in both DIPSS lowest and highest categories.

Summary/Conclusion: Altogether, our results confirmed that CALR mutation has a higher impact on the oxidative stress status in MF cells compared to JAK2 variant. Furthermore, plasma samples from CALR mutated patients have significantly lower TAC levels leading to a lower responsiveness to oxidative injury. On the other hand, increased TAC levels correlate with the presence of JAK2 mutation and several detrimental clinical features. MF patients with high plasmatic TAC display inferior survival and multivariate analysis demonstrated that increased TAC activity might represent a novel prognostic biomarker independent from DIPSS classification. We speculated that the high increase in oxidative stress in CALR mutated patients could be involved in the activation of protective mechanisms which ultimately promote cell death. On the contrary, in JAK2 mutated patients, the slight increase in oxidative stress can determine the persistence of cells with damaged DNA where the accumulation of mutations promotes the disease progression.

P988: LACTATE RESHAPES TUMOR MICROENVIRONMENT AND METABOLIC PROFILE IN MYELOFIBROSIS

S. Giallongo 1,* , D. Tibullo 1 , M. Spampinato 1 , C. Giallongo 2 , E. La Spina 3 , L. Longhitano 1 , A. Romano 3 , I. Dulcamare 3 , A. Barbato 3 , G. Scandura 3 , A. M. Amorini 1 , G. Lazzarino 1 , T. Zuppelli 2 , R. Caltabiano 2 , G. Li Volti 1 , F. Di Raimondo 3 , G. A. M. Palumbo 2

1 BIOMETEC; 2 Department of Medical, Surgical Sciences and Advanced Technologies G.F. Ingrassia, A.O.U. “Policlinico-Vittorio Emanuele; 3 Department of General Surgery and Medical-Surgical Specialties, Division of Hematology, A.O.U. “Policlinico-Vittorio Emanuele”, University of Catania, CATANIA, Italy

Background: Primary myelofibrosis (PMF) is myeloproliferative neoplasm depicted by bone marrow (BM) fibrosis, ineffective clonal hematopoiesis, and splenomegaly. These outcomes are related with changes in bone marrow microenvironment, sustaining cancer glycolytic metabolism, fibrosis, immune evasion, and thrombotic events. Lactate, a glycolysis byproduct, may represent the driving-force towards the acquisition of these features. For this reason, we investigated PMF CD34+ cells lactate production as factor prompting the metabolic rewire of BM niche.

Aims: In this work we aimed to target lactate metabolism as stricking factor improving PMF proliferative features.

Methods: Primary mesenchymal stem cells (MSCs) and HS-5 proliferation assays were performed in vitro . Protein expression level was assessed using immunoblot and immunohistochemical assays. Moreover, Cytokines were also investigated ex vivo , using Multiplex immunobead assay technology. These results were also mirrored in vivo on TPO high Zebrafish ( Danio rerio ) model.

Results: The monocarboxylate transporters (MCT-1 and -4), orchestrating lactate intake, were significatively upregulated in CD34 + cells and sera derived from MF-patients compared to healthy donor. MF-patients are characterized by increased percentage of circulating immunosuppressive cells. Incubation of peripheral blood mononucleated cells (PBMNCs) with MF-sera increased Treg and Myeloid-derived suppressor cells (M-MDSCs) expansion. This effect was disrupted by addition of MCT1 inhibitor AZD3965. The role of lactate on BM niche was evaluated by culturing healthy primary MSCs. Lactate (20 mM) and MF-sera exposition induced extracellular matrix (ECM) reorganization as supported by increased level of metalloproteases (MMP9, MMP2) together with reticulin and collagen deposition. Moreover, lactate induces calcium accumulation and soluble osteogenic molecular signals production (i.e. osteoprotegerin, osteonectin). On the epigenetic side, lactate treatment reshapes chromatin architecture eventually affecting histones lactylation, acetylation and methylation levels. These features were rescued upon AZD3965 treatment. Cancer-associated fibroblast (CAF) play an outstanding role in cancer development by ECM remodeling.Upon lactate exposure, expression of CAF markers α-SMA, FAP1 and TGFβ were upregulated in MSCs. The effect was reverted by AZD3965 treatment. The data achieved in vivo , and ex vivo were corroborated in a new pre-clinical TPO high Zebrafish model eventually showing increased Whole Kidney Marrow lactate.

Summary/Conclusion: In this work we report MCT1 inhibition as a strategy to treat invasive and metastatic cancers. Our results corroborate the idea that targeting lactate metabolism may represent a promising path to counteract inflammation, osteosclerosis and fibrosis in PMF patients.

P989: DYNAMIC CHANGES IN THE HISTONE H3 LYSINE 27 TRIMETHYLATION EPIGENETIC LANDSCAPE FOLLOWING RUXOLITINIB ADMINISTRATION

G. Greenfield 1,* , M. F. McMullin 2 , K. Mills 3

1 PGJCCR; 2 CME, Queen’s University Belfast; 3 PGJCCR, Queen’s Universtiy Belfast, Belfast, United Kingdom

Background: Dysregulation of normal epigenetic processes is increasingly recognised as a feature of myeloid malignancy generally and implicated in the pathogenesis of MPN. Epigenetic changes may directly result from the mutation of key epigenetic regulators including TET2, ASXL1 and EZH2 or indirectly from the activation of intracellular signalling cascades. We have previously observed that ruxolitinib, a JAK1/2 inhibitor, may alter the epigenetic landscape of histone modifications in MPN. CUT&RUN is a novel experimental approach providing high resolution profiling of epigenetic modifications and transcription factor binding.

Aims: To comprehensively profile the H3K27me3 epigenetic landscape of JAK2 V617F positive cells resulting from JAK inhibition and determine the dynamic changes occurring between short term and persistent ruxolitinib therapy to identify potential genetic contributors to pathogenesis and options for future therapeutics.

Methods: JAK2 V617F positive UKE1 cells were cultured in duplicate in the presence of 1000nM ruxolitinib or DMSO 0.1% vehicle control continuously for approximately five weeks. Samples were harvested at 37 days for CUT&RUN analysis using H3K27me3, EZH2 and IgG (negative control) antibodies. CUT&RUN performed using Epicypher CUTANA Kit according to manufacturer instructions. Libraries prepared using NEBNext® Ultra™ II DNA Library Prep kit with 50 base pair, paired end sequencing undertaken on Illumina NovaSeq 6000. Resulting FASTQ files were trimmed using Trim Galore! and aligned to reference genome hg38 using Bowtie2. Duplicate reads were removed using SAMtools rmdup and peaks called with MACS2 callpeak on the usegalaxy.eu platform. Differential peak analysis undertaken with Diffbind package in R. Samples were normalised to library size prior to differential analysis with DESeq2 methodology. Bigwig files generated using deepTools bamcoverage for visualisation.

Results: UKE1 cells remain sensitive to ruxolitinib after a period of treatment with high dose ruxolitinib lasting over 5 weeks. CUT&RUN libraries were sequenced to a minimum depth of 3.6 million unique reads per sample. Initial comparisons between Day 37 high dose treated cells and vehicle control revealed 1004 differentially enriched H3K27me3 peaks. Of these, 545 (54.2%) peaks were increased in the ruxolitinib treated cells and 459 (45.7%) peaks were increased in the control cells. Analysis of the difference between day 37 and day 1 high dose ruxolitinib treated cells revealed 290 peaks differentially enriched. Of these peaks, 279 (96.2%) were increased and 11 (3.8%) were decreased at day 37. Analysis of the overlap between these results identified 33 peaks increased at D37 in comparison to DMSO control and day 1 treatment samples.

Analysis of the differential binding patterns and direct visualisation of the results in a genome browser revealed particular genetic locations of interest including the promoter region of SPRY1, a potential regulator of erythropoiesis and JAK signalling, UGCG and BTBD11 . Figure 1 shows an example. Differential H3K27me3 was also noted at regions associated with PLXNA1 and NRP2 genes which act as co-receptors for class III semaphorin signalling.

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Summary/Conclusion: The dynamic changes in the H3K27me3 epigenetic landscape of JAK2 V617F positive cells following persistent high dose ruxolitinib administration has potential therapeutic implications and may sensitise cells to epigenetic therapies. Specific gene loci identified demonstrating differential H3K27me3 may offer some insight into JAK2 V617F mediated pathology and require further investigation.

P990: T CELL RESPONSE AND OMICRON VARIANT NEUTRALISATION FOLLOWING VACCINATION AGAINST SARS-COV-2 IN PATIENTS WITH CHRONIC MYELOID DISORDERS

P. Harrington 1,2,* , J. Saunders 1 , C. Saha 1 , A. Sheikh 1 , R. Dillon 1 , K. Raj 1 , L. Cadman-Davies 1 , T. Lechmere 3 , H. Khan 3 , C. Woodley 1 , S. Asirvatham 1 , N. Curto-Garcia 1 , J. O’Sullivan 1 , S. Kordasti 1 , D. Radia 1 , D. McLornan 1 , M. Malim 3 , K. Doores 3 , C. Harrison 1 , H. de Lavallade 1,2

1 Clinical Haematology, Guy’s & St Thomas’ NHS Foundation Trust; 2 School of Cancer & Pharmaceutical Science; 3 Infectious Diseases, King’s College London, London, United Kingdom

Background: Concerns remain over the response to vaccination against SARS-CoV-2 in patients with haematological malignancy.

Aims: Herein, we report a comprehensive immunological evaluation of the response to SARS-CoV-2 vaccination in patients with chronic myeloid neoplasms (MPN).

Methods: Antibody response was assessed using anti-Spike (anti-S) IgG with anti-nucleocapsid (anti-N) IgG used to determine previous infection. Neutralising antibody analysis was performed assessing inhibitory effect of plasma on entry of HIV-1 particles expressing Wuhan and omicron variant spike proteins into cells expressing ACE-2 receptor.

T cell response was assessed using flow cytometric evaluation of intracellular pro-inflammatory cytokines and surface exhaustion markers upon re-exposure to S peptides (Miltenyi) in T cell subsets. Subsequently, T cell response was evaluated using a fluorospot assay assessing IFNg/IL-2 secretion upon re-exposure to S peptides (Mabtech). Plates were analysed using the IRIS reader providing both spot-forming unit (SFU) frequency and relative spot volume (RSV).

Results: Samples were collected in 102 patients with MPN or post allo-SCT. Testing was performed in 37 MPN patients after one dose, 61 after 2 doses and 30 after 3 doses to date.

An anti-S IgG response was observed in 81.1% (30/37) of MPN patients after a first dose, increasing after two doses to 91.7% (55/60). Those with previous infection had higher anti-S IgG and neutralising antibody levels following two doses of vaccine (p=0.0002/p<0.0001). MPN patients receiving the BNT162b2 vaccine had higher anti-S IgG and neutralising antibody levels compared with those receiving ChAdOx-1 (p=0.05/p=0.04). The proportion of patients on ruxolitinib (rux) with a negative or borderline response after two doses was higher than observed in other patients (5/10 vs 5/47, p=0.01). Preliminary analysis in 20 patients showed reduced neutralisation of omicron compared with the Wuhan spike, with neutralising antibody ID50 of 93 vs 218 (p=0.06, Fig. 1a).

Using fluorospot analysis after two doses, an overall T-cell response was observed in 88.3% (53/60) patients. Patients on rux had lower SFUs for IFN and IL-2 and amongst those on treatment, RSV of IFNg was again lowest in patients on rux, with mean RSV of 2942 vs 7990 in patients on hydroxycarbamide and 6687 in peg-IFNa (p=0.016/p=0.0008). T cells expressed increased exhaustion markers upon re-exposure to S peptides in patients with CML following vaccination.

After a third dose, T cell response was assessed in 30 patients with a positive response in 90% (27/30). IFNg SFUs and RSV were significantly higher after 3 vs 2 doses at 242 and 9184 vs 72 and 5031 (p=0.0004/<0.0001, Fig. 1b,c). Patients on rux after 3 doses had lower SFU and RSV of IFNg than other patients at 79 and 7690 vs 302 and 9727 (p=0.035/0.04 Fig 1d,e). Patients on rux were more likely to have a negative T cell response than other patients (4/8 vs 20/22, p=0.0294). Patients with a diagnosis of MF had lower SFU and RSV for IFNg than patients with CML, PV and ET (SFU: 93 vs 294, 224 and 446, p=0.09/0.002/0.15, RSV: 7532 vs 9528, 10499 and 10630, p=0.08/0.001/0.01, Fig. 1f)

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Summary/Conclusion: We show overall high frequency of seroconversion and memory T cell responses but demonstrate the need for alternative strategies in some groups, including patients taking ruxolitinib. The increase in T cell reactivity after 3 doses is of particular significance in view of the reduced neutralisation of the now dominant omicron variant, with studies showing T cell functionality is maintained against omicron. Updated results will be presented.

P991: IMPAIRMENT OF TCR SIGNALLING IN PATIENTS WITH MYELOFIBROSIS TREATED WITH RUXOLITINIB

P. Harrington 1,2,* , R. Dillon 1 , D. Radia 1 , D. McLornan 1 , N. Curto-Garcia 1 , C. Woodley 1 , S. Asirvatham 1 , S. Kordasti 1,2 , C. Harrison 1,2 , H. de Lavallade 1,2

1 Clinical Haematology, Guy’s & St Thomas’ NHS Foundation Trust; 2 School of Cancer & Pharmaceutical Science, King’s College London, London, United Kingdom

Background: Ruxolitinib (rux) is a JAK1/JAK2 inhibitor with varied immune effects including on T cell, NK cell and dendritic cell function. Therapeutic effects are largely attributed to marked reduction in pro-inflammatory cytokines.

Aims: We hypothesised that impairment of T cell receptor (TCR) signalling through off-target binding of Src kinases, including Lck, would be an additional mechanism of immune dysfunction.

Methods: We performed phosphoflow cytometry in Tregs, T effectors and NK cells to assess the effect on signalling downstream from the TCR and activating NK cell receptors. 11-colour flow cytometry was performed after cells were activated with H2O2 for 15 minutes, due to its activity as a potent phosphatase inhibitor and cells were analysed for phosphorylation of ZAP70 as a surrogate of TCR signalling and STAT5. A gating strategy of CD4+/CD25+/FOXP3+/CD127 lo cells was used for identification of Tregs.

Results: Phosphoflow analysis was performed in 7 patients with a diagnosis of MF managed with rux and compared with 7 healthy controls (HC). 4 (57%) of patients were male and mean age was 61 (range 44-76). Patients were taking a median rux dosage of 30mg daily (10-50). Three patients had a DIPPS+ score of Int-2, 2 Int-1 and 2 low-risk.

Results of phosphoflow analysis are expressed as relative fluorescence intensity (RFI) defined as MFI in H2O2 stimulated sample/MFI in unstimulated sample. As expected, patients on rux had significantly reduced phosphorylation of pSTAT5 when compared with HC in all cell subsets evaluated. The mean RFI of pSTAT5 in CD4+ and CD8+ cells in HC was 21.9 and 31.1 respectively, compared with 6 and 8.5 in rux patients (p=0.001/p=0.002). In CD56+ cells this was 46.2 vs 7.7 (p=0.005) and in Tregs this was 21.9 vs 6.1 (p=0.019).

Of note, a difference in phosphorylation was also observed for pZAP70 indicative of inhibition of TCR proximal signalling. For pZAP70 mean RFI in CD4+ cells was 8.8 in controls compared with 2.2 in rux patients (p=0.05). In CD8+ cells mean RFI was 11.4 in controls and 2.4 in rux patients (p=0.04). In CD56+ cells mean RFI was 17.8 vs 2.4 (p=0.02), whilst in Tregs, significance was not reached with mean RFI of 6.3 vs 2.2 (p=0.13). Univariate analysis of rux patients showed no effect of age, comorbidities, DIPPS+ score or rux dosage on TCR or STAT5 signalling.

Findings were compared with those observed in 4 patients with CML and deep molecular response on nilotinib, in view of the highly specific ABL1 binding properties of this TKI. As expected, patients on nilotinib had significantly higher RFI for pSTAT5 compared with rux patients, at 23, 32.8, 21.2 and 46.5 in CD4+, CD8+, Treg and NK cells respectively (p=0.002/p=0.002/p=0.006/p=0.003). Interestingly, patients on nilotinib also had significantly higher RFI for pZAP70 in CD4+, CD8+ and NK cells at 6.7, 7.4 and 8.1 compared with rux patients (p=0.035/p=0.025/p=0.037).

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Summary/Conclusion: Studies have shown that rux inhibits T cell proliferation and reduces CD4+ cell cytokine secretion. Previous in vitro analysis of rux on CD4+ cells failed to identify an effect on TCR signalling after CD3/CD28 stimulation. In our ex-vivo analysis, using H2O2 stimulation, we observe TCR signalling inhibition in CD4+, CD8+ and NK cells, although reduced compared with effects on STAT5. It has been shown that rux inhibits Lck with an IC50 of 3.6uM. Whilst this is significantly less than the inhibitory effect on JAK kinases, there is ~34% sequency homology between Src and JAK family kinases in the kinase domain, suggesting off target kinase inhibition is implicated in the impairment of TCR signalling.

P992: OUTCOMES OF GLOBAL COAGULATION ASSAYS IN PATIENTS WITH PHILADELPHIA-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS WITH RESPECT TO THEIR CLINICAL AND GENETIC DETERMINANTS OF CLONAL EVOLUTION

A. Lucchesi 1,* , R. Napolitano 2 , M. T. Bochicchio 2 , G. Simonetti 2 , G. Micucci 1 , M. Poggiaspalla 1 , V. Di Battista 1 , G. Musuraca 1 , F. Foca 3 , G. Giordano 4 , L. Catani 5 , M. Napolitano 6

1 Hematology Unit; 2 Biosciences Laboratory; 3 Unit of Biostatistics and Clinical Trials, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola; 4 Internal Medicine Division, Hematology Service, Regional Hospital “A. Cardarelli”, Campobasso; 5 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Department of Experimental, Diagnostic and Specialty Medicine, School of Medicine, Institute of Hematology “Seràgnoli”, University of Bologna, Bologna; 6 Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties and Haematology Unit, University Hospital “P. Giaccone”, Palermo, Italy

Background: Classical Myeloproliferative Neoplasms (MPNs) are hematopoietic stem cell diseases characterized by inflammation, promotion of atherosclerosis, hypercoagulability, fibrosis, and clonal evolution. Our previous studies in cytofluorimetry have shown that the reduced expression of platelet fibrinogen receptor (PFR) - common to all MPNs - can be due to the hyperactivation of plasma-dependent mechanisms, such as tissue factor (TF) release, an unbalanced thrombin generation and the marked involvement of Protease Activated Receptors (PARs) and their signaling crosstalk with TGF-ß. Acetylsalicylic acid (ASA) seemed to be able to restore the expression of PFRs.

Aims: The aim of the present study is to verify the existence of imbalances in hemostatic function in rotational thromboelastometry (ROTEM), with respect to the different classical forms of MPN, their cell counts, the presence of genetic mutations in Next Generation Sequencing (NGS), PFR expression, the use of ASA and the history of vascular events.

Methods: We enrolled 53 patients affected by MPNs, naïve from any therapy, with the only exception of ASA (taken by 50% of patients). We used the samples to run assays of genetics (30-gene panel in NGS - SOPHiA Myeloid Solution™, SOPHiA Genetics, Switzerland), cytofluorimetric determination of PFRs, global coagulation by rotational tromboelastometry (ROTEM® Delta, Werfen, Spain) for INTEM, EXTEM, FIBTEM parameters.

Results: ROTEM parameters appear to exhibit substantial variations, depending on the type of disease under investigation, cell counts, and selected mutations. Since most of the values are in the normal range, the results should be read in terms of “efficiency” or “preferential use” of one pathway over another. In particular, essential thrombocythemia (ET) and CALR mutation seem to be related to an increased efficiency of both classical coagulation pathways, with significantly more contracted clot formation times (CFT). In contrast, primary myelofibrosis (PMF) and polycythemia vera (PV) show greater imbalances in the hemostatic system. PV patients have longer CFT (EXTEM 114 sec, p= 0.008, INTEM 82 sec, p=0.027) and at the same time a selective contraction of the parameters in INTEM for higher platelet (Plt), white blood cells (WBC) and hemoglobin (Hb) levels. PMF - on the contrary - seems to exploit more the extrinsic pathway (EXTEM) - figure A-B . In multivariate analysis, the major contribution to the alterations is credited to Hb values for PV, while only Plt retains statistical significance for PMF and ET.

Selected mutations are associated with changes in ROTEM parameters. Among the DTA mutations, the presence of DNMT3A shows a significant reduction in clotting time (CT) in EXTEM, while ASXL1 is associated with reduced maximum lysis (ML). EZH2 could be responsible for CFT elongations in the INTEM assay.

In addition, increased expression of PFRs is associated with bleeding history and sustained CT time at FIBTEM under ASA prophylaxis.

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Summary/Conclusion: In the most indolent MPNs (such as ET, especially if CALR mutated) the balance and efficiency of the classical pathways is preserved. In PMF, the hemostatic system is unbalanced towards the extrinsic pathway, confirming the connection between increasing fibrosis and plasma-dependent hypercoagulable states. In contrast, rheological characteristics and endothelial stress in PV seem to result in a greater involvement of the intrinsic pathway. ROTEM and PFR expression confirm the existence of dynamic states of hypercoagulability in MPNs and could be used to assess the efficacy of prophylaxis.

P993: SETD2 IS A BONA FIDE TUMOR SUPPRESSOR IN SYSTEMIC MASTOCYTOSIS

M. Mancini 1,* , C. Monaldi 2 , S. De Santis 2 , C. Papayannidis 1 , M. Rondoni 3 , C. Sartor 1 , S. Bruno 2 , A. Curti 1 , R. Zanotti 4 , M. Bonifacio 4 , L. Scaffidi 5 , L. Pagano 5 , M. Criscuolo 5 , F. Ciceri 6 , C. Elena 7 , P. Tosi 8 , P. Valent 9 , M. Cavo 10 , S. Soverini 2

1 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; 2 Department of Experimental Diagnostic and Specialty Medicine - DIMES, Institute of Hematology “L. e A. Seràgnoli”, University of Bologna, Bologna; 3 Azienda USL della Romagna, Ravenna; 4 Azienda Ospedaliera di Verona, Verona; 5 Fondazione Policlinico Universitario A. Gemelli, Roma; 6 Servizio Immunoematologia Trasfusionale (SIMT), IRCCS Ospedale San Raffaele, Milano; 7 Fondazione I.R.C.S.S. Policlinico San Matteo, Pavia; 8 U. O. di Oncologia ed Oncoematologia-Osp. Infermi Azienda Unità Sanitaria-Locale di Rimini, Rimini, Italy; 9 Department of Hematology, Internal Medicine I, Medical University of Vienna, Vienna, Austria; 10 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; Department of Experimental Diagnostic and Specialty Medicine - DIMES, Institute of Hematology “L. e A. Seràgnoli”, University of Bologna, Bologna, Italy

Background: The trimethylation of histone H3 at lysine 36 (H3K36me3), catalyzed by the SETD2 methyltransferase, is a post-translational modification involved in the fidelity of transcription and splicing and in the recruitment of DNA repair machinery. Recent studies have emphasized the tumor-suppressive role of SETD2, especially in renal cancer where SETD2 is often deleted or mutated. A novel mechanism of SETD2 non genomic loss of function due to proteasome-mediated degradation has recently been reported in advanced systemic mastocytosis (advSM). Proteasome inhibition has been found to inhibit clonogenic growth and induce apoptosis, but whether this is due to SETD2 re-expression remains to be demonstrated.

Aims: The aim of our study is to demonstrate unequivocally that forced expression of SETD2 in a cellular context in which it was lost, may restore proliferation control, clonogenic capacity and DNA damage repair mechanisms.

Methods: The SETD2-deficient HMC-1.2 mast cell leukemia cell line was used as in vitro model. Forced SETD2 re-expression was obtained by nucleofection: 10 6 HMC-1.2 cells were resuspended in 85μl of cell line Nucleofector Solution V and 2μg of a SETD2 (GFP-tagged) construct were added. An empty vector coding for GFP was used as negative control. Cells were transfected by using the Lonza Nucleofector 2b and the Lonza Amaxa Cell Line Nucleofector Kit V according to manufacturer’s instructions. GFP expression was assessed using a Cytoflex flow cytometer 24 and 48 hours post transfection; 78% of fluorescence positivity was observed at 48 hours. Neomycin selection (1mg/ml) was performed to obtain stable SETD2 expression. One month after selection, SETD2 and H3K36me3 were assessed by Western Blotting (WB). Clonogenic capacity was tested by clonogenic assays. Immunofluorescence experiments were performed to assess if SETD2 forced expression was able to restore DNA damage repair by using p-H2AX (S139), RAD51, MSH6 and THEX1 antibodies.

Results: We previously characterized the HMC-1.2 cell line as deficient for SETD2/H3K36me3, as virtually all patients with advSM. To investigate whether SETD2 may indeed play a tumor suppressor role in SM, we transfected HMC-1.2 cells with an ectopic SETD2 plasmid. After transfection, the morphology of cells dramatically changed; moreover, HMC-1.2tsSETD2 showed a 70% increase in doubling time. Co-immunoprecipitation demonstrated that tsSETD2 was able to interact with p53 and to restore its expression and activity. We thus observed an increase in p21 and p27 associated with an accumulation of cells at the G1/S checkpoint. Moreover, SETD2 stable transfection restored DNA damage responses, as demonstrated by an increase in H2AX phosphorylation and RAD51 (HR), THEX1 (DNA replication) and MSH6 (MMR) expression after UV exposure. Finally, clonogenic assays in control and HMC-1.2tsSETD2 cells showed that: 1) SETD2 re-expression restores cell proliferation control; 2) reduction of clonogenic growth observed after proteasome inhibition is indeed SETD2-dependent.

Summary/Conclusion: In advSM, SETD2 is a bona fide tumor suppressor and its loss impairs proliferation control and DNA damage responses. Its overexpression restores cell proliferation control by stabilizing p53 activity, and DNA damage repair by rescuing the H3K36me3 mark. This validates the therapeutic potential of interfering with the mechanisms responsible for SETD2 loss. Supported by AIRC IG 2019 grant (23001).

P994: HEDGEHOG PATHWAY INVOLVEMENT IN MASTOCYTOSIS DEVELOPMENT

L. Polivka 1,2 , V. Parietti 3 , J. Bruneau 4 , E. Soucie 5 , P. Dubreuil 5 , C. Bodemer 2 , O. Hermine 6 , L. Maouche Chretien 7,*

1 IMAGINE Institute, Paris-Centre University, INSERM U1163; 2 Department of Dermatology, Hôpital Necker-Enfants Malades; 3 Department of animal experimentations, Institut Universitaire d’Hématologie, Université Paris Diderot, Sorbonne Paris Cité; 4 Department of Pathology, Necker-Enfants Malades Hospital, Paris; 5 Centre de Recherche en Cancérologie de Marseille, INSERM U1068, Marseille; 6 Department of Hematology, Necker-Enfants Malades Hospital; 7 Imagine Institute, Paris-Centre University, INSERM 1163, Paris, France

Background: Mastocytosis is a rare and heterogenous disease characterized by abnormal accumulation and activation of mast cells in one or several organs. The clinical spectrum of the pathology varies from relatively mild forms with isolated skin lesions to very aggressive forms with wide systemic involvement, often fatal. The disease can affect both children and adults. While most pediatric forms resolve during or in late puberty, adult cases are persistent and may evolve into aggressive forms. A somatic KIT D816V mutation is detected in 85% of patients but all attempts to demonstrate its oncogenic effect have failed. There is strong evidence that the KIT D816V mutation alone cannot trigger the disease and that other genes may be involved.

Aims: The French national reference center of mastocytosis (CEREMAST) has recorded more than 2000 patients with sporadic mastocytosis and 60 familial cases. Since KIT mutations do not fully explain the pathophysiology of the disease, we sought to identify other mutations that could act synergistically with KIT D816V to induce the disease.

Methods: We focused on patients presenting with mastocytosis in particular syndromic contexts within the pediatric cohort followed at Necker hospital; we first investigated the molecular mechanisms underlying mastocytosis onset in patients with both congenital mastocytosis and an extremely rare polymalformative syndrome.

Results: From 3 children presenting with both Greig cephalopolysyndactyly syndrome (GCPS) and congenital mastocytosis, we could demonstrate the implication of the hedgehog (Hh) pathway in mastocytosis. GCPS is a very rare syndrome resulting from haploinsufficiency of GLI3 , the major repressor gene of the hedgehog (Hh) family. We showed that the Hh pathway is barely active in normal primary mast cells (MCs) and overactive in neoplastic MCs. Using a GCPS mouse model, we demonstrated that mutations in GLI3 and KIT have a synergistic, tumorigenic effect leading to mastocytosis onset. We also showed that Hh inhibitors suppress abnormal mast cell proliferation, in vitro, and extend the survival of mice with aggressive systemic mastocytosis (ASM).

Summary/Conclusion: Thus, for the first time, we revealed the involvement of Hh signaling pathway in the pathophysiology of mastocytosis and demonstrated the cooperative effects of the KIT and Hh oncogenic pathways in ASM. Finally, we provided the first evidence that Hh inhibitors represent a promising new therapeutic target.

P995: MYELOID NEOPLASMS-ASSOCIATED GENE VARIANTS IN 639 PATIENTS WITH POST-POLYCYTHEMIA VERA AND POST-ESSENTIAL THROMBOCYTHEMIA MYELOFIBROSIS: AN ANALYSIS OF THE MYSEC COHORT

B. Mora 1,2,* , P. Guglielmelli 3 , A. Kuykendall 4 , M. Maffioli 1 , G. Rotunno 3 , R. S. Komrokji 4 , F. Palandri 5 , J.-J. Kiladjian 6 , A. Iurlo 7 , G. Auteri 5 , D. Cattaneo 7 , V. De Stefano 8 , S. Salmoiraghi 9,10 , T. Devos 11 , F. Cervantes 12 , M. Merli 1 , A. Campagna 13 , G. Benevolo 14 , M. Brociner 1 , F. Albano 15 , J. Gotlib 16 , M. Caramella 17 , M. Ruggeri 18 , D. M. Ross 19 , F. Orsini 20 , C. Pessina 20 , I. Colugnat 20 , F. Pallotti 2,20 , T. Barbui 10 , L. Bertù 2 , M. G. Della Porta 13 , A. M. Vannucchi 3 , F. Passamonti 1,2

1 Hematology, Ospedale di Circolo, ASST Sette Laghi; 2 Department of Medicine and Surgery, University of Insubria, Varese; 3 Center for Research and Innovation of Myeloproliferative Neoplasms, University of Florence, Florence, Italy; 4 Malignant Hematology Department, Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America; 5 Institute of Hematology, Azienda Ospedaliero-Universitaria S.Orsola-Malpighi, Bologna, Italy; 6 Centre d’Investigations Cliniques, Hôpital Saint-Louis, Paris, France; 7 UOC Hematology, Foundation IRCCS Ca’Granda Ospedale Maggiore Policlinico, Milan; 8 Hematology, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome; 9 Hematology and Bone Marrow Transplant Unit; 10 FROM Research Foundation, ASST Papa Giovanni XXIII, Bergamo, Italy; 11 Hematology, University Hospitals Leuven, Rega Institute, Leuven, Belgium; 12 Hematology, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain; 13 Cancer Center, IRCCS Humanitas Clinical and Research Center, Rozzano; 14 Hematology, Azienda Universitario-Ospedaliera Città della Salute e della Scienza di Torino, Turin; 15 Hematology - Dept. of Emergency and Organ Transplantation, University of Bari, Bari, Italy; 16 Division of Hematology, Stanford Cancer Institute, Stanford University School of Medicine, Stanford, California, United States of America; 17 Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milan; 18 Hematology, San Bortolo Hospital, Vicenza, Italy; 19 Haematology Directorate, SA Pathology, Royal Adelaide Hospital and Flinders Medical Centre, Adelaide, Australia; 20 Laboratory of Cytogenetics and Molecular Biology, Ospedale di Circolo, ASST Sette Laghi, Varese, Italy

Background: Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms evolving to post-PV (PPV-) and post-ET (PET-) myelofibrosis (MF), called secondary MF (SMF). In primary MF, knowledge on non-driver myeloid neoplasms-associated gene variants (M-GVs) influences clinical decision-making. In SMF, information on M-GVs is scant.

Aims: The primary aim was to assess the pattern of distribution of M-GVs, their correlations with SMF subtype and with driver mutations, in a large cohort of SMF patients studied by next generation sequencing (NGS).

Methods: The study involved SMF patients of the MYSEC ( Myelofibrosis Secondary to PV and ET ) project. In 97% of cases, NGS were performed within one-year pre/post-SMF diagnosis. Characteristics of study cohort were described by standard statistic. Associations were investigated by Chi-square or Fisher exact test.

Results: The clinical features of the 639 NGS-annotated patients entering the analysis are reported in Table 1.

A total of 198 (31%) patients did not harbor any M-GV, with no imbalance as for SMF subtype. Among the 441 (69%) with M-GVs, 223 (51%) had one, 137 (31%) two, 52 (12%) three, 23 (5%) four and 6 (1%) five or more. PPV-MF subjects reported more frequently one M-GV, while those with PET-MF at least three ( p=0.02 ). Mean number of M-GVs was 1.4 per patient (range, 0-7). In detail, it was 1.2 (range, 0-4) and 1.5 (range, 0-7) per patient in PPV- and in PET-MF, respectively ( p=0.01 ). The most frequent (≥5% of dataset) M-GVs involved: ASXL1 (n=181, 41%), TET2 (n=145, 33%), DNMT3A (n=49, 11%), TP53 (n=43, 10%), EZH2 (n=39, 9%), SF3B1 (n=31, 7%), U2AF1 (n=29, ~7%), ZRSR2 (n=27, 6%), CBL and RUNX1 (n=21 each, 5%). In PET-MF there was a significantly higher frequency of M-GVs in ASXL1 (47% vs 34%, p=0.01 ), SRSF2 (5% vs 1%, p=0.01 ), U2AF1 (9% vs 4%, p=0.04 ) and CBL (7% vs 2%, p=0.01 ) compared to PPV-MF. The latter was significantly associated with ETV6 alterations (5% vs 1% in PET-MF, p=0.04 ). As regards driver mutations, we found an association between “triple negative” status (TN) and M-GVs in SETBP1 (38%, p=0.002 ), IDH2 (25%, p=0.02 ), EZH2 (25%, p=0.05 ), and SRSF2 (25%, p=0.01 ). Figure 1 shows the frequency of M-GVs found in the MYSEC cohort, distinguished by SMF subtype ( a ) and driver mutations ( b ).

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Summary/Conclusion: Among 639 NGS-annotated SMF cases, 69% presented at least one M-GV with a mean of 1.4 per patient. Overall, the most frequent (≥10%) M-GVs were in ASXL1, TET2, DNMT3A and TP53 genes. Different pathways of progression among PPV- and PET-MF have been disclosed. PPV-MF showed an increased rate of ETV6 alterations, opening the question of possible predisposing genetic factors. TN cases clustered with specific M-GVs, potentially targetable ( IDH2 ). This is the first study exploring the mutational landscape of a wide cohort of SMF patients, paving the way for further investigations on the topic.

P996: UTILITY OF KIT P.D816 IN MYELOID NEOPLASM WITHOUT DOCUMENTED SYSTEMIC MASTOCYTOSIS TO DETECT HIDDEN MAST CELLS IN BONE MARROW

D. H. Kim 1,* , F. Jia 1 , C. Y. Ok 1

1 Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: KIT p.D816 mutation is strongly associated with systemic mastocytosis (SM). Although it is not specific for SM, it is one of the diagnostic criteria for SM since its presence in the right morphologic context (i.e. presence of atypical mast cell clusters) is a strong support for neoplastic nature of mast cells. Next-generation sequencing (NGS) is now routinely performed in almost all bone marrow sample and KIT mutations are detected from patients who are not known or suspected to have SM.

Aims: Therefore, we wanted to assess if KIT mutations in this patient population are associated with unsuspected SM.

Methods: We searched NGS result in our institution between 1/1/2013 and 9/30/2021 with positive result for KIT mutation from patients with known/suspected myeloid neoplasms. Patients with previously documented history of systemic mastocytosis were excluded. Bone marrow biopsies from patients with KIT mutation were assessed with immunohistochemical stains for CD117 and mast cell tryptase (MST).

Results: A total of 49 patients who had KIT mutation and had available formalin-fixed paraffin embedded blocks were identified. Thirty-eight (77.6%) patients has acute myeloid leukemia (AML) and 6 patients had chronic myelomonocytic leukemia (CMML, 12.2%). The remaining patients had myelodysplastic syndrome (n=1), myelodysplastic/myeloproliferative neoplasm, unclassifiable (n=1), myeloproliferative neoplasm (MPN, n=1), AML in remission (n=1) and therapy-related myeloid neoplasm (n=1).

A total of 41 patients had a single KIT mutation and 8 patients had 2 or more KIT mutations. KIT p.D816V mutation was the most common mutation (n=38, 64.4%), followed by D816Y (n=8, 13.6%), D816H (n=5, 8.5%), Y418_D419insFF (n=2, 3.4%), R815_D816insVL (n=1, 1.7%), S197L (n=1, 1.7%), K558E (n=1, 1.7%), D419del (n=1, 1.7%), N822K (n=1, 1.7%) and R815_D816insIPP (n=1, 1.7%).

Immunohistochemical stains for CD117 and MST were performed in all 49 patients. The MST stain was more suitable since 10 patients (20%) had increased blasts with CD117 expression which obscured morphologic review. A total of 4 patient (8.2%) showed mast cell nodules where spindled shaped mast cells were present, meeting the criteria for SM. All four patients had KIT p.D816V mutation and had high mutant allelic frequency (~50%) except one patient (1%). These patients had MPN (n=1), AML (n=1), AML in remission (n=1) and CMML (n=1), respectively. One (2%) patient has increased (>10%) mast cells but scattered throughout the bone marrow space without nodules and mast cells were round-to-ovoid shape. Eleven patients (22.4%) showed rare scattered mast cells and 33 patients (67.3%) did not show mast cells at all.

Summary/Conclusion: We discovered approximately 8% of patients who had myeloid neoplasms with unexpected KIT mutations are met for systemic mastocytosis after additional immunohistochemical studies. Our data support that application of additional immunohistochemical studies are recommended to identify underrecognized SM when KIT mutations are unexpectedly found by molecular assays.

P997: PROTEOMIC ANALYSIS ON PLATELETS OF ESSENTIAL THROMBOCYTHEMIA PATIENTS UNDERSCORES THE ROLE OF MITOCHONDRIA IN JAK2 V617F PLATELET REACTIVITY AND FUNCTION

X. Guerrero-Carreño 1 , S. Smits 2 , A. Esteban Lasso 3 , J. A. Escudero García 3 , M. Samiotaki 4 , A. Alvarez-Larrán 5 , A. Angona 6 , A. J. Sáen Marín 7 , V. García Gutiérrez 7 , D. Dekkers 8 , J. Demmers 8 , C. M. Benavente Cuesta 9 , F. Ferrer-Marín 10 , J. C. Hernández-Boluda 11 , F. J. Iborra 12 , I. M. De Cuyper 13 , P. Vandenberghe 14 , P. Papadopoulos 1,*

1 Hematology, IdISSC, Madrid, Spain; 2 Human Genetics, KULeuven, Leuven, Belgium; 3 Sanidad Animal and VISAVET, Universidad Complutense de Madrid, Madrid, Spain; 4 Institute for Bioinnovation, BSRC “Al. Fleming”, Vari, Greece; 5 Hematology, Hospital Clínic; 6 Hematology, Hospital del Mar, Barcelona; 7 Hematology, Hospital Ramón y Cajal, IRICYS, Madrid, Spain; 8 Proteomics Center, ErasmusMC, Rotterdam, Netherlands; 9 Hematology, Hospital Clínico San Carlos, Madrid; 10 Hematology, Hospital Morales Meseguer, UCAM, IMIB, Murcia; 11 Hematology, Hospital Clínico Universitario, INCLIVA; 12 Biological Noise and Cell Plasticity, CIPF, Centro de Investigación Príncipe Felipe, Valencia, Spain; 13 Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Amsterdam, Netherlands; 14 Hematology, UZLeuven, Leuven, Belgium

Background: Essential thrombocythemia (ET) is a heterogeneous disease subdivided into five genetic groups according to WHO, based on the common MPN driver mutations ( JAK2 V617F, MPL W515K/L, and CALR Type I&II). Current treatment strategies include anti-platelet (e.g. acetylsalicylic acid (ASA)) or cytoreductive agents (e.g. hydrea (HU)) and target mainly complications related to platelet dysfunction ( i.e. hemorrhage/thrombosis). Platelet activation is an energy demanding process fueled mainly by a dynamic equilibrium between mitochondria oxidative phosphorylation and glycolysis 1 . Importantly, altering the platelet catabolic response to activation has been shown to prevent thrombus formation 2 . JAK2 V617F platelets have been reported to be more activated than CALR mutated platelets 3 , as well as JAK2 mutated patients have a greater thrombotic risk in comparison to other ET groups. However, the cause of this phenotype is not completely understood.

Aims: In this study we aimed to analyze the proteome of ET platelets and to characterize their functional properties according to the mutational background and treatment regimens.

Methods: 22 healthy donors (HD) and 67 ET patients have been included in the study. Most of the patients were treated with low dose aspirin (ASA), anagrelide (ANA) or hydrea (HU) or a combination of these. Specifically, 35 out of the 67 ET platelet samples and 8 out of 22 HD were subjected to label-free quantitation mass spectrometry (LFQ-MS). All HD and ET platelets were also analyzed for surface marker expression, degranulation and aggregation capacity by flow cytometry.

Results: Hierarchical clustering of the mass spectrometry data revealed different proteomic profiles between HD and ET mutational groups and specifically between JAK2 V617F and CALR I and CALR Type II treated and/or untreated platelets. In general, HD platelets were more enriched for proteins related to platelet activation, and degranulation as compared to ET platelets and clustered next to JAK2 V617F ASA-treated platelet samples (Figure 1). In particular, the JAK2 V617F ASA-treated platelets presented significant enrichment in platelet activation metabolic and mitochondrial proteins in contrast to the CALR Type I and Type II ET platelets. Of note, no peptides corresponding to the mutant CALR were detected using label-free MS methods. Surface marker expression was variable among ET patients. However, CD49B and CD36 surface markers were the most affected among the JAK2 V617F and CALR Type I and II platelets and their levels (mean fluorescence intensity-MFI-) were inversely correlated. With regards to platelet aggregation, JAK2 V617F platelets presented similar or lower levels when compared to HD platelets, in line with their proteomic profiles. Differences in aggregation and degranulation levels were also observed between CALR Type I and Type II platelets specifically in the untreated condition.

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Summary/Conclusion: ET platelets are functional but they show different capacities to respond upon various stimuli and treatment regimens. JAK2 V61F platelets are more activated than any other ET group according to their proteome profile, which is not fully reflected by the functional assays. Mitochondrial activity arises as an important factor in the control of platelet reactivity and it could be critical in disease management and treatment strategies, specifically for the JAK2 V617F patients.

References

1. Aibibula, M., et al. J Thromb Haemost. 2018; 16(11):2300-2314,

2. Nayak, M.K., et al. Blood Adv. 2018; 2(15):2029-2038,

3. Hauschner, H., et al. Am J Hematol, 2020. 95(4): p. 379-386.

P998: REGULATION OF BCL-X SPLICING BY SRC KINASES INHIBITORS IN MYELOPROLIFERATIVE NEOPLASMS

J. Petiti 1,* , F. Itri 1 , C. Fava 1 , D. Cilloni 1

1 Dept. of Clinical and Biological Sciences, University of Turin, Orbassano, Italy

Background: Myeloproliferative neoplasms (MPN) are a group of incurable diseases that include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Both ET and PV are associated with prolonged clinical courses, with symptoms that affect their quality of life, and risk of progression to MF and acute leukemia. MF is the MPN subtype with the most adverse prognosis and is associated with a high risk of leukemic transformation. The FDA approved the use of ruxolitinib, a JAK1/2 kinase inhibitor, that proved to be effective in reducing symptoms and spleen volume, but not in inducing complete molecular remission. Although hematopoietic stem cell transplantation involves a high risk of mortality, it is nowadays the only curative treatment for MF. Therefore, it is relevant to identify new therapeutic strategies to improve the clinical outcome of MPN. Recently, there has been a growing interest in Bcl‐xL, the long isoform encoded by alternative splicing of the Bcl‐x gene, that acts as an anti‐apoptotic regulator. Bcl-xL appears to be up-regulated in MPN patients and we previously showed that its modulation correlates with the clinical severity of MPN subgroups. We also demonstrated that ABT-737, a specific Bcl-xL inhibitor, has a synergistic effect in combination with Ruxolitinib in HEL cell lines and MPN patients’ cells, suggesting that Bcl-xL inhibition could increase the power of treatment (Petiti et al. J Cell Mol Med . 2020). Unfortunately, ABT-737 is characterized by poor bioavailability, so a new approach to target Bcl-xL must be investigated. Paronetto et al. found that tyrosine phosphorylation of Sam68 in cancer cells may protect them from apoptosis by altering the Bcl-xS/L ratio in favor of Bcl-xL. Intriguingly, c-Src is one of the tyrosine kinases that phosphorylate Sam68 and several papers indicated its involvement in the MPN pathogenesis, without explaining the molecular mechanism.

Aims: We aimed to molecular investigate the mechanism linking c-Src kinase activity with the deregulation of Bcl-x splicing in MPN, intending to consider c-Src kinase inhibitors, widely bioavailable, as indirect modulators of Bcl-xL to improve MPNs outcome.

Methods: HEL cell lines, JAK2 V617F-mutated in homozygosis, has been incubated with different concentrations of ruxolitinib (JAK1/2 inhibitor) and dasatinib (SRC kinases inhibitor) at different concentrations and time points. Then, proliferation, and apoptosis were evaluated. Subsequently, the c-Src pathway, Sam68, and Bcl-xL/S ratio were investigated at both RNA and protein levels.

Results: Performing in vitro study on HEL cell lines, we identified that dasatinib induced apoptosis and inhibition of proliferation in HEL cell lines, with a synergistic effect in combination with ruxolitinib. We also showed that dasatinib alone inhibited c-Src and Sam68 phosphorylation and down-modulated Bclx-L protein expression. Combination treatment with ruxolitinib and dasatinib increased both pro-apoptotic Cleaved Caspase 3 and BAX protein expression. Furthermore, we noted that treatment with ruxolitinib alone decreases the mRNA levels of Bclx-L, but also of the Bcl-xS isoform. On the contrary, combined drug therapy modulated the Bcl-xL/S mRNA ratio in favor of the pro-apoptotic Bcl-xS isoform.

Summary/Conclusion: Although further studies will be necessary to better understand the c-Src role in Bcl-x splicing regulation in MPN, our preliminary data suggest that indirect downmodulation of Bcl-xL through SRC kinases inhibitors in combination with standard therapy could be useful in the future for the treatment of MPN patients.

P999: ERK1/2 INHIBITION REDUCES OSTEOPONTIN PLASMA LEVELS AND BONE MARROW FIBROSIS IN A MYELOFIBROSIS MOUSE MODEL

S. Rontauroli 1,* , E. Bianchi 1 , L. Tavernari 1 , M. Dall’Ora 2 , G. Grisendi 3 , M. Mirabile 1 , S. Sartini 1 , E. Genovese 1 , C. Carretta 1 , S. Mallia 1 , S. Parenti 1 , L. Fabbiani 4 , N. Bartalucci 5 , L. Losi 6 , M. Dominici 3 , A. M. Vannucchi 5 , R. Manfredini 1

1 Centre for Regenerative Medicine, Life Sciences Department, University of Modena and Reggio Emilia, Modena; 2 Rigenerand S.R.L., Medolla (MO); 3 Division of Oncology, Laboratory of Cellular Therapy, Department of Medical and Surgical Sciences of Children & Adults; 4 Department of Medical and Surgical Sciences of Children & Adults, Pathology Unit, University of Modena and Reggio Emilia, Modena; 5 Department of Experimental and Clinical Medicine, and Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), University of Florence, Careggi University Hospital, Florence; 6 Department of Life Sciences, Pathology Unit, University of Modena and Reggio Emilia, Modena, Italy

Background: Primary myelofibrosis (PMF) is a stem cell disorder belonging to Philadelphia-negative myeloproliferative neoplasms (MPNs). The disruption of bone marrow (BM) microenvironment due to the extensive deposition of extracellular matrix fibers is the distinctive trait of PMF and is accompanied by hematopoietic stem cells (HSCs) mobilization and extramedullary hematopoiesis. BM fibrosis is caused by the complex interaction between stromal and hematopoietic cells belonging to the neoplastic clone, in particular megakaryocytes and monocytes play a pivotal role through the production of pro-fibrotic cytokines. Supporting this hypothesis, we have previously demonstrated that osteopontin (OPN) contribute to the development of BM fibrosis. OPN plasma levels are increased in PMF patients and correlate with higher BM fibrosis grade, circulating CD34+ cells and inferior survival. Megakaryocytes and monocytes turned out as the main source for OPN production that can induce fibroblasts and mesenchymal stromal cells proliferation, as well as collagen upregulation.

Aims: To determine whether OPN might represent a druggable target in PMF we assessed the inhibition of signaling pathways responsible for its production. In particular, we evaluated the effect of ERK1/2 inhibition over OPN expression and myelofibrosis development in a MF mouse model.

Methods: The activity of inhibitors of signaling pathways affecting OPN expression was evaluated in vitro in human primary monocytes, given their key role in OPN secretion. The effects on cell viability were assessed by XTT assay at 72 hours of treatment, while OPN expression and production were evaluated by qRT-PCR and ELISA, respectively, at 72 and 96 hours of treatment.

Inhibitors of OPN production selected from the in vitro screening were analyzed for their effect on OPN plasma levels in a MF mouse model induced by the treatment with a thrombopoietin receptor agonist Romiplostim (Rom). Hematological parameters and splenomegaly were monitored over time while BM and spleen fibrosis were evaluated at sacrifice.

Results: Our in vitro analysis demonstrated that drug inhibitors of ERK1/2, MEK1/2, p38 and a Ca2+ channel antagonist were able to reduce OPN expression in primary monocytes, both at RNA and protein levels, without affecting cell viability.

Next, we moved to a MF mouse model obtained through the stimulation of thrombopoietin receptor that develops bone marrow and spleen fibrosis, together with increased OPN expression, faster than JAK2V617F knock-in mice. ERK1/2 inhibition by Ulixertinib did not affect the development of thrombocytosis and splenomegaly but was able to significantly reduce OPN plasma levels. Interestingly, ERK1/2 inhibition also counteracted the development of bone marrow fibrosis in Rom-treated mice. The same results were observed in mice when Ulixertitnib was given together with the JAK inhibitor Ruxolitinib. Drug combination resulted in a reduction of both OPN plasmatic levels and bone marrow fibrosis development.

Summary/Conclusion: Our results demonstrated that Ulixertinib treatment reduced OPN production both in vitro and in vivo . Moreover, in a MF mouse model, the concurrent inhibition of ERK1/2 and JAK2 signaling pathways displayed synergistic effects by diminishing OPN plasma levels and constraining BM fibrosis. These results provide a rational for the development of novel combination therapeutic approach for PMF patients.

P1000: INCREASED PLASMA LEVELS OF LNCRNAS ARE POTENTIAL PROGNOSTIC BIOMARKERS IN MYELOFIBROSIS

S. Sartini 1,* , S. Fantini 1 , S. Rontauroli 1 , M. Mirabile 1 , E. Bianchi 1 , F. Badii 1 , M. Maccaferri 2 , P. Guglielmelli 3 , T. Ottone 4,5 , R. Palmieri 4 , E. Genovese 1 , C. Carretta 1 , S. Parenti 1 , S. Mallia 1 , L. Tavernari 1 , C. Salvadori 3 , F. Gesullo 3 , C. Maccari 3 , M. Zizza 3 , A. Grande 6 , S. Salmoiraghi 7 , B. Mora 8 , L. Potenza 9 , V. Rosti 10 , F. Passamonti 8 , A. Rambaldi 7 , M. T. Voso 4,5 , C. Mecucci 11 , E. Tagliafico 9,12 , M. Luppi 9 , A. M. Vannucchi 3 , R. Manfredini 1

1 Center for Regenerative Medicine, Life Sciences Department, University of Modena and Reggio Emilia; 2 Department of Laboratory Medicine and Pathology, Diagnostic Hematology and Clinical Genomics, AUSL/AOU Policlinico, Modena; 3 Department of Experimental and Clinical Medicine, and Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), University of Florence, Careggi University Hospital, Florence; 4 Department of Biomedicine and Prevention, University of Tor Vergata; 5 Neuro-Oncohematology, Santa Lucia Foundation, Istituto di Ricovero e Cura a Carattere Scientifico (I.R.C.C.S.), Rome; 6 Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena; 7 Hematology, ASST Papa Giovanni XXIII, Bergamo; 8 Division of Hematology, Ospedale ASST Sette Laghi, University of Insubria, Varese; 9 Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, AOU Policlinico, Modena; 10 Center for the Study of Myelofibrosis, Foundation Policlinico San Matteo, Istituto di Ricovero e Cura a Carattere Scientifico (I.R.C.C.S.), Pavia; 11 Department of Medicine and Surgery, Section of Hematology and Clinical Immunology, University of Perugia, Perugia; 12 Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy

Background: Myelofibrosis (MF) displays the worst prognosis among Philadelphia-negative myeloproliferative neoplasms and is characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and frequent transformation to acute myeloid leukemia.

Different therapeutic approaches are being used depending on the severity and the specific clinical manifestations of the disease in each patient. Unfortunately, no curative therapy is currently available for MF, except for bone marrow transplantation, which however has a consistent percentage of failure. There is therefore a great urgency to identify biomarkers correlated with the different stages of the disease and to treat patients with a more tailored treatment.

Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. Moreover, circulating lncRNAs have already been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis.

Aims: The aim of this study was to identify circulating lncRNAs whose plasmatic concentration differs between MF patients and healthy donors (HDs). Subsequently, we evaluated their potential role as non-invasive disease biomarkers in MF.

Methods: As a preliminary result we analyzed the expression of 38 lncRNAs in CD34+ cells collected from 83 MF patients and 26 HDs leading to the identification of 26 differentially expressed lncRNAs. To confirm these results and to move to a more accessible sample type we collected plasma from 143 MF patients and 65 HDs. RNA was extracted from these samples and the relative abundance of lncRNAs, including some of those deregulated in CD34+ cells or already described as involved in hematological malignancies and myeloid differentiation, was assessed using qRT-PCR. According to the plasmatic levels of each lncRNA patients were split into two groups (low- or high-) and this subdivision was used to unveil the potential correlation between the various lncRNAs and the clinical features of MF patients.

Results: Our analysis identified 7 lncRNAs significantly upregulated in MF patients’ plasma compared to HDs. Among these, high levels of LINC01268, MALAT1 or GAS5 correlated with several detrimental clinical features of MF, such as high counts of leukocytes and CD34+ cells, a severe grade of bone marrow fibrosis and the presence of splenomegaly.

Strikingly, high plasma levels of LINC01268 (Log-rank p-value = 0.0018) , GAS5 (Log-rank p-value = 0.0008) or MALAT1 (Log-rank p-value = 0.0348) were associated with a poor overall-survival (OS) while high levels of LINC01268 correlated also with a shorter leukemia-free-survival (LFS). Finally, multivariate analysis demonstrated that a high plasma concentration of LINC01268 was an independent prognostic variable for both OS (HR = 2.104; confidence interval (CI) = 1.08–4.12; p = 0.0297) and LFS (HR = 8.190; CI = 1.02–65.78; p = 0.0479).

Summary/Conclusion: To our knowledge, this is the first study describing the expression profile of circulating lncRNAs in MF patients’ plasma and focusing on their putative role as biomarkers in clinical practice. In particular, our results demonstrated that increased levels of circulating LINC01268 , GAS5 or MALAT1 are associated with disease detrimental features and correlate with an inferior OS in MF patients. Notably, multivariate analysis confirmed that LINC01268 plasma levels might improve the identification of patients with a poor prognosis. If the prognostic value of this lncRNA will be confirmed in independent patients’ cohorts it might be used to integrate contemporary prognostic models.

P1001: DNMT3A/TET2/ASXL1 MUTATIONS DETERMINE THROMBOTIC RISK IN POLYCYTHAEMIA VERA

A. Segura Diaz 1 , R. Stuckey 1,* , Y. Florido Ortega 1 , M. Sobas 2 , A. Álvarez Larrán 3 , F. Ferrer-Marín 4 , M. Pérez Encinas 5 , G. Carreño 6 , M. Fox 7 , B. Tazón Vega 7 , B. Cuevas 8 , J. López Rodriguez 1 , N. Farías Sánchez 1 , C. Bilbao Sieyro 1 , M. Gómez Casares 1

1 H.U.Dr. Negrin de GC., Las Palmas, Spain; 2 Wroclaw Medical University, Wrocław, Poland; 3 H. Clínic de Barcelona, Barcelona; 4 Hospital Morales Messeguer, IMIB, UCAM, Murcia; 5 Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela; 6 Hospital Universitario 12 de Octubre, Madrid; 7 , Hospital Universitari Vall d’Hebron, Barcelona; 8 Hospital Universitario de Burgos, Burgos, Spain

Background: Polycythaemia vera (PV) is the myeloproliferative neoplasm with the highest incidence of thrombosis, with cardiovascular events being the main cause of death in these patients. Recent studies have identified clonal haematopoiesis (CHIP) in healthy individuals, which is associated with older age and an increased risk of developing both myeloid neoplasms and vascular events.

Aims: In a previous study we saw an association between the presence of mutations in DTA genes ( DNMT3A, TET2 and ASXL1, the most frequently mutated genes in CHIP), and vascular events in patients with PV. We aimed to confirm this association in a consecutive series of PV patients from our centre and in an age-matched case control study with patients from various European centers.

Methods: All patients aged 18 years and above with a confirmed diagnosis of PV in our hospital were recruited consecutively between 2003 and 2018. NGS was performed on 200 ng genomic DNA extracted from peripheral blood at diagnosis. Sequencing was performed using a MiSeq (Illumina) with the 30-gene panel Myeloid Solution (SOPHiA Genetics). Only variants with an allelic frequency (VAF) ≥ 2% and annotated as pathogenic or probably pathogenic were considered. Additional mutations are defined as pathogenic mutations in any non-driver gene from the myeloid panel.

The case-control study included PV patients with a thrombotic event in their patient history and the results of a myeloid NGS panel available from 9 Spanish and 1 Polish hospital. Cases were gender- and age-matched with control patients (with a diagnosis of PV but no thrombotic event) from the consecutive series.

Results: A total of 79 patients with PV were analysed in our consecutive series. An association was observed between DTA mutation and thrombotic event (OR 4.5; p=0.002; χ2). An association was also observed between any non driver mutation from the myeloid panel and thrombotic event (OR 7.7; p=<0.0001; χ2). However, no association was seen between non-DTA mutation and thrombotic events (p=0.231; χ2). The association between DTA mutation and thrombotic event was confirmed in multivariate analysis (p=0.021, Table 1. Thrombotic event was associated with age (p=0.020) and marginally with leukocyte count at diagnosis (p=0.065) but not with JAK2 VAF. We saw that when the JAK2 mutation comes before DTA, there is a tendency for the risk of thrombosis to be higher than for DTA first.

We also observed a positive association between cardiovascular risk factors (CVRF) and thrombotic events (Table 1), as expected. However, its significance was lost in the multivariate analysis. The DTA and CVRF variables are closely related, with a positive association between them (OR 6.77; p=0.009; χ2). Fifty eight of 79 patients (73.4%) were hypertensive; however, an association was still found between thrombotic event and DTA mutation in this group of hypertensive patients (p=0.033).

The association between DTA and thrombotic events in the uni and multivariate analyses was confirmed by the case-control study of 47 cases and 47 gender- and age-matched controls (OR 2.7; p=0.036; χ2).

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Summary/Conclusion: We were able to confirm the previously observed association in PV between the presence of DTA mutations and higher risk of developing a vascular event. Thus, detection of DTA mutation by NGS could help predict thrombotic risk in PV patients, including those with pre-existing CVRF.

P1002: ANTI-FIBROTIC ACTIVITY OF BMP2 IN BONE MARROW-DERIVED MESENCHYMAL STROMAL CELLS OF MYELOPROLIFERATIVE NEOPLASMS

T. Subotički 1,* , E. Živković 1 , O. Mitrović Ajtić 1 , M. Vukotić 1 , D. Đikić 1 , T. Dragojević 1 , D. Šefer 2 , S. Bižić 2 , J. F. Santibanez 1 , M. Gotić 3 , V. P. Čokić 1

1 Department for Molecular Oncology, Institute for Medical Research, National Institute of Republic of Serbia; 2 Clinic of Hematology, University Clinical Center of Serbia; 3 Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Bone marrow fibrosis (BMF) is a shared feature of all MPN, although it is most pronounced in PMF and represents a major diagnostic criteria. Studies suggest a correlation between the grade of BMF and prognosis of MPN, with more fibrosis associated with worse outcome. A large body of evidence has suggested that transforming growth factor beta (TGF-β) is among the most prominent inducers of fibrotic processes. Bone morphogenetic proteins (BMPs) are major regulators of cell fate in tissue homeostasis. In MPN, bone marrow-derived mesenchymal stromal cells (BM-MSC) are identified as a major cellular source of fibrosis, but exact molecular mechanism involved have not been identified so far.

Aims: In addition to apoptosis and prolifration, we analyzed the effect of BMP2 and TGF- β / SMAD signaling pathway on the fibrotic phenotype of BM-MSC isolated from MPN patients and healthy donors.

Methods: Bone marrow aspirates from 5 newly diagnosed MPN patients (3 PMF and 2 PV patients) and 3 healthy donors were analyzed by immunofluorescence expression of fibronectin and alpha smooth muscle Actin (αSMA), after treatment with TGF-β and / or BMP2. Using immunocytochemistry, we analyzed HEL 92.1.7 cells with a homozygous expression of JAK2 V617F for proliferation (Ki67) and apoptosis (ssDNA) during exposure to BMP2 and selective BMP signaling inhibitor LDN-193189.

Results: Our results showed that TGF-β significantly increased fibronectin expression, in contrast to BMP2, in BM-MSC of healthy donors. Also, the joint treatment of TGF-β and BMP2 reduced the level of fibronectin expression relative to TGF-β. In addition, TGF-β increased αSMA expression in BM-MSC from healthy donors. In contrast, BMP2 reduces the expression of αSMA and fibronectin in BM-MSC of healthy donors.BMP2 significantly reduced fibronectin expression in BM-MSC compared to untreated cells of patients with MPN. BMP2 dose dependently and significantly (p<0.01) increased the proliferation of HEL 92.1.7 cells, while the BMP signaling inhibitor LDN-193189 also demonstrated dose dependence in stimulation of proliferation (p<0.001). Apoptosis of HEL 92.1.7 cells was slightly affected by BMP2 and LDN-193189.

Summary/Conclusion: Our results show that BMP2 has anti-fibrotic activity that is antagonistic to TGF-β. This indicate that BMP2 may modify the TGF-β signaling pathway.

P1003: PERIPHERAL BLOOD CYTOTOXIC T CELLS SHOW EARLY EXHAUSTED FEATURES IN MYELOFIBROSIS PATIENTS

L. Tavernari 1,* , S. Rontauroli 1 , M. Maccaferri 2 , B. Mora 3 , E. Bianchi 1 , S. Parenti 1 , E. Genovese 1 , P. Guglielmelli 4 , C. Carretta 1 , S. Mallia 1 , M. Mirabile 1 , S. Sartini 1 , C. Colasante 5 , L. Potenza 5 , F. Passamonti 3 , E. Tagliafico 5,6 , M. Luppi 5 , A. M. Vannucchi 4 , R. Manfredini 1

1 Centre for Regenerative Medicine, Life Sciences Department, University of Modena and Reggio Emilia; 2 Department of Laboratory Medicine and Pathology, Diagnostic Hematology and Clinical Genomics, AUSL/AOU Policlinico, Modena; 3 Division of Hematology, Ospedale ASST Sette Laghi, University of Insubria, Varese; 4 Department of Experimental and Clinical Medicine, and Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), University of Florence, Careggi University Hospital, Firenze; 5 Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, AOU Policlinico; 6 Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy

Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by megakaryocyte hyperplasia, bone marrow fibrosis and extramedullary hematopoiesis. Compelling evidence are suggesting that persistent antigen stimulation in the tumor microenvironment can differentiate T effector cells into terminally exhausted T cells, a functional state characterized by decrease in proliferation, cytotoxicity, and cytokine production. Cutting-edge therapies are focused on the reversion of exhausted state through immune checkpoint inhibition to recover the immune response against the tumor.

Aims: Despite MPNs are myeloid neoplasms, T cells are showing dysfunctional features. To assess if immune checkpoint inhibition could be a new effective therapy in combination with those currently approved, we investigated T cell exhaustion in MF.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 38 MF patients and 20 healthy donors (HD). The expression profile of the following inhibitory receptors (IRs): PD1, CD244, CD160, TIM3, LAG3, CTLA4 was characterized by flow cytometry in CD3+CD8+ T cells. Results were correlated with clinical features such as Dynamic International Prognostic Scoring System (DIPSS) classification, splenomegaly, hemoglobin level and other negative prognostic factors. For T cell activation assays we stimulated PBMCs overnight in the presence of coated anti-CD3, anti-CD28 and Brefeldin A, whilst cytokine production was then assessed by intracellular flow cytometry staining. Chi-squared and Mann Whitney tests were used for statistical analysis. Total cellular RNA was isolated from peripheral blood granulocytes of 141 primary and secondary MF patients and 28 HDs, and we performed Gene expression profiling (GEPs) by microarray platform.

Results: We reported expansion of PD1+CD244+, PD1+CD160+, PD1+CD57- cytotoxic T cell populations from MF patients, together with increased expression of all the IRs assessed on both total CD3+CD8+ T cells and PD1+CD57- T cells fraction. Furthermore, we observed decreased secretion of IFNɣ and TNFɑ by CD3+CD8+ T cells of MF patients, while significant reduction of Granzyme B production was observed mainly in patients with expansion of PD1+CD57- T cell population. GEPs analysis highlighted increased expression of IR-ligands like CD274, CEACAM1, CD48 on granulocytes from MF patients compared to HDs. The correlation analysis of IRs expression with patients’ clinical features showed that higher levels of PD1, CD244, CD160, CTLA4, LAG3 or TIM3 associates with detrimental features like splenomegaly, low hemoglobin levels, HMR mutation number and more severe DIPSS classification.

Summary/Conclusion: Our data evidenced in MF patients the presence of an impaired population of peripheral blood cytotoxic T cells expressing multiple IRs and with reduced cytokine production after in vitro activation. Moreover, granulocytes from MF patients display higher level of IR-ligands transcripts hinting an immunosuppressive interplay between the myeloid neoplastic clone and T cells. Lastly, clinical correlations suggest a more severe disease in patients with higher IRs expression on cytotoxic T cells. Taken together this data highlights an early exhausted state likely implicated in immune escape which will be investigated for immune checkpoint inhibition.

P1004: EFFECT AND MOLECULAR MECHANISM OF TQ05105, A NOVEL SMALL MOLECULE INHIBITOR OF JAK2 IN MYELOPROLIFERATIVE NEOPLASM

W. Zhang 1,2,3,4,* , J. Liu 1,2,3,4 , Z. Shi 4 , L. Yang 1,2,3,4 , B. Li 1,2,3,4 , Z. Xiao 1,2,3,4

1 State Key Laboratory of Experimental Hematology; 2 National Clinical Research Center for Blood Diseases; 3 Institute of Hematology & Blood Diseases Hospital, Tianjin; 4 Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China

Background: The JAK2V617F gene mutation leads to upregulation of the JAK/STAT signaling pathway, which causes excessive bone marrow cell proliferation, elevated inflammatory cytokines, constitutional symptoms and shortened survival in myeloproliferative neoplasms (MPN) patients. The JAK1/2 kinase inhibitor ruxolitinib can dramatically improve disease-related symptoms, but side effect such as anemia and thrombocytopenia limit its use in a substantial proportion of patients. Several JAK2 kinase inhibitor III Clinical trials have yielded unsatisfactory outcomes recently. TQ05105 is a new small molecule inhibitor that targets JAK2 kinase.

Aims: We aim to investigate the effect and molecular mechanism of TQ05105 on MPN cell lines and mouse models.

Methods: We systematically measured the function of TQ05105 in MPN cell lines, including UKE1 and SET2 cells, by performing proliferation, apoptosis and colony-forming assays. RT-PCR and inflammatory factor multi-assay kit was used to detect the secretion of inflammatory factors such as TNF-α, IL-1β, MCP-1 in mRNA level and protein level respectively. CFU-E and BFU-E assays were performed on MPN cell lines and primary cells. In vivo, TQ05105 was evaluated in JAK2V617F Jak2V617F transplant mouse model and MPL515 retroviral mouse model. Mice were orally administrated with placebo, 25 mg/kg (bid) and 50 mg/kg (bid) every day for 4 weeks. Flow cytometry analysis was performed to determine the proportion of different hematopoietic progenitor cell populations. In addition, HE staining and Gomori staining was used to detect the changes in the spleen and bone marrow.

Results: In UKE1 and SET2 cell lines, TQ05105 can inhibit the proliferation and induced cell apoptosis, notably reduce the expression of cytokines, including TNF-α, IL-1 and MCP-1. According to Western blot results, the phosphorylation of JAK/STAT signaling pathway associated proteins JAK2, STAT3 and STAT5 were significantly suppressed by TQ05105. Furthermore, compared to the control group, TQ05105 can strongly limit the ability of UKE1 cell lines, SET2 cell lines and primary cells from MPN patients (PMF, PV, ET) to form BFU-E and CFU-GM.

TQ05105 can striking reduce mouse spleen volume, along with favorable impact on leukocyte and erythrocyte counts in Jak2V617F transplant mouse model. The proportion of stem and progenitor cells, especially CMPs and MEPs, was considerably reduced in mice after treatment with TQ05105. HE staining showed remarkably Spleen response such as recovering structure, reduced infiltration of erythrocytes and suppressed proliferation of megakaryocytes. MPLW515L retroviral mouse model recapitulated the feature of myelofibrosis, application of TQ05105 in this model resulted in reduction of the spleen volume, extramedullary hematopoiesis, leukocyte and platelets count, bone marrow fibrosis and decreased fraction of CMPs and MEPs.

Summary/Conclusion: TQ05105, as a novel small molecule JAK2 inhibitor, can inhibit cell proliferation, induce cell apoptosis and decrease inflammatory cytokines by affecting the JAK/STAT signaling pathway. In vivo experiments further proved that TQ05105 can reduce spleen size and improve disease-related symptoms of MPN, indicating that it is a promising therapeutic drug for MPN.

P1005: A PHASE 1, OPEN-LABEL, DOSE-ESCALATION STUDY OF SELINEXOR PLUS RUXOLITINIB IN PATIENTS WITH TREATMENT-NAÏVE MYELOFIBROSIS

H. Ali 1,* , A. Kishtagari 2 , K. Maher 3 , S. Mohan 2 , A. Mazumder 4 , K. Chamoun 5 , I. Karasik 5 , E. Sbar 5 , L. Dugom 5 , S. Tamir 5 , X. Wang 5 , J. Prchal 6 , S. Tantravahi 6

1 City of Hope, Duarte; 2 Vanderbilt Ingram Cancer Center, Nashville; 3 VCU Massey Cancer Center, Richmond; 4 The Oncology Institute of Hope & Innovation, St. Petersburg; 5 Karyopharm Therapeutics, Newton; 6 Division of Hematology and Hematologic Malignancies, Huntsman Cancer Institute, University of Utah, Salt Lake City, United States of America

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm commonly associated with gene mutations in JAK2, CALR , or MPL caused by the unregulated proliferation of clonal myeloid precursors in the bone marrow. The JAK 1/2 inhibitor, ruxolitinib (RUX), has shown reductions in spleen volume and improvement in MF-related symptoms when used in the frontline. Despite the significant improvements of RUX, most patients (pts) eventually progress and lose response to treatment over time. Therefore, novel combinations are critical to improve responses and delay progression. Selinexor (SEL) is an oral selective inhibitor of nuclear export (SINE) compound, specifically inhibiting exportin-1 (XPO1), that has been approved for use in multiple myeloma and diffuse large B-cell lymphoma. Preclinical studies of the combination SEL and RUX have demonstrated significant activity. In clinical studies of MF refractory to JAK inhibitors, SEL monotherapy has exhibited robust clinical activity with a tolerable safety profile ( NCT03627403 ).

Aims: Here, we present the initial results of a phase 1 dose escalation study to determine the optimal dose and preliminary efficacy of once weekly (QW) SEL in combination with RUX in pts with treatment-naïve MF.

Methods: In the ongoing multicenter, open-label, Phase 1/2 study ( NCT04562389 ) using a 3 + 3 design, two dose levels of SEL were evaluated, 40 mg and 60 mg QW plus RUX twice daily (BID) as per label in 28-day cycles. For nausea prophylaxis, all pts received a 5-HT3 antagonist. Primary study objectives include safety, maximum tolerated dose (MTD), recommended Phase 2 dose (RP2D), and preliminary efficacy. Secondary objectives include spleen volume, symptom and anemia response, and overall survival (OS).

Results: As of 24 Feb 2022, 10 pts have been dosed in 2 dose levels 40 mg (n=3), and 60 mg (n=7) SEL QW plus RUX. The starting dose of RUX was 20 mg in 8 pts, 15 mg in one patient and 10 mg in one patient. The median age was 64 (range 45-76) and 7 pts had primary MF and 3 had post-essential thrombocythemia (ET) MF. The Dynamic International Prognostic Scoring System (DIPSS) risk category was int-1 (n=4), int-2 (n=4) and high risk (n=2). There were no dose limiting toxicities reported for either SEL dose levels. Due to dizziness, one patient had a dose interruption and after 5 months of therapy discontinued treatment from new onset of atrial fibrillation and pulmonary hypertension (unrelated to SEL and RUX). Currently, all other pts remain on study. Hemoglobin levels were maintained without significant worsening in majority of patients. Low grade nausea (30%) was the most common treatment-emergent adverse event. All pts experienced an improvement in their white blood cell count. Of the 6 evaluable pts, 5 had a ≥35% spleen volume reduction at week 12.

Summary/Conclusion: In pts with treatment-naïve MF, QW SEL in combination with RUX is well tolerated with a manageable side effect profile. Based on current data there have been no observed dose limiting toxicities in cohort 1 of QW oral SEL 40 and 60 mg with RUX.

P1006: NEXT GENERATION SEQUENCING (NGS) IN PEDIATRIC MASTOCYTOSIS

A. Angi 1,* , S. Bianchi 1 , G. Palumbo 1 , V. Filipponi 1 , M. Rousseau 1 , M. L. Moleti 1 , F. Giona 1

1 EMATOLOGIA PEDIATRICA, Policlinico Umberto I Università Sapienza Roma, Roma, Italy

Background: Mastocytosis is a clonal disorder characterized by the accumulation of mast cells in the skin and, less frequently, in other organs. The disease is limited to the skin in children and may occur in one of three cutaneous variants: maculopapular cutaneous mastocytosis (MPCM), diffuse cutaneous mastocytosis (DCM) and mastocytoma. Systemic mastocytosis (SM) typically occurs in adults with KIT D816V mutation (3). Additional genetic mutations (TET2, N-RAS, SF3B1, ASXL1, etc.) have been detected using NGS in adults with mastocytosis (4). Currently, there is no data concerning the use of NGS in pediatric mastocytosis.

Aims: The aims of the monocentric study, named MAS_PED1, that was approved by Ethics Committee, are to: a) identify patients (pts) with different cutaneous forms at risk of developing aggressive systemic disease; b) detect the known KIT D816V mutation using RT-PCR; c) identify different KIT, and/or additional gene mutations using NGS; and d) evaluate potential role of different mutations in the outcome of pts with mastocytosis, aged < 18 years at lesion onset.

Methods: Pts with pediatric onset of mastocytosis, followed at the study Center were included. Peripheral blood samples were collected to detect KITD816V mutation, using both RT-PCR and ddPCR techniques, and to investigate other molecular mutations using NGS panels for rare and myeloid genes.

Results: All 36 pts included in this study, 19 male and 17 female, with median age of lesion onset ranged from birth to 17.81 years (median 4.74 months), had a cutaneous mastocytosis (CM). Twenty-one of the 36 pts (58%) underwent cutaneous biopsy after a median from lesion onset of 3.77 months (range: 2.49 months – 11.6 years). According to the type of cutaneous lesions at diagnosis, pts were classified as: 20 (55%) MPCM, 10 (28%) DCM and 6 (17%) mastocytoma. The median tryptase value at the onset was 5 ng/ml, higher in MPCM (range: 1.2 - 141 ng/ml), than in DCM (range: 2.71 - 19.4 ng/ml), and in mastocytoma (range: 3.8 - 7.3 ng/ml). Two of the MPCM pts developed indolent SM (ISM) after 10 and 20 years from the onset disease. RT-PCR and NGS assessment were performed in 17 pts (47%), 15 CM and 2 ISM. RT-PCR identified KIT D816V mutation in 4 pts (2 MPCM, 1 DCM, 1 ISM) while NGS revealed KIT D816V in 3 pts and other KIT mutations, KIT D816Y and KIT Y553C, in 2 pts. An additional 10 myeloid gene mutations were detected by NGS technique: 5 were already known (ASXL1 G1397S; JAK2 L393V; KITD816Y; LNK E208Q; TET2 Y867H) while the other 5 have not been previously described (ETV6A215P; KIT Y553C; NFE2 1291T; SH2B3 G382D; SH2B3 L438V). Mutations in myeloid genes were prevalent in the signalling functional group. A single mutation was found in 7 pts (3MPCM, 3 DCM, 1ISM), while two or more mutations were found in 3 DCM pts, 2 of them had a spontaneous regression. Two different mutations (KIT Y553C and SH2B3 L438V) were found in two sisters. Overall, 9/36 pts (5 DCM, 3 MPCM, 1 mastocytoma) presented spontaneous complete regression of cutaneous lesions after a median time of 25 months (range: 17 months - 25 years).

Summary/Conclusion: KIT mutations resulted in 35% of the children tested. The RT-PCR technique resulted more sensitive in finding KIT D816V, while NGS in detecting other mutations. ASXL1, JAK2 and TET2 mutations, detected in our population, are different to those reported in adult pts with SM. The presence of multiple mutations in the DCM form, does not appear to influence the evolution to a systemic form, unlike in adults.

P1007: PHASE 2, OPEN-LABEL, MULTICENTER, SINGLE-ARM STUDY INVESTIGATING THE EFFICACY AND SAFETY OF ROPEGINTERFERON ALFA-2B IN JAPANESE PATIENTS WITH POLYCYTHEMIA VERA

K. Kirito 1,* , Y. Edahiro 2,3,4 , K. Ohishi 5 , A. Gotoh 6 , K. Takenaka 7 , H. Shibayama 8 , T. Shimizu 9 , K. Usuki 10 , K. Shimoda 11 , M. Ito 12 , S. A. VanWart 13 , O. Zagrijtschuk 14 , A. Qin 15 , H. Kawase 16 , N. Miyachi 16 , T. Sato 16 , N. Komatsu 2,3,4,16

1 Department of Hematology and Oncology, University of Yamanashi, Chuo-shi, Yamanashi; 2 Department of Hematology; 3 Laboratory for the Development of Therapeutics Against MPN; 4 Department of Advanced Hematology, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo; 5 Department of Transfusion Medicine and Cell Therapy, Mie University Hospital, Tsu-shi, Mie; 6 Department of Hematology, Tokyo Medical University, Shinjuku-ku, Tokyo; 7 Department of Hematology, Clinical Immunology, and Infectious Diseases, Ehime University Graduate School of Medicine, Toon-shi, Ehime; 8 Department of Hematology, National Hospital Organization Osaka National Hospital, Chuo-ku, Osaka; 9 Division of Hematology, Department of Medicine, Keio University School of Medicine, Shinjuku-ku, Tokyo; 10 Department of Hematology, NTT Medical Center Tokyo, Shinagawa-ku, Tokyo; 11 Division of Hematology, Diabetes, and Endocrinology, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Kiyotake-cho, Miyazaki; 12 Department of Pathology, Japanese Red Cross Aichi Medical Center Nagoya Daiichi Hospital, Nagoya-shi, Aichi, Japan; 13 Enhanced Pharmacodynamics, LLC, NY; 14 PharmaEssentia Corporation USA, MA, United States of America; 15 PharmaEssentia Corporation, Taipei, Taiwan; 16 PharmaEssentia Japan K.K., Minato-ku, Tokyo, Japan

Background: Polycythemia vera (PV) is a BCR–ABL-negative myeloproliferative neoplasms with a JAK2 gene mutation, that can be complicated by thrombosis and hemorrhage. It may also evolve to secondary acute myeloid leukemia, which has a poor prognosis and is potentially fatal. Ropeginterferon alfa-2b, a peg-proline-interferon alfa-2b with improved pharmacokinetic properties, allowing for less frequent administration and better tolerability, has been shown in clinical studies (PEGINVERA, PROUD-PV, CONTINUATION-PV) to be efficacious and have a favorable safety profile. However, its efficacy and safety have not been evaluated in Japanese PV patients.

Aims: The aim of this phase 2, single-arm study ( NCT04182100 ) was to investigate the efficacy and safety of ropeginterferon alfa-2b in Japanese patients with PV to assess the clinical utility of this cytoreductive therapy as a treatment option.

Methods: Japanese patients aged ≥20 years with a PV diagnosis according to World Health Organization 2008/2016 criteria and who were unsuitable candidates for current standard treatment were included, and patients being refractory to hydroxyurea (HU), symptomatic splenomegaly, or previous treatment with interferon alfa were excluded. Ropeginterferon alfa-2b was administered subcutaneously every 2 weeks for 12 months (M) at a starting dose of 100μg (50μg in patients receiving HU). This was increased by 50μg every 2 weeks until patients had achieved complete hematological response (CHR): hematocrit <45% without phlebotomy in the preceding 3M, platelets ≤400×10 9 /L, leukocytes ≤10×10 9 /L, or reached the maximum recommended single dose (500μg). The primary endpoint was phlebotomy-free CHR at 9M and 12M (end of treatment [EOT]). Secondary endpoints were change in hematologic parameters, CHR in patients with or without prior HU use, JAK2 V617F allele burden, molecular response, and frequency of phlebotomy. Safety endpoints were incidence of treatment-emergent adverse events (TEAEs) and AEs of special interest.

Results: A total of 29 patients comprised the intent-to-treat population (median age, 54 years; mean hematocrit, 46.85%; mean platelets, 747.3×10 9 /L; mean leukocytes, 17.07×10 9 /L at baseline). Twenty-seven patients had a JAK2 V617F mutation (mean allele burden, 72.19%). Two patients discontinued the study (one withdrew consent, the other due to an AE [silent thyroiditis]). The primary outcome of durable CHR without phlebotomy at 9M and EOT was achieved by 8/29 (27.6%) patients. Hematocrit, platelets, and leukocytes decreased over time (mean±SD change from baseline to EOT: –5.45±6.66%, –493.6±374.9×10 9 /L, –11.71±8.38×10 9 /L, respectively), and all parameters improved to their target value range. The proportion of patients with CHR at EOT was similar in patients with or without prior HU use (Table). JAK2 V617F allele burden decreased over time (change from baseline to EOT: mean ±SD, –19.17±22.64; Median [min, max], -11.62 [-74.80,33.80]; n=26). Molecular response increased over time. Eight (27.6%) patients did not require phlebotomy. TEAEs occurred in all patients; however, no event of grade ≥ 3 occurred. The most common TEAEs were alopecia (55.2%), fatigue (27.6%) and influenza-like illness (27.6%). AEs of special interest, which could be both disease and treatment-related, occurred in 9 (31.0%) of the patients and were related to the study treatment in 5 (17.2%) of the patients.

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Summary/Conclusion: Ropeginterferon alfa-2b is a safe and efficacious treatment option in Japanese patients with polycythemia vera.

P1008: ASSOCIATION BETWEEN FRAILTY AND CLINICAL OUTCOMES IN MYELOPROLIFERATIVE NEOPLAMS: A POPULATION-BASED STUDY FROM ONTARIO, CANADA

A. Bankar 1,* , W. Chan 2 , N. Liu 2 , M. Cheung 3 , S. Alibhai 4 , V. Gupta 1

1 Medical Oncology and Hematology, Princess Margaret Cancer Center, University Health Network; 2 ICES; 3 Sunnybrook Health Sciences Centre; 4 General Internal Medicine, Toronto General Hospital- University Health Network, Toronto, Canada

Background: Frailty independently predicts adverse outcomes in several different cancers and community-dwelling older adults. Its impact on clinical outcomes in myeloproliferative neoplasms (MPN) is unknown.

Aims: To measure the association between frailty and all-cause mortality and thrombosis in MPN patients.

Methods: Method: A retrospective, population-based study using province-wide administrative databases of Ontario.

Study population: We included MPN patients in the Ontario Cancer Registry from 2004 to 2019 ((Total n= 10,336: ET, n=5,108; PV, n=3,843; MF, n=1,385). Baseline frailty was measured during two years prior to date of MPN diagnosis using either (i) the Johns Hopkins Adjusted Clinical Groups ® frailty indicator (ACG-F), categorized as fit or frail if any of the ten frailty-defining diagnoses were present or (ii) the McIsaac’s cumulative deficit frailty index (mFI), categorized as fit, prefrail, or frail if mFI <0.10, 0.10-0.19, > 0.19 respectively.

Main outcome measures: Cox proportional hazard model was used to generate the Hazard Ratios (HRs) with 95% confidence interval (CI) for all-cause mortality comparing frail vs. prefrail vs. fit patients. Subdistribution hazard ratios (SHR) using the Fine-Gray models were used to evaluate the effect of frailty on development of thrombosis, taking death as the competing event. Analyses were adjusted for age, comorbidities, prior thrombosis, and level of marginalization.

Results: Results: The mean duration of follow-up for ET, PV and MF was 3.8, 4.0 and 2.9 years, respectively. 16% MPN cases were categorized as mFI-frail and 51% as mFI-prefrail. Patient with MF were more likely to be mFI-frail or mFI-prefrail compared to ET and PV (34%, 23%, and 20% respectively, p<0.001). In all MPN subtypes, frailty was independently associated with increased risk of mortality after adjusting for age, sex, and comorbidities. Compared to fit patients, the HRs for all-cause mortality for prefrail and frail patients were: 1.6 (1.3-1.9), and 3.6 (2.9-4.4) in ET; 1.3 (1.1-1.5) and 2.7 (2.1-3.4) in PV, and 1.2 (1.0-1.5) and 2.0 (1.5-2.7) in MF. Other predictors associated with increased all-cause mortality were advanced age (compared to age <40 years, HRs for 40-65 years, 65-75 years, >75 years: ET: 3.3 (2.0-5.5), 5.34 (3.2-8.7), 12.9 (7.9-21.2); for PV: 3.2 (1.6-6.7), 7.36 (3.6-15.0), 17.3 (8.5-35.2), for MF: 1.8 (0.8-3.8), 3.0 (1.4-6.5), 4.8 (2.2-10.4)) and comorbidities (1 and ≥ 2 comorbidities vs no comorbidities: ET: 1.3 (1.1-1.5), 2.4 (2.0-2.8); for PV: 1.0 (0.8-1.2), 1.2 (1.0-1.5); for MF: 1.2 (1.0-1.5), 1.4 (1.1-1.8)). With ACG-F, frailty was noted in 11% in MPN cases and was associated with increased mortality after adjusting for comorbidities and advanced age in all MFN subtypes [HRs in ET 2.5 (2.2-2.8), PV: 2.1 (1.8-2.4), and MF: 1.8 (1.5-2.2)].

In multivariable Fine-Gray regression, neither mFI-prefrail nor mFI-frail patients had an increased risk of thrombosis in all MPN subtypes. Compared to fit patients, frail patients using ACG-F had lower SHRs of thrombosis (ET: 0.6 (0.4-0.8), PV: 0.6 (0.4-0.8), MF: 0.2 (0.05-0.8), but higher SHRs for the competing event of death (ET: 3.0 (2.6-3.5), PV: 2.3 (1.9-2.9), MF: 1.9 (1.5-2.4)).

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Summary/Conclusion: Conclusions: Using large population-based databases, we found that a significant proportion of MPN patients are frail or prefrail at diagnosis despite younger age or less comorbidity burden. After adjusting for confounding from advanced age and increasing co-morbidity burden, frailty independently predicts increased risk of all-cause mortality in ET, PV, and MF.

P1009: PREDICTORS OF HOSPITALIZATION AND SEVERE OUTCOMES IN PATIENTS WITH MPN AND COVID-19

T. Barbui 1,* , A. Carobbio 1 , A. Masciulli 1 , A. Iurlo 2 , M. A. Sobas 3 , E. M. Elli 4 , E. Rumi 5 , V. De Stefano 6 , F. Lunghi 7 , M. Marchetti 8 , R. Daffini 9 , M. Gasior Kabat 10 , B. Cuevas 11 , M. L. Fox 12 , M. M. Andrade Campos 13 , F. Palandri 14 , P. Guglielmelli 15 , G. Benevolo 16 , C. Harrison 17 , M. A. Foncillas 18 , M. Bonifacio 19 , A. Alvarez-Larran 20 , J.-J. Kiladjian 21 , E. Bolanos Calderon 22 , A. Patriarca 23 , K. S. Quiroz Cervantes 24 , M. Griesshammer 25 , V. Garcia-Gutierrez 26 , A. Marin Sanchez 27 , E. Magro Mazo 28 , M. Ruggeri 29 , J. C. Hernandez-Boluda 30 , S. Osorio 31 , G. Carreno-Tarragona 32 , M. Sagues Serrano 33 , R. Kusec 34 , B. Navas Elorza 35 , A. Angona 36 , B. Xicoy Cirici 37 , E. Lopez Abadia 38 , S. Koschmieder 39 , E. Cichocka 40 , A. Kulikowska de Nałęcz 41 , M. Bellini 42 , D. Cattaneo 2,43 , C. Bucelli 2 , F. Cavalca 4 , O. Borsani 5 , S. Betti 6 , N. Curto-Garcia 17 , S. Carbonell 20 , L. Benajiba 21 , A. Rambaldi 42,43 , A. M. Vannucchi 15

1 FROM Research Foundation, Bergamo; 2 Hematology division, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 3 Department of Hematology, Blood neoplasms and Bone marrow transplantation, Wroclaw Medical University, Wroclaw, Poland; 4 Hematology division and Bone marrow transplant unit, San Gerardo Hospital, ASST Monza, Monza; 5 Department of Molecular medicine, University of Pavia, Pavia; 6 Fondazione Policlinico “A. Gemelli” IRCCS, Rome; 7 IRCCS Ospedale San Raffaele, Milan; 8 AOU SS. Antonio e Biagio e C. Arrigo, Alessandria; 9 ASST Spedali Civili, Brescia, Italy; 10 Hospital Univeristario La Paz, Madrid; 11 Hospital Universitario de Burgos, Burgos; 12 Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus; 13 Hospital del Mar, Barcelona, Spain; 14 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna; 15 Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), Department of Experimental and Clinical Medicine, Azienda Ospedaliera Universitaria Careggi, University of Florence, Florence; 16 AOU Città della Salute e della Scienza di Torino, Torino, Italy; 17 Guy’s and St. Thomas’ NHS Foundation Trust, London, United Kingdom; 18 Hospital Universitario Infanta Leonor, Madrid, Spain; 19 Ospedale Policlinico “G.B. Rossi”, Borgo Roma, Verona, Italy; 20 Hospital Clinic de Barcelona, Barcelona, Spain; 21 Hospital Saint-Louis, Paris, France; 22 Hospital Clinico San Carlos, Madrid, Spain; 23 AOU Maggiore della Carità, Novara, Italy; 24 Hospital Universitario de Mostoles, Madrid, Spain; 25 University Clinic for Hematology, Oncology, Hemostaseology and Palliative Care, Johannes Wesling Medical Center, Minden, Germany; 26 Hospital Ramon y Cajal, IRYCIS, Madrid; 27 Hospital General Universitario de Albacete, Albacete; 28 Hospital Universitario Principe de Asturias, Alcalà de Henares, Madrid, Spain; 29 Ospedale San Bortolo, Vicenza, Italy; 30 Hospital Clinico Universitario, INCLIVA, Valencia; 31 Hospital Gregorio Maranon; 32 Hospital Universitario 12 de Octubre, Madrid; 33 ICO L’Hospitalet-Hospital Moises Broggi, Sant Joan Despì, Barcelona, Spain; 34 Department of haematology, Clinic of internal medicine, University Hospital Dubrava-School of Medicine University of Zagreb, Zagreb, Croatia; 35 Hospital Moncloa, Madrid; 36 ICO Girona Hospital Josep Trueta, Girona; 37 ICO Hospital Germans Trias i Pujol, Badalona (Barcelona); 38 Hospital General de Elche, Elche (Alicante), Spain; 39 Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany; 40 Department of Hematology and Bone Marrow Transplantation, Nicolaus Copernicus Hospital, Torun; 41 Department of Hematology and Internal Diseases, State Hospital, Opole, Poland; 42 ASST Papa Giovanni XXIII, Bergamo; 43 Università degli Studi di Milano, Milano, Italy

Background: Risk factors for severe COVID-19 in myeloproliferative neoplasms (MPN) have been extensively explored. However, no information is available on risk factors to hospitalization at COVID-19 diagnosis.

Aims: To provide an evidence-based triage and inform for a timely and antiviral therapy prescription in early phases of viral replication.

Methods: The MPN-COVID study is still enrolling consecutive adult MPN patients with COVID-19 infection since February 15, 2020. Among 479 patients (ET n=175, PV n=158, MF n=91, and pre-PMF n=55) with COVID-19 from Feb 2020 to Jun 2021, 248 (52%) were managed at home and 231 (48%) were hospitalized.

Results: Univariate analysis

Compared to outpatients, those admitted to hospital were more likely to be men (58.9% vs. 45.2%, p=0.003), older than 70 years (61.3% vs. 29.0%, p<.001), with at least one comorbidity (79.7% vs. 55.5%, p<.001) and a history of thrombosis (26.5% vs. 16.6%, p=0.008). Overt myelofibrosis (MF) cases were more frequent in hospital (38.5% vs. 18.1%, p<.001) than PV, ET or pre-PMF. Ruxolitinib was more frequently used in patients who underwent to hospital than managed at home. (25.7% vs. 12.1%, p<.001). In comparison with outpatients, hospitalized cases had a significantly lower median values of hemoglobin (12.1 vs. 13.3 g/dL, p<.001), platelets (250 vs. 390 x10 9 /L, p<.001), absolute lymphocytes (0.8 vs. 1.4 x10 9 /L, p<.001) and higher neutrophil counts (5.1 vs. 4.5 x10 9 /L, p=0.022), leading to a significant increase of neutrophil to lymphocyte ratio (NLR) (6.6 vs. 3.2, p<.001). Compared to lymphocytopenia, the best sensitivity and specificity was found for NLR, whose AUC was 77.28% by ROC analysis.

Multivariate analysis

By adjusting for sex, comorbidity, fever, systemic symptoms, O2 saturation, previous thrombosis, MPN type, ruxolitinib exposure, and first vs. subsequent waves and vaccination period, three factors emerged as independent predictors of hospitalization: age over 70 years, (OR=3.02, p=0.038), dyspnea (OR=7.23, p<.001) and NLR ≥4, (OR=6.94, p<.001).

Interaction model of risk factors

In a model fitted to test the interaction terms of the three significant variables, we evaluated the marginal effect of NLR and dyspnea across different age classes (Figure) and found that in younger patients (i.e., from 50 to 70 years) dyspnea was the stronger predictor than increased NLR; conversely, both dyspnea and NLR showed a high and comparable marginal effect in age >80 years. Remarkably, the probability of hospitalization consistently exceeded 90% for any age group when dyspnea and NLR were concomitantly present, and their combination was more prevalent in MF (42%) than in the other phenotypes (24%, 25% and 29% in pre-PMF, PV and ET patients, respectively).

In addition to predict hospitalization, dyspnea and NLR≥4 were also associated with severity of COVID-19 illness in terms of respiratory support at hospitalization (OR=2.44, p=0.023). Of note, only age >70 years and NLR higher than 6 were predictors of survival in hospitalized patients (OR=3.24, p=0.007 and OR=5.41, p=0.041, respectively).

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Summary/Conclusion: For triage purposes of MPN patients tested positive for COVID-19, dyspnea, age and NLR are powerful predictors of hospitalization and identify patients at higher probability of invasive or non-invasive respiratory support and survival, particularly in overt MF. This MPN subgroup at high risk for progression to severe COVID-19 disease should be prioritized for antiviral therapy, even in the ambulatory setting.

P1010: RUXOLITINIB IN MYELODEPLETIVE MYELOFIBROSIS: RESPONSE, TOXICITY, AND OUTCOME

F. Palandri 1,* , D. Bartoletti 1,2 , M. Breccia 3 , G. Auteri 1,2 , E. M. Elli 4 , M. M. Trawinska 5 , N. Polverelli 6 , M. Tiribelli 7 , G. Benevolo 8 , A. Iurlo 9 , A. Tieghi 10 , F. H. Heidel 11 , G. Caocci 12 , E. Beggiato 13 , G. Binotto 14 , F. Cavazzini 15 , M. Miglino 16,17 , C. Bosi 18 , M. Crugnola 19 , M. Bocchia 20 , B. Martino 21 , N. Pugliese 22 , A. D. Romagnoli 1,2 , C. Mazzoni 1,2 , L. Scaffidi 23 , A. Isidori 24 , D. Cattaneo 9 , M. Krampera 23 , F. Pane 22 , D. Cilloni 25,26 , G. Semenzato 14 , R. M. Lemoli 16,17 , A. Cuneo 15 , E. Abruzzese 5 , N. Vianelli 1 , M. Cavo 1,2 , M. Bonifacio 23 , G. A. Palumbo 27

1 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; 2 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna; 3 A.O.U. Policlinico Umberto I, Università degli Studi di Roma “La Sapienza”, Rome; 4 Ospedale San Gerardo, ASST Monza, Monza; 5 Ospedale S. Eugenio, Università Tor Vergata, Rome; 6 ASST Spedali Civili di Brescia, Brescia; 7 A.O.U. Integrata di Udine, Udine; 8 A.O.U. Città della Salute e della Scienza, Torino; 9 Foundation IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano; 10 Azienda USL - IRCCS di Reggio Emilia, Reggio Emilia, Italy; 11 Innere Medicine C, Universitätsmedizin Greifswald, Greifswald, Germany; 12 Polo oncologico “A. Businco”, Università degli studi di Cagliari, Cagliari; 13 Dipartimento di Oncologia, Università di Torino, Torino; 14 A.O.U. di Padova, Padova; 15 A.O.U. Arcispedale S. Anna, Ferrara; 16 IRCCS Policlinico San Martino; 17 Dipartimento di Medicina interna e Specialità mediche, Università di Genova, Genova; 18 AUSL di Piacenza, Piacenza; 19 Azienda Ospedaliero-Universitaria di Parma, Parma; 20 Policlinico S. Maria alle Scotte, AOU Senese, Siena; 21 Division of Hematology, Azienda Ospedaliera ‘Bianchi Melacrino Morelli’, Reggio Calabria; 22 Dipartimento di Medicina clinica e Chirurgia, Università degli Studi di Napoli Federico II, Napoli; 23 A.O.U. Integrata Verona, Verona; 24 A.O. Ospedali Riuniti Marche Nord (AORMN), A.O. San Salvatore, Pesaro; 25 A.O. Ordine Mauriziano di Torino; 26 AOU San Luigi Gonzaga, Torino; 27 Dipartimento di Scienze Mediche, Chirurgiche e Tecnologie Avanzate “G.F. Ingrassia”, Università di Catania, Catania, Italy

Background: Around 30% of Myelofibrosis (MF) either primary (PMF) or secondary to polycythemia vera/essential thrombocythemia (SMF) may present a myelodepletive phenotype (MyD) (ie, thrombocytopenia, leukopenia, anemia). Pts with MyD MF represent a challenging population, as prognosis is poorer compared to pts with myeloproliferative (MyP) MF and therapeutic options, including the JAK1/2 inhibitor ruxolitinib (RUX), are limited or must be given at reduced doses.

Aims: In light of the upcoming new drugs that may be used in MyD MF, we explored prognostic correlates of MyD phenotype in RUX-treated MF pts.

Methods: After IRB approval, the “RUX-MF” retrospective real-world study collected 801 chronic phase MF pts treated with RUX in 26 Hematology Centers. MyD was defined as: WBC <4×109/L and/or Hb <11/<10 g/dL (males/females) and/or PLT <100×109/L with no increase of other blood cells (WBC >15×109/L, Hb >16.5/>16 g/dL in males/females, Plt >450×109/L). 219 (27.3%) had a MyD MF, including 140 (17.5%) PMF and 79 (9.8%) SMF. Spleen and symptoms response (SR/SyR) were defined according to IWG-MRT criteria. NGS mutational analysis was available for 167 pts.

Results: In multivariable analysis (MVA), PMF diagnosis (p=0.001) and unfavorable karyotype (p=0.01) confirmed their significant association with MyD. In PMF pts, MyD was due to leukopenia, anemia and thrombocytopenia in 7.1%, 52.2% and 9.3%, respectively; in SMF, corresponding figures were 5%, 51.9% and 11.4%. Two or more cytopenias were found in 31.4% and 31.7% of PMF and SMF patients, respectively.

In MVA, lower peripheral blast count (p=0.03), higher TSS (p=0.04) and BM fibrosis grade ≥2 (p=0.03) confirmed their association with MyD in PMF pts. In univariate/MVA, MyD SMF patients were more likely to have higher peripheral blast count (p=0.003/p=0.04), a higher MYSEC-PM risk (p<0.001/p=0.001), and to be triple negative (p=0.005/0.03).

RUX starting, median at 3 months, and median overall dose was more frequently ≤10 mg BID in MyD than in MyP pts (44.9% vs 67.5%, p<0.001; 39.5% vs 59.9%, p<0.001; 34.9% vs 57.3%, p<0.001, respectively). This was confirmed also in PMF and SMF separately.

The rate of SR was comparable in MyD and MyP patients. However, SR at 3 and 6 months was lower in pts with PLT<100 x 10 9 /l (p=0.02). In SMF, MyD pts had lower rates of SR (10.8% vs 28.0% at 3 mos, p=0.004; 20.0% vs 33.1% at 6 mos, p=0.05).

SyR was significantly lower in MyD MF (51.9% vs 62.5% in MyP at 3 mos, p=0.01; 59.8% vs 71.0% at 6 mos, p=0.008). In particular, anemia and thrombocytopenia were significantly associated with lower SyR.

After a median RUX exposure of 2.3 yrs (0.1-12.6), 364 (45.4%) pts stopped RUX, 110 (13.7%) had a blast phase and 366 (45.7%) died. After competing risk analysis, the cumulative incidence of RUX discontinuation was higher in MyD MF patients overall (p<0.001), only PMF (p=0.03) and only SMF (p<0.001) ( Fig.1a ). Incidence of RUX stop was significantly higher in MyD patients with ≥2 cytopenias ( p=0.03 ).

Leukemia-free survival was not influenced by MyD/MyP phenotype ( Fig.1b ).

In Cox regression analysis adjusted for DIPSS score, OS was significantly shorter in MyD vs MyP MF (median, 4.5 vs 5.7 yrs; p= 0.03) ( Fig.1c ). This was confirmed considering only SMF patients (p=0.02).

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Summary/Conclusion: MyD phenotype is associated with baseline high-risk clinical and molecular features, with lower responses to RUX, particularly in case of low PLT count, and higher risk of drug discontinuation and death. Newer strategies are warranted in this setting.

P1011: PREDICTORS OF COVID-19 DISEASE AND SURVIVAL TO COVID-19 IN MPN PATIENTS TREATED WITH RUXOLITINIB

F. Palandri 1,* , D. Bartoletti 1,2 , E. M. Elli 3 , G. Auteri 1,2 , M. Bonifacio 4 , G. Benevolo 5 , F. Heidel 6 , M. M. Trawinska 7 , E. Rossi 8,9 , C. Bosi 10 , A. Tieghi 11 , M. Tiribelli 12 , A. Iurlo 13 , N. Polverelli 14 , G. Caocci 15 , G. Binotto 16 , F. Cavazzini 17 , E. Beggiato 18 , D. Cilloni 19 , C. Tatarelli 20 , F. Mendicino 21 , M. Miglino 22,23 , M. Bocchia 24 , M. Crugnola 25 , C. Mazzoni 1,2 , A. D. Romagnoli 1,2 , G. Rindone 3 , S. Ceglie 8 , A. D’Addio 26 , E. Santoni 4 , D. Cattaneo 13 , R. M. Lemoli 22,23 , M. Krampera 4 , A. Cuneo 17 , G. Semenzato 27 , R. Latagliata 28 , E. Abruzzese 7 , N. Vianelli 1 , M. Cavo 1,2 , A. Andriani 29 , V. De Stefano 8,9 , G. Palumbo 30 , M. Breccia 31

1 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”; 2 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna; 3 Ospedale San Gerardo, ASST Monza, Monza; 4 A.O.U. Integrata Verona, Verona; 5 A.O.U. Città della Salute e della Scienza, Torino, Italy; 6 Innere Medicine C, Universitätsmedizin Greifswald, Greifswald, Germany; 7 Ospedale S. Eugenio, Università Tor Vergata; 8 Section of Hematology, Department of Radiological and Hematological Sciences, Catholic University School of Medicine; 9 Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome; 10 AUSL di Piacenza, Piacenza; 11 Azienda USL - IRCCS di Reggio Emilia, Reggio Emilia; 12 A.O.U. Integrata di Udine, Udine; 13 Foundation IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano; 14 ASST Spedali Civili di Brescia, Brescia; 15 Polo oncologico “A. Businco”, Università degli studi di Cagliari, Cagliari; 16 A.O.U. di Padova, Padova; 17 A.O.U. Arcispedale S. Anna, Ferrara; 18 Dipartimento di Oncologia, Università di Torino, Torino; 19 A.O. Ordine Mauriziano di Torino, Torino; 20 Azienda Ospedaliera Universitaria Sant’Andrea di Roma, Rome; 21 Unit of Hematology, Hospital of Cosenza, Cosenza; 22 IRCCS Policlinico San Martino; 23 Dipartimento di Medicina interna e Specialità mediche, Università di Genova, Genova; 24 Policlinico S. Maria alle Scotte, AOU Senese, Siena; 25 Azienda Ospedaliero-Universitaria di Parma, Parma; 26 Division of Hematology, Onco-hematologic Department, AUSL della Romagna, Ravenna; 27 Unit of Hematology and Clinical Immunology, University of Padova, Padova; 28 Hematology Unit, Ospedale Belcolle, Viterbo; 29 Hematology, Fabrizio Spaziani Hospital, Frosinone; 30 Department of Scienze Mediche, Chirurgiche e Tecnologie Avanzate “G.F. Ingrassia”, University of Catania, Catania; 31 Division of Cellular Biotechnologies and Hematology, University Sapienza, Rome, Italy

Background: Ruxolitinib (RUX) use and discontinuation are risk factors for severe COVID-19 and death in MPN patients (pts). In pts on RUX therapy, predictors for COVID-19 disease and survival (OS) to COVID-19 are unknown.

Aims: The aims of this study were to distinguish RUX-treated pts at higher risk of COVID-19 and to assess prognostic factors for OS.

Methods: We performed a sub-analysis of the RUX-MF and the PV-ARC observational studies that include consecutive adult pts with myelofibrosis (MF) and polycythemia vera (PV), respectively. Overall, 815 MF and 172 PV pts treated with RUX outside clinical trials have been registered. At pandemic start, 494 pts (359 MF and 135 PV) on RUX were included in this analysis.

Results: Among 66 (13.6%) pts (PV n=11, MF n=55) with COVID-19 from Feb 2020 to Jan 2022, 1 (1.5%), 14 (21.2%), 9 (13.6%), 17 (25.8%), 4 (6.1%) and 21 (31.8%) pts had an asymptomatic, mild, moderate, severe, critical, and fatal infection, respectively; 42 (63.7%) were hospitalized.

Overall, 14, 38 and 14 infections were observed during the 1st (Feb-Jun 2020), 2nd (Jul 2020-Jun2021) and 3rd (Jul 2021-Jan 2022) wave of the pandemic, with an overall incidence rate of 10.2 per 100 pt-yrs. Incidence rates in the 3 waves were 8, 10.2 and 7 per 100 pt-yrs respectively. Hospitalized cases were significantly less frequent during the 3rd wave (35.7% vs 64.3%/73.7% in the 1st/2nd wave, p=0.04).

Overall, 283/390 evaluable pts (72.6%) received ≥1 dose of Comirnaty vaccine (19/66 COVID-19 pts; 5, 7 and 7 pts had received 1, 2 or 3 vaccine doses, respectively). At COVID-19 diagnosis, RUX was reduced in 10 (15.1%) pts and discontinued in 9 (13.6%) pts, comparably in MF and PV.

In the total cohort, COVID-19 infection was more frequent in pts with MF (15.3% vs. 8.2% PV pts, p=0.04), with ≥1 comorbidity (15% vs. 8.7%, p=0.04). Also, COVID-19 infections after vaccine availability were more frequent in unvaccinated pts (37.4 vs. 6.3%, p<0.001).

COVID-19 requiring hospitalization was more frequently observed in pts ≥70 yrs (12.2% vs. 6.8% in pts <70 yrs, p=0.04), and without COVID-19 vaccine (32.4% vs. 2.9%, p<0.001). No additional predictors for COVID-19 were noted analyzing MF and PV separately.

In COVID-19 pts, hospitalized cases had a significantly lower median platelet count (275 vs. 168 x10 9 /L, p=0.02), were receiving lower RUX doses (33.3% <10 mg BID vs. 8.3%, p=0.02) and more frequently presented comorbidities (40.5% vs. 13.6%, p=0.03) compared to outpatients. MF vs. PV, median hemoglobin levels, age≥70 yrs and sex were not associated with hospitalization. MF pts who were not in spleen response at COVID-19 infection had higher risk of hospitalization (73% vs. 44.4% in responders, p=0.04).

After multivariable Cox analysis including previous anti-SARS-Cov-2 vaccine, need for hospitalization, age≥70 and male sex, OS to COVID-19 was significantly improved in pts who had previously received anti-SARS-Cov-2 vaccine (HR=0.10, p=0.02) (Fig.1 ), in pts with COVID-19 not requiring hospitalization (HR=0.19, p=0.03) and in patients <70 yrs (HR=0.38, p=0.03). The COVID-19 wave did not impact OS (p=0.53).

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Summary/Conclusion: Among RUX-treated pts, lower RUX doses, comorbidities and no spleen response are significant predictors of hospitalization. Vaccine was the most protective factor against COVID-19 disease, hospitalization, and mortality. RUX-treated pts, regardless of MPN type, should be sensitized to adherence to the vaccine program and prioritized for antiviral therapy in case of infection.

P1012: A NUMBER OF CONGENITAL ERYTHROCYTOSIS ARE MULTIGENIC

A. Benetti 1,* , G. Biagetti 1 , E. Cosi 1 , I. Bertozzi 1 , G. Ceolotto 1 , M. L. Randi 1

1 Department of Medicine, University of Padua, Padua, Italy

Background: Imbalance in erythropoiesis and oxygen homeostasis could lead to the onset of Erythrocytosis, a clinical condition characterized by persistently raised hemoglobin (Hb) and hematocrit (Ht) levels.

Nowadays, a high number of patients are classified as “Idiopathic Erythrocytosis” (IE) meaning that it is not possible to identify the cause of hematocrit increase.

Recently, HFE mutations has been found to be often present in idiopathic erythrocytic patients.

The HFE mutations could facilitate the increase in red cell mass, and this indicates the possible involvement of Iron Metabolism in causes of IE.

Aims: To identify the possible mutations of IE patients using our gene panel for Next Generation Sequencing (NGS).

Methods: In 118 patients with IE, regularly followed in our surgery, we used a gene panel for NGS composed by fifteen genes: JAK2 , EGLN1 ( PHD2 ), EPO-R , FTL , FTH , ASXL1 , HFE , HFE2 , TFR2 , HAMP , SLC40A1 , SLC11A2 , VHL , BPMG , EPAS1 ( HIF-2α ). We analyzed all the exons parts of these genes. The approach used for the library preparation was a multiplexing PCR and data were analyzed by bioinformatics tools. In all patients, the mutations found were confirmed with Sanger Sequencing.

Results: In 78 out of the 118 patients (66%) evaluated, we found one 1 mutated gene in 55 patients (7 EGLN1 , 1 VHL , 34 HFE , 6 TFR2 , 3 JAK2 and 4 EPO-R ), 2 mutated genes in 18 patients (1 EGLN1 / JAK2 , 4 EGLN1 / HFE , 2 EPAS1 / HFE , 1 EPAS1 / JAK2 , 1 EPAS1 / EGLN1 , 2 EPO-R / HFE , 2 HFE / JAK2 , 1 JAK2 / TFR2 , 2 with TFR2 / EGLN1 , 1 VHL / HFE , 1 TFR2 / HFE ) and 3 mutated genes in 5 patients (1 EGLN1 / EPO-R / HFE , 1 VHL / HFE / EGLN1 , 1 EPAS1 / EPO-R / HFE , 1 EPAS1 / HFE / EGLN1 and HFE / TFR2 / JAK2 ). All the mutations found were germline. Interestingly, 21 out of the 27 (78%) patients carrying HFE mutation displayed 2 or more mutations.

Summary/Conclusion: The use of NGS panels in various clinical conditions has frequently demonstrated that multiple mutations coexist in various patients and that the monogenic diseases are quite rare. This study is the first at our best knowledge that gives evidence of more than one mutated gene in erythrocytotic patients, and this is the results of the use of an appropriate NGS panel.

The data here reported suggest that we can confirm that HFE mutations are common in IE patients, in some cases associated with other mutations, but the present data ask some questions: HFE mutations alone can induce erythrocytosis? or other genetic imbalances are needed? The presence of multiple mutations could lead to a greater severity of the disease? A significant number of IE patients displayed TFR2 mutations, giving more relevance to the hypothesis of the iron metabolism implication in IE genesis. Finally, the presence of 11 patients carrying germlines JAK2 mutations is surprising, and we suppose it will be necessary to strictly watch these patients in the suspicion of a future appearance of a polycythemia vera.

The present study demonstrates the need to extend the biomolecular knowledge in patients with idiopathic erythrocytosis.

P1013: OVERALL SURVIVAL IN PATIENTS WITH SYSTEMIC MASTOCYTOSIS WITH ASSOCIATED HEMATOLOGIC NEOPLASM TREATED WITH AVAPRITINIB VERSUS BEST AVAILABLE THERAPY

A. Reiter 1,* , J. Gotlib 2 , I. Álvarez-Twose 3 , D. H. Radia 4 , J. Luebke 1 , P. J. Bobbili 5 , A. Wang 5 , C. Norregaard 6 , S. Dimitrijević 7 , E. Sullivan 6 , M. Louie-Gao 6 , J. Schwaab 1 , I. A. Galinsky 8 , C. Perkins 2 , W. R. Sperr 9 , P. Sriskandarajah 4 , A. Chin 5 , S. R. Sendhil 5 , M. S. Duh 5 , P. Valent 9 , D. J. DeAngelo 8

1 Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany; 2 Stanford Cancer Institute/Stanford University School of Medicine, Stanford, United States of America; 3 Institute of Mastocytosis Studies of Castilla La Mancha (CLMast) ─ Spanish Reference Center (CSUR) for Mastocytosis and CIBERONC, Virgen del Valle Hospital, Toledo, Spain; 4 Guy’s & St Thomas’ NHS Foundation Trust, Guy’s Hospital, London, United Kingdom; 5 Analysis Group, Inc., Boston; 6 Blueprint Medicines Corporation, Cambridge, United States of America; 7 Blueprint Medicines Corporation, Zug, Switzerland; 8 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America; 9 Department of Internal Medicine I, Division of Hematology and Hemostaseology, and Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, Vienna, Austria

Background: Avapritinib, a selective inhibitor of KIT D816V, was approved in the United States (US) for treatment of adults with advanced systemic mastocytosis (AdvSM), a rare myeloid neoplasm with poor overall survival (OS), based on data from the Phase 1 EXPLORER ( NCT02561988 ) and Phase 2 PATHFINDER ( NCT03580655 ) single-arm trials. Systemic mastocytosis with an associated hematologic neoplasm (SM-AHN) is both the most common and most heterogenous subtype of AdvSM. No randomized control trial (RCT) has been conducted comparing efficacy of avapritinib with best available therapy (BAT) in SM-AHN.

Aims: This study ( NCT04695431 ) compared OS between SM-AHN patients treated with avapritinib in the EXPLORER and PATHFINDER trials versus SM-AHN patients treated with BAT in standard clinical practice.

Methods: A multi-center, global, observational, retrospective chart review study was conducted at 6 study sites (4 European, 2 US) to identify and collect data from AdvSM patients who received BAT. SM-AHN patients were identified using inclusion/exclusion criteria similar to the EXPLORER and PATHFINDER trials. Patients receiving BAT could contribute data on multiple lines of therapy (LOTs) to the analysis; these data were compared with patient-level data from the EXPLORER and PATHFINDER trials. OS was defined as the time from avapritinib or BAT initiation to death from any cause; patients were censored at date of last follow-up if alive. Kaplan-Meier analysis was used to assess OS. Inverse probability of treatment weighting (IPTW) was used to adjust for differences in key variables between the treatment cohorts, including age, sex, region, anemia, thrombocytopenia, leukocyte count, skin involvement, number and type of prior treatments, performance status, serum tryptase level, and presence of SRSF2/ASXL1/RUNX1 mutations. An IPTW-weighted Cox proportional hazards model, adjusted for variables that remained unbalanced after weighting, was used to compare OS between treatment groups.

Results: This analysis included 119 avapritinib patients and 83 BAT patients with SM-AHN (the latter contributed data on 121 LOTs). Median (range) age of the avapritinib and BAT cohorts was 70 (45−88) and 71 (38−88) years, and 61% vs. 76% of patients were male, respectively. Prior systemic therapy was received by 58% of avapritinib and 44% of BAT patients. In the avapritinib cohort, 76% of patients from both trials were dosed at ≤200mg and 24% of patients, all from EXPLORER, were dosed at ≥300mg. BAT patients were most frequently treated with tyrosine kinase inhibitors (73 out of 121 LOTs, 60%) or cytoreductive therapies (46 out of 121 LOTs, 38%). Mean durations of follow-up were 17.6 and 18.1 months, with 29 (24%) and 56 (68%) deaths in avapritinib and BAT cohorts, respectively. Median OS for avapritinib was 46.9 months (95% CI: 44.9, not estimable) and 18.0 months (95% CI: 13.0, 26.8) for BAT (Figure 1). IPTW-adjusted median OS was 46.9 months (95% CI: 21.4, 49.0) for avapritinib and 19.5 months (95% CI: 13.0, 32.2) for BAT. Weighted Cox analysis showed that OS was significantly improved for avapritinib versus BAT (hazard ratio [95% CI]: 0.42 [0.24, 0.74]; P <0.001), even with further adjustment for unbalanced variables.

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Summary/Conclusion: The results of this analysis indicate that patients with SM-AHN treated with avapritinib in clinical trials had significantly longer OS compared to patients treated with BAT. With no RCTs, these data offer crucial insights into the survival benefit of SM-AHN patients treated with avapritinib compared to other treatments for AdvSM.

P1014: OVERALL SURVIVAL IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS RECEIVING AVAPRITINIB VERSUS MIDOSTAURIN OR CLADRIBINE

A. Reiter 1,* , J. Gotlib 2 , I. Álvarez-Twose 3 , D. H. Radia 4 , J. Luebke 1 , P. J. Bobbili 5 , A. Wang 5 , C. Norregaard 6 , S. Dimitrijević 7 , E. Sullivan 6 , M. Louie-Gao 6 , J. Schwaab 1 , I. A. Galinsky 8 , C. Perkins 2 , W. R. Sperr 9 , P. Sriskandarajah 4 , A. Chin 5 , S. R. Sendhil 5 , M. S. Duh 5 , P. Valent 9 , D. J. DeAngelo 8

1 Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany; 2 Stanford Cancer Institute/Stanford University School of Medicine, Stanford, United States of America; 3 Institute of Mastocytosis Studies of Castilla La Mancha (CLMast) ─ Spanish Reference Center (CSUR) for Mastocytosis and CIBERONC, Virgen del Valle Hospital, Toledo, Spain; 4 Guy’s & St Thomas’ NHS Foundation Trust, Guy’s Hospital, London, United Kingdom; 5 Analysis Group, Inc., Boston; 6 Blueprint Medicines Corporation, Cambridge, United States of America; 7 Blueprint Medicines Corporation, Zug, Switzerland; 8 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America; 9 Department of Internal Medicine I, Division of Hematology and Hemostaseology, and Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, Vienna, Austria

Background: Avapritinib, a selective KIT D816V inhibitor, was approved for the treatment of adults with advanced systemic mastocytosis (AdvSM) in the United States (US), based on two single-arm trials: Phase 1 EXPLORER ( NCT02561988 ) and Phase 2 PATHFINDER ( NCT03580655 ). No randomized controlled trial (RCT) has been conducted comparing efficacy of avapritinib versus alternative therapies for AdvSM.

Aims: This study ( NCT04695431 ) compared overall survival (OS) between AdvSM patients treated with avapritinib in the EXPLORER and PATHFINDER trials and those treated with midostaurin or cladribine in real-world clinical practice.

Methods: Pooled data from the EXPLORER and PATHFINDER trials were compared with data for patients with AdvSM obtained through a multi-center, global, observational, retrospective chart review study conducted at 6 study sites (4 European, 2 US). Patients treated with midostaurin or cladribine were identified using inclusion/exclusion criteria similar to the trials and could contribute multiple lines of therapy (LOTs) to the analysis. OS, assessed using Kaplan-Meier analysis, was defined as time from initiation of avapritinib, midostaurin, or cladribine to death from any cause; patients were censored at date of last follow-up if alive. Inverse probability of treatment weighting (IPTW) was used to adjust for differences in key variables between treatment cohorts, including age, sex, AdvSM subtype, anemia, thrombocytopenia, leukocyte count, skin involvement, number and type of prior treatments, performance status, serum tryptase level, and presence of SRSF2/ASXL1/RUNX1 mutations. IPTW-weighted Cox proportional hazards models, adjusted for variables that remained unbalanced after weighting, were used to compare OS between cohorts.

Results: This analysis included 176 patients treated with avapritinib, 94 treated with midostaurin (LOT, n=99), and 44 treated with cladribine (LOT, n=49). Median (range) age was 68 (31−88) for avapritinib, 69 (26−87) for midostaurin, and 66 (45−88) years for cladribine patients. 103 (59%) avapritinib patients were male, versus 64 (68%) in the midostaurin and 27 (61%) in the cladribine cohort. In the avapritinib cohort, 136 (77%) patients from both trials were dosed at ≤200mg and 40 (23%) patients, all from EXPLORER, were dosed at ≥300mg.

In unweighted analysis of avapritinib versus midostaurin, mean follow-up durations were 17.9 and 27.9 months, respectively, during which 34 (19%) avapritinib patients and 56 (60%) midostaurin patients died. Median OS was not reached (NR) (95% CI: 46.9, not estimable) in the avapritinib cohort and was 28.6 months (95% CI: 18.2, 44.6) in the midostaurin cohort (Figure 1). In weighted Cox analysis, OS was significantly improved in the avapritinib versus midostaurin cohort (hazard ratio [HR] [95% CI]: 0.59 [0.36, 0.97]; P <0.001).

The mean-follow-up duration for the cladribine cohort was 24.2 months, during which 29 (66%) patients died. The median OS in the cladribine cohort was 23.4 months (95% CI: 14.8, 40.6). Weighted Cox analysis showed that OS was significantly improved in the avapritinib versus cladribine cohort (HR [95% CI]: 0.32 [0.15, 0.67]; P =0.003).

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Summary/Conclusion: The results from this study indicate that AdvSM patients treated with avapritinib in clinical trials experienced significantly improved survival compared with patients treated with midostaurin or cladribine in the real world. Given the lack of RCTs, these data offer essential insights into the improved survival of patients treated with avapritinib compared to alternative therapies for AdvSM.

P1015: DURATION OF TREATMENT AND REDUCTION IN SERUM TRYPTASE LEVELS IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS TREATED WITH AVAPRITINIB VERSUS BEST AVAILABLE THERAPY

A. Reiter 1,* , J. Gotlib 2 , I. Álvarez-Twose 3 , D. H. Radia 4 , J. Luebke 1 , P. J. Bobbili 5 , A. Wang 5 , C. Norregaard 6 , S. Dimitrijević 7 , E. Sullivan 6 , M. Louie-Gao 6 , J. Schwaab 1 , I. A. Galinsky 8 , C. Perkins 2 , W. R. Sperr 9 , P. Sriskandarajah 4 , A. Chin 5 , S. R. Sendhil 5 , M. S. Duh 5 , P. Valent 9 , D. J. DeAngelo 8

1 Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany; 2 Stanford Cancer Institute/Stanford University School of Medicine, Stanford, United States of America; 3 Institute of Mastocytosis Studies of Castilla La Mancha (CLMast) ─ Spanish Reference Center (CSUR) for Mastocytosis and CIBERONC, Virgen del Valle Hospital, Toledo, Spain; 4 Guy’s & St Thomas’ NHS Foundation Trust, Guy’s Hospital, London, United Kingdom; 5 Analysis Group, Inc., Boston; 6 Blueprint Medicines Corporation, Cambridge, United States of America; 7 Blueprint Medicines Corporation, Zug, Switzerland; 8 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America; 9 Department of Internal Medicine I, Division of Hematology and Hemostaseology, and Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, Vienna, Austria

Background: Avapritinib, a selective and highly potent KIT D816V inhibitor, was approved in the United States (US) for treatment of adults with advanced systemic mastocytosis (AdvSM) based on results from two single-arm trials: EXPLORER (Phase 1; NCT02561988 ) and PATHFINDER (Phase 2; NCT03580655 ). There is no comparative response data for avapritinib versus best available therapy (BAT) for AdvSM.

Aims: This study ( NCT04695431 ) compared duration of treatment (DOT) and maximum reduction in serum tryptase levels between patients treated with avapritinib at a starting dose ≤200mg versus BAT.

Methods: A multi-center, global, observational, retrospective chart review study was conducted at 6 study sites (4 European, 2 US) to identify and collect data from AdvSM patients who received BAT. Patients were identified using inclusion/exclusion criteria similar to EXPLORER and PATHFINDER trials. AdvSM patients receiving BAT could contribute data on multiple lines of therapy (LOT); these data were compared with patient data from the trials. DOT was defined as time from initiation to discontinuation of each LOT. Maximum reduction in serum tryptase on treatment was defined during each LOT. DOT was compared between cohorts using a Cox proportional hazards model and reduction in serum tryptase levels was compared using a generalized estimating equation linear model. Both models were weighted by inverse probability of treatment weights (IPTW) to adjust for confounding by key variables: age, sex, region, AdvSM subtype, anemia, thrombocytopenia, leukocyte count, skin involvement, number and type of prior treatments, performance status, serum tryptase level, and presence of SRSF2/ASXL1/RUNX1 mutations. Both models further adjusted for key covariates that remained unbalanced after weighting. Subgroup analyses were conducted in patients who received ≥1 prior LOT (2L+), and a 200mg starting dose of avapritinib.

Results: The DOT analysis included 136 avapritinib patients and 137 BAT patients. Median (range) age for avapritinib and BAT patients was 68 (31−88) and 69 (21−88) years, and 59% versus 66% of patients were male, respectively. In the avapritinib group, 67%, 17%, and 16% of patients were diagnosed with SM with an associated hematological neoplasm (SM-AHN), aggressive SM (ASM), and mast cell leukemia (MCL), respectively, while in the BAT group, 55%, 29%, and 16% were diagnosed with SM-AHN, ASM, and MCL. Among 188 BAT LOTs with agent-level data available, most common treatments were midostaurin (53%) and cladribine (24%). Median DOT was 32.1 months (95% CI: 23.8, not estimable) for avapritinib and 5.5 months (95% CI: 5.1, 7.0) for BAT. In IPTW-weighted Cox analysis, DOT was significantly longer for avapritinib versus BAT (hazard ratio [95% CI]: 0.35 [0.23, 0.51]; P <0.001).

The analysis of reduction in serum tryptase included 135 avapritinib and 116 BAT patients. Unadjusted maximum percentage reduction in serum tryptase levels was -84.8% (standard deviation [SD]: 19.9%) for avapritinib versus -9.2% (SD: 161.4%) for BAT. The adjusted mean difference in maximum percentage reduction in serum tryptase comparing avapritinib to BAT was -69.8% (95% CI: -89.4%, -50.2%; P <0.001).

Results were similar in subgroups (Table 1).

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Summary/Conclusion: AdvSM patients treated with avapritinib experienced significantly longer DOT and greater reduction in serum tryptase levels compared to patients treated with BAT. In the absence of RCTs, these data offer important and meaningful insight into the improved effectiveness of avapritinib compared to alternative therapies for AdvSM.

P1016: POLYCYTHEMIA VERA AND IMMUNE THROMBOGENESIS

R. Cacciola 1,* , V. Vecchio 2 , E. Gentilini Cacciola 3 , E. CACCIOLA 4

1 Experimental and Clinical Medicine, Haemostasis Unit - University of Catania; 2 University of Catania, University of Catania School of Medicine, Catania; 3 Public Health and Infectious Diseases, Sapienza University, Roma; 4 Medical, Surgical Sciences and Advanced Technologies “G.F. Ingrassia”, Haemostasi Unit, Catania, Italy

Background: Polycythemia Vera (PV) is a Ph1-negative myeloproliferative neoplasm characterized by vascular thrombosis and poor survival. The polycythemic thrombogenesis is associated with erythrocytosis and red cell adhesiveness angiopathy. Recent studies reported a JAK/STAT-mediated reduction of the regulatory T cells (Tregs) (CD4+, CD25high, CD127low, FoxP3+) and loss of self-tolerance.

Aims: We investigated Tregs, anti-endothelial cell antibodies (AECA), and endothelial, platelet and coagulation activation, in Polycythemia Vera (PV) and thrombosis.

Methods: We enrolled 60WHO-defined PV patients (30 men, 30 women; mean age 45±10 years) without cardiovascular risk factors, autoimmune disease or thrombotic history. Of PV patients, 40/60 had thromosis includingmyocardial infarction (MI) (10/60) according to the WHO critera, deep vein thrombosis (20/60) and pulmonary embolism (10/60) on lower-limb ultrasonography and computed tomography angiography, respectively. All patients were evaluated for JAK2V617F allele burden, Tregs, AECA,Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1), Intercellular Adhesion Molecule-1 (ICAM-1),prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimer (DD). JAK2V617F allele burden were analyzed by Polymerase chain reaction, Tregs were measured by flow cytometry,AECA, ELAM-1 and ICAM-1 by ELISA, PT and APTT by coagulometric test, Fib using Clauss method, and DD using ELISA.Complete blood hemostasis was studied by PFA-100 on Collagen/ADP (CT-ADP) and Collagen/Epinephrine (CT-EPI) cartridges and Thromboelastometry method on Clotting Time (CT), Clotting Formation Time (CFT), Maximum Clot Firmness (MCF), and clot lysis at 30 minutes (LY-30).

Results: The patients with thrombosis had JAK2V616F allele burden higher (> 50%) compared to patients without thrombosis (< 50Kroll%), lower Tregs (1,5±0,5% vs 3.5±1%),higher AECA (200±20% vs 110±10%), ELAM-1 (80±10 ng/ml vs 45±5 ng/ml), and ICAM (170 ng/mL±10 vs 110±10 ng/mL), longer PT (30±10 s vs 20±2 s) and PTT (60±10 s vs 38±5 s), lower Fib (90±20 mg/dl vs 120±20 mg/dl), higher DD (650±100 mg/l vs 300±50 mg/l) and shorter C/ADP and C/EPI (C/ADP, n.v. 68-121 s (40±10 s vs 55±20 s) and C/EPI n.v. 84-160 s (35±5 s vs 60±10 s). The patients with thrombosis had shorter CT (INTEM 35±20 s vs 70±20 s, EXTEM 20±10 s vs 30±5 s), shorter CFT (INTEM 15±10 s vs 25±5 s EXTEM 18±10 s vs 28±5 s), longer MCF (INTEM 130±10 mm vs 90±10 mm, EXTEM 120±10 mm vs 82±10 mm), and lower LY-30 (INTEM 0.9% vs 15%, EXTEM 0.8% vs 15%). A positive correlation there was between Tregs and AECA, ELAM-1 and ICAM-1 and thrombosis.

Summary/Conclusion: These findings shed new light on thrombotic pathogenesis in patients with PV.

P1017: HARBOR: A PHASE 2/3 STUDY OF BLU-263 IN PATIENTS WITH INDOLENT SYSTEMIC MASTOCYTOSIS AND MONOCLONAL MAST CELL ACTIVATION SYNDROME

M. Castells 1,* , R. Scherber 2,3 , V. Bhavsar 2 , K. He 2 , C. Akin 4

1 Mastocytosis Center, Brigham and Women’s Hospital and Harvard Medical School, Boston; 2 Blueprint Medicines Corporation, Cambridge; 3 UT Health San Antonio, MD Anderson Cancer Center, San Antonio; 4 University of Michigan, Ann Arbor, United States of America

Background: The KIT D816V mutation plays a key role in the aggregation and accumulation of aberrant mast cells which characterize systemic mastocytosis (SM), a rare, clonal mast cell neoplasm. The disease subtype of indolent SM (ISM) is characterized by the presence of mast cell aggregates in bone marrow and other organs which can lead to chronic, debilitating, and potentially life-threatening symptoms. Mast cell infiltration in the skin is common in ISM but is not always present. Similarly, monoclonal mast cell activation syndrome (mMCAS) is a clonal KIT D816V–positive mast cell disease without mast cell aggregates which can be accompanied by similar symptoms. Neither ISM nor mMCAS currently have approved molecularly targeted therapies, and significant symptom burden exists despite best supportive care (BSC) symptom-based therapy. BLU-263 is a novel, oral, next-generation tyrosine kinase inhibitor, exhibiting potent inhibition of KIT D816V and an evolved preclinical profile with limited central nervous system penetration. In our phase 1 study in normal healthy volunteers, BLU-263 was safe, with linear pharmacokinetics across all tested doses and a half-life allowing once-daily dosing, and thus supporting continued development for patients with SM.

Aims: HARBOR ( NCT04910685 ) is a randomized, double-blind, placebo-controlled, phase 2/3 study assessing efficacy and safety of orally administered BLU-263 in patients with ISM whose symptoms are not adequately controlled by standard therapies.

Methods: Part 1 of the study includes 3 dose groups (25 mg, 50 mg, and 100 mg) and a placebo group combined with BSC therapies to determine the recommended dose (RD) of BLU-263 in approximately 40 patients with ISM (Figure). After determination of the RD and analysis of Part 1, Part 2 will open for enrollment of approximately 303 patients undergoing BSC therapies, randomized 2:1 to BLU-263 or placebo. The primary endpoint of Part 2 is the proportion of patients achieving a ≥30% reduction in total symptom score as assessed by the Indolent Systemic Mastocytosis Symptom Assessment Form (ISM-SAF). Patients completing Part 1 or Part 2 will roll over to Part 3 for open-label long-term evaluation of BLU-263 at the RD. The exploratory, open-label study Part M will explore BLU-263 at the RD in patients diagnosed with mMCAS. Other endpoints including but not limited to Mastocytosis Quality of Life scores, KIT D816V allele burden, and bone marrow mast cell involvement will also be assessed. Two pharmacokinetic (PK) groups will enroll patients with ISM prior to or concurrent with the Part 1 and Part 2 study cohorts to better characterize the PK and safety of BLU-263 in specific patient populations.

Results: Topline results of Part 1 of the study are anticipated in the second half of 2022.

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Summary/Conclusion: Overall, the goal of HARBOR is to investigate the safety and efficacy of the selective, targeted KIT inhibitor BLU-263 as a potential treatment option to reduce symptom burden for patients with ISM.

P1018: TREND OF CIRCULATING CD34+ CELLS IN MYELOFIBROSIS PATIENTS TREATED WITH RUXOLITINIB

D. Cattaneo 1,* , N. Galli 1 , C. Bucelli 2 , S. Artuso 3 , A. Iurlo 3

1 Oncology and Hemato-Oncology, University of Milan; 2 Hematology, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 3 Hematology, Foundation IRCCS Ca’ Granda Policlinico, Milano, Italy

Background: BCR-ABL1 -negative myeloproliferative neoplasms (MPNs) are variably characterized by an increase in peripheral blood (PB) and bone marrow (BM) progenitor cells. More specifically, in myelofibrosis (MF) patients (pts) it has already been reported that the number of circulating hematopoietic precursors is consistently high, with a relative circulating CD34+ cells count of 15 ×10 6 /L as the most frequently used criterion to discriminate between MF and other MPNs. Nowadays, ruxolitinib (RUX) is used for the treatment of splenomegaly and related symptoms in MF.

Aims: To evaluate CD34+ cells levels in a series of primary (PMF) and secondary (SMF) MF pts at baseline and during treatment with RUX and identify any possible correlation with response to treatment.

Methods: Between Oct 2014 and Dec 2021, CD34+ cells count from 49 consecutive pts with PMF or SMF (32 males and 17 females; median age at RUX start, 70.6 years) was retrospectively assessed at baseline and after 3, 6, 12 and 24 months (mts) from RUX start. All pts were treated with RUX according to current indications, requiring an IPSS risk of at least intermediate-1. At the time of CD34+ cells measurement, a complete blood cells count was available for all pts and their spleen measurement [expressed as the distance from the left costal margin (BCM) in cm] was taken.

Results: 24 pts were classified as having PMF (10 in the pre-fibrotic and 14 in the overt fibrotic stage) and 25 as having SMF (15 had PPV- and 10 PET-MF).

JAK2 V617F mutation was detected in 38 (77.6%) cases, CALR mutations in 8 (16.3%), and MPL in 1 (2%). The remaining 2 pts (4.1%) were defined as “triple-negative”.

The median absolute number of circulating CD34+ cells in the overall population at diagnosis was 83.5/mcL (range, 1-1528/mcL), with 31 (63.3%) pts showing >15 CD34+ cells/mcL.

At RUX start (after a median time from MF diagnosis of 33.9 mts), median absolute number of CD34+ cells was 123/mcL (range, 2-1528/mcL), with 43 (87.7%) cases showing higher than normal levels.

As expected, spleen measurements progressively decrease during the first mts of RUX (Fig. 1A): in particular, the spleen was palpable at a median of 10 cm BCM at RUX start, 5 and 3.5 cm after 3 and 6 mts of therapy, respectively. On the contrary, it increases up to 5.5 cm after 12 mts, without significant changes after 24 mts.

Interestingly, with the exception of a transient increase after 3 mts of RUX therapy, a progressive reduction in the absolute number of PB CD34+ cells after 6 and 12 mts was documented in the whole cohort, with only a slight increase after 24 mts (Fig. 1B). However, considering PMF and SMF separately, a different behavior of CD34+ cells was detected: in detail, in PMF pts circulating hematopoietic precursors gradually decreased from 3 to 24 mts of RUX therapy in parallel with the persistence of a reduction in the spleen size (Fig. 1C). On the contrary, in SMF pts both CD34+ cells and spleen size still improve up to 6 mts of RUX, while they both regrow after 12 and 24 mts of treatment (Fig. 1D).

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Summary/Conclusion: Preliminary results of our study confirm that PB CD34+ cells are increased in the majority of MF pts, both at diagnosis and during follow-up. To the best of our knowledge, we have first reported the changes in circulating CD34+ cells count during RUX, showing a parallel decrease in spleen diameter and circulating hematopoietic precursors.

These findings suggest that this tool could facilitate the assessment of RUX responses in MF pts, although this needs to be confirmed by further studies.

P1019: A PREDICTION RULE TO GUIDE JAK2 MUTATION TESTING IN PATIENTS WITH SUSPECTED POLYCYTHEMIA VERA: RESULTS FROM THE JAK2 PREDICTION COHORT (JAKPOT) STUDY

B. Chin-Yee 1,* , P. Bhai 2 , I. Cheong 2 , M. Matyashin 1 , C. Hsia 1 , E. Kawata 3 , J. Ho 1 , M. Levy 2 , A. Stuart 2 , H. Lin 2 , I. Chin-Yee 2 , M. Kadour 2 , B. Sadikovic 2 , A. Lazo-Langner 1

1 Hematology; 2 Pathology and Laboratory Medicine, Western University, London, Canada; 3 Hematology and Oncology, Kyoto Prefectural University of Medicine, Kyoto, Japan

Background: The widespread availability of molecular testing for JAK2 mutations in patients referred for elevated hemoglobin has facilitated the diagnosis of polycythemia vera (PV) but also raises concerns of test overuse. A prediction rule could be useful to improve test utilization.

Aims: In this study, we aimed to derive and validate of a simple rule using complete blood count and white blood cell differential (CBC) parameters to predict the likelihood of having a JAK2 mutation in patients referred for elevated hemoglobin.

Methods: We examined all adult patients with elevated hemoglobin (≥160 g/L for women, or ≥165 g/L for men) who underwent JAK2 mutation testing using either quantitative polymerase chain reaction (qPCR), single nucleotide polymorphism (SNP) allelotyping or Next Generation Sequencing (NGS) panel between 2015 and 2021 at London Health Sciences Centre in Ontario, Canada. We extracted data on age, sex, and complete blood count and white blood cell differential (CBC) at the time of testing. All CBCs were performed on a Sysmex XN Analyzer. Patients were randomly divided into derivation and validation cohorts including all methods of JAK2 mutation testing. JAK2 -positive and -negative groups were compared using Student’s t -tests or χ 2 tests, as appropriate. Continuous variables were dichotomized at optimal cut-off points using receiving operating characteristic curves. Potentially significant predictors were evaluated using multiple variable stepwise logistic regression analysis with JAK2 positivity as the dependent variable. A score was derived and internally validated using non-parametric bootstrapping. The model was tested in the validation cohort and sub-analyses for each method were conducted in a similar fashion. Test accuracy for the score was evaluated in all cohorts.

Results: The total study cohort included 901 patients (derivation n =616, validation n =285). Population characteristics are shown in Table 1 A . The final model included 1-point for any of the following: erythrocytes >6.45 × 10 12 /L, platelets >350 × 10 9 /L, and neutrophils >6.2 × 10 9 /L. Patients with a score of 0 were considered low-risk; all others were classified as high-risk. The percentage of JAK2 positive patients in patients with a score of 1-3 was 24% versus 0.8% in patients with a score of 0. The model had a sensitivity of 94.7% and a negative predictive value of 98.8% in the derivation cohort; both sensitivity and negative predictive value were 100% in the validation cohort. The percent of false negatives was 0.6% and 0% in the derivation and validation cohorts, respectively (Table 1 B ). The results were consistent for each testing method.

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Summary/Conclusion: We developed and validated a simple rule using CBC parameters to predict the likelihood of JAK2 mutation positivity in patients with elevated hemoglobin with demonstrated high sensitivity and negative predictive value across different JAK2 mutation testing platforms. In our cohort, the use of this rule to guide molecular testing would have resulted in over 50% fewer tests. Further studies to prospectively validate this prediction rule in different populations are planned.

P1020: SCREENING FOR PORTAL HYPERTENSION IN PATIENTS WITH PHILADELPHIA NEGATIVE MYELOPROLIFERATIVE NEOPLASMS

M. Davidson 1,* , F. Wong 1 , M. Atri 1 , H. Sibai 1 , D. Maze 1 , V. Cheung 1 , T. Nye 1 , J. Callum 2,3 , E. Atenafu 1 , V. Gupta 1

1 Princess Margaret Hospital; 2 University of Toronto, Toronto; 3 Queen’s Unviversity, Kingston, Canada

Background: Portal hypertension (PH) is a well-recognized complication of BCR-ABL negative myeloproliferative neoplasms (MPNs) and is associated with significant morbidity and mortality. However, the prevalence of PH and its associated risk factors have not been studied systematically in this patient population. Screening by esophagogastroduodenoscopy (EGD) can facilitate early diagnosis of PH and opportunity for intervention to prevent complications.

Aims: To investigate the prevalence of PH and its patient and disease-related risk factors.

Methods: From May 2013 to March 2019, MPN patients ≥ 18 years old with spleen >5 cm below the costal margin in myelofibrosis (MF) or any size in Polycythemia Vera (PV) or Essential Thrombocythemia (ET) were enrolled prospectively ( NCT01816256 ). Patients with a known history of portal or hepatic vein thrombosis, liver cirrhosis, and esophageal varices were excluded. Screening for PH was done by EGD to evaluate for esophageal varices (EV) or gastric varices (GV), and portal hypertensive gastropathy (PHG). Abdominal Doppler ultrasonography (USG) was performed to evaluate for portal vein thrombosis and spleen size. PH severity was based on the size of varices or severity of PHG. For sample-size calculation, a precision approach was used, considering scenarios where PH prevalence ranged from 5 to 10% and an upper limit for sample size of 100. At a prevalence estimate of 8%, the 90% confidence interval (CI) was 4-14%, with an acceptable lower limit for moving forward with a screening program. Prevalence of PH was calculated as a proportion and CI are exact using Clopper-Pearson method.

Results: Among 102 enrolled patients, EGD and USG were completed in 85 and 84, respectively. Primary and Post-PV/ET MF comprised the majority of the study population (93% [79/85]). Evidence of PH was found in 32 patients (38%, [90% CI 29-47]) of whom 12 (14% [90% CI 9-22]) had moderate to severe PH. EV, GV, and PHG were found in 81% (26/32), 13% (4/32), and 31% (10/32) of patients, respectively. The prevalence of PH among MF and PV/ET patients was 35% (28/79) and 67% (4/6), respectively. In MF patients, PH was moderate-to-severe in 13% (90% CI 8-20). No cases of portal vein thrombosis were identified on USG. Apart from a palpable liver observed more frequently in non-PH patients, there were no other significant differences in baseline characteristics among patients with and without PH. The frequency of JAK2 , MPL and CALR mutations among PH patients was 78% (25/32),13% (4/32), and 6% (2/32). There was a trend towards more JAK2 mutations in PH patients (78% [25/32] vs 64% [34/53], p=0.23), but no difference in DIPSS risk or frequency of high molecular-risk mutations. Overall survival was similar in both groups. PH-directed interventions were initiated in 59% (19/32); 28% (9/32) were started on beta blocker (BB) prophylaxis, 16% (5/32) were treated with BB and variceal banding, and 16% (5/32) continued on surveillance EGDs alone. Three patients with moderate PH at baseline developed variceal bleeding (2) and ascites (1). All 3 patients were on BB prophylaxis, and 1 had prior prophylactic banding. There were no adverse events related to study procedures EGD or USG.

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Summary/Conclusion: Given the study population, our results are generalizable to MF patients. Our study shows that clinically significant PH is common in MF patients, and led to additional clinical interventions in significant number of patients. These data support the inclusion of asymptomatic PH screening in the work up of MF patients.

P1021: ERYTHROCYTOSIS, THROMBOCYTOSIS AND RATE OF RECURRENT THROMBOEMBOLIC EVENT - A POPULATION BASED COHORT STUDY

A. Enblom-Larsson 1,* , B. Andreasson 2 , H. Holmberg 1 , M. Liljeholm 3 , A. Själander 1

1 Department of Public Health and Clinical Medicine, Umeå University, Umeå; 2 Department of Hematology, NU Hospital Group, Uddevalla; 3 Department of Radiation Sciences, Umeå University, Umeå, Sweden

Background: The diagnosis and management of myeloproliferative neoplasms (MPN), polycythemia vera (PV) and essential thrombocythemia (ET) are well established. Other conditions with elevated hemoglobin (Hb), hematocrit (HCT) or platelets (Plt) are not as studied and there are no international accepted guidelines of how to use phlebotomy and anticoagulants to reduce thromboembolic complications.

Aims: To assess the frequency of elevated blood values in patients diagnosed with a thromboembolic event, defined as myocardial infarction, ischemic stroke, transient ischemic attack, peripheral arterial thromboembolism, pulmonary embolism, deep venous thrombosis or abdominal thrombosis, and how many of these patients should be examined further to find an underlying MPN, according to the 2016 revised WHO classification. The second aim is to investigate if individuals with elevated Hb, HCT or Plt have a higher rate of first recurrent thromboembolic event depending on underlying condition.

Methods: All patients over the age of 18 years with a thromboembolic event as defined above during 2017 and 2018 in the county of Norrbotten (population n=250 230) were included (n=3931). From medical records data of elevated blood values according to the 2016 revised WHO criteria of PV and ET (Hb >16.5g/dL in men, Hb >16.0g/dL in women, HCT >0.49 in men, HCT >0.48 in women or Plt >450 x10 9 ) were retrieved, as well as underlying health conditions, recurrent thromboembolic events and death.

Results: In total 29.6% (1165/3931) had elevated Hb/HCT or Plt. Elevated Hb/HCT was seen in 12.7% (501/3931), 4% (159/3931) had elevated Hb/HCT at the time of initial thromboembolic event. Underlying conditions causing secondary erythrocytosis was found in 25.5% (128/501). A MPN-diagnosis was found in 1.8% (9/501), and 72.7% (364/501) had an unexplained erythrocytosis.

Elevated Plt were found in 17.3% (680/3931). Of these, 91.0% (619/680) had an obvious reactive thrombocytosis while 2.4% (16/680) were diagnosed with MPN. The remaining 6.6% (45/680) had unexplained thrombocytosis. Thrombocytosis at the time of initial thromboembolic event was found in 1.3% (51/3931).

In total 35% (405/1165) of the patients with elevated Hb/HCT or Plt should be investigated further to rule out MPN according to current guidelines. Of these, 41 patients had thrombocytosis, 360 erythrocytosis and 4 patients had both.

A recurrent thromboembolic event was found in 11% (434/3931), of these 9.7% (42/434) had elevated Hb or HCT and 18.7% (81/434) elevated Plt. The highest rate of recurrent events per 100 years was seen in the group with unexplained elevated platelets (8.11, 95% CI 2.37-13.84).

Patients with secondary erythrocytosis and unexplained thrombocytosis had the most inferior event-free survival, 2.30 years (95% CI 1.95-2.64) and 2.51 (1.98-3.04). On the other hand, patients with unexplained erythrocytosis displayed an event free survival even surpassing that of patients without erythrocytosis or thrombocytosis 3.72 (3.55-3.89) vs 3.27 (3.2-3.3) years respectively.

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Summary/Conclusion: Elevated Hb/HCT or Plt is common in patients with arterial or venous events, affecting almost one third of all patients (1165/3931), out of these one third (405/1165) should be investigated further to rule out MPN. Secondary erythrocytosis and unexplained thrombocytosis were associated with the worst event-free survival while patients with unexplained erythrocytosis were less prone to a recurrent thromboembolic event.

These results highlights the importance of finding underlying cause of elevated blood values to reduce risk of recurrent thromboembolic event.

P1022: OUTCOMES IN MYELOPROLIFERATIVE NEOPLASM PATIENTS WITH OR WITHOUT PRIOR HISTORY OF CANCER: A POPULATION-BASED STUDY FROM ONTARIO

J. T. England 1,* , A. Bankar 1 , W. C. Chan 2 , N. Liu 2 , M. C. Cheung 3 , V. Gupta 1

1 Medical Oncology/Hematology, Princess Margaret Cancer Centre; 2 ICES; 3 Hematology, Sunnybrook Health Sciences Centre, Toronto, Canada

Background: Therapy-related myeloid neoplasms (tMNs) exist as a distinct diagnostic entity in the 2016 WHO classification, and include MDS, MDS/MPN overlap, and AML that develop following iatrogenic exposure to mutagenic agents and ionizing radiation. Patients with the classical myeloproliferative neoplasms (MPNs), essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF), are excluded from the tMN category. Prior studies have observed an increased risk of other cancers in patients with MPNs, possibly from shared risk factors including older age and chronic inflammation. The impact of prior history of cancer on outcomes in MPN patients is not well understood. A single centre study of MF (Masarova et al, Blood Advances , 2017) found no survival difference between patients exposed to chemotherapy/radiation and those managed with surgery/observation/hormone therapy.

Aims: To evaluate the clinical outcomes of MPN patients with or without prior non-MPN cancer, and the impact of tMN-implicated therapy on survival following MPN diagnosis.

Methods: We conducted a population-based, retrospective cohort study using the ICES provincial health databases. Included patients were residents of Ontario aged ≥18 years with an MPN diagnosis from Jan 1, 2004 to Dec 31, 2019. Diagnoses of MPN and non-MPN (other cancers excluding AML) cancers were based on International Classification of Diseases for Oncology, third edition (ICD-O-3) codes. Therapy for prior non-MPN cancer were grouped as: therapy implicated in tMN per 2016 WHO criteria (alkylating agents, antimetabolites, topoisomerase inhibitors, antitubulin agents, and ionizing radiation), non-implicated therapies (targeted inhibitors, hormone therapy, other), and surgery/observation alone. The primary outcome was overall survival (OS) from the MPN diagnosis, with multivariable Cox regression adjusted for age and comorbidities.

In addition, to understand the patterns of non-MPN cancer type, therapy exposure, and latency period, we compared the MPN cohort with a control cohort with no MPN history matched (1:4) for age (±3 years), gender, geographic location, and neighbourhood income quintile.

Results: A total of 10 336 MPN patients (5108 ET, 3843 PV, and 1385 MF) matched to 41 344 controls were identified in the study. MPN patients more frequently had a history of non-MPN cancer compared to the control cohort (n=1421[13.5%] vs. n=5509[11.9%], P<0.001). The most common non-MPN cancers were male genitourinary (24%), breast (18%), gastrointestinal (18%), urothelial (9%), and melanoma (5%). Comparing MPN patients to matched controls, there was no difference in the types of non-MPN cancer, treatment exposures, or latency time.

After adjustment for age and comorbid conditions, the OS from PV/ET diagnosis was predicted by prior cancer managed with surgery/observation hazard ratio (HR)[95% confidence interval] 1.36 [1.22-1.52], tMN-implicated therapy HR1.83 [1.55-2.18] and non-implicated therapy HR1.88 [1.49-2.38] (Fig 1a). In MF patients the OS was predicted by tMN therapy, HR1.44 [1.04-1.98]; but not surgery/observation, HR1.17 [0.94-1.46], or non-implicated therapy, HR1.25 [0.83-1.87](Fig 1b). Lack of availability of cytogenetics and mutation profile in this population based dataset is the main limitation of this study.

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Summary/Conclusion: Compared with matched controls, patients with MPNs are more likely to have had a history of prior cancer. MPN patients with prior cancer have worse OS; and those treated with systemic therapies have shorter OS than patients managed with surgery/observation alone.

P1023: COHORT ANALYSIS OF MPN PATIENTS WITH CONCOMITANT MGUS DIAGNOSIS: A RETROSPECTIVE STUDY

C. Fatigati 1,* , F. Antonucci 1 , L. De Fazio 1 , R. Iannotta 1 , M. Lamagna 1 , A. Leone 1 , F. Muriano 1 , G. Scairati 1 , F. Pane 1 , N. Pugliese 1

1 University of Naples Federico II, University of Naples Federico II, Napoli, Italy

Background: Patients with a Ph-negative myeloproliferative neoplasm (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), may harbor or develop plasma-cell dyscrasia such as monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM), however, the clinical and molecular determinants of this co-occurrence are still uncertain.

Aims: The main aim of this study was to evaluate correlation between MGUS and MPN related features and clinical course.

Methods: We retrospectively analyzed clinical data from 448 patients with a diagnosis of MPNs according to 2016 WHO criteria, performed from 1989 to 2020 at our Institution. Here, we described the clinical and genetic features of MPN patients with concomitant MGUS. Data collection included NGS panel, cytogenetic analysis, clinical history (including thrombotic events) and pharmacological therapy during the entire MPNs follow up.

Results: Among the 448 consecutive patients analyzed, 33 (7.4%) displayed both MPN and MGUS.

Twenty-three of them were females while 10 males. MPN diagnosis were ET (12/33, 36%), MF (11/33, 33%), PV (8/33, 24%), or pre-MF (2/33, 6%) at a median age of 61 years (28-84); 25 patients showed JAK2 V617F mutation (75.8 %), 3 patients type I CALR mutation (9.1%), 1 patient harbored W515K MPL mutation (3%), and 4 were triple negative (12.1%). Half of them incurred a MPN diagnosis antecedent (19/33, 57.6%) or synchronous (11/33, 33%) with MGUS diagnosis, while only 3 patients (9.1%) had MGUS diagnosed before MPN. Serum M protein consisted in a majority of IgG (84.8%, 14 cases ʎ e 14 ƙ light chain), 2 cases of IgA and 1 case of IgM protein with a median count of 1.04 plasma-cells in bone marrow aspirate at diagnosis.

Eventually, after a median follow-up of 7 years (range 1-30 years), 5 patients (15.2%) originated a second hematological malignancy and 2 patients (6.1%) evolved in secondary myelofibrosis. In particular, 3 patients (10%) developed Non-Hodgkin lymphoma, 1 patient MM, and 1 patient developed acute myeloid leukemia (AML).

NGS, cytogenetic, clinical history and MPN specific therapies don’t seem to influence clinical course of analyzed patients.

Summary/Conclusion: In literature there are few publications that highlight the coexistence of MGUS in MPN patients. Currently little is known about the underlying molecular mechanisms and there are no guidelines clarifying whether a particular treatment or follow-up is necessary.

The incidence of MGUS in our MPN cohort is higher than general population matched for sex and age. Although, this data can be influenced by an accurate hematological follow-up, we can’t exclude a real increased rate of MGUS in MPN patient.

It is known MPN can predispose to an higher risk of developing second primary malignancies such as B cellular lymphoproliferative disorders. Interestingly, in our cohort the percentage of patients with coexisting MGUS and MPN who developed second hematological malignancies seemed slightly higher than literature data.

Further studies with case-control cohort are necessary to better understand the possible prognostic impact and mutual influence between MPN and MGUS.

P1024: IMPACT OF TP53 IN MYELOFIBROSIS UNDERGOING STEM CELL TRANSPLANTATION

N. Gagelmann 1,* , A. Badbaran 1 , V. Panagiota 2 , C. Wolschke 1 , F. Ayuk 1 , F. Thol 2 , M. Ditschkowski 3 , B. Cassinat 4 , M. Robin 4 , T. Schroeder 3 , M. Heuser 2 , H. C. Reinhardt 3 , N. Kröger 1

1 University Medical Center Hamburg-Eppendorf, Hamburg; 2 Medical School Hannover, Hannover; 3 University Hospital Essen, Essen, Germany; 4 APHP, Paris, France

Background: Most of myelofibrosis (MF) cases harbor somatic mutations in the driver genes JAK2 , CALR , or MPL . Other somatic nondriver mutations have been increasingly detected with the use of high-throughput sequencing. The role of TP53 in prognosis of MF has long been unclear, but recent first studies suggested worse survival for patients harboring this mutation.

Aims: While allogeneic stem cell transplantation still remains the only curative treatment option, outcome of patients with TP53 has not been evaluated yet.

Methods: Here, we analysed in 417 patients with PMF or post ET/PV MF incidence and impact on otcome of TP53. We detected 46 patients with detectable TP53 mutation using next-generation sequencing, and compared characteristics and outcome after allograft with 371 non- TP53 patients with primary or secondary MF. Samples were collected at time of transplantation. Primary end points were overall survival (OS) and nonrelapse mortality. Secondary objectives were relapse, relapse-free survival, and graft-versus-host disease (GVHD).

Results: Median follow-up from transplantation was 6.1 years for the TP53 group and 7.0 years for the non- TP53 group, respectively (P=0.50). Baseline characteristics were generally balanced, whereas more patients in the TP53 group had Karnofsky performance score <90 (57% versus 40%; P=0.04). Median OS was 2.1 years (0.1-5.4 years) for the TP53 group versus 15.4 years (11.5-19.3 years) for the non-TP53 group (P=0.002), and the 6-year OS was 40% (24-55%) for the TP53 group versus 65% (60-70%) for the non-TP53 group. 6-year nonrelapse mortality was 35% (21-49%) for the TP53 group versus 27% (22-32%) for the non-TP53 group (P=0.16) and 6-year incidence of relapse was 26% (13-39%) for the TP53 group versus 18% (14-21%) for the non-TP53 group (P=0.09). 6-year relapse-free survival was 35% (20-50%) versus 53% (48-58%; P=0.01). Acute GVHD occurred in 63% versus 57% (P=0.43). Of patients with relapse, 92% in the TP53 group versus 63% in the non- TP53 group died (P=0.05). Risk for death appeared to be associated with variant allele frequency (P=0.06).

Summary/Conclusion: MF patients with TP53 appeared to have higher nonrelapse mortality and relapse incidence but significantly worse relapse-free and overall survival, mainly because of worse survival after relapse.

P1025: RUXOLITINIB-ASSOCIATED CRYPTOCOCCOSIS: A SYSTEMATIC REVIEW AND META-ANALYSIS

M.-A. Gaman 1,2,*

1 Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy; 2 Hematology Department, Centre of Hematology and Bone Marrow Transplantation, Fundeni Clinical Institute, Bucharest, Romania

Background: The use of the JAK 1/2 inhibitor ruxolitinib (RUX) in myeloproliferative neoplasms (MPNs), namely primary (PMF) or secondary myelofibrosis (MF) and polycythemia vera (PV), has been associated with the development of opportunistic infections due to the immunosuppressive actions of this drug on cellular immunity. However, there is still controversy regarding the association between ruxolitinib treatment and the occurrence of cryptococcosis, a fungal infection caused by the yeast Cryptococcus neoformans or Cryptococcus gattii .

Aims: To report on cryptococcosis cases in RUX-treated MPNs and report on epidemiology, clinical manifestations, RUX associations, outcomes.

Methods: Systematic search computed in PubMed/Medline, Web of Science and SCOPUS, from the inception of these databases until 28.02.2022, to identify relevant English-written articles. Eligibility criteria: 1. Confirmed MPNs diagnosis, 2. Confirmed diagnosis of cryptococcosis, 3. MPNs patients receiving RUX therapy.

Results: The systematic search yielded 60 potentially relevant papers. After removal of duplicates (n=20) and screening of abstracts/titles (n=29), 11 manuscripts were selected for full-text review. After applying the eligibility criteria, 10 articles were entered into the qualitative and quantitative analysis. In total, 10 case reports on cryptococcosis occurring in RUX-treated MPNs patients were detected: 80.0% males, mean age 66.8±8.3 years, range 51-79 years. Most reports (70.0%) came from Asia (3 from Japan, 2 from Thailand, 1 from India and Taiwan each) and from the USA (30.0%) and were published between 2013 and 2021. In total, 70% were diagnosed with PMF, whereas cryptococcosis also occurred in PV, post-PV MF or post-ET MF (10.0%, 1 subject each). The average time from RUX initiation was 22.1±14.5 (6-48) months. HIV status was reported in 60.0% of cases and all subjects were apparently immunocompentent and had negative HIV screening. Cryptococcosis was caused by Cryptococcous neoformans only and occurred most commonly as disseminated infection (40.0%), followed by cryptococcal meningitis (30.0%) or pulmonary cryptococcosis (30.0%). Cryptococcosis co-occurred with tuberculosis (20.0%), disseminated histoplasmosis (10.0%) or Mycobacterium haemophilum cellulitis (10.0%). Most common presentations were fever (70.0%), neurological symptoms/signs (headache, consciousness/visual disturbances, 40.0%), shortness of breath (30.0%) or fatigue/lethargy (30.0%). Positive cryptococcosis diagnosis was established based on serum/cerebrospinal fluid (CSF) cryptococcal antigen positivity (80.0%) and/or positive CSF, blood or bronchoalveolar fluid cultures (70.0%). Specific treatment consisted in amphotericin B (50.0%) and/or fluconazole (70.0%). Amphotericin B was given in combination with flucytosine (20.0%) or fluconazole (20.0%) or in monotherapy (30.0%). Other combinations or drugs, i.e., isavuconazole, voriconazole, were used due to renal toxicity related to amphotericin B use. RUX was discontinued in 70.0% of cases and restarted in 20.0% of subjects. Cryptococcosis-related death occurred in 30.0% of cases.

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Summary/Conclusion: Cryptococcosis can developed during ongoing RUX therapy in MPNs subjects, even if apparently immunocompetent, potentially due to the immunosuppressive actions of this drug on cellular immunity. Screening for Cryptococcus neoformans in selected individuals might reduce the risk of disseminated infection and death. RUX discontinuation during antifungal treatment is advised due to reported progression of lesions and drug-drug interactions with antifungal agents.

P1026: COMPARATIVE GENOMIC PROFILING OF MYELOPROLIFERATIVE NEOPLASMS PRESENTING WITH AND WITHOUT SPLANCHNIC VEIN THROMBOSIS

M. Garrote 1,* , M. López-Guerra 1 , E. Arellano-Rodrigo 2 , M. Magaz 3 , A. Triguero 4 , S. Carbonell 4 , J. R. Alamo 1 , F. Turon 3 , V. Hernandez-Gea 3 , A. Baiges 3 , D. Colomer 1 , J. C. García-Pagán 3 , F. Cervantes 4 , A. Alvarez-Larrán 4

1 Pathology - Hematopathology Unit; 2 Hemotherapy and Hemostasis; 3 Hepatology; 4 Hematology, Hospital Clínic de Barcelona, Barcelona, Spain

Background: Splanchnic vein thrombosis (SVT) is the initial manifestation in 5% of myeloproliferative neoplasms (MPN). MPN patients presenting with SVT are usually younger, have less abnormal blood counts and lower JAK2V617F allele burden than other MPN patients. However, it is unknown whether they have a different genomic background.

Aims: To describe the clinical and genomic profile of MPN presenting with SVT and to compare it with a matched group of MPN without SVT.

Methods: Samples of 190 MPN patients were selected: 64 MPN presenting with SVT (SVT+) and 126 MPN without SVT (SVT-). SVT+ and SVT- were matched according to sex, age, MPN clinical subtype and driver mutation. Next Generation Sequencing was performed using Myeloid Solution by Sophia Genetics, which includes 30 genes recurrently mutated in MPN. Variants were selected using Sophia DDM software and were subsequently classified into 5 categories: 5 (definitely pathogenic), 4 (likely pathogenic), 3 (uncertain), 2 (likely benign), and 1 (benign). Only variants included into 4 and 5 categories were taken into account. Patients were classified according to the genomic classification proposed by Grinfeld and colleagues.

Results: One hundred and three patients were women (54.2%) and 87 patients were men (45.8%), without significant differences between SVT+ and SVT- groups. Median age was 44 years (range: 15-85) and 50 years (range: 15-87) in SVT+ and SVT-, respectively ( p not significant). Regarding MPN clinical subtype in SVT+ group, 39 (60.9%), 15 (23.4%) and 10 (15.6%) corresponded to Polycythemia Vera (PV), Essential Thrombocythemia (ET) and MPN unclassifiable, respectively. In the SVT- group, 75 (59.5%) and 51 (40.5%) corresponded to PV and ET, respectively.

Genomic classification in the SVT+ and SVT- groups is shown in Table 1. The proportion of patients with high molecular risk ( TP53 disruption/aneuploidy, chromatin/spliceosome mutation, or homozygous JAK2 mutation) was 14.1% and 26.2% for SVT+ and SVT-, respectively ( p =0.06). Median number of pathogenic mutations was 1 (range: 1-4) in both SVT+ and SVT- groups. Pathogenic mutations in TET2 , DNMT3A and ASXL1 were present in 10%, 3.7% and 3.7%, respectively, without significant differences between both groups.

With a mean follow-up of 10.1 years for cases and 9.7 years for controls, 27 patients died (13 SVT+ and 14 SVT-). Median survival was 24 years and not reached for SVT+ and SVT-, respectively ( p =0.1). On multivariate analysis, SVT+ group showed a higher risk of death (HR: 2.4, 95%CI: 1.1-5.5, p =0.03) after correction by age, sex and genomic classification. Transformation to myelofibrosis and acute leukemia was low and similar in both groups: 4.7% vs. 5.6% for myelofibrosis in SVT+ and SVT-, respectively ( p not significant); and 0% vs. 2.4% for acute leukemia ( p not significant). On multivariate analysis, patients with TP53 disruption/aneuploidy, chromatin/spliceosome mutation, or homozygous JAK2 showed a higher risk of disease progression to myelofibrosis or acute leukemia (HR: 14.5, 95%CI: 3.1-68.7, p=0.001) regardless of the presence of SVT at diagnosis, age and sex.

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Summary/Conclusion: MPN presenting with SVT and MPN-matched controls showed a low molecular complexity. Genomic classification was helpful in identifying a small proportion of MPN patients presenting with SVT who are at high risk of disease progression. SVT at MPN diagnosis is associated with a higher risk of death independent of sex, age and genomic classification.

P1027: RESPONSES TO AVAPRITINIB IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS: HISTOPATHOLOGIC ANALYSES FROM EXPLORER AND PATHFINDER CLINICAL STUDIES

T. George 1,* , K. H. Karner 1 , K. A. Moser 1 , A. Rets 1 , A. Reiter 2 , D. H. Radia 3 , M. Deininger 4 , H.-M. Lin 5 , S. Dimitrijević 6 , D. J. DeAngelo 7

1 Department of Pathology, ARUP Laboratories, University of Utah, School of Medicine, Salt Lake City, United States of America; 2 Department of Hematology and Oncology, University Hospital Mannheim, Heidelberg University, Mannheim, Germany; 3 Guy’s & St Thomas’ NHS Foundation Trust, London, United Kingdom; 4 Versiti Blood Research Institute, Milwaukee; 5 Blueprint Medicines Corporation, Cambridge, United States of America; 6 Blueprint Medicines Corporation, Zug, Switzerland; 7 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America

Background: Systemic mastocytosis (SM) is a mast cell (MC) neoplasm driven by the KIT D816V mutation in ~95% of cases. Diagnosis includes evaluation of MC aggregates in extracutaneous organs, atypical MC morphology, and MC immunophenotype. Avapritinib, a potent and selective KIT D816V inhibitor, has been approved in the US for patients with advanced SM (AdvSM) based on the phase 1 EXPLORER and phase 2 PATHFINDER studies.

Aims: Here, we describe a comprehensive evaluation of the effect of avapritinib on bone marrow (BM) pathology in patients treated in these studies.

Methods: Patients aged ≥18 years with diagnoses of AdvSM per local investigator were treated with once-daily avapritinib <200 mg (n=17), 200 mg (n=126), and ≥300 mg (n=50). Bone marrow biopsies (BMBs), aspirates (BMAs), and peripheral blood (PB) smears were obtained at screening, and after 8 and 24 weeks of treatment with avapritinib. Evaluations were performed using standard Wright-Giemsa and H&E staining, and IHC was performed on formalin-fixed EDTA-decalcified BMBs using standard techniques for CD25 and CD30. Changes in fibrosis were assessed by the European Consensus on grading of BM fibrosis (MF score).

Results: As of April 2021, 193 patients enrolled in EXPLORER (n=86) and PATHFINDER (n=107) were included in analyses with centrally confirmed diagnoses of aggressive SM (n=29), SM with an associated hematologic neoplasm (n=119), mast cell leukemia (n=28), indolent SM (n=14), smoldering SM (n = 2) or CMML (n=1). Median (range) age was 67 years (31–88), 56% were male and 88% had a KIT D816V mutation. Following treatment with avapritinib, a decrease in the proportion of patients with multifocal dense aggregates of MCs in BMBs was observed from 93% at screening to 50% by Week 8 (Table). Avapritinib treatment reduced the proportion of aberrant CD25+ and CD30+ MCs in BMBs by Week 8 and Week 24 (Table). In BMAs, avapritinib reduced the mean MC burden from 11% of total nucleated cells at screening to 2% at Week 8 and by Week 24, with reduced proportions of immature and spindle-shaped MCs (Table). Of 9 patients with circulating MCs at screening and post-screening sample measurements (6 with SM with associated hematologic neoplasm diagnoses and 3 with MCL), 8 had no detectable MCs in PB by Week 8. The proportion of patients with fibrosis in BMBs decreased from 96% (178/186) at screening to 90% (142/158) at Week 8 to 84% (103/123) through week 24, and MF scores were reduced during avapritinib treatment in those patients presenting with increased fibrosis at screening (Table).

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Summary/Conclusion: Patients treated with avapritinib were observed to have rapid (Week 8) and marked (Week 24) reductions in neoplastic BM MCs with a return to a normal morphologic appearance and immunophenotype. This was accompanied by an improvement of fibrosis and a decrease in circulating MCs. These improvements in disease histopathology provide further support that treatment with this highly selective and potent KIT D816V inhibitor is disease modifying in AdvSM.

P1028: PTG-300 (RUSFERTIDE) TREATMENT INTERRUPTION REVERSES HEMATOLOGICAL GAINS AND RESTORES THERAPEUTIC BENEFIT ON REINITIATION IN SUBJECTS WITH POLYCYTHEMIA VERA

A. Kuykendall 1,* , M. Kremyanskaya 2 , Y. Ginsburg 2 , N. Pemmaraju 3 , E. Ritchie 4 , J. Gotlib 5 , F. Valone 6 , S. Khanna 6 , S. Gupta 6 , R. Hoffman 2 , S. Verstovsek 3

1 Hematology and Med Oncology, USF, Moffitt Cancer Center, Tampa; 2 Hematology, Mt. sinai, New york; 3 Dept of Leukemia, M.D. Anderson, Houston, Tx; 4 Hematology, Weill Cornell medical College, New york; 5 Hematology, school of Medicine, Stanford, Palo Alto, Ca; 6 Clinical Research and Development, Protagonist Therapeutics, Newark, United States of America

Background: Polycythemia vera (PV) patients are treated with periodic therapeutic phlebotomy (TP) and if needed, cytoreductive therapy to maintain hematocrit (HCT) <45% to reduce the incidence of thrombosis. Constitutive erythropoiesis and repeated TP result in iron deficiency which further stimulates iron absorption and availability for robust erythropoiesis. Iron deficiency contributes to fatigue, which is the most severe and prevalent symptom for patients with PV. We have previously shown that adding Rusfertide, a hepcidin mimetic, to existing PV treatment, significantly, and effectively reverses iron deficiency and essentially eliminates TP requirement for >1 year in PV patients [Hoffman ASH 2021]. The ongoing PTG-300-04 ( NCT04057040 ) Phase 2 study of Rusfertide in TP-dependent PV patients experienced an FDA-mandated brief hold which resulted in dosing interruption of Rusfertide for all patients.

Aims: To examine the impact of temporary rusfertide withdrawal on iron homeostasis and TP requirements after temporary suspension in the ongoing PTG-300-04 study.

Methods: Eligibility criteria for PTG-300-04 study include PV diagnosis [by 2016 WHO criteria] and ≥3 phlebotomies with/without concurrent cytoreductive therapy in the 24 weeks before enrollment. Rusfertide 10-120 mg weekly SQ injection was added to existing cytoreductive treatment and adjusted to maintain HCT <45%. During the brief clinical hold patients were maintained on their cytoreductive regimens and had TP for HCT >45%. Most patients returned to Rusfertide add-on treatment 2-3 months after the dosing interruption and reinititation.

Results: Pre-enrollment, PV patients were treated with TP alone (30) or with TP+cytoreductive therapy (33). TP requirements in the 24 weeks before enrollment ranged from 3 to 9. Rusfertide treatment enabled consistent HCT control <45%, essentially eliminated TPs in all sub-groups, decreasing RBC count and increased MCV and MCH values. Pre-enrollment, mean iron-related parameters were consistent with systemic iron deficiency. Rusfertide treatment resulted in progressive normalization of serum ferritin, suggesting a redistribution of systemic iron. During Rusfertide dosing interruption, all patients had significant (p<0.01) increase in TPs, HCT, and RBC count; and a simultaneous decrease in MCV and serum ferritin. Patients also reported increase in PV-related symptoms (i.e., increased fatigue and worsening concentration). Reinitiating Rusfertide resulted in rapid normalization of hematologic parameters, elimination of TP, and improvement in symptoms demonstrating potential effectiveness of this approach. Most adverse events were grade 1-2. The most frequent AE was transient injection site reaction reported in 59% of patients.

Summary/Conclusion: The current results support Rusfertide as an effective treatment option in PV, reversing iron deficiency and essentially eliminating TP requirements in PV patients with/without cytoreductive agents. Reversal of hematological parameters during dosing interruption further confirms the effect of Rusfertide on both iron homeostasis and erythrocytosis. Taken together, Rusfertide is a very promising novel biologic agent in both low and high-risk PV patients. A global Phase 3 trial of rusfertide in PV, VERIFY, has been initiated.

P1029: MATCHING-ADJUSTED INDIRECT COMPARISON (MAIC) OF PELABRESIB (CPI-0610) IN COMBINATION WITH RUXOLITINIB VS RUXOLITINIB OR FEDRATINIB MONOTHERAPY IN PATIENTS WITH INTERMEDIATE OR HIGH-RISK MYELOFIBROSIS

V. Gupta 1,* , J. Mascarenhas 2 , M. Kremyanskaya 2 , R. K. Rampal 3 , M. Talpaz 4 , J.-J. Kiladjian 5 , A. Vannucchi 6 , S. Verstovsek 7 , G. Colak 8 , D. Dey 9 , C. Harrison 10

1 Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada; 2 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai; 3 Memorial Sloan Kettering Cancer Center, New York, NY; 4 University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, United States of America; 5 Hôpital Saint-Louis, Université de Paris, Paris, France; 6 Azienda Ospedaliero-Universitaria Careggi, University of Florence, Florence, Italy; 7 University of Texas MD Anderson Cancer Center, Houston, TX; 8 Constellation Pharmaceuticals a MorphoSys Company, Boston, MA, United States of America; 9 MorphoSys AG, Planegg, Germany; 10 Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom

Background: Pelabresib (CPI-0610), an investigational product, in combination with ruxolitinib (ruxo) has shown encouraging responses in terms of ≥35% reduction in spleen volume from baseline (SVR35) and ≥50% reduction in total symptoms score (TSS) from baseline (TSS50) in Janus kinase inhibitor (JAKi) treatment-naïve patients with intermediate or high-risk myelofibrosis (MF) in Arm 3 of the open-label Phase 2 MANIFEST study ( NCT02158858 ). A cross-trial comparison with ruxo or fedratinib monotherapy has limitations in the presence of the potential imbalances in baseline characteristics.

Aims: Here we report findings from matching-adjusted indirect comparisons (MAICs), conducted to correct for potential imbalances in baseline characteristics, comparing the primary endpoint SVR35 and secondary endpoint TSS50 at Week (Wk) 24 for the combination of pelabresib and ruxo (MANIFEST Arm 3) with Phase 3 ruxo monotherapy (COMFORT-I and II, SIMPLIFY-1) and fedratinib monotherapy (JAKARTA) data.

Methods: Individual patient level data were available for MANIFEST Arm 3, while only published summary data were available for COMFORT-I and II, SIMPLIFY-1 and JAKARTA. An unanchored MAIC was conducted wherein patients in MANIFEST Arm 3 were reweighted to adjust for imbalances in the following key prognostic baseline characteristics: gender, MF subtype, International Prognostic Scoring System risk status, previous hydroxyurea use, platelet count, hemoglobin levels, spleen volume and JAK2V617F status. Weighted mean outcomes of the treatment effects were estimated together with their 95% confidence interval (CI) using robust sandwich estimators for variance. The weights were also used to calculate the Effective Sample Size (ESS). Treatment effect outcomes for SVR35 and TSS50 at Wk 24 are presented in terms of Response Rates (RR) and Response Rate Ratio (RRR, defined as RR in MANIFEST Arm 3/RR in comparator arm). An RRR >1 favors the pelabresib + ruxo arm in MANIFEST Arm 3 over the JAKi monotherapy comparator arm. A limitation of a MAIC is that bias due to potential unmeasured differences in patient baseline characteristics between trials cannot be excluded.

Results: The MAIC analysis resulted in complete summary-level balance in the weighted distributions of the prognostic factors in MANIFEST Arm 3 versus comparators. The figure presents the weighted (or MAIC-adjusted) and unweighted (or unadjusted) treatment effect comparisons along with their 95% CI for SVR35 and corresponding ESS at Wk 24. Statistically significant MAIC-adjusted (weighted) RRRs of 1.57 (95% CI 1.10, 2.24; p-value: 0.012), 1.82 (95% CI: 1.17, 2.83; p-value: 0.008), 2.13 (95% CI: 1.51, 3.02; p-value: <0.001), 1.76 (95% CI: 1.16, 2.66; p-value: 0.008) and 1.55 (95% CI: 1.06, 2.27; p-value: 0.023) were observed for MANIFEST Arm 3 (pelabresib + ruxo) versus COMFORT-I and -II and SIMPLIFY-1 (ruxo only) and JAKARTA (fedratinib 400 mg and 500 mg), respectively. These weighted analyses are consistent with statistically significant results of unadjusted analyses. RRRs >1 were also observed for TSS50 at Wk 24 for all the comparisons.

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Summary/Conclusion: The MAIC analysis, adjusting for eight key baseline prognostic factors, suggests an improvement in SVR35 and TSS50 at Wk 24 for JAKi treatment-naïve patients with MF treated with a combination of pelabresib and ruxo over ruxo or fedratinib monotherapy. MANIFEST-2 ( NCT04603495 ), a Phase 3 double-blind, randomized study assessing the efficacy and safety of pelabresib or placebo in combination with ruxo in patients with MF, is currently open for enrollment.

P1030: MANIFEST-2, A GLOBAL, PHASE 3, RANDOMIZED, DOUBLE-BLIND, ACTIVE-CONTROL STUDY OF PELABRESIB (CPI-0610) AND RUXOLITINIB VS PLACEBO AND RUXOLITINIB IN JAK INHIBITOR-NAÏVE MYELOFIBROSIS PATIENTS

C. Harrison 1,* , R. K. Rampal 2 , V. Gupta 3 , S. Verstovsek 4 , M. Talpaz 5 , J.-J. Kiladjian 6 , R. Mesa 7 , A. Kuykendall 8 , A. Vannucchi 9 , F. Palandri 10 , S. Grosicki 11 , T. Devos 12 , E. Jourdan 13 , M. J. Wondergem 14 , H. K. Al-Ali 15 , V. Buxhofer-Ausch 16 , A. Alvarez-Larrán 17 , S. Akhani 18 , R. Muñoz-Carerras 19 , Y. Sheykin 19 , G. Colak 19 , M. Harris 19 , J. Mascarenhas 20

1 Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; 2 Memorial Sloan-Kettering Cancer Center, New York, NY, United States of America; 3 Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada; 4 Leukemia Department, University of Texas MD Anderson Cancer Center, Houston, TX; 5 University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, United States of America; 6 Hôpital Saint-Louis, Université de Paris, Paris, France; 7 Mays Cancer Center at UT Health San Antonio MD Anderson Cencer Center, San Antonio, TX; 8 Mofitt Cancer Center, Tampa, FL, United States of America; 9 Azienda Ospedaliero-Universitaria Careggi, University of Florence, Florence; 10 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy; 11 Medical University of Silesia in Katowice, Katowice, Poland; 12 University Hospitals Leuven and Laboratory of Molecular Immunology (Rega Institute), KU Leuven, Leuven, Belgium; 13 C.H.U, Nîmes, France; 14 Amsterdam University Medical Centers, Amsterdam, Netherlands; 15 University Hospital Halle, Halle, Germany; 16 KRANKENHAUS DER ELISABETHINEN Linz GmbH, Linz, Austria; 17 Hospital Clínic Barcelona, Barcelona, Spain; 18 MorphoSys AG, Planegg, Germany; 19 Constellation Pharmaceuticals a MorphoSys Company, Boston, MA; 20 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America

Background: Myelofibrosis (MF) is characterized by bone marrow fibrosis, anemia, splenomegaly and constitutional symptoms. Progressive bone marrow fibrosis results from aberrant megakaryopoeisis and expression of proinflammatory cytokines, both of which are heavily influenced by bromodomain and extraterminal domain (BET)-mediated gene regulation and lead to myeloproliferation and cytopenias. Pelabresib (CPI-0610) is an oral small-molecule investigational inhibitor of BET protein bromodomains currently being developed for the treatment of patients with MF. It is designed to downregulate BET target genes and modify nuclear factor kappa B (NF-κB) signaling. MANIFEST-2 was initiated based on data from Arm 3 of the ongoing Phase 2 MANIFEST study ( NCT02158858 ), which is evaluating the combination of pelabresib and ruxolitinib in Janus kinase inhibitor (JAKi) treatment-naïve patients with MF. Primary endpoint analyses showed splenic and symptom responses in 68% and 56% of 84 enrolled patients, respectively.

Aims: MANIFEST-2 ( NCT04603495 ) is a global, Phase 3, randomized, double-blind, active-control study of pelabresib and ruxolitinib versus placebo and ruxolitinib in JAKi treatment-naïve patients with primary MF, post-polycythemia vera MF or post-essential thrombocythemia MF. The aim of this study is to evaluate the efficacy and safety of pelabresib in combination with ruxolitinib. Here we report updates from a recent protocol amendment.

Methods: The MANIFEST-2 study schema is shown in Figure 1. Key eligibility criteria include a Dynamic International Prognostic Scoring System (DIPSS) score of Intermediate-1 or higher, platelet count ≥100 × 10 9 /L, spleen volume ≥450 cc by computerized tomography or magnetic resonance imaging, ≥2 symptoms with an average score ≥3 or a Total Symptom Score (TSS) of ≥10 using the Myelofibrosis Symptom Assessment Form v4.0, peripheral blast count <5% and Eastern Cooperative Oncology Group performance status ≤2. Patient randomization will be stratified by DIPSS risk category (Intermediate-1 vs Intermediate-2 vs High), platelet count (>200 × 10 9 /L vs 100–200 × 10 9 /L) and spleen volume (≥1800 cm 3 vs <1800 cm 3 ). Double-blind treatment (pelabresib or matching placebo) will be administered once daily for 14 consecutive days, followed by a 7-day break, which is considered one cycle of treatment. Ruxolitinib will be administered twice daily for all 21 days of the cycle. The primary endpoint is SVR35 response (≥35% reduction in spleen volume from baseline) at Week 24, and the key secondary endpoint is TSS50 response (≥50% reduction in TSS from baseline) at Week 24. Other secondary endpoints include safety, pharmacokinetics, changes in bone marrow fibrosis, duration of SVR35 response, duration of TSS50 response, progression-free survival, overall survival, conversion from transfusion dependence to independence and rate of red blood cell transfusion for the first 24 weeks.

Results: Study recruitment is ongoing; 400 patients (200 per arm) from North America, Europe, Asia and Australia will be enrolled. The study opened for enrollment in November 2020.

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Summary/Conclusion: MANIFEST-2 was initiated based on data from the ongoing Phase 2 MANIFEST study with the aim of assessing the efficacy and safety of pelabresib and ruxolitinib in JAKi treatment-naïve patients with MF. MANIFEST-2 is currently open for enrollment.

P1031: LOW-DOSE DOACS IN VERY HIGH-RISK MYELOPROLIFERATIVE NEOPLASMS: LESS BLEEDING BUT MORE ARTERIAL THROMBOTIC EVENTS

L. Herbreteau 1,* , L. Papageorgiou 2,3 , L. Le Clech 4 , G. Garcia 5 , C. James 5 , B. Pan-Petesch 1,6 , F. Couturaud 7 , E. Lippert 8,9 , G. Gerotziafas 2,3 , J.-C. Ianotto 1,6,9

1 Service d’Hématologie Clinique, Centre Hospitalier Universitaire de Brest, Brest; 2 Service d’Hématologie Biologique, Hôpital Tenon, Hôpitaux Universitaires de l’Est Parisien, Assistance Publique Hôpitaux de Paris; 3 Research Group “Cancer, Hémostase et Angiogenèse” INSERM UMR_S 938, Centre de Recherche Saint-Antoine Faculté de Médecine, Institut Universitaire de Cancérologie, Sorbonne Universités, Paris; 4 Service de Médecine Interne, Maladies Infectieuses et du Sang, Centre Hospitalier de Cornouaille, Quimper; 5 Laboratoire d’Hématologie, Centre Hospitalier Universitaire de Bordeaux, Bordeaux; 6 GETBO, Groupe d’Etude des Thromboses en Bretagne Occidentale, EA3878; 7 Département de Médecine Interne et de Pneumologie; 8 Laboratoire d’Hématologie, Centre Hospitalier Universitaire de Brest, Brest; 9 France Intergroupe des néoplasies Myéloprolifératives, Paris, France

Background: Direct oral anticoagulants (DOACs) have recently proven their efficacy and safety, as primary and secondary prevention agents, for thrombosis in cancer patients. Thrombotic and haemorrhagic events are major causes of morbidity and mortality in Philadelphia-negative myeloproliferative neoplasm (MPN) patients. However, the best agent to prevent thrombotic events without increasing the bleeding incidence among these patients remains uncertain.

Aims: In this study, we aimed to determine if DOACs might be an interesting choice to reduce the thrombotic risk in high-risk MPN patients.

Methods: We analysed a large multicentric cohort of MPN patients treated with DOACs for atrial fibrillation (AF) or thrombotic events. We evaluated the overall thrombosis and haemorrhage rates related to DOACs, and also by the DOAC type (apixaban or rivaroxaban) as well as the daily dosage prescribed.

Results: We included 135 MPN patients with a median follow-up of 23.8 months since DOAC initiation. Twenty patients (14.8%) developed 30 thrombotic events (28 arterial thromboses in 19 patients) for a global incidence of 6.5% patient-years. No difference was highlighted between apixaban and rivaroxaban in terms of thrombosis risk, but the incidence of arterial thrombosis was significantly higher on low-dose DOACs (11.9 vs. 4.5% patient-years, p=0.04). Bleeding events were more frequent in the full-dose group (15.2 vs. 41.2%, p=0.006). However, major and clinically relevant non major (CRNM) bleeding events occurred in 18 patients (13.3%), with no difference between the DOAC types or the DOAC doses. In multivariate analysis, age was the only identified thrombotic risk factor, whereas risk factors for major or CRNM bleeding were a full-dose treatment regimen and a combination of DOAC and low-dose aspirin.

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Summary/Conclusion: DOACs seem effective in preventing venous thrombosis in MPN patients with AF or thrombotic events. For these high-risk patients, low-dose DOACs exposed patients to more arterial thrombosis but fewer bleeding events. Prospective studies are now needed to evaluate and compare DOACs to the currently recommended antithrombotic drugs for high-risk MPN patients.

P1032: REAL-LIFE VALIDATION OF MIPSS70: A RETROSPECTIVE MULTICENTER AND NGS ANALYSIS FROM THE GEMFIN DATABASE

A. Hernández-Sánchez 1,2,* , Á. Villaverde-Ramiro 2 , A. Álvarez-Larrán 3 , E. Arellano-Rodrigo 3 , M. Pérez Encinas 4 , F. Ferrer Marín 5,6,7,8 , E. Such 9 , G. Carreño Gómez-Tarragona 10 , N. de las Heras Rodríguez 11 , M. A. Durán Pastor 12 , M. I. Mata Vázquez 13 , L. Fox 14 , J. M. Raya Sánchez 15 , E. Magro Mazo 16 , M. T. Gómez Casares 17 , M. J. Ramírez Sánchez 18 , B. Xicoy Cirici 19 , Á. Ramírez Páyer 20 , M. J. Fernández Llavador 21 , A. Á. Martín López 1,2 , A. Mosquera Orgueira 4 , J. Martínez Elicegui 2 , M. Garrote 3 , B. Bellosillo 22 , T. González 1,2,23 , J. Hernández-Rivas 1,2,23 , J. C. Hernández Boluda 24

1 Hospital Universitario de Salamanca; 2 Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca; 3 Hospital Clínic de Barcelona, Barcelona; 4 Complexo Hospitalario Universitario de Santiago, Santiago de Compostela; 5 Hospital Morales Meseguer; 6 Universidad Católica San Antonio de Murcia (UCAM); 7 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER); 8 Instituto Murciano de Investigación Biosanitaria, Murcia; 9 Hospital Universitari I Politècnic La Fe, Valencia; 10 Hospital Universitario 12 de Octubre, Madrid; 11 Hospital Universitario de León, León; 12 Hospital Universitari Son Espases, Palma de Mallorca; 13 Complejo Hospital Costa Del Sol, Marbella; 14 Hospital Universitari Vall D’Hebron, Barcelona; 15 Hospital Universitario de Canarias, Santa Cruz de Tenerife; 16 Hospital Universitario Príncipe de Asturias, Alcalá de Henares; 17 Hospital Universitario de Gran Canaria Dr. Negrín, Las Palmas de Gran Canaria; 18 Hospital Universitario de Jerez, Jerez de la Frontera; 19 Institut Català d’Oncologia-Hospital Universitari Germans Trias I Pujol de Badalona, Badalona; 20 Hospital Universitario Central de Asturias, Oviedo; 21 Hospital Universitario Dr. Peset, Valencia; 22 Hospital del Mar, Barcelona; 23 Centro de Investigación del Cáncer (CIC), Salamanca; 24 Hospital Clínico Universitario de Valencia, Valencia, Spain

Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the presence of driver mutations ( JAK2 , CALR or MPL ) in most of the patients. The development of next generation sequencing (NGS) has favored the incorporation of mutational information in novel prognostic scores. However, most of these risk stratification models also include cytogenetic information, which is only available for a fraction of patients with this disease, thus reducing their real-life applicability. Mutation-enhanced international prognostic scoring system for transplant-age patients (MIPSS70) is based on clinical and mutational risk factors and it is useful in the absence of cytogenetic information, but it has not been validated yet in a real-life clinical setting.

Aims: To validate MIPSS70 risk stratification model for transplant-age PMF patients from a multicenter database.

Methods: From the GEMFIN multicenter database, 668 myelofibrosis patients with enough information to calculate MIPSS70 were selected. As the score is intended for patients aged ≤70 years with PMF, the final analysis was carried out in 218 patients meeting these criteria. Patients who received allogeneic stem cell transplantation were censored on the date of transplantation.

Results: Median age was 61 years (range 18-70), 69% of patients were male and median follow-up time was 4.65 years. Regarding the presence of MIPSS70 variables at disease diagnosis, 45% of patients had constitutional symptoms, 43% had hemoglobin <100g/L, 15% leukocytes >25 (x10 9 /L) and 33% platelets <100 (x10 9 /L). Circulating blasts were ≥2% in 30% and most patients presented BM fibrosis grade ≥2 (74%). CALR type 1 mutation was detected only in 12%, while any high-molecular risk (HMR) mutation was present in 31% (8% for ≥2 HMR mutations). In brief, in comparison to MIPSS70 training cohort, the patients in the present series were older and presented a higher proportion of adverse risk factors (Figure 1 A ).

More than half of the patients were classified as high risk according to MIPSS70 (52%), while 35% were intermediate and 13% low risk. The 5-year overall survival (OS) was 44% (median 4.6 years), 77% (median 13 years) and 95% (median not reached) for high, intermediate and low risk patients respectively (p<0.001, Figure 1 B ). The 5-year leukemia-free survival (LFS) was 42%, 77% and 95% for high, intermediate and low risk groups (p<0.001).

Univariate analysis of each MIPSS70 variable on OS was carried out, with statistically significant differences being confirmed for all variables except fibrosis grade and HMR mutations. ASXL1 was mutated in 27%, but it did not impact OS in our patients (HR 0.96, p=0.89). SRSF2 was the only MIPSS70 mutated gene (8%) associated with inferior OS (HR 2.5, p=0.04), while EZH2 and IDH1/2 mutations were only present in 2.5% of patients each. However, the analysis of the complete NGS panel allowed the identification of other genes associated with shorter OS in our series: CBL (HR 3.7, p=0.014), SETBP1 (HR 4.9, p=0.034) and KRAS (HR 4.6, p=0.041), with a tendency for U2AF1 (HR 2.3, p=0.061) and NRAS (HR 3.6, p=0.083).

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Summary/Conclusion: This study constitutes the first real-life validation of MIPSS70 in a multicenter analysis. The score was able to stratify our cohort in three categories with significant differences in OS and LFS. Of note, mutation on ASXL1 gene was not associated with shorter OS in our patients. By contrast, several other genes with potential prognostic significance were identified. Further cooperative studies are needed in order to unravel PMF mutational landscape and its prognostic implications.

P1033: A PHASE 2 STUDY OF THE LSD1 INHIBITOR IMG-7289 (BOMEDEMSTAT) FOR THE TREATMENT OF ESSENTIAL THROMBOCYTHEMIA (ET)

F. Palandri 1,* , D. M. Ross 2 , T. Cochrane 3 , C. Tate 3 , S. W. Lane 4 , S. R. Larsen 5 , A. T. Gerds 6 , A. B. Halpern 7 , J. Shortt 8 , J. M. Rossetti 9 , K. M. Pettit 10 , J. Liang 11 , A. Mead 12 , M. Marchetti 13 , A. Vannucchi 14 , A. Wilson 15 , J. R. Göthert 16 , M. Hanna 17 , A. Jones 18 , J. Peppe 18 , G. Natsoulis 18 , T. McClure 18 , W.-J. Hong 18 , W. S. Stevenson 19 , C. N. Harrison 20 , M. Talpaz 10 , N. Vianelli 21 , H. Y. Rienhoff Jr. 18

1 Institute of Hematology “L. & A. Seràgnoli”, Sant’Orsola-Malpighi University Hospital, Bologna, Italy; 2 Department of Haematology, Royal Adelaide Hospital and SA Pathology, Adelaide; 3 Department of Haematology, Gold Coast University Hospital, Southport; 4 QIMR Berghofer Medical Research Institute, Brisbane; 5 Institute of Haematology, Royal Prince Alfred Hospital, Sydney, Australia; 6 Cleveland Clinic Taussig Cancer Institute, Cleveland; 7 Department of Medicine, Division of Hematology, University of Washington, Seattle, United States of America; 8 Monash Haematology, Monash Health, Clayton, Australia; 9 UPMC Hillman Cancer Center, Pittsburgh; 10 Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan, Ann Arbor, United States of America; 11 Middlemore Clinical Trials, Auckland, New Zealand; 12 MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 13 Azienda Ospedaliera Nazionale SS. Antonio e Biagio e Cesare Arrigo, Alessandria; 14 University of Florence, AOU Careggi, CRIMM, Center for Research and Innovation of Myeloproliferative Neoplasms, Florence, Italy; 15 University College Hospital, NHS Foundation Trust, London, United Kingdom; 16 Department of Hematology, West German Cancer Center (WTZ), University Hospital Essen, Essen, Germany; 17 North Shore Hospital. Waitemata District Health Board, Auckland, New Zealand; 18 Imago BioSciences, Inc., South San Francisco, United States of America; 19 Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia; 20 Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; 21 Institute of Hematology, “L. & A. Seragnoli”, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy

Background: Lysine-specific demethylase-1 (LSD1) is critical for the self-renewal malignant stem cells and maturation of megakaryocytes, the cell central to ET pathogenesis. Bomedemstat is an oral LSD1 inhibitor active in mouse models of MPNs including improved survival (Kleppe et al. 2015; Jutzi et al. 2018). IMG-7289-CTP-201 is an ongoing, global, open-label, Phase 2b study of bomedemstat taken QD for 24+ weeks in patients with ET who are resistant/intolerant to at least one standard treatment ( NCT04254978 ).

Aims: Key objectives are safety and response, defined as platelet (plt) count ≤400x10 9 /L without thromboses. Exploratory endpoints include durability of response (defined as plt ≤400x10 9 /L for ≥12 wks) symptom improvement (TSS) and reduction of WBCs and mutation burden.

Methods: Key eligibility criteria: cytoreduction required, platelet count >450x10 9 /L, haemoglobin (Hb) ≥10 g/dL. The starting dose is 0.6 mg/kg/d and titrated, if needed, to a platelet count of 200-400x10 9 /L.

Results: At data cut-off (3Feb’22), 44 patients had enrolled. Median age was 68 (42-92) yrs; of the patients most recently treated with hydroxyurea, 88% met ELN criteria for resistance/ intolerance. The Day 1 mean plt, WBC and Hb values were 825x10 9 /L (457-2220), 8.9x10 9 /L (3.9-30.6), and 13.1 g/dL (9.4-16.5), respectively. Median TSS at baseline was 15 (0-74). Sequencing 261 genes (N=42) identified mutations in JAK2 (45%), CALR (43%), and MPL (5%). Other mutations ( e.g ., EZH2, ASXL1, SF3B1, TP53 ) were present in 29%.

Median time on treatment is 29 weeks (0-68). For patients treated ≥12 weeks, 91% (31/34) achieved a plt count of ≤400x10 9 /L in a median time of 8.1 weeks. 83% of patients (20/24) treated for >24 weeks achieved a durable response as defined above. Of the 9/30 (30%) patients Day 1 WBC count ≥10x10 9 /L, 89% (8/9) had a WBC count <10x10 9 /L; all maintained a stable hemoglobin (see figures). In patients with baseline TSS ≥10 (29/43), at Week 12 69% (11/16) showed reductions; 38% (6/16) had improvements >10-points. The median baseline TSS score for fatigue was 5 (N=41). At Week 24, the median score for evaluable patients fell to 3 and dropped to a median of 1 at Week 48. At Week 24 (N=14), JAK2 and CALR mutations were equally sensitive: 87% had a decrease in allele frequencies (mean: -29%; [S.D. 5%]; range -3 to -91). No new mutations were found.

The most common AEs regardless of causality were dysgeusia (52%), fatigue (34%), constipation (32%), arthralgia (27%), thrombocytopenia (25%), and contusion (21%). Eight patients reported a total of 12 serious AEs (SAE), with two (thrombocytopenia and mouth haemorrhage reported in 1 patient) deemed related per the PI. Seven patients discontinued treatment, 6 due to AEs (fatigue, invasive ductal breast carcinoma, headache, respiratory failure, dysgeusia x 2) and one withdrew consent on Day 1. Similar to an ongoing MF study of bomedemstat ( NCT03136185 ), there have been no safety signals or deaths related to drug. At the censor date, 84% (37/44) of patients remain on study.

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Summary/Conclusion: To date, in this patient population, bomedemstat is generally well-tolerated, reduces platelets, improves symptoms, reduces the mutation burden, and moderates WBC counts without affecting haemoglobin. For those patients treated for at least 24 weeks, 79% achieved a durable reduction in platelet count to ≤400x10 9 /L without thromboembolic events.

A Phase 3 study of bomedemstat for the treatment of ET is being planned.

P1034: HEALTHCARE UTILIZATION OF PATIENTS WITH HYPEREOSINOPHILIC SYNDROME IN EUROPE

J. Hwee 1,* , N. Kwon 2 , R. Alfonso-Cristancho 3 , L. Baylis 4 , G. Requena 5 , S. Du 6 , A. Khanal 6 , L. Huynh 6 , M. S. Duh 6 , R. W. Jakes 5

1 Value Evidence & Outcomes, GSK, Mississauga, ON, Canada; 2 Respiratory Research & Development, GSK, Brentford, Middlesex, United Kingdom; 3 Value Evidence & Outcomes, GSK, Collegeville, PA, United States of America; 4 Global Medical Affairs, GSK, Durham, NC, United States of America; 5 Epidemiology, GSK, London, United Kingdom; 6 Analysis Group, Inc., Boston, MA, United States of America

Background: Hypereosinophilic syndrome (HES) is a rare group of blood disorders characterized by prolonged eosinophilia that can lead to tissue and organ damage. HES was previously considered to be largely idiopathic, but myeloid and lymphocytic variants are also now recognized. The disease may manifest in multiple organ systems, most commonly the heart, nervous system, gastrointestinal tract, skin, and lungs, due to eosinophilic infiltration. Treatment aims to reduce eosinophil counts, and the current standard of care consists of high-dose glucocorticosteroids, despite the risks associated with high doses or long exposure, and antineoplastic agents for those with more severe disease or who do not respond to steroids. Healthcare resource utilization (HCRU) in patients with HES is not well characterized, and data from clinical trials may not be representative of the general disease population encountered in real-world settings.

Aims: To describe HES-related HCRU among a real-world cohort of patients with a confirmed diagnosis of HES in Europe.

Methods: This retrospective chart review study included patients ≥6 years old with a confirmed diagnosis of HES and ≥1 year of follow-up data from index (first physician encounter: Jan 2015–Dec 2019). Chart review was conducted by physicians in 5 European countries (France, Germany, Italy, Spain, and the United Kingdom [UK]). Data from a similar number of patients were reviewed and included across each country. HES-related hospitalizations and outpatient and emergency room (ER) visits were assessed and reported descriptively across all patients and separately by country.

Results: Of the 280 patients included, the majority were male (65.0%) and had idiopathic HES (55.4%); patients had a mean (standard deviation [SD]) age at HES diagnosis of 42.4 (16.2) years. The most common comorbidity was asthma (45.0%). The mean disease duration was 4.0 (4.5) years, and the mean length of follow-up was 2.8 (1.4) years. Overall, 85 (30.4%) patients had ≥1 HES-related hospitalization. For patients with a HES-related hospitalization, the mean (SD) length of stay (LoS) was 11.0 (9.4) days. HES-related ER visits were reported for 72 (25.7%) patients, and HES-related outpatient visits were reported for 243 (86.8%) patients. Patients had a mean of 0.4 (1.2) hospitalizations, 0.3 (0.8) ER visits, and 4.3 (4.9) outpatient visits (of which 1.0 [3.3] were unscheduled outpatient visits) per year ( Table ). HCRU varied between countries. The proportion of patients requiring hospitalization was highest in the UK (43.5%) and lowest in Spain (11.5%). Mean (SD) days of LoS during a HES-related hospitalization was highest in Spain (15.3 [4.2]) and lowest in the UK (9.9 [11.0]) and France (9.9 [7.3]). The proportion of patients with outpatient visits was highest in France (93.4%) and lowest in Italy (78.8%). The proportion of patients with ER visits was highest in the UK (37.1%) and lowest in Italy (13.5%).

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Summary/Conclusion: These results demonstrate that real-world patients with HES have substantial HCRU, including lengthy hospitalizations, ER visits, and outpatient visits, which is higher than that reported in previous clinical trials for control (placebo-treated) patients with eosinophilic diseases such as EGPA and severe eosinophilic asthma. These observations highlight the need for novel approaches to treatment to relieve the disease-related burden of HES for patients and to ameliorate the associated HCRU burden.

P1035: COUNTRY DIFFERENCES IN THE BURDEN OF PATIENTS WITH HYPEREOSINOPHILIC SYNDROME IN EUROPE

J. Hwee 1,* , N. Kwon 2 , R. Alfonso-Cristancho 3 , L. Baylis 4 , G. Requena 5 , S. Du 6 , A. Khanal 6 , L. Huynh 6 , M. S. Duh 6 , R. W. Jakes 5

1 Value Evidence & Outcomes, GSK, Mississauga, ON, Canada; 2 Respiratory Research & Development, GSK, Brentford, Middlesex, United Kingdom; 3 Value Evidence & Outcomes, GSK, Collegeville, PA, United States of America; 4 Global Medical Affairs, GSK, Durham, NC, United States of America; 5 Epidemiology, GSK, London, United Kingdom; 6 Analysis Group, Inc., Boston, MA, United States of America

Background: Hypereosinophilic syndrome (HES) is a group of rare blood disorders characterized by peripheral eosinophilia that can lead to tissue and organ damage. Diagnosis and management of HES can be challenging owing to differences in patient characteristics, disease characteristics and clinical manifestations. At present, there is limited information available on the burden of HES in Europe.

Aims: To describe real-world patient demographics, disease characteristics, clinical manifestations, treatment patterns, and clinical outcomes for patients with HES across Europe.

Methods: This retrospective chart review study included patients ≥6 years old with a confirmed diagnosis of HES and ≥1 year of follow-up data from index (first physician encounter: Jan 2015–Dec 2019). Chart review was conducted by physicians in 5 European countries (France, Germany, Italy, Spain, and the United Kingdom [UK]). Data from a similar number of patients were reviewed and included across each country. Patients could be newly diagnosed during the patient identification window or could be diagnosed prior to their index date, allowing for the inclusion of patients with differing disease duration and organ involvement. Patient demographics, disease characteristics, clinical manifestations, treatment patterns, and clinical outcomes were summarized and described across the overall population and separately by country.

Results: Overall, 280 patients were included in the study. Most patients were male (65.0%), with the highest proportion of male patients in Spain (73.1%) and France (75.4%). The majority of patients (55.4%) had idiopathic HES. The most common comorbidity overall was asthma (45.0%); however, in the UK, anxiety/depression was more common than asthma (51.6% vs 41.9%), and in France, both anxiety/depression and hypertension were more common than asthma (37.7% vs 23.0% and 36.1% vs 23.0%, respectively) ( Table ). Most patients (88.6%) were newly diagnosed with HES during the study time frame. Italy had the highest proportion of patients (17.3%) diagnosed prior to the study while Germany had the lowest (1.9%). The overall mean (standard deviation [SD]) number of distinct clinical manifestations was 3.7 (3.7), with the highest mean (SD) numbers of clinical manifestations observed in Italy (4.6 [4.7]) and Spain (4.7 [3.7]). Patients across all 5 countries were treated with 4 classes of therapy: oral corticosteroids (OCS; 89.3%), immunosuppressants or cytotoxic agents (63.6%), and biologics (43.9%), other therapies (10.7%). Spain had the highest mean (SD) maximum daily dose of OCS (42.4 [19.6] mg). In total, 22.9% of patients experienced a flare, and the mean (SD) number of flares in patients with ≥1 flare was 0.5 (0.3) flares/year. Patients in France, Germany, and the UK had the highest mean (SD) numbers of flares (0.7 [0.6], 0.6 [0.3], and 0.6 [0.3] flares/year, respectively). The overall mean (SD) duration of flares was 2.9 (3.2) months, with the longest flare duration observed in France (4.8 [6.3] months) and the shortest in the UK (1.6 [1.2]).

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Summary/Conclusion: These results demonstrate that in patients with HES, there are differences in patient and disease characteristics as well as treatment strategies across Europe. These differences likely contribute to challenges in the diagnosis and management of HES and highlight the need for a common strategy to address these difficulties.

P1036: VOLUMETRIC SPLENOMEGALY IN PATIENTS WITH POLYCYTHEMIA VERA

M.-W. Lee 1,* , S.-H. Yeon 1 , H. Ryu 1 , I.-C. Song 1 , H.-J. Lee 1 , H.-J. Yun 1 , S. Y. Kim 2 , J. E. Lee 3 , K. S. Shin 3 , D.-Y. Jo 1

1 Department of Internal Medicine; 2 Department of Laboratory Medicine; 3 Department of Radiology, Chungnam National University College of Medicine, Daejeon, South Korea

Background: At present, palpable splenomegaly is relatively uncommon in patients with polycythemia vera (PV), possibly because of early diagnosis facilitated by a lowering of the diagnostic thresholds for hemoglobin and hematocrit levels as well as the wide application of driver gene mutation tests. Radiological volumetric analysis of spleen size is now readily available. However, non-palpable splenomegaly in patients with PV has seldom been addressed.

Aims: In this retrospective study, we evaluated non-palpable, volumetric splenomegaly defined based on age- and body surface area (BSA)–matched criteria in patients with PV diagnosed according to the 2016 World Health Organization diagnostic criteria.

Methods: Patients with PV who underwent abdominal computed tomography (CT) and who had palpable splenomegaly at diagnosis from January 1991 to December 2020 at Chungnam National University Hospital were enrolled in the study. The spleen volume of each patient was determined by volumetric analysis of abdominal CT and adjusted for the patient’s age and BSA. And then the degree of splenomegaly was classified as follows: no splenomegaly, spleen volume less than the mean plus 2 SD of the reference volume based on both age and BSA; overt volumetric splenomegaly, spleen volume greater than the mean plus 3 SD of the reference volume based on both age and BSA; borderline volumetric splenomegaly, a spleen volume between no splenomegaly and overt splenomegaly; or palpable splenomegaly, spleen palpable below the left costal margin.

Results: Of the 87 PV patients enrolled in the study, 15 (17.2%) had no splenomegaly, whereas 17 (19.5%), 45 (51.7%), and 10 (11.5%) had borderline volumetric, overt volumetric, and palpable splenomegaly, respectively. Spleen volume was significantly positively correlated with lactate dehydrogenase level ( r = 0.227, P = 0.042) and tended to be positively related to white blood cell count ( r = 0.209, P = 0.055) and monocyte count ( r = 0.210, P = 0.059) at diagnosis. Spleen volume was not correlated with hemoglobin level, platelet count, or JAK2 V617F burden at diagnosis. The degree of splenomegaly did not affect the cumulative incidence of thrombotic vascular events (10-year incidence: 7.7%, 0%, 22.3%, and 50.7%, respectively, P = 0.414). By contrast, splenomegaly tended to adversely affect myelofibrotic transformation (10-year cumulative incidence: 0%, 0%, 7.1%, and 30.3%, respectively, P = 0.062). Moreover, the cumulative incidence of myelofibrotic transformation was significantly higher in patients with overt volumetric or palpable splenomegaly than those with no or borderline volumetric splenomegaly (10-year incidence: 0% vs. 10.3%, respectively; 15-year incidence: 0% vs. 26.3%, respectively, P = 0.020). Overall survival (OS) differed among patients with different degrees of splenomegaly (15-year OS: 100%, 78.6%, 71.7%, and 51.9%, respectively, P = 0.021). The degree of splenomegaly was independent risk factor for both myelofibrotic transformation (hazard ratio [HR]: 7.75; 95% confidence interval [CI]: 1.87-21.10; P = 0.005) and OS (HR: 6.70; 95% CI: 1.89-23.72; P = 0.003).

Summary/Conclusion: The degree of splenomegaly, including volumetric splenomegaly, based on age- and BSA-matched reference spleen volumes at diagnosis reflects disease progression in PV patients. Therefore, volumetric splenomegaly should be evaluated at the time of PV diagnosis and taken into consideration when predicting the prognosis of patients with this disorder.

P1037: DOES EARLY INTERVENTION IN MYELOFIBROSIS IMPACT OUTCOMES? A POOLED ANALYSIS OF THE COMFORT I AND II STUDIES

C. Harrison 1,* , J.-J. Kiladjian 2 , A. M. Vannucchi 3 , R. A. Mesa 4 , R. M. Scherber 5 , J. Hamer-Maansson 5 , S. Verstovsek 6

1 Guy’s and St. Thomas’ NHS Foundation Trust, Guy’s Hospital, London, United Kingdom; 2 Hôpital Saint-Louis, Assitance Publique – Hôpitaux de Paris, Université de Paris, Inserm, Paris, France; 3 CRIMM, Center for Research and Innovation of Myeloproliferative Neoplasms, AOU Careggi, University of Florence, Florence, Italy; 4 Mays Cancer Institute at UT Health San Antonio MD Anderson, San Antonio; 5 Incyte Corporation, Wilmington; 6 The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Myelofibrosis (MF) is characterized by cytopenias, splenomegaly, burdensome symptoms, and poor overall survival (OS). Few studies have investigated if earlier intervention with targeted MF therapies affects response and OS. In a pooled analysis of the COMFORT I and II trials of ruxolitinib (RUX), patients (pts) who received RUX at randomization or after crossover from placebo (PBO) or best available therapy (BAT) had improved OS.

Aims: To assess the association of MF disease duration before treatment with disease outcomes using pooled COMFORT data

Methods: COMFORT I ( NCT00952289 ) and COMFORT II ( NCT00934544 ) were phase 3 trials of RUX vs PBO or BAT, respectively, in pts with intermediate-2 or high-risk MF who provided written informed consent. In this post hoc analysis, data from RUX-treated pts in both studies were combined (RUX group), and data from the PBO/BAT arms were pooled (control group). Pt subgroups were defined based on disease duration before treatment initiation (≤12 mo or >12 mo from diagnosis). Assessments included frequency of thrombocytopenia (platelets [PLT] <100 Gi/L or PLT transfusion) and anemia (hemoglobin <100 g/L or red blood cell transfusion) events, spleen volume response (SVR; spleen volume reduction ≥35% from baseline [SVR35]), symptom response (MF-Symptom Assessment Form total symptom score [TSS] reduction ≥50% from baseline [TSS50]; available in COMFORT I only), and OS. OS was assessed using the Kaplan-Meier method; pts randomized to PBO/BAT were included in the PBO/BAT group regardless of crossover. A multivariable analysis (MVA) using a logistic regression model was used to examine factors associated with SVR.

Results: There were 525 pts in the analysis (RUX: ≤12 mo, n=84; >12 mo, n=216; PBO/BAT: ≤12 mo, n=66; >12 mo, n=159). Median age across groups ranged from 65 to 70 y. Baseline clinical characteristics were generally similar across subgroups, but pts with shorter vs longer disease duration were slightly younger with higher blood counts. Among pts who received RUX, fewer thrombocytopenia events were observed among those treated earlier (≤12 mo vs >12 mo), with differences seen as early as Wk 4–8 on treatment (18% vs 33%) and sustained over time; a similar trend was observed for anemia events (Wk 4–8, 59% vs 72%). The proportion of pts with SVR35 was greater among those who initiated RUX earlier (≤12 mo vs >12 mo) at Wk 24 (48% vs 33%; P =0.0610) and 48 (44% vs 27%; P =0.0149; Table ). A numerically greater proportion of pts who initiated RUX at ≤12 mo vs >12 mo achieved TSS50 at Wk 24 (56% vs 40%; P =0.0829; Table ). OS at Wk 240 was improved among pts who initiated RUX at ≤12 mo vs >12 mo (63% [95% CI, 51%–73%] vs 57% [95% CI, 49%–64%]; P =0.0430; Table ). Comparatively, OS was longer with RUX vs PBO/BAT regardless of disease duration. A sensitivity analysis using a 24-mo cutoff was also conducted but yielded weaker associations between disease duration and SVR, TSS, and OS. In the MVA, a significantly greater binary SVR was seen among pts with shorter (≤12 mo) vs longer (>12 mo) MF disease duration (odds ratio, 2.1; P =0.022).

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Summary/Conclusion: These findings suggest that earlier RUX initiation in MF may improve clinical outcomes, including cytopenias, SVR, symptom burden, and OS. While “watch and wait” remains a common treatment approach for newly diagnosed patients, these data suggest that pts with MF may benefit from earlier intervention. Additional studies to further evaluate the impact of early intervention are warranted.

P1038: CLINICOPATHOLOGIC AND MOLECULAR CORRELATES OF ORGAN DAMAGE ACROSS THE SPECTRUM OF ADVANCED SYSTEMIC MASTOCYTOSIS

E. Liang 1,* , C. Perkins 2 , R. Lu 3 , H. Shi 4 , S. Dimitrijevic 5 , G. Hoehn 4 , J. Abuel 2 , C. Langford 2 , P. Abidi 2 , L. Fechter 2 , W. Shomali 2 , J. Gotlib 2

1 Department of Medicine, Stanford University School of Medicine; 2 Division of Hematology, Stanford Cancer Institute/Stanford University School of Medicine; 3 Quantitative Sciences Unit, Stanford University School of Medicine, Stanford, CA; 4 Blueprint Medicines Corporation, Cambridge, MA, United States of America; 5 Blueprint Medicines Corporation, Zug, Switzerland

Background: Advanced SM (AdvSM) comprises 3 subtypes: aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN), and mast cell leukemia (MCL). The patterns of organ damage across the spectrum of AdvSM have not been well characterized. The definition of hematologic and non-hematologic organ damage has evolved from World Health Organization (WHO) C-findings, to organ damage criteria specified by the International Working Group-Myeloproliferative Neoplasms Research and Treatment and European Competence Network on Mastocytosis (IWG). More recently, modified IWG (mIWG) criteria have been used to define eligible organ damage in trials of AdvSM.

Aims: 1) To describe the distribution of organ damage in AdvSM patients (pts) at initial presentation or at time of trial enrollment using WHO, IWG, and mIWG criteria; 2) to evaluate clinicopathologic and molecular correlates of organ damage.

Methods: Our cohort included 244 AdvSM pts: 68 pts from our Stanford IRB-approved MPN registry and 176 pts from the phase I EXPLORER ( NCT02561988 ) and phase II PATHFINDER ( NCT03580655 ) studies of avapritinib in AdvSM. Comparisons between continuous and categorial variables were performed using the Wilcoxon rank sum test and the Fisher’s exact test, respectively. The Benjamini and Hochberg method was used to control the false discovery rate in multiple comparisons.

Results: Median age at diagnosis was 68 years (range, 24-88) and 146 pts (60%) were male. Forty-one pts (17%) had ASM, 165 (68%) had SM-AHN, and 38 (16%) had MCL. The median number of prior anti-neoplastic therapies was 1 (range, 0-6), with 87 pts (36%) previously treated with midostaurin.

Comparison of AdvSM Subtypes: Pts with MCL had the highest transfusion requirement, with 13% and 8% of pts requiring RBC and platelet transfusions, respectively. SM-AHN pts had the highest median alkaline phosphatase (ALP) (214 U/L), while pts with MCL had the largest median liver (2860 cm 3 ) and spleen (1120 cm 3 ) volumes by imaging. Among the two largest SM-AHN subtypes, pts with SM-CMML (n = 72) had a lower ANC (p = 0.02) and tryptase levels (p = 0.04) than pts with MDS/MPN-U (n = 36). Pts with SM-AHN fulfilled the WHO thrombocytopenia criterion (<100 x 10 9 /L) more frequently than MCL and ASM pts, and more WHO hematologic (p = 0.02) and IWG non-hematologic (p = 0.008) criteria than ASM pts.

Prior Therapy: Pts who received ≥1 prior anti-neoplastic therapy (compared to none) had greater tryptase levels (p = 0.02) and bone marrow (BM) MC burden (p = 0.03), lower ALP (p = 0.04), and higher likelihood of meeting WHO malabsorption criteria (hypoalbuminemia and/or weight loss) (p = 0.03). Compared to midostaurin-naïve pts, those who had received midostaurin had a greater BM MC burden (p = 0.045), but less frequently met mIWG splenomegaly criteria (p = 0.04) and met fewer WHO non-hematologic criteria (p < 0.001).

Mutation Profiles: Compared to pts with KIT D816V only, pts who also had ≥1 SRSF2, ASXL1 , or RUNX1 mutations had a lower median hemoglobin (p = 0.02), platelet count (p = 0.02), and greater direct bilirubin (p = 0.005), ALP (p = 0.04), and spleen volume (p = 0.06). They were more likely to fulfill the WHO hypersplenism criterion (p = 0.03) and exhibited more WHO-defined cytopenias (p = 0.006).

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Summary/Conclusion: In AdvSM, patterns of organ damage reflect disease subtype, prior therapy, and mutation profile beyond KIT D816V ( Figure ). These data can help inform the clinical heterogeneity of AdvSM patients who are being evaluated for clinical trials which currently require 1 or more organ damage findings defined by the WHO or (m)IWG criteria.

P1039: PALPABLE SPLEEN SIZE IS PROGNOSTIC IN PRIMARY BUT NOT SECONDARY MYELOFIBROSIS

M. Lucijanic 1,* , I. Krecak 2 , E. Soric 1 , D. Galusic 3 , H. Holik 4 , V. Perisa 5 , M. Moric Peric 6 , I. Zekanovic 6 , R. Kusec 1

1 Hematology Department, University hospital Dubrava, Zagreb; 2 Internal medicine department, General Hospital Sibenik-Knin county, Sibenik; 3 Internal medicine department, University hospital center Split, Split; 4 Internal medicine department, Slavnoski Brod, General hospital Slavonski Brod; 5 Hematology Department, University hospital center Osijek, Osijek; 6 Internal medicine department, General hospital Zadar, Zadar, Croatia

Background: Splenomegaly is a clinical hallmark of primary (PMF) and secondary myelofibrosis (SMF). Although not a part of disease specific risk scores, spleen size represents disease burden and correlates with unfavorable disease features. Spleen size reduction is a function of baseline spleen size and was recently shown to be independent prognostic factor in composite PMF and SMF cohort of ruxolitinib treated patients.

Aims: To estimate prognostic properties of palpable splenomegaly and potential differences between PMF and SMF patients.

Methods: We analyzed a multicentric cohort of 191 patients from 6 Croatian centers with either PMF or post polycythemia vera (PV) and essential thrombocythemia (ET) SMF diagnosed in period from 2004-2021 who had available data on palpable spleen size. Diagnoses were established according to 2016 and 2008 criteria. Risk was stratified according to the DIPSS in PMF and according to the MySEC-PM in SMF patients. Palpable spleen size was assessed at the time of diagnosis or referral and stratified in three categories: non-palpable, <10 cm and ≥10 cm.

Results: A total of 191 patients were analyzed, among them 144 PMF (63 early, 81 overt fibrotic) and 47 SMF (27 post PV; 20 post ET). Median age was 67 years, there were 64% males. Intermediate-2 or high risk disease was present in 45% PMF and 49% SMF patients. Non-palpable spleen, <10 cm and ≥10 cm splenomegaly were present in 31%, 43% and 26% PMF patients, respectively, and 23%, 49% and 28% SMF patients, respectively. Palpable spleen size was similar between patients with PMF and SMF (median 3 vs 4 cm, P=0.325). Patients with palpable spleen ≥10 cm were significantly grouped among patients with higher risk disease in PMF (P=0.026) but not SMF (P=0.654). Degree of bone marrow fibrosis was significantly associated with higher spleen size in PMF (P=0.008) but not SMF (0.809). On the contrary, JAK2 mutational status had no significant association with spleen size in PMF (P=0.847) but was significantly associated with higher spleen size in SMF (P=0.035).

Regarding overall survival, larger spleen size was significantly associated with shorter overall survival in PMF (P<0.001) but not SMF (P=0.825) as shown in Figure. Time to thrombosis had no significant association with larger spleen size in neither PMF nor SMF (P>0.05). Time to bleeding was significantly shorter in PMF patients with both <10 cm and ≥10 cm in comparison to non-palpable spleen (P<0.05) whereas palpable spleen size had no significant association with in SMF patients (P>0.05).

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Summary/Conclusion: Various clinical features are differently associated with palpable spleen size in PMF and SMF patients. Larger spleen size is prognostic of worse survival and higher bleeding risk in PMF but not SMF patients. This might have repercussions on prognostic scores based on palpable spleen size that might differently perform in PMF and SMF patients.

P1040: HIGHER ESTIMATED PLASMA VOLUME STATUS IS ASSOCIATED WITH INCREASED THROMBOTIC RISK AND IMPAIRED SURVIVAL IN PATIENTS WITH PRIMARY MYELOFIBROSIS

M. Lucijanic 1,* , I. Krecak 2 , E. Soric 1 , D. Galusic 3 , H. Holik 4 , V. Perisa 5 , M. Moric Peric 6 , I. Zekanovic 6 , R. Kusec 1

1 Hematology Department, University hospital Dubrava, Zagreb; 2 Internal medicine Department, General hospital Sibenik-Knin county, Sibenik; 3 Hematology Department, University hospital center Split, Split; 4 Internal medicine Department, General hospital Slavonski Brod, Slavonski Brod; 5 Hematology Department, University hospital center Osijek, Osijek; 6 Internal medicine Department, General hospital Zadar, Zadar, Croatia

Background: Blood plasma experiences substantial changes in both volume and composition in patients with chronic myeloproliferative neoplasms (MPN) and represents large reservoir of cytokines and other mediators of inflammation. Higher estimated plasma volume status (ePVS) has been shown to correlate with increased thrombotic risk in polycythemia vera patients, but its clinical and prognostic associations are unknown in patients with other MPN subsets.

Aims: To estimate clinical and prognostic associations of ePVS in patients with primary myelofibrosis (PMF).

Methods: We retrospectively analyzed a cohort of 167 patients with myelofibrosis treated in 6 Croatian hematology centers in period from 2004-2021. Diagnosis was established according to the WHO 2016 criteria. ePVS was calculated using the Strauss derived Duarte formula: 100-hematocrit (%)/hemoglobin (g/dL) and expressed as dl/g. ePVS associations with baseline clinical parameters and its prognostic associations with overall survival (OS), time to thrombosis (TTT) and time to bleeding (TTB) were analyzed.

Results: There were 167 patients with PMF analyzed (72 pre-fibrotic and 95 overt fibrotic). Median age was 68 years, there were 65% males, 67% were JAK2 mutated. Intermediate-2 or high DIPSS risk was present in 48% of patients.

Median ePVS was 5.62 dl/g. Higher ePVS was significantly associated with higher degree of bone marrow fibrosis, absence of JAK2 mutation, presence of constitutional symptoms, presence of circulating blasts, transfusion dependency, larger palpable spleen size, higher Charlson comorbidity index, history of bleeding, lower WBC, lower platelet count, lower hemoglobin, higher RDW, higher LDH, higher CRP, higher ferritin, lower albumin, higher fibrinogen, higher risk DIPSS status (P<0.05 for all analyses).

Median follow-up of our cohort was 57 months. During follow-up period a total of 79 patients died, 18 experienced thrombotic event (14 arterial and 5 venous thromboses) and 14 experienced bleeding event. Higher ePVS stratified at the ROC curve analysis defined cut-off points was significantly associated with worse OS (ePVS>6.2 dl/g, HR 1.74, P=0.017) and shorter TTT (ePVS>6.6 dl/g, HR 2.69, P=0.047) as shown in Figure. There was no significant association with TTB. In a multivariate Cox regression analysis model association of higher ePVS with increased thrombotic risk (HR 3.22, P=0.019) was present independently of WBC (HR 1.02, P=0.003), whereas JAK2 mutational status (HR 2.74, P=0.080) and cardiovascular risk factors (HR 7.12, P=0.060) retained marginal significance. Association with OS diminished in multivariate analysis after adjustment for DIPSS.

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Summary/Conclusion: PMF patients with more advanced disease features (more pronounced cytopenias and splenomegaly) have higher ePVS indicative of expanded plasma volume. Higher ePVS is associated with higher thrombotic risk and impaired survival.

P1041: IMPACT OF FEDRATINIB ON SPLEEN VOLUME AND MYELOFIBROSIS SYMPTOMS IN PATIENTS WITH SUBSTANTIAL SPLENOMEGALY: POST HOC ANALYSES FROM THE JAKARTA AND JAKARTA2 TRIALS

J.-J. Kiladjian 1,* , A. Tefferi 2 , F. Passamonti 3 , A. Vannucchi 4 , M. Talpaz 5 , F. Cervantes 6 , C. N. Harrison 7 , R. A. Mesa 8 , J. Mascarenhas 9 , N. Schaap 10 , S. Verstovsek 11 , T. Devos 12 , S. Rose 13 , J. Zhang 13 , O. Sy 13 , A. Pardanani 2

1 Hôpital Saint-Louis; Université de Paris, INSERM, Paris, France; 2 Mayo Clinic of Rochester, Rochester, United States of America; 3 University of Insubria, Varese; 4 CRIMM, University of Florence, AOU Careggi, Florence, Italy; 5 University of Michigan Comprehensive Cancer Center, Ann Arbor, United States of America; 6 Hospital Clinic Provincial de Barcelona, Barcelona, Spain; 7 Guy’s and St Thomas’ Hospital, London, United Kingdom; 8 Mays Cancer Center at UT Health San Antonio MD Anderson Cancer Center, San Antonio; 9 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States of America; 10 Radboud University Medical Centre, Nijmegen, Netherlands; 11 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 12 University Hospitals Leuven and Laboratory of Molecular Immunology (Rega Institute), KU Leuven, Leuven, Belgium; 13 Bristol Myers Squibb, Princeton, United States of America

Background: In myelofibrosis (MF), splenomegaly is a marker of disease progression, and larger spleen size (SS) is correlated with poorer survival and debilitating symptoms. Fedratinib (FEDR) is an oral, selective Janus kinase 2 (JAK2) inhibitor approved as first-line (1L) treatment (Tx) of patients (pts) with MF and in pts previously treated with ruxolitinib (RUX). In the phase 3, placebo-controlled JAKARTA trial ( NCT01437787 ) and the phase 2, single-arm JAKARTA2 trial ( NCT01523171 ), pts experienced substantial improvements in spleen volume and MF symptoms after receiving 6 FEDR Tx cycles. Given the prognostic impact of splenomegaly, these analyses explore whether these clinical benefits were influenced by pre-Tx SS.

Aims: Investigate the efficacy of FEDR 400 mg/d in JAKARTA and JAKARTA2, in pt subgroups defined by baseline (BL) SS.

Methods: The JAKARTA trial assessed FEDR 400 mg/d, FEDR 500 mg/d, and placebo in pts with JAK inhibitor-naïve MF, and the JAKARTA2 trial evaluated FEDR 400 mg/d (starting dose) in pts resistant/intolerant to prior RUX. Both studies enrolled pts with intermediate- or high-risk MF, SS >5 cm from the left costal margin by palpation, platelets ≥50×109/L, and ECOG PS ≤2. These post hoc analyses included pts from both studies who were allocated to receive FEDR 400 mg/d (the approved dosage) in continuous 28-d Tx cycles.

Pt subgroups were divided according to BL median SS (mSS) in each trial. Changes from BL in spleen volume and Myelofibrosis Symptom Assessment Form (MFSAF) total symptom score (TSS) were measured at the end of cycle 6 (EOC6). Spleen volume was assessed by MRI/CT scan; MFSAF TSS was the sum of 6 MF symptom scores: night sweats, early satiety, pruritus, pain under ribs-left side, abdominal discomfort, and bone/muscle pain. Eligible pts must have had spleen volume and/or TSS data at BL and EOC6.

Results: Overall, 96 pts in JAKARTA and 97 pts in JAKARTA2 received FEDR 400 mg/d; mSS (range) at BL was 16 (5–40) cm and 18 (5–36) cm, respectively. In the JAKARTA <mSS and ≥mSS cohorts, median BL spleen volume was 1771 (316–3396) mL and 3329 (983–6430) mL, respectively, and median TSS was 13.0 (0.0–54.9) and 17.2 (0.4–57.0). In JAKARTA2, median BL spleen volume was 1937 (737–4305) mL and 4002 (1821–7815) mL for pts with <mSS and ≥mSS, respectively; BL TSS was 17.2 (0.7–48.0) and 23.6 (1.0–44.0).

Overall, 75 (78%) pts in JAKARTA and 51 (53%) pts in JAKARTA2 had spleen volume data available at BL and EOC6; the <mSS and ≥mSS cohorts, comprised 37 and 38 pts, respectively, in JAKARTA, and 31 and 20 pts in JAKARTA2. Median spleen volume reduction (SVR) from BL was similar between the <mSS and ≥mSS cohorts in each trial, and 97% of pts with ≥mSS in both trials achieved some degree of SVR. In JAKARTA, median SVR at EOC6 was −38% (95% CI, −43 to −31) in the <mSS and −40% (95% CI, −40 to −28) in the ≥mSS cohort; in JAKARTA2, median SVR was −37 (95% CI, −43 to −30) in the <mSS and −38.5% (95% CI, −49 to −14) in the ≥mSS cohort.

TSS data were available at BL and EOC6 for 71 (74%) pts in JAKARTA and 51 (53%) pts in JAKARTA2. Changes from BL in TSS at EOC6 were similar in the JAKARTA <mSS and ≥mSS cohorts, with median TSS reductions of −49% and −51%, respectively. Median TSS changes at EOC6 in JAKARTA2 were greater for pts with larger spleens, −36% and −49% in the <mSS and ≥mSS cohorts, respectively.

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Summary/Conclusion: Pts who completed 6 Tx cycles with FEDR 400 mg/d, whether used as 1L MF Tx or after RUX Tx, experienced substantial improvements in spleen volume and MF symptom severity regardless of the extent of splenomegaly at BL.

P1042: SAFETY AND TOLERABILITY RESULTS FROM THE PHASE 3B FREEDOM TRIAL OF FEDRATINIB (FEDR), AN ORAL, SELECTIVE JAK2 INHIBITOR, IN PATIENTS WITH MYELOFIBROSIS (MF) PREVIOUSLY TREATED WITH RUXOLITINIB (RUX)

V. Gupta 1,* , A. Yacoub 2 , S. Verstovsek 3 , R. A. Mesa 4 , C. N. Harrison 5 , G. Barosi 6 , J.-J. Kiladjian 7 , H. J. Deeg 8 , S. Fazal 9 , L. Foltz 10 , R. J. Mattison 11 , C. B. Miller 12 , V. Parameswaran 13 , C. Hernandez 14 , J. Zhang 14 , M. Talpaz 15

1 Princess Margaret Cancer Centre, Toronto, Canada; 2 University of Kansas Medical Center, Kansas City; 3 The University of Texas MD Anderson Cancer Center, Houston; 4 Mays Cancer Center at UT Health San Antonio MD Anderson Cancer Center, San Antonio, United States of America; 5 Guy’s and St Thomas’ Hospital, London, United Kingdom; 6 Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 7 Hôpital Saint-Louis, Université de Paris, INSERM, Paris, France; 8 Fred Hutchinson Cancer Center, Seattle; 9 Allegheny Health Network Cancer Institute, Pittsburgh, United States of America; 10 St. Paul’s Hospital, University of British Columbia, Vancouver, Canada; 11 University of Wisconsin Carbone Comprehensive Cancer Center, Madison; 12 Ascension Saint Agnes Hospital, Baltimore; 13 Avera Cancer Institute, Sioux Falls; 14 Bristol Myers Squibb, Princeton; 15 University of Michigan Comprehensive Cancer Center, Ann Arbor, United States of America

Background: FEDR is an oral, selective Janus kinase 2 (JAK2) inhibitor approved for treatment (Tx) of patients (pts) with MF, including those previously treated with RUX. In the single-arm, phase 2 JAKARTA2 trial in pts with MF resistant/intolerant to prior RUX, the most common adverse events (AEs) during FEDR Tx were gastrointestinal (GI) events. JAKARTA2 ended in 2013, when a temporary clinical hold was placed on FEDR due to suspected cases of Wernicke’s encephalopathy (WE). The safety and efficacy of FEDR in pts with MF previously treated with RUX are being further evaluated in the ongoing, single-arm, phase 3b FREEDOM trial ( NCT03755518 ), which includes prospective strategies for preventing or mitigating GI AEs, thiamine decreases, and potential WE.

Aims: To assess the safety of FEDR and the effectiveness of AE mitigation strategies in the FREEDOM trial.

Methods: Eligible pts had intermediate- or high-risk MF, platelets ≥50×10 9 /L, and spleen volume ≥450cm 3 by MRI/CT or palpable spleen ≥5 cm below the left costal margin. Pts must have received prior RUX for ≥3 mo, or for ≥28 d with development of red blood cell transfusion requirement (≥2 units/mo for 2 mo) or grade ≥3 thrombocytopenia, anemia, hematoma, or hemorrhage. All pts received FEDR 400 mg/d in continuous 28-d cycles. AE mitigation strategies include prophylactic and symptomatic use of anti-nausea/vomiting and anti-diarrheal Tx, thiamine supplementation, FEDR dosing modifications, and administration of FEDR with food.

Results: In all, 34 pts were enrolled and 16 pts continued to receive FEDR at data cutoff (April 9, 2021). Reasons for FEDR discontinuation (D/C) in >1 pt were lack of efficacy (n=5), AEs (n=4), disease progression (n=2), pt decision (n=2), and to undergo transplant (n=2). Median FEDR Tx duration at cutoff was 28.3 (range, 1.6–101.3) wk; 14 (41%) pts had completed >12 cycles. At baseline (BL), median (range) age was 68.5 (49–82) y, time from MF diagnosis was 3.4 (0.1–17.4) y, and spleen size was 15 (3–31) cm. Most pts (62%) had primary MF, and all pts had received ≥3 mo of prior RUX; the most common reason for RUX D/C was loss of response/Tx failure (41%).

During FEDR Tx, 22 (65%) pts received ondansetron and 11 (32%) pts received loperamide. GI AEs in >10% of pts were constipation (47%), diarrhea (35%), nausea (26%), abdominal pain (24%), and vomiting (18%). Nausea, vomiting, and diarrhea decreased as Tx continued. Most GI AEs were grade (G) 1/2 (including nausea, vomiting, and diarrhea) and there were no Tx-related G3/4 GI AEs. No pt required FEDR reduction, interruption, or D/C due to a Tx-related GI AE.

Tx-related G3/4 AEs were reported in 11 (32%) pts, including anemia in 7 (21%) pts, and neutropenia, thrombocytopenia, and hyperkalemia in 2 (6%) pts each.

At BL, 1 pt had thiamine below the 70 nmol/L lower limit of normal (LLN), which normalized before the pt received FEDR. During FEDR Tx, thiamine levels dropped below the LLN for 4 pts between cycles 2 and 3 (and for 1 pt at end of Tx); for these pts, levels returned to normal with thiamine supplementation and no FEDR interruption or reduction was required (Figure). Five other pts received prophylactic thiamine supplementation. There were no cases of WE.

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Summary/Conclusion: FEDR was generally well tolerated in pts with MF previously treated with RUX. The frequency and severity of GI AEs were substantially lower in FREEDOM than in previous FEDR clinical trials, likely due to early implementation of GI prophylaxis. Thiamine decreases were uncommon and easily managed with routine monitoring and oral supplementation as needed.

P1043: OUTCOME OF PATIENTS WITH PREFIBROTIC MYELOFIBROSIS FROM A LARGE ACADEMIC CENTER

L. Masarova 1,* , P. Bose 1 , N. Pemmaraju 1 , H. Chifotides 1 , L. Zhou 1 , Z. Estrov 1 , H. Kantarjian 1 , S. Verstovsek 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: The 2016 WHO classification includes prefibrotic primary myelofibrosis (pre-PMF; fibrosis grading 0-1) and overt PMF (fibrosis grading 2–3). Pre-PMF has favorable survival when compared to overt PMF, however pre-PMF patients with intermediate -2 or high risks IPSS still have poor outcome (Guglielmelli et al. Blood. 2017;129(24):3227–3236).

Aims: We aimed to characterize pre-PMF patients referred to our institution between the years of 2000-2020. We specifically investigated the role of known IPSS prognostic factors separated into i) clinical factors (CF): presence of either hemoglobin [hgb] < 10 g/dL / white cells [WBC] > 25 x10 9 /L] or peripheral blasts [bl] ≥1%]; ii) age > 65 years and iii) systemic symptoms (IPSS defined; Cervantes, et al. Blood. 2009;113(13):2895–901).

Methods: This retrospective study included 130 patients with newly diagnosed pre-PMF (median 1.5 months from diagnosis to presentation) and available bone marrow fibrosis grading (Thiele, J. et al. Haematologica. 2005. 90, 1128–1132). Demographics were expressed by descriptive statistics. Overall survival was estimated from the time of presentation using Kaplan Meier method with log rank test.

Results: Clinical characteristics are detailed in Table 1, PART I. Fibrosis degree included grade 0 in 9, grade 0/1 in 7 and grade 1 in 114 patients, respectively. IPSS risks were as follows: low (N 22), intermediate 1 (N 50), intermediate 2 (N 30) and high (N 28). Thirty-five patients had no i-iii) factors (27%), symptoms only were in 49 patients (38%), one CF without and with symptoms was noticed in 9 (7%) and 22 (17%) patients, respectively, and 15 (11%) patients had 2 CF +/- symptoms.

Median OS of the entire cohort was 68 months (95% CI 42-94) with 54% of patients being alive at 5 years. Overall survival (95% CI) per IPSS scores: low-intermediate 1 and 2 - high were not reached, 179 months (65-not expressed), 65 months (32-98) and 27 months (5-49), respectively. Median OS (95% CI) of patients without i-iii) factors (age < 65 years, no symptoms and no CF) and patients with only symptoms (all ages included) was not reached; median OS of patients with one CF with and without symptoms was of 48 months (33-63) and 47 months (37-57), respectively and median OS of patients with 2 CF +/- symptoms was of 16 months (8-24). Detailed separation of survivals per age categories and IPSS subgroups is provided in Table 1, PART II.

Over the median follow-up of 43 months (95% CI 28-59), 7 patients progressed to acute leukemia with time to progression of 40 months (95% CI 9-178). Forty-one percent of patients (n = 53) required MF therapy during their follow-up, including 35 patients with JAK inhibitors and 8 with stem cell transplantation, respectively. Patients without the need for therapy or those who received JAK inhibitor had median OS of 81 (95% CI 65-not estimated) and 100 months (95% CI 17-183), respectively. This was superior to treated patients without exposure to JAK inhibitors (median OS of 45 months, 95% CI 16-47).

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Summary/Conclusion: Presence of more adverse clinical factors is the strongest predictor of unfavorable outcome of patients with pre-PMF. Use of JAK inhibitors might have significantly improved the outcome of patients with constitutional symptoms.

P1044: REAL-WORLD RUXOLITINIB TREATMENT PATTERN IN MYELOFIBROSIS PATIENTS WITH THROMBOCYTOPENIA

J. Mei *1, , Y. Wang *2 , S. Kabir 1 , N. Ichikawa 3 , S. Iino 3 , C. Lebedinsky 1

1 Clinical Development, Sumitomo Dainippon Pharma Oncology, Inc, Cambridge; 2 Computational Research, Sumitovant Biopharma, New York, United States of America; 3 Oncology Clinical Development Unit, Sumitomo Dainippon Pharma Co., Ltd, Osaka, Japan

Background: Ruxolitinib (RUX) is the standard treatment for patients (pts) with myelofibrosis (MF) to reduce splenomegaly and improve MF-associated symptoms. RUX treatment is titrated based on platelet counts and is associated with high rates of cytopenia effect. The incidence of moderate thrombocytopenia (platelet count <100 x 10 9 /L) is about 25% in newly diagnosed MF pts; however, the prevalence is about 67% in all MF pts. Low platelet count has been identified as an important surrogate marker of survival in MF pts with associated poor clinical features. Thrombocytopenia is also an independent predictor in the Dynamic International Prognostic Scoring System plus (DIPPS plus), with an actual 1.4-fold increased risk of death. Thus, treatment of MF pts with thrombocytopenia confers a unique challenge and unmet medical need. Real world studies will provide a perspective on the current management of MF pts with thrombocytopenia.

Aims: To characterize the disease and RUX treatment pattern in MF pts with thrombocytopenia using de-identified insurance claims data in the US.

Methods: The IBM MarketScan ® Medicare Supplemental Database data was retrospectively analyzed to identify pts aged ≥18 years with ≥1 claim for RUX and ≥2 non-diagnostic medical claims for MF (ICD-10 D47.4, D7581) from 2017–2019. The first RUX claim on or after the first MF claim defined the index date. Pts who continuously enrolled in a health plan for 3 months before the index date and 6 months after the index date were included. Clinical characteristics and RUX treatments were compared in MF pts with or without a history of thrombocytopenia prior to RUX initiation.

Results: The database contained 136 MF pts who had received treatment with RUX. Median age was 61 years, 61% were male, 62% of pts had primary MF, and 38% had post-polycythemia vera or essential thrombocythemia MF. Prior to initial RUX treatment, 57% of pts had a diagnosis of splenomegaly, 56% had anemia, 25% had thrombocytopenia, 19% had received RBC transfusion, and 7% had neutropenia. During RUX treatment, 25% of pts had a new diagnosis of thrombocytopenia, 22% had neutropenia, and 19% had anemia. Pts with a history of thrombocytopenia also had more anemia (79% vs 48%, p=0.001) and more RBC transfusions (44% vs 11%, p<0.001) than those without a history of thrombocytopenia. History of thrombocytopenia was associated with a lower starting dose of RUX (average starting daily dose 20 mg vs 27 mg, p=0.005), whereas history of anemia, leukocytosis, neutropenia, MF type, and splenomegaly were found to have no difference in the RUX starting dose. In addition, MF pts with history of thrombocytopenia continued RUX treatment at lower doses compared with those without a history of thrombocytopenia. During RUX treatment, 57% of pts with history of thrombocytopenia experienced anemia compared with 42% in pts without history of thrombocytopenia (OR 1.88, p=0.43).

Summary/Conclusion: In this real-world data analysis, history of thrombocytopenia was a major factor influencing the RUX dose for treatment of MF pts, consistent with prescribing data that RUX dose is adjusted based on platelet counts. These data show that pts with thrombocytopenia were treated with lower doses of RUX in clinical practice. Additionally, history of thrombocytopenia was associated with history of anemia; thus, these MF pts may be more prone to develop anemia during RUX treatment compared with pts without a history of thrombocytopenia. Our findings underscore the need for more treatment options to manage MF pts with thrombocytopenia.

*Equal contribution.

P1045: IS JAK2V617F BUT NOT CALR AS DRIVER MUTATION ENOUGH BY ITSELF IN THE PATHOGENESIS OF UNUSUAL TYPE VENOUS THROMBOSIS IN MPN PATIENTS?

E. Morsia 1,* , E. Torre 1 , G. Tarantino 2 , G. Svegliati Baroni 2 , G. Goteri 3 , S. Mancini 1 , A. Tassoni 1 , F. Alessandro 1 , A. Olivieri 1 , S. Rupoli 1

1 Hematology; 2 Gastroenterology and hepatology; 3 Pathological Anatomy and Histopathology, Ospedali Riuniti di Ancona, Ancona, Italy

Background: Myeloproliferative neoplasms (MPN) are a heterogenous group of hematopoietic stem cell disorders and clinically they constitute the most frequent underlying cause of venous thrombosis (VTE) in unusual site, including splanchnic vein thrombosis (SVT) and cerebral vein thrombosis (CVT). This subgroup of patients has been showed to have distinct characteristics compared to MPN patients with VTE in usual site, including deep vein thrombosis and pulmonary embolism.

Aims: The aim of this study is analyzed in a retrospective cohort of patients with MPN clinical characteristics, molecular features and outcome data in patients who have experienced an unusual site thrombosis.

Methods: In our study, we retrospectively analyzed a cohort of 577 consecutive patients with MPN according to WHO 2016 criteria who referred to our institute between 2009 and 2020. In all, 113 patients (19.58%) had a vascular event during a median follow up of 94.3 months (range, 2.4-416.0). Usual and unusual site venous thrombosis occurred in 40.7% and 33.6% in MPN patients with thrombosis, respectively.

Results: From the 38 MPN patients with unusual site VTE, 19 (50%) were male. Twenty-seven patients had an SVT and 11 a CVT. The driven mutation was JAK2V617F in 79% with a median allelic burden of 20% using NGS. MPN patients with SVT and CVT are younger (ORR 0.87 (0.82-0.93), pValue < 0.0001), with higher PLT count at diagnosis (ORR 0.06 (0.01-0.38), pValue=0.0085), and higher splenomegaly rate (ORR 0.97 (0.90-0.99), pValue=0.0003) compared to MPN patients with usual site VTE. Globally survival data in our population reported an overall survival in MPN with thrombosis of 238 months (95% I.C. 177-336), poorer in patients with arteriosus events compared to VTE (usual and unusual sites; pValue=0.017). At all, only few patients experienced recurrence thrombosis during the follow up and the recurrence thrombosis free survival rate at 5 years in MPN with thrombosis was 85%. Moreover, older age and level of hemoglobin at time of thrombosis significantly influence survival in MPN patients with unusual site VTE. Using NGS on 20 MPN patients with unusual site VTE, molecular analysis identified the mutation in the hotspot exon 14 region of JAK2 in 13 patients, among them the 92% as isolated alteration. The most frequent additional mutations were found in CALR mutated patients, including high risk mutation as ASXL1 and U2AF1 (Figure 1).

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Summary/Conclusion: This finding underlies the central role of JAK2V617F mutation in the pathogenesis of unusual type VTE in MPN, which occurs as sole molecular alteration in more than 90% of cases. However, in our cohort most patients who carried CALR as driver mutation seem to need one or more additional mutations. Our interpretation on these findings is that JAK2V617F is per se a strong risk factor for unusual venous thrombosis while CALR mutation must be enriched for additional non-driver alterations, needing a second hit to drive to thrombosis.

P1046: SEVERE COMPLICATIONS IN JAK2 V617F POSITIVE PEDIATRIC PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS

W. Novak 1,* , C. Annamária 1 , R. Crazzolara 2 , J. Neil 3 , K. Pirolt 4 , M. Dworzak 1,5 , L. Kager 1,5

1 St. Anna Children’s Hospital, Department of Pediatrics, Medical University Vienna, Vienna; 2 Department of Pediatrics, Medical University of Innsbruck, Innsbruck; 3 Department of Pediatrics, Paracelsus Medical University, Salzburg; 4 Department of Pediatrics, Klinikum Klagenfurt, Klagenfurt; 5 St. Anna Children’s Cancer Research Institute (CCRI), Vienna, Austria

Background: Myeloproliferative neoplasms (MPNs) are very rare diseases in children and adolescents and neither detailed risk stratification nor treatment guidelines are available.

Aims: The aim was to evaluate disease characteristics, complications, and treatment response of pediatric patients diagnosed with MPNs.

Methods: We retrospectively analyzed clinical data from a cohort of 14 pediatric patients diagnosed and treated in Austria over the past 12 years.

Results: Of these 14 patients (female, N=9), 10 patients were diagnosed with essential thrombocythemia (ET), three patients with polycythemia vera (PV) and one patient with primary myelofibrosis (PMF). One of the three main driver mutations known to cause MPN ( JAK2 , MPL or CALR mutation) was found in nine patients, eight of whom carrying a JAK2 V617F mutation (ET, N=5; PV, N=3; female, N=5; male, N= 3) and the patient with PMF harbored a CALR type 1 mutation.

Thrombotic complications occurred in 3/5 female (0/3 male) JAK2 V617F positive patients; one patient with ET developed recurrent sinus vein thrombosis (SVT) and two patients with PV developed a Budd-Chiari syndrome (BCS). One patient with BCS died. A transformation into a pre-PMF was observed in the other patient with BCS. In addition, another JAK2 V617F positive patient showed transformation from ET to PV seven years after diagnosis. Although 7/11 tested patients developed an acquired von Willebrand syndrome, there were no major bleeding complications. Two symptomatic JAK2 V617F positive female ET patients were treated off-label with ropeginterferon alfa-2b. The indications for treatment were microvascular symptoms (headache) and recurrent SVT. To our knowledge, this new long-acting interferon has not previously been used in children and adolescents. The treatment was well tolerated, no complications occurred and both patients quickly resolved their clinical symptoms.

Summary/Conclusion: Our data support results from studies in adult MPN patient populations, which showed that JAK2 V617F positivity is a risk factor for thrombotic complications and female gender is an additional risk factor for atypical thrombosis (BCS and SVT).

P1047: REAL-WORLD SAFETY OF RUXOLITINIB IN PATIENTS WITH INTERMEDIATE OR HIGH RISK OF PRIMARY MYELOFIBROSIS, POST-POLYCYTHEMIA VERA MYELOFIBROSIS OR POST-ESSENTIAL THROMBOCYTHEMIA MYELOFIBROSIS IN CHINA

Z. Xu 1 , M. Duan 2 , Q. Jiang 3 , Q. Leng 4 , N. Xu 5 , Y. Zhang 6 , C. Zhao 7 , W. Wu 8 , Q. Zhang 9 , J. Fu 10 , J. Zhang 11 , R. Fu 12 , Z. Yan 13 , J. Zhang 14 , C. Lin 15 , G. Ouyang 16 , Z. Wang 17 , L. Ma 18 , H. Hao 19 , X. Li 20 , S. Ran 21 , Y. Chen 22 , T. Li 23 , Z. Xiao 1,*

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin; 2 Peking Union Medical College Hospital; 3 Peking University People’s Hospital, Beijing; 4 Anshan Central Hospital, Liaoning; 5 Nanfang Hospital, Southern Medical University, Guangdong; 6 Henan Provincial People’s Hospital, Henan; 7 Affiliated Hospital of Qingdao University, Shandong; 8 Ruijin Hospital, Shanghai Jiao Tong University, Shanghai; 9 Gansu Provincial People’s Hospital, Gansu; 10 Shaoxing People’s Hospital, Zhejiang; 11 The Second Hospital of Hebei Medical University, Hebei; 12 Tianjin Medical University General Hospital, Tianjin; 13 North China University of Technology Affiliated Hospital, Hebei; 14 Nanchang University Affiliated Ganzhou Hospital, Jiangxi; 15 Zhangzhou Affiliated Hospital of Fujian Medical University, Fujian; 16 Ningbo First Hospital, Zhejiang; 17 Wuxi Second People’s Hospital, Jiangsu; 18 Sun Yat Sen Memorial Hospital of Sun Yat Sen University, Guangdong; 19 Hebei General Hospital, Hebei; 20 Affiliated Hospital of Southwest Medical University, Sichuan; 21 Beijing Novartis Pharma Co., Ltd, Beijing; 22 China Novartis Institutes For Biomedical Research Co., Ltd; 23 China Novartis Institutes For Biomedical Research Co., Ltd., Shanghai, China

Background: Ruxolitinib, a potent and selective inhibitor of Janus kinase 1 (JAK1)/JAK2 tyrosine kinases, was approved for treatment of intermediate- and high-risk patients with myelofibrosis (MF) based on the findings from the pivotal studies, COMFORT I and II. Although an Asia regional study was conducted, the enrolled Chinese population was limited (N=63).

Aims: We aimed to evaluate the safety profile and the treatment of ruxolitinib in Chinese patients with MF under routine clinical practice.

Methods: This was a retrospective, non-interventional, multicenter, post-marketing surveillance study collecting data up to 48 weeks after ruxolitinib initiation. Patients aged ≥18 years with a confirmed diagnosis of intermediate or high-risk primary MF (PMF), post-polycythemia vera MF (PPV-MF) or post-essential thrombocythemia MF (PET-MF) who had received or were currently receiving ruxolitinib treatment per clinical judgment according to China approved label were included.

Results: In total, 480 patients from 20 sites were enrolled and 428 patients completed the study treatment. The most reported reasons for treatment discontinuation were death (17.3%), adverse events (AE, 7.7%) and abnormal laboratory value (5.8%). Gender distribution were balance (male 51.6% vs female 48.4%) with median age of 61.0 years and median BMI of 22.5 kg/m 2 . Majority of the patients were diagnosed with PMF (69.8%), followed by PPV-MF (15.7%), and PET-MF (14.5%).

The information on dosing and safety is detailed in Table 1. Ruxolitinib dosing was according to the local approved label (median initial daily dose 30.0 mg). The median time from first diagnosis to medication was 1.5 months. Overall, patients received ruxolitinib for a median of 11.1 months with median dose intensity of 28.7 mg/day. The most prescribed dose was 15 mg BID across varying durations of exposure to ruxolitinib (up to 48 weeks). A total of 196 (40.8%) patients had dose modification.

On-treatment AE and serious AE (SAE) were reported in 361 (75.2%) and 57 (11.9%) patients, respectively. Of those, 60.2% and 6.0%, were related to study drug, respectively. The severity of majority of the AE were grade 2 and 3 (26.7 & 25.2%, respectively) while SAE were grade 3 & 4 (3.5% & 4.4%, respectively). A total of 4.0% and 2.3% of patients reported AEs and SAEs leading to discontinuation. The most commonly reported AEs were anemia (47.3%), decreased platelet count (22.1%), and abnormal liver function (12.7%). The most commonly reported SAEs were anemia (3.8%), platelet count decreased (1.0%), infectious pneumonia and pulmonary inflammation (both 0.8%). On-treatment AESI was reported by 18 (3.8%) patients, consisted of bleeding events (2.3%), serious or opportunistic infections (0.8%), and second primary malignancies (SPM, 0.6%). The SPM were pulmonary malignant tumors (n=2) and liver cancer (n=1). A total of 18 on-treatment deaths (MF progression=9, AE=4, and others=5) were reported.

During the study period, we found that the number of patients with efficacy data were decreasing with time (baseline=183&116, Week 16=126 & 96, Week 32=90 & 62, Week 48=38 & 19 for spleen size measurement and total symptom score, respectively). This reflects the need for improve compliance in disease management.

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Summary/Conclusion: This was the largest ruxolitinib real-world study in China. The safety profile of ruxolitinib in patients with intermediate- or high-risk PMF, PPV-MF, or PET-MF was generally consistent with previous global and Chinese studies of MF. The incidence of SPM was lower than that reported previously 4 . No new unexpected safety signals have been identified.

P1048: MYF3001: A RANDOMIZED OPEN LABEL, PHASE 3 STUDY TO EVALUATE IMETELSTAT VERSUS BEST AVAILABLE THERAPY IN PATIENTS WITH INTERMEDIATE-2 OR HIGH-RISK MYELOFIBROSIS REFRACTORY TO JANUS KINASE INHIBITOR

J. Mascarenhas 1,* , C. N. Harrison 2 , J.-J. Kiladjian 3 , R. S. Komrokji 4 , S. Koschmieder 5 , A. M. Vannucchi 6 , T. Berry 7 , D. Redding 7 , L. Sherman 7 , S. Dougherty 7 , L. Peng 7 , L. Sun 7 , F. Huang 7 , Y. Wan 7 , F. M. Feller 7 , A. Rizo 7 , S. Verstovsek 8

1 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America; 2 Guy’s and St Thomas’ Hospital, London, United Kingdom; 3 Hôpital Saint-Louis, Université Paris, Paris, France; 4 H Lee Moffitt Cancer Center, Tampa, FL, United States of America; 5 RWTH Aachen University, Aachen, Germany; 6 AOU Careggi, University of Florence, Florence, Italy; 7 Geron Corporation, Parsippany, NJ; 8 The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America

Background: Myelofibrosis (MF) is a life-threatening myeloproliferative neoplasm. The Janus Kinase inhibitors (JAKi) ruxolitinib and fedratinib are the only FDA approved treatment options for MF. Despite benefits reported with ruxolitinib in the front-line setting, a high proportion of patients discontinue treatment (Abdelrahman 2015), and the median overall survival (OS) is 11-16 months (Kuykendall 2018; Newberry 2017; Schain 2019; Palandri 2019; Mascarenhas 2020), highlighting a great unmet need for pts non-responsive to a JAKi treatment. Imetelstat, a first-in-class telomerase inhibitor, has shown meaningful clinical improvement in IMbark, a Phase 2 study in patients with intermediate-2 or high-risk MF who have relapsed after or are refractory to JAKi (Mascarenhas JCO 2021). Treatment with 9.4 mg/kg imetelstat resulted in 32.2% symptom response (total symptom score [TSS] reduction ≥50%) at Week 24 and median overall survival (OS) of 29.9 months with overall study follow up of 27.4 months. Dose-dependent inhibition of telomerase with imetelstat resulted in on-target activity that correlated with clinical benefits; dose-dependent reduction in variant allele frequency of MF driver mutations indicated targeting of the underlying malignant clone.

Aims: The Phase 2 results support continued study of imetelstat 9.4 mg/kg in a Phase 3 randomized controlled study, for which the study design is described here.

Methods: Study MYF3001 (IMpactMF; NCT04576156 ) is an open label, randomized (2:1), multicenter, Phase 3 study of imetelstat compared with best available therapy (BAT) in ~320 patients with intermediate 2 or high-risk MF refractory to JAKi treatment. Patients will be randomized to receive imetelstat 9.4 mg/kg IV every 21 days or investigator selected BAT including hydroxyurea, thalidomide, interferon, danazol, hypomethylating agents, chemotherapy, or other non-JAKi containing therapy as appropriate). Eligible patients will be stratified based on a) Intermediate 2 or high-risk per Dynamic International Prognostic Scoring System; b) platelet count at entry (platelets ≥ 75 and < 150 x 10 9 /L vs ≥ 150 x 10 9 /L). Patients who meet progressive disease criteria and discontinue BAT may be eligible to crossover to imetelstat.

Results: The primary endpoint is OS and one interim analysis is planned when approximately >71% of death events have occurred. Secondary endpoints include symptom and spleen response rates at week 24, progression-free survival, clinical response assessment per modified 2013 International Working Group - Myeloproliferative Neoplasms Research and Treatment criteria, time to and duration of response, reduction in degree of bone marrow fibrosis, safety, pharmacokinetics and patient-reported outcomes. Biomarkers and mutation analyses will be performed to evaluate the impact of imetelstat on reduction/depletion of malignant clones. (Figure 1)

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Summary/Conclusion: Approximately 180 sites are planned in North and South America, Europe, Middle East, Australia and Asia. The study is open for enrollment.

P1049: A PHASE 2 STUDY OF BEZUCLASTINIB (CGT9486), A NOVEL, HIGHLY SELECTIVE, POTENT KIT D816V INHIBITOR, IN ADULTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS (APEX): METHODS, BASELINE DATA, AND EARLY INSIGHTS

D. J. DeAngelo 1,* , V. Pullarkat 2 , M. Piris-Villaespesa 3 , T. I. George 4,5 , J. L. Patel 4,5 , C. Ustun 6 , P. Bose 7 , M. L. Heaney 8 , A. Pilla 9 , M. Massaro 9 , B. Exter 9 , H. A. Jolin 9 , Z. Mikhak 9 , T. Tashi 5

1 Dana-Farber Cancer Institute, Boston; 2 City of Hope, Duarte, United States of America; 3 Hospital Universitario Ramón y Cajal, Madrid, Spain; 4 ARUP Laboratories; 5 Huntsman Cancer Institute, University of Utah, Salt Lake City; 6 Rush University Cancer Center, Chicago; 7 The University of Texas MD Anderson Cancer Center, Houston; 8 Columbia University Medical Center, New York; 9 Cogent Biosciences, Cambridge, United States of America

Background: Systemic mastocytosis (SM) is a rare disease characterized by accumulation of pathogenic mast cells in bone marrow and extracutaneous tissues with or without skin involvement. In approximately 95% of adult patients, SM pathogenesis is driven by a gain-of-function mutation (D816V) in exon 17 of the KIT gene. Advanced systemic mastocytosis (AdvSM) is an aggressive, life-threatening form of SM with three variants: aggressive systemic mastocytosis (ASM), SM with an associated hematologic neoplasm (SM-AHN), and mast cell leukemia (MCL). Overall survival ranges from <6 months to 3-4 years. Bezuclastinib (CGT9486) is an orally administered, highly selective and potent tyrosine kinase inhibitor (TKI) that targets KIT D816V, avoiding other closely related kinases with clinical liabilities such as PDGFRα, PDGFRβ, and CSF1R. Bezuclastinib has demonstrated low brain penetration in preclinical tissue distribution studies, potentially addressing challenges associated with other targeted agents such as CNS effects and intracranial hemorrhage. Bezuclastinib has been administered to >100 participants in clinical trials, including 51 patients with advanced solid tumors (e.g., GIST) in a Phase 1b/2a study. In that study, bezuclastinib led to a reduction in KIT exon 17 mutational burden, temporally correlated with a reduction in tumor burden, and an acceptable safety profile.

Aims: To describe the Apex study methods and present baseline data and preliminary findings from Part 1 of this Phase 2 clinical trial evaluating bezuclastinib in patients with AdvSM ( NCT04996875 ).

Methods: This open-label, 2-part, multicenter study aims to enroll approximately 140 adult patients with AdvSM per WHO criteria assessed with SM-related organ damage and baseline serum tryptase of ≥20 ng/mL. Part 1 patients are randomized 1:1:1:1 to 50, 100, or 200 mg of bezuclastinib twice daily, or 400 mg once daily. Data from Part 1 will help determine the optimal dose of bezuclastinib, which will be utilized to further assess safety, efficacy, PK, and PD in Part 2. The primary efficacy endpoint is overall response rate (% patients classified as confirmed responders; CR, CRh, PR and CI) according to mIWG-MRT-ECNM response criteria, assessed by a Central Response Review Committee.

Results: Patients diagnosed with ASM, SM-AHN, and MCL have been enrolled in Part 1 (dose optimization) at centers in Europe and the United States. Baseline values include median (min, max) serum tryptase of 170 ng/mL (95.7-303), KIT D816V variant allele fraction 8.7% (7.04-13.48) by ddPCR, and bone marrow mast cells of 70% (7-80) by IHC. Dosing continues in these patients and the study is actively enrolling. Preliminary safety, PK, and PD data from Part 1 will be presented. Safety data will include AEs/SAEs/AECIs and dose modification frequencies while PD biomarker data, as early evidence of target engagement and clinical activity, will include changes in mast cell and KIT D816V mutational burden.

Summary/Conclusion: There is a significant unmet need for novel, safe, and effective treatment options for patients with AdvSM. Bezuclastinib, an orally administered and highly selective TKI with potent activity against KIT D816V, may be a treatment option in AdvSM. Part 1 of the Apex study aims to determine the optimal dose to further evaluate in Part 2. Early insights into the safety and tolerability profile and biomarker data will further inform clinical development of bezuclastinib in AdvSM.

P1050: THROMBOCYTOPENIC MYELOFIBROSIS (MF) PATIENTS PREVIOUSLY TREATED WITH A JAK INHIBITOR IN A PHASE 3 RANDOMIZED STUDY OF MOMELOTINIB (MMB) VERSUS DANAZOL (DAN) [MOMENTUM]

A. Vannucchi 1,* , R. Mesa 2 , A. Gerds 3 , H. K. Al-Ali 4 , D. Lavie 5 , A. Kuykendall 6 , S. Grosicki 7 , A. Iurlo 8 , Y. T. Goh 9 , M. Lazaroiu 10 , M. Egyed 11 , M. L. Fox 12 , D. McLornan 13 , A. Perkins 14 , S.-S. Yoon 15 , V. Gupta 16 , J.-J. Kiladjian 17 , R. Donahue 18 , J. Kawashima 18 , S. Verstovsek 19

1 Center Research and Innovation of Myeloproliferative Neoplasms, AOU Careggi, University of Florence, Florence, Italy; 2 UT Health San Antonio Cancer Center, San Antonio, TX; 3 Cleveland Clinic Department of Hematology and Medical Oncology, Avon, OH, United States of America; 4 University Hospital of Halle, Halle, Hungary; 5 Hadassah Hebrew University Medical Center, Jerusalem, Israel; 6 Moffitt Cancer Center, Tampa, FL, United States of America; 7 Medical University of Silesia, Katowice, Poland; 8 Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 9 Singapore General Hospital, Singapore, Singapore; 10 Policlinica de Diagnostic Rapid Brasov, Brasov, Romania; 11 Somogy County Mór Kaposi General Hospital, Kaposvár, Hungary; 12 Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain; 13 Guy’s and Saint Thomas’ NHS Foundation Trust, London, United Kingdom; 14 Monash University, Melbourne, Australia; 15 Seoul National University Hospital, Seoul, South Korea; 16 Princess Margaret Cancer Centre, Toronto, ON, Canada; 17 Saint-Louis Hospital (AP-HP), Paris, France; 18 Sierra Oncology, Inc., San Mateo, CA; 19 The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America

Background: MMB, a novel oral ACVR1/ALK2 and JAK1/2 inhibitor, showed clinical activity on MF symptoms, red blood cell (RBC) transfusion requirements (anemia), and spleen volume in the SIMPLIFY trials, including in MF patients (pts) with thrombocytopenia.

Aims: MOMENTUM is a pivotal phase 3 study of symptomatic and anemic MF pts previously treated with a JAK inhibitor (JAKi) testing MMB vs DAN. This analysis evaluated MOMENTUM pts with baseline (BL) platelet counts (PLT) ≤150 x 10 9 /L on key symptom, anemia, and spleen volume endpoints at 24 weeks (wks).

Methods: Eligibility: Primary or post-ET/PV MF; DIPSS high risk, Int-2, or Int-1; MF Symptom Assessment Form Total Symptom Score (MFSAF TSS) ≥10; hemoglobin (Hgb) <10 g/dL; prior JAKi for ≥90 days, or ≥28 days if RBC transfusions ≥4 units in 8 wks or Gr 3/4 thrombocytopenia, anemia, or hematoma; palpable spleen ≥5 cm; PLT ≥25 x 10 9 /L. JAKi taper and washout was ≥21 days. Randomization: 2:1 to MMB 200 mg QD plus DAN placebo or DAN 600 mg QD plus MMB placebo for 24 wks. Primary endpoint: TSS response (≥50% reduction from BL) rate at wk 24. Key secondary endpoints, assessed sequentially at wk 24: RBC transfusion independence (TI) rate, splenic response rate (SRR; ≥25% reduction in volume from BL), change from BL in TSS, SRR (≥35% reduction from BL) and rate of zero transfusions since BL. Informed consent was obtained from all participants.

Results: 64% of the MOMENTUM pts had BL PLT of ≤150 x 10 9 /L. Of this subset, 60 (74%) of 81 MMB pts and 25 (58%) of 43 DAN pts completed the 24-week randomized treatment (RT) phase. In this subset, median BL TSS were 29 (MMB) and 24 (DAN), Hgb were 7.9 (MMB) and 8.0 (DAN) g/dL, and PLT were 67 x 10 9 /L (MMB) and 64 x 10 9 /L (DAN). BL mean spleen volume was 2504 (MMB) and 2282 (DAN) cm 3 . Prior JAKi was ruxolitinib in 124 pts (100%) and fedratinib in 6 pts (5%); mean duration of prior JAKi was 136 weeks. Efficacy results are in Table. These results are consistent with the overall intent-to-treat (ITT) analysis set (N=195). Most common Gr ≥3 treatment-emergent adverse events (TEAEs) in the RT phase were thrombocytopenia (MMB, 31%; DAN, 16%) and anemia (MMB, 7%; DAN, 14%); Gr ≥3 bleeding events occurred in 9% of MMB and 5% of DAN pts. TEAEs led to study drug discontinuation in 15% of MMB and 19% of DAN pts, and serious TEAEs were reported in 36% of MMB and 40% of DAN pts, in RT phase. A trend toward improved OS up to wk 24 was seen with MMB vs DAN [HR (95% CI)=0.490 (0.195, 1.235)]. Additional analyses of pts with BL PLT <100 x 10 9 /L (N=100) and BL PLT <50 x 10 9 /L (N=31) show similar treatment effects of MMB vs DAN.

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Summary/Conclusion: In thrombocytopenic MF pts who were symptomatic and anemic, MMB was superior to DAN for symptom responses, transfusion requirements, and spleen responses and showed comparable safety and favorable survival. MMB may address a critical unmet need in thrombocytopenic MF pts. NCT04173494 .

P1051: A PHASE 2 STUDY OF IMG-7289 (BOMEDEMSTAT) IN PATIENTS WITH ADVANCED MYELOFIBROSIS

H. Gill 1,* , A. Yacoub 2 , K. Pettit 3 , T. Bradley 4 , A. Gerds 5 , M. Tatarczuch 6 , J. Shortt 7 , N. Curtin 8 , J. Rossetti 9 , K. Burbury 10 , A. Mead 11 , J. Göthert 12 , S. Koschmieder 13 , A. Jones 14 , J. Peppe 14 , J. Dias 15 , G. Natsoulis 16 , T. McClure 17 , M. Kleppe 15 , W.-J. Hong 14 , W. Stevenson 18 , J. Ewing 19 , J. Chacko 20 , E. Rumi 21 , A. Halpern 22 , F. Palandri 23 , N. Vianelli 23 , F. Passamonti 24 , R. Mesa 25 , M. Marchetti 26 , C. Harrison 27 , A. Vannucchi 28 , J. Watts 29 , D. Ross 30 , M. Talpaz 31 , H. Rienhoff 32

1 Medicine, University of Hong Kong, Hong Kong, China; 2 Internal Medicine, The University of Kansas Cancer Center, Leawood; 3 Internal Medicine, University of Michigan, Ann Arbor; 4 Hematology and Medical Oncology, University of Miami, Sylvester Comprehensive Cancer Center, Miami; 5 Hematology and Medical Oncology, Cleveland Clinic, Cleveland, United States of America; 6 Haematology and Oncology, Monash Health; 7 Clinical Sciences, Peter MacCallum; 8 Monash Health, Monash University, Melbourne, Australia; 9 Medicine, UPMC Hillman Cancer Center, Pittsburgh, United States of America; 10 Oncology, Peter MacCallum Cancer Centre, Melbourne, Australia; 11 MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; 12 Hematology, University Hospital Essen, Essen; 13 Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Aachen University, Aachen, Germany; 14 Hematology; 15 Basic Sciences; 16 Genetics; 17 Biometrics, Imago BioSciences, South San Francisco, United States of America; 18 Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia; 19 Haematology, University Hospitals Birmingham, Birmingham; 20 Haematology, The Royal Bournemouth & Christchurch Hospitals, Bournemouth, United Kingdom; 21 Haematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 22 Medicine, University of Washington, Seattle, United States of America; 23 Azienda Ospedaliero, Universitaria di Bologna Policlinico S.Orsola-Malpighi, Bologna; 24 Haematology, University of Insubria, Varese, Italy; 25 UT Health San Antonio Cancer Center, University of Texas Health Science Center, San Antonio, United States of America; 26 Haematology, Azienda Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Alessandria, Italy; 27 Haematology, Guy’s and St Thomas’, London, United Kingdom; 28 Center for Research and Innovation of Myeloproliferative Neoplasms, University of Florence, Florence, Italy; 29 Medicine, University of Miami, Sylvester Comprehensive Cancer Center, Miami, United States of America; 30 Haematology, Flinders Medical Centre and University, Adelaide, Australia; 31 Medicine, University of Michigan, Ann Arbor; 32 Hematology, Imago BioSciences, San Carlos, United States of America

Background: Lysine-specific demethylase-1 (LSD1) is an activity critical for the self-renewal of malignant myeloid cells and maturation of megakaryocytes, cells central to the pathogenesis of MF. Bomedemstat is an orally active LSD1 inhibitor that in mouse models ameliorated the hallmarks of MPNs and improved survival (Kleppe et al. 2015; Jutzi et al. 2018).

Aims: IMG-7289-CTP-102 is an ongoing, global, open-label Phase 2 study evaluating bomedemstat dosed QD in MF patients ( NCT03136185 ). Key eligibility criteria include patients intolerant, refractory, resistant, or inadequately controlled by approved therapy, and platelet count ≥100 x 109/L. Key objectives are safety and reduction of spleen volume (SVR) by MRI/CT and total symptoms scores (TSS) using the MPN-SAF instrument.

Methods: Serial bone marrow (BM) biopsies and imaging studies are read centrally. 261 genes are serially sequenced to quantify changes in the allelic frequencies of mutations (MAF) and identify new mutations. The starting dose is 0.6 mg/kg/d titrating, as needed, to a platelet count of 50-75x109/L.

Results: At 89 patients, the study is now fully enrolled: 46% primary MF, 33% post-essential thrombocythaemia-MF, 21% post-polycythaemia vera-MF. Median age is 68 (35-88) with 52% males. Prior treatment with ruxolitinib was reported in 83% (74/89); 46% had also received at least 1 additional treatment. 30% of patients (27/89) had received ≥1 RBC transfusion prior to dosing. By IPSS, 53% were high-risk, 40% int.-2, and 7% int.-1. At screening (N=103), sequencing to a mean depth of >1000 bp, JAK2 was mutated in 69%, CALR in 21%, MPL in 6% ; 59 % had ≥2 mutations of which 71% were high-molecular risk mutations in ASXL1, IDH1/2, EZH2, U2AF1, TP53 and/or SRSF2.

At data cutoff (3 Feb 2022), the median duration of treatment is 28 weeks (2-131). Of patients for whom TSS data is available at 24 weeks in those with baseline values ≥20, 72% (18/25) had a reduction in TSS; 24% (6/25) reported a ≥50% reduction. Of patients evaluable for SVR (N=50), 64% had a reduction in spleen volume from baseline; in 28%, SVR was ≥20%. Of evaluable patients (N=41), 90% had stable ( ∆ <± 1.0 g/dL) or improved (≥1.0 g/dL) hemoglobin. Of patients with BM fibrosis scoring post-baseline (N=52), 31% improved by 1 grade and 50% were stable. CCL5 and S100A8/A9 cytokines were elevated at baseline in 50% (16/32) and 78%, respectively; at Day 84, 81% and 68%, respectively, showed reductions of at least 10% and of those, 100% (CCL5) and 47% (S100A8/A9) normalized. In follow-up sequencing at around Week 24, of 60 mutant alleles in 32 patients, the mean MAF fell by 39% in 48%. JAK2 MAFs fell by 31% [SD 5%] in 46% (N=24); ASXL1 MAFs fell 40% in 71%. Clones with JAK2 and/or ASXL1 mutations were most affected. No new mutations have been identified and no patient has transformed to AML.

The most common non-hematologic AEs reported by patients was dysgeusia in 36% (32/90) and diarrhoea in 34% (31/90). All dysgeusia events were grade 1/2 and 1 led to treatment discontinuation. Of 14 related SAEs, 4 were Grade 2, 9 Grade 3 and 1 Grade 4 (thrombocytopenia). Twenty-nine patients remain on bomedemstat. Early terminations due to AEs occurred in 18 (20%) patients (9 related to bomedemstat), and 13 discontinued for other reasons. There have been no safety signals, DLTs, or deaths related to drug.

Summary/Conclusion: In patients with advanced MF, bomedemstat alone had an acceptable tolerability profile, relieved symptoms, reduced spleen volume and mutation burden while improving fibrosis and anemia without safety signals.

P1052: IMPACT OF SF3B1 MUTATION IN MYELOFIBROSIS

J. Senapati 1,* , S. Verstovsek 1 , L. Masarova 1 , N. Pemmaraju 1 , G. Montalban Bravo 1 , S. Pierce 1 , L. Zhou 1 , G. Garcia-Manero 1 , H. Kantarjian 1 , P. Bose 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: Splicing factor 3B subunit 1 ( SF3B1 ) mutations have been shown to confer a unique phenotype in MDS and MDS/MPN overlap syndromes, with ring sideroblasts, thrombocytosis and a relatively favorable prognosis. In myelofibrosis (MF), the frequency of SF3B1 mutation is <10% and it may play a less important role in disease outcomes (Lasho et. al, Leukemia, 2011 ).

Aims: We aimed to characterize the phenotypic and genotypic associations of SF3B1 mutations in pts with MF and compare them to SF3B1 wild type MF.

Methods: We retrospectively analyzed all pts with WHO-defined MF (primary, including pre-fibrotic MF, or progressed from PV or ET) seen at our center from 1/2017 through 7/2021. All pts had a BM biopsy with karyotyping and most of the pts had an 81-gene myeloid mutation panel performed by next-generation sequencing (including JAK2, CALR, MPL and SF3B1) at presentation or at progression to MF from PV or ET. We compared the disease phenotypes, MPN driver and co-occurring mutations, cytogenetics, DIPSS, transfusion requirements, treatment characteristics and outcomes between pts with SF3B1 -mutated ( SF3B1 + ) and -wild type ( SF3B1 - ) MF.

Results: 381 pts with MF were identified, 29 (8%) of whom were SF3B1 + . There were similar frequencies of JAK2 , CALR and MPL driver mutations and “triple negative” MF in the SF3B1 + and SF3B1 - groups (Table 1). The median number of SF3B1 mutations was 1 (range, 1-2); K666N being the most common (52%), followed by K700E (14%). 10% SF3B1 + pts were DIPSS high risk, compared to 19% SF3B1 - pts ( p =0.3). There were no significant differences between the 2 groups in terms of baseline hemoglobin level, white blood cell count, platelets, co-occurring mutations, and symptom burden but more SF3B1 + pts were red blood cell (RBC)-transfusion dependent at presentation than SF3B1 - pts (38% vs. 15%, p =0.003). A total of 152 (43%) SF3B1 - pts and 14 (48%) SF3B1 + pts received ruxolitinib-based therapy ( p =0.7). At a median follow up (mFU) of 17.2 months (mos) for the entire cohort (range, 0.1- 52.2 mos; mFU 22.6 mos for SF3B1 + and 16.8 mos for SF3B1 - ), 41 pts had died [4 (14%) SF3B1 + and 37(10%) SF3B1 - ] and 7 pts (1 SF3B1 + and 6 SF3B1 - ) had experienced leukemic transformation. The median estimated OS was 230 mos for SF3B1 - vs. not reached for SF3B1 + pts ( p = 0.57, Fig. 1).

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Summary/Conclusion: SF3B1 mutation is an uncommon event in MF and does not substantially affect the disease phenotype and outcomes. SF3B1 mutated MF pts appear to be more be RBC transfusion dependent given the underlying biology of SF3B1 mutation.

P1053: ALBUMIN AND C-REACTIVE PROTEIN PROVIDE PROGNOSTIC INFORMATION INDEPENDENTLY FROM MIPSS70 IN OVERT MYELOFIBROSIS. A RETROSPECTIVE STUDY.

N.-M. Messerich 1 , T. Volken 2 , S. Cogliatti 3 , T. Lehmann 4 , A. Holbro 5 , I. Demmer 3 , R. Benz 6 , W. Jochum 3 , T. Silzle 4,*

1 Department of Intensive Care, Cantonal Hospital St. Gallen, St. Gallen; 2 Institute of Public Health, ZHAW School of Health Sciences, Winterthur; 3 Institute of Pathology; 4 Clinic for Medical Oncology and Hematology, Cantonal Hospital St. Gallen, St. Gallen; 5 Division of Hematology, University Hospital Basel, Basel; 6 Clinic for Hematology and Oncology, Spital Thurgau AG, Münsterlingen, Switzerland

Background: Accurate prognostication is essential for patients with myelofibrosis (MF). The Dynamic Prognostic Scoring System (DIPSS) relies on age and several clinical and laboratory parameters and can be refined by information about chromosomal and/or molecular aberrations. C-reactive protein (CRP) and albumin have been reported to add prognostic information to both DIPSS and DIPSS-plus, which incorporates certain chromosomal aberrations and to scoring systems including both molecular and cytogenetic data (e.g. Molecular International Prognostic Scoring System 70 (MIPSS70)-plus V2). The impact of CRP and albumin in the context of the MIPSS70, which is useful, if metaphase cytogenetics are not obtainable, but next generation sequencing (NGS)-data are on hand, has not yet been reported.

Aims: To define the prognostic impact of albumin and CRP in the context of the MIPSS70.

Methods: We performed a retrospective chart review of all patients diagnosed at our institution with MF between 2000 and 2020 and collected information from hospital records as documented at diagnosis. For cases lacking NGS during diagnostic-workup, NGS was performed retrospectively, if DNA was available from diagnostic samples (Oncomine Myeloid Research Assay, Thermo Fisher Scientific, Waltham, MA, USA).

Results: 79 patients (median age 72 years, range 28-87; primary MF n=54, MF following ET or PV: n=22) were evaluable. Thirty-eight (48.1%) died during follow-up (median 34 months; range 0-184). CRP-values were available for 71/79 (89.9%), albumin-values in 62/79 (78.5%) and both parameters in 57/79 (72.2%) patients.

A CRP-elevation (>8mg/l, n=24) was associated with a shorter overall survival (OS) compared to a CRP <8mg/l (n=47, median 44 vs. 89 months, p<0.001, see fig A), as was an albumin below the median of the population (</≥ 40 g/l, n=31 each, median 50 vs. 101 months, p=0.018; see fig B). In univariate analyses, both CRP >8mg/l and albumin <40 g/l were associated with a higher risk of death (HR 3.85, 95% CI 1.85-8.0, p<0.001 and HR 2.49, 95% CI 1.13-5.49, p=0.024).

A logistic regression model showed a continuously rising probability of death with lower albumin levels even in the range of normal (at our institution: 34-45 g/l; OR=0.85, 95% CI 0.73-0.99; p=0.043)

The MIPSS70 was available for 60/79 (76%) patients. Due to the low number of low-risk-patients (n=2) we split the cohort into a “MIPSS70dich low/intermediate ” risk group (MIPSS70-low and MIPSS70-intermediate, n=45) and a “MIPSSdich high ” risk group, comprising the 15 MIPSS70 high-risk patients, for further analyses.

CRP >8 mg/l and albumin <40 g/l retained their prognostic value, if included each into a bivariate model together with the MIPSS70dich (HR 2.498, 95% CI 1.13-5.52, p=0.0236 and HR 5.486, 95% CI 1.89-15.96, p=0.0018).

After inclusion of CRP >8 mg/l and albumin< 40 g/l together with MIPSS70dich into a multivariate model, only albumin <40 g/l added prognostic information (HR 3.8, 95% CI 1.27-11.48, p=0.0173).

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Summary/Conclusion: Albumin and CRP deserve further evaluation as prognostic factors in myelofibrosis, since they add prognostic information irrespective of the molecular risk profile. In a common view, CRP and hypalbuminaemia reflect the extent of MF-associated inflammation. However, already albumin-levels in the lower range of normal are associated with an increased mortality. Given its role as main antioxidant in the extracellular space and its potential anti-inflammatory/anti-thrombotic properties, higher-albumin levels could be associated with an increased capability to counteract MF-associated inflammation.

P1054: MYLOX-1: AN OPEN-LABEL, PHASE IIA STUDY OF THE SAFETY, TOLERABILITY, PHARMACOKINETICS AND PHARMACODYNAMICS OF ORAL LOXL2 INHIBITOR, GB2064, IN MYELOFIBROSIS

C. Harrison 1 , J. Mascarenhas 2 , R. Rampal 3 , D. Cilloni 4 , B. Lindmark 5,* , B. Singh 5 , B. Jacoby 5 , S. Verstovsek 6

1 Guy’s and St Thomas Hospital, London, United Kingdom; 2 Icahn School of Medicine at Mount Sinai; 3 Memorial Sloan Kettering, New York, United States of America; 4 University Hospital San Luigi Gonzaga, Orbassano, Italy; 5 Galecto Inc, Copenhagen, Denmark; 6 MD Anderson Cancer Center, Houston, United States of America

Background: GB2064 is a high-affinity, selective, pseudo-irreversible, small-molecule inhibitor of LOXL2, a secreted glycoprotein that crosslinks extracellular matrix collagens and elastin which contributes to stiffness and loss of function of fibrotic organs. GB2064 is being developed as an oral treatment for myelofibrosis (MF), a rare myeloproliferative disease with high morbidity and mortality. Janus kinase (JAK) inhibitor therapy has brought significant advancements in the treatment of MF, but a significant proportion of patients would eventually discontinue treatment, predominantly due to the development of cytopenia (Kyukendall et al Ann, Hematol 2018). Thus, there remains a substantial unmet need for developing well-tolerated disease-modifying treatments that reduce bone marrow fibrosis to improve haematologic parameters, splenomegaly, symptom burden and quality of life.

Aims: To assess the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and clinical effects of oral GB2064 (1000 mg twice daily [BID]) dosed for 9 months to participants with primary or secondary myelofibrosis (PMF/SMF).

Methods: Open-label study in 16 adult participants diagnosed with PMF or SMF in accordance with World Health Organization diagnostic criteria (Barbui et al , Blood Cancer 2018; Cruz et al, Expert Rev Hematol 2020), who are not taking a JAK inhibitor and therefore likely to be refractory, intolerant or ineligible for such inhibitors, with Eastern Cooperative Oncology Group performance status 0-2 and clinical laboratory parameters within appropriate limits per protocol. Primary endpoint is safety and tolerability. Safety and tolerability, PK, PD and appropriate MF-specific assessments will take place at all visits, except Day 7, Day 15 and Month 4 when only safety and tolerability will be assessed (Fig A). Bone marrow biopsies, magnetic resonance imaging (MRI) of spleen and quality of life measures, MPN-10 and EQ-5D-5L, are performed at prespecified timepoints within the protocol. Exploratory endpoints include LOXL2 binding assay in the circulation, relationships between PK plasma exposures, PD markers, and markers of clinical activity, fibrosis and inflammation biomarkers (YKL-40, PAI-1, PDGF, CCN2, collagen formation and degradation neoepitopes). The study is not formally powered. Participants who derive benefit may continue therapy for an additional 3 years (Fig B).

Results: More than half of the intended participants have been enrolled and are on treatment with GB2064 as of February 2022.There have been no significant safety concerns observed at a dose of 1000 mg BID to date.

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Summary/Conclusion: MYLOX-1 is designed to explore the safety and clinical effects of GB2064, a novel small-molecule LOXL-2 inhibitor, addressing bone marrow fibrosis as a main element of myelofibrosis, with the aim of decreasing extramedullary haematopoesis and improving haematological parameters, symptom burden and the quality of life for patients with MF.

P1055: CLINICAL AND GENETIC RESULTS OF THE PHASE IB/II TRIAL MPNSG-0212: RUXOLITINIB PLUS POMALIDOMIDE IN MYELOFIBROSIS WITH ANEMIA

F. Stegelmann 1,* , E. Jahn 1 , S. Koschmieder 2 , F. Heidel 3 , A. Hochhaus 4 , H. Hebart 5 , S. Isfort 2 , A. Reiter 6 , M. Bangerter 7 , C. F. Waller 8 , D. Wolleschak 9 , C. Scheid 10 , J. Göthert 11 , P. Schafhausen 12 , T. Kindler 13 , M. P. Radsak 13 , N. Gattermann 14 , R. Möhle 15 , N. von Bubnoff 16 , A. Schrade 1 , T. Brümmendorf 2 , H. Döhner 1 , M. Griesshammer 17 , K. Döhner 1

1 Internal Medicine III, University Hospital of Ulm, Ulm; 2 Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, RWTH Aachen University, Aachen; 3 Klinik und Poliklinik für Innere Medizin C, Universitätsmedizin Greifswald, Greifswald; 4 Klinik für Innere Medizin 2, Universitätsklinikum Jena, Jena; 5 Internal Medicine, Stauferklinikum, Mutlangen; 6 III. Medical Department, University Medical Centre Mannheim, Mannheim; 7 Hematology and Oncology, Practice, Augsburg; 8 Department of Internal Medicine I, University Hospital of Freiburg, Freiburg; 9 Medical Center, Otto-von-Guericke University, Magdeburg; 10 Department I of Internal Medicine, University of Cologne, Cologne; 11 Department of Hematology, University Hospital of Essen, Essen; 12 2nd Medical Clinic, University Hospital of Hamburg-Eppendorf, Hamburg; 13 Internal Medicine III, Johannes Gutenberg University, Mainz; 14 Hematology, Oncology and Clinical Immunology, Heinrich Heine University, Düsseldorf; 15 Hematology and Oncology, University Tübingen, Tübingen; 16 Hematology and Oncology, University Hospital of Schleswig-Holstein, Lübeck; 17 Hematology/Oncology, Universitätsklinik Minden, Minden, Germany

Background: Ruxolitinib (RUX) alleviates disease-associated symptoms including splenomegaly in patients (pts) with myelofibrosis (MF). However, management of cytopenia remains challenging.

Aims: As single-agent pomalidomide (POM) improved cytopenia in 14-29% of MF pts in our previous MPNSG-0109 trial, we sought to investigate the combination of RUX plus POM in MF pts with anemia (Hb <10 g/dL and/or RBC transfusion dependency [RBC-TD]).

Methods: MPNSG-0212 is a multicenter, open-label, phase-Ib/II trial ( NCT01644110 ) comprising 39 pts in cohort 1 (co1, recruited 2013-2017) and 52 pts in co2 (2017-2021). Co1 pts received RUX 10 mg BID plus POM 0.5 mg QD, while POM was intended to be increased in co2 to 1 and 2 mg QD after 3 and 6 28-day-cycles, respectively. Primary endpoint was response according to IWG-MRT and RBC-TI criteria at end of cycle 12 (EOC12). In addition, genomic landscape was characterized in all pts using targeted NGS of 269 candidate genes (Illumina NextSeq550).

Results: Co1 and co2 pts had similar characteristics: median age was 71 years (range 49-86), median Hb level 8.6 g/dL (5.4-11.7), and median spleen size 17.5 cm (11.4-36); 30% were RBC-TD, 66% intermediate-2, and 25% high-risk according to DIPSS; mutations (muts) in JAK2 , CALR , or MPL were identified in 57%, 23%, and 20%, respectively; 55% had ≥1 high-molecular risk (HMR) mut, with ASXL1 being the most common (41%), followed by SRSF2 (24%), EZH2 (10%) and IDH2 (9%). Of note, pts in co2 were more frequently pre-treated with RUX compared to co1 (44% vs 15%; p =.006).

Median treatment time at data cut-off was 12 cycles in co1 (2-98) and co2 (3-46); 3 pts of co2 have not yet reached EOC12.

In co1, 8/39 pts (21%) achieved response at EOC12: partial remission (PR, n=1), clinical improvement (CI, n=6), or RBC-TI (n=1); 18/39 pts (46%) were treated for >12 cycles due to response (n=8), clinical benefit (CB, n=5) defined as Hb increase ≥1 g/dL or >50% improvement of ≥1 quality of life (QoL) symptom according to MPN-SAF, or stable disease (SD, n=5). Median Hb level at EOC12 was 9.3 g/dL; 15/39 pts (38%) were on treatment for >30 cycles, and 3 pts remained on long-term treatment (cycle 76, 97, and 98).

In 73% and 40% of pts in co2, POM dose was increased to 1 mg and 2 mg QD after cycle 3 and 6, respectively. 5/49 pts (10%) showed response at EOC12 (CI, n=3 and RBC-TI, n=2), while 14 additional pts (29%) had CB; median Hb level at EOC12 was 8.6 g/dL; 22/49 pts (45%) were treated for >12 cycles; 3 pts were still on treatment (cycle 21, 36, and 46).

Targeted NGS did not reveal a distinct mutational pattern associated with treatment response. Median overall survival (OS) of co1 and co2 was 3.6 and 2.6 years, respectively ( p =.66). Among all, CALR , but not JAK2 or MPL muts were associated with better OS ( p =.04), whereas HMR muts were in trend prognostically adverse ( p =.057). Pts with more than 3 muts had a worse median OS (1.2 vs 4 years, p =.002).

Combination therapy was well tolerated in both cohorts. Most common grade 1/2 adverse events (AE) were dyspnea (28%) and fatigue (23%), while the most frequent grade 3 AE was worsening of anemia (32%) in the first weeks of combination treatment not limiting therapy.

Summary/Conclusion: Treatment with RUX and POM was safe and feasible in our study of advanced MF with an adverse genetic profile. Almost half of co1 and co2 pts were treated >12 cycles due to clinical benefit of the combination therapy with a subset of 38% of pts in co1 receiving treatment for >30 cycles. Dose increase of POM in co2 did not result into better anemia response or improvement of OS.

Authors FS and EJ contributed equally.

P1056: SCREENING OF JAK2 EXON 12 SOMATIC MUTATIONS BY HIGH-RESOLUTION MELTING CURVE ANALYSIS

D. Kurochkin 1,* , I. Maslyukova 1 , T. Subbotina 1,2 , A. Khazieva 3 , E. Dunaeva 4 , K. Mironov 4

1 Siberian Federal University; 2 The Federal State-Financed Institution «Federal Siberian Research Clinical Centre under the Federal Medical Biological Agency»; 3 Regional Clinical Hospital, Krasnoyarsk; 4 Central Research Institute of Epidemiology of The Federal Service on Customers’ Rights Protection and Human Well-being Surveillance, Moscow, Russia

Background: JAK2 exon 12 mutations are seen in about 2-5% of JAK2 V617F-negative cases of polycythemia vera (PV). Nowadays about 40 different JAK2 exon 12 mutations associated with PV have been identified and classified. To identify all possible variants, it is necessary to use sequencing. However, due to the high cost of sequencing, developing a two-stage algorithm for detect mutations in JAK2 exon 12 using inexpensive screening is of immediate practically necessity. We have previously proposed a two-stage algorithm for detect mutations in JAK2 exon 12 using inexpensive screening test by heteroduplex analysis (Subbotina T et al, Haematologica 2017).

Aims: The aim of this study was to demonstrate the feasibility of HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) as the preliminary screening test for detection of JAK2 exon 12 mutations.

Methods: DNA samples of 5 JAK2 exon 12 mutation positive PV patients were included in this study. The identification of the JAK2 exon 12 mutation types and allele burden measurement was carried out by pyrosequencing (Subbotina T et al, Haematologica 2014). All 5 patients have different mutation variant in the 12 exon of the JAK2 and different levels of allelic burden: c.1624_1629delAATGAA – 67%; с.1619_1627TCAgAAATg>AAA – 14%; c.1623_1628delAAATGA – 15%; c.1622_1627delGAAATG – 33%; c.1612_1616CACAA>TT – 21%. HRM analysis was performed using a Precision Melt Supermix reagent kit in the presence of Eva Green dye (Bio-Rad, USA). PCR with an additional high-resolution melting step was carried out on a CFX96 thermocycler (Bio-Rad, USA) according to the following program: denaturation at 95°C for 2 minutes, then 40 cycles at 95°C for 10 seconds, 58°C in for 30 seconds. The high resolution melting program consisted of denaturation at 95°C for 30 seconds, renaturation at 60°C for 1 minute, and melting at 65°C to 95°C with a 0.2°C gradient in 10 seconds. Each DNA sample was analyzed in duplicate. For the two out five JAK2 exon 12 mutations a threshold determination of the mutant allele presence was analyzed. To analyze the threshold for determining the proportion of the mutant allele, dilution of cloned wild-type and mutated samples was performed to obtain samples with different levels of allelic burden: 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%.

Results: Figure 1 shows the differential melting plots of the DNA fragments. The analyzed samples were divided into five clusters: first cluster – melting curves of wild type DNA; second cluster – melting curves of DNA from samples with deletion type mutations: c.1624_1629delAATGAA and c.1622_1627delGAAATG; the third cluster – the DNA melting curves from the sample with the combined mutation: c.1612_161616CACAA>TT; the fourth cluster – DNA melting curves from the sample with deletion type mutation: c.1623 _1628delAAATGA; fifth cluster – DNA melting curves from a sample with the combined mutation: c.1619_1627TCAGAAATG>AAA. The detection thresholds in case of the c.1624_1629delAATGAA and с.1619_1627TCAgAAATg>AAA mutation analysis are 6.25% of the presence of the mutant allele in the samples (data not shown).

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Summary/Conclusion: Therefore, the HRM analysis that was conducted on the CFX96 allows to screen highly specific for the PV diagnosis mutations in exon 12 of the JAK2 gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of PV.

P1057: REAL-WORLD UTILIZATION OF FEDRATINIB FOR MYELOFIBROSIS FOLLOWING RUXOLITINIB FAILURE

C. Harrison 1,* , J. Mascarenhas 2 , P. Abraham 3 , J. A. Nadal 3 , A. Balanean 4 , A. McBride 3 , J. K. Kish 4 , D. Liassou 4 , B. A. Feinberg 4 , A. T. Gerds 5

1 Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; 2 The Tisch Cancer Intitute, Icahn School of Medicine at Mount Sinai, New York; 3 Bristol Myers Squibb, Princeton; 4 Cardinal Health, Dublin; 5 Cleveland Clinic Taussig Cancer Institute, Cleveland, United States of America

Background: In August 2019, fedratinib (FEDR) became the second treatment, after ruxolitinib (RUX), to be approved for intermediate (Int)- or high-risk (HR) primary or secondary myelofibrosis (MF).

Aims: To characterize real-world patient characteristics and treatment patterns for patients with MF receiving FEDR after RUX therapy in US clinical practices.

Methods: Adults with Int- or HR MF initiating FEDR on or after August 16, 2019 (FEDR approval date) after discontinuing RUX were identified from community oncology practices. Eligible patients had ≥90 days of follow-up from FEDR initiation, completed ≥1 FEDR cycle, and had a spleen size assessment at FEDR initiation. Treating physicians completed electronic case report forms for the selected eligible patients, capturing patient characteristics at time of MF diagnosis and during RUX and FEDR therapy, including bone marrow fibrosis grade, biomarkers, risk score, performance status score, spleen size (cm above the costal margin), symptoms (per Modified Myelofibrosis Symptom Assessment Form version 4.0), and complete blood count data. Providers reported treatment dosing and modifications and dates/rationale for RUX treatment failure (defined a priori, including refractory or suboptimal response, disease progression [PD], or intolerance).

Results: Among 150 eligible patients, the median age at diagnosis was 68 years; 55% were male, 68% were non-Hispanic White, 85% had primary MF, 55% were JAK2 V617F-positive, and 9% were JAK2/MPL/CALR -negative. RUX initiation dosage was ≥20 mg twice/daily (bid) for approximately 50% of patients. At RUX initiation, 65% had ECOG scores of 0/1, and 90% had palpable spleen (Table). A total of 19 patients (13%) had RUX dosage reductions due to neutropenia (7), thrombocytopenia (6), other adverse event (AE)/toxicity (3), patient request (2), and anemia (1); 4 patients (3%) had treatment interruptions due to thrombocytopenia (2), neutropenia (1), other AE/toxicity (1), and patient request (1); and 18 patients (12%) had dosage increases due to titration (13) and persistent MF symptoms (5). Primary reasons for RUX discontinuation were PD (70%), refractory or suboptimal response (25%), or intolerance (5%). RUX discontinuation due to PD was most often attributed to MF symptom return (65%), increased spleen size to >50% vs best achieved response (23%), and other reasons (12%). Median RUX duration was 4.0 months (IQR, 3.0–6.1) for refractory or intolerant patients and 11.7 months (IQR, 6.5–17.1) for those with PD. FEDR was initiated at 400 mg daily for 74% of patients. At FEDR initiation, 43% had International Prognostic Scoring System (IPSS)/Dynamic IPSS HR, 37% Int-2 risk, and 88% had palpable spleen; mean spleen size was 16.0 cm, and median platelet count was 98.0×10 9 /L (Table). Symptoms reported at FEDR initiation included fatigue (72%), abdominal discomfort (51%), night sweats (44%), early satiety (25%), bone pain (18%), itching (13%), and pain under the left rib (13%). At the end of follow-up, 44 patients (29.3%) were deceased, and mean time to death among deceased patients was 6.5 (standard deviation, 2.7) months from initiation of FEDR.

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Summary/Conclusion: This study showed a short duration of RUX therapy for real-world patients with MF, and poorer clinical status at FEDR initiation than at RUX initiation. Patients at risk of early failure of RUX should be considered for earlier FEDR interventional treatment.

P1058: REAL-WORLD OUTCOMES WITH FEDRATINIB THERAPY IN PATIENTS WITH PRIMARY MYELOFIBROSIS POST-RUXOLITINIB DISCONTINUATION

F. Passamonti 1,* , Y. Lou 2 , M. Chevli 3 , P. Abraham 4

1 University of Insubria and ASST Sette Laghi, Ospedale di Circolo, Varese, Italy; 2 Bristol Myers Squibb, Lawrenceville, United States of America; 3 Bristol Myers Squibb, Uxbridge, United Kingdom; 4 Bristol Myers Squibb, Princeton, United States of America

Background: Dysregulation of the Janus kinase 2 (JAK2) hematopoiesis-signaling pathway in myelofibrosis (MF) disrupts bone marrow production of blood cells, causing anemia, fatigue, and splenomegaly. Dual JAK1/JAK2 inhibitor ruxolitinib (RUX) was the first drug approved in the USA for intermediate/high-risk MF, but many patients only have a partial response or develop cytopenia, ultimately discontinuing treatment. Fedratinib (FEDR), approved in August 2019, is active against wild-type and mutationally activated JAK2 and receptor tyrosine kinase FLT3, but its real-world effectiveness in patients discontinuing RUX has not been evaluated.

Aims: To compare baseline characteristics and survival outcomes for patients with MF receiving FEDR vs those not receiving FEDR after discontinuing RUX in routine US clinical practice.

Methods: Patients receiving RUX for primary MF were identified using Flatiron Health’s nationwide electronic health record-derived database. Eligible patients were those with ≥2 recorded visits in the database between Jan 1, 2011 and Oct 31, 2020, ≥18 years of age at the index date (start of FEDR therapy for patients receiving FEDR and last date of RUX therapy for those not receiving FEDR), with data ≥1 month before and after index, and no record of receiving unclassified clinical study drugs prior to index. Patients were stratified by treatment with FEDR post-RUX (FEDR and non-FEDR group); the non-FEDR group was further stratified by time of RUX discontinuation (before [subgroup A] and after [subgroup B] US approval of FEDR) to enable a contemporaneous comparison with the FEDR group. Demographics and clinical characteristics were assessed at index. Overall survival (OS) was defined as time from index until death or censoring and assessed by Kaplan–Meier analysis; landmark survival was defined as the proportion of patients who survived at a given point. Associations of baseline variables and survival were assessed by Cox proportional hazards model.

Results: A total of 229 patients were evaluated (FEDR group: n=70; non-FEDR group: n=159). Median age at index was 71.0 for the FEDR group and 70.0 years for the non-FEDR group. Baseline demographic characteristics were broadly similar for the 2 groups. Median follow-up from index in the FEDR and non-FEDR group was 7.0 and 6.0 months, respectively. At index, 90.2% (46/51) and 74.3% (84/113) of patients had an ECOG performance status (PS) score of 0–1, respectively. Median duration of FEDR therapy in the FEDR group was 3.7 (range, 0–12.2) months, and 47.1% (33/70) received 400 mg FEDR once/day. Median OS was not reached in the FEDR group (vs 17 months in the non-FEDR group). Landmark survival in the FEDR and non-FEDR groups was 71.6% and 53.5% at 12 months, respectively. In the non-FEDR subgroup B, the corresponding survival rate at 12 months was 47.9%. The Cox proportional hazards model suggested that being male, of ‘other’ race (non-White, non-Black/African–American, non-Asian, non-missing), and having Charlson comorbidity index ≥1, ECOG PS 2–4, and body mass index <18.5 kg/m 2 were significantly associated with poorer survival. Based on the risk-adjusted analysis, we witnessed an apparent trend toward increased OS with FEDR, but the observed differences did not achieve statistical significance (HR [95% CI]: 0.6 [0.3–1.2]; P =0.1466).

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Summary/Conclusion: FEDR treatment of real-world patients with primary MF post-RUX may offer improved likelihood of survival rate up to 1 year after index compared with non-FEDR therapy. These differences in survival rate were sustained when the FEDR group and the non-FEDR B subgroup were compared.

P1059: COMPARATIVE EFFICACY OF FEDRATINIB AND PACRITINIB FOR THE TREATMENT OF MYELOFIBROSIS IN PATIENTS WITH LOW PLATELET COUNTS: A SIMULATED TREATMENT COMPARISON STUDY

G. Tremblay 1,* , P. Daniele 1 , P. Abraham 2 , S. Rose 2 , A. McBride 2

1 Purple Squirrel Economics, Montreal, Canada; 2 Bristol Myers Squibbs, Princeton, United States of America

Background: Myelofibrosis (MF) is a Philadelphia chromosome-negative myeloproliferative neoplasm with an estimated prevalence of 4–6/100,000 persons in the United States (US). Among intermediate- to high-risk patients, Janus kinase 2 (JAK2) inhibitors are the primary treatment for MF. Fedratinib was approved by the US Food and Drug Administration (FDA) based on the results of the JAKARTA trials ( NCT01437787 ; NCT01523171 ), and a new drug application for pacritinib was submitted to the FDA for the treatment of MF in patients with severe thrombocytopenia.

Aims: To assess the comparative efficacy of fedratinib and pacritinib in patients with MF and thrombocytopenia (platelets < 100 × 109/L), in the absence of head-to-head clinical trials, an indirect treatment comparison (ITC) was conducted.

Methods: According to the results of a systematic literature review, JAKARTA, JAKARTA2, and PERSIST-2 ( NCT01773187 ) trials formed the basis of the ITC. A pooled analysis data set was developed using individual patient-level data from the fedratinib 400-mg arms of JAKARTA and JAKARTA2 including patients with platelets < 100 × 109/L. Published summary data from the pacritinib 200-mg arm of PERSIST-2 served as the comparator. Simulated treatment comparisons (STCs) were used to compare spleen volume reduction (SVR) ≥ 35% while adjusting for mutually reported baseline patient characteristics such as age, sex, Dynamic International Prognostic Scoring System status, Eastern Cooperative Oncology Group (ECOG) performance status (PS), JAK2 V617F mutation status, prior ruxolitinib exposure, and laboratory tests. Final adjustment models were selected based on fit statistics and the literature review. Indirect relative risk (RR) was estimated with 95% confidence intervals (CIs) using unanchored naive ITC (unadjusted) and STC (adjusted) methodologies. A naive ITC was also conducted for the subgroup of patients who received ruxolitinib in prior lines of therapy. In addition, a sensitivity analysis was conducted to evaluate the impact of differential median baseline platelet counts, which PERSIST-2 did not report. Using the full pooled population of the JAKARTA trials, outcomes were simulated at 3 median baseline platelet counts (25, 50, and 75 × 109/L) and compared using similar methodology to the main analysis.

Results: The main analysis suggests that fedratinib is associated with a greater proportion of SVR ≥ 35% than pacritinib. According to the naive ITC, fedratinib was numerically favored over pacritinib in terms of SVR ≥ 35% (RR, 1.67 [95% CI, 0.94–2.97]). After the STC adjustment, fedratinib was statistically favored relative to pacritinib for SVR ≥ 35% (RR, 1.76 [95% CI, 1.00–3.10]). The naive ITC results were similar for patients who had received ruxolitinib in prior lines of therapy (RR, 2.82 [95% CI, 1.02–7.82]). Results of the platelet-count-adjusted sensitivity analysis showed a significant difference in favor of fedratinib with respect to SVR ≥ 35%. Differential baseline platelet count had minimal impact on the ITC results, with similar RRs regardless of simulated median baseline platelet count.

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Summary/Conclusion: This analysis used a population-adjusted ITC to assess the comparative efficacy of pacritinib and fedratinib in patients with MF and thrombocytopenia. Following population adjustment, fedratinib was associated with a greater proportion of patients achieving SVR ≥ 35% than pacritinib. Further real-world evidence studies should be conducted to assess the effectiveness of these treatments in clinical settings.

P1060: REAL-WORLD CLINICAL OUTCOMES AFTER 3 AND 6 MONTHS OF TREATMENT WITH FEDRATINIB FOLLOWING RUXOLITINIB FAILURE

J. Mascarenhas 1,* , A. T. Gerds 2 , J. K. Kish 3 , P. Abraham 4 , A. Balanean 3 , J. A. Nadal 4 , D. Liassou 3 , B. A. Feinberg 3 , A. McBride 4 , C. Harrison 5

1 The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York; 2 Cleveland Clinic Taussig Cancer Institute, Cleveland; 3 Cardinal Health, Dublin; 4 Bristol Myers Squibb, Princeton, United States of America; 5 Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom

Background: Relief of symptoms and splenomegaly is an important treatment (Tx) goal for patients with myelofibrosis (MF). Fedratinib (FEDR) and ruxolitinib (RUX) are approved in the USA for Intermediate (Int)- or High-risk (HR) primary or secondary MF.

Aims: To describe spleen size, and hematologic and MF-specific symptoms, during the first 6 months of FEDR therapy after prior Tx with RUX in US clinical practices.

Methods: Adults with Int- or HR MF who initiated FEDR on or after August 16, 2019 (FEDR approval date) and with ≥90 days of follow-up were identified from community oncology practices. Eligible patients were those treated with RUX and then FEDR, who had spleen palpation at FEDR initiation, and who had completed ≥1 cycle of FEDR. Treating physicians completed electronic case report forms for the selected eligible patients, extracting patient characteristics, Tx pattern data, and clinical outcomes such as spleen size, MF-related symptoms per the Myelofibrosis Symptom Assessment Form version 4.0 (MFSAF v4.0), hemoglobin (Hb), and platelet and white blood cell (WBC) counts, at each visit in the first 6 months of FEDR Tx. Data were collected from February to March 2021. Mean spleen size, symptom count, Hb, and platelet and WBC counts at FEDR initiation vs at 3 and 6 months post-FEDR were compared via paired 2-sided t-test. Percentage reduction in spleen size and proportion of transfusion-dependent patients between FEDR initiation and 3 and 6 months post-FEDR were compared via 1-sided chi-square test. Only patients assessed at FEDR initiation (with palpable spleen or complete blood count available) and with a corresponding assessment at 3 or 6 months were included in the comparative analysis.

Results: Among 150 eligible patients, the mean age at FEDR initiation was 68 years (range, 36–85). Median duration of RUX Tx prior to FEDR was 7.6 months (range, 0.7–65.5). At FEDR initiation, MF risk was categorized as International Prognostic Scoring System (IPSS)/Dynamic IPSS HR in 43% of patients, Int-2 in 37%, Int-1 in 9%, and unknown in 10%. A total of 74% of patients started FEDR at the recommended dosage of 400 mg daily. Median duration of post-FEDR follow-up was 5.1 months (IQR, 3.0–17.3) for patients initiated at FEDR 400 mg daily and 4.4 months (IQR, 3.0–14.3) for those initiating at <400 mg daily. At data cutoff, 55% of patients were still on FEDR. The primary reason for FEDR discontinuation was disease progression (43%). Mean duration of FEDR Tx was 4.7 months. Of 67 patients who discontinued FEDR, 44 were deceased at data cutoff. At FEDR initiation, 88% of patients had palpable spleen; mean spleen size was 16.0 cm (standard deviation [SD] 5.8) above the costal margin, which decreased to 13.2 cm (SD 7.9) at 3 months ( P =0.0001) and 7.2 cm (SD 7.4) at 6 months ( P =0.012). Mean number of MF-related symptoms declined significantly at 3 and 6 months; mean platelet count increased significantly at 6 months, from 160×10 9 /L to 186×10 9 /L (Table). Overall, 65.2% of patients experienced spleen size reduction, 18.8% had no change, and 16.0% had an increase. Mean best percentage change in spleen size was −29.2% (median −9.8%); complete resolution of palpable spleen occurred in 21% of patients.

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Summary/Conclusion: This study illustrates the real-world effectiveness of FEDR after RUX failure in patients with Int- or HR MF. FEDR provided significant reductions in spleen size and reported number of MF-related symptoms after 3 months of Tx, including complete spleen responses. Greater benefits were observed with longer Tx duration.

P1061: INDIRECT TREATMENT COMPARISONS OF FEDRATINIB VERSUS NAVITOCLAX PLUS RUXOLITINIB: EFFECT ON SPLEEN VOLUME AND SYMPTOMS IN RUXOLITINIB-EXPOSED MYELOFIBROSIS PATIENTS

P. Abraham 1,* , X. Liao 2 , M. Chevli 3 , S. Smith 2

1 Bristol Myers Squibb, Princeton, United States of America; 2 BresMed Health Solutions Ltd, Sheffield; 3 Celgene, a Bristol-Myers Squibb Company, Uxbridge, United Kingdom

Background: Myelofibrosis (MF) is a life-threatening myeloproliferative neoplasm characterized by stem cell-derived clonal myeloproliferation, bone marrow fibrosis, anemia, and splenomegaly. The Janus kinase (JAK) pathway is the critical pathway in its pathogenesis. Ruxolitinib (RUX), a JAK1/2 inhibitor, was the first therapy for intermediate- and high-risk MF approved in the United States; however, a high unmet need remains for alternative treatment options for patients who discontinue or are no longer responding to RUX. The efficacy and safety of fedratinib (FEDR), a JAK2 inhibitor approved by the US Food and Drug Administration in 2019, was investigated post RUX in the single-arm JAKARTA2 trial ( NCT01523171 ). Clinical data from a similar population treated with navitoclax plus RUX (NAV+RUX) have been reported. The efficacy of FEDR relative to NAV+RUX in patients with MF previously treated with RUX has not been evaluated.

Aims: To explore the comparative efficacy of FEDR versus NAV+RUX in patients with MF previously treated with RUX for the binary endpoints of ≥35% spleen volume reduction (SVR) from baseline to the end of cycle 6 (EOC6; 24 weeks) and ≥50% reduction in total symptom score (TSS) from baseline to the EOC6.

Methods: Evidence for FEDR was informed by JAKARTA2 patient-level data, and evidence for NAV+RUX was informed by known reported evidence from the REFINE study ( NCT03222609 ). The suitability of these studies for indirect treatment comparison (ITC) was assessed by considering the comparability of study design, population, intervention, and outcomes. Given the lack of a common comparator in the identified studies, unanchored ITCs were performed for SVR using matching-adjusted indirect comparison (MAIC) and simulated treatment comparison (STC) methods. MAICs used effective sample size (ESS) of the FEDR cohort as an indicator of the amount of overlap. Univariable and multivariable regression models were used to identify potential prognostic factors to adjust for in the ITCs. Additionally, all Dynamic International Prognostic Scoring System (DIPSS)-Plus criteria reported were considered.

Results: A subgroup of 58 JAKARTA2 patients with an Eastern Cooperative Oncology Group performance status (ECOG PS) score of 0 or 1 and intermediate-2 or high-risk disease most closely aligned with the REFINE population (N=34) was used in the analyses. Baseline mean platelet count was similar between subgroups. Across all analyses, results suggested FEDR consistently increased the odds/risk of a spleen response compared with NAV+RUX. The MAIC, matching on ECOG PS score, suggested that the odds of having an SVR for patients in the FEDR group was 2.19 times that of the NAV+RUX group (95% confidence interval [CI], 1.26 to 3.66), and the risk of having an SVR for patients in the FEDR group was 17.6% higher (95% CI, −2.1 to 37.0) (Figure). The results from the MAIC that additionally matched on all possible DIPSS-Plus criteria (age, hemoglobin, and platelet count) were consistent. Results from the 2 methods (MAIC and STC) were also consistent. For TSS reduction, the sample size (N=20) in REFINE was considered too small to perform a meaningful ITC; however, the absolute response rates for TSS reduction were similar across the 2 groups (29% [16/56] in the FEDR group and 30% [6/20] in the NAV+RUX group).

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Summary/Conclusion: In patients with MF previously treated with RUX, these analyses suggest treatment with FEDR was associated with a greater proportion of patients achieving a spleen response compared with NAV+RUX. Limited data were available for comparison of TSS.

P1062: A REAL-WORLD EVALUATION OF THE ASSOCIATION BETWEEN ELEVATED BLOOD COUNTS AND THROMBOTIC EVENTS IN POLYCYTHEMIA VERA: AN ANALYSIS OF DATA FROM THE REVEAL STUDY

A. T. Gerds 1,* , R. Mesa 2 , J. M. Burke 3 , M. R. Grunwald 4 , R. Scherber 5 , J. Yu 5 , J. Hamer-Maansson 5 , S. T. Oh 6

1 Cleveland Clinic Taussig Cancer Institute, Cleveland; 2 UT Health San Antonio MD Anderson Cancer Center, San Antonio; 3 Rocky Mountain Cancer Centers, Aurora; 4 Levine Cancer Institute, Atrium Health, Charlotte; 5 Incyte Corporation, Wilmington; 6 Washington University School of Medicine, St. Louis, United States of America

Background: Polycythemia vera (PV) is characterized by clonal hematopoiesis leading to elevated peripheral blood counts and an increased risk of thrombotic events (TEs). Advanced age and TE history form the conventional risk model used to determine TE risk/treatment strategy. Associations between TEs and elevated hematocrit (HCT) levels exist, but associations with white blood cell (WBC) or platelet (PLT) counts have not been assessed consistently. The large, real-world, p r ospective Obs e rvational Study of Pts with Polycythemia Ve ra in US Clinic al Practices (REVEAL; NCT02252159 ) followed pts with PV treated in community or academic centers.

Aims: This analysis evaluated associations between elevated blood counts and TEs in pts with PV using data from REVEAL.

Methods: Eligible pts had ≥3 lab values (blood counts) post-enrollment; pts with a post-enrollment TE but no lab value <6 mo before that TE were excluded. The association between blood counts and TEs was assessed using a time-dependent covariate Cox proportional hazards model. Time to first post-enrollment TE was modeled with time censored at last known visit for pts with no TE. Each lab parameter was modeled with sex, age, disease duration, and TE history at enrollment as baseline covariates and treatment as a time-dependent covariate. Blood counts were included as binary time–dependent covariates using the following thresholds: HCT >45%, WBC >11×10 9 /L, PLT >400×10 9 /L. Linear interpolation was used to determine lab values between observed lab values. Alternative thresholds for WBC (<7, ≥7 to <8.5; ≥8.5 to <11, and ≥11×10 9 /L, and >12×10 9 /L with HCT controlled at ≤45%) and PLT counts (>600×10 9 /L) were evaluated. Statistical significance was considered at P <0.05.

Results: 2271/2510 pts were eligible (median age, 66 y [range, 22–95]; male, 54.1%). Median disease duration was 4.1 y (range, 0–56.3), 456 (20.1%) had TE history; 52.6% of pts received hydroxyurea. Of 106 pts who had TEs, 30 had arterial TEs (most commonly, transient ischemic attack [n=15]) and 76 had venous TEs (most commonly, deep vein thrombosis [n=37]).

Elevated HCT levels (>45%, hazard ratio [HR]=1.84 [95% CI, 1.234–2.749], P =0.0028), WBC (>11×10 9 /L, HR=2.35 [1.598–3.465], P <0.0001), and PLT counts (>400×10 9 /L, HR=1.60 [1.088–2.359], P =0.0170) were each associated with increased TE risk (Table 1). WBC count ≥11×10 9 /L is associated with the highest TE risk compared with WBC count <7×10 9 /L (HR=2.61 [95% CI, 1.594–4.262], P <0.0001). Elevated WBC >12×10 9 /L was significantly associated with increased risk of TE with HCT controlled at ≤45%. PLT count (>600×10 9 /L) increased TE risk (HR=1.37 [95% CI, 0.763–2.468]) compared with PLT count ≤600×10 9 /L, but this was not statistically significant ( P >0.05). In all models, advanced age, female sex, and TE history were associated with increased TE risk.

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Summary/Conclusion: This analysis of REVEAL, the largest real-world cohort of PV pts to date, demonstrated that elevated HCT levels (>45%), WBC (>11×10 9 /L), and PLT counts (>400×10 9 /L) were associated with increased TE risk. An association of elevated WBC >12×10 9 /L with increased risk of TE was also observed when HCT was controlled, indicating that TE risk may be reduced by controlling WBC as well as HCT. These data support the need to incorporate blood count into risk stratification and treatment strategies for pts with PV in clinical practice and to move beyond the conventional risk model. Further studies to understand the causal relationship between elevated blood counts and TEs are warranted.

P1063: EFFICACY AND SAFETY OF PARSACLISIB-RUXOLITINIB COMBINATION THERAPY IN MYELOFIBROSIS PATIENTS WITH LOW VS HIGHER BASELINE PLATELET COUNT: A SUBGROUP ANALYSIS OF DATA FROM A PHASE 2 STUDY

A. Yacoub 1,* , U. Borate 2 , R. Rampal 3 , H. Ali 4 , E. Wang 5 , A. Gerds 6 , G. Hobbs 7 , M. Kremyanskaya 8 , E. Winton 9 , C. O’Connell 10 , S. Goel 11 , S. Oh 12 , G. Schiller 13 , A. Assad 14 , S. Erickson-Viitanen 14 , F. Zhou 14 , N. Daver 15

1 University of Kansas Cancer Center, Westwood; 2 Oregon Health & Science University, Portland; 3 Memorial Sloan Kettering Cancer Center, New York; 4 City of Hope National Medical Center, Duarte; 5 Roswell Park Comprehensive Cancer Center, Buffalo; 6 Cleveland Clinic, Cleveland; 7 Massachusetts General Hospital, Boston; 8 Icahn School of Medicine at Mount Sinai, New York; 9 Emory University, Atlanta; 10 University of Southern California, Los Angeles; 11 Montefiore Medical Center, Bronx; 12 Washington University School of Medicine, St. Louis; 13 David Geffen School of Medicine, University of California Los Angeles, Los Angeles; 14 Incyte Corporation, Wilmington; 15 University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Despite the demonstrated efficacy of ruxolitinib (a potent and selective JAK1 and JAK2 inhibitor), patients with myelofibrosis (MF) often exhibit inadequate or loss of response to chronic ruxolitinib therapy, possibly due to persistent PI3K pathway activation. Parsaclisib (INCB50465) is a potent and highly selective next generation PI3Kδ inhibitor. In the ongoing phase 2 INCB 50465-201 trial ( NCT02718300 ), add on parsaclisib has shown preliminary efficacy in patients with MF who experienced a suboptimal response to ruxolitinib. JAK inhibitors, including ruxolitinib, are associated with thrombocytopenia; therefore, patients with low platelet count (PC) are commonly more difficult to treat.

Aims: Here we present a subgroup analysis of efficacy and safety data from the ongoing INCB 50465-201 study by baseline PC.

Methods: Eligible adults had primary or secondary MF with suboptimal response (palpable spleen >10 cm below left subcostal margin [LSM]; or palpable spleen 5–10 cm below LSM and active symptoms) after ≥6 months of ruxolitinib monotherapy (5–25 mg BID; stable dose ≥8 weeks). Patients remained on their last stable ruxolitinib dose and received add on parsaclisib (10 or 20 mg QD for 8 weeks; same dose QW thereafter) or parsaclisib (5 or 20 mg QD for 8 weeks; 5 mg QD thereafter). For this analysis, spleen volume (SV), total symptom score (TSS) assessed by Myelofibrosis-Symptoms Assessment Form (MFSAF) v3.0 daily diary, and safety, were evaluated by baseline PC (low PC, 50 to <100×10 9 /L; higher PC, ≥100×10 9 /L).

Results: At data cutoff (August 27, 2020), 67 patients were enrolled (low PC, n=21; higher PC, n=46; median age 68 years). Median prior duration of ruxolitinib use was 34.7 months for low PC compared with 14.9 months for higher PC; baseline symptoms were worse for patients with low PC than higher PC (median [range] MFSAF-TSS, 21.4 [0.6–47] vs 10 [0–43]). Table 1 summarizes the responder analysis for SV reduction (SVR) based on baseline PC. At week 12, slightly more patients with low PC achieved ≥10% SVR compared with patients with higher PC (50.0% vs 39.4%), whereas, at week 24, responses were similar between the 2 groups (35.2% vs 37.1%). Of patients with ≥10% SVR at week 24, 4/6 with low PC and 9/13 with higher PC were on all daily dosing regimens. Median (range) percentage change in MFSAF-TSS was −20.5 (−56.6 to +17.1) for patients with lower PC compared with −22.2 (−100 to +500) for patients with higher PC at week 12; and −26.1 (−54.7 to +2.4) compared to −23.1 (−91.3 to +222.5) at week 24, respectively. In both subgroups, nonhematologic treatment-emergent adverse events (TEAEs) were mostly grade 1 or 2. Most common (≥20%) TEAEs were dyspnea (33%), falls (33%), peripheral edema (29%), and nasal congestion (24%) for low PC; diarrhea (28%), nausea (24%), abdominal pain (24%), cough (20%), and fatigue (20%) for higher PC. For patients with low PC compared to higher PC, 9/21 (43%) compared to 3/46 (7%) patients had parsaclisib dose interruption due to thrombocytopenia. One patient with low PC had ruxolitinib interruption due to thrombocytopenia.

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Summary/Conclusion: Add-on parsaclisib showed efficacy in patients from both low and higher baseline PC groups. Given the acceptable safety profile and efficacy of add-on parsaclisib, MF patients with both low and higher PC may be able to benefit from parsaclisib-ruxolitinib combination therapy. Phase 3 trials in ruxolitinib-treated and ruxolitinib-naive patients are underway to further assess the combination of JAK and PI3K inhibitors.

P1064: CIRCULATING CYTOKINE LEVELS IN PATIENTS WITH PHILADELPHIA-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS – PROFILE AND CLINICAL CORRELATIONS – A SINGLE CENTER STUDY

G. Tsvetkova 1,* , S. Mihailova 2 , N. Ivanov 2 , M. Ivanova 2 , E. Bekirova 1 , G. Balatzenko 3 , A. Vlahova 4 , E. Hadjiev 1

1 Medical University - Sofia, Faculty of Medicine, Department of Internal diseases, Clinic of clinical hematology; 2 Medical University - Sofia, Faculty of Medicine, Department of Clinical Immunology, University Hospital “Alexandrovska”-Sofia; 3 Laboratory of Cytogenetics and Molecular Biology, National Specialized Hospital for Active Treatment of Hematological Diseases; 4 Medical University - Sofia, Faculty of Medicine, Department of General and Clinical Pathology - Sofia, University Hospital “Alexandrovska”-Sofia, Sofia, Bulgaria

Background: Myeloproliferative neoplasms(MPNs) are a group of disorders with heterogeneous features and often progressive clinical course. Over the past few years a remarkable advance has been made in the knowledge on the pathogenesis and therapeutic management of the Philadelphia(Ph)-negative MPNs. Despite the wide variety of studies in this field, there are still unsolved issues regarding their biology, disease course, prognosis and treatment. The improved recognition of molecular markers is not sufficient for a comprehensive explanation of the variable clinical symptoms and complications among these patients. The role of the chronic inflammation has been widely considered as a co-factor, involved in the pathogenesis and progression of MPNs.

Aims: To analyze the serum levels of a panel of cytokines, associated with the mechanisms of chronic inflammation, among patients with Ph-negative MPNs and healthy controls and the possible correlations of these cytokines with the disease’s features.

Methods: One hundred and fifty adults were included in the study, distributed in comparable groups, as follows: 36 patients with Essential thrombocytemia (ET), 49 patients with Polycythemia vera (PV), 30 patients with Myelofibrosis (MF) and 35 age-matched healthy controls. The patients with MPNs were diagnosed according to the latest WHO criteria. The cytokines’ levels were tested in serum samples with a specific kit Cytokine Human Panel for Luminex™. Assessments of the symptoms burden were performed with MPN-SAF- questionnaire.

Results: Among the 11 tested cytokines we detected significantly higher serum levels of Interleukin-2, Interleukin-5, Interleukin-6, Interleukin-8, Interleukin-18 and GM-CSF among the MF patients (p<0.05), in comparison with the healthy controls, as well as significantly higher levels of Interleukin-6 in the MF-group, compared to the PV-patients. Elevated serum Interleukin-6, Interleukin-8, Interleukin-18 and GM-CSF were observed in the ET-group versus the control group levels (p<0.05). The cytokine profile analysis also revealed higher serum levels of Interleukin-2, Interleukin-5, Interleukin-6 and Interleukin-18 in the patients with PV, than in the healthy subjects. In terms of estimation of the clinical impact of the circulating inflammatory cytokines among the analyzed MPN cohort - for the ET-patients a positive correlation was present between GM-CSF and the fatigue severity and between Interleukin-6 levels and the weight loss severity index, both assessed according to the MPN-SAF score. A positive correlation was detected between Interleukin-10 levels and the bone pain burden and the degree of weight loss for the patients with MF. Interleukin-18 values demonstrated a positive correlation with the weight loss among the patients with PV.

Summary/Conclusion: Our single center study results can give a ground to assume the potential role of the chronic inflammation and the pathogenetic impact of the circulating inflammatory cytokines on the MPNs clinical features and course. The cytokine profile of the different subgroups of MPNs is a topic of interest and can be monitored as a potential predictor of disease progression. It can motivate future research attempts, aiming to alter the clinical evolution of MPNs by affecting the pathogenetic mechanisms of the inflammatory process. It can create prerequisites for future studies regarding more effective therapeutic targets, related to both – the molecular pathways of the mutations in MPNs and the molecular mechanisms of the chronic inflammation.

P1065: CONGENITAL ERYTHROCYTOSIS DUE TO HETEROZYGOUS VARIANTS IN THE BISPHOSPHOGLYCERATE MUTASE GENE

M. J. van Dijk 1,* , B. A. van Oirschot 1 , M. C. Stam-Slob 1 , B. van der Zwaag 1 , E. J. van Beers 1 , J. J. Jans 1 , P. van der Linden 2 , J. M. Torregrosa Diaz 3 , B. Gardie 4,5,6 , F. Girodon 6,7,8 , R. Schots 9 , N. Thielen 10 , R. van Wijk 1

1 University Medical Center Utrecht, Utrecht; 2 Spaarne Gasthuis, Haarlem, Netherlands; 3 University Hospital of Poitiers, Poitiers; 4 L’institut du Thorax, Inserm, CNRS, University of Nantes, Nantes; 5 Ecole Pratique des Hautes Etudes (EPHE), Université Paris Sciences et Lettres; 6 Laboratory of Excellence GR-Ex, Paris; 7 Service d’Hématologie Biologique, Pôle Biologie, Centre Hospitalier Universitaire (CHU) de Dijon; 8 INSERM U1231, Université de Bourgogne, Dijon, France; 9 Universitair Ziekenhuis Brussel, Brussels, Belgium; 10 Diakonessenhuis, Utrecht, Netherlands

Background: Congenital erythrocytosis is an inherited condition characterized by increased red blood cell mass, reflected by persistently elevated hemoglobin (Hb) and hematocrit levels. Erythrocytosis can be primary due to an intrinsic defect of erythropoiesis, or secondary, including germline variants in genes involved in the oxygen-sensing pathway and Hb variants with altered oxygen affinity. A very rare cause involves variants in bisphosphoglyerate mutase ( BPGM ), which regulates the red blood cell concentration of 2,3-bisphosphoglycerate (2,3-BPG), an important modifier of Hb-oxygen affinity.

Aims: We report three European families with erythrocytosis and partial BPGM deficiency due to heterozygosity for variants in the BPGM gene, including one novel variant.

Methods: The study was performed on adults of three families with JAK2 -negative erythrocytosis. DNA analysis concerned sequencing of relevant coding exons, including flanking splice-site consensus sequences, of the eight major genes involved in secondary congenital erythrocytosis ( EPOR, HBB, HBA1, HBA2, VHL, EPAS1, ELGN1, and BPGM ). Identified variants were evaluated using Ensembl, gnomAD, PolyPhen-2, and SIFT. BPGM enzyme activity was determined as described by Beutler. Quantitative analysis of 2,3-BPG was performed using liquid chromatography tandem mass spectrometry. Hb-oxygen affinity, reflected by p50 levels (the oxygen pressure when Hb is 50% saturated with oxygen), was analyzed using the Hemox Analyzer (TCS Scientific).

Results: In the first family (Table 1), a novel missense variant (c.535C>T p.(Arg179Cys) was identified in BPGM . Both the 65-year-old mother with complaints of headaches, and her 39-year-old son with a medical history of retinal vein occlusion were heterozygous for this variant. Allele frequency in gnomAD was 3.98 -5 . The predicted effect ranged from either deleterious (SIFT) to benign (HumVar model of Polyphen-2). Subsequent functional analysis showed low-normal BPGM activity, whereas 2,3-BPG and p50 levels were decreased (Table 1). In the second and third families, three male individuals were heterozygous for the c.269G>A p.(Arg90His) missense variant in BPGM . This variant was only once previously reported, and has an allele frequency of 1.06 -5 . All affected subjects had mild erythrocytosis without any additional clinical features, and showed decreased BPGM activity, decreased 2,3-BPG levels and decreased p50 (Table 1).

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Summary/Conclusion: We describe five individuals from three families in which heterozygosity for missense variants in BPGM is associated with congenital erythrocytosis. Our findings further strengthen the hypothesis of an autosomal dominant inheritance pattern of BPGM variants. Our data also demonstrate the added value of functional analyses to validate gene panel and in silico analysis results for their clinical relevance. Future cases may help to interpret the impact of BPGM variants on hematological and clinical phenotype.

P1066: OUTCOMES OF COVID-19 IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS - AN OBSERVATIONAL COHORT STUDY FROM A LARGE ACADEMIC CANCER CENTER IN THE UNITED STATES

S. Venugopal 1,* , H. Song 2 , C. Young 2 , M. A. Cutherell 2 , J. A. Baganz 2 , S. Zatorsky 2 , S. E. Woodman 3 , N. Pemmaraju 1 , P. Bose 1 , L. Masarova 1 , L. Zhou 1 , S. Pierce 1 , H. Kantarjian 1 , S. Verstovsek 1

1 Leukemia; 2 Data Engineering-Analytics; 3 Genomic Medicine, The University of Texas, MD Anderson Cancer Center, Houston, United States of America

Background: Infections are one of the main causes of morbidity and mortality in patients with Myeloproliferative Neoplasms (MPN). Several studies from all over the world has reported on outcomes of the SARS-CoV-2 infection (COVID-19) pandemic in MPN pts but there is no robust data on characterizing the clinical outcomes of patients with MPN and COVID-19 in the United States (US).

Aims: Therefore, we aimed to investigate the effect of COVID-19 on MPN patients’ clinical outcomes including the effect of MPN-subtypes, and SARS-CoV-2 vaccination on COVID-19 infection.

Methods: We conducted a single center, retrospective, observational cohort study. Between April 2020 – December 2021, data was obtained from 388 patients flagged in the electronic health records system with MPN and COVID-19, of which 53 MPN patients with positive SARS-CoV-2 test were identified. COVID-19 infection was ascertained by a positive real-time reverse transcriptase polymerase chain reaction from nasal swab. Presenting clinical information was abstracted from review of unstructured notes as well as structured data from the foundry. Demographic and baseline parameters were summarized. The Kaplan-Meier method was utilized to compare the median time from date of diagnosis to date of last follow up or death.

Results: Among 53 patients with MPN and COVID-19 infection, the median age was 58 yrs (range,21-83) with equal distribution between men and women of which 46% had a body mass index ≥30. Most common comorbidities included hypertension (60%), and diabetes (19%). 17 (33%) patients required hospitalization of which all (33%) of them required oxygen supplementation. 12 pts (23%) had radiological evidence of pneumonia of which 4 pts (8%) required high flow oxygen and 3 pts (6%) required mechanical ventilation. 2 pts had new thrombosis of portal vein and coronary artery each. 10 pts (19%) received remdesivir and 4 (8%) received tocilizumab, and 9 pts (17%) received steroids. At a median follow up of 11.8 months, 7 pts died at a median of 24 days after COVID-19 diagnosis and the case fatality rate was 13%. Except for 1 pt,all the other 6 pts who died had BMI≥30.

In the US, COVID-19 predominantly occurred in pts with myelofibrosis (56%), polycythemia vera (29%), and essential thrombocythemia (15%). 54% received MPN directed therapy of which 17 (33%) pts received ruxolitinib, and 11 pts (21%) received hydroxyurea. At the time of infection, median WBC was 6.2 x 10 9 /µL (range,0.3-16.3),Hb was 11.6 gm/dL (range,6.8-15.8),platelet count was 178 x 10 9 /µL (range,2-766), and the median neutrophil to lymphocyte ratio was 4.1. In this cohort, 22 pts (42%) were infected prior to the availability of vaccination. 42% of the entire cohort has received vaccination of which 45% pts were infected after first dose of vaccination. Among the 7 deaths, 6 patients had underlying myelofibrosis, and one pt had polycythemia vera, and all deaths occurred in unvaccinated population except for one death that occurred post vaccination. Hospitalization rate was higher in the unvaccinated (33%) pts than those who were vaccinated (22%).

Summary/Conclusion: To date this is the largest cohort of MPN patients with COVID-19 infection in the US accounting for 14% of the MPN patients with COVID-19 in our center. BMI≥30 appear to be an important risk factor for COVID-19 infection and mortality in MPN pts. SARSCOV2-vaccination appear to decrease the risk of mortality and hospitalization. Patterns of inflammatory markers, and long term follow up of recovered MPN pts with COVID-19 will be presented in the meeting.

P1067: PHASE 2 STUDY OF ORAL THALIDOMIDE-CYCLOPHOSPHAMIDE-DEXAMETHASONE FOR RECURRENT/REFRACTORY ADULT LANGERHANS CELL HISTIOCYTOSIS

J.-N. Wang 1,* , T. Liu 1 , A.-L. Zhao 1 , B.-J. Pan 2 , J. Sun 2 , J. Li 1 , D.-B. Zhou 1 , M.-H. Duan 1 , X.-X. Cao 1

1 Department of Hematology; 2 Department of Pathology, Peking Union Medical College Hospital, Beijing, China

Background: Langerhans cell histiocytosis (LCH) is a clonal histiocytic neoplasm with various clinical manifestations and heterogeneous prognoses. No standard therapy is currently available for adults with recurrent/refractory LCH.

Aims: To determine the efficacy and safety of oral thalidomide combined with cyclophosphamide and dexamethasone (TCD) regimens in the treatment of recurrent/refractory LCH among adult patients.

Methods: This single-center, single-arm, phase 2 study enrolled 32 recurrent/refractory LCH patients between October 2019 and August 2021. The final follow-up date was October 31 st , 2021.The TCD regimen (thalidomide 100 mg daily, cyclophosphamide 300 mg/m 2 Day 1, 8, 15, and dexamethasone 40 mg Day 1, 8, 15, 22 every 4 weeks) was administered for 12 cycles and then thalidomide alone as maintenance for 12 months. The primary endpoint was event-free survival (EFS). Events were defined as progression during or after TCD therapy or death from any cause.

Results: The median number of prior lines of systemic treatment was 1 (range 1-4), and all patients received a cytarabine-based regimen as first-line or second-line therapy. After a median follow-up of 22 months (range 5-24 months), no patient died of all causes. The overall response rate was 87.5%, including 18 patients (56.3%) achieving complete remission and 10 patients (31.3%) as partial remission. The estimated 24-month EFS was 64.0%. Fourteen patients (43.7%) had risk organ involvement. Patients with risk organ involvement had a similar EFS compared to patients without risk organ involvement ( P =0.38). The common toxicities of the TCD regimen include grade 1-2 constipation (12.5%), grade 1-2 tiredness (9.4%) and grade 2 peripheral neuropathy (12.5%).

Summary/Conclusion: Oral thalidomide, cyclophosphamide and dexamethasone are effective and safe regimen for recurrent/refractory LCH patients, particularly for patients with risk organ involvement.

P1068: RISK-ADJUSTED SAFETY ANALYSIS OF PACRITINIB IN PATIENTS WITH MYELOFIBROSIS

A. Vannucchi 1 , N. Pemmaraju 2,* , B. Scott 3 , M. Savona 4 , S. Oh 5 , F. Palandri 6 , H. K. Al-Ali 7 , M. Sobas 8 , M. F. McMullin 9 , V. Gupta 10 , A. Yacoub 11 , R. Mesa 12 , S. Buckley 13 , K. Roman-Torres 13 , S. Verstovsek 2 , C. Harrison 14

1 Azienda Ospedaliera Universitaria Careggi, University of Florence, Florence, Italy; 2 The University of Texas MD Anderson Cancer Center, Houston, TX; 3 Fred Hutchinson Cancer Research Center, Seattle, WA; 4 Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN; 5 Washington University School of Medicine, St. Louis, MO, United States of America; 6 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy; 7 Krukenberg Cancer Center, University Hospital Halle, Halle, Germany; 8 Wroclaw Medical University, Wroclaw, Poland; 9 Queen’s University Belfast, Belfast, United Kingdom; 10 Princess Margaret Cancer Centre, Toronto, ON, Canada; 11 The University of Kansas Cancer Center, Kansas City, KS; 12 UT Health San Antonio Cancer Center, San Antonio, TX; 13 CTI BioPharma, Seattle, WA, United States of America; 14 Guy’s and St Thomas’ NHS Trust, London, United Kingdom

Background: Pacritinib is a novel JAK2/IRAK1 inhibitor that has shown clinically significant activity in patients with myelofibrosis, including those with platelet counts <50 x 10 9 /L. Recently, JAK inhibitors have come under increased scrutiny due to specific, emerging toxicities with drugs in this class.

Aims: This safety analysis focuses on these toxicities of interest for patients treated with pacritinib 200 mg twice daily (BID) and best available therapy (BAT), including ruxolitinib, on the phase 3 PERSIST-2 and phase 2 PAC203 studies. Data are presented as risk-adjusted incidences to account for differential time at risk for adverse events (AEs) between arms due to cross-over.

Methods: Patients treated with pacritinib 200 mg BID on PERSIST-2 and PAC203, and those treated with BAT, including ruxolitinib on PERSIST-2, were included. Risk-adjusted AEs, representing event rate per 100 patient-years, were calculated for the following: overall and fatal AEs, bleeding AEs (determined by Standardized Medical Dictionary for Regulatory Activities Query [SMQ]), cardiac AEs (by SMQ), major cardiac events (determined by major adverse cardiovascular events [MACE] classification), infections, thromboses, and secondary malignancies.

Results: A total of 160 patients were analyzed as the pooled pacritinib 200 mg BID group (n=106 in PERSIST-2; n=54 in PAC203) and 98 patients in the BAT group (44 on ruxolitinib). At baseline, the median platelet count was 57 x 10 9 /L for the pooled pacritinib group and BAT group; hemoglobin was 9.2 vs 9.7 g/dL and 66% vs 53% had prior JAK2 inhibitor therapy respectively.

The rate of AEs was higher on pacritinib versus BAT, while the rate of fatal AEs was lower (Table 1). Both bleeding and cardiac events occurred at slightly lower rates on pacritinib compared to BAT. There were no MACE for pacritinib, whereas there were for BAT, including in patients treated with ruxolitinib. Malignant neoplasms (excluding worsening myelofibrosis and leukemia) occurred at similar rates on pacritinib and BAT, though rate of non-melanoma skin cancers was lower in the pooled pacritinib group (3/100 patient-years) versus BAT (7/100 patient-years), including ruxolitinib (11/100 patient-years). Infection occurred more frequently on pacritinib versus BAT, though fungal and viral infections occurred less frequently, as did Herpes Zoster reactivation (pooled pacritinib: 0/100 patient-years vs BAT: 2.4/100 patient-years, including ruxolitinib: 5.5/100 patient-years). Thrombosis occurred at similar rates on pacritinib and BAT.

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Summary/Conclusion: Risk-adjusted analysis demonstrates that the safety profile of pacritinib 200 mg BID is comparable or superior to BAT, including ruxolitinib. Pacritinib 200 mg BID may represent a full-dose therapeutic option for patients with myelofibrosis, including those with thrombocytopenia.

P1069: RETROSPECTIVE COMPARISON OF PATIENT OUTCOMES ON PACRITINIB VERSUS RUXOLITINIB IN PATIENTS WITH MYELOFIBROSIS AND THROMBOCYTOPENIA

C. Harrison 1,* , P. Bose 2 , R. Mesa 3 , A. Gerds 4 , S. Oh 5 , J.-J. Kiladjian 6 , V. García-Gutierrez 7 , A. Vannucchi 8 , C. Scheid 9 , M. Sobas 10 , S. Verstovsek 2 , S. Buckley 11 , K. Roman-Torres 11 , J. Mascarenhas 12

1 Guy’s and St Thomas’ NHS Trust, London, United Kingdom; 2 The University of Texas MD Anderson Cancer Center, Houston, TX; 3 UT Health San Antonio Cancer Center, San Antonio, TX; 4 Cleveland Clinic Taussig Cancer Institute, Cleveland, OH; 5 Washington University School of Medicine, St. Louis, MO, United States of America; 6 Hôpital Saint-Louis, Université de Paris, Paris, France; 7 Hospital Universitario Ramón y Cajal, Madrid, Spain; 8 Azienda Ospedaliera Universitaria Careggi, University of Florence, Florence, Italy; 9 University of Cologne, Cologne, Germany; 10 Wroclaw Medical University, Wroclaw, Poland; 11 CTI BioPharma, Seattle, WA; 12 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America

Background: Pacritinib is a JAK2/IRAK1 inhibitor for patients with myelofibrosis and thrombocytopenia. Unlike the JAK1/2 inhibitor ruxolitinib, which is used at lower doses in patients with thrombocytopenia, pacritinib has been studied at full dose regardless of platelet count.

Aims: We retrospectively analyzed the phase 3 PERSIST-2 study, comparing pacritinib with ruxolitinib, with a focus on disease symptomatology.

Methods: PERSIST-2 enrolled patients with platelet counts ≤100 x 10 9 /L and randomized them 1:1:1 to pacritinib 200 mg twice daily (BID), pacritinib 400 mg once daily (QD), or best available therapy (BAT). This analysis focused on the 200-mg BID dose and patients who received ruxolitinib prior to week 24. Endpoints included the percentage of patients with ≥35% spleen volume reduction (SVR), ≥50% modified total symptom score (mTSS) reduction, and improvement in disease symptoms measured by Patient Global Impression of Change (PGIC). Safety analyses were based on all treated patients; efficacy analyses were based on the intention-to-treat population randomized at least 22 weeks prior to study end. The Fisher Exact test was used to describe differences in response. Logistic regression was used to adjust for differences in baseline characteristics.

Results: Safety analysis included 106 patients on pacritinib and 44 on ruxolitinib; efficacy analysis included 74 on pacritinib and 32 on ruxolitinib (median daily dose of 10 mg). Baseline characteristics were similar between the groups, including median platelet count (55 vs 61 x 10 9 /L) and percentage receiving RBC transfusion (46% vs 43%). The following differences in baseline characteristics were accounted for in multivariable modeling: percentage of patients with grade 3 fibrosis (58% vs 36%), primary myelofibrosis (77% vs 50%), ≥1% peripheral blasts (45% vs 61%), and prior JAK2 inhibitor (48% vs 73%).

Patients treated with pacritinib vs ruxolitinib achieved higher rates of SVR (22% vs 3%, p=0.02) and numerically higher rates of mTSS response (35% vs 19%; p=0.11) at week 24 (Figure 1 A ). A greater percentage of patients on pacritinib reported “much” or “very much” improved symptoms (35% vs 16%, p=0.06; Figure 1 A ). Among ruxolitinib-treated patients with an available PGIC measure at week 24, 50% reported either no improvement or worsening symptoms, while 76% of pacritinib-treated patients reported improvement (Figure 1 B ). After adjusting for imbalances in baseline characteristics, there was no diminution of treatment effect on SVR or mTSS, and the hazard ratio for survival on pacritinib vs ruxolitinib was 0.46 [95% CI: 0.15-1.43].

Fatal adverse events occurred at slightly lower rates on pacritinib vs ruxolitinib (8% vs 11%). Bleeding occurred at similar rates on pacritinib and ruxolitinib (43% vs 41%). There were low rates of herpes zoster reactivation (n=0 vs 1), fungal skin infection (n=0 vs 1), pulmonary aspergillosis (n=1 vs 0), deep venous thrombosis (n=0 vs 1), and pulmonary embolism (n=1 vs 0) on pacritinib and ruxolitinib, respectively.

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Summary/Conclusion: Pacritinib yielded higher response rates and a similar safety profile to lower-dose ruxolitinib in patients with myelofibrosis who have moderate or severe thrombocytopenia.

P1070: NAVITOCLAX MONOTHERAPY IN PATIENTS WITH MYELOFIBROSIS PREVIOUSLY TREATED WITH JAK-2 INHIBITORS: SAFETY AND TOLERABILITY

V. Pullarkat 1 , A. Cruz-Chacon 2 , S. Gangatharan 3 , A. Melnyk 4 , G. A. Palumbo 5 , M. Bellini 6 , S. K. Tantravahi 7 , Q. Qin 8 , J. Potluri 8 , P. Vachhani 9,*

1 Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, CA, United States of America; 2 Hospital Centro Comprensivo de Cancer Universidad de Puerto Rico, San Juan, Puerto Rico; 3 Department of Haematology, Fiona Stanley Hospital, University of Western Australia, Perth, Western Australia, Australia; 4 Texas Oncology-Abilene, Abilene, TX, United States of America; 5 Dipartimento di Science Mediche Chirurgiche e Tecnologie Avanzate “GF Ingrassia”, Università degli Studi di Catania, Catania; 6 ASST Papa Giovanni XXIII, Bergamo, Italy; 7 University of Utah and Huntsman Cancer Institute, Salt Lake City, UT; 8 AbbVie Inc., North Chicago, IL; 9 O’Neal Comprehensive Cancer Center at UAB, Birmingham, AL, United States of America

Background: Navitoclax is an oral, small-molecule inhibitor of anti-apoptotic B-cell lymphoma 2 family proteins (BCL-X L , BCL-2, BCL-W) that is being evaluated in the ongoing, multicenter, multi-cohort, phase 2 trial (REFINE; NCT03222609 ). Previous results from REFINE Cohort 1a indicated that addition of navitoclax to ruxolitinib resulted in clinically meaningful outcomes with acceptable safety profile in patients with myelofibrosis (MF) with progression or suboptimal response to ruxolitinib monotherapy (Pemmaraju et al. J Clin Oncol . 2022).

Aims: To report preliminary safety results of navitoclax monotherapy (Cohort 2) in patients with suboptimal response to prior Janus kinase inhibitors (JAKis).

Methods: This phase 2, open-label trial in patients with MF enrolled patients into 4 cohorts according to JAKi experience; all patients provided informed consent. Patients in Cohort 2 had discontinued prior JAKi therapy and received navitoclax monotherapy orally at the starting dose of 100 mg/day (QD) or 200 mg QD if baseline platelet count was 75–150 × 10 9 /L or >150 × 10 9 /L, respectively. Eligible patients had previously received JAKi for ≥12 weeks, or ≥28 days with red blood cell transfusion dependence (≥2 units/month for 2 months) or with ≥ grade 3 adverse event (AE) of thrombocytopenia or anemia, while on JAKi; all patients had splenomegaly. The primary endpoint was spleen volume reduction of ≥35% (SVR 35 ) from baseline (BL) assessed centrally at Week 24. Secondary endpoints included change in grade of bone marrow fibrosis according to the European consensus grading system and anemia response as per International Working Group criteria. Exploratory endpoints included duration of response of SVR 35 and overall survival. Safety and AEs were monitored throughout the study.

Results: As of Oct 4, 2021, 30 patients were enrolled and received navitoclax. A majority of patients were male (63%), median age was 68 years (range 55–84), with a median prior ruxolitinib exposure of 100 weeks (range 11–496). Fifteen patients (50%) had secondary MF, of which 8 patients (27%) had post–polycythemia vera MF and 7 patients (23%) had post-essential thrombocytopenia MF ( Table ). Fourteen patients (47%) started navitoclax at a dose of 100 mg QD and 16 patients (53%) started at 200 mg QD. The median follow-up time was 4.2 months (range 0.2–15.5). Median duration of navitoclax exposure was 9.4 weeks (range 0.1–67.1). Twenty-seven patients (90%) experienced ≥1 AE, the most common being: thrombocytopenia (n=16; 53%), diarrhoea (n=9; 30%), and nausea (n=8; 27%). Grade ≥3 AEs were experienced by 63% of patients, with thrombocytopenia (n=11; 37%) and anemia (n=7; 23%) being the most common. Three patients had serious AEs of dyspnea, hypoxia, and pulmonary hypertension. Navitoclax dose reductions and interruptions were experienced by 15 patients (50%) and 16 patients (53%), respectively. Three patients experienced AEs leading to navitoclax discontinuation (pulmonary hypertension, increased bilirubin, and low platelets; n=1 each). Eight patients discontinued navitoclax due to: AE (n=3), consent withdrawal (n=1), physician decision (n=1), disease relapse (n=1), and progressive disease (n=2). Two patients died >30 days after the last dose of navitoclax. Details will be included in the presentation.

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Summary/Conclusion: Navitoclax monotherapy in patients with MF after prior JAKi had a similar safety profile as previously reported in Cohort 1a. Efficacy analyses are underway to evaluate the activity of navitoclax monotherapy in patients with MF and will be available for the presentation.

P1071: IMPACT OF ACETYLSALICYLIC ACID DOSAGE ON INCIDENCE OF THROMBOSIS AND MORTALITY IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS.

A. Wild 1,2,* , S. Wimmerová 3

1 Haematology department, Teaching F.D.Roosevelt Hospital, Banská Bystrica; 2 Medical faculty; 3 Public Health Faculty, Slovak Medical University, Bratislava, Slovakia

Background: Bcr/abl – negative myeloproliferative neoplasms (MPN) are associated with a higher risk of thrombosis and higher vascular mortality. Acetylsalicylic acid (ASA) used in prevention decreases risk of vascular events in patients with MPN. There is some evidence that ASA in dose 100 mg once daily results in incomplete inhibition of a platelet function over the whole 24-hour interval in most patients with essential thrombocythemia (ET). High on-treatment reactivity (HOTR) was corrected with dosing ASA twice daily. Evidence of clinical consequences of these findings has been missing.

Aims: Authors compared thrombosis incidence, mortality and safety among cohorts of MPN patients with adequate on-treatment reactivity (AOTR) on ASA dosed once daily, twice daily and patients with HOTR.

Methods: Response to ASA in patients with MPN was evaluated by a platelet light transmission aggregometry induced by arachidonic acid. AOTR was defined as maximal amplitude up to 20% just before the next dose of ASA. In patients with HOTR, ASA dosing was changed to twice daily with a dose from 50 mg step by step up to 150 mg till AOTR was reached. A few patients didn´t change their doses at their own decision despite HOTR. The patients were regularly followed-up. Incidence of thrombosis, adverse events and mortality were compared among the cohorts.

Results: The platelet aggregation was tested in 69 patients with MPN on ASA. Patients with ET, polycythemia vera (PV), primary myelofibrosis, post-PV plus post-ET myelofibrosis and MPN not otherwise specified presented 16, 42, 36, 5 and 1%, respectively. The adequate responses (AOTR) were found in 57 (83%), cohort 1. The insufficient responses were found in 12 persons (17%), in 30% of patients on an enterosolvent form of ASA. In these 12 patients the dose of ASA was changed to 2x50mg in 4 and to 2x100mg in 1 patient (cohort 2) and remained unchanged in seven (cohort 3). There were no differences in frequency of risk factors besides higher frequency of diabetes mellitus in group 3 compared to group 1 (p=0,021). Non-adherence to regular administration of ASA was found in 10% of patients.

The follow-up was 36,8 months. All-cause mortality was higher in the cohort 3 compared to the cohort 1 (p=0,009), see fig. 1, but thrombotic episodes incidence not. The cohort 2 had not different incidence of both thrombotic episodes and deaths compared to the cohort 1. No proximal gastrointestinal bleeding and other gastropathies occurred. There was no bleeding episode on ASA in dose of 2x50mg daily. A non-severe bleeding dependant on any ASA dose were found.

Repeated measurement of platelet aggregation was done in 29 patients in median 49,5 months after previous measurements. There was no statistically significant difference of adequate/high platelet reactivity on ASA between the first and the last measurement.

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Summary/Conclusion: The frequency of MPN patients with higher platelet reactivity on ASA was lower than in the most data in literature. Correction of an ASA dosage to twice daily reaching AOTR resulted in lower mortality. Other studies are required to confirm these data. Initial dose 2x50mg seems to be effective and safer than higher dosage. The long-term reproducibility of platelet aggregation testing results was preserved.

P1072: NATURAL HISTORY OF JUVENILE MYELOMONOCYTIC LEUKEMIA

W. Yang 1,* , X. Zhu 1

1 Pediatric department, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical Colleg, Tianjin, China

Background: Juvenile Myelomonocytic leukemia (JMML) is a rare clonal disease that occurs in infants and young children, and most patients respond poorly to chemotherapy. Hematopoietic stem cell transplantation (HSCT) is the only way to cure this disease. The clinical heterogeneity of JMML is large, and the disease progresses rapidly in most patients, whereas a few patients progress slowly and survive for long periods without treatment.

Aims: This study retrospectively analyzed clinical data of untreated JMML patients from our department. Explore their natural course of disease and prognosis factors and provide clinical research data for screening the prognostic factors for natural history of JMML.

Methods: Patients who were diagnosed JMML from January 2008 to December 2021 in our center were included in this study. The clinical characteristics, bone marrow morphology, cytogenetics, mutated genes, hypersensitivity of GM-CSF, abdominal ultrasound and other laboratory test results were retrospectively analyzed.

Results: In this study，88 patients were diagnosed JMML from 2008 till now in our single institute, 44 cases (50%) without treatment (including chemotherapy and HSCT) in the whole course. The median age of onset was 13 months (range 2-48), and the incidence was mostly male, with the male to female ratio =32: 12, median time between onset and diagnosis was 2 (0.3-36.0) months. Among them, 15 cases (34.1%) had simple abnormal blood image as chief complaint, and 17 cases (38.6%) had non-specific rash on physical examination. Lymphadenopathy was found in 23 cases (52.3%) with a median subcostal value of 3.9 (0-11) cm in liver and 5.4 (1-16) cm in spleen. The median WBC count at initial diagnosis was 28.2 (range 9.1-128.6) ×10 9 /L, and the hemoglobin value was 88.8 (range 52.0-153.0) g/L. Platelet count(PLT) was 44.0 (range 1.0-389.0) ×10 9 /L, absolute value of monocyte was 4.5 (1.3-30.5) ×10 9 /L, absolute value of median lymphocyte was 8.2 (2.4-48.7) ×10 9 /L, and median fetal hemoglobin was 10 (0-88) %. Thirty-four patients underwent cytogenetic examination, of which 31 (91.2%) had normal karyotype and 3 patients had chromatid 7. Thirty patients underwent gM-CSF hypersensitive examination, with a median CFU-GM-3 (0-73) BMMNC. JMML mutations were detected in 36 children, including 6 patients with second-generation sequencing, and 30 patients with first-generation sequencing JMML hotspot mutations, including 13 (36.1%) PTPN11 mutations, 4 (11.1%) KRAS mutations, 9 (25.0%) NRAS mutations, and 1 (2.8%) NF1 mutations. One (2.8%) had mutations of NRAS and PTPN11, and 8 (22.2%) had no known JMML mutants. The study was followed up to 2022.1, with a median follow-up time of 20.6 (range 1-132) months. Among the 44 patients, 8 cases (18.2%) were lost to follow-up, 28 cases (63.6%) died, and 8 cases (18.2%) survived. To explore the influence of clinical characteristics and laboratory test results on patients’ OS, the study found that PLT < 40×10 9 /L, spleen subcostal > 5 cm and PTPN11 were important adverse factors affecting OS. The 5-year overall survival rate of 44 children was 40%, and the 5-year overall survival rate of those with PTPN11 mutation was significantly lower than that of other mutation gene types.

Summary/Conclusion: The natural history of JMML was significantly different among different mutated gene types, among which PLT, spleen size and PTPN11 mutated gene type were independent prognostic factors affecting the natural course of JMML.

P1073: MUTATIONAL LANDSCAPE AND CLINICAL OUTCOMES OF PATIENTS WITH MYELOID AND LYMPHOID NEOPLASMS WITH EOSINOPHILIA (MLN-EOS) AND ABNORMALITIES OF PDGFRA, PDGFRB, FGFR1, FLT3 AND JAK REARRANGEMENT

Y. Zhang 1,* , L. Nguyen 2 , C. Lu 3 , E. Wang 4 , M. Lauw 4 , S. Ball 1 , N. Dong 1 , L. Moscinski 5 , O. Chan 6 , S. Yun 6 , D. Sallman 6 , L. Sokol 6 , B. Shah 6 , J. Lancet 6 , R. Komrokji 6 , A. Kuykendall 6 , E. Padron 6 , L. Zhang 6

1 Moffitt Cancer Center; 2 Department of Pathology, James A Haley Veterans’ Hospital, Tampa; 3 Department of Pathology, Duke University School of Medicine, Durham; 4 Department of Pathology, University of California San Francisco, San Francisco; 5 Department of Pathology; 6 Department of Hematologic Malignancies, Moffitt Cancer Center, Tampa, United States of America

Background: To recognize the growing list of recurrent genetically defined eosinophilia driven by constitutively active tyrosine kinase fusion genes, WHO created a provisional entity of myeloid/lymphoid neoplasms with eosinophilia (MLN-Eo) with rearrangement of PDGFRA , PDGFRB, FGFR1 , or PCM1- JAK2.

Aims: We aim to describe the mutational landscape and outcome with upfront targeted tyrosine kinase inhibitor (TKI) therapy in patients (pts) with MLN-Eo.

Methods: We retrospectively reviewed clinical and molecular data on ptss with MLN-Eos with PDGFRA , PDGFRB , FGFR1 , JAK2 or FLT3 rearrangement at Moffitt Cancer Center, Duke Cancer Center, and UCSF Comprehensive Cancer Center. The next generation sequencing of the bone marrow or peripheral blood was either done using our in-house targeted 54 gene myeloid panel or the commercially available panel, FoundationOne TM Heme Genomic profile.

Pts were divided into two categories based on morphology at diagnosis: chronic and blastic phase disease. Median time to blastic phase and overall survival (mOS) were calculated using Kaplan Meier method and compared with log-rank test. Hazard ratio was calculated using the Cox proportional hazards (PH) model.

Results: A total of 41 pts were included in the analysis. Thirteen patients had available comprehensive NGS results. 23 pts had PDGFRA fusion (21) or activating mutations (2); 4 had PDGFRB ; 8 had FGFR1 ; 2 had JAK2 , PCM-JAK2 , and BCR-JAK2 ; 4 had FLT3 , ETV6-FLT3 (3) and FLT3-TRIPP (1). Twelve (92%) patients had at least one other coexisting mutation. The mutations in at least two patients included TET2 , RUNX1 , TP53 , and PTPN11 (Fig 1). Pathogenic PTPN11 mutation was present in all activating PDGFRA mutation but absent in other cases.

Among 23 patients in chronic-phase disease 14 patients (61%) received the corresponding TKI to their fusion in the frontline setting. None transformed into blastic-phase disease. Six patients in the de novo blastic phase received the upfront TKI alone or combined with chemotherapy. All achieved complete remission (CR) and remained alive at a median follow-up of 44 months.

In the OS analysis, upfront TKI use was associated with longer survival (HR 0.065; 95%CI, 0.008-0.50, p=0.008) in univariate analysis. The improved OS emained significant (HR 0.043, 95% CI 0.003-0.63, p =0.021) in multivariate analysis when adjusted for sex, age, hypereosinophilia in the blood, PDGFRA/B fusion status, complex cytogenetics at diagnosis, the blastic-phase at diagnosis, and alloHSCT.

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Summary/Conclusion: Our study showed the high frequency of secondary somatic mutations in MLN-eos and described the mutational landscape similar to other myeloid malignancies. Targeted TKI therapies in the upfront setting are associated with excellent outcomes in patients with MLN-Eos. The elucidation of potential mechanisms needs further exploration.

P1074: PRIMARY REFRACTORY HODGKIN’S LYMPHOMA: A MOROCCAN MONOCENTRIC RETROSPECTIVE STUDY

M. Ababou 1,* , A. Hammani 1 , A. Siham 1 , W. Elfaria 1 , E. M. Mahtat 1 , S. Jennane 1 , H. EL Maaroufi 1 , K. Doghmi 1

1 Clinical Hematology, Military hospital Mohammed v, Rabat, Morocco, Rabat, Morocco

Background: Primary refractory Hodgkin’s lymphoma (PRHL)is defined as disease progression during initial first-line therapy or early relapse of less than 3 months [1], this entity is a therapeutic challenge for hematologists, as the response rate to second-line therapy remains lower (51% vs. 83%, P < 0.0001)[2]

Aims: Compare different parameters between patients with PRHL and patients with non-PRHL to reveal factors that could predict the primary refractory character of our patients.

Evaluate the evolution in terms of therapeutic response and survival of our patients with PRHL compared to non-PRHL

Methods: This is a monocentric retrospective descriptive study that collected all the files of patients followed for Hodgkin Lymphoma at the clinical hematology department of the Military Hospital of Instruction Mohammed V in Rabat between January 2010 and December 2020. Their epidemiological, clinical and prognostic characteristics were recorded, and the efficacy and tolerance of the therapies used were evaluated. We used the t-student, Mann-Whitney, χ2 tests (or Fisher’s exact test) to compare the differences between PRHL and non-PRHL patients. To assess the difference in survival between the two groups, we used the Log-rank test on Kaplan-Meier survival curves.Treatment outcomes were measured in terms of response rate, overall survival (OS) and progression-free survival (PFS). Statistical analyses were performed using JAMOVI software (version 1.6)

Results: Classical Hodgkin lymphoma was diagnosed in 122 patients. 27 patients were primary refractory (22.1%). Our comparison between the group of PRHL and non-PRHL patients in terms of age, sex, time to consultation, performance status (PS) score, clinical signs at admission, histological subtype, presence of Bulky mass on chest X-ray, the Ann Arbor stage and the prognostic risk group did not show any significant difference, which makes the identification of primary refractory patients in pre-therapy a difficult challenge. In our serie 83(68%) patients were re-evaluated at 2 courses by PET-Scan (Positron Emission Tomography, 45(54.3%) patients were in Complete Remission (CR), 34(41%) in Partial Remission (PR). Comparing PRHL and non-PRHL patients according to the response at 2 courses, a highly significant difference was observed (p=0.001). Using a univariate logistic regression model, we found that a PR at 2 courses was more likely to be PRHL than a complete response (OR= 4.95; CI95% [1.55-15.78]; p=0.007). At the end of first-line treatment, 77.1% of all patients achieved complete remission, 11.5% achieved partial response, 1 patient had a stable response and 10.6% were in progression. The overall response at the end of treatment for PRHL patients was 44.4% vs 93.6% for the non-PRHL group (p<0.001). 10.7% of patients relapsed within a median of 23 months. With a median follow-up of 65.5 months, the OS and PFS of all patients at 5 years were 91.2% and 90.6% respectively. Comparing primary refractory and non-refractory patients, the OS at 5 years was 73% [56.2-94.8] vs 96.5% [92.7-100] (p<0.0001) (Figure 1).

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Summary/Conclusion: Primary refractory Hodgkin’s lymphoma remains a form with a poor prognosis, the overall 5-year survival of this form in our series is 73% compared to 96.5% for the non-PRHL patients. PET-2 may be a predictive step for this form, studies are needed to identify the place of immunotherapy in the management of these patients

P1075: EVALUATION OF CYTOKIN GENE POLYMOPHISMS AND GENE EXPRESSIONS IN PATIENTS WITH HODGKIN LYMPHOMA

H. Haydaroglu Sahin 1,* , D. D. Demirbas 1 , D. M. Akkurd 1 , V. Okan 1

1 Haematology Department, Gaziantep University Sahinbey Research and Practice Hospital, Gaziantep, Turkey

Background: In Hodgkin Lymphoma (HL), Reed-Sternberg (RS) cells play a role in the etiopathogenesis and clinical course of the disease and that is thought to be the result of expression of cytokines.

Aims: In this study; it was aimed to evaluate the role of gene polymorphisms and gene expression of five cytokines (tumour necrosis factor alfa (TNF-α), transforming growth factor beta-1 (TGF-β1), interferon gamma (IFN-γ), interleukin-6 (IL-6), and interleukin-10 (IL-10)) in the etiopathogenesis of the disease, clinical parameters and prognosis in HL patients.

Methods: A total of 70 patients and 70 control group who were diagnosed, treated and followed up for HL in the Hematology clinic of Gaziantep University Faculty of Medicine were included in the study. Cytokine gene polymorphisms and expression levels were analyzed by DNA isolation from peripheral blood of the patient and control groups and by using of Sequence Specific Primary polymerase chain reaction (PCR-SSP) method.

Results: TNF-α (-308) gene variant GA genotype (high expression), IFN-γ (+874) gene variant TT genotype (high expression), IL6 (-174) gene variant GG genotype (high expression), TGF-β1 (T10/C10) gene variant TT genotype, TGF-β1 (C25/G25) gene variant GC genotype, IL10 (-1082) gene variantGG genotype, IL10 (-819) gene variantCC genotype and IL10 (-592) gene variantCC genotype was significantly higher in HL patients compared to healthy controls (respectively; OR= 0.3636, p= 0.0259; OR= 0.3164, p= 0.0349, OR= 0.4189, p = 0.0299, OR= 0.4329, p= 0.0257, OR= 0.4889, p<0.001, OR= 0.4189, p= 0.0299, OR= 0.3951, p= 0.0193, OR= 0.3951, p= 0.0193). While IL6 (-174) G allele (p = 0.0302), TGF-β1 (T10 / C10) T allele (p = 0.0224), IL10 (-1082) G allele (p = 0.0232), IL10 (-819) C allele (p = 0.0156), IL10 (-592) C allele (p = 00156) was higher in HL patients compared to the control group, expression of IL6 (-174) C allele (p = 0.0302), TGF-β1 (T10 / C10) C allele (p = 0.0224), IL10 (-1082) A allele (p = 0.0232), IL10 (-819) T allele, IL10 (-592) A allele (p = 0.0156) was found to be low. In haplotype analysis; TGF-β1 (T / C10, C / G25) gene “TCGC, CCGG, TTGC” (moderately increased plasma TGF-β1 level) and IL10 (-1082, -819, -592) gene “GCC GCC” (high IL10 plasma level) haplotype variants was significantly higher in HL patients than in the control group (respectively; OR= 0.3406, p= 0.0161; OR= 0.4149, p= 0.0299).In multivariate analysis; Bone marrow involvement (p <0.001) and carrier of IL6 (-174) gene polymorphism CC variant genotype (p= 0.02) were detected as an independent risk factor associated with poor prognosis on 5-year PFS.

Summary/Conclusion: The results of the study suggests that TNF-α (-308) GA genotype, IFN-γ (+874) TT genotype, IL6 (-174) GG genotype, TGF-β1 (T10 / C10) TT genotype, TGF-β1 (C25 / G25) GC genotype, IL10 (-1082) GG genotype, IL10 (-819) CC genotype, IL10 (-592) CC genotype, TGF-β1 (T / C10, C / G25) gene “TCGC, CCGG, TTGC” and IL10 (-1082, -819, -592) gene “GCC GCC” haplotype may be a risk factor for HL development.

P1076: A RETROSPECTIVE ANALYSIS ON OLD AND EMERGING PROGNOSTIC FACTORS IN CLASSICAL HODGKIN’S LYMPHOMA IN THE PET-GUIDED ERA

A. Cellini 1,* , C. A. Cavarretta 1 , S. Pravato 1 , M. Pizzi 2 , M. Gregianin 3 , F. Crimì 4 , F. Piazza 1 , L. Trentin 1 , A. Visentin 1

1 Hematology and Clinical Immunology Unit, Department of Medicine; 2 Surgical Pathology and Cytopathology Unit, Department of Medicine, University of Padua, Padua; 3 Nuclear Medicine Unit, Veneto Institute of Oncology IOV-IRCCS, Castelfranco; 4 Institute of Radiology, Department of Medicine, University of Padua, Padua, Italy

Background:: Hodgkin’s Lymphoma (HL) has been commonly recognized as a chemo-sensitive and potentially curable neoplastic disease. However, some patients are refractory or relapse after frontline therapy, and their prognosis is severely impacted by such events. Baseline prognostic factors that can reliably identify patients with high-risk disease are currently lacking, as classical prognostic factors have lost their meaning in the modern era, where a PET-guided approach and novel therapies are being incorporated in the treatment of HL. Such therapeutic options, however, need to be directed to the right subset of patients to avoid overtreatment, unwanted toxic effects, or financial burden.

In recent years lymphocyte to monocyte ratio (LMR) and neutrophil to lymphocyte ratio (NLR) have been evaluated as potential outcome predictors in patients affected by HL. These factors, however, need to be further evaluated to be included in prognostic scores that reliably predict outcomes in HL patients treated in the current era.

Aims: This study’s aim was to identify potential predictors of worse overall survival (OS) and progression free survival (PFS) in patients affected by HL treated with a PET-adapted chemotherapeutic approach.

Methods: We included HL patients diagnosed at the University Hospital of Padova between 2004 and 2020. All patients were treated with ABVD, were staged with PET-TC at baseline, and evaluated after 2 cycles (interim, iPET) and at the end of therapy. iPET positive patients (Deauville score 4-5) were intensified with escalated BEACOPP. The number of cycles and the addition of radiation therapy as mandated by stage and local policy. Low LMR was considered as ≤2.1 and high NLR as ≥6. Survival curves were compared with Log-rank test, Cox proportional hazards test was used for multivariate analysis. 95% Confidence intervals (CI) were also reported.

Results: Of the 261 patients analyzed, 101 (39%) had early-stage and 160 (61%) had advanced-stage (IIB or higher) HL. Median age was 32 years, 127 (49%) of patients were male, 134 (51%) presented with B-symptoms and 91 (35%) had bulky disease.

After a median follow up of 59 months the 5-year OS and PFS for the whole population were 93.4% (CI 88.7–96.1) and 73.6% (CI 67.3–78.9), respectively. In univariate analysis stage ≥III, age ≥65 years, B symptoms, hemoglobin <105 g/L, low LMR, high NLR were associated with a shorter PFS (p<0.05), while stage ≥III, advanced age, low LMR and high NLR were associated with worse OS (p<0.05). In multivariate analysis, only stage ≥III significantly predicted worse PFS (HR 2.04; 1.13–3.69), while age ≥65 years (HR 5.38; 1.49–19.40) and high NLR (HR 4.14; 1.15–14.87) were associated with decreased OS.

Focusing on the 160 advanced stage patients’ subgroup, stage ≥III and low LMR predicted a decreased PFS both in univariate and multivariate analysis with a HR of 2.14 (CI 1.30–4.08) and 2.14 (CI 1.10-4.31), respectively. Age ≥65 years and high NLR were associated with a worse OS both in univariate and multivariate analysis, with a HR of 7.48 (CI 2.14–26.17) and of 6.57 (CI 1.70–25.48), respectively.

Summary/Conclusion: In this study, we provide evidence that current practice, based on a PET-guided therapy intensification, is unsatisfactory, since the survival of stage III-IV is worse than the one reached by earlier stage patients. We confirmed the reliability prognostic efficacy of NLR and LMR. Whether advanced stage patients with low LNR and high NLR might benefit from a frontline brentuximab or nivolumab-based therapies is unknown, and deserve further investigation.

P1077: HODGKIN LYMPHOMA IN PATIENTS WITH HIV: NATIONAL RETROSPECTIVE MULTICENTER STUDY

A. Chekalov 1,* , M. Popova 1 , I. Tsygankov 1 , Y. Rogacheva 1 , N. Volkov 1 , A. Beynarovich 1 , K. Lepik 1 , M. Demchenkova 2 , T. Schneider 3 , Y. Kopeikina 3 , V. Potapenko 4 , I. Zyuzgin 5 , M. Kolesnikova 6 , T. Pospelova 6 , E. Zinina 7 , A. Myasnikov 8 , K. Kaplanov 9 , T. Ksenzova 10 , E. Pavlyuchenko 11 , N. Mikhaylova 12 , V. Baykov 13 , A. Kulagin 13

1 Department of Oncology, Hematology and Transplantation for teenagers and adults, Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, Saint Petersburg; 2 Irkutsk Regional Cancer Center, Irkutsk; 3 Leningrad Regional Clinical Hospital, Saint Petersburg; 4 Municipal educational hospital №31, Saint-Petersburg; 5 National Medicine Research Center of oncology named after N.N. Petrov, Saint Petersburg; 6 Municipal Clinical Hospital No. 2 of Novosibirsk Region, Center of Hematology, Novosibirsk; 7 Surgut’s Clinical Hospital, Surgut; 8 Republican hospital named under V.A. Baranov, Petrozavodsk; 9 Volgograd Regional Clinical Oncology Dispensary, Volgograd; 10 Regional Clinical Hospital, Tumen; 11 II Mechnikov North-Western State Medical University; 12 Department of Oncology, Hematology and Transplantation for teenagers and adults, Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation; 13 Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, Saint Petersburg, Russia

Background: Patients with HIV infection have a significantly higher risk of developing cancer than the general population. In the era of antiretroviral therapy (ART) the cancer risk and mortality have been decreased, however, the risk of developing Hodgkin lymphoma (HL) increased. The ART allows treating HIV-infected patients with lymphomas with protocols for patients in the general population. Withal the data on the results of the treatment HL in patients with HIV controversy.

Aims: To study epidemiology and evaluate the results of the treatment of HL in patients with HIV in national multicenter study.

Methods: The study included 45 patients with HL in patients with HIV who received treatment in 9 Russian centers from 2007 to 2021. The median follow-up was 9 months (1-129). Patients and treatment characteristics were analyzed. Overall survival (OS) and progression-free survival (PFS) were calculated within two years from the diagnosis using the Kaplan-Meier method.

Results: The median age was 39 years (25-66), men - 25 (55.6%), women - 20 (44.4%). Histological variants of HL in most cases were represented by nodular sclerosis (56%) and mixed-cell variant (41%). The advanced stage of the disease (3-4 Ann Arbor) was observed in 72.7% of patients, B-symptoms at the onset of the disease - 68.2%. The majority of patients (97.7%) received ART at the diagnosis of HL. The median number of CD4+ cells/µl at the onset of HL was 352.8 (50-692) cells/μl. General somatic status at the start of chemotherapy ECOG 0-1 - 34 (82.9%), ECOG≥2 - 7 (17.1%). As the first line of therapy, patients with localized stages of HL received ABVD (75%) and BEACOPP (25%), with advanced stages - ABVD (61.3%) and BEACOPP (38.7%). The median courses of first-line therapy were 4 (1-10). Radiation therapy in first-line therapy was performed in 4 patients (8.9%). The structure of response to first-line therapy were complete response - 51.4%, partial response - 25.7%, disease stabilization - 2.9%, disease progression - 20%. Fourteen and 8 patients received second and third-line therapy, respectively. Autologous hematopoietic stem cell transplantation was performed in 6 patients - the only 42.8% of patients with relapsed / refractory HL. OS in the study group was 81%, PFS - 38% (median PFS - 23 months). The level of CD4+ cells at the onset of HL less than 250/µl was associated with a statistically significant worsening of OS during 1 year (50% vs 100%, p=0.014). Factors such as gender, age, stage of the disease, ECOG status, the presence of B-symptoms at the onset of the disease, and the treatment regimen did not statistically significantly affect the two years OS and PFS from the diagnosis of HL in patients with HIV.

Summary/Conclusion: The national multicenter study allowed characterization of HL in patients with HIV and evaluation of the efficacy of first-line therapy, which was found to be lower than in the general population. The level of CD4+ cells was the only factor that affect 2-year OS. The obtained data can form the basis for further prospective studies aimed at improving the results of HL treatment in HIV-infected patients.

P1078: PATIENTS WITH HODGKIN LYMPHOMA DEVELOP ADEQUATE HUMORAL SEROLOGICAL RESPONSE TO VACCINATION WITH TWO DOSES OF BNT162B2 AND THEIR IGG ANTIBODY LEVELS MARKEDLY INCREASE AFTER A THIRD VACCINE DOSE

E. Dann 1,2,* , T. Inbar 1 , T. Mashiach 1 , J. Eisa 1 , S. Ringelstein-Harlev 1 , N. Horowitz 1

1 Rambam Health Care Campus; 2 The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel

Background: In patients (pts) with hematological malignancies, COVID-19 is considered to be associated with a high risk of severe morbidity and mortality. While anti-COVID-19 vaccination of such pts has become the standard of care, pts undergoing lymphodepleting therapy fail to generate protective serological response due to either the nature of their underlying disease or exposure to therapy.

Aims: This study aimed to assess serological response to vaccination with BNT162b2 (Pfizer) as well as COVID-19-related morbidity and mortality in Hodgkin lymphoma (HL) pts.

Methods: The above vaccine was available in Israel from January 2021 and all pts with hematological malignancies were recommended to undergo vaccination with 2 doses of this vaccine, injected 21 days apart. Six months later a 3rd dose was recommended and in another 3 months a 4th dose was available for pts at risk. Serology tests were performed at least 2 weeks after the 2nd vaccination. The SARS-CoV-2 IgG II Quant (Abbott©) assay was used to measure levels of IgG antibodies (Abs) against the SARS-CoV-2 spike protein. A result was considered positive if the IgG level was ≥150 AU/ml, which was defined as an adequate serological response.

Results: The current non-interventional single-center study evaluated the outcome of 55 HL pts (median age 46 years, 53% females); 51% of pts had advanced HL. Study participants received 1-9 lines of therapy (median 1 line). Six pts had COVID-19 prior to vaccination, 49 were vaccinated: 9 with 2 doses, 36 with 3 doses and 4 with 4 doses of BNT162b2. Following initial 2 vaccine doses and after a 3rd dose Ab levels >150 AU/ml were developed in 85% and 89.5% of pts, respectively. At a median of 95 days post-2nd vaccination, Ab levels were 2024 (1-29400) and 4 (0-7539) in 48 patients with no background disease versus 7 pts treated with lymphodepleting drugs or having a background disease, respectively. During the follow-up, 5 vaccinated pts were diagnosed with COVID-19 when the Delta variant was prevalent and 9 - during the Omicron wave. Notably, similar Ab levels were observed in those infected with Omicron and in non-infected pts, reflecting the genetic drift of this variant. A further analysis was performed to compare findings in a subgroup of 7 pts who had an additional background disease along with HL, such as chronic lymphocytic leukemia, s/p kidney transplantation, solid tumor, or those who were heavily pretreated, including therapy with bendamustine, versus the values observed in the rest 48 pts. The median age in the former subgroup was 58 (31-80) years, which was significantly older than in the remaining pts [median 45 (18-78) years] and median Ab levels were 4 (0-7539) AU/ml and 2024 (1-2940) AU/ml, respectively. Notably, after the 3rd vaccination, the median Ab level in both groups was 7000 AU/ml.

Summary/Conclusion: The results of the current study show that at least 85% of HL pts develop a high titer of anti-spike antibodies after vaccination with 2 BNT162b2 doses. These titers substantially increased post the 3rd vaccine dose. Only a minority of HL pts who had additional background diseases or were heavily pretreated, failed to develop an adequate serological response; however, some of them had high Ab titers post-3rd and 4th vaccinations. In this study, morbidity and mortality rates of HL pts infected with COVID-19 were lower than those reported in pts with other lymphoma types.

P1079: COMPARISON OF NOVEL SALVAGE REGIMENS AND TRADITIONAL SALVAGE CHEMOTHERAPY IN RELAPSED AND REFRACTORY CLASSIC HODGKIN LYMPHOMA

D. Ermann 1,* , V. Vardell 2 , E. Zacholski 3 , A. Fegley 3 , D. Modi 4 , K. Fedak 4 , S. Sundaram 5 , S. Rajeeve 5 , G. Goyal 6 , H. Hatic 7 , P. Torka 8 , S. Ba Aqeel 8 , A. Borogovac 9 , K. MacDougall 9 , S. Kothari 10 , A. Kress 11 , E. Travers 12 , N. Chilakamarri 13 , E. Brem 14 , L. Fitzgerald 15 , C. Wagner 16 , B. Hu 15 , D. Stephens 15 , H. Shah 15

1 Hematology/Oncology, Huntsman Cancer Institute, University of Utah; 2 Internal Medicine, University of Utah, Salt Lake City; 3 Clinical Pharmacology, Virginia Commonwealth University, Richmond; 4 Hematology/Oncology, Karmanos Cancer Institute, Detroit; 5 Hematology/Oncology, Tisch Cancer Institute, Mount Sinai, New York; 6 Hematology; 7 Hematology/Oncology, University of Alabama, Birmingham; 8 Hematology/Oncology, Roswell Park Comprehensive Cancer Center, Buffalo; 9 Hematology/Oncology, University of Oklahoma, Oklahoma City; 10 Hematology; 11 Hematology/Oncology, Yale Comprehensive Cancer Center, New Haven; 12 Hematology, University of Kentucky, Lexington; 13 Internal Medicine, Pomona Valley Medical Center, Pomona; 14 Hematology/Oncology, University of California, Irvine; 15 Hematology; 16 Clinical Pharmacology, Huntsman Cancer Institute, University of Utah, Salt Lake City, United States of America

Background: Traditional salvage chemotherapy followed by autologous stem cell transplant (ASCT) is the standard second line approach for treatment of relapsed/refractory (r/r) Classical Hodgkin Lymphoma (cHL) and can cure approximately 50-60% of patients (pts). Novel agents such as brentuximab vedotin (BV) and check-point inhibitors (CPIs) have high response rates in the r/r setting and have recently been used as salvage regimens to induce remissions before ASCT. Limited data exists comparing efficacy of these two approaches.

Aims: Our aim was to compare outcomes of pts receiving salvage chemotherapy (CT) as opposed to novel treatments (NT) for their first salvage regimen for r/r cHL.

Methods: Adult pts with r/r cHL who received their first salvage regimen (SR1) between January 2018 and June 2020 were retrospectively identified across 9 academic centers in the United States. Baseline characteristics were compared across CT and NT cohorts based on SR1. Endpoints were to compare complete response (CR), overall response rate (ORR), and event free survival (EFS) between the CT and NT cohorts. ORR was defined as CR+ partial response (PR). Events were defined as stable disease (SD) or progressive disease (PD) after SR1, less than a complete response (CR) at 90 days post-ASCT, PD after ASCT, or death.

Results: In total, 120 pts were identified with a median age of 33 years (range 18-85). 68% had advanced disease and 13% were early stage unfavorable (NCCN) at diagnosis. 88% received ABVD based front-line regimen. Many pts had poor prognostic characteristics including 43% with primary refractory disease, 52% with relapse <12 months from diagnosis, 38% with extranodal disease, and 26% with B-symptoms at time of relapse (Table 1 ).

65% of pts (n=78) received CT and 35% (n=42) received NT for SR1. 90% of CT pts received Ifosfamide, Carboplatin, and Etoposide (ICE) as SR1. Regimens used for NT treated pts included BV + Bendamustine (43%), BV alone (36%), BV + CPI (12%), BV + CT (5%), and other CPI (4%). No significant difference was found in the ORR and CR rates according to pts treated with CT vs. NT of 67% vs. 69% and 47% vs. 55%, respectively. Of the 104 patients who received ASCT, 88% received CT and 83% received NT for SR1. At a median follow-up of 32 months, 1-year EFS was 57% vs 69% and 2-year EFS was 56% vs 66% between CT and NT cohorts (p=0.25) (Figure 1).

All pts who progressed after CT for SR1 (n=31) received NT for SR2; whereas 73% (n=11) of those progressing after NT for SR1 received CT for SR2. ORR for CT in SR2 was 91% vs 70% for NT (p=.48). The most common SR2 CT regimen was ICE (82%), and NT regiment for SR2 was BV alone (40%), BV + Bendamustine (31%), BV + CPI (23%), and other CPI (6%). 79% vs 89% remained progression-free post-ASCT at their last follow-up after receiving CT vs. NT at last salvage respectively (p=0.15).

Of the 16 pts who did not proceed to ASCT, 7 died within an average of 19.5 months from the receipt of SR1. Interestingly, pts who did not proceed to ASCT had a significantly improved 2-year EFS if they received NT (n=7) at SR1 (71% vs. 13%) when compared to CT at SR1 (n=9) (p=0.03).

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Summary/Conclusion: There was a numerical trend towards better CR and EFS for novel therapy compared to traditional chemotherapy for first salvage, however the outcomes were not statistically significant. This demonstrates that CT is still a useful salvage therapy that can effectively get patients to an ASCT for curative intent. Prospective studies comparing novel therapy to traditional chemotherapy for salvage are warranted.

P1080: COMPARISON OF NIVOLUMAB 40 MG EFFICACY VERSUS 3 MG/KG IN PATIENTS WITH RELAPSED AND REFRACTORY CLASSIC HODGKIN LYMPHOMA

L. Fedorova 1,* , K. Lepik 1 , N. Mikhailova 1 , E. Kondakova 1 , Y. Komarova 1 , M. Popova 1 , P. Kotselyabina 1 , E. Borzenkova 1 , V. Baykov 1 , I. Moiseev 1 , A. Kulagin 1

1 RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia

Background: Nivolumab 3 mg/kg was shown to be effective in patients with relapsed and refractory classic Hodgkin lymphoma (r/r cHL). However, previously obtained data on pharmacokinetics, as well as financial toxicity of therapy, raised the issue of studying necessity of efficacy of reduced doses of PD-1 inhibitors. Efficacy of nivolumab 40 mg has previously been demonstrated in patients with r/r cHL in a prospective phase 2 study (Lepik KV et al., 2020). Continued accumulation of experience in the use of low-dose PD-1 inhibitors in patients with r/r cHL is required to support previous data.

Aims: To compare the efficacy of nivolumab (Nivo) 40 mg therapy with 3 mg/kg for patients with r/r cHL.

Methods: The Nivo40 trial ( NCT03343665 ) expanded prospective cohort of patients (group 1, n=50) treated with Nivo 40 mg was compared with the retrospective group 2 (n=116) of patients treated with Nivo 3 mg/kg. Patients characteristics are demonstrated in the Table 1.

The response was evaluated every 3 months by PET-CT using LYRIC criteria. Adverse events (AE) were analyzed by NCI CTCAE 4.0.3.

Overall response rate, progression-free survival (PFS) and overall survival (OS) were compared between group 1 and 2. During the survival analysis the PFS was censored by the time of additional therapy initiation.

Results: Median follow up was 44 (11-55) months in group 1 and 60 (6-70) months in group 2. Median Nivo cycles was 19 (2-49) and 20 (1-32) respectively. The best response to Nivo therapy was detected at 6 (2-24) and 6 (1-27) cycles respectively.

Overall response rate was 66% in group 1 and 67% in group 2. The structure of response in group 1 was: complete response (CR) in 38% of patients, partial response (PR) in 28%, stable disease (SD) in 6%, indeterminate response (IR) in 22% and progressive disease (PD) in 6%; in group 2: CR in 34%, PR in 33%, SD in 5%, IR in 20% and PD in 8%. Median OS was not achieved in both groups, 3-year OS was 97,8% and 96,5% respectively (p=0.356). Median PFS was 21,9 months (95%CI: 16,75-27,05) in group 1 and 18,8 months (95%CI: 13,44-24,16) in group 2, 3-year PFS was 25,6% and 27% respectively (p=0.356).

Additional therapy after Nivo monotherapy was started in 78% of patients after Nivo 40 mg and in 84% after Nivo 3 mg/kg. Allogeneic stem cell transplantation after Nivo therapy was performed in 5 (10%) patients in group 1 and in 26 (22%) patients in group 2.

Any grade adverse events were detected in 66% of patients in group 1 and in 79% in group 2 (p=0.068), 3-4 grade AE were detected in 10% and 19% respectively (p=0.151).

Summary/Conclusion: Nivolumab 40 mg therapy is comparable to the standard dose of 3 mg/kg in terms of overall response rate as well as survival in patients with r/r cHL. However, a direct comparison of different doses of nivolumab in a prospective study is required.

P1081: PET-ADAPTED THERAPY AFTER THREE CYCLES OF ABVD FOR ALL STAGES OF HODGKIN LYMPHOMA: LONG TERM FOLLOW UP OF THE GATLA LH-05 TRIAL

A. Pavlovsky 1,* , N. L. Fiad 1 , M. V. Prates 1 , I. Fernandez 1 , N. Kurgansky 1 , A. Cerutti 1 , F. Sackmann 1 , F. Negri Aranguren 1 , P. Negri Aranguren 1 , G. Remaggi 1 , L. Ferrari 1 , R. Mariano 1 , L. Guanchiale 1 , J. Maradei 1 , F. Giuliani 1 , E. Roveri 1 , A. Enrico 1 , S. Zabaljauregui 1 , M. D. R. Cabrejo 1 , C. Gumpel 1 , A. I. Varela 1 , M. Riddick 1 , S. Pavlovsky 1

1 GATLA, Buenos Aires, Argentina

Background: PET-CT adapted treatment for first line Hodgkin Lymphoma has been widely studied in the last decades. Long-term follow-up is important to judge both effcacy and safety of this approach.

Aims: Evaluate the efficacy of the PET3 adapted treatment in patients with classical Hodgkin’s lymphoma (HL) in Ann Arbor stages I-IV.

Methods: We analyzed updated follow-up data on all patients (pts) treated within the LH-05 GATLA trial. Newly diagnosed pts with HL Stages I-IV were included. All patients received 3 ABVD and were evaluated with a PET-CT-3 (PET3). Pts with a negative PET3 (DS 1 and 2) were consideredin metabolic CR and received no further therapy. Pts with DS 3 and 4 completed 6 ABVD and IFRT on PET-CT positive areas. Pts with progressive disease (DS 5) after 3 ABVD received salvage chemotherapy. We present the updated results of 490 pts with a median follow-up of 10 years. With a median age of 35 yrs., 300 presented with localized stage and 190 with advanced stage.

Results:

In LH-05, of all pts, 338 (69%) achieved CR with negative PET3, 152 (31 %) were PET3 positive. With a median follow up of 120 months the PFS and OS for all pts at 5 years is 79.2%and 94.3% respectively. Pts with negative PET3 had an PFS of 89% and 80% for localizedand advanced stage, compared to 63% for all pts with positive PET3 (p <0.0001). We performed a multivariate analysis for PFS which included age, stage, IPS, bulky disease, extranodalareas and the result of the PET3. This last parameter, together with age, were the only ones with statistical significance (p=0.001 and 0.046 respectively). Stage at diagnosis was not significant.

With long term follow up the OS at 5 years is 97.3% and 87.3% for all PET3 negative vs PET3 positive pts.

When comparing the results LH-05 with our previous clinical trial (LH-96) there is no difference in PFS and OS at 5 years but in LH-05 only 31% received more than 3 cycles of ABVD and IFRT compared to 61% and 100% in LH-96. This PET adapted approach reduces exposure to chemo andradiotherapy with no negative effect on long term outcome.

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Summary/Conclusion:

This long term follow up data support the PET-CT adapted approach for all stages ofHL after a short course of ABVD. In the Cox regression model, PET-CT at completion of treatment was the most significant factor associated to PFS.

Treatment with 3 cycles of ABVD can be adequate for pts with negative PET3 regardlesstheir stage at diagnosis. Nevertheless, this long term follow up demonstrated that there is still a room for improvement trying to identify PET3 negative pts that will relapse and escalating treatment in PET3 positive pts to improve outcome. GATLA is designing a trial with the aim to improve these two different risk groups.

P1082: SAFETY AND EFFICACY ANALYSIS IN OLDER PATIENTS TREATED WITHIN THE GATLA LH-05 PROTOCOL: PET-ADAPTED THERAPY AFTER 3 CYCLES OF ABVD FOR ALL STAGES OF HODGKIN LYMPHOMA

N. L. Fiad 1,* , M. V. Prates 1 , I. Fernandez 1 , N. Kurgansky 1 , A. Cerutti 1 , F. Negri Aranguren 1 , L. Guanchiale 1 , F. Sackmann 1 , J. Maradei 1 , A. Enrico 1 , P. Negri Aranguren 1 , A. Pavlovsky 1

1 GATLA, Buenos Aires, Argentina

Background: Treatment for classic Hodgkin lymphoma (cHL) in older age population continues to be a challenge. Comorbidity prevalence, increase toxicity to the standard treatments and the lack of inclusion in clinical trials all contribute to this phenomenon. The obtained results are inferior in comparison to young adults and there is shortage of evidence regarding effective therapeutic strategies. Recently, the GATLA LH-05 protocol has published the long-term follow-up of the results of the PET/CT adapted strategy after 3 cycles of ABVD, regardless of the presentation stage and no upper age limit.

Aims: To assess the effectiveness and safety of the interim PET/CT adapted treatment in cHL patients older than 60 years old.

Methods: A retrospective analysis of the LH-05 database was performed. Patients were included ≥ aged 60 with recent diagnosis of cHL stage I-IV and HIV negative. All patients received 3 cycles of ABVD and were evaluated with PET-TC (PET3). Those patients with negative PET (Deauville score 1 and 2) were considered to be in complete remission (CR) and they finalized the treatment. Patients with DS 3 and 4 completed 6 ABVD cycles and involved-field radiotherapy in hypermetabolic areas in interim PET3. Patients with DS 5 were considered to have progressive disease. Progression-free survival (PFS) and overall survival (OS) were evaluated. Kaplan-Meier method and Log-rank test were used for survival analysis.

Results: Of a total of 490 patients included in the GATLA LH-05 protocol, 59 met the inclusion criteria. The mean age was of 66 years (range: 60-89), 90% presented a PS<2. The most frequent histological subtype was Nodular Sclerosis (54%) and 75% presented in localized stage (I-II). All patients received initial treatment with 3 cycles of ABVD and were evaluated by PET: 81% presented negative PET (DS 1-2) and 19% were positive. No GIII-IV toxicity or treatment-related deaths were recorded. With a median follow-up of 10 years, median PFS and OS were not reached. At 36 and 60 months, PFS was 86% and 78,9%, and OS was 90% and 85.5%, respectively. Negative PET3 patients had a PFS of 85.4%, while positive PET3 patients had a PFS of 43% (p=0,0001). In the multivariable analysis that included age (>60 vs. <60), stage (localized vs. advanced), IPS (<2 vs. >2), extranodal areas, bulky disease and PET3 result, only age and PET3 result had significant impact in PFS (p=0.046 and p=0,001 respectively). PFS of PET/TC- positive patients was significantly lower than in the cohort of patients younger than 60. OS at 36 and 60 months in PET negative patients was 97.5% and 94.5% respectively, versus 63.6% and 53% in PET/TC positive patients.

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Summary/Conclusion: With the PET/TC adapted treatment after 3 cycles of ABVD in 59 patients > 60 years old, 81% of patients achieved negative PET and therefore received no further treatment. These patients had an excellent result with a PFS of 85.4% at 3 years, similar to the younger patient population. However, a significant reduction in PFS was observed in PET3 positive patients > 60 years old compared to younger ones. Implementation of this PET/TC guided strategy, regardless of stage at diagnosis, resulted in reduced exposure to chemotherapy and radiotherapy, contributing to the absence of severe treatment-related morbidity and mortality.

P1083: BRENTUXIMAB VEDOTIN, ETOPOSIDE, SOLUMEDROL, HIGH DOSE ARA-C & PLATINUM FOLLOWED BY HDT & APBSCT FOR REFRACTORY/RELAPSED HODGKIN LYMPHOMA PATIENTS: LONG-TERM RESULTS OF THE GELTAMO GROUP BRESHAP STUDY

R. Garcia-Sanz 1,* , C. Martínez 2 , F. De la Cruz 3 , A. P. González 4 , A. Rodríguez 5 , B. Sánchez-González 6 , E. Domingo-Domenech 7 , M. Moreno 8 , J. López 9 , J. L. Piñana 10 , M. Bastos 11 , M. Canales 12 , A. Gutiérrez 13 , M. J. Rodríguez-Salazar 14 , A. Navarro 1 , A. Sureda 7

1 hematology, Hospital Universitario, Salamanca; 2 hematology, Hospital Clinic, Barcelona; 3 hematology, Hospital Virgen del Rocío, Sevilla; 4 hematology, Hospital Universitario Central de Asturias, Oviedo; 5 hematology, Hospital 12 de Octubre, Madrid; 6 hematology, Hospital del Mar; 7 hematology, Hospital Duran i Reynals, Barcelona; 8 hematology, Hospital Germans Trias i Pujol, Badalona; 9 hematology, Hospital Ramón y Cajal, Madrid; 10 hematology, Hospital Clínico, Valencia; 11 hematology, Hospital Gregorio Marañón; 12 hematology, Hospital La Paz, Madrid; 13 hematology, Hospital Son Espases, Palma de Mallorca; 14 hematology, Hospital Universitario de Canarias, Tenerife, Spain

Background: For Refractory/Relapsed Hodgkin Lymphoma (RRHL) patients, the combination of BRentuximab vedotin, Etoposide, Solumedrol, High dose Ara-C and Platinum (BRESHAP) as induction therapy prior to high-dose therapy and Autologous Stem Cell Transplantation (APBSCT) induces an overall (OR) and complete remission (CR) rate of 93% and 71% of patients with an initial 75% Time to Tumor Progression at three years (Garcia-Sanz et al, Ann Oncol 2019). In these patients, the primary efficacy endpoint is usually the CR pre-APBSCT, but long-term results including time to tumor progression (TTP) or overall survival are challenges that should be evaluated in these recently adopted strategies.

Aims: In this work, we evaluate long-term results of the phase II trial with the combination of BV and ESHAP [BRESHAP] as 2 nd line therapy for RRHL prior to APBSCT (ClinicalTrials.gov # NCT02243436 ).

Methods: Data update with an additonal follow-up period of four years compared with the initial publication in the EHA-2018 congress

Results: The trial included 66 patients with relapsed or refractory classical HL (cHL) who had failed to one prior line of therapy were eligible. There were 35 females & 31 males, with a median age of 36 years (18-66). Forty patients were primary refractory, 16 early relapses (CR ≤1 year) and 10 late relapses (CR >1 year). During the follow-up, excluding progressions, there were 39 Severe Adverse Events (SAEs) reported in 22 patients (hospitalizations and AEs around transplant were not considered SAEs), which means that no new SAEs were reported after the initial publication of the results. Transplant was done after BRESHAP in 61 patients with no problems reported about the engraftment in the long-term evaluation. As reported, pre-transplant OR was 93% (including a 71% CR).

At a mean follow-up of 75 months, 16 patients have progressed and 3 died without progression, providing 7-year time to treatment failure (TTP) and progression free survival (PFS) of 74% and 70%, respectively. Eight patients have died: 5 due to progression, and the 3 due to complications in the absence of relapse or refractory disease: pneumonia, sepsis and pulmonary embolism. The overall survival (OS) at seven year was 88% (figure 2). There were three variables related to a lower PFS and TTP: CR after BRESHAP, bone marrow infiltration at relapse/refractoriness and B symptoms at inclusion in the trial. In the multivariate analysis, only response to BRESHAP maintained the independent predictive capacity. Respect to OS, the only prognostic factor was CR to 2 nd line therapy

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Summary/Conclusion: BRESHAP followed by high dose therapy and autologous peripheral blood stem transplant is a safe and effective strategy for patient with refractory or relapsed Classical Hodgkin lymphoma with excellent long-term results.

P1084: CHECK-POINT INHIBITORS IN PATIENTS WITH RELAPSE/REFRACTORY HODGKIN’S LYMPHOMA: A RETROSPECTIVE ANALYSYS BY THE RETE EMATOLOGICA PUGLIESE (REP).

F. Gaudio 1,* , G. Loseto 2 , V. Bozzoli 3 , P. R. Scalzulli 4 , A. M. Mazzone 5 , L. Tonialini 6 , V. Fesce 7 , G. Quintana 8 , G. De santis 9 , P. Masciopinto 10 , E. Arcuti 10 , F. Clemente 2 , S. Scardino 3 , G. Tarantini 9 , D. Pastore 8 , L. Melillo 7 , V. Pavone 6 , P. Mazza 5 , A. M. Carella 4 , N. Cascavilla 4 , N. Di Renzo 3 , A. Guarini 2 , P. Musto 1,10

1 Unit of Hematology and Stem Cell Transplantation, AOUC Policlinico; 2 Hematology Unit, Giovanni Paolo II IRCCS Cancer Institute Oncology Hospital, Bari; 3 Hematology and Stem Cell Transplant Unit, “Vito Fazzi” Hospital, Lecce; 4 Hematology Unit, IRCCS “Casa Sollievo della Sofferenza”, S. Giovanni Rotondo (FG); 5 Hematology Unit, Department of Hematology-Oncology, Moscati Hospital, Taranto; 6 Hematology and Transplant Unit, Cardinal Panico Hospital, Tricase (LE); 7 Hematology Unit, Azienda Ospedaliero Universitaria-Ospedali Riuniti, Foggia; 8 Hematology Unit, A. Perrino Hospital, Brindisi; 9 Hematology Unit, Dimiccoli Hospital, Barletta; 10 Department of Emergency and Organ Transplantation, “Aldo Moro” University, Bari, Italy

Background: The majority of patients with either limited stage or advanced-stage classical Hodgkin lymphoma (cHL) can be cured with chemotherapy; however, 5–10% will have refractory disease to frontline therapy and approximately 10–30% will relapse. In recent years, checkpoint/PD-1 inhibitors have significantly changed the prognosis of patients with relapsing refractory cHL, demonstrating exceptional results in heavily pretreated patients. There is limited data on the real-world experience with PD-1 inhibitors in cHL and it is unknown whether fewer selected patients treated with these agents receive benefits similar to those observed in published trials.

Aims: We performed a retrospective multicentre analysis of the Rete Ematologica Pugliese (REP) of 66 patients with relapsing refractory cHL who received PD-1 inhibitors in the non-trial setting.

Methods: Forty-three patients (65%) were treated with nivolumab and 23 (35%) with pembrolizumab. Median age at diagnosis was 36 years (range, 18–81) and median age at initiation of PD-1 inhibitor therapy was 43 years (range 19–86).

Forty-four (67%) and 23 (35%) patients underwent autologous stem cell transplantation (SCT) and allogeneic SCT, respectively, prior to checkpoint inhibitor therapy.

The median lines of treatment attempted prior to PD-1 inhibitor therapy was 4 (range, 3 to 7). All patients were treated with brentuximab vedotin prior to receiving checkpoint inhibitor therapy.

Results: The best overall response rate to PD-1 inhibitor therapy was 70%: 47% complete remission (CR) and 23% partial remission (PR). Five patients (8%) achieved stable disease (SD), while 14 patients (21%) showed progressive disease (PD). In patients treated with nivolumab the response was: CR in 23 (54%), PR in 9 (21%), SD in 4 (9%), PD in 7 (16%) patients, respectively; in those treated with pembrolizumab CR occurred 8 (35%), PR in 6 (26%), SD in 1 (4%), PD in 8 (35%) patients, respectively. Twenty-four immune-related adverse events were documented (4 gastrointestinal, 4 hepatic, 6 fever, 4 hematological, 3 dermatological, 3 allergic rhinitis). Toxicity resolved in all patients and there were no deaths attributed to checkpoint inhibitor therapy.

After a median follow-up of 26 months (range 3-72 months), 54 patients (82%) are alive and 12 (18%) died. The cause of death was attributed to disease progression in 9 patients, and sepsis in 3 patients. After PD-1 inhibitor therapy, 22 patients (33%) relapsed or progressed. The overall survival and progression free survival at five years were 65% and 54%, respectively.

Summary/Conclusion: We demonstrate similar response rates, clinical outcome and toxicity profiles compared to clinical trials. These results support the effectiveness and tolerability of PD-1 inhibitors therapy in relapsing refractory cHL in a real-world setting.

P1085: EVALUATION OF GONADAL FUNCTION IN YOUNG WOMEN DIAGNOSED WITH HODGKIN AND NON-HODGKIN LYMPHOMA

A. N. Georgopoulou 1,* , A. Giannakou 1 , T. P. Vassilakopoulos 1 , M. Siakantaris 1 , N. A. Georgopoulos 2 , S. Kalantaridou 3 , E. Lalou 1 , K. Keramaris 1 , E. Loukari 1 , M. Arapaki 1 , I. Konstantinou 1 , A. Machairas 1 , A. Kopsaftopoulou 1 , I. Mammali 2 , I. Vassilopoulos 1 , D. Galopoulos 1 , M. K. Angelopoulou 1

1 Department of Hematology and BMT of the National and Kapodistrian University of Athens, Laikon, laikon general hospital, Athens; 2 Department of Endocrinology of the University Hospital of Patras, University Hospital of Patras, Patras; 3 B Obstetrics and Gynecology Department of the National and Kapodistrian University of Athens, Areteion Hospital, Athens, Greece

Background: ABVD chemotherapy (CT) is the standard treatment approach for Hodgkin lymphoma (HL), with a negative interim PET. R-DA-EPOCH and R-CHOP are the most frequently used regimens used in primary mediastinal/high grade B-NHL and DLBCL, respectively. Young women are often affected by these types of lymphoma and may be cured in >80% of the cases. Thus, treatment-related complications are being increasingly recognized, among which, gonadal insufficiency with its major psychological consequences. This is more prevalent in female patients, in whom collection and cryopreservation of oocytes/ovarian tissue are not applied in everyday clinical practice. Published data are scarce on this subject. Moreover, little is known about the kinetics of gonadal function and sex hormones during chemotherapy to safely guide contraceptive measures.

Aims: The aim of this study is the prospective evaluation of gonadal function in young women with malignant lymphoma who are receiving CT. We here present our preliminary results on 50 patients with HL and B-NHL.

Methods: This is a prospective study of gonadal function in female patients≤40 years. Hormonal measurements were performed at pre-specified time points: before treatment (t0), during CT(t1), at the end of CT(t2) and every six months(t6,t12) thereafter. The following hormones were measured: follicle-stimulating hormone (FSH), lutenizing hormone (LH), progesterone (PG), estradiol (Ε 2 ), anti-Mullerian hormone (ΑΜΗ). The study included:32 HL [median age: 29 years, 32 ABVD] and 18 B-NHL patients [median age:27 years, 9 RDAEPOCH, 7 RCHOP, 2 other]. FSH reflects gonadal function in women (increased levels indicate gonadal dysfunction). AMH is considered to be the most sensitive biomarker for gonadal reserve (decreasing values correlate with ovarian insufficiency). E2 and progesterone are the major sex hormones.

Results: HL-ABVD: FSH constantly increased from the beginning, peaking in the middle (fsh0-1 p<0.0001), remaining high until the end (fsh0-2 p<0.0001), without reaching normal levels at 6 months (fsh0-6 p=0.002), finally normalizing at 12 months after the end of treatment. Consistently with FSH, AMH sharply decreased during treatment: [median values: 2,85IU/mL(t0), 0,45IU/mL(t1), 0,62IU/mL(t2), p<0.0001 for both]. A rebound increase was observed at 6 months and slowly decreased again to normal values at 12 months after the end of CT. Estradiol and progesterone were not affected throughout the treatment.

B-NHL: Gonadal damage, reflected by the increase in FSH, was evident, though with slightly different kinetics compared to HL: [median values: 4,1IU/mL(t0), 8,3IU/mL(t2), fsh0-2 p=0,035]. FSH gradually increased during treatment, reaching a peak towards the end. FSH slowly reached normal values after six months from the end of treatment. In parallel to HL, AMH values sharply decreased during CT: [median values: 7,3IU/mL(t0), 0,17IU/mL(t1), 1,57IU/mL(t2), amh0-1 p=0.002, amh0-2 p=0,043). However, in contrast to HL, no clear rebound was seen and AMH values remained extremely low, even at 18 months after the end of treatment (median values: 0,23IU/mL), highlighting the gonadotoxic effect of CT. Estradiol and progesterone did not change significantly.

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Summary/Conclusion: Gonadal function in female patients with malignant lymphomas is affected during CT in both HL and B-NHL, though with different kinetics. In HL patients treated with ABVD, gonadal function normalized at 6 months, whereas, in B-NHL gonadal dysfunction remained even at 18 months after the end of CT, possibly indicating a chemotherapy-dependent genotoxic effect.

P1086: FAVEZELIMAB (ANTI–LAG-3) AND PEMBROLIZUMAB CO-BLOCKADE IN ANTI–PD-1–NAIVE PATIENTS WITH RELAPSED OR REFRACTORY CLASSICAL HODGKIN LYMPHOMA: AN OPEN-LABEL PHASE 1/2 STUDY

G. Gregory 1,* , J. Timmerman 2 , D. Lavie 3 , P. Borchmann 4 , A. F. Herrera 5 , L. Minuk 6 , V. Vucinic 7 , P. Armand 8 , A. Avigdor 9 , R. Gasiorowski 10 , Y. Herishanu 11 , C. Keane 12 , J. Kuruvilla 13 , J. Palcza 14 , P. Pillai 14 , P. Marinello 14 , N. A. Johnson 15

1 School of Clinical Sciences at Monash Health, Monash University, Melbourne, Australia; 2 UCLA Medical Center, Los Angeles, United States of America; 3 Hadassah Medical Center, Jerusalem, Israel; 4 University Hospital of Cologne, Cologne, Germany; 5 City of Hope, Duarte, United States of America; 6 CancerCare Manitoba, Winnipeg, Canada; 7 University of Leipzig Medical Center, Leipzig, Germany; 8 Dana-Farber Cancer Institute, Boston, United States of America; 9 Sheba Medical Center, Ramat Gan, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 10 Concord Hospital, University of Sydney, Concord, Australia; 11 Tel Aviv Sourasky Medical Center, Tel Aviv-Yafo, Israel; 12 Princess Alexandra Hospital, Brisbane, Australia; 13 Princess Margaret Cancer Centre, Toronto, Canada; 14 Merck & Co., Inc., Kenilworth, United States of America; 15 Jewish General Hospital, Montreal, Canada

Background: Programmed cell death 1 (PD-1) inhibitors are a standard of care in patients with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL), but clinical interest persists for new approaches to deepen and lengthen responses. Dual blockade of PD-1 and lymphocyte-activation gene 3 (LAG-3) has demonstrated antitumor activity in preclinical models.

Aims: The multicohort phase 1/2 MK-4280-003 study ( NCT03598608 ) evaluated the safety and efficacy of favezelimab (MK-4280), a humanized Immunoglobulin G4 LAG-3 inhibitor, plus pembrolizumab (a PD-1 inhibitor) in patients with R/R hematologic malignancies. This analysis focused on anti–PD-1–naïve patients with R/R cHL (cohort 1).

Methods: This study included a safety lead-in phase (part 1) to determine the recommended phase 2 dose (RP2D) followed by a dose-expansion phase (part 2). Eligible patients in cohort 1 must have had R/R cHL after autologous stem cell transplantation (ASCT) or been ineligible for ASCT and have had no prior anti–PD-1 therapy. In part 1, patients from all cohorts received intravenous (IV) pembrolizumab 200 mg every 3 weeks (Q3W) and favezelimab IV 200 mg or 800 mg Q3W. Dose-finding based on occurrence of dose-limiting toxicities (DLTs) was determined using a modified toxicity probability interval design. In part 2, patients received pembrolizumab + favezelimab at the established RP2D for up to 2 years (35 cycles), or until documented disease progression, adverse events (AEs), or withdrawal from the study. Primary end point was safety, including DLTs and AEs. Secondary end point was objective response rate (ORR). Duration of response (DOR), progression-free survival (PFS), and overall survival (OS) were exploratory end points.

Results: Only 1 DLT (autoimmune hepatitis [grade 4]) was identified among the first 6 patients from all cohorts included in part 1 at the favezelimab 200 mg dose; thus, the dose was escalated to 800 mg. No DLTs were observed in the 15 additional patients treated at the 800 mg dose. The RP2D for the combination was defined as 800 mg Q3W + pembrolizumab 200 mg Q3W. In cohort 1, 30 patients were enrolled; median age was 40 years, 53% had ECOG PS 0, and 80% had no more than 3 prior lines of therapy. As of the database cutoff (Nov 1, 2021), 9 of 30 (30%) patients had discontinued treatment either due to adverse events (n=3) or disease progression (n=6). After a median follow-up of 13.5 months, ORR for cohort 1 was 73% (95% CI, 54-88; complete response, 7 patients [23%]; partial response, 15 patients [50%]). 28 of 30 patients (93%) had a reduction from baseline in target lesions. Median DOR was not reached ([NR]; 95% CI, 0+ to 23+ months) and 6 patients (51%) had response ≥12 months. The median PFS was 19 months (95% CI, 8-NR) and the 12-month PFS rate was 57%. The median OS was NR (95% CI, NR-NR) and the 12-month OS rate was 94%. Treatment-related AEs (TRAE) occurred in 26 patients (87%); most common (≥10%) were hypothyroidism (27%), fatigue (20%), infusion-related reactions (20%), headache (17%), and arthralgia, hyperthyroidism, myalgia, and nausea (10% each). Grade 3 or 4 TRAEs occurred in 6 patients (20%) and 10% of patients discontinued due to TRAEs. No treatment-related deaths occurred.

Summary/Conclusion: Favezelimab 800 mg + pembrolizumab 200 mg Q3W demonstrated an acceptable safety profile and effective antitumor activity in anti–PD-1–naïve patients with R/R cHL. Further studies are merited to compare the activity of this combination to that of pembrolizumab alone.

P1087: FAVEZELIMAB (ANTI–LAG-3) AND PEMBROLIZUMAB CO-BLOCKADE IN PATIENTS WITH RELAPSED OR REFRACTORY CLASSICAL HODGKIN LYMPHOMA WHO PROGRESSED AFTER ANTI–PD-1 THERAPY: AN OPEN-LABEL PHASE 1/2 STUDY

A. F. Herrera 1,* , D. Lavie 2 , N. A. Johnson 3 , A. Avigdor 4 , P. Borchmann 5 , C. Andreadis 6 , A. Bazargan 7 , G. Gregory 8 , C. Keane 9 , T. Inna 10 , V. Vucinic 11 , P. L. Zinzani 12 , H. Zhang 13 , P. Pillai 13 , P. Marinello 13 , J. Timmerman 14

1 City of Hope, Duarte, United States of America; 2 Hadassah Medical Center, Jerusalem, Israel; 3 Jewish General Hospital, Montreal, Canada; 4 Sheba Medical Center, Ramat Gan, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 5 University Hospital of Cologne, Cologne, Germany; 6 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, United States of America; 7 University of Melbourne, Melbourne, and St Vincent’s Hospital, Fitzroy; 8 School of Clinical Sciences at Monash Health, Monash University, Melbourne; 9 Princess Alexandra Hospital, Brisbane, Australia; 10 Rambam Health Care Campus, Haifa, Israel; 11 University of Leipzig Medical Center, Leipzig, Germany; 12 IRCCS Azienda Ospedaliero-Universitaria di Bologna Istituto di Ematologia “Seràgnoli” and Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy; 13 Merck & Co., Inc., Kenilworth; 14 UCLA Medical Center, Los Angeles, United States of America

Background: PD-1 inhibitors are a standard of care for relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL), but optimal therapy is yet to be defined for patients whose disease progresses after anti–PD-1 treatment. Dual blockade of PD-1 and lymphocyte-activation gene 3 (LAG-3) has demonstrated antitumor activity in preclinical models.

Aims: This multicohort phase 1/2 study ( NCT03598608 ) evaluated safety and efficacy of favezelimab (MK-4280), a humanized IgG4 LAG-3 inhibitor, plus the PD-1 inhibitor pembrolizumab in patients with R/R hematologic malignancies. Cohort 2 focused on patients with R/R cHL refractory to anti–PD-1 therapy.

Methods: This study included a safety lead-in phase (part 1) to determine recommended phase 2 dose (RP2D) followed by a dose-expansion phase (part 2). Eligible patients in cohort 2 had R/R cHL, relapsed after or were ineligible for autologous stem cell transplantation (ASCT), and progressed after ≥2 doses of anti–PD-1 therapy (within 12 weeks of last dose). In part 1, patients from all cohorts received intravenous (IV) pembrolizumab 200 mg every 3 weeks (Q3W) and favezelimab IV 200 mg or 800 mg Q3W. Dose-finding based on occurrence of dose-limiting toxicities (DLT) was determined using a modified toxicity probability interval design. In part 2, patients received pembrolizumab + favezelimab at the established RP2D for up to 2 years (35 cycles), or until documented disease progression, adverse events (AEs), or withdrawal from study. Primary end point was safety, including DLTs and AEs. Secondary end point was objective response rate (ORR). Duration of response (DOR), progression-free survival (PFS), and overall survival (OS) were exploratory end points.

Results: Only 1 DLT (autoimmune hepatitis [grade 4]) was observed among the first 6 patients from all cohorts included in part 1 at the favezelimab 200 mg dose; thus, the dose was escalated to 800 mg. No DLTs were observed in the 15 additional patients at the 800 mg dose. Favezelimab RP2D was defined as 800 mg Q3W + pembrolizumab 200 mg Q3W. In cohort 2, 33 patients were enrolled; median age was 37 years, 64% had ECOG PS 0, and 94% had at least 4 prior lines of therapy. At database cutoff (Nov 1, 2021), 20 patients had discontinued treatment due to adverse events (n=7); disease progression or clinical progression (n=11); or withdrawal/physician decision (n=2). After a median follow-up of 16.5 months, ORR for patients receiving favezelimab 800 mg (n=29) was 31% (95% CI, 15-51; complete response, 2 [7%]; partial response, 7 [24%]). 19 of 29 responders (66%) had an anti–PD-1–based regimen as their most recent line of therapy at study entry. 23 of 29 patients (79%) had a reduction from baseline in target lesions. Median DOR for patients who received favezelimab 800 mg was not reached ([NR]; 95% CI, 0+ to 14+ months). For all patients in cohort 2, the median PFS was 9 months (95% CI, 5-15); 12-month PFS rate was 39%. Median OS was 26 months (95% CI, 26-NR); 12-month OS rate was 91%. Treatment-related AEs (TRAE) occurred in 28 patients (85%); most common (>10%) were hypothyroidism (18%), nausea and fatigue (15% each), and arthralgia and diarrhea (12% each). Grade 3 or 4 TRAEs occurred in 6 patients (18%) and 18% of patients discontinued treatment due to TRAEs. No treatment-related deaths occurred.

Summary/Conclusion: Favezelimab 800 mg + pembrolizumab 200 mg Q3W showed a tolerable safety profile and effective antitumor activity in heavily pretreated patients with R/R cHL whose disease had progressed after anti–PD-1 therapy, suggesting that the combination may reinduce a response in these patients.

P1088: UPDATED DATA FROM A PHASE 1/2 STUDY OF BRENTUXIMAB VEDOTIN COMBINED WITH CHEMOTHERAPY IN PEDIATRIC PATIENTS WITH ADVANCED STAGE CLASSICAL HODGKIN LYMPHOMA

F. Locatelli 1,* , F. Luisi 2 , M. Pianovski 3 , M. A. Salvino 4 , F. Fagioli 5 , S. Epelman 6 , L. Britto De Abreu Lima 7 , R. Norris 8 , V. Odone Filho 9 , M. Zecca 10 , C. Favre 11 , R. Kobayashi 12 , Y. Koga 13 , Y. Sidi 14 , X. Zhou 14 , X. Bai 14 , F. Campana 14 , E. J. Leonard 14 , A. R. Franklin 15

1 Department of Pediatric Haematology and Oncology and Cell and Gene Therapy, IRCCS Ospedale Pediatrico Bambino Gesù, Sapienza, University of Rome, Rome, Italy; 2 Pediatria, GRAACC/UNIFESP, São Paulo; 3 Department of Pediatric Oncology, Hospital Erastinho, Liga Paranaense de Combate ao Câncer, Jardim das Américas, Curitiba, Parana; 4 Hematology, IDOR, Salvador, Hospital São Rafael, Salvador, Brazil; 5 Department of Sciences of Public Health and Pediatrics, Regina Margherita Children’s Hospital, Turin, Italy; 6 Pediatric Oncology Department, Hospital Santa Marcelina, São Paulo; 7 Hematology, INCA - Instituto Nacional de Câncer, Rio de Janeiro, Brazil; 8 Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cancer and Blood Diseases Institute, Cincinnati, OH, United States of America; 9 ITACI – Instituto De Tratamento Do Câncer Infantil – Departament De Pediatria – Faculdade De Medicina Da Universidade De São Paulo, São Paulo, Brazil; 10 Oncoematologia Pediatrica, Fondazione IRCCS Policlinico San Matteo, Pavia; 11 Dipartimento di Oncoematologia, Azienda Ospedaliero Universitaria Ospedale Pediatrico Meyer, Firenze, Italy; 12 Department of Hematology/Oncology for Children and Adolescents, Sapporo Hokuyu Hospital, Sapporo; 13 Department of Pediatrics, Kyushu University Hospital, Fukuoka-shi, Fukuoka-Ken, Japan; 14 Takeda Development Center Americas, Inc. (TDCA), Lexington, MA; 15 Center for Cancer and Blood Disorders, Children’s Hospital Colorado, Aurora, CO, United States of America

Background: Brentuximab vedotin is a CD30-directed monoclonal antibody-drug conjugate approved for use in the first-line adult classical Hodgkin lymphoma (cHL) setting in combination with doxorubicin, vinblastine, and dacarbazine (A+AVD). The safety, tolerability, recommended dosing, immunogenicity, and antitumor activity of A+AVD have not previously been evaluated as a first-line therapy in treatment-naïve pediatric patients with cHL.

Aims: To provide updated safety and efficacy data from an open-label phase 1/2 study of A+AVD in previously untreated pediatric patients with stage III or IV cHL ( NCT02979522 ).

Methods: Patients with newly diagnosed stage III or IV cHL who were aged 5 to <18 years were treated with A+AVD on days 1 and 15 of 28-day cycles, for a maximum of 6 cycles. In phase 1, patients (n = 8) received brentuximab vedotin 48 mg/m 2 + AVD; dose-limiting toxicity (DLT) was assessed from day 1 of cycle 1 to day 56. In phase 2, 51 additional patients received the same treatment regimen. Progression-free survival (PFS) was defined as the time between first dose and time of disease progression, and event-free survival (EFS) was defined as the time between first dose and time of treatment failure; EFS events were disease progression, treatment withdrawal, and death. All patients had been on study for at least 2 years at the data cutoff (September 24, 2021). The data presented refer to phases 1 and 2 combined.

Results: All patients (n = 59) in phases 1 and 2 completed 6 cycles of A+AVD. Among 6 DLT-evaluable patients in phase 1, no DLTs were observed. The maximum tolerated dose of brentuximab vedotin was not reached and the recommended dose was determined to be 48 mg/m 2 . Treatment-emergent adverse events (TEAEs) occurred in all patients; the most common TEAEs were vomiting (85%), nausea (75%), and neutropenia (58%). Fourteen patients (24%) experienced treatment-emergent peripheral neuropathy (PN); of these, 10 patients had PN resolved by end of treatment (EOT), 13 patients had PN resolved by last contact, and one patient had ongoing grade 1 paraesthesia at last contact. No on-study deaths occurred. At the end of cycle 2, the PET-negative rate (Deauville score 1, 2, or 3) was 90%. Objective response rate (complete response [CR] + partial response) according to independent review facility was 88%, with 76% of patients achieving a CR. Median duration of response and duration of CR were not estimable. Median PFS, EFS, and overall survival were not reached, suggesting promising efficacy of A+AVD in pediatric patients. Overall PFS, EFS and overall survival data will be presented at the meeting.

Summary/Conclusion: Brentuximab vedotin 48 mg/m 2 plus AVD every two weeks was generally well tolerated with an acceptable safety profile, and demonstrated an efficacy benefit in CD30+ pediatric patients with previously untreated advanced cHL. These data support A+AVD as a suitable frontline therapy option in this patient population.

P1089: BRENTUXIMAB VEDOTIN, NIVOLUMAB, DOXORUBICIN, AND DACARBAZINE (AN+AD) FOR ADVANCED STAGE CLASSIC HODGKIN LYMPHOMA: PRELIMINARY SAFETY AND EFFICACY RESULTS FROM THE PHASE 2 STUDY (SGN35 027 PART B)

H. Lee 1 , I. W. Flinn 2 , J. Melear 3 , R. Ramchandren 4 , J. Friedman 5 , J. M. Burke 3 , Y. Linhares 6 , M. Raval 3 , R. Chintapatla 7 , T. A. Feldman 8 , H. Yimer 3 , M. Islas-Ohlmayer 3 , A. Dean 3 , V. Rana 9 , M. D. Gandhi 3 , J. Renshaw 3 , A. L. Gillespie-Twardy 3 , L. Ho 10,* , M. Puhlmann 10 , W. Guo 10 , C. A. Yasenchak 11

1 MD Anderson, Houston; 2 Sarah Cannon Research Institute and Tennessee Oncology, Nashville; 3 US Oncology Research, The Woodlands; 4 University of Tennessee Medical Center, Nashville; 5 University Hospitals Seidman Cancer Center, Cleveland; 6 US Oncology Research, Woodlands; 7 Kadlec Clinic, Kennewick; 8 Lymphoma Division, John Theurer Cancer Center at Hackensack Meridian Health, Hackensack; 9 University of Colorado Health Hematology and Oncology, Colorado Springs; 10 Seagen, Bothell; 11 Willamette Valley Cancer Institute and Research Center, Eugene, United States of America

Background: Brentuximab vedotin (BV) and nivolumab are both active and well-tolerated in patients (pts) with classical Hodgkin lymphoma (cHL) and were previously studied in first salvage (overall response rate [ORR] 85%; complete response [CR] 67%) (Advani 2021) and as firstline (1L) therapy in older adults (ORR 95%; CR 79%) (Yasenchak 2019). The combination of BV plus nivolumab demonstrated promising activity as a frontline treatment option for pts over 60 yrs of age with cHL (Friedberg 2018). Additionally, in pts with non-bulky Stage I or II cHL, treatment with BV plus doxorubicin and dacarbazine (AD) resulted in a CR rate of 97% at end of treatment (EOT), as well as a promising 4-year progression-free survival (PFS) estimate of 91%. There were no cases of ≥Grade 3 peripheral neuropathy and only 9% were Grade 2 (Abramson 2021).

Aims: This abstract will present the preliminary safety and efficacy results from Part B of the SGN35-027 study, which enrolled pts with Ann Arbor Stage II cHL with bulky mediastinal disease (defined as a single node or nodal mass ≥10 cm as determined by CT imaging), or Stage III or IV cHL.

Methods: SGN35-027 ( NCT03646123 ) is an open-label, multiple part, multicenter, phase 2 clinical trial. Pts received up to 6 cycles of AN+AD (consisting of BV 1.2 mg/kg, nivolumab 240 mg, doxorubicin 25 mg/m 2 , and dacarbazine 375 mg/m 2 ). All study drugs were administered by IV infusion on Days 1 and 15 of each 28-day cycle. The primary endpoint was CR rate at EOT. Key secondary endpoints included safety, tolerability, ORR, and PFS. Disease response and progression was assessed by investigators using the Lugano Classification Revised Staging System for nodal non-Hodgkin and Hodgkin Lymphomas (Cheson 2014) and Lymphoma Response to Immunomodulatory Therapy Criteria (LYRIC) (Cheson 2016) at Cycle 2 and EOT.

Results: In Part B, 58 pts were enrolled and 57 pts received at least 1 dose of study treatment. Of these 57 pts, 30 (53%) were male, 27 (47%) were female, 50 (88%) were white, 46 (81%) were not of Hispanic or Latino/a or Spanish origins, and 54 (95%) were <65 years old. Median age was 35 yrs (range: 19-78 yrs); 30% had Stage II cHL with bulky mediastinal disease, while 18% and 51% had Stage III and IV cHL, respectively.

Of 58 pts enrolled, 52 (90%) completed treatment and 4 (7%) discontinued due to AEs. The most frequently reported TEAEs were nausea, fatigue, and diarrhea (70%, 51%, and 46% of pts, respectively). Grade 3 or higher TEAEs were reported in 30 pts (53%), with neutropenia and increased ALT in 5 pts (9%) being the most frequent. Treatment-related AEs occurred in 98% of pts; the most frequent were nausea, fatigue, and peripheral sensory neuropathy (65%, 46%, and 39% of pts, respectively). Treatment-related SAEs occurred in 8 pts (14%), of which pneumonitis was the most common (3 pts [5%]). Immune-mediated AEs were observed in 18 pts (32%), and hypothyroidism (4 pts [7%]) was the most frequently reported. No febrile neutropenia was observed, and there were no Grade 5 AEs.

Preliminary results show at EOT, 52 pts had a response to treatment (ORR 93% [95% CI:82.7-98.0]), with 49 pts having a complete response (CR rate: 88% [95% CI: 75.9-94.8]).

Summary/Conclusion: Preliminary results demonstrate that AN+AD has promising clinical activity and is well-tolerated, with no new safety signals observed. The omission of bleomycin and vinblastine may have contributed to the absence of certain AEs, such as febrile neutropenia. AN+AD may provide another active treatment option for patients with 1L advanced cHL.

P1090: CAMRELIZUMAB (CAM) COMBINED WITH GEMOX CHEMOTHERAPY RESULTS IN HIGH COMPLETE METABOLIC RESPONSE RATES IN RELAPSED/REFRACTORY CLASSIC HODGKIN LYMPHOMA (CHL): A PHASE II TRIAL

Y. Xie 1,* , Y. Tang 1 , W. Zheng 1 , L. Ping 1 , N. Lin 1 , M. Tu 1 , C. Zhang 1 , Z. Ying 1 , W. Liu 1 , L. Deng 1 , M. Wu 1 , L. Mi 1 , T. Du 1 , X. Wang 1 , J. Zhu 1 , Y. Song 1

1 Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Lymphoma, Peking University Cancer Hospital & Institute, Beijing, China

Background: Approximately 30-35% of patients with classic Hodgkin Lymphoma will prove refractory to frontline therapy or relapse subsequently. Traditional second-line chemotherapy regimens result in complete response rates about 20-40%. Achievement of complete metabolic response (CMR) assessed by PET/CT imaging prior to hematopoietic stem cell transplant (HSCT) predicts favorable progression free survival (PFS) and overall survival (OS). PD‐1 antibody has shown remarkable efficacy in relapsed or refractory hodgkin lymphoma (R/R HL).

Aims: we designed a clinical trial to evaluate the efficacy and safety of PD‐1 antibody camrelizumab combined with GEMOX chemotherapy in R/R HL patients. The preliminary results had been obtained.

Methods: This is an open‐label, single‐center, non‐randomized, phase 2 study ( NCT04239170 ). The main inclusion criteria included age ≥ 18, ECOG < 2, histopathology confirmed classical HL, no more than 3 lines of previous chemotherapy treatment, having measurable lesion(s) assessed by Lugano 2014 criteria and preparing to receive autologous stem cell transplantation (ASCT). Patients were treated with 2 cycles of camrelizumab (CAM, 200 mg IV, q2w) followed by 2 cycles (28-day cycle) of CAM (IV, day 1 and day 15) combined with standard GEMOX (gemcitabine 1000 mg/m 2 and oxaliplatin 100mg/m 2 , IV, day 1 and day 15). Patients achieved CR were transferred to stem cell mobilization/collection stage; patients achieved PR/SD could have another cycle of CAM plus GEMOX, patients who achieved PR/CR underwent stem cell mobilization/collection. For patients waiting more than 4 weeks for ASCT, 1-2 cycles of CAM monotherapy were allowed. The primary endpoint is the proportion of patients who achieved PET‐CT‐confirmed complete response (CR) according to Lugano 2014 criteria.

Results: From March 2020 to December 2021, 30 patients were enrolled. One patient withdrew informed consent. The median age of remaining 29 patients was 34 years old (ranged from 22 to 63), with a male‐to‐female ratio of 16:13. At the first tumor response evaluation, 69% (20/29) patients achieved CR and 17% (5/29) patients achieved partial response (PR), resulting an overall response rate (ORR) of 86%. Only one patient had progression disease (PD). The patients who achieved PR or stable disease (SD) received an additional cycle of CAM plus GEMOX treatment, and one patient who had PR in the first evaluation was found to achieve CR. Up to now, collection of the autologous hematopoietic stem cells was completed in thirteen patients. With a cutoff date of February 10, 2022, the 1-year (progression-free survival) PFS rate was 74% and the 1-year (overall survival) OS rate was 100%, median PFS and OS were not reached with too few events (Figure). Most adverse events (AEs) were of grade 1 to 2. Common AEs included reactive capillary endothelial proliferation (RCEP), alanine aminotransferase (ALT)/ aspartate aminotransferase (AST) elevation, vomiting, nausea, hyperuricemia (all > 30%). Hematologic AEs included neutropenia (> 34%). No grade 4 or above AEs occurred. Grade 3 AE occurred in 6 patients, including 1 pulmonary infection, 1 nausea, 1 fever, 1 hypertriglyceridemia, 1 ALT elevation, and 1 neutropenia. Serious adverse events included 1 case of interstitial pneumonia (grade 2) and 1 case of fever (grade 3) resulting in hospitalization.

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Summary/Conclusion: Camrelizumab combined with GEMOX chemotherapy showed encouraging clinical efficacy and tolerable toxicity in R/R HL patients and merited further study.

P1091: FEASIBILITY OF ASCT AFTER ANTI-PD-1 THERAPY FOR R/R CLASSICAL HODGKIN LYMPHOMA

N. Mochkin 1,* , V. Sarzhevskiy 1 , Y. Protopopova 2 , E. Demina 1 , V. Melnichenko 1 , V. Bogatyrev 1 , A. Samoylova 1 , A. Mamedova 1 , A. Rukavitsyn 1 , A. Bannikova 1 , E. Smirnova 1 , N. Shorokhov 1

1 FSBI National Medical Surgical Center n.a. N.I. Pirogov; 2 I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia

Background: Immunotherapy with checkpoint inhibitors (anti-PD-1) is highly effective in relapsed/refractory (R/R) classical Hodgkin Lymphoma (cHL) and leads to more than 70% overall response rate. However, long-term progression-free survival rate is not sufficient. Consolidation with ASCT after anti-PD-1 could be a very promising option even among previously chemorefractory patients. Nevertheless, there is not enough experience about the safety and feasibility of ASCT after anti-PD-1 in the context of possible immune-related adverse events (AE).

Aims: to assess the safety and feasibility of ASCT after anti-PD-1 therapy in patients with R/R cHL.

Methods: We retrospectively analysed patients with cHL who previously underwent ASCT from November 2018 until December 2021 after treatment with anti-PD-1. A median patient age was 34 years (range, 20-55), 22 patients were female, 21 - male. Patients received anti-PD-1 as monotherapy (n=17), combination of anti-PD-1 and chemotherapy (n=4), anti-PD-1 monotherapy followed by combination of anti-PD-1 and chemotherapy (n=14), anti-PD-1 monotherapy followed by salvage therapy (n=3) and other (n=2). A median number of anti-PD-1 cycles was 6 (range, 1-45). The median time from the last dose of anti-PD1 was 69 days (range, 14-365). Conditioning regimen for ASCT consisted of BeEAM (n=12), BeAC (n=30) and other (n=1). Safety assessment was performed by Common Terminology Criteria for Adverse Events (CTCAE) (v5.0).

Results: Forty-three eligible patients were enrolled in this analysis. A median time to engraftment was 10.5 days (range, 9-26). A median time to platelet count > 20x109/L was 13.5 days (range, 7-43). A median use of G-CSF after high-dose chemotherapy followed by ASCT was 9 days (range, 0-26). PEG-G-CSF (empegfilgrastim 7.5 mg/ml once) was used in 10 patients (23.3%). Plerixafor was used in 6 patients (14%). Neutropenic fever was observed in 32 patients (74.4%). Documented infections were reported in 19 patients (44.2%) among which were 5 cases of clostridial colitis (11.6%). A median intravenous administration of antibiotics was 8 days (range, 0-23). Grade 3-4 mucositis and enteropathy was reported in 2 patients (4.6%) and 6 (14%), respectively. Other non-hematological toxicity was observed in 10 patients (23.4%): 8 patients (18.6%) developed engraftment syndrome (that was successfully treated with steroids), grade 4 autoimmune toxic myocarditis was reported in 1 patient (2.3%). One death due to grade 5 autoimmune myocarditis in combination with grade 5 autoimmune pneumonitis was reported (2.3%). A median duration of hospitalization was 25 days (range, 14-72). Transplant-related mortality (TRM) was reported in 2 patients (4.6%).

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Summary/Conclusion: The obtained data on safety of ASCT after anti-PD-1 treatment generally correspond to the data of international studies. Toxicity profile tends to be very similar to the published data about ASCT without anti-PD-1 previous treatment, especially in terms of TRM. However, we observe a high incidence of engraftment syndrome that can lead to fulminant immune-related AE (myocarditis and pneumonitis) probably. Attention should be paid to the defining of optimal “wash-out” period after last dose of anti-PD-1 and early defining of possible life-threatening immune related AE (especially myocarditis and pneumonitis).

P1092: NODULAR LYMPHOCYTE PREDOMINANT HODGKIN LYMPHOMA (NLPHL): HISTOLOGYCAL AND CLINICAL REVIEW IN A SINGLE CENTRE.

E. Rámila 1,* , N. Papaleo 2 , A. Arrieta 2 , C. Blazquez 2 , M. Solorzano 2 , M. Vidal 2 , X. Vilaseca 1 , J. Piedra 1 , J. I. Martinez 1

1 Department of Hematology; 2 Department of Pathology, Hospital Universitari Parc Taulí Sabadell (Barcelona), Sabadell, Spain

Background: NLPHL constitutes a subtype of Hodgkin lymphoma with distinct clinical characteristics and evolution.

Aims: To review the histopatologycal diagnosis and clinical data in patients with NLPHL in our institution between 1999-2021.

Methods: We retrospectively reviewed the histology of lymph node biopsy and clinical data, treatment and outcome of 21 adult patients consecutively diagnosed of a NLPHL between 1999 and 2021.

Results: Twenty-one adult patients with diagnosis of NLPHL (3 of which presented transformation areas to diffuse large B-cell lymphoma (t-DLBCL) in the biopsy), were identified. After the histological review of the biopsies 1 patient was reclassified as diffuse large B cell lymphoma (DLBCL) and another patient as NLPLH with t-DLBCL. Six (30%) patients showed a typical histological pattern (A-B) in the biopsy whereas 14 (70%) presented histopathological variants (pattern C-F) including the 4 patients with transformation areas to DLBCL. Thus, the clinical data of a total number of 20 patients with NLPLH (4 of them with transformation areas) were reviewed.

Twelve (60%) patients were males and 8 (40%) female with a median age of 46 years (range 17-79). Regarding the clinical stage at diagnosis, 9 (45%), 2 (10%), 5 (25%) and 4 (20%) were in favourable IA stage, favourable IIA stage, unfavourable I-IIA stage (according the German Study Hodgkin Group and European Organisation for Research and Treatment of Cancer criteria) and advanced III-IV stage respectively. Three of the 4 patients in advance stage presented variant histological patterns.

Among the 9 patients in favourable clinical IA stage the treatment administered was the following: 2 of them didn’t receive any further treatment (in one by rejection and other patient was in surveillance after lymph node resection), 5 received Radiotherapy and 2 received combined treatment (chemotherapy with ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) and radiotherapy). The 2 patients in favourable IIA stage received combined treatment. Among the 5 patients in unfavourable I-II clinical stage, 3 received combined treatment (ABVD and radiotherapy), 1 patient was treated with ABVD and 1 with R-CHOP (rituximab, ciclophosphamide, doxorubicin, vincristine and prednisone) due to areas of t-DLBCL in the biopsy. Patients (4) in advanced stage were treated with chemotherapy: 2 with ABVD, 1 with CVP (ciclophosphamide, vincristine and prednisone) and 1 with rituximab and platinum based regimen (transformation areas to DLBCL in the biopsy and previous episode of DLBCL).

Regarding to responses, 15 (83.3%) of the 18 treated patients achieved a complete remission and 3 (16.7%) were refractory (2 of them received second line therapy incuding rituximab with a complete remission). One patient relapsed; he was refractory to ABVD and achieved a complete remission with R-CHOP.

With a median follow-up of 5.9 years (range 0.8-18) the overall survival is 90%; 2 patients have died (1 of them, a 79 years old patient with comorbidities, due to lymphoma).

Summary/Conclusion: We have seen a 95% of concordance with the initial diagnosis after a histological review in our patients with NLPLH. 20% of the biopsies showed transformation areas to DLBCL. Most of the patients (80%) presented in a localized stage. Refractory patients responded well to antiCD20 treatment. The prognosis is good with an overall survival of 90%.

P1093: A NEW PROGNOSTIC MODEL FOR INDIVIDUAL OUTCOME PREDICTION IN ADVANCED-STAGE HODGKIN LYMPHOMA.

R. Rask Kragh Jørgensen 1,2,* , S. Eloranta 3 , M. Tang Severinsen 1,2 , A. Kiesbye Øvlisen 1 , K. Bjøro Smeland 4 , C. Kiserud 4 , J. Haaber Christensen 5 , M. Hutchings 6 , R. Bo-Dahl Sørensen 7 , P. Kamper 8 , I. Glimelius 3,9 , K. E. Smedby 3,10 , F. Bergström 3,11 , T. C. El-Galaly 1,2 , L. Hjort Jakobsen 1,2,12

1 Department of Hematology, Clinical Cancer Research Centre, Aalborg University Hospital; 2 Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; 3 Department of Medicine Solna, Division of Clinical Epidemiology, Karolinska Institutet, Stockholm, Sweden; 4 Department of Oncology, Oslo University Hospital, Oslo, Norway; 5 Department of Hematology, Odense Universitets Hospital, Odense; 6 Department of Hematology, Copenhagen University Hospital, Copenhagen; 7 Department of Hematology, Zealand University Hospital, Roskilde; 8 Department of Hematology, Aarhus University Hospital, Aarhus, Denmark; 9 Department of Immunology, Genetics and Pathology, Unit of Oncology, Uppsala University, Uppsala; 10 Department of Hematology, Karolinska University Hospital; 11 Department of Mathematics, Stockholm University, Stockholm, Sweden; 12 Department of Mathematical Sciences, Aalborg University, Aalborg, Denmark

Background: Patients diagnosed with advanced-stage Hodgkin lymphoma (aHL) are commonly risk stratified based on prognostic scores like the International Prognostic Score (IPS). These scores are often based on dichotomized clinical predictors, leading to loss of information and thus inaccurate prognoses.

Aims: This study investigated if a machine learning approach utilizing real world data from aHL patients can outcompete the IPS in predicting overall survival (OS).

Methods: This study included patients with classical aHL registered in the Danish Lymphoma Registry (LYFO). We developed a new prognostic model for predicting OS using stacking, which combined several predictive survival models into a single model. The models used in the stacking, were Cox porportional hazard (CPH), Flexible parametric survival (FPS), Random Survival forest and IPS models, with different combinations of covariates included depending on the model. The time-varying area under the curve (AUC), integrated Brier score (IBS), and concordance index (C-Index) were used to measure performance of the new model as well as the IPS and a simplified version containing three risk-factors (IPS-3). The performance measures were computed using 10-fold cross-validation.

Results: A total of 1,110 aHL patients were included and the 5-year OS was 81% (95% CI, 79-84%). The IBS for the stacking, IPS, and IPS-3 model was 0.078, 0.099, and 0.096, respectively, while the C-Index was 0.86, 0.73, and 0.71. The time-varying AUC was consistently higher for the stacking-model compared to IPS-models within the first five years after diagnosis (Figure).

According to the IPS-model, 143 (12.9%) patients were high-risk (IPS 5-7). The 5-year OS for these patients was 62% (95% CI, 54-71%). We defined an alternative high-risk group by including the 143 patients with the lowest predicted 5-year OS based on the stacking-model. This high-risk group had a 5-year OS of 40% (95% CI, 32-50%).

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Summary/Conclusion: The new prognostic model for aHL based on machine-learning technique (stacked model) demonstrated a substantial improvement in predictive performance compared to the IPS-models. The model also detected a high-risk group with worse OS than the IPS high-risk group. The model is planned for international validation.

P1094: CHARACTERISTICS, MANAGEMENT, AND OUTCOMES OF PATIENTS WITH T-CELL LARGE GRANULAR LYMPHOCYTIC LEUKEMIA AT A LARGE TERTIARY CARE CENTER

H. Audil 1,* , M. Shah 1 , K. Rabe 1 , W. Ding 1 , P. Hampel 1 , N. N. Bennani 1 , T. Call 1 , C. Hanson 1 , Y. Wang 1 , A. Koehler 1 , S. Schwager 1 , J. Leis 2 , S. Slager 1 , S. Kenderian 1 , A. Al-Kali 1 , G. Nowakowski 1 , M. Shi 1 , S. Parikh 1

1 Mayo Clinic, Rochester; 2 Mayo Clinic, Phoenix, United States of America

Background: Large granular lymphocytic leukemia (LGLL) comprises 2-6% of chronic lymphoproliferative disorders. T-LGLL accounts for approximately 85% of cases, while NK-LGLL represents 15% of cases. There is a significant knowledge gap regarding diagnosis, management, and outcomes of LGLL.

Aims: Our aims were to describe the clinical characteristics, time to first therapy (TTFT), type of first line therapy, response to treatment, and overall survival (OS) in patients with LGLL seen at our institution.

Methods: We identified LGLL patients from the Mayo Clinic Chronic Lymphoproliferative Disorders Database seen between 1/1995-7/2021. At least 3 of the following 4 criteria were used to diagnose LGLL (T-LGLL or NK-LGLL): (1) a distinct T-cell or NK-cell population by flow cytometry; (2) a clonal T-cell or NK-cell population; (3) intrasinusoidal cytotoxic T-cell or NK-cell infiltrates in bone marrow, spleen, or liver; and (4) persistence of the abnormal T-cell or NK-cell population or unexplained cytopenia for more than 6 months (Salama, BCJ , 2022). Baseline clinical characteristics, TTFT, first line therapies, and OS were analyzed for all patients. The Mayo Clinic IRB approved this study.

Results: We identified 217 patients who met inclusion criteria for LGLL diagnosis; 196 (90.3%) patients had T-LGLL and 21 (9.7%) patients had NK-LGLL. The median age at diagnosis was 65 years [range 21-86], and 120 (55.3%) were male. 159 (73.3%) patients had evidence of cytopenia at the time of diagnosis. Frequently associated clinical characteristics included dependence on red cell or platelet transfusions (44.3%), colony-stimulating factors (20.0%), or both (21.7%); splenomegaly (32.9%); history of autoimmune disease (24.9%); history of hematologic disorder (18.4%) or malignancy (5.6%); and constitutional symptoms (11.1%).

The median follow-up was 8.4 years; 133 patients died, and 111 patients were treated for LGLL during this period. The median TTFT after diagnosis was 47.0 months (42.8 for T-LGLL and 68.0 for NK-LGLL) with a median of 1 (range 0-11) line of treatment. First-line therapy consisted of methotrexate in 33 (29.7%) patients, cyclophosphamide in 25 (22.5%), cyclosporine in 6 (5.4%), chlorambucil in 6 (5.4%), rituximab in 5 (4.5%), steroids alone in 17 (15.3%), and splenectomy in 7 (6.3%); 5 (4.5%) patients received combination therapy and 2 (1.8%) received IVIG. Alemtuzumab, cladribine, fludarabine, lenalidomide, and vincristine were first-line therapy in 1 (0.9%) patient each. Response to first-line therapy (Lamy, Blood , 2011) consisted of complete response (CR) in 31%, partial response (PR) in 33%, and treatment failure or progressive disease in 36% patients. The median OS was 11.0 years: 11.0 years for T-LGLL and 8.1 years for NK-LGLL (p = 0.90; Figure 1).

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Summary/Conclusion: In this retrospective study of LGLL, we utilized stringent pathology inclusion criteria to allow for analyses of a homogenous cohort of patients seen at our institution. One in 4 patients with LGLL had a concomitant autoimmune condition, and 25% had a concomitant hematologic condition or hematologic malignancy at the time of initial diagnosis. Approximately a third of all patients who needed LGLL treatment did not achieve a response to frontline therapy, suggesting that newer treatments are needed to improve outcomes of this rare disease.

P1095: TREATMENT ATTRIBUTES FOR 3RD LINE FOLLICULAR LYMPHOMA TREATMENT DECISION-MAKING: PHYSICIAN PERSPECTIVES FROM A SURVEY ACROSS WESTERN EUROPE AND THE UNITED STATES

P. C. Johnson 1,* , A. Bailey 2 , N. Milloy 2 , E. Clayton 2 , R. G. Quek 3 , Q. Ma 3

1 Department of Medicine, Division of Hematology & Oncology, Massachusetts General Hospital Cancer Center & Harvard Medical School, Boston, United States of America; 2 Oncology, Adelphi Real World, Bollington, United Kingdom; 3 Health Economics & Outcomes Research, Regeneron Pharmaceuticals Inc., Sleepy Hollow, United States of America

Background: Follicular lymphoma (FL) is the second most common subtype of non-Hodgkin lymphoma (NHL), and is the most common of the clinically indolent NHLs. Treatment (tx) selection must integrate a myriad of clinical and patient factors, including disease resistance, cumulative toxicities and treatment efficacy, as well as patient quality of life (QoL) and the costs of therapies. However, real-world data regarding physician perspectives on tx selection is lacking, especially at later lines of therapy.

Aims: This study aims to assess physician perspectives regarding key tx selection attributes including those beyond efficacy and safety, as well as physician perception of patient’s tx preference, when selecting a 3 rd line (3L) FL tx.

Methods: Descriptive data were drawn from the Adelphi FL Disease-Specific Programme™, a point-in-time study that was fielded June 2021-Jan 2022. Haematologists, haem-oncologists and medical oncologists (med-onc) in EUR [France, Germany, Italy, Spain, United Kingdom (UK)] and the US completed online surveys regarding self-reported demographics and the top 7 tx attributes contributing to their rationale for 3L FL tx selection from a comprehensive prespecified list of 28 attributes derived from expert opinions. Physicians also provided their perception on patient’s preference regarding tx attributes for choosing 3L tx.

Results: Of 251 physicians surveyed in EUR (France: n=46; Germany: n=40; Italy: n=43; Spain: n=41; UK: n=31) and the US (n=50), 49% were haem-oncologists, 41% haematologists and 10% med-oncs. 52% primarily worked at academic hospitals.

Progression free survival (74.1%), overall survival (70.1%), duration of response (59.8%), evidence-based efficacy overall / overall response rate (57.4%) and ability to achieve complete response (46.2%) were most frequently physician reported top 7 attributes for 3L tx selection.

Notably, long-term safety and impact on patients’ QoL were reported by 38.6% and 34.3% of physicians, respectively.

Fewer physicians reported cost (4.8%), patient acceptability of lag time between leukapheresis and CAR-T infusion (7.2%), patient acceptability of frequency of administration (8.0%), less monitoring needed (8.8%) and fewer outpatient / inpatient consultations needed (5.6% and 7.6% respectively) as their top 7 3L tx attributes considered.

Frequency of some 3L tx selection attributes reported differed substantially between EUR and the US including long-term safety (35.8% vs 50.0%), impact on patients’ QoL (37.8% vs 20.0%), and suitability for patients aged over 60 years (10.9% vs 22.0%).

Summary/Conclusion: Treatment efficacy and improved survival were the most important attributes for physicians’ 3L FL tx selection whilst safety, impact on patient QoL, frequency of inpatient / outpatient consultations, monitoring requirements, and cost were amongst those attributes considered less important by physicians.

Despite literature indicating that patient QoL and cost of tx should both be key considerations for physicians when choosing FL tx, our study highlights these are less commonly reported attributes for treatment selection. Future study is warranted to further explore reasons for physician treatment selection.

P1096: TREATMENT PATTERNS AND OUTCOMES IN RELAPSED/REFRACTORY MANTLE CELL LYMPHOMA

A. Bock 1,* , K. Poonsombudlert 2 , M. Larson 3 , J. Gile 1 , R. Tawfiq 1 , S. Maliske 2 , M. Maurer 3 , J. Cerhan 4 , X. Andrade-gonzalez 1 , A. Saliba 1 , J. Paludo 1 , D. Inwards 1 , S. Ansell 1 , T. Witzig 1 , T. Habermann 1 , B. Link 2 , S. Ayyappan 2 , G. Nowakowski 1 , U. Farooq 2 , Y. Wang 1

1 Division of Hematology, Mayo Clinic, Rochester; 2 Division of Hematology, University of Iowa, Iowa City; 3 Division of Biostatistics; 4 Division of Epidemiology, Department of Quantitative Health Sciences, Mayo Clinic, Rochester, United States of America

Background: Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma with heterogeneous clinical presentation and no established standard of care. In the last two decades, the pattern of frontline therapy for MCL has evolved, and multiple new agents became available for relapsed/refractory (R/R) MCL. The pattern of treatment, its evolution with time, and the association with treatment outcomes in R/R MCL are not well understood.

Aims: To characterize second line (2L) treatment patterns and outcomes in patients with R/R MCL initially diagnosed in the rituximab era.

Methods: Patients with newly diagnosed MCL between August 2002 and April 2015 and followed through 2021 were identified from the Mayo Clinic/University of Iowa Molecular Epidemiology Resource (MER) prospective cohort study. Patients who initiated therapy for R/R MCL after relapse or progression to first line (1L) therapy were included in this analysis. Clinical characteristics and 1L and 2L therapies were abstracted from MER and by chart review; treatment outcomes were analyzed.

Results: Among a total of 343 MCL patients with a median follow-up of 11.8 years, 188 had documented progression/relapse to 1L therapy, of which 170 had 2L treatment information available for this analysis. At the time of 2L therapy, 123 (72%) patients had an age >60, 136 (80%) were male, 132 (78%) had stage III/IV, and simplified MIPI was 0-3 (low) in 37 (35%), 4-5 (intermediate) in 40 (37%), and 6-11 (high) in 30 (28%; simplified MIPI missing in 63 patients). The median time from diagnosis to 2L was 27 months (range 0.5-141).

The patterns of 1L and 2L treatment are shown in Figure 1. 1L treatment included HiDAC-based (n=31, 13 had autologous stem cell transplant [ASCT]), R-CHOP-like (n=79, 45 had ASCT), R-Bendamustine (BR, n=22, 2 had ASCT), other systemic (n=29), and non-systemic (n=9). 15 patients (9%) received rituximab maintenance after induction or ASCT. 2L treatment included salvage therapy followed by ASCT (n=13; salvage with HiDAC-based n=2, R-CHOP-like n=5, BR n=3, platinum-based n=2, other n=1) or Allogeneic SCT (AlloSCT, n=5), HiDAC-based n=9, R-CHOP-like n=6, BR n=33, BTK inhibitor (BTKi, n=21), other targeted therapy (lenalidomide, bortezomib, mTOR inhibitors, n=26), other systemic (n=47), and non-systemic (n=10).

The median follow-up from 2L therapy was 8.2 years. The objective response rate to 2L therapy was 71% (45% complete response). The median progression-free survival (PFS) and overall survival (OS) were 1.1 and 4.0 years, respectively. The 2-year PFS rate and 5-year OS rate were 55% (95% CI 36-84) and 71% (95% CI 49-100), respectively for patients treated with BTKi at 2L, and 42% (95% CI 28-63) and 46% (95% CI 32-68), respectively, for those treated with BR. In patients intended to undergo salvage therapy followed by ASCT (n=18, n=13 transplanted) or AlloSCT (n=5, all transplanted), the 2-year PFS rate and 5-year OS rate were 80% (95% CI 52-100) and 60% (95% CI 41-88), and 80% (95% CI 52-100) and 61% (95% CI 42-88), respectively. Patients treated with HiDAC-based, R-CHOP-like, other targeted therapy, or other systemic therapy had poorer outcomes (2-year PFS 0-27%, 5-year OS 0-35%).

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Summary/Conclusion: Second line treatment for MCL was very heterogenous in terms of treatment and outcomes and was likely impacted by 1L treatment and other clinical factors. The evolution of 1L and R/R MCL treatment patterns and the associated patient characteristics and outcomes requires further exploration in larger datasets, given extensive heterogeneity in practice patterns.

P1097: MEK-INHIBITORS IN TREATMENT OF LANGERHANS CELL HISTIOCYTOSIS

E. Burtsev 1,* , G. Bronin 1

1 HSCT and BMT, Morozov Children’s Hospital, Moscow, Russia

Background: There are increasing data of targeted therapy efficacy of different types of Langerhans cell histiocytosis (LCH) with inhibitors of BRAF-specific serin-threonine kinase (BRAF-inhibitors) in cases with BRAFV600e mutation published last years. At the same time there are lack of published data of MAPK/ERK pathway inhibitors (MEK-inhibitors) use in pediatric patients with BRAF-negative forms of LCH.

Aims: Aim of the study is to evaluate efficacy and safety of MEK-inhibitor (cobimetinib) in eight pediatric BRAFV600e-negative refractory LCH patients.

Methods: The study included 8 children with various forms of LCH. BRAFV600e mutation was not found in any case included in the trial. All patients received therapy according to the LCH IV protocol and were diagnosed with progression of LCH during or after termination of the treatment. Monotherapy with cobimetinib was used as a second line therapy in all cases. The response to the targeted therapy was assessed in accordance with the international scale Response Evaluation Criteria in Solid Tumors (RECIST v.1.1). The assessment of the toxicity was performed in accordance with the international scale of Common Terminology Criteria for Adverse Events (CTCAE v.5.0).

Results: Complete response to cobimetinib was not achieved in any patient. Partial response was established in 62,5% cases. One patient was diagnosed with disease progression in three months after termination of the therapy. The incidence of adverse events was high (75%). The most common side effects were diarrhea (75%), rush (50%) and electrolyte disturbances (37,5%).

Summary/Conclusion: Cobimetinib monotherapy is effective in BRAF V600e- negative refractory pediatric LCH patients. The response to the treatment can be delayed. All cases of the toxicity were dose dependant and successfully resolved after dose correction. Further research is needed to define duration of treatment and optimal dosage regimens.

P1098: OUTCOME OF FOLLICULAR LYMPHOMA PATIENTS IN MAINTENANCE TREATMENT WITH ANTICD20 MONOCLONAL ANTIBODIES IN SARS-COV2 ERA: RESULTS FROM A MULTICENTER, RETROSPECTIVE- PROSPECTIVE ITALIAN STUDY.

A. Castellino 1,* , C. Castellino 1 , C. Boccomini 2 , M. Clerico 3 , P. Nicoli 4 , A. Vanazzi 5 , F. Fanelli 6 , T. Perrone 7 , F. Marchesi 8 , F. Cocito 9 , M. Merli 10 , S. Bigliardi 11 , B. Mecacci 12 , V. Bozzoli 13 , G. Margiotta-Casaluci 14 , E. Meli 15 , A. Anastasia 16 , L. Farina 17 , O. Annibali 18 , D. Gottardi 19 , M. Zanni 20 , A. Conconi 21 , C. Ciochetto 22 , N. Cenfra 23 , F. Rotondo 24 , S. Ratotti 25 , A. Cuneo 26 , C. Selleri 27 , S. Galimberti 28 , M. Massaia 1

1 Hematology Unit, AO Santa Croce e Carle di Cuneo, Cuneo; 2 Hematology Unit, Citta’ della Salute e della Scienza; 3 Hematology Unit, Citta della Salute e della Scienza, Torino; 4 Hematology Unit, San Luigi Gonzaga Hospital and University, Orbassano; 5 Hematology Unit, Istituto Europeo di Oncologia, Milano; 6 Hematology Unit, San Camillo Hospital, Roma; 7 Hematology Unit, Policlinico Hospital, Bari; 8 Hematology Unit, IRCCS Regina Elena National Cancer Institute, Roma; 9 Hematology Unit, San Gerardo Hospital, Monza; 10 Hematology Unit, Ospedale di Circolo ASST Sette Laghi, Varese; 11 Hematology Unit, Ospedale Civile, Sassuolo; 12 Hematology Unit, University of Siena, Siena; 13 Hematology Unit, Vito Fazzi Hospital, Lecce; 14 Hematology Unit, Department of Translation Medicine, AOU Maggiore della Carità, Novara; 15 Hematology Unit, ASST Grande Ospedale Metropolitano Niguarda, Milano; 16 Hematology Unit, Ospedali Civili, Brescia; 17 Hematology Unit, Istituto Nazionale Tumori, Milano; 18 Hematology Unit, Università Campus Bio-Medico, Roma; 19 Hematology Unit, Ospedale Mauriziano, Torino; 20 Hematology Unit, SS Arrigo e Biagio Hospital, Alessandria; 21 Hematology Unit, Ospedale degli Infermi, Biella; 22 Hematology Unit, ASL TO4, Ivrea; 23 Hematology Unit, Ospedale Santa Maria Goretti, Latina; 24 Hematology Unit, Ospedale Papardo, Messina; 25 Hematology Unit, IRCCS Fondazione San Matteo, Pavia; 26 Hematology Unit, Sant’Anna Hospital, Ferrara; 27 Hematology Unit, AOU S. Giovanni di Dio e Ruggi D’Aragona Salerno, Salerno; 28 Hematology Unit, Santa Chiara Hospital and University, Pisa, Italy

Background: Maintenance in FL patients (pts) improves progression free survival (PFS). SARS-Cov2 pandemic posed unique challenges for immunocompromised pts.

Aims: The aim is to evaluate the outcome of FL pts in maintenance with antiCD20-MoAb during SARS-Cov2 pandemic and how suspension of therapy affected lymphoma outcome and the risk of SARS-Cov2 infection and its morbidity and mortality.

Methods: This is an observational, multicenter, retrospective and prospective study.

Results: A total of 420 from 18 Italian Hematological Centers were included in the analysis. Median age was 62 years old (range 27-91 years), 216 pts (51%) were male. Main clinical characteristics of the population were: histological grade 1-2 vs 3A in 288 (69%) vs 109 (26%), while not valuable in 23 (5%) pts; limited I-II vs advanced III-IV stage in 57 (14%) vs 361 (86%) pts, not reported in 2 cases. FLIPI score was low vs intermediate vs high in 71 (17%) vs 151 (36%) vs 192 (46%) patients, respectively, not valuable in 6 cases. All 420 patients included were in maintenance treatment with antiCD20 MoAb at the time of the onset of SARS-Cov2 pandemic (March 2020): 333 (79%) pts were receiving maintenance after a first line, while 87 (21%) after a second line. 342 (81%) pts were receiving Rituximab, while 75 (18%) Obinutuzumab, 3 patients did not start the planned maintenance because of pandemic spread. Status of disease after induction was complete remission (CR) in 374 (89%), partial response (PR) in 41 (10%), progressive disease (PD) in 1, not evaluated in 4 pts, respectively. At the end of maintenance was CR in 265 (63%), PR in 19 (4%), stable disease (SD) in one and PD in 14 (3%) patients, respectively, maintenance is stiil ongoing in 121 (29%) pts. Because of SARS-Cov2 pandemic from March 2020 consequences on maintenance treatment were: temporary suspension in 122 (29%), definitively interruption in123 (29%), no modification in 175 (42%) of pts, respectively. Median number of maintenance treatment administered at the time of SARS-Cov2 pandemic onset was 2 (range 1-12), median number of courses administered at the time of analysis was 8 (range 0-12), in patients who modified treatment because of pandemic median number of performed courses was 7 (range 0-11) and median number of lost cycles were 2 (range 1-12). Pts were divided into two groups according to type of approach to maintenance during pandemic: pts who interrupted maintenance (temporary or definitively): groups A (245 (58%) pts) vs pts who did not modified maintenance: group B (175 (42%) pts). No differences in clinical characteristics, type of therapy and response were observed between the two groups. 29(7%) relapses were observed: 16 (7%) vs 13 (7%) in group A vs B, respectively. 70 (17%) pts experienced SARS-Cov2 positivity: 47 (19%) vs 23 (13%) in group A vs B, respectively. 53 (76%) pts had symptomatic COVID syndrome and 43 (61%) were hospitalized, with no differences between the two groups. Anti-SARS-Cov2 vaccine was administered in 349 patients, serology assessment was done in 46% of cases, showing 21 (13%) reactive vs 138 (87%) not reactive pts, with no differences between the two groups. 21 (30%) pts died because of COVID: 9 (19%) vs 12 (52%) in groups A vs B, respectively.

Summary/Conclusion: Suspension of maintenance treatment during SARS-Cov2 pandemic did not show a protection in terms of SARS-Cov2 positivity and morbidity. A trend in lower mortality is suggested. No differences in terms of relapse rate were observed, but longer follow up is needed.

P1099: THE SPECTRUM OF SECOND PRIMARY MALIGNANCIES AND CAUSE-SPECIFIC MORTALITY AMONG PATIENTS WITH FOLLICULAR LYMPHOMA IN THE UNITED STATES: A POPULATION-BASED STUDY.

K. Chamarti 1,* , V. Rudraraju 2 , S. Kiani 1

1 Department of Internal Medicine, Texas Tech University Health Sciences Center at Permian Basin, Odessa, United States of America; 2 Department of Internal Medicine, Jawaharlal Nehru Medical College, Belgaum, India

Background: Follicular lymphoma (FL) is the second most common type of non-Hodgkin lymphoma (NHL). FL accounts for 35 percent of NHLs and has an estimated incidence of 3.18 cases per 1,00,000 people in the United States. The clinical course of FL is variable, and it has median overall survival of 10 to 12 years. In the era of targeted therapies with anti-CD20 antibodies, there is data lacking on the spectrum of second primary malignancies (SPMs) and cause-specific mortality in patients with follicular lymphoma.

Aims: In the current study, we aim to identify the risk of SPM and cause specific mortality in FL patients in the era of targeted therapies with anti CD-20 antibodies using a national registry from the United States.

Methods: Using the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER)-18, we conducted a retrospective study with patients diagnosed with FL (ICD-0-3 code 9690/3) between 2012 and 2018. We calculated the risk of SPMs developing ≥ 2 months using standardized incidence ratios (SIRs) and cause-specific mortality by standardized mortality ratios (SMRs) and absolute excess risk (AER)/10,000 population.

Results: A total of 6,215 patients with follicular lymphoma (FL) were included in this study. The median age at diagnosis was 66 years (range 3 to 85 years) and the median follow-up duration was 31 months (range 0 to 81 months). A total of 355 patients (5.7%) developed 376 SPMs, of which 56% were solid organ malignancies, and 44% were hematologic neoplasms. The latency period for SPM development was 20 months (range 2 to 80 months). The SPM risk for the cohort was significantly elevated (SIR= 1.99, 95% confidence interval [CI] = 1.79-2.2, P<0.05) when compared with the general population. The SIR was higher in females (SIR= 2.16, 95% CI=1.85-2.51) when compared to males (SIR= 1.86, 95% CI= 1.61- 2.13). The SIR for age groups were as follows: age < 65 years (SIR= 2.84, 95% CI= 2.43-3.31), age > 65 years (SIR= 1.59, 95% CI= 1.38-1.82). The risk for hematopoietic SPMs was higher (SIR= 8.66, 95% CI= 7.33-10.15) than solid organ tumors (SIR= 1.30, 95% CI= 1.13-1.49). Specific SPMs with the highest risk included NHL (SIR= 16.24, 95% CI= 13.62-19.22), Hodgkin lymphoma (SIR= 14.31, 95% CI= 5.25-31.15), followed by thyroid cancer (SIR= 4.63, 95% CI= 2.53-7.76), kidney and renal pelvis cancer (SIR= 1.43, 95% CI= 1.55-4.14), and lung cancer (SIR= 2.16, 95% CI= 1.63-2.79). The risk of developing Hodgkin lymphoma, thyroid, and kidney cancers was high in the first year of FL diagnosis. While, the risk for NHL was elevated throughout the follow-up period. At last follow-up, 724 (11.5%) patients had died, of which 65% were due to malignant neoplasms. The risk of mortality due to all malignant cancers combined was statistically significant (SMR= 6.26, 95% CI= 2.00-2.31), largely due to mortality from NHL (SMR= 139.5, 95% CI= 126-154, AER= 286.8), leukemia (SMR= 5.23, 95% CI= 2.99-8.49, AER= 9.63), and infectious diseases (SMR= 2.58, 95% CI= 1.04-5.31, AER=3.19).

Summary/Conclusion: Our study reveals that despite therapeutic advances in FL, hematologic neoplasms (including NHL and leukemia) remain the major cause of death in our cohort. Patients with FL are at higher risk of SPM than the general population, most notably for NHL, Hodgkin lymphoma, followed by thyroid, kidney, and lung cancer. These patients may benefit from cancer-specific screening during follow-up.

P1100: BRUIN MCL-321: A PHASE 3 OPEN-LABEL, RANDOMIZED STUDY OF PIRTOBRUTINIB VS INVESTIGATOR CHOICE OF BTK INHIBITOR IN PATIENTS WITH PREVIOUSLY TREATED, BTK INHIBITOR NAÏVE MCL (TRIAL IN PROGRESS)

T. A. Eyre 1 , N. N. Shah 2 , S. Le Gouill 3 , M. Dreyling 4,* , E. Vandenberghe 5 , W. Jurczak 6 , Y. Wang 7 , C. Y. Cheah 8 , M. Gandhi 9 , C. Chay 10 , J. P. Sharman 11 , D. J. Andorsky 12 , M. Yin 13 , M. Balbas 13 , J. Kherani 13 , M. L. Wang 14

1 Oxford University Hospitals NHS Foundation Trust, Churchill Cancer Center, Oxford, United Kingdom; 2 Medical College of Wisconsin, Milwaukee, United States of America; 3 Service d’hématologie clinique du CHU de Nantes, INSERM CRCINA Nantes-Angers, NeXT Université de Nantes, Nantes, France; 4 Medicine III, LMU University Hospital, Munich, Germany; 5 Hope Directorate St. James Hospital, Dublin, Ireland; 6 Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 7 Division of Hematology, Mayo Clinic, Rochester, United States of America; 8 Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 9 Virginia Cancer Specialists, Fairfax; 10 Messino Cancer Centers, Asheville; 11 Willamette Valley Cancer Institute and Research Center, US Oncology Research; 12 Rocky Mountain Cancer Centers, US Oncology Research, Eugene; 13 Loxo Oncology at Lilly, Stamford; 14 University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Covalent Bruton’s Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of relapsed mantle cell lymphoma (MCL), but these treatments are not curative and the majority of patients will require additional treatment. Covalent BTKi share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage especially in rapidly proliferating tumors with high BTK protein turnover such as MCL. To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. In the phase 1/2 BRUIN study, pirtobrutinib achieved pharmacokinetic exposures that exceeded its BTK IC96 at trough, was well tolerated, and demonstrated promising efficacy in heavily pretreated, poor-prognosis MCL patients, most of whom had prior treatment with a covalent BTKi.

Aims: The purpose of this randomized study is to determine whether pirtobrutinib is superior to investigator’s choice of covalent BTKi in patients with previously treated MCL.

Methods: BRUIN MCL-321 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus investigator’s choice of covalent BTKi monotherapy (ibrutinib, acalabrutinib, or zanubrutinib) in patients with previously treated, BTKi naïve MCL. Approximately 500 patients will be randomized 1:1. Randomization will be stratified by sMIPI risk (low/intermediate vs high), comparator BTKi (ibrutinib vs acalabrutinib/ zanubrutinib), and number of prior lines of therapy (1 vs ≥ 2).

Eligible patients are adults aged ≥18 years with a confirmed diagnosis of MCL (cyclin D1 overexpression, and ≥ 1 B-cell marker) who have received ≥ 1 prior line of systemic therapy for MCL that did not include a prior BTKi. Patients must have measurable disease per Lugano criteria and must have progressed on or relapsed following the most recent line of therapy prior to study enrollment. Key exclusion criteria include a history of current or prior central nervous system (CNS) involvement, significant cardiovascular disease, stroke, or intracranial hemorrhage within 6 months of randomization, and allogeneic stem cell transplant (SCT), autologous SCT or chimeric antigen receptor (CAR) T-cell therapy within 60 days of randomization.

The primary endpoint is progression-free survival (PFS) per Lugano criteria assessed by an independent review committee (IRC), with the goal of demonstrating superiority of pirtobrutinib over investigator’s choice of covalent BTKi. Secondary endpoints include overall response rate (ORR), duration of response (DoR), investigator-assessed PFS per Lugano criteria, overall survival, event-free survival, time to treatment failure, time to next treatment, PFS2 (time from randomization to disease progression on next line of treatment or death from any cause), safety and tolerability, and patient reported outcomes. This global study is currently enrolling patients ( NCT04662255 ).

Results: This study is a Trial in Progress. The results will be presented at a later date.

Summary/Conclusion: This study is a Trial in Progress. The conclusions will be presented at a later date.

P1101: PIRTOBRUTINIB, A HIGHLY SELECTIVE, NON-COVALENT (REVERSIBLE) BTK INHIBITOR IN PREVIOUSLY TREATED MANTLE CELL LYMPHOMA: UPDATED RESULTS FROM THE PHASE 1/2 BRUIN STUDY

T. A. Eyre 1,* , M. L. Wang 2 , N. N. Shah 3 , A. J. Alencar 4 , J. N. Gerson 5 , M. R. Patel 6 , B. Fakhri 7 , E. Vandenberghe 8 , W. Jurczak 9 , X. N. Tan 10 , K. L. Lewis 10 , T. Fenske 3 , Y. Wang 11 , C. C. Coombs 12 , I. Flinn 13 , D. Lewis 14 , S. Le Gouill 15 , M. Gandhi 16 , C. Chay 17 , M. L. Palomba 18 , J. A. Woyach 19 , J. M. Pagel 20 , N. Lamanna 21 , J. P. Sharman 22 , D. J. Andorsky 23 , J. B. Cohen 24 , M. A. Barve 25 , P. Ghia 26,27 , M. Yin 20 , P. L. Zinzani 28 , C. Ujjani 29 , Y. Koh 30 , K. Izutsu 31 , E. Lech-Maranda 32 , J. Kherani 20 , C. Tam 33 , S. Sundaram 34 , B. Nair 20 , D. E. Tsai 20 , M. Balbas 20 , A. R. Mato 18 , C. Y. Cheah 10

1 Oxford University Hospitals NHS Foundation Trust, Churchill Cancer Center, Oxford, United Kingdom; 2 MD Anderson Cancer Center, Houston; 3 Medical College of Wisconsin, Milwaukee; 4 Sylvester Comprehensive Cancer Center, Miami; 5 University of Pennsylvania, Philadelphia; 6 Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota; 7 University of California San Francisco, San Francisco, United States of America; 8 Hope Directorate St. James Hospital, Dublin, Ireland; 9 Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 10 Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 11 Division of Hematology, Mayo Clinic, Rochester; 12 University of North Carolina at Chapel Hill, Chapel Hill; 13 Sarah Cannon Research Institute, Nashville, United States of America; 14 University Hospitals Plymouth NHS, Plymouth, United Kingdom; 15 Service d’hématologie clinique du CHU de Nantes, INSERM CRCINA Nantes-Angers, NeXT Université de Nantes, Nantes, France; 16 Virginia Cancer Specialists, Fairfax; 17 Messino Cancer Centers, Asheville; 18 Memorial Sloan Kettering Cancer Center, New York; 19 The Ohio State University Comprehensive Cancer Center, Columbus; 20 Loxo Oncology at Lilly, Stamford; 21 Herbert Irving Comprehensive Cancer Center, Columbia University, New York; 22 Willamette Valley Cancer Institute and Research Center, US Oncology Research; 23 Rocky Mountain Cancer Centers, US Oncology Research, Eugene; 24 Winship Cancer Institute, Emory University, Atlanta; 25 Mary Crowley Cancer Research, Dallas, United States of America; 26 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 27 Medicine III, LMU University Hospital, Munich, Germany; 28 Institute of Hematology “Seràgnoli” University of Bologna, Bologna, Italy; 29 Fred Hutchinson Cancer Research Center, Seattle, United States of America; 30 Internal Medicine, Seoul National University Hospital, Seoul, South Korea; 31 National Cancer Center Hospital, Tokyo, Japan; 32 Institute of Hematology and Transfusion Medicine, Warsaw, Poland; 33 Peter MacCallum Cancer Center, Royal Melbourne Hospital, and University of Melbourne, Melbourne, Australia; 34 Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States of America

Background: Covalent BTK inhibitors (BTKi) have transformed the management of mantle cell lymphoma (MCL), but these treatments are not curative and the majority of patients (pts) will require additional treatment. Covalent BTKi share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage, for example in rapidly proliferating tumors with high BTK protein turnover such as MCL. To address these limitations, pirtobrutinib, a highly selective, non-covalent (reversible) BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed.

Aims: BRUIN is a multicenter phase 1/2 study ( NCT03740529 ) of oral pirtobrutinib monotherapy in pts with advanced B-cell malignancies who have received ≥2 prior therapies.

Methods: Pirtobrutinib was dose escalated in a standard 3 + 3 design in 28-day cycles. The primary objective for phase 1 was to determine the recommended phase 2 dose (RP2D) and the primary objective of phase 2 was overall response rate (ORR); secondary objectives included duration of response (DoR), progression-free survival (PFS), overall survival (OS), safety and tolerability, and pharmacokinetics. Efficacy evaluable pts included all dosed pts who underwent their first response evaluation or discontinued therapy. Response was assessed every 8 weeks from cycle 3, and every 12 weeks from cycle 13 and was measured according to Lugano Classification. Safety was assessed in all pts (CLL/SLL and NHL).

Results: As of 27 September 2020, 323 pts (170 CLL/SLL, 61 MCL, 26 WM, 26 DLBCL, 13 MZL, 12 FL, 9 RT, and 6 other NHL [other transformation, B-PLL and hairy cell leukemia]) were treated on 7 dose levels (25-300mg QD). Median age was 69 (range 50-87) years for MCL pts. Among the 61 MCL pts, median number of prior lines of therapy was 3 (range, 1-8) and a majority of them had received a prior BTKi (93%), an anti-CD20 antibody (98%) or chemotherapy (92%). No DLTs were reported and MTD was not reached (n=323). 200mg QD was selected as the RP2D. Fatigue (20%), diarrhea (17%) and contusion (13%) were the most frequent treatment-emergent adverse events regardless of attribution or grade seen in ≥10% of pts (n=323). The most common adverse event of grade ≥3 was neutropenia (10%). Treatment-related hemorrhage and hypertension occurred in 5 (2%) and 4 (1%) pts, respectively. Five (1%) pts discontinued due to treatment-related adverse events. At the efficacy cutoff date, 52 prior BTKi treated MCL pts were efficacy evaluable with an ORR of 52% (95% confidence interval 38-66; 13 complete response (CR) [25%], 14 partial response (PR) [27%], 9 stable disease (SD) [17%]), 11 progressive disease (PD) [21%] and 5 [10%] discontinued prior to first response assessment). Median follow up was 6 months (range 0.7-18.3+). Responses were observed in 9/14 pts (64%) with prior autologous or allogeneic stem cell transplant, and 2 of 2 with prior CAR-T cell therapy.

Summary/Conclusion: Pirtobrutinib demonstrated promising efficacy in heavily pretreated, poor-prognosis MCL following multiple prior lines of therapy, including a covalent BTKi. Pirtobrutinib was well tolerated and exhibited a wide therapeutic index. Updated data, including approximately 60 new pts with MCL and an additional 10 months since the prior data cut will be presented.

P1102: A PHASE 1 STUDY OF PARSACLISIB IN COMBINATION WITH RITUXIMAB, BENDAMUSTINE + RITUXIMAB, OR IBRUTINIB IN PATIENTS WITH PREVIOUSLY TREATED B-CELL LYMPHOMA (CITADEL-112): PRELIMINARY SAFETY RESULTS

J.-M. Sancho 1,* , A. Lopez-Guillermo 2 , P. Abrisqueta 3 , A. Kumar 4 , R. Cordoba 5 , M. Tani 6 , W. Zhao 7 , E. Rappold 7 , P. Langmuir 7 , M. Mims 8

1 Clinical Hematology Department, Hospital Germans Trias i Pujol, Institut Català d’Oncologia; 2 Clinic Barcelona; 3 Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), Hospital Universitari Vall d’Hebron, Barcelona, Spain; 4 University of Arizona, Tucson, United States of America; 5 Lymphoma Unit, Department of Hematology, Fundacion Jimenez Diaz University Hospital, Health Research Institute IIS-FJD, Madrid, Spain; 6 Haematology Unit, Santa Maria delle Croci Hospital, Ravenna, Italy; 7 Incyte Corporation, Wilmington; 8 Department of Medicine, Hematology/Oncology, Baylor College of Medicine, Houston, United States of America

Background: Rituximab-based chemoimmunotherapy regimens are backbone treatment (Tmt) for both indolent (follicular [FL], marginal zone [MZL]) and aggressive (diffuse large B-cell [DLBCL], mantle cell [MCL]) B-cell lymphomas. Standard of care (SoC) for relapsed or refractory (R/R) disease includes anti-CD20 in combination with chemotherapy and targeted therapies, such as Bruton’s tyrosine kinase inhibitors (eg, ibrutinib) and phosphoinositide 3-kinase (PI3K) inhibitors. Parsaclisib is a potent and highly selective next generation PI3Kδ inhibitor that is currently being investigated in hematological malignancies.

Aims: CITADEL-112 ( NCT03424122 ) is an open-label phase 1 study evaluating the safety and tolerability of adding parsaclisib to investigator choice SoC Tmt rituximab (RIT), RIT + bendamustine (BEN), or ibrutinib (IBR) in patients (pts) with R/R B-cell lymphoma.

Methods: Enrolled pts were ≥18 years and had histologically confirmed DLBCL, FL, MCL, or MZL, ECOG PS 0–2, were R/R to ≥1 (≥2 for FL) prior systemic therapy, and ineligible for stem cell transplant. Pts received parsaclisib 20 mg orally once daily (QD) for 8 weeks then 20 mg once weekly (QW) in combination with either: RIT 375 mg/m 2 IV QW for 4 doses in cycle 1 (± cycle 2) (Tmt A); RIT 375 mg/m 2 IV on day 1 + BEN 90 mg/m 2 on day 1 and day 2 of each 28-day cycle for ≤6 cycles (Tmt B); or IBR 560 mg QD (Tmt C). Pts received treatment until disease progression, unacceptable toxicity, or withdrawal.

Results: At data cutoff (May 14, 2021), 50 pts were treated (16 pts each in Tmt A and C, 18 pts in Tmt B) and 13 pts were ongoing treatment (3 pts in Tmt A, 8 pts in Tmt B, 2 pts in Tmt C). Most pts had received ≥2 prior systemic treatments (81.3%, 61.1%, and 68.8% in Tmt A [range 1–4], B [range 1–4], and C [range 1–7], respectively). The most common reasons for discontinuation were progressive disease (56.3%, 38.9%, and 50.0%) and adverse events (AEs) (12.5%, 11.1%, and 6.3% in Tmt A, B, and C, respectively). One pt in Tmt B experienced a dose-limiting toxicity of grade 4 neutropenia for >14 days. All pts experienced at least 1 treatment-emergent AE (TEAE); in Tmt A, 75.0% had grade ≥3 and 37.5% had serious TEAEs; Tmt B, 83.3% had grade ≥3 and 27.8% had serious TEAEs; and Tmt C, 62.5% had grade ≥3 and 43.8% had serious TEAEs. Common any-grade TEAEs (≥30%) included neutropenia (62.5%), diarrhea (37.5%), and anemia (31.3%) in Tmt A; neutropenia (50.0%), abdominal pain, asthenia, diarrhea, and nausea (each 33.3%) in Tmt B; neutropenia (50.0%) and increased ALT and increased AST (each 37.5%) in Tmt C. Most common grade ≥3 TEAEs (≥15%) were neutropenia (50.0%) and diarrhea (18.8%) in Tmt A, and neutropenia (38.9% and 25.0%) in Tmt B and Tmt C, respectively. Serious TEAEs occurring in >1 pt were COVID-19, diarrhea, and pneumonia (n = 2 each) in Tmt A, and atrial fibrillation (n = 2) in Tmt C. TEAEs with fatal outcome were reported in 2 pts in Tmt A (COVID-19 and COVID-19 pneumonia [n = 1], interstitial lung disease [n = 1]) and 1 pt in Tmt C (COVID-19, acute kidney injury). Parsaclisib dose interruption or dose reduction due to TEAEs occurred in 75.0% and 18.8% of pts, respectively, in Tmt A; 66.7% and 27.8% of pts, respectively, in Tmt B; and 56.3% and 18.8% of pts, respectively, in Tmt C.

Summary/Conclusion: Parsaclisib 20 mg QD for 8 weeks followed by 20 mg QW can be safely combined with RIT, RIT + BEN, or IBR in pts with R/R B-cell lymphomas. The tolerability profile of the combination regimens was manageable, with no unexpected safety concerns.

P1103: INMIND: A PHASE 3 STUDY OF TAFASITAMAB PLUS LENALIDOMIDE AND RITUXIMAB VERSUS PLACEBO PLUS LENALIDOMIDE AND RITUXIMAB FOR RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA (FL) OR MARGINAL ZONE LYMPHOMA (MZL)

L. H. Sehn 1,* , K. Hübel 2 , S. Luminari 3 , A. Salar 4 , B. E. Wahlin 5 , A. K. Gopal 6 , C. Bonnet 7 , S. Paneesha 8 , M. Trneny 9 , H. Mashegu 10 , C. Lihou 10 , D. Li 10 , C. W. Scholz 11

1 BC Cancer Centre for Lymphoid Cancer and The University of British Columbia, Vancouver, Canada; 2 Department of Internal Medicine I Oncology and Hematology, University Hospital Cologne, Cologne, Germany; 3 Hematology Unit, Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy; 4 Department of Haematology, Hospital del Mar-IMIM, Barcelona, Spain; 5 Department of Medicine, Unit of Hematology, Karolinska Institute, Stockholm, Sweden; 6 Division of Medical Oncology, University of Washington Medicine, Seattle, United States of America; 7 Clinical Hematology, Centre Hospitalier Universitaire, University of Liège, Liège, Belgium; 8 University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom; 9 First Department of Medicine, First Faculty of Medicine, Charles University, General Hospital, Prague, Czechia; 10 Incyte Corporation, Wilmington, United States of America; 11 Department of Hematology and Oncology, Vivantes Klinikum Am Urban, Berlin, Germany

Background: Patients (pts) with indolent non-Hodgkin lymphoma (NHL) subtypes FL or MZL respond to first-line treatment but relapse is common; there is no standard treatment for pts with relapsed/refractory (R/R) FL or MZL. Tafasitamab (TAFA) is an Fc-engineered humanized monoclonal antibody (mAb) against CD19, which is broadly expressed in FL and MZL and regulates proliferation via B-cell receptor signaling. In preclinical studies, TAFA has shown activity against NHL cell lines in combination with rituximab (anti-CD20 mAb) and lenalidomide (LEN). TAFA monotherapy demonstrated promising clinical activity in a phase 2a study in pts with R/R NHL ( NCT01685008 ), with an ORR of 29% (n/N=10/34) in pts with FL and 33% (n/N=3/9) in pts with MZL. In an ongoing phase 2, single-arm study (L-MIND, NCT02399085 ), TAFA + LEN followed by TAFA alone demonstrated an ORR of 57.5% (n/N=46/80) with CR of 40% (n/N=32/80) and median DOR of 34.6 mo (95% CI: 26.1–NR), in pts with R/R diffuse large B-cell lymphoma (FDA approved indication). These observations suggest a potential clinical benefit of TAFA + LEN and rituximab for pts with R/R FL or MZL.

Aims: Phase 3 double-blind, placebo-controlled, randomized study designed to investigate whether TAFA + LEN and rituximab provides improved clinical benefit compared with LEN and rituximab in pts with R/R FL or MZL.

Methods: Pts will be randomized 1:1 to receive TAFA (12 mg/kg IV on days 1, 8, 15, and 22 of 28-day cycle [cycles 1–3], then days 1 and 15 [cycles 4–12]) + LEN (20 mg PO QD [or starting dose 10 mg PO QD if creatinine clearance ≥30 to <60 mL/min], days 1–21/cycle for 12 cycles) and rituximab (375 mg/m 2 IV on days 1, 8, 15, and 22 of cycle 1, then day 1 of cycles 2–5), or placebo (0.9% saline solution IV) + LEN and rituximab. Stratification will include progression of disease within 24 mo (FL; yes vs no), refractoriness to prior anti-CD20 mAb therapy (FL; yes vs no), and number of prior lines of therapy (FL or MZL; <2 vs ≥2). Radiological (PET) assessment will be performed at baseline, every 12 (± 2) weeks in year 1, 16 (± 2) weeks in years 2–3, and 24 (± 3) weeks in years 4–6; additional assessment performed within 4 (± 2) weeks if progressive disease confirmed before end-of-treatment (EOT) visit. The primary study endpoint is PFS (investigator assessed [INV] by Lugano 2014 criteria) for pts with FL. Key secondary endpoints are PFS (INV) in the overall population (FL and MZL), PET-CR rate (INV) at EOT (90 days after last treatment) and OS in pts with FL. Other secondary endpoints include PET-CR rate (INV) at EOT and OS in the overall population, and ORR (INV), DOR (INV), health related quality of life, safety, and minimum residual disease-negativity rate at EOT in FL and the overall population. Inclusion criteria include age ≥18 y, histologically confirmed FL (grade 1, 2, or 3a) or MZL (nodal, splenic, or extranodal), documented R/R disease, ≥1 prior systemic anti-CD20 therapy (including anti-CD20 refractory disease), ECOG PS ≤2, adequate systemic organ function, and high tumor burden (per GELF criteria). Exclusion criteria include prior rituximab + LEN treatment, history of radiotherapy for other diseases (≥25% of bone marrow), nonhematologic malignancy, congestive heart failure (LVEF <50%), active systemic infection, known CNS lymphoma, or severe immunocompromised state. inMIND ( NCT04680052 , EudraCT2020-004407-13) is currently enrolling pts; planned enrollment is 528 pts with R/R FL and 60–90 pts with R/R MZL across North America, Europe, and Asia Pacific.

Results: Not applicable.

Summary/Conclusion: Not applicable.

P1104: A PHASE 1 STUDY EVALUATING SAFETY AND EFFICACY OF PARSACLISIB IN COMBINATION WITH BENDAMUSTINE + OBINUTUZUMAB IN PATIENTS WITH RELAPSED OR REFRACTORY FOLLICULAR LYMPHOMA (CITADEL-102)

M. Hamadani 1,* , M. Coleman 2 , R. Boccia 3 , J. Duras 4 , M. Hutchings 5 , P. L. Zinzani 6 , R. Cordoba 7 , M. B. Oreiro 8 , V. Williams 9 , M. Stouffs 9 , P. Langmuir 9 , J.-M. Sancho 10

1 Division of Hematology and Oncology, Medical College of Wisconsin, Milwaukee; 2 Clinical Research Alliance Inc, Westbury; 3 Center for Cancer and Blood Disorders, Bethesda, United States of America; 4 Department of Haematooncology, University Hospital Ostrava and Faculty of Medicine, University of Ostrava, Ostrava, Czechia; 5 Department of Haematology and Phase 1 Unit, Rigshospitalet, Copenhagen, Denmark; 6 Institute of Hematology “Seràgnoli”, University of Bologna, Bologna, Italy; 7 Lymphoma Unit, Department of Hematology, Fundación Jimenez Diaz University Hospital; 8 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; 9 Incyte Corporation, Wilmington, United States of America; 10 Clinical Hematology Department, Institut Català d’Oncologia-Hospital Germans Trias i Pujol, Barcelona, Spain

Background: Patients (pts) with follicular lymphoma (FL) generally respond well to first-line CD20-targeted therapies, such as obinutuzumab or rituximab-based regimens. However, many pts relapse and studies suggest that each subsequent relapse is associated with shorter durations of response to the next treatment. Parsaclisib is a potent and highly selective next generation PI3Kδ inhibitor. The combination of bendamustine + obinutuzumab is approved for pts with relapsed/refractory (R/R) FL. We hypothesized that adding parsaclisib may improve clinical benefit with a manageable safety profile in this pt population.

Aims: CITADEL-102 ( NCT03039114 ) is an open-label, phase 1, dose-finding study that investigated safety and efficacy of parsaclisib in combination with bendamustine + obinutuzumab in pts with R/R FL following rituximab-containing regimens.

Methods: Pts enrolled were ≥18 years with histologically confirmed CD20-positive FL, R/R to any prior rituximab-containing regimen, ECOG PS 0–2, ≥1 measurable lesion, and ≤4 prior therapies. Pts received parsaclisib 20 mg orally once daily (QD) for 8 weeks then 20 mg once weekly (QW); bendamustine 90 mg/m 2 infusion on days 1 and 2 of cycles 1–6; and obinutuzumab 1000 mg infusion on days 1, 8, and 15 of cycle 1, and day 1 of cycles 2–6, and on every second cycle of cycles 8–30 in pts having complete response/complete metabolic response (CR/CMR), partial response/partial metabolic response (PR/PMR), or stable disease/no metabolic response. Part 1 (safety run-in) used a 3 + 3 design with dose de-escalation to identify the maximum tolerated dose (MTD) of parsaclisib in combination with bendamustine + obinutuzumab. In Part 2 (dose expansion), the safety and efficacy of this combination were further evaluated. The primary study endpoint was safety and tolerability; secondary endpoints included efficacy outcomes (ORR, DOR, PFS, and OS).

Results: A total of 26 pts were enrolled and treated; median (range) age was 65.0 (44–80) years, 25 (96.2%) had ECOG PS ≤1, 11 (42.3%) had ≥2 prior systemic therapies, and 6 (23.1%) had received prior bendamustine. Median (range) parsaclisib exposure was 10.6 (0.4–32.8) months. Main reasons for treatment discontinuation included adverse events (AEs) (8 pts, 30.8%) and progressive disease (6 pts, 23.1%). All pts experienced treatment-emergent AEs (TEAEs); most common any-grade TEAEs (≥10 pts) were pyrexia (53.8%), neutropenia (50%), diarrhea (46.2%), thrombocytopenia, and nausea (each 38.5%). Grade ≥3 TEAEs were experienced by 88.5% of pts; most common grade ≥3 TEAEs (≥2 pts) were neutropenia (34.6%), febrile neutropenia (23.1%), thrombocytopenia (19.2%), ALT and AST increase (each 11.5%), and diarrhea, neutrophil count decreased, and rash maculopapular (each 7.7%). One of 6 evaluable pts in Part 1 had a DLT of grade 4 QTc elongation. The MTD was not reached, and parsaclisib 20 mg QD for 8 weeks then 20 mg QW was the selected dosage for dose expansion in Part 2. Treatment discontinuation due to TEAEs was 30.8%, 7.7%, and 15.4% for parsaclisib, bendamustine, and obinutuzumab, respectively. One fatal TEAE (COVID-19 pneumonia) occurred. ORR (95% CI) as reported by the investigator was 76.9% (56.4–91.0), with 17 pts (65.4%) achieving CR/CMR and 3 pts (11.5%) achieving PR/PMR as the best overall response. Median DOR, PFS, and OS were not reached.

Summary/Conclusion: Parsaclisib in combination with bendamustine + obinutuzumab appears to have a manageable safety profile and demonstrated promising efficacy in pts with R/R FL.

P1105: FINAL RESULTS OF THE PHASE I/II HOVON124/ECWM-R2 STUDY INCLUDING 2-YEAR RITUXIMAB MAINTENANCE AFTER INDUCTION WITH IXAZOMIB, RITUXIMAB AND DEXAMETHASONE IN RELAPSED WALDENSTRÖM’S MACROGLOBULINEMIA

M.-A. Dimopoulos 1,* , K. Amaador 2,3 , M. C. Minnema 4 , K. Nasserinejad 5 , M. Kap 5 , E. Kastritis 1 , M. Gavriatopoulou 1 , W. Kraan 3,6 , M. E. D. Chamuleau 7 , D. Deeren 8 , L. Tick 9 , J. K. Doorduijn 10 , F. Offner 11 , L. H. Böhmer 12 , R. D. Liu 2 , S. T. Pals 3,6 , J. M. Vos 2,3 , M. J. Kersten 2,3

1 Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 2 Department Of Hematology, Amsterdam UMC, University of Amsterdam, Cancer Center Amsterdam; 3 LYMMCARE (Lymphoma and Myeloma Center Amsterdam), Amsterdam; 4 Department of Hematology, University Medical Center Utrecht, University Utrecht, Utrecht; 5 HOVON Data Center, Department of Hematology, Erasmus MC Cancer Institute, Rotterdam; 6 Department of Pathology, Amsterdam UMC, University of Amsterdam, Cancer Center Amsterdam; 7 Department of Hematology, Amsterdam UMC, VU University, Amsterdam, Netherlands; 8 Department of Hematology, AZ Delta, Roeselare, Belgium; 9 Department of Hematology, Maxima Medical Center, Eindhoven; 10 Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 11 Department of Hematology, University Hospital Gent, Gent, Belgium; 12 Department of Hematology, Haga Teaching Hospital, The Hague, Netherlands

Background: In the phase I/II HOVON124/ECWM-R2 trial, induction treatment with the combination of ixazomib, subcutaneous (s.c.) rituximab and dexamethasone (IRd) showed promising efficacy with manageable toxicity in patients with relapsed Waldenström’s Macroglobulinemia (WM).

Aims: Here we report the final analysis of the trial after two years of rituximab maintenance and with a median follow-up (FU) on study of 45.6 months (range, 12.4-72.2).

Methods: In total, 59 patients were enrolled (median age, 69 years; range, 46-91 years) of which 48 patients completed at least six cycles of induction with IRd; 41 patients (median age 66 years, 66% male) with at least a minimal response (MR) continued to 2 years of rituximab maintenance (1400 mg s.c., q 3 months) starting 3 months after the last induction cycle. The primary endpoint of the study was overall response rate (ORR, ≥ MR)) after 8 induction cycles and was 71% (Kersten/Amaador et al, JCO, 2022). Secondary endpoints included progression-free survival (PFS), overall survival (OS) and ORR after 2 years of rituximab maintenance and improvement of response after maintenance.

Results: In total, 22 (54%) out of 41 patients completed 2 years of rituximab maintenance. The median number of cycles was 8 (range, 1-8). Nineteen patients did not complete maintenance treatment due to progression ( n =16), excessive toxicity ( n =1), non-compliance ( n =1), and other unknown reason ( n =1). The best ORR after maintenance was 85% (2% complete response [CR], 24% very good partial response [VGPR], 39% partial response [PR], and 20% MR). Improvement of response after maintenance occurred in 9 (22%) patients; VGPR to CR in one (2%), PR to VGPR in 6 (15%), and MR to PR in 2 (5%). A further decrease in IgM levels was seen after 2 years of maintenance (IgM 3.62 g/dl at baseline; 1.3 g/dl after induction and 0.42 g/dl after end of maintenance, p-value<0.001; Figure 1A), accompanied by a further increase in Hb levels (Hb 10.5 to 14.3 g/dl, p-value<0.001; Figure 1B). The median progression-free survival (PFS) was 23.6 months (95% CI, 13.4 to 43.2; Figure 1C), and median overall survival (OS) was not reached; at 45 months, 85% of patients were alive (95% CI, 0.72 to 0.92; Figure 1D). The median time to best response was 7 months, the median duration of response (DOR) was 34.8 months, and the median time to next treatment (TTNT) was 36 months[MM(1]. After a median FU of 45.6 months, 29 patients had received subsequent therapy and the median time to progression after subsequent therapy was 53.7 months[MM(2]. During maintenance, rituximab was administered i.v. instead of s.c. in 7 (17%) patients. None of the patients treated with s.c. rituximab developed systemic hypersensitivity reactions but 2 patients had an injection site reaction (1 grade 1, 1 grade 2). Grade 3/4 toxicity was seen in 10% and 5% of patients, respectively (grade 3/4 neutropenia ( n =1 and n =2), grade 3 chronic kidney disease (n=1), and grade 3 elevations of transaminases (n=2)). In the 41 patients who started maintenance, 8 SAEs were reported in 6 patients, of which 4 occurred during maintenance (mostly infections) and 4 (mostly secondary malignancies, considered unrelated to study drug) during FU. Three patients died during maintenance therapy due to progressive disease, intracranial bleeding, and acute myeloid leukemia, respectively.

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Summary/Conclusion: Rituximab maintenance after IRd induction is feasible and well tolerated. The ORR improved in 22% of patients, confirming the efficacy of this chemo-free regimen in combination with rituximab maintenance in relapsed/refractory WM.

P1106: PRELIMINARY RESULTS OF A PHASE 1 STUDY OF NOVEL BCL-2 INHIBITOR LISAFTOCLAX (APG-2575) IN CHINESE PATIENTS (PTS) WITH RELAPSED OR REFRACTORY (R/R) NON-HODGKIN LYMPHOMAS (NHLS)

M. Sun 1,* , J. Qi 1 , Y. Song 2 , K. Zhou 2 , H. Liu 3 , A. Shen 3 , L. Geng 3 , F. Zhou 4 , J. Huang 4 , Z. Chen 5 , H. Zhang 5 , M. Lu 6 , M. Ahmad 6 , L. Men 5 , D. Yang 7 , Y. Zhai 6 , J. Wang 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin; 2 Department of Hematology, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou; 3 First Affiliated Hospital of University of Science and Technology of China (USTC) Anhui Provincial Hospital, Hefei; 4 Zhongnan Hospital, Wuhan University, Wuhan; 5 Ascentage Pharma (Suzhou) Co., Ltd., Suzhou, China; 6 Ascentage Pharma Group Inc., Rockville, United States of America; 7 State Key Laboratory of Oncology in South China Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China

Background: Many B-cell malignancies evade apoptosis by overexpressing BCL-2 proteins. Although indicated for the management of certain hematological malignances (HMs), BCL-2 inhibitor (BCL-2i) venetoclax requires a slow dose ramp-up to reduce the risk of tumor lysis syndrome (TLS) and is associated with severe neutropenia. Investigational lisaftoclax is a novel, potent, selective BCL-2i, which was previously reported to have activity against HMs in US and Australian pts. Lisaftoclax also offers a daily (rather than weekly) ramp-up to the target dose.

Aims: The objective of this study is to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of lisaftoclax in Chinese pts with R/R NHLs.

Methods: In this study, pts were dosed with lisaftoclax orally once daily in 28-day cycles. A daily ramp-up schedule was used in pts with chronic lymphocyte leukemia (CLL) or NHL with medium/high-risk TLS. A regular schedule was used in pts without CLL and those with NHL with low-risk TLS.

Results: As of January 1, 2022, 40 pts (median age 55.5 [range 30-79] years; 72.5% male) had been treated with lisaftoclax (dose range 20-800 mg), including 20 who remain in the trial. The median number of prior therapies was 4 (range 0-14). NHL subtypes included: 12 CLL, 9 follicular, 7 mantle cell (MCL), 4 marginal zone (MZL), 4 T-cell, 2 diffuse large B-cell, and 2 Waldenström macroglobulinemia. DLT, MTD, and laboratory/clinical TLS were not observed, and the RP2D was 600 mg. Treatment-emergent adverse events (TEAEs) were reported in 36 pts (90%), of which most were grade 1-2 (67.5%). Common TEAEs (≥ 15% all grades) were hyperuricemia (38.6%), hypertriglyceridemia and anemia (each 36.4%), thrombocytopenia (29.5%), hyperphosphatemia (27.3%), neutropenia (25%), proteinuria (22.7%), leukopenia (18.2%), and diarrhea (15.9%). Common grade 3-4 TEAEs (> 5%) were neutropenia (13.6%) and thrombocytopenia (9.1%). A total of 4 (10%) pts experienced serious AEs, including 1 small intestinal obstruction, 1 pneumonia, 1 wound hemorrhage, and 1 case each of anemia and thrombocytopenia. One pt had dose reduction because of grade 2 colitis, and 1 pt receiving 100 mg discontinued because of grade 4 thrombocytopenia. With a medium treatment duration of 4 (range 4-20) cycles, 12 of 32 (37.5%) evaluable pts reached a complete response (CR; n = 4) or partial response (PR; n = 8), including 7 with CLL, 2 MCL, 2 MZL, and 1 T-cell NHL. In pts with CLL, the overall response rate (ORR) was 63.6%, CR 27.3%, and PR rate 36.4%. At doses ≥ 200 mg, the ORR was 87.5%. A total of 8 of 11 pts had at least 1 adverse prognostic factor ( TP53 mutation/17p-/IGHV unmutated/complex chromosomal abnormalities). Of 11 pts, 5 had disease that previously failed BTKi or CD20 monoclonal antibody therapy. Systemic exposure increased across lisaftoclax doses from 20 to 800 mg, with an average half-life of 4 to 6 hours. There was no significant accumulation following the once-daily dosing.

Summary/Conclusion: Here, we report for the first time clinical data for lisaftoclax in Chinese pts with R/R NHLs. Lisaftoclax was well tolerated at doses of up to 800 mg/day, with no TLS observed (despite the daily ramp-up), and was active against CLL/SLL, MZL, MCL, and T-cell NHL subtypes. Lisaftoclax may offer a more convenient treatment alternative, with a daily ramp-up schedule that may be more pt friendly. ClinicalTrials.gov: NCT03913949 .

P1107: CLINICAL OUTCOMES OF SOLID ORGAN TRANSPLANT PATIENTS WITH EBV+ PTLD WHO FAIL RITUXIMAB PLUS CHEMOTHERAPY: A MULTINATIONAL, RETROSPECTIVE CHART REVIEW STUDY

V. Dharnidharka 1 , D. Thirumalai 2 , U. Jaeger 3,* , W. Zhao 2 , D. Dierickx 4 , P. Xun 2 , P. Minga 5 , A. Sawas 6 , N. Sadetsky 7 , P. Chauvet 8 , E. Sundaram 9 , A. Barlev 7 , H. Zimmerman 10 , R. U. Trappe 10

1 Washington University School of Medicine & St. Louis Children’s Hospital, St Louis; 2 Atara Biotherapeutics, Thousand Oaks, United States of America; 3 Medical University of Vienna, Vienna, Austria; 4 Universitair Ziekenhuis Leuven, Leuven, Belgium; 5 Niguarda Ca’ Granda Hospital, Milan, Italy; 6 Herbert Irving Comprehensive Cancer Center, New York; 7 Atara Biotherapeutics, South San Francisco, United States of America; 8 CHU Lille, Lille, France; 9 Nashville Bioscences, Nashville, United States of America; 10 DIAKO Bremen, Bremen, Germany

Background: Post-transplant lymphoproliferative disease (PTLD) is a rare and often aggressive disease i n the setting of immunosuppression following solid organ transplant (SOT). Epstein-Barr virus (EBV) infection of B cells is responsible for about 50% of cases, either due to reactivation of the virus after transplantation or primary EBV infection. Although there is no approved therapy for patients (pts) with PTLD, guidelines include reduction of immunosuppression (RIS) as a part of initial treatment and may be sufficient for pts with early lesions. Rituximab, either as monotherapy or in combination with chemotherapy (CT), is used in addition to RIS as initial treatment. Pts with EBV + PTLD following SOT who fail rituximab plus CT have poor outcomes with limited treatment options. Published data on clinical outcomes of these pts remain limited and not well documented.

Aims: To characterize the outcomes for pts diagnosed with EBV + PTLD following SOT who fail initial rituximab plus CT in a multi- national real-world setting.

Methods: We conducted a large multinational, multicenter, retrospective chart review study of pts with EBV + PTLD following allogeneic hematopoietic cell transplantation (HCT) or SOT who received rituximab or rituximab plus CT between January 2000‒December 2018 and were refractory (failed to achieve complete response [CR] or partial response [PR]) or relapsed at any point after such therapy. Data were collected from 29 centers across North America (United States and Canada) and the European Union. This analysis includes pts with EBV + PTLD following SOT who were refractory/relapsed to rituximab plus CT. The Kaplan-Meier method was utilized to estimate the overall survival (OS).

Results: : A total of 86 pts with EBV + PTLD following SOT who failed rituximab plus CT were included in the analysis; 65 (75.6%) pts were refractory while 21 (24.4%) relapsed after initial response of CR or PR. Median age at PTLD diagnosis was 43 years (range 1‒78) and median time to PTLD onset from transplant was 1.7 years (range 0.1‒27.9). Median follow up time was 12.9 months from the date of PTLD diagnosis. PTLD histological subtypes were 66 (76.7%) monomorphic, 18 (20.9%) polymorphic, and 2 (2.3%) early lesions. The most common PTLD subtype was diffuse large B-cell lymphoma (DLBCL) (58, 67.4%).

Of the 86 pts, 49 (57%) received CT following rituximab monotherapy while 37 (43%) pts received CT concurrently with rituximab. Overall, 63 (73.3%) pts died. PTLD-specific mortality was observed in 41 (65.1%) pts, treatment-related mortality in 10 (15.9%) pts, mortality due to organ rejection/failure in 2 (3.2%) pts, mortality due to other causes in 7 (11.1%) pts, and mortality due to unknown causes in 3 (4.8%) pts. Median OS was 15.5 months (95% confidence interval [CI]: 8.3‒22.9) from PTLD diagnosis, and was 4.1 months (95%CI: 1.9‒8.5) from the earliest date when pts became refractory or relapsed following rituximab plus CT.

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Summary/Conclusion: The prognosis for pts with EBV + PTLD following SOT who fail rituximab plus CT remains poor, with an estimated median OS of about 4 months and a majority of pts dying from PTLD and related treatment. In this specific population, there remains a significant unmet medical need for effective and well-tolerated therapies.

P1108: HIGH COMPLETE RESPONSE RATE FOLLOWING POINT-OF-CARE ANTI CD19 CAR T-CELL THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA

S. Fried 1,2,* , M. J. Besser 3,4 , E. Shkury 1,2 , R. Yerushalmi 1,2 , N. Shem-Tov 1,2 , I. Danylesko 1,2 , E. Jacoby 2,5 , O. Itzhaki 4 , R. Shouval 1,2,6 , M. Kedmi 1,2,7 , A. Shimoni 1,2 , A. Nagler 1,2 , A. Avigdor 1,2

1 Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel Hashomer; 2 Sackler School of Medicine; 3 Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel-Aviv; 4 Ella Lemelbaum Institute for Immuno Oncology; 5 Department of Pediatric Hematology-Oncology, Safra Children’s Hospital, Chaim Sheba Medical Center, Tel Hashomer, Israel; 6 Adult BMT Service, Memorial Sloan Kettering Cancer Center, New York, United States of America; 7 The Mina and Everard Goodman faculty of life sciences, Bar Ilan University, Ramat Gan, Israel

Background: Patients with relapsed/refractory follicular lymphoma (R/R FL) often experience multiple relapses and require various lines of therapy. The ELARA and ZUMA-5 trials demonstrated high response rates along with acceptable safety profiles. We perform a phase 1b/2 single-center clinical trial of autologous point-of-care (POC) academic anti-CD19 chimeric antigen receptor (CAR) T-cells for patients with R/R FL treated with at least 2 lines of systemic therapy ( NCT02772198 ).

Aims: To report outcomes of POC CAR T-cell therapy in patients with R/R FL.

Methods: Adults with R/R FL underwent a single leukapheresis procedure. Fresh peripheral blood mononuclear cells were isolated, activated, and transduced with a gammaretrovirus encoding for a CD19 CAR (based on an FMC63-derived ScFv, a CD28 costimulatory domain, and a CD3-ζ signaling domain). Lymphodepletion included fludarabine 25 mg/m 2 over 3 days (days −4 to −2) and cyclophosphamide 900 mg/m 2 once (day −2), followed by infusion of 1×10 6 /kg CAR T-cells in the inpatient setting. Primary endpoints were response (by PET-CT, per Lugano criteria) at day 28, best response, and safety. Secondary endpoints included overall survival, progression-free survival (PFS), and production feasibility. Last follow-up was as of 02/2022.

Results: All 19 patients enrolled received CAR T-cell infusion in a median of 11 days (IQR 10-11) after leukapheresis. The median age was 61 years (IQR 52-66). Five (26%) patients had Karnofsky performance status < 90%. Disease stage at enrollment was III-IV in 16 (84%) patients. Two (11%) patients had bulky disease; 8 (42%) had LDH > upper limit of normal; and 16 (84%) had Follicular Lymphoma International Prognostic Index ≥ 3. Disease status at enrollment was progressive disease (n=14, 74%), stable disease (n=3, 16%), or partial response (PR; n=2, 11%). Twelve patients (64%) were refractory to last treatment. Disease grade at most recent lymph node biopsy was 1 (n=3, 16%), 2 (n=11, 58%), or 3a (n=5, 26%). The median time from FL diagnosis was 3.9 years (IQR 2.5-4.6). Sixteen (84%) patients had progression of disease within 24 months of initial therapy. The number of prior therapies was ≥ 4 in 6 (32%) patients; and 5 (26%) patients underwent prior autologous transplantation.

Grade III-IV cytokine release and immune effector cell-associated neurotoxicity syndromes occurred in 1 (5%) and 4 (21%) patients, respectively. One patient was infected with COVID-19 on the 5 th day following cell infusion and was admitted to the intensive care unit. One patient had grade 3 atrial fibrillation. Severe neutropenia (absolute neutrophil count <500/µL), thrombocytopenia (platelets <50K/µL) and anemia (hemoglobin <10g/dl) occurred in 15 (79%), 5 (26%), and 7 (37%) patients, respectively. No bleeding events or death were recorded following cell infusion.

Response was evaluated in all patients. Overall response rate on day 28 was 84% (79% complete response [CR]). One patient with PR on day 28 achieved a CR after a year of follow-up. Three patients (16%) continued to progress following CAR infusion.

All patients were alive at the last follow-up (median follow-up, 11.5 months [IQR 4-21]). One-year PFS was 74% (95% CI, 53-100). The median duration of response (DOR) was not reached (95% CI, 12.5-not reached). Estimated DOR at 1-year was 89% (95% CI, 71-100).

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Summary/Conclusion: Point-of-Care anti-CD19 CAR T-cell therapy, performed following a very short production time, induced high CR rate with an acceptable safety profile in a cohort of patients with high-risk R/R FL.

P1109: TRIAL IN PROGRESS: PHASE 1B/2 TRIAL OF TAZEMETOSTAT IN COMBINATION WITH VARIOUS TREATMENTS IN PATIENTS WITH RELAPSED OR REFRACTORY HEMATOLOGIC MALIGNANCIES

A. Bessudo 1,* , D. Greenwald 2 , M. H. Kazemi 3 , A. Mahindra 4 , L. Shunyakov 5 , M. Sondhi 6 , H. O’Connor 6 , Y. Chen 6 , J. Yang 6 , G. Salles 7

1 cCare, Encinitas; 2 UCLA, Santa Barbara; 3 Astera Cancer Care, East Brunswick; 4 Scripps MD Anderson Cancer Center, La Jolla; 5 Central Care Cancer Center, Bolivar; 6 Epizyme, Inc., Cambridge; 7 Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator, suppresses key cellular checkpoints and differentiation pathways to maintain the oncogenic characteristics of germinal center B cells. In addition to activity in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), EZH2 inhibitors induced cell cycle arrest and apoptosis in preclinical studies of multiple myeloma (MM). Tazemetostat (TAZ), an oral selective EZH2 inhibitor, is approved for treatment of relapsed or refractory (R/R) follicular lymphoma. Preclinical studies demonstrated antiproliferative and synergistic effects of TAZ + lenalidomide (LEN), a Bruton tyrosine kinase inhibitor (BTKi), and daratumumab, pomalidomide, and dexamethasone (mAbPD) in DBLCL, MCL, and MM cell lines, respectively. LEN is effective for DLBCL and is approved in combination with tafasitamab-cxix, a CD19-directed cytolytic antibody (CD19 antibody [Ab]), for R/R DLBCL. Acalabrutinib, a BTKi, is approved in MCL, and mAbPD showed clinical activity in R/R MM.

Aims: The aim of this study is to examine the efficacy and safety of TAZ + CD19 Ab, LEN, BTKi, or mAbPD in patients (pts) with R/R hematologic malignancies.

Methods: This trial is a 2-part, multicenter, open-label, safety, efficacy, signal-finding, multi-arm phase 1b/2 study of TAZ plus various treatment regimens for hematologic malignancies ( NCT05205252 ). Phase 1b is a dose escalation safety run-in; phase 2 is a dose expansion study. Currently, there are 4 treatment arms ( Table ). Written informed consent will be obtained upon enrollment. Eligible pts are adults (aged ≥18 y) with an ECOG performance status 0–1 (phase 1b) or 0–2 (phase 2) and measurable disease by Lugano classification for arms 1–3 or IMWG 2016 criteria for arm 4. Pts with severe concurrent disease are excluded from this study. During the phase 1b safety run-in portion of the study, pts will be enrolled in 3 dose escalation cohorts of TAZ 400 mg, 600 mg, and 800 mg. Pts in all novel treatment combinations (arms 1, 3, 4) will start at the lowest TAZ dose of 400 mg, and treatment combinations previously studied (arm 2) will start at TAZ 800 mg. All pts will receive TAZ orally twice daily in continuous cycles. All combination partners will be administered per their respective package insert or investigator brochure. The phase 1b portion endpoint summarizes treatment-emergent dose-limiting toxicities and adverse events to inform the recommended phase 2 dose for each arm. The primary endpoint for all arms in phase 2 is objective response rate; key secondary endpoints include progression-free survival, duration of response, overall survival, and safety.

Results: As of February 9, 2022, no pts are enrolled at 1 active site; additional sites are in startup.

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Summary/Conclusion: This phase 1b/2 study will provide insights into the efficacy and safety of TAZ + CD19 Ab, LEN, BTKi, or mAbPD in pts with R/R hematologic malignancies.

P1110: EFFICACY AND SAFETY OF IMMUNOCHEMOTHERAPY IN TREATMENT OF FOLLICULAR NON-HODGKIN’S LYMPHOMA DURING COVID-19 PANDEMIC: A STUDY OF KROHEM, THE CROATIAN COOPERATIVE GROUP FOR HEMATOLOGIC DISEASES

D. Galusic 1,2 , S. Basic - Kinda 3 , A. Pijuk 1 , V. Milunovic 4 , B. Dreta 3,* , N. Franjic 5 , B. Coha 6 , J. Sincic – Petricevic 7 , P. Gacina 8 , V. Pejsa 9 , M. Lucijanic 9 , I. Aurer 3,10

1 Division of Hematology, Department of Internal Medicine, University Hospital of Split; 2 School of Medicine, University of Split, Split; 3 Division of Hematology, Department of Internal Medicine, University Hospital Centre Zagreb; 4 Division of Hematology, Department of Internal Medicine, University Hospital Merkur, Zagreb; 5 Division of Hematology, Department of Internal Medicine, University Hospital Centre Rijeka, Rijeka; 6 Department of Internal Medicine, Dr. “Josip Bencevic” General Hospital, Slavonski Brod; 7 Division of Hematology, Department of Internal Medicine, University Hospital Centre Osijek, Osijek; 8 Division of Hematology, Department of Internal Medicine, Sestre Milosrdnice University Hospital Center; 9 Division of Hematology, Department of Internal Medicine, University Hospital Dubrava; 10 School of Medicine, University of Zagreb, Zagreb, Croatia

Background: Follicular lymphoma (FL) is a systemic neoplasm of the lymphoid tissue arising from B cell proliferation. The novel monoclonal anti-CD20 antibody obinutuzumab in combination with chemotherapy has been widely accepted as the first choice in front line treatment of FL. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), responsible for coronavirus disease 2019 (COVID-19) is causing increased mortality among patients with lymphoproliferative disorders compared with the general population. Furthermore, there are some concerns in terms of morbidity and mortality for patients with FL because of their immunocompromised status induced by recent exposure to cytotoxic chemotherapy, especially bendamustine and anti-CD20.

Aims: To investigate efficacy and safety of immunochemotherapy protocols for patients with newly diagnosed FL during COVID-19 pandemic.

Methods: We retrospectively investigated medical data of all patients with newly diagnosed FL grade 1, 2 or 3A from Croatian hematologic registry in period from April 2019 to March 2021. Only patients which required systemic treatment were included in the analysis. All patients received obinutuzumab (G) in combination with either CHOP, bendamustine (B) or CVP chemotherapy protocol. Treatment response was evaluated using international lymphoma response criteria.

Results: We analyzed a total of 114 FL patients treated with G-chemotherapy. Mean age was 62.4 ±10.5 years. Majority of patients were female (71/114 (62.3%)). FL grade I was present in 45/114 (39.5%), grade II in 28/114 (24.6%), grade III in 27/114 (23.7%) and not specified (but not IIIB) in 14/114 (12.3%) patients. A total of 61/114 (53.5%) patients were treated with G-B, 49/114 (43%) with G-CHOP and 4/114 (3.5%) with G-CVP immunochemotherapy. Similar rates of adverse events were observed in patients treated with G-CHOP and G-B Median follow up was 17 months. Overall response rate was 94%, complete remission (CR) in 68% and partial remission (PR) in 25% of patients. Median overall survival (OS) and progression free survival (PFS) were not reached with 12-months rates of 94% and 92%, respectively. Patients treated with G-CHOP had statistically significantly superior OS and PFS compared to patients treated with G-B (P=0.002 and P=0.006, respectively, Fig. 1). More favorable survival course associated with G-CHOP in comparison to G-B persisted in multivariate analysis (P=0,026, HR=15,12) after adjustment for age, sex, FLIPI grade and SARS-CoV-2 infection. Total of 12 patients died during the follow up and COVID-19 was cause of death in 5 patients. During the follow-up SARS-CoV-2 infection was diagnosed in 20/114 (17,5%) patients with overall mortality rate of 25%. All of the 7 patients treated with G-CHOP recovered from SARS-CoV-2 infection and mortality rate in infected group of patients treated with G-B was 33% (4/12 patients).

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Summary/Conclusion: Increased COVID-19 mortality in patients with lymphoproliferative disorders was observed in this study. Our group of patients had reduced OS and PFS compared to the GALLIUM trial and SARS‐CoV‐2 infection was the most pronounced risk factor for death. Even though in some studies bendamustine has shown to be less toxic and more effective than CHOP in FL, there are some important pandemic aspects that must be considered. Bendamustine exposure seems to be associated with worse outcome in case of the infection with SARS-CoV-2. These intriguing differences could play important role in treatment approach in COVID-19 pandemic. Future studies investigating hematological malignancies in COVID-19 pandemic are warranted.

P1111: COMPARATIVE EFFICACY AND SAFETY OF TISAGENLECLEUCEL AND AXICABTAGENE CILOLEUCEL IN RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA

M. Dickinson 1,* , J. Martinez-Lopez 2 , E. Jousseaume 3 , C. Anjos 4 , H. Yang 5 , X. Chai 5 , T. Wang 5 , R. Ramos 4 , C. Lobetti-Bodoni 3 , S. Schuster 6 , N. Fowler 7

1 Peter MacCallum Cancer Centre, Royal Melbourne Hospital and the University of Melbourne, Melbourne, VIC, Australia; 2 Hospital Universitario 12 de Octubre, Complutense University, CNIO, Centro de Investigación Biomédica en Red Cáncer (CIBERONC), Madrid, Spain; 3 Novartis Pharmaceuticals AG, Basel, Switzerland; 4 Novartis Pharmaceuticals Corporation, East Hanover, NJ; 5 Analysis Group Inc., Boston, MA; 6 Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA; 7 The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America

Background: The chimeric antigen receptor T-cell (CAR-T) therapies, tisagenlecleucel (tisa-cel) and axicabtagene ciloleucel (axi-cel) have demonstrated clinical benefits in treating relapsed/refractory follicular lymphoma (r/r FL).

Aims: To compare the efficacy and safety outcomes of tisa-cel and axi-cel in r/r FL using matching-adjusted indirect comparison (MAIC).

Methods: Individual patient-level data (IPD) in ELARA (tisa-cel; NCT03568461 ; 03/2021 data-cut; N=97 [infused set]; N=90 [efficacy-evaluable set]) were weighted to match the patient population in ZUMA-5 (axi-cel; NCT03105336 ; 03/2020 data-cut; N=124 [infused set]; N=84 [efficacy-evaluable set]). Because patients from ZUMA-5 did not use bridging chemotherapy, the primary analysis compared a patient subgroup not exposed to bridging chemotherapy in ELARA (N=53 [infused set]; N=50 [efficacy-evaluable set]) with patients in ZUMA-5. Baseline characteristics available in both trials were adjusted in MAIC including age, sex, ECOG performance status, Ann Arbor stage, FLIPI, tumor burden per GELF criteria, number of prior therapy lines, refractory status to the most recent regimen, progression of disease within 24 months from first anti-CD20 monoclonal antibody-containing therapy, and prior autologous stem cell transplant. Efficacy outcomes, including overall response rate (ORR), complete response (CR) rate, and progression-free survival (PFS), were compared among the efficacy-evaluable set, defined as patients with ≥12 months of follow-up in both trials. Selected safety outcomes, including cytokine release syndrome (CRS), neurological events (NE), and tocilizumab and corticosteroid use for CRS, were compared between the infused populations. A sensitivity analysis was conducted to compare patients with and without bridging chemotherapy in ELARA with ZUMA-5.

Results: After adjusting for differences in baseline characteristics, ORR, CR rate, and PFS were comparable between tisa-cel and axi-cel efficacy-evaluable patients (Figure 1 A ). In the primary analysis, the hazard ratio for PFS post-weighting was 0.90 (95% confidence interval: 0.39, 2.06; p=0.81) (Figure 1 B ). The adverse event rates for CRS and NE of any grade were 32.70% (p<0.01) and 47.70% (p<0.001) lower, respectively, in patients infused with tisa-cel compared to those infused with axi-cel. The proportion of patients who used tocilizumab and corticosteroids for CRS were 35.57% (p<0.001) and 12.69% (p<0.01) lower, respectively, in patients infused with tisa-cel (Figure 1 C ). Similar results were observed in the sensitivity analysis among patients with and without bridging chemotherapy in ELARA (Figure 1 A-C ).

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Summary/Conclusion: The MAIC results indicated that tisa-cel and axi-cel were comparable in response rates and PFS, while tisa-cel was associated with better safety outcomes than axi-cel. These results confirm and reinforce the relevance of CAR-T therapies for r/r FL. Future analyses using IPD from both trials and real-world data are warranted.

P1112: TREATING BING-NEEL SYNDROME USING A TAILORED APPROACH: EXPERIENCE OF A SPECIALIST NEUROHAEMATOLOGY CLINIC

J. Khwaja 1,* , D. Smyth 2 , A. Rismani 1 , C. Hoskote 3 , C. Kyriakou 1 , M. P. Lunn 2 , S. D’Sa 1

1 Department of Haematology, University College London Hospital; 2 Centre for Neuromuscular Disease; 3 Department of Neuroradiology, National Hospital for Neurology and Neurosurgery, London, United Kingdom

Background: Bing-Neel syndrome (BNS) is a rare complication of lymphoplasmacytic lymphoma (LPL) comprising LPL infiltration in the central nervous system (CNS). Clinical and radiological features are diverse; the diagnosis is confirmed by cerebrospinal fluid (CSF) analysis using immunological and molecular techniques. Rarely, a tissue biopsy is required. The pattern of presentation including systemic involvement and CSF features inform treatment strategies, which include CNS-penetrating therapies.

Aims: To evaluate the diagnostic characteristics of patients with BNS and their influence on therapy.

Methods: Data from patients referred between 2011-2021 for management of BNS to our academic neurohaematology centre were retrospectively reviewed. Those with imaging features alone or where it was not possible to distinguish from high-grade transformation were excluded.

Results: Thirty-five patients (22 male, 13 female) were identified. Median age at diagnosis of BNS was 65 years (range 48-85). All patients were symptomatic. In 12 patients (34%) BNS was the de novo presentation of the IgM-related disorder, of which 3 (25%) had no detectable bone marrow (BM) infiltration of LPL at diagnosis. Approximately half (17; 49%) had previously received therapy for LPL; median time to BNS diagnosis in these was 49 months (range 3-125). At BNS diagnosis, BM involvement with LPL ranged from 0-95%. More than half (14/26; 54%) had ≤ 10% infiltrate and almost a fifth (4/26) >60%.

All patients had leptomeningeal involvement and 8 (23%) additionally had parenchymal CNS disease. The majority had kappa light-chain predominance: IgMκ (n=26), non-IgMκ (n=5), IgMλ (n=3), one unknown. The BNS diagnosis was made on CSF analysis (n=28; 80%), leptomeningeal tissue biopsy (n=3; 9%) where CSF was non-informative, or by expert opinion based on supportive clinical, radiological and non-definitive CSF features (n=4; 11%). Of those with a diagnosis based on CSF studies, B-cell clonality was confirmed by flow cytometry (27/28; 96%), MYD88 L265P mutation (18/28; 64%) and immunoglobulin gene rearrangement (12/28; 43%). In 22 samples with a full dataset, median CSF white cell count was 25/ul (1-233), CSF protein 1.69g/l (0.35-6), CSF IgM 9.49mg/l (1.07-61.5).

The majority were treated with intensive regimens (rituximab, methotrexate (MTX), cytarabine (ARA-C) ± thiotepa/idarubicin; n=30) due to the presence of CNS disease bulk and clinical need, and less commonly ibrutinib (n=3), bendamustine-rituximab (BR, n=1); one patient had intrathecal therapy (MTX, ARA-C) at the height of the COVID pandemic. Of those who received 2 cycles of intensive chemotherapy, 3 had ≥ 4 cycles followed by BCNU/thiotepa autologous stem cell transplant; 10 proceeded to ‘consolidation’ (indefinite) ibrutinib to limit intensive chemotherapy or tackle systemic disease.

At a median follow up of 26 months (range 1-121), median survival was not reached; 2-year overall survival was 91% (95% CI 74-97). Three patients died during treatment (1 invasive fungal infection post COVID-19 during ibrutinib consolidation post MTX/ARA-C based therapy) and 2 during MTX-ARA-C based therapy; 7 patients relapsed or progressed and were treated with ibrutinib: 1 relapsed after ibrutinib use, 1 patient was intolerant of ibrutinib and switched to BR.

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Summary/Conclusion: Our cohort confirms that BNS may present with leptomeningeal disease and/or parenchymal disease, de novo and without systemic disease. Overall outcomes are excellent with intensive regimens, consolidated with or followed by ibrutinib; however, there are treatment-related toxicities emphasising the need for a tailored approach.

P1113: PROGNOSTIC ROLE OF GENETIC ABNORMALITIES IN MANTLE CELL LYMPHOMA

E. Kleina 1,* , S. Voloshin 1 , J. Vokueva 1 , O. Petukhova 1 , L. Martynenko 1 , M. Bakai 1 , J. Ruzhenkova 1 , S. Linnikov 1 , E. Kariagina 2 , O. Uspenskaia 3 , I. Ziuzgin 4 , S. Bessmelcev 1 , S. Sidorkevich 1 , I. Martynkevich 1

1 Russian Research Institute of Hematology and Transfusiology, Federal Medical and Biological Agency; 2 St. Petersburg GBUZ “City Hospital No. 15; 3 GBUZ Leningrad Regional Clinical Hospital; 4 ”The N.N. Petrov National Medicine Research Institute of oncology” Ministry of Health of Russia, Saint-Petersburg, Russia

Background: Mantle cell lymphoma (MCL) – B-cell lymphoma with a high frequency of genome instability associated with a recurrent clinical course, short non-progressive and overall survival. Special attention is paid to the “double-hit” MCL (simultaneous presence of translocations of CCND1 and C-MYC genes), characterized mainly by a pleomorphic/blastoid morphological variant, refractory clinical course and unfavorable prognosis.

Aims: To identify the spectrum and frequency of occurrence of genetic anomalies and their effect on the course of MCL.

Methods: The results of a standard cytogenetic study (G-banding) in 51 and FISH studies in 75 patients with MCL are presented.

Results: In a standard cytogenetic study, a pathological karyotype was found in 25/51 (49,0%) patients. Translocation t(11;14)(q13;q32) was detected in 23/51 (45,1%) patients. As part of the complex karyotype, t(11;14) was determined in 16/51 (31,4%) cases. The FISH study made it possible to additionally identify genetic aberrations in patients with MCL. In patients with normal karyotype t(11;14) was found in 18/51 (35,3%) cases. C-MYC gene rearrangements were detected in 16/75 (21,3%) cases. In this group, genetic abnormalities were found in 7/11 (63,6%) patients (in 5 patients, mitoses were not obtained). In all patients t(11;14) was detected either as a single genetic aberration (in 1/7 (14,3%)) or as part of a complex karyotype (in 6/7 (85,7%)) in combination with genetic abnormalities 7 and 17 chromosomes. In 8/16 (50,0%) patients, C-MYC gene rearrangements were combined with a deletion of the TP53 gene. In 59/75 (78,7%) cases, C-MYC gene rearrangements were not detected. Chromosomal aberrations were found in 18/39 (46,2%) patients, including complex karyotype changes in 10/18 (55,6%) patients. Translocation t(11;14) - in 16/18 (88,9%) cases. TP53 gene deletion was detected in 10/59 (16,9%) patients. Data analysis showed a significant adverse effect of the complex karyotype, TP53 deletion, C-MYC rearrangement on the clinical course of MCL compared with the standard risk group. The median progression-free survival in high molecular risk patients was 12,5 months; the median progression-free survival was not achieved in the standard risk group.

Summary/Conclusion: An integrated approach to the genetic diagnosis of MCL using standard cytogenetic and FISH studies makes it possible to identify a high-risk group: “double-hit” and with complex changes in the karyotype. It was also shown that in the group of patients with changes in the C-MYC gene, additional genetic anomalies were more often detected, which are an unfavorable prognostic factor: a complex karyotype (the presence of three or more aberrations) – 37,5% and 16,9%, and aberrations involving the gene TP53 – 50,0% and 16,9%. Changes in these genes are associated with shorter progression-free survival rates.

P1114: ZANDELISIB ON INTERMITTENT DOSING AS A SINGLE AGENT OR IN COMBINATION WITH RITUXIMAB OR ZANUBRUTINIB IN RELAPSED/REFRACTORY (R/R) FOLLICULAR LYMPHOMA (FL): RESULTS FROM A MULTI-ARM PHASE 1B STUDY

J. D. Soumerai 1,* , D. Jagadeesh 2 , H. Salman 3 , F. Samaniego 4 , K. Patel 5 , A. Stathis 6 , N. Reddy 7 , V. P. Kenkre 8 , A. Asch 9 , C. Diefenbach 10 , I. S. Lossos 11 , D. Persky 12 , F. Awan 13 , W. Huang 14 , K. Vandever 14 , A. D. Zelenetz 15

1 Massachusetts General Hospital, Boston; 2 Cleveland Clinic, Cleveland; 3 Stony Brook University, Stony Brook; 4 The University of Texas MD Anderson Cancer Center, Houston; 5 Swedish Cancer Institute, Seattle, United States of America; 6 Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 7 Vanderbilt University Cancer Center, Nashville; 8 University of Wisconsin Carbone Cancer Center, Madison; 9 Oklahoma University Health Sciences Center, Oklahoma City; 10 Perlmutter Cancer Center at NYU Langone Health, New York; 11 Sylvester Comprehensive Cancer Center, Miami; 12 University of Arizona, Tucson; 13 University of Texas Southwestern Medical Center, Dallas; 14 MEI Pharma, Inc., San Diego; 15 Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: PI3Kδ inhibitors administered daily in patients (pts) with FL were limited by immune-related adverse events (AEs) due to regulatory T cell (Treg) suppression following continuous on-target inhibition. Zandelisib is a structurally differentiated oral PI3Kδ inhibitor with high specificity and target-binding affinity. We hypothesized that zandelisib administered on an intermittent dosing (ID) schedule (one capsule daily on days 1-7 of a 28-day cycle) would enable Treg repopulation during treatment breaks and improve safety.

Aims: We conducted this analysis to assess the safety and efficacy of zandelisib administered by ID as a single agent or in combination therapy in pts with R/R FL enrolled in a 3-arm phase 1b study in various B-cell malignancies.

Methods: Eligible pts ≥18 years with FL Grade I-IIIA, ECOG PS 0-2, progressive disease after ≥1 prior therapy and no prior PI3Kδ inhibitor, provided consent prior to enrollment ( NCT02914938 ). A dose escalation stage assessed single-agent zandelisib daily continuously and is not reported here (Soumerai et al, J Clin Oncol 2018;36:#7519). Subsequent cohorts evaluated zandelisib 60 mg daily for two 28-day cycles then on ID as a single agent (Group 1) or in combination with rituximab 375 mg/m 2 weekly x4 in cycle 1 and on Day 1 of cycles 3-6 (Group 2). Group 3 evaluated zandelisib 60 mg on ID from cycle 1 and zanubrutinib 80 mg twice daily. Treatment was continued until disease progression or intolerance. Imaging scans were obtained after 2 and 6 cycles 6 and then every 6 months. Response was assessed by investigators using the Lugano Criteria.

Results: 69 FL pts were treated: 18 in Group 1, 19 in Group 2, and 32 in Group 3. Enrollment began in September 2017 for Group 1 and 2 and June 2019 for Group 3, and is completed in all 3 groups. Median age was 66 years (range 38-87), median prior therapies was 2 (range 1-5), 40 pts (58%) received ≥2 prior therapies, 45 pts (65%) were POD24, 21 pts (30.4%) were refractory to last therapy, and 28 pts (41%) had tumors ≥5 cm. With a median follow-up of 10.8 months (range 1.8-49.5), 6 pts (8.7%) have discontinued therapy due to an AE. Grade 3 AEs of special interest (AESI) were rash in 6 pts (8.7%), ALT increased in 6 (8.7%), diarrhea in 3 (4.3%), colitis in 2 (2.9%), and AST increased in 3 (4.3%). No grade 4-5 AESI were reported. 1 pt in Group 3 had reversible grade 4 drug rash with eosinophilia and systemic symptoms (DRESSS) syndrome. Grade 3-4 neutropenia was observed in 11 pts (16%). The overall response rate (ORR) in 65 evaluable pts was 84.6% (55/65), 95% CI 73.5-92.4, and the CR rate was 24.6% (16/65), 95% CI 14.8-36.9. The ORR was 77.8% (14/18) in Group 1, 94.7% (18/19) in Group 2, and 82.1% (23/28) in Group 3. The median duration of response (DOR) was 31.1 months in Group 1, 25.8 months in Group 2, and not yet mature in Group 3, with median drug exposure of 17.1, 20.5, and 7.1 months, respectively (Figure).

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Summary/Conclusion: Zandelisib was generally well tolerated when administered by ID as a single agent or in combination with rituximab or zanubrutinib, with a low rate (8.7%) of grade 3 AESI and discontinuations due to AEs. The high ORR and prolonged DOR are encouraging. This profile supports further evaluation of zandelisib on ID as a single agent and in combination in various B-cell malignancies, both in R/R disease and in earlier lines of therapy.

P1115: A MULTICENTER PHASE 1/2 TRIAL OF EO2463, A MICROBIAL-DERIVED PEPTIDE THERAPEUTIC VACCINE, AS MONOTHERAPY AND IN COMBINATION WITH LENALIDOMIDE AND RITUXIMAB, FOR TREATMENT OF PATIENTS WITH INDOLENT NHL

P. L. Zinzani 1,* , S. M. Ansell 2 , F. Bosch 3 , J. W. Friedberg 4 , J. P. Marolleau 5 , L. Arcaini 6 , R. Garcia-Sanz 7 , A. K. Gopal 8 , C. Grande 9 , R. Merryman 10 , A. Pinto 11 , S. D. Smith 12 , J. C. Villasboas 2 , D. Wallace 13 , J. Fagerberg 14 , J. G. Magalhaes 14 , P. Armand 10

1 Institute of Hematology “L. e A. Seràgnoli”, University of Bologna, Bologna, Italy; 2 Division of Hematology, Mayo Clinic, Rochester, United States of America; 3 Department of Hematology, Vall d’Hebron Institute of Oncology, Barcelona, Spain; 4 University of Rochester Medical Center, Rochester, United States of America; 5 CHU Amiens, Amiens, France; 6 Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 7 Hospital Universitario de Salamanca, Salamanca, Spain; 8 Division of Medical Oncology, University of Washington, Seattle, United States of America; 9 Universidad de Navarra, Madrid and Pamplona, Spain; 10 Dana-Farber Cancer Institute, Boston, United States of America; 11 Istituto Nazionale Tumori “Fondazione G.Pascale”- IRCCS, Naples, Italy; 12 University of Washington/Fred Hutchinson Cancer Research Center, Seattle; 13 Department of Medicine, University of Rochester, Rochester, United States of America; 14 Enterome, Paris, France

Background: EO2463 is a therapeutic vaccine designed to activate existing commensal bacteria-specific memory T cells that cross-react with B cell markers in order to drive anti-tumor immune activity against B-cell malignancies. EO2463 contains four microbial-derived, synthetically produced peptides (OMP72, OMP64, OMP65, and OMP66), which correspond to cytotoxic CD8 T cell HLA-A2 restricted epitopes, and exhibit molecular mimicry with the B cell markers CD20, CD22, CD37, and CD268 (BAFF-receptor), respectively. In pre-clinical models, these peptides can generate strong immune responses and specifically stimulate cross-reactive cytotoxic CD8 T cells to recognize the chosen B cell targets. EO2463 also contains a CD4 helper peptide referred to as universal cancer peptide 2, derived from the human telomerase reverse transcriptase catalytic subunit. The present study is a first-in-human clinical trial of this microbiome-derived peptide therapeutic cancer vaccine approach in patients with follicular lymphoma (FL) and marginal zone lymphoma (MZL).

Aims: The primary objectives of the phase 1 part of the trial are to define the recommended phase 2 dose (RP2D) for EO2463 monotherapy, and to confirm the safety of EO2463 at the monotherapy RP2D in combination with lenalidomide (EL), rituximab (ER), and lenalidomide/rituximab (ER2).

The primary objective of the phase 2 part of the trial is to estimate the objective response rate (ORR) according to the Lugano Classification 2014 during EO2463 monotherapy

Methods: This four-cohort phase 1/2 trial will investigate EO2463 monotherapy, and combinations of EO2463/lenalidomide (EL), EO2463/rituximab (ER), and EO2463/lenalidomide/rituximab (ER 2 ), for treatment of patients with FL and MZL. Cohort 1 is a safety lead-in dose-finding in patients with relapsed/refractory (RR) disease, with a 3-by-3 design to establish the recommended phase 2 dose (RP2D) for EO2463 monotherapy and to confirm the safety of the RP2D for combination schedules of EL, and ER 2 . Maximal eighteen patients will be included in cohort 1. After the recommended EO2463 monotherapy dose is established, cohorts 2, 3, and 4 will open to accrual. Fifteen patient will be included in each of these cohorts. Cohort 2 will investigate EO2463 monotherapy in patients with newly diagnosed FL/MZL who are not in need of treatment; cohort 3 will investigate EO2463 monotherapy, followed by ER in patients with limited tumor burden who need treatment, and cohort 4 will further investigate EL, followed by ER 2 in the RR setting. EO2463 will be administered subcutaneous 4 times at 2-week intervals, followed by continued booster administrations every 4 weeks for 9 (Cohorts 2 and 3) or 12 (Cohorts 1 and 4) months. Inclusion/exclusion criteria, and the design and schedule of the intense immune and safety monitoring will be presented.

Results: The safety lead-in dose-finding is currently ongoing, and no safety concerns have been observed thus far.

Summary/Conclusion: This is a first in human clinical trial to evaluate safety, determine the recommended phase 2 dose and estimate efficacy of a novel microbial-derived peptide therapeutic vaccine, EO2463, in patients with indolent non-hodgkin lymphoma as monotherapy and in combination with lenalidomide and rituximab.

P1116: SAFETY, PHARMACOKINETIC (PK), PHARMACODYNAMIC (PD) AND ACTIVITY OF THE HIGHLY SELECTIVE PHOSPHOINOSITIDE 3-KINASE INHIBITOR DELTA (PI3KΔ) INHIBITOR IOA-244 IN PATIENTS WITH FOLLICULAR LYMPHOMA (FL)

C. Carlostella 1,* , M. Lahn 2 , T. Hammett 2 , L. van der Veen 2 , Z. Johnson 2 , C. Pickering 2 , A. Santoro 1

1 IRCSS Humanitas Research Hospital, Milan, Italy; 2 iOnctura, Geneva, Switzerland

Background: IOA-244 is a highly selective inhibitor of PI3Kδ, and has a favourable safety and ADME profile in patients (pts) with solid malignancies. In pts with uveal melanoma, IOA-244 also reduced circulating Tregs counts at the biologically effective dose (BED) range.

Aims: As part of the First-in-human (FiH) dose escalation study, pts with FL received IOA-244 at the BED range established in patients with solid malignancies to confirm its favourable safety and PK profile in pts with haematologic malignancies.

Methods: IOA-244 was investigated in a two-part FIH study. Part A explored the continuous daily dosing of IOA-244 at 10, 20, 40 and 80 mg in pts with solid malignancies and at 20 mg and 80 mg in pts with FL. Part B consists of expansion cohorts of specific tumour indications, including pts with lymphoma. Primary objective: safety of the anticipated BED, or the recommended phase 2 dose (RP2D). Secondary objectives: PK; PD (e.g., inhibition of CD63 expression on basophils, changes in immune cell subsets in peripheral blood); Lugano-based responses; PFS and OS

Results: While Part A for solid malignancies have previously been reported (Di Giacomo et al 2021), we here report for the first time the safety, PK, PD and activity of pts with FL. This part of the study is still ongoing in patients with FL and is anticipated to be completed in 2022. Pts at the first cohort of 20 mg QD daily (4/4; 2 female and 2 males) had no DLT and no dose interruptions. Transient platelet reduction (G3) and AST/ALT elevation (G2) were observed in 1/4 pts, which improved while pt was on therapy. The PK and ADME profile was consistent with the ones observed in pts with solid malignancies. One patient had FL and DLBCL as prior co-primary malignancy with 7 prior lines of therapy. The other 3 pts had 1-3 prior lines of therapy. The best response was SD (1/3 pts) and all pts progressed after completing 2 cycles. LDH was initially elevated in 2/4 pts which subsequently decreased on therapy. 3/4 pts had low Treg counts at baseline. Additional pts are being recruited at the RP2D for solid malignancies, at 80 mg QD.

Reference:

Di Giacomo et al. Annals of Oncology (2021) 32 (suppl_7): S1428-S1457. 10.1016/annonc/annonc787

Summary/Conclusion: The PK and ADME profile of IOA-244 in pts with FL appears to match that in pts with solid malignancies. Additional pts at the 80 mg QD dose are expected to match the safety observed in pts with solid malignancies. In contrast to other PI3Kd inhibitors, IOA-244 is highly selective and may therefore target Tregs in pts with FL without off-target effects induced by inhibition of other PI3K isoenzymes.

P1117: THREE-YEAR FOLLOW-UP OF OUTCOMES WITH KTE-X19 IN PATIENTS WITH RELAPSED/REFRACTORY MANTLE CELL LYMPHOMA IN ZUMA-2

M. L. Wang 1,* , J. Munoz 2 , A. Goy 3 , F. L. Locke 4 , C. A. Jacobson 5 , B. T. Hill 6 , J. M. Timmerman 7 , H. Holmes 8 , I. W. Flinn 9 , D. B. Miklos 10 , J. M. Pagel 11 , M. J. Kersten 12 , R. Houot 13 , A. Beitinjaneh 14 , W. Peng 15 , X. Fang 15 , R. R. Shen 15 , R. Siddiqi 15 , I. Kloos 15 , P. M. Reagan 16

1 The University of Texas MD Anderson Cancer Center, Houston, TX; 2 Banner MD Anderson Cancer Center, Gilbert, AZ; 3 John Theurer Cancer Center, Hackensack University, Hackensack, NJ; 4 Moffitt Cancer Center, Tampa, FL; 5 Dana-Farber Cancer Institute, Boston, MA; 6 Cleveland Clinic Foundation, Cleveland, OH; 7 David Geffen School of Medicine at UCLA, Los Angeles, CA; 8 Texas Oncology, Dallas, TX; 9 Sarah Cannon Research Institute and Tennessee Oncology, Nashville, TN; 10 Stanford University School of Medicine, Stanford, CA; 11 Swedish Cancer Institute, Seattle, WA, United States of America; 12 Amsterdam UMC, University of Amsterdam, Amsterdam, Cancer Center Amsterdam, The Netherlands, on behalf of HOVON/LLPC, Amsterdam, Netherlands; 13 CHU Rennes, Université Rennes, INSERM & EFS, Renne, France; 14 University of Miami, Miami, FL; 15 Kite, a Gilead Company, Santa Monica, CA; 16 University of Rochester Medical Center, Rochester, NY, United States of America

Background: Brexucabtagene autoleucel (KTE-X19) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of patients (pts) with relapsed/refractory (R/R) mantle cell lymphoma (MCL). In ZUMA-2, a 93% objective response rate (ORR; 67% complete response [CR] rate) was reported with KTE-X19 in pts with R/R MCL (median follow-up: 12.3 mo; 60 efficacy-evaluable pts; Wang et al. N Engl J Med. 2020).

Aims: To present updated outcomes from ZUMA-2 with 2 years of additional follow-up.

Methods: Adult pts (≥18 years) with R/R MCL underwent leukapheresis and conditioning chemotherapy followed by a single infusion of KTE-X19. Minimal residual disease (MRD) was an exploratory endpoint (sensitivity 10 -5 ) evaluated in peripheral blood using next-generation sequencing. Updated results are reported for all 68 treated pts.

Results: After 35.6 mo median follow-up, the ORR (CR + partial response) was 91% (95% CI, 81.8-96.7), with a 68% CR rate (95% CI, 55.2-78.5). The median duration of response (DOR) was 28.2 mo (95% CI, 13.5-47.1), with 25 of 68 treated pts (37%) still in ongoing response (all CR) at data cutoff. Late relapse >24 mo post-infusion was infrequent (n=3). The medians for progression-free survival (PFS) and overall survival (OS) were 25.8 mo (95% CI, 9.6-47.6) and 46.6 mo (95% CI, 24.9-not estimable), respectively.

MRD was analyzed in 29 pts total; 24 of 29 were MRD-negative at mo 1, and 15 of 19 with available data were MRD-negative at mo 6. At data cutoff, the medians for DOR, PFS, and OS in the 15 MRD-negative pts were all not reached, vs 6.1, 7.1, and 27.0 mo, in the 4 MRD-positive pts, respectively. MRD-negative status at mo 1, 3, and 6 was associated with durable response, with 55%, 71%, and 69% of MRD-negative pts at those timepoints remaining in ongoing CR at data cutoff. Circulating tumor DNA analysis of MRD at mo 3 and 6 was predictive of relapse (AUC 0.80 and 0.75, respectively).

No new safety signals were observed. Only 3% of treatment-emergent adverse events (AEs) of interest occurred since the primary report. The most frequent Grade ≥3 AE was neutropenia (1 [1%] Grade 3; 7 [10%] Grade 4). Two pts had KTE-X19-related Grade 3 serious infections: pneumonia and upper respiratory tract infection (n=1); influenza (n=1). There were no new cytokine release syndrome AEs and 1 new serious neurologic AE of Grade 3 encephalopathy (13.0 mo post-infusion) that was considered not related to study treatment. Three new Grade 5 AEs occurred, none of which were considered related to study treatment: Salmonella bacteremia (24.9 mo post-infusion), myelodysplastic syndrome (25.2 mo post-infusion), and acute myeloid leukemia (37.5 mo post-infusion).

Summary/Conclusion: These data represent the longest follow-up of CAR T-cell therapy in pts with MCL to date and suggest that KTE-X19 induces durable long-term responses with manageable safety and low late relapse potential in R/R MCL.

P1118: PROGNOSTIC SIGNIFICANCE OF ABSOLUTE MONOCYTE COUNT AND LYMPHOCYTE TO MONOCYTE RATIO IN MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMA

Y. Li 1,* , C. Shang 1 , Y. Ren 1 , L. Wang 1 , J. Li 1 , W. Xu 1

1 Hematology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China

Background: Extranodal marginal zone B-cell lymphoma of the MALT lymphoma is a unique type of indolent lymphoma. It usually presents with local disease and exhibit an indolent clinical course, but some patients have disseminated systemic symptoms and require systemic treatment. Monocytes, as part of the important immune cells in tumor microenvironment, are closely linked to the pathogenesis and progression of malignancies. In recent studies, the absolute monocyte count (AMC) and lymphocyte to monocyte ratio (LMR) are shown to reflect the host systemic immunity states and have prognostic significance in different kinds of lymphoma.

Aims: Our study aims to explore the prognostic significance of AMC and LMR in MALT lymphoma, and to propose a more accurate prognostic index based on MALT-IPI.

Methods: Baseline clinical characteristics collected from the medical records at diagnosis. PFS and OS were analyzed with the Kaplan-Meier method and compared using the log-rank test. X-tile analysis was performed to identify cut-off value. Chi-square test was performed for comparisons between groups. Cox proportional-hazards regression models were used for univariate and multivariate analysis. Statistical analyses were performed with MedCalc 19.5.6, SPSS software 26.0, R software version 4.0.4 and Graphpad Prism 9.0.

Results: 316 MALT lymphoma patients were enrolled in this study. A statistically evident dominance showed that LMR was related to age, LDH level, β2-MG level, B symptom, ECOG PS and systemic therapy. With a median follow-up time of 39.1 months (1–237 months), median PFS was 146.4 months and median OS was not reached, estimated PFS rate at 3 years and 5 years were 84.1% and 79.6%, estimated OS rate at 3 years and 5 years were 94.9% and 92.4%, respectively. According to Figure 1, high AMC group (>0.6×109/L) and low LMR group (<1.8) were associated with poor outcomes. We performed Cox proportional-hazards regression for PFS and OS, MALT-IPI, ECOG PS and LMR were identified to have independent prognostic significance for PFS while MALT-IPI, β2-MG and LMR were independently associated with poor OS. Figure 2A showed that low level of LMR in subgroup was related to a poor PFS. The prognostic value of low level of LMR for PFS was more significant in age (p value for interaction =0.020). Across a subgroup analysis of OS (Figure 2B), the prognostic value of low level of LMR was consistent. Since we have confirmed AMC showed prognostic significance in MALT lymphoma survival. We combined MALT-IPI and AMC to generate a new prognostic index named ‘MALT-IPI-M’, which included four parameters: age≥70 years, Ann Arbor stage III or IV, serum LDH level >UNL, and LMR <1.8. MALT-IPI-M classified patients into low-risk group (the MALT-IPI-M=0), intermediate-risk group (the MALT-IPI-M=1) and high-risk group (the MALT-IPI-M≥2), the proportions of different stratifications in our cohort are 52.5%, 32.9% and 14.6%, respectively. As shown in Figure 3, we generated Receiver-operator characteristic (ROC) curves to compare the prognostic prediction capability of MALT-IPI and MALT-IPI-M. The area under the curves (AUCs) for MALT-IPI-M were 0.682 for PFS and 0.804 for OS, which was larger than AUCs for MALT-IPI, 0.654 for PFS and 0.788 for OS. This illustrated that MALT-IPI-M has a better capability of distinguishing MALT patients with different risk.

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Summary/Conclusion: In conclusion, our findings suggest that low level LMR at diagnosis might be associated with inferior PFS and OS in patients with MALT lymphoma. Incorporating LMR into MALT-IPI may permit a more accurate risk assessment of disease progression.

P1119: THE ORAL PI3KΔ INHIBITOR LINPERLISIB FOR THE TREATMENT OF RELAPSED OR REFRACTORY FOLLICULAR LYMPHOMA: A SINGLE-ARM MULTICENTER PHASE 2 CLINICAL TRIAL

T. wang 1 , X. sun 2 , L. qiu 3 , H. su 4 , J. cao 5 , Z. li 6 , Y. song 7 , L. zhang 8 , S. yi 9 , L. qiu 9,* , J. zhou 10 , H. wu 11 , W. zhang 12 , J. li 13 , K. zhou 14 , H. zhou 15 , Y. yang 16 , Z. li 17 , H. cen 18 , Z. cai 19 , Z. zhang 20 , W. fu 21 , J. jin 19 , F. li 22 , W. wu 23 , X. gu 24 , W. zhu 25 , L. liu 26 , Z. li 27

1 Department of Lymphoma and Myeloma, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin; 2 department of medical oncology, the second hospital of dalian medical university, dalian; 3 Department of Lymphoma, Tianjin Medical University Cancer Institute and Hospital, Tianjin; 4 Department of Lymphoma, The Fifth Medical Center of PLA General Hospital, Beijing; 5 Fudan University Shanghai Cancer Center, Shanghai; 6 Sun Yat-sen University Cancer Center, guangzhou; 7 Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing; 8 West China Hospital, Sichuan University, chengdu; 9 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin; 10 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology; 11 Department of Medical Oncology, Hubei Cancer Hospital, Wuhan; 12 Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing; 13 Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai; 14 Department of Hematology, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Zhengzhou; 15 Department of Lymphoma & Hematology, Hunan Cancer Hospital, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha; 16 Department of Lymphoma and Head and Neck Cancer, Fujian Provincial Cancer Hospital, The Affiliated Tumor Hospital of Fujian Medical University, Fuzhou; 17 Department of Hematology, The First Affiliated Hospital of Xiamen University and Institute of Hematology, School of Medicine, Xiamen University, Xiamen; 18 Department of Haematology/Oncology and Paediatric Oncology, Guangxi Medical University Affiliated Cancer Hospital, Nanning, China, Nanning; 19 Department of Hematology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou; 20 Department of Medical Oncology, Sichuan Cancer Hospital and Institute, Chengdu; 21 Department of Hematology, The Myeloma & Lymphoma Center, Shanghai Changzheng Hospital, The Second Military Medical University, Shanghai; 22 Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang; 23 Department of Internal Medicine and Oncology, Zhongshan Hospital, Xiamen University, Xiamen; 24 Department of Hematology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine; 25 Department of Oncology, Zhujiang Hospital of Southern Medical University, Guangzhou; 26 Department of Hematology, The Fourth Hospital of Hebei Medical Universit, Shijiazhuang; 27 Hematological Disorders, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

Background: The orally active phosphatidylinositol 3-kinase delta (PI3Kδ) inhibitor linperlisib which conveyed acceptable safety and notable efficacy for B-cell lymphoma medications in a previous phase 1 dose escalation study was investigated in this phase 2 study for the treatment of relapsed and/or refractory follicular lymphoma (FL) patients who had received at least two prior systemic treatments

Aims: To explore the satefy and efficacy of linperlisib in FL

Methods: Linperlisib was administered at 80 mg qd, po in a 28-day cycle until disease progression or intolerable toxicity occurred. The primary outcome was the objective response rate (ORR), while secondary outcomes included the duration of response (DOR), progression-free survival (PFS), overall survival (OS), disease control rate (DCR) and the drug safety profile.

Results: For 84 FL patients in the full analysis set (FAS) the ORR was 79.8% (95% CI: 69.6-87.8, 67 patients), with 13 patients achieving a complete response and 54 a partial response. The median DOR was 12.3 months (range, 9.3-15.9) with 6-month and 12-month DORs of 80.0% (95% CI: 67.4-88.1) and 55.3% (95% CI: 40.6-67.8). The median PFS was 13.4 months (95% CI: 11.1-16.7), with 6-month and 12-month PFS rates of 78.7% (95% CI: 67.5-86.3) and 53.1% (95% CI: 40.3-64.3). The median OS was not reached, while 6-month and 12-month OS rates were 97.6% (95% CI: 90.6-99.4) and 91.4% (95% CI: 82.7-95.8), respectively. The most frequent any-grade treatment related AEs (TRAE) were neutropenia (47%), hypertriglyceridemia (25%), while the most frequent TRAEs ≥ grade 3 were neutropenia (15%), infectious pneumonia (19.4%) and interstitial lung disease (6.5%). AEs that were potentially immune-mediated, such as diarrhea, colitis, pneumonitis, and transaminitis, were observed at significantly lower incidences of occurrence.

Summary/Conclusion: Linperlisib demonstrated compelling clinical efficacy and a promising safety profile, which has the potential to surpass the approved PI3K inhibitors for the treatment of relapsed or refractory FL patients after two prior therapies.

P1120: THE EFFICACY AND SAFETY OF ZANUBRUTINIB AND DEXAMETHASONE IN SYMPTOMATIC WALDENSTROM MACROGLOBULINNEMIA

A. Liu 1 , J. Yin 1 , J. Lu 2 , Y. Ma 3 , D. Gao 4 , L. Hua 5 , Y. Tian 1 , Y. Jian 1 , W. Chen 1,*

1 Beijing Chaoyang Hospital, affiliated to Capital Medical University; 2 Peking University People’s Hospital, Beijing; 3 Second hospital of Shanxi Medical University, Taiyuan; 4 The Affiliated Hospital of Inner Mongolia Medical University, Huhehaote; 5 Affiliated Hospital of Hebei University, Shijiazhuang, China

Background: Bruton tyrosine kinase (BTK) inhibition is an effective treatment approach for patients with Waldenström macroglobulinemia (WM)，but the efficacy of single drug is limited.

Aims: This single arm study (ChiCTR2000038140) evaluated the efficacy and safety of the combination of zanubrutinib, a novel, highly selective BTK inhibitor, and dexamethasone in patients with WM.

Methods: Symptomatic patients with WM were enrolled to the regimen of zanubrutinib and dexamethasone (ZD)*. The primary endpoint was objective response rate (ORR), progression-free survival (PFS). Key secondary endpoints included the proportion of patients achieving a complete or very good partial response (CR or VGPR), duration of response (DOR), Time to response (TOR), disease burden, and safety. The control group** were matched patients treated by chemotherapy or immunechemotherapy previously in Beijing Chaoyang Hospital.

Results: A total of 22 Patients with WM were enrolled in this study, median age 67(36-89); 68.2% males, 12 patients were untreated, others were treated patients. IPSS assessment grade1 23.8%; grade2 19.0%; grade3 57.2%. 90.9% (20/22) patients with MYD88L265P mutation, 27.3% (3/11) patients with CXCR4 mutation.

21 patients received ≥1 dose of study treatment. Median follow-up of 8.2 months, median DOR and PFS were not reached; 95% of patients were progression-free at 6 months. ORR was 95% in those (17/18) received ZD regimen more than 2 months. No patient achieved a CR. 33.3% of patients in ZD group achieved a VGPR, time to VGPR within 3 months in 57.1% of patients，a statistically significant difference with control group (0%, P = 0.001). Time to PR in ZD group was 2 months, much faster than control group (11 months) (P = 0.023) by K-M analysis.

The study-safety profile was consistent with previous BTK inhibitor clinical trial data. 45% of patients had any grade AEs. In which, the most frequent grade <=2 AEs were hemorrhage(18.2% all grade 1), rash(9.1%), hyperglycemia (13.6%), infection(9.1%), nausea and vomiting (9.1%), hypogammaglobulinemia(4.5%), neutropenia(9.1%). Grade 3/4 AEs were atrial fibrillation(4.5%), leading to treatment discontinuation. Other cause of treatment discontinuation is hyperglycemia, bowel obstruction by disease.

Comments

*The regimen of ZD: Zanubrutinib 240mg d1-28, dexamethasone 20mg D1-4,15-18. Patients more than 75 years old, Zanubrutinib 160mg d1-28, dexamethasone 10mg D1-4,15-18. After 8 cycle, Zanubrutinib use as maintenance.

** The control group: 22 treated patients with WM, median age 69.5(39-84); 68.2% males. IPSS Grade1 21.4%; Grade2 32.1%; Grade3 42.9%; Unknown 3.6%. 75%(9/12) patients with MYD88L265P mutation, 33%(1/3) patients with CXCR4 mutation. Treatment included chemotherapy(containing nitrogen mustard phenylbutyrate, fludarabine, cyclophosphamide), proteasome inhibitor regimens, rituximab regimens and immunomodulator regimens.

Summary/Conclusion: These results demonstrate that zanubrutinib and dexamethasone are quickly effective in the treatment of WM, with more deeper response and less toxicity.

P1121: TAKEAIM LYMPHOMA- AN OPEN-LABEL, DOSE ESCALATION AND EXPANSION TRIAL OF EMAVUSERTIB (CA-4948) IN COMBINATION WITH IBRUTINIB IN PATIENTS WITH RELAPSED OR REFRACTORY HEMATOLOGIC MALIGNANCIES

E. Joffe 1,* , G. Nowakowski 2 , H. Tun 3 , A. Rosenthal 4 , M. Lunning 5 , R. Ramchandren 6 , C.-C. Li 7 , L. Zhou 7 , E. Martinez 7 , R. von Roemeling 7 , R. Earhart 7 , M. McMahon 7 , I. Isufi 8 , L. Leslie 9

1 MSKCC, NY; 2 Mayo Clinic-Minnesota, Rochester, Rochester; 3 Mayo Clinic-Florida, Jacksonville; 4 Department of Hematology, Mayo Clinic- Arizona, Phoenix; 5 University of Nebraska, Omaha; 6 University of Tennessee Medical Center, Knoxville; 7 Curis, Lexington; 8 Yale New Haven Hospital, New Haven; 9 John Theurer Cancer Center, Hackensack, United States of America

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4), which is essential for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in B cell proliferation. IRAK4 forms a Myddosome complex with MYD88 adaptor protein and drives overactivation of nuclear factor-kappa B (NF-κB), causing inflammation and tumor growth. Emavusertib has been reported to be well tolerated and active as monotherapy in heavily pretreated patients with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL). Preclinical studies demonstrated that tumor resistance and survival via IRAK4 activation could be delayed or reversed. Emavusertib crossed the blood-brain barrier in a murine PDX model of pCNS lymphoma, resulting in tumor response and prolonged survival. In combination with Bruton tyrosine kinase (BTK) inhibitors, emavusertib showed in vivo synergy in B-cell NHL. Here we present an update on the preliminary efficacy data of emavusertib + ibrutinib in R/R hematologic malignancies.

Aims: Assessment of safety and clinical activity of emavusertib in combination with ibrutinib at full prescribed dose.

Methods: This is an ongoing open-label trial ( NCT03328078 ) of emavusertib as monotherapy and in combination with ibrutinib. Part A1 (completed) dose escalation of emavusertib as monotherapy; the recommended phase 2 dose (RP2D) is 300 mg BID with continuous oral dosing. Part A2 (dose escalation in combination with ibrutinib), and Part B (a basket design of 4 expansion cohorts of emavusertib and ibrutinib: BTK-naïve MZL, DLBCL, or PCNSL and NHL with adaptive resistance to ibrutinib). The primary endpoints of Parts A1 and A2 include safety, tolerability, and RP2D. The primary endpoints of Part B include CR or ORR, with key secondary endpoints of DOR, DCR, PFS and OS following treatment of emavusertib at dose levels of 200 (DL1) or 300 mg BID (DL2) with ibrutinib at full prescribed dose.

Results: As of December 7 th , 2021, 35 heavily pretreated NHL patients have received emavusertib monotherapy (median age 66 years, range 50-87), of which six patients have been on emavusertib for approximately 1 year or longer, suggesting emavusertib has a long-term acceptable safety and tolerability profile at RP2D (dose level of 300 mg BID). In Part A2 , 10 patients are treated with emavusertib + ibrutinib (median age 65 years, range 56-82). Median number of prior lines of anti-cancer therapies is 3 (range 1-8). No DLTs were observed at 200 or 300 mg dose levels to date. The preliminary efficacy data of seven evaluable patients with combination therapy showed 1 CR (MCL), 2 PR (MCL and MZL), 3 SD, and 1 PD, 3 of whom had failed prior ibrutinib. The preliminary data indicate the combination therapy may overcome ibrutinib resistance.

Summary/Conclusion: Emavusertib as a monotherapy and in combination with ibrutinib is well tolerated with an acceptable long term safety profile and promising efficacy. Part A2 is transitioning to Part B basket cohorts of MZL, ABC-DLBCL, PCNSL and NHL with adaptive resistance to ibrutinib.

P1122: EFFICACY OF ALPELISIB IN PI3K-DRIVEN LANGERHANS CELL HISTIOCYTOSIS

R. Mazor 1,* , B. Durham 2 , O. Abdel-Wahab 2 , E. Diamond 3 , G. Itchaki 4 , M. Ben-Sasson 5,6,7 , O. Hershkovitz-Rokah 1,8,9 , O. Shpilberg 1,9,10

1 Clinic of Histiocytic Neoplasms, Institute of Hematology, Assuta Medical Center, Tel Aviv, Israel; 2 Human Oncology and Pathogenesis Program; 3 Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, United States of America; 4 Department of Hematology, Rabin Medical Center, Petah Tikva; 5 The Institute for Pain Medicine, Rambam Medical Center; 6 The Rappaport school of medicine, Technion, Haifa; 7 Meuhedet Health Maintenance Organization, Zikhron Ya’akov; 8 Department of Molecular Biology, Faculty of Natural Sciences, Ariel University, Ariel; 9 Translational Research Lab, Assuta Medical Center, Tel Aviv; 10 The Adelson School of Medicine, Ariel University, Ariel, Israel

Background: Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm characterized by the accumulation of clonal dendritic cells expressing CD1a and CD207 (Langerin). LCH affects both children and adults and typically ranges from an indolent unifocal form to a progressive multisystemic disease. In the past decade, in depth molecular profiling of LCH as well as other histiocytic neoplasms demonstrated that these diseases are fundamentally dependent on somatic MAPK activating mutations, with the canonical V600E BRAF mutation emerging in more than 50% of LCH patients. Nonetheless, MAPK independent driver mutations were previously reported and anecdotal evidence of successful targeted treatment of various histiocytoses harboring non-canonical drivers such as RET, ALK and CSF1R were previously described in the literature.

Aims: To report a case of PI3K driven LCH treated with an isoform specific PIK3CA inhibitor.

Methods: Case summary: a 46 year-old mother of 3 presented with complaints of progressive headaches, polydipsia and polyuria. Following a pathological water deprivation test and identification of pituitary stalk thickening on MRI, a diagnosis of central diabetes insipidus (CDI) was established and desmopressin treatment was initiated. However, 10 months following her CDI diagnosis, the patient began to complain of sub febrile episodes of fever and worsening dry cough. Imaging studies including PET/CT disclosed extensive bilateral reticulo-nodular infiltration, nodules with cystic transformation and ground glass opacity, suspicious of pulmonary LCH. Histological examination of a wedge biopsy specimen obtained from the lungs confirmed the diagnosis of LCH and consequent molecular studies identified the M1043V PIK3CA mutation. During her medical investigation, the patient’s conditioned worsened, with new disease foci emerging – including cervical lymphadenopathy and a lytic vertebral lesion involving D11. The patient then received palliative radiotherapy directed at the D11 lytic lesion followed by escalating systemic therapy with single agent Alpelisib, via the Managed Access Program at Novartis. Response to treatment was assessed clinically and using PET/CT. At the molecular level, the effect of Alpelisib was assessed by measuring PTEN expression levels and cell cycle regulating non-coding RNA molecules by using quantitative real-time polymerase chain reaction (qRT-PCR).

Results: Treatment with Alpelisib resulted in rapid amelioration of night sweats within 6 days, decrease in back pain within 3 weeks and normalization of all abnormally FDG avid disease foci within 3 months of treatment. The complete metabolic response observed persists currently more than a year following treatment initiation. Treatment with Alpelisib is well tolerated except for a mild increase in HbA1C levels to 6.7%. Moreover, PTEN, the main regulator of the PI3K pathway was found to be upregulated following 3 and 9 months of treatment, in parallel to downregulation of small non-coding RNAs molecules that regulates its expression.

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Summary/Conclusion: This is the first study demonstrating that the alpha catalytic subunit of PI3K is a targetable non-canonical driver of LCH.

P1123: TEMPO: A PHASE 2, RANDOMIZED, OPEN-LABEL, 2-ARM STUDY COMPARING TWO INTERMITTENT DOSING SCHEDULES OF DUVELISIB IN SUBJECTS WITH INDOLENT NON-HODGKIN LYMPHOMA (INHL)

V. Vorobyev 1,* , D. H. Yoon 2 , M. Kaźmierczak 3 , S. Grosicki 4 , C. Tarella 5 , A. Genua 6 , J. S. Kim 7 , D. Cohan 8 , M. Daugherty 8 , I. W. Flinn 9 , P. L. Zinzani 10 , L. I. Gordon 11

1 S. P. Botkin City Clinical Hospital, Moscow, Russia; 2 University of Ulsan College of Medicine, Seoul, South Korea; 3 Poznań University of Medical Sciences, Poznań; 4 Silesian Medical University, Katowice, Poland; 5 IEO Istituto Europeo di Oncologia IRCCS, Milano; 6 Università degli studi di Perugia, Terni, Italy; 7 Yonsei University College of Medicine, Severance Hospital, Seoul, South Korea; 8 Secura Bio, Las Vegas; 9 Sarah Cannon Research Institute-Tennessee Oncology, Nashville, United States of America; 10 University of Bologna, Istituto Di Ematologia, Bologna, Italy; 11 Northwestern University Feinberg School of Medicine and the Robert H. Lurie Comprehensive Cancer Center, Chicago, United States of America

Background: Dosing of duvelisib at 25 mg orally BID has been shown to be active in iNHL, as the pivotal phase 2 DYNAMO study ( NCT01882803 ) in iNHL met its primary endpoint of overall response rate (ORR) assessed by an IRC.

Aims: The TEMPO study ( NCT04038359 ; supported by Secura Bio) examined the effects of prespecified 2-week dose holidays on tumor responses and safety/tolerability in patients (pts) with iNHL based on data from a phase 3 trial (DUO) in CLL/SLL that demonstrated no negative impacts of (unscheduled) dose interruptions on response (Flinn I et al. J Clin Oncol. 2019;37(15):7523).

Methods: In TEMPO, patients were randomized to receive duvelisib 25 mg BID for one 10-week cycle followed by 25 mg BID on Weeks 3 and 4 of each subsequent 4-week cycle (Arm 1) or duvelisib 25 mg BID on Weeks 1, 2, 5, 6, 9, and 10 of one 10-week cycle, and then on Weeks 3 and 4 of each subsequent 4-week cycle (Arm 2). Pts continued treatment until progressive disease (PD), unacceptable toxicity, or study withdrawal. ORR by investigator assessment, based on 2007 revised International Working Group (IWG) criteria was the primary endpoint.

Results: As of data cutoff on 5 May 2021, 65 treated pts had at least 4 months of follow-up on or after study drug treatment. Across both arms, the median age was 63 years (range 34-85), and 47 (72.3%) of patients remained on duvelisib at the time of this analysis (see Table). The patient population was heavily pretreated (total prior lines: 36.9% = 1; 26.2% = 2; 10.8% = 3; and 26.2% ≥ 4); 93.8% of pts received prior rituximab and 43.1% prior bendamustine. As of data cutoff, 27.7% of pts have discontinued duvelisib, 19.7% due to PD (21.2% in Arm 1; 18.8% in Arm 2). Four patients, all in Arm 1, had a TEAE that led to treatment discontinuation. Across both arms, the ORR was 60% (39 of 65 pts), and the complete response (CR) rate was 12.3% (8 of 65 pts). Results were similar in pts who received prior rituximab (59.0%) and those who received prior bendamustine (57.1%) and were relatively consistent regardless of the number of prior treatments received (62.5%, 64.7%, 54.2% for pts with 1, 2, or ≥ 3 prior lines of therapy, respectively).

Overall, the incidence of TEAEs was higher in Arm 1 vs Arm 2, including grade ≥ 3 AEs (ALT elevation 30.3% vs 3.1%; AST elevation 27.3% vs 3.1%; GI disorders 9.1% vs 0; infections 9.1% vs 3.1%; rash 6.1% vs 3.1%; and drug reaction with eosinophilia 3.0% vs 0); the incidence of grade ≥ 3 neutropenia was higher in Arm 2 (9.1% vs 18.8%). TEAEs leading to discontinuation of study drug occurred only in Arm 1 (15.2%; 5 of 33 pts) and included ALT/AST elevations (2 of 33 pts; 6.1%), skin and subcutaneous tissue disorders (2 of 33 pts; 6.1%), and pneumonia (1 of 33 pts; 3.0%). There was 1 death on study in Arm 1 due to disease progression. No TEAE-related deaths occurred in either arm.

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Summary/Conclusion: Based on this interim analysis, continuous followed by intermittent dosing (Arm 1) and intermittent dosing (Arm 2) are both safe and effective in patients with iNHL. Efficacy was seen irrespective of prior lines of therapy, including those heavily pretreated and/or treated with prior rituximab or bendamustine. The ORR and CR are consistent with those seen in DYNAMO. Because these are early data, the differences between arms in certain types of AEs or discontinuations due to AEs are difficult to interpret. The study confirms the overall therapeutic profile of duvelisib and demonstrates that intermittent dosing may decrease the incidence of AEs without compromising efficacy.

P1124: MOSUNETUZUMAB RETREATMENT IS EFFECTIVE AND WELL-TOLERATED IN PATIENTS WITH RELAPSED OR REFRACTORY B-CELL NON-HODGKIN LYMPHOMA

C. Y. Cheah 1,* , N. L. Bartlett 2 , S. Assouline 3 , S. J. Schuster 4 , W. Seog Kim 5 , M. Shadman 6 , I. Isufi 7 , S. Yin 8 , M. Y. Doral 8 , J. Sit 8 , V. Chen 8 , H. Huang 9 , M. Zhou 10 , M. C. Wei 8 , L. E. Budde 11

1 Linear Clinical Research, Sir Charles Gairdner Hospital and The University of Western Australia, Perth, Australia; 2 Washington University School of Medicine, Siteman Cancer Center, St Louis, MO, United States of America; 3 Jewish General Hospital, Montreal, Canada; 4 University of Pennsylvania, Philadelphia, PA, United States of America; 5 Samsung Medical Centre, Seoul, South Korea; 6 Fred Hutchinson Cancer Research Center, Seattle, WA, Seattle, WA; 7 Yale School of Medicine, New Haven, CT; 8 Genentech, Inc., South San Francisco, CA, United States of America; 9 Hoffmann-La Roche Ltd, Mississauga, Canada; 10 F. Hoffmann-La Roche, Shanghai, China; 11 City of Hope, Duarte, CA, United States of America

Background: The prognosis for heavily pretreated patients (pts) with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL) is poor and there is a need for effective treatment options (Batlevi, et al. 2020; Crump, et al. 2017). Mosunetuzumab (M) is a T-cell engaging CD20xCD3 bispecific monoclonal antibody that redirects T cells to eliminate malignant B cells (Sun, et al. 2015; Budde, et al. 2022). Results from a single-arm, Phase I/II study ( NCT02500407 ) of M in pts with R/R aggressive and indolent NHL showed durable complete responses (CR) and a manageable safety profile (Budde, et al. 2022). Specifically, in pts with R/R follicular lymphoma (FL) after ≥2 prior therapies, M demonstrated a 60% CR rate and an 80% objective response rate with a median duration of response (DoR) of 22.8 months (Budde, et al. ASH 2021). Initial M treatment had a fixed duration (8 cycles for pts who achieved CR; 17 cycles for pts with partial response or stable disease after 8 cycles); however, for pts who achieved a CR but had progressive disease (PD) after initial treatment completion, M retreatment could be provided. We report additional data from this ongoing study to describe the M retreatment experience of pts with R/R NHL.

Aims: To evaluate the efficacy and safety of M monotherapy (mono) retreatment in pts with R/R NHL from an ongoing Phase I/II study ( NCT02500407 ).

Methods: Pts with R/R NHL who achieved CR after initial treatment with intravenous (IV) M mono but subsequently developed PD after treatment completion were eligible for retreatment. Pts were retreated with IV M mono at a previously tested dose and schedule, which included step-up dosing for cytokine release syndrome (CRS) mitigation. All pts provided informed consent.

Results: At the clinical cut-off date, March 15, 2021, 12 pts received M retreatment; 6 pts completed retreatment and 6 pts discontinued retreatment (PD, n=5; start of another anti-lymphoma therapy, n=1). M retreatment step-up dose schedules were as follows (Cycle [C] 1, Day [D] 1/C1D8/C1D15/C2D1/C3+D1): 1/2/13.5mg (n=1), 1/2/20mg (n=1), 1/2/27mg (n=1), 1/2/40.5mg (n=1) and 1/2/60/60/30mg (n=8). Median number of M retreatment cycles received was 8.0 (range, 2–13). Overall, 8 pts with R/R FL, 2 pts with R/R diffuse large B-cell lymphoma (DLBCL), 1 pt with R/R transformed FL (trFL) and 1 pt with R/R mantle cell lymphoma (MCL) were included in the analysis. During retreatment, 9 (75.0%) pts had an objective response (6/8 pts with R/R FL; 2/2 pts with R/R DLBCL; 1/1 pt with trFL; and 0/1 pt with MCL; Table ). Six/12 pts achieved a CR (4/8 pts with R/R FL, 1/2 pts with R/R DLBCL; 1/1 pt with R/R trFL; and 0/1 pt with MCL). All pts who received M retreatment were included in the safety population and had ≥1 adverse event (AE). The most common any-grade AE and Grade 3–4 AE was hypophosphatemia (n=7, 58.3% [each]; none were serious and all events resolved with or without treatment). Four pts experienced ≥1 serious AE. No Grade 5 events were reported and no pts had an AE that led to treatment discontinuation. Three pts (25.0%) had low-grade CRS (by Lee, 2014; Grade 1, n=2; Grade 2, n=1) and there were no Grade 3–4 CRS events.

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Summary/Conclusion: Our results show that IV M mono retreatment was efficacious in heavily pretreated pts with R/R NHL who initially achieved a CR with M treatment, but subsequently developed PD. M retreatment had a manageable safety profile, consistent with the initial treatment.

P1125: CELESTIMO: A PHASE III TRIAL EVALUATING THE EFFICACY AND SAFETY OF MOSUNETUZUMAB PLUS LENALIDOMIDE VERSUS RITUXIMAB PLUS LENALIDOMIDE IN PATIENTS WITH RELAPSED OR REFRACTORY FOLLICULAR LYMPHOMA

L. Nastoupil 1,* , F. Morschhauser 2 , C. W. Scholz 3 , M. Bishton 4 , S.-S. Yoon 5 , P. Giri 6 , M. C. Wei 7 , A. Knapp 8 , C.-C. Li 7 , A. Bottos 8 , H. Li 9 , E. Purev 7 , N. L. Bartlett 10

1 Emory University, Atlanta, GA, United States of America; 2 University of Lille, CHU Lille, Lille, France; 3 Vivantes Klinikum am Urban, Berlin, Germany; 4 Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; 5 Seoul National University Hospital, Seoul, South Korea; 6 Royal Adelaide Hospital, Adelaide, Australia; 7 Genentech, Inc., South San Francisco, CA, United States of America; 8 F. Hoffmann-La Roche Ltd, Basel, Switzerland; 9 Hoffmann-La Roche Ltd, Mississauga, ON, Canada; 10 Washington University School of Medicine, St. Louis, United States of America

Background: Despite significant progress with first-line immunochemotherapy, most patients with follicular lymphoma (FL) will eventually relapse with increasing refractoriness and decreasing duration of response to subsequent therapy lines (Rivas-Delgado et al. 2019). Mosunetuzumab (M) is a CD20xCD3 bispecific antibody that engages and redirects T-cells to eliminate malignant B cells (Sun et al. 2015). In an ongoing, pivotal Phase I/II trial of M monotherapy, patients with relapsed/refractory (R/R) FL who have received ≥2 prior treatment lines achieve deep and durable responses ( NCT02500407 ; Budde et al. ASH 2021). Preliminary data from a Phase Ib study have suggested favorable safety and promising activity of M in combination with lenalidomide (Len), a potent immunomodulatory agent that has shown additive/synergistic activity with an anti-CD20 antibody in R/R indolent lymphoma (Leonard et al. 2019), in patients with R/R FL who have received ≥1 prior therapy (NCT4246086; Morschhauser et al. ASH 2021). The chemotherapy-free M-Len combination may represent a promising outpatient therapy option for future management of patients with R/R FL. The randomized, multicenter Phase III study has been initiated.

Aims: CELESTIMO ( NCT04712097 ) is a randomized, multicenter, open-label Phase III study evaluating the efficacy and safety of M-Len versus rituximab plus Len (R-Len) in patients with previously treated R/R FL.

Methods: Patients must have histologically documented CD20+ FL (Grades 1–3a) requiring systemic therapy and have received ≥1 prior line of systemic therapy. Patients are randomized (1:1) to receive M-Len (M intravenously [IV] on Days [D] 1, 8 and 15 of Cycle [C] 1 [21-day cycle] and D1 of C2–12 [28-day cycles], plus Len orally [PO] on D1–21 of C2–12) or R-Len (R IV on D1, 8, 15 and 22 of C1 then on D1 of C3, 5, 7, 9, and 11, plus Len PO on D1–21 of C1–12 [all 28-day cycles]), and stratified by disease progression within 24 months of initial treatment (yes/no), number of prior lines of therapy (1 versus ≥2), and refractoriness to anti-CD20 therapy (refractory/non-refractory). All patients must provide informed consent.

Results: The primary endpoint is progression-free survival (PFS) assessed by independent review committee; secondary endpoints include investigator-assessed PFS, complete and objective response, overall survival, and safety. Biomarkers predictive of response to M-Len and R-Len will also be investigated as exploratory endpoints.

Summary/Conclusion: The study started recruitment in 2021 and plans to enroll ~400 patients from approximately 16 countries and 150 sites globally. Further study details will be presented.

P1126: MOSUNETUZUMAB IS EFFICACIOUS AND WELL TOLERATED IN PATIENTS AGED <65 AND ≥65 YEARS WITH RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA AND ≥2 PRIOR THERAPIES: SUBGROUP ANALYSIS OF A PIVOTAL PHASE II STUDY

M. Matasar 1,* , N. L. Bartlett 2 , L. H. Sehn 3 , S. J. Schuster 4 , S. Assouline 5 , P. Giri 6 , J. Kuruvilla 7 , M. Canales 8 , S. Dietrich 9 , K. Fay 10 , M. Ku 11 , L. J. Nastoupil 12 , M. C. Wei 13 , S. Yin 13 , I. To 13 , D. Turner 13 , H. Huang 14 , J. Min 15 , E. Penuel 13 , L. E. Budde 16

1 Memorial Sloan Kettering Cancer Center, New York, NY; 2 Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, United States of America; 3 BC Cancer Centre for Lymphoid Cancer and University of British Columbia, Vancouver, BC, Canada; 4 Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, United States of America; 5 Jewish General Hospital, Montreal, QC, Canada; 6 Royal Adelaide Hospital, Adelaide, Australia; 7 Princess Margaret Cancer Centre, Toronto, ON, Canada; 8 Hospital Universitario La Paz, Madrid, Spain; 9 Universitat Heidelberg, Heidelberg, Germany; 10 St Vincent’s Hospital and Royal North Shore Hospital, Sydney; 11 St Vincent’s Hospital, University of Melbourne, Melbourne, Australia; 12 MD Anderson Cancer Center, Houston, TX; 13 Genentech, Inc., South San Francisco, CA, United States of America; 14 Hoffmann-La Roche Ltd, Mississauga, ON, Canada; 15 Roche Products Ltd, Welwyn Garden City, United Kingdom; 16 City of Hope, Duarte, CA, United States of America

Background: Advancing age is associated with a decline in immune function and an increase in the incidence of comorbidity (Castellino et al. 2017). Vulnerability to treatment-related toxicities may also increase with age. Mosunetuzumab is a T-cell engaging CD20xCD3 bispecific monoclonal antibody (Ab) that redirects T cells to eliminate malignant B cells. In a pivotal Phase II study ( NCT02500407 ), mosunetuzumab induced deep and durable remissions and had a favorable safety profile in patients (pts) with relapsed/refractory (R/R) follicular lymphoma (FL) and ≥2 prior therapies (Budde et al. ASH 2021).

Aims: In the current study, we evaluated the efficacy and safety of mosunetuzumab in pts aged <65 and ≥65 years (age stratification per ESMO Clinical Practice Guidelines for management of newly diagnosed and R/R FL; Dreyling et al. 2021) in the pivotal Phase II study.

Methods: All pts had Grade (Gr) 1–3a FL and ECOG performance status (PS) 0–1, and were R/R to ≥2 prior therapies, including an anti-CD20 Ab and an alkylating agent. Intravenous mosunetuzumab was given in 21-day cycles with Cycle (C) 1 step-up dosing: C1 Day (D) 1: 1mg; C1D8 2mg; C1D15 and C2D1: 60mg; D1 of C3+: 30mg. Pts with a complete response (CR) by C8 completed therapy; pts with a partial response or stable disease continued treatment for up to 17 cycles, unless disease progression or unacceptable toxicity occurred. The primary endpoint was CR (as best response) rate by PET/CT assessed by Independent Review Committee using standard response criteria (Cheson et al. 2007). Cytokine release syndrome (CRS) was graded using ASTCT criteria (Lee et al. 2019). All pts provided informed consent.

Results: As of August 27, 2021, 90 pts had received mosunetuzumab; 67% were aged <65 years and 33% were aged ≥65 years. Of 30 pts aged ≥65 years, median age was 71 years (range: 65–90) and 43% had ECOG PS 1 at entry, 53% had FLIPI 3–5, and 70% had Ann Arbor stage III/IV disease. Median number of prior therapies in the older age group was 3 (range: 2–8); 77% were refractory to a prior anti-CD20 Ab and 43% were double-refractory to a prior anti-CD20 Ab and an alkylating agent. Compared with younger pts, those aged ≥65 years had a numerically higher objective response rate (87% [95% CI: 69–96] vs 77% [95% CI: 64–87]) and CR rate (70% [95% CI: 51–85] vs 55% [95% CI: 42–68]). The 18-month event-free rate for duration of response was 54% (95% CI: 31–76) in pts aged ≥65 years and 59% (95% CI: 43–74) in those aged <65 years. The rate of Gr 3–4 adverse events (AEs) was comparable in the older and younger age groups (73% vs 68%, respectively). A lower rate of serious AEs of any Gr was observed in pts aged ≥65 years (37% vs 52%). One patient in each age group discontinued treatment due to mosunetuzumab-related AEs. CRS occurred less frequently in pts aged ≥65 years than in those aged <65 years (30% vs 52%), while CRS events were predominantly low-Gr in both groups (age ≥65 vs <65 years: Gr 1, 20% vs 28%; Gr 2, 7% vs 22%); high-Gr CRS was uncommon (one patient in each group). All CRS events resolved. No cases of aphasia, seizures, encephalopathy or cerebral edema occurred. Rates of serious AEs of infection (any Gr) were comparable in pts aged ≥65 and <65 years (17% vs 22%, respectively). The PK disposition of mosunetuzumab was also comparable in pts aged ≥65 and <65 years.

Summary/Conclusion: Mosunetuzumab is efficacious and well tolerated in older and younger pts with R/R FL and ≥2 prior therapies. In older pts (aged ≥65 years), numerically lower rates of CRS and serious AEs were observed.

P1127: WATCHFUL WAITING IS AN ACCEPTABLE TREATMENT OPTION FOR PRIMARY OCULAR ADNEXAL MUCOSA-ASSOCIATED LYMPHOID TISSUE LYMPHOMA: A RETROSPECTIVE STUDY

K. Mizuhara 1,* , T. Kobayashi 1 , M. Nakao 2 , R. Takahashi 3 , H. Kaneko 4 , K. Shimura 4 , K. Hirakawa 5 , N. Uoshima 6 , Y. Kamitsuji 6 , K. Wada 7 , E. Kawata 7 , R. Isa 1 , T. Fujino 1 , Y. Matsumura-Kimoto 1 , T. Tsukamoto 1 , S. Mizutani 1 , Y. Shimura 1 , M. Taniwaki 8 , J. Kuroda 1

1 Division of Hematology and Oncology, Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto; 2 Department of Hematology, Otsu City Hospital, Otsu; 3 Department of Hematology, Omihachiman Community Medical Center, Omihachiman; 4 Department of Hematology, Aiseikai Yamashina Hospital, Kyoto; 5 Department of Hematology, Fukuchiyama City Hospital, Fukuchiyama; 6 Department of Hematology, Japanise Red Cross Kyoto Daini Hospital, Kyoto; 7 Department of Hematology, Matsushita Memorial Hospital, Moriguchi; 8 Center for Molecular and Therapeutics, Kyoto Prefectural University of Medicine, Kyoto, Japan

Background: Primary ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma (POAML) is the most frequent subtype of ocular adnexal lymphomas. Radiotherapy (RT) is the standard of care for localized POAML, despite several complications, such as cataracts, retinitis, dry eye, and optic neuropathy. Watchful waiting (WW) is expected to be a therapeutic option for POAML, however, its efficacy for POAML remains to be verified.

Aims: The aim of the study was to clarify the validity of WW for POAML.

Methods: We retrospectively analyzed the background, disease status, treatments, and clinical outcomes of patients histologically diagnosed with POAML between 2008 and 2019 at seven institutes belonging to the Kyoto Clinical Hematology Study Group. The primary ocular adnexal disease sites were defined as involvement of the conjunctiva, orbit, eyelid, or lacrimal gland. Bilateral ocular adnexal lymphoma without other lymphomatous involvement was defined as stage IE disease.

Results: One hundred and three patients were diagnosed with POAML, of which 11 were excluded from analysis because of lost follow-up, 11 due to missing data, and 6 due to short follow-up within 1 year. As a result, 75 patients were analyzed in this study. The median age was 68 years, and 41 patients (54.7%) were females. The most common involved site was the conjunctiva (42.7%), followed by the orbit (36.0%), lacrimal gland (12.0%), and eyelid (8.0%). Sixty-nine patients (92.0%) presented with stage IE disease, and 61 patients (81.3%) presented with a unilateral ocular region. According to the International Prognostic Index, most patients (92.0%) were classified as low risk. The tumor was completely resected at diagnosis in 17 patients (22.7%), while a residual tumor existed after biopsy in 58 patients (77.0%). WW was the most frequent first-line modality, selected for 29 patients (38.7%), including 14 patients with complete tumor resection at diagnosis. Twenty-four patients (32.0%) were treated with RT and 19 (25.3%) with rituximab monotherapy (R). In the RT group, most patients had a residual tumor after the biopsy, and the orbit was the most frequent site of lymphoma, which caused treatment-emergent symptoms that potentially impair visual function. Complete response rates were 79.2% and 42.1% in the RT and R groups, respectively. With a median follow-up of 48.8 months, there were no significant differences in the time to start of new treatment between WW and RT groups (median: both not reached [NR], p = 0.187) and between WW and R groups (median: NR vs. 69.0 months, p = 0.554). At 60 months follow-up, the estimated proportions of POAML patients not requiring new treatment were 69.4%, 85.2%, and 53.8% in the WW, RT, and R groups, respectively. All patients enrolled in this study were alive at the time of analysis. In the WW group, 9 patients (31.0%) showed disease progression, seven of whom received treatments with a median time to next treatment of 15.6 months, but the remaining two patients continued WW even after disease progression because of no subjective symptoms. In the RT group, 16 patients (66.7%) developed adverse events, most of which were mild, while grade 3 cataracts occurred in five patients. We compared the clinical characteristics to identify predictive factors for disease progression in the WW group, however, all factors analyzed were not significantly associated with disease progression.

Summary/Conclusion: WW may be an acceptable treatment option for POAML, especially in asymptomatic patients.

P1128: CLINICAL FEATURES AND OUTCOME OF PATIENTS WITH CASTLEMAN DISEASE: A SPANISH MULTICENTRIC STUDY OF 134 PATIENTS FROM GELTAMO

J. T. Navarro 1,2,3,* , C. Celades 2,3 , O. García 1,2,4,5 , E. González-Barca 6,7,8 , F. Climent 7,9 , A. Feu 9 , A. Jiménez 10 , A. Gutiérrez de la Peña 10 , M. Bastos-Oreiro 11,12 , T. Aldamiz-Echevarria 13 , A. Gutiérrez 14,15 , L. Bento 14,15 , P. Abrisqueta 16,17 , C. M. Alonso 18 , C. Tejada Chavez 18 , E. M. Ocio 19,20 , N. Fernández Escalada 19,20 , M. B. Navarro Matilla 21 , J. M. Mateos Pérez 21 , A. López-García 22 , C. Castillo-Girón 23 , S. F. Pinzón 24 , E. Pérez Ceballos 25 , J. Á. Hernández Rivas 26,27 , R. del Campo García 15,28 , E. Pardal de la Mano 29 , R. García-Sanz 30,31,32,33 , J. Rovira 34 , J. M. Sancho 1,2,3 , G. Tapia 35,36

1 Hematology, Institut Català d’Oncologia-Germans Trias i Pujol Hospital; 2 Lymphoid Neoplasms, Josep Carreras Leukaemia Research Institute; 3 Medicine, Universitat Autònoma de Barcelona; 4 Institut Germans Trias i Pujol; 5 Universitat Autònoma de Barcelona, Badalona; 6 Hematology, Institut Català d’Oncologia, Hospitalet de Llobregat; 7 IDIBELL; 8 Universitat de Barcelona, Hospitalet; 9 Pathology, Hospital Universitari de Bellvitge, Hospitalet de Llobregat; 10 Hematology, Hospital 12 de Octubre; 11 Hematology, Hospital Gregorio Marañón; 12 Instituto de Investigación Sanitaria Gregorio Marañón; 13 Infectious Diseases-Clinical Microbiology, Hospital Gregorio Marañón, Madrid; 14 Hematology, Son Espases University Hospital; 15 IdISBa, Palma de Mallorca; 16 Hematology, Hospital Universitari Vall d’Hebron; 17 Vall d’Hebron Institute of Oncology (VHIO), Barcelona; 18 Hematology and Hemotherapy, Hospital Arnau de Vilanova, Valencia; 19 Hematology, Hospital Universitario Marqués de Valdecilla (IDIVAL); 20 Universidad de Cantabria, Santander; 21 Hematology, Hospital Universitario Puerta de Hierro Majadahonda; 22 Hematology, Hospital Universitario Fundación Jiménez Diaz, Madrid; 23 Amyloidosis and Myeloma, Hospital Clinic; 24 Hematology, Hospital del Mar, Barcelona; 25 Hematology, Hospital Universitario Morales Meseguer, Murcia; 26 Hematology and Hemotherapy, Hospital Universitario Infanta Leonor; 27 Universidad Complutense de Madrid, Madrid; 28 Hematology, Hospital Universitari Son Llàtzer, Palma de Mallorca; 29 Hematology and Hemotherapy, Hospital Virgen del Puerto, Plasencia; 30 Hematology, University Hospital of Salamanca & Cancer Research Center (CiC-IBMCC, CSIC/USAL); 31 Center for Biomedical Research in Network of Cancer (CIBERONC); 32 Institute of Biomedical Research of Salamanca (IBSAL); 33 Universidad de Salamanca, Salamanca; 34 Hematology, Hospital Joan XXIII-ICO Tarragona, Tarragona; 35 Pathology, Hospital Germans Trias i Pujol, Badalona; 36 Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain

Background: Castleman disease (CD) is a heterogeneous rare disorder, characterized by lymph node enlargement with a common histopathological spectrum. This disease comprises the subtypes unicentric (UCD), HHV-8 multicentric CD (HHV-8 MCD), POEMS-asociated MCD (POEMS MCD) and idiopathic multicentric CD (iMCD). 2 Whereas UCD is a localized reversible disease, the other three are systemic diseases with multiple lymphadenopathies. The TAFRO (thrombocytopenia, anasarca, fever, reticulin myelofybrosis, organomegaly) syndrome is an aggressive form of iMCD. Each CD subtype has different clinical features, prognosis and treatment.

Aims: This study aims to study a large series of patients diagnosed with CD to describe the clinical and biological characteristics, as well as the outcome, of the different CD subtypes.

Methods: Multicentric retrospective study which includes patients diagnosed with different subtypes of CD in 19 Spanish hospitals from 2006 to 2020. Clinical and biological data were retrieved from the clinical records. The project has been approved by the Ethical Committee of Germans Trias I Pujol Hospital (PI-20-103). Statistical analyses were performed using SPSS v24.0 (IBM, Somer, NY).

Results: One hundred and thirty-four patients with available data were included; 47 with UCD and 87 with any of the 3 subtypes of MCD. The median follow-up was 3.4 years. The main clinical and biological characteristics of the 4 CD subtypes are shown in Table 1 and differences in OS in figure 1. Most patients with HHV-8 MCD were HIV-infected (73%); median CD4 lymphocyte count 0.229x10 9 /L (range: 0.016, 1.2) and 46% of them had concomitant or prior Kaposi’s sarcoma. Three iMCD patients had TAFRO. All patients with UCD were treated with surgery. Most patients with HHV-8 MCD were treated at front-line with rituximab (69%) or polychemotherapy plus rituximab (21%). Patients with iMCD were treated with anti-IL-6 (25%), polychemotherapy (25%), rituximab (25%) and steroids (25%). Treatment of POEMS MCD was also diverse; 3 patients received lenalidomide plus dexamethasone, 2 polychemotherapy, 1 anti-IL-6 and 1 auto SCT (3 patients unrecorded). Eleven patients (8.2%) had concomitant or evolved to lymphoma (2 UCD, 2 iMCD and 7 HHV-8 MCD). Thirty-two patients are dead (20 HHV8-MCD, 7 iMCD, 2 UCD, 3 POEMS), 6 of them from CD (4 HHV8-MCD, 2 iMCD).

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Summary/Conclusion: Castleman Disease subtypes have different characteristics and outcomes. First-line treatment is heterogeneous in MCD subtypes pointing a need for national guidelines.

P1129: ACALABRUTINIB IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) MARGINAL ZONE LYMPHOMA (MZL): RESULTS OF A PHASE 2, MULTICENTER, OPEN-LABEL TRIAL

P. Strati 1,* , M. Coleman 2 , D. Stevens 3 , S. Ma 4 , C. Patti 5 , M. Levy 6 , I. S. Lossos 7 , P. Ramakrishnan Geethakumari 8 , S. Lam 9 , R. Calvo 10 , K. Higgins 11 , L. Budde 12

1 The University of Texas MD Anderson Cancer Center, Houston, TX; 2 Clinical Research Alliance/Weill Cornell Medicine, New York City, NY; 3 Norton Cancer Institute, Louisville, KY; 4 Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, United States of America; 5 A.O.O.R. Villa Sofia-Cervello, U. O. Ematologia I, Palermo, Italy; 6 Baylor University Medical Center, Dallas, TX; 7 Sylvester Comprehensive Cancer Center, University of Miami−Miller School of Medicine, Miami, FL; 8 Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Cente, Dallas, TX, United States of America; 9 London Health Sciences Centre, London, Ontario, Canada; 10 AstraZeneca, Gaithersburg, MD; 11 AstraZeneca, South San Francisco, CA; 12 City Of Hope National Medical Center, Duarte, CA, United States of America

Background: MZL is a rare indolent B-cell malignancy considered incurable at recurrent stage. Bruton tyrosine kinase (BTK) inhibitors have produced durable responses in patients (pts) with R/R MZL. Acalabrutinib (acala) is a potent next-generation BTK inhibitor with high selectivity for BTK.

Aims: We report data for acala monotherapy from the R/R MZL cohort (phase 2) of a phase 1b/2 clinical trial ( NCT02180711 ).

Methods: Pts with histologically confirmed MZL, ECOG performance status ≤2, and ≥1 prior therapy (including ≥1 CD20-directed regimen) received oral acala 100 mg twice daily until disease progression or unacceptable toxicity ± R. The primary objective was overall response rate (ORR; Lugano criteria as assessed by the investigator). Secondary objectives were duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Data were analyzed descriptively (no formal hypothesis testing).

Results: Forty-two pts received acala (median age 69 y [range 42–84]; median 2 prior systemic regimens [range 1–4]). MZL subtypes were extranodal (43%), nodal (31%), and splenic (26%). At data cutoff (Oct 15, 2021), median follow-up duration was 10.7 mo (range 0.4–42.8). Sixteen (38%) pts discontinued acala, most commonly due to disease progression (26%). Among pts evaluable for response (n=37; 3 pts had not reached the first assessment timepoint and 2 pts exited the study without response assessment), ORR was 54% (95% CI 37%–71%) with 6 complete (16%) and 14 partial (38%) responses; 17 (46%) pts had stable disease. ORRs in extranodal, nodal, and splenic subtypes were 65%, 44%, and 45%, respectively. Median time to initial response was 3.0 mo; median DOR was 19.3 mo (95% CI 8.4–not estimable). Median PFS was 27.4 mo with a 12-mo PFS rate of 66%. Four pts died (disease progression, n=2; transformation to diffuse large B-cell lymphoma after stopping treatment, n=1; adverse event [AE], n=1 [septic shock unrelated to treatment]); median OS was not reached. Treatment was well tolerated with most AEs being grade 1 or 2. Sixteen pts (38%) had grade ≥3 AEs, most commonly (in ≥2 pts) anemia, dyspnea, neutrophil count decrease (n=3 each), fatigue, thrombocytopenia, and neutropenia (n=2 each). AEs led to treatment discontinuation in 2 pts (grade 3 hypotension and grade 1 myalgia). Among AEs of clinical interest, hypertension was reported in 2 pts (both grade 2); no cases of atrial fibrillation/flutter or major hemorrhage were reported.

Summary/Conclusion: These early results indicate that acala is efficacious and well tolerated in pts with R/R MZL. The AE profile is consistent with the known safety profile of acala.

P1130: ACALABRUTINIB IN TREATMENT-NAIVE OR RELAPSED/REFRACTORY WALDENSTRÖM MACROGLOBULINEMIA: 5-YEAR FOLLOW-UP OF A PHASE 2, SINGLE-ARM STUDY

R. Owen 1,* , H. McCarthy 2 , S. Rule 3,4 , S. D’Sa 5 , S. Thomas 6 , O. Tournilhac 7 , F. Forconi 7 , M. José Kersten 8 , P. Luigi Zinzani 9 , S. Iyengar 10 , J. Kothari 11 , M. Minnema 12 , E. Kastritis 13 , B. Cheson 14 , H. Walter 15 , D. Greewald 16 , R. Calvo 17 , C.-C. Wun 18 , R. Furman 19

1 St. James’s University Hospital, Leeds; 2 Royal Bournemouth Hospital, Bournemouth; 3 Plymouth University Medical School, Plymouth, United Kingdom; 4 AstraZeneca, Mississauga, Ontario, Canada; 5 University College London Hospitals NHS Trust, London, United Kingdom; 6 MD Anderson Cancer Center, Houston, TX, United States of America; 7 University of Southampton Hospital Trust, Southampton, United Kingdom; 8 Amsterdam University Medical Centers, University of Amsterdam, Cancer Center Amsterdam, Amsterdam, Netherlands; 9 Institute of Hematology University of Bologna, Bologna, Italy; 10 Royal Marsden Hospital, London; 11 Churchill Hospital, Oxford, United Kingdom; 12 University Medical Center Utrecht, University Utrecht, Utrecht, The Netherlands; on behalf of the Lunenburg Lymphoma Phase I/II Consortium – HOVON/LLPC, Utrecht, Netherlands; 13 National and Kapodistrian University of Athens, Athens, Greece; 14 Georgetown University Hospital, Lombardi Comprehensive Cancer Center, Washington, DC, United States of America; 15 Ernest and Helen Scott Haematological Research Institute and Leicester Cancer Research Centre, University of Leicester, Leicester, United Kingdom; 16 Cancer Center of Santa Barbara, Santa Barbara; 17 AstraZeneca, Gaithersburg, MD; 18 AstraZeneca, South San Francisco; 19 Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY, United States of America

Background: Bruton tyrosine kinase (BTK) inhibitors are effective in the treatment of Waldenström macroglobulinemia (WM). Acalabrutinib is a highly selective, potent, covalent BTK inhibitor that demonstrated less toxicity and improved tolerability in a head-to-head trial versus ibrutinib in chronic lymphocytic leukemia.

Aims: The aim of this trial was to determine the efficacy and safety of acalabrutinib in patients with treatment-naive (TN) or relapsed/refractory (R/R) WM. Interim results at 25 months (mo) of median follow-up have been previously reported. At a 5-year median follow-up, we report the final results of this trial.

Methods: In this single-arm, multicenter, phase 2 trial, after informed consent was obtained, patients with TN or R/R WM received 100 mg oral acalabrutinib twice daily (or 200 mg acalabrutinib once daily, later switched to 100 mg twice daily). The primary endpoint was investigator-assessed overall response rate (ORR). Key secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Efficacy parameters were based on the modified 3 rd International Workshop on Waldenström Macroglobulinemia (IWWM) criteria (Kimby 2006). Mutation data were available for a minority of patients and therefore are not reported.

Results: Of 106 patients treated with acalabrutinib, 14 were TN and 92 were R/R; median age was 69 years (range 39–90); 94% had ECOG PS ≤1; median serum IgM was 3615 mg/dL (range 291–9740). Patients with R/R disease had a median of 2 prior therapies (range 1–7). ORR was 93% (95% CI: 66, 100) in the TN cohort and 95% (88, 98) in the R/R cohort; major response rates (≥partial response) were 79% (49, 95) and 82% (72, 89), respectively. Median DOR was not estimable (NE) (12, NE) in the TN cohort and 65 mo (55, NE) in the R/R cohort; the estimated 66-mo DOR was 90% (TN; 47, 99) and 45% (R/R; 27, 61). At a 63.7-mo median follow-up, median PFS rate was NE (TN; 19, NE) and 68 mo (R/R; 53, NE); estimated 66-mo PFS rate was 84% (TN; 48, 96) and 52% (R/R; 39, 63). Median OS rate was NE for the TN and R/R cohorts; estimated 66-mo OS rate was 91% (TN; 51, 99) and 71% (R/R; 60, 80). Of the patients who remained on treatment, 7 (50%) were in the TN cohort and 43 (47%) were in the R/R cohort. The main reasons for discontinuing acalabrutinib were adverse events (AEs) (4 [29%] patients in the TN cohort, 15 [16%] patients in the R/R cohort) and progressive disease (PD) (1 [7%] patient in the TN cohort, 20 [22%] patients in the R/R cohort). The most common AEs and selected events of clinical interest are listed in the Table. There were 12 grade 5 AEs (11 and 1 patients in the R/R and TN cohorts, respectively); 1 (intracranial hematoma [R/R]) was considered treatment-related.

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Summary/Conclusion: Acalabrutinib is a highly effective treatment that achieved durable responses with a favorable safety profile in patients with TN or R/R WM.

P1131: POST HOC ANALYSIS OF PATIENTS WITH HIGHLY PROLIFERATIVE VARIANTS OF MANTLE CELL LYMPHOMA TREATED WITH ACALABRUTINIB

S. Le Gouill 1,* , M. Długosz-Danecka 2 , S. Rule 3,4 , P. Luigi Zinzani 5 , A. Goy 6 , S. D. Smith 7 , J. K. Doorduijn 8 , C. Panizo 9 , B. Shah 10 , A. Davies 11 , R. Eek 12 , E. Jacobsen 13 , A. P. Kater 14 , T. Robak 15 , P. Jain 16 , R. Calvo 17 , H. Yang 18 , M. Wang 16

1 Institut Curie, Paris, France; 2 Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 3 Plymouth University Medical School, Plymouth, United Kingdom; 4 AstraZeneca, Mississauga, Ontario, Canada; 5 IRCCS Azienda Ospedaliero-Universitaria di Bologna Istituto di Ematologia “Seràgnoli,” Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale Università di Bologna, Bologna, Italy; 6 John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ; 7 University of Washington, Fred Hutchinson Cancer Research Center, Seattle, WA, United States of America; 8 Erasmus MC Cancer Institute, HOVON Lunenburg Lymphoma Phase I/II Consortium, Rotterdam, Netherlands; 9 Clínica Universidad de Navarra, Pamplona, Spain; 10 Moffitt Cancer Center, Tampa, FL, United States of America; 11 Cancer Research UK Experimental Cancer Medicines Centre, University of Southampton Faculty of Medicine, Southampton, United Kingdom; 12 Border Medical Oncology, Albury, NSW, Australia; 13 Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, United States of America; 14 Amsterdam University Medical Center, Amsterdam, on behalf of HOVON, Amsterdam, Netherlands; 15 Copernicus Memorial Hospital, Medical University of Lodz, Lodz, Poland; 16 MD Anderson Cancer Center, University of Texas, Houston, TX; 17 AstraZeneca, Gaithersburg, MD; 18 AstraZeneca, South San Francisco, CA, United States of America

Background: Blastoid and pleomorphic variants of mantle cell lymphoma (MCL) are considered high risk and have poor prognoses. Patients with these variants rarely achieve durable remission or prolonged clinical outcomes with currently available chemotherapies and targeted therapies. Final results from ACE-LY-004, an open-label, multicenter, single-arm phase 2 study of the Bruton tyrosine kinase (BTK) inhibitor acalabrutinib in adult patients with relapsed/refractory (R/R) MCL confirmed acalabrutinib was highly active in patients with R/R MCL, with a median overall survival of 59.2 months, a median progression-free survival (PFS) of 22.0 months, and a median duration of response (DOR) of 28.6 months after a median 38.1 months of follow-up. In a subgroup analysis, we found that patients with 1 prior line of therapy had unexpectedly shorter DOR than those who had ≥2 prior lines of therapy.

Aims: To investigate a possible reason for shorter DOR in patients with R/R MCL treated with acalabrutinib who received only 1 prior line of therapy, we conducted a post hoc analysis on a subgroup of patients with blastoid/pleomorphic disease.

Methods: ACE-LY-004 enrolled adult patients with confirmed R/R MCL and an Eastern Cooperative Oncology Group performance status (ECOG PS) of ≤2. Informed consent was obtained. Acalabrutinib 100 mg was administered orally twice daily until occurrence of progressive disease or unacceptable toxicity.

Results: A total of 124 patients were enrolled; median age was 68 years and 80% of patients were male. As of December 4, 2020, median follow-up was 38.1 months and median treatment exposure was 17.5 months (range 0.1–65.3). Fifty-nine of the 124 patients (48%) had 1 prior therapy. The remaining 65 patients (52%) had 2 prior therapies (n=37) or ≥3 prior therapies (n=28). Median DOR was longer in patients with ≥2 prior lines of therapy versus those with 1 prior therapy (33.8 and 25.6 months, respectively). Similarly, median PFS was longer in patients who received acalabrutinib in later lines of therapy than in those who received acalabrutinib as second-line therapy (24.8 and 19.2 months, respectively). Among the 59 patients with 1 prior therapy, 16 (27%) had blastoid or pleomorphic disease and 43 (73%) did not, compared with 10 patients (15%) with blastoid/pleomorphic disease and 55 (85%) who did not among the 65 patients with ≥2 prior therapies. Overall response rate (ORR) in those with 1 prior therapy was 88% (n=14; 95% CI 61.7–98.4) in those with blastoid/pleomorphic disease (complete response [CR] in 50%, n=8) versus 81% (n=35; 95% CI 66.6–91.6) in those without blastoid/pleomorphic disease (CR in 47%, n=20). Median DOR for these patients was 20.3 (95% CI 3.5–46.9) and 31.8 (95% CI 14.9–not estimable) months, respectively.

Summary/Conclusion: In this post hoc analysis of the ACE-LY-004 study of acalabrutinib in adult patients with confirmed R/R MCL, a shorter DOR was observed among patients who received acalabrutinib after 1 prior therapy compared with those who received acalabrutinib after ≥2 prior therapies, likely explained by a high proportion of patients with blastoid/pleomorphic variants in the group of patients receiving acalabrutinib after 1 prior therapy. While patients with blastoid/pleomorphic variant had comparable ORR, DOR was shorter but still substantial at 20.3 months, indicating that some patients with highly proliferative disease and poorer prognosis also achieve clinical benefit from acalabrutinib.

P1132: PET INTERIM RESULTS PREDICT PROGRESSION-FREE-SURVIVAL IN FOLLICULAR LYMPHOMA PATIENTS WITH ADVANCED STAGE

M. Poza 1,* , I. Zamanillo 1 , R. Íñiguez 1 , P. Sarandeses 2 , A. Rodriguez 1 , T. Baumann 1 , S. Barrio 3 , C. Grande 1 , A. Martin 3 , L. Rufian 1 , J. Martinez-Lopez 1 , A. Jiménez-Ubieto 1

1 Hematology; 2 Department of Nuclear Medicine, Hospital 12 Octubre; 3 Altum Sequencing Co,., Madrid, Spain

Background: Follicular lymphoma (FL) is the second most common NHL. It is generally associated with an indolent course, but a subgroup of patients has worse outcome, lower progression-free-survival (PFS) and higher risk of histological transformation (HT). Since Lugano classification, the Deauville criteria using the 5-point scale (D5PS) is validated as the interpretation method for response assessment PET scans in HL and NHL. Nevertheless, the prognostic value of D5PS in the interim evaluation in FL (PET INT ) is not well stablished. Additionally, the quantitative value of SUV max parameter in the response assessment is almost not studied.

Aims: The aim of this study is to evaluate the prognostic value of PET INT evaluation and quantitative mid-induction SUVmax parameter in a cohort of FL patients followed and treated in a single, academic centre with chemo-immunotherapy.

Methods: 179 consecutive FL patients were newly diagnosed between 2011 and 2020. 40 patients that did not require systemic treatment and 68 that did not have a PET INT evaluation were excluded. As well, patients with HT (9) and FL grade 3B (4) were excluded. From the 58 remaining patients, clinical, laboratory and PET examinations data was recorded. All patients were Ann-Arbor stage III-IV and were treated with chemo-immunotherapy. PET/CT examinations were acquired at baseline, mid-induction (after 4 cycles of treatment) and end of treatment (EOT). PET positive was considered when Deauville score (DS) was 4 or 5. Statistical analysis was performed with SPSS® using Cox regression, Kaplan-Meier survival curves and log-rank test.

Results: The median age at diagnosis was 64 years (26-95) and 29 (50%) patients were female. 33/58 (57%) patients had a high risk FLIPI and 18/55 (33%) had a high risk PRIMA-PI. 37 (64%) patients were treated with R-CHOP and 21 with R-bendamustine. 44 (76%) patients received maintenance with rituximab.

After a median follow-up of 43 months, 20 (34%) patients had progression. On PET INT , 19 (32%) patients were PET(+), and 10 of them remained PET EOT (+). Only one patient with PET INT (-), became PET EOT (+). PET INT (+) patients had a median PFS of 31 months compared with a median PFS of 88 months in PET INT (-) (p < 0.001), Figure 1. 38.2% PET INT (+) presented progression of disease in less than 24 months (POD24) vs 5.2% on those who were in PET INT (-), with a p<0.001. PET INT (-) presented a negative predictive value of 95%. No differences in OS was observed.

3/19 (16%) patients with PET INT (+) presented HT vs 1/39 (2%) with PET INT (-). Transformation-free survival was lower in patients with PET INT (+), with a trend towards significance (p= 0.057).

Similar PFS results were obtained independently of prognostic index, treatment or maintenance received.

Analysing the prognostic value of quantitative SUVmax, patients with SUV max < 3,8 (28/58; 48%) presented better outcomes, with larger PFS (p < 0.001) and less HT (p= 0.004). Figure 2.

Additionally, PET/CT findings at EOT were predictive of relapse, with a median PFS of 30 months in PET EOF (+) vs 88 months in PET EOT (-) (p = 0.005). PET EOT (+) presented a higher risk of HT (p= 0.004). Figure 3.

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Summary/Conclusion: This study suggests that response assessment by PET at mid-induction is predictive of relapse in advanced stage FL patients treated with 1 st line chemo-immunotherapy. With previous conflicting results, further studies are needed to confirm this preliminary data. Probably, new laboratory tests such as MRD monitored by liquid biopsy could improve the detection of this subgroup of high risk patients and help to develop a response-adapted precision therapy.

P1133: SUB-CUTANEOUS RITUXIMAB INDUCTION FOLLOWED BY SHORT RITUXIMAB MAINTENANCE IMPROVES PFS IN PATIENTS WITH LOW-TUMOR BURDEN FOLLICULAR LYMPHOMA. FINAL RESULTS OF FLIRT PHASE III TRIAL, A LYSA STUDY.

G. Cartron, MD, PhD, E. Bachy, MD, PhD, H. Tilly, MD 3 , N. Daguindau, MD 4 , G.-M. Pica, MD 5 , F. Bijou, MD 6 , C. Mounier, MD 7 , A. M. Clavert, MD 8 , G. L. Damaj, MD, PhD, B. Slama, MD 10 , O. Casasnovas, MD 11 , R. Houot, MD, PhD, K. Bouabdallah, MD 13 , D. Sibon, MD, PhD, O. Fitoussi, MD 15 , N. Morineau, MD 16 , C. Herbaux, MD, PhD, T. Gastinne, MD 18 , L.-M. Fornecker, MD 19 , C. Haioun, MD, PhD, V. Launay, MD 21 , C. Araujo, MD 22 , O. Benbrahim, MD 23 , L. Sanhes, MD 24 , R. Gressin, MD 25 , H. Gonzalez, MD 26 , F. Morschhauser, MD, PhD, L. Xerri, MD, PhD, K. Tarte, PhD 12 , D. Pranger, MD 29

1 Haematology, CHU de Montpellier, MONTPELLIER Cedex 5; 2 CHU Lyon Sud, Pierre Benite; 3 Centre Henri Becquerel, Rouen; 4 Centre Hospitalier Annecy-Genevois - Site d’Annecy, Pringy Cedex; 5 CH de Chambéry, Chambéry; 6 Institut Bergonié, Bordeaux; 7 Institut de Cancérologie Lucien Neuwirth, St-Priest-en-Jarez; 8 CHU Angers, Angers; 9 CHU Côte de Nacre, Caen; 10 CH d’Avignon - Hôpital Henri Duffaut, Avignon; 11 CHU Dijon - Hôpital d’Enfants, Dijon; 12 CHU Rennes, Rennes; 13 CHU Bordeaux, Bordeaux; 14 Hôpital Necker, Paris; 15 Polyclinique Bordeaux Nord Aquitaine, Bordeaux; 16 CH Départemental Vendée, La Roche sur Yon; 17 CHU Montpellier, Montpellier; 18 CHU de Nantes - Hôtel Dieu, Nantes; 19 Hôpitaux Universitaires de Strasbourg - Hôpital de Hautepierre, Strasbourg; 20 Hôpital Henri Mondor, Créteil; 21 CH de Saint-Brieuc - Hôpital Yves Le Foll, Saint- Brieuc; 22 Centre Hospitalier de la Côte Basque - Hôpital de Bayonne, Bayonne; 23 CHR de la Source, Orléans; 24 Hôpital Saint Jean, Perpignan; 25 CHU de Grenoble - Hôpital Albert Michallon, Grenoble; 26 CH René Dubos, Pontoise; 27 CHRU de Lille - Hôpital Claude Hurriez, Lille; 28 Departement de bio-pathologie, Institut Paoli Calmette, Marseille, France; 29 GHdC, Charleroi, Belgium

Background: In low–tumor burden follicular lymphoma (FL), maintenance rituximab (MR) has been shown to improve progression-free survival (PFS) when compared with observation. RESORT study (Kahl et al. 2014) clearly showed that Rituximab (R) retreatment strategy provided similar time to treatment failure that maintenance strategy with less rituximab use.

Aims: It is not known whether short MR using sub-cutaneous (sc) route could improve PFS while reducing R infusions.

Methods: Patients with the diagnosis of low-tumor burden FL (GELF criteria) within the last 4 months before signing informed consent were randomly assigned to either Iv-Arm: 4-weekly iv infusions of R 375 mg/m 2 or Sc-Arm: one iv infusion of R (D1, 375 mg/m 2 ) followed by 3 sc infusions of R (1400 mg, on days 8, 15 and 22) followed by Rsc maintenance on months (M) 3, 5, 7 and 9. The primary endpoint was PFS and secondary endpoints included safety, response rates (M3, M12), duration of response (DOR), time to next anti-lymphoma treatment (TTNTL) and overall survival (OS).

Results: A total of 202 patients were included, 102 in Iv-Arm and 100 in Sc-Arm and constitute the intent to treat population. The median uses of R were 4 infusions (range: 1-4, Iv-Arm) and 8 infusions (range: 2-8, Sc-Arm). With a median follow-up of 50.2 months (95% CI: 48.3-54.5), 4‐year PFS was 41.2% (95% CI: 30.6%; 51.6%) in Iv-Arm and 58.1% (95% CI: 47.5%; 67.4%) in Sc-Arm. Median PFS was then 36.1 months (95% IC: 23.9-52.6) in Iv-Arm and 73.8 months (95% CI: 39.4-NA) in Sc-Arm (Fig 1.) (HR: 0.58; 95% CI: 0.39-0.87; P = 0.0076). Patients with at least one AE grade ≥ 3 were 8 (7.8%) and 12 (12.4%) in Iv-Arm and Sc-Arm, respectively. According to Cheson criteria, ORR at M3 were: 83% and 80% including 38% and 29% of CR/CRu, in Iv-Arm and Sc-Arm, respectively. According to Lugano criteria, 36.3% (Iv-Arm; 95% CI: 27.0%. 46.4%) and 59.0% (Sc-Arm; 95% CI: 48.7%; 68.7%) were in CMR at M12. The median DOR was 32.7 months (95% IC: 20.6-49.7) and 70.8 months (36.4-NR) (HR: 0.56; 95% IC: 0.37-0.84) in Iv-Arm and Sc-Arm, respectively. 4-year TTNLT was 54% (95% CI: 42.9%; 63.8%) in Iv-Arm and 61.8% (95% CI: 50.8.6%; 71.0%) in Sc-Arm (HR: 0.81, 95% IC: 0.53-1.24). 4-year TTNLT chemotherapy was 60.8 % (95% CI: 49.6%; 70.3%) in Iv-Arm and 71.4% (95% CI: 60.7%; 79.8%) in Sc-arm (HR: 0.69, 95% IC: 0.42-1.12) (Fig 2.). OS was not different according to treatment arm, 4-year OS was 95.0% (95% CI: 88.5%; 97.9%) in Iv-Arm and 96.7% (95% CI: 89.9%; 98.9%) in Sc-Arm.

Summary/Conclusion: This phase III study met its primary endpoint and demonstrated that Rsc induction followed by a short MRsc improves PFS of patients with low-tumor burden. MRsc did not however improved TTNLT. R in low-tumor burden FL allowed to avoid cytotoxic use in most patients 6 years after treatment initiation.

P1134: OUTCOMES FOR PATIENTS WITH MANTLE CELL LYMPHOMA POST-CBTK INHIBITOR THERAPY IN THE UNITED STATES AND JAPAN: A STUDY OF TWO REAL-WORLD DATABASES

S. Rai 1,* , L. Hess 2 , Y. Chen 2 , P. Abada 2 , H. Konig 2 , R. Walgren 2 , Y. Tanizawa 3 , Z. Cai 3 , M. Tajimi 3

1 Kindai University Faculty of Medicine, Osakasayama, Japan; 2 Eli Lilly and Company, Indianapolis, United States of America; 3 Eli Lilly Japan K.K, Kobe, Japan

Background: Patients with mantle cell lymphoma (MCL) have limited treatment options following covalent Bruton’s tyrosine kinase inhibitor (cBTKi) therapy, with no standard regimens defined.

Aims: The aim of this study was to investigate real-world treatment patterns and the outcomes for patients with MCL following cBTKi treatment utilizing two separate datasets—one from the US and one from Japan.

Methods: De-identified patient-level electronic medical record data from two real-world databases (ConcertAI in the US and Medical Data Vision in Japan) were utilized for this study. Eligible patients were ≥18 years old and were diagnosed with MCL between January 2011 and October 2020 in the US or between December 2010 and July 2020 in Japan. Data were available through 2020 to allow a minimum of 1 year of follow-up for all patients. Patients were required to have completed treatment with at least one cBTKi during the first through third lines of therapy. Time-to-event analyses utilized the Kaplan-Meier method.

Results: 946 US patients (median age 72 years, male 75.1%) and 196 Japanese patients (median age 78 years, male 73.5%) met the eligibility criteria. 352 (37.2%) patients in the US and 103 (52.6%) in Japan received subsequent post-cBTKi therapy. The remaining 387 (40.9%) patients in the US and 93 (47.5%) patients in Japan did not receive further treatment. In the US, immediate post-cBTKi regimens included rituximab (with or without other chemotherapy agents, n=152, 43.2%) or additional cBTKi-based 146 (41.5%) or B-cell lymphoma 2 inhibitor–based (n=30, 8.5%) therapy. In Japan, immediate post-cBTKi regimens were nearly exclusively chemotherapy based (n=92, 89.3%), and only 11 (10.7%) patients were retreated with cBTKi-containing regimens. Median time from the end of cBTKi therapy to discontinuation of the immediate post-cBTKi therapy or death was 3.9 months (95% confidence interval [CI]: 3.3-4.6) in the US and 2.4 months (n=196, 95% CI: 1.7-3.0) in Japan. Among those who received post-cBTKi therapy, the duration of therapy was 2.6 months (95% CI: 2.1-3.3) in the US and 1.8 months (n=103, 95% CI: 1.1-2.3) in Japan. Median overall survival (OS) from discontinuation of cBTKi therapy was 10.3 months (95% CI: 8.0-13.0 in US patients and 7.1 months (n=196, 95% CI: 4.6-12.7) in Japanese patients.

Summary/Conclusion: Patients with MCL previously treated with cBTKi experience very poor outcomes, as measured by their duration of subsequent therapy and OS. Data were generally consistent between the US and Japan. The development of new safe and effective therapies after cBTKi is needed to address the growing unmet medical need in this treatment setting.

P1135: SUBCUTANEOUS EPCORITAMAB IN COMBINATION WITH RITXUIMAB + LENALIDOMIDE IN RELAPSED OR REFRACTORY FOLLICULAR LYMPHOMA: PHASE 1/2 TRIAL UPDATE

D. Belada 1,* , S. Leppä 2 , B. E. Wahlin 3 , M. Nijland 4 , J. H. Christensen 5 , S. de Vos 6 , H. Holte 7 , K. M. Linton 8 , A. Abbas 9 , L. Wang 9 , M. Dinh 10 , B. Elliott 9 , L. Falchi 11

1 4th Department of Internal Medicine – Hematology, University Hospital and Faculty of Medicine, Hradec Králové, Czechia; 2 Helsinki University Hospital Comprehensive Cancer Center and University of Helsinki, Helsinki, Finland; 3 Karolinska Institutet, Stockholm, Sweden; 4 University Medical Center Groningen and University of Groningen, Groningen, Netherlands; 5 Odense University Hospital, Odense, Denmark; 6 Ronald Reagan University of California Los Angeles Medical Center, Los Angeles, United States of America; 7 Oslo University Hospital and KG Jebsen Center for B-cell Malignancies, Oslo, Norway; 8 The Christie NHS Foundation Trust and Manchester Cancer Research Centre, Manchester, United Kingdom; 9 Genmab, Princeton; 10 AbbVie, North Chicago; 11 Lymphoma Service, Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Patients (pts) with relapsed or refractory (R/R) follicular lymphoma (FL) experience less frequent and shorter responses with each line of treatment. Although the rituximab + lenalidomide (R 2 ) regimen is effective and has an acceptable safety profile in R/R FL, FL remains incurable; therefore, better treatment options are needed to help pts achieve durable responses. Epcoritamab is a subcutaneously administered bispecific antibody that binds to CD3 on T cells and CD20 on malignant B cells. In the dose-escalation portion of the first-in-human phase 1/2 trial (EPCORE NHL-1), epcoritamab monotherapy (0.76–48 mg) resulted in an overall response rate (ORR) of 90% and a complete response rate of 50% in pts with R/R FL. Because epcoritamab and R 2 have distinct mechanisms of action and generally non-overlapping toxicity profiles, combining these treatments may safely enhance antitumor response.

Aims: To present updated results for epcoritamab with R 2 in pts with R/R FL (EPCORE NHL-2 arm 2; NCT04663347 ).

Methods: Adult pts with R/R CD20 + FL received subcutaneous epcoritamab + R 2 for 12 cycles (28 d/cycle). In the dose-escalation part of the trial, epcoritamab was administered at 24 or 48 mg; in the expansion part, the dose was 48 mg. Pts received their assigned epcoritamab dose as follows: every wk, cycles 1–3; every 2 wk, cycles 4–9; and every 4 wk, cycles ≥10 up to 2 y. Corticosteroid prophylaxis and step-up epcoritamab dosing were required during cycle 1 to mitigate CRS. PET-CT was used to assess response. All pts provided informed consent before enrollment.

Results: As of the data cutoff date of December 1, 2021, 30 pts had received epcoritamab (24 mg, n=3; 48 mg, n=27) + R 2 . Median age was 68 y. Overall, 70% of pts had stage IV disease, 67% had FLIPI scores 3–5, 30% had primary refractory disease, and 40% had disease progression within 24 mo after starting first-line treatment (20% within 24 mo after starting immunochemotherapy). The median number of prior lines of therapy was 1 (range, 1–5). With a median follow-up of 5.1 mo (range, 0.8–12.3), 25 pts (83%) remained on study treatment; 5 pts had discontinued due to disease progression (n=2), AEs (n=2), or withdrawal of consent (n=1). Frequent treatment-emergent AEs (TEAEs; any grade [G]) included infections (57%), injection-site reactions (50%), constipation (37%), fatigue (37%), and neutropenia (37%). CRS was reported in 15 pts (50%; G1 30%, G2 13%, G3 7%). Most CRS events occurred in cycle 1, and all CRS events resolved with standard management (median time to resolution, 3 d; range, 1–8). Tocilizumab was used in 3 pts with CRS, and 1 pt discontinued study treatment due to CRS. ICANS (G2) was reported in 1 pt and resolved. There were no fatal TEAEs. Response profiles for individual pts are shown in Figure 1. Among the efficacy-evaluable pts, the ORR was 100% (27/27), with 93% (25/27) achieving a complete metabolic response (CMR) and 7% (2/27) achieving a partial metabolic response. As of the data cutoff date, the longest duration of response was 7.0+ mo and ongoing.

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Summary/Conclusion: Subcutaneous epcoritamab + R 2 demonstrated promising efficacy in pts with R/R FL, with a high CMR rate. The safety profile remained consistent with prior data. CRS events occurred mostly in cycle 1 and were generally low grade. Updated data with 30 additional pts will be presented at the meeting.

P1136: INTERIM IMAGING DOES NOT PREDICT CLINICAL EVOLUTION IN FOLLICULAR LYMPHOMA IN A REAL-LIFE CLINICAL SETTING

V. Ramos De Ascanio 1,* , L. Sánchez Paz 1 , M. S. Infante 1 , J. Churruca Sarasqueta 1 , M. Á. Foncillas García 1 , E. Landete Hernández 1 , K. D. C. Marín Mori 1 , C. Muñoz Novas 1 , J. Á. Hernández Rivas 1 , I. González Gascón y Marín 1

1 Hematology, Infanta Leonor University Hospital, Madrid, Spain

Background: Interim scan evaluation is a common practice during the treatment of some types of lymphomas with (immuno)-chemotherapy (ICT). In follicular lymphoma (FL) its role remains controversial and few studies have looked at this topic specifically. However, it is performed in clinical practice, clinical trials and recommended by some international guidelines (ESMO).

Aims: In this study, we analyzed the clinical and prognostic value of interim imaging (iPET/iCT) during the first line-treatment of patients diagnosed with FL.

Methods: In order to execute this, we carried out a retrospective analysis of all FL patients that received a first line treatment (ICT) at our institution and underwent an iPET/iCT between 2008 and 2021. Patients with Grade 3b or histologic transformation were excluded from the analysis. All data was extracted from electronical medical records. The Lugano response criteria were used to assess disease response. Statistical analysis was performed using SPSS version 25.0 and survival was estimated by Kaplan-Meier curves and log rank tests.

Results: Baseline characteristics of patients included in this study are described in Table 1. Median follow-up was 71 months (6-153). All patients underwent iPET/iCT (iCT=52 (81.3%); iPET=12 (18.8%)). Most iPET/iCT showed a partial response (PR) (n=48, 75%), and a minority a complete response (CR) (n=14, 21.9%). Two patients (3.1%) did not respond to induction. The 14 patients with CR maintained the response at the end of induction; 34 of 48 PR patients (70.8%), achieved a CR on final imaging; whereas 14 patients (29.2%) maintained a PR. Out of the 64 patients, 17 (26.6%) had disease progression during the observation period. In 6 patients (9.4%) interim imaging conditioned a treatment modification (premature discontinuation of therapy, increase/decrease in the number of ICT cycles or ICT dose attenuation). No significant differences were found in progression-free survival (PFS) (p=0.3) between the 14 patients with CR and the 48 patients who showed PR or less in the interim imaging. Nevertheless, patients in PR who converted to CR at the end of therapy had a significantly higher PFS than patients who did not achieve CR at the end of induction (p<0.01). Consequently, patients who reached a CR at the end of therapy achieved a significantly higher PFS compared to those who did not (p<0.01).

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Summary/Conclusion: Obtaining an early CR on interim imaging did not predict PFS in FL patients treated with IQT. In addition, clinical decisions based on iPET/iCT were only made in a minority of the patients, with the detection of early refractoriness being residual. Conversely, end of treatment imaging assessment was a useful tool capable of predicting PFS.

P1137: ACCURACY AND PROGNOSTIC IMPACT OF FDG PET/CT AND BIOPSY IN BONE MARROW ASSESSMENT OF FOLLICULAR LYMPHOMA AT DIAGNOSIS: A NATION-WIDE STUDY.

I. Ródenas Quiñonero 1,* , T. Chen-Liang 1 , T. Martín-Santos 2 , A. Salar 3 , M. Fernández-González 2 , C. Celades 4 , J. T. Navarro 5 , A. B. Martinez 6 , R. Andreu 7 , A. Balaguer 7 , A. Martín 8 , M. Baile 8 , J. López-Jiménez 9 , J. Marquet 9 , A. I. Teruel 10 , M. J. Terol 10 , C. Benet 11 , L. Frutos 12 , J. L. Navarro 12 , J. Uña 13 , M. Suarez 14 , M. Cortes 15 , J. Contreras 16 , C. Ruiz 17 , P. Tamayo 18 , J. Mucientes 19 , P. Sopena 20 , L. Reguilón-Gallego 1 , J. J. Sánchez-Blanco 1 , E. Pérez-Ceballos 1 , A. Jerez 1 , F. J. Ortuño 1

1 Hematology, H. Morales Meseguer, Murcia; 2 Hematology, H. Universitario de Canarias, Tenerife; 3 Hematology, H. del Mar, Barcelona; 4 Hematology, Josep Carreras Leukaemia Research Institute (IJC); 5 Hematology, ICO-H. Germans Trias i Pujol, Badalona; 6 Hematology, H. Santa Lucía, Cartagena; 7 Hematology, H. La Fe, Valencia; 8 Hematology, H. Clínico Universitario, Salamanca, Salamanca; 9 Hematology, H. Ramon y Cajal, Madrid; 10 Hematology, H. Clínico, Valencia; 11 Hematology, H. Arnau de ViIlanova, Valencia; 12 Nuclear Medicine, H. Virgen de la Arrixaca, Murcia; 13 Nuclear Medicine, H. Universitario N.S. de la Candelaria, Tenerife; 14 Nuclear Medicine, H. del Mar; 15 Nuclear Medicine, H. Universitari de Bellvitge-IDIBELL, Barcelona; 16 Nuclear Medicine, H. Santa Lucía, Cartagena; 17 Nuclear Medicine, H. La Fe, Valencia; 18 Nuclear Medicine, H. Clínico Universitario de Salamanca/IBSAL, Salamanca; 19 Nuclear Medicine, H. Puerta de Hierro, Madrid; 20 Nuclear Medicine, H. 9 de Octubre, Valencia, Spain

Background: In the workout of Follicular Lymphoma (FL), bone marrow biopsy (BMB) assessment is a key component of FLIPI and FLIPI2, the most widely used outcome scores. During the previous decade, several studies explored the role of FDG-PET/CT for detecting nodal and extranodal disease, with only one large study comparing both techniques.

Aims: The aim of this study was to evaluate the diagnostic accuracy and the prognostic impact of both procedures in a retrospective cohort of 299 FL patients.

Methods: Two hundred and ninety-nine, ≥ 18 y.o. patients diagnosed with FL between June 2005 and December 2018 were included. We used the Kaplan-Meier and the Cox method to analyze overall survival (OS) and progression free survival (PFS). To avoid collinearity in the multivariate regressions, we deconstructed the FLIPI2 composite in its foundational factors, considering bone marrow involvement (BMI) by mean of different measures in four models: BMB+, PET/CT+, “BMB+ and PET/CT+” and “BMB+ or PET/CT+”. In Cox regressions, examination of log (-log) survival plots ant partial residuals was performed to assess that the underlying assumption of proportional hazards was met.

Results: The median follow-up was 57.3 months (range 3.6-185.8 months). The PET/CT was positive in 58 patients and negative in 238. Among those positive, 37 also had a BMB+, whereas 151 patients had a BMB+ with a PET/CT-. The BMB was positive in 124 patients and negative in 172. Among those negative, 21 had a PET/CT+. Considering PET/CT, the sensitivity was 63.7% (95% confidence interval(CI);50.5-77.0), specificity was 63.4% (95%CI;57.1-69.7), negative predictive value (NPV) was 87.7% (95%CI;82.6-92.9), accuracy 63.5% (95%CI:57.8-69.1). Focusing BMB, the sensitivity was 29.8% (95%CI;21.3-38.3), specificity was 87.7% (95%CI;82.6-92.9), NPV was 63.4% (95%CI;57.1-63.7) and accuracy was 63.5% (95%CI:57.8-69.1).

Either beta2-microglobulin (B2M) over the upper normal limit (ULN), a diameter of the largest involved node (LoDLIN) over 6cm and a hemoglobin lower than 120g/L, were significantly associated with a shorter PFS in the univariate analysis. Also in univariate, a positive BMB and the combined “PET/CT or BMB positive”, were significantly associated with a shorter PFS. Neither BMI positive or the combined “PET/CT or BMB positive”, could add an independent prognostic value to the two factors than remained significant in the multivariate model: B2M higher than ULN and a LoDLIN over 6cm.

Regarding OS, an elevated B2M, a LoDLIN over 6cm, a hemoglobin lower than 120g/L and an age older than 60y.o., were significantly associated with a shorter OS in the univariate analysis. A positive result by BMB, a positive PET/CT and the combined “PET/CT or BMB positive” result, were significantly associated with a shorter OS in univariate regression. The combined “PET/CT or BMB positive” and an age older than 60y.o. remained significant for a shorter OS in the multivariate model (p=0.030, HR 2.318, 95%CI 1.083-4.961 and p= 0.020, HR 2.473, 95%CI 1.151-5.314, respectively).

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Summary/Conclusion: In our FL series, the combined “PET/CT or BMB positive” was the only BMI variable that showed an independent prognostic value in FL. Therefore, our data support that both procedures should be carried out in the upfront work of all suitable FL patients.

P1138: COPANLISIB + RITUXIMAB VS RITUXIMAB + PLACEBO IN PATIENTS WITH RELAPSED INDOLENT NON-HODGKIN LYMPHOMA (NHL): UPDATED SAFETY AND EFFICACY FROM THE PHASE III CHRONOS-3 TRIAL

P. L. Zinzani 1,2,* , M. Özcan 3 , K. Sapunarova 4 , W. Jurczak 5 , A. Hamed 6 , K. Bouabdallah 7 , G. Saydam 8 , K. Geissler 9 , Á. Szomor 10 , M. Lazaroiu 11 , A. Salar 12 , A. Tempescul 13 , M. Nalcaci 14 , L. Gercheva 15 , M. Egyed 16 , P. Panayiotidis 17 , L. Mongay Soler 18 , A. Cao 18 , C. Phelps 18 , B. H Childs 18 , M. J Matasar 19

1 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seragnoli”; 2 Dipartimento di Medicina Specialistica, Diagnostica e Sperintale, Universita di Bologna, Bologna, Italy; 3 Ankara University School of Medicine, Ankara, Turkey; 4 Medical University, Plovdiv, Bulgaria; 5 Maria Skłodowska Curie National Research Institute of Oncology, Krakow, Poland; 6 Petz Aladár Megyei Oktató Kórház, Gyor, Hungary; 7 Hematology and Cellular Therapy Department, University Hospital of Bordeaux, Bordeaux, France; 8 Ege Üniversitesi Tip Fakültesi, Izmir, Turkey; 9 Sigmund Freud University, Vienna, Austria; 10 Pécsi Tudományegyetem Klinikai Központ, Pécs, Hungary; 11 S.C. Policlinica de Diagnostic Rapid S.A., Brasov, Romania; 12 Hospital del Mar, Barcelona, Spain; 13 Hôpital Morvan - Brest, Brest, France; 14 Istanbul Universitesi İstanbul Tip Fakultesi, Istanbul, Turkey; 15 MHAT Sveta Marina EAD, Varna, Bulgaria; 16 Somogy Megyei Kaposi Mor Oktato Korhaz, Kaposvar, Hungary; 17 First Department of Propaedeutic Internal Medicine, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 18 Bayer HealthCare Pharmaceuticals, Inc., Whippany, NJ; 19 Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America

Background: The Phase III CHRONOS-3 trial of copanlisib plus rituximab (C+R) vs placebo plus rituximab (P+R) in patients (pts) with indolent NHL (iNHL) showed a significant improvement in progression-free survival (PFS; hazard ratio [HR] 0.52; Matasar et al. Lancet Oncol 2021). At the time of data cut-off (August 31, 2020), 23% of pts were ongoing on treatment in the C+R arm and 43% of pts had discontinued treatment and entered active follow-up; corresponding numbers in the P+R arm were 19% and 17%.

Aims: Here we report an updated 1-year follow-up analysis of efficacy and safety for all pts from CHRONOS-3 based on a data cut-off of August 6, 2021.

Methods: Eligible pts with iNHL who relapsed after the last R-containing regimen and were progression-free and treatment-free for ≥12 months (mo) after the last R-containing regimen, or for >6 mo if unwilling/unfit to receive chemotherapy, were randomized 2:1 to receive C+R or P+R. All pts provided informed consent. Treatment continued until progression or unacceptable toxicity and was administered i.v. on a 28-day cycle, with C 60 mg/P given on days 1, 8, and 15 and R 375 mg/m 2 given on days 1, 8, 15, and 22 of cycle 1 and on day 1 of cycles 3, 5, 7, and 9. Primary endpoint was centrally assessed PFS and secondary efficacy endpoints included overall survival (OS), objective response rate (ORR), duration of response (DoR), complete response rate (CRR), and treatment-emergent adverse events (TEAEs).

Results: 307 pts were randomized to C+R and 151 to P+R. As of the August 2021 cut-off, 46 pts (15.0%) receiving C+R and 16 (10.6%) receiving P+R were ongoing on treatment. The most common reason for discontinuation was adverse events (AEs) unrelated to clinical progression (35.8%) for C+R and radiologic progression (49.7%) for P+R. Similar to the initial disclosure, C+R reduced the risk of progression or death vs P+R by 47%, with median PFS of 22.3 mo (95% confidence interval [CI] 19.4, 30.7) for C+R and 13.8 mo (10.8, 17.5) for P+R (HR 0.53 [95% CI 0.41, 0.70]; p=0.000001). PFS benefit was preserved across all cancer subtypes (HR [95% CI]): follicular lymphoma (n=275; 0.57 [0.41, 0.80]); marginal zone lymphoma (n=95; 0.53 [0.28, 0.99]); small lymphocytic lymphoma (n=50; 0.22 [0.10, 0.49]); lymphoplasmacytic/Waldenström macroglobulinemia (n=38; 0.42 [0.15, 1.16]). Updated ORRs were 80.8% vs 49.7% and CRRs were 34.9% vs 14.6% for C+R vs P+R, respectively. Median OS was not estimable and no significant benefit was seen for C+R over P+R, although the risk of death was reduced (HR 0.86 [95% CI 0.55, 1.32]) vs the August 2020 disclosure (HR 1.07 [0.63, 1.82]). Median DoR was 25.7 mo (17.6, 31.5) for C+R vs 17.3 mo (12.6, 25.3) for P+R; for pts with complete responses, median DoR was 42.3 mo (29.2, not estimable) vs 24.7 mo (10.2, 29.7) (HR 0.46 [0.24, 0.87]). Safety profiles for C+R and P+R were mostly unchanged from the initial disclosure, and the most common treatment-related AEs for pts receiving C+R were hyperglycemia (69.4%), hypertension (49.5%), and diarrhea (33.9%). For C+R, 6 additional pts (2.0%) discontinued C due to an AE, bringing the total to 33.2%. The incidence of grade 3/4 treatment-related TEAEs was generally unchanged with the longer follow-up (2 new grade 4 for C+R; 1 new grade 4 for P+R), with no new grade 5 events.

Summary/Conclusion: 1 year after the primary disclosure of CHRONOS-3, C+R continued to demonstrate acceptable safety and superior efficacy with durable complete responses vs P+R in pts with relapsed iNHL. These data support the long-term use of C+R in pts with relapsed iNHL.

P1139: A POPULATION-BASED STUDY OF ISOLATED ADULT PULMONARY LANGERHANS CELL HISTIOCYTOSIS IN THE UNITED STATES: 2010-2017

G. Ruan 1,* , J. Abeykoon 1 , A. Hazim 2 , A. Ravindran 3 , K. Rech 3 , J. Young 4 , C. Cox 4 , J. Ryu 5 , M. Koster 6 , O. Tobin 7 , M. Shah 1 , N. Bennani 1 , R. Vassallo 5 , G. Goyal 1,8 , R. Go 1

1 Hematology; 2 Internal Medicine; 3 Laboratory Medicine and Pathology; 4 Radiology; 5 Pulmonology; 6 Rheumatology; 7 Neurology, Mayo Clinic Rochester, Rochester; 8 Hematology, University of Alabama, Birmingham, United States of America

Background: Isolated adult pulmonary Langerhans cell histiocytosis (pLCH) is a rare histiocytic neoplasm that involves the lungs as single system disease. Retrospective studies have suggested that patients with isolated adult pLCH have better overall survival compared to multisystem LCH. While population-based studies have been done for LCH with multisystem disease, there has been no epidemiologic studies done to date on isolated adult pLCH.

Aims: To characterize the incidence and outcomes of isolated adult pLCH in the United States.

Methods: We queried the Surveillance, Epidemiology, and End Results Program (SEER) 18 Registries (2010-2017) using ICD-0-3 code 9751/3 with the primary site of involvement being the lung. Patients were included if they were ≥ 18 years and had localized pulmonary involvement. Patients with extrapulmonary disease or unknown staging were excluded. The primary outcome was overall survival (OS), which was analyzed using the Kaplan-Meier method. Data on incidence rates (IR) and relative survival (RS) were calculated using the SEER*Stat software. IR (cases/1,000,000) was age-adjusted to the U.S. 2000 standard population. RS was defined as the ratio of the proportion of observed survivors in a cohort of isolated adult pLCH to the proportion of expected survivors in a comparable set of individuals that do not have isolated adult pLCH adjusting for the general survival of the US population for race, sex, age, and time when the diagnosis was established.

Results: In SEER, there were a total 147 patients with isolated adult pLCH. Between the years 2010-2017, the median IR of isolated adult pLCH was 0.3 (range 0.1-0.5) and did not change significantly. The median IR from 2010-2013 was 0.2 (range 0.1-0.2) and 2014-2017 was 0.5 (range 0.3-0.5). The median age at diagnosis was 53 years (range 23-79) and 54 (37%) were male. 115 (78%) were white, 21 (14%) were black, and 11 were labeled as other. 116 (79%) were diagnosed on histopathologic evaluation while the remainder were diagnosed without microscopic confirmation. No patients were documented to have secondary malignancies.

With a median follow up of 35 months, the median OS was not reached (95% CI: not reached-not reached) and the 3-year OS was 89%. The 3-year OS between males and females was 93% and 87% (p=0.16) respectively. The 3-year OS between white and non-white was 90% and 87% (p=0.98) respectively. In the general U.S. population, the expected survival for 1, 2, 3 years were 99.2%, 98.5%, 97.8% respectively. In contrast, the RS for patients with isolated adult pLCH at the same time points were 98.3%, 94.8%, 90.9% (Figure ). A total of 14 patients died. 3 patients died from isolated adult pLCH while 11 patients died of other causes, including chronic obstructive pulmonary disease (3), cardiac disease (3), chronic liver disease (1), and unknown (4).

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Summary/Conclusion: The incidence of isolated adult pLCH has not changed over the years from 2010-2017. Patients with isolated adult pLCH have lower relative survival compared to the general population. Among patients who died, the majority died from cause other than pLCH.

P1140: TIME TO TUMOR, SYMPTOMATIC AND LABORATORY RESPONSES FOLLOWING SILTUXIMAB TREATMENT IN IDIOPATHIC MULTICENTRIC CASTLEMAN DISEASE

F. van Rhee 1,* , A. Rosenthal 2 , K. Kanhai 3 , R. Martin 3 , K. Nishimura 2 , A. Hoering 2 , D. Fajgenbaum 4

1 University of Arkansas for Medical Sciences, Little Rock; 2 Cancer Research And Biostatistics, Seattle, United States of America; 3 EUSA Pharma, Hemel Hempstead, United Kingdom; 4 University of Pennsylvania, Philadelphia, United States of America

Background: Idiopathic multicentric Castleman disease (iMCD) is a rare, heterogeneous disorder involving multicentric lymphadenopathy, systemic inflammation, and cytokine-driven organ dysfunction. Siltuximab—a monoclonal antibody against interleukin-6—is the only FDA- and EMA-approved treatment for iMCD. A phase 2, randomized, double-blind, placebo-controlled trial of siltuximab for the treatment of iMCD ( NCT01024036 ) showed that 18/53 patients (34%) responded to treatment, compared with 0% in the placebo group, but it is not known how long patients should be maintained on siltuximab before deciding whether the treatment is beneficial.

Aims: To determine the time to and sequence of normalization of laboratory, clinical, and lymph node responses in patients who responded to siltuximab.

Methods: The phase 2 trial enrolled adults with symptomatic iMCD who were human immunodeficiency virus seronegative and human herpesvirus 8 negative. In this post hoc analysis, we aimed to determine the sequence of and time to normalization of laboratory, clinical, and lymph node responses in patients who responded to siltuximab. The Kaplan–Meier and cumulative incidence with competing risks methods with corresponding log-rank tests were produced to estimate and compare the progression-free survival (PFS), overall survival, time to treatment failure, time to lymph node response, time to durable symptomatic response, and time to normalization of laboratory values.

Results: Seventy-nine patients were enrolled in the trial (n=53 siltuximab, n=26 placebo; all patients received best supportive care). The median duration of follow-up was 422 days (range 55‒1051). Siltuximab treatment improved PFS compared with placebo (p=0.0001). The median PFS was 14.5 months (95% CI 13.6–upper bound not reached) for patients on placebo, whereas it was not reached for patients on siltuximab. The 2-year estimates for overall survival were 93% (95% CI 85–100%) for the siltuximab arm and 77% (95% CI 55–98%) for the placebo arm (p=0.11). Time to treatment failure in the placebo arm was 4.8 months and was not reached with siltuximab (p=0.005). In the 18 siltuximab-treated patients who achieved the primary endpoint of durable tumor (radiologic) and symptomatic response, most first achieved rapid normalization of various laboratory parameters and symptomology (median of 0.8 months to normalization based on a 34-point MCD symptom score) (Table 1). Lymph node responses occurred later (median of 4.1 months to normalization).

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Summary/Conclusion: Significant improvements in biochemical parameters such as platelet count, albumin, and symptomatic responses occur within 3 months in the majority of responders, showing them as early indicators of response. Lymph nodes were slower to show an improvement in response to treatment, and so involution of these should not be used to drive early therapeutic decisions in iMCD patients following siltuximab. The present study shows the recovery sequence of different parameters over time following initiation of treatment with siltuximab.

P1141: IBRUTINIB AND RITUXIMAB AS FIRST LINE THERAPY FOR MANTLE CELL LYMPHOMA: A MULTICENTRE, REAL-WORLD UK STUDY

A. Tivey 1,* , R. Shotton 1 , T. A. Eyre 2 , D. Lewis 3 , N. Crosbie 3 , E. Nga 4 , R. Guerrero Camacho 5 , W. Swe 6 , H. Marr 7 , C. Rees 8 , S. Moule 8 , T. Sutton 9 , D. Wrench 10 , N. Thomas 11 , M. Wilson 11 , J. Bailey 12 , M. Prahladan 13 , A. Hodson 13 , M. Koppana 13 , S. Smith 14 , S. Jones 14 , F. Miall 15 , J. Norman 16 , E. Davies 16 , C. Hildyard 17 , L. Lowry 18 , S. Paneesha 19 , I. Qureshi 19 , A. Beech 20 , C. Bedford 21 , A. Everden 21 , D. Tucker 21 , J. Wright 22 , J. Goddard 22 , T. Nicholson 23 , J. Wilson 24 , A. Lord 25 , B. Jackson 25 , M. Flont 26 , A. Gibb 27 , K. Linton 27

1 Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester; 2 Oxford University Hospitals NHS Foundation Trust, Oxford; 3 University Hospitals Plymouth NHS Foundation Trust, Plymouth; 4 Airedale NHS Foundation Trust, Airedale; 5 Blackpool Teaching Hospitals NHS Foundation Trust, Blackpool; 6 Calderdale and Huddersfield NHS Foundation Trust, Huddersfield; 7 The Freeman Hospital, Newcastle; 8 Frimley Health NHS Foundation Trust, Frimley; 9 Gateshead Health NHS Foundation Trust, Gateshead; 10 Guy’s and St Thomas’ NHS Foundation Trust, London, London; 11 Harrogate and District NHS Foundation Trus, Harrogate; 12 Hull University Teaching Hospitals NHS Trust, Hull; 13 East Suffolk and North Essex NHS Foundation Trust, Ipswich; 14 Sherwood Forest Hospitals NHS Foundation Trust, Sutton in Ashfield; 15 University Hospitals of Leicester NHS Foundation Trust, Leicester; 16 Manchester University NHS Foundation Trust, Manchester; 17 Milton Keynes University Hospital, Milton Keynes; 18 Somerset NHS Foundation Trust, Taunton; 19 University Hospitals Birmingham NHS Foundation Trust, Birmingham; 20 Nottingam University Hospitals, Nottingham; 21 Royal Cornwall Hospitals NHS Trust, Truro; 22 Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield; 23 St Helens & Knowsley Teaching Hospitals NHS Trust, Whiston; 24 University Hospitals Sussex NHS Foundation Trust, Chichester; 25 Torbay and South Devon NHS Foundation Trust, Torbay; 26 York and Scarborough Teaching Hospitals NHS Foundation Trust, York; 27 Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, Manchester, United Kingdom

Background: Ibrutinib (IBR) is an oral covalent Bruton tyrosine kinase inhibitor (BTKi), licensed for treatment of relapsed or refractory mantle cell lymphoma (MCL). Under NHS interim Covid-19 agreements in England, IBR with or without rituximab (R) was approved for the frontline treatment for MCL patients (pts) as a safer alternative to conventional immunochemotherapy. Although recent phase 2 studies have reported high response rates in low-risk patients for this combination in the frontline setting, randomised phase 3 and real-world data are currently lacking.

Aims: To describe the real-world response rates (overall response rate (ORR), complete response (CR) rate) and toxicity profile of IBR +/- R in adult patients with previously untreated MCL.

Methods: Following institutional approval, adults commencing IBR +/- R for untreated MCL under interim Covid-19 arrangements were prospectively identified by contributing centres. Hospital records were interrogated for demographic, pathology, response, toxicity and survival data. ORR/CR were assessed per local investigator according to the Lugano criteria using CT and/or PET-CT.

Results: Data were available for 66 pts (72.7% male, median age 71 years, range 41-89). Baseline demographic and clinical features are summarised in Table 1. 23/66 pts (34.8%) had high-risk disease (defined as presence of TP53 mutation/deletion, blastoid or pleomorphic variant MCL, or Ki67%/MiB-1 ≥30%). IBR starting dose was 560mg in 56/62 pts (90%) and was given with R in 22/64 pts (34%). At a median follow up of 8.7 months (m) (range 0-18.6), pts had received a median of 7 cycles of IBR. 19/60 pts (32%) required a dose reduction or delay in IBR treatment. New atrial fibrillation and grade ≥3 any-cause toxicity occurred in 3/59 pts (5.8%) and 8/57 (14.0%) respectively.

For the whole population and high-risk pts only, ORR was 74.4% and 64.7% respectively (p=0.2379), with a median time to response of 3.8m, coinciding with the first response assessment scan. Seven pts (16.7%), of whom 2 had high-risk disease, attained CR at a median of 6.0m. ORR for pts receiving vs not receiving R were 84.2% and 66.7% respectively (p=0.1904). IBR was discontinued in 20/61 pts (32.8%) at a median time to discontinuation of 4.1m, due to progressive disease (PD, 19.7%), toxicity (4.9%), death (3.3%; 1 pt each of Covid-19 and E. coli infection), pt choice (3.3%) and other unspecified reasons (1.6%). 15/66 pts (22.7%) overall and 7/23 (30.4%) with high-risk disease progressed on IBR at a median time to PD of 4.0m. No pts underwent autologous stem cell transplantation consolidation during the study period. 12/57 pts (21.1%) received second line treatment (R-chemotherapy n=7, Nordic MCL protocol n=2, VR-CAP n=2, pirtobrutinib n=1). Response to second line treatment was CR in 4/11 pts, PD in 7/11. Of the 2 Nordic-treated patients, 1 had CR after cycle 2 and 1 PD. Fourteen pts (21.2%) died during the follow up period, due to MCL (n=11), Covid-19 (n=2) and congestive cardiac failure (n=1). Overall survival was lower for patients with high-risk disease (HR 0.55, p=0.038).

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Summary/Conclusion: In this real-world UK cohort of pts receiving first-line IBR +/-R for MCL, including older and high-risk pts, we report high ORR rates in a similar range to the phase II Geltamo IMCL-2015 study of combination IBR-R in an exclusively low-risk population. Documented CR rates were lower, possibly reflecting a low usage of rituximab in the Covid-19 pandemic as well as CT assessment of response. Treatment was generally well tolerated, with low rates of toxicity-related treatment discontinuation. The study is ongoing.

P1144: RADIOIMMUNOTHERAPY (RIT) VERSUS AUTOLOGOUS HEMATOPOIETIC STEM-CELL TRANSPLANTATION (ASCT) IN RELAPSED/REFRACTORY (R/R) FOLLICULAR LYMPHOMA: A FONDAZIONE ITALIANA LINFOMI (FIL) PHASE III TRIAL .

M. Ladetto 1,* , R. Tavarozzi 1 , A. Evangelista 2 , M. Zanni 3 , A. Tucci 4 , A. Anastasia 4 , B. Botto 5 , C. Boccomini 6 , S. Bolis 7 , S. Volpetti 8 , V. R. Zilioli 9 , B. Puccini 10 , A. Arcari 11 , V. Pavone 12 , G. Gaidano 1 , P. Corradini 13 , M. Tani 14 , S. Ferrero 15 , F. Cavallo 15 , G. Milone 16 , C. Ghiggi 17 , A. Pinto 18 , D. Pastore 19 , A. J. Ferreri 20 , G. Latte 21 , C. Patti 22 , F. Re 23 , L. Arcaini 24 , F. Benedetti 25 , S. V. Usai 26 , S. Luminari 27 , D. Mannina 28 , A. Pulsoni 29 , C. Stelitano 30 , E. Pennese 31 , G. Pietrantuono 32 , F. Gherlinzoni 33 , F. Pomponi 34 , A. Olivieri 35 , T. Perrone 36 , D. Rota Scalabrini 37 , C. Califano 38 , B. Falini 39 , G. Ciccone 2 , U. Vitolo 37

1 Department of Translational Medicine, University of Eastern Piedmont, Novara; 2 Unit of Clinical Epidemiology, A.O.U. Città della Salute e della Scienza, Torino; 3 SCDU of Hematology, AO SS Antonio e Biagio e Cesare Arrigo, Alessandria; 4 Hematology, ASST Spedali Civili, Brescia; 5 Hematology, A.O.U. Città della Salute e della Scienza; 6 Hematology, A. O. U. Città della Salute e della Scienza, Torino; 7 Hematology Department, ASST San Gerardo University Hospital, Monza; 8 Institute of Hematology, University and Hospital of Udine, Udine; 9 ASST Grande, Ospedale Metropolitano Niguarda, Milano; 10 Hematology Department, AOU Careggi, Firenze; 11 Hematology, Ospedale Guglielmo da Saliceto, Piacenza; 12 Hematology, Card. Panico Hospital S.Pio X, Tricase; 13 Division of Hematology and Stem Cell Transplantation, University of Milano Istituto Nazionale Tumori, Milano; 14 Hematology, Santa Maria delle Croci Hospital, Ravenna; 15 Department of Molecular Biotechnologies and Health Sciences, Division of Hematology, University of Turin, Torino; 16 Hematology and BMT Unit, Azienda Policlinico Vittorio Emanuele, Catania; 17 Clinic of Hematology, IRCCS Ospedale San Martino, Genova; 18 Hematology-Oncology and Stem-Cell Transplantation Unit, National Cancer Institute, Fondazione ‘G. Pascale, Napoli; 19 Ospedale Antonio Perrino, Brindisi; 20 Onco-Hematology Department, Fondazione Centro San Raffaele, Milano; 21 Unità di Ematologia e Trapianto di Midollo Osseo, San Francesco hospital, Nuoro; 22 Divisione di Oncoematologia, Azienda Villa Sofia-Cervello, Palermo; 23 Hematology, A.O.U. di Parma, Parma; 24 Department of Onco-Hematology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Pavia; 25 Department of Medicine, Section of Hematology and Bone Marrow Transplant Unit, University of Verona, Verona; 26 Ospedale Businco, Cagliari; 27 Hematology, IRCCS Reggio Emilia, Reggio Emilia; 28 Hematology, Azienda Ospedaliera Papardo, Messina; 29 Hematology, Department of Translational and Precision Medicine, Sapienza University of Rome, Roma; 30 Hematology, Azienda Ospedaliera Bianchi-Melacrino-Morelli, Reggio Calabria; 31 Lymphoma Unit, Department of Hematology, Ospedale Spirito Santo, Pescara; 32 IRCCS Centro Oncologico Della Basilicata, Rionero In Vulture; 33 Division of Hematology, Ca’ Foncello Hospital, Treviso; 34 ULSS8 Berica, Vicenza; 35 Hematology, AOU Ospedali Riuniti, Ancona; 36 Unit of Hematology with Transplantation, Dept. of Emergency and Organ Transplantation, University of Bari, Bari; 37 Candiolo Cancer Institute, FPO-IRCCS, Candiolo; 38 U.O.C. Ematologia, P.O. “Andrea Tortora”, Pagani; 39 Department of Medicine, Division of Hematology and Clinical Immunology, University of Perugia, Perugia, Italy

Background: Optimal consolidation for young R/R FL in the rituximab age remains uncertain and the benefit of ASCT is not clearly established.

Aims: The FIL FLAZ12 trial ( NCT01827605 ) is a prospective, multicenter, randomized, phase 3 trial, comparing RIT versus ASCT, as consolidation after chemoimmunotherapy, both followed by R maintenance in R/R FL.

Methods:

Pts aged 18-65 yrs, with R/R FL after 1 or 2 lines of chemoimmunotherapy, without significant comorbidities were enrolled. Patients received 3 courses of therapy chosen by the investigator among RCHOP, R-DHAP, R-FM, R-ICE, R-IEV or R-B. Pts achieving at least PR (according to Cheson et al. 2007) were randomized 1:1 to either RIT or ASCT before CD34+ collection. Conditioning for ASCT was BEAM or TEAM. RIT was given as previously described (Morschhauser et al., 2008). After consolidation, pts received R maintenance every 3 months for eight courses. Primary endpoint was PFS. Considering ASCT toxicity, it was hypothesized to be a superior choice, if capable of increasing 3-years PFS from 40% to 60% (two-side log-rank test with alpha of 5% and a power of 85%). Clinical secondary endpoints were ORR, CRR, OS, EFS and TTF.

Results:

Between Aug 2012, and Sep 2019, 164 pts were screened and 159 enrolled by 38 FIL Centers (enrolled population). Unfortunately, the study was prematurely closed due to low accrual. The data were analyzed on an ITT basis on May 2, 2021 with a median follow-up (mFU) from enrollment of 43 months and 75 PFS events. The two arms were clinically well balanced, with median age of 57 yrs (IQR 49- 62), 55% male, 57% stage IV, 20% bulky disease. Tumor re-biopsy was performed in 79% pts. POD-24, retrospectively assessed was observed in 32% of pts. Two pts (1%) did not start treatment (non-confirmed histology and withdrawal). Sixteen (10%) pts discontinued before randomization (7 SD, 3 PD, 3 AE, 1 withdrawal, 2 poor compliance) and 141 (89%) were randomized to either RIT (71) or ASCT (70) (randomized population). Of these 19 (13%) (RIT 8, ASCT 11) did not receive the planned consolidation due to 7 PD, 4 AE, 1 medical decision, 2 poor mobilization, 2 withdrawals, 1 poor compliance, 2 protocol breaches, while 63 (89%) received RIT and 59 (84%) ASCT. After RIT, 61% of pts achieved CR and 23% PR, while after ASCT these were 70% and 9%.

Estimated PFS at 3 yrs was 60% (95% CI: 46%-71%) in the RIT arm vs. 59% (95% CI:45%-70%) in the ASCT arm, p = 0.8613 (HR 0.96, 95%CI: 0.57,1.59). (Figure 1) 3yrs-OS was again superimposable in the two arms: 83% (95%CI: 69%-91%) in the RIT vs 85% (95% CI: 72%-91%) in the ASCT, p = 0.8310 (HR 1.10, 95%CI: 0.45,2.72). Grade ≥ 3 hematological toxicity was 46% in the RIT vs 94% in the ASCT arm (p < 0.001). For ASCT vs RIT grade ≥3 neutropenia occurred in 94% vs 41% of pts (p < 0.001). During follow-up, 4 pts died in remission: 1 AML (RIT), 2 SARS- COV2 infections (RIT) and 1 pneumonia (ASCT). Second cancers occurred in 3 pts after RIT and 7 after ASCT (p = 0.480). Multivariable analysis for PFS indicated POD-24, male sex, LDH and refractory disease as adverse parameters. Subgroup analysis for PFS including gender, age, LDH, POD-24 and extranodal disease show no subgroup favoring RIT nor ASCT.

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Summary/Conclusion: Even if prematurely interrupted, our study demonstrated no meaningful difference in efficacy between ASCT and RIT, but ASCT was more toxic and more demanding for pts and health service. Both strategies induced a similar and favorable long-term outcome suggesting that consolidation programs milder than ASCT require further investigation in R/R FL.

P1145: OUTCOMES AND TREATMENT PATTERNS AFTER FIRST RELAPSE IN PATIENTS WITH WALDENSTRÖM MACROGLOBULINEMIA

R. Tawfiq 1,* , J. Abeykoon 2 , S. Zanwar 2 , J. Paludo 2 , P. Kapoor 2

1 Department of Medicine; 2 Division of Hematology, Mayo Clinic, Rochester, United States of America

Background: Waldenström macroglobulinemia (WM) is a rare, B cell lymphoma, with a relapsing-remitting course. Despite expansion of therapeutic options, WM remains incurable. Data on outcomes of patients (pts) after first relapse or primary refractory disease are sparse. Frequently used salvage therapies include chemoimmunotherapy (CIT), proteasome inhibitor (PI) based regimens and Bruton tyrosine kinase inhibitors (BTKi).

Aims: Analyze the outcomes of pts with WM after initial relapse and/or refractory (RR) disease and the impact of the type of therapy used in the second line setting.

Methods: ecords of pts with WM seen at Mayo Clinic between 2000 and 2021 were reviewed. Pts with RRWM requiring second line therapy were included in the primary analysis. Response rates were assessed per the modified IWWM-6 criteria. All time to event analyses, barring progression free survival 2 (PFS2), were calculated from the start of the second line treatment, using the Kaplan Meier method. Pts were grouped based on sequence of therapy and response to frontline treatment for additional subgroup analysis.

Results: Records of 220 pts with WM were reviewed; 92 had RRWM and were treated with a second line therapy. For the entire cohort, frontline regimens included dexamethasone, rituximab, cyclophosphamide (DRC) (n=93, 42%), bendamustine rituximab (BR) (n=71, 32%), bortezomib, dexamethasone, rituximab (BDR) (n=33, 15%), BTKi (n=20, 9%), and other (n=3, 1%). Median follow-up of RRWM cohort from second line therapy was 5.1 years (95% CI: 4.5-6.3). Pts with RRWM received the following therapies at first relapse: BR (n=24, 26%), BTKi (n=24, 26%), PI (n=17, 19%), DRC (n=6, 7%), autologous transplant (n=4, 4%), and other (n=17, 18%). Following second-line treatment, the overall response rate and major response rate (MRR) were 72% and 67% (CR=5%, VGPR=15%, and PR=47%), respectively. The median PFS was 2.7 years (CI: 1.7-4). 2-year overall survival (OS) rate was 87%. The median PFS2 from frontline therapy was 7.03 years (CI: 5.5-9.8).

Baseline characteristics were similar for subgroups in all the following subgroup analyses. With first-line therapy, 54% of pts (n=50) achieved MRR vs. 46% (n=42) who did not. The former group had similar progression-free survival (PFS) rates as well as overall survival (OS) rates and response rates (MRR and ORR) to the second-line treatment compared to their counterparts who did not achieve MRR with the frontline regimen ( Table ).

Thiry one pts received BTKi as either first or second line and 61 pts received no BTKi in either line. As expected, the BTKi group was enriched for pts with MYD88 L265P mutation (n=29, 94%) vs 49% (n=30) in the no BTKi group, p<0.001. The outcomes between the 2 groups were comparable ( Table ). The group that received frontline BTKi followed by CIT/PI based Rx as second line (n=7) was compared to that receiving frontline CIT/PI based Rx followed by BTKi (n=21). The outcomes were similar ( Table ).

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Summary/Conclusion: Currently available second line therapies are effective in RRWM. Achieving MRR with frontline regimen does not significantly affect overall outcomes with the second-line therapy. Although follow up is short, using a BTKi as the first- or second-line therapy appears to show comparable outcomes after second line treatment as compared to not treating with a BTKi in the upfront setting or in second-line setting. Similarly, using frontline BTKi followed by CIT as second line shares similar outcomes to treating with CIT initially followed by a BTKi. These findings require confirmation in prospective studies.

P1147: SERUM ALBUMIN AND NEUTROPHIL-TO LYMPHOCYTE RATIO TWO INDEPENDENT FACTOR PREDICTING SURVIVAL IN FOLLICULAR LYMPHOMA. MULTI-INSTITUTIONAL 763 COHORT LA

M. A. Torres Viera 1,*

1 LYMPHOMAS, Hematologia Oncologia 360 Clinica Sta Sofia, Caracas, Venezuela

Background: Introduction: The neutrophil-lymphocyte ratio (NLR) is a measure of systemic inflammation that appears prognostic in different cancers. Although the exact mechanism remains to be elucidated, reduced lymphocyte intratumoral infiltration coupled with the formation of neutrophil extracellular traps (or NETosis) have been postulated as endogenous mechanisms for tissue damage and inflammation. Along this line, serum albumin has also been studied as a biomarker of inflammation and has been associated to prognosis in certain cancers. We have previously reported on the prognostic value of the NLR and serum albumin in diffuse large B-cell lymphoma (Villela, ASH meeting, 2019; Castro, ASH meeting, 2019) and peripheral T-cell lymphoma, not otherwise specified (Idrobo, ASH meeting, 2019), but nothing on follicular lymphoma (FL) yet.

Aims: Therefore, we aim to investigate the role of different biomarkers on the prognosis of patients with FL diagnosed and managed in Latin America

Methods: We analyzed patients with FL diagnosed between 2010 and 2020 from 30 centers in 10 Latin American countries. The study outcomes were overall survival (OS) and progression-free survival (PFS) in relation to different biomarkers. Kaplan-Meier and log-rank test were used for survival analysis. Univariate and multivariate Cox regression analysis were used to estimate hazard ratios (HR) with a 95% confidence interval (CI) and adjusted to the Follicular Lymphoma International Prognostic Index (FLIPI) score. Outcomes with a p-value <0.05 were considered statistically significant

Results: We identified 939 FL patients; 741 were included for the final analysis (median age 58 y, female 52%). There was no significant correlation between the NLR and other clinical factors such as: age, clinical stage, histological FL grading, and chemotherapy regimen used. A cutoff of 2.15 for NLR was defined as the maximum point for sensitivity and specificity based on ROC analysis. Table 1 and 2 summarizes the results from the univariate and multivariate analysis for 2 years OS and PFS, respectively. Both, serum albumin <3.5 g/dL and a NLR >2.15 were independently associated with worse OS (adjusted, aHR 2.48 [1.26-4.91], p=0.009; and 2.55 [1.21-5.37], p=0.014) and PFS (aHR 1.62 [1.03-2.55], p=0.038; 2.22 [1.45-3.40], p<0.001), respectively. The lymphocyte:monocyte ratio (LMR) was not found to be prognostic for OS or PFS, although with a trend for worse PFS with a LMR ≤2.5. With a median follow of 43 months, (95% CI: 40-47), the survival rates in patients with FL and albumin <3.5 were OS of 83% (vs. 95%) and PFS of 70% (vs. 83%); whereas in patients with NLR >2.15 the survival rates were OS of 91% (vs. 96%) and PFS of 75% (vs. 88%) (Figures 1 and 2; Table 3).

Summary/Conclusion: In this study, serum albumin and NLR emerge as reliable predictors for survival for FL patients in Latin America. Although these markers have been associated to an increased inflammatory state in cancer patients; other factors such as poor nutritional status, and advanced disease stage due to delayed access to specialized cancer care in our region may have contributed to the observed outcome. Further studies are needed to better understand the role of these biomarkers on lymphoma care and to validate our findings. Lastly, we are currently working on evaluating these biomarkers on existing prognostic models and to improve prognostication for FL patients in Latin America

P1148: REAL WORLD DATA (RWD) AMONG FOLLICULAR LYMPHOMA (FL) PATIENTS IN GERMANY WITH AT LEAST TWO PRIOR LINES OF SYSTEMIC THERAPY AND COMPARISON WITH CLINICAL DATA FOR MOSUNETUZUMAB

N. Marschner 1,* , J. Hanselmann 2 , S. McGough 3 , S. Braun 4 , S. Pfizler-Dempfle 5 , R. Sandner 6 , J. Mohm 7 , M. Jaber 8 , D. Hamm 9 , N. Hamm 10 , M. Jaenicke 11 , A. Shewade 3

1 Outpatient Centre for Interdisciplinary Oncology and Haematology; 2 Biostatistics, iOMEDICO, Freiburg, Germany; 3 Roche Global Product Development Data Sciences, Genentech, Inc., South San Francisco, CA, United States of America; 4 Biometrics and Epidemiology. Roche Pharma AG, Grenzach-Wyhlen; 5 Oncology and Haematology Outpatient Centre, Kaiserslautern; 6 MVZ for Haematology and Oncology, Passau; 7 Oncology and Haematology Outpatient Centre, Dresden, Germany; 8 Roche Global Access, F. Hoffmann-La Roche Ltd, Basel, Switzerland; 9 Medical Affairs Excellence, Roche Pharma AG, Grenzach-Wyhlen; 10 Medical Department, iOMEDICO; 11 Clinical Epidemiology, iOMEDICO, Freiburg, Germany

Background: FL is characterized by its indolent nature, with patients (pts) experiencing multiple relapses and successively poorer outcomes with each relapse (Link et al. 2019). Treatment with immunochemotherapy regimens has significantly improved outcomes in pts with newly diagnosed FL, but limited treatment options are available in Europe, with no standard of care for pts with multiple relapses, such as those who have received at least two prior lines of systemic therapy (3L+ FL). Mosunetuzumab (M) is a T-cell engaging CD20xCD3 bispecific antibody that has shown high rates of complete response and favorable safety in pts with 3L+ FL in a single-arm Phase II trial ( NCT02500407 ; Budde et al. ASH 2021). RWD can provide relevant context to interpret data from the single-arm trial (SAT) of M as a treatment option in 3L+ FL.

Aims: This study summarizes RWD in pts with 3L+ FL utilizing the Tumor Registry Lymphatic Neoplasms extension (TLNext) and plans to perform a matching-adjusted indirect comparison (MAIC) between the M SAT and TLNext.

Methods: TLN was a multicenter, longitudinal, observational, prospective cohort study collecting data from medical charts of pts with lymphoid B-cell neoplasms receiving care at office-based hematology practices in Germany ( NCT00889798 ; Knauf et al. 2019). Patients were enrolled between 2009–14 and followed for up to 5 years. TLNext collected additional data up to November 2021 from consented pts originally enrolled in TLN as well as additional newly-consented pts with 3L+ FL. Baseline characteristics, treatment patterns, and outcomes were summarized for pts with 3L+ FL in TLNext. A MAIC is planned between the M SAT and a TLNext cohort selected by applying key eligibility criteria from the M SAT. An index line of therapy (LoT) will be selected for all pts with more than one recorded LoT. Imbalance adjustment will be performed by re-weighting the TLNext cohort using weights determined from distributions of prognostic clinical factors observed in M SAT.

Results: TLNext included 69 pts who had received 112 documented LoTs in 3L+ FL. Median follow-up was 40 months from the start of the 3rd LoT. 65 pts had received an anti-CD20 therapy and an alkylator therapy prior to index therapy and had a known relapse or refractory status (Table). Latest recorded LoT was selected as the index therapy for every patient. Compared to M SAT, the TLNext cohort was slightly older and had more female pts. A higher proportion of pts in TLNext had received only 2 prior LoT. Overall, M SAT had a higher proportion of pts with factors known to be associated with poorer outcomes in 3L+ FL, e.g. refractory to prior anti-CD20 therapy, double-refractory to prior anti-CD20 therapy and alkylator therapy. The most common regimens in the TLNext cohort were bendamustine-like regimens (34%), PI3K-like regimens (15%), and anti-CD20 monotherapy (14%). Comparative MAIC analyses are ongoing.

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Summary/Conclusion: TLNext represents a real-world 3L+ FL cohort from Germany that provides relevant context for the M SAT. TLNext outcomes data will be re-weighted using the MAIC approach to generate a descriptive benchmark for outcomes observed in M SAT. While comparisons between clinical trials and RWD carry several limitations pertaining to measurement, reporting of data, and choice of analytical approach to conduct the comparison, RWD from TLNext may be useful to describe a benchmark for clinical practice in 3L+ FL in Germany and globally prior to the availability of randomized evidence on newer treatment options like mosunetuzumab.

P1149: THE EVOLVING STATE OF PLAY IN 1000 PATIENTS WITH WALDENSTRÖM’S MACROGLOBULINAEMIA IN THE UNITED KINGDOM (UK): A REAL-WORLD DATA ANALYSIS FROM THE WMUK RORY MORRISON REGISTRY PROJECT

E. Uppal 1,* , A. Otamas 1 , J. Khwaja 1 , H. McCarthy 2 , J. Kothari 3 , A. Rismani 1 , D. El-Sharkawi 4 , C. Kyriakou 1 , S. D’Sa 1

1 Department of Haematology, University College London Hospitals NHS Foundation Trust, London; 2 Department of Haematology, Royal Bournemouth Hospital, Bournemouth; 3 Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford; 4 Department of Haematology, The Royal Marsden Hospital NHS Foundation Trust, London, United Kingdom

Background: Waldenström’s Macroglobulinaemia (WM) is a rare B-NHL, with an incidence of 0.55 per 100,000 per year in the UK (1). As novel therapies move from the bench to the clinic, real world data (RWD) is important to understand trends and supplement data from clinical trials. The Rory Morrison Registry (RMR) collects sequential data on diagnosis, treatment landscape and outlook for patients from 25 UK centres.

Aims: To characterise therapy trends in the UK by an analysis of disease and treatment characteristics, and survival status.

Methods: The RMR was searched for all patients with a diagnosis of WM. Information regarding demographics, disease characteristics, bone marrow (BM) results, treatment and survival status were collected retrospectively; the earliest diagnosis was in 1978 and the data lock was on 25/2/22.

Results: 1007 patients with a diagnosis of WM using consensus criteria (2) at 25 UK Centres (94.5% academic, 5.5% district centres) were identified. The following data pertain to diagnosis; median age was 64 years (21-93); M:F ratio 1.6:1, 90% of white ethnicity. Diagnostic sub-entities included peripheral neuropathy (116), cryoglobulinaemia (68), amyloidosis (22), Bing-Neel syndrome (18) and Schnitzler’s syndrome (9); 53% (501/945) were symptomatic at diagnosis. The median IPSSWM for 496 patients was 2. MYD88 L265P by RT-PCR was found in 87.1% (304/349) and CXCR4 mutations in 28.9% (26/90).

Of the total, 690 (68.5%) patients received treatment with a median time from diagnosis to first line (L1) treatment of 3 months (0-312). Indications for L1 were recorded as: 274/690 lymphoma related, 248/690 paraprotein-related, 65/690 B-Symptoms, 101/690 other indications. Regarding later lines, 402 (39.9%) patients received a 2 nd treatment (L2); median time from end L1 to beginning of L2 was 15.1 months (0-163). A 3 rd line (L3) was administered in 226 (22.4%) patients; median time from end of L2 to start of L3 was 9 months (0-158); 125 (12.4%) patients received ≥4 treatments.

Between 1984 and 2021 the three most popular treatments received at L1 were: Dexamethasone-Rituximab-Cyclophosphamide (DRC) 20.1% (144/690), R-Bendamustine (BR) 15.2% (105/690), Fludarabine based (±Cyclophosphamide, Rituximab) 9.0% (62/690) [Figure 1].

The three most popular treatments at L2 were: Ibrutinib 24.9% (100/402), BR 9.5% (38/402), DRC 5.0% (20/402). Ibrutinib is the leading 2 nd , 3 rd , 4 th line treatment option from 2018 across all centres, due to its availability on the national Cancer Drugs Fund from Nov 2017. The most received treatments from 2020 at: Ibrutinib 31.1% (60/193), R-Bendamustine 12.4% (24/193), DRC 10.4% (20/193). 38 patients underwent autologous and 7 allogeneic stem cell transplants between 2002 to 2020 at a median of L3 (range 1-7).

At the time of data lock, 184 patients were labelled deceased. Cause of death was available for 78 patients: 15 due to WM, 7 patients high grade transformation, 3 amyloid, 3 during ASCT, 2 AML/MDS.

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Summary/Conclusion: This RWD analysis of >1000 UK patients shows that inter/national consensus guidelines are resulting in more streamlined treatment approaches in WM. As targeted therapies have become available, there is a paradigm shift towards their use; however there is still much to learn about the optimal selection and sequencing of therapies in WM patients whose diseases are heterogenous by nature. Patient-related outcome studies are underway to complement ongoing follow-up of clinically meaningful remission duration and tolerance.

P1150: REAL WORLD DATA ON BORTEZOMIB-BASED THERAPY IN WALDENSTRÖM’S MACROGLOBULINAEMIA: EFFECTIVE EVEN IN MULTIPLY TREATED PATIENTS INCLUDING PRIOR BTK-INHIBITORS

E. Uppal 1,* , J. Khwaja 1 , A. Rismani 1 , C. Kyriakou 1 , S. D’Sa 1

1 Department of Haematology, University College London Hospitals NHS Foundation Trust, London, United Kingdom

Background: Waldenström macroglobulinemia (WM) is an indolent lymphoma with a prolonged disease course which typically follows a remitting and relapsing trajectory, eventually leading to treatment resistance. Several treatment options exist including Bruton tyrosine kinase inhibitors (BTKi), rituximab-containing regimens, and bortezomib-containing regimens. Treatment selection is based on patient performance status, disease characteristics, drug tolerability and availability.

Aims: To assess the effectiveness and tolerability of bortezomib-based regimens in WM.

Methods: Data for patients who had Bortezomib-containing regimens between 2010 and 2021 from 6 centres in the United Kingdom were retrospectively reviewed. Data was acquired from the WMUK Rory Morrison Registry. Research ethics approval was obtained.

Results: Thirty-four patients were identified: 32/34 had Bortezomib-containing regimens once and 2/34 had >1 Bortezomib-containing regimens on separate occasions, giving a total of 38 subcutaneous Bortezomib-containing regimens administered at bi-weekly and weekly schedules. Median age was 62 years (37-87), with a median of 2 prior lines of therapy (0-7), at a median duration of 49.6 months from date of WM diagnosis (0.7-422). 5 patients received a prior BTKi, with the Bortezomib regimen prescribed following a median of 3 prior lines of therapy (2-5) in this group. Patients who were treated at first line had elected for non-chemotherapy regimens. Median performance status was 1 (0-2) in 23 evaluable patients. The median M-protein at initiation was 34.5g/l (8-60) with bone marrow infiltration 70%, and haemoglobin 94g/l (88-107). A median of 5 cycles (1-8) were delivered and 65% (13/20) received a dose of 1.6mg/m 2 and 35% (7/20) received 1.3mg/m 2 .

Grade (G) 1 to 2 neuropathy occurred in 19% (5/26) of evaluable patients but did not result in treatment cessation in any case. Six of 25 (24%) needed a dose reduction, the majority due to G1-2 neuropathy (67%; 4/6). Gastrointestinal disturbance occurred in 12% (3/26) patients, 1 required admission with G4 diarrhoea and remaining cases were G1.

Of 34 evaluable cases, major response rate (≥ PR) was 74% (5 CR, 6 VGPR, 14 PR). 62% (8/13) of patients receiving 1.6mg/m 2 achieved a major response and 86% (6/7) of those who received 1.3mg/m 2 . Three of 5 patients who had prior BTKi achieved PR, 1 MR, 1 SD.

Two patients had treatment discontinued due refractory disease. The overall median time to best response was 81 days from end of treatment. Six of 19 (26%) evaluable patients achieved best response during therapy. Two patients died during treatment due to infection (COVID; respiratory sepsis), not attributable to disease relapse. Eighteen patients (60%; 18/30) had treatment after bortezomib regimens at a median of 5.3 months (0-75) and are alive. Median follow up was 30 months (1-111). Twenty-one evaluable patients (72%; 21/29) were alive at the end of follow up.

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Summary/Conclusion: This retrospective real-world analysis shows that bortezomib-containing regimens have utility in WM with effective major response rates even in those with multiple prior lines of therapy and heavy marrow infiltration including BTKi failures. Lower bortezomib doses are effective, and GI and neurotoxicity are manageable with dose reductions but no treatment discontinuations in this real-world cohort indicating an acceptable safety profile.

P1151: METABOLIC TUMOR VOLUME IMPROVES OUTCOME PREDICTION IN UNTREATED MANTLE CELL LYMPHOMA

V. K. Vergote 1,* , G. Verhoef 1 , A. Janssens 1 , F. S. Woei-a-jin 2 , A. Laenen 3 , T. Tousseyn 4 , D. Dierickx 1 , C. M. Deroose 5

1 Hematology; 2 General Medical Oncology, UZLeuven; 3 Biostatistics and Statistical Bioinformatics Center, KULeuven; 4 Pathology; 5 Nuclear Medicine, UZLeuven, 3000, Leuven, Belgium

Background: Mantle cell lymphoma (MCL) is a rare, incurable subtype of B-cell non-Hodgkin’s lymphoma, with heterogenous clinical behavior ranging from indolent to very aggressive disease course with rapid relapses and short survival.

Aims: The aim of this retrospective study is to analyze the prognostic value of baseline [ 18 F]FDG-PET/CT metabolic parameters in untreated MCL.

Methods: We retrospectively analyzed baseline [ 18 F]FDG-PET/CT of patients in our institution (University Hospitals Leuven, Belgium) with histopathological confirmed newly diagnosed MCL between January 1 st 2004 and 31 st December 2020. Maximum standardized uptake value (SUV max ), mean standardized uptake value (SUV mean ), peak standardized uptake value (SUV peak ), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were analyzed. Lesion dissemination (Dmax) and lesion dissemination standardized to body surface area (SDmax) were calculated. Univariate and multivariate analysis were performed using Cox proportional hazard models. Kaplan Meier survival curves were constructed.

Results: We included 83 patients with a median age of 66 years. The median MTV was 141.5 ml and the median Dmax was 0.6 m. A median follow-up of 47 months was achieved. In total 42 patients died. Higher MTV was an adverse factor for overall survival (OS) (p=0.0137; hazard ratio (HR): 1.48; 95% confidence interval (CI): 1.08-2.03), progression-free survival (PFS) (p=0.0106; HR: 1.41; 95% CI: 1.08-1.84) and disease-specific survival (DSS) (p=0.0366; HR: 1.56; 95% CI: 1.03-2.36). Furthermore, higher age at diagnosis, MCL International Prognostic Index-score (MIPI) and Eastern Cooperative Oncology Group Performance status (ECOG PS) were adverse factors for OS, PFS and DSS. We found no significant correlation between Dmax, SDmax and any of the outcomes considered. In multivariate analysis accounting for clinical characteristics, MTV was significantly associated with DSS (p=0.0072; HR: 1.53; 95% CI: 1.12-2.08), but not with OS and PFS. OS in the patients with MTV above the median (high, ≥141 ml) was significantly worse compared to the group with low MTV (<141 ml) (median OS 6.1 years versus 7.6 years; p=0.0077). Furthermore, there was a significant difference in PFS (median 2.9 versus 5.0 years; p=0.0055) and DSS (median 7.3 years versus not reached; p=0.0219) between the groups with high and low MTV. MTV spleen was an adverse factor for OS (p=0.0103; HR: 1.36; 95% CI: 1.08-1.72) and PFS (p=0.0019; HR: 1.40; 95% CI: 1.13-1.72) in multivariate analysis. Ann Arbor stage was not withheld as a significant factor for outcome prediction in univariate nor multivariate analysis.

Summary/Conclusion: MTV is an important prognostic tool and can further improve patient risk stratification at staging of untreated MCL. We found no correlation between lesion dissemination (Dmax, SDmax) on [ 18 F]FDG-PET/CT and outcome.

P1152: RESULTS FROM A PHASE I PHARMACOKINETIC (PK) AND SAFETY STUDY OF TRPH-222, A NOVEL CD22-TARGETING ANTIBODY-DRUG CONJUGATE, IN PATIENTS WITH RELAPSED/REFRACTORY B-CELL NON-HODGKIN LYMPHOMA (R/R NHL)

F. J. Hernandez-Ilizaliturri 1 , J. Kuruvilla 2 , B. A. Christian 3 , I. W. Flinn 4 , S. E. Assouline 5 , M. L. Ulrickson 6 , D. J. Landsburg 7 , M. Stuart 8 , H. Lowman 8 , N. Levin 8 , D. Maetzel 9 , N. N. Viller 9 , A. MacLaren 8,*

1 Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, United States of America; 2 The Princess Margaret Hospital, Toronto, ON, Canada; 3 The Ohio State University, Columbus, OH; 4 Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, United States of America; 5 Division of Hematology, Sir Mortimer B. Davis-Jewish General Hospital, Department of Oncology, McGill University, Montreal, QC, Canada; 6 Banner MD Anderson Cancer Center, Gilbert, AZ; 7 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA; 8 Triphase Accelerator, La Jolla, CA, United States of America; 9 Triphase Accelerator, Toronto, ON, Canada

Background: TRPH-222 is a novel antibody-drug conjugate (ADC) comprised of a humanized anti-CD22 monoclonal antibody and maytansine, a potent microtubule inhibitor, joined by a novel third-generation linker-conjugation technology (SMARTag ® ). This approach enables site-specific conjugation of the maytansine payload while tightly controlling drug:antibody ratio (DAR), resulting in a highly stable anti-CD22 ADC with a non-cleavable linker designed to widen the therapeutic window.

Aims: The primary objectives of this first in human study are to determine the safety, tolerability and pharmacokinetics (PK) of TRPH-222 monotherapy in patients with R/R NHL.

Methods: TRPH-222-100 is an open-label, multicenter study comprised of dose-escalation and dose-expansion stages. TRPH-222 was administered IV once every 3 weeks. 22 patients were enrolled in dose-escalating cohorts of TRPH-222 (0.6 mg/kg to 10 mg/kg) from DLBCL, FL, TFL, MCL and MZL histologies, and 10 patients in a dose-expansion cohort (7.5 mg/kg) focusing on DLBCL and FL histologies.

Results: As of January 7, 2022, 32 NHL patients have been enrolled: 15 indolent (14 FL and 1 MZL) and 17 aggressive histologies (15 DLBCL, 1 TFL and 1 MCL). Patients had a median age of 64.5 years, a median of 4 prior lines of therapy including 7 patients receiving prior CAR-T treatment.

Three DLTs occurred in 2 patients during the study and comprised Grade 3 and 4 transaminase elevations; one each at 4.2 and 10 mg/kg and one Grade 3 thrombocytopenia at 4.2 mg/kg. Treatment-related serious adverse events (AEs) occurred in 2 patients (6.3%), caused by thrombocytopenia and pyrexia (both at 7.5 mg/kg TRPH-222). AEs were more prevalent at doses ≥7.5 mg/kg and less prevalent at lower doses. There was a trend to higher grade AEs in patients with aggressive histologies, compared to indolent ones. The most frequent (≥ 5%) treatment-emergent related AEs (Grade ≥3) included thrombocytopenia (34%), neutropenia (22%), ALT/AST elevation (6%), dry eye (6%) and blurred vision (6%). Cytopenias were non-febrile, infrequent, asymptomatic and resolved without significant intervention. Ocular findings were consistent with known epithelial keratopathy of ADCs and were generally low grade and resolved to ≤ Grade 1 with dose interruptions and/or reductions. Overall, TRPH-222 demonstrated a favourable safety profile with most AEs being predominantly low grade, tolerable, easily managed and reversible.

Preliminary efficacy results suggest evidence of anti-tumor activity, most notably in patients with R/R FL. Of the 13 response-evaluable FL patients, 4 complete responses (CR) and 2 partial responses (PR) were observed, with an overall response rate (ORR) of 46% and a complete response rate (CRR) of 31%. Four patients with metabolic CRs maintained these CRs for long periods off treatment; 3 patients remain in CR with responses maintained for up to 25 months. Responses were generally early, durable and CRs were maintained off-therapy. Beyond FL, CRs were also observed in 1 DLBCL patient and in 1 MCL patient.

Summary/Conclusion: TRPH-222 was found to be well tolerated at higher dose levels than evaluated for other ADCs. TRPH-222 monotherapy resulted in robust and durable CRs in FL across dose levels where patients were able to discontinue TRPH-222 while remaining in remissions. Collectively, these characteristics of TRPH-222 are favorable for further development in the indolent lymphoma setting either as monotherapy or in combination with other anti-tumor agents in B-cell lymphoma patients.

P1153: LONG-TERM SURVIVAL OUTCOMES OF PATIENTS WITH PRIMARY OCULAR ADNEXAL MALT LYMPHOMA: A LARGE SINGLE-CENTER COHORT STUDY

Y. Liang 1 , R.-Y. Fu 1 , X. Li 1 , Y.-S. Piao 2 , J.-M. Ma 3 , L. Wang 1,*

1 Hematology; 2 Pathology; 3 Eye center, Beijing Tongren Hospital, Capital Medical University, Beijing, China

Background: Primary ocular adnexal extranodal marginal zone mucosa-associated lymphoid tissue lymphoma (OAML) is a rare subtype of non-Hodgkin’s lymphoma, and no consensus has been defined concerning the optimal treatment strategies.

Aims: This study aims to investigate the associations of disease characteristics and different treatments with long-term outcomes of patients with localized OAML.

Methods: A large retrospective cohort study was conducted in a single-center of China, and 166 patients with newly diagnosed primary localized OAML between April 2008 and August 2021 were enrolled in our analysis. Detailed data of disease characteristics at diagnosis and treatments were collected for all patients, and treatment response was evaluated at one month after therapy and every six months thereafter. We compared treatment response and progression-free survival (PFS) among patients with different characteristics and treatments.

Results: Of the 166 patients, 52 received complete resection of neoplasm, whereas 114 had residual lesion after surgery. Among the 114 patients, 61 underwent watchful waiting and 53 received further treatment including localized radiotherapy, chemotherapy (including immunochemotherapy), or combined radiotherapy and chemotherapy. Median follow-up of these patients was 49 months (range: 2-156). A total of 31 patients had disease progression or relapse during follow-up, including four patients with such event more than five years after initial treatment. The 5-year PFS was 73.9%, 70.6% and 85.9%, whereas the 10-year PFS was 69.3%, 59.2% and 79.3%, among patients with complete resection of neoplasm, patients in the watchful waiting group and patients with further treatment, respectively. Patients with further treatment had longer PFS, compared with patients in the watchful waiting group (P=0.011), while no difference in PFS was noted between patients with further treatment and patients with complete resection of neoplasm (P=0.127). Bilateral involvement at diagnosis was associated with significantly inferior PFS (P=0.029), whereas age, IPI score, or TNM staging was not associated with PFS. No serious adverse reaction was reported among patients with further treatment.

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Summary/Conclusion: Bilateral involvement was associated with poor prognosis. Among patients with residual lesions after surgery, further treatment was associated with improved survival. Patients with OAML might experience disease progression or relapse more than five years after initial treatment.

P1154: STUDY ZILO-301: A PHASE 3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, MULTICENTER STUDY OF ZILOVERTAMAB PLUS IBRUTINIB VS IBRUTINIB IN PATIENTS WITH RELAPSED OR REFRACTORY MANTLE CELL LYMPHOMA

S. Yazji 1,* , S. Hamburger 1 , Y. Wang 1 , S. Yavrom 1 , A. Pietrofeso 1 , R. Bliss 2 , J. B. Breitmeyer 1 , M. Dreyling 3 , M. Wang 4

1 Oncternal Therapeutics, Inc., San Diego, CA; 2 Veristat, LLC, Southborough, MA, United States of America; 3 University of Munich, Munich, Bavaria, Germany; 4 Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America

Background: Zilovertamab (ZILO) is a humanized monoclonal antibody that inhibits the tumor promoting activity of ROR1 and has demonstrated additive/synergistic activity with anti-cancer agents including ibrutinib (Ibr). In the Phase 1/2 Study CIRM-0001, ZILO in combination with Ibr was well tolerated and demonstrated promising efficacy in heavily pretreated, relapsed and refractory (R/R) patients (pts) with mantle cell lymphoma (MCL). As of 1 Oct 2021, 26 evaluable pts were enrolled, the objective response rate (ORR) was 80.8 % of which 34.6% achieved complete response (CR), with a median progression free survival (mPFS) of 35.9 months (mos). The rationale for the novel enrichment design (Figure 1) of Study ZILO-301 is (1) there is an unmet medical need for pts with R/R MCL that only achieve a partial response (PR) or have stable disease (SD) on Ibr, (2) there is an early opportunity for marketing authorization based upon an accepted surrogate endpoint (namely ORR), and (3) a confirmatory study for regular approval is built into the study design based on PFS. The timeframe from initial accelerated marketing authorization to regular approval is shorter than historical approaches for this patient population. Additionally, in Study ZILO-302, the safety and efficacy of ZILO rescue therapy can be evaluated in pts that progress early on Ibr in Study ZILO-301.

Aims: To evaluate the efficacy and safety of ZILO plus Ibr vs Ibr in R/R MCL pts.

Methods: Study ZILO-301 is a Phase 3 randomized, double-blind, placebo-controlled, multicenter study under which pts with R/R MCL will initially receive single agent Ibr (560 mg daily). After 4 mos, pts who only have PR or SD will be randomized (1:1) to receive an infusion of ZILO or matching placebo (Pbo) and continue to receive Ibr. Study ZILO-301 key inclusion criteria: Aged ≥18 years, confirmed diagnosis of MCL, received ≥ 1 prior treatment for MCL (other than Bruton’s tyrosine kinase inhibitor), have measurable disease, and signed informed consent. Key exclusion criteria: History or current CNS involvement, significant cardiovascular disease, prior stem cell transplant requiring immunosuppressive therapy, or graft versus host disease. Efficacy assessments will be evaluated by a Blinded Independent Central Review Committee utilizing Lugano Classification (Cheson 2014).

Results: Three analysis timepoints are planned. Analysis 1 is a futility assessment focused on ORR. Analysis 2 is an analysis for potential early marketing authorization (using accelerated assessment pathways by FDA and EMA), based upon ORR, duration of response (DOR), and safety. This analysis is expected to occur approximately 2 years after the first pt is enrolled in Study ZILO-301. Regardless of the Analysis 2 result, the study will proceed without modification. Analysis 3 is the end of study analysis for the primary endpoint of PFS and secondary endpoints of CR rate, duration of CR, time to CR on therapy, OS, proportion of pts experiencing Grade ≥ 3 neutrophil or platelet decrease, and overall safety. Regular marketing approvals would be sought based on Analysis 3. Figure 1 provides a schematic and subject flow for Study ZILO-301. Study ZILO-302 data may also support additional labeling for the combination of ZILO plus Ibr.

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Summary/Conclusion: Study ZILO-301, a phase 3, multinational, double-blind, placebo-controlled study is open for enrollment in June 2022.

P1155: BREXUCABTAGENE AUTOLEUCEL FOR RELAPSED/REFRACTORY MANTLE CELL LYMPHOMA IN ROUTINE PRACTICE: UPDATED REPORT FROM THE US LYMPHOMA CAR T CONSORTIUM

Y. Wang 1,* , P. Jain 2 , F. Locke 3 , M. Maurer 1 , M. Frank 4 , J. Munoz 5 , S. Dahiya 6 , A. Beitinjaneh 7 , M. Jacobs 8 , J. Mcguirk 9 , J. Vose 10 , A. Goy 11 , C. Andreadis 12 , B. Hill 13 , K. Dorritie 14 , O. Oluwole 15 , A. Deol 16 , B. Shah 3 , J. Paludo 1 , T. Wang 7 , R. Banerjee 12 , S. Neelapu 2 , D. Miklos 4 , A. Rapoport 6 , L. Lekakis 7 , A. Ghobadi 8 , Y. Lin 1 , M. Wang 2 , M. Jain 3

1 Mayo Clinic, Rochester; 2 The University of Texas MD Anderson Cancer Center, Houston; 3 Moffitt Cancer Center, Tampa; 4 Stanford University Medical Center, Stanford; 5 Mayo Clinic, Phoenix; 6 University of Maryland School of Medicine, Greenebaum Comprehensive Cancer Center, Baltimore; 7 University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami; 8 Washington University School of Medicine, Siteman Cancer Center, St Louis; 9 University of Kansas Medical Center, Kansas City; 10 University of Nebraska Medical Center, Buffett Cancer Center, Omaha; 11 John Theurer Cancer Center, Hackensack Meridian Health, Hackensack; 12 University of California San Francisco, San Francisco; 13 Cleveland Clinic, Cleveland; 14 UPMC Hillman Cancer Center, Pittsburg; 15 Vanderbilt-Ingram Cancer Center, Nashville; 16 Wayne State University, Karmanos Cancer Institute, Detroit, United States of America

Background: The ZUMA-2 trial and real-world studies showed high efficacy of brexucabtagene autoleucel (brexu-cel) in relapsed/refractory mantle cell lymphoma (MCL). We previously reported an objective response rate (ORR) of 89% in 95 patients receiving standard-of-care brexu-cel in the US Lymphoma CAR T Consortium (Wang et al, ASH 2021).

Aims: To update the safety and efficacy results of the Consortium study with more patients and longer follow-up.

Methods: Patients who underwent leukapheresis for standard-of-care brexu-cel manufacture at one of the 16 Consortium centers between 8/1/2020 and 12/31/2021 were included. Baseline characteristics, bridging therapy, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and treatment outcome data were collected. Eligibility for ZUMA-2 was retrospectively determined based on characteristics at leukapheresis. Time-to-event data were analyzed using the Kaplan-Meier method.

Results: At the data cut-off date of 1/31/2022, 189 patients underwent leukapheresis; 167 (88%) completed infusion and 22 (12%) did not (manufacture failure n=7, death n=6, disease progression n=5, organ dysfunction n=2, complete response [CR] to bridging therapy n=1, patient decline n=1).

The median age of the 167 infused patients was 67 years (range 34-89) and 76% were male. 16% had high risk simplified MIPI, 57% had Ki-67 ≥50%, 41% had blastoid or pleomorphic variant, 49% had TP53 alteration, 31% had complex karyotype, 14% had bulky disease (≥10 cm), and 10% had CNS involvement. The median prior lines of therapy was 3 (range 1-10), and 86% were BTKi-exposed (89% of which were refractory). 130 (78%) patients would not have met ZUMA-2 eligibility criteria, and the most common reasons were prior therapies (eg, anthracycline- or bendamustine-naïve [16%], BTKi-naïve [14%], >5 lines of prior therapy [11%]), renal dysfunction (21%), cardiac comorbidities (14%), cytopenias (11%), ECOG PS ≥2 (11%), and CNS involvement (10%).

113 (68%) patients received bridging therapy, which included immunochemotherapy (n=45), BTKi (n=45), venetoclax (n=24), radiotherapy (n=23), corticosteroid (n=18), and lenalidomide (n=6). The median time from leukapheresis to lymphodepletion chemotherapy was 28 days (range 17-140).

The rate of CRS was 90% (8% grade ≥3, 1 grade 5), and the rate of ICANS was 61% (32% grade ≥3, 0 grade 5). Medications used to manage CRS and/or ICANS included tocilizumab (76%), corticosteroid (68%), anakinra (16%), and siltuximab (3%). 20% of patients required ICU admission, with a median stay of 3 days (range 1-12); 18% required vasopressors, 3% required mechanical ventilation, and 3% required dialysis.

Among 159 evaluable patients, the day 30 ORR was 89%, with 70% CR and 19% partial response (PR). With continued follow-up, the best ORR was 89%, with 80% CR and 9% PR. The best ORR/CR rate were 89%/77% for high Ki-67% (≥50%), 88%/79% for blastoid or pleomorphic variant, 88%/73% for complex karyotype, 90%/72% for TP53 altered, 81%/75% for CNS involved, 89%/79% for BTKi-exposed, 91%/83% for BTKi-naïve, and 89%/78% for ZUMA-2 ineligible.

With a median follow-up of 5.6 months (range 0.2-15.3), the 6-month estimates for duration of response, progression-free survival, and overall survival were 67% (95% CI 57-75), 63% (95% CI 54-71) and 85% (95% CI 77-90), respectively.

Summary/Conclusion: This updated analysis demonstrated favorable safety and efficacy results of brexu-cel in R/R MCL that are consistent with the initial report. Despite 78% of patients being ineligible for ZUMA-2, the responses, CRS, ICANS and survival outcomes were comparable to ZUMA-2 data.

P1156: MAGNIFY PHASE 3B STUDY OF LENALIDOMIDE + RITUXIMAB (R2) FOLLOWED BY MAINTENANCE IN RELAPSED/REFRACTORY INDOLENT NON-HODGKIN LYMPHOMA: COMPLETE INDUCTION PHASE ANALYSIS

F. Lansigan 1,* , D. J. Andorsky 2 , M. Coleman 3 , A. Yacoub 4 , J. M. Melear 5 , S. R. Fanning 6 , K. S. Kolibaba 7 , C. Reynolds 8 , G. S. Nowakowski 9 , M. Gharibo 10 , J. R. Ahn 10 , J. Li 10 , M. J. Rummel 11 , J. P. Sharman 12

1 Dartmouth–Hitchcock Medical Center, Lebanon, NH; 2 Rocky Mountain Cancer Centers, US Oncology Research, Boulder, CO; 3 Clinical Research Alliance Inc, Weill Cornell Medicine, New York, NY; 4 University of Kansas Cancer Center, Westwood, KS; 5 Texas Oncology — Austin, US Oncology Research, Austin, TX; 6 Prisma Health, US Oncology Research, Greenville, SC; 7 US Oncology Research, Vancouver, WA; 8 IHA Hematology Oncology Consultants — Ann Arbor, Ypsilanti, MI; 9 Mayo Clinic, Rochester, MN; 10 Bristol Myers Squibb, Princeton, NJ, United States of America; 11 Justus-Liebig-Universität, Giessen, Germany; 12 Willamette Valley Cancer Institute and Research Center, US Oncology Research, Eugene, OR, United States of America

Background: Patients with relapsed indolent NHL (iNHL) have limited standard treatment options. Lenalidomide combined with rituximab (R 2 ) has shown complimentary clinical activity and is a tolerable regimen in both untreated and relapsed or refractory (R/R) patients with iNHL (RELEVANCE : N Engl J Med 2018;379:934 and AUGMENT: J Clin Oncol. 2019;37:1188).

Aims: These analyses examine the MAGNIFY interim primary endpoint of overall response rate (ORR; 1999 IWG) for induction R 2 in efficacy-evaluable patients receiving ≥ 1 treatment and who have available baseline and post-baseline assessments.

Methods: MAGNIFY is a multicenter, phase 3b trial in patients with R/R follicular lymphoma (FL) grades 1–3b, transformed FL (tFL), marginal zone lymphoma (MZL), or mantle cell lymphoma (MCL; NCT01996865 ) exploring optimal lenalidomide duration. In the induction phase, lenalidomide 20 mg PO on days 1–21 of a 28-day cycle + rituximab IV at 375 mg/m 2 /week cycle 1 and then every 8 weeks starting with cycle 3 (R 2 ) are administered for 12 cycles. Patients with stable disease, partial response, or complete response/complete response unconfirmed (CR/CRu) were randomized 1:1 to R 2 vs rituximab maintenance for 18 months. Data presented here are the complete analysis from the induction phase in efficacy-evaluable patients with FL grades 1–3a or MZL (FL grade 3b, tFL, and MCL not included).

Results: As of March 5, 2021, 394 patients (318 [81%] FL gr1–3a; 76 [19%] MZL) were enrolled. The median follow-up was 40.6 mo (range, 0.6–79.6). Median age was 66 y (range, 35–91), 328 (83%) had stage III/IV disease, with a median of 2 prior therapies (94% prior rituximab-containing). ORR was 71% (n = 279) with 42% (n = 164) CR/CRu (Table). All patients have completed R 2 induction (n = 232, 59%) or discontinued study treatment (n = 162, 41%). 141 patients (36%) prematurely discontinued both lenalidomide and rituximab, primarily due to adverse events (AEs) (n = 54, 14%) or progressive disease (n = 42, 11%). The majority of patients who have completed induction have been randomized and entered maintenance (n = 217). Median duration of response in the induction period was not reached (95% CI, 43.9 mo–NR), and median progression-free survival in the induction safety population (n = 393) was 50.5 mo (95% CI, 39.5–NR). Efficacy results are reported in the table by histology subgroups (FL vs MZL), and rituximab-refractory, double-refractory, and early relapse statuses. Most common all-grade treatment emergent AEs (TEAEs) were 47% fatigue, 43% neutropenia, 37% diarrhea, 30% nausea, and 30% constipation. Grade 3/4 AEs occurring in ≥ 5% of patients included 37% neutropenia (10 patients [3%] had febrile neutropenia), 8% leukopenia, 6% thrombocytopenia, 5% anemia, and 5% fatigue. TEAEs led to discontinuation of lenalidomide in 19% of patients and rituximab in 12% of patients; reduction or interruption of lenalidomide in 64% of patients; and to interruption of rituximab in 30% of patients (dose reduction for rituximab was not allowed). Neutropenia was the most common TEAE leading to lenalidomide discontinuation in 6% and reduction/interruption in 32%, and rituximab discontinuation in 3%. Infusion-related reaction was the most common TEAE leading to rituximab interruption in 8%.

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Summary/Conclusion: These data represent complete analysis of all patients in the induction phase of MAGNIFY which continue to support that R 2 is active with a tolerable safety profile in patients with R/R FL grade 1–3a and MZL, including rituximab-refractory, double-refractory, and early relapse patients.

P1157: REAL-WORLD TREATMENT PATTERNS AND COMPARATIVE EFFECTIVENESS OF BRUTON TYROSINE KINASE INHIBITORS IN PATIENTS WITH MANTLE CELL LYMPHOMA

B. Shah 1,* , K. Yang 2 , A. Klink 3 , T. Liu 2 , T. Zimmerman 2 , A. Gajra 3 , B. Tang 2

1 H. Lee Moffitt Cancer Center & Research Institute, Tampa; 2 Beigene USA, San Mateo; 3 Cardinal Health, Dublin, United States of America

Background: Bruton tyrosine kinase inhibitor (BTKi) therapies, approved for relapsed or refractory (R/R) mantle cell lymphoma (MCL), have not been comprehensively evaluated in real world populations.

Aims: This study aimed to assess patient characteristics, treatment patterns and associated outcomes in real world BTKi-treated MCL patients.

Methods: The retrospective multicenter chart review was conducted in the Cardinal Health Oncology Provider Extended Network. EMR data were extracted for eligible patients diagnosed with MCL who initiated any of the approved BTKi (ibrutinib, acalabrutinib, zanubrutinib) from 2018 to 2021; patients enrolled in trials were excluded. Index date was defined as the use of any of the BTKis. Patients were required to have 12-month pre-index for medical history, and from index to last follow-up or death. Descriptive analyses were conducted to assess demographic/clinical characteristics, MCL baseline features, BTKi treatment patterns, adverse events (AE), and response rates by BTKi. Multivariable logistic regression was performed to assess factors associated with response and AE.

Results: The study cohort consisted of 300 MCL patients (59% male; 69% white); most (64%) patients were covered by Medicare, 34% had commercial insurance. BTKis were given mainly as monotherapy (93%) and in R/R setting (86%). Patients in zanubrutinib group were significantly older (n = 100, median age = 71, range = 50-91) than patients in ibrutinib (n = 100, median age = 69, range = 39-87) and acalabrutinib (n = 100, median age = 70, range = 51-86) groups. Significantly fewer patients in the zanubrutinib group had baseline Ann Arbor stage I-II (4%) than ibrutinib (10%) or acalabrutinib (13%), while more zanubrutinib patients had presence of B symptoms (67%) than ibrutinib (44%) or acalabrutinib (57%). Patients in the zanubrutinib group also had significantly less with ECOG of 3+ (4%) compared to ibrutinib (8%) or acalabrutinib (6%). At BTKi initiation, significantly more patients in zanubrutinib group (18%) had history of atrial fibrillation than ibrutinib (1%) or acalabrutinib (5%). BTKis were given mainly as second-line (86%) and as monotherapy (93%). Most patients were started at an on-label BTKi dose. In zanubrutinib patients, the dose of 160 mg BID was more commonly administered (64%) than 320 mg QD (31%). Multivariable regression reported a significant association of age, gender, extranodal/splenic involvement, and timing of BTKi initiation with response and AE (Table).

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Summary/Conclusion: This study provides the first real world evidence on comparative effectiveness of ibrutinib, acalabrutinib, and zanubrutinib in MCL patients. While patients treated with zanubrutinib were older and had more complex MCL baseline features at initiation, multivariable regression suggested a trend favoring zanubrutinib over ibrutinib or acalabrutinib for both response and AE. Frontline initiation of BTKi therapy was also associated with improved tolerability. Future real world studies are needed to discern long-term outcomes.

P1158: REAL-WORLD TREATMENT PATTERNS AND ECONOMIC BURDEN OF PATIENTS WITH MARGINAL ZONE LYMPHOMA

K. Yang 1 , T. Liu 1 , B. Tang 1 , B. Shah 2,*

1 Beigene USA, San Mateo; 2 H. Lee Moffitt Cancer Center & Research Institute, Tampa, United States of America

Background: Marginal zone lymphoma (MZL) is an indolent non-Hodgkin lymphoma that is treatable, yet incurable with a remitting and relapsing disease course. Given its disease rarity and underlying heterogeneity, MZL remains understudied with limited real-world evidence on how current treatment pattern conform to clinical guidelines, and the economic outcomes associated with current treatments.

Aims: This study aimed to assess real-world treatment patterns, costs, and healthcare resource utilization (HRU) in US MZL patients.

Methods: A retrospective, observational study was conducted using the IBM MarketScan® commercial and Medicare supplemental claims dataset (2017-2020). Newly diagnosed adult MZL patients (≥18 years) continuously enrolled 6 month pre- and 3-month post-index date, defined as the first diagnosis date, were included. Treatment regimens were identified by line of therapy and mutually exclusively categorized as rituximab monotherapy (R-mono), bendamustine + rituximab (BR), rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), ibrutinib, or other. Descriptive analyses were conducted to assess patient sociodemographic and clinical characteristics, and treatment utilization patterns including the frequency, duration, and discontinuation of each treatment regimen. Costs and HRU assessed included inpatient, outpatient, and pharmacy visits per-patient-per-month (PPPM). Treatment regimens, costs, and hospitalizations were examined overall, and by line of therapy. Multivariable logistic regression was conducted to examine predictors of costs and HRU.

Results: Among the 2491 newly-diagnosed MZL patients (median age = 63 years; 52% male), 59% were commercially insured (median age = 57 years) and 41% in Medicare (median age = 76 years). The most common comorbidities were hypertension (44%), diabetes (17%), atrial fibrillation (AF; 16%), and gastroesophageal reflux disease (15%). Mean time from diagnosis to treatment initiation was 223 days. A total of 1,781 (72%) patients received first-line (1L), 518 (29%) patients received second-line (2L) and 239 (13%) patients received third-line (3L) therapies. R-mono was the most common regimen across both commercial and Medicare patients and all treatment lines (Table). R-CHOP and BR were the second most used regimen in 1L with decreased use in 2L+. Ibrutinib was used more in 2L+ setting but had the lowest 1L PPPM cost (median $2958.9) than other regimens. Overall MZL patients had PPPM 4.6 outpatient visits, 0.5 hospitalization, and mean length of stay of 2.6 days. Total PPPM healthcare cost was $19,895.8. Multivariable regression showed that baseline comorbidities (AF, renal disease, neutropenia) and treatment discontinuation were significant predictors of higher costs and HRU.

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Summary/Conclusion: This real-world data suggested that the overall US MZL real-world treatment pattern across lines of therapy follows the regimen recommendations by the National Comprehensive Cancer Network clinical practice guidelines and that MZL patients incur high economic burden. Future studies are needed to evaluate long-term outcomes and the impact of heterogenous MZL subtypes.

P1159: A MULTICENTER, INTERNATIONAL COLLABORATIVE STUDY EVALUATING FRONTLINE THERAPY WITH BENDAMUSTINE RITUXIMAB FOR WALDENSTRÖM MACROGLOBULINEMIA

S. Zanwar 1,* , J. Abeykoon 1 , J. Castillo 2 , E. Durot 3 , E. Kastritis 4 , E. Uppal 5 , P. Morel 6 , R. Tawfiq 7 , L. Montes 8 , J. Paludo 1 , S. Sarosiek 2 , S. Kumar 1 , O. Ogunbiyi 9 , P. Cornillet-Lefebvre 10 , R. Kyle 1 , A. Delmer 3 , M. Gertz 1 , M. Dimopoulos 11 , S. Ansell 1 , S. Treon 2 , S. D’Sa 12 , P. Kapoor 1

1 Hematology, Mayo Clinic, Rochester, Rochester; 2 Hematology, Dana Farber Cancer Institute, Boston, United States of America; 3 Hematology, University Hospital of Reims and UFR Médecine, Reims, France; 4 Department of Clinical Therapeutics, National and Kapodistrian University of Athens, Athens, Greece; 5 Hematology, University College London Hospitals (UCLH) NHS Foundation Trust, London, United Kingdom; 6 Hematology, Hôpital Schaffner, Lens, France; 7 Internal Medicine, Mayo Clinic, Rochester, Rochester, United States of America; 8 Service d’Hématologie Biologique, CHU Amiens, Amiens, France; 9 Hematology, Imperial College of London, London, United Kingdom; 10 Laboratory of Hematology, University Hospital of Reims and UFR Médecine, Reims, France; 11 Department of Clinical Therapeutics, National and Kapodistrian University of Athens, Athens, Greece; 12 Hematology, University College London Hospitals, London, United Kingdom

Background: Bendamustine rituximab (BR) is a frequently used chemoimmunotherapy for indolent lymphomas, including Waldenström macroglobulinemia (WM), a rare B-cell malignancy. The promising results of a small subset analysis (n= 41) of the Study group indolent Lymphomas (StiL) trial, demonstrating a median progression-free survival (PFS) of 69.5 months in the frontline setting served as the basis for widespread adoption of BR. Whether the more recently identified somatic mutations within the MYD88 and CXCR4 genes impact the outcomes of patients (pts) treated with BR remains less clear.

Aims: To study a large cohort of BR-treated pts with active WM through an international, multicenter collaborative effort.

Methods: Records of pts with newly diagnosed active WM who received BR between January 2012 and July 2021, in the US and Europe, were reviewed. The MYD88 L265P and CXCR4 mutation status were captured, if available. All time-to-event analyses were performed from the frontline therapy initiation, using the Kaplan-Meier method.

Results: Among 248 pts who were treated with BR, 208 pts received BR induction without rituximab maintenance, and were included in the primary analysis. The median age at treatment initiation was 65 (range 40-86) years; 64 % were males. The baseline characteristics are outlined in Table 1. The median follow-up was 4 (95% CI: 3.6-4.6) years.

The estimated median PFS was 5.9 years [95% CI: 5.3-not reached (NR)]. The estimated 5-year overall survival (OS) rate was 90%. Among 174 pts evaluable for response, the overall response rate (ORR), major response rate (MRR) and very good partial response (VGPR) rates were 95%, 93% and 31%, respectively, per the modified IWWM-6 criteria based on serum IgM level alone. Pts with progression of disease (POD) within 24 months of BR therapy (11%) had an inferior subsequent survival compared to those without POD within 24 months, the reference group [5-year subsequent survival rate, 71 % versus (vs) 86%, p=0.02].

Among 131 (63%) pts with a known MYD88 L265P status, 88% (n=116) had MYD88 L265P genotype. The 4-year PFS was 71% for both pts with MYD88 L265P and MYD88 WT genotypes (p=0.44), Figure 1 A . The VGPR rates were also comparable between the two groups (41% for MYD88 L265P and 50% for MYD88 WT genotypes, p=0.55). Among 42 (20%) pts with a known CXCR4 mutation status, 28% harbored a CXCR4 mutation. The ≥VGPR rate for pts with CXCR4 MUT genotype was numerically lower; 33% vs 57% for those with CXCR4 WT genotype, p=0.3. A trend towards shorter PFS among pts with CXCR4 MUT genotype [estimated median PFS for 3.9 years (95% CI: 0.8-NR) vs 5.5 years (95% CI 5.3-NR) for pts with CXCR4 WT genotype, p=0.05, Figure 1 B ] was observed. The PFS rates for pts without a known MYD88 or CXCR4 mutational status was comparable to their counterparts with known genotypes, respectively.

Among 40 (16%) pts who had received rituximab maintenance following BR, the median age at initiation of therapy was 67 years (range 44-88). After 1:1 matching for age, the 4-year PFS for the rituximab maintenance group was 89% vs 73% for pts who did not receive rituximab maintenance (p=0.09); OS was comparable (5-year OS rate, 85% for both groups, p=0.99).

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Summary/Conclusion: Fixed-duration BR is a highly effective regimen for pts with previously untreated symptomatic WM, irrespective of the MYD88 L265P mutation status, although early POD, within 2 years of initiation of BR, is associated with inferior subsequent survival. Our preliminary analysis, suggesting that the presence of CXCR4 mutation confers resistance to BR, warrants confirmation in prospective studies.

P1160: RESULTS OF A PHASE 2 EXPANDED ACCESS STUDY OF ZANUBRUTINIB IN PATIENTS WITH WALDENSTRÖM MACROGLOBULINEMIA

J. J. Castillo 1,* , E. C. Kingsley 2 , M. Narang 3 , H. A. Yimer 4 , C. A. Dasanu 5 , J. M. Melear 6 , M. Coleman 7 , C. M. Farber 8 , M. Gupta 9 , J. Shulman 10 , E. H. Mantovani 11 , X. Zhang 11 , A. Cohen 11 , J. Huang 11

1 Dana-Farber Cancer Institute, Boston, MA; 2 Comprehensive Cancer Centers of Nevada, Las Vegas, NV; 3 US Oncology Research, Maryland Hematology Oncology, Columbia, MD; 4 Texas Oncology, US Oncology Research, Tyler, TX; 5 Lucy Curci Cancer Center, Eisenhower Health, Rancho Mirage, CA; 6 US Oncology Research, Texas Oncology, Austin Midtown, Austin, TX; 7 Clinical Research Alliance, New York, NY; 8 Atlantic Hematology Oncology, Morristown Medical Center, Morristown, NJ; 9 Ridley-Tree Cancer Center at Sansum Clinic, Santa Barbara, CA; 10 Icahn School of Medicine at Mount Sinai, New York, NY; 11 BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America

Background: Bruton tyrosine kinase (BTK) inhibition is an emerging standard of care for Waldenström macroglobulinemia (WM). Zanubrutinib (ZANU; BGB-3111) is a next-generation BTK inhibitor designed to maximize BTK occupancy and minimize off-target inhibition of TEC- and EGFR-family kinases. ZANU was recently approved by the United States (US) Food and Drug Administration, Health Canada, and the European Union at a dose of 320 mg once daily (QD) or 160 mg twice daily (BID) for the treatment of adult patients (pts) with WM. BGB-3111-216 ( NCT04052854 ) is a single-arm, expanded access study of ZANU for treatment-naïve (TN) pts who are unsuitable for standard chemoimmunotherapy or pts with relapsed refractory (R/R) WM.

Aims: To provide real-world experience with ZANU in pts with WM.

Methods: Eligible pts with TN or R/R WM were assigned to receive ZANU at a dose of 320 mg QD or 160 mg BID. The primary endpoint was the number of pts enrolled/treated and the number of enrolling sites. Secondary endpoints of safety and efficacy included treatment emergent adverse events (TEAEs) of special interest, disease response (overall response rate [ORR] and very good partial response or better [VGPR+]), progression-free survival (PFS), and overall survival (OS). Response was evaluated by investigator-assessment according to the 6th International Workshop on WM ( Br J Haematol. 2013;160(2):171-6) every 6 months at minimum. The study was closed by the sponsor in July 2021 and active pts were transitioned to commercial ZANU via a patient assistance program.

Results: Fifty pts with WM (17 TN; 33 R/R), were enrolled from December 2019 to June 2021 across 10 academic and community medical centers in the US. At study entry, median age was 72 years, 54% had intermediate-risk disease, 40% had high-risk disease, and median number of prior therapies for R/R pts was 2. Median treatment exposure was 9.2 months (range 1.4 to 20.0). Thirty-eight (76%) pts had ≥1 TEAE, and 36 (72%) experienced ≥1 TEAE of special interest ( Table ). Grade ≥3 TEAEs of special interest included hypertension (8%), infection (8%), atrial fibrillation or flutter (2%), neutropenia (2%), and second primary malignancy (2%). No new safety signals were observed. In the 41 pts with ≥1 response evaluation, 39.0% achieved a best overall response (BOR) of VGPR (16; 95% CI, 24.2-55.5). ORR was 85.4% (35; 95% CI, 70.8-94.4), and major response rate was 73.2% (30; 95% CI, 57.1-85.8). Of the 4 pts who achieved a BOR of progressive disease, 3 had IgM values that met partial response criteria before the first 6 months response assessment. PFS and OS were immature due to short follow-up, and the median was not met.

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Summary/Conclusion: The results of this real-world expanded access study were consistent with the established ZANU profile in WM and other B-cell malignancies when administered as monotherapy at a daily dose of 320 mg orally (either as 160 mg BID or 320 mg QD) in pts with intermediate or high-risk R/R or TN WM.

P1161: ASPEN: LONG-TERM FOLLOW-UP RESULTS OF A PHASE 3 RANDOMIZED TRIAL OF ZANUBRUTINIB (ZANU) VS IBRUTINIB (IBR) IN PATIENTS (PTS) WITH WALDENSTRÖM MACROGLOBULINEMIA (WM)

M. Dimopoulos 1,* , S. Opat 2 , S. D’Sa 3 , W. Jurczak 4 , H.-P. Lee 5 , G. Cull 6 , R. G. Owen 7 , P. Marlton 8 , B. E. Wahlin 9 , R. Garcia-Sanz 10 , H. McCarthy 11 , S. Mulligan 12 , A. Tedeschi 13 , J. J. Castillo 14 , J. Czyz 15 , C. Fernandez De Larrea Rodriguez 16 , D. Belada 17 , E. Libby 18 , J. Matous 19 , M. Motta 20 , T. Siddiqi 21 , M. Tani 22 , M. Trneny 23 , M. Minnema 24 , C. Buske 25 , V. Leblond 26 , S. P. Treon 14 , J. Trotman 27 , W. Y. Chan 28 , J. Schneider 28 , H. Allewelt 28 , A. Cohen 28 , J. Huang 28 , C. S. Tam 29

1 National and Kapodistrian University of Athens, Athens, Greece; 2 Monash Health and Monash University, Clayton, Victoria, Australia; 3 Centre for Waldenström’s Macroglobulinemia and Associated Disorders, University College London Hospital Foundation Trust, London, United Kingdom; 4 Maria Sklodowska-Curie National Institute of Oncology, Krakow, Poland; 5 Flinders Medical Centre, Adelaide, SA; 6 Sir Charles Gairdner Hospital, University of Western Australia, Perth, WA, Australia; 7 St. James University Hospital, Leeds, United Kingdom; 8 Princess Alexandra Hospital, University of Queensland, Brisbane, Queensland, Australia; 9 Karolinska Universitetssjukhuset and Karolinska Institutet, Stockholm, Sweden; 10 Hospital Universitario de Salamanca, Salamanca, Spain; 11 Royal Bournemouth and Christchurch Hospital, Bournemouth, United Kingdom; 12 Royal North Shore Hospital, Sydney, New South Wales, Australia; 13 ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 14 Dana-Farber Cancer Institute, Boston, MA, United States of America; 15 Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland; 16 Hospital Clinic de Barcelona, Barcelona, Spain; 17 FN Hradec Kralove, Hradec Králové, Czechia; 18 University of Washington/Seattle Cancer Care Alliance - Clinical Research, Seattle, WA; 19 Colorado Blood Cancer Institute, Denver, CA, United States of America; 20 AO Spedali Civili di Brescia, Lombardia, Italy; 21 City of Hope National Medical Center, Duarte, CA, United States of America; 22 Ospedale Civile S.Maria delle Croci, AUSL Ravenna, Italy; 23 Vseobecna fakultni nemocnice v Praze, Prague, Czechia; 24 University Medical Center Utrecht, Utrecht, Netherlands; 25 CCC Ulm - Universitätsklinikum Ulm, Ulm, Baden-Württemberg, Germany; 26 Sorbonne University, Pitié Salpêtrière Hospital, Paris, France; 27 Concord Repatriation General Hospital, Sydney, New South Wales, Australia; 28 BeiGene USA, Inc., San Mateo, CA, United States of America; 29 Royal Melbourne Hospital, Parkville, Victoria, Australia

Background: ZANU is a potent and selective next-generation Bruton tyrosine kinase inhibitor (BTKi) designed to have greater affinity to BTK while minimizing off-target inhibition of TEC-and EGFR-family kinases. ASPEN ( NCT03053440 ) is a randomized, open-label, phase 3 study comparing ZANU with the first-generation BTKi IBR in pts with WM. We present data with a median follow-up of 43 months.

Aims: To compare the efficacy and safety of ZANU vs IBR in pts with MYD88 mutant ( MYD88 mut ) WM and ZANU in pts with wild-type MYD88 ( MYD88 wt ) WM.

Methods: Pts with MYD88 mut WM were assigned to cohort 1 and randomized 1:1 to receive ZANU 160 mg twice daily or IBR 420 mg once daily. Pts with MYD88 wt were assigned to cohort 2 and received ZANU 160 mg twice daily until disease progression. Randomization was stratified by CXCR4 mutational status by Sanger sequencing and lines of prior therapy (0, 1-3, or >3). All pts gave informed consent. The primary endpoint was proportion of pts achieving very good partial response or better (VGPR + complete response [CR]). Primary analysis occurred at 19 months median follow-up, and final analysis is planned to occur ~4 years after the first pt enrolled.

Results: A total of 201 pts (102 ZANU; 99 IBR) were enrolled in cohort 1 and 28 pts in cohort 2. Baseline characteristics in cohort 1 differed between pts treated with ZANU vs IBR in CXCR4 mutations by next-generation sequencing (32% vs 20%, or 33 of 98 vs 20 of 92 available samples, respectively) and pts aged >75 years (33% vs 22%, respectively). Median duration of treatment was 42 months (ZANU) and 41 months (IBR), with 67% and 58% remaining on treatment, respectively. The VGPR+CR rate by investigator was 36% with ZANU vs 22% with IBR (descriptive p = 0.02) in cohort 1, and 31% in cohort 2. One pt in cohort 2 obtained a CR. In pts with wild-type (65 ZANU; 72 IBR) or mutant CXCR4 (33 ZANU; 20 IBR) from cohort 1, VGPR+CR rates with ZANU vs IBR were 45% vs 28% ( p = 0.04) and 21% vs 5% ( p = 0.15), respectively. Median progression-free survival and overall survival were not reached.

Consistent with less off-target inhibition, rates of atrial fibrillation, diarrhea, hypertension, localized infection, hemorrhage, muscle spasms, pneumonia, and adverse events (AEs) leading to discontinuation or death were lower with ZANU vs IBR (Table); neutropenia (including grade ≥3) was the only AE of interest that was higher with ZANU (33.7%) vs IBR (19.4%). Rate of grade ≥3 infection was lower with ZANU (20.8%) vs IBR (27.6%). AE incidence with ZANU was similar across cohorts 1 and 2.

Annual prevalence analysis of cohort 1 AEs showed reduced prevalence of hemorrhage over time and lower prevalence with ZANU vs IBR at all intervals. In pts treated with ZANU, neutropenia and infection prevalence decreased over time. Prevalence of infection was lower in pts treated with ZANU vs IBR, and neutropenia was similar between arms (8.8% vs 9.7%, respectively) at >24-36 months of treatment. Prevalence of atrial fibrillation remained ≤5% and hypertension remained stable with ZANU, each with a lower prevalence at all intervals vs an increasing trend seen with IBR.

Consistently, exposure-adjusted incidence rates of atrial fibrillation/flutter and hypertension were lower with ZANU vs IBR (0.2 vs 0.8 and 0.5 vs 1.0 persons per 100 person-months, respectively; p < 0.05).

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Summary/Conclusion: ASPEN is the largest phase 3 trial with head-to-head BTKi comparison in WM. At a median follow-up of 43 months, ZANU was associated with a higher VGPR+CR rate and demonstrated clinically meaningful advantages in long-term safety and tolerability vs IBR.

P1162: ZANUBRUTINIB IN OLDER PATIENTS (PTS) WITH RELAPSED/REFRACTORY (R/R) MARGINAL ZONE LYMPHOMA (MZL): SUBGROUP ANALYSIS OF THE MAGNOLIA STUDY

S. Opat 1,* , B. Hu 2 , A. Tedeschi 3 , K. M. Linton 4 , P. McKay 5 , H. Chan 6 , J. Jin 7 , M. Sun 8 , M. Sobieraj-Teague 9 , P. L. Zinzani 10 , M. Coleman 11 , P. Browett 12 , X. Ke 13 , C. A. Portell 14 , C. Thieblemont 15 , K. Ardeshna 16 , F. Bijou 17 , P. Walker 18 , E. A. Hawkes 19 , S.-J. Ho 20 , K.-S. Zhou 21 , M. Co 22 , J. Xu 22 , Z. Liang 22 , J. Anderson 22 , C. Tankersley 22 , J. Huang 22 , J. Trotman 23

1 Monash Health and Monash University, Clayton, Victoria, Australia; 2 Levine Cancer Institute/Atrium Health, Charlotte, NC, United States of America; 3 ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 4 The Christie Hospital NHS Foundation Trust, Manchester; 5 Beatson West of Scotland Cancer Centre, Glasgow, United Kingdom; 6 North Shore Hospital, Auckland, New Zealand; 7 The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang; 8 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; 9 Flinders Medical Centre, Bedford Park, South Australia, Australia; 10 Institute of Hematology “Seràgnoli” University of Bologna, Bologna, Italy; 11 Clinical Research Alliance/Weill Cornell Medicine, Lake Success, NY, United States of America; 12 Auckland City Hospital, Grafton, New Zealand; 13 Peking University Third Hospital, Beijing, China; 14 University of Virginia Health System, Charlottesville, VA, United States of America; 15 APHP, Hôpital Saint-Louis, Hemato-oncology, Paris University Diderot, Paris, France; 16 University College London Hospitals, London, United Kingdom; 17 Institut Bergonié, Bordeaux, France; 18 Peninsula Private Hospital, Frankston, Victoria; 19 Box Hill Hospital, Box Hill, Victoria; 20 St. George Hospital, Kogarah, New South Wells, Australia; 21 Henan Cancer Hospital, Zhengzhou, Henan, China; 22 BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA, United States of America; 23 Concord Repatriation General Hospital, University of Sydney, Concord, New South Wales, Australia

Background: MZL is the second most common lymphoma in older pts. Choosing an optimal treatment can be challenging because of patient- or disease-related risk factors and treatment-related toxicities ( Curr Opin Oncol. 2019;31(5):386-393). Zanubrutinib is a potent, irreversible next-generation Bruton tyrosine kinase (BTK) inhibitor designed to maximize BTK occupancy and minimize off-target kinase inhibition, which may improve efficacy outcomes and minimize toxicities, such as cardiac arrythmias and bleeding events. Zanubrutinib received accelerated approval from the United States FDA for the treatment of pts with R/R MZL ( Haematologica . 2022;107(1):35-43).

Aims: We aim to present a subgroup analysis of efficacy and safety of zanubrutinib in pts aged ≥65 years with R/R MZL enrolled in MAGNOLIA (BGB-3111-214; NCT03846427 ).

Methods: MAGNOLIA is a phase 2, multicenter, single-arm study of adults with R/R MZL who had received ≥1 line of therapy including ≥1 CD20-directed regimen. All were treated with zanubrutinib 160 mg twice daily until disease progression or unacceptable toxicity. Use of long-term antiplatelet and anticoagulation agents was permitted. The primary endpoint was overall response rate (ORR; complete response [CR] and partial response [PR]) determined by an independent review committee (IRC) in accordance with the Lugano classification. Secondary endpoints include ORR by investigator assessment (INV), duration of response (DOR), progression-free survival (PFS), and safety. All pts gave informed consent.

Results: As of 18 January 2021, a total of 68 pts were enrolled (Table). Forty (61%) pts were ≥65 years old with a median age of 73 (range, 65-85); 18 pts were ≥75 years old. Median number of prior therapies was 2 (range, 1-6) and 10 (25%) pts were refractory to last therapy. Most pts received prior rituximab + cyclophosphamide + vincristine + prednisone (48%) or bendamustine + rituximab (30%), while 5 (13%) pts received rituximab monotherapy. MZL subtypes included extranodal (n=17, 43%), nodal (n=14, 35%), and splenic (n=8, 20%). Median duration of treatment was 14.4 months (mo; range, 0.9-19.6). At a median follow-up of 15.8 mo (range, 2.8-21.8), ORR by IRC was 75% (CR 25%, PR 50%; Table). Responses were observed in all subtypes, with an ORR of 71%, 86%, and 75% in extranodal, nodal, and splenic subtypes, respectively (CR 41%, 21%, and 0%, respectively). Median DOR and PFS were not reached; 15-month PFS was 87% and 12-month DOR was 93%. Most (63%) pts are continuing zanubrutinib. Treatment discontinuation due to disease progression was 28% by INV. Most common treatment-emergent adverse events (AEs) observed in ≥20% of pts include contusion (28%), diarrhea (25%), and constipation (20%). Grade ≥3 neutropenia occurred in 5% of pts. The most common infection was upper respiratory tract infection (10%). Two (5%) pts discontinued zanubrutinib due to unrelated fatal AEs (COVID-19 pneumonia and myocardial infarction in a patient with pre-existing coronary artery disease). Atrial fibrillation/flutter and hypertension occurred in 2 (5%) pts each and did not lead to treatment discontinuation. No pts required dose reductions, or experienced major or serious hemorrhage.

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Summary/Conclusion: The safety profile of zanubrutinib observed in older pts was consistent with previously published results ( Clin Cancer Res . 2021;27(23):6323-6332). Zanubrutinib was well tolerated and effective, as demonstrated by a high response rate and durable disease control in older pts with R/R MZL.

P1163: OUTCOMES OF HIGH-RISK LARGE B-CELL LYMPHOMA TREATED WITH STANDARD OF CARE CHEMOIMMUNOTHERAPY: A NON-RANDOMIZED COMPARISON TO FRONTLINE CAR-T CELL THERAPY

O. Albanyan 1,* , O. Castaneda-Puglianini 1 , J. Chavez 2

1 Blood and Marrow Transplant and Cellular Immunotherapy; 2 Department of Malignant Hematology, Moffitt Cancer Center, Tampa, United States of America

Background: High-risk LBCL is associated with poor prognosis after standard of care (SOC) first-line anti-CD20 mAb-containing chemoimmunotherapy (CIT), therefore here is a need for novel approaches. ZUMA-12 reported high overall response (ORR) and complete response (CR) rates in high risk LBCL defined as IPI score of ≥3 and/or double or triple-hit status [MYC and BCL2 and/or BCL6 translocations (double-hit/triple-hit lymphoma – DHL/THL)] and positive interim PET scan (iPET+)

Aims: Here, we report the outcomes of these high risk LBCL patients (pts) who did not qualify due to a negative (-) iPET or declined or were ineligible to be treated in the ZUMA-12 clinical trial.

Methods: Eligible patients were older than 18 and had high-risk LBCL, defined as DHL/THL status and/or an IPI score ≥3. Patients could have had an available iPET (or CT scan). Patients did not pursue ZUMA-12 due to trial ineligibility (i.e., iPET-) or did not want to pursue the trial. Patients continued SOC CIT per discretion of treating physician. The primary endpoint was end of treatment ORR and CR. Secondary endpoints included progression-free survival (PFS), overall survival (OS).

Results: During the time period ZUMA-12 was open, 61 pts were approached for the trial, 8 patients were excluded from the final analysis due to incomplete data. The remaining 53 pts did not qualify to ZUMA-12 due to iPET- (n=31, 58.4%), declined (n=10, 18.8%)), ineligible (n= 8, 15.1%) and other (n= 4). Median age was 67 (26 – 78), 11 (20.1 %) had DHL/THL, 46 (86.8 %) had IPI score 3-5. Patients were treated with DA-EPOCH-R (n=11, 21%) and R-CHOP (n=41, 77%). The ORR and CR rates was 83 and 79.2%, respectively in this cohort. Four patients did not complete therapy due to PD (n=3) and death for other causes (n=1). The ORR and CR rates for the ZUMA-12 study were reported at 90 and 80%, respectively. In our study, the median PFS and OS was 27.4 months (95% CI: 23.7 – 31.1) and 30.8 months (95% CI: 27.7 – 33.8), and the 30-months PFS and OS were 60 and 71%, respectively. In comparison, the ZUMA-12 reported a PFS and OS that was not reached.

Summary/Conclusion: In this nonrandomized study of high risk LBCL, the efficacy of axi-cel in the ZUMA-12 was numerically higher than SOC CIT. The PFS and OS in our cohort was lower despite majority of patients achieving early CR per iPET. This analysis supports a potential trial of frontline CAR-T versus SOC CIT for high risk LBCL.

P1164: IMAGE-BASED DETECTION OF HIGH-GRADE B CELL LYMPHOMAS (DH-L)

I. Avivi 1,2,* , C. Perry 1,2 , O. Ahron 2,3 , N. Hershkovitz 4 , D. Hershkovitz 2,5 , A. Avinoam 6 , I. Gazy 6 , N. Paz-Yaacov 6

1 Hematology department, Tel Aviv Sourasky Medical Center; 2 Sackler Faculty of Medicine., Tel Aviv University; 3 Pathology department; 4 Hematology department, Tel Aviv Medical Center; 5 Pathology department, Tel Aviv Sourasky Medical Center; 6 Imagene AI, Tel Aviv, Israel

Background: Aggressive B cell Non-Hodgkin Lymphomas (B-NHL) are divided into two main categories: diffuse large B cell lymphoma (DLBCL) accounting for 90% of cases, and high-grade B-cell lymphoma (HGBL). Diagnosis of high-grade lymphoma with MYC and BCL2 and/or BCL6 rearrangements (double-hit lymphoma-DHL) is confirmed by fluorescence in situ hybridization (FISH) analysis, demonstrating c-MYC -rearrangement in combination with BCL-2 / BCL-6 rearrangement in lymphoma cells. Accurate and rapid diagnosis of DHL is obligatory, when considering more aggressive treatment regimens (other than R-CHOP), suggested in these patients.

Aims: To establish a novel tool for diagnosing DLBCL and DHL, directly on the scanned Hematoxylin and Eosin (H&E) biopsy slides, by applying digital imaging technologies supported by machine learning algorithms.

Methods: H&E whole slide images, prepared from biopsies obtained mainly from lymph nodes, but also from extra-nodal organs of patients with aggressive B cell lymphoma histology, were collected from the pathology department at Tel-Aviv Medical center (TASMC). Cases were randomly divided into a training (n=43) and a validation (n=35) set. On-the-fly augmentation was applied to images, together with advanced Convolutional Neural Network (CNN) analysis, to generate the aggressive B-NHL classifier (powered by Imagene-AI). Proprietary multiple instance learning (MIL) algorithms and training scheme, based on cases ranking, rather than absolute values, were applied. The classifier was validated through a testing set in a blinded study scheme, and results were compared to the FISH results for c- MYC and BCL2 / 6 rearrangements (Figure 1A).

Results: The classifier training set was composed of 36 DLBCL NOS and 7 DHL cases. The aggressive B-NHL classifier was validated on a cohort of 35 cases, including 14 DHL cases (diagnosed by FISH) and 21 DLBCL cases. The model demonstrated 100% sensitivity and 90.48% specificity, with an accuracy rate of 94.29% and Area Under the Curve (AUC) of 0.99 (Figure 1B). Two cases were concluded as false positive, including one case that demonstrated rearrangements in substantial percentages of cells, just below the required positivity threshold.

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Summary/Conclusion: Herein, we demonstrate for the first time a machine learning-based genomic testing solution for the detection and classification of aggressive B-NHL, based on H&E stained slide images. Implementation of this solution in clinical practice can support the diagnosis of DHL, which currently has both technical and financial challenges. Applying the proposed accessible and standardized AI-based method, can improve and facilitate the diagnosis of DHL, thereby directly improving patient care.

P1165: BETALUTIN® IN PATIENTS WITH RELAPSED/ REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) NOT ELIGIBLE FOR AUTOLOGOUS STEM CELL TRANSPLANT

T. Illidge 1,* , M. Beasley 2 , J. G. Codina 3 , F. Cavallo 4 , V. Pascal 5 , V. Wills 6

1 Haematology/Oncoloy, The Christie NHS Foundation Trust, Withington; 2 Haematology/Oncoloy, Bristol Haematology and Oncology Centre, Bristol, United Kingdom; 3 Hospital Universitari i Politècnic La Fe, Hospital Universitari i Politècnic La Fe, Valencia, Valencia, Spain; 4 Division of Hematology, Department of Molecular Biotechnology and Health Sciences, University of Torino, University of Torino, A.O.U. Città della Salute e della Scienza di Torino, Turin, Italy; 5 Bioanalytics; 6 Clinical Sciences, Nordic Nanovector ASA, Oslo, Norway

Background: There remains a significant unmet medical need for the treatment of patients with relapsed/refractory DLBCL. Betalutin® (lilotomab labelled with the beta-emitter 177 Lu), targets the CD37 antigen which permits delivery of a therapeutic dose of radiation directly to the DNA of tumour cells. Betalutin® is under development for the treatment of relapsed/refractory Non-Hodgkin’s Lymphoma (NHL).

Aims: This was an open label Phase I, multi-centre, single ascending dose study of four different treatment regimens of Betalutin® administered after rituximab pre-treatment and lilotomab pre-dosing. The purpose of this study was to define the maximum tolerated dose (MTD) of Betalutin®.

Methods: Eligible participants received 375 mg/m 2 rituximab on Day -14 and escalating doses of both lilotomab (60 and 100 mg/m 2 ) and Betalutin® (10, 15 and 20 MBq/kg) on Day 0. Participants were followed-up for dose limiting toxicities (DLTs) for up to 12 weeks. Participants were followed-up until disease progression, the start of further anticancer treatment or until withdrawal from the study for any reason. Tumour response was determined using Cheson et al, 2014. Clinical benefit was assessed using the ECOG performance and quality of life (QoL) assessments using the Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) scale.

Results: A total of 16 participants (8 males and 8 females) with median age 75 years (range: 45 to 94 years) received lilotomab and Betalutin® after pre-treatment with rituximab. Overall ECOG scores at screening was 0 in 5 (31.3%), 1 in 7 (43.8%) and 2 in 4 (25.0%) participants. DLBCL stage at screening was Stage III or IV in 13 (81.3%). Median time since last relapse was 1.3 months, and 11 (68.8%) participants were refractory to the last anti-lymphoma therapy. Cell surface expression of CD37 on tumour cells was demonstrated for all patients before treatment.

A single case of DLT (grade 4 neutropenia) was reported in an 83-year-old female at the dose of 100 mg/m 2 lilotomab and 20 MBq/kg Betalutin® on day 43; there was no fever or infections associated with neutropenia; the event was treated with filgastrim and resolved after 2 weeks on day 57. A total of 14 (87.5%) participants had at least one treatment emergent adverse event (TEAE). Most events occurred between Days 1-92 and the most commonly reported events (in 4 [25.0%] participants each) were diarrhoea, decrease in lymphocyte count, and decrease in neutrophil count. Grade ≥3 TEAEs were reported in 10 (62.5%) participants, of which only four (25.0%) participants had treatment related events (decrease in lymphocyte count - 2 participants, decrease in neutrophil count - 1 participant and decrease in white blood cell count -1 participant). No SAEs assessed were deemed to be related to lilotomab and Betalutin®, by the investigators. Nine (56.3%) deaths were reported, all were determined to be related to disease progression and none was considered related to the study drugs.

Two participants (one each from the 2 highest dose cohorts) had complete responses that lasted 2.8 and 3.0 months, respectively.

Summary/Conclusion: Betalutin® is well tolerated in relapsed/ refractory DLBCL and a MTD was not reached in this study. The initial efficacy and safety findings from this study warrant further investigation of Betalutin® as a single agent or in combination with a standard of care treatment in relapsed/refractory DLBCL.

References: Cheson et al, J Clin Oncol 2014:32: 3059-67

P1166: REPEAT BIOPSY IN RELAPSED OR REFRACTORY DIFFUSE LARGE B CELL LYMPHOMA: A NATIONWIDE SURVEY AND RETROSPECTIVE STUDY

T. Berger 1,2,* , K. R. Geiger 1,2 , M. Yeshurun 1,2 , A. Gafter-Gvili 1,2,3 , T. Shochat 4 , R. Gurion 1,2 , P. Raanani 1,2 , O. Pasvolsky 1,2

1 Hematology division, Davidoff cancer center, Beilinson hospital, Rabin medical center, Petah Tikva; 2 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; 3 Department of Medicine A; 4 Bio-Statistical Unit, Rabin Medical Center, Beilinson Campus, Petah-Tikva, Israel

Background: Almost half of patients with diffuse large B-cell lymphoma (DLBCL) will have relapsed/refractory (R/R) disease after frontline immunochemotherapy. Although guidelines recommend histological confirmation of R/R disease, repeat biopsies are not always performed.

Aims: To better appreciate the extent and reasoning for not performing repeat biopsies in R/R DLBCL.

Methods: We conducted a two-part study: (1) using a nationwide electronic questionnaire consisting of 8 clinical vignettes, we evaluated the views of practicing hematologists in Israel regarding the need for repeat biopsies in suspected R/R DLBCL; (2) a single center retrospective study was utilized to describe real-life clinical practice patterns of performing procedures aimed at achieving tissue diagnosis for relapsed/refractory aggressive NHL (aNHL) patients: we included all consecutive adult (≥18 years of age) patients diagnosed with aNHL treated at a tertiary center in Israel, between 1/1/2013 and 1/1/2020, we identified patients who were diagnosed with R/R disease and analyzed data pertaining to utilization of histological confirmation via repeat biopsy. Each timepoint of management decision regarding biopsy performance was considered as a separate “episode of R/R lymphoma”. We extracted data regarding reasons for not performing the biopsies from the computerized system, according to the treating physician.

Results: In the survey part, all 64 participating physicians opted not to perform a repeat biopsy in at least one clinical case scenario. Physicians’ age, gender, tenure or clinical experience with DLBCL patients did not correlate with the decision to perform a biopsy, whereas more physicians chose to perform biopsies in relapsed compared to refractory disease cases.

In the retrospective part, 116 episodes of R/R aNHL among 61 patients were identified. In 72% of these episodes a repeat biopsy was not performed, mostly due to low likelihood of an alternative diagnosis or problematic location for biopsy. Patients with relapsed disease were more likely to undergo biopsy, as compared to those with refractory disease (47% vs 19%, p=0.002). Focusing on the group of patients with DLBCL (excluding primary CNS lymphoma, primary mediastinal B cell lymphoma and Burkitt lymphoma), there were 43 patients with refractory (n=26) or relapsed (n=17) DLBCL. In 61.4% out of the 83 episodes of R/R disease in these 43 patients, a biopsy was not performed: 74.5% of refractory DLBCL episodes versus 35.7% of relapsed DLBCL episodes (p=0.001). Stratifying biopsy utilization for each patient according to the timing of event, i.e first, second or third event of R/R disease, revealed that less biopsies were performed at more advanced relapse episodes. The most common reason for not performing a repeat biopsy was having stable radiological findings and other diagnoses deemed unlikely, as depicted in Figure 1.

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Summary/Conclusion: Our study suggests that contrary to guideline recommendations, in clinical practice many patients do not undergo repeat biopsy in R/R DLBCL, especially in refractory cases. Since this is a common dilemma faced in clinical practice, future studies and recommendations should address the necessity of repeat biopsy, according to patient and disease related characteristics.

P1168: CHARACTERISTICS PREDICTIVE OF PROGRESSION DURING FRONTLINE TREATMENT IN AGGRESSIVE B-CELL LYMPHOMAS

A. Bock 1,* , R. Mwangi 2 , M. Maurer 2 , J. Cerhan 3 , W. L. Ng 1 , R. King 4 , N. N. Bennani 1 , S. Ansell 1 , T. Habermann 1 , T. Witzig 1 , Y. Wang 1 , G. Nowakowski 1

1 Division of Hematology; 2 Division of Biostatistics; 3 Division of Epidemiology, Department of Quantitative Health Sciences; 4 Department of Pathology, Mayo Clinic, Rochester, United States of America

Background: Primary refractory diffuse large B-cell lymphoma (DLBCL) represents 10-15% of DLBCL cases. The subgroup of refractory patients who have no response or progressive disease (PD) by the end of frontline treatment (EOT) is a high-risk group with particularly poor outcomes. It is challenging to identify such patients at diagnosis.

Aims: To identify clinical, pathological, and cytogenetic characteristics of patients with progression during frontline treatment.

Methods: Adult patients with newly diagnosed DLBCL, including patients with high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (DHL/THL), between 2002 and 2019 and seen at Mayo Clinic Rochester were identified from the prospective Molecular Epidemiology Resource (MER) cohort study of the University of Iowa/Mayo Clinic Lymphoma SPORE. We analyzed patients who had no response or PD during treatment or by EOT (early PD group) and compared to non-early PD (non-ePD) patients (including relapsed patients). We compared demographics, clinical characteristics, cell of origin (COO), and MYC rearrangements using Chi-square analysis.

Results: Out of 1,505 patients with DLBCL, there were 66 patients (4.3%) with early PD (N=20 PD/stable disease at interim PET-CT, N=46 PD by EOT). At baseline, 61% were male and the median age was 60. Early PD patients had more advanced stage III-IV (72.7% vs 53.3%), elevated serum lactate dehydrogenase (LDH) (64.0% vs 47.3%), >1 extranodal site involved (36.4% vs 23.3%), and International Prognostic Index (IPI) ≥ 3 (52.0% vs 33.6%)(all p <0.05 compared to non-ePD). COO distribution was similar between early PD and non-ePD patients. Among patients with complete pathological and cytogenetic data available (early PD, n=47; non-ePD, n=813), DHL or THL was observed in 23.4% of early PD patients compared to 9.2% in non-ePD patients (p=0.001). Of the DHL patients in the early PD group (n=8), MYC/BCL2 rearrangement was present in 87.5%, MYC/BCL6 in 12.5%. Dual Myc/Bcl2 expressor (DEL) was present in 42.2% of early PD patients (n=45) compared to 25.5% in non-ePD patients (n=736)(p=0.013). First-line treatment regimens in the early PD group included R-CHOP (74.2%), R-EPOCH (7.6%), and other immunochemotherapy (IC)(18.2%), which was similar to non-ePD patients.

Summary/Conclusion: High-risk characteristics including DHL/THL and DEL are significantly associated with progressive disease during frontline treatment, suggesting that MYC-signaling may mediate chemoresistance. Known high-risk clinical features including advanced stage, elevated LDH, >1 extranodal site, and IPI ≥ 3 are also associated with early PD. Early identification of patients at high risk for early PD through clinical, pathological, and cytogenetic features is of utmost importance given the availability of clinical trials incorporating novel therapies in both frontline and consolidative approaches.

P1169: ELEVATED CRP AT INFUSION OF AXICABTAGENE CILOLEUCEL PREDICTS HIGH-GRADE CAR-T TOXICITY AND OUTPERFORMS MORE COMPLEX SCORES

S. Boyle 1,* , A. Kuhnl 1 , M. Correia de Farias 1 , P. E. Patten 1,2 , D. Yallop 1 , R. Benjamin 1 , V. Potter 1 , R. Sanderson 1

1 Dept of Haematology, King’s College Hospital; 2 King’s College London, London, United Kingdom

Background: Predicting CRS and ICANS prior to CAR-T infusion is desirable as it may help to plan management. Two groups 1,2 have recently shown that a modification of the Endothelial Activation and Stress Index (EASIX) score, developed to predict toxicity in bone marrow transplant recipients 3 , can predict CRS and ICANS severity in CAR-T patients, however these have not been externally validated or are difficult to apply in practice.

Aims: We aimed to determine whether the EASIX or related scores could predict CAR-T toxicity in our patients and if not, then to identify other potential predictors from the available clinical information.

Methods: We reviewed the medical records of adult patients who received axicabtagene ciloleucel (axi-cel) as standard of care at our centre over a 2yr period, collecting data on timing and severity of CRS and ICANS as well as baseline demographic and biochemical factors. We calculated an EASIX score for all patients and an ‘EASIX-F’ score based on the values provided by Greenbaum et al (EASIX-F1) and our own interquartile values (EASIX-F2). We then performed a linear regression of each of: the EASIX score; EASIX-F1; EASIX-F2; and other variables of interest with maximum grade of CRS, with a plan to perform a multivariate analysis with any significant variables (p<0.05).

Results: 67 patients were eligible for analysis. Baseline demographics were similar to those described on the licensing trial 4 . On univariate analysis (UVA), neither EASIX score (p=0.10), nor either iteration of the EASIX-F score (p= 0.08 and 0.15 respectively), was associated with severity of CRS for our patients. Pre-infusion C-Reactive Protein (D0 CRP) however appeared strongly associated with CRS severity, both on UVA (p=0.001) and by multiple linear regression, where it was the only variable identified that showed any association with CRS severity (p=0.003). Given the strength of the D0 CRP and CRS association, we evaluated the association of D0 CRP with ICANS severity which was significant (p=0.012) and D0 CRP with a combined outcome of ‘high grade CAR-T toxicity’ (HGT), defined as development of either grade 3+ CRS or grade 3+ ICANS, where it was again strongly associated (p<0.001). We then dichotomised D0 CRP to determine whether we could identify a practically useful cut-off to predict HGT. We looked at D0 CRP values of ≥50, ≥100 and ≥150 individually and all cut-offs performed well with increasing likelihood of HGT with higher CRP values. With D0 CRP ≥50, probability of developing HGT was 42% vs 13% (OR 5.09, p=0.007); with CRP ≥100 it was 55% vs 14% (OR 7.2, p=0.006); and with CRP ≥150 it was 100% vs 13% (OR undefined, p<0.001).

Finally, given these unexpected findings we performed a prospective review of the 19 axi-cel patients treated from our initial data-cut off to 1 Feb 2022 and found that for these patients, a D0 CRP ≥100 or ≥150 predicted HGT well. HGT incidence was 75% vs 7% (OR 42, Fisher’s p=0.016) for both cut-offs because 3 of the 4 patients with HGT had a CRP of >150 (Table 1). The single patient with HGT and D0 CRP <100 interestingly was a non-responder. For the full 86-patient cohort, D0 CRP ≥150 was the best cut-off, predicting a 90% incidence of HGT vs 12% if CRP <150 (OR 42, p<0.001).

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Summary/Conclusion: On retrospective analysis of 67 axi-cel patients, D0 CRP was more strongly associated with CRS severity than more complex prognostic scores. D0 CRP level, especially if ≥150, showed strong association with high grade CAR-T toxicity and remained predictive in a 19-patient validation cohort. This has potential implications for management of axi-cel patients.

P1170: INCIDENCE, TREATMENT AND OUTCOME OF PATIENTS WITH RICHTER’S SYNDROME: A POPULATION-BASED COHORT STUDY IN THE NETHERLANDS

F. Huisman 1 , D. Al-sarayfi 1 , M. Bellido 1 , R. Mous 2 , J. S. Vermaat 3 , A. P. Kater 4 , M. Nijland 5 , M. Brink 6,*

1 Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen; 2 Department of Hematology, UMC Utrecht Cancer Center, Utrecht; 3 Department of Hematology, Leiden University Medical Center, Leiden; 4 Department of Hematology, Academic Medical Centre, Amsterdam; 5 Department of Hematology, University Medical Center Groningen, Groningen; 6 Department of Research and Development, Netherlands Comprehensive Cancer Organisation, Utrecht, Netherlands

Background: Richter’s syndrome is a rare aggressive transformation of chronic lymphocytic leukaemia (CLL) into a diffuse large B-cell lymphoma (RS-DLBCL) or Hodgkin lymphoma. Patients with RS-DLBCL have an average overall survival (OS) of 6-12 months. Survival of RS-DLBCL patients previously treated for their CLL is inferior as compared to patients with a previously untreated CLL. In recent years, management of CLL has changed with the introduction of novel agents.

Aims: This study evaluates the clinical characteristics, treatment and survival of RS-DLBCL patients in a contemporary era of CLL management in the Netherlands.

Methods: DLBCL patients ≥18 years diagnosed in 2014-2020 were identified in the Netherlands Cancer Registry (NCR), with survival follow-up through February 1 st , 2021. A prior CLL, diagnosed in 2014-2019, was identified through cross-linkage with the NCR. For these patients, detailed information on CLL and RS-DLBCL treatment was available. Patients with CLL and DLBCL diagnosis within a time interval of 3 months were defined as concurrent RS-DLBCL (concurrent RS). The primary endpoint was overall survival (OS). OS was defined as the time between diagnosis and death from any cause and presented for three subgroups of RS-DLBCL patients, 1) concurrent RS, 2) RS-DLBCL following treated CLL (treated RS), and 3) RS-DLBCL following untreated CLL (untreated RS).

Results: A total of 110 patients with RS-DLBCL (median age 72 years, range 43-89 years; 61% males) were selected, including 22 (20%) concurrent RS, 38 (35%) treated RS, and 50 (45%) untreated RS. The majority of patients were diagnosed with stage III/IV RS-DLBCL, i.e., 55% of the concurrent RS were diagnosed with advanced disease as compared to 70% and 79% of the untreated and treated RS, respectively. Median time from CLL to RS diagnosis was for untreated RS 23.2 months (range, 3.4-74.4 months) and for treated RS 17.4 months (range, 4.7-67.5 months).

Of the 38 treated RS patients, 30 patients had received rituximab in combination with either chlorambucil (n=11), purine analogues (n=8) or chemotherapeutic regimens such as cyclophosphamide-vincristine-prednisolonie (n=11). Seven patients received treatment including ibrutinib or venetoclax, and 3 patients received treatment not including one or more of the abovementioned regimens.

Regarding primary treatment of all RS-DLBCL patients, 19 (86%) out of 22 concurrent RS patients received R-CHO(E)P, 2 patients received another R-based chemotherapy, and 1 patient did not receive therapy. Of the 88 (un)treated RS patients, 59 (67%) received R-CHO(E)P of whom 6 patients were consolidated with autologous stem cell transplantation (SCT) and 2 patients with allogenic SCT. Of the 29 remaining patients, 17 (19%) were treated with a R-based chemotherapy regimen including R-PECC and R-bendamustine, and 12 (14%) patients did not receive therapy.

The overall response rate (≥partial remission) of concurrent RS patients was superior as compared to untreated and treated RS patients (86% vs. 70% vs. 55%, respectively; p <0.01). With a median follow-up of 9.5 months (range 0.3-78.3), 3-years OS was 64% for patients with concurrent RS, 53% for untreated RS patients and 5% for treated RS patients (Figure; p <0.01).

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Summary/Conclusion: In this contemporary population-based study, concurrent RS patients have superior OS, while survival among treated RS patients is very poor. Our data underline the importance of development of new strategies, specifically aiming at treated RS patients.

P1171: REFLECT: PROSPECTIVE NON-INTERVENTIONAL STUDY ON THE EFFECTIVENESS AND SAFETY OF SANDOZ RITUXIMAB (SDZ-RTX) WITH CHOP FOR PATIENTS WITH PREVIOUSLY UNTREATED CD20-POSITIVE DIFFUSE LARGE B-CELL LYMPHOMA

M. Welslau 1,* , B. Kubuschok 2 , J. Topaly 3 , B. Otremba 4 , T. Wolff 5 , G. Bryn 6

1 Department of Oncology, Klinikum Aschaffenburg, Aschaffenburg; 2 Department of Hematology/Oncology, Augsburg University Medical Center, Augsburg; 3 Klinik für Hämatologie und Onkologie, CaritasKlinikum Saarbrücken, Saarbrücken; 4 Onkologische Praxis Oldenburg, Oldenburg; 5 OncoResearch Lerchenfeld, Hamburg; 6 Sandoz, Holzkirchen, Germany

Background: Sandoz biosimilar rituximab (SDZ-RTX) was approved in the EU for all the indications of reference rituximab. REFLECT is a real-world, multicenter, open-label, single-arm, non-interventional trial, and is the first prospective, post-approval study of SDZ-RTX in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) as a curative therapy for patients with CD20-positive diffuse large B-cell lymphoma (DLBCL).

Aims: The aim of the REFLECT study was to evaluate the 2-year effectiveness and safety of SDZ-RTX as first-line treatment for DLBCL.

Methods: Treatment-naïve, CD20-positive adult patients (aged ≥18 years) with DLBCL eligible for therapy with R-CHOP were treated with SDZ-RTX-CHOP every 2 or 3 weeks for 6–8 cycles according to the product label, as per routine clinical practice. Data was collected at enrollment, at every patient contact during the 12-month treatment period, and at one time point ≥30 days after the last dose of R-CHOP. Complete response was defined as the best result, as assessed by the treating physician using the Response Evaluation Criteria in Lymphoma 2017 (RECIL criteria). Kaplan–Meier estimates of progression free survival (PFS) were assessed at 24 months. No imputation for missing data was used; endpoints were summarized descriptively. All patients provided written informed consent prior to study entry. The cut-off for data collection was March 31, 2021.

Results: A total of 169 patients (52.1% female, mean [standard deviation] age 67.3 [13.4] years) with DLBCL were included in the full analysis set. At baseline, 19.5% and 24.3% of patients had Ann Arbor disease stage III or IV, respectively, and most patients (80.5%) had Eastern Cooperative Oncology Group performance score of 0 or 1. In terms of International Prognostic Index, 92 (54.5%) patients had a score of ≥2 at baseline. A total of 100 (59.2%) patients completed the 24-month observation period. In total, 110 (65.1%; 95% confidence interval [CI] 57.4–72.3) patients achieved complete response as the best response and 50 (29.6%; 95% CI 22.8–37.1) patients achieved partial response. The overall response rate was 94.7% (95% CI 90.1–97.5). One-year PFS was 84.9% (95% CI 78.2–89.6), while 2-year PFS was 78.5% (95% CI 70.9–84.4) (Figure 1); median PFS was not reached within the observational period. A total of 143 (84.6%) patients experienced at least one adverse event (AE), 53 (31.4%) of which were suspected to be related to the study drug. The most common AEs were anemia (24.3%), fatigue (20.7%), polyneuropathy (17.2%), and nausea (12.4%). Serious AEs were reported in 63 (37.3%) patients, with those suspected to be related to the study drug reported in 11 (6.5%) patients. AEs requiring dose interruption and/or dose change were reported in 24 (14.2%) patients. There were 8 (4.7%) deaths recorded during the study, of which 3 (1.8%) occurred during the on-treatment period.

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Summary/Conclusion: This real-world, post-approval study reconfirms that first-line treatment of CD20-positive DLBCL with R-CHOP using SDZ-RTX is effective over a 24-month observation period, with no unexpected safety signals. These data may help to broaden patient access to rituximab-based chemotherapy and support the sustainability of cancer care.

P1172: DUVELISIB IN PATIENTS WITH RELAPSED/REFRACTORY PERIPHERAL T-CELL LYMPHOMA FROM THE PHASE 2 PRIMO TRIAL: UPDATED EXPANSION PHASE ANALYSIS

P. L. Zinzani, MD, PhD, J. Zain, MD 2 , M. Mead, BS, MD, C. Casulo, MD 4 , E. D. Jacobsen, MD 5 , G. Gritti, MD, PhD 6 , L. Pinter-Brown, MD 7 , K. Isutzu, MD 8 , D. Cohan, MD 9 , M. Daugherty, PharmD 10 , J. E. Brammer, MD 11 , N. Mehta-Shah, MD 12 , B. Pro, MD 13 , S. M. Horwitz, MD 14

1 Institute of Hematology “Seràgnoli”, University of Bologna, Bologna, Italy; 2 Department of Hematology and Hematopoietic Cell Transplantation, City of Hope Comprehensive Cancer Center, Duarte; 3 Department of Medicine, Division of Hematology/Oncology, UCLA Medical Center, Santa Monica; 4 University of Rochester Medical Center, James P. Wilmot Cancer Center, Rochester; 5 Dana-Farber Cancer Institute, Boston, United States of America; 6 Hematology and Bone Marrow Transplant Unit, Ospedale Papa Giovanni XXIII, Bergamo, Italy; 7 University of California-Irvine, Irvine, United States of America; 8 National Cancer Center Hospital, Tokyo, Japan; 9 Secura Bio, Inc.; 10 SecuraBio, Inc, Las Vegas; 11 Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus; 12 Washington University School of Medicine in St. Louis, Saint Louis; 13 Columbia University Herbert Irving Comprehensive Cancer Center; 14 Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Peripheral T-cell lymphoma (PTCL) is a family of aggressive lymphomas, with a short median overall survival when relapsed or refractory (R/R). Current single-agent therapies for R/R PTCL have modest overall response rates (ORR) of <30%. Use of duvelisib (DUV), an oral dual inhibitor of phosphatidylinositol 3-kinase (PI3K)-δ and PI3K-γ isoforms, in PTCL is as an investigational agent only. Both Phase 1 data and prior interim analyses of this Phase 2, open-label, multi-center, parallel cohort PRIMO Trial ( NCT03372057 ; supported by Secura Bio) demonstrated promising efficacy (ORR ~ 50%) of DUV in patients (pts) with R/R PTCL. Based on interim PRIMO data, DUV was added to the National Comprehensive Cancer Network® T-cell Lymphoma Guidelines® (Version 1.2022, 12/22/21) as a Category 2A other recommended regimen for pts with R/R PTCL of all subtypes. Here we present the latest PRIMO Expansion Phase (EP) data.

Aims: The primary objective of the PRIMO EP is to determine the efficacy of DUV given at a recommended dose in pts with R/R PTCL; secondary objectives include additional efficacy measures, safety, and pharmacokinetics.

Methods: EP eligibility criteria includes adults with pathologically confirmed PTCL, defined per WHO criteria, after ≥ 2 cycles of 1 prior standard regimen, and a CD4 lymphocyte count of ≥ 50/mm 3 . Based on the dose optimization results, the dose in the EP is DUV at 75 mg BID for 2 cycles, to maximize disease control, followed by 25 mg BID, to mitigate late toxicities, until progressive disease (PD) or unacceptable toxicity. Pneumocystis jirovecii prophylaxis is required; herpes simplex and varicella zoster virus prophylaxis are strongly recommended. The primary endpoint is ORR by IRC assessment using the Lugano 2014 criteria; efficacy is assessed in all pts that received at least 1 dose of DUV.

Results: This updated analysis of the EP included 101 pts (data cutoff Oct 1, 2021); median 10.4 months follow-up from first dose. Pts had a median age of 67.0 years (range, 21-92) with a median of 3 prior lines of therapy (range 1-9). Fourteen pts remain on treatment with 87 pts withdrawn from treatment for: PD (44), clinical deterioration due to PD (4), adverse events (AE; 20), death (5), other (13), and withdrawal of consent (1). The ORR by IRC was 49% with a CR rate of 34%; responses were seen across multiple subtypes (Table 1 Median PFS was 3.6 months, though not yet fully mature. Five pts discontinued therapy to undergo stem cell transplantation. There were 3 treatment-related AEs associated with death (1 each): pneumonitis, Epstein-Barr associated lymphoproliferative disorder, and sepsis. AEs of special interest ≥ Grade 3 (all causality, number of events) were transaminase elevation increased (23), neutropenia (19), infections (10), cutaneous reactions (9), diarrhea (7), pneumonia (2), and pneumonitis (1). There was 1 event of colitis (Grade 1). ALT and/or AST elevations were the most common AEs leading to treatment discontinuations (15).

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Summary/Conclusion: This updated analysis of 101 patients in the PRIMO EP demonstrated an ORR of 49% and a CR rate of 34%, which compares favorably to currently available single-agent options. Duvelisib was generally manageable with per-protocol dose modifications in this population, showing an AE profile consistent with what has been observed previously and no unexpected or novel toxicities. These data confirm duvelisib to be a promising agent for a disease set with high unmet needs, poor prognoses, and limited effective treatment options.

P1173: CLINICAL APPLICABILITY OF TARGETED NEXT-GENERATION SEQUENCING FOR PRECISION MEDICINE IN DIFFUSE LARGE B CELL LYMPHOMA

D. Huang 1,* , J. Li 1 , T. Yang 1 , Y. Nan 1 , L. Zhang 1 , Y. Xiang 1 , Q. Li 1 , Y. Liu 1

1 Department of Hematology-Oncology, Chongqing University Cancer Hospital, Chongqing, China

Background: The cure rate of diffuse large B-cell lymphoma is only 60-70%, and new treatment methods need to be found.

Aims: Comprehensive genomic analysis based on next-generation sequencing (NGS) allows a better molecular characterization of diffuse large B-cell lymphoma, offering a roadmap toward precision medicine.

Methods: We analyzed somatic alterations of 37 DLBCL tissue or blood samples by a hybridization capture-based NGS panel targeting 188 lymphoma-related genes.

Results: Totally, 279 single-nucleotide variants and small insertions or deletions affecting 103 genes were identified, with at least 1 mutation detected in 100% (37/37) of cases. The most frequently mutated genes were those involved in epigenetic modifiers ( DNMT3A , KMT2B and KMT2D ), activated signaling ( TYK2 , MYD88 , NOTCH2 and BCR ) and transcription factors/transcriptional regulator ( RUNX1 , SPEN , IRF4 , KLF2 and ASXL1 ). Significant co-occurrences were shown between RUNX1 mutations with NOTCH2 , IRF4 , ARID1A and BCR ( P <0.05, respectively), BCR and IRF4 ( P <0.01), etc . Additionally, we analyzed the association of genomic aberrations with clinicopathological features in DLBCL. Patients aged >60 years harbored less LRP1B mutations than those aged ≤60 years (5.3% vs. 33.3%, P < 0.05). The mutation frequency of TP53 was significantly higher in LDH-high (>244 U/L) group than that in LDH-low (≤244 U/L) group (40% vs. 6.25%, P <0.05). Compared with germinal center B-cell (GCB), the mutation frequency of TYK was significantly lower in non-GCB (5% vs. 36.4%, P <0.05). New potential therapeutic targets ( CREBBP , EP300 , EZH2 , SMARCB1 , CD79A/B , BTK , CARD11 , MYD88 , THFAIP3, PIK3CA, PTEN and JAK ) were identified in 55% (11/20) of newly diagnosed patients and in 41.2% (7/17) of relapsed/refractory patients. Ibrutinib, as a single agent, has demonstrated limited activity in DLBCL with mutant MYD88 and wildtype CD79 . In this study, we identified 3 patients with mutant MYD88 and wildtype CD79A/B (8.1%), among whom 2 harbored MYD88 L265P variants and 1 had MYD88 S219C mutation.

Summary/Conclusion: The utility of mutational profiling may facilitate the identification of potential drug targets, which in turns may represent new therapeutic possibilities.

P1174: THE COMBINATION OF QUALITATIVE AND QUANTITATIVE METABOLIC PARAMETERS PREDICTS PROGRESSION-FREE SURVIVAL IN PATIENTS WITH LARGE B-CELL LYMPHOMAS (LBCL) TREATED WITH ANTI-CD19 CAR-T CELLS.

A. Dodero 1,* , A. Guidetti 1 , A. Lorenzoni 2 , S. Pizzamiglio 3 , G. Argiroffi 2 , P. Verderio 3 , A. Chiappella 1 , F. Bagnoli 4 , C. Carniti 1 , M. Pennisi 1 , A. Alessi 2 , P. Corradini 1

1 Hematology; 2 Nuclear Medicine; 3 Unit of Bioinformatics and Biostatistics, Fondazione IRCCS Istituto Nazionale dei Tumori; 4 Hematology, Policlinico di Milano, Ospedale Maggiore Ca’ Granda, Milano, Italy

Background: Autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is an effective therapy for 40% of patients (pts) with relapsed/refractory (R/R) LBCL. Qualitative and quantitative metabolic parameters from the 18-FDG PET-CT scan are tools able to predict outcomes in lymphomas, but their value in the CAR-T setting is still under investigation.

Aims: Prognostic role of PET-CT performed before lymphodepletion and 1 month after CAR-T cell infusion, analyzed by qualitative and quantitative parameters

Methods: We conducted an observational a prospective study on pts affected by R/R aggressive lymphomas receiving axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). PET-CT was evaluated before lymphodepletion, at 1 month (M1) and at 3 months (M3) post-infusion using visual assessment with a 5-point Deauville score (DS). Quantitative PET parameters were evaluated with PET-VCAR software (GE Healthcare). The contour of pathological lesions was delineated using semiautomatic contouring systems including a gradient-based algorithm. SUVmax, SUVmean, Metabolic tumor volume (MTV), and Total lesions glycolysis (TLG) were automatically recorded. Total MTV (TMTV) and total TLG (TTLG) were calculated as the sum of all individual lesions. The percentage change in FDG uptake of each parameter between baseline and M1 was defined as Δ=(baseline-M1)/baseline x 100%. Logistic and Cox regression models were applied to evaluate the association between PET parameters at baseline, at M1 and in terms of Δ with respect to clinical response and progression free survival (PFS), respectively.

Results: 47 pts were analyzed: median age was 55 years (range, 22-70); histologies included diffuse-large B-cell lymphomas (n=33) and primary mediastinal B-cell lymphomas (n=14); pts received treatment with axi-cel (n=31) or tisa-cel (n=16). Before infusion, 18 (38%) had high LDH, 14 (30%) had extranodal disease and 16 (34%) had bulky disease. Majority of pts [n=41 (84%)] received bridging therapy. At a median follow-up of 12 months (range, 3-29 months), 38 patients are alive and 9 died of disease progression with an estimated 1-year PFS and OS of 46% (95%CI; 30%-60%) and 83% (95%CI; 66%-92%), respectively. Baseline values of SUVmax, but not SUVmean, TTLG, TMTV correlated with poor clinical response [HR 1,49; (95%CI; 1,01-2,2), p=0,04]. By univariate logistic model, a higher ΔSUVmean between baseline and M1 was associated with better response at M3 [odds ratio 3,49, (95%CI; 1,25-9,77), p=0.02]. Univariable Cox analyses showed that a higher ΔSUVmean and ΔTMTV between baseline and M1 were associated with a better PFS [HR 0,63, (95%CI; 0,41-0,96), p=0,03; HR 0,67 (95%CI; 0,48-0,94), p=0,02], respectively. In terms of visual assessment, pts with DS 4-5 at M1 had a significantly higher risk of failure as compared to those with DS 1-3 [HR 2,92, (95%CI; 1,12-7,64), p=0,02]. A combination of M1 DS score and ΔSUV mean showed a better prognosis for patients with DS1-3 (PFS 60%), an intermediate prognosis for those with DS4/5 and high ΔSUVmean (PFS 49%) and a poor prognosis for those with DS4/5 and low ΔSUVmean (PFS 22%), [p=0,009]. In a multivariable analyses, evaluating clinical prognostic factors and PET parameters, a higher risk of failure was observed in pts with high LDH [HR 3,2 (95%CI; 1,30-8,14), p=0,01] whereas a higher ΔSUVmean was associated to a better PFS [HR 0,61 (95%CI, 0,41-0,92), p=0,02].

Summary/Conclusion: Assessment of DS combined with variation of SUVmean at M1 provides informations about early risk of CAR-T failure. A validation group are needed to extend and confirm our observations.

P1175: REAL LIFE OUTCOMES OF RELAPSED/REFRACTORY DIFFUSE LARGE B CELL LYMPHOMA: A SINGLE CENTRE EXPERIENCE

I. Dogliotti 1,* , M. Clerico 1 , F. Vassallo 2 , S. Ragaini 1 , V. Peri 1 , B. Botto 2 , C. Consoli 1 , L. Orsucci 2 , R. Freilone 2 , S. Ferrero 1 , A. Lonardo 1 , G. De Luca 1 , D. Ferrero 1 , F. Cavallo 1

1 Division of Hematology 1U, Department of Biotechnology and Health Sciences, University of Torino; 2 Division of Hematology 2, University Hospital AOU Città della Salute e della Scienza, Torino, Italy

Background: Nearly 40% diffuse large B cell lymphoma (DLBCL) patients relapse after first line treatment (Crump, Blood 2017) and around 15% are chemorefractory (Feugier, JCO 2005). Despite modern treatments availability, outcome of these patients remains suboptimal (Crump, Blood 2017).

Aims: With the observational retrospective study STRIDER, we aimed at determining real-life outcome of relapsed/refractory (R/R) DLBCL patients.

Methods: Patients were selected based on local diagnosis of DLBCL between Jan 2010 and Dec 2019 within Hematology Units at the University Hospital Città della Salute e della Scienza, Torino, Italy. R/R disease was documented by biopsy, imaging, or clinical evaluation; refractoriness was defined as reappearance or progression of DLBCL within 12 months from initial diagnosis. Demographics, laboratory, imaging, pathological and clinical data were collected retrospectively from hospital health records.

Results: Overall, 413 DLBCL patients aged >18 years old were included. 151 (36%) patients had an event of relapse (n=51 patients, 12%) or refractory disease (n=100, 24%). Median age was 67 (range 21-89) years for non R/R patients (NRR) vs 72 (19-89) for R/R group (p <0.001). Advanced stage (stage 3-4: 68% vs 87%, p <0.001), baseline B symptoms (28 vs 43%, p <0.001), higher LDH level (p <0.001) and >1 extranodal site involvement (39 vs 53%, p =0.007) were linked to R/R disease, and consequently, IPI score highly affected R/R disease distribution. Kidney or adrenal involvement was less frequent in the NRR group (6% vs 11%, p =0.042). Central nervous system (CNS) involvement was present at diagnosis in only 8/413 (2%) patients, while median CNS-IPI score was 2 vs 3 for the 2 groups (p <0.001). Expression of immunohistochemical markers was not significantly different between groups. Treatment intent was curative (R-CHOP/COMP, R-Magrath, R-DA-EPOCH, MATRIX) for 391/413 (95%) patients; R/R group included reduced dose or palliative intent treatments (11 vs 2%, p <0.001). For CNS prophylaxis, intrathecal methotrexate was used in 78/413 (19%) patients, while intravenous in 22 (5%) patients. After first line completion, 99/413 (24%) patients received consolidative radiotherapy on previous bulky areas, while 4 (1%) received upfront autologous stem cell transplantation (ASCT). Interim response was not different, but end of treatment response was inferior for R/R group (p <0.001). After a median follow up of 50 months, median overall survival (OS) from initial diagnosis was NR for NRR patients, 38 months (IQR 25-64) for relapsed, 11 (5-20) for refractory patients (Figure 1). Median progression free survival (PFS) was 21 (IQR 16-30) months for relapsed vs 6 (4-9) for refractory patients. Treatments performed in II line were platinum-based (rituximab + OxDHA/DHAP/ICE/GemOx) for 50 patients (22 relapsed, 28 refractory), lenalidomide for 17 patients (5 vs 12); 14 patients were enrolled in clinical trials. Palliative intent treatments (oral chemotherapy +/- rituximab) were administered to 17 patients. II line consolidation with RT was performed in 11, ASCT consolidation in 6 patients. After II line, best response was CR in 29%, PR in 17% (ORR 45%); 45 patients received a third line, 10 more than three.

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Summary/Conclusion: Our retrospective series confirmed good survival for NRR patients, but 36% patients were R/R after first line, with refractory patients showing a dismal outcome despite intensive treatments. Survival analyses after II line and multivariate analysis are ongoing and will be presented at the meeting.

P1176: RITUXIMAB IN PRIMARY CNS LYMPHOMA – LONG TERM FOLLOW-UP OF THE PHASE III HOVON 105/ALLG NHL 24 STUDY

J. Doorduijn 1,* , S. Issa 2 , B. van der Holt 3 , M. Minnema 4 , T. Seute 5 , M. Durian 6 , G. Cull 7 , M. van der Poel 8 , W. Stevens 9 , J. Zijlstra 10 , M. Nijland 11 , K. Mason 12 , A. Beeker 13 , D. Brandsma 14 , M. van den Bent 15 , M. Gonzalez 16 , D. de Jong 17 , J. Bromberg 15

1 Hematology, Erasmus MC Cancer Institute Rotterdam, Rotterdam, Netherlands; 2 Hematology, Middlemore Hospital, Auckland, New Zealand; 3 HOVON Data Center, Erasmus MC, Rotterdam; 4 Hematology; 5 neurology, University Medical Center Utrecht, Utrecht; 6 Hematology, ETZ Hospital, Tilburg, Netherlands; 7 Hematology, Sir Charles Gairdner Hospital and PathWest Laboratory Medicine, Nedlands, Australia; 8 Hematology, University Medical Center, Maastricht; 9 Hematology, Radboud University Medical Center, Nijmegen; 10 Hematology, Amsterdam UMC, Amsterdam; 11 Hematology, University Medical Center Groningen, Groningen, Netherlands; 12 Hematology, Royal Melbourne Hospital, Melbourne, Australia; 13 Hematology, Spaarne Gasthuis, Haarlem; 14 neurology, Netherlands Cancer Institute, Amsterdam; 15 neurology, Erasmus MC Cancer Institute Rotterdam, Rotterdam, Netherlands; 16 pathology, Royal Melbourne Hospital, Melbourne, Australia; 17 pathology, Amsterdam UMC, Amsterdam, Netherlands

Background: The efficacy of rituximab in Primary CNS Lymphoma (PCNSL) is still under debate. We performed an international randomized phase III study to investigate the efficacy of rituximab when added to methotrexate, BCNU, teniposide and prednisolone (MBVP) in PCNSL. The primary endpoint, event-free survival (EFS) at one year, was similar in both treatment groups and was previously reported (Bromberg et al, Lancet Oncology 2019; 20: 216-228).

Aims: Here we present long-term follow up results with a median follow-up of 82 months.

Methods: Between August 2010 and May 2016 200 newly-diagnosed, non-immunocompromised patients with PCNSL aged 18-70 years and WHO performance status 0-3 were randomized between treatment with MBVP chemotherapy with (arm B) or without (arm A) rituximab. The rituximab was given weekly in the first MBVP cycle, fortnightly in the second (in total 6 rituximab administrations). Responsive patients received consolidation with high-dose cytarabine, and patients aged ≤ 60 were subsequently treated with low-dose WBRT if in CR/CRu; in case of PR with an additional boost on the tumor. Patients > 60 were not irradiated. All patients gave written informed consent.

Results: The modified intention-to-treat (m-ITT) population consisted of 199 eligible patients, 55% were men. The median age was 61 yrs (range 26-70), the median WHO performance status 1 (range 0-3). The primary endpoint EFS at one year was 49% (95% CI 39-58)(MBVP) vs 55% (95% CI 44-64) (R-MBVP). The EFS at 5 years was 25% (17-34) vs 36% (27-46) respectively, hazard ratio (HR) 0.85, 95% CI 0.61-1.18, p=0.33 (adjusted for age and WHO performance status). The progression-free survival (PFS) at one and 5 years were 58% (47-67) and 29% (21-39) (MBVP) and 65% (54-73) and 43% (33-53) (R-MBVP)) (HR 0.73, 95% CI 0.52-1.02, p=0.07).

80 patients were still alive. Overall survival (OS) at 5 years for MBVP and R-MBVP was 49% (39-59) and 53% (43-63) respectively. The median OS was 57 months (95% CI 38-75) in the MBVP arm and 85 months (95% CI 43–104) in the R-MBVP arm (HR 0.87, 95% CI = 0.61-1.26, p=0.47). A total of 111 patients had progression or relapse, 63 after MBVP and 48 after R-MBVP. 79% of these patients received further treatment. The median OS after progression/relapse was 9.7 months (5.9-19.9) in the MBVP arm, and 6.1 months (2.4-13.1) in the R-MBVP arm (HR 1.25, 95% CI 0.83-1.87, p=0.29).

119 patients died, 64 in the MBVP arm and 55 in the R-MBVP arm. Causes of death were PCNSL in 69% of the patients (both arms), complication of treatment (6% vs 5%), secondary malignancy (5% vs 2%) and other or unknown causes (20% vs 24%). Age was the strongest prognostic factor for EFS, PFS and OS in multivariate analysis.

An unplanned subgroup analysis by age ( ≦ 60 vs > 60 yrs) showed a significant different EFS at one year for younger patients with R-MBVP, not found in patients > 60 yrs (Bromberg et al, 2019). With longer follow up the 5-year EFS in the younger group was 28% (16-41)(MBVP) vs 55% (40-68) (R-MBVP), HR 0.48, 95% CI 0.28-0.81, p=0.006. 5-year PFS was 31% (18-44) versus 57% (42-70), HR 0.47, 95% CI 0.27-0.81, p=0.007. The OS at 5 years was 54% (39-68) vs 70% (55-81) respectively (HR 0.65, 95% CI 0.36-1.19, p=0.16).

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Summary/Conclusion: In the modified-ITT population we found no statistically significant benefit of the addition of rituximab to MBVP on EFS, PFS and OS in patients with PCNSL, even after a long follow-up of median 82 months. Therefore, the results of this study do not support the use of rituximab with MBVP in the treatment of primary CNS lymphoma.

P1177: PROGNOSTIC VALUE OF TP53 VARIANTS IN CHILDREN WITH MATURE B-CELL LYMPHOMA TREATED ACCORDING TO REDUCED-INTENSITY B-NHL-2010M PROTOCOL

V. Egor 1,* , A. Yuliya 1 , K. Brenning 1 , A. Itov 1 , D. Abramov 2 , A. Sharlai 2 , M. Senchenko 2 , D. Konovalov 2 , G. Novichkova 1 , N. Miakova 1

1 Oncohematology; 2 Pathology, Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology, Moscow, Russia

Background: Reduced-intensity rituximab-based therapy leads to long-term remission in more than 80-85% of pediatric patients with B-cell non-Hodgkin lymphoma (B-NHL), but in case of relapse or refractoriness the prognosis in poor with less then 20% survival rate. Therefore, the identification of adverse prognostic factors is critically important. Recent data show that the status of the TP53 gene may be of prognostic value in children with B-NHL. We evaluated the clinical significance of TP53 abnormalities in children with B-NHL enrolled in the Russian-Belorussian B-NHL-2010mab multicenter clinical trial.

Aims: To investigate the prognostic value of TP53 gene status in pediatric B-NHL receiving reduced-intensity therapy

Methods: Due to reference status of pathology department in Dmitry Rogachev National Medical Research Center for Pediatric Hematology, Oncology, and Immunology, samples from 79 pediatric B-NHL patients (77 with Burkitt lymphoma and 2 with diffuse large B-cell lymphoma) treated according to B-NHL-2010mab protocol in 2012-2021 were included in this study. The median age was 9 years (range 2-17 years), m:f ratio was 6:1. 19 patients (24%) had limited stage and 60 patients (76%) had advanced-stage of disease. DNA was extracted from FFPE tissue, TP53 variants were detected using Sanger sequencing of exons 5 to 8 according to the International Agency for Research on Cancer (IARC) protocol. B-NHL samples were obtained following informed consent from participants or their parents/guardians.

Results: Median follow up was 16 months (range 5-117 months). The 5-year event free and overall survival for all patients was 86,3% (95%CI=78, 3-94%) and 86,5% (95%CI=78,3-94,8%) respectively. DNA suitable for sequencing was obtained from 66 samples. TP53 pathogenic variants were present in 26% of cases and were associated with a significantly inferior event free and overall survival compared to those without a TP53 abnormality: 45,3% (95%CI = 21-69,6%) vs 97,9% (95%CI=93,9-100%) (p < 0.001) and 47,1% (95%CI=21,8-72,4%) vs 97,9% (95%CI= 93,9-100%) respectively (p = 0.001). 13 patients with unknown TP53 status are alive without relapsed of disease.

Summary/Conclusion: Our findings show the independed prognostic value of TP53 pathogenic variants in pediatric B-NHL patients treated with rituximab-based reduced-intensity therapy. Therefore, given the poor survival rate of patients with TP53 mutations, this marker can be used to define high risk groups within pediatric B-NHL, those who need new therapeutic approaches to achieve better results. It is necessary to study this prognostic factor in large groups of patients.

P1178: BRENTUXIMAB VEDOTIN IN COMBINATION WITH LENALIDOMIDE AND RITUXIMAB IN PATIENTS WITH RELAPSED/REFRACTORY DLBCL: SAFETY AND EFFICACY RESULTS FROM THE SAFETY RUN-IN PERIOD OF THE PHASE 3 ECHELON-3 STUDY

N. L. Bartlett 1,* , C. A. Yasenchak 2 , K. K. Ashraf 3 , W. N. Harwin 4 , J. Orcutt 5 , P. Kuriakose 6 , P. L. Zinzani 7,8 , A. Mamidipalli 9 , K. Fenton 9 , C. Glenn 9 , G. Nowakowski 10

1 Washington University School of Medicine, St. Louis, MO; 2 Willamette Valley Cancer Institute and Research Center/US Oncology Research, Eugene, OR; 3 Hem-Onc Assocs, Birmingham, AL; 4 Florida Cancer Specialists and Research Institute, Fort Myers, FL; 5 Charleston Hematology Oncology Associates, Charleston, SC; 6 Henry Ford Health System, Detroit, MI, United States of America; 7 Institute of Hematology “L. e A. Seràgnoli”, University of Bologna; 8 Department of Specialized Medicine, Diagnostic and Experimental, University of Bologna, Bologna, Italy; 9 Seagen Inc., Bothell, WA; 10 Division of Hematology, Mayo Clinic, Rochester, MN, United States of America

Background: Patients (pts) with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) who relapse after or are ineligible for hematopoietic stem cell transplant (HSCT) or chimeric antigen receptor T cell (CAR-T) therapy generally have poor outcomes. Preclinical data provide the rationale for combining brentuximab vedotin (BV), lenalidomide (len), and rituximab for the treatment of R/R DLBCL. BV+len showed promising clinical activity in a Phase 1 trial with a 57% overall response rate (ORR), 10.2-month median progression-free survival, and 14.3-month overall survival (Ward 2021).

Aims: We report the results of the open-label safety run-in that was conducted prior to the randomized portion of the study.

Methods: ECHELON-3 ( NCT04404283 ) is a randomized, double-blind, placebo-controlled, active-comparator, multicenter Phase 3 study. Prior to randomization, an open-label safety run-in was conducted; pts received BV (1.2 mg/kg) and rituximab (375 mg/m 2 ) both q3w, and len 20 mg qd. Pts must have received ≥2 prior lines of therapy and be previously treated with or were ineligible for HSCT or CAR-T, ECOG score ≤2, and PET-avid, bidimensional measurable disease (>1.5 cm by CT). Pts with negligible CD30 expression (<1%) were eligible. Response was assessed by the investigator according to the Lugano Classification Revised Staging System (Cheson 2014).

Results: 10 pts with R/R DLBCL were enrolled. Median age was 70.5 years, 7 pts were male, and all had an ECOG ≤1. Median prior lines of therapy was 3 (range, 2 to 6); no pts received prior HSCT and 6 pts received prior CAR-T.

At a median follow-up of 6.9 months (range, 2.3 to 14.1), the most common treatment emergent adverse events (TEAE) were fatigue (n=5), anemia (n=4) and constipation (n=4). Grade ≥3 events were experienced by 8 pts, most commonly anemia and pneumonia (n=3 each), and neutropenia and thrombocytopenia (n=2 each). Serious adverse events were observed in 7 pts. Seven pts each had BV and rituximab dose modifications, and 6 pts had a len dose modification. The most common reasons for any dose modification were anemia (n=3), neutropenia, peripheral neuropathy, or pneumonia (n=2 each). 4 pts discontinued treatment due to an AE, 2 of which were treatment related (n=1 each of Grade 2 fatigue; Grade 3 anemia). There was 1 death due to a TEAE that was not treatment related.

The ORR (best response) was 70%, including 5 pts with a complete metabolic response and 2 pts with a partial metabolic response; 3 pts had progressive disease. Responses were seen in both CD30 (+) and (-) pts, as well as in 4 pts who received prior CAR-T.

Summary/Conclusion: This novel triplet regimen appears active in R/R DLBCL with an acceptable safety profile. The randomized portion of the study is currently enrolling.

P1179: EFFECTIVENESS OF CENTRAL NERVOUS SYSTEM PROPHYLAXIS STRATEGIES IN DIFFUSE LARGE B-CELL LYMPHOMA

F. Z. Eraslan 1 , T. Toptas 2,* , T. Tuglular 2 , I. Atagunduz 2

1 Internal Medicine; 2 Hematology, Marmara University School of Medicine Hospital, İSTANBUL, Turkey

Background: Central nervous system recurrence is seen in 5% of patients diagnosed with DLBCL. CNS relapse is associated with survival limited to months. Therefore, determining the patients who are at high risk of CNS relapse and selecting the most efficient prophylactic approach is one of the mainstays of treatment. IV-MTX and IT-MTX are widely used in CNS prophylaxis. Nevertheless, the efficiency of IV-MTX and IT-MTX in preventing CNS relapse was not proven in large study groups or in prospective studies.

Aims: Our study aims to verify the efficiency of current CNS prophylaxis in preventing CNS relapse in DLBCL patients. Additionally, association of CNS relapse with survival and the correlation of CNS-IPI score, age, albumin, origin of cell, and DHL/DEL status with cumulative CNS relapse incidence.

Methods:

The data from 190 patients were analyzed retrospectively. Among the 89 patients who had been classified as high-risk group for CNS relapse, seven were administered IT-MTX, 17 received IV-MTX, and remaining 65 patients received no prophylaxis.

Results: Median age was 57(18-88) and male to female ratio was almost equal (male: 48.8%). At the time of diagnosis, 66.8% of the patients were defined to have advanced stage disease. All of the cohort included 89 patients (46.8%) who were at high risk for CNS relapse. Cumulative CNS relapse risk at one-year was estimated as 12.2%, 9.5%, 4.5% for patients received IV-MTX, IT-MTX and no prophylactic treatment, respectively. Two-year cumulative CNS relapse risk was 24.5%, 12.2%, 6.0% for patients who received IV-MTX, IT-MTX and no prophylaxis, respectively (p=0.008). The frequency of DEL or DHL were higher in patients who received IV-MTX (41.1%). CNS relapse rate was 25% and 50% for patients who did not receive prophylaxis and who did, respectively. In patients with DEL, CNS relapse rate (33.3%) did not differ for those who received prophylaxis and those who did not. In our study, we found overall CNS relapse incidence 6.8% in DLBCL. Median time to CNS relapse was 12 months in all cohort. Median overall survival after occurrence of CNS relapse was two (95% CI: 0-4.9) months. We found that the patients who had high CNS-IPI score were at six times greater risk of CNS relapse than those with low and intermediate CNS-IPI score (9.0% and 1.5% after 12 months, 11.6% and 2% after 24 months) (p=0.03). Despite the univariate analyses revealed a statistically significant association, multivariate analysis has shown that albumin level is not an independent predictor for CNS relapse. Additionally, we have shown that CNS-IPI score, DHL/DEL status are strong predictors of CNS relapse in DLBCL patients.

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Summary/Conclusion: Independent predictors of CNS relapse in DLBCL are DHL/DEL status and CNS-IPI score. No predictive relationship was proven to exist between CNS relapse and gender, albumin levels, cell of origin classification by Hans-algorithm. Patients with CNS relapse had poor prognosis (median survival two months). IV-MTX prophylaxis has been shown to have no preventive effect on CNS relapse in high risk DLBCL population. Due to this data, significant changes in prophylactic approach against CNS relapse in high risk DLBCL patients is required.

P1180: ARE EXTRANODAL LIMITED-STAGE DIFFUSE LARGE B-CELL LYMPHOMA MORE AGRESSIVE THAN NODAL FORMS?

C. Fernandes 1,* , S. Alves 1 , F. Moita 1 , I. Dlouhy 1 , S. Esteves 2 , M. Gomes da Silva 1

1 Hematology; 2 Clinical Research Unit, Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal

Background: Limited-stage diffuse large B-cell lymphoma (DLBCL) is associated with favourable outcomes with overall survival of 70-80% at 10 years. Stage-modified IPI (sm-IPI) predicts the risk of relapse and death based on clinical factors that do not include extranodal (EN) involvement, although in some studies it has been associated with worse outcomes.

Aims: To analyse the characteristics and outcomes of limited-stage DLBCL in a single center series and evaluate the prognostic impact of EN involvement.

Methods: We conducted a retrospective analysis of consecutive patients with newly diagnosed stage I-II DLBCL treated with curative intent at a single centre from 2015 to 2019. Primary central nervous system (CNS) lymphoma, primary testicular lymphoma, primary mediastinal B-cell lymphoma and transformed lymphoma were excluded. Response to therapy was assessed according to Lugano criteria. Overall (OS) and progression free survival (PFS) were calculated by Kaplan-Meier method and comparisons done by the log rank test. Cumulative incidence of relapse was calculated using competing risk models and Gray test for group comparisons. Cox regression was used to identify prognostic factors for PFS.

Results: Of 152 patients identified, 53 (35%) had EN disease and 99 (65%) were nodal (N). The most common EN sites were stomach (30%), sinus/nasal cavity (13%), skin and soft tissue (9%) and breast (9%). Seventy-two (47%) had low risk sm-IPI (45 N; 27 EN). Clinical characteristics were similar between both groups, except for stage (stage I in 50.9% of EN vs 32.3% of N cases) and bulky disease (EN 15.1% vs N 37.4%).

All patients received R-CHOP (median 6 cycles; range 1-8). Eight patients, all with EN involvement (3 sinus/nasal cavity, 2 breast, 2 bone and 1 orbit), received CNS prophylaxis. Seventeen out of 152 patients received additional radiotherapy (RT), 71% of whom had nodal involvement; 16/17 were irradiated due to bulky disease and/or treatment with abbreviated immunochemotherapy. Globally, 89% patients had a complete response (CR) to treatment: 85% in the N and 96% in the EN group ( p =0.212). Nine patients relapsed at a median of 18 (7-52) months after obtaining CR (6 from the N and 3 from the EN group). The 4-year cumulative incidence of relapse was similar: 11.2% (95%CI 9.0-13.9%) in EN and 6.7% (95%CI 5.9-7.5%) in N group ( p =0.778). No gastric lymphomas relapsed. The most common sites of relapse were lymph nodes (3), soft tissue (3) and CNS (2). Nineteen (14 N; 5 EN) patients died at a median of 12 (0-47) months after starting treatment, mostly (52.6%) due to lymphoma progression. With a median follow-up of 43 months, 4-year OS and PFS were 87% and 82%, respectively. No significant survival differences were observed in patients with N or EN presentation with a 4-year PFS of 81.8% and 81.3% ( p =0.428), respectively. On multivariable analysis adjusting for RT, sm-IPI and bulky disease, EN involvement was not associated with a decreased PFS compared with N involvement (HR 0.77; 95%CI 0.31-1.90; p =0.570).

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Summary/Conclusion: Our study corroborates the very favourable outcomes of localized DLBCL. We could not find differences in PFS according to N and EN presentations when controlling for other prognostic factors. These results do not support different treatment strategies for EN disease; however, due to limited sample size the role of RT and the prognostic impact of specific locations cannot be fully ascertained.

P1181: REAL-WORLD CHARACTERISTICS AND CLINICAL OUTCOMES IN RELAPSE/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA POST CAR-T FAILURE

C. Flores Avile 1,* , L. Liao 2 , L. Wilson 1 , A. Lau 2 , L. Chen 2

1 Genesis Research, Hoboken; 2 ADC Therapeutics, New Providence, United States of America

Background: Progressive disease following chimeric antigen receptor T-cell (CAR-T) therapy for diffuse large B-cell lymphomas (DLBCL) is a common scenario. There are limited treatment options after CAR-T failure with a poor prognosis for patients at this stage in their disease. The effectiveness of existing treatment options following CAR-T failure is still being investigated in the real-world setting.

Aims: To further understand the clinical outcomes of CAR-T failure in relapse/refractory (RR) DLBCL patients in the real-world setting.

Methods: This retrospective analysis identified adult patients diagnosed with RR-DLBCL [01/01/2014 – 03/31/2021] who received CAR-T therapy and experienced a subsequent disease progression or death. COTA’s Real World Evidence (RWE) database is comprised of longitudinal, HIPAA-compliant data abstracted from electronic health records (EHR) from over 200 sites of care in US (60% academic, 40% community). The first post CAR-T therapy was categorized as checkpoint inhibitor +/- other therapies (CPI), investigational therapies, tafasitamab +/- lenalidomide, polatuzumab-containing regimen (pola-containing), lenalidomide +/- anti-CD20, BTK inhibitors (BTKi), chemotherapy/chemoimmunotherapy (CT/CIT), allogenic stem-cell transplant (allo-SCT), or anti-CD20 monoclonal antibody. Overall response rate (ORR), complete response (CR), and overall survival (OS) were reported for treatment groups with at least 5 patients.

Results: Of the 97 CAR-T patients identified, 57 (59%) patients failed CAR-T therapy due to receiving subsequent line of therapy, having documented progression event, or death. Patients who failed CAR-T were 67% male and on average 59 years old. Within a median follow-up of 12.4mo, 44 (77%) initiated further therapy whereby 7 (16%) initiated investigational therapies, 9 (20%) CPI, 7 (16%) tafasitamab +/- lenalidomide, 6 (14%) pola-containing, 5 (11%) lenalidomide +/- anti-CD20, 3 (7%) CT/CIT, 3 (7%) anti-CD20 monoclonal antibody, 2 (5%) BTKi, and 2 (5%) allo-SCT as their first post CAR-T therapy. Of these patients, 48% received more than two lines of therapies after CAR-T. Response rates of select first therapies received after CAR-T are detailed in Table 1. Outside clinical trials, ORR and CRs were highest for lenalidomide +/- anti CD20 (60% & 20%), followed by CPI (33% & 0%), pola-containing (33% & 0%) and tafasitamab +/- lenalidomide (14% & 0%). OS of first post CAR-T therapy was highest for lenalidomide+/- anti CD20 (12.5 mo), then CPI (11 mo), pola-containing therapy (6.37 mo), and tafasitamab +/- lenalidomide (2.7 mo).

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Summary/Conclusion: There is no existing standard of care after patients fail CAR-T therapy. Although further research is warranted in a larger sample population, poor clinical outcomes in treatment response and longevity were observed with existing treatment options. There is still a high unmet need for more effective therapies after CAR T-cell therapy has failed.

P1182: REAL-WORLD CHARACTERISTICS AND CLINICAL OUTCOMES IN RELAPSE/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA PATIENTS WHO RECEIVED CAR-T THERAPY

L. Liao 1 , L. Nastoupil 2 , L. Wilson 3 , C. Flores Avile 3,* , T. Kilavuz 1 , L. Chen 1

1 ADC Therapeutics, New Providence; 2 Department of Lymphoma–Myeloma, Division of Cancer Medicine, University of Texas, Houston; 3 Genesis Research, Hoboken, United States of America

Background: A significant proportion of patients diagnosed with diffuse large B-cell lymphoma (DLBCL) experience refractory or relapse (RR) disease. Approval of chimeric antigen receptor T-cell (CAR-T) therapies has resulted in a novel therapeutic option for eligible patients with RR-DLBCL. However, progressive disease post CAR-T remains a common scenario as patient identification, timing, and effectiveness of CAR-T in the real-world setting is still evolving.

Aims: To further understand clinical outcomes of standard of care CAR-T in RR-DLBCL in clinical practice.

Methods: This retrospective analysis identified adult patients diagnosed with RR-DLBCL [01/01/2014 – 03/31/2021] who received CAR-T therapy. COTA’s Real World Evidence (RWE) database is comprised of longitudinal, HIPAA-compliant data abstracted from the electronic health records (EHR) of healthcare provider sites, representing diverse treatment U.S settings from over 200 sites of care; roughly 60% of patients are seen at academic sites and 40% are seen at community sites. Patients were categorized as having received CAR-T therapy in 2L, 3L, 4L, or 5L. Baseline characteristics was reported for CAR-T patients. Best response rate, treatment failure, and overall survival (OS) were reported by line of therapy. Disease characteristics were derived from the EHR, including the presence of high-grade lymphoma (positive rearrangement in C-MYC and BCL-2 or BCL-6 biomarkers) and primary refractory disease (2L started within 6 months not due to patient preference, drug shortage, insurance reasons, toxicity, or pandemic reasons). CAR-T treatment failure was defined as the earliest of death, initiation of subsequent line of therapy, or documented progression event after CAR-T.

Results: A total of 97 CAR-T patients were identified whereby 17 received CAR-T therapy in a clinical trial setting and were excluded from this real-world evidence study. Of the 80 patients that remained, 10 (13%) received CAR-T in 2L, 31 (39%) in 3L, 24 (30%) in 4L, and 15 (19%) in 5L+. CAR-T patients had a mean age of 57 years, most were male (58%), 16% were diagnosed with high-grade lymphoma, and 60% were primary refractory. Median time from diagnosis to initiation of CAR-T was 15 months. Overall, 38% of patients achieved a complete response with a decrease in response in later lines (2L: 70%, 3L: 58%, 4L: 29%, 5L+: 33%). Within a median follow up of 13.5 months (2L: 13.5 mo, 3L: 15.2 mo, 4L: 12.8 mo, 5L+: 9.5 mo), treatment failure occurred in 45% of patients, with an increase in later lines (2L: 20%, 3L: 48%, 4L: 63%, 5L+: 80%). Median OS was 31.2 months (Not reached (NR); 2L: NR, 3L: NR, 4L: 26.3 mo, 5L+: 13.5 mo) with unequal survival probabilities across lines of therapy (Log-rank test: p=0.004) (Figure 1).

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Summary/Conclusion: CAR T-cell therapies are considered a major advance in DLBCL, yet over half of those patients eventually fail. Outcomes are inferior in later lines with a decrease in complete response rates and shorter survival by line of therapy, thus, highlighting the need to provide CAR T-cell therapies in earlier settings.

P1183: CAR-T MORPHOLOGICAL CHARACTERISTICS AND DYNAMICS IN PERIPHERAL BLOOD, EVALUATED BY A NOVEL MICROSCOPY PLATFORM

G. Fridberg 1,* , B.-Z. Katz 1 , D. Benisty 1 , C. Perry 1 , R. Ram 1 , I. Avivi 1

1 Hematology Division, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

Background: Chimeric Antigen Receptor T-cells (CAR-T) therapy becomes a preferable therapeutic approach in patients with relapsed and refractory (R/R) non-Hodgkin’s large B cell lymphomas (LBCL) who failed ≥2 therapeutic regimens. However, there are no commercial assays for routine CAR-T measurements and clinical studies often employ PCR or FACS, both expensive and relatively complicated. Detection of CAR-T in peripheral blood smear (PBS) is challenging, mostly due to the lack of data regarding their morphology prior to transfusion and the low sensitivity of currently available technologies. As oppose to the currently available digital morphology, Scopio Labs Full Field Morphology (FFM) is a novel digital microscopy platform that provides high-resolution images combined with a full field analysis, incorporating artificial intelligence capabilities, thereby enabling the detection and classification of rare cells in PBS, including CAR-T cells.

Aims: The dynamics of CAR-T quantity and subtypes in PBS were evaluated, and correlated with responsiveness to treatment and treatment-related toxicities.

Methods: Morphological library of CAR-T cells was established from cells obtained directly from the CAR-T transfusion-bag (Fig. 1A). FFM analysis of consecutive PBS samples obtained from 26 R/R DLBCL patients, treated either with tisagenlecleucel (tisa-cel) or axicabtagene ciloleucel (axi-cel) at the Tel Aviv Sourasky Medical Center between October 2019-October 2020 was performed.

Results: We identified in the CAR-T cell transfusion bags 5 distinct CAR-T morphological sub groups prior to transfusion: regular, activated, apoptotic, multinucleated and cells in mitosis (Fig. 1A).

First, 79 smears, obtained from 18 patients, were examined with the FFM platform vs the CellaVision DM1200 system. Overall, 24,247 WBC were detected by the FFM platform (average 303 cells per PBS), compared with 4,335 detected by CellaVision DM1200 system (average 54.4 cells per PBS). Detection of CAR-T was superior with the FFM platform (median difference per PBS - 2, IQR 0-6, p<0.001). Then, PBS from 26 patients, 21 (80.7%) treated with tisa-cel, and 5 (19.2%) treated with axi-cel, were analyzed by the FFM platform. The absolute number of CAR-T cells in the PBS post infusion was higher in patients treated with axi-cel (across days 1 to 15). The average numbers of total, regular, and activated CAR-T cells, measured on day 5 post CAR-T-transfusion, were all higher in patients that obtained CR vs those who did not (Fig. 1B). Longer duration of cytokine release storm (CRS) was associated with higher number of CAR-T cells with activated morphology, and a lower presence of apoptotic cells on day 14. Higher number of apoptotic cells on day 14 were associated with a deferred CRS onset, while lower number of activated morphology cells on day 14 was associated with earlier onset of CRS. There was a positive correlation between the number of cells, measured on day 7 by FACS, and time to peak of regular morphological CAR-T cells (r=0.67, p=0.034, n=10).

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Summary/Conclusion: In this study, we show that CAR-T cells has heterogeneous morphology identified in PBS. Specific morphological features in specific days were associated with outcome and therapy related toxicities, emphasizing that CAR-T monitoring require quantitative, qualitative and repeated measurements. Thus, morphological CAR-T surveillance using FFM might serve as an available, simple and inexpensive method to provide clinically relevant insights.

P1184: PHASE I TRIAL OF MB-CART2019.1 IN PATIENTES WITH RELAPSED OR REFRATORY B-CELL NON-HODGKIN LYMPHOMA: 2 YEAR FOLLOW-UP REPORT

P. Borchmann 1,* , A. Lohneis 1 , P. Gödel 1 , H. Balke-Want 1 , C. Schmid 2 , F. Ayuk 3 , S. Holtkamp 4 , L. Hanssens 4 , G. Zadoyan 4 , B. Friedrichs 4 , M. Assenmacher 5 , I. Bürger 5 , D. Schneider 6 , T. Overstijns 4 , C. Scheid 7 , U. Holtick 7 , S. Miltenyi 5 , M. Hallek 1

1 Department of Internal Medicine I, University Hospital of Cologne, Cologne; 2 II. Med. Klinik Hämatologie/Onkologie, Universitätsklinikum Augsburg, Augsburg; 3 Department of Stem Cell Transplantation, Universitätsklinikum Hamburg-Eppendorf, Hamburg; 4 Miltenyi Biomedicine GmbH; 5 Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany; 6 Lentigen Technology Inc., Gaithersburg, United States of America; 7 Leukapheresis and Stem Cell Transplant Unit, Department of Internal Medicine I, University Hospital of Cologne, Cologne, Germany

Background: A tandem chimeric antigen receptor (CAR) targeting CD20 and CD19 (pLTG1497) has shown superior efficacy as compared to each single-CAR in pre-clinical models. MB-CART2019.1 consists of autologous CD4 and CD8 enriched T-cells, which are transduced with a lentiviral vector that encodes the CAR construct for both CD20 and CD19 incorporating a 4-1BB co-stimulatory domain.

Aims: This first-in-human, Phase I study had the objective to assess feasibility, safety and toxicity of ex vivo generated MB-CART2019.1 in mainly elderly patients (pts) with relapsed or refractory (r/r) CD20 and CD19 positive aggressive B-cell Non-Hodgkin lymphoma (B-NHL) without curative treatment option ( NCT03870945 ).

Methods: The trial included two predefined dose levels (DL1 1.0x10 6 and DL2 2.5x10 6 CAR T-cells/kg body weight (BW), respectively). In DL1 pts with multiple prior treatment lines were included while in DL2 the patient population was limited to pts with r/r DLBCL and 1 prior line of treatment who were transplant-ineligible. Fludarabine and cyclophosphamide were used for lymphodepletion. Infusion of fresh MB-CART2019.1 was scheduled 14 days after leukapheresis. The primary endpoint was to evaluate the maximum tolerated dose of MB-CART2019.1 as determined by dose limiting toxicities (DLT). Secondary endpoints included adverse events, overall response rate (ORR), maximum concentration (C max ), area under the curve (AUC d0-d28 ), and persistence of CAR T-cells, measured by flow-cytometry.

Results: A total of 12 pts, 6 per dose level, have been treated in the trial after obtaining informed consent. Median age was 72 y (range 20, 78 y), with 8 pts >70 y. Histologies included aggressive B-NHL (9), transformed follicular lymphoma (2), and mantle cell lymphoma (1). No grade ≥3 cytokine release syndrome (CRS) or neurotoxicity were observed. Haematotoxicity was very limited with no anemia or thrombocytopenia ≥grade 3 beyond day 28 and intermittent neutropenia ≥grade 3 in only two pts beyond week 8 after treatment. No DLT were observed.

The ORR was 75% (3/6 pts in DL1 and 6/6 pts in DL2) with 5/12 pts (3/6 pts in DL1 and 2/6 pts in DL2) achieving PET-CT negative complete remission (CR) (CR-group). Among those 5 pts, all had ongoing CR as radiologically documented by PET-CT or CT at 12 months and all completed a 2-year follow-up visit without evidence of relapse as per investigator assessment. Patients with only partial response or stable disease as best overall response ultimately progressed.

All pts with CR had a C max ≥450 cells/µL with mean C max 1,092.5 cells/µL (range 460.1, 3,147.0) while pts with no CR had lower values with mean C max of 111.0 cells/µL (3.9, 458.0). Mean AUC d0-d28 was 7,901 d*cells/µL (2,399.2; 19,574.7) in the CR-group as opposed to mean AUC d0-d28 of 942.0 d*cells/µL (39.3; 3,471.62) in the non-CR group. Mean time of CAR T-cell persistence in the CR-group was 491 days (274, 736 d) and all 5 CR pts had detectable CAR T-cells beyond month 6.

Summary/Conclusion: As reported previously, recommended dose of MB-CART2019.1 is 2.5x10 6 CAR T-cells/kg BW. Importantly, all 5 pts with CR were still in CR 12 months after treatment and have now completed the 2-year follow-up visit without evidence of relapse. MB-CART2019.1 is currently challenging conventional immune-chemotherapy in a randomized Phase II trial for elderly, non-transplant eligible pts with first progression or relapse of aggressive B-NHL ( NCT04844866 ).

P1185: ANTITUMOR ACTIVITY AND SAFETY OF ZANUBRUTINIB COMBINED WITH R-CHOP REGIMEN IN THE TREATMENT OF NEWLY DIAGNOSED NON-GCB DLBCL WITH EXTRANODAL INVOLVEMENT: A PROSPECTIVE PHASE II STUDY.

C. Li 1,2,* , H. Geng 1 , X. Zong 1,2 , J. Zhou 1,2 , Y. Zhang 1 , J. Li 1 , S. Jia 1 , D. Wu 1,2

1 National Clinical Research Center for Hematologic Diseases, Jiangsu Institute of Hematology, The First Affliated Hospital of Soochow University; 2 Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, Soochow University, suzhou, China

Background: Approximately one-third of Diffuse large B-cell lymphoma(DLBCL) arises primarily from extranodal sites,less than 70% showed extranodal involvement in at least 1 site.Patients with multiple extranodal involvement indicates adverse clinical outcomes,and non-GCB subtype is another poor prognostic factor.Study shows that the rates of complete remission(CR),five-year OS,five-year PFS were significantly lower for the non-GCB subtype(77.5% vs 52.6%,78% vs 54%,76% vs 48%).Patients who failed in initial treatment had poor prognosis and short overall survival.In the genetic taxonomy, with non-GCB tumors enriched for MCD and BN2 subtype are suitable for the treatment of BTK inhibitor.Zanubrutinib has shown very promising future in newly diagnosed and relapsed/refractory B-cell lymphoma.

Aims: We conducted a single-arm,phase II trial for newly diagnosed non-GCB DLBCL with extranodal involvement to evaluate the efficacy and safety of the zanubrutinib combined with R-CHOP(number NCT 04835870 ).

Methods: 20 patients enrolled in this prospective single-center study with newly diagnosed non-GCB DLBCL from October 2020 to February 2022.Patients received therapies:R-CHOP(intravenously rituximab(375mg/m2 on Day0), cyclophosphamide(750mg/m2on Day1),doxorubicin(50mg/m2 on Day1),vincristine(1.4mg/m2 on Day1),and oral prednisone(50mg/day Day1-5) with Zanubrutinib(160mg bis in die orally) regimen. Particularly,patients with a weak constitution or older than 70 years old were administered ZR-miniCHOP.

Results: 20 patients received ZR-CHOP regimen treatments, Patients had a median age of 60 years(31~83years) and 4 patients were over 70 years old,11 patients (55%) had IPI-PS ≥3 score,16 patients (80%) had stage III/IV disease,13 patients (65%) had ≥2 extranodal organs involved,2 patients (10%) had bulky disease and 9 patients (45%) were double-expression lymphoma confirmed by immunohistochemistry and FISH (Table 1). The median follow-up time of all patients was 8.7 months(0.4~16.1months) and there are 12 patients received 6 ~ 8 cycles of treatment who can evaluate the efficacy. The objective response rate(ORR) of 12 patients was 91.67%(11/12),11 patients(91.67%) reached complete remission(CR),of which 9 achieved CR at the interim evaluation, and 1 patient(8.3%) reached progression disease(PD).The median OS and PFS of the 12 patients who finished treatment were 10.7months(5.6~16.1months) and 10.2months(5.6~16.1months).One patient relapsed(Figure 1).13(65%) patients reduced the dose during treatment, the most common AEs were hematological toxicity, fatigue and apositia. all-grade and grade 3 or higher hematological toxicity were 100% and 65%,11 patient observed grade 3 or higher neutropenia and 3 patients had severe pneumonia during treatment. Hemorrhage were observed in 3 patients,2 patients developed atrial fibrillation after the first medication.No patient had fatal adverse events(Table2).

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Summary/Conclusion: Our results showed that Zanubrutinib plus R-CHOP(ZR-CHOP) is a safe and effective scheme for this part of non-GCB DLBCL patients with extranodal involvement.It indicate that the addition of zanubrutinib to the standard first-line treatment can enable these patients to achieve remission in the early stage of treatment and without significantly increasing toxicity.

P1186: A LARGE FRENCH REAL WORLD MULTICENTRIC PROSPECTIVE COHORT OF PATIENTS WITH LYMPHOMA (REALYSA STUDY): DESCRIPTION OF THE DIFFUSE LARGE B CELL LYMPHOMA PATIENTS IN REAL WORLD IN FRANCE

H. Ghesquieres 1,* , F. Cherblanc 2 , A. Belot 2 , V. Camus 3 , K. Thokagevistk 4 , K.-K. Bouabdallah 5 , C. Esnault 4 , L.-M. Fornecker 6 , S. Micon 4 , F. Bijou 7 , C. Haioun 8 , N. Morineau 9 , L. Ysebaert 10 , G. Damaj 11 , S. Le Gouill 12 , S. Guidez 13 , F. Morschhauser 14 , C. Thiéblemont 15 , A. Chauchet 16 , R. Gressin 17 , F. Jardin 3 , C. Fruchart 18 , G. Labouré 19 , L. Fouillet 20 , P. Lionne-Huyghe 21 , A. Bonnet 22 , L. Lebras 23 , S. Amorim 24 , C. Leyronnas 25 , G. Olivier 26 , R. Guieze 27 , T. Lamy 28 , V. Launay 29 , B. Drenou 30 , O. Fitoussi 31 , L. Detourmignies 32 , J. Abraham 33 , C. Soussain 34 , F. Lachenal 35 , P. Fogarty 2 , P. Cony-Makhoul 2 , A. Bernier 2 , S. Le Guyader-Peyrou 36 , A. Monnereau 36 , F. Boissard 37 , C. Rossi 38

1 Hopital Lyon Sud, Pierre Benite; 2 LYSARC, Pierre-Benite; 3 Centre Henri Becquerel, Rouen; 4 Roche, Boulogne-Billancourt; 5 CHU Haut Lévêque, Pessac; 6 ICANS, Strasbourg; 7 Institut Bergonié, Bordeaux; 8 Hôpital Henri Mondor, Créteil; 9 CHD Vendée, La Roche sur Yon; 10 IUCT Oncopôle, Toulouse; 11 Institut d’Hématologie de Basse Normandie, Caen; 12 Centre Hospitalier Universitaire de Nantes, Nantes; 13 Centre Hospitalier Universitaire de Poitiers, Poitiers; 14 CHRU de Lille, Lille; 15 Hôpital Saint-Louis, Paris; 16 CHU Jean Minjoz, Besancon; 17 Centre Hospitalier Universitaire Michallon, La Tronche; 18 Centre Hospitalier de Dunkerque, Dunkerque; 19 Centre Hospitalier de Libourne, Libourne; 20 Institut de Cancérologie Lucien Neuwirth, Saint-Priest-en-Jarez; 21 Centre Hospitalier d’Arras, Arras; 22 Centre Hospitalier Bretagne Atlantique, Vannes; 23 Centre Léon Bérard, Lyon; 24 Hôpital Saint-Vincent-de-Paul, Lille; 25 Groupe Hospitalier Mutualiste de Grenoble, Grenoble; 26 Centre Hospitalier de Niort, Niort; 27 CHU de Clermont-Ferrand, Clermont-Ferrand; 28 CHU Pontchaillou, Rennes; 29 Centre Hospitalier Yves Le Foll, Saint-Brieuc; 30 Groupe Hospitalier Mulhouse et Sud Alsace, Mulhouse; 31 Polyclinique Bordeaux Nord Aquitaine, Bordeaux; 32 Centre Hospitalier de Roubaix, Roubaix; 33 Centre Hospitalier Universitaire de Limoges, Limoges; 34 Institut Curie, Saint-Cloud; 35 Centre Hospitalier Pierre Oudot, Bourgoin-Jallieu; 36 Université de Bordeaux, Bordeaux, France; 37 Roche - F.Hoffman-La Roche Ltd, Basel, Switzerland; 38 Centre Hospitalier Universitaire Francois Mitterand, Dijon, France

Background: In France, there were an estimated 18.000 incident lymphoma cases in 2018. Currently, most of the knowledge comes from clinical trials (CT), with stringent inclusion criteria and poor representativeness. Real world data (RWD) is essential to complement CT data, but major challenges such as feasibility and data quality remain. In this context, the REal world dAta in LYmphoma and Survival in Adults (REALYSA) multicentric prospective cohort started in 2018 ( NCT03869619 ), with a recruitment objective of 6000 patients and a 9-year follow-up (FU).

Aims: The aims of this study are (i) to evaluate the capacity of a real-world program to provide high-quality data that translate into meaningful clinical endpoints, and (ii) to describe the demographic and clinical characteristics as well as therapeutic management and treatment effectiveness of 1 st line (1L) DLBCL patients.

Methods: Patients diagnosed with 7 histological lymphoma subtypes are included in REALYSA at diagnosis after signing informed consent. Patients receive standard routine care. Data collection includes demographic, clinical, quality of life and epidemiological data at inclusion and every 6 to 12 months. A biobank is also constituted. A strong data validation system inspired from clinical research standards, including care pathway visualisation tools, is regularly running. Meaningful real-life endpoints were derived, such as end of treatment (EoT) evaluation and event-free survival (EFS).

We present here the results of a proof-of-concept analysis for REALYSA patients with a DLBCL diagnosis and who received a 1L treatment.

Results: Thirty-five hospitals/clinics are currently participating to REALYSA across France. As of Jan 31st 2022, 1217 patients with DLBCL were included in REALYSA with a mean number of 60 patients per month; 645 epidemiological questionnaires were collected; and there were more than 1500 samples in the biobank.

The analysis was conducted on 645 DLBCL patients included in REALYSA before March 31 st 2021. Median age was 66.3 years [54-75]. Most patients were male (344; 53.3%), with advanced-stage disease (Ann-Arbor stage III/IV for 472 patients (73.3%)), extra-nodal locations (499; 77.4%) and elevated LDH level (402; 64.0%). The international prognostic index was 2 to 5 for 486 patients (76.2%). Treatments received at 1L were R-CHOP (482; 74.7%), R-miniCHOP (86; 13.3%), high dose anthracycline-based regimen (62; 9.6%) and non-anthracycline based regimen (15; 2.3%). Among patients with EoT evaluation done (603), 483 (80.1%) had a complete response, 51 (8.5%) a partial response, 7 (1.2%) a stable disease and 62 (10.3%) a progressive disease. Of note, EoT evaluation was performed by PET/scan for 92% of patients. Median FU was 9.9 months [4.5-17.2]. Results for effectiveness at 12 months were: (i) EFS of 77.9% [95% CI 73.8-81.4]; (ii) PFS of 79.7% [75.6-83.1]; and (iii) overall survival of 90.0% [86.5-92.5]. With the limitation of the short FU, and using backward stepwise regression model, we confirmed that EFS was associated with age, ECOG PS, LDH level and bulk disease.

Summary/Conclusion: This analysis showed that REALYSA, the first nationwide real-life cohort on lymphoma, is able to provide high-quality data, with results for 1L DLBCL patients consistent with the literature, including recent phase 3 trials. These data will be useful for numerous purposes, including a better characterization of lymphoma population in France (clinic, epidemiology, biology), as well as innovative study designs (e.g. new outcome endpoints, synthetic control arm).

P1187: END OF TREATMENT 18F FDG-PET/CT RESPONSE IS PROGNOSTIC FOR OVERALL AND PROGRESSION-FREE SURVIVAL IN PERIPHERAL T-CELL LYMPHOMA: RESULTS OF THE UK NCRI PHASE 2 RANDOMISED CHEMO-T TRIAL PET/CT SUBSTUDY

M. Gleeson 1,2,* , C. Arias 2 , D. Cunningham 2 , C. Peckitt 2 , Y. Du 2 , N. Hujairi 2 , Y. M. To 2 , H.-C. Chen 2 , S. Patel 2 , I. Chau 2 , P. Johnson 3 , K. M. Ardeshna 4 , A. Wotherspoon 2 , A. Attygalle 2 , E. A. Hawkes 5,6 , M. P. Macheta 7 , G. P. Collins 8 , J. Radford 9 , A. Forbes 10 , A. Hart 11 , S. Montoto 12 , P. McKay 13 , K. Benstead 14 , N. Morley 15 , N. Kalakonda 16 , Y. Hasan 17 , D. Turner 18 , S. Chua 2

1 Guy’s and St. Thomas’ NHS Foundation Trust, London; 2 The Royal Marsden NHS Foundation Trust, London and Surrey; 3 Cancer Research UK Centre, University of Southampton, Southampton; 4 University College London Hospitals, London, United Kingdom; 5 Olivia Newton John Cancer Research Centre, Austin Health; 6 Monash University, Melbourne, Australia; 7 Blackpool Victoria Hospital, Blackpool; 8 Oxford Cancer and Haematology Centre Churchill Hospital, Oxford; 9 University of Manchester and the Christie NHS Foundation Trust, Manchester; 10 Royal Cornwall Hospital, Truro; 11 New Victoria Hospital, Glasgow; 12 St Bartholomew’s Hospital, London; 13 Beatson West of Scotland Cancer Centre, Glasgow; 14 Gloucestershire Hospitals NHS Foundation Trust, Cheltenham; 15 Nick.morley@sth.nhs.uk, Sheffield; 16 The Clatterbridge Cancer Centre NHS Foundation Trust, Liverpool; 17 Sandwell and West Birmingham Hospitals NHS Trust, Birmingham; 18 Torbay Hospital, Torquay, United Kingdom

Background: The role of 18 F FDG PET (PET) in peripheral T-cell lymphoma (PTCL) remains under evaluation. The UK NCRI phase 2 randomised CHEMO-T trial compared cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) with gemcitabine, cisplatin and methylprednisolone (GEM-P) in previously untreated PTCL patients. The primary endpoint, to demonstrate a superior complete response (CR) rate for GEM-P by contrast-enhanced CT (CECT) at end of treatment (EOT) was not met.

Aims: To investigate the role of PET in determining baseline bone marrow (BM) involvement in PTCL and as a prognostic tool at EOT.

Methods: Patients required a baseline PET (acquired as per protocol) within 28 days of randomisation and a PET at EOT at least 28 days post chemotherapy. Site accreditation, data collation, and quality control for PET imaging was performed by the UK PET Core Lab. BM biopsy was required within 6 weeks of randomisation. PET scans were centrally reviewed at The Royal Marsden by 2 expert nuclear medicine physicians (YD and NH). Baseline BM involvement by PTCL on PET was evaluated. PET response was assessed according to the revised response criteria for malignant lymphoma and by the Deauville score (DS) 5-point scale. Where discordance existed between reviewers, a third reviewer (SC) adjudicated. EOT PET response and PET status (DS 1-3=PET-negative, DS 4-5=PET-positive) were correlated with PFS and OS.

Results: Baseline PET data was available for 82/84 patients, 80/82 (98%) had FDG-avid disease (n=2 with non-avid disease (n=1 AITL, n=1 EATL) were excluded). Clinical characteristics were similar to the main trial cohort: median age=63 yrs, male=73% and high-risk IPI=23%. PTCL subtypes were AITL n=33 (41%), PTCL-NOS n=32 (40%), ALK- ALCL n=14 (18%) and EATL n=1 (1%). For 20/79 (25%) patients with biopsy-proven BM infiltration 9/19 (47%) had BM uptake on baseline PET. Of 20/80 patients (25%) with BM uptake on baseline PET, 9/20 (45%) had biopsy-confirmed infiltration. PET had a sensitivity of 47% and a specificity 81% for determination of BM involvement. EOT PET was available for 69/80, n=4 were excluded as data was acquired following a later line (n=3) or complete imaging was not retrievable (n=1); therefore 65/80 had evaluable EOT PET data. The median number of days from EOT to PET was 41. At EOT, PET response was CR n=41 (63%), partial response (PR) n=15 (23%), stable disease n=0 and progressive disease (PD) n=9 (14%); 41 (63%) were PET-negative and 24 (37%) were PET-positive. At a median follow-up of 27 months, PET-negative vs. PET-positive patients at EOT had a 2-yr OS of 72% vs. 52% [HR 0.58; 95% CI 0.24–1.41), p=0.231] and 2-yr PFS of 55% and 29% [HR 0.45 (95% CI 0.23–0.88), p=0.021] respectively. EOT PET response correlation with 2-yr OS was CR=72%, PR=67%, PD=29%; and with 2-yr PFS was CR=55%, PR=32%, PD=25%. A CR by PET (vs. PD) was associated with improved OS [HR 0.245 (95% CI 0.083–0.730), p=0.012] (Figure 1) and PFS [HR 0.265 (95% CI 0.102–0.687), p=0.006]. Adjusting for stratification parameters at randomisation (IPI risk group, PTCL subtype) PET response remained prognostic for both OS and PFS while EOT PET status (positive vs. negative) did not. CECT response remained prognostic for PFS only.

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Summary/Conclusion: Our findings indicate that PET has insufficient sensitivity for detection of BM involvement for baseline staging. Furthermore, while both EOT CECT and PET response were prognostic for PFS and OS, only PET response remained prognostic for OS following adjustment for stratification factors suggesting it is a superior prognostic tool in PTCL.

P1188: PERSONALIZED TREATMENT ACCORDING TO BIOLOGICAL PROFILE IMPROVES OUTCOME OF NEWLY DIAGNOSED PATIENTS AFFECTED BY DIFFUSE LARGE B-CELL LYMPHOMA: A REAL-LIFE EXPERIENCE

A. Guidetti 1,2,* , R. Pasquale 3 , A. Dodero 1 , A. Chiappella 1 , L. Devizzi 1 , L. Farina 1 , C. Rusconi 1 , M. Pennisi 1 , P. Corradini 1,2

1 Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori; 2 Pathophysiology and Transplantation, University of Milano; 3 Hematology, Ospedale Maggiore Policlinico, Milano, Italy

Background: Diffuse Large B-Cell Lymphoma (DLBCL) is a heterogeneous disease characterized by biological and clinical aspects conditioning different prognosis. Chemo-immunotherapy treatment with R-CHOP proved to be less effective in patients with double hit (DH)/triple hit (TH) or double expressor (DE) DLBCL who require more intensive regimens

Aims: To investigate survival outcomes of DLBCL patients treated according to biological profile in the real-life clinical practice at Istituto Nazionale dei Tumori in Milano. Primary objective was to verify whether personalized treatment could overcome well known differences in Progression Free Survival (PFS) observed between DE, DH/TH and non DE/DH/TH patients when they are uniformly treated with R-CHOP

Methods: We retrospectively collected clinical and survival data of all newly diagnosed DLBCL patients referred at our Hematology Division from January 2013 to August 2021. All DLBCL patients are evaluated for BCL2, BCL6 and MYC expression in immunohistochemistry and gene rearrangements by fluorescent in situ hybridization, and they are assigned to treatment based on the biologic profile: non DE/DH/TH patients receive 6 cycles R-CHOP-21 + 2R, DE patients receive 6 cycles R-DA-EPOCH and DH/TH receive R-CODOX-M/R-IVAC. Central Nervous System (CNS) prophylaxis with lumbar puncture and/or intravenous high dose methotrexate is reserved for DE or DH patients and for those with CNS-IPI 3-5

Results: Since 2013, 243 patients affected by DLBCL at diagnosis were consecutively treated according to biological risk: 139 non DE/DH/TH patients received R-CHOP, 80 DE patients received R-DA-EPOCH and 24 high risk patients received R-CODOX-M/R-IVAC (n= 13 DH/TH patients and n=11 DE patients with CNS IPI 3-5). Median age was 62 years (range, 21-86), 178 patients (73%) presented in stage 3-4 and 97 patients (40%) had an IPI score of 3-5. The three groups were not significantly different in terms of clinical characteristics, except for median age, which was higher in the R-CHOP group (66 years vs. 58 and 57 years in R-DA-EPOCH and R-CODOX-M/R-IVAC groups, respectively). Despite the toxicity expected with an intensified therapy, 95% of patients completed the treatment and no toxic deaths were registered. Febrile neutropenia was observed in 3%, 15% and 29% of patients in RCHOP, RDAEPOCH and RCODOX-M/R-IVAC, respectively.

At a median follow-up of 42 months (range, 2-106 months), the estimated 3 years PFS and OS for the entire population were 80% and 89%, respectively. Patients with low IPI, limited stage and germinal center B cell of origin had significant better PFS as compared to the rest. Three years PFS and OS according to biological entity and relative treatment group were not significantly different, being 84%, 77% and 66%, (p=ns) and 93%, 85% and 74%, (p=ns) in non DE/DH/TH, DE and high risk patients, respectively. Similar PFS between non DE/DH/TH, DE and high risk patients were observed also in the subgroup of patients with IPI3-5 with PFS of 77%, 65% and 57%, respectively (p=ns). CNS prophylaxis was administered to 177 patients (73%) and 9 cases of CNS recurrence (4%) were observed

Summary/Conclusion: In this study, we describe the feasibility of a personalized treatment according to biological risk in a real-life setting. This approach seems to overcome the poor outcome of DLBCL patients at high risk of relapse and ameliorate the prognosis of the overall DLBCL population. Biological characteristics should be carefully considered at diagnosis in order to give patients a personalized program and improve their outcome

P1189: INITIAL SAFETY RUN-IN RESULTS OF THE PHASE III POLARGO TRIAL: POLATUZUMAB VEDOTIN PLUS RITUXIMAB, GEMCITABINE, AND OXALIPLATIN IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA

A. McMillan 1,* , C. Haioun 2 , J.-M. Sancho 3 , A. Viardot 4 , A. Rodriguez Izquierdo 5 , E. M. Donato Martin 6 , A. M. García-Sancho 7 , J. Sandoval-Sus 8 , H. Tilly 9 , E. Vandenberghe 10 , J. Hirata 11 , P. Choudhry 11 , Y. M. Chang 12 , L. Musick 11 , M. Matasar 13

1 Centre for Clinical Haematology, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; 2 Lymphoid Malignancies Unit, Groupe Hospitalier Henri Mondor, Créteil Cedex, France; 3 ICO-HU Germans Trias I Pujol, Barcelona, Spain; 4 University Hospital of Ulm, Ulm, Germany; 5 Hospital Universitario 12 de Octubre Servicio de Hematología, Madrid; 6 Hospital Universitario Dr Peset Servicio de Hematología, Valencia; 7 Hospital Clínico Universitario de Salamanca Servicio de Hematología, Salamanca, Spain; 8 Moffitt Cancer Center at Memorial Healthcare Institute, Pembroke Pines, United States of America; 9 Centre Henri Becquerel and University of Rouen, Rouen, France; 10 St James Hospital Cancer Clinical Trials Office, Dublin, Ireland; 11 Genentech, Inc., South San Francisco, United States of America; 12 Hoffmann-La Roche Ltd, Mississauga, Canada; 13 Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Transplant-ineligible patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) have a poor prognosis (Gisselbrecht C, et al. Br J Haematol 2018). Several treatment options are available, including platinum-based chemotherapies such as oxaliplatin plus rituximab and gemcitabine (R-GemOx). Adding polatuzumab vedotin to R-GemOx (Pola-R-GemOx) may improve outcomes for patients with a continued unmet medical need. Pola-R-GemOx is an investigational combination. The safety of polatuzumab vedotin and platinum-based therapy combinations must be considered as both are associated with neuropathy. POLARGO ( NCT04182204 ; MO40598) is a Phase III, multicenter, open-label, randomized trial evaluating the safety and efficacy of Pola-R-GemOx vs R-GemOx in patients with R/R DLBCL.

Aims: To present the results from the safety run-in stage of POLARGO.

Methods: The primary endpoint is the safety and tolerability of polatuzumab vedotin (1.8 mg/kg) + R-GemOx (R, 375 mg/m2; Gem, 1000 mg/m2; Ox, 100 mg/m2) given every 21 days for up to 8 cycles. Safety was assessed by the incidence, nature, and severity of adverse events (AEs; NCI CTCAE v5.0), with a focus on peripheral neuropathy (PN). Dose interruptions and reductions were used to assess tolerability. Granulocyte-colony stimulating factor was given as primary prophylaxis with each cycle (C) of therapy; anti-infective prophylaxis for pneumocystis and herpes virus was mandatory. All patients provided informed consent.

Results: As of October 26, 2021, 15 patients were enrolled, and 11 (73%) received ≥4 cycles of Pola-R-GemOx. Median age was 76 (range 47–87) years, 10 (67%) patients had an IPI score of 3–5, 7 (47%) had ≥2 prior therapy lines, and 8 (53%) were refractory to last treatment. Grade 3–4 AEs were reported in 5 (33%) patients: thrombocytopenia (20%) and neutropenia (13%) were the most common. Two (13%) patients had serious AEs (grade 3 febrile neutropenia and grade 3 infection [n=1 each]). There were no grade 5 AEs or AEs leading to drug discontinuation. Eight (53%) patients had grade 1 or 2 PN; there were no cases of grade ≥3 PN. Three (20%) patients had drug interruptions due to PN. Two (13%) patients had a dose reduction of polatuzumab vedotin and oxaliplatin due to PN at C5 and C8, respectively; one patient (7%) had a dose reduction of polatuzumab vedotin due to grade 4 thrombocytopenia at C2.

End-of-treatment (EOT) objective response rate was 40% (95% confidence interval [CI]: 16–68) and complete response rate was 27% (95% CI: 8–55); 7 (47%) patients had progressive disease at EOT. Ten (67%) patients received subsequent therapies following Pola-R-GemOx, including CAR T-cell therapy (n=3) and stem-cell transplant (n=1).

Summary/Conclusion: In the safety run-in stage, Pola-R-GemOx was safe and tolerable. PN was manageable with dose interruptions and reductions; no cases of grade ≥3 PN were observed. The toxicity of this combination did not compromise delivery of subsequent treatments. POLARGO is currently enrolling patients to receive Pola-R-GemOx vs R-GemOx; results will be presented at a future meeting.

P1190: OUTCOMES BY BCL2 AND MYC EXPRESSION AND REARRANGEMENTS IN UNTREATED DIFFUSE LARGE B-CELL LYMPHOMA FROM THE POLARIX TRIAL

F. Morschhauser 1,* , Y. Jiang 2 , F. Jardin 3 , A. F. Herrera 4 , L. H. Sehn 5 , C. Herbaux 6 , C. Flowers 7 , T. Phillips 8 , A. López Guillermo 9 , C. Diefenbach 10 , G. P. Gregory 11 , A. Kim 12 , A. M. Barbui 13 , S. Balasubramanian 2 , W. Harris 2 , J. Hirata 2 , J. N. Paulson 2 , C. Lee 2 , G. Lenz 14

1 Lille University Hospital Center, Lille Cedex, France; 2 Genentech, Inc., South San Francisco, United States of America; 3 University of Rouen, Rouen, France; 4 City of Hope, Duarte, United States of America; 5 BC Cancer Centre for Lymphoid Cancer and University of British Columbia, Vancouver, Canada; 6 University of Montpellier, Montpellier, France; 7 M.D. Anderson Cancer Center, Atlanta; 8 University of Michigan Medical School, Ann Arbor, United States of America; 9 Hospital Clínic de Barcelona, Barcelona, Spain; 10 Perlmutter Cancer Center at NYU Langone Health, New York, United States of America; 11 Clinical Sciences at Monash Health, Monash University, Melbourne, Australia; 12 Dana-Farber Cancer Institute, Boston, United States of America; 13 Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy; 14 University Hospital Münster, Münster, Germany

Background: Overexpression of BCL2 and MYC, and translocation of MYC , BCL2 , and BCL6, are associated with poorer outcomes in patients with diffuse large B-cell lymphoma (DLBCL; Horn et al. Blood 2013). We previously reported progression-free survival (PFS) from POLARIX in subgroups of patients with DLBCL receiving Pola-R-CHP or R-CHOP, including patients with double expressor lymphoma (DEL; favoring Pola-R-CHP: hazard ratio [HR] 0.64, 95% confidence interval [CI] 0.42–0.97), and double- or triple-hit lymphoma (DHL/THL; favoring R-CHOP: HR 3.81, 95% CI 0.82–17.64) (Tilly et al. NEJM 2022).

Aims: This is a prespecified exploratory analysis, to further analyze immunohistochemistry (IHC) expression status of BCL2 and MYC, and rearrangements (R) of BCL2 , BCL6 and MYC as independent prognostic markers. The prognostic impact of DEL, and BCL2 and MYC overexpression within treatment arms was also explored.

Methods: BCL2 and MYC protein expression were assessed by IHC and identified as IHC+ (≥50% [BCL2]/≥40% [MYC]) or IHC−; MYC -R, BCL2 -R, and BCL6 -R were detected by fluorescence in situ hybridization (Tilly et al. NEJM 2022). All patients provided informed consent. Pola-R-CHP is an investigational combination. Exploratory multivariate Cox regression models were adjusted for treatment, stratification factors (International Prognostic Index, bulky disease, geographic region), age >60 years, cell of origin, and biomarker evaluated, as appropriate.

Results: The prevalence, univariate HR, and 2-year PFS estimates of BCL2+/MYC+ and BCL2 -R/ MYC -R/ BCL6 -R subgroups are presented (Table) except for BCL6 -R, because all four PFS events in this subgroup were with Pola-R-CHP. Multivariate analyses results for Pola-R-CHP vs R-CHOP in patients with BCL2+ (HR 0.60, 95% CI 0.43–0.86) and those with MYC+ (HR 0.63, 95% CI 0.45–0.89) were similar to results of univariate analyses. Multivariate analyses of other subgroups were not performed due to the low patient number with BCL2 -R/ MYC -R/ BCL6 -R.

While patients with DEL treated with Pola-R-CHP had improved PFS vs patients treated with R-CHOP (HR 0.64, 95% CI 0.42–0.97), the prognostic impact of DEL vs non-DEL was identified in the R-CHOP arm (univariate HR 1.53, 95% CI 1.06–2.21; multivariate HR 1.29, 95% CI 0.88–1.91) vs the Pola-R-CHP arm (univariate HR 1.10, 95% CI 0.72–1.69; multivariate HR 1.42, 95% CI 0.89–2.28). Similarly, patients with BCL2+ had inferior PFS vs patients with BCL2 – in the R-CHOP arm (univariate HR 1.96, 95% CI 1.31–2.93; multivariate HR 1.74, 95% CI 1.14–2.66); however, no prognostic difference was detected in the Pola-R-CHP arm (univariate HR 1.30, 95% CI 0.85–2.01; multivariate HR 1.56, 95% CI 0.99–2.47). No prognostic impact of MYC+ vs MYC – was detected in either arm (R-CHOP: univariate HR 1.17, 95% CI 0.77–1.77; multivariate HR 1.00, 95% CI 0.65–1.55; Pola-R-CHP: univariate HR 0.83, 95% CI 0.54–1.27; multivariate HR 0.91, 95% CI 0.58–1.42).

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Summary/Conclusion: Multivariate analyses support the benefit of Pola-R-CHP in patients with BCL2+ and MYC+ DLBCL. The poor prognostic impact associated with DEL, which was mainly driven by BCL2+, appears reduced in Pola-R-CHP- vs R-CHOP-treated patients.

P1191: ANALYSIS OF PERIPHERAL NEUROPATHY USING CLINICIAN- AND PATIENT-REPORTED OUTCOMES IN THE POLARIX STUDY

M. Trněný 1,* , L. Fogliatto 2 , F. Gardner 3 , I. Flinn 4 , J.-F. Larouche 5 , C. Haioun 6 , M. Canales 7 , H. Goto 8 , R. Hajek 9 , G. Salles 10 , J. Qiu 11 , A. Campinha-Bacote 11 , M. Yan 12 , R. Kothari 11 , G. Man 11 , D. Sahin 13 , J. Hirata 11 , C. Lee 11 , H. Tilly 14 , C. Flowers 15

1 Charles University General Hospital, Prague, Czechia; 2 UFRGS, Porto Alegre, Brazil; 3 Florida Cancer Specialists, Cape Coral; 4 Sarah Cannon Research Institute, Nashville, United States of America; 5 CHU de Quebec, Quebec, Canada; 6 Groupe Hospitalier Albert Mondor, Créteil Cedex, France; 7 Hospital Universitario La Paz, Madrid, Spain; 8 Hokkaido University Hospital, Sapporo, Japan; 9 University of Ostrava, Ostrava, Czechia; 10 Memorial Sloan Kettering Cancer Center, New York; 11 Genentech, Inc., South San Francisco, United States of America; 12 F. Hoffmann-La Roche Ltd, Mississauga, Canada; 13 F. Hoffmann-La Roche Ltd, Basel, Switzerland; 14 Centre Henri Becquerel, Rouen, France; 15 M.D. Anderson Cancer Center, Atlanta, United States of America

Background: Peripheral neuropathy (PN) is an identified risk of anti-microtubule agents, including polatuzumab vedotin and vincristine. POLARIX ( NCT03274492 ), a Phase III randomized, double-blind, placebo-controlled study comparing Pola-R-CHP with R-CHOP, demonstrated improved progression-free survival (PFS) with Pola-R-CHP (Tilly et al. NEJM 2022).

Aims: To evaluate the impact of Pola-R-CHP vs R-CHOP on PN using clinician- and patient-reported outcomes (ClinRO and PRO) data.

Methods: Patients with previously untreated diffuse large B-cell lymphoma (DLBCL) received Pola-R-CHP or R-CHOP. All patients provided informed consent. Pola-R-CHP is an investigational combination. ClinRO data were based on PN grading according to the NCI CTCAE v4.0. PRO data were generated from assessment of patient-reported PN symptoms at baseline and Day 1 of each cycle using the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group – Neurotoxicity (FACT-GOG/NTX) subscale, ranging from 0–44, with higher scores representing lower levels of PN (minimal clinically important difference: 1.38–3.68 [Cheng et al. Health Qual Life Outcomes 2020]).

Results: At baseline, ClinRO and PRO symptom scores showed low PN burden (Table). Overall incidence of PN was comparable between treatment arms (Pola-R-CHP: 52.9%; R-CHOP: 53.9%); most events were grade 1, and incidence of grade 2 (Pola-R-CHP: 12.2%; R-CHOP: 15.5%) and grade 3 (Pola-R-CHP: 1.6%; R-CHOP: 1.1%) events were comparable between treatment arms. FACT-GOG/NTX survey completion was high in both arms (96% at baseline; >80% at other timepoints). When evaluated by cycle, ClinRO and PRO demonstrated that more patients experienced earlier onset PN with R-CHOP than with Pola-R-CHP (Table), with ~10% more R-CHOP- than Pola-R-CHP-treated patients having clinician-reported PN in Cycle (C) 2–5, and a ~+1-point difference (i.e. fewer symptoms) in PRO symptom scores in C3–6 with Pola-R-CHP vs R-CHOP; by C8+, and during follow up, rates and symptoms of PN were similar. PN symptoms resulted in fewer dose reductions (3.9% vs 8.2%) and drug discontinuations (0.7% vs 2.1%) with Pola-R-CHP vs R-CHOP. Duration of PN was similar for both treatments.

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Summary/Conclusion: In the POLARIX study, Pola-R-CHP did not result in different rates or severity of PN vs R-CHOP. According to ClinRO and PRO data, PN occurred later following initial exposure to Pola-R-CHP than to R-CHOP, and there were fewer dose modifications with Pola-R-CHP than with R-CHOP. Overall, the risk of PN was manageable.

P1192: ASIA SUBPOPULATION ANALYSIS FROM THE PHASE III POLARIX TRIAL

Y. Song 1,* , J. Jin 2 , H. Tilly 3 , H. Zhang 4 , J. Cao 5 , J. Feng 6 , L. Zhang 7 , S. Rai 8 , H. Goto 9 , Y. Terui 10 , W. S. Kim 11 , H.-J. Shin 12 , J.-P. Gau 13 , C.-H. Tsai 14 , J. P. Sharman 15 , L. Bu 16 , J. Hirata 16 , C. Lee 16 , K. Izutsu 17 , J. Zhu 1

1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, Beijing; 2 The First Affiliated Hospital of College of Medicine, Zhejiang University, Zhejiang, China; 3 Centre Henri Becquerel and University of Rouen, Rouen, France; 4 Tianjin’s Clinical Research Center for Cancer, Tianjin Medical University, Tianjin; 5 Fudan University Shanghai Cancer Center, Shanghai; 6 Jiangsu Cancer Hospital, Nanjing; 7 Union Hospital Tongji Medical College Huazhong University of Science and Technology, Wuhan, China; 8 Kindai University Hospital, Osaka; 9 Hokkaido University Hospital, Sapporo; 10 The Cancer Institute Hospital of JFCR, Tokyo, Japan; 11 Samsung Medical Center; 12 Pusan National University Hospital, Seoul, South Korea; 13 Taipei Veterans General Hospital; 14 National Taiwan University Hospital, Taipei, Taiwan; 15 Willamette Valley Cancer Institute/US Oncology, Eugene; 16 Genentech, Inc., South San Francisco, United States of America; 17 National Cancer Center Hospital, Tokyo, Japan

Background: In the pivotal Phase III POLARIX trial ( NCT03274492 ), Pola-R-CHP demonstrated significantly improved progression-free survival (PFS) compared with R-CHOP, with a similar safety profile in patients with previously untreated diffuse large B-cell lymphoma (DLBCL; Tilly et al. 2022). An Asia subpopulation analysis was included as part of the trial design.

Aims: To analyze patients enrolled in Asia for the purpose of registration of the POLARIX trial in China.

Methods: Patients from mainland China, Hong Kong, Japan, South Korea, and Taiwan (enrolled during the global phase), and from the China extension cohort were included in the Asia subpopulation. All patients provided informed consent. POLARIX methods were previously described (Tilly et al. 2022). Briefly, patients with untreated DLBCL were randomized 1:1 using the same stratification factors to receive six cycles of Pola-R-CHP or R-CHOP, plus two cycles of rituximab alone. Pola-R-CHP is an investigational combination. The purpose of this analysis was to evaluate consistency of PFS (defined as ≥50% risk reduction in PFS) in the Asia subpopulation with the global population.

Results: Overall, 281 patients (intent-to-treat population; 150, 85, 31 and 15 patients in mainland China, Japan, South Korea and Taiwan, respectively) were analyzed (141 received Pola-R-CHP and 140 received R-CHOP); 160 patients from the global population and 121 from the China extension cohort. Median age was 63 (range 19–79) years, and most patients had an International Prognostic Index of 3–5 (61.9%). At the data cut-off of June 28, 2021 (median follow-up of 24.2 months), PFS was superior with Pola-R-CHP vs R-CHOP (hazard ratio [HR] 0.64; 95% confidence interval [CI]: 0.40–1.03) and met the consistency definition with the global population. The 2-year PFS rate was 74.2% (95% CI: 65.7–82.7) with Pola-R-CHP vs 66.5% (95% CI: 57.3–75.6) with R-CHOP. Other key efficacy results were similar to the global study results (Table 1). The safety profile was generally comparable for Pola-R-CHP vs R-CHOP, including rates of grade 3–4 adverse events (AEs; 72.9% vs 66.2%), serious AEs (32.9% vs 32.4%), grade 5 AEs (1.4% vs 0.7%), AEs leading to discontinuation of any study treatment (7.2% vs 5.0%), and incidence of peripheral neuropathy (all grades, 44.3% vs 50.4%), respectively.

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Summary/Conclusion: The results from the Asia subpopulation of POLARIX were consistent with the global study, with a clinically meaningful improvement in PFS (risk of disease progression, relapse or death reduced by 36%) following treatment with Pola-R-CHP vs R-CHOP, and a comparable safety profile in Asian patients with previously untreated DLBCL.

P1193: A POPULATION PHARMACOKINETIC MODEL OF HIGH-DOSE METHOTREXATE IN ADULTS WITH CANCER IDENTIFIES BASELINE ALBUMIN, BODY WEIGHT, AND TIME-DEPENDENT CREATININE AS PREDICTORS OF ACUTE KIDNEY INJURY

S. Howard 1,* , M. Ibarra 2 , R. Combs 3 , P. Schaiquevich 4

1 University of Tennessee Health Science Center, Arlington, United States of America; 2 Universidad de la Republica, Montevideo, Uruguay; 3 Resonance, Arlington, United States of America; 4 Consejo Nacional de Investigaciones Cientificas y Tecnicas, Buenos Aires, Argentina

Background: High-dose methotrexate (HDMTX) is an essential part of the treatment of several adult and pediatric malignancies. Despite meticulous supportive care during HDMTX administration, severe toxicities can occur, including acute kidney injury (AKI) which can delay methotrexate elimination, prolong hospital stay, delay subsequent anticancer therapy, and contribute to morbidity. Population pharmacokinetics provide a powerful tool to predict the time to clear HDMTX and adjust doses for subsequent courses.

Aims: We developed and validated pharmacokinetic models for adults with diverse malignancies and related their systemic exposure to methotrexate to the incidence of serious toxicity.

Methods: Anonymized, de-identified data were provided from 101 US oncology practices that participate in the Guardian Research Network, a non-profit clinical research consortium with access to entire electronic medical records, including clinical information, drug treatments with dose and route of administration, laboratory values, procedures, notes, and reports. Modeled variables included clinical, laboratory, demographic, and pharmacological data. Population pharmacokinetic analysis was performed on all adults who received HDMTX using nonlinear mixed effects modeling with MonolixSuite.

Results: A total of 708 HDMTX courses from 258 adults were analyzed, of which 90 (13%) were associated with acute kidney injury within 48 hours of the start of HDMTX and 31% with delayed methotrexate elimination (>2 standard deviations above the mean). A three-compartment model adequately fit the data, and a combination of time-dependent serum creatinine, baseline albumin, and allometrically scaled body weight best predicted methotrexate clearance. The 3-compartment model was validated using two reference datasets from adults receiving HDMTX as short or long infusions. Dose-normalized methotrexate concentration at 24 and 48 hours correlated with AKI. Patients with AKI who did not receive glucarpidcase experienced significantly longer hospital length of stay (p<0.01) and time to methotrexate elimination (p<0.01).

Summary/Conclusion: We developed a population pharmacometric model of HDMTX elimination that considers weight, albumin, and time-dependent creatinine to predict AKI and guide supportive care in patients with delayed HDMTX elimination.

P1194: A PHASE IB TRIAL OF ITACITINIB COMBINED WITH ALEMTUZUMAB IN PATIENTS WITH T-CELL PROLYMPHOCYTIC LEUKEMIA (T-PLL)

T. Kadia 1,* , F. Ravandi 1 , G. Borthakur 1 , E. Jabbour 1 , G. Montalban-Bravo 1 , L. Adewale 1 , S. Pierce 1 , C. Lacke 1 , M. Brandt 1 , N. Short 1 , N. Jain 1 , J. Burger 1 , A. Ferrajoli 1 , W. Wierda 1 , H. Kantarjian 1

1 MD Anderson Cancer Center, Houston, United States of America

Background: T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm with an aggressive clinical course including rapid lymphocyte proliferation, progressive organomegaly, lymphadenopathy, pleural and pericardial effusions, skin manifestations, and severe B-symptoms. Overall prognosis has been poor, with inadequate response to conventional chemotherapy and limited efficacy of allo stem cell transplant (SCT). The CD52 monoclonal antibody alemtuzumab (CAMPATH) has been the most effective therapy for T-PLL, but responses are not durable. Three quarters of cases of T-PLL harbor activating mutations in the JAK-STAT pathway ( JAK1, JAK3, STAT5b, IL2RG ).

Aims: We designed astudy to evaluate the combination of the JAK1 inhibitor itacitinib with CAMPATH to target activated JAK-STAT signaling.

Methods: 15 pts were enrolled (Table 1). 8 pts (53%) were newly diagnosed and 7 pts (47%) were previously treated, with a median of 1 (1-3) prior therapies, including 2 (33%) with prior SCT. 10 pts (67%) had chromosome 14 abnormality by conventional cytogenetics, and 6/6 (100%) pts tested by FISH had TCL1 positivity. 5/7 (71%) tested pts having activating mutations in the JAK-STAT pathway. Pts received a median of 4 cycles (1-12) of therapy, including a median of 2 (1-4) combination cycles and a median of 3 (0-10) maintenance cycles. Of 8 evaluable frontline pts, there were 4 (50%) complete remissions (CR), all MRD (-) by flow cytometry, and 3 (38%) partial remissions (PR) for an overall response rate of 88%. Of 7 evaluable previously-treated pts, there were 3 (43%) CR, all MRD (-) by flow cytometry, 1 (14%) PR, 2 non responders (29%), and 1 (17%) early death in a pt that declined transfusions, for an ORR 57%. The median number of cycles to best response were 2 (1-4) and 1 (1-2) in the frontline and salvage cohorts, respectively. While no objective responses were observed in WBC or lymph nodes during the 2 week window of single-agent itacitinib, pts experienced improvement in B-symptoms, splenomegaly, and rash during this period prior to CAMPATH. The regimen was well tolerated. The most common grade 3/4 AEs on study were lung infection (4), allergic reaction (3), other infection (2), neutropenic fever (1), and kidney injury (1). No AEs were attributed to single-agent itacitinib. 3 of the 6 (50%) responding frontline pts went on to allogeneic SCT, 1 patient (81 years old) died after 11 cycles on protocol of unknown cause, and 2 pts are ongoing on protocol. The median OS was 18.6 m and 8.5 m in the frontline and salvage cohorts, respectively.

Results: Single-agent itacitinib was associated with patient reported improvements in B-symptoms, rash, appetite, and reduced splenomegaly. Response rates with the combination were encouraging in frontline and salvage, with favorable OS. Further study of the combination without a 2-week window (of itacitinib alone) and with objective measurement of patient reported symptoms is planned.

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Summary/Conclusion: The combination of itacitinib 200 mg daily and CAMPATH in pts with T-PLL was safe and well tolerated with no dose-limiting toxicities. Single-agent itacitinib was associated with patient reported improvements in B-symptoms, rash, appetite, and exam findings of reduced splenomegaly. Response rates with the combination were encouraging in frontline and salvage, with favorable OS. Further study of the combination without a 2-week window (of itacitinib alone) and with objective measurement of patient reported symptoms is planned.

P1195: A REAL WORLD EXPERIENCE OF COMBINED TREATMENT WITH ROMIDEPSIN AND AZACITIDINE IN PATIENTS WITH PERIPHERAL T-CELL LYMPHOMA.

M. Kalac 1,* , C. Tam 2 , M. Goldfinger 3 , Z. Xiao 3 , S. Rajeeve 1 , F. Montanari 4 , J. Kanakry 5 , O. O’Connor 6 , E. Marchi 6

1 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States of America; 2 Department of Hematology, Peter MacCallum Cancer Centre, Melbourne, Australia; 3 Department of Medicine, Albert Einstein College of Medicine; 4 Department of Hematology, Yale University Cancer Center, New York; 5 Experimental Transplantation & Immunotherapy Branch, National Cancer Institute, Bethesda; 6 Division of Hematology and Oncology, University of Virginia Cancer Center, Charlottesville, United States of America

Background: Patients with peripheral T-cell lymphoma (PTCL) lack good treatment options, particularly in the relapsed and refractory setting (Mak V et al. J Clin Oncol 2013). Our work suggested that epigenetic therapies can be safe and effective for patients with PTCL, particularly those with T-cell lymphomas with a follicular helper (TFH) phenotype (Marchi E et al. Br. J Haematol 2015; O’Connor O.A. et al; Blood 2019; Falchi L et al. Blood 2020). It is thought that recurrent mutations in epigenetic factors, including Ten-Eleven Translocation-2 (TET2), DNA methyl transferase-3A (DNMT3A) and isocitrate dehydrogenase-2 (IDH2) may contribute to PTLC vulnerability to epigenetic drugs (Couronné L. et al. N Eng J Med 2012; Lemonnier F et al. Blood 2012).

Aims: Objective of this study was to evaluate the merits of romidepsin plus subcutaneous azacitidine in patients with PTCL when administered in a ‘real-world’ scenario. We retrospectively identified PTCL patients that were treated with azacitidine and romidepsin outside of a clinical trial based upon queries regarding off study use. The study was reviewed and approved by each Medical Center Institutional Review Board.

Methods: We have identified 17 patients world-wide whose pretreatment characteristics are shown in Table 1. These patients were treated using 3 different schedules: Schedule A: azacitidine 75mg/m2 s.c. on days 1-7, romidepsin 14 mg/m2 on day 1, 8 and 15 of a 28 day cycle (total of 6 patients); Schedule B: azacitidine 75mg/m2 s.c. on days 1-5, romidepsin 14 mg/m2 on day 8, 15 and 22 of a 35 day cycle; and Schedule C (total of 2 patients): azacitidine 75mg/m2 s.c. on days 1-7, romidepsin 12-14 mg/m2 on day 8, 15 and 22 of a 28 day cycle (total of 5 patients).

Results: Eleven patients had angioimmunoblastic lymphoma (AITL), 2 had adult T-cell leukemia/lymphoma (ATLL), 2 had TFH PTCL, 1 had PTCL-NOS and 1 had composite lymphoma (TFH PTCL with diffuse large B cell lymphoma). Ten of the 17 patients had next generation sequencing performed. Most common mutations found were those of TET2 (7 pts), RHOA (4pts), IDH2 (3pts) and DNMT3A (1 pt). One ATLL patient had mutations in TRAF3, FAT1 and MED12. Among these 17 patients, overall response rate (ORR) was 76% and the complete response rate (CR) was 52%. Median number of cycles was 4 (range 1-12). Treatment was well tolerated but notable adverse effects included nausea, fatigue, rash, neutropenia and thrombocytopenia. One patient experienced febrile neutropenia while another had pulmonary infiltrates (differential diagnosis included drug toxicity versus infection). Thrombocytopenia was the most common reason for dose reduction of romidepsin (to 12mg/m2) or its omission on day 8, 15 or 22. In 4 patients, azacitidine and romidepsin were used to achieve remission prior to allogeneic transplant (range of cycles 1-3), with all 4 patients were in CR at their last disease assessment.

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Summary/Conclusion: Subcutaneous azacitidine and romidepsin administered in a ‘real-world’ situation is highly effective in patients with relapsed PTCL with tolerable toxicity, and can be used to successfully bridge patients to stem cell transplant. Notably, the efficacy was similar to the one reported on a clinical study with oral azacitidine and romidepsin.

P1196: DIFFERENT MUTATIONS IN PATIENTS WITH PRIMARY LARGE B-CELL LYMPHOMA

I. Kamaeva 1,* , I. Lysenko 2 , N. Nikolaeva 2 , O. Kit 3 , I. Novikova 3 , Y. Gaysultanova 2

1 Hematology, National Medical Research Centre for Oncology, Rostov-on-Don; 2 Гематология; 3 Национальный медицинский исследовательский центр онкологии, Ростов-на-Дону, Russia

Background: Primary mediastinal large B-cell lymphoma (PMBCL) is a type of aggressive large B-cell lymphoma which affects mainly young patients and is characterized by a local spread and development of a tumor conglomerate in the mediastinum. One of the most important pathogenetic mechanisms of the development of PMBCLs is their genetic heterogenety. There are a lot of similarities between molecular profiles of diffuse large B-cell lymphoma, Hodgkin’s lymphoma, and gray zone lymphoma, which are described in the literature. However, there is no clear molecular-genetic characteristic of PMBCL. Despite the successful treatment of patients with modern methods of immunochemotherapy, a large number of patients are resistant to the therapy due to the tumor heterogeneity. The aim of this study was the detection of a clinically significant mutation for patients with PMBCL by next-generation sequencing.

Aims: To identify significant mutations in the genes in patients with PMBCL for the further studies.

Methods: The study included 47 patients diagnosed with different stages of PMBCL who received treatment from 2013 to 2021. NGS was performed using the AVENIO Tumor Expanded Panel, Roche, USA, which includes 77 genes. DNA was isolated using the Gene Read DNA FFPE Kit panel, Qiagen, USA. The data were processed using the AVENIO Oncology Analysis Software to search for clinically significant mutations. The mutations were also confirmed by Sanger sequencing.

Results: The genetic material of selected patients was analyzed for the presence of clinically significant mutations among the known databases: COSMIC: v83, TCGA 9.0, ExAC:1.0, dbSNP:150, 1000 Genomes: phase_3_v5b, SnpEff:4.2. A missense variant of clinically significant mutations in the FGFR3, PIK3CA, ALK, EZH2, RNF43, MET genes was identified in patients with refractory PMBCL. For example, an activating driver mutation in the FGFR3 gene was detected in the 9th exon ( rs121913479 ), which is pathogenic. The frequency of alleles was 25.54%. An activating driver mutation in the PIK3CA gene was detected in the 10th exon ( rs121913273 ), which is also pathogenic. The frequency of alleles was 10.88%. The aim of the study was to identify signaling pathways which were related to mutated genes. They were PI3K-AKT, JAK-STAT, RAP1, RAS, MAPK, stem cell regulation signaling pathway, which were also interpreted. According to the bioinformatics analysis, FGF/FGFR3 was the most significant signaling pathway for PMBCL in our study, because it is able to activate all listed pathways via FGFR-receptor activation.

Summary/Conclusion: NGS revealed clinically significant mutations in the genes in patients with PMBCL, the therapeutic potential of which requires the further studies.

P1197: AXICABTAGENE CILOLEUCEL (AXI-CEL) IN COMBINATION WITH RITUXIMAB FOR THE TREATMENT OF RELAPSED/REFRACTORY (R/R) LARGE B-CELL LYMPHOMA (LBCL): OUTCOMES OF THE PHASE 2 ZUMA-14 STUDY

P. Strati 1,* , L. Leslie 2 , P. Shiraz 3 , E. Budde 4 , O. O. Oluwole 5 , M. Ulrickson 6 , A. Ramakrishnan 7 , J. Sun 8 , R. Shen 8 , J. Kanska 8 , P. McCroskery 8 , J. Dong 8 , M. Schupp 8 , H. Xu 8 , K. Patel 9

1 The University of Texas MD Anderson Cancer Center, Houston, TX; 2 John Theurer Cancer Center, Hackensack, NJ; 3 Stanford University, Stanford, CA; 4 City of Hope National Medical Center, Duarte, CA; 5 Vanderbilt University Cancer Center, Nashville, TN; 6 Banner MD Anderson Cancer Center, Gilbert, Arizona; 7 Texas Transplant Physician Group, Austin, TX; 8 Kite, a Gilead Company, Santa Monica, CA; 9 Swedish Cancer Institute, Seattle, WA, United States of America

Background: Despite the success of axi-cel, ≈60% of patients have no response or relapse within ~2 y after treatment (Jacobson C, et al. ASH 2021. #1764), highlighting the need for more therapeutic strategies. In preclinical studies, rituximab augmented CD19 chimeric antigen receptor (CAR) T-cell function and increased tumor reduction and survival in murine models via synergistic targeting with CAR T-cells (Mihara K, et al. Br J Haematol. 2010).

Aims: Here, we report outcomes of ZUMA-14, a Phase 2, multicenter study of axi-cel in combination with rituximab in pts with R/R LBCL after ≥2 lines of systemic therapy.

Methods: Eligible patients were ≥18 y with R/R LBCL. Patients received one rituximab dose (375 mg/m 2 ) on Day -5, a conditioning regimen of cyclophosphamide and fludarabine on Days -5, -4, and -3, and a single axi-cel infusion of 2×10 6 CAR T cells/kg on Day 0. Starting on Day 21 post–axi-cel infusion, patients received 1 rituximab dose every 28 days for up to 5 doses. The primary endpoint was investigator-assessed complete response (CR) rate. Secondary endpoints included objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), safety, and biomarker assessments. The analysis reported here occurred after all treated patients had ≥12 months of follow-up.

Results: As of 12/2/21, 27 patients were enrolled, and 26 received axi-cel and ≥1 rituximab dose (15 patients received all 6 rituximab doses); 1 patient discontinued treatment due to an adverse event (AE). Median age was 63 y (range, 38-82), 54% were male, 81% had stage III/IV disease, 62% had extranodal disease, 38% had elevated lactate dehydrogenase, and 85% had an age-adjusted International Prognostic Index (aaIPI) ≥1 (35% aaIPI=2).

The CR rate was 65% (95% CI, 44%-83%), and the ORR was 88% (95% CI, 70%-98%). With a median follow-up of 17 months, 65% of the patients had ongoing response, with 57% ongoing in CR. Medians for DOR, PFS and OS were not reached. The estimated DOR and PFS rates at 12 months were 64% and 56%, respectively. The estimated 12-month OS rate was 76%, and 6 patients (23%) died of progressive disease. Most patients (92%) experienced Grade ≥3 AEs. Grade ≥3 cytopenias were reported in 85% of patients, with 38% ongoing on Day 30. Grade ≥3 neurologic events (NEs) occurred in 4 patients (15%); there was no Grade ≥3 cytokine release syndrome (CRS). Median times to onset of CRS and NEs were 4 days (range, 1-7 days) and 6 days (range, 3-32 days), respectively, with median durations of 5 days (range, 2-15 days) and 7 days (range, 1-39 days). No patients experienced myelodysplastic syndrome.

Median peak CAR T-cell levels were comparable to the ZUMA-1 pharmacokinetic profile. Immune-modulating cytokines, including granzyme B, IL-6, CXCL10, IFN-γ and IL-2, were induced in patients following axi-cel and rituximab infusion and were more prominently elevated in responders vs non-responders. Peak rituximab levels were also elevated in responders vs non-responders.

Summary/Conclusion: Results from ZUMA-14 demonstrated that axi-cel in combination with rituximab elicited a high CR rate and durable PFS with no new safety signals detected in patients with R/R LBCL.

P1198: ASSOCIATION OF PRETREATMENT TUMOR CHARACTERISTICS AND CLINICAL OUTCOMES FOLLOWING SECOND-LINE AXICABTAGENE CILOLEUCEL VS STANDARD OF CARE IN PATIENTS WITH RELAPSED/REFRACTORY LARGE B-CELL LYMPHOMA

F. L. Locke 1 , J. Chou 2 , S. Vardhanabhuti 2 , R. Perbost 3 , P. Dreger 4 , B. T. Hill 5 , C. Lee 6 , P. L. Zinzani 7,* , N. Kröger 8 , A. López-Guillermo 9 , H. Greinix 10 , W. Zhang 2 , G. Tiwari 2 , C. To 2 , P. Cheng 2 , A. Bot 2 , R. Shen 2 , S. Filosto 2 , J. Galon 11

1 Moffitt Cancer Center, Tampa, FL; 2 Kite, a Gilead Company, Santa Monica, CA, United States of America; 3 HalioDx, Marseille, France; 4 Heidelberg University Hospital, Heidelberg, Germany; 5 Cleveland Clinic Foundation, Cleveland, OH; 6 Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, United States of America; 7 University of Bologna, Bologna, Italy; 8 University Medical Center Hamburg, Hamburg, Germany; 9 Department of Hematology, Hospital Clinic, Barcelona, Spain; 10 Division of Hematology Medical University Graz, Graz, Austria; 11 INSERM, Sorbonne Université, Université de Paris, Centre de Recherche des Cordeliers, Equipe Labellisée Ligue Contre le Cancer, Laboratory of Integrative Cancer Immunology, F-75006, Paris, France

Background: The Phase 3 randomized ZUMA-7 trial in second-line (2L) relapsed/refractory (R/R) large B-cell lymphoma (LBCL) showed axicabtagene ciloleucel (axi-cel) superiority to standard of care (SOC; salvage chemotherapy and HDT-ASCT) in event-free survival (EFS; hazard ratio [HR], 0.398; P<.0001; Locke et al. N Eng J Med . 2021).

Aims: We report results of exploratory analyses of tumor characteristics, including pretreatment (preTx) tumor burden (TB), tissue hypoxia-related lactate dehydrogenase (LDH) level, and tumor microenvironment (TME).

Methods: TB was calculated as the sum of product diameters of ≤6 reference lesions (Locke et al. Blood Adv . 2020). Serum LDH was assessed. PreTx tumor samples were assessed for RNA expression by the NanoString IO 360™ panel and prespecified immune contexture indexes related to T-cell involvement (Immunosign 15 [IS15] and 21 [IS21]; Galon et al. ASCO 2020. #3022). ZUMA-1 data were used for comparison to patients with 3L R/R LBCL. CD19 protein expression was assessed by immunohistochemistry (H-score). Associations between biomarkers and clinical outcomes were assessed using descriptive statistics (P<.05 was significant).o

Results: Axi-cel EFS was superior to SOC for both high and low TB (HR, 0.29 and 0.49, respectively; P<.001 for both) and elevated and nonelevated LDH (HR, 0.32 and 0.5, respectively; P<.001 for both). EFS in axi-cel patients was not associated with preTx TB (HR, 1.01 [95% CI, 0.88-1.16]; P=.89) or LDH (HR, 0.98 [95% CI, 0.74-1.29]; P=.86), but was worse in SOC patients with higher preTx TB (HR, 1.17 [95% CI, 1.03-1.32]; P=.01) or higher LDH (HR, 1.29 [95% CI, 1.02-1.63], P=.03). PreTx TB was lower in SOC patients with ongoing response vs nonresponders and patients who relapsed (P=.07), but not in axi-cel patients (P=.99). Non-germinal center B-cell (GCB) cell-of-origin subtypes is a poor prognostic factor for EFS in SOC (EFS was significantly worse in SOC patients with non-GCB vs GCB; HR, 1.82 [95% CI, 1.12-2.96]; P=.02) but not in axi-cel. In IO360 analysis, gene expression of B-cell lineage antigens (CD19, CD20, BCMA) and markers highly expressed by tumor cells (CD45RA, IRF8, BTLA) positively associated with objective and durable responses to axi-cel. While axi-cel remained superior to SOC with high (>median) or low CD19 expression level, the probability of an ongoing response increased with a higher CD19 H-score. PreTx TME IS15 and IS21 scores were generally higher in 2L vs 3L.H (HR, 1.29 [95% CI, 1.02-1.63], P=.03). PreTx TB

was lower in SOC patients with ongoing response vs nonresponders and patients

who

Summary/Conclusion: Axi-cel was superior to SOC in all subgroup analyses, including higher TB and LDH. Durable responses with axi-cel were greatest in tumors with prominent B-cell features, but were superior to SOC regardless of these features. Axi-cel intervention in 2L is supported by durable response rates not impacted by high TB, as seen in 3L axi-cel or 2L SOC. Higher preTx immune involvement in 2L vs 3L tumors suggests high TB may be overcome with axi-cel in patients with a more favorable immune contexture.

P1199: MACHINE-LEARNING ALGORITHM TO DETECTION OF LYMPHOMA WITH BONE MARROW INVOLVEMENT VIA FDG-18 PET/CT

C.-H. Lee 1,* , C.-L. Ho 2

1 Hematology and Oncology, Tri-Service General Hospital/National Defense Medical Center; 2 Hematology and Oncology, Tri-Service General Hospital, Neihu Dist, Taiwan

Background: Lymphoma is the commonest hematological malignancy in the world. The evaluation of bone marrow (BM) status is a key process in the initial workup of patients diagnosed with lymphoma. The BM is the most common site of extranodal involvement in lymphoid malignancies and the frequency of BM involvement varies according to the lymphoma subtype. Currently, noninvasive clinical staging by computed tomography (CT), or positron emission tomography (PET)-CT cannot fully assess BM involvement, which is possible only by pathological staging (aspiration and trephine biopsy). However, the invasive procedure is not indicated for fragile, critical illness patients and poor performance status patients. 18F-FDG PET / CT is used clinically for the determination of disease status in patients with lymphoma. However, the visual analysis of 18F-FDG diffuse bone marrow uptake in detecting bone marrow involvement (BMI) in routine clinical practice is still challenging.

Aims: To create a machine learning algorithm of 18F-FDG PET/CT for identifying bone marrow involvement in lymphoma patients.

Methods: Lymphoma patients underwent both bone marrow biopsy (BMB) and 18F-FDG PET/CT within one week were eligible. We took BMB results as the gold standard for bone marrow involvement. The bone marrow 18F-FDG uptake was visually diagnosed as positive and negative by two senior nuclear medicine experts. Totally, 1118 PET radiomic features were extracted to provide a more thoroughly understanding of the embedded pattern automatically. To select the most valuable and predictive features, an unsupervised consensus clustering method was first performed to analyze the feature correlations and then used to guide a random forest supervised machine learning model for feature importance analysis. We set 80% of PET images as training dataset and the rest as independent validation dataset. Cross-validation and independent validation were conducted to justify the performance of our model.

Results: Totally, 529 consecutive lymphoma patients with 1,118 PET images were performed in this model. The representative example of deep learning detection showed at figure. In terms of bone marrow involvement images, 227 images were for training, 44 images and 51 images were for validation and test, respectively. As non-bone marrow involvement, 555 images were as training set, 84 images and 157 images were took as validation and test.

The sensitivity, specificity, and accuracy of visual analysis for BMI diagnosis were 63.6%, 68.7%, and 71.2%, respectively. With the cross-validation on the training group, the machine learning model correctly predicted 93/112 patients in BMI. The sensitivity, specificity, and accuracy of the machine learning model in BMI detection were 91.3%, 80.8%, and 83.3%, respectively, significantly higher than the ones in visual analysis ( P < 0.05). The evaluation on the independent test group showed that the machine learning model could achieve 85.1% accuracy.

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Summary/Conclusion: 18F-FDG PET/CT radiomic analysis with machine learning model provided a quantitative, objective and efficient mechanism for identifying BMI in the patients with suspected lymphoma. It is suggested in particular for the diagnosis of BMI in the patients with 18F-FDG diffuse uptake patterns. Our algorithm could provide a potential simply, non-inferior and rapid detection ability of lymphoma with bone marrow involvement to replace invasive bone marrow biopsy for patients who are ineligible for invasive procedure.

P1200: RELAPSE AND OCCURRENCE OF SECOND PRIMARY MALIGNANCIES IN PATIENTS WITH DIFFUSE LARGE B CELL LYMPHOMA IN RITUXIMAB ERA, A NATIONWIDE OBSERVATIONAL STUDY

H. J. Lee 1,* , M. H. Chang 2 , S. V. Anh 3 , D. W. Kim 4 , Y. E. Lee 1

1 Internal Medicine, Dongguk University Ilsan Hospital; 2 Internal Medicine, National Health Insurance Service Ilsan Hospital, Goyang-si; 3 Health Convergence, Ewha Womans University, Seoul; 4 Information and Statistics, Gyeongsang National University, Jinjoo, South Korea

Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of B cell lymphoma. Rituximab, Cyclophosphamide, Adriamycin, Vincristine, and prednisolone (R-CHOP) is the standard of care in newly diagnosed DLBCL and has been covered by National Health Insurance Service (NHIS) of Korea since October 2004. The Korean government has established the National Health Information Database (NHID) in 2006. NHID contains identified claims, health screening data and mortality data and has been provided to researchers since 2010.

Aims: We conducted retrospective study using data from NIHD to describe the type and timing of relapse and occurrence of second primary malignancies (SPM) in patients with DLBLC who were treated with R-CHOP.

Methods: All patients who received inpatient care between 2004 and 2013 for an initial diagnosis of DLBLC in NHID were included. Eligible patients with DLBLC in 2004~2013 were identified after excluding potential pre-existing cares of DLBLC (n=9,610). Event Free Survival (EFS) was defined as time from the 1st day of R-CHOP therapy until relapse or progression, unplanned next treatment, or death as a result of any cause. SPM incidences were evaluated in patients who survived more than 1 year after the 1st day of R-CHOP therapy. The patients were followed up until December 2018 to confirm relapse, death, or occurrence of SPM using cox-proportional hazard model.

Results: From 2004 to 2013, a total of 9610 patients who had not been previously diagnosed with another type of cancer and received R-CHOP treatment for the first time were identified. The average age of the patients at the time of diagnosis was 57.5 years (standard deviation 15.04), and the male to female ratio was 56.5% and 43.5%, respectively. 11.53% (n=1108) of patients who received R-CHOP chemotherapy also received radiation therapy. During the entire follow-up period, 5-year survival rate of patients treated with R-CHOP chemotherapy was 65% (95% CI, 64.1 – 66.0), and the 5-year Event Free Survival (EFS) rate was 56.5% (95% CI, 55.5 – 57.4) with time showed a continuously decreasing pattern. DLBCL specific relapse free survivals were 83.7% (95% CI, 82.9 – 84.4) at 1 year and 78.4% (95% CI, 77.5 – 79.3) at 2 year. It was confirmed that there were rare DLBLCL specific relapse cases after 2 years of R-CHOP treatment (Figure 1). Among the 7384 patients who survived 1 year or more after completing R-CHOP therapy, SPM occurred in a total of 493 (6.7%) patients. Hematologic malignancy (34.1%, n=168) was the most common type of SPM, followed by stomach cancer in 49 (9.9%) patients, lung cancer in 44 (8.9%) patients, and colon cancer in 31 (6.3%) patients.

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Summary/Conclusion: The recurrence was uncommon after 2 years in DLBLC patients treated with R-CHOP therapy as the initial treatment. However, the occurrence of SPM was higher than the cancer prevalence of normal population. SPM could be a factor that affect the life expectancy of the long term survivors from DLBCL. Close monitoring for the occurrence of SPM may be necessary in DLBLC survivorship care.

P1201: ZANUBRUTINIB PLUS RCHOP(ZR-CHOP) REGIMEN ACHIEVES HIGH COMPLETE RESPONSE RATE IN THE TREATMENT OF NEWLY-DIAGNOSED DOUBLE-EXPRESSION DIFFUSE LARGE B CELL LYMPHOMA

Q. He 1 , L. Xie 1 , R. Zhao 1 , J. Ma 1 , C. Liu 1 , C. Chen 2 , H. Wang 1 , K. Lu 1 , J. Li 2 , C. Zhao 2 , N. Shan 3 , L. Xing 1 , Z. Li 1,*

1 Department of Hematology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences; 2 Qilu Hospital of Shandong University; 3 Shandong Provincial Hospital, jinan, China

Background: Diffuse large B cell lymphoma with double expression of MYC/BCL2(DE-DLBCL), is usually responsive poorly to standard therapy of R-CHOP regimen, and the long-term survival rate of patients with DE-DLBCL is only 30% - 40%. Recently, Phoenix study demonstrate Ibrutinib plus R-CHOP improved EFS and OS (versus placebo plus R-CHOP) for DE-DLBCL younger patients. While for elder patients, the outcome makes no difference between two groups, due partly to the poor tolerance in Ibrutinib group. The novel BTK inhibitor, zanubrutinib, have better safety profile. So we suppose zanubrutinib plus R-CHOP (ZR-CHOP) may improve the outcome of DE-DLBCL even in older patients.

Aims: To investigate the response rate, progression free survival (PFS) and safety of Zanubrutinib plus RCHOP (ZR-CHOP) regimen in the first-line treatment of DE-DLBCL patients.

Methods: We initiate a prospective, single arm, phase II clinical trial. Standard dose of Zanubrutinib plus R-CHOP regimen (ZR-CHOP) was adopted in our center as first-line treatment for patients (18-75yrs) with DE-DLBCL. DE-DLBCL is defined as ≧40% positive rate for MYC and ≧50% for BCL2 by immunohistochemistry, and double hit cases were excluded through FISH test (MYC, BCL2 and BCL6). The patients in Ann Arbor stage I were also excluded. The 6 cycles of ZR-CHOP plus 2R were administered as induction therapy, with no maintenance. The primary endpoint was two-year-PFS. Response was assessed after 2 cycles of therapy using CT scanning, and after 4 and 6 cycles of therapy using PET-CT.

Results: From November 6, 2020 to February 16, 2022, 28 patients with DE DLBCL were enrolled and completed 6 cycles of ZR-CHOP regimen therapy. The baseline characteristics of the patients are listed in Table 1. After 2 cycles of therapy, 2/28 patients achieved CR (7.1%), 25/28 PR (89.3%) and 1/28 not evaluated. After 4 cycles of therapy, 23/28 got CR (82.1%), 3/28 PR (10.7%), and 2/28 PD (7.1%). After 6 cycles of therapy, 24/28 CR (85.7%), and 4/28 PD (14.3%) (Figure 1). The response rate after 6 cycles of ZRCHOP regimen was obviously higher than that of the previous report after R-CHOP regimen for DE-DLBCL (85.7%VS 70%). After median follow-up of 6 month (2-14m), the estimated PFS rate is 88.2%（95%,CI 81.7-94.7, Figure 2）.Totally four patients progressed during or shortly after the induction therapy. NGS analysis revealed 3/4 patients has the mutation of TP53, solely or with other cytogenetic disorders, and the other one has the mutation of ATM. All the four patients are still alive after second line therapy including CART therapy and auto-stem cell transplantation.

About the safety of the study, grade 3 or more severe AEs were recorded in 15/28 patients (42.9% vesus 89.9% in PHOENIX study), including 12 patients with grade 3-4 hematological toxicities and 3 patients with pneumonia. No patients withdraw permanently from the study due to toxicity. No atrial fibrillation, tumor lysis syndrome or massive hemorrhage were reported.

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Summary/Conclusion: Despite the small sample size and short time of follow-up, we found that ZR-CHOP regimen achieved very high CR rate for DE DLBCL, and the toxicities were acceptable, which may indicate a promising therapy for this subset of DLBCL. Further studies are required to prove the benefit of zanubrutinib when added to R-CHOP for newly-dignosed DE-DLBCL. TP53 mutation is probably the main reason for disease progression.

P1202: INTENDED USE OF GLUCARPIDASE IN PATIENTS WITH CENTRAL NERVOUS SYSTEM LYMPHOMA AND IMPAIRED RENAL FUNCTION – THE VALIDATE STUDY

S. Liebig 1,* , S. Burock 2 , I. Merz 1 , F. Knörnschild 1 , P. Kießling 3 , K. Kappert 3,4 , U. Keller 1,5,6 , S. Schwartz 1

1 Department of Hematology, Oncology and Tumorimmunology, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Campus Benjamin Franklin; 2 Charité Comprehensive Cancer Center, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin; 3 Labor Berlin, Charité Vivantes GmbH; 4 Institute of Diagnostic Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin; 5 German Cancer Research Center (DKFZ); 6 German Cancer Consortium (DKTK), Heidelberg, Germany

Background: Central nervous system lymphoma (CNSL) could occur as a primary disease or as a secondary manifestation of a systemic lymphoma; most patients (pts) with primary CNSL are diagnosed at an age >60 years. High-dose Methotrexate (HD-MTX) is an essential backbone of established treatment regimens, but chronic renal failure inevitably results in delayed MTX elimination, prolonged drug exposure and enhanced toxicities. Thus, chronic renal failure limits MTX-based therapies in adequate doses in pts with CNSL. Glucarpidase, approved for use in pts with delayed MTX clearance, is a zinc-dependent enzyme, which rapidly cleaves MTX into non-toxic metabolites and offers an alternative route of MTX elimination.

Aims: The ongoing phase I/II VALIDATE study evaluates the safety as well as pharmacological efficacy (primary study endpoints) of intended and repeated use of Glucarpidase following HD-MTX in pts with CNSL and impaired renal function. The pharmacological efficacy of Glucarpidase will be assessed by determining the immediate and sustained reduction of MTX plasma concentrations after Glucarpidase administration, in addition to leucovorin.

Methods: Pts with histologically confirmed CNSL and renal insufficiency, defined as either i) (estimated) glomerular filtration rate of 40–80 mL/min, or ii) experienced renal failure during a previous HD-MTX treatment, defined as doubling of blood creatinine compared to the baseline value, are eligible for study inclusion. Study pts may receive up to 6 cycles of HD-MTX (additional Rituximab prior HD-MTX is allowed as standard of care treatment) at MTX dose levels of 3.0 or 3.5 or 4.0 g/m 2 body surface area administered over 4 hours (6 pts per dose level). Treatment cycles are repeated every 14 days (maximum interval 28 days). A fixed dose of 2000 units Glucarpidase or 20 units/kg in pts weighing > 100 kg is injected 24 hours after start of the HD-MTX administration. MTX and MTX metabolites are closely monitored in acidified plasma samples using combined high performance liquid chromatography and mass spectrometry (LC-MS).

Results: Currently, 4 pts have been enrolled and treated at the MTX dose level of 3.0 g/m 2 . Of these, 2 pts completed 6 HD-MTX courses and 2 pts have completed so far 2 and 4 courses, respectively. Glucarpidase administration resulted in a rapid and pronounced reduction of MTX blood levels (> 97%) within 15 minutes and in all 12 treatment cycles. Treatment intervals ranged from 12 to 32 days, including 2 intervals longer than 28 days due to infectious complications (respiratory syncytial virus pneumonia, N=1; urinary tract infection, N=1). Noteworthy, no deterioration of kidney function was observed. Hypersensitivity reactions occurred in 3 of 4 pts in treatment cycles 3 or 4 onwards, requiring therapeutic intervention and subsequent prophylaxis. The 2 pts, who completed 6 HD-MTX courses, experienced a complete remission of their lymphoma.

Summary/Conclusion: These first data demonstrate that treatment with HD-MTX and Glucarpidase is feasible in pts with CNSL and impaired renal function. However, the available data are currently limited and the first HD-MTX escalation step has not yet been reached. The observed hypersensitivity reactions are possibly due to antibody formation, which will be further investigated and prophylactic measures at least beyond the first treatment cycle will be considered.

P1203: THE OPTIMIZED HLH INFLAMMATORY (OHI) INDEX IDENTIFIES LYMPHOMA PATIENTS WITH UNEXPECTEDLY HIGH MORTALITY NOT RELATED TO DISEASE PROGRESSION

A. Zoref-Lorenz 1,2,3,* , J. Murakami 4 , L. Hofstetter 3,5 , U. Abadi 1,3 , S. Iyer 6 , S. Mohamed 7 , P. G. Miller 8 , A. E. H. Natour 3,9 , S. Weinstein 3,10 , J. Yacobovich 3,11 , S. Izraeli 3,11 , S. Nikiforow 3,8 , B. Ebert 8 , R. Gurion 5,12 , I. Cohen 3,5 , O. Pasvolsky 5 , P. Raanani 3,5 , A. Nagler 3,13 , N. Berliner 14 , N. Daver 7 , M. Ellis 1,3 , M. Jordan 15

1 Hematology Institute, Meir Medical Center, Kfar Saba, Israel; 2 Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States of America; 3 Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 4 Clinical Laboratory Transfusion Medicine and cell therapy, University of Toyama, Toyama, Japan; 5 Institute of Hematology, Davidoff Cancer Center, Rabin Medical Center, Petah- Tikva, Israel; 6 Department of Lymphoma; 7 Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston; 8 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America; 9 Internal Medicine A, Meir Medical Center, Kfar Saba; 10 Internal Medicine D, Sheba Medical Center, Ramat Gan; 11 Hematology-Oncology Department, Schneider Children’s Medical Center of Israel, Petah Tikva; 12 Sackler School of Medicine, Sackler School of Medicine, Tel Aviv; 13 Hematology Institute, Sheba Medical Center, Ramat Gan, Israel; 14 Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston; 15 Divisions of Immunobiology and Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, United States of America

Background: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening inflammatory syndrome that may complicate hematologic malignancies (HM). We recently developed a simplified diagnostic and prognostic index termed the ‘optimized HLH inflammatory’ (OHI) index comprising the combined elevation of sCD25 (> 3,900 U/mL) and serum ferritin (> 1,000 ng/mL), which in HM patients both identifies HLH and predicts mortality more accurately than conventional criteria for HLH.

Aims: In this study, we examined whether mortality in our cohort is directly related to progressive malignancy vs. HLH-associated causes in OHI+ and OHI- patients.

Methods: We performed a multicenter, retrospective study of patients with newly diagnosed Lymphoma from Israel, the USA, and Japan for whom sCD25 and ferritin levels were measured either as routine surveillance or during investigation for HLH classified patients by their OHI status. The International Prognostic Index, International Prognostic Score, and Follicular Lymphoma International Prognostic Index were used to estimate the predicted prognosis of T/B cell non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, and follicular Lymphoma, respectively. The predicted five-year overall survival was calculated for each patient based on the relevant prognostic index and compared between OHI+ and OHI- patients using the unpaired t-test. The actual survival at five years/last follow-up was recorded, as was the cause of death. Finally, the odds ratios (ORs) for observed vs. predicted mortality and HLH- vs. malignancy-related death were calculated using the Chi-square test.

Results: 100 lymphoma patients were studied: 65% with B cell NHL, 18% with natural killer/ T cell lymphoma, 17% with Hodgkin’s Lymphoma; 37 were OHI+, and 63 were OHI-. The disease-relevant international prognostic index-predicted five-year survival did not differ between OHI + and OHI- patients (a mean of 58% n OHI+ and 57% in OHI- p = 0.62). However, the observed five-year survival in OHI+ patients was lower (12%) than predicted, reflecting a mortality incidence that was four times higher than predicted by the relevant prognostic score (OR 3.9; CI 1.3-12.1). By contrast, OHI- patients had better survival (79%) than predicted by their prognostic scores (OR 0.15; CI 0.07-0.34). More than half of the OHI+ patients died from non-malignant causes (39% multi-organ dysfunction or HLH, 18% infection), while most OHI- patients (92%) died from progressive malignancy. The likelihood of dying from multi-organ dysfunction or HLH was 26 times higher in OHI+ vs. OHI- patients (OR 26.2; CI 4.1-286.7).

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Summary/Conclusion: OHI index status strongly correlated with mortality in patients with lymphoma within our cohort, and death in OHI+ patients was largely due to causes other than progressive malignancy. The OHI index appears to identify a harmful inflammatory state and deserves further prospective study.

P1204: TREATMENT PATTERNS AND REAL-WORLD OUTCOMES IN PATIENTS (PT) WITH LARGE B-CELL LYMPHOMA (LBCL) WHO RECEIVED SECOND-LINE (2L) THERAPY

M. Lunning 1,* , M. Che 2 , T. Gu 2 , T. K. Le 2 , F. F. Liu 2

1 University of Nebraska Medical Center, Omaha; 2 Bristol Meyers Squibb, Princeton, United States of America

Background: Pts with LBCL who fail first-line (1L) treatment may be candidates for high-dose chemotherapy (HDCT)/hematopoietic stem cell transplantation (HSCT) if they respond to salvage chemotherapy. However, pts unable to undergo HDCT/HSCT have limited treatment options and often have poor outcomes.

Aims: To characterize the demographics, clinical characteristics, treatment patterns, and outcomes in pts with LBCL receiving 2L therapy with or without HDCT/HSCT in the real-world setting.

Methods: A retrospective observational study was performed in adult pts with relapsed or refractory LBCL in the United States using electronic health record data from the Flatiron Health database from 01/01/2011 to 09/30/2021. The index date was defined as the 2L therapy start date. Pts were followed from index date until the end of study or death, whichever occurred first. Eligibility criteria included a diagnosis of LBCL, 1L treatment with an anti-CD20 therapy and anthracycline, 2L therapy with or without HDCT/HSCT, and ≥ 12 mo of follow-up. Pt demographics, clinical characteristics, treatment patterns, and overall survival (OS) were examined. Survival probabilities were estimated by Kaplan-Meier (KM) methods. Risk of death between groups was assessed by Cox proportional hazards regression models.

Results: Out of 881 pts who met the eligibility criteria, 142 (16.1%) received HDCT/HSCT and 739 (83.9%) did not. Median follow-up time was 17.3 mo. Of all pts, median (range) age was 67.0 yr (19–85), 58.6% were male, 61.9% had stage III or IV disease, and 84.7% received treatment in a community setting. Of pts with/without HDCT/HSCT, median (range) age was 60.0 yr (19–77)/69.0 yr (23–85), 65.5%/57.2% were male, and 69.8%/60.3% had stage III or IV disease, respectively. Among pts with HDCT/HSCT, the most common 2L treatments were rituximab plus ifosfamide, carboplatin, and etoposide (R-ICE)-based (72.5%) and rituximab plus gemcitabine, dexamethasone, and cisplatin (6.3%); the most common third-line (3L) treatments were bendamustine plus rituximab (BR; 12.2%) and lenalidomide plus rituximab (12.2%). Among pts without HDCT/HSCT, the most common 2L treatments were R-ICE (25.3%) and BR (12.2%); the most common 3L treatments were rituximab plus gemcitabine and oxaliplatin (R-GemOX, 11.9%) and R-ICE (8.2%). Pts who received R-ICE might have been intended to proceed to HDCT/HSCT but did not due to lack of response to R-ICE. The median (95% confidence interval [CI]) KM estimates of OS were 22.1 mo (17.9–29.0), 100.0 mo (71.3–not reached), and 15.4 mo (12.9–19.7) in all pts and pts with or without HDCT/HSCT, respectively ( P < 0.0001); OS probabilities at 12/24 mo were 60.2%/48.4%, 85.9%/77.3%, and 55.2%/42.8%. Of the 881 pts, 645 (73.2%) and 236 (26.8%) relapsed in ≤ 12 mo and > 12 mo from 1L, respectively. The median (95% CI) KM estimate of OS was 14.3 mo (12.1–18.7) and 47.8 mo (35.8–not reached) in pts who relapsed in ≤ 12 mo versus > 12 mo from 1L, respectively ( P < 0.0001; Figure ); OS probabilities at 12/24 mo were 54.0%/42.4% and 77.1%/64.9%. The risk of death was significantly greater among pts who relapsed in ≤ 12 mo versus > 12 mo from 1L therapy (hazard ratio, 1.81; 95% CI, 1.45–2.26; P < 0.0001).

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Summary/Conclusion: Pts with LBCL who did not receive HDCT/HSCT in 2L (including those who were intended but failed to proceed to HSCT) or relapsed in ≤ 12 mo of 1L therapy initiation have a poor prognosis. Significant unmet need exists in pts who were unable to derive the benefit from HDCT/HSCT.

P1205: PROGNOSTIC ROLE OF TUMOR P53 MUTATION IN DIFFUSE LARGE B CELL LYMPHOMA: A SYSTEMATIC REVIEW AND META-ANALYSIS

J. Ma 1,* , H. Peng 1 , Q. He 1 , J. Shi 1

1 Medical Oncology, Shandong Cancer Hospital and Institute, Jinan, China

Background: TP53 gene is the most commonly mutated gene in human cancers, and dysregulation of the TP53 pathway is essential in cancer pathogenesis, predominantly in diffuse large B-cell lymphoma (DLBCL). In 2011, Lenka Stefancikova first reported the effect of TP53 mutation (21.3%) and treatment sensitivity prognosis in DLBCL patients. A 2012 article in BLOOD showed that missense mutations in the DNA binding domain of the TP53 gene, rather than deletes or heterozygous deletes, lead to poorer survival in DLBCL patients. In 2017, Wang et al. found that patients with TP53 gene mutation had a worse prognosis than patients with double-hit / double expression. In the same year, Thorsten Zenz retrospectively analyzed TP53 subgroup data from the Recover-60 trial, and TP53 mutations were associated with reduced complete response, event-free, progression-free, and overall survival.

Aims: We hope to find favorable evidence of whether P53 mutation affects diffuse large B lymphoma prognosis. Therefore, we conducted a systematic review and meta-analysis of P53 mutation rate, response rate, and prognostic significance in diffuse large B-cell tumors.

Methods: A literature search was conducted on the prognosis of P53 mutation in diffuse large B-cell tumors in the PubMed database. Data on the efficacy and prognosis of P53 mutation in diffuse large B-cell tumor patients were extracted, and the associations between P53mutations and clinicopathologic parameters were examined. Hazard ratio (HR) and 95% confidence interval (CI) were calculated and combined using the random-effect Mantel-Haenszel model. The effect of P53 mutation on outcome parameters was estimated with a 95% confidence interval odds ratio.

Results: Twenty studies with 2,704 patients were eligible for inclusion. The mutation rate of P53 in 19 studies was 21%. In 6 studies, the CR rate of P53 mutation patients was 36% (95% confidence interval 0.32-0.41); the total HR of CR was 0.39 (95% confidence interval 0.29-0.53). Data on overall survival (OS) and progression-free survival (PFS) were obtained from 16 and 8 studies. When comparing patients with P53 mutant DLBCL and P53 wild-type DLBCL, the total HR of OS and PFS were 2.26 (95% confidence interval 1.90-2.69) and 1.90 (95% confidence interval 1.49-2.42).

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Summary/Conclusion: Our fundings suggest that P53 mutation shows negative associations with CR and harms OS and PFS prognosis. Clinical trials targeting TP53 mutations are needed to solve the problem.

P1206: GASTROINTESTINAL BLEEDING SIGNIFICANTLY REDUCED THE EFFICACY AND SURVIVAL OF CD19 CAR-T CELL THERAPY IN PATIENTS WITH GASTROINTESTINAL TRACT INVOLVEMENT OF REFRACTORY/RELAPSED B-CELL LYMPHOMA

L. Ma 1 , Y. Dou 1 , R. Liu 1 , T. Xu 1 , F. Yang 1 , F. Xue 1 , P. Zheng 1 , S. Feng 1 , Y. Guo 1 , H. Shi 1 , B. Deng 2 , X. Ke 1 , K. Hu 1,*

1 Department of Adult Lymphoma; 2 Cytology Laboratory, Beijing GoBroad Boren Hospita, Beijing, China

Background: CD19 Chimeric Antigen Receptor T cell(CAR-T)therapy has been successfully used to treat refractory/relapse B-cell lymphoma (r/r B-NHL). However,the risk was significantly increased due to gastrointestinal bleeding of CAR-T cell therapy for gastrointestinal (GI) lymphoma.

Aims: To explored the risk factors of GI bleeding and the efficacy of CAR-T cell therapy for GI lymphoma.

Methods: From May 2019 to December 2021,26 patients with r/r B-NHL involvement of the GI tract were enrolled. The male/female ratio was 1.8:1, with a median age of 40.5 years (range, 19-69 years).The diagnosis included diffuse large B-cell lymphoma (DLBCL) (n=21) and Burkitt lymphoma (n=5). All patients were in advanced stage (stage III-IV), and received a median of 7 lines of prior treatment.The disease status was progressive disease (PD)in all patients who failed to multi-line therapies.The median International Prognostic Index (IPI) was 3 (range, 1-4).Most patients (16/26,61.5%) received bridging therapy before CAR T cell infusion.The most common site of involvement was the colonic(10/26,38.5%), followed by small intestinal (8/26,30.8%), gastric (5/26,19.2%) and multiple GI tract sites (3/26, 11.5%).Before the trial, the expression of CD19 antigen in tumor tissue was positive confirmed by pathology. Patients received FC regimen (fludarabine and cyclophosphamide) before CAR-T cells infusion. The kinetics and function of CAR-T cells was monitored by quantitative PCR and flow cytometry. The efficacy was evaluated by PET-CT every 3 month after CAR-T infusion.

Results: GI complications occurred in 21/26(80.7%), including GI bleeding in 11/26(42.3%), intestinal obstruction in 2/26 (7.7%), vomiting in and diarrhea in 8/26 (30.8%) respectively.The median GI bleeding time was 4 days(range,1-33days) after CAR-T infusion.The number of patients in bleeding group who received bridging therapy before CAR-T were significantly more than those in non-bleeding group (10/11, 90.9% vs. 6/15, 40%) (P=0.0259). The proportion of patients with ECOG score greater than 3 in bleeding group before CAR-T therapy was significantly higher than that in non-bleeding group (6/11,54.5% vs 2/15, 13.3%)(P=0.0384).There was no significant difference between dose of CAR-T cell infusion (P=1.0) and the median peak lever of CAR-T cells in vivo (P=0.8565) in two groups.The incidence of cytokine release syndrome(CRS)was 84.6%(22/26), of which 27.3%(6/22) was severe CRS (≥grade3).The incidence of ≥grade 3 CRS in GI bleeding group was higher than that in non-bleeding group (5/11, 45.5% vs 1/15, 6.7%) (P=0.0538). Altogether,the best overall response rate (ORR) and complete response rate (CR) rate were 61.5%(16/26) and 26.9%(7/26),respectively.The ORR of GI bleeding group was significantly lower than that of non-bleeding group (3/11,27.3% vs 13/15,86.7%) (P = 0.0076)).With the median follow-up time was 5.8 months (range, 0.5-30.4months), the overall survival(OS) and progression free survival(PFS) in one year were also 50%. OS and PFS rate at 1 year after infusion in GI bleeding group were significantly lower than those in non-bleeding group (OS 27.3% vs 57% (P = 0.0198), PFS 27.3% vs 60.9% (P = 0.0293)).3 cases(3/26,11.5%) died of GI bleeding and 10 cases (10/26,38.4%) died of disease progression.

Summary/Conclusion: Our study showed promising efficacy of CAR-T cell therapy in r/r B-NHL involving the GI tract.However the GI bleeding significantly reduced the efficacy and survival.ECOG score greater than 3 and receiving bridging therapy before CAR-T may be the risk factors of GI bleeding.

P1207: MULTICENTER RANDOMIZED CONTROLLED (COMPARATIVE) OPEN PROSPECTIVE STUDY TO EVALUATE THE EFFICACY OF THE R-DA-EPOCH-21 AND R-MNHL-BFM-90 ± AUTO- HSCT PROGRAMS IN PATIENTS WITH DE NOVO DIFFUSE B-CELL LAR

A. Magomedova 1,* , S. Kravchenko 1 , A. Misyurina 1 , Y. Mangasarova 1 , O. Margolin 1 , E. Fastova 1 , F. Babaeva 1 , L. Gorenkova 1 , M. Bagova 1 , E. Nesterova 1 , K. Sychevskaya 1 , T. Moiseeva 1 , A. Kovrigina 1 , T. Obukhova 1 , V. Dvirnyk 1 , I. Galtseva 1 , V. Galuzyak 1 , S. Maryina 1 , K. Danishyan 1 , I. Kostina 1 , A. Sudarikov 1 , G. Klyasova 1 , S. Kulikov 1 , Y. Chabaeva 1 , E. Zinina 2 , I. Kryuchkova 3 , S. Sizikova 3 , V. Sergeyevicheva 3 , T. Ksenzova 4 , E. Anikina 4 , T. Shelekhova 5 , O. Arzhanukhina 5 , K. Kaplanov 6 , T. Konstantinova 7 , O. Serdyuk 8 , T. Sychyova 9 , T. Chagorova 10 , I. Nechunayeva 11 , L. Anchukova 12 , O. Dzarasova 13 , E. Parovichnikova 1 , V. Savchenko 1

1 Federal State Institution “National Research Centre for Hematology” Ministry of Health Russian Federation, Moscow; 2 Regional Clinical Hospital, Surgut; 3 Research Institute of Fundamental Clinical Immunology, Hematology Department, Novosibirsk; 4 Regional Clinical Hospital No. 1, Hematology Department, Tyumen; 5 Clinic of Occupational Pathology and Hematology, Hematology Department, Saratov; 6 Regional Oncological Clinic No. 1, Hematology Department, Volgograd; 7 Regional Hematology Centre, Yekaterinburg; 8 Clinic of Occupational Pathology and Hematology, Saratov; 9 Regional Clinical Hospital, Hematology Department, Astrakhan; 10 Regional Oncological Clinic, Hematology Department, Penz; 11 City Hematology Centre, Novosibirsk; 12 Regional Clinical Hospital, Hematology Department, vologda; 13 Republican Clinical Hospital, Hematology Department, Vladikavkaz, Russia

Background: S hort-pulse NHL-BFM-90 chemotherapy is highly effective in pediatric aggressive B-cell lymphomas. We modified R3 arm of NHL-BFM-90 program (named R-mNHL-BFM-90) for adults under 60 years with high-risk de novo DLBCL and conducted the randomized study compared to well-known R-DA-EPOCH-21 chemotherapy

Aims: to evaluate R-mNHL-BFM-90 and R-DA-EPOCH-21 efficacy and toxicity and auto-H SCT reasonability as well in de novo adult DLBCL patients with 2 or more signs of poor prognosis

Methods: Treatment protocol was registered as NCT02842931 at www.clinicaltrials.gov . Inclusion criteria were newly diagnosed DLBCL, not otherwise specified (NOS), according WHO, 2008 and 2017 criteria; two or more signs of poor prognosis (i.e. intermediate and high aaIPI risk); age 18-60. Complete remission (CR), partial remission (PR) or disease progression (DP) corresponded Lugano 2014 criteria. The study design intended randomization for four parallel arms. The first one was 6-cycle R-DA-EPOCH-21 as described earlier. The second, 6-cycle R-DA-EPOCH-21 plus auto-HSCT. The third, 6-cycle (A-B-A-B-A-B) R-mNHL-BFM-90. In addition, the fourth one was 6-cycle R-mNHL-BFM-90 plus auto-HSCT. If CR was not achieved after 6-cycle initiating treatment in every arm additionally 2-cycle R-DHAP chemotherapy were completed before auto-HSCT if intended. G-CSF after chemotherapy was administrated according clinical guides. A total of 140 patients from 13 medical Russia clinics were included from February 2015 to June 2021. Randomization was done according “intention-to-treat” as follows: R-DA-EPOCH-21 (35 patients); R-DA-EPOCH-21+auto-H SCT (30 patients); R-mNHL-BFM-90 (37 patients); R-mNHL-BFM-90+auto-H SCT (38 patients). Ten patients were excluded from the cohort due various reasons. Therefore, the first-step analysis was carried out for 130 patients according “intention-to-treat”. In cases of PR after 6 cycles or progression at any treatment stage or relapse (a total 14 patients) we performed the second tumor biopsy. In all cases, the final diagnosis was assessed as indolent lymphoma transformation into DLBCL or other “non-DLBCL” lymphomas. So, the second-step analysis was performed for 116 “proved” de novo DLBCL patients.

Results: Evaluation of comparative efficacy was carried out in R-DA-EPOCH-21±auto-HSCT and R-mNHL-BFM-90±auto-HSCT cohorts. Detailed results are presented in Table 1. Hematological/non-hematological toxicity was acceptable in all groups. However, neutropenic fever and grade 3-4 thrombocytopenia differed statistically significantly (χ 2 test, p<0.05) between R-DA-EPOCH-21 and R-mNHL-BFM-90 arms. The value of auto- auto-HSCT will be determined later.

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Summary/Conclusion: Thus, de novo DLBCL NOS is potentially curable disease with no relapses. R-mNHL-BFM-90 program is highly effective in de novo DLBCL NOS adult patients. R-mNHL-BFM-90 therapy toxicity is acceptable. The estimated CR duration probability within 10 months after treatment completion in the high-risk group is 100% in R-mNHL-BFM-90 group compared to 57% in R-DA-EPOCH group (χ 2 test, p=0.0047).

P1208: CHARACTERISTICS AND CLINICAL OUTCOMES OF PATIENTS WITH ALK-POSITIVE ANAPLASTIC LARGE CELL LYMPHOMA: REPORT FROM THE PROSPECTIVE INTERNATIONAL T-CELL LYMPHOMA PROJECT

C. Chiattone 1,* , M. Civallero 2 , T. Fischer 3 , E. Miranda 4 , M. Manni 2 , N. P. C. Zing 3 , S. A. Pileri 5 , S. Montoto 6 , S. M. Horwitz 7 , M. E. Cabrera 8 , C. A. De Souza 4 , A. Nagler 9 , S. Luminari 2,10 , A. J. M. Ferreri 11 , K. R. Carson 12 , A. Re 13 , L. Rigacci 14 , L. Nassi 15 , M. Federico 2 , G. Inghirami 16

1 Santa Casa Medical School, Sao Paulo and Samaritano Hospital, Sau Paulo, Brazil; 2 CHIMOMO, University of Modena and Reggio Emilia, Modena, Italy; 3 Santa Casa Medical School, of Sao Paulo and Samaritano Hospital; 4 Hematology and Hemotherapy Center, University of Campinas, Sau Paulo, Brazil; 5 Division of Diagnostic Haematopathology, European Institute of Oncology, IRCCS, Milan, Italy; 6 Department of Haemato-Oncology, St Bartholomew’s Hospital, Barts Health NHS Trust, London, United Kingdom; 7 Department Medicine, Memorial Sloan-Kettering Cancer Center, New York, United States of America; 8 Sección Hematología, Hospital del Salvador, Universidad de Chile, Santiago de Chile, Chile; 9 Hematology Division BMT and Cord Blood, Bank Chaim Sheba Medical Center Tel-Hashomer, Tel-Hashomer, Israel; 10 Hematology, AUSL-IRCCS di Reggio Emilia, Reggio Emilia; 11 Lymphoma Unit, Department of Onco-Hematology, IRCCS San Raffaele Scientific Institute, Milan, Italy; 12 Oncology Saint Louis, Internal Medicine, Washington University School of Medicine, Washington, United States of America; 13 UO Ematologia A.O, Spedali Civili di Brescia, Brescia; 14 S.O.D. Ematologia Dipartimento Area Critica Medico Chirurgica, A.O.U. Careggi, Firenze; 15 Department of Translational Medicine, AOU Maggiore della Carità and University of Eastern Piedmont, Novara, Italy; 16 W Pathology and Laboratory Medicine, New York Presbyterian Hospital, Weill Cornell Medicine, New York, New York.eill Cornell Medical College, New York, United States of America

Background: Anaplastic large cell lymphoma kinase-positive (ALCL, ALK+) is a distinct lymphoid neoplasm whose malignant cells express CD30 (TNFR8), and carrying translocations of the anaplastic lymphoma kinase. Downstream ALK signaling cascades modulate both anti apoptotic signals and growth patterns fostering lymphoma proliferation and promoting an aggressive clinical behavior. T-cell Lymphoma Project (TCP) is the largest prospective multicenter cohort study conducted in newly diagnosed PTCL patients. Herein, we report the analysis of demographics, initial clinical characteristics, treatment patterns, and outcomes in ALCL, ALK+ patients enrolled into the TCP database.

Aims: We report the analysis of demographics, initial clinical characteristics, treatment patterns, and outcomes in ALCL, ALK+ patients enrolled into the TCP database.

Methods: Seventy-four institutions from 14 countries in North America, South America, Europe, and Asia collected data on patients diagnosed and treated at respective centers between September 2006 and February 2018. One hundred thirty-one patients (8.4% of the total cohort) were confirmed to have anaplastic large cell lymphoma - kinase positive (ALCL, ALK+), among a total of 1553 PTCL patients

Results: Patients’ median age was 39 years (18-84). Sixty-five patients (66%) had advanced-stage disease, although the majority (45 patients, 54%) had a low-risk International Prognostic Index score (0-1). Of 81 patients treated with chemotherapy, 97% received anthracycline-containing regimens. The overall response rate was 81%, with 69 patients (70%) achieving complete remission. OS and PFS estimated in 3 years were 77% (95% CI 55-99%) and 68% (95% CI 46-90%), respectively, and in 5 years were very similar 77% of OS (95% CI 62-92%) and 64% of PFS (95% CI 34-94%). Multivariate analysis for PFS the results were advanced stage (HR 4.72 95%CI: 1.43-23.9, P=0.015), elevated LDH (HR 4.85 95%CI: 1.73-13.60, P=0.014) and ECOG-PS≥2 (HR 5.25 95%CI: 1.68-16.4, P=0.024). Whereas for OS resulted elevated LDH (HR 3.77 95%CI: 1.98-14.17, P=0.014) and ECOG-PS ≥2 (HR 4.59 95%CI: 1.46-14.39, P=0.004).

Summary/Conclusion: Considering the low incidence and PTCL heterogeneity, diagnostic algorithms and therapeutic guidelines remain challenging. Indeed, although CHOP-like regimens of ALCL ALK+‘s treatment lead to favorable responses, ~30% need a second-line therapy (refractory or relapsed patients). Developing more specific/effective first/second-line protocols to achieve long-term response rates/eradication and preserve safety profiles remain highly desirable. We anticipate that only international-based studies will improve our collective knowledge and ameliorate outcomes.

P1209: INCIDENCE, TREATMENT AND OUTCOME OF BREAST IMPLANT-ASSOCIATED ANAPLASTIC LARGE CELL LYMPHOMA: A POPULATION-BASED COHORT STUDY IN THE NETHERLANDS

F. O. Meeuwes 1,2,* , M. Brink 3 , M. W. Van der Poel 4 , M. J. Kersten 5 , M. J. Wondergem 5 , P. G. Mutsaers 6 , L. H. Böhmer 7 , F. J. S. H. Woei-A-Jin 8 , O. Visser 9 , R. Oostvogels 10 , P. M. Jansen 11 , W. J. Plattel 2 , G. A. Huls 2 , J. S. Vermaat 12 , M. Nijland 2

1 Hematology, Martini Ziekenhuis; 2 Hematology, University Medical Center Groningen, Groningen; 3 Research and Development, Netherlands Comprehensive Cancer Organization (IKNL), Utrecht; 4 Hematology, Maastricht University Medical Center, Maastricht; 5 Hematology, Amsterdam University Medical Centers, Amsterdam; 6 Hematology, Erasmus MC Cancer Institute, Rotterdam; 7 Hematology, Haga Teaching Hospital, The Hague, Netherlands; 8 General Medical Oncology, University Hospitals Leuven, Leuven, Belgium; 9 Hematology, Isala Hospital, Zwolle; 10 Hematology, University Medical Center Utrecht, Utrecht; 11 Pathology; 12 Hematology, Leiden University Medical Center, Leiden, Netherlands

Background: Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare peripheral T-cell lymphoma (PTCL) that develops in the fluid of the seroma cavity or the pericapsular fibrotic tissue surrounding a breast implant. The first case of BIA-ALCL was reported in 1997. According to the Food and Drug Administration (FDA), 733 unique cases of BIA-ALCL have been reported worldwide as of 2020. Involvement of locoregional lymph nodes or other organs outside of the breast occurs infrequently. The management of patients with limited stage disease BIA-ALCL is primarily surgical, but the optimal treatment of patients with advanced stage disease is largely unknown.

Aims: The aim of our study was to describe the incidence, treatment and survival of all reported BIA-ALCL cases in the Netherlands.

Methods: All patients diagnosed with BIA-ALCL since 1997 were identified in in the Netherlands Cancer Registry (NCR) with survival follow-up through February 1 st , 2021. Baseline characteristics, a history of cancer, time between breast cancer (due to possible breast reconstruction with an implant following cancer surgery) and BIA-ALCL, treatment modality, best response to treatment and survival outcomes were collected. Overall survival (OS) was defined as the time from BIA-ALCL diagnosis to all-cause-death.

Results: Between 1997 and 2020, a total of 42 patients have been diagnosed with BIA-ALCL in the Netherlands. The incidence was constant with 1 to 2 patients per 2 years until 2017. From 2018 onwards, the number of patients increased fourfold and 24 patients have been diagnosed with BIA-ALCL since then. The median age of patients was 51 years (range 28-87 years). The majority of patients were diagnosed with stage I disease (76%; n=32). Stage II was seen in 17% (n=7) and stage III/IV in 7% (n=3) of patients. Of the 3 patients with advanced stage disease, one patient had leptomeningeal involvement, one had involvement of the manubrium sterni and one had liver involvement. Fourteen patients (31%) had a prior diagnosis of breast cancer. The median time between the breast cancer diagnosis and BIA-ALCL was 13,3 years (range 6,7-31,8). Treatment for all BIA-ALCL patients consisted of surgery (55%, n=23), chemotherapy (14%, n=6); chemotherapy and radiotherapy (7%, n=3); chemotherapy and autologous stem cell transplant (7%, n=3), surgery and chemotherapy (5%, n=2), surgery, chemotherapy and radiotherapy (2%, n=1) or radiotherapy (2%, n=1). For 3 patients (7%), the treatment was watchful waiting. Treatment modality according to disease stage is presented in Table 1. Of the 15 patients who received chemotherapy, 10 patients received CHOP with number of cycles varying between 3 and 8, 1 patient received 4 cycles of CHOP followed by 4 cycles of CEOP, 1 patient received 6 cycles of CHOEP, 1 patient received 3 cycles of CHOEP followed by 3 cycles of CEOP, and for 2 patients the exact regimen was unknown. Overall, with a median follow-up of 2,9 years (range 0,3-24,2), three patients had died and the 3-years OS was 98%.

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Summary/Conclusion: In the Netherlands, 42 cases of BIA-ALCL have been reported since 1997, with 24% of the patients diagnosed with stage II-IV. Although the incidence of breast cancer, and thereby the number of breast reconstructions, is on the rise, it is likely that the rapid increase in the number of BIA-ALCL cases since 2018 is due to increased awareness of this disease. The treatment of BIA-ALCL has been heterogeneous and there is an urgent need for uniformity, making evaluation of different strategies possible. Nevertheless, the prognosis is excellent with a 3-year OS of 98%.

P1210: IMMUNE CORRELATES OF RESPONSE TO GLOFITAMAB: BIOMARKER FINDINGS FROM A PIVOTAL PHASE II EXPANSION STUDY IN PATIENTS WITH RELAPSED OR REFRACTORY (R/R) DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL)

E. C. Piccione 1,* , A. Belousov 2 , H. Hamidi 1 , C. Carlo-Stella 3 , M. Dickinson 4 , F. Morschhauser 5 , L. Lundberg 2 , K. Humphrey 6 , S. Tracy 1 , M. Hutchings 7

1 Genentech, Inc., South San Francisco, CA, United States of America; 2 F. Hoffmann-La Roche Ltd, Basel, Switzerland; 3 Humanitas University and IRCCS Humanitas Research Hospital, Milan, Italy; 4 Peter MacCallum Cancer Centre, Royal Melbourne Hospital and The University of Melbourne, Melbourne, VIC, Australia; 5 Hôpital Claude Huriez and CHU de Lille, Lille, France; 6 Roche Products Ltd, Welwyn Garden City, United Kingdom; 7 Rigshospitalet, Copenhagen, Denmark

Background: Glofitamab is a T-cell-engaging bispecific antibody with a novel 2:1 configuration that confers bivalency for CD20 (B cells) and monovalency for CD3 (T cells). In an ongoing Phase I/II study ( NCT03075696 ), glofitamab demonstrated durable complete responses (CR) and a favorable safety profile in patients (pts) with R/R B-cell lymphomas (Phase I dose-escalation part; Hutchings, et al. 2021) and in pts with R/R DLBCL (pivotal Phase II expansion).

Aims: To evaluate tumor and peripheral blood (PB) biomarkers associated with clinical response to glofitamab in the pivotal Phase II expansion.

Methods: Pts with DLBCL (DLBCL, NOS, HGBCL, PMBCL, or trFL; n=107) and ≥2 prior therapies received intravenous (IV) obinutuzumab pretreatment (Gpt; 1000mg) 7 days before the first glofitamab dose. IV glofitamab was then given as step-up doses on Day (D) 1 (2.5mg) and D8 (10mg) of Cycle (C) 1 and at the target dose (30mg) on D1 of C2–12 (21-day cycles). Response was assessed by Independent Review Committee (Lugano 2014 criteria). All pts provided informed consent. Baseline (BL) tumor biopsies were assessed by CD20/PAX5 immunohistochemistry (n=64) and CD8/Ki67 immunofluorescence (IF) (n=61). PB biomarkers were evaluated by flow cytometry (n=87) and plasma cytokines by ELISA (n=70). Immune and stromal cell type were inferred from bulk tumor RNAseq samples (n=55) using xCell cell type enrichment analysis. All BL samples were collected prior to Gpt.

Results: Analysis of BL tumor biopsies showed all pts were CD20 positive (range: ~35–98% CD20+ PAX5+ B cells) and CD20 levels were not associated with response. A trend towards higher CD8 counts was observed in pts with CR (by IF in total CD8 and CD8+Ki67- T cells). Gene expression analysis showed that responders had higher tumor microenvironment abundance enrichment scores, including higher CD4+ and CD8+ T-cell subsets scores ( Figure ).

PB immune profiling revealed a positive association of BL counts of B cells (p<0.01), CD4 T cells (p=0.02), and CD4 effector memory (EM) cells (p=0.03) with response. There were no significant differences in BL counts of CD8 subsets, regulatory T cells, natural killer cells or monocytes. In pts with progressive disease (PD), there was a trend towards higher expression of the checkpoint marker PD1 on CD8 cells (p=0.05). Pts with CR had lower levels of IL-6 and C-reactive protein (CRP) at BL (test for association with CR: p=0.1 and p=0.01, respectively). Early on-treatment changes in PB were associated with response to glofitamab: induction of IL-6, IFNγ, IL-10, and TNFα in the 6 hours after the first glofitamab dose was strongest in pts with CR (IL-6, IFNγ, IL-10: 6- to 16-fold induction; TNFα: 1.5-fold induction). Transient margination of CD4 and CD8 T cells was strongest in pts with CR. Additional analyses, including ctDNA, are ongoing and updated data will be presented.

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Summary/Conclusion: Biomarker results from the expansion cohort in pts with R/R DLBCL were consistent with those from the dose-escalation (Broeske et al. 2021), including comparable BL tumor CD20 expression across response categories, a trend towards higher BL tumor CD8 levels in pts with CR, and lower BL IL-6 and CRP in pts with CR. T-cell margination was strongest in pts with CR. Several novel response-associated biomarkers were identified in PB including higher BL B cell, CD4, and CD4 EM counts in responders and higher PD1 expression on CD8 in pts with PD. These data suggest a model where the clinical activity of glofitamab may be dependent upon immune contexture in the tumor and PB at the start of treatment.

P1211: CLINICAL HETEROGENEITY OF HIGH-GRADE B-CELL LYMPHOMA. EDGE OF TRANSFORMATION.

A. Misyurina 1,* , S. Kravchenko 1 , A. Magomedova 1 , A. Kovrigina 2 , T. Obukhova 3 , K. Sychevskaya 1 , E. Zakharko 4 , E. Rubkina 4 , V. Misyurin 5 , F. Babaeva 1

1 Intensive high-dose chemotherapy of hemablastosis; 2 Pathology department; 3 Laboratory of cariology; 4 Flow cytometry laboratory, National Research Center for Hematology; 5 Laboratory of experimental biotherapy of tumors, “N.N. Blokhin National Medical Cancer Research Center”, Moscow, Russia

Background: In the revised version of WHO classification of 2017 th the group of high-grade B-cell lymphoma double-hit (HGBL DH) was distinguished from high-grade B-cell lymphoma not otherwise specified (HGBL NOS) based on revealing of rearrangements of genes c-MYC and BCL2 and/or BCL6 . Nowadays we have more data telling us that these provisional entities are represented by different types of lymphomas which can develop from low-grade: follicular (FL) or marginal zone lymphoma (MZL).

Aims: To show heterogeneity of HGBLs based on clinical features and to distinguish the group of transformed low-grade-lymphomas (LGLs) from de novo HGBLs.

Methods: We have analyzed the data of 99 patients (pts) with HGBLs that have been treated since 2010 till 2022 years. Six pts had HGBL TH, 26 pts - c-MYC/BCL2 HGBL DH, 16 pts - c-MYC/BCL6 HGBL DH and 51 pts - HGBL NOS. The diagnosis was confirmed by histological and immunohistochemical study in all cases. FISH was performed in all pts to reveal translocation of genes c-MYC (8q24), BCL2 (18q21) and BCL6 (3q27). We suggested an integral transformation index (ITI) indicating the probability of HGBL development from LGL (FL or MZL). It included following: histologically confirmed transformation (by two consequent biopsies or morphological signs of high- and low-grade lymphomas in one biopsy sample), lymphoma history longer than 4 months, discordant bone marrow involvement (infiltration by small B-cells), paraprotein secretion, leukemic blood count, submitted by flow cytometry. Compassion of groups was carried out using χ2 test. Differences were considered as significant at p-value < 0,05 (STATISTICA 12.0).

Results: Morphological signs of transformation from LGL were revealed: in 3/6 (50%) of pts with HGBL TH (from FL); in 11/26 (42%) of pts with c-MYC/BCL2 HGBL DH (from FL), in 6/16 (38%) of pts with c-MYC/BCL6 HGBL DH (from MZL) and in 4/51 (8%) of pts with HGBL NOS (1 – from FL, 3 - from MZL). When we estimated all HGBL pts regarding on ITI we revealed that: 34/51 (67%) of pts with HGBL NOS, 16/26 (62%) of pts with c-MYC/BCL2 HGBL DH, 13/16 (81%) of pts with c-MYC/BCL6 HGBL DH and 5/6 (83%) of pts with HGBL TH had at least one sign of transformation. ITI significantly increased the probability to reveal the transformation from LGL in pts with c-MYC/BCL6 HGBL DH (p=0,01) and HGBL NOS (p<0,001) compared with morphological study only. We hadn’t observed paraprotein secretion in group of pts with c-MYC/BCL2 HGBL DH or HGBL TH that could highlight pathogenic differences between c-MYC/BCL2 HCBL DH and HGBL TH from c-MYC/BCL6 HGBL DH and HGBL NOS. Furthermore, groups of patients with c-MYC/BCL2 HGBL DH and HGBL TH were predominantly presented by GCB-subtype (93%) while c-MYC/BCL6 HGBL DH – by non-GCB (44%), (p=0,03). We found a strong correlation between CNS involvement (at the time of disease onset or in relapse/progression) and having ITI score at least 1 point (from 1 to 4), (p=0,008).

Summary/Conclusion: We suggested a powerful Integral Transformation Index to distinguish cases of low-grade lymphomas transformation into high-grade. c-MYC/BCL2 HGBL DH and HGBL TH are more homogenous group of GCB-lymphomas in majority of cases represented by transformation of FL. C-MYC/BCL6 HGBL DH and HGBL NOS are more heterogenous and in some cases can be represented by transformation of MZL with paraprotein secretion. Cases of transformed FL without c-MYC rearrangements also can be involved into HGBL NOS basket. Involvement of the CNS is predominantly characteristic of transformed low-grade lymphomas.

P1212: PERSONALIZED TREATMENT FOR ELDERLY PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA

H. Nukariya 1,* , K. Miura 1 , H. Takahashi 1 , M. Nakagawa 1 , T. Hamada 1 , S. Itho 1 , T. Ichinohe 1 , T. Endo 1 , K. Kurihara 1 , T. Koike 1 , K. Iizuka 1 , S. Ohtake 1 , U. Yoshihito 1 , N. Iriyama 1 , Y. Hatta 1 , H. Nakamura 1

1 Division of Hematology and Rheumatology, Department of Medicine, Nihon University School of Medicine, Japan, Tokyo, Japan

Background: The optimal dose intensity of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for elderly patients with diffuse large B-cell lymphoma (DLBCL) has been controversial because the standard dose of CHOP is too toxic for some older adults. We previously developed Age, Comorbidity, Albumin (ACA) Index, a host-dependent prognostic model that can stratify the tolerability to R-CHOP treatment among patients aged ≥65-years with DLBCL.

Aims: This study aimed to evaluate the efficacy of applying the ACA Index to real-world clinical practice.

Methods: This was a retrospective, observational cohort study. Patients with DLBCL aged 65 years or older treated with R-CHOP with curative intent and written informed consent between January 2016 to February 2022 were included. The initial dose intensity (DI) was determined by International Prognostic Index (IPI), ACA Index, and organ functions by the team members of oncology physicians and pharmacists in our related institutions. ACA Index consisted of Age (>75 years), Comorbidity (Charlson Comorbidity Index score ≥3), and Albumin (<3.7 g/dL): patients were categorized into either Excellent (0 points), Good (1 point), Moderate (2 points), or Poor (3 points) group. Primary outcomes were treatment completion rate for full 6-cycles R-CHOP and estimated 2-year overall survival (OS). Secondary outcomes included initial DI; relative dose intensity (RDI) (i.e., regular-dose drugs delivered six cycles with just 21 days intervals equals 100%); and incidence of unplanned hospitalization, febrile neutropenia (FN), and treatment-related mortality (TRM).

Results: A total of 158 patients were treated with R-CHOP during the study period but 20 patients were excluded because of poor records ( N = 8), planned R-CHOP cycle number other than six ( N = 7, e.g., abbreviated cycles plus radiation therapy), and treatment incompletion ( N = 5). Thus, 138 patients with median 75 (range 65–91) years of age ware analyzed. Of these, 79 (57%) were high or high-intermediate risk in the IPI. Most patients had DLBCL not otherwise specified ( N = 126), but patients with double-hit lymphoma ( N = 5), intravascular lymphoma ( N = 4), and transformed lymphoma ( N = 3) were also included. In total, treatment discontinuation was observed in 27 patients due to adverse events ( N = 16), progressive disease ( N = 8), or patient’s request ( N = 3). Consequently, treatment completion rate and 2-year OS for all patients were 80% and 76%, respectively. For ACA Index, 30, 45, 45, and 18 patients were categorized into Excellent, Good, Moderate, and Poor group, respectively. Treatment completion rates and 2-year OSs of these groups were 97%, 89%, 69%, and 61% ( P = 0.0009) and 95%, 84%, 67%, and 46% ( P = 0.0003), respectively. Among them, mean initial DIs were 94%, 83%, 71%, and 66% ( P <0.0001) (Figure); mean RDIs were 89%, 75%, 59%, and 48% ( P <0.0001); incidences of unplanned hospitalization were 10%, 16%, 22%, 44% ( P = 0.03845); incidences of FN were 7%, 9%, 16%, and 28% ( P = 0.1712); and incidences of TRM were 0%, 0%, 2%, and 17% ( P = 0.0155). In an exploratory analysis for patients aged ≥80 years (N = 39%), treatment completion rate and 2-year OS were 72% and 69%, respectively, with mean initial DI and RDI of 62% and 53%, respectively.

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Summary/Conclusion: Our risk-oriented, personalized strategy conferred an excellent outcome among non-selected, population-based elderly patients with DLBCL. The study results were comparable or even favorable compared to those of major clinical trials, in which one-size-fits-all strategies were adopted.

P1213: SUBCUTANEOUS EPCORITAMAB WITH GEMOX INDUCED HIGH RESPONSE RATES IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA INELIGIBLE FOR AUTOLOGOUS STEM CELL TRANSPLANT

B. E. Wahlin 1,* , J. Brody 2 , T. Phillips 3 , R. Costello 4 , P. Lugtenburg 5 , R. Cordoba 6 , L. Wang 7 , J. Wu 8 , B. Elliott 7 , A. Abbas 7 , J. Jørgensen 9

1 Karolinska Institutet, Stockholm, Sweden; 2 Icahn School of Medicine at Mount Sinai, New York; 3 University of Michigan Comprehensive Cancer Center, Ann Arbor, United States of America; 4 Assistance Publique – Hôpitaux de Marseille (AP-HM), Marseille, France; 5 Department of Hematology, On behalf of the Lunenburg Lymphoma Phase I/II Consortium-HOVON/LLPC, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 6 Fundacion Jimenez Diaz University Hospital, Health Research Institute IIS-FJD, Madrid, Spain; 7 Genmab, Princeton; 8 AbbVie, North Chicago, United States of America; 9 Aarhus University Hospital, Aarhus, Denmark

Background: Outcomes are poor with standard palliative chemotherapy in patients (pts) with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) who fail or are ineligible for autologous stem cell transplant (ASCT). Rituximab with gemcitabine + oxaliplatin (GemOx) was associated with a complete response rate of 33% (Cazelles et al, Leuk Lymphoma 2021); new approaches are needed. Epcoritamab, a subcutaneously administered bispecific antibody, targets CD3 on T cells and CD20 on B cells. Epcoritamab had a manageable safety profile and antitumor activity in heavily pretreated B-cell non-Hodgkin lymphoma (NHL), including DLBCL, in the EPCORE NHL-1 dose-escalation trial. Epcoritamab + standard B-cell NHL therapies are being evaluated in the phase 1/2 EPCORE NHL-2 trial ( NCT04663347 ); results from arm 5 are shown here.

Aims: To evaluate the safety and preliminary efficacy of epcoritamab + GemOx in pts with R/R DLBCL who are ineligible for ASCT.

Methods: Adults who had R/R CD20 + DLBCL and had failed or were ineligible for ASCT were enrolled. Pts received epcoritamab (weekly, cycle [C] 1–3; every 2 wk, C4–9; every 4 wk, C≥10) and GemOx (every 2 wk, C1–4) until disease progression or unacceptable toxicity (28 d/C). To mitigate CRS, step-up epcoritamab dosing and prophylactic corticosteroids were required in C1. Response was assessed by PET-CT according to Lugano 2014 criteria. Informed consent was obtained.

Results: As of the data cutoff date (December 1, 2021), 27 pts (median age, 71 y; range, 47–87) had received epcoritamab + GemOx (epcoritamab 24 mg, n=3; 48 mg, n=24). Most pts were stage IV (56%), primary refractory (56%), and/or refractory to last therapy (70%). Number of prior therapies ranged from 1–13, with a median of 2 prior therapies. Treatment was ongoing in 16 pts (59%), with a median follow-up of 6.0 mo (range, 1.0–11.1). Treatment-emergent AEs (any grade [G]) in >50% of pts were CRS (70%), thrombocytopenia (70%), neutropenia (56%), anemia (52%), and infections (52%). Most cases of CRS occurred in C1; all were G1/2 and resolved (median time to resolution, 2 d). No pts discontinued epcoritamab due to CRS. ICANS (G3) and tumor lysis syndrome (G3) were reported in 1 pt each. Six pts had G5 AEs; the contribution of epcoritamab could not be ruled out by the investigator in 2 pts: small bowel perforation in 72-year-old with transformed DLBCL and extensive gastrointestinal involvement (achieved complete metabolic response [CMR]); acute hepatitis/multiorgan failure in 68-year-old with transformed DLBCL and liver involvement (achieved CMR). Of the 4 pts who had fatal AEs unrelated to epcoritamab according to the investigator: 2 pts aged 87 y had primary refractory disease and 2 pts aged 74 y had multiple comorbidities. Antitumor response is shown in Table 1. All 3 pts previously treated with CAR T-cell therapy remained on study treatment and in response (2 CMR, 1 partial metabolic response).

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Summary/Conclusion: No unexpected safety findings were observed for epcoritamab + GemOx when considering the individual safety profiles of epcoritamab and GemOx. Given the high unmet need in this R/R pt population, these initial data are encouraging; further clinical evaluation is warranted.

P1214: SUBCUTANEOUS EPCORITAMAB + R-CHOP FOR FIRST-LINE TREATMENT OF PATIENTS WITH HIGH-RISK DIFFUSE LARGE B-CELL LYMPHOMA: PHASE 1/2 UPDATE

M. R. Clausen 1,* , F. Offner 2 , D. Belada 3 , J. Brody 4 , K. M. Linton 5 , Y. Karimi 6 , R. Cordoba 7 , S. Snauwaert 8 , A. Abbas 9 , L. Wang 9 , J. Wu 10 , B. Elliott 9 , L. Falchi 11

1 Vejle Hospital, Vejle, Denmark; 2 Universitair Ziekenhuis Gent, Ghent, Belgium; 3 4th Department of Internal Medicine – Hematology, University Hospital and Faculty of Medicine, Hradec Králové, Czechia; 4 Icahn School of Medicine at Mount Sinai, New York, United States of America; 5 The Christie NHS Foundation Trust and Manchester Cancer Research Centre, Manchester, United Kingdom; 6 University of Michigan Comprehensive Cancer Center, Ann Arbor, United States of America; 7 Fundacion Jimenez Diaz University Hospital, Health Research Institute IIS-FJD, Madrid, Spain; 8 Department of Hematology, AZ Sint-Jan Hospital, Bruges, Belgium; 9 Genmab, Princeton; 10 AbbVie, North Chicago; 11 Lymphoma Service, Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Patients (pts) with newly diagnosed diffuse large B-cell lymphoma (DLBCL) with high/poor risk according to the revised International Prognostic Index (IPI) who are treated with immunochemotherapy (IC) have a 4-y survival rate of 55% (Sehn et al, Blood 2007). Epcoritamab is a subcutaneously administered, well-tolerated, bispecific antibody with single-agent antitumor activity in relapsed/refractory (R/R) aggressive B-cell non-Hodgkin lymphoma (NHL). As its mechanism of action and safety profile are distinct from IC, epcoritamab is well suited for use in combination therapy and in earlier lines of therapy. The suboptimal prognosis of high-risk pts may be improved with the addition of a novel agent to standard IC.

Aims: To present updated results of epcoritamab + rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in previously untreated pts with DLBCL and IPI 3–5 (EPCORE NHL-2 arm 1; NCT04663347 ).

Methods: Adult pts with previously untreated CD20 + DLBCL (IPI ≥3) received subcutaneously administered epcoritamab (every wk, cycles 1–4; every 3 wk, cycles 5–6) + R-CHOP for 6 cycles of 21 d, followed by epcoritamab monotherapy (every 4 wk, in cycles of 28 d, up to 1 y). Corticosteroid prophylaxis and step-up dosing of epcoritamab were required in cycle 1 to mitigate CRS. PET-CT with contrast was used to assess response per Lugano 2014 criteria. Informed consent was obtained.

Results: As of the data cutoff date of December 1, 2021, 33 pts had received epcoritamab + R-CHOP (epcoritamab 24 mg, n=4; 48 mg, n=29). Median age was 66 y (range, 19–82); median time from diagnosis to first dose of epcoritamab was 25 d (range, 5–70). At least 24% of pts had double- or triple-hit DLBCL; all had IPI ≥3. Median number of total cycles initiated was 5 (range, 1–13), and median follow-up was 3 mo (0–9.7). Among the 33 pts enrolled, 31 (94%) remained on treatment. The most common treatment-emergent adverse events (TEAEs) were neutropenia (48%; febrile neutropenia in 9% of all pts), CRS (45%), infections (42%), anemia (39%), injection-site reactions (36%), nausea (33%), constipation (30%), and pyrexia (30%). No pts discontinued epcoritamab due to TEAEs. AEs of special interest were CRS (24% grade [G] 1, 18% G2, 3% G3; resolved) and ICANS (3% G2; resolved); tumor lysis syndrome was observed in 1 pt (3% G3). Most CRS events were associated with the first full dose. Median time to resolution of CRS events was 2 d (range, 1–11); 4 pts with CRS were treated with tocilizumab. Epcoritamab was not discontinued or delayed due to CRS. There were no fatal TEAEs. Individual pt response profiles are shown in Figure 1. For efficacy-evaluable pts, irrespective of whether they had yet completed 6 cycles of therapy, the overall response rate (ORR) was 96% (24/25). Among the 10 pts who completed 6 cycles of R-CHOP and had a subsequent response assessment, the ORR and complete metabolic response (CMR) rate were 100% and 90%, respectively. These 10 pts remained in response as of the data cutoff date. The longest duration of response was 7.1+ mo and ongoing.

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Summary/Conclusion: Epcoritamab is the first subcutaneously administered bispecific antibody to be evaluated in combination with standard IC in pts with previously untreated DLBCL. These results show that epcoritamab + R-CHOP has a manageable safety profile. CRS events were observed to be mostly of low grade and were not associated with treatment discontinuation. Rates of overall response and CMR were high, and no relapses were reported as of the data cutoff date. Updated data will be presented at the meeting.

P1215: PRELIMINARY PHASE 1/2 RESULTS OF SUBCUTANEOUS EPCORITAMAB + R-DHAX/C IN PATIENTS WITH RELAPSED OR REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA ELIGIBLE FOR AUTOLOGOUS STEM CELL TRANSPLANT

R. Cordoba 1,* , L. Falchi 2 , T. Phillips 3 , S. de Vos 4 , M. Nijland 5 , F. Offner 6 , I. Bykhovski 7 , J. Wu 8 , L. Wang 7 , A. Rana 7 , P. Abrisqueta 9

1 Fundacion Jimenez Diaz University Hospital, Health Research Institute IIS-FJD, Madrid, Spain; 2 Lymphoma Service, Memorial Sloan Kettering Cancer Center, New York; 3 University of Michigan Comprehensive Cancer Center, Ann Arbor; 4 Ronald Reagan University of California Los Angeles Medical Center, Los Angeles, United States of America; 5 University Medical Center Groningen and University of Groningen, Groningen, Netherlands; 6 Universitair Ziekenhuis Gent, Ghent, Belgium; 7 Genmab, Princeton; 8 AbbVie, North Chicago, United States of America; 9 Hospital Universitario Vall d’Hebron, Barcelona, Spain

Background: Salvage immunochemotherapy, such as rituximab, dexamethasone, cytarabine, and oxaliplatin or carboplatin (R-DHAX/C), followed by consolidation with high-dose therapy (HDT) and autologous stem cell transplant (ASCT), is part of the current standard of care for patients (pts) with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) who are eligible for ASCT; however, this approach fails in many pts. To improve response and long-term outcomes, novel treatment options are needed. Epcoritamab is a subcutaneously administered bispecific antibody that targets CD3 on T cells and CD20 on B cells. With epcoritamab treatment, meaningful single-agent antitumor activity and a manageable safety profile were demonstrated in pts with heavily pretreated B-cell non-Hodgkin lymphoma (NHL) in the dose-escalation portion of the EPCORE NHL-1 trial. Initial results from arm 4 (epcoritamab + R-DHAX/C) of the EPCORE NHL-2 phase 1/2 trial ( NCT04663347 ) are presented here.

Aims: To evaluate safety and preliminary efficacy of epcoritamab + R-DHAX/C in pts with R/R DLBCL eligible for ASCT.

Methods: Adult pts with R/R CD20 + DLBCL who were eligible for HDT-ASCT received standard R-DHAX/C and subcutaneous epcoritamab (every wk, 21-d cycles 1–3). Pts who had HDT-ASCT postponed could continue epcoritamab monotherapy (28-d cycles: every wk, cycle 4; every 2 wk, cycles 5–9; every 4 wk, cycles ≥10) until disease progression or unacceptable toxicity. To mitigate CRS, step-up epcoritamab dosing and corticosteroids were required in cycle 1. Response was assessed by PET-CT using Lugano 2014 criteria. Informed consent was obtained.

Results: As of the data cutoff date (January 26, 2022), 27 pts had received epcoritamab + R-DHAX/C. Median age was 59 y (range, 30–75), 74% of pts had received 1 prior line of therapy, 26% had received 2–3 prior therapies, and 3 pts were previously treated with CAR T-cell therapy (CAR T). Most pts (70%) had primary refractory disease. Thrombocytopenia (67%), neutropenia (48%; febrile neutropenia, 19% of all pts), infections (37%), nausea (37%), and anemia (33%) were the most common treatment-emergent adverse events of any grade (G). Regarding adverse events of special interest, CRS occurred in 8 pts (30%; all G1/2) and ICANS occurred in 1 pt (G2; discontinued epcoritamab, pt with prior CAR T). Clinical tumor lysis syndrome was not observed. CRS events were reported early in treatment and resolved in a median time of 2 d. Of the 23 evaluable pts, 11 pts received ASCT; prior to ASCT, the overall response rate (ORR) was 100% (82% [9/11] complete metabolic response [CMR]; 18% [2/11] partial metabolic response [PMR]). HDT-ASCT was postponed/canceled and epcoritamab monotherapy continued for 12 pts; among these pts, 5 achieved CMR (42%, including 2 with prior CAR T) and 3 achieved PMR (25%). For the entire evaluable population, CMR and PMR rates were 61% (14/23) and 22% (5/23), respectively.

Summary/Conclusion: Subcutaneous epcoritamab had a manageable safety profile when combined with R-DHAX/C in pts with R/R DLBCL. When CRS events occurred, they were low grade and resolved following standard management. A high ORR and CMR rate were observed. As of January 26, 2022, 70% of pts had either undergone ASCT or continued epcoritamab treatment in CMR.

P1216: ZANUBRUTINIB PLUS R-CHOP(RI-CHOP) AS THE TREATMENT FOR DOUBLE EXPRESSION OF MYC/BCL2 IN PATIENTS WITH DIFFUSED LARGE B-CELL LYMPHOMA

K. Ren 1,* , L. zou 1 , M. jiang 1 , W. wu 1 , X. chen 1 , T. hai 1 , Q. luo 1

1 Department of Head and Neck Oncology, Department of Radiation Oncology, Cancer Center and State Key Laboratory of Biotherapy, sichuan university west china hospital, chengdu, China

Background: Diffuse large B cell lymphoma (DLBCL) with double expression of MYC and BCL2 proteins (DE-DLBCL) is an aggressive disease. The incidence of double expressor status in DLBCL was 20～30%.BCL2 overexpression was more frequent in Chinese compared with Western cases in the non-GCB subgroup. MYC/BCL2 co-expression is an adverse predictive factor for response to chemotherapy regimens, and associateswith a significantly lower probability of complete remission.

Aims: To investigate a prognostic significance of MYC/BCL2 double expression in DLBCL patients treated with intensive chemotherapy RI-CHOP, we conducted this single-arm prospective study.

Methods: We enrolled 31 patients aged 18 to 70 years old with double expressor status in DLBCL (DE-DLBCL) underwent modified protocol of intensive chemotherapy – zanubrutinib plus R-CHOP(RI-CHOP) in West China Hospital between 2020 and 2021 years were included in the study. We used the cutoff values of protein co-expression are 50% for BCL2 and 40% for MYC. This study excluded DE-DLBCL With c-MYC 、 BCL2 or BCL6 rearrangements and Central nervous system involvement. Oral zanubrutinib was given continuously (160mg twice daily) from Day 1. Patients were administered intravenously rituximab, cyclophosphamide doxorubicin, vincristine,and prednisone. All patients were recommended to receive 6 cycles of RI-CHOP. Patients received oral ciprofloxacin (500 mg daily on Day 3-13) to prevent bacterial infection.CT/PET-CT scans were conducted to assess efficacy in 2,4,6 cycles. Four cycles of intrathecal methotrexate administration (15 mg) were performed for patients with a high CNS-IPI score and/or lymphoma-risk factors warranting CNS prophylaxis.The primary endpoint was overall response rate (ORR), after mid-term and 6 cycles. The secondary endpoint was complete response ratio (CR) and adverse events (AE). AEs were graded according to CTCAE (version 5.0).

Results: 31 treatment-naïve DE-DLBCL patients diagnosed in West China hospital were enrolled in this cohort between April 2020 and December 2021. The median age of patients was 56 years old, all patients had ECOG performance ≤2.Germinal center B cell-like DLBCL (GCB) and non-GCB subtypes were found in (5/31) and (26/31), respectively. Extranodal involvement was found in 17 patients (54.8%), which the sites of extranodal involvement were the gastrointestine (3/31), bone marrow (5/31), nasal cavity (5/31),breast(2/31),testicle (1/31), renal(1/31),Vertebral body(1/31),Uterine appendage(1/31). 7 patients were classified as high-intermediate and high-risk group according to CNS-IPI. At data cutoff (December, 2021), the median follow-up was 10.5 months (2.2-19.1months) with all patients have completed at least 2 cycles. Patients received a median of 5 cycles (range 2–6 cycles),and midterm assessment could be conducted.The ORR was 100.0%, with 19 patients achieved CR (19/31),14 patients achieved PR (12/31). 2 patients developed tumor recurrence after achieved CR within one year.The most common 3-4 level AEs with RI-CHOP were neutrophil count decreased(14/31), thrombocytopenia(5/31) and typically occurred within the 4~6 cycles. Other 1-2 level toxicity events were subcutaneous hemorrhage(4/31),nausea(18/31),fatigue(10/31)and reactivation of varicella zoster virus(3/31). Two patients discontinued oral Zanubrutinib due to oral ulceration and hematuria.

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Summary/Conclusion: RI-CHOP could be one of the promising choice for DE-DLBCL patients with fair physical condition,and could achieve high ORR and CRR. This study demonstrated a manageable safety profile of the combination regimen.

P1217: INTRATHECAL METHOTREXATE PROPHYLAXIS AND CENTRAL NERVOUS SYSTEM RELAPSE IN PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA TREATED WITH INTENSIFIED R-ACVBP STRATEGY

L. Renaud 1,* , P. Stéphan 2 , S. durand 3 , G. Escure 4 , M. Franck 5 , V. camus 3 , H. tilly 3 , O. casasnovas 2 , S. Chevret 6 , C. Thieblemont 7

1 Hemato-oncologie, DMU DHI; Université de Paris, AP-HP, Hôpital Saint-Louis, F-75010 Paris; 2 Department of Hematology, University Hospital F. Mitterrand and Inserm UMR 1231, Dijon; 3 Department of Hematology, Centre Henri Becquerel, University of Rouen, Rouen; 4 Department of Hematology, CHU Lille, Université de Lille 2; 5 Department of Hematology, CHU Lille, Unité GRITA, Université de Lille 2, Lille; 6 Biostatistiques, AP-HP, Hôpital Saint-Louis,Université de Paris – Paris Diderot, paris; 7 Hemato-oncologie, DMU DHI; Université de Paris, AP-HP, Hôpital Saint-Louis, F-75010 Paris, France

Background: Central nervous system (CNS) relapse of diffuse large B-cell lymphoma is relatively uncommon (2-5%) and remains catastrophic with a median survival of 2 to 5 months. Intrathecal (IT) or intravenous high-dose methotrexate (HD MTX) have limited if any prophylactic impact on CNS relapse. We recently demonstrated in a cohort of 2203 patients included in multicenter clinical trials conducted by LYSA and GLA/ DSHNHL, that only 4 (1.6%) CNS relapses were seen with the R-ACVBP strategy in pts with aaIPI 2, 3 (and intermediate/high CNS-IPI), while 15 (3.9%) relapses did occur after R-(Mega)CHO(E)P.

Aims: To address the role of IT in this intensified strategy R-ACVBP, we took the advantage of real world data of patients treated in four centers, and compared CNS relapses occurring in patients treated with R-ACVBP strategy with or without IT.

Methods: The LYSA R-ACVBP strategy include four cycles of R-ACVBP (75 mg/m 2 doxorubicin, and 1200 mg/m 2 cyclophosphamide; 2 mg/m 2 vindesine and 10 mg bleomycin on days 1 and 5) with four IT, two cycles of high dose methotrexate (3g/m 2 ), four cycles of R-etoposide (300 mg/m 2 ) and ifosfamide (1500 mg/m 2 ) and 2 cycles of cytarabine(100 mg/m 2 ) given sub cutaneously for 4 days. 233 patients with previously untreated diffuse large B-cell lymphoma (DLBCL) aged 18 to 59 years old received this intensified strategy between 2010 and 2020. We excluded 30 patients who received ASCT in first line and 10 others who received less than four cycles of R-ACVBP. We assessed the risk for of relapse and CNS-relapse. We also assesed the impact of the 2 courses of subcutaneous aracytine on overall survival (OS) and progression free survival (PFS).

Results: A total of 193 pts were included. Median age was 44.4 years (IQR, 30.8-53, range: 17-62). Distribution of CNS IPI was not significantly different comparing IT (n=132) vs non-IT (n=61) groups within aaIPI categories (Table 1). 92 patients did not receive the two courses of subcutaneous Aracytine (no AraC group) for consolidation and 101 patients received the two courses (AraC group).

With a median follow-up at 57.2 months, the 3 year OS and 3 year PFS of the whole cohort was 93.6% and 86.2%, respectively, without significant difference between IT and non IT groups in terms of OS (3yr-OS, = 92.3% versus 95.1%, HR= 1.29 (95%CI, 0.41 to 4.04, p=0.67) and PFS (3yr-PFS, 84.4% versus 90.1%, HR= 1.26 (95%CI, 0.59 to 2.72, p=0.55).

The 3y-cumulative incidence of CNS relapse for pts treated IT group was 3.2% (95%CI, 1.0 to 7.4), and 3.3% (95%CI, 0.6 to 10.2) in the non-IT group (p= 0.93). In pts with aaIPI 1, two patients experienced a CNS relapse, one in the IT group and one in the non-IT group. In pts with aaIPI 2,3 and intermediate/high CNS IPI, 4 patients experienced a CNS relapse, three out of four in the IT group.

92 patients did not receive the two courses of subcutaneous Aracytine (no AraC group) for consolidation and 101 patients received the two courses (AraC group). Receiving or not AraC did not have any impact on the CNS relapse with a risk of CNS relapse at 4.1% (95%CI, 1.3 to 9.5) for patients receiving AraC and 2.2% (95%CI, 0.4 to 6.9) for patients not receiving AraC (p= 0.48).

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Summary/Conclusion: CNS relapse was rare in younger DLBCL treated with this intensified strategy. Only 6 CNS relapses were observed without difference between IT and non-IT groups. The results presented at the EHA will included the total cohort of more than 600 patients.

P1218: FIRST-IN-HUMAN STUDY OF CD19/BCMA DUAL-TARGETING FAST CAR-T GC012F FOR PATIENTS WITH RELAPSED/REFRACTORY B-CELL NON-HODGKIN’S LYMPHOMA

X. Chen 1,* , Y. Ping 2 , L. Li 2 , L. Zhang 2 , X. Zhang 2 , J. Huang 2 , M. Sersch 3 , Z. Wang 4 , D. Song 4 , H. Wang 4 , L. Shen 4 , W. Zhao 4 , H. Zhang 4 , M. Zhang 2 , Y. Zhang 2

1 The First Affiliated Hospital of Zhengzhou University, Zhengzhou; 2 The First Affiliated Hospital of Zhengzhou University, Zhengzhou; 3 Gracellbiotechnologies Ltd; 4 Gracell Biotechnologies Ltd, Shanghai, China

Background: Relapse due to antigen escape remains a challenge for CD19-targeted chimeric antigen receptor T(CAR-T) cell therapy in B cell malignancies including B-cell non-Hodgkin’s lymphoma (B-NHL). In addition, complete response (CR) rates of 40-54%, as reported in CD19-targeted CAR-T cell therapy for relapsed/refractory(r/r) large B-cell lymphoma, should be further improved. Aiming to optimize response rate and depth of response in B-NHL, a dual targeted CD19 and B cell maturation antigen (BCMA) CAR-T manufactured on the novel FasT CAR-T platform enabling 22-36 hours manufacturing was developed.

Aims: We report early results of a phase 1 investigator initiated (IIT) dose escalation study of GC012F in r/r B-NHL (ChiCTR2100047061) to evaluate safety and preliminary efficacy.

Methods: Patients (pts) with r/r B-NHL were eligible to be enrolled. Pts with active CNS involvement were excluded. The primary objectives were safety and tolerability, secondary objectives were pharmacokinetics and efficacy. Expansion of CAR-T cells was monitored by CAR copy number and flow cytometry. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded by ASBMT, AE according to CTCAE 5.0. To date 3 Pts were enrolled and treated with a single dose of GC012F in escalating dosing cohorts after a standard LD regimen over 3 days (20-30 mg/m 2 /day Flu, 250-300 mg/m 2 /day Cy).

Results: Preclinical studies have shown that GC012F CAR-T cells specifically and effectively killed CD19+, BCMA+ and double positive tumor cell lines in vitro . In xenograft NOG mouse models, GC012F eliminated B cell lymphoma including DLBCL, MCL and Burkitt lymphoma cell lines. As of data cute-off February 22, 2022, with a median of 90 (range 29-92) days of follow-up, 3 pts with r/r DLBCL had received one GC012F infusion at dose levels 3.7*10 4 CAR- T/kg and 2-3*10 5 CAR- T/kg and completed at least 28 days of follow-up. Median age was 52 years (range 31-60). All pts presented with Ann Arbor stage IV disease. Two pts had IPI score 3, one had IPI score 2. One pt had dual expression of MYC and BCL2. Pts had received a median of 2 (range 2-3) prior lines of therapy. All pts presented with rapidly progressive disease especially pt 3 had a bulky nodal lesion with 12.2 cm in the long axis before lymphodepletion (8.6 cm at screening stage).

No dose-limiting toxicities were observed. 2 pts experienced gr 1 CRS, 1 pt in DL 2 (3*10 5 CAR- T/kg) had gr 3 CRS over 2 days. No ICANS were observed. The median Tmax was 14d (range 14-14d), median peak copy number (Cmax) in peripheral blood was 89,120 (range 55,222-90,174) copies/mgDNA. CAR-T geometric mean AUC 0-28 for pts was 753,873 (range 685,879-834,124) copies/mg DNA×day. All three pts achieved CR by PET-CT at day 28 after GC012F infusion, and response is ongoing at 3-months follow-up in the two assessable patients.

Summary/Conclusion: The preliminary safety and tolerability of GC012F in r/r B-NHL at three different dose levels ranging from 3.7*10 4 CAR-T/kg-3*10 5 CAR-T/kg showed promising early and potent activity in r/r B-NHL pts with 100% CR rate at 1-month which was maintained in pts that were available to be assessed at the 3-month visit including r/r DLBCL pts and pts with bulky disease whose treatment is often challenging. Additional pts and longer follow up are needed to further evaluate these promising early results of GC012F in r/r B-NHL and to determine the RP2D for phase 2.

P1219: ZANUBRUTINIB, LENALIDOMIDE PLUS R-CHOP (ZR2-CHOP) AS THE FIRST-LINE TREATMENT FOR NON-GCB DIFFUSED LARGE B-CELL LYMPHOMA (DLBCL)

H. Zhu 1,2,* , Y. Sha 1,2 , Y. Miao 1,2 , S. Qin 1,2 , W. Wu 1,2 , J. Qiu 2 , H. Mi 2 , Y. Yang 2 , C. Peng 2 , C. Ding 3 , Z. Wang 4 , L. Fan 1,2 , W. Xu 1,2 , J. Li 1,2

1 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University; 2 Department of Hematology, Pukou CLL Center, Pukou division of Jiangsu Province Hospital; 3 Department of Nuclear Medicine; 4 Department of Pathology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China

Background: R-CHOP is the standard first-line therapy of DLBCL. Although over half of patients have been cured under R-CHOP, the remaining patients shared poor prognosis with median OS less than 1 year when progression after front-line treatment.

Aims: To evaluate the safety and efficacy of zanubrutinib, lenalidomide plus R-CHOP (ZR 2 -CHOP) as the first-line treatment for non-GCB DLBCL patients, we updated the results in our cohort.

Methods: We enrolled patients aged 18 to 75 with high-risk non-GCB DLBCL (including double expression and other molecular characteristics). Oral zanubrutinib was given continuously (160mg twice daily) from Day 0, lenalidomide 25mg daily Day 1-7. Patients were administered intravenously rituximab (375mg/m 2 Day 0), cyclophosphamide (750mg/m 2 Day 1), doxorubicin (50mg/m 2 Day 1), vincristine (1.4mg/m 2 Day 1), and oral prednisone (100mg Day 1-5). All patients were recommended to receive 6 cycles of ZR 2 -CHOP (R-CHOP or R 2 -CHOP were allowed in cycle 1 due to poor physical condition or inaccessible molecular characteristics at treatment) and patients older than 70 years old were administered ZR 2 -miniCHOP. Prophylaxis was mandatory with G-CSF in all patients and entecavir in patients with seropositive occult HBV (HBsAg negative but HBcAb positive). CT or PET-CT scans were applied to mid-term efficacy and PET-CT scan was conducted after 6 cycles. ctDNA was dynamically detected baseline, after 3 and 6 cycles to evaluate tumor mutational burden. The primary endpoint was complete response ratio (CRR) at mid-term and after 6 cycles. The secondary endpoint was overall response rate (ORR), ctDNA dynamics and adverse events (AE). AEs were graded based on CTCAE (version 5.0).

Results: 18 treatment-naïve non-GCB DLBCL patients were enrolled in this cohort between July 2020 and December 2021. The median age was 57.5 and all patients had ECOG-PS ≤2. 14 patients were diagnosed as double-expression (Table 1). At data cutoff (1st February, 2022), the median follow-up was 11 (2-19) months with 16 patients have completed 6 cycles. The ORR was 100.0%, with 15 patients achieved CR and 1 patients achieved PR (R-CHOP in cycle 1 due to poor physical condition) after 3 cycles and all of them achieved CR after 6 cycles (Figure 1). 4 patients have received autologous stem cell transplantation for consolidation after 6 cycles. ctDNA was dynamically detected in 14 patients (2 MCD subtype patients, 1 N1 patient, 1 BN2 patient, 1 A53 patient and 5 patients sharing TP53 mutation or deletion, Figure 2). 13 patients showed undetectable ctDNA after 3 cycles while 1 patient with TP53 p.R248W mutation remained detectable after 3 cycles and vanished after 6 cycles (Figure 3). During end of treatment follow-up, all patient remained CR. The most common hematological toxicity events were lymphocytes count decreased, neutrophil count decreased, thrombocytopenia and anemia, with 3-4 level occurrence rate 68.8%, 43.8%, 43.8% and 25.0%. The most common non-hematological toxicity events were nausea, fatigue and anorexia. Two patients discontinued ZR 2 -CHOP regimen during treatment, one with R-CHOP in last two cycles due to drug rash and HBV reactivation. Another one with Rituximab in last cycle due to lumbar vertebra fracture.

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Summary/Conclusion: ZR 2 -CHOP for high-risk non-GCB DLBCL patients with fair physical condition could achieve high CRR and early-stage undetectable ctDNA. The overall tolerability was under control. The multi-center, prospective, phase II clinical trial to evaluate safety and efficacy of ZR 2 -CHOP as first-line treatment of intermediate- to high-risk non-GCB DLBCL is now in progress ( NCT05200312 ).

P1220: CHARACTERISTICS OF SECONDARY CENTRAL NERVOUS SYSTEM INVOLVEMENT IN PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA – A SUPPLEMENTARY ANALYSIS OF JCOG0601

K. Shimada 1,* , K. Ohmachi 2 , R. Machida 3 , S. Ota 4 , H. Itamura 5 , H. Tsujimura 6 , N. Takayama 7 , T. Shimada 8 , M. Kurosawa 9 , T. Tabayashi 10 , T. Shimoyama 11 , K. Ohshima 12 , D. Maruyama 13 , T. Kinoshita 14 , K. Ando 2 , T. Hotta 15 , K. Tsukasaki 16 , H. Nagai 15

1 Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya; 2 Department of Hematology and Oncology, Tokai University School of Medicine, Isehara; 3 Japan Clinical Oncology Group Data Center/Operations Office, National Cancer Center Hospital, Tokyo; 4 Sapporo Hokuyu Hospital, Sapporo; 5 Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga; 6 Department of Hematology-Oncology, Chiba Cancer Center, Chiba; 7 Department of Hematology, Kyorin University Faculty of Medicine, Mitaka; 8 Division of Clinical Oncology/Hematology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo; 9 Department of Hematology, National Hospital Organization Hokkaido cancer Center, Sapporo; 10 Department of Hematology, Saitama Medical Center, Saitama Medical University, Kawagoe; 11 Department of Medical Oncology, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo; 12 Department of Pathology, Kurume University School of Medicine, Kurume; 13 Department of Hematology Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research, Tokyo; 14 Department of Hematology and Cell Therapy, Aichi Cancer Center Hospital; 15 Department of Hematology, National Hospital Organization Nagoya Medical Center, Nagoya; 16 Department of Hematology, National Cancer Center Hospital East, Kashiwa, Japan

Background: Secondary central nervous system involvement (sCNSi) during the clinical course of diffuse large B-cell lymphoma (DLBCL) develops in approximately 5% of patients, and has a poor prognosis. The features of sCNSi in patients identified as higher risk according to international prognostic index (IPI) and CNS-IPI have been well evaluated, however those in patients with sCNSi who are lower risk by IPI or CNS-IPI are not yet fully understood.

Aims: To explore the characteristics of sCNSi in patients who participated in JCOG0601 and were considered mainly low risk according to IPI.

Methods: Of 409 eligible patients in the JCOG0601 randomized controlled trial, we evaluated patients who developed sCNSi at their first progression. The JCOG0601 trial compared standard R-CHOP-21 (arm A, n = 204) and RW-CHOP-21 in which rituximab is administered 8 times per week from the commencement of treatment (arm B, n = 205) for untreated patients with DLBCL (Ohmachi K, et al. Blood Adv. 2021;5:984). Patients were grouped according to IPI as follows: in arm A, 92 in lower risk (L), 67 in low-intermediate risk (L-I), 33 in high-intermediate risk (H-I), and 15 in high risk (H); in arm B, 112 in L, 67 in L-I, 18 in H-I, and 8 in H. Patients at lower risk of IPI accounted for 82% of the total. The risk of CNS-IPI in each arm was as follows: in arm A, 91 in L, 96 in intermediate (I), and 17 in H; in arm B, 112 in L, 84 in I, and 9 in H. The protocol did not allow CNS prophylaxis. The cumulative incidence (CI) of sCNSi was calculated with consideration to competing risks of death and non-CNS progression or relapse.

Results: Median follow-up was 4.9 years, during which 21 patients (5.1%) developed sCNSi. The 5-year overall CI of sCNSi was 5.1%, and that in L, I, and H of CNS-IPI was 4.0%, 5.3%, and 11.5%, respectively. Five-year CI of sCNSi was 7.3% in arm A and 2.9% in arm B (Gray’s test, p = 0.11). The CI of sCNSi by CNS-IPI was 5.6% in L, 6.8% in I, and 17.6% in H in arm A; and 2.7% in L, 3.6% in I, and 0% in H in arm B. Of the 21 patients with sCNSi, 8, 10, and 3 were categorized as L, I, and H of CNS-IPI, respectively. Common locations of extranodal lesions at the time of registration in patients with sCNSi were stomach (n = 4), paranasal cavity (n = 3), and bone marrow (n = 2). In particular, 2 of 3 patients with paranasal cavity lesions were categorized as L or I of CNS-IPI. In univariable analysis, paranasal cavity lesion was a high-risk factor for sCNSi (subdistribution hazard ratio, 4.34 [95% confidence interval, 1.28–14.73], p = 0.018). Kidney and adrenal gland, which are high-risk organs in CNS-IPI, were not high risk in this analysis. Median overall survival (OS) after sCNSi was 1.3 years, with 2-year OS of 39.3%.

Summary/Conclusion: sCNSi in DLBCL patients at lower risk of CNS-IPI was low, as previously reported, but those with paranasal cavity lesions developed sCNSi despite the lower risk of CNS-IPI.

P1221: DIFFUSE LARGE B-CELL LYMPHOMA RICHTER SYNDROME IN CHRONIC LYMPHOCYTIC LEUKEMIA – A RETROSPECTIVE ANALYSIS OF TREATMENT OUTCOMES IN POLISH ADULT LEUKEMIA STUDY GROUP

O. Sośnia 1,* , M. Danecki 2 , E. Wąsik-Szczepanek 3 , J. Tryc-Szponder 4 , E. Iskierka-Jażdżewska 5 , A. Majeranowski 6 , K. Pruszczyk 1 , H. Krzemień 7 , L. Bołkun 8 , E. Paszkiewicz-Kozik 9 , J. Drozd-Sokołowska 10 , J. Kwiatkowski 11 , D. Wiśniewska-Organek 12 , B. Budziszewska 1 , K. Jamroziak 10 , I. Hus 1 , E. Lech-Marańda 1 , B. Puła 1

1 Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw; 2 Department of Clinical Oncology, Maria Sklodowska-Curie National Research Institute of Oncology, Cracow; 3 Department of Hematooncology and Bone Marrow Transplantation, Medical University, Lublin, Lublin; 4 Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan; 5 Department of Hematology, Medical University of Lodz, Copernicus Memorial Hospital, Lodz; 6 Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk; 7 Department of Hematology and Bone Marrow Transplantation, Medical School of Silesia, Silesian Medical University, Katowice; 8 Department of Hematology, Medical University, Bialystok; 9 Department of Lymphoid Malignancies, Maria Sklodowska-Curie National Research Institute of Oncology; 10 Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw, Warsaw; 11 Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Wroclaw; 12 Department of Hematology, Copernicus Hospital, Torun, Poland

Background: Richter syndrome (RS) is a recognized uncommon manifestation and evolution of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with a dismal prognosis. It occurs in 2-10% of CLL patients. It is associated with a clonal evolution and progression, generally with a transformation of the original CLL clone to diffuse large B-cell lymphoma (DLBCL) or, less frequently, to Hodgkin Lymphoma (HL). Despite the advances in understanding pathogenesis of the disease, the DLBCL variant of RS (DLBCL-RS) is associated with a dismal prognosis with a median survival < 1 year. The treatment options and risk factors of worse outcome for RS still need to be further expanded.

Aims: To study the characteristic and treatment outcomes of patients with DLBCL-RS.

Methods: The retrospective analysis of 99 patients diagnosed with DLBCL-RS treated in hematology centers of the Polish Adult Leukemia Study Group (PALG) between 2011 and 2021.

Results: Clinical data of 124 patients with Richter syndrome was collected. Ninety-nine patients were diagnosed with DLBCL-RS (79.8%) and further analyzed. Median time since CLL diagnosis to Richter transformation was 43.0 months (range 0-252.0). At the time of transformation 31.3% patients had CLL Rai stage IV, 74.7% were ECOG< 2, while the median Cumulative Illness Rating Scale (CIRS) was 4 (range 0-16). Thirty-seven patients transformed during CLL treatment, 56.7% of them were receiving novel agents (Brutons tyrosine kinase inhibitor or BCL2 inhibitor). Median time of observation after transformation was 10.7 months (range 0-98.6). Median overall survival (OS) after DLBCL-RS diagnosis was 17.3 months (95% Cl 11.9-46.8) and median progression-free survival (PFS) 13.5 months (95% Cl 9.6-22.7). Seventy-six patients (76.8%) had RS treated with intensive chemotherapy protocols, mainly R-CHOP-like regimens for the first line treatment. Only 42.3% had response to the treatment (25/76 complete responses and 11/76 partial responses). Worth mentioning is that in the group of patients with response two patients had ibrutinib monotherapy and one had rituximab-ibrutinib.

In forty patients RS was primarily resistant to administered treatment and 65% of them died due to progression or treatment related complications. In the primary resistant DLBCL-RS p53 pathway aberration was noted in 47.5% of patients (17p deletion in 13 patients or TP53 mutation in 6 patients). Better results were achieved for patients who proceeded to autologous hematopoietic stem cell transplantation (auto-HSCT; n=8) and allogeneic HSCT (allo-HSCT; n=10). The median OS in auto-HSCT recipients was 46.1 months, while in allo-HSCT the median OS reached 18.3 months (3 patients died due to allo-HCST-related complications). Significantly lower OS (3.4 months, 95% Cl 14.13-not reached) was observed for RS in heavily pretreated CLL patients (>3 lines of therapy preceding transformation) compared to those treated with up to 3 lines of therapy (23.7 months) (p=0.0002). Median OS for treatment naïve patients at DLBCL-RS diagnosis was not reached. Other factors identified as predictors of poor OS in univariate analysis were ECOG ≥2 (p=0.0014), hemoglobin level <10 g/dl (p=0.017) and platelet count <100x10 9 /L (p=0.0065).

Summary/Conclusion: Our study represents the largest dataset of DLBCL-RS patients in Polish hematology centers and confirms the poor outcomes and prognosis associated with RS. The worst treatment results were observed in RS receiving >3 lines of CLL therapy preceding transformation. Only proceeding to hematopoietic stem cell transplantation may improve the prognosis.

P1222: PACIFIC: BRENTUXIMAB VEDOTIN AND NIVOLUMAB ALONE AND THEN COMBINED WITH RITUXIMAB, CYCLOPHOSPHAMIDE, DOXORUBICIN AND PREDNISONE FOR UNTREATED PRIMARY MEDIASTINAL LARGE B-CELL LYMPHOMA PATIENTS

R. Steiner 1,* , P. Strati 1 , C. Flowers 1 , S. Neelapu 1 , M. Green 1 , L. Nastoupil 1 , F. Hagemeister 1 , L. Feng 2 , S. Ahmed 1 , R. Nair 1 , L. Fayad 1 , H. Lee 1 , M. A. Rodriguez 1 , J. Westin 1

1 Lymphoma & Myeloma; 2 Biostatistics, MD Anderson Cancer Center, Houston, United States of America

Background: Primary mediastinal large B-cell lymphoma (PMBL) is a rare subtype of aggressive B-cell lymphoma and affects primarily young adults. PMBL has a distinct genomic profile that has similarities to classic Hodgkin lymphoma with CD30 expression and genomic alterations in the programmed T-cell death-ligand 1. The trial CheckMate-436, including relapsed/refractory (rr) PMBL patients treated in at least 3 rd line with nivolumab and brentuximab vedotin (BV) showed an overall response rate (ORR) of 73% and complete response rate (CRR) of 43% (Zinzani et al. JCO 2019). Although 70-94% of PMBL patients can be cured with frontline modality, the outcome of patients having rr-PMBL treated with intensive regimens is generally unfavorable. The discovery of novel frontline regimens to decrease chemoresistance and toxicities represents an urgent unmet clinical need for PMBL patients.

Aims: We aim to evaluate the efficacy, safety, and quality of life of PMBL patients treated in the PACIFIC trial with BV and Nivolumab alone and then combined with Rituximab, Cyclophosphamide, Doxorubicin, and Prednisone (R-CHP). We hypothesize that BV+Nivolumab in the frontline setting will show an ORR at least as high as in the rr setting.

Methods: The investigator-initiated trial PACIFIC ( NCT04745949 ) is a phase II, open-label, single-center clinical trial for patients with previously untreated PMBL. Patients 18 years or older with untreated PMBL, stage I to stage IV disease are eligible. However, patients with an urgent need for systemic cytotoxic treatment will be excluded. Patients will start with an immune lead-in of BV (1.8 mg/Kg IV) and Nivolumab (240 mg flat dose IV) day 1 for cycles 1 and 2, in a 21-day cycle. During cycles 3 and 4, R-CHP will be added to BV-Nivolumab. Patients who have achieved complete response (CR) at PET/CT before cycle 5 will receive 2 more cycles of BV-Nivolumab+R-CHP (cycle 5 and 6) and BV-Nivolumab only for cycles 7 and 8. In case of CR on PET/CT after cycle 8, therapy will be considered completed. Patients in PR on PET/CT before cycle 5 will receive 4 more cycles of BV-Nivolumab+R-CHP (cycles 5-8). The primary endpoint is CRR at the end of therapy (EOT). The maximum sample size for the cohort is 40 patients, with a target CRR at EOT of 70%. The null hypothesis is that the true CRR at EOT is 50%, and the alternative hypothesis is that the true CRR at EOT is 70%. The Simon’s optimal two-stage design controls the one-sided type I error rate at 0.06 and yields the power of 0.8. Exploratory analyses include assessing molecular response by sequencing cell-free DNA and Multiplexed ion beam imaging to analyze the tumor microenvironment.

Results: The trial is actively accruing at MD Anderson Cancer Center. To date, 2 out of 40 patients have been enrolled and are receiving therapy. Both patients (28 and 58 years old) presented a partial response (Lugano Classification) after 2 cycles of BV+Nivolumab, and one of them was evaluable after 2 cycles of BV-Nivolumab+R-CHP (cycles 3 and 4) with CR. Adverse events (AE) were similar to what is expected with the used drugs (nausea Grade 1 (n=1), fatigue Grade 1 (n=1), hypophosphatemia Grade 1 (n=1), hyperglycemia Grade 1 (n=1), leukocytosis Grade 1 (n=1), anemia Grade 1 (n=1)), including immune-related AE (fever Grade 1 (n=1), rash Grade 3 (n=1)).

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Summary/Conclusion: The preliminary data of the PACIFIC trial with BV and Nivolumab alone and then combined with R-CHP are encouraging. Completion of this trial and further studies are needed to optimize frontline therapy of PMBL patients.

P1223: PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA: A RETROSPECTIVE STUDY OF THE ZRMA REGIMEN（ZANUBRUTINIB, RITUXIMAB, METHOTREXATE AND CYTARABINE)

Y. Tang 1,*

1 clinical oncology, Xiangya hospital, Central south University, changsha, China

Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive non-Hodgkin lymphoma that affects the brain, eyes, cerebrospinal fluid (CSF), or spinal cord without systemic involvement. Current treatments including surgery, chemotherapy and whole-brain radiotherapy often fail to achieve satisfactory effect, especially in elderly. Novel insights into the pathophysiology of PCNSL have identified key mechanisms in tumor pathogenesis including activation of the B-cell receptor pathway.Bruton’s tyrosine kinase (BTK) inhibitor zanubrutinib has been tested in clinical trials against R/R PCNSL and shows potential to be used in first-line treatment of PCNSL.

Aims: To evaluate efficacy and safety of Zanubrutinib, rituximab, methotrexate and cytarabine(ZRMA regimen) in patients with primary CNS diffuse large b cell lymphoma.

Methods: Retrospective study was done on patients (aged 18-70 years) with newly diagnosed primary CNS lymphoma and measurable disease who received ZRMA regimen in our hospital.Patients received four courses of ZRMA regimen every 21 days a cycle: Zanubrutinib 80mg twice daily, rituximab 375 mg/m( 2 ) on days 0,methotrexate 3·5 g/m( 2 ) on day 1 plus cytarabine 2 g/m( 2 ) once daily on days 2. Patients with responsive were then received whole-brain radiotherapy, autologous stem cell transplantation or lenalidomide maintenance.

Results: Between Aug 18, 2020, and Feb 28, 2022, 15 enrolled patients were assessable. At median follow-up of 10 months（3-18）, patients had a complete remission rate of 93%(14/15). The median progression-free survival (PFS) and overall survival (OS) was not reached. The only two grade 3-4 adverse events were neutropenia(7%,1/15) and f infections(7%,1/15). The ZRMA regimen was well tolerated in these treatment-naïve PCNSL patients with an acceptable safety profile.

Summary/Conclusion: This retrospective study provides an evidence supporting the use of ZRMA regimen for further research in patients aged up to 70 years with newly diagnosed primary CNS lymphoma.

P1224: EFFICACY OF RITUXIMAB AND BIOSIMILAR RITUXIMAB IN UNTREATED DIFFUSE LARGE B-CELL LYMPHOMA, A DANISH POPULATION BASED ANALYSIS

A. Haujir 1,* , M. Tøstesen 2 , T. C. El-Galaly 3 , T. S. Larsen 4 , J. Starklint 5 , C. B. Poulsen 6 , P. L. Josefsson 7 , E. Clasen-Linde 7 , A. Dessau-Arp 8 , M. R. Clausen 1 , J. M. Jørgensen 2

1 Department of hematology, Sygehus Lillebælt, Vejle; 2 Department of hematology, Aarhus University Hospital, Aarhus; 3 Department of hematology, Aalborg Universitetshospital, Aalborg; 4 Department of hematology, Odense Universitetshospital, Odense; 5 Department of hematology, Hospitalesenheden Vest, Gødstrup; 6 Department of hematology, Sjællands Universitetshospital, Roskilde, Roskilde; 7 Department of hematology, Rigshospitalet, Copenhagen; 8 Department of hematology, Sydvestjysk Sygehus, Esbjerg, Denmark

Background: The standard treatment for diffuse large B-cell lymphoma (DLBCL) was for decades conventional chemotherapy eg. CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone). Around the year 2000 it became possible to add the monoclonal CD20 antibody rituximab, resulting in improved overall survival [1]. In 2017 biosimilars of rituximab were approved by the European Medicine Agency for the treatment of DLBCL, after showing non-inferiority to rituximab in randomized clinical trials[2–6]. Biosimilar rituximab is a cheaper alternative, and was implemented in danish hospitals for the treatment of non-Hodgkin lymphoma and DLBCL during 2018. Biosimilar rituximab has shown non-inferiority in randomized trials, but population-based analyses of the efficacy are still lacking.

Aims: In this study we assessed the efficacy of biosimilar rituximab compared to rituximab on progression free survival (PFS) and overall survival (OS) in danish patients with previously untreated DLBCL.

Methods: We used the population-based lymphoma registry in Denmark, LYFO, to identify adult patients with newly diagnosed DLBCL from January 2015 until January 2022. Patients treated with CHOP or CHOP-like treatment were included and we analyzed outcomes depending on type of rituximab. Primary CNS-lymphoma was excluded due to different treatment and prognosis. From pharmacies of the treating hospitals, we gathered information on the date of change to biosimilar rituximab. The primary endpoint was progression free survival defined as either registered relapse or death. We used Kaplan Meier curves and multivariable Cox regression analysis was used to evaluate outcomes. In the multivariable model we adjusted for the International Prognostic Index (IPI), sex and age as a continuous variable. Further we registered the type of chemotherapy in the first line of treatment and categorized these data as either CHOP/CHOEP or other. The proportional hazards assumption was evaluated graphically using log minus log plots.

Results: We identified 2.372 patients with a median age of 70 years (range:18-95 years). There were 1.049 in the rituximab group with a median age of 69 years and 1.343 in the biosimilar rituximab group, median age 71 years. The International Prognostic Index distribution was comparable between groups. The fraction of DLBCL patients receiving CHOP/CHOEP based regimen was lower in the biosimilar rituximab group, 94% vs. 98% respectively (Table 1). The 3-year PFS was 71.8% (95% confidence interval[CI] 69.1-74.3) and 72.2% (95% CI 69.3-74.9) for rituximab and biosimilar rituximab treated patients (Figure 1). In the multivariable analyses we found no difference in PFS or OS with a hazard ratio of 0.94 (95% CI 0.81 - 1.10) and 0.99 (95% CI 0.83 - 1.17).

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Summary/Conclusion: In this population-based study we found no significant difference in PFS or OS in patients treated with chemotherapy in combination with rituximab or biosimilar rituximab. Fewer patients were treated with CHOP/CHOEP in the biosimilar group due to an increased use of mini-CHOP and more intensive regimens. These results substantiate the continued use of biosimilar rituximab in patients with DLBCL.

P1225: SECONDARY PRIMARY MALIGNANCIES (SPM) AFTER HIGH-DOSE CHEMOTHERAPY (HDT) WITH AUTOLOGOUS STEM CELL TRANSPLANT (ASCT) FOR LYMPHOMA: A DANISH NATIONWIDE COHORT STUDY

T. Trab 1,* , J. Baech 1 , L. Hjort Jakobsen 1,2 , S. Husby 3 , M. Tang Severinsen 1,2 , S. Eloranta 4 , J. S. Gørløv 3 , J. M. Jørgensen 5 , S. Gudbrandsdottir 6 , T. Stauffer Larsen 7 , P. Brown 3 , K. E. Smedby 4 , T. Christoffer El-Galaly 1,2

1 Department of Hematology, Clinical Cancer Research Center, Aalborg University Hospital; 2 Department of Clinical Medicine, Aalborg University, Aalborg; 3 Department of Hematology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark; 4 Department of Medicine Solna, Division of Clinical Epidemiology, Karolinska Institutet, Stockholm, Sweden; 5 Department of Hematology, Aarhus University Hospital, Aarhus; 6 Department of Hematology, Zealand University Hospital, Roskilde; 7 Department of Hematology, Odense University Hospital, Odense, Denmark

Background: High-dose chemotherapy (HDT) with autologous stem cell transplant (ASCT) have resulted in more long-term lymphoma survivors. However, HDT-ASCT is potentially associated with an increased risk of secondary primary malignancies (SPM) leading to loss of lifetime for these patients.

Aims: To investigate the risk and types of SPM in lymphoma patients treated with HDT-ASCT compared to the risk of malignancies in a matched background population and to explore risk factors for SPM in HDT-ASCT treated lymphoma patients.

Methods: In a nationwide matched cohort study, The Danish Lymphoma Register was used to identify all adult patients treated with HDT-ASCT in Denmark between 2001-2017 for diffuse large B-cell lymphoma, Hodgkin lymphoma, peripheral T-cell lymphoma, or mantle cell lymphoma, excluding patients with other prior cancers. Patients were matched in a ratio of 1:5 to cancer-free comparators from the Danish background population. Malignancies were identified in the Danish National Patient Register using ICD-10 codes and compared between the two groups using the Aalen-Johansen estimator, Cox regression, and incidence rate ratios (IRRs).

Results: A total of 856 HDT-ASCT treated lymphoma patients and 4,280 matched comparators were followed for a median of 8 years. The cumulative risk of SPM with death as competing risk was higher for patients compared to comparators with risks at 5, 10, and 15 years of 8.9% [95% confidence interval 6.9;11.0], 16.3% [13.3;19.3], and 20.1% [16.1;24.2] for patients compared to 4.8% [4.1;5.5], 10.5% [9.3;11.7], and 16.6% [14.6;18.5] for comparators (Fig. 1 a ). The crude hazard ratio (HR) of SPM among patients vs comparators was 2.26 [1.83;2.79]. After adjusting for sex, educational level (low/high), chronic obstructive pulmonary disease (COPD), and alcohol-related diseases (neurological, mental, cardiovascular, or chronic use), the HR was 2.50 [2.02;3.10]. Compared to comparators, patients had increased rates of non-melanoma skin cancer (IRR 3.47 [2.18;5.38]) and myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) (IRR 35.32 [15.73; 94.26]) (Table 1). Rates of other malignancies (oral, gastrointestinal, respiratory, breast, female- or male genital, urinary, soft tissue, and melanoma) were similar between the two groups. The 15-year cumulative risk of MDS/AML was 4.6% [2.8;6.3] for patients (Fig. 1 c ) and the median overall survival following secondary MDS/AML was only 7 months. In comparison, median overall survival was 40 months in a sex- and age matched background population with newly diagnosed MDS/AML (Fig. 1 d ). Risk factors for SPM in lymphoma patients were older age (HR 1.06 [1.04;1.08] per year) and COPD (HR 3.15 [1.16;8.57]). Prior therapy (number of lines, intensive chemotherapy, radiotherapy), time since diagnosis, Ann Arbor stage, performance status, alcohol-related disease, and education were not associated with SPM.

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Summary/Conclusion: Lymphoma patients treated with HDT-ASCT have an increased risk of SPM compared to a matched background population, especially for non-melanoma skin cancer and MDS/AML. Risk factors for SPM in patients were older age and COPD. These findings are important in the light of new potential treatment alternatives, such as CAR-T.

P1226: A RETROSPECTIVE REVIEW OF SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS FROM A TEACHING HOSPITAL IN SINGAPORE.

A. C. Y. Tso 1,* , S. Z. Fong 1 , L. K. Lee 1 , V. S. Sampath 1 , B. E. Fan 1 , S. S. W. Chan 1 , K. H. Ong 1

1 Haematology, Tan Tock Seng Hospital, Singapore, Singapore

Background: Secondary Hemophagocytic Lymphohistiocytosis (HLH) is an aggressive life-threatening syndrome leading to uncontrolled immune hyperactivation and end organ failure. Real world data on the outcome of secondary HLH amongst Asians are limited to small case series.

Aims: The purpose of this paper is to describe the patient profiles, aetiologies and survival outcomes of adult patients with secondary HLH in a tertiary hospital in Singapore.

Methods: This is a retrospective medical records review of adult patients with secondary HLH between March 2015 and June 2020. Ethics approval was obtained (DSRB 2020/00833). Survival probabilities over time were estimated using the Kaplan–Meier method and their distributions between malignancy and non-malignancy-associated HLH were compared using log rank test. Hazard ratios (HR), along with 95% confidence intervals (CI), were estimated using Cox proportional hazard models.

Results: 36 patients (18 females, 18 males) aged 23 to 80 were followed up for a median 14 months. 16 had underlying aggressive lymphomas (B cell (N=5), T cell (N=5), NK/T cell (N=5), unknown subtype (N=1)). 15 had underlying viral infections (11 Dengue, 2 EBV, 1 parvovirus, 1 unknown). No cause was found in 5 patients. Of the 30 patients who had tissue biopsies performed, 28 (93.3%) demonstrated hemophagocytosis. The mean HSCORE was 234.3 indicating >96% probability for HLH.

Treatment: 65% patients without an underlying malignancy required HLH-directed therapy and blood product transfusion. This is in contrast with 100% patients with an underlying malignancy. 13 of 16 patients with aggressive lymphomas were also treated with systemic chemotherapy. 2 patients with aggressive T cell lymphoma underwent stem cell transplantation.

Overall survival: Malignancy-associated HLH patients had poorer survival compared to non-malignancy-associated HLH (median OS 1.5 months vs not reached, p value 0.01). The 1-year survival rates of patients with malignancy-associated HLH, HLH with unknown aetiologies and infection-associated HLH were 0.312 (95% CI: 0.151 – 0.646), 0.400 (95% CI: 0.137 – 1.000) and 0.867 (95% CI: 0.711 – 1.000), respectively. Regression analysis revealed that malignancy significantly increased the risk of death when compared to infection-associated HLH (HR 7.74, 95% CI: 1.72 – 34.8, p value 0.007). These remained significant after adjusting for differential distribution in age, gender and ethnicity. Within different subtypes of malignancy, we found that NK/T cell lymphoma had the worst survival outcome, followed by T cell and B cell lymphoma (median OS time 0.6, 2 and 19 months, respectively). The adjusted hazard ratios suggested the risk of death among patients with NK/T cell lymphoma was as big as seven times compared to B cell lymphoma, but it didn’t reach to statistical significance (HR 7.33, 95% CI: 0.96 – 55.98, p value 0.055).

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Summary/Conclusion: Our case series highlight the stark contrast in the overall survival of secondary HLH patients with and without an underlying malignancy. The outcome of malignancy-associated HLH in our study was similar to MD Anderson Cancer Center’s experience (25% and 20% overall survival respectively; with a median survival of 1.5 months in both). Prompt recognition amongst physicians likely to encounter HLH, early referral to hematologists, prompt initiation of HLH-directed and supportive therapies and tackling underlying triggers, are key to improving the outcome of secondary HLH.

P1227: POLATUZUMAB VEDOTIN, RITUXIMAB, AND BENDAMUSTINE COMBINATION IN RELAPSED OR REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA: A REAL-WORLD DATA FROM TURKEY

M. S. Dal 1 , B. Uncu Ulu 1,* , A. Uzay 2 , O. M. Akay 3 , S. Besisik 4 , M. N. Yenerel 4 , S. Celik 5 , L. Kaynar 6 , O. K. Yucel 7 , B. Deveci 8 , M. Sonmez 9 , O. Mehtap 10 , H. S. Bekoz 6 , C. Sunu 11 , O. Salim 7 , T. Ulas 12 , S. Karti 2 , F. Altuntas 1 , B. Ferhanoglu 13 , T. Firatli Tuglular 14

1 Hematology and Bone marrow transplantation, Ankara Oncology Training and Education Hospital, Ankara; 2 Hematology and Bone marrow transplantation, Acibadem University Atakent Hospital; 3 Hematology, Koc University, School of Medicine; 4 Hematology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul; 5 Hematology, Erciyes University School of Medicine, Kayseri; 6 Hematology and Bone marrow transplantation, Medipol University Faculty of Medicine, Department of Internal Medicine, Istanbul; 7 Hematology, Akdeniz University Faculty of Medicine; 8 Hematology and Bone marrow transplantation, Medstar Antalya Hospital, Antalya; 9 Hematology, Department of Internal Medicine, Karadeniz Technical University, Trabzon; 10 Hematology, Kocaeli University Faculty of Medicine, Kocaeli; 11 Hematology, Faculty of Medicine, Sakarya University, Sakarya, Turkey; 12 Hematology, Near East University School of Medicine, Nicosia, Cyprus; 13 Hematology, Department of Internal Medicine, Koc University School of Medicine and V.K.V. American Hospital; 14 Hematology, Marmara University, School of Medicine, Istanbul, Turkey

Background: Polatuzumab vedotin (Pola) in combination with Bendamustine and Rituximab (BR) is a promising option for patients with relapsed/ refractory (R/R) diffuse large B-cell lymphoma (DLBCL).

Aims: Given reduced survival in patients who are unresponsive to subsequent lines of therapy and the toxicity involved, there is an unmet need in the R/R DLBCL setting. We present a retrospective, multicenter, real-world data study consisting of 71 R/R DLBCL patients treated with Pola-BR in the named patient program in Turkey.

Methods: We analyzed the data of 71 R/R DLBCL patients who had been treated with Pola-BR through their participation in the named patient program from March 2018 to April 2021 from 32 hematology centers in Turkey. All patients received Polatuzumab vedotin 1.8 mg/kg intravenously (IV) on day 1 of each cycle, Rituximab 375 mg/m 2 IV on day 1 of each cycle, and Bendamustine 90 mg/m 2 IV on days 1 and 2 of each cycle in any order. Patients were treated for up to six cycles. Each cycle was applied every 21 days.

Results: The median age at Pola-BR initiation was 55 (range,19-84). The patients had undergone a median of three lines (range, 2-5 lines) of treatment before Pola-BR treatment (Table 1). The overall response rate was 47.9%, including 32.4% complete response rate when a median of three cycles was applied. With a median follow-up of 5 months, the median OS of our cohort was 5 months. Patients who were chemorefractory to Pola-BR and patients who had ECOG score ≥2 had a worse survival which was almost double in the multivariate analysis (p=0.042 and p=0.029, respectively). The most common adverse events of grades 3-4 were infections and cytopenias. Grade 1-2 anemia, and grade 3-4 neutropenia and thrombocytopenia were the most common hematological toxicities of Pola-BR treatment. Six patients (8.4%) received allogeneic stem cell transplantation after Pola-BR treatment.

Summary/Conclusion: The real-world data from our cohort of hard-to-treat and aggressive R/R DLBCL showed the Pola-BR combination is a promising treatment option with a manageable toxicity profile.

P1229: RITUXIMAB-DOSE-ADJUSTED EPOCH (R-DA-EPOCH) IN PRIMARY MEDIASTINAL LARGE B-CELL LYMPHOMA (PMLBCL): REAL-LIFE EXPERIENCE ON 225 PATIENTS FROM 4 COUNTRIES

T. Vassilakopoulos 1,* , B. Ferhanoglu 2 , N. A. Horowitz 3 , J. Apostolidis 4 , Z. Mellios 5 , L. Kaynar 6 , M. Zektser 7 , A. Symeonidis 8 , A. Piperidou 9 , C. Kalpadaki 10 , O. M. Akay 11 , S. C. Atalar 11 , A. Sayyed 4 , E. Katodritou 12 , T. Leonidopoulou 13 , S. Papageorgiou 14 , T. Tadmor 15 , O. Gutwein 16 , S. Karakatsanis 17 , C. Ganzel 18 , G. Karianakis 19 , G. Isenberg 3 , G. Gainaru 19 , E. Vrakidou 19 , M. Palassopoulou 20 , M. Ozgur 21 , M. Siakantaris 22 , S. Paydas 23 , P. Tsirigotis 24 , M. Tsirogianni 25 , E. Hatzimichael 26 , T. F. Tuglular 27 , C. Chatzidimitriou 28 , A. Liaskas 22 , M. A. Lefaki 22 , N. Kanellias 29 , P. Zikos 30 , A. Koumarianou 31 , A. Gafter-Gvili 32 , M. Angelopoulou 33 , T. Karmiris 5 , R. Gurion 34

1 Department of Hematology and Bone Marrow Transplantation, National and Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece; 2 American Hospital, Istabul, Turkey; 3 Rambam Medical Center, Haifa, Israel; 4 Lymphoma Program, Department of Adult Hematology & Stem Cell Transplantation, King Fahad Specialist Hospital, Dammam, Saudi Arabia; 5 Department of Hematology and Lymphoma, Evangelismos General Hospital, Athens, Greece; 6 Erciyes University, Kayseri, Turkey; 7 Soroka Medical Center, Beer Sheba, Israel; 8 Hematology Division, Department of Internal Medicine, University of Patras, Patras; 9 Haematology and Bone Marrow Transplantation, “Laikon Hospital”, National and Kapodistrian University of Athens, Athens; 10 Department of Hematology, University Hospital, University of Crete, Heraclion, Greece; 11 Koc University, Istabul, Turkey; 12 Department of Hematology, Theagenion Anticancer General Hospital, Thessaloniki; 13 Department of Hematology, Sismanoglion General Hospital; 14 Second Propedeutic Department of Internal Medicine,National and Kapodistrian University of Athens, ATTIKON General Hospital, Athens, Greece; 15 Bnai Zion Medical Center, Haifa; 16 Shamir Medical Center, Be’erYa’akov, Tel Aviv, Israel; 17 Third Department of Internal Medicine, National and Kapodistrian University of Athens, Sotiria Hospital, Athens, Greece; 18 Shaare Zedek Medical Center, Jerusalem, Israel; 19 HYGEIA Hospital, Athens; 20 Department of Hematology, University Hospital, University of Thessaly, Larissa, Greece; 21 Kocaeli University, izmit, Turkey; 22 Department of Hematology and Bone Marrow Transplantation, National and Kapodistrian University of Athens, Laikon General Hospital, Athens, Greece; 23 Cukurova University, Adana, Turkey; 24 Second Propedeutic Department of Internal Medicine,National and Kapodistrian University of Athens, ATTIKON General Hospital; 25 Department of Hematology and Bone Marrow Transplantation, Saint Savvas Regional Cancer Hospital, Athens; 26 Department of Hematology, University of Ioannina, Ioannina, Greece; 27 Marmara University, Istabul, Turkey; 28 Department of Hematology and Bone Marrow Transplantation, National and Kapodistrian University of Athens, Laikon General Hospital; 29 Department of Therapeutics, National and Kapodistrian University of Athens, Athens; 30 Hematology Department, GeneralHospitalof Patras “Agios Andreas”, Patras; 31 Department of Hematology, METROPOLITAN Hospital, Athens, Greece; 32 Rabin Medical Center, PetachTikva, Israel; 33 Department of Hematology and Bone Marrow Transplantation, National and Kapodistrian University of Athens, Athens, Greece; 34 Rabin Medical Center, PetachTikva, Israel

Background: Real-life studies of moderate size have shown satisfactory but less impressive results compared with the initial study of R-da-EPOCH in PMLBCL. However, the strategies regarding the use of consolidative radiotherapy (RT) and the compliance with the strict instructions on dose escalation have not been studied.

Aims: To assess the clinical outcomes and prognostic factors after R-da-EPOCH, the use of consolidative RT and protocol compliance in a multinational real-life setting.

Methods: 225 patients (≤60 years) were enrolled from 18 Greek (n=114), 6 Israeli (n=57), 6 Turkish (n=34) and a single Saudi Arabia center (n=20). Consolidative RT was given at the treating physician’s discretion and was highly affected by PET/CT results.

Results: The median age of the patients was 33 years (16-60), 139 (62%) were females, 40% had B-symptoms, 32% extranodal involvement (E or stage IV), 18% PS≥2, 84% elevated LDH (35% highly elevated≥2xnormal). The median follow-up was 32.5 months (2-97). Among 200 evaluable patients with conventional response, who would be potentially eligible for RT, RT was spared in 169 (84%); 31 received RT mainly for Deauville 4 (or rarely Deauville 5) residuals. The 5-year Freedom From Progression (FFP) was 86%. However, 4 patients developed therapy-related (t-) AML at 10.5-22 months from treatment initiation, while in 1 st remission, and one Hodgkin lymphoma, for a 2-year event-free survival of 83%, if considered as events. The 5-year overall survival (OS) was 92% with 14 disease-related deaths (1 toxic). Protocol violations were common (52%), mainly consisting of insufficient dose escalation despite the absence of prohibitive toxicity. Among 209 patients with available data, 58% reached level ≥3 and 29% ≥4 (73% and 46% among those with strict protocol adherence). The 5-year FFP was 89% vs 84% for patients with strict protocol adherence or not (p=0.37); OS was not also different. FFP did not differ according to the final level reached (≥3 or ≥4; both p>0.20). A more detailed analysis of outcome according to the degree of protocol violations is currently ongoing. The prognostic systems including any extranodal involvement (E/IV) and highly elevated LDH (≥2x) or bulk marginally or significantly affected prognosis: Considering the E/IV-LDH model, the 5-year FFP was 89%, 84% and 73% for patients with 0, 1 or 2 adverse factors (p=0.14), while 5-year OS was 97%, 91% and 81% (p=0.08). The corresponding figures for the E/IV-bulk model were 90%, 89% and 75% (p=0.026) for FFP and 98%, 94% and 83% for OS (p=0.01). Thus, patients with both risk factors had 5-year FFP rates 73-75% and 5-year OS 81-83%.

Summary/Conclusion: In the largest series reported so far for R-da-EPOCH in PMLBCL, FFP appeared somewhat but not impressively better than the expected with R-CHOP. However, consolidative RT was safely omitted in ~85% of responders mainly based on PET findings. OS was 92%. However, the appearance of 4 cases of t-AML among 225 patients is worrisome. Significant dose-escalation violations were recorded in the real-life; their impact on outcomes appears to be modest and is further evaluated in detail while the series is also expanded with inclusion of even more patients. Patients with multiple adverse factors appear to do better than with R-CHOP, while those with 0-1 factor have similar outcomes to R-CHOP but with minimal consolidative RT.

P1230: PET/CT IMAGING FOR RESPONSE ASSESSMENT AND TREATMENT GUIDANCE AFTER RITUXIMAB-DOSE-ADJUSTED-EPOCH(R-DA-EPOCH) IN PRIMARY MEDIASTINAL LARGE B-CELL LYMPHOMA (PMLBCL):THE GREEK EXPERIENCE ON 107 PATIENTS

T. Vassilakopoulos 1,* , A. Piperidou 1 , Z. Mellios 2 , E. Verigou 3 , C. Kalpadakis 4 , E. Katodritou 5 , S. Papageorgiou 6 , C. Chatzidimitriou 1 , C. Giatra 2 , T. Leonidopoulou 7 , V. Xanthopoulos 8 , S. Karakatsanis 9 , G. Gainaru 8 , E. Vrakidou 8 , A. Koumarianou 10 , E. Hatzimichael 11 , E. Verrou 5 , M. Tsirogianni 12 , I. Drandakis 1 , M. A. Lefaki 1 , A. Liaskas 1 , A. Kopsaftopoulou 1 , P. Lampropoulou 13 , N. Kanellias 12 , P. Zikos 14 , D. Liapi 15 , G. Karianakis 8 , D. Grentzelias 6 , E. Papadaki 4 , M. Siakantaris 1 , P. Tsirigotis 6 , F. Panitsas 1 , E. Plata 1 , M. Bakiri 2 , I. Datseris 16 , S. Chatziioannou 17 , V. Prassopoulos 18 , E. Skoura 19 , M. Angelopoulou 1 , A. Symeonidis 3 , T. Karmiris 2 , P. Rontogianni 16

1 Haematology and Bone Marrow Transplantation, “Laikon Hospital”, National and Kapodistrian University of Athens; 2 Department of Haematology, Evangelismos Hospital, Athens; 3 Department of Haematology, University of Patras, Rio; 4 Department of Haematology, University of Crete, Heraclion; 5 Department of Haematology, Theagenion Anticancer Hospital, Thessaloniki; 6 2nd Propedeutic Dept of Internal Medicine, National & Kapodistrian University of Athens; 7 Department of Haematology, Sismanogleion Hospital; 8 Department of Haematology, HYGEIA Hospital; 9 3rd Dept of Internal Medicine, National & Kapodistrian University of Athens; 10 Department of Haematology, IASO Hospital, Athens; 11 Department of Haematology, University of Ioannina, Ioannina; 12 Department of Haematology, Agios Savvas Anticancer Hospital, Athens; 13 Anticancer Hospital “Metaxa”, Pireaus; 14 Department of Haematology, Hospital of Patras “Agios Andreas”, Patras; 15 Department of Haematology, University of Crete, “Venizeleio” Hospital, Heraclion; 16 Department of Nuclear Medicine, Evangelismos Hospital; 17 Division of Nuclear Medicine, Foundation for Biomedical Research of the Academy of Athens; 18 Department of Radiology and Nuclear Medicine, HYGEIA Hospital; 19 Department of Nuclear Medicine, «BIOIATRIKI», Athens, Greece

Background: The prognostic significance of end-of-treatment PET/CT (EOT-PET) has been already evaluated in PMLBCL under R-CHOP or R-MACOP-B. Patients with clearly positive scans have inferior outcomes even after consolidative radiotherapy (RT). However, after R-da-EPOCH, the prognostic significance of PET/CT may be modulated, since most patients with positive scans achieve durable remissions without consolidative RT, pointing out to false-positive results. The omission of RT in patients with Deauville 5-point scale score (D5PSS) 4 often causes anxiety to the treating physicians, while handling of patients with D5PSS5 is controversial (RT vs high dose chemotherapy and autologous stem cell transplantation). Accumulation of real-world experience is required to provide answers to these dilemmas.

Aims: To assess the clinical and prognostic significance of EOT-PET imaging and its effect on further treatment guidance of patients with PMLBCL under R-da-EPOCH in a real-life setting.

Methods: Among 119 patients with PMLBCL treated with R-da-EPOCH in 19 Greek Centers (2014-2021), 107 were evaluated with EOT-PET-scan after 6 cycles (2/107 with clearly progressive disease did not undergo PET/CT and they were considered as D5PSS-5), 10 patients have not completed therapy yet and 2 did not undergo EOT-PET (technical issues/early death). D5PSS-4 category was evaluated visually (visual assessment) and additionally according to the criterion of “1.4x”.

Results: The median follow-up after EOT-PET was 21.8 months (1-85). Among 107 evaluable patients, 17 had an EOT-PET D5PSS-1 (16%), 21 D5PSS-2 (20%), 33 D5PSS-3 (31%), 21 D5PSS-4 (20%) and 15 D5PSS-5 (14%). D5PSS-1-3: Only one relapse was reported among 71 D5PSS-1-3 patients during a median follow-up of 24.2 months (0.5-85). D5PSS-4: Among 19 D5PSS-4 patients, who were evaluable according to the “1.4x” criterion, 12 (63%) remained D5PSS-4 (SUVmax>1.4xSUVmax liver ) and 7 (37%) were reclassified as D5PSS-3 (SUVmax<1.4x). 5/21 patients with D5PSS4 (24% of total) actually received RT, without significant difference based on the “1.4” criterion” (29% vs 17%, p=0.54). The median SUVmax of D5PSS-4 patients who received RT or not was 4.3 (2.9-6.0) vs 4.0 (2.5-5.6) (p=0.84), respectively. After a 35-month median follow-up (2-65), 2/16 patients who did not receive RT relapsed (13% or 10% of D5PSS4 patients) and 1/2 received later on treatment RT and achieved complete remission (CR). Overall, RT was omitted in 84/92 (91%) patients with D5PSS-1-4 and only 2/92 developed disease progression. D5PSS-5: 10/15 patients with D5PSS-5 received salvage chemotherapy [9 with stable/progressive disease (SD/PD) and 1 with partial response (PR)], whereas 5 patients with PR underwent RT. Median SUVmax values for these 2 groups was 17.2 (12.8-27.7) vs 10.4 (7.7-11.7) respectively (p<0.001). All 5 patients with PR who received RT remain in remission compared to 3/10 patients who received salvage chemotherapy after SD/PD (6/10 died).

Summary/Conclusion: PET/CT is a valuable tool for response assessment in PMBCL after R-da-EPOCH, facilitating clinical decision making regarding further consolidative RT, which can be minimized. In a real-life setting, RT was safely omitted in the vast majority (91%) of patients with EOT-PET D5PSS-1-4 with only 2% treatment failure. Handling of D5PSS-4 subgroup requires further study in larger case series. Patients with D5PSS5 are rather rare; consolidative RT appears extremely effective in conventional responders but those with SD/PD still have poor outcomes irrespectively of treatment.

P1231: ZILOVERTAMAB VEDOTIN (MK-2140) FOR THE TREATMENT OF NON-HODGKIN LYMPHOMA: THE PHASE 1 DOSE ESCALATION AND COHORT EXPANSION WAVELINE-001 STUDY OF AN ANTI-ROR1 ANTIBODY-DRUG CONJUGATE

M. Wang 1,* , M. Mei 2 , P. M. Barr 3 , J. Barrientos 4 , S. de Vos 5 , R. Furman 6 , K. Patel 7 , P. A. Thompson 1 , M. Choi 8 , A. Kallam 9 , Y. Zhu 10 , S. Chakraborty 10 , P. Marinello 10 , S. E. Spurgeon 11

1 MD Anderson Cancer Center, Houston; 2 City of Hope Cancer Center, Duarte; 3 University of Rochester Medical Center, Rochester; 4 Northwell Health, Inc., New Hyde Park; 5 UCLA Santa Monica Medical Center, Santa Monica; 6 Weill Cornell Medical College, New York; 7 Swedish Cancer Institute, Seattle; 8 University of California San Diego, San Diego; 9 University of Nebraska Medical Center, Omaha; 10 Merck & Co., Inc., Kenilworth; 11 Oregon Health and Science University, Portland, United States of America

Background: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transmembrane protein overexpressed in hematological malignancies. Zilovertamab vedotin (MK-2140) is an antibody-drug conjugate comprising a humanized IgG1 monoclonal antibody, a proteolytically cleavable linker, and the antimicrotubule cytotoxic agent monomethyl auristatin E (MMAE). The cytotoxicity of zilovertamab vedotin in hematologic cell lines has been demonstrated in preclinical studies.

Aims: This first human phase 1 dose escalation study, WAVELINE-001 ( NCT03833180 ), evaluated safety and efficacy of zilovertamab vedotin at various doses in patients (pts) with relapsed/refractory hematologic malignancies.

Methods: Eligible pts aged ≥18 years with an ECOG PS of 0-2 and histological diagnosis of chronic lymphocytic leukemia/small lymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia, acute lymphoid leukemia, acute myeloid leukemia, or non-Hodgkin lymphoma (NHL; mantle cell lymphoma [MCL], follicular lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma [DLBCL], Richter transformation, Burkitt lymphoma, and T-cell-NHL) were enrolled. Pts received zilovertamab vedotin intravenously at starting doses of 0.50 mg/kg (up to 2.5 mg/kg) on day 1 every 3 weeks (Q3W) (Schedule 1), 1.0 mg/kg (planned up to 2.25 mg/kg) on day 1 and 8 Q3W (Schedule 2), or 1.0 mg/kg (planned up to 2.25 mg/kg) on days 1, 8, and 15 Q4W (Schedule 3) using an accelerated plus 3 + 3 dose-escalation design. Primary end point was determination of the maximum tolerated dose (MTD). Secondary end points included safety, objective response rate (ORR), and duration of response (DOR). Data for pts with NHL enrolled in Schedule 1 are presented.

Results: A total of 51 pts were enrolled in Schedule 1 (starting dose 0.5 [n=1], 1.00 [n=3], 1.50 [n=3], 2.00 [n=3], 2.25 [n=11], or 2.50 [n=30] mg/kg) as of the data cutoff of May 18, 2021. Median age was 70 (range, 44-91) years, 54.9% of pts were male, 49.0% had an ECOG PS of 0, and 41/51 (80%) were diagnosed with NHL; 13/51 (25.5%) were diagnosed with DLBCL and 17/51 (33.3%) were diagnosed with MCL. 15 pts (29.4%) received prior CAR-T/CAR-NK. Enrollment in Schedules 2 and 3 is currently ongoing. The MTD for Schedule 1 was determined to be 2.5 mg/kg. Treatment-related adverse events (AEs) occurred in 37 pts (72.5%), most commonly (≥20%) peripheral neuropathy (47.0%), fatigue (37.3%), neutrophil count decrease (31.4%), nausea (27.5%), and diarrhea (21.6%); 24 pts (47.1%) experienced a grade ≥3 treatment-related AE. One pt died due to acute respiratory failure; however, it was not considered treatment-related by the investigator. For Schedule 1, after a median (range) follow-up of 17.3 (range, 1.2-25.8) mo, ORR was 36.6% (15/41 [95% CI, 22.1%-53.1%]; 5 CR; 10 PR) among all pts with NHL. For pts in the NHL group who had DLBCL (n=13), ORR was 38.5% (95% CI, 13.9%-68.4%; CR 3; PR 2). For pts from the NHL group who had MCL (n=17), ORR was 52.9% (95% CI, 27.8%-77.0%; CR 2; PR 7). For pts with DLBCL and MCL, 9/13 (69.0%) and 9/17 (53%) had any decrease in target lesions, respectively. Median DOR was 7.8 mo (range, 2.1-17.6+ mo) among all pts in Schedule 1 with NHL who achieved a response; 4.6 mo (range, 2.9-17.6) for DLBCL and 10 mo (range, 2.1-15.7) for MCL. For pts who received prior CAR-T/CAR-NK, the ORR was 40.0% (95% CI, 16.3%-67.6%); 2 pts had a CR and 4 had a PR.

Summary/Conclusion: Targeting the ROR1 pathway with zilovertamab vedotin is associated with a tolerable safety profile and promising antitumor activity in pts with relapsed/refractory NHL.

P1232: PRINCIPLES OF PRECISION ONCOLOGY IN PRIMARY REFRACTORY HIGH-GRADE B-CELL LYMPHOMA WITH MYC-, BCL2 AND/OR BCL6 REARRANGEMENTS (HGBL-DH/TH)

H. Witte 1,* , A. Faehnrich 2 , J. Riedl 3 , A. Kuenstner 2 , J. Ketzer 3 , N. von Bubnoff 3 , H. Merz 4 , H. Busch 2 , A. Feller 4 , N. Gebauer 3

1 Hematology and Oncology, German Armed Forces Hospital Ulm, Ulm; 2 Medical Systems Biology Group, University of Luebeck; 3 Hematology and Oncology, UKSH Campus Luebeck; 4 Haematopathology, Haematopathology Luebeck, Luebeck, Germany

Background: The optimal treatment strategy in primary refractory high-grade B-cell lymphomas with MYC-, BCL2 and/or BCL6 rearrangements (prHGBL-DH/TH) remains insufficiently addressed in the light of unfavorable prognosis and recent findings concerning the mutational pathogenesis of this provisional entity. Although there is growing evidence supporting multidisciplinary molecular tumor-boards (MTB) in solid tumors, only a minor subset of MTB recommendations affects hematologic malignancies.

Aims: In the present study, we aimed to emphasize the potential of MTB recommendations in HGBL-DH/TH patients (n=19) based on previously published whole exome sequencing (WES) data.

Methods: For the annotation of genomic alterations, the institutional MTB pipeline (University Cancer Center Schleswig-Holstein: UCCSH), certified for routine clinical diagnostics, was employed. Consecutive data-base research and annotation according to NCT/DKTK MASTER evidence levels was performed in order to identify molecularly stratified treatment strategies.

Results: Median age was 72 (range 35-79) and 68.4% (n=13) were male. Cytogenetically 7/19 cases presented with MYC/BCL2 , 5/19 with MYC/BCL6 double-hit-constellation and 7/19 triple-hit lymphomas were included. The standard turn-around time for the MTB pipeline after sampling was 21 days. The applied LymphGen algorithm proposed by Wright et al. revealed the majority of cases (n=13) to correspond to the C3/EZB cluster whereas three cases corresponded to the C1/BN2 cluster and the remaining three cases were not classifiable. Our virtual institutional MTB pipeline identified molecular stratified treatment approaches in 16/19 HGBL-DH/TH cases with NCT/DKTK evidence level of m2A or higher. As proposed by Horak et al. , we classified significant mutations in accordance with biomarker/treatment baskets and detected four targetable cell cycle (CC) alterations, two targetable tyrosine kinases (TK) and five others (OTH). Noticeable, 14 mutations affecting chromatin and/or epigenetic modifiers such as EZH2 or IDH1 were annotated as promising therapeutic targets. Moreover, one case presented with relevant BRCAness features suggesting the administration of a PARP-inhibitor such as Olaparib. Each recommended drug was either approved by the FDA and/or EMA or at least designated for FDA/EMA fast track development.

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Summary/Conclusion: The applied virtual MTB approach revealed molecularly stratified treatment options alongside targetable genomic signatures in the majority of primary refractory HGBL-DH/TH patients. The present results underline the potential relevance of MTB consultations in this difficult-to-treat entity.

P1233: PRIMARY REFRACTORY PLASMABLASTIC LYMPHOMA: A PRECISION ONCOLOGY APPROACH

H. Witte 1,* , A. Fähnrich 2 , J. Riedl 3 , A. Kuenstner 2 , N. Hertel 3 , N. von Bubnoff 3 , H. Merz 4 , H. Busch 2 , A. Feller 4 , N. Gebauer 3

1 Hematology and Oncology, German Armed Forces Hospital Ulm, Ulm; 2 Medical Systems Biology Group, University of Lübeck; 3 Hematology and Oncology, UKSH Campus Lübeck; 4 Haematopathology, Haematopathology Luebeck, Luebeck, Germany

Background: The comprehensive implementation of standardized multidisciplinary molecular tumor boards (MTB) and synchronous advancements in genomic profiling diversified the spectrum of treatment strategies in cancer patients, predominantly focusing on those with solid tumors. However, hematologic malignancies remain underrepresented in MTBs.

Aims: The current study aimed to assess the potential of precision oncology for primary refractory plasmablastic lymphoma (PBL), a highly lethal blood cancer.

Methods: We re-evaluated previously published whole exome sequencing (WES) data of 14 primary refractory PBL cases from initial diagnosis. For this proof-of-concept study, the institutional MTB pipeline (University Cancer Center Schleswig-Holstein: UCCSH) which is certified for routine clinical diagnostics has been conducted for the individual annotation of mutations aiming to virtually recommend molecularly stratified treatment strategies. Evidence levels for MTB-recommendations were defined in accordance with the NCT/DKTK MASTER program.

Results: Median age was 76.5 years (range 56-91) and 78.6% patients were male. The composition of given cytoreductive regimens was heterogenous and the overall prognosis was dismal. The standard turn-around time for the MTB pipeline after sampling was 21 days. Potential recommendations on a molecular stratified treatment option with a NCT/DKTK evidence level of at least m2A were detected for each PBL case. Based on immunohistochemical assessment (CD19, CD30, CD38 and CD79B), data-base research revealed targeted treatment recommendations in 11/14 (78.6%) cases. Genetic alterations were classified in accordance with biomarker/treatment baskets proposed by Horak et al . According to baskets, we identified tyrosine kinases (TK; n=7), PI3K-MTOR-AKT pathway (PAM; n=3), cell cycle alterations (CC; n=2), RAF-MEK-ERK cascade (REM; n=2) and others (OTH; n=7) for targeted treatment recommendations. Moreover, immunotherapeutic approaches seemed feasible in 6 PBL cases due to the predominant affection of DNA-damage repair genes. Each recommended drug was either approved by the FDA and/or EMA or at least designated for FDA fast track development.

Summary/Conclusion: The presented proof-of-concept study demonstrates the applicability of precision oncology approaches in PBL patients. Due to the aggressive course of the disease, such patients are in urgent medical need for personalized treatment approaches instead of conventional chemotherapeutic options and should be considered for MTB inclusion at the earliest time.

P1234: REAL-WORLD EXPERIENCE OF OUTCOMES WITH GLOFITAMAB SALVAGE THERAPY FOR RELAPSE/REFRACTORY B-CELL LYMPHOMA IN TAIWAN: MINIMAL SAFETY CONCERN WITH HEPATITIS B REACTIVATION

S.-J. Wu 1,* , C.-K. Liao 2 , Y.-W. Wang 1 , M.-C. Wang 2

1 National Taiwan University Hospital, Taipei City; 2 Kaohsiung Chang Gung Memorial Hospital, Kaohsiung City, Taiwan

Background: Glofitamab, a T-cell-engaging bispecific antibody (Ab) with a novel 2:1 configuration conferring robust bivalency for CD20 and monovalency for CD3, has recently shown promising activity in its phase I/II trial as a salvage therapy in various major subtypes of B-cell lymphoma. Few studies, however, have described the use of glofitamab in a real-world clinical setting.

Aims: We aimed to depict the efficacy and safety profiles of glofitamab as a salvage therapy for patients with diffuse large B-cell lymphoma (DLBCL) and other subtype of B-cell lymphoma in the real-world setting with a special focus on the risk of hepatitis B virus (HBV) reactivation.

Methods: The clinical data of patients with DLBCL or other B-cell lymphomas who had received ≥3 lines of prior therapies and received glofitamab salvage therapy in a compassionate use program in Nov. 2020 - Dec. 2021 were retrospectively analyzed. The treatment protocol included obinutuzumab pretreatment 1000mg given 7 days before the first glofitamab dose, followed by step-up dose of glofitamab administration: 2.5mg on Day 1 and 10mg on Day 8 of cycle 1, and then 30mg on Day 1 of cycle 2-12 (21-day cycles).

Results: As of the end of 2021, 26 patients from two medical centers in Taiwan, 11(43%) of whom were females, were enrolled. Among them, 18 were diagnosed with de novo DLBCL, 3 with transformed large B-cell lymphoma, and 5 with other subtypes of B-cell lymphoma. The median age was 57 years (26-75). Among the 18 cases with de novo DLBCL, 10(56%) had non-germinal center subtype, and 9(50%), double-hit/triple-hit lymphoma. Extra-nodal lesions were observed in 12(46%) patients, and 7(27%), marrow involvement. Elevated LDH levels occurred in 15 (58%) patients. Median prior therapies were 5 (3–10) with 24(92%) patients having received ≥4 prior therapies. All patients had received prior rituximab therapy, 6(23%), autologous or allogeneic stem cell transplantation (SCT), and 9(35%), polatuzumab salvage. Nineteen (79%) patients were refractory to their most recent regimen. Median cycles of glofitamab treatment were 5 (1-12). With the median follow-up of 9 months, the median overall survival was 6 months. The overall response and complete response rates were 50% and 23%, respectively. The remissions are durable (Figure 1): among 13 responders, 8 are continuing the treatment, 1 finished the planned 12 cycles of therapy, 1 had consolidated autologous SCT in remission status, and 2 passed away because of infections in remission status; only one case had disease relapse after achieving remission. No case has had disease progression after achieving an initial response. Cytokine releasing syndrome (CRS) occurred in 15(58%) patients; most of them occurred in the initial two cycles and were mostly of grade 1/2 severity except two cases (8%) with grade 3 CRS, both resulted from transaminitis. No grade 4/5 CRS, neurological toxicity, or fatal toxicity was observed. In terms of HBV infection, 7 patients were HBs antigen(+) and 6 of them had concurrent prophylactic HBV therapy during glofitamab treatment, whereas 11 patients were anti-HBc antibody(+)/HBs antigen(-) and 2 of them received prophylactic HBV therapy. No HBV reactivation event was observed in this cohort of patients.

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Summary/Conclusion: In the real-world setting, glofitamab remains an effective salvage treatment for patients with heavily pretreated relapse/refractory DLBCL and B-cell lymphomas. No new safety issues were observed. With optimal prophylaxis, the risk of HBV reactivation would not be of concern during glofitamab treatment.

P1235: GENOTYPING ON CIRCULATING TUMOR DNA IMPROVES MUTATION DETECTION RATE IN HIGH-RISK DIFFUSE LARGE B-CELL LYMPHOMA

Y. Xia 1 , L. Wang 1 , J. Liang 1 , H. Shen 1 , J. Wu 1 , H. Yin 1 , H. Zhu 1 , J. Li 1 , W. Xu 1,*

1 Jiangsu province hospital, Nanjing, China

Background: Diffuse large B cell lymphoma (DLBCL) is one of the most common lymphomas with heterogeneous clinical outcome. The circulating tumor DNA (ctDNA) represents a novel non-invasive source of tumor genetic material that overcomes anatomical biases of tissue biopsy, and thus has been widely used in aggressive lymphomas.

Aims: In this study, we collected a total of 170 samples from 104 DLBCL patients with different disease status, including 62 with paired ctDNA and tissue biopsy genomic DNA (gDNA) samples, to determine the clinical relevance of ctDNA genotyping as well as its concordance with tissue biopsy gDNA genotyping.

Methods: A targeted sequencing gene panel including all exons and critical introns of 112 genes that are recurrently mutated in DLBCL was used for this study (Burning Rock Biotech Co. Ltd, Guangzhou, China). The samples were divided into 4 groups according to the disease status at enrollment: ① treatment-sensitive (TS), representing samples from 39 treatment-naïve DLBCLs who responded to frontline treatment; ② primary refractory (Pri-R), representing samples from 10 treatment naïve DLBCLs who failed to achieve partial remission (PR) at the interim evaluation or complete remission (CR) at the end of frontline treatment; ③ early-relapse/refractory (ERR), representing samples collected at the time of relapse from 45 patients who relapsed within 12 months after diagnosis or refractory to prior treatment; ④ late-relapse (LR), representing samples collected at the time of relapse from 13 patients who relapsed more than 12 months after diagnosis.

Results: With a mean sequencing depth of 16140× (range: 8377×−23356×) in ctDNA and 1868× (range: 1009×−2881×) in gDNA, variants were identified in 88.2% (90/102) ctDNAs and profiles were similar to that of gDNA genotyping. The mean VAF of ctDNA mutations showed a good correlation with elevated lactate dehydrogenase (LDH, P=0.0008) and β2-microglobulin (β2-MG, P=0.039), as well as the metabolism tumor volume (MTV, R 0.38; P=0.0024) and the total lesion glycolysis (TLG, R 0.40; P=0.0012) assessed by PET/CT. Besides, patients with a mean VAF of ≥1.5% (determined by X-tile) had significantly shorter PFS (P=0.0004) and OS (P=0.0024). In the 62 patients who had paired ctDNA and gDNA samples, we found ctDNA genotyping outperformed gDNA genotyping in ERR DLBCLs. The proportion of ctDNA-specific variants in ERR DLBCLs was significantly higher than that of the other three groups (TS: 7.3%, 29/398; Pri-R: 10.3%, 4/39; ERR: 25.9%, 225/870; LR: 3.2%, 4/126; P<0.001). We further investigated the profile of ctDNA-specific mutations in ERR DLBCLs and found that a considerable amount of unfavorable gene mutations, including 33.3% (4/12) of TP53 mutations, 66% (4/6) of PRDM1 mutations, and 62.5% (5/8) of EP300 mutations.On the other hand, the detection rate of genetic subtypes was 51.6% (32/62) and 50% (31/62) in ctDNA and gDNA in the 62 patients with paired samples.When the two sample types were combined together, the detection rate of genetic subtypes could reach 58.1% (36/62). Interestingly, it was found that all but one (6/7) ctDNA-specific subtype belonged to the MCD subtype. In fact, 35.3% (6/17) MCD DLBCLs were only identified by ctDNA but not by gDNA, suggesting the addition of ctDNA genotyping substantially improved the detection rate of MCD subtype (figure 1).

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Summary/Conclusion: Our study highlights the clinical application of ctDNA, in particular in patients with high-risk and early relapsed or refractory diseases, where ctDNA analysis represents a complementary approach to tissue biopsy gDNA genotyping.

P1236: EFFICACY AND TOXICITY FOR CD7 CHIMERIC ANTIGEN RECEPTOR T-CELL THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY T-CELL LYMPHOMA

K. Hu 1,* , F. Yang 1 , H. Shi 1 , R. Liu 1 , P. Zheng 1 , S. Feng 1 , Y. Guo 1

1 Beijing GoBroad Boren Hospital, Beijing, China

Background: The prognosis of refractory/relapsed T-cell lymphoma is extremely poor, especially for the patients who failed to allogeneic hematopoietic stem cell transplantation(allo-HSCT).

Aims: For r/r T-cell lymphoma, a clinical trial using CD7 chimeric antigen receptor T-cell(CD7 CAR-T) has been registered, and the safety and efficacy will be evaluated.

Methods: From August 2017 to January 2022, 10 patients were enrolled. The median age was 32(19-72) years old. The diagnosis included T cell lymphoblastic lymphoma(T-LBL,n=8),hepatosplenic T-cell lymphoma(HSTL,n=1)and monomorphic epitheliotropic intestinal T-cell lymphoma(MEITL,n=1). The disease status was progressive disease (PD)in all patients who failed to multi-line therapies, including allo-HSCT (7/10,70%), local irradiation (2/10,20%).Three patients(3/10, 30%)had central nervous system involvement.In order to further reduce the tumor burden, all patients were treated with bridging therapy before CAR-T cell infusion. Before the trial, the expression of CD7 antigen in tumor tissue was positive confirmed by pathology. Patients received FC regimen (fludarabine and cyclophosphamide) before CAR-T cells infusion. The kinetics and function of CAR-T cells was monitored by quantitative PCR and flow cytometry. The efficacy was evaluated by PET-CT as well as bone marrow puncture every 3 month after CAR-T infusion.

Results: The median CAR-T cells infused were 1.06×10 5 /kg (range,0.08-24×10 5 /kg). For CAR T cell expansion,the peak time in vivo was on median 14(range,11-29) days after CAR-T cell infusion.The median peak lever of CAR-T cell was 7.94 (range,1.61-49)×10 7 /L, which was no correlation with the number of CAR-T infused. The incidence of cytokine release syndrome (CRS) was 90%(9/10), of which 20% (2/10)was grade 3, as the incidence of severe immune effector cells associated neurologic toxicity (ICANS) (grade 4)was 10%(1/10). Levels of CAR-T cells were very low after the first 1 months postinfusion which detected persistently in 6/10(60%) patients, and the longest lasting time was 355 days post-CAR-T. Although patients’CD7-positive normal T cells were depleted, CD7-negative T cells expanded in all patients.Cytopenias occurred in all patients.Six patients had grade 3 or higher lymphocytopenia, anemia (n=5), thrombocytopenia (n=4), and neutropenia (n=6).Four patients（4/10,40%）had prolonged cytopenias (1 month).1/10(10%) patient of grade IV acute GVHD (intestine) were seen.Viremia and herpes zoster occurred in 4/10(40%) and 2/10(20%) patients respectively. 1/10(10%) patient developed posttransplant lymphoproliferative disorders(PTLD)associated with EBV infection.Altogether,the overall response (ORR) was100% (10/10). Nine patients achieved complete response(CR) (9/10; 90%), one patient of MEITL achieved partial response (PR)(1/10; 10%).With the median follow-up 4.8months (range,3.2-14months), 8 patients survived(8/10,80%),7 patients remained in complete remission at the cutoff date(7/10,70%),and 1 patient of T-LBL achieved PD and survival with tumor(1/10,10%).Two patients(2/11,20%) with T-LBL died of infection.

Summary/Conclusion: Our study showed promising efficacy of CD7 CAR-T cell therapy in r/r T-cell lymphoma. CRS is manageable.We need to pay close attention to the incidence of prolonged cytopenias and infection.Long-term follow-up is needed.

P1237: PRELIMINARY RESULTS OF A PHASE Ⅱ STUDY OF ZANUBRUTINIB COMBINED WITH IMMUNOCHEMOTHERAPY IN PATIENTS WITH CD79A/CD79B-MUTANT DIFFUSE LARGE B-CELL LYMPHOMA

Z. Yan 1 , S. Yao 1 , H. Wang 1 , J. Chu 1 , S. Zhao 1 , J. Ma 2 , B. Wei 2 , J. Zheng 2 , Q. Lin 3 , Z. Xu 4 , Z. yao 1,* , Y. liu 1

1 Department of Internal Medicine, Affiliated Cancer Hospital of Zhengzhou University; 2 Department of Molecular Pathology, Affiliated Cancer Hospital of Zhengzhou University; 3 Department of Hematology, Affiliated Cancer Hospital of Zhengzhou University, zhengzhou; 4 Department of Hematology, Xinyang Central Hospital, xinyang, China

Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous hematologic malignancy with highly variable genetic abnormalities and clinical outcomes, highlighting the importance of individualized treatment. Previous studies showed that CD79B mutation was associated with higher risk of relapse and shorter survival after standard treatment (2020 ASH Abstract 2923), and both CD79B - or CD79A -mutant lymphoma cells were sensitive to BTKi in vitro (Nat Med. 2015;21:922-926). Moreover, a recent pooled analysis revealed that CD79B -mutant lymphoma patients (pts) treated with Zanubrutinib alone or in combination with anti-CD20 antibody had better response (2020 EHA Abstract EP1246). Thus, we inferred that BTKi combined with immunochemotherapy might be a favorable option for pts with CD79A / CD79B -mutant DLBCL. Here, we present preliminary data from the phase 2 trial of Zanubrutinib combined with immunochemotherapy in pts with CD79A / CD79B -mutant DLBCL ( NCT04668365 ).

Aims: To investigate the efficacy of Zanubrutinib combined with immunochemotherapy in pts with CD79A / CD79B -mutant DLBCL. The primary objective is complete response rate (CRR).

Methods: Pts aged 18-80 years with CD79A / CD79B -mutant DLBCL and adequate organ function are being enrolled. This study consisted of treatment naïve (TN) cohort and relapsed/refractory (R/R) cohort. Zanubrutinib (160 mg po bid) plus R-CHOP (ZR-CHOP) was administered in TN cohort, and Zanubrutinib (160 mg po bid) combined with investigator-determined conventional salvage chemotherapy (CSC, including ICE, DHAP, GDP, or GemOx, +/- rituximab) was administered in R/R cohort. A total of 7 cycles of treatment was planned for TN patients. After 5 cycles of induction treatment, responders in R/R cohort undergo autologous stem cell transplantation (ASCT) or Zanubrutinib maintenance for 12 months, based on the patient’s fitness and preference.

Results: From July 2020 to 22 February 2022, 91 pts had been screened, including 65 TN pts and 26 R/R pts. CD79A / CD79B mutation was identified in 14 (21.5%) TN pts and in 11 (42.3%) R/R pts, of which 7 TN pts and 8 R/R pts agreed to participate in this trial. Their baseline characteristics are displayed in Table 1.

By 22 February 2022, 6 TN pts and 7 R/R pts had been evaluated for response. All of the 6 (100%) TN pts achieved CR. In the R/R cohort, the responses were CR in 4 (51.7%) pts, partial response (PR) in 2 (28.6%) pts, and progression disease (PD) in 1 (14.3%) patient. The median follow-up time was 9.7 (1.2 – 19.2) months for the TN cohort and 3.8 (0.3 – 9.6) months for the R/R cohort. None of the patients had PD in the TN cohort. Two pts (1 R/R and 5 R/R ) in the R/R cohort experienced PD and subsequently succumbed to lymphoma, both of which harbored TP53 mutations.

The most common adverse events in both cohorts were hematologic toxicities. Grade 3/4 AEs occurred in ≥20% pts were neutropenia, leukopenia, anemia, and thrombocytopenia. Bleeding events and cardiac events were not observed.

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Summary/Conclusion: CD79A / CD79B mutation was frequent in DLBCL patients, especially in R/R cases. Zanubrutinib combined with immunochemotherapy showed encouraging activity and acceptable tolerance in pts with CD79A / CD79B -mutant DLBCL. TP53 mutation seems to be a detrimental factor. The study is still ongoing and it is worth looking forward to updating the long-term survival data.

P1238: EFFICACY ANALYSIS OF PEGASPARGASE, GEMCITABINE, OXALIPLATIN, DEXAMETHASONE COMBINED WITH TISLELIZUMAB IN FIRST-LINE TREATMENT OF PLASMA EBV-DNA POSITIVE EXTRANODAL NK/T CELL LYMPHOMA

X. Yuan 1 , Z. Zhu 1,* , R. Ma 1 , L. Wang 2 , H. Li 1 , L. Jiang 1 , K. Sun 1

1 Department of Hematology; 2 Department of pathology, Henan Provincial People’s Hospital, Zhengzhou, China

Background: Positive circulating EBV-DNA is associated with a very poor outcome in Natural killer/T cell lymphoma (NKTCL). Efforts to further improve treatment of plasma EBV positive NKTCL are urgently needed.

Aims: To investigate the clinical efficacy, plasma EBV-DNA load and safety of P-GEMOXD combined with anti-programmed death 1 (anti-PD-1) antibody Tislelizumab in first-line treatment of plasma EBV-DNA positive NKTCL.

Methods: In this prospective, single-arm, phase 2 trial, we recruited patients aged 18–70 years with newly diagnosed NKTCL who were EBV-DNA positive in plasma in Henan People’s Hospital. Patients received four courses of P-GEMOXD plus Tislelizumab treatment (pegaspargase 2500U/m 2 [day3], gemcitabine 800mg/m 2 [day1 and 8]; oxaliplatin100 mg/m 2 [day1], dexamethasone 20mg/d [day1-4]; Tislelizumab 200mg /d [day9] every 21days). Thirteen patients were enrolled from January 2018 to August 2021 December, the median follow-up was 26 (12-46) months. The short-term and long-term clinical efficacy, load of EBV-DNA and adverse reactions were observed.

Results: Among the 13 patients, there were 9 males and 4 females, the median age was 54 years old (range 25-64). After 4 courses of treatment, 10 patients (76.9%) had complete response, 3 patients (23.1%) had partial response, and the objective response rate (ORR) was 100%. The 2-year progress-free survival (PFS) rate was 76.9%, and the 2-year overall survival (OS) rate was 85.7%. Among them, there were 6 patients with advanced stage (III and IV), 4 patients (66.7%) had complete response, 2 patients (33.3%) had partial response, 5 patients (83.3%) plasma EBV-DNA converted negative; the 2-year progression-free survival (PFS) rate was 66.7%, and the 2-year overall survival (OS) rate was 66.7%. Treatment-related AEs of any grade occurred in 84.6% (11/13) of all patients. The main adverse reactions were albumin reduction (8/13), fibrinogen reduction (9/13), autoimmune diseases (3 cases：1 case of hypopituitarism， 1 case of Hashimoto’s thyroiditis, and 1 case spondylitis).

Summary/Conclusion: P-GEMOXD plus Tislelizumab can significantly improve the prognosis of patients with NKTCL, improve the rate of plasma EBV-DNA negative conversion, and the adverse reactions can be well tolerated.

P1239: TISLELIZUMAB, A PD-1 INHIBITOR FOR RELAPSED/REFRACTORY MATURE T- AND NK-CELL NEOPLASMS: RESULTS FROM A PHASE 2 STUDY

E. Bachy 1,* , K. J. Savage 2 , H. Huang 3 , Y. L. Kwong 4 , G. Gritti 5 , Q. Zhang 6 , A. M. Liberati 7 , J. Cao 8 , H. Yang 9 , S. Hao 10 , J. Hu 11 , K. Zhou 12 , F. Russo 13 , H. Zhang 14 , W. Sang 15 , J. Ji 16 , A. J. M. Ferreri 17 , G. L. Damaj 18 , H. Liu 19 , W. Zhang 20 , X. Ke 21 , C. Ghiggi 22 , S. Huang 23 , X. Li 23 , H. Yao 23 , J. Paik 24 , W. Novotny 24 , W. Zhou 23 , H. Zhu 23 , J. Huang 24 , P. L. Zinzani 25

1 Hematology Department, Lyon Sud Hospital and Claude Bernard University Lyon 1, Lyon, France; 2 Department of Medical Oncology, BC Cancer, Vancouver, Canada; 3 Sun Yat-Sen University Cancer Center, Guangzhou; 4 Queen Mary Hospital, Hong Kong, China; 5 ASST Papa Giovanni XXIII, Lombardia, Italy; 6 Harbin Medical University Cancer Hospital, Harbin, China; 7 Azienda Ospedaliera Santa Maria Terni, Terni, Italy; 8 Fudan University Shanghai Cancer Center, Shanghai; 9 The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Hangzhou; 10 Xin Hua Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai; 11 Fujian Medical University Union Hospital, Fujian Institute of Hematology, Fuzhou; 12 Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, China; 13 Ospedale Maggiore, Ematologia e Centro Trapianti Midollo Osseo (CTMO), AOI Parma, Italy; 14 Tianjin Medical University Cancer Institute & Hospital, Tianjin; 15 The Affiliated Hospital of Xuzhou Medical University, Xuzhou; 16 West China Hospital, Sichuan University, Chengdu, China; 17 Lymphoma Unit, IRCCS San Raffaele Scientific Institute, Milano, Italy; 18 Institut d’Hematologie de Basse Normandie, Caen, France; 19 Beijing Hospital, Beijing; 20 Peking Union Medical College Hospital; 21 Peking University Third Hospital, Shanghai, China; 22 Ospedale Policlinico San Martino, Genova, Italy; 23 BeiGene (Shanghai) Co., Ltd., Shanghai, China; 24 BeiGene USA, Inc., San Mateo, CA, United States of America; 25 Institute of Hematology “Seràgnoli”, University of Bologna, Bologna, Italy

Background: Effective treatment choices for patients (pts) with relapsed/refractory (R/R) mature T- and NK-cell neoplasms after failure of standard therapies are limited. Tislelizumab (TIS), a humanized anti–PD-1 mAb, demonstrated outstanding efficacy and favorable safety in pts with R/R classical Hodgkin lymphoma or solid tumors.

Aims: To evaluate the safety and efficacy of TIS in pts with R/R mature T- and NK-cell neoplasms.

Methods: This was a global, multicenter, single-arm, open-label, phase 2 study ( NCT03493451 ). Pts were enrolled into 3 cohorts stratified by the type of T- and NK-cell neoplasms to receive TIS 200 mg intravenously every 3 weeks until disease progression or intolerable toxicity. Eligible pts had ≥1 prior systemic therapy, disease progression during or after most recent therapy completion or refractory disease, Eastern Cooperative Oncology Group performance score ≤2, and life expectancy ≥6 mo. All pts gave informed consent. Primary endpoint was investigator-assessed overall response rate (ORR). Secondary endpoints included duration of response (DOR), complete response (CR) rate, progression-free survival (PFS), overall survival (OS) in cohorts 1 and 2, and safety.

Results: A total of 77 pts were treated. Cohort 1 included 22 pts with R/R extranodal NK and T-cell lymphomas; cohort 2 included 44 pts with R/R mature T-cell neoplasms (21 peripheral T-cell lymphoma not otherwise specified, 11 angioimmunoblastic T-cell lymphoma, and 12 anaplastic large cell lymphoma); and cohort 3 included 11 pts with R/R cutaneous T-cell lymphomas (CTCL; stage ≥1B; 8 mycosis fungoides and 3 Sézary syndrome). Of all pts, 76.6% had advanced-stage disease, 51.9% had refractory disease, and 49.4% had ≥3 prior systemic regimens. Median treatment cycles for cohorts 1, 2, and 3 were 5 (range 1-37), 4.5 (range 1-38), and 17 (range 3-25), respectively. Cohort 3 had promising efficacy (median study follow-up [FU] 16.6 mo); ORR 45.5%; CR 9.1%; median DOR 11.3 mo (95% CI, 2.76-11.30); median PFS 16.8 mo (95% CI, 2.60-16.82); and median OS not reached (NR) (95% CI, 4.90-not estimable [NE]). Modest efficacy was reported in cohort 1 (median study FU 8.4 mo): ORR 31.8%; CR 18.2%; median DOR NR (95% CI, 2.66-NE); median PFS 2.7 mo (95% CI, 1.45-5.32); and median OS 8.8 mo (95% CI, 3.25-NE). Modest efficacy also was noted in cohort 2 (median study FU 9.3 mo): ORR 20.5%; CR 9.1%; median DOR 8.2 mo (95% CI, 2.50-NE); median PFS 2.7 mo (95% CI, 2.56-4.76); and median OS 13.3 mo (95% CI, 7.66-26.22). Most frequent adverse events (AEs) were pyrexia (32.5%), anemia (18.2%), arthralgia (18.2%), and diarrhea (15.6%); most frequent grade ≥3 AEs were anemia (7.8%), pneumonia (6.5%), and neutropenia (5.2%). Any-grade immune-mediated AEs occurred in 22 (28.6%) pts, most frequently hypothyroidism (10.4%), hyperglycemia (5.2%), and rash (5.2%). Grade ≥3 AEs occurred in 4 (5.2%) pts and included increased blood creatine phosphokinase, hepatitis, hypothyroidism, rash, and urticaria (1 pt each). No treatment-related AEs led to death.

Summary/Conclusion: TIS was well tolerated, achieving modest efficacy in R/R mature T- and NK-cell neoplasms, with some long-lasting remissions particularly in CTCL. Further studies are needed to determine the biologic features associated with response and explore optimal combination therapies.

P1240: ANTI-PD-1-ANTIBODY (TISLELIZUMAB) COMBINED WITH DEACETYLASE INHIBITOR (CHIDAMIDE), LENALIDOMIDE AND ETOPOSIDE FOR THE TREATMENT OF REFRACTORY/RELAPSED EXTRANODAL NATURAL KILLER/T CELL LYMPHOMA, NASAL

L. Zhang 1,* , X. Liu 1 , X. Wang 1 , Y. Chang 1 , X. Fu 1 , X. Li 1 , L. Li 1 , X. Zhang 1 , J. Yan 1 , F. Nan 1 , L. Zhu 1 , Z. Sun 1 , H. Yu 1 , S. Guo 2 , W. Sang 3 , L. Wang 4 , M. Zhang 1

1 Department of Oncology, the First Affiliated Hospital of Zhengzhou University, zhengzhou; 2 Luoyang central hospital affiliated to zhengzhou University, luoyang; 3 Affiliated Hospital of Xuzhou Medical University, Xuzhou; 4 Department of Hematology,Beijing Tongren Hospital,Capital Medical University, Beijing, China

Background: Extranodal Natural Killer/T Cell Lymphoma (ENKTL) is a highly aggressive non-Hodgkin lymphoma (NHL), with higher incidence in Asia, which is related to Epstein-Barr virus (EBV) infection. Patients with r/r-ENKTL have a poor prognosis, and is lack of effective treatment. Anti-PD-1-Antibody plus chidamide was effective and well tolerated on r/r-ENKTL (SCENT Trial, 2020 ASH). Lenalidomide is an immunomodulatory agent, which has been shown to be effective in treatment of r/r-lymphoma with an acceptable safety. Etoposide is an important chemotherapeutic agent in the treatment of ENKTL. Here we present preliminary results of anti-PD-1 antibody combined with chidamide, lenalidomide and etoposide for the treatment of r/r-lymphoma.

Aims: To explore the efficacy and safety of anti-PD-1-Antibody (Tislelizumab) combined with Deacetylase Inhibitor (Chidamide), Lenalidomide and Etoposide for the treatment of refractory/relapsed Extranodal Natural Killer/T Cell Lymphoma, Nasal Type (r/r-ENKTL).

Methods: This was a prospective, single-arm, open-label, multi-center, phase II clinical trial. This trial enrolled eligible patients with histologically confirmed r/r-ENKTL failing from at least 1-line treatment; ECOG performance status ≤ 2; adequate organ function and bone marrow function; and at least one measurable or evaluable lesion. Patients received 6 cycles of Tislelizumab (200mg), Chidamide (20mg biw), Lenalidomide (25mg d1-10) and Etoposide (100mg/m 2 d1-3), every 21 days cycle. Then received 10 cycles of Tislelizumab (200mg) maintenance treatment, every 21 days cycle. The primary endpoints were objective response rate (ORR) based on Lugano 2014 criteria, duration of response (DOR), progression-free survival (PFS), overall survival (OS) and disease control rate (DCR). Secondary endpoints were median survival time (MST) and safety. Adverse events (AE) were rated according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) 5.0. This trial is registered at ClinicalTrials.gov ( NCT04038411 ).

Results: From July 2020 to March 2022, 23 patients were screened and 20 eligible patients were enrolled from 4 centers in China, with median age at 36.5 years (range 17-60), 16 (80.0%) male, 15 (75.0%) patients with stage Ⅲ-Ⅳ of disease at screening, 10 (50.0%) patients received ≧ 2 lines of prior systemic therapy. Of 19 response evaluable patients, 18 (94.7%) achieved response including 9 (50%) patients with CR. The median time to initial response was 6.0 weeks (6-12 weeks). The 12-month PFS rate was 86.8%. 19 patients reported treatment-related AEs (TRAEs). The most frequently observed TRAEs were anemia (15 patients,78.9%), neutropenia (14 patients, 73.6%), leukopenia(11 patients, 57.9%), nausea (10 patients, 52.6%), thrombocytopenia (9 patients, 50%). The most frequent Grade (G) ≥3 TRAEs were neutropenia (9 patients, 50%). Immune-related AEs were reported in 4 patients (21.1%) with G1 hypothyroidism. No death was related to the study.

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Summary/Conclusion: Tislelizumab combined with chidamide, lenalidomide and etoposide regimen have a very high response rate in r/r-ENKTL for the first time, and the safety is under control. It is a promising therapeutic option for this population, although further investigation is warranted.

P1241: THE INCIDENCE AND SURVIVAL OUTCOMES OF PATIENTS WITH PRIMARY GASTRIC T-CELL LYMPHOMA: A SEER POPULATION-BASED STUDY

M. Zhang 1,* , F. Xiao 1 , J. Hou 1 , H. Huang 1

1 Division of Hematology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China

Background: Primary gastric T-cell lymphoma (PG-TCL) is a rare neoplasm with a handful of sporadic case reports or small retrospective series study in the literature. Due to its extreme rarity, the clinicopathological entity and survival of PG-TCL have not been well characterized.

Aims: We aimed to investigate the epidemiology, clinical features and prognosis of PG-TCL.

Methods: A total of 164 patients diagnosed with PG-TCL from 1975 to 2016 extracted from the Surveillance, Epidemiology, and End Results Program (SEER) database were analyzed. Overall survival (OS) and cancer-specific survival (CSS) curves were explored using the Kaplan-Meier method. Cox proportional hazard regression was applied to investigate the independent prognostic factors of OS and CSS. Nomograms were established to predict survival possibilities.

Results: The age-adjusted incidence rate of PG-TCL was 0.0091 per 100,000 person-years and increased with age. The median age at diagnosis was 65 years old with male predominance. The major histological type was peripheral T-cell lymphoma, NOS (63.4%). The 1-, 2-, and 5-year OS were 45.5%, 34.7%, and 23.5%, respectively while the 1-, 2-, and 5-year CSS were 47.4%, 37.3%, and 29.6%%, respectively. Multivariate Cox regression analysis revealed that age at diagnosis, chemotherapy and radiotherapy were the independent prognostic factors for OS. Chemotherapy combined with radiotherapy could significantly improve patients’ OS compared with chemotherapy alone. Moreover, age at diagnosis was also the independent prognostic factor for CSS. Nomograms for PG-TCL were developed to predict 1-, 2-, and 5-year OS possibility (Figure A). The accuracy of nomograms was verified by high concordance index and good agreement with the predicted value in calibration plots.

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Summary/Conclusion: PG-TCL is a rare neoplasm with low incidence. Patients with PG-TCL generally exhibited poor prognosis. Use of Chemotherapy plus radiotherapy was associated with favorable OS.

P1242: ZANUBRUTINIB AND RITUXIMAB REGIMEN COMBINED WITH INTRAVITREAL METHOTREXATE FOLLOWED BY ZANUBRUTINIB MAINTENANCE FOR TREAT-NAIVE PRIMARY VITREORETINAL LYMPHOMA: A PROSPECTIVE PHASE II STUDY

Y. Zhang 1,* , X. Zhang 2 , W. Wang 1 , Y. Zhang 3 , M.-F. Zhang 2 , W. Zhang 1 , D. Zhou 1

1 Department of Hematology; 2 Department of Ophthalmology, Peking Union Medical College Hospital; 3 Department of Hematology, Beijing Longfu Hospital, Beijing, China

Background: Primary vitreoretinal lymphoma (PVRL) is a rare variant of extra-nodal lymphoma. The best therapeutic strategy for PVRL without CNS involvement remains undefined due to the paucity of high-quality trials in this rare disorder. Local treatments alone, including intravitreal or radiation, are related to a higher CNS relapsing rate, and systemic intensive regimens have demonstrated serious adverse effects. Many efforts have been made to balance toxicity and sustained disease control. However, rituximab and lenalidomide (R2) regimen was failed as front-line treatment in PVRL.

Aims: Zanubrutinib is a new-generation Bruton kinase inhibitor (BTKi) that had good blood-brain-barrier permeability and activities in CNS lymphoma. So we conducted a single-arm, phase II trial for newly diagnosed B-cell PVRL to evaluate the efficacy and safety of ZR (zanubrutinib plus rituximab) regimen with intravitreal MTX.

Methods: This is a prospective multi-center, open-label, phase II trial ( NCT 04899453 ). Immunocompetent patients aged 18–75 years with newly diagnosed B-cell PVRL were eligible. Eligible patients received intravitreal MTX injection and the ZR regimen for 6 cycles as induction therapy, followed by zanubrutinib monotherapy for maintenance for 2 years, or until disease progression, intolerable toxicity, or death. The treatment schedule was: MTX (400 μg) intravitreally injection, weekly×4, biweekly×2, and monthly×10; ZR regimen: rituximab 375 mg/m2 d1, zanubrutinib 160mg bid, 21 days per cycle; Zanubrutinib maintenance: 160mg bid in patients who achieved CR/PR. The primary point was 1-year progression-free survival (PFS). The safety and toxicity were also investigated. Intraocular CR was defined as the intraocular lesion disappearance and undetectable IL-10 concentration in the aqueous humor.

Results: From August 2020 to January 2022, 10 patients were enrolled, and the cut-off date was February 25, 2022. The median age was 55 yr (range 39 to 70) and seven (70%) were female. Most patients (90%) had bilateral eyes involvement and increased baseline interleukin-10 in CSF (range 20.7~118ng/ml, normal range <5ng/ml). 9 patients have completed 6 cycles of ZR regimen and all achieved complete remission; 1 patient was under her first induction cycle. After a median follow-up of 12.4 (0.3-18.1)months, 2 patients suffered CNS relapsing at 9.5 and 11.7 months. The estimated 1 year PFS was 68.5%(95%CI 21.3% to 92.2%). Compared to the historical data (R2 regimen) in our institution, there was a trend that ZR regimen prolonged PFS (unreached vs 389 days, HR 0.47, 95%CI 0.13 to 1.75, p 0.289, see Fig. 1). The toxicities were quite mild. The most common adverse events were bleeding events, including bruising(grade 1, n=4), hypermenorrhea(grade 2, n=1); vitreous hemorrhage after intravitreal injection (grade 3, n=1). ≥10% Grade 1-2 AE included pruritus, fatigue, hypertension, and anemia. No other grade 3-4 AEs were observed.

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Summary/Conclusion: Integration of ZR regimen and intravitreal MTX demonstrated excellent responses rate in newly diagnosed PVRL with very mild toxicity. To our knowledge, this is the first prospective trial to evaluate BTKi in PVRL. Although preliminary, these results provided evidence and support the use of ZR in PVRL. More clinical data will be updated from this ongoing study.

P1243: RUXOLITINIB COMBINED WITH DEXAMETHASONE IN ADULT PATIENTS WITH NEWLY DIAGNOSED HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS: INTERIM ANALYSIS OF A PROSPECTIVE, SINGLE-CENTER, SINGLE-ARM, PHASE 2 CLINICAL TRIAL

D. Zhou 1,* , X. Huang 1 , X. Li 1 , L. Zhu 1 , M. Xie 1 , X. Huang 1 , L. Ma 1 , X. Yang 1 , J. Sun 1 , L. Li 1 , J. Zhu 1 , X. Zheng 1 , S. Zhao 1 , W. Yu 1 , W. Xie 1 , H. Tong 1 , J. Jin 1 , H. Zhu 1 , X. Ye 1

1 Department of Hematology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

Background: Hemophagocytic lymphohistiocytosis (HLH) in adults is a major clinical challenge due to its rapid progress and high mortality. The current first-line therapy strategies such as HLH-94 or HLH-2004 regimens are largely extrapolated from paediatric HLH; however, the prognosis of adult HLH is worse than that of children. Ruxolitinib (an oral JAK inhibitor) has shown favorable efficacy and safety in murine models and clinical trials in adult HLH. Moreover, Lauren K. Meyer et al. demonstrate that ruxolitinib can reverse the resistance of CD8 positive T cells to dexamethasone in hypercytokinemia of HLH. We were very interested in these results and preliminarily revealed the high response rate and good short-term survival of ruxolitinib combined with dexamethasone (Ru-D) in a pilot study involving 8 adult HLH patients. On these bases, we conducted this prospective, single-center, single-arm, Phase 2 clinical study.

Aims: This phase 2 clinical trial (ChiCTR2100049996) aims to evaluate the efficacy and safety of Ru-D regimen as first-line therapy in adults with newly diagnosed HLH.

Methods: This is an interim analysis of an ongoing phase 2 clinical trial of a planned 28 newly diagnosed adult HLH patients. Inclusion criteria were patients who met HLH-2004 diagnostic criteria, older than 18 years old, and had not received HLH first-line therapy. Patients in this study received an 8-week period of treatment. The Ru-D regimen consists of ruxolitinib (15 mg twice daily for 8 weeks) and dexamethasone (initially 10 mg/m 2 for 2 weeks followed by 5 mg/m 2 for 2 weeks, 2.5 mg/m 2 for 2 weeks, 1.25 mg/m 2 for one week, and one week of tapering). The primary endpoint is the 2-month overall survival rate (OS), and the secondary endpoints are overall response rate (ORR), progression-free survival (PFS), safety and changes in biomarkers.

Results: From August 15, 2021 to January 15, 2022, there were 15 patients enrolled in this study with a median follow-up of 78 (31-171) days. The 15 patients included 8 cases of lymphoma-associated HLH (LAHS), five case of Epstein-Barr virus-associated HLH (EBV-HLH), and 2 cases of unknown etiology. It should be mentioned that 4 of 15 patients carried heterozygous HLH-related gene mutations. Patients were followed up until February 15, 2022 or until death, with none lost to follow-up. The primary endpoint, 2-month OS, was 83.3% in 12 evaluable patients, both patients died due to lymphoma progression. The overall mortality rate was 26.7% (4/15), 75% (3/4) deaths occurred in LAHS patients and there was no death directly related to HLH until data cutoff. The ORR to Ru-D regime was 86.7% (13/15), with 13.3% (2/15) in complete response (CR), 73.3% (11/15) in partial response (PR), and 13.3% (2/15) in no response (NR). The treatment was well tolerated, with no toxicities leading to dose reduction or discontinuation of treatment. All LAHS patients were successfully bridged to chemotherapy after Ru-D therapy. Table 1 shows the response outcomes in detail.

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Summary/Conclusion: In our study, the Ru-D regimen initially demonstrated favorable efficacy in newly diagnosed adult patients with HLH. For patients with LAHS, it is an efficient and safe bridging therapy while waiting for pathological diagnosis. Therefore, Ru-D represents a novel and promising therapeutic option for HLH patients that may provide clinical benefit.

P1244: ROLE OF SELECT T HELPER CELL MONONUCLEAR CELL SUBSETS PROMOTING EPSTEIN-BARR VIRUS-DRIVEN LYMPHOPROLIFERATIVE DISEASE IN THE HU-PBL-SCID MODEL

E. H. Ahmed 1,* , M. Lustberg 2 , C. Hale 1 , S. Sloan 1 , C. Mao 1 , X. Zhang 1 , H. G. Ozer 1 , S. Schlotter 1 , F. Jeney 1 , W. Chan 1 , B. Harrington 3 , C. Weigel 1 , L. Alinari 1 , C. Oakes 1 , T. Abebe 4 , G. K. Behbehani 1 , P. Shindiapina 1 , M. A. Caligiuri 5 , R. A. Baiocchi 1

1 The Ohio State University, Columbus; 2 Yale University, New Haven; 3 Michigan State University, East Lansing, United States of America; 4 Addis Ababa University, Addis Ababa, Ethiopia; 5 City of Hope National Medical Center, Duarte, United States of America

Background: The Epstein-Barr virus (EBV) is a gamma herpes virus that infects over 90% of the adult population worldwide. EBV is trophic to human B-cells and is capable of driving benign (mononucleosis) or malignant B-cell lymphoproliferative disorder (EBV-LPD). Infected B-cells are cleared by a highly efficient, EBV-specific, memory cytotoxic T lymphocyte (CTL) response in immunocompetent individuals. EBV ultimately assumes latency and establishes life-long persistence in the memory B-cell compartment. EBV can reactivate a lytic cycle program leading to the production of infectious virus, placing immunocompromised individuals, including HIV patients, organ transplant recipients, and patients with primary immune deficiency at the highest risk for EBV-LPD. Despite the ubiquity of EBV, a small percentage of immunocompromised patients (2-20%) develop EBV-LPD. Such clinical heterogeneity may reflect the presence of host factors that predispose certain individuals to EBV-LPD.

Engraftment of severe combined immune deficient (SCID) mice with peripheral blood leukocytes (PBMCs) from EBV seropositive donors leads to spontaneous EBV-LPD of human B-cell origin. PBMCs from EBV+ human donors show distinct rates of EBV-LPD in the Hu-PBL-SCID model. Of all EBV seropositive PBMC donors, only about 20% of donors will induce EBV-LPD in 100% of engrafted mice (High-Incidence, HI donors) and another 20% of donors will never generate EBV-LPD (No-Incidence, NI donors). Development of spontaneous EBV-LPD in the Hu-PBL-SCID model displays an absolute requirement for autologous CD4+ T cells in engrafted PBMC.

Aims: Here we aimed to identify immune factors that contribute to the development EBV-LPD in the hu-PBL-SCID model. We hypothesized that distinct phenotypes of PBMCs from HI and NI exist and that specific subsets of mononuclear cells promote EBV-LPD in this model.

Methods: To address this hypothesis, we studied a pool of EBV seropositive donors (HI donor n= 4; NI Donor n=5).

Results: Immunophenotypic analysis of CD4+ T-helper cell subsets showed HI donors have higher basal levels of T follicular helper (Tfh) and regulatory T-cells (Treg) cells compared to NI donors (p-values = 0.0499 and 0.0309 respectively). Mice engrafted with PBMC depleted of Tfh or Treg subsets from HI donors showed improved survival compared to mice engrafted with non-depleted PBMC. Furthermore, mass-cytometry analysis revealed a significantly higher percentage of myeloid cells in the PBMCs of HI donors compared to NI donors (p-value = 0.0018). Further analysis confirmed a higher percentage of myeloid-derived suppressor cells in the PBMCs of HI donors that, in in vitro EBV-LCL co-cultures, prevented memory CTL proliferation.

Summary/Conclusion: Our findings point toward potential immune cell subsets that contribute to the development of EBV-LPD. Further studies will enable us to define immune profile characteristics associated with a high risk of EBV-LPD and point toward potential strategies to prevent these disorders in select patient populations.

P1245: CLINICAL IMPACT OF IMMUNOGLOBULIN HEAVY CHAIN REPERTOIRE IN MANTLE CELL LYMPHOMA: A STUDY FROM THE FONDAZIONE ITALIANA LINFOMI (FIL) PHASE III MCL0208 TRIAL.

B. Alessandria 1,* , E. Genuardi 1 , A. M. Civita 1 , D. Drandi 1 , B. Mantoan 1 , M. Ferrante 1 , M. Borriero 1 , S. Ragaini 1,2 , G. Bondielli 1 , G. M. Zaccaria 1,3 , D. Barbero 1 , V. Peri 1,2 , S. Hohaus 4,5 , G. Musuraca 6 , N. Cascavilla 7 , C. Ghiggi 8 , M. Tani 9 , G. Gaidano 10 , S. Volpetti 11 , S. V. Usai 12 , N. Di Renzo 13 , S. Cortelazzo 14 , M. Ladetto 10,15 , S. Ferrero 1,2

1 Department of Molecular Biotechnologies and Health Sciences, University of Torino; 2 Città della Salute e della Scienza di Torino, Hematology Division, Torino; 3 Unit of Hematology and Cell Therapy, IRCCS-Istituto Tumori ‘Giovanni Paolo II’, Bari; 4 Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario A. Gemelli, IRCCS; 5 Dipartimento di Scienze Radiologiche ed Ematologiche, Università Cattolica del Sacro Cuore, Rome; 6 IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Hematology unit, Meldola; 7 IRCCS, Casa Sollievo della Sofferenza, Hematology Division, San Giovanni Rotondo; 8 IRCCS San Martino, U.O. Ematologia e Terapie cellulari, Genova; 9 UOC. Ospedale Santa Maria delle Croci, Hematology Unit, Ravenna; 10 Department of Translational Medicine, University of Eastern Piedmont, Division of Hematology, Novara; 11 Centro trapianti e terapie cellulari, Clinica ematologica, Ospedale Santa Maria della Misericordia- ASUFC, Udine; 12 Ospedale Oncologico Armando Businco, Division of Hematology, Cagliari; 13 Vito Fazzi Hospital, Unit of Hematology, Lecce; 14 Humanitas/Gavazzeni Clinic, Oncology Unit, Bergamo; 15 Azienda Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Struttura Complessa di Ematologia, Alessandria, Italy

Background: Mantle cell lymphoma (MCL) is characterized by a highly restricted immunoglobulin heavy chain (IGH) gene repertoire with stereotyped IGHV CDR3s, strongly implying a role for antigen-driven selection. Nonetheless, differently from chronic lymphocytic leukemia, where the prognostic role of IGH parameters is known since decades, published data are less consistent in MCL. Recently, LRPAP1 autoantibodies were detected in 13% of patients from the European MCL Network trials and were associated with superior outcome, while the autoreactivity of IGHV 4-34 antibodies is largely known.

Aims: We aimed at characterizing the IGH VDJ repertoire and its putative clinical relevance in a large, prospective, trial of the Fondazione Italiana Linfomi (FIL) enrolling younger MCL patients.

Methods: IGH clonal rearrangements detection was performed by Sanger sequencing on bone marrow or peripheral blood samples collected for minimal residual disease purposes in FIL MCL0208 trial ( NCT02354313 ). This is a prospective, randomized phase III trial comparing lenalidomide maintenance vs observation after an intensive cytarabine containing chemo-immunotherapy followed by autologous transplantation in frontline <66 year-old MCL patients. IMGT/V-QUEST tool was used to annotate IGHV-D-J and mutational status (cut-off 98%) according to ERIC guidelines. Finally, these biological data were integrated with published clinical, pathological, and mutational data from the trial (current median follow-up 38 months).

Results: A clonal IGH productive rearrangement was detected in 209/300 patients (70%), resulting in 159 (76%) unmutated (UM) and in 50 (24%) mutated (M) cases, as expected. Overall, VDJ usage included 29 IGHV families, with IGHV 3-21 (#45, 22%), 4-34 (#33, 16%), 3-7 (#15, 7%), 3-23 and 5-51 (#12 each, 6%), 4-39 and 4-59 (#10 each, 5%) as the most frequent groups, together representing the 66% of the analyzed series (Figure 1A). Interestingly, patients carrying IGHV 3-21 were often characterized by low Ki-67 index (Ki-67 <30%: 86% vs 64% of other cases, p<0.01) and low risk MIPI score (73% vs 43%, p=0.001), while no case of blastoid morphology was registered among the IGHV 4-34 patients (vs 89%, p<0.05). Moreover, by grouping together these two rearrangements, statistically significant associations were found with several biomarkers of favorable prognosis (low Ki-67, p<0.01; non-blastoid, p<0.05; absence of TP53 aberrations, p<0.05, or KMT2D mutations, p<0.05, and low-risk MIPI, p<0.0001). Accordingly, both IGHV 3-21 and 4-34 patients showed better long-term outcomes, with longer PFS and OS if compared with all the other cases, both singularly and when grouped together (3 years PFS: 75% vs 53%, p<0.05; 3 years OS: 91% vs 76%, p<0.01, Figure 1B, C). Finally, these results were independent from IGHV mutational status (p=0.09) and lenalidomide maintenance (p=0.46).

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Summary/Conclusion: This is the first study assessing the clinical impact of VDJ IGH repertoire in a large prospective trial in MCL. As expected, 76% of patients resulted UM, with 3-21 and 4-34 representing more than one third of all the IGHV rearrangements. Notably, in our series, no significant correlation between mutational status and outcome was recorded. On the other hand, patients carrying either IGHV 3-21 or 4-34 rearrangements were characterized by favorable clinical and biological known prognosticators and were associated with both PFS and OS improvement, if compared to other patients. Deeper translational insights are needed to clarify the biological (autoreactive?) basis of this observation.

P1246: DIFFERENTIAL ROLE OF MICROENVIRONMENT AND STROMAL-CELL INDUCED VENETOCLAX RESISTANCE IN MANTLE CELL LYMPHOMA

P. Andersen 1,2,* , A. Granau 1,2 , T. Jakobsen 3 , L. Sommer Kristensen 3 , J. Bay Mogensen 1,2 , K. Dimopoulos 4 , K. Grønbæk 1,2

1 Department of Hematology, Rigshospitalet; 2 Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen; 3 Department of Biomedicine, University of Aarhus, Aarhus; 4 Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark

Background: Venetoclax is an inhibitor of the anti-apoptotic protein BCL-2 and a promising drug for MCL treatment. However, resistance towards venetoclax has already been reported in several MCL studies. There is growing evidence suggesting that interactions between lymphoma cells and their surrounding microenvironment might play a key role in not only promoting tumor cell survival but also facilitating resistance to certain chemotherapeutic agents, but little is known about the role of microenvironment in drug resistance in MCL.

Aims: To understand the cellular and molecular resistance mechanisms mediated by the lymphoma microenvironment to prevent MCL patients from developing resistance to venetoclax treatment. To investigate the interactions of MCL cell lines with Mesenchymal Stromal Cells (MSCs), a key component of the microenvironment, in an in vitro co-culture system.

Methods: MCL cell lines (MAVER-1, MINO) were cultured alone or in co-culture with HS-5, a human MSC cell line, or MS-5, a murine MSC cell line and exposed to different drugs, including venetoclax. Flow cytometry was used to assess cell viability. RNA sequencing of suspension and adherent MCL cells was used to identify differentially expressed genes. ELISA multiplex analysis was used to identify human or murine pro-inflammatory cytokines in co-culture supernatant.

Results: Co-culture of MCL cell lines with both HS-5 and MS-5 induced resistance in MCL cells to doxorubicin and vincristine. However, only co-culture with MS-5 induced venetoclax resistance in MCL cells. Interestingly, conditioned media alone from MS-5 cultures was sufficient to induce venetoclax resistance, suggesting that soluble factors (such as cytokines and microvesicles) might mediate the venetoclax resistance mechanisms in MCL cells.

Induction of apoptosis through MCL-1 inhibition with drug S6338 was also abrogated by co-culture with both HS-5 and MS-5, while BCL-xL inhibition using drug WEHI-549 was only ineffective in MINO cells in MS-5 co-culture. MCL cells in HS-5 co-cultures exhibited upregulation of genes involved in inflammatory response, cytokine signaling, and apoptosis, compared to MS-5 co-cultures. Accordingly, we identified unique sets of genes that were significantly upregulated in adherent MCL cells in HS-5 co-cultures, mainly involved in cell adhesion and cytokine signaling. Moreover, HS-5 co-cultures contained high levels of IL-6 and IL-8, corresponding with the increased gene expression levels of genes involved in the IL-6 signaling pathway in MCL cells. On the contrary, MS-5 co-cultures contained only high levels of murine KC/GRO, corresponding to human chemokine CXCL1.

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Summary/Conclusion: We show that HS-5 provides MCL cells resistance to drugs, such as vincristine and doxorubicin, but not venetoclax, reflecting the different mechanisms of action of these therapies as well as their differential effects in the MCL microenvironment. MCL cells were resistant to venetoclax only in murine MSCs co-cultures, and further transcriptome and cytokine analysis supported this difference. Among others, our data questions how reliable murine MSCs are for obtaining translatable results, as MS-5 is the most used MSC cell line for co-cultures studies. These findings together provide a rationale to further investigate the role of MSCs in MCL drug resistance, as understanding the role of microenvironment in venetoclax sensitivity in MCL will potentially allow for more effective and targeted therapy.

P1247: GB5121 IS A NOVEL, IRREVERSIBLE, COVALENT BTK INHIBITOR WITH HIGH SELECTIVITY AND CNS-PENETRANCE FOR TREATMENT OF CNS MALIGNANCIES

I. Yusuf 1,* , D. Guimond 1 , Z. Naiman 1 , T. Schiff 1 , B. Clemons 1 , B. Paulson 1 , K. Hou 1 , K. Taylor Meadows 1 , M. Rose 1 , L. Carter 1

1 Gossamer Bio, Inc., San Diego, CA, United States of America

Background: Inhibitors of Bruton’s tyrosine kinase (BTK) are approved treatments for several B cell lymphomas. However, they are characterized by modest selectivity and/or limited central nervous system (CNS) penetration. GB5121 is an orally available, selective, irreversible, covalent small molecule BTK inhibitor that was designed to address these limitations.

Aims: To profile GB5121 for selectivity, potency, inactivation kinetics and CNS penetrance in comparison to ibrutinib.

Methods: Selectivity of GB5121 and ibrutinib were assessed in kinome scans against 349 kinases at 1 µM compound concentration with 1mM ATP. Irreversible and covalent nature of GB5121 was evaluated using enzymatic assays compared to analogs that lacked an active covalent warhead. Both biochemical and cell-based assays were employed for evaluation of potency and inactivation kinetics of GB5121. For assessment of BTK target occupancy within the CNS, naïve mice with an intact blood brain barrier were dosed with either GB5121 or ibrutinib followed by perfusion, brain dissection and processing into lysates that were analyzed for unoccupied BTK in a probe-based ELISA assay. The pharmacokinetic profile of GB5121 was assessed in multiple species for both intravenous (IV) and oral routes of dosing.

Results: In a kinome scan, GB5121 exhibited high kinase selectivity against 349 kinases with only TEC/TXK demonstrating >50% inhibition at 1 µM; additionally, GB5121 did not inhibit phosphorylation of EGFR in a cell-based assay. Importantly, GB5121 demonstrated rapid BTK inactivation kinetics (Kinact/Ki) in both peripheral and CNS tissues, a critical parameter for covalent irreversible inhibitors. When compared with ibrutinib, GB5121 showed superior CNS target occupancy in the brain of mice receiving three daily oral doses and demonstrated significantly higher brain to plasma ratio. In non-human primates, a 1:1 brain to plasma concentration ratio was demonstrated for GB5121 for up to 8 hours with both oral (30 mg/kg) and IV (2 mg/kg) doses.

Summary/Conclusion: The unique profile of GB5121 provides differentiation from both FDA-approved BTK inhibitors and those currently under clinical investigation. Our data supports the use of GB5121 in clinical trials where BTK is a known driver of malignancies, including in CNS lymphoma.

This research has been previously presented at the AACR Annual Meeting 2022.

P1248: CTP SYNTHASE 1 IS A NOVEL TARGET IN T CELL CANCERS, WITH SMALL MOLECULE INHIBITION INDUCING DEATH OF NEOPLASTIC HUMAN T CELLS IN VITRO AND INHIBITION OF THEIR GROWTH IN AN IN VIVO XENOTRANSPLANT MODEL

P. Beer 1,* , H. Asnagli 1 , N. Minet 2 , E. Hoeben 1 , A. Parker 1 , A. Fischer 3 , S. Latour 2

1 Step Pharma, Saint-Genis-Pouilly; 2 Laboratory of Lymphocyte Activation and Susceptibility to EBV infection, Imagine Institute; 3 Université de Paris, Paris, France

Background: T cell lymphomas are rare haematological cancers characterised by poor response to cytotoxic chemotherapy with resulting unmet clinical need. Recent studies have identified targeted therapies as a promising approach to improve outcomes. CTPS1, which catalyses the rate limiting step in pyrimidine synthesis, plays an essential and non-redundant role in B and T cell proliferation but is complemented by the homologous CTPS2 isoform outside of the haemopoietic system [1].

[1] E. Martin et al. , Nature. 2014 Jun 12; 510(7504):288-292.

Aims: To assess the impact of selective CTPS1 inhibition on the proliferation and survival of human neoplastic T cells.

Methods: Recombinant CTPS1 and CTPS2 enzymatic activity was assessed by quantitating cytidine triphosphate by RapidFire Mass Spectrometry. HEK cells lacking either CTPS1 or CTPS2 were generated using CRISPR technology; cell viability was measured after 3-5 days incubation with test compounds by CellTiter-Glo 2.0. For the cellular complementation assay, Jurkat cells lacking CTPS1 and CTPS2 were transduced with lentivirus carrying different CTPS1 and CTPS2 constructs cloned into a pLVX-EF1α-IRES-mCherry backbone; transduced cells were cultured with or without test compound and cell viability was assessed 72 hours later by CellTiter-Blue. For in vitro cancer cell line studies, cells were incubated for 72 hours with or without test compound; cell viability was assessed by CellTiterGlo and cell death by CellTiterTox Green. For in vivo experiments, sub-lethally irradiated female NOD-SCID mice were transplanted subcutaneously with 10 7 Jurkat cells embedded in Matrigel. Mice were randomised to n=8 per treatment group when tumours reached mean 150 mm 3 . Test compound and vehicle control were administered subcutaneously every two days. Tumour volume was measured twice weekly.

Results: STP938 is a potent and reversible low molecular weight inhibitor of CTPS1, which shows >1,300-fold selectivity for human CTPS1 over CTPS2. Selectively for CTPS1 inhibition was confirmed in a cellular system using HEK cells lacking either CTPS1 or CTPS2, which are consequently dependent on CTPS2 or CTPS1, respectively, for proliferation (Figure A). A cellular complementation assay identified the ATP pocket of CTPS1 as the binding site for the compound series and highlighted residues critical for the selectivity over CTPS2. STP938 exhibited anti-proliferative activity against a broad range of human cancer cells when tested in vitro ; haematological malignancies were significantly more sensitive than solid tumours cell lines, with T cell malignancies being the most sensitive (Figure B). Exposure of human neoplastic T cells (Jurkat and MOLT-4) to STP938 resulted in concentration dependent induction of cell death (Figure C). In an in vivo model of T cell neoplasia, where human Jurkat cells were transplanted subcutaneously into immunodeficient mice, STP938 showed dose dependent inhibition of tumour growth (Figure D).

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Summary/Conclusion: CTPS1 inhibition by STP938, a novel small molecule with favourable pharmaceutical properties, inhibits the growth of human neoplastic T cells in vitro and in vivo . CTPS1 represents a novel target in T cell cancers. STP938 is currently being prepared for a first in human study in patients with relapsed or refractory lymphoma.

P1249: INHIBITION OF THE PENTOSE-PHOSPHATE-PATHWAY (PPP) MEDIATES MACROPHAGE REPROGRAMMING FROM SUPPORTIVE BYSTANDER FUNCTION TOWARDS ANTIBODY-DEPENDENT LYMPHOMA CELL CLEARANCE AND LYMPHOMA SUPPRESSION

A. Beielstein 1,* , E. Izquierdo-Alvarez 2 , N. Nickel 3 , S. Blakemore 3 , D. Vorholt 3 , S. Chawan 3 , H.-H. Bartel 3 , J. Wiederstein 3 , R. Linke 3 , R. Brinker 3 , M. Michalik 3 , C. R. Costa Picossi 2 , A. Villasenor 2 , C. Barbas 2 , M. Krüger 3 , M. Hallek 1 , C. Pallasch 1

1 Department I of Internal medicine, University Hospital Cologne, Cologne, Germany; 2 Universidad CEU San Pablo, Madrid, Spain; 3 CECAD Research Center, University Hospital Cologne, Cologne, Germany

Background: Treatment response in lymphoma depends on the interaction and functional status of macrophages as important effector cells for tumor cell clearance by antibody-dependent cellular phagocytosis (ADCP). In the tumor microenvironment (TME) there is a limited supply of nutrients which implicates metabolic reprograming of immune cells such as changed phagocytic capacity of macrophages.

Aims: We aimed to identify metabolic pathways regulating macrophages in the TME to improve macrophage effector cell function in lymphoma therapy.

Methods: We addressed macrophage lymphoma cell co-cultures using macrophage cell lines, primary murine macrophages and human monocyte-derived macrophages. For pathway inhibition specific compounds as well as shRNA-mediated knockdown were used for functional read-out of ADCP and viability in lymphoma co-culture. Macrophage reprogramming was assessed by microscopy, FACS-immunophenotyping, ELISA, (phospho)-proteomic and metabolomic assessment. C57BL/6 mice and the humanized aggressive lymphoma model hMB were used for in vivo treatment with the pentose phosphate pathway (PPP) inhibitor S3 and analysis of macrophage programming and overall survival.

Results: Inhibition of AMP kinase, glycolysis or mitochondrial ATP-production did not increase the phagocytosis rate in ADCP co-cultures. Inhibiting the PPP induced an increased lymphoma cell phagocytosis by macrophages. Under PPP-inhibition, the oxygen consumption and the glycolysis of macrophages was increased and the cells developed a more activated morphology with formation of filopodia, which are needed for phagocytosis initiation. Moreover, a down-regulation of immune-inhibitory surface proteins PD-L1 and SIRP1α was seen and cytokine secretion was shifted from anti-inflammatory IL10 to pro-inflammatory IL6 secretion. PPP-inhibition of macrophages significantly reduced CLL-cell support in vitro abrogating the “nurse-like” effect of macrophages. The increased lymphoma cell clearance and phenotypic alterations of macrophages were also observed in PPP-enzyme knockdown of transketolase (TKT) and 6-phosphogluconatedehydrogenase (6PGD) in macrophages and by inhibiting the PPP in primary murine and human macrophages.

In a multiomics assessment of PPP-inhibition on proteome, phosphoproteome and metabolome level, we found protein expression alterations regulating metabolism and immunity. A connection between PPP inhibition and changed immune profile by modulation of the Stat1-Irg1-itaconate axis was identified and validated.

In a humanized lymphoma mouse model the addition of PPP-inhibitor S3 led to significant prolonged survival of lymphoma bearing mice and an increased maturation and pro-inflammatory polarization of macrophages.

Summary/Conclusion: An increased maturation, activation and repolarization of macrophages towards a pro-inflammatory and phagocytic active phenotype was observed by inhibiting the pentose-phosphate-pathway (PPP). This led to a significant survival benefit in in vivo lymphoma mouse model. We were able to point out the underlying Stat1-Irg1-itaconate signalling axis, connecting metabolic regulation and immune-phenotype of macrophages. We hypothesize the PPP as a key regulator of macrophage function determining the support of malignant B cell growth versus the capacity of lymphoma cell clearance and thereby therapy outcome. By that, we have identified the PPP as a targetable modulator of macrophage polarization to improve efficacy of B cell lymphoma and CLL therapy.

P1250: EU-SOX11CCND1: A NOVEL IMMUNOCOMPETENT MURINE MODEL OF MANTLE CELL LYMPHOMA

F. Brown 1,* , S. Sloan 1,2 , J. Helmig-Mason 1 , C. Hinterschied 1 , M. Long 1,2 , W. K. Chan 1 , P. Shindiapina 1 , V. Leshchenko 3 , D. Edwards 3 , K. Vaddi 4 , P. Scherle 4 , R. Lapalombella 1 , L. Alinari 1 , S. Parekh 3 , R. A. Baiocchi 1

1 Hematology; 2 Veterinary Biosciences, The Ohio State University, Columbus, OH; 3 Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY; 4 Prelude Therapeutics, Wilmington, DE, United States of America

Background: Mantle cell lymphoma (MCL) is an incurable B cell malignancy, comprising 5% of non-Hodgkin lymphomas (NHL) diagnosed annually and associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. The average overall survival of patients with MCL is 4-6 years and for most patients who progress on targeted agents, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life.

Immunotherapy has shown great promise in MCL, with the recent FDA approval of Brexucabtagene autoleucel for R/R MCL. In order to optimize the preclinical testing of immunotherapy for MCL, immunocompetent mouse models are essential; there are currently fewer than five available making new models very valuable for MCL researchers.

Aims: The C57BL/6J- Tg ( Eµ-sox11-GFP, Eµ-ccnd1 ) or Eµ-SOX11CCND1 transgenic mouse model utilizes two key genes in MCL. SOX11 has been shown to be a driver of MCL, with overexpression in 80-90% of cases, and is highly correlated with more aggressive disease. CCND1 overexpression is a hallmark of MCL, where the gene translocation to the IgH promotor is frequently used to diagnose MCL from other NHLs. We show this model recapitulates an MCL-like tumor burden, displays an altered immune environment, and test the effects of the selective PRMT5 inhibitor PRT382.

Methods: This model spontaneously develops CD19+/CD5+/CD23- lymphoma which progresses with splenomegaly, cytopenias, lymphadenopathy, and pre-mature death. Lymphoma cells from a founder were passaged through additional wild type, immune competent C57bl/6j mice through adoptive transfer and 100% of engrafted animals developed MCL-like tumor burden. This model can be utilized as a systemic model of MCL via intravenous (iv) tail vein or subcutaneous (sc) flank engraftment and reach early removal criteria by approximately day 21 showing disease pathology consistent with MCL. We created a luciferase positive clone that allows us to detect tumor burden by IVIS as early as one week post engraftment and to track disease progression. PRT382 was dosed orally and spectral flow was used to comprehensively monitor the immune response.

Results: PRT382, in this model of MCL provided a significant survival advantage (18 vs 67 days, p<0.0001) as well as a reduction in disease burden as determined by flow cytometry. This data supports the results we have seen with PRMT5 inhibition in pre-clinical models of human MCL and reinforce the MCL disease characteristics of this model.

Comprehensive immune monitoring was performed on control and diseased mice. Analysis blood, spleen, bone marrow, and tumor showed distinct immune signatures within each compartment. We observed significant shifts in the ratio of Ly-6C+/Ly-6G- and Ly-6C(low/-)/Ly-6G+ myeloid cells, enrichment of CD8+ T cells, and depletion of mature B cells in the spleen. Markers of immune exhaustion including PD1 and PDL1 were increased on T cells, CD11b+, and lymphoma cells in MCL burdened mice. These expression patterns have also been described in human samples, validating the immune landscape of this model. PRMT5 inhibition alters several of these population, details of which will be shared.

Summary/Conclusion: These results support the Eµ-SOX11CCND1 as an attractive model to understand the pathology of MCL and how disease burden and treatment influences host adaptive and innate immune networks. We will use this model to explore mechanisms of resistance to immunotherapy and create new treatment strategies for MCL.

P1251: BONE-BASED 3D SCAFFOLD AS AN IN-VITRO MODEL OF MICROENVIRONMENT-LYMPHOMA INTERACTION

J. Ceccato 1,* , M. Pizzi 1 , I. Caputo 1 , M. Piazza 1 , S. Manni 1 , F. Cinetto 2 , R. Scarpa 1 , F. Piazza 1 , L. Trentin 1 , F. Vianello 1

1 Medicine, University of Padova, Padova; 2 Ca’ Foncello Hospital, Treviso, Italy

Background: Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) and it accounts for about 30-40% of new NHL cases annually. Although immunochemotherapy is highly effective in DLBCL, yet about 30% of patients will relapse or exhibit refractory disease (r/r DLBCL). Moreover, the presence of bone marrow (BM) involvement in those patients is associated with a higher risk of aggressive features and worse outcome, likely due to the interaction between neoplastic cells and BM tumor microenvironment (TME).

Aims: The TME is a complex network in which a multitude of cell types interact with an intricate web of macromolecules, the extracellular matrix (ECM). In order to provide a tool for tumor cells and TME interactions studies, we aimed to develop an innovative and versatile three-dimensional (3D) in-vitro model which accounts for the diverse components of TME. The 3D model will be useful to highlight mechanisms behind drug resistance and to test patient-specific therapies.

Methods: The 3D in-vitro model was developed starting from human decellularized femoral bone fragments, isolated from the trabecular zone, as structural 3D scaffold and biological ECM provider. Specific seeding techniques, together with the construction of a PDMS device, were developed to adapt and expand MSC and DLBCL cells in the scaffold. Adhesion and growth capability of OCI-LY18 DLBCL cell were evaluated in decellularized scaffold (ECM model) and in BM-MSC recellularized one (ECM/MSC model). 3D spatial configuration of the models was studied by histological analysis, confocal microscopy and two photon techniques which allowed the 3D digital reproduction of the model structure. 3D models underwent doxorubicin treatment and viability of DLBCL cells was measured by Annexin V assay and compared to canonical cultures.

Results: Both HS-5 cell line and BM-MSC effectively adhered to the bone scaffold without synthetic matrix addition. Moreover, the PDMS device we developed accelerate cell adaptation and homogeneous recellularization.

3D confocal microscopy and histological analysis of recellularized model showed the generation of MSC niches which fostered growth and adaptation of lymphoma cells. Both MSC and ECM were effective in hosting tumor cells. Interestingly, neoplastic cells were able to strongly adhere, adapt and grown not only in ECM/MSC model but also to the bare ECM scaffold, suggesting an active interaction between OCI-LY18 and the ECM. Both MSC and lymphoma cells maintained viability and growth capacity when harvested from the scaffold and readapted to canonical 2D cultures.

We found that chemosensitivity to doxorubicin was hampered when neoplastic cells were grown in ECM and ECM/MSC model. Results showed that the levels of apoptosis were significantly lower if compared to conventional 2D cultures (Figure. Statistical analysis: non parametric t-test; *=0.06; **= p<0.005; ****= p<0.0001; n=4). Moreover, the presence of BM-MSC in the 3D model increased OCI-LY18 resistance to apoptosis both in adherent and in non-adherent cells. Interestingly, the protective effect was not provided from BM-MSC in 2D co-culture in those conditions.

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Summary/Conclusion: The 3D bone-based scaffold model we proposed is a powerful toll for exploring cell-cell and cell-ECM interaction in the setting of DLBCL. By recreating a 3D microenvironment, the model may help to clarify the mechanisms behind drug resistance in r/r DLBCL. Nevertheless, this approach may be exploited to create patient-specific models in order to improve personalized therapies.

P1252: PREDICTING THE RESPONSE OF DLBCL CELLS TO BH3-MIMETICS USING SYSTEMS BIOLOGY

I. Cloete 1 , V. Smith 2 , R. Jackson 2 , A. Pepper 1 , C. Pepper 1 , M. Dyer 2 , S. Mitchell 1,*

1 Brighton & Sussex Medical School, Brighton; 2 The Ernest and Helen Scott Haematological Research Institute, Leceister, United Kingdom

Background: Bcl-2 proteins are prominent regulators of mitochondrial-dependent apoptosis in healthy cells and lymphoma. In healthy cells, finely balanced interactions between family members within the Bcl-2 family of proteins ensure normal cell functions. However, in cancerous cells, anti-apoptotic Bcl-2 family members are often overexpressed, tipping the scale towards cell survival.

In recent years, therapeutic drugs targeting anti-apoptotic Bcl-2 proteins (BH3-mimetics) have been developed. However, these BH3-mimetics have shown heterogeneous clinical activity in different cancer subtypes, despite Bcl-2 overexpression. Recent experimental studies have suggested that complex interactions between the Bcl-2 family of proteins mediate the response to BH3-mimetics. Therefore, assigning the correct BH3-mimetic to the DLBCL cells in which they will be effective requires predicting the response of the apoptotic molecular signalling network. This response is an emergent property of the abundance of the signalling network’s components along with the binding affinities, regulatory interactions and kinetic rates.

Aims: Construct a computational model that enables BH3-mimetics to be tested in virtual DLBCL cell lines and accurately predicts cellular responses, as measured experimentally.

Methods: By combining published data including protein abundances, binding affinities, mutational profiles and kinetic rates we construct a computational model of the distinct apoptotic signalling network in multiple DLBCL cell lines. The simulation incorporates experimentally-measured Bcl-2 expression profiles, generating a set of virtual DLBCL cell lines. Bcl-2 (ABT-199), Mcl-1 ( S63845 ) and Bcl-x L (A1331852) inhibitors are simulated to predict cell viability and compared to experimental results.

Results: Our virtual DLBCL cell lines recapitulate the Bcl-2 protein expression profiles in DLBCL cell lines. Our computational cell line models recreate how cells expressing distinct abundances of Bcl-2 proteins respond to BH3-mimetics through complex shifts in binding partners between pro-and anti-apoptotic Bcl-2 family proteins. The result of this work is a computational tool that, by simulating the regulatory interactions governing apoptosis, can predict the sensitivity of DLBCL cells to BH3-mimetics (Fig. 1). We validate predicted heterogeneous response of DLBCL cells to BH3-mimetics with experimental data from DLBCL cell lines.

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Summary/Conclusion: The cellular response to BH3-mimetics is a complex emergent behaviour resulting from a combination of affinity, abundance and genetics, and is therefore challenging to predict. Computational systems biology provides a tool to overcome this challenge. Using a combination of in silico and in vitro experiments, we show that a computational model can predict the sensitivity of a cell to targeted therapies. Future work will test the ability of the model to predict patient responses to targeted therapies in order to enable a personalised medicine approach.

P1253: MAVORIXAFOR DISRUPTS THE CROSS TALK BETWEEN WALDENSTRÖM’S MACROGLOBULINEMIA CELLS AND THE BONE MARROW MICROENVIRONMENT RESULTING IN ENHANCED SENSITIVITY TO B-CELL TARGETED THERAPIES

H. Monticelli 1,* , T. Kruitwagen 1 , M. Tardelli 1 , T. Rebelo 1 , B. Maierhofer 1 , S. Maier-Munsa 1 , K. Zmajkovicova 1 , A. G. Taveras 2 , C. Nguyen 1

1 X4 Pharmaceuticals (Austria) GmbH, Vienna, Austria; 2 X4 Pharmaceuticals, Boston, United States of America

Background: Waldenström’s macroglobulinemia (WM) is a rare B-cell malignancy characterized by infiltration of bone marrow (BM) and lymphoid tissues with lymphoplasmacytic cells along with hypersecretion of monoclonal immunoglobulin M (IgM) by progenitor B cells. Studies have shown that more than 90% of patients with WM have an L265P mutation in the myeloid differentiation primary response gene 88 ( MYD88 ), and 30% to 40% of those patients show mutations in the carboxyl terminus of C-X-C receptor 4 (CXCR4). The CXCR4/C-X-C motif ligand 12 (CXCL12) axis plays an important role in the homing and retention of WM cells in the BM. The interaction of WM cells and the BM microenvironment, via cell-to-cell contacts or indirectly through secreted factors (e.g. interleukin 6 [IL-6], C-C motif ligand 5 [CCL5]), is thought to be essential for the pathogenesis of WM and to confer drug resistance. A clinical trial evaluating the efficacy of the CXCR4 antagonist, mavorixafor, in combination with the BTK inhibitor, ibrutinib in patients with MYD88 L265P and CXCR4 WHIM WM is currently ongoing ( NCT04274738 ). However, the effects of mavorixafor with ibrutinib and other B-cell targeted therapies on WM cells harboring only the single mutation in MYD88 L265P and without a CXCR4 mutation have not been evaluated.

Aims: This study was designed to elucidate the crosstalk between WM cells and the BM stromal cell (BMSC)-based microenvironment and to explore the effect of mavorixafor in combination with different B-cell targeted therapies in a WM/BMSC co-culture model.

Methods: WM cells (MWCL-1 cell line, MYD88 L265P CXCR4 WT ) pretreated with mavorixafor and B-cell targeted therapies (BTK antagonists: ibrutinib, zanubrutinib, evobrutinib, pirtobrutinib, nemtabrutinib; or BCL2 inhibitor venetoclax) were co-cultured with established BMSCs (HS27a cells). Apoptosis, IL-6 production, and IgM release were measured after 48 or 72 hours. The effects of mavorixafor on calcium mobilization, cell migration, and adhesion to BMSCs were also measured.

Results: Co-culture of WM cells with established BMSCs led to increased IgM secretion and enhanced surface expression of CXCR4 in WM cells by activation of the IL-6 receptor (IL-6R)/ Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Co-culture with established BMSCs protected WM cells from the effects of B-cell targeted therapies, reducing apoptosis of tumor cells and decreasing the IgM-reducing efficacy of the targeted therapies. Mavorixafor alone reduced IgM secretion, blocked CXCL12-induced cell migration and Ca 2+ mobilization and disrupted the adhesion of WM cells to established BMSCs. Mavorixafor in combination with B-cell targeted therapies synergistically enhanced antitumor activity in WM cells, inhibited BMSC-induced IgM hypersecretion, and overcame the protective effects of BMSCs on tumor cells.

Summary/Conclusion: Our studies provide preliminary evidence for the potential use of mavorixafor in disrupting the interaction of WM cells with the BMSC-based microenvironment, enhancing the efficacy of B-cell targeted therapies in the treatment of WM and potentially other lymphomas. Further studies using additional WM cell lines and/or primary patient cells are warranted to support these findings.

P1254: RESPONSE TO DA-EPOCH-R IN PATIENTS WITH HIGH GRADE B CELL LYMPHOMA IS ASSOCIATED WITH A REDUCTION OF PD1 POSITIVE EXHAUSTED CD8 T CELLS

V. de Jonge 1,* , C. Duetz 1 , W. Bruins 1 , E. van Werkhoven 2 , M. Nijland 3 , K. de Heer 4 , M. van der Poel 5 , Y. Sandberg 6 , C. Klerk 7 , D. de Jong 8 , J. Zijlstra 1 , M. J. Kersten 1 , T. Mutis 1 , M. Chamuleau 1

1 Department of Hematology, Amsterdam UMC, Amsterdam; 2 HOVON Data Center, Department of Hematology, Erasmus MC Cancer Institute, Rotterdam; 3 Hematology, University Medical Center Groningen, Groningen; 4 Department of Internal Medicine, Flevoziekenhuis, Almere; 5 Department of Internal Medicine, Maastricht University Medical Center, Maastricht; 6 Department of Internal Medicine, Maasstad Hospital, Rotterdam; 7 Department of Internal Medicine, Dijklanderziekenhuis, Hoorn; 8 Department of Pathology, Amsterdam UMC, Amsterdam, Netherlands

Background: Patients with high grade B cell lymphoma (HGBL) who harbor a MYC rearrangement with BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) have a poor prognosis upon treatment with R-CHOP [Rosenwald, JCO 2019]. Although disease free survival may be prolonged with intensification of chemotherapy with DA-EPOCH-R, improvement of overall survival has not been demonstrated [Dunleavy, Lancet Haematol 2018].

Aims: Preclinical data suggest that high MYC expression impairs anti-tumor immune responses [Casey, Science 2016]. Therefore, we designed the HOVON-152 trial to investigate whether survival can be improved by nivolumab consolidation after DA-EPOCH-R induction. We also investigated whether DA-EPOCH-R induction influences the immune system to have an impact on the response.

Methods: In the HOVON-152 single arm, phase II trial ( NCT03620578 ) HGBL-DH/TH patients received 1 cycle of R-CHOP followed by 5 cycles of DA-EPOCH-R induction treatment. Patients in complete metabolic remission (CMR) after induction (Deauville score (DS) 1-3 or a negative lymphoma biopsy in case of DS 4) proceeded with nivolumab consolidation (480 mg iv every 4 weeks) for one year. Peripheral blood was sampled after one cycle of R-CHOP (enroll), before start of the 3 rd DA-EPOCH-R cycle (midterm) and after the last DA-EPOCH-R cycle (end-of-induction, EOI).

As of January 2022, 55/90 enrolled patients completed DA-EPOCH-R induction, of whom 31 achieved PET-CT based CMR, 24 did not achieve CMR or went off study prematurely. Multiparameter flow cytometry of unseparated full blood was used to enumerate major immune cell subsets. Cryopreserved peripheral blood mononuclear cells were used to determine the frequencies of T cell subsets. Computational flow cytometry analyses (i.e UMAP and FlowSOM) and appropriate unpaired (Mann-Whitney U) or repeated (Friedman) statistical tests were performed in R version 4.0.3.

Results: In full blood, CD3 T cell frequencies and numbers decreased midterm DA-EPOCH-R treatment and restored at EOI. Remarkably, the proportion of CD4 T cells decreased (p=0.048) and that of CD8 T cells increased (p=0.01), resulting in lower CD4/CD8 ratios at EOI. In depth analysis revealed a significant decrease of CD4 naïve cells (p=0.00002) and increase of CD4 central memory (CM) cells (p=0.0005). Over time, CD8 cells showed a more senescent phenotype (CD28-KLRG1+CD57+, p=0.013), but the proportion of PD-1 positive exhausted CD8 cells decreased (p=0.007). When stratified for response, it appeared that patients achieving CMR after DA-EPOCH-R had significantly higher CD3 T cells (p=0.04) at enroll than patients not achieving CMR. T cell differentiation states (CD4 or CD8 naïve cells, stem cell memory cells, CM, effector memory (EM) or effector memory RA (EMRA) cells) and the proportion of TIGIT and TIM3 positive cells did not significantly differ between patients achieving CMR or not achieving CMR. Most interestingly, the decrease in PD-1 positive exhausted CD8 T cells was most outspoken in the CMR group (p=0.0008) (Figure 1) in CD8 CM, EM and EMRA cells (all p<0.008). Thus, at EOI, a lower proportion of CD8 PD-1 cells was associated with achieving CMR (p=0.04).

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Summary/Conclusion: Treatment of HGBL-DH/TH patients with DA-EPOCH-R results in a shift towards a lower CD4/CD8 ratio. Response to DA-EPOCH-R is associated with higher T cell frequencies at enroll and a significant decrease in PD-1 positive exhausted CD8 T cells during DA-EPOCH-R. Additional analyses on NK cells are ongoing. Future analyses of the nivolumab consolidation phase of this study will indicate whether additional blockade of PD-1 prolongs survival.

P1255: TUMOR-ASSOCIATED MONOCYTIC MYELOID DERIVED SUPPRESSOR CELLS IS A POTENTIAL PROGNOSTIC BIOMARKER, PROMOTING MULTI-DRUG RESISTANCE IN NHL PATIENTS BY MODULATING IL-6/IL-10/IL-1Β AXIS

S. Dhar 1,* , M. Chakravarti 1 , S. Bera 1 , N. Ganguly 1 , S. Dasgupta 1 , A. Sarkar 1 , A. Saha 1 , S. Banerjee 1 , B. Saha 2 , R. Baral 1 , K. K. Mukherjee 3 , A. Bose 1

1 IMMUNOREGULATION & IMMUNODIAGNOSTICS, CHITTARANJAN NATIONAL CANCER INSTITUTE, KOLKATA; 2 Pathogenesis & Cell Responses, NCCS, Pune, Pune; 3 Medical Oncology, CHITTARANJAN NATIONAL CANCER INSTITUTE, KOLKATA, India

Background: Non-Hodgkin-Lymphoma (NHL), the most prevalent hematologic malignancies in the world has majorly originates from B cells. R-CHOP represents the new-standard treatment regimen for NHL. Most NHL patients initially respond to chemotherapy yielding complete response rates of 40–50%. Unfortunately, a substantial population of patients undergo relapse, resulting in poor clinical ramifications. The onset of NHL relapse evolves from several months (early relapse) to years (late relapse) after the initial remission. However, the majority of relapse occurs within two years of initial treatment. Despite considerable advancements in therapeutic concepts and techniques, disease relapse with limited response rate remains a major challenge and depicts poor prognosis in successful clinical management. Patient’s response to chemotherapy varies widely from static disease to cancer recurrence and the later is primarily associated with generation of multi drug resistance (MDR) phenotypes that ultimately promotes disease progression and metastasis. However, the causes of differential responses to standard chemotherapeutic regimens and therapy failure in NHL patients are yet to be elucidated.

Aims: To understand the influence of immune cells in differential response in NHL patients following R-CHOP-therapy.

Methods: Peripheral blood was collected from 51 CD20 + NHL patients before and after frontline chemotherapy and at the time of relapse. Clinical variables at diagnosis (age, performance status, stage of the disease, number of extra nodal lesions) were obtained to calculate prognostic indices (IPI). A panel of immune cells CD4 + T cells, CD8 + T cells, Cytotoxic T cells, CD8 + CD45RO + CD45RA + Memory T cells, CD14 + CD80 + M1 macrophage, CD4 + CD25 + FoxP3 regulatory T cells(Treg), CD33 + CD11b + CD14 -/+ CD15 -/+ Myeloid Derived Supressor Cells(MDSCs), CD14 + CD163 + Tumor associated Macrophages (TAM), MDR phenotype P-gp(ABCB1) and MRP1(ABCC1) were studied by flow-cytometry at different phases of treatment. In vivo and in vitro doxorubicin resistance model were developed with murine Dalton’s lymphoma and Raji (B cell), Jurkat cell (T cell) lines respectively and impact of responsible immune cells on generation of drug resistance were studied by RT-PCR, qPCR, flow-cytometry, colorimetric assay, gene silencing and ChIP assays.

Results: A strong positive correlation between elevated levels of CD33 + CD11b + CD14 + CD15 - monocytic MDSCs, but not CD33 + CD11b + CD14 - CD15 + granulocytic MDSC and MDR was depicted in non-responder patients compared to responder cohorts. Moreover, in vitro supplementation of MDSCs in murine or human lymphoma culture increases the expression of mrp1 , pgp and cellular GSH level from early passage than passage without MDSCs, which is correlated well with in vitro drug retention and generation of drug resistance phenotypes and tumor progression. MDSC secreted cytokines IL-6, IL-10, IL-1β are the dominant constituent in regulating expression of multidrug-resistance pump by modulating STAT3, STAT1 and NF-κβ signalling axis.

Summary/Conclusion: Observed correlation between monocytic MDSCs with the relapse of NHL patients following R-CHOP-therapy along in vitro data from lymphoma cells suggest monocytic MDSCs might be considered as a new potential biomarker and therapeutic target to enhance the relapse free survival in NHL patients.

P1256: A NOVEL DROP-OFF DIGITAL PCR ASSAY FOR CXCR4 MUTATION SCREENING IN IGM GAMMOPATHIES: FIRST DATA FROM THE FONDAZIONE ITALIANA LINFOMI BIO-WM STUDY

D. Drandi 1,* , M. Ferrante 1 , S. Zibellini 2 , L. Marcheselli 3 , E. Cappello 2 , M. Borriero 1 , S. Ragaini 1 , I. Dogliotti 4 , C. Varraso 2 , F. Cavallo 1,5 , A. Ferrari 6 , M. Merli 7 , G. Zamprogna 8 , L. Laurenti 9 , S. Tomasetti 10 , E. Cencini 11 , G. Loseto 12 , S. Finotto 13 , M. Marchetti 14 , F. Re 15 , A. Sica 16 , J. Olivieri 17 , C. Jimenez 18 , N. Puig 18 , C. Cavalloni 19 , R. García-Sanz 18 , M. Varettoni 19 , S. Ferrero 1

1 Department of Molecular Biotechnologies and health sciencies, University of Torino, Torino; 2 Division of Hematology, Fondazione IRCCS Policlinico San Matteo, Pavia; 3 Fondazione Italiana Linfomi- c/o Centro Oncologico Modenese (COM), Università di Modena e Reggio Emilia, Modena; 4 Stem Cell Transplant Unit, University Hospital AOU Città della Salute e della Scienza; 5 Division of Hematology, A.O.U. Città della Salute e della Scienza di Torino, Torino; 6 Ematologia, AO Arcispedale Santa Maria Nuova - IRCCS, Reggio Emilia; 7 UOC Ematologia, Ospedale di Circolo e Fondazione Macchi – ASST Sette Laghi, Varese; 8 SC Ematologia, ASST Grande Ospedale Metropolitano Niguarda, Milano; 9 S. Ematologia, Dipartimento Scienze Radiologiche Radioterapiche ed Ematologiche, Fondazione Policlinico Universitario A Gemelli, Roma; 10 Ematologia, Ospedale degli Infermi di Rimini, Rimini; 11 U.O.C. Ematologia, AOU Senese, Siena; 12 U.O.C Ematologia, IRCCS Istituto Tumori Giovanni Paolo II, Bari; 13 Oncologia 1 - I.R.C.C.S., Istituto Oncologico Veneto, Padova; 14 S.O.S. Ematologia, Ospedale Cardinal Massaia, Asti; 15 UO Ematologia e CTMO, Azienda Ospedaliera Universitaria di Parma, Parma; 16 Oncologia Medica ed Ematologia, AOU Università degli Studi della Campania Luigi Vanvitelli, Napoli; 17 Clinica Ematologica, Centro Trapianti e Terapie Cellulari “Carlo Melzi”, Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy; 18 Hospital Universitario de Salamanca (HUSAL), IBSAL, IBMCC (USAL-CSIC), CIBERONC, Salamanca, Spain; 19 Division of Hematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

Background: In IgM gammopathies: Waldenström’s Macroglobulinemia (WM) and IgM gammopathy of uncertain significance (IgM-MGUS), MYD88 L265P and CXCR4 S338X are the most frequent mutations, and both have clinical implications in WM. Recent studies demonstrated that, in unselected bone marrow (BM) samples, allele specific polymerase chain reaction (AS-qPCR) was superior in detecting CXCR4 S338X compared to next generation sequencing, and digital PCR (dPCR) was more sensitive than AS-qPCR for MYD88 L265P detection, highlighting the lack of a gold standard molecular method in WM diagnostics.

Aims: The aim of the present study was to evaluate a new sensitive approach based on a drop-off dPCR assay for CXCR4 S338X mutation detection in BM and peripheral blood (PB) samples from WM and IgM-MGUS patients, enrolled between 2018 and 2020 in the multicenter observational trial from the fondazione italiana linfomi (“BIO-WM”, NCT03521516), and to correlate the mutational status with clinical features.

Methods: Genomic DNA from 240 patients (184 WM, 56 IgM-MGUS) was analyzed by dPCR: 51 BM-CD19 selected cells (CD19+), 189 BM and 239 PB unselected white blood cells (WBC). Allele frequency (AF) of CXCR4 mutations at p.S388 locus (CXCR4 MUT ) was detected by a drop-off dPCR assay, with a sensitivity of 0,001%. For all patients, MYD88 L265P mutation data were also available.

Results: Forty-one out of 184 WM patients (22%) showed CXCR4 MUT in BM (median AF 1.6%, range: 0.14%-23.1%) while only 8 were positive also in PB (20% of the BM mutated cases, median AF: 0.6%, range: 0.23%-35.2%). Moreover, 39/41 (95%) were MYD88 L265P , too. Conversely, only 5 out of 56 IgM-MGUS patients (9%) scored CXCR4 MUT in BM (median AF 0.65%, range: 1.1%-0.2%) and none in PB: all patients were MYD88 L265P , too. Interestingly, 38 BM WBC samples (36 WM, 2 IgM-MGUS) mutated for both CXCR4 MUT and MYD88 L265P showed correlation in mutational levels between the two mutations (R 2 =0.8). On the contrary, no correlation was observed between the two mutations in CD19+ cells (R 2 =0.06). Overall, clinical features of CXCR4 MUT vs CXCR4 WT patients did not differ significantly, except for lower hemoglobin levels (median 11.6 g/dl vs 12.8 g/dl, p=0.002) and higher serum IgM monoclonal component (median 1.76 g/dl vs 1 g/dl, p=0.016). Only 15/41 WM CXCR4 MUT patients (37%) received immediate treatment at the time of trial enrollment (7 DRC, 3 R-Benda, 5 others: 2 single agent and 3 R-chemo based therapy). These cases showed higher disease and mutational burden compared to the 26 CXCR4 MUT patients still in watch and wait (WW) at the time of the present analysis. Indeed, median histologic BM infiltration was 80% vs 30% (p<0.001), median IgM monoclonal component was 3.6 g/dl vs 1.6 g/dl (p=0.003) and median CXCR4 MUT AF was 9.5% vs 0.8% (p<0.001), respectively. Finally, at the present the median follow-up (FU) of the trial is relatively short (34 months) and no statistically significant differences in outcome parameters were recorded between CXCR4 MUT and CXCR4 WT cases.

Summary/Conclusion: We here describe a sensitive, easily applicable and standardizable approach for detection of the most common CXCR4 mutations on BM WBC, with no need for CD19+ selection. A lower mutational AF was observed in IgM-MGUS compared to WM and, similarly to what was previously reported for MYD88 L265P , PB resulted suboptimal for CXCR4 MUT detection. A statistically significant difference in CXCR4 MUT level was observed between patients treated at the time of enrollment and those still in WW. However, a longer FU is needed to better clarify the clinical implications of CXCR4 mutations in our series.

P1257: MOLECULAR CHARACTERIZATION AND CLONAL EVOLUTION OF MYCOSIS FUNGOIDES

L. Fléchon 1,* , I. Arib 2 , A. Dutta 3 , R. Sklanvenitis Pistofidis 4 , C. Stewart 5 , R. Dubois 6 , S. Poulain 6 , M.-C. Copin 7 , S. Javed 8 , M. Nudel 2 , L. Hasan Bou Issa 1 , A. Ouelkite-Oumouchal 1 , S. Faiz 9 , O. Carpentier 9 , L. Mortier 9 , S. Mitra 1 , M. Figeac 10 , F. Morschhauser 2 , B. Quesnel 1,2 , I. Ghobrial 11 , S. Manier 1,2

1 Université de Lille, Laboratoire Canther, INSERM UMR-S1277 - CNRS UMR9020; 2 Department of Hematology, CHU Lille, Lille, France; 3 Harvard Medical School, Dana-Farber Cancer Institute, Boston, United States of America; 4 Harvard Medical School, Dana-Farber Cancer Institute, Boston, France; 5 Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, United States of America; 6 Biology and Pathology Department, CHU Lille, Lille; 7 Departement of Pathology, Université d’Angers, CHU Angers, INSERM, CRCI2NA, Angers; 8 Department of Oncology, CHU Valenciennes, Valenciennes; 9 Department of Dermatology, CHU Lille; 10 Université de Lille, CHU Lille, CNRS, INSERM, Inst Pasteur Lille, US 41 – UAR 2014 - PLBS, Lille, France; 11 Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States of America

Background: Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma with a frequency of over 50% of all cases. MF has an indolent progression over several years through different stages. Symptoms evolve from a non-aggressive “low-risk” (LR) clinical form to an aggressive and treatment-resistant “high-risk” (HR) form. Approximately 20% of patients with MF evolve to a HR MF with transformed cells (tMF), which is associated with a poor prognosis.

Aims: At the molecular level, the genomic landscape of MF remains poorly studied. Furthermore, the mechanisms that determine the progression to HR and tMF are still unknown.

Methods: We performed whole exome sequencing (WES) of 70 samples from 48 patients at the University Hospital Center (CHU) of Lille, France, between 2007 and 2018. Some patients have multiple sequential samples over several years, with or without progression from LR to HR. Sequencing was performed on the Illumina NovaSeq 6000 system at the Broad Institute (BI) Genomics Platform. The bioinformatics analysis was performed on the Terra platform (BI), which includes tools for the analysis of genomic data. Copy number variations (CNVs) are detected with ModelSegments software in Tumor-Only mode and common large and focal CNVs in the cohort are highlighted by GISTIC2.0. Single nucleotide variants (SNVs) were detected with the CGA WES Characterization Pipeline (Getz lab, BI). Significantly mutated genes were determined by MutSig2CV. The mutational signature was performed with SignatureAnalyzer software. Subsequently ABSOLUTE software is used to calculate the cancer cells fraction of each variant and CNVs in order to follow the clonal evolution of sequential samples.

Results: Somatic mutation analysis at the time of diagnosis detected genes involved in T-cell oncogenesis, by order of frequency: the transcription factor JUNB, the MAP kinase member MAPK1, the JAK/STAT member STAT5A and the epigenetic regulators of DNA SUZ12 and TET2. Mutational signatures were represented by the COSMIC SBS1, SBS7, and SBS15, characterized by aging, ultraviolet (UV) exposure, and defective DNA mismatch repair and microsatellite instability, respectively, suggesting their role in the mutational process of MF oncogenesis. The analysis of the CNVs revealed focal deletions affecting genes involved in ER stress TMEM259 (cytoband 19p13.3), the tumor suppressor gene TP53 (17p13.1), the transcriptional repressor gene ZEB1 (10p11.22) which inhibits interleukin 2 IL-2 gene expression, the TNFAIP3 gene (6q16.3) which inhibits nuclear factor kappa B (NF-kB), and the CDKN2A and MTAP genes (9p21.3) which are frequently co-deleted in cancers. The single focal amplification identified a small cytoband in 10p15.1 that carries the genes encoding the interleukin 2 receptor alpha ( IL2RA ) and interleukin 15 receptor alpha ( IL15RA ) which have a central role in T cell survival and proliferation. Clonal evolution analysis established that del10p11.22, amp10p15.1 and triplication of chromosome 7 are present only in HR stage, suggesting their role in the tumor progression. Overall survival analysis shows that amp10p15.1 and del10p11.22 – both associated with IL2 pathway - are significantly associated with reduced survival.

Summary/Conclusion: WES data from MF identified key CNVs events of oncogenesis and clonal evolution of MF that could serve as predictive markers in clinical practice. Our findings clarify MF genetics and provide novel insights into the disease progression.

P1258: MOLECULAR CYTOGENETICS CHARACTERIZATION OF CLASSICAL HODGKIN LYMPHOMA. ROL OF 9P24.1 (PD-L1/PD-L2) POLYSOMIES

M. García-Montenegro 1,* , M. Narbaitz 2 , M. F. Metrebian 2 , A. Pavlovsky 3 , I. Slavutsky 4

1 Lymphoid Neoplasms Genetic Laboratory, Institute of Experimental Medicine - National Academy of Medicine; 2 Hematological Research Institute “Mariano Castex”, National Academy of Medicine; 3 Hemathology, Fundaleu; 4 Lymphoid Neoplasms Genetic Laboratory, Institute of Experimental Medicine - National Academy of Medicine, Ciudad de Buenos Aires, Argentina

Background: Classical Hodgkin lymphoma (CHL) is characterized by scarce Reed-Sternberg/Hodgkin (RS/H) cells surrounded by a dense immune cell infiltrate belonging to the tumor microenvironment (TME). Chromosomal alterations on 9p24.1 (PD-L1/PD-L2) locus, especially amplifications and gains, are considered as recurrent abnormalities in RS/H cells as well as a potential prognostic and predictive marker of immunotherapy.

Aims: To characterize genomic imbalances at 9p24.1 locus by FISH on formalin-fixed paraffin embedded primary diagnostic biopsies of CHL patients, in both RS/H and TME cells, and correlate them with histopathological, immunohistochemically and clinical parameters.

Methods: A retrospective analysis of randomly chosen biopsies of patients with CHL enrolled in the GATLA LH05 study was performed. For FISH analysis, the SPEC CD274/PDCD1LG2/CEN9 probe (9p24.1) (ZytoVision, Germany) was used. In each patient, 50 RS/H and 100 TME cells were analyzed and the frequency of 9p24.1 alteration were determined. Nuclei with a target:control ratio ≥3:1 was defined as amplification (A), >1:1 but <3:1 as gains (G) and 1:1 as polisomy (P). According to the magnitude and percentage of cells with A, G and P, patients were divided in: AGP group (with A), GP-G-rich group (G>50%) and GP-P-rich group (P≥50%). Protein expression of PD-L1 was evaluated by immunohistochemistry (IHC) using anti-PD-L1/CD274 antibody clone RBT-PDL1 (BioSB, USA); the H-score modified (HSM) (range: 8-300) was applied. EBV integration was evaluated using LMP1 antibody clone CS1-4 (Cell Marque, Germany) as positive/negative. The study was approved by the Institutional Ethics Committee, in compliance with the Helsinki Declaration.

Results: A total of 28 patients with CHL (14 males; mean age: 31 years, range: 18-54 years; early stages: 64.3%; advanced stages: 35.7%) were analyzed. All cases showed PD-L1/PD-L2 copy number alterations, with huge intratumoral heterogeneity, belonging 32% to AGP group, 36% to GP-G-rich and 32% to GP-P-rich. In addition to the A, the number of P were significantly different between groups (p<0.01). All cases showed PD-L1 expression; differences in the HSM mean between groups were observed: AGP: 266, GP-G: 123 and GP-P: 60 (p<0.01). Negative correlation between the HSM and the number of P for each group was found (p<0.01). No association with LMP1 expression or CHL histological subtype was observed. TME cells showed chromosome 9 (Ch9) monosomy in 100% of patients from the GP-P-rich group compared to 40% of the remaining cases. In relation with the clinical data, GP-P-rich group showed a higher mean age at diagnosis (38 years) than AGP and GP-G-rich groups (29 years) (p=0,019). In addition, GP-P group had higher frequency of advanced stage (p=0,035), extranodal involvement and B symptoms compared to the other groups, which showed association with localized stage and bulky disease. Interim PET scan after 3 cycles of ABVD (PET3) was positive (Deauville score 3-4-5) in 55.5% of cases with A compared to 26.3% of patients without A (Figure 1).

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Summary/Conclusion: Our results confirm the high recurrence of 9p24.1 alteration in CHL, associated with highly intratumoral heterogeneity. In spite of the limited number of our series, we found differences in clinical parameters, PD-L1 protein expression and TME cells with Ch9 monosomy in GP-P-rich cases compared to the other groups. These findings suggest an important role for polysomies in the biologic characterization of CHL patients.

P1259: CHARACTERIZATION OF VENETOCLAX RESISTANCE IN CELL MODELS OF MANTLE CELL LYMPHOMA

A. Granau 1,2,* , P. Andersen 2 , T. Jakobsen 3 , L. Sommer Kristensen 3 , J. Bay Mogensen 1 , G. Dias Correa Guldbergsen 1 , K. Dimopoulos 4 , K. Grønbæk 1,2

1 Faculty of Health and Biomedical Sciences, University of Copenhagen; 2 Department of Hematology, Rigshospitalet, Copenhagen; 3 Department of Biomedicine, University of Aarhus, Aarhus; 4 Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark

Background: Venetoclax (Ven), a BH3 mimetic inhibiting BCL2, appears to be highly cytotoxic to most Mantle Cell Lymphoma (MCL) cells due to their high BCL2 levels. However, resistance to Ven in both MCL and other lymphomas is being observed. Characterizing underlying mechanisms of acquired Ven resistance can hopefully highlight potential treatment options for patients who develop resistance or prolong the responsive phase of patients receiving Ven.

Aims: To investigate and characterize drivers of acquired Ven resistance in cell models of Mantle Cell Lymphoma with an exploratory approach to potentially elucidate drivers of resistance development.

Methods: Using two Ven-sensitive MCL cell lines (MAVER1 and MINO) we established Ven resistant cell lines (MAVER1-R and MINO-VR). Changes between the maternal and the VR cells were characterized using full exome and transcriptome sequencing and by Acce SssI ble, which investigates changes to CpG methylation and genomic accessibility. BH3 profiling was performed to investigate the degree of dependance on antiapoptotic BCL2 family proteins and mitochondrial priming. Finally, we performed a drug screen which targeted pathways identified through GO analysis of the transcriptomic data, targeting both oncogenic and epigenetic targets.

Results: In both MAVER1-VR and MINO-VR we observed an increase in genome-wide CpG methylation concurrently with decreased overall genomic accessibility associated with Ven resistance (figure 1A). However, 2-day pre-treatment with epigenetic drugs (Vorinostat, Tazemetostat, Valemostat and Azacytidine) and 7-day pre-treatment with Tazemetostat + Azacytidine had no effect on Ven sensitivity. Based on RNA sequencing, we observed clustering of the -VR cell lines together, separate from their maternal cell lines indicating common transcriptional changes associated with Ven resistance (figure 1B). GO analysis performed on the differentially expressed genes (DEGs) highlighted PLCb mediated events, JAK-STAT- and PI3K-AKT signaling in development of Ven resistance (figure 1C). Inhibition of JAK1 or JAK2/3 showed no change to Ven sensitivity in neither maternal nor VR cells. Inhibition of mTORC1 with Everolimus abrogated the toxic effect of Ven in the maternal cell lines (figure 1E), whereas TIC-10, an AKT/MEK inhibitor, partially restored Ven sensitivity in MAVER1-VR and MINO-VR cell lines (figure 1D). PLC inhibition didn’t affect Ven sensitivity, however it was more cytotoxic to VR cells than to the maternal cells (figure 1F).

BH3 profiling showed a decreased response to the apoptotic activators BIM and PUMA in VR cell lines (figure 1G), indicating lowered mitochondrial priming.

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Summary/Conclusion: We found a combination of epigenetic and transcriptomic changes associated with Ven resistance development in MCL cell lines; however, the observed epigenetic alterations might not drive Ven resistance alone, since pretreatment with epigenetic drugs did not affect Ven sensitivity. The PI3K-AKT-mTOR pathway appears to be involved in Ven resistance development, since interestingly, mTOR inhibition leads to a decreased response to Ven treatment in maternal cell lines but AKT inhibition leads to an increased response in -VR cell lines. Ven resistance in MINO-VR appears to be driven primarily by decreased mitochondrial priming, whereas MAVER-VR appears to be driven by decreased mitochondrial priming combined with a switch from BCL2 to MCL1 dependence.

P1260: UNRAVELING THE GENETICS OF TRANSFORMED SPLENIC MARGINAL ZONE LYMPHOMA

M. Grau 1,* , C. López 1,2 , A. Navarro 1,2 , G. Clot 1,2 , F. Nadeu 1,2 , G. Bastidas 3 , M. Alcoceba 2,4 , M. J. Baptista 5 , M. Blanes 6 , F. Climent 7 , D. Colomer 1,2,8 , D. Costa 1,8 , E. Domingo-Domènech 9 , P. Forcada 10 , A. Enjuanes 11 , L. Escoda 12 , G. Frigola 8 , E. Giné 1,2,3 , M. Lopez-Guerra 1,8 , A. Rivas-Delgado 1,3 , L. Vicente-Folch 13 , A. Wotherspoon 14 , E. Campo 1,2,8 , A. López-Guillermo 1,2,3 , E. Matutes 3 , S. Beà 1,2,8

1 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona; 2 Centro de Investigación Biomédica en red de Cáncer (CIBERONC), Madrid; 3 Hematology, Hospital Clínic de Barcelona, Barcelona; 4 Hematology, Cancer Research Institute of Salamanca-IBMCC (USAL-CSIC), University Hospital of Salamanca (HUS/IBSAL), Salamanca; 5 ICO-Hospital Germans Trias I Pujol- Josep Carreras Leukaemia Research Institute (IJC), Barcelona; 6 Hospital General Universitario de Elda, Alicante; 7 Pathology, Hospital Universitari de Bellvitge-IDIBELL, L’Hospitalet de Llobregat; 8 Hematopathology Unit, Hospital Clínic de Barcelona, Barcelona; 9 Hematology, Institut català d’oncologia-IDIBELL, L’Hospitalet de Llobregat; 10 Hospital Universitari Mútua Terrassa, Terrassa; 11 Genomics Unit, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona; 12 ICO-Hospital Joan XXIII, Tarragona; 13 Consorci Sanitari de Terrassa, Terrassa, Spain; 14 Royal Marsden NHS Foundation Trust, London, United Kingdom

Background: Splenic marginal zone lymphoma (SMZL) is an indolent non-Hodgkin lymphoma but about 10-15% of patients may undergo transformation to an aggressive lymphoma, commonly diffuse large B-cell lymphoma (DLBCL). Although SMZL genomic profile has been largely investigated, the genetic mechanisms leading to this transformation are still unknown.

Aims: To characterize the genomic landscape of transformed SMZL in order to establish the underlying mechanisms associated with transformation and the potential clonal evolution during this event.

Methods: We investigated 42 SMZL that underwent DLBCL transformation. Tumor material was obtained at diagnosis and transformation in 19 patients, only at transformation in 14 patients, and only at diagnosis in 9 (61 total samples). The pathology and flow cytometry data of all cases were reviewed. Samples were categorized in two groups: i) at diagnosis (SMZL, n=28 samples), and ii) at transformation (SMZL-T, n=33 samples). DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue (n=45), frozen tissue biopsies (n=10), or PB (n=6). Single nucleotide variants (SNV) and indels were analyzed in all samples (n=61) using a SMZL-panel capturing 37 genes and custom bioinformatic pipelines. Copy number alterations (CNA) were assessed using OncoScan TM CNV FFPE (n=48) or CytoScan HD (n=3) arrays, and analyzed using Nexus Biodiscovery software. Whole genome sequencing (WGS) was performed in one case (diagnostic and transformed tumor and germline DNA samples). The IGH clonal relationship between samples at diagnosis and transformation was examined in 17/19 cases.

Results: Integrating mutations and CNA, the most frequently altered genes in the 33 SMZL-T samples were TNFAIP3 (57.6%), KMT2D (48.5%), KLF2 (33.3%), TP53 (33.3%), and ARID1A (30.3%). The most frequent CNA were gains of 1q (39.3%), 6p (28.6%) and 3q (27.3%); and losses of 9p21.3 ( CDKN2A / B ) (39.3%), and 7q31-q32 (33.3%). SMZL-T had higher number of CNA than SMZL at diagnosis (8 vs 5; P <0.001). Moreover, genetic alterations of TNFAIP3 , 9p21.3 ( CDKN2A / B), TP53 and 6p were significantly enriched at transformation as compared to diagnosis (57.6% vs 28.6%, P <0.001; 39.3% vs 10.7%, P =0.001; 33.3% vs 14.3%, P =0.04; and 28.6% vs 13%, P =0.05, respectively). All paired samples with available results (n=12) were clonally related. We evaluated the clonal evolution of SMZL/SMZL-T with complete NGS and CNA data (n=14) and identified a divergent evolution pattern in the majority of the cases (13/14, 93%), with only one case showing a linear evolution. Finally, the WGS analysis revealed the following alterations in SMZL and SMZL-T samples respectively: 62 and 76 coding SNVs/indels, 9 and 11 CNA, 14 and 19 interchromosomal translocations and insertions. About half of the aberrations (52%) were shared by diagnostic and transformation samples. Interestingly, we identified an IGH::BCL3 translocation, displaying the breakpoints at the switch region IGHG2 and downstream of BCL3 in the diagnostic and transformation samples. This case did not have any of the characteristic alterations of SMZL.

Summary/Conclusion: Taken together, SMZL-T are characterized by higher genomic complexity than SMZL and have specific genomic alterations acquired during transformation including aberrations in TNFAIP3 and TP53 , focal losses at 9p21.3 ( CDKN2A / B ) and 6p gains, suggesting that these alterations may represent key players in SMZL transformation. A divergent evolution pattern was observed in virtually all cases.

P1261: ALLG LABORATORY SCIENCE STUDY LS21: MOLECULAR CORRELATES OF RESPONSE IN RELAPSED/REFRACTORY MARGINAL ZONE LYMPHOMA (RRMZL) PATIENTS TREATED WITH ZANUBRUTINIB IN THE MAGNOLIA TRIAL

M. Tatarczuch 1,2,* , M. Waltham 1,2 , J. Shortt 1,2 , E. Hawkes 3,4,5 , S.-J. Ho 6,7 , J. Trotman 8,9 , D. Brasacchio 1,2 , M. Co 10 , J. Li 10 , V. Ramakrishnan 10 , K. Dunne 11 , S. Opat 1,2 , G. Gregory 1,2

1 Clinical Sciences at Monash Health, Monash University; 2 Monash Haematology, Monash Health; 3 Eastern Health; 4 Olivia Newton John Cancer Research Institute at Austin Health; 5 School of Public Health and Preventive Medicine, Monash University, Melbourne; 6 Haematology, St George Hospital; 7 St George & Sutherland Clinical School, University of New South Wales; 8 Haematology, Concord Repatriation General Hospital; 9 Concord Clinical School, University of Sydney, Sydney, Australia; 10 BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, United States of America; 11 Australasian Leukaemia & Lymphoma Group, Melbourne, Australia

Background: The MAGNOLIA trial demonstrated the safety and efficacy of zanubrutinib, a second generation BTK inhibitor (BTKi), in patients with rrMZL (ORR 68.2%, CR 26%, 15-month PFS 83%). An exploratory analysis of the ibrutinib-treated rrMZL study showed mutations in genes regulating NF-kB signalling pathway: TNFAIP3 ( A20 ) and MYD88 predicted response, whereas KMT2D ( MLL2 ) and CARD11 were associated with primary resistance. The impact of acquired mutations in BTK and PLCg2 which mediate resistance to BTKi in CLL is unknown in MZL.

Aims: This Australasian Leukaemia and Lymphoma Group (ALLG) laboratory science correlative study LS21 seeks to determine if the baseline molecular profile by whole exome sequencing (WES) can predict primary resistance to zanubrutinib, and whether the emergence of resistance mutations in circulating tumour DNA (ctDNA) can herald clinical progression.

Methods: DNA was extracted from baseline FFPE tumour samples from 18 patients. Longitudinal monitoring to detect the acquisition of resistance mutations was monitored in 7 patients at multiple time points using cell-free DNA (cfDNA) isolated from plasma. NGS libraries were constructed using Agilent XTHS. Unique molecular barcoding was used to facilitate error correction. Agilent WES Ver7 was used for tumour sequencing. A bespoke bait capture of 48 genes set was used for ctDNA and buccal analysis. Sequencing was performed by NovaSeq 6000 (Illumina, SP flow cell, 2x150bp chemistry) and data processing of FASTQ files via in-house bioinformatic pipeline incorporating fgbio (for UMI de-duplexing, Fulcrumgenomics), VarDict for variant calling and CNVkit for copy number analysis. Variant calls in patient samples (identified by VarDict ) were manually curated from a predetermined list of 48 candidate genes by inspecting BAM files in Integrative Genomics Viewer (IGV). Mutational analysis was correlated with clinical data and survival analysis calculated using the Kaplan-Meier (log-rank) method.

Results: Baseline WES identified mutations in 33/48 (69%), with a median of 5 genes affected per sample (range: 0-8). In patients treated with zanubrutinib (screen failure n=1), NFkB and NOTCH pathway genes were implicated in 13 (73%) and 10 (56%) patients respectively. KMT2D was most affected (n=7) followed by NOTCH1, NOTCH2, TNFAIP3 and MYD88 (all n=4). Patients with either MYD88 or TNFAIP3 mutations had improved median PFS (not reached (NR) vs 11.1 months, p: 0.009, HR: 0.09, 95% CI: 0.02-0.55); patients with KMT2D showed a trend to worse outcomes (PFS: 13.40 months vs NR, p: 0.06, HR 6.5, 95%CI: 1.06-38.76) [Figure 1a-c]. NOTCH mutations did not associate with outcome. CARD11 mutations were not detected. PLCy2 (H244R, L704V) mutations were detected in 2 patients prior to BTKi commencement but neither was associated with primary resistance.

Within the ctDNA cohort, 8/14 (57%) mutations detected in tumour samples were present in ctDNA at study entry. Furthermore, ctDNA also harboured mutations not present in tumour WES in 4 patients, likely reflective of tumour heterogeneity. Two patients had persistent MYD88 mutations detectable in ctDNA 15 months after treatment commencement (to sensitivity of AF 0.13%). Acquired resistance mutations, BTK (C515Y/515F) and PLCy2 (R665W/R742P) were detected in 2 patients who progressed on therapy.

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Summary/Conclusion: Mutations in genes associated with the NFkB pathway present at baseline are predictive of response to zanubrutinib in rrMZL patients. Detection of acquired BTK and PLCy2 mutations on therapy is feasible and may herald clinical disease progression.

P1262: EZH2 INHIBITORS MEDIATE PLATINUM RESISTANCE BY ENHANCED EFFLUX

E. Groß 1,* , R.-A. Hilger 2 , J. Wehde 1 , F. L. Schümann 1 , C. Rohde 3 , E. Willscher 1 , J. Lützkendorf 1 , L. P. Müller 1 , B. Edemir 1 , T. Müller 1 , M. Binder 1 , G. Posern 4 , T. Weber 1

1 Department of Hematology and Oncology, University Hospital Halle (Saale), Halle (Saale); 2 West German Cancer Center, University Hospital Essen, Essen; 3 Department of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Heidelberg; 4 Institute for Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany

Background: T-cell neoplasms are a heterogeneous and rare group of lymphomas with unfavorable prognosis. Polychemotherapy has cure rates of only 30 %. Consequently, new strategies of therapies are needed (Vose et al, J Clin Oncol, 2008, 26(25): 4124-30). EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 and responsible for the trimethylation of Lysine 27 of Histone 3 (H3K27me3). The protein is overexpressed in several tumor entities including T-cell neoplasms and leads to epigenetic dysregulation by H3K27me3 (McCabe et al, PNAS , 2012, 109(8): 2989–2994). Thus, EZH2 inhibition (EZH2i) might be a promising target and is in clinical evaluation in B- and T-cell malignancies showing favorable results (Duan et al, J Hematol Oncol, 2020, 13(1): 104)).

Aims: We aimed to investigate EZH2i in a characterized panel of Peripheral T-Cell Lymphoma (PTCL) and T-cell leukemia cell lines in combinational therapy with widely used salvage chemotherapeutics.

Methods: The T-cell leukemia and PTCL cell lines Jurkat, Loucy, HH, and DERL-2 were characterized regarding the canonical EZH2 signaling. They were treated with the EZH2 specific inhibitors GSK126 or EPZ6438 in combination with the common second line chemotherapy platinum analogue Oxaliplatin. The change in cytotoxic effects under pharmacological EZH2i or EZH2 Knockdown was evaluated. Cell viability was analyzed by Resazurin assay after 72 h of incubation. Target genes of chemical EZH2i that are responsible for the change in Oxaliplatin sensitivity were uncovered by RNA sequencing and validated by qPCR and western blotting. The effect of inhibiting EZH2 downstream targets on Oxaliplatin sensitivity modulation were analyzed.

Results: EZH2i led to a drastic increase in Oxaliplatin resistance in all tested cell lines regardless of cell type or EZH2 and H3K27 methylation status. This result could not be accomplished by EZH2 knockdown. RNA sequencing revealed an increase of expression in the transporters ABCG1 and ABCG2 by EZH2i that are associated with chemotherapy resistance. In addition, SRE binding factors SREBF1 and SREBF2 were overexpressed under EZH2i. Coherently, SREBP1 is known to activate ABCG1 transcription (Ecker et al, Biochem Biophys Res Commun, 2007, 352(3): 805-11). Under EZH2i, platinum was less present in the cells proven by inductive coupled plasma mass spectrometry. The EZH2i effect on Oxaliplatin resistance was lowered by additional inhibition of SRE binding proteins or ABC transporters.

Summary/Conclusion: We have shown that EZH2i is not suitable in combination with the common chemotherapeutic Oxaliplatin in T-cell neoplasms. EZH2i leads to increased expression of SREBF1 and SREBF2 as well as ABCG1 and ABCG2 . These transcriptional changes lead to increased Oxaliplatin efflux resulting in increased resistance. This effect was an EZH2-independent off-target effect of chemical EZH2i. Our data do not support the combination of platinum derivates and EZH2i in PTCL.

P1263: A SENSITIVE NGS ASSAY TO DETECT MEASURABLE RESIDUAL DISEASE (MRD) IN B-CELL LYMPHOPROLIFERATIVE DISEASES

L. Hebert 1 , P. Shah 1,* , O. Diaz 1 , A. A. Street 1 , J. Li 1 , E. Rudenko 1 , A. M. Zlotnicki 2 , C. Morrison 3 , R. Bomben 4 , Y. Huang 1 , A. Medina Herrera 5 , A. Wong 6 , S. Gleen 3

1 R&D; 2 BioInformatics, Invivoscribe, San Diego; 3 Center for Personalized Medicine, Roswell Park Cancer Institute, Buffalo, United States of America; 4 Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico di Aviano, Aviano, Italy; 5 Biología molecular y celular de hemopatías, Hospital Universitario de Salamanca, Salamanca, Spain; 6 Lahey Hospital, Lahey Hospital & Medical Center, Burlington, United States of America

Background: MRD assessment in B-cell lymphoproliferative diseases has proven utility in assessing response to therapy, refining treatment and predicting clinical outcome in patients previously treated with a combination of treatments (Garcia-Marco et al. Haematologica 2019; Pui et al. Leukemia 2017). Next-generation sequencing (NGS) based approaches for MRD detection have provided high sensitivity and specificity to identify and track minimal residual quantities of clonal immunoglobulin heavy chain ( IGH ) gene rearrangements (Rawstron, Leukemia, 2016). Here, we report a sensitive NGS assay to detect MRD in B-cell malignancies such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM) and mantle cell lymphoma (MCL).

Aims: To develop B-cell MRD Assay, a CAP/CLIA-accredited NGS-based MRD Laboratory Developed Test (LDT) to detect malignant B-cells at sensitivity up to 1.0 x 10 -6 .

Methods: The workflow of the B-cell MRD Assay consists of two parts, sample preparation and sequencing. Within the sample preparation, DNA from 1-2 mL of bone marrow (BM) samples were isolated and concentrated to allow up to 20 µg DNA as the input for the downstream PCR. Prior to sequencing, the library was prepared during PCR using proprietary multiplex master mixes (MMx) targeting either IGH Framework 1 (FR1) or Framework 3 (FR3). Followed by amplicon purification, equimolar pooling and sequencing on Illumina’s NextSeqDx ® platform. The sequencing data FASTQ output file was analyzed using Invivoscribe B-Cell MRD Software-NextSeqDx ® . Contrived DNA panels were prepared by diluting DNA from either cell lines or clinical BM samples (ALL, CLL, MM and MCL) into DNA from healthy BM samples. CLSI guidelines were used to design analytical studies and assess the limit of detection (LoD), limit of quantitation (LoQ), limit of blank (LoB), linearity, precision and accuracy of the B-cell MRD Assay and to determine the analytical performances of the Assay.

Results: Using FR1 targeted PCR MMx, the B-Cell MRD Assay exhibited a LoB at 0, LoD at 2.3x10 -6 , LoQ at 2.4x10 -6 , good linearity with R 2 >0.88 across 4.5 logs and precision with %CVs (coefficient variation) ranging from 27.6% to 63.5% at the LoD. Using FR3 targeted PCR MMx, the B-Cell MRD Assay exhibited a LoB at 0, LoD at 1.8x10 -6 , LoQ at 1.7x10 -5 , good linearity with R 2 >0.78 across 4.5 logs and precision with %CVs ranging from 46.6% to 89.4% at the LoD. In addition, the assay demonstrated 100% agreement in an accuracy study that included 20 clinical specimens and 10 healthy donors.

Summary/Conclusion: The turn-around-time (TAT) for B-Cell MRD Assay is about 5 days starting from specimens to MRD result. The assay has demonstrated good linearity, specificity and sensitivity to meet clinical threshold requirement of 10 -5 (FR1 LoD is 2.3x10 -6 , FR3 LoD is 1.8x10 -6 ) and showed excellent concordance in detection of patient specific clonal sequences at MRD levels. These results demonstrate that the B-Cell MRD Assay is a highly sensitive tool for detecting MRD in B-cell malignancies.

P1264: HEMATOPOIETIC CLONES WITH TP53 MUTATIONS EXPAND IN PATIENTS WITH MCL DURING LENALIDOMIDE-BASED THERAPY

S. Husby 1,* , C. Bæch-Laursen 2 , F. Favero 3 , J. S. Jespersen 4 , C. W. Eskelund 1 , M. Hutchings 1 , L. B. Pedersen 1 , C. U. Niemann 1 , J. Weischenfeldt 3 , C. Geisler 1 , R. Räty 5 , T. S. Larsen 6 , A. Kolstad 7 , M. Jerkeman 8 , K. Grønbæk 1

1 Hematology, Rigshospitalet, Copenhagen; 2 Hematology, Rigshospitalet, Copenhagen N; 3 Biotech Research & Innovation Center (BRIC), University of Copenhagen, Copenhagen; 4 Biotech Research & Innovation Center (BRIC), University of Copenhagen, Copenhagen N, Denmark; 5 Hematology, Helsinki University Hospital, Helsinki, Finland; 6 Hematology, Odense University Hospital, Odense, Denmark; 7 Oncology, Sykehuset Innlandet, Lillehammer, Norway; 8 Oncology, Lund University Hospital, Lund, Sweden

Background: Lenalidomide is widely used to treat mantle cell lymphoma (MCL) and multiple myeloma. It is however associated with development of therapy-related leukemia (t-AML/MDS) and the most frequent serious adverse events are hematological toxicities. It is unclear how lenalidomide affects the hematopoietic system in patients with mantle cell lymphoma.

To answer this question, we investigated somatic myeloid mutations, also called clonal hematopoiesis (CH), in sequential blood and bone marrow samples from patients with MCL treated with Lenalidomide-Ibrutinib-Rituximab on the Nordic Lymphoma Group (NLG) Philemon trial ( NCT02460276 ).

Aims: To investigate how a lenalidomide-based therapy impact hematopoietic clones in patients with malignant lymphoproliferative disease.

Methods: A total of 62 sequential blood samples (at pre-treatment and at 6 months treatment follow-up) were analyzed from 31 patients with relapsed/refractory MCL treated in the NLG Philemon trial. Patients received treatment in 28 day cycles with lenalidomide (Len; 15mg days 1-21), ibrutinib (Ibr; 560mg days 1-28), and rituximab (R; 375mg/m2 every 8 weeks). MCL clone-specific minimal residual disease (MRD) in patients bone marrow and blood was measured according to EuroMRD criteria.

DNA from the collected MRD samples were analyzed for somatic mutations with a custom capture-based NGS panel of 31 genes related to CH and t-AML/MDS. MCL tumor mutations were identified with a MCL NGS panel and excluded from CH analyses. Since TP53 mutations occur in both MCL and CH, de novo TP53 variants were considered as of MCL origin if 1) the patient was MRD positive at the same timepoint OR 2) the patient had a relapse in the following six months after the blood sample was taken. Somatic non-MCL mutations (CH) were identified with a variant allele frequency (VAF) down to 0.01.

Results: A total of 40 somatic mutations in myeloid related genes, i.e. CH mutations, were identified in 22 patients (71% of cohort) with the majority being in DNMT3A and DNA repair pathway genes ( TP53 and PPM1D ; Figure 1 A ). Number of CH mutations were associated with development of Grade 3/4 hematological toxicity during Len-based therapy (Figure 1 B ). Patients with pre-existing CH mutations tended to more likely develop neutropenia compared to patients without CH (Fischer’s exact test p=0.11). CH mutations were not associated with age or number of prior therapies in a linear regression model.

During treatment with Len-Ibr-R there was a significant overall increase in CH clone size (median VAF= +44%, p=0.005), but heterogenous growth patterns of the respective mutated genes was noted (Figure 1 C ). Mutations in TP53 increased significantly in size (median +153%, p=0.008). Mutations in ASXL1 , TET2 , and PPM1D had marked median increases (median +2363%, +49%, +94%, respectively) though the increase did not reach statistical significance. On the contrary, mutations in DNMT3A were stable during treatment with Len-Ibr-R (median +6%, p=1.0).

There was no significant difference in overall or progression-free survival with regards to pre-existing clonal hematopoiesis.

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Summary/Conclusion: To our knowledge, we here present the first description of CH evolution during treatment with lenalidomide-based therapy. Pre-treatment presence of somatic mutations were associated with development of grade 3/4 hematological toxicity. Therapy with Len-Ibr-R caused hematopoietic clones with TP53 mutations to significantly expand over time, while clones with DNMT3A mutations were remarkably stable.

P1265: TMEM244 GENE EXPRESSION AS A POTENTIAL BLOOD DIAGNOSTIC MARKER THAT CAN DISTINGUISH SÉZARY SYNDROME FROM MYCOSIS FUNGOIDES AND BENIGN ERYTHRODERMA

K. Iżykowska 1,* , K. Rassek 1 , M. Żurawek 1 , K. Nowicka 1 , M. Joks 2 , K. Olek-Hrab 3 , B. Olszewska 4 , M. Sokołowska–Wojdyło 4 , G. Przybylski 1

1 Institute of Human Genetics PAS; 2 Department of Hematology and Marrow Transplantation; 3 Department of Dermatology, University of Medical Sciences, Poznań; 4 Department of Dermatology, Venereology and Allergology, Medical University of Gdansk, Gdańsk, Poland

Background: Sézary syndrome (SS) is a leukemic form of cutaneous T-cell lymphoma (CTCL) characterized by erythroderma, intractable pruritis, and the presence of circulating malignant CD4+ T cells called Sézary cells. The diagnosis of SS is challenging and requires the integration of clinical presentation, histopathologic evaluation, and molecular studies. The immunophenotype of Sézary cells is heterogeneous and often varies among patients. What’s more, SS can resemble inflammatory dermatoses as well as another CTCL type Mycosis Fungoides (MF). Therefore, there is a need for a relevant diagnostic tool, that will also allow sensitive disease monitoring and accurate assessment of treatment response.

Aims: This study aimed to analyze and compare the expression of the TMEM244 gene in peripheral blood mononuclear cells (PBMC), CD4+ T-cells from peripheral blood, and lymphocytes from skin biopsies, from SS, MF, and benign erythroderma (E) patients. Moreover, TMEM244 expression was measured in different subpopulations of PBMC from healthy donors (HD).

Methods: TMEM244 expression was evaluated in 18 SS, 5 MF, 8 E, and 44 HD samples. PBMC were separated from the whole blood by density gradient centrifugation and the CD4+ T-cells were separated using negative selection on magnetic beads. The skin biopsies were cut into 1 mm3 pieces, treated overnight with Collagenase P, and passed through nylon strainers (100-40 µm). The following subpopulations of PBMC from 6 HD: CD4, CD8, CD45RO, CD45RA, CD31, CD56, CD14, CD19 were separated by PE-conjugated antibody staining and positive selection on magnetic beads. RNA was extracted by TRI Reagent, reverse transcribed, and TMEM244 expression was analyzed by qRT-PCR. All samples were assayed in triplicates, the relative gene expression was calculated (2^-ΔCT). Statistical significance was determined with the unpaired t-test with Welch’s correction and the Mann–Whitney test.

Results: Within the PBMC samples mean TMEM244 expression fold change in SS was 46.9 relative to HD and 52.24 relative to MF/E (p-values of < 0.0001 respectively) (Fig. 1A; 2A). The relative fold TMEM244 expression in CD4+ T cells was even higher than in the whole population of PBMC, in SS it was 359.2 relative to HD and 42.4 relative to MF/E (p-values of < 0.0001 respectively) (Fig. 1B; 2B). The experiment failed to show differential relative TMEM244 expression in SS skin samples compared to MF/E samples (p-values > 0.05). In blood cells, relative TMEM244 expression in T cells, both CD4+ (4.7E-04) and CD8+ (6.9E-04) was higher than in any other population of PBMC, including CD19+ B-cells (2.7E-05), CD56+ NK cells (1.2E-04), and CD14+ monocytes (8.8E-05). Moreover, the analysis of naïve CD45RA+ (5.2E-05) and memory CD45RO+ (3.8E-04) populations, as well as the youngest subset of naïve T cells CD31+ (7.6E-05) showed that the expression of TMEM244 is associated more with the later stages of T cell activation.

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Summary/Conclusion: The analysis showed that elevated TMEM244 expression is only present in the blood cells of Sezary syndrome patients but not in healthy individuals. What is more, the expression of TMEM244 in the blood can differentiate Sezary syndrome patients from those suffering from benign erythroderma or other types of CTCL. Moreover, both CD4+ and CD45RO+ markers, a characteristic immunophenotype of Sezary cells, represented subpopulations with a higher basic expression of TMEM244, suggesting the involvement of this gene in the disease onset.

P1266: FLOW CYTOMETRY COMBINED WITH SYSTEMS BIOLOGY ENABLES RATIONAL TARGETING OF NFKB IN DLBCL

E. Jayawant 1,* , A. Pepper 1 , C. Pepper 1 , S. Mitchell 1

1 Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton, United Kingdom

Background: NFκB signalling plays a crucial role in lymphoid malignancies and is frequently mutated leading to aberrant activation. Broad targeting of NFκB has failed due to on-target toxicity, and the roles of specific NFκB molecules in lymphoma remain poorly understood. Previous studies have identified high abundance of NFκB-cRel in GC-like lymphomas, and a subset of DLBCL cases dependent on NFκB-RelB. This suggests that characterisation of NFκB composition and regulatory network state may unlock the ability to rationally target NFκB in lymphoma cells, whilst avoiding systemic toxicity. Systems biology approaches, using computational models, have provided insight into the roles and regulation of specific NFκB components. Capturing the heterogeneous state of NFκB signalling in DLBCL in computational models enables prediction of the efficacy of targeting NFκB in subsets of DLBCL, this drastically reduces laboratory time.

Aims: We aimed to develop a library of computational simulations, informed by flow cytometry data from different DLBCL cell lines, capturing the heterogeneous state of NFκB signalling in DLBCL. We aim to establish these models as tools that enable the prediction of the impact of targeted therapeutics on NFκB.

Methods: Using a bespoke flow cytometry analysis pipeline, we have collected intracellular flow cytometry data for four NFκB subunits in two DLBCL cell lines. The flow cytometric data were validated using NFκB subunit ELISAs. We further analysed two subclones found within U2932 cells (R1 and R2). These data informed ordinary differential equation (ODE) computational models, allowing us to change parameters encoding expression rates of NFκB subunits to reflect the heterogeneity between DLBCL cell lines (Figure 1A).

Results: We validated our flow cytometry pipeline using published data in multiple myeloma cells and characterised the relative abundance of NFκB subunits in DLBCL cell lines. Key differences in subunit abundances, not explained by cell-of-origin classifications, were revealed. SUDHL8 cells showed increased canonical activity compared to U2932 cells. Even within a single cell line (U2932), two subclones had distinct NFκB signalling states, with increased canonical activity in R1. These differences in NFκB signalling state result in distinct predicted response to NFκB targeting therapeutics. Our models were able to recapitulate the vast heterogeneity within these cell lines with only minor changes to expression levels, consistent with epigenetic heterogeneity (Figure 1B). We found NFκB signalling states in DLBCL to be a complex emergent property resulting from competition between monomers, dimers and trimers. We used computational cell lines to predict the impact of targeted therapeutics on NFκB signalling.

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Summary/Conclusion: NFκB is an exciting target but challenging drug target due to its heterogenous activity in DLBCL cases and importance in normal cellular functions. Systems biology approaches can enhance our understanding of NFκB and the roles of specific subunits in DLBCL. Using a combination of flow cytometry and ODE modelling, we show that a computational model can recapitulate the heterogeneity of the different NFκB subunit abundances observed in DLBCL cell lines and predict the impact of targeted therapies on NFκB. We are now expanding the pipeline from flow cytometry to computational model and back to laboratory experiments, to include patient samples. Long term this work provides a foundation for a personalised medicine approach in which models enable the prediction of which drugs will be most effective for individual DLBCL patients.

P1267: HSP110 FAVORS BCR SIGNALING THROUGH SYK PHOSPHORYLATION IN DIFFUSE LARGE B CELL LYMPHOMA

V. Cabaud Gibouin 1,* , C. Boudesco 1 , M. Durand 1 , F. Hermetet 1 , C. Garrido 2 , G. Jego 1

1 UFR Sciences de Santé, University of Burgundy; 2 INSERM U1231, INSERM, Dijon, France

Background: Heat-shock proteins (HSPs) are molecular chaperones highly expressed in hematological malignancies. They promote proliferation, immune escape, and resistance to chemotherapies. Our team and others identified HSP110 as a pro-survival factor in diffuse large Bcell lymphomas (DLBCL). In particular, HSP110 sustains aberrant NFkB signaling in activated B-cell DLBCL (ABC-DLBCL) through MyD88 stabilization (Boudesco et al. Blood 2018 Aug 2;132(5):510-520). These findings highlighted HSP110 as a new potential therapeutic target in DLBCL. However, the role of HSP110 in the global signaling pathway of DLBCL has not been studied yet. In addition, the impact of HSP110 inhibition in in vivo xenograft models has been hampered by the absence of specific inhibitors.

Aims: We recently identified two foldamers by their capacity to specifically disrupt HSP110 functions in colorectal cancer models (Gozzi et al. Cell Death Differ. 2020 Jan;27(1):117-129). These first HSP110 inhibitors have given us the opportunity to gain more insight into the signaling pathways controlled by HSP110 in DLBCL in vitro and in vivo .

Methods: We treated DLBCL cell lines in vitro , and in vivo in xenografted NSG mice. Analyses were done using Western blot, IHC, proximity ligation assay, and in vitro phosphorylation assay.

Results: We first validated the two HSP110 inhibitors in DLCBL since they efficiently disrupted the binding of HSP110 to MyD88 in DLBCL cell lines. They impaired cell growth and led to cell death in the Myd88 L265P cell lines OCI-Ly10, HBL1, TMD8 and OCI-Ly3 in vitro . Treatment of TMD8-xenografted NSG mice with increasing doses of HSP110 inhibitors limited tumor growth, without tissue toxicity. Analysis of xenografted tumors and in vitro treatments of DLCBL cell lines revealed that the MyD88/NFkB pathway was altered as expected, but also the BCR pathway since we observed a decrease in the activation of BTK, SYK and PLCy2.

Using immunoprecipitations and Duolink TM assays, we identified an in vitro and in cellulo physical interaction between HSP110 and SYK (but not BTK) in several DLBCL cell lines. Furthermore, HSP110 inhibitors disrupted the binding of HSP110 with SYK in vitro , suggesting that the foldamers may act through this mechanism in vivo . We confirmed this assumption using Duolink TM assays, which showed fewer interactions in xenografted tumors after treatment.

Moreover, the phosphorylation of SYK recombinant protein is increased in vitro in the presence of a HSP110 recombinant protein, suggesting that HSP110 acts as a molecular chaperone for optimal activation of SYK through phosphorylation.

In addition to the MyD88 L265P promotion of the NFkB pathway, the chronic BCR activation in DLBCL has been shown to mediate the activation of both the SYK/BTK and PI3K/Akt pathways, converging to NFkB. Targeting these pathways with ibrutinib and copanlisib, respectively, synergistically suppresses tumor growth in xenograft models (Paul et al. Cancer Cell 31, 64–78, January 9, 2017). We showed that the combination of HSP110 inhibitors with copanlisib also decreases SYK/BTK and AKT phosphorylation synergistically, leading to less NFkB activation in vitro and in vivo . Accordingly, the drug combination stopped the progression of the xenograft.

Summary/Conclusion: In conclusion, we identified HSP110 as a regulator of BCR signaling through SYK chaperoning in DLBCL. This finding highlights the major potential of HSP110 as a therapeutic target in DLBCL and suggests a broader role in immune responses.

P1268: DELINEATING THE NEOANTIGEN BURDEN AND TUMOUR MICROENVIRONMENT IN PATIENTS WITH DIFFUSE LARGE B CELL LYMPHOMA (DLBCL).

Y. Jiang 1,* , D. Jin 2 , C. Henneges 3 , E. Penuel 3 , H. J. Hiew 1 , J. N. Paulson 3 , A. Bazeos 1

1 Product Development, Roche Products Limited, Welwyn Garden City, United Kingdom; 2 Cancer Genomics Research, Foundation medicine, Inc., Cambridge, MA; 3 Genentech Inc., South San Francisco, CA, United States of America

Background: DLBCL is a heterogeneous neoplasm classified into 2 main cell-of-origin (COO) subtypes; activated B cell (ABC) and germinal centre B cell (GCB), with ABC having a worse clinical prognosis. Neoantigens are highly selective and immunogenic tumour antigens which result from somatic hypermutations in malignant cells.

Aims: The neoantigen landscape in DLBCL has been relatively underexplored. Therefore the aim of this study was to characterise the neoantigen burden (NAB) in DLBCL by COO subtype as well as elucidate the relationship between NAB, tumour microenvironment and disease pathogenesis.

Methods: Data were obtained from patients enrolled with previously untreated DLBCL in the Phase III GOYA study ( NCT01287741 ). COO was determined by standard gene expression profiling. Neoantigens were identified and analysed using the Roche/Foundation Medicine neoantigen pipeline where NAB is defined as the total number of predicted neoantigens per patient. The immunedeconv R package was used to analyse the immune cell fractions from bulk RNA-seq data. Post-hoc, exploratory data analysis was performed in R.

Results: A total of 404 patients with both COO and NAB data recorded were available for analysis; 272 GCB (67%) and 132 ABC (33%). Overall, the median NAB was 2 with no significant difference between the two COO subgroups (Kruskal wallis chi-squared 0.63, p= 0.42).

Next, 449 patients with COO and immune cell fractions data were assessed. A greater proportion of M1 macrophages were present in patients with GCB compared to ABC COO subtype (Kruskal Wallis chi-squared 7.66, p value <0.01). Conversely, higher fraction of M2 macrophages were identified in the ABC compared to GCB subtype (Kuskal Wallis chi-squared 31.39, p <0.001). Of note, there were no significant COO differences in the proportions of CD4+ T cells, CD8+ T cells or T regulatory cells.

Of the 444 patients with NAB and immune cell fraction data available. There was a negative correlation between NAB and CD8+ T cell (Pearson correlation -0.162, 95% CI (-0.25, -0.07), adjusted p <0.01; Spearman correlation -0.134, 95% CI (-0.22, -0.04), adjusted p = 0.05). No statistically significant correlation with other immune cell subsets, including macrophages, CD4+ T or T regulatory cells were identified.

Summary/Conclusion: To our knowledge this is the first study exploring the complex interplay between neoantigen formation and the tumour microenvironment in DLBCL patients. We report that a greater neoantigen burden is correlated with lower CD8+ T cell fraction; this could represent a potential tumour evasion mechanism. In addition, we find that whilst there is no difference in neoantigen burden between cell of origin subtypes, a significant variation exists in the tumour microenvironment composition, particularly with respect to M1 and M2 macrophage proportions. Further studies are needed to understand the prognostic impact and clinical significance of these findings.

P1269: VIP152 IS A NOVEL CDK9 INHIBITOR WITH IMPROVED SELECTIVITY, TARGET MODULATION, AND CARDIAC SAFETY IN PATIENTS WITH LYMPHOMA

M. Frigault 1,* , S. Sher 2 , J. Ravikrishnan 2 , R. Izumi 1 , J. Ware 1 , H. Wong 1 , A. Johnson 1 , B. Stelte-Ludwig 3 , J. Byrd 4 , D. Sampath 5 , K. Rogers 2 , J. Woyach 2 , R. Lapalombella 2 , A. Hamdy 1

1 Vincerx Pharma, Palo Alto; 2 The Ohio State University, Columbus, United States of America; 3 Vincerx Pharma, Monheim, Germany; 4 University of Cincinnati, Cincinnati; 5 MD Anderson, Houston, United States of America

Background: Cyclin dependent kinase 9 (CDK9) controls transcriptional elongation, its inhibition may be effective in controlling cancer. To date, no drugs specifically targeting CDK9 have been approved due to challenges with therapeutic window. VIP152 was designed to be a highly potent and selective CDK9 inhibitor with favorable pharmacokinetic (PK) and pharmacodynamic (PD) effects to improve efficacy in patients with lymphoma.

Aims: - To demonstrate the pharmacodynamic effects and safety profile associated with a selective CDK9 inhibitor with a favorable pharmacokinetic profile.

- To demonstrate the utility of a selective CDK9 inhibitor for treatment of hematologic patients with an unmet medical need such as high-grade B cell lymphoma (HGBL) and chronic lymphocytic leukemia who relapse or are refractory to ibrutinib and venetoclax (R/R CLL).

Methods: VIP152, fadraciclib, alvocidib (flavopiridol), KB-0742, and AZD4573 are evaluated in a CDK9/Cyclin T assay in the presence of low and high ATP and in a kinome scan at 1 µM. Differential expression (DE) analysis from VIP152, atuveciclib and KB-0742 treatment of 2 lymphoma cell lines by RNA seq is compared to DE of samples from 7 VIP152 treated HGBL patients. VIP152 cytotoxicity is evaluated in CRISPR/Cas9 edited HG-3 CLL cell line with homozygous TP53 edits R175H or R248Q, and in samples from 8 R/R CLL patients who relapsed or were refractory to ibrutinib and venetoclax.

Results: In the presence of low ATP, the IC50 for VIP152 is 4.5 nM where others have IC50s from 3.2-29.4 nM. With high ATP, VIP152, AZD4573 and alvocidib maintain potency in low nM range, while fadraciclib and KB-0742 have 760 nM-1.7 µM IC50s. The kinome scan shows VIP152, KB-0742 and fadraciclib have the least number of hits. K d values show VIP152 is the most selective CDK9 inhibitor versus all other CDKs. In patients at the current clinical dose of 30 mg administered once weekly, the average VIP152 unbound plasma concentration over a 24h period is 18 nM. The VIP152 IC50 of 4.5 nM unbound is maintained for 15h indicating target coverage. VIP152 PK exposure is dose-linear and proportional with low intra/inter-subject variability. Analysis of triplicate ECG data from 58 solid and heme cancer patients demonstrates that VIP152 does not prolong QTc interval after single or multiple doses (5-30 mg). DE analysis of MYC+ DLBLC cell lines with VIP152, atuveciclib or KB-0742 1µM treatment indicate that 85%, 76% or 68% of DE genes are downregulated, respectively and only VIP152 and atuveciclib achieve significant DE of MYC. DE is reproducible in HGBL patients’ whole blood at Cycle 1 Day 1 and Day 15 (r=0.9627) and findings will be validated in the ongoing clinical trial. VIP152 treatment demonstrates significant cytotoxicity in both WT and TP53 mutant CLL lines. R/R CLL samples treated with VIP152 showed a significant reduction in viability in a concentration-dependent manner.

Summary/Conclusion: VIP152 is a potent and selective CDK9 inhibitor currently in development with a favorable cardiac safety profile. PK/PD demonstrates unbound plasma concentrations exceed in vitro IC50. Selective transcriptional downregulation is observed with 30mg IV weekly. Samples from patients with DLBCL or CLL are sensitive to VIP152 and updated PK, PD and ctDNA dynamics from the ongoing phase 1 trials ( NCT02635672 , NCT04978779 ) will be presented.

P1270: IMMUNE REGULATORY CELLS IN B-CELL LYMPHOMAS: SIGNIFICANT HETEROGENEITY ACROSS HISTOLOGIC TYPES.

M. STAVROULAKI 1 , A. DAMIANAKI 2 , K. CHAIKOU 1 , A. KAVIDOPOULOU 1 , P. VERGINIS 2 , C. KALPADAKIS 2,*

1 UNIVERSITY HOSPITAL OF CRETE; 2 MEDICAL SCHOOL, UNIVERSITY OF CRETE, HERAKLION, Greece

Background: Immune regulatory cell populations play a fundamental role in tumor development and tumor immune evasion. Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) considered as dominant suppressor cells which are significantly enriched in blood, bone marrow (BM), or tumor sites of patients with hematologic malignancies and often associate with high tumor burden, adverse clinical features, and outcome. Studies in lymphoma patients however, have generated conflicting results, regarding the function and phenotype of MDSC and Tregs mainly due to large heterogeneity of the disease as well as due to the lack of a unanimous nomenclature of these cell subsets in human samples. Furthermore, the molecular mechanisms via which MDSCs and Tregs contribute to clinical outcomes of disease and how they interfere with response to therapy remain elusive.

Aims: To investigate the prevalence of immune suppressive population in B-cell lymphomas according to histologic, subtype providing the basis for further analysis of their biologic properties and association with clinical features and outcome of specific lymphoma subtypes.

Methods: Peripheral blood (PB) and BM samples were collected from healthy individuals (n= 22) and lymphoma patients (n=50). Specifically, lymphoma subtypes were as follows:Marginal Zone (MZL, N=19), Diffuse Large B-cell Lymphoma (DLBCL, N=18) and Follicular Lymphoma (FL, N=13). Peripheral blood mononuclear cells (PBMCs) were isolated with ficoll gradient and subjected to flow cytometry analysis. MDSC characterization was based on HLADR - /lowCD11b + CD33 - expression with CD14 + CD15 - for M-MDSCs and CD14 - CD15 + for PMN-MDSCs. In addition, Treg cells were denoted as CD4 + CD25 high CD127 - Foxp3 + . Expression of PDL1 and PD1 on MDSCs and Tregs respectively, was also determined by flow cytometry. M-MDSCs and PMN-MDSCs were sorted from BM samples, RNA was extracted in order to subjected to transcriptomic analysis (RNAseq). Informed consent was obtained.

Results: Frequencies of MDSCs were significantly increased in PB of all lymphoma patients compared to healthy individuals, with MZL subtype exhibiting the most prominent increase of MDSCs (p<.0001). Notably numbers of M-MDSCs were increased in FL and MZL patients and to a lesser extend in DLBCL patients while PMN-MDSCs were elevated in all subtypes with MZL displaying the higher frequencies. Regarding the Treg cells, although all lymphoma patients exhibited increased absolute numbers of CD4 + CD25 high CD127 - Treg cells, Foxp3-expressing Tregs were significantly increased in the PB of DLBCL and MZL patients, but not in FL patients. Of interest, CD4+ and CD8+ T cells in the PB of DLBCL patients exhibited increase expression of PD1 compared to respective cell subsets from healthy individuals.

Summary/Conclusion: Our findings comprehensively demonstrate enhanced numbers of MDSC and Treg immune suppressive cells in the PB of B-cell lymphoma patients while an increased heterogeneity was observed among the different histological lymphoma types. Functional characterization of MDSCs and Tregs by transcriptomic (RNAseq) and proteomic analysis are ongoing.

P1271: ELUCIDATING TUMOR SUPPRESSOR FUNCTIONS OF TET2 IN B CELL LYMPHOMAS

N. Kinz 1,* , S. Spoeck 1 , M. Leone 1 , M. Lohmueller 1 , A. Villunger 1 , V. Labi 1

1 Department of Developmental Immunology, Medical University of Innsbruck, Innsbruck, Austria

Background: Remodeling of the epigenetic landscape via DNA cytosine methylation directs cell lineage specification, establishing and maintaining gene expression programs that uniquely define given cell types. During tumorigenesis the epigenome becomes corrupted, impairing lineage-specific gene expression programs. TET2, an enzyme that contributes to the erasure of cytosine methylation via oxidation of the 5’ methyl group, is among the most frequent somatically mutated genes in human hematological tumors, commonly identified as loss-of-function versions. In mouse models, TET2 loss on its own is only weakly tumorigenic, but rather facilitates leukemic transformation driven by prototypic oncogenes such as AML-ETO or FLT3 ITD . However, TET2 LOF is not limited to leukemias. In B cell malignancies, TET2 is found mutated in a fraction of diffuse large B cell lymphomas (DLBCL), which display a distinct gene expression profile.

Aims: Having established a role for TET2 loss-of-function in enhancing MYC-driven lymphomas, our overall aim is to understand how TET2 loss conspires with MYC to overcome the barriers that maintain cell identity and function.

Methods: Using pre-clinical mouse models and in vitro culture systems, we establish that TET2 loss boosts the formation of B cell lymphomas driven by the MYC oncoprotein.

Results: Thus, (1) TET2 deficiency specifically in B cells facilitates MYC-mediated transformation in a dose dependent manner, and (2) TET2 deficiency early on in hematopoietic stem and progenitor cells enhances the transformative potential later on in antigen-activated B cells. Our data suggest that TET2 may exert tumor suppressor functions via controlling the expression of DNA repair genes, or via direct functions of oxidized cytosine species. In addition, TET2-deficient lymphomas select for high expression of the pro-survival BCL2 family protein BCLX L , potentially providing protection from MYC-driven apoptosis.

Summary/Conclusion: Altogether, our data concerning the impact of TET2 loss-of-function on DNA repair and apoptosis mechanisms creates understanding of how TET2 deficiency nudges transformation, and reveals testable therapeutic vulnerabilities.

P1272: GP130/STAT3 AXIS IS A POTENTIAL THERAPEUTIC TARGET FOR HISTONE DEACETYLASE INHIBITOR-RESISTANT CUTANEOUS T-CELL LYMPHOMA

Y. Takahashi 1,* , A. Kitadate 2 , S. Ikeda 2 , S. Iwama 1 , K. Abe 2 , Y. Matsuda 1 , H. Tagawa 2 , H. Wakui 1 , N. Takahashi 2

1 Department of Life Science, Graduate School of Engineering Science, Akita University; 2 Department of Hematology, Nephrology, and Rheumatology, Akita University Graduate School of Medicine, Akita, Japan

Background: Histone deacetylase inhibitors (HDACis), such as vorinostat and romidepsin, are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, and adult T-cell lymphoma/leukemia. Despite the promising anti-lymphoma activity of HDACis, resistance remains a significant clinical problem. Therefore, novel therapeutic strategies to overcome HDACi resistance are required. In this study, we generated HDACi-resistant CTCL cell lines and investigated the mechanisms of HDACi resistance.

Aims: The aim of the study was to identify the mechanism of resistance to HDACis in CTCLs and to explore new therapeutic targets.

Methods: CTCL cell lines My-La, MJ, and Hut78 were used in the study. Vorinostat-resistant cell lines MyLa resistant, HUT78 resistant, and MJ resistant were prepared by repeatedly exposing cells to increasing concentrations of vorinostat. To maintain drug resistance, cells were continuously cultured in a medium containing 2.5 μM vorinostat. Following this, we conducted a comprehensive gene expression analysis using a microarray platform to investigate deregulated genes in the resistant cell lines.

Results: CTCL cell lines resistant to vorinostat were also resistant to romidepsin, suggesting that vorinostat-resistant cell lines were cross-resistant to other HDACis. Next, we selected genes whose expression was upregulated more than two-fold in the three resistant cell lines and performed pathway analysis. Pathway analysis revealed that upregulated genes were significantly enriched in cytokine-mediated signaling pathways. Among the upregulated genes, we focused on IL6ST/gp130, which forms a heterogeneous complex with IL6 and IL6 receptor and activates the JAK/STAT pathway. Further, flow cytometric analysis revealed that surface gp130 expression was significantly upregulated in resistant cell lines. Moreover, resistant cell lines showed an increased sensitivity to a gp130 inhibitor (SC144), and gp130 activation could activate the JAK/STAT pathway. Accordingly, we examined the expression level of the activated form of STAT3, that is p-STAT3, and found that p-STAT3 was significantly upregulated in resistant cell lines. To investigate whether p-STAT3 upregulation is involved in the acquisition of vorinostat resistance, we performed STAT3 knockdown using an siRNA. We confirmed that STAT3 knockdown significantly decreased the survival of resistant cell lines. Moreover, resistant cell lines showed increased sensitivity to the STAT3-selective inhibitor, C188-9. We also examined the expression level of p-STAT5; however, it was downregulated in resistant cell lines, which was opposite to that of p-STAT3. Finally, we examined gp130 and p-STAT3 protein expression in CTCL skin samples, which were obtained from the same patients before and after vorinostat treatment. We found that gp130 and p-STAT3 expression significantly increased in the specimens at the time of relapse after vorinostat treatment.

Summary/Conclusion: Activation of STAT3 in HDACi-resistant CTCL cells was enhanced via gp130, which contributed to their resistance. These results suggested that STAT3 could be a new therapeutic target in HDACi-resistant CTCL, and gp130/STAT3 inhibition could be an important therapeutic strategy.

P1273: INVESTIGATING THE ONCOGENIC POTENTIAL OF STAT5BN642H IN NK CELLS

K. Klein 1,* , M. Randhawa 1 , A. Witalisz-Siepracka 1,2 , J. Trifinopoulos 1 , P. Kudweis 1 , V. M. Knab 1 , D. Gotthardt 1 , V. Sexl 1

1 Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna; 2 Department of Pharmacology, Physiology and Microbiology, Division Pharmacology, Karl Landsteiner University of Health Sciences, Krems, Austria

Background: The signal transducer and activator of transcription 5B (STAT5B) regulates development, survival and function of innate(-like) lymphocytes. STAT5B gain-of-function (GOF) mutations, including the most common STAT5B N642H mutation, are found in different hematopoietic malignancies. These include aggressive NK-cell malignancies and CD56+ T-cell large granular lymphocyte leukemia (T-LGLL). A previously established mouse model expresses human STAT5B N642H under the Vav1 promoter and thus in the entire hematopoietic system. These Vav1 -driven STAT5B N642H transgenic mice develop severe CD8+ T-cell malignancies. In a transplantation setting devoid of CD8+ T cells, Vav1 -driven STAT5B N642H also transforms NKT cells and promotes aggressive NKT-cell leukemia, resembling CD56+ T-LGLL.

Aims: We aim to investigate whether STAT5B N642H also acts as an oncogenic driver in NK cells and establish an NK-cell leukemia model, which would enable the development of treatment options for highly aggressive NK-cell malignancies.

Methods: While Vav1 -driven STAT5B N642H caused a temporary expansion of NK cells, no NK-cell disease was observed with this model. To avoid competition with more potently transformed cell types, we generated a novel mouse model in which STAT5B N642H expression is restricted to the NK (NKp46-positive) cell lineage (N642H NK/NK mice). NK cells were characterized by flow cytometry in 8-12-week-old mice. N642H NK/NK and control mice were aged to investigate whether mutant STAT5B has an oncogenic potential in NK cells. Furthermore, NK-cell functionality was assessed in vitro upon IL-2 culture and in vivo with the subcutaneous NK cell-sensitive RMA-S tumor model to study effects of enhanced STAT5B signaling on NK-cell activity.

Results: STAT5B N642H expression in NKp46+ cells resulted in an increased number of NK cells in young and aged mice but failed to cause signs of an aggressive NK-cell malignancy up to an age of 10-14 months. Ageing experiments will be continued until an age of 15-18 months to investigate an oncogenic potential of mutant STAT5B in NK cells at a later timepoint. Overexpression of non-mutant STAT5B or STAT5B N642H decreased the expansion capacity of NK cells in vitro, associated with a “senescence-like” state and reduced cytotoxic capacity. On the contrary, overexpression of non-mutant STAT5B or STAT5B N642H in NKp46+ cells enhanced tumor surveillance of RMA-S tumors in vivo .

Summary/Conclusion: Our current data suggest that STAT5B N642H on its own is not able to transform NK cells, in contrast to other cell types. Additional alterations might be required to allow for NK-cell transformation by STAT5B N642H and to cause a symptomatic disease. Enhanced STAT5B signaling has opposing effect on NK-cell functionality in vitro compared to in vivo . Exploring the underlying mechanisms will contribute to a better understanding of the complex regulation of NK-cell activity. This might have implications for immunotherapeutic approaches using cytokines that activate STAT5 to expand and stimulate NK cells.

P1274: EARLY DETECTION OF NON-HODGKIN LYMPHOMA IN PREDISPOSED PATIENT GROUPS THROUGH IMMUNOGENETIC SEQUENCING

P. M. Kolijn 1,2,* , K. Boer 3 , O. C. Manintveld 4 , C. C. Baan 3 , P. L. van Daele 5 , M. A. Versnel 6 , R. C. Vermeulen 2 , A. W. Langerak 1

1 Department of Immunology, Laboratory Medical Immunology, Erasmus MC, Rotterdam; 2 Division of Environmental Epidemiology and Veterinary Public Health, Institute for Risk Assessment Sciences, Utrecht; 3 Department of Internal Medicine, Division of Nephrology and Transplantation; 4 Department of Cardiology, Thorax Center, Erasmus MC Transplant Institute; 5 Department of Internal Medicine, Division of Clinical Immunology; Department of Immunology; 6 Department of Immunology, Erasmus MC, Rotterdam, Netherlands

Background: Since chronic lymphocytic leukemia development can be detected up to 16 years prior to diagnosis through immunogenetic sequencing, we aim to evaluate whether early detection through immunogenetic sequencing translates to other types of non-Hodgkin lymphoma (NHL). A particularly interesting group with potential clinical utility consists of patients with underlying conditions that confer an increased risk of lymphoma and leukemia. Generally, this group is characterized by dysregulation of the immune system, in the form of autoimmune diseases, primary immunodeficiencies, immunosuppressive treatment and several infectious diseases.

Aims: To evaluate the capacity of immunogenetic sequencing for early detection of NHL and leukemia in patient groups at increased risk.

Methods: We selected two patient groups with an increased risk of NHL and leukemia: solid organ transplant recipients and patients with auto-immune disease. For each group, we selected patients who had developed NHL and controls with the same underlying condition matched on sex, age and sampling window. Currently, we have included 16 cardiac and 10 renal transplant recipients and 10 primary Sjögren’s syndrome (SjS) patients (in total 18 cases and 18 controls). A total of 114 longitudinal pre-diagnostic samples were included in the study, taken a median of 1.2 years before diagnosis (IQR 3.5 years). Genomic DNA was isolated from the PBMC samples of the participants and the immunoglobulin heavy chain (IGH) was sequenced on the Illumina Miseq platform. All PBMC samples were taken after SjS diagnosis or solid organ transplantation and prior to NHL diagnosis.

Results: In 7 out of 18 NHL patients we identified clonotypes in prediagnostic PBMC samples that were present at an abnormal frequency compared to the healthy background clonotypes. Notably, distribution of the IGH gene repertoire in controls was normal. Prevalence of IGH gene repertoire skewing was particularly pronounced among SjS patients (3 out of 5 patients) and cardiac transplant recipients (4 out of 8 patients) compared to renal transplant recipients (1 out of 5 patients). IGH gene repertoire skewing was restricted to samples taken within 5 months to two years before diagnosis for the majority of the cases. In solid organ transplant recipients with an abnormal clonotype, the degree of skewing of the IGH gene repertoire increased over time towards the moment of NHL diagnosis or remained stable at elevated levels.

For the SjS patients, two out of the three abnormal clonotypes utilized the IGHV1-69/IGHJ4 or IGHV4-59/IGHJ5 rearrangements stereotypic for rheumatoid factors, which have previously been associated with MALT lymphoma development in SjS. Unbiased marker identification has yielded putative prediagnostic lymphoma markers for 7 out of 18 patients.

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Summary/Conclusion: We conducted a pilot study investigating a novel method of early detection of NHL development in 36 patients at increased risk of hematological malignancy. In total, we observed an abnormal distribution of the IGH repertoire between 5 months to 2 years prior to NHL diagnosis in 7 out of 18 patients, compared to 0 out of 18 of the matched controls. While our results suggest potential merit for early detection of NHL development through immunogenetic sequencing, the current cohort size does not yet permit drawing definitive conclusions. Therefore, inclusion of additional patients will be a priority during the continuation of the study.

P1275: PERCENTAGE OF TUMOR INFILTRATING T LYMPHOCYTES MEASURED BY FLOW CYTOMETRY AT DIAGNOSIS PREDICTS SURVIVAL AFTER SALVAGE CHEMOTHERAPY IN RELAPSED/ REFRACTORY DIFFUSE LARGE B CELL LYMPHOMA PATIENTS.

L. Korin 1,* , M. Fuente 1 , S. Cranco 1 , A. Vitriu 1 , P. Ochoa 1 , E. Babuin 1 , O. Gelo 1 , L. Ayala 1 , P. Tempra 2 , M. Venegas 2 , A. Novoa 2 , A. Vijnovich 3 , N. Tartas 1 , M. D. R. Custidiano 1 , M. C. Foncuberta 1 , J. Sanchez Avalos 1

1 Hematology; 2 flow cytometry, Instituto Alexander Fleming; 3 Pathology, Cepacit, Buenos Aires, Argentina

Background: Tumor microenvironment (TME) in diffuse large B-cell lymphoma (DLBCL) is considered to play a major role in lymphomagenesis and tumor progression. In relapsed/refractory (R/R) patients (pts), data regarding outcomes of salvage chemotherapy according to easily acquired TME variables such as percentage (%) of infiltrating T- lymphocytes (TL) is scarce. This information may be of particular interest in this era where standard of care in the 2 nd line (2L) setting in DLBCL is being challenged.

Aims: To determine the prognostic impact of the % of TL in lymph node (LN) biopsies at diagnosis, on event free and overall survival after 1 st line (EFS 1 and OS 1 ) and 2 nd line treatment (EFS 2 and OS 2 ) in a cohort of patients with R/R DLBCL.

Methods: We identified pts with R/R DLBCL and available tissue biopsy flow cytometry (FC) data from diagnosis who received treatment at our center from 2012-2021.

FC analysis was performed with 8-color FC panels according to international Euroflow protocols. % of TL in LN biopsy by FC was compared to normal values determined by Battaglia et al (Immunology 2003) and analyzed as dichotomized variables: low vs. normal-high.

Survival analysis was estimated with Kaplan-Meier and Cox regression and the comparison between variables was performed with log-rank test.

Results: 43 pts were included in this retrospective study. At diagnosis, median age was 63 years. 79% had advanced stage disease and 69.5% poor R-IPI. All pts were treated with immunochemotherapy regimens. Complete response (CR) rate was 67.4%. Median EFS 1 was estimated at 12.3 months (m) and median OS 1 at 34.3 m, with a median follow up time of 27.8 m (range: 6-188). 44.2% had primary refractory disease or early relapse. All pts were treated with immunochemotherapy regimens as 2L. 24 pts (55.8%) achieved 2 nd CR and the remaining were chemorefractory. Only 34.9% received consolidation with ASCT, 30/43 due to progressive disease and 11/43 for ineligibility. Median EFS 2 and OS 2 were 7.6 and 14.2 m, respectively.

Median EFS 1 did not statistically differ between pts with low LN TL % vs those with normal-high values (10 vs 13.6 m respectively, p: 0.41). In contrast, a low % of TL in LN biopsy samples was associated with inferior EFS 2 (median of 5.4 vs. 47.3 m, p: 0.028, figure 1). The risk of progression or death after 2L treatment was 2.44 higher in pts with low LN TL % (CI 95%: 1.07-5.57, p: 0.03). Primary refractory pts had similar adverse outcomes to 2L therapy when stratified according to LN TL % (p: 0.8). Conversely, relapsed pts with low LN TL % had a median EFS 2 of 6 vs 77m for the normal-high subgroup (p: 0.017).

Low LN TL % was also associated with inferior OS 1 (median 22.5 vs. 120.4 months, p: 0.007). Moreover, survival after 2L chemotherapy was significantly decreased in pts with low LN TL % with a median of 7.3 vs 101.6 m in those with normal-high values, p: 0.004. The risk of death after salvage treatment was 3.31 times higher in the low LN TL % subgroup, p: 0.007.

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Summary/Conclusion: Tumor infiltrating TL in LN in DLBCL measured by FC showed prognostic impact in our cohort of R/R pts. Although timing of progression or relapse did not significantly differ between subgroups, EFS 2 and consequently OS were markedly decreased in pts with low LN TL. These results suggest that outcomes of 2L chemotherapy in this subgroup are disappointing with only a third of pts receiving consolidation with ASCT mainly due to refractory disease. Changing 2L treatment paradigms in DLBCL with the introduction of CAR-T cells may prove beneficial in this high risk subset of pts with low intratumoral immunity.

P1276: DOSE RESPONSE PROFILE OF IGM-2323, A CD20XCD3 IGM BISPECIFIC T CELL ENGAGER, IN TRANSLATIONAL MODELS SUPPORTS PHASE 2 DOSE SELECTION IN NON-HODGKIN’S LYMPHOMA

M. K. Leabman 1 , G. Hernandez 1 , C. M. Ng 1 , J. Tang 1 , D. Pandya 1 , K. C. Hart 1 , K. Li 1 , P. R. Hinton 1 , J. So 1 , I. Qazi 1 , C. Y. Cheah 2 , W. S. Kim 3 , E. Budde 4 , A. K. Gopal 5 , T. Manley 1 , C. H. Takimoto 1 , A. M. Sinclair 1 , S. F. Carroll 1 , B. A. Keyt 1 , M. F. Kotturi 1,*

1 IGM Biosciences, Inc., Mountain View, United States of America; 2 Linear Clinical Research and Sir Charles Gairdner Hospital, Perth, Australia; 3 Samsung Medical Center, Seoul, South Korea; 4 Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte; 5 University of Washington, Fred Hutchinson Cancer Research Center, Seattle Cancer Care Alliance, Seattle, United States of America

Background: IGM-2323 is an engineered high-affinity, high-avidity bispecific anti-CD20 IgM antibody T cell engager (TCE) that is currently being studied as monotherapy in a Phase 1 clinical trial for relapsed/refractory non-Hodgkin’s lymphoma (NHL) ( NCT04082936 ). IGM-2323 offers a novel treatment strategy in NHL through multiple mechanisms: the recruitment of T cells to kill CD20-expressing tumor cells through T cell dependent cellular cytotoxicity (TDCC), complement-dependent cytotoxicity (CDC), and enhanced immune modulation via IFNγ-dominant cytokine stimulation. Preliminary results from the first-in-human study of IGM-2323 show a 60% overall response rate at a titration dose level of 100 mg, and an excellent safety and tolerability profile up to 1000 mg with low cytokine release syndrome (CRS), no immune effector cell-associated neurotoxicity syndrome (ICANS) and limited neutropenia. Interestingly, lower response rates of IGM-2323 were observed at the highest dose levels of 600 mg and 1000 mg.

Aims: An optimal bispecific antibody concentration may be required for effective synapse formation between a T lymphocyte and tumor cell. For bispecific IgG TCEs, low or excess concentrations have been described to result in impaired synapse formation and consequently reduced T cell activation in vitro . The aims of this study were to (1) characterize the concentration versus response relationship of IGM-2323 in vitro and compare with bispecific IgG TCEs, and (2) build a mechanistic binding model based on preclinical in vitro data to aid in prediction of an optimal dose of IGM-2323 in the clinic.

Methods: To evaluate the dose response of IGM-2323, a concentration range that encompassed the peak and trough plasma levels up to the 1000 mg dose of IGM-2323 and equivalent concentrations of bispecific IgG TCEs were tested in preclinical in vitro assays. T cell-mediated activity was assessed using an engineered Jurkat T cell line, and in TDCC assays with primary healthy donor T cells. CDC was evaluated with human B lymphoma cell lines in the presence of normal human serum as a source of complement. Additionally, the activation status of peripheral T cells in patients that were dosed with 100 mg and 1000 mg of IGM-2323 was evaluated by flow cytometry. The in vitro data were fit to a novel mechanistic binding model of synapse formation.

Results: A bell-shaped dose response was observed for IGM-2323 and bispecific IgG TCEs in the in vitro T cell activation assay, and to a lesser extent in TDCC and CDC assays. IGM-2323 exhibited peak T cell activation over a broad range of antibody concentrations which eventually declined at the highest concentrations tested. These data were well-described by the model. Interestingly, the concentration range of IGM-2323 resulting in peak activity corresponded to the peak and trough plasma levels in patients following 100 mg dose. The high concentrations associated with reduced activity are similar to plasma levels following 1000 mg dose. A trend for higher frequency of activated CD69+ peripheral T cells was detected in patients treated with 100 mg of IGM-2323 at 24 hours post dose compared to 1000 mg dose.

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Summary/Conclusion: IGM-2323 shows a bell-shaped dose versus response relationship preclinically, with high concentrations associated with reduced T cell activity. The in vitro data support the decision to move forward with our randomized Phase 2 design testing 100 mg and 300 mg dose levels in NHL patients.

P1277: MUTATIONAL LANDSCAPE AND COPY NUMBER ALTERATIONS IN TESTICULAR LARGE B-CELL LYMPHOMA

C. López 1,2,* , A. Rivas-Delgado 1,3 , F. Nadeu 1,2 , M. Grau 1 , A. Rivero 3 , J. Boschs-Schips 4 , M. Alcoceba 5,6 , G. Tapia 7 , L. Luizaga 8 , C. Bárcena 9 , N. Kelleher 10 , M. Pablo 1,3 , O. Balague 1,2,11 , G. Frigola 11 , N. Villamor 1,2,11 , L. Magnano 1,2,12 , T. Baumann 2,13 , A. Muntañola 14 , J. M. Sancho-Cia 15 , A. M. García-Sancho 5,6 , E. Gonzalez Barca 4 , F. Climent 4 , E. Campo 1,2,11 , E. Giné 1,2,12 , A. López-Guillermo 1,2,12 , S. Beà 1,2,11

1 IDIBAPS; 2 Centro de Investigación Biomédica en red de Cáncer (CIBERONC); 3 Department of Hematology, Hospital Clínic de Barcelona; 4 Pathology, Hospital Universitari de Bellvitge, IDIBELL, Barcelona; 5 Centro de Investigación Biomédica en red de Cáncer (CIBERONC), Madrid; 6 Hospital Universitario de Salamanca, (HUS/IBSAL), and Centro de Investigación del Cáncer-IBMCC (USAL-CSIC), Salamanca; 7 Pathology, Hospital Universitari Germans Trias i Pujol, Badalona; 8 Pathology, Hospital Universitari Mutua de Terrassa, Terrassa; 9 Pathology, Hospital Universitario 12 de Octubre, Madrid; 10 Hematology, Hospital Universitari Josep Trueta, Girona; 11 Hematopathology Unit; 12 Hematology, Hospital Clínic de Barcelona, Barcelona; 13 Hematology, Hospital Universitario 12 de Octubre, Madrid; 14 Hematology, Hospital Universitari Mutua de Terrassa, Terrassa; 15 Hematology, Institut Català d’Oncologia, Hospital Germans Trias i Pujol, Barcelona, Spain

Background: Testicular large B-cell lymphoma (T-LBCL) is an infrequent and aggressive lymphoma accounting for 1-2% of all non-Hodgkin’s lymphomas. T-LBCL develops in an immune privileged site behind the blood-testis barrier and has a molecular profile similar to primary central nervous system lymphoma (PCNSL). Around 75% of T-LBCL are of ABC subtype and display frequent genetic alterations of BCR signaling ( CARD11, MYD88 and CD79B ). Nevertheless, comprehensive molecular studies in large series of T-LBCL and comparison with nodal ABC-DLBCL or PCNSL are still missing.

Aims: Herein we aimed: i) to characterize the genomic profile of T-LBCL to investigate if they are all molecularly homogeneous and to determinate if they could belong to the same entity to PCNSL; ii) to analyze the clinical features and outcome of a series of T-LBCL.

Methods: We collected 63 patients with T-LBCL diagnosed between 2002 and 2021. DNA was extracted from formalin-fixed paraffin-embedded tissue biopsies. Copy number alterations (CNA) were studied using OncoScanTM CNV FFPE arrays (Thermofisher) (n=40) and gene mutations by a B-cell malignancy-oriented targeted next generation sequencing (NGS) panel capturing 121 genes (SureSelectXT, Agilent Technologies) (n=37). The genomic status of BCL2 , BCL6 , and MYC locus was investigated by FISH (n= 50). Cell-of-origin (COO) was assessed by immunochemistry (Hans algorithm) and/or digital expression (Lymph2Cx assay, Nanostring technologies).

Results: Median age at diagnosis was 70 (30-89) years, 67% had stages I/II, and 73% low- or low-intermediate-risk IPI. All but five patients received first-line treatment with immunochemotherapy, followed by radiotherapy in 62%. Complete response rate was 80%. Progression-free survival and overall survival at 5 years were 49% (95% CI: 36-65) and 51% (95% CI: 40-69), respectively.

Eighty-four percent (45/53) showed a non-GCB phenotype by Hans algorithm and 71% ABC subtype by gene expression. Preliminary data showed mutations in MYD88 L265P (60%), PIM1 (57%), CD79B (54%), KMT2D (49%), OSBLP10 (41%), and TBL1XR1 (32%). Thirty-two percent of the cases had concomitant mutations in MYD88 L265P , CD79B , and PIM1. MYC , BCL2 , and BCL6 rearrangements were observed in 8%, 4%, and 38% cases, respectively. Two cases showed MYC and BCL6 rearrangement simultaneously. We detected CNA in all samples with a median of 13.5 alterations per case (range 1-43), including a median of 6 gains (0-24), 6 losses (0-28), and 2 copy neutral loss of heterozygosity (CN-LOH) (0-8). The most frequent CNA were trisomy 18 (33%); gains of 1q (43%), 3 (35%), 6p (28%), 9p13.2 (29%), 18q (30%), and 19q13-q14 (50%); and focal losses of 9p21.3 ( CDNK2A/B ) (73%), 6q21 ( PRDM1 ) (68%), as well as larger losses of 6q22.31-q24.3 ( TNFAIP3 ) (63%), and 17p ( TP53 ) (20%), CN-LOH of 9p region was recurrent in 35% of cases. Loss of 6p22 ( HLA ) previously described in PCNSL, was detected in 12.5% of the T-LBCL cases.

Summary/Conclusion: Although these data warrant further investigation, we identified that T-LBCL is a poor-risk type of DLBCL with mainly ABC subtype and peculiar genetic characteristics.

P1278: ACTIVATION OF THE NECROPTOSIS PATHWAY PLAYS A ROLE IN SYNERGISM WITH COMBINATION OF THE IAP ANTAGONIST, TOLINAPANT AND HYPOMETHYLATING AGENTS (HMA) IN IN VITRO MODELS OF T-CELL LYMPHOMA (TCL).

J. S. Manavalan, MD 1,* , I. Pal, PhD 1 , A. Pursley 1 , G. Ward, PhD 2 , T. Smyth, PhD 2 , M. Sims, PhD 2 , D. Feith, PhD 1 , T. P. Loughran Jr, MD 1 , O. A. O’Connor MD, PhD 1 , E. Marchi MD, PhD 1

1 Medicine/Hematology/Oncology, University of Virginia, Charlottesville, United States of America; 2 Astex Pharmaceuticals, Cambridge, United Kingdom

Background: Peripheral T-cell lymphoma (PTCL) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas that often carry a poor prognosis with standard chemotherapy. Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In an ongoing Phase 2 trial ( NCT02503423 ), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma. HMAs have also shown clinical responses in some subsets of PTCL. Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. We have also shown that decitabine and tolinapant as single agent induced necroptosis in mouse TCL cell lines with the activation of the RIPK1/RIPK3/MLKL necroptosis pathway. In addition, re-expression of RIPK3 in CT26 cells by CRISPR increased lytic cell death on treatment with tolinapant indicating its essential role in the necroptosis pathway.

Aims: We explored the sensitivity of a range of TCL lines to tolinapant, established the synergy coefficient and interrogated the role of necroptosis in the mechanism of synergy between tolinapant and drugs active against PTCL, the HMAs; azacytidine and decitabine

Methods: A panel of 10 human TCL lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10 ng/ml of TNFa. For combination studies, each drug was tested at the IC 10 , IC 20 and IC 40 concentrations, in the presence or absence of TNFa. Synergy coefficient was calculated using the Excess over Bliss model with SynergyFinder. Additionally, the on-target effects of the drugs were measured by analyzing levels of the IAPs, DNMTs and key apoptosis and necroptosis markers, such as PARP and RIPK3 by Western blotting.

Results: TCL Lines demonstrate varying sensitivities to tolinapant with the most sensitive cell line, ALK+ ALCL SUP-M2, having an IC 50 as low as 20 nM ± 1 nM while a resistant CTCL cell line HH had an IC 50 of over 20 µM. In combination experiments using both cell lines, tolinapant plus azacytidine or decitabine displayed a very high degree of synergism; Average synergy score of 50.9 and 49.8 with azacytidine and 22.6 and 31.3 with decitabine for HH and SUP-M2, respectively. Of note, a high degree of synergism was also seen with concentrations 10 fold lower of azacytidine or 100 fold lower of decitabine, added daily (Figure 1). The combination of tolinapant and the HMAs led to a decrease in the levels of cIAP1 and DNMT3a in TCL lines, demonstrating on-target activity of tolinapant and the HMAs, respectively. In addition, both azacytidine and decitabine increased the levels of RIPK3 up to 3-4 fold and 6-8 fold respectively, in a concentration dependent manner at 72Hrs by western blot analysis, indicating activation of the necroptosis pathway.

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Summary/Conclusion: Tolinapant has demonstrated varying cytotoxic effects against a range of TCL lines as a single agent. Tolinapant, in combination with the HMAs, azacytidine and decitabine, displays a very high degree of synergism, in vitro, even at very low concentrations of both HMA drugs. The HMAs were able to activate the necroptosis pathway, as shown by the increase in RIPK3. Thus, activation of the necroptosis pathway, is a possible mechanism for the high degree of synergism displayed when HMAs are used in combination with tolinapant in the TCL lines in vitro . These data provide the rationale to initiate clinical trials with the combination of tolinapant and decitabine.

P1279: INHIBITION OF PROTEIN KINASE CK2 AND BCL-2: A NOVEL THERAPEUTIC STRATEGY TO ANTAGONIZE MANTLE CELL LYMPHOMA CELL GROWTH

S. Manni 1,2,* , Z. Spinello 1,2 , M. Pesavento 1,2 , A. Fregnani 1,2 , L. Saggin 1,2 , L. Quotti Tubi 1,2 , G. Scapinello 1 , F. Vianello 1,2 , R. Zambello 1,2 , C. Gurrieri 1 , G. Semenzato 1,2 , L. Trentin 1,2 , F. Piazza 1,2

1 Department of Medicine, University of Padova; 2 Veneto Institute of Molecular Medicine, VIMM, Padova, Italy

Background: Mantle cell lymphoma (MCL) is a B-cell tumor which often relapses. BCR inhibitors (Ibrutinib, Acalabrutinib) and antiapoptotic BCL2-family members blockers BH3-mimetics (Venetoclax, ABT-199) are effective drugs to fight MCL. However, the disease remains incurable, due to therapy resistance, even to the promising Venetoclax and Ibrutinib combination. Therefore, there is a profound need to explore novel useful therapeutic targets. CK2 is a S/T kinase overexpressed in several solid and blood tumors. We demonstrated that CK2, operating through a “non-oncogene addiction” mechanism promotes tumor cell survival, and counteracts apoptosis, by activating pro-survival signaling cascades, such as NF-κB, STAT3 and AKT. CK2 could regulate also BCL2 family members. The CK2 chemical inhibitor CX-4945 (Silmitasertib, Sil) is already under scrutiny in clinical trials in relapsed multiple myeloma, solid tumors and COVID-19.

Aims: In this work, we tested the effect of CK2 chemical inhibition or knock down on Venetoclax (Ven)-induced cytotoxicity in MCL pre-clinical models to effectively reduce MCL cell growth and clonal expansion.

Methods: CK2 expression and BCR/BCL2 related signaling components were analyzed in MCL cells and control cells by Western blotting. CK2 and BCL2 inhibition was obtained with Sil and Ven, respectively and with CK2 gene silencing through the generation of anti-CK2 shRNA IPTG-inducible MCL cell clones. Survival, apoptosis, mitochondrial membrane depolarization and proliferation were investigated by FACS analysis of AnnexinV/PI and JC-10 staining. The synergic action of Ven and Sil was analyzed by the Chou-Talalay combination index (CI) method. CK2 knock down in vivo was obtained in xenograft NOD-SCID mouse models

Results: CK2 inactivation (with Sil or CK2 silencing) determined a reduction in the activating phosphorylation of S529 p65/RelA and S473 and S129 AKT, important survival cascades for MCL. Sil or CK2 silencing caused BCL2 and related MCL1 protein reduction, causing cell death. Importantly, we confirmed these results also in an in vivo xenograft mouse model of CK2 knockdown in MCL.

Sil +Ven combination increased MCL cell apoptosis, as judged by the augmented frequency of Annexin V positive cells and expression of cleaved PARP protein, and JC-10 mitochondrial membrane depolarization, with respect to the single treatments. Captivatingly, Sil or CK2 gene silencing led to a substantial reduction of the Ven-induced increase of MCL-1, potentially counteracting a deleterious Ven-induced drawback. Analysis of cell cycle distribution confirmed an increased frequency of apoptotic cells in the sub G1 phase in CK2-silenced cells and a modulation of the other phases of the cell cycle. Remarkably, the calculated CI less than 1 suggested a strong synergic cell-killing effect between Sil and Ven, on all the cell lines tested, including those less sensitive or resistant to Ven

Summary/Conclusion: We demonstrated that the simultaneous inhibition/knock down of CK2 and BCL2 synergistically cooperates in inducing apoptosis and cell cycle arrest of MCL malignant B-lymphocytes and has the potential of reducing MCL clonal growth, also counterbalancing mechanism of resistance that may arise with Ven. Therefore, CK2 is a rational therapeutic target for the treatment of MCL to be tested in combination with Ibrutinib or Ven.

P1280: TARGETED INHIBITION OF DHODH IS SYNERGISTIC WITH BCL2 BLOCKADE IN LYMPHOMA WITH CONCURRENT MYC AND BCL2 REARRANGEMENT

W. Mo 1 , L. Liu 1 , M. Chen 1 , Y. Qu 1 , P. Wang 1 , Y. Liang 1 , X. Yan 1,*

1 Department of Hematology, The First Affiliated Hospital of China Medical University, Shenyang, China

Background: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, can be cured in approximately two-thirds of patients after initial therapy. The remaining one-third of patients have inferior survival outcomes due to relapse or refractoriness despite salvage chemotherapy with or without stem cell transplantation. The dual dysregulation of MYC and BCL2 is one of the most important pathogenic mechanisms. Thus, new therapies are needed to improve the prognosis of patients, and combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine and has been shown to be a potential therapeutic target for multiple diseases. Recent data have demonstrated that DHODH inhibitors can target MYC in some tumors.

Aims: To develop and assess new therapeutics, we investigated the effects of the DHODH inhibitor brequinar in DHL lymphoma cell lines and the synthetic effects of brequinar and venetoclax.

Methods: We first investigated the activity of DHODH inhibition in genetically defined double-hit DLBCL(DHL) cell lines. We then investigated whether c-MYC could be repressed by brequinar in DHL cells. Then we tried to uncover the synergistic mechanism of brequinar and venetoclax. To understand the genome-wide effects and target genes of brequinar and the combination with venetoclax, we performed an RNA-seq of SU-DHL4 cells treated with brequinar, venetoclax, or the combination of both agents versus DMSO-treated cells. At last, we confirmed the synergistic effect of DHODH inhibition and BCL2 blockade in vivo.

Results: DHODH inhibition could repress DLBCL cell lines with MYC and BCL2 abnormalities. The protein level of c-MYC was upregulated 6 hours after brequinar treatment and then downregulated gradually with prolonged treatment time. The gene levels of c-MYC decreased over time. The synergistic mechanism of brequinar and venetoclax is the complementarity of different pathways. RNA-seq of SU-DHL4 cells treated with different agents further implied the possible synergistic mechanism of the two agents. Finally, in vivo study confirmed targeted inhibition of DHODH is synergistic with BCL2 blockade in lymphoma with concurrent MYC and BCL2 rearrangement

Summary/Conclusion: Our study demonstrates that combined blockade of DHODH and BCL2 could synergistically inhibit DLBCL with concurrent MYC and BCL2 abnormalities through multiple pathways. Thus, it can be reasonably expected that a clinical-grade DHODH inhibitor combined with venetoclax might improve outcomes for patients with DHL or double-expression lymphoma and other cancers with high expression of MYC and BCL2 or MYC and MCL-1. Our findings provide new insights into the molecular basis of this effect and offer opportunities to design new targeted treatments for lymphoma.

P1281: LIQUID BIOPSY PROVIDES COMPLEMENTARY INFORMATION TO TISSUE BIOPSIES FOR THE MOLECULAR CLASSIFICATION OF DLBCL PATIENTS

R. Moia 1,* , C. Favini 1 , D. Talotta 1 , S. Sagiraju 1 , R. Bruna 1 , R. Dondolin 1 , I. Della Starza 2 , R. Soscia 2 , A. Andorno 3 , F. Mercalli 3 , C. Deambrogi 1 , S. Rasi 1 , L. Petrucci 2 , T. Bellissimo 2 , R. Luciano Boldorini 3 , A. Di Rocco 2 , I. Del Giudice 2 , M. Martelli 2 , R. Foà 2 , G. Gaidano 1

1 Division of Hematology, Department of Translational Medicine, Università del Piemonte Orientale, Novara; 2 Division of Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome; 3 Division of Pathology, Department of Health Sciences, Università del Piemonte Orientale, Novara, Italy

Background: Diffuse large B-cell lymphoma (DLBCL) displays a high degree of molecular heterogeneity and may be classified into molecular clusters harboring specific lesions and therapeutic vulnerabilities. To date, DLBCL molecular clusters are identified on the tissue biopsy and do not consider mutations deriving from different anatomical compartments.

Aims: The aim of this study is to evaluate the potential contribution of liquid biopsy to the identification of DLBCL molecular clusters.

Methods: A multicenter cohort of newly diagnosed DLBCL provided with circulating tumor DNA (ctDNA) from the plasma and with genomic DNA (gDNA) from the lymph node (LN) biopsy represented the basis of this study. The LyV4.0 CAPP-Seq assay that comprised a panel of 59 genes relevant to B-cell malignancies was used. The LymphGen tool v1.0 was utilized for cluster analysis.

Results: The cohort included 77 newly diagnosed DLBCL patients treated with R-CHOP. After a median follow up of 27.1 months, the 36-month PFS and OS were 62.0% and 79.5%. Mutation analysis identified at least one somatic non-synonymous mutation in 92.2% (71/77) of patients in the LN biopsy and in 87.0% (67/77) in the ctDNA. Higher levels of ctDNA (≥2.5 log hGE/mL) were associated with a significantly worse PFS ( p =0.025) and OS ( p =0.004). Mutation analysis of different compartments, i.e. lymph node and plasma, allowed to identify mutations with potential clinical impact, that otherwise would have been missed by analyzing only one compartment. In particular, GRHPR ( p =0.035) and SGK1 ( p =0.039) mutations identified only on the liquid biopsy, and MYC mutations identified only on the LN biopsy ( p =0.021) were associated with a shorter PFS. Based on the mutational landscape identified in each compartment, DLBCL were analyzed with the LymphGen tool, that allows the cluster assignment in approximately 40-50% of DLBCL. In our cohort, 46.5% (33/71) of cases were assigned to a specific molecular cluster on the LN biopsy and 40.3% (27/67) on the liquid biopsy. Interestingly, one case was classified as EZB on the LNF biopsy and as MCD on the ctDNA (Fig. 1A). In all other cases, if not unclassified, the cluster identified on the ctDNA reflected the cluster identified on the LN biopsy. The combination of mutational data from LN and ctDNA improved DLBCL assignment to a specific cluster, thus classifying 48.7% (36/74) of cases. Two patients were assigned to two different clusters and defined as genetically composite. From a clinical perspective, by combining mutational data from the LN and from ctDNA, patients belonging to the BN2 and ST2 clusters showed a favorable outcome with a 36-month PFS of 100% compared to 62.3% for patients belonging to the MCD or EZB clusters (p=0.040) (Fig. 1B-C).

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Summary/Conclusion: The combination of mutational data from the LN biopsy and from the liquid biopsy provides complementary information for the molecular classification and prognostic stratification of newly diagnosed DLBCL patients.

P1282: DISEASE-SPECIFIC U1 SPLICEOSOMAL RNA MUTATIONS IN MATURE B-CELL NEOPLASMS

F. Nadeu 1,2,* , S. Shuai 3 , G. Clot 1,2 , L. K. Hilton 4 , A. Diaz-Navarro 2,5 , S. Martín 1,2 , R. Royo 6 , T. Baumann 7 , M. Kulis 1,2 , I. López-Oreja 1,2,7 , M. Cossio 1 , J. Lu 8 , V. Ljungström 9 , E. Young 10 , K. Plevova 11,12 , B. A. Knisbacher 13 , Z. Lin 13,14 , C. K. Hahn 13,15 , P. Bousquets 5 , M. Alcoceba 2,16 , M. González 2,16 , E. Colado 17 , M. Aymerich 1,2,7 , M. J. Terol 18 , A. Rivas-Delgado 1,7 , A. Enjuanes 1 , S. Ruiz-Gaspà 1 , T. Chatzikonstantinou 19,20 , D. Hägerstrand 10 , C. Jylhä 10,21 , A. Skaftason 10 , L. Mansouri 10 , K. Stranska 11,12 , M. Doubek 11,12 , E. J. van Gastel-Mol 22 , Z. Davis 23 , R. Walewska 24 , L. Scarfò 25 , L. Trentin 26 , A. Visentin 26 , S. A. Parikh 27 , K. G. Rabe 28 , R. Moia 29 , M. Armand 30 , D. Rossi 31 , F. Davi 30 , G. Gaidano 29 , N. E. Kay 27 , T. Shanafelt 32 , P. Ghia 25 , D. Oscier 24 , A. W. Langerak 22 , S. Beà 1,2,7,33 , A. López-Guillermo 1,2,7,33 , D. Neuberg 34 , C. J. Wu 13,15,35,36 , G. Getz 13,35,37,38 , S. Pospisilova 11,12 , K. Stamatopoulos 10,19,20 , R. Rosenquist 10,21 , W. Huber 8 , T. Zenz 39 , D. Colomer 1,2,7,33 , J. I. Martín-Subero 1,2,33,40 , J. Delgado 1,2,7,33 , R. D. Morin 4,41 , L. D. Stein 42,43 , X. S. Puente 2,5 , E. Campo 1,2,7,33

1 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona; 2 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain; 3 Department of Human Cell Biology and Genetics, School of Medicine, Southern University of Science and Technology, Shenzhen, China; 4 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, Canada; 5 Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo; 6 Barcelona Supercomputing Center (BSC); 7 Hospital Clínic of Barcelona, Barcelona, Spain; 8 European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 9 Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala; 10 Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 11 University Hospital Brno & Medical Faculty, Masaryk University; 12 Central European Institute of Technology (CEITEC) - Masaryk University, Brno, Czechia; 13 The Broad Institute of MIT and Harvard; 14 Harvard University, Cambridge; 15 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States of America; 16 Biología Molecular e Histocompatibilidad, IBSAL-Hospital Universitario, Centro de Investigación del Cáncer-IBMCC (USAL-CSIC), Salamanca, Spain; 17 Servicio de Hematología y Hemoterapia, Hospital Universitario Central de Asturias, Oviedo, Sweden; 18 Servicio de Hematología, Hospital Clínico Universitario, INCLIVA, Universidad de Valencia, Valencia, Spain; 19 Institute of Applied Biosciences, Centre for Research and Technology Hellas; 20 Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece; 21 Clinical Genetics, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden; 22 Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, Netherlands; 23 Department of Molecular Pathology; 24 Department of Haematology, University Hospitals Dorset, Bournemouth, United Kingdom; 25 Università Vita-Salute San Raffaele, Milan - Strategic Research Program on CLL, Division of Experimental Oncology, IRCCS San Raffaele Hospital, Milan; 26 Department of Medicine, Hematology and Clinical Immunology Section, University of Padua, Padua, Italy; 27 Division of Hematology; 28 Department of Quantitative Health Sciences, Division of Clinical Trials and Biostatistics, Mayo Clinic, Rochester, United States of America; 29 Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy; 30 Department of Laboratory Medicine and Pathology, Hospital Pitie-Salpetriere, Sorbonne University, Paris, France; 31 Department of Hematology, Oncology Institute of Southern Switzerland and Institute of Oncology Research, Bellinzona, Switzerland; 32 Stanford School of Medicine, Stanford, United States of America; 33 Universitat de Barcelona, Barcelona, Spain; 34 Department of Data Science, Dana-Farber Cancer Institute; 35 Harvard Medical School; 36 Department of Medicine, Brigham and Women’s Hospital; 37 Center for Cancer Research; 38 Department of Pathology, Massachusetts General Hospital, Boston, United States of America; 39 Department of Medical Oncology and Hematology, University Hospital and University of Zürich, Zürich, Switzerland; 40 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; 41 Canada’s Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver; 42 Department of Molecular Genetics, University of Toronto; 43 Computational Biology Program, Ontario Institute for Cancer Research, Toronto, Canada

Background: Recurrent mutations in the third base of U1 spliceosomal RNA responsible for marked splicing and expression abnormalities have been described in chronic lymphocytic leukemia (CLL) and some solid tumors (Shuai et al. Nature, 2019; Suzuki et al. Nature, 2019). However, the clinical significance of these mutations in large CLL cohorts and their presence in other B-cell neoplasms is unknown.

Aims: To provide a comprehensive map of U1 mutations across a wide spectrum of mature B-cell neoplasms and to assess their transcriptional and clinical implications.

Methods: We first performed an unbiased characterization of U1 mutations in 762 mature B-cell neoplasms analyzed by whole-genome sequencing (WGS) and complemented with RNA-seq data. This WGS cohort comprised 399 CLL, 155 diffuse large B-cell lymphomas (DLBCL), 110 Burkitt lymphomas (BL), 61 mantle cell lymphomas (MCL), and 37 follicular lymphomas (FL). WGS data were analyzed using a bioinformatic pipeline designed to call mutations in any of the 11 canonical U1 genes found in the human genome. Second, we expanded the characterization of recurrent U1 mutations in 1,670 CLL patients from two independent cohorts.

Results: Our WGS analyses uncovered that the majority of U1 mutations were present between positions 3 and 10 of the gene, the region responsible for 5’ splice site recognition via base-pairing. In line with this, 10.5% CLL, 17.4% DLBCL, and 15.5% BL cases carried mutations in this region, which contrast with only 3.3% MCL and 5.4% FL cases. We observed substantial differences in the mutated sites of CLL, DLBCL, and BL. In CLL, the most frequently mutated site was the position 3 with 7.3% of the whole WGS cohort carrying the previously identified A>C mutation (g.3A>C). In addition, we also identified a novel C>T mutation at position 9 (g.9C>T) in 2% of CLL cases, which was associated with splicing abnormalities both in primary cases and transduced CLL cell lines. In DLBCL, the most frequent mutation was a novel C>T mutation at position 4 (g.4C>T) found in 8.4% of cases. This g.4C>T mutation showed a non-significant enrichment in germinal center B-cell like (GCB) (12.8%) compared to activated B-cell (3.8%) DLBCL ( p =0.12). In GCB-DLBCL, this mutation was associated with 1,902 differentially spliced introns and 397 differentially expressed genes. In BL, the most frequent mutation was an A>G mutation at position 7 (g.7A>G), which was found in 10.9% of cases. Of note, this mutation was detected in 29.5% EBV-negative BL while only in 1.4% EBV-positive BL ( p <0.001). The g.7A>G mutation was associated with 6,970 introns differentially spliced in EBV-negative BL.

In the extended CLL cohorts, the g.3A>C was enriched in unmutated IGHV (7.4%) and naïve-like (10.9%) subtypes of the disease, whereas the g.9C>T was more often detected in mutated IGHV/memory-like CLL (2%). These two mutations did not significantly co-occur with several CLL driver alterations of prognostic relevance and retained independent prognostic value for time to first treatment in multivariable models.

Summary/Conclusion: This study expands our understanding of the biologic and clinical consequences of U1 mutations in CLL and reveals novel U1 mutations in DLBCL and BL. U1 mutations and their downstream effects were specific of different entities and subtypes with distinct mutations shaping the transcriptome of CLL (g.3A>C and g.9C>T), DLBCL (g.4C>T) and BL (g.7A>G). Based on its downstream effects, mutation prevalence among distinct B-cell neoplasms, and prognostic value in CLL, U1 represents a new pan-B-cell malignancy driver gene.

P1283: CIRCULATING IMMUNE CELLS IN CLASSICAL HODGKIN LYMPHOMA IN RELATION TO TUMOR BURDEN AND TREATMENT

T. A. Mulder 1,* , M. L. Andersson 1,2 , K. Heimersson 1 , I. Xagoraris 1,3 , B. E. Wahlin 2,4 , G. Rassidakis 1,3,5 , L. Hansson 1,2 , M. Palma 1,2

1 Oncology-pathology, Karolinska Institutet; 2 Hematology; 3 Clinical Pathology and Cancer Diagnostics, Karolinska University Hospital; 4 Medicine (Huddinge), Karolinska Institutet, Stockholm, Sweden; 5 Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Classical Hodgkin lymphoma (cHL) has a peculiar histology with pathognomonic Hodgkin and Reed-Sternberg (HRS) cells representing only a small fraction of the tumor mass. HRS orchestrate a lymphocyte-dominated tumor microenvironment (TME) that supports their survival and growth, suggesting that even circulating immune cells might be affected.

Aims: To comprehensively characterize circulating lymphocytes in cHL patients (pts) in relation to their frequency in the tumor tissue, the tumor load and the effect of standard first-line treatment.

Methods: Peripheral blood (PB) samples were obtained from 48 consecutive newly diagnosed pts before start of treatment (BL), right at the end of treatment (EoT) and at 6 months´ follow-up after EoT (FU). Twenty-eight pts had limited-stage (I-IIA) (LIM) and 20 had advanced-stage (IIB-IV) (ADV) disease. All the pts in the FU analysis were in complete remission. Twenty healthy individuals were included as controls. Cells from PB and lymph node (LN) biopsies were freshly analyzed by flow cytometry. Plasma concentrations of CCL17/TARC were measured by Enzyme-Linked ImmunoSorbent Assay.

Results: At BL, the numbers of B cells were lower in both LIM and ADV cHL compared to controls (median 0.075 (p<0.0001) and 0.067 (p<0.0001) vs. 0.17 x10 9 cells/L, respectively), while T cells were normal. Higher frequencies of intracellular Ki67 expression were observed in LIM and ADV cHL pts compared to controls, among CD4 + (median 2.77 (p<0.0001) and 3.36, (p=0.0004) vs. 0.92 %, respectively) and CD8 + T cells (median 1.85 (p=0.0009) and 3.66 (p<0.0001) vs. 0.91 %, respectively). PD-1 + CD4 + T cells were higher in LIM cHL than in controls (median 31.75 vs. 23.05 %, respectively, p=0.0030), while PD-1 + CD8 + T cells were higher in both LIM and ADV cHL (median 32.05 (p=0.0041) and 32.20 (p=0.0135) vs. 21.15 %, respectively). CD8 + naive T cells (T N ) cells were lower in LIM and ADV cHL than in controls (median 27.40 (p=0.0005) and 20.15 (p=0.0003) vs. 53.25 %, respectively).

Advanced-stage pts had less NK cells than controls (median 0.14 vs. 0.20 x10 9 cells/L, respectively, p=0.0135). The plasma concentration of CCL17/TARC was elevated in both LIM and ADV cHL compared to controls (median 5256 (p=0.0002) and 9165 (p<0.0001) vs. 61.03 pg/mL, respectively) (Figure 1).

The frequencies of T and B cells positively correlated between the PB and LN compartments (r=0.3855, p=0.0243, and r=0.5203, p=0.0016, respectively).

Patients with a high inflammatory status (i.e. ESR ≥50) had higher percentages of PD-1 + CD4 + T N cells and lower percentages of CD4 + effector memory (T EM ) cells, NKG2D + CD56 dim NK cells and total NK cells in their PB. Pts with a high tumor burden (i.e. bulky tumor and/or >2 nodal sites involved and/or stage IV) had higher plasma levels of CCL17/TARC, higher percentages of CTLA-4 + CD4 + T cells, activated regulatory T (Treg) cells, Ki67 + CD4 + T cells and Th2 cells in their PB.

T-cell exhaustion and NK-cell depletion were reversed by standard first-line treatment and CCL17/TARC concentrations also dropped back to control levels.

Pts who received radiotherapy (RT) involving the mediastinum had lower T-cell numbers, specifically Th2 cells, CD4 + T N cells and Tregs, at EoT and FU than those who were not subjected to RT.

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Summary/Conclusion: Immunosuppression of lymphocytes in the TME of cHL might be reflected in the PB. Most immunological changes are reverted after successful standard primary treatment. Patients who receive RT involving the mediastinum have a prolonged T-cell deficit and might benefit from longer Pneumocystis pneumonia prophylaxis.

P1284: LIQUID BIOPSY BY ULTRA-DEEP SEQUENCING PLUS PET-TC MONITORING IN REAL-LIFE FOLLICULAR LYMPHOMA PATIENTS

A. Jiménez-Ubieto 1,1,* , M. Poza Santaella 1 , A. Martin 2 , Y. Heredia 2 , S. Dorado 2 , G. Figaredo 3 , L. Rufian 1 , A. Rodriguez 1 , P. Sarandeses 4 , T. Baumann 1 , C. Grande 1 , C. Bárcena 5 , J. Martinez-Lopez 1 , J.-M. Rosa 2 , I. Rapado 1 , R. Ayala 1 , M. Gallardo 6 , R. Sánchez 1 , J. Carrillo 2 , M. Calbacho 1 , L. Parrilla 3 , A. Juarez 2 , M. Mollejo 3 , L. F. Casado 3 , C. Wang 7 , M. Rodriguez 2 , P. De Toledo 8 , S. Barrio 2

1 Hematology, Hospital 12 Octubre; 2 Altum Sequencing Co,., Madrid; 3 Hematology, Hospital Universitario de Toledo, Toledo; 4 Department of Nuclear Medicine; 5 Pathology, Hospital 12 Octubre; 6 Unidad de Investigación Clínica de Tumores Hematológicos H12O-CNIO,., Madrid, Spain; 7 Hosea Precision Medical Technology Co, Weihai, Shandong, China; 8 Computational Science Department, Carlos III University, Madrid, Spain

Background: Follicular lymphoma (FL) is considered an indolent disorder with a relatively favorable course. With modern day treatments, long remissions are often achieved both in front-line and relapsed setting. However, a subset of patients has a more aggressive course and a poorer outcome. Although understanding prognosis is important, ultimately the goal in FL should be to guide therapy. Studies of risk-adapted therapy based on MRD remains investigational.

Aims: To prospectively evaluate the predictive value of a liquid Biopsy MRD (LiqBio-MRD) SNV-NGS method in combination with the Deauville criteria using the 5-point scale (D5PS) in the evaluation of response in a cohort of 72 FL patients that require treatment from 2018-2021.

Methods: 57 patients received first-line therapy (39 R-CHOP, 10 R-B, 5 Rituximab in monotherapy and 3 radiotherapy) and 15 patients salvage treatment. Genomic DNA from formalin-fixed paraffin embedded (FFPE) lymph node biopsies and/or cell-free DNA (cfDNA) was obtained before start treatment. These samples were sequenced with short length Ampliseq Custom Panel targeting 56 important genes in B-cell dyscrasias. Somatic mutations were selected as disease biomarkers, and cfDNA samples were analysed at mid-induction (n = 26) and at the end of treatment (n= 41). PET/CT examinations were usually performed after 4 cycles (n = 44) and at the end of treatment (n = 63). PET positive was considered when D5PS was 4 or 5. Statistical analysis was performed with SPSS® using Cox regression, Kaplan-Meier survival curves and log-rank test.

Results: Median age at diagnosis was 66 years (35 – 90) and 58% were female. At diagnosis only the lymphocyte to monocyte ratio < 2.5 had prognostic value (p=0.04).

Median PFS was 13 months for PET EOT (+) cases and 10 months for MRD EOT (+) ones. 71% of patients with PET EOT (+) presented POD24, in contrast to only 9% of patients with PET EOT (-), p < 0.001. Regarding MRD, 73% of patients with MRD EOT (+) presented POD24 vs 12% of patients with MRD EOT (-), (p < 0,001; HR 8.30, 95%CI 1.70–40.38). The sensitivity (SE) and specificity (SP) of both tests to predict POD24 were: PET EOT (SE77%, SP68%) and MRD (SE77%, SP86%).

40 patients had MRD EOT and PET EOT available and results were concordant in 80% of cases. In a stratified analysis combining the information on MRD and PET, the 2-yr PFS were; 96% for MRD/PET -/-, 71% for MRD/PET -/+, and 58% MRD/PET +/-. Strikingly, patients that were positive by both tests presented a 2-yr PFS of only 10%. Using only concordant results, SE91% and SP96% was achieved.

Similar results in PFS and in predicting POD24 were obtained when including only patients with first line therapy. Figure 1.

When interim results were analysed, also PET INT (+) and MRD INT (+) were associated with higher risk of relapse (p<0,001 and p=0.009, respectively). The negative predictive value of PET INT results was 96%. Concordance between both tests was clearly inferior (47%), although no patient with PET INT (-) and MRD INT (-) results relapsed.

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Summary/Conclusion: Our results demonstrate for the first time that NGS based MRD quantification is feasible in Liquid Biopsies from FL. The achievement of negative MRD after treatment, but also in an interim analysis, enhances prognostic information on patient outcome. PET/CT and cfDNA are complementary increasing SE and SP to predict progression and POD 24 with a high accuracy. Although, more patients are necessary to draw meaningful conclusion, the combination of these techniques could aid to the develop clinical trials with a response-adapted precision therapy.

P1285: IN VIVO IMPACT OF ABERRANT OVEREXPRESSION OF C-REL NF-ΚB SUBUNIT IN GERMINAL CENTER B-LYMPHOCYTES: A TUMOR ONCOGENIC EVENT IN GCB-DLBCLS

L. Prévaud 1,* , C. Vincent-Fabert 1 , J. Feuillard 1 , N. Faumont 1

1 Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR7276/INSERM1262), University of Limoges, Limoges, France

Background: Diffuse large B-cell lymphomas (DLBCLs) are the most NF-κB-related aggressive non-Hodgkin’s lymphomas. DLBCLs are clinically, molecularly and genetically heterogeneous, suggesting different oncogenic mechanisms. Gene-expression profiling studies led to the proposal of two main cell-of-origin molecular subtypes known as germinal center B-cell-like (GCB) DLBCLs, and activated B-cell like (ABC) DLBCLs. With the Lymphoma Study Association LYSA, we previously published that strong c-Rel DNA-binding activity was mostly found in GCBs and was associated with increased REL mRNA expression. These cases with both overexpression of REL mRNA and c-Rel related gene expression profile were correlated with various recurrent GCB-DLBCLs genetic events, including REL gains, BCL2 translocation, MEF2B , EZH2 , CREBBP , and TNFRSF14 mutations and with the EZB GCB genetic subtype.

Aims: We propose that c-Rel is a major oncogene of GCB-DLBCLs. We speculate that c-Rel confers a selective advantage in the germinal center (GC)-specific environment. We wish to study the consequences of aberrant overexpression and activation of c-Rel in vivo that could promote tumorigenesis in DLBCL precursor cells by disrupting the normal dynamics of B-cell development in the GC.

Methods: We have generated the fl_STOP-REL mouse model: the murine REL transgene preceded by a STOP-Neomycin cassette flanked of loxP sites was introduced in Rosa26 locus on chromosome 6. The REL transgene is followed by an IRES to allow YFP marker concomitant expression. To reach GC B-cell specific expression, we used AID_Cre-ert2 mice: the Cre-ert2 transgene was at the AICDA gene locus on chromosome 6. On the same chromosome, the tdTomato marker preceded by a STOP cassette flanked by loxP sites was introduced at the Rosa26 locus. A pool of REL overexpressing GC B-cell was generated after tamoxifen administration and T-cell-dependent antigen stimulation with sheep red blood cells (SRBC). The fate of these YFP and tdTomato positive GC B-cells was followed over-time until 8 months. The first four months, a SRBC immune booster was performed to maintain the GCs.

Results: As expected, our model fl_STOP-REL | AID_Cre-ert2 ( i.e. REL mice) allowed for in vivo competition between c-Rel overexpressing (tdTomato+/YFP+) and normal (tdTomato+/YFP-) GC B-cells. In blood, the percentage of YFP positive B-lymphocytes among the tdTomato positive cells was increased over-time: 30% at 14 days, 65% at 2 months and about 80% at 8 months. From blood, spleen and lymph nodes, phenotyping analysis by flow cytometry of REL/YFP positive B-cells indicated an activated profile compared to normal GC-derived B-cells: CD19+, IgM+, IgD-, CD21+, CD80+, and B220low. At 8 months after generation of GC B-cells pool, approximatively 3-5% of B-cells were tdTomato and YFP positives in spleen of REL mice, whereas tdTomato positives cells in control mice were rare hardly detectable (less than 0.8%). Altogether, our result indicated that c-Rel reprograms B-cell to be activated and to better survive in vivo for very long time.

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Summary/Conclusion: We bring strong experimental arguments showing that increased c-Rel expression in GC B-cell favors their survival for several months, and would thus be a primary event. We suspect that additional oncogenic events are probably needed for tumor clonal expansion such as BCL2 , EZH2 , CREBBP , TNFRSF14 and/or c-Myc dysregulation.

P1286: IMPAIRED ACTIVITY OF NATURAL KILLER CELLS IN DIFFUSE LARGE B-CELL LYMPHOMA MOST LIKELY ORIGINATES FROM LYMPHOMA-INDUCED MODULATION OF THEIR SURFACE RECEPTOR EXPRESSION

S. Ringelstein-Harlev 1,2,* , T. Azoulay 1 , I. Slouzkey 1 , M. Karmona 1 , M. Filatov 1 , G. Sarig 1

1 Rambam Health Care Campus; 2 The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel

Background: Natural killer (NK) cells play a role in cancer cell elimination; however, modifications in their activity induced by malignant cells can lead to solid and hematological cancer immune evasion. Diffuse large B-cell lymphoma (DLBCL) extensively infiltrates the lymphoid tissue, where environmental stimuli mediate the development of NK cell subpopulations.

Aims: The aim of this study was to explore phenotypic and cytotoxic activity of DLBCL patient NK cells that could support lymphoma immune escape.

Methods: Given that peripheral blood (PB) NK cells reflect lymph node subpopulations, CD56 + PB NK cells, derived from 26 DLBCL patients at staging, and from 13 healthy age-matched healthy controls (HC), were collected and analyzed for the surface expression of NK-cell specific C-type lectin receptors (CLRs), killer cell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs) and other co-stimulatory receptors, using FACS. To assess NK cell activation, degranulation (evaluated by CD107a staining) and intracellular IFNg and TNFα production were examined following co-culture with the K562 malignant cell line. The percentage of CFSE stained target K562 cells that underwent necrosis/apoptosis upon co-incubation with NK cells was used to measure cytolytic activity of the latter cells. All statistical analyses were conducted with the Mann Whitney U test.

Results: The percentage of NK cells among lymphocytes was comparable in patients and HC. As all the patient mature NK subsets were found to be CD56 dim , all further experiments were conducted on this fraction only. The mean expression of the inhibitory CLR NKG2A was significantly higher in DLBCL than in HC samples (42%±5.1 vs. 27%±2.3; p<0.001). Conversely, the levels of activating CLRs NKG2D and CD161 were significantly lower in patients (15%±8 vs. 40.2%±4; p<0.01 and 47.8±7 vs. 82.7±5, respectively for both antigens; p=0.006). Patient NK cells demonstrated decreased levels of KIRs (CD158a and CD158b) compared to HC (16.2±5 vs 33.8±2.1; p<0.01 and 17.6±6 vs 32.4±3.1; p<0.01, respectively), despite being exclusively CD56 dim , and as such expected to highly express KIRs.

Degranulation was found to be lower in patient than in HC cells (CD107a of 2.8% vs. 14.6%; p<0.01), as was intracellular cytokine production [2.4% vs. 6.9% for TNFα (p=0.03) and 3.2% vs. 11.3% for IFNγ (p=0.04)]. This difference maintained upon IL-2 supplementation, which enhanced NK cell responses in both patient and HC cells. Non-receptor dependent activation with PMA/inomycin led to a marked and comparable increase in degranulation and cytokine production, in patient and HC cells. Dose-dependent lysis of target K562 cells was significantly lower with patient NK cells. At the highest effector:target cell ratio of 5:1, patient cells induced lysis of only 16.3% of target cells, whereas 24.2% of these cells were lysed following exposure to HC NK cells (p=0.001). IL-2 supplementation increased cytotoxicity, that still remained inferior in patient cells (20.7% vs. 28.1% target cell lysis; p=0.04).

Summary/Conclusion: NK cells derived from DLBCL patients are found to display an aberrant surface receptor repertoire leading to receptor-dependent attenuated degranulation and cytokine production, along with diminished ability to promote lysis of malignant target cells. The demonstrated functional impairment of NK cells is likely to be driven by their exposure to the DLBCL environment and could promote lymphoma immune evasion.

P1287: DISTINGUISHING BETWEEN PRIMARY MEDIASTINAL LARGE B-CELL LYMPHOMA AND DIFFUSE LARGE B-CELL LYMPHOMA USING THE LYMPH3CX GENE EXPRESSION PROFILING ASSAY

R. S. Robetorye 1,* , C. A. Ramsower 1 , C. L. McKinney 1 , L. M. Rimsza 1

1 Molecular Diagnostics Arizona Laboratory (MDAZL), Department of Laboratory Medicine and Pathology, Mayo Clinic Arizona, Phoenix, United States of America

Background: Primary mediastinal large B-cell lymphoma (PMBL) is a distinct lymphoid neoplasm arising in the mediastinum that is morphologically similar to diffuse large B-cell lymphoma (DLBCL), but has distinct clinical, immunophenotypic, genotypic, and molecular features. Although differentiation of PMBL from DLBCL can be important for guiding subsequent therapy, interpretation of morphologic findings and associated immunohistochemical stains in small, and often crushed, mediastinal biopsies can be difficult.

Aims: Therefore, we developed and fully validated a clinical gene expression profiling assay in the Molecular Diagnostics Arizona Laboratory (MDAZL) using the Lymph3Cx gene expression signature to distinguish these lymphomas using formalin-fixed paraffin-embedded (FFPE) tissue specimens. This assay includes and builds upon the Lymph2Cx expression signature and can subtype DLBCL into germinal center B-cell (GCB)-type, activated B-cell (ABC)-type, and unclassifiable (UNC) categories as well as accurately and robustly differentiate PMBL from DLBCL using the NanoString nCounter ® platform.

Methods: A hematopathologist identifies a large B-cell lymphoma, and additional testing is ordered for further classification of the lymphoma (including Lymph3Cx analysis). Pathological assessment of tumor content is performed on the H&E-stained slide, and unstained sections are sent to MDAZL for further processing. Total RNA is isolated and quantitated in MDAZL, and the RNA is hybridized overnight to fluorescently labeled probes in the Lymph3Cx assay panel. The probe/RNA complexes are quantitated on a NanoString nCounter Digital Analyzer. Algorithmic analysis is performed (R-script) for quality control (QC) and subtype determination and resulting data are verified by the pathologist and loaded directly into the EMR for correlation with clinical data and final diagnosis.

Results: So far, 40 clinical specimens have been subjected to Lymph3Cx testing in the MDAZL Laboratory with the following results: PMBL: 19 (47.5%); DLBCL: 15 (37.5%); Unclear: 5 (12.5%); Failed QC: 1 (2.5%). Of the 15 cases with gene expression signatures consistent with DLBCL, cell-of-origin results showed the following results: GCB: 8 (53.33%); ABC: 5 (33.33%); UNC: 2 (13.33%). Ordering locations included Mayo Clinic Arizona: 14 (35%); Mayo Clinic Minnesota: 7 (17.5%); Mayo Clinic Florida: 5 (12.5%); other external sites: 14 (35%). Average in-laboratory turn-around-time for these cases was 1.8 days, with an average of 3.3 days from sample receipt to final report.

Summary/Conclusion: The Molecular Diagnostics–Arizona Laboratory (MDAZL) has performed a full clinical validation of the performance characteristics of the Lymph3Cx assay and is the first molecular diagnostics laboratory to offer the Lymph3Cx assay as a clinical molecular diagnostic test for distinguishing PMBL from DLBCL and molecular cell-of-origin subtyping of DLBCL to patients both inside and outside of the Mayo Clinic. With a relatively rapid turn-around-time, the Lymph3Cx assay is suitable for use in routine hematopathology practice for distinguishing PMBL and DLBCL and is also suitable for definitive identification of PMBL cases for possible use in ongoing clinical trials to identify more specific therapeutics for treatment of PMBL.

P1288: ALTERED AMINO ACID HOMEOSTASIS CAN ELICIT AN ADAPTIVE RESPONSE VIA UPREGULATION OF UFMYLATION IN HODGKIN LYMPHOMA

A. Romano 1,* , G. Scandura 1 , D. Tibullo 2 , A. M. Amorini 2 , E. La Spina 1 , A. Barbato 1 , C. Giallongo 3 , C. Cerchione 4 , G. Simonetti 4 , G. Martinelli 4 , F. Di Raimondo 1

1 Dipartimento di Chirurgia e Specialità Medico Chirurgiche; 2 DIPARTIMENTO DI SCIENZE BIOMEDICHE E BIOTECNOLOGICHE; 3 DEPARTMENT OF MEDICAL, SURGICAL SCIENCES AND ADVANCED TECHNOLOGIES, Università degli Studi di Catania, CATANIA; 4 IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST S.r.l., Meldola, Italy

Background: Our previous work showed that Arginase (ARG-1) and Indoleamine Deoxigenase (IDO-1) are increased in tumour microenvironment of classic Hodgkin Lymphoma (HL), but the consequences of arginine (arg) and tryptophan (trp) deprivation on neoplastic cells fitness are largely unknown.

Aims: To identify the adaptive response to arg and trp deprivation at metabolic and transcriptional level in HL.

Methods: In three human HL cell lines (L428, L540 and KM-H2) individually cultured with customized complete media or deprived of arg/trp, we performed an integrative metabolic and transcriptome profiling, respectively by Metabolon Inc and Illumina RNA-seq platforms. Findings about the amino acid metabolism were validated in both lymph-node and peripheral blood of 64 consecutive newly-diagnosed patients affected by advanced stage HL.

Results: In vitro, specific-amino acid deficiency leads to an increase in uptake of free fatty acids from the media to maintain membrane integrity. Consistent with a metabolic rewiring to maintain mitochondrial integrity (the pyruvate is an important intermediary in the conversion of carbohydrates into fatty acids), the adaptive response to arg deprivation was associated to increased oxidative stress (reduced glutathione, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine), while no significant changes were observed in trp deprivation.

Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements in KMH2 and HDMYZ cell lines upon arg deprivation, involving upregulation of genes required for the unfolded protein response p53 pathway and networks and proteosome degradation, with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation. Upon both arg and trp deprivation, all cell lines tested showed down-regulation of CCNI2, LCROL, MKI67, NCAPG, PEX10 and UFSP2, confirmed by increased ribosome stalling. Thus, we disclosed how amino acid starvation engages the UFMylation pathway, an adaptive response never investigated before in HL.

Despite ARG-1 and IDO-1 cells were significantly enriched in HL tissue samples as compared with control reactive lymph node samples, peripheral blood HL patients showed dysregulated distribution of aminoacids, with significant increase of arginine, tryptophan, kynurenine, glutamate (specular to reduced amounts in the lymph node) and reduction of glutamine, asparagine, histidine, threonine, alanine, valine and methionine, confirming in vivo the relevance of amino acid deprivation in HL.

Summary/Conclusion: In HL there is a dysregulation in amino acid metabolism as consequence of the complex crosstalk between neoplastic cells and their microenvironment. However, compensatory pathways can be elicited to maintain arginine and tryptophan availability in peripheral blood, where several amino acids, other than arginine and tryptophan, are reduced. Further studies are required to unveil the adaptive response to amino acid deprivation in HL.

P1289: COMPUTATIONAL SIMULATIONS SHED LIGHT ON THE 3D CONSEQUENCE OF SUPER-ENHANCER HIJACKING IN B CELL MALIGNANCIES

L. Russell 1,* , D. Kent 1 , A. Mikulasova 1 , N. Karataraki 1 , B. Walker 2 , B. Javierre 3 , D. Rico 1 , C. Brackley 4

1 Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom; 2 Melvin and Bren Simon Comprehensive Cancer Center, Indiana University, Indianapolis, United States of America; 3 Josep Carreras Leukaemia Research Institute, Barcelona, Spain; 4 School of Physics and Astronomy, The University of Edinburgh, Edinburgh, United Kingdom

Background: Chromosomal translocations are important drivers of haematological malignancies whereby proto-oncogenes are activated by juxtaposition with super-enhancers. The immunoglobulin (IG) and T-cell receptor loci are enhancer-rich regions that are commonly hijacked via translocation in many subtypes of lymphoid malignancies. However, the epigenomic landscape and 3D genome structure remains poorly understood. We previously analysed the epigenomic consequence of rearrangements between the super-enhancers of the IG heavy chain locus ( IGH ) and proto-oncogene CCND1 1 . By integrating BLUEPRINT epigenomic data with DNA breakpoint detection, we observed a broad H3K4me3 domain (BD) within the IGH locus of healthy B-cells that was absent in samples with IGH-CCND1 translocations. Upon chromosomal translocation, we observed the appearance of an H3K4me3-BD over CCND1 concomitant with its overexpression and an extensive increase in chromatin accessibility of this locus. In addition, we observed similar cancer-specific H3K4me3-BDs associated with super-enhancer hijacking of other common oncogenes in B-cell ( MAF , MYC and FGFR3/NSD2 ) and T-cell malignancies ( LMO2, TLX3 and TAL1 ) 1 .

Aims: To understand and predict the 3D genome consequence of IGH-CCND1 translocation events using the highly predictive heteromorphic polymer model (HiP-HoP).

Methods: Using methods from polymer physics, HiP-HoP uses data on DNA accessibility, chromatin states, and protein binding to generate an ensemble of simulated 3D chromosome structures. After confirming that the model gives good predictions of Hi-C data for a healthy human B-cell derived cell line, we generated predictions for two B-cell malignant cell lines (U266, Z-138) possessing IGH-CCND1 rearrangements.

Results: We confirmed that the insertion of a single IGH super enhancer ( Eα1 ) in U266 leaves the local topologically associated domain (TAD) structure intact. Simulations of the reciprocal IGH-CCND1 translocation in Z-138, suggested the TAD boundaries are preserved, creating an oncogenic fusion TAD encompassing CCND1 and all four IGH super-enhancers.

Further simulations suggested that it is the downstream epigenetic changes which drive the 3D structural changes, rather than the genomic rearrangement itself. This points to a scenario where the translocated IGH super-enhancers first recruit chromatin remodelers to the region; this then drives epigenomic changes which in turn disrupts the wider 3D structure leading to dysregulation of cyclin D1. The model was able to predict how the structure of CCND1 changes in these different cell lines, could simulate different breakpoints, and could predict the impact on gene expression. Using qPCR and 3D-FISH we were able to validate the predicted high mRNA expression levels and increase in volume of the CCND1 locus.

Summary/Conclusion: Taken together, our analysis suggests that H3K4me3-BDs are created by super-enhancers and supports the new concept of epigenomic translocation , where the relocation of H3K4me3-BDs accompanies the translocation of super-enhancers, altering the local 3D genome.

References

1. PMID:34933939

P1290: A TISSUE-BASED EX VIVO CULTURE SYSTEM TO ANTICIPATE TEHRAPY RESPONSE IN LYMPHOMA.

A. Santamaria-Martínez 1,2,* , J. Epiney 1,2,3 , D. Srivastava 1,2,4 , D. Tavernari 1,2,4 , D. Milowich 5 , I. Letovanec 5 , G. Ciriello 2,4 , A. Cairoli 3 , E. Oricchio 1,2

1 Swiss Institute for Experimental Cancer Research, EPFL; 2 Swiss Cancer Center Léman; 3 Hemato-oncology department, CHUV; 4 University of Lausanne (UNIL), Lausanne; 5 ICH, Hôpital du Valais, Sion, Switzerland

Background: B-cell malignancies are a heterogenous group of malignancies that acquire multiple genomic alterations over time and in which the interactions between tumor cells and the tumor microenvironment (TME) are often important for tumor growth. Modeling lymphoma ex vivo has been hampered by the lack of suitable 3D models and the complexity of translating organoid technology from other cancer types into lymphoma. Hence, research mainly relies on the use of transgenic and xenograft animal models.

Aims: We wanted to develop a method to maintain lymphoma tissue explants in culture which could be used to study lymphoma pathogenesis and to test response to anti-cancer therapies directly on patient tumor tissues. We call these tumor tissue fragments lymphomoids .

Methods: We have designed an ex vivo culture system to maintain lymphoma tissues in culture for several weeks. We have tested this technology on murine models of lymphoma and on tissue biopsies obtained from 9 different patients diagnosed with different lymphoma subtypes. To confirm that our method supports tissue architecture for several weeks we have performed multiparametric FACS analyses, single-cell RNA sequencing, high-plex spatial proteomics profiling, and spatial transcriptomics analyses.

Results: With our approach, we are able to preserve the tissue composition and the interactions between tumor cells and their microenvironment ex vivo for three to four weeks. The transcriptional profile of tumor tissue biopsies was highly conserved in lymphomoids, indicating that we are able to maintain the tissue heterogeneity present in the original tumors. To anticipate sensitivity to anti-cancer therapies, we have tested and compared response to targeted therapies and immunotherapy on lymphomoids obtained from the 9 human primary lymphomas. Histopathological and spatial transcriptomic analyses showed patient-specific sensitivity to particular compounds and revealed features on the tumor tissue composition associated with resistance or response to therapies.

Summary/Conclusion: Overall, we have developed a system to maintain patient-derived lymphomoids in culture which allows to anticipate sensitivity and resistance to anti-cancer therapies, and it could potentially be used to identify the best therapeutic options for each patient in a clinical setting.

P1291: BTM-3566, A NOVEL ACTIVATOR OF THE MITOCHONDRIAL STRESS RESPONSE ERADICATES DIFFUSE LARGE B-CELL LYMPHOMA IN MICE

A. Schwarzer 1,2,* , M. Oliveira 3 , M.-J. Kleppa 2 , A. Anantha 4 , A. Cooper 4 , T. Hembrough 4 , J. Levine 4 , M. Luther 4 , M. Stocum 4 , M. Hannink 5 , M. Liesa-Roig 3 , M. Kostura 4

1 Dept. of Hematology, Hemostaseology, Oncology and Stem Cell Transplantation; 2 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 3 Department of Medicine, UCLA. David Geffen School of Medicine, Los Angeles; 4 Bantam Pharmaceutical, Durham; 5 Biochemistry Department, University of Missouri, Columbia, United States of America

Background: Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or after CAR-T-cell therapy.

Aims: We describe and characterize BTM-3566, a first-in-class compound that activates the OMA1-dependent mitochondrial stress pathway. BTM-3566 is active against a variety of B-cell malignancies but has the greatest effect in DLBCL, inducing robust therapeutic responses in vitro and in vivo .

Methods: Chemical biology, animal models, functional genetics

Results: BTM-3566 is a small molecule based on a pyrazolothiazol-backbone. BTM-3566 induces apoptosis and complete cell killing in DLBCL lines with an IC 50 of ~300 nM, including ABC, GCB, double-hit and triple-hit lymphoma lines. BTM-3566 has > 50% of oral bioavailability and 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. In a dose-finding study using the DLBCL line SUDHL-10, once daily oral treatment with 20 mg/kg BTM-3566 for 21 days resulted in complete regression in all tumor-bearing animals. Importantly, no subsequent tumor growth was observed for 2 weeks after cessation of therapy. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1).

Transcriptome analyses revealed that BTM-3566 activates the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and upregulation of the stress transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined that HRI (EIF2AK1) was uniquely required for BTM-3566 eIF2a phosphorylation, activation of the ATF4-ISR and induction of apoptosis. HRI is activated by mitochondrial-related stress, including heme depletion, ROS generation or blockage of mitochondrial ATP synthesis which all result in the activation of the mitochondrial protease OMA1. We found that BTM-3566 activates OMA1 in a manner unrelated to changes in mitochondrial ATP synthesis, reactive oxygen species generation or electron transport chain inhibition. OMA1 activation was required for the therapeutic effect and CRISPR-Cas9 depletion of OMA1 eliminated BTM-3566’s apoptotic activity. Substrates of OMA1 include the mitochondrial GTPase OPA1 and DELE1, a mitochondrial protein recently shown to act as a sensor of mitochondrial dysfunction and to signal through HRI kinase and ATF4. We show that downstream of OMA1 the apoptotic stress signal is relayed to HRI by DELE1, whereas OMA1-dependent OPA1 cleavage and mitochondrial fragmentation were dispensable for the therapeutic effect.

Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a poorly characterized mitochondrial membrane protein, negatively correlated with response to BTM-3566. Overexpression of FAM210B prevented OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis and cell cycle arrest. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation.

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Summary/Conclusion: We describe a novel antitumor mechanism in DLBCL, where BTM-3566 induces mitochondrial stress, activating the OMA1-DELE1-HRI-eIF2a-ATF4 pathway leading to apoptosis and tumor regression. An IND application in DLBCL has been completed with initiation of first in human clinical trials planned in 2022.

P1292: MOLECULAR CHARACTERIZATION OF DIFFUSE LARGE B-CELL LYMPHOMA ASSOCIATED TO HEPATITIS C VIRUS INFECTION

R. Sciarra 1,2,* , M. Merli 3 , C. Cristinelli 2 , M. Lucioni 2,4 , S. Zibellini 1 , R. Riboni 4 , S. Uccella 5 , D. Furlan 6 , C. Zerbi 2 , B. Bianchi 3 , M. Gotti 1 , V. V. Ferretti 7 , C. Varraso 1 , S. Fraticelli 2 , T. Lazic 2 , I. Defrancesco 1,8 , M. Frigeni 1 , B. Mora 3 , L. Libera 6 , M. Bonfichi 1 , F. Sessa 6 , F. Passamonti 3 , M. Paulli 2,4 , L. Arcaini 1,2

1 Division of Hematology, Fondazione IRCCS Policlinico San Matteo; 2 Department of Molecular Medicine, University of Pavia, Pavia; 3 Division of Hematology, University Hospital Ospedale di Circolo and Fondazione Macchi-ASST Sette Laghi, University of Insubria, Varese; 4 Anatomic Pathology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia; 5 Pathology Unit, Department of Medicine and Surgery; 6 Pathology Unit, Department of Medicine and Surgery, University of Insubria, Varese; 7 Service of Clinical Epidemiology and Biometry, Fondazione IRCCS Policlinico San Matteo; 8 Department of Medical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Pavia, Italy

Background: HCV positive DLBCL has distinct clinical and pathological characteristics compared to its negative counterpart; however, in the era of DLBCL genomic profiling, its molecular landscape has been scarcely outlined.

Aims: To investigate the clinico-pathological and molecular features of a well characterized bicentric series of 54 patients (pts) with HCV positive DLBCL.

Methods: With Illumina HiSeq 2500, we applied targeted next generation sequencing (NGS) of 184 genes on DNA extracted from archived formaline fixed paraffin embedded tissue; the core panel probes was designed using IDT tool and libraries were prepared using Illumina DNA-prep-with enrichment. We applied fluorescence in-situ hybridisation (FISH) for MYC, BCL2 and BCL6 rearrangements detection and NanoString technology (Lymph2Cx assay) for cell-of-origin (COO) determination. We performed cluster analysis with LymphGen genetic classifier.

Results: Median age was 71 (33-84). Stage was III/IV in 34 pts (63%). Extranodal sites and spleen were involved in 22 pts (41%) each. R-IPI was intermediate in 22 pts (40%) and poor in 30 (56%). HCV-RNA was detectable in 52 pts (96%). Overall, 19 out of 37 pts with available data (51%) received an antiviral therapy course, including 7 (19%) with interferon-based regimens and 12 (32%) with direct-acting antivirals. Twenty-seven pts (50%) received rituximab-based regimens, 23 (43%) chemotherapy alone and 1 (2%) surgery alone. With a median follow up of 7.7 years (yrs) (IQR: 4.6-10.6), 5-yrs overall survival (OS) was 49.3% (95%CI 34.1-62.8%) and 5-yrs progression free survival (PFS) 39.5% (95%CI 25.5-53.3%).

FISH analysis showed rearrangements involving BCL2 in 2/54 pts (4%), BCL6 in 27/53 pts (51%), MYC in 6/53 pts (11%). Hans algorithm classified 25 pts as Germinal Center B Cell (GCB) and 27 as non-GCB. Lymph2Cx-based assay was successful in 38 cases and classified 16 cases each (42%) as Activated B Cell (ABC) or GCB subtypes, while 6 cases resulted unclassified.

NGS showed mutations in 158 out of the 184 analyzed genes. All pts harbored at least one oncogenic variant with a median mutation load (MML) of 13 mutated genes per case (2-26; IQR: 9-16). Most frequently mutated genes were the epigenetic regulators KMT2D (n=23; 42.6%) and SETD1B (n=17; 31.5%), followed by FAS, PIM1 and RERE (n=15; 27.8%), TBL1XR1 (n=14; 26%), BCL11A (n=13; 24%) and SGK1 (n=12; 22%). Mutated pathways involved the epigenetic regulation (94% of pts; MML: 3; range 1-7; IQR: 1.25-4), apoptosis (90% of pts; MML: 2; 1-7; IQR: 1-3), BCR/NFkB signaling (77% of pts; MML: 2; 1-7; IQR: 1-3), immune regulation (56% of pts; MML: 1.7; 1-5; IQR: 1-2) and the NOTCH pathway (25% of pts; MML: 1.2; range 1-2). The LymphGen tool classified 29 out of 54 pts (54%) in defined clusters, including 14 in BN2 (48%), 7 in ST2 (24%), 4 in MCD (14%) and 4 in EZB subtype (14%). Notably, all assessable MCD cases (3/3) displayed an ABC signature, while all EZB tumors (3/3) showed a GCB COO. Conversely, ST2 and BN2 subtypes comprised mixed COO subgroups. No significant differences in terms of OS or PFS were detected according to either genetic clusters or COO.

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Summary/Conclusion: Our study elucidates molecular landscape of HCV-positive DLBCL, showing scarcity of BCL2 translocations, an equal distribution of COO subgroups according to NanoString and high prevalence of mutated genes within the epigenetic and immune regulation pathways. BN2 cluster prevalence and enrichment of mutations of NOTCH pathway genes seem to indicate a preferential marginal-zone origin for a consistent subgroup of HCV-positive DLBCL cases.

P1293: COMBINING THE IAP ANTAGONIST, TOLINAPANT, WITH A DNA HYPOMETHYLATING AGENT ENHANCES ANTI-TUMOUR MECHANISMS IN PRECLINICAL MODELS OF T-CELL LYMPHOMA.

M. Sims 1,* , G. Ward 1 , Z. Zhang 1 , S. Jueliger 1 , M. Davis 2 , A. Boxall 2 , J. Taylor 3 , A. Biondo 1 , J. Lyons 1 , T. Smyth 4

1 Translational Sciences; 2 Bioinformatics, Astex Pharmaceuticals Limited, Cambridge, United Kingdom; 3 Clinical Development, Astex Pharmaceuticals Inc, Pleasanton, United States of America; 4 DMPK, Astex Pharmaceuticals Limited, Cambridge, United Kingdom

Background: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In a Phase 2 trial ( NCT02503423 ), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al ., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in preclinical studies.

Aims: Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death, such as necroptosis, by rational combination of our clinical compounds in preclinical models of TCL.

Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key necroptosis biomarkers (RIPK3, MLKL) were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. Karpas-299 cells genetically-manipulated to express RIPK3 were used to demonstrate involvement of necroptosis in drug-induced cell death (Cytotox NIR) in vitro . Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key immunomodulatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma. Levels of methylation in RIPK3 promoter regions were measured by pyrosequencing after bisulfite conversion. Comparative changes in gene expression were measured by RT-qPCR.

Results: TCL cell lines treated with tolinapant, decitabine or both led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating target engagement of both agents. The combination of tolinapant and decitabine synergistically reduced viability of some human T-cell lymphoma cell lines.

Some cell lines, including Karpas-299, were resistant to tolinapant treatment and showed low expression of RIPK3 , which was found to be due to promoter methylation. Increased expression of RIPK3 in Karpas-299 by genetic manipulation or by decitabine treatment resulted in enhanced lytic cell death upon tolinapant treatment.

Decitabine and tolinapant treatments resulted in expression of cytokines, chemokines and DAMPs, suggesting potential for immune activation and the effects were enhanced when combined. Furthermore, normally silenced cancer/testis antigen expression was increased by decitabine, potentially increasing the immunogenicity of the cells.

Evaluation of the combination of agents in mouse models suggested that increased necroptosis signal and immune-potentiating biomarker modulation can be achieved in vivo .

Summary/Conclusion: These data demonstrate that hypomethylating agents enhance immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. In addition, modulation of cytokine response and cancer/testis antigen expression could enhance anti-tumour response. These findings provide a strong rationale to explore this combination clinically.

P1294: SINGLE-CELL RNA SEQUENCING REVEALS TUMOR CELL HETEROGENEITY AND COMPREHENSIVE IMMUNE PROFILE OF T FOLLICULAR HELPER CELL LYMPHOMA

S. Suma 1,* , M. Fujisawa 2 , Y. Abe 1 , Y. Suehara 2,3 , D. Kaji 4 , T. Sugio 5 , K. Kato 6 , K. Akashi 6 , K. Matsue 7 , N. Nakamura 8 , A. Suzuki 9 , Y. Suzuki 9 , S. Chiba 2,3 , M. Sakata-Yanagimoto 2,3,10

1 Department of Hematology, Graduate School of Comprehensive Human Sciences; 2 Department of Hematology, Faculty of Medicine, University of Tsukuba; 3 Department of Hematology, University of Tsukuba Hospital, Tsukuba; 4 Department of Hematology, Toranomon Hospital, Tokyo, Japan; 5 Department of Medicine, Division of Oncology, Stanford University, Stanford, United States of America; 6 Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Science, Fukuoka; 7 Division of Hematology/Oncology, Department of Internal Medicine, Kameda Medical Center, Kamogawa; 8 Department of Pathology, Tokai University School of Medicine, Isehara; 9 Department of Computational Biology and Medical Sciences, The University of Tokyo, Tokyo; 10 Division of Advanced Hemato-Oncology, Transborder Medical Research Center, University of Tsukuba, Tsukuba, Japan

Background: Angioimmunoblastic T-cell lymphoma (AITL) and other nodal lymphomas of T follicular helper (T FH ) cell origin (T FH lymphoma) are associated with immune dysregulation. However, comprehensive immune profiles of T FH lymphoma have not been fully investigated. The RHOA G17V mutation (G17V) is found in 60-70% of T FH lymphoma and considered to gain a novel function through binding to VAV1 protein. Nevertheless, the prevalence of homozygous (Homo) and heterozygous (Hetero) G17V mutations and the difference in their oncogenicity due to its zygosity remain unclear.

Aims: We performed single-cell RNA sequencing (scRNA-seq) for immune and tumor cells of T FH lymphoma to determine (1) the specific immune profiles, (2) the characteristics of tumor cells and (3) the mechanisms of clonal evolution from G17V Hetero to Homo.

Methods: We collected 29 PB (12 at initial diagnosis [ID], 17 at relapsed/refractory diagnosis [RR]) and 10 LN (6 ID, 4 RR) from 25 patients with T FH lymphoma, 7 homeostatic LN (HLN) from patients with solid cancers. scRNA-seq and single-cell T cell receptor (TCR) sequencing were performed by the Chromium system (10x Genomics) from 16 PB and 9 LN of T FH lymphoma patients and 7 HLN. Publically available scRNA-seq data from 5 healthy donors (HD) were also integrated as controls. scRNA-seq data analysis was performed by Seurat, Scater and Batchelor for quality control, integration, and clustering; SingleR for annotation; Monocle3 and Slingshot for trajectory analysis; Immunarch for TCR repertoire analysis; gene set variation analysis (GSVA) for pathway analysis. VarTrix pipeline was used to detect mutation distribution in scRNA-seq data.

Results: In total, 98,891 cells from PB and 68,738 cells from LN were analyzed. Unsupervised clustering identified 19 T/NK-, 2 B-, and 9 myeloid-cell clusters in PB (Figure 1). Tumor cells were identified by the expanded TCR clonality and the T FH -like gene expression signatures. (1) Subsequent clustering and GSVA of each cell type identified dysfunctional CD8 T cells (Dys CD8) in PB and LN of patients, some of which had enriched the proliferation signature (Pro CD8) (Figure 2). In some samples, Dys/Pro CD8 shared their TCRs between PB and LN, suggested that they have the same cellular origin. The proportion of Pro CD8 in LN correlated with that of T REG in PB; moreover, T REG effector function was activated in both PB and LN of patients compared to HD and HLN, respectively. By sub-clustering of myeloid subset, C1QA/B/C ( C1Q ) + macrophages and LAMP3 + PD-L1 + dendritic cells (DCs) increased in LN of RR patients, whereas C1Q + monocytes increased and CLEC9A + conventional DCs and plasmacytoid DCs decreased in PB. (2) Differential expression gene (DEG) analysis identified five tumor-specific gene candidates and the cell surface protein PLS3 was validated by flow cytometry (Figure 3). (3) The G17V RHOA -mutated cells were detected in 6 PB and 3 LN. The G17V Hetero- versus Homo-mutated cells were 657 vs 59 in PB, while 1529 vs 288 in LN. DEG and GSVA in LN identified that the regulatory signature was significantly activated in Homo, whereas the proliferation signature was up-regulated in Hetero (Figure 4).

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Summary/Conclusion: These results clarified the comprehensive immune structures including novel tumor-specific markers in T FH lymphoma. PLS3, one of the new tumor markers, could be detected by flow cytometry, a viable method in clinical practice. In the clonal evolution process from the G17V Hetero to Homo, the proliferative signaling switch to the immune escape signaling.

P1295: DECIPHERING THE ROLE OF MSI2 AS A REGULATOR OF MANTLE CELL LYMPHOMA STEM-LIKE PROPERTIES

M. Sureda-Gómez 1,* , P. Balsas 1,2 , M.-L. Rodríguez 1 , F. Nadeu 1,2 , A. De Bolòs 1 , Á. Eguileor 1 , M. Kulis 1 , G. Castellano 1 , E. Giné 3 , S. Demajo 1 , J. I. Martín-Subero 1,2,4 , P. Jares 1,2,5 , E. Campo 1,2,5 , V. Amador 1,2

1 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona; 2 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid; 3 Department of Hematology, Hospital Clinic; 4 Institució Catalana de Recerca i Estudis Avançats (ICREA); 5 Hematopathology Section, Department of Anatomic Pathology, Hospital Clinic, Barcelona, Spain

Background: Mantle cell lymphoma (MCL) is a mature B cell neoplasm with two distinct biological subtypes regarding clinical, molecular and pathological features. Conventional MCL (cMCL) subtype is characterized by high aggressiveness and poor outcome with frequent relapses after initial response to treatment, suggesting the presence of a population of cells able to self-renew and responsible for drug resistance. SOX11 transcription factor, expressed in embryonic stem cells (ESC), is aberrantly overexpressed mostly in cMCL and has an oncogenic role in MCL. MCL-cancer stem cells have been isolated from MCL cases by different groups. However, its relationship to SOX11 expression and contribution to MCL pathogenesis and clinical evolution remain unknown.

Aims: We hypothesized that SOX11 may promote stem cell-like properties through the activation of stem cell-related genes, leading to an aggressive and incurable tumor in MCL patients. We aimed to identify potential mediators of stemness directly regulated by SOX11 in MCL.

Methods: Here, we integrated the differential gene expression profile between SOX11+ and SOX11- MCL primary cases with stem cell-related genes, specific SOX11 ChIP-chip bound genes and epigenomic data of MCLs.

Results: SOX11+ MCL showed enrichment in leukemic (LSC) and hematopoietic stem cells (HSC) gene signatures compared to SOX11- MCL primary cases. Musashi-2 (MSI2), an RNA binding protein that maintains self-renewal and prevents differentiation in HSC and ESC, emerged as one of the most significant upregulated stem cell-related genes in SOX11+ MCLs. SOX11 directly bound to MSI2 regulatory regions increasing its expression in vitro in MCL cells. MSI2 enhancers located in intronic regions were strongly activated in SOX11+ but not in SOX11- MCL cell lines and primary cases. MSI2 upregulation significantly associated with chemoresistance and poor overall survival, independently of common risk factors (SOX11 expression, high copy number alterations, 17p/ TP53, 11q/ ATM and 9p/ CDKN2A alterations), in MCL patients. MSI2 knockdown MCL cell lines had a gene expression profile enriched in proapoptotic-related genes; whereas HSC and LSC gene signatures were downregulated compared to its control cells. MSI2 knockdown or MSI2 inhibition with Ro 08-2750 treatment decreased clonogenic growth, tumor survival and chemoresistance in MCL cells. SOX11+/MSI2 high MCL primary leukemic samples showed higher percentage of ALDH+ cells compared to SOX11-/MSI2 low samples, which decreased after Ro 08-2750 treatment. Besides, MSI2 silencing delayed tumor growth in vivo in MCL xenograft mice models.

Summary/Conclusion: Overall, our results suggest that MSI2 might play a key role in sustaining stemness, chemoresistance and tumor cell survival in MCL. MSI2 represents a potential novel target for therapeutic interventions in MCL.

P1296: SUPPRESSING SYNTHESIS OF THE LONG ISOFORM OF THE PROLACTIN RECEPTOR IS A TARGETED STRATEGY TO TREAT AUTOIMMUNE B-LYMPHOPROLIFERATIVE DISEASES AND B CELL MALIGNANCIES

A. Taghi Khani 1 , A. Kumar 1 , K. Radecki 2 , S. J. Lee 1 , A. Sanchez Ortiz 1 , M. Lorenson 2 , X. Wu 3 , J. Koff 4 , S. Forman 5 , Z. Gu 1 , A. Walker 2 , S. Swaminathan 1,*

1 Systems Biology, Beckman Research Institute of City of Hope, Monrovia; 2 Biomedical Sciences, University of California Riverside, Riverside; 3 Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte; 4 Hematology and Medical Oncology, Emory University School of Medicine, Atlanta; 5 Hematology & Hematopoietic Cell Transplantation, Beckman Research Institute of City of Hope, Duarte, United States of America

Background: Developing early interventions for diffuse large B cell lymphoma (DLBCL) and B cell acute lymphoblastic leukemia (B-ALL) requires an understanding of the molecular mechanisms underlying the initiation, establishment, and sustenance of these high-risk B cell malignancies. B cell cancers are initiated 3-8-fold more frequently in patients suffering from refractory autoimmune B-lymphoproliferative disorders such as systemic lupus erythematosus (SLE), making SLE a relevant disease model to study initiation of B cell cancers. The hormone prolactin (PRL) exacerbates the symptoms of SLE, enhances lymphoid cell survival, and promotes the expression of MYC and BCL2 protooncogenes in lymphocytes. PRL signals via prolactin receptors (PRLRs). PRLRs have long (LF), intermediate (IF, only in humans) and short (SF) isoforms generated by alternative splicing. Increased expression of the LF/IF relative to the SF PRLR on cells leads to cell proliferation and survival, whereas increased expression of SF relative to LF/IF inhibits proliferation and induces apoptosis.

Aims: Whether PRL drives initiation, establishment, and sustenance of B cell malignancies was unknown. We hypothesized that PRL, by signaling specifically through the pro-proliferative, anti-apoptotic LF/IFPRLR drives the three stages of B cell malignancy development, namely, initiation ( Stage 1 ), establishment ( Stage 2 ), and sustenance ( Stage 3 ). To test this, we measured the effects of LF/IFPRLR knockdown on B cells in vivo in transgenic mouse models of SLE (Stage 1/ Aim 1 ), DLBCL (Stage 2/ Aim 2 ), and in vitro in malignant human B cells (Stage 3/ Aim 3 ).

Methods: To knockdown LFPRLR in mice and LF/IFPRLR in human cells without suppressing expression of the SFs, we used a patented splice-modulating DNA morpholino oligomer (SMO), termed LFPRLR SMO, that prevents the splicing event needed to produce LF in mice and LF/IF in humans.

Results: LFPRLR knockdown reduced splenic B cell numbers in SLE-prone mice. Consistent with reduced B cell numbers, splenic B cell proliferation and subsequent generation of antibody-producing plasma cells was significantly lowered after LFPRLR knockdown. LFPRLR SMO-treated SLE-prone mice had reduced frequencies of deleterious splenic B cells with long immunoglobulin heavy chain ( IGH ) complementary determining region 3 (CDR3) >20 amino acids and B cells with non-functional IGH that are prone to malignant transformation. LFPRLR knockdown in SLE-prone mice also significantly reduced the expression of the activation induced cytidine deaminase (AID) enzyme, known to induce malignant transformation of B cells. Therefore, we infer that PRL-LFPRLR signaling exacerbates SLE B cell pathology and promotes malignant transformation of SLE B cells ( Stage 1 of B cell malignancy development). In DLBCL-prone mice carrying pre-malignant B cell clones, LFPRLR knockdown strikingly reduced splenic B cell counts and expression of BCL2 in B cells. Thus, PRL-LFPRLR signaling drives stage 2 of B cell malignancy development, i.e., establishment of overt B cell neoplasms from pre-malignant B cells. Finally, in overt human DLBCL and B-ALL ( Stage 3 ), we found that knockdown of LF/IFPRLR reduced the expression of MYC and BCL2 and impaired viability of malignant B cells.

Summary/Conclusion: LF/IFPRLR is a safe and isoform-specific prophylactic and therapeutic target to alleviate pathology of B cell subsets and concurrently lower the incidence of B cell malignancies in SLE patients ( Stage 1 ), reduce the risk of B cell malignancies in people with pre-malignant and indolent B cell clones ( Stage 2 ), and treat overt B cell neoplasms ( Stage 3 ).

P1297: ABERRANT MYCN EXPRESSION DRIVES ONCOGENIC HIJACKING OF EZH2 AS A TRANSCRIPTIONAL ACTIVATOR IN PERIPHERAL T CELL LYMPHOMA

M. Vanden Bempt 1,* , K. Debackere 1,2 , S. Demeyer 3 , Q. Van Thillo 3 , N. Meeuws 1 , J. Cools 3 , D. Dierickx 1,2

1 Department of Oncology, KU Leuven; 2 Department of Hematology, UZ Leuven; 3 Center for cancer biology, VIB-KU Leuven, Leuven, Belgium

Background: Peripheral T cell lymphoma (PTCL) is a group of rare but aggressive hematological cancers arising from the malignant transformation of mature post-thymic T cells. PTCL patients currently face an unfavorable prognosis: approximately 75% of all PTCL patients relapse after first-line chemotherapy treatment. Consequently, 5-year survival rates are as low as 10-30%, which underscores the urgent medical need for new therapeutic options.

Aims: The aim of this study was to characterize the oncogenic role of MYCN in PTCL development, and to identify novel targets for therapy.

Methods: We performed RNA-sequencing on a cohort of 28 PTCL cases. In addition, we have generated a bone marrow transplant model of MYCN-driven T cell lymphoma, and we performed a thorough characterization of this model using RNA-sequencing and ChIP-sequencing. Downstream targets were validated using luciferase experiments, immunoprecipitation followed by mass spectrometry, flow cytometry and ex vivo treatments.

Results: We observed aberrant overexpression of MYCN in 5/28 PTCL cases. In addition, we found that approximately 50% of the PTCL tumors is characterized by high expression of MYC target genes. Inducible expression of MYCN in lymphoid cells in a mouse model caused T cell lymphoma that recapitulated human PTCL with a MYC expression signature. Integration of mouse and human expression data identified EZH2 as a key downstream target of MYCN. Remarkably, EZH2 was found to be an essential co-factor for the transcriptional activation of the MYCN-driven gene expression program, which was independent of methyltransferase activity, but dependent on phosphorylation by CDK1. MYCN-driven T cell lymphoma was sensitive to EZH2 degradation or CDK1 inhibition, which displayed synergy with FDA-approved HDAC inhibitors.

Summary/Conclusion: In conclusion, we identified MYCN as a recurrent aberrantly overexpressed gene in PTCL, and characterized the oncogenic mechanisms underlying MYCN overexpression, which led to the identification of novel targeted treatments for PTCL.

P1298: THE IRAK-4 INHIBITOR EMAVUSERTIB (CA-4948) FOR THE TREATMENT OF PRIMARY CNS LYMPHOMA

C. Von Roemeling 1,* , M. Iqbal 2 , B. Doonan 3 , L. Hoang-Minh 1 , K. Klippel 1 , H. Tun 2 , D. Mitchell 1

1 Neurosurgery, University of Florida, Gainesville; 2 Internal Medicine, Mayo Clinic, Jacksonville; 3 Medicine, University of Florida, Gainesville, United States of America

Background: Primary central nervous system lymphoma (PCNSL) is an aggressive brain tumor accounting for 3% of all CNS malignancies and is usually associated with poor prognosis. 5-year survival is less than 30% with current treatment options. Standard of care treatment includes induction with high-dose methotrexate based chemoimmunotherapy followed by consolidation with autologous stem cell transplant or whole brain radiation. However, more than half of PCNSL patients are not eligible for this type of intensive therapeutic intervention. Novel treatment strategies with better efficacy and toxicity profile are urgently needed. Toll-like receptor signaling pathway via MyD88/IRAK-4 signalosome is constitutively active in PCNSL secondary to MYD88 L265P mutation and represents an excellent therapeutic target. Emavusertib (CA-4948) is an oral first-in-class small molecule inhibitor of IRAK4 that has demonstrated clinical activity in patients with systemic Non-Hodgkin’s Lymphoma, however it has not been evaluated in the CNS space.

Aims: In this study we examine translational and clinical therapeutic applicability and efficacy of emavusertib for the treatment of PCNSL.

Methods: Using multiparameter immunohistochemistry (IHC) and proteomics analysis, IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient PCNSL tissues as compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for emavusertib drug concentration following single oral high dose in a murine model. Using an aggressive preclinical A20 immunocompetent CNSL model, preclinical therapeutic activity of emavusertib was evaluated with assessment of downstream biomarker response to treatment including phospho-NF-κB, phospho-ERK1/2, and phospho-P38 in control and treated animals by multi-parameter IHC. We also report therapeutic response in a relapsed/refractory PCNSL patient enrolled in a Phase I/II clinical trial (TakeAim) assessing the combination of emavusertib and Ibrutinib.

Results: Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient PCNSL. We also demonstrate that emavusertib can achieve therapeutically relevant concentrations in the brain parenchyma and shows single-agent activity in an aggressive preclinical model of PCNSL. We further confirm decreased ERK1/2, MAPK and NF-κB activation in response to preclinical emavusertib treatment, indicative of downstream effect of IRAK-4 inhibition. Finally, one patient who could not tolerate standard therapies and was resistant to Ibrutinib achieved complete response when treated with the combination of emavusertib and Ibrutinib, providing early clinical evidence of CNS penetration and anti-tumor activity of emavusertib.

Summary/Conclusion: Herein we confirm for the first time that targeted inhibition of IRAK-4 with the oral small molecule inhibitor emavusertib demonstrates preclinical and clinical therapeutic activity against CNS lymphoma. Further evaluation of this agent for the treatment of PCNSL is warranted.

P1299: LOSS OF TYROSINE PHOSPHATASE SHP1 SENSITIZES DLBCL TO IBRUTINIB: A CANDIDATE PREDICTIVE BIOMARKER

Y. L. Wang 1,* , W. Wu 1 , J. Ma 2 , R. Nejati 1

1 Blood Cell Development and Function, Fox Chase Cancer Center, Philadelphia, United States of America; 2 Department of Medical Oncology, Shandong Cancer Hospital and Institute, Jinan, China

Background: B-cell receptor (BCR) signaling plays a key role in B-cell non-Hodgkin lymphoma (NHL). Protein tyrosine kinases such as LYN, SYK and BTK are essential in the activation of BCR leading to B-cell survival, proliferation and differentiation. SHP1 is a cytosolic protein tyrosine phosphatase functioning as a negative regulator of the BCR signaling cascade. It dephosphorylates and counteracts the activities of the tyrosine kinases, including CD79A/CD79B, SYK and BLNK. Ibrutinib is a selective BTK inhibitor that has been approved for the treatment of many B-cell malignancies, but not yet for diffuse large B-cell lymphoma.

DLBCL is the most common type of aggressive NHL. Based on cell-of-origin, DLBCL is subclassified into Germinal Center B cell-like (GCB) and activated B-cell like (ABC) DLBCL where chronic active BCR signaling plays a particularly important role in the pathogenesis of the ABC-DLBCL. However, only a subset of ABC-DLBCL respond well to ibrutinib-containing regimen (Wilson et al., 2021, Cancer Cell 39, 1643–1653). New predictive biomarkers are needed to identify patients who likely benefit from ibrutinib.

Aims: We aim to test the hypothesis that SHP1 may play a role in defining the level of the activity of the BCR pathway and impact on the response to ibrutinib.

Methods: A variety of methods was used to demonstrate the roles of SHP1 loss in DLBCL cell lines and primary tumors, including meta-analysis, immunohistochemistry on tissue microarray, promotor methylation, and genetic editing with CRISPR-Cas9 and lenti-viral transduction.

Results: We first reviewed 17 published literatures on SHP1 in NHL using a meta-analysis. The analysis of the SHP1 protein expression by immunohistochemistry revealed that lack of SHP1 expression is strongly associated with NHL including DLBCL. Likewise, the analysis of the methylation studies revealed a strong association between SHP1 promoter hypermethylation and NHL.

To further understand the range of variability of SHP1 expression in subtypes of DLBCL tumors, we constructed a tissue microarray with a large number of primary DLBCL tumors and cell lines. We found a wide range of variability and no substantial difference between the GCB and non-GCB subtypes regarding the extent of SHP1 expression.

We identified a strong reverse linear correlation between SHP1 protein expression and promoter methylation (r = 0.85, P=0.0002) suggesting that promoter hypermethylation may be responsible for reduced SHP1 expression in DLBCL cells. SHP1 was knocked out in both BCR-dependent GCB and ABC cell lines using CRISPR-Cas9 system. The phosphorylation of early BCR components, BTK, SYK and LYN, were increased in the SHP1 knockout clones suggesting that SHP1 depletion causes increased proximal BCR signaling activity. Moreover, the knock out clones demonstrated increased sensitivity to ibrutinib. We further showed that re-introduction of SHP1 vector to the knockout clones via stable lentiviral transduction restored normal sensitivity of cells to ibrutinib.

Summary/Conclusion: Our results show that SHP1 expression is suppressed in a substantial fraction of DLBCL tumors regardless of cell-of-origin subclassifications. Using genetic approaches, we demonstrated that loss of SHP1 increases BCR signaling activity and sensitizes tumor cells to the inhibition by ibrutinib. Our results suggest that loss of SHP1 may be used as an alternative biomarker to cell-of-origin to identify patients who potentially benefit from ibrutinib treatment.

P1300: EPIGENETIC CHARACTERISTICS AND PROGNOSIS OF FOLLICULAR LYMPHOMA IN A SINGLE JAPANESE INSTITUTION

M. Watanabe 1,2,* , N. Gouda 3 , J. Kanda 4 , A. Yoshizawa 3 , M. Sakurada 1 , M. Nishikori 1 , H. Hironori 3 , A. Takaori-Kondo 1

1 Department of Hematology, Kyoto University Hospital, Kyoto; 2 Division of Hematopoietic Stem Cell Transplantation, National Cancer Center Hospital, Tokyo; 3 Department of Diagnostic Pathology, Kyoto University Hospital, Kyoto; 4 Department of Hematology, National Cancer Center Hospital, Tokyo, Japan

Background: The use of epigenetic modifier drugs such as EZH2 inhibitors has recently expanded. However, since the clinical course of follicular lymphoma (FL) is heterogenous, there are still challenges in identifying patients who will benefit from EZH2 inhibitors in general hospital settings without next-generation sequencing.

Aims: We aimed to validate the epigenetic characteristics of EZH2 expression and H3K27 methylation status using immunohistochemistry staining. We also examined the usefulness of FLIPI, POD24, and cell-free DNA concentration in a recent cohort.

Methods: Altogether, 164 patients (female, n=91; male, n=73) diagnosed with FL at Kyoto University Hospital between 2005 and 2020 were included. Immunohistochemical (IHC) EZH2 expression and H3K27 methylation profiles were evaluated in 105 patients. EZH2 expression was assessed using the H-score (positivity rate (%) x staining intensity: negative, 0; weak, 1; moderate, 2; strong, 3), and H3K27 methylation profile was assessed by two independent estimators using the H3K27me3/me2 IHC scoring system, based on a previous report. Cell-free DNA concentration was examined at diagnosis or prior to the first chemotherapy in a total of 18 patients, out of whom 12 patients were assessed sequentially at 1, 3, 6, and 12 months after the first examination.

Results: The median age at diagnosis was 61 years (range, 33-86) and the median follow-up period was 7.5 years. FLIPI at diagnosis was high in 46 patients (28.3%), intermediate in 60 patients (37.0%), and low in 47 patients (29.0%). A total of 90 patients received rituximab (RTX)-containing chemotherapy (BR, n=10; R-CHOP/R-CVP, n=68; RTX monotherapy, n=9; other regimen, n=3), 62 patients were observed without any intervention (watchful waiting), and 5 patients underwent resection of the tumors. Of those with watchful waiting, 27 experienced lymphoma progression and received chemotherapy. H-scores of EZH2 ranged from 0 to 300 (mean score, 92) and H3K27me3/me2 score ranged from -4.2 to 5.0 (mean score, 2.3). A high H3K27me3/me2 score (≥ 2) was associated with superior OS, and the combination of low H3K27me3/me2 score (<2) and high EZH2 score (≥ 100) was associated with inferior OS (p=0.009) with high frequency rapid progression (62.5%) within 2 years after the 1 st immunochemotherapies (Figure 1A, B). Cell-free DNA concentration ranged from 0.45 ng/µl to 85.60 ng/µl (median conc, 0.67 ng/µl), which showed no apparent correlation with the serum level of LDH, sIL-2R, or imaging status. POD24 was detected in 38 patients, and its impact on OS was not statistically significant (HR 8.1, p=0.070). No patients with FLIPI-Low died during the follow-up, and only FLIPI-Int was associated with inferior OS (6/60 died), but not FLIPI-High (1/46 died).

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Summary/Conclusion: Our study suggested that H3K27me3/me2 and EZH2 scores of the tumor samples at diagnosis could predict their outcomes after immunochemotherapy. As high H3K27me3/me2 is known to be associated with EZH2 mutations, good prognosis of those with high H3K27me3/me2 scores might reflect the superior impact of EZH2 mutations, as reported in m7FLIPI. Meanwhile, tumors presenting low H3K27me3/me2 scores with high EZH2 scores have the potential to rely on other driver mutations for pathogenicity, although genetic analysis remains under investigation. FLIPI and POD24 were not good predictors of survival in our cohort, which might be due to our regimen choice and the frequency of imaging follow-up. More experiments are needed to apply cell-free DNA for clinical use in patients with FL.

P1301: BIOLOGICAL SIGNIFICANCE OF UCK2 IN HTLV-1-INFECTED CELLS IN ATL LEUKEMOGENESIS AND ACQUIRED RESISTANCE TO AZACITIDINE

T. Watanabe 1,* , Y. Yamamoto 2 , K. Kamachi 1,2 , Y. Kurahashi 1,2,3 , N. Yoshida-Sakai 1,2 , H. Ureshino 1,2 , Y. Fukuda-Kurahashi 1,3 , H. Nakamura 4 , E. Sueoka 5 , S. Kimura 1,2

1 Department of Drug Discovery and Biomedical Sciences; 2 Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga; 3 OHARA Pharmaceutical Co., Ltd., Shiga; 4 Department of Transfusion Medicine, Saga University Hospital; 5 Department of Clinical Laboratory Medicine, Saga University, Saga, Japan

Background: Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). We have reported that regional DNA hypermethylation in HTLV-1-infected T-cells reflects the disease status of ATL and the anti-ATL effects of DNA demethylating agents, azacitidine (AZA) and decitabine ( Blood 2020, 136: 871-884). We recently generated AZA-resistant (AZA-R) cells from ATL cell lines via long-term drug exposure and found down-regulation of uridine cytidine kinase2 (UCK2) correlated with lower susceptibility to AZA ( Int J Cancer 2022, 150: 1184-1197). UCK1 and UCK2 are involved in mono-phosphorylation of uridine and cytidine in pyrimidine nucleotide biosynthesis. Both proteins also catalyze mono-phosphorylation of AZA. Overexpression of UCK2 has been observed in several types of solid tumor tissues.

Aims: Here, we aimed to understand the role of UCK2 in HTLV-1-infected cells during multi-step carcinogenesis of ATL and the biological significance of inactivation of UCK2 in AZA-R cells.

Methods: All studies using human samples were performed in accordance with the guidelines set out in the Declaration of Helsinki and were approved by the institutional review board at Saga University (2018-03-02). Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples from healthy volunteers, HTLV-1 carriers and patients with ATL. ATL cell lines were cultured in RPMI-1640 medium containing 10% FBS.

Results: We found that UCK2 gene expression was higher in HTLV-1-infected T-cells than in normal T-cells using the gene expression datasets GSE55851 and GSE33615 . UCK2 protein expression was increased in HTLV-1-infected T-cells (CADM1+ cells) isolated from patients with ATL compared with uninfected T-cells (Fig. A). We also found UCK2 gene expression was upregulated via activation of T-cell receptor (TCR) signaling pathway in normal CD4+ T-cells (Fig. B). Somatic alterations and epigenetic modifications activating TCR signaling have been identified in ATL cells. Therefore, we think UCK2 protein is overexpressed through dysregulated TCR signaling in HTLV-1-infected cells during ATL leukemogenesis. We have successfully established AZA-R cell lines, whose UCK2 expression was lost or decreased. Although they proliferated normally in vitro , the knockdown of UCK2 dramatically suppressed cell growth of ATL cell lines (Fig. C). Since UCK2 is involved in pyrimidine nucleotide biosynthesis, we then performed metabolome analysis in AZA-R cells. Although uridine and cytidine were accumulated in AZA-R cells, possibly due to loss of UCK2, the amount of UTP and CTP was almost same with parental cells. On the other hands, dihydroorotic acid (DHO) was decreased and orotate was increased in AZA-R cells (Fig. D). DHODH catalyzes the oxidation of DHO to orotic acid and is a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. AZA-R cells were more susceptible to BAY2402234, a DHODH inhibitor (Fig. E), indicating the activation of pyrimidine de novo synthesis in AZA-R cells.

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Summary/Conclusion: Taken together, we think UCK2 supports the vigorous cell proliferation not only in normal TCR-stimulated T-cells but also in HTLV-1-infected cells. Inactivation of UCK2 makes cells resistant to AZA but suppresses the cell growth possibly due to the pyrimidine nucleotide starvation. AZA-R cells with reduced UCK2 expression proliferate normally in vitro by the activation of pyrimidine de novo synthesis.

P1302: INTEGRATION OF ROUTINE RNA AND WHOLE-EXOME SEQUENCING AND FLOW CYTOMETRY IMPROVES THE DIAGNOSTIC WORKUP OF PEDIATRIC LYMPHOMAS

L. Westera 1,* , M. Scheijde-Vermeulen 1 , L. Kester 1 , B. Tops 1 , F. Meyer-Wentrup 1

1 Princess Máxima Center for pediatric oncology, Utrecht, Netherlands

Background: Lymphomas are traditionally diagnosed by histology and immunohistochemistry, complemented by molecular methods like fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) to detect the genetic abnormalities that define certain malignancies.

Aims: To improve both diagnostic accuracy and efficiency, we aimed to implement RNA sequencing (RNAseq) and Whole Exome Sequencing (WES) and flow cytometry into the routine diagnostic workup of pediatric lymph node biopsies.

Methods: Within a year we analyzed 122 lymph node biopsies of all 110 children referred to our new Dutch National Pediatric Cancer Center because of suspected malignant lymphoma, using a standardized workflow combining histology and immunohistochemistry, flow cytometry, and next-generation sequencing technologies.

Results: Flow cytometry was performed with fresh tissue directly after biopsy in 83% of specimens (101/122) and allowed rapid diagnosis of mature and immature T- and B-cell non-Hodgkin lymphomas (NHL), enabling early treatment initiation in ill patients. RNAseq was performed on all biopsies of NHL and in a subset of Hodgkin lymphoma (HL) samples. RNAseq detected the vast majority of translocations found by traditional methods. In some cases IgH::MYC translocations were not detected by RNAseq, but for these cases expression analysis showed high MYC expression indicative of the underlying IgH::MYC translocation. In addition, both RNAseq and WES identified additional genetic abnormalities not found by conventional approaches. In several cases WES and/or RNA sequencing resolved remaining diagnostic uncertainty. Machine learning analysis of the RNA sequencing data allowed correct lymphoma subtype classification in >90% of NHL. Classification of HL was less accurate due to the low tumor content of HL.

Summary/Conclusion: The successful integration of histology and immunohistochemistry, flow cytometry, and next-generation sequencing technologies into one standardized workflow depends on close multidisciplinary collaboration of different clinical and laboratory specialists, which has been greatly facilitated by the recent centralization of diagnostics and care for pediatric cancer patients. We highlight a few cases that are exemplary of how the synergy between different diagnostic techniques and specialists can improve diagnostic accuracy and speed and thus benefit patients.

P1303: POTENTIATING ANTI-CD20 MONOCLONAL ANTIBODY THERAPY BY TARGETING COMPLEMENT C3 ACTIVATION FRAGMENTS COVALENTLY DEPOSITED ON LYMPHOMA CELLS

A. Wiestner 1,* , S. Baskar 1 , H. Peng 2 , E. Gaglione 1 , M. Lindorfer 3 , R. Taylor 3 , C. Rader 2

1 Hematology Branch, NHLBI, NIH, Bethesda; 2 Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter; 3 Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, United States of America

Background: The addition of monoclonal antibodies (mAbs) to chemotherapy or targeted agents improves survival of lymphoma patients. Fc receptor-dependent effector mechanisms achieve tumor cell killing but also can induce antigen loss through trogocytosis, which is mediated by Fc receptors on acceptor cells, contributing to treatment failure. Initiation of the complement cascade by cell-bound mAbs also deposits complement C3 activation fragments on the cell surface and this can promote downstream lysis of target cells by the membrane attack complex of complement. However, in the case of chronic lymphocytic leukemia (CLL) it is well established that not all of the mAb-targeted B cells are killed during these reactions. Instead, substantial numbers of surviving B cells that have lost CD20 due to trogocytosis and are covalently tagged with the complement breakdown product C3d are readily demonstrable in blood and bone marrow. Antigen loss due to trogocytosis with several types of mAbs has been demonstrated in lymphoma as well as in multiple myeloma.

Aims: We sought to test the hypothesis that covalently attached C3d fragments deposited on B cells by anti-CD20 mAbs constitute a neoantigen suitable for targeting antigen loss variants and, when used in combination with a complement fixing mAb, can increase efficacy of mAb therapy.

Methods: We generated mouse and rabbit mAbs specific for C3d and selected three mAbs that bind distinct epitopes to construct human IgG1 chimeric mAbs for further study. We obtained primary CLL cells from patients before and during treatment with ofatumumab on a clinical study conducted at the NIH ( NCT01145209 ) and used lymphoma cell lines HBL2 (Mantle Cell Lymphoma) and SUDHL6 (Diffuse Large B cell Lymphoma) to test in vitro and in vivo efficacy of anti-C3d antibodies. Lymphoma cell lines were injected subcutaneously, mAbs were administered i.p. starting one week after tumor challenge and continued for 4-8 weekly injections and mice were sacrificed when pre-determined limits of tumor burden were reached.

Results: For initial proof-of-concept, we demonstrated binding of a murine anti-C3d mAb (C8) to CLL cells from patients obtained 24 hours after ofatumumab administration; these cells had lost CD20 (negative on Western blot) and carried covalently-bound C3d. C8 selectively bound to C3d-opsonized CLL cells at low nanomolar concentrations and effectively killed these cells in a patient-derived xenograft mouse model. However, C8 did not react with murine C3d. Thus, for further in vivo studies, we used rabbit human IgG1 chimeric mAbs that cross-react with murine C3d. All 3 mAbs were able to mediate phagocytosis by monocyte derived human macrophages. In lymphoma xenografts, co-administration of a rabbit anti-C3d mAb with either rituximab or ofatumumab was more effective than the anti-CD20 mAb alone: in HBL xenografts median survival increased from 35 days with ofatumumab alone to 63 days for the combination (P=0.012). In SUDHL6 xenografts, median survival was 114 days for anti-CD20 (either rituximab or ofatumumab) treated mice and not reached for mice treated with anti-CD20 combined with an anti-C3d mAb (P=0.008), and tumor-free survival at 6 months was 20% for mice treated with an anti-CD20 mAb alone and 75% for mice treated with the combination. In surviving mice, there was no evidence of any adverse side effects or tissue damage.

Summary/Conclusion: Our findings indicate that C3d-targeting with a specific mAb can provide a decisive “second hit” to enhance the efficacy of complement fixing mAbs commonly used in lymphoma therapy.

P1304: THE NOVEL TUMOR SUPPRESSOR SAMHD1 CONTRIBUTES TO ANTI-TUMOR RESPONSES IN A CGAS-STING-DEPENDENT OR INDEPENDENT PATHWAY IN HODGKIN LYMPHOMA (HL)

I. Xagoraris 1,* , G. Kokaraki 1 , K. Stathopoulou 1 , P. Xyderou 1 , A. Kurzay 1 , N. Herold 2 , A. Lundqvist 1 , G. Z. Rassidakis 1

1 Department of Oncology-Pathology; 2 Department of Women’s and Children’s Health, Karolinska Institute, Stockholm, Sweden

Background: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which depletes the intracellular dNTP substrates. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS) and have been detected in a subset of chronic lymphocytic leukemia, mantle cell lymphoma, and T-prolymphocytic leukemias. Therefore, SAMHD1 may play a role in lymphomagenesis as a tumor suppressor. We have recently reported (Xagoraris et al, Br J Haematol 2021;193(3):488) that SAMHD1 is expressed by the neoplastic Hodgkin and Reed Sternberg cells (HRS) and correlates with unfavourable clinical outcome in classical Hodgkin lymphoma (cHL). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses through production of the second messenger cGAMP, which activates the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB, which induce expression of interferons and other cytokines involved in anti-tumor immune responses. SAMHD1 may reduce cytosolic DNA of either exogenous or endogenous origin, which can trigger activation of the cGAS-STING immune response pathway.

Aims: This study aimed to investigate the potential role of SAMHD1 in anti-tumor immune responses in cHL.

Methods: The in vitro system included 5 cHL cell lines (MDA-V, L-1236, HDLM-2, L-428 and KM-H2). Expression and activation (phosphorylation) of cGAS-STING pathway proteins at baseline and experimental conditions were analysed by quantitative RT-PCR and Western blot analysis at the RNA and protein level, respectively. The cHL cell lines were treated with VPX (or control DX) viral proteins to eliminate SAMHD1 as previously described (Herold et al, Nat Med 2017;23(2):256), and with a CDK inhibitor (Palbociclib) to restore SAMHD1 activity through dephosphorylation. Treatment with a STING agonist and TBK inhibitor were also used alone or in combination with other agents. Silencing of STING gene was performed using transient transfection of the cells with specific STING siRNA. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of interferon beta (IFN-β), CXCL10, interferon gamma (IFN-γ), STING, as well as, a control gene (GAPDH) with qRT-PCR.

Results: STING expression at the mRNA and protein level was substantially higher in L-1236, HDLM-2, and L-428 compared to other cHL cell lines. Depletion of SAMHD1 by VPX resulted in significantly lower levels of IFN-β and CXCL10 in cHL cells, which was linked to decreased NK cell killing of the cHL cells in vitro . Inversely, treatment with Palbociclib resulted in SAMHD1 dephosphorylation thus restoring SAMHD1 activity, which was associated with increased but differential expression of IFN-β and CXCL10 genes among the cHL cell lines. These effects were associated with activation of the cGAS-STING pathway (TBK1 phosphorylation) in cHL cells. Treatment with STING agonist alone stimulated immune responses by increased IFN-β and CXCL10 levels indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Inversely, STING gene silencing resulted in decreased CXCL10 but not IFN-β gene expression.

Summary/Conclusion: SAMHD1 contributes to anti-tumor immune responses in cHL, in part, through modulation of the cGAS-STING pathway. As STING agonists and Palbociclib are currently used in clinical trials for certain malignancies, our findings might have direct therapeutic implications in patients with cHL.

P1305: HSA_CIRC_0001599 REGULATES IL18 LEVELS IN AGVHD VIA CERNA MECHANISM

M. Xia 1,* , J. Zhang 2 , Y. Dong 3 , X. Lv 3 , X. Cui 3

1 The First School of Clinical Medicine, Shandong University of Traditional Chinese Medicine; 2 Department of Hematology, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine; 3 The Oncology Center, The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China

Background: AGVHD (acute graft-versus-host disease) is one of the major complications after hematopoietic stem cell transplantation, and severe aGVHD not only seriously affects the outcome of transplantation but also may pose a threat to the patient’s life.The gastrointestinal system is one of the areas most strongly affected by aGVHD.

Aims: This study was dedicated to investigating the pathogenic mechanism of hsa_circ_0001599 in intestinal acute graft-versus-host disease (aGVHD).

Methods: Four patients with intestinal aGVHD and four healthy volunteers were invited to have peripheral blood drawn early in the morning on an empty stomach. After treatment, peripheral blood mononuclear cells (PBMCs) were obtained, and after further testing and purification, RNAs that met the experimental requirements were obtained from them. After screening circRNAs with abnormal expression levels by high-throughput sequencing as well as data processing, the levels of specific circRNAs in 20 patients were verified by qRT-PCR. Then, the ceRNA interaction network was constructed by bioinformatics analysis using GO, KEGG and miRanda. Finally, clinical patients’ correlations were analyzed to determine the sensitivity and specificity of disease-related indicators for disease diagnosis.

Results: 1. High-throughput sequencing results showed that a total of 330 circRNAs showed bidirectional expression in the aGVHD group relative to the HC group, of which 8 were up-regulated and 322 were down-regulated (FC≥2.0, P≤0.05) (Figure 1 A).

2. PCR validation of high level expression of hsa_circ_0001599

The PCR results showed that the mean expression level of hsa_circ_0001599 in the aGVHD group was more than twice that of the control group. (Figure 1 B)

3. Analysis of differentially expressed genes involved in intestinal aGVHD and pathway prediction

Analysis by GO and KEGG enrichment indicated that the upregulation of circRNA expression may have an impact on the pathogenesis of intestinal aGVHD (Figure 1 C).

4. Construction of circRNA-miRNA-mRNA interaction network

The hsa_circ_0001599-targeted mRNA: IL18 was predicted using miRanda, TargetScan and other databases, and the ceRNA interaction network was constructed (Figure 1 D).

5. We analyzed the relationship between clinical characteristics, hsa_circ_0001599 and IL18 in 15 patients with intestinal aGVHD. The results showed that the level of hsa_circ_0001599 was positively correlated with IL18 level in intestinal aGVHD patients.(R2=0.832, P<0.05). (Figure 1 E)

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Summary/Conclusion: The aberrant expression of hsa_circ_0001599 in PBMCs may be a new diagnostic marker and therapeutic target for intestinal aGVHD.

P1306: CLINICAL AND GENOMIC ANALYSIS OF HIGH-GRADE TRANSFORMATION OF MARGINAL ZONE LYMPHOMA

C. Yannakou 1,2,* , D. Villa 3,4 , C. Gould 2,5 , Y. Kankanige 2,5 , J. Lickiss 2,5 , G. Ryland 2,5 , S. Lade 2,5 , G. Slack 3 , W. Limvorapitak 6 , C. Tam 2,7 , J. Seymour 7 , D. Westerman 2,5 , P. Blombery 2,5

1 Department of Molecular Oncology and Cancer Immunology, Epworth HealthCare; 2 Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia; 3 BC Cancer Centre for Lymphoid Cancer; 4 University of British Columbia, Vancouver, Canada; 5 Pathology Department, Peter MacCallum Cancer Centre, Melbourne, Australia; 6 Faculty of Medicine, Thammasat University, Pathumthani, Thailand; 7 Department of Haematology, Peter MacCallum Cancer Centre & Royal Melbourne Hospital, Melbourne, Australia

Background: Marginal zone lymphoma (MZL) is an indolent B-cell non-Hodgkin lymphoma with splenic, nodal and mucosa-associated (MALT) subtypes that may undergo high-grade transformation (tMZL).

Aims: We aimed to describe the clinical features, treatment outcomes and genomic characteristics of tMZL.

Methods: Patients with a diagnosis of tMZL managed at the Peter MacCallum Cancer Centre (PMCC) and the British Columbia Cancer Agency from 2002–2018 were identified from institutional databases. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tMZL tissue biopsies of >60% tumour burden. DNA sequencing was performed using the PMCC PanHaem hybridisation capture panel of genes recurrently mutated in haematological malignancy. Expression of immune-related genes was assessed using the NanoString nCounter ® Immunology Panel (V2).

Results: 43 patients with tMZL (21 males, median age 66 years (range 40-86), 74% advanced stage) were identified. MZL subtypes were: 12 splenic, 15 nodal and 16 MALT. 35% had tMZL at first presentation, the remainder had a median time from diagnosis to transformation of 54.5 months (range 3-270). 39 were diffuse large B-cell lymphoma (DLBCL), 1 was Hodgkin lymphoma (HL) and 3 were composite lymphomas (2 DLBCL/HL, 1 DLBCL/follicular lymphoma). Of the 26 cases in which cell of origin was assessed by immunohistochemistry 69% were non-GCB.

First line treatment of tMZL was with anthracycline containing chemoimmunotherapy in 84% of cases. 40% of patients received consolidation with autologous stem cell transplantation.

For the 41 patients treated intensively the complete remission rate to first line treatment was 80%. Median progression free survival and overall survival were 37 and 76 months respectively.

DNA sequencing was performed in 25 cases. The most recurrent aberrations (>15% cases) involved genes involved in DNA damage repair, cell cycle regulation, Ras/Raf/MAPK signalling, epigenetic regulation, lymphocyte development/signalling, apoptosis and Notch signalling (Figure 1A). TP53 loss of function aberrations were detected in 24% of sequenced tMZL cases while genes involved in cell cycle regulation were mutated and/or deleted in 40%. Concurrent MYC and BCL2 translocations (evaluated in 26 cases) were detected by FISH and/or sequencing in only a single case (GCB origin).

To evaluate the immune landscape of tMZL we performed targeted gene expression profiling. 23 tMZL were compared to 55 de novo DLBCL and 16 transformed follicular lymphoma (tFL) cases. Unsupervised clustering did not stratify cases by disease (tMZL/DLBCL/tFL) but did identify two distinct groups characterised by ‘hot’ and ‘cold’ tumour immune microenvironment (TIME) profiles. This dichotomy remained evident within tMZL when cases were stratified by disease (Figure 1B). When focussing on genes involved in the immune response, ‘hot’ tMZL samples were characterised by overexpression of genes related to immune cells, immune checkpoints and antigen presentation.

The most upregulated genes in tMZL compared to de novo DLBCL were TNFRSF13 and CXCL13 (both logFC ≥2.0, p<0.05) while the most downregulated gene was FN1 (logFC -1.6, p=0.0057). When tMZL was compared to tFL the most upregulated genes were CCR6 , TBX21 and IFNG (all logFC ≥1.7, p<0.05) while MME was the most downregulated (logFC -2.6, p=0.012).

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Summary/Conclusion: We have described the clinical features, treatment outcomes and genomic characteristics of tMZL. Genomic data implicate TP53 and cell cycle dysregulation as contributors to the pathogenesis of tMZL, as is the case in tFL. ‘Hot’ and ‘cold’ tMZL TIME profiles were identified.

P1307: TESTING THE IMPORTANCE OF ANTIOXIDANTS ON THE PATHOGENESIS OF B CELL LYMPHOMA

H. Yao 1,* , M. Bergo 1

1 BioNut, Karolinska Institutet, Huddinge, Sweden

Background: Our group found that antioxidant supplementation of the diet – in doses that reduce reactive oxygen species (ROS) levels – markedly accelerates progression and metastasis of malignant melanoma and lung cancer in mice. These findings can help explain the puzzling outcome of clinical trials with antioxidants and led to a heated debate about the safety of supplements for cancer patients. The findings, combined with those of dozens of clinical antioxidant trials, suggest that cancer patients and survivors should avoid antioxidant supplements. Although multiple other cancer types including breast, colon, and pancreatic cancer has been found to respond similarly to antioxidants, little is known about the effects in hematologic malignancies. In the latter, high doses of VitC – that increase oxidative stress – have shown therapeutic effects in some studies, including in B cell lymphoma. However, other studies have raised doubts about high-dose VitC therapy. Thus, many questions remain about the impact of antioxidant administration for B cell lymphoma progression, in particular in ROS-lowering doses.

Aims: Define the impact of antioxidant administration on B cell lymphoma cell proliferation and viability in vitro and on the progression and survival of lymphoma in vivo.

Methods: For in vitro experiment, we performed analyses of cell proliferation, cell cycle, apoptosis, and ROS levels; RNA from cells following interventions was isolated for RNAseq analysis; real-time PCR was performed to confirm candidate genes; and ChIP-qPCR to study protein-DNA binding. For in vivo experiments, we used xenograft models by subcutaneous injection of human B lymphoma cell lines, and used λMYC mice as a model of endogenous B cell lymphoma (these mice harbor a mutant human MYC gene controlled by a reconstructed Igλ locus). In vivo antioxidants were administered both orally in the drinking water, and was injected in different doses and combinations.

Results: The antioxidants N-acetylcysteine (NAC), vitamin C (VitC), L-Cystein, and glutathione in doses that reduce ROS levels markedly reduced the viability of human B cell lymphoma cell lines in a MYC-dependent fashion. Intraperitoneal injections of NAC and VitC dose-dependently reduced tumor growth in xenograft experiments. Knockdown of MYC restored tumor growth in the presence of antioxidants and switching-on MYC expression in B cells rendered them sensitive to NAC and VitC. Molecular analyses revealed that antioxidants increased the binding of EGR1 to MYC and stimulated EGR1/MYC binding to apoptotic gene promotors—thus stimulating apoptosis. Intraperitoneal injections or life-long oral antioxidant administration reduced tumor growth and improved survival in λMYC transgenic mice.

Summary/Conclusion: High MYC expression renders B lymphoma cells sensitive to therapy with common soluble antioxidants and indicates that this strategy might be useful—alone or in combination with lower doses of standard chemotherapy—in patients with MYC-driven lymphomas such as the diffuse large B cell variant.

P1308: SELECTIVE SMALL MOLECULE INHIBITION OF CTP SYNTHASE 1 (CTPS1) SUPPRESSES T CELL PROLIFERATION AND CYTOKINE RELEASE, HIGHLIGHTING A NOVEL THERAPEUTIC TARGET FOR GRAFT-VERSUS-HOST DISEASE

P. Beer 1,* , A. Parker 1 , H. Asnagli 1

1 Step Pharma, Saint-Genis-Pouilly, France

Background: Graft-versus-host disease (GvHD) significantly increases both the morbidity/mortality and economic cost of allogeneic haematopoietic stem cell transplantation. Key to successful therapy is effective disease control whilst maintaining a competent anti-infection response. Acute GvHD (aGvHD) is driven by the rapid proliferation of, and cytokine release from, donor-derived T cells. Standard of care therapy with systemic steroids is associated with broad and deleterious immunosuppression, and fails to achieve adequate disease control in a third of patients. There is currently a lack of consensus around therapy beyond steroids [1]. CTP synthases catalyse the conversion of UTP to CTP, the rate limiting step in pyrimidine synthesis. Humans have two isoforms of this enzyme (CTPS1 and CTPS2); human genetic studies have identified an essential and non-redundant role for CTPS1 in the proliferation of B and T cells [2].

[1] Blood. 2020 May 7;135(19):1630-1638. [2] Nature. 2014 Jun 12;510(7504):288-92. [3] J Transl Med. 2010 Oct 26;8:104.

Aims: To assess the impact of selective CTPS1 inhibition on the proliferation of, and cytokine release from, human T cells.

Methods: Aliquots of whole blood or isolated peripheral blood mononuclear cells (PBMC) from healthy volunteer donors underwent T cell activation with CD3/CD28 beads and incubation with or without a selective CTPS1 inhibitor for 24-72 hours. Levels of intracellular CTP, ATP and GTP nucleotides were quantitated by liquid chromatography–mass spectrometry; cell proliferation was assessed by tritiated thymidine incorporation, Ki-67 by flow cytometry and cytokine release by analysis of plasma using the Luminex platform.

Results: STP938 is a potent and reversible low molecular weight small molecule inhibitor of CTPS1, which shows >1,300-fold selectivity for human CTPS1 over CTPS2. Exposure of stimulated PBMCs to STP938 resulted in a concentration dependent depletion of CTP but had no effect on the levels of GTP or ATP, confirming selective inhibition of CTP synthase by STP938. CTPS1 inhibition by STP938 elicited a concentration dependent inhibition of CD3/CD28-induced T cell proliferation in whole blood samples after 72 hours incubation, resulting in full suppression of proliferation at the highest concentration tested (1 µM) (Figure A). Exposure to STP938 also reduced the proportion of T cells in cell cycle, as evidenced by a decrease in the expression of Ki-67 (Figure B). STP938 exposure induced a concentration dependent inhibition of T cell cytokine release in whole blood samples, with higher STP938 concentrations producing near-complete suppression (Figure C). Notably, significant inhibition of T cell cytokine release was observed after only 24 hours’ exposure of stimulated T cells to STP938 (Figure D, 1 μM STP938). Given that human T cells do not undergo cell division until at least 40 hours after CD3/CD28 stimulation is initiated [3], these findings indicate that the ability of CTPS1 inhibition to prevent cytokine release is independent of its effects on cell proliferation.

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Summary/Conclusion: CTPS1 inhibition by STP938 induces a rapid suppression of T cell cytokine release along with suppression of T cell proliferation; both of these effects are concentration dependent and near maximal at exposures of STP938 that are likely to be achievable in the clinic. CTPS1 represents a novel target with the potential to effect rapid and specific disease control in aGvHD. STP938 is currently on the path to clinical development in lymphoid malignancy.

P1309: DYSREGULATED EXPRESSIONS OF INHIBITORY CHECKPOINT MOLECULES AND THEIR LIGANDS ON T-CELLS AND BLASTS IN AML RELAPSES AFTER STEM CELL TRANSPLANTATION (SCT)

S. Bohlscheid 1,* , G. Filippini Velazquez 2 , H. Aslan 1 , T. Baudrexler 1 , H. Schmetzer 1 , C. Schmid 2

1 Medical Department III, Department for Hematopoietic Cell Transplantation, Munich University Hospital, Munich; 2 Department of Hematology and Oncology, Section for Stem Cell Transplantation, Augsburg University Hospital, Augsburg, Germany

Background: Upregulation of inhibitory checkpoint molecules (ICM) on T-cells and their ligands on leukemic blasts may be a mechanism of acute myeloid leukaemia (AML) relapse after allogeneic stem cell transplantation (SCT).

Aims: Better understanding of relapse biology could improve treatment efficacy.

Methods: We examined peripheral blood (PB) and bone marrow (BM) samples of 5 AML patients (PTs) relapsing after SCT, and PB of 5 healthy individuals (H), including 2 stem cells donors. ICM (PD-1, CTLA-4) expressions on T-cells and their ligands (CD86, PD-L1, PD-L2) on leukemic blasts were assessed through flow cytometry. PTs’ PB was cultivated with and without “KitM” (GM-CSF+PGE-1) to generate leukemia-derived dendritic cells (DC leu ), followed by MLC, enriched by PTs’ or donors’ T-cells. After MLC, immune activation and functionality (degranulation, intracellular cytokine production, blast lysis) was assessed.

Results:

1. Whilst all patients showed high expressions of PD-1 on their T-Cells, additional overexpression of CTLA-4 was negatively correlated with responses to relapse treatment. Expression of ICM was low on T-cells from 4/5 healthy individuals (Figure 1).

2. Influence of KitM on ICM/ligand expressions on T-cells/blasts

- ICM/ligand expressions on uncultured T-cells/blasts: In contrast to H, PTs presented high co-expressions of CTLA-4 and PD-1 on PB T-cells (Figure 1). In addition, PTs showed high frequencies of PB/BM blasts co-expressing CD86.

- DC/DC leu in PB: Generation of DC leu in AML as well as generation of DC in H was successful with KitM pretreated PB vs. Control.

- ICM co-expression on T-cells after MLC with KitM pretreated PB: MLC of KitM treated PB enriched with unstimulated PTs T-cells resulted in reduced frequencies of ICM-positive T-cells in 3/5 PTs, and increased frequencies of activated (leukemia specific) T-cells in 3/5 PTs. Beyond, blast lysis was improved in 4/5 samples treated with KitM. Both findings were not observed when PB cells were cultivated in the absence of KitM.

3. Possible impact of ICM profiles on clinical outcome in a particular case PT1 suffered from early relapse both after 1 st and 2 nd SCT from her healthy father. A role of ICM in relapse mechanisms was suggested by CTLA-4/PD-1 expression on her T-cells and CD86 expression on AML blasts. In addition, >90% of the healthy father’s T-cells expressed CTLA-4/PD-1, which might have contributed to treatment failure. In contrast, T-cells from PT1’s mother presented with low ICM levels (Figure 1), suggesting that the mother might have been a better donor. Stimulation of PT1’s PB cells with KitM generated DCleu, decreased ICM-expressions and increased T-cell activity. KitM pretreated samples also revealed improved blast lysis after MLC.

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Summary/Conclusion: T-cells and blasts of AML PTs relapsing after SCT uniformly co-expressed ICM and their ligands, which could be a reason for inferior immune responses. High aberrant ICM-expressions, in particular CTLA-4, on donor T-cells could be responsible for relapse after SCT by inactivating antileukemic immune reactions. Hence, checkpoint-inhibitory antibodies might represent a specific therapeutic option in these patients. Further, interactions between ICM-expressions on healthy (fresh) donor T-cells could have an impact in therapy responses and should be evaluated in donor selection. Finally, generation of DC leu by treatment with KitM triggers immune responses in MLC alongside with reduced ICM-expressions on T-cells, possibly reducing their inhibitory effects and therefore improving antileukemic responses.

P1310: A ROBUST APPROACH FOR THE GENERATION OF FUNCTIONAL HEMATOPOIETIC PROGENITOR CELL LINES TO MODEL LEUKEMIC TRANSFORMATION

E. Doma 1,* , I. Mayer 1 , A. Hoelbl-Kovacic 1 , R. Grausenburger 1 , K. Kollmann 1 , V. Sexl 1

1 Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Vienna, Austria

Background: Preclinical research aims at developing robust methods to maintain, expand and manipulate haematopoietic stem/progenitor cells (HSPC) ex vivo , as these cells are infrequent and their immortalization is hardly achieved. The transcription factor, LIM homeobox protein 2 (LHX2), has a critical role in maintaining self-renewal and has been reported to facilitate ex vivo expansion of immature HSPCs.

Aims: We aimed to establish a robust and low-cost murine HSPC in vitro model system that allows maintenance and expansion of functional LSK (lin - , Sca-1 + , c-Kit + ) cells, and guarantees long-term multipotency.

Methods: We generated HPC LSK cell lines from high-purity sorted murine LSK cells by Lhx2 overexpression. SCF/IL-6 dependent HPC LSK cells are kept on agarose-coated plates to prevent adherence-induced myeloid differentiation. HPC LSK cells can be genetically modified e.g. by retroviral transduction, which serves as a source to generate LSCs harboring hematopoietic stem cell oncogenes including BCR/ABL p210+ , MLL-AF9 or FLT3-ITD/NRAS G12D . Immunophenotyping, transcriptome analysis and functionality tests of HPC LSK and LSC lines were carried out.

Results: HPC LSK cells preserve LSK markers despite continuous proliferation. HPC LSK cells efficiently home to the BM, blood, spleen, and thymus and are able to differentiate into myeloid and lymphoid lineages in vitro and in vivo . Numbers of HPC LSK –derived myeloid and lymphoid progenitors in the BM and differentiated blood cells are comparable to BM-injected mice. Transcriptome analysis of HPC LSK lines showed a ST-HSC/MPP1 signature, which corresponds to the earliest proliferating HSPCs.

Injection of transformed HPC LSK -LSC (BCR/ABL p210 , MLL-AF9, FLT3-ITD/NRAS G12D ) lines in immunocompromised mice induced myeloid leukemia. All diseased animals displayed elevated WBC counts, blast-like cells in the blood and suffered from splenomegaly. To test the applicability of the model system for drug sensitivity tests we employed Imatinib mesylate (IM). As in patients, HPC LSK -BCR/ABL p210 cells show partial IM resistance and leads to the enrichment of undifferentiated LSK cells.

Summary/Conclusion: We created a robust method of culturing functional HSPCs, which are immortalized and can be expanded indefinitely. HPC LSK lines can be rapidly generated from transgenic mouse models and are a useful system for functional assays that require high cell numbers. By oncogenic transformation we created leukemia triggering LSCs. These findings support the relevance of HPC LSK cell system that represents a unique tool to compare LSCs to non-transformed HPC LSK cells in vitro and in vivo , which can be adapted for high-scale preclinical compound screening.

P1311: GRANULOCYTE COLONY-STIMULATING FACTOR MOBILIZATION FACILITATES ANTI-GRAFT-VERSUS-HOST DISEASE AND GRAFT-VERSUS-LEUKEMIA EFFECTS OF MUCOSA-ASSOCIATED INVARIANT T CELLS THROUGH DISTINCT FUNCTIONAL SUBSE

M. Gao 1,* , Y. Sun 1 , J. Kong 1 , Y. Chang 1 , Y. Wang 1 , X. Huang 1 , X. Zhao 1

1 Peking University People’s hospital, Beijing, China

Background: Graft-versus-host disease (GVHD) and relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are the main reasons leading to transplantation failure and affecting the survival of patients. Granulocyte-colony stimulating factor (G-CSF) is used to mobilize stem cells in healthy donors and promote T cell hyporesponsiveness and polarization of T cells from Th1 to Th2. Mucosal-associated invariant T cells (MAITs) are a group of multifunctional innate immune T cells, which can be activated through TCR-dependent or independent pathways, and tight integration of these signals is critical for triggering one function versus another. MAITs can exert anti-gut GVHD effects by expressing mucosal protective factors such as IL-17, while activated MAITs have certain anti-tumor effects by expressing cytotoxic factors. The two seemingly contradictory functions appear to be segregated in two distinct subsets expressing T-bet or RORγt. Taken together, G-CSF mobilization in transplantation is a critical factor affecting the cellular composition of the infused grafts. However, the effects of G-CSF on the differentiation of MAITs to various subsets and the exertion of different effector functions remain to be further elucidated. Thus, we aimed to explore the effect of G-CSF mobilization on the anti-GVHD or GVL effect of MAITs.

Aims: We aimed to explore the effect of G-CSF mobilization on the anti-GVHD or GVL effect of MAITs.

Methods: MAITs from peripheral blood (PB) of 4 healthy donors before and after G-CSF mobilization were analyzed by single-cell RNA sequencing (RNA-seq) and PB from 16 healthy donors before and after G-CSF mobilization was tested by flow cytometry (FCM). In vitro experiment was performed to observe the effect of G-CSF mobilization on the anti-tumor effect of MAITs.

Results: First, data showed that MAITs were clustered in 13 clusters (Figure 1A and 1B), of which Cluster 1 exhibited high expression of cytotoxic molecules, including GZMA, GZMB, GZMK, and NKG7, which might be the most important anti-tumor effector subsets. And T-bet+IFN-γ+MAITs were mainly enriched in Cluster 1, which also suggested that Cluster 1 was the main effector MAIT1 subset. Second, G-CSF mobilization may promote the distribution of MAITs to cluster 1, and prompt MAITs to differentiate into T-bet+MAIT (MAIT1) that exert GVL effects and GATA3+MAIT (MAIT2) that have immunomodulatory effects, but the GVL effect of MAIT1 was limited to a certain extent, showing a state of hyporesponsiveness or inhibitory that the main effectors IFN-γ was at low expression and the inhibitory markers PD-1 and TIGIT was at high expression compared with pre-G-CSF. In terms of effector expression, G-CSF mobilization had a trend toward MAIT2 polarization (Figure 1C). In contrast, compared with pre-G-CSF mobilization, there was no significant change in RORγt+MAIT (MAIT17), but the increased expression of chemokine receptors (CCR6, CXCR3) after mobilization, which suggested that MAIT17, once activated by specific stimulatory signals, enhanced chemotaxis to inflammation or specific tissues. Finally, in vitro mixed culture of MAITs and K562, results showed that the stimulated MAITs had a certain anti-tumor effect, but G-CSF mobilization had no obvious effect on the enhancement or weakening of anti-tumor function.

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Summary/Conclusion: G-CSF mobilization facilitates the segregation of MAITs anti-GVHD and GVL dual effects, inducing immune tolerance while promoting differentiation of GVL effector subsets, while maintaining or increasing the initiation of anti-GVHD effect.

P1312: FORODESINE AMPLIFIES HOST INNATE IMMUNE RESPONSE THROUGH TOLL-LIKE RECEPTOR 7 ACTIVATION WHILE PREVENTING EXPERIMENTAL GRAFT-VERSUS-HOST DISEASE

T. Ikeda 1,* , K. Sato 1 , S.-I. Kawaguchi 1 , H. Nakano 1 , J. Izawa 1 , N. Takayama 1 , H. Hayakawa 2 , T. Nagayama 1 , K. Umino 1 , K. Morita 1 , K. Matsumoto 3 , K. Ushijima 4 , Y. Kanda 1

1 Division of Hematology, Department of Medicine; 2 Core Center of Research Apparatus, Jichi Medical University, Tochigi; 3 Department of clinical pharmaceutics, Faculty of pharmaceutical sciences, Doshisha Women’s College of Liberal Arts, Kyoto; 4 Sanyo-Onoda City University, Sanyoonoda, Yamaguchi, Japan

Background: Forodesine, a novel inhibitor of purine nucleoside phosphorylase (PNP), causes nucleotide pool imbalances by accumulating intracellular deoxyguanosine, thereby resulting in selective apoptosis of T-cells. Recent evidence indicates that specific types of nuclear acids including guanosine and its derivatives act as natural ligands for toll-like receptor 7 (TLR7). So far, it is unclear whether and how forodesine can modulate host innate immunity, including antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), which express TLR7 abundantly.

Aims: The aim of this study is to investigate the role of forodesine in host APC function as well as donor T-cell activation both in vivo graft-versus-host disease (GVHD) models and in vitro cell assays.

Methods: NOG mice were subjected to 250 cGy total body irradiation followed by intravenous injection of human isolated T-cells. Mice were treated with forodesine (20 mg/kg) or vehicle daily after transplantation.

Results: Despite the mice without treatment developing severe GVHD after transplantation, guanosine and deoxyguanosine were hardly detected in the plasma, suggesting that purine nucleosides released from damaged tissues were rapidly degraded by PNP. In fact, inhibition of PNP activity by forodesine markedly increased plasma guanosine levels, whereas deoxyguanosine was still undetectable. This was also the case in GVHD target organs, including liver and lungs. At the same level of guanosine in the liver, forodesine promoted TNF-a production from in vitro alveolar macrophage cultures. Preincubation with a TLR7 antagonist, ODN20958 , attenuated the TNF-a production from macrophages stimulated by guanosine and forodesine. Nuclease treatment did not reduce macrophage TNF-a production, suggesting that single-strand RNA, a well-known TLR7 ligand, is not involved in this response. Similar results were observed in Flt3-ligand-induced plasmacytoid DCs. Not only TNF-a but also IL-12 production was induced by the combination of guanosine and forodesine, and was comparable to that in pDCs pulsed with R848, a potent agonist of TLR7/8. These observations suggest that a sustained high level of guanosine caused by forodesine treatment lead to continuous activation of TLR7 signaling, resulting in cytokine production from APCs. Meanwhile, as reported previously, forodesine inhibited the proliferation of human T-cells activated with CD3 and CD28 antibodies in the presence of deoxyguanosine. Of note, however, this combination did not impair but rather stimulated IFN-g production from activated T-cells cocultured with pDCs. This suggests that T-cell priming capacity mediated by the interaction with pDCs is conserved through TLR7 activation. Finally, we investigated the in vivo effects of forodesine on survival, body weight change, and disease severity of GVHD mice. Forodesine treatment significantly prolonged survival of the mice compared with control mice, with a median survival of 17.5 days vs 14 days, respectively ( p = 0.029). The mean body weight loss percentage at day 9 was 11.5% vs 19.0% in the forodesine group and control, respectively ( p = 0.003). Furthermore, the clinical GVHD scores were also lower in the forodesine group vs control (4.9 vs 0.6 points, p = 0.001).

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Summary/Conclusion: This study demonstrates that forodesine amplifies host innate immune response through TLR7 signaling, while ameliorating T-cell GVH reaction. Further studies are needed to investigate whether forodesine can provide host protection against infections in a transplant setting.

P1313: PREDICTING OUTCOMES IN AML PATIENTS CONSOLIDATED BY AN ALLOGENEIC STEM CELL TRANSPLANTATION USING THE KNOWLEDGE BANK APPROACH

M. Jentzsch 1,* , L. Herrmann 1 , L. Bischof 1 , D. Backhaus 1 , D. Brauer 1 , G.-N. Franke 1 , V. Vucinic 1 , U. Platzbecker 1 , S. Schwind 1

1 Medical Clinic and Policlinic 1, Hematology, Cellular Therapy and Hemostaseology, Leipzig University Hospital, Leipzig, Germany

Background: Current risk stratification systems in acute myeloid leukemia (AML), e.g. by the European LeukemiaNet (ELN) 2017, help to inform treatment decisions, including those for an allogeneic stem cell transplantation (HSCT) in first remission. Recently Gerstung et al. (Nature Genetics, 2017) developed a knowledge bank (KB)-based algorithm to in order to more precisely predict individual outcomes based on demographic, clinical, and genetic data. Two studies validated the feasibility of the KB prediction in AML patients (pts) consolidated with chemotherapy. However, a validation in a HSCT-treated cohort - crucial with respect to informed decisions towards HSCT - is lacking.

Aims: To validate the feasibility of KB outcome prediction in a HSCT-treated cohort.

Methods: We analyzed 545 AML pts (median age at diagnosis 62, range 21-77 years) consolidated by reduced-intensity (23%) or non-myeloablative (77%) allogeneic HSCT. 60% of pts were in first remission. All clinical variables included in the KB were available for this cohort, while our gene mutation panel did not cover 17/58 of genes included in the KB. KB predictions 3 years after diagnosis were retrospectively calculated by using the adapted transplant strategy and compared to the observed outcomes using receiver operating characteristics (ROC) curves. Additionally, the measurable residual disease (MRD) status at HSCT - based on NPM1 mutation and BAALC , MN1 , and WT1 expression - was evaluated by digital droplet PCR in pts with material available.

Results: The area under the curve (AUC) to predict 3-year overall survival (OS) produced by the KB value was 0.69 (95% CI 0.62-0.72), not significantly different compared to the AUC based on ELN2017 risk groups (0.66 [95% CI 0.57-0.71], P = 0.23, Figure 1A), and worse compared to the published results in chemotherapy consolidated pts (AUC KB = 0.80, Bill et al, J Hematol Oncol 2021). Still, in a multivariate analysis the KB prediction for 3-year OS significantly impacted OS (OR 6.25, CI 2.9-13.2) after adjustment for the MRD-corrected remission status at HSCT. Aikaike Information criterion comparison with a model including the ELN2017 risk groups and the MRD-corrected remission status at HSCT demonstrated the model containing the KB prediction as preferable. When introducing cut-offs according to the KB value for OS at 3 years similar to Fenwarth et al. (Blood 2021), we observed a clear separation of OS curves according to a KB value of <20, 20-39, and ≥ 40 with higher values indicating a higher likelihood for OS ( P < 0.001, Figure 1B). Regarding additional endpoints, the KB algorithm had the highest probability to correctly predict death without previous achievement of a remission (AUC KB = 0.75, Figure 1C, black), restricted prediction for death in first remission (AUC KB = 0.61, Figure 1C, red) or after relapse (AUC KB = 0.63, Figure 1C, blue), but good prediction for being alive in first remission (AUC KB = 0.69, Figure 1D, red) or after relapse (AUC KB = 0.77, Figure 1D, blue).

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Summary/Conclusion: For HSCT treated AML pts the KB-based outcome prediction was inferior compared to previous studies of pts receiving chemotherapy. The likely reason for this inferiority is the introduction of confounders ( e.g donor selection or conditioning regimens), that may impact treatment-related mortality, and are not integrated in and not predictable by the current KB algorithm. Future versions of the KB that also consider HSCT-associated factors might further improve outcome prediction by this promising clinical tool.

P1314: PURIFICATION AND FUNCTIONAL CHARACTERIZATION OF CD34+ CELLS FROM CRYOPRESERVED APHERESIS PRODUCTS OF PATIENTS WITH BONE MARROW INFILTRATING MALIGNANCIES

F. Muller 1,* , K. Wendland 1 , S. Petkovic 1 , J. Strobel 2 , A. Mackensen 1

1 Department of Internal Medicine 5, Haematology and Oncology; 2 Transfusion Medicine, University Hospital of Erlangen, Erlangen, Germany

Background: In vivo models to study activation of human T cells or NK cells against malignancies depend on immune compromised mice in which a xeno -transplanted tumor is treated with allogeneic T cells not matching the HLA of the tumor. Immunologically, these models are far from representative. With the goal of developing a lymphoma-matching, humanized model system, we tested the potency of cryo-preserved, human CD34 + hematopoietic stem cells to engraft the murine bone marrow niche of immune compromised mice, which are known to take human tumor grafts at high rates, thus, allowing for autologous primary tumor grafts following BM engraftment.

Aims: To develop and characterize a lymphoma-matching, humanized mouse model with funtinal immune cell subsets.

Methods: We developed a method to isolate viable human CD34 hi and CD34 int cells from cryopreserved apheresis products of patients with BM infiltrating disease such as multiple myeloma and B-cell NHL. Colony-forming potential was assessed using CFU assays. Engraftment potential was tested by injecting irradiated NOD. Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice with CD34 hi or CD34 int populations and engraftment quality and duration was assessed by flow cytometry of murine peripheral blood and of spleen and BM post mortem .

Results: Initial frequency of CD34 + cells in cryopreserved apheresis products ranged from 1-5% and was enriched to > 96% CD34 hi cells after CD34 MACS separation. A large population of CD34 int cells not selected by CD34 MACS was purified by column-free CD3 T cell depletion. While CD34 hi cells gave rise to all lineages in in vitro CFU assays, CD34 int cells produced fewer colonies overall, with a less efficient differentiation towards myeloid lineage compared to CD34 hi . In vivo engraftment of CD34 hi cells from 3 of 3 patients gave rise to human CD45 + immune cells. In contrast, none of CD34 int cells resulted in more than 20% human cells in peripheral blood. Engraftment of immune cell subsets varied between the three distinct donors. Generally, neutrophils and monocytes were detected from week 3 to 4 and rates declined quickly to undetectable at week 9, B cell engraftment showed highest variability between donors.T-cell engraftment followed from week 4 to 6. From 9 weeks, human T-cells further expanded without signs of GvHD. Functional T cell analysis showed enrichment of effector memory T cells and low numbers of naïve T cells which frequently showed exhausted or senescent phenotype.

Summary/Conclusion: Cryopreserved apheresis products present a possible source of CD34 + stem cells that can be further differentiated into CD34 hi and CD34 int cells with distinct stemness features and engraftment potential. Long-term engraftment of T-cells from cryopreserved, disease-bearing BM from patients is feasible and may allow e.g. testing of T-cell recruiting agents such as bispecific antibodies or CAR T cells.

P1315: ENGRAFTMENT AND MOBILIZATION OF HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS ALIGNS WITH DYNAMIC OSCILLATION OF TETRASPANIN CD9

W. H. Ng 1,* , C. Zhang 1 , H. Wang 1 , K. Y. Y. Chan 1 , K. T. Leung 1

1 Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong, Hong Kong

Background: Entry and exit of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche require highly orchestrated molecular programs. Our group previously demonstrated the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis controlling homing of human CD34+ HSPCs but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remain largely unknown.

Aims: This study was designed to (1) map the fate of HSPC engraftment following CD9 neutralization; (2) track the changes of CD9 expression during hematopoietic reconstitution and mobilization; (3) validate the oscillation of CD9 on donor HSPCs residing in the BM and circulation; and (4) address the mechanisms of CD9 governing HSPC trafficking.

Methods: Cord blood (CB) CD34+ cells were exposed to a CD9 neutralizing antibody prior to infusion into irradiated NOD/SCID mice, with HSPC engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitor) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was determined by flow cytometry and compared with those before infusion or those undergone G-CSF-induced mobilization. The expression of CD9 were further characterized on HSPCs from normal donors in pairwise BM and peripheral blood (PB) samples, and on those in G-CSF-mobilized peripheral blood (MPB). Activities of the integrin VLA-4 (an established binding partner of CD9) were evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay.

Results: A short exposure of CB CD34+ cells to the CD9 neutralizing antibody substantially reduced their engraftment in the BM by 64.2% denoted by the human CD45+ population ( P =0.003). No bias in lineage commitment was reflected upon CD9 blockade, suggesting engraftment of HSPCs is affected in general but not limited to specific subset of hematopoietic precursors.

Comparing with those before transplantation, CD9 expression was significantly elevated on engrafted CD34+ cells retaining in the BM from 9.6% to 91.4% ( P <0.001). Contrarily, G-CSF administration downregulated CD9 expression on BM CD34+ cells from 73.4% to 33.5% ( P <0.001) where the decline was observed in both primitive stem cells and committed progenitors, indicating that the oscillation of CD9 is highly associated with HSPC trafficking.

Concordantly, analysis of steady-state HSPCs from healthy donors showed that BM-resident CD34+ cells expressed a higher level of CD9 than those in the circulation (46.1% vs. 26.5%, P =0.004; n=13). In addition, a significantly higher CD9 expression was witnessed on BM CD34+ cells (47.7%; n=16) than those from MPB donors (32.3%, n=25; P =0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure ( P =0.006), revealing a dynamic regulation.

Subpopulation analysis of BM CD34+ cells dictated preferential binding of CD9+ cells to soluble VCAM-1 comparing with CD9- cells (51.4% vs. 25.9%, P <0.001; n=10). BM CD34+ cells also had a superior VCAM-1 binding capacity than the MPB counterpart (37.7% vs. 4.1%, P =0.014), suggesting CD9 may regulate HSPC trafficking via modulation of integrin activities.

Summary/Conclusion: HSPC engraftment and mobilization coincide with dynamic oscillation of CD9 expression and integrin VLA-4 activities within the BM microenvironment. The current study has added impetus to our understanding on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.

P1316: ASSESSING GENOMIC SAFETY OF ANTIVIRAL NUCLEOSIDE ANALOGS IN HEMATOPOIETIC STEM CELLS

F. Peci 1,* , J. de Kanter 1 , N. Groenen 1 , A. Rosendahl-Huber 1 , L. Derks 1 , E. Bertrums 1 , S. Middelkamp 1 , M. J. van Roosmalen 1 , R. van Boxtel 1

1 Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands

Background: Nucleoside analog (NAs) drugs are used for the treatment of a variety of diseases, such as cancers and viral infections. After phosphorylation of viral and host kinases, NA drugs compete with the corresponding naturally occurring nucleotide during DNA replication of the infected cell. After incorporation, they can lead to mutations, or chain termination. However, because of their mechanism of action, NA are also potentially mutagenic to the genome of physiologically normal cells. Indeed, we have shown that treatment with the antiviral NA ganciclovir (GCV) after stem cell transplantation induces an increased mutation burden in the hematopoietic stem and progenitor cells (HSPCs) of pediatric leukemia patients. Using mutational signature analysis, we provided evidence that GCV-induced mutagenesis contributes to development of relapses and second malignancies in pediatric patients by inducing driver mutations. Over 30 NA drugs have been approved for clinical use and millions of people receive antiviral treatment worldwide to treat viral infections, including COVID-19. However, the mutagenicity in normal cells and potential carcinogenicity is unclear.

Aims: Here, we aimed to systematically assess the mutational consequences of antiviral NAs in human HSPCs and identify underlying mechanisms.

Methods: By combining in vitro treatment of umbilical cord blood-derived HSPCs with whole-genome sequencing (WGS) analyses, we provide a compendium of mutational consequences of antiviral NAs in a relevant human tissue (i.e., toxicity to the hematopoietic system is often dose-limiting). We treated HSPCs with IC40-60 concentration of the assessed compound followed by clonal expansions to obtain sufficient DNA for WGS. Using established bioinformatic pipelines, we catalogued the somatic mutations and mutational signatures in these cells.

Results: At time of writing, 5 out of 7 tested antiviral NAs induce an enhanced mutation burden in exposed HSPCs. For some of this antiviral NAs we were able to identify unique unreported mutational signatures. Of note, the thymidine analog brivudine showed the highest increase in single base substitutions, which were characterized by a T>C signature, depleted for flanking cytosines. Furthermore, like GCV, we also observed a signature characterized by C>ApA substitution after treatment with the penciclovir, a molecule nearly identical to GCV.

Currently we are working on machine learning approach to identify relevant mutation characteristics and modes of action as well as to screen cancer genome databases for mutational signature occurrence.

Summary/Conclusion: Many compounds of the NA class currently prescribed for the treatment of viral infections are mutagenic to healthy cells. This calls for more thorough screening of these drugs, incorporation of information on mutagenicity to healthy cells in drug safety guidelines and patient surveillance over time.

P1317: TIME SERIES CLUSTERING OF T CELL SUBSETS DISSECTS HETEROGENEITY IN IMMUNE RECONSTITUTION AND SURVIVAL AMONG RECIPIENTS OF MUD-HCT WITH ATG OR PTCY

A. Turki 1,* , S. Leserer 2 , T. Graf 1 , U. Buttkereit 2 , E. Arrieta-Bolanos 2 , N. Leimkühler 1 , K. Fleischhauer 2 , D. Beelen 2 , C. Reinhardt 1

1 Department of Hematology and Stem Cell Transplantation; 2 Universitätsklinikum Essen, Essen, Germany

Background: Proliferating alloreactive T cells have a central role in the induction of acute GVHD (aGVHD) making them a promising target in preventing alloreactivity. T cell depleting regimens such as anti-T-lymphocyte globulin (ATG) or post-transplant cyclophosphamide (PTCy) effectively eliminate these cells and reduce aGVHD after hematopoietic cell transplantation (HCT) with marginal differences in clinical outcome. Comparative immune reconstitution analyses could contribute to answer the question, which agent would be most appropriate for an individual patient. However, not only effects relating to the different T cell depleting regimens, i.e. ATG or PTCy may be relevant but the general heterogeneity of T cell reconstitution.

Aims: Here, we aimed at dissecting this heterogeneity with an approach called time-series clustering to better understand the impact of both regimens in individual patients and to consequently identify distinct patient subsets which benefit the most from each protocol.

Methods: We retrospectively compared immune reconstitution data of 339 recipients of matched-unrelated donor (MUD) HCT with either ATG (n=304) or PTCy (n=35) as T cell depletion via conventional analysis on the cohort level and for its individual heterogeneity via time-series clustering. This analysis leveraged the approach of dynamic time warping (DTW) to determine the distance measure later used for the partitional clustering of individual patient time-series data. The performance of this methods was evaluated by the silhouette coefficient (Sil) which indicates a separation of clusters with values between 0 (overlapping clusters) and +1 (best separation); A 10-fold resampling was used to assess model robustness. Cluster information were used for subsequent analysis of clinical outcomes. All analysis were performed using R packages R stats , dtwclust , survival , survminer and cmprsk.

Results: Comparative analysis of cellular reconstitution revealed distinct prominent T cell population after each protocol. While patients receiving PTCy as GVHD prophylaxis presented with higher levels of regulatory T cells, ATG patients had higher levels of γδ T- or NK-T cells. Time-series clustering of T cell subpopulations that associate with GVHD successfully dissected each population’s heterogeneity. For ATG patients the clustering revealed two distinct clusters with its optimal model configuration showing a good and robust silhouette coefficient (7_1: Sil =0.524) and a balanced patient distribution (Fig. 1A). In this model, clustering of ATG patients was driven by αβ- and activated T cells as those revealed higher absolute counts and a greater difference in shape over time as compared to regulatory- and γδ T cells. Patients in ATG cluster 1, showing higher absolute counts of αβ- and activated T cells compared to cluster 2 were associated to a significantly higher 1-year OS (98% vs. 79%, p =0.0023) due to lower NRM ( p =0.032) and relapse ( p =0.01). The clustering of PTCy patients distinguished two clusters with lower silhouette coefficients had a strong impact of αβ- and activated T cells (Fig.1B). Comparing the overall survival probability of both ATG clusters with the PTCy cohort revealed a significant difference driven by the ATG clusters (Fig 1C).

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Summary/Conclusion: Beyond a differential impact of ATG and PTCy on immune reconstitution our analysis identified phenotypes that reproducibly associated with impaired clinical outcomes within the same T cell depletion platform. This provides guidance for individually choosing the most appropriate agent in the MUD setting.

P1318: THE IMPACT OF MELPHALAN DOSING IN COMBINATION WITH FLUDARABINE IN CLINICAL OUTCOMES OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES RECEIVING ALLO-HCT

O. Albanyan 1,* , D. Kalos 2 , J. Kim 2 , R. Faramand 1 , H. Elmariah 1 , A. Mishra 1 , T. Nishihori 1 , L. Perez 1 , M. Nieder 1 , J. Pidala 1 , N. Bejnayan 1

1 Department of Blood and Marrow Transplantation – Cellular Immunotherapy; 2 Department of Biostatistics and Bioinformatics, Moffitt Cancer Center, Tampa, United States of America

Background: Allogenic hematopoietic cell transplant (allo-HCT) is the only curative treatment option for many adults with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Fludarabine (Flu) and melphalan (Mel) reduced intensity conditioning (RIC) regimen is frequently used for older patients and those with comorbidities. Mel dosing based on body surface area raised concerns for patient under- or overdosing in prior report.

Aims: Here we studied the impact of Mel dose per kilogram of actual body weight (mg/kg) on clinical outcomes of allo-HCT.

Methods: We retrospectively evaluated patients with AML or MDS who underwent allo-HCT from an 8/8 HLA matched donor and received Flu/Mel (Mel dose of 100-140 mg/m 2) . The objectives were to compare overall survival (OS), relapsed free survival (RFS), non-relapse mortality (NRM), relapse, grade II-IV acute graft vs host disease (aGVHD), chronic GVHD (cGVHD), mucositis and gastrointestinal (GI) toxicity based on Mel mg/kg dosing. We calculated the median Mel mg/kg dose and compared the outcomes of patients receiving median and above versus below median dose.

Results: From January 2008 to December 2020, 345 patients underwent allo-HCT with Flu/Mel, all patients received peripheral blood graft, 209 patients transplanted for AML and 136 for MDS. Patient characteristics are presented in Table 1. Median dose of Mel was 3.28 mg/kg (range, 1.61-6.21 mg/kg);173 patients received Mel ≥ 3.28 mg/kg and 172 patients received Mel < 3.28 mg/kg. Median follow up time for the entire study cohort was 36.5 months. For the Mel ≥ 3.28 mg/kg vs. Mel < 3.28 mg/kg groups, median OS was 57.5 months (95% CI: 39.5 – N.E) vs. 46.6 months (95% CI: 28.2 – 109.5, p=0.49) and median RFS was 51.4 months (95% CI: 30.7 – 70.4) vs. 29.7 months (95% CI: 18.1 – 98.0, p=0.36). Similarly, there were no significant differences between the Mel ≥ 3.28 mg/kg and Mel < 3.28 mg/kg groups in 2-year rates of relapse (19.9% vs. 23.8%, p=0.3) and NRM (20.9% vs. 25.4%, p=0.94; Figure 1D). The cumulative incidences of day 100 grade II-IV aGVHD (39.9% vs. 39.0%, p=0.86), grade III-IV aGVHD (9.2% and 9.3%, p=0.99) and moderate/severe cGVHD (67.6% vs.58.4%, p=0.19) were also similar for patients receiving Mel ≥ 3.28 mg/kg vs. Mel < 3.28 mg/kg.

On multivariable analysis, the Mel dose/kg had no independent impact on OS, RFS, relapse, NRM, acute and chronic GVHD outcomes. OS was worse with KPS ≤ 80 (HR, 1.51, p=0.014) and HCT-CI ≤ 3 (HR, 1.68, p=0.004), while RFS was worse with KPS ≤ 80 (HR, 1.5, p=0.012), HCT-CI ≥ 3 (HR, 1.72, p=0.002) and high/very high DRI (HR, 1.36, p=0.046). KPS ≤ 80 (HR1.75, p=0.006) and HCT-CI ≥ 3 (HR,1.67, p=0.023) were also associated with higher NRM.

Cumulative incidence of any grade mucositis for Mel ≥ 3.28 mg/kg vs. Mel < 3.28 mg/kg was 33.5% vs. 29.1% (p=0.37) and of grade III mucositis was 12.1% vs. 24.0%. None of the study patients experienced grade IV mucositis. Incidence of GI toxicity with non-infectious diarrhea following Flu/Mel chemotherapy of any grade was 65.3% vs. 60.5%, (p=0.35) and of grade III was 16.8% vs. 19.2% for patients received Mel ≥ 3.28 mg/kg vs. Mel < 3.28 mg/kg, respectively. We observed no grade IV GI toxicity on our study patients.

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Summary/Conclusion: Our study showed no significate influence of Mel mg/kg dose on clinical outcomes of allo-HCT in patients with AML and MDS receiving Flu/Mel RIC. Similarly, mucositis and GI toxicities were not affected by the Mel mg/kg dose. These findings support Mel dosing based on body surface area when combined with Flu as RIC regimen used for allo-HCT.

P1319: T AND B LYMPHOCYTES SUBSETS AFTER BNT162B2 ANTI-SARS-COV-2 M-RNA VACCINATION: A COMPARISON BETWEEN HEMATOPOIETIC STEM CELL TRANSPLANTATION PATIENTS AND HEALTHY CONTROLS.

I. Attolico 1,* , F. Tarantini 2 , C. P. Schifone 2 , A. Mestice 2 , G. De Tullio 1 , C. Derosa 3 , F. Carbone 3 , A. Negri 4 , P. Carluccio 1 , M. Delia 1 , F. Albano 2 , A. M. V. Larocca 5 , P. Stefanizzi 6 , L. Vimercati 7 , S. Tafuri 6 , P. Musto 8

1 Hematology and Stem Cell Transplantation Unit, AOUC Policlinico, Bari; 2 Department of Emergency and Organ Transplantation, “Aldo Moro” University School of Medicine, Bari; 3 Department of Clinical Pathology, Miulli General Hospital Acquaviva delle Fonti; 4 Haematology Unit, National Cancer Research Center “Giovanni Paolo II”, Bari; 5 Hygiene Unit, AOUC Policlinico, Bari; 6 Department of Biomedical Sciences and Human Oncology, “Aldo Moro” University School of Medicine, Bari; 7 Interdisciplinary Department of Medicine/Occupational Medicine Unit; 8 Department of Emergency and Organ Transplantation/Hematology and Stem Cell Transplantation Unit, “Aldo Moro” University School of Medicine, Bari/AOUC Policlinico, Bari, BA, Italy

Background: We previously demonstrated that 76% of allogeneic (ALLO) and 94% of autologous (AUTO) hematopoietic stem cell transplantation (HSCT) recipients develop a serological response after two doses of BNT162b2 anti SARS-Cov-2 mRNA vaccine (Attolico et al, BJH 2021). However, less is known about the lymphocyte (Ly) immune profile as possible predictor of response to the vaccine in these immunocompromised patients.

Aims: Aim of the study was to evaluate absolute count and maturation profile of T and B Ly subsets in transplanted patients and to compare them to healthy controls (HC).

Methods: We evaluated 39 transplanted patients (24 M, 15F; median age: 55 years, range 26-71; 20 AUTO, 19 ALLO) and 78 age and sex matched HC, after completion of the vaccination program. Samples were collected one month after the second and the third dose. Eight out of 39 patients (21%) did not respond to vaccination (NR: anti Spike titer<50 AU/ml): 6 ALLO, 2 AUTO. Responders (R: ≥50 AU/ml) were 31 (79%). T, B and NK Ly counts were performed with Multitest 6-color TBNK reagent, using FACSCanto cytofluorimeter and Canto software (Becton Dickinson). B and T cell maturation was evaluated with DuraClone IM B and T cells kits, using Navios cytofluorimeter and Kaluza software (Beckman Coulter). Comparison between groups was performed using the Mann-Whitney and Kruskall Wallis tests.

Results: No differences were observed in total Ly count between all patients and HC (Fig.1A). However, the analysis of Ly subsets revealed that HC had significantly higher CD4+ count; by contrast, R exhibited higher CD8+, CD16/56+ and CD19+ counts (Fig.1B). When comparing HC to AUTO and ALLO subgroups, we observed that only AUTO patients had a significantly lower CD4 count (p 0.0006). CD4/CD8 Ratio was higher in HC than in both R and NR patients (Fig.1C). We compared Ly subsets before and after the completion of the two doses of vaccination schedule: interestingly, after the second dose, R exhibited significantly higher total Ly counts, CD3+, CD4+ and CD19+ (Fig.1I). We also evaluated T and B maturation subsets. HC had significantly higher Marginal Zone (MZ), unswitched memory and transitional B cells, while R had higher B naïve counts (Fig.1D). The comparison of T CD4+ maturation pattern showed that HC had significantly higher central memory, naïve counts and exhaust CD279+ counts, while R had higher effector memory, senescent CD57+ and senescent CD27-.(Fig.1E) When analyzing the T CD8+ maturation subsets, we found that HC had significantly higher central memory, naïve and exhaust CD279+ while patients had higher effector memory, effector T, senescent CD57+ and CD27-.(Fig.1F) No differences were observed between AUTO and ALLO patients regarding B and T maturation patterns. Despite booster dose recovered, in terms of antibody titer, all the 8 NR after the first two doses, we did not notice any difference when comparing Ly subsets before and one month after the third dose (Fig.1G). Likewise, no differences were shown in Ly subsets of R before and one month after the third dose (Fig.1H).

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Summary/Conclusion: Overall, our preliminary evaluations show differences in Ly subsets amounts and T and B maturation profiles between HC and transplant patients. Furthermore, no changes were observed before and after the booster dose even in NR who subsequently developed an antibody response. Finally, our study confirms the predictive role of CD4+/CD8+ ratio for the response to vaccination.

P1320: INCIDENCE AND IMPACT OF COMPLICATIONS OF ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) IN PATIENTS OVER 60 YEARS. RETROSPECTIVE EXPERIENCE OF TWO SPANISH CENTERS IN THE 2015-2020 PERIOD

M. Baile 1,* , M. Hidalgo Soto 2 , S. Marcos Asensio 1 , M. Cabrero Calvo 1 , R. Martínez Fernández 3 , A. Avendaño Pita 1 , A. Á. Martín López 1 , A. Cabero Martínez 1 , M. Cortés Rodríguez 1 , E. Pérez López 1 , L. López Corral 1 , F. Sánchez Guijo 1 , L. Vázquez López 1 , M. D. Caballero Barrigón 1

1 Hematology, Hospital Clínico Universitario de Salamanca (CAUSA/IBSAL), Salamanca; 2 Hematology, Hospital Universitario 12 de Octubre, Madrid; 3 Hematology, Hospital Universitario Príncipe de Asturias, Alcalá de Henares, Madrid, Spain

Background: Not fully known if allogeneic HCST complications have a more impact on patients over 60 years and this may limit their access to the procedure.

Aims: To describe the incidence of the main complications and to analyze their relationship with age, the transplant modalities, and also their impact on survival and transplant-related mortality (TRM).

Methods: Retrospective study of 142 patients over 60 years with hematological neoplasms undergoing allogeneic HSCT.

Results: Patient characteristics and survival curves are in Image 1. Graft Versus Host Disease (GVHD) results are presented in the abstract EHA-1952. Post-chemotherapy mucositis was the 2nd complication (60%) after aGVHD and only 5.5% were grade IV. The use of myeloablative conditioning (MAC) (86% vs 57% in reduced intensity (RIC); p-value 0.04) and Methotrexate on the immunosuppression regimen (100%; p-value <0.001) were the two factors with more impact on its appearance. A higher incidence was observed in patients >70 years (82%; p-value 0.19) and it was no impact on survival or TRM. CMV reactivation was the 3rd complication (38%) and it appeared early (median 5 weeks after infusion). Patients with severe aGVRD presented more reactivation (73.5%/p-value 0.005) and also the unrelated transplant with MAC regimen (66.5%) or haploidentical (RIC 63%/MAC 59 %) with a low incidence in related transplant with RIC regimen (19.5%/p-value 0.001). There was also more in seropositive recipients (41%; p-value 0.07), especially if there was a discrepancy recipient +/donor- (46%/p-value 0.3). CMV disease had a lower incidence (7%) and 100% were gut forms. Prior reactivation and aGVHD were the two most important factors related (18% and 100%; p-value <0,001). CMV disease had impact on survival (median OS 6 months vs 40 if no disease; p-value 0.005/HR 0.36 (CI 0.17-0.76)). There were a 20% bacterial sepsis and 11.5% invasive fungal infections (IFI), (90% Aspergillus) and both were early complications (median onset of 2 and 4 months). Once again, severe aGVHD was associated with a higher incidence (60% sepsis; p-value <0.001 and 26.5% IFI; p-value 0.001) and an increase was also observed in patients with graft failure (33,5% sepsis, p-value 0.26 and 22% IFI, p-value 0.04). No differences were observed between the different age groups. Both present an unfavorable impact on survival (median OS of 6 months; p-value<0.001/HR 1.8 (CI 0.63-5.2). Sinusoidal obstruction syndrome (SOS) had an incidence of 7.5%, the median onset was 4 weeks and 72.5% were mild cases. All patients were CMV seropositive and it was more frequent in patients with ALL (16.5%) and in haploidentical transplant recipients (14%). The only case in >70 years was severe (in < 69 years; 90% mild cases; p-value 0.13) and was related to lower survival (median OS of 6 months vs 40 if no SOS; p-value 0.001/HR 0.06 (CI 0.17-0.47) with 100% mortality in patients with severe SOS. Primary graft failure was the least frequent complication (6.5%). Patients with cMPN and those transplanted after to 2 years from diagnosis had a higher incidence (28.5% and 13%; p-value 0.03). No differences were found of older age and other known risk factors. Its impact was very high (mortality of 100% with median OS of 1 month). Although SOS and graft failure were infrequent, they were a very early cause of TRM (80% of deaths before day +100).

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Summary/Conclusion: Age is not a risk factor for the development of complications, given that an incidence similar to that reported in younger patients and related to known risk factors is observed. However, comparative studies are needed to confirm these results.

P1321: INCIDENCE AND IMPACT OF GRAFT-VERSUS-HOST DISEASE (GVHD) IN PATIENTS OVER 60 YEARS OF AGE UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT). RETROSPECTIVE EXPERIENCE OF TWO SPANISH CENTERS

M. Baile 1,* , M. Hidalgo Soto 2 , S. Marcos Asensio 1 , M. Cabrero Calvo 1 , R. Martínez Fernández 3 , A. Avendaño Pita 1 , A. Á. Martín López 1 , A. Cabero Martínez 1 , M. Cortés Rodríguez 1 , E. Pérez López 1 , L. López Corral 1 , F. Sánchez Guijo 1 , L. Vázquez López 1 , M. D. Caballero Barrigón 1

1 Hematology, Hospital Clínico Universitario de Salamanca (CAUSA/IBSAL), Salamanca; 2 Hematology, Hospital Universitario 12 de Octubre, Madrid; 3 Hematology, Hospital Universitario Príncipe de Asturias, Alcalá de Henares, Madrid, Spain

Background: Acute and chronic GVHD are of the main complications of HSCT and their frequency and impact in patients over 60 years is not fully understood.

Aims: To describe their incidence and to analyze the influence of age and modalities of HSCT on their appearance, as well their impact on survival.

Methods: Retrospective study of 142 patients over 60 years with hematological neoplasms undergoing allogeneic HSCT

Results: Patient characteristics and survival curves are shown in Image 1. Acute GVHD was the most common complication (incidence of 72.5%) although only 10.5% were severe cases (Grades 3-4). It was an early complication (median onset 28 days). A higher incidence was observed in unrelated donor transplant recipients (82%/100% if mismatch) compared to related (62%) and haploidentical (66%) (p-value 0.28). There were no differences with the intensity of conditioning (Myeloblative 78.5% and reduced intensity 72%) although the highest severe cases was observed of myeloablative unrelated transplant (66.5%; p-value 0.03). The incidence was higher with older age (91% in >70 years; 74% in 65-69 and 69% in <65; p-value 0.15) and in CMV serological discrepancy (88.5% in recipient+/donor-; p-value 0.19). In relation to immunosuppression, there was a higher incidence with Tacrolimus/Sirolimus (82%) and less with Cyclosporine/ Mycophenolate of Mofetil (MMF) (40%): p-value 0.2; however they were the regimen most and least used, respectively. Severe aGVHD had an unfavorable impact on survival: median Progression-Free Survival (PFS) of 5 months vs 25 and 27 if not aGVHD or if mild (p-value 0.008/HR 2.3 (CI 1.2-4.6)); and median overall survival (OS) of 7 months vs 40 and 45 if not aGVHD or if mild (p-value 0.002/HR 2.6 (CI 1.3-5.4)). Severe aGVHD was associated with a higher frequency of infectious complications: more CMV reactivation (73.5% vs 37.5% if mild and 25.5% if not aGVHD; p-value 0.005), CMV disease (40% vs 4.5% if mild or 0% if not aGVHD; p-value <0.001), bacterial sepsis (60% vs 13.5% if mild or 18% if not aGVHD; p-value <0.001) and invasive fungal infection (26.5% vs 1 and 5% if mild or not aGVHD; p-value 0.001). And, these complications were associated with worse survival (median OS of 6 months vs 34 at the global serie). Together (aGVHD and related infections) caused 15% of deaths and were the first cause of mortality related to treatment. Chronic GVHD was less frequent (31%), only 13.5% were severe cases, and it occurred later (median onset of 7.5 months). A higher incidence was in unrelated-mismatch donor recipients (60%; p-value 0.04) and lower in haploidentical (15%) and no differences were observed with the intensity of conditioning (mieloablatyve 35,5% and reduced intensity 30.5%). There was a higher incidence with older age (36.5% in >70 years; 33.5% in 65-69 and 28.5% in <65; p-value 0.61) and if serological CMV discrepancy recipient -/donor+ (50%; p-value 0.83). The incidence was again higher with Tacrolimus / Sirolimus regimen and the lowest with Tacrolimus/MMF/Ciclofosfamide-post-transplant (p-value 0.09). Mild-moderate cGVHD had a protective effect on survival: median PFS of 38 months vs 8 if not cGVHD (p-value 0.03/HR 0.55 (CI 0.32-0.94)); and median OS of 19 months vs 11 if not cGVHD (p-value 0.001/HR 0.47 (CI 0.27-0.84)).

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Summary/Conclusion: GVHD is a very relevant complication: the aGVHD due to its high incidence and impact on survival and appearance of infectious complications; and the protective effect of the cGVHD. Although a higher incidence is observed with older age, longer series and comparative studies are needed to confirm these results.

P1322: ALLOGENEIC HEMATOPOIETIC STEM TRANSPLANTATION (HSCT) IN PATIENTS OVER 60 YEARS. RETROSPECTIVE EXPERIENCE OF TWO SPANISH CENTERS BETWEEN 2015-2020

M. Baile González 1,* , M. Hidalgo Soto 2 , S. Marcos Asensio 1 , M. Cabrero Calvo 1 , R. Martínez Fernández 3 , A. Avendaño Pita 1 , A. Á. Martín López 1 , A. Cabero Martínez 1 , M. Cortés Rodríguez 1 , E. Pérez López 1 , L. López Corral 1 , F. Sánchez Guijo 1 , L. Vázquez López 1 , M. D. Caballero Barrigón 1

1 Hematology, Hospital Clínico Universitario de Salamanca (CAUSA/IBSAL), Salamanca; 2 Hematology, Hospital Universitario 12 de Octubre, Madrid; 3 Hematology, Hospital Universitario Príncipe de Asturias, Alcalá de Henares, Madrid, Spain

Background: The improvement in support treatment and the implementation of reduced intensity conditioning (RIC) makes that Allogeneic-HSCT in patients over 60 years is a standard clinical practice.

Aims: To describe the results in overall survival (OS), progression-free survival (PFS), mortality and treatment-related mortality (TRM), and the factors involved in them.

Methods: Retrospective study of 142 patients over 60 years with hematological neoplasms undergoing allogeneic HSCT.

Results: Median age was 64 years, 8% were over 70 years old and 68% were male. 86% had positive CMV serology with donor/patient discrepancy in 18.5%. According to HCT-CI index, 27% were at high risk and with the HCT-CI-age, 5.5% were at very high risk. The principal indications (69.5%) were Acute Myeloblastic Leukemia and Myelodysplastic Syndrome and the median time from diagnosis to transplant was 7 months. 37% received HSCT with active disease and according to DRI-index, 27% were high or very high risk. The hematopoietic source was peripheral blood in all cases. 48% undergoing a related donor trasplant and 33%, haploidentical and in 90%, RIC regimens was used. Immunosuppression regimen was determined by the intensity of conditioning and donor type and the two most used were Tacrolimus/Sirolimus (43%) and Tacrolimus/Mycophenolate Mofetil/Cyclophosphamide post- transplant (34.5%). Median number of days of hospitalization was 30 and 93% had an optimal graft with a median of 17 days. The relapse/progression and mortality rate was 35% and 51%, respectively. Both events occurred more frequently in the first year after transplant (78% of relapses and 68% of deaths). With a median follow-up of 16.5 months from infusion, PFS was 43% (median 19 months) and OS was 49% (median 34 months). The first cause of death was relapse/progression (24.5%) and TRM was 22.5% (2nd cause of death). Within this, Graft Versus Host Disease and related infections were the predominant cause (65.5%) and globally were the 3rd cause of death (15%). Factors involved in OS and TRM are in Image 1. Diagnosis of chronic myeloproliferative disease, the donor/patient CMV serological discrepancy and undergoing unrelated trasplant with MAC regimen have unfavorable impact in OS. Although not significant, age >65 years, scoring high-risk in prognostic index (HCT-CI and DRI) were also associated with worse OS. Active disease at transplant did not have an apparent impact. No significant differences were observed in PFS. Higher relapse was observed in related recipients transplant (47%; p-value 0.04) and in patients with high and very high risk in DRI-index (54.5 and 60% respectively; p-value 0.001). Age was not related to a higher risk of relapse or worse PFS. A higher TRM was observed in haploidentical and unrelated trasplant compared with related donor (specially if RIC regimen). There were no differences with age, disease status to HSCT, and prognostic indexs. The specific modality of transplant (donor and conditioning intensity) maintained impact in the multivariate analysis. Non CR or progression at +100 day and early relapse (< 12 months) had a very negative impact on OS (median of 9 months: Non CR/progression p-value < 0.001/ HR 3, 3 (CI 1.9-6); early relapse p-value <0.001/HR 8.7 (CI 4.8-16.4).

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Summary/Conclusion: Allogeneic HSCT in patients over 60 years achieves comparable results to younger patients with an acceptable TRM. The absence of response at day +100 and early relapsed have a wrong prognosis. Related donor trasplant with RIC regimen seems the best option dice less toxicity although comparative studies are necessary to confirm these results.

P1323: PREDICTION OF NON-RELAPSE MORTALITY BY EASIX AND HCT-CI SCORES IN PATIENTS WITH CHRONIC MYELOMONOCYTIC LEUKEMIA UNDERGOING ALLOGENEIC STEM CELL TRANSPLANT

A. Baranwal 1,* , A. Mangaonkar 1 , M. Shah 1 , M. Litzow 1 , W. Hogan 1 , M. Patnaik 1 , H. Alkhateeb 1

1 Mayo Clinic, Rochester, United States of America

Background: The endothelial activation and stress index (EASIX) score has been shown as a predictor of non-relapse morality (NRM) after allogeneic stem cell transplantation (alloSCT) [Luft, et al. Bone Marrow Transplant. 2020;55(3):553–561]. However, data in patients with chronic myelomonocytic leukemia (CMML) is limited.

Aims: Our goal was to study the risk of NRM and the impact on overall survival (OS) by EASIX and HCT-CI scores in patients with CMML undergoing alloSCT.

Methods: We retrospectively evaluated patients with CMML who underwent alloSCT. We reviewed patient demographics, disease characteristics, and post-transplant outcomes. EASIX score was calculated using the formula: LDH (U/L) × Creatinine (mg/dL) / platelet count (10^9/L) and analyzed based on log2 transformed values. EASIX score was calculated within 45 days of alloSCT, but prior to starting conditioning therapy. The optimal log2 EASIX score cutoff was determined using maximally selected log-rank statistics. Patients with an HCT-CI score ≥ 3 were considered to have a high HCT-CI score.

Results: Between, January 1992 to February 2019, a total of 70 CMML patients underwent alloSCT, 13 patients were excluded due to incomplete records and 57 patients (39 males and 18 females) were evaluated for the purposes of the study. CMML-0 was the most common subtype (21 patients, 36.8%), followed by CMML-2 (19 patients, 33.3%)) and CMML-1 (17 patients, 29.8%). Seventeen (29.8%) patients had progressed to CMML blast phase before alloSCT. Of the entire cohort, 18 (31.6%) patients were in complete remission at the time of alloSCT.

Median age at transplant for the entire cohort was 58 years (range: 18 – 73 years). Median time of follow-up was 2 years (range: 11 days – 16 years). Median OS was 2 years, with a 3-year OS rate of 49.1%. Eighteen (31.6%) patients were alive at last follow-up.

The median log2 EASIX score was 0.88 (range -2.29 to 5.91). The optimal log2 EASIX score cutoff using the maximally selected LogRank statistics was found to be 2.33. Patients with log2 EASIX score > 2.33 were considered to have a high EASIX score. Thirteen (22.8%) patients had a high EASIX score and 44 (77.2%) had a low EASIX score. The cumulative incidence of NRM among patients with high EASIX score was significantly higher compared to those with low EASIX score at 100 days (30.8% vs 0%, p < 0.001), at 1-year (46.2% vs 9.1%, p = 0.002) and at 3-years (53.8% vs 15.9%, p = 0.003) after alloSCT (Figure 1A).

A total of 55 patients were evaluated for HCT-CI score, of whom 30 (54.54%) were found to have a low HCT-CI score, i.e., HCT-CI < 3, and 25 (45.45%) were found to have a high HCT-CI score. The median HCT-CI score was 2 (range: 0-7). NRM after alloSCT among patients with high HCT-CI score was not significantly different compared to those with low HCT-CI scores at 100 days (8% vs 6.7%, p = 0.85), at 1 year (24% vs 13.3%, p = 0.33), and at 3 years (24% vs 26.7%, p = 0.92) after alloSCT (Figure 1B).

In multivariate competing risk regression analysis, a high EASIX score significantly increased NRM at 3 year (HR 4.14, 95% CI: 1.42 – 12.01, p = 0.009).

Patients with a high log2 EASIX score had significantly worse OS at 3 years (median 8 months vs. NA, p = 0.007). OS at 3 years did not differ among patients with high vs. low HCT-CI scores (median 1.02 years vs. NA, p = 0.38).

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Summary/Conclusion: In CMML, EASIX predicted NRM following alloSCT. EASIX was a better predictor of NRM compared to HCT-CI in this patient population. Larger patient cohort is required to confirm our findings.

P1324: A RISK-BASED STRATEGY FOR THE USE OF LETERMOVIR IN CMV-POSITIVE RECIPIENTS OF ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION

D. Beauvais 1,2,* , M. Sourisseau 1 , H. Béhal 1 , S. Alfandari 3 , N. Simon 1,4 , E. Faure 1,5 , I. Yakoub-Agha 1,2

1 Centre Hospitalier Regional et Universitaire de Lille; 2 Univ. Lille, Inserm, U1286 - Infinite, Lille Cedex; 3 Centre Hospitalier Gustave Dron, Tourcoing; 4 Univ. Lille, ULR 7365-GRITA; 5 Univ. Lille, Inserm, CNRS, U1019-UMR 9017-CIIL, Lille Cedex, France

Background: Letermovir has been approved since 2018 for the prevention of CMV infection and disease in adult CMV-seropositive recipients after allogeneic hematopoietic cell transplant (allo-HCT). It is an orally administered treatment but expensive and a potential source of drug interactions. Its benefit could therefore vary according to the risk of CMV infection, which depends on the transplantation modalities and the characteristics of the donor/recipient couple.

Aims: The objective of this study was to evaluate the interest of a letermovir administration strategy based on the risk of CMV infection in allo-HCT recipients.

Methods: The study was led according to the Declaration of Helsinki, and patients provided informed consent about the retrospective use of their clinical data. All consecutive CMV-seropositive adult patients who underwent allo-HCT from January 1, 2015, to July 31, 2021 at the University Hospital of Lille, France, were included. Patients were divided into two periods based on the date of transplantation: a first one (P1: 2015-2017) during which letermovir was not administered and a second one (P2: 2018-2021) during which letermovir was administered based on the risk of CMV infection, calculated according to a previously published score ( Beauvais et al, BMT 2021 ). Specifically, during P2 in high-risk patients, letermovir was administered systematically, whereas in low-risk patients, letermovir was administered only in those receiving systemic corticosteroid therapy.

Results: Three hundred and sixteen CMV positive patients were included (186 in P1, 130 in P2). The median age was 56 years (IQR: 43.5-64.0) and 52.5% were male. Patient characteristics and transplantation modalities between the 2 periods were comparable. The median follow-up was 60.3 months for P1 and 18.0 months for P2.

In high-risk patients, with systematic letermovir use in P2, clinically significant CMV infection decreased substantially compared with P1: 10.5% [IC95: 4.6-19.2] vs 51.6% [41.0-61.3] at 14 weeks, 16.9% [8.9-27.0] vs 52.7% [42.0-62.3] at 24 weeks, and 53.8% [43.0-63.3] vs 28.2% [17.3-40.2] at 1 year post allo-HCT, p < 0.0001. CMV disease was not significantly different between the 2 periods: 0% vs 3.2% [0.9-8.4] at 14 weeks, 1.5% [0.1-7.2] vs 4.3% [1.4-9.9] at 24 weeks, and 3.3% [0.6-10.3] vs 4.3% [1.4-9.9] at 1 year post allo-HCT, p = 0.69.

In low-risk patients, only 18/63 (28.6%) patients received letermovir due to the initiation of systemic corticosteroid therapy. The median time to initiation from allo-HCT was 28 days (range: 16-50). Despite this limited use of letermovir, clinically significant CMV infection also decreased substantially: 7.9% [2.9-16.3] vs 29.0% [20.2-38.5] at 14 weeks, 11.2% [4.9-20.5] vs 33.3% [23.9-43.0] at 24 weeks, and 12.9% [6.0-22.6] vs 34.4% [24.9-44.1] at 1 year post allo-HCT, p = 0.0025. A majority of low-risk patients (45/63; 71.4%) did not receive letermovir. Most had blips, all of which resolved spontaneously by week 14. CMV disease was not significantly different between the 2 periods: 0% vs 0% at 14 weeks, 1.7% [0.1-8.0] vs 1.1% [0.1-5.3] at 24 weeks, and 1.7% [0.1-8.0] vs. 2.2% [0.4-6.9] at 1 year post allo-HCT, p = 0.85.

For high-risk and low-risk patients, overall survival, progression-free survival, incidence of relapse, and relapse-free mortality were not significantly different between the 2 periods.

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Summary/Conclusion: A risk-based strategy for the use of letermovir, rather than systematically, is feasible in CMV-positive recipients of allo-HCT without increasing the risk of clinically significant CMV infection and CMV disease.

P1325: ACCURACY OF CRYOTUBE METHOD TO ASSESS PRETHAWING RICHNESS OF CRYOPRESERVED CD34+ HEMATOPOIETIC STEM CELLS FOR AUTOLOGOUS TRANSPLANTATION

D. Belloumi 1,* , I. Ben Yaiche 1 , J. Fekih 2 , A. Ghorbel 1 , L. Torjemane 1 , M. Chaabane 2 , S. Mekni 1 , N. Ben Abdeljelil 1 , I. Turki 1 , S. Ladeb 1 , S. Hmida 2 , T. Ben Othman 1

1 Centre national de greffe de moelle osseuse, Tunis, Tunisia; 2 Unité de thérapie cellulaire, Centre national de transfusion sanguine, tunis, Tunisia

Background: Cryopreservation of hematopoietic stem cells (HSC) is associated with variable loss of CD34+ graft richness.

Aims: Our aim was to examine the validity of measuring CD34 richness on flow cytometry from cryotubes before thawing infusing bags in order to reduce risks associated with low-quality grafts.

Methods: CD34+ cells richness was analyzed on flow cytometry in leukapheresis procedures before freezing and after thawing. Data from control cryotubes were compared to those from infusing bags using Wilcoxon signed-rank test.

Results: Between October 2017 and November 2020, 129 patients underwent HSC autologous transplantation in our department for multiple myeloma (n=94) or Hodgkin and non-Hodgkin lymphoma (n=35). The mean time from mobilization to HSC transplantation was 3.3 months (0-11).The median age was 52 years (11-66) with a sex ratio of 1.4. We analyzed 154 infusion bags. The mean amount of CD34+ cells on flow cytometry was respectively 3.2 x 10⁶/kg (0.39-19.95) and 1.86 x 10⁶/kg (0.16-21.58) per bag before cryopreservation and after thawing, corresponding to a cell loss of 40% (p=0.008). The mean CD34+ cell richness found in cryotubes after thawing was 1.7x 10⁶/kg (0-21.09), concordant with that found in the infusing bags after thawing (p=0.132).

The positive predictive value (PPV) of cryotubes (CD34+ cells richness in infusing bag greater than 2 x 10⁶/kg when cryotube richness is greater than 2 x 10⁶/kg) was 70% and the negative predictive value (NPV) was 71%. Cryopreservation duration >3 months did not alter the predictive value of cryotubes (PPV=71%, NPV=68%). The PPV was lower for patients older than 50 years (60%) and for patients treated for lymphoma (50%) compared to those younger than 50 years (82%) and treated for multiple myeloma (75%).

No delayed engraftment was noted in our cohort regardless of CD34+ richness in the cryotube.

Summary/Conclusion: Cryotube used to evaluate CD34+ richness prior to reinfusion is not informative enough although its predictive value may differ according to the age and the underlying disease.

P1326: FACTORS ASSOCIATED WITH THE OUTCOME OF ALLO-HSCT FOR ADVERSE-CYTOGENETIC RISK ACUTE MYELOID LEUKEMIA

J. M. Cerezo-Martin 1,* , A. Avendaño 2 , S. Fernandez-Luis 1 , J. A. Calvo 1 , E. Ocio 1 , M. Colorado 1 , A. Bermudez 1

1 Hematology, Marques de Valdecilla University Hospital, santander; 2 Hematology, Salamanca University Hospital, Salamanca, Spain

Background: Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) remains the unique intention-curative consolidation strategy in patients with Acute Myeloid Leukemia (AML) with unfavorable cytogenetic risk. It is a high-cost procedure with significant transplant-related mortality (TRM), therefore, the correct selection of candidates for allo-HSCT resembles as a crucial issue.

Aims: To describe the outcome of this procedure and to identify those patients who could obtain a better benefit. The primary endpoint was Overall Survival (OS) and secondary endpoints were relapse-free survival (RFS), cumulative incidence of Graft-versus-host disease (GVHD), cumulative incidence of relapse and (TRM).

Methods: We conducted an observational retrospective study in patients with adverse-risk cytogenetically defined AML (Upon the European LeukemiaNet 2017 criteria and the results of the Next Generation Sequencing) who underwent a first allo-HSCT in two tertiary referral centers between 2010 and 2020.

Results: We analyzed 82 patients with a median age at the moment of transplant of 55 (R 22-73) years. The median time from diagnosis to transplant was 5 (IR 4-7) months. Forty-two (51,2%) were women. Twenty-six patients (31,7%) had an identical HLA sibling donor and 37 (45,1%) had an unrelated HLA identical donor. A myeloablative conditioning was performed in 60 (51%) patients and in 48 (59,8%) the source of HSC was bone marrow. The majority (81,7%) had a first complete remission (CR1) at the moment of transplant and 13,4% had a partial response or a refractory disease (PR/RD). The 41,5% patients had a low comorbidity index score (HCT-CI<3).

With a median follow up of 36 months, the OS at 1 and 3 years was 67% (95% CI 64-66%) and 48% (95% CI 45-51%) respectively. The RFS at 1 and 3 years was 62% (95% CI 61-63%) and 45% (95% CI 43-47%), the cumulative incidence of relapse at 1 and 3 years was 18% (95% CI 11-28%) and 29% (95% CI 19-40%) and the TRM at 1 and 3 years was 21% (95% CI 13-30 %) and 28% (95% CI 19-39%) respectively.

The cumulative incidence of acute GVHD grade III-IV at day +100 was 17% (95% CI 9-26%). The cumulative incidence of moderate-severe chronic GVHD at 1 and 3 years was 21% (95% CI 13-30 %) and 31% (95% CI 21-42%) respectively.

There were no differences in OS regarding age (p=0,2), sex (p=0,9), HCT-CI score (p=0,7), type of donor (p=0,4), acute GVHD (p=0,5), HSCT source (p=0,06), conditioning (p=0,04) and subtype of ELN2017 (p=0,9).

A worse OS was observed in patients with PR/RD (HR 1,5 95% CI 1,2-1,9) (p<0,01) compared to those in first complete remission. The OS at two years was 28% (95% CI 26-30) and 59% (95% CI 58-60) respectively (Figure 1).

Sixty-six patients were analyzed for OS depending on the development of cGVHD. The OS at 2 years of the patients who did not develop cGVHD (n= 32) was 58% (95% CI 56-60) of those with a mild cGVHD (n=10) was 85% (95% CI 83-87) of those with a moderate cGVHD (n=15) was 79% (95% CI 77-81) and of those who develop a severe cGVHD (n=9) was 64% (95% CI 61-67). Therefore, a worse OS was observed in patients who did not develop cGVHD or had a severe grade compared to those who had a mild or moderate cGVHD HR 5 (95% CI 1,8-15) (p<0,01).

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Summary/Conclusion: In our series, Allo-HSCT in adverse-cytogenetic risk AML are associated with better OS when it is performed in first complete remission and its benefit is unclear performed in partial response or in a refractory disease, where other strategies should be considered. The development of mild or moderate cGVHD was associated with a better overall survival.

P1327: RUXOLITINIB COMBINED WITH GLUCOCORTICOID IN THE FIRST-LINE TREATMENT OF GRAFT-VERSUS-HOST DISEASE IN CHILDREN: A SINGLE CENTER RETROSPECTIVE STUDY

K. Chen 1,* , B. Zou 1 , J.-B. Shao 1 , Q. Liu 1 , N. Zhang 1 , P. Fu 1 , X.-H. Sun 1 , H. Jiang 1

1 Hemato-Oncology, Shanghai Children’s Hospital, shanghai, China

Background: Ruxolitinib has certain advantages and potential in the treatment of GVHD. It can inhibit the secretion of inflammatory factors by T cells, increase the number of regulatory T cells（Tregs）in the recipient, and then mediate immune tolerance. It can also reduce T cell surface related chemokines or chemokine receptors, and then change T cell migration. A multicenter retrospective study showed that the effective rate of ruxolitinib on glucocorticoid resistant aGVHD was 81.5%, and the effective rate of glucocorticoid resistant cGVHD was 85.4%. At present, there is no report on the first-line treatment of GVHD in children with glucocorticoid combined with glucocorticoid.Our center tried to use this combined scheme as the first-line treatment option in the early stage, in order to reduce the application time of glucocorticoid and reduce glucocorticoid related complications.

Aims: To investigate the efficacy and safety of ruxolitinib combined with glucocorticoid in the treatment of graft-versus-host disease (GVHD) in children.

Methods: A retrospective analysis of 17 cases of graft-versus-host disease（GVHD） after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in our center was conducted. The clinical efficacy and adverse reactions of early application of ruxolitinib combined with glucocorticoid were observed.

Results: The median treatment time of 17 children was 47 (24-380) days, and the median onset time was 7 (4-90) days. The overall effective rate was 94.1% (16/17), the complete remission rate was 58.8% (10/17), the partial remission rate was 35.3% (6/17). The incidence of hemocytopenia during the combination therapy was 41.1% (7 /17).The incidence of EBV infection was 35.2% (6 /17), and the incidence infection of CMV was 29.4% (5/17).The incidence of hemorrhagic cystitis was 11.8% (2/17). The median follow-up time was 230 (74-545) days. One child died of grade IV acute graft versus host disease (aGVHD), and the other 16 children survived. The 6-month overall survival rate was 94.1%.

Summary/Conclusion: Ruxolitinib combined with corticosteroid is effective and safe in the treatment of children with GVHD. It is expected to become a first-line anti GVHD therapy.

P1328: SUCCESFUL TREATMENT OF GRAFT FAILURE IN AUTOLOGOUS STEM CELL TRANSPLANTATION WITH CYCLOSPORINE

D. Cubillas 1,* , M. Quesada Sanchez 1 , P. Martinez Sanchez 1 , M. Calbacho Robles 1 , J. M. Sanchez Pina 1 , T. Baumann 1 , A. J. Saez 1 , M. Poza 1 , R. Iñiguez 1 , I. Zamanillo 1 , J. Martinez Lopez 1 , A. Jimenez Ubieto 1

1 Hospital 12 de Octubre, Madrid, Spain

Background: Graft failure is an unusual complication of autologous stem cell transplantation (ASCT). It is a life threatening complication where most patients (pts) die because of infections or bleeding. It is defined by absence of neutrophil recovery (>500 ANC) on day +28. It can be primary (there wasn´t a previous neutrophil recovery) or secondary (if there was). Ruling out infections, toxicity of conditioning or poor quality of cells infused, the main cause is still unknown. There is no much literature about GF in ASCT. Treatments proposed are cellular reinfusion or search of a donor for allogeneic stem cell transplantation with poor results.

Aims: The aim of this study is to describe our experience in treating such a rare ASCT complication. Primary endpoint is the obtainment of neutrophils (>500) and the platelet engraftment (>20.000) with no transfusion-dependence.

Methods: Between 2010 and 2021 a total of 706 pts underwent an ASCT in our institution. 5 pts presented a primary (n=4) or secondary (n=1) GF. We collect the data from these patients.

Results: Our incidence of GF is 0.71%, making GF a rare complication of ASCT. 4 pts had a primary GF and 1 had a secondary GF. Peripheral blood was the source in the pts; who were mobilized with G-CSF without any complication. All the pts were infused with more than 2 x10 6 CD34+ cells /kg (median number of 2.7 (2.2 -3.4)) without any manipulation of the graft. The median viability was 96% (92-99.7). None of the pts had splenomegaly or bone marrow infiltration at the moment of ASCT. These 5 pts had different hematological diseases (3 Multiple Myeloma (MM), 1 composite lymphoma (CL) and 1 primary nervous system lymphoma (PCNSL)). The status of response at ASCT was 3 CR, 1 VGPR (1 MM) and 1 PR (PCNSL). The conditioning regimen used was BUMEL (2 MM), MEL200 (1 MM) BEAM (1 CL) and TT-BCNU (1 PCNSL). All the pts received G-CSF from day +5 after cell infusion. Bone marrow biopsy was made in 3 of these pts, with severe hypoplasia without fibrosis. After diagnosis of GF, in all the pts, the first line treatment was increased dose of G-CSF. 4 pts were also treated with corticoids without any response. 2 pts additionally received previously cryopreserved cells without response. We started empiric treatment with CsA at an initial dose of 1.5mg/kg twice daily in these pts and the median time to reach>500 ANC was 5 days (4-9). Three of these pts could stop CsA treatment after 30 days due to recovery of neutrophil and platelet count. Another one had to be under treatment 150 days. The last one is still on treatment (after 2 months). With a median follow up of 90 months (2-91) all the pts are alive and with a good neutrophil and platelet engraftment. In terms of security, 2 pts developed mild renal failure, managed by dose adjust. No other adverse events were seen in these pts.

Summary/Conclusion: CsA is an immunosuppressive drug widely used in aplastic anemia and other bone marrow failure syndromes, due to suppression of haematopoiesis by autoreactive T- lymphocytes. This can also be the pathogenic mechanism of GF. We successfully used empirical CsA treatment in these pts. All the pts but one had an ANC>500 in the first week of treatment with CsA and were able to discontinue the drug soon. Severe infections due to neutropenia are the first causes of death in pts with GF so improvement of ANC decrease is the main goal in the treatment of GF. As CsA is a secure drug, we propose to urgently include this treatment as a curative option in the algorithm of GF management to be taking into account before the allogeneic stem cell trasplantation option.

P1329: POST-TRANSPLANT CYCLOPHOSPHAMIDE (PT-CY) VERSUS ANTITHYMOCYTE GLOBULIN (ATG) AS GVHD PROPHYLAXIS FOR MATCHED UNRELATED HEMATOPOIETIC STEM CELL TRANSPLANTATION.

F. Dachy 1,* , S. Furst 1 , B. Calmels 2 , T. Pagliardini 1 , S. Harbi 1 , B. Bouchacourt 1 , A. Calleja 1 , C. Lemarie 2 , A. Collignon 1 , G. Morel 1 , F. Legrand 1 , E. Bekrieva 1 , A. Granata 1 , P. J. Weiller 1 , C. Chabannon 2 , J. M. Schiano de Collela 1 , N. Vey 1 , D. Blaise 1 , R. Devillier 1

1 Hematology; 2 Cell therapy facility, Institut Paoli-Calmettes, Marseille, France

Background: In recent years, post-transplant cyclophosphamide (PT-Cy) has become an essential component for GVHD prophylaxis in T-cell replete haploidentical (HRD) stem cell transplantation. It is now being evaluated in unrelated donor (UD) transplants.

Aims: Our objective is to evaluate the efficacy of PT-Cy as part of GVHD prophylaxis in the setting of 10/10 UD allo-HSCT.

Methods: We reported the outcomes of a HLA-10/10 matched unrelated donor (MUD) allo-HSCT cohort, treated with PT-Cy (n=30) between April 2020 and July 2021, and performed a retrospective comparison with an historical control group treated with ATG (n=64).

Results: Among the whole 94 patients, the median age was 57.5 (18-75), 43 (45%) patients had acute myeloid leukemia, 12 (13%) had myelodysplastic syndrome, 12 (13 %) had acute lymphoid leukemia, 27 (29%) had lymphoma. Median HCT-CI score was 3. All patients received peripheral blood stem cells as graft source. There was no significant difference in these baseline characteristics between both groups. In the PT-Cy group, 21 (70%) and 9 (30%) patients received non-myeloablative (fludarabine cyclophosphamide 2 Gray TBI) and thiotepa busulfan fludarabine based conditioning regimens, respectively. They were given cyclosporine A and mycophenolate mofetil as additional GVHD prophylaxis. All patients in the ATG group homogeneously received a reduced intensity conditioning regimen with fludarabine, 2-day bulsulfan. GVHD prophylaxis was ATG (5mg/kg thymoglobulin) and cyclosporine A. G-CSF from day+5 to neutrophil recovery was given in the PT-Cy group while no patient in the ATG received G-CSF.

The day-100 cumulative incidence of grade 2-4 acute GVHD was significantly lower in the PT-Cy group (23% vs. 45%, p=0.014, Figure A). We observed a trend for a lower 2-year cumulative incidence of all grades and moderate to severe chronic GVHD in the PT-Cy group (all grades: 13% vs 33%, p=0.063, Figure B; moderate to severe: 10% vs. 27%, p=0.079). No significant difference was observed in the incidence of relapse (20% in the PT-Cy group vs 11% in the ATG group, p=0.382), non-relapse mortality (3% vs 11%, p=0.203), progression free survival (77% vs 78%, p=0.912) and overall survival (86% vs 83%, p=0.58).

All patients engrafted in both groups. Median time from allo-SCT to neutrophil (>0.5G/L) and platelet (>20G/L) recovery was significantly delayed in the PT-Cy group (neutrophil: 19 vs 17 days, p=0.049; platelets: day 26 vs 10, p<0.001). Finally, we followed the GVHD and the immunosuppressive treatment (IST) prevalences in disease free patients as a surrogate marker of quality of life. At 6 months post allo-HSCT, 5% and 36% of the disease-free patients in the PT-Cy group were living with GVHD and IST, respectively. Corresponding values for the ATG group were 26% and 64% for the prevalence of GVHD and IST, respectively.

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Summary/Conclusion: These data suggest that the PT-Cy platform is an effective GVHD prophylaxis for the 10/10 UD allo-HSCT patients. It significantly decreases the risk of acute GVHD compared with standard prophylaxis with ATG, without increasing the risk of relapse. This study adds to the experience in the HRD and MMUD allo-HSCT, where PT-Cy seems to counterbalance the impact of HLA disparity, leading to promising survivals such as after HLA matched allo-HSCT.

P1330: IMPACT OF COVID-19 IN HSCT ACTIVITY. A SURVEY FROM THE INFECTIOUS DISEASES WORKING PARTY (IDWP) OF EBMT

R. De La Camara 1,* , Z. Szmit 2 , P. Ljungman 3 , N. Knelange 4 , L. Wendel 4 , G. Tridello 5 , K. Kalwak 6

1 haematology, Hospital de la princesa, Madrid, Spain; 2 Anesthesiology, Wroclaw Medical University, Wroclaw, Poland; 3 Cellular Therapy and Allogeneic Stem Cell Transplantation, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden; 4 Dept. Medical Statistics & Bioinformatics, EBMT Data Office, Leiden, Netherlands; 5 Paediatric Haematology Oncology, Azienda Ospedaliera Universitaria Integrata, Verona, Italy; 6 Pediatric Hematology/Oncology and BMT, Wroclaw Medical University, Wroclaw, Poland

Background: The SARS-CoV-2 pandemic has impacted nearly all the aspects of HCT. One of those has been historical: the decrease for the first time in 30 years in the whole transplant activity (Passweg J. BMT 2022)

Aims: To analyse the impact of the pandemic on HCT activity in 2020 compared to pre-pandemic 2019

Methods: In April 2021, a survey was sent to all EBMT centres to collect the HCT activity, reporting the number of transplant patients by month for 2020 and 2019

Results: A total of 168 centres participated: 136 adult and 75 paediatric centres (43 did both)

1.- HCT activity in adults

1.1 Global (allo & auto): a marked decrease in activity from March-May, with the biggest drop in April 2020 (-40.6%) (Fig 1)

1.2 Allogeneic : a marked decrease in activity from March-May 2020, the biggest drop in April (-39%), and a moderate total decrease (-6.1%). - Matched related donor (MRD) (1,201 vs 1,434): a marked decrease in activity from April-July, the biggest drop in April (-46%), and a marked total 2020 decrease (-16%) - Mismatched related/haploidentical donor (MMRD) (1,005 vs 869): no decrease in activity, with a final 15% total increase. - Unrelated donors (URD) with PB/BM (2,348 vs 2,558): a marked decrease in activity from March-May, the biggest drop in April (-46%), and a moderate total decrease (-8%). - URD Cords (57 vs 50): no decrease in activity. However, too few cases

1.3 Autos : a marked decrease in activity from March-May, the biggest drop in April (-42%), but a moderate total decrease (-7%)

1.4 Non-malignant diseases (47% auto and 53% allo-HCT): a marked total decrease, higher in allo-HCT (-19.7%) than in auto-HCT (-5.6%)

2.- HCT activity in children

2.1 Global (allo & auto): a discrete increase in the activity in nearly all months, with a 5.2% total increase.

2.2 Allogeneic -Total: marked decrease in activity in April (-29.3%), but nearly identical total activity. - MRD (532 vs 611): a marked decrease in activity from March-May, with the biggest drop in April (-56%), and a marked total decrease (-13%). - URD PB/BM ( 631 vs 651): a marked decrease in activity for URD from April-May, with the biggest drop in April (-46%), but a mild total decrease (-3%). - MMRD (446 vs 373): an increase in activity in all months, with a final 19.5% increase. - URD Cords (69 vs 37): an increase in activity in nearly all months, however few cases.

2.3 Autologous HCT: an increase in activity in nearly all months, with a 21% total increase

2.4 Non-malignant diseases (4% auto and 96% allo-HCT): a marked total decrease of 15.9%, due to a marked decrease in allo-HCT (-18.2%) not compensated by a 64% increase in auto-HCT

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Summary/Conclusion: The SARS-CoV-2 pandemic had a significant impact on HCT activity in 2020 compared to 2019, with substantial differences between adults and children . Globally there was a mild decrease in HCT activity in 2020 (-4.7%) due to a moderate decrease in adult HCTs (-6.6%) not compensated by a 5.2% increase in pediatric transplants. Non-malignant diseases had a marked decrease (-14.9%), both in adults and children

In adults, a similar decrease was observed in allo and auto-HCT (-6.1% vs -7%). MRD suffered the greatest decrease (-16%), followed by URD with PB/BM (-8%). In contrast, URD Cords and MMRD showed a marked increase (+14% and +15%)

In children , auto-HCT showed an unexpected marked increase (21%) with no variation in allo-HCT. MRD suffer the greatest decrease (-13%), followed by URD with PB/BM (-3%). In contrast, URD Cords and MMRD showed a marked increase (+86% and +19.5%)

The increase in cords and MMRD HCT was probably due to the easier access of these donors in a pandemic situation

P1331: CENTRAL AND PERIPHERAL VENOUS ACCESSES SHOW NO DIFFERENCES IN EFFICIENCY OF CD34 + CELLS COLLECTION BY APHERESIS

J. C. De La Rosa García 1,* , B. Diaz Roldan 1 , A. J. Palma Vallellano 1 , A. R. Carranco Falcón 2 , R. Zapata Bautista 2

1 Hematology; 2 Hospital Juan Ramon Jiménez, Huelva., Huelva, Spain

Background: Cytokine-mobilized peripheral blood stem cells (PBSCs) are the most common source in autologous and allogenic hematopoietic stem cell transplantation (HSCT) at present, displacing bone marrow harvest. Through apheresis we can obtain PBSCs, process that may be performed by different types of vascular accesses, either peripheral veins (PV) or central venous catheter (CVC). Nowadays there is no consensus on the superiority of one access over another in terms of quality of harvesting.

Aims: The goal of this study is to compare both techniques in order to discover differences between them in the scope of efficiency.

Methods: Our single-center study retrospectively revised and compared 155 apheresis process performed between July/2016 and October/2021. We defined collection efficiency, as the CD34+ cellularity obtained in relation to the expected (according to the processed volemia and the CD34+ pre-apheresis cells concentration in blood determined on a flow cytometer).

Haematocrit, leukocytes, platelets and total proteins were compared in both groups to rule out differences, since several studies have shown that can influence in the collection efficiency. In our study CVC employed were Shaldon or Hickman.

Results: Of 155 apheresis, 71 (45.81%) were performed through CVC, and 84 (54.19%) through PV. We also divided our sample according to sex and pathology (“sample” of Table 1). Differences in Haematocrit, leukocytes, platelets and total proteins are also showed at “pre-apheresis results” in Table 1.

Finally, we focused in post-apheresis data. We analyzed: the efficiency of the procedure, the volume of collected product and the final count of CD34+ collected. We also analyzed the possible differences between both groups of patients, using the Student’s T technique, looking for statistically significant differences (p) between them (“post-apheresis results” of Table 1).

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Summary/Conclusion: The results of our study reveal a similar efficiency regardless of the type of access used for apheresis. Therefore, taking into account the possible complications of central catheter venous access, as a higher rate of infection, thrombosis, pneumothorax, bleeding or the recirculation rate risk, it should be used only in patients with poor peripheral accesses.

P1332: PROMISING OUTCOME OF PATIENTS OLDER THAN 70 YEARS WHO UNDERWENT PERIPHERAL BLOOD HAPLOIDENTICAL HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR ACUTE MYELOID LEUKEMIA OR HIGH RISK MYELODYSPLASTIC SYNDROME

S. Harbi 1 , L. Brac de la perriere 1 , S. Garciaz 1 , T. Pagliardini 1 , B. Calmels 2 , M. Cecile 3 , Y. Hicheri 1 , B. Bouchacourt 1 , A. Calleja 1 , F. Dachy 1 , S. Fürst 1 , C. Lemarie 2 , C. Braticevic 3 , G. Morel 1 , F. Legrand 1 , E. Bekrieva 1 , A. Granata 1 , P. J. Weiller 1 , C. Chabannon 1 , N. Vey 1 , D. Blaise 1 , R. Devillier 1,*

1 hematology; 2 cell therapy facility; 3 oncogeriatry, institut paoli calmettes, Marseille, France

Background: Allogeneic stem cell transplantation is a curative option of patients older than 60 years of age with AML and high risk MDS. In this situation, the development of haploidentical transplantation (Haplo-SCT) using post transplantation cyclophosphamide (PT-Cy) allowed extending the feasibility of Allo-HSCT. However, there is few data specifically focusing on Haplo-SCT in patients over 70 years of age with AML and MDS, for whom the benefit-risk balance of Haplo-SCT is still a matter of debate.

Aims: To evaluate the outcome of patients older than 70 years with AML or high risk MDS who underwent Haplo-SCT.

Methods: This retrospective study included consecutive AML or MDS patients older than 70 years who received first Haplo-SCT from 2013 to 2021 in our center. All patients had the same transplantation modalities: 1) Non-myeloablative conditioning regimen as described by the Baltimore’s group (Fludarabine, cyclophosphamide and 2-Gray TBI); 2) Peripheral blood stem cells (PBSC) as graft source; 3) GVHD prophylaxis using PT-Cy, cyclosporine A and mycophenolate mofetil; 4) G-CSF from day+5 to neutrophil recovery. Prophylactic donor lymphocyte infusion (DLI) was initially planned after day+60 in absence of GVHD.

Results: We analyzed 45 patients who met inclusion criteria, 23 with AML and 22 with MDS. The median age was 72 years (range: 70-75). HCT-CI was >= 3 in 22 patients (49%). At the time of Haplo-SCT, 22/23 AML patients were in CR (17 CR1 and 5 CR2) and one patient had refractory AML. Disease status at Haplo-SCT was CR, PR, marrow CR and active disease in 12, 2, 1 and 7 patients, respectively. All patients received pre transplantation treatment with intensive chemotherapy and/or azacytidine, except one MDS patients who underwent upfront Haplo-SCT. Disease risk index was high or very high in 11 patients (24%), and intermediate in 34 patients (76%). Median follow up using the reverse Kaplan Meier method was 36 months (95%CI: 25-43).

Grade 2-4 and 3-4 acute GVHD at day+100 was 33% and 7%, respectively. There was no early death (at day+100: OS=100%; NRM=0%). At 3 years after Haplo-SCT, the cumulative incidence of all grades and moderate to severe chronic GVHD was 37% and 28%. Half of the cases of chronic GVHD occurred after prophylactic DLI. The 3-year cumulative incidence of NRM was 20%. In AML patients, the 3-year CIR, PFS and OS were 17%, 61% and 64% respectively. Corresponding values for MDS patients were 26%, 56% and 66%, respectively. Ten patients received prophylactic DLI. Among them, one died from late NMR (urothelial metastatic cancer after 2 years post Haplo-SCT) and one patient relapsed and subsequently died from AML (complex karyotype, CR2). The 8 remaining patients who received prophylactic DLI are still disease free at last follow up. As surrogate marker of quality of life, we evaluated the prevalence of GVHD and immunosuppressive treatment (IST) in disease free patients. At 2 years after Haplo-SCT, 87% of disease free patients are living without GVHD and without IST.

Summary/Conclusion: Haplo-SCT in patients older than 70 years with AML or high risk MDS is feasible without early NRM. The use of PBSC and prophylactic DLI may explain the low incidence of relapse and long-term survival. Our results support that selected patients older than 70 years can achieve promising outcomes after non-myeloablative Haplo-SCT using PBSC, confirming that age by itself and the absence of an HLA-matched donor should not be considered as firm contraindication for transplantation.

P1333: PLASMA EXCHANGE, IMMUNOGLOBULIN & PRE-TRANSPLANT IMMUNOSUPPRESSION: AN EFFECTIVE DESENSITISATION STRATEGY FOR DSA ALLOANTIBODIES IN HAPLOIDENTICAL HCT FOR PAEDIATRIC RED CELL DISORDERS

D. Dexter 1,* , S. Bremathas 1 , K. Hennessy 1 , D. Johnson 1 , T. Petterson 1 , K. Lund 1 , A. Gassas 1 , L. Karnik 1 , J. de la Fuente 1,2

1 Paediatric Haematology, Imperial College NHS Foundation Trust; 2 Paediatric Haematology, Imperial College London, London, United Kingdom

Background: Near universal availability of HCT is possible due to the feasibility of haploidentical transplantation [haplo HCT] with the use of post-transplantation cyclophosphamide or TCRαβ CD+19 depletion, enabling HCT for haemoglobinopathies and rare inherited anaemias for whom donors are underrepresented in registries. Patients with these disorders are at increased risk of developing anti-HLA antibodies having often received multiple transfusions pre-transplantation. HLA antibodies constituting donor specific antigens [DSA] affect approximately 15% of haplo HCT [Leffel, 2013] representing a significant cause of primary graft failure [Ciurea, 2009]. An effective desensitisation strategy is necessary as alternative donors for haploidentical HCT are often lacking. Several desensitisation approaches have been developed based on strategies used for solid organ transplantation, yet there exists no consensus in their application to haplo HCT for paediatric haemoglobinopathies where there is a particularly high risk of graft failure [de la Fuente, BBMT 2019, de la Fuente, ASH 2020].

Aims: To assess the efficacy of plasma exchange, high dose immunoglobulin and pre-transplant immunosuppression as a desensitisation strategy for DSA alloantibodies in paediatric patients undergoing haploidentical HCT for red cell disorders.

Methods: Seven [17.5%] patients were found to have HLA class I DSA in 40 consecutive related reduced-intensity conditioning haplo HCT with post-transplant cyclophosphamide for red cell disorders performed at St Mary’s Hospital from 2013 to December 2021. The MFI cut off used was 2,000U. Transplant indications included homozygous sickle cell disease [n=5], HbSβ0 thalassaemia [n=1] and Diamond-Blackfan anaemia [n=1]. Median age was eight years [range 3-15 years]. Five children were female. Two patients had two different DSA. Each patient received a regimen based on the John Hopkins protocol [Leffel, 2013], comprising a single dose of rituximab 375mg/kg, four alternate day cycles of single volume plasmapheresis [replacing 100% volume with 5% albumin] and anti-CMV hyperimmune immunoglobulin 100mg/kg on days -16, -14, -12 and -10 before the initiation of the conditioning regimen and a further cycle of single volume plasmapheresis on D -1 and anti-CMV hyperimmune immunoglobulin 100mg/kg, and immunosuppression with tacrolimus and MMF to Day 0. HLA antibodies were measured on Day 0 prior to HSC infusion. If the MFI had not decreased <2000U, two further cycles of single volume plasmapheresis and anti-CMV hyperimmune immunoglobulin 100mg/kg were given on D+1 and D+2.

Results: The median MFI at baseline was 5,974 U [range 1,544-22806U]. The protocol was well tolerated and completed without serious adverse events in all patients. Four patients required additional cycles on days +1 and +2. Median MFI at protocol completion was 230U [range 0-769U]. No patient suffered primary graft failure. Median time to neutrophil engraftment was 21 days [range 15-24]. All patients achieved full donor whole blood chimerism on D+28 and wheras there was mixed chimerism in the T cell fraction in three of the patients, this improved over time in all patients and no patient suffered secondary graft failure.

Summary/Conclusion: The use of a desensitisation protocol comprising the combination of plasma exchange, high dose immunoglobulin and pre transplant immunosuppression constitutes an effective desensitisation strategy for DSA and abrogates the risk of graft failure in multiply transfused paediatric patients undergoing haploidentical HCT for red cell disorders, enabling HCT where no alternative donor exists.

P1334: THE IMPACT OF IMMUNOSUPPRESSION ON RECONSTITUTION OF CMV-SPECIFIC T CELLS AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

A. Dmitrova 1,* , M. Drokov 2 , T. Tupoleva 3 , D. Tikhomirov 3 , L. Kuzmina 1 , M. Vagida 4 , G. Efimov 4 , E. Parovichnikova 5

1 Bone marrow transplantation department; 2 Immunotherapy and Post-BMT complications department; 3 Laboratory of virological diagnostics; 4 Laboratory of transplantation immunology; 5 Chief of the National Research Center for Hematology, National Research Center for Hematology, Moscow, Russia

Background: Cytomegalovirus infection (CMV infection) is the most frequent and life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The development of CMV infection is controlled by T-cell immunity. The type of the immunosuppressive regimen is the most important reason of decreased CMV-specific CD8+ immune response following allo-HSCT.

Aims: In our study we try to assess the influence of different types of immunosuppressive regimens on recovery of the CMV-specific CD8+ T cells in patients after allo-HSCT.

Methods: Analysis of peripheral blood CMV-specific CD8+ T cells from 108 patients performed by flow cytometry at + 30, +90, +180 days after allo-HSCT. We used fluorochrome-labeled monoclonal antibodies against CD3, CD8, CD45 molecules, viability reagent and tetramers which were formed from MHC class I monomers loaded with immunodominant epitopes of cytomegalovirus pp65 protein (HLA-A*0201/NLV, HLA-B*0702/TPR, HLA-B*0702/RPH) and Streptavidin, R-Phycoerythrin conjugate (SAPE). For graft-versus-host disease (GVHD) prophylaxis 22 (20,3%) patients received anti-thymocyte globulin (ATG), 52 (48,2%) – posttransplant cyclophosphamide (PT-Cy), 12 (11,1%) – ATG + PT-Cy, 13 (12%) – T-cell depletion protocol and 9 (8,4%) patients received other immunosuppressive regimens. Detailed patients’ characteristics are presented in Table 1.

Results: The data shown in figure 1 indicate that the use of PT-Cy (PT-Cy 50 mg/kg/day intravenously on days +3, +4) and T-cell depletion protocols as GVHD prophylaxis leads to lower CMV-specific T cells counts during 6-months follow-up period after allo-HSCT. The combination of PT-Cy and ATG also shows reduced absolute numbers of CMV-specific CD8+ T cells compared to ATG protocols (ATG 40 mg/kg/day intravenously for 4 days).

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Summary/Conclusion: Our results show that patients who received PT-Cy as GVHD prophylaxis and T-cell depletion-grafts have highest risk for developing CMV infection after allo-HSCT. The management of CMV infection by monitoring of CMV by polymerase chain reaction, use of antiviral prophylaxis and prompt use of pre-emptive therapy are very important for such patients’ groups.

P1335: SECONDARY MALIGNANCIES AFTER AUTOLOGOUS STEM CELL TRANSPLANTATION. SINGLE HOSPITAL EXPERIENCE.

A. Dorado López 1,* , M. Calviño Suárez 1 , E. Lavilla Rubira 1 , P. Sar Fuentes 1 , J. A. Arias Sampedro 1 , L. Lopez Gonzalez 1 , M. J. Sánchez Sánchez 1 , M. Varela Pérez 1

1 Haematology and Hemotherapy Department, University Hospital Lucus Augusti, Lugo, Spain, Lugo, Spain

Background: Autologous stem cell transplantation (ASCT) has a key role in the treatment of haematological malignancies, improving results and survival outcomes. Acute toxicity is well known and support therapy has improved over the years, with an overall procedure-related mortality currently being at 2-3%. However, there are long-term complications with high morbidity and mortality, such as secondary neoplasms, which limit the benefit of the procedure. The reported incidence ranges around 9-17% at 15 years after transplantation and they are responsible for 5-10% of late deaths.

Aims: The main objective was to estimate the incidence and identify the risk factors related to second malignancies.

Methods: We submit a retrospective study of 242 patients who underwent a total of 269 ASCT in our Institution over a period of 24 years (1995-2019). Multiple variables were collected from a clinical care database and statistically analysed using IBMSPSS-Stadistic-2019 and the proper comparative methods.

Results: Of all the variables collected, it is worth mentioning the following: median age 56 (range 16-73), female (52.5%), diagnosis [LPDs (50.8%), MM (35.5%), AL (8.7%), other (4.8%)], >1 previous line of chemotherapy (44.2%) and second transplant (16%).

The overall mortality (OM) at 2, 5 and 10 years was 21.5, 29.3 and 38% respectively, with a corresponding non-relapse OM of 10.3, 13.6 and 16.7%.

Fifteen (7.2%) out of the 209 patients who were alive after 6 months from the transplant, developed a secondary cancer after transplantation: 11 solid neoplasms [oral (1), urologic (1), digestive (4), lung (3), breast (2)] and 4 hematologic [acute myeloid leukaemia (2), acute lymphoid leukaemia (1) and T lymphoma (1)] with the characteristics set out in Table 1. Additional risk factors (such as smoking and/or alcoholism) unrelated to the hematologic malignancy or previous therapies were identified in only 3 patients. The latency period from transplantation to onset of the malignancy was 8 years (range 3-13), a period that is shortened by half in the case of acute leukaemia (median 4 years, range 3-5).

More than half of these patients (n=8; 53.3%) died as a direct cause of the second neoplasm, within a period ranging from 1-48 months with a median of 3 months. Therefore, it becomes the 2nd cause of death at 2- and 5-years following relapse of the underlying disease and the first cause at 10 years after transplantation. No differences were observed between patients with or without secondary neoplasm (Figure 1) in terms of overall survival neither according to the type of cancer.

Data’s statistical univariant comparison (including age, sex, diagnosis, previous therapies, PH mobilization, conditioning regimen, radiotherapy...) identified only the additional execution of allogeneic transplantation (p value 0.001) as significantly associated with the development of the neoplasia.

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Summary/Conclusion: The study verifies the incidence and morbidity and mortality associated with the development of secondary neoplasms and highlights the need for systematic follow-up of the long-term survivor in order to identify this complication as soon as possible. This is a complex process that requires the joint participation of a multidisciplinary team and the patient involvement in order to realize the risks still present many years after ASCT regardless the underlying disease’s remission.

Given the heterogeneous population and the study’s retrospective nature we have faced some limitations, hence it is important to continue exploring this situation for a better understanding.

P1336: ANTIVIRAL EXPOSURE TO GANCICLOVIR AND VALGANCICLOVIR IS NOT ASSOCIATED WITH INCREASED INCIDENCE OF RELAPSE OR SECONDARY MALIGNANCIES IN HEMATOPOIETIC STEM CELL TRANSPLANTATION RECIPIENTS

D. Duarte 1,2,* , S. Duarte 1 , C. Sol dos Reis 1 , P. Silva Coelho 1 , L. Leite 1 , R. Branca 1 , S. Roncon 3 , C. Pinho Vaz 1 , A. Campos 1

1 Bone Marrow Transplantation Service, Instituto Português de Oncologia (IPO)-Porto; 2 Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto; 3 Celular Therapy Service, Instituto Português de Oncologia (IPO)-Porto, Porto, Portugal

Background: Hematopoietic stem cell (HSC) transplantation (HSCT) is used in the treatment of hematologic malignancies, bone marrow failure and immunodeficiencies and is often associated with cytomegalovirus (CMV) reactivation/infection. Ganciclovir or its prodrug valganciclovir (herein GCV) are guanine analogs that compete with dGTP for DNA incorporation, thereby inhibiting viral DNA replication. A recent study by de Kanter et al. (Cell Stem Cell. 2021) demonstrated that GCV induces mutagenesis in human cells, including HSC. In particular, GCV induces C > A changes at CpA dinucleotides. In contrast, the antiviral foscarnet was not associated with mutations in HSCs. The authors found the GCV mutational signature in relapses and second neoplasias in transplantation recipients, raising the possibility of increased incidence of secondary cancers in patients (pts) receiving GCV treatment.

Aims: To evaluate the incidence of secondary neoplasia in HSCT recipients treated with GCV.

Methods: Unicentric retrospective analysis of HSCT recipients receiving GCV between 2007 and 2021. HSCT pts not receiving GCV were also included as controls (C). Pts who had documented CMV reactivation but no complete information about antiviral exposure were excluded. Statistical analysis was performed in SPSS.

Results: A total of 930 pts were included, 522 (56.1%) receiving GCV and 408 (43.9%) control pts not exposed to GCV (C). Median follow-up was 23 months (0-170) (20 for GCV and 30 for C, p=0.124). CMV reactivation was detected in 68% of pts (96.9% for GCV and 19.8% for C, p<0.001). Median age was 42 years old (0-69) (43 for GCV and 39 for C, p=0.001) and 59% were man (56.7% for GCV and 59.3% for C, p=0.517). Myeloablative conditioning was used in 32.9% of pts (34.5% in GCV and 30.6% in C, p=0.208). HSC sources were similar between groups (p=0.133) and peripheral blood was the most common (86% in GCV and 81.6% in C). Donor kinship was also similar (p=0.056) and donors were HLA-identical in 69.5% of the cases (66.3% in GCV and 73.3% in C, p=0.062). Within the GCV group, 411 (44.2%) pts were exposed to ganciclovir alone, 276 (29.7%) pts to valganciclovir alone and 159 (17.1%) pts to both drugs.

We detected a total of 228 (24.5%) relapses, 115 (22.0%) in GCV and 113 (27.7%) in C (p=0.048). We also detected a total of 43 (4.6%) secondary malignancies, 28 (5.4%) in GCV and 15 (3.7%) in C (p=0.222). Subtypes of secondary malignancies were similar (p=0.205) and included basocellular carcinoma and post-transplant lymphoproliferative disorders. No donor-cell leukemias were detected in our cohort. Combined incidence of relapse and secondary cancers was similar between groups (26.1% vs 30.6%, p=0.127). We next scored relapses and secondary cancers as events. The median event-free survival was 133 months in GCV and not reached in C (p=0.339; HR 1.126). Global overall survival (OS) was 48 months, with median OS of 33 months for GCV vs 97 months for C (p=0,025, HR 1,238).

Summary/Conclusion: In our study, GCV exposure in HSCT was not associated with combined incidence of relapse and secondary cancers. In contrast, an excess of relapses was detected in HSCT pts not exposed to GCV. Shorter OS in GCV-treated pts likely translates other HSCT complications not related with relapse/cancer. Our study is limited by its retrospective and unicentric nature. A longer follow-up might be required to detect more subtle differences. In conclusion, we suggest that although GCV induces in vivo mutagenesis of human cells, it does not translate in increased risk of cancer in HSCT.

P1337: SURVIVAL EFFICACY OF MDS/AML PATIENTS WITH TP53 GENE ALTERATION RECEIVED ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

D. Feng 1,* , E. Jiang 1

1 Hematological Hospital, Chinese Academy of Medical Sciences, Hematological Hospital, Chinese Academy of Medical Sciences, Tianjin, China

Background: Although not yet included in guidelines, TP53 mutations are undoubtedly one of the undesirable factors in the risk prognosis of medullary tumors in the hematologic system. Different TP53 gene states have different prognostic effects on MDS and AML, and whether allogeneic hematopoietic stem cell transplantation can overcome this undesirable factor needs to be further explored.

Aims: To investigate the survival efficacy of allo-geneic hematopoietic stem cells in patients with MDS/AML with TP53 state changes (deletion, monoallelic mutation, multi-hit). Analyzing the clinical characteristics in patients with different TP53 gene alterations, and further exploring the influencing factors of clinical prognosis of TP53-change MDS/AML after allo-HSCT.

Methods: A retrospective analysis was conducted on 42 patients with TP53-MDS/AML who underwent allo-HSCT from 2006.1 to 2021.7.These patients may have deletion of the TP53 gene and/or mutations in the TP53 gene.The 42 patients were divided into 3 groups,TP53 deletion MDS/AML group (group A), TP53 mono-mutation MDS/AML group (group B), and TP53 multiple-hits group (group C), and the differences in clinical features and the effect on the prognosis of inhibition were analyzed.The changes in the characteristics of second-generation sequencing of 137 genes in bone marrow specimens of 29 patients with mutations were also discussed.

Results: There were 42 MDS/AML patients, 30 males (71.4%), 12 females (28.6%), 21 MDS patients, 1 MDS-AML patient, and 20 AML patients. The average age of patients at the time of transplantation was 42 years, of which 11 patients with only TP53 deficiency (26.1%), 25 patients with TP53 single mutation (59.5%), and 6 patients with TP53 multiple hits (14.2%). Among the 42 patients, 14 (33.3%) died, 10 cases relapsed (23.8%), and the overall patients who received hematopoietic stem cell transplantation had 3 years OS＞40%,and there was no statistical difference in 3 years OS in group A, B and C. Univariate analysis shows,hemoglobin、age of HSCT、complex karyotype、pre-HSCT MRD were risk factors for survival (p=0.003,p=0.042,p=0.009,p=0.004), and TP53 stage、co-mutation、treatment before HSCT、type of Diseases not. Complex karyotype、hemoglobin、pre-HSCT MRD were also risk factors for relapse (p=0.038,p=0.02,p=0.023),besides,pre-HSCT BM blast% also have an impact on relapse,p=0.027.Among the 42 patients, 12 patients (28.5%) with complex karyotype, including 2 patients with TP53 deletion alone (16.6%), 5 patients with TP53 single mutation (41.6%), and 5 patients with TP53 multiple hit (41.6%).Of the 12 patients with complex karyotypes, 7 died, 5 relapsed, and 4 of the 7 deaths were patients with TP53 multi-hits. Survival and relapse of TP53 mutations may be associated with complex karyotype (p=0.009,p=0.038).

Summary/Conclusion: Allo-hematopoietic stem cell transplantation can overcome the poor changes of MDS/AML patients with TP53 mutation/deletion, but complex karyotype is still a poor prognostic factor for TP53-MDS/AML patients receiving allo-HSCT.

P1338: INFUSION OF DIMETHYL SULFOXIDE (DMSO) - DEPLETED AUTOLOGOUS PERIPHERAL BLOOD PROGENITOR CELLS REDUCES POST-AUTOGRAFTING SEVERE GASTRIC TOXICITIES AND HOSPITALIZATION LENGTH

V. Filipponi 1,* , M. Antonacci 1 , M. C. Scerpa 2 , U. Coppetelli 2 , S. Perrone 2 , N. Cenfra 2 , S. Mecarocci 2 , L. Fiori 2 , F. Brinchi Giusti 2 , P. Giovangrossi 3 , F. Equitani 3 , E. Ortu La Barbera 2 , G. Cimino 1,2

1 Department of Translational and Precision Medicine, Hematology, Sapienza University of Rome, Rome; 2 Hematology Unit Santa Maria Goretti Hospital; 3 Immunoematology and Trasfusion Medicine Service Santa Maria Goretti Hospital, ASL Latina, Latina, Italy

Background: High-dose chemotherapy (HD CHT) and autologous blood stem cell transplantation (ABSCT) are the cornerstone in the treatment of several hematologic malignancies. Cryopreservation of stem cells needs the use of DMSO as cryoprotectant. Unfortunately, DMSO may induce some severe adverse effects during and after autograft infusion, justifying the attempts to deplete DMSO. Despite several studies have demonstrated that DMSO depletion did not affect engraftment of peripheral blood progenitor cell (PBPC), there is still few and inconclusive data on its impact on post-infusion toxicities.

Aims: Since from March 2021 we started to remove DMSO, we are comparing infusion and post-infusion outcome in 55 patients consecutively autografted with DMSO depleted (25 pts; group1) or undepleted (30 pts; group 2) at the hematology unit of the S.M. Goretti Hospital of Latina (Italy) from January 2019 and December 2021, aiming to evaluate whether post-infusion toxicities and clinical outcome may differ between the two groups.

Methods: The DMSO was removed by rinsing the units with the Sepax S-100 (Biosafe S.A. Eysins, Switzerland) cellular centrifugation instrument that automatically processes blood or haematic components in a closed and sterile environment followed by the PBSC washing, using the dedicated CS-600 kit. The clinical characteristics, biological parameters of autografted PBPC and post-infusion outcome of the 55 patients entered this study are reported in Table 1. In group 1, 12 patients had a diagnosis of multiple myeloma (MM) and 13 of non Hodgkin’s lymphoma (NHL), whereas in group 2, 17 patients were affected by MM, 6 by Hodgkin’s lymphoma (HL) and 7 by NHL.

Results: As reported in Table 1, the amount of harvested and infused CD34+ cells were similar in the two groups. To evaluate viability in group 1 we used a different ISHAGE protocol with lower thresholds. This consideration explains the lower viability rates observed in group 1 respect to those of group 2. No differences between the two groups were observed in the average time to neutrophil (N≥0.5 x 10 9 /L) and platelet (PLTs ≥20x 10 9 /L) recovery (10.4 vs 10 days and 12.5 vs 11.7 days, respectively), as well as in the number of transfused RBCs and PLTs units. No grade 3-4 infusion reactions were observed in any case. By contrast, as concern the post-infusion adverse events, in group 2 we observed a significant higher incidence of grade 3 mucositis (37% in group 2 vs 4% in group 1, p=0.003) and of episodes of nausea associated with vomiting (40% in group 2 vs 8% in group 1, p=0.007). Occurrence of febrile neutropenia, sepsis and other documented infections were similar between the two groups. Overall, the average hospitalization length after the infusion was 17 days (median=16) for group 2 and 14 days (median=13) for group 1 (p=0.059).

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Summary/Conclusion: Present findings for the first time show a significant lower incidence of severe post infusional gastrointestinal adverse effect in patients receiving depleted DMSO autograft that may result in a reduced hospitalization length. As reported in animal model, this finding may suggest a direct gastrotoxicity of DMSO, that can potentiate that of chemotherapy. Whether these data may be confirmed in future larger studies they further suggest the usefulness of DMSO depletion from PBPC autografts.

P1339: CLINICAL OUTCOMES OF PATIENTS WITH EBV+ PTLD FOLLOWING HEMATOPOIETIC STEM CELL TRANSPLANTATION WHO FAIL RITUXIMAB: A MULTINATIONAL, RETROSPECTIVE CHART REVIEW STUDY

J. Sanz-Caballer 1 , J. Storek 2 , G. Socié 3 , D. Thirumalai 4 , N. Guzman-Beccera 4 , P. Xun 4 , D. Kumar 5 , N. Sadetsky 6 , D. Dierickx 7 , J. Reitan 8 , A. Barlev 6 , M. Mohty 9,*

1 Servicio de Hematología, Hospital Universitari i politècnic La Fe, Valencia, Spain; 2 Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Canada; 3 Service d’Hématologie-Greffe, Assistance Publique Hôpitaux de Paris-Hôpital Saint Louis, Paris, France; 4 Atara Biotherapeutics, Thousand Oaks, United States of America; 5 Ajmera Transplant Centre, University Health Network, Toronto, Canada; 6 Atara Biotherapeutics, South San Francisco, United States of America; 7 Universitair Ziekenhuis Leuven, Leuven, Belgium; 8 RJM Group, LLC, Crown Point, United States of America; 9 Hôpital Saint Antoine, Paris, France

Background: Post-transplant lymphoproliferative disease (PTLD) occurs following allogeneic hematopoietic stem cell transplantation (HCT) as a consequence of immunosuppression. In most cases following HCT, PTLD is associated with Epstein-Barr Virus (EBV) infection of B cells, either due to reactivation, or from primary EBV infection (Styczynski J, Haematol . 2016; Allen UD, Am J Transplant , 2019; Nijand M, Transplant Direct , 2016). Clinical practice treatment guidelines recommend rituximab as preemptive therapy for EBV reactivation (based on EBV virus load) and for treatment of EBV-driven (EBV + ) PTLD following HCT. However, EBV + PTLD patients (pts) who fail rituximab have very poor outcomes with limited treatment options. Published evidence on the clinical outcomes of these pts who fail rituximab is also limited.

Aims: To describe the outcomes for pts diagnosed with EBV + PTLD following HCT who fail rituximab in a multinational real-world setting.

Methods: We conducted a large multinational, multicenter retrospective chart review study of EBV + PTLD pts following HCT or solid organ transplantation who received rituximab or rituximab plus chemotherapy (CT) between January 2000-December 2018 and were refractory (failed to achieve complete response [CR] or partial response [PR]) or relapsed at any point after such therapy. Data was collected from 29 centers across North America (United States and Canada) and the European Union. This analysis includes pts with EBV + PTLD following HCT who were refractory or relapsed after rituximab ± CT as first line of therapy. The Kaplan-Meier (KM) method was used to estimate the overall survival (OS). Rituximab failure date was defined as the earliest date when pts became refractory or relapsed following rituximab ± CT.

Results:: A total of 81 pts with EBV + PTLD following HCT who failed rituximab ± CT were included in the analysis. Median age at PTLD diagnosis was 49 years (interquartile range [IQR]: 33‒57) and median time to PTLD onset from transplant was 3 months (IQR: 1.9‒4.2). Median follow-up time was 1.7 months (IQR: 0.6‒3.4) from the date of PTLD diagnosis. Of all the PTLDs, 52 (64.2%) were monomorphic, 18 (22.2%) polymorphic, 2 (2.5%) early lesions, and 9 (11.1%) were unknown. The most common PTLD subtype was diffuse large B-cell lymphoma (DLBCL) (46, 56.8%).

Sixty-eight (84%) pts received rituximab monotherapy and 13 (16%) pts received rituximab plus CT as first line of therapy. Seven out of 13 pts who received rituximab plus CT had received preemptive rituximab treatment for EBV viremia prior to PTLD treatment. Median OS was 0.7 months (95% CI: 0.3‒1; IQR: 0.1‒2.7) for 81 pts from rituximab failure date (Figure 1). Median OS from PTLD diagnosis was 1.7 months (95% CI: 1.1‒2.3; IQR: 0.6‒3.4).

Seventy-four (91.4%) out of the 81 pts ultimately died. Causes of death comprised 50 (67.6%) related to PTLD and therapy, 10 (13.5%) graft-versus-host disease (GvHD), 5 (6.8%) from sepsis/infection, 3 (4.1%) due to primary disease leading to HCT, 2 (2.7%) organ failure, 1 (1.4%) graft failure, 1 (1.4%) from hepatic failure, and 2 (2.7%) unknown.

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Summary/Conclusion: The prognosis of EBV + PTLD pts following HCT who fail rituximab ± CT remains very poor with an estimated median OS of less than 1 month, highlighting the significant unmet need in this population.

P1340: RESPONSE RATES AND PROGNOSTIC FACTORS IN ACUTE GASTROINTESTINAL GRAFT-VERSUS-HOST DISEASE

J. Freitas 1,* , M. Pimenta 1 , E. Couto 1 , P. Coelho 1 , R. Branca 1 , C. Pinho Vaz 1 , L. Leite 1 , A. Campos 1

1 Bone marrow transplant, IPO Porto, Porto, Portugal

Background: Graft-versus-host disease (GVHD) continues to be the major lethal complication of allogeneic hematopoietic stem cell transplantation (HCT). The standard first-line therapy for acute GVHD is high-dose steroids, but 50% of cases are steroid refractory (SR), typically involving the gastrointestinal (GI) tract. Unfortunately, there is no established second-line therapy for GVHD which the response rates are typically low (20–40%) and the overall survival is poor, highlighting the urgent need for better therapies.

Aims: Evaluate the response rates and prognostic factors of GI-GVHD patients

Methods: GI-GVHD was diagnosed clinically and/or histologically and graded by MAGIC criteria. Patients whose GI-GVHD progressed within 3 days after the initiation of therapy or who failed to improve by at least one stage within 5 days were defined as non-responders. The overall response rate (ORR) to GVHD treatment was evaluated retrospectively at 4 weeks from the first day of first-line therapy. Survival outcomes were assessed by the Kaplan-Meier method. In the univariate analysis, OS were compared using the Log rank test. In multivariate analyses, the Cox proportional hazard regression model was performed for OS. A p<0.05 was considered to be statistically significant.

Results: 351 patients (pts) were submitted to allogeneic HCT and 67pts (19.1%) had GI-GVHD at our institution between 2016 and 2021. The median time of onset of GI-GVHD was 37 days [7-92]. According to grading, 15pts (22.4%) were grade 4, 20 (29.9%) grade 3, 13 (19.4%) grade 2 and 19 (28.4%) grade 1. Although the ORR to first line (corticosteroid therapy) was 68.6%, 34pts (50.8%) were treated with 2 nd line therapies, whom 20pts (30%) with anti-thymocyte globulin (ATG), 9 pts (13.4%) with infliximab and 5pts (7.5%) with ruxolitinib. The response with ruxolitinib was 80%, infliximab 55.6% and ATG 35%. The median OS was 4months [0-69] and was significantly inferior in GI-GVHD grade 3-4 vs grade 1-2 (2 vs 11 months, p=0.018, fig.1). In non-responders, the OS was significantly inferior vs responders (1-year overall survival 3.7 vs 41.2%, P<0.001, fig.2). In univariate analysis, the gender, non-responders patients and grade 3-4 of GI-GVHD were significantly associated with an increased risk of death, but in multivariate analysis only the non-responders patients were independently prognostic factor in OS.

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Summary/Conclusion: In conclusion, patients with GI-GVHD who did not respond to the first-line therapy and grade 3-4 GI-GVHD had a worse clinical outcome, and the non-responders patients were independently associated with an inferior OS.

The first two authors contributed equally to this work.

P1341: HAPLO-CORD VS HAPLOIDENTICAL TRANSPLANT IN 49 CHILDREN – A RETROSPECTIVE SINGLE CENTER ANALYSIS

P. Fu 1,* , K. Chen 1 , J. Shao 1 , Q. Liu 1 , N. Zhang 1 , B. Zou 1 , X. Sun 1 , H. Jiang 1

1 Hematology and Oncology, Shanghai Children’s Hospital, Shanghai, China

Background: Haplo-cord transplantation means that umbilical cord blood grafts from unrelated donors are infused together with CD34 + -selected cells selected from Haploidentical relatives, which avoids the delayed implantation and long initial hospital stay caused by umbilical cord blood transplantation, and provides an effective alternative for patients without suitable donors. Some studies showed that haplo-cord transplantation could lead to faster neutrophil recovery and lower incidence of chronic GVHD compared with haplo transplantation. In order to make full use of the advantages of easy access to grafts and possibly reducing the incidence of chronic GVHD, we have adopted haplo-cord program to some children lacking suitable donors, and reported the outcome of haplo-cord transplantation, comparing Virus reactivation,the incidence rate of acute and chronic GVHD, and 3-year overall survival rate with haplo-SCT.

Aims: To compare the outcome of haplo-cord transplantation program with haplo transplantation.

Methods: 35 children with Haplo-cord transplantation and 14 children with Haplo-SCT in our hospital were followed up between May 2017 and May 2021, the engraftment, immune reconstitution,Virus reactivation, incidence of GVHD and 3-year overall survival rate were observed.

Results: The median time of neutrophil recovery in both groups was 13 days, and the cumulative recovery rate of neutrophils on the 30th day was 100%. By day 15,85.7% of the haplo-cord recipients had recovered neutrophil counts vs 92.7% of haplo recipients (P= 0.8). The median time of platelet recovery in both groups was 17 days, and no primary graft failure was observed in all patients. Secondary graft failure was documented in 5 haplo-cord recipients and 2 haplo recipients. 17 patients developed CMV-DNAemia in haplo-cord transplant and 9 in haplo patients. 31 cases had EB-DNAemia in haplo-cord vs 10 in haplo patients.By day 100, the cumulative incidence of acute GVHD in Haplo-cord patients was 25.7% vs 28.5% in haplo patients (P=1), and the cumulative incidence of grade 3-4 acute GVHD by day 100 was 11.4% vs 21.4% respectively (P = 0.65). There were no acute GVHD related deaths. Chronic GVHD was significantly reduced in haplo-cord with a cumulative incidence of 5.7% vs 28.6% in haplo patients by 1 year (P =0.04), but no extensive chronic GVHD was observed. The 3-year overall survival rate was 84.5% in haplo-cord transplant vs 74.3% in haplo transplant(P=0.98).

Summary/Conclusion: Haplo-cord transplantation has high implantation rate and good long-term prognosis, and the incidence of chronic GVHD is significantly reduced, which provides an alternative for patients lacking Human leukocyte antigen-matched sibling donor and HLA-matched unrelated donor.

P1342: A SCREENING TOOL TO DETECT CHRONIC OCULAR GRAFT VERSUS HOST DISEASE IN A HEMATOLOGY/ ONCOLOGY OUTPATIENT SETTING

E. Greenan 1,2,* , E. Vandenberghe 3 , E. Conneally 3 , J. Ní Gabhann-Dromgoole 1,4 , C. C. Murphy 1,2

1 Ophthalmology, Royal College of Surgeons Ireland; 2 Ophthalmology, Royal Victoria Eye and Ear Hospital; 3 Haematology/ Oncology, St James Hospital; 4 Pharmacy and Biomolecular Sciences, Royal College of Surgeons Ireland, Dublin, Ireland

Background: Chronic ocular GvHD (oGvHD) is reported to occur in 30-60% of patients after hematological stem cell transplantation (HSCT). Presently, there is no diagnostic test available nor a single ‘gold standard’ sign or symptom for the disease. Instead the diagnosis is confirmed through criteria set by the National Institute of Health (NIH) and the International Chronic Ocular Graft-vs-Host-Disease Consensus Group (ICCGvHD), which place a heavy reliance on findings found on slit lamp examinations. Furthermore, after HSCT patients can suffer from non-GvHD related complications, such as cataracts, leading to compound presentations. Thus, this need for a specialist examination and confounding presentation means that often patient’s oGvHD may be advanced before the diagnosis is established and subsequent referral to specialist care. Such delays cause unnecessary suffering and, in some cases, permanent damage.

Aims: We aimed to develop an effective and efficient screening tool for chronic oGvHD that could be integrated into a Hematology/ Oncology outpatient clinic.

Methods: All post-HSCT patients attending the Hematology/ Oncology outpatient department clinic 18 months were invited to partake in the study. In PHASE I the Ocular Surface Disease Index (OSDI) questionnaire was used to screen the participants for chronic oGvHD and a score of >33 was chosen as the threshold. In PHASE II if participants scored >33 in the OSDI they were then asked a further ‘yes/ no’ question relating to daily use of ocular lubricants. If both criteria were fulfilled, chronic oGvHD was considered likely. In case instance, questionnaires were completed before the ophthalmic examination to prevent bias, and examination was performed by a trained ophthalmologist. Participants were considered to have a diagnosis of chronic oGvHD if they fulfilled one or both of the NIH or ICCGvHD criteria. The efficacy of was measured by sensitivity and specificity along with PPV and NPV. LR were used to assess the screening algorithm as it is less likely to change with disease prevalence. Pearson r formulation was used to determine degree of correlation between OSDI scores and disease severity.

Results: PHASE I (n=100) resulted in a sensitivity of 100%, specificity of 86.9%, PPV of 0.59, NPV of 1.0 and a LR of 7.3 (p <0.0001). PHASE II (n=68) resulted in 100% sensitivity and specificity with a PPV and NPV of 1.00 (p <0.0001). Disease severity and ocular surface disease strongly correlated (n=65, 0.67, p <0.0001).

Summary/Conclusion: We propose that a simple algorithm using the self-assessment OSDI questionnaire and a polar ‘yes/ no’ question relating to ocular lubricant usage that could be widely implemented in hematology/oncology settings to screen for oGvHD with minimal cost and inconvenience. The algorithm also provides an accurate prediction of disease severity, enabling ophthalmology services to triage patients so that timely care is provided to those in greatest need.

P1343: TRANSPLANT OUTCOMES OF LYMPHOMA PATIENTS UNDERGOING STEM CELL MOBILIZATION WITH PLERIXAFOR

A. K. Gunes 1,* , S. Dagdas 1 , F. Ceran 1 , C. Sunu 2 , G. Korkmaz 1 , G. Ozet 1

1 Hematology, Ankara City Hospital, Ankara; 2 Hematology, Sakarya Training and Research Hospital, Sakarya, Turkey

Background: Autologous stem cell transplantation is a standard treatment modality widely used in relapsed refractory Hodgkin and Non-Hodgkin Lymphoma. In order to perform an autologous stem cell transplantation, it is necessary to collect sufficient stem cells. The accepted minimal number of stem cells that will provide the reconstruction of hematopoiesis after high-dose chemotherapy is >2x106/kg CD34(+), and the optimal number is >5x106/kg CD34(+) stem cells.

Aims: In this study, we aimed to compare the engraftment kinetics and long-term survival data after autologous stem cell transplantation in lymphoma patients who underwent stem cell mobilization with plerixafor.

Methods: Between 2006 and 2020, 138 lymphoma patients who underwent autologous stem cell transplantation in our clinic were retrospectively analyzed. The last follow-up and survival data of the patients were obtained in January 2022. First-line peripheral stem cell mobilization with chemotherapy+G-CSF (5-10 μg/kg/day) was planned for all patients. Patients whom were mobilized at least 2x106/kg CD34(+) stem cells with this regimen were defined as “good mobilizers”. Patients who could not be mobilized with chemotherapy + G-CSF in the first line were mobilized with Plerixafor + G-CSF who could collect at least 2x106/kg CD34(+) stem cells with this collection regimen was defined as “poor mobilizers”.

Results: While 110 of the patients (79.8%) were considered as “good mobilizers”, 28 patients (20.2%) took place in the “poor mobilizer” group. The mean age was 37.6 years in the good mobilizer group, while the mean age was 48.3 years in the poor mobilizer group.The good mobilizers group were significantly younger than poor mobilizer group (p<0.001). There was no significant difference between the two groups in terms of gender, histological diagnosis, stage, presence of B symptoms, bone marrow involvement, Bulky disease, prior chmo lines, disease status at the time of transplant, and ECOG performance score. The amount of collected and infused stem cells in the poor mobilizers group was significantly lower than in the good mobilzers arm (4.54x106/kg vs 3.6 x106/kg, p<0.001). While no significant difference was observed in the median neutrophil engrafment day(11 vs 10 day, p=0.06), median platellet engrafment day was significantly longer in the poor mobilizers group (12 vs 13 day, p=0.011). After a median 38 month follow up time; The 5-year progression-free survival rate was 49.7% in the good mobilizers arm and 48.6% in the poor mobilizers arm, with a median PFS of 73 months in the good mobilizers arm and 59 months in the poor mobilizers arm, and no significant difference was found between the two groups in terms of PFS. (p=0.912).The 5-year Overall Survival was 62.1% in the good mobilizers arm and 58.5% in the poor mobilizers arm with a median OS was not reached in the good mobilizers arm, the median OS was 83 months in the poor mobilziers group. There was no significant difference in overall survival between the two groups (p=0.748)

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Summary/Conclusion: In our study, no difference was found between poor mobilizers and good mobilizers in terms of progression-free survival and overall survival. However, the amount of mobilized stem cells were lesser and the median platelet engraftment day was longer in the poor mobilizers arm. We conclude that plerixafor administration is safe and efficient for mobilization in lymphoma patients who were poor mobilizers.

P1344: METHODOLOGY OF AN INTERNATIONAL MULTI-CENTER RETROSPECTIVE REVIEW OF TRANSPLANT-ASSOCIATED THROMBOTIC MICROANGIOPATHY (TA-TMA) BY THE TA-TMA WORKING GROUP

V. Ho 1,* , R. F. Duarte 2 , J.-J. Boelens 3 , M. S. Cairo 4 , R. E. Champlin 5 , S. Giralt 3 , M. Hamadani 6 , C. S. Higham 7 , J. H. Jerkins 6 , J. Laurence 8 , S. Mayer 8 , R. Nakamura 9 , O. Penack 10 , M.-A. Perales 3 , G. Rondón 5 , M. Scordo 3 , S. Hingorani 11 , S. Vasu 12

1 Dana-Farber Cancer Institute, Boston, United States of America; 2 Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain; 3 Memorial Sloan Kettering Cancer Center, New York; 4 New York Medical College, Valhalla; 5 MD Anderson Cancer Center, Houston; 6 Medical College of Wisconsin, Milwaukee; 7 UCSF Benioff Children’s Hospital, San Francisco; 8 Weill Cornell Medicine, New York; 9 City of Hope Comprehensive Cancer Center, Duarte, United States of America; 10 Charité Universitätsmedizin Berlin, Berlin, Germany; 11 Seattle Children’s Hospital, Seattle; 12 The James Cancer Center, Columbus, United States of America

Background: Transplant-associated thrombotic microangiopathy (TA-TMA) is a serious and potentially life-threatening complication after allogeneic hematopoietic cell transplantation (alloHCT). Multiple sets of clinical diagnostic criteria for TA-TMA have been developed in adult and pediatric settings based on single-institution chart review or expert panels, but none have been rigorously evaluated across multiple centers to assess validity or reproducibility. The TA-TMA Working Group is conducting a multi-national, multi-center, retrospective study to identify and confirm potential adult and pediatric cases of TA-TMA. The aim of creating a large cohort of adjudicated TA-TMA cases is to better understand the incidence, risk factors, clinical outcomes, and healthcare resource utilization (HRU) associated with this condition.

Aims: To describe the multiple-adjudication methodology employed to confirm diagnosis of TA-TMA across adult and pediatric transplant centers.

Methods: Twelve US and EU centers retrospectively screened medical records of patients who received alloHCT between 2015 and 2017 using 5 criteria: schistocytosis (>2 per high-power field); LDH level above ULN; doubling of baseline creatinine; platelet refractoriness (50% decrease in platelet count and/or platelet count less than individual center’s transfusion threshold x 4 days despite transfusions); and decreased haptoglobin. Cases meeting ≥3 criteria within a 10-day period during the first 12 months after alloHCT were considered potential TA-TMA and were further evaluated by 2 clinicians from each transplant center. Patients who had a positive biopsy, were diagnosed with TA-TMA or aHUS, or had received eculizumab following alloHCT were considered to have definite TA-TMA.

Of all positive cases determined by local adjudications, 30% will be randomly selected for central adjudication by independent adult and pediatric adjudication committees. Central adjudicators have access to relevant laboratory and radiologic data. In cases with 2 discordant decisions, a third central adjudicator will be assigned to break the tie. In cases of 3 discordant decisions regarding the diagnosis of TA-TMA, the case will be forwarded for discussion led by the central adjudication chair. All cases determined as positive TA-TMA after local and central adjudication will be aggregated and analyzed to evaluate clinical outcomes, risk factors for TA-TMA, disease course, survival and non-relapse mortality, and HRU.

Results: As of March 1, 2022, 3,732 cases have been screened at 11 transplant centers and 836 were considered potential TA-TMA that met ≥3 criteria. To date, local adjudication at 8 centers determined 179 cases were positive TA-TMA.

Due to variations in laboratory standards at different centers, a challenge encountered in this study was harmonization of screening criteria. For example, labs at several centers did not quantify number of schistocytes as part of their CBC, which restricted screening of potential TA-TMA cases to 4 criteria, leading to some cases being categorized as “unknown.” These outcomes highlight the differences in assessing TA-TMA at multiple centers.

Summary/Conclusion: This is the first multi-center comprehensive retrospective study to evaluate TA-TMA with a formalized process for independent local and central adjudication to verify TA-TMA diagnosis. The large cohort of TA-TMA cases identified in this study should enable validation of current and future diagnostic criteria, improve our understanding of prognostic/risk factors, and enhance awareness of this disease.

P1345: IMMUNE RECONSTITUTION AFTER BMT FOR NON-MALIGNANT HEMATOLOGICAL CONDITIONS FOR RELATED, UNRELATED AND HAPLOIDENTICAL DONORS. A STUDY IN THE PEDIATRIC DEPARTMENT AT ST MARY’S HOSPITAL BETWEEN 2019&2020

A. Khoder 1,* , A. Gassas 1 , S. Bremathas 1 , O. Rabanal 1 , K. Hennesy 1 , S. Loaiza 2 , K. Lund 1 , T. Petterson 1 , L. Karnik 1 , J. Josu de la Fuente 1

1 Paediatric Haematology/Blood &Marrow Transplantation Department, St Mary’s Hospital, Imperial College Healthcare NHS Trust; 2 John Goldman Centre for Cellular Therapy, Imperial College Healthcare NHS Trust, London, United Kingdom

Background: Allogeneic blood & marrow transplant (BMT) is a curative option for a wide range of non-malignant childhood hematological conditions. Significant comorbidity and mortality is associated with delayed immune reconstitution (IR). Previous studies on the main included patients with malignant conditions with previous exposure to chemotherapy and different kinetics of chimerism are likely to have an impact on IR

Aims: To study IR in a single pediatric BMT Center, St Mary’s Hospital, Imperial College London between 2019 and 2020, which exclusively undertakes BMT for non-malignant hematological conditions (hemoglobinopathies and bone marrow failure) comparing matched sibling (Sib), haploidentical (Haplo) and matched unrelated donor (MUD

Methods: Peripheral blood was collected monthly for lymphocyte subsets: CD4+, CD8+,CD56+,CD19+ cells and IgG, IgA and IgM levels in 49 consecutive patients. IR is defined as median cell count reaching ≥ lower normal limit or > a cut-off of clinical significance e.g CD4+ cell>300/μL. Conditioning regimens varied per indication, donor source and degree of HLA matching but were maintained consistently throughout the study period. Thymoglobulin (rabbit ATG, Genzyme, dose range 4.5 -11.25 mg/kg) was the T cell depletion used in all conditioning regimens except for Fanconi anaemia (FA), where alemtuzumab 0.3 and 0.9 mg/kg was used for Sib and unrelated transplantation, respectively. Thymoglobulin stopped on day -2 except in Haplo was given upfront and alemtuzumab on day -4. TBI 2-3 Gy was incorporated in conditioning for mismatched unrelated BMT for FA and in all haplo. Haplo BMT was performed with PTCy.

Results: Type of BMT was as follows: 44% Sib, 27% Haplo and 19% MUD; for following indications 37% sickle cell anaemia, 35% thalassaemia, 12% AA, 8% DBA, 6% FA and 2% DC. Median age was 8.5 y (3-19). Almost half the patients were ≤6 years at time of BMT with 3/49 ≥18 years. Any form of aGVHD occurred in 29%, 54% and 55% of Sib, Haplo and MUD, and cGVHD in 5%, 23% and 27%, respectively. Innate immunity CD56+NK and CD3+CD8+cytotoxic cells were first to reconstitute. Median CD56+cell count >100 (100-1200 /μL) in Sib and MUD were observed by 1 st month while in Haplo in 2 nd month post BMT. Median CD8+ >200 (200-1300/μL) was first observed 4 months post BMT in Sib and Haplo and 7 months in MUD. Adoptive immunity CD4+and CD19+cells expectedly took longer to reconstitute. Median CD4+count>300 (700-4000/μL) was noted at 7 months post BMT in Sib, and 8-10 months in MUD and Haplo. However, reaching lower normal limit was only achieved by end of 1st year for Sib and not reached in the Haplo at 12 months. Median CD19+B cell count reached >100 (100-1200/μL) at 2 to 4 months in Sib; continued to rise throughout first year, and 8 to 10 months in MUD while in Haplo, this was not reached by end of 1st year. IgG level remained >5 g/L throughout post BMT in Sib. Although B cell reconstitution was delayed, level remained >5 g/L throughout the 12 months post BMT in Haplo; this may reflect the use of IVIg treatment or replacement. In MUD, IgG levels were lower than other two cohorts at or close to 5 g/L most of the time. IgM and IgA levels have remained mostly below lower normal limit throughout the first-year post BM.

Summary/Conclusion: IR for non-malignant hematological conditions in children is reached at different time point following BMT. Haplo is associated with delayed IR therefore poses higher risk of infection. Comprehensive phenotypic analysis of IR is required to help assess infection and risk and guide effective prophylaxis

P1346: EVALUATION OF CURRENT NATIONAL PRACTICE IN THE PREVENTION OF PTLD POST PAEDIATRIC HSCT

E. Kissane 1 , I. Sulevani 1,* , D. B. James 2

1 University of Leeds; 2 Paediatric Haematology, Leeds Teaching Hospitals, Leeds, United Kingdom

Background: Post-transplant lymphoproliferative disorder (PTLD) is a rare but significant cause of mortality in paediatric patients undergoing haematopoietic stem cell transplantation (HSCT). It is typically caused by reactivation of the Epstein-Barr virus (EBV), a virus present in 95% of the adult population. Immunocompromise post-transplant enables EBV, typically passively acquired from the donor, to proliferate, with the potential for malignant transformation. HSCT recipients are at greatest risk of PTLD in the first year after transplant. Pre-emptive Rituximab, given to those with EBV reactivation, reduces the risk of PTLD. However, the level of EBV viraemia at which Rituximab should be initiated is not defined in any national or international guidelines.

Aims: This prospective project aimed to understand current national practice in all twelve centres within the UK and Ireland which carry out paediatric allogenic HSCTs. These centres are: Birmingham, Bristol, Dublin, Glasgow, Great Ormond Street, Leeds, Manchester, Newcastle, Sheffield, The Royal Marsden, St.Mary’s and University College Hospital.

Methods: An online survey was sent to each centre which included six questions focusing on: patient monitoring, frequency of testing, pre-emptive Rituximab thresholds and general threshold rules.

Results: The survey had a 92% response rate. Regarding monitoring, nine centres monitored EBV PCR levels once weekly and two centres monitored twice weekly. Seven centres reduced the frequency of monitoring when patients reached a degree of immune reconstitution, two centres reduced monitoring when patients were discharged, and one centre had no fixed rules. Nationally there is no agreed threshold for starting pre-emptive Rituximab but 46% of centres initiated Rituximab when EBV PCR reached 40,000 copies/ml, with the second most common threshold being 20,000 copies/ml used by 18%. In patients that were symptomatic ten out of eleven centres used a lower threshold to initiate Rituximab, with nine of the ten centres beginning Rituximab on clinical symptoms alone and the remaining one centre having a threshold of 10,000 copies/ml alongside clinical symptoms.

Summary/Conclusion: National practice varies, including when to initiate pre-emptive Rituximab, with the most common threshold being 40,000 copies/ml. Notably, there is no nationally agreed guideline defining EBV viraemia. Future research is needed comparing PTLD incidence between centres who use a higher threshold and those who use a lower one.

P1347: THE POTENTIAL INFLUENCE OF KILLER CELL IMMUNOGLOBULIN-LIKE RECEPTOR–LIGAND MISMATCH ON THE PROGNOSIS OF HAPLOIDENTICAL HEMATOPOIETIC STEM CELL TRANSPLANTATION IN ACUTE LEUKEMIA PATIENTS

Z. Konova 1,* , E. Khamaganova 2 , I. Uribin 2 , M. Drokov 3 , I. Galtseva 4 , U. Maslikova 5 , M. Dovidenko 1 , N. Popova 3 , F. Omarova 5 , A. Dmitrova 1 , O. Starikova 5 , E. Mikhaltsova 3 , O. Koroleva 5 , D. Dubnyak 5 , E. Kolgaeva 1 , V. Vasilyeva 5 , L. Kuzmina 1 , E. Parovichnikova 6

1 Department of Intensive High-Dose Chemotherapy and Bone Marrow Transplantation; 2 Tissue typing laboratory; 3 Sector for the Study of Immune Effects and Complications after BMT; 4 Laboratory of immunophenotyping of blood cells and bone marrow; 5 Department of immunochemotherapy with a day hospital for patients after BMT; 6 Head of National Research Center for Hematology, National Research Center for Hematology, Moscow, Russia

Background: Natural killer cells (NK) are a component of innate immunity and are capable of cytotoxic lysis and cytokine secretion without prior antigen presentation. They are among the first to recover after transplantation, so they can play a decisive role in preventing early relapses and infectious complications. The killer immunoglobulin-like receptor (KIR), which is expressed on NK cells, mediates the immune response, both anti-tumor and directed against infectious agents. In the case of haploidentical hematopoietic stem cell transplantation (haplo-HSCT), if the donor has inhibitory KIR, to which the recipient has no ligand, donor NK cells can be activated against residual leukemic cells.

Aims: To determine the effect of killer cell immunoglobulin-like receptor (KIR)–ligand mismatch (KIR-Lmm) on the results of haplo-HSCT in patients with acute leukemia.

Methods: The analysis included 62 patients with acute myeloid leukemia (AML, n=30) and acute lymphoblastic leukemia (ALL, n=32) who underwent haplo-HSCT at the National Medical Research Center for Hematology in the period from May 2018 to February 2021. HLA genotyping of donors and recipients was performed by PCR-SBT. KIR genotyping of genomic DNA was performed using KIR-Type SSP Kits. The analysis of long-term results of haplo-HSCT was performed according to the Kaplan-Meier method. Comparison of survival rates in groups was performed using the log-rank test. Differences were considered statistically significant at p<0.05.

Results: In our study, 41 (66%) patients (17 patients with AML and 24 with ALL) out of 62 had KIR/KIR–ligand mismatch (KIR-Lmm), so these patients could potentially develop an NK-cell alloreactive immune response, which, in statistical analysis, significantly improved OS (93% vs. 47%, p = 0.0263, Fig. 1A) and DFS (80% vs. 44%, p = 0.0245, Fig. 1B), and also significantly reduced the probability of relapse (PR) (12% vs. 42 %, p=0.1) in patients with AML. At the same time, the improvement in DFS and the decrease in PR did not lead to an increase in the probability of developing GVHD, neither acute (“KIR-Lmm “ - 12% vs. “no KIR-Lmm” - 33%, p = 0.2, Fig. 1C), nor its chronic form. (6% vs. 17%, p=0.3, Fig. 1D). The long-term results of haplo-HSCT in ALL patients, depending on the absence/presence of a ligand for inhibitory KIR, did not differ (OS: 74% vs. 75%, p=0.9, Fig. 1E; DFS: 65% vs. 62%, p=0.7, Fig. 1F).

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Summary/Conclusion: NK cell alloreactivity due to the absence of a ligand for an inhibitory KIR donor may improve long-term haplo-HSCT results in patients with AML, but not ALL, which may be due to the inability of NK cells to destroy residual leukemic cells in ALL due to their lack of expression molecules that provide intercellular communication, such as ICAM-1.

P1348: COMPARISON OF NATURAL KILLER CELL RECONSTITUTION AFTER HAPLOIDENTICAL HEMATOPOIETIC STEM CELL TRANSPLANTATION DEPENDING ON THE VARIANT OF T-CELL DEPLETION

Z. Konova 1,* , I. Galtseva 2 , N. Kapranov 2 , J. Davydova 2 , M. Drokov 3 , M. Dovidenko 1 , N. Popova 3 , A. Dmitrova 1 , U. Maslikova 4 , O. Starikova 4 , E. Mikhaltsova 3 , O. Koroleva 4 , D. Dubnyak 4 , E. Kolgaeva 1 , F. Omarova 4 , V. Vasilyeva 4 , L. Kuzmina 1 , E. Parovichnikova 5

1 Department of Intensive High-Dose Chemotherapy and Bone Marrow Transplantation; 2 Laboratory of immunophenotyping of blood cells and bone marrow; 3 Sector for the Study of Immune Effects and Complications after BMT; 4 Department of immunochemotherapy with a day hospital for patients after BMT; 5 Head of National Research Center for Hematology, National Research Center for Hematology, Moscow, Russia

Background: Natural killer cells (NK) can play a decisive role in preventing early relapses and infectious complications after haploidentical hematopoietic stem cell transplantation (haplo-HSCT) due to their comparatively fast reconstitution after HSCT. And when performing haplo-HSCT with depletion of TCRαβ-positive T-lymphocytes, the graft versus leukemia (GVL) effect is carried out only at the expense of donor NK cells. NK differentiate from immature (CD56bright) to mature (CD56dim) and terminally differentiated. NK differentiation to the subpopulation CD56dimCD16+ is necessary to implement an alloreactive NK cell immune response.

Aims: To study the rate and features of NK cell reconstitution after haplo-HSCT.

Methods: The study included 16 patients with acute myeloid leukemia (AML) and 13 patients with acute lymphoblastic leukemia (ALL) who underwent haplo-HSCT at the National Medical Research Center for Hematology from April 2020 to June 2021. 15 patients - with the use of post-transplant cyclophosphamide (PT-Cy), 14 - with previous TCRαβ-depletion. To assess the reconstitution of NK, all patients underwent immunophenotypic studies of peripheral blood samples at +14, +30, +60, +90 and +180 days after haplo-HSCT using a 12-color flow cytometer (CytoFlex Beckman Coulter, USA). Monoclonal antibodies to human differentiation antigens CD3, CD8, CD158a, CD158b, CD158e, CD159a, CD57, CD16, CD56, CD14, CD45, CD62L were used in the analysis of subpopulations of NK cells. The SAS 9.4 software (Sas institute inc., Cary, NC, USA) was used for statistical data processing. Differences were considered statistically significant at p<0.05.

Results: NK reached a mature immunophenotype faster in the case of haplo-HSCT with TCRαβ-depletion: by day +30 after HSCT, the median proportion of CD56dimCD16+ NK cells with TCRαβ-depletion was 57% versus 9% when using PT-Cy, p= 0.0045. Significant differences persisted up to +60 (74% vs. 41% (p=0.0079)) and +90 days after haplo-HSCT (81% vs. 46% (p=0.0076)) (Fig. 1).

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Summary/Conclusion: The rate of reconstitution and the acquisition of an alloreactive phenotype by NK depends on the variant of T-cell depletion; faster recovery occurs when performing haplo-HSCT with TCRαβ-depletion. When using PT-Cy, the recovery of the mature NK cell population occurs at a later time after haplo-HSCT (from +3 to + 6 months) and such patients may benefit from transfusions of donor NK cells at the post-transplant stage. This approach can be considered as a worthy alternative to donor.

P1349: EFFECT OF TRANSPLANTED CD184-POSITIVE CELLS (WITH EXPRESSION CXCR4) FOR A LONG SURVIVAL AFTER AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION IN MULTIPLE MYELOMA – ONE CENTER PROSPECTIVE STUDY.

A. Kopinska 1,* , A. Wieczorkiewicz 1 , A. Koclega 1 , K. Jagoda 1 , J. Dziaczkowska-Suszek 1 , G. Helbig 1

1 HEMATOLOGY AND BONE MARROW TRANSPLNATATION, SPSK-M, KATOWICE, Poland

Background: Autologous hematopoietic stem cell transplantation (AHSCT) remains the treatment of choice in multiple myeloma (MM) patients (pts). The role of chemokine CXCR4 knows also as CXCR4 in hematopoietic stem cell homing engraftment, and in hematopoietic recovery after AHSCT is still not fully understood as well.

Aims: In this prospective study we would like to investigate the influence CD184+ cells for overall survival (OS) and progression free survival (PFS) after AHSCT in MM pts.

Methods: Forty eight pts with MM with median age 56 (range 21-76) were undergoing AHSCT in our center in years 2011-2013. Conditioning regimen was Melphalan. Number of all CD184+ cells were measured in harvested material.

Results: All pts successfully engrafted. The median follow-up was over 8 years. Currently 21 pts (44%) are alive, including 8 pts (17%) in disease’s remission, and 13 are treated for MM progression. Twenty-seven pts died including 19 pts due to disease progression. The median OS was 7.8 years. In the study group a statistically significant correlation was found between OS, PFS and the number of CD184+ cells. In the context of OS and PFS, number of CD184+ cells was found to have a negative impact (correlation coefficient R = -0.32, p=0.0261, and correlation coefficient R = -0.41, p=0.0039 for OS and PFS retrospectively). As OS and PFS increased, the number of CD184+ cells decreased.

Summary/Conclusion: The number of CD184+ cells could have a negative effect on both OS and PFS in multiple myeloma patients.

P1350: THE EFFICACY OF UPFRONT HIGH-DOSE CHEMOTHERAPY WITH AUTOLOGOUS STEM CELL TRANSPLANTATION IN DECREASING RELAPSE RATE IN IV STAGE AND HIGH-INTERMEDIATE/HIGH IPI RISK GROUPS DLBCL

A. Koviazin 1,* , L. Filatova 1,2 , I. Zyuzgin 1 , A. Artemyeva 3 , M. Motalkina 1 , Y. Chudinovskikh 1 , S. Volchenkov 1 , I. Poliatskin 3 , E. Dobrovolskaya 1 , S. Shalaev 1 , I. Ishmatova 1 , A. Zverkova 1 , D. Burda 3 , S. Elkhova 1 , T. Semiglazova 2,4

1 Department of Hematology and Chemotherapy with intensive care unit, NMRC of Oncology named after N.N.Petrov of MoH of Russia; 2 North-Western State Medical University named after I.I.Mechnikov; 3 Department of Pathology; 4 Department of Innovative Methods in Therapeutic Oncology and Rehablitation, NMRC of Oncology named after N.N.Petrov of MoH of Russia, Saint-Petersburg, Russia

Background: Approximately 30% of patients(pts) with IPI≥2 diffuse large B-cell lymphoma(DLBCL) have relapsed after the frontline regimens. Upfront high-dose chemotherapy(HDCT) with autologous hematopoietic stem cell transplantation(auto-HSCT) can be an effective option to decrease the relapse rate and increase the response rate in these patients.

Aims: To determine the significance of the upfront HDCT with auto-HSCT in decreasing relapse rate and increased response rate for IV stage DLBCL pts with unfavorable prognosis.

Methods: Included 105 pts: DLBCL NOS, age 18-65, stage IV, IPI≥2, CR/PR after x6 CHOP/EPOCH+R from 2010 to 2019 at NMRC of Oncology named after N.N.Petrov of MoH of Russia were retrospectively analyzed. HSCT group (G1) includes pts with upfront HDCT followed by auto-HSCT (n=35). The control group (G2) includes pts with non-invasive follow-up after induction only (n=70).

Groups had equal mean (53,4±21.2 mon in G1 vs 48,9±12.5 mon in G2, p=0.71) observation period and mean (48.0±11.7 yrs in G1 vs 48.9±12.5 yrs in G2, p=0.72) age. No difference was in sex (51% (18/35) men in G1 vs 45.7% (32/70) in G2, p = 0.67), IPI (71.4% (25/35) IPI≥3 pts in G1 vs 58.5% (41/70) IPI≥3 pts in G2, p=0.28) or extranodal (E) involvement (77.1% (27/35) pts with >1 E in G1 vs 71.4% (50/70) in G2, p=0.69). Extra with lung lesions (EL) was detected in 20% (7/35) pts in G1 vs 21,4% (15/70, p=0.86) in G2; with adrenal lesions (EA) in 20% (7/35) pts in G1 vs 6% (4/66, p=0.055) in G2. MYC and BCL-2 coexpression were analyzed in 100% (35/35) pts samples in G1 and in 74.2% (52/70) samples in G2 and were present in 37% (13/35) G1 pts and 31/52 G2 pts, p=0.0503.

Between-group comparisons were conducted using a χ 2 , χ 2 with Yates correction(Yates), Fisher’s exact test or McNemar criteria with Yates in paired samples for categorical variables and a two-sided independent t-test for continuous variables. A two-sided p-value of <0.05 significance level was considered to be statistically significant in all analyses.

Results: Upfront HSCT significantly increased number of complete responses(CR) from 63% (22/35) after induction to 100% (35/35) after HSCT, p = 0.0009.

Early relapse rate (RR) was significantly lower in G1 pts (2.8%(1/35) in HSCT vs 21.4%(15/70) in G2, p=0,027). Overall RR was borderline (8.5%(3/35) in G1 vs 27.1%(19/70) in G2, p=0,051).

In combined group analysis, early RR was significanty lower in pts with double-expressor lymphomas (DEL) treated by upfront HSCT - 0%(0/13) in G1 vs 57.1%(12/21) in G2, p<0.001. Also, DEL was a worsening factor in overall RR(0% (0/13) in G1 vs 66.6%(14/21) in G2, p=0.013). Lung E significantly increased overall RR in control group (0%(0/7) in G1 vs 60%(9/15) in G2, p=0.021).

In subgroup analysis no significant factors for overall and early RR were detected in HSCT group. In control group early RR was higher if pts had DEL (57.1%(12/21) DEL+ vs 16.1%(5/31) DEL-, p=0.003) or EA (75%(3/4) EA+ vs 24.2%(16/66) EA-, p=0.041). Overall RR was significantly increased in pts with DEL (66.6%(14/21) DEL+ vs 16.1%(5/31) DEL-, p<0,001) and with EL(60%(9/15) EL+ vs 18.1%(10/55) EL-, p=0.04).

Summary/Conclusion: 1. Complete response rate significantly increased in 18-65 yrs, DLBCL NOS, IV stage, IPI≥2 pts treated by upfront HSCT.

2. Upfront HSCT significantly decreasing early RR and have tend to decrease overall RR in 18-65 yrs, DLBCL NOS, IV stage, IPI≥2 pts.

3. Double-expressor lymphoma increasing overall and early RR in 18-65 yrs, DLBCL NOS, IV stage, IPI≥2 pts. More than one site extranodal involvement with lung or adrenal lesions increasing overall and early RR correspondingly.

P1351: CHARACTERIZING ALLOGENEIC STEM CELL TRANSPLANTATION SAFETY IN PATIENTS WITH HEMATOLOGIC MALIGNANCIES AFTER PEMBROLIZUMAB THERAPY

J. Kuruvilla 1,* , P. Armand 2 , A. F. Herrera 3 , V. Ribrag 4 , P. Brice 5 , C. Thieblemont 5 , B. Von Tresckow 6 , E. Kim 7 , P. Marinello 7 , S. Chakraborty 7 , R. Orlowski 7 , P. L. Zinzani 8

1 Princess Margaret Cancer Centre, Toronto, Canada; 2 Dana-Farber Cancer Institute, Harvard Medical School, Boston; 3 City of Hope Medical Center, Duarte, United States of America; 4 Institut Gustave Roussy, Villejuif; 5 APHP, Hôpital Saint-Louis, Hemato-Oncologie, Université de Paris, Paris, France; 6 University Hospital Cologne, University of Cologne, and West German Cancer Center, Essen University Hospital, University of Duisburg-Essen, Essen, Germany; 7 Merck & Co., Inc., Kenilworth, United States of America; 8 IRCCS Azienda Ospedaliero-Universitaria di Bologna Istituto di Ematologia “Seràgnoli” and Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy

Background: Although allogeneic stem cell transplantation (allo-SCT) and programmed cell death 1 (PD-1) blockade have separately shown efficacy in some lymphoid malignancies, the safety of allo-SCT after anti–PD-1 therapy in these patients remains a high clinical interest.

Aims: We present data from an analysis from 4 KEYNOTE phase 1-3 studies to describe predetermined complications in patients who received allo-SCT following pembrolizumab therapy.

Methods: The analysis included patients from the KEYNOTE-013 ( NCT01953692 , n=20), KEYNOTE-087 ( NCT02453594 , n=31), KEYNOTE-170 ( NCT02576990 , n=5), and KEYNOTE-204 ( NCT02684292 , n=14) trials who have a known allo-SCT transplantation date received within 2 years of the last dose of study pembrolizumab. Descriptive statistics were used for predefined post–allo-SCT complications of interest. The cumulative incidence of acute grade 2-4 graft-versus-host disease (GVHD), acute grade 3-4 GVHD, and chronic GVHD was estimated. The corresponding competing risk events were death without acute grade 2-4 GVHD, death without acute grade 3-4 GVHD, and death without chronic GVHD, respectively. The cumulative incidence of post–allo-SCT transplant-related mortality (TRM) was estimated with relapse as a competing risk. Progression-free survival (PFS) and overall survival (OS) were estimated by the Kaplan-Meier method.

Results: Seventy patients were included in the analysis. Median age was 30 y (range, 18-65), 57 (81.4%) had classical Hodgkin lymphoma (cHL), and the rest had non-Hodgkin lymphoma. 69 of 70 (98.6%) patients received pembrolizumab monotherapy; 1 patient received pembrolizumab + lenalidomide. Median duration on study treatment was 5.3 months (range, 0.7-29.6) and median time from last pembrolizumab dose to first allo-SCT was 4.6 months (range, 1-20). Before transplantation, 49 patients (70.0%) had intervening anticancer regimen; 34 (48.6%) had active disease, 31 patients (44.3%) were in remission at time of transplant, and 5 patients (7.1%) had unknown disease status. Overall, 55 patients (78.6%) developed GVHD (acute, 38; chronic, 17). The estimated 6-month post–allo-SCT cumulative incidence was 0.41 (95% CI, 0.30-0.53) for grade II-IV acute GVHD and 0.20 (95% CI, 0.12-0.30) for grade III-IV acute GVHD; 1 year post–allo-SCT was 0.21 (95% CI, 0.12-0.31) for chronic GVHD. Other predetermined complications, including critical illness, immune-mediated adverse events, pulmonary complications, and veno-occlusive liver disease, occurred in 32 patients (45.7%). After a median follow-up (defined as the time from allo-SCT to data cutoff) of 40.1 months (range, 5.1-71.2), the post–allo-SCT median PFS was not reached (NR) (95% CI, 14.5-NR) and the 30-month post–allo-SCT PFS rate was 56.8% (95% CI, 42.9-68.5) (Figure A); median OS was NR (95% CI, NR-NR) and the OS rate at 12 months was 82.2% (Figure B). The estimated post–allo-SCT cumulative incidence for TRM at 6 months was 0.09 (95% CI, 0.03-0.17) and for relapse at 26 months was 0.27 (95% CI, 0.16-0.38). PFS and OS outcomes were comparable in the subgroup of patients with cHL.

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Summary/Conclusion: The incidence of GVHD in this population is comparable to historical data (40%-80% and 30%-70%, respectively). The incidence of severe acute GVHD may be higher than a typical modern allo-SCT series, although TRM was low. The observed PFS and OS outcomes compare favorably to historical benchmarks (2-year PFS ranging from 45%-48%; 1-year OS >80%). Altogether, this analysis lends confidence that allo-SCT is feasible for patients with lymphoid malignancies after PD-1 blockade.

P1352: THE INTRA-POPULATION HLA DIVERSITY IN CRETE AND THE IMPORTANCE OF THE REGIONAL PUBLIC UMBILICAL CORD BLOOD BANK IN HLA-BASED DONOR SELECTION FOR ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

H. Latsoudis 1,* , M. Loulakaki 2 , P. Pavlidis 1 , A. Kanterakis 1 , I. Mavroudi 3 , I. Fragiadaki 3 , I. Gontika 3 , H. A. Papadaki 3

1 Institute of Computer Sciences, Foundation for Research and Technology Hellas; 2 Computer Sciences Department, University of Crete; 3 Public Umbilical Cord Blood Bank of Crete, University Hospital of Heraklion, Heraklion, Crete, Greece

Background: Allogeneic hematopoietic stem cell transplantation is the elective treatment for several life-threatening hematologic diseases. The high genetic diversity of HLA across populations, combined with the complexity of its molecular variation and the limitations of traditional HLA typing, confines the effectiveness of matched unrelated donor registries and public umbilical cord blood banks (UCBB), particularly in populations with ethnic minorities.

Aims: Prompted by our recent study describing a considerable HLA diversity between a representative Cretan cohort and the donors of DKMS, we set out to unravel the effect of potential intra-population diversity on HLA distribution. We used samples stratified by a specific Prefecture of origin aiming to signify the possible existence of a severe shortage of genetically HLA compatible samples and to justify the importance of the regional UCBB for an HLA-based donor selection in Crete.

Methods: A combined cohort of 1,744 representative samples from the four Prefectures of Crete (Heraklion, Lasithi, Rethymno, Chania) were typed for HLA Class I ( -A, -C, -B ) and Class II ( -DRB1, -DQB1 ) loci using 2nd field NGS. Fisher’s exact test was used for the comparison of g-grouped alleles with frequencies corrected by Bonferroni and of haplotypes estimated by Hapl-o-Mat with frequencies f≥1/2n. The average probability of finding a >6/10 compatible donor within and across the four Prefectures was also estimated using custom scripts in R.

Results: In the combined cohort, HLA-B exhibited the highest allelic variability (n=74) and number of unique alleles (n=21/39). While the diversity of all loci, except for HLA-C , was attributed to the largest Prefecture, Heraklion, the high number of unique HLA-C alleles in Chania (n=4/8) contributed to both intra- and inter-population diversity. While the distribution of the most frequent common alleles across the 5 loci were similar between all Prefectures, significant differences (n=27 alleles) were revealed when each Prefecture was compared to the remaining pooled Cretan cohort. Such analyses substantiated the diversity of Chania (37.04%) mostly versus the geographically and genetically isolated Prefectures of Lasithi and Rethymno. Significant diversity was also observed among the 1,406 inferred haplotypes, with 80.2% being unique. The distribution of the top 10 out of the 21 common haplotypes in the combined cohort was 0.63%-1.38%. However, the most common haplotypes A*24:02~B*35:02~C*04:01~DRB1*11:04~DQB1*03:01 (1.38%) and A *33:01g~B*14:02g ~C*08:02g~DRB1*01:02g~DQB1*05:01g (1.38%) were mainly attributed to Rethymno with frequencies of 3.35% and 1.96%, respectively. The low frequency of A*24:02~B*35:02~C*04:01 ~DRB1*11:04~DQB1*03:01 in Heraklion (0.97%) and Chania (0.73%) resulted in a significant difference (p=5.8e-05) when both were compared to Rethymno. The diversity of Chania may underlie the higher probability of not finding a compatible donor in Lasithi and Rethymno (i.e. >30%) or Heraklion (i.e. >20%). Besides the lower distribution of the most common haplotypes, Heraklion seems a good pool for finding potential donors since 46.8-49.1% of common HLA haplotype needs are met between the former and each Prefecture.

Summary/Conclusion: The in-depth immunogenetic profile of the Cretan population justifies the reported HLA diversity compared to the European populations of DKMS. The significant differences in the estimated haplotype frequencies between the Cretan Prefectures substantiates the importance of geographically or genetically isolated population clusters in the regional UCB banking and donor selection.

P1353: EFFICACY OF SARSCOV2 VACCINATION IN PATIENTS UNDERGOING AUTOLOGOUS STEM CELL TRANSPLANTATION. A MULTICENTER EXPERIENCE

L. Stirparo 1 , F. Autore 2 , I. Innocenti 2 , E. Papa 3 , F. Marchesi 3 , C. Togni 4 , S. Mariani 4 , L. Torrieri 5 , M. Salvatori 5 , F. Fazio 5 , E. Metafuni 2 , S. Giammarco 2 , F. Sorà 1,2 , P. Falcucci 3 , A. Ferrari 4 , S. M. Trisolini 5 , A. Tafuri 4 , P. Chiusolo 1,2 , S. Sica 1,2 , L. Laurenti 1,2,*

1 Sezione di Ematologia, Dipartimento di Scienze Radiologiche ed Ematologiche, Università Cattolica del Sacro Cuore; 2 Sezione di Ematologia, Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario A.Gemelli IRCCS; 3 Hematology and stem cell transplant Unit, Regina Elena National Cancer Institute IRCCS; 4 Hematology Institute, La Sapienza University of Rome, Sant’Andrea Hospital; 5 Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy

Background: COVID-19 disease have strongly hit patients with hematological diseases and the ones receiving haematopoietic stem cell transplantation (HSCT) are a particularly vulnerable group. However, the effectiveness of vaccination in self-transplanted patients seems to be variable. The article by Chiarucci et al compared serological data of 38 autologous recipient and 45 healthy subjects, collected 30 days after their vaccination against SARS-CoV-2. In patients receiving high-dose therapy and auto-HSCT, prior administration of Rituximab was associated with a lower rate of immunization against SARS-CoV-2 (54%) and a significantly lower IgG antibody.

Aims: Confirm and implement the data found out by Chiarucci et al. with a wider database derived from our four centers.

Methods: This multicenter retrospective observational study included 82 patients who underwent aHSCT from January 2020 to October 2021, with 58 vaccinated after the autologous procedure (group A) and 24 patients before getting transplantation (group B). We divided patients of group A in 2 subgroups according to the hematological disease: 29 patients affected by plasma cell neoplasms (28 multiple myeloma MM and 1 plasma cell leukemia) and 29 affected by lymphomas (26 non Hodgkin lymphoma, NHL, and 3 Hodgkin lymphoma, HL). Than, we compared patients according to the conditioning regimen and the previous treatment. In group B, twelve patients were tested the first day of hospitalization, the day of discharge and one month after the transplantation.

Results: In group A, 39 (67%) patients had a positive serology and 19 (33%) did not develop any antibody response. In particular, we noticed that the rate of positivity increased with the distance from the aHSCT. When vaccinated within 6 months after the procedure, only 57% of patients were positive, but it increased to 89% after 12 months. The subgroup of patients affected by lymphomas had a lower rate of positivity (52%). In this group, indeed, the distance from the aHSCT was particularly significant in terms of response: in fact, the positivity registered was 30% when the vaccination was done within 6 months, 54% after 6 and within 12 months and 83% after 12 months. When considering NHL the distance from the administration of RTX had a particular impact on the vaccination response: patients receiving the monoclonal antibody within 6 months had a poorer response to the vaccination (13% of positivity) than those who were treated with RTX after 6 and within 12 months (50%) and after 12 months (87%). This result was statistically significant (p= 0.01). In the plasma cells neoplasms subgroup, the rate of positivity was higher (83% overall), and it was not particularly affected by the distance from the transplant. In group B, 15 (63%) were positive at least 1 month after the procedure, while 9 (37%) were negative. We also noted that, in the subgroup of patients tested also at the hospitalization and at the discharge, no initially positive patient became negative after the transplantation.

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Summary/Conclusion: Our data on 82 patients affected by lymphoproliferative disorders who underwent aHSCT confirmed the role of rituximab as negative predictor of response to anti-SARS-CoV-2 vaccination, as already reported by Chiarucci et al. We therefore showed the scanty importance of conditioning and transplantation procedure to the response of vaccination. According to this data, it would be more fruitful consider the vaccination at least 6 months from the last rituximab administration, even though it’s necessary to take into account patient conditions and the epidemiological context.

P1354: A SYSTEMATIC LITERATURE REVIEW (SLR) OF THE MANIFESTATIONS OF VENO-OCCLUSIVE DISEASE/SINUSOIDAL OBSTRUCTION SYNDROME (VOD/SOS) AFTER HEMATOPOIETIC CELL TRANSPLANTATION (HCT) IN ADULTS VERSUS CHILDREN

J. Angus 1,* , W. Letton 2 , S. Connolly 2 , J. Bilimoria 2 , C. Martin 2

1 Jazz Pharmaceuticals, Oxford; 2 Crystallise Ltd., Essex, United Kingdom

Background: VOD/SOS is a potentially fatal complication of HCT. Its diagnosis can be challenging due to the many patient- and transplant-related VOD/SOS risk factors, its sometimes slow, progressive onset, and the dynamic presentation of signs and symptoms, some of which may differ by age.

Aims: This SLR evaluated differences in the clinical manifestations of VOD/SOS post-HCT in adults compared to children.

Methods: Medline and Embase were searched to 4 March 2021 for studies of VOD/SOS post-HCT and were supplemented with guidelines on VOD/SOS diagnosis and management. English language reports of observational and database studies were included if they studied adults or children with any HCT-related disease, therapies aimed at preventing/treating VOD/SOS, or HCT-associated VOD/SOS outcomes. Publications were evaluated based on inclusion of the 5 cardinal features related to VOD/SOS diagnostic criteria (i.e., ascites, hepatomegaly, bilirubin ≥2 mg/dL, weight gain of ≥5%, and right upper quadrant [RUQ] pain) and changes in diagnostic features over time. In cohort studies, an unweighted mean was calculated for the proportion of patients with each VOD/SOS feature; for case studies, the number of case reports in which the feature was present was divided by the total number of cases in which any features were reported.

Results: Overall, 204 publications were included in the SLR. Weight gain of ≥5% and hepatomegaly were more common at diagnosis in children compared to adults, whereas the presence of ascites or RUQ pain was similar between age groups ( Figure ). While only 40% of cases had all 5 cardinal features of VOD/SOS, age did not determine the likelihood of missing any single feature. When weight gain was present, it appeared as the first manifestation more often in adults than children (54% vs 22%, respectively); it was more likely to appear second in children, suggesting VOD/SOS-related weight gain may be less easily detected or develops later in children. Similarly, hepatomegaly appeared first in >90% of adults compared to <50% of children. While RUQ pain was more likely to be the second or third feature in children, it appeared first in around 85% of adults compared to 50% of children, suggesting it presents later in children in relation to other symptoms or is more difficult to detect. Ascites was absent in 22.7% of all cases, making it the feature most frequently missing; however, when it was present, it appeared as the first manifestation more frequently in children than adults (40% and 6%, respectively). There was no difference between adults and children on the day post-HCT that the first VOD/SOS features were detected.

Hyperbilirubinemia was more common at diagnosis in adults than children ( Figure ). Importantly, hyperbilirubinemia was absent in 18.2% of children and 6.7% of adults, and analysis of individual VOD/SOS features showed younger age was associated with an absence of hyperbilirubinemia compared to other features. The averaged time series data for bilirubin levels appeared to peak earlier and to have a higher peak for adults than children, suggesting more rapid onset in adults.

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Summary/Conclusion: Presentation of VOD/SOS symptoms post-HCT, as identified and reported in publications of patients diagnosed with VOD/SOS, varied between adults and children. Notably, VOD/SOS without elevated bilirubin may be more common at diagnosis in children compared to adults. VOD/SOS features are not reported in a distinct sequence, requiring a high index of suspicion for VOD/SOS and continuous vigilance by those involved in patient monitoring post-HCT.

P1355: VENO-OCCLUSIVE DISEASE/SINUSOIDAL OBSTRUCTION SYNDROME (VOD/SOS) AFTER AUTOLOGOUS HEMATOPOIETIC CELL TRANSPLANTATION (HCT): OUTCOMES OF DEFIBROTIDE-TREATED ADULT PATIENTS FROM THE DEFIFRANCE STUDY

M. Mohty 1,* , I. Yakoub-Agha 2 , J.-H. Dalle 3 , M. Labopin 4 , J. Cornillon 5 , A. Huynh 6 , T. Lamy 7 , C. Soussain 8 , K. Asubonteng 9 , D. Gutierrez 10 , R. Peffault de Latour 11

1 Department of Clinical Hematology and Cellular Therapy, Hôpital St Antoine, Sorbonne University, INSERM UMRs 938, Paris; 2 CHU de Lille, Université Lille, INSERM U1286, Infinite, Lille; 3 Hôpital Robert-Debré, GHU AP-HP Nord, Université of Paris; 4 Hôpital St Antoine, Sorbonne University, INSERM UMRs 938, Paris; 5 Department of Clinical Hematology, CHU de St-Étienne, Saint-Étienne; 6 Département d’Hématologie, Institut Universitaire du Cancer de Toulouse – Oncopole, Toulouse; 7 Service d’Hématologie Clinique, CHU de Rennes, Rennes; 8 Hématologie, Institut Curie, Saint-Cloud, France; 9 Jazz Pharmaceuticals, Philadelphia, PA, United States of America; 10 Jazz Pharmaceuticals, Lyon; 11 Hôpital Saint-Louis, Paris, France

Background: Hepatic VOD/SOS is a potentially fatal complication of HCT conditioning. Although allogeneic HCT (allo-HCT) and autologous HCT (auto-HCT) conditioning regimens often contain the same drugs, their protocols differ. VOD/SOS is more common after allo-HCT, but it also occurs after auto-HCT. Defibrotide is approved for the treatment of severe VOD/SOS post-HCT in patients aged >1 month in the European Union and for VOD/SOS with renal or pulmonary dysfunction post-HCT in the United States. The DEFIFrance study collected real-world data on the effectiveness and safety of defibrotide from HCT centers in France.

Aims: This post-hoc analysis presents patient characteristics and effectiveness and safety outcomes in adult patients who received defibrotide treatment for VOD/SOS after auto-HCT.

Methods: This post-marketing registry study collected retrospective and prospective real-world data on patients receiving defibrotide at 53 HCT centers in France. Diagnosis of VOD/SOS was at the investigator’s discretion using standard criteria, per typical clinical practice. Disease severity was categorized using adult EBMT severity criteria in patients aged ≥18 years. The primary endpoints were Kaplan-Meier (KM)–estimated Day 100 post-HCT survival and complete response (CR; total serum bilirubin <2 mg/dL and multiorgan failure resolution per investigators’ assessment) in patients with severe/very severe VOD/SOS. A secondary endpoint was the evaluation of treatment-emergent serious adverse events (SAEs) of interest: hemorrhage, coagulopathy, immunogenicity, injection-site reaction, septicemia, infection, and thromboembolic event, irrespective of relationship to treatment.

Results: Of 249 adult patients in the post-HCT population treated with defibrotide, 22 (9%) underwent auto-HCT. Median age of this subgroup was 53.7 (range: 22, 66) years. The most common primary disease was lymphoma/chronic lymphocytic leukemia (n = 18 [82%]), and 1 (5%) patient each had either ALL, AML, neuroblastoma/solid tumor, or myeloma. The majority of patients with available data (20/21 [95%]) received intensive, myeloablative conditioning. The median time from auto-HCT to VOD/SOS diagnosis was 14.0 (range: 0, 185) days. At diagnosis, 17 (77%) patients had ascites, 16 (73%) had refractory thrombocytopenia, and 10 (45%) had anicteric VOD/SOS. Seven (32%) patients had mild/moderate VOD/SOS, 9 (41%) had severe, and 4 (18%) had very severe. The median duration of defibrotide treatment was 15.0 (range: 3, 55) days. By Day 100 post-HCT, the KM–estimated survival rate was 73% (95% CI: 49%, 87%). The CR rate at Day 100 was 55% (95% CI: 32%, 76%). Treatment-emergent SAEs of interest occurred in 4 (18%) patients, specifically: catheter site hemorrhage, gastrointestinal hemorrhage, coagulopathy, and hypotension, each occurring in 1 patient.

Summary/Conclusion: VOD/SOS can develop after auto-HCT and can be severe. Defibrotide treatment has previously demonstrated efficacy among patients with VOD/SOS after auto-HCT. The DEFIFrance study represents the largest collection of real-world data on post-registration use of defibrotide. The effectiveness and safety observed in this post-hoc analysis support the real-world use of defibrotide in adult patients who develop VOD/SOS after auto-HCT.

P1356: PATIENT-SPECIFIC DIGITAL DROPLET PCR ASSAYS FOR MINIMAL RESIDUAL DISEASE DETECTION OF RUNX1 MUTATIONS IN ACUTE MYELOID LEUKEMIA PATIENTS UNDERGOING ALLOGENEIC STEM CELL TRANSPLANTATION

M. Jentzsch 1,* , L. Bischof 1 , D. Backhaus 1 , D. Brauer 1 , G.-N. Franke 1 , V. Vucinic 1 , U. Platzbecker 1 , S. Schwind 1

1 Medical Clinic and Policlinic 1, Hematology, Cellular Therapy and Hemostaseology, Leipzig University Hospital, Leipzig, Germany

Background: The persistence of non-clonal-hematopoiesis mutations (mut) in remission prior to a consolidating allogeneic stem cell transplantation (HSCT) in acute myeloid leukemia (AML) patients (pts) has been associated with a higher relapse probability. RUNX1 mut are frequent in AML and associate with adverse outcomes, often indicating the need for an allogeneic HSCT. They may also represent suitable targets for measurable residual disease (MRD) detection, but have not yet been evaluated for this purpose separately.

Aims: To develop patient-specific digital droplet PCR (ddPCR) assays for sensitive RUNX1 mut detection and evaluate their applicability for predicting relapse in AML pts undergoing allogeneic HSCT.

Methods: We retrospectively analyzed 171 AML pts consolidated by allogeneic HSCT (non-myeloablative 64%, 24% reduced-intensity, or myeloablative 12%) for their diagnostic RUNX1 mut status. At diagnosis, cytogenetics, and additional 53 recurrently mutated genes in AML were analyzed. Adequate material at and/or following HSCT was available for 22 RUNX1 mut pts (up to 28 days prior to HSCT [n=19], during post-HSCT follow up [61 samples in 11 pts] and at relapse [n=3]). For these, patient-specific ddPCR assays were developed using a competitive probe approach to reach MRD levels of at least 0.01% VAF. Median follow up after HSCT was 1.5 years.

Results: At diagnosis, 22 pts had one and 4 pts had two RUNX1 mut (16%, median VAF 36.5, range 3.8-95.1%), 10 were frameshift, 12 were missense, and 8 were nonsense. Most frequent co-mut were found in ASXL1 (31%), DNMT3A (29%), and NRAS (26%). One patient was transplanted with active AML and had a RUNX1 VAF of 30.4% at HSCT. For all 19 measurable pts in morphologic remission at HSCT, RUNX1 mut had decreased compared to VAFs at diagnosis (median VAF at HSCT 2.3, range 0.06-10.9%), but persisted in 11 (58%) of the pts. Noteworthy, 7/8 RUNX1 MRDneg, but only 2/11 RUNX1 MRDpos pts were female (P=.005). There were no other characteristics or mutated genes at diagnosis that associated with RUNX1 MRDpos at HSCT (Figure 1A). RUNX1 MRDpos pts at HSCT had a higher cumulative incidence of relapse (P=.05) and by trend shorter overall survival (P=.07, Figure 1B,C). At day 28 after HSCT, all 11 pts with material available were MRDneg, 3 of them relapsed. In 2 of those pts, RUNX1 MRD predicted relapse at VAFs of 0.09% and 0.04% 10 and 5.5 months earlier, respectively, while in the third patient the last MRD sample taken 4 months prior to relapse remained RUNX1 MRDneg. All 3 pts with relapse material available relapsed with the RUNX1 mut present at diagnosis (at VAFs of 37%, 8.7%, and 36.2%). All 8 pts with material available that remained in remission had no detectable RUNX1 mut at any time during post-HSCT follow up.

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Summary/Conclusion: RUNX1 -mut based patient-specific ddPCR assays could be developed for all RUNX1 mut AML pts. MRD determination by these assays is a feasible tool to determine AML MRD with a relatively short turn-around time during disease course at high sensitivity and relatively low cost. RUNX1 MRDpos at HSCT associated with high relapse risk. Within 28 days after HSCT, all pts became RUNX1 MRDneg, identifying this timepoint as least meaningful for MRD assessment in our cohort. In sequential analyses after HSCT RUNX1 -mut based MRD was a stable marker allowing for a reliable and clinically meaningful MRD detection, as they predicted relapse when rising as well as persisting remissions when remaining undetectable in the majority of analyzed AML pts.

P1357: IMPAIRED CELLULAR RESPONSES AGAINST SARS-COV-2 AFTER COVID-19 INFECTION IN ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANT RECIPIENTS

A.-C. Mamez 1,* , A. Pradier 1 , C. Stephan 2 , F. Giannotti 1 , S. Masouridi-Levrat 1 , S. Morin 1 , D. Neofytos 3 , D.-L. Vu 3 , A. Melotti 4 , I. Arm 3 , C. S. Eberhardt 5 , J. Tamburini 4 , L. Kaiser 6 , Y. Chalandon 7 , F. Simonetta 7

1 Division of Hematology, Department of Oncology; 2 Département de médecine, unité de médecine transfusionnelle et d’immunohématologie; 3 Division of Infectious Diseases, Geneva University Hospitals; 4 Translational Research Centre in Onco-Haematology, Faculty of Medicine, University of Geneva; 5 Center for Vaccinology; 6 Division of Infectious Diseases; 7 Division of Hematology, Department of Oncology, University Hospitals of Geneva, Geneva, Switzerland

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) recipents have a higher risk to develop severe forms of COVID-19 after infection with SARS-CoV2 compared to the general population.

Aims: To gain further insights into the immune defects leading to increased severity of COVID-19 infection in allogeneic HSCT recipients, we evaluated B and T cells responses in convalescent healthy controls and allogeneic HSCT recipients after COVID-19 infection.

Methods: Peripheral blood samples were obtained from allogeneic HSCT recipients (n=11) between 1 and 8 months after COVID-19 infection. Median time of COVID-19 infection since HSCT was 17 months (range 8-70). Healthy controls recruited by the Geneva Blood Transfusion Center 1 to 3 months after mild COVID-19 infection served as controls (n=11). None of the HSCT recipients nor healthy controls has received anti-SARS-Cov2 vaccination before infection. Antibody responses against the SARS-CoV-2 spike protein (anti-S) and nucleocapside phosphoprotein (anti-N) were measured in plasma using the semi-quantitative Elecsys® Anti-SARS-CoV-2 immunoassay (Roche). SARS-CoV-2-specific T cell responses were quantified based on IFN-γ release against a range of peptides from the SARS-CoV-2 Spike protein (S) as well as from the membrane glycoprotein (M) and the nucleocapside phosphoprotein (N) using an Enzyme-Linked ImmunoSpot (ELISpot) assay. SARS-CoV-2- specific T cell clonotypes were identified by genomic DNA T-cell receptor (TCR) sequencing (immunoSEQ® T-MAP™ COVID, Adaptive).

Results: We observed no difference in anti-S and anti-N antibody titers between convalescent healthy controls and HSCT recipients (Figure 1A). We observed significantly lower numbers of IFN-γ spot forming units after stimulation with peptides from both the S protein (p=0.0068) and the M plus N proteins (p=0.0067; Figure 1B) in the transplant group as opposed to the control group. Importantly, such differences were not merely due to T cell lymphopenia in HSCT recipients as similar numbers of IFN-γ producing T cells after phytohemagglutinin (PHA) stimulation and similar percentages of CD3+ T cells among PBMCs were measured in PBMCs from HC and HSCT recipients. SARS-CoV-2- specific T cell clonotypes were detectable in both HC and HSCT recipients (Figure 1C). The degree of diversity of the SARS-CoV-2-specific T cell clonotypes differed between the two groups, with HSCT recipients displaying a significantly reduced number of SARS-CoV-2-specific T cell clonotypes compared with HC (p=0.0037; Figure 1D). Such a difference was maintained when T cell clonotypes specific for the S protein (p=0.0011) or the N and M proteins (p=0.036) were analyzed (Figure 1E). As predicted, HSCT recipients displayed a less diverse TCR repertoire compared with HC as revealed by higher Simpson clonality (p= 0.0079). Interestingly, the Simpson clonality negatively correlated with the number of different SARS-CoV-2-specific T cell clonotypes (R 2 =0.86, p=4.8e−06; Figure 1F).

Summary/Conclusion: Our results indicate that allogeneic HSCT recipients display a defect in cellular but not humoral SARS-CoV-2-specific responses after COVID-19 infection. Such impairment was both quantitative, as revealed by reduced IFN-γ release upon stimulation with SARS-CoV-2 peptides, and qualitative as demonstrated by the reduced clonotypic diversity of SARS-CoV-2-specific T cell clonotypes. These findings provide insights into our understanding of post-HSCT immune-dysfunction and into the increased risk of HSCT recipients of developing severe forms of COVID-19 after infection by SARS-CoV-2.

P1358: WILL COVID-19 PANDEMIC CHANGE MOBILIZATION STRATEGY OF HEMATOPOIETIC STEM CELLS IN MULTIPLE MYELOMA PATIENTS FOR THE BETTER?

B. Marques 1,2,3,4,* , A. Roque 1,5 , L. Ruzickova 1 , J. P. Carda 1,3,4,6 , M. Gomes 1,2 , A. B. Sarmento Ribeiro 1,2,3,4,6 , C. Geraldes 1,2,3,4,6

1 Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra; 2 Laboratory of Oncobiology and Hematology and University Clinic of Hematology; 3 Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO); 4 Center for Innovative Biomedicine and Biotechnology; 5 Institute of Physiology; 6 Clinical Academic Center of Coimbra, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Background: Autologous hematopoietic stem cell transplantation (ASCT) is the current standard of care for younger patients (pts) with multiple myeloma (MM), improving overall survival (OS) and progression free survival (PFS). According to EBMT, use of chemotherapy, typically cyclophosphamide (Cy), in combination with granulocyte colony-stimulating factor (G-CSF) is the preferred strategy for hematopoietic stem cell (HSC) mobilization to peripheral blood (PB). However, major disadvantages are therapy-related toxicity and higher mobilization failure. Plerixafor plus G-CSF has been shown to be superior to G-CSF alone, leading to higher CD34+ yields in fewer apheresis sessions; however there are few data in comparison to chemomobilization. Since the beginning of COVID-19 pandemic, our center adopted G-CSF ± Plerixafor as initial strategy to mobilize HSC in MM pts.

Aims: To evaluate the efficacy, length of hospitalization and safety of G-CSF ± Plerixafor mobilization of HSC in MM pts, compared to chemomobilization.

Methods: We performed a retrospective study of MM pts submitted to mobilization and 1 st ASCT between 2018 and 2021. Pts who underwent mobilization with Cy (1.5 g/m 2 ) plus pegylated G-CSF were included in group A; pts who received G-CSF ± Plerixafor were included in group B. Group B only received Plerixafor if they presented PB CD34 + cells < 15/mL on day 4 of mobilization with G-CSF.

Results: We identified a total of 126 pts, excluded 4 (2 under hemodialysis and 2 receiving different protocols) and included 122 pts, 83 (68%) in group A and 39 (32%) in group B. Demographic characteristics and disease stage (ISS and R-ISS) were similar between the groups, presenting a male predominance (n=73, 60%) and a median age of 61 years. Since frontline protocol in MM changed recently, induction therapy in group A and B was, respectively, VCD 60 vs 26%; VRD 30 vs 75%; VTD 10 vs 0% (P<0.001). No differences were observed in pre-ASCT quality of responses (89.2 vs 89.7% ≥VGPR; P=0.532).

The impact of apheresis in terms of length of hospitalization (4 vs 2 days, P<0.001) and median time from the 4 th day of mobilization with G-CSF until first apheresis was lower in group B; 2 vs 1 days, P<0.001). There was no difference in the number of apheresis sessions (1 vs 1; P=0.260). The volume of DMSO was higher in group B (22 vs 30 mL, P=0.001).

In group B, 16 (41%) pts did not need Plerixafor and there were no differences in length of hospitalization or the number of apheresis sessions compared with those who received Plerixafor.

Mobilization failure was only seen in group A, (n=14, 17%, P=0.006). Poor mobilizers were remobilized with G-CSF + Plerixafor and presented longer hospitalization (4 vs 2 days, P=0.001) and number of apheresis sessions (2 vs 1 days, P=0.001) when compared to group B.

During mobilization, grade ≥3 complications were observed in 8.4% (n=3) in group A, being febrile neutropenia the most frequent; mobilization was interrupted in 2 pts.

All pts presented neutrophil and platelet engraftment and there was no difference in time of engraftment after ASCT between groups A and B (12 days in both, P=0.711).

No differences were observed in PFS (74.5 vs 40 months, HR 0.8[0.2-2.9], P=0.782) or OS (82.6 vs NR, HR 1.1[0.2-6.0], P=0.839) between groups.

Summary/Conclusion: The mobilization protocol changed, due to COVID-19 pandemic, from chemomobilization to G-CSF ± Plerixafor and resulted in less hospital resource utilization and less febrile neutropenia, without compromising ASCT success. Unlike prior reports, number of apheresis sessions after mobilization with G-CSF alone was similar to G-CSF + Plerixafor.

P1359: USING GENE-MODIFIED AUTOLOGOUS STEM CELLS PROGENY TO REEDUCATE THE TUMOR IMMUNE MICROENVIRONMENT OF SOLID TUMORS (TEM-GBM STUDY)

F. Farina 1,* , B. Gentner 2 , M. Eoli 3 , G. Finocchiaro 4 , A. Capotondo 2 , V. Cuccarini 5 , M. M. Naldini 2 , M. Barcella 2 , E. Anghileri 6 , V. Brambilla 7 , M. G. Bruzzone 8 , M. Carrabba 1 , G. D’Alessandris 9 , F. Di Meco 10 , V. Ferla 1 , A. Franzin 11 , P. Ferroli 10 , F. Gagliardi 12 , F. Legnani 10 , S. Mazzoleni 7 , P. Mortini 12 , A. Olivi 13 , R. Pallini 9 , M. Patanè 14 , R. Paterra 6 , B. Pollo 14 , M. Saini 10 , S. Snider 12 , L. Naldini 2 , C. Russo 7 , F. Ciceri 1

1 Hematology and Bone Marrow Transplant Unit; 2 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), San Raffaele Hospital; 3 Neuro-Oncology Unit, Istituto Neurologico Carlo Besta; 4 Neuro-Oncology Unity, San Raffaele Hospital; 5 Neuroradiology Unit; 6 Neuro-Oncology Unity, Istituto Neurologico Carlo Besta; 7 Genenta Science; 8 Neuroradiology Unit Istituto Neurologico Carlo Besta – Milano, Istituto Neurologico Carlo Besta, Milan; 9 Neurosurgery Unit, Policlinico Gemelli, Rome; 10 Neurosurgery Unit, Istituto Neurologico Carlo Besta, Milan; 11 Neurosurgery Unit, Fondazione Poliambulanza, Brescia; 12 Neurosurgery Unit, San Raffaele Hospital; 13 Neurosurgery Unit, Policlinico Gemelli; 14 Neuropathology Unit, Istituto Neurologico Carlo Besta, Milan, Italy

Background: Immunotherapies represent powerful tools that are transforming the treatment of many cancers. However, immune dysfunction in cancer is multifactorial requiring multiple points of action, especially in immunologically-cold tumors

Aims: The aim of TEM-GBM study is to evaluate the safety and the efficacy of Temferon in up to 21 newly diagnosed

patients with glioblastoma and unmethylated MGMT promoter

Methods: We have developed a genetically modified, autologous hematopoietic stem cell-based platform designed to deliver Interferon-alpha (IFNa) specifically into the tumor microenvironment through Tie-2 expressing monocytes (Temferon), in order to activate the immune system in an agnostic way against the tumor and re-establish immunosurveillance

Results: As of Jan 2022, 3 escalating doses of Temferon (from 0.5 to 2.0x10 6 /kg) were tested across 15 patients assigned to 5 cohorts affected by newly diagnosed, unmethylated MGMT glioblastoma (GBM). The follow-up range from surgery is 5 – 27 mo (3 – 24 mo after Temferon). In all patients, we observed rapid engraftment of gene modified progenitors and fast recovery from sub-myeloablative conditioning (median engraftment across all the cohorts: Neu D+13, PLT D+14). Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA, were found at increasing proportions in blood and bone marrow, reaching up to 30% at 1 mo for the highest dose cohorts tested and persisting up to 18 mo, albeit at lower levels. Despite the substantial proportion of engineered cells, only very low median concentrations of IFNα were detected in the plasma (D+30, 5.9; D+90, 8.8pg/mL) and in the CSF (D+30, 1.5; D+90, 2.4pg/mL), indicating tight regulation of vector expression. SAEs were mostly attributed to conditioning chemotherapy (e.g. infections) or disease progression (e.g. seizures). 1 SUSAR (persistent GGT elevation) has occurred. Median OS is 14.7 mo from surgery (range 6.1-28.4 mo; 10.8 mo post Temferon). Of the 15 pts treated so far, 4 pts belonging to low dose cohorts underwent 2nd surgery. Homing of transduced cells from BM to the tumor site was demonstrated by the significant content of gene-marked cells in the specimens collected from 3 of the 4 analyzed pts. Single-cell RNA seq performed on CD45+ cells purified from the TME of Temferon-treated pts compared to recurrent tumors belonging to GBM pts treated as per the current standard of care, highlighted a Temferon signature defined by the induction of markers of IFNa responses and macrophage repolarization. A patient from cohort 3, had at D+120 disease progression with two distant enhancing lesions, and increased tumor necrosis. One year following Temferon, with no 2nd line therapy added, there was approximately 40% reduction in enhancing tumor volume compared to D+180.

Summary/Conclusion: Our interim results show that Temferon is well tolerated, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon’s potential to modulate the TME of GBM patients.

P1361: TREATMENT OF SEVERE STEROID-REFRACTORY ACUTE GVHD WITH MESENCHYMAL STROMAL CELLS: RESULTS OF THE PHASE III RANDOMIZED DOUBLE-BLIND MULTI-CENTER HOVON-113 TRIAL

L. Oosten 1,* , M. van Pel 2 , B. van der Holt 3 , E. Bach 4 , M. Cross 4 , H. Roelofs 5 , P. Meij 5 , B. Wieles 2 , J. J. Zwaginga 1 , A. Lankester 6 , H. Veelken 1 , U. Platzbecker 4 , F. Sanchez-Guijo 7 , M. Algeri 8 , F. Locatelli 8 , T. Devos 9 , J. Cornelissen 10 , D. Niederwieser 4 , W. Fibbe 2

1 Hematology; 2 Nephrology, Leiden University Medical Center, Leiden; 3 HOVON Data Center, Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 4 Medical Clinic and Policlinic 1, Hematology and Cellular Therapy, University Hospital Leipzig, Leipzig, Germany; 5 Clinical Pharmacy and Toxicology; 6 Pediatrics, Leiden University Medical Center, Leiden, Netherlands; 7 Hematology, IBSAL-Hospital Universitario de Salamanca, CIC and CIBERONC, University of Salamanca, Salamanca, Spain; 8 Pediatric Hematology and Oncology, Cellular and Gene Therapy, Bambino Gesù Children’s Hospital, Sapienza, University of Rome, Rome, Italy; 9 Hematology, University Hospitals Leuven and Department of Microbiology and Immunology, Laboratory of Molecular Immunology (Rega Institute), KU Leuven, Leuven, Belgium; 10 Hematology, Erasmus MC Cancer Institute, Rotterdam, Netherlands

Background: Severe steroid-refractory acute graft-versus-host disease (SR-aGvHD) following allogeneic stem cell transplantation (HSCT) is associated with a high mortality rate. Mesenchymal stromal cell (MSC) therapy has emerged as a promising treatment option for SR-aGvHD, yet its clinical efficacy has never been conclusively demonstrated.

Aims: The randomized double-blind placebo-controlled HOVON-113 trial was designed as an international effort to evaluate whether addition of MSC to standardized second line treatment of SR-aGVHD improves clinical outcome. Patients developing aGvHD grade II to IV after alloSCT or donor lymphocyte infusions (DLI) with gut and/or liver involvement and stable or progressive disease or mixed response after 5 to 10 days of 2 mg/kg steroids or equivalent, were eligible for the study.

Methods: Patients were randomized to receive 2 intravenous infusions at a 7-day interval of either MSC 2 x 10 6 /kg body weight or placebo. In addition, all patients received mycophenolate mofetil (MMF) as standardized second line treatment. Patients who developed aGvHD while on prophylactic MMF were ineligible for inclusion. The trial’s primary endpoint was complete or partial response at 28 days following the 1 st MSC or placebo infusion, reported as overall response (OR). Secondary endpoints included overall survival (OS), cumulative incidence of chronic GvHD, and adverse events. A sample size of 150 patients was calculated to detect an increase in the aGvHD response rate from 40% to 65% with a power of 0.80 and a 2-sided significance level of 0.05.

Results: In the course of 5 years, 41 eligible patients were included in the Netherlands, Germany, Spain, Italy, and Belgium. 7 of these were < 18 years (age range 1-16 ). 20 patients were randomized to the MSC arm and 21 to the placebo arm. At diagnosis, 12% of patients had grade II aGvHD, 68% grade III, and 20% grade IV. In 63% of cases, aGvHD had been elicited by alloSCT and in 37% of cases by DLI. These variables were distributed evenly between the treatment arms. Of the 41 patients, 36 (85%) received both infusions. 3 did not receive any infusions and 2 received only 1 infusion due to refusal (1) or swift clinical deterioration and death (4). No infusion toxicity was observed.

OR at day 28 was reached by 8 (38%) of patients in the placebo arm and by 12 (60%) in the MSC arm (odds ratio = 2.48, 95% confidence interval (CI) 0.65-9.51, p = 0.19). 7 patients died before day 28 and could not be evaluated, 6 (29%) in the placebo arm and 1 (5%) in the MSC arm, and were considered as non-responders in the primary analysis. OS at 1 year after randomization was 33% (95% CI 15-53) in the placebo arm and 45% (95% CI 23-65) in the MSC arm (HR = 0.81, 95% CI 0.37-1.77, p = 0.60). Infectious complications were comparable between treatments arms; placebo arm: 25% grade 3, 45% grade 4; MSC arm: 22% grade 3, 39% grade 4. The main causes of death were GvHD and infections. The median follow-up of the 15 patients still alive was 24.0 months (range 9.4 – 73.8 months).

Summary/Conclusion: Due to regulatory issues and insufficient accrual, the trial could only include 41 patients and did not have sufficient power to detect the anticipated difference between the two treatment arms. No statistically significant effect of MSC infusions on aGvHD response on day 28, overall survival, or safety could be demonstrated. An extensive biomarker analysis, immunological monitoring, quality of life analysis, health technology assessment, and ethical examination are still ongoing.

P1362: FEASIBILITY OF HAEMATOPOIETIC STEM CELL MOBILIZATION AFTER CONSOLIDATION WITH GEMTUZUMAB OZOGAMICIN AND INTERMEDIATE-DOSES ARA-C AND DAUNORUBICIN IN AML

S. Perrone 1,* , E. Ortu La Barbera 1 , S. Capria 2 , F. Marchesi 3 , S. M. Trisolini 2 , S. Sorella 2 , M. Antonacci 2 , C. Minotti 2 , V. Filipponi 2 , M. Shafii Bafti 4 , F. Equitani 5 , M. C. Scerpa 1 , M. Martelli 2 , G. Cimino 1,2

1 Hematology Unit, ASL LATINA, Latina; 2 Department of Translational and Precision Medicine, Haematology, Sapienza University of Rome; 3 Hematology and Stem Cell Transplant Unit, IRCCS Regina Elena National Cancer Institute; 4 Immunoematologia e medicina trasfusionale, Policlinico Umberto 1, Rome; 5 Department of Transfusion Medicine, ASL LATINA, Latina, Italy

Background: Gemtuzumab ozogamicin (GO), an anti-CD33 immunoconjugate Antibody is currently approved in combination with 7 + 3 in low- and intermediate risk acute myeloid leukaemia (AML). These patients are candidate for consolidation with autologous stem cell transplantation (ASCT) particularly when MRD- is obtained. GO can improve the rate of MRD negativity. There are limited data on the effect of its addition on the mobilization of Hemopoietic Stem Cells (HSC).

Aims: To assess the feasibility of mobilization of HSC after re-introduction into market of GO at 3mg/m 2 in 2019.

Methods: We retrospectively studied AML patients undergoing 3 + 7 + GO induction and Ara-C + Daunorubicine + GO, consolidation (doses are derived from label instructions and ALFA0701 study) and mobilization on day +20 using G-CSF 10µg/kg. CD34+ were monitored, and patients were harvested when a threshold of 20 cells/µL was reached in peripheral blood.

Results: In 2020 and 2021, also considering constrains caused by COVID-19 pandemics, we attempted mobilization in our 3 Italian centres of 14 patients with a diagnosis of CD33+ de novo -AML. The median age was 52 years (range 29-65 yrs.), 4 were males and 10 females; 11 patients carried a mutation of NPM1 and all had a normal karyotype except one with t(10p12;11q14) (Table 1). All received 3 + 7+GO induction and achieved a CR. Therefore, we started consolidation (total ARA-C 8g/m 2 ) + GO as inpatient.

Ten patients (71%) reached the established threshold of 20 CD34+ /µL and were successfully harvested, while 4 patients (29%) failed mobilization. The median day of apheresis was D+26 from the start to chemotherapy (range 22-39). The median number of circulating CD34+ cells on the day of collection was 35.9 cells/µL (range 20-2153 cells/µL). The median CD34+ harvested was 4.65 x 10 6 /kg (range 1.8- 44.6 x 10 6 /kg). In our cohort, 4 patients (28% of the entire cohort and 40% of the harvested patients) underwent ASCT, 3 achieved favourable engraftment, while in the last patient ASCT is ongoing. Several reasons prevented ASCT in the remaining 6 patients: 3 patients underwent allogeneic SCT (2 had positive MRD on harvested apheresis; 1 was reclassified as high-risk ELN2017 due to RUNX1 mutation resulting from NGS panel), 2 refused ASCT and one suffered early relapse.

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Summary/Conclusion: In our patients, the addition of GO did not impair HSC mobilization and harvesting that was reached in about 71% of cases, similarly to the AML-10 trial of the EORTC and GIMEMA Leukemia Groups where 70% of patients were successfully harvested.

Our data are particularly interesting because in the pivotal ALFA0701 study, only one patient underwent Autologous-SCT, but in the control arm. An important limit of our case-series is that only 4 patients were auto-transplanted, so we have scant data on engraftment. In particular, evaluating day to engraftment of platelets would be interesting, given the known increase of thrombocytopaenia in patients treated with GO. In conclusion, mobilization with GO is feasible and further studies are warranted to evaluate the effects of fractioned doses of GO on HSC mobilization and ASCT outcome; the ongoing trial GIMEMA AML1819 - EudraCT number 2019-003871-20 - will prospectively assess the effect of GO, but with lower doses of ARA-C (total ARA-C 6 g/m 2 ).

P1363: OUTCOME OF ADULT HEMATOPOIETIC TRANSPLANT RECIPIENTS AFTER ADMISSION TO INTENSIVE CARE UNIT (ICU): SINGLE CENTRE EXPERIENCE.

A. Puchol 1,* , C. Mayor 1 , S. Díaz 1 , I. Iturrate 1 , Á. Figuera 1 , A. Alegre 1 , B. Aguado 1

1 Haematology and hemotherapy, Hospital La Princesa, Madrid, Spain

Background: Intensive care unit (ICU) admission during hematopoietic stem cell transplant (HSCT) is associated with poor prognosis 1,2 . Published series report a range of ICU admission rates from 24-40% of transplant patients, most frequent reasons involving septic shock, respiratory failure and veno-occlusive disease 3 . In addition, patients undergoing HSCT are at a high risk of severe morbidity and mortality associated with COVID-19 4 .

Aims: The aim of this study was to analyze outcome of HSCT patients requiring ICU admission in our center.

Methods: We retrospectively analysed outcome of 752 patients who underwent HSCT in our centre from January/2008 to June/2021. Data were collected from patients’ clinical histories.

Results: 103 (14%) patients required ICU admission (baseline and HSCT characteristics on table). Median time to ICU admission was 42 days (-2-1765). Seven of these patients were admitted to ICU on two occasions giving a total of 110 consecutive ICU admissions available for analysis.

Main reason for ICU admission was respiratory distress (74; 67%), mainly due to pneumonia (53%) including a 3% caused by COVID19, pulmonary edema (26%) and pulmonary haemorrhage (8%). Septic shock was second most common cause for ICU admission (26; 24%) due to gram-negative bacilli (47%), fungal (15%) gram-positive bacteria (13%), virus (10%) and others/idiopathic (16%). Other less frequent causes were veno-occlusive disease (11; 10%), hepatic failure/encephalopathy (8; 7%), haemorrhagic complications (6; 5%), cardiorespiratory arrest (2%), GVHD (2%), cardiogenic shock (2%).

Of the 110 ICU admissions, 37 (34%) required hemofiltration, of which 30 (81%) died; and 77 (70%) required orotracheal intubation, of which 59 (77%) died.

During the 110 ICU admissions, 67 patients (61%) died in the ICU; of these, 40 (37%) received unrelated donor HSCT, 36 (33%) sibling donor, 16 (15%) haploidentical and 17 (16%) autologous. Median ICU length of stay of these patients was 13 days (range 1-76). The cause of death was the same reason for ICU admission. Eighteen (16%) patients were discharged from ICU and died prior to hospital discharge and 24 (22%) survived to hospital discharge and were classified as post-discharge survivors. Of these 24 cases, 19 (79%) remain alive while the others (5; 21%) succumbed to underlying disease or complications post-HSCT.

Off note, both patients with COVID19 pneumonia (haploidentical and autologous HSCT respectively) were discharged from ICU and remain alive to date, without major complications.

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Summary/Conclusion: In our study 14% of transplant recipients required ICU admission, slightly lower than previous reports. Most common cause of admission was respiratory failure, consistent with reported. Mortality rate during ICU admission was 61%; higher death rate observed in allogeneic transplantation and those requiring aggressive ICU treatments such as mechanical ventilation or hemofiltration. Although patients with COVID19 pneumoniae who require ICU admission are usually associated with adverse outcome, in our series they responded successfully to intensive treatment. ICU admission following HSCT is associated with poor prognosis, but should not be considered futile.

P1364: HLA MISMATCHED UNRELATED STEM CELL TRANSPLANTATION USING ANTITHYMOCYTE GLOBULIN IS A SUITABLE ALTERNATIVE FOR PATIENTS LACKING HLA-MATCHED UNRELATED DONORS: SINGLE CENTER LONG-TERM FOLLOW-UP DATA ANAL

L. Wang 1 , P. Kong 1 , L. Zhu 1 , Y. Feng 1 , T. Chen 1 , J. Liu 1 , C. Zhang 1 , L. Gao 1 , S. Gao 1 , H. Liu 1 , L. Gao 1,* , X. Zhang 1

1 Medical center of hematology, Xinqiao hospital, Army medical university, Chongqing, China

Background: Antithymocyte globulin (ATG) can improve the outcome of HLA mismatched unrelated transplantation, but there is limited data to compare HLA mismatched unrelated transplantation with matched unrelated transplantation both under condition of ATG-containing regimen.

Aims: To compare HLA mismatched unrelated transplantation with matched unrelated transplantation both under condition of ATG-containing regimen.

Methods: A total of 186 patients with hematological malignancies who underwent a first allo-SCT from an HLA-matched (10/10) or mismatched (9/10 or 8/10) unrelated donor from Chinese bone marrow bank and Taiwan Tzu Chi bone marrow bank following ATG-containing condition was retrospectively analyzed. A total dose of 10mg/kg ATG- Genzyme or 20mg/kg ATG- Fresenius was given from days -5 to days -2. 114 patients received HLA 10/10 fully matched, while 68 received a 9/10 and 4 received an 8/10 mismatched unrelated donor allo-SCT. OS, RFS and GRFS was calculated by the Kaplan-Meier method and the differences between groups were assessed by the log-rank test, and multivariate analysis was carried out by Cox regression model. The cumulative incidence of relapse, NRM, grade II-IV aGVHD, cGVHD and extensive cGVHD was estimated by competing risk model and compared by Gray test. Multivariate analysis was conducted by Fine and Gray regression model.

Results: the median follow-up time of HLA matched transplantation and mismatched transplantation were 24 months and 21 months, respectively (P = 0.333). Univariate analysis showed that there was no significant difference in OS (78.7% vs 73.9%, P = 0.766), RFS (61.9% vs 54.8%, P = 0.464) and GRFS (46.6% vs 30.7%, P = 0.105) between HLA matched and mismatched transplantation. Multivariate analysis showed that HLA mismatch did not affect OS, RFS or GRFS (P = 0.654, P = 0.811, and P = 0.114, respectively). Univariate analysis showed no significant difference in CIR (26.1% vs 32.6%, P = 0.531), NRM (12.0% vs 12.6%, P = 0.800), cumulative incidence of cGVHD (45.3% vs 27.7%, P = 0.293) and cumulative incidence of extensive cGVHD (26.7% vs 23.4%, P = 0.635) between matched and mismatched transplantation. The cumulative incidence of grade II-IV aGVHD was higher in HLA mismatched transplantation (4.4% vs 12.3%, P = 0.046). Multivariate analysis showed that HLA mismatch was not a risk factor for CIR, NRM, cGVHD and extensive cGVHD (P = 0.44, P = 0.88, P = 0.32, P = 0.9, respectively). HLA mismatched transplantation had higher risk of grade II-IV aGVHD (HR = 2.98, P = 0.047).

Summary/Conclusion: Although the incidence of aGVHD in HLA mismatched unrelated hematopoietic stem cell transplantation is higher, ATG can improve the outcome of HLA mismatched transplantation, and the survival is equivalent to that of HLA matched unrelated hematopoietic stem cell transplantation.

P1365: DONOR LYMPHOCYTE INFUSION IN ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION: RESULTS FROM A RETROSPECTIVE STUDY

D. Rochate 1,* , F. Vasconcelos 1 , E. Couto 1 , P. Silva Coelho 1 , L. Leite 1 , R. Branca 1 , S. Roncón 2 , C. Pinho Vaz 1 , A. Campos 1

1 Bone Marrow Transplantation Service; 2 Cellular Therapy Service, Instituto Português de Oncologia do Porto IPO Porto, Porto, Portugal

Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a therapeutic option with curative intent in hematological malignancies. Despite improvements in alloHSCT, relapse of the underlying disease is still one of the main causes of mortality. Donor lymphocyte infusion (DLI) is an immunotherapy that can be used after alloHSCT in patients with high-risk disease (prophylaxis), mixed donor chimerism or minimal residual disease (preemptive) and relapse (therapeutic). Donor derived graft versus tumor effects induce a durable remission after DLI.

Aims: To characterize the use of DLI as prophylaxis, preemptive or therapeutic strategy in alloHSCT and evaluate overall survival (OS).

Methods: A retrospective unicenter study was conducted to evaluate the use of DLIs in a cohort of 50 patients (91 DLIs), treated in a Bone Marrow Transplantation Unit, between January 2005 and December 2021. The chi-square test (categorical variables) and t-test or Wilcoxon-Mann-Whitney test (continuous variables) were used. OS was estimated according to Kaplan-Meier and compared by the log-rank test. Statistical analysis was made using SPSS28®.

Results: Our cohort showed male predominance (n= 29, 58%) and a median age of 40 years (4-65), with 4 pediatric patients. Patients were affected by acute myeloid leukemia (n=20), acute lymphoblastic leukemia (n=12), chronic myeloproliferative leukemia (n=6), Hodgkin lymphoma (n=2), myelodysplastic/myeloproliferative syndrome (n=4), non-Hodgkin lymphoma (n=3), chronic lymphocytic leukemia (n=1), mixed phenotype leukemia, (n=1), multiple myeloma (n=1). Conditioning regimen was myeloablative in 19 (38%) patients and reduced-intensity in 31 (62%). All 50 donors were HLA-matched, and 8 (16%) were HLA-matched unrelated (MUR). DLIs use was prophylactic in 2 (4%) patients, preemptive in 7 (14%) and therapeutic in 41 (82%) patients. The median number of DLI/patient was 1 (1-8) and the median dose of total CD3+ cells infused was 1,0x107/Kg (0,1-13,48). The median time between alloHSCT and the first DLI was 17 months (1-133). Complete Response (CR) was defined by completed donor chimerism or no MRD detected. CR was maintained in 1/2 (50%) patients undergoing prophylactic DLIs. Patients who received preemptive and therapeutic DLIs achieved CR in 2/7 (29%) and 9/41 (22%) cases, respectively. Overall, 11 patients (22%) developed GVHD after DLIs. Presence of GVHD was associated with higher number of DLIs (2,45 vs 1,64) and with CR (p=0,041) No significant differences were observed in age (p=0,154), number of CD3+ infused (p=0,414), DLIs strategy (p=0,433), time between alloHSCT to first DLI (p=0,668) and CR. Death occurred in 30 (60%) patients. OS survival was defined as time from date of first DLI to date of last follow-up (FU) or date of death. With a median FU of 141 months, the OS was superior in patients who achieved CR compared to relapsed patients (34 months), Log-rank p=0.109. OS was significantly superior in preemptive and prophylaxis DLIs compared with therapeutic DLIs (Log-rank p<0,01). Overall, 8/19 (42%) patients are currently alive in CR.

Summary/Conclusion: DLI has been widely applied to the therapeutic intervention, with promising results in prevention and preemptive strategy. Although with the limits of a relatively small and heterogeneous cohort, we describe our experience in the use of DLI and demonstrate that it is an effective prophylactic and preemptive treatment option in alloHSCT patients. Prospective studies on homogeneous patient cohorts are needed improve the effectiveness of DLI.

P1366: NEW LYMPHOABLATIVE CONDITIONING REGIME FOR MULTIPLE SCLEROSIS – FIRST RESULTS FROM RUSSIAN PILOT STUDY

A. Rukavitcyn 1,* , D. Fedorenko 1 , V. Sarzhevskij 1 , A. Samoylova 1 , N. Mochkin 1 , V. Bogatyrev 1 , E. Smirnova 1 , A. Bannikova 1 , A. Mamedova 1 , V. Melnichenko 1

1 NMSC named after N.I. Pirogov, Ministry of Health Russian Federation, Moscow, Russia

Background: Intermediate-intensity chemotherapy (IDCT) followed by autologous transplantation of hematopoietic stem cells (ASCT) is the standard of treatment for the part of patients with hightly aggressive relapse-remitting multiple sclerosis (RRMS). There are several most used conditioning regimens, previous EBMT recommendations recommended the use of ‘intermediate intensity’ regimen namely cyclophosphamide 200 mg/kg with T-cell depleting. However, there are currently no data on the conduct of randomized studies that would compare the effectiveness and toxicity of different regimens.

Aims: Comparison toxicity conditioning regimens Cph+R and Flu/Cph+R as a conditioning regimen before auto-HSCT for the treatment of and MS

Methods: In active study with retrospective control included 94 patients with MS, median age 43 years; 64 men and 30 women, RRMS – 31 patients, PPMS – 39 patients, SPMS -24 patients. EDSS score was 1.5 – 7.5 (average 4.5). All patients received HDCT and ASCT in NMSC named after N.I. Pirogov (2018 - 2021). Conditioning regimens: Cph200 (cyclophosphamide 200 mg/kg 4 days + Rituximab 500 mg/m 2 1 day) – 49 patients, Flu/Cph (Fludarabine 150mg/m2 + 6 days + cyclophosphamide 100 mg/kg 2 days + Rituximab 500 mg/m 2 1 day) – 45 patients.

Results: Hematologic toxicity of different regimens Cph+R, Flu/Cph+R : Duration of neutropenia was– 7-12 days (average 9.6) in Cph+R group and 3-10 (average 6.3) in Flu/Cph+R group. Duration of thrombocytopenia in Cph+R regimen – 7.7 days, Flu/Cph+R -3.4 days. There are 31 (63%) patients received platelet transfusion in Cph+R group and 6 (12%) patients in Flu/Cph+R group. Anemia Grade II (CTCAE 5.0, 2017) was identified in booth regimens. Anemi grade III was most commonly appear in Flu/Cph+R regime (n=4 – 8%).

Range of immunosuppression : The minimal count of lymphocytes was significant lower in Flu/Cph+R group 0,0001 – 0,1 х10 9 /l (average 0.008х10 9 /l ), than in Cph+R group witch minimal count of lymphocyte was ( 0,001 – 0,1 х10 9 /l (average 0.03х10 9 /l ) (p<0.05) (pic. 1).

Non-hematologic toxicity .

Oral mucositis (WHO criteria) was observed at 13 (26%) patients Cph+R group (Grade I - 61,5 %; Grade II - 38,5 %) in Flu/Cph+R 4 (9%) patients (Grade I - 100 %).

Enteropathy more common at Cph+R group - 16 % (n=8), Grade I -10%, Grade II – 4%, Grade III – 2%, in Flu/Cph+R – 2 (4%) patients had enteropathy (Grade II).

Cardiotoxic effects was observed once at Cph+R group (atrial fibrilation), no cases in Flu/Cph+R group.

Hepatic toxicity (CTCAE 5.0, 2017) was observed at 9 (18%) patients Cph+R group (Grade I - 10%; Grade II - 8%) in Flu/Cph+R 9 (20%) patients (Grade I - 15 %, Grade II – 5%).

Infection in Cph+R group 13 (26%) patients had infection complications (7 local infections, 6 blood stream infections), in Flu/Cph+R – 5 (10%) patients had infection complications complications -1 local infection, 1 catheter –related infection, 3 neutropenic fever).

There are no cases of transplant-related mortality in booth regimes.

Summary/Conclusion: The effectiveness of the IDCT method in the treatment of aggressive forms of multiple sclerosis has been proven, at the same time, this method has significant limitations due to high toxicity. Our regime ( Flu/Cph+R ) showed a significantly deeper lymphodepletion, with less toxicity and a reduced incidence of infectious complications. Thus, this method can be applied to the larger number of patients.

P1367: COMPARISON CONDITIONING REGIMENS (BEEAC,LEAM,CLV) BEFORE AUTOLOGOUS STEM CELL TRANSPLANTATION FOR TREATMENT OF PRIMARY-REFRACTORY AND RELAPSED FORMS HODGKIN’S LYMPHOMA

A. Samoylova 1,* , V. Sarzhevskiy 1 , V. Melnichenko 1 , N. Mochkin 1 , A. Rukavitsyn 1 , E. Smirnova 1 , V. Bogatyrev 1 , A. Bannikova 1

1 Department of Hematology and Chemotherapy with a room of bone marrow and hematopoietic stem cells transplantation, NMSC named after N.I. Pirogov, Moscow, Russia

Background: High-dose chemotherapy (HDCT) followed by autologous transplantation of hematopoietic stem cells (ASCT) is the gold standard of treatment for patients with primary-refractory and relapsed forms of Hodgkin’s lymphoma (R/R HL).There are several most commonly used conditioning regimens for HDCT followed ASCT.However, there are currently no data on the conduct of randomized studies that would compare the effectiveness and toxicity of different regimens.

Aims: Comparison HDCT (CLV, LEAM, BeEAC) as a conditioning regimen before ASCT for the treatment of R/R HL.

Methods: In retrospective study were included 279 patients with HL, median age 30 years;121 men and 158 women. All patients received HDCT and ASCT in NMSC named after N.I.Pirogov (2006 - 2018). Conditioning regimens: CLV (cyclophosphamide, lomustine, etoposide) – 78 patients, LEAM (lomustin, etoposide, cytarabine, melphalan) – 129 patients, BeEAC (bendamustine, cytarabine, etoposide, cyclophosphamide) – 72 patients.

Results: Hematologic toxicity of different regimens (CLV, LEAM, BeEAC). All patients developed grade IV neutropenia, anemia with/without transfusion necessity, severe thrombocytopenia with transfusion requirements in most cases. Duration of neutropenia was the same – 9 days. Duration of thrombocytopenia in CLV regimen – 9 days, LEAM and BeEAC -11 days. Anemia Grade II (median) was identified in CLV, Grade III (median) in LEAM and BeEAC regimens.

Non-hematologic toxicity . Presented in Table 1.

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Summary/Conclusion: HDCT followed ASCT is the best therapeutic approach for a R/R HL. HDCT is of utmost importance, with BeEAC, LEAM and CLV conditioning regimens being considered as viable alternatives. Our results suggest a comparable efficacy of BeEAC, LEAM and CLV conditioning in terms of survival and disease control for HL patients treated with HDCT and ASCT. However, we also observed higher rates of gastrointestinal and cardiac toxicities in patients transplanted after LEAM and BeEAC. The worst OS in patients received LEAM can be explained by the fact that the regimen was used in our hospital earlier than others, when such drugs as Nivolumab and Brentuximab vedotin were not available to the patients with relapse after HDCT and ASCT.

P1368: OUTPATIENT AUTOTRANSPLANTS FOR PERSONS WITH MULTIPLE MYELOMA

J. M. Sánchez-Raga 1,* , M. Jiménez 1 , L. García-Maño 1 , N. Beltran 1 , T. Ros 1 , C. Martorell 2 , J. Ginés 2 , M. Batle 3 , L. Casado 1 , S. A. Jiménez 1 , S. Pérez-León 1 , E. Medina 1 , J. O. Díaz-Carbonero 1 , A. Pérez-Montaña 1 , L. Bento 1 , A. Sampol-Mayol 1

1 Hematología y Hemoterapia; 2 Farmacia; 3 Hospital de Día, Hospital Universitari Son Espases, Palma, Spain

Background: Patients younger than 70 years diagnosed with multiple myeloma (MM) and without associated comorbidities routinely receive consolidation treatment with hematopoietic Stem Cell Transplantation (SCT) conditioned with high-dose melphalan after several induction cycles. In the last two decades, numerous experiences have been published from different countries of results obtained through home and outpatient transplants. The difference between the two is that in the first model, the nurse usually goes to the patient’s home on a daily basis to administer medication, health care, and take blood tests, and in the second model, the patient goes to the hospital on certain days to receive this care and medical visits.

Aims: We report the results of the first patients included since we started the program in 2019.

Methods: We reviewed the electronic medical records in the computer system of our center (Millennium) of all patients with MM who have received an autologous SCT in our center from January 2019 to 2022, accounting for all those who have been specifically assessed for the outpatient program and we collect in an Excel the reasons for exclusion from the program, day of recovery of neutrophils (>500 neutrophils/uL) and platelets (>20,000/uL), number of face-to-face visits, readmissions, mucositis G III and IV, fever, as well as microbiological isolates obtained in an excel.

Results: Since the implementation of the outpatient SCT program in our center in January 2019, 57 autologous SCT have been performed on patients with Multiple Myeloma. 23 of them have not been candidates due to interruption of the program due to the pandemic situation (34 remain). Of these 17 (50%) were assessed in the pre-HSCT consultation: 2 patients rejected due to comorbidities (1 with a history of complications due to multiresistant bacteria), 4 patients preferred the hospitalized model, 3 patients could not due to the absence of a caregiver and finally 10 agreed to perform the ambulatory HPT, and 9 were finally able to perform it. 8 were male. Median age was 50 years (range 41-64). 8 were in the first line of treatment (induction with bortezomib-lenalidomide-dexamethasone scheme). Median neutrophil recovery was 11 days and median platelet recovery was 13 days. 1 patient was readmitted on day 5 due to mucositis and oral intolerance and remained hospitalized until discharge. 4 patients presented fever on day 9 and were admitted a median of one day for this reason. No microorganisms were isolated in the microbiological studies and it was suspected that it could be a seizure syndrome. The median outpatient follow-up of 12 days and the median number of Day Hospital visits was 5. The transplant-related mortality in the outpatient cohort was 0%.

Summary/Conclusion: Outpatient transplantation is a safe procedure with efficacy and safety results similar to hospitalization with a significant decrease in the costs of the procedure associated with admission. The selection of the patient and caregiver is fundamental, since not all patients will be candidates for the same for health, social reasons and/ or their preference. The most frequent reason for readmission is fever, and they can be discharged with empirical intravenous antibiotic treatment in the first 24 hours in most cases. With our results, supported by the experience of other centers, we are considering the cancellation of the prophylactic administration of G-CSF to reduce the incidence of engrafment syndrome.

P1369: PRETRANSPLANT MODIFIED ENDOTHELIAL ACTIVATION AND STRESS INDEX (M-EASIX-7): A PREDICTOR OF PROGNOSIS IN ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

E. M. Savaş 1,* , S. Yildiz 1 , M. I. Kök 1 , M. B. Erdemir 2 , B. Bostankolu Değirmenci 1 , Z. N. Özkurt 1 , Z. A. Yeğin 1

1 Department of Hematology; 2 Department of Internal medicine, Gazi University Faculty of Medicine, Ankara, Turkey

Background: Endothelial damage is considered to be the major underlying mechanism in the physiopathogenesis of transplant related complications including graft versus host disease (GVHD), thrombotic microangiopathy (TMA), sinusoidal obstruction syndrome (SOS) and sepsis. A novel scoring index, Endothelial Activation and Stress Index (EASIX), is a simple method consisting of endothelial activation parameters, which was indicated to predict GVHD and posttransplant survival. Similarly, modified EASIX (mEASIX) is a marker of endothelial dysfunction and has been shown to be associated with the development of cytokine release syndrome after CAR-T cell therapy.

Aims: In this study, we investigated the role of EASIX and mEASIX scores in the prediction of transplant related complications and survival in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHCT).

Methods: A total of 398 alloHCT recipients [median age: 43(17-71) years; male/female: 243/155] were included in this retrospective study. EASIX [lactate dehydrogenase (U/L) x creatinine (mg/dl) / platelets (10 9 /L)] and mEASIX [lactate dehydrogenase (U/L) x C reactive protein (CRP) (mg/dl) / platelets (10 9 /L)] scores were calculated on days -7, 0, +3 and +7. Statistical analysis was performed based on log2 transformed values. Patient and transplant characteristics are summarized in Table 1.

Results: Median follow-up time was 573.5 (4-3877) days. Probability of OS was estimated to be 35.1% (1343 ± 351.7; 95% CI: 653.6-2032.3), while the probabilities of day-30 mortality, day-100 mortality and NRM were 6%, 12.5% and 49% respectively. In multivariate analysis EBMT score (p=0.026, HR: 1.177; 95% CI: 1.019-1.359), CRP (p=0.033, HR: 1.004; 95% CI: 1.000-1.008), EASIX -7 (p=0.042, HR: 0.182; 95% CI: 0.035-0.941) and mEASIX -7 (p=0.016, HR: 2.668; 95% CI: 1.202-5.924) were found to have significant impact on OS. The predictive effect of mEASIX -7 was confirmed for day-30 mortality (p=0.038; HR: 1.7; 95% CI: 1.030-2.805). A positive correlation was demonstrated between mEASIX -7 and SOS (p=0.013; r=0.249). On the other hand, mEASIX +7 was found to have a significant impact on the development of engraftment syndrome (p=0.028; HR: 1.403; 95% CI: 1.038-1.896), EASIX +3 on TMA (p=0.027; HR: 2.361; 95% CI: 1.100-5.066) and mEASIX 0 on TMA (p=0.011; HR: 2.209; 95% CI: 1.199-4.068). Based on the optimal cutoff value of mEASIX -7 for the prediction of day-30 mortality, which was determined to be 2.71 [AUC: 0.747 (0.599-0.896);p=0.021], the study group was divided into two subgroups as low-mEASIX -7 and high-mEASIX 7 groups. Probability of OS was significantly higher in the low-mEASIX -7 group compared to high-mEASIX -7 group (37% vs 25.2%; p=0.008; HR: 2.057; 95% CI: 1.208-3.504). Day-30 mortality (2.9% vs 19.4%; p=0.017; HR: 7.028; 95% CI: 1.418-34.836) and day-100 mortality (9% vs 33%; p=0.004; HR: 4.469; 95% CI: 1.619-12.336) were significantly lower in the low-mEASIX -7 group. Furthermore, the probability of NRM was significantly lower in the low-mEASIX -7 group compared to high-mEASIX -7 group (44.8% vs 61.4%; p=0.005; HR: 2.551; 95% CI: 1.318-4.941).

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Summary/Conclusion: Pretransplant risk assessment is essential to guide the mainstays of the transplant procedure and optimize supportive care measures. In consideration with the basic role of endothelial activation in the development of life threatening complications of alloHCT, mEASIX -7 is indicated to have a predictive prognostic impact on survival and transplant related complications.

P1370: EFFECTS OF CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL FROM HEALTHY DONORS ON RECIPIENTS AFTER TRANSPLANTATION AND RELATED RESEARCH

B. Shen 1,* , E. Jiang 2

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; 2 The department of hematopoietic stem cell transplantation, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Allogeneic hematopoietic stem cell transplantation is an important method for treating hematologic malignant. However, Finding an HLA-matched donor has always been a significant obstacle and limited the therapy. Still, with the development of haploid-HSCT, this problem has been effectively solved. However, some donors had been found carrying malignant hematopathy-related mutation genes when they had a physical examination. But these donors do not have any clinical manifestations of hematopathy or related history. To distinguish it from clonal hematopoiesis, We called this Clonal hematopoiesis of indeterminate potential（CHIP）. There is little research on the effects of transplanting cells carrying mutated genes on patients, so we explored this.

Aims: To analyze the situation of Clonal hematopoiesis of indeterminate potential（CHIP） in healthy donors, to investigate the effects of CHIP-carrying hematopoietic cells on the survival and relapse of recipients after transplantation, and to screen out the types of gene mutations associated with poor prognosis of recipients.

Methods: Healthy donors who were examined at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College from August 2015 to August 2020 were screened for the hematological tumor gene mutations, The effects of CHIP+ and CHIP- hematopoietic cells on graft engraftment, patient survival, disease recurrence and acute graft-versus-host disease (aGVHD) were compared. SPSS22.0 and GraphPad Prism 8 were used to analyze the data.

Results: A total of 351 donors underwent hematological tumor genetic testing, including 223 males (63.53%) and 128 females (36.47%). 147 (41.88%) were positive for gene mutation and 204 (58.12%) were negative for gene mutation. Among the patients with positive gene mutation, there were 93 males with a composition ratio of 41.70% and 54 females with a composition ratio of 42.18%. The top five mutant genes carried by donors were FAT1, NOTCH1, RELN, SETBP1 and KMT2D; The proportion of CHIP among donors in different age groups was similar, about 25%. There were no statistical differences in post-transplant survival, cumulative relapse rate, graft engraftment time, and incidence of aGVHD between chip group and non-CHIP group (p>0.05); Survival analysis was performed on the gene mutations carried by the donors, JAK2 and CREBBP were associated with poor prognosis of patients, (p= 0.001 and p= 0.01, respectively); The variables with univariate analysis P<0.1 were included in multivariate analysis, and the results showed that JAK2 was an independent risk factor for poor survival prognosis, p=0.03.

Summary/Conclusion: About 42% of the healthy donors carry CHIP; CHIP exists in all age groups, and the incidence is similar; Donor CHIP has no significant effect on graft engraftment, disease relapse, and aGVHD occurrence; JAK2 gene mutation carried by donors is associated with a poor survival prognosis of patients.

P1371: GRAFT-VERSUS-HOST DISEASE OFFERS NO GRAFT-VERSUS-LEUKEMIA ADVANTAGE IN PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA AFTER HAPLO-IDENTICAL STEM CELL TRANSPLANTATION WITH POST-TRANSPLANT CYCLOPHOSPHAMIDE

A. Shimoni 1,* , C. Peczynski 2 , M. Labopin 2 , E. Angelucci 3 , Y. Koc 4 , M. Arat 5 , J. Tischer 6 , S. Sica 7 , Z. Gülbas 8 , G. Socié 9 , D. Blaise 10 , P. Pioltelli 11 , H. Ozdogu 12 , J. Vydra 13 , F. Ciceri 14 , A. Nagler 15 , M. Mohty 16

1 bone marrow transplant and cellular therapy, Chaim Sheba Medical Center, Tel-Hashomer, Israel; 2 EBMT ALWP, Paris, France; 3 Ospedale San Martino, Genova, Italy; 4 Medicana International Hospital; 5 Demiroglu Bilim University, Istanbul, Turkey; 6 Klinikum Grosshadern, Munich, Germany; 7 Universita Cattolica S. Cuore, Istituto di Ematologia, Rome, Italy; 8 Anadolu Medical Center Hospital, Kocaeli, Turkey; 9 Hopital St. Louis, Paris; 10 Institut Paoli Calmettes, Marseille, France; 11 Ospedale San Gerardo, Monza, Italy; 12 Baskent University Hospital, Adana, Turkey; 13 Institute of Hematology and Blood Transfusion, Prague, Czechia; 14 Ospedale San Raffaele, Milano, Italy; 15 Chaim Sheba Medical Center, Tel-Hashomer, Israel; 16 Hôpital Saint-Antoine, Paris, France

Background: The graft-versus-leukemia (GVL) effect after HLA- matched allogeneic stem-cell transplantation (SCT) in patients with acute lymphoblastic leukemia (ALL) is closely associated with graft-versus-host disease (GVHD). Patients with GVHD have a lower incidence of relapse, but at high grades a higher incidence of non-relapse mortality (NRM). The GVL effect after T-cell depleted haplo-identical SCT is mostly related to natural killer cell alloreactivity in patients with acute myeloid leukemia but not in ALL. The use of haplo-identical SCT has increased rapidly over the last decade due to the introduction of non T-cell depleted methods, in particular with post-transplant cyclophosphamide (PTCy), with expected outcomes that are similar to other donor sources. We have previously shown that in this setting GVL is not associated with GVHD in AML but there are currently no data on this association in ALL.

Aims: To define the association of GVHD and GVL in ALL following non T- cell depleted haplo-identical SCT with PTCy

Methods: We assessed the impact of acute and chronic GVHD on SCT outcomes in patients with ALL following non T- cell depleted haplo-identical SCT with PTCy, by using GVHD rates as time-dependent variables.

Results: The study included 516 patients with ALL in first complete remission (CR1, 68%) or CR2 (32%) after non T-cell depleted haplo-identical SCT with PTCy, given during the years 2010-2019. The immune-phenotype was B-cell (75%) or T-cell (25%) and 36% were Philadelphia positive. The median age was 36 years (range, 18-75). 81% had myeloablative conditioning and 19% had reduced-intensity conditioning. The stem cell source was bone marrow (BM, 47%) or peripheral blood (PB, 54%) The 180-day cumulative incidence rates of acute GVHD grade II-IV and III-IV were 33.3% and 11.7%, respectively. The 2-year rates of chronic GVHD all grades and extensive were 35.3% and 11.8%, respectively. The 2-year rates of relapse and NRM were 27.1% and 17.3% and the rates of leukemia-free survival (LFS) and overall survival (OS), 2 years after SCT were 55.7% and 64.4%, respectively. Multivariate analysis of factors predicting relapse after SCT did not show a statistically significant effect of GVHD of any type or grade. The hazard ratios (HRs) for acute GVHD grade II, grade III-IV and limited and extensive chronic GVHD were 0.92 (0.58-1.48, P=0.74), 0.57 (0.27-1.22, P=0.15), 1.06 (0.62-1.82, P=0.83) and 0.95 (0.42-2.17, P=0.91), respectively. CR2 vs. CR1 at SCT and T-cell vs. B-cell ALL predicted for higher relapse rates with HRs of 3.18 (2.15-4.7, P<0.0001) and 1.93 (1.29-2.89, P=0.001). Relapse rates were lower in more recent years; 1-year HR 0.88 (0.81-0.95, P=0.001), respectively. The more severe GVHD grades were associated with higher NRM with HRs for acute GVHD grade III-IV and extensive chronic GVHD of 1.95 (1.09-3.48, P=0.0024) and 3.3 (1.41-7.73, P=0.006), respectively. Lower grade GVHD had no statistically significant association with NRM. Acute GVHD grade III-IV, limited and extensive chronic GVHD predicted OS with HRs of 1.91 (1.07-3.39, P=0.028), 2.29 (1.07-4.94, P=0.0034) and 3.27 (1.4-7.66, P=0.006), respectively.

Summary/Conclusion: GVHD of any type or grade is not associated with reduced relapse rate after haplo-identical SCT with PTCy in ALL. Higher grades of GVHD are associated with higher NRM and lower OS. GVHD offers no GVL advantage in this SCT setting and future novel strategies for prevention of significant GVHD are warranted.

P1372: REAL-WORLD RETROSPECTIVE ANALYSIS OF INFECTIOUS COMPLICATIONS AFTER RUXOLITNIB USE IN ACUTE AND CHRONIC GRAFT-VERSUS-HOST DISEASE

C. Sol Reis 1,* , D. Duarte 1 , I. Brito 1 , P. Silva Coelho 1 , L. Leite 1 , R. Branca 1 , S. Roncon 2 , C. Pinho Vaz 1 , A. Campos 1

1 Bone Marrow Transplantation Service; 2 Celular Therapy Service, Instituto Português de Oncologia do Porto IPO-Porto, Porto, Portugal

Background: Graft-versus-host disease (GvHD) is a major complication of allogenic stem-cell transplantation. Recent randomized trials (REACH2 and REACH3) demonstrated efficacy of the selective JAK1/2 inhibitor ruxolitinib (Ruxo) in steroid-refractory acute (aGvHD) and chronic GvHD (cGvHD) treatment (tr). JAK/STAT signalling is key in the immune response against infection. Therefore there is a putative additional risk of clinically relevant infections in GvHD patients (pts) receiving Ruxo.

Aims: Real life assessment of Ruxo toxicity in GvHD pts, focusing on infection.

Methods: Unicentric retrospective review of GvHD pts who started Ruxo from January 2019 to August 2021.

Results: Included 47pts, median age at Ruxo start 43years [4-66], 7 paediatric, 67%man. Primary disease was haematological malignancy in 90%; even distribution of donor type; myeloablative conditioning in 55%. Ruxo was started for aGvHD in 4pts and for cGvHD in 43pts – these 77% had previous aGvHD. The median follow-up since Ruxo start was 9months [0-37].

Of the 4 pts with aGvHD: 4 had digestive and 3 skin involvement; in 2pts, Ruxo was started after steroid failure, other 2pts as third line. Of the 43pts with cGvHD: 20 had skin, 12 pulmonary, 11 hepatic, 11 ocular, 9 digestive, 8 articular and 3 mucosal involvement with most pts (77%) experienced multiple GvHD involvement. On average, 3 or more treatments per pts [1-5] were attempted before initiating Ruxo.

The median duration of Ruxo exposure was 7months [0-37]. 75%pts (n=35) developed 76 episodes of infectious complications, 58% (n=44) with grade≥3 severity. The median time to first infection grade≥3 was 1,5months. In general, most infections had respiratory origin (37%) followed by urinary tract (16%). In 61% of episodes no infectious agent was identified. A total of 5 episodes resulted in septic shock. Cytomegalovirus (CMV) reactivation occurred in 18pts (38%) and 1pt also had systemic adenovirus infection. None did prophylactic antiviral for CMV reactivation. Invasive fungal infection was observed in 7pts (15%; 2 probable and 5 confirmed) - 3 of these pts did antifungal prophylaxis.

Other toxicities included haematological (n=14, 29%; n=3 grade≥3), increased AST/ALT (n=8, 17%; n=3 grade≥3) and increased GGT (n=4, 8.5%; n=2 grade≥3).

Treatment discontinuation occurred in 17pts (36%) – most commonly due to toxicity (n=8; 17%) and lack of efficacy (n=4; 8.5%). A total of 20pts (43%) died. Most deaths were attributed to GvHD progression (n=10; 21%) followed by infection (n=6; 13%). On multivariate analysis, only infection grade≥3 had statistical impact on mortality (p=0.027; HR 10.037).

Summary/Conclusion: Infectious complications in our cohort were higher than the incidence reported in REACH2 and REACH3 – particularly severity grade≥3 infections which may reflect Ruxo use in a previously heavily treated population, with higher steroid doses than used in clinical trials. Infections included bacterial, viral and fungal, likely due to Ruxo interference with innate immunity. This subset of pts might benefit from CMV-directed prophylaxis. A careful assessment of infectious risk and antimicrobial prophylaxis could be beneficial in some.

Our study is limited by its retrospective design. Well-designed studies focused on Ruxo-associated infection risk are warranted to correctly evaluate this risk and to define a solid evidence-based infectious prophylactic strategy.

P1373: TACKLING IMMUNOLOGICAL FINDINGS AS SURROGATE MARKERS OF HISTOLOGICAL SEVERITY AND RESPONSE TO THERAPY IN ACUTE GASTROINTESTINAL GRAFT-VERSUS-HOST DISEASE

M. Sousa-Pimenta 1,* , J. Freitas 1 , E. Couto 1 , P. Coelho 1 , R. Branca 1 , C. Pinho Vaz 1 , L. Leite 1 , A. Campos 1

1 Bone Marrow Transplantation Unit, IPO Porto, Porto, Portugal

Background: Acute digestive tract graft-versus-host disease (GI-GVHD) is a potential complication for patients receiving allogeneic hematopoietic stem cell transplants (HSCT). The disease burden and response to therapy rely on a dynamic system: the hematopoietic stem cells (HSC) receptor characteristics (baseline degree of immune dysfunction, bone marrow microenvironment, bone marrow and gut microbiomes); the HSC donor characteristics and graft relative cellular composition.

Aims: To retrospectively evaluate the role of immune reconstitution kinetics and conventional inflammatory biomarkers in the burden and response to therapy of patients with acute GI-GVHD.

Methods: Cases of acute GI-GVHD after allogeneic HSCT occurring between 2016 and 2021 were assessed. The ratio of CD3 + /CD34 + infused cells, the neutrophil-to-lymphocyte (NLR), lymphocyte-to-monocyte (LMR) and fibrinogen to albumin (FTA) ratios at the onset of GI-GVHD were analyzed. Central tendency measures were plotted against histological and clinical MAGIC (Mount Sinai Acute GVHD International Consortium) scores related to GI-GVHD and a multivariate analysis was performed to exclude confounding factors. Only patients with engraftment and diagnosed with GI-GVHD were included.

Results: Forty-seven patients were included in the analysis (males=30), 57.4% receiving HSC from unrelated donors (n=27). At the diagnosis of GI-GVHD, the NLR was 3.4±2.3 for stage II histological grading (n=14); 6.4±6.5 for stage III (n=14) and 21.6±23.6 for stage IV (n=4); whilst LMR averaged 3.4±1.8; 1.6±1.4 and 0.7±0.4, respectively. These ratios did not correlate with the MAGIC score at diagnosis; however, the ratio of infused CD3+/CD34+ cells varied from 0.51±0.31x10 2 (if 3 points were achieved in gastrointestinal clinical grading) to 1.65±2.23x10 2 (in patients scoring 4), showing a tendency yet it did not achieve statistical significance. No significant correlation with response to steroid therapy was found.

Summary/Conclusion: Overall, the higher the GI-GVHD histological score at diagnosis, the higher the NLR and the lower the LMR. In our analysis, the ratio of CD3 + /CD34 + infunsed cells lost significance after adjustment for provenience of HSC. The FTA ratio had no significance as a GI-GVHD severity and/or response-related biomarker. Even though general parameters of immune reconstitution kinetics allow predicting the severity of the colon mucosa damage, they do not relate with clinical severity and response to therapy.

The first two authors contributed equally to this work.

P1374: EASIX ASSESSED AT 1 YEAR AFTER ALLOGENIC HEMATOPOIETIC CELL TRANSPLANTATION PREDICTS THE ONSET OF SUBSEQUENT NON-RELAPSE MORTALITY

R. Takeda 1,* , Y. Shibasaki 1 , T. Katagiri 1 , K. Fuse 1 , H. Sone 1,2 , M. Masuko 2

1 Department of Hematology, Niigata University Faculty of Medicine, Niigata, Japan; 2 Division of Hematopoietic Cell Transplantation, Niigata University Hospital, Niigata, Japan, Niigata, Japan

Background: Delayed non-malignant complications after allogeneic hematopoietic cell transplantation (allo-HCT) affect quality of life and increase the risk of premature death over 10 fold. Although about half of all deaths after allo-HCT are due to the primary disease, non-relapse mortality (NRM) is also a major issue for long-term follow-up.

Transplant endothelial activation and stress index prior to allo-HCT (EASIX-pre) were reported to be predictive markers for NRM after allo-HCT. However, risk factors of late NRM after over 1 year from allo-HCT, which is caused by organ damage affected by allo-HCT, have not been elucidated. In the present study, we evaluated late NRM after allo-HCT by assessing EASIX 1 year after allo-HCT (EASIX-1year).

Aims: The purpose of this study was to clarify the usefulness of EASIX-pre and EASIX-1year as predictive markers of NRM and overall survival (OS).

Methods: We included 210 patients with hematological disease who underwent a first allo-HCT between 2006 and 2019 at our facility. We evaluated EASIX-1year in 102 (53 males) patients who were alive after 1 year without relapse. The patients consisted of acute myeloid leukemia (n = 44), acute lymphoblastic leukemia (n = 24), myelodysplastic syndrome (n = 14), malignant lymphoma (n = 8), and other diseases (n = 12). The median age of the patients was 40 years (range: 16-66 years). Fifty-seven patients received myeloablative conditioning,77 patients received total body irradiation, and 44 patients developed chronic graft-versus-host disease. The HCT-comorbidity index (HCT-CI) risk groups consisted of low-risk (n = 51), intermediate-risk (n = 28), and high-risk (n = 23).

EASIX was calculated as lactate dehydrogenase (U/I) × Cre (mg/dL) / Plt (nL). EASIX-pre was evaluated 7-10 days before conditioning, and EASIX-1year was evaluated within 1 month of 1 year after allo-HCT. NRM and OS that developed 1 year after allo-HCT were defined as late NRM and late OS, respectively. Analyses were performed using landmark analysis. Late NRM and late OS assessments were performed based on the cutoff values from the receiver operating characteristic curve. The study was approved by the Ethical Committee of our facility.

Results: Median EASIX-pre was 0.98 (0.12–24.1). The C-statistics of EASIX-pre for 1-year NRM and OS were 0.579 and 0.698 (both cutoff values: 1.108), and those for late NRM and late OS were 0.561 and 0.591 (cutoff value: 0.595 and 0.766), respectively. Univariate analysis revealed that a high EASIX-pre value was associated with higher 1-year NRM (18.6% vs. 8.0%, p<0.05), and significantly lower 1-year OS (56.3% vs. 82.8%, p<0.01). However, a high EASIX-pre value was not significantly associated with both late NRM and late OS (5-year NRM 3.4% vs. 0%, p=0.24, 5-year OS 88.9% vs. 96.9%, p=0.06). HCT-CI was not an indicator of late NRM (5-year NRM 10.2 % vs. 0%, p=0.29).

Median EASIX-1year was 0.98 (0.15–21.8). The C-statistic of EASIX-1year for late NRM and late OS were 0.63 and 0.663 (cutoff value: 0.595 and 1.159), respectively. Univariate analysis revealed that a high EASIX-1year value was significantly associated with both late NRM and late OS (5-year NRM 10.1% vs. 1.3%, p<0.05, 5-year OS 88.7% vs. 95.1%, p 0.05).

Multivariate analysis was performed by adjusting age, donor source, HCT-CI, chronic graft-versus-host disease, and conditioning. A high EASIX-1year was extracted as a risk factor for late NRM (Hazard ratio 4.26, p<0.05).

Summary/Conclusion: This study showed that EASIX-1year is a useful prognostic indicator for late NRM occurring after over 1 year after allogeneic HCT.

P1375: SECOND PRIMARY MALIGNANCY AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION

T.-D. Tan 1,* , L.-W. Chiou 1

1 Hematology and Medical Oncology, Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan

Background: Hematopoietic stem cell transplantation is a curative treatment for a variety of hematologic malignancies and some benign hematologic diseases. Besides chronic GVHD, second primary malignancy is also one of long-term adverse effect.

Aims: We analyze the incidence and type of second malignancy in a single institute in 20 years.

Methods: We retrospectively searched our post-transplant patients long-term follow up data between 2001 and 2021 and analyzed the incidence and pattern of second primary malignancy in our cohort.

Results: Between 2001 and 2021, in our 380 patients cohort (184 auto- and 196 allo-transplant) with long-term survival 126 (68%) in autologous and 100 (51%) in allogeneic transplant, we had 12 patients of second primary malignancy including 5 of head and neck squamous cell carcinoma, 3 of MDS/AML, 1 of ALL, 1 of esophageal squamous cell carcinoma, 1 of breast cancer, and 1 of papillary thyroid cancer. Eight of them underwent allogeneic hematopoietic stem cell transplant had head and neck SCC, esophageal cancer, breast cancer, and papillary thyroid cancer. Four of whom underwent autologous transplant are MDS/AML and ALL. All 5 patients with head and neck cancer and one esophageal cancer patients had extensive chronic GVHD. Ten of our patients survived with 8 free of disease and one low risk MDS and one AML undergoing treatment. One hypo-pharyngeal and one esophageal cancer passed away after definitive cancer treatment. The accumulative incidence of second malignancy was 6% in 10 years and 16% in 19 years and post-autologous and post-allogeneic transplant were 5% vs 7% and 15% vs 17% in 10 years and 19 years, repectively.

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Summary/Conclusion: Second primary malignancy in 20-year follow-up in our post-transplant cohort was 6% and 16% in 10 years and 19 years, respectively, and extensive chronic GVHD is associated with head and neck and esophageal squamous cell carcinoma.

P1376: LENALIDOMIDE–BASED MAINTENANCE AFTER AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANT FOR PATIENTS WITH HIGH RISK MULTIPLE MYELOMA

O. Pasvolsky 1,* , D. Milton 2 , M. Rauf 1 , M. Tanner 1 , Q. Bashir 1 , S. Srour 1 , N. Saini 1 , J. Ramdial 1 , Y. Nieto 1 , H. Lee 3 , K. Patel 3 , P. Kebriaei 1 , S. Thomas 3 , D. Weber 3 , R. Orlowski 3 , E. Shpall 1 , R. Champlin 1 , M. Qazilbash 1

1 Stem Cell Transplantation & Cellular Therapy; 2 Biostatistics; 3 Lymphoma/Myeloma, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Maintenance therapy with single-agent lenalidomide (Len) after autologous hematopoietic stem cell transplantation (autoHCT) for multiple myeloma (MM) is associated with improved progression-free survival (PFS). However, patients with high-risk chromosomal abnormalities may need a more intense regimen.

Aims: We determined if adding another anti-myeloma drug to Len would lead to improved outcomes in patients with MM following autoHCT.

Methods: We conducted a retrospective single-center chart review analysis of adult MM patients with high-risk cytogenetic abnormalities that received autoHCT between 2008-2018, followed by Len-based maintenance therapy. High-risk cytogenetics were defined as del17p, t(4;14), t(14;16), 1q21 gain or amplification by fluorescence in situ hybridization (FISH). We divided patients into those who received either single-agent Len maintenance (Len-only) or Len-based combinations (Len-combo). We compared PFS, overall survival (OS), non-relapse mortality (NRM), day 100 and best post-transplant responses, and minimal residual disease (MRD) status between the two groups. We performed sensitivity analyses using inverse probability weights to correct for potential bias due to nonrandomization of the two groups.

Results: A total of 231 patients with HRMM were included in our analysis, with a median age of 62.4 (range 33.5-79.9) years, and 55% were male. There were 153 patients in the Len-only group, and 78 in the Len-combo group. Len-combo regimens were either doublets (Len with either dexamethasone [dex: n=10], elotuzumab [n=28] or ixazomib [n=14]) or triplets (Len with either bortezomib/dex [n=10], ixazomib/dex [n=10] or carfilzomib/dex [n=6]). More patients in the Len-combo group had ≥2 high risk cytogenetic abnormalities compared to the Len-only group (32% vs. 12%: p <0.001). Busulfan + melphalan (Bu-Mel) conditioning was used in 12% vs. 18% in Len-only vs. Len-combo groups (p=0.32). The final best response post-transplant of sCR/CR was 50% in the Len-only group vs. 54% in the Len-combo group (p=0.72). Median follow up time was 40.7 and 35.3 months, in the Len-only and Len-combo groups, respectively. Median PFS and OS for all patients were 25.5 and 82.6 months, respectively. There was no significant difference in PFS (HR: 1.01, CI: 0.71-1.44, p=0.94) or OS (HR: 0.84, CI: 0.49-1.43, p=0.52) between the Len-only and the Len-combo groups, respectively. Similarly, there was no difference in outcomes between the two groups whether they received conditioning with melphalan-only or Bu-Mel. However, for patients with HR cytogenetic abnormalities other than 1q+, there was a trend towards a better PFS in the Len-combo group (HR 0.59, CI 0.32-1.09, p=0.09; Figure 1), without a difference in OS (HR: 0.79, CI: 0.37-1.65, p=0.53).

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Summary/Conclusion: In this single center retrospective analysis, intensification of post-transplant Len maintenance did not show an improvement in the outcomes for patients with HRMM. However, there was a trend towards improved PFS in patients with HR abnormalities other than 1q+.

P1377: THE IMPACT OF THE TRAVELED DISTANCE TO THE TRANSPLANT CENTER ON THE CLINICAL OUTCOME OF ALLOGENIC BONE MARROW TRANSPLANT PATIENTS

J. El Cheikh 1,2,* , K. Terro 1 , L. Sharrouf 1 , S. Chehayeb 2 , T. Sebai 3 , D. Karam 3 , A. Zahreddine 2 , R. Nehme 2 , I. Abou Dalle 1,2 , N. Moukalled 1 , A. Bazarbachi 1,2

1 Internal Medicine; 2 Bone Marrow Transplantation Program; 3 Faculty of Medicine, AUBMC, Beirut, Lebanon

Background: Several factors affect the outcome of allogeneic stem cell transplantation (Allo-SCT) in hematologic malignancy patients. The disease type, treatment modality and patients’ clinical profile largely determine the outcomes of Allo-SCT; however, the latter may vary among patients with similar aforementioned variables. Proximity to the transplant center could be an independent factor that affects the clinical outcome of Allo-SCT in patients. This is a retrospective study conducted at American University of Beirut Medical Center (AUBMC).

Aims: The aim of this study is to determine the differences in the clinical outcome between Allo-SCT patients residing at different distances from the transplant center.

Methods: We identified a total of 275 adult patients receiving Allo-SCT between January 2007 and June 2021. The patients were divided into three groups: patients residing within 20 km from AUBMC N=120 (44%) labeled as Urban, patients residing beyond 20 km from AUBMC N=111 (40%) labeled as Rural and patients who arrived from countries abroad N=44 (16%) labeled as Abroad. All patients received Allo-SCT along with the required follow up medical care at AUBMC. Using univariate and multivariate analysis, we examined the impact of distance from the transplant center along with other independent variables on the outcomes of Allo-SCT. Kaplan-Meier curves were also exploited to identify overall survival (OS), progression free survival (PFS) and transplant-related-mortality (TRM).

Results: Male predominance is seen in all three groups. The distribution of disease types was similar in all three groups, where acute leukemia was the dominant hematological disease. However, a significant difference in age at transplant was noted, with the Urban group being the eldest (median age=43) and the Abroad group being the youngest (median age=35) (P=0.011). Also, the three groups differed in their median follow up whereby the Abroad group had the shortest (30 months) while the Urban group had the longest (55 months) (P<0.001). Concerning the pre-transplant conditioning regimen, there is no difference in the intensity in all three groups (P=0.052). 89% of patients of the Abroad group, 61% of the Urban and 65% of the Rural received their stem cells from a matched related donor (P=0.047). However, both Urban and Rural groups (35% and 34%) respectively received stem cells from a haplo-identical donor unlike the Abroad group only 9% (P=0.047).

On multivariate analysis (Table 2) there was no statistically significant difference at 3 years in terms of OS, TRM and acute GVHD (aGVHD) between the three groups. However, the 3 years PFS was higher among patients from the Abroad group (26.8 months) compared to the Urban group (19.2 months) (P=0.035). Moreover, chronic GVHD (cGVHD) was higher the closer the patient was to the transplant center with a hazard’s ratio (HR) = 2.183 (P=0.044).

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Summary/Conclusion: Distance didn’t show any significant difference in OS, TRM and aGVHD. On the other hand, the closer patients from the transplant center had higher risk for cGVHD and lower 3 years PFS most probably because they have more frequent and stricter follow up compared to others. More studies should be done on the current matter with adjustments for confounding variables.

P1378: PLERIXAFOR AND GRANULOCYTE COLONY STIMULATING FACTOR FOR POOR MOBILIZERS IN PATIENTS UNDERGOING AUTOLOGOUS PERIPHERAL HEMATOPOIETIC STEM CELL TRANSPLANTATION: SINGLE INSTITUTION STUDY

J. El Cheikh 1,2,* , K. Terro 1 , S. El Warrak 1 , L. Sharrouf 1 , M. A. Timonian 3 , F. Ismail 4 , A. Zahreddine 2 , N. Kreidieh 2 , I. Abou Dalle 1,2 , N. Moukalled 1 , A. Bazarbachi 1,2

1 Internal Medicine; 2 Bone Marrow Transplantation Program; 3 Faculty of Medicine; 4 Department of Pharmacy, AUBMC, Beirut, Lebanon

Background: Autologous hematopoietic stem cell transplantation (ASCT) has become the mainstay treatment for many hematological malignancies and solid tumors. An adequate number of stem cells must be collected for better ASCT outcomes, which is challenging in 5-30% of patients. To improve mobilization, plerixafor is used along with granulocyte colony-stimulating factor (GCSF).

Aims: Our aim is to examin the efficacy of Plerixafor in improving PSC mobilization, identify the factors influencing mobilization outcomes and point out the differences in the outcomes post PSC infusion.

Methods: We conducted a retrospective single center study involving patients who received plerixafor pre-ASCTs between January 2013 and December 2020 at a tertiary care center in Lebanon. We identified a total of 84 consecutive adult patients. All patients identified were poor mobilizers and have eventually received Plerixafor either as pre-emptive use before first apheresis in those with peripheral CD34+ stem cells (PSC) <2.5 x10 6 cells/Kg, or after failure of first apheresis in those with PSC ≥2.5 x10 6 cells/Kg. The primary end point of the study was to evaluate the efficacy of Plerixafor and to identify factors associated with better mobilization. Secondary endpoints included evaluating characteristics of poor mobilizers, stem cell yield pre and post-plerixafor, as well as the time to neutrophils and platelets engraftment. Using one sample T test, univariate and multivariate analysis, we examined the efficacy of Plerixafor in improving PSC mobilization, identified the factors influencing mobilization outcomes and pointed out the differences in the outcomes post PSC infusion.

Results: The median age at ASCT was 52.7 years (22-74) with 61% male predominance. Multiple myeloma was the most prevalent disease 64% followed by Lymphoma 32%. The majority of patients were in complete remission 64% at the time of ASCT. Most patients received proteasome inhibitor-based induction therapy 67% and Melphalan-based conditioning therapy 68%. The median follow up from ASCT was 9 months (1-59). It was noted that greater BMI is a significant factor for better PSC collection whether premobilization (P=0.003), or post Plerixafor mobilization (P=0.024). Moreover, Multiple Myeloma patients showed better mobilization using Plerixafor (P=0.049). Using Plerixafor along with GCSF in poor mobilizers post GCSF alone showed a statistically significant increase in the collected PSC mean from 0.67 x10 6 cells/Kg to 4.90 x10 6 cells/Kg (P< 0.001) with a failure rate only for 12 patients (15%). The infusion of PSC > 2.5x10 6 cells/Kg has shown a 3 days decrease in time to platelet engraftment (P=0.021) and a 36% decrease in progression/relapse rate (P=0.025).

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Summary/Conclusion: Plerixafor is effective in increasing the PSC yield in poor mobilizers. Low BMI and hematologic malignancies other than Multiple Myeloma are risk factors for poor mobilization. More studies should be performed to establish more risk factors, helping us to identify poor mobilizers more accurately and initiate Plerixafor mobilization early on, to be able to improve the engraftment and to decrease the relapse rate accordingly.

P1379: OPTIMIZATION OF FLUDARABINE PHARMACOKINCETICS TO REDUCE THE RELAPSE INCIDENCE IN AML PATIENTS AFTER ALLOGENEIC STEM CELL TRANSPLANTATION

T. Thavayogarajah 1,* , G. Nair 1 , S. Balabanov 1 , M. G. Manz 1 , D. Müller 2 , U. Schanz 1

1 Department of Medical Oncology and Hematology; 2 Institute of Clinical Chemistry, University Hospital Zurich, Zurich, Switzerland

Background: Acute myeloid leukemia (AML) is a hematological, clonal malignancy of the myeloid stem cell precursors, which is proliferative and is characterized by clonal evolution and genetic heterogeneity. For adverse risk acute myeloid leukemias, allogeneic stem cell transplantation (allo HCT) is one of the most potent curative options. Unfortunately, disease relapse is still around 40% in the first year after HSCT.

In allo HCT, fludarabine is a frequently used agent, mainly in reduced intensity conditioning regimes. It is often combined with busulfan for which dose individualization based on pharmacokinetics with area under the curve (AUC) determination is used successfully for many years.

It has already been shown that fludarabine exposure might be predictive for the survival in allo HCT and it is suggested that individualized dosing can improve the survival after transplantation within the first year. Actually for fludarabine dose calculation in adults, only the body surface area is used, which might either result in too high exposition causing increased toxicity or with too low exposure and increased relapse incidence (RI).

With the aim to reduce RI we established pharmacokinetic measurements for fludarabine in addition and combined to the established ones for busulfan.

Aims: Definition of the pharmacokinetic target for optimal fludarabine exposure during conditioning prior to HCT and establishment of a pharmacokinetic population model to support target attainment in individual patients.

Methods: Fludarabine was measured with a validated LC-MS/MS method. The exposure, expressed as AUC, was calculated for each patient using a three-compartmental model (adapted from Langenhorst et al.) in n=19 consecutive patients receiving a conditioning regime with fludarabine being diagnosed with acute myeloid leukemia. Fludarabine was given on days -7 to -2 with a dose calculation based on 30mg/m 2 infused over 30 minutes. In addition patients received peroral busulfan 4mg/kg bw on days -3 and -2 and ATG 10mg/kg bw (Grafalon®) on days -4 to -1.

Results: So far, there is no dose individualization based on pharmacokinetic parameters for fludarabine specifically suggested in AML patients. The study published by Langenhorst et al . mixed up benign and malignant hematological malignancies, suggests lower optimal fludarabine AUC than we observed in our patients.

We have seen that the fludarabine AUC were higher within our patients. In the analysis of Langenhorst et al. the optimal AUC was postulated to be 20 mg*h/l. In our (n=19) AML patients the median of the AUC was 42.97 (+/- 11.5) mg*h/l, twice as high than the suggested level for an optimal toxicity profile. However, none of our patients experienced any acute toxicity (within 30 days after transplant). Our 1 year TRM is stable for many years with 2-4%.

Moreover, there was also no major toxicity (e.g. cytopenias, infections) seen at day 100. So far, we did not see a low retrospective RI (currently 35% at 1 year) despite the AUC was much higher than the suggested optimal levels.

Summary/Conclusion: In a regimen with more (6 vs 4), but lower single fludarabine dosage (30mg/m 2 vs 40mg/m 2 ) and a shorter infusion duration (30min vs 60min) resulting in higher AUC (40mg*h/l vs 20 mg*h/l) than previously published. Within our small cohort, a low RI or high TRM seem not to be observed compared with a retrospective cohort. Further prospective data within an observational study are needed.

P1380: DEGREE OF CELL PACKING OF CRYOPRESERVED AT -80ºC HEMATOPOIETIC STEM CELLS DOES NOT AFFECT THEIR VIABILITY AND HEMATOLOGIC RECOVERY OF TRANSPLANTED MYELOMA PATIENTS

I. Tonev 1,* , S. Simeonov 1 , K. Milcheva 1 , K. Venkov 1 , P. Ganeva 1 , G. Arnaudov 1 , M. Mincheff 1

1 National Specialized Hospital for active treatment of hematological diseases, Sofia, Bulgaria

Background: Dimethylsulfoxide (DMSO) is toxic to both cryopreserved cells and patients. We are using a method for freezing at - 80ºC with 5% final DMSO concentration.

Aims: In an attempt to further decrease the amount of infused DMSO we tested viability of cells and hematologic recovery of patients following cryopreservation with different degree of cell packing.

Methods: We compared two groups of myeloma patients with low and high cryopreserved cells concentration. Cell suspension in the first group of 88 patients contained average 175x10 6 cells/ml (maximum 250x10 6 cells/ml) and in the second group of 175 patients the cells were completely packed by total plasma removal (average 391x10 6 cells/ml and maximum 597 x10 6 cells/ml) before adding the cryoprotectants resulting in a 60% decrease of the volume to be frozen. Cells were non-programmed frozen and stored at -80ºC in a solution with final concentrations of 5% DMSO, 3.6% hydroxyethyl starch (HES 450 000 mw) and 3% of human serum albumin.

Stem cell viability was evaluated by trypan blue exclusion test. Cell dose in the first patient group was 2,62x10 6 /kg (0,8–6,6), while in the second group it was 2,67x10 6 /kg (1,5-6,8).

Results: There was no statistically significant difference between the two myeloma patient groups. Viability was 97,0% (85-99) and 96,5 % (70-99) respectively. The neutrophil recovery was on day 11 (9-19) for the first and on day 11 (9-18) for the second group. Platelets recovered after 12 days for the first and the second group.

Summary/Conclusion: Dimethylsulfoxide is toxic and induces many side effects following transplant (cardiac, neurologic, respiratory, etc.), which are dose dependent. Reducing them could be achieved by lowering the final DMSO concentration (5%) and by decreasing the volume of the frozen suspension. Our approach does not affect the cellular viability or the hematologic recovery of the patients following transplantation.

P1381: EARLY FULL DONOR CHIMERISM AFTER FLAMSA RIC BASED ALLOGENEIC HSCT BUT NOT PRESENCE OF MORPHOLOGICAL REMISSION ASSOCIATES WITH OUTCOMES IN R/R AML PATIENTS

J. Ussmann 1,* , S. Hell 1 , B. Schetschorke 1 , G. Friedrich 1 , C. Pfrepper 1 , S. Schwind 1 , M. Jentzsch 1 , V. Vucinic 1 , M. Herling 1 , U. Platzbecker 1 , G.-N. Franke 1

1 Medical Department I, Hematology, cellular therapies and Hemostaseology, University of Leipzig Medical Center, Leipzig, Germany

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is still the only potentially curative treatment for patients (pts) with high-risk acute myeloid leukemia (AML). Despite recent advances, a significant number of pts are primary refractory or have early relapse (r/r) with <6 months (m) remission duration. One strategy to improve the outcomes of r/r AML is to perform HSCT after aplasia-inducing chemotherapy. Here, we report on a series of pts receiving a sequential chemotherapy regimen (fludarabine 30 mg/m 2 , amsacrine 100 mg/m 2 , cytarabine 2 g/m 2 (FLAMSA)) and reduced-intensity conditioning (RIC) followed by HSCT as well as prophylactic donor lymphocyte infusions (DLI) as described by Schmid et al (JCO 2005).

Aims: We aimed to retrospectively analyze the outcome of 82 consecutive pts with r/r AML who underwent FLAMSA RIC based HSCT between 2006 and 2020 at our center.

Methods: Median age of the 82 pts was 55 years (y) and 38 pts (46%) were male. ELN risk and revised MRC prognostic classification at diagnosis were favorable in 8% and 4%, intermediate in 28% and 56%, and adverse in 64% and 40% of the pts, respectively. Median time from diagnosis to HSCT was 5 months. At the time of HSCT, only 3 pts (3%) were in complete remission (CR). RIC following the FLAMSA chemotherapy was 4Gy TBI and cyclophosphamide (73%), busulfan and cyclophosphamide (11%), melphalan (13%) or 2Gy TBI only (2%). 95% of pts received peripheral blood stem cells and 5% bone marrow (BM) from a matched unrelated donor (MUD, 63%), matched sibling donor (MSD, 10%), mismatch unrelated donor (MMUD, 23%) or a haploidentical donor (4%). Immunosuppression (IS) consisted of cyclosporine A, mycophenolate mofetil and antithymocyte globuline (MUD and MMUD only) or post transplantation cyclophosphamide, tacrolimus and MMF (haplo-donors only). 12 pts (15%) received ≥ 1 DLI preemptively past d+120 as planned.

Results: Median overall survival (OS) and event-free survival (EFS) were 276 and 135 days (d). 33 pts (39%) had an OS >1y, and 22 (27%) an event free survival (EFS) >1y. 49 pts (59%) relapsed within the 1 st y. Age at HSCT, type of AML (de novo vs secondary), remission status at time of HSCT, ratio of blasts in blood to bone marrow, donor type, type of RIC, mismatch and MRC risk classification had no significant impact on OS and EFS, whereas the number of treatment lines before HSCT significantly influenced OS (p=.05). Pts with ELN favorable/intermediate risk had longer EFS (p=.04, Figure 1A), but not OS (p=.12). On d+28 after HSCT, 43 pts (52%) had reached morphologic leukemia free state (MLFS), CR with incomplete count recovery (CRi) or CR, which was not associated with better OS or EFS (Figure 1B). In contrast, pts (n=49, 60%) with a donor chimerism (DC) ≥98% in BM had a significantly longer EFS (p<.01, Figure 1C) and OS (p<.01) than those with <98%. At d+100 32 pts (39%) were in MLFS, CRi or CR, and had a significant better for EFS (p<.01, Figure 1D) and OS (p<.05). Pts with a d+100 DC ≥98% in BM also had improved EFS (p=.02, Figure 1E) but not OS (p=.16). Acute Graft-versus-Host-Disease (GvHD) °II-IV occurred in 21 pts (26%). 13 pts (16%) developed any grade of chronic GvHD. Incidence of GvHD did not increase after DLI.

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Summary/Conclusion: Despite the poor prognosis of r/r AML, a significant number of pts after FLAMSA RIC HSCT survived >1 y. Except ELN risk classification, there was no strong outcome predictor prior to HSCT. D+28 DC but not morphological remission in BM identifies pts at high risk for relapse after HSCT, allowing for additional post-HSCT strategies.

P1382: RUXOLITINIB USE IN A REAL WORLD ACUTE AND CHRONIC GRAFT VERSUS HOST DISEASE COHORT IN THE NETHERLANDS: A RETROSPECTIVE STUDY

L. Van Der Wagen 1,* , S. van Dorp 2 , M. Bellido Cassado 3 , F. Verheij 4 , K. Nijssen 4 , W. van der Velden 2 , A. Biswana 3 , N. Blijlevens 2 , J. Kuball 4

1 Hematology, UMC Utrecht, Utrecht; 2 Hematology, Radboud umc, Nijmegen; 3 Hematology, UMCG, Groningen; 4 Hematology, UMCU, Utrecht, Netherlands

Background: Preclinical data showed that Janus Kinase (JAK) signaling plays an important role in the pathogenesis of Graft versus Host Disease (GVHD) and several clinical studies showed remarkable clinical efficacy in both acute and/or chronic steroid-refractory (SR)-GVHD patients. However, these clinical results have not yet been repeated in real world cohorts with long term follow-up. Furthermore, infectious complications have not been highlighted in published clinical trials whilst these remain a significant problem in daily practice.

Aims: To assess the response of acute and/or chronic SR-GVHD patients to ruxolitinib treatment and to assess side-effects of ruxolitinib treatment occurring in adult acute and/or chronic SR-GVHD patients.

Methods: We performed a retrospective analysis of 121 consecutive patients treated with ruxolitinib for either acute or chronic GVHD. Patients were included from 3 academic centers in the Netherlands from 2014 to 2019. Patients provided written informed consent, with the exception of deceased patients in accordance with the local ethics committee. Patient data were extracted by personnel from an independent CRO to minimize reviewer bias. Patient data were anonymized.

Results: 121 patients were included in the study of whom 54 were diagnosed with acute and 67 patients with chronic GVHD. Selected baseline characteristics are depicted in Table 1. The overall response rate at day 28 of acute GVHD was 70% (38/54) with a complete response in 50% of patients (27/54).

At timepoint 6 months after initiation of ruxolitinib for chronic GVHD, the majority of patients showed stable disease. The overall response rate at 6 months in chronic GVHD was 28% (19/67) with a complete response in 13% of patients (9/67).

Ruxolitinib use was mainly complicated by infections and hematological toxicity. Of the reported (S)AE’s possibly related to ruxolitinib, 38% were infectious and 13% were hematological. Severity of infections ranged from grade 1 to grade 5, median grade 2. Most infections were viral (43%), followed by bacterial (19%) and fungal or mould infections (14%). During follow up mortality was 14,9% (18 patients) of which 6 cases were due to uncontrolled GVHD, 5 cases due to infections and 3 patients died of relapse of their primary malignancy, 4 patients died of other causes.

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Summary/Conclusion: In a real world cohort of GVHD patients treated with ruxolitinib we observed in patients with acute GVHD a complete response rate at day 28 of 50%. Overall response rate at 6 months in cGVHD patients was 28%. Infectious adverse events were common in both acute and chronic GVHD patients and were mainly caused by viral pathogens.

P1383: EXTRAMEDULLARY RELAPSE OF ACUTE MYELOID LEUKEMIA AND ACUTE LYMPHOBLASTIC LEUKEMIA AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION - A SINGLE-CENTER EXPERIENCE

F. Vasconcelos 1,* , D. Rochate 1 , E. Couto 1 , P. Coelho 1 , L. Leite 1 , R. Branca Ferreira 1 , C. Pinho Vaz 1 , A. Campos 1

1 Bone Marrow Transplantation Service, Instituto Portugues de Oncologia do Porto, Porto, Portugal

Background: Acute leukemia relapse after allogeneic hematopoietic cell transplant (allo-HCT) is challenging. Allo-HCT is a potentially curative treatment for acute myeloid leukemia (AML) and Acute lymphoblastic leukemia (ALL). Extramedullary relapse (EMR) can occur in various extramedullary tissues such as the central nervous system (CNS), breast, bone, testis, and skin, and data on EMR after allo-HCT is limited.

EMR is a long-term complication of stem cell transplantation with a poor prognosis.

Aims: To characterize EMR in the setting of allo-SCT for AML and ALL.

Methods: Retrospective analysis of relapsed patients (pts) submitted to allo-SCT for AML and ALL at our center from January 2011 to December 2021. Disease characteristics, type of transplant, outcome-related data were collected from clinical files. All cases were confirmed by biopsy. Survival was estimated by Kaplan-Meier method.

Results: Between 2011 and 2021, three hundred and seventy one AML and ALL pts were transplanted in our center. After the allo-HCT, 106 patients had a relapse, of which 86 were bone marrow (BM) relapses and 20 were EMR.

Among the 20 EMR pts there were: 14 males and 6 females with a median age of 38 (range, 19 – 59) years. Of these, 13 had AML (AML Not Otherwise Specified (NOS) (n=9), AML with myelodysplasia-related changes (n=2), Myeloid sarcoma (n=1) and AML with recurrent genetic abnormalities: AML with inv(16)(p13.1q22) (n=1)), 7 had ALL: ALL-B (n=4), ALL-T (n=2), ALL-B with recurrent genetic abnormalities t(9;22)(q34;q11) (n=1). Twelve pts had a related donor, 1 pt had an HLA-haploidentical donor and 7 pts had a non-related donor (NRD). All patients with NRD were transplanted with a myeloablative regimen. Among patients with related donors, 11 were transplanted with a myeloablative regimen and 2 with a non-myeloablative regimen. The EMR site varied widely, including the CNS (n=3), skin or soft tissue (n=3), bone (n=1), testis (n=2), lymph nodes (n=2), breast (n=3). Multiple sites of EMR (≥2) were observed in 6 patients.

Among the 86 BM relapsed pts there were: 42 males and 44 females with a median age of 43 (range: 1-66) years. Of these, 57 had AML (AML Not Otherwise Specified (NOS) (n=45), AML with myelodysplasia-related changes (n=12). Twenty nine pts had ALL: ALL-B (n=19), ALL-T (n=3), ALL-B with recurrent genetic abnormalities t(9;22)(q34;q11) (n=7). 40 pts had a related donor, 1 pt had an HLA-haploidentical donor and 45 pts had a non-related donor (NRD). Thirty seven patients with NRD were transplanted with a myeloablative regimen and 8 with a non-myeloablative regimen. Among patients with related donors, 22 were transplanted with a myeloablative regimen and 19 with a non-myeloablative regimen.

Relapse free survival (RFS) of EMR pts was 12 months (CI 95%: 0-26.6) and the median overall survival (OS) after EMR was 4 months (CI 95%: 0-13.2)

RFS of pts with BM relapse was 2 months CI 95%: 1.1-2.9) and the median OS after BM relapse was 3 months (CI 95%: 1.5-4.4).

Summary/Conclusion: Although our study includes a small number of pts experiencing EMR, results show that EMR was more frequent in men, AML NOS, and EMR is frequent in multiple sites (6 of 20 patients). The median OS after EMR was 4 months, the same reported in the literature.

Results suggest that EMR has a different behavior from BM relapse, associated with better RFS and OS.

P1384: EFFECT OF NUTRITIONAL SUPPORT ON THE INCIDENCE OF MUCOSITIS IN AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION (AUTOHSCT) IN PATIENTS WITH HODGKIN’S LYMPHOMA. (INTERIM RESULTS)

S. Volchenkov 1,* , L. Filatova 1 , T. Semiglazova 1 , S. Lyubimov 1 , I. Zyuzgin 1 , I. Ishmatova 1 , A. Zverkova 1 , S. Elkhova 1 , M. Motalkina 1 , Y. Chudinovskikh 1 , S. Shalaev 1 , A. Koviazin 1

1 hematology, NMRCC named after N.N.Petrov, St.Petersburg, Russia

Background: Correction of metabolic disorders is one of the important point’s supportive care during high-dosechemotherapy with autologous stem cell transplantation (autoHSCT) in patients with Hodgkin’s lymphoma (HL). The impact of nutritional support on recovery during autoHSCT for this group of patients is not clearly understood. To assess the effect of combined nutritional support on the frequency of mucositis, grade of mucositis, number of nasogastric tube in patients group, and frequency of blood transfusions during high-dose chemotherapy with autoHSCT in patients with HL.

Aims: The study analyzed 68 patients with Hodgkin’s lymphoma who received high-dose chemotherapy with only parenteral nutritional support (NS) between 2013 and 2016 and 71 patients treated between 2016 and 2020 who received combined NS: combination of parenteral and enteral sipping nutrition. The primary endpoints were the frequency of mucositis, number of nasogastric tube in patients group.

Methods: Group of patient without combined NS group received nutritional support using parenteral nutrition in case of a decrease in the level of total protein or albumin, which was assessed by a biochemical blood test. Patients in the combined nutritional support group received combined nutritional support with enteral sips in combination with parenteral nutrition, regardless of total protein and albumin levels.

Results: The relative risk (RR) of developing stomatitis in the combined NS group was lower than in the group without combined NS: RR=0.67 (95% CI 0.49-0.92), p=0.015. Enteral nutritional support was associated with a reduced risk of stomatitis when adjusted for age, sex, and BMI: odds ratio (OR)=0.33, 95% CI 0.14-0.78, p=0.012. The RR of developing colitis in the combined NS group was lower: RR=0.58 (95% CI 0.36-0.94), p=0.026. Enteral nutritional support was associated with a reduced risk of colitis after adjusting for age, sex, and baseline BMI: OR=0.38, 95% CI 0.17-0.86, p=0.021. A nasogastric tube was placed statistically less frequently in the combined NS group: 3.0% (n=2) versus 19.6% (n=10), p=0.015. Enteral nutritional support was associated with a reduced frequency of nasogastric tube insertion when adjusted for age, sex, and baseline BMI: OR=0.16, 95%CI 0.03-0.83, p=0.029. In patients with an installed nasogastric tube, the duration of its stay did not statistically significantly differ between groups with and without combined NS: median 6 days (IQD: 4-8) vs. 5 days (IQD: 3-6), respectively (p=0.382).

Summary/Conclusion: The data obtained show that combined nutritional support reduces the risk of mucositis, reduces the during frequency of nasogastric tube insertion autoHSCT in patients with Hodgkin’s lymphoma.

P1385: THE GUIDING SIGNIFICANCE OF IMMUNE CELL SUBSET MONITORING IN IMMUNE RECONSTITUTION IN PATIENTS WITH CAR-T BRIDGED ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

W. zhao 1,* , M. chen 2 , Y. an 2 , P. hu 3 , M. chen 3 , H. Wang 4,4 , P. Lu 2 , Y. zhao 2

1 Beijing Lu Daopei Hospital, Beijing; 2 Hebei Yanda Lu Daopei Hospital, Langfang, sanhe; 3 Beijing Lu Daopei Hospital, Beijing; 4 clinical lab, Hebei Yanda Lu Daopei Hospital, Langfang, sanhe, China

Background: Immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) can effectively prevent infection and graft-versus-leukemia effect. Monitoring the lymphocyte subsets of patients after transplantation has certain guiding value for understanding the immune function status.

Aims: study the immune reconstitution after CAR-T bridging HSCT.

Methods: A total of 83 patients with acute lymphoblastic leukemia who received allo-HSCT in Beijing Lu Daopei Hospital from August 2018 to December 2021 were included in this study (61 cases of B-ALL and 22 cases of T-ALL,54 males and 29 females) with a median age of 29 years (1-57). 33 were CAR-T patients and 50 were non-CAR-T patients. MFC was applied to detect PB lymphocyte subsets of the patients 1 month before and 1, 2, 3, 6, 8, 10, and 12 months after HSCT, as well as the transplants.

Results: Before transplantation, we recorded primary disease CR in 76 cases, PR or NR in 7 cases. The HSCT types are 71 haploids, 6 unrelated donors, and 6 siblings. The difference analysis of pre-transplant status between CAR-T or non-CAR-T groups showed that the CAR-T group had a longer medical history before transplantation than the non-CAR-T group (P=0.000). Furthermore, in the CART group, the serum IgA/IgG/IgM (P= 0.000/0.008/0.000), peripheral blood total lymphocytes, T lymphocytes, helper T cells, cytotoxic T cells, B lymphocytes counts, and other results were lower than those of the non-CAR-T group(P=0.000-0.002). All of the above have significant differences (P<0.05). 2、The difference analysis results of the treatment regimen and immune status between the two groups after transplantation showed that: The dose of rabbit anti-human thymocyte immunoglobulin in the conditioning regimen of the CAR-T group was lower than that of the non-CAR-T group (P=0.023);The CD34+ count in the reinfused graft was higher than that in the non-CAR-T group (P=0.003);The leukocyte/neutrophil engraftment time was earlier in the CAR-T group after transplantation (P=0.038/0.043) and the plasma cytomegalovirus DNA level was higher (P=0.002), the application time of cyclosporine was significantly shorter than that of the non-CAR-T group (P=0.000). 3、The difference analysis of the results of peripheral blood immune cell subsets between the two groups at 1, 2, 3, 6, 8, 10, and 12 months after transplantation showed that: There were no significant differences in the detection results of total lymphocytes, T lymphocytes cells, helper T cells, cytotoxic T cells, B lymphocytes, NK and NKT cells, and CD4/CD8 ratio (P>0.05). Furthermore, there were no significant differences in other biomarkers.

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Summary/Conclusion: For CAR-T patients with low immune function before transplantation, to achieve the same immune reconstitution level as non-CAR-T patients, we reduced the dose of ATG in the conditioning regimen, increased the CD34+ reinfusion count of the graft, and stopped cyclosporine as soon as possible after transplantation. Dynamic monitoring of immune cell subsets in peripheral blood of patients after transplantation helps grasp the status of postoperative immune function and immune reconstitution and guides clinical treatment.

P1386: A REVIEW OF HEMATOPOIETIC CELL TRANSPLANTATION IN TAIWAN: DATA AND TRENDS DURING 2009–2018

Y.-H. Wang 1,* , S.-P. Yeh 2 , J.-P. Gau 3 , P.-N. Wang 4 , Y.-C. Liu 5,6 , Y.-C. Chen 7 , C.-L. J. Teng 8 , T.-Y. Chen 9 , S.-C. Lin 10 , T.-D. Tan 11 , T.-F. Wang 12 , M.-C. Ma 13 , S.-J. Lin 13 , C. Wang 14 , M.-Y. Lee 15 , C.-C. Chen 16,17 , T.-Y. Chao 18 , S.-T. Jou 19 , M. Yao 1 , B.-S. Ko 1,20

1 Internal Medicine, National Taiwan University Hospital, Taipei; 2 Internal Medicine, China Medical University Hospital, Taichung; 3 Internal Medicine, Taipei Veterans General Hospital, Taipei; 4 Internal Medicine, Chang Gung Medical Foundation, Taoyuan; 5 Internal Medicine, Kaohsiung Medical University Hospital; 6 Faculty of Medicine, Kaohsiung Medical University, Kaohsiung; 7 Internal Medicine, Tri-Service General Hospital, Taipei; 8 Internal Medicine, Taichung Veterans General Hospital, Taichung; 9 Internal Medicine, National Cheng Kung University Hospital, Tainan; 10 Internal Medicine, Far Eastern Memorial Hospital, New Taipei City; 11 Hematology and Medical Oncology, Koo Foundation Sun Yat-Sen Cancer Center, Taipei; 12 Hematology and Oncology, Buddhist Tzu-Chi General Hospital, Hualien; 13 Internal Medicine, Kaohsiung Chang-Gung Memorial Hospital, Kaohsiung; 14 Hematology and Oncology, Changhua Christian Hospital, Changhua; 15 Internal Medicine, Chia-Yi Christian Hospital; 16 Internal Medicine, Chang Gung Memorial Hospital, Chia-Yi; 17 College of Medicine, Chang Gung University, Tao-Yuan; 18 Hematology and Oncology, Taipei Medical University-Shuang Ho Hospital; 19 Pediatrics, National Taiwan University Hospital; 20 Hematological Oncology, National Taiwan University Cancer Center, Taipei, Taiwan

Background: Despite the advent of novel gents and CAR-T therapies, haematopoietic cell transplantation (HCT) remains the curative treatment for many malignant and non-malignant diseases involving the hematopoietic system and some solid tumours.

Aims: To decipher the patient and donor pools in Taiwan and provide valuable information for clinicians, investigators, and patients worldwide, we performed this cross-sectional survey, which adopted a standardized registry structure, to present and discuss trends and outcomes of HCTs in Taiwan during 2009-2018.

Methods: Currently, 17 hospitals contribute to the registry, and the collection and analysis of data from the Taiwan Blood and Marrow Transplantation Registry is approved by the institutional review board of each participating hospital. All methods were carried out in accordance with relevant guidelines and regulations. Patient numbers corresponding to the number of patients receiving their first HCTs between 2009 and 2018 and transplant numbers reflecting the total number of transplants performed are presented. Haploidentical transplants are being described as any family member with two or more (but not more than 5) loci mismatches.

Results: The record number of HCTs was 4927 HCT in 4738 patients, comprising 2688 (54.6%) allogeneic and 2239 (45.4%) autologous, reported by 17 centres. The main indications were lymphoid malignancies 3038 (64.1%), myeloid malignancies 1675 (35.4%), and non-malignant disorders 258 (5.4%). HCT trends showed growth in transplant activity, a rapid increase in the number of autologous HCT, remarkable growth in older (>60-year-old) patients receiving HCT, an increase in haploidentical donors (HID), and an expansion of harvesting stem cells from both the peripheral blood (PB) and the bone marrow (BM). HCTs have been implemented increasingly and most frequently for acute myeloid leukaemia, non-Hodgkin lymphomas, and multiple myeloma (MM). Amongst them, MM showed a particularly noticeable expansion in the number of HCT, comprising 15.7% in 2009 and 19.4% in 2018, respectively. The proportion of older patients receiving HCT, autologous and allogeneic, increased from 5.5% and 1.7% in 2009 to 17.7% and 10.5% in 2018, respectively. During this period, the proportion of autologous HCT rose from 44.3% in 2009 to 52.9% in 2018. Meanwhile, HID also showed a magnificent increase, accounting for 0.9% in 2009 and 6.5% in 2018, respectively. On the other hand, stem cells harvested from PB+BM increased from 0.3% in 2009 to 2.9% in 2018, while the use of PB stem cells remained stable during this time frame, spanning from 92% to 98%.

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Summary/Conclusion: Aside from the significant elaboration of HCT technology, most worthy of mention developments include the success in haploidentical HCT and the use of reduced-intensity conditioning in older patients. Overall, this review of the TBMTR provides a snapshot of the current transplant state and highlights important HCT trends in Taiwan.

P1387: MONITORING OF DONOR CHIMERISM ON RED BLOOD CELLS ANTIGENS AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION

A. Yovdiy 1,* , M. Smolnikova 1 , E. Butina 1 , E. Poponina 1 , N. Zorina 1

1 The Federal State-Financed Scientific Institution Kirov Research Institute of Hematology and Blood Transfusion under the Federal Medical Biological Agence, Kirov, Russia

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment for diseases of the blood system. One of the indicators reflecting the functioning of donor hematopoietic tissue is posttransplant chimerism.

Aims: To evaluate the effect of ABO belonging of the donor and recipient on the timing of the formation of red blood cells (RBS) donor chimerism after HSCT.

Methods: The study included 57 patients who underwent HSCT in 2013-2021. Patient age ranged from 3 to 60 years (median - 34). Diagnosis of the patients: acute leukemia - 42 persons, Hodgkin’s lymphoma - 6, aplastic anemia - 4 and other blood diseases - 5. Before HSCT, the study of RBC antigens of the ABO, Rh, Kell systems in donors and recipients was performed by gel agglutination using reagents and equipment BioRad (USA). Antigens identified in the recipient’s phenotype and absent in the donor’s phenotype were considered informative, or vice versa. For statistical data processing, the nonparametric Kruskal-Wallis test was used.

Results: Thirty-two (56.1%) of 57 pairs differed in ABO system antigens. Of these, 16 pairs (28.1%) had a major ABO incompatibility, 16 pairs (28.1%) had a minor incompatibility. The time of appearance of donor’s RBS in patients with ABO incompatibility had a wider range (from 14 to 201 days) and occurred significantly later (Me - 28 days) than in recipients ABO identical with donors (Me - 20 days), and from minor ABO incompatibility (Me - 19 days) (Table 1). The periods of occurrence of 50% and 100% donor chimerism did not differ significantly in all groups. In the group of recipients with major ABO incompatibility a case of pure red cell aplasia was revealed. The first appearance of RBC carrying donor antigens was recorded on day 201. 100% donor chimerism was formed on day 278. Antibodies of the ABO system (anti-A) IgG were present in the recipient’s blood for 189 days, IgM - for 230 days.

Summary/Conclusion: In recipients with a major ABO incompatibility the first appearance of donor’s RBS after HSCT occurs significantly later than in couples ABO identical and couples with a minor ABO incompatibility.

P1388: A SINGLE-ARM, PROSPECTIVE MULTICENTER CLINICAL STUDY OF RELATED HLA HAPLOIDENTICAL PERIPHERAL BLOOD HEMATOPOIETIC STEM CELL TRANSPLANTATION WITHOUT IN VITRO T-CELL DEPLETION FOR HEMATOLOGICAL MALIGNAN

H. Yuan 1 , S. Wang 2 , J. Li 3 , H. Bai 4 , B. Zhang 5 , C. Wang 6 , L. Liu 7 , L. Su 8 , M. Jiang 1,*

1 The First affiliated Hospital of Xinjiang Medical university, Urumuchi; 2 The 920th Hospital of the PLA Joint Logistics Support Force, Kunming; 3 Jiangsu Provincial People’s Hospital, Nanjing; 4 The 940th Hospital of the PLA Joint Logistics Support Force, Lanzhou; 5 Harbin Institute of Hematology, Harbin; 6 Shaihai General Hospital, Shanghai; 7 Tangdu Hospital of The fourth Military Medical University, Xian; 8 Shanxi Cancer Hospital, Taiyuan, China

Background: Although haplo-HSCT has made great progress, how to better improve engraftment, reduce infection、GVHD and relapse incidence still requires continuous research. We believe that haplo-HSCT requires a higher dose of hematopoietic stem cells to solve the problem of engraftment and immune reconstitution, which is difficult to achieve simply by harvesting bone marrow as graft. To solve this problem, we designed a protocol using a high-dose PBSC without T-cell depleted in vitro as graft to undergo MAC haplo-PBSCT.Since 2004, our center has been carrying out this protocol to treat hematological malignancies. The primary engraftment was very good, the incidence rate of 100-day severe aGVHD was not high. PFS and OS are similar to those of other transplant centers at home or abroad. On this basis, to validate this protocol, we regisrered a voluntary single-arm, prospective multi-center clinical study in China. The incidence of 100-day aGVHD is the primary endpoint.From 2014 to 2019,a total of 8 hematopoietic stem cell transplantation centers in tertiary hospitals participated in this prospective multi-center clinical study.

Aims: Taking aGVHD as the primary endpoint, engraftment, relapse rate, PFS and OS as the secondary endpoints. To verify the safety and feasibility of our unique haplo-PBSCT protocol.

Methods: Haplo-PBSCT is based on ATG, ABuCy as a conditioning regimen, an intensive GVHD conditioning regimen containing anti-CD25 monoclonal antibody and early application of glucocorticoids, and enhanced infection prevention and control,without in vitro T-cell depletion, high-dose peripheral blood MNC infusion.

Results: 102 haplo-PBSCT patients, 1 patient had primary failure of engraftment, and 3 patients had platelet non-engraftment. The median of transplanted MNC was 14.3 (8.2-27.8) × 10 8 /kg, and the median of CD34+ cells was 8 (3.96-21.00) × 10 6 /kg. The follow-up ended on January 31, 2022, and the median follow-up time was 66 (1-112) months. All patients could be assessed for aGVHD. 56 cases developed aGVHD, with a cumulative incidence of 52.8%. The cumulative incidence of aGVHD of grade II-IV was 25.1%, the cumulative incidence of grade III-IV aGVHD was 12.4%, the cumulative incidence of aGVHD in 27 standard-risk patients was 48.1%, and the aGVHD in 75 high-risk patients was 43.4%; 97 evaluable patients, 49 patients developed cGVHD, the 3-year cumulative incidence of cGVHD was 55%, the cumulative incidence of localized cGVHD was 46%, and the cumulative incidence of extensive cGVHD was 14.3%.The 3-year cumulative recurrence rate was 27.1%. The cumulative recurrence rates of ALL, AML, and MDS patients were 38.2%, 23.9%, and 16%, respectively. A total of 35 cases died, 10 cases of severe pulmonary infection, 4 cases of multiple organ failure (MOF), 2 cases of cerebral hemorrhage, and 19 cases of recurrence and death. The 3-year expected overall OS of AML, ALL and MDS patients was 72.7%, 57.1% and 58.3%, respectively.

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Summary/Conclusion: This study shows that the severe aGVHD of our unique haplo-PBSCT is not high, which is comparable to the other ATG-based research datas. The relative long term follow-up observation indicates that a better clinical efficacy is achieved, the incidence of viral infection and recurrence of hematological malignancies are lower.

P1389: RUXOLITINIB DEMONSTRATES A GREATER CORTICOSTEROID-SPARING EFFECT THAN BEST AVAILABLE THERAPY IN PATIENTS WITH CORTICOSTEROID-REFRACTORY/DEPENDENT CHRONIC GRAFT-VS-HOST DISEASE

R. Zeiser 1,* , D. Russo 2 , R. Ram 3 , S. Hashmi 4 , R. Chakraverty 5 , J. Moritz Middeke 6 , S. Giebel 7 , R. Sarkar 8 , M. Gowda 9 , S. Gunes 10 , T. Stefanelli 11 , S. J. Lee 12 , T. Teshima 13 , F. Locatelli 14

1 Department of Medicine I, Faculty of Medicine, Medical Center, University of Freiburg, Freiburg, Germany; 2 University of Brescia, ASST-Spedali Civili Brescia, Brescia, Italy; 3 BMT Unit, Tel Aviv (Sourasky) Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 4 Department of Medicine, Sheikh Shakhbout Medical City, Mayo Clinic, Abu Dhabi, United Arab Emirates; 5 UCL Cancer Institute, Institute of Immunity and Transplantation, London, United Kingdom; 6 Universitätsklinikum “Carl Gustav Carus” der Technischen Universität Dresden, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), and National Center for Tumor Diseases (NCT) Dresden, Dresden, Germany; 7 The Department of Bone Marrow Transplantation and Onco-Hematology, Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice, Poland; 8 Novartis Healthcare PVT. LTD, Hyderabad, India; 9 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 10 Novartis Farma SPA, Lombardy, Italy; 11 Novartis Pharma AG, Basel, Switzerland; 12 Fred Hutchinson Cancer Research Center, Seattle, United States of America; 13 Department of Hematology, Hokkaido University Faculty of Medicine, Sapporo, Japan; 14 Dipartimento di Oncoematologia Pediatrica, IRCCS, Ospedale Pediatrico Bambino Gesú, Sapienza, Università di Roma, Rome, Italy

Background: Chronic graft-vs-host disease (cGVHD) is a major complication in recipients of allogeneic stem cell transplants. The recommended treatment for moderate/severe cGVHD is systemic corticosteroids (CS) ± calcineurin inhibitor (CNI). However, limiting exposure to CS is advised due to high morbidity and poor quality of life associated with CS treatment. Additionally, ≈50% of patients (pts) become CS refractory/dependent (SR/D), requiring second-line treatment. Ruxolitinib (RUX), a JAK1/2 inhibitor, demonstrated superior efficacy over best available therapy (BAT) in the phase 3 REACH3 study ( NCT03112603 ) in pts with SR/D cGVHD (Zeiser NEJM 2021).

Aims: To evaluate the impact of RUX vs BAT on CS use in pts with SR/D cGVHD in REACH3.

Methods: Pts aged ≥12 years with moderate or severe SR/D cGVHD were randomized 1:1 to receive either RUX 10 mg twice daily (n=165) or investigator-selected BAT (n=164). Pts received randomized treatment along with CS (prednisone or equivalent) ± CNI for 24 weeks. In this post hoc, descriptive analysis, data were collected from baseline (BL) to week 24, and changes in CS use over time were analyzed by treatment and best overall response (BOR; responders [complete or partial response by 2014 NIH criteria at any time] vs nonresponders).

Results: BL pt characteristics, including body mass index (BMI) and CS dose, were generally balanced between groups—median BMI (RUX vs BAT): responders, 22.7 vs 23.3; nonresponders, 22.4 vs 22.1; median CS dose (mg/day, RUX vs BAT): responders, 30.0 vs 30.0; nonresponders, 33.9 vs 35.0. A greater proportion of RUX vs BAT responders had severe disease (56.3% [71/126] vs 44.4% [44/99]). Overall, median CS dose decreased over time in the RUX and BAT arms and was similar between groups. RUX-treated pts experienced a faster and greater median percentage decrease from BL in CS dose over time vs BAT-treated pts. When analyzed by BOR, the median percentage change (decrease) from BL in CS dose was similar in RUX and BAT responders but was smaller in BAT vs RUX nonresponders (Figure). RUX responders had the greatest median percentage decrease in CS dose over time.

The proportion of pts achieving a ≥50% reduction from BL in CS dose at any time was similar in the RUX and BAT arms (81.2% [134/165] vs 79.9% [131/164]) and RUX and BAT responders (81.7% [103/126] vs 86.9% [86/99]). This proportion was numerically lower in BAT vs RUX nonresponders (69.2% [45/65] vs 79.5% [31/39]). Pts in the RUX arm achieved a ≥50% reduction in CS dose earlier than pts in the BAT arm, with a median time of 1.9 months (95% CI, 1.4-1.9 months) vs 2.4 months (95% CI, 1.9-2.8 months).

The proportion of pts who achieved a CS dose of ≤7.5 mg/day at any time was higher with RUX vs BAT (71.5% [118/165] vs 61.6% [101/164]). This proportion was similar between RUX and BAT responders (69.8% [88/126] vs 71.7% [71/99]) and numerically lower in BAT vs RUX nonresponders (46.2% [30/65] vs 76.9% [30/39]). RUX also led to higher rates of CS discontinuation vs BAT overall (48.5% [80/165] vs 35.4% [58/164]), in responders (42.9% [54/126] vs 33.3% [33/99]), and nonresponders (66.7% [26/39] vs 38.5% [25/65]).

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Summary/Conclusion: RUX led to a faster and larger reduction in CS dose vs BAT, with a greater proportion of RUX-treated patients discontinuing CS treatment. Median decreases in CS dose were similar among RUX-treated pts, regardless of response. In contrast to RUX, BAT did not demonstrate a substantial CS-sparing effect in nonresponders. Further analyses assessing the impact of the CS-sparing effect observed with RUX are warranted.

P1390: THE EFFICACY AND SAFETY OF MODIFIED MELPHALAN AND BUSULFAN-BASED REGIMEN FOR ALLOGENEIC TRANSPLANTATION IN REFRACTORY/RELAPSED AML PATIENTS

S. Chen 1 , X. Zhang 1 , W. Zhai 1 , Y. He 1 , S. Feng 1 , M. Han 1 , T. Niu 2 , Y. Xu 3 , G. Gao 4 , S. Fan 5 , Z. Zhou 6 , F. Zhou 7 , F. Li 8 , L. Liu 9 , W. Yang 10 , Q. Liu 11 , X. Zhang 12 , E. Jiang 1,*

1 Department of hematopoietic stem cell transplantation, Institute of Hematology and Blood Diseases Hospital, CAMS and PUMC, Tianjin; 2 West China Hospital Of Sichuan University, Sichuan; 3 Xiangya Hospital Central South University, Changsha; 4 Xijing Hospital of Air Force Military Medical University, Xian; 5 The First Affiliated Hospital of Harbin Medical University, Haerbin; 6 The Second Affiliated Hospital of Kunming Medical University, Kunming; 7 The 960th Hospital of Chinese People’s Liberation Army Joint Logistic Support Force, Jinan; 8 The First Affiliated Hospital of Nanchang University, Nanchang; 9 Second Affiliated Hospital of Air Force Military Medical University, Xian; 10 Shengjing Hospital of China Medical University, Shenyang; 11 Nanfang Hospital, Southern Medical University, Guangzhou; 12 The Second Affiliated Hospital of Army Medical University, Shanghai, China

Background: Allogeneic Hematopoietic stem cell transplantation (Allo-HSCT) offers the only survival opportunity for refractory/relapsed (R/R) AML patients, as those patients responded insufficiently to chemotherapy. However, the overall survival (OS) rate after HSCT is reported to be only 20~40%.

Aims: In this study, we made refinements in the conditioning regimen with both melphalan and busulfan, namely MCBC (the combination of melphalan, cladribine, busulfan, and cyclophosphamide). We aim to investigate the efficacy and safety of MCBC conditioning regimen for allo-HSCT in R/R AML patients.

Methods: This prospective multi-center clinical trial was conducted in 12 tertiary hospitals in China, and enrolled R/R AML patients underwent allo-HSCT from March 2020 to January 2022(ChiCTR Registration ID: ChiCTR2000029936). The protocol was approved by institutional review boards at participating centers. All patients enrolled received MCBC preconditioning regimen: melphalan 60mg/m2/d, day -9~-8, cladribine 5mg/m2/d, day -7~-5, busulfan 3.2mg/kg/d, day -5~-3, cyclophosphamide 30mg/kg/d, day -2~-1. Rabbit ATG was added in haploid-identical and unrelated-matched donor transplantation. FK506/CsA +short course MTX±MMF was mostly used to prevent graft-versus-host disease（GVHD）. A post-transplantation consolidation treatment was provided whenever possible, including low-dose azacytidine, venetoclax, or FLT3 inhibitors. Probabilities of OS, DFS were calculated by Kaplan-Meier method. COX regression model was used for multivariate analysis.

Results: This study included 56 R/R AML patients, including 48 refractory and 8 relapsed AML. Only 7 patients achieved hematology remission before transplantation, and the blast count, measure by flow MRD pre-HSCT, of 25 patients was ≥ 20%. It is inspiring that hematopoietic reconstitution was achieved in all 56 patients, and all patients (100%) achieved hematology complete remission (CR) on day +14. With a median follow-up of 186（48-555）days, the estimated 1-year overall survival (OS) and 1-year disease-free survival (DFS) were 77.8±7.72% and 71.9±7.46%, respectively. 11 patients relapsed within the 1 st year, and the median time from HSCT to relapse was 105 (83-184) days. The probability of relapsed rate was 32.4±8.3%, and non-relapse mortality (NRM) was 3.7±2.6%. Infection is the leading NRM cause. The incidence of Ⅲ and Ⅳ aGVHD was 10.71%. We furtherly performed univariant and multivariant analyses to identify the independent predictors of poor outcomes. Univariate analysis results revealed blast count ≥ 20% before transplantation, and absence of maintenance treatment post-HSCT were the risk factors that affected prognosis. Competitive risk modal analysis for multivariate analysis showed both high blast count pre-HSCT (HR 1.496, p=0.0015) and absence of maintenance treatment after HSCT (HR 2.050, p=0.0170) was the independent factor. Mucositis was the main reported regimen-related toxicity, and it was well tolerated.

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Summary/Conclusion: Our study confirms the excellent anti-leukemic capacity and good acceptable toxicity of MCBC conditioning regimen in R/R AML, that more than 70% patients could achieve leukemia free survival within the 1 st year after allo-HSCT. Furthermore, it is important to decrease leukemic blast burden pre-HSCT and perform maintenance treatment after transplantation as soon as possible.

P1391: COMPARISON OF AUTOLOGOUS AND ALLOGENEIC STEM CELL TRANSPLANTATION FOR ADULT PATIENTS WITH PHILADELPHIA CHROMOSOME-NEGATIVE ACUTE LYMPHOBLASTIC LEUKEMIA IN FIRST COMPLETE REMISSION

R. Zhang 1,* , M. Lv 1 , D. Yang 1 , A. Pang 1 , W. Zhai 1 , X. Chen 1 , Q. Ma 1 , Y. He 1 , J. Wei 1 , E. Jiang 1

1 National Clinical Research Center for Blood Diseases, State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, TIANJIN, China

Background: Adult Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph - ALL) is a high-risk subtype of acute leukemia and allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is still the standard consolidation therapy for patients with available donors. With the intensive consolidation chemotherapy and the accuracy of minimal residual disease (MRD)，auto-SCT also can be used for eligible patients.

Aims: To analyze the outcomes of patients who received autologous stem cell transplantation (auto-SCT) or Allo-HSCT and provide the basis for the choice of transplantation method in Ph - ALL in first complete remission.

Methods: We retrospectively investigated the outcomes of 139 adult patients PH - ALL who received stem cell transplantation with myeloablative condition regimen from January 2014 to September 2021 in our center. The overall survival (OS) rate, leukemia-free survival (LFS) rate, cumulative incidence of relapse (CIR) rate and non-relapse mortality (NRM) rate were explored.

Results: The median follow-up time was 23 (2-96.5) months with a total of 33 patients dead and 34 patients relapsed among the 139 patients and the 3 years OS, LFS, CIR and NRM rates after transplantation were 71.7 ± 4.6%, 61.3 ± 4.5%, 27.2 ± 4.1%,11.2 ± 2.9%, respectively. Both positive MRD after 3 courses of chemotherapy and MRD-positive before transplantation were poor prognostic factors for LFS and RR. For the 98 patients who achieved MRD negative after 3 courses of chemotherapy, there was no significant difference in OS(82.8% vs 72.3%， P =0.119)，LFS(66.4% vs 64.3%， P =0.690), CIR(27.1% vs 17.1%， P =0.237）between auto-SCT group and allo-HSCT group, while auto-SCT group showed lower NRM (2.5% vs. 18.6 %, P= 0.028 ) than the allo-HSCT group. However, for the 41 patients who remained MRD positive after 3 courses of chemotherapy, the auto-SCT group had higher CIR (66.7% vs 36.8%, P = 0.021) than the allo-HSCT group.

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Summary/Conclusion: For Ph-negative ALL patients who obtain MRD-negative after 3 courses of chemotherapy, autologous hematopoietic stem cell transplantation is an effective consolidation therapy option and maintenance chemotherapy after transplantation is important toreduce recurrence, and for patients with MRD-positive after 3 courses of chemotherapy, allogeneic hematopoietic stem cell transplantation is an effective means to reduce relapse and improve survival.

P1392: MACHINE LEARNING ALGORITHM AS A PROGNOSTIC TOOL FOR VENOUS THROMBOEMBOLISM IN ALLOGENIC TRANSPLANT PATIENTS

R.-X. Deng 1,2,3,4,* , Y. He 1,2,3,4 , X.-L. Zhu 1,2,3,4 , H.-X. Fu 1,2,3,4 , X.-D. Mo 1,2,3,4 , J.-Z. Wang 1,2,3,4 , K.-Y. Liu 1,2,3,4 , X.-J. Huang 1,2,3,4 , X.-H. Zhang 1,2,3,4

1 Peking University People’s Hospital, Peking University Institute of Hematology; 2 Collaborative Innovation Center of Hematology, Peking University; 3 Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation; 4 National Clinical Research Center for Hematologic Disease, Beijing, China

Background: As a serious complication following allogenic haematopoietic stem cell transplantation (allo-HSCT), venous thromboembolism (VTE) is significantly related to poor survival and increased nonrelapse mortality. Therefore, distinguishing patients at high risk of death who should receive specific therapeutic management is key to improving survival. Here, we established the first machine-learning-based model to predict posttransplant VTE prognosis to facilitate physician decision-making.

Aims: This study aimed to establish a machine-learning-based prognostic model for selecting posttransplant VTE patients who are at a higher risk of death.

Methods: We retrospectively evaluated 256 consecutive VTE patients who underwent allo-HSCT at a single centre between 2008 and 2019. These patients were further randomly divided into 1) a derivation (80%) cohort of 205 patients and 2) a test (20%) cohort of 51 patients. The least absolute shrinkage and selection operator (LASSO) approach was utilized to choose the potential predictors from the primary dataset. Eight machine learning classifiers were utilized to produce eight candidate models. A 10-fold cross-validation (CV) procedure was used to internally evaluate the eight models and to select the best-performing model for external assessment using the test cohort. We assessed the prognostic model performance by evaluating the discrimination [area under the curve (AUC)], calibration (calibration plot), and net benefit [decision curve analysis (DCA)].

Results: In total, 256 of 7238 patients (3.5%) were diagnosed with VTE after transplantation. Among them, 118 patients (46.1%) had catheter-related venous thrombosis (CRT), 107 (41.8%) had isolated deep-vein thrombosis (DVT), 20 (7.8%) had isolated pulmonary embolism (PE), and 11 (4.3%) had concomitant DVT and PE. The two-year overall survival (OS) rate was 68.8%.

Using LASSO regression analysis, eight potential features were selected from the 54 candidate variables. The optimal algorithm based on the validation cohort with 10-fold CV runs was a logistic regression classifier. Therefore, a prognostic model was then established to predict 2-year outcome, which was named BRIDGE ( b acteraemia, r elapse, I FI and F I B, grade II-IV aGVH D and F D P, a g e, and history of strok e ). The AUCs of the BRIDGE model were 0.883 (95% CI 0.831-0.937), 0.871 (0.720-0.994), and 0.858 (0.740-0.976) for the training, validation, and test cohorts, respectively. The calibration plots showed a high agreement between the predicted and observed outcomes. Decision curve analysis indicated that VTE patients could benefit from the clinical application of the prognostic model. Patients were further classified into low-risk, intermediate-risk, and high-risk categories while considering the observed death distribution of the total cohort by tertiles of 2-year outcome probability according to the BRIDGE score. Significant differences in Kaplan–Meier estimations of overall survival were obtained. A BRIDGE risk score calculator for predicting the study result is available online (47.94.162.105:8080/bridge/).

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Summary/Conclusion: We explored the BRIDGE risk score as a machine-learning-based tool that combines clinical and laboratory risk factors to precisely predict the risk for all-cause death in VTE patients after allo-HSCT. Identifying VTE patients who are at a high risk of death can help physicians treat them in advance, which will improve patient survival and prognosis.

P1393: HIGHER DOSE OF CD34+ CELLS PROMOTES EARLY RECONSTITUTION OF NATURAL KILLER CELLS AND IS ASSOCIATED WITH BETTER OUTCOMES AFTER UNMANIPULATED HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR MYELOID MALIGNAN

F. Zhao 1,* , E. Jiang 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China, TianJin, China

Background: Natural killer (NK) cells are potent effectors for eliminating myeloid leukemia and this effect is particularly robust after T-cell-deplete allogenic hematopoietic stem cell transplantation (allo-HSCT). The significance of NK cells after unmanipulated transplantation is less clear and factors affecting early NK reconstitution remain elusive.

Aims: To evaluate early NK reconstitution and its effects on HSCT outcomes and related factors.

Methods: This study retrospectively analyzed a cohort of 180 allo-HSCT recipients who received unmanipulated grafts between June 2012 and May 2020. All patients have acute myeloid leukemia or myelodysplastic syndrome, and most of the patients and donors (99%) are serologically positive for cytomegalovirus (CMV). NK counts in the peripheral blood at 30 days after allo-HSCT (NK30) are strongly associated with all-cause mortality and a cut-off value at 90 cell/µl is determined for further prognostic analysis.

Results: Higher NK30 is associated with reduced disease relapse (HR 0.56, P = 0.02), lower CMV reactivation (HR 0.41, P = 0.001) and better survival (HR 0.43, P = 0.01). Further analysis finds that NK30 is associated with the dose of CD34 + cells ( r = 0.739, P < 0.001) and not the numbers of mature NK cells in the graft. In flow cytometric analysis of cryopreserved day +30 bone marrow samples, patients in continuous complete remission ( N = 6) demonstrate higher frequencies of CD34 + CD7 + progenitor cells and CD56 bright NK cells as compared to patients with disease relapse within 1 year ( N = 6). No suppressive effect of T cells on NK recovery is found since the majority of patients (131/180, 72.8%) received rabbit anti-thymocyte immunoglobulin (ATG) in the conditioning regimen.

Summary/Conclusion: Higher dose of CD34 + cells promotes early recovery of NK cells after allo-HSCT with unmanipulated graft and NK30 represents a valuable prognostic marker of better outcomes. Our data thus provide new information for the therapeutic manipulation of NK cells for the promotion of transplant outcome.

P1394: DIFFERENT DOSES OF ANTITHYMOCYTE GLOBULIN (ATG) AS PART OF THE PREPARATIVE REGIMEN IN HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT)

S. Zhou 1 , M. xiong 1,* , G. wang 2 , W. ling 2 , J. zhu 2

1 BeiJing Ludaopei Hospital, BeiJing; 2 Hebei yanda Ludaopei Hospital, hebei, China

Background: Antithymocyte Globulin (ATG) are often used as a component of the preparative regimen in allogeneic HSCT to reduce the incidence and severity of GVHD. However, the appropriate dose of ATG is yet unclear.

Aims: The aim of the present study is to report comparing outcomes of patients who receiving allo-HSCT with different doses of ATG in their conditioning regimen.

Methods: We retrospectively analyzed the clinical characteristics of 237 patients with AML or B-ALL who underwent allo-HSCT in our hospital from May 2013 to August 2019. The median age was 21.63 years (range: 1-69 years). The median disease course from diagnosis to transplant was 28 months (range: 13-42 months). 81 patients with acute leukemia in first complete remission were considered to have low-risk disease. All other 156 (65.8%) patients were placed in the high-risk. Thirty nine(16.5%) patients underwent a matched unrelated donor (MUD) HSCT. Others underwent a haplo-HSCT. The conditioning regimen was Ara-C/Bu/cyclophosphamide or fludarabine/ATG for person with AML, Ara-C/TBI/cyclophosphamide or fludarabine /ATG for patients with B-ALL. ALL the patients received different doses of ATG: 61 patients received rabbit ATG (Thymoglobulin, Genzyme) at a total dose of 6 mg/kg and 100 patients received 5mg/kg total dose ATG (Thymoglobulin) and 76 patients received total dose of 16mg/kg ATG(Fresebius S). We compared the aGVHD, cGVHD, relapse, and overall survival (OS) of the three groups.

Results: All patients tolerated the pre-treatment well. The total implantation rate was 100%. The median time of neutrophil engraftment (14 days,14days,13.5days, respectively) and platelet engraftment(13 days, 13days, 12days, respectively) were not significantly different between the three groups.The incidences rates of grade I-II aGVHD in 5mg/kg ATG, 6mg/kg ATG and 16mg/kg ATG-F patients were 39%, 36% and 21%, respectively. The grade III-IV aGVHD rates of three groups patients were 16%, 6.5% and 9.2%, respectively. The incidence rates of cGVHD were 30% in 5mg ATG, 21.3% in 6mg ATG and 32.9% in16mg ATG-F.There was no significant difference in either incidence rates of aGVHD or cGVHD. Futher, there were no statistically significant differences in morbidity of CMV between 5mg ATG Vs 6mg ATG and 6mg ATG Vs 16mg ATG-F(39%Vs36.1%, 36.1%Vs21.1%), but incidence rate of CMV was statistically significant difference in 5mg ATG 39% Vs 16mg ATG-F 21.1%(P=0.011). The cumulative incidence of EBV and BK-virus associated hemorrhagic cystitis between the three groups were no statistically significant.During the median follow-up period of 19.31 months (range: 1-74 months), OS were 66% in 5mg ATG patients,68.9% in 6mg ATG patients and 56.6% in16 mg F-ATG group, respectively. And,the OS were not significantly different between three groups.The relapse rate of 16mg ATG-F patients were higher than 6mg ATG group(8.2%Vs21.1%), and it was statistically significant(P=0.038).But there was no difference with the 5mg ATG group.

Summary/Conclusion: Our data showed that: ATG dose of 6 mg/kg group was associated with a similar proportion of GVHD, Viral infections, relapse, and (OS) as 5 mg/kg ATG.Hower,the incidence rates of relapse in 16mg ATG-F patients higher than 6mg ATG group, but similar with ATG dose of 5mg group. In the future studies, the correct dose and preparation of ATG should be further discussed through multi-factor analysis.

P1395: POSTERIOR REVERSIBLE ENCEPHALOPATHY SYNDROME AFTER PEDIATRIC HEMATOPOIETIC STEM CELL TRANSPLANTATION: SINGLE-CENTER EXPERIENCE

S. Zhumatayev 1,2,* , K. Yalcin 3 , S. Celen 3 , N. Azizova Gurbanzade 2 , M. Mazen Jazazi 2 , G. Karasu 2,4 , V. Uygun 4 , M. A. Yesilipek 2,4

1 Pediatric Hematology and Oncology, Nazarbayev University, University Medical Center, Nursultan, Kazakhstan; 2 Pediatric Hematology and Oncology, Medical Park Goztepe Hospital; 3 Pediatric Hematology and Oncology, Bahcesehir University, Istanbul; 4 Pediatric Hematology and Oncology, Medical Park Antalya Hospital, Antalya, Turkey

Background: Posterior reversible encephalopathy syndrome (PRES) is a reversible clinical-neuroradiological complication seen after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Aims: This study aims to predict the risk factors that cause PRES in pediatric patients undergoing allo-HSCT and to evaluate the management of PRES.

Methods: Forty-four patients were diagnosed with PRES out of 641 patients who underwent hematopoietic stem cell transplantation in Göztepe Medical Park Hospital Pediatric Bone Marrow Transplantation Unit, between April 2014 and November 2020, were evaluated retrospectively.

Results: Characteristics of transplantation were presented in Table-1. The median PRES day of the patients was found to be 40 (1-363 days), and PRES occurred in the first 100 days in 84.4% of the cases.Thirty-three patients (75%) were using steroids at the time of PRES diagnosis, and concomitant hypertension was detected in 29 of these patients. Forty-three patients were using calcineurin inhibitors (CNI) as immunosuppressive therapy at the time of PRES. The median plasma cyclosporine (CSA) level measured closest to the time of PRES was detected 191 (65-650) ng/ml, and the median tacrolimus level was 15 (2.6-30) ng/ml. Antiepileptic, antihypertensive, and supportive therapy were initiated just after PRES. Thirteen patients (28.8%) were followed up in the intensive care unit. CNI was discontinued in 40 patients and instead of CNI, sirolimus was started in 8 patients, mycophenolate mofetil (MMF) in 22 patients, and ruxolitinib in 4 patients. In 2 of the 3 patients whose CNI was not discontinued, another type of CNI was used and the seizure did not recur. In one patient, the preparation was interrupted because the blood level was excessively high, and when the blood level was restored, the same preparation was continued, and seizures did not recur. Twenty-three patients (52.2%) had graft versus host disease (GvHD) at the time of PRES. Twelve patients (27.2%) who had no sign of GvHD at the time of PRES developed GvHD in the follow-up after the treatment alteration, and 5 of these patients died due to severe GvHD during the follow-up. When CNI treatments were discontinued in 7 patients with low-grade GvHD at the time of PRES, their GvHD stages increased and 4 patients died due to severe GVHD. Three patients who were followed up with severe GvHD during PRES died due to complications of GvHD.

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Summary/Conclusion: As a result of this study, it is seen that the concomitant use of steroids with calcineurin inhibitors may increase the possibility of PRES. Therefore, the possibility of PRES development should be considered in cases where steroid addition is considered for the treatment of GvHD. The median level of calcineurin inhibitors measured during PRES is within the upper limits of normal and PRES findings can be seen even at low grades, suggesting that PRES may develop independently of the drug level. In addition, it has been observed that discontinuation of CNI in patients who develop PRES may lead to deterioration in GvHD findings and increase mortality. Therefore, in patients with high-grade GvHD, reintroduction of calcineurin inhibitors may be considered after PRES has been stabilized.

P1396: EFFICACY AND SAFETY COMPARISON OF CALCINEURIN INHIBITORS USED FOR GVHD PROPHYLAXIS IN HEMATOPOIETIC STEM CELL TRANSPLANTATION IN CHILDREN WITH THALASSEMIA

S. Zhumatayev 1,2,* , K. Yalcin 3 , S. Celen 3 , N. Azizova Gurbanzade 2 , M. Mazen Jazazi 2 , G. Tezcan 2,4 , V. Uygun 4 , M. A. Yesilipek 2,4

1 Pediatric Hematology and Oncology, Nazarbayev University, University Medical Center, Nursultan, Kazakhstan; 2 Pediatric Hematology and Oncology, Medical Park Goztepe Hospital; 3 Pediatric Hematology and Oncology, Bahcesehir University, Istanbul; 4 Pediatric Hematology and Oncology, Medical Park Antalya Hospital, Antalya, Turkey

Background: For the prophylaxis of graft versus host disease (GVHD), the calcineurin inhibitors are the backbone of the therapy and cyclosporine is preferred more frequently than tacrolimus in pediatric hematopoietic stem cell transplantation. However, although the use of tacrolimus has increased recently, the number of studies comparing these two drugs in the pediatric patient group is limited.

Aims: In this study, the efficacy and safety of the cyclosporine and tacrolimus were compared in pediatric patients transplanted for thalassemia.

Methods: In this study, 129 pediatric patients who underwent hematopoietic stem cell transplantation with the diagnosis of thalassemia in our center between January 2017 and December 2020 were divided into two groups according to the type of calcineurin inhibitor used in GVHD prophylaxis. In the prophylaxis of GVHD, the patients in the first group used cyclosporine (n=68), the patients in the second group used tacrolimus (n=61), and the effects of the groups on the transplantation process were compared retrospectively. In all patients, methotrexate and anti-thymocyte globulin (ATG) were used in addition to cyclosporine or tacrolimus for GVHD prophylaxis. A myeloablative regimen was used in all transplants. Patients who were followed up for at least 7 months after transplantation were evaluated in terms of transplantation related complications and possible calcineurin inhibitor related side effects.

Results: Median age of patients was 81 months (10-263 months), female/male ratio was 1.1 (69/60), donor types MUD 72 patients (56%), MSD 50 patients (39%), and MFD 7 patients (5%) was detected. Bone marrow in 65 (50%) patients, peripheral stem cells in 53 (41%) and bone marrow-cord blood combinations in 11 (9%) patients were used as stem cell source. There was no statistically significant difference between the two groups in terms of age, gender, donor type and stem cell source. The median follow-up period of all patients was 31 months (7-54 months).

Despite the use of GVHD prophylaxis, it was determined that at least grade 2 acute GVHD developed in 29 patients. Of these patients, 12 had only gut, 10 had only skin, 6 had combined gut and skin, and one had only liver GVHD. There was no significant difference between the groups in terms of acute GVHD development, GVHD stage and involvement sites. During the acute GVHD follow up period, only 3 of all patients developed chronic GVHD, and all of these patients were in the cyclosporine group. There was no significant difference between the groups in terms of engraftment syndrome, veno-occlusive disease, CMV reactivation, PRES and graft rejection.

Neurotoxicity in 12 (cyclosporine n=7, tacrolimus n=5) and nephrotoxicity in 18 (cyclosporine n=4, tacrolimus n=14) patients were observed as possible side effects associated with the use of calcineurin inhibitors. While there was no difference between the two groups in terms of neurotoxicity, more nephrotoxicity developed in patients using tacrolimus (p=0.013).

Summary/Conclusion: There was no difference in efficacy between the use of cyclosporine or tacrolimus for GVHD prophylaxis in pediatric stem cell transplantation for thalassemia. In terms of side effects, more nephrotoxicity was observed in patients receiving tacrolimus. In the selection of calcineurin inhibitor, this difference should be taken into account according to the clinical condition of the patient.

P1397: CHRONICALLY REDUCED LEVELS OF THROMBOPOIETIN IMPAIR HEMATOPOIETIC STEM CELL FUNCTION AND MEGAKARYOCYTE BONE MARROW NICHE

A. Aprile 1,* , M. Storto 1 , A. Malara 2,3 , A. Gulino 4 , L. Raggi 1 , S. Sighinolfi 1,5 , S. Beretta 1 , I. Merelli 1,6 , S. Marktel 7 , M. Ponzoni 8 , C. Tripodo 4 , A. Balduini 2,3 , G. Ferrari 1,5

1 San Raffaele-Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan; 2 Department of Molecular Medicine, University of Pavia; 3 Laboratory of Biochemistry, Biotechnology and Advanced Diagnostics, IRCCS San Matteo Foundation, Pavia; 4 Tumor Immunology Unit, Human Pathology Section, Department of Health Sciences, University of Palermo, Palermo; 5 Vita-Salute San Raffaele University, Milan; 6 Institute for Biomedical Technologies, National Research Council, Segrate; 7 Hematology and Bone Marrow Transplantation Unit; 8 Pathology Unit; Unit of Lymphoid Malignancies, IRCCS San Raffaele Scientific Institute, Milan, Italy

Background: In the last decade many studies unraveled the regulation of the bone marrow (BM) niche and hematopoietic stem cells (HSC) by using transgenic knock-out or reporter mice, in steady state or upon acute stimulation. However, HSC-niche interactions are still underexplored in disease condition associated to chronic stress. Beta-thalassemia (BT) is a severe congenital anemia with ineffective erythropoiesis and multi-organ secondary complications and may represent an ideal model to study HSC in a chronically altered BM microenvironment. We recently demonstrated an impaired function of HSC due to the defective crosstalk with stromal BM niche in BT mice ( Aprile et al., Blood 2020 ). In addition to the BM stroma, we found altered levels of multiple local and systemic factors, including reduction of systemic thrombopoietin (TPO).

Aims: Further investigation is pivotal to define the role of chronically reduced stimulation of TPO signaling on HSC and BM microenvironment.

Methods: Gene expression profiling of HSC, megakaryocytes (Mk) and spleen macrophages from Hbb th3/+ ( th3 ) BT mice was assessed by RNAseq analysis. Flow cytometry characterization, in vitro Mk maturation, histological analysis on the BM of BT mice and patients, in vivo platelet (Plt) biogenesis, half-life and phagocytosis were performed. In vivo stimulation of TPO was evaluated.

Results: Since TPO is a key regulator of both HSC and Mk, we investigated the dual role of TPO defect in the disease model of BT. RNAseq profiling revealed a downregulation of TPO signaling and target stemness genes in th3 HSC, including Cdkn1a , Hoxa9 and Hoxb4 , negatively affecting HSC function. The decreased TPO causes a reduced commitment of HSC towards the Mk lineage, with under-expression of Mk-biased genes and lower frequency of CD41 + CD9 high HSC. In vivo stimulation of TPO axis in th3 mice restored the pool of quiescent HSC, thus demonstrating the contribution of defective TPO signaling in altering BT HSC.

Consistently, histopathological analyses of th3 mice showed dysmegakaryopoiesis and this defect was confirmed in BM sections from BT patients. The decreased maturation of th3 Mk, with loss of the mature polyploid profile, correlated with a reduced in vivo Plt biogenesis and impaired in vitro differentiation of th3 Mk. Sorted BT Mk showed the downregulation of niche factors, as Pf4 , Cxcl12 , TnC , relevant for HSC maintenance and reduced expression of extracellular matrix molecules, contributing to the impaired HSC-niche crosstalk.

We explored the origin of TPO defect: TPO levels fluctuate in response to Plt number and its reduced production by hepatocytes in th3 mice is associated to the increased count of Plt. A negative correlation between Plt and TPO was confirmed in BT patients. Consistently, acute Plt depletion in th3 animals is sufficient to restore normal TPO levels, thus excluding an intrinsic defect. In vivo labeling demonstrated a higher half-life of BT Plt, which accumulate in the circulation. Phagocytosis assays proved a reduced Plt clearance by th3 spleen macrophages, whose activity is impaired by iron overload and increased erythrophagocytosis associated to the disease.

Summary/Conclusion: Our results unraveled the dual role of TPO on HSC and Mk BM niche in a disease condition of chronically reduced TPO stimulation. This research elucidates the multiple mechanisms of the BM niche regulation in a model of chronic hematopoietic stress, with a potential relevance in improving HSC transplantation approaches for hematological diseases.

P1398: INSIGHT INTO ACUTE MYELOID LEUKEMIA INDUCED MSC SECRETOME TO REROUTE THE COURSE OF LEUKEMIA BONE MARROW NICHE

G. Borella 1,* , A. Da Ros 1 , G. Borile 2 , G. Longo 1 , E. Campodoni 3 , M. Montanari 3 , M. Benetton 1 , M. Sandri 3 , C. Tregnago 1 , F. Locatelli 4 , M. Pigazzi 1

1 Department of Women’s and Children’s Health, University of Padova; 2 Onco-Hematology, Stem Cell Transplant and Gene Therapy, Istituto di Ricerca Pediatrica Foundation–Citta della Speranza, Padova; 3 Institute of Science and Technology for Ceramics, National Research Council (ISTEC-CNR), Faenza; 4 Department of Pediatric Hematology and Oncology, IRCCS Bambino Gesu Children’s Hospital, Sapienza University of Rome, Rome, Italy

Background: Bone marrow (BM) niche sustains hematopoiesis and hematopoietic stem cells (HSCs) homeostasis, but at Acute Myeloid Leukemia (AML) onset it undergoes to drastic remodeling to support the disease, thus impeding HSCs functions. To date, the underlying mechanisms and interactions within BM niche are not clear, but several line of evidences suggest that mesenchymal stromal cells (MSCs)-derived soluble factors play a key role in suppressing HSCs activity strongly contributing to the emergence of an AML-infiltrated BM niche thus increasing treatment complications and mortality rates.

Aims: We aim to characterize the secretome profile of MSCs derived from BM of AML patients at diagnosis (AML-MSCs) with respect to MSCs derived from healthy donors (h-MSCs), and h-MSCs when co-cultured with AML blasts (induced, iAML-MSCs) in a predictive three-dimensional (3D) AML-niche model, including the mechanism whereby normal hematopoiesis is impaired and leukemia advantaged.

Methods: Gene expression of AML-MSCs and iAML-MSCs showed an enrichment of upregulated genes related to inflammation and tumor-associated soluble factors with respect to h-MSCs. We explored AML-, h- and iAML-MSCs secretome in 3D by a SILAC-label proteomic analysis. Then, we dissected the role of 3 candidate secreted proteins by evaluating the effects when exogenously expressed in AML or HSCs co-cultures (CD34 + sorted cells), and explore their role during chemotherapy treatment.

Results: 3D co-cultures of h- or AML-MSCs and AML blasts or HSCs showed that AML-MSCs strongly supported AML cells proliferation (by Ki67), but decreased normal HSCs proliferation (reduction of 32%). Moreover, we observed an increased number of CD34 + colony-forming cells (CFCs) when co-cultured with AML-MSCs with respect to h-MSCs, suggesting the maintenance of more immature hematopoietic cells. Same effects on CD34 + block of differentiation was observed when co-cultures of AML-MSCs and HSCs were separated via a transwell-insert. These latter findings suggested that AML-MSCs secretome exerted a suppressive role on HSCs regulation. We applied Mass-Spectometry analysis of AML blasts and AML- or h-MSCs co-cultured for 21 days in 3D, finding a total of 15 differentially new synthesized secreted factors in the 3D medium of co-cultures with the AML blasts, related to inflammation, angiogenesis and cancer pathways. Through a validation by ELISA assay, we selected PAI-1, CHI3L1 and PTX3 as the 3 top up-secreted factors in the AML niche for further functional studies. Recombinant proteins PAI-1, CHI3L1 and PTX3, used in combination or alone, were shown to reduce CD34 + proliferation (of 30% when used in combination, and 25%, 17% and 10%, respectively when used alone), including the support of a more immature phenotype (CD34+/CD33-) than when cultured alone or with h-MSCs. Interestingly, adding the recombinant proteins to the co-culture of CD34 + with h-MSCs we recovered same features mediated by AML-MSCs in terms of proliferation (reduced of 15%), CFCs (2-fold increase) and CD34 + expression (increased of 17%). However, recombinant proteins did not showed effects on AML blasts neither on proliferation, nor under chemotherapy treatments supporting these factors being fundamental in the control of HSCs.

Summary/Conclusion: We characterized the secretome profile within 3D AML niche identifying PAI-1, CHI3L1, and PTX3 being upregulated. These factors were shown to modulate HSCs differentiation and proliferation capabilities in 3D providing a potential route for novel secretome-targeted strategies to favor the healthy hematopoiesis, and disadvantage AML progression.

P1399: EXTRAMEDULLARY HEMATOPOIESIS IN THE ADULT ADRENAL GLAND AS A MODEL OF THE MINIMAL HEMATOPOIETIC NICHE

A. Alonso Calleja 1,2,* , F. Schyrr 1,2 , S. F. Lopes 2 , S. Gebhard 3 , R. Sarkis 1,2 , A. Oggier 1 , O. Naveiras 2

1 SV, EPFL; 2 DSB, UNIL; 3 Service de Psychiatrie de l’adulte, CHUV, Lausanne, Switzerland

Background: Hematopoietic Stem and Progenitor Cells (HSPCs) reside in the hematopoietic niche, a structure that regulates the balance of cellular quiescence, self-renewal and commitment towards progenitor cells in a demand-adapted manner. The bone marrow (BM) hematopoietic niche is formed by several cellular players, mainly endothelial cells, osteoblasts, adipocytes, and a variety of stromal cells. While the BM niche contains a complex stromal population, evidence exists for simpler, albeit functional, extramedullary hematopoietic (EMH) niches. EMH can be found associated to adipose tissue. An example of adipose-associated EMH is myelolipoma, an adrenal tumor composed of myeloid and adipose tissue. Myelolipoma is frequent in patients with congenital adrenal hyperplasia, an endocrine disorder that associates an increase in ACTH.

Aims: We aimed to produce an EMH animal model that would recapitulate myelolipoma to study a minimalistic, boneless hematopoietic niche.

Methods: We designed a strategy to induce EMH in the adrenal gland. For this, we injected hematopoietic cytokines (Granulocyte colony-stimulating factor (G-CSF)) and pituitary axis hormones (ACTH) as well as androgens (testosterone) in splenectomized C57BL/6J mice and studied the hematopoietic-supportive capacity of the induced adrenal glands.

Results: Here, we show that the adult adrenal gland can be transformed into a hematopoietic supportive environment. Upon hormonal stimulation, the adult adrenal gland can be induced to recruit and host hematopoietic cells, including serially transplantable HSCPs. The adrenal stroma supports HSPC proliferation and function; furthermore, the adrenal stroma contains a CXCL12+ population, reminiscent of BM CXCL12-Abundant Reticular (CAR). These findings are mirrored by the presence of CXCL12+ cells in human myelolipoma samples, a benign adrenal tumor composed of adipose and hematopoietic tissue.

Summary/Conclusion: We present our model as a novel tool to increase our understanding of the physiology of hematopoietic support and to facilitate the development of a boneless niche model.

P1400: BOOSTING MITOCHONDRIAL RECYCLING REJUVENATES HEMATOPOIETIC AND IMMUNE SYSTEMS

Y.-H. Chiang 1,* , M. Girotra 1 , M. Charmoy 2 , H. Carrasco Hope 1 , P. Ginefra 1 , F. Schyrr 3,4 , F. Franco 1 , P.-C. Ho 1 , O. Naveiras 3,4,5 , J. Auwerx 6 , W. Held 2 , N. Vannini 1

1 Department of Oncology, Ludwig Institute for Cancer Research, University of Lausanne; 2 Department of Oncology, University of Lausanne, Epalinges; 3 Laboratory of Regenerative Hematopoiesis, Department of Biomedical Sciences, University of Lausanne; 4 ISREC, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL); 5 Hematology Service, Department of Oncology, Centre Hospitalier Universitaire Vaudois (CHUV); 6 Laboratory of Integrative and Systems Physiology, Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

Background: Hematopoietic stem cells (HSCs) generate all blood lineages and ensure the correct homeostasis between myeloid and lymphoid lineages during the entire life of an organism. However, HSCs decrease their regenerative potential and undergo toward a myeloid-biased fate skewing with age. This phenomenon leads to a reduction of B and T cell compartments and immune dysfunction. Consequently, elderly patients are more susceptible to severe viral and fungal infections. It was reported that HSC aging is associated with defective autophagy and accumulation of damaged mitochondria. And emerging studies showed that dysregulation of mitochondria metabolism correlates closely with hematopoietic aging process. However, the clear causal relationship between mitochondrial metabolism and HSC aging remains unknown.

Aims: We were wondering if metabolic modulator can rejuvenate aged HSCs, rescue loss-of-function of aged HSCs and improve overall immune response.

Methods: We used mito-QC reporter mice and mitochondrial stain to analyze mitochondrial network upon in vitro treatment of metabolic modulator. In vivo long-term blood reconstitution assays and LCMV infection model were used to estimate the effect of metabolic modulator on HSC functionality.

Results: Here we reported that interventions aimed to modulate mitochondria recycling and metabolism can rejuvenate aged HSCs and thus preserving the lymphoid compartments and immune function. In vitro administration of the mitophagy inducer Urolithin A on aged HSCs restore their blood and immune reconstitution capability. Moreover, Urolithin A supplemented in the mouse diet, improves bone marrow functionality, expands the lymphoid compartment and boost immune response against viral infection in old mice. Finally, we discovered that HSCs rejuvenation is associated with mitophagy induction and re-establishment of mitochondria homeostasis and fitness in aged HSCs.

Summary/Conclusion: In summary, we identified mitophagy as potent regulator of aging process and as valuable target for rejuvenating approaches in HSCs.

P1401: UNBIASED, LONGITUDINAL ANALYSIS OF THE INFLAMMATORY RESPONSE OF HSPCS AT THE SINGLE CELL LEVEL RESOLVES CONTROVERSIES REGARDING THE HSPCS STRESS RESPONSE

Y. Demerdash 1,2,* , B. J. Bouman 3 , L. Haghverdi 3 , M. Essers 1,2

1 Inflammatory Stress in Stem Cells, DKFZ; 2 HI-STEM gGmbH, Heidelberg; 3 Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany

Background: Research investigating inflammatory stress responses in the hematopoietic system have mainly focused on one time-point and one defined cell population. Surface markers typically used to isolate hematopoietic stem and progenitor cells (HSPCs) massively change upon inflammation, which questions the reliability of using flow cytometry to study the system upon inflammation.

Aims: Here we will investigate the dynamics of stress responses in the hematopoietic stem and progenitor compartment at the single-cell level, independent of the use of defined surface markers.

Methods: Hence, we performed a time series single-cell sequencing experiment on sorted HSPCs (Lin neg cKit + ), 3, 24 and 72 hours after injection of mice with the pro-inflammatory cytokine IFNa to investigate the acute inflammation response of bone marrow hematopoietic stem and progenitor cells (HSPCs) in time.

Results: Marker independent abundance analysis in our dataset based on the transcriptional profile of the cell clusters highlight both an HSC enrichment as well as a myeloid depletion over time. The dramatic depletion of myeloid progenitors was not restored within 72h post treatment and is possibly linked to impaired myeloid differentiation programs in the HSCs, suggested by decreased expression of myeloid differentiation factors (ex. MPO, Elane and Ctsg). A longitudinal single-cell dataset covering multiple timepoints after treatment poses new challenges for the computational analyses. Current single-cell algorithms studying the progression of cells in time rely on the assumption that proximity of two cell states is a measure for the typical transition time between them. However, in a time series which covers both response and recovery this assumption is violated. Therefore, we introduce a new approach for the identification of pseudotime. Our method allows us to explore the expression of genes in continuous time, rather than looking at the four experimental timepoints only. Using our pseudotime approach, we discover a variety of different gene responses of the HSPCs to the treatment. Additionally, we found that most differentially expressed genes show the most extreme response in the HSCs. Thus, even though the whole system is sensing the treatment, the underneath response is very heterogeneous, with the earliest, strongest response in the HSC clusters themselves.

Summary/Conclusion: In conclusion, using single cell sequencing analysis over time, for the first time we have been able to study the impact effect of IFNa treatment on the whole HSPCs compartment at the single cell level in an unbiased manner. Moreover, adding the dimension of time helped us infer the dynamics of the inflammatory process in the HSPCs compartment. Our study not only provides new insights into the impact of inflammatory stress on the HSPC compartment but also serves as a guideline for the analysis of longitudinal response datasets in the hematopoietic system.

P1402: ALTERATIONS OF HUMAN BONE MARROW NICHE IN CLONAL HEMATOPOIESIS AND AML

Y. Dragieva 1,2,* , M. Rothenberg-Thurley 2,3 , B. Ksienzyk 2,3 , A. Hadzic 2 , A. Paulus 4 , B. Tast 2,3 , M. Subklewe 2,3 , K. Spiekermann 1,2,3 , S. Theurich 2 , K. Metzeler 5 , M. Solovey 6 , F. Ziemann 1,2,3

1 Laboratory for Experimental Leukemia and Lymphoma Research (ELLF); 2 Department of Medicine III, LMU University Hospital; 3 Laboratory for Leukemia Diagnostics (LfL); 4 Department of Orthopedic Surgery, LMU University Hospital, Munich; 5 Department of Hematology and Cell Therapy, Leipzig; 6 Institute of Computational Biology, Helmholtz Zentrum München, Munich, Germany

Background: Acute myeloid leukemia (AML) is a heterogeneous disease, predominantly caused by acquisition of mutations in the hematopoietic stem and progenitor cells. Some of these mutations are acquired in a preleukemic state termed clonal hematopoiesis of indeterminate potential (CHIP). Recent studies show that alterations in the bone marrow niche can support progression and therapy resistance of myeloid neoplasia. To date, it is unclear when these niche alterations occur and how they contribute to disease progression. In our study, we aim to elucidate alterations in the bone marrow niche, in particular in mesenchymal stem cells (MSC), during the evolution from healthy to CHIP and further to AML.

Aims: Primary goal of this project is to examine how hematopoietic and niche cells from different health status (healthy, CHIP and AML) interact with each other and to what extent the presence of clonal hematopoiesis and leukemia have an impact on function and structure of the bone marrow niche.

Methods: We collected primary human bone marrow (BM) and peripheral blood (pB) from hematologically healthy individuals who underwent hip replacement surgery at the LMU University Hospital. BM samples from AML patients are provided by the Laboratory for Leukemia Diagnostics, LMU University Hospital. Individuals are assigned to non-CHIP or CHIP cohorts based on targeted mutation analyses on pB samples. To study transcriptional changes in the bone marrow compartments, non-CHIP, CHIP and AML BM samples were FACS-sorted for 10 hematopoietic and non-hematopoietic cell types, and further processed for bulkRNA sequencing. MSC from donor BM samples were isolated via plastic adherence over 10-14 days of in vitro culture. MSC were characterized regarding their surface marker expression (FACS), differentiation (osteogenic, adipogenic, chondrogenic) potential and metabolic activity (Seahorse XF).

Results: So far, we have included 98 healthy individuals and 64 have undergone targeted sequencing, revealing 19 individuals (30%) with CHIP. While there was no difference between the gender of the non-CHIP and CHIP groups, individuals with CHIP tended to be of older age (median, 64 y non-CHIP vs. 73.5 y CHIP). Most common mutations were DNMT3A (10/19), TET2 (7/19), ASXL1 (2/19), with variant allele frequencies varying between 1.0% and 16.8%, and 1.42 mutations per individual on average. The amount of mutations per individual increased with age. Analysis of MSCs from non-CHIP (n=5) and CHIP (n=5) individuals revealed no difference in surface marker expression over 10 passages. Differentiation assays of MSCs (non-CHIP=4, CHIP= 2) showed no difference in adipogenic and chondrogenic differentiation after 21 days. For osteogenesis, we noticed a trend towards an increased differentiation potential in the MSCs of CHIP individuals. To gain deeper insights into the BM niche we sorted 4 non-hematopoietic and 6 hematopoietic cell populations from non-CHIP (n=3), CHIP (n=4) and AML (n=4) individuals and performed bulkRNA sequencing on each population. So far changes in the composition of the bone marrow niche were only detected in the AML samples.

Summary/Conclusion: Currently, we are analyzing the bulkRNA sequencing data and performing further experiments on cultured MSC to increase group sizes, as well as MSC and HSPC co-cultures in vitro to gain more insight into the bone marrow niche in non-CHIP, CHIP and AML. Additionally, we are continuously recruiting new individuals into our study. A comprehensive update on our cohort, sequencing data and MSC characterization will be shown at the conference.

P1403: ENDOMUCIN IS A MARKER OF QUIESCENT HAEMATOPOIETIC STEM CELLS WITH HIGH LONG-TERM ENGRAFTMENT CAPACITY

S. Engelhard 1,2,* , M. Drilsvik 1,2 , F. G. Rodriguez Gonzalez 1,2 , C. Nerlov 3 , J. Weischenfeldt 1,2 , K. Reckzeh 1,2 , K. Theilgaard-Mönch 1,2,4

1 Biotech Research and Innovation Centre, University of Copenhagen; 2 The Finsen Laboratory, Rigshospitalet/ National University Hospital, University of Copenhagen, Copenhagen, Denmark; 3 MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, United Kingdom; 4 Department of Hematology, Rigshospitalet/ National University Hospital, University of Copenhagen, Copenhagen, Denmark

Background: The purification of haematopoietic stem cells (HSCs) is crucial for studies on normal and leukaemic stem cell biology. We recently identified Endomucin (EMCN) as a novel HSC marker, a transmembrane protein conserved in mouse and human throughout development, but not in lineage-committed haematopoietic progenitor cells. Therefore, EMCN can be potentially used in combination with “classic” HSC markers to refine the immuno-phenotype, thus improving HSC purification for studies in HSC biology.

Aims: Our aim was to characterise EMCN as a novel complementary marker for purification of HSCs and to investigate its biological function in an EMCN knock-out (KO) mouse model.

Methods: Using flow cytometry, we sub-classified HSCs by combining “classic” HSC markers (CD150 + CD48 - LSK) with EMCN into EMCN + HSCs and EMCN - HSCs. We characterised these populations (i) functionally by transplantation and in vitro assays, molecularly by (ii) RNA-sequencing and by (iii) flow cytometry-based cell cycle analysis. We further analysed “classic” HSCs molecularly and functionally in EMCN KO mice.

Results: Gene set enrichment analysis (GSEA) indicated that EMCN + HSCs are more quiescent than EMCN - HSCs, since gene signatures associated with DNA replication were upregulated in the EMCN - HSCs vs EMCN + HSCs. Consistently,EMCN + HSCs demonstrated upregulation of RhoGTPase signatures associated with low migration activity. Accordingly, genes associated with mitochondrial biogenesis were upregulated in EMCN - HSCs as compared to EMCN + HSCs, indicating that these cells are metabolically more active. In addition, EMCN - HSCs demonstrated upregulation of genes regulated by Runx1, a key transcription factor involed in haematopoietic differentiation, highlighting that EMCN - HSCs are primed for proliferation and differentiation. Thus, our data suggest that EMCN expression on HSCs is associated with quiescence and reduced migration potential, which are characteristics of HSCs residing in the stem cell niche.

Flow cytometry analysis showed that EMCN + HSCs were more quiescent as compared to EMCN - HSCs (G 0 : p = 0.001; G 1 : p < 0.0001; S-G 2 M: p = 0.014; n = 10), and single cell cultures demonstrated that EMCN + HSCs initiate their first division in vitro later than EMCN - HSCs (undivided cells after 2 days- EMCN + HSCs: 18.6% vs EMCN - HSCs: 2.6%; p = 0.04, n= 3). Consistent with the more quiescent phenotype of EMCN + HSCs, limiting dilution transplantation experiments showed that EMCN + HSCs contained a higher frequency of long-term repopulating HSCs as compared to EMCN - HSCs (estimated HSC frequency according to limiting dilution analysis: EMCN + 1 in 7, EMCN - 1 in 42; p < 0.001).

Functional and molecular characterisation of HSCs in EMCN KO mice revealed no effect of the loss of EMCN on HSC function during steady-state haematopoiesis or multi-lineage bone marrow reconstitution after two rounds of serial transplantation.

Summary/Conclusion: In conclusion, EMCN can be used in combination with “classic” HSC markers (CD150 + CD48 - LSK) to enrich for quiescent HSCs with high long-term engraftment capacity. However, functionally, EMCN seems to be dispensable for steady-state haematopoiesis in adult mice, and its loss does not affect the long-term engraftment capacity of HSCs in mice.

P1404: HDAC6 REGULATES HUMAN ERYTHROID DIFFERENTIATION THROUGH MODULATION OF JAK2 SIGNALING

P. Vong 1 , K. Messaoudi 1 , N. Jankovsky 1 , A. Caulier 2 , Y. Demont 1 , T. Boyer 1 , J.-P. Marolleau 1 , J. Rochette 1 , H. Houled Haddou 1 , L. Garcon 1,*

1 University Picardie Jules Verne, CHU Amiens, Amiens CEDEX1, France; 2 Boston Children Hospital, Harvard Medical School, Boston, United States of America

Background: Histone desacetylases (HDAC), through regulation of their acetylation level, modify the physico-chemical properties of proteins and modulate their activity, stability, localization and affinity for partners. Among them, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of HDAC6 (HDAC6i) that has been shown to be of value in treating multiple myeloma or lymphoma but was associated with secondary off target effects Among those, anemia is one of the most frequent, arguing for a particular importance of HDAC6 during erythropoiesis. So far, HDAC6 function during human erythropoiesis remains unknown.

Aims: We present here an extensive study of HDAC6 expression and function during human erythropoiesis.

Methods: We used two cell models: CD34 + progenitor cells obtained from apheresis from healthy donor, driven into in vitro erythroid diffenretiation and the leukemic cell line UT7/EPO. HDAC6 inhibition was obtained chemically using ACY-1215 and using an shRNA-based knockdown.

Results: We quantified HDAC6 expression during erythroid differentiation and observed a progressive increase in HDAC6 RNA expression raising from Day 11 to D18. Using indirect immunofluorescence and flow imaging, we observed that HDAC6 expression was mainly cytoplasmic arguing for non-histone cytoplasmic targets. We then cultured CD34 + -cells into in vitro erythroid differentiation in the presence of ACY-1215. We observed that 0.5µM ACY-1215 gave the best result in terms of HDAC6 inhibition, leading to an increased acetylated α-tubulin without affecting histone H3 acetylation level. ACY-1215 exposure on CD34 + -cells driven in vitro towards the erythroid lineage led to a decreased cell count, an increased apoptotic rate and a delayed erythroid differentiation with accumulation of weakly hemoglobinized immature erythroblasts. shRNA-mediated HDAC6 knockdown in the erythroleukemic cell line UT7/EPO also decreased GPA expression at cell surface. In primary cells, the delay in erythroid differentiation was accompanied by drastic changes in the transcriptomic profile of primary cells as shown by RNAseq. We observed 1920 differentially expressed genes including 1134 upregulated and 786 downregulated. Rank-based GSEA analysis showed a (i) significant enrichment of downregulated genes related to bone marrow erythroblast associated with a downregulation of genes involved in heme metabolism, (ii) a significant enrichment of downregulated GATA1 targets and (iii) an enrichment of downregulated genes related to cell cycle. In UT7/EPO and in primary cells, HDAC6i decreased JAK2 phosphorylation in response to EPO as well as STAT5 activation, as assessed using immunoblot and phosphoflow. Using acetylome, we identified 14-3-3ζ, known to interact directly with the JAK2 negative regulator LNK, as a potential HDAC6 target in erythroid cells. We confirmed that 14-3-3ζ was hyperacetylated after ACY-1215 exposure. We analyzed LNK/14-3-3ζ and LNK/JAK2 direct interactions using co-immunoprecipitation assays. ACY-1215 decreased the 14-3-3ζ/LNK interaction while increasing LNK/JAK2 interactions.

Summary/Conclusion: Here, we show that LNK availability to interact with JAK2 in erythroid cells is regulated by HDAC6-dependent 14-3-3ζ acetylation level. Thus, we identified here a new mechanism of HDAC6-dependent control of human erythropoiesis through 14-3-3ζ acetylation level, LNK availability and JAK2 activation in response to EPO.

P1405: ARGININE-DEPENDENT REGULATION OF THE EUKARYOTIC TRANSLATION INITIATION FACTOR (EIF)5A CONTROLS HUMAN HEMATOPOIETIC PROGENITOR FATE TO THE ERYTHROID LINEAGE

P. Gonzalez-Menendez 1,* , I. Phadke 1 , M. Olive 2 , J. Platon 3 , K. McGraw 4 , J. Papoin 5 , H. Yan 6 , N. Udeshi 2 , R. G. Mirmira 7 , A. Narla 8 , S. Carr 2 , M. Sitbon 1 , L. Da Costa 9 , N. Mohandas 6 , L. Blanc 5 , S. Kinet 1 , N. Taylor 1

1 Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique, Montpellier, France; 2 Broad Institute, Boston, United States of America; 3 University of Picardy Jules Verne, Amiens, France; 4 National Cancer Institute, National Institute of Health, Bethesda; 5 Feinstein Institutes for Medical Research; 6 New York Blood Center, New York; 7 University of Chicago, Chicago; 8 Stanford University School of Medicine, Standford, United States of America; 9 Hospital Robert Debre, Reims, France

Background: The production of 200x10 9 red cells per day in the human bone marrow is highly dependent on iron, glucose, fatty acid and amino acid metabolism. Amino acids support multiple aspects of cell metabolism, ranging from precursors of nucleic acids, conversion to glucose and/or lipids, stimulation of the mTOR signaling pathway, production of TCA cycle intermediates, and maintenance of intracellular redox, amongst others. Arginine––a semi-essential dibasic, cationic amino acid––is one of the most versatile molecules, promoting the synthesis of urea, nitric oxide, proline, creatine, agmatine, and polyamines. These diverse properties contribute to arginine’s critical role in a myriad of physiological and pathological processes such as immune function, insulin sensitivity, wound healing, hormone secretion, endothelial function, and cancer proliferation. Notably though, our understanding of the function of arginine metabolism in erythropoiesis is limited.

Aims: This study aimed to investigate the role of SLC7A1/CAT1-mediated uptake of arginine and its intracellular catabolism in the commitment of human hematopoietic stem and progenitor cells (HSPC) to the erythroid lineage as well as in terminal erythroid differentiation. This study further evaluated the mechanisms via which arginine regulates erythropoiesis in both physiological and pathological contexts.

Methods: EPO-induced differentiation of human CD34 + progenitors was evaluated following alterations in arginine transport and metabolism by different pharmacological inhibitors and shRNA-mediated knockdown of identified genes. Polyamine-mediated regulation of eukaryotic translation factor 5A (eIF5A) activity was evaluated by direct hypusination, metabolic assays were performed as indicated, and proteomics profiling was performed by LC-MS/MS. The role of arginine in disordered erythropoiesis was evaluated in human HSPC with shRNA-mediated downregulation of RPS19 and RPL11 , as well as in del(5q)-MDS progenitors and in murine haploinsufficient Rps14 +/- progenitors.

Results: We find that SLC7A1/CAT1-mediated arginine uptake and its catabolism to spermidine control erythroid commitment and differentiation. Spermidine serves as a precursor for hypusine, required for activation of the eIF5A translation factor. Hypusine levels increased during early erythroid differentiation and importantly, attenuated hypusine synthesis skewed the differentiation of EPO-stimulated HSPCs from an erythroid to a myeloid cell fate. LC-MS/MS analyses together with metabolic assays revealed mitochondrial protein translation and function to be critical downstream effectors of hypusinated eIF5A. Finally, within the hypusine network, ribosomal proteins (RPs) are highly enriched. As genetic alterations in RPs drive anemia in Diamond-Blackfan anemia and myelodysplastic syndromes with chromosome 5q deletions, we evaluated eIF5A hypusination in different models of these bone marrow failure syndromes. Abnormal hypusination and attenuated mitochondrial metabolism were hallmarks of progenitors with RP haploinsufficiency, highlighting RP-eIF5A interactions in regulating erythroid differentiation.

Summary/Conclusion: Our data show that HSPC commitment to the erythroid lineage requires SLC7A1/CAT1-mediated arginine uptake––driving polyamine metabolism and hypusination of the eIF5A translation elongation factor. Furthermore, our study reveals a novel link between hypusination and RPs in the regulation of erythropoiesis, identifying aberrant eIF5A activity in ribosomal protein-linked disorders of ineffective erythropoiesis.

P1406: TIMING OF EXPOSURE TO INFECTIOUS MICROBIOTA DETERMINES HSC FITNESS

J. Jayarajan 1,2,3,* , M. Ball 3 , D. Shah 3 , S. Lux 1,3 , F. Grünschläger 2,4,5 , E. Rodriguez-Correa 1,2,3 , F. Fotopoulou 1,2,3 , J. Knoch 3 , A.-M. Mikecin 1,3 , S. Haas 6 , K. Schmidt 4 , S. Rosshart 7 , M. Milsom 1,3

1 Experimental Hematology, German Cancer Reserach Center (DKFZ); 2 Heidelberg University; 3 Experimental Hematology, Heidelberg Institute of Stem Cells and Experimental Medicine (HI-STEM); 4 German Cancer Reserach Center (DKFZ); 5 Heidelberg Institute of Stem Cells and Experimental Medicine (HI-STEM), Heidelberg; 6 Systems Hematology, Stem Cells & Precision Medicine, Max Delbrück Center for Molecular Medicine, Berlin; 7 Translational Microbiome Research, Uniklinik Freiburg, Freiburg, Germany

Background: Chronic infections in mice have been shown to lead to an increased proliferation of the hematopoietic stem and progenitor cell (HSPCs) compartment and a subsequent loss of stem cell function. However, many of these infection-models involve challenging mice with a very high dose of purified pathogen administered via a non-physiologic route, thereby not recapitulating a natural infection/colonization process. Recent studies from Rosshart et al (PMID: 31371577) has led to the generation of so-called “wildling” mice that carry a full spectrum of microbiota/pathogens found in wild mice, but on a full C57BL/6 genetic background.

Aims: To compare the effects of horizontal (co-housing-mediated) versus vertical transmission of wild microbiota and pathogens into C57BL/6 mice with a focus on generation of mature cells from HSPCs.

Methods: SPF C57BL/6 mice were co-housed with wildling mice for a period of 3 weeks. 6 months from the start of co-housing, the bone marrow of co-housed mice and age-matched wildling mice was analyzed using flow cytometry, and HSC function was assessed by competitive bone marrow transplantation assays. Additionally, BrdU labelling experiments were performed to assess HSC proliferation rate. Mice housed under SPF hygiene status were used as controls for all experiments.

Results: 3 weeks of co-housing resulted in a robust transfer of most viral and bacterial pathogens, and parasites such as mites into co-housed mice. These co-housed mice showed sustained elevated peripheral blood counts in response to the transmitted microbiota. However, neither the co-housed mice nor the wildling mice exhibited any changes in HSPC proliferation or numbers in the bone marrow. Competitive bone marrow transplantation using bone marrow from wildling mice that have neonatal microbial colonization, showed no defect in long-term HSC engraftment suggesting uncompromised HSC function. In contrast, bone marrow from adult co-housed mice showed a significant reduction in long-term engraftment suggesting a reduction in HSC function even though no changes in HSC proliferation were perceivable at the time point of analysis.

Summary/Conclusion: Our study has shown that horizontal transmission of microbiota/pathogens to immunologically naïve adult mice leads to a reduction in HSC function similar to that shown by other groups with more severe infection models. However, the lack of reduction in HSC function in wildling mice suggests that neonatal exposure and colonization of pathogens maintains HSC fitness in adults. This suggests there is a context-specific window of opportunity for infection-mediated functional attrition of HSCs. We are currently analysing scRNA-seq data from bone marrow hematopoietic and niche cells to dissect the molecular differences in steady state hematopoiesis of mice subject to horizontal versus vertical transfer of pathogens/microbiota, to better understand why the HSC function might be compromised in co-housed mice but not in wildling mice.

P1407: CDK6 IN HEMATOPOIETIC STEM CELLS: MORE THAN A CELL CYCLE KINASE

I. Mayer 1,* , E. Doma 1 , M. Prchal-Murphy 1 , T. Klampfl 1 , K. Kollmann 1 , V. Sexl 1

1 Department of Biomedical Sciences, Univeristy of Veterinary Medicine, Institute of Pharmacology and Toxicology, Vienna, Austria

Background: Cell cycle kinase CDK6 acts as a transcriptional regulator and links cell cycle progression to differentiation and cellular homeostasis. CDK6 has been implicated in the initiation and progression of lymphoid and myeloid malignancies and regulates the balance of long-term hematopoietic stem cell (LT-HSC) dormancy and activation. CDK6 acts in a kinase-dependent and kinase-independent way to regulate haematopoiesis.

Aims: As CDK4/6 inhibitors have entered the clinics, we aim at understanding and dissecting kinase-dependent and independent-functions of CDK6 in adult HSCs with application of different stress conditions.

Methods: To investigate kinase-independent effects of CDK6 in HSCs, we made use of a mouse model harbouring a kinase inactivated mutant of CDK6 ( Cdk6 KM/KM ). We performed in vivo serial transplantation and homing assays to study HSC re-population capacity. Low-input RNA-Seq of LT-HSCs after the 2 nd serial transplantation round was used to understand transcriptional effects. Stressed haematopoiesis was further induced by chemical treatment with polyI:C.

Results: Cdk6 -/- HSCs are impaired in their ability to re-populate. This was not the case for Cdk6 KM/KM hematopoietic cells evidenced by re-plating assays in vitro and serial transplantation settings in vivo . Cdk6 KM/KM derived LT-HSCs maintain a stem-cell signature and express genes indicative for HSC dormancy at high levels compared to wild type LT-HSCs. Transcriptional analysis pointed to an altered and improved homing of Cdk6 KM/KM LT-HSCs, which was confirmed in homing assays in vivo . This indicates a repressive role of CDK6 in stem cell homing. Upon polyI:C treatment, Cdk6 KM/KM mice reacted with decreased stimulation as more Lin - Sca-1 + c-kit + (LSK) cells were residing in the bone marrow.

Summary/Conclusion: Our data uncovers kinase-dependent and kinase-independent functions of CDK6 for HSC self-renewal and homing. Whereas serial transplantation settings and homing depend on kinase-inactive CDK6, poly-I:C treatment relies on kinase-activity.

P1408: MONOALLELIC AND BIALLELIC GERMLINE MUTATIONS AFFECTING THE TRANSCRIPTION FACTOR HELIOS CAUSE PLEIOTROPIC DEFECTS OF IMMUNITY

D. Mayr 1,* , T. Shahin 1 , M. Shoeb 1 , H. S. Kuehn 2 , B. Hoeger 1 , S. Giuliani 1 , L. Gawriyski 3 , Ö. Yüce Petronczki 1 , J. Hadjadj 4 , S. Köstel Bal 1 , S. Zoghi 1 , M. Haimel 1 , R. Jimenez Heredia 1 , D. Boutboul 5 , M. Triebwasser 6 , F. Rialland-Battisti 7 , S. Tangye 8 , T. Fleisher 2 , N. Rezaei 9 , N. Romberg 6 , S. Latour 5 , M. Varjosalo 3 , F. Halbritter 1 , F. Rieux-Laucat 4 , I. Castanon 1 , S. Rosenzweig 2 , K. Boztug 1

1 St. Anna Children’s Cancer Research Institute, Vienna, Austria; 2 Immunology Service, Department of Laboratory Medicine, National Institutes of Health, Bethesda, United States of America; 3 Institute of Biotechnology, Helsinki Institute of Life Science, Proteomics Unit, University of Helsinki, Helsinki, Finland; 4 Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Institut Imagine, INSERM Unité Mixte de Recherche (UMR) 1163; 5 Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, INSERM UMR 1163, Paris, France; 6 Division of Immunology and Allergy, The Children’s Hospital of Philadelphia and Department of Pediatrics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States of America; 7 Pediatric onco-hematology department, CHU de Nantes, Nantes, France; 8 Garvan Institute of Medical Research, Darlinghurst, Australia; 9 Research Center for Immunodeficiencies, Children’s Medical Center, Tehran, Iran

Background: Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies.

Aims: Although germline mutations in IKZF1 and IKZF3, encoding Ikaros and Aiolos, have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains to be elucidated.

Methods: We performed whole exome sequencing in six patients with immune dysregulation and their family members. We then applied Co-immunoprecipitation, electrophoretic mobility shift assay and immunofluorescence microscopy techniques to assess dimerization and DNA binding capabilities of the mutations. Proteomic analysis by proximity-dependent Biotin Identification (BioID) was used to assess affinity of mutant Helios to its interaction partners, while single-cell RNA sequencing was applied to peripheral blood mononuclear cells (PBMCs) of two patients to study the transcriptional effect of the truncating and biallelic IKZF2 mutations.

Results: We identified six patients with monoallelic germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) and one patient with a biallelic germline IKZF2 mutation (p.I325V). While patients with heterozygous variants were affected by immune dysregulation (systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis), the patient with the homozygous variant suffered from recurrent respiratory tract infections complicated by bronchiectasis. Immunophenotype analysis revealed reduced propensities of NK cells, CD4+ T-cells with an inverted CD4/CD8 ratio, T-follicular helper cells and hypogammaglobulinemia across patients, while the p.I325V patient also had a reduction in relative and absolute B-cells and low relative proportions of Treg, MAIT and iNKT populations. Single-cell RNA sequencing of PBMCs from the patients carrying the monoallelic R291X truncating variant or the biallelic I325V variant revealed upregulation of pro-inflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Our biochemical and proteomic analysis has revealed that only the truncating R291X mutation results in loss of Helios DNA binding and dimerization properties. However, BioID analysis revealed aberrant interaction of 4/6 Helios mutants with core components of the Nucleosome Remodeling histone Deacetylase (NuRD) complex conveying Helios-mediated epigenetic and transcriptional dysregulation.

Summary/Conclusion: Thus, germline mutations in IKZF2 cause a novel syndrome associating immunodeficiency and profound immune dysregulation and disrupt interaction with the NuRD complex.

P1409: MACROPHAGES PROVIDE ESSENTIAL SUPPORT FOR ERYTHROPOIESIS DURING CHRONIC PSYCHOLOGICAL STRESS

S. Momčilović 1,* , A. Bogdanović 2 , M. Milošević 1 , S. Mojsilović 3 , A. Jauković 3 , D. Bugarski 3 , S. Vignjević Petrinović 1

1 Group for Neuroendocrinology, Institute for Medical Research, National Institute of Republic of Serbia, University of Belgrade; 2 Clinika for Hematology, University Clinical Centre of Serbia; 3 Group for Hematology and Stem Cells, Institute for Medical Research, National Institute of Republic of Serbia, University of Belgrade, Belgrade, Serbia

Background: Under steady-state conditions, a population of tissue-resident macrophages within erythroblastic islands is required for erythroid homeostasis. In addition to tissue resident macrophages, newly recruited monocyte-derived macrophages also contribute to erythropoietic niche during stress erythropoiesis. However, recent evidence suggests that the extent of macrophage contribution to stress erythropoiesis is likely to be both tissue- and stress-type dependent. Using a murine model of psychological stress, we have previously shown that chronic stress induces erythropoiesis both in the bone marrow and the spleen, but tissue-specific contribution of macrophages to erythropoiesis during chronic psychological stress remains unknown.

Aims: The purpose of this study is to examine the effects of macrophage depletion on bone marrow and splenic erythropoiesis under chronic stress conditions.

Methods: Adult male BALB/c mice were subjected to 2h daily restraint stress for 7 consecutive days. Clodronate liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to first restraint procedure, as well as newly recruited macrophages every third day for the duration of the experiment. Mice were randomly assigned to following groups: (1) R - restraint group exposed to daily restraint stress; (2) CLOD + R group, received clodronate liposomes (i.p. 200μl /20g), and subjected to daily restraint; (3) CLOD group, treated with clodronate liposomes only; (4) CTRL + R received control liposomes (i.p. 200μl /20g) and subjected to daily restraint; and (5) control, untreated group. In the bone marrow and spleen number of erythroid progenitors burst forming units-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) were determined using colony asays and CD71/Ter119 profiles of erythroid cells were analyzed by flow citometry.

Results: Chronic stress induced a great increase in the number of BFU-E and CFU-E progenitors in the bone marrow and spleen. Macrophage depletion did not significantly alter the number of erythroid progenitors under basal conditions, but completely abolished the effect of repeated stress on BFU-E and CFU-E cells in both the bone marrow and spleen. Flow cytometric analysis of erythroid precursors revealed that macrophage depletion under steady-state conditions substantially reduced the percentage of CD71 + /Ter119 + cells in the bone marrow, while the percentage of CD71 + /Ter119 + cells in the spleen was not significantly altered. Chronic exposure to daily restraint resulted in considerably increased percentage of CD71 + /Ter119 + cells in the bone marrow and spleen, and macrophage depletion fully abrogated the effect of stress on these cells. The treatment with clodronate liposomes under basal conditions increased the percentage of CD71 - /Ter119 + cells in the spleen, while the percentage of these cells in the bone marrow remained almost unchanged. The percentage of CD71 - /Ter119 + cells was significantly increased in the bone marrow and spleen of chronically stressed mice, and the depletion of macrophages has prevented the effect of stress on these cells.

Summary/Conclusion: Under basal conditions, treatment with clodronate liposome induced tissue-specific changes in the percentage of CD71 + /Ter119 + and CD71 - /Ter119 + cells in the bone marrow and spleen. Macrophage depletion completely abolished the stimulatory effect of repeated stress on erythroid progenitors and precursors in both the bone marrow and spleen, pointing towards a critical role for macrophages in erythropoiesis under chronic stress conditions.

P1410: RKER-050, A NOVEL INHIBITOR OF TGF-Β SUPERFAMILY SIGNALING, INDUCED PLATELET PRODUCTION IN HEALTHY MOUSE MEGAKARYOCYTES

B. Moses 1,* , J. LaMora 1 , R. Nathan 1 , F. Fisher 1 , C. Tseng 1 , J. Seehra 1 , J. Lachey 1

1 Keros Therapeutics, Lexington, United States of America

Background: Transforming growth factor-beta (TGF-β) superfamily signaling is vital in regulating hematopoiesis, including normal platelet (PLT) production. Megakaryocytes (MKs) are platelet producing cells that are controlled, in part, by this signaling pathway. Dysfunctional signaling can lead to abnormal PLT production causing thrombocytopenia as well as myeloproliferative diseases. KER-050 is an investigational modified ActRIIA ligand trap designed to inhibit a subset of TGF-β superfamily ligands, including activin A (ActA), activin B, GDF8, and GDF11. In a clinical study in healthy volunteers, KER-050 increased red blood cell and PLT levels. Understanding the mechanism of action of KER-050 in the context of PLT production is important to ascertain its potential clinical benefits.

Aims: To investigate the effects of KER-050 on PLT biology in mice.

Methods: All studies used RKER-050, a research form of KER-050. Murine studies were carried out in 8-14-week-old C57BL/6 mice. For PLT number, % CD41 + (MK marker) composition and ploidy assessment, a single intraperitoneal dose of RKER-050 (10 mg/kg) was given with multiple timepoints post-dose assessed. Ploidy levels of the CD41 + population were analyzed using a DNA stain. For in vitro ploidy assays, bone marrow (BM) from untreated mice was isolated and cultured with vehicle, ActA, RKER-050, or RKER-050+ActA for 6 days and analyzed as described above. For proplatelet formation assays, lin - (lineage negative) cells were isolated from BM of untreated mice and cultured with thrombopoietin and hirudin for 3 days. MKs were enriched using a BSA gradient and then cultured with vehicle, RKER-050, ActA, or RKER-050+ActA for 16-18 hrs and the percentage of MKs producing proplatelets assessed. For gene expression analysis, qPCR was performed on RNA from BM-derived CD41 + cells isolated from untreated mice.

Results: Circulating PLT counts and percent CD41 + BM cells were elevated >2-fold relative to vehicle 12 hrs post-RKER-050 treatment. The rapidity of these changes suggests that RKER-050 may affect terminal stages of platelet production, while the increase in CD41 + cells suggests that RKER-050 may also expand MKs in BM. At 24 hr post-dose, CD41 + cell ploidy was increased, suggesting RKER-050 may promote MK maturation. To more directly assess changes elicited by RKER-050 on the terminal stages of platelet formation, proplatelet formation was assessed. RKER-050 increased the percentage of MKs producing proplatelets, indicative of RKER-050 enhancing these terminal stages. Treatment with ActA reduced proplatelet formation. In contrast, co-treatment of ActA with RKER-050 partially rescued the inhibitory effects of ActA on proplatelet formation, resulting in more mature, proplatelet-forming MKs. Additionally, a qPCR panel was conducted on CD41 + cells isolated from untreated mouse BM to assess the TGF-β superfamily ligands and receptors that may be involved in regulating thrombopoiesis.

Summary/Conclusion: In these preclinical studies, we determined that RKER-050 acts on multiple stages of thrombopoiesis resulting in increased PLTs in mice. Concordant with the increase in PLT output, the percentage and ploidy of PLT progenitor CD41 + cells were also increased. In addition, we demonstrated that the effect of RKER-050 could be, at least in part, through its ActA inhibition. These data support that RKER-050 may be promoting MK maturation and the terminal stages of thrombopoiesis. Overall, these data support that KER-050 has the potential to treat diseases of thrombocytopenia.

P1411: DUAL EPIGENETIC AND GENETIC EDITING OF PRIMARY HUMAN HAEMATOPOIETIC STEM AND PROGENITOR CELLS

E. Saunderson 1,* , H. Encabo 2 , K. Rouault-Pierre 1 , M. Piganeau 2 , D. Bonnet 2 , J. Gribben 1 , G. Ficz 1

1 Centre for Haemato-Oncology, Barts Cancer Institute; 2 Haematopoietic Stem Cell Laboratory, Francis Crick Institute, London, United Kingdom

Background: The epigenome is perturbed in haematological malignancies including localized DNA hypermethylation at the gene promoter CpG islands. The impact of these hypermethylation events on normal cell function is unknown due to a lack of appropriate methods to edit the epigenome in human primary haematopoietic stem and progenitor cells (HSPCs). For example, INK4b (p15) promoter hypermethylation is frequently found in acute myeloid leukaemia (AML), but whether this can contribute to disease causation is not known. It is also unknown whether sporadic age-associated DNA methylation is maintained during HPSC differentiation.

Aims: To address this, we edited the epigenome of human HSPCs using a modified form of CRISPR/dCas9 that targets DNA methylation rather than mutations in cells. Our aim was to target DNA methylation to key cell cycle regulators, p15 ( INK4b ) and p14 ( ARF ), test maintenance of DNA methylation as cells differentiate in vitro and in vivo, and assess the biological impact. We also aimed to multiplex CRISPR/Cas9 editing to generate a TET2 mutation and p15 hypermethylation in the same HSPC.

Methods: We nucleofected human HSPCs from umbilical cord blood with an in vitro transcribed form of CRISPR/dCas9 containing a portion of the DNA methyltransferase enzyme 3A (DNMT3A) and DNMT3L, along with guide RNAs (gRNAs) to target the p15 and p14 promoters. In some experiments we also nucleofected HSPCs using the nuclease form of Cas9 with a gRNA targeting TET2. To assess the level of editing and the biological impact we seeded edited HSPCs in methylcellulose and 14 days later harvested either bulk or individual colonies to analyse DNA methylation and/or genetic editing using targeted sequencing, gene expression using q-PCR and RNA-sequencing, and genome-wide DNA methylation analysis using the Illumina 850K array. We also engrafted the CRISPR/Cas9 edited human cells into NSG mice to check the repopulating capabilities of these cells.

Results: Starting from as few as 50,000 cells using the CRISPR/Cas9 system we demonstrate dual epigenetic and genetic editing in the same CD34+ cell with DNA methylation targeted at p15 and p14 as well as mutating TET2. We find that despite using a transient delivery system, DNA methylation is maintained with some CpGs in myeloid colonies having up to 99% DNA methylation, and this results in inhibition of gene expression. We further show that HSCs with CRISPR targeted hypermethylation at p15 can repopulate the bone marrow of NSG mice and that p15 hypermethylation is maintained in vivo.

Summary/Conclusion: We have demonstrated the feasibility of combining epigenetic and genetic editing in HSPCs, to recapitulate disease-relevant hits using CRISPR/dCas9 to help us study the cellular processes that may underlie myeloid cancer development.

P1412: UNCOUPLING INK4 PROTEINS FROM CDK4/CDK6 IN HEMATOPOIETIC STEM CELLS

A. Schirripa 1,* , M. Prchal-Murphy 1 , R. Grausenburger 1 , K. Kollmann 1 , V. Sexl 1

1 Veterinärmedizinische Universität Wien, Vienna, Austria

Background: Cell-cycle progression is governed by regulatory proteins including cyclin-dependent kinases (CDKs), cyclins and CDK inhibitors. The INK4 family comprises p16 INK4a , p15 INK4b , p18 INK4c and p19 INK4d which are CDK4/6 specific inhibitors. Different tumor types display deletion, mutation, or promoter hypermethylation of the INK4 proteins resulting in enhanced CDK4/6 activity. The INK4a-ARF-INK4b locus encodes for p16 INK4a and p15 INK4b and for the tumor suppressor protein p14 ARF (p19 ARF in the mouse) and is one of the most frequently mutated or epigenetically silenced site in human malignancies. CDK4 R24C /CDK6 R31C double knock-in mice, where R24C and R31C mutations make CDK4/6 fully insensitive to INK4 mediated inhibition, show a shortened survival caused by the onset of a variety of tumors including hematopoietic malignancies. This shows the vital importance of a tight controlled inhibition of CDK4/6 activity in hematopoiesis.

Aims: We here explore the consequences of INK4 inhibitory loss on CDK4/6 on the hematopoietic stem cell (HSC) compartment using CDK4 R24C /CDK6 R31C mice. We propose that INK factors regulate important functions in HSCs by direct binding to CDK4/6. This knowledge is required to understand how to best employ CDK inhibitors in therapeutic settings.

Methods: We analyzed the composition of the hematopoietic compartment of CDK4 R24C /CDK6 R31C double knock-in mice under homeostatic conditions. Long-term self-renewal capacity of CDK4 R24C /CDK6 R31C bone marrow (BM) cells was assessed by serial bone marrow transplantation (BMT). RNAseq was used to investigate the molecular mechanisms underlying the improved self-renewal of CDK4 R24C /CDK6 R31C HSCs. Using the stem/progenitor cell line HPC LSK enabled us to perform ChIP qPCR to understand the involvement of CDK4 R24C /CDK6 R31C for Id1 transcriptional regulation.

Results: Upon inhibition of CDK4/6 binding to INK4 proteins, HSC numbers in the BM of CDK4 R24C /CDK6 R31C mice were reduced. Despite the cell number reduction, CDK4 R24C /CDK6 R31C HSCs displayed an enhanced self-renewal potential during serial BMT. Transcriptome analysis pointed to the involvement of Id1 in enhanced self-renewal. We indeed found Id1 down-regulated in CDK4 R24C /CDK6 R31C HSCs at the 3 rd round of BMT. CDK6 ChIP experiments confirmed that INK4 binding alters binding of CDK6 to the Id1 promoter, which is significantly reduced in CDK6 R31C cells.

Summary/Conclusion: Despite reduced qHSCs/MPP1 content under homeostatic conditions, CDK4 R24C /CDK6 R31C HSCs possess an enhanced self-renewal potential elucidated in serial BMT. We propose that a reduced Id1 expression in qHSCs/MPP1 CDK4 R24C /CDK6 R31C cells explains this phenotype and underlines improved repopulation ability of CDK4 R24C /CDK6 R31C BM cells. Our data indicate that INK4 binding to CDK6 dictates its function in regulating transcription.

P1413: TRANSCRIPTION FACTOR-DRIVEN COORDINATION OF CELL CYCLE EXIT AND SPECIFICATION OF CELL IDENTITY IN VIVO DURING GRANULOCYTIC DIFFERENTIATION - IN MEMORIAM PROF. NIELS BORREGAARD

K. Theilgaard-Mönch 1,2,3,4,* , S. Pundhir 1,2,4,5 , K. Kristian Reckzeh 1,2,4 , J. Su 1,2,4 , M. Tapia 1,2,4 , B. Furtwängler 1,4,6 , J. Jendholm 1,2,4 , J. Schou Jakobsen 1,2,4

1 Biotech Research and Innovation Center, Univ. of Copenhagen; 2 The Finsen Laboratory; 3 Dept. of Hematology, Rigshospitalet, Univ. of Copenhagen; 4 Novo Nordisk Foundation Center for Stem Cell Biology, DanStem; 5 The Bioinformatics Centre, Department of Biology; 6 The Finsen Laboratory, Univ. of Copenhagen, Copenhagen, Denmark

Background: Differentiation of multi-potent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues and organs such as the hematopoietic system. This process requires temporal coupling of cell cycle exit and differentiation and is tightly coordinated by regulatory networks inhibiting cell cycle master regulators concomitant with activation of distinct transcriptional programs defining the identity of tissue-specific cell types.

Aims: The current study investigated the regulatory mechanisms of how tissue-specific transcription factors (i.e. CEBPA and CEBPE) coordinate cell cycle exit, lineage-specification and terminal dofferentiation during in vivo hematopoietic development (i.e. granulocytic differentiation).

Methods: For the current study we implemented a novel hierarchy of fifteen immunophenotypically defined differentiation stages, which refined the granulocytic and monocytic differentiation hierarchies at a hitherto unprecedented resolution. We purified these differentiations stages from CEBPA/CEBPE WT and KO mice and conducted global mRNA-seq and CEBPA/CEBPE ChIP-seq analyses, as well as complementary mechanistic validation experiments.

Results: Our study demonstrates that CEBPA promotes lineage-specification by launching a full enhancer-primed differentiation program and by activation of CEBPE expression. Subsequently, CEBPE takes over and directs sequential promotor-driven cell cycle exit by (i) launching a MYC – MAD switch leading to inhibition of G1/S gene expression, (ii) repression of E2F-meditated G2/M gene expression, and (iii) up-regulation of Cdk1/2/4 inhibitors. After cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program at the enhancer level to generate fully mature granulocytes.

Summary/Conclusion: Our study highlights that tissue-specific transcription factors regulate cell cycle exit at the promotor level and lineage-specification and terminal differentiation at the enhancer level during hematopoietic development. In a broader perspective, our study contributes to the understanding of the temporal regulatory mechanisms of normal tissue development and homeostasis, which might have impact on the development of novel differentiation therapies for patients suffering from malignant and non-malignant diseases with impaired transcriptional differentiation programs.

P1414: MARROW ADIPOCYTE-ENRICHED NICHE HOSTS AND INFLUENCES A FRACTION OF HEMATOPOIETIC PROGENITORS IN ADULT HIP BONE

D. Trivanovic 1,2,* , K. Horas 2,3 , B. Schlierf 1,2 , M. Rudert 2,3 , M. Herrmann 1,2

1 IZKF Research Group Tissue Regeneration in Musculoskeletal Diseases Orthopedic Center for Musculoskeletal Research, University Clinics Wuerzburg; 2 Bernhard-Heine-Center for Locomotion Research; 3 Department of Orthopaedic Surgery, Koenig-Ludwig-Haus, University of Wuerzburg, Wuerzburg, Germany

Background: The hematopoietic process is tightly regulated by the bone marrow (BM) microenvironment. Emerging studies recognized a regulatory role of mouse BM adipose tissue (BMAT) on self-renewal and differentiation of hematopoietic stem cells (HSC) and progenitors, while results on human BMAT are still scarce.

Aims: Although hip arthroplasty-derived BM serves as an available resource for non-malignant hematopoietic cell research, the BMAT component of this BM and its’ impact on hematopoietic progenitors is not yet revealed. Therefore, our aims were to explore the contribution of human BMAT to the maintenance of hematopoietic progenitors.

Methods: BM samples of acetabular bones were obtained from patients undergoing hip arthroplasty (n=13, 65.7±12.5 years, Ethical approval 187/18). Centrifugation was applied to separate the fatty layer containing BMAT from the BM cell suspension. The BM fraction was used for isolation of BM mononuclear (BMC) and expansion of BM mesenchymal stromal cells (BM-MSC). Digestion of BMAT was applied to separate the non-adipocytic cells of BMAT from mature adipocytes, expand them, and to investigate their functions and transcriptional activity by RNA sequencing. Multiparametric flow cytometry was applied to explore surface marker expression. Hematopoietic activity was investigated by short-term colony assays of cells ex vivo and in vitro upon co-culture with BM-MSC or sub-cultured BMAT adipocytes. Results are presented as mean ±SD and statistical significance was estimated by paired tests.

Results: We found that the total colony forming cell number in BMC (0.147±0.068) was significantly higher than in BMAT-associated cells (0.0684±0.062%, p=0.0002, n=11). The strongest reduction within BMAT was observed in colony forming unit (CFU)-macrophages and CFU-granulocyte-erythroid-macrophage/monocyte-megakaryocytes. The content of CD45 + CD34 + cells was higher in BMC (0.901±1.009%) than in BMAT (0.357±0.505%), while interestingly no differences were found in CD33 + cell frequencies. Both, expanded BMAT and sub-cultured BMAT adipocytes showed stronger adipogenic potential than BM-MSC. When compared to BM-MSC, sub-cultured BMAT adipocytes showed weaker potential to support growth of committed hematopoietic progenitors within BMC ex vivo , favorizing the growth of early and late erythroid progenitors at the expense of other hematopoietic cells. Transcriptomic analyses showed that HSC niche factors ( Angptl4, Wnt5b, Il7, Dkk3 ), but also LepR were upregulated in BM-MSC compared to expanded BMAT cells.

Summary/Conclusion: Our findings indicate a yet unrevealed participation of the BMAT niche in compartmentalization of adult hip bone hematopoietic progenitors by hosting a set of hematopoietic cells and potentially, BMAT progenitors in the close proximity of BM adipocytes. Since a behavior of BM populations is dependent on local microenvironmental context and bone region, obtained results might reflect pathologic settings in arthroplasty patients. Further studies have to address the potential coupling of marrow adipogenesis and hematopoietic progenitor maintenance during aging and diseases.

P1415: HIGH AND LOW MYELOID SKEWING IN THE GENERAL POPULATION AND THE ASSOCIATION WITH CLONAL HEMATOPOIESIS

M. van Bergen 1,* , P. Kamphuis 2 , I. van Zeventer 2 , J. Salzbrunn 2 , J. J. Schuringa 2 , B. van der Reijden 1 , A. de Graaf 1 , G. Huls 2 , J. Jansen 1

1 Laboratory medicine, laboratory of hematology, Radboud university medical center, Nijmegen; 2 Hematology, University medical center Groningen, Groningen, Netherlands

Background: The blood cell production represents a balance between myeloid and lymphoid progeny. During aging an increase in the absolute numbers of HSCs is observed and their differentiation potential declines. One of the hallmarks of the aging HSC is a myeloid differentiation bias and decreased lymphopoiesis. A common denominator of aging is the development of somatic mutations within the hematopoietic stem cell compartment. Clonal outgrowth of a subpopulation of these cells is called clonal hematopoiesis (CH) and is measurable in >10% of the elderly over 60 years of age.

Aims: We studied high myeloid skewing and low myeloid skewing in the general, population-based Lifelines cohort (n=167729). As the contribution of CH to high or low myeloid skewing is currently unknown, individuals >60 years were selected with the highest and lowest myeloid percentages, together with age and sex matched controls.

Methods: All individuals above >18 years old with available myeloid and lymphoid peripheral blood counts were selected to study myeloid skewing (n=144676). CH was assessed in individuals above 60 years (n=21727) with the highest myeloid percentage (>99%, n=218) and lowest myeloid percentage (<1%, n=218) and age and sex matched controls (n=436). Targeted error-corrected next-generation sequencing was performed on 27 genes recurrently mutated in myeloid malignancies with a variant allele frequency (VAF) >1%.

Results: High myeloid and low myeloid skewing is present in all age categories, and not restricted to the older population. Overall, CH was not enriched in cases with high myeloid skewing compared to matched controls. In contrast, CH was depleted in cases with low myeloid skewing. A significant enrichment of mutations involved in the spliceosome ( SF3B1, U2AF1 and SRSF2 ) was observed in cases with high myeloid skewing (p=0.02). The VAF of detected mutations did not correspond to the number of myeloid cells for both cohorts. Cases with high myeloid skewing had a significantly worse overall survival compared to controls (HR, 2.6; 95% CI, 1.98-3.41; p =<0.001), and this was a consistent finding in all age categories >30 years old. Overall survival in cases with myeloid skewing above 60 years was significantly decreased in combination with anemia (HR, 2.27; 95% CI, 1.16-4.47; p =0.017), however, no specific mutational pattern was observed in these cases. Low myeloid skewing did not significantly affect survival, but in combination with TET2 mutations (n=15) these cases had a significantly worse overall survival (HR, 3.42; 95% CI, 1.22-9.64; p =0.02). From these, nine individuals with low myeloid skewing and a TET2 mutation carried an additional neutropenia.

Summary/Conclusion: High or low myeloid skewing is a common finding in the general population, and not restricted to individuals >60 years. The presence of CH is not associated with high or low myeloid skewing. Mutations in the spliceosome were enriched in cases with myeloid skewing, possibly representing cases with early oncogenic transformation. Although CH was not enriched in low myeloid skewing cases, mutations in TET2 had a negative impact on survival of these cases. In combination with neutropenia, these cases may be at risk of developing lymphoid malignancies. Overall, mutations in genes involved in myeloid malignancies are less frequently observed in cases with low myeloid skewing compared to matched controls.

P1416: DNA DAMAGE TRIGGERS A P53 INDUCED AGEING RESPONSE IN HAEMATOPOIETIC STEM CELLS

M. Wang 1,2,* , L. Brandt 2 , X. Wang 1,3 , J. Garaycoechea 4 , N. Wilson 1 , B. Göttgens 1 , K. J. Patel 5

1 Wellcome - MRC Cambridge Stem Cell Institute; 2 MRC - Laboratory of Molecular Biology, Cambridge, United Kingdom; 3 School of Public Health, School of Medicine, Shanghai JiaoTong University, Shanghai, China; 4 Hubrecht Institute, Utrecht, Netherlands; 5 MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

Background:

Ageing of haematopoietic stem cells (HSC) has been frequently observed as reduced self-renewal capacity and differentiation bias towards the myeloid and away from lymphoid lineages. However the physiological drivers, and the molecular mechanism of HSC ageing is poorly understood. We have previously discovered that mammalian cells produce toxic levels of aldehydes that are a physiological source of DNA damage to HSCs. Protection from genotoxic aldehydes critically rely on detoxifying enzymes (ALDH2 and ADH5) and the Fanconi anaemia (FA) DNA repair pathway. Children born with genetic deficiencies in ALDH2 and ADH5, or the FA pathway develops bone marrow failure and leukaemia due to attrition of HSCs. Given the ubiquitous nature of aldehydes in our own body, can aldehydes contribute to the ageing of HSCs in haematopoiesis?

Aims:

1. Assess whether genotoxic aldehydes accelerate and recapitulate features of ageing in haematopoiesis.

2. Dissect the molecular mechanism of how aldehyde-DNA damage triggers ageing in haematopoiesis.

Methods:

We utilised the Aldh2 -/- Fancd2 -/- mouse model, where combined deficiency in the aldehyde detoxification enzyme Aldh2, and the FA DNA repair pathway (Fancd2) causes accumulation of aldehyde-induced DNA damage in HSCs. To assess for evidence of aged HSCs, we subjected Aldh2 -/- Fancd2 -/- mice to:

1. Single cell RNA sequencing (scRNAseq) of haematopoietic stem and progenitor cells to characterise for transcriptome features of ageing and to identify differentially regulated pathways that may underpin such changes.

2. Direct quantification of aged HSCs through immunophenotyping and use of genetic reporters in Aldh2 -/- Fancd2 -/- mice.

3. Functional interrogation of the renewal and differentiation capacity of Aldh2 -/- Fancd2 -/- HSCs using single HSC transplantation analysis.

Results:

Analysis of 8 weeks old Aldh2 -/- Fancd2 -/- HSCs revealed features of accelerated ageing, harbouring an aged transcriptome signature similar to 24 months old wild-type HSCs, and myeloid biased haematopoiesis. To investigate the origins of the myeloid biased stem and progenitor cells, we introduced the Vwf-GFP genetic reporter into Aldh2 -/- Fancd2 -/- mice to find enrichment for Vwf+ longterm HSCs previously shown to be biased towards megakaryocytic and myeloid differentiation. We found strong activation the p53 pathway in Aldh2 -/- Fancd2 -/- HSCs, which enabled the definition of an single cell transcriptome atlas of the p53 network in haematopoiesis. Strikingly, genetic deletion of p53 in Aldh2 -/- Fancd2 -/- mice rescued the accelerated ageing and myeloid bias shown by both scRNAseq and transplantation of single HSCs.

Image:

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Summary/Conclusion:

DNA damage from endogenous aldehydes in HSCs activates p53 to trigger accelerated ageing and drives differentiation towards myeloid lineage. This pathway may have evolved to cleanse DNA damaged stem cells and maintain the HSC pool.

P1417: PROPHYLACTIC NAC PROMOTED HEMATOPOIETIC RECONSTITUTION BY IMPROVING ENDOTHELIAL CELLS AFTER HSCT: A PHASE 3, OPEN-LABEL RANDOMIZED TRIAL

Y. Wang 1,* , Y. Kong 1 , H.-Y. Zhao 1 , Y.-Y. Zhang 1 , Y.-Z. Wang 1 , L.-P. Xu 1 , X.-H. Zhang 1 , K.-Y. Liu 1 , X.-J. Huang 1,2

1 Peking University People’s Hospital, Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University; 2 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

Background: Poor graft function (PGF) or prolonged isolated thrombocytopenia (PT), which are characterized by pancytopenia or thrombocytopenia, have become serious complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our previous single-arm trial suggest that N-acetyl-L-cysteine (NAC) prophylaxis reduced PGF or PT after allo-HSCT.

Aims: Investigate the efficacy and tolerability of NAC prophylaxis to reduce PGF or PT after allo-HSCT.

Methods: A phase 3, open-label randomized trial was performed. Based on the percentage of CD34+VEGFR2(CD309)+ endothelial cells (ECs) in bone marrow (BM) detected by flow cytometry at 14d before conditioning, patients aged 15 to 60 years with acute leukemia undergoing haploidentical HSCT were categorized as low-risk(EC≥0.1%) or high-risk(EC<0.1%); patients at high risk were randomly assigned(2:1) to receive NAC prophylaxis or nonprophylaxis. The primary endpoint was PGF and PT incidence at +60d post-HSCT. Secondary endpoints included cumulative incidences of leukemia relapse(CIR), GvHD, nonrelapse mortality (NRM), leukemia-free survival (LFS), overall survival (OS), and adverse events. This clinical trial was registered at www.clinicaltrials.gov as # NCT03967665 .

Results: Between April 18, 2019, and June 24, 2021, 120 patients with BM EC <0.1% were randomly assigned for NAC (Group A, N=80) or nonprophylaxis (Group B, N=40), and 105 patients with EC≥0.1% (Group C) were also analyzed. The +60d incidence of PGF and PT was 7.5% (95% CI, 1.7% to 13.3%) and 22.5% (95% CI, 9.1% to 35.9%) in Group A and Group B (hazards ratio, 0.317; 95% CI, 0.113 to 0.890; P = .021) and 11.4% (95% CI, 5.2% to 17.6%) in Group C (hazards ratio, 0.643; 95% CI, 0.242 to 1.715; P = .373) (Figure A). Consistently, NAC prophylaxis gradually improved BM ECs and CD34+ cells in Group A, whereas reduced their ROS levels post-HSCT. With a median follow-up of 347 days (range, 60-844days) post-HSCT, the 1-year probabilities relapse, NRM, OS (Figure B), and LFS (Figure C) were similar among the three groups. Within 60days post-HSCT, the most common grade 3 to 5 adverse events for the NAC and control groups were infections (19/80 [24%] vs. 10/40 [25%]) and gastrointestinal adverse events (16/80 [20%] vs. 7/40 [18%]). There were no treatment-related deaths.

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Summary/Conclusion: N-acetyl-L-cysteine prophylaxis can prevent the occurrence of poor hematopoietic function and is well tolerated in haploidentical HSCT. It may offer a potential pathogenesis-oriented therapeutic approach for patients with poor hematopoietic function.

P1418: EXPANDED COMMON MYELOID PROGENITORS ACT IN CONCERT WITH THE SPLEEN AND SUPPORT EARLY HEMATOPOIETIC RECOVERY

T. Yogo 1,* , H. J. Becker 2 , S. Yamazaki 2

1 Division of Stem Cell Biology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo; 2 Laboratory of Stem Cell Therapy, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

Background: Hematopoietic stem cells (HSC) represent the apex of the hematopoietic hierarchy and their engraftment post-transplantation is essential for long-term donor hematopoiesis. After intravenous injection, HSCs are thought to home directly toward the bone marrow niche, and their homing mechanisms and interactions with the microenvironment have been intensively studied. However, the time course of the transplanted HSCs to reconstitute the entire blood system and the spatial transition within the whole body have remained a mystery.

Aims: We aim to elucidate the spatiotemporal dynamics of hematopoiesis in the same individual in a murine HSC transplantation model.

Methods: Current methods for tracking transplanted HSCs in vivo are limited due to their low sensitivity and their restriction to observe only previously known niches. To address these issues, we used the recently published, highly sensitive AkaLuc bioluminescence imaging system to visualize HSC migration in vivo. We combined Akaluc 3D images with CT scans to spatially capture early hematopoiesis after transplantation on a whole-body scale, and continuously monitored bioluminescence signals to clarify the dynamics of cellular distributions over time.

Results: Surprisingly, our data show that the spleen acts as a major site of hematopoiesis and shows unique hematopoietic patterns with dynamic changes. Splenic hematopoiesis was distinct from that occurring in the bone marrow and we found that common myeloid progenitors (CMPs) use the spleen as a site for hematopoiesis, producing many cells early after transplantation. Based on these findings, we developed expanded CMPs (exCMPs) that leverage the spleen environment to improve early hematopoietic recovery after transplantation. exCMPs, which can be produced in large quantities in polyvinyl-alcohol (PVA)-based cultures (Wilkinson et al., Nature 2019), had lower CXCR4 expression, different integrin expression patterns, better cytokine responsiveness and mitogenic potential, as well as higher expression of platelet-related genes compared to fresh CMP. Therefore, exCMPs are more likely to migrate to the spleen and proliferate more rapidly than fresh CMPs. Exploiting these properties, we could show that weekly administration of exCMP allowed for the long-term survival of lethally irradiated mice in a spleen-dependent manner.

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Summary/Conclusion: Our studies of post-transplant hematopoiesis using a next-generation luminescence imaging system showed that the spleen functioned as a major hematopoietic tissue and that CMPs and the spleen cooperated to contribute to early hematopoiesis post-SCT, which led to the concept of utilizing the spleen as an alternative hematopoietic site. These findings refine our understanding of the dynamics of hematopoietic reconstitution, reveal the unique collaboration of the spleen and transplanted cells and establish the therapeutic potential of exCMPs for early hematopoietic recovery.

P1419: APEX1 NUCLEASE AND REDOX FUNCTIONS ARE BOTH ESSENTIAL FOR ADULT HEMATOPOIETIC STEM AND PROGENITOR CELLS.

S. Zaunz 1,* , L. Lauwereins 1 , J. De Smedt 1 , L. Cleuren 1 , M. Bajaj 1 , J. Marteijn 2 , K. De Keersmaecker 3 , C. Verfaillie 1

1 Stem Cell Institute, Department of Development and Regeneration, KU Leuven, Leuven, Belgium; 2 Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands; 3 Laboratory for Disease Mechanisms in Cancer, Department of Oncology, KU Leuven, Leuven, Belgium

Background: Hematopoietic stem (and progenitor) cells (HS(P)Cs) are highly vulnerable to enhanced reactive oxygen species levels, potentially leading to early hematopoietic senescence and/or malignant transformation. Apurinic/apyrimidinic endonuclease/redox factor 1 (APEX1) is a multifunctional protein that is essential for Base Excision Repair (BER), but also for gene transcription regulation, mainly involving the APEX1 redox signaling activity (Ref-1). Although BER is involved in repairing oxidized bases, as well as other spontaneous and frequent single base lesions, the role(s) of the core BER genes, such as Apex1 , in maintaining adult HSPC functionality remain(s) to be elucidated. Similarly, its Ref-1 function, which reduces oxidized transcription factors and thereby regulates transcriptional networks, has not been studied in primary adult hematopoietic cells.

Aims: We aim to study the role of the APEX1, often dysregulated in cancer, and its 2 functionally distinct domains in murine adult HSPCs.

Methods: We assessed the role of the Apex1 gene in murine HSPCs using CRISPR-Cas9 knockout with 2 Apex1 targeting sgRNAs. The effect thereof was evaluated on hematopoietic repopulation in vivo as well as on HSPC expansion during long-term in vitro culture, using a HSPC maintaining medium (Wilkinson et al , 2020).

Next, we delineated the role of the 2 functionally different domains of APEX1 in adult HSPCs, by culturing BM HSPC for 1 week in the continuous presence of different chemicals blocking the nuclease (involved in BER) or the Ref-1 function.

To shed light on the general transcriptional changes caused by APEX1 deficiency, bulk RNA sequencing was performed on progeny of HSPC treated with or without inhibitors. Weighted gene co-expression analysis (WGCNA) was used to identify common and distinct gene clusters modified by different concentrations of the inhibitors.

Results: We showed that full APEX1 KO HSPCs failed to repopulate irradiated recipient mice. Already after 2 weeks, significantly less APEX1 deficient cells were detected in the different blood lineages and nearly no KO cells could be detected from 4 weeks onwards. This was confirmed in vitro , where reduced expansion of APEX1 KO cells was observed.

We demonstrated that both nuclease and Ref-1 APEX1 inhibitors lead to an ex vivo expansion defect of HSPCs, enhanced apoptosis and decreased cell proliferation rates. Bulk RNAseq analysis demonstrated that following nuclease and Ref-1 chemical inhibitions, immune response and interferon signaling were downregulated, whereas oxidative stress response genes were upregulated. Upon nuclease inhibition, genes involved in RNA and DNA metabolism were dysregulated, and integrin and thrombin signaling genes were upregulated. Although no specific module could be linked uniquely to Ref-1 inhibition, the majority of differentially expressed genes were involved in immune response and interferon signaling in contrast to the nuclease inhibition. Currently, we have completed single cell RNA sequencing which should identify the transcriptional networks regulated by the different APEX1 functions in the individual HSPC sub-populations.

Summary/Conclusion: APEX1 and both its nuclease and its Ref-1 functions are essential for functional HSPCs. Moreover, our studies are defining the transcriptional networks regulated by the APEX1 nuclease and Ref-1 functions in primary HSPCs.

P1420: PRE-CLINICAL PROOF OF CONCEPT DEMONSTRATES ROBUST ACTIVITY OF UCART20X22 DUAL CAR T-CELLS FOR THE TREATMENT OF B-CELL MALIGNANCIES

B. Aranda Orgilles 1,* , I. Chion-Sotinel 2 , S. Grudman 1 , B. Mumford 1 , C. Dixon 1 , P. Duchateau 2 , R. Galetto 2 , A. Gouble 2 , L. Poirot 2

1 Cellectis Inc, New York, United States of America; 2 Cellectis SA, Paris, France

Background: Autologous CAR T-cell therapies have been transformative in the treatment of selected blood cancers. Despite this remarkable success, long term studies on patients treated with CD19 or CD22 CAR T-cells revealed the emergence of relapses that can be due to antigen loss. The therapeutic options after CAR T-cell relapses are limited, underscoring the urgent need to develop novel therapies to improve current survival rates. In addition, there is a need to develop allogeneic “off-the-shelf” therapies that are readily available at the time of treatment decision and that overcome development limitations of current autologous approaches.

Aims: To develop efficient dual allogeneic CAR T-cells with the potential to overcome current challenges in the treatment of B-cell malignancies.

Methods: We generated UCART20x22, allogeneic CAR T-cells targeting CD20 and CD22, using a bicistronic lentiviral construct to express both CARs. TALEN® gene editing technology was used to mediate inactivation of the TRAC and CD52 genes: TRAC KO is used to prevent Graft-vs-Host Disease, and CD52 KO to allow deeper host lymphocyte depletion by conferring resistance to lymphodepletion regimens including an anti-CD52 monoclonal antibody, such as alemtuzumab. To test UCART20x22 activity, we use a combination of in vitro cytotoxicity assays against tumor cell lines, along with pre-clinical in vivo models assessing efficient dose/response and recapitulating antigen escape. We also performed in vitro activity assays against primary non-Hodgkin Lymphoma cells expressing different levels of the targeted antigens.

Results: We demonstrate that UCART20x22 displays strong activity against tumor cell lines expressing either a single antigen, CD20 or CD22, or both simultaneously. UCART20x22 also specifically releases IFNg in response to antigen specific stimulation. We also show that the specific activity of UCART20x22 persists over time against tumor cells expressing either one or both antigens. Our preclinical models illustrate that UCART20x22 dual CAR T-cells provide efficient in vivo clearance of tumor cells in a dose dependent manner. UCART20x22 efficiently eradicates tumors expressing both or a single antigen, and we observe persistence of UCART20x22 cells in the bone marrow after tumor clearance. Furthermore, in vitro assays against primary cells from Non-Hodgkin Lymphoma patients with diverse CD22 and CD20 antigen levels demonstrate that UCART20x22 has potent and specific cytotoxic activity as well as IFNg release against these panel of samples.

Summary/Conclusion: We present a robust pre-clinical proof of concept of a potent allogeneic dual CAR T-cell product candidate, UCART20x22, with the potential to 1) overcome common mechanisms of resistance in B-NHL, 2) enable the development of allogeneic CART option for B-NHL patients, 3) reduce the time from treatment decision to infusion.

P1421: BIMODAL TARGETING OF CYTOKINE RECEPTOR-LIKE FACTOR 2 (CRLF2) WITH JAK INHIBITION AND CHIMERIC ANTIGEN RECEPTOR T CELL IMMUNOTHERAPY IN DOWN SYNDROME ACUTE LYMPHOBLASTIC LEUKEMIA

A. Bagashev 1,* , J. Loftus 1 , L. Niswander 1 , S. Ross 2 , C. D. Falkenstein 3 , J. Junco 4 , K. R. Rabin 4 , T. Fry 2 , S. K. Tasian 3

1 Oncology, Children’s Hospital of Philadelphia, Philadelphia; 2 Children’s Hospital Colorado, Denver; 3 Children’s Hospital of Philadelphia, Philadelphia; 4 Texas Children’s Hospital, Houston, United States of America

Background: CRLF2 ( cytokine receptor-like factor 2 ; encoding the thymic stromal lymphopoietin receptor [TSLPR]) rearrangements occur in 50% of children with Down Syndrome-associated B-acute lymphoblastic leukemia (DS-ALL). These genetic alterations induce constitutive JAK/STAT and other kinase signaling that may be targetable by the JAK1/2 inhibitor ruxolitinib. Patients with DS-ALL experience substantial toxicity with conventional chemotherapy and particularly with hematopoietic stem cell transplantation (HSCT). The Children’s Oncology Group AALL1521 clinical trial ( NCT02723994 ) is investigating the potential superior efficacy of ruxolitinib addition to chemotherapy for pediatric patients with CRLF2 -rearranged/JAK pathway-mutant BCR-ABL1-like ALL, but children with DS-ALL are not eligible to participate in this study Successful development of precision medicine approaches that can increase cure, reduce treatment toxicity, and avoid HSCT remain a priority for children with D-ALL.

Aims: We hypothesized that dual CRLF2 targeting with ruxolitinib and cellular immunotherapy could improve anti-leukemia activity against CRLF2 -rearranged (R) DS-ALL.

Methods: We previously reported preclinical activity of TSLPR-redirected chimeric antigen T cells with 4-1BB/CD3z costimulatory domains [TSLPRCART] against CRLF2 -overexpressing ALL cells (Qin Blood 2015). In the present studies, we investigated the effects of TSLPRCART +/- ruxolitinib co-treatment in vitro and in vivo against CRLF2 -R human ALL cell lines and childhood DS-ALL patient-derived xenograft (PDX) models (n=4).

Results: In vitro incubation of CD28/CD3 bead-activated normal human T cells or TSLPRCART with ruxolitinib significantly diminished IL-2 and IFN-ɣ cytokine production, although did not induce overt apoptosis of normal or engineered T cells. As expected, treatment of CRLF2 -R DS-ALL PDX models with TSLPRCART potently inhibited in vivo leukemia proliferation and induced long-term ‘cure’ of animals. However, simultaneous co-treatment of engrafted mice with ruxolitinib and TSLPRCART led to markedly diminished in vivo T cell proliferation and persistence, blunted cytokine production, and facilitated eventual leukemia relapse (representative data in Figure 1). These deleterious effects could be abrogated by 14-day delayed sequencing of ruxolitinib after TSLPRCART activation and expansion and were further fully reversible after ruxolitinib withdrawal, resulting in renewed TSLPCART functionality over time and upon leukemia rechallenge.

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Summary/Conclusion: TSLPRCART is a promising cellular immunotherapeutic approach for DS-ALL and for BCR-ABL1-like ALL given shared features of CRLF2 rearrangement and cell surface protein overexpression. In the present studies, we demonstrate that ruxolitinib co-administration in close proximity impaired in vivo TSLPRCART-induced DS-ALL cell killing but was also beneficial in protection against life-threatening hyperinflammation/cytokine release syndrome (CRS) in dual-treated animals. These results suggest potential for both anti-ALL efficacy and CRS dampening when JAK inhibition and TSLPRCART are properly time-sequenced and may have broader applicability for toxicity mitigation with other cellular immunotherapies. Studies of CAR T cell immunotherapy and other kinase inhibitors in additional CRLF2 -rearranged and non-rearranged DS-ALL models are ongoing.

P1422: IN VIVO G-CSF TREATMENT ACTIVATES THE GR-SOCS1 AXIS TO SUPPRESS IFN-Γ SECRETION BY NATURAL KILLER CELLS

X. Cao 1 , X. Zhao 1,* , T. Peng 2 , Y. hou 2 , R. Li 2 , Z. Fan 1 , M. Zhao 1 , Y. chang 1 , H. chen 2 , C. li 2 , X. Huang 1

1 Peking university of people‘s hospital; 2 peking university, Beijing, China

Background: Natural killer (NK) cells are lymphocytes of the innate immune system that are involved in controlling tumors or microbial infections through the production of interferon gamma (IFN-γ). Granulocyte-colony stimulating factor (G-CSF) inhibits IFN-γ secretion by NK cells, but the mechanism underlying this effect remains unclear.

Aims: To explore the mechanism of G-CSF on NK cells.

Methods: Here, by comparing the multi-omics profiles of human NK cells before and after in vivo G-CSF treatment, we identified a pathway that was activated in response to G-CSF treatment, which suppressed IFN-γ secretion in NK cells. Specifically, our integrative genomic strategy revealed glucocorticoid receptor (GR) activation in NK cells that mediated the genomic response to G-CSF treatment.

Results: Activated GRs can inhibit secretion of IFN-γ by promoting interactions between suppressor of cytokine signaling 1 (SOCS1) promoter and enhancer, as well as increase the expression of SOCS1. Experiments in mice confirmed that G-CSF in vivo treatment significantly down-regulated IFN-γ secretion and up- regulated GR and SOCS1 expression in NK cells. In addition, GR blockade (RU486) significantly

reversed the effects of G-CSF, demonstrating that GR up-regulates SOCS1 and inhibits the production of IFN-γ by NK cells.

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Summary/Conclusion: In this study, based on a HSC transplantation platform, we collected human bone marrow NK cells before and after G-CSF in vivo mobilization. By comparing the multi-omics profiles of NK cells before and after G-CSF treatment, we demonstrate that the glucocorticoid receptor (GR) mediates the genomic response to G-CSF treatment, and the GR- SOCS1 signal axis participates in reducing IFN-γ production by NK cells. Our work provides new potential avenues for therapeutic intervention for inflammatory diseases.

P1423: CIRCULATING CELL-FREE DNA KINETICS MEASURED BY DIGITAL PCR IN LYMPHOMA PATIENTS UNDERGOING CD19-CAR-T THERAPY

M. López-Esteban 1,* , D. Carbonell 1,2 , M. Bastos-Oreiro 1,2 , I. de la Iglesia 1,2 , A. Pérez-Corral 1,2 , M. Chicano 1,2 , P. Muñiz 1,2 , L. Sanz-Villanueva 2 , R. Bailén 1,2 , G. Oarbeascoa 1,2 , J. Anguita 1,2 , M. Kwon 1,2 , J. L. Díez-Martín 1,2,3 , I. Buño 1,2,4,5 , C. Martínez-Laperche 1,2

1 Hospital General Universitario Gregorio Marañón; 2 Instituto de Investigación Sanitaria Gregorio Marañón; 3 Department of Medicine; 4 Department of Cell Biology, Complutense University of Madrid; 5 Genomics Unit, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain

Background: Monitoring of CD19-targeted chimeric antigen receptor (CAR)-T cell levels is essential for an optimal follow-up of patients. In this regard, methodologies such as multiparametric flow cytometry (MFC) or qPCR are commonly performed. In addition, the use of new approaches, such as digital PCR (dPCR), has improved the analysis of cell kinetics. Nevertheless, the role of CAR circulating cell-free DNA (ccfDNA) dynamics during CAR-T cell treatment is not clear.

Aims: To evaluate the usefulness of CAR ccfDNA measurement by dPCR in patients treated with CD19-CAR-T cell therapy and its correlation with CAR-T cell levels measured by MFC and dPCR.

Methods: Forty patients diagnosed with a B-cell lymphoma who underwent CAR-T therapy (28 Axicabtagen Ciloleucel and 12 Tisagenlecleucel) between May 2019 and May 2021 were included in the analysis. One hundred and five peripheral blood (PB) samples were collected in EDTA tubes at days +1, +7, +14, +30 (10, 31, 33, and 31 samples, respectively) after CAR-T administration. Plasma was obtained from 20 mL of PB by centrifugation at 2500g for 30 minutes at 4°C. DNA was purified from PB samples using the Maxwell 16 Blood DNA Purification Kit ( Promega ) and ccfDNA was extracted from 5 mL of plasma, using QIAamp® Circulating Nucleic Acid ( Qiagen ). dPCR was carried out in DNA and ccfDNA samples, using specific primers and probes for FMC63 and a reference gene. MFC was performed on a DxFLEX cytometer ( Beckman Coulter ), using CD19 (20-291) protein-FITC ( ACRO Biosystems ). Continuous variables were expressed as median and range. Correlations between CAR copies in plasma determined by dPCR and CAR-T cells measured in PB by MFC and dPCR were analyzed using Pearson’s test. Comparison between number of copies and clinical variables (cytokine release syndrome -CRS-, immune effector-cell associated neurotoxicity syndrome -ICANS-, and relapse) was analyzed using Mann-Whitney U test. All statistical analyses were performed using GraphPad Prism 8.0.1.

Results: Median values of CAR copies of ccfDNA per milliliter measured by dPCR were 39, 38, 25 and 2 at days +1, +7, +14 and +30. Similarly, median reference gene values were 3079, 1799, 2588 and 2183 (Figure 1). Thus, we validate previous observations of an initial peak concentration followed by a constant decrease in plasma CAR copies kinetics. Moderate correlation was found between CAR copies/mL of plasma and CAR-T cells in PB determined by MFC and dPCR ( R =0.49, p <0.001, Figure 2A; R =0.51, p <0.001, Figure 2B, respectively). So, although a part of plasma CAR copies variation is explained by changes in whole blood CAR content, there might be other influencing factors. Regarding the association between CAR copies in plasma at +7 and clinical variables, an association was only found in the case of development of CRS<2 and CRS grade ≥2 (28 [0-220] vs 168 [11-1479], p <0.01, Figure 3). Further experiments are needed to evaluate whether this reflects the overall CAR-T cell kinetics.

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Summary/Conclusion: The analysis of multiple variables is important for an optimal monitoring of patients undergoing CAR-T cell therapy. In this sense, ccfDNA dynamics may play an important role during the follow-up and even have relevant clinical implications as we have demonstrated in the present study. However, more studies are necessary for validating our data.

P1425: ACTIVATED AND EXPANDED NATURAL KILLER CELLS TRANSDUCED WITH AN NKG2D CHIMERIC ANTIGEN RECEPTOR AGAINST ACUTE MYELOID LEUKEMIA

L. Córdoba 1,* , E. Castellano 1 , A. Valeri 1 , P. Rio 2 , A. García-Ortiz 1 , J. Encinas 1 , E. Maroto 1 , L. Fernández-Casanova 3 , A. Pérez Martínez 4 , D. Lee 5 , D. J. Powell Jr. 6 , A. Leivas 1 , J. Martínez-López 1

1 H12O-CNIO Hematological Malignancies Clinical Research Unit, Hospital 12 de Octubre; 2 Unidad de Innovación Biomédica, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; 3 H12O-CNIO Hematological Malignancies Clinical Research Unit, CNIO; 4 Unidad de Investigación en Onco-hematologia Infantil, Hospital Universitario La Paz, Madrid, Spain; 5 The Research Institute, Nationwide Children’s Hospital, Columbus, OH; 6 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, United States of America

Background: Acute Myeloid Leukemia (AML) is a hematological malignancy incurable for almost all patients. Chimeric Antigen Receptor (CAR) therapy is showing promising results in other hematological disorders but remains challenging in AML since no specific antigens have been described yet. Using NKG2D as a CAR, a natural NK receptor with 8 ligands overexpressed in several tumors, could surmount AML targeting limitations. T cells are considered the gold standard immune effector cells for CAR therapy, but they show toxicities that could be overcome using other cells such as activated and expanded natural killer cells (NKAE), that can be used in an allogeneic context with no GvHD, have natural anti-tumor properties and a short lifespan within the organism. In this project we analyze the in vitro efficacy and security of peripheral blood NKAE cells lentivirally transduced with an NKG2D-41BB-CD3z CAR.

Aims: The primary objective of this project is to assess the efficacy and security of a cell immunotherapy against AML based on the use of NKAE cells transduced with an NKG2D CAR.

Methods: NKAE cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors (HD) and/or AML patients after 7 days of coculture with the irradiated cell line k562-mb21-41BBL by magnetic immunodepletion, when they were transduced with lentiviral vectors carrying NKG2D-41BB-CD3z CAR construct. CAR expression was measured up to 15 days post-transduction by flow cytometry. Cytotoxic activity against AML cell lines was performed by Propidium Iodide and Annexin V staining. Toxicity was evaluated by Europium-TDA assays.

Results: AML can be targeted with an NKG2D CAR since all patients studied expressed at least one NKG2DL (Fig.1). Primary NK cells can be lentivirally transduced with an NKG2D CAR, showing a modest but stable CAR expression up to 15 days after transduction (23% ±4,7% NKG2D+, 33,35% ±4,25% GFP+). CAR-NKAE cells perform robust cytotoxicity towards MOLM-13 AML cell line after 24h of coculture at an effector:target ratio of 1:1, exerting a quasi-total lysis of AML cells (Fig.2A). This represents a significant increase in anti-leukemia effect compared to untransduced NKAE (92,6% ±0,3% vs 72,2% ±10%, p= 0,0138). Surface expression of relevant molecules for NK activity showed that NKp30, NKp44 (natural cytotoxicity receptors), CD69 (early activation marker), CD25 (IL2R alpha chain), FasL (mediates FasL-mediated cytotoxicity), NKp80 (C-type lectin-like surface-activating receptor) and TRAIL (TNF-Related Apoptosis Inducing Ligand) were more expressed by transduced NKAE (Fig.2B). Cytokine release profile revealed a higher production of IL-6, IL-17A, sFasL and IFNg by CAR-NKAE (Fig.2C). We did not observe relevant toxic effects of none of the cells against healthy PBMCs and very low toxicities were found against NL-20 lung cell line, but there were no differences between NKAE and CAR-NKAE.

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Summary/Conclusion: Our preliminary results show that primary NK cells can be lentivirally transduced with a second generation NKG2D CAR, exerting a robust anti-leukemia activity towards AML cell lines. Transduced cells show a more cytotoxic immunophenotype as well as a higher release of cytolytic molecules and proinflammatory and proapoptotic proteins, that could be responsible of the increased anti-tumor response. Hence, NKG2D-CAR transduced NKAE cells could be a suitable approach to treat AML. Other costimulatory domains are being studied in order to assess which one triggers a higher cytotoxic activity, thus allowing decreasing the effector:target ratios and therefore, on-target off-tumor effect.

P1426: WU-NK-101, AN ALLOGENEIC MEMORY NK CELL, FOR THE TREATMENT OF RELAPSE OR REFRACTORY (R/R) ACUTE MYELOID LEUKEMIA (AML)

J. J. Muth 1 , M. E. Mathyer 1 , B. Cruz 1 , C. DuFour 1 , T. Leedom 2 , A. S. Hamil 2 , S. Pouyanfard 2 , M. Berrien-Elliot 3 , L. Luukkonen 2 , M. Tran 2 , B. Tumala 1 , N. Pomper 1 , L. Arthur 1 , M. Foster 4 , J. Gorrochategui 5 , P. Comune Pennacchi 5 , D. Primo 5 , J. Ballesteros 5 , V. Tolstikov 6 , M. A. Kiebish 6 , P. Shah 6 , J. Vadakekolathu 7 , S. Rutella 7 , T. A. Fehniger 3 , R. Sullivan 1 , A. Kabakibi 2 , M. L. Cooper 1 , J. Davidson-Moncada 1,*

1 Wugen, St Louis; 2 Wugen, San Diego; 3 Washington University St Louis, St Louis, United States of America; 4 Washington University St Louis, St Louis, United Kingdom; 5 Vivia Biotech, Madrid, Spain; 6 Berg Health, Framingham, United States of America; 7 Nottingham Trent University, Nottingham, United Kingdom

Background: Memory NK cells have emerged as being safe and effective for the treatment of AML. Memory NK are significantly more potent than conventional NK cells (cNK) with enhanced activity and functional persistence. WU-NK-101 is an ‘off-the-shelf’ Memory NK cell therapy product derived from healthy donor cells, generated using Wugen’s proprietary MONETA™ process and scaled for commercial utility. WU-NK-101 overcomes several limitations associated with NK cell therapy in AML and furthermore displays enhanced trafficking, metabolic fitness and functionality within the TME.

Aims: To characterize WU-NK-101 and evaluate its anti-tumor activity in preclinical models of AML

Methods: WU-NK-101 cells were phenotypically characterized using multidimensional mass cytometry (CyTOF). Functionality was evaluated using in vitro cytotoxicity assays against AML cell lines (TF-1, THP-1, and HL-60). Cells were characterized in conventional and tumor microenvironment (TME)-aligned media. Proteomic analysis of WU-NK-101 was performed using tandem-mass spectrometry. In vivo efficacy was evaluated in NSG mice bearing THP-1 AML tumors.

Results: A recent clinical study of CIML-NK cells in R/R AML ( NCT01898793 ) showed a CR/CRh/CRi rate of 47% with a median DOR of 9.4 months following a single dose. As a scaled-up off-the-shelf product, multiple infusions of WU-NK-101 may be administered to increase the area under the curve, enhance persistence, and improve depth of remission. WU-NK-101 displays elevated expression levels of activating receptors (NKG2D, NKp30, NKp44, DNAM-1, and TRAIL), as well as cytotoxic and functional markers (CD25, CD69, Ki67, and granzyme B), compared with cNK cells. In addition, WU-NK-101 exhibits enhanced in vitro potency against multiple leukemic cell lines versus cNK cells. In culture conditions that mimic the TME, an immunosuppressive, acidic and hypoglycemic environment, cNK cell function was strongly inhibited, while WU-NK-101 maintained its enhanced cytotoxic activity. Furthermore, WU-NK-101 exhibited enhanced glycolytic and mitochondrial oxidative phosphorylation capacity (i.e., metabolic fitness) compared with cNK cells. Proteomic analysis revealed that glucose metabolism pathways were augmented at basal state in conventional media whereas amino acid metabolism was upregulated in hypoglycemic and nutrient depleted TME conditions. These findings suggest that WU-NK-101 cells are highly resistant to immunosuppression and can overcome adverse tumor microenvironments.

In vivo activity of WU-NK-101 was confirmed in an AML THP-1 xenograft model, where a single dose effectively reduced tumor burden relative to vehicle controls. Long-term persistence of WU-NK-101 and anti-THP-1 efficacy could be further enhanced upon repeated dosing and using fluorescently labeled WU-NK-101 cells, homing and penetration into the BM was observed.

Summary/Conclusion: CIML-NK cells have been shown to be clinically safe, tolerable, and effective in patients with AML. WU-NK-101 is an optimized off-the-shelf Memory-like NK cell therapy being developed for AML and will soon be tested in patients. Pre-clinically, WU-NK-101 displayed improved homing to the BM, resistance to an adverse and immunosuppressive TME, and activity against AML cell lines both in vitro and in vivo. Repeated dosing further enhanced efficacy in vivo. The data presented here augur well for R/R AML patients who will be treated in the upcoming clinical study, WUN101-01. Furthermore, the functional characteristics of WU-NK-101 described here support consideration of additional indications including solid tumors.

P1427: MONITORING CAR-T CELL IN B-CELL LYMPHOMA PATIENTS THROUGH SINGLE CHAIN VARIABLE FRAGMENT BY DIGITAL PCR

I. De La Iglesia San Sebastián 1,2,* , D. Carbonell 1,2 , A. Pérez Corral 1,2 , M. Bastos 1,2 , R. Bailén 1,2 , M. Silvia 1 , M. Chicano 1,2 , P. Muñíz 1,2 , G. Oarbeascoa 1,2 , J. Anguita 1,2 , M. Kwon 1,2 , J. L. Díez-Martín 1,2,3 , I. Buño 1,2,4,5 , C. Martínez-Laperche 1,2

1 Department of Hematology, Hospital General Universitario Gregorio Marañón; 2 Instituto de Investigación Sanitaria Gregorio Marañón; 3 Department of Medicine; 4 Department of Cell Biology, Complutense University of Madrid; 5 Genomics Unit, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain

Background: CD19-targeted chimeric antigen receptor (CAR)-T cell is a treatment for B-cell lymphoma patients. CAR-T cell monitoring is important to ensure a correct follow-up, being multiparametric flow cytometry (MFC) the actual gold standard technique. However, other molecular techniques such as digital PCR (dPCR) could add complementary information that could be valuable to predict CAR-T response. Both available commercial antiCD19 CAR-T cell therapies, Tisagenlecleucel (tisa-cel) (Kymriah®) and Axicabtagen Ciloleucel (axi-cel) (Yescarta®) share the single variable fragment domain ( FMC63 ), which makes possible the measurement by dPCR.

Aims: To evaluate the usefulness of digital PCR for monitoring CAR-T cell levels in comparison to multiparametric flow cytometer.

Methods: Forty-five patients diagnosed with diffuse large B-cell lymphoma (36), transformed-follicular lymphoma (7) and mediastinal primary large B-cell lymphoma (2), treated consecutively with anti-CD19 CAR-T cell therapy between June 2019 and May 2021 were included in this study. One hundred and forty-two peripheral blood (PB) samples were collected at days +7, +14, +30, +90 (40, 39, 40 and 23 samples, respectively) after infusion. DNA was purified using the Maxwell® RSC Whole Blood DNA Kit (Promega, USA). dPCR assays were performed on the QIAcuity One platform (QIAgen, Germany). MFC analysis was performed on a DxFLEX cytometer (Beckman Coulter), using CD19 (20-291) protein-FITC (ACRO Biosystems). To assess the sensitivity of molecular methodologies, serial dilutions from 100% to 0.001% of CAR-T were analyzed by dPCR. Correlation of CAR-T cell detection between MFC and dPCR was calculated using Pearson’s test.

Results: A high correlation between dPCR and MFC was found ( r = 0.90), demonstrating the usefulness of dPCR to quantify absolute number of CAR copies (Figure 1A and 1B). dPCR improved the detection capacity of MFC, detecting CAR-T in 15 samples that were negative by MFC because of low CAR-T cell presence in PB (Figure 1C), as MFC measures CAR-T events in total PB the low content of CAR-T could reduce MFC sensitivity.

Median number of CAR-T cells detected by dPCR was greater than that quantified by MFC in each days of the follow-up (Figure 1D). Different CAR-T cell products (Yescarta and Kymriah) differ in its expansion rates, therefore tisa-cel expand before axi-cel (near day 7 th and day 14 th , respectively) (Figure 1C).

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Summary/Conclusion: FMC63 quantification through dPCR was effective for CAR-T measurement, even improving MFC sensitivity, especially when CAR-T counting in PB is low in late monitoring days. dPCR monitoring could complement data given by MFC and being a very sensible gold standard technique in late monitoring as its sensitivity is very superior to MFC in those follow-up days.

P1428: IMPLICATIONS OF THE RUXOLITINIB (JAK1/JAK2 INHIBITOR) ON GRAFT-VERSUS-LEUKAEMIA EFFECT MEDIATED BY THE NK CELL.

C. Durán 1,* , C. Martín Cortázar 1 , C. Ferreras 1 , A. Navarro Zapata 1 , L. Clares Villa 1 , K. Al-akioui 1 , A. Escuredo 2 , A. Pernas 2 , V. Galán 3 , D. Bueno 3 , L. Sisinni 3 , A. Pérez Martínez 1,3

1 Translational Research in Pediatric Oncology, Hematopoietic Transplantation & Cell Therapy Hospital La Paz Institute for Health Research (IdiPAZ) Madrid Spain.; 2 Institute of Medical and Molecular Genetics (INGEMM) La Paz University Hospital, Madrid, Spain.; 3 Paediatric Haematology and Oncology Service La Paz University Hospital, Madrid, Spain., Madrid, Spain

Background: Haematopoietic stem cell transplantation (HSCT) is one of the therapeutic options for high-risk paediatric leukaemia. One of the adverse effects of HSCT is graft-versus-host disease (GvHD). Ruxolitinib is one of the drugs used as GvHD treatment. In this context, alloreactive NK cells have been correlated with a lower relapse rate, decreasing GvHD with maintenance of beneficial graft-versus-leukaemia (GvL) effect.

Aims: We aimed to evaluate the role of Ruxolitinib on the NK cell mediated GvL effect.

Methods: Purified NK cells were obtained from healthy donors. NK cells were incubated for 16h with LPS and CpG in presence or absence of Ruxolitinib (0.1, 1, 10 µM). Their gene expression and production of pro-inflammatory cytokines IL-1β, TNFα, IFNγ and IL-6 were analysed by RT-qPCR and ELISA, respectively. Functional capacity was also assessed using DELFIA Cell Cytotoxicity Assay and CD107a degranulation assay against leukaemic cell lines by flow cytometry. To evaluate proliferative capacity, activated and expanded NK cells (NKAEs) were monitored in presence or absence of Ruxolitinib. Phosphorylation of STAT3 and STAT5 of JAK/STAT pathway was studied by western blot.

Results: Stimulated NK cells treated with increasing doses of Ruxolitinib show decreased mRNA expression of TNFα, IFNγ and IL-6 in a dose-dependent manner. In terms of proinflammatory cytokine release, activated NK cells produce lower levels of pro-inflammatory cytokines (TNFα, IFNγ and IL-6) in the presence Ruxolitinib in a dose-dependent manner. The cytotoxic and degranulation capacity of stimulated NK cells was reduced in the presence of increasing doses of Ruxolitinib but not completely abolished, versus K562, SEM and MV-4-11 cell lines. Treatment with 1 and 10 uM Ruxolitinib impairs NKAES cell proliferation, while at low doses of Ruxolitinib NKAES cells proliferate until day 14. Ruxolitinib inhibits phosphorylation of the JAK/STAT pathway with no effect on Toll-Ligand Receptors signalling pathway (activated by LPS and CpG ligands).

Summary/Conclusion: The JAK1/JAK2 inhibitor Ruxolitinib decreases functional and proliferative capacities of NK cells from healthy donor, inhibiting their GvL effect in vitro.

P1429: MULTIPLEX DELETION OF MYELOID ANTIGENS CD33 AND CLL-1 BY CRISPR/CAS9 IN HUMAN HEMATOPOIETIC STEM CELLS HIGHLIGHTS THE POTENTIAL OF NEXT-GENERATION TRANSPLANTS FOR AML TREATMENT.

J. Xavier-Ferrucio 1 , C. Luo 1 , G. Angelini 1 , S. Krishnamurthy 1 , N. Patel 1 , M. Pettiglio 1 , T. Collingsworth 1 , M. Isik 1 , A. Ghodssi 1 , A. Halfond 1 , J. Etchin 1 , Y. Keschner 1 , G. Mundelboim 1 , P. Tavares 1 , C. Cummins 1 , A. Falla 1 , D. Hazelbaker 1 , E. Paik 1 , J. Sanchez 1 , G. Ge 1 , J. Lydeard 1 , M. I. Lin 1,* , T. Chakraborty 1

1 Vor Biopharma, Cambridge, United States of America

Background: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by abnormal clonal expansion and is the most common form of adult acute leukemia. Though hematopoietic stem cell transplant is the standard of care for high-risk AML patients, relapse post-transplantation occurs in 40% of these patients, highlighting the need for new therapeutic approaches such as immunotherapy.

Flow cytometric profiling of 26 AML patient samples demonstrated that CD33 is expressed on >94% of blasts/LSCs (leukemic stem cells) while CLL-1 is expressed on >85% of blasts/LSCs at density levels targetable by immunotherapies (1283-2260 molecules per blast), suggesting that immuno-targeting both CD33 and CLL-1 can address AML heterogeneity and reduce chances of tumor resistance. Targeting these antigens, however, can lead to cytopenia due to shared expression on normal hematopoietic cells.

Aims: We propose to delete both CD33 and CLL-1 from hematopoietic stem cell grafts, thereby restricting these antigens to AML cells only to enable subsequent immunotherapy without risk of on-target off-tumor toxicities.

Methods: Mobilized peripheral blood CD34 + human hematopoietic stem and progenitor cells (hHSPCs) were gene edited using CRISPR/Cas9 with gRNAs against CD33 and CLL-1, resulting in biallelic deletion of both genes in >80% cells.

Results: The viability of multiplex-edited hHSPCs was >90% with no impact on the distribution of hematopoietic stem cells, multi-potent progenitors, myeloid or lymphoid progenitors. The editing frequencies were maintained in each of these HSPC compartments. Loss of CD33 and CLL-1 proteins did not impact myeloid and erythroid potential or differentiation into granulocytic or monocytic lineages in vitro . Additionally, myeloid cells derived from multiplex-edited hHSPCs also retained their function, demonstrating similar phagocytotic capacity and cytokine secretion compared to unedited control cells. Multiplex-edited hHSPCs xenotransplanted into NOD- scid IL2Rgamma null (NSG) mice showed no defect in long-term engraftment. No impact was observed in lymphoid and myeloid lineage reconstitution (10 hematopoietic lineages analyzed) while CD33 and CLL-1 antigens were simultaneously absent from >95% of cells due to genome editing (CD33 + /CLL-1 + monocytes: 0.9±0.4% for multiplex-edited and 71.5±1.4% unedited arms; CD33 - /CLL-1 - monocytes: 92.2±2.5% for multiplex edited and 1.1±0.3% unedited arms). This data highlights the long-term persistence of high levels of biallelic editing at both genes with no impact to myeloid differentiation in vivo . Quantification of on-target editing by NGS amplicon sequencing revealed no loss of total editing frequencies after engraftment and most importantly, no detectable translocations due to our temporally staggered editing strategies to reduce simultaneous double stranded breaks at both loci. Together, these pre-clinical data indicate that gene modifications in dual engineered cells can persist long-term post-engraftment and that there were no counterselection for these cells. Lastly, CD33 and CLL-1 dual edited cells also showed significant protection from CD33 and/or CLL-1 CAR-T cells.

Summary/Conclusion: Here we show that CD33 and CLL-1 multiplex edited hHSPCs maintain robust hematopoiesis with high levels of editing at both loci. Pairing multiplex-edited hHSPCs with subsequent multi-specific immunotherapy can obviate concerns around tumor heterogeneity and escape mechanisms related to single antigen downregulation, transforming the current treatment approach for AML.

P1430: GUT MICROBIOTA DIVERSITY AND BUTYRATE PRODUCERS IMPACTS ON NON-HODGKIN LYMPHOMA PATIENTS RESPONSE TO CD19 CAR-T THERAPY

R. Garcia-Vicente 1,* , A. Rodriguez-Garcia 1 , R. Gomez-Gordo 2 , A. Ortega-Hernandez 2 , R. Ancos 1 , S. Serrano 1 , J. Modrego 2 , A. Leivas 1 , A. Valeri 1 , A. Jimenez-Ubieto 1 , J. M. Sanchez-Pina 1 , M. L. Paciello 1 , D. Gomez-Garre 2 , J. Martinez-Lopez 1,3 , M. Linares 1,4

1 Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12), Hematological Malignancies Clinical Research Unit H120-CNIO, CIBERONC; 2 Microbiota and Vascular Biology Laboratory, Hospital Clínico San Carlos-Instituto de Investigación Sanitaria San Carlos (IdISSC); 3 Department of Medicine, Medicine School, Universidad Complutense de Madrid; 4 Department of Biochemistry and Molecular Biology, Pharmacy School, Universidad Complutense de Madrid, Madrid, Spain

Background: The gut microbiota is involved in the regulation of normal hematopoiesis and the correct development of the immune system. Particularly butyrate, a short chain fatty acid (SCFA) derived from the microbial fermentation of dietary fibers, affects immune cell proliferation, activation, apoptosis and cytokine expression. Recent studies have focused on understanding the microbiota as a modulator of the initiation and progression of hematological neoplasms. In addition, gut microbiota plays a key role in the efficacy and toxicity of antitumor treatments, especially immunotherapy.

Aims: The aim of this study is to analyse the prognostic role and mechanism of action of the gut microbiota, and specifically of SCFAs, in NHL patients treated with CD19 CAR-T.

Methods: Stool and serum samples were collected from NHL patients at the time of screening prior to CAR-T treatment, categorizing them according to the primary response. Microbial DNA was extracted with the AllPrep PowerFecal DNA/RNA kit and 7 hypervariable regions of the 16S rRNA gene were sequenced using the Ion 16S Metagenomics kit on an Ion S5 System. The sequences were analysed using QIIME2 and the SILVA138 database. PICRUSt2 was used to predict metagenomic functions based on as Kyoto Encyclopaedia of Genes and Genomes (KEGG) ortholog. SCFAs concentration were measured by LC-MS in an LC-ESI-QQQ 8030 (Shimadzu) instrument after acetonitrile precipitation and derivatization. The antitumor potential of SCFAs was evaluated in NHL human (DoHH2) and murine (A20) cell lines through the elaboration of dose-response curves after exposure to 48 hours. Finally, molecular pathways altered were analysed by western blot or qPCR in DoHH2 treated with butyrate 1mM.

Results: Responder patients exhibited higher microbial richness, equity and α-diversity indexes than non-responder´s ones (Fig 1A). The β-diversity analysis determined the existence of a significantly different microbiome between both groups (Fig 1B).

Interestingly, the taxonomic comparison revealed a significantly higher relative abundance of butyrate producer groups as Butyricicoccaceae (p=0.008) , Clostridiaceae (p=0.009) and Oscillopiraceae (p=0.042) families in responders (Fig 1C). Indeed, the metagenomic analysis, which allows inference of the functional profile, revealed that the microbiome of responders is enriched in metabolic pathways related to the fatty acid metabolism, especially butyrate and propionate (Fig 1D). Similarly, serum butyrate levels were higher in responders. (Fig 1E). Recently it has been shown that in vitro pre-treatment of cytotoxic lymphocytes with this metabolite increases the antitumor cellular immunity in the context of adoptive immunotherapy (Luu et al., Nat Commun 2020).

Finally, supplementing this property of butyrate we have also characterized a direct effect on tumour cells. Butyrate and other SCFAs inhibit in vitro lymphoma cell growth in the millimolar range (Fig 1F) by the dysregulation of MAPK pathway, but also by the enhanced expression of p21 (Fig 1G).

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Summary/Conclusion: These preliminary results reveal a higher diversity and a different microbiome profile in the gut microbiota of NHL patients that response to CD19 CAR-T cell therapy. Particularly, the taxonomic analysis, and subsequent validation in patient samples, reveals that butyrate is an important metabolite that may impact in the treatment response. Finally, the antitumor potential of this bioactive SCFAs found could promote their use as adjuvant therapy or chemical modifier of CAR-T.

P1431: MOLECULAR CHARACTERIZATION AND LENTIVIRAL CORRECTION OF IN VITRO ERYTHROPOIEIS IN PATIENTS WITH A NOVEL RECESSIVE CONGENITAL DYSERYTHROPOIETIC ANEMIA TYPE III (CDA TYPE IIIB).

G. Hernández 1,2,* , L. Romero-Cortadellas 1 , X. Ferrer-Cortès 1,2 , V. Venturi 1 , M. Dessy-Rodriguez 3,4 , M. Olivella 5 , A. Husami 6,7 , C. Pérez de Soto 8 , R. M. Morales-Camacho 9 , A. Villegas 10 , F.-A. González-Fernández 10 , M. Morado 11 , T. A. Kalfa 7,12 , O. Quintana-Bustamante 3,4 , S. Pérez-Montero 2 , C. Tornador 2 , J.-C. Segovia 3,4 , M. Sánchez 1,2

1 Department of Basic Sciences, International University of Catalonia, Sant Cugat; 2 BloodGenetics S.L. Diagnostics in Inherited Blood Diseases., Esplugues de Llobregat; 3 Cell Technology Division. Biomedical Innovative Unit. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER); 4 Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez, Madrid; 5 Biosciences Department, Faculty of Sciences and Technology (FCT), Universitat de Vic – Universitat Central de Catalunya (UVic-UCC), Vic, Spain; 6 Division of Human Genetics, Cincinnati Children’s Hospital Medical Center; 7 Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, United States of America; 8 Service of Pediatric Hematology, Hospital Universitario Virgen del Rocío, UGC HH. HHUUVR; 9 Department of Hematology, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS/CISC/CIBERONC). Universidad de Sevilla, Sevilla; 10 Service of Hematology, Hospital Clínico San Carlos. Universidad Complutense; 11 Service of Hematology, Hospital La Paz, Madrid, Spain; 12 Division of Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States of America

Background: Congenital dyserythropoietic anemia type III (CDA III) is one of the rarest types of CDA. The autosomal dominant form of CDA III, originally described as familial erythroid multinuclearity and so far identified in two families, is due to a monoallelic missense mutation in the KIF23 gene. We previously identified three different families affected with a recessive form of CDA III with missense mutations in the RACGAP1 gene, the partner of KIF23 .

Aims: To further characterize the erythropoyesis of these patients and to perform lentiviral transduction of the WT gene to observe the rescue of the phenotype observed in the patients (multinucleation, macrocytosis...).

Methods: We have isolated CD34 + cells from peripheral from two of the patients and performed an in vitro erythroid differentiation. For the characterization of the differentiation process we have used flow cytometry to differentiate the pupulations of differentiating erythroid cells by immunostaining. By cytospin preparations we have studied the multinucleation, enucleation and size of the cells.

Results: Cells from patients showed a marked growth impairment compared with healthy donors. Cells at day 9 and 14 of the differentiation process show a high percentage of multinucleation with more than 2 nuclei, nuclear deformities and mitotic problems. At the final stage of erythroid differentiation, in the patient-derived cultures we observe a decrease in the production of erythrocytes, and the ones that are being produced show a clear macrocytic phenotype. The observed multinucleation and macrocytosis observed in the in vitro differentiation is in line with the observation in the bone marrow and peripheral blood from the patients, indicating that we can successfully recapitulate in vitro the erythropoiesis process from the patients.

Additionally, flow-cytometry assessment of the erythroid differentiation shows a delay in the process where we observe an increased presence of erythroid progenitors (CD71 + only) at later stages and a reduced erythroid (CD235a + only) production. Importantly, lentiviral transduction of the WT RACGAP1 gene in vitro could rescue the defects in erythroblast growth, multinucleation, macrocytosis, and enucleation.

Summary/Conclusion: These results supports that mutations in the RACGAP1 gene are the genetic origin of the CDA IIIb disease.

P1432: THE NOVEL DUAL T/B-CELL SIGNALING MOLECULE CONFERS THE GREATER TUMORICIDAL ACTIVITY AND T-CELL PERSISTENCE OF CD19 CHIMERIC ANTIGEN RECEPTOR T-CELL

W. Khopanlert 1,* , N. Jangphattananont 2 , K. Maneechai 1 , S. Ung 1 , P. Viboonjuntra 2 , S. Terakura 3 , J. Julamanee 2

1 Biomedical Sciences and Biomedical Engineering; 2 Internal Medicine, Prince of Songkla University, Hatyai Songkhla, Thailand; 3 Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan

Background: Chimeric antigen receptor (CAR) T-cell targeting CD19 incorporating either CD28 or 4-1BB co-stimulatory domains have achieved impressive clinical outcomes for adoptive immunotherapy in B-cell malignancies. However, the recurrences after therapies still occur due to the poor CAR-T persistence. Various approaches have been studied to extend the efficacy including signaling domain modification. The nuclear factor-κB (NF-κB) is a critical signaling pathway for CAR-T cell persistence and anti-tumor activity. Interestingly, this downstream signaling cascade exists in both T- and B-cells. Here, we adopted the third generation CD19CAR incorporated dual T- and B-cell costimulatory molecules; CD28 and CD40 (CD19.28.40z), to improve CAR-T cell functions.

Aims: To generate the novel dual T/B-cell signaling domain of CD19CAR (CD19.28.40z CAR) enhancing cytotoxicity and persistence.

Methods: The dual T/B-cell costimulatory domain; CD28/CD40, was fused into the anti-CD19CAR backbone; CD19scFv-CD28TM-CD3z-tEGFR, to generate the third generation CAR. The CAR gene was cloned into retroviral packaging cells and transduced into primary human CD3 cells. The CAR + -T cells were enriched using biotinylated anti-EGFR mAb and further expanded with anti-CD3/CD28 beads or gamma-irradiated EBV-LCL cells. The CAR + -T cells were then used for downstream experiments compared with control CD19CARs including CD19.28z, CD19.BBz, and CD19.40z CAR-T cells.

Results: All CD19CAR genes were successfully transduced into primary CD3 + cells with the transduction efficacy of 40–60% and slightly higher in CD19.28.40z CAR-T cells. We next investigated the downstream T-cell signaling and found the higher baseline pNF-κB and pRelB of CD19.28.40z CAR-T cells compared to the others. The novel CD19.28.40z CAR-T cells exhibited robust T-cell proliferation after stimulation once with gamma-irradiated CD19-expressing K562 cells regardless of IL-2 supplementation. In terms of cytokine secretion, we observed similar level of IL-2, TNF-α, and IFN-γ concentrations among the CAR constructs after stimulated with target cells. To assess the long-term cytotoxicity, we performed co-culture assay and found that CD19.28.40z CAR-T cells effectively eradicated tumor cell growth throughout the end of the experiment at an effector to target cell ratio of 1:16, compared to control CAR T-cells (Figure 1). Notably, CD19.28.40z CAR-T cells sustained greater proliferation after repetitive weekly stimulation without influencing T-cell fate and exhaustion (Figure 2). The preliminary in vivo murine experiment demonstrated that CD19.28.40z CAR-T cells profoundly suppressed the tumor cell growth in Nalm-6-inoculated NSG mice compared to other control T-cells.

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Summary/Conclusion: The novel CD28/CD40 signaling domain enhances CD19CAR-T cell proliferative capacity and persistence which translated into the greater anti-tumor efficacy both in vitro and xenograft model.

P1433: USE OF A FOAMY-VIRUS VECTOR SYSTEM TO PRODUCE AN ‘OFF-THE-SHELF’ ANTI-CD19 CAR T CELL PRODUCT

I. Lazana 1,2,* , E. Simantirakis 1 , M. Daniil 1 , I. Tsironis 1 , G. Vassilopoulos 1,2

1 Cell and Gene therapy, Biomedical Research Foundation of the Academy of Athens, Athens; 2 Department of Hematology, University of Thessaly Medical School, Larissa, Greece

Background: Anti-CD19 chimeric antigen receptor (CAR) T cells have transformed the field of cancer immunotherapy. Current CAR T cell trials use autologous cells, with several limitations (prolonged production time, costly, manufacturing failure), whereas the Lentiviral vectors (LV), used for commercial products, are endowed with extra limitations (packaging limits and mutagenesis risk).

Aims: Our group has developed an in-house anti-CD19 CAR-T cell product, using a safer to LV, foamy virus vector (FV).

Methods: FV vectors expressing the anti-CD19 CAR from an EFP1 promoter were constructed. 2nd generation LV vector backbones were purchased from a commercial vendor. Peripheral blood from healthy individuals were used as T cell sources. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 CAR-T, LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM). FV and LV CAR-T cells were expanded with a modified Rapid Expansion Protocol (REP). Immunophenotypic analysis of CAR T cells was performed by FCM. Their cytotoxic effect was evaluated against the CD19+ Raji and Daudi cell lines and against the CD19- cell line, HL60. Cytotoxicity was assessed by FCM and calculated as: [1-live targets (sample)/live targets (control)]x100. CAR-T cell activation was also assayed by INF-γ ELISA.

Results: LV and FV vector titers were between 3-5x10^5 TU/ml and 4-5x10^5 TU/ml, respectively. Transduction efficiency ranged from 45-83% at MOI 3-5 with FV vectors and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10-20). Following REP, the vast majority of cells consisted of CAR T cells, regardless of the vector used or the T cell source and isolation method. Immunophenotypic analysis of CAR T cells revealed a central memory phenotype (CD45RO+/CD45RA-/CCR7+), (>99% cells). For cytotoxicity assays, CAR-T cells were incubated with CFSE-labelled Raji and/or Daudi cells at different ratios (5:1, 10:1) for 18 hours. At the end of the incubation period, the % cell lysis was 87.3 (SD 6.38) and 92.4 (SD 3.2) at 5:1 and 10:1 ratio, respectively (n=3). Similar results were obtained for LV vectors. When CD19- HL60 cells were used as targets, no/minimal lysis was noted, indicating a specific anti-CD19 cytolytic effect of CAR T cells. IFN-γ levels were measured at the end of the co-culture period in the supernatant to assess CAR T cell activation. Results showed a significant increase both in the FV- and LV- CAR T cells, compared to untransduced cells (mean 890.2 and 3,141pg/ml and 1,553 and 2,225pg/ml for FV- and LV- CAR T cells, in ratios 5:1 and 10:1, respectively). Integration site analysis of FV vectors in the T-cells is under way.

Summary/Conclusion: Our group has developed for the first time a FV vector for anti-CD19 CAR T cell production, with an efficient gene transfer to human T cells and with potent in vitro cytotoxic properties, similar to their LV-derived counterpart. Overall, we provide a proof of concept that allogeneic, in-house CAR T cells derived from an alternative viral backbone such as the FV, could be a safe, efficient and affordable vehicle to LV-derived vectors for immunotherapy.

P1434: CORRECTION OF CONGENITAL NEUTROPENIA-RELATED GENES IN NAÏVE IPS CELLS DERIVED FROM PATIENTS USING VIRAL-FREE CRISPR/CAS9 GENE-EDITING SYSTEM

D. Krzyzanowski 1,* , Z. Rydzynska 1 , B. Pawlik 1 , W. Dobrewa 1 , D. Grzela 1 , W. Mlynarski 1 , J. Madzio 1

1 Department of Pediatric, Oncology and Hematology, Medical University of Lodz, Lodz, Poland

Background: Severe congenital neutropenia (SCN) is a rare haematological condition with a heterogeneous genetic background that often leads to severe impairment of the immune system. For patients who do not respond to standard treatment, the only possible therapy is allogeneic haematopoietic stem cell transplantation. SCN patients have an increased risk of the development of myelodysplastic syndrome or acute myelogenous leukaemia. Rare genetic disorders, such as SCN, require prioritising bone marrow failure diagnosis and therapy tailored to individual patients. Human induced pluripotent stem (iPS) cells are a promising prospect for future cell therapy in a wide range of diseases like congenital neutropenia for which there are currently no cures or effective therapies.

Aims: The aim of this study was to develop potential treatment procedure based on the use of iPS cells generated from epithelial cells of patients with congenital neutropenia. Using mutation-specific gRNA and CRISPR-Cas9 system, we made an attempt to repair patients-derived iPS cells which may have potential for renewal of the corrected granulocyte population in the bone marrow.

Methods: Patient-derived epithelial cells were reprogrammed with the EBV-derived oriP/EBNA-1 system carrying transcription factors. The transition of these cells into the naïve state was accomplished with the use of cell culture media supplemented with Leukaemia inhibitory factor, an inhibitor of Protein kinase C, small molecule activators of WNT, and inhibitors of ERK1/2 and JNK signaling pathways. CRISPR-Cas9 was used to repair mutations in patients-derived naïve iPS cells. Into the target cells, a specific sgRNA combined with eSpCas9 protein and HDR template was introduced via nucleofection with Lonza 4D Nucleofector, as a CRISPR enhancer, brefedrin A was used. To verify the efficiency of transfection and gene edition, the targeted region were analysed with Sanger sequencing (Fig. 1).

Results: We have generated primed and naïve iPS cells from 7 patients with different severity of neutropenic phenotype and genetic background, 4 another samples are pending. The morphology of the iPSCs line (primed cells) displayed a typical ESC-like colony appearance comprised of tightly packed cells. Alkaline phosphatase and immunocytochemical staining confirmed that the reprogrammed cells express pluripotent stem cell markers and can differentiate into the three germ layers.

We successfully repaired a mutation in ELANE gene in 4 out of 30 screened colonies as well as TCIRG1 gene in 3 out of 14 screened colonies. Additionally, for further functional studies, we generated a homozygotic ELANE p.Cys55Arg mutant, that might serve as a unique model of the genotype-phenotype gradient of elastase activity.

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Summary/Conclusion: We have successfully generated and repaired iPS cells derived from patients with congenital neutropenia which might serve as a procedure with a great potential in the field of cell therapy tailored to individual patients.

P1435: PRECLINICAL EVALUATION OF ZILOVERTAMAB-BASED ANTI-ROR1 CHIMERIC ANTIGEN RECEPTORS IN NK AND T CELLS

S. Meinke 1,* , N. Kadri 1 , S. Bi 2 , B. Bostancioglu 1 , C. Markey 2 , J. B. Breitmeyer 2 , A. Hussain 1 , R. Krishnan 2 , G. F. Kaufmann 2 , E. Alici 1

1 Department for Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden; 2 Oncternal Therapeutics, San Diego, CA, United States of America

Background: The receptor tyrosine kinase-like orphan receptor ROR1 is a promising target for immunotherapies of many cancers, including mantle cell lymphoma, diffuse large B-cell lymphoma, B-cell acute lymphocytic leukemia and chronic lymphocytic leukemia. Normally only expressed during embryonic development and only minimally, if at all, in adult normal tissues, ROR1 is overexpressed on malignant cells. Zilovertamab is a monoclonal antibody binding ROR1 that is currently in Phase 1/2 clinical trials. The antibody blocks survival signals mediated by Wnt5a via the ROR1 receptor. A different approach is the use of the antigen binding domain of the antibody in chimeric antigen receptors (CARs) expressed in NK or T cells to induce cellular cytotoxicity against ROR1-expressing cancer cells.

Aims: This study aimed to evaluate different CAR designs in NK cells and T cells for cytotoxicity and cytokine production in vitro and control of cancer growth in a murine in vivo model.

Methods: Two CARs using different linkers between the single chain antibody fragment (scFv) and the transmembrane domain were designed and cloned into lentiviral vectors. The two constructs were expressed in the cell line NK92 and the cells’ response against ROR1-positive and negative cancer cell lines was measured by degranulation and interferon-γ production. The construct that showed a stronger activation was tested in an in vivo model. Immune deficient NSG mice were injected with ROR1-positive JEKO-1 cells transgenic for luciferase. The cancer cell burden was monitored by intravital imaging. When cancer cell growth was established, the mice received either 1, 3, or 9 x 10 6 human T cells expressing the CAR or untransduced T cells.

Results: NK92 cells expressing either of the two CARs showed enhanced degranulation and IFN-γ production in response to the ROR1-expressing cancer cell lines RPMI 8226 and JEKO-1. NK cells expressing the CAR containing the shorter linker between the scFv and the transmembrane domain exhibited higher activation levels. Primary human T cells expressing this CAR construct demonstrated degranulation and cytokine production during co-incubation with the ROR1-expressing JEKO-1 cell line but not the ROR1-negative Jurkat cell line.

Injection of anti-ROR1 human CAR-T cells into NSG mice with established cancer growth greatly reduced the cell burden of JEKO-1 while untransduced T cells were unable to control cancer cell growth.

Summary/Conclusion: The zilovertamab-based CAR tested in this study enhances the NK cell response towards ROR1-expressing cancer cells and enables T cells to clear fast-growing cancer cells in an in vivo model. These findings support the development of anti-ROR1 cell therapies for the potential treatment of ROR1-positive hematological malignancies.

P1436: DEVELOPMENT OF A BETA-GLOBIN GENE REPLACEMENT STRATEGY AS A THERAPEUTIC APPROACH FOR ΒETA-THALASSEMIA

B. Wienert 1,* , K. Wallace 1 , C. Bandoro 1 , A. Churi 1 , J. Partridge 1 , R. Sharma 1 , W. Matern 1 , S. Treusch 1 , D. Dever 1

1 Graphite Bio, Inc., South San Francisco, CA, United States of America

Background: β-thalassemia is a genetic disorder characterized by reduced production of β-globin, a protein that forms functional, oxygen-carrying hemoglobin with α-globin (HbA, α 2 β2). In its most severe form, β-thalassemia is caused by mutations in both alleles of the β-globin gene. These patients fail to produce functional β-globin, resulting in severe anemia. More than 300 β-thalassemia mutations are known; most are small nucleotide insertions, substitutions, or deletions within or directly adjacent to the β-globin ( HBB ) gene. A gene editing platform using homology directed repair (HDR) that replaces the HBB gene to achieve a normal or β-thalassemia trait phenotype with HbA expression similar to healthy individuals is an ideal strategy. The challenge is achieving high levels of gene replacement that result in physiologic HbA expression.

Aims: We designed a homologous DNA donor that can replace a mutated HBB gene with a functional HBB gene and overcomes the two main challenges with gene replacement at this locus. First, to avoid sequence homology of the insert with the endogenous locus, we used a diverged HBB coding sequence. Second, because introns are required for high HBB expression, we hypothesized that incorporation of non-homologous introns would result in physiologic HbA production.

Methods: We developed an HBB gene replacement strategy using a high-efficiency gene editing platform that combines: 1) a high fidelity Cas9 precomplexed with chemically modified guide RNA, 2) a DNA template delivered by AAV6, and 3) CD34+ hematopoietic stem and progenitor cell culture optimization. To identify DNA templates resulting in high HBB expression, we screened 39 versions of T2A-EGFP-tagged HBB coding sequences containing various heterologous introns and polyadenylation signals. Primary CD34+ hematopoietic stem and progenitor cells (HSPCs, n = 3 healthy donors) were edited with different β-globin homologous DNA donors, differentiated into red blood cells (RBCs) in vitro , and then evaluated for EGFP mean fluorescence intensity (MFI) by flow cytometry as a surrogate for beta-globin expression. To evaluate if the MFI positively correlated with HbA expression, the T2A-EGFP sequence was removed from the DNA donors and HbA expression was measured by HPLC in HSPC-derived RBCs. To increase gene replacement frequencies, the top DNA donors were further optimized by truncating the introns to create a smaller insertion cassette

Results: Adding heterologous introns to the HBB coding sequence significantly increased EGFP MFI by three-fold (p=0.0008). MFI and HbA output were positively correlated, and the top DNA donors resulted in HbA expression on par with physiologic levels. Using this gene replacement strategy, we achieved HDR frequencies of up to 40% in CD34+ HSPCs. Optimized smaller DNA templates resulted in higher HDR and maintained high levels of HbA expression. Using HSPCs from patients with sickle cell disease as a therapeutically relevant model, we investigated if this DNA donor can replace the non-functional HBB gene that produces HbS. Gene replacement of the sickle allele using the optimized DNA donor restored physiologic HbA expression (by HPLC) comparable to an HDR gene correction strategy that corrects the HbS point mutation. Future experiments are investigating in vitro and in vivo stem cell repopulation capacity of HSPCs targeted for HBB gene replacement.

Summary/Conclusion: In summary, we developed a precise HBB gene replacement strategy that is highly efficient and results in high HbA production, offering a potential differentiated approach for treating β-thalassemia.

P1437: NOVEL ERYTHROID ENHANCERS IMPROVE GENE THERAPY VECTORS FOR BETA-HEMOGLOBINOPATHIES

N. Psatha 1,2,* , P. Sova 2 , G. Georgolopoulos 2 , M. Iwata 2 , K. Paschoudi 3 , A. Kirtsou 4 , T. Ulyanova 5 , J. A. Stamatoyannopoulos 2 , E. Yannaki 4 , T. Papayannopoulou 5 , J. Vierstra 2

1 Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece; 2 Altius Institute for Biomedical Sciences, Seattle, United States of America; 3 Aristotle University of Thessaloniki; 4 Gene and Cell Therapy Center, George Papanikolaou Hospital, Hematology Clinic-BMTU, Thessaloniki, Greece; 5 Hematology, University of Washington, School of Medicine, Seattle, United States of America

Background: Regulation of gene expression during cell development and differentiation is chiefly orchestrated by distal noncoding regulatory elements. Massively parallel reporter gene assays (MPRAs) have provided tremendous insights into transcriptional enhancers’ function and principles of regulation.

Aims: Understanding the enhancer’s mode of function serves an important role for contemporary medicine as they constitute a prominent feature of therapeutic vectors. In the present study we developed a screening assay using a clinically relevant viral vector while simultaneously maintaining genomic context and cell type relevance.

Methods: We designed a library of 15,000 short sequences (200bp) derived from a set of developmentally active DHS elements during human ex vivo erythropoiesis and cloned them into a GFP reporter lenti-viral vector. These elements proved to not only be highly cell type restricted but also stage specific. Subsequently, two of these newly identified, compact, strong erythroid enhancers were cloned in a BCL11A-shRNA vector similar to what is currently used in clinical trials for beta-hemoglobinopathies to replace the widely applied, yet of considerable size, β-globin μLCR.

Results: The novel gene therapy vectors yielded 2-5 times higher viral titers during production due to their small size, while they proved to be more efficient in transducing normal CD34+ cells at the same multiplicity of infection. Their ability to reactivate the endogenous gamma globin expression within the transduced population was equivalent to that of the μLCR-BCL11a-shRNA vector. Engraftment and multilineage reconstitution of the transduced cells after xenotransplantation in a mouse model was similar in all groups, with the new vectors yielding higher transduction and HbF levels over time. Finally, transduction of CD34+ cells from patients with beta-thalassemia major with the new vectors resulted in higher HbF expression coupled with phenotypic correction of their ex vivo generated erythroid cells.

Summary/Conclusion: Collectively, our data provide further insights into enhancer biology in erythropoiesis with wider implications into the development of a new generation of highly cell type-specific and efficacious viral vectors for human gene therapy.

P1438: PROCALCITONIN IS ELEVATED IN SEVERE INFECTIONS AFTER CD19 CAR-T IN R/R B-NHL AND ENABLES DISCRIMINATION OF EARLY INFECTIONS FROM CRS – PARTICULARLY IN COMBINATION WITH THE CAR-HEMATOTOX SCORE

K. Rejeski 1,2,3,* , V. Blumenberg 1,2,3 , F. Hildebrand 1 , L. Frölich 1,3 , P. Karschnia 4 , M. Dreyling 1 , M. von Bergwelt-Baildon 1,3 , C. Schmidt 1 , M. Subklewe 1,2,3 , V. Bücklein 1,2

1 Department of Medicine III - Hematology/Oncology, LMU University Hospital; 2 Laboratory for Translational Cancer Immunology, LMU Gene Center, Munich; 3 German Cancer Consortium (DKTK), Munich Site, and German Cancer Research Center, Heidelberg; 4 Department of Neurosurgery, LMU University Hospital, Munich, Germany

Background: Infections substantially contribute to the toxicity burden of CD19 CAR-T and represent the most common cause of non-relapse mortality. Determining if an episode of fever represents the onset of infection or cytokine release syndrome (CRS) remains a major clinical challenge. Most patients thus receive broad-spectrum antibiotics, which can confer resistance and may negatively influence the microbiome.

Aims: We sought to characterize dynamic changes in serum inflammatory markers for infection vs. CRS and to develop clinical tools that discriminate for severe infections.

Methods: Early infection events (day 0-30) and day-by-day CRS dynamics were characterized in 62 patients treated with CD19 CAR-T for relapsed/refractory B-NHL in a real-world setting (23 Axi-cel, 30 Tisa-cel, 9 KTE-X19). Severe infections were defined as requiring i.v. anti-infectives and/or hospitalization in the presence of microbiologic evidence or distinct clinical signs of infection; CRS was graded according to ASTCT criteria. Serum inflammatory markers (CRP, interleukin-6, procalcitonin) were recorded daily, and the baseline CAR-HEMATOTOX score (Rejeski et al Blood 2021) was assessed for each patient.

Cumulative incidence curves were calculated as time-to-event-analysis for CRS 1°, ≥2° and severe infections. Index events were defined per patient for each toxicity category (= day of event). Inflammatory markers were tested using binary logistic regression for severe infections vs. CRS index events. Discriminatory thresholds were determined by optimizing the Youden J statistic.

Results: The cumulative 30-day incidence of CRS 1°, CRS ≥2° and severe infections was 47%, 43%, and 25% respectively. Tocilizumab was applied in the majority of CRS patients (1°: 90%, ≥2°: 100%). We did not find a significant correlation between CRS grade and severe infections (G 2 = 0.13, p = n.s.). Median onset of CRS and infection was 3 and 8 days, respectively. The majority of events occurred in the setting of coincident severe neutropenia (ANC <500/µl).

Inflammatory markers were assessed both relative to CAR-T transfusion and the respective index event. CRS ≥2° was characterized by higher day 0-21 levels of interleukin-6 compared to CRS 0-1° (p<0.001). On the other hand, patients with severe infections exhibited both higher baseline inflammation (reflected by higher CRP levels on days 0-3), and higher peak procalcitonin levels (p<0.001) compared to CRS controls. These trends were particularly evident on the day of the index event (Fig. 1). Procalcitonin was significantly elevated for severe infections vs. CRS controls (median 1.6 vs. 0.3 µg/l, p<0.001), and an optimal discriminatory threshold of 1.4 µg/l was established.

An elevated baseline CAR-HEMATOTOX was noted for patients with severe infections (median 5 vs. 2, p=0.002). By incorporating index-event procalcitonin levels with the score, the discriminatory capacity for severe infection events was improved. On ROC analysis, the ‘HT10 score’ (addition of 10 points to the patient-specific CAR-HEMATOTOX in case procalcitonin ≥1.4 µg/l on event day) exhibited superior discrimination for severe infections (AUC 0.92, p<0.0001, sens. 80%, spec. 89%).

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Summary/Conclusion: CRS and infections are characterized by distinct patterns of inflammation after CD19 CAR-T (CRS: high IL-6; Infections: high procalcitonin). The high discriminatory capacity of the HT10 score for infections emphasizes the utility of procalcitonin in routine inflammatory panels. If validated prospectively, the score will enable more patient-individual decisions on antibiotic use.

P1439: IMMUNE RESPONSE TO ANTI-SARS-COV-2 MRNA VACCINES IN MULTIPLE MYELOMA AND CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

J. Zaleska 1,* , P. Kwaśnik 1 , M. Paziewska 1 , J. Purkot 1 , A. Szabelak 1 , M. Jurek 1 , N. Masny 1 , I. Dziatkiewicz 1 , P. Własiuk 1 , K. Skórka 1 , G. Stasiak 1 , B. Pronobis-Szczylik 1 , A. Piebiak 2 , A. Szymczyk 2,3 , K. Jarosz-Chudzik 1,2 , L. Bołkun 4 , K. Kozłowska 4 , J. Piszcz 4 , E. Subocz 5 , J. Hałka 5 , M. Bator 6 , E. Kalicińska 6 , T. Wróbel 6 , L. Usnarska-Zubkiewicz 6 , J. Rybka 6 , I. Dereń-Wagemann 7 , M. Szyca-Śmieszniak 7 , J. Dybko 7 , I. Hus 3,8 , B. Puła 8 , E. Cichocka 9 , M. Rymko 9 , D. Zduńczyk 10 , M. Ziarkiewicz 10 , G. Basak 10 , L. Bullinger 11,12 , K. Giannopoulos 1,2

1 Department of Experimental Hematooncology, Medical University of Lublin; 2 Department of Hematology, St John’s Cancer Centre; 3 Department of Clinical Transplantology, Medical University of Lublin, Lublin; 4 Department of Hematology, Medical University of Bialystok, Bialystok; 5 Department of Hematology, Warmian-Masurian Cancer Center of the Ministry of the Interior and Administration’s Hospital, Olsztyn; 6 Department of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University; 7 Department of Hematology and Transplantation, Lower Silesian Center of Oncology, Wroclaw; 8 Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw; 9 Department of Hematology and Bone Marrow Transplantation, Nicolaus Copernicus Hospital, Torun; 10 Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw, Warsaw, Poland; 11 Department of Hematology, Oncology, and Tumor Immunology, Charité – Universitätsmedizin Berlin; 12 German Cancer Consortium, Berlin, Germany

Background: Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients have increased morbidity and mortality rates of COVID-19 due to immunosuppression associated with the disease and ongoing therapy. The same immune impairment accompanying CLL and MM also affects suboptimal vaccine response. The analyses of the effectiveness of the post-vaccination response may lead to the optimization of vaccination.

Aims: The study assessed the effectiveness of the humoral and T cell-mediated immunity as well as characterized immune system populations following mRNA COVID-19 vaccination using either BNT162b2 or mRNA-1273 in the short-term (2-5 weeks after 2nd dose) and long-term follow-up (12 weeks after vaccination).

Methods: Blood samples were obtained from 62 CLL and 60 MM patients from eight hematology departments in Poland and were assessed at pre and post-vaccination in three time points. Anti-SARS-CoV-2 antibodies (IgM, IgG, and IgA) were evaluated in qualitative and quantitative (IgG) ELISA. Flow cytometry was used to characterize changes in the immune system of patients after anti-SARS-CoV-2 mRNA vaccination. Evaluation of specific cytotoxic T cell response after vaccination was performed in HLA-A*0201 positive CLL and MM patients with the most common and the highest binding score SARS-CoV-2 specific epitope - HLA-A*0201/YLQPRTFLL 269-277 .

Results: Total neutralizing antibodies were detected in 37% MM patients before vaccination, increased to 91% and 94% in short- and long-term follow-up, respectively. In CLL, serological responses were detectable in 21% of patients before vaccination and increased to 45% in the short-term and 71% in long-term observation.

We detected a tendency to higher frequencies of specific CD8+ T cells against SARS-CoV-2 after vaccination compared to samples before vaccination in MM patients (median: 0.18% vs 0.11%, P <.06), and no changes in frequencies of specific T cells in CLL patients.

Comparison between MM and CLL serological response showed a significant increase in OD values in MM samples in the short-term (median: 1.71 vs 0.12, P <.0001) and long-term (median: 3.57 vs 0.90, P <.005) follow-up. COVID-19 naïve patients treated for CLL demonstrated significantly lower serological response early after the 2nd dose (median 0.08 vs 2.2, P<.001) as well as in long-term follow-up (median: 0.21 vs 2.86, P<.05) than untreated COVID-19 naïve patients.

Flow cytometry characteristics of immune system subpopulations showed a moderate positive correlation between IgG antibodies levels and percentage of iNKT cells ( P <.04, r=0.59) in short-term follow-up in CLL patients. Interestingly, we found a significant decrease in levels of iNKT PD-1+ cells after mRNA vaccination in CLL patients in short-term (median: 50% vs 65.22%, P <.05) as well as in long-term response (median: 46.67% vs 66.67%, P <.03, compared to baseline in paired samples. Analysis of the impact of frequent infections in patients with CLL showed higher levels of NKT (median: 6.37% vs 1.36%, P<.02) in short-term follow-up and levels of NKT (median: 7.4% vs 1.93%, P<.02), iNKT (median: 0.021% vs 0.007%, P<.02) and CD8PD-1+ (median: 34.2% vs 20.14%, P<.02) cells in long-term follow-up in patients without infections.

Summary/Conclusion: This study provides novel insights into mRNA vaccination efficacy in immunocompromised MM and CLL patients. Our findings highlight that specific CD8+ T cells against SARS-CoV-2 might be induced by vaccination but do not correlate positively with serological responses.

This study was supported by research grants from the Medical University of Lublin PBsd 251 and DS 174.

P1440: BTK INHIBITORS IMPROVE CART19 CELL THERAPY BY MODULATING IMMUNE SYSTEM

W. Luo 1,2 , C. Li 1,2 , M. Du 1,2 , F. Zhou 2,3 , Y. Zhang 1,2 , L. Tang 1,2 , J. Wu 1,2 , H. Jiang 1,2 , Q. Wei 1,2 , C. Lu 1,2 , H. Kou 1,2 , H. Mei 1,2,* , Y. Hu 1,2

1 Institute of Hematology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology; 2 Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease; 3 Department of Pediatrics, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Background: CD19-targeted chimeric antigen receptor-T (CART19) cell therapy has achieved extraordinary success in B-cell lymphoma and leukemia. However, recurrence and adverse effects, including cytokine release syndrome (CRS) and immune cell-associated neurotoxicity syndrome (ICANS), remain its obstacles. Recurrence, even resistance to CART19 cell therapy, might be attributed to CAR-T cell dysfunction and suppressor cells in the microenvironment. Cytokines required for CRS and ICANS are predominantly derived from monocytes and macrophages. Given that Bruton’s tyrosine kinase (BTK) is expressed on myeloid cells, we considered that BTK inhibitors (BTKi) could improve CAR-T cell therapy by modulating immune system.

Aims: To systematically evaluate the effects of three BTKi, ibrutinib (IB), zanubrutinib (ZB) and orelabrutinib (OB), on T cells, CART19 cells and tumor microenvironment.

Methods: T and CART19 cells were cultured with the three BTKi for 4 days, and the immune and differentiated phenotypes (PD1, TIM3, CTLA4, CD25, CD69 and CD28, CD45RA and CD62L) were analyzed by flow cytometry (FCM). The effects of BTKi on CART19 cytotoxicity against NALM-6 were assessed for 24-hour coculture.

CART19, NALM-6 and macrophages differentiated from THP-1 upon PMA stimulation were cocultured and IL-6 in the supernatants was detected by cytometry bead assays.

Lymphoma mouse models were established with BALB/c mice by subcutaneous injection of 5x10 6 A20 cells. IB, ZB and OB were administered to the BALB/c mice at 25, 10 and 10mg/kg respectively by oral gavage once daily for 28 days. Vehicle treatment was performed using 0.5% carboxymethylcellulose sodium. Mice in healthy controls were fed with no additional treatment. Splenocytes, bone marrow (BM) samples and tumor specimens were harvested, and single-cell suspensions were prepared to analyze immune phenotypes by FCM.

Results: Compared with control T cells, IB-supplemented T cells significantly reduced the expression of PD-1 (62.28% vs 14.95%, P = 0.0024), TIM-3 (71.38% vs 7.88%, P <0.0001) and CTLA-4 (92.65% vs 15.5%, P <0.0001) following CD3/CD28 stimulation (Figure 1A). The downregulation of the suppressive markers was also observed in T cells treated with ZB, but not with OB (Figure 1A). In parallel, IB and ZB decreased activation markers expression and increased effector memory cell subsets, while decreased naïve T cells (Figure 1A and B). BTKi decreased IL-6 level in the supernatants of CART19, NALM-6 cells and macrophages (Figure 1C), and decreased CTLA-4 expression on CART19 (Figure 1D). The effects of BTKi on CART19 differentiated phenotypes were not observed (Figure E). Considering strong CD3/CD28 stimulation and tonic signaling could drive terminal differentiation and even apoptosis of CAR-T cells, we supposed BTKi could protect CART19 from excessive activation in long-term cultivation and stimulation (not shown). The three BTKi did not show synergistic effects on cytotoxicity of CART19 against NALM-6 cells (not shown). In lymphoma mice models, the three BTKi all increased the ratio of type 1 macrophages (M1) to type 2 macrophages (M2) and Th1 to Th2 in tumor, and reduced tumor-infiltrating macrophages (Figure 1F-H). BTKi induced significant decreases in exhaustion markers expression on T cells in BM tissues, but not in tumor specimens (Figure I).

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Summary/Conclusion: Differentiation and exhaustion of CAR-T cells highly activated upon CD3/CD28 stimulation and tonic signaling can be prevented by BTKi. Moreover, BTKi can alter the tumor microenvironment and downregulate IL-6 level to improve the efficacy and safety of CART19 cell therapy.

P1441: SINGLE-CELL TRANSCRIPTOMIC CHARACTERIZATIONS OF BONE MARROW MONONUCLEAR CELLS AND CAR-T PRODUCTS ASSOCIATED WITH EFFICACY AND TOXICITY IN B-ALL PATIENTS

M. Du 1,2 , C. Li 1,2 , F. Zhou 2,3 , W. Luo 1,2 , Y. Zhang 1,2 , L. Tang 1,2 , J. Wu 1,2 , H. Jiang 1,2 , Q. Wei 1,2 , C. Lu 1,2 , H. Kou 1,2 , H. Mei 1,2,* , Y. Hu 1,2

1 Institute of Hematology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology; 2 Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease; 3 Department of Pediatrics, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Background: Chimeric antigen receptor-T (CAR-T) cell therapy has transformed B-cell acute lymphoblastic leukemia (B-ALL) treatment, yielding outstanding clinical results. However, less than half of patients achieve long-term remission, with treatment-associated adverse events a clinical challenge.

Aims: This study aims to investigate the transcriptomic characterizations of CAR-T products and bone marrow mononuclear cells, and identify predictive biomarkers for efficacy and toxicity.

Methods: Seventeen B-ALL patients were enrolled in this study. Single-cell RNA sequencing was performed on BMMCs (baseline and post-infusion) and IPs in 4 patients, and 120,780 residual cells were collected for bioinformatic analysis to identify biomarkers for the early clinical forecast. Another 13 patients-cohort was used for bulk RNA-sequencing validation.

Results: An overview of our approach for sc-RNA-seq is shown in Figure A. Twenty-one distinctly transcriptional cell clusters were identified after extensive, unbiased clustering of all BMMCs and IPs (Figure B). Cell clusters were classified into five broad cell types -- IPs, HSPCs, NK/T cells, myeloid cells, and B-ALL cells (Figure C). For severe CRS prediction, 4 patients were divided into two groups based on tumor burden. Baseline cytokine-mediated signaling increased in high-tumor burden patients, mainly in B-ALL blasts, HSPCs (Pt 10 vs Pt 8); and low tumor burden patients, primarily in NK/T and Myeloid cells (Pt 11 vs Pt 9) (Figure D). IFITM1, a gene in IFN-related signaling pathways, was significantly upregulated in the above-mentioned cell types and validated in the bulk-RNA cohort (Figure E). Unlike complete response patients, NR (Pt 8) patients had diminished SRGN expression in every BMMC-1 cell type (Figure F), with probable arrests of IPs at the G1 phase (Figure G). In the NR patient tumor subsets, treatment-resistant tumor clusters (C1) in the IFN-I signaling pathway were enriched, whereas treatment-sensitive tumor cells (C3/5/10) underwent apoptosis (Figure H/I/J). Other ways, including the extracellular structure organization that predicts extramedullary relapse, were amplified (Figure K). The bulk RNA-sequencing cohort produced identical results.

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Summary/Conclusion: IP and BMMC transcriptional feature heterogeneity possibly contributes to diverse clinical outcomes post-anti-CD19 CAR-T cell therapy and could be predictive.

P1442: DYNAMIC LANDSCAPE OF PLASMA PROTEOME AND METABOLOME IN B-ALL PATIENTS DURING CAR-T CELL THERAPY

J. Wu 1,2 , L. Tang 1,2 , M. Du 1,2 , C. Li 1,2 , H. Kou 1,2 , H. Jiang 1,2 , W. Luo 1,2 , Y. Zhang 1,2 , Z. Huang 1,2 , W. Xiong 2 , H. Mei 1,2 , Y. Hu 1,2,*

1 Institute of Hematology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology; 2 Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, China

Background: Poor understanding of plasma proteomic and metabolomic features during chimeric antigen receptor (CAR) T cell therapy may affect the exploitation of therapeutic potential in the management of hematological malignancies.

Aims: This word was designed to illustrate the dynamic landscape of plasma proteome and metabolome in B-ALL patients during CAR-T cell therapy and detailed the kinetics of proteins and metabolites during cytokines storm.

Methods: In this study, we performed proteomic and metabolomic profiling of plasma from 20 patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) on CAR-T cell therapy and 22 healthy controls.

Results: We identified molecular changes in the plasma of B-ALL patients compared to healthy controls. Many of the changes involved actin cytoskeleton organization, acute-phase responses, autophagy, branched-chain amino acid, and lipid metabolism (Figure A). Furthermore, CAR-T cell therapy reshaped plasma proteome and metabolome with discrete kinetics, as represented by upregulated acute-phase response and triglycerides metabolism and downregulated actin cytoskeleton organization, axon extension, retrograde endocannabinoid signaling, autophagy, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis during the therapy (Figure B). Moreover, we identified sets of covarying molecules involved in the acute inflammatory response, epithelial-mesenchymal transition, glutathione metabolism, and tryptophan metabolism and change in concert with the dynamics of circulating IL-6 in patients with cytokines storm (Figure C). Subgroup analysis further revealed numerous prognostic biomarkers, such as proteins related to allograft rejection, which were down-regulated in patients who achieved stable complete remission.

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Summary/Conclusion: This study provided a dynamic landscape of plasma proteome and metabolome in B-ALL patients during CAR-T cell therapy and offered novel insights into the pathogenesis of B-ALL and CAR-T cell therapy-related toxicities.

P1443: AUTOLOGOUS NK CELLS AS CONSOLIDATION AFTER FRONT-LINE STEM CELL TRANSPLANTATION IN MULTIPLE MYELOMA: A LONG-TERM FOLLOW-UP

J. Lund 1,2,* , H. Nahi 1,2 , S. Meinke 1,2 , P.-H. Holmqvist 3 , H.-G. Ljunggren 1,4 , J. Aschan 3 , E. Alici 1,2

1 Department of Medicine Huddinge; 2 Center for Hematology and Regenerative Medicine, Karolinska Institutet; 3 XNK Therapeutics; 4 Center for Infectious Medicine, Karolinska Institutet, Huddinge, Sweden

Background: Immunotherapies have shown great anti-tumour effect in a variety of malignancies. In multiple myeloma (MM) antibodies, including bispecific T cell engagers, antibody-drug conjugates, and more recently, CAR T cells have reported exciting clinical results and many of them have been incorporated in the treatment armamentarium. Less is reported for NK cells, in particular autologous NK cells. Initial results from our first-in-human trial ACP-001 with autologous NK cells given as consolidation after front-line autologous stem cell transplantation (ASCT) were recently reported (Nahi et al., 2022, Cell Reports Medicine 3 , 100508 February 15, 2022). It showed that the concept was feasible with good safety, including no SAEs, and promising efficacy. We here report the long-term clinical follow-up.

Aims: The aim of this report was to investigate long-term safety, disease progression, second-line therapy and survival after autologous NK cell treatment.

Methods: This first-in-human trial included six patients with newly diagnosed MM eligible for ASCT at the Karolinska University Hospital Huddinge in Sweden. After standard induction treatment (bortezomib, cyclophosphamide and dexamethasone (VCD) at the time) and ASCT with melphalan 200 mg/m 2 (MEL200) the patients received three escalating doses of autologous activated and expanded NK cells. Clinical data on long-term safety, disease progression, second-line therapy and survival were captured from the patients’ electronical charts.

Results: No subject were lost to follow-up or had withdrawn from the study. With a median follow-up from study inclusion of 6.9 (6.1-7.2) yrs, and 6.2 (5.4-6.7) yrs from the first NK cell infusion, there are no new reported AE related to the NK cell infusions. All six patients are alive with one subject still in first remission 6.8 yrs after inclusion. Five patients had disease progression 13-51 months after study inclusion and four of them underwent reinduction (VRD (n=2), KRD (n=1), KRD+dara (n=1)) followed by a second ASCT with MEL200. The fifth patient progressed within the first yr after ASCT and received VRD+dara. All five patients responded to second line treatment with PR (n=2), VGPR (n=2) and MRD negative (n=1) but subsequently progressed with a PFS2 ranging from 11.5 to 26.3 months.

Summary/Conclusion: This long-term follow-up confirms the safety and feasibility of autologous NK cell therapy as consolidation after front line ASCT in MM. Autologous NK cell treatment in first line did not negatively impact the possibility to administer, or the outcome, of later treatments which is important with all new treatment options for patients with advanced MM.

P1444: IMPACT OF CO-MORBIDITIES ON NON-RELAPSE MORTALITY AND CYTOKINE-RELEASE SYNDROME AFTER CD19 CAR-T CELL THERAPY - A RETROSPECTIVE STUDY FROM THE TCWP OF THE EBMT AND GOCART COALITION

G. Basak 1,* , C. Peczynski 2 , C. Koenecke 3 , S. Terwel 4 , Y. Cabrerizo 5 , O. Penack 6 , I. Moiseev 7 , H. Schoemans 8 , N. Fegueux 9 , V. Potter 10 , S. Zeerleder 11 , I. Yakoub-Agha 12 , M. Collin 13 , U. Schanz 14 , P. Dreger 15 , D. Blaise 16 , C. Besley 17 , B. Glass 18 , M. Mohty 19 , C. Chabannon 20 , J. Kuball 21 , J. Snowden 22 , A. Sureda 23 , N. Kröger 24 , Z. Peric 25

1 Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw, Warsaw, Poland; 2 EBMT Transplant Complications Working Party, EBMT, Paris, France; 3 Clinic for Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; 4 The GoCART Coalition, EBMT Executive Office, EBMT, Leiden; 5 Tthe GoCART Coalition, EHA Executive Office, EHA, Hague, Netherlands; 6 Medical Clinic, Department of Haematology, Oncology and Tumor Immunology, Charité Universitätsmedizin Berlin, Berlin, Germany; 7 RM Gorbacheva Research Institute, Pavlov University, St Petersburg, Russia; 8 Department of Hematology, Department of Public Health and Primary Care, University Hospitals Leuven, University of Leuven, Leuven, Belgium; 9 Département d`Hématologie Clinique, CHU Lapeyronie, Montpellier, France; 10 Dept. of Haematological Medicine, Kings College Hospital, London, United Kingdom; 11 Paediatric Hematology/Oncology, University Hospital Bern, Bern, Switzerland; 12 CHU de Lille, LIRIC, INSERM U995, Université de Lille, Lille, France; 13 Adult HSCT unit, Northern Centre for Bone Marrow Transplantation, Freeman Hospital, Newcastle Tyne, United Kingdom; 14 Clinic of Medical Oncology and Hematology, University Hospital and University of Zurich, Zurich, Switzerland; 15 Medizinische Klinik u. Poliklinik V, University of Heidelberg, Heidelberg, Germany; 16 Programme de Transplantation & Therapie Cellulaire, Centre de Recherche en Cancérologie de Marseille, Institut Paoli Calmettes, Marseille, France; 17 Dept. of Paediatric Oncology/BMT, Bristol Royal Hospital for Children, Bristol, United Kingdom; 18 AbteilungHämatologie und Stammzelltransplantation, AsklepiosKlinik St Georg, Hamburg, Germany; 19 Service d’hématologie clinique et thérapie cellulaire, Hôpital Saint-Antoine, INSERM UMRs 938 and Université Sorbonne, Paris; 20 Centre d’Investigations Cliniques en Biothérapies, Institut Paoli-Calmettes Comprehensive Cancer Center, Aix-Marseille Université School of Medicine & Inserm CBT1409, Marseille, France; 21 Department of Haematology, University Medical Centre Utrecht, Utrecht University, Utrecht, Netherlands; 22 Department of Haematology, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom; 23 Institut Català d’Oncologia-Hospitalet, Institut d’Investigació Biomedica de Bellvitge, Universitat de Barcelona, Barcelona, Spain; 24 Bone Marrow Transplantation Centre, University Hospital Eppendorf, Hamburg, Germany; 25 Department of Hematology, University Hospital Centre Zagreb, Zagreb, Croatia

Background: CAR T-cell therapies have been associated with a profile of significant toxicities and previously unknown side effects. For that reason, the registration of clinical trials with commercial and non-commercial products included very strict inclusion and exclusion criteria, to minimize severe adverse events and maximize therapeutic effect. The real-life analyses revealed that a proportion of patients are treated outside of these specifications, with variable outcomes.

Aims: The goal of this study was to determine the cumulative incidence of non-relapse mortality (NRM) and cytokine-release syndrome (CRS) after anti-CD19 CAR T-cell therapy, and impact of patients’ co-morbidities expressed as the hematopoietic cell transplantation-co-morbidity index (HCT-CI) scores, on the major outcome measures.

Methods: This is a retrospective multicentre analysis from the Transplant Complications Working Party (TCWP) of the EBMT using the data of the GoCART coalition. Eligibility criteria for this analysis include diffuse large B-cell lymphoma (DLBCL) and acute lymphocytic leukemia (ALL) patients that underwent CD19 CAR T-cell therapy in an EBMT member centre before 31 January 2021. Exclusion criteria were lack of information on comorbidities, survival or relapse status, or age <18 years. Outcomes were measured from the date of CAR T-cell infusion for all endpoints. NRM was defined as death without relapse/progression. Cumulative incidence functions were used to estimate the incidence of complications. Multivariate analyses were performed using the Cox cause-specific model for all endpoints. The primary endpoint was NRM at 3 and at 12 months post CAR T-cell therapy, while secondary endpoints included cumulative incidence of CRS as well as NRM and CRS incidence in subgroups based on HCT-CI. Statistical analyses were performed with R 3.4.2 software packages.

Results: The study identified 586 patients treated with CAR T-cells in the years 2018-2021 for DLBCL (96.4%) or ALL (3.6%). The median age was 60 (range, 18-84). 166/437 (38%) patients had a Karnofsky performance status <90%, while the HCT-CI was available for 483 (82%) patients and was intermediate (1-2) in 17.6% and high (≥3) in 13.9% of these patients. The most common co-morbidities were pulmonary (16.1%), cardiac (6.8%), obesity (6.8%), diabetes (6.4%), arrythmia (5.5%), history of solid tumor (5.3%), and active infection before treatment (4.3%). The median follow up was 12.5 months. The NRM was reported to be very low: 1.7% (95%CI 0.9-3) at 3 months and 2.9% (95%CI 1.7-4.5) at 12 months. The cumulative incidence of CRS (all grades) was 35.3% (95%CI 30.6-40.1) at 3 days and 82.7% (95%CI 78.5-86.1) at 12 days. Unexpectedly, no significant impact of HCT-CI on NRM, CRS incidence, or overall survival was observed (Fig. 1) both in univariate and multivariate analyses. Most deaths were caused by the original disease (75.4%) with concomitant infection (5.9%) or with multiorgan failure (4.3%).

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Summary/Conclusion: Our real-life data from the GoCART registry revealed the profile and frequency of co-morbidities of patients undergoing CAR T-cell treatments at EBMT centers. While the HCT-CI score was high in a substantial proportion of patients, it did not influence any major outcome. The incidence of CRS was also high and not influenced by the HCT-CI. Our preliminary results might support application of more liberal criteria for patient qualification for CAR T-cell therapies based on co-morbidities, but this needs to be confirmed in further studies.

P1445: MYTCELL®: AN INTERACTIVE SMARTPHONE APPLICATION FOR CARS AND BITES INCREASES GUIDELINE ACCESSIBILITY AND REDUCES TIME TO APPLY EVIDENCE-BASED PATIENT CARE

V. Blumenberg 1,2,3,* , L. Siegmund 1,2 , L. Frölich 1,3 , K. Rejeski 1,2,3 , M. Warm 1 , F. Hildebrand 1 , C. Schmidt 1 , M. von Bergwelt 1,3 , V. Bücklein 1,2 , M. Subklewe 1,2,3

1 Department of Medicine III, University Hospital LMU Munich; 2 Laboratory for Translational Cancer Immunology, Gene Center LMU Munich, Munich; 3 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

Background: The Bispecific T-cell engager (BiTE) Blinatumomab and CD19-specific Chimeric Antigen Receptor (CAR) T-cell products are approved for the treatment of relapsed and refractory B-cell neoplasms. Several ongoing clinical trials and further development of different drug formats will lead to a substanially increased number of eligibile patients and a broader application in the near future. However, the complex pre-treatment workflow, particularly with regard to timelines of apheresis, bridging therapies, and CAR T-cell transfusion, and accompanying toxicities still pose a challenge for health care professionals.

Aims: To support the optimal management of these patients, we developed an interactive smartphone application (myTcell ® ), which provides guidelines on pre-treatment logistics and toxicity management in an actionable format.

Methods: We initiated a multi-step content development process researching guidelines consented by the ASTCT, SITC, NCCN and EBMT as well as the prescribing information. Findings were integrated into an information platform to provide diagnostic and therapeutic recommendations as well as algorithms for interactive toxicity grading tools. We could successfully implement an app (myTcell ® ) guiding health workers in a disease- and product-specific step by step process through the clinical workflow of CAR and BiTE therapy. This includes recommendations for patient screening and stopping rules prior to leukapheresis and CAR T-cell transfusion. Upon entering relevant clinical data for grading of CRS, ICANS and HLH, interactive tools display toxicity grade and grade-specific management. Further, myTcell ® assists with the diagnosis and treatment of pancytopenia and infections. The app includes an overview of important publications with direct links to respective abstracts to support evidence-based decision-making. A prototype was validated at five Geman treatment centers through a questionnaire, which measured the advantage of the app compared to common clinical practice.

Results: 38 physicians validated the app protoype in a real-world setting of CAR T-cells or BiTEs at five German treatment centers with an excellent overall rating (University Hospitals of LMU Munich, Cologne, Göttingen, Erlangen, Tübingen). 78.9% of the users most valued the content about toxicity management. In addition, 63.2% reported to dominantly use the interactive tools and calculators to assess severity of toxicities and to instantly receive grade-specific management recommendations. Most of the users agreed, that the interactive tools improved toxicity management (94.7%) and saved time during their clinical practice (81.6%). Based on these results, myTcell ® has been certified as a European medicinal product class I and has been released in the app stores and for desktop application ( www.myTcell.de ) on July 15, 2021. Since then, myTcell ® has achieved a great visibility with 3790 pages views and up to 267 users of five different European countries. The release for USA and China is expected for 2022 and will come with further interactive tools estimating toxicity risk such as the Hematotox and the EASIX Score.

Summary/Conclusion: myTcell ® is a smartphone app supporting the application of CAR T cells and BiTEs. Health care professionals validated feasibility and in particular appreciated fast and easy assessment and management of toxicities. Thus, myTcell ® has the potential to improve guideline adherence and accelerate broader and safer application of CARs and BiTEs.

P1446: PHASE I STUDY DATA UPDATE OF PHE885, A FULLY HUMAN BCMA-DIRECTED CAR-T CELL THERAPY MANUFACTURED USING THE T-CHARGETM PLATFORM FOR PATIENTS WITH RELAPSED/REFRACTORY (R/R) MULTIPLE MYELOMA (MM)

A. S. Sperling 1,* , S. Nikiforow 1 , B. Derman 2 , O. Nadeem 1 , C. Mo 1 , J. Laubach 1 , K. Anderson 1 , A. Alonso 1 , S.-Y. Im 1 , S. Ikwgawa 1 , R. Prabhala 1 , D. Hernandez Rodriduez 1 , H. Daley 1 , K. L. Shaw 1 , Y. Arihara 1 , S. Ansari 3 , D. S. Quinn 3 , D. Pearson 4 , A. Hack 5 , L. Treanor 3 , D. Bu 3 , J. Mataraza 3 , L. Rispoli 3 , M. Credi 3 , J. Ritz 6 , A. Jakubowiak 2 , S. De Vita 3 , N. Munshi 1

1 Dana-Farber Cancer Institute, Harvard University, Boston; 2 Section of Hematology/ONcology, University of Chicago Medical Center, Chicago; 3 Novartis Institutes for BioMedical Research, Cambridge, United States of America; 4 Novartis Institutes for BioMedical Research, Basel, Switzerland; 5 Novartis Pharmaceuticals Corporation, East Hanover; 6 Harvard University, Boston, United States of America

Background: Chimeric antigen receptor (CAR)-T cell therapy (tx) is approved for treatment of r/r MM. However, prolonged product manufacturing times and a need for longer duration of response are a major obstacle in this population, necessitating fast manufacture of a reliable and durable CAR-T cell product.

Aims: Herein we report updated clinical data from a Phase I trial assessing the fully human B-cell maturation antigen (BCMA) CAR-T cell product PHE885 in adult patients (pts) with r/r MM.

Methods: This multicenter, open-label study ( NCT04318327 ) characterizes the safety and efficacy of PHE885 manufactured using the novel T-Charge TM platform, which takes <2 d to manufacture the final product and relies on in vivo expansion after CAR-T cell infusion. Pts with MM r/r to ≥2 prior lines of tx, including an immunomodulatory drug, proteasome inhibitor, and anti-CD38 monoclonal antibody, were eligible. Pts received fludarabine and cyclophosphamide for lymphodepletion prior to a single PHE885 intravenous injection. Primary objectives were safety, including dose-limiting toxicities (DLTs) and adverse events (AEs). Secondary objectives included clinical response, T-Charge TM process evaluation, and cellular kinetics.

Results: At abstract submission, 22 pts were infused with PHE885 at the following fixed doses: 2.5×10 6 (n=4), 5×10 6 (n=12), 10×10 6 (n=5), and 14.3×10 6 (n=1) CAR-T cells; 15 pts (4 at 2.5×10 6 , 10 at 5×10 6 , and 1 at 14.3×10 6 ) were evaluable for safety and efficacy at the data cutoff for the abstract (October 13, 2021). Longer follow up of these 15 pts and data from additional pts treated at doses of 5×10 6 and 10×10 6 will be presented at the annual meeting. Of the 15 pts evaluable at data cutoff, DLTs were reported in 3 pts and included asymptomatic grade (gr) 3 transaminitis (n=1, 14.3×10 6 ), asymptomatic gr ≥3 elevated pancreatic enzymes (n=1, 5×10 6 ), and gr 4 neutropenia (n=1, 2.5×10 6 ). Most frequent tx-related gr ≥3 AEs included neutropenia (n=15, 100%), anemia (n=13, 87%), and thrombocytopenia (n=11, 73%). All pts experienced cytokine release syndrome (CRS) per Lee 2014 criteria; 2 had gr ≥3 CRS (n=1, 5×10 6 ; n=1, 14.3×10 6 ). Median times to CRS onset and resolution were 7 d (range, 4-12 d) and 11 d (range, 9-22 d), respectively. Tocilizumab (n=13, 87%), steroids (n=11, 73%), anakinra (n=4, 27%), and pressors (n=3, 20%) were used to manage CRS. Gr 2 neurotoxicity occurred in 4 pts (27%). Pts had an overall response rate of 93%, stringent complete response in 33%, very good partial response (VGPR) in 20%, and PR in 40%. Of evaluable pts at 3 mo post infusion, 43% (3/7) had minimal residual disease (MRD) at <10 –5 sensitivity[JC(1], while 33% (2/6) were MRD negative at <10 –6 by next-generation sequencing. Robust expansion of BCMA CAR-T cells was observed in all pts via qPCR and flow cytometry; maximum expansion (geometric mean C max ) was 316,000 copies/μg by qPCR and 76% of circulating T cells by flow cytometry. Maximum expansion was reached at a median of 14.2 d by qPCR (13.9 d by flow cytometry). Naive-like T cells present in the leukapheresis were preserved during manufacturing of most PHE885 products as measured by flow cytometry, and an increase in naive/T scm populations is observed in ≥VGPR pts post-infusion.

Summary/Conclusion: Low doses of PHE885 manufactured by T-Charge TM in <2 d have a manageable safety profile and encouraging clinical activity in pts with r/r MM. PHE885 expands rapidly in vivo, persists for prolonged periods, and demonstrates a relatively naive T-cell phenotype.

P1447: INFERIOR OUTCOMES OF EU VS. US PATIENTS WITH RELAPSED/REFRACTORY LARGE B-CELL LYMPHOMA AFTER CD19 CAR T-CELL THERAPY ARE IMPACTED BY BASELINE RISK FACTORS AND CAR PRODUCT CHOICE

V. Bücklein 1,2,* , A. Perez 3,4 , G. Iacoboni 5 , K. Rejeski 1,2,6 , U. Holtick 7 , O. Penack 8 , S. Kharboutli 9 , V. Blumenberg 1,2,6 , J. Ackermann 1 , L. Frölich 1 , G. Johnson 10 , K. Patel 10 , B. Arciola 10 , C. Schmidt 1 , O. Albanyan 4 , P. Gödel 7 , E. Hoster 11 , L. Bullinger 8 , A. Mackensen 9 , F. Locke 4 , M. von Bergwelt 1,6 , P. Barba 5 , M. D. Jain 4 , M. Subklewe 1,2,6

1 Department of Medicine III, University Hospital, University of Munich; 2 Laboratory for Translational Cancer Immunology, Gene Center, LMU Munich, Munich, Germany; 3 Blood & Marrow Transplant Program, Miami Cancer Institute, Miami; 4 Department of Blood and Marrow Transplant and Cellular Immunotherapy, Moffitt Cancer Center, Tampa, United States of America; 5 Department of Hematology, University Hospital Vall d’Hebron, Vall d’Hebron Institute of Oncology (VHIO), Department of Medicine, Universitat Autònoma of Barcelona (UAB), Barcelona, Spain; 6 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg; 7 Department I of Internal Medicine, Medical Faculty and University Hospital, Cologne; 8 Medical Clinic, Department for Haematology, Oncology and Tumorimmunology, Charité Universitätsmedizin Berlin, Berlin; 9 Department of Internal Medicine 5, Hematology and Oncology, University of Erlangen-Nuremberg, Erlangen, Germany; 10 USF Health Morsani College of Medicine, Tampa, United States of America; 11 Institute for Medical Information Processing, Biometry, and Epidemiology, LMU Munich, Munich, Germany

Background: CD19 CAR T cells have significantly improved the prognosis of patients with relapsed/refractory (r/r) large B-cell lymphoma (LBCL). Real-world evidence (RWE) in the US has confirmed outcome data from pivotal trials. In contrast, several European consortia reported inferior real-world response rates and survival.

Aims: To understand these divergent outcomes, we sought to characterize differences in patients treated with CD19 CAR T cells in Europe vs. the US.

Methods: In this retrospective observational study, we assessed CAR T cell logistics, patient/disease characteristics, toxicities, response, and survival of r/r LBCL patients treated with Axi-cel or Tisa-cel at five EU and one US CAR T center. We evaluated variables for their association with PFS by univariate and multivariate stepwise Cox regression.

Results: A total of 386 patients were included (EU: 175, USA: 211). Indeed, the objective response rate was lower in the EU (65% vs. 82% in the US, p<0.001). Median PFS (Fig. 1, 3 vs. 8 months, p<0.001) was significantly shorter for EU compared to US patients. Tisa-cel was applied more frequently in the EU cohort (74% vs. 14%, p<0.001). While severe CRS rates did not significantly differ between cohorts, grade ≥3 ICANS was more common in US patients (25% vs. 9%, p<0.001), likely reflecting the more frequent use of of Axi-cel.

In terms of baseline characteristics, median LDH (321 vs. 272 U/l, p=0.02) and ferritin levels (682 vs. 449 ng/ml, p=0.002) were significantly higher in the EU cohort. EU patients received fewer treatment lines (p=0.004), though the percentage of prior auto-SCT (35% vs. 18%, p<0.001) and active/history of CNS disease (17% vs. 7%, p=0.005) was higher. Analysis of CAR-T logistics revealed longer EU vein-to-vein intervals (43 vs. 28 days, p<0.001). The proportion of patients receiving “holding” therapy, defined as lymphoma treatment between indication-to-CAR and apheresis, was comparable between EU and US centers (22% vs. 27%, p=0.32). However, significantly more patients were treated with bridging therapy (between apheresis and transfusion) in the EU (89% vs. 69%, p<0.001).

In univariate analyses, shorter PFS was associated (p<0.05) with lack of response to the latest therapy, longer indication-to-CAR intervals, higher ECOG, stage III/IV disease, IPI, extranodal disease (END), higher LDH, ferritin, CRP levels, and Tisa-cel use. On multivariate regression (n=319), ECOG (adjusted HR 1.4), ferritin (HR 1.5), refractory disease (HR 1.5), END (HR 1.4), and Tisa-cel use (HR 1.5) represented independent risk factors.

Of note, the aggregate of these four independent baseline risk factors (ECOG, ferritin, refractory disease, END) did not significantly differ between EU vs. US patients (p=0.18), indicating that product choice likely influenced the observed differences in PFS. Interestingly, although Axi- vs. Tisa-cel did not confer different outcomes for patients with 0-1 risk factors (p=0.47), Axi-cel led to significantly improved PFS compared to Tisa-cel in patients with 2-4 risk factors (p<0.001).

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Summary/Conclusion: This is the first analysis to comprehensively compare CAR T cell treatment characteristics in Europe vs. the US in a large real-world cohort. Response rates and PFS were significantly inferior in the EU, potentially due to the use of Tisa-cel in adverse-risk patients. Our data suggest that Tisa- and Axi-cel are equally effective in low-risk patients (≤1 risk factor - ECOG, ferritin, refractory disease, END), but that Axi-cel facilitates improved outcomes in patients with 2-4 risk factors, with a representative toxicity profile.

P1448: LONG-TERM SURVIVORS AFTER FAILURE OF CAR-T CELL THERAPY FOR R/R LARGE B-CELL LYMPHOMA (LBCL): A GLA/DRST STUDY

P. Derigs 1,* , W. Bethge 2 , U. Holtick 3 , B. von Tresckow 4 , F. Ayuk 5 , O. Penack 6 , V. Vucinic 7 , M. von Bonin 8 , C. Baldus 9 , D. Mougiakakos 10 , G. Wulf 11 , U. Schnetzke 12 , M. Stelljes 13 , S. Thomas 14 , R. Schroers 15 , N. Kroeger 5 , P. Dreger 1,16

1 Department of Internal Medicine V, University Hospital Heidelberg, Heidelberg; 2 Department of Internal Medicine II, University Hospital Tuebingen, Tuebingen; 3 Department of Internal Medicine I, University Hospital Cologne, Cologne; 4 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen; 5 Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg; 6 Department of Hematology, Oncology and Tumorimmunology, University Hospital Charité Berlin, Berlin; 7 Medical Department for Hematology, Cell Therapy and Hemostaseology, University Hospital Leipzig, Leipzig; 8 Department of Internal Medicine I, University Hospital Dresden, Dresden; 9 Department of Internal Medicine II, University Hospital Schleswig-Holstein, Kiel; 10 Department of Internal Medicine V, University Hospital Erlangen, Erlangen; 11 Department of Hematology and Medical Oncology, University Medical Center Goettingen, Goettingen; 12 Department of Internal Medicine II, University Hospital Jena, Jena; 13 Department of Medicine A, University Hospital Muenster, Muenster; 14 Department of Internal Medicine III, University Hospital Regensburg, Regensburg; 15 Department of Medicine, Hematology and Oncology, University Hospital Bochum, Bochum; 16 On behalf of the German Lymphoma Alliance (GLA), and the German Stem Cell Transplantation Registry (DRST), Germany

Background: Information on the long-term outcome of patients who relapse or progress after CD19-directed CAR-T therapy with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) for >2L treatment of LBCL is still limited.

Aims: The purpose of this study was to investigate characteristics, relapse patterns and management strategies in patients with LBCL surviving one year or longer after having failed a preceding standard of care CAR-T therapy.

Methods: The study sample consisted of all patients who had a relapse/progression event in a prior GLA/DRST real world analysis (Bethge et al, ASH 2022 #3822). Baseline patient, disease, and treatment data were collected from MED-A cellular therapy forms. Centers were approached to provide additional treatment and follow-up information for each patient with a documented event.

Results: Altogether, relapse/progression (REL) after CAR-T therapy was reported for 214 of 356 patients (60%) of the original study. For 143 of 214 patients (67%) a full dataset as needed for the purpose of this study was available. OS at 12 months post REL of these 143 patients was 22% with no significant difference between axi-cel and tisa-cel failures ( Figure ). 26 of 143 patients (18%) survived 12 months or longer from REL, 109 (76%) died within the first year after REL, and 8 patients (6%) were alive but had not reached the 12-month landmark. Patients surviving and not surviving 12 months post REL, respectively, were comparable for age, gender, IPI at lymphodepletion, PS, prior HCT, and 1 st salvage regimens used. However, long-term survivors had less often bridging failure prior to CAR-T therapy, met less often ineligibility criteria for ZUMA1, had less often REL within the first 30d and more often beyond 100d post CAR-T therapy, and had more often received alloHCT for consolidation of treatment responses post REL (Table). 12-month OS post REL of the 40 patients who proceeded to alloHCT was 34% (19-39%).

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Summary/Conclusion: Long-term survival after failure of CAR-T therapy for LBCL is possible in about 20% of the patients. Predictors of a favorable survival post REL are good-risk pre CAR-T features and a long interval between CAR-T therapy and REL. AlloHCT can successfully consolidate salvage treatment responses in a sizable proportion of patients and may improve outcome of CAR-T failure.

P1450: AUTOLOGOUS STEM CELL TRANSPLANTATION INDUCES HIGHER LEVEL OF EXHAUSTED T-CELLS IN DLBCL PATIENTS BEFORE LEUKAPHERESIS FOR CAR-T CELL THERAPY.

M. Farina 1,* , M. Chiarini 2 , C. Almici 3 , A. Beghin 4 , E. Accorsi Buttini 1 , A. Galvagni 2 , E. Ferrari 3 , A. Bianchetti 3 , F. Zuccalà 5 , S. Piva 6 , I. Volonghi 7 , L. Poli 7 , D. Moratto 2 , A. Leoni 1 , F. Re 18 , K. Bosio 1,8 , S. Bernardi 1,8 , N. Polverelli 1 , A. Turra 1 , E. Morello 1 , C. Cattaneo 9 , A. Lanfranchi 4 , M. Malagola 1 , D. Brugnoni 2 , A. Re 9 , D. Russo 1

1 Unit of Blood Diseases and Bone Marrow Transplantation, Cell Therapies and Hematology Research Program, Department of Clinical and Experimental Sciences, University of Brescia; 2 Clinical Chemistry Laboratory, Flow Cytometry Section, Diagnostics Department; 3 Laboratory for Stem Cells Manipulation and Cryopreservation, Department of Transfusion Medicine; 4 Stem Cell Laboratory; Section of Hematology and Blood Coagulation Clinical Chemistry Laboratory; Diagnostics Department; 5 First Division of Anesthesiology and Critical Care Medicine; 6 Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia; University Division of Anesthesiology and Critical Care Medicine; 7 U.O.C. Neurology Center for Neuromuscular Diseases, Department of Clinical and Experimental Sciences, University of Brescia; 8 Research Center Ail (CREA), Chair of Hematology-Department of Clinical and Experimental Sciences, Unit of Blood Diseases and Stem Cell Transplantation, University of Brescia; 9 U.O.C. Hematology, Department of Clinical Oncology, ASST Spedali Civili Brescia, Brescia, Italy

Background: Chimeric antigen receptor T-cells (CAR-T) are an highly effective therapy in relapse/refractory DLBCL, PMBCL and ALL, but almost half of treated patients (pts) still relapse. CAR-T cells generated from T-lymphocytes (T-Ly) enriched for early lineage T cells has shown durable persistence and replicative potential both in animal and in vivo models, while CAR-T cells from “exhausted” T-Ly have been related with poorer response. In the prospective BioCART BS study, we assessed the T-cell repertoire before the Ly-apheresis in DLBCL patients for evaluating the impact of previous treatments and efficacy of CAR-T therapy.

Aims: To evaluate T-cells repertoire before the Ly-apheresis in high risk DLBCL pts.

Methods: Between April and December 2021, a total of 11 DLBCL pts underwent Ly-apheresis for CAR-T manufacturing. Since cryopreservation of the apheresed Ly is allowed for Tisa-cel, DLBCL pts with poor prognostic risk factor (refractory to first line of treatment; PET positivity before Autologous Stem Cell Transplantation [ASCT]; first complete response less than 12 months) were enrolled in an early/“pre-emptive” Ly-apheresis program, scheduling leukapheresis as soon as possible, with the aim to collect more “fit” Ly.

Combinations of monoclonal antibodies directed against CD45RA, CCR7, CD3, CD4, and CD8 were used by Flow Cytometry to evaluate the following CD4+/CD8+ T-ly subsets: T-naïve (CD45RA+CCR7+); central memory (CM, CD45RA-CCR7+); effector memory (EM, CD45RA-CCR7-); terminally differentiated (TD; CD45RA+CCR7-).

For evaluating CAR-T cells expansion, pts’ cells were tested with a specific biotinilated CD19 CAR reagent followed by incubation with an anti-biotin PE conjugated MoAb. The time points were: at day +1, +3, +7, +10, +14, +21 after CAR-T cells infusion, and then monthly until 1 year or at any time for relapse onset. All stained samples were acquired on a Canto II (BD Bioscience) flow cytometer and analyzed using DIVA software version 8.0.2. Continuous and categorical variables were compared using an unpaired T-test, Mann-Whitney U test, or Chi-square test, as appropriate.

Results: Table 1 shows the main patients’ characteristics. 5 out of the 11 (45%) pts performed Ly-apheresis before ASCT. At the time of Ly-apheresis, no significative differences were observed between pts who previously underwent ASCT and those who did not, except for age. ASCT induced a more “exhausted” T-cells repertoire. Indeed, pts who previously underwent ASCT had lower CD4+/CD8+ ratio (p 0.04) (Fig 1C) as well as lower levels of CD4+ Naïve T cells, both in terms of absolute count (p 0.05) and as percentage of CD4+ T cells (p 0, ), and lower prevalence of CD8+ Naïve T cells (p 0.03) (Fig 1A). Moreover they also presented higher prevalence of CD4+ EM T cells (p 0.01) and of CD4+ TD T-cells (p 0.05), as well as higher level of both CD8+ CM (p 0.03) and TD (p 0.05) T cells (Fig 1B). 3 out of the 6 pts who underwent ASCT before Ly-apheresis and 2 of the 5 who did not, sent Ly for manufacturing. To date, no differences were apparently observed on CAR-T cells expansion comparing the two groups, but the number of pts analyzed is too small to make any consideration (Fig 1D for pts with longer follow up).

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Summary/Conclusion: ASCT before Ly-apheresis may result in a higher prevalence of “exhausted” T-cells. In DLBCL pts primary refractory or at high risk of relapse after second line of treatment, timely pre-ASCT Ly-apheresis would be considered.

P1451: PRELIMINARY RESULTS FROM THE FLU/CY/ALEMTUZUMAB ARM OF THE PHASE I BALLI-01 TRIAL OF UCART22, AN ANTI-CD22 ALLOGENEIC CAR-T CELL PRODUCT, IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) CD22+ B-CELL ALL

N. Jain 1,* , G. Roboz 2 , M. Konopleva 1 , H. Liu 3 , G. Schiller 4 , E. Jabbour 1 , P. Desai 2 , D. Whitfield 5 , A. Haider 5 , O. Zernovak 5 , M. Frattini 5 , C. Brownstein 5 , R. Larson 3

1 Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 2 Medicine, Weill Cornell Medicine/New York Presbyterian Hospital, New York; 3 Medicine, The University of Chicago, Chicago; 4 Medicine, David Geffen School of Medicine at UCLA, Los Angeles; 5 Cellectis, Inc., New York, United States of America

Background: UCART22 is a genetically modified allogeneic T-cell product manufactured from non-HLA matched healthy donor cells. Donor-derived T-cells are transduced with a lentiviral vector to express the anti-CD22 chimeric antigen receptor (CAR; anti-CD22 scFv-41BB-CD3ζ) and are modified using Cellectis’ TALEN ® technology to disrupt the T-cell receptor α constant (TRAC) and CD52 genes to minimize risk of graft-vs-host disease (GvHD) and allow use of anti-CD52 drugs for lymphodepletion (LD).

Preliminary results from the phase 1, open-label, dose-escalation BALLI-01 study ( NCT04150497 ) in patients (pts) with R/R B-ALL showed that UCART22 is tolerable and shows anti-leukemic activity after LD with fludarabine and cyclophosphamide (FC) (Jain, ASH 2020). Host T-cell recovery was observed in all pts (days 7-28).

Aims: Subsequent UCART22 dose-finding cohorts utilized a new LD regimen that included addition of alemtuzumab to FC (“FCA”), which could deepen and sustain host T-cell depletion and promote CART-cell expansion and persistence.

Methods: After informed consent, eligibility criteria include age 15‒70 yrs, adequate organ function, ECOG PS ≤ 1, B-ALL blast CD22 expression ≥ 70% by flow cytometry. Pts must have received ≥ 2 Tx regimens. After LD with FCA (F=30 mg/m2 × 3d, C=0.5 g/m 2 × 3d, A=20 mg/d × 3d), pts received one infusion of UCART22 at 1 ×10 6 cells/kg (FCA-DL2) or 2.5 ×10 6 cells/kg (FCA-DL2i). DLTs are assessed over a 28d observation period after UCART22 infusion. The primary endpoint is the safety, tolerability, and MTD of UCART22. Additional endpoints are anti-leukemic activity (investigator assessed), and expansion, trafficking, persistence of UCART22 in peripheral blood [PB] and bone marrow [BM] assessed by flow cytometry and vector copy number [VCN] and immune reconstitution assessed by flow cytometry.

Results: As of October 1, 2021, 12 pts received LD; 11 were administered UCART22, of which 6 received UCART22 after FCA. Enrollment in FC-DL1/DL2 (n=5), FCA-DL2 (n=3), and FCA-DL2i (n=3) was complete. Median age was 30 yrs (20-61) and 7/11 (58%) had B-ALL with recurrent genetic abnormalities (WHO classification). Pts were heavily pretreated, having received a median of 3 (2–6) prior Tx including blinatumomab 8/11 (73%); inotuzumab 5/11 (45%); CD19 CAR-T therapy 3/11 (27%); and HSCT 3/11 (25%). Median BM blast % prior to LD was 60 (3.5-97). UCART22 at DL2 and DL2i administered after FCA LD regimen was well tolerated. No DLTs nor ICANS were observed. Three patients experienced CRS (grade [G]1, n=2; G2, n=1). Two patients experienced G ≥3 infections that were not related to UCART22. One serious AESI of G2 GvHD limited to the skin was reported in the setting of reactivation of prior allogeneic transplant donor stem cells.

Anti-leukemic activity (blasts < 5% by day 28) was observed in 2 pts in the FCA cohort; 1 in DL2 and 1 in DL2i. Host lymphocytes on average remained suppressed throughout the 28-day DLT observation period for all pts in the FCA cohorts, correlating with UCART22 proliferation and increases in inflammatory cytokines.

Summary/Conclusion: UCART22 after FCA LD was well tolerated and associated with improved host lymphocyte suppression and UCART22 expansion. UCART22 expansion correlated with antileukemic activity with changes in relevant inflammatory markers. Overall, the data support the safety and antileukemic activity of UCART22 after FCA LD in pts with R/R B-ALL. The study is enrolling pts at FCA-DL3 (5 × 10 6 UCART22 cells/kg).

P1452: THE PRELIMINARY SAFETY AND EFFICACY STUDY OF SC-U02, A NON-VIRAL GENOME TARGETING, ANTI-CD19 UNIVERSAL CAR-T PRODUCT, IN RELAPSED/REFRACTORY (R/R) DIFFUSE LARGE B-CELL LYMPHOMA PATIENTS

M. Hao 1 , D. Yin 1 , Y. Li 1 , Y. Gi 1 , J. Tian 1 , H. Guo 1 , D. Fan 1 , W. Qin 1 , Q. Liu 1 , W. Wang 1 , L. Li 2 , Z. Peng 2,3 , L. Liu 1,*

1 Hematology, Tangdu Hospital, Fourth Military Medical University; 2 Sunnycell Therapeutics Co., Ltd; 3 Faculty of Medicine, Northwest University, Xi’an, China

Background: Allogeneic CAR-T therapy has made exciting progresses and represents the future trend of this T cell therapy. However, the classic way of using CD52 antibody for lymphodepletion, which aim to overcome Host versus Graft reaction (HvG), would put patients at risk of infection and longer in-patient care duration. In this study, we conducted a Non-viral Genome Targeting approach for producing the allogeneic anti-CD19 CAR-T cells (nvGT UCART19), SC-U02. To avoid Graft versus Host Disease (GvHD), TCR gene was knocked-out. And to prevent T cell rejection, B2M gene was disrupted. Meanwhile, the anti-CD19 CAR gene together with an artificial B2M-HLA-E gene were inserted at the same TRAC locus (Figure 1), which prevents NK cell rejection triggered by the loss of surface expression of HLA class I due to B2M gene knock-out (Figure 2). Thus, it prolongs the persistence of SC-U02 in patients’ body without the aid of CD52 antibody, which reduces the infection risk of patients in UCAR-T treatment process. The efficacy of SC-U02 had been demonstrated in our previous pre-clinical studies. In this single-arm, single center, dose-finding, investigator initiated trial (IIT) (ChiCTR2100051028), its safety and efficacy in relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) patients were explored.

Aims: The primary objective of this trial is to assess safety profile of SC-U02, including evaluation of adverse events (AEs) and AEs of special interests, such as Cytokine Release Syndrome (CRS), neurotoxicity, GvHD and infection. Secondary objective is to evaluate efficacy as measured by the ratio of overall response (ORR) and complete response (CR). The persistence of SC-U02 in human body is observed as an exploratory endpoint.

Methods: The primary objective of this trial is to assess safety profile of SC-U02, including evaluation of adverse events (AEs) and AEs of special interests, such as Cytokine Release Syndrome (CRS), neurotoxicity, GvHD and infection. Secondary objective is to evaluate efficacy as measured by the ratio of overall response (ORR) and complete response (CR). The persistence of SC-U02 in human body is observed as an exploratory endpoint.

Results: Up to December 1 st 2021, two patients with r/r DLBCL had been enrolled in this study and completed their treatment. The median age was 56 years (range, 52 to 59), and the number of previous treatment course is from 3 to 13. They all went through at least 2 lines of systematic chemotherapy, and both were refractory to last therapy. Baseline characteristics refer to Table 1.

CRS occurred in one patients (50%) with grade 2 CRS. Tocilizumab was administered during CRS for this patient, and the symptom was reversed afterwards. The time of CRS onset was D8 and it lasted for 11 days. Data including body temperature (Figure 3), Interleukin-6 (IL-6) (Figure 4) are in accordance with the trend of CRS. Neither of them developed neurotoxicity and infection. No GvHD was observed.

Until D56 post nvGT UCART19 cell infusion, 1/2 (50%) cases achieved PR (Figure 5) and the other subject did not response (Table 1). CAR-positive T cell percentage showed a peak around D14 (Figure 6) for the responded patient.

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Summary/Conclusion: In this trial, we see patient responded to this novel allogeneic CAR-T therapy which probably shows promise of the approach. However, due to limited cases reported, SC-U02’s safety and efficacy profile still needs to be decided. The research is ongoing and 6×10 8 cell dose level will be assessed later on.

P1453: STABLE TRANSDUCTION OF FETAL HEMOGLOBIN IN PATIENTS WITH SICKLE CELL DISEASE IN THE PHASE 1/2 MOMENTUM STUDY OF ARU-1801 GENE THERAPY AND REDUCED INTENSITY CONDITIONING

M. Grimley 1 , M. Kent 2,* , M. Asnani 3 , A. Shrestha 4 , S. Felker 1 , C. Lutzko 1 , P. Arumugam 1 , S. Witting 1 , J. Knight-Madden 3 , O. Niss 1 , C. Quinn 1 , C. Lo 4 , C. Little 4 , A. Dong 4 , P. Malik 1

1 Cincinnati Children’s Hospital Medical Center, Cincinnati; 2 Atrium Health Levine Children’s Hospital, Charlotte, United States of America; 3 Caribbean Institute for Health Research, Kingston, Jamaica; 4 Aruvant Sciences, New York, United States of America

Background: Sickle cell disease (SCD) is a genetic red blood cell disorder that causes chronic hemolytic anemia, progressive organ damage, and painful vaso-occlusive crises. ARU-1801 is a gene therapy for SCD, designed to produce HbF G16D expression in autologous CD34+ hematopoietic stem cells (HSCs). The ongoing Phase 1/2 MOMENTUM study ( NCT02186418 ) is evaluating the safety and efficacy of ARU-1801, which is administered following reduced-intensity conditioning (RIC) and has demonstrated clinically meaningful improvements with drug product vector copy number (DP VCN) at and below 1.

Aims: Here, we demonstrate stable, high levels of HSC engraftment in patients treated with ARU-1801 gene therapy after RIC melphalan.

Methods: Adults (18-45 years old) with severe SCD (as defined by recurrent vaso-occlusive events [VOE] and acute chest syndrome) were screened for eligibility. Prior to infusion of ARU-1801, all patients received a single IV dose of RIC melphalan (140 mg/m 2 ). Endpoints included measures of melphalan pharmacokinetics, safety, engraftment, peripheral blood (PB) VCN, hemoglobin, and SCD-related outcomes.

Results: As of Feb 1, 2022, five patients (mean age [range], 26 [19-35] years old) have been treated with ARU-1801 gene therapy for SCD. Transient thrombocytopenia and neutropenia lasted a mean of 6 and 8 days, respectively. There have been no other serious adverse events related to chemotherapy or ARU-1801 to date. To quantify the proportion of genetically modified HSCs that engrafted in the setting of RIC, the ratio of PB VCN to DP VCN was measured over time (Figure 1). Four patients (Patients 1, 3, 4, and 5) had adequate RIC melphalan exposure (AUC = 6.8-10.1 mg.h/L) following a 140 mg/m 2 dose, maintaining a ratio of 72-97% at 6 months, which has remained stable. Anti-sickling globin expression has correlated with PB VCN and also remains stable (16-25% HbF G16D , 22-39% total anti-sickling globin). One patient (Patient 2) had renal hyperfiltration (eGFR = 200 mL/min/1.73 m 2 ) resulting in sub-therapeutic melphalan exposure (AUC = 5.7 mg.h/L) and a decline in PB VCN which stabilized at ~10% of DP VCN. The protocol has since been amended to adjust the dose of melphalan for patients with renal hyperfiltration.

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Summary/Conclusion: High engraftment in this trial compares favorably to other clinical trials of SCD gene therapy which have observed marked decreases in VCN shortly after infusion and is particularly significant in the setting of RIC, where a population of patients’ unmodified HSCs remain and compete with the genetically modified HSCs. The ARU-1801 manufacturing process uses a proprietary methodology to prevent HSC differentiation in culture, which may favor transduction of long-term HSCs over short-term progenitor cells. Overall, preliminary data suggest ARU-1801 achieves stable, high engraftment of transduced HSCs even in the setting of RIC, resulting in robust long-term maintenance of therapeutic hemoglobin levels. We have previously shown (1) ARU-1801 can reach effective levels of anti-sickling hemoglobin at VCN ≤1, with 42% HbF G16D expression per DP VCN and (2) HbF G16D may have a more potent anti-sickling effect than endogenous HbF. Along with these effects, strong long-term HSC engraftment (as demonstrated by high, stable ratios of PB to DP VCN) requiring only RIC makes ARU-1801 a promising gene therapy alternative to treatments that require myeloablative conditioning, offering amelioration of SCD phenotype without the toxicities and resource utilization of full myeloablation.

P1454: REAL-WORLD OUTCOMES OF BREXUCABTAGENE AUTOLEUCEL (BREXU-CEL) FOR THE TREATMENT OF RELAPSED OR REFRACTORY (R/R) MANTLE CELL LYMPHOMA (MCL) IN THE UNITED STATES (US)

F. Locke, MD 1,* , Z.-H. Hu, MPH 2 , J. Gerson, MD 3 , M. J. Frank, MD, PhD, L. E. Budde, MD, PhD, M. Wang, MD 6 , B. Logan, PhD 7 , I. Kloos, MD, PhD, R. Siddiqi, PhD 2 , J. Shah, MD 2 , H. Xu, MD, PhD, M. Pasquini, MD, MS

1 H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; 2 Kite, a Gilead company, Santa Monica, CA; 3 University of Pennsylvania, Philadelphia, PA; 4 Stanford Unversity, Stanford, CA; 5 City of Hope National Medical Center, Duarte, CA; 6 The University of Texas MD Anderson Cancer Center, Houston, TX; 7 Medical College of Wisconsin, Milwaukee, WI, United States of America; 8 Kite, a Gilead company, Boulogne-Billancourt, France, France

Background: Brexucabtagene autoleucel (brexu-cel) is a CD19 CAR T-cell therapy approved in the US for adult patients (pts) with r/r MCL. In the primary analysis of the pivotal ZUMA-2 trial, 93% of the pts receiving brexu-cel achieved objective response with a 67% CR rate. Estimated 1-year OS and PFS were 83% and 61% respectively. Grade ≥ 3 CRS and neurologic events occurred in 15% and 31% of the pts. A post-authorization safety study (PASS) for brexu-cel is ongoing in the US using the Center for International Blood and Marrow Transplant Research (CIBMTR) data registry to collect long-term safety and efficacy information for up to 15 years.

Aims: We present the first report on real-world outcomes of pts in the PASS receiving commercial brexu-cel for r/r MCL, including pts with no prior exposure to BTK inhibitors (BTKi-naïve) who would otherwise be ineligible for inclusion in the reported ZUMA-2 cohort.

Methods: From July 2020 to December 2021, 254 pts (planned total 500 pts) with r/r MCL have been enrolled in the PASS from 70 US centers. Of these, 135 pts had at least one scheduled follow-up at Day 100 and were included in this analysis. Median follow-up was 6 mo. Efficacy outcomes of interest were ORR and CR as best response, DOR, PFS and OS. Adverse events included CRS (Lee 2014 criteria for comparison with ZUMA-2), ICANS (ASTCT consensus grade), prolonged cytopenia, infection and subsequent neoplasm. ORR and CR were evaluated among pts with PET/CT or CT scan assessment. Percentages and 95% Clopper-Pearson exact CIs were calculated for dichotomous outcomes. DOR, PFS and OS were estimated using the Kaplan-Meier estimator. Cumulative incidence functions were used for CRS and ICANS resolution rates.

Results: Of the 135 pts, 53% were 65 years or older (median 65.6 years), 79% were male, 87% had an ECOG performance score of 0 or 1, and 61% were chemo-resistant prior to infusion. Extranodal CNS involvement prior to infusion occurred in 5 (4%) pts while clinically significant comorbidities (Sorror 2013) were present in 71% of the pts. Prior to leukapheresis, pts received a median of 4 lines of prior therapy (range 2-12) and 30% had an ASCT. Median time from leukapheresis to infusion was 28 days (range 18-96). Bridging therapy was given to 19% of the pts. Of all, 12 (9%) pts were BTKi-naïve vs 120 (89%) BTKi-exposed.

Overall response rate for all pts and pts with ≥ 180 days of follow-up were 84% (95% CI 77-90%) [CR 71% (95% CI 63-79%)] and 78% (95% CI 67-87%) [CR 68% (95% CI 56-78%)], respectively. Of those who achieved CR/PR, 85% (95% CI 75-92%) remained in response at 3 mo. OS and PFS at 6 mo were 79% (95% CI 71-86%) and 66% (95% CI 56-75%), respectively. Grade ≥ 3 CRS and ICANS occurred in 9% (95% CI 5-15%) and 29% (95% CI 22-38%) of the pts. Median time to onset of any grade CRS and ICANS were 4 and 7 days, respectively. Tocilizumab and corticosteroids were given to 92% and 86% of pts with both CRS and ICANS. Most CRS [95% (95% CI 88-98%)] and ICANS [75% (95% CI 63-83%)] resolved by 21 days since onset. For pts who survived Day 30, 22% experienced prolonged cytopenia. Infections requiring treatment occurred in 32% of the pts (20% bacterial, 7% fungal and 17% viral). A total of 4 (3%) pts developed subsequent neoplasms (1 MDS/MPN + squamous cell carcinoma, 1 genitourinary cancer and 2 sarcoma).

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Summary/Conclusion: Despite short follow-up, this is the largest report on brexu-cel use in the real-world setting. Early results demonstrated consistent safety and efficacy profile of brexu-cel as observed in ZUMA-2 in a population with broader demographics, disease and treatment profile.

P1455: FIRST-IN-HUMAN TRIAL OF CB-010, A CRISPR-EDITED ALLOGENEIC ANTI-CD19 CAR -T CELL THERAPY WITH A PD-1 KNOCK OUT, IN PATIENTS WITH RELAPSED OR REFRACTORY B CELL NON-HODGKIN LYMPHOMA (ANTLER STUDY)

L. J. Nastoupil 1,* , S. O’Brien 2 , H. E. Holmes 3 , L. Dsouza 1 , D. Hart 4 , E. Matsuda 5 , T. Satterfield-Ledbetter 6 , J. Skoble 7 , E. Garner 8 , M. Bryan 9 , S. Kanner 10 , E. Li 11 , C. T. Thomas 12 , J. Essell 13

1 Lymphoma/Myeloma, UT MD Anderson Cancer Center, Houston; 2 Department of Hematology and Oncology, University of California Irvine, Irvine; 3 Oncology, Texas Oncology/Baylor Sammons Cancer Center, Dallas; 4 Research, Oncology Hematology Care, Cincinnati; 5 UCI Cancer Center, University of California Irvine, Irvine; 6 Clinical Oncology Research Coordination, Baylor Research Institution, Dallas; 7 Technical Operations; 8 T Cell Therapeutics; 9 Program Management/Operations; 10 R&D, Caribou Biosciences, Inc., Berkeley; 11 EffectStats LLC, Cambridge; 12 Clinical Development, Caribou Biosciences, Inc., Berkeley; 13 Blood Cancer, Oncology Hematology Care, Cincinnati, United States of America

Background: Autologous CAR-T cell therapies have shown significant benefit in the treatment of patients (pts) with relapsed/refractory B cell NHL (r/r B-NHL). Autologous CAR-T cells may have inherent functional deficiencies or yield an inconsistent product and their manufacturing is lengthy and complex. CB-010 is an allogeneic, anti-CD19 CAR-T cell therapy derived from healthy donor T cells. Pts receiving CB-010 do not require leukapheresis or bridging therapy. A next-generation CRISPR technology (Cas9 chRDNAs) that significantly reduces off-target editing was used to generate 3 genome edits in the manufacture of CB-010: knockout (KO) of the TRAC gene to eliminate TCR expression to reduce the risk of GvHD; site-specific insertion of an anti-CD19 CAR into the TRAC locus; and KO of the gene encoding PD-1, designed to limit CAR-T cell exhaustion. Statistically significant preclinical survival benefit across B-NHL subtypes and longer-duration efficacy in vivo compared to similar CAR-T cells without a PD-1 KO support the clinical evaluation of CB-010.

Aims: To report the preliminary safety, tolerability, and initial antitumor efficacy of CB-010.

Methods: ANTLER is a multicenter, Phase 1 trial in pts with r/r B-NHL. A 3 + 3 dose escalation design followed by expansion at the maximum tolerated dose (MTD) and/or the recommended Phase 2 dose (RP2D) is used. Primary objectives are to determine the RP2D and the safety and tolerability of CB-010. Additional objectives include preliminary antitumor activity, and PK. After serially receiving lymphodepletion therapy with cyclophosphamide (60mg/kg/day x 2 days) and fludarabine (25mg/m 2 /day x 5 days), pts receive a single dose infusion of CB-010.

Results: As of the data-cut-off date of 23 Feb 2022, 6 pts with r/r B-NHL (DLBCL: 2, PMBCL: 1, FL: 2, MCL: 1) were treated with CB-010 at the initial dose level of 40 x10 6 CAR-T cells. Pts received a median of 3 prior therapies (range 2-8), and all had relapsed on their last therapy. As of the data cut-off, 5 of 6 pts completed the 28-day DLT period. One CRS (Grade 1) has been reported; this pt experienced concurrent CRS and Grade 3 ICANS, characterized as a DLT. The pt received tocilizumab and steroids and recovered from the DLT within 39 hours. There were no cases of GvHD. In addition, 3 of 6 pts developed Grade 3 or 4 AEs within the first 28 days: neutropenia (50%), thrombocytopenia (33%), anemia (16.7%), and hypogammaglobulinaemia (16.7%). Results are preliminary and unaudited.

Five out of 6 pts had post baseline tumor assessments and achieved complete response (4) or partial response (1). With a median follow up of 4.5 months (range: 0.6-8.6 months), 4 pts have ongoing responses, 1 pt relapsed at the 6-month evaluation and 1 patient is in the DLT evaluation period.

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Summary/Conclusion: CB-010, an allogeneic CD19-directed CAR-T cell therapy with a PD-1 KO, demonstrated promising preliminary safety and efficacy in pts with r/r B-NHL at the initial dose level . The study is ongoing, and additional safety and efficacy data will be provided at the meeting. The trial is registered on clinicaltrials.gov ( NCT04637763 ).

P1456: EARLY SAFETY AND CLINICAL COURSE OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND MEASURABLE RESIDUAL DISEASE RECEIVING GTA002, AN OFF-THE-SHELF, EX VIVO-CULTURED ALLOGENEIC NK CELL PREPARATION

M. Heuser 1,* , W. Fiedler 2 , A. Block 2 , D. C. de Leeuw 3 , A. van de Loosdrecht 3 , A. Astrid Tschan-Plessl 4 , J. Passweg 4 , A. Kloos 1 , A. Schwarzer 5 , L. Venturini 1 , A. D. Duru 6 , K. Pinkernell 6 , A. Ganser 1

1 Department for Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover; 2 II. Medizinische Klinik und Poliklinik, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 3 Department of Hematology, Amsterdam University Medical Center, Amsterdam, Netherlands; 4 Clinical and Diagnostic Hematology, University Hospital Basel, Basel, Switzerland; 5 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 6 Glycostem Therapeutics, Oss, Netherlands

Background: Measurable residual disease (MRD) is a predictor of adverse prognosis in patients with Acute Myeloid Leukemia (AML). Elimination of MRD in patients having reached complete remission (CR) could therefore improve their long-term outcomes.

Aims: In the WiNK trial ( NCT04632316 ), AML patients, who are in CR/CRi with MRD positivity, but are not undergoing allogeneic hematopoietic stem cell transplantation and who have signed informed consent, are treated with an “off-the-shelf” (readily available), allogeneic (unmatched), cryopreserved NK cell preparation (GTA002), which is generated from CD34 + hematopoietic stem and progenitor cells derived from umbilical cord blood.

Methods: The WiNK trial is an ongoing, prospective, open-label, single arm, multicenter Phase I/IIa trial to evaluate the safety and efficacy of GTA002. The Phase I portion follows a 3 + 3 dose escalation design with 3 cohorts testing 1, 2 or 3 infusions (dose levels (DL) 1 to 3) with up to 1 x 10 9 viable NK cells/dose, spaced 4 days apart, resulting in a cumulative dose range of 1 (lowest) to 3 x 10 9 (highest) viable NK cells. GTA002 is infused after a conditioning regimen of cyclophosphamide (Cy) and fludarabine (Flu) from day -5 to -3 at 300 and 30 mg/m 2 , respectively, with no subsequent planned antileukemic treatment. MRD is assessed by multiparametric flowcytometry (MRD-MFC) and error corrected next generation sequencing (MRD-NGS), while chimerism analysis is assessed by duplex qPCR.

Results: To date, three patients have been treated at DL1 and two at DL2. Median age of all 5 patients was 73 years (range 69 to 81) with an ELN risk category of favorable in one, intermediate in three and adverse in one patient. Infusions with GTA002 were well tolerated at DL1, with no serious adverse events (SAE) or adverse events (AEs) related to the IMP. One SAE Grade (G) 3 febrile neutropenia related to the conditioning was reported, resulting in a protocol change to a lower dose of Cy/Flu. No ICANS, CRS or GvHD was reported. Severe AEs were almost all (8 of 9) related to the conditioning regimen: anemia G3 (2x), leukopenia G4 (2x), neutropenia G3 (1x) and G4 (1x), thrombocytopenia G4 (1x), febrile neutropenia G3 (1x), lactic acidosis G3 (1x). At DL2, no SAE or dose limiting toxicity was reported up to the date of data cut off.

At DL1, all three patients achieved changes of their MRD levels with MRD negativity of 9, 1 and 2 months (ongoing) by MRD-MFC. MRD-NGS showed MRD negativity for 6 ( NPM1 ), 4 ( PTPN11 clone only, out of 3 clones: PTPN11, IDH2, SRSF2 ) and 0 months (mutated TP53 ). At DL1, GTA002 could be detected by chimerism analysis in peripheral blood at levels up to 2.4% with trace amounts present up to 4 weeks after infusion.

Summary/Conclusion: GTA002 treatment of AML patients in CR/CRi with MRD was well tolerated with manageable side effects of the conditioning therapy. Even at the lowest dose level, periods of MRD negativity could be documented by MFC and NGS adding to the excitement of the evaluation of allogeneic, off the shelf NK cell-based immunotherapies in hematological malignancies.

P1457: DEMOGRAPHICS AND TREATMENT OUTCOMES IN PATIENTS WITH EBV+ PTLD TREATED WITH OFF-THE-SHELF EBV-SPECIFIC CTL (TABELECLEUCEL) UNDER AN ONGOING EXPANDED ACCESS PROGRAM IN EUROPE: FIRST ANALYSES

S. Choquet 1 , B. Uttenthal 2 , S. Chaganti 3 , P. Comoli 4 , R. Trappe 5 , A. Friedetzky 6 , B. Xing 7 , X. Li 7 , T. Polak 8,9 , L. Gamelin 7 , J.-H. Terwey 6 , D. Dierickx 10,*

1 Hématologie clinique, Sorbonne Université, Hôpital de la Pitié-Salpêtrière, Paris, France; 2 Department of Haematology, Cambridge University Teaching Hospitals NHS Foundation Trust, Cambridge; 3 Department of Haematology, Queen Elizabeth Hospital, Birmingham, United Kingdom; 4 Cell Factory and Pediatric Hematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 5 DIAKO Evangelisches Diakonie-Krankenhaus Bremen, Bremen, Germany; 6 Atara Biotherapeutics, Zug, Switzerland; 7 Atara Biotherapeutics, Thousand Oaks, United States of America; 8 Biostatistics & Epidemiology, ErasmusMC, Rotterdam; 9 Real-World Data, myTomorrows, Amsterdam, Netherlands; 10 Department of Hematology, University Hospital Leuven, Leuven, Belgium

Background: Patients undergoing allogeneic hematopoietic cell transplant (HCT) or solid organ transplant (SOT) are at risk of developing Epstein–Barr virus driven post-transplant lymphoproliferative disorder (EBV + PTLD), a rare hematologic malignancy, which is often aggressive and life-threatening.

Patients with relapsed or refractory (r/r) EBV + PTLD have few treatment options with poor outcomes, demonstrating a clear unmet medical need.

Tabelecleucel is an investigational, off-the-shelf, allogeneic EBV-specific T-cell immunotherapy being studied in patients with serious EBV + diseases ( NCT04554914 & NCT03394365 ) that has demonstrated clinical benefit and favorable safety profile in the treatment of EBV + PTLD after failure of rituximab (R) ± chemotherapy (Prockop, EBMT 2021, ATC 2021, ASH 2021).

Aims: First analyses of demographics, efficacy and safety results of r/r EBV + PTLD patients following SOT or HCT who presented between Jul 2020 and Nov 2021 and consented to research.

Methods: Atara Biotherapeutics supports an ongoing expanded access program (EAP) in Europe for patients with EBV + diseases who have no other treatment options.

Demographics, efficacy and safety data of r/r EBV + PTLD patients following SOT or HCT who presented to the EAP between Jul 2020 and Nov 2021 and consented to research were analyzed.

Results: A total of 48 EAP requests from 9 countries for patients with EBV + diseases were received.

Twenty-two patients from 7 countries consented to this research: 16 EBV + PTLD and 6 EBV + non-PTLD. Of the 16 PTLD patients 15 received at least one dose of tabelecleucel. Overall, 9 out of 15 (60%) patients achieved a response as assessed by the treating physician, with 6 complete responses and 3 partial responses. Eight out of nine responses were seen after the first cycle.

No adverse events were reported as related to tabelecleucel by the treating physician.

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Summary/Conclusion: The successful execution of this European EAP demonstrates the feasibility to deliver an off-the-shelf allogeneic EBV + T-cell therapy in time-sensitive clinical situations when no other treatment options exist. These data show clinically meaningful outcomes for patients with r/r EBV + PTLD post-SOT or post-HCT treated with tabelecleucel consistent with previously reported favorable safety and efficacy profile (Prockop, ASH 2021).

P1458: PATTERNS OF LONG-TERM HEMATOPOIETIC RECOVERY IMPACT SURVIVAL OUTCOMES AFTER CD19-DIRECTED CAR T-CELL THERAPY FOR R/R LBCL

K. Rejeski 1,2,3,* , O. Albanyan 4 , G. Iacoboni 5,6 , V. Bücklein 1,2 , V. Blumenberg 1,2,3 , O. Penack 7 , W. Bethge 8 , S. Völkl 9 , A. Perez 4,10 , C. Carpio 5,6 , C. Schmidt 1 , N. Müller 1 , K. Reid 4 , R. Faramand 4 , M. Davila 4 , L. Bullinger 7 , F. Locke 4 , M. von Bergwelt-Baildon 13 , A. Mackensen 9 , P. Barba 5,6 , M. Jain 4 , M. Subklewe 1,2,3

1 Department of Medicine III - Hematology/Oncology, LMU University Hospital; 2 Laboratory for Translational Cancer Immunology, LMU Gene Center, Munich; 3 German Cancer Consortium (DKTK), Munich Site, and German Cancer Research Center, Heidelberg, Germany; 4 Department of Blood and Marrow Transplant and Cellular Immunotherapy, Moffitt Cancer Center, Tampa, United States of America; 5 Department of Hematology, University Hospital Vall d’Hebron, Vall d’Hebron Institute of Oncology (VHIO), Barcelona; 6 Department of Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain; 7 Department of Hematology, Oncology and Tumorimmunology, Charité Universitätsmedizin Berlin, Berlin; 8 Department of Hematology, Oncology, Immunology and Rheumatology, University Hospital Tübingen, Tübingen; 9 Department of Internal Medicine 5, Hematology and Oncology, University of Erlangen-Nuremberg, Erlangen, Germany; 10 Blood & Marrow Transplant Program, Miami Cancer Institute, Miami, United States of America

Background: Real-world evidence has underlined the role of hematological toxicity and infections in driving the toxicity burden of CD19 CAR-T. We have recently developed a classification system for post-CAR-T hematotoxicity, identifying three unique phenotypes of neutrophil recovery (Rejeski et al Blood 2021). However, their prognostic impact remains poorly understood.

Aims: We sought to understand potential pathomechanisms underlying hematopoietic recovery patterns, and characterize their influence on toxicity and clinical outcomes.

Methods: In this multicenter retrospective observational study, we analyzed 316 pts. receiving Axicabtagene ciloleucel (n=194) or Tisagenlecleucel (n=122) for R/R LBCL in a real-world setting. The phenotypes of neutrophil recovery were defined as follows:

1) Quick Recovery: sustained neutrophil recovery without a second dip below an ANC<1000/µl.

2) Intermittent Recovery: neutrophil recovery (ANC>1500/µl) followed by a second dip with an ANC<1000/µl after day 21.

3) Aplastic: continuous severe neutropenia (ANC<500/µl) ≥14 days.

Multivariate analysis was performed as stepwise binary logistic regression for the aplastic vs. non-aplastic phenotypes. Variables with a p<0.2 on univariate analysis were included. Clinical outcomes were studied via Kaplan-Meier estimates.

Serum cytokines were analyzed using the Ella automated immunoassay system (ProteinSimple) in 70 pt. from Moffitt Cancer Center. The plasma proteome was characterized in a further 38 pts. from the LMU Munich across four time points (day 0, 4, 14, 28) using a 92-protein multiplex proximity extension assay (Olink Bioscience).

Results: The overall distribution of the ‘quick’ (Q), ‘intermittent’ (I) and ‘aplastic’ (A) phenotypes was 40%, 41%, and 19%, respectively. Prior to lymphodepletion, ‘aplastic’ patients displayed increased systemic inflammation (median ferritin 1168 ng/ml), impaired hematopoietic function (median platelet count 101 G/l) and high tumor burden (median LDH 344 U/l).

‘Aplastic’ patients displayed a longer median duration of severe neutropenia, (A vs. I vs. Q: 28 vs. 10 vs. 5 days, p<0.001), higher rate of severe infections (A vs. I vs. Q: 40% vs. 22% vs. 18%), and a trend towards higher 1-year non-relapse mortality (A vs. I: 10% vs. 4%, p=0.09). Progression-free (PFS) and overall survival (OS) was poor in the ‘aplastic’ patients (Fig. 1, 1-yr PFS 24%, 1-yr OS 48%). On the other hand, ‘intermittent’ patients exhibited the best survival outcomes (HR for PFS, I vs. A: 0.46, 95% CI 0.32-0.69). Median PFS was 3 vs. 4 vs. 20 months (A vs. Q v. I, p<0.001); median OS was 8 vs. 23 months vs. not reached (A vs. Q v. I, p=0.0025).

On multivariate regression (n=316), the aplastic phenotype was primarily driven by baseline ECOG (OR=1.6, p=0.05), ferritin (OR=2.5, p=0.01), ANC (OR=0.3, p=0.01), and hemoglobin (OR=0.8, p=0.04). Of note, high-grade CRS/ICANS and CAR product were not associated with the aplastic phenotype. Initial analyses of inflammatory patterns revealed higher peak IL-15 levels and an increased ratio of ANG2 to ANG1 in ‘aplastic’ patients in both cohorts, with ANG-1 levels decreasing over time.

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Summary/Conclusion: Hematopoietic recovery patterns impact disease outcomes after CD19 CAR-T. Surprisingly, a phenotype associated with recurrent neutrophil dips (i.e. prolonged cytopenia) was associated with improved survival outcomes. The poor-risk ‘aplastic’ phenotype was driven by baseline inflammation and hematopoietic function, and characterized by progressive endothelial dysfunction. Further mechanistic exploration is warranted and ongoing.

P1459: SAFETY AND EFFICACY FINDINGS OF AUTO1, A FAST-OFF RATE CD19 CAR, IN RELAPSED/REFRACTORY B-CELL NON-HODGKIN’S LYMPHOMA (B-NHL), AND CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) / SMALL LYMPHOCYTIC LYMPHOMA (SLL)

C. Roddie 1,* , J. Dias 1 , M. O’Reilly 1 , M. Mitsikakou 1 , E. Charalambous 1 , L. Green 1 , M. Vaughan 1 , G. Agliardi 1 , J. Garcia 1 , E. Lewin 1 , M. Lowdell 1 , M. Marzolini 2 , L. Wood 2 , H. Holmes 3 , Y. Ngai 3 , B. Popova 3 , W. Wilson 3 , S. Kunaseelan 3 , V. Spanswick 4 , H. Lowe 4 , L. Ensell 4 , J. Hartley 4 , S. Domning 5 , S. Morley 6 , T. Marafioti 7 , A. Bloor 8 , D. Irvine 9 , K. Orchard 10 , A. Khwaja 1 , D. Linch 1 , M. Pule 1 , K. Peggs 1

1 Research Department of Haematology, University College London; 2 Dept. Haematology, University College London Hospitals; 3 UCL Cancer Trials Centre; 4 UCL Cancer Institute, University College London; 5 RCTS, Kings College London; 6 Dept. Imaging, University College London Hospitals; 7 Research Dept. Pathology, University College London, London; 8 Dept. Haematology, Christie Hospital Manchester, Manchester; 9 Dept. Haematology, Queen Elizabeth Hospital Glasgow, Glasgow; 10 Dept. Haematology, University Hospital Southampton, Southampton, United Kingdom

Background: We have previously described AUTO1, a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce CAR T-cell immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL (Ghorashian S et al., Nat Med 2019; Roddie C et al., JCO 2021). This data confirms the intended function of the receptor, with low levels of CRS/ICANS and long-term engraftment of CAR T-cells observed in both patient groups.

Aims: We have initiated testing of AUTO1 in the setting of B-NHL and CLL/SLL ( NCT02935257 ).

Methods: Manufacturing: CAR T-cell products were generated using a semi-automated closed process from non-mobilised patient leukapheresate.

Study design: Subjects ≥ 16y underwent lymphodepletion with fludarabine (30mg/m x3) and cyclophosphamide (60mg/kg x1) prior to AUTO1 infusion, with the exception of the DLBCL cohort who additionally received a single dose of pembrolizumab (200mg) on day -1 to potentiate CAR-T expansion. AUTO1 dose varies based on the indication. Split dosing of 230 x10 6 CD19 CAR T-cells at day 0 and day 9 is employed in the CLL cohort. A single dose of 200 x10 6 CD19 CAR T-cells is delivered to patients with B-NHL. Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months.

Results: As of 8 th February 2022, we enrolled 23 patients: 11 low grade NHL (LG-NHL:7 with FL and 3 with MCL), 7 DLBCL and 5 CLL. Apheresis was successful in all 23 patients and product manufacture was successful in 22 (pending in the last). 19 patients were infused: 10 with LG-NHL, 6 with DLBCL and 3 with CLL. 1 CLL patient was pending infusion at time of data cut-off and 2 patients died pre-infusion: 1 MCL patient, from COVID-19 and 1 CLL patient, from intracerebral haemorrhage. Patients treated with AUTO1 had a median age of 60 years (range 39-79), had received a median of 3 prior lines of treatment (range 2-8). Grade 1 CRS was reported in 6/19 and Grade 2 CRS in 3/19. No ICANS was observed in the B-NHL and CLL cohorts. CAR engraftment was observed in 13/13 patients evaluated by qPCR with ongoing persistence in 12/13 patients at last follow-up.

In the LG-NHL and DLBCL cohorts 10/10 and 4/5 evaluable patients respectively were in CMR by 18FDG PET-CT post-treatment. Responses were ongoing in 9/10 LG-NHL at 12 months and in 4/4 DLBCL at months 1, 3, 3 and 6. In the CLL cohort, 2/3 evaluable patients achieved MRD negative remission in the bone marrow with residual small volume lymph nodes by CT at 6 and 3 months of follow-up respectively. 1 CLL patient did not engraft and had SD at month 1.

Summary/Conclusion: AUTO1 has a tolerable safety profile in patients with r/r B-NHL and CLL despite high disease burden. Early data shows excellent complete remission rates and excellent CAR engraftment/expansion. Additional patients, updated data and longer follow up will be presented.

P1460: SAFETY AND EFFICACY FINDINGS OF AUTO1, A FAST-OFF RATE CD19 CAR, IN RELAPSED/REFRACTORY PRIMARY CNS LYMPHOMA

C. Roddie 1,* , J. Dias 1 , M. O’Reilly 1 , L. Green 1 , M. Vaughan 1 , G. Agliardi 1 , J. Garcia 1 , E. Lewin 1 , M. Lowdell 1 , M. Mitsikakou 1 , E. Charalambous 1 , A. Hotblack 1 , H. Dreau 2 , M. Marzolini 3 , L. Wood 3 , C. Every-Clayton 3 , A. Lal 4 , Y. Ngai 4 , B. Popova 4 , A. Malhi 4 , S. Kunaseelan 4 , V. Spanswick 5 , H. Lowe 5 , L. Ensell 5 , J. Hartley 5 , S. Domning 6 , L. Thorne 7 , H. Hyare 8 , P. Murphy 9 , D. Linch 1 , C. Fox 10 , K. Peggs 1 , K. Cwynarski 1 , M. Pule 1

1 Research Department of Haematology, University College London, London; 2 Oxford Molecular Diagnostics Centre, University of Oxford, Oxford; 3 Dept. Haematology, University College London Hospital; 4 UCL Cancer Trials Centre; 5 UCL Cancer Institute, University College London; 6 RCTS, Kings College London; 7 Dept. Neurosurgery, National Hospital for Neurology and Neurosurgery, Queen’s Square; 8 Dept. Neuroimaging, University College London Hospital; 9 Dept. Clinical Psychology, National Hospital for Neurology and Neurosurgery, Queen’s Square, London; 10 Research Department of Haematology, Nottingham University Hospital, Nottingham, United Kingdom

Background: Relapsed/refractory (r/r) primary central nervous system lymphoma (PCNSL) has a median overall survival of 2-8 months and few therapeutic options. CD19 CAR-T therapy is highly effective for systemic B-cell lymphoma, but associated neurotoxicity has led to exclusion of PCNSL patients from pivotal clinical trials. We have previously described AUTO1, a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL, NHL and CLL, with high remission rates, low incidence of CRS/ICANS despite CAR trafficking into the cerebrospinal fluid (CSF) and long-term CAR-T engraftment.

Aims: Based on these favourable characteristics, we initiated the CAROUSEL study of AUTO1 in PCNSL ( NCT04443829 ), testing both intravenous (IV) and intraventricular (I-VEN) routes of CAR-T delivery. We incorporated anti-PD1 into the conditioning strategy with the aim of enhancing CAR expansion and preventing PD1-mediated CAR silencing in the PCNSL microenvironment.

Methods: Manufacturing : CAR-T products were generated using a semi-automated, closed process from non-mobilised patient leukapheresate. Study Design: Subjects>/=16y underwent lymphodepletion with fludarabine (Flu;30mg/m2x3) and cyclophosphamide (Cy;60mg/kgx1) and Pembrolizumab (200mgx1) prior to IV AUTO1 infusion (250x10^6 CAR-T). In the event of failure to achieve PR or a frank relapse event, a 2nd dose of AUTO1 was administered into the CSF via ommaya reservoir (25x10^6 CAR-T) following Flu/Cy at an interval of >/=28d post-IV dosing. Study endpoints include manufacture feasibility, grade 3-5 toxicity and remission rates at 1 and 3 months.

Results: As of 14 February 2022, we enrolled 6 patients, apheresed and manufactured 5 products and infused 5 patients with IV AUTO1 and 1 patient with I-VEN AUTO1. Of enrolled patients, median age was 43y (range 23-50), median ECOG was 1 (range 1-2) and median prior lines of therapy was 2 (range 1-3). All patients had parenchymal disease and all received bridging therapy. Following IV CAR-T, grade 1 and 2 CRS affected 1 and 3 patients respectively and any grade ICANS was observed in 2 patients, with 2 grade 3 events reported. AUTO1 engraftment and response was evaluable in 4 patients at month 1 following IV infusion. Excellent AUTO1 engraftment was observed in all patients in blood and CSF, and all 4 patients had ongoing CAR-T persistence at last follow-up. CSF and blood cytokines were also evaluated.

2/4 evaluable patients had no measurable disease at 2 and 6 months of follow-up respectively. 1 patient had PD at month 1 and was unable to receive further trial treatment. 1 patient had SD at month 1 but progressed at month 2. This patient had an ommaya reservoir implanted to receive I-VEN AUTO1, following which the patient developed grade 2 CRS for 5 days, without overt ICANS. The patient achieved PR at month 1 but progressed at month 2.

Summary/Conclusion: In r/r PCNSL, AUTO1 shows encouraging response rates and excellent CAR-T engraftment/expansion in the blood and CSF. I-VEN administration was well-tolerated and AUTO1 demonstrated activity via direct CSF infusion in a patient who failed IV therapy. Additional patients, updated biological data and longer follow-up will be presented.

P1461: CAR-T VS. STANDARD OF CARE AS SECOND LINE TREATMENT FOR LARGE B CELL LYMPHOMA: A SYSTEMATIC REVIEW AND META-ANALYSIS

L. Shargian 1,* , P. Raanani 1 , M. Yeshurun 1 , A. Gafter-Gvili 2 , R. Gurion 1

1 Hematology; 2 Internal Medicine A, Rabin Medical Center, Beilinson Hospital, Petah-Tikva, Israel

Background: Treatment with high dose chemotherapy followed by autologous stem cell transplantation (ASCT) is considered the standard of care (SOC) second line treatment for relapsed or refractory (R/R) large B-cell lymphoma (LBCL) in patients responding to salvage therapy. However, more than half of the patients will not proceed to ASCT due to progressive disease. Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has revolutionized the treatment of R/R LBCL. With unprecedented overall response rates of 50-80% and durable responses of 30-40% at 4-5y, CAR T-cell therapy is now considered SOC for patients with R/R LBCL after 2 or more lines of therapy. Randomized prospective studies have recently examined whether earlier admission of CAR T-cell therapy after failure of 1 line of therapy could improve outcomes.

Aims: The aim of present meta-analysis was to compare the efficacy and safety of CAR-T vs. high dose chemotherapy followed by ASCT as second line treatment for R/R LBCL.

Methods: Systematic review and meta-analysis of randomized controlled trials including patients with R/R LBCL, comparing second line treatment with high dose chemotherapy followed by ASCT to CAR-T cell therapy. Cochrane Library, PubMed, conference proceedings and references were searched until January 2022. Two reviewers appraised the quality of trials and extracted data. Primary outcome was overall survival (OS). Secondary outcomes included overall response rate (ORR), complete response (CR) rate, event free survival (EFS), progression free survival (PFS) and safety.

Results: Our search yielded 3 trials conducted between the years 2018 and 2021, including 865 patients (one abstract, and two published in peer review journals). Age of patients enrolled ranged between 19 to 81 years. Each trial tested one of the three FDA approved anti-CD19 CAR-T products: Axicabtagene Ciloleucel (Axi-cel, “ZUMA-7”), Tisagenlecleucel (Tisa-cel, “BELINDA”) and Lisocabtagene Maraleucel (Liso-cel, “TRANSFORM”). All trials included LBCL patients either refractory to frontline therapy or relapsed within 12 months. OS was significantly improved with CAR-T as compared to SOC, HR 0.77, [95% confidence interval (CI) 0.60-0.98, 865 patients, 3 trials] (figure 1). CAR-T therapy was associated with significantly better ORR and CR rate, RR 1.55 [95% CI 1.12-2.13, 865 patients, 3 trials] and RR 1.49 [95% CI 1.09-2.05, 865 patients, 3 trials]) respectively. EFS and PFS were significantly improved as compared to SOC, HR 0.57 [95% CI 0.49-0.68, 865 patients, 3 trials] and HR 0.47 [95% CI 0.37-0.60, 543 patients, 2 trials] respectively. Regarding safety, all trials reported grade 3 or 4 adverse events (N=739). The risk of any grade 3 or 4 adverse event was comparable between the two arms, RR=1.03 [95% CI 0.93–1.14, 865 patients].

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Summary/Conclusion: This systematic review and meta-analysis shows that CAR-T therapy has superior outcomes compared to SOC in LBCL refractory to frontline therapy or relapsing within 12 months, without excess toxicity. CAR-T therapy was associated with statistically significant improved ORR and CR rates and longer EFS and PFS, translating into improved OS. However, results are quite preliminary with median follow up of trials ranging between 6-24 months. Longer follow-up is needed to confirm these results and determine the optimal sequencing of CAR T-cell therapy in the management of R/R LBCL.

P1462: POPULATION-BASED REAL WORLD RESULTS OF CD19-DIRECTED CAR T-CELL THERAPY FOR PATIENTS WITH RELAPSED OR REFRACTORY LARGE B-CELL LYMPHOMA: A DUTCH CAR T-CELL TUMORBOARD EXPERIENCE

A. M. Spanjaart 1,* , E. Pennings 1 , P. Mutsaers 2 , S. van Dorp 3 , M. Jak 4 , J. van Doesem 5 , J. de Boer 5 , J. Vermaat 6 , A. Sijs-Szabo 6 , M. van der Poel 7 , I. Kuipers 1 , M. Chamuleau 1 , I. Nijhof 1 , M. Minnema 4 , P. Lugtenburg 2 , T. van Meerten 5 , M. J. Kersten 1

1 Dept. of Hematology, Amsterdam University Medical Centers, Amsterdam; 2 Dept. of Hematology, Erasmus MC Cancer institute, Rotterdam; 3 Dept. of Hematology, Radboud University Medical Center, Nijmegen; 4 Dept. of Hematology, University Medical Center Utrecht, Utrecht; 5 Dept. of Hematology, University Medical Center Groningen, Groningen; 6 Dept. of Hematology, Leiden University Medical Center, Leiden; 7 Dept. of Hematology, Maastricht University Medical Center, Maastricht, Netherlands

Background: CD19-directed CAR T-cell therapy has become standard of care for patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL) and durable responses are seen in up to 40% of treated patients. Optimal selection of patients who will benefit from CAR T-cell therapy is important. In the real world reasons for ineligibility of patients who meet the registration label are often not reported. Additionally, data on health-related quality of life (HR-QoL) after infusion are scarce. Real world (in)eligibility and HR-QoL can play an important role in guiding treatment choices with novel therapeutic options becoming available. In the Netherlands, population-based referral and eligibility assessment for CAR T-cell therapy are centralized in a national tumorboard. A multicenter expert team discusses cases and data are collected in a national registry from time of referral. The aims of the tumorboard and registry are to capture population-based data on patients potentially eligible for CAR T-cell therapy and to optimize (cost-) effective, high quality care.

Aims: To evaluate real world results of axicabtagene ciloleucel (axi-cel) for R/R LBCL on a population level, including reasons for ineligibility, efficacy, toxicity and (in a subset of patients) HR-QoL.

Methods: For this prospective multicenter cohort study, all adult patients referred to the tumorboard for treatment of R/R LBCL with axi-cel were included from May 1 st 2020 – October 19 th 2021. Descriptive statistics were used for baseline characteristics, Kaplan Meier methods for survival analysis, regression models for univariate/multivariate analysis and linear mixed models for HR-QoL over time.

Results: In total, 186 patients were referred to the tumorboard, 57 were not approved for axi-cel during the online tumorboard meeting, mostly (n=23) due to rapid progression (RP). The 129 approved patients were screened at a CAR T center, 32 were rejected (n=21 RP). Ninety-seven patients underwent apheresis of whom 7 did not receive axi-cel, mostly due to RP (n=6). Ninety patients were infused with CAR T-cells. Data of 74 of these patients are currently available for toxicity/efficacy analysis and updated results, including HR-QoL, will be presented during the EHA meeting. Median age was 59-years (min-max 27-79), 81% received bridging therapy. Best overall response rate was 82.4 %, including 66.2% complete remissions (CR). Median time to response was 1 month and all patients achieved their best response within 3 months. Four patients with PR at 1 month achieved a CR at month 3. The PFS and OS curves are shown in Figure 1. All grades CRS occurred in 89%, including ≥grade 3 in 2.7%, median time to onset was 3 days (min-max 0-14) and median duration 6 days (min-max 1-17). All grades ICANS occurred in 58%, including ≥ grade 3 in 27%, with a median time to onset of 7 days (min-max 1-28) and a median duration of 7 days (min-max 1-28). Non-relapse mortality was 1.4% due to one grade 5 ICANS event.

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Summary/Conclusion: The merit of a national tumorboard is to enable prospective, in-dept evaluation of rejected as well as CART-infused patients. Twenty-eight percent of the referred patients in the Dutch population that fulfilled the indication criteria for R/R LBCL treatment with axi-cel could not receive the therapy due to rapidly progressive disease. A relatively low percentage of patients, 7%, underwent leukapheresis but eventually did not receive axi-cel. Efficacy and toxicity in our cohort are comparable with ZUMA-1 study results and other large real world cohorts.

P1463: PHASE I DOSE ESCALATION OF LAVA-051, A NOVEL BISPECIFIC GAMMA-DELTA T-CELL ENGAGER (GAMMABODY™), IN RELAPSED/REFRACTORY HEMATOLOGICAL MALIGNANCIES

A. Broijl 1,* , N. van de Donk 2 , F. Bosch 3 , M. V. Mateos 4 , P. Rodriguez-Otero 5 , A. Tucci 6 , P. Ghia 7 , A. Adang 8 , P. Parren 8 , I. Tuinhof 8 , S. Umarale 8 , B. Winograd 8 , H. van der Vliet 8 , A. Kater 2

1 Department of Hematology, Erasmus MC Cancer Institute, Rotterdam; 2 Department of Hematology, Cancer Care Amsterdam, Amsterdam UMC, Amsterdam, Netherlands; 3 Department of Hematology, Hospital Vall d’Hebron, Barcelona; 4 Institute of Cancer Molecular and Cellular Biology, University Hospital of Salamanca, Salamanca; 5 Department of Hematology, Clínica Universidad De Navarra, Pamplona, Spain; 6 Department of Hematology, ASST Spedali Civili di Brescia, Brescia; 7 Division of Experimental Oncology, Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milano, Italy; 8 LAVA Therapeutics N.V., Utrecht, Netherlands

Background: LAVA-051 is a 27kD humanized bispecific single domain antibody (VHH) that directly engages CD1d and the Vδ2-TCR chain of Vγ9Vδ2-T cells and additionally stabilizes the interaction between CD1d and type 1 NKT cells (Nature Cancer 2020;1:1054-1065) to mediate potent killing of CD1d-expressing tumor cells. By engaging innate-like T cell subsets with inherent antitumor activity, namely Vγ9Vδ2-T and type 1 NKT cells, LAVA-051 has the potential to combine high therapeutic efficacy with limited off-tumor toxicity. CD1d is expressed by tumor cells in the majority of patients with CLL (chronic lymphocytic leukemia) and MM (multiple myeloma), while expression in AML (acute myeloid leukemia) is most pronounced on (myelo)monocytic subtypes. Tolerability studies in non-human primates with cross-reactive surrogate bispecific γδ-T cell engagers showed a good safety profile without signs of CRS (cytokine release syndrome).

Aims: To provide initial dose escalation findings from a First In Human Study of LAVA-051 in relapsed/refractory hematologic malignancies.

Methods: This is an open label, accelerated titration design, phase 1/2a study in patients with relapsed/ refractory CLL, MM, and AML ( NCT04887259 ). The phase 1 study starts with single-patient cohorts for the first 3 dose levels followed by a 3 + 3 design. The primary objectives of the study are to investigate the safety and tolerability of LAVA-051 and to determine its recommended Phase 2a dose (RP2D). Secondary objectives of the study include evaluation of PK, PD, immunogenicity, and preliminary antitumor activity. The (flat) starting dose was determined to be 0.45 µg. LAVA-051 is administered by IV infusion over 2 hours twice a week. Informed consent has been obtained from all participating patients.

Results: As of Feb 14, 2022, a dose level of 45 µg has been reached with no reports of CRS (ASTCT grading) or dose limiting toxicities (DLTs). Six patients in total have been treated with LAVA-051; 4 patients (2 MM, 2 CLL) were evaluable for DLTs during cycle 1 at dose levels 0.45 µg, 3 µg, 15 µg and 45 µg. Treatment emergent AEs (TEAEs) (CTCAEv5.0) were predominantly of grade 1 and 2 severity (e.g. fever, chills, and headache). One patient (45 µg) reported a grade 2 infusion related reaction (IRR) that lasted less than 12 hours following the first administration only. Frequency and severity of TEAEs did not increase with escalating doses and all TEAEs were reversible. One patient with CLL developed symptoms consistent with a tumor flare reaction during cycle 1, and continues on cycle 5 of treatment with stable disease at 15 µg. PK data indicate increasing drug exposure in correlation with increasing doses of LAVA-051, and PD data indicate a dose-dependent increase in LAVA-051 receptor occupancy of the Vγ9Vδ2-T cell receptor. Importantly, no consistent or significant changes in measured cytokines have been observed. Updated results will be presented at the congress.

Summary/Conclusion: LAVA-051 has been well tolerated early in dose escalation with on-mechanism pharmacodynamics consistent with Vγ9Vδ2-T cell engagement.

P1464: DECREASING HPK1 EXPRESSION IN CD19 CAR-T CELLS: A NOVEL STRATEGY TO OVERCOME CHALLENGES OF CELL THERAPY FOR ADULT (R/R) B-ALL

N. ZHANG 1 , J. SI 2 , G. LI 3 , Y. WANG 4 , F. LONG 4 , T. WANG 2 , Y. SONG 3 , D. S. Dimitrov 5 , X. LIAO 2 , G. GAO 1,*

1 Department of Hematology, Xijing Hospital, Xi’an; 2 School of Pharmaceutical Sciences, Tsinghua University, Beijing; 3 Institute of Hematology, Xi’an Central Hospital; 4 Xi’an Yufan Biotechnologies Co., Ltd, Xi’an, China; 5 Center for Antibody Therapeutics, University of Pittsburgh, Pittsburgh, United States of America

Background: Since the FDA approved the first CD19 directed chimeric antigen receptor (CAR) T-cell therapy (Kymriah) for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (ALL), cytokine release syndrome (CRS) has still been a life-threatening toxicity that limits the full therapeutic potential in adults [1,2]. So far, Tecartus was the first and only approved CAR-T-cell therapy for adult (r/r) B-ALL, which was based on results from ZUMA-3 study. CRS occurred in 92% patients, and neurologic toxicities occurred in 87% patients in ZUMA-3 study[3], suggesting that therapeutic developments are needed to ensure more safe and potent options for adult B-ALL.

Aims: Our preclinical data demonstrated that CAR-T cells with decreased HPK1 expression had significantly enhanced antitumor effects and did not induce higher grade CRS [4]. Thus, the first-in-human clinical study is ongoing, to test CD19 CAR-T cells with decreased HPK1 expression (XYF19 CAR-T cells) for adult (r/r) B-ALL ( NCT04037566 ).

Methods: Dose-escalation study was initiated to explore the safety and efficacy of XYF19 CAR-T cells in adult (r/r) B-ALL. 11 eligible adult (r/r) B-ALL patients with a median age of 37 years (range 19 - 61 years) were enrolled. The cell dosage of XYF19 was significantly lower than Tecartus and Kymriah, participants were infused with XYF19 CAR-T cells: 3 at low dose (2.0×10 5 /kg), 5 at medium dose (5.0×10 5 /kg), and 3 at high dose (1.0×10 6 /kg). All patients received a conditioning regimen of IV fludarabine (30mg/m 2 /d) for 4 days and cyclophosphamide (500mg/m 2 /d) for 2 days.

Results: 30 days after infusion with XYF19 CAR-T cells, 8 of the 11 (72.7%) patients had achieved complete remission (CR) or CR with incomplete count recovery (CRi). Considering the low dosage of XYF19 and that the CR/CRi rate is 71% with Tecartus [2] and 69% with Kymriah [3], the clinical response of XYF19 is similar or even the highest in these three studies. Strikingly, in this clinical trial, no patient developed neurotoxicity and none has yet experienced dose-limiting toxicity. No grade 3 or higher CRS occurred in XYF19 treated patients, while Grade 3 or higher CRS was observed in 26% of patients with Tecartus [2], and 72% of patients with Kymriah[3]. No neurologic events occurred in the current study, while the incidence of neurological events was 51% with Tecartus [2] and 43% with Kymriah[3]. Thus, XYF19 is a strong potential novel treatment for CD19 CAR-T cell therapy in adult B-ALL.

Summary/Conclusion: Treatment with XYF19 CAR-T cells promises a novel treatment that can alleviate some of the stern challenges of CD19 CAR-T cell therapy for adult patients with r/r B-ALL. In the current study, this treatment showed robust activity against adult (r/r) B-ALL without >Grade 2 toxicity. This efficacy and safety will be demonstrated in further trials with larger sample sizes.

References:

[1] Blood. 2021; 19;138 (7): 531–543.

[2] Lancet.398(10299):491-502.

[3] J. Biol. Chem. 287,34091–34100.

[4] Cancer Cell. 2020; 38; 1-16.

P1465: INITIAL SAFETY AND EFFICACY STUDY OF RM-001, AUTOLOGOUS HBG1/2 PROMOTER-MODIFIED CD34+ HEMATOPOIETIC STEM AND PROGENITOR CELLS, IN TRANSFUSION-DEPENDENT ΒETA-THALASSEMIA

L. Wang 1,* , H. Xu 2 , J. Liang 2 , Y. Li 1 , L. Shi 2 , B. Xiao 1 , J. Huang 3 , X. Xu 4 , X. Zhang 5

1 Department of Pediatrics, 923rd Hospital of the People’s Liberation Army, Nanning; 2 Reforgene Medicine; 3 School of Life Sciences, Sun Yat-sen University; 4 Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou; 5 Department of Hematology, 923rd Hospital of the People’s Liberation Army, Nanning, China

Background: Natural mutations that cause hereditary persistence of fetal hemoglobin (HPFH) alleviate the symptoms of β-thalassemia. It is now known that small natural deletions and mutations in the γ- globin gene ( HBG1/HBG2 ) promoters disrupt the binding of the transcriptional repressors BCL11A, causing HPFH. Using gene editing to mimic these mutations should reactivate γ- globin in patients with transfusion-dependent β-thalassemia (TDT) and ameliorate the debilitating effects of mutations in β-globin.

Aims: To induce HbF production in erythrocytes, we used ex vivo CRISPR-Cas9–based gene editing to modify HBG1/HBG2 promoter in hematopoietic stem and progenitor cells (HSPCs), producing RM-001. We initiated initial safety and efficacy study of RM-001 in TDT patients. Here, we present initial results from the first 2 patients treated with RM-001.

Methods: Patients (6–35 y of age) with TDT receiving packed red blood cell (pRBC) transfusions of ≥100 mL/kg/y or ≥10 units/y in the previous 2 y were eligible. Peripheral CD34+ HSPCs were collected by apheresis after mobilization with G-CSF and plerixafor. CD34+ cells were edited with CRISPR-Cas9 using a guide RNA specific for the binding site of BCL11A on the HBG1/HBG2 promoter. Prior to RM-001 DP infusion, patients received myeloablative conditioning with Busulfan from day-7 to day-3. Patients were monitored for stem cell engraftment/hematopoietic recovery, adverse events (AEs), Hb production, HbF and F-cell expression, pRBC transfusion requirements.

Results: Data presented here for the first 2 TDT patients treated with RM-001. Both patients have β 0 /β 0 genotype (CD17/CD41-42, CD41-42/CD41-42), with an annualized packed red blood cell (pRBC) transfusion history of 39 units/y and 54.8 units/y over 2 y prior to consent, respectively. Both patients received a single dose of RM-001 cells, ceased pRBC transfusions within 1 month after RM-001 infusion and remained transfusion-free through the reported period. The first patient (CD17/CD41-42, 9y) achieved neutrophil and platelet engraftment on Day(D+)19 and D+21; the other (CD41-42/CD41-42, 13y) achieved neutrophil and platelet engraftment on D+16 and D+21, respectively. No serious adverse event (SAE) occurred. The safety profile was generally consistent with busulfan myeloablation and autologous hematopoietic stem cell transplantation. Total Hb, HbF and F-cell percentage increased over time in both patients (Figure). Both patients received 6 units of pRBC before transfusion independent (TI) achieved (Hb stably ≥ 90g/L), with last pRBC transfusion on D+15 and D+28, respectively. The first patient achieved TI on D+28 and the other achieved TI on D+39. At 3 month post-RM-001 infusion, total Hb exceed 110 g/L in both patients, with 97-98 g/L HbF. Approximately 89% peripheral erythrocytes are F-cells at 3 month post-RM-001 infusion (Figure). Data will be updated for the presentation.

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Summary/Conclusion: The first 2 patients treated with RM-001 demonstrated successful engraftment and both have been transfusion free within two month. Although both patients have β 0 /β 0 genotype, they ceased pRBC transfusions within 1 month after RM-001 infusion and remained transfusion-free through the reported period. This is the first-in-human study of autologous HBG1/2 promoter-modified CD34+ HSPCs (RM-001) in TDT and both patients were treated successfully without SAE. Our studies provide novel and initial clinical evidence supporting the safety and efficacy of RM-001 for treating TDT.

P1466: CD7 CHIMERIC ANTIGEN RECEPTOR T CELLS FOR ADULT PATIENTS WITH REFRACTORY AND RELAPSED T CELL MALIGNANCIES

Y. Zhang 1,2 , W. Luo 1,2 , C. Li 1,2 , M. Du 1,2 , F. Zhou 2,3 , L. Tang 1,2 , J. Wu 1,2 , H. Jiang 1,2 , Q. Wei 1,2 , C. Lu 1,2 , H. Kou 1,2 , D. Wu 4 , A. H. Chang 5 , Y. Hu 1,2 , H. Mei 1,2,*

1 Institute of Hematology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology; 2 Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease; 3 Department of Pediatrics, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan; 4 Beijing GoBroad Hospital Management Co.,Ltd, Beijing; 5 Shanghai YaKe Biotechnology Ltd., Shanghai, China

Background: Although first-line chemotherapies had a satisfying response in primary T cell malignancies, those with refractory or relapsed diseases still presented dismal outcomes, especially in adults. CD7 is a potential target highly expressed both in malignant and normal T cells. CD7-knockout anti-CD7 CAR-T cells have been proven effective in pediatric T-ALL, but its administration in adult patients has not been reported.

Aims: This study aims to evaluate the safety and efficacy of anti-CD7 CAR-T cells in adults with R/R T cell malignancies and the distinctions between autologous and allogeneic CAR-T cells.

Methods: In this trial ( NCT04823091 ), anti-CD7 CAR T cells were manufactured from HLA-matched sibling donors or autologous mononuclear cells and administrated to 10 participants at a dose of 1×10 6 (n=5) or 2×10 6 (n=5) cells/kg. The primary endpoint was safety with efficacy secondary. Four participants were chosen to match patients receiving CD19 CAR-T according to their baselines. Both CAR-T had humanized antigen recognition and 4-1BB costimulatory. Subtype and expression of immune checkpoints of infusion products were detected.

Results: Ten patients received CAR-T cells after lymphodepleting chemotherapy, five with autologous and five with allogenic CAR-T. Adverse events including cytokine release syndrome grade 1-2 occurred in 70% and grade 3-4 in 10%, cytopenia grade 4 in 100%, graft-versus-host disease grade 1-2 in 20% and multiple infections in 60%. No neurotoxicity was observed (Figure 1B). All adverse events were reversible after prompt treatment, except in one patient who died of fungal pneumonia at 2.5 months. As of Feb 22, 2022, the median follow-up was 4 months (range 2-9.5). Eight patients responded to CAR-T therapies and 100% (n=6) of patients achieved MRD-negative hematological remission. Extramedullary lesions were eliminated partially in 80% of 5 patients. Pt1 with mycosis fungoides bridging lradiation obtained a long-term CR over 9.5 months (Figure 1A).

CD7 CAR T-cells expanded to peak level at 14 days (7-23) post-infusion and persisted in a small quantity for up to 4 months (Figure 1C). A transient reduction of lymphocytes was observed in all patients and recovered in about one month with CD7-negative. There was no difference in the proportion of cell subtypes and expression of exhausted biomarkers between CD7- and CD7+ T cells.

There was a greater likelihood of developing mild CRS and prolonged leukopenia in the allogenic group, which could be resolved spontaneously. The administration of allogenic CAR-T did not increase the risk of viral infection or GVHD. Patients in two groups presented the same overall response rate but a higher possibility of complete remission was observed in allogenic ones. Peak numbers of CAR-T cells were not associated with the origins of T cells.

CD7 CAR-T cell contained significantly lower percentages of CD8+T cells (P=0.0017), naive T cells (P=0.0477), higher percentages of Treg (P=0.0206), effector memory T cells (P=0.018), and CTLA-4 positive T cells (P=0.0226) compared to CD19 CAR-T (Figure 1D). The characters of CAR-T cell phenotypes may explain the significant reduction of CD7 CAR-T cells in one month.

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Summary/Conclusion: These results show that CD7 CAR-T cells can produce anti-tumor responses with relatively controlled adverse events. Auto- or allo-CAR-T have similar efficacy and safety in the process, thus optimal solutions can be selected depending on their situations. CD7 CAR-T presents a potential therapeutic option for adults with R/R T cell malignancies, but the long-term prognosis remains to be observed and improved.

P1467: HEALTHCARE PROFESSIONALS (HCP) OPINIONS ON TREATMENTS FOR SICKLE CELL DISEASE (SCD) AND THE HCP–PATIENT RELATIONSHIP: RESULTS FROM THE INTERNATIONAL SICKLE CELL WORLD ASSESSMENT SURVEY (SWAY)

B. Andemariam 1,* , J. James 2 , C. Minniti 3 , B. Inusa 4 , F. El Rassi 5 , A. Nero 6 , C. Trimnell 7 , M. R. Abboud 8 , J.-B. Arlet 9 , R. Colombatti 10 , M. de Montalembert 11 , S. Jain 12 , W. Jastaniah 13 , E. Nur 14 , M. Pita 15 , L. DeBonnett 16 , T. Bailey 17 , O. Rajkovic-Hooley 17 , I. Osunkwo 18

1 New England Sickle Cell Institute, Division of Hematology-Oncology, Neag Comprehensive Cancer Center, UCONN Health, University of Connecticut, Farmington, United States of America; 2 Sickle Cell Society, London, United Kingdom; 3 Montefiore Health System, Albert Einstein College of Medicine, New York, United States of America; 4 Paediatric Haematology, Evelina Children’s Hospital, Guy’s and St Thomas’ Hospital, London, United Kingdom; 5 Department of Hematology and Medical Oncology, Emory University School of Medicine and Georgia Comprehensive Sickle Cell Center at Grady Health System, Atlanta; 6 University of Texas Southwestern Medical Center, Dallas; 7 Sickle Cell 101, San Jose, United States of America; 8 American University of Beirut Medical Center, Beirut, Lebanon; 9 Hôpital Européen Georges-Pompidou, Paris, France; 10 Clinica di Oncoematologia Pediatrica, Dipartimento della Salute della Donna e del Bambino Azienda Ospedaliera, Università di Padova, Padova, Italy; 11 Hôpital Universitaire Necker-Enfants Malades, Paris, France; 12 Thalassemia And Sickle Cell Society, Hyderabad, India; 13 Department of Oncology, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia; 14 Department of Clinical Hematology, Amsterdam University Medical Centers, Academic Medical Center, Amsterdam, Netherlands; 15 Department of Hematology and BMT, Samaritano Hospital, São Paulo, Brazil; 16 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 17 Adelphi Real World, Bollington, United Kingdom; 18 Forma Therapeutics, Watertown, United States of America

Background: SCD is an inherited blood disorder that has a significant impact on morbidity and mortality. Care for patients (pts) with SCD may be influenced by HCP specialty and care setting (Smeltzer et al . BMJ Open 2021).

Aims: To assess potential differences in treatment and management of SCD by HCPs participating in SWAY, based on HCP specialty and care setting.

Methods: SWAY was a cross-sectional survey developed by international SCD experts, pt advocates and Novartis to assess pt and HCP experiences of SCD. Pts with SCD ≥6 years old (N=2145) and HCPs (N=365) completed the survey between Apr and Oct 2019. Eligible HCPs had qualified in their primary specialty by 2014 and were managing ≥10 SCD pts at the time of survey (≥5 pts per HCP in Canada; ≥2 pts per HCP in the Netherlands). Data reflect only the experiences of the surveyed HCPs (recruited by Adelphi Real World Network).

Results: HCPs were grouped by specialty (hematology, n=150; hematology–oncology, n=101; pediatric, n=31; general practitioner [GP], n=50 and other [internal medicine specialist, emergency department physician, physician assistant, nurse/nurse practitioner, other], n=33) and care setting (SCD center, n=46; university/teaching hospital, n=105; regional center/community hospital, n=57; private hospital/office, n=109 and other [health center and other], n=48).

Most hematologists (82%), hematologist–oncologists (79%) and pediatricians (87%) reported acute (vaso-occlusive crisis [VOC]) pain as one of the most common symptoms discussed with their pts vs 54% of GPs and 42% of other HCPs (chronic pain was reported by 78% of GPs and 70% of other HCPs). Accordingly, 97% of hematologists, 88% of hematologist–oncologists and 90% of pediatricians reported ever initiating hydroxyurea (HU) – a treatment for VOCs – vs 14% of GPs and 30% of other HCPs. More hematologists, hematologist–oncologists and pediatricians ranked reducing VOCs as a top treatment goal (other than a cure for SCD) than reducing chronic pain levels, while the opposite was true for GPs or other HCPs (Table). More HCPs at SCD centers and university/teaching hospitals initiated standard and exchange blood transfusions to help manage their pts SCD than other care settings (Table), yet fewer HCPs at SCD centers than at other care settings reported iron overload as a complication their pts’ experienced (SCD center, 26%; university/teaching hospital, 40%; regional center/community hospital, 47%; private hospital/office, 31% and other, 42%). HCPs at SCD centers were also more likely to provide information to pts in the form of leaflets (SCD center, 70%; university/teaching hospital, 60%; regional center/community hospital, 49%; private hospital/office, 59% and other, 60%) or direct them to pt support groups (SCD center, 63%; university/teaching hospital, 47%; regional center/community hospital, 30%; private hospital/office, 33% and other, 33%).

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Summary/Conclusion: Hematologists, hematologist–oncologists and pediatricians in SWAY were more likely to discuss VOCs (the hallmark of SCD) during consultation with their pts than GPs or other HCPs. This may be a reason why fewer GPs and other HCPs reported initiating HU, the mainstay of SCD therapy, compared with the other groups analyzed. Moreover, HCPs at SCD centers vs other care settings appeared to be more likely to initiate certain SCD treatments. These results support a multidisciplinary and collaborative approach to SCD care in order to optimize pt outcomes, where each HCP speciality and care setting fulfills distinct but vital aspects of pts’ needs.

P1468: SLEEP DISORDERED BREATHING IN SICKLE CELL DISEASE: RELATION TO PULMONARY HYPERTENSION AND STROKE

N. B. E.-D. Farghal 1,* , A. Tantawy 1 , F. S. E. Ebied 1 , N. El-Sherif 1 , N. Salah Eldeen 1 , N. Soliman 1 , S. Makkeyah 1

1 pediatrics, Ain Shams University hospitals, cairo, Egypt

Background: Sleep disordered breathing (SDB) is a common underdiagnosed sequela of sickle cell disease (SCD) characterized by daytime sleepiness, behavioral changes, and cognitive deficits. Nocturnal desaturations have also been linked to the frequency of vaso-occlusive crises (VOCs), acute chest syndrome (ACS), cardiac abnormalities, central nervous system (CNS) events, and nocturnal enuresis.

Aims: To determine the frequency of SDB in patients with SCD and its association with SCD-related complications.

Methods: Thirty children and adolescents with SCD were evaluated using overnight polysomnography after completing the modified STOP-Bang questionnaire. Transcranial doppler (TCD) and echocardiography with assessment of tricuspid regurge velocity (TRV) were performed. Data of SCD, and related complications were collected from the patients’ records.

Results: The median age of the studied cohort was 10.2 years (range 6-17 years), with male: female ratio 1.7:1. Six patients (20%) had high-risk for obstructive sleep apnea (OSA), nine (30%) had intermediate risk, and fifteen (50%) at low risk. Sleep apnea, defined as respiratory disturbance index (RDI) > 1 event/hour, was found in 18/30 (60%) of patients (14 males and 4 females). Patients with RDI > 5 (moderate to severe OSA) had significantly higher TRV (p = 0.007) and left MCA flow velocity (p = 0.049) when compared to those with RDI < 5. Although right MCA flow velocity was higher in the moderate to severe OSA group, this did not reach statistical significance (p = 0.054). Other variables including BMI-SDS (0.07), presence of cardiac dysfunction (p = 0.26), history of stroke (p = 0.17), or presence of nocturnal enuresis (p = 0.33) were comparable between both groups. Patients with RDI > 5 were at higher risk of OSA according to the modified STOP-Bang questionnaire (p = 0.02). RDI positively correlated with TRV (r = 0.53, p = 0.003), right MCA flow velocity (r = 0.45, p = 0.013) and left MCA flow velocity (r = 0.55, p = 0.002), while it showed negative correlation with BMI-SDS (r = -0.48, p = 0.008).

Summary/Conclusion: Sleep apnea is a common problem in patients with SCD associated with risks of pulmonary hypertension and alterations in cerebral blood flow which highlights the need for early diagnosis and management.

P1469: LONGITUDINAL SICKLE CELL DISEASE COHORT-STUDY: IN CHILDREN WITH SPLENIC SEQUESTRATION HISTORY OR HYPERSPLENISM, PARTIAL VS TOTAL SPLENECTOMY BEFORE STEM CELL TRANSPLANTATION PRESERVES SPLEEN FUNCTION

F. Bernaudin 1,* , C. Arnaud 1 , A. Kamdem 1 , C. Pondarré 1 , I. Hau 2 , N. Bayani 3 , M. Kuentz 4 , E. Gluckman 5 , R. Peffault de Latour 5 , N. Dhédin 5 , H. Lezeau 6 , J.-H. Dalle 7

1 Pediatrics, Referral Center for SCD, CHIC Hospital; 2 Pediatrics; 3 Hematology, CHIC Hospital; 4 Transplant unit, Mondor Hospital, Creteil; 5 Transplant Unit, St-Louis Hospital, Paris; 6 Pediatric Surgery, CHIC Hospital, Creteil; 7 Transplant Unit, Debré Hospital, Paris, France

Background: The presence of Howell-Jolly bodies (HJB) is associated with splenic dysfunction that appears progressively during aging or after splenectomy in patients with sickle cell disease (SCD). Improvement of splenic function has been found in several SCD patients after transplantation, prompting our team to propose a partial splenectomy prior to transplantation to patients with hypersplenism and available matched-sibling donor (MSD)

Aims: To evaluate splenic function from birth to adulthood, the impact of intensive treatments: hydroxyurea (HU), chronic transfusion (CT), transplantation and compare the impact of splenectomy (partial/total) in SCD patients when transplanted or not

Methods: Scores for HJB (0 to 3) were assessed on blood smears in the pediatric Créteil SCD-cohort and prospectively recorded at each annual check-up. This study included 4103 annual check-ups with available HJB score and all other biological parameters: Hb, HbA%, HbS%, HbF%, platelets etc. performed in 907 SCD patients (111 SC, 47 Sb+, i.e 158 SC/Sb+) and (725 SS, 21 Sb0 and 3 SDPunjab, i.e. 749 SCA-children) from January 1991 to January 2014

Results: HJB scores increased with aging in SCD patients, but were significantly lower in SC/Sb+ than in SS/Sb0 patients at each assessment age, P<0.001 (Figure 1). Multivariate logistic regression analysis showed that genotype (SCA vs SC/Sb+), splenectomy history and age at check-up were all strong significant (P<0.001 ) and independent risk factors for HBJ score >1 (>0.2% RBCs containing HJBs).

Among 749 SCA (SS/Sb0) children, 86 were splenectomized at the mean age (SD) of 6.4 years (3.9). As expected, HJB score was significantly higher at check-ups in splenectomized than in non-splenectomized SCA-patients, P <0.001 (Figure 2).

Among SCA-children, 138 were transplanted with MSD at a mean age (SD) of 9.0 years (4.9). At each age of assessment, HJB score was strongly significantly lower in transplanted than in non-transplanted children ( p <0.001), (Figure 3) and the score in transplanted children was significantly positively correlated with the age at transplant (r=0.335; p=0.002). In non-splenectomized SCA-children, mean (SD) HJB scores were unexpectedly significantly higher (P<0.001) in patients on HU 1.39 (0.91) than in not-intensified 1.11 (0.82), in those on CT 1.09 (0.86) and in transplanted children 0.35 (0.7) (Figure 4). Multivariate analysis showed that on HU, HbF and MCV were inverse independent significant risk factors for HBJ score>1 (p<0.001).

Seven children with splenic sequestration history or hypersplenism and available MSD-donor had partial splenectomy before transplant performed at mean (SD) age of 5.9 years (1.8). The mean (SD) HJB score was 1.83 (1.2) before and was null after transplant in the seven children

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Summary/Conclusion: The higher number of RBCs containing HJBs observed on HU is intriguing and could be related to altered erythropoisesis kinetics, drug-induced genotoxicity or altered splenic function because of drug-induced high MCV.

This cohort study clearly shows that splenic dysfunction increases with aging in SCA children and that stem cell transplantation allows to preserve splenic function, especially if the transplant is performed early. In children with indication of splenectomy for recurrent splenic sequestrations or hypersplenism who have an available MSD, the present study suggests that partial splenectomy just before stem cell transplantation may be useful to preserve splenic function.

P1470: NATURAL HISTORY AND RATE OF PROGRESSION OF RETINOPATHY IN PATIENTS WITH SICKLE CELL DISEASE: AN 11-YEAR RETROSPECTIVE FOLLOW-UP ANALYSIS

R. Brandsen 1,* , R. Diederen 1 , S. Bakhlakh 2 , E. Nur 2 , R. Schlingemann 1 , B. Biemond 2

1 Ophthalmology; 2 Haematology, Amsterdam UMC, Amsterdam, Netherlands

Background: Sickle cell disease (SCD) is characterized by chronic haemolysis and recurrent vaso-occlusion, resulting in tissue ischemia and organ damage. The retina is very sensitive to ischemic damage, which can result in sickle cell retinopathy (SCR). SCR can be divided into non-proliferative sickle cell retinopathy (NPSCR) and proliferative sickle cell retinopathy (PSCR). The proliferative form can be complicated by severe visual impairment due to vitreous haemorrhage and retinal detachment. Knowledge of risk factors for progression of SCR and subsequent complications is limited.

Aims: The aim of our study is to describe the natural history of SCR in a systematically analysed cohort of patients with SCD and to identify risk factors for the development of PSCR.

Methods: In our retrospective cohort study, we recruited all patients with SCD (HbSS, HbSC, HbSβ 0 and HbSβ + ) attending the departments of Haematology and Ophthalmology between 2006 and 2011. Patients lost to follow-up were excluded. Clinical, ophthalmologic and laboratory data were systematically collected and retrospectively analysed. Univariate and multivariate binary logistic regressions were used to identify associations between clinical and laboratory variables and progression to PSCR.

Results: 129 SCD patients were included with a median follow-up of 11 years (IQR = 8.5-12). The median age at the end of follow-up was 34 years (IQR = 28-45). HbSS/HbSβ 0 genotype was present in 54.3% (n = 70) of the patients and HbSC/HbSβ + genotype was present in 45.7% (n = 59). Progression of SCR during follow-up was seen in 28.7% (n = 37) of the patients. Age (aOR: 1.060, 95% CI: 1.016-1.106, p = .007) and HbSC/HbSβ + genotype (aOR: 17.487, 95% CI: 3.494-87.519, p <.001) were associated with PSCR at the end of follow-up. No signs of SCR at the end of follow-up was significantly associated with female sex (aOR: 3.113, 95% CI: 1.418-6.834, p = .005) and HbSS/HbSβ 0 genotype (aOR: 3.516, 95% CI: 1.095-11.288, p = .035).

Summary/Conclusion: Progression of SCR was seen in more than a quarter of the patients. Age and HbSC/HbSβ + genotype were associated with progression to PSCR at end of follow-up. Female SCD patients and patients with HbSS/HbSβ 0 genotype were at significant lower risk of developing SCR than other patients. Given the high rate of progression of SCR, screening of SCR remains important, but differentiated strategies for screening and follow-up of SCR could be considered for low-risk or high-risk patients.

P1471: CURRENT INDICATIONS AND RISKS OF SPLENECTOMY IN A LARGE MODERN COHORT OF CHILDREN WITH SCA

A. Mechraoui 1 , G. Ithier 1 , J. Pages 2 , Z. Haouari 1 , L. Ali 3 , A. Bonnard 3 , M. Benkerrou 1,4 , F. Missud 1 , B. Koehl 1,5,6 , L. Holvoet 1 , E. Le Roux 2,4 , V. Brousse 1,5,6,*

1 Site de référence labellisé sur la drépanocytose, filière MCGRE, Service d’Hématologie-Immunologie; 2 Unité d’épidémiologie clinique, Inserm, CIC 1426, F-75019; 3 Service de Chirurgie, Hôpital Universitaire Robert Debré; 4 Inserm, UMR-1123 ECEVE; 5 Biologie Intégrée du Globule Rouge, Unité Mixte de Recherche S1134, INSERM; 6 Laboratoire de Référence GR-Ex, Université de Paris, Paris, France

Background: In children with sickle cell anemia (SCA), the spleen is altered early in life and may be the site of serious complications such as acute splenic sequestration (ASS) or hypersplenism. Surgical splenectomy may be necessary in those who experience such complications, but both the consequent infectious and thrombo-embolic risks, and the age at which splenectomy may be safely performed, remain unclear.

Aims: The objective of our study was to determine the post-splenectomy incidence of invasive bacterial infections and thrombo-embolic complications in a large cohort of children with SCA. The secondary objective was to analyze the effect of age at splenectomy on these risks.

Methods: Retrospective review of children with SCA (SS or SB 0 -thalassemia) splenectomized during the 2000-2018 period at Robert Debré University Hospital (Paris, France) was undertaken. The study received ethical approval. The main clinical characteristics of patients, indications for splenectomy and outcomes were analyzed.

Results: A total of 188 children were included, with 164 (87.2%) SS children. Median (Q1-Q3) age at splenectomy was 4.1 yrs (2.5-7.3). Median duration of post splenectomy follow-up (FU) was 5.9 yrs (2.7-9.2) yielding 1192.6 patients-years (PY) of observation. One death (0.5%) occurred during FU, unrelated to splenectomy. Indications for splenectomy were ASS in 101 (53.7%) cases, hypersplenism in 75 (39.9%) and diverse reasons in the remaining children (splenomegaly n=5; delayed hemolytic post transfusion reaction n=5, other n=2). Pneumococcal immunization coverage was good with 166 (95.9%) eligible children immunized with polysaccharidic pneumococcal vaccination at the time of splenectomy and 7 (4.0%) after the procedure. Regarding the conjugated pneumococcal vaccination, 158 (91.9%) of eligible patients had received at least one injection at the time of splenectomy. All patients received penicillin prophylaxis. Overall incidence of invasive bacterial infection was 0.005 /PY (n=6 in 5 patients, no pneumococcal infections). Overall incidence of thrombo-embolic events was 0.003 /PY (n= 4 in 4 patients).

In this cohort, 123 (65.4%) and 65 (34.6%) children were splenectomized > or < 3 years of age, respectively. Overall incidence of invasive bacterial infection and thrombo-embolic events was not different according to the age at splenectomy (0.005/ PY in both groups) and 0.004/ PY versus 0.003/ PY (p= 0.7), respectively. Likewise, there was no difference between these groups in the incidence of acute chest syndrome or vaso-occlusive events. Conversely, a significant increase in the overall incidence of cerebral vasculopathy (including abnormal TCD, stroke and cerebral stenosis) was found in children splenectomized before 3 (0.037/PY) versus after 3 (0.011/ PY), p<0.01).

Patients who presented with ASS (n=101) were splenectomized at a median age of 3.3 yrs (2.4-5.4), following a median number of 3 (2-4) episodes of ASS and therefore contributed to a large proportion of patients splenectomized under 3 (n=44/65, 67.7 %).

Summary/Conclusion: To date this is the largest pediatric cohort study of splenectomized patients with SCA. We show that surgical splenectomy does not result in a significant increased risk of complications, notably invasive bacterial infections or thrombo-embolic complication. Splenectomy should not be delayed in patients once there is an indication for surgical removal, and the children have received recommended vaccines and prophylactic penicillin therapy. The relationship between ASS, splenectomy and cerebral vasculopathy needs to be further assessed.

P1472: INTERIM RESULTS OF SAFETY, TOLERABILITY, PHARMACOKINETICS, AND PHARMACODYNAMICS FROM AN ONGOING OPEN-LABEL STUDY INVESTIGATING FTX-6058 IN ADULTS LIVING WITH SICKLE CELL DISEASE

P. Bruno 1 , J. Kanter 2,* , J. Ziegler 1 , B. Stuart 1 , J. Ahn 1 , J. Dunn 1 , G. Allen 1 , C. Morabito 1

1 Fulcrum Therapeutics, Inc., Cambridge; 2 University of Alabama Medical Center, Birmingham, United States of America

Background: FTX-6058 is a potent and selective oral small-molecule inhibitor of Embryonic Ectoderm Development (EED) that has demonstrated robust fetal hemoglobin (HbF) protein expression in cell and murine models of Sickle Cell Disease (SCD). SCD is a genetic disorder of the red blood cells caused by a mutation in the HBB gene, which results in red blood cell sickling, hemolysis, vaso-occlusive crises (VOCs), and other complications. HbF (α 2γ2 ) prevents the pathologic polymerization of HbS in deoxygenated environments. Individuals with SCD who also have hereditary persistence of HbF (HPFH) have attenuated pathology and may present as asymptomatic when HbF levels are >25%. HPFH provides genetic and clinical evidence that increasing HbF has the potential to prevent or reduce disease-related pathophysiology, including hemolysis, pain, and VOCs. Preclinically, FTX-6058 has demonstrated increases in HbF levels up to approximately 40% of total hemoglobin, which has the potential to positively impact important clinical outcomes in SCD. Clinically, multiple ascending dose (MAD) data from a Phase 1, randomized, double-blind, placebo-controlled study of FTX-6058 in healthy volunteers demonstrated robust target engagement (H3K27me3 reduction) and potent induction of HBG (hemoglobin subunit γ) mRNA, translation of which is required for HbF expression.

Aims: To share interim results from the ongoing open-label study investigating FTX-6058 in adults living with SCD.

Methods: The ongoing Phase 1b open-label study investigating FTX-6058 is a multiple cohort trial that is being conducted in adults with SCD +/- hydroxyurea. In the first cohort, up to 10 volunteers will receive FTX-6058 6 mg once daily orally for 4 weeks. Eligible volunteers may choose to continue dosing for an additional 8 weeks at the same dose via an extension study. Up to two additional cohorts with doses selected between 2 – 20mg inclusive may be added to the study. The primary endpoints of the study are the safety and tolerability of FTX-6058 as measured by the frequency of adverse events and the pharmacokinetic profile of FTX-6058 in subjects with SCD. Secondary and exploratory endpoints include the effect of FTX-6058 on HbF induction in peripheral blood and other relevant SCD biomarkers and clinical endpoints.

Results: To date, FTX-6058 has been evaluated in 7 healthy volunteer (HV) SAD cohorts (2 to 60 mg), 5 HV MAD cohorts (2 to 30 mg QD, 14-day dosing), and an open-label food-effect cohort. Treatment-related adverse events have been mild (Grade 1 or 2) and resolved. There have been no SAEs or discontinuations. PK profiles have demonstrated dose proportionality across all doses. Robust target engagement was observed across the entire dose range in the MAD cohorts. Additionally, exposures achieved in the 6 – 30mg MAD cohorts resulted in dose-proportional 2.4 – 6.2 fold placebo-adjusted increases from baseline in HBG mRNA and no decrease in HBB mRNA.

Safety, tolerability, pharmacokinetics, and pharmacodynamics data from the ongoing, open-label trial in adults with SCD will be presented. In people with SCD, measurable increases in HbF (HPLC) may be observed by 4 weeks and may achieve near-maximal levels by 12 weeks. Pharmacodynamic reporting will focus on HbF changes.

Summary/Conclusion: FTX-6058 is an investigational, potential first-in-class oral HbF inducer. To date, FTX-6058 has demonstrated favorable safety and tolerability profile, with PK linearity and time- and dose-dependent increases in HBG mRNA that support longer term dosing in people living with SCD.

P1473: CREATING A RANDOM FOREST CLASSIFIER TO PREDICT HBF LEVELS IN SICKLE CELL DISEASE PATIENTS

C. Rakhit 1,* , S. Menzel 2 , A. Nielsen Nardo-Marino 2,3 , J. Strouboulis 2 , D. Rees 4,5 , B. Clark 1,2

1 Precision Medicine, Viapath LLP; 2 Red Cell Biology Group, King’s College London, London, United Kingdom; 3 Department of Haematology, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark; 4 Red Cell Biology Group, Kings College London; 5 Haematological Medicine, King’s College Hospital, London, United Kingdom

Background: Sickle cell disease (SCD) is caused by a genetic variant in the HBB gene, forming HbS. Expression of fetal globin (HbF), reduces the severity of SCD. Three main quatitative trait loci (QTL) have been identified that influence HbF expression; chr 2 ( BCL11A ), Chr 6 and the globin loci. Drugs such as Hydroxyurea are given to SCD patients as they increase HbF levels and reduce clinical symptoms.

Being able to predict the HbF level of an individual is complex as the number of QTLs is large. Having an accurate HbF prediction may enable triage of affected children, starting effective treatments and management earlier.

We have built a Random Forest model to predict the HbF level of an individual using genome wide SNP array data. This creates decision trees to find the most informative markers that are associated with the trait (HbF). With successive decision trees the data set is split until as many individuals with the same HbF level are grouped. Moving down a decision tree, the entropy level is reduced and the information gain increased, this is quantifiable. Genetic markers that have high predictive value of HbF are likely to be in LD to genomic regions affecting HbF expression. Therefore the predictive model can be used to identify novel regions associated with HbF levels.

Aims: (1) Create a Random Forest machine learning model that is able to predict the HbF level of an individual affected by sickle cell disease using genome wide SNP array data. (2) Assess the accuracy of the models prediction (3) Review the genetic loci that are contributing to the prediction of HbF

Methods: Random Forest analyses were primarily performed in the R programming language using the h2o.randomForest algorithm. We created a prediction model using an unaffected twin cohort (1825 individuals) in which both HbF and Illumina MEGA array data was held.

A different model was created for a SCD cohort where HbF values and array data was available. A total of 752 individuals were used to create the sickle model. Both models were adapted to remove overfitting, reducing the number of decision trees to 8 and only using the top 1000 predictive markers.

Results: In a non-sickle model, 653 cases had HbF prediction. The observed HbF level was plotted against the predicted and the model linearity assessed, R 2 = 0.45. The three main QTLs, involved in HbF regulation were identified in the top 10 ranked variants, plus other published variants, with a weak association. Plotting the relative importance of each SNP showed the top 10 variants contributed substantially to the model prediction.

A separate sickle model was created. The number of genetic variants that contributed to the HbF prediction in the sickle model was increased compared to the non-sickle cohort. Although the model was more complex the overall prediction accuracy was similar, R2=0.38. The genetic loci used in the prediction included BCL11A but also included NOS1 and ARG2 . In addition, intragenic regions were identified at loci very distal to any know gene. Re-running the model, consistently saw the same top predicting SNPs, indicating they were significant findings. We have mapped and described the top performing variants used in the sickle model.

Summary/Conclusion: We created a Random Forest classification model that can predict HbF levels using genome-wide SNP array data (R2=0.38). The model identified known and novel loci, potentially associated with HbF expression. The model may prove useful for predicting HbF% clinically. We are currently validating the model on a second sickle cell cohort.

P1474: MOLECURAL MECHANISM OF CHEMOTHERAPEUTIC HYDROXYUREA IS MEDIATED BY NOS2

T. Dragojević 1,* , O. Mitrović Ajtić 1 , M. Diklić 1 , T. Subotički 1 , D. Đikić 1 , E. Živković 1 , V. Čokić 1 , M. Vukotić 1

1 Department of Molecular Oncology, Institute for Medical Research, National Institute of Republic of Serbia, Belgrade, Serbia

Background:: Hydroxyurea (HU) is a chemotherapeutic agent that reduces ribonucleotide reductase, stops DNA synthesis and repar, and therefore causes cell proliferation inhibition and apoptosis. Due to its cytostatic properties, HU is frequently used for treatment of myeloproliferative neoplasms, ovarian cancer, and sickle cell anemia. Nitric oxide (NO), produced by nitric oxide synthase (NOS) enzymes is a potent signaling molecule involved in blood flow regulation, neutrotransmission, and immunity. Although HU treatment increases NO levels, up to date it is not clear whether it originates from activation of NOS enzymes or HU degradation.

Aims: The aim of this study was to determine the involvement of NOS2 enzyme in the cytostatic effect of HU.

Methods: To examine the involvement of the NOS2 enzyme in the molecular mechanism of HU, we treated erythroleukemic HEL92.7.1 cells with pan-selective NOS inhibitor L-NAME (200µM, 1mM, and 5mM), NOS2 specific inhibitor 1400W (1, 10, and 100µM), or NOS2/NOS3 inhibitor DPI (1, 5, and 10µM), in combination with hydroxyurea (200µM), and monitored their effect on proliferation and cell cycle. Immunocytochemistry for the proliferation marker Ki67 was performed to assess proliferation, while cell distribution in cell cycle phases was determined by flow cytometry after propidium iodide staining. Colony forming assay have been performed with the bone marrow cells of Nos2 null mice after oral HU treatment to corroborate the data obtained by enzymatic inhibition.

Results: In this study, we demonstrated that treatment of HEL92.7.1 cells with HU induces a dose-dependent increase in NOS2 protein levels and two products of the enzyme NOS - NO and citrulline. HU-induced citrulline levels can be reduced by treatment with the NOS inhibitor L-NAME, indicating that NO is produced de novo by the NOS enzyme rather than HU degradation. Inhibition of the NOS2 enzyme by L-NAME, 1400W, or DPI was sufficient to abolish HU-mediated inhibition of proliferation. While HU increased the number of cells in S-phase of the cycle at the expanse of the G0/G1 due to blocked DNA synthesis, combined treatment with HU and L-NAME or DPI inhibitor resulted in decreased G0/G1 phase and increased S and G2/M phases pointing to increased proliferation. These data indicate that the cytostatic properties of HU are mediated by the NOS2 enzyme. A colony formation assay showed that Nos2 deficient bone marrow cells isolated from mice treated orally with HU (200mg/kg) formed significantly more erythroid colonies (BFU-E and CFU-E) and granulocyte/macrophage progenitors (CFU-GM) compared to HU treated wild-type and untreated Nos2 null mice showing the involvement of Nos2 in the molecular mechanism of HU in vivo .

Summary/Conclusion: Our results show that HU induces the enzymatic activity of the NOS2 protein which in turn is involved in the HU regulation of proliferation and cell cycle. Comprehensive knowledge of the molecular mechanism of HU might help to improve its beneficial properties and decrease adverse effects.

P1475: THE ROLE OF BCL11A AND HBS1L-MYB SINGLE NUCLEOTIDE POLYMORPHISMS IN EGYPTIAN SICKLE CELL DISEASE PATIENTS

A. El Maraashly 1,*

1 Clinical Pathology, Kasr Al Ainy Medical School, Cairo, Egypt

Background: Sickle cell disease (SCD) is a debilitating blood disorder with a highly variable phenotype. Inter-individual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. The only currently FDA-approved HbF inducing drug for sickle cell disease (SCD) is hydroxyurea (HU). Genome wide association studies (GWAS) have identified three major quantitative trait loci (QTL) (intergenic region of HBS1L-MYB, BCL11A gene and XmnI site) associated with HbF. Several single nucleotide polymorphisms (SNPs) with persistence of HbF and amelioration of disease severity of β-thalassemia (β-thal) and SCD have been identified.

Aims: The aim of the current study is to find the influence of these genetic modifiers, especially BCL11A (rs4671393) and HBS1L-MYB intergenic region (rs9399137) SNPs on the level of HbF, severity of SCD and response to hydroxyurea in Egyptian sickle cell disease patients.

Methods: The present study included 100 patients with SCD; 56 patients were diagnosed as homozygous sickle cell anemia (SS) patients, while 44 patients were diagnosed as sickle beta thalassemia patients (Sβ). Genotyping for the genes was done by Real-Time PCR (RT-PCR) technique.

Results: BCL11A (rs4671393) SNP showed higher hemoglobin and HbF levels when induced with HU, with significantly higher increment in HbF level post HU therapy as well as a milder clinical phenotype in our Sβ patients’ group. In our SS patients’ group it showed a lower incidence of pneumonia as well as a more effective response to HU treatment. HBS1L-MYB intergenic region (rs9399137) SNPs showed lower reticulocytic counts and serum ferritin levels in our Sβ patients’ group.

Summary/Conclusion: In conclusion, our study shows that BCL11A (rs4671393) SNP showed higher hemoglobin and HbF levels when induced with HU, with significantly higher increment in HbF level post HU therapy as well as a milder clinical phenotype in our Egyptian study.

P1476: REVEALING THE POWER OF DISEASE REGISTRIES IN REAL-WORLD PATIENT CARE AND RESEARCH – THE SAUDI NATIONAL SICKLE CELL DISEASE REGISTRY SUCCESS STORY

H. Ezzat 1,* , A. Alghamdi 1 , S. Abuthnain 1 , M. Al-Abdulaali 1

1 Ministry of Health, Riyadh, Saudi Arabia

Background: The transformation in healthcare driven by the expansion of digital health is reforming clinical practice and medical research. Real-world health data is playing an important role in this transformation. National registries are recognized as one of the main sources for real-world data that is utilized to enhance practice and conduct of medical research. The value of national registries is profound when collecting health data for rare diseases as Sickle Cell Disease (SCD). In Saudi Arabia, the prevalence of sickle cell trait ranges from 4.5% to 27% and up to 1.4% for SCD. 1-3 Lacking data about SCD hinder informed decision making on healthcare, research and health policies in Saudi Arabia. As a result, the Saudi Ministry of Health has acknowledged the need for a national SCD registry to serve as valuable resource for real-world data. In 2019, the Saudi MOH launched national SCD registry to collect data for all SCD patients treated within MOH hospitals. This abstract aims to describe the registry, its current status and future plans.

Aims: The registry aims to collect data on SCD patients from all hematology treating units within MOH hospitals. Within that scope and serving relevant stakeholders (patients, providers, and payers), the registry intends to achieve multiple aims as describing baseline clinical and demographic characteristics of patients with SCD, disease patterns, prevalence, clinical outcomes, effectiveness of therapy, Quality of life (QoL), patient reported outcomes and healthcare resource utilization, with the goal of enhancing patient care and value-based outcomes.

Methods: Following established guidelines for registries, Saudi MOH implemented multiple steps in building the registry which include establishing registry team, defining the scope, objectives, and data sets, establishing a governance and oversight plan, and developing a project plan and timelines. 4-6 The registry is composed of a developed digital platform that collects, manages, and analyzes the data. For sites with paper based medical records, data entered into the platform manually by site coordinators while for sites with Electronic Medical Records (EMRs), data captured using integration solutions within the platform. The registry captures data on patient demographics, medical history, procedures and interventions, laboratories results, medications, blood transfusion, SCD complications and pain crisis.

Results: Between 2019 and 2021, the registry collected data on 22,956 patients from 15 MOH sites. The registry generated important insights such as patient average age (28 years), average number of missing days from school and work in one year (16.04 and 90.35 days, respectively) and pain assessment (96% of patients who experienced pain, of which 29% was mild, 51% moderate, 19% severe and 1% very severe).

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Summary/Conclusion: The Saudi MOH established a national SCD registry, with the long-term aim of developing value-based healthcare and enhancing medical research at the community and country level. After successfully completing the initial phase of the project, the Saudi MOH is planning for a second phase to expand the number of hospitals and patients. The registry platform will be modified and upgraded to include other features such as remote monitoring, patient reported outcomes and outcome-based agreement execution with the vision to leverage the registry data for AIML implementations. Lastly, the registry data will be leveraged to support multiple MOH systems such as Seha virtual hospital, NHCC, national call center (937) and appointments.

P1477: CHARACTERIZING SICKLE CELL DISEASE AND BURDEN OF ILLNESS IN THE UNITED STATES OF AMERICA: A RETROSPECTIVE ANALYSIS OF REAL-WORLD DATA

E. L. J. Clay 1,* , J. Paulose 2 , C. Rowe 3 , M. Tierney 3 , Y. Zhou 4 , T. Pattipaka 5 , D. Wormser 5 , J. M. Fermont 5

1 Johns Hopkins University, Johns Hopkins All Children’s Hospital, St Petersburg; 2 Novartis Pharmaceuticals Corporation, East Hanover; 3 PicnicHealth, San Francisco; 4 Genesis Research, Hoboken, United States of America; 5 Novartis Pharma AG, Basel, Switzerland

Background: Despite advances in screening, management and treatment, gaps remain in our understanding of sickle cell disease (SCD) in the real-world setting. Most observational studies rely on structured data (ie predefined clinical concepts such as ICD9/10 codes) from electronic medical records (EMRs), but this neglects clinically relevant information stored in unstructured narrative sections of physician notes. Advances in data analytic methods allow for the combination of physician notes and structured data, potentially providing a more accurate and complete picture of disease manifestation.

Aims: To evaluate disease manifestation in terms of vaso-occlusive crisis (VOC) and SCD-related organ damage, as well as current management approaches, and to evaluate the clinically relevant information that can be captured from physician notes.

Methods: 403 patients with confirmed SCD, who had ≥1 hospitalization due to VOC (H-VOC) in the 12 months before recruitment, were enrolled in this retrospective, non-interventional observational study. Data were analyzed from 235 patients (172 women and 63 men) aged ≥16 years in the year of index date (ie the first day after baseline [the first 12 months of the observation period]) and who had ≥1 year of follow-up data after the index date. The observation period (baseline plus follow-up) required ≥2 clinical document records ≥30 days apart, and ≥1 clinical document every 6 months. Each patient’s longest observation period was used for this analysis. Event rates are adjusted for exposure time and reported as rates per person-year with 95% confidence intervals (CI). Human-curated natural language processing with assistance from machine learning algorithms was used to extract EMR data.

Results: During the baseline period, 102 (43%) patients had ≥1 H-VOC (including emergency department visits), 88 (37%) had ≥1 outpatient VOC, and 32 (14%) had been hospitalized for SCD-related organ damage, primarily related to bone necrosis (9%) and pulmonary disease (8%). During follow-up (median 3.6 years), 218 (93%) patients experienced ≥1 VOC (H-VOC rate [95% CI] of 4.25 [3.64–4.95] per person-year; outpatient VOC rate of 2.39 [1.97–2.91]), and 143 (61%) were hospitalized for SCD-related organ damage (0.97 [0.79–1.21]). Of these, 764 (19%) H-VOCs, 1351 (52%) outpatient VOCs, and 423 (50%) hospitalizations due to SCD-related organ damage were extracted exclusively from physician notes. The overall diagnostic procedure rate was 20.36 (18.54–22.36), with blood tests being the most common (14.07 [12.17–16.25]) followed by X-rays (7.26 [6.50–8.10]) and urinalysis (2.43 [2.08–2.83]). The blood transfusion rate was 0.99 (0.77–1.27), and most patients received hydroxyurea (78%) and supportive treatments (95%). Patients mostly visited (ie inpatient and outpatient visits, and visits related to diagnostic procedures and treatment) specialist medical practitioners (15.46 [13.73–17.40]), while relatively few visited general medical practitioners (1.72 [1.46–2.02]).

Summary/Conclusion: To our knowledge, this is the first documented use of physician notes complementing structured data to characterize SCD in a real-world setting. We report a high level of healthcare consumption, predominantly involving specialist care and diagnostic procedures; only a small proportion of patients utilized outpatient-only care for VOC management. Our findings emphasize the importance of preventing and minimizing VOCs. Importantly, we demonstrate the considerable contribution that physician notes can make to better characterizing disease manifestation in SCD.

P1478: INTEGRATIVE DIAGNOSIS OF SICKLE CELL DISEASE PATIENTS FOR PERSONALIZED MEDICINE

A. Idrizovic 1,2,* , A. Collado Gimbert 1,3 , E. van Beers 4 , R. Colombatti 5 , P. Bartolucci 6 , M. de Montalembert 7 , M. P. Boaro 5 , D. Beneitez 8 , A. Ortuño 8 , A. Ruiz 9 , I. Isola 9 , E. Cela 10 , R. Van Wijk 11 , M. Rab 11,12 , M. D. M. Mañú Pereira 1

1 Cancer and blood disorders in children, Vall d’Hebron Research Institute; 2 Genetic and Microbiology Department, Universitat Autonoma de Barcelona; 3 Pediatric Hematology and Oncology department, Vall d’Hebron Hospital, Barcelona, Spain; 4 Center for Benign Hematology, Thrombosis and Hemostasis - Van Creveldkliniek, University Medical Center Utrecht, Utrecht, Netherlands; 5 Department of Woman’s and Child’s Health, University of Padova, Padova, Italy; 6 Red Cell Genetic Disease Unit, Universite Paris Est, Institut Mondor de Recherche Biomedicale, Creteil; 7 Department of pediatrics, Hopital Necker, Paris, France; 8 Department of Eritropathology, University Hospital Vall d’Hebron; 9 Department of Oncology and Hematology, Hospital Sant Joan de Déu, Barcelona; 10 Department of Oncology and Hematology, Hospital Materno Infantil Gregorio Marañón, Madrid, Spain; 11 Central Diagnostics Laboratory, University Medical Center Utrecht, Utrecht; 12 Department of Hematology, University Medical Center Utrecht, Utecht, Netherlands

Background: Sickle cell disease (SCD) is a chronic life threatening autosomal recessive disorder, caused by the presence of structurally abnormal adult hemoglobin S (HbS). Under low oxygen saturation, HbS forms hemoglobin polymers that deform the red blood cell structure, referred to as ‘sickling’. Sickled erythrocytes result in hemolytic anemia and recurrent vaso-occlusive crisis, which lead to long-term morbidity and ultimate early death. The patient specific pO2 at which sickling starts (PoS) along with RBC deformability at normoxia (EImax) and upon deoxygenation (EImin) can be measured by oxygen gradient ektacytometry (Laser Optical Rotational Red Cell Analyzer (LoRRca)). In the GenoMed4ALL project, oxygen gradient ektacytometry data will be integrated with genomics, metabolomics and clinical data of 1000 SCD patients. This will allow better characterization of SCD and development of Artificial Intelligence (AI) algorithms for personalized medicine.

Aims: To analyze the correlation of Lorrca parameters (PoS, EImax and EImin) with SCD genotypes (SS, Sβ o , SC and Sβ + ), hydroxyurea treatment (HU-, HU+) and HbF.

Methods: SCD patients with SS, Sβ o , SC and Sβ + genotypes, older than 1yo at steady state with no transfusion in the last 3 months were analyzed by oxygen gradient ektacytometry. All samples were processed in duplicates. Derived parameters were correlated with genotypes, and for severe SCD (SS, Sβ o ) with hydroxyurea options (HU-, HU+) and HbF. An Anova analysis has been carried out to test the relation of the parameters among groups. The post-hoc test Scheffe has been used to test which groups are different two by two.

Results: : 49 samples were analyzed: 32 SS, 2 Sβ o , 12 SC and 3 Sβ + . Mean and standard deviation values for PoS, EImax and EImin are shown in Table 1. PoS and EImin allowed patients’ clustering in 2 groups according to genotype: SS PoS: 34.34±5.0 Elmin: 0.23±0.1 vs Sβ o , SC and Sβ + SS PoS: 24.62±3.7 and Elmin: 0.34±0.1. EImin showed statistically significant Scheffe test value in discriminating SS vs Sß o and SS vs SC, while PoS was statistically significant only in discriminating SS vs SC.

31 samples from severe SCD patients (SS, Sβ o ) were further analyzed for correlation with HU (7 HU-, 24 HU+) and HbF. Mean and SD values are shown in Table 1. The differences in mean values for PoS, EImax and EImin between HU- and HU+ were of -27%, 54% and 135% respectively. The best correlation between Lorrca parameters and HbF was found in group of patients without treatment (HU-). EImin showed the highest correlation being 93% in HU- and 81% in HU+ group.

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Summary/Conclusion: Our results demonstrate the value of oxygen gradient ektacytometry to the characterization of SCD patients. PoS and Elmin were able to distinguish between SS and other genotypes, regardless of HU treatment. In addition, PoS, EImax and EImin all showed differences between HU+ and HU- groups, being Elmin the most valuable one. Elmin also showed the highest correlation with HbF. It is highly likely that PoS could be impacted by other variables: 2,3 DPG, pH, RBC hydration, genetic variants or metabolomics. Pooling data of 1000 SCD patients from at least 9 EU centers in the GenoMed4LL project is needed to improve results robustness. Standardization of Oxygenscan data generation should be ensured for data comparison and development AI algorithms for personalized medicine.

P1479: A BASELINE AUDIT OF THE USE A VALIDATED PSYCHOMETRIC TOOL (ASCQ-ME) TO ASSESS THE UNMET PSYCHOLOGICAL NEEDS OF A 2021 COHORT OF PATIENTS LIVING WITH SICKLE CELL

K. Kelleher 1,2,* , V. Garcia 3 , R. Auturally 3 , R. Jolley 3 , R. Marks 1 , M. Besser 3

1 Clinical Health Psychology Service, North Middlesex University Hospital, London; 2 Psychology, Durham University, Durham; 3 Haematology, Addenbrookes Hospital, Cambridge, United Kingdom

Background: Adults living with inherited red cell disorders face myriad social and emotional challenges. While the literature highlights higher incidence rates of depression and anxiety in those with sickle cell disease (SCD) compared to the general population, granular data on the specific emotional experience of these people is missing, resulting in boundaries to accessing mental health care. New disease modifying drugs can now preferentially increase haemoglobin with relatively small impact on crisis frequency or reduce crisis frequency with minimum impact on haemoglobin. Current management of patients still focuses on pain crisis and crisis prevention. At the time of this study, in the UK, hydroxycarbamide and exchange transfusion were the only available disease modifying therapies.

Aims: This study aimed to quantify psycho-social functioning and more accurately identify multifactorial level of need in patients living with SCD.

Methods: The present study used a validated, detailed, psychometric test, the Adult Sickle Cell Questionnaire Measure (ASCQ-Me), to identify need in a population of adults living with SCD in Cambridge. Self-report ASCQ-Me data was collected from patients (N=59) first on how SCD has affected 5 domains of psych-social impact (emotional, pain, sleep, social, stiffness), and second about the frequency and severity of sickle crisis. Demographic data was recorded based on sickle genotype, age, sex, disease modification, whether patients had fatigue, and whether patients requested counselling at the time of annual review.

Results: Patients who reported fatigue had significantly lower scores across all 5 domains of psychological impact than patients that did not report fatigue. However, there was no difference in Hb or HbF levels. When patients were split by disease modification (hydroxycarbamide, exchange transfusion, or no modification) there seemed to be a trend towards worsening psychological impact in all 5 domains for patients in the exchange group, with better outcomes from patients on hydroxycarbamide. There was no significant difference in psychological impact scores when patients were split by sex or sickle genotype. In particular, there was a trend towards higher frequency, but reduced intensity of pain crisis in patients with SC disease, but this did not reach significance. Patients who stated that they would like to be referred for psychological counselling also showed significantly worse scores across all 5 domains of psychological impact.

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Summary/Conclusion: There is an unmet need in providing psychological support for patients with complex diseases like sickle cell. Our finding that the subjective feeling of fatigue is associated with a multidimensional lower performance in the ASCQ-Me, in addition to disease modifying therapy, psychological support will remain very important and should not be attributed to anaemia alone.

P1480: SPLENIC SEQUESTRATION IS MORE COMMON IN SCD-S/Β0-THAL THAN IN SCD-S/S PATIENTS: RESULTS FROM THE GERMAN SCD REGISTRY

P. Allard 1 , L. Tagliaferri 1 , S. Lobitz 2 , H. Cario 3 , A. Jarisch 4 , R. Grosse 5 , S. Ebert 5 , M. Bleeke 5 , L. Oevermann 6 , D. Hakimeh 6 , A. Kulozik 1 , J. Kunz 1,*

1 Pediatric Hematology, Oncology and Immunology, University Hospital Heidelberg, Heidelberg; 2 Pediatric Hematology and Oncology, Gemeinschaftsklinikum Mittelrhein, Koblenz; 3 Klinik für Kinder- und Jugendmedizin, Pädiatrische Hämatologie und Onkologie, Universitätsklinikum Ulm, Ulm; 4 Zentrum für Kinder- und Jugendmedizin, Klinikum der Johann-Wolfgang-Goethe-Universität, Frankfurt; 5 Klinik und Poliklinik für Pädiatrische Hämatologie und Onkologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg; 6 Klinik für Pädiatrie m. S. Onkologie/Hämatologie/KMT, Charité- Universitätsmedizin Berlin, Berlin, Germany

Background: The national German SCD registry collects data on the genotype, the presentation, complications and treatment of patients with SCD both retrospectively and prospectively. Currently, 820 patients are enrolled. Compared to other cohorts of patients with SCD, these patients are ethnically more heterogeneous and more frequently treated with hydroxyurea (HU) [1, 2].

Aims: We aim at characterising the genotypes, complications, treatment and laboratory parameters in patients with heterozygous SCD (SCD-S/C, SCD-S/β 0 -thal, SCD-S/β + -thal) enrolled in the German SCD Registry.

Methods: The German SCD registry documents β-globin genotype, α-thalassemia genotype and four genetic modifiers of HbF synthesis ( BCL11A rs1427407, rs7606173; HMIP rs66650371; HBG2 rs7482144). The genotypes were correlated with laboratory parameters (total hemoglobin, MCV, reticulocytes, fetal hemoglobin, bilirubin, LDH), complications (prospectively collected: vasoocclusive crises (VOC), acute chest syndrome (ACS), red blood cell (RBC) transfusions; retrospectively collected: VOC, splenic sequestration, splenectomy) and treatment (HU, red blood cell transfusions). For the analysis of laboratory parameters and complications, patients of two years of age or older were included.

Results: We identified 88 patients with SCD-S/C (10,7% of all patients enrolled), 37 with SCD-S/β 0 -thal (4,5%), 50 with SCD-S/β + -thal (6,1%), and six patients (0,7%) with other genotypes (3 SCD-S/HPFH, 2 SCD-S/D Punjab, 1 SCD-S/OArab). The most recurrent β + -thalassemia mutation was IVS-I-110 G>A (n=18 patients). In contrast to patients with SCD-S/C, who originated almost exclusively from sub-Saharan Africa, patients with SCD-S/β 0 -thal and SCD-S/β + -thal originated mostly from the mediterranean region and carried HbF-boosting alleles more frequently.

The majority of patients were prescribed HU (SCD-S/β 0 -thal 97%, SCD-S/β + -thal 79%, SCD-S/C 60%). The prospectively documented frequency of VOC, ACS and red blood cell transfusions per patient year was lowest in SCD-S/C (Table 1).

For all patients aged between five and 18 years at registration, we analysed retrospectively collected data for the proportion of patients who had ever experienced typical complications of SCD, stratified according to the genotype. Splenic sequestration, splenectomy and pain crises were rare in SCD-S/C compared to SCD-S/S. In contrast, patients with SCD-S/β 0 -thal had suffered from splenic sequestration more frequently and had undergone splenectomy more often compared to SCD-S/S (Table 1).

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Summary/Conclusion: The overall frequency of complications of SCD is highest in homozygous SCD and lowest in SCD-S/C. With the most frequent β + -mutation being IVS-I-110 G>A among our patients, further data on the rare complications such as sepsis and stroke are required in order to guide the decision which patients will likely benefit from penicillin prophylaxis and screening by transcranial Doppler ultrasound.

P1481: THE INTERNATIONAL HEMOGLOBINOPATHY RESEARCH NETWORK (INHERENT): AN INTERNATIONAL INITIATIVE TO STUDY THE ROLE OF GENETIC MODIFIERS IN HEMOGLOBINOPATHIES

P. Kountouris 1,* , C. Stephanou 1 , N. Archer 2 , F. Bonifazi 3 , V. Giannuzzi 3 , K. Kuo 4 , A. Maggio 5 , J. Makani 6 , M. D. M. Mañú Pereira 7 , K. Michailidou 8 , S. W. Nkya 9 , O. Nnodu 10 , S. Trompeter 11 , L. Tshilolo 12 , A. Wonkam 13 , B. A. Zilfalil 14 , B. Inusa 15 , M. Kleanthous 1

1 Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; 2 Pediatric Hematology-Oncology, Dana-Farber/Boston Children’s Cancer and Blood Disorder Center, Boston, United States of America; 3 Fondazione per la ricerca farmacologica Gianni Benzi onlus, Bari, Italy; 4 Division of Hematology, University of Toronto, Toronto, Canada; 5 Campus of Hematology Franco and Piera Cutino, Azienda Ospedaliera Ospedali Riuniti Villa Sofia, Palermo, Italy; 6 Department of Haematology and Blood Transfusion, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania; 7 Translational Research in Child and Adolescent Cancer, Vall d’Hebron Institut de Recerca, Barcelona, Spain; 8 Biostatistics Unit, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; 9 Hematology and Blood Transfusion, Muhimbili University of Health and Allied Sciences, Dar Es Salaam, Tanzania; 10 Department of Haematology & Blood Transfusion, University of Abuja, Abuja, Nigeria; 11 Department of Haematology, University College London Hospitals NHS Foundation Trust, London, United Kingdom; 12 Centre Hospitalier Monkole, Kinshasa, Congo, The Democratic Republic of the; 13 Division of Human Genetics, University of Capetown, Cape Town, South Africa; 14 Universiti Sains Malaysia - Kampus Kesihatan, Kubang Kerian, Malaysia; 15 Paediatric Haematology, Guy’s and St Thomas’ NHS Trust, London, United Kingdom

Background: Haemoglobinopathies, including sickle cell disease (SCD) and thalassaemia syndromes, represent the commonest monogenic diseases in the world. Although their pathogenicity is well established, the diverse clinical manifestations and the varying degree of severity are less understood and are thought to be governed, in part, by genetic modifiers. Despite the identification and characterisation of a few genetic modifiers by previous studies, these are as yet insufficient to guide treatment recommendations or stratify patients reliably. Larger, multi-ethnic studies are needed to identify and validate further disease modifiers that can be used for patient stratification and personalised treatment. There is a growing need for deeper insight with the availability of novel targeted therapies and potentially curative options like gene therapy in both SCD and thalaessemia.

Aims: The International Haemoglobinopathy Research Network (INHERENT) is a recently established network with the aim of investigating the role of genetic modifiers in haemoglobinopathies, through a large-scale, multi-ethnic genome-wide association study (GWAS).

Methods: INHERENT brings together nine existing international or regional consortia in the field of haemoglobinopathies, namely ITHANET, RADeep, ARISE, SPARCO, SADaCC, REDAC, the HVP Global Globin Network, the International Health Repository, and the ClinGen Haemoglobinopathy VCEP. The activities of INHERENT are currently divided into five working groups, as follows: clinical, genotyping, data management and analysis, ethics, and knowledge translation. Participation in INHERENT is open for any group that can submit a minimum number of samples with their core phenotypic description. INHERENT membership is international and interdisciplinary and, currently, includes over 160 experts from 89 organisations, spanning 36 countries worldwide. INHERENT aims to recruit over 30,000 hemoglobinopathy patients, which is larger than any previous GWAS in the field.

Results: A survey amongst 67 (80%) of the participating centers revealed that INHERENT has the potential to recruit over 73 500 patients, of which 53 755 are SCD, 15 691 are β thalassemia (62% transfusion dependent), 4 179 (α thalassemia). A sample size of 10 000 individuals can detect associations down to MAF=0.05 with an OR=1.5 in binary phenotypes assuming a case/control ratio of 0.3 and p value 5x10 8 . For survival endpoints, 5000 individuals will be sufficient to detect a HR=1.5 and MAF 0.05 if we assume an event rate of 30%.

Summary/Conclusion: We demonstrate that the current membership of INHERENT has the potential to reach this sample size target. The large increase in the sample size and the diversity in the studied populations will enable novel discoveries and expand knowledge on haemoglobinopathy genetics, thus paving the way for advancing the science of personalised diagnosis and treatment.

P1482: PATIENTS WITH SEVERE SICKLE CELL DISEASE ON STANDARD OF CARE TREATMENT ARE VERY UNLIKELY TO BECOME VOC FREE FOR ONE YEAR: A COHORT STUDY OF MEDICAID ENROLLEES

M. Mahesri 1,* , S. B. Lee 1 , R. Levin 1 , S. Imren 2 , L. Zhang 2 , T. Beukelman 2 , L. Titievsky 2 , R. J. Desai 1

1 Division of Pharmacoepidemiology and Pharmacoeconomics, Brigham and Women’s Hospital & Harvard Medical School; 2 Vertex Pharmaceuticals, Boston, United States of America

Background: Sickle cell disease (SCD) patients have frequent vaso-occlusive crises (VOCs), which impact their quality of life, contribute to organ damage and failure, and are associated with increased mortality. An ongoing, open-label Ph3 clinical trial with CTX001, an investigational, autologous, ex vivo CRISPR/Cas-9-based gene edited therapy, in patients with severe SCD, is evaluating the proportion of patients who become VOC-free for at least 1 year after CTX001 treatment. Thus, it is important to know the proportion of patients with severe SCD on standard-of-care (SOC) who become VOC-free over time to provide context for the efficacy of CTX001 in eliminating VOCs in severe SCD.

Aims: To contextualize the efficacy of CTX001 in eliminating VOCs in patients with severe SCD, we used nationwide Medicaid claims data from 2000 to 2014 to assess the proportion of patients on SOC who became VOC-free during a 1-year follow-up. The inclusion/exclusion criteria were similar to those of the Ph3 clinical trial of CTX001 in patients with severe SCD.

Methods: A cohort of patients 12-35 years old with SCD was identified based on ≥1 inpatient claim or ≥2 outpatient claims for SCD after at least 2 years of continuous Medicaid enrollment. The date patients met the SCD case algorithm was defined as the cohort entry date. The 2-year period preceding the cohort entry date was defined as the baseline period. Consistent with the inclusion criteria in the CTX001 Ph3 trial, we restricted this population to severe SCD defined by ≥2 VOCs per year in each of 2 consecutive years (total of ≥4 VOCs over 2 years) at baseline. A VOC was identified using ICD diagnosis codes in hospitalization and ER visit claims for acute pain crisis, acute chest syndrome, priapism, and splenic sequestration. Additional exclusion criteria that were operationalizable using claims data included prior bone marrow transplant, moyamoya syndrome or history of stroke, low left heart function, low lung diffusing capacity (including pulmonary hypertension), advanced liver disease, decreased kidney function, significant bleeding disorder, prior malignancy or myelodysplasia, and select infections. We further restricted the cohort to patients with 1-year of continuous Medicaid enrollment (or less if any deaths) following the cohort entry. The primary outcome was the proportion of patients with 0 VOCs during a 1-year follow-up.

Results: A total of 5,874 patients with severe SCD were identified [mean (SD) age: 20.5 (6.0) years, 54.4% female, 87% African Americans]. Patients had a mean of 5.1 VOCs, 4.7 ER visits and 3.6 all-cause hospitalizations per year during the 2-year baseline period. Hydroxyurea was used in 37.5%, opioid analgesics in 88.7%, and NSAIDs in 71.9%. The proportion of patients with 0 VOCs during the 1-year follow-up was 9.1% (95% CI: 8.4, 9.9). When stratified by the number of VOCs at baseline, patients with higher numbers of VOCs had a lower proportion of 0 VOCs in the 1-year follow-up (Table).

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Summary/Conclusion: Fewer than 10% of patients with severe SCD with at least 2 VOCs per year in the prior 2 years at baseline became VOC-free for 1 year with SOC in this nationwide cohort of Medicaid enrollees. This demonstrates that patients with severe SCD are very unlikely to become VOC-free over a 1-year period and provides context for the efficacy of gene therapies in eliminating VOCs. These data also highlight the urgent need to develop effective therapies for patients with severe SCD as ~90% of patients continue to have VOCs, which are life-limiting and life-threatening events.

P1483: EPELEUTON, A NOVEL SYNTHETIC SECOND GENERATION W-3 FATTY ACID, PROTECTS HUMANIZED SICKLE CELL MICE AGAINST HYPOXIA/REOXYGENATION ORGAN DAMAGE

E. Federti 1 , A. Matte’ 1 , M. Hamza 2 , A. Lafferty 2 , D. Coughlan 2 , M. Weissbach 2 , D. L. Bhatt 3 , V. Riccardi 1 , R. Perissinotto 1 , A. Siciliano 1 , J. Climax 2 , C. Brugnara 4 , L. De Franceschi 1,*

1 Dept of Medicine, University of Verona, Verona, Italy; 2 Afimmune Ltd, Dublin, Ireland; 3 Brigham and Women’s Hospital Heart & Vascular Center; 4 Dept of Laboratory Medicine, Harvard Medical School, Boston, United States of America

Background: Sickle cell disease (SCD) is a hereditary red cell disorder with high mortality and morbidity. Although crizanlizumab, a P-selectin inhibitor, and voxelotor, an oral anti-sickling agent, have been recently approved for clinical management of SCD, therapeutic options to limit acute events and to reduce disease progression are still required. An attempt to target inflammatory vasculopathy and to modulate inflammatory response has been made based on evidence in other diseases such as in cardiovascular disease looking to administration of ω-3 fatty acids. In humanized SCD mice, we previously showed that polyunsaturated fatty acid supplementation protects against acute sickle cell-related lung and liver damages during hypoxia/reoxygenation (H/R) induced vaso-occlusive crisis (VOCs) (Kalish B et al. Haematologica 2016). This agrees with the observation that the resolution process actively controlled by specialized pro-resolving lipid mediators is defective in SCD (Matte A et al, Blood 2019).

Epeleuton (15-HEPE, 15 hydroxy eicosapentaenoic acid) is a novel synthetic second generation w-3 fatty acid which (i) has a favorable clinical safety profile, (ii) modulates systemic inflammation and triglyceride synthesis in patients with cardiometabolic disease (Climax J et al J Am Heart As 2020) and (ii) beneficially affects red cell indices in long-term studies in rats.

Aims: To evaluate the impact of epeleuton on acute organ damage in a humanized mouse model of SCD.

Methods: 3-4 month old male and female mice healthy control (Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) and Tim Townes sickle mice (SCD, Hba tm1(HBA)Tow Hbb tm2(HBG1,HBB*)Tow ) were used. Animals were treated for 6 weeks with epeleuton 1,000 mg/kg once daily or vehicle by gavage. Mice underwent 10 hours of hypoxia (8% oxygen), followed by 3 hours of reoxygenation (21% oxygen) to mimic acute VOCs (Matte A et al, Blood 2019). Hematologic parameters and molecular analysis of target organs for SCD such as lung were carried out.

Results: In SCD mice exposed to H/R stress, we found that epeleuton (i) reduces H/R induced hemolysis; (ii) normalizes tyrosine-phosphorylation profile of red cell membrane proteins; and (iii) decreases neutrophil count. In lungs of SCD mice exposed to H/R stress, we observed that epeleuton prevents the H/R induced activation of inflammatory and redox related transcription factors NF-kB, p65 and Nrf2 local inflammatory response compared with vehicle-treated animals. Epeleuton treated SCD mice exposed to H/R stress showed down-regulation of VCAM-1 and E-selectin, markers of inflammatory vasculopathy as well as reduction of lung protein oxidation and expression of anti-oxidant systems such as heme-oxygenase-1 (HO-1) or peroxiredoxin-2. Similar changes were also observed in livers of epeleuton-treated SCD mice exposed to H/R stress compared with vehicle-treated SCD animals. Of note, epeleuton prevented the H/R induced up-regulation of both VCAM-1 and ICAM-1, suggesting a direct impact of treatment on vascular activation and inflammation.

Summary/Conclusion: In humanized SCD mice exposed to H/R stress to mimic acute VOCs, epeleuton acts as a multimodal agent targeting red cells, hemolysis, inflammatory response, and vascular dysfunction. Our data provide the rationale for epeleuton as a potential novel therapeutic option for clinical management of patients with SCD.

P1484: UNDERSTANDING STAKEHOLDERS’ KNOWLEDGE ON SICKLE CELL DISEASE AND THEIR PERCEPTION ON THE PATIENT JOURNEY BY SOCIAL MEDIA LISTENING ACROSS EUROPE

D. Bras 1,2 , C. Bento 3,4,* , S. Aasaithambi 5 , J. Chauhan 5 , I. Moital 1

1 Novartis, Produtos Farmacêuticos SA, Porto Salvo; 2 National School of Public Health, Universidade NOVA de Lisboa, Lisboa; 3 Department of Haematology, Centro Hospitalar e Universitário de Coimbra; 4 Research Centre for Anthropology and Health (CIAS), Universidade Coimbra, Coimbra, Portugal; 5 Novartis Healthcare Pvt. Ltd., Hyderabad, India

Background: Sickle cell disease (SCD) is a common hematological disorder that affects millions of people worldwide. SCD poses a significant disease and financial burden, not only on patients but also on their families. Vaso-occlusive crisis are a common painful complication. However, despite their severe pain, patients with these crises are often undertreated. Social media platforms are increasingly being used in healthcare, including in the context of SCD, for generating content and sharing experiences, feelings, and perspectives between peers.

Aims: To gain understanding on patient awareness on SCD to help map perceptions around patient journey through social media analysis across Europe.

Methods: Data was collected retrospectively from April 2019 to April 2021 on SCD specific terms in 14 European countries (Figure 1A). Data sources were blogs, forums, and social networking sites (including Twitter, public Facebook, YouTube, and Instagram). The Talkwalker tool was used to collect and aggregate publicly available data.

Results: A total of 317.9K conversations on SCD were detected, mainly on Twitter (93%). Most conversations were among patients (61%), followed by their family and friends (16%), caregivers (4%), healthcare professionals (HCP) (3%), and others (16%). A total of 945 posts of relevant patient-centric conversation were analyzed. The highest share in conversation came from the UK (56%), followed by France (24%), and Germany (4%). Most patients were females (73%) and ≤30 years old (75%) (Figure 1B). Patient journey stages (Figure 1C) were addressed in 52% of the conversation. Among these stages, symptoms (56%) were the most discussed, where pain episodes, pain in general, and vaso-oclusive crises (often associated with pain) were the most common topics. The second most discussed stage of patient journey was treatment (44%); blood transfusions was the most mentioned treatment (34%), which was generally found to be effective. Hospital visits or hospitalization were mentioned in 11% of the conversations, and 62% of them included crisis that patients faced due to symptoms. Impact on quality of life was also extensively discussed among patients, with emotional impact (56%) being the most prevalent, followed by physical (47%), social (30%), and financial (10%) impact. Unmet needs were derived from 24% of the conversations. Most frequent unmet needs were lack of awareness of SCD by HCP and the general public (42%), and lack of empathy and support from HCP (24%). Racial bias was highlighted as a concern by patients who had their symptoms questioned or dismissed by their care team.

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Summary/Conclusion: Social media, especially Twitter, are mainly used by young patients with SCD in Europe. Approximately half of the conversation between users were related to the stages of patient journey, being symptoms and treatment the most discussed topics. One-fourth of the conversations mentioned unmet needs, where both a low level of awareness of SCD by HCP and the general population and a lack of support from the HCP was perceived by patients. SCD patients actively campaigned to raise awareness of the disease, whilst also expressing the physical and emotional impact that SCD had on their lives.

P1485: A MULTICENTER, RETROSPECTIVE STUDY ON REAL-WORLD EXPERIENCE OF PATIENTS WITH SICKLE CELL DISEASE TREATED WITH VOXELOTOR

B. Andemariam 1,* , M. Idowu 2 , N. Shah 3 , R. Drachtman 4 , A. Sharma 4 , A. Glaros 5 , M. Achebe 6 , A. Nero 7 , S. Curtis 8 , C. Minnitti 8

1 New England Sickle Cell Institute, University of Connecticut Health, Farmington; 2 University of Texas Health, Houston; 3 Duke University School of Medicine, Durham; 4 Rutgers Robert Wood Johnson Medical School, New Brunswick; 5 Division of Pediatric Hematology/Oncology, Central Michigan University, Detroit; 6 Brigham and Women’s Hospital, Boston; 7 University of Texas Southwestern Medical Center, Dallas; 8 Albert Einstein College of Medicine, Bronx, United States of America

Background: Sickle cell disease (SCD) is an inherited systemic disorder, with pathology driven by polymerization of sickle hemoglobin (HbS). Voxelotor, a HbS polymerization inhibitor, is approved in the United States for treatment of SCD in adults and pediatric patients aged ≥4 years and in the European Union for the treatment of hemolytic anemia due to SCD in adult and pediatric patients ≥12 years of age as monotherapy or in combination with hydroxycarbamide. Efficacy and safety data from the randomized, placebo-controlled HOPE trial demonstrated the effectiveness of voxelotor in increasing hemoglobin (Hb) levels and reducing markers of hemolysis. Real-world studies complement and expand upon information gathered in randomized clinical trials by providing evidence of treatment safety and efficacy in clinical practice.

Aims: The Retrospective Study to Evaluate Outcomes in Patients With Sickle Cell Disease Treated With Oxbryta (RETRO) aims to characterize real-world safety and effectiveness of voxelotor in adults and adolescents (aged ≥12 years) with SCD treated with voxelotor as part of their usual care.

Methods: RETRO is a multicenter, post-marketing, retrospective study that collected laboratory and clinical data from patients’ medical records 1 year before and 1 year or more after initiation of voxelotor treatment. Patients with documented SCD who received voxelotor for ≥2 consecutive weeks were included in this analysis.

Results: Data from 216 patients across 9 US sites were collected and analyzed. The mean (SD) patient age was 33.5 (14.2) years, and the mean (SD) duration of voxelotor treatment was 51.1 (25.6) weeks. Reasons for voxelotor prescription (n, %) included reducing the following: anemia (151, 69.9%), pain (51, 23.6%), frequency of vaso-occlusive crises (45, 20.8%), and the need for blood transfusions (17, 7.9%); multiple reasons may have been selected. Most patients were prescribed an initial voxelotor dose of 1500 mg (n=187, 86.6%), and 68.1% (n=147) of patients used hydroxyurea concomitantly. A total of 25.0% (n=54) of patients had a dosage interruption or adjustment. Reasons for dosage change (n, %) included adverse event (AE; 37, 17.1%), other (22, 10.2%), pill burden (2, 0.9%), and lack of efficacy (1, 0.5%); multiple reasons may have been selected.

A total of 198 patients had recorded baseline and post-treatment Hb values. In these patients, the mean (SD) peak observed post-treatment Hb level increased from baseline by 1.4 (1.6) g/dL, from 7.8 (1.5) g/dL to 9.2 (2.0) g/dL ( Figure ). In patients with recorded baseline and post-treatment indirect bilirubin levels (n=80) and reticulocyte percentages (n=178), the mean (SD) minimum observed post-treatment value for indirect bilirubin decreased from baseline by 1.1 (1.9) mg/dL, from 3.1 (2.0) mg/dL to 1.9 (1.9) mg/dL, and reticulocyte percentage decreased from baseline by 3.8% (5.8%), from 11.6 % (6.8%) to 7.7% (5.1%). The safety and tolerability of voxelotor in the real-world setting will be presented. The most common non-SCD-related treatment-emergent AEs were diarrhea, headache, and rash; 37.0% (n=80) of patients reported ≥1 non-SCD-related AE, and most AEs were mild in severity.

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Summary/Conclusion: RETRO is the first multicenter study to collect and analyze retrospective data from patients with SCD treated with voxelotor in a real-world setting. These interim results are consistent with the HOPE trial, showing that voxelotor treatment was associated with increased Hb levels and decreased hemolytic markers. The safety data are also consistent with those from the HOPE trial.

P1486: TRIALS IN PROGRESS: THE THRIVE STUDIES EVALUATING THE EFFICACY, SAFETY, AND LONG-TERM TREATMENT WITH INCLACUMAB, A P-SELECTIN INHIBITOR, IN PATIENTS WITH SICKLE CELL DISEASE

B. Andemariam 1,* , A. Inati 2 , R. Colombatti 3 , C. Minniti 4 , C. Brown 5 , M. Hottmann 6 , S. Gray 6 , C. Hoppe 6 , M. Davis 6 , P. Yue 6

1 New England Sickle Cell Institute, University of Connecticut Health, Farmington, United States of America; 2 Lebanese American University, Byblos and Beirut, Nini Hospital, Tripoli, Lebanon; 3 Clinic of Pediatric Hematology Oncology, Department of Woman’s and Child’s Health, Azienda Ospedale – Università di Padova, Padua, Italy; 4 Albert Einstein College of Medicine, Bronx; 5 Aflac Cancer and Blood Disorder Center of Children’s Healthcare of Atlanta and Department of Pediatrics, Emory School of Medicine, Atlanta; 6 Global Blood Therapeutics, South San Francisco, United States of America

Background: Sickle cell disease (SCD) is a group of autosomal recessive red blood cell (RBC) disorders caused by a single point mutation in the β-globin gene, resulting in the production of hemoglobin S. Hemoglobin S polymerization within deoxygenated RBCs contributes to hemolysis, vaso-occlusion, and end-organ damage. Abnormal heterocellular adhesive interactions between RBCs and endothelial cells, leukocytes, and platelets play a central role in triggering painful vaso-occlusive crises (VOCs) in patients with SCD. Inclacumab is a recombinant, fully human monoclonal antibody directed against human P-selectin, a cell adhesion molecule produced by endothelial cells and platelets. Inclacumab binding of P-selectin, and prevention of P-selectin ligand binding, interferes with the adhesion of sickle RBCs, platelets, and leukocytes to endothelium and is the putative mechanism by which inclacumab may potentially reduce the incidence of VOCs.

Aims: We aim to conduct two global, multicenter, phase 3 studies and an open-label extension (OLE) study to evaluate the safety and efficacy of inclacumab in individuals with SCD.

Methods: THRIVE-131 ( NCT04935879 ) is a randomized, double-blind, placebo-controlled, multicenter trial in which ~240 participants aged ≥12 years experiencing 2 to 10 VOCs in the previous 12 months will be randomized 1:1 to receive intravenous (IV) inclacumab or placebo every 12 weeks for 48 weeks (Figure). The primary endpoint is the rate of VOCs during the 48-week treatment period. THRIVE-132 ( NCT04927247 ) is a randomized, double-blind, placebo-controlled, multicenter trial in which ~280 participants aged ≥12 years experiencing 2 to 10 VOCs in the previous 12 months will be randomized 1:1 to receive a single dose of IV inclacumab or placebo within 5 days of resolution of an index VOC that required admission to a healthcare facility and treatment with parenteral pain medication. The primary endpoint is the proportion of patients with readmission for a VOC within 90 days of randomization. After completion of their participation in THRIVE-131 or THRIVE-132, participants may enroll in THRIVE-133 OLE , evaluating the long-term safety of inclacumab. In THRIVE-133 OLE, participants will receive IV inclacumab every 12 weeks as long as the clinical benefit of treatment outweighs the risk and until access to inclacumab from an alternative source (eg, through commercialization or a managed-access program) becomes available.

Results: Recruiting for both THRIVE-131 and THRIVE-132 began in October 2021 and is ongoing.

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Summary/Conclusion: The THRIVE-131 and THRIVE-132 phase 3 studies will examine the efficacy of inclacumab in reducing VOCs and readmissions due to VOCs, and the THRIVE-133 OLE study will examine the long-term safety of inclacumab in individuals with SCD.

P1487: SICKLE CELL HEALTH AWARENESS, PERSPECTIVES AND EXPERIENCES (SHAPE) SURVEY: FINDINGS ON THE BURDEN OF SICKLE CELL DISEASE ON PATIENTS AND THEIR UNMET NEEDS AS REPORTED BY HEALTHCARE PROFESSIONALS

M. de Montalembert 1,* , A. Anderson 2 , F. Costa 3 , W. Jastaniah 4 , J. B. Kunz 5 , I. Odame 6 , A. Beaubrun 7 , B. Lartey 8 , B. P. Inusa 9

1 Department of Pediatrics, Necker-Enfants Malades Hospital, Paris, France; 2 Department of Pediatric Hematology-Oncology, PRISMA Health-Upstate Comprehensive SCD Program, Greenville, United States of America; 3 School of Medicine, University of Campinas - UNICAMP, Campinas, São Paulo, Brazil; 4 King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia; 5 Department of Pediatric Oncology, Hematology and Immunology, Hopp Children’s Cancer Center, University of Heidelberg, Heidelberg, Germany; 6 Department of Hematology/Oncology, The Hospital for Sick Children, Toronto, Canada; 7 Global Blood Therapeutics, South San Francisco, United States of America; 8 Ipsos Healthcare; 9 Department of Paediatric Haematology, Guy’s and St Thomas’ Hospital, London, United Kingdom

Background: Sickle cell disease (SCD) has a substantial emotional and physical burden on patients. There is currently limited research on the impacts of SCD on patient quality of life (QOL). Recently, research into treatment options to improve QOL for these patients has increased. To complement this drive to improve patient QOL, there needs to be a more detailed understanding of what it means to live with SCD in different communities, especially considering the health inequalities faced by different groups around the world. In addition to surveying patients and caregivers, viewpoints from healthcare professionals (HCPs) should also be considered, as the perspectives of HCPs may impact or reflect patient QOL.

Aims: To understand the perspectives of HCPs on the patient burden of SCD, the impacts of SCD on patient QOL, and the needs of patients with SCD.

Methods: The SHAPE survey is a multinational study composed of quantitative online surveys of patients, their caregivers, and HCPs. The surveys require participants to answer a range of closed-ended questions about their circumstances and experiences in order to build a robust and reliable dataset, on which descriptive statistics were performed. HCPs were included if they had at least 10 patients with SCD under their care and had been practicing for 3 to 35 years. Informed consent was obtained from all participants, and all identifiable information was kept private and secure. The study protocol was reviewed and approved by an independent institutional review board.

Results: Interviewed HCPs (n=219) were composed of 48% hematologists, 37% hematologist-oncologists, 11% pediatric hematologists, 1% pediatricians, 1% internists, and <1% general or family practitioners. Participating HCPs were from the US (23%), Germany (15%), Brazil (14%), France (14%), UK (14%), Canada (7%), Saudi Arabia (7%), and United Arab Emirates (7%). The mean (range) number of patients under their care was 32 (16-65). Most HCPs (82%) agreed that SCD causes financial difficulties for patients, and many (73%) agreed with the statement that “patients of lower socioeconomic status are more difficult to treat” (Figure). Nearly 1 in 3 HCPs (31%) found it challenging to understand their patients’ needs, with more than 2 in 5 (43%) citing difficulties due to having different ethnic backgrounds from their patients. Most HCPs (78%) felt confident in their knowledge of the long-term effects of SCD, but 83% wanted more support in educating their patients on this topic. Almost two-thirds (59%) of HCPs believed that end-organ damage is unavoidable and that SCD greatly impacts patients’ long-term health prospects (64% for patients <18 years; 60% for patients ≥18 years) and their ability to attend and succeed at work or school (53% for <18 years; 49% for ≥18 years).

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Summary/Conclusion: Almost one-third of HCPs found it challenging to understand their patients’ needs. Based on these findings, additional support for HCPs in their communication with and education of their patients could result in more effective SCD treatment. HCPs also noted an unmet need for treatments that fully address end-organ damage in SCD. These findings highlight the complex environment that HCPs face when treating patients with SCD, including differences in socioeconomic status and ethnic background, the need for education, and a lack of resources. The patients and caregivers portion of the SHAPE survey will complement the data presented here by providing additional perspectives on the burden of SCD.

P1488: ASSOCIATION BETWEEN HEMOGLOBIN LEVELS AND END-ORGAN DAMAGE IN SICKLE CELL DISEASE: A RETROSPECTIVE LINKED PRIMARY AND SECONDARY CARE DATABASE ANALYSIS IN ENGLAND

P. Telfer 1,* , S. Carvalho 2 , J. Ruzangi 3 , M. Arici 4 , M. Binns 4 , A. Beaubrun 4 , C. Rice 2 , J. J. Were 2

1 Queen Mary, University of London; 2 Health iQ, CorEvitas; 3 London School of Hygiene and Tropical Medicine, London, United Kingdom; 4 Global Blood Therapeutics, South San Francisco, United States of America

Background: Sickle cell disease (SCD) is a chronic, multifaceted blood disorder that affects the oxygen-carrying protein hemoglobin (Hb). Along with chronic hemolysis and vaso-occlusion that often accompanies SCD, low Hb levels may contribute to downstream end-organ damage (EOD). Emerging evidence suggests an association between chronic hemolytic anemia in SCD and both short-term and long-term adverse outcomes, including EOD. Therefore, treatments that directly increase Hb may improve patients’ acute symptoms while reducing the risk for long-term disease-related effects known to contribute to morbidity and mortality.

Aims: This study aims to ascertain the association between Hb level variation and EOD and clinical conditions typically associated with SCD (stroke, pulmonary hypertension, chronic kidney disease [CKD], end-stage renal disease [ESRD], leg ulcers, and pneumonia) in a real-world data set.

Methods: The Clinical Practice Research Datalink (CPRD) and the Hospital Episode Statistics (HES) databases in England were used to retrospectively identify linked Hb data among patients with SCD (1 April 2007 to 31 March 2019). Recurring results in any 90-day period were averaged. Logistic regression adjusted with demographic and clinical variables was used to determine odds ratios (ORs) of developing each EOD outcome or clinical complication. ORs, P values, 95% confidence intervals (CIs), and areas under the receiver operating curve (AUC-ROCs) and corresponding 95% CIs were reported.

Results: Of 11,936 patients identified, 5379 (45%) had Hb results recorded in the CPRD. The logistic model for risk assessment of all outcomes studied indicated that an increase in Hb among patients with SCD was associated with a statistically significant ( P <0.001) decrease in adjusted ORs per 1 g/dL increase in Hb (Table). Except for ESRD, leg ulcers, and pneumonia, the adjusted models showed statistically significant increases in AUC-ROC compared with the unadjusted models, signifying that the adjusted models were more robust and further verified a statistical OR after adjustment.

Increased Hb was associated with decreased risk of respiratory-related events. Pulmonary hypertension had the strongest effect with the most robust model (OR adjusted of 0.66). For pneumonia, OR adjusted was 0.77. For renal insufficiency, ORs for developing CKD or ESRD were significantly lower than 1 (OR adjusted =0.73 and OR adjusted =0.75, respectively). After adjusting for clinical and demographic factors, the model predicted a lower risk for stroke (OR adjusted =0.89). The estimated AUC-ROCs suggest that the adjusted model is more robust in stroke, hypertension, and CKD. These findings did not change when testing for other covariates such as deep vein thrombosis.

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Summary/Conclusion: Among patients with SCD, an increase in Hb of 1 g/dL was associated with a statistically significant reduction in risk for 6 common EOD outcomes and clinical conditions. The results were more pronounced after adjusting for demographic and clinical covariates (based on clinical opinion and literature review) and further assessed with a bivariate analysis. The results were obtained from a representative real-world evidence dataset analyzed over a 12-year period, which was sufficient to observe EOD events, and are generalizable to the UK and similar populations. Our findings support the use of therapeutics that increase Hb in patients with SCD to protect against deleterious organ damage.

P1489: MANAGEMENT OF SICKLE CELL DISEASE WITH HYDROXYUREA: A SYSTEMATIC REVIEW FROM LOW- AND MIDDLE-INCOME COUNTRIES

V. Paintsil 1,* , K. Marfo 2 , F. Adisa 3 , S. Khan 4 , L. M. Tshilolo 5

1 Kwame Nkrumah University of Science and Technology/Komfo Anokye Teaching Hospital, Kumasi, Ghana; 2 Novartis Pharma AG, Switzerland, Basel, Switzerland; 3 Novartis Pharmaceuticals Inc., United states, Cambridge, United States of America; 4 Novartis Healthcare Pvt Ltd, Hyderabad, India; 5 Centre Hospitalier Mère-Enfant, CEFA/Monkole, Kinshasa, Democratic Republic of the Congo, Kinshasa, Congo, The Democratic Republic of the

Background: Clinical trials conducted in Africa have demonstrated the safe and effective use of hydroxyurea (HU) for sickle cell disease (SCD) children in low-resource settings. Despite WHO listing of HU as essential medicine, it’s clinical use is limited in LMICs with high disease burden.

Aims: We aim to assess the available literature on the clinical efficacy, safety and real-word effectiveness with HU therapy in LMICs. Here we present the preliminary observation from the clinical studies.

Methods: This systematic literature review was performed using Embase, PubMed, OVID and Web of Science, in English between January 2000–2022. Clinical and real-world studies assessing therapy with HU in patients with SCD were considered.

Results: Of 3709 studies identified in the search, 124 unique studies with 20 clinical trials were included (Figure). There is a dearth of studies from Asian countries, and majority of the included studies are from sub-Saharan Africa (SSA) and Latin America. In these regions the initial dose of HU ranged from 10–25 mg/kg/day and the most common initial dose was 15–20 mg/kg/day. The maximum tolerated dose (MTD) of HU studied was 25–35 mg/kg/day; however, few studies on stroke prevention used MTD of 40 mg/kg/day. As anticipated, significant increases in haemoglobin (Hb), foetal haemoglobin (HbF) and mean corpuscular volume levels were reported both with fixed (20 mg/kg/day) as well as escalated dose (≤35 mg/kg/day) across various subgroups. Treatment with HU at doses 15–30 mg/kg/day was shown to reduce the need for transfusions, and few studies reported that no transfusions were required during the treatment period. HU treatment initiation also reported reduction in vaso-occlusive crisis (VOC)–related pain episodes; treatment for only 6 months across fixed doses of 15–20 mg/kg/day has resulted in significant reduction in VOC-related pain. HU therapy reduced abnormally high transcranial Doppler velocities, was associated with a significantly lower risk of conversion to abnormal time-averaged maximum mean velocities and prevented the risk of primary stroke in SCD patients. The use of low-dose HU therapy (10 mg/kg/day) for children with SCD was also beneficial in primary stroke prevention. The HU dose of 30–40 mg/kg/day in children for a period of 3.5–9.3 years was effective in preventing secondary stroke with no risk of toxicity. Treatment with HU for a year with fixed dose of 20 mg/kg/day was associated with lower stroke recurrence rates if started within 2 months of initial stroke. Significant reduction in malaria events and related hospitalisations were associated with HU. Treatment for 2 years at a dose of 20 mg/kg/day in young infants (median age 15 months) was also reported to be well tolerated and effective in improving Hb and HbF levels and delaying functional asplenia. Clinical safety of HU is long well-established, and no new safety signals were reported. The myelosuppression observed with HU treatment was mostly transient in nature but required regular monitoring which can be challenging in LMICs.

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Summary/Conclusion: Our preliminary findings showed that in patients with SCD from LMICs especially SSA and Latin American regions, HU was well tolerated and effective in improving haematological outcomes, lowering VOC and malaria events, and reducing primary/recurrent stroke risks. HU treatment was also effective and safe in young infants (≥ 9 months of age). Treatment initiation with HU reduced the need for transfusions, which can be beneficial for SCD management in these resource-limited countries.

P1490: CHILDREN WITH SICKLE CELL DISEASE AND MOYAMOYA SYNDROME: A 10-YEAR EXPERIENCE

A. Pina Monteiro 1,* , A. R. Henriques 2 , M. Mota 3 , C. Salgado 4 , J. Coelho 5 , S. Quintas 5 , M. J. Palaré 4 , A. Ferrão 4

1 Hematologia, Instituto Português de Oncologia de Lisboa; 2 Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte (CHULN); 3 Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte (CHULN), Lisboa; 4 Unidade de Hematologia Pediátrica, Serviço de Pediatria, Departamento de Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte (CHULN), Lisboa; 5 Unidade de Neurologia Pediátrica, Serviço de Pediatria, Departamento de Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte (CHULN), Lisboa, Lisboa, Portugal

Background: Sickle cell anemia (SCD) is a multiorgan disease. Involvement of the central nervous system (CNS) is associated with increased mortality. Cerebrovascular accidents, especially when associated with permanent neurological deficits, are one of the most devastating potential outcomes of SCD. SCD can be complicated by Moyamoya syndrome (MMS), which is characterized by progressive stenosis of the supraclinoid segment of carotid arteries and development of typical collaterals, resulting in increased risk for both ischemic and hemorrhagic strokes.

Aims: We describe the clinical characteristics of children with SCD and MMS treated at our center over the last 10 years and define the prevalence of MMS in our setting.

Methods: We conducted a retrospective, descriptive study of all patients with SCD who were diagnosed with cerebrovascular disease and with MMS in our institution from 2012 to 2021. Indications for neuroimaging [(magnetic resonance imaging (MRI) or magnetic resonance angiography (MRA)] in our center include recurrent episodes of syncope, epileptic seizures, neurocognitive changes, time-averaged maximum mean velocity (TAMMV) of the middle cerebral or intracranial internal carotid arteries (ICA) measured ≥200 cm/s or before starting regular transfusion regime. Relevant clinical data was collected and summarized using descriptive statistics.

Results: The study included 109 patients with SCD and 19,3% of them (21 patients) required neuroimaging. Within these patients, 6 presented with acute focal neurological signs that resulted in a diagnosis of ischemic stroke in four patients, and posterior reversible encephalopathy syndrome diagnosis in the other two. Besides these, 2 patients (9,5%) were diagnosed by neuroimaging with MMS and 1 (4,8%) had a pattern suggestive of MMS on brain MRI not confirmed by angiography. Furthermore, whereas four (19%) patients had no imaging findings suggestive of cerebrovascular disease, they presented with TAMMV ≥200cm/s, so they were proposed to regular transfusion regimen for primary prevention of stroke. Regarding the patients diagnosed with SCD and MMS, MMS was diagnosed at 9 years old in one patient and at 10 years old in another. Both patients had a SS genotype and were diagnosed following an abnormal surveillance transcranial doppler study, since they were asymptomatic for the disease. One of the patients was under erythrocytapheresis protocol and achieved a HbS level <20%. Currently she is without critical stenosis or cerebrovascular events, maintaining only imaging follow-up. The other patient was under regular transfusion regimen with HbS levels between 40 and 50%, and has been proposed for sibling bone marrow transplantation and encephaloduroarteriosynangiosis.

Summary/Conclusion: The prevalence of MMS in our center (1,8%) is lower than described in other studies. Recent guidelines recommend at least a one-time MRI screening in early-school-age children to detect cerebrovascular disease earlier and reduce the burden of CNS complications in children and adults with SCD. By following these new recommendations, we expect an earlier and more frequent diagnosis of MMS, thus reducing the morbidity of these patients.

P1491: REAL-WORLD INCIDENCE OF VASO-OCCLUSIVE CRISES IN PATIENTS WITH SICKLE CELL DISEASE (SCD) AND A HIGH BASELINE DISEASE BURDEN TREATED WITH CRIZANLIZUMAB: RESULTS FROM A MANAGED ACCESS PROGRAM (MAP)

A. C. Silva-Pinto 1,* , R. Colombatti 2 , A. Pasanisi 3 , F. Arcioni 4 , L. DeBonnett 5 , W. Soliman 6 , R. Sarkar 7 , R. D. Cançado 8

1 Regional Blood Center, Department of Medical Imaging, Hematology and Oncology, Ribeirão Preto School of Medicine, São Paulo, Brazil; 2 Azienda Ospedale-Università di Padova, Padova; 3 Centro Microcitemia-UOC, Ematologia con trapianto, PO Perrino, Brindisi; 4 Pediatric Hematology and Oncology with Bone Marrow Transplantation, Azienda Ospedaliera di Perugia, Perugia, Italy; 5 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 6 Novartis Saudi Ltd, Riyadh, Saudi Arabia; 7 Novartis Healthcare Pvt. Ltd., Hyderabad, India; 8 Department of Hematology and Oncology, Santa Casa de São Paulo School of Medicine, São Paulo, Brazil

Background: Vaso-occlusive crises (VOCs) are the hallmark of SCD and can lead to serious complications and organ damage. P-selectin is a cell adhesion protein that plays a key role in the multicellular interactions that can lead to VOCs. Crizanlizumab, a first-in-class humanized monoclonal antibody that blocks P-selectin, is approved in several regions to prevent/reduce VOCs for SCD patients (pts) aged ≥16 years. Since June 2018, pts in some countries have received crizanlizumab before health authority approval via a MAP ( NCT03720626 ).

Aims: To describe the rate of VOCs and use of opioids for VOC-related pain relief 12 months before crizanlizumab initiation and after ≥12 months of crizanlizumab treatment in SCD pts participating in the MAP, in countries where publication of these data is allowed.

Methods: The MAP was designed to provide access to crizanlizumab for pts with a serious or life-threatening disease (SCD) for which no comparable or satisfactory alternative to crizanlizumab was available in their country. Other eligibility criteria included: aged 16–70 years (18–70 years in Italy); history of VOCs as determined by the treating physician (including recurrent VOCs while taking preventative therapies eg hydroxyurea [HU]); and ineligibility for a crizanlizumab clinical trial. Pts’ disease burden in the 12 months prior to crizanlizumab initiation (baseline period) was provided by the treating physician. This analysis reports VOC and opioid use frequency after ≥12 months of crizanlizumab treatment, overall and stratified by SCD genotype and history of HU use, compared with baseline.

Results: As of Feb 2022, 188 pts have received crizanlizumab in the MAP, of whom 87 have been treated for ≥12 months (Brazil, n=79 [91%]; Italy, n=5 [6%]; Spain, n=2 [2%]; Israel, n=1 [1%]). Median (interquartile range [IQR]) age of the 87 pts was 33 (25–40) years, 57% were female, 45% were African American, 8% Caucasian, 20% Hispanic and 28% of ‘other’ ethnicity, and 82% had an HbSS genotype. History of HU use was reported for 41/56 (73%) pts with available data. During the baseline period, 85% (n=74/87) of pts were hospitalized for a total of 220 SCD-related complications.

At baseline, 100% (n=87) and 93% (n=81) of pts had ≥1 home- and ≥1 healthcare-managed VOC, respectively, vs 79% and 63% of pts after ≥12 months of crizanlizumab treatment. Overall, crizanlizumab led to a median (IQR) absolute reduction from baseline of –3.0 (–6.0 to –1.0) home-managed and –2.0 (–4.0 to 0) healthcare-managed VOCs after ≥12 months of treatment. Similar reductions in VOC rates post- vs pre-crizanlizumab were observed when stratifying the data by SCD genotype/prior HU use (Figure), although some groups only contain a small number of pts.

Opioids were taken for VOC-related pain relief by 95% of pts (n=83/87) at baseline and by 69% (n=60/87) in the 12 months after crizanlizumab treatment; the most common was morphine (48% [n=40/83] and 40% [n=24/60], respectively).

Adverse events were consistent with those reported in other crizanlizumab studies.

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Summary/Conclusion: Pts in the crizanlizumab MAP had significant disease burden at baseline, as evidenced by the high rate of home- and healthcare-managed VOCs, proportion of patients with SCD-related complications and use of opioids for VOC-related pain relief, despite many pts reporting prior HU use. Crizanlizumab treatment led to clinically relevant reductions from baseline in the median annualized rates of home- and healthcare-managed VOCs and use of opioids in this real-world setting, consistent with results from SUSTAIN.

P1492: PHARMACODYNAMIC EFFECTS OF AG-946, A HIGHLY POTENT NOVEL ACTIVATOR OF PYRUVATE KINASE, IN EX VIVO TREATMENT OF RED BLOOD CELLS FROM SICKLE CELL DISEASE PATIENTS

M. Rab 1,* , M. van Dijk 1 , J. Bos 1 , B. van Oirschot 1 , J. Gerrits 2 , P. Kosinski 3 , C. Kung 4 , J. Jans 2 , E. van Beers 5 , L. Dang 3 , R. van Wijk 1

1 Central Diagnostic Laboratory Research; 2 Metabolic Diagnostics, University Medical Center Utrecht, Utrecht, Netherlands; 3 Agios Pharmaceuticals inc., Cambridge, United States of America; 4 Agios Pharmaceuticals inc., Cambridge; 5 Van Creveldkliniek, University Medical Center Utrecht, Utrecht, Netherlands

Background: Sickle cell disease (SCD) is a monogenetic red blood cell (RBC) disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of RBC, which is the direct result of the polymerization of abnormal hemoglobin S. Sickling is one of the core factors that cause vaso-occlusion and sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP). Previously we showed that RBC pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling ( Rab et al , Blood 2021). Currently, mitapivat is being investigated in phase 1, phase 2, and phase 2/3 trials in patients with SCD (# NCT04000165 , EudraCT#2019-003438-18, and NCT05031780 ).

Aims: Recently, AG-946, a novel PK activator, has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat.

Methods: Buffy coat depleted whole blood obtained from five patients with SCD was incubated for 20-24 hours in absence or presence of mitapivat (100 mM) or AG-946 (1 mM, 5 mM, 50 mM). After ex vivo treatment, enzymatic activities of PKR and PKR thermostability were measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured with the Hemox Analyzer. RBC sickling was analyzed with the oxygenscan, a method that characterizes individual sickling behavior by oxygen gradient ektacytometry. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO 2 at which RBCs start to sickle during deoxygenation under shear stress.

Results: PKR activity was increased compared to vehicle (DMSO) to a similar extent in the presence of mitapivat or AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5 mM (mean 66%, SD 23%), and AG-946 50 mM (mean 95%, SD 17%, Figure 1B). After incubation with mitapivat or AG-946, 2,3-DPG decreased (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). Accordingly, p50 decreased significantly after incubation with mitapivat 100 mM (mean 95%, SD 2%), as well as AG-946 1 mM (mean 96%, SD 2%), AG-946 5 mM (mean 94%, SD 2%), and AG-946 50 mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS compared to vehicle in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency in vitro (Figure 1F).

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Summary/Conclusion: Ex vivo treatment of SCD RBCs with the novel PK activator AG-946 activates and stabilizes PKR, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Taken together, these results are the first in an ex vivo model to demonstrate that the novel PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PKR-stabilizing properties and, hence, might represent a potential novel therapeutic option in addition to mitapivat for the treatment of SCD.

P1493: CHRONIC COMPLICATIONS IN THE EAST LONDON SICKLE NEWBORN COHORT STUDY (ELSNCS)

C. Sangarappillai 1 , M. Soriano 1,* , J. Bestwick 2 , D. Kafuko 3 , K. Newell 3 , B. Kaya 3 , S. Bennett 3 , F. Barroso 3 , D. Tsitsikas 4 , P. Telfer 1

1 Centre of Genomics and Child Health; 2 Wolfson Institute for Population Health, Queen Mary University of London; 3 Haematology, Barts Health NHS Trust; 4 Haematology, Homerton University hospital NHS Foundation Trust, London, United Kingdom

Background: Current standard of care for sickle cell disease (SCD) has resulted in a significant reduction in childhood mortality. It is likely that health is improved also for adults, but the expected residual chronic morbidity and reduced life expectancy is not clear, and accurate prognosis is important for planning health care services and in evaluating the impact of new therapies. The ELSNCS has been recruiting consecutive infants diagnosed within a universal newborn screening programme since 1983. Subjects have been managed in a relatively seamless paediatric and adult specialised service in the UK National Health Service during the era where standard of care has evolved.

Aims: This study provides an opportunity to make an early estimate of the risk of recognised chronic morbidities.

Methods: Eligible patients were born in the London Boroughs of Hackney and Tower Hamlets, diagnosed by newborn screening, with initial referral to Royal London Hospital (RLH) Paediatric Sickle Cell Service and seen at least once. Permission to study all patients without individual consent was obtained from the UK National Confidentiality Advisory Group and National Research Ethics Committee. All clinical data on these patients has been captured in a designated clinical and research database maintained in our service over the past 22 years. A set of definitions for each complication was developed based on published literature and consensus group discussion taking into account the nature of the cohort data. For each subject, clinical data was validated against source records up to death, stem cell transplant, or follow-up cut set at December 31,2018. Risks of developing specific complications (95%CI) were estimated at age 25 by Kaplan Meier survival analysis.

Results: There were 404 subjects (268 HbSS , 118 HbSC , 17 HbS beta thal, 1 HbS beta 0). 200 were female. At time of data cut, mean age was 13.3 years (age range 0.3 – 35.4),11 had died, 6 had BMT. 172 (42.6%) were under follow-up in paediatrics at RLH, 93 (23.0%) in adult at RLH, 95 (23.5%) had transferred care to another clinic, 12 (3.0%) were known to be alive but not under follow-up and 15 (3.7%) had been lost to follow-up. During follow-up 93 (23.0%) had been treated with chronic transfusion for more than 6 months and 95 (23.5%) with Hydroxyurea for more than 6 months Risks of chronic complications at age 25 are shown in the table.

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Summary/Conclusion: Estimated survival for HbSS at age 25 is 92.1%, and there is an increase in mortality in adolescents and young adults as reported in other cohorts. Most mortality is due to acute complications and all genotypes are affected. The ELSNCS provides unique data to inform an unbiased estimate of risks for chronic complications of SCD in the context of current standard of care. Although not sufficiently mature to estimate risks of SCD-related severe heart, kidney and liver failure, there is already significant morbidity associated with pulmonary hypertension, cerebrovascular disease, and avascular necrosis in the HbSS genotype despite standard therapy, and this suggests the need to intensify current treatment, and consider new therapeutic options which might mitigate the risk of chronic complications.

P1494: INDICATIONS OF HEMATOPOIETIC STEM CELL TRANSPLANT ACCORDING TO DIFFERENT SETS OF INDICATIONS AND ANALYSIS OF COMORBIDITY INDEX SCORES IN BRAZILIAN PATIENTS WITH SICKLE CELL DISEASE

G. Santos 1,* , K. Tozatto-Maio 1 , L. Alves 1 , G. Fonseca 2 , G. Melo 2 , L. Suganuma-Otsuka 2 , F. Nogueira 2 , A. Cardoso 2 , V. Rocha 2 , S. Gualandro 2

1 Serviço de Hematologia, Hemoterapia e Terapia Celular, Hospital das Clínicas da Faculdade de Medicina de São Paulo; 2 Serviço de Hematologia, Hemoterapia e Terapia Celular, Hospital das Clinicas da Faculdade de Medicina de Sao Paulo, São Paulo, Brazil

Background: Hematopoietic stem cell transplantation (HSCT) is the only curative therapy currently available for sickle cell disease (SCD). However, HSCT indications might vary across countries, and who should not be transplanted is still a matter of debate since HSCT mortality scores lack validation in SCD settings.

Aims: To identify how many patients currently followed at the SCD outpatient service of the Hospital das Clínicas de São Paulo meet at least one indication for HSCT, either according to the Brazilian Ministry of Health (BMH) or to an international expert panel (Angelucci et al , 2014); to characterize the distribution of the hematopoietic cell transplantation comorbidity index (HCT-CI) and Charlson comorbidity index (CCI) scores in this cohort, comparing the scores between transplantable and non-transplantable population to correlate them with the severity of the disease.

Methods: Single-center, retrospective study with 219 patients with SCD. Using the set of HSCT indications either from the BMH or Angelucci, we determined the frequency of patients with >1 indication. The concordance between the two sets of indications was assessed using Cohen’s kappa coefficient. HCT CI and CCI were used to assess risk of HSCT-related severity; median scores in transplantable and non transplantable populations were compared using Mann-Whitney.

Results: We included 219 patients, of which 143 were SS genotype, 47 SC and 29 Sβ;138 were female. The median age of the cohort was 38(range,20-74) years. 33 patients had ischemic stroke, 2 had hemorrhagic stroke and 10 had Moya-moya. 125 patients had acute chest syndrome and 210 had vaso-occlusive crisis (VOC), of whom 74 had >3 VOC in the last 12 months. 11 patients had pulmonary arterial hypertension, 73 had avascular necrosis, 35 priapism, 65 proliferative retinopathy and 184 proteinuria. 103 patients were under hydroxyurea and 48 had previously used. 186 received blood transfusion, 47 being under exchange transfusion program; 72 were alloimmunized. 168 patients had at least one HSCT indication, 108 by the BMH and 166 by Angelucci; 60 met an indication only by Angelucci and 2 only by the BMH. The overall agreement rate between the 2 sets was 0.71, with a Cohen’s kappa coefficient of 0.44. When only SS and SB0 patients are considered, the Cohen’s kappa coefficient remains moderate (0.50). The main indication by both sets was alloimmunization, followed by CNS event (BMH) and recurrent VOC (Angelucci). Among age groups, the number of patients with >1 TCTH indications was:18-30 years:46; 31-40 years:50;41-50 years:31;51-60 years:24;+60 years:17. The median HCT-CI score in the transplantable population was 2(range,0-12) and in the non-transplantable population was 1(range,0-5)(p=0.004); median CCI was respectively 1(range,1-9) and 0(range,0-3) (p<0.001).

Summary/Conclusion: HSCT indications according to the BMH are stricter than those proposed by Angelucci et cols, which explains the moderate agreement on Cohen’s kappa coefficient.The rate of indications is higher than in previously reported similar cohorts, mainly due to the higher rate of alloimmunization and older median age of our cohort, as HSCT indications increase over time in SCD. Median HCT-CI and CCI were significantly higher in the population with >1 HSCT indication, reflecting the higher severity of these patients.Although this might mean that such severity scores can be used to predict severity in SCD patients, further validation in prospective HSCT cohorts is warranted.

P1495: ETAVOPIVAT TREATMENT FOR UP TO 12 WEEKS IN PATIENTS WITH SICKLE CELL DISEASE IS WELL TOLERATED AND IMPROVES RED BLOOD CELL HEALTH

S. Saraf 1,* , R. C. Brown 2 , R. W. Hagar 3 , M. Idowu 4 , I. Osunkwo 5 , T. A. Kalfa 6 , F. A. Kuypers 7 , J. Geib 8 , P. Schroeder 8 , E. Wu 8 , P. Kelly 8 , M. J. Telen 9

1 University of Illinois at Chicago, Chicago; 2 Children’s Healthcare of Atlanta, Atlanta; 3 UCSF Benioff Children’s Hospital San Francisco, Oakland; 4 UTHealth McGovern Medical School, Houston; 5 Levine Cancer Institute, Charlotte; 6 Cincinnati Children’s Hospital Medical Center, Cincinnati; 7 University of California, San Francisco; 8 Forma Therapeutics, Inc., Watertown; 9 Duke University Medical Center, Durham, United States of America

Background: Etavopivat, an investigational, once-daily, selective, activator of erythrocyte pyruvate kinase (PKR) increases PKR activity, resulting in decreased 2,3-DPG and increased ATP in red blood cells (RBCs) of healthy volunteers and patients (pts) with sickle cell disease (SCD). 1,2

Aims: We report results of an open-label (OL) extension cohort from a Phase 1 study ( NCT03815695 ) designed to characterize the safety and clinical activity of etavopivat at a maximal pharmacodynamic dose in pts with SCD.

Methods: 15 pts were enrolled to receive etavopivat 400 mg once daily for 12 wks, followed by a 4-wk follow-up. Assessments included safety, pharmacokinetics, pharmacodynamics, RBC health parameters and systemic markers of SCD pathophysiology.

Results: Of 15 pts (age 32.3 ±10.1 yr; HbSS/SC n=13/2), 14 completed 12-wks of treatment (tx); 1 pt discontinued tx after ~2 wks. Etavopivat 400 mg once daily was generally well tolerated. Adverse events (AEs) reported during tx and follow-up were commonly low grade (Gr) and consistent with pts’ SCD. Gr1-2 AEs in >2 pts (n [%]) were sickle cell pain events (9 [60%]); headache (5 [33%]); and upper respiratory tract infection (3 [20%]). The Gr3-4 AE in >1 pt was sickle cell vaso-occlusion (VOC; 4 [27%]). On-tx serious adverse events (SAEs; 1 pt each) were Gr3 VOC post Gr3 COVID (not tx-related) and Gr3 left femoral deep vein thrombosis (possibly related, resulting in tx discontinuation as stated above). SAEs (1 pt each) during the 4-wk follow-up were Gr3 acute chest syndrome + Gr3 VOC, Gr3 non-cardiac chest pain and Gr3 syncope (all unrelated).

Observed increases in ATP and decreases in 2,3-DPG were durable over 12 wks of etavopivat tx. Etavopivat tx normalized hemoglobin (Hb)S-oxygen affinity to that of HbA. Etavopivat tx over 12 wks improved overall sickle RBC health, demonstrated by a reduction in point of sickling as well as improved measures of deformability and hydration of sickle RBCs (all P <0.01; Figure). Etavopivat tx over 12 wks was associated with a sustained significant increase in Hb compared with baseline (BL; P <0.0001), with mean maximal increase of 1.5 (range 0.7-2.3) g/dL. On-tx increase in Hb >1g/dL was achieved in 11 (73%) pts, for whom the mean maximal Hb increase was 1.8 (1.2-2.3) g/dL. Absolute reticulocytes, indirect bilirubin and lactate dehydrogenase significantly decreased from BL and were sustained over the 12wk period (all P <0.05), indicative of increased RBC lifespan and decreased hemolysis. Several markers of disease activity significantly decreased from BL during daily etavopivat tx, including the inflammatory marker tumor necrosis factor-α, which decreased by 35% ( P <0.001). Based on preliminary exploratory analysis with an aggregate duration of etavopivat exposure of 3.32 pt-yrs in the OL cohort, a decrease in the trend for VOC hospitalizations was observed: annualized historical and on-tx VOC hospitalization rates were 0.93 and 0.30, respectively; the 1 on-tx VOC hospitalization was COVID-related.

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Summary/Conclusion: Etavopivat 400 mg once daily for up to 12 wks demonstrated a tolerable safety profile and showed improvements in various markers of RBC health in pts with SCD. Rapid and sustained increases in Hb were associated with decreases in reticulocyte counts and markers of hemolysis, supporting increased sickle RBC lifespan and improved anemia. Together, these results support further evaluation of etavopivat in the Phase 2/3 Hibiscus Study ( NCT04624659 ) currently enrolling pts.

1 Kalfa et al, Blood 2019.

2 Brown et al, Blood 2020.

P1496: OPERATOR-INDEPENDENT, FLUORESCENCE-BASED QUANTIFICATION OF POCKED RED CELLS CORRELATES WITH SPLEEN SIZE AND FUNCTION IN SICKLE CELL DISEASE

A. Sissoko 1,* , A. Fricot 2 , L. Dumas 3 , C. Roussel 1,2 , S. Manceau 4,5 , C. Capito 4 , S. Allali 4 , N. Yekkache 2 , M. Dussiot 6 , Y. NGUYEN 4 , A. Lefort 4 , B. Aussilhou 4 , M. Tichit 7 , D. Hardy 7 , B. Maître 8 , A. Michel 8 , M. De Montalembert 4 , M. Cavazzana 4 , L. Joseph 4 , P. Buffet 2,4

1 UMR-S 1134 BIGR, APHP, GREx; 2 UMR-S 1134 BIGR “biologie intégrée du globule rouge”, Université de Paris, UMR-S 1134 BIGR “biologie intégrée du globule rouge”; 3 UMR-S 1134 BIGR “biologie intégrée du globule rouge”, Inserm/Université de Paris, UMR-S 1134 BIGR “biologie intégrée du globule rouge”; 4 APHP, Paris, Assistance Publique Hôpitaux de Paris; 5 GR-Ex, Paris, France., Laboratoire d’Excellence GR-Ex, Paris, France.; 6 Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications, INSERM, Paris, France, Université de Paris, U1163; 7 Experimental Neuropathology unit, Paris, France, Institut Pasteur, Université de Paris; 8 Etablissement Français du Sang-Grand Est, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, France., Université de Strasbourg, UMR_S1255, INSERM, Paris, France

Background: The spleen protects against blood-borne infections, contributes to the maturation of reticulocytes. Defective spleen function (hyposplenism) is caused by splenectomy or by immunological or hematological diseases. It occurs very soon in Sickle Cell Disease (SCD), without clear correlation with spleen size. Counting vacuole-containing RBC (pocked RBC) by Differential Interference Contrast microscopy ( DIC ) is a robust marker of defective splenic function but it relies on the expertise of fewer and fewer specialized laboratory technicians, and poorly reproducible from site to site.

Aims: Find an operator-independent method to quantify the proportion of pocked red blood cells in the circulation.

Methods: We developed an automated method using a common fluorescent cytoplasmic marker, and imaging flow cytometry to quantify pocked RBC in 128 adult patients with Sickle Cell Disease (SCD). Spleen size was determined by 2 members of the team and scored as 4 categories: Splenomegaly, Normal, Small and Atrophic.

Six children with SCD who had partial splenectomy for hypersplenism were also included. Spleen were retrieved immediately after surgery, histology and electronic microscopy were performed. A venous blood sample was collected at the time of surgery and the splenic blood was retrieved from spleen fragments.

Results: The proportion of RBC containing one or more fluorescent dots by IFC correlated with results of the conventional method, i.e., the operator-dependent quantification of pocked RBC by differential interference contrast microscopy. Proportions of pocked RBC in circulation correlated with spleen size, which varied widely from splenomegaly to atrophic spleen. Histology of post-splenectomy samples from 3 SCD children with normal, moderately, or markedly elevated pocked RBC counts, showed either an almost normal aspect with congestion, intense sickling, or widespread fibrosis, respectively. Electronic microscopy of the spleen from the child with normal pocked RBC counts showed persistent RBC filtration through inter-endothelial slits. Intrasplenic blood was enriched in RBC displaying a peripheral fluorescent dot, highly suggestive of ongoing intrasplenic expulsion of vacuoles (pitting). Operator-independent, fluorescent-based quantification of pocked RBC correlates with the reference method and confirms that spleen function is partially preserved in many adults with SCD.

Summary/Conclusion: In summary, this new fluorescence-based quantification of pocked-RBC, based on the spontaneous accumulation of the dye in vacuoles, correlates with the reference method, opening the way to a robust, operator-independent quantification of spleen function. We show that spleen function is partially preserved in many adults with SCD. We also show that the fluorescent vacuoles accumulating in RBC from SCD patients are indeed expelled by the spleen-specific pitting process, as long as sinusal structures persist in the partially damaged SCD spleens.

P1497: BURDEN OF SICKLE CELL DISEASE: RESULTS FROM THE REAL WORLD ASSESSMENT SURVEY FOR SICKLE CELL DISEASE IN SAUDI (ROARS)

W. Jastaniah 1,* , A. Al Zayed 2 , H. Al Saeed 2 , M. AlDarwish 2 , M. Albagshi 3 , H. Malhan 4 , A. Tarawa 5 , A. AlManea 6 , I. AlHazmi 6 , M. Qari 7 , A. Al Jefr 8 , A. Alzahrani 9 , M. Alzahran 10 , W. Soliman 11 , F. Askar 11 , H. Ezzat 12

1 King Faisal Specialist Hospital & Research Center (Gen. Org.), Jeddah; 2 Qatif Central Hospital, Qatif; 3 Almana General Hospital-Al Ahsa, Hofouf; 4 Prince Mohamed ben Naser, Jizan; 5 King Salman Medical City Al Madinah, Madinah; 6 King Fahad Armed Forces Hospital; 7 King Abdulaziz University Hospital, Jeddah; 8 King Faisal Specialist Hospital; 9 Prince Sultan Military Medical City; 10 King Abdulaziz Medical City; 11 Novartis, Riyadh; 12 Johns Hopkins Aramco Healthcare, Dahran, Saudi Arabia

Background: The Kingdom of Saudi Arabia (KSA) has one of the highest prevalence of sickle cell disease (SCD; an inherited hemoglobinopathy) in the world, with up to 2.6% of the population affected. The severity of SCD in KSA varies widely, likely due to existing haplotype variations. This creates a unique opportunity to understand the impact of SCD on patients’ (pts’) quality of life (QoL) and available treatments, and to inform management strategies that can reduce disease burden and improve QoL.

Aims: The Real World Assessment Survey for Sickle Cell Disease in Saudi (ROARS) is a cross-sectional survey of pts with SCD and treating healthcare professionals (HCPs) which was designed to understand the burden of SCD, including QoL, symptoms, treatment strategies, treatment goals, and management approaches, in KSA.

Methods: Between September and December 2021, pts with SCD (age: 5-65 years) and HCPs (>2 years SCD experience, SCD load >50 pts, >50% of practice time spent treating SCD pts) were surveyed (in person or via caregiver if pts were between 5-14 years) using the structured, bilingual ROARS questionnaires (English/Arabic) through personal quantitative web-assisted telephone interviews. ROARS, developed by SCD experts from KSA and Novartis, includes the global health domain of Patient-Reported Outcomes Measurement Information System (PROMIS), a validated tool for assessing QoL among SCD pts and caregivers of different age ranges. Questions covered topics such as disease impact on QoL, symptoms, disease complications, treatment goals, and treatment strategies.

Results: Overall, 1011 pts (pediatric: n=504; adult: n=507) and 64 HCPs (specialists: n=31; non-specialists: n=33) completed the survey. The mean (standard deviation; SD) age was 20.6 (13.2) years, 52% of pts were female, and 60% were homozygous for hemoglobin S (self-reported genotype). Data were collected from 11 cities, with 90% of respondents from Eastern and South-Western provinces of KSA where prevalence of SCD is the highest. Burden of the disease was significant regardless of the region; QoL was rated as poor/fair by 27% of adult and 38% of pediatric pts (Figure). Pts reported that SCD caused a negative impact on emotions (28%), school achievement (19%), and a reduction in work hours (16%). The top 5 symptoms reported by both pediatric and adult pts were bone ache (83%), fatigue/tiredness (68%), headache (56%), poor appetite (47%), and anxiety (45%). The mean (SD) incidence of vaso-occlusive crises (VOCs) in the 12 months prior to survey completion was 6.5 (4.6) and severe VOCs resulting in heathcare facility visits was 5.5 (4.3). Reducing the number of VOCs and increasing the number of days free of severe pain was an important treatment goal in 84% of pediatric pts and in 71% of adult pts. Overall, 51% of pts managed their VOCs at home. The most important factors in treatment decision were improving QoL and preventing end organ damage, reported by 56% and 45% of HCPs, respectively. Top treatments that the pts are currently receiving are antibiotics (48%), opioids (33%), hydroxyurea (33%), blood transfusion (31%), and iron chelation therapy (26%). Innovative treatments were reported by 5% of pts and hematopoietic stem cell transplantation in only 2%. Overall, 68% of pts reported dissatisfaction with their current treatments for SCD.

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Summary/Conclusion: Based on ROARS data, which incorporated pts’ voices, there is a significant burden of SCD among pts in KSA. This study highlights the need for national-specific plans for effective management strategies and allocation of resources.

P1498: INHIBITION OF POLYCOMB REPRESSIVE COMPLEX 2 THROUGH EED INDUCES FETAL HEMOGLOBIN IN HEALTHY AND SICKLE CELL DISEASE MODELS

B. Stuart 1,* , P. Bruno 1 , D. Polioudakis 1 , M. Roth 1 , M. Wertz 1 , A. Edelstein 1 , I. Efremov 1 , D. Matson 1 , J. Dunn 1

1 Fulcrum Therapeutics, Inc, Cambridge, United States of America

Background: Sickle Cell Disease (SCD) and β-thalassemias are caused by mutations in the hemoglobin β (HBb) gene locus, resulting in dysfunctional or diminished adult hemoglobin (HbA) production that is the root cause of the increased morbidity and mortality associated with these diseases. Individuals with SCD and β-thalassemias who harbor additional genetic alterations that maintain elevated fetal hemoglobin (HbF) expression during adulthood demonstrate improved disease prognosis or, at sufficient HbF amounts, restore normal hemoglobin functions and are asymptomatic. Elucidating the regulatory networks that control HbF expression is critical to the discovery of new therapies for SCD and β-thalassemia.

Aims: To elucidate the mechanism by which Polycomb Repressive Complex 2 regulates HbF expression

Methods: Genetic and pharmacologic targeting of PRC2 components in healthy and SCD CD34+ derived erythroid cells in vitro and wildtype and Townes HbSS in vivo models. RNA-sequencing was used to look at response to PRC2 modulation and impact on HbF levels.

Results: We show that modulation of PRC2 potently downregulates expression of master fetal hemoglobin regulator BCL11A, and thus is a putative upstream regulator of HBG gene expression. Specifically, we demonstrate negative modulation of PRC2 activity via multiple different pharmacologic or genetic knockdown approaches results in decreased expression of BCL11A and concomitant and robust increases in both HBG mRNA and HbF protein in vitro and in vivo. Downregulation of BCL11A is mechanistically distinct from the current standard of care, Hydroxyurea, and pre-clinical HbF induction via FTX-6058 exceeds that observed with Hydroxyurea.

Summary/Conclusion: These novel findings further elucidate the regulatory network that underlies fetal hemoglobin gene expression and has important implications for the use of inhibitors of PRC2 as potential therapies for SCD and β-thalassemia. The translation of these findings is underway as FTX-6058, an investigational, novel and potent inhibitor of Embryonic Ectoderm Development (EED), is currently being evaluated in clinical studies as a potential treatment for select hemoglobinopathies ( NCT04586985 , NCT05169580 ).

P1500: 2,3-DIPHOSPHOGLYCERATE DETECTION VIA DIRECT INFUSION HIGH RESOLUTION MASS SPECTROMETRY CORRELATES WITH QUANTITATIVE DETECTION IN BLOOD OF PATIENTS WITH SICKLE CELL DISEASE

S. van der Veen 1,2,* , M. van Dijk 1,3 , J. Jans 2 , N. Verhoeven-Duif 2 , R. van Wijk 3 , M. Bartels 4 , M. Mañú Pereira 5 , R. Colombatti 6 , M. Martella 6 , V. Munaretto 6 , M. Boaro 6 , P. Bartolucci 7,8 , M. Cnossen 9 , B. Biemond 10 , E. van Beers 1

1 Center for Benign Hematology, Thrombosis and Hemostasis - Van Creveldkliniek, University Medical Center Utrecht, Utrecht University; 2 Section Metabolic Diagnostics, Department of Genetics; 3 Department of Central Diagnostic Laboratory - Research, University Medical Center Utrecht; 4 Paediatric Haematology Department, Wilhemina Children’s Hospital, University Medical Centre Utrecht, Utrecht, Netherlands; 5 Group of cancer and blood disorders in children, Vall d’Hebron Research Institute, Barcelona, Spain; 6 Department of Woman’s and Child’s Health, University of Padova, Padova, Italy; 7 Univ Paris Est Créteil, Hôpitaux Universitaires Henri Mondor, APHP, Sickle cell referral center – UMGGR; 8 Univ Paris Est Créteil, IMRB, Laboratory of excellence LABEX, Créteil, France; 9 Department of Pediatric Hematology, Erasmus MC- Sophia Children’s Hospital, Erasmus University Medical Center, Rotterdam; 10 Department of Hematology, Amsterdam University Medical Center, AMC, Amsterdam, Netherlands

Background: Sickle cell disease (SCD) is a hereditary and chronic life-threatening disorder, characterized by haemolytic anaemia. Increased 2,3-diphosphoglycerate (2,3-DPG) concentrations, along with decreased oxygen affinity of hemoglobin, may be related to the variability of clinical outcomes in SCD. Furthermore, genomic health data holds promise to improve the prediction of disease severity in SCD. Based on the integration of genomics, metabolomics and clinical data from 1000 SCD patients, to be included in 2022, GenoMED4all aims to develop Artificial Intelligence (AI) based deep learning algorithms to improve the prediction of disease severity and phenotype in SCD.

Aims: To correlate non-quantitative metabolomics data obtained from dried blood spots (DBS), one of the inputs for GenoMED4all, to quantitative measurement of 2,3-DPG. Aiming to improve the potential of non-quantitative metabolomics from DBS to asses 2,3-DPG.

Methods: In snap frozen blood samples from 37 SCD patients and 29 healthy controls, 2,3-DPG was quantified by liquid chromatography mass spectrometry. 2,3-DPG was also detected in DBS from the same subjects by direct infusion high resolution mass spectrometry (DIHRMS). The oxygen tension at 50% Hb saturation (p50)was determined using a Hemox Analyzer (TCS). Statistical analysis were performed by Spearman’s correlation coefficients (SPSS v26.0.0.1) and Mann Whitney testing (GraphPad Prism v9.3.0).

Results: After correcting for Hb, 2,3-DPG concentrations were higher in SCD patients than in controls (p<0.001) and Z-scores for 2,3-DPG, as assessed by DIHRMS, were similar in patients and controls. The Z-scores positively correlated with 2,3-DPG concentrations (Fig.1A, 0.353, p=0.004). Because of the anaemia in SCD, RBCs and plasma make up lower and higher volumes in the DBS, respectively, compared to healthy controls. DIHRMS detects a wide range of RBC and plasma metabolites, whereas the quantitative measurement is restricted to measuring 2,3-DPG of RBCs. To correct for those differences between methods, we applied a correction factor to the DIHRMS data using the formula (1/Ht)*(1-Ht/1), correcting for the RBC volume (1/Ht) and the plasma volume (1-Ht/1). This resulted in a trend towards higher Z-scores in patients than controls (p=0.0597). Moreover, the positive correlation with 2,3-DPG concentrations increased to 0.526 (Fig 1B, p<0.001). As 2,3-DPG affects the oxygen affinity of Hb, all measurements were correlated to p50. Expectedly, 2,3-DPG concentrations positively correlated with p50 (0.842, p<0.001). After applying the correction factor to the DIHRMS data, p50 correlations increased from 0.361 (p=0.003) to 0.529 (p<0.001).

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Summary/Conclusion: Strongest correlation between quantitative and non-quantitative methods for 2,3-DPG detection were observed after correcting for both the RBC and plasma volume in non-quantitative metabolomics. After correction, DIHRMS can be used to assess 2,3-DPG concentrations in DBS. This translation of non-quantitative to quantitative metabolomics for 2,3-DPG and potentially other RBC metabolites adds significant value to the use of DIHRMS, as DIHRMS is far more efficient in obtaining extensive information about the total metabolome than quantitative methods. The large population size and the AI based deep learning algorithms of GenoMED4all will enable to evaluate the potential of non-quantitative metabolomics in SCD.

Funding: Horizon2020, GenoMED4all ( https://genomed4all.eu/ ), Agios Pharmaceuticals Inc., ERN-EuroBloodNet

P1501: FOLLOW-UP RESULTS OF A PHASE 2 STUDY ASSESSING THE SAFETY AND EFFICACY OF MITAPIVAT TREATMENT, AN ORAL PYRUVATE KINASE ACTIVATOR, FOR UP TO 60 WEEKS IN SUBJECTS WITH SICKLE CELL DISEASE

M. J. van Dijk 1,* , M. A. Rab 1 , B. A. van Oirschot 1 , J. Bos 1 , C. Derichs 1 , A. W. Rijneveld 2 , M. H. Cnossen 2 , E. Nur 3,4 , B. J. Biemond 3 , M. Bartels 1 , J. J. Jans 1 , W. W. van Solinge 1 , R. E. Schutgens 1 , R. van Wijk 1 , E. J. van Beers 1

1 University Medical Center Utrecht, Utrecht; 2 Erasmus University Medical Center, Rotterdam; 3 Amsterdam University Medical Center; 4 Sanquin Research, Amsterdam, Netherlands

Background: Sickle cell disease (SCD) is a devastating red blood cell (RBC) disorder. The root cause of SCD is polymerization of sickle hemoglobin (HbS) upon deoxygenation, resulting in poorly deformable sickled RBCs with a shortened lifespan. 2,3-Diphosphyglycerate (2,3-DPG), a glycolytic intermediate in RBCs, promotes deoxygenation by lowering hemoglobin (Hb)-oxygen affinity. Mitapivat (AG-348) is an oral allosteric activator of pyruvate kinase (PK), a key enzyme in RBC glycolysis generating adenosine triphosphate (ATP) and reducing 2,3-DPG levels.

Aims: To report follow-up data of the safety and efficacy of mitapivat treatment in subjects with SCD enrolled in the currently ongoing ESTIMATE study ( www.trialregister.nl NL8517; EudraCT 2019-003438-18).

Methods: The ESTIMATE study is a phase 2, investigator initiated, open-label study. Subjects ≥16 years with SCD (HbSS, HbS/β 0 or HbS/β + ), 1-10 vaso-occlusive crises (VOCs) in the prior year and/or prior SCD-related complications, a Hb level >6.1 g/dL and ≤11.1 g/dL, and not receiving chronic transfusions are eligible. In the 8-week mitapivat dose finding period, initial dosing is 20 mg twice daily escalated to max 100 mg twice daily, and followed by ≤52-week fixed dose extension period (FDEP). Primary endpoints are safety, based on adverse events (AEs), and efficacy of mitapivat treatment based on RBC sickling assessed as the Point of Sickling (PoS) using oxygen gradient ektacytometry. Key secondary endpoints are changes in Hb level, markers of hemolysis, ATP and 2,3-DPG levels, and Hb-oxygen affinity (p50, Hemox Analyzer).

Results: Until Jan. 2022, nine subjects were treated with mitapivat. Baseline characteristics were: median age 22 years (range 16-59 years), 6/9 (67%) female, and 6/9 (67%) used hydroxyurea. 7/9 (78%) had HbSS, 1/9 (11%) had HbS/β 0 , and 1/9 (11%) had HbS/β + . One subject with a non-treatment related serious AE (SAE) of a urinary tract infection (grade 4), was lost to follow-up shortly after first dosing. No other SAEs or treatment-emergent AEs (TEAEs) grade ≥3 occurred. The remaining eight subjects had a median treatment duration of 38 weeks (range 11-60 weeks). 6/8 (75%) took 100 mg mitapivat twice daily in the FDEP, and 2/8 (25%) took 50 mg mitapivat twice daily after a single dose reduction because of transaminase increase. The most common TEAEs were (n>2 subjects): ALT or AST increase (both 5/9, 56%; all grade 1), and headache (4/9, 44%; grade 1-2). Three VOCs occurred: one related to excessive alcohol consumption with no need for hospitalization, and two in subjects with documented noncompliance the week before hospitalization. Mean annual VOC rate and SCD-related hospital admission days were, respectively, 1.5 ± 1.3 and 5.9 ± 7.1 days at baseline, and reduced to 0.5 ± 0.7 and 1.6 ± 3.1 days when weighting cases by follow-up duration (p=0.021 and p=0.134, respectively). Table 1 summarizes mean changes in parameters of primary and key secondary endpoints of scheduled visits in the FDEP versus baseline. Hb level significantly increased, accompanied by a decrease in markers of hemolysis (absolute reticulocyte count, total bilirubin, and lactate dehydrogenase). Mean PoS decreased, and p50, 2,3-DPG level and ATP/2,3-DPG ratio significantly improved.

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Summary/Conclusion: Treatment with mitapivat for up to 60 weeks in subjects with SCD showed no treatment related TEAEs grade ≥3. Improvements in anemia, markers of hemolysis, oxygen affinity, 2,3-DPG level and ATP/2,3-DPG ratio were seen. Follow-up data will be collected in this ongoing study.

P1502: LONG-TERM FOLLOW-UP OF DUTCH PATIENTS WITH SICKLE CELL DISEASE DIAGNOSED BY NEONATAL SCREENING – EFFECT ON THE MORBIDITY AND MORTALITY IN THE NETHERLANDS.

C. Vuong 1,* , L. de Ligt 1 , H. Heijboer 1 , E. Rettenbacher 1 , K. Fijnvandraat 1 , C. L. de Groot-Eckhardt 1

1 Pediatric Hematology, Amsterdam University Medical Centers, Amsterdam, Netherlands

Background: Sickle cell disease (SCD) is associated with increased morbidity and mortality compared to the general population. To initiate early preventive measures, newborn screening for SCD has been introduced in January 2007 in the Netherlands.

Aims: The objective of this study is to assess the effectiveness of this neonatal screening for SCD by describing the residual risks of death and major disease-related events during the first fourteen years of life in children diagnosed with SCD at birth in the Netherlands.

Methods: Here we report the first data of the largest center (Amsterdam UMC) of this prospective, national multicenter cohort study. Following informed consent, data were collected from medical files of all children diagnosed by neonatal screening. Descriptive data on SCD genotype and occurrence of major disease-related events are presented, including hospitalization for vaso-occlusive crisis (VOC), acute chest syndrome, severe infections and neurological complications. Overall survival and survival without specific SCD-related complications were analyzed by Kaplan-Meier curves.

Results: Up until now, 98 (56%) out of 174 eligible subjects from this institution were included, with a total follow-up of 805 patient-years. The majority (55%) had the severe genotype (HbSS/ beta0-thalassemia), the remainder had genotypes HbSC or HbS/beta+-thalassemia. Survival by the age of 14 was 98.9%, with 1 death at the age of 1 years due to sepsis, most likely related to SCD. Seven patients (7.1%) had a severe infection (meningitis, sepsis, osteomyelitis) caused by Streptococcus Pneumoniae in 3/7 cases. Antibiotic prophylaxis was prescribed for all 7 patients before they reached the age of 1 year, and 6/7 patients had been fully vaccinated with the 23 valent pneumococcal vaccine. Two patients experienced a symptomatic cerebral infarction at the age of 11 months and 1.5 years, respectively. At the age of 10 years the survival without hospitalization for vaso-occlusive crisis was 27% (95% CI: 12.7 – 43.14%) and 51% (25.3 – 72.0%) for the SS/Sβ0 and SC/Sβ+ genotype, respectively.

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Summary/Conclusion: In this cohort of neonatally screened patients with SCD, the SCD-related mortality and morbidity is still impressive with 1% mortality, 7 severe infections, and 2 patients with a CVA. A final analysis of the effect of neonatal screening for SCD will follow after completion of data collection in all participating centers in the Netherlands.

P1503: REAL WORLD DATA ON EFFICACY OF PHARMACEUTICAL-GRADE L-GLUTAMINE IN PREVENTING SICKLE CELL DISEASE-RELATED ACUTE COMPLICATIONS AND HEMOLYSIS IN PEDIATRIC AND ADULT PATIENTS.

N. Elenga 1 , M. Etienne-Julan 2 , G. Loko 3 , R. AlOkka 4 , A. Adel 4 , M. Yassin 5,*

1 pediatric, CHU Cayenne, Cayenne, French Guiana; 2 CHU Guadeloupe, Point a pitre; 3 CHU de Fort de France, Fort de France, France; 4 pharmacy; 5 Hematology, National Centre for Cancer Care and Research - Hamad Medical Corporation, doha, Qatar

Background: Oxidative stress is one of the key contributors to the pathophysiology of sickle cell disease (SCD) and related complications including acute pain (vaso-occlusive crisis or VOC), tissue ischaemia, stroke, and acute chest syndrome (ACS). L-glutamine (L-gln), a precursor of nicotinamide adenine dinucleotide (NAD), has been shown to play an important role in the regulation of oxidative stress. In the pivotal Phase 3 clinical trial, L-gln demonstrated significant reduction in VOC, hospitalizations, and ACS events compared to placebo in patients with SCD, with or without hydroxyurea (HU) use, over a 48-week period. In September 2021, a re-analysis of the Phase 3 trial data revealed that L-gln decreased the number of VOCs by 45%.

Aims: To confirm the efficacy on clinical observations and hemolysis markers of oral therapy with pharmaceutical-grade L-gln in pediatric and adult patients with SCD at follow-up time points of 24, 48 and 72 weeks.

Methods: In the observational study conducted from October 2019 through April 2021, 19 patients (4 patients from Qatar and 15 patients from French Guiana) were treated with L-gln (0.3mg/kg) twice daily. Laboratory parameters (hemoglobin (Hb) levels, hematocrit, WBC counts, reticulocyte counts, and LDH levels) were measured at baseline and follow-up time points. Clinical parameters (number of VOCs, hospitalizations, days hospitalized, ACS events, and blood transfusions) were documented for the year prior to treatment initiation as baseline values. These parameters were also collected at 24, 48, and 72 weeks from treatment initiation. Adverse events (AEs) were also collected during the treatment period. The data values at 24, 48, and 72 weeks have been annualized. Statistical analysis was performed using MedCalc 20.015.

Results: Compared to baseline, patients had significantly fewer number of VOCs at 24, 48, and 72 weeks following L-gln therapy (median change from 3.0 to 0;p<0.00001). Compared to baseline, there were fewer hospitalizations (median change from 3.0 to 0;p<0.00001) and patients spent fewer days in hospital (median change from 15.0 to 0;p<0.00001). Furthermore, at 24, 48, and 72 weeks, the number of blood transfusions was considerably lower than at baseline (median, from 3.0 to 0.0;p<0.00001). Following treatment with L-gln, the mean Hb level increased significantly from baseline to 72 weeks (8.2 to 8.8 g/dL;p<0.001) with peak mean increase from baseline of 11.2% at 48 weeks. A similar increasing trend was observed for hematocrit proportions from baseline to 72 weeks (24% to 27%;p<0.001) with highest mean improvement from baseline of 15.5% at 48 weeks. Conversely, mean reticulocyte counts and LDH levels were significantly reduced at follow-up time points compared to baseline (p=0.003 and p<0.001, respectively).

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Summary/Conclusion: This study demonstrated for the first time that L-gln therapy in SCD patients from Qatar and French Guiana resulted in significant improvements in clinical outcomes (number of VOCs, number and duration of hospitalizations, and number of blood transfusions) accompanied by a noteworthy increase in Hb levels and a reduction in markers of hemolysis (reticulocyte counts and LDH levels). Moreover, while 11 ACS events were reported the year before starting L-gln, only 2 ACS events occrred after 12 months of initiation of L-gln. Treatment with L-gln resulted in clinically significant outcomes combined with significant improvement of the hemolysis parameters from baseline through 72 weeks suggesting sustained long-term efficacy. Only a few number of AEs occurred and were mild. No compliance issue was reported.

P1504: AN AUDIT OF PAIN EDUCATION IN PATIENTS WITH SICKLE CELL DISEASE

Y. Zhou 1,* , R. Auturally 1 , V. Garcia 1 , R. Jolley 1 , M. Besser 1

1 Dept Haematology, Box 217, Cambridge University Hospital, Cambridge, United Kingdom

Background: Pain crises represent the primary reason for hospital care-seeking in individuals with sickle cell disease (SCD). The NICE guideline CG143 emphasises that patient should be treated as experts in the pain management of their sickle cell disease.

Aims: To assess the performance of acute sickle cell pain crisis management at Cambridge University Hospital (CUH) and assess methods of secondary prevention of admission for pain crisis through patient pain education.

Methods: Patient admission records during the year of 2021 were reviewed for a population of 85 SCD patients registered at Cambridge University Hospital (CUH). For these encounters, patient assessment at presentation, primary analgesia and ongoing management were audited against CG143 standards. All SCD patients registered with CUH were contacted for a telephone interview to discuss home pain management.

Results: In a population of 85 patients living with SCD, there were 22 admissions in 17 patients during 2021, of which 5 patients had multiple admissions. Auditing CUH performance against the NICE CG143 guidelines, it was found that initial pain relief was provided within 30 minutes of presentation in 15 out of the 21 relevant admissions (71%). However, none of the patients had documented discussion of pain control education, any psychological support they may need nor were encouraged to use their own coping mechanisms for dealing with acute pain in their hospital notes.

Following on from this, we conducted telephone interviews and received a total of 51 responses of which 13 patients were admitted to hospital with sickle cell pain in 2021. In the 46 patients who experience sickle cell pain crises, 20 had strong opiates, 27 had weak opiates, and all 46 had a supply of paracetamol/non-steroidal anti-inflammatory drugs (NSAIDs). 8/20 (40%) patients with a home supply of strong opiates were admitted to CUH with an acute sickle cell pain crisis in 2021 compared to 5/26(19.2%) in those who did not have strong opiates at home.

Notably, only 6/46 patients were familiar with the World Health Organisation pain ladder. 2 of 6 patients who were aware of the WHO pain ladder had an admission to CUH in 2021, while 11 of 40 patients who were not aware had an admission to CUH in 2021. The findings are summarised in Table 1.

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Summary/Conclusion: Our audit has demonstrated a significant gap in the management of acute sickle cell pain crises at CUH with the absence of patient pain education. This may have contributed to the number of admissions of SCD patients to CUH. The limited patient knowledge of optimal use of pain control in line with WHO pain ladder is significant, given that most episodes are managed at home. Frequent painful crises are associated with psychological issues and poor engagement in normal daily activities. Better home pain control should be a future priority for our service.

P1505: USING ARTIFICIAL INTELLIGENCE NEURAL NETWORKS TO OBTAIN AUTOMATED LIVER IRON CONCENTRATION MEASUREMENTS USING MAGNETIC RESONANCE IMAGING – A MULTI-SCANNER VALIDATION STUDY

T. St Pierre 1,* , Y. Aydinok 2 , A. El-Beshlawy 3 , S. Bayraktaroglu 2 , A. Ibrahim 4 , M. Hamdy 5 , W. Pang 6 , S. Khorshid 7 , S. Bangma 8 , M. House 6

1 Department of Physics, The University of Western Australia, Perth, Australia; 2 Department of Paediatric Hematology & Radiology, Ege University Hospital, Izmir, Turkey; 3 Department of Paediatric Hematology, Cairo University; 4 Radiology Department, Ain Shams University; 5 Faculty of Medicine, Cairo University, Cairo, Egypt; 6 Resonance Health, Burswood; 7 Three Springs Technology; 8 Moodle, Perth, Australia

Background: Because of the limitations of serum ferritin and liver biopsy for measuring liver iron concentration (LIC), magnetic resonance imaging (MRI) methods have been developed. Reliable use of these methods requires extra training of radiologists or outsourcing of the data analysis. While the cost of outsourcing data analysis is acceptable to many patients or providers in high income countries, the majority of hemoglobinopathy patients reside in low-income countries. Therefore, automated LIC measurements using artificial intelligence (AI) or deep-learning-assessed (DLA) algorithms could provide the solution for globally affordable and reliable patient monitoring of LIC.

Aims: The aim of this study was to evaluate the performance of an automated deep-learning-based medical device (DLA R2-MRI) for measuring liver iron concentration (LIC) from MRI using an independent multi-scanner dataset.

Methods: The tested device was assessed prospectively on 1395 eligible consecutive MRI datasets from 66 different scanners submitted for expert manual analysis using spin-density projection assisted (SDPA) R2-MRI (the reference standard) between August 2017 and July 2020. The aetiologies for iron overload reported by the radiologists submitting the image data were thalassaemias (498), hereditary haemochromatosis (161), sickle cell disease (145), MDS (11), other (309), unknown (271). The bias and limits of agreement between the automated and manual measurements of LIC were assessed. In addition, diagnostic performance was assessed using sensitivity and specificity.

Results: The distribution of LIC values measured by the reference method was 0-1.8 mg Fe/g (17.6%), 1.8-3.2 mg Fe/g (16.7%), 3.2 – 5.0 mg Fe/g (14.8%), 5.0 – 7.0 mg Fe/g (11.4%), 7.0-15.0 mg Fe/g (18.6%), >15.0 mg Fe/g (21.0%). Automated LIC results from the DLA R2-MRI had geometric mean ratios to manual results from SDPA R2-MRI of 0.98 (95% CI 0.94 – 1.01) below 3 mg Fe/g dry tissue and 0.93 (95% CI 0.92 – 0.95) above 3mg Fe/g dry tissue were recorded. The sensitivities and specificities of the automated system for predicting LIC values above clinically relevant thresholds of 3.0, 3.2, 5.0, 7.0, and 15.0 mg Fe/g dry tissue were > 90%.

Summary/Conclusion: While there is an overall bias between DLA R2-MRI and SDPA R2-MRI, the bias does not result in unacceptable sensitivities and specificities of DLA R2-MRI for predicting SDPA R2-MRI results above the clinically relevant LIC thresholds. However, the bias between the automated and manual methods indicates that the two techniques should not be used interchangeably.

P1506: REAL-WORLD DATA ON THE USE OF LUSPATERCEPT IN GREEK PATIENTS WITH TRANSFUSION DEPENDENT THALASSEMIA

P. Delaporta 1,* , S. Delicou 2 , A. Kouraklis-Symeonidis 3 , M. Diamantidis 4 , E. Vlachaki 5 , A. Xydaki 6 , V. Labropoulou 3 , V. Lazaris 7 , M. Michalitsis 4 , P. Klonizakis 8 , K. Toutoudaki 9 , M.-I. Chatzieleftheriou 10 , D. Kyriakopoulou 11 , A. Kattamis 11

1 Thalassemia Unit, First Department of Pediatrics, National and Kapodistrian University of Athens, Agia Sophia Children Hospital; 2 Thalassemia and Sickle Cell Unit Hippocratio, General Hospital Athens, Athens; 3 Thalassemia and Hemoglobinopathies’ Unit, Hematology Division, Dept Int Medicine, University Hospital of Patras, Patra; 4 Thalassemia and Sickle Cell Disease Unit, General Hospital of Larissa, Larissa; 5 Adults Thalassemia Unit, Hippokration Hospital Thessaloníki, Thessaloniki; 6 Thalassemia and Sickle Cell Unit, Hippocratio General Hospital Athens, Athens; 7 Thalassemia and Hemoglobinopathies’ Unit, Hematology Division, Dept Int Medicine, University Hospital of Patras, Patras; 8 Adults Thalassemia Unit Hippokration, Hospital Thessaloníki, Thessaloniki; 9 Thalassemia Unit, First Department of Pediatrics, National and Kapodistrian University of Athens, Aghia Sophia Children’s Hospital; 10 Thalassemia Unit, First Department of Pediatrics, National and Kapodistrian University of Athens, Aghia Sophia Pediatric Hospital; 11 Thalassemia Unit, First Department of Pediatrics, National and Kapodistrian University of Athens, Aghia Sophia Children’s Hospital, Athens, Greece

Background: The EMA-approved, erythroid maturation agent, luspatercept, has been shown to decrease transfusion burden in patients with transfusion-dependent thalassemia (TDT).

Aims: In this multicenter, retrospective cross-sectional study, real-world data from the use of luspatercept in TDT patients, managed in 5 major Thalassemia Centers in Greece are presented.

Methods: Inclusion criteria included TDT patients, having received luspatercept as per approved indications for at least 3 months. Data cut-off date was 31/1/2022. We estimated the quantity of blood (in cc of Packed Red Blood Cells -PRBC) received over 12 weeks for the intervals: 12 weeks before starting treatment (baseline); 1-12 weeks; 5-16 weeks and 13-24 weeks post starting therapy. Changes in mean pre-transfusion hemoglobin (Hb), uric acid, creatinine, lactic acid dehydrogenase (LDH), white blood cells (WBC) and platelets counts were analyzed for these same respective periods. Adverse events (AE) were recorded. Statistical analysis performed with RStudio v.3.6.2.

Results: Main results of the study are shown in Table 1. Forty-nine patients (median age: 46 years range:15-64, sex: M:F/33:16) received Luspatercept every 21 days. Data for weeks 5-16 and 13-24 were available for 30 and 16 patients, respectively. The initial dose of luspatercept was 1 mg/kg and increased selectively up to 1.25mg/kg based on tolerability and efficacy and according to guidelines. A statistically significant (p<0.005) decrease in PRBC transfused in all 12-weeks’ intervals analyzed compared to baseline was observed. No statistically significant decrease of PRBC transfused was found between the intervals 5-16weeks and 13-24 weeks compared to the initial interval of 1-12 weeks. There was not a significant change for the mean pre-transfusion Hb compared to baseline.

A statistically significant ( p <0.005) increase in uric acid, creatinine, LDH, WBC and platelets was observed for the interval 1-12 weeks in comparison with baseline (mean: 6.42±1.64mg/dl vs 5.48±1.6mg/dl, 0.87±0.16mg/dl vs 0.83±0.18mg/dl, 352±205mg/dl vs 204±62mg/dl, 11.78x10^9 ±5.29x10^9/L vs 10.36x10^9±4.35x10^9/L, and 450x10^9±221x10^9/L vs 415x10^9±202x10^9/L, respectively. Similar difference was observed between the 13-24 weeks interval and baseline only for uric acid, LDH and platelets (mean: 6.28±1.41mg/dl vs 5.48±1.6mg/dl p=0.003, 406±301mg/dl vs 204±62mg/dl p =0.014, 467x10^9±226x10^9/L vs 415x10^9±202x10^9/L p<0.008 respectively).

Twenty five out of 49 patients reported AE. The most common AE included bone pain 16/49 (32.6%) and fatigue 7/49 (14.2%). Frequent urination, headache, swelling at injection site, blurry vision, tearing, libido decrease, tachycardia, periorbital oedema, dizziness, and exacerbation of manic-depressive episodes were also reported.

Twelve patients discontinued treatment. Reasons for discontinuation included: non-response to treatment (7 patients), adverse events (3 patients), non-compliance (1 patient), death due to COVID19 infection (1 patient).

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Summary/Conclusion: Real world data on the use of luspatercept in TDT parallel results from the trial, showing heterogeneous and lasting efficacy and acceptable toxicity. Longer follow up and increased number of patients are required to validate these initial observations.

P1507: CLINICAL PROFILE OF COVID-19 INFECTION AND IMMUNE RESPONSE AFTER 3AND6 MONTH VACCINATION AGAINST SARS-COV-2 IN ADULT PATIENTS WITH TRANSFUSION-DEPENDENT THALASSAEMIA-THE EXPERIENCE OF A GREEK CENTER

P. Delaporta 1,* , E. Terpos 2 , E. E. Solomou 1 , S. Gumeni 3 , M.-I. Chatzielefteriou 1 , K. Toutoudaki 1 , D. Kyriakopoulou 1 , N. Ioakeimidou 1 , I. Ntanasis-Stathopoulos 4 , I. Papassotiriou 5 , I. P. Trougakos 6 , M. A. Dimopoulos 7 , A. Kattamis 8

1 Thalassemia Unit, First Department of Pediatrics, National and Kapodistrian University of Athens, Agia Sophia Children Hospital; 2 Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens; 3 Department of Cell Biology and Biophysics, Faculty of Biology; 4 Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens; 5 Department of Clinical Biochemistry, “Aghia Sophia” Children’s Hospital; 6 Department of Cell Biology and Biophysics, Faculty of Biology; 7 Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens; 8 Thalassaemia Unit, First Department of Pediatrics, School of Medicine, National and Kapodistrian University of Athens, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece

Background: Patients with transfusion-dependent-thalassaemia (TDT) are considered as increased risk population for severe and/or morbid COVID-19 infection. Timely vaccination is the main preventive method for severe COVID-19.

Aims: To provide an overview of the clinical profile and outcome of COVID-19 infection in patients with TDT as well as to study the immune response after 3 and 6 months after vaccination against COVID-19 in adult patients with transfusion-dependent thalassaemia.

Methods: This analysis focused on the evaluation in TDT patients on the long-term immune response post vaccination and on the course of COVID-19 infection and its correlation with immunization status. Serum was collected at 4 pre-defined time points, namely, just before 1st dose (TP1), 7 weeks after the 1st dose (TP2), 3 months (TP3) and 6 months (TP4) after 2 nd dose. Neutralizing antibodies (NAbs) against SARS-CoV-2 were measured using FDA-approved methods. According to manufacturer, the scale of NAbs titer is 0-100%, with ≥30% considered as positive and ≥50% as clinically relevant viral inhibition. Age-matched healthy volunteers (median age: 46 years, range: 24-64 years, 24 males / 53 females) who received mRNA vaccines served as the control group for NAbs evaluation.

Results: 340 (170female/170male) TDT patients older than 18 years (mean 43.6±11.5 years) followed in a single unit were included in the analysis.

270 patients (79%) were vaccinated with 2 or 3 doses. Immune response to vaccination was evaluated in 90 patients (median age: 46 years, range: 19-63 years, 40 males / 50 females). NAbs were at the level of non-immunity in all the patients at baseline (TP1) (mean 16.57% ±11.85) and showed a significant increase after the second dose (TP2) mean 86.96%±12.95 ( p <0.0001). At TP3 and TP4 Nabs showed a significant decrease but remained in protective levels for the majority of the patients (mean 88.75% ±9.7 and 74.64% ±17.2 respectively( p <0.0001). The kinetics of NAbs were similar to controls except for levels at TP4 ( p= 0.02) (Figure 1).

Up to 10/FEB/2022, 43 TDT patients (median age 43.52 range 18.6-57.9 years) were diagnosed with COVID-19, with 1 of them being infected twice. Of them, 17 were unvaccinated, 18 had received 2 doses of vaccine, while 8 had received 3 doses of the vaccine. Τhe incidence rate was 9.6% and 24.3% for vaccinated and unvaccinated patients, respectively. The severity of the COVID-19 for vaccinated and unvaccinated patients were as follows, respectively,; Grade 1 (asymptomatic): 0 and 1, Grade 2 (mild symptoms, symptomatic therapy, no COVID19 specific therapy): 23 and 9, Grade 3 (mild symptoms, symptomatic therapy, with COVID19 specific therapy): 1 and 3, Grade 4 (moderate: pneumonia, thrombophlebitis, hospitalization): 2 and 3, Grade 5 (hospitalization requiring ICU, death): 0 and 1. Thrombotic event was documented in 1 patient. All patients except one from unvaccinated group are alive.

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Summary/Conclusion: Immune response to vaccination may wean faster in TDT patients. in Unvaccinated TDT patients were more likely to be infected and to develop more serious COVID-19 infection compared to vaccinated patients.

P1508: LUSPATERCEPT REDUCES THE BURDEN OF RED BLOOD CELL TRANSFUSION IN PATIENTS WITH BETA -THALASSEMIA AND COMORBIDITIES: THE EXPERIENCE OF A SINGLE THALASSEMIA AND SICKLE CELL UNIT IN A UNIVERSITY HOSPITAL

S. Delicou 1,* , M. Triantafyllou 2 , A. Xydaki 1 , C. Savvidis 3 , K. Aggeli 4 , I. Koskinas 5

1 Thalassemia and Sickle Cell Unit; 2 Statistics Office; 3 Hemoglobinopathies Endocrinology Outpatient Clinic; 4 Hemoglobinopathies Cardiology Outpatient Clinic; 5 Hemoglobinopathies Hepatology Outpatient Clinic, Hippokratio General Hospital Athens, ATHENS, Greece

Background: An erythroid maturation agent, Luspatercept is approved to treat adults with β-thalassemia. Its availability in Greece coincided with severe blood shortages due to the COVID outbreak, making its administration even more necessary.

Aims: Luspatarept’s usage in patients with comorbidities.

Methods: Between May and December 2021, luspatercept was administered for a period of 12 to 24 weeks to twelve individuals with confirmed β-thalassemia (β0/β+: 4/12, β+/β+:3/12, β0/β++:1/12, β0/β0:1/12,β+/β++:1/12) and significant comorbidities such as chronic heart failure (3/12), osteoporosis (3/12), atrial fibrillation (2/12), extramedullary hematopoiesis (2/12) and cirrhosis (1/12). The average age was 48.3 years; 7male /5 female. Prior to initiating luspatercept, the patient’s medical history was reviewed for risk factors for thrombophilia (9/12 had low protein S and C and 2/12 had also low ATIII) as well as anticoagulant (4/12 on acenocoumarol) and antiplatelet (3/12 on aspirin) medication related to splenectomy (10/12) or past thrombotic episodes (4/12).Transfusion requirements, transfusion intervals, mean hemoglobin and LDH values were documented for 12 weeks prior to and during luspatercept initiation. To maintain stable blood volumes in each transfusion, prestorage leukoreduced RBCs with an average volume per unit of 320 ml was used.

Results: Table 1 summarizes the study’s main results.Statistical significance is p <0.05.

Throughout treatment, all but one patient (11/12) received a dose of 1 mg/kg. One patient did not respond to a dose increase of 1.25 g / kg and discontinued on week 12. Additionally, two other luspatercept responders discontinued treatment after the 12th and 24th week, respectively, due to significant fatigue. After 12 and 24 weeks, all patients who continued in luspatercept had a significant decrease in transfusion blood needs, approximately -29.4% and -36.1 % compared to baseline. They also showed significant increase in transfusion intervals for an average of 20.7 days.

In addition, it became apparent that, although the volume of blood supplied was reduced and the interval between transfusions increased, the patients’ hemoglobin levels remained adequately high in luspatercept treated patients. LDH as hemolysis biomarker, did not reveal any significant changes. During the follow-up period, no patient reported progression of existing comorbidities or the development of new ones. As far as their cardiovascular disease is concerned, the patients clinical status was stable NYHA II despite the reduction of transfusions. Six months after taking luspatercept, one patient showed an improvement in extramedullary hematopoiesis as evaluated by magnetic resonance imaging. Additionally, despite the presence of predisposing factors and the significant increase of platelets, no new thrombosis developed.

Summary/Conclusion: In patients with significant comorbidities, luspatercept significantly decreased transfusion burden and prolonged transfusion intervals, without any observed worsening of their comorbidities or development of new ones.

P1509: BONE MARROW TFR2 GENETIC DELETION ABROGATES BLOOD TRANFUSION REQUIREMENT IN THE HBBTH1/TH2 Β-THALASSEMIC MURINE MODEL

S. M. Di Modica 1,* , E. Tanzi 1,2 , V. Olivari 1,3 , M. R. Lidonnici 4 , M. Pettinato 1,3 , A. Pagani 1 , F. Tiboni 4 , G. Ferrari 4 , L. Silvestri 1,3 , S. Rivella 5 , A. Nai 1,3

1 Division of Genetics and Cellular Biology, San Raffaele Scientific Institute; 2 University of Milan Bicocca; 3 Vita-Salute San Raffaele University; 4 San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), San Raffaele Scientific Institute, Milan, Italy; 5 Division of Hematology, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, United States of America

Background: β-thalassemia is a genetic disorder caused by mutations in the β-globin gene, characterized by anemia, due to defective production of hemoglobin (Hb) and red blood cells (RBC), ineffective erythropoiesis (IE) and iron overload, whose management still requires improvement. People affected by the most severe transfusion-dependent (TDT) form of the disease require lifelong blood transfusions (BT), treatment associated to several complications. Allogenic bone marrow (BM) transplantation is the only definitive cure, but it is limited by the availability of suitable donors. Gene therapy and Luspatercept are recently approved therapeutic options applicable to selected patients.

Transferrin receptor 2 (TFR2) is an activator of the iron-regulatory hormone hepcidin in the liver and a brake of Erythropoietin signalling in erythroid cells, balancing RBC production according to iron availability. The potentiality of BM Tfr2 deletion for the treatment of β-thalassemia has been already proven in non-TDT mice, both as a monotherapy (Artuso et al., Blood 2018) and, even more strikingly, in combination with iron-restricting approaches (Casu, Pettinato et al., Blood 2020).

Aims: Here, we investigate whether BM Tfr2 targeting might represent a therapeutic option for TDT.

Methods: TDT mice ( Hbb th1/th2 ) with heterozygous ( Tfr2 BMhetero / Hbb th1/th2 ) and homozygous ( Tfr2 BMKO / Hbb th1/th2 ) BM Tfr2 deletion were generated by transplantation of fetal liver cells (FLT) from day E14.5 Hbb th1/th2 , Tfr2-hetero / Hbb th1/th2 and Tfr2-ko / Hbb th1/th2 embryos. A cohort of mice was sacrificed 8 weeks after FLT for complete phenotypic analysis before the onset of transfusion-dependence. Blood transfusion requirement was monitored on a second cohort of mice through a weekly evaluation of Hb levels performed from 12 to 30 weeks after FLT. Animals were transfused when Hb dropped ≤5,5 g/dL.

Results: Eight weeks after FLT, BM Tfr2 deletion improved anemia of Hbb th1/th2 mice with a gene-dosage effect, increasing RBC count and Hb levels and reducing the percentage of circulating reticulocytes. In addition, Tfr2 BMKO / Hbb th1/th2 animals had a more effective erythropoiesis both in the BM and in the spleen, as shown by an increased proportion of the most mature erythroblasts (orthochromatic, reticulocytes and RBCs). The expression of α-globin was reduced in the BM and spleen of Tfr2 -deficient mice: this partially rescued the unbalance with β-globin, contributing to the more effective erythropoiesis and leading to an improved RBCs morphology.

BM Tfr2 deficient mice maintained RBC count and Hb levels higher than controls until 30 weeks after FLT without any transfusion. On the contrary, Hbb th1/th2 mice became transfusion-dependent 14 weeks after FLT, with some animals suffering from very severe anemia and requiring weekly BT. Among these, 2 out 7 mice died, while no mortality was recorded among BM Tfr2 deficient mice. At the end of the follow-up Tfr2 BMKO / Hbb th1/th2 animals had lower hepatic iron content than Hbb th1/th2 controls, showing that BM Tfr2 deletion may prevent secondary iron-loading in these mice.

Summary/Conclusion: Hematopoietic Tfr2 deletion increased the levels of functional Hb and RBCs and improved IE in TDT mice, thus mitigating the severity of the disease. This effect was sufficient to avoid long-term blood transfusions, to abolish mortality due to severe chronic anemia or BT complications, and to strongly reduce iron loading, showing that BM TFR2 targeting might represent a therapeutic option also for TD β-thalassemia. Further studies are ongoing to elucidate the exact mechanism.

P1510: VAMIFEPORT PREVENTED THE FORMATION OF NON-TRANSFERRIN-BOUND IRON IN A MOUSE MODEL OF ΒETA-THALASSEMIA WITH BLOOD TRANSFUSIONS

N. Kalleda 1,* , A. Flace 1 , P. Altermatt 1 , G. Ingoglia 1 , C. Doucerain 1 , N. Nyffenegger 1 , F. Dürrenberger 1 , V. Manolova 1

1 Research and Non-clinical development, Vifor (International) Ltd., St. Gallen, Switzerland

Background: β-thalassemia is an inherited anemia caused by mutations in the β-globin gene of hemoglobin (Hb) resulting in ineffective erythropoiesis, formation of red blood cells (RBCs) with short lifespan and organ iron overload. Patients with the most severe form-transfusion-dependent β-thalassemia (TDT), receive regular blood transfusions (BTs) leading to secondary iron overload requiring iron chelation therapy. Moreover, TDT patients often show increase in non-transferrin bound iron (NTBI), released by macrophages recycling damaged RBCs via a ferroportin-dependent mechanism leading to oxidative stress and vascular damage. Previously, we showed that limiting iron availability with the oral ferroportin inhibitor vamifeport (also known as VIT-2763) ameliorated anemia, ineffective erythropoiesis, and the dysregulated iron homeostasis in the Hbb th3/+ murine model of β-thalassemia under non-transfused conditions 1 .

Aims: The aim of the study was to determine the effect of the oral ferroportin inhibitor vamifeport on plasma NTBI levels and ineffective erythropoiesis following a single or repeated BTs in the Hbb th3/+ mouse model of b-thalassemia.

Methods: Hbb th3/+ mice were transfused with 200 µl of blood collected from wild-type mice ubiquitously expressing GFP (UBC-GFP mice). Vamifeport (120 mg/kg) was administered in Hbb th3/+ mice by oral gavage 30 min before a single BT. Repeated BTs were performed in one-week interval for four times. Transfused Hbb th3/+ mice were treated with 120 mg/kg vamifeport once daily for 4 weeks. Hematological parameters were analyzed using an automated analyzer, erythropoiesis by flow cytometry, and α-globin aggregates by TAU gel electrophoresis.

Results: A single BT significantly increased plasma NTBI levels in Hbb th3/+ mice as compared to non-transfused mice. NTBI levels were elevated for about 3h and returned to the baseline at 6h post transfusion. Vamifeport significantly reduced plasma NTBI levels in transfused Hbb th3/+ mice to the levels of non-transfused controls.

Vamifeport as a single therapy or in combination with repeated BTs increased Hb, RBC counts, hematocrit and decreased spleen size, reticulocyte counts, plasma erythropoietin and α-globin aggregates in RBCs of Hbb th3/+ mice. Both, vamifeport and BTs administered as single therapy improved the ineffective erythropoiesis in spleens of Hbb th3/+ mice. Importantly, the combination therapy of vamifeport and BTs resulted in lower proportion of immature erythrocytes and higher percentage of mature erythrocytes as compared to either treatment alone, suggesting additive effects of vamifeport and repeated BTs. As expected, repeated BTs increased the transferrin saturation (TSAT), whereas vamifeport decreased TSAT levels either in transfused or non-transfused Hbb th3/+ mice, reflecting the ability of vamifeport to inhibit ferroportin-mediated iron efflux into the plasma.

Summary/Conclusion: Vamifeport prevented formation of BT-mediated NTBI in the plasma of Hbb th3/+ mice, suggesting iron restriction by ferroportin inhibition prevents formation of potentially harmful reactive plasma iron species. Vamifeport ameliorated anemia, ineffective erythropoiesis, and the dysregulated iron homeostasis in Hbb th3/+ mice either as a single therapy or in combination with repeated BTs.

P1511: EFFICACY AND SAFETY OF COMBINATION IRON CHELATION THERAPY WITH DEFERIPRONE AND DEFERASIROX IN PATIENTS WITH Β-THALASSEMIA MAJOR: A SYSTEMATIC LITERATURE REVIEW

A. Piga 1,* , S. Sheth 2 , A. Kattamis 3 , F. Tricta 4 , E. Igboekwe 5 , C. Fradette 4 , J. Kwiatkowski 6

1 University of Turin, Turin, Italy; 2 New York Presbyterian Hospital/Weill Cornell Medical College, New York, United States of America; 3 National and Kapodistrian University of Athens, Athens, Greece; 4 Chiesi Canada Corporation, Toronto, Canada; 5 Chiesi USA, Inc, Boston; 6 The Children’s Hospital of Philadelphia and Perelman School of Medicine of the University of Pennsylvania, Philadelphia, United States of America

Background: Iron overload can occur in patients with thalassemia syndromes because of hyperabsorption and/or frequent blood transfusions. Currently, 3 iron chelators are approved for clinical use: deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX). Combination iron chelation therapy may be prescribed for patients who do not adequately respond to monotherapy, have increased heart and/or liver iron overload, or have dose-dependent toxicity. It is important to optimize chelation to reduce iron-associated morbidity and early mortality and to improve quality of life. Oral DFP is typically administered as a monotherapy or in combination with DFO or DFX. Combination therapy was approved for clinical use by the European Medicines Agency in 2016; additionally, a recent British Society for Haematology guideline states that DFP+DFX is a highly effective combination therapy, although the safety and efficacy of DFX in combination with other iron chelators is not well established.

Aims: A systematic literature review was conducted to evaluate the efficacy and safety profile of combination chelation therapy with DFP+DFX in patients with transfusion dependent β-thalassemia major (TDT).

Methods: A systematic search was performed of the Embase and MEDLINE databases to identify trials related to the use of combination iron chelation therapy (DFP+DFX) in patients with TDT. The literature search was conducted in May 2021 and was not restricted to a specific start date. The search strings were established by logical association of appropriate words and MESH terms. The publications were screened based on a priori exclusion and inclusion criteria (eg, case-report exclusion).

Results: Eight clinical study publications (7 prospective, 1 retrospective) of 1725 screened articles assessed the DFP+DFX combination for a total of 181 patients (Table). One publication was a pharmacokinetic (PK) study.

Of the 7 studies, 6 reported a consistent reduction in at least 1 of the 3 studied endpoints (serum ferritin, liver, and cardiac iron). The PK study reported an additive effect in the total iron excretion in comparison with monotherapy. The adverse events reported with DFP+DFX were consistent with what has previously been reported for DFP or DFX monotherapy; no new adverse events were observed. The most common adverse events across the 7 studies included gastrointestinal symptoms, elevation in alanine aminotransferase and/or aspartate aminotransferase, arthralgia or joint symptoms, an increase in creatinine levels, proteinuria, and red-colored urine. The numbers of patients reported with neutropenia and thrombocytopenia were low (neutropenia, n=7 patients; thrombocytopenia, n=8 patients); agranulocytosis was not observed. Two studies reported serious adverse events, of which 1 (acute cholecystitis) was related to treatment.

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Summary/Conclusion: This systematic literature review suggests that DFP+DFX combination iron chelation therapy can be a highly effective therapeutic option for improving iron overload, with an acceptable tolerability profile. This review supports the safety and efficacy of the combination, as approved by the EMA. Limitations of this analysis include small sample size and nonrandomization in most studies.

P1512: HEART FAILURE IN THALASSEMIA MAJOR: TIME FOR NEW CARDIOVASCULAR MAGNETIC RESONCE MARKERS?

A. Meloni 1,* , L. Pistoia 1 , N. Martini 1 , D. Visceglie 2 , P. M. G. Sanna 3 , M. Murgia 4 , A. Barone 5 , P. Giovangrossi 6 , G. Alberini 7 , V. Positano 1 , F. Cademartiri 1

1 Department of Radiology, Fondazione G. Monasterio CNR-Regione Toscana, Pisa; 2 Servizio di Immunoematologia e Medicina Trasfusionale, A.S.L. di Bari, Ospedale “Di Venere”, Bari; 3 Servizio trasfusionale Aziendale, Azienda Ospedaliero-Universitaria di Sassari, Sassari; 4 Centro Provinciale per le Microcitemie, Ospedale San Martino di Oristano, Oristano; 5 Unità Operativa di Pediatria e Oncoematologia Dipartimento Materno-Infantile, Azienda Ospedaliero-Universitaria di Parma, Parma; 6 Servizio di Immunoematologia e Medicina Trasfusionale, Ospedale S. M. Goretti, Latina; 7 U.O.C. INFOTEL Translational BioInformatics and eHealth, Fondazione G. Monasterio CNR-Regione Toscana, Pisa, Italy

Background: Altough T2* cardiovascular magnetic resonance (CMR) represents the non-invasive gold standard for myocardial iron overload (MIO) assessment, native myocardial T1 mapping was demonstrated more sensitive in presence of small amounts of iron. The myocardial extracellular volume (ECV) by CMR has been introduced as a surrogate marker of diffuse interstitial myocardial fibrosis, significantly associated with MIO.

Aims: We explored the clinical impact of ECV mapping for detecting heart failure in thalassemia major (TM).

Methods: 108 β-TM patients (62 females, 40.16±8.83 years), consecutively enrolled in the Extension-Myocardial Iron Overload in Thalassemia Network, underwent CMR. T2* and native and post-contrast T1 values were assessed in all 16 myocardial segments. Segmental ECV values were calculated with input of native and post-contrast myocardial segmental and blood pool T1 values and same-day hematocrit. Global value was the mean of all segmental values.

Results: Patients without and with at least one cardiovascular risk factor showed comparable global ECV values (31.89±5.65% vs 33.00±7.46%; P=0.404).

N-terminal pro-BNP levels, a proven diagnostic biomarker for the diagnosis and prognosis of patients with heart failure, were significantly correlated with global ECV values (R=0.237; P=0.016) but not with global heart T2* or T1 values.

Significant MIO (global heart T2*<20 ms) was found in 9 (8.3%) patients. Global ECV values were significantly higher in patients with significant MIO than in patients without significant MIO (40.07±10.62% vs 31.85±5.71%; P=0.049).

Ten (9.3%) patients had a history of heart failure. Compared to patients without heart failure, patients with a history of heart failure showed significantly lower global heart T1 values (882.68±125.46 ms vs 962.66±81.90 ms; P=0.047) and significantly higher global heart ECV values (36.63±9.41 vs 32.12±6.14 ms; P=0.039) (Figure). Global heart T2* values tended to be lower in patients with heart failure than in patients free of heart failure, but the difference was not significant (30.44±14.53 vs 38.24±8.63 ms; P=0.077).

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Summary/Conclusion: In our population of well treated patients, generally not heavily loaded at the cardiac level, native T1 and ECV values turned out to be a associated with heart failure, more strongly than global heart T2* values. So, when possible, the ECV assessment should be included in the routine CMR of TM patients. Longitudinal prospective studies are needed to clarify the temporal association between MIO and diffuse fibrosis. Moreover, the potential of treatments targeted at interstitial fibrosis to reduce the risk of heart failure should be investigated, especially in TM patients refractory to iron chelating therapy.

P1513: LOW VITAMIN D LEVELS ARE ASSOCIATED WITH INCREASED CARDIAC IRON UPTAKE IN THALASSEMIA MAJOR.

A. Meloni 1,* , L. Pistoia 1 , C. Vassalle 2 , S. Maffei 3 , I. Fotzi 4 , R. Rosso 5 , M. Serra 6 , A. Quota 7 , P. P. Bitti 8 , V. Carrai 9 , V. Positano 1 , F. Cademartiri 1

1 Department of Radiology; 2 Medicina di laboratorio; 3 Endocrinologia Cardiovascolare Ginecologica ed Osteoporosi, Fondazione G. Monasterio CNR-Regione Toscana, Pisa; 4 Centro Talassemie ed Emoglobinopatie, Ospedale “Meyer”, Firenze; 5 Unità Operativa Talassemie ed Emoglobinopatie, Azienda Ospedaliero-Universitaria Policlinico “Vittorio Emanuele”, Catania; 6 Day Hospital di Talassemia, Ospedale “V. Fazzi”, Lecce; 7 Servizio di Talassemia, Ospedale “V. Emanuele III”, Gela (CL); 8 Ambulatorio Trasfusionale, Presidio Ospedaliero “San Francesco” ASL Nuoro, Nuoro; 9 Ematologia, Azienda Ospedaliero - Universitaria Careggi, Firenze, Italy

Background: Decreased vitamin D (VitD) levels stimulate the expression of the transmembrane L-type voltage-dependent calcium channels (LVDCC), which, in addition to calcium, absorb iron. However, there is little and controversial data on the correlation of VitD levels with cardiac iron.

Aims: We investigated the association between vitamin D levels and cardiac iron and function in thalassemia major (TM) patients.

Methods: We considered 278 TM patients (39.04±8.58 years, 56.8% females), consecutively enrolled in the Extension-Myocardial Iron Overload in Thalassemia Network. Myocardial iron overload (MIO) was quantified using a T2* segmental approach. Left ventricular (LV) function parameters were quantified by cine images. Measurement of serum 25(OH)D was carried out using a chemiluminescent immunoassay.

Results: Vitamin D supplements were taken by 61.4% of our patients. VitD levels were deficient (<20 ng/dl) in 107 (38.5%) patients, insufficient (20–30 ng/dl) in 96 (34.5%) patients and adequate (>30 ng/dl) in 75 (27.0%) patients. Patients with deficient vitD levels were significantly younger than patients with adequate (36.96±7.64 yrs vs 41.26±8.19 yrs; P<0.0001) as well as insufficient vitD levels (36.96±7.64 yrs vs 39.63±9.39 yrs; P=0.042) while no difference was detected in male to female ratio.

MIO (global heart T2*<20 ms) was significantly more frequent in patients with deficient VitD than in patients with adequate and insufficient VitD levels (Figure). Patients with deficient vitD levels had a significant higher risk of MIO than patients with adequate vitD levels (odds ratio-OR=20.62, 95%CI=2.67-153.72; P=0.004) and patients with insufficient VitD levels (OR=8.49, 95%CI=2.46-29.29; P=0.001). At receiver operating characteristic curve analysis, a vitD level≤ 17.3 ng/dl predicted MIO with a sensitivity of 81.5% and a specificity of 75.3% (P<0.0001). The area under the curve was 0.79 (95% confidence interval: 0.74–0.84).

Compared to patients with normal VitD levels, patients with deficient vitamin levels showed significantly higher LV end-diastolic volume index (87.61±19.19 ml/m2 vs 79.17±14.27 ml/m2; P=0.003) and LV mass index (63.39±13.55 g/m2 vs 57.45±12.90 g/m2; P=0.006). The LV ejection fraction tended to be lower in patients with VitD deficiency, but no statistical difference versus the other groups was detected.

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Summary/Conclusion: In thalassemia major vitamin D deficiency is associated with and increased risk of cardiac iron overload. Periodic and regular assessment of VitD levels can be useful in the prevention not only of bone disorders but also of cardiac iron accumulation and subsequent overt dysfunction.

P1514: MULTI-PARAMETRIC CARDIAC MAGNETIC RESONANCE FOR RISK PREDICTION OF HEART FAILURE DEATH IN THALASSEMIA MAJOR

A. Meloni 1,* , L. Pistoia 1 , E. Facchini 2 , S. Armari 3 , G. Roccamo 4 , R. Renni 5 , R. Mattei 6 , A. Vitucci 7 , S. Renne 8 , A. Vallone 9 , V. Positano 1 , F. Cademartiri 1

1 Department of Radiology, Fondazione G. Monasterio CNR-Regione Toscana, Pisa; 2 Unità Operativa di Pediatria, Azienda Ospedaliero-Universitaria di Bologna - Policlinico “S. Orsola-Malpighi”, Bologna; 3 Reparto di Pediatria, Azienda Ospedaliera di Legnago, Legnago (VR); 4 Unità di Prevenzione e Cura delle Microcitemie, Ospedale “Civile”, S. Agata di Militello (ME); 5 Day Hospital, Ospedale Civile “F. Ferrari”, Casarano (LE); 6 Unità Operativa Pediatria, A.U.L.S.S. 5 Polesani, Adria (RO); 7 Ematologia con Trapianto-Servizio Regionale Talassemie Dipartimento dell’Emergenza e dei Trapianti d’Organo (DETO), Azienda Ospedaliero-Universitaria-Policlinico di Bari, Bari; 8 Struttura Complessa di Cardioradiologia-UTIC, Presidio Ospedaliero “Giovanni Paolo II”, Lamezia Terme (CZ); 9 Reparto di Radiologia, Azienda Ospedaliera “Garibaldi” Presidio Ospedaliero Nesima, Catania, Italy

Background: Iron-induced heart failure (HF) is the main cause of mortality in patients with thalassemia major (TM). Cardiovascular Magnetic Resonance (CMR) plays a key role in the management of these patients, assessing myocardial iron overload (MIO), biventricular function, and myocardial fibrosis.

Aims: We evaluated the prognostic value of CMR parameters in predicting death for HF.

Methods: We followed prospectively 1681 white TM patients who performed a baseline CMR exam within the Myocardial Iron Overload in Thalassemia (MIOT) network. All prognostic variables associated with the outcome at the univariate Cox model were placed in the multivariate model and were ruled out if they did not significantly improve the adjustment.

Results: At baseline the mean age was 31.11±9.01 years and 874 (52.0%) patients were females. During a mean follow-up of 4.76±2.07 years, 47.3% of the patients changed at least once the chelation regimen, that is switched to a different type of chelator or underwent dose/frequency modification.

Sixteen (1.0%) patients died for heart failure.

No association was detected between age or gender and HF mortality. Patients who died for HF had significantly lower baseline global heart T2* values (19.45±15.11 ms vs 29.52±12.02 ms, P=0.006) and significantly higher baseline MR liver iron concentration (LIC) values (19.17±15.95 mg/g dw vs 8.72±10.63 mg/g dw, P=0.005) than patients still alive or died for a different cause, but the presence of a pathologic MRI LIC (>3 mg/g dw) was not associated with an increased risk of HF mortality.

Heart iron, ventricular dysfunction, and ventricular dilatation were significant univariate prognosticators. In the multivariate analysis the independent predictive factors were a global heart T2*<10 ms (compared to a global heart T2*≥20 ms) (hazard ratio-HR=5.38, 95%CI=1.79-16.22, P=0.003) and ventricular dilatation (HR=4.59, 95%CI=1.55-13.59, P=0.006). The Figure shows the Kaplan–Meier survival curves. The log-rank test revealed a significant difference in the curves for each predictor (severe MIO: P<0.0001 and ventricular dilatation: P<0.0001).

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Summary/Conclusion: We detected few HF deaths thanks to a MR-guided patient-specific adjustment of the chelation therapy. Severe MIO and ventricular dilatation identified TM patients at high risk for heart failure death. So, a widespread program using CMR exploiting its multi-parametric potential can have considerable power for further opening the prognosis of TM patients.

P1515: IMPACT OF GENOTYPE ON PANCREATIC IRON OVERLOAD AND IMPAIRED GLUCOSE METABOLISM IN THALASSEMIA MAJOR

A. Meloni 1,* , L. Pistoia 1 , M. Santodirocco 2 , L. Rigoli 3 , C. Bosi 4 , F. M. Pasin 5 , C. Argento 6 , G. Peritore 7 , P. Fina 8 , V. Positano 1 , F. Cademartiri 1

1 Department of Radiology, Fondazione G. Monasterio CNR-Regione Toscana, Pisa; 2 Centro Microcitemia – Day Hospital Thalassemia Poliambulatorio “Giovanni Paolo II”, Ospedale Casa Sollievo della Sofferenza IRCCS, San Giovanni Rotondo (FG); 3 Unità Operativa Complessa di Pediatria d’Urgenza, Policlinico “G. Martino”, Messina; 4 U. O. Ematologia e CTMO, Ospedale “G. Da Saliceto”, Piacenza; 5 DH Medicina Trasfusionale, Ospedale S. Pertini, Roma; 6 Centro di Talasssemia, Ospedale “San Giovanni Di Dio”, Agrigento; 7 Unità Operativa Complessa di Radiologia, “ARNAS” Civico, Di Cristina Benfratelli, Palermo; 8 Unità Operativa Complessa Diagnostica per Immagini, Ospedale S. Pertini, Roma, Italy

Background: β-thalassemia major (TM) is characterized by a wide spectrum of clinical manifestations and laboratory findings and the disease phenotype largely depends on the underlying mutations of the β gene. These mutations cause a reduced (β+) or absent (β0) production of the β-globin chain. No study has evaluated the link between genotype and pancreatic iron, representing a powerful predictor for glucose metabolism and cardiac iron and complications.

Aims: The objective of this multicenter study was to evaluate the impact of genotype on pancreatic iron levels and glucose metabolism in TM.

Methods: We considered 549 TM patients (36.23±10.63 years, 46.3% females), consecutively enrolled in the Extension-Myocardial Iron Overload in Thalassemia Network. T2* measurements were performed over pancreatic head, body and tail and global value was the mean. The pattern of disturbances of glucose metabolism was assessed by means of the oral glucose tolerance test (OGTT).

Results: Three groups of patients were identified: homozygous β+ (N=158), compound heterozygous β 0 β + (N=206), and homozygous β0 (N=145). The three groups were homogeneous for age, sex, age at start of regular transfusions and chelation, and frequency of splenectomy.

The homozygous β0 group showed significantly lower global pancreas T2* values than the homozygous β+ group (9.91±8.13 ms vs 14.45±10.72 ms; P<0.0001) and the heterozygous β0β+ group (9.91±8.13 ms vs 14.69±10.76 ms; P<0.0001). Pancreatic iron overload (T2*<26 ms) was found in 81.6% of β+β+ patients, 82.5% of β0β+ patients, and 95.1% of β0β0 patients (P<0.0001) (Figure 1A). β0β0 patients were more likely to have pancreatic iron overload than both β+β+ patients (odds ratio-OR=4.39, 95%CI=2.01-9.61; P<0.0001) and β0β+patients (OR=4.14, 95%CI=1.94-8.86; P<0.0001).

An altered OGTT was found in 154 patients (34 impaired fasting glucose, 54 impaired glucose tolerance, and 66 diabetes mellitus), all showing pancreatic iron overload. The prevalence of impaired OGTT was significantly higher in the homozygous β0 group than in the homozygous β+ group (34.9 vs 22.4%; P=0.042) (Figure 1B). β0β0 patients had around twice the risk of alterations of glucose metabolism (OR=1.86, 95%CI=1.33-3.07; P=0.014) than β+β+ patients.

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Summary/Conclusion: In TM the homozygous β0 genotype is associated with higher pancreatic iron levels and prevalence of impaired glucose metabolism. Therefore, the knowledge of the genotype can be useful in the prediction of some phenotypic features and in the clinical and instrumental management of TM patients.

P1516: SCREENING FOR BETA-THALASSEMIA MINOR IN PREGNANT FEMALES PREVIOUSLY DIAGNOSED WITH IRON DEFICIENCY ANEMIA: WHAT ARE THE RATES OF MISDIAGNOSIS AND THE NEED FOR AN INCREASED UPTAKE OF CARRIER SCREENING

S. Menakuru 1,2,* , S. Kalla 3

1 Internal Medicine, Indiana University Health, Muncie, United States of America; 2 Internal Medicine, Narayana Medical College, Nellore; 3 Internal Medicine, Maharajah’s Institute Of Medical Sciences, Vizinagaram, India

Background: In rural India, iron deficiency anemia is a common diagnosis in pregnant women; however, several females are possibly misdiagnosed due to lack of follow-up. There are high rates of consanguineous marriages, which may be a cause for the higher rates of beta-thalassemia major in these populations. The rates of beta-thalassemia major may be prevented by proper screening for beta-thalassemia minor during the prenatal period. Uptake of premarital screening in India is poor, and therefore the effectiveness of a prenatal screen was evaluated in those with a prior history of iron deficiency anemia (IDA). The reason for choosing patients with a history of IDA was that the patients already had a diagnosis of microcytic anemia, and the authors wanted to evaluate for possible rates of misdiagnosis.

Aims: To study the prevalence and the need to screen for beta-thalassemia minor in India’s pregnant rural population, and to help prevent couples at risk from producing a fetus with beta-thalassemia major. In addition, an assessment of the rates of misdiagnosis of IDA was done to check the accuracy of the current estimates of beta-thalassemia in India.

Methods: 800 pregnant women, previously diagnosed with IDA, in their first trimester from various rural villages around Andhra Pradesh, India were selected. These 800 females were screened for beta-thalassemia minor during their prenatal screening period. The mean gestational age for screening was 12 ± 4 weeks. Screening was done utilizing a complete iron profile with complete blood count and the Mentzer index, defined as the mean corpuscular volume divided by the red blood cell count. Greater than 13 on the Mentzer index indicated that iron deficiency anemia was more likely, while less than 13 indicated probable beta-thalassemia. Those individuals with a Mentzer index less than 13 were then screened with the Naked Eye Single Tube Red cell Osmotic Fragility Test (NESTROFT) and then confirmed with High-Performance Liquid Chromatography (HPLC). The individuals who were positive for beta-thalassemia minor then had their husbands screened.

Results: Of the 800 pregnant women screened 47 came back positive for beta-thalassemia minor, equating to 5.87% of the sample size. At-risk couples, defined as both husband and wife being positive for the beta-thalassemia minor was found in 7 couples. The 7 couples were informed about the risk of development of beta-thalassemia major in their fetus, and a prenatal diagnosis was offered to them. Of the 7 patients, 5 elected to terminate the pregnancy after a positive prenatal diagnosis of beta-thalassemia major, and the remaining 2 couples did not respond and were lost to follow-up.

Summary/Conclusion: There was a moderately high prevalence of undiagnosed beta-thalassemia minor, and presently the uptake of the current voluntary screening program is limited in India. The ultimate goal would be to detect carrier couples with a premarital screening program; however, it has been challenging to implement thus far. Therefore the authors propose the utilization of a prenatal carrier screening method ideally as early as possible as this may help prevent cases of beta-thalassemia major. There should be more campaigns to increase awareness about the need to screen pregnant females, and if they are positive, then their husbands should be screened as well to detect at-risk couples. The authors also conclude that the rates of misdiagnosis of IDA were equally alarming as there may be more cases of beta-thalassemia minor in India than previously suspected.

P1517: REAL-WORLD COMPLICATION BURDEN AND DISEASE MANAGEMENT IN TRANSFUSION-DEPENDENT ADULTS WITH ΒETA-THALASSEMIA (Β-THAL) IN GREECE: FINAL RESULTS OF THE EPIDEMIOLOGICAL CROSS-SECTIONAL ULYSSES STUDY

A. Kattamis †1, , E. Voskaridou †2 , S. Delicou 3 , E. Klironomos 4 , I. Lafiatis 5 , F. Petropoulou 6 , M. D. Diamantidis 7 , S. Lafioniatis 8 , L. Evliati 9 , E. Kapsali 10 , K. Karvounis-Marolachakis 11 , D. Timotheatou 11 , C. Deligianni 12 , P. Viktoratos 12 , A. Kourakli 13

1 Thalassemia Unit, First Department of Pediatrics, National and Kapodistrian University of Athens; 2 Expertise Center in Rare Haematological Diseases-Haemoglobinopathies, “Laikon” General Hospital of Athens; 3 Thalassemia and Sickle Cell Unit, “Hippocrateion” General Hospital of Athens, Athens; 4 Thalassemia and Sickle Cell Unit, “Venizelion” General Hospital of Heraklion, Heraklion; 5 Thalassemia and Sickle Cell Unit, “Vostanio” General Hospital of Mytilene, Myrtilene; 6 Thalassemia Unit, General Hospital of Athens “Georgios Gennimatas”, Athens; 7 Thalassemia and Sickle Cell Disease Unit, General Hospital of Larissa “Koutlimbaneio & Triantafylleio”, Larissa; 8 General Hospital “Achilopouleio” of Volos, Volos; 9 Thalassemia and Sickle Cell Unit, General Hospital of Athens “Evaggelismos”, Athens; 10 Department of Hematology, University Hospital of Ioannina, Ioannina; 11 Medical Department, Genesis Pharma SA; 12 Medical Department Bristol-Myers Squibb Company SA, Athens; 13 Hematology Division, Department of Internal Medicine, University General Hospital of Patras, Patras, Greece

Background: Despite improved prognosis, patients with transfusion-dependent β-thal commonly experience treatment and disease complications.

Aims: To assess the prevalence and severity of treatment and disease complications, capture disease management, and identify predictors of complications in a representative sample of patients with transfusion-dependent β-thal in routine settings in Greece.

Methods: ULYSSES is an epidemiological, multicenter, retrospective cross-sectional study of adults diagnosed with β-thal ≥12 months before enrollment who received ≥6 red blood cell (RBC) units (excluding elective surgery) with no transfusion-free period ≥35 days in the 24 weeks before enrollment. Hemoglobinopathies S/β- and α-thalassemia were excluded. Primary data were collected at a single visit and through chart review.

Results: From Oct 21, 2019 to June 15, 2020, a total of 201 eligible patients were enrolled by 12 major thalassemia centers; 64.2% were female, median age was 45.7 years (interquartile range [IQR], 40.2–50.5; 75.6% >40 years), mean (standard deviation [SD]) time since diagnosis was 42.9 (7.8) years.Overall, 44.3% were β + β + , 36.8% β + β 0 , 13.4% β 0 β 0 , and 5.5% carried β + , δβ-Sicilian, Corfu δ 0 β + , Turkish (δβ) 0 , and hemoglobin Lepore combinations. The most common β-globin mutations were IVS1-nt110 G>A (63.7%), Codon 39 C>T (25.4%), and IVS1-nt6 T>C (22.9%). Median (IQR) age at diagnosis and RBC transfusion initiation was 0.8 (0.4–2.8) and 1.3 (1.0–5.0) years, respectively. During the 48 weeks prior to enrollment, patients received a mean (SD) of 34.2 (9.5) RBC units from a median (IQR) of 23.0 (18.0–28.0) RBC transfusion events. Before enrollment, all patients had received iron chelation therapy (ICT), started at a median (IQR) age of 6.4 (3.2–12.9) years (median [IQR] exposure 35.6 [30.0–40.0] years), and 53.2% had received other therapies, including folic acid (48.3%); 53.2% had a splenectomy at a median (IQR) age of 22.0 (14.0–29.0) years. From diagnosis to enrollment, patients developed 1443 cumulative complications (median 6 [range, 1–55]; 585 ongoing at enrollment; 19.6% [237/1211] known Grade ≥3). Disease-related complications related to β-thal were experienced by all patients (total 1089 [median 5 (range, 1–21)]; 22.1% were Grade ≥3). Over half of patients (53.2%) experienced complications related to treatment (total 354; [median 1 (range, 0–35)]; 13.0% Grade ≥3; Figure), of which 43.4% (n=273), 25.4% (n=73), and 2.0% (n=8) were related to RBC transfusions, ICT, or other treatments, respectively. Of patients 18–40 years, 36.7%, 30.6%, and 16.3% experienced ≥1 treatment-related, RBC transfusion,or ICT complication, respectively, compared with 58.6%, 48.7%, and 28.3% of those >40 years. The 48-week pre-enrollment hospitalization rate due to disease or treatment complications was 0.076 per patient-year (95% confidence interval [CI], 0.045–0.128). Medical/surgical history (odds ratio[OR], 5.16; 95% CI, 1.91–13.92; P =0.001), splenectomy (OR, 2.86; 95% CI, 1.16–7.02; P =0.022), and >24 transfusion events in 48 weeks prior to enrollment (OR, 2.78; 95% CI, 1.15–6.74; P =0.023) were associated with increased odds for developing treatment-related complications. In contrast, splenectomy was associated with lower odds (OR, 0.27; 95% CI, 0.08–0.89; P =0.031) of having either treatment- or disease-related complications.

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Summary/Conclusion: Real-world data generated by ULYSSES underscore the substantial complication burden of patients with transfusion-dependent β-thal routinely managed in Greece, which worsens with advanced age.

† First and second author have contributed equally.

P1518: LONG-TERM SAFETY RESULTS OF THE BELIEVE STUDY OF LUSPATERCEPT IN ADULTS WITH ΒETA-THALASSEMIA

V. Viprakasit 1,* , M. D. Cappellini 2 , J. B. Porter 3 , K. H. M. Kuo 4 , T. D. Coates 5,6 , E. Voskaridou 7 , V. M. Pinto 8 , I. Tartaglione 9 , A. Khelif 10 , A. Lal 11 , A. Kattamis 12 , A. Piga 13 , O. Hermine 14,15 , N. Holot 16 , F. Lersch 17 , J. K. Shetty 17 , J. Zhang 16 , L. Bueno 16 , A. C. Giuseppi 16 , D. Miteva 17 , A. T. Taher 18

1 Siriraj Hospital, Mahidol University, Bangkok, Thailand; 2 Fondazione IRCCS Ca’ Granda Policlinico Hospital, University of Milan, Milan, Italy; 3 University College London, University College London Hospitals, London, United Kingdom; 4 Division of Medical Oncology and Hematology, Department of Medicine, University Health Network, Division of Hematology, Department of Medicine, University of Toronto, Toronto, ON, Canada; 5 Children’s Center for Cancer and Blood Diseases, Children’s Hospital Los Angeles; 6 USC Keck School of Medicine, Los Angeles, CA, United States of America; 7 Thalassemia and Sickle Cell Center, Laiko General Hospital, Athens, Greece; 8 Centro della Microcitemia e Anemie Congenite e del Dismetabolismo del Ferro, Ospedale Galliera, Genoa; 9 Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Università della Campania, Luigi Vanvitelli, Naples, Italy; 10 Farhat Hached Teaching Hospital, Sousse University, Sousse, Tunisia; 11 University of California San Francisco Benioff Children’s Hospital, Oakland, CA, United States of America; 12 First Department of Pediatrics, National and Kapodistrian University of Athens, Athens, Greece; 13 University of Turin, Turin, Italy; 14 Department of Hematology, Hôpital Necker, Assistance Publique Hôpitaux de Paris, University Paris Cité; 15 INSERM U1163 and CNRS 8254, Imagine Institute, Université Sorbonne Paris Cité, Paris, France; 16 Bristol Myers Squibb, Princeton, NJ, United States of America; 17 Celgene International Sàrl, a Bristol-Myers Squibb Company, Boudry, Switzerland; 18 Department of Internal Medicine, American University of Beirut Medical Center, Beirut, Lebanon

Background: The phase 3, placebo-controlled BELIEVE trial was shown luspatercept to be well tolerated in patients (pts) with transfusion-dependent β-thalassemia. Among the treatment-emergent adverse events (TEAEs) more commonly associated with luspatercept were bone pain, arthralgia, dizziness, hypertension, and hyperuricemia.

Aims: To report long-term BELIEVE trial safety data and compare them with safety data from the primary data cut (May 11, 2018).

Methods: Eligible pts were ≥18 y of age; had β-thalassemia or hemoglobin E/β-thalassemia diagnosis; required regular red blood cell transfusions (RBCT) (6–20 RBC units/24 wk before randomization; no RBCT-free period >35 d). Pts were randomized 2:1 to luspatercept or placebo, 1 mg/kg (up to 1.25 mg/kg allowed) subcutaneously every 3 wk. Pts (N=92) crossed over from placebo to luspatercept after study unblinding. Safety analyses were performed on the safety population (N=332) and summarized using descriptive statistics. TEAEs of special interest were thromboembolic events (TEEs), premalignancies and malignancies, bone pain, and hypertension. Combined luspatercept and crossover arms (N=315) from herein will be referred to as the luspatercept arm.

Results: As of Jan 5, 2021, median (range) duration of treatment was 103.0 (1.7–215.0) vs 74.7 (8.9–104.0) wk for luspatercept vs placebo arms (Table). At least 50% of pts in each treatment group received the maximum dose of study drug (1.25 mg/kg). Treatment-related TEAEs were reported in 175 (55.6%) pts receiving luspatercept vs 31 (28.4%) receiving placebo; 23 (7.3%) pts in luspatercept vs 1 (0.9%) in placebo arm discontinued treatment due to TEAEs. Rate of discontinuation was comparable to the primary data cut (Table). Three of 5 pts died during the study due to TEAEs; no deaths were study drug-related. TEAEs more frequent with luspatercept included headache, arthralgia, bone pain, dizziness, nausea, hypertension, jaw pain, and hyperuricemia; similar to TEAEs reported in the primary data cut. No premalignant conditions were reported; the 2 reported malignancies (acute erythroid leukemia and hepatocellular carcinoma) were unrelated to the study drug. Median (range) time to TEE was 474.0 (75–1184) vs 238.0 (238–238) d with luspatercept vs placebo, respectively. TEEs occurred in 13 (4.1%) vs 1 (0.9%) pts in the luspatercept vs placebo arms, respectively; all pts were splenectomized and had ≥1 other TEE risk factor. Similar rates of TEEs with luspatercept were reported in the primary data cut (8 pts [3.6%]). Bone pain was more frequent with luspatercept vs placebo (21.6% vs 8.3%), comparable with the primary data cut (19.7%). Bone pain was mild and occurred more commonly in wk 1–24. Median (range) time to first bone pain event was 24.5 (1–947) vs 16.0 (1–342) d in the luspatercept and placebo arms, respectively. Hypertension was reported in 8.9% vs 2.8% of pts in the luspatercept and placebo arms, respectively; median (range) time to first hypertension event was 209.5 (2–1136) vs 176.0 (148–464) d. Rates of hypertension with luspatercept were comparable between the 2 data cutoffs.

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Summary/Conclusion: In this long-term analysis, treatment discontinuations due to TEAEs were more common in the luspatercept arm compared with placebo, as expected due to longer treatment exposure, and similar to the discontinuation rate reported in the primary data cut. No new safety concerns were reported and occurrence of TEAEs of special interest (bone pain, hypertension, and TEE) was comparable with previous reports. Overall, long-term safety analysis showed results consistent with the safety profile of luspatercept.

P1519: EXPLORING THE ROLE OF FGF23 IN BETA-THALASSEMIA: A NOVEL THERAPEUTIC TARGET TO AMELIORATE ALTERED INTERACTIONS BETWEEN HSC AND BONE MARROW NICHE

L. Raggi 1,* , A. Aprile 1 , S. Bolamperti 2 , I. Villa 2 , M. Storto 1 , G. Morello 3 , S. Marktel 4 , C. Tripodo 3 , M. D. Cappellini 5,6 , I. Motta 5,6 , A. Rubinacci 2 , G. Ferrari 1,7

1 San Raffaele-Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute; 2 Bone Metabolism Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan; 3 Tumor Immunology Unit, Human Pathology Section, Department of Health Sciences, University of Palermo, Palermo; 4 Hematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute; 5 UOC General Medicine, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 6 Department of Clinical Sciences and Community Health, University of Milan; 7 Vita-Salute San Raffaele University, Milan, Italy

Background: Beta-thalassemia (BT) is a congenital blood disorder resulting in severe anemia and ineffective erythropoiesis. Therapeutical approach for BT is represented by the correction of the erythropoietic defect by allogeneic hematopoietic stem cells (HSC) transplantation from healthy donors or by autologous HSC upon gene therapy. In both settings bone marrow (BM) niche and functional HSC are key elements for successful engraftment and reconstitution. Our group showed that chronic condition of hematopoietic stress and secondary alterations associated to BT interfered with HSC-niche interaction having a negative impact on HSC function. HSC defect is reversible by targeting the BM stromal niche (Aprile et al., Blood 2020 ). We showed a reduced bone mineral density (BMD) in th3 mice, associated to lower levels of parathyroid hormone (PTH), consistent with osteoporosis and hypoparathyroidism in BT patients.

To investigate the molecular causes of bone defect, we focused on fibroblast growth factor 23 (FGF23), a phosphaturic hormone negatively affecting BMD and PTH production. Bone and BM erythroid cells are the primary sources of FGF23, which is secreted as active intact protein (iFGF23) and is inactivated by cleavage in c-terminal fragment (cFGF23). Recent publications showed erythropoietin (EPO) as a novel inducer of FGF23 production. In this scenario, we hypothesized that high EPO levels, characteristic of BT, contribute to stimulate FGF23 synthesis, thus negatively affecting bone and BM niche homeostasis.

Aims: We investigate the molecular mechanisms and the role of EPO in enhancing FGF23 levels, focusing on the impact of high FGF23 levels on bone and HSC-BM niche crosstalk in th3 mice.

Methods: We measured FGF23 levels in th3 mice and BT patients by ELISA. We blocked EPO pathway by monoclonal anti-EPO antibody. We performed in vitro stimulation and inhibition of EPO signaling. We inhibited FGF23 signals by cFGF23 administration in BT mice and we evaluated bone, BM niche and HSC function upon treatment.

Results: We found increased systemic levels of FGF23 and an unbalanced iFGF23/cFGF23 ratio in favor of cFGF23. FGF23 is also enhanced in th3 BM fluids associated to its increased expression by bone and BM erythroid cells. In vivo neutralization of EPO pathway normalized FGF23 levels demonstrating that EPO acts upstream of FGF23. To dissect the molecular mechanism linking EPO to FGF23, we modelled in vitro EPO stimulation on bone and BM erythroid cells, showing an increase of both Fgf23 transcription and post-translational cleavage with decreased expression of Galnt3 , involved in stabilizing iFGF23. Inhibition of specific EPO signaling showed that Erk1/2 and Stat5 pathways are involved in enhancing Fgf23 and decreasing Galnt3 transcription in EPO-stimulated bone and BM erythroid cells, suggesting a transcriptional regulation.

FGF23 inhibition strategy in th3 mice rescued bone mineralization and bone deposition following 38h and 13 days, respectively. FGF23 inhibition was efficacious in normalizing the expression of the niche factors osteopontin, Jagged-1 and CXCL12, and restored HSC function. Further analysis will unravel the impact of high FGF23 levels on other BM niche populations involved in the crosstalk with HSC.

Summary/Conclusion: Our findings unveil FGF23 as the missing link between hematopoiesis and bone homeostasis in a congenital hemolytic anemia, as a condition of chronic EPO stimulation. FGF23 inhibition provides a novel strategy to target bone defects and HSC-BM niche interactions and could be a valuable strategy to improve HSC transplantation and gene therapy approaches for BT.

P1520: AN ACTIVIN RECEPTOR IIB LIGAND TRAP, IN COMBINATION WITH TMPRSS6 INDUCED IRON-RESTRICTION, IS A SUPERIOR TREATMENT FOR CORRECTING Β-THALASSEMIA IN MICE

A. Guerra 1 , P. Demsko 2 , S. Sinha 1 , P. McVeigh 1 , C. Castruccio Castracani 1 , L. Breda 1 , C. Casu 1 , S. Guo 3 , S. Rivella 1,*

1 Pediatrics, Children’s Hospital of Philadelphia; 2 Pediatrics, University of Pennsylvania, Perelman School of Medicine, Philadelphia; 3 Pediatrics, Ionis Pharmaceuticals, Inc., Carlsbad, United States of America

Background: The hallmarks of β-thalassemia (BT) include ineffective erythropoiesis (IE), splenomegaly and iron overload (IO). There are several new promising therapetutics to treat BT under clinical investigation. One strategy employs iron restriction (IR) using antisense oligonucleotide technology and targets the matriptase-2 ( TMPRSS6 ) gene (T-ASO). This mode of IR has the potential to be more tolerated by BT patients than oral chelators. In mouse studies, T-ASO has been shown to improve anemia, red blood cell (RBC) life span, spenomegaly, and several IO markers (Casu et al., 2016, 2020; Guo et al., 2013). Another exciting therapeutic approach targets the TGF-β pathway. Luspatercept, an FDA approved TGF-β ligand trap designed against the extracellular binding domain of activin receptor IIB. It has been shown to improve anemia in transfusion dependent BT(Cappellini and Taher, 2021). Mouse studies using the murine analog (RAP-536) was shown to promote erythropoietin (EPO)-independent maturation of late-stage erythroid cells and to increase RBC and hemoglobin (Hb) in a dose-dependent manner (Suragani et al., 2014).

Aims: Our aim was to investigate if this strategy would successfully target distinct morbidities associated with BT.

Methods: We generated a Luspatercept-like protein (RAP-GRL) and treated th3/+ mice in combination with T-ASO (RAP-GRL+T-ASO). First, we designed, cloned, and purified RAP-GRL from a mammalian cell line. Then we tested the efficacy of our design, using RBC and Hb as our ready-out, by injecting wild-type (WT) and th3/+ mice with 10mg/kg of RAP-GRL subcutaneously (s.c.) for 3 weeks. For our combination therapy experiments we administered RAP-GRL s.c. along with T-ASO (5mg/kg) via intraperitoneal (i.p.) injection to th3/+ mice. We evaluated IE and spenomegaly by flow cytometry using CD71, Ter119 and CD44 analysis of bone marrow (BM) and and spleen (SPL). Serum iron, EPO, hepcidin and erythroferrone (ERFE) measurements were done by ELISA.

Results: Our results showed that treatment of WT or th3/+ with RAP-GRL increased RBC and Hb in both WT and th3/+ mice (n=3-9), evidence that our construct and purification methods were viable to study the combination therapy strategy. In our combination therapy experiments, RBC and Hb RBC parameters were increased in all treatment groups except vehicle controls, with the best results achieved in the RAP-GRL+T-ASO treated group (Figure 1). Serum assessments of iron (Figure 1), transferrin saturations (TfSAT), and ERFE showed that only animals treated with the T-ASO exhibited significant changes in iron metabolism. Additionally, flow cytometric analysis showed improved IE in th3/+ treated with single agents (T-ASO or RAP-GRL) in the BM and SPL, but additive improvements in the combination groups. Interestingly, EPO levels were increased only in the group treated with RAP-GRL only. Messnger RNA analysis of the kidney did not correlate with our EPO serum findings.

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Summary/Conclusion: In conclusion, our results provide pre-clinical support for combining IR and TFG-β ligand-trap strategies for the treatment of BT, and also evidence that the modes of action of these two drugs act indefependently. How activin ligand traps are able to increased RBCs remains a mystery. However, our data provides evidence that IR, in conjunction with the erythroid maturation action of drugs like Luspatercept, may offer an additive, more effective and more tolerable therapeutic strategy for BT patients.

P1521: A SEVERE MOUSE MODEL OF ALPHA-THALASSEMIA SHOWS ABNORMAL IRON METABOLISM, ERYTHROPOIESIS AND COAGULATION, AND CAN BE RESCUED BY A NOVEL GENE THERAPY APPROACH

S. Rivella 1,* , M. Chappell 1 , D. Jarocha 1 , L. Breda 1 , V. Ghiaccio 1 , M. Fedorky 1 , M. Triebwasser 1 , A. Guerra 1 , K. Gollomp 1 , N. Teawtrakul 2 , S. Glentis 3 , A. Kattamis 3 , O. Abdulmalik 1

1 Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, United States of America; 2 Internal Medicine, Khon Kaen University, Khon Kaen, Thailand; 3 Pediatrics, University of Athens, Athens, Greece

Background: Clinical presentation of deletional a-thal varies from an asymptomatic condition (one inactivated a-globin gene) to a complete knockout (Hb Bart’s Hydrops Fetalis). In patients with severe a-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation.

Aims: The aims of this study are to develop a novel adult mouse model of a-thal and a gene therapy approach for this disease.

Methods: We generated adult animals that do not produce a-globin chains (a-KO) through transplantation of homozygous B6.129S7-Hba tm1Paz /J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice.

To generate a gene therapy tool, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human a-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human a-globin genes.

Results: The a-KO animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only b-globin chains (HbH). RBC show aberrant morphology and aggregation of b-globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events, associated with neutrophil infiltration, fibrinogen staining, von Willebrand factor (vWF) released, platelet recruitment and activation. These animals also show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney.

We identified ALS20a, a vector where a-globin is under control of the b-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Use of ALS20a resulted in correction of the disease phenotype in a dose-dependent manner in a-KO mice. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels.

We tested ALS20a in CD34 cells isolated from four patients with both deletional and non-deletional HbH disease. We measured the change of b/a-globin mRNA ratio (b/aR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial b/aR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a b/aR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β 4 ), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced a-thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2).

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Summary/Conclusion: We generated an adult mouse model of lethal a-thal and, in preliminary experiments, we rescue it with ALS20a. Furthermore, ALS20a successfully improves a-globin levels in patient cells.

P1522: LONG-TERM EFFICACY AND SAFETY OF THE ORAL PYRUVATE KINASE ACTIVATOR MITAPIVAT IN ADULTS WITH NON–TRANSFUSION-DEPENDENT ALPHA- OR BETA-THALASSEMIA

K. H. M. Kuo 1,* , D. M. Layton 2 , A. Lal 3 , H. Al-Samkari 4 , J. Bhatia 5 , P. A. Kosinski 5 , B. Tong 5 , M. Lynch 5 , K. Uhlig 5 , E. P. Vichinsky 3

1 Division of Hematology, University of Toronto, Toronto, ON, Canada; 2 Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom; 3 Division of Hematology, UCSF Benioff Children’s Hospital Oakland, Oakland, CA; 4 Massachusetts General Hospital, Harvard Medical School, Boston, MA; 5 Agios Pharmaceuticals, Inc., Cambridge, MA, United States of America

Background: Thalassemia is characterized by ineffective erythropoiesis and hemolysis that occur due to imbalanced production and precipitation of globin chains. Thalassemic red blood cells (RBCs) have insufficient levels of adenosine triphosphate (ATP) to meet increased energy demands associated with globin chain imbalance, protein degradation, and cellular oxidative stress responses. Mitapivat is a first-in-class, small molecule, oral activator of RBC pyruvate kinase (PKR), a key glycolytic enzyme regulating ATP production. In a phase 2, open-label trial of mitapivat in adults with α- or β-non–transfusion-dependent (NTD) thalassemia ( NCT03692052 ), 80% (16/20) of patients (pts) met the primary endpoint of a hemoglobin (Hb) response (≥1.0 g/dL increase from baseline [BL] at ≥1 assessments between Weeks (Wks) 4–12, inclusive). Improvements in markers of hemolysis and ineffective erythropoiesis were also observed and mitapivat was generally well tolerated.

Aims: To report data from the ongoing long-term extension (LTE) period (≤Wk 72; data cutoff 27Mar2021).

Methods: Pts aged ≥18 years (yrs) with a known medical history of α- or β-thalassemia, Hb concentration ≤10.0 g/dL, and ≤5 RBC units transfused in prior 24 wks and none in 8 wks prior to study drug were eligible. All pts started mitapivat 50 mg twice daily (BID), escalating to 100 mg BID based on individual safety and Hb assessments. After completion of the 24-wk core period, pts with a Hb response or a delayed Hb response (after Wk 12), with no ongoing study drug-related grade ≥3 treatment-emergent adverse events (AE), continued on mitapivat in the LTE at the same dose as the Wk 24 visit. Study visits occur every 12 wks for ≤10 yrs.

Results: Of 19 pts who completed the core period, 17 entered the LTE (mitapivat 100 mg BID, n=16; 50 mg BID, n=1). As of the data cutoff, 1 pt discontinued (pt decision). Median duration of treatment for pts in the LTE was 70.9 wks (range 54.7, 105.6); 8 pts received ≥72 wks of treatment as of data cutoff. Median pt age in the LTE was 44 yrs (range 29, 67). Mean (standard deviation [SD]) BL Hb, total bilirubin and lactate dehydrogenase (LDH) were 8.1 (1.2) g/dL, 40.1 (26.2) μmol/L, and 272.4 (121.7) U/L, respectively. Median BL erythropoietin (EPO) was 70.5 (range 15, 11191) IU/L. Hb improvements achieved in the core period were sustained in the LTE (Figure). Mean (SD) Hb increase from BL to Wk 60 (α-thalassemia, n=4; β-thalassemia, n=9) and Wk 72 (β-thalassemia, n=8) were 1.5 (0.4) and 1.7 (0.5) g/dL, respectively. Improvements in markers of hemolysis and ineffective erythropoiesis observed in the core period were maintained in the LTE up to Wk 72 (mean [SD] bilirubin and LDH, –15.8 [16.6] μmol/L and –63.6 [216.0] U/L, respectively; median [range] EPO, –33.0 [–72.0, –16.0] IU/L).

The safety profile was consistent with that observed in the core period. AEs in ≥15% of pts were headache (5/17) and back pain (3/17), none were grade ≥3. No trends for decreases in bone mineral density were observed. No treatment-related serious AEs occurred.

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Summary/Conclusion: In pts with either α- or β-thalassemia, a favorable efficacy-safety profile was observed with long-term treatment with mitapivat. Data show sustained improvements in Hb, hemolysis, and ineffective erythropoiesis despite globin genotypic heterogeneity, and no new safety findings. Mitapivat, through its unique mechanism of action, may represent a novel therapeutic approach for this condition. Two phase 3 trials of mitapivat in α- and β-thalassemia for both NTD (ENERGIZE, 2021-000211-23) and transfusion-dependent (ENERGIZE-T, 2021-000212-34) pts are enrolling.

P1523: DEMOGRAPHICS, CLINICAL CHARACTERISTICS, AND REAL-WORLD TREATMENT PATTERNS AMONG PATIENTS WITH BETA-THALASSEMIA: A RETROSPECTIVE MEDICAL RECORD ABSTRACTION STUDY

M. D. Cappellini 1,* , A. Yucel 2 , J. Meyers 3 , M. Jimenez 3 , T. Nham 3 , M. B. Glassberg 4 , F. Alashkar 5

1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy; 2 Bristol Myers Squibb, Princeton, NJ; 3 RTI Health Solutions, Research Triangle Park, NC; 4 Bristol Myers Squibb, Summit, NJ, United States of America; 5 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital of Essen, Essen, Germany

Background: Beta-thalassemias (BTs) are genetic disorders of hemoglobin (Hb) synthesis defined by deficient or absent synthesis of the beta-globin subunit. As a result, patients with BT experience hypochromic anemia and excess alpha-globin chain, causing ineffective erythropoiesis and low-grade hemolysis; however, patient characteristics and treatment patterns are not well understood.

Aims: This retrospective medical record abstraction study evaluated patients prior to introduction of luspatercept with transfusion-dependent (TD) and non-transfusion-dependent (NTD) BT for demographics, clinical characteristics, and treatment patterns.

Methods: Medical records of adults with TD or NTD BT in the United Kingdom (UK), France, Germany, Spain, and Canada with ≥5 years of visit history within the practice were evaluated. The target sample size was 50 patients in all countries (except Canada: N=20 patients), with an even distribution of patients with TD and NTD BT. Information on demographics, clinical characteristics, and treatment patterns was abstracted from the medical records.

Results: In total, 118 patients with TD and 96 patients with NTD BT were identified. Among patients with TD BT, mean (standard deviation [SD]) patient age was 36.1 (11.9) years, and 28.8% were female; among patients with NTD BT, mean (SD) patient age was 36.6 (9.8) years, and 38.5% were female (Table). Table shows characteristics and results per country. Among patients with TD BT, 90/118 (76.3%) were receiving transfusions at least once every 4 weeks, with 5.1% receiving transfusions weekly, 16.1% every 2 weeks, 28.8% every 3 weeks, 26.3% every 4 weeks, 3.4% every 5 weeks, 17.8% every 6 weeks, and 2.5% other or unknown. Patients with TD BT had an average (SD) of 2.4 (0.6) units of blood transfused per session (range, 1.9 in Spain to 2.7 in UK), with an average (SD) Hb level before the transfusion of 6.9 (1.3) g/dL (range, 5.9 in Canada to 7.3 in Germany). Patients with TD BT had been on the transfusion regimen for an average of 72.6 months (range, 45.2 in Canada to 104.9 in UK) during the observation period. All patients with NTD BT had previously had ≥1 transfusion at any point in time that the physician was aware of, with an average (SD) of 2.9 (0.9) transfusions per patient (range, 2.6 in Canada to 3.1 in Germany). Among patients with NTD BT with detailed transfusion data available post-index date (N=81; 84.4%), the mean (SD) number of units of blood per transfusion was 2.2 (0.6) (range, 1.9 in Spain to 2.7 in France), and the mean (SD) Hb level before transfusion was 7.4 (1.2) g/dL (range, 6.2 in Canada to 8.2 in Spain). Iron chelation therapy was received in 70.3% of TD patients (range, 44.4% in the UK and 92.6% in Germany) and in 45.8% of NTD patients (range, 37.0% in France to 80.0% in Canada). The percentages of patients with hepatitis B, C, and D were low (ie, all <2%) for both the TD and NTD cohorts, as were the percentages of patients with cerebrovascular disease (2.5% for TD and 1.0% for NTD) and congestive heart failure (5.1% for TD and 2.1% for NTD).

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Summary/Conclusion: Real-world data on clinical practice and outcomes in patients with BT are lacking. This study found that NTD and TD patients with BT have similarly low pre-transfusion Hb levels. A high number of patients, especially TD patients, are not treated according to the current recommendations, highlighting the importance of national reference centers to improve outcomes and quality of life in these patients.

P1524: EFFECTS OF CORONAVIRUS DISEASE 2019 (COVID-19) VACCINATION ON HEMOGLOBIN LEVELS IN THALASSEMIA PATIENTS

R. Vanavanitkun 1,* , P. Watanaboonyongcharoen 1 , N. Uaprasert 2

1 Laboratory medicine; 2 Internal Medicine, Faculty of medicine, Chulalongkorn University, Bangkok, Thailand

Background: COVID-19 vaccines are effective in reducing the risks of hospitalization and death, especially for people with comorbidities and impaired immunity. Systemic reactions such as fever, headache and myalgia are frequently reported after COVID-19 vaccination, especially adenoviral vector vaccines followed by mRNA and inactivated vaccines. Thalassemia disease is the most common monogenic disease worldwide. Among patients with thalassemia diseases, high fever or systemic reactogenicity after vaccination may trigger hemolysis, resulting in a sudden decrease in hemoglobin levels.

Aims: To determine whether COVID-19 vaccination significantly reduces hemoglobin (Hb) levels in Thalassemia patients by comparing pre- and post-vaccinated Hb levels after individual vaccination.

Methods: A retrospective cohort study was performed using medical records of thalassemia disease patients, who were followed up at King Chulalongkorn Memorial Hospital (KCMH) from January 2021 to January 2022. Four Hb values; the most recent pre-vaccinated Hb levels (Hb1) and post-vaccinated Hb levels (Hb2) after the first COVID-19 vaccination and the most recent pre-vaccinated Hb levels (Hb3) and post-vaccinated Hb levels (Hb4) after the second COVID-19 vaccination of individual patients were retrieved. Comparison between pre- and post-vaccinated Hb levels of each vaccination was computed using paired t-test.

Results: Among 220 transfusion-dependent thalassemia patients who were regularly followed up at Hematology Department, KCMH, 199 had COVID-19 vaccination records. There were 184 fully vaccinated patients. The first dose vaccinations were being comprised of 115 viral vector vaccines (ChAdOx1 nCoV-19), 42 inactivated vaccines (BBIBP-CorV and CoronaVac)) and 32 mRNA vaccines (BNT162b2 and mRNA-1273). The proportions of the second vaccinations were almost similar. Only 9 patients first vaccinated with inactivated vaccines were administered viral vector vaccines in the second vaccination. Of 120 patients with available Hb data after the first vaccination, the mean difference of Hb levels after first vaccination was 0.03±0.9 g/dL ( p = 0.685). The median interval between obtained Hb1 and the first vaccination was 50.6 (IQR: 22-70) days, and between obtained Hb2 and the first vaccination was 28.5 (IQR: 14-38) days. Of 71 patients with available Hb data after the second vaccination, the mean difference of Hb levels after the second vaccination was 0.13±1.0 g/dL ( p = 0.292). The median interval between obtained Hb3 and the second vaccination was 27.6 (IQR: 14-34) days, and between obtained Hb4 and the second vaccination was 38.2 (IQR: 18-52) days. There were 14 patients (11.7%) whose Hb levels were decreased over 1 g/dL after the first vaccination, while there were 7 patients (9.9%) whose Hb levels were decreased over 1 g/dL after the second vaccine. Most of these patients were administered viral vector vaccines.

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Summary/Conclusion: COVID-19 vaccines generally do not significantly affect Hb levels in thalassemia disease patients. Approximately 10% of patients had a clinically significant reduction in Hb levels after vaccination, especially those administered viral vector vaccines probably due to prevalent systemic reactogenicity. Although the benefit of COVID-19 vaccine protection is still outweighed this risk, thalassemia patients should be educated about this possible reaction and to observe symptoms and signs of hemolysis after COVID-19 vaccination.

P1525: MANAGEMENT OF AUTOIMMUNE HAEMOLYTIC ANAEMIA DURING PREGNANCY OR POST-PARTUM: AN INTERNATIONAL MULTI-CENTRE EXPERIENCE.

M. Bortolotti 1,2,* , N. N. Fantini 3 , A. Glenthoj 4 , M. Michel 5 , M. Napolitano 6 , S. Raso 7 , F. Chen 8 , V. McDonald 8 , I. Murakhovskaya 9 , J. M. I. Vos 10 , A. Patriarca 11 , M. E. Mingot-Castellano 12 , D. Prati 3 , W. Barcellini 1 , B. Fattizzo 1,2

1 Haematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 2 Department of Oncology and Oncohaematology, University of Milan; 3 Department of Transfusion Medicine and Haematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 4 Department of Hematology, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark; 5 Centre de Référence Maladies Rares sur les Cytopénies Auto-Immunes de l’Adulte, Centre Hospitalier Universitaire Henri Mondor, Assistance Publique-Hôpitaux de Paris, Créteil Université Paris-Est Créteil, Paris, France; 6 Haematology Unit, Thrombosis and Haemostasis Reference Regional Center, University of Palermo; 7 Department of Hematology and Rare Diseases, Azienda Ospedaliera Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy; 8 Clinical Haematology, Barts Health National Health Service Trust, Queen Mary University, London, United Kingdom; 9 Department of Hematology and Oncology, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY, United States of America; 10 Department of Hematology, Amsterdam University Medical Center, University of Amsterdam; Sanquin, Amsterdam, Netherlands; 11 Department of Translational Medicine, Azienda Ospedaliera-Universitaria “Maggiore della Carità”, University of Eastern Piedmont, Novara, Italy; 12 Servicio de Hematología, Hospital Universitario Virgen del Rocio, Seville, Spain

Background: Autoimmune haemolytic anaemia (AIHA) during pregnancy is a rare finding, and few is known about maternal and foetal outcomes. AIHA may either develop or relapse during gestation and postpartum or be an issue in a patient on active therapy who becomes pregnant. AIHA management during pregnancy and lactation is not standardized and drug use is often limited by safety concerns.

Aims: We studied AIHA impact on pregnancy focusing on disease severity, treatment need and maternal/foetal outcome.

Methods: Through a multicentric retrospective cohort study, we identified 38 pregnancies occurred in 28 women from 1997 to 2021 in 10 European centres in Italy, Denmark, France, the Netherlands, USA, and Spain. All included patients had a previous AIHA history or developed/exacerbated AIHA during gestation or postpartum. AIHA was classified according to the direct antiglobulin test.

Results: We registered 18 warm AIHA (10 IgG; 8 IgG+C3d), 2 cold agglutinin disease, 3 mixed and 5 atypical forms (Table 1). Evans syndrome (i.e., association of AIHA and immune thrombocytopenia or neutropenia) was present in 4. Mean age at AIHA diagnosis was 27 (3-39) and at pregnancy 32 (21-41) years.

AIHA diagnosis predated pregnancy in 15 women and had required at least 1 therapy line in all of them, and ≥2 lines in 12 (rituximab, N=7; cytotoxic immunosuppressants, N=6; splenectomy, N=5). Among these 15 patients, 6 had a relapse during pregnancy, 3 during postpartum and 9 were on active treatment at the time of pregnancy (steroids, N=8; cyclosporine, N=1; azathioprine, N=1; the latter stopped after positive pregnancy test). A patient with a previous AIHA, relapsed as immune thrombocytopenic purpura during pregnancy. Further 8 patients had an AIHA onset during gestation and 2 postpartum. A patient had AIHA onset during the postpartum of the 1 st pregnancy and relapsed during the 2 nd one.

In the 20 women experiencing AIHA during pregnancy/postpartum, median Hb and LDH levels were 6,4 g/dL (3,1 – 8,7) and 588 UI/L (269-1631), respectively. Management consisted in blood transfusions (N=10) and prompt establishment of steroid therapy+/-IVIG (N=20), all with response (complete N=13, partial N=7). After delivery, rituximab was necessary in 4 patients and cyclosporine was added in one. Anti-thrombotic prophylaxis was given in 7 patients.

Overall, we registered 10 obstetric complications (10/38, 26%), including 4 early miscarriages, a premature rupture of membranes, a placental detachment, 2 preeclampsia, a postpartum infection and a biliary colic. Apart from the case of biliary colic and one of the two cases of preeclampsia, 8/10 complications occurred during active haemolysis and treatment for AIHA. Nine foetal adverse events (9/38, 24%) were reported: a transitory respiratory distress of the new-born in a mother with active AIHA, 3 cases of foetal growth restriction, a preterm birth, an infant reporting neurologic sequelae, a case of AIHA of the new-born requiring intravenous immunoglobulins, blood transfusions and plasma exchange, and 2 perinatal deaths. The latter both occurred in women on active AIHA therapy and were secondary to a massive placental detachment and a symptomatic SARS-CoV-2 infection.

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Summary/Conclusion: AIHA developing/reactivating during pregnancy or postpartum is rare (about 5%) but mainly severe requiring steroid therapy and transfusions. Importantly, severe maternal and foetal complications may occur in up to 26% of cases mostly associated with active disease, pinpointing the importance of maintaining a high level of awareness. Passive maternal autoantibodies transfer to the foetus seems a rare event.

P1526: AUTOIMMUNE HEMOLYTIC ANEMIAS: RELATIONSHIP BETWEEN SINGLE NUCLEOTIDE POLYMORPHISMS OF CYTOKINE GENES AND CLINICAL/HEMATOLOGICAL PARAMETERS

A. Zaninoni 1 , B. Fattizzo 1,* , J. Giannotta 1 , E. Fermo 1 , P. Bianchi 1 , N. Cecchi 1 , W. Barcellini 1

1 Hematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

Background: Autoimmune hemolytic anemia (AIHA) is highly heterogeneous and involves several pathogenic mechanisms including autoantibody production, complement activation, and cytokine dysregulation. Hb levels at baseline and the degree of bone marrow compensation are the main predictors of outcome. The contribution of high, intermediate, and low producer genetic profile cytokine genes may give further insights into the pathogenesis of the disease.

Aims: To evaluate the frequency of cytokine single nucleotide polymorphisms (SNPs) in warm (wAIHA) and cold agglutinin disease (CAD) and their relationship with clinical and hematological parameters, including the degree of anemia, treatment refractoriness and number of therapies lines.

Methods: We investigated SNPs of five cytokine genes [tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, interleukin (IL)-6 and IL-10] in 72 Italian warm AIHA patients, using the Cytokine Tray (One Lambda, Canoga Park, CA) according to the manufacturer’s recommendations. Allele and genotype frequency results were compared to those described in the Italian population by Uboldi de Capei et al. (2003) and Poli et al. (2002).

Results: One hundred and twenty AIHA patients have been evaluated: median age 63 years (range 17-90), classified as wAIHA (n=75) and CAD (n=45). Demographic and hematological characteristics of patients are described in Figure 1A. Regarding cytokine SNP frequencies (Figure 1B), we observed that the TNF-alpha-308 high producers genotypes (G/G and G/A) were significantly more frequent in AIHA patients (37% vs 16%, p=0.0003 or 37% vs 23%, p=0.01) and in wAIHA (43% vs 16%, p<0.0001 or 43% vs 23%, p=0.0014) compared to controls. No differences were observed in CAD patients versus controls. Likewise, no significant differences between AIHA (including wAIHA and CAD subtypes) and controls were noted regarding other cytokine SNPs. Concerning the clinical severity at presentation, the frequency of TGF-beta (82% vs 58%, p=0.01) and IL-10 (17% vs 5%, p=0.03) high producers was higher in patients with Hb ≤7.5 g/dL (median of all AIHA) versus those with less severe presentation. Similarly, the frequency of high/intermediate IFN-gamma producers was higher within the group with Hb levels ≤7.5 g/dL at onset (75% vs 58%, p=0.03). No characteristic patterns were observed regarding IL-6 and TNF-alpha. Finally, the patterns of TNF-alpha and TGF-beta production were differently distributed considering the number of therapy lines: the first both in all AIHA and in CAD patients (p=0.004 and p=0.01, respectively), the second only in CAD patients (p=0.03). No differences in genotype frequencies of cytokine SNPs were observed regarding the number of relapses.

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Summary/Conclusion: These preliminary data show that AIHA patients display a more pro-inflammatory cytokine signature at molecular level (TNF-alpha high producer genotype) as compared to the Italian control population. Moreover, cytokine SNPs were also associated with clinical severity at onset, and requirement of multiple treatments.

P1527: NEXT GENERATION SEQUENCING IN HEREDITARY SPHEROCYTOSIS DIAGNOSIS

A. García Feria 1,* , J. Marco Buades 1 , A. Zuñiga Cabrera 2 , A. López Gabaldon 1 , S. Broseta Tormos 1 , M. I. Villa Velazquez 1 , I. Fernandez Gayete 1 , S. Gasco Clar 1 , P. Rivera Garcia 1 , E. Frances Aracil 1 , E. Meseguer Martinez 1 , O. Cortes Ortega 1 , E. Donato Martinez 1 , M. Fernandez Zarzoso 1 , M. L. Juan Marco 1 , A. Tolosa Muñoz 1 , E. Gomez Beltran 1 , M. J. Cejalvo Andujar 1 , M. Panero Ruiz 1 , P. Ribas Garcia 1 , M. J. Fernandez Llavador 1 , M. J. Sayas Lloris 1

1 Hematología, Hospital Doctor Peset; 2 Unidad de Genética, Hospital La Fe, Valencia, Spain

Background: Hereditary Spherocytosis (HS) is the most frequent cause of hereditary hemolytic anemia, clinically very heterogeneous. Diagnosis is made by family history, clinical history and presence of spherocytes in peripheral blood smear, but sometimes a combination of additional techniques is used for an adequate diagnosis, such EMA test, osmotic fragility test by flow cytometry (OFT), and recently, next generation sequencing (NGS) is being applied, which consists in the genetic study of the coding region and flanking intronic regions of the genes included in the TROMB1 V2 NGS panel, by the of ultrasequencing techniques.

Aims: Show possible relationship between clinical behavior and NGS study in HS.

Methods: Data has been collected from a total of 23 patients with a previous diagnosis of HS, of hematimetry tests such hemoglobin (Hb), mean corpuscular volumen (MCV), spherified MCV (SMCV), mean corpuscular hemoglobine (MCH), hemolysis (bilirubin, LDH, haptoglobin, reticulocytes), iron metabolism, % spherocytes, erythrocyte osmotic fragility (EOF), EMA test, OFT, and all these data have been compared together with possible patterns of anemia or hemolysis with NGS results.

Ilumina (MiSeq) technology was used for sequencing. The Surelect custom gene panel with capture technology - Agilent Technologies- has been used. (genes panel is shown in table 1 ).

Results: Results showed that all 23 patients had a pathological result on the NGS study. Among the multiple genes that code for erytrocyte membrane proteins, significant disease-related mutations were observed in four genes: SPTB (spectrin, beta), ANK1 (ankyrin 1), SLC4A1 (band 3/solute carrier family 4, member 1) and SPTA1 (spectrin, alpha 1). 16 significant mutations were found, of which 7 were previously known and 9 variants were novel mutations. The highest number of mutations was observed in SLC4A (n=10), followed by SPTB (n=6), ANK1 (n=6) and double heterozygosis for SPTB/SPTA1 (n=1). According to population databases (1000 genomes; ExAc; gnom AD), 11 mutations were pathological, 3 probably pathological and 2 of uncertain clinical significance.

We observed that in the group of splenectomized patients (n=6) (26%)the most prevalent mutated gene was SLC4A1 (n=4), the group with slightly lower Hb concentration was ANK1 (Hb: 11.4 gr/dL and EOF 0.84%) and the pattern of hemolysis was higher in the SPTB gene group (reticulocytes 292.8 x103 mm3, total bilirubin 2.18 mg/dL, SMCV 63 fL and % spherocytes in peripheral blood: 9.6/field).

The results of the genetic study were compared against OFT and EMA, these tests could be performed in all patients. OFT were negative in 4 patients (17.3%), of which 50% were in SLC41 group and 50% in SPTB group, being one of the mutations known with uncertain clinical degree and novel mutations in the other 3 patients, with pathological significance in one patient and with probably pathological repercussion in the other two.

EMA tests were also negative in 4 patients (half of them also OFT negative, with one patient having a uncertainly significance in SLC4A gene and one patient in SPTB gene, a novel variant that was probably pathological). The other 2 patients had positive OFT, one patient with a known pathological variant in SLC4A and the other in ANK1 with an unknown mutation of uncertain significance.

Summary/Conclusion: These results point out that there could be a relationship between the clinical diagnosis and the genetic profile of HS, according to the studied literature, and that NGS study can complement conventional tests to reach a more precise diagnosis of HS.

P1528: GENETIC AND PHENOTYPIC CHARACTERIZATION OF HEREDITARY SPHEROCYTOSIS AND RELATED DISORDERS – DESCRIPTION OF 9 NEW GENETIC VARIANTS

F. Haeuser 1,* , H. Rossmann 1 , A. Adenaeuer 1 , B. Lämmle 2,3,4 , C. Paret 5 , K. J. Lackner 1 , J. Faber 5 , O. Beck 5

1 Institute of Clinical Chemistry and Laboratory Medicine; 2 Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany; 3 Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, Bern, Switzerland; 4 Haemostasis Research Unit, University College London, London, United Kingdom; 5 Department of Pediatric Hematology, Oncology & Hemostaseology, Center for Pediatric and Adolescent Medicine, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

Background: Erythrocyte membrane defects are a group of hereditary diseases with highly variable severity of symptoms and caused by a set of different variants of genes encoding for red cell membrane and skeleton proteins. Although the most common hereditary hemolytic disease in Caucasians, hereditary spherocytosis (HS) is underdiagnosed, because mild clinical course is common. While mostly variants of structural proteins of the erythrocyte membrane (i.e. ANK1 (ankyrin), SLC4A1 (band 3), SPTA1 (α-spectrin) or SPTB (β-spectrin)) can be causative for the phenotype, only the minority of patients have been genetically characterized so far.

Aims: We aimed to develop a simple strategy to confirm the suspected diagnosis of HS using an NGS (next generation sequencing)-based approach.

Methods: 12 patients with clinical and laboratory signs of HS and one patient with massive, unclear hemolysis and initially inconclusive laboratory results, were genetically and phenotypically characterized. In 12 patients, hemolytic spherocytic anemia was confirmed by blood smear, complete blood count, haptoglobin, bilirubin, osmotic resistance test, and flow cytometric eosin-5-maleimide binding test. Acquired hemolysis was excluded. In two patients, a membrane protein defect was suggested by SDS-PAGE. Patient 13 showed massive hemolytic anemia with reduced osmotic resistance, but without elevation of MCHC. No spherocytes but poikilocytes were observed. All patients were subjected to exome sequencing (NGS). Family members of the index patients were studied by Sanger sequencing.

Results: In all 13 patients, pathogenic variants fulfilling ACMG-criteria were found limited to only 4 genes: ANK1, SLC4A1, SPTA1, and SPTB. In the 12 HS patients, variants were heterozygous, consistent with the predominantly dominant inheritance. The predescribed SLC4A1 variant Band 3 Bicêtre I was detected twice, a nonsense variant in ANK1 and one in SPTB once. The new variants were detected once each. We detected two splice-site variants in ANK1 as well as one in SPTB, one missense variant in SLC4A1, two nonsense variants in ANK1 as well as one in SPTB, and one frameshift variant with a premature termination codon in SPTB. One of the splice defects located in ANK1 and the nonsense variant in SPTB were de novo variants. Only in patient 13, in SPTA1 the low expression allele α-LELY was detected, located in trans to a missense variant. Such a compound heterozygosity in SPTA1 is typical for pyropoikilocytosis and is consistent with the clinical presentation of the patient.

Medical history and standard laboratory tests (blood count including MCHC, blood smear, hemolysis parameters and osmotic resistance) are usually sufficient to establish the diagnosis of HS. More sophisticated methods such as SDS- and native PAGE or ectacytometry can elucidate unclear cases. However, in our cases, all relevant variants were located in just four genes, suggesting that a reduced panel of four to five genes (incl. EPB42 (band 4.2)) may be sufficient to characterize most of the patients.

Summary/Conclusion: The diagnosis of HS and hereditary pyropoikilocytosis was confirmed in 12 and one patient respectively by NGS. Panel sequencing of the five most common genes is nowadays a comparatively rapid and inexpensive method to confirm the diagnosis of HS and related disorders. Time-consuming or low-specificity tests can be avoided, and family members can be easily genotyped.

P1529: HEMOGLOBIN RESPONSE TO ZINC SUPPLEMENTATION IN PATIENTS WITH ZINC DEFICIENCY AND CHRONIC ANEMIA

Z. Hanson 1,* , S. Kaur 1 , S. Lee 2 , M. Akhtari 3

1 Graduate Medical Education; 2 Internal Medicine; 3 Hematology and Medical Oncology, Loma Linda Medical Center, Loma Linda, United States of America

Background: Zinc is a necessary micronutrient used as a catalyst, structural element, and regulatory ion in many metabolic processes including erythropoiesis. While zinc fortification has been studied in developing countries and has been associated with a decrease in the overall prevalence of anemia within the pediatric and maternal populations ( Ara et al., PLoS One . 2019;14(1); Saaka, Ghana Med J . 2012;46(4):225-233), there have been no studies in adults with chronic anemic in the developed world. This descriptive, retrospective, study describes zinc deficient patients with chronic anemia, who underwent zinc supplementation, and their changes in hemoglobin (HGB) over a 6-month period, in an academic outpatient hematology center in California, United States of America.

Aims: Primary endpoints: determine if zinc supplementation in zinc deficient patients with chronic anemia will improve HGB by at least 0.5 g/dL by 1, 3, and 6 month time points. Secondary endpoints: subgroup analysis for HGB improvement by 1, 3, and 6 month time points between all patients versus those with rheumatologic disorders, active malignancy, and patients with chronic kidney disease (CKD) including end stage renal disease (ESRD).

Methods: Twenty-Nine patients with chronic anemia (HGB <12 for women, HGB <13 for men for >3 months) over the age of 18 were identified to have zinc deficiency (plasma zinc concentration <65 ug/dL). Patients did not have elevated inflammatory markers (C-reactive protein (CRP) >5 mg/L, and erythrocyte sedimentation rate (ESR) >22 mm/hr for males, >29 mm/hr for females) at time of plasma zinc collection nor started new treatment for malignancy or rheumatologic disorder 3 months preceding, and within the data collection period.

A retrospective data collection was completed for prior medical history, HGB, hematocrit (HCT), platelets (PLTS), mean corpuscular volume (MCV), plasma zinc concentration, CRP, ESR, and glomerular filtration rate (GFR). HGB was collected at the time markers of 1, 3, and 6 months following initiation of zinc supplementation. Collected data was analyzed with ANOVA and CHI-squared for response in all patients. Subgroup analysis was then completed if patients had been diagnosed with rheumatologic disorders (systemic lupus erythematosus and Sjogren disorder), active malignancy, or CKD including ESRD.

Results: Prior to supplementation, average hemoglobin among patients was 9.95 g/dL, WBC was 5.0 g/dL, MCV was 92.3 fl, PLTS were 220 g/dL. Following 1 month of supplementation average hemoglobin was 10.9 g/dL (p = 0.029, n = 23), at 3 months average hemoglobin was 11.0 g/dL (p = 0.016, n = 24), and at 6 months average hemoglobin was 11.5 g/dL (p = 0.001, n = 20).

Primary endpoint of HGB improvement by at least 0.5 g/dL was found in 61% of patients at 1 month, 66% of patients at 3 months, and 80% of patients at 6 months. Hemoglobin improved by at least 1.0 g/dL in 48% of patients at 1 month, 42% of patients at 3 months, and 65% of patients at 6 months. Resolution of anemia was reached in 36% of patients within 6 months.

The secondary endpoint with subgroup analysis for patients with rheumatologic disorders, active malignancy, or CKD including ESRD versus the group as a whole showed no significant differences in response.

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Summary/Conclusion: Zinc supplementation significantly improved hemoglobin levels in patients with zinc deficiency and chronic anemia, independent of their underlying comorbidities, with resolution of anemia in 36% of patients. A large prospective study is needed to evaluate and correlate zinc supplementation and hemoglobin improvement in patients with chronic anemia.

P1530: PERITRANFUSIONAL C1-INHIBITOR IN PATIENTS WITH SEVERE COMPLEMENT-MEDIATED AUTOIMMUNE HEMOLYTIC ANEMIA: AN OPEN LABEL PHASE 2 TRIAL.

E. C. W. de Boer 1,2 , M. Jalink 3,4,5,* , L. Delvasto-Nuñes 1 , E. M. Meulenbroek 6 , I. Baas 1 , S. R. Janssen 4 , E. E. Dijkman 4 , K. A. Gelderman 7 , D. Wouters 8 , M. D. Engel 4 , M. de Haas 3,9,10 , M.-J. Kersten 11 , I. Jongerius 1,2 , S. Zeerleder 12 , J. M. Vos 9,11

1 Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Centre, Amsterdam Infection and Immunity Institute; 2 Department of Pediatric Immunology, Rheumatology, and Infectious Diseases, Emma Children’s Hospital, Amsterdam University Medical Center; 3 Center for Clinical Transfusion Research, Sanquin Research; 4 Department of Hematology, Amsterdam University Medical Center; 5 Department of Transfusion Medicine, Sanquin Blood Supply; 6 Janssen Vaccines & Prevention B.V., former employee of Sanquin; 7 Sanquin Diagnostic Services, Amsterdam; 8 Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven; 9 Department of Immunohematology Diagnostics, Sanquin, Amsterdam; 10 Department of Hematology, Leiden University Medical Center, Leiden; 11 Department of Hematology, Amsterdam University Medical Center, University of Amsterrdam, Cancer Center Amsterdam and LYMMCARE, Amsterdam, Netherlands; 12 Abteilung für Hämatologie, Luzerner Kantonsspital, Luzern und Universität Bern, Bern, Switzerland

Background: Complement-mediated autoimmune hemolytic anemia (AIHA) is characterized by destruction of red blood cells (RBCs) due to autoantibodies that initiate complement activation via the classical pathway. This may result in fulminant intra- and extravascular hemolysis with need for RBC transfusions, of which the efficacy can be compromised by the antibodies reacting with donor RBCs. Hemolysis induces a pro-inflammatory response that can contribute to hypercoagulability, as seen in patients with other complement-mediated hemolytic disorders such as paroxysmal nocturnal hemoglobinuria.

C1-inhibitor (C1-INH; Cinryze) is an endogenous plasma regulator of the classical and lectin complement pathway approved for patients with hereditary angioedema. In a single case report and in vitro , we observed that C1-INH administration attenuated complement deposition on RBCs and subsequent hemolysis. Therefore, we hypothesized that peritransfusional administration of C1-INH in complement AIHA would reduce complement activation and hemolysis and thus improve transfusion outcome.

Aims: To assess the efficacy and safety of intravenous C1-INH in patients with transfusion-dependent complement-mediated AIHA on parameters for hemolytic activity, complement activation and inflammation.

Methods: We conducted a prospective, single center, phase 2 open label trial. Patients with AIHA and confirmed C3c/d-positive direct antiglobulin test, baseline hemoglobin (Hb) of ≤8 g/dL and an indication for ≥ two RBC transfusion units were included after informed consent. Treatment consisted of four peritransfusional doses of C1-INH intravenously (6000, 3000, 2000 and 1000 U) with 12 hour (hr) intervals. Blood samples at 0, 12, 24, 36, 48, 72 hrs, 7 and 14 days were analyzed for hemolytic parameters, RBC opsonization and markers of complement activation and systemic inflammation (nucleosomes and human neutrophil elastase-α1-antitrypsin complexes (HNE-α1-ATc)). Safety was monitored and scored (CTCAE version 4.0).

Results: Ten patients were included in the study. At baseline, we found increased complement C3d, IgG and IgM deposition on RBCs compared to healthy controls (p=0.002, 0.005 and 0.002). Circulating C3b/c levels, nucleosomes and HNE-α1-ATc were increased compared to healthy controls (p<0.001, p=0.046 and p=0.001). C1-INH administration resulted in an increase of C1-INH antigen and -activity, peaking at 48 hrs (Fig. A). At 48 hrs after first administration, treatment significantly reduced C3d deposition on RBCs (-21%, p=0.016), but not systemic complement activation as shown by C3b/c levels (-14.96 nM, p=0.874; Fig. B). With declining C1-INH levels, C3 deposition increased again. Shortly after transfusion, Hb increased (1.59 g/dL at 12 h; p=0.008), but at 48 hrs, hemolytic and inflammation parameters did not improve (LDH -169.5 U/L, p=0.572; bilirubin total -4.306 µmol/L, p=0.987; Hb -1.217 g/dL; p=0.056; nucleosomes -17%, p=0.938; HNE-α1-ATc +21%;, p=0.297; Fig. C, D). No serious adverse events were reported.

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Summary/Conclusion: Peritransfusional C1-INH in AIHA temporarily reduced local complement activation as evidenced by C3d deposition on RBCs, but did not affect hemolysis or systemic complement activation. Increased Hb levels at later timepoints were observed, but this effect might be confounded by additional treatments and transfusions. In conclusion, we found no (clinically) significant effect of C1-INH on hemolysis and systemic complement activation. This may be explained by the dosing of C1-INH, which possibly was too low to counter the severe complement-mediated AIHA and additional transfusion effects.

P1531: HETEROGENEITY OF DISEASE-CAUSING GENOTYPES BETWEEN FAMILIES WITH HEREDITARY SPHEROCYTOSIS: A SINGLE-CENTER COHORT STUDY

R. Jimenez Heredia 1,2,3,* , L. Kager 1,4 , P. Zeitlhofer 5 , W. Novak 4 , S. Eder 1,4 , A. Segarra Roca 1,2 , K. Nebral 5 , M. Haimel 1,2,6 , R. Geyeregger 1 , O. Haas 1,3,4 , K. Boztug 1,2,3,4,6

1 St. Anna Children’s Cancer Research Institute (CCRI); 2 Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases; 3 Department of Pediatrics and Adolescent Medicine; 4 St. Anna Children’s Hospital, Department of Pediatrics and Adolescent Medicine, Medical University of Vienna; 5 Labdia, CCRI; 6 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

Background: Hereditary spherocytosis (HS) is a phenotypically and genetically heterogeneous disease, in which erythrocytes are unable to maintain their normal biconcave shape due to genetic mutations in membrane/cytoskeletal proteins that play a role in structural morphologic stability. Recent progress in genomic analyses technologies has helped to shed light on the complexity of genotype/phenotype correlations within this orphan hemolytic red blood cell disorder.

Aims: In this retrospective investigation, we aim to investigate the genotype-phenotype correlation based on the clinical severity of the disease and the gene-wise predicted pathogenicity comparison. Furthermore, we want to highlight the importance of next generation sequencing (NGS) technologies in the diagnosis of HS.

Methods: We provide the results of a single-center study in Austria on a total of 69 patients (core group, N=35; affected family members, N=34) with HS, focusing on NGS results (both exome and panel sequencing), ancestry, laboratory findings, clinics, as well as genotype-phenotype correlations.

Results: A pathogenic (N=59/69; core group, N=29/35) or likely pathogenic variant (N=6/69; core group N=3/35) was identified in 94% (core group 91%) of the patients, including 18 novel variants. Variants in ANK1 (46%; European ancestry 37%, non-European 9%) were most encountered, followed by SPTB (31%, all European) and SLC4A1 (14%; European 11%, non-European 3%). In three patients (9%, all European) we were unable to identify the genetic cause unequivocally. Except for a heterozygous intronic ANK1 c.1504-9G>A variant, which was identified in two unrelated European families, there was no overlap on causative variants. The majority of mutations (N=25; 81%) were of high impact and include frameshifts, N=14 (45%); nonsense, N=8 (26%); CNVs, N=2 (6%) and splice-site variants, N=1 (3%). Sufficient data for phenotype-genotype correlation testing were available for 56 patients (core group, N=35), and phenotypes were milder in the patients with SLC4A1 variants. Patients with ANK1 and SPTB variants had comparable phenotypes.

Summary/Conclusion: To our knowledge, this is the first study describing genomics (N=69) and genotype-phenotype correlation (N=56) in patients with HS in Central-Europe. We were able to identify causative variants in 94% of the 69 patients, including atypical disease-causing genetic findings such as intronic and CNV mutations. This evidenced the compelling need of a combination of standardized NGS analysis and clinical deep investigation in order to accurately characterize and treat HS patients.

P1532: SUTIMLIMAB AFTER PRIOR RITUXIMAB USE IN PATIENTS WITH COLD AGGLUTININ DISEASE (CAD): POOLED POST-HOC ANALYSES FROM THE CARDINAL AND CADENZA TRIALS

A. Röth 1 , A. Karaouni 2 , J. Msihid 3 , I. Hemim 3 , F. Joly 3 , F. Shafer 4 , T. Sourdille 5 , Q. Hill 6,*

1 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, Essen, Germany; 2 BMAPS SARL, Geneva, Switzerland; 3 Sanofi, Chilly-Mazarin, France; 4 Sanofi, Bridgewater; 5 Sanofi, New York, United States of America; 6 Department of Clinical Hematology, Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom

Background: Cold agglutinin disease (CAD) is a rare hemolytic anemia. Sutimlimab, a humanized monoclonal anti-C1s antibody, received approval in the United States following results of the Phase 3 CARDINAL ( NCT03347396 ) trial.

Aims: This post-hoc analysis aimed to describe the characteristics of patients with CAD who received prior off-label rituximab treatment versus rituximab-naïve patients and present the efficacy of sutimlimab in these subgroups, using pooled data from the Phase 3 CARDINAL and CADENZA ( NCT03347422 ) trials.

Methods: CARDINAL was an open-label, single-arm, multicenter study for patients with a recent blood transfusion. CADENZA was a randomized, double-blind, placebo-controlled, multicenter study in patients without a recent blood transfusion. Included patients had a hemoglobin (Hb) level of ≤10 g/dL and were excluded if they received rituximab monotherapy <3 months, or combination therapy <6 months, prior to enrollment. For this analysis, composite efficacy endpoint was response rate; responders received no blood transfusions or prohibited medications from Week 5 to 26, and had an improvement in Hb from baseline of ≥2.0 g/dL (level chosen for post-hoc analysis) or a Hb level of ≥12 g/dL, at an average of Weeks 23, 25, and 26 (treatment assessment timepoint [TAT]). Secondary endpoints included mean change from baseline to TAT in Hb, bilirubin, lactate dehydrogenase (LDH), and FACIT-Fatigue score. Endpoints were analyzed descriptively according to prior rituximab use (previous monotherapy and/or combination therapy).

Results: Overall, 46 patients receiving sutimlimab were included in this analysis (CARDINAL, n=24; CADENZA, n=22) and mean (SD) time between last rituximab intake and sutimlimab treatment start date was 26.4 (24.0) months. At baseline, 54% (n=25) of patients had prior rituximab use; mean number of rituximab courses received prior to trial enrollment was 1.92. Mean (SD) age was 67.8 (9.7) for patients with prior rituximab use and 69.2 (10.4) years for rituximab-naïve patients. Baseline clinical and laboratory characteristics were similar between subgroups, including Hb, bilirubin, LDH, and FACIT-Fatigue score; however, in CADENZA, immunoglobulin M level and cold hemagglutinin titer were greater in the rituximab-naïve subgroup versus the prior rituximab group (Table).

Accounting for differences in blood transfusion inclusion criteria between CARDINAL and CADENZA, in both trials at baseline there were a greater number of blood transfusions in patients with versus without prior rituximab use (Table). CAD disease duration was longer and corticosteroid use was greater in patients with prior rituximab versus those without. In CARDINAL, CAD-related hospitalizations were greater in prior rituximab users (84.6%) versus those with no prior use (45.5%).

The response rate was 60% in patients with prior rituximab use (n=15/25) and 67% in rituximab-naïve patients (n=14/21). Mean (SD) change from baseline to TAT in Hb level was 2.6 (1.8) and 2.7 (1.8) g/dL for patients with versus without prior rituximab use, respectively. Corresponding values for bilirubin were -28.5 (18.9) versus -25.6 (11.9) µmol/L, and for LDH were -183.8 (267.8) versus -58.9 (178.2) U/L. Mean (SD) change in FACIT-Fatigue score was similar in both subgroups (9.9 [13.0] and 9.8 [11.4], respectively).

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Summary/Conclusion: In this post-hoc analysis, response rates were similar between subgroups of patients with prior off-label rituximab treatment versus rituximab-naïve patients. These results suggest that sutimlimab is effective in patients with CAD, irrespective of prior off-label rituximab use.

P1533: TRIAL IN PROGRESS: A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED PHASE 3 STUDY OF THE EFFICACY AND SAFETY OF PARSACLISIB IN PATIENTS WITH PRIMARY WARM AUTOIMMUNE HEMOLYTIC ANEMIA

W. Barcellini 1,* , K. Butler 2 , S. Moran 2 , K. Bibeau 2 , I. Dzhindzhikhashvili 2 , S. Wei 2 , U. Jäger 3

1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2 Incyte Corporation, Wilmington, DE, United States of America; 3 Medical University of Vienna, Vienna, Austria

Background: Autoimmune hemolytic anemia (AIHA) is a rare acquired disorder characterized by autoantibodies targeted against erythrocyte membrane antigens, leading to accelerated red blood cell destruction. The estimated incidence of AIHA is 1.3–2.9 per 100,000/year in North America and Western Europe, of which warm AIHA (wAIHA) accounts for 60%–70% of cases. In wAIHA, autoimmunity is typically immunoglobulin G mediated with a warm thermal reactivity range (~37°C). Approximately 50% of wAIHA cases are characterized as primary disease, in which hemolysis occurs without other coexisting disorders. There are few available treatment options for wAIHA beyond corticosteroids and rituximab. Parsaclisib is a potent inhibitor of phosphoinositide 3-kinase (PI3K)-δ that has demonstrated efficacy and safety in a phase 2 study of patients with primary AIHA.

Aims: To describe the study design of an ongoing double-blind, randomized, multicenter, placebo-controlled phase 3 trial evaluating the safety and efficacy of parsaclisib in patients with primary wAIHA (PATHWAY; NCT05073458 ).

Methods: Approximately 100 patients will enroll in the study and provide written informed consent before initiation of any study-related procedures. Eligible patients are ≥18 years old and must have a wAIHA diagnosis based on the presence of hemolytic anemia and serologic evidence of anti-erythrocyte antibodies; be inadequately controlled with, intolerant to, or have a contraindication to other therapies; have a hemoglobin (Hgb) level ≥7–<10 g/dL with symptoms of anemia; and have a Functional Assessment of Chronic Illness Therapy–Fatigue (FACIT-F) score ≤43 at screening. Exclusion criteria include other AIHA subtypes, suspected secondary AIHA, splenectomy <3 months before randomization, and prior treatment with a PI3Kδ or pan-PI3K inhibitor for any indication. Patients will be screened for up to 32 days and randomized in a 1:1 ratio to parsaclisib 2.5 mg once daily (QD) or placebo QD for 24 weeks ( Figure ). Concomitant treatment with low-dose corticosteroids (equivalent to ≤20 mg/d of prednisone) is permitted during the study. Patients who experience worsening wAIHA (defined as a ≥1-g/dL decrease in Hgb from prior assessment or development of new or worsening symptoms) can use rescue therapy (new or increased dose of corticosteroids, transfusions, intravenous immunoglobulin, or epoetin alfa); those who continue on rescue therapy after Week 6 will be considered nonresponders in the primary efficacy analysis. Dose reductions to 1 mg are allowed for adverse events or a rapid rise in Hgb. Patients who complete the 24-week treatment period will enter an open-label extension period and receive parsaclisib 2.5 mg QD for an additional 24 weeks; patients will be monitored for 12 weeks after the last study dose with visits every 4 weeks.

The primary endpoint is the proportion of patients attaining a durable Hgb response, defined as Hgb ≥10 g/dL with a ≥2-g/dL increase from baseline not attributed to rescue therapy at ≥3 of 4 available visits at Week 12 or later during the 24-week double-blind treatment period. The key secondary endpoint is the proportion of patients with a ≥3-point increase from baseline in FACIT-F score at Week 24. Safety will be assessed by monitoring adverse events from informed consent until ≥12 weeks after the last dose of study drug.

Results: The study is currently open and recruiting.

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Summary/Conclusion: This phase 3 study will provide valuable insight into the safety and efficacy of parsaclisib as a potential treatment for patients with primary wAIHA.

P1534: SAFETY AND TOLERABILITY OF NIPOCALIMAB ADMINISTERED AT DIFFERENT RATES OF INTRAVENOUS INFUSION IN HEALTHY ADULTS: A PHASE 1 PLACEBO-CONTROLLED SINGLE-DOSE STUDY

J. Leu 1,* , A. Vermeulen 2 , L. Ling 3

1 Janssen Research & Development, LLC, Spring House, PA, United States of America; 2 Janssen Research & Development, a division of Janssen Pharmaceutica NV, Beerse, Belgium; 3 Janssen Research & Development, LLC, Cambridge, MA, United States of America

Background: Warm autoimmune hemolytic anemia (wAIHA) is a rare, life-threatening disorder characterized by the destruction of red blood cells by pathogenic IgG autoantibodies. There is currently no approved treatment for wAIHA; patients with severely symptomatic anemia are typically managed with corticosteroids and rituximab, both of which are associated with potential relapse and adverse events. Nipocalimab is a fully human, aglycosylated, effectorless IgG1 monoclonal antibody that targets the IgG binding site on the neonatal Fc receptor (FcRn) with high affinity, which is in clinical development for the treatment of wAIHA. In a prior phase 1 study in healthy adults receiving single and multiple ascending doses infused intravenously (IV) over 2 hours, nipocalimab was well-tolerated with an adverse event profile comparable to placebo. However, shorter durations of nipocalimab infusion may be more convenient for patients.

Aims: To assess the safety and tolerability of single doses of nipocalimab administered at different IV infusion rates in healthy adults to support the potential use of shortened infusions in future studies.

Methods: The trial was a single dose, sequential, randomized, double-blind, placebo-controlled, escalating dose and infusion rate study. Eligible participants were males or females aged 18-55 years with no clinically significant medical or physical conditions. Participants were randomized to 1 of 5 cohorts (n=8 per cohort [6 nipocalimab, 2 placebo]) to receive nipocalimab 30 mg/kg IV infused over 60, 30, 15 or 7.5 min (0.5, 1, 2, or 4 mg/kg/min), nipocalimab 60 mg/kg IV infused over 15 min (4 mg/kg/min) or matching placebo. Escalation to the next dose level was permitted following review by a safety monitoring committee. Safety was assessed by the frequency and nature of treatment-emergent adverse events (TEAEs), as well as abnormalities in laboratory assessments and vital signs. Pharmacokinetics of nipocalimab were also assessed.

Results: A total of 40 participants (70% female, 93% white) received study drug and were included in the safety analysis; 39 completed the study. A total of 12 participants (40%) experienced TEAEs across all nipocalimab dosing cohorts and 1 (10%) participant experienced TEAEs following administration of placebo. The most frequently reported TEAE was headache, reported by 6 participants with nipocalimab and 1 participant with placebo. Nausea was reported by 3 participants receiving nipocalimab. Grade 2 injection site reactions were reported by 2 participants but were considered unrelated to the investigational agent. None of the TEAEs were severe, and no participants discontinued treatment due to TEAEs; there were no serious adverse events or deaths. Only 4 participants experienced AEs that occurred within 24 hours after the infusion that were considered related to the study drug. Lower rates of nipocalimab infusion were associated with fewer TEAEs, while participants receiving 4 mg/kg/min (as either 30 mg/kg infused over 7.5 min or 60 mg/kg infused over 15 min) reported more TEAEs (Table).

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Summary/Conclusion: Single doses of nipocalimab, when administered at doses up to 60 mg/kg and infusion rates up to 4 mg/kg/min were safe and well-tolerated in healthy adults. The frequency of reported TEAEs was lower in participants receiving IV nipocalimab at rates of 1 or 2 mg/kg/min, providing a target infusion rate for current and future studies.

P1535: SAFETY AND TOLERABILITY OF SINGLE ESCALATING DOSES OF INTRAVENOUS NIPOCALIMAB IN HEALTHY JAPANESE ADULTS: A RANDOMIZED, PHASE 1 PLACEBO-CONTROLLED STUDY

J. Leu 1,* , A. Vermeulen 2 , L. Ling 3

1 Janssen Research and Development, LLC, Spring House, PA, United States of America; 2 Janssen Research & Development, a division of Janssen Pharmaceutica NV, Beerse, Belgium; 3 Janssen Research & Development, LLC, Cambridge, MA, United States of America

Background: Autoimmune hemolytic anemia (AIHA) is a rare, life-threatening disorder characterized by the destruction of red blood cells (RBCs) by autoantibodies. In Japan, the estimated prevalence of AIHA is 3-10 cases per 1 million person-years. Of these, approximately 90% of patients present with warm AIHA (wAIHA), which occurs when pathogenic IgG antibodies bind to RBCs at a temperature of ~37 °C. There is currently no approved treatment for wAIHA, and patients with severe symptomatic anemia are generally managed with corticosteroids, immunosuppressants, rescue therapies, and immune cell depleting biologic therapies. Nipocalimab is a fully human, effectorless IgG1 monoclonal antibody in clinical development for wAIHA. Nipocalimab targets the IgG binding site on the neonatal Fc receptor (FcRn) to reduce circulating IgG including anti-RBC autoantibodies. In a prior phase 1 study in healthy adults receiving single and multiple ascending doses administered by intravenous (IV) infusion, nipocalimab was well-tolerated with an adverse event profile comparable to placebo. However, the proportion of Asian participants in that study was small, and therefore the safety of nipocalimab required further investigation in this population.

Aims: To assess the safety and tolerability of single escalating doses of nipocalimab administered as IV infusions in healthy Japanese adults.

Methods: The trial was a randomized, double-blind, placebo-controlled, escalating single dose study. Eligible participants were healthy Japanese males or females aged 18-64 years with no clinically significant medical or physical conditions. Dosing cohorts were: nipocalimab 10 mg/kg, 30 mg/kg, and 60 mg/kg or matching placebo, infused IV over 30 min. Escalation to the next dose level only proceeded following review of safety and tolerability by the principal investigator. Safety was assessed by the frequency of treatment-emergent adverse events (TEAEs), as well as abnormalities in clinical laboratory assessments, physical examinations, or vital signs.

Results: A total of 24 Japanese participants (92% female) were randomized, received study drug, and completed the study. A total of 3 participants (12.5%) experienced 1 or more TEAE: 2 in the 30 mg/kg nipocalimab cohort and 1 in the 60 mg/kg nipocalimab cohort (Table). Mild (grade 1) dizziness was the most common TEAE, which was reported by 2 participants, 1 each in the 30 mg/kg and 60 mg/kg nipocalimab cohorts. Mild influenza-like illness was reported by 1 participant who received 30 mg/kg nipocalimab and mild diarrhea was reported by 1 participant who received 60 mg/kg nipocalimab; these TEAEs were considered by the investigator as unrelated to study drug. One participant in the 30 mg/kg nipocalimab cohort discontinued study drug infusion after 22 minutes due to a moderate infusion-related reaction, which was considered to be related to study treatment. This AE was managed with an antihistamine, and the participant received only 22 mg of nipocalimab. There were no clinically significant changes in laboratory parameters or vital signs, no injection site reactions, serious adverse events, or deaths.

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Summary/Conclusion: Single doses of nipocalimab, administered at doses between 10 mg/kg and 60 mg/kg by IV infusion, were generally safe and well-tolerated in healthy Japanese adults, consistent with the prior phase 1 study in a general population, which included Asian and non-Asian participants.

P1536: ERYTHROCYTES’ OXIDATIVE STRESS AND ANTIOXIDANT RESPONSE IN PATIENTS WITH HEREDITARY SPHEROCYTOSIS AND Β-THALASSEMIA MINOR – A SMALL NUMBER CASE STUDY

D. Melo 1,2,* , F. Ferreira 3 , M. J. Teles 4,5,6,7 , G. Porto 5,7,8,9 , S. Coimbra 1,2,10 , S. Rocha 1,2 , A. Santos-Silva 1,2

1 UCIBIO, REQUIMTE, Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto; 2 Associate Laboratory i4HB - Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto; 3 Hematology Service, Centro Hospitalar e Universitário de São João, Porto; 4 Clinical Pathology, Centro Hospitalar e Universitário de São João, Porto; 5 Clinical Hematology, Santo António Hospital – Centro Hospitalar do Porto; 6 Anemia Working Group Portugal; 7 Institute for the Health Research and Innovation (i3S), University of Porto; 8 Center for Predictive and Preventive Genetics (CGPP), University of Porto; 9 Abel Salazar Institute of Biomedical Sciences, University of Porto, Porto; 10 TOXRUN – Toxicology Research Unit, University Institute of Health Sciences, CESPU, CRL, Gandra, Portugal

Background: Hereditary spherocytosis (HS) and β-thalassemia (β-tal) are both associated with oxidative stress, despite their different etiologies. The pathologic red blood cells (RBC) are metabolically stressed to maintain cell integrity, accumulating oxidative stress-induced damage within the cells. To counteract the oxidative modifications, the RBC are equipped with an efficient antioxidant system, which includes several enzymes, such as catalase (CAT) and some non-enzymatic antioxidants, namely ascorbic acid (AA). In a previous work (DOI: 10.1016/j.freeradbiomed.2020.10.084) we hypothesized that AA could be an important player in the RBC antioxidant defense in patients with non-immune hemolytic anemias (NIHAs) and that its mobilization from plasma to the RBC’s cytosol could be an adaptive mechanism to face erythrocyte’s oxidative stress, in these patients.

Aims: Our aim was to test that hypothesis, by studying the redox status and antioxidant system of RBC from non-splenectomized patients with HS and β-tal minor separately.

Methods: In 13 healthy individuals (control group), 14 non-splenectomized patients with HS and 11 patients with β-tal minor, we evaluated the total antioxidant status (TAS), AA levels and CAT activity in both plasma and RBC’s cytosol by spectroscopy methods.

Results: When compared with the control group, both HS and β-tal patients presented statistically significant increased AA levels and lower TAS values in RBC. CAT activity showed similar values in RBC from HS, β-tal and control group. No differences were found for TAS and AA levels in plasma among all groups. We observed significant negative correlations between RBC AA with plasma AA in both HS (r= -0.741, p =0.002) and β-tal ( r = -0.742, p = 0.009), not observed in the control group. Additionally, RBC TAS was positively correlated with RBC AA in HS ( r =0.534, p =0.049) and RBC TAS was positively correlated with RBC CAT activity in β-tal ( r = 0.743, p =0.009).

Summary/Conclusion: The finding of a significant increase in RBC AA levels and the significant lower RBC TAS values alongside with a significant negative correlation between RBC AA and plasma AA levels in both HS and β-tal, support the hypothesis that AA may play an important role in the erythrocyte’s adaptive response to oxidative stress and, thereby, in its antioxidant defense system. We speculate that the mobilization of AA from plasma to the RBC is an adaptive mechanism to oxidative stress that occurs in both HS and β-tal. Moreover, the RBC TAS appears to result from different antioxidant responses, as shown by the significant positive correlations found between RBC TAS and RBC AA levels or RBC TAS and RBC Cat activity for HS and β-tal, respectively. These findings might be related to the distinct etiology of the studied anemias. Further studies are needed to better explore about the role of other players in the antioxidant response, not only in HS and β-tal, but also in other oxidative stress associated congenital anemias.

Acknowledgments: Financial support from FCT/MCTES through national funds to Applied Molecular Biosciences Unit (UCIBIO), UIDP/04378/2020 and UIDB/04378/2020 and the project LA/P/0140/2020 of Associate Laboratory Institute for Health and Bioeconomy (i4HB). and Daniela Melo’s PhD Grant (SFRH/BD/139622/2018).

P1537: HEREDITARY ANEMIA CAUSED BY MULTILOCUS INHERITANCE OF PIEZO1, SLC4A1 AND ABCB6 MUTATIONS: A DIAGNOSTIC AND THERAPEUTIC CHALLENGE.

B. E. Rosato 1,2,* , S. L. Alper 3 , G. Tomaiuolo 2,4 , R. Russo 1,2 , A. Iolascon 1,2 , I. Andolfo 1,2

1 Dip. di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli - Federico II; 2 CEINGE - Biotecnologie Avanzate, Napoli, Italy; 3 Dept. of Medicine, Harvard Medical School, Boston, Massachusetts, USA, Renal Division and Molecular and Vascular Medicine Division, Beth Israel Deaconess Medical Center, Boston, United States of America; 4 Dip. di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università degli Studi di Napoli - Federico II, Napoli, Italy

Background: Red blood cell (RBC) disorders encompass a vast group of hereditary hemolytic anemias (HHA) that differ in clinical, morphologic, and molecular features. Subtypes include RBC disorders caused by altered (i) membrane structural organization and (ii) transport function. The most common form of the first group is Hereditary Spherocytosis (HS) presenting anemia, reticulocytosis and splenomegaly due to mutations in SLC4A1, ANK1 , SPTA1 , SPTB , and EPB42 genes. The second group is represented by Dehydrated Hereditary Stomatocytosis (DHS) characterized by dehydration of RBC and iron overload, due to mutation in PIEZO1 gene (90% of cases). DHS may present as syndromic form with familial pseudohyperkalemia (FP), a red cell trait characterized by loss of K + at low temperatures. FP can be isolated and due to mutations in ABCB6 gene. The heterogeneity of these disorders often hampers correct clinical diagnosis. Genetic testing is now a suitable approach for differential diagnosis and identification of polygenic conditions.

Aims: We here investigate a patient with HHA using genetic and functional tests to demonstrate the complexity of multilocus inheritance and the need for precise genetic diagnostics.

Methods: Targeted Next Generation Sequencing (t-NGS), Western Blot (WB), ektacytometry, ion flux assay.

Results: The 24 years old proband, presented moderate anemia, jaundice, and splenomegaly. Blood smear revealed the presence of stomatocytes, target cells, and rare ovalocytes. Pink and acidified glycerol lysis tests were positive, leading to an initial clinical diagnosis of HS. The patient was treated by total splenectomy but, after surgery, she experienced portal vein thrombosis and anemia worsened. Furthermore, she developed severe iron overload and hyperkalemia. Indeed, studies of RBC cation content revealed high intracellular Na + and low intracellular K + , with increased leak flux at 37°C, suggesting a FP.

The proband was referred to our unit at 38 years old and tested for SLC4A1 gene. We found a heterozygous missense variant p.Thr686Met, predicted to impair splicing, and reported as likely pathogenic. Analysis of RBC membranes by WB, revealed a reduction of Band 3 expression, confirming the pathogenicity of the variant.

The worsening of anemia prompted re-evaluation of the case by t-NGS for RBC defects. A heterozygous nonsense variant in the ABCB6 gene p.Tyr471*, and a heterozygous missense variant p.Val598Met in the PIEZO1 gene were found. Thus, the occurrence of the ABCB6 variant explained the hyperkalemia and the temperature studies, while the presence of the PIEZO1 variant explained the altered permeability of the RBC, the hepatosiderosis, and the thrombosis after splenectomy. Finally, the ektacytometry analysis confirmed the multilocus inheritance, revealing a left shifted osmotic curve (typical of DHS) with a slightly decreased DiMax (typical of HS).

Summary/Conclusion: After 20 years, the patient received a complete diagnosis of multilocus inheritance of HS, DHS and FP due to mutations in SLC4A1, PIEZO1, and ABCB6 genes. This paradigmatic clinical case underlines the importance of a correct clinical assessment and genetic diagnosis to guide the personalized clinical management of the patient. An early diagnosis of DHS would have prevented the splenectomy and the following complications. This case confirms that the use of NGS-based testing in the diagnostic workflow of hereditary anemias is crucial to make a complete differential diagnosis.

P1538: INHIBITION OF COMPLEMENT C1S WITH SUTIMLIMAB IN PATIENTS WITH COLD AGGLUTININ DISEASE (CAD): RESULTS FOLLOWING 9-WEEK WASHOUT PERIOD IN PHASE 3 CARDINAL STUDY ( NCT03347396 )

A. Röth 1,* , W. Barcellini 2 , S. D’Sa 3 , Y. Miyakawa 4 , C. M. Broome 5 , M. Michel 6 , D. J. Kuter 7 , B. Jilma 8 , T. H. A. Tvedt 9 , I. C. Weitz 10 , T. Sourdille 11 , J. Wang 11 , D. S. Vagge 12 , K. Kralova 13 , F. Shafer 14 , M. Wardecki 15 , M. Lee 11 , S. Berentsen 16

1 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 3 UCLH Centre for Waldenström’s Macroglobulinemia and Related Conditions, University College London Hospitals NHS Foundation Trust, London, United Kingdom; 4 Thrombosis and Hemostasis Center, Saitama Medical University Hospital, Saitama, Japan; 5 Division of Hematology, MedStar Georgetown University Hospital, Washington DC, United States of America; 6 Henri-Mondor University Hospital, Assistance Publique-Hôpitaux de Paris, UPEC, Créteil, France; 7 Division of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, Boston, MA, United States of America; 8 Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria; 9 Section for Hematology, Department of Medicine, Haukeland University Hospital, Bergen, Norway; 10 Keck School of Medicine of USC, Los Angeles, CA; 11 Sanofi, Cambridge, MA, United States of America; 12 IQVIA, Bangalore, India; 13 Sanofi, Paris, France; 14 Sanofi, Bridgewater, NJ, United States of America; 15 Sanofi, Warsaw, Poland; 16 Department of Research and Innovation, Haugesund Hospital, Haugesund, Norway

Background: CAD is a rare, chronic autoimmune hemolytic anemia characterized by classical complement pathway (CP)-mediated hemolysis. Sutimlimab is a first-in-class humanized monoclonal antibody and C1s inhibitor that selectively targets the classical CP while leaving lectin and alternative pathways intact.

Aims: To report results from the 9-week follow-up visit after the last sutimlimab dose in the CARDINAL study.

Methods: CARDINAL is a two-part, open-label, single-arm study. In Part A, sutimlimab was administered on Days 0 and 7, then biweekly through Week 25. Patients then entered a Part B open-label extension and received biweekly sutimlimab for up to 3 years (N=22). Upon completion of dosing, or if a patient terminated the study early, a follow-up visit was completed 9 weeks after administration of the last dose of sutimlimab. Efficacy measures included hemoglobin (Hb) levels, hemolytic markers (total bilirubin, lactate dehydrogenase [LDH] and reticulocyte count); quality of life was assessed using the FACIT-Fatigue Score; PK/PD markers included Wieslab CP and total C4; adverse event data were collected. Descriptive statistics, frequency, or percentage were used to analyze outcomes.

Results: Of 22 patients who entered study Part B, 20 were assessed at the 9-week follow-up visit; 2 patients were not assessed due to death during the 9-week follow-up period. From baseline to last available on-treatment value, mean Hb improved to >12 g/dL, a mean (SD) change from baseline of 3.6 (2.19) g/dL (n=22); the mean (SD) Hb level at 9-week follow-up was 10.1 (1.6) g/dL (n=20), a decrease of 2.28 (1.8) g/dL from the last available on-treatment value (Figure 1). The mean levels of selected hemolytic markers (including bilirubin, LDH, reticulocytes), were decreased compared to baseline at the last available on-treatment time point, returning towards baseline 9 weeks after the last sutimlimab dose (Figure 1). The mean (SD) FACIT-Fatigue score increased from 32.4 (10.9) at baseline to 40.5 (9.3) at the last available on-treatment time point and returned to 34.1 (14.3) points at the 9-week follow-up (Figure 1). From Week 1, total C4 was consistently increased from baseline during treatment and returned towards baseline at the 9-week follow-up. The near-complete inhibition of CP activity observed throughout the treatment period was reversed at follow-up after discontinuation of sutimlimab. 45 treatment-emergent adverse events (TEAE) occurred after the last dose of sutimlimab in 11 patients. 10 of the 11 patients experienced TEAEs that were attributed to exacerbation of underlying CAD and included worsening of CAD in 3 patients, hemolysis in 1 patient, acrocyanosis in 2 patients, and anemia in 4 patients. 5 events were reported as serious TEAE (TESAE) in 3 patients, including 2 fatal events. TESAEs included: non-cardiac chest pain in 1 patient; acrocyanosis and fatal event of klebsiella pneumonia in 1 patient; and a femoral neck fracture and death due to CAD exacerbation (approximately 1.5 months after the last dose of sutimlimab) in 1 patient with complex medical history including myelodysplastic syndrome.

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Summary/Conclusion: In the 9-week washout period following the final dose of sutimlimab, classical complement pathway inhibition was reversed, and hemolytic markers and fatigue scores approached pre-sutimlimab values. Most of the TEAEs were due to exacerbation of underlying CAD.

P1539: SEASONALITY OF HEALTHCARE RESOURCE UTILIZATION AMONG COLD AGGLUTININ DISEASE AMONG PATIENTS IN DENMARK: A RETROSPECTIVE ANALYSIS

J. Fryzek 1 , N. Hooda 1 , B. Öztürk Esen 2 , T. Sourdille 3 , E. Horváth-Puhó 2 , E. K. Vago 2 , C. Broome 4 , A. Röth 5,*

1 EpidStrategies, a Division of ToxStrategies, Inc., Rockville, MD, United States of America; 2 Department of Clinical Epidemiology, Aarhus University, Aarhus, Denmark; 3 Sanofi, Cambridge, MA; 4 Division of Hematology, MedStar Georgetown University Hospital, Washington, DC, United States of America; 5 Department of Hematology and Stem Cell Transplantation, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany

Background: Cold agglutinin disease (CAD) is a rare form of autoimmune hemolytic anemia characterized by monoclonal autoantibodies targeting red blood cell (RBC) surface structures, preferentially at lower temperatures (Berentsen et al. Hematol Oncol Clin North Am , 2015). This results in both agglutination of RBCs and complement-mediated hemolysis. Patients with CAD were previously shown to have a greater healthcare utilization compared to the general population (Vago. CurMedRes , 2021). Considering exposure to cold temperatures may precipitate both the circulatory symptoms of CAD and hemolysis (Berentsen. Br J Haematol , 2018), understanding seasonal variation of CAD manifestations may be important to guide nonpharmacological management strategies and prophylactic recommendations (Lyckholm et al. N Engl J Med , 1996; Berentsen et al. Hematology , 2007). A previous retrospective cohort in the United States demonstrated persistent hemolysis independent of the season with disease manifestations, as evaluated by healthcare resource utilization (HRU) (Röth et al. Transfusion , 2022).

Aims: This study sought to elucidate the effects of seasonal temperature on the HRU of patients with CAD by comparing fall (September-November), winter (December-February), spring (March-May), and summer (June-August) values for inpatient admissions and outpatient visits in the first year after diagnosis or cohort entry and for the entire follow-up period with a matched comparison cohort in Denmark.

Methods: Patients were retrospectively identified using ICD-10 codes from the Danish National Patient Registry with linkage to multiple databases including the Danish Civil Registration System and the Danish Register of Causes of Death. Crude numbers of HRU were determined by the number of inpatient admissions and outpatient visits in the first year after diagnosis or cohort entry and for the entire follow-up period for each season.

Results: We identified 72 patients diagnosed with CAD and 720 matched comparisons, matched for age, gender, and region of residence, between 1999 and 2013. Inpatient stays and outpatient visits were higher for CAD patients than their matched comparisons for all seasons. Further there were no substantial differences between spring, summer, fall, and winter in CAD-related inpatient stays (40.3%, 31.9%, 36.1% and 36.1%, respectively) and outpatient visits (72.2% and 70.8%, 77.8% and 73.6%, respectively) during the entire duration of follow-up (Table 1). Results remained consistent in the first year after diagnosis or cohort entry wherein proportion of CAD patients with inpatient visits ranged from 19.4% in summer to 23.6% in winter and outpatient visits ranged from 62.5% in spring and fall to 63.9% in winter.

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Summary/Conclusion: Patients with CAD in this cohort had evidence of persistent HRU independent of the season. These data do not support perceptions of CAD as a seasonal disorder; recommendations such as moving to regions with warmer climates may not provide meaningful clinical benefit. The lack of seasonal variability in this cohort suggests that treatment considerations for patients with CAD should be season-independent.

P1540: STUDY OF MIRNA EXPRESSION PROFILES IN GAUCHER PATIENTS AND THEIR RELATIONSHIP WITH THE SEVERITY OF BONE INVOLVEMENT

I. Serrano-Gonzalo 1,2,* , L. López de Frutos 1,3 , M. Roca-Espiau 1 , R. Köhler 4 , P. Giraldo 1,3,5

1 FEETEG; 2 Grupo de Investigación de Enfermedad de Gaucher (GIIS-012), Instituto de Investigación Sanitaria de Aragón (ISSA); 3 Grupo Español de Enfermedades de Deposito Lisosomal (GEEDL), Sociedad Española de Hematología y Hemoterapia; 4 Fundación Agencia Aragonesa para la Investigación y el Desarrollo (ARAID); 5 Servicio de Hematología, Hospital QuirónSalud, Zaragoza, Spain

Background: Bone manifestations are one of the most prevalent comorbidities in Gaucher disease (GD) and, compromise the quality of life. They are evaluated by DEXA and MRI and several scores are available to assess its severity. Nowadays, no good biomarkers are available for this complication. miRNA are small non-coding RNA molecules whose function is regulate gene expression, and several of them were related to bone disease.

Aims: Identify different miRNA expression patterns, depending on the severity of bone involvement in patients with GD and, evaluate its predictive value.

Methods: 60 GD naïve patients, were selected and classified according to their bone disease severity using the S-MRI score, considering mild bone disease (MiBD; S-MRI<5), moderate (MoBD; S-MRI: 5-11) and, severe (SBD; S-MRI>11). miRNA was obtained from plasmatic exosomes using miRCURY Exosome Serum/Plasma and miRNeasy Serum/Plasma Advanced kits (QIAGEN) following manufacturer instructions. NGS technology and principal component analysis, corrected by sex and age, were applied to determine miRNA expression profiles.

Results: Patients were distributed proportionally in each group (20-20-20), gender distribution was equal between groups, and age was [median (Q1-Q3)]: 19.0 (4.00-40.00) – 40.5 (28.25-56.00) – 37.5 (31.25-47.00) years for MiBD, MoBD and SBD respectively. When compared MiBD against MoBD 3 miRNA were downexpressed in the MoDB group meanwhile 1 was increased. When MiBS vs SBD were compared 4 miRNA were downregulated and 5 increased on the SBD group. Only one miRNA is upregulated on MoDB and even more so in the SBD.

Summary/Conclusion: One miRNA, related inversely with osteoblastic differentiation is increased according with the bone severity. So, those preliminary results show an altered miRNA expression depending on the bone severity. Nowadays, we are validating them and trying to predict target-genes and the biological pathway they are involve.

Disclosure : Project founded by a Sanofi’s grant (SGZ-2019-12810).

P1541: PRELIMINARY RESULTS OF TWO YEARS FOLLOW-UP OF TYPE 1 GAUCHER DISEASE PATIENTS TREATED WITH ELIGLUSTAT IN TRAZELGA PROJECT.

I. Serrano-Gonzalo 1,2,* , L. López de Frutos 1,3 , I. Arévalo-Vargas 1,2 , M. Á. Fernández-Galán 3,4 , M. Morales-Conejo 5 , M. V. Calle-Gordo 3,6 , I. Vitoria-Miñana 7 , P. Correcher-Medina 7 , A. Madinaveitia-Ochoa 8 , J. Á. Hernández-Rivas 3,9 , J. García-Frade 3,10 , D. Ibarretxe-Gerediaga 11 , F. Delgado-Mateos 3,12 , M. D. M. Tormo-Díaz 3,13 , M. Á. Ruíz-Guinaldo 3,14 , E. Mora-Casterá 3,15 , M. S. Noya-Pereira 3,16 , A. Albarracín-Arraigosa 17 , M. López-Dupla 18 , J. Balanzat-Muñoz 3,19 , M. L. Lozano-Almela 3,20 , F. Labbadia 3,21 , M. Andrade-Campos 1,3,22 , P. Giraldo 1,3,23

1 FEETEG; 2 Grupo de Investigación de Enfermedad de Gaucher (GIIS-012), Instituto de Investigación Sanitaria de Aragón (IISA); 3 Grupo Español de Enfermedades de Depósito Lisosomal (GEEDL), Sociedad Española de Hematología y Hemoterapia, Zaragoza; 4 Servicio de Hematología, Hospital Virgen del Puerto, Plasencia; 5 Servicio de Medicina Interna, Hospital Universitario 12 de Octubre, Madrid; 6 Servicio de Hematología, Hospital de Torrecardenas, Almería; 7 Unidad de Nutrición y Metabolopatías, Hospital Universitari i Politècnic La Fe, Valencia; 8 Servicio de Hematología, Hospital Comarcal de Albarracín, Teruel; 9 Servicio de Hematología, Hospital Universitario de Salamanca, Salamanca; 10 Servicio de Hematología, Hospital Universitario Río Hortega, Valladolid; 11 Unitat de Medicina Vascular i Metabolisme, Hospital Universitari San Joan, Reus; 12 Servicio de Hematología, Hospital Punta de Europa, Cádiz; 13 Servicio de Hematología, Hospital Clínico Universitario de Valencia; 14 Servicio de Hematología, Hospital Comarcal Francesc de Borja; 15 Servicio de Hematología, Hospital Universitari i Politècnic La Fe, Valencia; 16 Servicio de Hematología, Hospital Teresa Herrera, A Coruña; 17 Servicio de Medicina Interna, Hospital Serranía de Ronda, Málaga; 18 Servicio de Medicina Interna, Hospital Universitari Joan XXIII, Tarragona; 19 Servicio de Hematología, Hospital Can Misses, Ibiza; 20 Servicio de Hematología, Hospital General Universitario Morales Meseguer; 21 Servicio de Hematología, Hospital de la Vega Lorenzo Guirao, Murcia; 22 Servicio de Hematología, Hospital del Mar, Barcelona; 23 Servicio de Hematología, Hospital QuirónSalud, Zaragoza, Spain

Background: Eliglustat (Cerdelga®, Sanofi-Genzyme), is a substrate reduction therapy approved in European Union in 2015 for Gaucher disease type-1 (GD1) adult patients. TRAZELGA (GEE-ELI-2017-01) project is a prospective follow-up post-authorization study designed as a tool to uniformly study the response to eliglustat therapy.

Aims: In this study, it is intending to determine if the drug administration maintains or improve clinical goals and/or plasmatic biomarkers as well as to evaluate eliglustat therapy response in terms of safety, effectiveness and patient´s satisfaction.

Methods: Clinical parameters, image bone assessment, and plasmatic biomarkers were analyzed for two years on follow-up: chitotriosidase [ChT] activity was evaluated by fluorimetry, glucosylsphingosine [GluSph] concentrations by LC-MSMS and CCL18/PARC, C5a, C3, YKL-40, Cathepsin S, Hepcidin and Lipocalin concentrations by immunoquantification assay. A quality of life study (SF-36), disease severity score index evaluation (GD-DS3) and compilation of side effects are included. Non-parametric statistical analysis was performed and p<0.05 was considered as statistically significant.

Results: 29 Spanish GD1 patients were included, 16 were males and 13 female. The median age was 42.0 (30.00-55.50) years. All patients were treated before starting eliglustat: 26 with enzyme replacement therapy, 2 with substrate reduction therapy and 1 with a combined therapy. The median age of treatment before eliglustat was 17.0 (10.00-21.50). A statistically significant decrease on GluSph and CCL18/PARC (p=0.0393 and p=0.0019, respectively) was observed meanwhile ChT was maintain. Clinical data and the analysis of the rest of the biomarkers is going on.

Summary/Conclusion: Eliglustat mantains or improve biochemical biomarkers after two years treatment.

Disclosure: TRAZELGA (GZ-2017-11713) received a grant from Sanofi Genzyme for its realization.

P1542: COMORBIDITIES AND COMPLICATIONS ACROSS GENOTYPES IN ADULT PATIENTS WITH PYRUVATE KINASE DEFICIENCY: ANALYSIS FROM THE PEAK REGISTRY

A. Glenthøj 1,* , R. F. Grace 2 , E. J. van Beers 3 , J.-L. Vives Corrons 4 , B. Glader 5 , K. H. M. Kuo 6 , C. Lander 7 , D. Pospíŝilová 8 , J. Williams 9 , Y. Yan 9 , B. McGee 9 , P. Bianchi 10

1 Department of Hematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 2 Dana-Farber/Boston Children’s Cancer and Blood Disorder Center, Boston, United States of America; 3 Van Creveldkliniek, Department of Internal Medicine, University Medical Center Utrecht, Utrecht, Netherlands; 4 Institute for Leukaemia Research Josep Carreras ENERCA Coordinator, University of Barcelona, Barcelona, Spain; 5 Stanford University School of Medicine, Palo Alto, United States of America; 6 Division of Hematology, University of Toronto, Toronto, Canada; 7 Metabolic Support UK, Wrexham, United Kingdom; 8 Deparment of Pediatrics, Palacky University and University Hospital, Olomouc, Czechia; 9 Agios Pharmaceuticals, Inc., Cambridge, United States of America; 10 UOC Ematologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

Background: Pyruvate kinase (PK) deficiency is a rare, congenital, glycolytic enzymopathy caused by mutations in the PKLR gene, which leads to lifelong hemolytic anemia and may result in complications such as iron overload, pulmonary hypertension, and gallstones. PK deficiency has wide genetic heterogeneity, with >300 mutations reported, and previous data suggest that disease complications may be common regardless of genotype.

Aims: To further characterize comorbidities/complications across genotypes in adult patients (pts) with PK deficiency enrolled in the Peak Registry ( NCT03481738 ).

Methods: The Peak Registry is a global retrospective and prospective observational study of adult and pediatric pts diagnosed with PK deficiency. For this analysis, adults (≥18 years [yrs]) with classifiable PKLR genotype data were grouped into 3 cohorts: missense/missense (M/M); missense/non-missense (M/NM); non-missense/non-missense (NM/NM). NM mutations include nonsense, frameshift, in-frame small indels, large deletions, and splicing variants (including R479H). Genotype classifications are aligned with those previously reported from the PK deficiency Natural History Study. Data on demographics, laboratory values, and medical history inclusive of comorbidities and complications were summarized descriptively.

Results: As of 29Jun2021, 90 of 103 adult pts in the registry had classifiable PKLR genotype data; 57 (63.3%) were classified as M/M, 28 (31.1%) as M/NM, and 5 (5.6%) as NM/NM. Median age (range) of PK deficiency diagnosis was 21.0 yrs (0–68) for M/M pts, 12.0 yrs (0–40) for M/NM pts, and 0.0 yrs (0–0) for NM/NM pts (Table). Among the 87 pts with known transfusion status, 36.8% had never been transfused (M/M: 44.4%; M/NM: 28.6%; NM/NM: 0%). Splenectomy had been performed in almost half of M/M pts (47.3%), the majority of M/NM pts (60.7%), and all NM/NM pts (100%). At registry enrollment, median hemoglobin (range) in the M/M, M/NM, and NM/NM cohorts was 9.9 g/dL (7.1–14.2), 9.1 g/dL (6.7–14.1), and 7.3 g/dL (6.9–8.1), respectively. History of iron overload was observed in substantial numbers of pts, regardless of genotype; 40.0% in M/M pts, 46.4% in M/NM pts, and 60.0% in NM/NM pts. Of these pts, 25.0% of M/M pts and 12.5% of M/NM pts had a history of iron overload despite never having been transfused. Jaundice was common for pts across genotypes (M/M: 32.7%; M/NM: 44.4%; NM/NM: 25.0%). Other comorbidities/complications included biliary events (M/M: 30.4%; M/NM: 33.3%; NM/NM: 0%) and bone health problems (M/M: 23.2%; M/NM: 22.2%; NM/NM: 0%). Of 7 pts who experienced 13 thromboembolic events, timing of the events relative to splenectomy was known for 6 pts, and in all 6 pts, the thromboembolic events occurred after splenectomy. Overall, 41.1% of pts experienced ≥2 distinct comorbidities/complications, as defined in the Table (M/M: 38.6%; M/NM 50.0%; NM/NM 20.0%).

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Summary/Conclusion: This analysis reveals that pts across all PKLR genotypes experienced a wide range of serious comorbidities/complications across multiple systems. In addition to the breadth of comorbidities presented, these data highlight the existence of multiple complications in individual pts with PK deficiency and the need for appropriate monitoring and management of these pts, regardless of genotype.

P1543: MITAPIVAT DECREASES THE NEED FOR TRANSFUSIONS SECONDARY TO POORLY TOLERATED ANEMIA AND ACUTE EVENTS COMPARED TO PLACEBO IN PATIENTS WITH PYRUVATE KINASE DEFICIENCY WHO ARE NOT REGULARLY TRANSFUSED

H. Al-Samkari 1,* , J. Morales-Arias 2 , R. Xu 2 , V. Beynon 2 , R. F. Grace 3

1 Division of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston; 2 Agios Pharmaceuticals, Inc., Cambridge; 3 Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, United States of America

Background: Pyruvate kinase (PK) deficiency is a rare, hereditary, chronic hemolytic anemia characterized by reduced activity of the red blood cell (RBC) PK (PKR) enzyme. A subset of patients (pts) require regular transfusions; however, transfusions may occasionally be needed in pts who are not regularly transfused due to hemolytic crisis, infections, and other acute events. Although transfusions can temporarily increase hemoglobin (Hb), they are associated with acute and chronic complications and can have a negative impact on quality of life. Mitapivat (AG-348) is a first-in-class, oral, allosteric activator of PKR which has demonstrated significant improvements in Hb, markers of hemolysis and hematopoiesis, and reduction in disease burden in pts who were not regularly transfused (ACTIVATE, NCT03548220 ) and significant reduction in transfusion burden in pts who were regularly transfused (ACTIVATE-T, NCT03559699 ) with PK deficiency.

Aims: Determine the effect of mitapivat on the number of transfusion episodes and RBC units transfused in pts with PK deficiency who were not regularly transfused, and randomized in the ACTIVATE trial.

Methods: ACTIVATE was a phase 3, global, double-blind, placebo (PBO)-controlled study of mitapivat in adults with PK deficiency who were not regularly transfused (≤4 transfusion episodes in prior year [yr]; none in prior 3 months). The proportion of pts requiring transfusions and the number of RBC units transfused were analyzed during the 24-week (wk) study (exploratory endpoint).

Results: 80 pts were randomized (mitapivat N=40; PBO N=40). The population was balanced between the mitapivat and PBO arms: mean age 36.6 yrs, 40% male, mean baseline Hb was 8.6 (mitapivat) and 8.5 (PBO) g/dL (Table). One pt in the PBO arm discontinued the study before being dosed. During the study, RBC transfusions were indicated for clinically significant or poorly tolerated anemia in 2 (5.0%) pts in the mitapivat arm (2 units in 1 pt and 4 units in the other pt) and 5 (12.8%) pts in the PBO arm (2 units per pt in 4 pts and 1 unit in 1 pt) (Table). Four (10.3%) pts in the PBO arm had transfusions (1–2 units per pt) due to an adverse event (AE) (viral upper respiratory tract infection, dyspnea, fatigue, presyncope). Two pts in the PBO arm received transfusions due to both an AE and clinically significant or poorly tolerated anemia. No pts in the mitapivat arm had transfusions due to an AE. One out of 11 (9.0%, mitapivat) and 6 out of 10 (60%, PBO) pts who received a transfusion in the 52 wks prior to study treatment required an on-study transfusion (Table).

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Summary/Conclusion: Mitapivat has the potential to decrease transfusion needs secondary to poorly tolerated anemia and acute events in pts with PK deficiency who are not regularly transfused, which is consistent with the decrease in transfusion burden previously observed in pts who are regularly transfused. This may in turn have a beneficial effect on reducing iron overload and improving the long-term disease burden and quality of life of pts with PK deficiency, across the disease spectrum. This analysis further adds to previously reported data showing beneficial effects of PK activation through mitapivat, the only disease-modifying drug therapy for adult pts with PK deficiency.

P1544: BONE MINERAL DENSITY REMAINS STABLE IN PYRUVATE KINASE DEFICIENCY PATIENTS RECEIVING LONG-TERM TREATMENT WITH MITAPIVAT

H. Al-Samkari 1,* , R. F. Grace 2 , A. Glenthøj 3 , O. Andres 4 , W. Barcellini 5 , F. Galactéros 6 , K. H. M. Kuo 7 , D. M. Layton 8 , M. Morado Arias 9 , V. Viprakasit 10 , Y. Dong 11 , F. Tai 11 , L. Grekas 11 , K. Khoja 11 , S. Gheuens 11 , B. McGee 11 , J. B. Porter 12 , E. J. van Beers 13

1 Division of Hematology, Massachusetts General Hospital, Harvard Medical School; 2 Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, MA, United States of America; 3 Department of Haematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 4 Department of Paediatrics, University of Würzburg, Würzburg, Germany; 5 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 6 Unité des Maladies Génétiques du Globule Rouge, CHU Henri Mondor, Créteil, France; 7 Division of Hematology, University of Toronto, Toronto, ON, Canada; 8 Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom; 9 Hematology Department, Hospital Universitario La Paz, Madrid, Spain; 10 Siriraj-Thalassemia Center, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; 11 Agios Pharmaceuticals, Inc., Cambridge, MA, United States of America; 12 Haematology Department, University College London Hospitals, London, United Kingdom; 13 Van Creveldkliniek, Department of Internal Medicine, University Medical Center Utrecht, Utrecht, Netherlands

Background: Hereditary pyruvate kinase (PK) deficiency results in chronic hemolysis that may lead to reduced bone mineral density (BMD). Mitapivat is an oral activator of PK in red blood cells (RBC) that improves hemoglobin concentration and reduces transfusion burden in patients (pts) with PK deficiency. Although mitapivat has mild aromatase inhibition effects, a study of pts with PK deficiency who were not regularly transfused showed that mitapivat did not appear to promote progression of BMD abnormalities for up to 56 months (mos).

Aims: Report BMD over time in a broad PK deficiency population (independent of transfusion) receiving long-term treatment with mitapivat.

Methods: This study included pts ≥18 years (yrs) with PK deficiency enrolled in DRIVE-PK ( NCT02476916 ; N=52, Phase [Ph] 2), ACTIVATE ( NCT03548220 ; N=80, Ph 3), ACTIVATE-T ( NCT03559699 ; N=27, Ph 3), and the long-term extension (LTE) of ACTIVATE and ACTIVATE-T ( NCT03853798 ). Pts in DRIVE-PK and ACTIVATE were not regularly transfused (≤3 RBC units and ≤4 transfusion episodes in the prior yr). Pts in ACTIVATE-T were regularly transfused (≥6 transfusion episodes in the prior yr). Data from 90 pts who received mitapivat for >12 mos were pooled from these trials along with pts from the LTE who elected to continue or crossover (if on placebo) to mitapivat. BMD changes were measured using dual-energy X-ray absorptiometry (DXA) scans at 3 locations (total femur [combined femoral neck and total hip], femoral neck, and spine). Based on DXA T-scores, pts were classified according to standard definitions: normal BMD (≥–1.0 at all locations), osteopenia (>–2.5 to <–1.0 at ≥1 locations), or osteoporosis (≤–2.5 at ≥1 locations). DXA scans were obtained locally for all studies and interpreted locally for DRIVE-PK and centrally for ACTIVATE, ACTIVATE-T, and the LTE. Scans were collected as follows: baseline (BL), between Week (Wk) 24 and 28, every 6 mos to Mo 30, then annually (DRIVE-PK); BL, Wk 24 (ACTIVATE); BL, Wk 16, Wk 40 (ACTIVATE-T); BL, every 24 wks to Wk 96, then every 48 wks (LTE).

Results: 90 pts were assessed: median age 34.5 yrs (range 18–71); 50.0% female; median mitapivat exposure 20 mos (range 12.2–64.9); concomitant bisphosphonate medication, 8 pts (8.9%); prior medical history of fracture, 8 pts (8.9%); fractures/joint injuries during study period, 6 pts (6.7%). At BL, 24 pts (26.7%) had normal BMD, 48 pts (53.3%) had osteopenia, 16 pts (17.8%) had osteoporosis, and 2 pts (2.2%) were missing BL T-score data. At last BMD assessment (data cut-off 04Mar2021), 76/88 pts (86.4%) remained stable, 7 pts (8.0%) improved, and 5 pts (5.7%) worsened (Figure). At last assessment, 27 (30.0%), 46 (51.1%), and 17 (18.9%) of 90 pts had a T-score indicating normal BMD, osteopenia, and osteoporosis (Table). Of 24 pts with BL T-scores ≥–1.0, 21 pts remained stable, and 3 pts had worsening BMD. Of 48 pts with a BL T-score >–2.5 to <–1.0, 6 pts improved to a T-score for normal BMD, 40 pts remained stable, and 2 pts worsened. Of 16 pts with a BL T-score of ≤–2.5, 1 pt improved to a T-score of >–2.5 to <–1.0.

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Summary/Conclusion: In this analysis, BMD remained largely stable over time in adults with PK deficiency receiving long-term mitapivat (up to 65 mos). Pts had substantially low BMD at BL which did not progress, and it is hypothesized that mitapivat may halt bone loss in pts with PK deficiency by decreasing hemolysis and ineffective erythropoiesis and stabilizing iron homeostasis. BMD data will continue to be monitored as part of the ongoing LTE study to further understand this process.

P1545: DURABILITY OF HEMOGLOBIN RESPONSE AND REDUCTION IN TRANSFUSION BURDEN IS MAINTAINED OVER TIME IN PATIENTS WITH PYRUVATE KINASE DEFICIENCY TREATED WITH MITAPIVAT IN A LONG-TERM EXTENSION STUDY

R. F. Grace 1,* , A. Glenthøj 2 , W. Barcellini 3 , M. Verhovsek 4 , J. A. Rothman 5 , M. Morado Arias 6 , D. M. Layton 7 , O. Andres 8 , F. Galactéros 9 , E. J. van Beers 10 , K. Onodera 11 , V. Viprakasit 12 , S. Chonat 13 , J. B. Porter 14 , M. P. Judge 15 , P. A. Kosinski 15 , P. Hawkins 15 , S. Gheuens 15 , R. Xu 15 , B. McGee 15 , V. Beynon 15 , H. Al-Samkari 16

1 Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, MA, United States of America; 2 Department of Haematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 3 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 4 McMaster University, Hamilton, ON, Canada; 5 Duke University Medical Center, Durham, NC, United States of America; 6 Hematology Department, Hospital Universitario La Paz, Madrid, Spain; 7 Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom; 8 Department of Paediatrics, University of Würzburg, Würzburg, Germany; 9 Unité des Maladies Génétiques du Globule Rouge, CHU Henri Mondor, Creteil, France; 10 Van Creveldkliniek, Department of Internal Medicine, University Medical Center Utrecht, Utrecht, Netherlands; 11 Tohoku University Hospital, Sendai, Japan; 12 Siriraj Hospital, Mahidol University, Bangkok, Thailand; 13 Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta and Department of Pediatrics, Emory University, Atlanta, GA, United States of America; 14 Haematology Department, University College London Hospitals, London, United Kingdom; 15 Agios Pharmaceuticals, Inc., Cambridge, MA; 16 Division of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States of America

Background: Mitapivat (AG-348), a first-in-class, oral, allosteric activator of red blood cell (RBC) PK enzyme, demonstrated improvements in hemoglobin (Hb) and markers of hemolysis and hematopoiesis (ACTIVATE, NCT03548220 ), and reduction in transfusion burden (ACTIVATE-T, NCT03559699 ) in patients (pts) with PK deficiency.

Aims: Report data from ACTIVATE, ACTIVATE-T, and their long-term extension (LTE) study ( NCT03853798 ).

Methods: In the double-blind, placebo (PBO)-controlled ACTIVATE study (12-week [wk] dose-optimization period [5/20/50 mg twice daily [BID]]; 12-wk fixed-dose period [FDP]), 80 pts (≥18 years [yrs]) with PK deficiency who were not regularly transfused (≤4 transfusion episodes in prior yr; none in prior 3 months [mos]) were randomized 1:1 to mitapivat or PBO. Primary endpoint was Hb response (≥1.5 g/dL increase in Hb from baseline [BL] sustained at ≥2 assessments at Wks 16, 20, and 24 in the FDP). In the single-arm, open-label ACTIVATE-T study (16-wk dose-optimization period [5/20/50 mg BID]; 24-wk FDP), 27 pts (≥18 yrs) with PK deficiency who were regularly transfused (≥6 transfusion episodes in prior yr) were treated with mitapivat. Primary endpoint was transfusion response (≥33% reduction in number of RBC units transfused during the FDP, compared with pt’s individual historical transfusion burden standardized to 24 wks). Secondary endpoint was achieving transfusion-free status (no transfusions in FDP). Pts who completed the FDP of ACTIVATE/ ACTIVATE-T were eligible to continue in the LTE, where all received mitapivat. The ACTIVATE/LTE analysis assessed duration of Hb response in 2 cohorts: 1) pts assigned to mitapivat who achieved a Hb response and continued to the LTE (mitapivat-to-mitapivat arm [M/M]); 2) pts assigned to PBO who switched to mitapivat in the LTE (PBO-to-mitapivat arm [P/M]) and then met Hb response criteria. The ACTIVATE-T/LTE analysis assessed transfusion response in the LTE, and transfusion-free duration among pts from ACTIVATE-T who achieved transfusion-free status.

Results: In ACTIVATE, 40% of pts treated with mitapivat (N=40) achieved a Hb response; in the LTE, pts randomized to PBO in ACTIVATE showed similar improvements in Hb levels after switching to mitapivat. These improvements were sustained with continued treatment (Figure 1a). All 16 pts assigned to mitapivat in ACTIVATE who achieved Hb responses continued to the LTE; 15 M/M pts were evaluable for Hb assessment in the LTE. 13/15 M/M pts (86.7%) maintained ≥1.5 g/dL Hb increase from BL up to 19.5 mos at all time points; 2 pts maintained ≥1 g/dL Hb increase from BL at all time points. None of the pts assigned to PBO in ACTIVATE (N=40) had a Hb response; 17 P/M pts had sufficient time (24 wks of treatment) in the LTE for Hb response assessment. 6/17 pts (35%) achieved Hb responses in the LTE, and all maintained Hb responses during follow-up. In ACTIVATE-T (N=27), 37% of pts achieved a transfusion response and 22% of pts achieved transfusion-free status. In the LTE, 9 pts (33.3%) met criteria for a transfusion response. All 6 pts who achieved transfusion-free status in ACTIVATE-T maintained the status in the LTE up to 21.9 mos (Figure 1b). One additional pt, who met the primary endpoint, but was not transfusion free in ACTIVATE-T, did not receive any transfusions in the LTE.

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Summary/Conclusion: Mitapivat improved Hb and reduced transfusion burden in pts with PK deficiency over time, demonstrating the consistency and long-term durability of treatment with mitapivat.

P1546: ACTIVATE-KIDST: MITAPIVAT IN CHILDREN WITH PYRUVATE KINASE DEFICIENCY WHO ARE REGULARLY TRANSFUSED

R. F. Grace 1,* , P. D. Tyler 2 , M. Little 2 , P. A. Kosinski 2 , V. Beynon 2

1 Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston; 2 Agios Pharmaceuticals, Inc., Cambridge, United States of America

Background: Pyruvate kinase (PK) deficiency is a rare hereditary chronic hemolytic anemia caused by PKLR gene mutations. Clinical management includes regular red blood cell (RBC) transfusions; some children require splenectomy, which carries a risk of sepsis and thrombosis. PK deficiency is associated with serious complications, regardless of transfusion status. There are no approved pharmacotherapies in children; there is an unmet need for therapies targeting the underlying cause of hemolysis. Mitapivat (AG-348), a first-in-class, oral, allosteric activator of the RBC PK enzyme, demonstrated significant reduction in transfusion burden in adults with PK deficiency who are regularly transfused (ACTIVATE-T NCT03559699 ); 37% achieved a transfusion reduction response and 22% achieved a durable transfusion-free status that was maintained in the long-term extension study ( NCT03853798 ). Safety and efficacy data support the evaluation of mitapivat in children with PK deficiency who are regularly transfused.

Aims: Report the design of the phase 3 ACTIVATE-KidsT study ( NCT05144256 ), which will evaluate the efficacy and safety of mitapivat in children with PK deficiency who are regularly transfused.

Methods: ACTIVATE-KidsT, a global, multicenter, double-blind, placebo-controlled study, will stratify 45 children by age and splenectomy status. A minimum of 6 patients per age group (1 to <6, 6 to <12, and 12 to <18 years) will be randomized (2:1) to receive mitapivat or placebo. The double-blind portion includes an 8-week dose titration period and a 24-week fixed-dose period; a 5-year open-label extension will follow. Study drug will be administered at a range of 1–50 mg twice daily, depending on age and weight; dosing categories are derived from pharmacokinetic modeling data of adults with PK deficiency. Key inclusion criteria: ≥2 mutant PKLR alleles with ≥1 missense mutation, ≥6 transfusion episodes in the past year. Key exclusion criteria: R479H homozygosity, presence of 2 non-missense mutations in PKLR without a missense mutation, or prior stem cell transplantation. The primary endpoint is transfusion reduction response, defined as ≥33% reduction in transfusion volume during the fixed-dose period normalized by weight and actual study drug duration, compared with historical transfusion volume standardized by weight and to 24 weeks. Secondary endpoints will assess the effect of mitapivat on transfusion-free status, iron metabolism and overload, safety, quality of life, and pharmacokinetics.

Results: Global site recruitment is in-progress; an update on recruitment and patient enrollment will be provided.

Summary/Conclusion: The ACTIVATE-KidsT study of children with PK deficiency who are regularly transfused will evaluate the efficacy and safety of mitapivat, a potential disease modifying pharmacotherapy that targets the underlying enzymatic defect of PK deficiency.

P1547: ACTIVATE-KIDS: MITAPIVAT IN CHILDREN WITH PYRUVATE KINASE DEFICIENCY WHO ARE NOT REGULARLY TRANSFUSED

R. F. Grace 1,* , P. D. Tyler 2 , M. Little 2 , P. A. Kosinski 2 , V. Beynon 2

1 Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA; 2 Agios Pharmaceuticals, Inc., Cambridge, MA, United States of America

Background: Pyruvate kinase (PK) deficiency is a rare, inherited disorder caused by PKLR gene mutations that is associated with chronic hemolytic anemia and lifelong complications. Clinical management includes red blood cell (RBC) transfusions and splenectomy, which only partially improves anemia. No pharmacotherapies are approved for use in children, and therapies targeting the underlying cause of hemolysis are needed. Mitapivat (AG-348), a first-in-class, oral, allosteric activator of the RBC PK enzyme, demonstrated significant and durable improvements in hemoglobin (Hb) and markers of hemolysis, hematopoiesis, and iron metabolism in adults with PK deficiency who were not regularly transfused (ACTIVATE NCT03548220 , long-term extension study NCT03853798 ). Safety and efficacy data support the evaluation of mitapivat in children with PK deficiency who are not regularly transfused.

Aims: Report the design of ACTIVATE-Kids ( NCT05175105 ), a phase 3 study, which will evaluate the efficacy and safety of mitapivat in children with PK deficiency who are not regularly transfused.

Methods: ACTIVATE-Kids, a global, multicenter, double-blind, placebo-controlled study, will stratify 30 children by age (1 to <6, 6 to <12, and 12 to <18 years); a minimum of 6 patients in each age group will be randomized (2:1) to mitapivat or placebo. The double-blind period comprises an 8-week dose-titration period and a 12-week fixed-dose period. A 5-year open-label period will follow the double-blind period. The study drug will be dosed (1–50 mg twice daily) according to patient age and weight, based on pharmacokinetic modeling data of adults with PK deficiency. Key inclusion criteria: ≥2 PKLR mutant alleles with ≥1 missense mutation, ≤5 RBC transfusions in the past year, Hb concentrations of ≤10 g/dL (12 to <18 years) or ≤9 g/dL (1 to <12 years). Key exclusion criteria: R479H homozygosity, 2 non-missense mutations without a missense mutation in PKLR , or prior stem cell transplantation. The primary endpoint is Hb response, defined as a ≥1.5 g/dL increase in Hb concentration from baseline that is sustained at ≥2 scheduled assessments at Weeks 12, 16, and 20 in the double-blind period. Secondary endpoints will evaluate the effect of mitapivat on Hb, hemolysis, erythropoiesis, iron metabolism and overload, safety, quality of life, and pharmacokinetics.

Results: Global site recruitment is in-progress; an update on recruitment and patient enrollment will be provided.

Summary/Conclusion: The phase 3 ACTIVATE-Kids study of children with PK deficiency who are not regularly transfused will evaluate treatment with mitapivat, a potential disease-modifying pharmacotherapy.

P1548: LONG-TERM TREATMENT WITH ORAL MITAPIVAT IS ASSOCIATED WITH NORMALIZATION OF HEMOGLOBIN LEVELS IN PATIENTS WITH PYRUVATE KINASE DEFICIENCY

W. Barcellini 1,* , R. F. Grace 2 , H. Al-Samkari 3 , A. Glenthøj 4 , J. A. Rothman 5 , M. Morado Arias 6 , D. M. Layton 7 , O. Andres 8 , M. DiBacco 9 , P. Hawkins 9 , M. P. Judge 9 , F. Tai 9 , J. Morales-Arias 9 , V. Beynon 9 , E. J. van Beers 10

1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2 Dana-Farber/Boston Children’s Cancer and Blood Disorders Center; 3 Division of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States of America; 4 Department of Haematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 5 Duke University Medical Center, Durham, NC, United States of America; 6 Hematology Department, Hospital Universitario La Paz, Madrid, Spain; 7 Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom; 8 Department of Paediatrics, University of Würzburg, Würzburg, Germany; 9 Agios Pharmaceuticals, Inc., Cambridge, MA, United States of America; 10 Van Creveldkliniek, Department of Internal Medicine, University Medical Center Utrecht, Utrecht, Netherlands

Background: Pyruvate kinase (PK) deficiency is a rare hereditary anemia caused by mutations in the PKLR gene encoding the red blood cell PK enzyme (PKR). Defects in PKR lead to chronic hemolytic anemia, which is associated with serious complications regardless of transfusion status. Mitapivat (AG-348), a first-in-class, oral, allosteric activator of PKR, has been shown in a placebo (PBO)-controlled, phase 3 clinical trial (ACTIVATE, NCT03548220 ) to significantly improve hemoglobin (Hb) concentration and markers of hemolysis and hematopoiesis in patients (pts) with PK deficiency who were not regularly transfused. These improvements were sustained in a long-term extension (LTE, NCT03853798 ) study that included pts who completed the fixed-dose period of the ACTIVATE study.

Aims: This analysis focuses on normalization of Hb levels in pts treated in the ACTIVATE and ongoing LTE studies.

Methods: The randomized, double-blind, PBO-controlled ACTIVATE study consisted of a 12-week dose-optimization period (5/20/50 mg twice daily) and a 12-week fixed-dose period. Eighty pts (≥18 years) with a diagnosis of PK deficiency who were not regularly transfused (≤4 transfusion episodes in prior year; none in the prior 3 months) were randomized 1:1 to receive mitapivat or PBO. The primary endpoint was Hb response, defined as ≥1.5 g/dL increase in Hb from baseline sustained at ≥2 scheduled assessments at weeks 16, 20, and 24 in the fixed-dose period. Pts who completed the fixed-dose period of ACTIVATE were eligible to continue in the LTE study, where all pts received mitapivat. In this analysis, the proportions of pts who achieved normal Hb at any time while receiving mitapivat and within specified time intervals were assessed, with data cutoff of 21Sep2021. Normal Hb levels achieved within 61 days after a transfusion were excluded.

Results: In ACTIVATE, 16 of the 40 pts (40%) treated with mitapivat and none of the 40 patients randomized to PBO achieved an Hb response as defined in the study protocol. Across the ACTIVATE and LTE studies, 28/78 (35.9%) pts achieved a normal Hb level at least once during treatment with mitapivat. Among pts treated with mitapivat in both the ACTIVATE and LTE studies (M/M arm, mean Hb at baseline 8.66 g/dL), 15/40 (37.5%) achieved a normal Hb level at least once during treatment, the majority within the first few months. Similarly, among patients randomized to PBO in the ACTIVATE study and treated with mitapivat in the LTE study (P/M arm, mean Hb at baseline 8.47 g/dL), 13/38 (34.2%) achieved a normal Hb level at least once during the LTE, all within the first 4 months of treatment with mitapivat. Of the 16 M/M pts who achieved Hb response, 14 (87.5%) achieved a normal Hb level at least once during treatment with mitapivat either in the ACTIVATE study or the LTE; of the 15 P/M pts who achieved a protocol-defined Hb response during mitapivat treatment in the LTE, 12 (80.0%) achieved a normal Hb at least once. Seven pts had normal hemoglobin measurements at each time point assessed while on mitapivat treatment (Figure).

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Summary/Conclusion: In this study, treatment with mitapivat was associated with early and robust Hb responses with approximately one-third of pts achieving normal Hb levels at least once. These results add to the evolving data demonstrating potential real-world benefits of mitapivat for patients with PK deficiency and supports mitapivat as an effective and disease-modifying therapy for this condition.

P1549: NOVEL SLC4A1 GENE MUTATIONS IN HEREDITARY SPHEROCYTOSIS BAND 3 PROTEIN-DEFICIENT TUNISIAN PATIENTS

N. TRABELSI 1,* , I. Kraiem 1 , S. Menif 1 , S. Abbes 1

1 Institut Pasteur de Tunis, Tunis, Tunisia

Background: Band 3 is the most abundant transmembrane protein in the erythrocyte, which is encoded by the SLC4A1 gene. Several pathogenic mutations in the SLC4A1 gene, affecting both the cytosolic and the transmembrane domains, are linked to Hereditary Spherocytosis (HS). This inherited disorder causing hemolytic anemia, due to membrane proteins abnormalities. HS is the result of defects that interrupt the vertical interaction including the band 3-ankyrin-spectrin complex.

Aims: We aimed to study HS in band 3 protein-deficient patients, identify the causative gene mutations and investigate phenotype-genotype correlations.

Methods: Ten HS Tunisian patients were investigated. We assessed red cell fragility parameters and eosine-5-maléimide (EMA), band 3 protein-binding dye, as an indicator of a membrane protein deficiency. We confirmed the EMA flow cytometry analysis by the erythrocyte membrane protein electrophoresis. Finally, we explored SLC4A1 gene mutations through PCR-Sanger sequencing. Different computational methods were used for the interpretation of the novel and known genetic variations effects.

Results: We identified eight novel SLC4A1 gene mutations: c.877-1G>A in intron 9, c.1031G>C;p.(Arg344Pro) and c.1034-1036delinsATA;p.(Arg345-Arg346delinsAsnSer) detected in exon 10, c.1192A>T;p.(Thr398Ser) and c.1193C>G;p.(Thr398Arg) in exon 11, c.1364G>A;p.(Gly455Glu) in exon 13, c.2354del;p.(Leu785Argfs*44) in exon 18 and c.2531del;p.(Leu844Argfs*7) in exon 19, as well as twenty-one polymorphisms in HS band 3 protein-deficient patient. The deleterious effect of the new mutations on the protein structure and on splicing process was confirmed using In silico analysis.

Summary/Conclusion: Our findings throw light on the molecular basis of HS-band 3 deficient Tunisian patients. The continuous characterization of the molecular defects responsible for membrane protein deficiency improves the diagnosis accuracy and helps to better understand the genetic mechanisms of HS for the early care of each affected individual.

P1550: NEW GENERATION EKTACYTOMETRY STUDY OF RED BLOOD CELLS RHEOLOGICAL PARAMETERS IN HEMOGLOBINOPATHIES

E. Krishnevskaya 1 , M. Molero Magariño 1 , Á. Ancochea Serra 2 , J.-L. Vives Corrons 1,* , I. Hernandez 3 , V. Leguizamón 1

1 Red Blood Cell and Hematopoietic Disorders, Josep Carreras Leukaemia Research Institute (IJC); 2 Haematology-Haemotherapy Service, Catalan Institute of Oncology. Hospital Universitari Germans Trias i Pujol; 3 Red Blood Cell and Hematopoietic Disorders, ICO-Hospital Universitari Germans Trias i Pujol, Badalona, Spain

Background: Next Generation Ektacytometry is up to now one of the best complementary diagnostic tool for hereditary rare anemias due to red cell defects. Impairment of red cell deformability leading to the decrease of red cell survival rate, is the common trait of hereditary haemolytic anemias and some hemoglobinopathies In general this is the consequence of the abnormal cell shape, increased rigidity or dehydration. Classical examples of this situation are HS, unstable hemoglobinopathies, sickle cell disease and homozygous b-thalassemia (BT) but up to now, the OGE experience with structural hemoglobinopathies and thalassemia is scarce

Aims: We have evaluated the OGE profiles in 70 patients with different hemoglobinopathies, both structural and thalassemia, with the aim to analyse their usefulness for the early diagnosis of these disorders either individual or in coinheritance with other hereditary RBC defects. In addition this study aims to improve our knowledge of the contribution of red cell deformability, osmotic fragility and intracellular viscosity to the pathophysiology of hemolysis especially when these disorders are a cause of rare anemia

Methods: The patients included in this study have been studied via a stepwise process including RBC morphology, high performance liquid chromatography (HPLC) complemented by Hb electrophoresis, and the measurement of common RBC enzyme activities.When necessary, the genetic diagnosis of hemoglobinopathies was performed by t-NGS. OGE provided by the osmoscan module of the Laser Optical Rotational Red Cell Analyser (LoRRca) MaxSis provides four main parameters1. The elongation index (EImax), that depends mostly of the cytoskeleton mechanics 2. The osmotic fragility (Omin) that depends on the surface-to-volume ratio (S/V), 3. The RBC hydration status (Ohyper) that depends of the intracellular viscosity or hemoglobin concentration (MCHC) and 4. The area under the curve (AUC) or the distance between the starting point in the hypo-osmolar region (Omin) and an ending point in the hyper-osmolar region (Ohyper) that partially depends of red cell deformability

Results: Exception made of HbE, in all the structural hemoglobinopathies and thalassemia the tail of the osmoscan curve shifts to the left suggesting the existence of a decreased osmotic fragility and dehydration. Significantly decreased (p<0.01) parameters have been found for EImax in HbC, HbSS and BT, Omin in HbC, HbS, HbSS, HbSC and BT, Ohyper in all hemoglobinopathies and AUC in HbC, HbSS and BT. The OGE curves of all the hemoglobinopathies are shown in the FIGURE

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Summary/Conclusion: The RBC shape and, hemoglobin concentration are important factors that contribute to maintaining the hydration/dehydration equilibrium, the osmotic fragility and cell rigidity. A characteristic of all structural hemoglobinopathies, exception made of HbE, and of BT is the decreased red cells osmotic fragility (Omin) and dehydration (Ohyper) that is specially marked for HbC, HbSS and BT. In HbC and HbSS this effect is probably due to the ion transport dysfunction created by the Hb polymerization and Hb crystallization respectively,whereas in BT it may be the consequence of an excess of alpha chains due to the imbalance of beta and alpha chains synthesis. We can conclude that, in contrast with RBC membrane defects, the Osmoscan profile do not allow a clear differentiation between hemoglobinopathies including BT. However, it provides a complementary information on red cells rheological behavior characterized by a decreased deformability and dehydration

P1551: CONFORMATIONAL STUDY OF PYRUVATE KINASE DEFICIENT VARIANTS WITH HEMOLYTIC ANEMIA. THE RELATIONSHIPS BETWEEN THE LOSS OF ENZYME FUNCTION AND STRUCTURAL DISRUPTION

L. Jorda 1,* , L. Montllor 2 , E. Krisnevskaya 2 , V. Leguizamón 2 , I. Hernandez 3 , J. L. Gelpi 4 , J.-L. Vives Corrons 2

1 Supercomputing Center, BSC, Barcelona; 2 Red Blood Cell and Hematopoietic Disorders, Josep Carreras Leukaemia Research Institute (IJC); 3 Hematology Department, ICO-Hospital Universitari Germans Trias i Pujol, Badalona; 4 Dept. Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain

Background: Hereditary pyruvate kinase (PK) deficiency is a known cause of rare anemia characterized by accelerated destruction of red blood cells in the spleen or hemolysis. This enzymopathy shows a high clinical and genetic heterogeneity; thus, its diagnosis is sometimes difficult. The administration of an allosteric PK activator (Mitapivat) that stabilizes the enzyme and increases its enzymatic activity is recently being tested. This has led to a renewed interest in the study of the genotype-phenotype relationship in patients with PK deficiency. The assessment of the impact of a given pathogenic mutation on the conformational structure and dynamics (and thus the enzymatic activity) of the protein can enable a prediction of the possible response to treatment with the allosteric activator Mitapivat

Aims: The objective of our study is to analyze the effect of the genetic mutation on the 3D structure of the enzyme and to investigate the impact on its activity and function with the insight provided by conformational modeling techniques

Methods: 15 patients, homozygous or double heterozygous for PK deficiency have been included in this study. The promoter region, the exomic regions and the flanking intronic regions of the PKLR gene have been sequenced. The functional impact of the 12 identified missense mutations has been studied on the basis of the 3D structure of the enzyme. The human PK protein in the R-state was used as the starting point of the study (Protein Data Bank entry “2VGB”). Each amino acid replacement was computationally modeled. Molecular dynamics (MD) simulations were then performed on each molecular system (without ligands). The local disturbances of the mutation spots in comparison with those of the native protein during the simulations were examined as follows. Firstly, by describing the major differences in interactions between the amino acids in the vicinity of the mutation spot. Secondly, by comparing their dynamical flexibility profile in terms of the root-mean-square fluctuation metric

Results: The majority of the analyzed variants display altered flexibility features in comparison with the native enzyme. A missense mutation in a given position alters the local network of amino acidic contacts. The type of functional impact of each mutation depends on the protein region where the replacement occurs

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Summary/Conclusion: Clinical studies and structural bioinformatics should work together towards precision medicine. This joint effort will enable more accurate treatments, early diagnosis and even genetic counselling. The study of PK deficiency using computational modeling helps confirm the disruptive mechanism of pathogenic variants and predict whether the administration of an allosteric activator like Mitapivat could boost enzymatic activity. For instance, variants that affect positions involved in the binding of ligands would likely not be responsive to such treatment

P1552: A PHASE 2, OPEN-LABEL STUDY TO ASSESS SAFETY, TOLERABILITY, AND CLINICAL EFFECT OF ANX005 IN PATIENTS WITH WARM AUTOIMMUNE HEMOLYTIC ANEMIA AND EVIDENCE OF COMPLEMENT ACTIVATION

U. Jaeger 1,* , C. Broome 2 , R. Go 3 , A. Patriarca 4 , A. Roeth 5 , Q. Chang 6 , N. Weedin 6 , P. Adams 6 , E. Cahir-McFarland 6 , H.-A. Kroon 6

1 Medical University of Vienna, Vienna, Austria; 2 MedStar Georgetown University Medical Center, Washington, DC; 3 Mayo Clinic, Rochester, MN, United States of America; 4 AOU Maggiore della Carita, Novara, Italy; 5 Klinik für Hämatologie und Stammzelltransplantation, Essen, Germany; 6 Annexon Biosciences, South San Francisco, CA, United States of America

Background: Autoimmune hemolytic anemia (AIHA) is caused by autoantibody destruction of red blood cells (RBCs) and is divided into warm autoimmune hemolytic anemia (wAIHA) and cold agglutinin disease. In wAIHA, which accounts for 60-70% of AIHA cases, polyclonal immunoglobulin G (IgG) antibodies cause hemolysis of RBCs at core body temperatures. However, approximately 50% of wAIHA cases are not caused by pathogenic IgG alone. The correlation between autoimmune hemolysis and presence of complement on RBCs in wAIHA patients suggests a role for the complement system in the pathogenesis of wAIHA, which can be triggered by C1q binding to autoantibodies bound to RBCs. ANX005 is a humanized IgG4 monoclonal antibody that selectively binds and inhibits C1q, blocking activation of the classical complement pathway, with the potential to inhibit complement-mediated hemolysis in wAIHA.

Aims: The goal of this clinical study is to evaluate two once-weekly IV infusions of ANX005 in patients with wAIHA having evidence of complement pathway activation to understand the effect of C1q inhibition on pathway markers and objective disease parameters.

Methods: This is an open-label, repeat-dose, Phase 2 study ( NCT04691570 ) of ANX005 in patients with wAIHA and evidence of classical complement pathway activation. Up to 12 patients will be enrolled in this study and undergo a screening period of up to six weeks prior to Day 1. Patients will receive two doses of ANX005 on Days 1 and 8. Safety and pharmacokinetic (PK)/pharmacodynamic (PD) evaluations will be completed on Days 4, 15, 22, 29, 36, 43, 50, 57, and 71. Individual study duration will be up to 16 weeks, and patients will complete study participation on Day 71. The primary objective is to evaluate the safety and tolerability of ANX005. Secondary objectives include evaluating the PK and PD profile of ANX005, examining markers of classical pathway activation, and determining the impact of classical complement inhibition on hemoglobin levels and biomarkers of hemolysis through Day 71 in this subset of patients.

Results: Results from the Phase 2 trial are expected by the end of 2022.

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Summary/Conclusion: In this Phase 2 study of ANX005 in patients with wAIHA and evidence of classical complement pathway activation, patients will receive two IV doses of ANX005 one week apart with the goal of evaluating its safety, tolerability, PK/PD, and clinical effect to further understand the role of classical complement in this heterogeneous disease as a target for effective therapy.

P1553: PREVALENCE AND IMPACT OF IRON DEFICIENCY ANEMIA IN A COHORT OF PATIENTS WITH CROHN’S DISEASE

A. Abomhya 1,* , W. Tai 1 , W. Saadedeen 1 , O. Ajala 1 , Z. Abdulsada 2 , A. Sadeghi 3 , J. Shim 4 , R. Mohamed 5

1 Internal Medicine, The Brooklyn Hospital Center, Brooklyn; 2 Internal Medicine, Meharry Medical College School of Medicine, Nashville; 3 The Brooklyn Hospital Center, Brooklyn, United States of America; 4 St. George’s University School of Medicine, West Indies, Grenada; 5 Digestive diseases and nutrition department, University of Kentucky, Lexington, United States of America

Background: Iron deficiency anemia (IDA) is the most common cause of anemia worldwide. Crohn’s disease (CD) is a chronic inflammatory disease that affects the digestive system and is associated with a significant risk of chronic blood loss. USA has one of the highest prevalences of CD worldwide, with 214 per 100,000 diagnosed with CD. The nature of CD puts the patients at risk of IDA as well as anemia of chronic disease. Few studies evaluated the association between IDA and CD.

Aims: We aimed to estimate the prevalence of IDA in patients with CD. Secondary outcomes included the effect of IDA on mortality, the length of stay, all-cause 30-day non-elective readmission rate, and total cost of hospitalization.

Methods: We queried the Nationwide Readmission Database 2018 using ICD-10-CM codes to identify all adult patients admitted with a diagnosis of CD. Patients with a comorbid diagnosis of IDA were identified. The case selection process is summarized in Figure 1. Median and IQR were used to describe Continuous variables, and proportions were used with categorical variables. Comparison between groups was performed by Mann Whitney test for continuous variables and Chi-Square test for Categorical variables. Multivariate regression analysis was performed to study the impact of IDA on inpatient mortality and non-elective readmissions. Statistical analyses were performed using SPSS Version 25 (IBM Corporation, Armonk, NY, USA).

Results: We extracted 72,076 index hospitalization records for CD, 8.1% had IDA. CD patients with IDA had increased length of stays in days (4; Interquartile Range (IQR): 2-6 vs. 3; IQR: 2-5, P <0.001), increased median total charges ($35,160; IQR: $19,786 - $64,126 vs. $31,299; IQR: $17,226 - $59,561, P <0.001) and were more common to require blood transfusion during hospitalization (13.6% vs. 3.4%, P <0.001) compared to CD patients without IDA, respectively. There was no statistically significant difference in mortality between CD patients with and without IDA (Odds ratio (OR): 0.79; 95% confidence interval (CI): 0.595-1.049, P= 0.103). IDA was independently associated with increased odds of all-cause 30-day non-elective readmission (OR: 1.254; 95% CI: 1.154-1.363, P <0.001).

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Summary/Conclusion: IDA is a common extraintestinal manifestation of Crohn’s disease. We observed a significant association between IDA and worse hospitalization outcomes in our nationwide cohort. Early recognition and management of IDA among patients with CD might be important to improve hospitalization outcomes. Further research studies are needed to improve screening, diagnosis, and management of IDA in this patient population.

P1554: INCREASED PREVALENCE OF THROMBOEMBOLIC EVENTS IN PATIENTS WITH CONGENITAL DYSERYTHROPOIETIC ANEMIA TYPE I

M. Asleh 1,* , A. Khalaila 1 , Y. Eshel 1 , H. Miskin 1 , J. Kapelushnik 1

1 Pediatric Hemato-oncology, Soroka University Medical Center, Be’er Sheva, Israel

Background: Congenital Dyserythropoietic Anemia Type I (CDA-I) is a rare disorder of erythropoiesis. All CDA-I patients are expected to suffer from iron overload and chronic hemolysis. Some patients with severe anemia may undergo splenectomy. Hemochromatosis, chronic hemolysis and splenectomy are all found to increase the risk for thromboembolism in thalassemic patients. Studies on thalassemic patients report an increased risk of thromboembolism, with an incidence ranging between 1.7 and 9.2% in thalassemic patients, compared to an annual incidence of 0.75-2.69 per 1000 in the general population. As a disease of ineffective erythropoiesis, CDA-I shares few pathophysiologic characteristics with thalassemia. Yet, there are no reports on the prevalence of thromboembolism in CDA-I patients, with only one case of pulmonary embolism reported in an adult patient

Aims: This study sought to evaluate prevalence of thromboembolic events in a group of CDA-I patients, and try to ascertain if this group of patients is at an increased risk of thromboembolism requiring a special follow-up.

Methods: a retrospective case-control study including 110 CDA-I patients (study group) and 326 age and sex-matched patients of the same ethnicity diagnosed with iron deficiency anemia (control group). . Medical charts of all patients in both groups were retrieved from the hospital’s main archive and reviewed for the presence of venous and/or arterial thrombosis. Charts were screened to determine risk factors for thromboembolic events. Patients were risk-stratified using the 2010 Caprini Risk Assessment Model for thromboembolism.

Continuous variables were expressed as means and standard deviations (SD). The Student’s t-test was used to compare the distribution of continuous variables. Correlation between continuous variables were assessed using Pearson correlations, where r>0.5 and p value < 0.05 indicated significant correlation.To analyze the association between exposure variables and thromboembolic events, logistic regression models were used to estimate unadjusted and adjusted odds ratios (ORs) with their 95% confidence intervals (CI 95%). The chi-square test of independence was used to determine if there is a significant relationship between categorical variables.

Results: We identified three cases (2.7%) with thromboembolic events in the CDA group and one case (0.3%) in the control group. All of them were female patients. Caprini risk assessment for venous thromboembolism was low to moderate for the three CDA patients and high for the IDA patient. When compared to the control group, patients with CDA-I were nine times more likely to develop a thromboembolic event (OR 9.11, 95% CI=1.15-185.27, p=0.057). All three CDA patients had a history of remarkable hemolysis and iron overload. Two of them underwent splenectomy. Gender was strongly associated with increased risk of thromboembolism, as all cases with thromboembolism in this study were female patients. Caprini scores ranged from mild to moderate risk (score =2-4) for CDA patients and was high (score =7) for the IDA patient. Mean values of ferritin, iron, LDH and bilirubin were significantly higher (p<0.001), and hemoglobin level was significantly lower (p<0.001) in CDA group compared to control group. As expected, iron was significantly correlated with ferritin (r=0.617, p<0.001), and bilirubin was significantly correlated with LDH (r=0.631, p<0.001).

Summary/Conclusion: These findings show that CDA patients appear to be at increased risk for thromboembolic events.

A high index of suspicion may help in early diagnosis and intervention in these patients.

P1555: IRON CHELATION THERAPY IN CONGENITAL HEMOLYTIC ANEMIAS: AN ITALIAN SINGLE CENTER STUDY

A. Bosi 1,* , B. Fattizzo 2 , J. A. Giannotta 3 , I. Papetti 4 , W. Barcellini 5

1 University of Milan, Milano, Italy; 2 Department of Hemato-oncology, University of Milan; 3 Department of Hematology, IRCCS Fondazione Ca’ Granda Policlinico Ospedale Maggiore, Milano; 4 University of Milan; 5 Department of Hematology, Fondazione IRCCS Ca’ Granda Policlinico Ospedale Maggiore, Milan, Italy

Background: Congenital hemolytic anemias (CHAs) can be associated with iron overload due to chronic hemolysis, ineffective erythropoiesis and, in some cases, red blood cell transfusions. Because of the rarity of these conditions, the appropriate management of iron chelation is poorly known and mainly based on thalassemia experience.

Aims: To evaluate frequency, duration, efficacy and tolerability of iron chelation in patients with CHAs followed at our reference center.

Methods: Clinical and laboratory data were retrospectively collected from patients who had documented history of iron chelation. Data were collected at time of diagnosis, at time of starting chelation and at variable times from treatment beginning. Follow-up data were censored at February 1 st 2022.

Results: 206 patients were retrospectively reviewed (151 with membrane defects, 31 with enzyme defects and 24 with congenital dyserythropoietic anemias – CDAs, Table 1). Twenty-six patients (13%) received iron chelation, mostly those with enzyme defects (35%) or CDAs (38%); at variance, only 4% of patients with membrane defects required chelation (p < 0.0001). Transfusion status showed that 31% of patients had history of transfusion-dependence (TD) (5 with Pyruvate kynase deficiency and 3 CDAs), 38% had received occasional transfusions and 31% had never received transfusions. Iron chelator was parenteral deferoxamine (DFA) or oral deferasirox (DFX), with a median daily dose of 573 mg for DFA and 584 mg for DFX. Median duration of treatment was 91 months with a wide range (3 – 372), including patients who received intermittent dosing (42%) and those on continuous chelation (58%). Concerning efficacy, median variation of serum ferritin from baseline was -51%, ranging from -94% to +6% (the latter in a patient who received a suboptimal dose). Overall, 64% of evaluable patients reduced ferritin below 500 ng/mL. Notably, a 33 year-old woman affected by CDA type II, with history of transfusion dependence since birth to age 9 y/o (time of splenectomy), developed cardiac iron overload with a cardiogenic shock. After a 4-year course of DFA, laboratory and imaging tests for iron overload markedly improved and her cardiac function recovered. Regarding toxicity, 5 out of 22 (23%) patients reported reversible renal (n = 2) and gastrointestinal adverse events (n = 2); one patient developed a non-fatal anaphylactic shock, leading to switch to a different chelator (from DFA to DFX).

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Summary/Conclusion: Iron chelation was required in 13% of patients with CHAs, mostly among CDAs and enzyme defects, independently from transfusion requirements. It allowed a reduction of iron parameters in about 2/3 of cases with a good tolerability.

P1556: HYPER -FERRITINEMIA IN PEDIATRIC ONCOLOGY PATIENTS SECONDARY TO TREATMENT: DIAGNOSTIC APPROACH AND OUTCOME

I. Grafakos 1,* , V. Papadakis 1 , K. Antoniadi 1 , M. Ampatzidou 1 , S. Papargyri 1 , C. Kelaidi 1 , V. Tzotzola 1 , S. Polychronopoulou 1

1 Department of Pediatric Hematology and Oncology (TAO), Agia Sofia Children’s Hospital, Athens, Greece

Background: Multiple red blood cell transfusions during cancer therapy are the leading cause of iatrogenic hyper- ferritinemia which of notice is often overlooked. Patients with genetic predisposition for hemochromatosis have a higher chance of developing hyper-ferritinemia, with a potential to promote significant organ dysfunction.

Aims: The aim of this study was to identify pediatric oncology patients with high levels of ferritin post cancer treatment, and refer their management and outcome.

Methods: Selected patients’ charts of our Department were retrospectively reviewed. Patient and treatment characteristics, ferritin levels and their progression over time, genetic testing for hemochromatosis genes, treatment and outcome were recorded and analyzed.

Results: Twelve (12) patients were identified with increased (>600 ng/ml) ferritin levels, diagnosed with Acute Lymphoblastic Leukemia (N=5), Acute Myeloid Leukemia (N=1), Non-Hodgkin Lymphoma (N=1), Neuroblastoma (N =2), Medulloblastoma (N=1), Rhabdomyosarcoma (N=2) from 1995 to 2020. Mean patient age at diagnosis was 7.1 years (range 3.0-15.2 years). Three patients had undergone Hematopoietic Stem Cell Transplantation, 2 of them twice. Mean blood transfusions per patient were 42 (range: 9-62), with 6 patients receiving more than 50 transfusions each (10-15 ml/kg). Of 6 patients tested for hemochromatosis genes, 2 were negative for the common mutations of HFE, TfR2 FPN1 genes, 1 was found double heterozygote for the His63Asp and Cys282Tyr HFE mutations and 1 patient carried the HFE H63D mutation. Eight (8/12) patients were observed only, and ferritin reduction of 5.7% to 69.7% was observed, within 12 months of follow-up, but none normalized ferritin levels, the lowest value being 560 ng/ml (Image). One patient received chelation therapy (deferasinox po) for 13 months, 1 patient (heterozygous for beta-thalassemia trait) received 37 phlebotomy sessions and 2 received combination of chelation (deferasirox only or with deferoxamine) and short-course phlebotomies. Out of the later intervention group of patients, none achieved normal ferritin levels after an observation period of 12 months. With a mean follow up of 3.7 years for all patients (range 1,1-15,7 years), mean ferritin levels after cancer therapy and at +12 months, were 2056 ng/ml (range: 621-4750 ng/ml) and 1227 ng/ml (range: 590-3550 ng/ml), respectively. Only 1 patient achieved ferritin levels under 500 ng/ml following 3-years of phlebotomies, and she has remained so, for another 4 years of follow-up (b-thalassemia heterozygote and Η63D HFE heterozygote for the hemochromatosis genes).

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Summary/Conclusion: Iatrogenic iron overload post antineoplastic therapy in pediatric patients is a common and often overlooked diagnosis. Prompt evaluation and appropriate treatment is necessary. Patients presenting with hyper-ferritinemia for extended time period could bear hemochromatosis gene mutations. Since the symptoms are absent initially, pediatric patients after cancer treatment should be screened for ferritin levels. If persistently high levels are documented, evaluation of relevant mutations and appropriate treatment is necessary. The long-term effect of high Ferritin levels is not fully documented, but it has the potential to promote significant organ dysfunction. Further research is necessary in this field.

P1557: DISTINGUISHING OF IRON REFRACTORY IRON DEFICIENCY ANEMIA FROM IRON DEFICIENCY ANEMIA AND THALASSEMIA TRAIT BY SYSMEX RESEARCH RED CELL PARAMETERS

E. Yilmaz 1 , Z. Kaya 1,* , S. Kirkiz 1 , Ü. Koçak 1

1 Pediatric Hematology, Gazi University School of Medicine, ANKARA, Turkey

Background: Iron refractory iron deficiency anemia (IRIDA), iron deficiency anemia (IDA), and Thalassemia trait (TT) are the most common causes of microcytic anemia. It is possible to discriminate between IDA and TT by routine complete blood count (CBC), hemoglobin (Hb) electrophoresis and iron parameters; however, it is very difficult to distinguish IRIDA from IDA and TT using only routine CBC and iron parameters.

Aims: The aim was to distinguish IRIDA from IDA and TT using Sysmex research red cell parameters.

Methods: The study included a total of 25 patients ( n =10, TT; n =10, true IDA; n = 5, genetically confirmed IRIDA). Traditional [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell count (RBC), and red cell distribution volume (%RDW)] and new red cell parameters [percentage hypochromic RBCs (%Hypo-He); percentage microcytic RBCs (%Micro-R)] were measured in a Sysmex-XN ® 2000 (Sysmex Corporation, Kobe, Japan), along with iron parameters. Participants’ disorders were categorized based on patients’ ferritin, Hb electrophoresis, genetic results, and CBC values.

Results: Compared with the IDA patients, IRIDA patients had significantly higher mean levels of ferritin, RBC, %Hypo-He, %Micro-R and significantly lower mean levels of serum iron level MCV, MCH, and MCHC values ( p <0.05 for all). Compared with TT patients, IRIDA patients had significantly higher mean levels of ferritin, %Hypo-He and %RD,W and significantly lower mean levels of serum iron level and MCH values ( p <0.05 for all). The RBC, Micro-R, Hypo-He, MCV and MCH cut-off values for discriminating IRIDA from IDA patients were 4.9µL, %60, 50%, 60fL, and 16.0pg, respectively. The RDW, Hypo-He, and MCH cut-off values for discriminating IRIDA from TT patients were %19, %30, and 17.0pg, respectively.

Summary/Conclusion: This first study has examined the utility of new red cell parameters in patients with IRIDA. Our findings should be confirmed in larger studies; however, we believe that higher percentage of hypochromic microcytic erythrocytes with an elevated RBC value may be useful predictors for distinguishing IRIDA from IDA patients. As well, a moderate percentage of hypochromic erythrocytes with high RDW value can guide physicians in discriminating IRIDA from TT patients

P1558: EARLY-START DEFERIPRONE IN INFANTS/YOUNG CHILDREN WITH TRANSFUSION-DEPENDENT BETA THALASSEMIA: EVIDENCE FOR IRON SHUTTLING TO TRANSFERRIN—RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED TRIAL (START)

F. Tricta 1,* , C. Fradette 1 , N. Temin 1 , A. Rozova 1 , D. Lee 1 , I. Cabantchik 2

1 Chiesi Canada Corporation, Toronto, Canada; 2 Alexander Silberman Institute of Life Sciences, Jerusalem, Israel

Background: Children with transfusion-dependent beta thalassemia will develop iron overload unless treated with an iron chelator such as deferiprone (DFP), deferasirox (DFX), or deferoxamine (DFO). Current practice delays chelation therapy until patients reach the iron overload criteria of serum ferritin (SF) levels ≥1000 µg/L or >10 transfusions. This “late-start” strategy was thought to minimize toxicity risks of iron depletion from biological stores or iron-bound proteins, such as transferrin, observed during “early-start” studies with DFO. This delay may increase the risk of iron overload toxicities later in life due to labile iron accumulation in organs difficult to reach with chelation. DFP is a membrane-permeable iron chelator with a lower iron affinity (pFe 3+ =19.3) than DFO (pFe 3+ =26.6), DFX (pFe 3+ =23.5), or transferrin (pFe 3+ =22.3). These pharmacological properties suggest “early-start” DFP could reduce the risks/toxicities associated with early iron overload and iron depletion by shuttling labile iron to transferrin. Previously, we reported that “early-start” DFP can be safely initiated in infants and young children with beta-thalassemia (START study NCT03591575 ) and maintain subthreshold SF levels in significantly more patients than placebo for up to 12 months.

Aims: To evaluate the ability of “early-start” DFP to shuttle iron to transferrin in infants and young children with transfusion-dependent beta thalassemia using transferrin-saturation (TSAT) measurements from the START study.

Methods: Infants or children with beta thalassemia who received ≥2 blood transfusions and had SF levels between 200–600 μg/L were randomly assigned 1:1 to DFP or placebo. Double-blinding continued until study end. Treatment continued until SF levels were ≥1000 μg/L at 2 consecutive visits (SF threshold) or 12 months. Patients started at 25 mg/kg/d DFP, escalated to 50 mg/kg/d DFP, with some to 75 mg/kg/d DFP, depending on iron load. The efficacy endpoint included imputation of % patients not reaching the SF threshold each month. DFP’s ability to shuttle iron to transferrin was evaluated at each month by comparing: 1) proportion of patients with ≥60% TSAT and 2) mean TSAT levels for DFP vs placebo groups. All iron measured in serum is total bound iron, which is the basis for calculating TSAT. The patient’s parent or legal guardian provided written informed consent before any study intervention.

Results: Mean (SD) age was 3.0 (2.4) and 2.6 (1.7) years in the DFP (n=32; 63% male) and placebo (n=32; 66% male) groups, respectively. At month 12, 66% of patients receiving DFP remained below the SF threshold versus 39% of patients receiving placebo (p=0.045).

Significantly more patients had TSAT ≥60% early with DFP than placebo at months 3–6 (each p<0.05); range: DFP 56%–66%, placebo 16%–34%. Similarly, mean TSAT levels with DFP were significantly higher than with placebo at months 3, 4, 6, 9, and 11 (each p<0.05); range: DFP 60%–69%, placebo 41%–55%. At 12 months, no DFP-treated patients were iron-depleted or reported iron depletion-associated adverse events (AEs). There were no significant differences between DFP and placebo in number of AEs (p=1.00), serious AEs (p=0.43), or AEs possibly related to DFP (p=1.00).

Summary/Conclusion: Early-start DFP chelation therapy initiated before reaching current iron-load criteria was safe and efficacious in preventing iron overload in most transfusion-dependent children with beta thalassemia. START provides the first clinical evidence supporting preclinical studies that DFP can shuttle iron from labile pools to transferrin.

P1559: DISC-0974, A FIRST-IN-HUMAN ANTI-HEMOJUVELIN MONOCLONAL ANTIBODY, REDUCES SERUM HEPCIDIN LEVELS AND MOBILIZES IRON IN HEALTHY PARTICIPANTS

N. Novikov, MD, PhD 1 , H. Yang, PhD 1 , S. Nguyen, MS 1 , A. Buch, PhD 1 , S. Tuller, JD, RAC 1 , M. Andruk, MBA 1 , K. Chan, PhD, MPH, MBA 1 , M. Wu, PhD 1 , R. Rodriguez 1 , R. Panwar, PhD 1 , H. Howell 1 , B. MacDonald, MBChB, PhD 1 , W. Savage, MD, PhD 1

1 Disc Medicine, Watertown, United States of America

Background: Hepcidin-induced iron restriction results from inflammatory stimuli and can cause anemia of inflammation. Hemojuvelin (HJV) is a glycosylphosphatidylinositol-anchored membrane protein expressed in iron-loading tissues that binds Bone Morphogenetic Protein (BMP) family ligands as a central regulator of hepcidin expression. DISC-0974 is a monoclonal antibody developed to target HJV and block binding of HJV with BMP ligands to reduce hepcidin production and treat anemia of inflammation.

Aims: This is a first-in-human, Phase 1a, double-blind, placebo-controlled single-ascending dose study of intravenous (IV) and subcutaneous (SC) DISC-0974 in healthy volunteers to evaluate safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD).

Methods: Eligible participants include healthy females of non-reproductive potential and males 18-65 years old with normal baseline red blood cell parameters, serum iron, and Total Iron Binding Capacity (TIBC), morning transferrin saturation (TSAT) <30%, and serum ferritin ≥30 ng/mL. Dosing is planned at the 7 mg IV, 14 mg SC, 28 mg SC, and 56mg SC doses, with Safety Review Committee assessment of data through Day 15, followed by unblinding in each cohort. Treatment is allocated in a 3:1 ratio with 8 participants planned per cohort. The primary endpoints are safety and tolerability. Secondary endpoints include PK of DISC-0974 and PD response evaluated using serum iron, TSAT, and hepcidin-25. Endpoints are summarized using descriptive statistics.

Results: The 7mg IV and 14mg SC cohorts have been unblinded, with a total of 18 participants dosed. Fourteen participants received DISC-0974, and 4 received placebo. In the active treatment group, the median age was 54 years [range: 19-62], 50% were females, and median baseline hemoglobin (Hgb) was 14.0 g/dL [11.7 – 17.3]. The placebo group had median age of 47 years [29 -64], 25% females, and median baseline Hgb of 15.1 [13.6 – 16.2]. No serious AEs, Grade >= 2 AEs, and AEs leading to study withdrawal were reported. Four Grade 1 AEs were seen in two placebo participants. No AEs related to DISC-0974 were reported.

Based on preliminary analysis, PK profiles showed anticipated patterns, with lower Cmax and longer Tmax in SC as compared to IV. Dose-related increases in serum iron and TSAT as well as hepcidin reduction were seen in participants treated with DISC-0974 as compared to placebo. The 7mg IV group had no appreciable difference from placebo, whereas the 14mg SC group had increased mean and median serum iron and TSAT between baseline and Day 8 as compared to placebo (Fig 1A). TSAT escalation stopping rules were not met in any subject, but in the 14mg group, two subjects had a majority of individual TSAT values >40% through Day 15, as compared to none in the 7mg group. Consistent with the iron data, a dose related decrease in hepcidin, as measured by area under the curve (AUC) calculations between baseline and Day 15, was observed (Fig 1B). Updated data will be presented at the meeting.

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Summary/Conclusion: Preliminary analysis of data from the 7 mg IV and 14 mg SC DISC-0974 cohorts shows an acceptable safety and tolerability profile, as well as evidence of target engagement and iron mobilization. Higher doses of DISC-0974 are under investigation and potentially represent a new therapeutic approach for patients with functional iron deficiency in a variety of disease states.

P1560: IMR-261, A NOVEL, ORAL NRF2 ACTIVATOR, REDUCES IRON OVERLOAD IN AN EXPERIMENTAL MOUSE MODEL BY ACTIVATING BMP6-MEDIATED HEPCIDIN SYNTHESIS

T. Trovati Maciel 1,* , C. Carvalho 1 , R. Rignault 1 , A. Slimane 1 , H. Drakesmith 2 , J. Isler OCain 3 , R. Ballal 3

1 Imagine Institute, Paris, France; 2 University of Oxford, Oxford, United Kingdom; 3 Imara, Boston, United States of America

Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates several cellular defense mechanisms, including the oxidative stress response. Recent studies elucidated a protective role for Nrf2 under conditions of iron overload. Accumulation of toxic iron activates Nrf2, which activates Bmp6-mediated synthesis of hepcidin, thereby inhibiting ferroportin and improving iron homeostasis (Lim et al , Nat Metab 2019). IMR-261 is a novel, oral Nrf2 activator which has been tested in Phase 2 clinical trials (previously as CXA-10). Our previous work in mouse models of sickle cell disease and b-thalassemia showed that activation of Nrf2 by IMR-261 increases fetal hemoglobin (HbF) and hemoglobin (Hb), decreases markers of hemolysis and adhesion, reduces VOCs and improves ineffective erythropoiesis.

Aims: The objective of this study was to evaluate the ability of IMR-261 to activate Bmp6-mediated synthesis of hepcidin (encoded by the Hamp gene), regulate iron stores and modulate transferrin saturation (TSAT) in a mouse model of iron overload.

Methods: b-thalasssemic mice (Hbb th1/th1 ) were treated with IMR-261 at 37.5 or 50 mg/kg BID for 4 weeks. To assess activation of the Bmp6-hepcidin pathway, hepatic mRNA and plasma protein levels were measured. Iron concentrations in serum and tissue (liver, kidney and spleen) as well as markers of iron overload were also evaluated, and standard hematologic assessments were performed.

Results: IMR-261 showed a significant, dose-dependent increase in hepatic mRNA levels of Bmp6 and Hamp (~3-fold increase; high dose vs vehicle) as well as Bmp6 target genes, Smad7 and Id1 . A corresponding significant increase in Bmp6 and hepcidin protein levels in plasma was also observed. IMR-261 significantly decreased iron in serum by 32% (high dose vs vehicle) and in all tissues tested (34% in liver, 39% in spleen and 24% in kidney; high dose vs vehicle). Mice treated with IMR-261 also showed a significant decrease in TSAT and ferritin levels (up to 30% and 27%, respectively), as well as a reduction in GDF15 and erythropoietin (up to 72% and 48%, respectively) relative to vehicle. IMR-261 improved hematologic markers including a 30% decrease in reticulocytes and significant increases in RBCs, hemoglobin and hematocrit (consistent with our previous data).

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Summary/Conclusion: Pharmacologic activation of Nrf2 by IMR-261 in a mouse model of iron overload activates hepcidin synthesis (likely via the Bmp6 signaling pathway) and decreases iron in serum, liver, spleen and kidney. IMR-261 also showed significant reductions in TSAT, ferritin, GDF15 and erythropoietin, which further support its role in iron homeostasis. Induction of hepcidin synthesis via Nrf2 activation is a novel therapeutic approach, and these results support further clinical development of IMR-261 as a treatment for conditions associated with iron dysregulation, including hereditary hemochromatosis.

P1561: IS IRON OVERLOAD ASSOCIATED WITH WORSE OUTCOMES IN PATIENTS WITH CHRONIC LIVER DISEASE UNDERGOING LIVER TRANSPLANT?

S. Rodriguez-Rodriguez 1,* , J. Delgado-de la Mora 2 , G. Zavala-Garcia 3 , B. Martinez-Benitez 2 , I. Garcia-Juarez 4 , R. Demichelis-Gomez 1

1 Hematology; 2 Pathology; 3 Internal Medicine; 4 Gastroenterology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Tlalpan, Ciudad de Mexico, Mexico

Background: Iron overload (IO) is found in up to 78% of patients with chronic liver disease (CLD), secondary to local iron deposits and hepcidin dysregulation. An increase in infections and non-relapse related mortality has been reported in patients with IO who underwent hematopoietic cell transplantation (HCT). The effect of IO in patients without hereditary hemochromatosis (HH) who underwent liver transplant (LT) is unknown.

Aims: To determine the clinical impact of IO in patients who underwent LT in a tertiary referral center.

Methods: Retrospective cohort study that included patients who underwent LT from 2015 to 2017 at the Instituto Nacional de Nutricion (INCMNSZ), excluding those with HH. Explant liver biopsies were analyzed by two pathologists, using Perls Prussian blue stain to detect iron deposits in hepatocytes, sinusoids, and portal areas. The sum of this achieved the total iron score (TIS), divided by age in order to obtain the histochemical hepatic iron index (HHII), considering a score ≥ 0.15 as IO. Group comparisons were made with Pearson chi-square and Mann-Whitney U test, a logistic regression was used to analyze factors associated with IO, and survival was analyzed with the log-rank test.

Results: A hundred and five patients were included. Kappa index between pathologists was 0.923 (IC 95% 0.849 – 0.997). Forty-seven patients had IO (44.7%), predominantly men (57.4%). Divided by etiology, IO was more frequent with viral (62.5%) and metabolic (53.6%) etiologies, compared to autoimmune and other causes (p=0.012). A multivariate analysis concluded that obesity (OR 4.35, IC 95% 1.52 – 12.45, p = 0.006) and alcohol consumption (OR 6.68, IC 95% 2.15 – 20.82, p=0.001) were associated with IO (w/IO). Median saturation index and ferritin were higher in patients w/IO, 47% (interquartile range (IQR) 74 – 84%) vs 25% (IQR 19 – 53%) without IO (w/o IO), and 347 ng/mL vs 61.2 ng/mL, respectively (both, p<0.001). Compared to liver biopsy, serum iron tests (SIT) had a sensitivity of 72.2% and a specificity of 87.1%. Regarding early (< 90 days) complications post LT, a trend towards a higher rate of complications was found in patients with IO (91.5 vs 77.6%, p=0.07), mainly due to metabolic complications (68.1 vs 46.6%, p=0.344). We found no difference in the patient’s length of stay or in intensive care unit stay, nor in infections, rejection or rehospitalization rates (Figure 1). Median follow-up was 61.5 months (IQR 49.7 – 72.9 months). All deaths occurred during the first 60 days, with an overall survival (OS) at 60 days of 92.4%, 97.9% in patients w/IO vs 87.9% w/o IO (p=0.006). Relapse occurred in 25.7% of the patients, 31.9% in the group w/IO and 20.7% w/o IO (p=0.26). Four-year disease-free survival (DFS) was 66.7% in patients w/IO vs 75.7% w/o IO (p=0.26).

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Summary/Conclusion: IO is frequent in patients with CLD, particularly in patients with viral and metabolic etiologies, and is associated with obesity and alcohol consumption. SIT had a specificity of 87.1% to exclude IO in patients with CLD. A trend towards a higher rate of metabolic complications was found, with a statistically significant higher OS rate at 60 days. No difference was found in relapse rate or DFS. Contrary to what has been described in other scenarios, in our cohort, IO was not associated with worse outcomes.

P1562: CHARACTERIZING IRON OVERLOAD BY AGE IN PATIENTS DIAGNOSED WITH PYRUVATE KINASE DEFICIENCY – A DESCRIPTIVE ANALYSIS FROM THE PEAK REGISTRY

P. Bianchi 1,* , R. F. Grace 2 , J.-L. Vives Corrons 3 , B. Glader 4 , A. Glenthøj 5 , H. Kanno 6 , K. H. Kuo 7 , C. Lander 8 , D. M. Layton 9 , D. Pospíŝilová 10 , V. Viprakasit 11 , J. Williams 12 , Y. Yan 12 , B. McGee 12 , E. J. van Beers 13

1 Foundation IRCCS Ca’ Granda, Milano, Italy; 2 Children’s Hospital Boston, Boston, United States of America; 3 Rare Anemias Research Unit, Institute for Leukaemia Research Josep Carreras, Barcelona, Spain; 4 Division of Pediatric Hematology/Oncology/Stem Cell Transplant and Regenerative Medicine, Stanford University School of Medicine, Stanford, United States of America; 5 Department of Hematology, Rigshospitalet, Copenhagen, Denmark; 6 Department of Transfusion Medicine and Cell Processing, Tokyo Woman’s Medical University, Tokyo, Japan; 7 Division of Medical Oncology and Hematology, University of Toronto, Toronto, Canada; 8 Thrive with PK Deficiency, Bath; 9 Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom; 10 Department of Pediatrics, Palacky University and University Hospital, Olomouc, Czechia; 11 Siriraj Hospital, Mahidol University, Bangkok, Thailand; 12 Agios Pharmaceuticals Inc., Cambridge, United States of America; 13 Van Creveldkliniek Department of Internal Medicine, University Medical Center Utrecht, Utrecht, Netherlands

Background: Pyruvate kinase (PK) deficiency is a rare, congenital, glycolytic enzyme defect that leads to chronic hemolytic anemia and other significant complications, including iron overload (FeO). FeO in PK deficiency is clinically underappreciated and may cause long-term organ damage and impact growth in children and adults.

Aims: Describe characteristics of patients (pts) with PK deficiency and history of FeO by age (pediatrics, <18 years [yrs]; adults, ≥18 yrs) enrolled in the Peak Registry ( NCT03481738 ).

Methods: The Peak Registry is a global retrospective and prospective observational study of adult and pediatric pts diagnosed with PK deficiency. For this analysis, pts were considered to have a history of FeO if they had ever received 1) chelation therapy; or 2) phlebotomy for removal of iron; or within 3 months of enrollment had 3) ferritin >1000 ng/mL; or 4) liver MRI (including FerriScan ® ) >3 mg Fe/g dry weight; or 5) cardiac T2* MRI ≤20 ms. Data collected at study enrollment were summarized descriptively among pts with a history of FeO.

Results: As of 24Mar2020, 57/140 (40.7%) enrolled pts had a history of FeO (28 adult and 29 pediatric pts); data from these 57 pts were used for this analysis. PKLR genotype information was available for 21 adult and 15 pediatric pts; among adults, 13 (61.9%) had two missense mutations (M/M), 7 (33.3%) had one missense/one non-missense mutation (M/NM), and 1 (4.8%) had two non-missense mutations (NM/NM); among pediatric pts, 3 (20%) had M/M, 7 (46.7%) had M/NM, and 5 (33.3%) had NM/NM (Table). Among FeO pts with known transfusion status, 6/56 (10.7%) had never been transfused, with all other pts having received ≥1 prior transfusion (ever-transfused). Median hemoglobin (Hb) level at the time of enrollment was 8.6 g/dL (range: 6.7–12.5) in ever-transfused adults and 10.9 g/dL (9.3–11.3) in never-transfused adults. In pediatric pts (all ever-transfused), median Hb at enrollment was 8.3 g/dL (6.8–10.8). Most ever-transfused pts had received chelation therapy (18/20 [90.0%] adults, 28/29 [96.6%] pediatrics). Of the 6 pts who had never been transfused, 3 (50.0%) had received chelation therapy. Transfusion details for the 12 months prior to enrollment were available for 23 adults, of whom 5 (21.7%) had been regularly transfused (≥6 transfusions) and 18 (78.2%) had not been regularly transfused (0–5 transfusions). In the pediatric cohort, 8/24 (33.3%) pts had been regularly transfused and 16/24 (66.7%) had not. In the prior 12 months, regularly transfused adult and pediatric pts each received an average of about 10 transfusions whereas non-regularly transfused adult and pediatric pts received about 1 transfusion. Additionally, 24/28 (85.7%) adult and 15/29 (51.7%) pediatric pts with FeO had a splenectomy, with median age (range) at splenectomy of 6.5 yrs (1–23) and 6.0 yrs (4–12), respectively. Chelation therapy had been utilized by 20/23 (87.0%) splenectomized adults and 2/4 (50.0%) non-splenectomized adults, while 15/15 (100%) splenectomized pediatric pts and 13/14 (92.9%) non-splenectomized pediatric pts had received chelation therapy.

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Summary/Conclusion: FeO is a common complication affecting 41% of pts with PK deficiency in this cohort, regardless of age, genotype, transfusion status, or splenectomy status. FeO still occurs in both adult and pediatric pts without splenectomies, in pts who are not regularly transfused, and even in those who have never been transfused. Evaluation and regular monitoring of FeO in all pts with PK deficiency should begin early in life and continue throughout adulthood.

P1563: INTRACELLULAR IRON OVERLOAD IMPAIRS HSC FUNCTION BY AFFECTING MITOCHONDRIAL FITNESS IN ΒETA-THALASSEMIA

S. Sighinolfi 1,2,* , A. Aprile 1 , L. Cassina 3 , M. Panzeri 4 , S. Beretta 1 , M. Storto 1 , A. Boletta 3 , I. Merelli 1,5 , G. Ferrari 1,2

1 San Raffaele-Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy; 2 Vita-Salute San Raffaele University, Milan, Italy; 3 Division of Genetics and Cell Biology, Dibit, San Raffaele Scientific Institute, Milan, Italy; 4 Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC), San Raffaele, Milan, Italy; 5 Institute for Biomedical Technologies, National Research Council, Segrate, Milan, Italy

Background: β-Thalassemia (BT) is a genetic hemolytic anemia caused by reduced or absent synthesis of hemoglobin β-chains. Iron overload (IO), associated with ineffective erythropoiesis and therapeutic blood transfusions, is one of the main complications, leading to morbidities in various organs. Despite the improvement in chelation therapies over the past years, IO toxicity is an important issue for a successful therapeutic outcome. Recently, we demonstrated an impaired function and activated transcriptional responses to stress in HSCs from thalassemic th3 mice. We hypothesized that IO and the resulting oxidative stress might impair the bone marrow (BM) niche, thus interfering with the maintenance of HSCs. We also reported that IO reduces the hematopoietic supportive capacity of BM mesenchymal stromal cells in BT patients. However, there is no evidence of the direct effect of IO on HSCs in BT.

Aims: To understand the key factors affecting BT HSC function it is essential to gain novel insight into BT pathophysiology. To this aim, we investigated the role of iron on HSCs.

Methods: Gene expression profiling of HSCs from th3 mice was assessed by RNAseq. IO and oxidative stress were analyzed by flow cytometry. Mitochondria were evaluated by flow cytometry and transmission electron microscopy. We studied HSC metabolism by ATP analysis after in vitro inhibition of the oxidative phosphorylation (OXPHOS) using oligomycin and glucose uptake assay by flow cytometry. HSC function was evaluated following in vivo treatment with the mitochondria-targeted antioxidant MitoQ.

Results: We found a positive enrichment of genes involved in iron homeostasis, such as TfR1 , Steap3 , Fth1 and Ftl1 , in th3 HSC transcriptome suggesting iron uptake and intracellular storage. These findings are corroborated by high levels of free reactive iron (Fe 2+ ) in th3 HSCs. Interestingly, we reported an accumulation of Fe 2+ in the mitochondria, correlating with a 2-fold increase in mitochondrial reactive oxygen species (ROS) content. As a result, mitochondria in th3 HSCs are impaired, with low mass and activity, as assessed by decreased mitochondrial membrane potential. Moreover, th3 HSCs showed a reduced expression of mitochondrial biogenesis and mitophagy genes, thus suggesting an accumulation of damaged mitochondria.

In line with mitochondrial dysfunction, in vitro inhibition of OXPHOS did not affect ATP levels in th3 HSCs. Interestingly, they preferentially rely on glycolysis rather than OXPHOS for their energy demands, as shown by the positive enrichment of glycolytic genes and by the 1.4-fold higher glucose uptake of th3 HSCs.

Finally, in vivo reduction of mitochondrial ROS by MitoQ administration rescues mitochondrial activity and th3 HSC quiescence. Ongoing studies on iron chelation will reveal how HSCs sense iron and the molecular mechanism leading to intracellular IO, providing clues for improving BT HSC function.

Summary/Conclusion: Our study unveils that IO directly affects HSCs in BT. IO promotes oxidative stress, which in turn induces mitochondrial dysfunction. Alterations in mitochondrial activity and metabolic profile, in response to IO, are expected to negatively impact HSC function.

This research will add novel contribution to the field of iron regulation and HSC biology by unraveling how intracellular iron and ROS affect the metabolic and transcriptional programs underlying HSC function and fate.

P1564: FAMILIAL ERYTHROCYTOSES IN THE CZECH REPUBLIC - GENETIC CHARACTERIZATION AND HEPCIDIN REGULATION

L. Sochorcová 1,* , K. Hlusickova Kapralova 1 , O. Jahoda 1 , J. Manakova 2 , E. Kriegova 2 , B. Kralova 1 , J. Fialova Kucerova 1 , M. Divoka 3 , D. Prochazkova 4 , D. Pospisilova 5 , V. Divoky 1 , M. Horvathova 1

1 Department of Biology; 2 Department of Immunology, Faculty of Medicine and Dentistry, Palacky University; 3 Department of Hemato-Oncology, Faculty Hospital Olomouc, Olomouc; 4 Department of Pediatrics, Faculty of Health Studies, J.E. Purkyne University, Usti nad Labem; 5 Department of Pediatrics, Faculty Hospital Olomouc, Olomouc, Czechia

Background: Absolute erythrocytosis is a condition characterized by elevated erythrocyte mass. Familial erythrocytoses represent a group of rare inherited red blood cell disorders caused by mutations in genes involved in erythropoietin receptor (EPOR) signal transduction, hypoxia signaling pathway, or hemoglobin-oxygen affinity regulation. Nevertheless, up to 70% of patients have unknown etiology. Similarly, the coordination of erythropoietic activity with iron homeostasis in different subtypes of familial erythrocytoses needs to be clarified.

Aims: We performed molecular-genetic analysis of 13 pediatric patients with inherited erythrocytosis from the Czech Republic and assessed co-regulation of erythropoiesis and iron metabolism, including evaluation of hepcidin and its negative erythroid regulator erythroferrone (ERFE).

Methods: Genomic DNA was used for Sanger sequencing, next generation sequencing (NGS), or whole exome sequencing (WES). The sensitivity of erythroid progenitors to erythropoietin (EPO) was determined in vitro . Serum EPO levels and iron status parameters were measured; hepcidin and ERFE levels were determined with commercial ELISA kits.

Results: Using targeted Sanger sequencing we found a causative mutation in 5/13 patients from the cohort. Three unrelated patients with low EPO levels and hypersensitivity of erythroid progenitors to EPO harbored mutations in EPOR. A known heterozygous c.5967insT EPOR mutation was identified in two of them; the third patient carried a novel heterozygous c.11092delTG EPOR mutation. Both these mutations lead to a truncation of EPOR. A previously reported heterozygous p.G537R mutation in hypoxia-inducible factor alpha (HIF2A), encoded by EPAS1, was detected in two unrelated patients with normal EPO levels and EPO-hypersensitive erythroid progenitors. Targeted NGS of erythrocytosis-associated genes was applied in the other 6/13 patients. The analysis was negative in all but one patient in whom a rare germline JAK2 variant (p.N1108S) was identified, known from triple-negative myelofibrosis patients and presumably predisposing to acute myeloid leukemia. The remaining 2/13 patients, siblings with plethora, splenomegaly, normal sensitivity of erythroid progenitors to EPO, and elevated/normal EPO, were subjected to WES. A known mutation in a cryptic exon of VHL gene (c.340 + 770T>C) was found in a homozygous state in both of them; their parents were heterozygous asymptomatic carriers of this mutation.

We then focus on the assessment of iron metabolism; 6 patients from the cohort (2 EPOR-mutant, 2 HIF2A-mutant, and 2 VHL-mutant) were available for hepcidin and ERFE measurements. All EPOR-mutant and HIF2A-mutant patients had normal serum iron and ferritin levels, and reduced/subnormal transferrin saturation; both VHL-mutant patients were iron deficient. Slightly elevated ERFE and low/undetectable hepcidin levels were observed in HIF2A- and VHL-mutant patients. On the other hand, EPOR-mutant patients had normal hepcidin and ERFE levels below the detection threshold, suggesting that ERFE is not the main regulator of hepcidin in EPOR-mutated erythrocytosis.

Summary/Conclusion: We identified disease-causing mutations in 7/13 erythrocytic patients from our cohort. Despite the use of targeted NGS, almost half of the cases remained idiopathic. In these patients, WES or WGS should be used. We also suggest that EPOR-mutant erythrocytoses differentiate from HIF2A- and VHL-mutant erythrocytoses not only in low EPO levels, but also in the regulation of systemic iron homeostasis.

Grants support: NV19-07-00412; IGA_LF_2022_003.

P1565: MITAPIVAT IMPROVES INEFFECTIVE ERYTHROPOIESIS AND REDUCES IRON OVERLOAD IN PATIENTS WITH PYRUVATE KINASE DEFICIENCY

E. J. van Beers 1,* , H. Al-Samkari 2 , R. F. Grace 3 , W. Barcellini 4 , A. Glenthøj 5 , M. P. Judge 6 , P. A. Kosinski 6 , R. Xu 6 , V. Beynon 6 , B. McGee 6 , J. B. Porter 7 , K. H. M. Kuo 8

1 Van Creveldkliniek, Department of Internal Medicine, University Medical Center Utrecht, Utrecht, Netherlands; 2 Division of Hematology, Massachusetts General Hospital, Harvard Medical School; 3 Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, MA, United States of America; 4 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 5 Department of Haematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 6 Agios Pharmaceuticals, Inc., Cambridge, MA, United States of America; 7 Haematology Department, University College London Hospitals, London, United Kingdom; 8 Division of Hematology, University of Toronto, Toronto, ON, Canada

Background: Pyruvate kinase (PK) deficiency is a rare hereditary disease resulting in chronic hemolytic anemia, which is associated with serious complications, including iron overload (FeO). Ineffective erythropoiesis is linked to FeO in patients (pts) with hemolytic anemias. Mitapivat (AG-348), an oral, allosteric activator of the red blood cell PK enzyme (PKR), has demonstrated improvement in hemoglobin, hemolysis, and transfusion burden in pts with PK deficiency.

Aims: Assess effect of mitapivat on markers of erythropoiesis and FeO in pts (≥18 years [yrs]) with PK deficiency enrolled in 2 phase 3 studies, ACTIVATE ( NCT03548220 ) and ACTIVATE-T ( NCT03559699 ), and their long-term extension (LTE) study ( NCT03853798 ).

Methods: In ACTIVATE (double-blind, placebo [PBO]-controlled study), 80 adult pts with PK deficiency who were not regularly transfused (≤4 transfusion episodes in prior yr; none in prior 3 months) were randomized 1:1 to receive mitapivat or PBO. In ACTIVATE-T (open-label, single-arm study), 27 adult pts with PK deficiency who were regularly transfused (≥6 transfusion episodes in prior yr) were treated with mitapivat. Pts completing either trial (24 weeks [wks] [ACTIVATE], 40 wks [ACTIVATE-T]) were eligible to continue in the LTE, where all received mitapivat. Erythropoiesis markers included erythropoietin (EPO), erythroferrone, reticulocytes, and soluble transferrin receptor (sTfR). Markers of FeO included hepcidin, iron, transferrin saturation (TSAT), ferritin, and liver iron concentration (LIC) by magnetic resonance imaging. Pts from ACTIVATE were categorized into mitapivat-to-mitapivat arm (M/M) or PBO-to-mitapivat arm (P/M). The ACTIVATE-T/LTE analysis includes pts who achieved transfusion-free status in ACTIVATE-T. The ACTIVATE/LTE analysis assessed change in markers from baseline (BL) over time in both study arms.

Results: 80 pts were included in the ACTIVATE/LTE analysis (M/M=40; P/M=40). Pts in both arms had abnormal BL erythropoiesis markers consistent with underlying ineffective erythropoiesis, and abnormal BL markers of FeO. In the M/M arm, mean (SD) EPO, erythroferrone, reticulocytes, and sTfR decreased from BL to Wk 24 of mitapivat treatment: –32.9 IU/L (62.47), –9834.9 ng/L (13081.15), –202.0 10 9 /L (246.97), and –56.0 nmol/L (82.57), respectively, they remained stable or increased in the P/M arm on PBO. 24 wks after starting mitapivat in the LTE (Wk 48 post BL), pts in the P/M arm had comparable beneficial decreases in mean (SD) EPO, erythroferrone, reticulocytes, and sTfR: –11.6 IU/L (30.74), –9246.1 ng/L (8314.17), –283.7 10 9 /L (374.27), and –38.7 nmol/L (48.37), respectively. Improvements in hepcidin, iron, TSAT, and LIC were also observed with mitapivat treatment; ferritin remained stable (Table). Mean (SD) hepcidin increased in the M/M arm at Wk 24 and in the P/M arm 24 wks after starting mitapivat (Wk 48 post BL). At Wk 24, mean (SD) iron and TSAT, and median (Q1, Q3) LIC decreased in the M/M arm, while they increased on PBO. In the P/M arm, iron, TSAT, and LIC decreased 24 wks after starting mitapivat (Wk 48 post BL). Transfusion-free responders from ACTIVATE-T (n=6) also experienced improvements in markers of erythropoiesis and FeO in the LTE.

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Summary/Conclusion: These data indicate that activation of PKR with mitapivat improves markers of ineffective erythropoiesis and iron homeostasis in PK deficiency, thereby decreasing FeO in these pts. Mitapivat is the only approved pharmacotherapy in PK deficiency, and may have the potential to improve iron metabolism and reduce FeO in pts with this condition.

P1566: PROTON PUMP INHIBITION FOR SECONDARY HEMOCHROMATOSIS IN HEREDITARY ANEMIA, A PHASE III PLACEBO-CONTROLLED RANDOMIZED CROSS-OVER CLINICAL TRIAL

A. Van Vuren 1,* , J. Kerkhoffs 2 , S. Schols 3 , A. Rijneveld 4 , E. Nur 5,6 , D. Peereboom 7 , Y. Gandon 8 , P. Welsing 9 , R. van Wijk 10 , R. Schutgens 1 , W. van Solinge 10 , J. Marx 11 , T. Leiner 12,13 , B. Biemond 5 , E. van Beers 1

1 Center for Benign Haematology, Thrombosis and Haemostasis, Van Creveldkliniek, University Medical Center Utrecht, Utrecht; 2 Department of Hematology, HagaZiekenhuis, Den Haag; 3 Department of Hematology, Radboud university medical center, Nijmegen; 4 Department of Hematology, Erasmus Medical Center Cancer Institute, Rotterdam; 5 Department of Hematology, Amsterdam University Centers; 6 Department of Blood Cell Research, Sanquin Research, Amsterdam; 7 Contactgroep Pyruvaatkinasedeficiëntie, Stichting Zeldzame Bloedziekten, Medemblik, Netherlands; 8 Department of Radiology, CHU Rennes, Rennes, France; 9 Division of Internal Medicine and Dermatology; 10 Central Diagnostic Laboratory - Research, Division of Laboratories, Pharmacy and Biomedical Genetics; 11 University Medical Center Utrecht, Utrecht, Netherlands; 12 Department of Radioloogy, Mayo Clinic, Rochester, United States of America; 13 Department of Radioloogy, University Medical Center Utrecht, Utrecht, Netherlands

Background: Iron overload is a severe general complication of hereditary anemias. Treatment with iron chelators is hampered by important side-effects, high costs and the lack of availability in many countries with high prevalence of hereditary anemias.

Aims: To investigate the safety and efficacy of esomeprazole in treatment of iron overload in patients with non-transfusion-dependent hereditary anemias.

Methods: Design Phase III randomized placebo-controlled cross-over trial.

Setting Enrollment from March 2018 through April 2019 in five centers of expertise in the Netherlands.

Participants Thirty adult patients with mild-to-moderate iron overload were enrolled (non-transfusion-dependent thalassemia (N=13), pyruvate kinase deficiency (N=8), congenital sideroblastic anemia (N=3), congenital dyserythropoietic anemia (N=3), sickle cell disease (N=2), and hereditary elliptocytosis (N=1)).

Intervention Esomeprazole 40mg twice daily and placebo for 12 months in a cross-over design.

Main Outcome and Measure Change of liver iron content measured by MRI.

Results: Treatment with esomeprazole resulted in a statistically significant reduction in liver iron content that was 0.55mg Fe/g dw larger than after treatment with placebo (95% CI [0.05 to 1.06]; p=0.03). Baseline liver iron content at start of esomeprazole was 4.99mg Fe/g dw (IQR 3.47; 7.21), versus 4.49mg Fe/g dw (IQR 2.96; 6.35) at start of placebo. Mean delta liver iron content after esomeprazole treatment was -0.57mg Fe/g dw (SD 1.20; N=24) versus -0.11 (SD 0.75; N=23) after placebo treatment. Changes in serum ferritin values were non-significant between treatment arms.

Esomeprazole was well tolerated, reported adverse events were mild and none of the patients withdrew from the study due to side effects.

Summary/Conclusion: Esomeprazole 40mg twice daily resulted in a significant reduction in liver iron content when compared to placebo in a heterogeneous group of patients with non-transfusion-dependent hereditary anemias with mild-to-moderate iron overload. From an international perspective this result can have major implications given the fact that proton pump inhibitors may frequently be the only realistic therapy for many patients without access to or not tolerating iron chelators. Longer follow-up is needed to see whether this effect will last for a longer period.

Trial Registration Netherlands Trial Register NL6659 – PPI in secondary hemochromatosis ( www.trialregister.nl ).

P1567: ALK2 INHIBITION LOWERED HEPCIDIN AND LIBERATED SPLEEN IRON FOR ERYTHROPOIESIS IN ANEMIA OF INFLAMMATION

C.-Y. Wang 1,* , N. Medeiros 1 , E. Melgar-Bermudez 1 , T. Nurse 1 , E. Lema 1 , C. Tseng 1 , F. Fisher 1 , J. Seehra 1 , J. Lachey 1

1 Keros Therapeutics, Lexington, United States of America

Background: Anemia of inflammation (AI) caused by persistent inflammation is common in chronic disease. Inflammatory cytokines, including interleukin 6 (IL-6), induce expression of hepcidin, an iron homeostasis regulator, which retains tissue iron and limits circulating iron, leading to anemia. Iron and inflammation regulate hepcidin through the bone morphogenetic protein (BMP)-SMAD and IL6-STAT3 pathways, respectively. BMP2 and 6 bind BMP receptors, including ALK2, to phosphorylate SMAD1/5/9, while IL-6 signals through STAT3. Activated SMAD1/5/9 or STAT3 bind independently to hepcidin promoter regions to initiate transcription. Evidence suggests that targeting BMP-SMAD to reduce hepcidin may ameliorate anemia in AI. We developed two investigational neutralizing antibodies against ALK2, KTI-m216 (m216) and KTI-m218 (m218), with unique variable regions but observed to have similar affinity for ALK2 and effect on hepcidin suppression in mice and monkeys.

Aims: We investigated the mechanism of ALK2 inhibition-mediated hepcidin suppression in acute inflammation and chronic AI mouse models.

Methods: C57BL/6 mice were dosed with 3 mg/kg m216 or isotype control (Ctrl) for 1 h, followed by 1 mg/kg lipopolysaccharide (LPS) or PBS for 6 h. For an AI model, C57BL/6 mice were fed a control or 0.2% adenine diet with 40 ppm iron for 6-7 weeks to induce chronic kidney disease (CKD), followed by 3 mg/kg m218 (CKD-m218) or isotype (CKD-Ctrl) twice weekly for 3 more weeks. In a separate experiment, CKD mice were switched to a similar adenine diet with low 3 ppm iron when receiving m218 or Ctrl for 9 days.

Results: Serum IL-6 was similarly induced in the Ctrl-LPS and m216-LPS mice, compared to respective PBS controls. Consistent with previous findings, m216 reduced serum hepcidin in PBS-treated mice (Ctrl-PBS 509 ng/ml; m216-PBS 78 ng/ml). While LPS induced serum hepcidin in Ctrl-LPS (801 ng/ml) and m216-LPS (247 ng/ml) mice, compared to respective PBS controls, absolute hepcidin was lower in the m216-LPS mice, suggesting that m216 reduced serum hepcidin by lowering basal transcription but not preventing LPS induction. To examine potential crosstalk between the two pathways, phosphorylation of liver SMAD5 (PSMAD5) and STAT3 (PSTAT3) were examined by Immunoblot. PSTAT3 was increased similarly in Ctrl-LPS and m216-LPS mice, compared to respective PBS controls, which is consistent with the similar serum IL-6 in these mice. PSMAD5 was not induced by LPS but, as expected, was decreased by m216 compared to Ctrl-PBS mice. These data support that BMP regulation of hepcidin may be distinct from regulation by IL-6. Importantly, ALK2 inhibition alone was sufficient for hepcidin reduction in inflammation.

CKD resulted in increased serum hepcidin, decreased serum iron and increased liver and spleen iron retention, compared to mice on a control diet. CKD-m218 mice had an 82% reduction in serum hepcidin and a 73% increase in serum iron after 3 weeks of treatment compared to CKD-Ctrl mice. Additionally, CKD-m218 mice had a 47% reduction in spleen iron and a 1.4 g/dL increase in hemoglobin compared to CKD-Ctrl mice. Similar responses were observed in CKD-m218 mice fed a low iron diet, suggesting that the increased iron in CKD-m218 mice was mostly from the spleen rather than diet.

Summary/Conclusion: These data suggest that IL-6-induced hepcidin is largely independent of ALK2, but ALK2 inhibition may be sufficient to result in a net hepcidin reduction and improved erythropoiesis. Data also suggest that, in AI with adequate iron stores, ALK2 inhibition potentially acts by liberating iron from the recycling pathway.

P1568: EARLY, BUT NOT LATE USE OF REMDESVIR AND CONVALESCENT PLASMA REDUCES MORTALITY OF COVID 19 IN PATIENTS WITH HEMATOLOGIC MALIGNANCIES – A STUDY OF KROHEM

I. Aurer 1,2,* , S. Basic-Kinda 1 , A. Grubesic 3 , O. Jaksic 2,4 , S. Mrdenovic 5 , S. Novakovic-Coha 6 , D. Lozic 7 , M. Mikulic-Pisk 1 , R. Serventi-Seiwerth 1 , D. Dujmovic 1 , A. Ostojic 1 , B. Dreta 1 , G. Pavlisa 2,8 , M. Narancic 9 , I. Hude-Dragicevic 1 , T. Valkovic 3,10

1 Division of Hematology, Department of Internal Medicine, University Hospital Centre Zagreb; 2 Medical School, University of Zagreb, Zagreb; 3 Division of Hematology, Department of Internal Medicine, University Hospital Centre Rijeka, Rijeka; 4 Division of Hematology, Department of Internal Medicine, University Hospital Dubrava, Zagreb; 5 Division of Hematology, Department of Internal Medicine, University Hospital Centre Osijek, Osijek; 6 Division of Hematology, Department of Internal Medicine, University Hospital Centre Sisters of Mercy, Zagreb; 7 Division of Hematology, Department of Internal Medicine, University Hospital Centre Split, Split; 8 Department of Pulmology, University Hospital Centre Zagreb, Zagreb; 9 Division of Hematology, Department of Internal Medicine, General Hospital Zadar, Zadar; 10 Medical School, University of Rijeka, Rijeka, Croatia

Background: Patients with hematologic malignancies have a high risk of dying from COVID19 due to inability to mount humoral and cellular immune responses to the virus. Remdesvir is an inhibitor of viral RNA-polimerase. Some, but not all studies suggest it hastens recovery and reduces mortality in patients with COVID19. In a large randomized trial, convalescent plasma obtained from persons recovering from the infection was not proven to be useful in treatment of COVID19 in the general population, but other studies suggest it is useful in hematologic patients unable to produce antibodies against the virus. Recommendations on the use of these two drugs vary, some recommend its use only in severely immunocompromized individuals, some only in serious cases and some not at all. Reflecting these differences, the practice of using them varied between Croatian centers during the current pandemic.

Aims: To analyze the effect of remdesvir and convalescent plasma on mortality in patients with hematologic malignancies by performing a matched-pair analysis.

Methods: KroHem, the Croatian Cooperative Group for Hematologic Diseases, collected data on the outcome of patients with hematologic malignancies who became infected with SARS-COV2 while on concurrent systemic antineoplastic therapy during 2020 and 2021, before the appearance of the omicron strain. Patients treated with remdesvir and/or convalescent plasma were matched to those untreated according to age, disease type and antineoplastic therapy, factors found in our previous analyses to be related to outcome. Patients with Hodgkin lymphoma and myeloproliferative neoplasms were excluded, due to low risk of COVID19 mortality. Death during infection was considered as due to COVID19.

Results: We identified 119 patients fulfilling the entry criteria. Three could not be matched, 2 with T-PLL treated with alemtuzumab and one with plasmablastic lymphoma and newly diagnosed HIV infection. All three died. In the remaining 116 patient pairs remdesvir significantly reduced the mortality: 36 out of 106 treated patients died, in comparison to 54 untreated (p=0.0207, McNemar’s test). The effect of plasma was not significant: 26 of 73 treated patients died, in comparison to 33 untreated (p=0.2812). Therapy was substantially more effective in patients who received treatment within a week from symptom onset; 11 of 58 patients treated with remdesvir died in comparison to 33 untreated (p<0.0001) and 8 out of 35 treated with plasma in comparison to 20 untreated (p=0.0095). Patients treated with remdesvir only had similar outcomes as those treated with remdesvir and plasma (15% vs. 19% respectively).

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Summary/Conclusion: Our study suggests that patients with hematologic neoplasia, who are at a high risk of dying from COVID19, should receive treatment with remdesvir and convalescent plasma as soon as possible, resulting in a 2.5-3 times reduction in mortality. The effect of later treatment, if any, is less prominent.

P1569: COVID19 AND ITS IMPACT ON HEMATO-ONCOLOGICAL PATIENTS. DIFFERENCES IN OUTCOMES IN RELATION TO OMICRON CIRCULATION AND VACCINATION STATUS?

A. Addiego 1 , A. I. Prado 2 , V. Moraes 2 , P. Cano 2 , I. Gervaz 1 , L. Perez 1 , R. De Giuda 2 , A. Flores 1 , E. Riva 1 , M. Ferrando 2 , S. Grille 1 , G. Borelli 2 , A. Vazquez 2 , M. Stevenazzi 1 , R. Gabus 2 , A. I. Landoni 2 , C. Guillermo 1 , M. Boada 1,*

1 Cátedra de Hematología, Hospital de Clínicas. Dr. Manuel Quintela; 2 Servicio de Hematología y Trasplante de Progenitores Hematopóiéticos., Hospital Maciel, Montevideo, Uruguay

Background: COVID-19 has a pleomorphic presentation that goes from asymptomatic patients to critical disease with pneumonitis as the lead complication. Main risk factors for critical illness comprise age, cardiovascular disease and obesity. In Hemato-oncological patients, immune response is impaired due in part to their disease and also oncological treatment. Lethality rates reported for these patients are higher than general population and there is a trend towards a greater impact in patients diagnosed in the last 5 years. Our country has experienced two COVID-19 waves since the first cases in march 2020. There is national evidence of less Iethality and critical cases with omicron circulation

Aims: To present an interinstitutional registry of COVID-19 patients with underlying hemato-oncological diseases.

Methods: We included patients with hemato-oncological diseases assisted at Hospital de Clínicas and Hospital Maciel from Montevideo Uruguay, who were diagnosed with COVID-19 between march 2020 to February 2022. Risk factor presence was established as one of the following: age older than 60, cardiovascular disease and obesity. Full vaccination was defined as two doses and 14 days after last injection. Omicron circulation refers to the period between December 2021 and February 2022 when Omicron variant was responsible for more than 99% of cases in Uruguay.

Results: 110 patients were included with a median age of 53 years. Diagnosis comprises 51.8% (n=57) chronic lymphoprolipherative diseases, 20% (n=22) monoclonal gammapaty, 14.5% (n=16) chronic myeloprolpherative diseases, and 13.6% (n=15) acute leukemias and myelodysplastic syndromes. A 34.5 % of the patients were fully vaccinated. Hospital admission was required in 23.6% and there was 14.5% of critical cases. Global lethality rate was 14.5% (n=16), higher than global lethality reported for Uruguay in the same period (0.84%, with a total of 815704 infections). Figure 1 shows data adjusted by disease, illness status, hospital admission, risk factors, vaccination status, and omicron circulation. Requirement of hospital admission, and presence of risk factors were associated with higher lethality (p=0.0001. and p=0.0022), whereas active disease was associated with more lethality but with no statistical difference (p=0.058). Lethality was inferior in full vaccinate state (5.3% vs.19.4%), but with no statistical difference (p=0.0504). Our data reveals a trend towards inferior lethality during omicron wave (9% vs 18%), however not statistical difference was shown.

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Summary/Conclusion: We present an interinstitutional registry from the main hematologic public centers in Uruguay. Patients included present a broad spectrum of hemato-oncological diseases. The proportion of vaccinated patients is significantly inferior to general population in our country (34.5% vs. 77.1%). Admission requirement and presence of risk factors were associated with higher lethality. Although full vaccination and omicron circulation seem to be associated with inferior lethality, difference was not statistically significant. That may be in relation to the number of patients included and ought to be evaluated prospectively. COVID-19 outcomes in hemato-oncological patients are substantially inferior to general population and our data suggest that impact is greater in patients with active disease. We believe that hemato-oncological patients constitute a vulnerable population in regards to COVID-19 justifying solid directed actions such as booster vaccination.

P1570: SARS-COV-2 HUMORAL AND CELLULAR VACCINATION RESPONSE IN PATIENTS WITH LYMPHOID HAEMATOLOGICAL MALIGNANCIES

L. S. Boerenkamp 1,2,* , C. R. Pothast 3 , R. C. Dijkland 3 , I. H. van Loo 4 , L. Wieten 5 , C. J. Halkes 6 , M. H. Heemskerk 3 , C. H. Van Elssen 2,7

1 Internal medicine; 2 GROW School for Oncology and Reproduction, Maastricht University, Maastricht; 3 Experimental hematology, Leiden University Medical Centre, Leiden; 4 Medical microbiology; 5 Transplantation immunology, Maastricht University Medical Centre, Maastricht; 6 Hematology, Leiden University Medical Centre, Leiden; 7 Internal medicine, Maastricht University Medical Centre, Maastricht, Netherlands

Background: Patients diagnosed with hematologic malignancy (HM) are at a high risk of severe or fatal coronavirus disease 19 (COVID-19). COVID-19 is the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A large-scale vaccination program was launched globally in the battle against this virus. However, the safety and efficacy of the vaccines was only tested in healthy individuals. HM patients often suffer from malignantly transformed immune cells and/or are treated with immune depleting/suppressive treatment leading to altered immune responses to infections and vaccination. We hypothesize that a part of HM patients will have an altered, attenuated, or absent immune response to SARS-CoV-2 vaccination. Therefore, data on response and protection rates is needed to provide correct information to patients.

Aims: The aim of this study is to assess the efficacy of SARS-CoV-2 vaccination, in terms of SARS-COV-2 spike antibody and T cell responses in patients diagnosed with and/or under treatment for chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL) or multiple myeloma (MM).

Methods: This is a multicenter prospective phase IV intervention study. Vaccine responses were evaluated pre-SARS-CoV-2 vaccination and 2 weeks, 3- and 6-months post SARS-CoV-2 vaccination. Humoral responses were evaluated by testing SARS-CoV-2 spike receptor binding domain (RBD) specific Ig. T cell responses were evaluated by CD4 and CD8 T cell stimulation with SARS-CoV-2 spike peptide followed by antibody staining for T cell activation and cytokine production analyzed by flow cytometry. Additionally CD8 T cell responses were analyzed by SARS-CoV-2 specific HLA-tetramer binding assays.

Results: In total 160 patients were included, 31 were diagnosed with CLL, 39 with aggressive NHL, 57 with indolent NHL, 30 with MM and 3 with other HM. All patients initially received two doses of mRNA-1273 or BNT162b2 and part received a booster during the study. Seroconversion rates two weeks after initial vaccination were 55% in CLL, 70% in aggressive NHL, 72% in indolent NHL and 100% in MM. The rates were stable at 3 months post vaccination and increased at 6 months post vaccination for CLL and NHL patients when boostered (Table 1). Two weeks post vaccination anti-SARS-CoV-2 S-RBD titers were above the 75 th percentile of the measuring area in 37% of CLL, 62% of aggressive NHL, 60% of indolent NHL and 93% of MM patients (Table 1). Seroconversion rates for NHL patients that have received anti-CD20 therapy in the last 6 months before vaccination, 6-12 months before vaccination or more than 12 months before vaccination were respectively 16%, 60% and 95% at two weeks after initial vaccination.

Spike specific CD4+ T cell responses, defined by activation markers (CD137 and CD154), were confirmed in 91% of the seronegative patients at two weeks after initial vaccination. CD8 spike specific tetramer binding was also observed in seronegative patients.

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Summary/Conclusion: Seroconversion rates are lower in patients diagnosed with CLL or NHL, but normal for MM patients. NHL patient on current or recent rituximab treatment had very low seroconversion rates, however this almost normalized when treatment was finished more than 12 months before vaccination. Antibody titers of seropositive patients with NHL or MM are mostly above the 75 th percentile of the measuring area, whereas this is lower for patients with CLL. Spike specific CD4+ T cell activation and presence of SARS-CoV-2 spike specific CD8 T cells was observed despite seronegativity. Additional T cell responses are currently being analyzed and will be presented.

P1571: EVALUATION OF THE CLINICAL IMPACT OF BONE MARROW CULTURES IN CURRENT MEDICAL PRACTICE

G. Sharvit 1,* , D. Schwartz 1 , G. Heering 1 , A. Shulman 1 , A. Avigdor 1 , G. Rahav 1 , A. Toren 1 , A. Nagler 1 , J. Canaani 1

1 Sheba Medical Center, Givatayim, Israel

Background: The clinical yield and benefit of performing bone marrow cultures for various clinical indications has been challenged and their clinical necessity remains debatable.

Aims: To characterize a large cohort of patients who had bone marrow cultures performed in terms of baseline clinical, laboratory, microbiological, and imaging data, and determine whether these parameters were predictive of a diagnostic bone marrow culture.

Methods: A single center retrospective analysis of all patients who underwent a bone marrow study comprising bone marrow cultures from January 1, 2012, through March 1, 2018. Data on comorbidities, baseline laboratory parameters, and indication for performing a bone marrow culture were extracted from the institution’s electronic medical records system.

Results: The analyzed cohort consisted of 139 patients admitted to our institution between the years 2012-2018 with a median age of 46 years (range 4 months- 85 years). The most common indication for a bone marrow study was workup of a fever of unknown origin (105 patients, 76%) while investigation for infection in immunocompromised patients accounted for 22 cases (16%) and suspected tuberculosis was the reason for acquisition of bone marrow cultures in 6 patients (4%). Of all patients who underwent bone marrow examinations with culture studies, only 3 patients had positive bone marrow cultures yielding in 2 patients a diagnosis of Mycobacterium avium and in one patient a microbiologically unclassifiable fungal infection. A univariate analysis revealed that mean age, hemoglobin level, platelet count, c-reactive protein levels, gender, indication for bone marrow study, yield of blood cultures, and contribution of imaging studies and bone marrow pathology results were not significantly different between patients with diagnostic bone marrow cultures and those with non-diagnostic bone marrow cultures. Mean white blood cell count was found to be significantly lower in patients with diagnostic bone marrow cultures (2.4 ×10 3 /µL versus 8.7 ×10 3 /µL; P =.038).

Summary/Conclusion: For most patients, performance of bone marrow cultures holds limited clinical value.

P1572: IMPACT OF GUT COLONIZATION BY GRAM NEGATIVE MULTIRESISTANT BACTERIA IN PATIENTS WITH ACUTE LEUKEMIA UNDERGOING INTENSIVE ANTINEOPLASTIC TREATMENT

C. Castañon Fernandez 1,2,* , P. López Ugarriza 2,3 , A. Fernández Moreno 2,3 , N. Diaz Varela 3 , A. M. Fernández Verdugo 4 , J. Fernández 4 , W. J. Zambrano 1 , L. Escalada 1 , P. C. Tamayo 3 , T. Bernal del Castillo 2,3

1 Hematology Department, Hospital Universitario San Agustín de Avilés; 2 ISPA, Instituto de Investigación Sanitaria del Principado de Asturias; 3 Hematology Department; 4 Servicio de Microbiología, Hospital Universitario Central de Asturias, Oviedo, Spain

Background: Gut colonization by Gram negative multiresistant bacteria (GNMRB) occurring during chemotherapy treatment for acute leukemia (AL) reduces overall survival (OS) by increasing early non-relapse mortality (NRM). The impact of colonization once patients have finished antineoplastic treatment is unknown.

Aims: To evaluate long-term consequences of GNMRB colonization on AL surviving patients.

Methods: Retrospective study including all consecutive AL patients treated with standard intensive chemotherapy with or without Hematopoietic Stem Cell Transplant (HSCT) who completed first line antineoplastic treatment. A landmark analysis was performed including all patients alive 60 days after last chemotherapy cycle of HSCT. Primary end point was OS. Secondary end points were non-relapse mortality (NRM) and acute (a) and chronic (c) Graft Versus Host Disease (GVHD) in allogeneic transplanted patients. Data are presented as median (interquartile range) or proportion. Cumulative incidence of NRM was calculated accounting for the competing risk of leukemic-death. Survival curves were built using the Kaplan-Meir method and univariate analysis was performed using the Log-Rank test. Statistical analysis was performed using the R 4.1.1 software package.

Results: From April 2016 to March 2021, 142 patients from a single center were treated with intensive chemotherapy, of whom 96 were alive 60 days after chemotherapy or HSCT. Median age was 55 (42-52) years with 50% (48/96) of female patients. ECOG performance status at diagnosis of the disease was 0-1 in 98% (94/96) patients. A diagnosis of Acute Myeloid Leukemia (AML), high-risk Myelodysplastic Syndrome and Acute Lymphoblastic Leukemia (ALL) was present in 85% (82/96), 1% (1/96) and 14 (14/96) patients, respectively. Hematopoietic Transplant Comorbidity Index (HCTCI) was low, intermediate and high in 68% (66/96), 16% (16/96) and 16% (16/96) patients, respectively. Within AML patients, ELN17 genetic risk was favourable, intermediate and adverse in 48% (38/79), 18% (14/79) and 34% (27/79) patients. Prophylactic fluoroquinolones were used in 65% (62/96) patients, significantly more used in AML/MDS than in ALL patients: 69% (57/83) vs 38% (5/13), respectively, P=0.03. Throughout the first line treatment, 48% (46/96) patients were colonized by GNMRB, being the recovered microorganisms: Klebisella sp. 61% (28/46), Enterobacter sp: 17%, (8/46), Citrobacter sp: 11% (5/46), E. Coli: 9% (4/46), and Pseudomonas aeruginosa 2% (1/46). With a median follow up of 17 (7-34) months, there were no differences in univariate analysis of OS between colonized compared to non-colonized patients: [53 months (95% CI: 26-NR) vs NR (95% CI: 26-NR), P= 0.9], (Figure). On the contrary, age and ELN17 genetic risk were significantly associated with OS (Table). Additionally, no differences in aGVHD [34% (10/29) vs 45% (10/22)], P=0.4, or cGVHD [18% (4/22) vs 14% (4/29), P=0.9] were observed between colonized and non-colonized patients.

Regarding NRM, a non-significant increase in non-leukemic deaths were observed in colonized patients compared to non-colonized patients: 15% (7/46) vs 8% (4/46) P= 0.6.

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Summary/Conclusion: Gram negative multiresistant bacteria gut colonization is not associated with worse long-term outcomes in acute leukemia patients compared to non-colonized patients.

P1573: VARIATION IN USE OF IMMUNOGLOBULIN AND IMPACT ON SURVIVAL IN MULTIPLE MYELOMA: A REPORT FROM THE AUSTRALIA/NEW ZEALAND (ANZ) AND ASIA-PACIFIC (APAC) MYELOMA AND RELATED DISEASES REGISTRIES (MRDR)

K. L. Chai 1,* , C. Wellard 1 , N. Aoki 1 , E. M. Moore 1 , B. Augustson 2 , A. Bapat 3 , H. A. Blacklock 4 , W. J. Chng 5 , R. Cooke 6 , C. Forsyth 7 , N. Hamad 8,9,10 , S. J. Harrison 11 , P. J. Ho 12 , J. Hocking 13 , I. H. Kerridge 14 , J. S. Kim 15 , K. Kim 16 , T. King 12 , G. J. McCaughan 8,9 , P. Mollee 17 , C. O. Morrissey 18 , N. Murphy 3 , H. Quach 19,20 , X. N. Tan 2 , A. Tso 21 , K. Wong 13 , A. Spencer 22 , E. M. Wood 1,23 , Z. K. McQuilten 1,23

1 Transfusion Research Unit, School of Public Health and Preventive Medicine, Monash University, Melbourne; 2 Department of Haematology, Sir Charles Gairdner Hospital, Perth; 3 Department of Haematology, Royal Hobart Hospital, Hobart, Australia; 4 Department of Haematology, Middlemore Hospital, Auckland, New Zealand; 5 Department of Haematology, National University Hospital, Singapore, Singapore; 6 Department of Haematology, Northern Hospital, Melbourne; 7 Department of Haematology, Gosford Hospital, Gosford; 8 Department of Haematology, St Vincent’s Hospital Sydney; 9 St Vincent’s Clinical School, University of New South Wales; 10 School of Medicine, University of Notre Dame, Sydney; 11 Department of Haematology, Peter MacCallum Cancer Centre and the Royal Melbourne Hospital, Melbourne; 12 Department of Haematology, Royal Prince Alfred Hospital, Sydney; 13 Department of Haematology, Box Hill Hospital, Melbourne; 14 Department of Haematology, Royal North Shore Hospital, Sydney, Australia; 15 Division of Hematology, Department of Internal Medicine, Yonsei University College of Medicine, Severance Hospital; 16 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Seoul, South Korea; 17 Department of Haematology, Princess Alexandra Hospital, Brisbane; 18 Department of Infectious Diseases, The Alfred Hospital and Monash University; 19 Department of Haematology, St Vincent’s Hospital Melbourne; 20 University of Melbourne, Melbourne, Australia; 21 Department of Haematology, Tan Tock Seng Hospital, Singapore, Singapore; 22 Department of Haematology, The Alfred Hospital; 23 Department of Haematology, Monash Health, Melbourne, Australia

Background: Serious infection is common in patients with multiple myeloma (MM), and associated with major morbidity and mortality. Immunoglobulin (Ig) replacement is frequently used to reduce infections, but this is based on small trials conducted >20 years ago. Guidelines for Ig use in MM patients vary between countries. Use of Ig and associated costs are increasing worldwide.

Aims: This study aims to outline Ig use in the “real-world MM” setting, identify variation in practice and predictors of use, and describe association with survival outcomes.

Methods: Retrospective review of patients registered on the ANZ and APAC MRDR with a diagnosis of MM or plasma cell leukaemia from sites with verifiable data regarding Ig. Survival and cause of death data (COD) was augmented via linkage with national death registries for ANZ patients. Patient/disease characteristics, therapy and survival were compared between patients who received Ig by 24 months of diagnosis and patients who did not, using chi-square tests for categorical variables and rank sum tests for continuous variables. Variation in Ig use across sites was compared. Kaplan-Meier survival analysis was used to estimate duration of Ig use. Time-dependent Cox analysis was used to compare survival for patients whilst on and off Ig therapy. All statistical analysis was completed on STATAv16.1.

Results: 2445 patients with a median follow-up time of 30 months were included in this analysis - 1744 from Australia (12 sites), 313 Korea (3 sites), 281 New Zealand (1 site) and 107 Singapore (3 sites). Of patients reaching 12-month follow-up post MM-diagnosis, 6.4% received Ig and of patients reaching 24-month follow-up, 7.0% received Ig. 69% of these Ig-users were estimated to receive over 24 months duration of Ig. Administration varied by country – between 0 to 17% of patients by country who reached 24 months follow-up received Ig. Patients who received Ig by 24 months of MM-diagnosis were younger, had lower baseline IgG and Ig levels, more likely to have abnormal FISH, receive first-line IMiDs, anti-CD38 therapy and ASCT ( Table 1).

Ig use was not associated with overall survival benefit (HR=0.72, 0.46-1.14, p=0.16). Baseline Ig levels were also not found to be associated with overall survival. At time of last follow-up, there were 623 deaths (25.4%). Available data on COD was available for 175 deaths in ANZ patients. Of these, 65 deaths (37.1%) had infection listed as primary/secondary COD. 64 of these 65 infection-related deaths occurred in patients who did not receive Ig. In patients who received Ig (at any time), 12.5% had infection as primary/secondary COD, compared to 38.5% in non-recipients (p=0.14).

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Summary/Conclusion: In this real-world cohort, use of Ig varied between countries and was associated with lower baseline IgG, abnormal FISH, and first-line therapy with IMiD, anti-CD38 and ASCT. Ig use was not associated with overall survival, highlighting the need for contemporary studies to inform patient selection, especially with rising use of Ig and targeted anti-myeloma therapies, and a high burden of infection-related mortality.

P1574: ANTINEOPLASTIC THERAPY FOR HEMATOLOGIC MALIGNANCIES DURING COVID-19

T. Chudnova 1,* , E. Baryakh 1 , Y. Polyakov 1 , T. Tolstykh 1 , O. Kochneva 1 , D. Ivanova 1 , E. Misyurina 1 , K. Yatskov 1 , E. Zhelnova 1 , M. Litvinenko 1 , K. Kaluzhskaya 1

1 heamatology, Moscow City Clinical Hospital 52, Moscow, Russia

Background: During the COVID-19 pandemic patients with hematologic malignancies frequently have to interrupt specific antineoplastic therapy as a result of a positive SARS-COV-2 RNA PCR result and for evidence of viral pneumonia on chest CT. However, delaying or withholding specific therapy for significant time may cause progression of a hematologic malignancy and increase the risks of tumor resistance.

Aims: Analyze in-hospital overall survival and causes of death in COVID-19 patients during specific therapy for hematologic malignancies.

Methods: During the period of March 2020 – November 2021, 192 patients with hematologic malignancies having coronavirus infection underwent specific antineoplastic therapy at the hematology unit of Moscow clinical hospital #52. We analyzed in-hospital overall survival and the causes of death, depending on the disease entity of a hematologic malignancy, disease status, patients’ age, Charlson comorbidity index and the intensity of specific antineoplastic therapy. The study included 93 patients with acute leukemias, 51 patients with non-Hodgkin’s lymphoma, 30 patients with chronic lymphocytic leukemia and 18 patients with multiple myeloma. Median age was 55 years (19-92 years).

Results: In the acute leukemias group mortality was 25-82%, depending on the disease entity, disease status, age and intensity of the antineoplastic treatment. The most significant unfavorable prognostic factors were as follows: lack of acute leukemia remission (p=0.003), age above 65 years (p=0.002), Charlson comorbidity index > 6 (p<0,001). Infectious complications (24%) and severe coronavirus disease (60%) were the main causes of death in the acute leukemias group. In the group of patients who received chemotherapy for acute leukemia according to standard treatment protocols the rate of remission was higher, mortality was the lowest, and the risk of severe coronavirus disease was lower compared to patients who received specific antineoplastic therapy of lower intensity. In the non-Hodgkin’s lymphoma group mortality was 30%. In 90% cases of death patients had no remission of their underlying disease. Among the causes of death, the most common were lymphoma progression (46%) and severe COVID-19 (47%). In the chronic lymphocytic leukemia and multiple myeloma group in-hospital mortality was 27% and 17% respectively, and in 100% cases deaths were caused by severe coronavirus disease.

Summary/Conclusion: The main risk factor for severe COVID-19 and death in patients with hematologic malignancies was lack of remission of the underlying disease. Therefore, specific antineoplastic therapy results in control over the neoplastic process, eventually reducing the risk of severe COVID-19. However, individual approach must be applied in the selection of antineoplastic treatment and its timing, and in evaluating the risks of coronavirus infection reactivation during immunosuppressive therapy.

P1575: DEVELOPMENT OF A RISK PREDICTION MODEL OF SUBSEQUENT BLOOLDSTREAM INFECTION AFTER CARBAPENEM-RESISTANT ENTEROBACTERIACEASE ISOLATED FROM PERIANAL SWABS IN PATIENTS WITH HEMATOLOGICAL DISEASES

J. Liu 1,* , E. Jiang 1

1 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Hematological patients are at higher risk of developing carbapenem-resistant Enterobacteriaceae (CRE) bloodstream infection (BSI) and associated with high mortality. Prediction model for a subsequent CRE BSI in hematological patients with CRE isolated from previous perianal swab samples can provide timely and useful target treatment strategies.

Aims: To developing a quantified risk prediction model based on the risk factors of subsequent BSI in CRE carriers will help clinicians better identify specific individuals who would truly benefit from early, empiric CRE-targeted treatment.

Methods: The data were extracted from patients with CRE isolated from perianal swab samples at the Hematopoietic Stem Cell Transplantation Center of Blood Diseases Hospital, Chinese Academy of Medical Sciences between July 2017 to October 2020 January. Patients who developed subsequent CRE BSI were compared with those who did not develop BSI. Univariate logistic analysis, multivariate logistic analysis and stepwise regression analysis were carried on a variety of clinical factors.

Results: A total of 215 cases were included and 29 (13.4%) patients of them with CRE isolated from perianal swab samples developed CRE BSI subsequently. Multivariate analysis showed that C-reactive protein(CRP)﹥30mg/l(OR 10.613，95%CI 2.965~37.985， P =0.000）, perianal infection(OR 6.450, 95%CI 2.223~18.714, P =0.001), concomitant gastrointestinal symptoms (OR 4.175，95%CI 1.476~11.813， P =0.007），age﹥44 years (OR 3.415，95%CI 1.222~9.541， P =0.007）and neutrophil count﹤0.025×10 9 /L (OR 4.583，95%CI 0.939~22.369， P =0.060）were risk factors for CRE BSI in patients with CRE isolated from perianal swab samples ( P<0.01 ). They were included in the Logistic regression model to predict BSI. According to receiver operating characteristic (ROC) curve analysis, the cut-off value of the model was 0.921 (0.851-0.968， P<0.001 ).

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Summary/Conclusion: Hematological patients with CRE isolated from perianal swab samples have a relatively low incidence of subsequent BSI but a relatively high risk of death. The risk prediction model based on CRP, perianal infection, concomitant gastrointestinal symptoms, age and valley absolute neutrophil may be used for predicting subsequent CRE BSI in patients with isolated from perianal swab samples and provide timely and effective treatment reference for this kind of patients.

P1576: A PHASE 3 RANDOMIZED, DOUBLE-BLIND, COMPARATOR-CONTROLLED STUDY TO EVALUATE SAFETY, TOLERABILITY AND IMMUNOGENICITY OF V114 PNEUMOCOCCAL VACCINE IN HEMATOPOIETIC CELL TRANSPLANT RECIPIENTS (PNEU-STEM)

M. Wilck 1,* , O. Cornely 2 , P. Ljungman 3 , C. Cordonnier 4 , J. Diego Velez 5 , R. Dagan 6 , P. Richmond 7 , Q. Chen 1 , C. Daus 1 , K. Geddie 1 , T. Sterling 1 , T. Shekar 1 , L. Musey 1 , U. Buchwald 1

1 Merck & Co., Inc., Kenilworth, United States of America; 2 University Clinic Cologne, Cologne, Germany; 3 Karolinska University Hospital, Stockholm, Sweden; 4 Henri Mondor Hospital, Creteil, France; 5 Fundacion Valle del Lili, Cali, Colombia; 6 Ben-Gurion University, Beer-Sheva, Israel; 7 University of Western Australia, Perth, Australia

Background: Allogeneic hematopoietic cell transplant (allo-HCT) represents a definitive therapy for a variety of hematological diseases. Immunosuppressive therapy following allo-HCT and graft-vs-host disease (GVHD) render the patient susceptible to infectious complications, including invasive pneumococcal disease. Post-allo-HCT pneumococcal vaccination is recommended 3-6 months after transplant in a multi-dose series to help prevent pneumococcal disease.

Aims: V114 is a 15-valent pneumococcal conjugate vaccine (PCV) approved in adults, containing all 13 serotypes in Prevnar 13™ (PCV13; contains serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) as well as epidemiologically important serotypes 22F and 33F. This study evaluated safety and immunogenicity of V114 when given to allo-HCT recipients.

Methods: Individuals ≥3 years of age were randomized 1:1 to receive 3 doses of V114 or PCV13 in one-month intervals starting 3-6 months after allo-HCT. At 12 months after HCT, participants received either PNEUMOVAX TM 23 or a fourth dose of PCV if they experienced chronic GVHD. All participants or their legal representatives provided informed consent at enrollment. Primary safety objective was to evaluate the proportion of participants with adverse events (AEs) within each vaccination group for 3 to <18 years old and ≥18 years old age groups. Primary immunogenicity objective was to evaluate serotype-specific immunoglobulin G (IgG) geometric mean concentrations (GMCs) for all 15 V114 serotypes at 30 days following the third PCV dose (Day 90) in each vaccination group. A key secondary objective was to evaluate opsonophagocytic activity (OPA) geometric mean titers (GMTs) for all V114 serotypes at Day 90.

Results: A total of 274 subjects (14 children [3 to <18 years of age] and 260 adults ≥18 years old) were enrolled and vaccinated in the study. The proportion of participants with systemic AEs and serious AEs after any dose of study vaccine were generally comparable between intervention groups in the pediatric and adult cohorts. V114 recipients reported a higher proportion of injection-site AEs and vaccine-related systemic AEs compared to PCV13 in the adult cohort. Of all vaccinated participants, 32.4% experienced serious AEs during the study (28.8% in V114 group, 36.3% in PCV13 group). Seventeen participants (5.8% in V114 group, 6.7% in PCV13 group) died during the study. None of the deaths were determined by the investigator to be related to study vaccines. For both IgG GMCs (Table 1) and OPA GMTs, V114 was generally comparable to PCV13 for the 13 shared serotypes, and higher for serotypes 22F and 33F at Day 90.

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Summary/Conclusion: V114 was well tolerated in allo-HCT recipients with a generally comparable safety profile to PCV13 for both pediatric and adult participants. Despite the immunocompromised status of the participants, the study vaccines induced serotype-specific immunogenicity and V114 induced generally comparable responses to PCV13 by IgG GMCs and OPA for the 13 shared serotypes at Day 90. In addition, V114 induced higher antibody responses than PCV13 for serotypes unique to V114 (22F and 33F). Study results support the use of V114 in allo-HCT recipients.

P1577: RISK FACTORS FOR MORTALITY IN CRITICALLY ILL PATIENTS WITH HEMATOLOGICAL MALIGNANCIES IN A POLYVALENT INTENSIVE CARE UNIT: A RETROSPECTIVE STUDY

A. R. Dias 1,* , J. Gonçalves 2 , F. Seganfredo 3 , J. Gramaça 2 , M. Rebelo 4 , E. Carmo 4

1 Hematology, Hospital do Divino Espírito Santo de Ponta Delgada, Lisboa; 2 Oncology, Centro Hospitalar Barreiro Montijo, Barreiro; 3 Hematology, Instituto Português Francisco gentil, Lisboa; 4 Intensive Care Unit, Hospital Egas Moniz, Centro Hospitalar Lisboa Ocidental, Lisboa, Portugal

Background: Advances in critical care medicine have increased survival and quality of life in patients admitted to Intensive Care Unit (ICU). However, critically ill hemato-oncological (HO) patients still have a high risk of mortality, and stigma of poor prognosis may affect triage to ICU admission and clinical decisions. The available scoring systems are not specific for that population and the risk factors for adverse outcomes in HO patients remain poorly defined.

Aims: The aim of our study is to analyze the risk factors for ICU mortality of HO patients.

Methods: This retrospective, single-center study included 124 critically ill patients with HO diseases admitted in a polyvalent ICU between January 1st 2008 and December 31st 2021. Data regarding demographics, HO disease and therapy, disease severity scores, clinical and laboratorial parameters at admission, and organ support therapies instituted during ICU stay were collected from the ICU database. We compared data collected between survivors (n= 93) and non-survivors (n = 49). Univariate and multivariate logistic regression models were used to evaluate the potential risk factors for ICU mortality. Statistical tests were two-sided; p-values <0.05 were considered significant.

Results: Median age was 65,5 [27-92] years and 60,5% (75) of patients were male. The majority of patients were admitted from other hospitals (39,5%), followed by medical hospital wards (29,8%). Most patients had non-Hodgkin lymphoma (37,9%), Acute Myeloid Leukemia (18,5%) and Hodgkin lymphoma (12.1%). 63,7% of patients were under treatment, 40,3% of which were under intermediate chemotherapy (Cht), and 23,4% under intensive Cht. The average time of hospital stay pre-ICU was 11,4 days (d) [CI 95% 8,9-13,8], and in the ICU was 8,9 [7,1-10,8]. The main reason for admission in the ICU was septic shock (47,6%), followed by respiratory failure (23,4%). Invasive mechanical ventilation (IMV) in the ICU was used for 62,1% of the patients, vasopressors for 66,9%, and renal replacement therapy (RRT) was used for 36,3%. ICU mortality rate was 39,5%. On univariate analysis, we identified as risk factors in the ICU for death the following factors: neutropenia (HR 1,73, CI 95% 0,98-3,1, p=0.06), sepsis or septic shock at ICU admission (HR 2,15, CI 95% 1,18-3,9, p=0.012), length of pre-ICU hospitalization >13 d (HR 1,43, CI 95% 0,81-2,55, p=0,22), Acute Physiology And Chronic Health Evaluation (APACHE) > 26 (HR 5,68, CI 95% 2,54-12,72, p<0,001), Simplified Acute Physiology Score II (SAPSII) score > 58 (HR 4,01, CI 95% 2,04-7,89, p<0,001), need of IMV (HR 3,60, CI 95% 1,41-9,81, p=0,007), vasopressors (HR 18,39, CI 95% 2,56-133,98, p=0,004) and RRT (HR 2,54, CI 95% 1,42-4,55, p=0,002). Factors including gender, ECOG, higher number of comorbidities, type of HO disease, need of ChT in the ICU or pre-admission ChT and fever at admission showed no significant impact (HR 10,45, CI 95% 1,40-78,00, p=0,022).

Summary/Conclusion: In this study, characteristics of the underlying hematological malignancy didn’t have influence in the mortality, while the severity of infection, namely septic shock, and the need of organ supportive therapies correlated with mortality. Our findings reinforce that triage decisions solely based on the age or type of HO disease are not justified, but instead should be based on a multidisciplinary approach with intensivists and experienced hemato-oncologists.

P1578: CLINICAL CHARACTERISTICS AND PROGNOSTIC ANALYSIS OF 88 PATIENTS WITH HEMOPHAGOCYTOSIS DETECTED BY BONE MARROW SMEAR

L. Ran 1,* , Z. Yang 1 , T. Yang 1 , Y. Nan 1 , Y. Xiang 1 , Y. Liu 1

1 Department of Hematology-Oncology, Chongqing University Cancer Hospital, Chongqing, China

Background: The clinical characteristics, laboratory examination and prognostic characteristics of patients with hemophagocytes detected in bone marrow smears were analyzed retrospectively to explore the differences in etiology and prognosis of patients with hemophagocytes detected in bone marrow smears.

Aims: To investigate the clinical characteristics, laboratory examination and prognostic characteristics of patients with hemophagocytes detected in bone marrow smears.

Methods: A total of 88 patients with hemophagocytosis detected by bone marrow smear in four hospitals were collected, and their clinical data were analyzed. They were divided into two groups: the group meeting the diagnostic criteria of hemophagocytic syndrome (HLH) and the group not meeting the diagnostic criteria of hemophagocytic syndrome (non HLH), and their survival was followed up.

Results: Among the 88 patients, 37 patients met the diagnostic criteria for hemophagocytic syndrome (42.05%), 51 patients did not meet the diagnostic criteria (57.95%), 68 patients had fever (77.28%), 37 patients showed splenomegasis (42.05%), 59 patients had cytopenia of 2 series or above (67.05%), Serum ferritin was elevated in 44 cases (50.00%). Etiological analysis showed that 58 of 88 patients were tumor-related (65.91%), including 32 cases of lymphoma (36.36%). 20 cases (22.73%) were infection-related. For the HLH group and the non-HLH group, there was no statistical difference in the proportion of macrophages in bone marrow smears between the two groups, and the phagocytes were white blood cells, platelets, red blood cells, etc. No significant difference was observed between the two groups. Follow-up showed that 18 patients in the HLH group died, with a mortality rate of 48.65%, and 19 patients in the non-HLH group died, with a mortality rate of 37.25%.

Summary/Conclusion: Due to the detection of bone marrow hemophagocytes, the diagnostic coincidence rate of HLH is only 42.05%. The main cause of HLH is malignant tumor. However, whether HLH can be diagnosed or not, there is a high mortality. The clinical situation of patients should be evaluated more comprehensively as soon as possible and intervened as soon as possible.

P1579: PHOTOBIOMODULATION FOR CHEMOTHERAPY-INDUCED ORAL MUCOSITIS IN LEUKEMIC CHILDREN: A RANDOMIZED CONTROLLED CLINICAL TRIAL

Y. El Chazli 1,* , F. Reyad 2 , N. Elsayed 2

1 Hematology/Oncology unit, Pediatric Department; 2 Oral Medicine, Periodontology, Oral Diagnosis, and Oral Radiology, Alexandria University, Alexandria, Egypt

Background: Oral mucositis (OM) comes on the top of the most serious and frequent complications in children with cancer, with incidence rates ranging from 52% to 100% of patients receiving high-dose CT. Rapid cellular mitosis makes children more susceptible to CT and hence to OM than adults. Another risk factor for OM in leukemic children is the associated neutropenia. Low-level laser therapy (LLLT) or “Photobio-modulating (PBM) lasers is a therapeutic modality having three main effects: reducing inflammation and edema, promoting wound healing, and treating pain.

Aims: Evaluation of the effectiveness of low-level laser treatment (LLLT) for chemotherapy-induced oral mucositis in children suffering from acute leukemia.

Methods: A randomized controlled clinical study including forty-four leukemic children diagnosed with chemotherapy-induced OM at the Hematology/Oncology inpatient unit at Alexandria University Children’s Hospital, Alexandria, Egypt. Patients were randomly assigned to either the control or test groups with a 1:1 ratio. The control group received conventional symptomatic treatment, while the test group was treated with LLLT in addition to the symptomatic treatment. The response to both treatment modalities was evaluated according to the reduction of pain and lesions severity from baseline to 5, 10, and 14 days after treatment.

Results: The patients’ age ranged from 4 to 11 years. They were all diagnosed with ALL, 79.5% had B-ALL, and 20.5% had T-ALL. They were all treated according to the Total XV study protocol. A significant reduction of pain was recorded on day 10 in the test group compared to the control group ( p <0.001 ). Furthermore, there was a significant decline in the mean VAS among the test group starting from day5. There was also a significant decline in the OM grades between the two groups on day14 (p = 0.003 ). No adverse events were reported.

Summary/Conclusion: The use of LLLT along with the conventional treatment was effective in reducing pain and in the recovery of OM lesions in children receiving chemotherapy for the treatment of ALL. It was also safe and applicable to children.

P1580: MANAGEMENT OF PATIENTS WITH MASTOCYTOSIS DURING THE COVID-19 PANDEMIC: A SINGLE CENTER EXPERIENCE

V. Filipponi 1,* , G. Palumbo 1 , M. Ribersani 1 , M. L. Moleti 1 , A. Angi 1 , M. C. Molinari 1 , R. Maglione 1 , F. Giona 1

1 Department of Translational and Precision Medicine, Hematology, Sapienza University of Rome, Rome, Italy

Background: The risk of developing severe symptoms caused by SARS-CoV2 infection in patients (pts) with cutaneous mastocytosis (CM), or indolent systemic mastocytosis (ISM) is like that of healthy people. Unfortunately, the risk may increase in pts with aggressive systemic mastocytosis (ASM), or mastocytosis associated to a hematologic neoplasm (SM-AHN). SARS-CoV2 vaccination reduces the risk of a potentially fatal infection. Considering the increased risk of allergic reactions, it is crucial for these pts to be safely vaccinated. The incidence of vaccine-related anaphylaxis has been reported to be 5 times higher for mRNA vaccines than others. The Italian Ministry of Health (MH) suggests mRNA vaccines for frail individuals. In pts with mastocytosis, it recommended prophylaxis with antihistamines from the day prior to the vaccination to 5 days after, and an observation of 60 min after the vaccination. The administration of the vaccine in a hospital setting was recommended in those with a history of anaphylaxis episodes. In our hospital a vaccination campaign for frail pts in 2 phases was organized.

Aims: The aim of this study is to retrospectively evaluate the incidences of SARS-CoV2 infection in our pts with mastocytosis, the complications that may arise, incidence and type of vaccination, and the frequency of adverse reactions to vaccines.

Methods: Sixty-three pts with mastocytosis, 30 adults (14 male, 16 female; median age 57.1 years) and 33 children (17 male, 16 female; median age 4.7 years, 2 of them >16 years) were considered. According to the WHO 2016 classification, out of 30 adults, 5 had isolated CM, 15 ISM, 1 had smouldering mastocytosis (SSM), 5 had ASM, and 4 SM-AHN. Out of 33 pediatric pts, 1 had ISM, 18 maculopapular cutaneous mastocytosis, 8 diffuse cutaneous mastocytosis and 6 mastocytoma. Seven adults had a history of anaphylaxis.

Results: Overall, 5 adult pts (3 ISM, 2 ASM), and none of the pediatric pts, got SARS-CoV2 infection (Table 1). Four of them refused vaccination when it became available. Out of 8 frail mastocytosis pts (4 ASM, 4 SM-AHN) without previous SARS-CoV2 infection, 4 (2 ASM, 2 SM-AHN), in treatment with kinase inhibitors (KI), accepted it, and 4 refused. Overall, 21/32 pts (65%) eligible to have the SARS-CoV2 vaccination received it. Fifteen out of 21 pts (1 CM, 6 ISM, 1 SSM, 4 SM-AHN, 3 ASM) received the mRNA vaccine according to the Italian MH recommendations, 5 at our hospital, and 10 elsewhere. Patients were treated with cetirizine 10 mg twice a day as antihistamine prophylaxis. Out of these 15 pts, 4 had previous episodes of anaphylaxis, and 5 (3ASM, 2 SM-AHN) were in treatment with KI (2 Imatinib, 3 Midostaurin). The remaining 6 pts (2 CM, 3 ISM, 1 ASM) received vaccination (5 mRNA and 1 viral vector vaccine) without any prophylaxis and/or caution. One of these patients had had previous episodes of anaphylaxis. Both doses of vaccine were well tolerated in all pts. None showed adverse events, anaphylactic vaccine-related reactions, or developed COVID-19 infection after vaccination.

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Summary/Conclusion: In our experience, 35% of pts with mastocytosis eligible to receive the SARS-CoV2 vaccination, refused it. This is in line with the general population. None of the vaccinated pts had adverse events or anaphylaxes, regardless of prophylaxis and type of vaccine. None of the pts that received vaccination developed SARS-CoV2 infection. Pts affected by SARS-CoV2 infection (8%) completely recovered, including those with an advanced form of mastocytosis in treatment with KI.

P1581: FEEDING IMMUNITY‘S CLOCK. THE REGULATION OF CIRCADIAN IMMUNE RESPONSES BY NUTRITIONAL CUES.

S. Geiger 1,* , R. Siegel 2 , M. Essers 1

1 Inflammatory Stress in Stem Cells, DKFZ Heidelberg, Heidelberg, Germany; 2 Novartis, Basel, Switzerland

Background: Disruption of circadian rhythm is associated with inflammatory diseases, metabolic syndrome and cancer. In mice, the time of day strongly influences lethality in response to LPS, with survival greatest at the beginning compared to the end of the light cycle (Halberg et al. 1960; Marpegan et al. 2009; Nguyen et al. 2013). Myeloid-cell intrinsic circadian clock components control inflammatory cytokine production (Gibbs et al. 2012; Curtis et al. 2015; Bellet et al. 2013), and metabolic inputs influence lethality in response to LPS (Wang et al. 2016; Weis et al. 2017; Traba et al. 2017), but the relative contributions of these inputs to daily changes in sepsis susceptibility are not known.

Aims: In this study we aim at better understanding the relative contributions of the light- and feeding cycle to daily changes in sepsis susceptibility and the metabolic requirements of hematopoietic stem cells as the origin of immune cells.

Methods: Using time restricted feeding, we have dissociated light from feeding cues to investigate sepsis survival in mice. Using animals deficient for BMAL1 in myeloid cells or hepatocytes we have further characterized a critical role for a functional clock in the liver to convey time-of-day dependent sepsis susceptibility. Further, we have generated several RNAseq datasets to better understand the molecular mechanisms in the liver upon time restricted feeding, but also the metabolic changes that occur in hematopoietic stem cells upon inflammatory stimulation. In addition we are complementing this transcriptional profiling with functional metabolic assays.

Results: Our work shows that feeding, rather than light, controls time-of-day dependent LPS sensitivity. Mortality following LPS administration after 12 hours of food deprivation was associated with hypoglycemia, rather than inflammatory cytokine production, and this was independent of the clock regulator BMAL1 expressed in myeloid cells. In contrast, deletion of BMAL1 in hepatocytes globally disrupted the transcriptional response to the feeding cycle in the liver and resulted in constitutively high LPS sensitivity. Using RNAseq and functional validation studies we identified hepatic farnesoid X receptor (FXR) signaling as a BMAL1 and feeding-dependent regulator of LPS susceptibility. These results show that hepatocyte-intrinsic BMAL1 and FXR signaling integrate nutritional cues to regulate survival in response to innate immune stimuli.

As the origin of all immune cells, hematopoietic stem cells (HSCs) undoubtedly play a critical role in the inflammatory response. We are therefore currently extending our research to investigate the effects of nutritional cues on HSCs, and the metabolic changes that are critical to mount appropriate stress responses. To this end, we have generated time resolution bulk and single cell RNA sequencing data of in vivo stimulated HSCs and are complementing this transcriptional information with functional assays to better profile metabolic cues.

Summary/Conclusion: Targeting specific pathways will allow us to assess their relevance in the context of inflammation and shed light on previously published contradictory findings. At the same time, understanding the molecular programs operational in response to these cues has the potential to identify novel pathways for targeting to enhance endotoxemia resistance.

P1582: CLINICAL CHARACTERISTICS AND INFECTION-RELATED OUTCOMES IN PATIENTS WITH LYMPHOID MALIGNANCIES WITH OR WITHOUT SECONDARY IMMUNODEFICIENCIES: RESULTS FROM A RETROSPECTIVE MULTI-DATABASE STUDY

M. S. Davids 1,* , J. Richter 2 , C. Anderson-Smits 3 , M. Kamieniak 4 , K. Ren 3 , M. Hull 5 , J. Multani 5 , D. Shah 3 , C. Siffel 3

1 Dana-Farber Cancer Institute, Boston; 2 Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York; 3 Takeda Development Center Americas, Inc., Lexington, United States of America; 4 Takeda Pharma Sp.z.o.o., Warsaw, Poland; 5 IQVIA, Falls Church, United States of America

Background: Patients with lymphoid malignancies are at particularly high risk of developing secondary immunodeficiencies (SID). Current guidelines do not provide an adequate framework for clinicians to identify patients at highest risk of developing SID and subsequent infections; nor do they provide clear treatment pathways once such patients are identified. Additional data in these areas may help to optimize outcomes for patients with lymphoid malignancies who experience SID.

Aims: To understand the demographics, clinical characteristics and burden of infection in patients with lymphoid malignancies and SID, compared with those in patients without SID.

Methods: In this observational, retrospective cohort study, data were extracted from two large electronic record databases – Optum-Humedica (H-DB) and Guardian Research Network (G-DB) – for the period Oct 1, 2015 to Mar 10, 2020. This period included a 6-month pre-index period (PIP) and a 12-month follow-up period following patient identification. Patients aged ≥18 years were included if they had a confirmed diagnosis of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), multiple myeloma (MM) or non-Hodgkin lymphoma (NHL; H-DB only) in the PIP. Using an algorithm that included diagnosis codes, immunoglobulin G (IgG) serum levels and presence of key infections/treatments, eligible patients were stratified into two cohorts: those with SID (SID cohort) and those without SID (no-SID cohort). Patients with SID or primary immunodeficiencies in the PIP were excluded. The SID index date was defined as the first occurrence of a hypogammaglobulinemia ICD-10 code, or a record of low (<5.0 g/L) serum IgG levels. The primary outcome was severe bacterial infection over 12 months of follow-up. Secondary outcomes included overall survival (Kaplan–Meier analysis).

Results: In total, 2,221 patients with SID were included (H-DB/G-DB: n=1,959/262 [25.6%/33.6% CLL or SLL, 44.4%/66.4% MM, 30.0%/0% NHL]), as were 19,141 patients without SID (H-DB/G-DB: n=17,598/1,543 [22.3%/50.2% CLL or SLL, 21.4%/49.8% MM, 56.3%/0% NHL]). Mean patient age and the proportion of men were similar in both cohorts (H-DB/G-DB SID cohort: 67.3/70.6 years, 54.8%/56.1% men; no-SID cohort: 67.9/72.2 years; 52.8%/53.6% men).

At 12-month follow-up, across both databases, significantly more patients with SID had experienced ≥1 infection than those without SID (both p <0.001; Table ). Bacterial infections were the most frequently reported type of infection in both databases and across cohorts (H-DB/G-DB SID cohort: 55.1%/54.6%; no-SID cohort: 24.0%/25.7%). Significantly more patients with SID experienced ≥1 severe bacterial infection than those without SID (both p <0.001); H-DB/G-DB mean (standard deviation) number of severe bacterial infections was 7.6 (9.9)/2.9 (2.7) for the SID cohort versus 5.2 (6.8)/2.4 (2.2) for the no-SID cohort ( Table ). The proportions of patients experiencing ≥1 hospitalization associated with infection and of those using anti-infective treatment were also significantly greater in the SID cohort than in the no-SID cohort (both p <0.001; Table ). Overall survival at 24 months was lower for patients with SID (H-DB/G-DB: 76.3%/77.3% of patients alive) than for those without SID (86.9%/87.8%).

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Summary/Conclusion: With comparable results in two large databases, this study confirms that patients with lymphoid malignancies and SID face a significantly higher burden of infectious complications and have shorter overall survival than those without SID.

P1583: EVALUATION OF THE KNOWLEDGE OF HEMATOLOGISTS ABOUT THE MANAGEMENT OF INFECTIOUS COMPLICATIONS IN HEMATOLOGIC PATIENTS

M. Guaraná 1,* , M. Nucci 1

1 Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Background: Infection is a serious complication among patients with hematologic malignancies (HM) and in hematopoietic cell transplant (HCT) recipients. In most centers, the management of these complications is provided by the hematologist itself, thus demanding a knowledge of basic aspects of infection.

Aims: To evaluate the level of knowledge about infectious complications by hematologists, and to identify possible gaps that could help to design educational activities.

Methods: We invited clinicians to answer two questionnaires with 20 multiple-choice questions covering areas of the epidemiology, prophylaxis, diagnosis and treatment of infection in patients with HM and HCT. Basic sociodemographic data of hematologists were also collected.

Results: We obtained 289 answers: 223 in survey 1 (febrile neutropenia) and 66 in survey 2 (infection in HCT). The median score was 5.0 in both surveys (range 0.5-9.0). In survey 1, the questions with the lowest number of correct answers were Q3 (7%), about cefepime dose and Q1 (9%) which asked about the epidemiologic link between the use of high-dose cytarabine and viridans streptococcal bacteremia. In survey 2, two questions about cytomegalovirus (CMV) infection had the lowest percentage of correct answers (Q4, 12% and Q11, 18%). Clinicians taking care of HCT recipients had higher scores compared with clinicians taking care of patients with HM only (median score of 5.0 and 4.5, p=0.01 in survey 1, and 6.0 and 4.5, p=0.001 in survey 2). In both surveys staff clinicians, residents and professors had similar scores.

Summary/Conclusion: This is the first study in Brazil assessing the knowledge of haematologists about infectious complications. The low median score overall indicates an urgent need for continuous education. Such initiatives will eventually result in better patient care.

P1584: CELLULAR IMMUNE RESPONSE TO THE BNT162B2 VACCINE IN LYMPHOMA PATIENTS

R. Gurion 1,2,* , U. Rozovski 1,2 , G. Itchaki 1,2 , A. Gafter-Gvili 1,2,3 , C. Leibovitch 1,2 , P. Raanani 1,2 , M. Szwarcwort 4 , M. Taylor-Abigadol 5 , E. Dann 5,6 , N. Horesh 5 , T. Inbar 5 , I. Tzoran 5,6 , N. Lavi 5,6 , E. Bar Haim 7 , H. Cohen 7 , I. Slouzky 8 , T. Azoulay 8 , M. Karmona 8 , S. Ringelstein-Harlev 5,6 , N. A. Horowitz 5,6

1 Hematology, Rabin Medical Center, Petach Tikva; 2 Hematology, Tel Aviv University, Tel Aviv; 3 Internal Department A, Rabin Medical Center, Petach Tikva; 4 Virology Laboratory; 5 Hematology, Rambam Health Care Campus; 6 Hematology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Haifa; 7 Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona; 8 Hematology laboratories, Rambam Health Care Campus, Haifa, Israel

Background: In most individuals, protective humoral and cellular immunity develops after two doses of the BNT162b2 Pfizer vaccine. In patients with lymphoma, humoral response is weaker and almost universally abrogated in patients who received anti-CD20 monoclonal antibodies. Whether cellular immune response is also abrogated is unknown.

Aims: To determine whether patients with lymphoma develop specific T-cell mediated cellular response to BNT162b2 Pfizer vaccine.

Methods: We included patients with lymphoma above the age of 18 years who received two doses of the BNT162b2 Pfizer vaccine and collected clinical and demographics data. T-cell immune response to the vaccine was analysed in patients’ blood samples stimulated by spike antigen and quantified by two methods: (1) Interferon-gamma (IFNg)-release assay (IGRA, EuroImmun, Germany)- IFNg was quantified by ELISA (DuoSet, R&D Systems, Minneapolis, Minnesota, USA) and response above 50 pg/ml was considered positive. (2) Flow cytometry- Quantification of the T cell activation markers, CD134+ CD25+CD4+ T-cells was performed (Act-T4 Cell TM kit, Cytognos, Spain), and any response above 0 was considered positive. Humoral response was measured by SARS-CoV-2 IgG II Quant (Abbott©) assay. The positive cut-off was set at 50AU/ml. Blood samples were drawn approximately 4 months after the second vaccination.

Results: Sixty-nine lymphoma patients, treated with two vaccine doses, were included in this study. Median age was 66 (range: 30-84) and 39 (57%) were males. Sixty-two patients (90%) had non-Hodgkin lymphoma (NHL) including 18 with DLBCL, 26 with follicular lymphoma and 14 with marginal zone lymphoma. Seven (10%) patients had Hodgkin lymphoma. In this cohort, 70% (n=49) of the patients received anti CD20 MoAb, and 35% of them (n=27) were still on anti CD20 treatment. Thirteen patients received bendamustine-based immunochemotherapy. At the time of assessment (median 4.8 months after the 2 nd vaccine) anti-spike antibodies were detected in only 42% (N = 29) of patients. In comparison, there was an increase in specific T cell response by any assay (IGRA and Flow) in 49% of patients (n = 34). The correlation between the IGRA and flow data was 0.7 (pearson correlation, P = 0.01). However, no correlation between humoral (qualitative and quantitative) and T cell response was shown, regardless of the assay applied. Cellular response was not corelated with the time elapsing from last immunochemotherapy. In the anti-CD20 MoAb treated cohort, of which 27 patients were still on active treatment at the time of vaccination, only 2 patients (7%) developed a humoral immune response, while cellular immunity was elicited in 52% (N = 15) patients (ELISA assay). In the Bendamustine treated cohort, with a median time from end of treatment to vaccination of 23 months (1-106 months), humoral but not cellular response correlated positively with the time from treatment completion to vaccination (p=0.04).

Summary/Conclusion: The rate of cellular and humoral response to two doses of the BNT162b2 Pfizer vaccine in lymphoma patients was found to be significantly abrogated. In this small cohort, 49% of patients developed a cellular response despite a severely abrogated humoral immunity. These findings suggest that vaccine administration should be considered even early after anti CD20 therapy despite the reduced humoral immunity. These findings should be validated in studies with a higher number of patients.

P1585: THE ROLE OF INTESTINAL MICROBIOME IN SYSTEMIC INFECTIONS IN PATIENTS WITH MULTIPLE MYELOMA

1. Institute Of Hematology And Transfusiology, St.Pet 1 , V. Burylev 1 , I. Kostroma 2 , E. Kiseleva 1 , N. Stizhak 1 , E. Kaytandzhan 1 , S. Gritsaev 2 , S. Bessmeltsev 3 , V. Chebotkevich 1,*

1 Department of microbiology; 2 Department of bone marrow transplantation; 3 Russian Research Institute of Haematology and Ttransfusiology, St.Petersburg, Russia

Background: Intensive cytostatic therapy, used in the treatment of hemoblastosis, as well as conditioning therapy before hematopoietic stem cell transplantation (HSCT) lead to severe intestinal epithelial disorders, and massive antibacterial therapy leads to profound changes in the composition of the intestinal microbiome.

Aims: to study the intestinal microbiome during cytostatic therapy and HSCT in patients with hemoblastosis and their role as predictors of the development of systemic bloodstream infections

Methods: We studied the intestinal microbiome of 18 adult patients (8 women, 10 men, aged 54-66 years) with multiple myeloma (n=15) and nonHodgkin’s lymphoma (n=3), who underwent autoHSCT. From each patient we obtained the stool speciments before and after HSCT. Among the eligibility criteria for the study was accepted the presence of ≥ 3 consecutive high quality fecal specimens in the one patient. From each fecal specimen, DNA was extracted and purified and was sequenced using the MiSeq Illumina platform. The species diversity of the microbiome was assessed using the Shannon index. The index of microbial diversity (Shannon index) was calculated in different periods before and after autoHSCT. The significance of the differences was determined using the paired Student’s test.

Results: A significant (p = 0.0305) decrease in the microbiota diversity index was revealed in the period after autoHSCT. . The characteristics of changes in the composition of the microbiome at the Phylum level before and after autoHSCT were studied. It was found that a significant change in the microbiome at the Phylum level was revealed only in relation to the Actinobacteria group. No significant changes in the level of Bacteroides, Firmicutes, and Proteobacteria were found.

In this group of patients the bloodstream infection was determined in one case. During the observation in post transplant period we reveled the increase of E. coli in his stool specimen from 10 7 up to 10 9 which was regarded as a predictor of the development of a systemic infection of the bloodstream. Earlier we demonstrated the domination of E. coli in gram-negative systemic infections in immunocompromised oncohematological patients. [Chebotkevich V.N et al., Cellular Therapy and Transplantation. 2021. Т. 10. № 1. С. 63-68].

Summary/Conclusion: The study of the microbiome by sequencing using the MiSeq Illumina platform made it possible to identify important patterns of changes in the microbiota in oncohematological patients with autoHSCT. The most severe bloodstream infections in patients with hemoblastosis are associated with gram-negative microbes, of which E. coli is dominated.

P1586: IN-HOSPITAL MORTALITY AND POOR PROGNOSIS FACTORS ASSESSMENT IN PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA AND COVID-19.

D. Ivanova 1,* , E. Baryakh 1 , T. Tolstykh 1 , T. Chudnova 1 , Y. Polyakov 1 , O. Kochneva 1 , M. Litvinenko 1 , E. Misyurina 1 , K. Yatskov 1

1 Moscow Clinical Hospital 52, Moscow, Russia

Background: Acute lymphoblastic leukemia (ALL) belongs to a heterogeneous, highly aggressive group of hematologic malignancies. In the context of the epidemic of a new coronavirus infection (COVID-19), patients with hemoblastoses have a more severe course of the viral infection and high mortality rate due to COVID-19. The management protocols of patients with ALL in the context of the COVID-19 pandemic has not been developed, and approaches to both the treatment of a new coronavirus infection and the antitumor treatment of ALL in patients with COVID-19 have not been defined.

Aims: To analyze in-hospital overall survival of patients with acute lymphoblastic leukemia and coronavirus infection, to assess the impact of poor prognostic factors on the outcome of the disease, and to determine the therapeutic tactics for ALL and COVID-19.

Methods: From April 21, 2020 to November 30, 2021 46 patients with ALL (24 men, 22 women) with a median age of 44.5 (18-74) years were observed on the basis of the hematological service of the City Clinical Hospital No. 52 (Moscow). According to the immunophenotypic variant of ALL, 27 (59%) were patients with B-ALL, of which Ph-positive ALL was diagnosed in 8 (30%), Ph-negative - in 19 (70%); T-ALL - 16 (35%), biphenotypic - 2 (4%). Patients with newly diagnosed ALL (20%) underwent induction antitumor therapy, 67% - convalescents and PCR positive for SARS-CoV-2 infection. To determine the factors of hospital mortality, a statistical analysis was performed, including: age, hematological disease status, degree of lung tissue damage according to computer tomography (CT), stay in the intensive care unit due to severe respiratory failure requiring mechanical ventilation (ALV), the presence of neutropenia of the IV degree, transfer from another hospital, the formation of an antiviral immune response after a coronavirus infection.

Results: Hospital mortality in ALL patients was 30%. Statistically significant factors for poor prognosis were: age (>40 years), p=0.033, hematological disease status (newly diagnosed ALL, progression/relapse), p=0.004, high (≥50%) degree of lung tissue damage according to CT (p =0.003), neutropenia IV degree (p=0.004), their transfer to another hospital, stay in the intensive care unit and mechanical ventilation (p=0.004).

Summary/Conclusion: The main adverse factors affecting hospital mortality were the absence of ALL remission and the presence of severe infectious complications. Carrying out antitumor therapy allows to stabilize the status of ALL and is an important aspect in the simultaneous course of coronavirus infection. In patients with ALL and confirmed coronavirus infection, the decision to conduct induction therapy should be discussed on an individual basis, depending on ALL status, the activity of coronavirus infection and comorbidity status.

P1587: IS IT SAFE FOR OUR PATIENTS TO COME OUT?

M. Jabbar Al-Obaidi 1,* , A. Chew 1 , C. Liu 1

1 Haematology, Chelsea and Westminister NHS Trust, West Middlesex University hospital, Isleworth, United Kingdom

Background: Vaccination against COVID-19 commenced in England in December 2020, most haematology patients iwere included in the first wave as they are identified as extremely vulnerable.

A second dose was administered 12 weeks after the initial dose, and booster doses became available in September 2021.

Severely immune compromised patients started to be contacted in December 2021 to offer them a fourth dose of vaccine.

With the availability of treatments there arose a need for early confirmation of presence or absence of antibodies against COVID-19 and testing for antibody was introduced into routine practice for patients about to commence chemotherapy, or those who had yet to complete the four-dose protocol.

On the 1st of March most restrictions imposed during the pandemic were removed, in the expectation that the majority of people would have protective antibodies from vaccination.

Aims: Over a period of six months from October 2021 to February 2022 we tested 90 patients attending routine haematology clinics, with a number of conditions, mainly malignancy under follow-up or recieving treatment.

We reviewed the results of these tests, to assess degree of protection

Methods: Retrospective analysis of all requests for SARS-Cov antibody tests carried out in our patient populations.

Results: There were 55 males, and 35 females, with an average age of 65y (range 24-90)

The majority attended clinic for management of haematological malignancy (Plasma cell disorders, Chronic Leukaemia, Lymphomas) with a smaller number of patients being managed for congenital or immune conditions.

Summary/Conclusion: Despite early availability of vaccines, and delivery of more than two doses to the majority of our patients, only half our patients had significant antibody response, and almost one quarter still had no detectable antibody.

Although the number of new cases is reducing, in the vicinity of our hospital there were 483 new cases per 100,000 on the last day of February, and during the six months of this study two fully vaccinated patients with CLL with no detectctable antibody died in hospital with covid-19 pneumonitis.

For some haematology patients particulalry those with CLL it is still not safe to come out.

P1588: A SINGLE CENTER COMPARATIVE CLINICAL ANALYSIS OF THE SARS-COV-2 INFECTION BEFORE AND AFTER VACCINATION IN PATIENTS DIAGNOSED WITH HEMATOLOGIC MALIGNANCIES

M. Jiménez 1,* , S. Novoa 1 , C. Fernandez-Naval 2 , V. Navarro 3 , C. Andrés 4 , M. Martínez-Gallo 5 , A. Antón 2 , L. Fox 1 , E. Roldán 1 , A. Cabirta 1 , M. Gironella 1 , G. Orti 1 , P. Barba 1 , D. Medina 6 , S. Peralta 6 , G. Pujadas 6 , C. Hernández 6 , C. Pages 6 , T. Pumarola 2 , M. Valentin 1 , A. Marín-Niebla 1 , D. Valcárcel 1 , M. Hernández 5 , I. Ruiz-Camps 7 , M. Crespo 6 , J. Esperalba 2 , F. Bosch 1 , P. Abrisqueta 1

1 Hematology; 2 Microbiology, Vall d’Hebron Hospital Universitari; 3 Oncology Data Science (ODysSey) Group; 4 Microbiology, Vall d’Hebron Institute of Oncology (VHIO); 5 Immunology, Vall d’Hebron Hospital Universitari; 6 Experimental Hematology, Vall d’Hebron Institute of Oncology (VHIO); 7 Department of Infectious diseases, Vall d’Hebron Hospital Universitari, Barcelona, Spain

Background: An impaired humoral and cellular immunogenicity to the mRNA SARS-CoV-2 vaccines has been reported in patients diagnosed with hematologic malignancies. Data regarding the effectiveness of SARS-CoV-2 vaccination on COVID-19 evolution an immunocompromised population are still scarce.

Aims: To compare disease severity and mortality in a real-life cohort according to the vaccination status of patients diagnosed with hematologic malignancies developing COVID-19 disease.

Methods: We retrospectively collected clinical data from the patients with a current or prior history of hematologic malignancy in the last 5 years at Vall d´Hebron Hospital whom suffered a SARS-CoV-2 infection. Patients were unvaccinated between March 2020 and May 2021, and partially or completely vaccinated between May 2021 and January 2022.

The primary endpoint was COVID-related mortality according to vaccination status. Secondary endpoints included rate and length of hospitalization, ICU admission, need for supplemental oxygen including high flow oxygen therapy and mechanical ventilation, and severity of COVID-19 disease.

Results: Overall, 113 vaccinated and 81 unvaccinated patients developed COVID-19. Both groups were similar in age (median 69 and 72 years, respectively), gender predominance, diagnosis (mainly lymphoproliferative diseases [58% and 53%]), and administered active treatment (mostly anti-CD20 monoclonal antibodies [38% and 37%]). In contrast, more patients received active treatment (78% vs. 48%; p=<0.001) in the vaccinated cohort.

Vaccinated patients developed infection by the delta variant in 27% (17/62) and by the omicron variant in 73% of the sequenced cases (45/62). Genotype analysis of the variant in unvaccinated cases is ongoing. Despite the fact that 96% of patients received 2 vaccine doses and 73% also a third boost dose, in only 67% (59/88) of patients basal IgG anti-spike antibodies were detected after vaccination.

In comparison with the unvaccinated cohort of patients, vaccinated patients had less severe (17% vs. 42%; p=0.01) and critical (13% vs 28%; p=0.03) COVID-19 infection, lower rates of hospitalization (42% vs. 90%; p=<0.001) with shorter length of stay (median 13 days (IQR 7-34) vs. 11 days (IQR 6-16), and fewer patients needed supplemental oxygen (34% vs. 73%; p=0). Admission rates to ICU were similar between both cohorts. Finally, mortality rate was lower in the vaccinated patients in comparison with the unvaccinated cohort (HR: 0.19 (95%CI 0.09, 0.38); p=<0.001). Of note, in hospitalized patients only, the mortality of the vaccinated cohort was also lower (HR: 0.38 (95%CI 0.19, 0.76); p=0.007). Among vaccinated patients who suffered a severe/critical infection, 52% did not have vaccine seroconversion. Of the vaccinated patients who died (11/113), 6/8 had negative anti-S IgG, 5/9 were infected by the delta variant and 4/9 by omicron, and 10/11 were on active treatment (5/11 with monoclonal antibodies).

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Summary/Conclusion: In our series, despite the heterogeneity of infection by different viral variants and treatment availability for COVID-19 during the evolution of the pandemic, vaccination has been widely effective at reducing need of hospitalization, severity and mortality of the SARS-CoV-2 infection in hematological patients. Nevertheless, a subgroup of vaccinated patients remains with an adverse outcome and requires further identification and management improvement.

P1589: AGENTS CONTRIBUTING TO SECONDARY IMMUNODEFICIENCY DEVELOPMENT IN PATIENTS WITH MULTIPLE MYELOMA, CHRONIC LYMPHOCYTIC LEUKEMIA AND NON-HODGKIN LYMPHOMA: A SYSTEMATIC LITERATURE REVIEW

S. Jolles 1 , S. Giralt 2 , T. Kerre 3 , H. M. Lazarus 4 , S. S. Mustafa 5 , R. Ria 6,* , D. C. Vinh 7

1 University Hospital of Wales & Noah’s Ark Children’s Hospital for Wales Cardiff, Cardiff, United Kingdom; 2 Memorial Sloan Kettering Cancer Center, New York, United States of America; 3 Ghent University Hospital, Ghent, Belgium; 4 Case Western Reserve University, Cleveland; 5 Rochester Regional Health, Rochester, NY & University of Rochester, Rochester, United States of America; 6 University of Bari Aldo Moro Medical School, Bari, Italy; 7 McGill University Health Centre, Montreal, Canada

Background: Patients with hematological malignancies (HM), such as multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL), have a higher risk of secondary immunodeficiency (SID), SID-related infections and mortality, due to both the intrinsic pathophysiology of the disease and certain cancer treatments. Here, we report the results of an interim analysis from a systematic literature review on the contribution of multiple cancer treatments to SID development.

Aims: To provide an overview of the cancer treatments associated with SID, including the incidence of SID and infections.

Methods: This review is in accordance with PRISMA guidelines on reporting reviews of the literature. On January 19, 2022, a systematic literature search was performed using the PubMed database to search for studies that mentioned in the title and/or abstract selected cancer treatments (Figure 1). Search strings included the MeSH terms for three types of HM: CLL, MM, or NHL; types of HM that are more chronic than others. Further inclusion criteria were applied, such as: published between 2011 and 2022; English language; included humans; and labeled as a clinical trial in PubMed. As shown in Figure 1, this initial search resulted in 721 entries, which were then further refined to include Phase III, Phase IV, and observational studies only, resulting in 236 studies. Publications had to report percentages of patients with any grade or grade ≥3 infections, any grade or grade ≥3 neutropenia, or hypogammaglobulinemia to be considered relevant. Of the 236 studies, 82 were considered relevant. For this interim analysis, we included the open-access publications only (n=53). The poster will report data from the full analysis, including all relevant publications.

Results: Out of the 53 studies, 11 included patients with CLL, 25 with MM and 17 with NHL. No studies reported the proportion of patients with hypogammaglobulinemia. Of note, not all studies reported values for both any grade and grade ≥3 events; therefore, in some instances the proportion of patients with grade ≥3 events might be higher than the proportion of patients with any grade events. Grade ≥3 infections were reported in 7.8–39% of patients with CLL, 0–50.2% of patients with MM, and 8.8–38% of patients with NHL. Across patients treated with an anti B-cell monoclonal antibody monotherapy or in combination, any grade and grade ≥3 infections were reported in 14.4–69.1% and 7.8–39% of patients with CLL, and 10–23.8% and 12–23% of patients with NHL, respectively. In the only study on MM in which patients were treated with an anti B-cell monoclonal antibody monotherapy or in combination, any grade and grade ≥3 infections were reported in 50.2% and 13.3% of patients, respectively. Comparable percentage of infections were reported in patients treated with a tyrosine kinase inhibitor or a proteasome inhibitor monotherapy or in combination across HM. The highest grade ≥3 infection rate was 50.2% in patients with MM treated with lenalidomide maintenance therapy.

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Summary/Conclusion: This systematic literature review will help shed light on the actual proportion of patients who might develop SID due to various cancer treatments. These results highlight the importance of prophylactic treatment approaches, which aim to reduce the risk of infections due to SID in patients with HM.

P1590: OUTCOME OF HYPERBARIC OXYGEN THERAPY IN PATIENTS WITH HEMATOLOGIC MALIGNANCIES –A TERTIARY CANCER CENTER EXPERIENCE

K. Kim 1,* , H. Ahaneku 1 , M. Deborah 2 , K. Sasaki 1 , D. Hammond 1 , K. Chien 1 , J. Adachi 3 , A. Ferrajoli 1

1 Department of Leukemia; 2 Division of Pharmacy; 3 Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, United States of America

Background: Hyperbaric oxygen therapy (HBO) has been used for various type of refractory infections in patients with hematologic malignancies. Despite of its prevalent use, efficacy of HBO in such population has been poorly studied.

Aims: Here, we reviewed our clinical experience with HBO use for patients with hematologic malignancies including patients that had undergone a stem-cell transplant.

Methods: The study patients were identified by insurance authorization data related to HBO treatment between December 2012 and October 2019 at a single tertiary cancer center. Patients with the underlying diagnosis of hematologic malignancies were included. Demographic and clinical data were collected retrospectively.

Results: 49 patients were included. Median age at HBO therapy was 51 years (range 22-86). Most common underlying disease was acute myeloid leukemia (53%), followed by acute lymphoblastic leukemia (18%), myelodysplastic syndrome (10%) and non-Hodgkin’s lymphoma (8%). 34 (68%) patients had active disease including 28 (57%) patients with relapsed/refractory disease. Thirty (61%) patients were stem-cell transplant (SCT) recipients. Median time from SCT to HBO was 102 days. Majority of patients (88%) were SCT recipients within 6 month or having active malignancies at time of HBO therapy.

Twenty-five (51%) patients had neutropenia at HBO initiation, only 5 patients recovered counts at the end of HBO. Treatment indications included: BK cystitis (37%), fungal sinusitis (33%), cellulitis (20%), non-BK cystitis (8%) and other fungal infection (2%). Median number of HBO sessions was 5 (range 1–60), and median HBO duration was 17 days (range 1–109 days).

Forty-four patients were evaluable for response of their infection to HBO treatment: 52% had improvement, 6% had stable infection, and 41% failed to respond. Twenty-four (49%) patients had uncontrolled infection at the last follow-up, including 8 out of 25 patients who achieved some improvement from HBO.

Median survival from HBO was 3.0 months (range 0.2-81.1). Sixty-day and 6-month mortality were 33% and 65%, respectively. Mortality from indicated infection with or without underlying disease was 37%, death was attributed to underlying disease progression in 66% of the patients and to infections in 34% of the patients. Twenty-seven (55%) patients were discharged from the hospital to outpatient care after HBO, 15 (31%) patients were discharged to hospice, and 7 (14%) patients died in the hospital.

Fourteen (32%) patients had recovery that we defined as being discharged from hospital with improved or stable infections after HBO responses. Patients with non-BK cystitis (OR 7.01, p=.087) or patients in remission of underlying hematologic malignancies without recent (within 6 months) SCT (OR 5.6, p=.049) were more likely to be recovered. Patients with recovery had superior survival than rest of patients (median 14.0 months, HR 0.28, p=.001)

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Summary/Conclusion: Majority of patients who received HBO therapy were severely immunocompromised with active hematological disease, receiving chemotherapy or recent SCT receipt. Overall these patients had a dismal outcome with short median survival at 3.0 months, death was not only from infection but also from underlying disease. A subset (32%) of patients achieved control of the infection and a longer survival after HBO therapy; these patients were more likely to have achieved a remission of their hematological malignancy without recent SCT history. Additional studies are needed to identify the right population who would benefit from HBO treatment.

P1591: HIGH RISK OF BLOOD STREAM INFECTION OF CRE CARRIERS IN NEUTROPENIC CHILDREN WITH HEMATOLOGICAL DISEASES

L.-P. Liu 1,* , Y. Guo 1 , X.-F. Zhu 1

1 Division of Pediatric Blood Diseases Center, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Neutropenic children with hematological diseases were associated with higher morbidity of carbapenem-resistant enterobacteriaceae (CRE) blood-stream infection (BSI) or colonization.

Aims: We aimed to identify the clinical characteristics and clinical outcome caused by CRE-BSI.

Methods: 2465 consecutive neutropenic children were enrolled. Antimicrobial susceptibility of CRE strains were explored and potential factors associated with subsequent CRE-BSI and 30-day survival probability were assessed.

Results: CRE-carriers were identified in 59/2465 (2.39%) patients and19/59 (32.2%) developed CRE-BSI, while 12/2406 (0.5%) of non-carriers developed CRE-BSI ( P <0.001). The 30-day survival probability was significantly lower in patients with CRE-BSI than non-BSI (73.9% vs. 94.9%, P =0.050). Moreover, 30-day survival probability was also poorer in CRE-carriers versus non-carriers (49.7% vs. 91.7%, P =0.048) among CRE-BSI patients. Tigecycline and amikacin exhibited the satisfactory antimicrobial activity against all isolated strains. Fluoroquinolone sensitivity was lower in E. coli (27.1%) versus satisfactory susceptibility of E. cloacae strains (93.1%). CRE-BSI accompanying intestinal mucosal damage were independent risk factors for 30-day survival probability (both P <0.05), while mucositis was also prone to developed CRE-BSI ( P =0.034).

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Summary/Conclusion: CRE-BSI was regarded as an independent predictor predisposing to high mortality in neutropenic children. Moreover, individualized antimicrobial therapy should be adopted due to the different features of patients with separate CRE strains.

P1592: MATCHED CONTROL COMPARISON OF SURVIVAL OF HOSPITALIZED COVID-19 PATIENTS WITH HEMATOLOGICAL MALIGNANCIES, DATA FROM A LARGE TERTIARY CENTER REGISTRY

M. Lucijanic 1,* , E. Soric 1 , M. Sedinic 1 , A. Sabljic 1 , S. Tomasinec 2 , A. Pasaric 2 , M. Ivic 1 , T. Vasilj 1 , D. Cicic 1 , Z. Jonjic 1 , M. Pirsic 1 , Z. Mitrovic 1 , Z. Prka 1 , V. Pejsa 1 , R. Kusec 1 , O. Jaksic 1

1 Hematology Department; 2 Clinical Immunology, Allergology and Rheumatology Department, University hospital Dubrava, Zagreb, Croatia

Background: There is an increasing pool of evidence describing detrimental outcomes of patients with hematological malignancies with acute COVID-19.

Aims: to evaluate risk of death with acute COVID-19 in patients with various hematological malignancies in comparison to matched control patients from a large single institution registry.

Methods: We retrospectively analyzed all consecutive hospitalized COVID-19 patients treated in period 3/2020-3/2021 in our institution (University hospital Dubrava) that served as a regional COVID-19 referral center during the pandemic. Data from a hospital Registry project were used. Patients with hematological malignancies were compared to the case-control matched group of patients without malignant disease. Matching was performed according to age, sex and modified Charlson comorbidity index (with subtracting points provided for hematological malignancy in hematological malignancy patients).

Results: Out of total 4014 consecutive hospitalized COVID-19 patients, there were 154 patients with hematological malignancies. There were 81/154 (52.6%) male patients. Median age was 71 years and median Charlson comorbidity index was 5 points. Most represented hematologic malignancy subtype was chronic lymphocytic leukemia (CLL) present in 37/154 (24%) patients, followed by aggressive lymphomas (30/154 (19.5%)), plasma cell dyscrasias (28/154 (18.2%)), acute leukemias (21/154 (13.6%)), indolent non Hodgkin lymphomas (18/154 (11.7%)), myelodysplastic syndromes (MDS, 12/154 (7.8%)) and chronic myeloproliferative leukemias (8/154 (5.2%)).

Disease course significantly differed between disease subsets (P=0.004) as shown in Figure. Patients with CLL and indolent lymphomas experienced more favorable disease course comparable to matched control groups of COVID-19 patients without malignancies, whereas other subsets of patients experienced more rapid deterioration. Recent treatment (actual or in 3 months prior to admission) did not affect survival outcomes (P=0.402), as well as active disease status (P=0.076) but where tendency for worse survival was evident. Patients previously receiving autologous or allogeneic stem cell transplantation also did not significantly differ in survival in comparison to non-transplanted patients (P=0.425).

In a multivariate survival analysis, acute leukemias (HR 5.88; P<0.001), MDS (HR 2.91; P=0.006), plasma cell dyscrasias (HR 4.68; P<0.001) and aggressive lymphomas (HR 2.53; P<0.001) were associated with worse survival in comparison to matched control patients independently of age (HR 1.04; P<0.001), modified Charlson comorbidity index (HR 1.1; P=0.017), and COVID-19 severity on admission (HR 5.98; P=0.003 for severe and HR 11.06; P<0.001 for critical vs mild disease).

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Summary/Conclusion: Hospitalized COVID-19 patients with acute leukemias, MDS, plasma cell dyscrasias and aggressive lymphomas experience higher mortality irrespectively of age, comorbidity burden and COVID-19 severity on admission.

P1593: IMMUNE BIOMARKERS TO PREDICT SARS-COV-2 VACCINE EFFECTIVENESS IN PATIENTS WITH HEMATOLOGICAL MALIGNANCIES

L.-E. Tamariz-Amador *1 , A. M. Battaglia *1,2 , C. Maia *1 , A. Zherniakova *1,3 , C. Guerrero *1 , A. Zabaleta 1 , L. Burgos 1 , C. Botta 2 , M.-A. Fortuño 1 , D. Alignani 1 , L. Blanco 1 , C. Grande 1 , A. Manubens 1 , J.-M. Arguiñano 4 , C. Gomez 5 , E. Perez-Persona 6 , I. Olazabal 7 , I. Oiartzabal 8 , C. Panizo 1 , F. Prosper 1 , J. F. San Miguel 1 , P. Rodriguez-Otero 1 , E. Martín-Sánchez #1, , B. Paiva #1 , A. V.-N. H.-H. ASOVASNA 9

1 Clinica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA), IdiSNA, Pamplona, Spain; 2 University “Magna Graecia” of Catanzaro, Catanzaro, Italy; 3 Russian Research Institute of Hematology and Transfusiology, Saint-Petersburg, Russia; 4 Hospital Universitario de Navarra, Pamplona; 5 Hospital Universitario de Galdakao, Galdakao; 6 Hospital Universitario de Araba, Vitoria-Gasteiz; 7 Hospital Universitario de Donostia, San Sebastian; 8 Hospital Universitario de Cruces; 9 Asociacion Vasco-Navarra de Hematología y Hemoterapia, Bilbao, Spain

Background: The great heterogeneity observed in the efficacy of vaccines for COVID-19 among hematological patients is probably associated with both therapy-related immunosuppression and disease-related immune dysregulation. We hypothesized that these features can be depicted in peripheral blood, and that immune profiling prior to vaccination can help predict immunogenicity.

Aims: Determine the immune landscape of patients with a mature B-cell and a plasma cell neoplasm prior to vaccination and its relationship with antibody response after two doses of SARS-CoV-2 vaccine.

Methods: We performed a comprehensive immunological characterization of 83 hematological patients before vaccination, and measured IgM, IgG and IgA antibody response to 4 viral antigens (RBD, S, N and Mpro) at day+7 after second-dose COVID-19 vaccination using multidimensional and computational flow cytometry. Health care practitioners (HCP) of similar age were the control group (n=102). Overall, 59 immune cell-types within the granulocytic, antigen presenting cell, T-cell and B-cell compartments, were systematically evaluated in peripheral blood of patients and HCP.

Results: When compared to HCP, 44 of the 59 (75%) immune cell-types were significantly altered in hematological patients; those with monoclonal gammopathies showed greater immunosuppression than patients with B-cell disorders and Hodgkin lymphoma. When comparing the relative distribution of the 59 immune cell-types to that of HCP, patients that were on and off-treatment showed more frequent alterations than those that were never treated (39/59 [66%], 33/59 [56%] and 20/59 [34%], respectively). Thus, immune dysregulation emerged before treatment, peaked while on-therapy, and did not return to normalcy after stopping treatment. One week after the second dose of the vaccine, the percentage of patients that tested positive for IgG against the RBD antigen was significantly inferior to that of HCP (71% vs 96%, P <.001). Seropositivity was more frequent in patients with Hodgkin lymphoma (86%) and B-cell lymphoproliferative disorders (75%), when compared to those with monoclonal gammopathies (61%). Seroconversion rates were greater in patients who never received treatment (82%), dropped considerably in those on-therapy (35%), particularly with anti-CD38 monoclonal antibodies, and bounced back once patients were off-treatment (80%). The frequencies of 43 immune cell-types were significantly associated with immunogenicity. Lasso regression was subsequently performed for feature selection, and six immune cell-types were selected by the algorithm for logistic regression multivariate analysis: neutrophils, classical monocytes, CD4 and CD8 effector memory CD127low T cells, as well as naïve CD21+ and IgM+D+ memory B cells. On logistic regression multivariate analysis, the six immune cell-types and two clinical parameters (no prior therapy and anti-CD38 therapy) predicted immunogenicity independently of patient’s demographics and other clinical characteristics.

Summary/Conclusion: This comprehensive immunological characterization of hematological patients prior to vaccination exposed the extent of immunosuppression induced by anticancer treatment and uncovered that immune dysregulation is present before and persists after therapy. Our results translated into cutoffs for broad use of new immune biomarkers to predict antibody response after vaccine for COVID-19 in hematological patients. Our study urges reflection on whether immune profiling before boosting is warranted to identify optimal timing of vaccine boosters.

P1594: LACTATE DEHYDROGENASE / ALBUMIN TO-UREA RATIO, A NEW PROGNOSTIC MARKER FOR THE PREDICTION OF COVID-19 INFECTION ASSOCIATED CLINICAL COMPLICATIONS.

H. Shokr 1 , M. Marwah 2 , H. Siddiqi 3 , F. Wandroo 3 , S. Marwah 3,*

1 Pharmacy Division, University of Manchester, Manchester; 2 Aston Medical School, Aston University, Birmingham; 3 SWBH NHS trust, West Bromwich, United Kingdom

Background: SARS-COV-19 is an RNA virus that spreads rapidly via droplets, aerosols, and contaminated surfaces. Infection by the new corona virus strain (COVID-19) and its related syndrome has been associated with more than 6 million deaths worldwide. Most infected patients suffer from mild to moderate illness, however some patients progress rapidly to acute respiratory distress syndrome, respiratory failure, sever sepsis, multiorgan failure and death. Early identification of those at risk of progression to critical illness can assist appropriate provision of early intensive interventions and priority access to the intensive care units. Currently high neutrophil to lymphocyte ratio has been suggested as a short-term disease prognostic indicator in critically ill patients, however, to date there are no available indictors to predict critical illness.

Aims: To investigate differences in biomedical markers and routine haematology indices to provide a potential novel ratio as predictor for COVID-19 induced critical illness and death.

Methods: This retrospective study involved 1139 COVID-19 infection survivors as derivation cohort and 349 COVID-19 deaths as a validation cohort. Peripheral blood samples were collected from all the study population and routine laboratory indices, inflammatory markers and organ function tests were analysed on admission.

Results: Compared to COVID-19 infection survivors, patients who didn’t survive showed lower haemoglobin concentrations (p=<0.001), platelets count (p=0.0001) and higher mean corpuscular volume, neutrophil count, neutrophil/lymphocytes ratio (NLR), and lactate dehydrogenase/albumin to urea ratio (LAU) (p=0.001, p=0.0013, p=<0.001, p=0.0126 respectively). They also exhibited higher infection related clinical complications expressed as higher international normalized ratio (INR), D-dimer, urea, total bilirubin levels, alkaline phosphatase (ALK), creatinine, c-reactive protein (CRP), and serum ferritin levels (p<0.05 in all). Lactate dehydrogenase/Albumin ratio was identified as an independent risk factor for end organ damage and clinical complications in COVID-19 positive patients. The LAU ratio was significantly and positively correlated with infection-related clinical complications prognostic parameters including INR (r=0.171), D-dimer (r=0.176), serum urea (r=0.424), total bilirubin (r=0.107), ALK (r=0.115), creatinine (r=0.365), CRP (r=0.268), ferritin (r=0.385) and negatively correlated with serum albumin levels (r=-0.114) (p=<0.05 in all). Additionally, The LAU ratio had an area under receiver operating characteristic (ROC) of 0.67 (95% confidence interval 0.56 to 0.79) compared to 0.60 (95% confidence interval 0.54 to 0.68) using NLR.

Summary/Conclusion: We found that LAU ratio is a new predictive factor for COVID-19 induced clinical complications and death. Patients having a LAU ratio above 54.7 are predicted to die from the infection thus close monitoring and early intensive intervention should be offered to these patients to control infection-related complications.

P1595: IFISTRATEGY: A SPANISH NATIONAL SURVEY ON STRATEGIES FOR MANAGEMENT OF INVASIVE FUNGAL INFECTION AND GUIDELINES APPLICATION IN HIGH RISK ONCO-HEMATOLOGICAL PATIENTS

C. Vallejo 1,* , I. Jarque 2 , J. Fortún 3 , J. Pemán 4 , Á. Muñoz 5 , T. I. Study Group 6

1 Hematology Service, Hospital Clínico Universitario Santiago de Compostela, Santiago de Compostela; 2 Hematology Service, Hospital Universitario y Politécnico La Fe, Valencia; 3 Infectious Disease Service, Hospital Universitario Ramón y Cajal, Madrid; 4 Microbiology Service, Hospital Universitario y Politécnico La Fe, Valencia; 5 Pharmacoeconomics & Outcomes Research Iberia (PORIB); 6 31 Spanish hospitals, Madrid, Spain

Background: The use of antifungals in prophylaxis and the appearance of azole-resistant Aspergillus fumigatus have conditioned an epidemiological change that, together with the interactions of targeted therapies, has modified the management strategies of onco-hematological patients with invasive fungal infections (IFI).

Aims: To know the strategies of clinicians in the management of IFI in onco-hematological patients and the role of guidelines and recommendations in decision making.

Methods: Cross-sectional multicenter survey with hematology or infectious diseases specialists, belonging to Spanish hospitals and experienced in treating IFI. Data collection was carried out through an electronic platform in February 2022. The survey questionnaire consisted of 12 items.

Results: Fifty-five experts from 31 hospitals completed the survey (64% hematologists, 78% of them for adult population; average experience: 21 years). Based on the latest epidemiological studies, 63% of participants indicated that there has been an increase in Aspergillus resistance to azoles. The possible coexistence of mixed infection (resistant/susceptible Aspergillus ) was a concerned to 100% of experts. For 75% of participants these resistances in onco-hematological patients are mainly of clinical origin, due to the routine use of prophylaxis. Only 5.5% stated that there were no azole resistant Aspergillus infections in this population. Regarding breakthrough IFI, 65% indicated an increase in the incidence of Mucorales and that proven IFI were usually resistant to previously administered antifungals. In case of persistent febrile neutropenia (5 days), 59% indicated that they would perform early treatment (even in presence of nonspecific or absence of lung infiltrates in CT-SCAN). In the case of suspected resistance in a patient being treated for aspergillosis, 82% of surveyed professionals would change the antifungal drug family to another broad-spectrum antifungal therapy (with one or two drugs). An 87% indicated that when the percentage of resistance of A. fumigatus against an azole is ≥10% (as per the IDSA and ECCMID guidelines), a change should be made in the choice of early treatment. Respect a possible interaction of targeted therapy drugs (midostaurin or venetoclax) with antifungal agents, 40% would use broad-spectrum azoles and 38% echinocandins as prophylaxis. In the case of using echinocandins and development of suspected breakthrough IFI, 67% would administer liposomal amphotericin B. For antifungals failing to reach levels during the first days and suspected invasive aspergillosis, the most appropriate strategy for 62% of experts would be to associate it to an antifungal from another family.

Summary/Conclusion: The present study shows that a high percentage of clinicians implicated in the management of onco-hematological patients at high risk of IFI follows the recommendations of the national and international and guidelines. Most of the experts agree on: a) Early treatment is the best option in case of persistent febrile neutropenia (even in the presence of nonspecific or absence of lung infiltrate in CT-SCAN). b) If resistance of Aspergillus to azoles is suspected, switching to another broad-spectrum antifungal family would be the best option. c) Broad-spectrum azoles and echinocandin can be used as prophylaxis in patients receiving new targeted therapies. Liposomal amphotericin B was the preferred option after prophylaxis with echinocandins.

P1596: PREDIAGNOSTIC ANTIMICROBIAL USE IN CLL, MM, FL AND DLBCL

E. M. Packness 1,* , M. A. Andersen 1,2 , K. Rostgaard 3,4 , H. Hjalgrim 3,4,5 , C. U. Niemann 1 , C. Brieghel 1,6

1 Department of Hematology, Copenhagen University Hospital, Rigshospitalet; 2 Department of Clinical Pharmacology, Copenhagen University Hospital, Bispebjerg; 3 Danish Cancer Society Research Center, Danish Cancer Society; 4 Department of Epidemiology Research, Statens Serum Institut; 5 Department of Clinical Medicine, Copenhagen Univeristy, Copenhagen; 6 Department of Hematology, Zealand University Hospital, Roskilde, Denmark

Background: Chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and multiple myeloma (MM) are B-cell lineage malignancies all sharing an immune dysfunction leading to an increased risk of infection. CLL, FL and MM are preceded by indolent precursor states; more specifically monoclonal B-cell lymphocytosis (MBL), in situ follicular neoplasia (ISFN), and monoclonal gammopathy of uncertain significance (MGUS), respectively. MBL, ISFN and MGUS may be detected decades prior to overt malignancy. Although CLL and FL may transform into DLBCL, evidence for a premalignant DLBCL precursor is scarce. We have previously demonstrated an increased use of antibiotics up to 20 years prior to CLL diagnosis (Andersen et al. Leukemia. 2020). Whether patients with DLBCL, FL or MM share the same trait as patients with CLL remains unknown.

Aims: To assess use of antimicrobials for patients with FL, DLBCL and MM prior to diagnosis

Methods: We included all patients diagnosed with CLL since 2008, small lymphocytic lymphoma (SLL), DLBCL, FL and symptomatic MM since 2005 through Danish national patient registries (DCLLR, LYFO and DaMyDa; https://www.rkkp.dk/ ). We analyzed patients with CLL and SLL jointly, as they are recognized as the same disease entity and treated similarly. Likewise, patients with FL grade 3b are clinically similar to DLBCL, thus patients with FL treated with R-CHOP-like regimens within 3 months of diagnosis were considered grade 3b and analyzed together with DLBCL.

Data on antimicrobial (AM) prescriptions (Rx) were retrieved from the Danish national database for reimbursed prescriptions ( DNDRP ) using WHO ATC codes. To characterize the use of prediagnostic AM use, we included AM Rx from January 2004 until date of diagnosis. Patients with multiple malignancies were classified according to the first occurring diagnosis. Statistical analyses were performed using R software.

Results: We followed 21,822 patients for a mean of 9.8 person-years prior to diagnosis (SD+/- 4.8), from which a total of 138,805 AM Rx were identified for 18,840 (86.3%) distinct patients. Within the cohort, 6377 (29.2%) were diagnosed with CLL accounting for 42,107 (30.33%) AM Rx, 5803 (26.6%) were diagnosed with MM accounting for 38,417 (27.7%) AM Rx, 6735 (30.9%) were diagnosed with DLBCL accounting for 41,623 (30.0%) AM Rx, and 2907 (13.3%) were diagnosed with FL accounting for 16,658 (12.0%) AM Rx. Thus, 2982 (13.7%) patients had no Rx prior to diagnosis of whom 989 (33.2%) had DLBCL, 775 (26.0%) had CLL, 722 (24.2%) had MM, and 496 (16.6%) had FL.

Stratified by disease, patients later diagnosed with CLL and MM accounted for more AM Rx than patients later diagnosed with DLBCL and FL (45.0% vs 41.6% vs 37.8% vs 35.4% with >3 AM Rx, respectively; Table 1). In particular, more penicillin and macrolide were prescribed prior to diagnosis of CLL and MM compared to patients later diagnosed with DLBCL and FL.

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Summary/Conclusion: These preliminary results indicate a higher rate of infections, as assessed by number of AM Rx, in individuals later developing CLL and MM compared to individuals later developing DLBCL and FL. Thus, MM and CLL seems to be prone to immune dysfunction also prior to diagnosis. Whether immune dysfunction plays a role in development of the disease or is due to the premalignant MBL and MGUS warrants further investigations.

P1597: THE DYNAMIC INTERACTION BETWEEN INFLAMMATION AND IRON SENSORS IN MALARIA-INDUCED ANEMIA

A. Pêgo 1,* , I. Lima 1 , G. Martins 1,2 , M. Vujic 3 , A. Nai 4 , L. Silvestri 5 , R. Gozzelino 1,2

1 Inflammation and neurodegeneration, CEDOC-NMS/UNL, Lisboa, Portugal; 2 Universidade Técnica do Atlântico, São Vicente, Cape Verde; 3 Hormonal regulation of iron homeostasis, Institute of Comparative Molecular Endocrinology, Ulm, Germany; 4 Regulation of iron metabolism; 5 San Raffaele Scientific Institute, Milan, Italy

Background: Severe malarial anemia (SMA) is a complication developed during Plasmodium infection. It is often associated with increased rates of malaria-related morbidity and mortality, especially in children and pregnant women. Hence, it is fundamental to investigate new parameters to prevent this lethality.

Aims: The occurrence of ineffective erythropoiesis, caused by a dysregulated erythropoietin (EPO)-erythropoietin receptor (EPOR) signaling pathway, is one of the main features of SMA. Therefore, to gain new insights into the pathogenesis of SMA, our study aims to better understand the dynamic interaction between erythropoiesis and inflammation in mice exposed to single or multiple infections, mimicking what occurs in endemic regions.

Methods: Erythroid cell differentiation and immune cells activation were evaluated by flow cytometry. Changes in iron metabolism were assessed by the differences in expression of genes regulating iron homeostasis by qRT-PCR.

Results: Flow cytometry analyses suggested that additional exposure to the parasite enhances erythropoiesis. Comparing infected with reinfected mice, we observed a faster erythropoietic response, induced by the bone marrow. It was also found that reinfected mice presented an earlier and robust immune response activation, as developing innate immune memory. Due to the fundamental role of iron in erythropoiesis and immune response activation, the expression of iron sensors, like HFE, was analyzed, detecting a strict correlation between disease severity and multiple infections exposure. The action of HFE in the pathogenesis of SMA and its role on the immune response activation, elicited to combat malaria, was also investigated. Preliminary data shows that when HFE knockout mice were infected with the parasite, their anemic profile improves.

Summary/Conclusion: These reveals HFE as a potential target for drug therapy during hemolytic disorders, such as malaria infection.

P1598: SARS-COV2 INFECTION IN FULLY VACCINATED PATIENTS WITH LYMPHOID MALIGNANCIES DURING THE “THIRD WAVE”: A SINGLE-CENTER EXPERIENCE OF 57 PATIENTS.

V. Strafella 1,* , T. Perrone 2 , P. Masciopinto 1 , A. Giordano 2 , F. Gaudio 2 , M. C. Longo 2 , O. Battisti 1 , E. Arcuti 1 , L. Strippoli 1 , G. Brindicci 3 , A. Saracino 3 , P. Stefanizzi 4 , S. Tafuri 4 , F. Albano 1 , P. Musto 1

1 Department of Emergency and Organ Transplantation, University School of Medicine, Bari; 2 Unit of Hematology and Stem Cell Transplantation; 3 Clinic of Infectious diseases, AOUC Policlinico; 4 Department of Biomedical Sciences and Human Oncology, University School of Medicine, Bari, Bari, Italy

Background: COVID-19-related mortality rates are increased in hematological patients and those with lymphoid malignancies look particularly vulnerable to SARS-CoV-2 infection, due to the characteristics of their disease and the anti-neoplastic treatments used. Vaccines against SARS-CoV-2 have been rapidly developed. However, patients treated with anti-CD20 monoclonal antibodies have shown lower seroconversion rates after receiving mRNA SARS-CoV-2 vaccines. Several antiviral agents, such as molnupiravir and remdesivir, and some monoclonal antibodies have been also used for the management of COVID-19 in order to reduce the risk of progression to severe disease.

Aims: The aim of this study was to evaluate the outcome of SARS-Cov2 infection in a population of patients affected by chronic lymphocytic leukemia (CLL), Hodgkin lymphoma (HL), B- and T-cell non-Hodgkin lymphomas (NHLs) during the so-called “third wave” of SARS-CoV-2 pandemic.

Methods: We performed a retrospective analysis of 57 patients affected by CLL (n. 12), HL (n. 8), B (n. 34) and T-cell (n. 3) NHLs who experienced SARS-CoV2 infection between December, 1, 2021, and February, 26, 2022. All patients had received a complete cycle of anti-SARS-Cov2 vaccine before infection. They were diagnosed by RT-PCR or by antigen rapid test on nasopharyngeal swabs. We collected data about sex, age, histological features, anti-neoplastic regimens, symptoms, hospitalization, mortality, use of antiviral drugs or monoclonal antibodies for the treatment of COVID-19, time to SARS-CoV-2 negativity.

Results: The median age of the whole group was 58 (18-94) years. Thirty-two patients (56%) were male, while 26 (44%) were female. As far as histological features are concerned, follicular lymphoma was the most frequent one (35%). Within the last 12 months before SARS-CoV-2 infection, 40 patients (70%) had received treatment for their hematological disease, 17 (30%) were on anti-CD20 maintenance, 12 (21%) on treatment with immuno-chemotherapy (ICT), 6 (11%) on ibrutinib, 3 (5%) on checkpoint inhibitors and 3 (5%) were on chemotherapy only. All patients had received two doses of anti-SARS-CoV-2 vaccines, and 77% of them a further, booster dose. The most common symptoms at presentation were fever, cough, muscle pain, headache, fatigue. Overall, 15 (26%) patients were hospitalized, and, among them, 4 (7%) were admitted to intensive care units (ICUs). Fifteen (26%) patients received an antiviral drug, while 4 (7%) hospitalized patients received monoclonal antibodies for the treatment COVID-19. Average time length to reach a negative test for SARS-CoV-2 was 17 days. Sixteen (28%) patients remained positive after 5-to-61 days and 6 (11%) patients died. At univariate analysis, CLL and Diffuse Large B-Cell Lymphoma (DLBCL) (p 0.026), as well as treatment with ICT or BTK inhibitors (p 0.002), were significantly associated to a worse overall survival (OS). Notably, maintenance therapy with anti-CD20 monoclonal antibodies as single agents had no impact on OS. Moreover, the cumulative incidence of nasopharyngeal swabs turned negative was significantly lower in patients treated with ICT (p 0.001). Booster dose and antiviral treatments did non influence neither OS, nor time to a negative viral test.

Summary/Conclusion: In our study histological subtype and type of treatment, but not antiviral therapies, had prognostic impact on the outcome of SARS COV 2 infection in patients with lymphoproliferative disorders during the current phase of pandemic. These findings warrant to be further investigated in larger series.

P1599: THERAPY AND HOSPITAL MORTALITY PREDICTORS IN PATIENTS WITH LYMPHOPROLIFERATIVE DISORDERS AND CONCOMITANT COVID-19 INFECTION

Y. Polyakov 1,* , K. Kaluzhskaya 1 , E. Baryakh 1,2,3 , E. Gemdzhian 4 , E. Misyurina 1 , E. Zhelnova 1 , K. Yatskov 1 , O. Kochneva 1 , S. Andreev 1 , T. Chudnova 1 , D. Ivanova 1 , T. Tolstykh 1

1 Moscow City Clinical Hospital 52; 2 Russian Medical Academy of Continuous Medical Education; 3 Federal State Budgetary Educational Institution of Higher Education “Russian National Research Medical University named after N.I. Pirogov” of Ministry of Health of RF; 4 National Research Center for Hematology, Moscow, Russia

Background: COVID-19 infection increases the risk of hospital mortality in patients with lymphoproliferative disorders (LPD).

Aims: The aim of the study was to identify the predictors of hospital mortality in adult patients with LPD and concomitant COVID-19.

Methods: 335 patients (53% of whom were male) aged 18 to 92 years (median age = 57) with LPD and COVID-19 were followed up by the hematology service of Moscow City Clinical Hospital No.52 (Moscow) from April 21, 2020 to December 31, 2021. The overall survival curve was constructed and multivariate analysis using a Cox regression model was performed to determine the predictors of hospital mortality.

Results: This retrospective study included patients with the following disorders: diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma, marginal zone lymphoma, hairy cell leukemia, Hodgkin’s lymphoma, Burkitt lymphoma, and T-cell lymphoma. The Charlson Comorbidity Index score was 5 points, and median time from the first symptoms to hospital admission was 7 days. The disease status included remission (n=143; 43%), relapse/progression (n=76; 23%), stable disease (n=96; 29%), newly diagnosed hematologic malignancy (n=16; 5%). The majority of patients had moderate to severe lung tissue involvement. Grade 4 neutropenia was reported in 150 patients who had a high level of C-reactive protein (median=115 mg/L), a significantly elevated D-dimer (median = 912 ng/mL) and fibrinogen (median=6.5 g/L) levels. Mean ICU length of stay was 9 days, and 21% of patients required mechanical ventilation. 182 patients were receiving combination chemotherapy for LPD within a month prior to the onset of COVID-19. 209 patients were treated with anti-CD20 monoclonal antibodies within a year prior to the onset of COVID-19.

Hospital therapy included IL-6 receptor antagonists, antimicrobial therapy (aminopenicillin/beta-lactamase inhibitor + fluoroquinolone as the first line therapy and ureidopenicillin or a fourth-generation cephalosporin/beta-lactamase inhibitor + aminoglycoside as the second line therapy), and antifungal therapy (38% of patients).

Hospital mortality rate was 21% (70/335) (Fig. 1). According to the results of the multivariate (cox regression) analysis, significant predictors of hospital mortality included the age (>65 years), 2.0 (1.1-5.2), р=0.05; hematologic malignancy status (relapse or progression/remission), 2.4 (1.3-4.3), р=0.008; CT severity score (4/0), HR=2.9 (95% CI: 1.2-7.1), р=0.004 and grade 4 neutropenia (yes/no), 1.5 (0.9-2.7), р=0.09 (Fig. 2).

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Summary/Conclusion: Mortality rate in patients with LPD was 21%. The following significant predictors of hospital mortality have been identified: age >65 years, relapse/disease progression, CT severity score index of 4, grade 4 neutropenia.

P1600: ANTIGENS OF THE DUFFY BLOOD GROUP SYSTEM AND RISK OF COVID-19 INFECTION

E. Poponina 1,* , E. Butina 2 , A. Yovdiy 2 , N. Minaeva 1

1 Federal State Budget Institution of Science “Kirov research Institute of Hematology and Blood Transfusion of the Federal Medical and Biological Agency of Russia”, Kirov, Russia; 2 laboratory of immunohematology, Federal State Budget Institution of Science “Kirov research Institute of Hematology and Blood Transfusion of the Federal Medical and Biological Agency of Russia”, Kirov, Russia

Background: The Duffy blood group antigens are transmembrane glycoproteins, represented by two specificities (Fy a and Fy b ), also known as atypical chemokine receptor 1 (ACKR1). These structures are located not only on the red blood cell membrane, but also on the vascular endothelium, alveoli, epithelium of the renal tubules, and cerebellar neurons. The biological role of the Duffy system is to bind inflammatory chemokines and inhibit excessive leukocyte activation. The antigens also serve as receptors for viruses and malaria plasmodia to enter the cell. Therefore, the study of the participation of the Duffy blood group system in the development of COVID-19 infection is of particular interest.

Aims: The aim of study was to compare the distribution of Duffy blood group antigens in individuals with and without COVID-19.

Methods: The distribution of antigens of the Duffy blood group system was studied in 329 donors of blood components using equipment and reagents from BioRad (USA), OrthoClinicalDiagnostics (USA). The study was held from February 2020 to February 2021 (before the start of vaccination). 71 people had COVID-19, 258 didn’t. Age ranged from 20 to 63 years, median 38 years. Reliability of the diagnosis of COVID-19 is confirmed by a positive PCR test result and/or the presence of class G antibodies to the SARS-Cov2 virus. Statistical processing was performed using Fisher’s exact test.

Results: The Fy a antigen was detected in 75.2% of those who did not become ill and in 60.6% of those who had COVID-19 (p<0.05). The frequency of Fy b in donors with and without a new coronavirus infection was comparable, 78.9% and 77.5% respectively. The disease was less common in the individuals with the Fy a phenotype. The COVID-19 incidence in them was 18.1%, while without the Fy a antigen it reached 30.4% (p=0.02). Carriage of the Fy b antigen had no effect on the incidence of the disease, 21.9% in individuals with the presence of Fy b and 20.5% without it.

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Summary/Conclusion: The Fy a antigen showed a protective effect against COVID-19, its absence increased the risk of the disease by 1.5 times. The obtained results indicate the impact of blood group systems on the risk of COVID-19 infection, but the issue requires further study.

P1601: THE ASSESSMENT OF PREDICTIVE ROLE OF PROTEIN C AND S FOR CRITICAL FORM OF COVID-19

V. Popov 1,* , M. Andreescu 2 , O. Patrinoiu 1 , M. Popescu 1 , M. Omer 1 , F. Mihai 1 , G. Ofiteru 1 , C. Despan 1 , O. Constantin 3 , C. Pirvu 3 , A. Grigorie 3 , A. Rus 4 , M. D. Badoiu Niculae 3 , A. Badea 3 , L. Iliescu 4

1 Hematology, Colentina Hospital Bucharest; 2 HEMATOLOGY, COLENTINA CLINICAL HOSPITAL; 3 Intensive Care Unit, Colentina Hospital Bucharest; 4 Intensive Care Unit, Colentina Clinical Hospital, Bucharest, Romania

Background: COVID-19 is characterized by an excessive immune response in the host “cytokine storm”, followed by extensive tissue damage, dysfunctional coagulation and thrombotic complication. The presence of thrombosis worsens the evolution of COVID-19 caused by refractory hypoxemia, respiratory failure, disseminated intravascular coagulation or death.

Aims: The main objective of our study was to evaluate the presence of any correlation between anticoagulants (protein S, protein C, antithrombin III) and clinical/laboratory features of critical forms of COVID 19 admitted in intensive care unit (ICU).

Methods: This observational study (retrospective and prospective) included 268 COVID-19 patients hospitalised Colentina Clinical Hospital Bucharest 2020-2021 and confirmed by real-time reverse transcriptase polymerase chain reaction (PCR) test. The study group was divided into two groups depending on the type of medical care required - medical care (non-ICU group) and intensive care (ICU group).

Results: The level of protein C and S is lower in COVID-19 patients with thrombosis compared with patients without thrombotic complication (median value: protein S: 43.5 (8-102) vs. 56 (18-144), p=0.05; protein C: 71 (15-169) vs. 95 (16-200), p=0.02). Critical forms of COVID 19 is associated with low level of protein S (ICU: 48 (8-122) vs. non-ICU: 63 (18-144), p=0.001); protein C (ICU: 82 (15-170) vs non-ICU: 96.5 (55-200), p=0.02). There are significant correlation between low level of protein S and duration of hospitalization in ICU department, rho=-0.367 (-0.541- -0.163) p=0.0007. In survival analyses only protein C level was predictive for unfavourable evolution, Wald 8.55, p=0.003. This parameter associated with high level of feritine and IL-6, are very important in evaluation of COVID-19 patients with unfavourable evolution. Prolonged positivity is associated with low level of protein S, rho -0.517 (-0.75- -0.171), p=0.006.

Summary/Conclusion: Evaluation of level of protein S and C in COVID-19 patient is very important at the admission moment in ICU. Low level of protein S is associated with prolonged positivity, unfavourable evolution and hiperinflamation. It also associated with thrombotic complication and cytokine storm (IL-6), all these modification contributed in high rate of ICU admission

P1602: THE ASSESSMENT OF LYMPHOPENIA AS RISK FACTOR FOR UNFAVORABLE EVOLUTION OF PATIENTS WITH SEVERE FORM OF COVID 19

V. Popov 1,* , L. Iliescu 2 , A. Grigorie 3 , C. Pirvu 3 , O. Constantin 3 , A. Badea 3 , M. D. Badoiu Niculae 3 , A. Rus 3 , A. Calen 3 , H. Bumbea 4,5 , B. Mastalier 6,7 , M. Andreescu 1,8

1 Hematology, Colentina Hospital Bucharest; 2 Intensive Care Unit, Colentina Clinical Hospital; 3 Intensive Care Unit, Colentina Hospital Bucharest; 4 Hematology, Emergency University Hospital; 5 Hematology, UMF Carol Davila; 6 Surgery, Colentina Hospital Bucharest; 7 Surgery, UMF Carol Davila; 8 Hematology, Titu Maiorescu University, Bucharest, Romania

Background: Coronavirus 2019 (COVID-19), the new infectious respiratory disease caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection is frequently associated with lymphopenia. Many studies involved lymphopenia as prognostic factor for unfavourable evolution of COVID-19 patients.

Aims: The main objective is to evaluate the impact of lymphopenia in survival analyses of patients with severe and critical form of COVID-19. The secondary objectives is to evaluate another predictor variables that influenced the survival rate of COVID-19 patients.

Methods: The retrospective observational study enrolled 261 COVID-19 patients with critical forms of COVID-19, who were admitted in Intensive Care Unit (ICU) between March2020-Sptember 2021. All patients have inform consent signed and RT-PCR test positive for SARS-Cov2. We consider lymphopenia, lymphocytes count bellow 1.0 x 1000/uL, the patient group was splitted by this factor in 2 groups. The analyses was done using clinical and lab data in order to identify parameter that could influence the evolution of COVID-19 in ICU-patients.

Results: The COVID-19 patients group with lymphopenia (group1) have median age greater than COVID-19 patients without limphopenia (group2): 65 (min 26, max 89) vs. 54.5 (min 17, max 94), p=0.00002. Lymphopenia analysed as predictive factor of COVID-19 patients admitted in ICU is not important for survival analyses when was analysed alone. Patients with age < 65 years old and lymphopenia, with obesity (IMC>30) and oncological diseases have severe evolution, these patients developed severe pneumonia (more than 30-40% of pulmonary field evaluated CT scan); Wald 9.34, p=0.002; 0 p<0.0001. The inflammation assessment using feritina, CRP and Fibrinogen results was more severe in patient with lymphopenia: feritina- group 1-1031 (576,25-1749.25) vs group 2- 344.5 (134.25-848.75) uFEU, p< 0.0001; CRP group 1- 86.02 (40.04-159.2) mg/dl vs. group 2-12.18 (2.17-57.06) mg/dl, p<0.0001; fibrinogen group 1-554 (408-712) mg/dl vs group 2-396 (335-541) mg/dl, p<0.0001. These results could be explained by higher median time of viral clearance that is higher in patients with lymphopenia: group 1- 17 days (13.25-29) vs. group 2- 16 (9-25), p=0.03. The thrombotic complications are more frequent in severe form of COVID-19 patients who associated lymphopenia, group 1-28.22% vs group 2- 13.12%, Chi-squared 10.03, p=0.001, thrombosis was an important covariate in Cox proportional hazard regression, Wald 6.72, p=0.01 . In COVID 19 patients group 1 D Dimers level is higher than second group, 1.71 (0.80-4.36) vs. 0.64 (0.36-1.68), p<0.0001, the scoring system for disseminated intravascular coagulation (DIC) being higher in the first group; interquartile range 2-3 in group 1 vs 0-2 in group 2, p<0.0001.

Summary/Conclusion: The inflammation and thrombotic complications are more frequent in COVID-19 patients with lymphopenia. Persistent viral clearance in patients with lymphopenia is likely to contribute to the clinical severity and adverse outcome of COVID-19. The results obtained require evaluation in a larger group of patients

P1603: CLINICAL OBSERVATION OF PATHOGEN MICROBIAL NEXT-GENERATION SEQUENCING ASSISTED IN INFECTION DIAGNOSIS AND TREATMENT OF PATIENTS WITH HEMATOLOGICAL TUMORS

X. Xiang 1 , H. Liu 1 , Y. Zeng 1 , S. Zhou 1 , J. Li 1 , J. Rao 1 , L. Gao 1 , X. Zhang 1,*

1 Medical center of hematology, Xinqiao hospital, Army medical university, Chongqing, China

Background: Infection is still the most important complication in patients with blood tumors and one of the most common causes of death. Timely finding of the pathogenic microorganisms is the key to control the infection.

Aims: Rapid identification of pathogenic microorganisms by pathogen microbial next-generation sequencing (NGS), analysis of their accuracy and effectiveness, as well as the impact on disease treatment.

Methods: Retrospective analysis of hematological tumor patients hospitalized from September 2019 to February 2021 in the Hematology Department of the Second Affiliated Hospital of the Army Military Medical University,70 patients developed recurrent fever after chemotherapy for a week, with empirical anti-infection symptoms uncontrolled. The pathogen basis could not be found through blood culture and humoral culture, and the pathogenic microorganisms were submitted for NGS examination to analyze the pathogenic microorganisms. Of these, 40 had acute leukemia, 8 lymphoma, 8 myelodysplastic syndrome, 5 multiple myeloma, and 9 patients with other diseases who received chemotherapy. 13 patients had used immunosuppressive agents such as glucocorticoids. The infection site was mainly respiratory tract and blood flow, and 2 cases had a cutaneous soft-tissue infection. The NGS test was mainly blood, 9 were bronchoalveolar lavage fluid, 3 with puncture tissue fluid, 1 sputum, 1 cerebrospinal fluid and 1 paraffin tissue section.

Results: In this study, 63 (90%) patients confirmed the pathogenic microorganisms based on NGS test. 18 bacterial infections, the most was Klebsiella pneumoniae (n=5). 19 fungal infections, at most Aspergillus (n=9), and 4 patients were infected with Candida; 24 viral infections were detected, followed at most by herpesvirus (n=19), followed by CMV infection (n=8). There were 2 cases of pulmonary tuberculosis. 11 of these patients had a carbapenem exposure.The mean time from initial infection symptoms to NGS delivery was 8.2 days.The median time for temperature control for the adjusted anti-infection regimen was 5.2 days. 61 patients (87.1%) were controlled by treatment, 2 patients died, and 7 patients delayed tumor treatment due to severe infection.

Summary/Conclusion: Pathogenic microorganism NGS has high sensitivity to the detection of pathogenic microorganisms, which can be used as an auxiliary means of infection diagnosis and treatment, and has a positive role in timely control of infection.

P1604: DISTRIBUTION AND ANTIBIOTIC RESISTANCE PROFILE OF PATHOGENIC MICROORGANISMS CAUSING NOSOCOMIAL INFECTION IN PATIENTS UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION

J. Tao 1 , F. Wu 1 , L. Gao 1 , A. Sun 1 , Y. Feng 1,* , X. Zhang 1

1 Medical center of hematology, Xinqiao hospital Army Medical University, Chongqing, China

Background: In recent years, with the wide use of antibiotics and extensive invasive procedures, the drug resistance of bacteria causing nosocomial infection is becoming more and more serious. Due to the characteristics of hematopoietic stem cell transplantation patients and the use of large doses of chemotherapeutic drugs, a variety of immunosuppressants and a large number of broad-spectrum antibiotics, the immune function of patients is very low. As a result, the infection rate of hematopoietic stem cell transplantation patients in hospital is rising and new drug-resistant strains are emerging, which poses a great challenge to the control of nosocomial infection.

Aims: To approach the distribution and antibiotic resistance profile of pathogenic microorganisms causing bloodstream infection in patients undergoing hematopoietic stem cell transplantation, so as to provide evidences for rational clinical use of antibiotics.

Methods: The clinical data, including the types of pathogenic microorganisms and their resistance profile to commonly used antibiotics, of patients developing bloodstream infection during hematopoietic stem cell transplantation from January 2020 to December 2021 were analyzed retrospectively.

Results: A total of 68 pathogenic strains were isolated from 405 patients undergoing hematopoietic stem cell transplantation, including 24 (35.3%) Gram-positive strains, primarily Staphylococcus and Enterococcus ; 42 (61.8%) Gram-negative strains, primarily Escherichia coli and Klebsiella pneumoniae ; and 2 (2.9%) fungus strains which were both Trichosporon asahii . Escherichia coli had the highest resistance rate (100%) to piperacillin, cotrimoxazole, and ampicillin, and the highest susceptibility rate (100%) to tigecycline, amikacin, imipenem, and meropenem. Klebsiella pneumoniae had the highest resistance rate (100%) to ampicillin, and the highest susceptibility rate (0) to tigecycline. The primary Gram-positive bacteria were 100% susceptible to teicoplanin, quinupristin/dafopentine, rifampicin, vancomycin, linezolid, nitrofurantoin, and amikacin; vancomycin-resistant enterococci (VRE) were not identified. Trichosporon asahii was 100% susceptible to amphotericin B, nystatin, caspofungin, and voriconazole.

Summary/Conclusion: There are various pathogenic microorganisms that could cause bloodstream infection in patients undergoing hematopoietic stem cell transplantation, with high multi-drug resistance rate. The proportion of Gram-negative bacteria is increasing. During hematopoietic stem cell transplantation, it is expected to strengthen monitoring of the pathogenic microorganisms, so as to provide effective prevention to control infection with the pathogenic microorganisms, thus improving the patients’ survival rate.

P1605: OUTCOME OF SARS-COV-2 INFECTION IN HAEMATOLOGICAL CANCER PATIENTS (PTS) IN TWO DIFFERENT PANDEMIC PHASES AT A SINGLE CENTRE

R. Rodrigues 1,* , A. C. Freitas 1 , S. Esteves 2 , M. Gomes da Silva 1

1 Hematology; 2 Clinical Investigation Unit, Portuguese Institute of Oncology, Lisbon, Portugal

Background: The outcome of haematological cancer pts with SARS-CoV-2 infection has been shown to be dismal since the beginning of the COVID-19 pandemic. Risk factors for serious infection may include recent antineoplastic treatment, type of treatment and active disease, besides age and comorbidities. The onset of vaccination reduced the risk of severe COVID-19 in the general population, but it is unclear whether the same is true in immunocompromised pts. The impact of different viral variants on outcomes is also a matter of debate.

Aims: To evaluate and compare the outcomes of COVID-19 measured by the severity of infection and short-term mortality, in pts with haematological cancer during two different pandemic phases.

Methods: Retrospective data was obtained from clinical charts of pts followed at a tertiary cancer centre and diagnosed with SARS-CoV-2 infection by RT-PCR in December 2020/January 2021 (series A) and December 2021/January 2022 (series B). Clinical characteristics collected were age, gender, tumour type, phase of neoplastic disease, comorbidities, concurrent antineoplastic treatment (grouped as shown on Table 1), need for suspension of active treatment, severity of infection and death due to COVID-19 within the first 30 days of infection. We used multivariable logistic regression analysis (MVA) to compare COVID-19 severity and 30-day mortality between the two periods, controlling for concurrent active antineoplastic treatment and number of comorbidities, which differed between the groups.

Results: We identified 127 pts infected with SARS-CoV-2 in December 2020/January 2021, and 136 in December 2021/January 2022. Patients’ characteristics are shown in Table 1. The two groups were comparable except for a higher incidence of multiple comorbidities in series A (33% vs 25% in series B) and an increased proportion of cases under active treatment in series B (61% vs 39% in series A). The majority of pts had at least one comorbidity with known impact for severe COVID-19, most frequently chronic cardiopathy. Low (22%) and high grade (26%) lymphoma were the predominant diagnoses in series A and B respectively. The most common antineoplastic treatments were immunotherapy/immunochemotherapy. 88% pts in series B were vaccinated against SARS-CoV-2, 42% with a 3-dose scheme. As a consequence of viral infection 48% pts in series B required suspension of active antineoplastic treatment , as opposed to 43% in series A. COVID-19 was classified as asymptomatic, mild, severe (requiring hospitalization) or critic (requiring ICU). Severe/critic infection was most frequent in series A (32% vs 15%). We noted a statistically significant reduction of 74% in the odds for severe/critic infection in series B compared to A (OR 0.26; CI95% 0.13-0.51; p<0.001), when controlling for number of comorbidities and concurrent antineoplastic treatment. COVID-19-related 30-day mortality was reduced from 9% to 4% between series A and B. However, this difference was not significant on MVA (OR 0.36; CI95% 0.10-1.09; p=0.084).

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Summary/Conclusion: In this single-centre analysis of a heterogeneous haematologic cancer population infected in two different pandemic phases, the severity of COVID-19 was significantly reduced in the most recent period. Whether this reduction is due to vaccination, a dominant less harmful viral variant or differences in supportive care is unknown. Also, the population analysed may not be representative of all haematological cancer pts, being subject to referral bias. Larger, homogeneous series with longer follow-up are needed to better understand the factors that lead to such different outcomes.

P1606: CENTRAL VEIN CATHETERISATION IN ONCOHEMATOLOGICAL PATIENTS: COMPLICATION, ASSOCIATED WITH PROCEDURA AND CARE

N. Romanenko 1,*

1 Clinical of Hematology, Russian Research Institute of Hematology and Transfusiology, Saint-Petersburg, Russia

Background: The central vascular access needs to provide a large volume of infusion therapy, including transfusions of blood, long term infusion of immunotherapy and chemotherapy in oncohematological patients (OHP). Nevertheless, the procedure and care of central vein catheter (CVC) may lead to a number of serious complications

Aims: To access the frequency of various complications associated with CVC in OHP

Methods: There were studied the frequency of complications associated with procedure and care of CVC in OHP (n=3025) for the period of above 18 years (2003-2021 years). In the most cases CVC were performed by subclavian access: in the right subclavian vain (v.) were performed 2495 (82.5%) procedures, in the left subclavian v. – 521 (17.2%), and so in in the right internal jugular v. 9 cases (0.3%). After CVC procedure was X-ray control. The bacteriological and fungal analyses and so identification of microbial pathogen were done in every cases if it was suspected an infectious

Results: The immediate hemorrhagic complications after procedure of CVC were found the most frequent. These were: 1) hematoma – in 121 cases (4.0%), 2) puncture of an artery – in 83 (2.7%), and one case of catheterization the right subclavian arteria, 3) bleeding from the catheter passage area – in 82 (2.7%), 4) clinically significant lymphorrhea – in 42 cases (1.4%). Delayed complication in generally connected with care or related to the course of the disease were little rear. So, the thrombotic complications included thrombophlebitis, infiltrate with thrombosis of subclavian vein and they were counted 79 cases (2.6%). A number of thrombotic complications were associated with bacterial infections. Besides we observed rarely such complications as pain, numbness, paresthesia of the upper limb in 50 patients (1.7%). As well as weakness, allergic Quincke’s edema, fainting or collapse associated with anesthesia were observed in 35 ones (1.2%). Among the dangerous clinically significant complications that required additional measures were pneumothorax (n=4; 0.13%) and pneumothorax with hemoptysis (n=2; 0.07%).

The complications associated with bloodstream infection in the form of bacteremia or fungemia, which were observed by bacteriological culture, were detected in 74 (2.4%) patients. Positive bacterial cultures from venous blood were detected in 92 cases, and 12 blood probes of them had mixed infection (2 or 3 bacterial pathogens). Analysis of the different microorganisms indicated the prevalence of Gram-positive pathogens – 63.1% (n=58): the most part was Coagulase-negative Staphylococcus – 51.1% (n=47), S. aureus – 8.7% (n=8), Streptococcus viridance 1.1% (n=1), Enterococcus spp. – 1.1% (n=1) and Micrococcus spp. – 1.1% (n=1). Gram-negative microorganisms were detected in the whole group 29.4% (n=27) of cases: Escherichia coli – 15.2% (n=14) patients, Enterobacter spp. – 8.6% (n=8), Acinetobacter spp. – 1.1% (n=1), Pseudomonas aeruginosa – 2.2% (n=2), Neisseria spp . – 2.2% (n=2). Causative agents of fungal infections were found for 7.6% of patients (n=7). They included fungi of the genus Candida – 6.5% ( C. albicans – 5.4% (n=5), C. crusei – 1.1% (n=1)) and Rhodotorula spp. – 1.1% (n=1).

Summary/Conclusion: In was found the most of CVC’s complications were associated with hemorrhagic as hematoma (4.0%), puncture of an artery (2.7%), and bleeding from the catheter passage area (2.7%) and they were connected with technic of procedure and hemostasis disorders. Blood stream infections associated with CVC were caused by gram-positive microbes in general and dependent on care and neutropenia

P1607: EPICOVIDEHA KIDS: A READY TO USE PLATFORM FOR EPIDEMIOLOGICAL STUDIES IN PEDIATRIC HEMATOLOGICAL PATIENTS WITH COVID-19

J. Salmanton-García 1,* , A. Busca 2 , O. A. Cornely 1 , M. Hoenigl 3,4 , N. Klimko 5 , P. Koehler 1 , F. Marchesi 6 , A. Pagliuca 7 , F. Passamonti 8 , T. E. Zaoutis 9 , L. Pagano 10 , Z. D. Pana 11

1 Department I of Internal Medicine, Excellence Center for Medical Mycology (ECMM), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany; 2 Stem Cell Transplant Center, AOU Citta’ della Salute e della Scienza, Turin, Italy; 3 Division of Infectious Diseases and Global Public Health, Department of Medicine, University of California San Diego, San Diego, CA, United States of America; 4 Division of Infectious Diseases, Department of Internal Medicine, Medical University of Graz, Graz, Austria; 5 North-Western State Medical University named after Iliá Ilich Méchnikov, Saint-Petersburg, Russia; 6 Hematology and Stem Cell Transplant Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy; 7 Department of Hematological Medicine, King’s College Hospital NHS Foundation Trust, London, United Kingdom; 8 Department of Medicine and Surgery, University of Insubria and ASST Sette Laghi, Ospedale di Circolo of Varese, Varese, Italy; 9 National and Kapodistrian University of Athens, Athens, Greece; 10 Hematology, Fondazione Policlinico Universitario Agostino Gemelli - IRCCS, Rome, Italy; 11 European University of Cyprus, Nicosia, Cyprus

Background: The coronavirus disease 2019 (COVID-19) pandemic has direct impact on patients with active hematological malignancies (HM), who are at a continuous risk for severe disease course and delay of chemotherapy. The COVID-19 study of the European Hematology Association (EPICOVIDEHA) was initiated in February 2020 by an international steering committee. As of November 2021, more than 4,500 adult cases from 35 countries in five continents have been registered. From January 2022, EPICOVIDEHA will broaden its target population, in order to include children too, establishing the EPICOVIDEHA Kids registry.

Aims: The aim is to enhance the knowledge of the outcome and clinical management of these patients. The incidence of severe COVID-19, admission to intensive care units (ICU), estimation of pre-existing comorbidities, or associated mortality rate will also be evaluated.

Methods: The EPICOVIDEHA Kids survey will be online accessible at www.clinicalsurveys.net . All data will be collected anonymously: demographics, underlying diseases, hematological malignancy, COVID-19 infection, and outcome. Inclusion criteria will be: age below 18 years, active HM within the last 5 years prior to COVID-19, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive test. Each documented patient will be reviewed for data completeness and coherence by a panel of experts.

Results: A general publication with the data from all patients will be performed at the end of 2022. Additionally, subset analyses on specific cutting-edge topics might be run after the proposal of any participant with the previous approval of the steering committee of the survey.

Summary/Conclusion: EPICOVIDEHA Kids is a ready-to-use survey, whose focal point is to gather epidemiological data from pediatric hematological populations and COVID-19. This study will help to better understand the features of these patients and to ameliorate their clinical management. Additionally, EPICOVIDEHA Kids will enable the partnership of international groups of hematologists and infectious diseases specialists, in order to improve the clinical management of HM patients with COVID-19.

P1608: EXPERIENCE AND COMPLICATIONS WITH THE USE OF PICCS IN HEMATOLOGIC PATIENTS

L. Sanchez-Paz 1,* , M. J. Solaeta Gómez 2 , N. Varona Torralvo 2 , P. Molina Mejías 2 , E. Valencia Ospina 2 , M. A. Rodriguez Calderita 2 , V. Díez Viñas 2 , V. Ramos de Ascanio 1 , C. Muñoz Novas 1 , J. Churruca 1 , M. S. Infante 1 , E. Landete 1 , K. Marín 1 , M. Á. Foncillas 1 , J.-Á. Hernández-Rivas 1 , I. González-Gascón-y-Marín 1

1 Hematology Department; 2 Day-Hospital Unit, Infanta Leonor University Hospital, Madrid, Spain

Background: In the last few years the widespread use of peripherally inserted central catheter (PICCS) has provided a benefit for patients with hematological disorders. PICCS can be used for administration of supportive therapy, chemotherapy or parenteral nutrition. In some cases, they represent a better alternative to other catheters because of their easier and safer insertion/removal and its capacity to provide long-term venous access. Nevertheless, infection and thrombosis are two of the main complications associated with their use, but their rates and risk factors are highly variable.

Aims: To review the experience with PICCs in patients with hematologic diseases as well as to describe and analyze the possible complications associated with their use.

Methods: This is a retrospective unicenter study that included all adult patients with hematologic diseases that required a PICC insertion between June 2019 and December 2021. This procedure was performed by specifically trained nurses, who from this date began to tunnel PICCs guided by ultrasonography, at our hospital day care units. These nurses were in charge of weekly monitoring and cleaning the devices. Demographic and clinical data were obtained from the electronic medical records. Results were analyzed using SPSS Statistics program (v.21).

Results: A total of 99 PICCs were inserted into 82 different patients (68 (1 PICC); 12 (2PICCs); 1 (3 PICCs); 1 (4 PICCs)). Table 1.a summarizes baseline characteristics of the cohort. The median duration of the devices was 123 days (interquartile range (IR) 48-176) with 13,203 in situ days. By the time of this analysis, 87 PICCs have been removed. The reasons for PICC insertion and removal are shown in Table 1.b. Interestingly, 41.4 % of the PICCs were inserted in patients receiving any type of antithrombotic therapy due to their underlying condition (low molecular weight heparin= 31.3%; oral anticoagulation=7.1% and low dose aspirin=3%). The incidence of PICC related thrombosis (PRT) was 4.04% with a rate of 0.30/1000 PICC days. One PICC was removed after the diagnosis of PRT, 2 could be safely maintained until end of treatment and 1 patient died due to disease progression 3 days after PRT. No patient had thrombus progression. All PRT occurred in patients that were not receiving antithrombotic therapy. Hodgkin lymphoma (HL) compared with other diagnosis was found as the only significant risk factor for PRT (p=0.03). The incidence of PICC related infection (PRI) was 7.1% with a rate of 0.53/1000 PICC days. The median onset of PRI was 22 days (IR 4-89) and 6/7 PICCs were removed after PRI. Microbial isolates of PIIC bloodstream infections were: Coagulase-negative staphylococci (N=5); Staphylococcus aureus (N=1); Bacillus thuringiensis (N=1). Age (p=0.4); sex (p=0.8); type of disease (p=0.8); steroid use (p=0.9)/neutropenia (p=0.4) at the time of line placement; parenteral nutrition (p=0.1); type of PICC (p=0.8) and days with PIIC (p=0.5) were not significant risk factors for PRI. No early PICC-related complications were documented and PICC complications were not the primary cause of death for any patient.

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Summary/Conclusion: In our experience, PICCs are safe devices for patients with hematological diseases and can be used for a prolonged period. We did not find early complications during PICC insertion. We found a low rate of PRT, with a higher predisposition in HL patients. A favorable low rate of PRI was also observed in this series which could be explained by the adequate management of the devices at our institution with a weekly follow-up nurse control.

P1609: VERY LOW MORTALITY IN DOUBLE VACCINATED IMMUNOCOMPROMISED HAEMATOLOGY PATIENTS INFECTED WITH SARS-COV-2.

B. Shaw 1,* , J. Shortt 1,2 , B. Rogers 2,3 , Z. Kaplan 1,2 , M. Low 1 , G. Gregory 1 , S. Vilcassim 1,2 , G. Grigoriadis 1,2 , P. Fedele 1 , M. Gilbertson 1 , S. Opat 1

1 Haematology, Monash Health; 2 Monash University; 3 Infectious Diseases, Monash Health, Clayton, Australia

Background: Patients with immunocompromising haematological neoplasms (HN) are reported to be at high risk of death from SARS-CoV-2 (COVID19). However, many studies were conducted prior to widespread immunisation and availability of effective antiviral strategies. Here, we report outcomes of patients with HN presenting with COVID19 infection in the setting of high vaccination rates and effective COVID19 therapies, in a population that had low prior exposure to COVID19 due to national border controls and strict isolation measures.

Aims: To ascertain the rate of COVID19 mortality and severe disease in a cohort of patients with HN, and identify factors associated with severity, including, immunisation status, use of antiviral therapies and likely viral strain.

Methods: An observational study was conducted at an Australian tertiary hospital network servicing a population of 1.3 million. Adults with HN who tested positive for COVID19 were included. Patient and disease characteristics, vaccination status, therapy, hospital admission data and outcomes were extracted from hospital records. Associations with disease severity were examined using Chi-squared with p-values adjusted for multiple testing by Benjamini-Hochberg procedure.

Results: Between January 2020- February 2022, 75 patients with HN and COVID19 infection were identified. Median age 70 y (range 18-91) with 60% male. Underlying HN included: indolent lymphoma 26, myeloma 25, aggressive lymphoma 7, myelodysplastic syndrome 6, myeloproliferative neoplasm 4, chronic myeloid leukaemia 4 and acute myeloid leukaemia 3. Grade 3+ neutropenia <3 months prior to infection was identified in 15, anti-CD20 antibody therapy < 6 months in 14 and AuSCT <2 years in 4. At COVID19 diagnosis, 10 were unvaccinated for COVID19, 3 had received a single dose, 43 had received 2 doses and 19 had received 3 doses. 54 patients were diagnosed with COVID19 when Omicron strain was the dominant strain circulating. 21 tested positive to COVID19 when the Delta strain was dominant. Other COVID19 strains were very unlikely to be present given the low prevalence in Victoria during the period of study. 37 patients were admitted to hospital with a median length of stay of 8 days (range 3 -33). Of these patients, 4 were admitted to ICU with 2 patients requiring intubation. Another patient received non-invasive ventilation in a high dependency ward. The median length of stay in ICU was 5 days (range 2-11). Therapies used in hospitalised patients were administered according to institutional protocol and included sotrovimab, dexamethasone, remdesivir and baricitinib. 4 patients died with 3 (4%) of these directly attributed to COVID19 infection and one due to myeloma. Of 4 patients admitted to ICU, three were unvaccinated. Factors associated with hospitalisation included age ≥70 (p = 0.002), receiving <2 vaccinations against COVID19 (p = 0.012) and likely non-Omicron strain (p = 0.012). The underlying haematological condition, prior anti-CD20 therapy, presence of recent neutropenia or use of Sotrovimab was not found to impact on the likelihood of severe disease or hospital admission.

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Summary/Conclusion: Within our cohort of patients with haematological cancer diagnosed with COVID19 infection, the role of immunisations and presence of the Omicron strain within the community has resulted in a mortality rate of 4% which is lower than the rates observed in previous studies. Hospitalisation occurred in 48% of the patients, with risk factors of older age, being non- or partially vaccinated and likely non-Omicron strain of COVID19 identified.

P1610: IMPACT OF COVID-19 ON PATIENTS TREATED WITH AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION - A RETROSPECTIVE COHORT STUDY

T. Silfverberg 1,* , H. Cherif 2 , K. Carlson 2 , B. Wahlin 3

1 Medical Sciences, Center for Clinical Research Dalarna/Uppsala University; 2 Medical Sciences, Uppsala University, Uppsala; 3 Division of Hematology Dept of Medicine, Karolinska Institutet, Stockholm, Sweden

Background: Early studies of COVID-19 reported high mortality rates in patients with malignancy and who were immunocompromised. The immunosuppression related to autologous hematopoietic stem cell transplantation (ASCT) together with the risk of contracting severe SARS-CoV-2 lead to modifications in the therapy recommendations and, in many cases, to postponed treatment.

Aims: To describe how Coronavirus Disease 2019 (COVID-19) affect patients with hematological malignancies treated with ASCT.

Methods: This retrospective observational cohort study includes all patients with hematological malignancies treated with ASCT in Sweden from 1 January 2020 until 31 December 2020. Patients who subsequently tested positive for SARS-CoV-2 until 31 March 2021 were analyzed for morbidity, mortality, need for supportive care and risk factors involved in COVID-19.

Results: The study identified 442 patients who underwent ASCT in Sweden in 2020, among whom 20 (4.5%) subsequently contracted COVID-19. The overall mortality was 15% and the COVID-19 related mortality 10%. The absolute risk of COVID-19 related mortality was 0.45% among patients treated with ASCT. Six (35%) patients were hospitalized, among which four (24%) needed supplementary oxygen and two (12%) intensive care.

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Summary/Conclusion: ASCT patients have a higher risk of severe outcome of COVID-19 compared to the general population. The risk of death, need for hospital care, oxygen and intensive care was lower in this study compared with most previous studies, possibly because of less missing asymptomatic patients. The risk of contracting SARS-CoV-2 was comparable with the general population. This study support performing ASCT for treatment of hematological malignancies despite the COVID-19 pandemic.

P1611: INSIGHTS INTO FACILITATED IMMUNOGLOBULIN (FSCIG) USE IN PATIENTS WITH SECONDARY IMMUNODEFICIENCY DISEASES: FINAL RESULTS FROM THE FIGARO STUDY

M. Dimou 1,* , M. Speletas 2 , C. Milito 3 , D. Huscher 4 , M. Kamieniak 5 , D. Pittrow 6 , M. Borte 7,8

1 First Department of Propaedeutic Internal Medicine, National & Kapodistrian University of Athens Medical School, General Hospital “LAIKO”, Athens; 2 School of Health Sciences Faculty of Medicine, Department of Immunology and Histocompatibility, Medical School Campus - University of Thessaly, Thessaly, Greece; 3 Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy; 4 Institute of Biometry and Clinical Epidemiology, Charité-Universitätsmedizin Berlin, Berlin, Germany; 5 Global Medical, Takeda Pharma Sp.z.o.o., Warsaw, Poland; 6 Institute for Clinical Pharmacology, Medical Faculty, Technical University of Dresden, Dresden; 7 Hospital for Children and Adolescents, St. Georg Hospital; 8 Academic Teaching Hospital of the University of Leipzig, Leipzig, Germany

Background: Secondary immunodeficiency diseases (SID) are common among patients with hematological malignancies such as chronic lymphocytic leukemia (CLL) and multiple myeloma, putting patients at increased risk of severe, recurrent/persistent infections and contributing to morbidity and mortality. Pharmacotherapeutic options available to patients with SID include antimicrobial treatment and immunoglobulin replacement therapy (IGRT). Facilitated subcutaneous immunoglobulin (fSCIG) is a dual-vial unit of immunoglobulin G 10% and recombinant human hyaluronidase (rHuPH20) approved in the European Union for patients with SID. rHuPH20 depolymerizes hyaluronan in the extracellular matrix to transiently increase local subcutaneous tissue permeability, allowing for fSCIG infusion at rates, volumes, and frequencies comparable to intravenous immunoglobulin with better systemic tolerability.

Aims: To provide insights on the real-world utilization and tolerability of fSCIG for up to 3 years in a broad population of patients with SID and assess any impact of older age on use.

Methods: FIGARO ( NCT03054181 ) was a European prospective, observational study in 14 sites. Patients (n=156) who received ≥1 fSCIG infusion for primary immunodeficiency diseases or SID and provided informed consent were included and followed for up to 3 years. Study initiation was December 2016; database closure was May 2021. Here we report final data for the overall cohort of patients with SID, together with further subgroup analysis for patients aged 18–64 years [adult] and ≥65 years [older adult].

Results: The SID cohort included 31 patients; 1 pediatric (not reported as age subgroup), 15 adult, and 15 older adult patients. At study close, 1-, 2-, and 3-year follow-up data were available for 25, 13, and 11 patients, respectively. The most common indications for IGRT were CLL (n=20) and non-Hodgkin lymphoma (n=6); the most common supportive therapies included Pneumocystis jirovecii pneumonia prophylaxis, virostatics, and antibiotics. In the year prior to inclusion, 10 (32%) patients had experienced ≥1 acute severe bacterial infection.

At the inclusion visit, fSCIG was most recently infused at home (61%) or a medical facility (39%) by the patient (58%) or a nurse (42%), with a tendency of adult patients to more self-application than older adults. Most patients received fSCIG every 4 weeks (71%), infused into a single site (most commonly upper abdomen in adults, and lower abdomen in older adults). Over the 3-year observation period, the median monthly dose of fSCIG, and median monthly infusion volume per patient remained consistent at 30 g and 300 mL, respectively, in both the adult and older adult groups. All patients received the full dose as planned, except for 1 patient at the 36-month follow-up visit who experienced infusion/technical problems. Two patients (in the adult group) each reported 1 adverse drug reaction (ADR; infusion site inflammation and headache) at the inclusion visit, with no ADRs reported at any of the follow-up visits. No acute severe bacterial infections were reported during the study.

Summary/Conclusion: These data provide insights into the long-term feasibility and tolerability of fSCIG administration at home or in a medical facility in patients with SID primarily due to hematologic malignancies, including in older patients aged ≥65 years.

P1612: ACUTE MYELOID LEUKEMIA IN ADULT PATIENTS: FEATURES OF TREATMENT IN CONTEXT OF THE COVID-19 PANDEMIC.

T. Tolstykh 1,* , E. Baryakh 1 , D. Ivanova 1 , T. Chudnova 1 , M. Misyurina 1 , E. Medvedovskaya 1 , M. Litvinenko 1 , Y. Polyakov 1 , O. Kochneva 1

1 Moscow Clinical Hospital 52, Moscow, Russia

Background: Acute myeloid leukemia (AML) is a disease with a poor prognosis and a high mortality rate, while patients with acute myeloid leukemia are at increased risk of complications from COVID-19 infection.

Aims: To investigate the treatment effectiveness and survival of patients with AML who had the COVID-19 diagnosis confirmed, as well as evaluating predictors of hospital mortality among this group of patients.

Methods: From April 2020 to November 2021, Moscow City Clinical Hospital 52 treated 149 patients with AML who had the COVID-19 diagnosis confirmed, with a median age of 61 years (range 18-91), while 56% of patients were over 60 years old. An equal ratio of men and women was performed, 70 and 79 respectively. 13% were diagnosed with APL, while the remaining 87% had other variants of AML. At the time of hospitalization the most of the patients admitted had first- or second-degree pulmonary lesions (28 and 35%, respectively), while 31% of patients performed more severe lesions and the other 6% had no pulmonary damage detected, but they had a positive PCR. At the time of hospitalization, 10 out of 149 patients (7%) achieved complete remissions (CR) and were withdrawn from therapy; 41 patients (28%) were diagnosed with AML for the first time in our clinic; and 98 patients (65%) were already in process of AML treatment in other clinics. The effectiveness of treatment was assessed by the frequency of CR, as well as by the amount of cases of lethality and in-hospital survival rates. The analysis of treatment results was performed as at 01 November 2021.

Results: 60 patients (38%) with AML (excluding APL) and confirmed COVID19 received anti-tumor treatment. Palliative care was received by 23 patients (38%), these were predominantly elderly patients with a median age of 71 years (range 45-91 years), with severe COVID-19. 19 patients (32%) with a median age of 38 years (range 21-61 years) received the 7 + 3 treatment protocol. While 18 elderly patients (30%) with a median age of 70 years (range 60-85 years) received the low intensity therapy (low doses of cytarabine, hypomethylation therapy and venetoclax containing regimens). As a result of COVID-19, 79 patients (54%) died. The main factor determining the high mortality rate in our study was the absence of AML CR. The Survival of patients in CR was 96.3±3.6%, which means almost all patients have recovered from COVID-19 and were discharged to continue their treatment in other medical institutions. For patients out of remission and primary patients with AML the survival rate was 38.0±6.5% and 33.7±8.7%, respectively. For the purposes of analyzes of predictors of survival, we divided our patients into 2 groups, due to a great variety of prognosis. Remission status essentially outweighs all other factors, including presence of significant comorbidities, AML variants, isolated cytopenias, severity of COVID-19 (CT, CRP, D-dimer, etc.), nature of AML therapy and other factors. In the group of patients out of AML remission the negative predictive value for hospital survival was shown by age over 60 years, as well as by agranulocytosis, CT 3-4, the use of GCS and transfer to the ICU.

Summary/Conclusion: The diagnosis of COVID-19 in patients with AML greatly worsens their life prognosis. The leading predictor of mortality becomes the lack of remission of AML. Thus, antitumor treatment allows achieving CR, which in turn reduces the risk of developing a severe course of coronavirus infection.

P1613: SINGLE-CELL ANALYSIS FOLLOWING SARS-COV-2 MRNA VACCINE IN ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION RECIPIENTS

E. Tranter 1,2,* , B. Obermayer-Wasserscheid 3 , M.-L. Hütter-Krönke 1 , C. Junkuhn 4 , I.-W. Blau 1 , G. L. Vuong 1 , L. Bullinger 1,5,6,7 , I.-K. Na 1,2,6,8,9

1 Department of hematology, oncology and tumor immunology, Charité Universitaetsmedizin Berlin; 2 Berlin Institute of Health (BIH); 3 Core Unit Bioinformatics – CUBI, Berliner Institut für Gesundheitsforschung in der Charité/Berlin Institute of Health at Charité (BIH); 4 Charité Universitaetsmedizin Berlin; 5 Core Unit Bioinformatics – CUBI, Berlin Institute of Health (BIH); 6 German Cancer Consrtium (DKTK); 7 Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association; 8 Berlin Institute of Health Center for Regenerative Therapies, Charité Universitaetsmedizin Berlin; 9 Experimental and Clinical Research Center, Berlin, Germany

Background: Allogeneic stem cell transplantation (alloHSCT) recipients are at particularly high risk to suffer from a severe course of disease when infected with SARS-CoV-2. It has been shown that cellular and humoral responses to available vaccines are significantly impaired in this vulnerable patient cohort. However, detailed data on the adaptive immune response is still lacking.

Aims: In the current study, the evolution of the adaptive immune response against SARS-CoV-2 mRNA vaccine BNT162b2 in patients after alloHSCT was longitudinally characterized.

Methods: We used single-cell RNA sequencing (scRNAseq) to integrate transcriptomic information and clone identification of about 75,880 single cells from 3 AML patients who underwent alloHSCT 188 days (± 16) prior immunization with mRNA COVID-19 vaccine BNT162b2. Blood from before, after the first and after the second vaccine was collected and analyzed. Further, serological vaccine responses were assessed.

Results: T cell compartment of alloHSCT recipients shows a strong distortion towards CD8+ T cells, indicating an incomplete immune cell reconstitution in the investigated patients. Upon first vaccination, newly appearing CD8+ clones can be detected in all patients. These newly appearing CD8+ clones can be traced until after the second vaccination, possibly signifying an evolving SARS-CoV-2 specific response. Differential gene expression analysis of vaccine-induced CD8+ clones revealed an enrichment in genes related to T cell activation, particularly detectable after the second vaccination. Although CD4+ T cells showed an increase in relative abundance, gene expression analysis of CD4+ clones appearing after vaccination did not show significant alteration in gene expression. The B cell compartment was diminished in all alloHSCT recipients. However, two of the three patients did achieve a protective serological titer of anti-IgG-Spike antibodies.

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Summary/Conclusion: In this study, we show a traceable, clonal expansion of CD8+ T cells in alloHSCT recipients after vaccination with BNT162b2, signifying an antigen specific T cell response. Only after the second dose, vaccine-induced CD8+ clonotypes show an enrichment of T-cell activation related genes, emphasizing the relevance of repeated vaccination. This data sheds light on the evolution of adaptive immune response of alloHSCT patients towards SARS-CoV-2 mRNA vaccine at a single cell level, giving crucial insights beyond serological assessment and in vitro T cell activity analysis.

P1614: MOLECULAR EPIDEMIOLOGY OF INFLUENZA VIRUS IN PATIENTS WITH HEMATOLOGIC DISORDERS

S. Ünal 1,* , J. Tabatabai 1 , N. Giesen 2 , M. Wedde 3 , P. Schnitzler 1

1 Virology; 2 Internal Medicine V, University Hospital Heidelberg, Heidelberg; 3 National Reference Centre for Influenza, Robert Koch Institute, Berlin, Germany

Background: Whereas influenza virus infections usually present as self-limiting upper respiratory tract infections, they are also one of the most common causes of viral lower respiratory tract infections. Especially immunocompromised patients, such as ones suffering from malignant diseases, are at a higher risk of severe influenza virus infection, thus leading to increased morbidity and mortality. With influenza viruses possessing the ability of antigenic shift and antigenic drift, consequently altering the viral genome frequently and avoiding long-term immunity of the host, the necessity for regular data collection is clear.

Aims: With this study we want to retrospectively assess influenza virus epidemiology in patients with hematological malignancies and investigate clinical and virological risk factors of severe infection to be able to apply information about improvement of hygiene and care.

Methods: We included clinical data from 159 influenza positive patients from the Department of Haematology of the University Hospital Heidelberg from the seasons of 2014 to 2019 to analyse occurrence of nosocomial transmission, prolonged viral shedding and associated risk factors for severe infection. Virological data was obtained by nasopharyngeal swabs, which were collected routinely, then Sanger sequencing of the hemagglutinin gene was performed for phylogenetic and mutational analysis. Combination of clinical and virological data was assessed to evaluate specific susceptibility to influenza subtype, subgroup or amino acid mutation.

Results: 42 patients (26%) developed LRTI. Associated risk factors with development of lower respiratory tract infection (LRTI) (p<0.05) were nosocomial infection (p=0.008), IgG levels lower than 6g/dl (p=0.046), bacterial (p=0.001), fungal (p<0.001) and bacterial and fungal (p=0.017) coinfection. 9 of 31 (29%) of bacterial copathogens belonged to multi-resistant strains. LRTI associated mortality rate in our study population was noticeably high with 38%. 67% of patients with consecutive testing (n=54) presented with prolonged viral shedding (median 9.5 days). Epidemiological comparison with national surveillance data mostly showed no divergence of our patient group regarding influenza type and subtype. In 115 patients, sequencing of influenza hemagglutinin gene was successful, and 13 virus isolates revealed significant substitutions: In A/H1N1 samples P137S, D187N and D222V; in A/H3N2 samples D7N, T10M, D101E, I140M, Q197K, Q197R and I214T; in B/Yamagata samples Y165H, P171Q and N202R and none in B/Victoria samples. Most of these substitutions could be assigned to an antigenic site or receptor binding site.

Summary/Conclusion: While no specific susceptibility to influenza subtypes or subgroups could be observed, high LRTI associated mortality rate and occurrence of nosocomial transmission as well as multi-resistant copathogens illustrate the need for better hospital hygiene and early evaluation of disease severity. To understand detected mutations and their impact on the course of infection as well as virus evolution and vaccination efficiency, further collection of data is needed.

P1615: AN ANALYSIS ACROSS ABO BLOOD GROUPS IN PATIENTS WITH COVID-19 INFECTION AND CORRELATION WITH HAEMATOLOGICAL BIOMARKERS, A SINGLE CENTRE STUDY

H. Shokr 1 , M. Marwah 2 , S. Marwah 3 , F. Wandroo 3,*

1 Pharmacy Division, University of Manchester, Manchester; 2 Aston Medical School, Aston University, Birmingham; 3 SWBH NHS trust, West Bromwich, United Kingdom

Background: The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the infectious disease coronavirus disease 2019 (COVID-19), has rapidly spread around the world and continues to be a global public health emergency. Currently worldwide over 5 million people have died due to this virus. Several studies observed that differences in blood groups may be linked to COVID-19 susceptibility and clinical outcomes; however, the results are controversial and there is a need for further research and validation. It is well known that ABO antigens are expressed on respiratory epithelial cells, however these antigens called type-1 antigens which differ from type-2 antigens expressed on red blood cells. Recent evidence has shown that receptor binding protein (RBD) of the SARS-CoV-2 binds preferentially to blood group A antigen on respiratory epithelial cells.

Aims: This retrospective study aimed to analyse the haematological laboratory parameters and clinical manifestations in COVID-19 infected patients: we hypothesised that certain routine haematological indices may be altered in across the ABO blood groups, and that pattern identification would facilitate prediction of those at high risk of severe disease.

Methods: A total number of 194 patient with COVID-19 confirmed by a real-time polymerase chain reaction (RT-PCR) were included in this study. Patients were classified into 4 groups according to their blood group (blood group A, 71 patients: blood group B, 30 patients; blood group AB, 8 patients and blood group O, 85 patients). Standard routine haematology laboratory indices were analysed for all the study participants.

Results: Mortality rates were assessed in all four groups; of blood group A, 36.0% died, of blood group B, 25.65% died, of blood group AB, 23.5% died and of those with blood group O, 33.2% died. Deceased patients with blood group AB exhibited lower white blood cells, neutrophils, monocyte counts compared to patients with blood group A, B and O. Similarly, D-Dimer, urea, bilirubin, and C-reactive protein were significantly lower in the AB group compared to the rest of the blood groups. Positive correlations were found between Neutrophil/lymphocyte ratio and D-dimer and urea blood levels in all the study population (p=0.022, r=0.227 and p=0.254, r= 0.0001 respectively). Likewise, a positive correlation was found between neutrophil/ eosinophils ratio and blood urea, bilirubin, and C-reactive protein (p=0.047, r= 0.205; p=0.021, r= 0.249 and p= 0.0001, r= 0.377 respectively).

Summary/Conclusion: This study observed patients with blood group AB have a lower risk of organ dysfunction or failure due to COVID-19. Although we found no significant statistical difference in mortality between different ABO groups, certain haematological indices and biochemical markers of inflammation were favourable in patients with blood group AB with a tendency to lower mortality.

P1616: NGS-BASED PATHOGEN DIAGNOSTIC IN FEBRILE HAEMATOLGY PATIENTS – A SINGLE-CENTRE EXPERIENCE

S. Wolf 1,* , C. S. Schmidt 1 , T. M. Schmitt 1 , E. Siegel 2 , C. Hamm 2 , P. Stevens 3 , M. Theobald 1 , D. Teschner 1,4

1 Department of Hematology, Medical Oncology, and Pneumology; 2 Institute of Clinical Microbiology and Hygiene, University Medical Center of the Johannes Gutenberg University, Mainz; 3 Noscendo GmbH, Duisburg; 4 Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany

Background: Bloodstream infections are frequent and serious complications in immunocompromised patients mainly in haemato- and oncology. The established gold standard in pathogen diagnostic for microbial infections is the analysis of blood cultures (BC). By BC, bacterial pathogens can be detected in approximately 30% of blood samples from neutropenic patients only. Additionally, viral or fungal infections are even more challenging to detect and require special procedures or invasive diagnostics. To close this diagnostic gap and to increase the yield of pathogen detection, NGS-based approaches were developed to detect cell-free DNA in plasma samples and evaluate the findings using elaborated mathematical algorithms.

Aims: The aim of this study was to compare the new NGS-based diagnostic approach with established conventional microbial diagnostic in febrile high-risk cancer patients.

Methods: In a first step, patients covered by selected health insurances were included in the analysis after informed consent between September ‘20 and July ‘21. Afterwards, all patients with a related underlying disease were included between August ’21 and November ’21.

From these patients, blood samples were drawn during a first episode of fever. These blood samples were analysed by NGS-based DISQVER®-diagnostic developed and performed by Noscendo GmbH. Addition, conventional pathogen diagnostic was performed according to our local standard of care. This includes mainly BCs but also specific viral or histopathologic diagnostic, when clinically indicated. The analysis of conventional diagnostic samples was performed by our Institute of Clinical Microbiology. After analysis, we reviewed the different results and correlated them with clinical information’s and vice versa.

Results: Diagnostic Samples were collected in a total of 71 febrile episodes (events) in 47 patients. In these patients, an underlying malignant hematologic disease was present in n=67 (87%). 30 events (42%) were post allogeneic stem cell transplantation. Neutropenia (granulocyte count <500/µl) was present during 37 febrile episodes (51%). Overall, microbial pathogen detection could be obtained in 45/71 events (63%). In 25/71 febrile episodes (35%), pathogens were detected by NGS-based diagnostic only and the corresponding conventional diagnostics remained negative. Positive conventional and positive NGS-based diagnostic were present in 18 events. In these, identical pathogens were detected NGS-based and in the corresponding BC only in 4 events while in the remaining 14 febrile episodes conventional pathogens could only be identified by additional assays or more invasive diagnostic procedures (endoscopy, biopsy and others). In 2 events, pathogens were detected by conventional diagnostics but NGS-based was negative.

Overall, 42 different pathogens could be identified by NGS-based diagnostic (24 bacterial, 12 viral and 6 fungal pathogens [including zygomycetes]).

Summary/Conclusion: In our study, combination of NGS-based and conventional microbial diagnostic identified substantially more pathogens than conventional diagnostic alone. Moreover, NGS-based diagnostic revealed a higher sensitivity and diagnostic yield compared to conventional and even more invasive diagnostic approaches. Additionally, the NGS-based diagnostic approach seems to detect more and/or even difficult-to-detect microbial pathogens compared to standard diagnostic procedures. However, not every NGS-based detected pathogen turned out to be clinically relevant and led to a therapeutic consequence.

P1617: CLINICAL SIGNIFICANCE OF CYTOMEGALOVIRUS DRUG RESISTANCE GENE MUTATIONS IN CYTOMEGALOVIRUS INFECTION AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

D. yang 1 , J. wen 2,* , Y. zhang 1 , G. li 1 , Y. zheng 1 , S. chen 1 , A. pang 1 , Y. he 1 , J. wei 1 , Q. ma 1 , W. zhai 1 , R. zhang 1 , X. chen 1 , S. feng 1 , M. han 1 , E. jiang 1

1 Department of hematopoietic stem cell transplantation, State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical Colleg, Tianjin; 2 Department of hematology, State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, CAMS & PUMC; Mianyang Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Mianyang, China

Background: Cytomegalovirus (CMV) infection is the most important complication after allo-HSCT and threatens the prognosis of patients seriously. Currently, there are limited data on CMV drug-resistant gene mutations in CMV infection after transplantation in China.

Aims: In order to further understand the expression of CMV drug-resistant gene mutations in patients with poor efficacy of CMV infection in single center tertiary hematology hospital in china.

Methods: The clinical data of 41 patients with CMV infection after allo-HSCT were retrospectively analyzed from January 2019 to July 2021 in transplantation Center of Hematology Hospital, Chinese Academy of Medical Sciences, due to refractory and probable refractory CMV infection, who were tested for CMV resistance gene mutation. The current recommended definitions are based on Guidelines for the management of cytomegalovirus infection from ECIL 7. A total of 21 patients with AML, 8 patients with ALL, 6 patients with AA, 5 patients with MDS and 1 patients with Fanconi anemia are included. Donors were HLA haploid-identical related ( N = 36), HLA-identical siblings and un-related ( N = 5).

Informed consent was obtained.

Results: ① Of All 41 patients included, 1 patient (2.4%) had UL97 mutation, 19 patients (46.3%) had UL54 mutation, no patients occur simultaneously with UL54 and UL97 mutation. The overall mutation frequency was 48.8% (20/41), and all mutation types were missense mutation.

② UL54 gene were detected 8 cases of T691S missense mutation (8/41, 19.5%), 3 cases of M827I missense mutation (3/41, 7.3%) and 1 case of A786V missense mutation (1/41, 2.4%). All above these 3 mutation sites had been reported, but the drug resistance significance was unknown. There were 1 site mutation (A543S) of UL97 gene and 19 sites mutation of UL54 gene that had not been reported. The most common mutation site is the T691S mutation in UL54 gene. In all the 41 patients, only one patient exhibited a mutation with definite anti-CMV drug resistance significance: Q578H mutation of UL54 gene, and finally was successfully treated with twice CMV-CTL infusions (figure).

③ Compared with patients without UL54 mutation, patients with UL54 mutation strains had a higher proportion of CMV-DNA re positive rate in 10 days after remission of DNAemia (42.1% vs.9.5%, χ2=5.647, P=0.028), and hadthe significantly longer complete clearance time of CMV DNAemia (40 days vs. 24 days, Z=2.198, P=0.027).

④ Compared with those patients without CMV gene mutation, the patients with T691S mutation had a higher incidence in terms of CMV-DNA turned re positive in 10 days and 14 days after remission of DNAemia (71.4% vs. 19.0%, P=0.008; 71.4% vs. 9.5%, P=0.001; 71.4% vs. 9.5%, P=0.001, respectively). The median time of CMV-DNA reaching the peak was significantly later (14 days vs. 10 days, P=0.016). The median clearance time of CMV DNAemia was significantly longer (56 days vs. 24 days, P=0.002), and the differences were statistically significant. Compared with the non-T691s UL54 mutation, the patients with T691S mutation also had similar results.

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Summary/Conclusion: CMV gene missense mutations were detected in 48.8% of patients infected with CMV after transplantation, most of which occurred in UL54 gene and T691S mutation is a common missense mutation. The incidence of missense mutations with clear drug resistance was low (2.4%) and might be induced by antiviral therapy. The median clearance time of CMV DNAemia in UL54 mutated patients was significantly longer, and the proportion of CMV-DNA turned re positive was increased, which might be caused by T691S mutation in UL54 gene.

P1618: CLINICAL OUTCOME OF COVID-19 CONVALESCENT PLASMA TRANSFUSION IN CHRONIC KIDNEY DISEASE PATIENTS ON RENAL REPLACEMENT THERAPY WITH SEVERE TO CRITICAL COVID-19 DISEASE

J. Ahorro 1,*

1 Internal Medicine, National Kidney and Transplant Institute, Quezon City, Philippines

Background: Convalescent plasma therapy (CPT) has been explored for Coronavirus disease 2019 (COVID-19), but limited data exists for Chronic Kidney Disease (CKD) patients. Since the effects of COVID-19 therapies are still under study, CPT remains an option for these patients in the absence of other effective therapy.

Aims: This study aimed to compare the clinical outcome of severe to critical COVID-19 patients with CKD on RRT who received COVID-19 CPT versus those who did not.

Methods: Retrospective study. Primary outcome was clinical improvement defined as mortality or whether discharged alive or improvement in WHO 6-point severity scale within 28 days. Secondary outcomes include factors associated with clinical improvement such as CPT’s effect on pulmonary status, timing of CPT, effects on hematologic profile and inflammatory markers.

Results: Total of 44 CKD patients were included. Half were given CPT, half were not. Mean age was 59±12.7 and 51±15.6 respectively; 68.2% male, 31.8% female. No significant difference was found in the clinical outcome of severe to critical COVID-19 CKD patients who received CPT and those who did not. However, significant reduction in HsCRP, procalcitonin and ferritin pre and post CPT was found. This provide support in the role of CPT in reducing the inflammatory cytokine course of COVID-19 in CKD patients.

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Summary/Conclusion: Convalescent plasma transfusion did not show significant difference in the clinical outcome of severe to critical COVID-19 CKD patients, but was associated with decreasing trends of HsCRP, procalcitonin and ferritin, which are poor prognosticators of COVID-19 disease.

P1619: PLATELET TRANSFUSION THRESHOLDS FOR PEDIATRIC PATIENTS; ARE WE FALLOWING THE GUIDELINE?

H. Alsulami 1,*

1 AGU, Riyadh, Saudi Arabia

Background: Platelet threshold of 10x109 /L is recommended for clinically stable thrombocytopenic pediatric patients. Transfusions at a higher level (given the absence of research evidence, as determined by clinical circumstances, generally at threshold of 40x109 /L) may be required for patients with signs of bleeding, high fever, hyper-leukocytosis, rapid fall in platelet count, concomitant coagulation abnormality, critically ill patients, and those with impaired platelet function (including drug induced). Transfusions at a higher level may be also required for patients undergoing invasive procedures.

Aims: the aime of this study is to check if the phycisions follow the guidelines for platelet transfusion

Methods: This study is a retrospective observational analysis of platelet transfusion thresholds in a single secondary pediatric hospital in Riyadh. From the blood bank database, the list of the patients who received platelet transfusions in the second half of 2018 was retrieved. Patients were divided into two groups; group A, those belong to the category of high platelet level for transfusion (such as those with bleeding, high fever, rapid fall in platelet count, impaired platelet function or undergoing invasive procedures) and group B, those who were not. Then we looked at the pre and post transfusion platelet levels for each group. The data was analyzed using GraphPad software and the data expressed as Mean ± SD.

Results: A total of 112 of transfusion episodes in 61 patients (38% female) were analyzed. The age ranged from 24 days to 8 years. The distribution of platelet transfusion episodes was 64% (n=72) for group A and 36% (n= 40) for group B. The mean pretransfusion platelet count was 46x103 ± (11x 103) for group A and 28x103 ± (6x103) for group B. the post-transfusion mean platelet count was 61 x 103 ± (14 x 103) and 60 x103 ± (24 x 103) for group A and B respectively. Among the groups the rise in the mean platelet count after transfusion was significant among stable patients (group B) compared to unstable patients (group A) (P < 0.001).

Summary/Conclusion: he platelet count threshold for transfusion varied with the clinical condition and is higher among unstable patients’ group which is expected. For stable patients the threshold was higher than what it should be which means that the clinicians don’t follow the guidelines in this regard. The rise of platelet count after transfusion was higher among stable patients.

P1620: RED CELL ALLOIMMUNIZATION IN MULTI-TRANSFUSED TURKISH PATIENTS WITH HEMATOLOGIC DISORDERS.

S. Oztaskin 1 , M. Yanasik 2 , M. Ozbalak 3 , M. Guzel Mastanzade 3 , O. Ozluk 3 , T. O. Tiryaki 3 , S. Erdem 3 , S. Besisik 3,*

1 INTERNAL MEDICINE; 2 BLOOD CENTER; 3 INTERNAL MEDICINE HEMATOLOGY, ISTANBUL UNIVERSITY ISTANBUL MEDICAL FACULTY, ISTANBUL, Turkey

Background: The red blood cell (RBC) alloimmunization rate varies with underlying disorder among genetic diversity of the population and environmental influences.

Aims: The aim of this study was to determine the RBC alloimmunization rate in multi-transfused patients with hematological disorders in Turkey as a country with different ethnic groups covering a mixture of European and Asian DNA.

Methods: The study was designed retrospectively. The research group consists 148 newly diagnosed hematological malignancy patients as candidates for multiple transfusion. Pre-transfusion tests included indirect anti-globulin test (IAT) with gel centrifugation among ABO Rh blood grouping and cross-match. Antibody identification test was applied to patients with positive IAT after direct antiglobulin tests was found to be negative.

Results: Of the 148 patients 53.4% (n=79) were male and 46.6% (n=69) were female. The mean age of the patients was 53.03±17.78. IAT proved to be in 22 patients (14.9%) positive. In 6 of them alloantibody determining test was not performed (not ordered by the clinician). In 9 patients alloantibody type could not be determined. In the remaining’s (43.8%) the most frequent alloantibody types were against Rh antigens and anti-E was found to the most common encountered (12.5%) antibody. In three patients, more than one antibody type was detected.

Summary/Conclusion: Our study is from the largest city from Istanbul as a tertiary hospital in Turkey. Apart of different antigenic exposure and microenvironment of underlying disorder, the general ethnic background of the country may be an influencer for the results. New molecular researchs including HLA and immune response analysis would be more definitely clear the alloimmunization development.

P1621: ANAPHYLACTIC SHOCK TO A HEMATOLOGICAL DISORDER PATIENT DURING TRANSFUSION OF A PLATELET APHERESIS UNIT STORED IN PLATELET ADDITIVE SOLUTION: A RARE CASE REPORT

B. Bhasker 1,*

1 Sparsh Hospital, BANGALORE, India

Background: Adverse transfusion reactions from transfusion of apheresis platelets (AP) stored in Platelet additive solution (PAS) are uncommon, as PAS is a balanced electrolyte solution developed to replace plasma from platelet concentrates as the storage medium. No anaphylactic reactions have ever been reported due to transfusion of AP stored in PAS.

Aims: The main objective of the study is to understand the reaction occurred due to apheresis platelet transfusion.

Methods: An AP product was donated by a blood group A male, processed using PAS. The patient developed severe Hypotension with Tachycardia immediately after 10 minutes of transfusion. Pre-transfusion and Post transfusion recipient vitals showed a marked decrease in Blood Pressure from 110/70 mmHg to 70/50 mm Hg with Pulse increased from 88 bpm to 118 bpm. Oxygen saturations were maintained normal without any changes in respiratory parameters. Serological testing included an antibody screen using gel technology, a direct antiglobulin test (DAT) using immunoglobulin G and C3d, and a Sterility testing of the transfused platelet product.

Results: A 61-year-old blood group O patient with Aplastic anemia developed severe hypotension within the first 10 minutes of the start of AP in PAS transfusion despite premedication with Inj. Avil and Inj.Hydrocort. The post-transfusion reaction evaluation was non-significant showing negative antibody screen and also negative DAT, both for immunoglobulin G and C3d; No bacterial growth was seen in the transfused product and patient blood culture was sterile. Ruling out hemolytic and septic transfusion reactions. The patient was resuscitated with Inj. Adrenaline and got shifted to ICU for further follow-up. The patient vitals got stabilized after 4 hours and got discharged in hemodynamically stable condition. Notably, a group O patient at a different hospital received a split of the same apheresis unit, with no reaction.

Summary/Conclusion: To our knowledge, this is the first case with anaphylactic shock reported from Apheresis platelet transfusion stored in PAS. The fact that one transfusion recipient had anaphylactic shock whereas another did not have any reported reaction highlights the potential importance of recipient variables in adverse transfusion reactions.

P1622: ANTIPHOSPHOLIPID ANTIBODIES IN CONVALESCENT PLASMA OF DONORS RECOVERED FROM MILD COVID-19

D. Blickstein 1,* , M. Izak 2 , T. Filipovich- Rimon 3 , O. Garach- Jehoshua 3 , N. Rahimi- Levene 4 , E. Shinar 5 , A. Bar- Chaim 6 , M. Koren-Michowitz 7

1 Hematology, Shamir Medical Center, Beer Yakov; 2 Magen David Adome, National Blood Services, Ramat Gan; 3 Hematology laboratory; 4 Blood bank, Shamir Medical Center, Beer Yaakov; 5 Magen David Adome, National Blood Services, Ramat Gan; 6 Clinical laboratories; 7 Hematology, Shamir Medical Center, Beer Yaakov, Israel

Background: High prevalence of antiphospholipid antibodies (APLA) in patients with COVID-19 has been reported. A meta-analysis concluded that APLA were detected in nearly half of hospitalized patients with COVID-19, and even higher prevalence was found in patients with severe disease.These reports raised questions about the true prevalence and clinical impact of APLA on disease course, long term complications and the prothrombotic influence of COVID-19 convalescent plasma (CCP) transfused to COVID-19 patients.

Aims: To evaluate the prevalence of APLA in CCP samples collected from otherwise healthy donors who recovered from mild COVID-19.

Methods: CCP was collected from volunteer donors in two time periods (September 2020-January 2021 [early period] and April- May 2021 [late period]). The study was approved by the local ethics committee and all donors and controls signed informed consent. The study was supported by a grant from the Laboratory Research Fund, Shamir Medical Center. Convalescence was supported by studying serum anti COVID-19 antibodies (Ab): Anti- N (nucleocapsid protein) Ab during the early period, and anti- S (Spike1 protein) Ab during the late period.Anti- N Ab were tested by a chemiluminescent immunoassay. Positive results were defined as sample/cutoff (S/CO) ≥1.4. Anti-S1 Ab were tested using ELISA. Results ≥ 50 arbitrary units per milliliter (AU/ml) were considered positive. Plasma samples were tested for APLA: Lupus anticoagulant (LAC)testing was performed using dilute Russell Viper Venom Time (dRVVT) and Silica Clotting Time (SCT). Results were reported as normalized ratios. APLA IgG, IgM and anti-β2-glycoprotein IgG, IgM antibodies were detected using an ELISA method and reported as SGU u/ml or SMU u/ml.

Results: Table 1 summarizes demographic data and laboratory results of plasma collected from donors. Plasma from 10 healthy volunteers were used as controls (8 women, median age 29 years [range 20-42]). None of the participants had an history of thrombosis. Early period CCP donors were significantly older compared to late period CCP donors, and approximately 80% of donors were male. Two donors had elevated APLA values and 5 had elevated normalized LAC SCT ratio, for a total prevalence of 6%. LAC was positive in the late period while APLA were found in the early period. All controls were negative for LAC and 9 were APLA negative. One woman had an elevated APLA anti-β2GP1 IgM (40.1 SMU u/ml).There was no association between anti-SARS-CoV-2 antibodies titer and the presence of LAC / APLA.

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Summary/Conclusion: Our results demonstrate low frequency (6%) of APLA / LAC in plasma collected from donors who have fully recovered from mild COVID-19.The low frequency of APLA/LAC can be explained by the mild COVID-19 disease prior to plasma donation or the rapid disappearance of autoantibodies after the acute disease. The presence of LAC in the plasma of late period CCP donors and APLA in the early period donors can be explained by the changed in SARS-CoV-2 viral variants between the two time periods (wild type alpha and non-alpha variants in the early and late periods, respectively).The age of plasma donors in early CCP period was older than the age of those who donated in late period, reflecting the fact that the alpha variant was more common in elderly people during the early pandemic or that there was a careful selection of CCP donors during the course of the pandemic. The fact that convalescent donors had no thromboembolic disease at the time of plasma collection and the low prevalence of APLA/LAC in this cohort, reassures the safety of CCP use.

P1623: ROLE OF HLA GENES IN DEVELOPMENT OF HLA-ALLOIMMUNIZATION IN BLOOD RECIPIENTS

E. Butina 1,* , F. Sherstnev 2 , A. Yovdiy 1 , O. Maksimov 3

1 immunohaematology; 2 Transfusiology; 3 Hematology, Federal State Institute of Science “Kirov research Institute of Hematology and Blood Transfusion of the Federal Medical and Biological Agency of Russia”, Kirov, Russia

Background: The main adverse effects of HLA-alloimmunization are transfusion failure and the need for additional platelet transfusions, febrile reactions, and the threat of fatal bleeding. The individual prediction of the risk of alloimmunization remains an unresolved problem. It seems relevant to study the relationship between the HLA genotype and the probability of alloimmunization of recipients as a result of blood transfusions.

Aims: Assess the genetic predisposition of recipients to the development of HLA alloimmunization.

Methods: Immunohematological and genetic studies were performed in 61 patients (22 men and 39 women) who received from 6 to 32 transfusions of blood components (median 11) during the previous treatment in a hematology clinic in 2019-2021. All blood components were leukoreduced. Patient age ranged from 3 to 78 years (median 48 years). Acute leukemia was diagnosed in 34 patients, myelodysplastic syndrome - in 10, aplastic anemia - in 8, other blood diseases - in 9. Typing of HLA DRB1, DQA1, DQB1 gene was performed by real-time polymerase chain reaction using equipment and reagents from DNA- technology (Russia). Statistical analysis was performed using the chi-square test.

Results: Based on the results of the study of antilymphocyte antibodies, patients were divided into two groups: HLA-nonimmunized (n=31) and HLA-immunized (n=30). The Panel Reactive Antibody in 3 out of 30 alloimmunized patients was 25-49%, in 12 - 50-74%, in 15 patients - 75-100%.

When analyzing the results of immunogenetic examination, statistical differences were established in the frequency of detection of HLA-alleles and haplotypes in the genotypes of HLA-alloimmunized and nonimmunized recipients (Table).

Summary/Conclusion: A genetically determined predisposition to the development of HLA-alloimmunization as a result of blood transfusions is associated with the presence of HLA-alleles DRB1*04, DQA1*03:01, DQA1*05:01 and HLA-haplotype DRB1*04-DQA1*03:01-DQB1*03:02 in the genotypes of recipients. The probability of antibody production is reduced in patients with HLA-alleles DRB1*16, DQA1*01:02, DQB1*05:02 and HLA-haplotype DRB1*16-DQA1*01:02-DQB1*05:02.

P1624: FRESH FROZEN PLASMA AND THE PRACTICAL MANAGEMENT: A COMPREHENSIVE AUDIT INCLUDING A SYNOPSIS OF CHALLENGES.

H. Fernandez Leyva 1,*

1 Haematology, NHS, Licoln, United Kingdom

Background: There is universal concern about the inappropriate use of fresh frozen plasma (FFP). Earlier investigations have indicated that FFP may be associated with high rates of inappropriate transfusion, with some studies indicating rates of up to 50% non-compliance with established guidelines. The current British Committee for Standards in Haematology (BCSH) guidelines on the use of FFP aim to reinforce the message regarding avoidance of its inappropriate use.

Aims: Audit the use of appropiate FFP in the context of the COVID pandemia.

Methods: Retrospective analysis of requests for plasma transfusion in adults, in our institution, between January/2020 and January/2022, This audit was undertaken to determine current use of FFP and to see if any improvements in the use of this component in relation to recommended guidance. May be we use more FFP due to covid situation. The data was obtained by consulting the Integrated System of Blood Banks.

Results: During the study period, 98 units of FFP were used for “acute” episodes including management of acute COVID complications. Only 67% of these transfusion episodes were deemed appropriate, based on the BSH criteria. This also means that 13 units of FFP may have been wasted. FFP is used by both medical and surgical specialties with general surgery, general medicine and neurosurgery being the main users.

During the study period, 98 units of FFP were used for “acute” episodes including management of acute COVID complications. Only 67% of these transfusion episodes were deemed appropriate, based on the BSH criteria. This also means that 13 units of FFP may have been wasted. FFP is used by both medical and surgical specialties with general surgery, general medicine and neurosurgery being the main users.

159 requests for plasma were registered in the analyzed period. In most cases (98), the reason was hemorrhage due to multiple factor deficiencies, including liver disease, trauma, lung disease or a massive transfusion. Prophylactic/therapeutic replacement of congenital factor XI deficiency, associated with bleeding episodes, was indicated in 2 situations. In 1 case, the indication was treatment of HUS and 2 cases of HELLP syndrome. In 7 cases, there was an overlapping of indications. In 7 cases, plasma was administered in unforeseen situations, such as for the correction of laboratory abnormalities without clinical manifestations (4 cases) or for prophylaxis of invasive techniques without abnormal coagulation tests (2 cases).

Summary/Conclusion: The proportion of inappropriate requests is similar between surgical and non-surgical specialties and also when broken down into individual departments, the number of inappropriate requests consistently outnumbers appropriate requests across all departments. FFP continues to be frequently used in the absence of bleeding and / or evidence of abnormal coagulation, thus highlights the need for concerted efforts in the education of clinicians who prescribe FFP. In the analysis of the indications for plasma prescription, it is concluded that in 17 % cases the indication was not complete adequately or justifable. 7% of cases given FFP had no record of post-transfusion coagulation data. Following this work, dissemination coupled with education was undertaken. The use of FFP for Warfarin reversal has correctly indicated (> 95%). it may suggest that an education programmes directed at specific aspects of practice do influence clinical behaviour over needs to be addressed.

P1625: STATE OF CERTIFICATION IN TRANSFUSION SERVICES IN ANDALUSIA. LOW RATE OF CERTIFICATION IN COMMUNITY HOSPITALS

J. R. Garcia Fernandez 1,*

1 Hematology, Hospital de Baza, Baza, Spain

Background: A quality management system certified bya an externel entity allows the development of a safe and efficient transfusion activity, while contributing to the involvement of all hospital staff to improving a continous quality system. In Spain there are two entities accredited by ENAC for certification in transfusion: Transfusion Accreditation Commitee (CAT) and the Agency for Health Quality of Andalusia (ACSA), although the latter with a specific program limited to transfusion centers not available for hospital practice.

Aims: Describe the situation related to quality management system in transfusion medicine in Andalusia (a región in the south of Spain) in 2021.

Methods: On the basis of data published by the CAT and ACSA we exposed the number of certified centers in Andalusia compared with to national data.

Results: .-Although almost half transfusion centers in Andalusia are certified by CAT, 100 % of them are validated by the ACSA specific program. 2.- 29 % of centers accredited in Spain are accredited in Andalusia. 3.-The percentage of accreditation is related to the type of centers: 100 % in cord blood banks and transfusion centers; 75 % in big hospitals and only 12 % in little community hospitals. 4.-The relationship with the referral hospital and transfusion center seems to be the most important predisposing factor to maintain a certification program. Related to the certification program in the form of mangement, it´s not fully adapted to “little” hospitals, because if it´s true that there is a hospital responsable for haemovigilance, hemotherapy isn´t an exclusive dedication for him.

Summary/Conclusion: The implementation of a management system certified quality by an external entity is a clear need in all transfusion centers, although the percentage of certified centers is less tahn 40 %, represented a 29 % of the certified centers nationwide. 2. Certification of transfusion services by CAT is the only feasible option, but its adaptation to “little” community hospitals is so difficult which is evident in view of the low number of certified centers. 3.Continuous quality improvement represents an additional workload sometimes unacceptable, it would be required a limited version adapted to general hospitals with low rate of transfusion and no specific staff transfusion related.

P1626: MANAGEMENT OF POST-TRANSFUSION COMPLICATIONS IN PATIENTS WITH HEMATOLOGICAL DISEASES. PREVENTION IS BETTER THAN CURE–DATA FROM A SINGLE INSTITUTION

Y. Lazarova 1,* , G. Balatzenko 1 , E. Aleksov 2 , M. Stoyanova 2 , K. Venkov 2 , K. Milcheva 2 , G. Arnaudov 2 , M. Donchev 2 , K. Ignatova 2 , B. Spassov 2

1 Laboratory of Transfusion Hematology; 2 Clinical Hematology, Specialized Hospital for Active Therapy of Hematological Diseases, Sofia, Bulgaria

Background: Post-transfusion complications (PTC) include acute (AHTR) and delayed (DHTR) hemolytic reaction, chill-rigor reaction, febrile non-hemolytic reaction (FNHR), Transfusion-related acute lung injury (TRALI), transfusion-associated circulatory overload (TACO), allergic (ATRs), anaphylactic reactions– are the risk of transfusion support. According to American Society of Hematology the frequency of adverse effects of transfusion is approximately: AHTR–1:76,000, FNHR–0,1–1%, ATRs–1–3%, anaphylactic reactions–1:20, 000–50, 000, TRALI–1:10, 000, TACO-1%. The safety and effectiveness of transfusion medicine, foremost in treatment in oncohematology, undoubtedly depends equally on the quality of transfusion therapy and the chosen specificity in transfusion approach in different hematological diseases.

Aims: Analyzing the frequency of PTC in pts with hematological diseases–chill-rigor reaction, AHTR, DHTR, FNHR, TACO, TRALI, ATRs, anaphylactic shock, bacterial sepsis.

Methods: Routine manual technique and column-agglutination technique for blood typing, screening and identification of anti-erythrocyte antibodies (agglutination, enzyme method, direct and indirect antiglobulin test), in vitro compatibility tests on all methods and techniques as necessary.

Materials : The number of all transfused pts and units of blood components in the Specialized Hospital for Active Therapy of Hematological Diseases for the 2020– 2021 period were: pts–2,521 adults (665 transfused only with red cell concentrates–RCCs,98 of whom with Beta Thalassemia); all blood components(units)–20,040, of which –9,743 RCCs, 3,084 platelet concentrates by apheresis (PCAP), 5,090 PC from whole blood(PCWB), 2,099 fresh frozen plasma(FFP), (severe clinical cases with post-transfusion complications–delayed hemolytic transfusion reactions, anaphylactic reactions–will be outlined), alloimmunization from RCCs–26 pts.

Results: Complications of transfusion were found in 80cases in total (0.4% of all transfused blood units; 3.2% of all transfused pts). The frequency of complications within the 80 total cases was as follows–AHTR–0, DHTR–1(1.25%), chill-rigor reactions–19(23.75%), FNHR–13(16.25%), ATRs–20(25%), anaphylactic shock–2(2.5%), TRALI–0, TACO–0, septic reaction–0. The frequency of post-transfusion reactions of transfused blood components was respectively: chill-rigor reactions in RCCs–9(0.09%), in FFP–3(0.14%), in PC–7(0.09%); FNHR in RCCs–10(0.1%), in PC–2(0.02%); ATRs in PC–19(0.23%), in FFP–1(0.05%); anaphylactic reactions in PCAP–2(0.06%). In 59 AIHA treated in our center, 29(49.2%) transfused with RCCs and 14(23.7%) alloimmunizated–the frequency of specificity was: anti-E (42.9%), anti–E+ c (14.3%), anti–Kell (21.4%), anti–C w , Jk a , Le a (7.1% vs 7.1% vs 7.1% respectively). Post-transfusion hemolytic reactions were not observed.

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Summary/Conclusion: In this analysis from our center the frequency of PTC was the same as the world frequency reported. Fatal post-transfusion reaction or acute hemolytic reaction was not observed. A longer period of research is needed to determine the frequency of TACO and TRALI.

P1627: EXPERIENCE OF THE USE OF CONVALESCENT PLASMA FOR THE MANAGEMENT OF COVID 19 IN PATIENTS WHO REQUIRED HOSPITALIZATION IN QUITO-ECUADOR, HOSPITAL METROPOLITANO, DURING THE 2020 PERIOD

J. Rojas 1,* , H. Jaramillo 1 , F. Avalos 1 , P. Velasco 1

1 Hematology, Hospital Metropolitano, Quito, Ecuador

Background: Convalescent plasma as a therapeutic alternative in COVID-19 could provide passive immunity creating neutralizing antibodies, potentially decreasing viral entry into host cells; however, the exact mechanism of viral elimination in COVID 19 is not yet known and the clinical data are not conclusive. Ecuador is a low-income country so access to Tocilizumab was delayed, that is why the goverment approved the use of convalescent plasma on april 2020 as an alternative of therapy specially with critically ill patients.

Aims: Describe our experiencua of the use of convalescent plasma for the management of COVID-19 in patients who required hospitalization in Hospital Metropolitano in Quito-Ecuador.

Methods: This is a retrospective, descriptive study based on patients data diagnosed with COVID- 19 in Quito-Ecuador in Hospital Metropolitano who required general hospitalization and/or intensive care unit; each of them received convalescent plasma from patients with previous infection. Sociodemographic characteristics, use of plasma in general hospitalization and intensive care unit, adverse effects and mortality were included in the analysis.

Results: During april 15 th and december 31 st 2020, 60 patients received convalescent plasma. 42 of them were male and 18 females, with an age range between 30-65 years old. Just 5 of them died during hospitalization.

At the time they received plasma, 25% of the patients (15) were anticoagulated and half of them were D-dimer positive. In relation to the PAFI (ratio of arterial oxygen pressure/inspired fraction of oxygen), the ranges vary with minimum values of 70 and maximum 240, with an average of 187 millimeters of mercury.

17 patients received plasma during their stay in the Intensive Care Unit (ICU), and the remaining 43 approximately on their third day of general hospitalization.

Finally, 40% presented severity criteria at the time of receiving plasma, these criteria correspond to lymphopenia less than 800 K/uL, C reactive protein (CRP) more than 100 mg/L and ferritin greater than 500 ng/ml.

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Summary/Conclusion: The use of convalescent plasma does not allow us to demonstrate it as a curative therapy and not even show a decrease in severity in patients. It is no posible to prove the efficacy of this therapy, but we can conclude with our data, that there is a safe alternative with no transfusions reactions, and a death rate of 12%. Randomized trial data are still needed to optimize the convalescent plasma indications.

P1628: ALLOIMMUNIZATION DURING ASSISTED REPRODUCTIVE TECNOLOGY: CASE REPORT OF A PREGNANCY WITH IRREGULAR ANTIBODIES PROBABILY RELATED TO EGG DONOR ANTIGEN.

M. Rolli 1,* , V. Lara 1 , L. Bensi 1 , P. Artusi 1 , M. Savarino 1 , G. B. Ceccherelli 1 , D. Venturelli 1

1 Immuno-Hematology and Transfusion Medicine Unit, Azienda Ospedaliero-Universitaria di Modena and University of Modena and Reggio Emilia, Modena, Italy

Background: Alloimmunization is a well known complication during pregnancy. It consists in an immune response of the mother against non-self antigens expressed by the fetus, which derive from the male partner, and its manifestation is an hemolytic disease of the fetus and newborn (HDFN).

The most common antigen involved in alloimmunization came from the AB0 group and affect 15% to 25% of all pregnancies. However, the response caused by AB0 incompatibly is modest most likely due to the expression of AB0 blood type antigen in several organs.

Other red cell antigens have been implicated in the HDFN; the Rh(D) antigen is the most immunogenic and the main cause of HDFN because the antigen is well expressed on the surface of the erythrocytes in the fetus.

Previously, HDFN was known to cause fetal death in 1% of all pregnancies; with the advent of immunoprophylactic therapies, HDFN is currently well managed with fewer complications.

In Italy, RBC iantibody screenings are typically performed at first and third trimester of pregnancy. Affected women are followed up to ensure the timely identification of those antibodies who require therapeutic interventions to prevent perinatal morbidity and mortality.

The italian guidelines aim to direct these women to third-level centers where management protocols are shared by Immuno-hematology and Obstetrics Units in order to monitor the pregnancy with laboratory tests and clinical interventions.

Aims: Identification of risk factor for alloimmunization during pregnancy

Methods: Blood group, undirect and direct antiglobulin test during prregnancy mounthly based.

After delivery blood group and DAT on Newborn

Results: We report a case of alloimmunization during pregnancy of a woman that underwent a medically assisted procreation with egg donation.

The subject, in apparently good health, had no chronic pathologies and was not taking medications. Her blood group was B Neg ccdee kk and the partner was 0 Pos CcDee Kk

During the last triemster of the pregnancy, the woman developed alloimmunization towards many antigens of different blood systems,Rh (anti-D, antiC, anti-E), and the Kell (anti-Kell) systems. at low titers

At week 37 of the pregnancy, she was admitted to the hospital for obstetrical problems and delivery was induced without clinical complications, neither for her nor for the fetus. Newborn blood group was 0 Pos CcDee Kk and Direct antiglobulin test was positive (++++). Anti-RHD and Kell antibodies were identified after elution.

Additional studies are underway to characterize the egg donor RBC Phenotype to verify the presence of E antigen

Summary/Conclusion: Alloimmunization during pregnancy is a condition caused by a response of the mother to paternal antigenes exhibited by the fetus and can be monitored with standard laboratory and clinical approaches

However, the possibility to carry out pregnancies with egg donor that has a different RBC antigen profile of the mother open a new immuno-hematological scenarios previously not pointed. This could lead to potentially increased risk for the mother and the newborn.

P1629: ADMINISTRATION OF CONVALESCENT PLASMA FOR THE TREATMENT OF SEVERE COVID-19: RESULTS OF A MULTICENTER PHASE II TRIAL

T. Thomopoulos 1,* , A. Bouchla 1 , A. Antoniadou 2 , E. Terpos 3 , M. Politou 4 , K. Stamoulis 5 , E. Koromboki 3 , S. Papageorgiou 1 , A. Kotanidou 6 , I. Kalomenidis 6 , E. Jahaj 6 , S. Grigoropoulou 2 , M. Pagoni 7 , E. Grouzi 8 , G. Poulakou 9 , I. Trontzas 9 , S. Labropoulou 10 , A. Mentis 10 , A. Bamias 1 , S. Tsiodras 2 , M.-A. Dimopoulos 3 , V. Pappa 1

1 Hematology Unit, Second Propaedeutic Department of Internal Medicine and Research Institute, School of Medicine National and Kapodistrian University of Athens, University General Hospital “Attikon”; 2 Fourth Department of Internal Medicine, University General Hospital “Attikon”, School of Medicine, National and Kapodistrian University of Athens; 3 Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens; 4 Hematology Laboratory-Blood Bank, Aretaieion Hospital, School of Medicine, National and Kapodistrian University of Athens; 5 Hellenic National Blood Transfusion Center; 6 First Department of Critical Care Medicine and Pulmonary Services, Evangelismos General Hospital, National and Kapodistrian University of Athens; 7 Haematology-Lymphomas Department and BMT Unit, Evangelismos Hospital; 8 Transfusion Service and Clinical Hemostasis of Saint Savvas, Oncology Hospital of Athens; 9 3rd Department of Internal Medicine, Sotiria General Hospital, School of Medicine, National and Kapodistrian University of Athens; 10 National Influenza Reference Laboratory of Southern Greece, Hellenic Pasteur Institute, Athens, Greece

Background: COVID-19 is an ongoing pandemic with high morbidity and mortality. Despite meticulous research, optimal management of patients with severe COVID-19 remains a subject of vigorous debate. On this ground, several studies have assessed the efficacy and safety of convalescent plasma (CP) administration for the management of COVID-19; however, their results have been conflicting. A recent well-designed meta-analysis has highlighted the effectiveness of CP 1 , albeit other meta-analyses have demonstrated no survival benefit. Nonetheless, a large retrospective study of more than 3000 patients have shown that higher-titer CP administration provides a survival benefit for patients with COVID-19 2 .

Aims: The aim of this multicenter phase II study ( NCT04408209 ), conducted at five tertiary hospital of Athens, Greece, was to evaluate the safety and efficacy of CP transfusion in patients with severe COVID-19.

Methods: Patients with severe COVID-19 (WHO grade ≥4), confirmed by RT-PCR on nasopharyngeal swabs, were enrolled in the study and received three doses of single-donor CP on days 1, 3 and 5. Donors were eligible if they tested positive for anti-SARS-Cov-2 antibodies determined by the Euroimmun Assay and were symptom free and PCR negative for the virus. All participants were followed for at least 28 days. Clinical and laboratory parameters as well as antibody titers against S1 domain of SARS-CoV-2 were determined in the recipients on days 1–7 and on days 14, 21 and 28. Primary endpoint of the study was survival on day 28. Secondary endpoints included duration of hospital stay, duration of mechanical ventilation and oxygen support, time to PCR negativity and safety.

Results: As of the end of March 2021, 106 patients with severe COVID-19 were enrolled and received CP at a median of 7 days from symptoms onset. Notably, 79.2% of patients received high-titer CP (anti-S1 IgG ≥3.5). The median age of patients was 61 years and 56.6% of them were male. Median SOFA score of patients was 4. At baseline, 82.3% of patients needed oxygen support ranging from nasal cannula to mechanical ventilation in 4.8% of patients. During a median follow-up of 25 days, 23 patients needed mechanical ventilation and 16 patients were discharged from Intensive Care Unit (ICU) at a median of 12 days. Median duration of oxygen support and total hospital stay for the entire cohort was 6 and 10 days respectively. 67.7% of participants achieved negative PCR result at a median of 14 days. Mortality at day 28 and end of follow-up was 3.3% and 6.6% respectively. Serious adverse events emerged only in one patient as hypoxemia after first CP infusion that although easily alleviated by conventional measures perturbated further CP administration. No association between anti-SARS CoV-2 anti-S1 antibody titer and survival or clinical status at any time-point or duration of hospital stay was noted; however, administration of CP with anti-S1 IgG above the 80 th percentile was predictive of shorter duration of mechanical ventilation (HR: 3.36, 95%CI: 1.07-10.5, p: 0.04). The corresponding Kaplan-Meier curve is provided in Figure 1. The finding remained significant in a multivariate Cox regression analysis including age, sex, baseline SOFA score, and baseline percentage of pulmonary infiltrates in CT imaging.

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Summary/Conclusion: CP can be administered safely to patients with severe COVID-19 without severe adverse events. Early administration of high-titer CP might be associated with an increased probability of earlier weaning from mechanical ventilation of intubated patients with COVID-19. Validation of this finding in larger cohorts is needed.

P1630: WEARABLES IN TRANSFUSION MEDICINE: CHALLENGES AND FUTURE PERSPECTIVE

R. Tonino 1,2,* , M. Schipperus 2,3,4 , J. J. Zwaginga 1,2,4

1 Hematology, LUMC; 2 Research, TRIP, Leiden; 3 Hematology, UMCG, Groningen; 4 CCTR, Sanquin, Amsterdam, Netherlands

Background: The digital era has granted us new techniques like photoplethysmogram or ECG-based wearable devices, that can remotely monitor physiological parameters of patients, including heart rate and activity measures continuously. These developments yield many opportunities but also great challenges. For instance, in the Myelodysplastic syndrome (MDS), most patients will become RBC transfusion dependent during the course of their disease. It has been well established that Hb-level alone is an insufficient marker to guide in RBC transfusions. Nonetheless, we base transfusion regiments on Hb-thresholds combined with a subjective interpretations of the patient’s quality of life. This leads to great variability in transfusion strategies causing patients to often be over- or undertransfused. We therefore need objective parameters to evaluate and guide our transfusion strategies.

Aims: We intend to share our practical knowledge of the encountered challenges.

Methods: Following pilots with the Withings Steel HR and VitalPatch (Table 1.), we have started the REMOTE 2 trial (NTR: NL9289) and are collaborating internationally to initiate larger trials. Here we have gathered the experiences we deem important for researchers new to the field.

Results: When choosing the device, first establish the desired outcomes. Is a true continuous heart rate collection necessary, or will a semi-continuous measurement suffice? Within an elderly MDS population a more manageable device with a long battery-life can help reduce data missingness. On the other hand, with only daily averages of parameters, a deeper understanding of interaction of the collected parameters is challenging. Also, product placement will impact the outcome, as wrist-worn devices are more susceptible to noise than chest-worn devices. Importantly, inter-device variability has been reported to be high. Therefore, it is strongly preferable to use one device only in a study.

Many wearable biosensors are not medically validated (yet). We believe, however, this should not discourage the investigator to use such a device. Unvalidated continuous heart rate measurements have been shown to produce very accurate data. And importantly, there are currently no validated – or unvalidated – parameters to evaluate these patients.

Physiological parameters are highly impacted by numerous factors like weather and daily schedules. Large sample sizes should reduce this natural variability, but trials involving MDS-patients have thus far been unable to collect more than 38 patients in one study. Combining groups, like thalassemia and myelofibrosis, will increase the sample size. However, subgroup analysis will still lack power. Therefore, international collaborations are necessary to acquire sufficient sample sizes to answer questions like: What parameters are of value to evaluate transfusion strategies? How do these parameters respond to different 1) amounts of RBCs; 2) thresholds; and 3) timing of transfusion? After answering these first questions, we can look at how these parameters change with longer-standing changes, and how deleterious the fluctuations in Hbmass are to the patient.

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Summary/Conclusion: Depending on the research context, intervention and the desired outcomes, one should carefully choose the required device. While various devices may mork, the trade-off between ease of use and data granularity can make a substantial difference when analysing the data. For sufficient sample sizes, international collaborations are required to answer the many questions in order to improve the care of chronic transfusion dependent patients.

P1631: PLATELET DYSFUNCTION DISORDERS; EMERGING COMPLICATIONS OF INFLAMMATORY BOWEL DISEASES

A. Abomhya 1,* , L. Attal 1 , R. Garcia Sturgill 1 , A. Frondella 2

1 Internal Medicine, The Brooklyn Hospital Center, Brooklyn, United States of America; 2 St. George’s University School of Medicine, West Indies, Grenada

Background: Apart from platelets’ role in homeostasis, they have pro-inflammatory functions and play a significant role in mucosal healing. Inflammatory bowel diseases (IBD) have been associated with Qualitative and quantitative platelet disorders.

Aims: While reactive thrombocytosis is an established feature of IBD flares, few studies investigated the association between IBD and platelet dysfunction disorders. We aimed to estimate the prevalence of platelet dysfunction disorders in patients with IBD. Secondary outcomes included mortality, length of stay, all-cause 30-day non-elective readmission rate, and total cost of hospitalization.

Methods: This is a retrospective cohort study. We queried the Nationwide Readmission Databases 2016-2018 using ICD-10-CM codes to identify all adult patients admitted for IBD. Patients with missing information and duplicate admission records were excluded. Patients with a comorbid diagnosis of one of the following platelet dysfunction disorders (qualitative platelet defects, Purpura, primary or secondary thrombocytopenia, and unspecified thrombocytopenia) were identified. The case selection process is summarized in Figure 1. Median and IQR were used to describe Continuous variables, and proportions were used with categorical variables. Before comparing IBD patients with and without platelet dysfunction disorders, we first excluded those with Thrombocythemia. Comparison between groups was performed by Mann Whitney test for continuous variables and Chi-Square test for Categorical variables. Multivariate regression analysis was performed to study the impact of platelet dysfunction disorders on inpatient mortality and non-elective readmissions. Statistical analyses were performed using SPSS Version 25 (IBM Corporation, Armonk, NY, USA).

Results: We extracted 365,152 index hospitalization records for IBD, 15,434 (4.3%) had a platelet dysfunction disorder. Platelet dysfunction disorders identified included Allergic purpura (n=85), Qualitative platelet defect (n=62), Immune thrombocytopenic purpura (n=755), other nonthrombocytopenic purpura (n=241), other primary thrombocytopenia (n=45), secondary thrombocytopenia (n=2,541) and unspecified thrombocytopenia (n=11,943). IBD patients with platelet dysfunction disorders were older (64; Interquartile Range (IQR): 51-75 vs. 56; IQR: 38-70, P <0.001), had increased length of stays in days (5; Interquartile Range (IQR): 3-10 vs. 3; IQR: 2-6, P <0.001), increased median total charges ($52,580; IQR: $26,101 - $119,629 vs. $31,965; IQR: $17,580 - $60,540, P <0.001), were more common to have lower GI bleeding (9.2% vs. 8.2%, P <0.001) and more common to require a blood transfusion during hospitalization (15% vs. 5.4%, P <0.001) compared to IBD patients without platelet dysfunction disorders, respectively. On multivariate analysis; having a platelet dysfunction disorder was associated with higher odds of inpatient mortality (Odds ratio (OR): 2.97; 95% confidence interval (CI): 2.75-3.2, P <0.001). and higher odds of all-cause 30-day non-elective readmission (OR: 1.19; 95% CI: 1.12-1.26, P <0.001).

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Summary/Conclusion: Platelet dysfunction disorders are emerging extraintestinal manifestations of IBD and are associated with worse hospitalization outcomes in this patient population. Revealing the burden of platelet disorders in patients with IBD might incite new research studies to investigate the implications of platelet disorders in IBD outcomes and potential novel targets for therapy.

P1632: PROSPECTIVE OBSERVATIONAL STUDY IN PATIENTS WITH IDIOPATHIC THROMBOCYTOPENIC PURPURA (ITP) TREATED WITH TPO-R AGONISTS (TPO-RA): ELTROMBOPAG (EPAG) AND ROMIPLOSTIM (ROMI) - THE PEPITE STUDY

S. Chèze 1,* , P. Quittet 2 , D. Adoue 3 , J.-F. Viallard 4 , P. Sève 5 , B. Bonnotte 6 , K. Laribi 7 , S. Tardy 8 , H. Henique 9 , M. Hamidou 10 , M. Hacini 11 , A. Santagostino 12 , S. Leclerc-Teffahi 13 , M. Aroichane 14 , A. Filipovics 15 , F. Brini 16 , H. Velasquez Giron 17 , A. Malatesta 18 , M. Michel 19

1 Institut d’Hematologie de Basse-Normandie, CHU de Caen, Caen; 2 Hematologie clinique, CHRU Montpellier, Montpellier; 3 Service de médecine interne, IUCT Oncopôle, Toulouse; 4 Médecine interne, Hôpital Haut-Lévêque, Pessac; 5 Service de médecine interne, Hôpital de la croix rousse, Lyon; 6 Médecine interne, CHU de Dijon, Dijon; 7 Hématologie clinique, CH Le Mans, Le Mans; 8 Service d’hématologie clinique, Centre Hospitalier Annecy Genevois, Annecy; 9 Service de médecine interne, CHRU Bretonneau, Tours; 10 Médecine interne, CHU de Nantes, Nantes; 11 Hematologie, Centre Hospitalier Métropole Savoie, Chambéry; 12 Hematologie, CH de Troyes, Troyes; 13 Médical hématologie, Novartis pharma, Rueil-Malmaison; 14 Médical hématologie, Novartis; 15 Statistiques; 16 Pharmacovigilance, Novartis pharma, Rueil-Malmaison Cedex; 17 RCTs Lyon, Lyon, France; 18 Statistiques, RCTs Lyon, Lyon; 19 Service de médecine interne, Hôpital Henri Mondor, Paris, France

Background: Initially, the marketing authorization (MA) of EPAG and ROMI was to adult patients (p.) with ITP ≥12 months (m.) and refractory to other treatments (t.), splenectomized or contraindicated to splenectomy. The MA was extended for EPAG in 2019 to p. aged ≥1 year with ITP ≥6 m., refractory to other t. (corticoids (CS), IgIV). In 2017, French national guidelines suggested the use of TPO-RA as an option of t. in 2 nd line (L).

Aims: The PEPITE study, still ongoing, aims to assess the modalities of use, effectiveness and safety of TPO-RAs in real-life.

Methods: Prospective, observational, multicenter study including adult p. who initiated TPO-RA with persistent (pITP) or chronic (cITP) ITP. Inclusions occurred between 12/21/2018 and 07/17/2020. Here’s the interim analysis, cut-off date: 03/22/2021. Characteristics at baseline were presented in 114 p. (analyzed pop). Efficacy analysis of TPO-RA was assessed in p. with a platelet count (PLAT) <100 G/L at TPO-RA initiation (efficacy pop 113 p.). Responses were defined as: response (R) = PLAT ≥30 G/L, complete response (CR) = PLAT ≥100 G/L and non-response (NR) = PLAT <30 G/L.

Results: 123 p. included through 40 centers by 25 hematologists and 15 internists, and 77 p. were still on TPO-RA at 6 m.

At baseline, mean age 62.7 ± 20.1 years, 55% men, 29% with at least 1 cardiovascular risk factor. At diagnosis: median PLAT = 26 G/L [0 to 134 G/L], 31% of p. with bleedings.

97% of p. received at least one L of t. before TPO-RA: CS 96%, IVIG 56%, rituximab 47%, dapsone 18%, hydroxychloroquine 11%, danazol 6% and 7% of p. were splenectomized. Median number L of t. = 2 and 8% of p. had more than 4 L. Median time between diagnosis and TPO-RA initiation was 2.6 years [0.3 to 49.3 years], 33% of p. with pITP (n=21 with ITP 3 -<6 months, n=16 with ITP 6 - <12 months) and 67% with cITP.

At TPO-RA initiation: 9% of p. were on CS and 48% p. had PLAT <30 G/L (median PLAT = 30 G/L), 95 p. (83%) received EPAG and 19 p. (17%) ROMI.

For the 77 p. still on TPO-RA at 6 m., R rate = 97% and CR = 60%. Within 6 m., 10 p. had permanently (perm.) discontinued TPO-RA, main causes were therapeutic effect deemed sufficient (TEDS) for 6 p. and NR for 2 p.

For the 27 p. still treated with TPO-RA at 18 m., R rate = 93% and CR = 48%. Within 18 m., 12 p. had perm. stopped TPO-RA, including 7 p. for TEDS and 1 p. NR.

P. initiated TPO-RA with ITP 3 -<6 months (N = 21), 9 (43%) p. were still on TPO-RA at 6 months, 5 (56%) in CR.

Over the entire follow-up, 24p. (21%) perm. discontinued TPO-RA, main causes were TEDS for 9 p., adverse event (AE) for 5 p. and absence of R for 4 p.

Of the 105 p. treated with EPAG at least once, 62 (59%) experienced at least one AE, and 26 SAE occurred in 17 p. The most common AEs were respectively 6% for headache and 3% for SARS-CoV-2 infections, diarrhea, asthenia, insomnia, arthralgia and alopecia. Of the 40 p. treated with ROMI at least once, 19 (48%) experienced at least one AE and 17 SAEs occurred in 10 p. The most common AEs: SARS-CoV-2 infections (5%) and arthralgias (5%). No deaths related to TPO-RA was reported.

Summary/Conclusion: Preliminary data from the PEPITE study show that TPO-RA are prescribed in early ITP, including 33% with pITP (18% with ITP 3-<6 m.) and are used in 7% of cases after splenectomy. At 6 m. R on t. was 97% and CR on t. was 60%. Within 6 m., 6 p. had perm. stopped TPO-RA due to TEDS. The real-life effectiveness and safety data for EPAG and ROMI are consistent with data reported in extension studies, with the specificity of occurrence of SARS-CoV-2. The final analysis is scheduled after 24 m. follow-up

P1633: LOW SERUM BDNF LEVELS ARE ASSOCIATED WITH LOWER PLATELETS’ CD62P REACTIVITY AND INCREASED BLEEDING TENDENCY IN PATIENTS WITH GAUCHER DISEASE

D. Azoulay 1,2,* , M. Naamad 3 , D. Frydman 4 , E. Broide 3 , A. Zimran 4 , G. Stemer 1,2 , S. Revel-Vilk 4,5,6

1 Hematology, Galilee Medical Center, Nahariya; 2 Azrieli Faculty of Medicine, Zefat; 3 Flow Cytometry Unit; 4 Gaucher Unit; 5 Pediatric Hematology/Oncology unit, Shaare Zedek Medical Center; 6 Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

Background: Recent studies suggest possible coagulative role for Brain Derived Neurotrophic Factor (BDNF), a neuronal growth factor that is stored in alpha granules of circulating platelets. Bleeding tendency, a prominent feature of patients with Gaucher disease (GD) have been shown to be associated with abnormal platelet function.

Aims: We studied the serum BDNF levels in patients with GD and their correlation with platelets’ function and bleeding tendency.

Methods: Platelet function was analyzed in 50 patients with type I GD (F:M [%] 50:50, age [mean ± SD, range] 42.67 ± 17.92 years, 8.94 - 86.55) by an established assay designed in Shaare Zedek Medical Center, using unstimulated and stimulated [TRAP (1.5 or 20 µM), ADP (0.5 or 20 µM), ADP+Epinephrine (0.5 + 0.1 µM) and Collagen (0.25µg/mL)] flow cytometry measurement of platelet surface-activated expression of αIIbβ3 integrin (PAC1), P-selectin (CD62P), and lysosomal-associated membrane protein (LAMP3/CD63). The response to each stimulant was calculated by the delta between the unstimulated and stimulated surface-activated expression level in paired samples from the same individual patient. Total serum BDNF level was quantified using the commercial DuoSet ELISA Development System kit (R&D System DY278). Patients were divided by a high and low BDNF levels according to the normal distribution of BDNF using cutoffs points that are above the upper 25 quartiles and below the lower 25 quartiles of BDNF level. Bleeding history was conducted by bleeding tendency questioner

Results: Serum BDNF levels were normally distributed in GD patients ( p 0.17) and showed with similar mean and range with those of healthy controls (BDNF [mean, range] 10.72 ng/ml, 3.2-18 vs. 10.3, 1.34-25.1 in GD and healthy controls, respectively). No correlation between BDNF and Glucosylsphingosine (Lyso-Gb1) levels was detected. We found a positive correlation between BDNF levels and platelets count (r=0.39 p=0.005) and with unstimulated, Epinephrine+ADP stimulated and Collagen stimulated CD62P expression in platelets (r=0.322, p=0.02; r=0.30, p=0.03 and r=0.29, p=0.04, respectively). Patients with high serum BDNF levels (i.e. > 14 ng/ml) showed significantly higher CD62P response to TRAP, ADP+Epinephrine and Collagen as compared to patients with low serum BDNF levels (i.e.<7.8 ng/ml) (mean response in patients with high vs. low BDNF levels; 76.57 vs. 65.45, p=0.02; 33.16 vs. 18.6, p=0.04; and 80.38 vs. 70.5, p=0.03 respectively). No correlation between BDNF and expression or response in PAC1 and CD63 was observed. Patients with more than one bleeding manifestation were shown with lower BDNF levels (serum BDNF ng/ml mean ± SD in >1 vs. ≤ 1 positive bleeding history; 7.66 ± 3.21 vs. 10.98 ± 4.0 p=0.03), albeit similar platelets numbers (171 ± 97.56 vs. 183 ± 57.85, p=N.S).

Summary/Conclusion: These preliminary observations demonstrate the first link between low BDNF and bleeding tendency in GD. The association between low serum BDNF levels, CD62P mediated platelets dysfunction and increased bleeding manifestation should be further studied in patients with GD as well as in other settings.

P1634: EFFICACY AND SAFETY OF ELTROMBOPAG IN THE TREATMENT OF CHRONIC HEPATITIS B VIRUS INFECTION PATIENTS WITH THROMBOCYTOPENIA（HBV-TP）: A PROSPECTIVE, MULTI-CENTER, SINGLE ARM, PHASE II TRAIL

Y. Chen 1,* , Y. Huang 2 , Y. Lai 3 , J. Peng 4 , Z. Zhou 5 , H. Zhou 6 , R. Zhou 7 , R. Yang 1 , L. Zhang 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin; 2 The First Affiliated Hospital of Xiamen University, Xiamen; 3 The Second Affiliated Hospital of Guangxi Medical University, Nannin; 4 Qilu Hospital of Shandong University, JInan; 5 The Second Affiliated Hospital of Kunming Medical University, Kunmin; 6 Henan Cancer Hospital, Zhengzhou; 7 The Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjin, China

Background: The standard treatment of chronic hepatitis B virus infection patients with thrombocytopenia (HBV-TP) is not well established. Long-term glucocorticoid therapy and rituximab therapy could probably activate hepatitis virus replication and aggravate the disease. Thrombopoietin-receptor agoinst such as eltrombopag shows good efficacy and safety profile in immune thrombocytopenia（ITP） without immunosuppression, while the data of eltrombopag in HBV-TP is limited.

Aims: To evaluate the efficacy and safety of eltrombopag in the treatment of chronic hepatitis B virus infection patients with thrombocytopenia.

Methods: This is a prospective multicenter single-arm Phase 2 study with two stages. Adult HBV-TP patients with baseline platelets count ＜30x10 9 /L，who were not accompanied by decompensated manifestations such as cirrhosis and splenomegaly were eligible for recruitment. By the end of stage 1（week1-6）, patients who responded to eltrombopag could enter a 16-week prolonged stage (stage 2) to evaluate the long-term efficacy and safety. The initial daily dose of eltrombopag is 25 mg and could be adjusted to 75 mg as maximum to maintain platelet counts between 50 × 10 9 /L and 150 × 10 9 /L. Patients were assessed for platelet counts weekly in Stage1 and every 4 weeks in the Stage2. Response to treatment was defined as a platelet count of ≥ 50 × 10 9 /L, and complete response was defined as platelet count ≥ 100 × 10 9 /L. The primary endpoint is the proportion of responders at the end of week 6. Subgroup analysis was performed by disease duration (newly diagnosed (0-3 months), persistent (> 3-12 months), or chronic (> 12 months)).

Results: By this data cut-off before December 31,2021, 43 patients were screened and 38 patients were recruited. Among them 18 patients were male and 20 were female, the median age was 47(20-67) years old and the median disease duration was 6.5(1-360) months. The median baseline platelet count was 16.5(3-29) × 10 9 /L, with newly diagnosed group 14.5(8-27) × 10 9 /L(n=6), persistent group 13(3-29) × 10 9 /L(n=15), chronic group 19(4-27) × 10 9 /L(n=17), respectively. The response rate and complete response rate was 70.3% and 37.8% at week 6 after eltrombopag treatment within 37 patients. The response rate and complete response rate in newly diagnosed group(n=6), persistent group(n=14), and chronic group(n=17) was 33.3% and 33.3%, 78.6% and 28.6%, 76.5% and 47.1%, respectively. The median duration of response in Stage 2 was 18(3-21) weeks (n=28). Treatment-related AEs were mainly of CTCAE grade 1 or 2 in intensity. There were two SAEs requiring additional medical intervention or discontinuation of eltrombopag. One had myocardial infarction during the experiment, while continued with eltrombopag and antiplatelet drugs after cardiac stent implantation to complete this study. One patient stopped taking elrombopag due to obvious liver laboratory abnormalities and lower extremity venous thrombosis.

Summary/Conclusion: Eltrombopag is effective in chronic hepatitis B virus infection with thrombocytopenia, and adverse reactions are tolerable. It is necessary to monitor platelet counts and liver function during eltrombopag treatment to manage the risk of thrombosis.

P1635: REAL-WORLD DATA OF THROMBOPOIETIN RECEPTOR AGONISTS (TPO-RA) USE IN IMMUNE THROMBOCYTOPENIA (ITP)

S. Dangudubiyyam 1,* , T. Seth 1 , R. Dhawan 1 , M. Aggarwal 1 , P. Kumar 1 , J. Dass 1 , G. Viswanathan 1 , S. Tyagi 1 , M. Mahapatra 1

1 Hematology, All India Institute of Medical Sciences, New Delhi, India

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by increased platelet destruction and impaired platelet production. Over the past decade, use of thrombopoietin receptor agonists (TPO-RA) is steadily rising in ITP.

Aims: In this study, we explore the clinical profile of ITP patients on TPO-RA therapy, response rates to TPO-RA therapy, and analyze various factors that can predict outcomes in one of the largest public-sector teaching hospitals in India.

Methods: This retrospective observational study included consecutive patients diagnosed with ITP and initiated on TPO-RA therapy (Romiplostim and Eltrombopag) in our clinic from January 1, 2016, to December 31, 2021. Response was defined according to standard international criteria.

Results: Over the past six years, 166 patients were initiated on TPO-RA therapy and had a total of 183 treatments (92 eltrombopag, 74 romiplostim, and 17 trailed on both). The median age in our cohort was 28.5 years (range, 1.5-70 years). Pediatric cohort (age < 18 years) included 57 patients. Almost half (N=93; 56%) of the patients had chronic ITP prior to TPO-RA initiation. Baseline characteristics were shown in Table 1.

Response rates were significantly higher in the romiplostim group when compared to the eltrombopag group (91% vs. 60%; p <0.001) in the pediatric cohort, which was not observed in the adult cohort (77% vs. 84%; p =0.28). Response rates were significantly higher in the pediatric cohort when compared to the adult cohort in the romiplostim group (91% vs. 77%; p = 0.008); however, adults responded better in the eltrombopag group (84% vs. 60%; p <0.001). There was a significant reduction in bleeding manifestations at 1 month when compared with baseline (20% vs. 61%; p <0.001). On univariate analysis, prior splenectomy was a predictor of poor response to TPO-RA therapy. However, on multivariate analysis, age, sex, ITP duration, prior splenectomy, prior lines of therapy were not predictive of response.

Adverse events: Thrombocytosis was noted in 12 (7%) patients with no evidence of thrombosis, and 2 (1%) patients progressed to myelodysplastic syndrome.

Of the 17 patients who were trailed on both therapies, 6 (3.5%) patients failed both TPO-RA therapies. Among the 54 (31%) patients who discontinued therapy, 11 (6.6%) patients achieved durable response off treatment. The median follow-up duration was 13 months (range, 0.8-79 months).

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Summary/Conclusion: Response rates to TPO-RA therapy in our cohort are consistent with those reported from the West. We observed that response rates were significantly higher in our pediatric cohort when compared to our adult cohort.

P1636: PATIENT PREFERENCES AND EXPERIENCES REGARDING THROMBOPOIETIN-RECEPTOR AGONISTS FOR IMMUNE THROMBOCYTOPENIA IN THE NETHERLANDS (TRAPEZE NL STUDY).

A. G. Jansen 1,* , V. McDonald 2 , A. Newland 3 , M. Morgan 4 , S. Visser-Peereboom 5 , K. Wilson 5 , D. Eriksson 5 , E. Geldman 6 , O. Daykin-Pont 6 , J. J. Zwaginga 7

1 Department of Haematology, Erasmus MC, Rotterdam, Netherlands; 2 Department of Clinical Haematology, The Royal London Hospital, Barts Health NHS Trust; 3 Academic Haematology Unit, Blizzard institute, Barts & The London School of Medicine & Dentistry, Queen Mary Institute of London, London; 4 ITP Support Association, Bolnhurst, United Kingdom; 5 Swedish Orphan Biovitrum AB, Stockholm, Sweden; 6 Wickenstones Ltd, Abingdon, United Kingdom; 7 Department of Haematology, Leiden University Medical Centre, Leiden, Netherlands

Background: Thrombopoietin-receptor agonists (TPO-RAs) are standard of care in immune thrombocytopenia (ITP) patients refractory to first-line treatment with glucocorticoids. Treatment of ITP with TPO-RAs is progressing towards integrating patient preference into management strategy. Currently, quantitative evidence evaluating the influence of therapy characteristics on patient preference has been generated in the UK via the multinational TRAPeze study (Thrombopoietin-Receptor Agonist Patient experience survey).

Aims: The TRAPeze study aims to identify patient experience and preference towards specific TPO-RAs in the treatment of ITP.

Methods: Presented here are results from the TRAPeze study in The Netherlands. Patients were recruited by the Dutch ITP patient association, which publicised the study via its website, social media and mailing list. The TRAPeze study used a discrete choice experiment (DCE) to elicit patient preferences regarding mode of administration, dosing frequency, drug-food interactions, platelet response, monitoring requirements and potential side effects. Additionally, a patient burden survey (PBS) determined patient demographics, disease characteristics, previous and current treatment patterns, treatment satisfaction, healthcare resource utilisation and social impact.

Results: 76 respondents from The Netherlands completed the DCE. 69 respondents completed both the DCE and PBS (mean [range] age 53.3 [19-83] years, 65% female). Of those currently or most recently receiving eltrombopag (n=43) or romiplostim (n=26), 98% and 88% respectively reported satisfaction with mode of administration. 30% (n=20) previously received another TPO-RA, with loss (29%, n=6) and lack (29%, n=6) of response most commonly reported as the reason for switching.

The TPO-RA characteristics which were most likely to influence patient preference were method of administration (odds ratio [OR] 4.33; 95% confidence interval [CI] 2.88–6.52), frequency of dosing (OR 2.33; 95% CI 1.86–2.92) and drug-food interactions (OR 1.91; 95% CI 1.54–2.37). Patients were more likely to prefer an oral tablet over subcutaneous injection (OR 4.22; 95% CI 2.76–6.46), a dosing frequency of once weekly over once daily (OR 2.37; 95% CI 1.58–3.54) and a therapy without food restrictions over with restrictions (OR 1.90; 95% CI 1.52–2.38).

The most commonly reported ITP symptoms were fatigue (83%), bruising (81%), petechiae (71%), anxiety about platelet count (49%) and unexplained nosebleeds (29%). Fatigue was the most negatively impactful symptom for 55% of patients. 47% of employed patients reported reducing their weekly working hours, of which 60% cited fatigue as the primary reason. Although 78% of patients reported their TPO-RA effectively managed symptoms, only 28% reported increased energy levels. Changes to daily routine and diet were both reported by 22% of patients, however only 3% of patients reported an inability to adhere to their treatment consistently.

Summary/Conclusion: In conclusion, patients prefer therapies with less frequent dosing, and, in concordance with previously published UK results, oral tablets and treatments without food restrictions. Despite treatment efficacy, improvement in energy levels is not guaranteed and many patients’ lives are impacted by fatigue. Furthermore, some patients must accommodate their treatment with changes to daily routine and diet. These findings further highlight the importance of shared decision-making between clinicians and patients to ensure the prescribed TPO-RA has the best impact on patients’ quality of life.

P1637: RISKS FOR A PLATELET COUNT DROP: COVID-19 & ITP DATA FROM THE PLATELET DISORDER SUPPORT ASSOCIATION (PDSA) PATIENT REGISTRY

J. DiRaimo 1,* , C. Kruse 1 , A. Kruse 1,2 , M. Lambert 3 , J. B. Bussel 4

1 Platelet Disorder Support Association, Cleveland; 2 Tulane University School of Medicine, New Orleans; 3 Hematology, Children’s Hospital of Philadelphia, Philadelphia; 4 Pediatric Hematology/Oncology, New York Presbyterian Hospital - Weill Cornell Medicine, New York, United States of America

Background: Immune thrombocytopenia purpura (ITP) is an autoimmune bleeding disorder with low platelet counts (PC). Both vaccines and viral infections can further lower PC in ITP patients. While fear of COVID-19 influences many to receive protective vaccinations, these same vaccinations greatly concern ITP patients, since they might lower the PC.

Aims: We describe data collected from the COVID-19 & ITP survey within the ITP Natural History Patient Registry on PC drops following both viral infection and vaccination to compare the effects of each on individuals with ITP.

Methods: Individuals with primary or secondary ITP completed the survey. For individuals under the age of 18, the survey was completed by a legally-authorized representative (LAR). Data was collected through January 4, 2022.

Results: Of 527 survey respondents, 78% were female (390); 83% were under age 65 years (418, range 5-87); and 16 (3%) were younger than 18; 170 (32%; n = 524) participants were in remission and 114 (22%; n = 524) had had previous splenectomy. Seventy participants tested positive for COVID-19. 387 reported vaccination with 282 fully vaccinated. Of 70 who were infected, 65 reported their PC change with A PC drop in 21 (32%). Recovery to baseline was within 3 months in 91% and within 1 month in 67%. Among 387 who reported receiving vaccine dose 1 (D1), 119 (31%) reported a PC drop. Recovery to baseline was within 3 months (77%) and 1 month (57%). Among the 282 who received dose 2 (D2), 87 (31%) had a PC drop. Recovery to baseline was within 3 months (83%) and 1 month (62%).

Of 119 participants with a PC drop after (D1), 68 (57%) received D2. Of these 68, 45 (66%) reported a PC drop after both D1+D2, more than the entire group (282) who received D2 (X 2 = 29.1, p = 0.00001). PC drops were not necessarily greater with subsequent doses. Among 45 participants who experienced PC drops following D1+D2, PC drops were larger with: a) D1 in 13 (28%) b) D2 in 7 (16%), and c) the same following D1 and D2 in 25 (56%). Of 45 participants reporting a PC drop post D1+D2, most were females (34; 76%) between 22-64 years (33; 73%), similar to the overall survey demographics. Eight (18%) participants with a PC drop following D1+D2 had had a previous splenectomy (same as the 22% overall).

Summary/Conclusion: One-third of participants experienced a PC drop following either viral infection or vaccine receipt which further supports the safety of COVID-19 vaccination in patients with ITP. For the 31% of patients that had a PC drop following D1, there was an increased probability of a PC drop with D2, however for more than half of those patients the degree of PC drop was not greater with D2. Age, gender, and splenectomy status did not influence the risk for PC drops with either the D1 or D2. PC changes following both infection and vaccination were short-lived and generally recovered within 1 month. These findings should further reduce vaccine hesitancy among ITP patients.

P1638: EARLY USE OF ELTROMBOPAG IN ADULT PATIENTS WITH ITP WHO ARE REFRACTORY OR RELAPSED AFTER FIRST-LINE CORTICOSTEROID THERAPY – AN AD HOC ANALYSIS OF THE TAPER TRIAL RESULTS

W. Ghanima 1,* , F. Zaja 2 , J. Maier 3 , J. Haenig 3 , J. Lee 4 , N. Cooper 5

1 Department of Medicine, Østfold Hospital Trust, Kalnes, Norway; 2 Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy; 3 Novartis Pharma AG, Basel, Switzerland; 4 Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States of America; 5 Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, Hammersmith Hospital, London, United Kingdom

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by reduced platelet count (<100×10 9 /L) and increased bleeding risk in the absence of other causes. It is categorized by disease duration from diagnosis: newly diagnosed (ndITP; ≤3 months [mo]), persistent (pITP; 3-12 mo), and chronic (cITP; >12 mo). Eltrombopag (EPAG), a thrombopoietin receptor agonist, is approved in Europe for the treatment of ITP lasting ≥6 mo in patients (pts) refractory to other treatments. The TAPER trial ( NCT03524612 ) was designed to assess sustained response off treatment following EPAG tapering in adults with ITP of any duration, who were refractory/had relapsed after first-line corticosteroids.

Aims: To conduct an ad hoc analysis of the ongoing TAPER trial, assessing initial response to EPAG therapy according to time since ITP diagnosis (ndITP, pITP, cITP).

Methods: Pts with ≥6 mo of data, or pts who discontinued the study prior to Month 6 were included. Newly enrolled pts with <6 mo of data were excluded. Pts were grouped according to time since ITP diagnosis at baseline (<3, 3 to <6, 6 to ≤12, and >12 mo). The main analysis of hematologic response (ie, percentage of pts with platelet count thresholds of ≥30, ≥50, and ≥100 ×10 9 /L achieved at least once without rescue therapy) was restricted to data from the first 2 mo (~Day 57) to ensure pts were on treatment. Safety analyses were restricted up to and including Month 6.

Results: A total of 82/105 (78%) pts from TAPER were included. Median (range) age was 49 (18-88) years and 58.5% were female. In the first 6 months, the median (interquartile range [IQR]) duration of EPAG exposure was 5.3 (2.6-6.0) mo, and median (IQR) dose was 53.2 (37.5-69.6) mg/day. The proportions of pts with platelet counts equal to or above the specified thresholds were comparable across the time-since-ITP-diagnosis groups, ranging from 69% to 100% (Fig. 1). The median time to a platelet count of ≥50×10 9 /L was also comparable across groups, ranging from 8 to 17 days, and the median (IQR) increase in platelet count to ~Day 57 was 84.5×10 9 /L (36.5-165.6). The percentages of pts without World Health Organization Grade 1-4 bleeding events increased from Day 1 to ~Day 57 across all groups (<3 mo: 39.5%→84%; 3 to <6 mo: 61%→100%; 6 to ≤12 mo: 50%→88%; >12 mo: 70%→89%). The most common all-grade adverse events (AEs) were headache (pts: 18% [<3 mo], 22% [3 to <6 mo], 12.5% [6 to ≤12 mo], 10% [>12 mo]) and thrombocytopenia (pts: 21% [<3 mo], 17% [3 to <6 mo], 12.5% [6 to ≤12 mo], 0% [>12 mo]). One pt (6%; 3 to <6 mo) had grade ≥3 headache; 5 (13%; <3 mo), 2 (11%; 3 to <6 mo), and 2 (12.5%; 6 to ≤12 mo) pts had grade ≥3 thrombocytopenia. There were 3 treatment-related severe AEs (intentional product misuse [<3 mo], infectious diarrhea secondary to EPAG, and pulmonary embolism [both 6 to ≤12 mo]). Two deaths were reported by the cut-off date (central nervous system hemorrhage [n=1; <3 mo]; intracranial hemorrhage [n=1; 3 to <6 mo]) that were not considered study drug related.

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Summary/Conclusion: The data show that EPAG achieved comparable rates of platelet response and reduced the frequency and severity of bleeding events in pts with ndITP, pITP, and cITP. Safety findings were comparable across the 4 groups, and aligned with the well-established safety profile of EPAG. These findings suggest that EPAG is as efficacious and well tolerated in pts who receive it early following diagnosis as in those who receive it later.

P1639: UTILITY OF NEXT GENERATION SEQUENCING TECHNIQUES TO EVALUATE POSSIBLE CAUSES OF REFRACTORINESS TO TREATMENT WITH ELTROMBOPAG IN ADULT PATIENTS WITH PRIMARY IMMUNE THROMBOCYTOPENIA (ITP) IN SPAIN.

T. J. Gonzalez-Lopez 1,2,* , R. Sanchez 3 , C. Pastoriza 4 , B. Sanchez Gonzalez 5 , P. Olivera 6 , S. Bernat 7 , F. Fernandez-Fuertes 8 , I. Caparrós Miranda 9 , E. Mingot 10 , G. Pérez-Rus 11 , I. Jarque 12 , M. E. Moreno Beltrán 13 , E. López Abadía 14 , E. De Cabo 15 , S. Marcellini 16 , G. Pérez Segura 3 , C. Fernández-Miñano 17 , M. J. Peñarrubia 18 , M. Alvarez 19 , M. A. Fuertes Palacio 20 , J. Martinez Lopez 3,21,22

1 Hematology, Hospital Universitario de Burgos; 2 Health Sciences Department, Universidad de Burgos, Burgos; 3 Hematology, Hospital Universitario 12 de Octubre, Madrid; 4 Hematology, Complejo Hospitalario Universitario de Orense., Orense; 5 Hematology, Hospital del Mar; 6 Hematology, Hospital Universitario Sagrado Corazón - Grupo Quirónsalud, Barcelona; 7 Hematology, Hospital Universitario de la Plana, Castellón; 8 Hematology, Hospital Universitario Insular de Gran Canaria, Las Palmas; 9 Hematology, Hospital Universitario Virgen de la Victoria de Málaga.; 10 Hematology, Complejo Hospital Regional de Málaga, Malaga; 11 Hematology, Hospital Universitario Gregorio Marañón de Madrid., Madrid; 12 Hematology, Hospital Universitario y Politécnico La Fe de Valencia, Valencia; 13 Hematology, Hospital Costa del Sol, Marbella (Malaga); 14 Hematology, Hospital General de Elche, Elche; 15 Hematology, Hospital del Bierzo, Ponferrada (León); 16 Hematology, Hospital General de Segovia, Segovia; 17 Hematology, Hospital Vega Baja, Orihuela (Alicante); 18 Hematology, Hospital Clinico de Valladolid, Valladolid; 19 Hematology, Hospital Universitario La Paz, Madrid; 20 Hematology, Hospital Clínico Lozano Blesa, Zaragoza; 21 Medicine Department, Universidad Complutense; 22 Spanish Center of Oncology Research, Madrid, Spain

Background: Primary immune thrombocytopenia (ITP) is an autoimmune acquired disease characterized by low production and increased platelet destruction. Thrombopoietin receptor agonists (TPO-RAs) i.e. eltrombopag, romiplostim and avatrombopag are currently used as second line treatment for ITP. Eltrombopag has demonstrated high response rates (≥80%) both in clinical trials and in routine practice studies. Nevertheless, some patients are refractory to this drug and do not attain a response when using the maximum eltrombopag approved dose of 75 mg per day after at least four weeks of treatment. Next generation sequencing (NGS) may help us discover underlying molecular biology variants that may be involved in eltrombopag refractoriness.

Aims: To evaluate potential molecular markers of refractoriness to eltrombopag in ITP in routine clinical practice through the use of most recent molecular biology techniques (NGS).

Methods: Nationwide multicenter, retrospective NGS study to investigate most important cell-signaling pathways that are involved in eltrombopag mechanism of action. So, 108 genes directly related to the downstream signaling of eltrombopag, cytokine signaling pathways, cytotoxic pathways, growth factors, adhesion molecules, apoptotic mechanisms and physiological mechanisms of platelet production are analyzed in peripheral blood from 35 refractory and 35 controls who responded to the drug.

Results: Our refractory population involved 18/35 (51.4%) of male with a median age at diagnosis of 48 [range, 38-70] years and a median platelet count of 8 [range, 4-16] x 10 9 /µL. At eltrombopag initiation, median time from diagnosis was 14 [range, 6-103] months with a median age of 60 [range, 41-75] years while median platelet count was 8 [range, 5-30] x 10 9 /µL. 78.3% and 13.0% of cases were chronic and persistent ITP respectively. They have received a median of only two previous treatment lines [range, 2-4] (100% steroids, 42.8% IVIG, 40% splenectomy, 31.4% rituximab, 37.1% romiplostim). Eltrombopag treatment was maintained during 3 [range, 1-9] months as a median before discontinuation. No major grade 3-4 side effects were reported. On the other hand, characteristics of respondent controls were similar to refractory population. Statistical differences were observed regarding prior treatment lines, response and complete response rates (p=<0.001).

Of the total sum of the NGS variants found and after artefacts eliminated a list of 53 variants were found. 13 variants with statistical significance (p≤0.05) between case and controls were relevant being some of them potentially associated with cancer and others considered benign instead. Variant two and seven are described in gastric and lung cancer respectively. Variants one, four, five, six, nine, eleven and thirteen belong to the second group. Variants three, eight, ten and twelve are not previously described and its significance is unknown (Table 1).

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Summary/Conclusion: To the best of our knowledge, none of the 13 statistically significant variants reported here have ever been correlated with eltrombopag refractoriness although some of them have been previously associated with different cancers. Here we describe four non-previously published DNA variants and other nine non-previously described in ITP which role in megakaryopoesis and ITP drug refractoriness must be elucidated. It is needed more studies (including functional) to establish a more robust correlation between these variants and eltrombopag refractoriness.

P1640: SINGLE NUCLEOTIDE POLYMORPHISMS OF THE HIF1A GENE ARE ASSOCIATED WITH SENSITIVITY OF GLUCOCORTICOID TREATMENT IN PEDIATRIC ITP PATIENTS

H. Gu 1,* , X. Xie 1 , J. Ma 1 , L. Fu 1 , J. Ma 1 , R. Wu 1 , Z. Chen 1

1 Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China

Background: Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in both innate and adaptive immunity. Emerging evidence indicates that HIF-1α is associated with the inflammation and pathologic activities of autoimmune diseases，suggesting that HIF1α may be involved in immune dysregulation in patients with immune thrombocytopenia (ITP).

Aims: The purpose of this study was to evaluate whether single nucleotide polymorphisms (SNPs) of the HIF1A gene are associated with susceptibility to ITP and its clinical prognosis including incidence of chronic immune thrombocytopenia (CITP) and glucocorticoid sensitivity.

Methods: This study involved 197 Chinese ITP pediatric patients (discovery cohort) and 220 healthy controls. The Sequenom MassArray system (Sequenom, San Diego, CA) was used to detect three SNPs genotypes in the HIF1A gene: rs11549465, rs1957757 and rs2057482. We also employed another ITP cohort (N = 127) to validate the significant results of SNPs found in the discovery cohort.

Results: The frequencies of the three SNPs did not show any significant differences between the ITP and healthy control groups. The CT genotype at rs11549465 was significantly higher in ITP patients sensitive to glucocorticoid-treatment than in those insensitive to glucocorticoid-treatment (P = .025). These results were validated using another ITP cohort (N =127, P = .033). Moreover, the CC genotype was a risk factor for insensitive to GT the OR (95% confidence interval) was 5.96(5.23-6.69) in standard prednisone (PDN) (P=.0069) and 6.35(5.33-7.37) in high-dose dexamethasone (HDD) (P=.04).

Summary/Conclusion: Although HIF1A gene polymorphisms were not associated with susceptibility to ITP, the CT genotype at rs11549465 was associated with the sensitivity to glucocorticoid-treatment of ITP patients, suggesting that the rs11549465 SNP may contribute to the sensitivity of glucocorticoid treatment in pediatric ITP patients.

P1641: ELTROMBOPAG TREATMENT, OUTCOMES AFTER DISCONTINUATION IN ADULT PATIENTS WITH PRIMARY IMMUNE THROMBOCYTOPENIA, AND EXPLORATION OF POTENTIAL PREDICTIVE FACTORS: A REAL-WORLD CLINICAL EXPERIENCE

X. Liu 1,* , R. Fu 1 , H. Li 1 , Y. Chen 1 , W. Liu 1 , F. Xue 1 , L. Zhang 1 , R. Yang 1

1 Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by accelerated platelet destruction and impaired platelet production. Eltrombopag has been widely used in ITP in clinical practice for over a decade, however, there is limited evidence on predictors of response and successful discontinuation.

Aims: To ascertain the optimal initial dose for East Asian descent, and explore the clinical and biological markers for adequate response. Besides, to investigate the potential for successful discontinuation in ITP patients treated with eltrombopag in a real-world setting and its clinical predictors.

Methods: We retrospectively evaluated 244 adult primary ITP patients. All patients were hospitalized at our institution between January 2016 and October 2020 and treated with eltrombopag. Patient and disease characteristics, clinical outcomes, and adverse events (AEs) during eltrombopag treatment, were investigated. Blood samples were also obtained at baseline to detect the number of lymphocytes and the expression of subsets and other autoantibodies.

Patients achieved overall response were divided into three groups (R1: 30-49, R2: 50-99, R3: ≥100 × 10⁹/L) according to platelet counts, which was used to analyze the potential predictors of response and successful discontinuation. Sustained response off-treatment (SRoT) was defined as platelet count ≥30 × 10⁹/L for at least 1 month with no ITP medication.

Results: The initial daily dose was 25 mg for 20 patients (8.2%), 50 mg for 129 patients (52.9%), 75 mg for 95 patients (38.9%), respectively. The overall response rate was 187/244 (76.6%), with 26/244 achieving R1 (10.7%), 49/244 achieving R2 (20.1%), and 112/244 achieving R3 (45.9%). The median of time to response (TTR) was 5 days (IQR: 3-11). Multivariable logistic analysis revealed that significant predictors of adequate response included female gender (OR=3.10, p=0.0377), less prior ITP therapies (OR=1.552, p=0.0103), less combination medication (OR=1.53, p=0.0328), lower maximum dose (OR= 6.637, p=0.0041), and more naive Treg/T cell % (OR=3.79, p=0.0120). Higher baseline megakaryocyte count was found to be significant correlated with less TTR (r=-0.191, p=0.022), while there was no significant correlation between the megakaryocytes count and the response rate (r=-0.009, p=0.894). A total of 60 treatment-related AEs was reported, which were mainly of CTCAE grade 1 or 2 in intensity. No thromboembolic events were observed during hospitalization; no thrombotic events, stroke or myocardial infarction requiring medical intervention were reported during follow-up period.

Eltrombopag was discontinued in 81 responders. Patients who failed to maintain platelet counts, who switched to other medications or underwent splenectomy were all considered relapse. A total of 29 patients (35.8%) observed SRoT after discontinuing eltrombopag. The multivariable analysis identified that response degree was a predictive factor of SRoT (p = 0.0007, Table 1), in which the SRoT rate was up to 53.2% in patients achieved R3.

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Summary/Conclusion: In clinical practice, most East Asian patients with higher initial dose obtained better short-term response and had an acceptable safety profile. Patients treated with eltrombopag in earlier lines, with less combination medication, with lower maximum dose, and higher Naive Treg/T cell (%) may achieve an adequate response. The higher number of megakaryocytes could potentially be associated with faster response. The higher SRoT rate in R3 patients encourages to taper eltrombopag in such patients.

P1642: OUTCOMES IN HOSPITALIZED PATIENTS WITH COMPLEMENT MEDIATED THROMBOTIC MICROANGIOPATHY

C. Logothetis 1,* , K. Finley 1 , S. Huang 1 , A. Wood 2 , D. Laber 2 , J. Eatrides 2

1 Department of Internal Medicine, University of South Florida; 2 Department of Satellite Oncology, Moffitt Cancer Center and Research Institute, Tampa, United States of America

Background: Complement-mediated (CM) thrombotic microangiopathy (TMA), also known as atypical hemolytic uremic syndrome is a serious and potentially life-threatening disorder characterized by dysregulation of the complement immune system. The advent of anti-C5 monoclonal antibodies (C5-mab) revolutionized the treatment of aHUS greatly reducing the morbidity and mortality. Experience outside clinical trials has been limited due to the rarity of the condition.

Aims: The aim of this study was to examine the outcomes of hospitalized patients diagnosed with CM-TMA. All patients received C5-mab at our institution, hence, we aim to assess the safety, efficacy and toxicity of therapy.

Methods: This was a retrospective study. The inclusion criteria comprised of diagnosis of CM-TMA (laboratory values consistent with TMA syndrome or TMA on a biopsy, with normal ADAMTS13 and absent shiga toxin). We used the following definitions through our data collection and analysis to assess treatment efficacy. Platelet response (PR): an increase in platelet count (PC) from baseline. Hematologic response (HR): PC and LDH normalization for at least 4 weeks. Complete TMA Response (CR): HR plus a decrease in Cr by 25% for at least 4 weeks without renal replacement therapy.

Results: Thirty-four patients with CM-TMA received C5-mab (eculizumab and/or ravulizumab) at our institution between 2013-2021. The median age at diagnosis was 51 years. Twenty-seven (79%) patients were white, 6 (18%) were African American and (3%) were Asian. Presenting symptoms included fever in 4 (12%), neurologic deficit (weakness, fatigue, and altered mental status) in 14 (41%) and anasarca in 6 (18%). Renal replacement therapy was initiated in 20 (58.8%) subjects while receiving C5-mab. Six (18%) patients had kidney transplants prior to the diagnosis of CM-TMA and 4 (12%) patients required renal transplant after receiving C5-mab. Median (range) laboratory values prior to C5-mab demonstrated: PC 59/uL (8-308), LDH 890 U/L (420-4053), Cr 3.9 mg/dL (0.5-12.9) and Hemoglobin 7.1 g/dL (4.6-11.7).

Therapy: Initiation of C5-mab occurred at a median of 11 (0-379) days after the initial TMA event.

Outcomes: All patients responded: PR 30 (88%) and CR 4 (12%). Time to PR and CR in days were 9 (0-268) and 57.5 (29-120), respectively. Adverse events while on C5-mab were infections (medical equipment, pneumonia, and abscess) in 10 (29%) and headache in 2 (6%) individuals. Three (9%) patients died during their hospitalization. The causes of death were multi-organ failure from lupus flare, acute hypoxic respiratory failure from pulmonary hypertension and septic shock in a subject who had a severe E. faecium bacteremia prior to initiating C5-mab.

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Summary/Conclusion: This is one of the largest real-world studies of hospitalized patients with CM-TMA treated with C5-mab. Limitations to this study include the retrospective nature and sample size, but the inclusion of all consecutive patients limited selection bias. We acknowledge that some hospitalized individuals with CM-TMA might not be diagnosed due to confounding illnesses. Since the diagnosis of CM-TMA usually takes several days to make, we speculate that patients with rapidly deteriorating organ function or with early demise might not reach diagnosis and therapy. Our findings confirm the efficacy of C5-mab at treating CM-TMA. We believe early diagnosis and initiation of therapy might avoid adverse outcomes. Large, long-term and prospective studies evaluating diagnostic criteria, therapy and outcomes in CM-TMA are warranted.

P1643: EFFICACY OF RECOMBINANT HUMANTHROMBOPOIETIN ON CHILDREN IMMUNE THROMBOCYTOPENIA AND ITS INFLUENCING FACTORS

J. Ma 1,* , Z. CHEN 1 , H. GU 1 , R. WU 1

1 Hematology Oncology Center, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China

Background: RhTPO can promote the production of platelets and its clinical value has been proved in adult ITP. However, there are little data on efficacy and safety in children ITP and still lacks predictive marker for efficacy. Considering THPO and MPL encode endogenous TPO and c-MPL, which participate in the biological efficacy of rhTPO, we speculate that the SNPs in THPO and MPL genes may have an impact on the efficacy of rhTPO.

Aims: To verify the efficacy of rhTPO in children ITP and to explore the effects of THPO SNPs (rs6141 and rs956732) and MPL SNP (rs176067 and rs190983971) on the efficacy of rhTPO, in order to find predictive factors for rhTPO.

Methods: We collect data from children ITP who received rhTPO and grouped them into severe newly-diagnosed ITP and ITP without response to first-line therapy. We evaluated the efficacy of rhTPO based on their platelet count and the severity of bleeding. NGS was used to test four targeted SNPs. The differences in SNPs distribution between different efficacy groups were compared.

Results: Fifty-eight ITP were collected, 22 were severe newly diagnosed ITP and 36 were those with poor response to first-line therapy. The ORR and CR were respectively 79% and 58%. All had their bleeding grade significantly decreased after treatment, including those non-responders. The genotype distribution of THPO rs6141 is statistically different between the 2 groups, the CC genotype had a significantly increased tendency to develop into non-responders (CC/TT: OR=30.000, 95%CI=2.871-313.470). Besides, AA genotype of THPO rs956732 is also significantly increased in non-responders (AA/AG: OR=7.467, 95%CI=1.400-39.836).

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Summary/Conclusion: rhTPO can effectively increase the platelet count and reduce bleeding severity of children ITP. The CC genotype at the rs6141 and AA genotype at the rs956732 of THPO are risk factors for the ineffectiveness of rhTPO.

P1644: NOVEL VARIANTS IN GALE CAUSED SYNDROMIC MACROTHROMBOCYTOPENIA DISRUPTING THROMBOPOIESIS AND GLYCOSYLATION

A. Marin Quilez 1,* , C. A. Di Buduo 2 , L. Díaz-Ajenjo 1 , P. M. Soprano 2 , E. Vuelta 1 , I. Serramito-Gómez 1 , S. Santos-Mínguez 1 , C. Miguel-García 1 , S. Tocino-Antonio 1 , V. Palma-Barqueros 3 , A. Zamora-Canovas 3 , P. Ruiz-Sala 4 , M. J. Peñarrubia 5 , E. Pardal 6 , J. M. Hernández-Rivas 1,7 , J. R. González-Porras 7 , R. Benito 1 , J. Rivera 3,8 , I. García-Tuñón 1 , A. Balduini 2,9 , J. M. Bastida 7,10

1 IBSAL, CIC, IBMCC, Universidad de Salamanca-CSIC, Salamanca, Spain; 2 Department of Molecular Medicine, University of Pavia, Pavia, Italy; 3 Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER-U765, Murcia; 4 Centro de Diagnóstico de Enfermedades Moleculares, Universidad Autónoma de Madrid, CIBERER, IdIPAZ, Madrid; 5 Department of Hematology, Hospital Clínico Universitario de Valladolid, Valladolid; 6 Department of Hematology, Hospital Virgen del Puerto, Plasencia; 7 Department of Hematology, Complejo Asistencial Universitario de Salamanca (CAUSA), Instituto de Investigación Biomédica de Salamanca (IBSAL), Universidad de Salamanca (USAL), Salamanca; 8 On behalf of “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Sociedad Española de Trombosis y Hemostasia (SETH), Murcia, Spain; 9 Department of Biomedical Engineering, Tufts University, Medford, United States of America; 10 On behalf of “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Sociedad Española de Trombosis y Hemostasia (SETH), Salamanca, Spain

Background: Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme UDP-galactose-4-epimerase, encoded by GALE , is involved in protein glycosylation. There is currently scarce information about the performance of megakaryopoiesis in patients with thrombocytopenia associated with GALE variants.

Aims: Assessing the clinical and platelet phenotype in three patients with syndromic macrothrombocytopenia associated to GALE variants, and to investigate the role of GALE in glycosylation and thrombopoiesis.

Methods: Three patients from two unrelated families with lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice were enrolled in the Spanish Project of Inherited Platelet Disorders. Biallelic variants in GALE were identified by whole-exome sequencing (WES) (Figure 1). Platelet phenotyping included full blood count, blood film, light transmission aggregometry, flow cytometry analysis of major surface glycoproteins, and agonist-induced granule secretion. UDP-galactose-4-epimerase enzymatic activity was measured by HPLC/MS/MS. GALE and N-acetyl-galactosamine (LacNac) levels were evaluated by immunoblotting. Human megakaryocyte (Mk) culture from peripheral blood samples were assessed in one proband.

Results: WES revealed four variants affecting GALE , three of them previously unreported (Figure 1). Patients showed giant and/or grey platelets, impaired platelet aggregation with several agonists, severely reduced alpha (P-selectin) and dense granule (CD63) secretion, and significant decrease of GPIbα-IX-V complex in the platelet surface. UDP-galactose-4-epimerase enzymatic activity was severely decreased in these patients. Immunoblotting showed reduced GALE in platelets and significant reduction of LacNAc, suggesting a hypoglycosylation pattern. In proband B.II.2, CD34+ derived Mks exhibited normal ploidy, and maturation, but impaired proplatelet formation. These Mks showed, similarly to patient platelets, normal surface levels of β3 integrin, but remarkable reduction in the glycosylation and externalization of GPIbα, and delocalization of vWF in the megakaryocyte membrane.

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Summary/Conclusion: This study expands our knowledge on the GALE related thrombocytopenic disorder, emphasizing its critical role in platelet glycosylation, and providing further evidence that thrombocytopenia derives from altered proplatelet formation developing from GPIbα impairment.

Funding

PI20/00926, GRS2135/A/2020, GRS2314/A/2021, FMM AP172142019

P1645: THE SEVERITY OF HEMORRHAGIC SYNDROME AND THE WEAKENING OF THE PROCOAGULANT ACTIVITY OF PLATELETS IN PATIENTS WITH WISKOTT-ALDRICH SYNDROME.

D. Polokhov 1,* , A. Ignatova 1 , E. Ponomarenko 1 , A. Poletaev 1 , P. Zharkov 1

1 Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia

Background: Patients with Wiskott-Aldrich syndrome (WAS) have a reduced platelet count. Bleeding occurs in most patients and may result in death in 4–10%. The role of impaired procoagulant activity of platelets in WAS has not been studied enough.

Aims: To study the role of the weakening of the platelets procoagulant activity in the development of hemorrhagic syndrome.

Methods: We examined 25 patients with WAS, observed from June 2017 to January 2020. The median age was 2 years (9 months to 18 years). Patients complained of hemorrhagic manifestations. The severity of hemorrhagic syndrome was assessed using the Pediatric bleeding questionnaire (PBQ). Patients were divided into 2 subgroups depending on the severity of bleeding from 0 to 2 score (n=12) and 3-6 score (n=13). The control group (CG) included 42 healthy children, median age 10 years (from 2-17 years). Flow cytometry was used to determine the total number of platelets and the proportion of phosphatidylserine-positive (PS+) platelets at rest and after activation with a mixture of TRAP-6 and CRP. Statistical differences were analyzed using the Mann-Whitney test with a significance level of p<0.05.

Results: The platelet count was lower in patient subgroups compared with CG (p<0.001), but did not differ between patient subgroups (p=1). In the subgroup of patients with bleeding from 0 to 2 points, the proportion of PS+ platelets at rest did not differ from CG (p=0.89), and after activation it was reduced (p<0.001). In the subgroup of patients with bleeding from 3 to 6 points, the proportion of PS+ platelets was lower compared with CG at rest (p=0.02) and after activation (p<0.001). After activation, the proportion of PS+ platelets in CG was ≥ 5%. In 10 (40%) patients, a decrease in platelet PS+ <5% correlated negatively with an increase in the severity of bleeding (corr.= -0.65, p=0.04).

Summary/Conclusion: The weakening of the ability to generate procoagulant platelets in patients with Wiskott-Aldrich syndrome was related with an increase in the severity of hemorrhagic syndrome.

P1646: PRIMARY IMMUNE THROMBOCYTOPENIA (ITP) IN PATIENTS OLDER THAN 75 YEARS: REAL WORLD DATA FROM THE ITP REGISTRY OF THE HELLENIC SOCIETY OF HEMATOLOGY

C. Pontikoglou 1,* , A. Matheakakis 1 , E. Stavroulaki 2 , T. Chatzilygeroudi 3 , A. Kourakli 3 , A. Symeonidis 3 , M. Dimou 4 , P. Panayiotidis 4 , G. Drakos 5 , A. Koudouna 5 , A. Galanopoulos 5 , V. Kaliafentaki 1 , P. Kanellou 2 , D. Liapi 2 , G. Tsirakis 6 , A. Kolovou 6 , E. Gavriilaki 7 , A. Syrigou 7 , I. Sakellari 7 , S. Chatzileontiadou 8 , M. Papaioannou 8 , M. Bobola 9 , P. Diamantopoulos 10 , M. Mantzourani 10 , N.-A. Viniou 10 , M. Dellatola 11 , A. Souravla 11 , M. Pagoni 11 , A. Megalakaki 12 , I. Christodoulou 13 , E. Vlachaki 13 , S. Giannouli 14 , V. Gkalea 15 , C. Matsouka 15 , I. Kotsianidis 16 , G. Vassilopoulos 17 , M. Protopappa 18 , E. Hatzimichael 19 , P. Zikos 20 , G. N. Chalkiadakis 21 , H. A. Papadaki 1

1 Department of Hematology, University of Crete School of Medicine, Heraklion, Greece; 2 Department of Hematology, Venizeleio-Pananeio General Hospital, Heraklion, Crete; 3 Hematology Division, Department of Internal Medicine, University of Patras Medical School, Patras; 4 First Propedeutic Department of Internal Medicine, National and Kapodistrian University; 5 Clinical Hematology, G.Gennimatas District General Hospital, Athens; 6 Department of Hematology, General Hospital “St. George”, Chania; 7 Hematology Department-BMT Unit, George Papanicolaou Hospital; 8 First Internal Medicine Department, Aristotle University of Thessaloniki, AHEPA University Hospital, Thessaloniki; 9 Department of Hematology-Blood donation, Pamakaristos General Hospital; 10 First Department of Internal Medicine, Laikon General Hospital; 11 Hematology and Lymphoma, Bone Marrow Transplant Unit, Evangelismos Hospital, Athens; 12 Department of Hematology, Metaxa Anticancer Hospital, Piraeus; 13 Second Department of Internal Medicine, Aristotle University of Thessaloniki Hippokration General Hospital, Thessaloniki; 14 Second Department of Internal Medicine, National and Kapodistrian University; 15 Hematology Departement, Alexandra General Hospital, Athens; 16 Department of Hematology, Democritus University of Thrace, University Hospital, Alexandroupoli; 17 Department of Hematology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Larissa; 18 Department of Hematology, General Hospital of Serres, Serres; 19 Department of Hematology, University of Ioannina, Ioannina; 20 Department of Hematology, General Hospital of Patras “St.Andrew”, Patras; 21 Information Systems Unit Center of Information and Communication Technologies, University of Crete, Heraklion, Crete, Greece

Background: ITP incidence is known to increase in adults aged >60 years and even more in men aged >75 years (Moulis G et al., Blood 2014; 124(22):3308). As population aging and life-expectancy continues to increase worldwide, ITP in the elderly is a growing concern in everyday clinical practice. There are only a few studies regarding the features of ITP in patients (pts) aged >60-65 years and data are even scarcer in pts aged ≥75 years

Aims: To describe real world characteristics at diagnosis and evaluate disease outcome in a group of pts aged 60-74 years (Group-1) as compared to very old pts (≥75 years; Group-2) with primary ITP, using data from the national database (ITP registry) operated under the auspices of the Hellenic Society of Hematology

Methods: The Greek ITP registry recruits pts (n=1560, to date) nationally through a network of 25 sites. In the present study we retrospectively analyzed data from pts with primary ITP aged ≥60 years, who were diagnosed from 1979 to 2021.

Results: The total number of evaluable pts was 298. Group-1 consisted of 180 and Group-2 of 118 pts. The mean age at diagnosis was 68 years (60.1-74.9) in Group-1 and 81 years (75.2-97.1) in Group-2. The female to male ratio did not differ between the 2 groups. The median platelet count at diagnosis was significantly higher in Group-1 (20x10 9 /L, interquartile range: 5-44x10 9 /L) than in Group-2 (11x10 9 /L, interquartile range: 5-32x10 9 /L; P=0.0096). Comorbidity rates did not differ between Group-1 and Group-2, with the exception of hypertension and chronic kidney disease, which were less frequently reported in Group-1 (P=0.0092 and P=0.0179, respectively). Concurrently used medications as a whole and also vitamin-K antagonists were reported less frequently in Group-1 (P=0.03 and P=0.049, respectively). Bleeding manifestations at diagnosis were comparable across the two age-groups. The choice of diagnostic procedure did not differ between Goup-1 and Group-2, with the exception of anti phospholipid antibody and anti nuclear antibody testing which were performed more frequently in Group-1 (P=0.048 and P=0.0002, respectively). Similar rates of positive laboratory test results were reported in the two age-groups. Fewer pts received treatment at diagnosis in Group-1 (P=0.023). Overall response rate did not differ between the two Groups. A similar proportion of Group-1 and Group-2 patients were treated with corticosteroids, intravenous IgG or both, rituximab, anti-D, immunoglobulin or thrombopoietin receptor agonists. Splenectomy was performed in 2.8% of Group-1 pts but in none of Group-2 pts. At 1 year after diagnosis, a similar proportion of Group-1 and Group-2 pts had developed chronic ITP.

Summary/Conclusion: Very elderly pts (Group-2) presented with lower platelet counts at diagnosis and required ITP treatment more frequently than elderly pts (Group-1). Furthermore, Group-2 pts suffered more often from hypertension and chronic kidney disease and used more often anticoagulant agents. On the other hand, the 2 age-groups did not differ in the frequency or the location of bleeding. Age did not seem to particularly influence the diagnostic workup or the choice of treatment, with the exception of splenectomy which was not performed in Group-2 patients. The outcome of the disease was comparable in the 2 age-groups, as suggested by the similar treatment response rates and chronic ITP frequencies. Further investigation is warranted to full characterize the very elderly pts with ITP and unravel the unmet need of this cohort in order to optimize management.

P1647: CONTRIBUTION OF NEXT GENERATION FLOW (NGF) CYTOMETRY IN PRIMARY IMMUNE THROMBOCYTOPENIA (ITP): UTILITY FOR THE DIFFERENTIAL DIAGNOSIS WITH MYELODYSPLASTIC SYNDROMES

T. J. Gonzalez-Lopez 1,2,* , S. Matarraz 3,4 , I. Caparros 5 , M. F. Lopez-Fernandez 6 , M. E. Mingot-Castellano 7 , A. Jimenez Martin 8 , F. Fernandez-Fuertes 9 , J. I. Sánchez Gallego 3 , P. Leoz 3 , B. Cuevas Ruiz 1 , M. V. Cuevas Ruiz 1 , C. Martin Marín 10 , J. Olazabal Herrero 1 , P. de Vicente Cámara 1 , A. Orfao 3

1 Department of Hematology, Hospital Universitario de Burgos; 2 Department of Health Sciences, University of Burgos, Burgos; 3 Cancer Research Center (IBMCC, USAL-CSIC), Cytometry Service (NUCLEUS) and Department of Medicine, University of Salamanca; 4 Institute of Biomedical Research of Salamanca (IBSAL), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Salamanca; 5 Department of Hematology, Hospital Virgen de la Victoria, Málaga; 6 Department of Hematology, Complexo Hospitalario Universitario A Coruña, A Coruña; 7 Department of Hematology, Complejo Hospitalario Regional de Málaga, Málaga; 8 Department of Hematology, Hospital Universitario Ramón y Cajal, Madrid; 9 Department of Hematology, Hospital Universitario Insular de Gran Canaria, Las Palmas de Gran Canaria; 10 Department of Public Health, Consejeria de Sanidad, Junta de Castilla y León, Valladolid, Spain

Background: Primary immune thrombocytopenia (ITP) is an immune-mediated acquired disorder characterized by impaired production and increased destruction of platelets with an elevated risk of bleeding. At present, diagnosis of primary ITP still remains one of exclusion with a need to discard other causes of isolated thrombocytopenia, in the absence of robust and accurate clinical and laboratory diagnostic criteria. For the diagnosis of ITP, a bone marrow (BM) study may be useful to differentiate between ITP and other diseases, such as myelodysplastic syndromes (MDS). Next generation flow (NGF) has emerged as a potential useful tool in these settings.

Aims: In FCR-PTI-2017-01 study we prospectively evaluated the potential utility of NGF analysis of BM for more accurate diagnosis of ITP vs MDS.

Methods: Multicenter, prospective, Spanish study to investigate morphological and/or immunophenotypic profiles among BM cell compartments from patients ≥ 18 years with newly diagnosed ITP or MDS. A multiparameter characterization of multiple compartments of BM cells was performed using the 8-color panel designed and validated by the EuroFlow Consortium for the diagnosis and characterization of MDS and acute myeloid leukaemia. NGF BM blinded results were then compared with the BM cytomorphological diagnosis.

Results: 62 patients, median age 71 years, 59% male, were included. Main comorbidities were neoplasms (19%), diabetes (17%), chronic respiratory disease (12%) and cardiac insufficiency (10%). Median platelet count at diagnosis was 45 x 10 9 /µL (range: 4-98 x 10 9 /µL) and 20.6% of patients had bleeding at diagnosis. By cytomorphology, expert hematologists were able to conclude an ITP or MDS diagnosis in only 31 cases of the 53 samples that could be analyzed (58.5%) [ITP (n=18), MDS (n=10) or inconclusive, i.e. unclassifiable, (n= 25)]. 62 BM samples evaluated by NGF were allocated in 4 immunophenotypic groups attending to: maturation blockades, abnormal antigen expression and cross lineage markers. We observed normal phenotype cases (n=10), isolated (unilineage) alterations (n=24), mild multilineage (>1) alterations (n=20) and severe multilineage (MDS-like) phenotypes (n=8). Cytomorphology diagnosed our cases as ITP, MDS or unclassifiable with a median number of alterations observed by BM NGF of 4 (IQR, 3-6), 2 (IQR, 1-6) and 4 (IQR, 3-5) respectively. For ITP cytomorphology group, NGF demonstrated numerous BM alterations being monocytic alterations (n=17, 94%) the most frequent finding observed. MDS presumed cases were also associated with monocytic alterations (n=5, 50%) with a frequent decrease in neutrophil precursors (n=4, 40%). On the contrary, when cytomorphology was not capable to establish a diagnosis, NGF showed a mixture of alterations with no clear predominance of none of them (Table 1).

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Summary/Conclusion: Limitations of BM cytomorphology when facing an isolated thrombocytopenia were demonstrated here. However, normal or unilineage BM NGF alterations may lead us to an ITP diagnosis while mild or severe multilineage BM phenotypes may correlate good with MDS.

P1648: ANALYSIS OF ANKRD26 GENE 5’UTR VARIANTS IN A COHORT OF CZECH PATIENTS WITH SUSPECTED HEREDITARY HEMATOLOGICAL DISORDER

K. Stano Kozubik 1,2,* , J. Trizuljak 1,2,3 , Z. Vrzalova 1,2 , L. Radova 2 , I. Blahakova 1,2 , J. Stika 2 , P. Smejkal 4,5 , V. Bergerova 6 , J. Hynst 2 , S. Pospisilova 1,2,3 , M. Doubek 1,2,3

1 Department of Internal Medicine, Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University; 2 Center of Molecular Medicine, Central European Institute of Technology, Masaryk University (CEITEC MU); 3 Institute of Medical Genetics and Genomics; 4 Department of Clinical Hematology, University Hospital Brno and Faculty of Medicine, Masaryk University; 5 Department of Laboratory Methods; 6 Faculty of Medicine, Masaryk University, Brno, Czechia

Background: Variants in the 5‘UTR region c.-116C - c.-140C of ANKRD26 gene were described as causal for ANKRD26-related thrombocytopenia (THC2). This autosomal dominant disorder presents as mild to moderate thrombocytopenia with minimal symptoms but for the carrier means increased risk of malignancy, mainly myeloproliferation. Recently, the c.-140C>G variant was described in dbSNP in 6.2% non-Finnish European population.

Aims: This fact raises the question whether the ANKDR26 c.-140C>G variant is a pathogenic mutation responsible for the THC2 phenotype or, given its frequency, a population polymorphism.

Methods: Exome sequencing (WES) was performed on a cohort of patients with suspected inherited hematological disorder. WES results were confirmed by Sanger sequencing. Subsequently, the cohort of patients with a variant in 5’UTR in ANKRD26 gene was analyzed with respect to clinical symptoms. Finally, variants in the 5’UTR of ANKRD26 gene were searched in the genomes of the healthy Czech population.

Results: WES analysis was performed on the cohort of 53 patients from 31 families with following results: 12 individuals from 5 families carry heterozygous, 1 homozygous c.-140C>G variant, and 3 individuals from 1 family heterozygous c.-118C>T variant of ANKRD26 gene. No other gene variant was detected that was causal for their phenotype except for that found in the 5’UTR of ANKRD26 gene. The c.-118C>T variant was already published by several authors and was always associated with thrombocytopenia. From clinical data: Thrombocytopenia was present in 4 individuals: 1 with c.-140C>G variant and 3 with c.-118C>T variant. Moreover, 3 individuals with c.-140C>G variant were diagnosed with the oncologic disorder: 2 with essential thrombocythemia and 1 with multiple myeloma. Aggregation test results were available from 9 individuals from 4 families with c.-140C>G variant: 5 individuals showed platelet hypoaggregation after Collagen, ADP, and Epinephrine – Table 1. Besides, one individual showed hypoaggregation similar to von Willebrand disease IIB. Analysis of 176 healthy Czech individuals showed presence of only one 5’UTR ANKDR26 variant: c.-140C>G in 21 individuals (11.9%).

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Summary/Conclusion: We can conclude that most individuals in our cohort (7 out of 9) with the c.-140C>G variant in the 5’UTR of ANKRD26 gene show pathology in platelet aggregation, but only one of them has thrombocytopenia. In addition, three individuals with this gene variant developed malignancy. To answer the above question, whether the ANKRD26 c.-140C>G variant is pathogenic or population polymorphism, functional analysis of this variant must be performed. Our current data show that the ANKRD26 c.-140C>G variant is probably not that pathogenic, as was published.

P1649: GFI1B AND LSD1 REPRESS A MYELOID DIFFERENTIATION PROGRAM DURING INDUCED PLURIPOTENT STEM CELL DERIVED MEGAKARYOCYTE DIFFERENTIATION

J. Venhuizen 1,* , M. van Bergen 1 , D. Gilissen 1 , L. Wingens 2 , E. van den Akker 3 , J. Jansen 1 , J. Martens 2 , B. van der Reijden 1

1 Department of Laboratory Medicine, Laboratory of Hematology, RadboudUMC; 2 Department of Molecular Biology, Radboud Institute for Molecular Life Sciences, Nijmegen; 3 Department of Hematopoiesis, Sanquin Research, Amsterdam, Netherlands

Background: Megakaryocytic development is driven by the transcription factor Growth Factor Independence 1B (GFI1B) and its interaction with the chromatin remodeler Lysine Specific Demethylase 1 (LSD1/KDM1A). GFI1B aberrations are linked to inherited bleeding disorders, resulting in macro-thrombocytopenia, gray platelets and increased expression of CD34. One such mutation, which was previously show to be the disease-causing mutation in a family with gray-platelet syndrome, introduces a premature stop codon (Q287*) in the fifth zinc finger.

Aims: Here, we study the effect of the Q287* mutation on gene expression regulation, as well as GFI1B interaction with LSD1, during induced pluripotent stem cells (IPSC) derived megakaryocytic development.

Methods: We differentiated IPSCs that contain wildtype GFI1B or GFI1B Q287* into megakaryocytes. During differentiation, megakaryocytes derived from GFI1B wildtype IPSCs were also treated with an LSD1 inhibitor that disrupts GFI1B-LSD1 interaction for 24 hours. The transcriptomic landscape of differentiated IPSCs was explored using single-cell RNA sequencing and downstream computational analysis. We applied a novel filtering strategy, removing cells based on doublet annotation, mitochondrial gene count and exclusive differential expression of ribosomal or mitochondrial genes.

Results: After filtering and data integration we retained 14,848 cells. Within this population we were able to annotate six different cell types using single-cell GSEA and publicly available reference datasets. Megakaryocytes and megakaryocyte progenitors made up roughly 75% of all cells differentiated from IPSCs containing wildtype GFI1B. They are characterized by approximately 220 differentially expressed genes (p-value < 0.05, log-fold change > 0.69), including common megakaryocyte markers such as CD41 and CD42b. Strikingly, we also observed a population of myeloid and myeloid-progenitor cells after differentiation towards megakaryocytes. This population specifically expressed common myeloid markers, such as VIM, CD81, and SPI1 as well as approximately 200 other genes (p-value < 0.05, log-fold change > 0.69) and made up around 10% of the entire GFI1B wildtype cell population. This population was expanded two-fold in cells differentiated from GFI1B-wildtype IPSCs treated with the LSD1 inhibitor and three-fold in GFI1B Q287* IPSCs. Notably, in the GFI1B Q287* cell population megakaryocytes were reduced by 2-fold. Pseudotime analysis showed a differentiation trajectory starting at megakaryocytes and going towards myeloid cells. Megakaryocyte and platelet associated genes, such as CD41, CD42b and PF4, are gradually downregulated along this trajectory while myeloid associated genes exhibited a mirrored expression pattern. Thus, LSD1i treated and GFI1B Q287* IPSC-derived cells showed an increase in myeloid marker gene expression after differentiation and downregulation of coagulation-associated genes, hinting at a decreased potential to differentiate into megakaryocytes.

Summary/Conclusion: Our data show that GFI1B and LSD1 function to repress a myeloid cell fate during IPSC-induced megakaryocytic development. Disruption of the interaction between GFI1B and LSD1 and a dominant-negative GFI1B mutation results in myeloid differentiation at the expense of megakaryocyte development.

P1650: METABOLIC ABNORMALITY AND GLUCOCORTICOID RECEPTOR MODULATION OF MYELOID-DERIVED SUPPRESSOR CELLS IN IMMUNE THROMBOCYTOPENIA PATIENTS AND AN ACTIVE MURINE MODEL

S. Wang 1,* , J. Peng 1

1 Department of hematology, Shandong University, Jinan, China

Background: Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder in which pathologic antiplatelet antibodies bind to platelets and mark them for phagocytic breakdown, and imbalanced T cell subsets are also involved in its pathogenesis. Myeloid-derived suppressor cells (MDSCs) are circulating immature myeloid cells that suppress T-cell responses and play an important role in the immunopathogenesis of ITP. Glucocorticoids have been widely used for ITP treatment, however, approximately 30% of patients fail to respond to the glucocorticoid treatment or relapse in a short period. The biological action of glucocorticoids is mediated by glucocorticoid receptor (GR); however, the underlying mechanism of glucocorticoid-resistance remains unknown.

Aims: To investigate the role of GR in abnormal metabolism of MDSCs in glucocorticoid-resistant ITP patients.

Methods: We enrolled 36 ITP patients with newly diagnosed ITP who had received glucocorticoid treatment. For immunostaining of MDSCs’ mitochondria, human MDSCs were generated in vitro, stained with MitoTracker Red and anti-CD33 antibody. In addition, quantitative real-time RT-PCR and immunoblotting analysis were performed to analyze the expression of GR and CPT-1 at the mRNA and protein levels, respectively. And we prepared GR-silence MDSCs to explore the function of GR in MDSCs metabolism and transferred them into an active ITP murine model. Flow cytometry analysis was performed to detect T cell subsets of active ITP mice.

Results: The mean fluorescence intensity of MitoTracker Red was significantly weaker in CD33 + cells from patients with glucocorticoid-resistant ITP, indicating lower levels of mitochondrial physiology than healthy controls and glucocorticoid-sensitive patients. The expression of GR and CPT-1 were also decreased at both the mRNA and protein levels in those with poor response to glucocorticoids. Moreover, we performed an siRNA transfection test, targeting GR in WT murine MDSCs. Results showed that the expression level of CPT-1 was significantly lower in GR-siRNA treated MDSCs (GR-MDSCs) than that in NC-siRNA treated MDSCs (NC-MDSCs), indicating that the decreased expression of CPT-1 was affected by down-regulated GR in MDSCs. The active ITP murine model exhibited profound thrombocytopenia, and their platelet count decreased significantly by the second week after irradiation. Mice received GR-MDSCs treatment displayed significantly lower platelet levels than mice treated with NC-MDSCs. Subsequently, spleens were harvested from ITP mice at day 28 after irradiation, flow cytometry analysis revealed that the percentage of CD8 + T cells in mononuclear cells and Th1 cells in CD4 + T cells decreased significantly in the spleen of mice who received NC-MDSCs than GR-MDSCs, while the percentage of Tregs and Th2 in CD4 + T cells were increased in NC-MDSCs treated mice.

Summary/Conclusion: This study shed new light on the molecular mechanism of glucocorticoid-resistant of ITP patients that GR regulated mitochondrial metabolism of MDSCs, which modulated the immunosuppressive functions of MDSCs. This could possibly provide novel therapeutic targets for patients with poor response to glucocorticoids.

P1651: HDAC3 RS2530223 IS ASSOCIATED WITH INCREASED SUSCEPTIBILITY AND SEVERITY OF PRIMARY IMMUNE THROMBOCYTOPENIA

S. Wang 1,*

1 Department of hematology, Shandong University, Jinan, China

Background: Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder characterized by a low platelet count and increased risk of bleeding. We previously reported that low-dose chidamide, a histone deacetylase (HDAC) inhibitor, restores immune tolerance in patients with ITP.

Aims: To evaluate the association of a single nucleotide polymorphism (SNP) rs2530223 in the HDAC3 gene with susceptibility to ITP and its clinical features.

Methods: Patients with ITP and age-matched healthy participants were recruited for this case-control study. Accordingly, from September 2017 to February 2021, 209 patients, diagnosed with primary ITP according to the ITP international guidelines were recruited. The genotyping of the SNP of HDAC3 (rs2530223) was conducted by the time-of-flight mass spectrometry system. And SPSS software was used for statistical analyses.

Results: Our results revealed that the T allele exhibited a 1.472-fold increased risk of ITP susceptibility (OR 1.472; 95% CI 1.100–1.969; p = 0.009), the TT genotype under the codominant and recessive models, and the TC/TT genotypes under the dominant model all revealed increased risk of ITP susceptibility (dominant OR 1.965; 95% CI: 1.046–3.656; p = 0.036; codominant OR 2.264; 95% CI 1.175–4.360; p = 0.015; and recessive OR 1.512; 95% CI 1.028–2.224; p = 0.036, respectively). Regarding platelet counts in ITP patients, we observed that the TC/TT genotypes exhibited a 3.932-fold increased risk for PLT <30×10 9 /L (OR 3.932; 95% CI 1.426–10.842; p = 0.008).

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Summary/Conclusion: This study indicates that HDAC3 rs2530223 may be an important genetic factor related to ITP susceptibility and platelet count in ITP patients, providing new perspectives on disease progression, new therapeutic targets, and severity prediction.

P1652: MACHINE-LEARNING-BASED MORTALITY PREDICTION OF ICH IN ADULTS WITH ITP: A NATIONWIDE REPRESENTATIVE MULTICENTRE STUDY

X.-H. Zhang 1 , S. Chong 1,* , P. Zhao 1 , R.-B. Huang 2 , H. Zhou 3 , J.-N. Zhang 4 , Q.-S. Huang 4 , H.-X. Fu 4 , Q. Jiang 1 , H. Jiang 1 , J. Lu 1 , X.-J. Huang 1

1 Peking University People’s Hospital, Beijing; 2 The First Affiliated Hospital of Nanchang University, NanChang; 3 Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou; 4 Peking University Cancer Hospital and Institute, Beijing, China

Background: ITP is more likely to be persistent or chronic in adults, whereas it is a self-limiting disease in children. Although mortality in patient with ITP is only slightly higher than in the general population, severe bleeding events such as intracranial haemorrhage (ICH) are often considered to be associated with a poor prognosis. Bleeding is considered to be one of the important clinical outcomes in patients with ITP. According to previous studies, ICH occurs in approximately 1% of ITP patients, with a mortality rate of 24.0%-31.2%, and is considered a fatal complication of ITP. (Blood, 2009; Platelets, 2021) However, large-scale clinical studies on mortality and associated risk factors are still lacking.

Aims: To identify 30-day mortality and associated risk factors for ICH in ITP adults. To develop and validate a machine learning model to predict 30-day mortality. To establish an application for mortality prediction.

Methods: A national real-world study of ICH in adult ITP patients was conducted using data from 27 centres. The clinical characteristics and mortality of these patients were summarized. In addition, we identified risk factors for 30-day mortality by least absolute shrinkage and selection operator (Lasso) regression in a training cohort of 16 centres. We developed 10 machine learning models with algorithms including support vector machine (SVM), k-nearest neighbour (kNN), logistic regression, linear discriminant analysis (LDA), decision tree, random forest, gradient boosted decision tree (GBDT), adaptive boosting (AdaBoost), extreme gradient boosting (XGBoost), and light gradient boosting machine (LGBM). We then evaluated the performance of the models with metrics including the receiver operating characteristic (ROC) area under the curve (AUC), accuracy, sensitivity/recall, specificity, positive predictive value (PPV)/precision, negative predictive value (NPV), and F1 score by 10-fold cross-validation in the training cohort and independent external test cohort with data from 11 other geographically separate centres. We selected the best-performing model and further established an application to predict 30-day mortality for ICH in adults with ITP.

Results: The mortality rate of ICH was 33.8% in 142 adults with ITP. Ninety (65.7%) patients had a platelet count of /L or less at the time of ICH. The parenchyma was the most commonly affected region (N=75, 58.14%). Intraparenchymal haemorrhage, the platelet count at the time of ICH, the coexistence of severe infection, prior severe bleeding events, and an absence of head trauma were considered potential risk factors for death, adjusted for a parameter (λ) of 1 standard error of the minimum criterion: 0.076 by lasso regression. In internal validation, the SVM model exhibited an optimal AUC of 0.879 ± 0.145 and an optimal F1 score of 0.748 ± 0.183. In independent external validation, the SVM model also exhibited an optimal sensitivity of 0.600 and an F1 score of 0.667. Therefore, a corresponding application (47.94.162.105:8080/ich/) was established for users to predict 30-day mortality in adult ITP patients at the time of ICH. The specific study process is depicted in Figure 1.

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Summary/Conclusion: ICH is a rare and life-threatening bleeding event in adult ITP patients. We developed a machine learning model using an SVM algorithm and validated its prediction of 30-day mortality.

P1653: CLINICAL EFFICACY AND SAFETY OF SWITCHING ELTROMBOPAG AND RH-TPO IN PATIENTS WITH IMMUNE THROMBOCYTOPENIA DURING THE COVID-19 PANDEMIC

X. Cai 1,* , X.-H. Zhang 1

1 Peking University Institute of Hematology, Peking University People’s Hospital, Beijing, China

Background: The current COVID-19 pandemic poses several dilemmas for the management of immune thrombocytopenia (ITP). Patients taking immunosuppressive agents have been considered potentially at risk for severe acute respiratory syndrome coronavirus 2 infection. In addition, clinical studies have reported significantly higher fatality rates and more severe complications in COVID-19 patients on immunosuppressive therapy (Lancet, 2020). Thus, a new prescription or increase in dose of an immunosuppressant is not proposed for ITP patients (Br J Haematol, 2020). Recommendations have been made that nonimmunosuppressive treatments, such as thrombopoietin (TPO) agents, be the first choice for ITP during the COVID-19 pandemic (ASH, 2020). The recombinant human TPO (rh-TPO) and eltrombopag are two distinct TPO receptor agonists (TPO-RAs) with different treatment mechanisms, side effects and administration routes. Based on previous clinical evidence, switching from one TPO-RA to another provides favorable outcomes.

Aims: Therefore, we proposed a prospective observational study to evaluate the response and safety of switching between eltrombopag and rh-TPO in adult ITP patients during the pandemic for the first time.

Methods: Adult ITP patients who received rh-TPO and then needed to switch to eltrombopag or vice versa were screened for eligibility. After enrollment, patients were followed up prospectively on administering the second TPO-RA.

Results: Between January 2020 and January 2021 at Peking University People’s Hospital, 53 patients switching from rh-TPO to eltrombopag and 43 patients switching from eltrombopag to rh-TPO were enrolled in our study after screen. Overall, 61/96 patients (63.5%) achieved a response at 6 months after switch. The outcomes were comparable regardless of the direction of the switch. Response rates at six months after switch (the primary endpoint) varied significantly among patients in different clinical settings (Figure 2). In the inefficacy group, 13 of 27 patients (48%) responded to eltrombopag, and 11 of 22 patients (50%) responded to rh-TPO at 6 months after switch. In the non-efficacy-related group, 85% and 71% of patients in eltrombopag and rh-TPO group maintained their response rates, respectively. The primary outcomes were comparable between eltrombopag and rh-TPO group. In the inefficacy group, there were no differences in the secondary outcomes of platelet counts, time to response, duration of response and bleeding events after switch between eltrombopag and rh-TPO group. In the non-efficacy-related group, the increase of the platelet counts after switch were also comparable between eltrombopag and rh-TPO group. Similar to the platelet counts, the response rates at 6 months, time to response, duration of response and incidence of bleeding after switch were also comparable between the 2 groups. A total of 13 patients underwent switch due to side effects, and most of these patients had their adverse events resolved during treatment with the second TPO-RA. Both after-switch treatments were well tolerated and adverse events were all grade 1–2 in severity. None of these patients were diagnosed with COVID-19 infection during the follow-up period. We did not identify any risk factor that could predict better outcomes at 6 months after switch.

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Summary/Conclusion: In the current COVID-19 pandemic, our study demonstrated the efficacy and safety of switching between eltrombopag and rh-TPO as treatments for ITP. Thus, switching to the alternate TPO-RA may be a desirable option in patients refractory or intolerable to their first TPO-RA.

P1654: COMPLEMENT ACTIVATION REGULATES PLATELET HOMEOSTASIS BY ARRESTING THE ANTI-INFLAMMATORY EFFECTS OF INTERLEUKIN-35 IN IMMUNE THROMBOCYTOPENIA

R.-Y. Gui 1 , Y. Wu 1,* , Y.-X. Chen 1 , F.-Q. Liu 1 , Q.-Y. Qu 1 , X.-J. Huang 1 , X.-H. Zhang 1

1 Peking University Institute of Hematology, Peking University People’s Hospital, Beijing, China

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune haemorrhagic disease. Enhanced complement activation has long been considered one of the major pathogenesis of ITP. Bone marrow (BM) mesenchymal stem cells (MSCs) are multipotent stromal cells with multidifferentiation capacities, haematopoiesis support and immunomodulatory effects. Our previous study confirmed that enhanced complement activation could induce apoptosis and dysfunction of MSCs in ITP and alterations in the location of megakaryocytes (MKs) in the thrombopoietic niche in BM ( Haematologica , 2019). The results strongly indicated that enhanced complement activation in the BM niche of ITP plays a role in the pathogenesis of ITP by influencing platelet formation. However, the roles of the complement system in the maturity and differentiation of MKs or in peripheral platelet destruction remain poorly understood.

Aims: To explore the effects of complement activation on platelet homeostasis in ITP.

Methods: Thirty consecutive patients with ITP and 10 healthy donors were enrolled in our study. C1q, C4d, C3b and C5b-9 on the surface of MSCs and in peripheral blood from ITP patients and healthy volunteers were detected by indirect enzyme linked immunosorbent assay (ELISA). MSCs were isolated from BM samples and cocultured with human naive conventional T (Tconv) cells in Transwell culture dishes. iTr35 subsets were measured by flow cytometry. Enzyme-linked immunosorbent assay kits were used for the quantification of IL-35. The key transcription factors of T cell differentiation were detected by real-time quantitative-PCR analysis.

Results: Sixteen ITP patients were assigned to the group with sublytic C5b-9 assembly on MSC membranes (MSC-ITP-C+ group), while the remaining 14 patients were assigned to the group without complement activation (MSC-ITP-C- group). The MSC-ITP-C+ group showed fewer iTr35 subsets ( P <0.05) in BM than the MSC-ITP-C- group and healthy donors (MSC-Control group). Coculture with MSCs-ITP-C+ and Tconv cells resulted in a relatively lower number of iTr35 subsets and lower IL-35 levels. The addition of prostaglandin E2 restored Tconv cell proliferation in a dose-dependent manner and suppressed differentiation. Peripheral blood CD34+ progenitors from the MK-ITP group and MK-Control group were differentiated into megakaryocytes in vitro. The IL-35 receptors gp130 and IL-12Rβ were found to be expressed on human MKs, suggesting a role for IL-35 during megakaryopoiesis. When blood CD34+ cells were cultured for 10 days, the gp130 level in the MK-ITP group was significantly lower than that in the MK-Control group ( P <0.0001), and the IL-12Rbeta level in the MK-Control group was significantly higher than that in the MK-ITP group ( P <0.0001). Decreased CD41+ cells and CFU-MKs were also observed in the MK-ITP group. In vitro, the effect of IL-35 on CD34+ progenitor cells (MK-ITP+IL-35 group) was measured. Compared to the MK-ITP group, gp130 was higher on Day 10 in the MK-ITP+IL-35 group ( P =0.017). The addition of IL-35 increased the total number of viable cells and CD41+ cells by approximately 10% on average through the phosphorylation of STAT1 and STAT3. Similar effects were also observed in ITP mice.

Summary/Conclusion: Sublytic C5b-9 deposition on MSCs displayed a defective role in regulating iTr35 subsets, resulting in the downregulation of IL-35 in ITP. Defective megakaryopathies in ITP were also correlated with the downregulation of the IL-35 receptor. This study sheds light on a novel mechanism of ITP pathogenesis and provides potential therapeutic targets in ITP patients.

P1655: PERSONALIZED MACHINE-LEARNING-BASED PREDICTION FOR CRITICAL IMMUNE THROMBOCYTOPENIA BLEEDS: A NATIONWIDE DATA STUDY

Z.-Y. An 1,* , Y.-J. Wu 1 , R.-B. Huang 2 , H. Zhou 3 , Q.-S. Huang 4 , H.-X. Fu 4 , Q. Jiang 1 , H. Jiang 1 , J. Lu 1 , X.-J. Huang 1 , X.-H. Zhang 1

1 Peking University People’s Hospital, Beijing; 2 The First Affiliated Hospital of Nanchang University, NanChang; 3 Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou; 4 Peking University Cancer Hospital and Institute, Beijing, China

Background: The rare but important critical bleeding events in primary immune thrombocytopenia (ITP) are life-threatening complications in ITP patients, severely affecting their prognosis, quality of life, and treatment decisions. However, critical ITP bleeds, including intracranial haemorrhage and some gastrointestinal bleeding and respiratory bleeding, have not been adequately studied. This suggests an unmet need for personalized patient management strategies and an urgent need for effective tools to predict the risk of critical ITP bleeds in hospitalized patients in medical practice.

Aims: The aim of this study was to develop a clinical prediction model based on a nationwide multicentre study in China on behalf of the National Cooperative ITP Working Group, integrating clinical and laboratory data.

Methods: We first conducted a multicentre, retrospective cohort study with consecutive unselected inclusion of ITP inpatients from 8 medical centres in China from December 2002 to September 2019. After the development and external validation of the model using machine learning algorithms, we established a prospective observational cohort of 37 medical centres across China from November 2021 to January 2022 to further assess model efficacy. The study was registered at ClinicalTrials.gov ( NCT05116423 ).

Results: We built a model including 9 simple predictors, including the initial PLT count, age, age at onset, infection, ITP type, skin and mucosa bleeding, low PLT count (below 20*10 9 ), uncontrolled diabetes, and cardiovascular disease. Through the development and validation of nine different machine learning algorithms, the random forest algorithm outperformed the others. The random forest algorithm reached an area under the receiver operating characteristic (ROC) curve (AUC) of 0.901 in independent external validation. The model showed good calibration capabilities, and the decision curve analysis showed a high average of predicted net benefit in the risk threshold range of 0-95%. The model is presented in the form of a web application ( http://47.94.162.105:8080/mavenitp/ ). The model in further prospective validation at 37 medical centres nationwide showed an AUC of 0.776, demonstrating good predictive efficacy.

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Summary/Conclusion: Our findings suggest that this machine-learning-based model based on 9 simple and easily available predictors provides good performance in predicting the occurrence of critical bleeds in ITP patients and has demonstrated good efficacy in a national multicentre prospective validation. A web-based online tool based on this algorithm rapidly identifies the bleeding risk profile of ITP patients to support clinical decision-making and reduce the occurrence of adverse outcomes.

P1656: EVALUATION OF PLATELET FUNCTION IN RARE OR UNDIAGNOSED BLEEDING DISORDERS

P. Acuña Butta 1,* , E. Monzón Manzano 1 , E. García Arias-Salgado 1 , E. García Pérez 1 , M. T. Álvarez Román 1 , M. Martín Salces 1 , M. I. Rivas Pollmar 1 , A. dos Santos 1 , V. Jimenez Yuste 1 , N. V. Butta Coll 1

1 Hematology, Hospital Universitario La Paz, Madrid, Spain

Background: Qualitative and/or quantitative platelet defects promote bleeding and its identification requires a careful clinical evaluation and a rational use of diagnostic laboratory assays.

Aims: To study platelet function by different methods in patients with hemorrhagic symptoms who are suspected of thrombopathy to compare results obtained with each system and validate usefulness of methods employed.

Methods: We included 72 patients with bleeding suspected of thrombopathy, which in some cases was confirmed by genetic study (Table 1). Patients with coagulation disorders were excluded. We used the following methods to test platelet function: Light aggregometry in platelet-rich plasma (PRP) using ADP, collagen, epinephrine and arachidonic acid as stimulating agonists. Flow cytometry in PRP using TRAP and ADP to induce platelet activation, and PAC1 monoclonal antibody (mAb) to recognize activated fibrinogen receptor, and anti P- selectin and anti-CD63 mAbs to test, respectively, release from alpha and dense granules. PFA-100 ® in whole citrated blood with collagen +ADP and collagen+Epinephrine cartridges. Total Thrombus-formation Analysis System (T-TAS ® 01, Zarcos, Japan) with T-TAS ® PL-chip (capillaries covered with collagen / under flow) in blood samples drawn in BAPA, a potent synthetic anticoagulant which inhibits Factor Xa and thrombin.

Results: Some bleeding due to platelet dysfunction was diagnosed by any of the methods used, but others can only be detected with some of the techniques tested (Table 2). This difference in the sensitivity of the methods may be due to the different experimental conditions used in each of them: type of sample used, platelets’ activators, and flow vs static conditions. Forty % of undiagnosed bleedings did not seem to be related to platelet dysfunction.

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Summary/Conclusion: All methods are complementary and their sensitivity depends on the pathway evaluated by the technique used and the mechanism involved in platelet dysfunction.

P1657: PATTERNS AND PATIENT SPECIFIC FEATURES CHARACTERIZING BLEEDING EVENTS IN PATIENTS ON ANTIPLATELET AND ANTICOAGULANT THERAPY: A TERTIARY CENTER EXPERIENCE

H. Awada 1,* , A. Hajj Ali 1 , H. Nassereldine 1 , M. Zeineddine 1

1 American University of Beirut Medical Center, Beirut, Lebanon

Background: Acute coronary syndrome and atrial fibrillation are among the major causes of morbidity and mortality in society. Treatment of these conditions requires the prescription of oral anticoagulants (OAC) and antiplatelets (AP). These drugs cause an increased risk of developing bleeding, a condition associated with a 60% increased risk of in-hospital death.

Aims: Physicians often have to find the balance between preventing the formation of thrombi without causing a significant increase in the bleeding risk of the patient. Here, we report our findings with regards to the incidence and patterns of bleeding admissions at a tertiary care center. Our goal is to identify patient-specific factors that can predict sites of bleeding if it occurs.

Methods: We retrospectively reviewed the electronic charts of patients who were admitted for bleeding events at the American University of Beirut Medical Center (AUBMC). Baseline and admission demographic, social and clinical characteristics were obtained. The patients’ history of antiplatelets and anticoagulants was obtained. Sites of bleeding were categorized as Intracranial, Upper gastrointestinal (Upper GI), Lower gastrointestinal (Lowe GI), Respiratory tract, and Genitourinary (GU). Patients who had bleeding with trauma were excluded. Continuous variables are reported as means with standard deviations, while categorical variables are presented as percentages. Categorical variables were compared using the chi-squared test while the t test and ANOVA test were used for analysis of continuous variables.

Results: We identified 343 adult patients who were admitted for bleeding at AUBMC. The average age was 72.44 ± 12.53 with a Body Mass Index (BMI) of 27.94 ± 5.17. Off all patient, 17.8% were alcohol users while 28.9% had a history of smoking. Bleeding sites included 23.24% Intracranial, 11% Respiratory, 22.32% Upper GI, 29.35% Lower GI, and 14.06% GU bleeding. Medications included single antiplatelet in 114 patients, dual antiplatelets in 31 patients, single antiplatelet and anticoagulant in 23 patients, and anticoagulant in 74 patients. Patient with older age with significantly more likely to experience Lower GI bleed while on antiplatelet or anticoagulants that at any other site (p=0.009). In contrast, patients with alcohol use were more likely to experience intracranial bleeding events (p=0.012). No difference in bleeding location was noted amongst current smokers, ex-smokers or never smokers (p=0.103) or with BMI levels (p=0.3). Medical history of Hypertension, Diabetes Mellitus or Atrial Fibrillation did not affect bleeding sites. Patients only on anticoagulation were more likely to have lower GI bleed than at other sites (p=0.001), however; no difference in bleeding sites was found in patients on a single antiplatelet, dual antiplatelet, or single antiplatelet and anticoagulation.

Summary/Conclusion: Location of bleeding events in patients on antiplatelets or anticoagulants is influenced by age and alcohol use but not smoking. Patient on anticoagulants are most likely to bleed from Lower GI, while no difference in bleeding site exists in patients on single antiplatelet, dual antiplatelet, or single antiplatelet and anticoagulation.

P1658: DELAYED REVERSAL OF FACTOR V INHIBITOR ACTIVITY POST-IMMUNOSUPPRESSIVE THERAPY, FOLLOWED BY A SUCCESSFUL PREGNANCY: A CASE REPORT

A. Cegledi 1,* , I. Bodo 2 , J. Dolgos 1 , A. Varkonyi 1 , B. Vilimi 1 , G. Mikala 1

1 Department of Hematology and Stem Cell Transplantation, South Pest Central Hospital, National Institute of Hematology and Infectious Diseases; 2 Department of Internal Medicine and Hematology, Semmeweis University, Budapest, Hungary

Background: Acquired factor V inhibitor (AFVI) is a very rare autoimmune hemostatic disorder that leads to hemorrhagic manifestations of variable severity.

Aims: We describe a case of a 35-year-old Caucasian female patient presenting with severe AFVI with life-threatening hemorrhages. The AFVI exhibited spontaneous reversal over several years after discontinuation of ineffective combination immunosuppressive therapies and subsequently a successful pregnancy.

Methods: Diagnosis of AFVI was based on a marked prolongation of prothrombin time-international normalized ratio (INR) and activated partial thromboplastin time (APTI) not corrected by the addition of normal plasma (1:1 ratio), with FV activity reduced to <1.0%. Other coagulation factor activity levels were unchanged, and the presence of an FV inhibitor was revealed by the Bethesda inhibitor assay.

Results: Over the course of 2.5 years, plasmapheresis plus immunoglobulins and different combination immunosuppressive regimens were adminstered (dexamethasone+rituximab; cyclophosphamide+dexamethasone + rituximab + cyclosporine; cyclosporine + sirolimus + cyclophosphamide + dexamethasone; bortezomib+sirolimus+methylprednisolone; sirolimus+mycophenolate mofetil). Coagulation could be effectively maintained using recombinant FVIIa.

The combination immunosuppressive therapies resulted in intermittent reversal of AFVI over 2.5 years, which then became therapy-resistant. After discontinuation of the unsuccessful immunosuppressive therapies, the patient exhibited partial spontaneous remission: her FV activity gradually increased from ~1% to ~33% over the course of 5 years. The patient became pregnant and during her pregnancy, FV activity increased to 54%, and her coagulation parameters were normalized. She gave birth to a healthy child by Caesarean section without any bleeding complications.

Summary/Conclusion: Patients with AFVI presenting with hemorrhagic manifestations should be treated aiming for hemostatic control as well as inhibitor eradication. The use of an activated bypassing agent for hemostatic control is effective in AFVI. The presented case is unique because the treatment regimens used included combinations of multiple immunosuppressive agents and also suggest that patients with AFVI resistant to combination immunosuppressive protocols receiving no therapy may undergo spontaneous remission. Long-term monitoring of the coagulation status and the anti-FV inhibitor levels is warranted.

P1659: UNIQUE GENOTYPE-PHENOTYPE RELATIONSHIP IN VON WILLEBRAND DISEASE: A SINGLE CENTRE EXPERIENCE

M. Elshinawy 1,2,*

1 Pediatric Hematology, SULTAN QABOOS UNIVERSITY HOSPITAL, Muscat; 2 Pediatric Hematology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt

Background: von Willebrand Disease (VWD) is the most common hereditary coagulation disorder. Generally, coagulation disorders are more prevalent in communities with high rate of consanguinity. Due to segregation of multiple alleles, combinations of abnormal variants causing unique clinical presentations can occur.

Aims: We aim to present our single centre experience of VWD in pediatrics.

Methods: Retrospective data analysis of all pediatric patients diagnosed with (VWD) throughout a period of 10 years, dating from January 2012 till December 2021. In addition, molecular testing using next generation sequencing (NGS) of a family, with a spectrum of bleeding phenotypes was done.

Results: Out of 455 cases with hereditary coagulation disorders, 219 (48%) were diagnosed with VWD throughout the study period (141 girls and 78 boys), with an age of presentation ranging from 18 months to 14 years. Significant bleeding episodes were encountered in 42/219 patients (19%). History of consanguinity was documented in 175 cases (80%). The most common presenting feature was mucocutaneous bleeding, followed by menorrhagia and then post-traumatic/post-surgical hemorrhage. Factor replacement therapy was used in 34 cases and desmopressin (DDAVP) in 22 cases. Hormonal therapy was offered to 18 girls presenting with menorrhagia. However, only 7 of them were compliant.

The index case is a 4-year-old girl, presented with profuse post-adenotonsillectomy hemorrhage. Her past history was negative for any significant bleeding, but no previous surgical challenges. Parents are cousins, and family history was irrelevant. Routine preoperative investigations didn’t reveal any abnormalities apart from raised mean platelet volume (14 fL), and a platelet count of 180 × 10ˆ9/L. Bleeding didn’t respond optimally to FFP, but controlled with multiple platelet transfusions and recombinant activated factor VII.

Molecular studies using NGS revealed two different heterozygous variants in the CD36 gene. The first heterozygous variant was NM_001001547.2 :c338_339del (p.Ser113Phefs*20), a frameshift variant which results in loss of function of the protein product of CD36 gene. The second was deletion of exons 2 and 3 ( NM_001001547.2 :c.1_120del), which results in loss of a significant portion of the coding region of CD36 gene. To our knowledge, both novel variants have not been reported in the literature. Biallelic mutations in CD36 are associated with platelet glycoprotein IV deficiency (PG4D), characterized by macrothrombocytopenia with variable bleeding phenotype (PubMed: 7533783; OMIM: 173510). In addition, A heterozygous variant in the VWF gene, NM_000552.4 :c.3692A>G (p.Asn1231Ser), was identified.

This heterozygous variant was reported in a case with von Willebrand disease type 1 (PubMed: 28971901). The distinctive combination of these three variants explains the severe bleeding phenotype. NGS identified the same heterozygous frameshift variant of CD36 in the mother, while the father carries both the deletional variant of CD36 and the missense variant of VWF gene. Two sisters (ages: 8 & 15 years) harbor the heterozygous VWF gene variant. On further enquiries, the eldest was found to have mild to moderate menorrhagia, that didn’t require any medical advice. Family pedigree is shown in figure 1.

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Summary/Conclusion: Genotype-phenotype correlation is mandatory in coagulation disorders, presenting with significant bleeding episodes.

P1660: PERIPROCEDURAL MANAGEMENT IN COMBINED DEFICIENCY OF FACTOR V AND FACTOR VIII: EXPERIENCE FROM A SINGLE CENTER IN SPAIN

E. FERNANDEZ MELLID 1,* , P. MELERO VALENTIN 1 , P. CADAHIA FERNANDEZ 1 , A. DE ANDRES Y JACOB 1 , L. BAO PEREZ 1 , A. ABUIN BLANCO 1 , R. FERREIRO FERRO 1 , A. PELETEIRO RAINDO 1 , M. RODRIGUEZ RUIZ 1

1 HEMATOLOGIA Y HEMOTERAPIA, COMPLEJO HOSPITALARIO UNIVERSITARIO DE SANTIAGO DE COMPOSTELA, SANTIAGO DE COMPOSTELA, Spain

Background: Inherited combined factor V (FV) and factor VIII (FVIII) deficiency is a rare disorder with typically mild bleeding symptoms. There is limited data about replacement therapy which is usually only required before surgical procedures or traumatic hemorrhages.

Aims: Revision of hemostatic treatment and bleeding control in FV+FVIII deficiency patients after surgical procedures. Evaluation of effectiveness and possible adverse events.

Methods: We described 3 patients with combined mild-moderate FV+FVIII deficiency. Characteristics of the patients are described in Table 1. There were 8 relevant surgical procedures between 2014 and 2021 performed with previous administration of frozen fresh plasma and/or FVIII replacement. Antifibrinolytic agents were used depending on the patient comorbidities and surgical site. Characteristics of procedures are described in Table 2. Table 1

Results: No relevant bleeding events were documented and all procedures were successfully completed. FFP dosage has been adapted not only to the FV level but also to patient comorbidities to avoid congestive cardiac symptoms. Desmopressin has only been used in one patient for a minor procedure.

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Summary/Conclusion: FFP and FVIII factor replacement is a safe measure for periprocedural preparation, and the correspondent dose can be adapted to patient characteristics including bleeding phenotype, comorbidities and surgical site without increasing hemorrhagic risks. Desmopressin maybe underused.

P1661: THROMBOPATHY IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES AND AUTOIMMUNE DISEASE

E. Garcia Perez 1,* , E. Garcia Arias-Salgado 1 , P. Acuña Butta 1 , E. Monzon Manzano 1 , M. T. Alvarez Roman 1 , M. Gutierrez Alvariño 1 , M. I. Rivas Pollmar 1 , M. Martin Salces 1 , A. Dos Santos Ortas 1 , N. V. Butta Coll 1 , V. Jimenez Yuste 1

1 Hematology, Hospital Universitario La Paz, Madrid, Spain

Background: Many patients with myelodysplastic syndromes (MDS) have associated autoimmune diseases due to the presence of clonality. The presence of non specific antibodies in these patients can alter hemostasis.

Aims: We aim to study if there were differences in hemostasis of patients with MDS, depending on the coexistence or not of an autoimmune disease (AD) employing global coagulation tests, and flow cytometry to evaluate platelet function.

Methods: This is an observational and prospective study. We included 21 patients with MDS without any associated autoimmune disease and 15 patients with autoimmune disease (Table 1). 46 healthy controls were also recruited. The following studies were carried out:

- Platelet ability to be activated was tested by flow cytometry in platelet rich plasma using TRAP and ADP to induce platelet activation, and PAC1 monoclonal antibody (mAb) to recognize activated fibrinogen receptor, and anti P- selectin and anti-CD63 mAbs to evaluate, respectively, release from alpha and dense granules.

- Global assessment of clot formation by calibrated automated thrombinography (CAT) and rotational thromboelastometry (ROTEM®).

Results: Platelets from MDS patients, with and without AD, showed less capacity of activation compared to the control group. Despite there were no significant differences between the groups of patients with MDS, platelets from patients without AD showed a clear decrease in their ability to be activated by TRAP and was almost absent in three of them. Kinetics of clot formation evaluated by ROTEM® showed that all MDS patients had a hypocoagulable profile. Moreover, thrombin generation was lower in patients with MDS compared to controls. We observed that in the group of patients with AD the peak of thrombin and the endogenous thrombin potential were much lower than in patients without AD (p<0.01). Accordingly, patients with AD presented a longer lag time and time to peak than patients without AD (p<0.05).

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Summary/Conclusion: Our results suggested the presence of non-specific antibodies in plasma of patients with MDS and AD that impaired hemostasis resulting in a hypocoagulable profile.

P1662: OPTIMIZED PROPHYLAXIS BASED ON INDIVIDUALIZED TARGET TROUGH FVIII LEVEL IN PEDIATRIC PATIENTS WITH SEVERE HEMOPHILIA A

K. Huang 1,* , Z. Chen 2 , R. Wu 2 , Y. Zhen 2 , G. Li 2

1 Beijing Children’s Hospital, Capital Medical University; 2 Beijing Children’s Hospital, Beijing, China

Background: Due to the great inter-individual variability of pharmacokinetics and bleeding phenotype among children with severe hemophilia A(SHA), individualized prophylaxis is urgent and vital.

Aims: To investigate the effectiveness of our new-proposed PK-guided, trough level escalating dosing regimen.

Methods: Pediatric patients with SHA were enrolled. They followed a PK-guided, trough level escalating protocol. PK tests were conducted with a five-point assay to calculate individualized PK profiles. During the two-year period, clinical assessments were conducted every six months, including joint bleeds, ultrasound(US) scores and Hemophilia Joint Health Score(HJHS) of ankles, knees and elbows. A quantitative scoring system based on the above-mentioned clinical assessments was used to determine the necessity of trough level’s escalation.

Results: Fifty-eight SHA boys were finally analyzed. Their age and body weight were 5.3(2.8,6.9)years and 21.5(16,25)kg respectively. At baseline, 34 of them had a trough level of <1IU/dL and the number remained 10 at study exit. During the study period, 47 escalations were observed. The most frequent reason was joint bleeds(59.6%, N=28). Significantly reduced annualized bleeding rate[0(0,6) vs. 4(0,8), P <0.0001] and annualized joint bleeding rate[0(0,0.25) vs. 0(0,2), P <0.0001] was observed at study exit as well as the continuous trend of decreased bleeding rates. Zero bleeding rate and zero joint bleeding rate raised from 27.6% to 69% and 46.5% to 81.3% respectively. In addition, 85%(6/7) of the target joints vanished at study exit. Statistical improvements of US scores( P =0.04) and HJHS scores( P =0.02) were also reported at end of study.

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Summary/Conclusion: This protocol individualized target trough level and optimized clinical outcomes in pediatric patients with severe hemophilia A.

P1663: THE EFFECTS OF OBESITY ON BLEEDING FREQUENCY AND PARAMETERS OF FIBRINOLYSIS IN PATIENTS WITH NON-SEVERE HEMOPHILIA

O. Königsbrügge 1,* , J. Rejtö 1 , D. Kraemmer 1 , G. Schuster 2 , C. Feistritzer 3 , J. Gebhart 1 , C. Ay 1 , I. Pabinger 1

1 Medical University of Vienna, Vienna; 2 Red Cross Upper Austria, Linz; 3 Medical University of Innsbruck, Innsbruck, Austria

Background: Obesity leads to increased strain on muscles and joints and could be a contributing factor to the burden of disease in patients with non-severe hemophilia. Fatty tissue, however, also plays an active part in the production of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) and is considered to transition the balance of hemostasis towards a reduction of fibrinolysis.

Aims: We aimed to investigate the effects of obesity on the frequency of bleeding and changes in parameters of fibrinolysis in persons with non-severe hemophilia.

Methods: Persons with non-severe hemophilia A or B (PwH) were recruited into a cross-sectional cohort study after informed consent. Frequency of bleeding in the past 5 years was retrospectively recorded. Body-mass index (BMI), FVIII or IX activity, t-PA antigen and activity, and PAI-1 activity were measured.

Results: The cohort includes 90 PwH (74 hemophilia A, 16 hemophilia B) with a median age of 49.6 years (25 th to 75 th percentile 34.5–59.9), and a median FVIII/FIX level of 12% (8–19%). The median BMI was 25.7 kg/m^2 (23.3–28.3), and 14 patients (15.5%) had BMI>30. The median number of bleeding episodes in the past 5 years was 0 (0-1).

In age-adjusted negative binomial regression, factor VIII/IX level (incidence rate ratio [IRR] 0.93 per 1% increase, 95% confidence interval [CI] 0.88-0.98, p<0.001), and BMI (IRR 0.82 per 1kg/m^2 increase, 95%CI 0.74-0.91, p<0.001) were independently associated with frequency of bleeding episodes. Factor VIII/IX levels (IRR 0.88, 95%CI 0.80-0.96, p=0.005) and BMI (IRR 0.68, 95%CI 0.55-0.82) were also independently associated with the frequency of joint and muscle bleeding.

BMI correlated with PAI and t-PA antigen levels (rho 0.241, p= 0.025 and 0.257, p= 0.017) and correlated inversely with t-PA activity (rho -0.240, p=0.026).

Summary/Conclusion: While level of FVIII or FIX are the main determinants of bleeding severity, there is considerable heterogeneity in the bleeding phenotype irrespective of factor level. We found that in non-severe hemophilia patients, increasing BMI decreased the number of bleeding episodes and joint and muscle specific bleeding episodes. The bleeding severity was potentially mitigated by increasing PAI levels and decreasing t-PA activity.

P1664: IMPACT OF A PROTOCOL USING RITUXIMAB FOR PREVENTION OF RELAPSE IN PATIENTS WITH ACQUIRED THROMBOTIC THROMBOCYTOPENIC PURPURA (ATTP)

E. Marín Sánchez 1,* , E. Fernandes 2 , J. López Marín 2 , F. Tarín 2 , L. Hernandez Mateo 2

1 Hospital General Universitario Alicante, Alicante, Spain; 2 Hematology, Hospital General Universitario Alicante, Alicante, Spain

Background: aTTP is a life threatening disease mediated by auto-antibodies directed against ADAMTS13, with reduced activity and high expression of von Willebrand factor large multimers, microthrombi and thrombopenia. Since ADAMTS13 activity is used as a marker for aTTP, it can be used to monitor the emergence of relapses.

Aims: To establish a follow-up protocol to anticipate and prevent clinical relapsing disease.

Methods: Since February 2015, a protocol has been developed consisting of regular monitoring of ADAMTS13 activity every six months. Rituximab was started when activity was less than 10%, without waiting for the clinical relapse.

24 patients were enrolled, 2 were excluded because of congenital TTP and 2 died in the first 24 h. A total of 20 patients with aTTP have been followed up by the HGUA Hematology Service.

The number of relapses before and after the application of the protocol was analized, calculating the annual rate of relapses for each of the follow-up periods. A statistical hypothesis testing was carried out (Wilcoxon).

Results: Patients had a mean age of 49.55 ± 14.15, 30% (6) male and 70% (14) female. Cumulative period pre-protocol (years, mean): 5.90 ± 7.89 and cumulative period post-protocol (years, mean): 5.65 ± 1.98. Pre-protocol relapse rate: 0.26 ± 0.46 and post-protocol relapse rate: 0.02 ± 0.08. Clinical relapses prevented (treated with rituximab): 9. Hypothesis contrast (Wilcoxon): p value 0.007

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Summary/Conclusion: The use of a protocol for ADAMTS13 monitorization, and the administration of preventive rituximab reduce the number of clinical relapses in patients with acquired TTP under follow-up.

P1665: WHOLE-BLOOD ROTATIONAL THROMBOELASTOMETRY (ROTEM) AS A DIAGNOSTIC TOOL IN PATIENTS WITH BLEEDING OF UNKNOWN CAUSE (BUC)

D. Mehic 1,* , B. Jilma 2 , C. Schörgenhofer 2 , M. Colling 1 , C. Ay 1 , I. Pabinger 1 , J. Gebhart 1

1 Clinical Division of Hematology and Hemostaseology; 2 Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

Background: A majority of patients with a mild to moderate bleeding tendency (mild bleeding disorders, MBD) remains without a diagnosis, despite extensive laboratory investigations (bleeding of unknown cause, BUC). Patients with BUC do not differ in their bleeding phenotype to patients with an established MBD like von Willebrand disease (VWD) or platelet function defects (PFD). Whole-blood thromboelastometry (ROTEM) depicts diverse aspects of coagulation including hyper-fibrinolysis and captures the influence of erythrocytes, leucocytes and platelets to hemostasis, which might contribute to BUC.

Aims: To analyze clinical risk factors for ROTEM parameters and compare results between patients with BUC and an established MBD.

Methods: Patients from the Vienna bleeding biobank (VIBB, EC no. 603/2009) were analyzed (Gebhart et al, Haemophilia, 2018). Viscoelastic clot measurement was performed with non-activated ROTEM® (EM International, Munich, Germany): clotting time (CT, sec, time from analysis start until start of clot formation), clot formation time (CFT, sec, time needed to form a clot), maximum clot firmness (MCF, mm, peak amplitude of the clot) and maximal lysis (ML, %, expression of clot lysis in % of MCF).

Results: ROTEM was performed in 513/714 (71.8%) patients included in the VIBB. The mean(SD) age was 41.4 (15.0) years, 425 (80.3%) were women and 252 (47.6%) patients had blood group O. In total, 318 patients had BUC, the reaiming patients hat PFD(n=122, 23.7%), VWD (n=51, 9.9%), mild clotting factor deficiencies (CFD, n=18, 3.5%) and 4 had an other bleeding disorder (0.8%).

Clinical factors that were independently associated with ROTEM values in BUC patients were sex, blood group O (vs. non-O) and the bleeding score (multivariable linear regression, Table 1).

BUC patients had comparable ROTEM results as patients with platelet function defects or VWD, after adjusting for blood group O and sex, whereas patients with clotting factor deficiency had longer CT and CFT, a lower MCF and higher ML (Table 2).

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Summary/Conclusion: Hemostatic capacity measured by ROTEM is similar in patients with BUC and those with mild primary hemostatic bleeding disorders. Our data support the hemostatic imbalance in patients with BUC.

P1666: EFFECT OF AGONISTS TO THROMBOPOIETIN RECEPTORS ON PLATELET GLYCOME OF PATIENTS WITH IMMUNE THROMBOCYTOPENIA

E. Monzón Manzano 1,* , M. T. Alvarez Román 1 , P. Acuña 1 , E. G. Arias Salgado 1 , A. Ramírez 1 , M. Martín Salces 1 , M. I. Rivas Pollmar 1 , A. González Ceberino 1 , N. V. Butta 1 , V. Jiménez Yuste 1

1 Hematology, La Paz University Hospital, Madrid, Spain

Background: Platelet destruction and suppression of platelet production are involved in immune thrombocytopenia (ITP). Recently, attention has been drawn to the composition of platelet glycome in etiopathogenesis of ITP. We have previously reported that exposure of glycosides that might be involved in platelet destruction are increased in platelets from ITP patients (Figure 1A). Agonists to thrombopoietin receptors (TPO-RAs) have been shown to produce sustained platelet responses in ITP. Nevertheless, little is known about the effect of TPO-RAs on platelet’s features.

Aims: We aimed to study whether the treatment with TPO-RAs modified platelet function and glycoside composition of platelets from ITP patients

Methods: ITP patients were studied before and after achieving a response (defined as a platelet count ≥30×109 platelets/L and at least two fold increases over the baseline count) to TPO-RA therapy. Platelet activation markers (activation of fibrinogen receptor determined through PAC1-binding, and TRAP-induced P-selectin and CD63 exposure) and active caspase-3, -7, -8 or -9 were evaluated. Platelet surface glycan exposure was analysed by determining the binding of lectins to specific glycosidic residues. Samples were analyzed by flow cytometry. Results were expressed as the ratio between values obtained after responding/before treatment with TPO-RA.

Results: 16 patients were included [53.8% women, 58 (22–81) years, 46.2% with eltrombopag, 53.8% with romiplostim]. Increment of platelet count was variable and was accompanied by the recovery of platelet´s ability to be stimulated, especially the activation of fibrinogen receptor. TPO-RA treatment seemed to have a negligible effect on caspase activity. Regarding glycoside residues, exposure of ɑ-mannose, galactose and ß-GlcNAc showed a tendency to decrease (Figure 1).

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Summary/Conclusion: Our results showed that TPO-RA treatment appeared to recover platelet function and to restore a glycome composition similar to that found in a healthy state.

P1667: ALTERED COAGULATION SCREENING TESTS AT DIAGNOSIS ARE A COMMON FINDING IN ACUTE LEUKEMIAS OF BOTH LYMPHOID AND MYELOID LINEAGES, AND NOT JUST IN ACUTE PROMYELOCYTIC LEUKEMIA

M. Pereira 1,* , G. Marques 2 , J. Pêgo 2 , E. Cortesão 1 , C. Geraldes 1 , F. Rodrigues 2

1 Clinical Hematology Department; 2 Clinical Pathology Department, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal

Background: The acute leukemias (AL) often present with both pro-haemorrhagic and pro-thrombotic complications, through a variety of different mechanisms. Life-threatening consumptive coagulopathy with hyperfibrinolysis is well described in acute promyelocytic leukemias (APL), but non-promyelocytic myeloid leukemias (AML, most notably of monocytic/monoblastic lineage) and lymphoblastic leukemias (ALL) can also develop coagulopathy, often worsened by underproduction thrombocytopenia.

Aims: To characterize the presence of altered coagulation screening tests (CST) in (non-APL) AL at diagnosis.

Methods: We reviewed all patients (pts) over 18 years old (yo) who presented to our Center with a diagnosis of AL over a 6.5 year period up to 22.02.2022, and who had synchronous diagnostic smears, flow cytometry and CST including fibrinogenemia (Fib) and D-dimers (DD). Fib under 150 mg/dL was considered low (LowFib); DD over 230 ng/mL were considered increased (HiDD). Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were analysed as ratios compared to a control (CTR), and were considered increased (>) when over 120%.

Results: We studied 188 pts (52% male); 63.3% had AML, 19.7% ALL, 15.4% APL and 1.6% (3 pts) had a biphenotypic AL and were excluded. Ages ranged from 19.2-95.7 yo (mean: 60.3±17.4); AML patients were on average a decade and a half older (65.6±15.3; 51.8±15.3 in APL; 50.9±18.1 in ALL, p<0.001).

Within the cohort, 50.5% had a >PT ratio, 11.3% a >aPTT ratio and in 9.7% both were increased; 9.5% of pts had normal Fib and DD, 76.8% had HiDD with normal Fib, and 13.7% had both LowFib and HiDD. Overall, 92.6% of pts had at least one altered test out of four.

The mean PT ratio was 1.35±0.75 (1.36±0.31 in APL, 1.39±0.90 in AML, 1.18±0.27 in ALL, p=NS). The proportion of pts with >PT increased from ALL (21.6%) to AML (51.4%) to APL (65.5%), p<0.001. The mean aPTT ratio was 1.00±0.20 (0.94±0.14 in APL, 1.03±0.23 in AML and 0.96±0.13 in ALL, p=0.03); the proportion of pts with >aPTT was not significantly different. ALL pts were twice as likely to have both normal times (80.6%) than AML (39.8%) or APL (34.5%), p=0.001.

Mean Fib was lower in APL (178.6±93.4) than AML or ALL (415±199.5 and 351±154.5, p<0.001), with 42.3% of pts with LowFib (7.5% in AML and 0% in ALL, p<0.001). Although the proportion of pts with HiDD was similar in the 3 groups (95.2%, 86.0% and 94.1% in APL, AML and ALL, p=NS), APL pts were more likely to have DD>500 (95.2%, 66.7% and 70.6%, p=0.04), with mean DDs higher in APL (23510±32664) than AML or ALL (3464±6203 and 4399±7248, p<0.001). Overall, 84.7% of pts (including all ALL pts) with HiDD had normal Fib; APL pts were more likely to have both HiDD and LowFib than AML pts (42.9% and 7.0%, respectively, p<0.001).

Summary/Conclusion: We found that altered CST were almost universal in AL at diagnosis, irrespective of promyelocytic lineage, with >90% of pts presenting with at least one altered test, most often a prolonged PT – which was found in over half of pts, with its likelihood increasing from one-fifth of pts with ALL to two-thirds of pts with APL, and with ALL pts twice as likely to have normal coagulation times than AML or APL.

In concordance with the characteristic coagulopathy of APL, fibrinogenemia in our cohort was 50% lower in APL than in AML or ALL, with almost half of APL pts (but less than 10% of AML pts and no ALL pts) presenting with levels under 150 mg/dL. APL pts were also more likely to have DD over 500 ng/mL (with a mean value that was over five-fold higher than in AML or ALL)¸ and to have both HiDD and LowFib.

P1668: SYNERGY OF RED BLOOD CELLS AND TRANEXAMIC ACID IN THE INHIBITION OF FIBRINOLYSIS

A. Raska 1,2,* , K. Kalman 3 , B. Egri 3 , I. Roberts 4 , K. Kolev 3 , N. Wohner 1,2

1 Department of Hematology and Internal Medicine; 2 HCEMM-SU Thrombosis and Haemostasis Research Group; 3 Department of Biochemistry, Semmelweis University, Budapest, Hungary; 4 Clinical Trials Unit, London School of Hygiene & Tropical Medicine, London, United Kingdom

Background:

Postpartum haemorrhage (PPH) is the leading cause of maternal death world-wide. Maternal anaemia strongly increases the risk of PPH. The Woman trial showed that the antifibrinolytic tranexamic acid (TXA) reduces PPH deaths. The Woman-2 trial is now assessing whether TXA can prevent PPH in anaemic women. Low RBC-counts promote fibrinolysis by altering fibrin structure and plasminogen activation. We explore the effect of low RBC counts on the potency of TXA.

Aims: To examine if the RBC content of fibrin clots affects the anti-fibrinolytic potency of TXA.

Methods:

We used ball sedimentation assay (BS) and elastic motion thromboelastography (ClotPro) to monitor the lysis of fibrin containing plasminogen and tissue-type plasminogen activator (tPA) at various RBC-counts and TXA concentrations. We examined the anti-fibrinolytic potency of TXA with parallel line bioassay analysis of lysis times and with dose-response curves of lysis inhibition, over a range of tPA and TXA concentrations.

Results:

Compared to RBC-free fibrin, the anti-fibrinolytic potency of 4–64 µM TXA was increased in the presence of RBC at 10–40% (V/V). This effect was consistent at all TXA concentrations and RBC counts in both BS and ClotPro assays. The maximal increase in TXA potency was 2.7-fold at 64 µM TXA and 20% (V/V) RBC (Fig.1A). The prolongation of lysis time in response to varying TXA concentration indicated that RBC increased the number of target sites available for TXA binding (Lt max in Fig.1B) without any significant change in the TXA concentration needed to achieve a half-maximal effect (K s in Fig.2B). These extra TXA

binding sites are probably presented by the higher fraction of plasmin residing in unbound form in a fibrin meshwork of thinner fibers which is formed in the presence of RBCs.

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Summary/Conclusion:

RBCs increase the anti-fibrinolytic potency of TXA. Higher doses of TXA may be required to inhibit fibrinolysis in severely anaemic women.

P1669: AUTOANTIBODY-MEDIATED PLATELET DESIALYLATION IN ADULTS WITH PRIMARY IMMUNE THROMBOCYTOPENIA

T. Schramm 1,* , C. Ungerböck 1 , M. Fillitz 2 , B. Dixer 2 , D. Mehic 1 , C. Ay 1 , T. Backchoul 3 , J. Zlamal 3 , I. Pabinger 1 , J. Gebhart 1

1 Department of Internal Medcinie 1, Division of Hematology and Hemostaseology, Medical University of Vienna; 2 Department of Internal Medcinie, Hanusch Krankenhaus, Vienna, Austria; 3 Transfusion Medicine, Medical Faculty of Tübingen, University Hospital Tübingen, Tübingen, Germany

Background: Primary immune thrombocytopenia (ITP) is an acquired thrombocytopenia. Its clinical symptoms are increased bleeding tendency and paradoxically also increased risk of thrombosis has been reported. Autoantibodies can bind and destruct platelet surface glycoproteins (GP), leading to Fc-independent platelet clearance. Refractory ITP patients show an increased tendency of platelet desialylation, whereas its impact on bleeding phenotype and thrombosis incidence is not completely elucidated yet.

Aims: To link bleeding phenotype or thrombosis incidence to desialylated platelets in adults with primary ITP.

Methods: Serum samples of 86 adult primary ITP patients and 20 healthy controls (HC) included in the Vienna ITP Biobank (EC 1843/2016) were analyzed. A lectin binding assay was performed to investigate the platelet desialylation status. Therefor healthy fresh washed platelets were incubated with patient or HC serum, stained with erythrina cristagalli lectin (ECL) and ricinus communis agglutinin (RCA ) and analyzed via flow cytometry. Mean fluorescence intensity (MFI) cutoffs (ECL: MFI=1.43; RCA: MFI=1.10) were calculated based on HCs. Patients MFIs above the respective cutoffs were considered positive for lectin binding.

Results: Table 1 depicts demographic and laboratory data of 86 primary ITP patients. Platelet desialylation with binding of ECL and/or RCA was detected in 9 (10.5%) out of 86 ITP patients (3 ECL only, 4 RCA only, 2 ECL and RCA). Of them, autoantibodies (AAbs) against GPIIb/IIIa were detected in 1 patient, whereas 5 patients did not express AAbs against GPIIb/IIIa or GPIb/IX and 3 patients had an unknown AAb status. The 2 ITP patients testing positive for binding both lectins were diagnosed with chronic ITP and did not express AAbs against GPIIb/IIIa or GPIb/IX. In comparison of patients with versus without platelet desialylation, no significant differences in relevant clinical characteristics, bleeding phenotype, or history of thrombosis were found (Table 1).

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Summary/Conclusion: In 10.5% of the investigated patient cohort lectin binding was observed, representing mainly chronic ITP patients. No clear link of platelet desialylation with multi-refractory ITP, the bleeding phenotype or thrombosis history was found.

The biobank from which this data derives is supported by Novartis, Swedish Orphan Biovitrum (SOBI), MedMedia Verlag und Mediaservice GmbH and AMGEN GmbH.

P1670: LONG TERM SAFETY AND EFFICACY OF RECOMBINANT HUMAN COAGULATION FACTOR VIII (SCT800) IN PREVIOUSLY TREATED PATIENTS WITH SEVERE HEMOPHILIA A

F. Xue 1,* , X. Zhao 2 , J. Sun 3 , X. Zeng 4 , F. Yang 5 , M. Xu 6 , Z. Yu 7 , W. Gu 8 , Y. Feng 9 , W. Li 10 , C. Zheng 11 , H. Bi 12 , C. Ma 13 , W. Gai 13 , L. Xie 13 , R. Yang 1

1 Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin; 2 Xiangya Hospital Central South University, Changsha; 3 Nanfang Hospital Affiliated to Southern Medical University, Guangzhou; 4 The affiliated Hospital of Guizhou Medical University, Guizhou; 5 Fujian Medical University Union Hospital, Fuzhou; 6 Chengdu Women’s & Children’s Central Hospital, Chengdu; 7 The First Affiliated Hospital of Soochou University, Suzhou; 8 he First People’s Hospital of Changzhou, Changzhou; 9 The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou; 10 Qinghai Provincial People’s Hospital, Xining; 11 Anhui Provincial Hospital, Hefei; 12 The Second Affiliated Hospital of Kunming Medical University, Kunming; 13 Sinocelltech Ltd., Beijing, China

Background: SCT800 (Omfiloctocog alfa) is the first domestic recombinant factor VIII approved in China based on pivotal study NCT03815318 . The extension study ( NCT03947567 ) is ongoing to evaluate the long term safety and efficacy in previously treated patients (≥12 years) with severe hemophilia A.

Aims: To report the interim data for the extension study.

Methods: Patients who completed the main study enrolled in the open‐label extension study and continued receiving 25-50 IU/kg SCT800 every other day or three times weekly. The primary endpoint was development of inhibitors; the key secondary endpoints included median annualized bleeding rate (ABR).

Results: As of 24 Feb 2022, no inhibitors were observed in total 69 enrolled patients with a median exposure of 332 days (range 79-432 days). Adverse reactions occurred in 4.3% of the patients and SAEs were reported in 4 patients (5.8%). Median ABR (IQR) for all bleeds was 2.00 (4.10) in the pivotal study and 0.48 (1.46) during the extension (Table). Of the 178 reported bleeds, hemostatic efficacy was rated as “Excellent” or “Good” for 88.8% of the events. Improved joint function was observed in 67 patients with a mean total HJHS scores decreased by 3.4.

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Summary/Conclusion: The interim data demonstrate favorable long-term safety of SCT800 and the maintenance of low ABR in patients with severe hemophilia A.

P1671: GENES POLYMORPHISM OF THROMBOPHILIA AND PROTHROMBOTIC CONDITIONS IN WOMEN WITH VARIOUS OBSTETRIC ANAMNESIS

A. Zagarskikh 1,* , E. Butina 2

1 laboratory of cellular and molecular immunology; 2 laboratory of immunohematology, KRIHBT, Kirov, Russia

Background: Reproductive failure is one of the leading problems in modern obstetrics and gynecology. Causes of fertility disorders are diverse, but a particular place among them is thrombophilia, as hereditary and acquired. Given the peculiarities of the physiological adaptation of the haemostatic system to pregnancy, the vast majority of genetic thrombophilia is clinically manifested during the gestational process, both in the form of thrombosis, as well as in the form of common obstetric complications.

Aims: Specify the role of genes for thrombophilia and prothrombotic conditions ( F2: 20210 G>A, F5: 1691 G>A, F7:10976 G>A, F13: 103 G>T, FGB: -455 G>A, ITGA2: 807 C>T, ITGB3: 1565 T>C, PAI-1: -675 5G>4G ) in the development of reproductive failure in women.

Methods: The distribution of the hemostasis system genes in 198 women was analyzed: 84 women with recurrent pregnancy loss (ICD-10: N96) with 3 or more missed abortions (anatomical, gynecological, infectious and endocrine aggravating factors are excluded). The comparison group consisted of 114 women without obstetric and gynecological complications, with two or more urgent deliveries. The women’s age ranged from 27 to 49 years (median 38 years). The study was carried out with reagents and equipment of “DNA Technology”, Russia). For statistical processing of data was used criterion χ 2 .

Results: The frequency of occurrence of thrombophilia and prothrombotic genes polymorphism are presented in the Table.

The data of our study showed that there were no differences in the distribution of polymorphism of genes associated with thrombophilia and prothrombotic conditions between the group with recurrent pregnancy loss and the comparison group ( p >0.05).

Summary/Conclusion: The study did not establish the influence of polymorphism of the hemostasis system genes ( F2: 20210 G>A, F5: 1691 G>A, F7: 10976 G>A, F13: 103 G>T, FGB: -455 G>A, ITGA2: 807 C>T, ITGB3: 1565 T>C, PAI-1: -675 5G>4G ) for miscarriage. Thus, the role of allelic variants of the hemostasis system genes in fertility disorders, in our opinion, is overestimated, and these pathologies are associated with other causes.

P1672: ARTERIAL EMBOLISM IN A NATIONWIDE COHORT OF PATIENTS WITH INFLAMMATORY BOWEL DISEASES

A. Abomhya 1,* , Z. Abdulsada 2 , H. Hamad 3

1 Internal Medicine, The Brooklyn Hospital Center, Brooklyn; 2 Internal Medicine, Meharry Medical College School of Medicine, Nashville, United States of America; 3 Faculty of Medicine, Al-Azhar University, Cairo, Egypt

Background: Inflammatory bowel disease (IBD) patients are at a significantly high risk of thromboembolism events. Arterial embolism is a rare but dire condition with only a few reported cases among IBD patients. Systemic chronic inflammation, thrombocytosis, abnormal platelet functions, proinflammatory cytokines, hypertension, obesity, diabetes, and dyslipidemia contribute to the elevated risk of thromboembolic events.

Aims: This retrospective cohort study aimed to estimate the prevalence of arterial embolism among hospitalized IBD patients. Secondary outcomes included mortality, length of stay, all-cause 30-day non-elective readmission rate, and total cost of hospitalization.

Methods: We queried the Nationwide Readmission Databases 2016-2018 using ICD-10-CM codes to identify all adult patients admitted for IBD. Patients with a comorbid diagnosis of arterial embolism (ICD-10-CM: I74.x) were identified. Median and IQR were used to describe Continuous variables, and proportions were used with categorical variables. Comparison between groups was performed by Mann Whitney test for continuous variables and Chi-Square test for Categorical variables. Multivariate regression analysis was performed to study the impact of comorbid arterial embolism on inpatient mortality and non-elective readmissions. Statistical analyses were performed using SPSS Version 25 (IBM Corporation, Armonk, NY, USA).

Results: We extracted 365,152 index hospitalization records for IBD. Of whom, 774 (0.2%) had a comorbid arterial embolism. IBD patients with arterial embolism were older (63; Interquartile Range (IQR): 53-74 vs. 56; IQR: 38-70, P <0.001), more common to be females (53% vs. 47%, P= 0.028), more common to have hypertension (38.4% vs. 31.6%, P <0.001), dyslipidemia (35.8% vs. 24.7%, P <0.001), diabetes (20.8% vs. 17.2%, p= 0.008), ESRD (4% vs. 1.6%, p <0.001) and hepatic failure (4.9% vs. 1%, p <0.001) compared to IBD patients without arterial embolism respectively. Essential thrombocythemia (3.9% vs 1.2%, P <0.001) and hepatic failure (4.9% vs. 1%, p <0.001) were more common among IBD patients with arterial embolism compared to those without arterial embolism respectively. Having arterial embolism was associated with increased length of stays in days (7; Interquartile Range (IQR): 4-17 vs. 3; IQR: 2-6, P <0.001), increased median total charges ($112,712; IQR: $48,807 - $245,548 vs. $32,581; IQR: $17,826 - $62,251, P <0.001). On multivariate analysis and after adjusting for comorbid conditions, having arterial embolism was associated with increased mortality (Odds ratio (OR): 5.575; 95% confidence interval (CI): 4.369-7.114, P <0.001) and higher odds of all-cause 30-day non-elective readmission (OR: 1.294; 95% CI: 1.028-1.627, P= 0.028).

Summary/Conclusion: In a large nationwide cohort of patients hospitalized for IBD, we identified 774 cases of an arterial embolism which was associated with significantly worse hospitalization outcomes. We highlighted rare but significant comorbidity that can have multiple clinical presentations. Clinicians should be aware of the increased risk of thromboembolic events in IBD patients which include venous thromboembolism and arterial embolism.

P1673: THROMBOTIC COMPLICATIONS IN MERS-COV COMPARED TO COVID-19

A. Al Raizah 1,* , M. Alzahrani 1 , A. Hejazi 1 , M. Almegren 2 , T. Alshuaibi 3 , A. Alaskar 1

1 Oncology, King Abdulaziz Medical City, Ministry of National Guard Health Affairs. King Abdullah International Medical Research Center.King Saud bin Abdulaziz University of Health Sciences, Saudi society of bone marrow transplant; 2 Medicine, College of Medicine, Imam Mohammad Ibn Saud Islamic University, Riyadh; 3 Medicine, King Fahd Hospital, Jeddah, Saudi Arabia

Background: Several observational studies have reported the rate of thrombotic events in patients infected with coronavirus disease 2019 (COVID-19), with conflicting results. Middle east respiratory syndrome (MERS-COV) is another coronavirus had been initially reported in Saudi Arabia in 2012. A genome scan has shown a 50% similarity between covid-19 and. Several common features of covid-19 and MERS-COV including transmissibility, and clinical presentation have been identified. However, data about thrombotic complications in patients with MERS-COV are limited.

Aims: To compare the rate of thrombotic events between COVID-19 and MERS-COV.

Methods: We included all confirmed COVID-19 patients who were admitted to intensive care unit (ICU) in 3 major hospitals in Saudi Arabia between February to July 2020. We included all confirmed cases of MERS-COV who were admitted to ICU from these centers between March to May 2014. Patients were excluded if they were transferred in or out from one of these three hospital to another hospitals. Patients also excluded is they were admitted for less than 24 hours. Data were collected retrospectively from the first day of admission until discharge or death.

The primary outcome was the rate of venous thromboembolism (VTE). The secondary outcomes were the rate of arterial events, the rate of composite events (venous and arterial) and the rate of bleeding. VTE included all symptomatic or incidentally diagnosed cases of pulmonary embolism (PE), deep vein thrombosis (DVT) and thrombosis in unusual sites (cerebral, mesenteric, portal, splenic, hepatic, and renal veins). Screening for VTE in asymptomatic patients was not performed. If more than on type of VTE occurred in the same patient, it was considered one event. Arterial events included cerebrovascular accidents (CVAs), mesenteric ischemia, and limb ischemia and were confirmed by the appropriate imaging modality. Myocardial infarction (MI) was diagnosed based on the suspicion of the attending physician using clinical criteria as well as biomarker elevations or electrocardiographic changes. Composite events were defined as any VTE or arterial event. Bleeding events were classified as major and nonmajor based on the definition of international society of thrombosis and hemostasis (ISTH) 5 . Informed consent was waived.

Results: After exclusion, 234 COVID-19 and 58 MERS-COV patients were included. The majority of patients with COVID-19 (97%, n=230) and more than (67%,n =39) of those with MERS-COV group received pharmacological prophylaxis according to local hospital practice. The most frequently prescribed regimen in both groups was enoxaparin (40 mg twice per day). Over a median length of stay in the COVID-19 group of 22 days, the rate of VTE 9.8 (6.64–14.3) and was 3.4 (0.95–11.7) in the MESR-COV group over median length of stay of 10 days.

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Summary/Conclusion: Conclusions: To our knowledge, this is the first study compared thrombotic risk between COVID-19 and MERS-COV critically ill patients. We found unexpected similar rate of composite thrombotic events (venous and arterial) between COVID-19 and MERS-COV with higher rate of venous thrombosis in COVID-19 and higher arterial thrombosis in MERS-COV. This may indicate that thrombotic complication is a unique complication of coronaviruses. These result needs to be confirmed in studies with larger sample size.

Helms J, et al. Intensive Care Med. 2020;46:1089-98.

Schulman S, et al. J Thromb Haemost. 2005;3:692-4.

P1674: CLINICAL IMPACT OF NON-ADHERENCE TO HIT GUIDELINES: A DOUBLE HIT IN HIT

A. Al Raizah 1,* , A. Almozaini 1 , R. Abu Eishah 1 , K. Alharbi 1 , M. almufarrih 1 , M. Al Shuaibi 2 , O. K. Alkhairy 3 , M. Alzahrani 1 , A. Hejazi 1 , A. Alaskar 1

1 Oncology, King Abdulaziz Medical City, Ministry of National Guard Health Affairs. King Abdullah International Medical Research Center.King Saud bin Abdulaziz University of Health Sciences, Saudi society of bone marrow transplant, Riyadh; 2 Internal medicine, King Abdulaziz hospital, Ministry of National Guard Health Affairs. King Abdullah International Medical Research Center.King Saud bin Abdulaziz University of Health Sciences, Alahsa; 3 Pathology and Laboratory Medicine, King Abdulaziz Medical City, Ministry of National Guard Health Affairs. King Abdullah International Medical Research Center.King Saud bin Abdulaziz University of Health Sciences, Riyadh, Saudi Arabia

Background: Heparin induced thrombocytopenia (HIT) is a pro-thrombotic complication of heparin. In patients with suspected HIT,guidelines recommend to use 4Ts score to estimate the probability of HIT and recommend against HIT testing in patients with a low probability. Use of this score would limit overtesting, unnecessary anticoagulation, bleeding, and thrombosis from holding heparin. In patients with an intermediate or high probability, guidelines recommend holding heparin and starting a non-heparin anticoagulant.

Aims: Describe clinical management and consequences of non-adherence to HIT guidelines

Methods: This is a retrospective study at single tertiary hospital. All inpatients 16 years or older who have been tested for HIT between june 2019 to end of june 2021 were included. Patintes were exculded if the reason for sending HIT was a repeat test. Data were collected retrospectively by manual chart review. HIT pretest probability was calculated using the 4Ts score by 2 independent hematologist. Any variations in 4Ts score were solved by discussion. Score of 0 to 3 considered low, 4-5 intermediate and 6-8 high probability.

Outcomes:

Low probability:

1. Frequency of requesting HIT test.

2. Thrombosis secondary to holding heparin defined as venous thrombosis (VTE) from time of holding heparin till death or 90 days after discharge.

3. Frequency of using alternative anticoagulant and consequent bleeding defined as bleeding from start of alternative anticoagulant till 24 hours after last dose.

Moderate and high probability:

1. Frequency of not stopping heparin and starting alternative anticoagulant.

2. Thrombosis (VTE or arterial) in patients not started on alternative anticoagulant defined as thrombosis from sending HIT till 90 days after discharge or death. VTE included pulmonary embolism, deep vein thrombosis and thrombosis in unusual sites. Arterial events included cerebrovascular accidents, mesenteric, limb ischemia and myocardial infarction. HIT test was done using Stago Asserachrom HPIA‐IgGAM ELISA with level of < 0.4 considered negative. Informed consent was waived.

Results: After exclusion of the repeat test (13), 302 patients were included in this study. Most common reason to request HIT test was low platlet 288 (95%). Most of the patients 183 (60.6 %) were low probability, 108 (35.8%) intermediate and the rest 11(3.6%) were high probability. In the low probability group,166(90.7%) of patients had negative HIT Eliza (Table 1 & 2).

Management and outcome:

Low probability ( table 3): Heparin was stopped in 85(46.4 %). Three out of these patients developed thrombosis (3.5%). Alternative anticoagulant was started in 12(6.5 %) patients. Bleeding developed in 9(5%) patients none of these events were secondary to the alternative anticoagulant.

Moderate and high probability ( table 4): Heparin stopped in 60 patients (50.4%).Majority 95(79.8 %)were not started on alternative anticoagulant.Thirteen (13.6%) of these patients developed thrombosis.

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Summary/Conclusion: We found that nonadherence to HIT guidelines is common and had a substantial impact. In the low probability group, overtesting for HIT is highly prevalent. This exposed patients to unnecessary anticoagulation. In the intermediate and high probability group, majority of patients were not started on alternative anticoagulant which may explain the high rate of thrombosis. Quality initiatives which incorporate education about evidence-based management of HIT, consistently applying these in practice, and incorporating them into electronic systems could minimize these consequences and lead to safer, more cost-effective care.

Cuker, A, et al. Blood advances 2.22 (2018 )

P1675: POST-HOSPITAL DISCHARGE EVALUATION OF COVID-19 SURVIVORS WHO SUFFERED ACUTE VENOUS THROMBOEMBOLISM (VTE) DURING HOSPITALIZATION: THE BERGAMO EXPERIENCE

C. Ambaglio 1,* , S. V. Benatti 2 , C. J. Tartari 1 , C. Giaccherini 1 , L. Russo 1 , M. Marchetti 1 , A. M. Palladino 3 , F. Schieppati 1 , S. Venturelli 2 , L. Barcella 1 , M. Rizzi 2 , A. Falanga 1,3

1 Division of Immunohematology and Transfusion Medicine; 2 Division of Infectious Diseases, Papa Giovanni XXIII hospital, Bergamo; 3 Medicine and Surgery, University of Milan-Bicocca, Milan, Italy

Background: Acute COVID-19 manifestations are well known, while post-COVID clinical and laboratory consequences are less clear. Due to the strict association of COVID-19 with thrombosis, the Hemostasis Service of Bergamo hospital had a central role in guiding anticoagulant treatments in patients during the acute and post-discharge phases.

Aims: In the post-hospital discharge setting, in a cohort of COVID-19 survivors who experienced VTE during the acute phase and were followed up at our outpatient clinic, we aim to evaluate whether alterations in coagulation and inflammatory parameters were occurring and were possibly associated with disease severity.

Methods: An outpatient service specifically dedicated to follow-up of COVID-19 survivors was set up at our Institution. A retrospective analysis of a cohort of 1563 patients enrolled from May 2020 to November 2020 was performed. Data were obtained on the occurrence or not of acute COVID-19 -associated VTE, the site of thrombosis, ongoing anticoagulant treatments, and the results of laboratory tests performed at the first post-discharge assessment visit, including blood cell count, D-dimer, fibrinogen, CRP and LDH. The severity degree of COVID-19 at the onset was defined by the need of Continuous Positive Airway Pressure devices (C-PAP) or tracheal intubation as compared to no-oxygen need, or use of nasal flow, reservoir mask, or Venturi Mask. Statistical analyses were performed using SPSS v21 software. Results of lab tests were expressed as median values (5th-95th percentile).

Results: Of the 1563 enrolled outpatient COVID-19 survivors [974 M/589 F, median age of 60 years (35.5-81)], 56 subjects [43M/13F; median age 62 years (42-80.5)] had had VTE during COVID-19 acute phase (3.58%), as follows: 9 isolated proximal DVT (4 in upper extremity), 45 isolated PE (34 subsegmental and/or segmental PE; 11 central pulmonary artery thrombosis), 2 DVT+PE. Concerning COVID-19 severity, 33 of 56 patients needed C-PAP or tracheal intubation. At the follow-up visit, patients were still on anticoagulation (76% with DOAC). Laboratory data analyses revealed that 25% of VTE-subjects had increased levels of both D-dimer and fibrinogen, and 11% had elevated levels of CRP, while no abnormalities were observed in the platelet and leucocyte counts. CRP significantly correlated with fibrinogen and D-Dimer levels. According to COVID-19 severity, only platelet count was significantly higher in patients with severe disease, who needed C-PAP or tracheal intubation, as compared to those with less severe disease. No patients had VTE recurrence or bleeding complications at the time of follow up.

Summary/Conclusion: Our data suggest persistence of pro-coagulant and inflammatory laboratory patterns in a proportion of patients with COVID-19-related VTE, in spite of anticoagulant therapy. Further studies are needed to establish on whether monitoring these parameters may help in the decision on the duration of anticoagulation in this condition.

P1676: COVID VACCINE INDUCED IMMUNO THROMBOCYTOPENIA AND THROMBOSIS - OBSERVATIONAL STUDY

S. Gopaluni 1 , C. Ranjith Kumar 2 , S. Koganti 3 , A. Aribandi 2,*

1 Nephrology, Citizens Hospital; 2 Haemato-oncology & BMT, American oncology Institute; 3 Cardiology, Citizens Hospital, Hyderabad, India

Background: Vaccination has been an important strategic tool in helping to bring the COVID pandemic to an end. A multitude of vaccines are available, a few of which have undergone phase 3 trials and been approved for use across the world. In India, Covishield, a viral vector-based RNA vaccine (ChAdOX1) was the most used vaccine. Viral vector vaccines were reported to cause serious thrombotic complications (Vaccine induced thrombocytopenia and thrombosis), especially cerebral sinus venous thrombosis in young females, by generating antibodies against PF4 antigen. Here we report three cases of non-fatal but serious thrombotic complications in patients that received Covishield vaccine.

Aims: In this observational study we aimed to study the thrombotic adverse effects following COVID vaccination.

Methods: Patients admitted to our 250 bedded multi-specialty hospital with any thrombotic complications were assessed for temporal relationship with Covid vaccination.

Results:

Summary/Conclusion: Here were described three different cases of thrombosis that presented within 6 week of receiving first dose of Covishield vaccine. A common feature with all of the patients was relatively mild thrombocytopenia, and possibly an underlying predisposition for thrombosis. In one patient we could conclusively prove the presence of HIT antibodies as much was known about VITT by then. The aim of this article is to bring awareness to the wider community about the possible association of vaccination in the context of acute thrombosis. It is imperative that heparin be avoided in these patients. Fondaparinux is a suitable alternative. Further anti-coagulation could be with either warfarin or novel oral anti-coagulants. IVIG was also used in the treatment of some patients with cerebral venous sinus thrombosis.

P1677: THROMBOSIS IN PREGNANT CANCER PATIENTS: A SYSTEMATIC REVIEW OF THE LITERATURE

S. Bos 1,* , H. Rier 2 , H. Duvekot 3 , I. Boere 2 , M. Kruip 1

1 Haematology; 2 Oncology; 3 Gynaecology, Erasmus MC, Rotterdam, Netherlands

Background: Cancer during pregnancy is a rare event, yet managing pregnant women with an active malignancy is challenging in many ways. One of these challenges is how to deal with the risk of venous thrombo-embolic events (VTEs). The risk of VTEs is increased in pregnancy, as well as during active malignancy. Because cancer during pregnancy is rare, little is known about the prevalence and risk of VTEs in these women.

Aims: The objective of this study was to identify the prevalence of VTEs in pregnant patients with an active malignancy, and to see if there is a risk difference in various cancer types.

Methods: A systematic search was performed in the databases of Embase, Medline, Web of Science, Cochrane, and Google Scholar from inception until the 5 th of march 2021. The outcome was reviewed for title and abstract independently by the two first authors. Discrepancies were discussed to be included or excluded. From the included studies we reported the VTE incidence among pregnant cancer patients during and if available to 6 months post-partum.

Results: Fifteen studies reported data on VTEs in pregnant women with an active malignancy. Underlying malignancy’s varied in the different studies. Eight of the studies reported data in patients with myeloproliferative diseases, and seven on other or multiple malignancies. In some studies preventive measures of VTEs were reported and consisted mainly of anticoagulant therapies. The incidence of VTEs ranged between 0.2% to 15%, and was highest in patients with myeloproliferative diseases, gynaecological malignancies, and acute leukaemia.

Summary/Conclusion: Pregnant cancer patients seem to have a severe increased risk of VTEs, especially in certain subgroups. Because of the rare occurrence of malignant disease during pregnancy, these women should be monitored in a tertiary hospitals. Additionally thrombosis prophylaxis with low molecular weight heparin should be considered ante-partum and at least 6 weeks post-partum.

P1678: ENDOVASCULAR TREATMENT FOR UPPER AND LOWER EXTREMITY DEEP VEIN THROMBOSIS, CASE REPORTS

A. Casamayor Garcia 1,* , M. V. Aznar Moreno 1 , J. Coll Vallier 1 , M. E. Sola Aparicio 1 , E. Gomez Arteta 1 , M. J. Paloma Mora 1 , M. Rodriguez Calvillo 1 , I. Insausti Gorbea 2 , M. I. Moreno Izco 3 , M. Lopez San Martín 4 , R. Monreal Beortegui 2 , J. Anniccherico Sánchez 3 , I. Mugica Muñagorri 1 , D. G. Gabaldon Limas 1 , J. Illarramendi Esteban 1 , R. J. Breeze 1 , C. Ceballos Bolaños 1 , A. M. Redondo Izal 1

1 HEMATOLOGY; 2 CARDIOVASCULAR RADIOLOGY; 3 INTERNAL MEDICINE; 4 VASCULAR SURGERY, HOSPITAL UNIVERSITARIO DE NAVARRA, PAMPLONA, Spain

Background: Guidelines suggest anticoagulation therapy alone over thrombolytic therapy in addition to anticoagulation in the treatment of deep vein thrombosis (DVT). However, endovascular treatment for iliofemoral and subclavian-axillary DVT has been shown useful in reducing post-thrombotic syndrome,with minimal complications. It is reasonable to be considered in young patients with low risk for bleeding.

Aims: To evaluate the efficacy and safety of endovascular treatment in patients with iliofemoral or subclavian-axillary DVT.

Methods: Retrospective descriptive study that included patients with upper or lower extremity DVT who received endovascular treatment, most of them with AngioJet Thrombectomy System,between October 2020 and January 2022 at Hospital Universitario de Navarra.

Results: A total of 20 patients were assessed. Patients were males (n=13) and females (n=7) with mean age of 47,8 years. 13 patients presented lower extremity DVT (65%) while 7 patients (35%) presented upper extremity DVT. 4 patients had pulmonar embolism(PE) at diagnosis. Risk factors for DVT were found in 13 patients: 5 patients had venous Thoracic Outlet syndrome, 3 patients had May-Thurner Syndrome, 2 events happened after surgery, 2 had active cancer and 1 patient had congenital iliac vein stenosis and antiphospholipid syndrome. Initially 14 patients (70%) were treated with low molecular weight heparin (LMWH) at therapeutic doses and 6 patients (30%) with unfractioned heparin (UH). After the first procedure, complete venous patency was achieved in 3 patients (15%), 11 patients (55%) almost complete, partial recanalization in 4 patients (20%) and failure in recanalization in 2 patients(10%).After 24 hours of thrombolytic infusion with alteplase or urokinase: 11patients required additional angioplasty and 6 patients needed stent placement. Radiological improvement was achieved in 18 patients. After the procedure, 5 patients continued UH infusion and 15 patients received LMWH, switching to oral anticoagulation when possible:2 patients were treated with a direct oral anticoagulant indefinitely and 16 patients with antivitamin K, 12 indefinitely and 4 switching to antiaggregant. In 2 patients LMWH was kept because active cancer treatment. Before the endovascular therapy, the average of hemoglobin level was 13,12 g/dl and the corresponding 95% confidence interval 13,12±1,45,and 24 hours after the treatment the average was 12,49 g/dl and the confidence interval 12,49±2,02. An important drop in hemoglobin was observed in 3 patients: 2 had cancer and one presented hemorrhagic shock with acute renal failure secondary to cave vein haematoma which needed intensive care. Low fibrinogen level (<60 mg/dl) was detected in 3 patients, after endovascular treatment. One of them suffered from congenital hypofibrinogenemia. We reported 3 cases of acute renal failure due to hemolysis as a consequence of acute tubular necrosis after procedure.

Summary/Conclusion: In our study, 45% patients had DVT due to anatomic factors (venous thoracic Outlet syndrome and May-Thurner syndrome). After endovascular therapy, 90% of patients achieved radiological improvement. 80% required long-term anticoagulation therapy.

The main complications were anaemia, renal failure due to hemolysis, hypofibrinogenemia and one case of cave vein haematoma. Endovascular therapy should be performed in selected patients who present a low risk of bleeding. It is important to implement a close monitoring of laboratory parameters.

P1679: GENETIC POLYMORPHISM OF PLASMA HEMOSTATIC FACTORS AND THE RISK OF DEEP VEIN THROMBOSIS IN ELDERLY AND SENILE PATIENTS

A. Chechulova 1,*

1 Vascular surgery, Dzhanelidze Research Institute of Emergency Medicine, St. Petersburg, St. Petersburg, Russia

Background: Hereditary thrombophilia is a risk factor for venous thrombosis (VT) at young age. However, the risk of VT in patients older than 60 years is greatly increasing. The role of hereditary risk factors in development of VT in elderly and senile patients has not been studied enough.

Aims: To assess the role of genetic variations in several plasma hemostatic factors in development of deep vein thrombosis (DVT) in elderly and senile patients.

Methods: We examined 76 patients (37 men, 39 women) over 60 years of age with acute episode of DVT. Patients with cancer were excluded from the study. All the patients were divided into two age-dependent groups according to the WHO classification: elderly (60-74 years old), n=67 (78.4%) and senile (75-90 years old), n=9 (21.6%). The control group (CG) consisted of 191 healthy persons without a history of thrombotic episodes. Genotyping for DNA polymorphism of 8 genes encoding plasma hemostatic factors was performed by PCR-RFLP: fibrinogen α- and β-subunits (Thr312Ala and −455 G/A, respectively), FII (20210 G/A), FV (1691 G/A), FXII (46 C/T), FXIII A-subunit (Val34Leu), PAI-1 (−675 4G/5G), TPA (311 bp Ins/Del). Intergroup differences in genotypes distribution were assessed by Fisher’s exact test. The odds ratios (OR) with their 95% confidence intervals (CI) and p-values were determined by using the SPSS statistical package (version 23.0; SPSS Inc, Chicago, IL, USA).

Results: The frequencies of heterozygous genotypes FV 1691 GA and FII 20210 GA were 3.8 and 3.5 times higher in DVT patients than in the CG (14.5% vs. 4.4%; OR=3.8; 95% CI: 1.5–9.7; p=0.004 and 5.3% vs. 1.6%; OR=3.5; 95% CI: 0.7–15.6; p=0.098, respectively). Homozygotes for the FII 20210 G/A mutation were not found in any group, and the FV 1691 AA variant was present in 1 (1.3%) patient with DVT. The heterozygous FXIII 34 Val/Leu variant was underrepresented in DVT group (32.9% vs. 47.7% in CG; OR=0.5; 95% CI: 0.3–0.9; p=0.03), whereas the genotype FXIII 34 Val/Val was prevalent among DVT patients (61.8% vs. 47.1% in CG; OR=1.9; 95% CI: 0.9–3.1; p=0.03). No differences were found between the compared groups for other genetic variants. However, when comparing patient groups depending on age, heterozygotes for the FV 1691 G/A (22.2% vs. 13.4%; OR=1.8; 95% CI: 0.3–10.3; p=0.58) as well as for the α- and β-subunits of fibrinogen (66.7% vs. 37.3%; OR=3.4; 95% CI: 0.7–14.5; p=0.093 and 44.4% vs. 28.4%; OR=2.0; 95% CI: 0.5–8.3; p=0.23, respectively) were more prevalent in senile patients. In contrast, heterozygotes for the TPA Ins/Del polymorphism were almost 4 times more common in elderly patients with DVT (40.3% vs. 11.1% in senile patients; OR=5.4; 95% CI: 0.7–45.3; p=0.089).

Summary/Conclusion: The heterozygous genotype FV 1691 GA as well as the FXIII 34 Val/Val variant are associated with increased VTE risk in elderly and senile patients from North-Western Russia.

P1680: INFLUENCE OF D-DIMERS ON THROMBOEMBOLIC RISK IN PATIENTS WITH SARS-COV-2 IN AN INTENSIVE CARE UNIT

A. Coelho 1,* , P. Moura 2 , I. Santos 3 , P. Martins 1,3 , J. Carda 2,4,5,6,7,8 , A. B. Sarmento-Ribeiro 2,4,5,6,7,8

1 Faculty of Medicine, University of Coimbra; 2 Clinical Hematology Department; 3 Intensive Care Unit, Centro Hospitalar e Universitário de Coimbra; 4 Laboratory of Oncobiology and Hematology and University Clinic of Hematology; 5 Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO); 6 Center for Innovative Biomedicine and Biotechnology; 7 Institute of Physiology; 8 Clinical Academic Center of Coimbra, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Background: Patients with COVID-19 have a higher incidence of thromboembolic events (TE) probably due to endothelial injury caused by the binding of SARS-CoV-2 to the ACE-2 receptors. The current literature showed that these patients have a tendency to a procoagulation state, and D-dimers (D-d) may be a global marker of hemostasis activation. In this way, the increase in D-d levels prompted the need to assess them at hospital admission and start anticoagulation (AC) prophylaxis, especially in intensive care unit (ICU) hospitalizations. Further, there is no consensus about the levels of D-d that could predict new TE.

Aims: This study intends to determine whether D-d levels at hospital admission are related with an increased risk for TE during ICU hospitalization and determine an eventual cut-off level that could predict the risk of these events.

Methods: Retrospective, observational, single-center study of patients with an established diagnosis of COVID-19, who underwent hospitalization in the ICU of a tertiary care hospital between December 2020 and January 2021. Clinical, analytical and demographic data were collected through clinical medical records. The data analysis was executed using IBM SPSS Statistics 28. To evaluate the predictive value of D-d on TE risk and survival in patients at ICU admission, a logistic regression and a ROC analysis were performed.

Results: A total of 116 patients were included, 38 (32.8%) females and 78 (67.2%) males, with a median age of 62 (27-86) years old, 15 (12.9%) of them with a previous AC therapy. The mean number of days spent in the ICU was 16 (13-18) days. Out of the 116 patients, 46 (39.7%) patients passed away during ICU stay and 17 (14.7%) patients presented with thromboembolic events (2 of them with 2 TE). Out of the 19 total TE, there were 8 (42.1%) deep vein thrombosis, 3 (15.8%) pulmonary embolisms, 3 (15.8%) arterial strokes, 3 (15.8%) upper limb infarctions and 1 (5.3%) myocardial infarction. The logistic regression model shows that the D-d levels at ICU admission are statistically significant on the likelihood to have TE (χ 2 (2)=9.089, p=.011). The model explained 13.9% (Nagelkerke R 2 ) of the variance in TE and correctly classified 86.1% of cases. Increasing levels of D-d at an ICU admission were associated with a slightly increased likelihood of exhibiting those events (relative risk=1.000051750). Another logistic regression was performed to evaluate whether D-d levels at ICU admission influenced prognosis (survivor vs non-survivor), however we didn´t find any differences statistically significant (p=0.727). Furthermore, the ROC analysis executed to determine the cut-off D-d level at ICU admission that would better predict the TE determined a value of 763.5 ng/mL with a sensibility of 76.5% and a specificity of 63.7%.

Summary/Conclusion: D-d levels at ICU admission are a risk factor to account for on the likelihood of future TE on COVID-19 patients, although the relative risk is low. Despite the lack of consensus for predicting D-d levels, our results suggest that D-d values, at ICU admission, can be predictive of future TE. In spite of this finding, D-d levels at admission are not correlated to survival. At the moment, despite AC therapy during ICU stay, there were still patients with TE, which means that the current prophylaxis isn’t totally effective. Further investigation is needed to establish criteria for an upgraded AC, for a lower ICU stay time and to improve the prognosis.

P1681: FXI LEVELS AS INDEPENDENT RISK FACTOR FOR THROMBOSIS/OBSTETRIC EVENTS IN PATIENTS WITH ANTIPHOSPHOLIPID ANTIBODIES

M. E. De La Morena-Barrio 1,* , J. Pagan 2 , A. Miñano 1 , V. Roldan 1 , A. Lopez-Arribas 3 , J. Lozano 2 , M. J. Hernandez-Vidal 2 , I. de la Morena-Barrio 4 , V. Vicente 1 , M. L. Lozano 1 , M. T. Herranz 2 , J. Corral 1

1 Internal Medicine, Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer; 2 Internal Medicine, Servicio de Medicina Interna, Hospital Universitario Morales Meseguer, Murcia; 3 Werfen; 4 Sevicio de Reumatología, Hospital Clínico, Valencia, Spain

Background: Antiphospholipid antibody (aPL) is the most important acquired thrombophilia. Thrombotic risk assessment in these patients must be improved by evaluating new biomarkers. FXI is an excellent candidate, as it is a phospholipid binding protein involved in coagulation and inflammation, high FXI levels increases the risk of thrombosis, and FXI deficiency protects against thrombosis.

Aims: To evaluate the prognostic role of FXI in aPL

Methods: Cross-sectional and observational study of aPL patients ≥ 18 years recruited in a single center. Clinical, analytical, and epidemiological variables were analyzed. Biochemical and functional evaluation of plasma FXI was done by western blotting and two coagulation assays (FXI:C). Molecular analysis of F11 gene (sequencing and MLPA) was done in cases with FXI deficiency. Primary study outcome was to identify a role for FXI levels in the thrombotic risk of aPL patients.

Results: 194 participants were included, 82 asymptomatic aPL (AaPL) and 112 with antiphospholipid syndrome (APS). FXI:C values were significantly higher in patients with APS than AaPL. Cases with high FXI (>150%) were 14 times higher in the APS group (OR= 14,95; 95% CI: 1.95-114.83; p= 0.009). In contrast, cases with FXI deficiency (<70%) were 4 times higher in the AaPL group (OR= 4.93; 95% CI: 1.31-18.54; p= 0.018). FXI deficiency in aPL may be caused by genetic defects, but it is mainly caused by an inhibitor.

Summary/Conclusion: This is the first study showing a prognostic role of FXI levels in patients with aPL. High levels increase the risk of APS while deficiency, either congenital or acquired by the aPL, protects against thrombosis.

P1682: MTHFR C677T GENOTYPE IN CORRELATION WITH THROMBOSIS AND ASSOCIATION WITH FVIII COAGULANT ACTIVITY IN GREEK PAEDIATRIC POPULATION

S. Saslis 1 , A. Dettoraki 1,* , Z. Kapsimali 1 , A. Michalopoulou 1 , S. Thymianou 1 , M. Mazarakis 1 , H. Pergantou 1

1 Haemophilia Centre/Haemostasis and Thrombosis Unit, “Aghia Sophia” Children’s Hospital, Athens, Greece

Background: The polymorphism C>T substitution in position 677 (C677T) of the encoding gene of methylenetetrahydrofolate reductase (MTHFR), creates a thermolabile enzyme with reduced activity that may predispose to hyperhomocysteinemia, an established risk factor for arterial thrombosis and premature atherosclerosis and a probable risk factor for venous thromboembolic disease and its recurrence in the general population. There are conflicting results as to the role of the MTHFR C677T polymorphism as a risk factor for thrombosis. Although the homozygous substitution is often associated with hyperhomocysteinemia, mainly in folate deficiency, a clear association between this genetic marker and thrombosis has not yet been established.

Aims: Primary aim was to evaluate the prevalence of mutant C677T MTHFR and its association with thrombosis in the Greek paediatric population. Secondary aim was to evaluate whether different MTHFR genotype is associated with evidence of activation of coagulation cascade through increased factor VIII activity.

Methods: Data were retrospectively collected from children referred to our Haemostasis and Thrombosis Centre for laboratory screening of thrombophilia between 2018 and 2020. Some of them were checked due to history of thrombosis. Children were categorized according to MTHFR C677T genotype. FVIII Coagulant activity (%) was measured by One Stage Assay, away from acute thrombosis. The identification of mutations was based on polymerase chain reaction (PCR) and reverse- hybridization. Statistical analysis was performed with t-test and ANOVA.

Results: A total of 688 children (boys: 49 %) of mean age 8.9± 8 years (0-18) were investigated. Twelve percent (81/688) of them had history of thrombosis, mainly venous thromboembolic disease. Children were categorized as follows: homozygous MTHFR C677T (15%), heterozygous (47%) and normal (38%). There was thrombosis history in 12% of homozygous MTHFR C677T, 11% heterozygous and 12 % of normal, fact showing that there is no association between MTHFR C677T genotype with thrombosis. Children with homozygosity MTHFR C677T, compared to children with heterozygosity and normal MTHFR C677T showed increased factor VIII values (132.8 vs 126.4 and 131.6, %, respectively), but without statistically significant difference. However, independently of MTHFR C677T genotype, mean factor VIII values were significantly increased in all children, with thrombosis history when they were compared to children without thrombosis history (161.3 vs 125.1, %, p<0.001). Interestingly, in children with thrombosis, mean factor VIII level was statistically increased in heterozygous and normal MTHFR C677T in contrast to children with MTHFR C677T homozygosity, in whom no statistically significant increase was found (166.9 vs 121.4, %-p<0.05, 160.2 vs 127.6, %-p<0.05 and 148.3 vs 130.6, %-p>0.05, respectively). Finally, it should be noticed that during the year 2020 mean factor VIII values were much increased in all children in comparison to the years 2018, 2019 and 2021 (137 vs 129, 123 and 127, %, respectively), probably reflecting the effects of unknown pandemic COVID-19 which initiated in 2020, and showing that factor VIII could be an evidence of stress condition.

Summary/Conclusion: There is no probably association between MTHFR C677T genotype and thrombosis in our paediatric population. Moreover, it was not proved that different MTHFR genotypes affect factor VIII activity.

P1683: PERSISTENT HYPERCOAGULABILITY, ENDOTHELIOPATHY AND INFLAMMATION IN COVID-19 PATIENTS APPROXIMATING 1 YEAR AFTER RECOVERY.

B. E. Fan 1,* , S. W. Wong 2 , C. L. L. Sum 3 , G. H. Lim 4 , B. P. Leung 5 , R. Dalan 6 , B. E. Young 7 , D. C. Lye 7 , C. Cheung 8 , K. Ramanathan 9 , C. W. Tan 10 , E. S. Yap 11 , Y. W. Chia 2

1 Department of Haematology; 2 Department of Cardiology; 3 Department of Laboratory Medicine; 4 Clinical Research and Innovation Office; 5 Department of Rheumatology, Allergy and Immunology; 6 Department of Endocrinology, Tan Tock Seng Hospital; 7 Department of Infectious Diseases, National Centre for Infectious Diseases; 8 Lee Kong Chian School of Medicine, Nanyang Technological University; 9 Cardiothoracic Intensive Care Unit, National University Heart Centre, National University Health System; 10 Department of Haematology, Singapore General Hospital; 11 Department of Haematology-Oncology, National University Health System, Singapore, Singapore

Background: Sustained hypercoagulability and endotheliopathy has been demonstrated in convalescent COVID-19 patients for up to 4 months from recovery in previous studies.

Aims: We performed a prospective, observational study assessing for hypercoagulability, endotheliopathy and inflammation in recovered COVID-19 patients beyond 6 months of recovery.

Methods: The haemostatic (coagulation tests, global haemostatic tests), endothelial function and inflammatory profiles of recovered COVID-19 patients were evaluated at least 6 months after diagnosis of COVID-19. 39 patients were reviewed at a mean of 12.7±3.6 months following recovery from SARS-CoV-2 infection. These values were compared with a control group of healthy volunteers (n=124) who did not have COVID-19.

Results: Among the 39 patients (71.8% males, median age 43 years, 11 having asymptomatic COVID-19, 19 having mild to moderate COVID-19, 2 having severe COVID-19 and 7 having critical COVID-19 requiring ICU care), 2 patients had pulmonary embolism during acute SARS-CoV-2 infection, and 1 patient without cardiovascular risk factors had post COVID-19 acute ischaemic limb. COVID-19 patients had significantly higher biomarkers of hypercoagulability, endothelial dysfunction and inflammation as compared to controls (Figure 1). Elevated D-dimer and Factor VIII levels above the normal ranges were noted in 17.9% (7/39) and 48.7% (19/39) of COVID-19 patients respectively, with a higher median D-dimer 0.34 μg/mL (IQR 0.28, 0.46) (p < 0.001) and Factor VIII 150% (IQR 171, 203) (p=0.004), compared to controls. Thrombin generation (Thromboscreen) also showed higher median endogenous thrombin potential (ETP) 1352nM*min (IQR 1152, 1490)(p=0.002), peak height 221.4nM (IQR 170.2, 280.4)(p=0.01) and a delayed lag time 2.4min (1.42-2.97)(p=0.0002) compared to controls. Raised endothelial biomarkers such as von Willebrand factor antigen (VWF:Ag) and ICAM-1 were observed in 17.9% (7/39) and 7.7% (3/39) of COVID-19 patients respectively, with a higher median VWF:Ag 117%(IQR 86, 154)(p=0.02) and ICAM-1 54.1%(IQR 43.8, 64.1)(p=0.004) than controls. Interleukin-6 (IL-6) was noted to be raised in 35.9% (14/39) of recovered COVID-19 patients, with a higher median IL-6 of 1.5pg/ml (IQR 0.6, 3.0) compared to controls. Subgroup analysis stratifying COVID-19 patients by age (≥50 years old), COVID-19 mRNA vaccination before acquiring SARS-CoV-2 infection, or pre-existing cardiovascular risk factors did not show any statistically significant differences across haemostatic, endothelial or inflammatory parameters. Patients who had experienced mild or asymptomatic COVID-19 compared with moderate to severe COVID-19 also demonstrated raised markers of hypercoagulability, endothelial dysfunction and inflammation.

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Summary/Conclusion: Persistent hypercoagulability, endotheliopathy and inflammation is still detectable approximately 1 year from recovery in COVID-19 patients, with prior COVID-19 mRNA vaccination not appearing to reduce this risk.

P1684: NO INCREASE IN THROMBIN GENERATION OR D-DIMER LEVELS AFTER ANTI-SARS-COV-2 VACCINES INCLUDING IN THOSE WITH ANTI-PLATELET FACTOR 4 ANTIBODIES

L. Garabet 1,2,* , A. Eriksson 1 , E. Tjønnfjord 1 , M. Kringstad Olsen 3 , H. K. Jacobsen 3 , C. Tøvik Jørgensen 1 , Å.-B. Mathisen 1 , M.-C. Mowinckel 4 , M. T. Ahlen 5 , I. Hausberg Sørvoll 5 , K. Daae Horvei 5 , S. Leiknes Ernstsen 5 , I. Jenssen Lægreid 5 , P. M. Sandset 6 , W. Ghanima 1

1 Østfold Hospital, Grålum; 2 Akershus University Hospital, Lørenskog; 3 Ostfold Hospital, Grålum; 4 Oslo University Hospital, Oslo; 5 University Hospital of North Norway, Tromsø; 6 University in Oslo, Oslo, Norway

Background: Anti-SARS-CoV-2 adenoviral-vectored-DNA vaccines have been linked to a rare but serious thrombotic post-vaccine complication called the vaccine-induced immune thrombotic thrombocytopenia (VITT). VITT has raised concerns regarding the possibilities of increased thrombotic risk and thrombocytopenia after anti-COVID-19 vaccines.

Aims: To investigate whether anti-SARS-CoV-2 vaccines can cause thrombocytopenia, coagulation activation and increased thrombin generation leading to a hypercoagulable state.

Methods: In this study, 567 healthcare personnel were included from two hospitals in Norway after obtaining informed consent. Of these, 521 were recruited 11-57 days post-vaccination with the first dose of ChAdOx1-S (Vaxzevria®, AstraZeneca, UK) vaccine, and 46 were recruited prospectively prior vaccination with an mRNA vaccine, either elasomeran (Spikevax, Moderna, n=38) or tozinameran (Comirnaty, Pfize-BioNTech, n=8). In the latter group, samples were acquired before and 1-2 weeks after vaccination. In addition to pre-vaccination samples, 56 unvaccinated healthy blood donors were recruited as controls (total n=102). Thrombin generation and D-dimer were analyzed.

Results: None of the participants developed thrombosis/VITT. 12% reported cutaneous bleeding after vaccination, however, none had thrombocytopenia with platelet count <100·10 9 /L. There were no significant differences in D-dimer or the parameters of thrombin generation between the two vaccine groups and the controls (Table 1). Anti-PF4/polyanion antibodies (optical density ≥0.4) were found in 11 of 513 individuals vaccinated with ChAdOx1-S vaccine (2.1%). None of the controls had anti-PF4/polyanion antibodies. Thrombin generation and D-dimer were not found to be higher in the ChAdOx1-S vaccinated individuals with anti-PF4 antibodies than in those without anti-PF4/polyanion antibodies. No differences in thrombin generation between the ChAdOx1-S group and the mRNA group. The median D-dimer level was slightly higher in the ChAdOx1-S group than the mRNA group, but both were within the normal range (Table 1). Thrombin generation and D-dimer showed no changes after mRNA vaccination compared with baseline.

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Summary/Conclusion: Anti-COVID-19 vaccines, both ChAdOx1-S and mRNA vaccines, did not lead to an increase in thrombin generation or D-dimer compared with controls, not even in the ChAdOx1-S vaccinated individuals with anti-PF4/polyanion antibodies. No differences were found between ChAdOx1-S and mRNA vaccines, and no increase in thrombin generation or D-dimer was found after mRNA vaccines compared with baseline levels. Our results are reassuring in the sense that no subclinical activation in the coagulation system was observed with these vaccines.

P1685: CHEMOTHERAPY-INDUCED ENDOTHELIAL DAMAGE AS UNDERLYING CAUSE FOR PERIPHERAL NEUROPATHY AND REDUCED METABOLISM IN STATIC PRESSURE-ADJUSTED COMPRESSION FOR CHEMOTHERAPY-INDUCED PERIPHERAL NEUROPATHIES

A. Haspel 1,* , R. Rheimann 2 , J. Mohammad 2 , B. Lischke 3 , H. Habazettl 2 , J. A. Schmidt-Lucke 4 , S. Ochsenreither 1 , U. Keller 1 , C. Schmidt-Lucke 3

1 Dpt of Hematology, Oncology and Tumor Immunology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Campus Benjamin Franklin; 2 Institute of Physiology, Charité - Universitätsmedizin Berlin; 3 MEDIACC GmbH; 4 Practice of Internal Medicine Prof. André Schmidt-Lucke, Berlin, Germany

Background: Chemotherapy-induced peripheral neuropathy (CIPN) as common side effect is caused by impaired microvascular neuronal perfusion as shown in animal models. In contrast, several authors unsubstantiate postulate reduced perfusion induced by compression therapy responsible for neuroprotection. We, thus, tested the hypothesis of chemotherapy-induced endothelial damage (CIED), increased microvascular perfusion and reduced metabolism in response to pressure-adjusted static compression (PSC) in patients undergoing chemotherapy (CTX) with and without CIPN.

Aims: Aim of the study is to test the hypothesis of chemotherapy-induced endothelial damage (CIED), increased microvascular perfusion and reduced metabolism in response to pressure-adjusted static compression (PSC) in patients undergoing chemotherapy (CTX) with and without CIPN.

Methods: Metabolic oxygen demand (dh), tissue oxygenation (StO 2 ), oxygen supply (O 2 h) and microvascular perfusion (th) were measured with quantitative time-resolved near infra-red spectroscopy (NIRS) and temperature with thermography following standard procedures in CTX (n=22) with and without clinical CIPN (n=11 each, 46% male; age 64 ± 11 years) and compared to healthy controls (n= 20, 50% male; age 24.5 ± 4.3 years) and patients with peripheral arterial disease (PAD, 75% male, age 68 ± 9 years, 42%, positive controls) on upper extremities. Endothelial microvascular function was quantified during post-ischaemic reactive hyperaemia (PIRH). PSC was applied with commercially available compression products and patented palmar pads for up to 90 minutes under standardised and controlled conditions.

Results: Patients with CTX with CIPN showed significant endothelial dysfunction compared to controls (StO 2 downslope -7 ± 2%, T 95 (s) 13 ± 5, PIRH-AUC 9 ± 6, p<0.05 each) and CTX without CIPN (p<0.05 each), much like PAD. Significant reduction of skin temperature in healthy controls as well as CTX patients of 2°C to 6°C were achieved with PSC (p<0.05 each) and cryoapplication (up to -15°C, p<0.001).

In controls, PSC instantly reduced metabolic oxygen demand of the intervened hand (dh: -4.6 ± 2.4%, p<0.05) that remained on a low level only influenced by opening of the capillary bed after 5 to 30 minutes (th: +5.6 ± 7.5%, p<0.01). Likewise, cryoapplication significantly decreased metabolic oxygen demand (p<0.05). In contrast to controls with constant increase of local tissue oxygenation (StO 2 : +11.2 ± 7.0%, p<0.001) for the entire 90 minutes driven by increased oxygen supply (O 2 h: +11.7 ± 7.3 µM/s, p<0.001) and microvascular perfusion (th: 7.6 ± 8.7%, p<0.005), patients with CTX showed significant reduced th and dh (p<0.05 each) only, but no increase of StO 2 with PSC.

Summary/Conclusion: Reduced local temperature and metabolic oxygen demand indicate metabolic reduction as an underlying mechanism of action of PSC. CIPN is associated with marked endothelial dysfunction comparable to PAD, hampering increase of O 2 -supply and microvascular tissue oxygenation in result to PSC. This indicates that optimisation of microvascular perfusion maybe a further player of the therapeutic effect of PSC. These finding open the field for well-designed RCT to develop novel treatment options for CIPN targeting CIED.

P1686: VARIATION IN DIAGNOSTIC REQUESTS AND OUTCOMES FOR HEREDITARY THROMBOPHILIA -OVER TESTING AND UNDER TESTING

S. isaacs 1,* , W. Cerfontein 1 , S. Isaacs 1 , N. Mashigo 1 , C. Swanepoel 1

1 Pathology, Haematology, Stellenbosch University, Cape Town, South Africa

Background: Heritable thrombophilia is a multiple gene disorder with high mortality and morbidity. Genetic risk factors associated with Venous Thromboembolism have been linked to mutations in the genes of the coagulation/anticoagulation system. The more prevalent inherited risk factors are the gain-of-function mutations in coagulation factors V and II, namely factor V-Leiden (FVL and/or F5G1691A (FVR506Q,)) and prothrombin/Factor II (FII) F2G20210A. The epidemiology of the FVL and FII mutations has not been well studied within either the South African or African context.

Aims: To conduct a retrospective audit to determine the total number of FVL and FII tests requested by clinicians and the prevalence of FII mutations at Tygerberg hospital, and screen a novel gain of function mutation, Prothrombin Yukuhashi, in exon 14 of the FII gene.

Methods: Test requests were analysed over a period of 11 years (2007-2017). 50 patients were randomly selected whose peripheral blood samples were received by the National Health Laboratory Services (NHLS) Molecular haematology diagnostic laboratory from clinicians requesting the FVL and FII assay. Exon 14 was amplified using reference primers and verified by PCR followed by bidirectional sequencing to identify the presence of the mutation. Statistical anlysis of all data was performed with the help of a statistician.

Results: For the audit, 80.4% of the requests were for FVL, 3.8% were for FII and 15.8% of the requests were for FVL and FII together. The most tests were requested during 2013 when the implementation of a multiplex assay occurred. The mutational frequency for FVL was shown to be 3.3%, compared to FII which was 6.9%.

Summary/Conclusion: The frequency of the FII mutation was more than double that of the FVL mutation, despite more than 80% of requests being for FVL alone. The study also showed that the Prothrombin Yukuhashi mutation was not prevalent in our cohort.

P1687: THROMBOEMBOLISM IN COVID-19 PATIENTS ON ECMO

C. Jones 1,* , K. Chen 1 , G. Cheng 2 , V. Narendran 3

1 Internal Medicine, University of Miami / Palm Beach Regional GME Consortium, West Palm Beach; 2 Department of Physiology, University of California, Los Angeles, Los Angeles; 3 Hematology/Oncology, JFK Medical Center, Atlantis, United States of America

Background: Venous thromboembolism (VTE) is a common complication of COVID-19 (coronavirus disease 2019), which often leads to sudden deterioration and death. There are multiple mechanisms contributing to this phenomenon. Endothelial injury from COVID-19 triggers platelet activation and adhesion, leukocyte aggregation, cytokine storm and complement activation. Cytokine storm triggers coagulation activation and thrombin generation. Complement activation is also thought to trigger the formation of systemic thrombus through recruiting inflammatory cytokines and possible complement-mediated thrombotic microangiopathy .

Patients on extracorporeal membrane oxygenation (ECMO) are at risk of developing thromboembolism. Thrombus formation within the extracorporeal circuit is the main reason for systemic thromboembolism. Possibly that by contacting blood and nonendothelial surfaces, ECMO triggers activation of coagulation pathway and inflammatory response.

Aims: Thromboembolic prophylaxis is critical in managing COVID-19 patients on ECMO. Anticoagulation is recommended to all hospitalized COVID-19 patients unless there are contraindications. However, patients are still found to develop VTE while on anticoagulation and the prevalence of VTE in COVID-19 patients on ECMO is still unclear. We aim to investigate the VTE incidence and contribute to anticoagulation strategy and management in this specific population.

Methods: We retrospectively reviewed the data of 23 patients who were diagnosed with COVID-19 and managed with ECMO. All patients received thromboembolic prophylaxis since admission. We report our findings of the incidences of thromboembolism.

Results: Twenty-three adult patients who were diagnosed with COVID-19 received ECMO support. Sixteen patients were minorities, and seven patients were Caucasians. The mean age of patients was 44.8-year-old. Seventeen patients were males, and eleven patients had at least one of the following pre-ECMO comorbidity: ten (43.5%) patients had hypertension, eleven (47.8%) patients had type 2 diabetes and four (17.3%) patients had hyperlipidemia. None of the patients were active smokers or had chronic lung disease.

During the hospital course, all patients received heparin for thromboembolic prophylaxis. The overall VTE rate was 34.7%. Six patients developed deep vein thrombosis (DVT) (26%) with lower extremities induration. Two patients were found to have pulmonary embolism (PE) (8.7%). Four patients had clotted circuit that requiring exchange. No stroke or myocardial infarction (MI) was diagnosed in these patients. Heparin-induced thrombocytopenia (HIT) was excluded in all cases.

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Summary/Conclusion: Based on our study, the overall VTE rate of COVID-19 patients on ECMO was 34.7% with 26% incidence of DVT and 8.7% incidence of PE. According to Jenner’s recent systemic review of 28 studies, 34% of 2928 ICU-managed COVID-19 patients developed VTE. PE was found in 12.6% of patients and DVT was detected in 16.1% of patients. 529 patients (18.0%) received ECMO in the cohort. When compared to our study, there were no statistically significant differences of the incidences of VTE, DVT or PE between these two studies, although all our patients were on ECMO support. Further investigation into the prevalence, implications, and management of thromboembolism in COVID-19 patients on ECMO will lead to significantly improved outcomes for this specific patient population.

P1688: VKORC1 -1639 G/A GENE POLYMORPHISM CAN MODULATE THE RISK OF VENOUS THROMBOEMBOLISM IN ELDERLY PATIENTS WITH FV LEIDEN MUTATION

S. Kapustin 1,* , A. Chechulova 2 , J. Drijun 1 , S. Svitina 1 , V. Burakov 1 , V. Soroka 3 , V. Soldatenkov 1 , L. Papayan 1

1 Russian Research Institute of Hematology and Tranfusiology, Saint-Petersburg; 2 Dzhanelidze Research Institute of Emergency Medicine, Saint Petersburg; 3 Dzhanelidze Research Institute of Emergency Medicine, Saint-Petersburg, Russia

Background: Vitamin K plays important role in hemostasis by activating both procoagulant (FII, VII, IX, X) and anticoagulant (proteins C, S, Z) factors. Vitamin K-epoxide reductase 1 (VKORC1) G-1639A gene polymorphism affects enzyme activity and bioavailability of vitamin K. Little is known about the role of the VKORC1 G-1639A variation in venous thromboembolism (VTE) development, in particular, in patients with inherited thrombophilia.

Aims: To study the role of the VKORC1 -1639G/A gene polymorphism in VTE development in patients from North-Western Russia.

Methods: We included 600 VTE patients (294 men and 306 women, mean age 43.6±15.3 years) originated from the North-Western region of Russia in the study. The control group (CG) consisted of 200 healthy individuals of the same origin. Genotyping for the VKORC1 -1639 G/A, FII 20210 G/A and FV 1691 G/A variations was performed by PCR-RFLP. Intergroup differences in genotype frequencies were assessed by Fisher’s exact test. The study was approved by the local ethical committee.

Results: Distribution of the VKORC1 G-1639A gene variants was similar in both VTE patients and controls. In the group of patients without FII and FV gene mutations, the frequency of the VKORC1 -1639AA genotype was almost equal to that in CG (17.1% vs. 16.5%, respectively). VKORC1 -1639AA genotype frequency in patients with the FV Leiden was 4-fold higher than in those having the FII G20210A mutation (19.6% vs. 4.4%, respectively; OR=5.2; p=0.021). Notably, among patients having FV Leiden variant, the VKORC1 -1639AA genotype frequency was much higher in the group with late-onset disease (after 45 years) compared to young VTE patients (31.6% vs. 11.9%; OR=3.4; p=0.02). Thus, in elderly VTE patients with FV Leiden, the VKORC1 -1639AA variant was almost 2-fold overrepresented when compared to CG (31.6% vs. 16.5%; OR=2.4; p=0.033).

Summary/Conclusion: We suggest that the VKORC1 -1639AA genotype could increase VTE risk in elderly patients with the FV Leiden mutation.

P1689: TISSUE-SELECTIVE PHARMACOLOGICAL TARGETING OF TUMOR INTRINSIC PROTHROMBIN (F2) EXPRESSION USING AN INTEGRATED GENETIC REPORTER SYSTEM

E. Khan 1,* , J. Nourse 1 , S. Tokalov 1 , S. Khokhar 1 , D. Arnold-Schild 2 , L. Schott 1 , L. Eder 1 , S. Danckwardt 1

1 Center for Thrombosis and Hemostasis; 2 Johannes Gutenberg-University Medical Center Mainz, Mainz, Germany

Background: Thrombin is the central protease involved in blood coagulation. Beyond its well-established role in stemming blood loss, it has a detrimental function in various (patho)physiological conditions including sepsis and various cancers. Around 17 million deaths occur worldwide due to cancer annually and blood coagulation, specifically thrombin, has been shown to play an important role. Prothrombin (F2), the precursor of thrombin is primarily synthesized in the liver.

Aims: To decipher and target tissue-selective pharmacological targeting of tumor intrinsic F2 expression using an integrated genetic reporter system

Methods: By using an innovative newly generated knock-in reporter mouse model combined with non-invasive molecular imaging, extra-hepatic expression of F2 in tumors was discovered, recapitulating findings in cancer patients.

Results: Strikingly, tumor-intrinsic F2 not only contributes to a thrombophilic state, it also exerts a protumorigenic function. We next set out to define modalities to selectively interfere with tumor-derived F2 without altering the natural source of hemostatic F2 production (i.e., in the liver). In a pilot screening using syngenic reporter lines, we identified pathways that allow selective pharmacological targeting of tumor-derived F2.

Summary/Conclusion: This confirms the in principle selective druggability of gene expression of this central serine protease in a target-tissue specific manner. It thereby opens potentially new therapeutic avenues to interfere with detrimental tumor-intrinsic F2 expression without impairing the normal hemostatic function of liver-derived prothrombin.

P1690: NEUTROPHIL EXTRACELLULAR TRAP (NET) LEVELS IN COVID-19 PATIENTS

L. López De Frutos 1,2,3,* , I. Serrano-Gonzalo 2,4 , I. Arevalo-Vargas 2,4 , B. Menéndez-Jandula 5 , E. Franco-García 6 , C. Lahoz 2 , P. Latre 2,7 , R. Köhler 1,4,8 , P. Giraldo 2,3,9

1 Grupo de Investigación Mecanismos de Enfermedad Crónica e Investigación Traslacional (MECIT), Instituto de Investigación Sanitaria Aragón (IIS Aragón); 2 Fundación Española para el Estudio y Terapéutica de la Enfermedad de Gaucher y otras lisosomales (FEETEG); 3 Grupo Estudio de Enfermedades de Depósito Lisosomal (GEEDL), Sociedad Española de Hematología y Hemoterapia (SEHH); 4 Grupo de Investigación en Enfermedad de Gaucher (GIIS-012, Instituto de Investigación Sanitaria Aragón (IIS Aragón); 5 Servicio de Hematología, Hospital Universitario Miguel Servet; 6 Servicio de Hematología, Hospital Provincial Nuestra Señora de Gracia; 7 Medicina de Familia, Centro de Atención Primaria; 8 Fundación Aragonesa para la Investigación y el Desarrollo (ARAID); 9 Servicio de Hematología, Hospital QuironSalud, Zaragoza, Spain

Background: Innate and adaptative immune responses are triggered by the SARS-CoV-2 infection and induces an exacerbate cytokine storms responsible from a septic shock, acute respiratory distress and/or multiple organs failure. As part of the response against infection, neutrophils release the neutrophil extracellular traps (NETs), as a mechanism to encapsulate the infective agent and reduce it, but an overproduction is related with several inflammatory and thrombotic complications.

Aims: As the production of microthrombi and endothelial damage is one of the complications due to SARS-CoV-2 infection we would evaluate several molecules linked to the NETosis process in patients infected during the first wave and compare them with levels in general population (pre-Covid) and patients during January 2021 wave.

Methods: Two plasma samples from 60 patients with SARS-CoV-2 infection were provided by the Aragon Health System Biobank (30 for each wave), the first sample just after the diagnosis and the second one seven days later, as well as 100 control samples obtained before May 2019. Demographic data, clinical manifestations, associated comorbidities and thrombotic complications were collected. Myeloperoxidase (MPO), neutrophil elastase (NE), S100A8/S1009A heterodimer (MPR) and P-Selectine (P-SEL) were evaluated by immunoquantification meanwhile circulating free DNA (cfDNA) and DNAses concentration were assessed by fluorometry, always following the manufacturer instructions. Statistical analysis was performed by SPSS software using non-parametric test for independent and related samples.

Results: Infected patients have a significantly increase of MRP (p<0.001) and NE (p<0.001) meanwhile P-SEL and DNAsa concentration was significantly decreased (p<0.001). The patients did not show any differences between first and second sample. But, differences between patients from each wave were significant for NE, MPO, and P-SEL (p>0.001), with lower levels of NE and higher MPO and P-SEL in those patients affected in the first wave.

Summary/Conclusion: In conclusion, the study demonstrates a NETs activation in infected patients that can be related with the increased risk of thrombotic events and vascular damage in infected patients. The differences between groups of patients are under analysis and can be influenced by age at infection. The correlation with the demographic data, comorbidities and complications is ongoing.

This work is ongoing with the support of a research grant from FEETEG.

P1691: COMPARISON OF IMPROVE, MODIFIED IMPROVE, IMPROVEDD AND PADUA RISK SCORES FOR VENOUS THROMBOEMBOLISM PREDICTION IN HOSPITALIZED COVID-19 PATIENTS

M. Lucijanic 1,* , I. Jurin 2 , M. Sedinic 1 , E. Soric 1 , A. Sabljic 1 , I. Hadzibegovic 2 , T. Lucijanic 3 , R. Kusec 1

1 Hematology Department; 2 Cardiology Department; 3 Endocrinology Department, University hospital Dubrava, Zagreb, Croatia

Background: Venous thromboembolism (VTE) imposes a significant burden of morbidity on COVID-19 patients. It is very challenging to predict and prevent these events from occurring due to high thrombogenicity of the disease and overlapping clinical presentations, despite of regular use of pharmacologic thromboprophylaxis.

Aims: To evaluate predictive properties of several VTE prediction scores developed prior to COVID-19 era in a large cohort of hospitalized COVID-19 patients.

Methods: We retrospectively evaluated all consecutive COVID-19 patients hospitalized in University Hospital Dubrava, Zagreb, Croatia in the period from 3/2020-6/2021. Data used were recorded as a part of the hospital Registry project (ClinicalTrials.gov Identifier: NCT05151094 ). VTE (deep venous thromboses (DVT) and pulmonary embolism (PE)) were considered if documented by objective imaging methods. We considered IMPROVE, modified IMPROVE, IMPROVEDD and Padua VTE risk scores. Scores were indirectly compared based on their AUC values obtained through ROC curve analysis. Cut-offs with highest Youden index were shown.

Results: A total of 5959 patients were analyzed. Median age was 72 years and median Charlson comorbidity index was 4 points. There were 56.2% males, majority of patients had either severe (70.5%) or critical (15.3%) severity of COVID-19 symptoms at the time of hospital admission. There were 365 (6.1%) VTE, among them 137 (2.3%) DVT and 262 (4.4%) PE.

Regarding VTE, all investigated scores demonstrated modest predictive properties with highest AUC observed for IMPROVEDD score (AUC 0.623, >3 points sensitivity 69% and specificity 49%) followed by modified IMPROVE (AUC 0.590, >1 point sensitivity 87% and specificity 29%), Padua (AUC 0.589, >4 points sensitivity 87% and specificity 29%) and IMPROVE (AUC 0.584, >1 point sensitivity 74% and specificity 43%) as shown in Figure. IMPROVEDD had significantly higher AUC in comparison to all other scores, whereas other scores had comparable AUC values. All scores could significantly discriminate patients with and without VTE.

Overall higher AUC values were achieved for DVT than for PE prediction, with IMPROVEDD score having highest AUC values for both DVT and PE prediction (DVT AUC 0.650, >3 points sensitivity 75% and specificity 48%; PE AUC 0.603, >2 points sensitivity 88% and specificity 29%).

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Summary/Conclusion: Predicting VTE in hospitalized COVID-19 patients cannot be relied on pre-COVID era developed VTE risk scores. D-dimer based score (IMPROVEDD) had best diagnostic properties in comparison to other scores.

P1692: PREDICTIVE MODEL FOR VEIN THROMBOEMBOLIC EVENTS DEVELOPMENT IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

M. Mitrovic 1,2,* , M. Virijević 1,2 , N. Pantic 1 , Z. Pravdic 1 , N. Sabljic 1 , Z. Bukumiric 3 , M. Cvetkovic 2 , J. Rajić 1 , M. Todorovic Balint 1,2 , V. Ana 1,2 , N. Suvajdzic Vukovic 1,2 , D. Antic 1,2

1 Clinic for Hematology, UCCS; 2 Faculty of medicine, University of Belgrade, Belgrade, Serbia; 3 Institute of medical statistics and informatics, Faculty of medicine, University of Belgrade, Belgrade, Serbia

Background: Patients with acute myeloid leukemia (AML) are at increased risk of vein thromboembolic events (VTE). However, thromboprophylaxis is largely underused, mainly due to increased risk of bleeding and limited number of studies analizing risk factors for VTE development.

Aims: To assess potential risk factors for the VTE development and develop a simple predictive model, based on patients and disease-related parameters.

Methods: We conducted a retrospective cohort study of adult patients with newly diagnosed AML who were treated in our institution between January 2009 and December 2021. Patients with AML-M3 were excluded. Primary end point of our study was imaging-confirmed VTE including simptomatic central venous catheter -related thrombosis (CVC). Participants were followed from the AML diagnosis to death or 6 months after the last cycle of therapy. We analyzed demographic factors (age, gender), Eastern Cooperative Oncology Group performance status (ECOG PS), body mass index (BMI), smoking status, comorbidities, Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI), baseline laboratory findings (complete bood counts, fibrinogen, PT, PTT, D dimer, LDH) leukemia related paremeters (cytogenetics, molecular genetics ( FLT3, NPM ), flow cytometry), Khorana score (KRS) as potential risk factors for the VTE development. Univariate and multivariate logistic regression model was used as the method for analyzing binary outcome (VTE development) and potential predictors. Based on our final model we cretaed a nomogram to enable calculalation of probability of VTE development.

Results: The study population included 608 eligible AML patients, mean age 55 (range: 18-81) years, male/female 341/267 (56.1%/43.9%). During the follow-up period 81/608 (13.3%) patients developed VTE: CVL 61/608 (10.0%), deep vein thrombosis 16/608 (2.63%), pulmonary embolism 4/608 (0.01%). Before therapy, during induction, consolidation, transplantation and relapse VTE were developed in 5/81 (6.2%), 36/81 (44.4%), 30/81 (37%), 4/81 (4.9%), 6/81 (7.4%) patients respectively.

Among all tested parameters five which are independent predictictors were included in predictive model: male sex (p=0.020, OR 1,92 95% CI: 1.11-3.31), prior history of TE (p=0.001, OR 3.83, 95% CI: 1.72-8.55), PT (p=0.107, OR 1.01, 95% CI: 0.99-1.03), ECOG PS (p=0.038, OR 0.72, 95% CI: 0.53-0.98), HCT-CI (p=0.032, OR 0.79, 95% CI: 0.64-0.98). All other patient and disease related paremeters, including Khorana score were not predictive. Our nomogram provides a visual depiction of the relative contribution of each prognostic factor to the total point score and, thus, the weight of factors regarding probability for VTE development (Picture 1).

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Summary/Conclusion: Our study point out that: 1. A KRS score has no predictive value in AML; 2. Male patients and patients with history of prior thrombosis have 1.92 and 3.83 higher odds of developed VTE; 3. Increasing of ECOG and HCT-CI for one point decrease probability of develope thrombosis for 28% and 21%; 4. Thromboprophylaxis should be considered in patients with prior history of TE, especially with male sex, good PS and low HCT-CI. Our nomogram may help clinicians in clinical decision making. However, validation of our predictive model through prospective study with a larger number of patients are required.

P1693: THROMBOMODULIN RESISTANCE: A NEW PROTHROMBOTIC PATHWAY IN COVID-19

A. Montaño 1,2,* , A. Marco 3 , F. J. López Jaime 1 , I. Fernández Bello 1 , I. Márquez Gómez 4 , J. M. Reguera Iglesias 4 , M. I. Muñoz Pérez 1 , P. Marco 3

1 Unidad de Hemostasia y Trombosis, Hospital Universitario Regional de Málaga, IBIMA, Málaga; 2 Universidad de Salamanca, Salamanca; 3 Hospital General Universitario Dr. Balmis, Alicante; 4 Servicio de Enfermedades Infecciosas, Hospital Universitario Regional de Málaga, Málaga, Spain

Background: Hypercoagulability and endothelial dysfunction have been shown to be closely related and both are considered as one of the main factors influencing the severity of COVID-19 patients. Thrombomodulin, among other factors, plays a key role as an anticoagulant factor through activation of protein C and subsequent limitation of thrombin generation. The endothelium also exerts anticoagulant activity under normal conditions. However, in COVID-19 patients, especially the most severe ones, the inflammatory process has been shown to trigger the release of the cytokine ANGPT2 through the endothelium, leading to the arrest of thrombomodulin and thus contributing to the prothrombotic state. Indeed, recent studies suggest that ANGPT2 inhibition may be explored as a therapeutic target for COVID-19 patients and other hypercoagulable diseases. However, to date there are no studies evaluating the role of thrombomodulin in COVID-19 hypercoagulability.

Aims: To analyze thrombomodulin resistance in hospitalized COVID-19 patients by thrombin generation test and correlation with disease severity.

Methods: Forty-two hospitalized COVID-19 patients on admission were included in the study. Blood samples were anticoagulated by trisodium citrate, and immediately centrifugated for platelet poor plasma, and frozen at -80ºC until analysis. The hemostatic profile of patients was determined by routine laboratory techniques and the Quantra ® Hemostasis Analyzer. Thrombomodulin resistance was assessed by comparing Endogenous Thrombin Potential (ETP) with/without thrombomodulin using GENESIA ® analyzer. Student’s t-test or Mann-Whitney U-test were used for comparison of means. Correlation was evaluated by Spearman correlation coefficient. Need for mechanical ventilation, ICU admission, encephalitis, thrombosis or death were considered as adverse events.

Results: 23.8% of patients (n=10) had an adverse event during hospitalization. Patients with and without adverse events showed similar ETP (1615 vs 1486; P =0.257). However, the thrombin generation study with thrombomodulin revealed a higher ETP in patients with adverse event (1237.8 vs 786.9 mA; P =0.002). Consistent with this result, these patients showed a higher resistance to thrombomodulin with a lower % inhibition of ETP compared to patients without adverse event (24.3 vs 47.6; P =0.003). In fact, 100% of patients with adverse events showed <40% inhibition of ETP with thrombomodulin ( P <0.001). Patients with a poor prognosis also showed a higher thrombin-peak (352.9 vs 310.6 nM; P =0.038), with an even more notable difference in the presence of thrombomodulin (308.6 vs 197.9 nM; P =0.006). In addition, these patients had a significantly higher thrombin generation velocity (247.8 vs. 144.3 nM/min; P =0.004) and consequently a shorter time to peak (4.4 vs 5.4 min; P =0.042). Notably, lower % inhibition by thrombomodulin and higher thrombin peak correlated significantly with higher clot stiffness measured by Quantra ® (R=0.372, P =0.017 and R=0.320, P =0.042, respectively). ETP in the presence of thrombomodulin proved to be a good predictor of an adverse event during admission with an AUC of 0.791 ( P <0.001).

Summary/Conclusion: Measurement of thrombin generation can be a powerful tool for the analysis of patients with COVID-19 on admission and thus for risk stratification. Increased thrombomodulin resistance is associated with the presence of an adverse event in patients and may be considered as new independent prognostic marker.

P1694: THROMBOSIS RISK ASSESSMENT IN LYMPHOMA PATIENTS - PROSPECTIVE PILOT SUDY

D. Antic 1,2,* , V. Otasevic 1 , N. Milic 3 , V. Vukovic 1 , B. Mihaljevic 1,2

1 Lymphoma Center, Clinic for Hematology, Univeristy Clinical Center of Serbia; 2 Faculty of Medicine, University of Belgrade, Belgrade, Serbia; 3 Institute for medical statistics and informatics, Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Background: The incidence of thromboembolism (TE) varies throughout the literature, because of several reasons. Firstly, lymphomas are characterized by pronounced heterogeneity, with distinctive features depending on the biology of lymphoma subtype. More importantly, the scarcity of proper disease specific guidelines for TE diagnostics and the lack of wide accepted usage of disease specific risk assessment model(s) (RAMs), combined with underestimation of frequency of TE in lymphoma patients, consequently results in inadequate standard of care for TE in this specific group of patients.

Aims: Therefore, we aimed to assess the predicting power of different RAMs for TE development, coupled with diagnostic algorithms that distinguish the objective need for radiological diagnostic procedures (colour duplex scan (CDS) and computerized tomography pulmonary angiography (CTPA)).

Methods: In a prospective cohort study a total of 36 patients with lymphoma were included. We used proper validated algorithms that define the demand for radiologic procedures. For suspected deep vein thrombosis (DVT) combined algorithm of Wells score and D-dimer was used, and for suspected pulmonary embolism (PE) we used YEARS algorithm. Prior to initiation of specific hematological treatment, all patients included in the study were subjected to the predefined study protocol which outlined the requirement of radiologic studies for TE evaluation. Blood samples were taken for D-dimer measurement (unit mg/L). TE events in lymphoma patients have been diagnosed from the time of initial hospitalization up to 3 months after the last cycle of therapy. Moreover, we used following RAMs for TE risk estimation: ThroLy score, Khorana score and Vienna CATS score. The patients at risk were classified by ThroLy score (>1), by Khorana score (≥2), and Vienna CATS score (expressed as predicted 6 months risk for venous TE (VTE) development).

Results: The median patients’ age was 62 years (range, 25-76 years); 52.8% were females. The diagnosis of aggressive non-Hodgkin lymphoma (NHL) was predominant (59.5%). The majority of the patients had limited or intermediate stage of disease: Ann Arbor stage I/II 51.4%, stage III/IV 48.6%, respectively. 5 TE events (13.8%) were diagnosed, 4 lower extremity DVT (11.1%) and 1 PE (2.7%). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of algorithm for DVT assessment based on Wells score and D-dimer level were 60%, 90.3%, 50% and 93.3% whilst for YEARS algorithm were 60%, 64.5%, 21.4% and 90.9%, respectively. Median D-dimer level in lymphoma patients with TE event was 2.19 mg/L (range, 0.54-6.64) comparing to patients without thrombosis 0.7 mg/L (range, 0.17-5.89). Median predicted 6 months risk for VTE development by Vienna CATS score in patients with thrombosis was 6.5% (range, 4.9-9.1) vs. patients without thrombosis 5.1% (range, 4.4-8.8). Sensitivity, specificity, PPV and NPV of ThroLy score were 80%, 38.7%, 17.4% and 92.3%, while for Khorana score were 40%, 83.9%, 28.6% and 89.7%, respectively.

Summary/Conclusion: The use of disease specific RAMs and TE diagnostic approach algorithms should be encouraged considering their acceptable statistic performance in risk assessment of TE complications in lymphoma patients. Further and larger studies, that would potentially incorporate D-dimer level in existing RAMs could possibly result in risk prediction enhancement.

P1695: CEREBRAL VENOUS THROMBOSIS AND ANTITHROMBOTIC THERAPY: A SYSTEMATIC REVIEW

V. Papageorgiou 1,* , E. Lefkou 1 , G. Xiromerisiou 2 , F. Girtovitis 3 , M. Matsagkas 4

1 PERIGENESIS - Institute of Obstetric Hematology, Thessaloniki; 2 Department of Neurology - Faculty of Medicine - School of Health Sciences, Thessaly University, Larissa; 3 Department of Blood Transfusion, AHEPA University Hospital, Thessaloniki; 4 Department of Vascular Surgery - Faculty of Medicine - School of Health Sciences, Thessaly University, Larissa, Greece

Background: Cerebral venous thrombosis (CVT) is a cause of stroke especially in the young. The anticoagulant treatment remains controversial as it prevents neurological deterioration, new venous infarcts and death but there is a risk of bleeding complications

Aims: The aim of the study was to systematically review the available studies on the efficacy and safety of anticoagulant therapy in CVT

Methods: A literature search was performed (PubMed) by a combination of the terms: 1. cerebral vein thrombosis 2. cerebral venous thrombosis 3. antithrombotic therapy 4. anticoagulant therapy 5. heparin 6. warfarin 7. VKA 8. DOAC. The references of the studies were checked to obtain additional studies. The search included studies: a) related to humans b) in English and c) period:1991-2021. The following data were extracted: study characteristics (year of publication, country, study-type, sample size), demographic characteristics, type/dose of anticoagulant therapy, median follow-up, major bleeding complications, other complications during follow-up. The inclusion criteria were: 1) CVT confirmed by imaging (angiography,CT,MRI,MRV) 2) any type of anticoagulation 3) sample size>5 & 4) age>16 years

Results: 25 studies were included with a total number of 2320 patients. The percentage of female patients was 70%. The age ranged 16-86 years. A total of 6 RCT, 9 prospective observational studies, 9 retrospective observational studies and 1 ambidirectional observational study were detected. Based on the limited data the anticoagulant therapy appears safe and effective, as in the acute phase it improves the clinical outcome. It is recommended that patients with acute CVT are treated with heparin. It is not contraindicated even in hemorrhagic infarcts but should be used with caution in extensive hemorrhagic infarcts, in-depth localization of the infarct and coma. LMWH appears to be associated with better outcome and fewer new intracranial hemorrhages than UFH. There is no study evaluating the role of antiplatelets. There are also insufficient data on the optimal duration of anticoagulation therapy. The use of DOAC is not recommended for the treatment of CVT, especially in the acute phase, due to the poor available evidence. Nevertheless, the limited data on the use of DOAC in the treatment of CVT imply adequate safety and efficacy. There is no study comparing different DOAC for CVT treatment. There are also insufficient data to support the use of thrombolysis in patients with CVT, as only case-reports and patient-series have been published. There is only 1 RCT in which “high-risk” patients were randomized to intravascular thrombolysis or heparin-controlled treatment. According to the European Guideline for the diagnosis and management of CVT it is not possible to provide recommendation for thrombolysis. It is suggested though not to use thrombolysis in patients with acute CVT with a low risk of poor outcome. The present systematic review highlights the lack of adequate data on optimal anticoagulation therapy in CVT

Summary/Conclusion: Anticoagulant therapy for CVT appears to be safe and effective. It is recommended that patients with acute CVT are treated with heparin even cases with bleeding elements at diagnosis. LMWH appears to be associated with better outcomes and fewer new intracranial hemorrhages. The position of DOACs is an object of future research. Thrombolysis in CVT should be reserved for high-risk patients. Given the low incidence of the disease the cooperation of centers at international level is necessary. The subject of anticoagulant therapy in the treatment of CVT remains open for further research

P1696: HEMOSTATIC ACTIVATION MARKERS AND SEROLOGICAL RESPONSE IN SUBJECTS RECEIVING ANTI-COVID-19 VACCINATION

F. Schieppati 1,* , S. Gamba 1 , E. Galimberti 2 , L. Russo 1 , C. Giaccherini 1 , S. Bolognini 1 , C. J. Tartari 1 , C. Ticozzi 1 , A. M. Palladino 2 , O. C. Cretu 2 , L. Barcella 1 , M. Marchetti 1 , A. Falanga 1,2

1 Immunohematology and Transfusion Medicine, Hospital Papa Giovanni XXIII, Bergamo; 2 Medicine and Surgery, University of Milano Bicocca, Milano, Italy

Background: SARS-COV2 infection is associated with inflammation, hypercoagulability and endothelial damage. Anti-SARS-COV2 vaccines have radically changed the course of the pandemic, however, reports on rare thrombotic events raise concern in the scientific community and the general population.

Aims: In a prospectively enrolled cohort of adult subjects undergoing mRNA or adenovirus vector vaccination, we wanted to longitudinally evaluate the changes in levels of hemostatic biomarkers (i.e. activation of blood coagulation and perturbance of endothelium and fibrinolysis), together with the serological response, and occurrence of manifest thrombotic complications.

Methods: Peripheral venous blood samples were collected at enrollment (day 0, D0) before the 1st vaccine dose, and on 15 (D15), 60 (D60), 90 (D90) and 180 (D180) days after the 1st dose. At each time point, hemostatic markers (i.e., fibrinogen, D-dimer, FVIII, von Willebrand Factor [vWF] antigen and activity, F1 + 2, thrombomodulin, protein C, protein S, FXIII, tPA, and PAI-1), and anti-Spike receptor-binding-domain protein (anti-S/RBD) IgG were measured. Follow up is currently continuing.

Results: Fifty-three subjects (57% males) with a median age of 50 years (range 23-86) were enrolled into the study and followed-up for 6 months: 36 (68%) received BNT162b2, 6 (11%) mRNA-1273, and 8 (15%) ChAdOx1 nCoV-19 vaccines, in 2 doses over 21, 30 and 77 days, respectively; while 3 (6%) subjects received Ad26.COV2.S as single shot. Twenty individuals (38%) reported previous history of COVID-19, with a mean time from infection to vaccination of 10 months (4-18); only 1 required hospitalization. Nine subjects presented cardiovascular risk factors and 4 a prior, non-active, cancer; 3 were on anticoagulation for atrial fibrillation. The evaluation of the hemostatic biomarkers at the different time points showed variations in some of the parameters evaluated, with median values remaining within normal range levels. Specifically, compared to baseline, we observed a significant increase in thrombomodulin at D90 (p=0.001) and D180 (p=0.03), in parallel to a significant decrease in fibrinogen (D60), vWF-Ag (D60 and D180), FVIII (D60, D90 and D180), and TPA (D60 and D90) levels. The reduction of these biomarkers was particularly evident in individuals with a history of COVID-19. Of interest, this group of subjects was also characterized by significantly lower levels of PAI-1 both at baseline (7.18 ng/mL vs 17.53 ng/mL; p<0.0001), and at other time points (p<0.0001), and by an increase in F1 + 2 at D90 (p=0.02). The association between lower baseline PAI-1 levels with history of COVID-19 was confirmed by linear regression analysis (B= -10.351, p=0.013), and was independent by the time of infection resolution. Notably, no differences were observed in the hemostatic biomarkers according to vaccine types. All subjects positively responded to vaccination with a significant increase in anti-S/RBD IgG from baseline (D0) to each time point, especially COVID-19 subjects (D15, D60, and D90:p<0.0001; D180:p=0.031). No thrombotic or cardiovascular complications occurred during follow-up.

Summary/Conclusion: No hypercoagulable state elicited by COVID-19 vaccination was observed, contrarily we detected an overall persistent reduction of coagulation activation over time. Subjects with previous SARS-COV2 infection had persistently low levels of PAI-1, supporting enhanced fibrinolysis activation. Compared with recent studies, our results provide a longer observation follow-up with all vaccine types and reassure on the safety of anti-SARS-COV2 vaccination.

P1697: EVALUATION OF THE DIAGNOSTIC PERFORMANCE OF THE TINA-QUANT D-DIMER GEN. 2 IN PATIENTS WITH SUSPECTED DEEP VEIN THROMBOSIS

B. Tonne 1,* , M. H. Pedersen 1 , S. G. Fronas 2,3 , C. T. Jorgensen 3,4 , A.-B. Mathisen 5 , W. Ghanima 2,3 , L. Garabet 1,5

1 Department of Multidisciplinary Laboratory Medicine and Medical Biochemistry, Akershus University Hospital, Lørenskog; 2 Department of Internal Medicine, Østfold Hospital, Grålum; 3 Institute of Clinical Medicine, University of Oslo, Oslo; 4 Department of Emergency Medicine; 5 Center of Laboratory Medicine, Østfold Hospital, Grålum, Norway

Background: Combined with clinical pretest probability evaluation, D-dimer is a central element in the diagnostic work-up of suspected deep vein thrombosis (DVT). Different assays are available for the measurement of D-dimer. However, D-dimer assays are not standardized and the results are not comparable among the different assays. Therefore, the cut-off used to discriminate between positive and negative test results should be evaluated assay-wise in each institution. According to our best knowledge, only one study has evaluated the diagnostic performance of Tina-quant D-Dimer Gen. 2 assay in proximal DVT, and this study excluded patients with recurrent DVT.

Aims: The aim of our study was to evaluate the diagnostic performance of the Tina-quant D-Dimer Gen. 2 assay (Roche Diagnostics) in the exclusion of all types of DVT, and to compare its performance with the STA-Liatest D-Di Plus (Stago).

Methods: In the Ri-Schedule study (Rivaroxaban for Scheduled Work-up of Deep Vein Thrombosis; NCT 02486445 ), which was a prospective outcome study, D-dimer was measured with STA-Liatest D-Di Plus in 1653 adult patients referred to the Emergency Department at Østfold Hospital with suspected DVT. Exclusion criteria were failure to consent, and previous inclusion in the Ri-Schedule study within the past 3 months. Patients with D-dimer levels ≥ 0.5 µg/mL fibrinogen equivalent units (FEU) were referred to whole-leg compression ultrasonography (CUS) to confirm/exclude the DVT diagnosis. All patients were followed up after 90 days. To evaluate the diagnostic performance of the Tina-quant D-Dimer Gen. 2 assay, stored frozen plasma samples acquired from 1097 patient in the Ri-Schedule study were analyzed on Cobas t711. These samples were withdrawn simultaneously as the sample that were analyzed with the STA-Liatest D-Di Plus on STA-R. The diagnostic performance of the two assays were depicted and compared using test performance parameters and ROC curve.

Results: Of the 1097 patients, 730 patients had a positive D-dimer with the Tina-quant D-Dimer Gen. 2, and 775 patients had a positive D-dimer with the STA-Liatest D-Di Plus; cut-off for both tests was ≥ 0.5 µg/mL FEU. DVT diagnosis was confirmed in 210 (19.2%) by CUS examination. The sensitivity and negative predictive value (NPV) for the Tina-quant D-Dimer Gen. 2 assay were 96.2% (95% CI 92.6–98.3) and 97.8% (95% CI 95.8–98.9) respectively; the specificity and positive predictive value (PPV) were 40.5% (95% 37.2–43.8) and 27.7% (95% CI 26.5–28.9) respectively (Table 1). The sensitivity and NPV for the STA-Liatest D-Di Plus assay were 98.1% (95% CI 95.2–99.5) and 98.8% (95% CI 96.8–99.5) respectively (Table 1). ROC curve analysis showed an AUC of 0.85 for both assays, with no significant differences between the assays. Of those with D-dimer value below the cut-off, 8 patients had DVT diagnosed by CUS at time of initial work up or during the subsequent 90 day with Tina-quant as compared with STA-D-Di Plus.

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Summary/Conclusion: The Tina-quant D-Dimer Gen. 2 assay on Cobas t711 showed an overall good and comparable performance for the exclusion of DVT when compared with STA-Liatest D-Di Plus assay.

P1698: INCREASED COMPLEMENT ACTIVATION AND DECREASED ADAMTS13 ACTIVITY IN PATIENTS WITH PRE-ECLAMPSIA COMPARED TO HEALTHY PREGNANCIES

T. M. Venou 1,* , C. Varelas 2 , A. Gardikioti 1 , E. Vetsiou 1 , P. Klonizakis 1 , A. Daniilidis 3 , I. Christodoulou 1 , E.-E. Koravou 2 , M. Koutra 2 , I. Mavrikou 2 , T. Touloumenidou 2 , A. Papalexandri 2 , I. Sakellari 2 , E. Mandala 4 , E. Vlachaki 1 , E. Gavriilaki 2

1 Hematological Laboratory, Second Department of Internal Medicine, Aristotle University, Hippokration Hospital; 2 Hematology Department, G. Papanicolaou Hospital; 3 Second Department of Obstetrics and Gynaecology, Faculty of Medicine, Aristotle University of Thessaloniki; 4 Fourth Department of Internal Medicine, Hippokration Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece

Background: Preeclampsia is a progressive multi systemic disorder, characterized by either proteinuria or critical organ damage in addition to new-onset hypertension. Although ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats) and the complement system have been implicated in the pathogenesis of the disease, there are currently no specific biomarkers for diagnosis and severity prediction of preeclampsia.

Aims: We investigated whether ADAMTS13 and the complement system could play a role in the development of preeclampsia.

Methods: We studied consecutive preeclamptic and control pregnant women of similar age and gestational weeks that provided a written informed consent. Preeclampsia was diagnosed according to American College of Obstetricians and Gynaecologists criteria: one instance of systolic blood pressure (SBP)>160 or diastolic blood pressure (DBP)>110 or two instances of SBP>140 or DBP>90 with proteinuria or evidence of end-organ damage. Patients with pre-existing hypertensive conditions or a history of pre-eclampsia, HELLP syndrome, antiphospholipid syndrome, or other autoimmune disorders and complement-mediated disease were excluded. Peripheral blood samples were collected to perform genetic sequencing of complement regulatory genes ( Complement factor H/CFH, CFH-related, CFI, CFB, CFD, C3, CD55, C5, MCP, thombomodulin/THBD, ADAMTS13 , MiniSeq System, Illumina). The modified Ham test was used as a functional in vitro assay to test for complement activation. ADAMTS13 activity and soluble C5b-9 were measured with commercially available ELISA kits.

Results: We studied 22 Caucasian preeclamptic pregnant women aged 34±7 years, at median 32 gestational weeks, as well as a control group of 15 healthy pregnancies aged 31±6 years, at median 30 gestational weeks. Healthy pregnancies had similar baseline characteristics and otherwise uncomplicated pregnancies with no sign of gestational hypertension until normal birth. As expected, serum creatinine and urine protein excretion were significantly increased in preeclampsia (p=0.029 and p:0.025). Interestingly, patients with preeclampsia had decreased ADAMTS13 activity (median 70%, range 1-96% versus 92% [54-104%], p=0.012) and increased C5b-9 (311[25-1909] versus 35[11-22], p=0.007), with a trend toward significant association between them (p=0.058). Importantly, the modified HAM test was found to be positive only in preeclamptic women (8/22, 36%) with a statistically significant difference between the two groups (p=0.008). Regarding the genetic analysis, risk-factor variants were found in ADAMTD13 (rs2301612), C3 (rs2230199), and CFH (rs800292). Double heterozygocity in these genes was found only in preeclamptic women (12/22, 54%, p<0.001). Focusing on patients with worse outcomes, only one patient with a positive mHam assay (C5b-9 1232 ng/ml, ADAMTS13 31%) and double heterozygous variants had renal failure.

Summary/Conclusion: Our study indicates that a portion of patients with preeclampsia has increased complement activation and decreased ADAMTS13 activity. Given the potential association of these markers with worse outcomes, further studies are needed to determine whether they can serve as reliable biomarkers or predictive factors of the disease course.

P1699: ANTITHROMBIN DEFICIENCY IS ASSOCIATED WITH PROTHROMBOTIC FIBRIN CLOT PHENOTYPE

M. Zabczyk 1,* , J. Natorska 1 , A. Undas 1

1 Jagiellonian University Medical College, Krakow, Poland

Background: Antithrombin is the key inhibitor of coagulation factors and its deficiency is associated with increased risk of venous thromboembolism (VTE).

Aims: This study was aimed to investigate whether antithrombin deficiency affects fibrin clot structure or function, and which factors determine fibrin clot properties in these patients.

Methods: We evaluated VTE patients with genetically confirmed antithrombin deficiency (n=40) aged 41±16 years compared to age- and sex-matched individuals with normal antithrombin activity following VTE (n=40). All patients evaluated >3 months since the first VTE episode were treated with non-vitamin K antagonist oral anticoagulants (NOACs). Blood was drawn 24 hours after NOAC intake. Fibrin clot permeability (K s ), clot lysis time (CLT), clot microstructure, along with thrombin generation markers and fibrinolysis inhibitors were assessed. The study was approved by the Ethical Committee and conducted in accordance with the Declaration of Helsinki.

Results: Subjects with antithrombin deficiency had 35% reduced antithrombin activity compared to controls (p<0.001). They did not differ with regard to levels of fibrinogen, thrombin-antithrombin complex or prothrombin fragments 1 + 2 (all p>0.05) compared to controls, while endogenous thrombin potential was 40% higher in antithrombin-deficient patients. Moreover, they were characterized by reduced K s (-22%, p<0.001) and prolonged CLT (+30%, p<0.001). Antithrombin activity correlated with K s (r=0.39, p<0.01) and CLT (r=-0.44, p<0.05) in antithrombin-deficient patients. ETP (r=-0.37, p=0.017) was associated with antithrombin levels (r=-0.37, p=0.017) but not with fibrin clot features. Type I plasminogen activator inhibitor (PAI-1) levels were reduced by 48% (p<0.001), while thrombin-activatable fibrinolysis inhibitor (TAFI) levels were 9% lower (p=0.028) in patients with antithrombin deficiency compared to patients with normal antithrombin activity. In vitro addition of human antithrombin resulted in thrombin generation inhibition in a dose-dependent manner (p<0.001 for trend), with a slight effect on fibrin clot properties.

Summary/Conclusion: Prothrombotic fibrin clot phenotype in antithrombin-deficient patients is associated with impaired plasma fibrinolytic potential, not related to increased levels of fibrinolysis inhibitors, even after antithrombin supplementation.

This work was supported by the Polish National Science Centre (UMO-2018/31/D/NZ5/01299).

P1700: EARLY APPEARANCE OF CARDIOTOXICITY, IN CHILDREN WITH HEMATOLOGICAL MALIGNANCIES (HM)

K. Antoniadi 1,* , C. Kelaidi 1 , V. Tzotzola 1 , M. Ampatzidou 1 , E. Karanasios 2 , M. Drakopoulou 3 , V. Papadakis 1 , P. Nihoyannopoulos 3 , D. Tousoulis 3 , K. Toutouzas 3 , N. Thomaidis 4 , S. Polychronopoulou 1

1 Department of Pediatric Hematology-Oncology,Aghia Sophia Children’s Hospital,Athens,Greece; 2 Department of Cardiology, Aghia Sophia Children’s Hospital; 3 First Department of Cardiology, Hippokration Hospital,Athens,Greece; 4 Department of Chemistry, National and Kapodistrian University of Athens, Athens, Greece

Background: Cardiotoxicity is the most serious non-hematological toxicity of cancer treatment and is defined as acute(<1 month) and early onset(< 1year from the initiation of treatment).

Aims: Aim of the study is the prospective analysis of the acute and early onset cardiotoxicity and outcome of patients with HM.

Methods: Cardiotoxicity was defined by any combination of 1) reduction of the ejection fraction> 10% or at a value <50%) 2) electrophysiological dysfunction 3) other abnormal ultrasound, clinical or enzymatic findings. During the period 2018-2021, 47 patients with HM treated in our Department were studied, due to early cardiotoxicity. Patiens’ characteristics:30/47 boys, median age 8.0 years old (range 2.5-16), 25 with ALL, 7 with AML, 2 with CML and 13 with Lymphoma. All patients underwent a complete clinical, laboratory and imaging examination (ECG, echocardiogram) at diagnosis, prior each regimen and at the onset of signs of cardiotoxicity.

Results: At admission 44/47 patients had a free medical history and normal tests. Two patients (1 with Hodgkin’s lymphoma and 1 with AML) had an abnormal troponin value before starting chemotherapy, without further clinical manifestations. The third patient, 3.5 years old with ALL, had hypertrophic cardiomyopathy from birth due to inherited Noonan syndrome.

Thirteen patients (9/13 boys) showed cardiotoxicity, 2 after the first regimen of chemotherapy (acute form) and 11 within the 1st year after onset (subacute form). Acute cardiotoxicity occurred in two patients (1 with high-risk ALL, 1 with Burkitt Lymphoma) with a decrease in ejection fraction (EF <55%) along with bradycardia and abnormal ECG. Subacute form of cardiotoxicity occurred in 11/13 patients. 4 patients (1 with AML high-risk monosomy 7, 1 with Hodgkin’s Lymphoma, 1 with ALL and 1 with T-Lymphoblastic Lymphoma) showed reduced in ejection fraction (<55%) and concomitant increase in troponin value (max 35.85 pg/ml). Two patients with ALL showed an increase in cardiac enzymes (max value 98.05 pg / ml) with concomitant ECG disturbance and normal echocardiographic study. Two high-risk AML patients (1 due to FLT3 positivity), presented with pericardial fluid and QTc prolongation, respectively. One intermediate risk AML patient developed abnormal ventricular contractions with normal echocardiogram and cardiac enzymes and was treated with propranolol. Two patients, one with Hodgkin’ Lymphoma and one with Burkitt Lymphoma, developed chest pain with elevated troponin but with normal ECG and echocardiographic study. Forty five patients received anthracyclines in a total dose of anthracyclines <360mg / m2 and no-one was irradiated. All thirteen patients showed gradual recovery over a period of 1 week to 2 months after the event. 5 patients were treated with ACE inhibitor or b-blocker. All patients continued their chemotherapy in full doses (2/13 with minor delay). In one patient the total dose was reduced to 1/3.

Summary/Conclusion: The early results of our ongoing study, show that moderate or severe cardiotoxicity occurred in 30% of patients with hematologic malignancies within the first year of diagnosis and 10% of them needed pharmaceutical intervention. The degree and course of cardiotoxicity seem to differ significantly between our patients, suggesting that both the underlying genetic predisposition and the presence of various other risk factors contribute to the severity of its manifestation.

P1701: A COMPARATIVE STUDY OF THE IMPACT OF COUNTRIES’ HEALTH EXPENDITURE ON THE DIAGNOSIS EXPERIENCES OF PATIENTS WITH LYMPHOMA AND CLL IN EUROPE

F. Bamigbola 1,* , L. Warwick 2 , N. Bolaños 2

1 Research; 2 Management, Lymphoma Coalition, Mississauga, Canada

Background: Lymphomas are a mixed group of cancers that have a variety of clinical presentations ranging from an indolent course to an aggressive disease. As such, they are often a challenge to diagnose. A long time to diagnosis of lymphoma or CLL can result in increased patient anxiety and may impact patients’ survival and quality of life. In addition, communication with healthcare professionals is a key part of the patient experience at diagnosis as patients will use the information to make sense of their diagnosis and care.

Aims: Using data from the Lymphoma Coalition (LC) 2020 Global Patient Survey (GPS) on Lymphomas and CLL, this study examines the impact of countries’ health expenditure (as a percentage of the Gross Domestic Product (GDP)) on the diagnostic experiences of patients with lymphoma in Europe.

Methods: Globally, there were 11,878 respondents to the LC 2020 GPS, comprised of 9,179 patients and 2,699 caregivers. 4,346 patients responded from 36 European countries. Using the 2019 World Health Organisation (WHO) Global Health Expenditure Database, LC calculated the average percentage of health spending by these European countries’ governments - 8.2% . A comparison was then done between GPS respondents from countries with below-average health expenditure (BA-HE) (n=452 ) and countries with above-average health expenditure (AA-HE) (n=3894).

Demographics of the two groups were examined, and univariate, bivariate and multivariate analyses of questions relating to patients’ diagnosis experience were performed in IBM SPSS v27.

Results: Both groups differed significantly in the time taken from patients’ first appointment with a GP about their symptoms to getting a lymphoma or CLL diagnosis (Table 1). 40% of patients in the BA-HE countries received their diagnosis in less than 3 months compared to 51% of patients in the AA-HE countries. Over 10% of patients in both country groups (BA-HE- 14%; AA-HE-13%) reported getting a diagnosis more than 12 months after their first GP visit for their symptoms (Table 1).

Regarding information experience at diagnosis, patients in AA-HE countries were 2.5 times more likely to have been told their lymphoma subtype at diagnosis than not remember if they were told their subtype at diagnosis when compared to patients in BA-HE countries (Table 1). Patients in AA-HE countries were also 67% more likely to report that they were given the right amount of information than not given enough information when compared to patients in BA-HE countries (Table 1).

Patients in AA-HE countries were 48% less likely to need more information about treatment options and 58% less likely to need more information about support for self-care compared to patients in BA-HE countries. These differences were statistically significant (Table 1).

There were no significant differences in both groups in the need for more information in the following areas: psychological support, support for their families, side effects of treatment, diagnosis and what it means, and fertility (Table 1).

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Summary/Conclusion: Both groups differed in the time taken to get a diagnosis of lymphoma or CLL and in their information experiences at diagnosis. On the other hand, both groups showed an equal need for more information in areas such as psychosocial support and the side effects of treatment.

LC advocates for increasing awareness of lymphoma among the general populace and the medical community to accelerate diagnosis. Healthcare providers should also provide patients with credible, understandable information to support the best possible patient experience.

P1702: IMPACT OF CENTER CHARACTERISTICS AND MACROECONOMIC FACTORS ON THE OUTCOME OF ADULT PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA TREATED WITH PEDIATRIC-INSPIRED PROTOCOLS

P. Barba 1,* , M. Morgades 2 , P. Montesinos 3 , J. Gonzalez Campos 4 , A. Torrent 2 , C. Gil 5 , T. Bernal 6 , M. Tormo 7 , S. Mercadal 8 , S. Saumell 9 , I. García-Cadenas 10 , M. Queipo de Llano 11 , M. Cervera 12 , R. Coll 13 , A. Bermudez 14 , M. Amigo 15 , S. Monsalvo 16 , J. Esteve 17 , R. Garcia Boyero 18 , A. Novo 19 , J. Hernandez Rivas 20 , A. Cladera 21 , P. Martinez-Sanchez 22 , J. Serrano 23 , M. T. Artola 24 , B. Soria 25 , E. Abella 26 , F. Vall-Llobera 27 , J. Bergua 28 , P. Herrera 29 , D. Barrios 30 , J. M. Ribera 2

1 Hematology, Vall Hebron University Hospital, BARCELONA; 2 Hospital Germans Trias i Pujol, Badalona; 3 Hospital La Fe, Valencia; 4 Hospital Virgen del Rocío, Sevilla; 5 Hospital General Alicante, Alicante; 6 Hospital General Asturias, Oviedo; 7 Hospital Clinico, Valencia; 8 Hospital Duran i Reynals, Hospitalet Llobregat; 9 Vall Hebron University Hospital; 10 Hospital de Sant Pau, BARCELONA; 11 Clinica Universitaria Virgen de la Victoria, Malaga; 12 Hospital Joan XXIII, Tarragona; 13 Hospital Trueta, Girona; 14 Hospital Marqués Valdecilla, Santander; 15 Hospital Morales Messeguer, Murcia; 16 Hospital Gregorio Marañon, Madrid; 17 Hospital Clinic, BARCELONA; 18 Hospital General Castellon, Castellon; 19 Hospital Universitari Son Espases, Palma Mallorca; 20 Hospital Universitario Salamanca, Salamanca; 21 Hospital Son Llatzer, Palma Mallorca; 22 Hospital 12 de Octubre, Madrid; 23 Hospital Reina Sofia, Cordoba; 24 Hospital Donostia, Donostia; 25 Hospital Universitario Canarias, Las Palmas; 26 Hospital del Mar, BARCELONA; 27 Hospital Mutua Terrassa, Terrassa; 28 Hospital San Pedro Alcántara, Caceres; 29 Hospital Ramon y Cajal, Madrid; 30 Hospital Carlos Haya, Malaga, Spain

Background: Several patient and disease characteristics have been identified as prognostic factors for several outcomes in adult patients diagnosed with acute lymphoblastic leukemia (ALL). However, the impact of socio-economic variables on the outcome of adult ALL has been little studied.

Aims: To analyze the potential impact of socio-economic variables on the outcome of newly diagnosed ALL in adult patients included in 4 consecutive trials of the Spanish Program for Treatment of Hematological Malignancies (PETHEMA) Group.

Methods: Patients with newly diagnosed Philadelphia chromosome (Ph) positive or negative ALL treated within one of 4 consecutive trials of the PETHEMA group from 2003-2018 were included in this study. Patients were treated with risk-adapted pediatric-inspired protocols. Allogeneic stem cell transplantation (allo-SCT) was indicated in all patients with Ph-positive ALL or in those with Ph-negative ALL showing slow clearance of measurable residual disease. Most relevant clinical variables as well as center-, demographic- and economic-related variables (generally named as socio-economic variables) were collected for all patients. A univariable and multivariable analyses (MVA) adjusted for relevant clinical variables were conducted to assess the impact of socio-economic variables on several outcomes. Collinearity among socio-economic variables was taken into account in the analysis.

Results: 830 ALL adult patients were treated in 59 centers during the study period and were enrolled in the study. Patients were included in protocols HR-03 (n=326, 39%), IR-08 (n=89, 11%), HR-11 (n=283, 34%) and PH-08 (n=132, 16%). Two-hundred and twenty-six patients (38% of the evaluable patients) received allo-SCT in first CR, according to treatment protocols. Main characteristics of patients at diagnosis as well as socio-economic variable distribution are summarized in Table 1.

Probability of CR ranged from 87% in the HR-03 to 96% in the IR-08 and PH-08 trials. Cumulative incidence of relapse (CIR) at 5 years among the 752 patients who achieved complete remission was 41% (95%CI 37-45). In the MVA adjusted model for socio-economic variables, the only variable associated with a higher CIR was a higher number of inhabitants in the city where the treatment center was located (HR 1.40 [95%CI 1.08- 1.8], p=0.01). High WBC at diagnosis and Ph-negative ALL were also associated with higher CIR.

Probability of DFS and OS at 5 years for the whole cohort was 43% (95%CI 39-47) and 46% (95%CI 42-50), respectively. In the MVA adjusted model for socio-economic variables, none of them were associated with a lower PFS or OS. ECOG PS> 1, higher WBC at diagnosis, Ph negative ALL were associated with lower PFS. These three variables and older age and earlier ALL diagnosis were associated with lower OS.

Regarding toxicity, cumulative incidence of treatment-related mortality (TRM) (including transplant-TRM) at 5 years was 17% (95% CI 14-20). Variables associated with higher TRM were older age, Ph negative ALL and earlier diagnosis. None of the socio-economic variables was associated with higher TRM in the MVA adjusted.

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Summary/Conclusion: Socio-economic variables do not seem to have an impact on outcomes in newly diagnosed adult patients with ALL in the setting of a public health system with clearly defined and structured treatment protocols. Based on our results, these protocols can be administered to ALL patients in all hospitals adhered to a PETHEMA cooperative ALL group.

P1703: ECONOMIC BURDEN OF TRANSFUSION-DEPENDENT BETA-THALASSEMIA IN THE UNITED STATES

C. Udeze 1,* , R. McKenna 2 , Y. Yang 3 , K. Evans 2 , P. Smith 2 , B. Andemariam 4

1 Vertex Pharmaceuticals, Boston; 2 IBM Watson Health, Cambridge; 3 Health Economics & Outcomes Research, Vertex Pharmaceuticals, Boston; 4 New England Sickle Cell Institute, Division of Hematology-Oncology, Neag Comprehensive Cancer Center, UCONN Health, University of Connecticut, Farmington, United States of America

Background: Transfusion-dependent beta-thalassemia (TDT) is a genetic blood disorder whose treatment is characterized by lifelong red blood cell transfusions (RBCTs) and iron chelation therapy (ICT).

Aims: This study aims to describe the economic burden of patients with TDT in the United States (US).

Methods: This retrospective cohort study used administrative claims from US MarketScan Commercial, Medicare, and Medicaid Multi-state databases to identify patients with ≥1 inpatient or ≥2 outpatient claims for beta-thalassemia or Hemoglobin E/beta-thalassemia between March 1, 2010 – March 1, 2019. Eligible patients with TDT were required to have ≥8 RBCTs in any 12-month period on or after the date of the earliest qualifying beta-thalassemia claim (first RBCT date = index date) and at least 12 months of post-index continuous enrollment with medical and pharmacy benefits. At least 3 days between service dates of RBCT claims were required to be considered discrete RBCTs. Patients were followed from index date to insurance disenrollment, death, or end of study period (March 1, 2020), whichever came first. Patients with hematopoietic stem cell transplant or sickle cell disease claims were excluded from this analysis. Baseline demographics, RBCT frequency, healthcare resource utilization, and costs were summarized per patient per year (PPPY).

Results: A total of 4,504 patients with a diagnosis of beta-thalassemia were identified, of which 198 met criteria for TDT and other inclusion/exclusion criteria. Mean patient age at index date was 22.6 years and 56.6% were female; 81.8% of patients were in the Commercial database, 16.7% in Medicaid, and 1.5% in Medicare. Patients received a mean 12.52 RBCTs annually. Mean total annual healthcare costs were $137,987 PPPY. Mean annual costs of RBCTs were $27,887 and ICT were $70,402 PPPY.

Summary/Conclusion: There is a significant economic burden with care of TDT patients in the US driven by chronic RBCTs and ICTs.

P1704: PROJECTED LIFETIME ECONOMIC BURDEN OF SEVERE SICKLE CELL DISEASE IN THE UNITED STATES

C. Udeze 1,* , K. Maruszczyk 2 , M. Atter 2 , A. Lopez 1

1 Vertex Pharmaceuticals, Boston, United States of America; 2 HCD Economics, The Innovation Centre, Daresbury, United Kingdom

Background: Sickle cell disease (SCD) is a rare hereditary blood disorder affecting approximately 100,000 individuals in the United States (US). SCD is associated with severe symptom burden, frequent hospitalizations, organ damage and increased risk of mortality. Vaso-occlusive crises (VOCs) are a hallmark clinical manifestation of SCD and often require emergency room visits and inpatient admissions. While there is some evidence describing the lifetime direct healthcare costs for SCD, the economic burden for patients with severe SCD, defined as experiencing recurrent VOCs, is currently unknown.

Aims: To estimate the per-patient lifetime direct healthcare cost of severe SCD from a US payer perspective using an economic model developed based on published model frameworks.

Methods: An individual-level simulation model was used to estimate the lifetime direct healthcare cost for patients with severe SCD in the US. The model followed patients with severe SCD, with an average of 7 VOCs per year, from birth until death. Similar to previously published economic models, mortality was estimated based on the occurrence of VOCs and other acute and chronic complications of SCD. Cost inputs were derived from published literature. Wholesale acquisition costs were utilized for all drug cost inputs. Scenario analyses were conducted to determine the impact of disease severity, defined by the number of VOCs per year, on the estimates of lifetime cost. The cost and impact of recently approved chronic, non-curative SCD therapies were not included in analyses.

Results: The model followed patients with severe SCD for an average of 35 years, over which time each patient experienced an average of 246 VOCs. The total undiscounted cost of SCD over the lifetime was estimated to be $5.2 million. The majority of costs (77%, $4.0 million) were attributable to the care associated with acute complications, which includes VOCs, acute chest syndrome and stroke. The lifetime cost of chronic complications, including pulmonary hypertension, chronic kidney disease, retinopathy, and neurocognitive impairment, was estimated as $760,000. Scenario analyses demonstrated the lifetime costs of SCD are highly sensitive to the annual number of VOCs; when varying the average number of VOCs per year from 5 to 9, the lifetime costs ranged from $4.2 million to $6.2 million. Patients with SCD who have a significantly elevated number of VOCs (14 annually) were projected to have $8.6 million in lifetime direct healthcare costs.

Summary/Conclusion: Model projections show significant lifetime economic burden with severe SCD in the US, with per-patient cost estimates substantially higher than those previously published for the overall SCD population. Treatments that can eliminate the occurrence of VOCs in this population would significantly lower this economic burden.

P1705: PATIENT-REPORTED OUTCOMES IN MYELOPROLIFERATIVE NEOPLASMS IN CLINICAL TRIALS: A SYSTEMATIC REVIEW

E. N. Oliva 1,* , S. Salek 2 , E. Laane 3 , M. Andreas 4 , M. Csenar 4 , N. Skoetz 4 , T. Ionova 5

1 Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio Calabria, Italy; 2 School of Life and Medical Sciences, University of Hertfordshire, Hatfield, United Kingdom; 3 Department of Hematology and Oncology, University of Tartu, Tartu, Estonia; 4 Department I of Internal Medicine, Evidence-based Oncology, University Hospital of Cologne, Cologne, Germany; 5 St. Petersburg State University Hospital, Saint-Petersburg, Russia

Background: The management of myeloproliferative neoplasms (MPNs) has substantially evolved over the past decade. Including patient-reported outcomes (PROs) to supplement clinical outcomes is becoming increasingly important in clinical trials of patients with MPNs, to ensure the intended benefits based on patients’ perspectives. PRO measurement is essential to understand the impact of treatment on their physical and psychosocial functional behaviour and evaluate the benefit-risk balance of specific treatments. However, it is unclear which PRO instruments are implemented in MPN trials, how often they are used and whether results are reported consistently.

Aims: This systematic review aimed to explore the use and frequency of PRO instruments in randomised controlled trials (RCTs) for MPNs and assess the consistency of their reporting.

Methods: Under the auspices of the European Hematology Association (EHA), the development of guidelines for the use of PROs in adult patients with haematological malignancies was conceptualised. MEDLINE and CENTRAL were systematically searched for RCTs investigating MPNs between January 1st, 2015 and August 20th, 2021. Study design, disease and treatment characteristics, primary outcome, PRO instrument(s) as well as the methods of their reporting were extracted using a pre-defined template. In order to assess the consistency of PRO reporting, study registries were compared with publications and study protocol, where available.

Results: Overall, 2626 records were found. Following screening for RCTs, 287 ongoing, completed or published studies were included for review. Sixty-seven studies planned the use of PROs: 25 were completed, and 32 studies were ongoing (23 recruiting; 4 not yet recruiting; 7 active; 6 terminated but unpublished; 2 unknowns). For 12 studies, trial protocols were available. Thereof, 7 reported PROs inconsistently. In 2 trials, PROs were assessed as both primary and secondary outcomes. In most trials, they were reported as secondary outcomes (51 trials) and were a primary outcome in only 10 trials, while they were exploratory in 2 and not specified in 1. Twenty-six PRO measures (PROMs) were reported. The most frequently used (in 15 trials) generic PROMs were the EQ-5D (generic) and the EORTC QLQ-30 (disease-specific - created to assess health-related quality-of-life/ HRQoL in patients with solid tumours). The most frequently used (in more than 10 trials) MPN-specific PROMs were MPN-SAF TSS (or MPN-10) and MF-SAF (including v2.0 and v4.0). The version of MPN-SAF was not specified in 11 trials and that for MF-SAF in 2 trials. Noteworthy, the most frequently used MPN-specific instruments were created by the same authors, who also were involved in clinical trials.

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Summary/Conclusion: The findings indicate that there is increasing interest in the use of PROs as an outcome in MPNs with the availability of MPN-specific PROMs. The use of such instruments has been important for drug approval. Thus, guidelines for the appropriate use of PROs are needed to ensure standardisation in the selection and reporting of PROs.

P1706: CLINICAL UTILITY OF ONLINE MONITORING OF PATIENT-REPORTED SYMPTOMS IN NEWLY DIAGNOSED PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN REAL-LIFE PRACTICE

F. Efficace 1,2,* , F. Cottone 1 , B. Yanez 2 , F. Castagnetti 3 , G. Caocci 4 , M. Bonifacio 5 , A. Patriarca 6 , I. Capodanno 7 , M. C. Miggiano 8 , M. Tiribelli 9 , M. Breccia 10 , L. Luciano 11 , V. Giai 12 , A. Iurlo 13 , E. Abruzzese 14 , C. Fava 15 , G. Rosti 16 , J. Cortes 17 , V. Kota 17 , M. Vignetti 1 , D. Cella 2

1 Data Center and Health Outcomes Research Unit, Italian Group for Adult Hematologic Diseases (GIMEMA), Rome, Italy; 2 Department of Medical Social Sciences, Northwestern University Feinberg School of Medicine, Chicago, United States of America; 3 Department of Experimental, Diagnostic and Specialty Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Bologna; 4 Department of Medical Sciences and Public Health, University of Cagliari, Businco Hospital, Cagliari; 5 Section of Hematology, Department of Medicine, University of Verona, Verona; 6 Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara; 7 Hematology Unit, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Reggio Emilia; 8 Hematology Department, San Bortolo Hospital, Vicenza; 9 Division of Hematology and BMT, Department of Medical Area, University of Udine, Udine; 10 Hematology, Department of Translational and Precision Medicine, Azienda Ospedaliera Policlinico Umberto I, Sapienza University of Rome, Rome; 11 Haematology Unit “Federico II”, University of Naples, Naples; 12 Hematology Unit, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza, Torino; 13 Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano; 14 Hemoglobinopathies Unit, Hematology Department, S. Eugenio Hospital (ASL Roma 2), Rome; 15 Department of Clinical and Biological Sciences, University of Turin, Turin; 16 IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) “Dino Amadori”, Meldola, Italy; 17 Georgia Cancer Center, Augusta University Medical Center, Augusta, United States of America

Background: Adherence to tyrosine kinase inhibitors (TKIs) is critical to maximize clinical effectiveness of drugs in patients with chronic myeloid leukemia (CML). However, adverse events (AEs) are one of the most frequent causes for non-adherence to TKI therapy.

Aims: We herein report patients’ and physicians’ perception of using a newly developed online tool for patient-reported AEs monitoring in newly diagnosed CML patients in real-life practice. We also report health-related quality of life (HRQoL) and fatigue of patients up to six months.

Methods: An online platform (EMPATHY) for systematic monitoring of patient-reported AEs (including a set 22 items mainly derived from the PRO-CTCAE Item Library) was developed for the purpose of this study. Newly diagnosed CML patients within 4 weeks of first line TKI therapy were eligible. At the time of a clinical visit (after having signed informed consent), and just before the consultation with the physician, patients were provided a tablet PC to self-rate (touchscreen) their symptoms. The local clinical research staff instructed patients on study procedures and on how to complete self- reported AEs via tablet. Patient-reported AEs were displayed graphically and available, in real time, to physicians’ PC during the clinical consultation. At 3- and 6-months both patients and physicians completed a Survey assessing acceptability, satisfaction and clinical utility of using this platform. At these time points, patient-reported HRQoL and fatigue were also assessed with the FACT-G and PROMIS-Fatigue questionnaires (via paper copies), respectively. Trajectories over time were estimated using a linear mixed model for repeated measures.

Results: Ninety-four newly diagnosed CML patients were enrolled between July 2020 and August 2021 in 15 centers across US and Italy. There were 43% of patients treated with imatinib and 31%, 24% and 2%, treated with nilotinib, dasatinib and bosutinib, respectively. Median age of patients was 57 years (range, 19-82 years) and 55% were male. Sokal-risk was low in 42% of patients and 34% had at least one comorbidity. The majority of them (73%) used internet on a regular basis. The platform was generally well received by patients, for example, 99% (77/78) and 94% (73/78) of them agreeing or strongly agreeing (at 3 months) that it was easy to use and improved quality of communication with their physician, respectively. Of the 21 physicians (median age of 42 years) involved, nearly all (20/21; 95%) agreed that it was a valuable tool for improving accuracy of symptomatic AE of their patients. Similar high percentages were observed at 6 months. Compliance with patient-reported HRQoL and fatigue assessments was 99%, 93% and 93% at baseline, 3 and 6 months, respectively. No statistically significant differences were observed for the FACT-G and the PROMIS-Fatigue scores over time in the overall population.

Summary/Conclusion: Current data suggest that the EMPATHY platform is well accepted by patients and physicians and its use may contribute to enhance physicians’ ability to better understand symptom burden of their patients.

P1707: FACTORS ASSOCIATED WITH A LYMPHOMA DIAGNOSIS PRIOR TO BIOPSY

A. Ejaz 1,2,* , C. Harris 2 , B. Tseu 2 , A. A. Kirkwood 3 , S. Bhuva 4 , N. Taylor 4 , C. A. Johnson 4 , L. Wing 4 , G. P. Collins 2

1 University of Oxford; 2 Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford; 3 Cancer Research UK & UCL Cancer Trials Centre, University College London, London; 4 Department of Radiology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom

Background: Lymphoma is usually diagnosed on biopsy, but clues to a possible diagnosis may be found earlier in the diagnostic pathway when patients undergo CT imaging. Often CT scans are reported as showing likely lymphoma, leading to specialty referral before tissue diagnosis. A proportion of these patients have alternative diagnoses on biopsy, with a subsequent delay in referral to the most appropriate specialty. In the UK NHS Delivering Cancer Waiting Times: Good Practice Guide (2014), a top priority from patients for improving their experience was to be told they did not have cancer as soon as possible and, where they did have cancer, for a treatment plan to be made early. With the pressure of cancer waiting times and 2-week wait targets, any factors which can aid in earlier and more appropriate referral for patients will be valuable for health service providers and patients.

Aims: Our aim was to identify factors that may predict a lymphoma diagnosis at the time of CT, prior to diagnostic biopsy. Our long-term goal is to create a diagnostic model which can be used to identify patients who are more likely to have lymphoma and who should be referred early to the appropriate team, leading to a more streamlined diagnostic pathway and improved patient experience.

Methods: Between 1st July 2019-30th June 2021, 441 consecutive patients were identified who had undergone a CT scan reported as showing ‘likely lymphoma’. Patients were excluded if they were <18 years old or had a history of lymphoma. 311 patients had a biopsy with a tissue diagnosis. Training and validation datasets were randomly selected (2:1), balanced for biopsy outcome (lymphoma, non-cancer, other cancer), extent of disease on CT and presence of bulk (defined as mass ≥5 cm). Univariable logistic regression and Receiver Operating Characteristic Area Under the Curve (ROC-AUC) analysis were performed on the training set. Multivariable analysis, model building and validation is being undertaken.

Results: Our patient cohort were 45.1% female with median age 67 years. Of the 311 patients with a tissue diagnosis, 48.9% (152/311) had a final diagnosis of lymphoma; 24.1% (75/311) had a diagnosis of another malignancy. Non-malignant diagnoses were heterogeneous and included granulomatous disease including sarcoid (19/311) and infection/reactive causes (30/311). There were 208 and 103 patients in the training and validation sets; demographics were well-balanced.

Univariable analysis was undertaken (Table 1) with outcome lymphoma/non-lymphoma. Factors significantly associated with lymphoma diagnosis were all radiological: size of largest lesion (odds ratio, OR, for a 1cm increase: 1.21 (1.09–1.35), p<0.001), presence of bulk (OR 3.29 (1.63–6.64), p=0.001) and para-aortic lymphadenopathy (OR 2.92 (1.57–5.42), p = 0.001). No blood parameters were significantly associated; raised LDH showed some predictive ability for cancer vs not but could not discriminate between other cancer and lymphoma.

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Summary/Conclusion: In univariable analysis, we found parameters associated with an increased chance of a biopsy-proven lymphoma diagnosis when lymphoma was reported as likely on a CT scan. These factors alone showed only moderate predictive ability. Our next step is to assess their role in a multivariable model and test their predictive efficacy within our validation dataset. We plan to present this data at the EHA meeting. If successful, we hope to use this diagnostic model to streamline the diagnostic pathway for patients presenting with a scan suggestive of lymphoma, improving the efficiency of the patient pathway and improving patient experience.

P1708: PREGNANCIES IN YOUNG ADULTS WITH A HISTORY OF ACUTE LYMPHOBLASTIC LEUKEMIA IN ITALY. A CAMPUS ALL SURVEY.

F. Giglio 1,* , A. Bruno 1 , C. Papayannidis 2 , M. Dargenio 3 , M. Cerrano 4 , F. Forghieri 5 , M. Fumagalli 6 , M. Lunghi 7 , M. Annunziata 8 , M. Bonifacio 9 , F. Ferrara 8 , F. Ciceri 1 , G. Pizzolo 9 , A. Vitale 10 , R. Foà 10

1 Haematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milano; 2 Dipartimento di Oncologia e Ematologia, Policlinico S. Orsola Malpighi, Bologna; 3 Unità Operativa di Ematologia e Trapianto, Ospedale Vito Fazzi, Lecce; 4 S.C. Ematologia 2, AO Città della Salute e della Scienza, Torino; 5 Unità Operativa Complessa di Ematologia, Azienda Ospedaliero-Universitaria di Modena, Modena; 6 Hematology, San Gerardo Hospital, Monza; 7 Division of Hematology, Department of Translation Medicine, Università del Piemonte Orientale, Novara; 8 UOC di Ematologia, AORN Cardarelli, Napoli; 9 Dipartimento di Medicina, Sezione di Ematologia, Università di Verona, Verona; 10 Hematology, Department of Traslational and Precision Medicine, Sapienza University, Roma, Italy

Background: Survival after acute lymphoblastic leukemia (ALL) has greatly improved in the last 20 years, particularly in children, adolescents and young adults (AYA). Fertility impairments are among the main concerns for the long-term survivors. Few studies have focused on fertility issues within this selected population and the existing data are difficult to interpret due to different treatment regimens, small sample sizes and unavailability of data concerning fertility issues such as pregnancies in many Centers. Overall, fertility outcomes seem to be comparable between childhood ALL survivors and controls, except for a higher proportion of miscarriages, but very little is known for adult survivors.

Aims: Register fertility outcomes of male and female adult ALL long-term survivors.

Methods: We conducted a survey within the Italian Campus ALL group about pregnancies in adult ALL survivors. Seventeen centers responded, accounting for 803 AYA patients (489 males, 315 females) treated between 2005 and 2020. All country geographic areas were represented. Our survey consisted in a first questionnaire concerning patients, disease characteristics and treatments including allogeneic stem cell transplant (allo-HSCT). A second questionnaire was specific on conception and pregnancies such as use of medical help to become pregnant and pregnancy outcomes (positive or negative such as birth defects, miscarriages, medical abortions or stillbirths).

Results: Ten out of 17 centers referred pregnancies. We registered 31 pregnancies in 11 female and 14 male patients (median age at ALL diagnosis 24 years; range 16-38). All patients had received chemotherapy (pediatric-like in half of the patients), while 9 patients received also allo-HSCT. 15 patients were diagnosed with B-lineage ALL, 7 with T-ALL, 3 with Ph+ ALL. 27 children were naturally conceived, while medical help was necessary for 4 pregnancies. Only 2 pregnancies were achieved through cryopreserved gametes (all male patients). The median age at the time of the delivery was 33 years (range 25-46), with a median time from the diagnosis of ALL of 9 years. We registered 6 patients having 2 children and 2 patients having twin pregnancies (1 complicated with the death of one foetus). The incidence of pregnancy diseases and birth defects was low: 1 pregnancy was complicated by diabetes, 1 with neonatal jaundice, 1 with urethral obstruction necessitating surgical correction at birth.

Summary/Conclusion: Our survey shows that pregnancies in AYA ALL survivors are fairly uncommon but possible, even after allo-HSCT. The major limit of the study is the selection bias related to missing information in most centers about pregnancies and even more about miscarriages and medical abortions (none were registered). Most pregnancies were uncomplicated and we didn’t document a high incidence of pregnancy-related diseases or birth defects. Interestingly, only a small fraction of pregnancies were carried out with medical help or cryopreserved gametes. More efforts are needed in both patients’ information and medical care to improve fertility rates in AYA ALL survivors, particularly as the survival rates are improving. Having more precise data about fertility is of paramount importance to give better information about gametotoxicity in newly diagnosed patients. Fertility issues and pregnancies should become part of the medical follow-up, considering the higher rates of survival and even more with the possibility of new chemo-free approaches. The institution of specific national guidelines/recommendations is needed to promote this cultural shift.

P1709: FERTILITY PRESERVATION FOR YOUNG ADULT ACUTE LYMPHOBLASTIC LEUKEMIAS IN ITALY: A CAMPUS ALL SURVEY.

F. Giglio 1,* , A. Bruno 1 , M. Dargenio 2 , M. Cerrano 3 , M. I. Del Principe 4 , F. Forghieri 5 , M. Fumagalli 6 , F. Guolo 7 , A. Imovilli 8 , M. Lunghi 9 , C. Mazzone 10 , C. Papayannidis 11 , C. Pasciolla 12 , S. Trappolini 13 , D. Lazzarotto 14 , M. Bonifacio 15 , A. Candoni 14 , F. Ciceri 1 , G. Pizzolo 15 , A. Vitale 16 , R. Foà 16

1 Haematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milano; 2 S.C. Ematologia, Ospedale Vito Fazzi, Lecce; 3 S.C. Ematologia 2, AO Città della Salute e della Scienza, Torino; 4 Ematologia, Dipartimento di Biomedicina e Prevenzione, Università di Roma Tor Vergata, Roma; 5 S.C. Ematologia, Azienda Ospedaliero Universitaria di Modena, Modena; 6 Hematology, San Gerardo Hospital, Monza; 7 Ematologia e Terapie Cellulari, IRCCS Policlinico San Martino, Genova; 8 Hematology, AUSL-IRCCS di Reggio Emilia, Reggio Emilia; 9 Division of Hematology, Department of Translation Medicine, University of Eastern Piedmont, Novara; 10 Haematology Unit, St. Eugenio Hospital, Roma; 11 Dipartimento di Oncologia e Ematologia, Policlinico S. Orsola Malpighi, Bologna; 12 U.O. di Ematologia, IRCCS Istituto Tumori Giovanni Paolo II, Bari; 13 SOD Clinica Ematologica, Azienda Ospedaliero - Universitaria Ospedali Riuniti Umberto I, Ancona; 14 Clinica Ematologica, Azienda Sanitaria Universitaria Friuli Centrale, Udine; 15 Dipartimento di Medicina, Sezione di Ematologia, Università di Verona, Verona; 16 Ematologia, Dipartimento di Medicina Traslazionale e di Precisione, “Sapienza” Università di Roma, Roma, Italy

Background: Survival rates have improved in the last decades in children and adolescent and young adults (AYA) with acute lymphoblastic leukemia (ALL). This is rising concerns about the late effects related to both cancer itself and its treatment. Fertility issues are well recognized in cancer survivors. In a study on AYA women with cancer, pregnancies were 38% less likely than in the general population. Moreover, an American survey showed that only 25% of patients with cancer had a documented discussion about fertility issues and preservation with their treating physicians, with less than 15% of patients being referred to a specialist. Lower rates were recorded among patients who already had children. A recent British survey showed inconsistencies in the same country about fertility preservation management, despite being recommended in local and international guidelines. Moreover, new emerging techniques for harvesting and preserving gametes are changing the fertility preservation scenario, especially for women.

Aims: Track down fertility preservation management in AYA with ALL.

Methods: We conducted a survey within the Italian Campus ALL group about gamete preservation. 16 centers responded to the survey, for a total of 803 AYA patients (489 males, 315 females) treated between 2005 and 2020. All country geographic areas were represented. The survey consisted in a first part about the existence of center-specific fertility preservation guidelines, the presence of a specialized medical counseling for fertility and a dedicated evaluation at diagnosis (AMH/semen analysis/etc). A second part was gender-specific, with questions on gamete cryopreservation habits, percentage of successful procedures and age limits for cryopreservation. For women, we asked whether a hormonal contraceptive therapy was routinely performed during chemo. The third part was about the public health system coverage of procedures and if the gamete cryopreservation of gametes was easily accessible in the same center where the disease was managed.

Results: The survey showed that only 50% of centers have specific guidelines for gamete cryopreservation and while in all of them fertility is discussed with the hematologist, only in 50% of centers the patient is referred to a fertility specialist. Moreover, while sperm cryopreservation is offered in all centers with high success rates (median 85%), only 25% of centers routinely offer oocyte or ovarian tissue harvesting and storage, with marked differences with regard to both age limits and success rates. Moreover, in 25% of centers the cryopreservation costs are not covered by the public health system. Only 50% of centers have the facilities to perform the preservation of gametes in their own institution.

Summary/Conclusion: We found relevant differences between centers in the referral to specialists, fertility evaluation at diagnosis, access to fertility preservation procedures by sex and age, and economic coverage by Italian public health system. Great inconsistencies were noted even in the same geographic area, probably because of the regional management of public health in Italy. The first step towards improving the situation is the institution of national guidelines for fertility preservation in AYA patients with cancer and specifically ALL. Guidelines should aim at standardizing and improving communication with patients about gonadotoxicity and at standardizing gamete preservation procedures particularly for women in light of the available and future techniques. A centralized funding of fertility preservation is needed in order to reduce inequality among regions.

P1710: LISOCABTAGENE MARALEUCEL (LISO-CEL) AS SECOND-LINE TREATMENT FOR R/R LARGE B-CELL LYMPHOMA (LBCL) IN PATIENTS NOT INTENDED FOR HSCT: PATIENT-REPORTED OUTCOMES (PRO) FROM THE PHASE 2 PILOT STUDY

L. I. Gordon 1,* , D. Hoda 2 , L. Shi 3 , S. Guo 3 , F. F. Liu 4 , J. Braverman 4 , R. Dubowy 5 , L. Peng 5 , A. Sehgal 6

1 Northwestern University, Feinberg School of Medicine, Robert H. Lurie Comprehensive Cancer Center, Chicago; 2 Intermountain Healthcare, Loveland Clinic for Blood Cancer Therapy, Salt Lake City; 3 Evidera, Waltham; 4 Bristol Myers Squibb, Princeton; 5 Bristol Myers Squibb, Seattle; 6 University of Pittsburgh Medical Center, Hillman Cancer Center, Pittsburgh, United States of America

Background: The PILOT study ( NCT03483103 ) evaluated liso-cel, an autologous, CD19-directed, chimeric antigen receptor T cell product, as second-line treatment in patients with relapsed or refractory (R/R) LBCL not intended for hematopoietic stem cell transplantation (HSCT).

Aims: To analyze changes in health-related quality of life (QOL) with respect to functioning and symptoms in PILOT.

Methods: Adults with R/R LBCL after first-line treatment were eligible. Patients were deemed not candidates for high-dose chemotherapy and HSCT by their physician and met ≥ 1 frailty criteria as follows: age ≥ 70 years, Eastern Cooperative Oncology Group performance status of 2, diffusing capacity for carbon monoxide ≤ 60%, left ventricular ejection fraction < 50%, creatinine clearance < 60 mL/min, or alanine aminotransferase/aspartate aminotransferase > 2 × the upper limit of normal. Patients completed the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire - 30 items (EORTC QLQ-C30), Functional Assessment of Cancer Therapy - Lymphoma Subscale (FACT-LymS), and EQ-5D-5L health utility index (HUI) and visual analog scale (VAS) questionnaires at screening (baseline), pretreatment (within 7 days before lymphodepletion), preinfusion on the day of liso-cel infusion (Day 1), posttreatment on Days 29, 60, 90, 180, 270, 365, 545, and 730/end of study after lisocel infusion, or after disease progression. The PRO-evaluable set included all liso-cel treated patients with baseline and ≥ 1 postbaseline assessments. Linear mixed-effects models for repeated measures assessed the least squares (LS) mean change from baseline for visits with ≥ 10 patients. Meaningful change from baseline was calculated using responder definitions of 10 points for EORTC QLQ-C30, 3 points for FACT-LymS, 0.08 points for EQ-5D-5L HUI, and 7 points for EQ-VAS.

Results: Among the PRO-evaluable set (EORTC QLQ-C30, n = 56; FACT-LymS, n = 49; EQ-5D-5L HUI, n = 55; EQ-VAS, n = 54), completion rates were high (≥ 80%) across most visits for all measures. For the EORTC QLQ-C30, mean baseline fatigue was meaningfully worse than in a general population (difference of > 10 points). Overall LS mean changes through Day 545 showed significant improvements in EORTC QLQ-C30 fatigue, pain, and appetite loss, FACT-LymS, and EQ-VAS ( Table ). Improvement for FACT-LymS was also clinically meaningful. Fatigue improvement was clinically meaningful with a more sensitive minimal important difference of 4 (Cocks et al, 2012). Significant worsening was not observed for any domain. In patient-level analyses, a majority of patients remained stable or achieved clinically meaningful improvement at Month 6 in EORTC QLQ-C30 global health status/QOL (93% of patients), physical functioning (80%), role functioning (90%), cognitive functioning (83%), fatigue (87%), pain (80%), FACT-LymS (96%), EQ-5D-5L HUI (86%), and EQ-VAS (93%). Further, 70% and 60% of patients achieved meaningful improvements in FACT-LymS and EORTC QLQ-C30 fatigue, respectively.

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Summary/Conclusion: Liso-cel meaningfully improved fatigue and FACT-LymS scores without negatively impacting other QOL measures. These data support the clinical evidence of liso-cel as a potential new second-line treatment in patients with R/R LBCL not intended for HSCT.

P1711: LOCKDOWNS AND CANCER DIAGNOSIS: IMPACT OF COVID19 PANDEMIC ON NEW HAEM-ONC PRESENTATIONS

D. Discalicau 1,* , P. Greaves 2

1 Queen Mary University of London; 2 Haematology, Barking Havering and Redbridge University Hospitals NHS Trust, London, United Kingdom

Background: National lockdowns of various stringency were adopted to reduce spread of COVID19 through 2020/2021. The effect of lockdowns and changed social behaviour impacted presentation to healthcare for all conditions, particularly new cancers, with potential harm through late or non-presentation. The UK adopted its most stringent lockdowns affecting London between March and June 2020, and December 2020 and April 2021. Patients urgently referred by GP for suspected cancer fell by 50% nationally during the first lockdown. The effect of reduced cancer presentation requires further examination to assist harm/benefit analysis of future lockdown strategies.

Aims: Examine the effect of the pandemic and lockdowns on new haem-onc diagnoses at a hospital in Greater London, UK, serving an ethnically and socioeconomically diverse population of around 850,000.

Methods: All new adult haem-onc diagnoses captured through an integrated electronic database at the hospital were reviewed. Years were defined as financial rather than calendar years (starting 1 st April) both conventionally and since this coincided approximately with lockdowns allowing easy comparison with preceding years.

Results: The department recorded a mean of 346 (SD 14.6) haem-onc diagnoses per year between 2015/16 and 2019/20. In 2020/21 only 272 diagnoses were recorded – 21% below average or 5 SD below the mean.

In the pre-pandemic 5 years, average quarterly diagnoses Q1&Q2 (Spring/Summer) were significantly lower than Q3&4 (Autumn/Winter) at 81 vs 92 per quarter (p=0.01). During 1 st lockdown only 58 diagnoses were captured (Q1 2020/21 – 72% of the Spring/Summer average). During 2 nd lockdown only 68 diagnoses were captured (Q4 2020/21 – 74% of the Autumn/Winter average).

During Q2 2020/21 when lockdowns eased, there were markedly reduced numbers of COVID cases and deaths, but an ongoing reduction in new cancer diagnoses (81% Spring/Summer average). During Q3 of 2020/21 when lockdowns were only regional and partial, but COVID case and deaths were increasing, new diagnoses approached normal levels (87% Autumn/Winter average).

During the first 3 quarters of 2021/22, new diagnoses had returned to pre-pandemic levels.

Impact on haem-onc cancers detected through blood tests (paraprotein and leukaemias) appeared more marked than those presenting as symptomatic (lump/pain/bleeding, etc) tumours – i.e. lymphoma.

During both lockdowns 35% fewer blood test detected diagnoses (defined as leukaemia or paraprotein) were captured (35 vs 55 per quarter). Between lockdowns there was only a 6% reduction. In contrast there was no reduction in new lymphoma diagnoses during or between lockdowns compared to 5 preceding years’ average (25 per quarter on average)

Summary/Conclusion: The COVID pandemic led to around 1/4 reduction in new haem-onc diagnosis with the most significant impact occurring during times of national lockdown. Causes will be complex and multifactorial including fear of healthcare environments and public places in general, loss of normal hospital and primary care services and death and illness from COVID prior to reaching healthcare. However, the impact seemed to be predominantly in diagnoses picked up on blood tests, rather than symptomatic ‘lumps’ and we might hypothesize that lymphomas, presenting conspicuously and symptomatically trigger patient and healthcare provider behaviour that allows access to diagnosis, and is less likely to be ignored or missed. The impact of missed and late cancer diagnoses on survival and excess deaths continues to be explored as an essential investigation into impact of both pandemic and public health responses.

P1712: CORONAVIRUS ANXIETY LEVEL AND COVID 19 VACCINE ATTITUDE AMONG HEMATOLOGICAL MALIGNANCY PATIENTS

Z. Güven 1,* , M. Keklik 1 , A. Ünal 1

1 Department of Hematology, Erciyes University Medicine Faculty, Kayseri, Turkey

Background: The COVID-19 vaccine is the most essential tool for altering the pandemic’s trajectory. The pandemic’s control is complicated by society’s unwillingness to vaccination.

Aims: The aim of this cross-sectional study was to assess patients with hematological malignancies’ attitudes regarding the COVID-19 immunization and to investigate COVID-19 anxiety in this susceptible population

Methods: In this study, 165 patients with hematological malignancies were included. The COVID-19 anxiety situation was evaluated with the Coronavirus Anxiety Scale (CAS). COVID- 19 vaccine attitude was evaluated with the Vaccine Attitudes Review (VAX) Scale.

Results: The mean CAS score was 2.42 (0 - 17). There were 22 (13%) participants with a mean CAS score of ≥9. Half of the participants had a CAS score of 0. The CAS score was higher in females (p= 0.023). Similarly, it was significantly higher in patients who were not in remission for hematological malignancy and who received active chemotherapy (p= 0.010). The mean VAX score was 49.07 ± 8.76 (27-72). Most of the participants (64%) had a neutral attitude towards COVID-19 vaccination. In a survey of 165 patients, 55% said that they were skeptical about vaccination safety, and 58% said that they were concerned about unintended side effects. In addition, 90% expressed moderate concerns about commercial profiteering. Natural immunity was preferred by 30% of the participants. There was no statistically significant correlation between CAS scores and Vaccine Attitudes Review (VAX) Scale.

Summary/Conclusion: This study draws attention to the level of anxiety in patients with hematological malignancies of the COVID-19 pandemic. Negative attitudes towards the COVID-19 vaccine are worrisome for at-risk patient groups. We think that patients with hematological malignancies should be informed to eliminate their hesitations about COVID-19 vaccines.

P1713: VENETOCLAX IN COMBINATION WITH OBINUTUZUMAB IN FIRST LINE CHRONIC LYMPHOCYTIC LEUKEMIA IN ARGENTINA: A COST-EFFECTIVENESS ANALYSIS

D. Kanevsky 1,* , J. Elgart 2 , M. Zerga 3 , M. Glancszpigel 4 , P. Morisset 5

1 Market Access, Abbvie, Buenos Aires; 2 CENEXA, Center of Experimental and Applied Endocrinology (UNLP-CONICET, La Plata; 3 Instituto de Oncología “Angel Roffo”. UBA.; 4 3eff, Buenos Aires, Argentina; 5 Abbvie, Chicago, United States of America

Background: The efficacy of venetoclax plus obinutuzumab (VenG) was examined in the CLL14 clinical trial, in unfit Chronic Lymphocytic Leukemia (CLL) patients who have not been previously treated.

Aims: The aim of the study was to estimate the cost-effectiveness of VenG in the treatment of first-line unfit CLL patients in Argentina

Methods: A three-state partitioned-survival model (with health states of progression-free survival (PFS), post-progression survival (PPS) and death), developed in accordance with NICE and ISPOR decision modelling guidelines, was adapted to Argentina setting and was used to estimate the accumulated costs and outcomes in terms of life years (LY) and quality-adjusted life years (QALY). A 30-year time horizon and the Social Security’s payer’s perspective were assumed. Cost-effectiveness was estimated by comparing a 12-month fixed duration of VenG versus (vs.) chlorambucil-obinutuzumab (ClbG) based on the CLL14 clinical trial. Other comparators included treat-to-progression therapies, such as ibrutinib (IBR), and a 6-month course of bendamustine + rituximab (BR). Using a network meta-analysis, the relative efficacy of VenG and ClbG vs. other selected comparators was estimated. Health state utilities and adverse event (AE) disutilities were derived from a systematic literature review and published health-technology assessment reports. To generate total quality-adjusted life years (QALYs), these health state utilities and AE disutilities were applied to the relative efficacy data. The cost of medication was based on country level drug prices. The healthcare resources used were validated by local experts through a Delphi panel methodology. Costs of resources used for routine medical attention and the management of adverse events were based on tariffs from social security system of Argentina. Costs were expressed in Argentine Pesos (AR$) (exchange rate 1 US dollars ($) = 100.75AR$, July 2021). A 5% annual discount rate was applied for costs and outcomes. Cost-effectiveness results are presented in terms of incremental cost per QALY. A new treatment that is both lower in total cost and more efficacious (in QALYs) vs. identified comparator treatments is described as being “dominant”. Uncertainty in the model was tested through one-way sensitivity analyses (OWSA) and probabilistic sensitivity analyses (PSA)

Results: Over a 30-year time horizon, the total discounted costs were, for VenG, $117,250.19; ClbG, $167,675.26; BR, $206,951.64; and IBR, $432,243.36. The benefits in the cost-effectiveness model (CEM) were measured in terms of total discounted QALYs which were 5.634 for VenG, 5.350 for ClbG, 4.996 for BR and 5.161 for IBR. The incremental discounted QALYs of VenG was: 0.284 vs. ClbG, 0.639 vs. BR, and 0.473 vs. IBR. Thus, VenG with a 12-month fixed duration, has lower total costs and is more efficacious (“dominant”) over all comparators in the CEM. OWSA analyses show that the results are robust, and in the PSA VenG is dominant over all the comparators considered, ICER is ≤$ 6,011.20 (1 GDP per capita for Argentina, 2020), /QALY in 97% of the iterations

CE Results

Summary/Conclusion: This study shows that in Argentina, VenG would be dominant treatment option (better results and lower costs) compared with ClbG, BR and IBR in the treatment of first-line unfit CLL patients.

P1714: HEALTHCARE RESOURCE UTILIZATION IN PATIENTS WITH LIGHT CHAIN AMYLOIDOSIS IN EUROPE

A. Jaccard 1 , F. Bridoux 2 , W. Roeloffzen 3 , M. C. Minnema 4 , R. Bergantim 5 , R. Hájek 6 , C. João 7 , M. T. Cibeira 8 , G. Palladini 9 , S. Schönland 10 , G. Merlini 9 , P. Milani 9 , M. A. Dimopoulos 11 , S. Ravichandran 12 , U. Hegenbart 10 , H. Agis 13 , B. Gros 14 , A. Asra 15 , E. Dergarabetian 15 , V. Magarotto 16 , A. Leonidakis 17 , G. Cheliotis 17 , P. Sonneveld 18 , A. Wechalekar 12 , E. Kastritis 11,*

1 National Amyloidosis Center and Hematology Unit, CHU Limoges, Limoges; 2 Nephrology Unit, CHU Poitiers, Poitiers, France; 3 Amyloidosis Centre of Expertise, Department of Internal Medicine, Faculty of Medical Sciences, University Medical Center Groningen, Groningen; 4 Department of Hematology, University Medical Center Utrecht, Utrecht, Netherlands; 5 Department of Hematology, Hospital São João, Porto, Portugal; 6 Department of Haematooncology, University Hospital Ostrava, Ostrava, Czechia; 7 Hematology Department, Champalimaud Center for the Unknown, Lisbon, Portugal; 8 Amyloidosis and Myeloma Unit, Department of Hematology, Hospital Clinic, IDIBAPS, Barcelona, Spain; 9 Amyloidosis Research and Treatment Center, Foundation “Instituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo”, Department of Molecular Medicine, University of Pavia, Pavia, Italy; 10 Medical Department V, Amyloidosis Center Heidelberg, University of Heidelberg, Heidelberg, Germany; 11 Department of Clinical Therapeutics, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece; 12 National Amyloidosis Centre, University College London, London, United Kingdom; 13 Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University Vienna, Vienna, Austria; 14 Janssen-Cilag S.A., Madrid, Spain; 15 Janssen-Cilag S.A., London, United Kingdom; 16 Janssen-Cilag S.A., Rome, Italy; 17 Health Data Specialists, Dublin, Ireland; 18 Erasmus MC Cancer Institute, Rotterdam, Netherlands

Background: Systemic light chain (AL) amyloidosis is a rare plasma cell disease characterized by the accumulation of amyloid fibrils in various tissues and organs throughout the body, leading to multiorgan dysfunction. Beyond anti-clonal therapy, this disease treatment requires multidisciplinary teams and resources to manage its multiorgan complications, however, the burden of the disease for health care system and health care resource use (HCRU) remain underexplored.

Aims: To assess the HCRU in patients (pts) with AL amyloidosis across several European countries.

Methods: EMN23, a retrospective, multicenter study, investigated the demographic and clinical characteristics, the treatment pathways, and the HCRU of adult pts with AL amyloidosis who initiated treatment in 2004–2018 from 13 sites across 10 European countries. The HCRU analysis on the 2011-2018 period included patient data from 7 countries (Greece, France, the Netherlands, Portugal, the Czech Republic, Austria, and Spain [only dialysis information available]). Patients who participated in clinical trials at any line were excluded. HCRU measures included hospitalizations, imaging examinations, adverse events of special interest (AESIs; Intensive Care Unit admission, Heart Failure, Need for pacemaker, Infections requiring hospitalization, Peripheral neuropathy), and dialysis information.

Results: In 2011-2018, HCRU data and safety information were available for 462 pts (531 pts. with dialysis data). Most (63.4%) pts were males ≥65 years old (52.4%). At baseline, 41.6% (192/462) pts had 1 organ involved, 36.1% (167/462), had 2 and 20.8% (96/462) pts had ≥3 organs involved. The proportions of pts per the Eastern Cooperative Oncology Group (ECOG) status of 0, 1, 2, and 3 were 20.8% (96/462), 24.9% (115/462), 19.0% (88/462), and 7.8% (36/472), respectively; (not reported for 26.4% (122/462) pts). At diagnosis, Mayo 2004/European cardiac stages I, II, IIIa, and IIIb distribution was 75 (16.2%), 126 (27.3%), 127 (27.5%), and 59 (12.8%), respectively (Table 1) and was unknown for 75 (16.2%) pts. In 2011-2018, the number of pts with ≥1 hospitalization was 280 (60.6%). Duration of hospitalization is longer for patients stage IIIa and IIIb compared to lower cardiac stages (Table). Ultrasound and X-rays were the most common imaging exams across most cardiac stages (Stage I: 45.3%; Stage II: 42.1%, Stage IIIa: 47.2%; Stage IIIb: 39.0%), but MRIs and scintigraphy were also used commonly (see Table). At first-line, the proportions of pts treated with immunomodulatory (IMiD)-based regimens, bortezomib-based regimens, chemotherapy, and autologous stem cell transplantation were 2.2% (10/462), 62.3% (288/462), 12.6% (58/426) and 11.5% (53/462), respectively. The proportions of pts with ≥1 SAE and ≥1 AESI were 49.4% (228/462) and 28.8% (133/462), respectively. Infectious complication and heart failure were common reasons for hospitalization, with, half of the AESI records for cardiac stage IIIb patients being cardiac failure and ~1/10 AESI records being ICU admissions. A median number of 70.5-139.6 dialyses per pt per year were received by 70 (13.2%) pts, across cardiac stages.

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Summary/Conclusion: The current analysis shows that in the recent years (post-2010), the burden of HCRU for pts with AL Amyloidosis was considerable, across all Mayo2004/European cardiac stages. The main burdensome components were hospitalizations and dialysis. A cost-of-illness study from the healthcare perspective will be conducted, to reveal the cost burden on the healthcare system of pts with AL amyloidosis in Europe.

P1715: KNOWLEDGE, ATTITUDES, ANXIETY, AND COPING STRATEGIES OF PEDIATRIC PATIENTS, PARENTS, AND HEALTH CARE PROVIDERS AFTER A LEUKEMIA DIAGNOSIS

Y. Yaşar 1 , Z. Kaya 1,* , E. Güney 2 , S. Kirkiz 1 , Ü. Koçak 1

1 Pediatric Hematology; 2 Department of Child and Adolescent Psychiatry, Gazi University School of Medicine, ANKARA, Turkey

Background: Acute leukemia is easily recognized by physicians through bone marrow examination; however, it is not easy to inform pediatric patients and their parents about a leukemia diagnosis. Most information to date has been obtained from adults studies. As well, data on issues related to informing children about leukemia diagnosis are lacking.

Aims: The aim was to evaluate the knowledge, attitudes, anxiety, and coping strategies of pediatric leukemia patients, their parents, and health care providers related to a child’s leukemia diagnosis.

Methods: Of the 350 participants, 100 were pediatric patients, 100 were patients’ parents, and 150 were healthcare providers (50 pediatricians, 50 nurses, and 50 interns). Each participant answered a short questionnaire regarding the appropriate person, time, and place for conveying a child’s leukemia diagnosis. The questions assessed the knowledge level, attitudes, anxiety, and coping strategies of the participant groups after this diagnosis. Patients’ and parents’ quality of life and anxiety levels were also evaluated using the 36-item Short-form Health Survey (SF-36) and the Revised Child Anxiety and Depression Scale.

Results: The response rate was 100%. Patients’ parents ( n =98; 98%) and health care providers ( n =145; 97%) strongly agreed that a child’s leukemia diagnosis should be disclosed directly to the pediatric patient. Compared with health care providers, patients’ parents were significantly more likely to opt for disclosure by the parents in the doctor’s office after the start of chemotherapy ( p <0.05). All 100 patients were asked detailed questions about their personal knowledge of their leukemia diagnosis. Sixteen (16%) responded that they were unaware of their diagnosis at the time of the survey. Of these patients, 13 (82%) were younger than 7 years old at the time of their initial diagnosis, and three (18%) were older than 7 years. All 16 of these patients responded during the survey that they wished to know their leukemia diagnosis. The remaining 84 patients (84% of total) stated that they were aware of their diagnosis at the time of the survey. Nine (10%) of them indicated that they wished they did not know about their leukemia diagnosis. These nine were all older than 12 years at the time of diagnosis. They had received antidepressant treatment and psychological support. Four (45%) of them had received chemotherapy. The mean anxiety score for the nine patients who wished they did not know their diagnosis was significantly higher than that for the 16 patients who desired to but did not know their diagnosis ( p <0.05). Compared with patients, their parents had significantly higher anxiety levels and significantly lower quality of life ( p <0.05). P Thirty-two percent of the pediatricians, 16% of the nurses, and 86% of the interns responded that they had received training in delivering bad news.

Summary/Conclusion: Detailed information about leukemia diagnosis should be avoided for adolescent patients, whereas patients who are initially diagnosed younger than 7 years of age should be re-informed of their diagnosis at an older age. Psychological support for parents and adolescents would enhance adherence to chemotherapy and would increase the quality of life and the ability to cope with anxiety. Providing targeted medical information about leukemia diagnosis and how to deliver bad news could positively affect the knowledge and attitudes of health care providers.

P1716: REGULAR HOSPITAL VISITS IMPROVE THE PROGNOSIS OF MULTIPLE MYELOMA PATIENTS

X. Li 1,* , J. Liu 1 , B. Huang 1 , J. Gu 1 , J. Li 1

1 The first affiliated hospital of sun yat-sen university, Guangzhou, China

Background: Multiple myeloma（MM）is a common hematological cancer. The overall survival rate of individuals with MM has risen significantly in the previous decade, primarily as a result of the advent and advancements of innovative treatments. As a consequence, given the possibility that cancer will progress to the stage of chronic illness, the post management and regular hospital visit of MM patients are particularly important. Although it seems that long term chronic disease management by regular hospital visit has increased medical expenditure, from the perspective of whole process treatment, it is the best way to achieve standardized treatment and monitoring and may improve the prognosis of MM patients The regular hospital visits means regular hematologist visits with regular treatment and monitor. Irregular hospital visits means visits to other hospital divisions or with no hospital visits.

Aims: The aims of this study were to investigate the relationship between regular hospital visits and prognosis of MM and to suggest methods to avoid poor prognoses in MM.

Methods: We performed a multicenter retrospective analysis of data from 832 MM patients across 9 centers in China from 2006 to 2020. Sociodemographic (including whether regular hospital visits) and clinical factors at diagnosis and treatment regimens were recorded, and univariate and multivariate analyses were performed.

Results: Overall, 30.3% of patients had irregular hospital visits. Patients with irregular hospital visits levels differed from those with regular hospital visits in that they were more likely to be older, and a higher proportion lived in rural areas, were unemployed, and with lower education levels, had lower annual incomes and lacked insurance. Additionally, compared to patients with regular hospital visits, patients with irregular hospital visits had a higher proportion of international staging system (ISS) stage III classification, time to diagnosis is longer and underwent transplantation less often. Patients with regular hospital visits had a median progression-free survival (PFS) of 52.04months, which was better than that of patients with irregular hospital visits (20.3 months, p < 0.001). Similarly, patients with egular hospital visits had a median overall survival (OS) of 107.4months, which was also better than that of patients with irregular hospital visits (37.1 months, p < 0.001). In the multivariable analysis, patients with regular hospital visits had lower relapse rates and higher survival rates than did those with irregular hospital visits in terms of PFS and OS (hazard ratio (HR) = 1.95, p < 0.001; HR =2.80, p < 0.001, respectively).

Summary/Conclusion: Irregular hospital visits may independently predict poor survival in MM patients in China.

P1717: HEALTH-RELATED QUALITY OF LIFE AND TOLERABILITY OF LONCASTUXIMAB TESIRINE IN HIGH-RISK PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA TREATED IN A PHASE 2 CLINICAL TRIAL (LOTIS 2)

A. Spira 1 , L. Liao 2,* , X. Zhou 3 , A. Gnanasakthy 3 , L. Chen 2 , D. Ungar 2 , E. Yu 2 , T. Kilavuz 2 , J. Radford 4 , M. Hamadani 5

1 Virginia Cancer Specialists Research Institute, Fairfax, VA; 2 ADC Therapeutics America Inc., New Providence, NJ; 3 RTI Health Solutions, Research Triangle Park, NC, United States of America; 4 NIHR Clinical Research Facility, the Christie NHS Foundation Trust and University of Manchester, Manchester Academic Health Centre, Manchester, United Kingdom; 5 Medical College of Wisconsin, Division of Hematology & Oncology, Milwaukee, WI, United States of America

Background: Loncastuximab tesirine (loncastuximab tesirine-lpyl; Lonca) has shown antitumor activity with an acceptable toxicity profile and provides stable or improved health-related quality of life (HRQOL) in adult patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) after ≥ 2 prior therapies. Lonca has also shown promising results for patients who are at high risk of a poor prognosis (i.e., with double/triple hit, primary refractory, or transformed disease).

Aims: This analysis evaluates the impact of Lonca on HRQOL and patient tolerability to treatment stratified by high-risk group.

Methods: The LOTIS 2 study ( NCT03589469 ) is a single-arm, open-label, phase 2 study of adult patients with R/R DLBCL after ≥ 2 prior treatments. Patients received Lonca as an intravenous infusion on day 1 of each 3-week treatment cycle. The EuroQol EQ-5D visual analog scale (VAS) was used to measure overall health state, and the Functional Assessment of Cancer Treatment–Lymphoma (FACT-Lym) was used to measure HRQOL, including physical, social/family, emotional, and functional well-being, and lymphoma-specific symptoms and concerns. Mean change from baseline scores during the treatment period were summarized by visit and risk group. Analysis of covariance (ANCOVA) models were conducted to adjust for age, sex, race, and baseline score. Treatment tolerability was reported by patients in FACT-Lym item GP5 (“I am bothered by side effects of treatment”). This single item has been used to measure overall side effect impact on patients. All patients provided written informed consent to participate the study.

Results: Patients with a baseline score and at least one postbaseline score were included in the analysis. Through cycle 9, the completion rate among patients treated at each cycle was ≥ 92% for EQ-5D and ≥ 88% for FACT-Lym. Of the 127 patients included in analysis, 16% were aged ≥ 75 years, 58% were male, 88% were White, and 39% were high risk. In the high-risk group, both unadjusted and adjusted analyses showed the VAS overall health score was improved after 2 cycles of treatment (figure and table); the FACT-Lym total score was also improved at some visits. In the non–high-risk group, the VAS and FACT-Lym total scores remained stable or improved except later cycles with small sample sizes. When asked how much they were bothered by treatment side effects, a majority of patients reported “a little bit” or “not at all” at most cycles (71%-89% in the high-risk group and 46%-86% in the non–high-risk group).

Age, sex, race, and baseline score were adjusted in the ANCOVA model. Visits after C9D1 are not displayed due to small sample sizes. A change of 7 points for the EQ VAS or FACT-Lym total is considered a minimally important difference. A positive change indicates improvement.

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Summary/Conclusion: The overall health state and HRQOL were stable or improved in high-risk patients during the treatment period of Lonca. Patients in the high-risk group tolerated the treatment just as well, if not better, as other patients. The findings further support the clinical use of Lonca for the treatment of R/R DLBCL in patients with high risk.

P1718: A PROSPECTIVE MULTICENTER SURVEY ABOUT THE USE OF NEW TECHOLOGIES AND DIGITAL HEALTH TOOLS IN PATIENTS (PTS) WITH HEMATOLOGICAL MALIGNANCIES

A. Lopez-Garcia 1,* , C. Diaz-Aizpun 2 , B. Gallego-Diaz 1 , C. Miranda-Castillo 3 , M. Yuste-Platero 4 , P. Beltran-Alvarez 5 , C. Carames-Sanchez 6 , J. Garcia-Foncillas 6 , P. Llamas-Sillero 1 , M. del Olmo Rodriguez 7 , J. Short Apellaniz 7 , J. Arcos Campillo 7 , R. Cordoba 1,7

1 Hematology, Fundacion Jimenez Diaz, Madrid; 2 Hematology, Regional University Hospital, Malaga; 3 Hematology, Rey Juan Carlos University Hospital Madrid; 4 Hematology, Villalba General Hospital; 5 Hematology, Infanta Elena Universtiy Hospital; 6 Medical Oncology, Fundacion Jimenez Diaz; 7 Clinical and Organizational Innovation Unit (UICO), Quironsalud Public Hospital Network, Madrid, Spain

Background: Telemedicine has landed on the care of pts with hematological malignancies enhanced by the COVID-19 pandemic

On the other hand, the use of eHealth and new forms of communication technologies requires technological skills on the part of pts

In order to guarantee the success of communication through these new tools, patients must be willing to and have access to this form of communication.

Aims: We hypothesize that telemedicine and eHealth have great acceptance among pts with hematololgical malignancies increasing their interest during the pandemic

Methods: Pts with any oncohaematological malignancy receiving oral and/or intravenous treatment in daytime hospital were included in our study between 1 st February and 30 th November 2021 in 4 hospitals.

We conducted a cross-sectional, multicenter, prospective study, based on the validation design of a survey offered to these patients, where demographic and social characteristics, tumor characteristics, management characteristics of the technologies of information and communication, as well as the patient portal and data on the COVID-19 pandemic were included.

Our endpoint was to describe the pattern of access and use of new technologies of patients treated in Hematology Services for some hematological malignancy and Medical Oncology for a solid tumor.

Results: Two hundred patients were included in our study. Median age was 60 years (range 21-87), and 119 (59.5%) were male. About the use of information and communication technologies (ICTs), 136 (69.5%) patients reported daily use of the internet and 172 (86%) have a smartphone. 85 (43.1%) of patients included in the study do health research on the internet, and 181 (90.5) think e-health tools may help them to improve the communication with the medical team during their treatment. The most chosen way to communicate is the mobile phone (45%).

When we analyzed the use of digital health and ITCs, patients older than 60 years old, without superior education and retired are the ones who never use internet, never do health research on the internet and do not think the use of these tools may help them to improve their communication with the medical team, with statistically significant differences (Table 1). No differences in gender were found.

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Summary/Conclusion: Although in the past years e-Health solutions development has exponentially increased motivated by COVID-19 pandemic, preferences and needs of patients remain unknown. In our study, most of them believe new technologies are accessible, useful, and the preferred way to use them is the mobile phone.

We can differentiate a group of patients older than 60 years, without superior studies and retired, who are out of this trend. Efforts to adapt digital solutions to this group´s needs and limitations should be made in order to avoid the digital gap.

P1719: A PATIENT ADVOCACY PERSPECTIVE ABOUT CHRONIC MYELOID LEUKEMIA INEQUITIES IN ACCESS TO THERAPIES AND PATIENT JOURNEY IN THE LATIN AMERICAN REGION.

J. L. Castro Aguilar 1,* , F. Tapia Mathus 1 , F. Piotrowski 1 , N. Brown 1 , C. Marin 1

1 CML Advocates Network, Bern, Switzerland

Background: The dramatic health-access inequalities of the population, even greater for chronic patients, have a high impact limiting the social and economical development of people and countries. Chronic myeloid leukemia (CML) patient organizations representing countries of the CML Advocates Network Latin American region have come to the conclusion that they share critical issues in patient access to treatment and monitoring tools, which results in health inequities arising from the location where people are born, live and age.

Aims: Although the study covers many areas, its underlying aim was to highlight health inequities between CML patients as well as explaining the patient journey in the different countries of the region.

Methods: A team consisted of four patient advocates designed the questionnaire to collect data and interviews, communication and website platform. Information was also collected from both the official sites of each country, the World Health Organization (WHO), the Panamerican Health Organization (PAHO) and other non-governmental organizations (NGOs) in a way that clearly reflects the reality of the CML patients in the region, from January 2019 and lasted until January 2020, covering the following topics: Population; National Health Law – Constitution; Health Expenditure in relation to GPD; Patient out-of-pocket expenses; Per capita gross domestic product; CML treatments available; Rate of estimated CML cases.

Through a questionnaire and online interviews with the leaders of the CML national patient advocacy organisations, data were provided by fifteen patient organisations based in thirteen countries.

The team carried out ten interviews with leaders of Costa Rica, Uruguay, Paraguay, Panama, Guatemala, Mexico, Argentina, Chile, and Bolivia.

An interactive map was developed and published, showing the data collected for the different countries, and interviews were additionally published on the website.

Presented data as of January 2020.

Results: Collected data about availability of the different CML therapies shows substantial differences between countries in the region:

14 countries reported Imatinib available. 8 countries reported Dasatinib available: Argentina, Colomboa, Chile, Dominican Republic, Mexico, Nicaragua, Peru, and Uruguay. All countries except Venezuela reported Nilotinib available thourgh donations. Only 5 countries reported Bosutinib available: Argentina, Colombia, Chile, Nicaragua and Venezuela. Ponatinib was reported as an available treatment for patients in 5 countries: Argentina, Colombia, Chile, Ecuador and Venezuela.

The study identified disparities in patient journey of people living with CML in different countries. Reported data evidence 9 diverse process defining the entire sequence of events that a CML patient experiences within a national healthcare system, from the diagnosis to receiving treatment for the disease.

The study also shows the variety of the patient advocacy groups in the region, listing twenty patient advocacy organisations from sixteen countries.

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Summary/Conclusion: Availability of the different types of CML treatments in Latin America is quite uneven: from countries in which patients can access all existing therapies including clinical trials, to others in which access is limited to one treatment achieved only through donations.

Inequities are not only present in the availability of the different therapies to treat CML, they are also evidenced in the differences existing in time to patient access to be effectively treated and monitored.

P1720: HOW TO IMPROVE THE TIME IN THERAPEUTIC RANGE (TTR) OF ANTICOAGULATED CHILDREN DURING THE COVID-19 PANDEMIC: TELECONTROL VERSUS TRADITIONAL HOSPITAL CONTROL

R. MARTOS MARTÍNEZ 1,* , L. BERMEJO MARTÍNEZ 2 , D. CORNEJO CORRALES 1 , M. YUSTE PLATERO 1 , E. GONZALEZ ARIAS 1 , P. BELTRÁN ALVAREZ 2 , D. NAYA ERREA 2 , E. HERREROS MONTERO 1 , B. VILLA GONZÁLEZ 1 , L. SASTRE MARTIN 1 , P. LLAMAS SILLERO 1

1 HEMATOLOGÍA Y HEMOTERAPIA, HOSPITAL UNIVERSITARIO GENERAL DE VILLALBA; 2 HEMATOLOGÍA Y HEMOTERAPIA, HOSPITAL UNIVERSITARIO INFANTA ELENA, MADRID, Spain

Background: The Covid-19 pandemic generated added difficulty in carrying out oral anticoagulation controls in children, due to limited travel to health centers for the mobility restrictions. Children there is greater difficulty in maintaining adequate coagulation times, measured in times in therapeutic range (TTR) due to the significant individual variability in anticoagulant medication that can cause thrombotic or hemorrhagic complications. We proposed carrying out controls at home telematically as an alternative to traditional hospital controls, avoiding travel during the pandemic and evaluating the safety of the results and the quality of life (QoL)

Aims: To assess the efficacy of remote control in children with chronic oral anticoagulation compared to patients with hospital control based on the analysis of TTR during covid-19 pandemic as well as QoL

Methods: Observational and longitudinal study comparing 2 cohorts: remote control group and hospital control group. During 2020, the patients were included in the remote control group in accordance with the usual clinical practice, keeping in hospital control those children who had to follow specific treatments in the hospital (cancer treatments). Classified the patients according INR ranges and age: 3 groups INR ranges established (1.8-2.2; 2.2-2.5 and 2.5-3) according to medical criteria and 3 age groups (0-6 yrs, 7-12 yrs, and 13-16 yrs). In January 2021, TTR data defined in both groups were collected according to Rosendaal’s methodology. Given the complexity of the TTR due to the pharmacokinetic variability of the antivitamin K drugs, which means that the specific determination in one day does not correspond to the INR values ​​that the patient will present on the days between the controls, we calculated the TTR of each patient by the fraction (number of INR in therapeutic range/total number of INR). We distributed patients into 4 groups: Group A: patients with less than 25% TTR. Group B: patients who have been between 25%-50% of TTR. Group C: patients who have been between 50%-75% of TTR. Group D:patients who have more 75% of TTR

Results: 44 patients were included: 23 remotely controlled (mean age 8.4 yrs) vs. 21 hospitalized (mean age 9.1). Reasons for anticoagulation: surgery, catheters, cancer, heart disease and vasculitis. Controls outside the therapeutic range were on average higher in the hospital group (15.1 vs 3.3), maintaining these differences for all ages and INR ranges. 91% children in remote control spend more than 75% in TTR compared to 13% of the hospital group, being significant in children older than 7 years, with INR 2.2-2.5 and with catheters or heart disease. 5 children in the remote-control group with 100% of their determinations in TTR. 16 bleeding events were documented, 12 in hospital group(4 major bleedings)and 4 bleedings (all minor) in remote control. 15 thrombotic events (14 hospital control vs 1 in remote control).

81 doses of low molecular weight heparin (LMWH) and 31 doses of vitamin K (VK) were administered to hospital control children vs. 15 doses of LMWH and 6 vials VK to remote control children

Summary/Conclusion: Remote control of anticoagulation in children is an effective and safe alternative to traditional hospital control, with very high TTR results. Using this type of control to avoid travel during mobility restrictions due to the covid-19 pandemic led to improved results, with fewer emergency visits related to anticoagulation, with a lower rate of bleeding or thrombotic complications, and less need for LMWH support. or VK compared with children with hospital control

P1721: PROGNOSTIC FACTORS IN PATIENTS IN THE TERMINAL PHASE OF HEMATOLOGICAL MALIGNANCIES WHO RECEIVED HOME MEDICAL CARE

N. Miyashita 1,2,* , M. Onozawa 3 , M. Fujita 4 , T. Hosoda 2,5 , Y. Kawasaki 2,6 , M. Takimoto 2,7 , S. Okina 2,8,9 , K. Ohashi 2,10

1 Department of Hematology, HOME CARE CLINIC N-CONCEPT, Sapporo, Hokkaido; 2 NPO Hemato-Homecare Network, Komae, Tokyo; 3 Department of Hematology, Hokkaido University Hospital, Sapporo, Hokkaido; 4 Medical Home Care Center, Tenri Hospital, Tenri, Nara; 5 hamorebi Clinic, Kamagaya, Chiba; 6 Kaedenokaze Medical Clinic, Machida, Tokyo; 7 Kawasaki Nanafuku Clinic, Kawasaki, Kanagawa; 8 Yaguchi Medical Clinic; 9 Department of Hematology, Kitasato University Hospital, Sagamihara, Kanagawa; 10 TOTUS home care clinic, Komae, Tokyo, Japan

Background: In the current global COVID-19 pandemic, terminal care in a patient’s home has been expanded as a positive choice even for patients suffering from hematological malignancy (HM). Although there are many tools for predicting the prognosis of patients in the terminal phase of solid tumors, there is little information about prognostic factors in patients in the terminal phase of HM, especially patients receiving home medical care (HMC). In comparison to patients with solid tumors, those with HM are more likely to have acute diseases such as acute bleeding and acute infection leading to death. A previous report revealed that HM was a factor associated with aggressive end-of-life care. Because of the various complications associated with HM, it was reported to be difficult to predict the prognosis for patients with HM. Providing patients with accurate information about prognosis is important for them to consider how to spend their remaining life.

Aims: In patients in the terminal phase of HM who received HMC, we aimed to validate the usefulness of two prognostic models: Palliative Prognostic Index (PPI), which is an established prognostic model for patients in the terminal phase of a solid tumor, and the prognostic model reported by Kripp et al., which is a prognostic model for patients with HM in a palliative care unit. In addition, we aimed to determine prognostic factors for patients in the terminal phase of HM who received HMC and to develop a more detailed prognostic scoring system.

Methods: We retrospectively evaluated 136 patients in the terminal phase of HM who were receiving HMC provided by 6 clinics between 2008 and 2022. Medical records relevant to prognosis were collected by a chart review. The effects of possible factors associated with overall survival (OS) were determined by the Kaplan-Meier method and univariate and multivariate Cox regression models. This study was approved by the IRB of Hokkaido University Hospital.

Results: Patients characteristics were as follows: male/female, 78/58; age, 25 to 94 years; median age, 79 years; AML, 50 patients; B-NHL, 32; MDS, 24; MM, 13; T-NHL 6; ALL, 5; ATL 2, CMML 2, and PV, 2. According to PPI, there was no significant difference in OS between the intermediate-risk group and the low-risk group (panel A; P = 0.15). By using the prognostic model reported by Kripp et al., we could stratify the patients into 3 risk groups with significantly different survival times (panel B; P < 0.01). However, there was a wide range of survival times in the high-risk group (OS, 0 to 125 days; median OS, 24 days). In our investigation of factors associated with OS, multivariate analyses revealed that there were 7 factors associated with poor OS (panel C). For the development of our prognostic scoring system, each variable was weighed according to the value of the hazard ratio (panel C) and 4 risk groups were shown to clearly discriminate survival ( P < 0.01): low-risk group (n = 25, median OS of 434 days), intermediate-low risk group (n = 60, median OS of 112 days), intermediate-high risk group (n = 31, median OS of 31 days), and high-risk group (n = 20, median OS of 9 days).

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Summary/Conclusion: This is the first investigation of prognostic factors that influence the overall survival of patients in the terminal phase of HM who received home medical care. In comparison to previously reported prognostic models, our scoring system could stratify patients in more detail. Providing patients and medical staff with accurate information about prognosis will lead to a higher quality of life in the terminal phase and better support by medical staff.

P1722: CHANGES IN ACUTE HEPATIC PORPHYRIA HEALTH IMPACTS SINCE INITIAL DIAGNOSIS: RESULTS FROM THE PORPHYRIA WORLDWIDE PATIENT EXPERIENCE RESEARCH (POWER) STUDY

D. Nance 1,* , D. Lyon 2 , S. Hegarty 3 , R. Falchetto 4 , J. Barman-Aksözen 4,5 , T. Mnif 6 , S. Meninger 7 , S. Lombardelli 8 , A. Dickey 9

1 Banner Health, Gilbert; 2 American Porphyria Foundation, Bethesda, United States of America; 3 Global Porphyria Advocacy Coalition, Durham City, United Kingdom; 4 Swiss Society for Porphyria; 5 Stadtspital Zürich, Triemli, Institute of Laboratory Medicine, Zürich, Switzerland; 6 Cerner Enviza, Paris, France; 7 Alnylam Pharmaceuticals, Cambridge, United States of America; 8 Alnylam Pharmaceuticals, Maidenhead, United Kingdom; 9 Massachusetts General Hospital, Boston, United States of America

Background: Acute hepatic porphyria (AHP) is a rare and potentially life-threatening inherited disease caused by genetic mutations in the heme biosynthetic enzymes affecting the liver. Patients can experience acute neurovisceral attacks, chronic symptoms, long-term complications, and negative impact on many quality-of-life domains. Limited evidence is available on disease changes and impacts on patient health over time.

Aims: This study evaluated AHP patient perceptions of changes in their disease characteristics and impacts in quality-of-life domains since AHP diagnosis among the overall study population and in subgroups based on time since first symptoms/diagnosis.

Methods: Adults with >1 AHP attack within the past 2 years or receiving intravenous hemin and/or glucose for attack prevention were recruited via patient advocacy groups and physician referral from the United States, Italy, Spain, Australia, Mexico, and Brazil and were administered an online survey from January 19, 2021, to April 26, 2021. Patients taking givosiran were excluded. Patient perceptions of changes in overall health and disease characteristics since diagnosis/disease onset were evaluated. Subgroup analyses were conducted to evaluate differences in patients experiencing active disease for 0–5 years versus those experiencing active disease for ≥6 years. Patient-reported outcomes to evaluate anxiety and depression were assessed among subgroups using the Generalized Anxiety Disorder-7 (GAD-7) scale (0–21) and the Patient Health Questionnaire (PHQ-8) scale (0–24), respectively.

Results: A total of 92 patients with AHP completed the survey. Mean age was 41.1 years, and 90% of patients were women. Mean time to diagnosis was 6.4 years, and mean duration of disease was 16.9 years. Most patients experienced negative (very or somewhat negative) impacts on emotional health (90%), physical health (87%), financial health (75%), social health (70%), and cognitive health (66%) since their diagnosis. Patients described disease characteristics as worsening (much worse or somewhat worse) since their diagnosis, including chronic pain (66%), kidney disease (55%), and acute pain (48%) ( Figure ). In subgroup analyses, 22% (N=20) of patients were found to have had active disease for 0–5 years, and 73% (N=67) had active disease for ≥6 years. Patients with active disease for 0–5 years were younger on average than patients with active disease for ≥6 years (33.9 vs 43.4 years, respectively). Patients with active disease for 0–5 years experienced a median of 2.5 attacks in the past 2 years, whereas patients with active disease for ≥6 years experienced a median of 5 attacks in the past 2 years. Thirty-five percent of patients experiencing AHP for 0–5 years reported a GAD-7 score ≥10, compared with 51% of patients experiencing AHP for ≥6 years. Severe anxiety (GAD-7 score ≥15) was reported in 20% and 28% of these patients, respectively. On the PHQ-8 scale, 30% of patients experiencing AHP for 0–5 years reported a score ≥10, indicating moderate to severe depression, compared with 66% of patients experiencing AHP for ≥6 years.

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Summary/Conclusion: Patients with AHP experience negative impacts across multiple health domains and worsening in some disease characteristics since initial diagnosis. Longer duration of disease is associated with poorer perceived health status.

P1723: A FEASIBILITY STUDY OF PEER SUPPORT IN FAMILY CAREGIVERS OF PATIENTS NEWLY DIAGNOSED WITH HEMATOLOGIC MALIGNANCY

I. H. Nielsen 1,* , K. Piil 2,3 , L. Kjeldsen 1 , K. Grønbæk 1,4 , M. Jarden 1,4

1 Department of Hematology; 2 Department of Oncology, Copenhagen University Hospital, Rigshospitalet, Copenhagen; 3 Department of Public Health, Aarhus University, Aarhus; 4 Department of Clinical Medicin, University of Copenhagen, Copenhagen, Denmark

Background: Informal family caregivers play a pivotal role in supporting patients with hematologic malignancies during their treatment. Due to the toxicities of treatment patients often have serious treatment-related side-effects, and with more treatment being placed in the outpatient settings, family caregivers are often the primary source of support between hospital visits assisting the patient and managing symptoms. The early phase of treatment has been found particularly stressful, and evidence shows that family caregivers are at higher risk of psychological distress and have unmet support needs impacting their ability to support the patient. Thus, finding new ways to support family caregivers is important within a health care context. Peer to peer support has shown great promise in hematologic patients, and there is a need to investigate psychosocial interventions such as peer to peer support in family caregivers.

Aims: To investigate the feasibility of a one-on-one peer support intervention in family caregivers of newly diagnosed hematologic patients.

Methods: A one-arm feasibility study was conducted. The intervention consisted of 12-weeks of one-on-one support provided by volunteer family caregivers of a hematologic cancer survivor (named caregiver ambassadors) (n = 17) to a family caregiver of a newly diagnosed patient (n =26). Support was delivered based on the needs and preferences of the family caregiver via telephone, text message, e-mail or face-to-face. The intervention also included a preparatory course for the caregiver ambassadors and available support during the intervention. Primary outcomes were feasibility parameters and secondary outcomes were collected at three time points and included anxiety, depression, sleep disturbances, self-efficacy, and quality of life.

Results: The peer support intervention was feasible and safe, and participants reported very high satisfaction with the support (75%). Dropout rate was low (7%) and no adverse events were reported. Over time, family caregivers improved their psychosocial well-being, and caregiver ambassadors overall maintained their levels from baseline. Participants were satisfied with the preparatory course enhancing their role preparation as peer supporters and 14 caregiver ambassadors attended the available support network meetings (n = 6). During the intervention, the total number of contacts between participants were 389 with each dyad having an average of 15 contacts during the 12-weeks. Support was most frequently delivered by telephone and text-messages. In-person meetings were less used due to geographical distances, personal preferences including the patients’ illness situation, and covid-19.

Summary/Conclusion: We tested the feasibility of a new peer to peer intervention in family caregivers of newly diagnosed hematologic patients. The results demonstrated that the intervention was feasible, safe, and satisfactory in both the caregiver ambassador and family caregiver groups. Flexibility in the delivery and frequency of support was essential and accommodated the individual support needs and preferences. Providing preparation and support for caregiver ambassadors was important. This family caregiver peer to peer intervention has the potential to be a new model of support incorporated in the clinical setting.

P1724: QUALITY OF LIFE AND UNMET NEEDS AMONG PH-NEGATIVE MYELOPROLIFERATIVE NEOPLASM PATIENTS: RESULTS OF THE NATIONWIDE SURVEY

T. Ionova 1,2,3,* , E. Andreevskaya 4 , E. Babich 5 , N. Bulieva 6 , O. Vinogradova 7,8,9 , E. Volodicheva 10 , S. Voloshin 11,12,13 , N. Glonina 14 , S. Dubov 15 , N. Esefieva 16 , E. Zinina 17 , M. Ivanova 18 , T. Klitochenko 19 , A. Kopylova 20 , A. Kulagin 21 , G. Kuchma 22 , O. Li 23 , E. Lomaia 24 , A. Melikyan 25 , V. Melnichenko 3 , S. Menshakova 26 , T. Mitina 27 , E. Morozova 21 , E. Osipova 28 , O. Ochirova 29 , A. Polyakov 12 , T. Pospelova 30 , A. Proydakov 31 , O. Rukavitsyn 32 , G. Safuanova 33,34 , I. Subortseva 25 , M. Fominykh 35 , M. Frolova 36 , T. Shelekhova 37 , D. Sherstnev 37 , T. Shneyder 38 , V. Shuvaev 11,39 , E. Laane 40 , E. Oliva 41 , S. Salek 42 , T. Nikitina 1,2

1 Multinational Center for Quality of Life Research; 2 Saint Petersburg State University Hospital, Saint-Petersburg; 3 Pirogov National Medical and Surgical Center, Moscow; 4 Regional Clinical Hospital №1, Chita; 5 Regional Clinical Hospital, Khanty-Mansiysk; 6 Regional Clinical Hospital of the Kaliningrad Region, Kaliningrad; 7 Hematology Center of Botkin Hospital; 8 Pirogov Russian National Research Medical University; 9 Dmitry Rogachev National Research Center of Pediatric Hematology/Oncology and Immunology, Moscow; 10 Regional Clinical Hospital, Tula; 11 Russian Research Institute of Hematology and Transfusiology; 12 SM Kirov Military Medical Academy; 13 North Western State Medical University named after I.I. Mechnikov, Saint-Petersburg; 14 Regional Clinical Hospital № 1 named after Professor S.I. Sergeev, Khabarovsk; 15 Regional Hematological Center of the Regional Clinical Hospital № 2, Vladivostok; 16 Regional Clinical Hospital, Ulyanovsk; 17 Surgut Regional Clinical Hospital, Surgut; 18 Clinical and Diagnostic Center, Pavlov University, Saint-Petersburg; 19 Volgograd Regional Clinical Oncology Dispensary, Volgograd; 20 Lipetsk Municipal Hospital № 3 “Svobodnyi Sokol”, Lipetsk; 21 RM Gorbacheva Research Institute, Pavlov University, Saint-Petersburg; 22 Regional Clinical Hospital, Orenburg; 23 Regional Clinical Hospital, Yuzhno-Sakhalinsk; 24 Almazov National Medical Research Centre, Saint-Petersburg; 25 National Research Center for Hematology, Moscow; 26 Regional Clinical Hospital, Tver’; 27 Moscow Regional Research and Clinical Institute (“MONIKI”), Moscow; 28 The Federal State-Financed Scientific Institution Kirov Research Institute of Hematology and Blood Transfusion under the Federal Medical Biological Agency, Kirov; 29 Republican Clinical Hospital named after N.A. Semashko, Ulan-Ude; 30 Federal State Budgetary Educational Institution of Higher Education «Novosibirsk State Medical University» of the Ministry of Health of the Russian Federation, Novosibirsk; 31 Komi Republican Oncology Center, Syktyvkar; 32 N.N. Burdenko Main Military Clinical Hospital, Moscow; 33 Bashkir State Medical University; 34 Republical Clinical Hospital named after G.G. Kuvatov, Ufa; 35 The Scandinavia AVA-PETER Clinic, Saint-Petersburg; 36 Regional Hospital № 1, Vologda; 37 Saratov State Medical University named after V.I. Razumovsky, Saratov; 38 Leningrad Regional Clinical Hospital, Saint-Petersburg; 39 City Clinical Hospital n.a. V.V. Veresaev Department of Healthcare of Moscow, Moscow, Russia; 40 Department of Hematology and Oncology, University of Tartu, Tartu, Estonia; 41 Hematology Unit, Grande Ospedale Metropolitano BMM, Reggio Calabria, Italy; 42 School of Life and Medical Sciences, University of Hertfordshire, Hatfield, United Kingdom

Background: Patients with Philadelphia-negative myeloproliferative neoplasms (MPNs), including myelofibrosis (MF), polycythemia vera (PV) and essential thrombocythemia (ET), experience troublesome symptoms leading to impaired functioning (physical and psychosocial) and quality of life (QoL). Understanding symptom burden, QoL and unmet patient needs across disease subgroups is of value to aid optimal treatment and rehabilitation decision-making for patients with different MPNs. It is also worthy to explore perceptions on the impact of the disease and its treatment among MPN patients and hematologists to ensure patient-centered care.

Aims: The national survey MPN-QoL-2020 was aimed to evaluate QoL and symptom burden in patients with MPN, as well as to examine the perceptions of patients and physicians about the impact of MPN and its treatment in a real world setting in Russia.

Methods: A survey of patients with MPN and their treating physicians was conducted from September to December 2020. This survey included in- or out-patients aged ≥18 years with a confirmed diagnosis of MF, PV, or ET. All the patients completed the HM-PRO, a hematological malignancy (HM) specific patient-reported outcomes (PRO) measure, the MPN10 symptom assessment tool, and a patient’s survey checklist. The HM-PRO consists of two scales: Part A measuring the ‘impact on patients’ QoL’; Part B measuring ‘signs and symptoms’ experienced by the patients. Part A has 4 domains: physical behaviour (PB), social well-being (SW), emotional behaviour (EB) and eating and drinking habits (ED). Physicians completed a physician’s survey checklist which included a record for each patient. Both paper and electronic versions of the survey forms were available. ANOVA and χ 2 test were applied to examine the differences between groups.

Results: 1100 patients with MPNs (MF, n=355, PV, n= 408 and ET, n=337; mean age = 58±14 yrs; female = 61%) and 100 hematologists (mean age = 42±12 yrs, female = 85%) from 37 medical centers completed the survey. The HM-PRO Parts A & B total scores were significantly worse (higher scores) in MF and PV patients than in ET (Table 1). Impact on PB and EB was higher in MF and PV as compared to ET: PB = 28.6 and 21.4 vs 14.3; EB = 31.8 and 27.3 vs 22.7, respectively. Impact on ED was higher in MF as compared to PV and ET. SF was less impaired and similar across different MPNs. Among MF there were more patients with moderate/large effect on QoL as compared to PV and ET – 40% vs 34% and 28% (p=0.003). The vast majority of MPN patients experienced symptoms (95%); more than 80% had fatigue and inactivity. The MPN10 Total Symptom Score was the highest in MF, intermediate in PV and the lowest in ET: 24 vs 20 vs 14 (p<0.001). Notably, 29% MF, 34% PV and 44% ET patients did not attribute their symptoms to MPN. Physicians and patients had a discordant perspective of the most bothersome symptoms. There was agreement between MF patients and their physicians about the impact of the disease on QoL and symptom burden. For PV and ET, physicians underestimated patient’s concerns; the discrepancies were pronounced for QoL impact (PV, p=0.028; ET, p<0.001) and symptom burden (PV, p=0.018; ET, p<0.001). Across MPNs, 41.5% MF, 37.8% PV and 35.2% ET agreed that there were areas of patient-physician relationship needed to be improved.

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Summary/Conclusion: The findings of this nationwide survey demonstrate differences in the impact on QoL and symptom burden across MPNs, identify the areas of different perspective between patients and physicians about MPN and its treatment as well as highlight the unmet needs among patients with MPN.

P1725: OBSERVATIONAL STUDY TO EVALUATE PATIENT-REPORTED OUTCOMES IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

E. Urciuoli 1 , E. Di Bona 2 , S. Greve 3 , A. De Vivo 4 , S. Sica 5 , R. Fontana 6 , W. Barcellini 7 , L. Marano 1 , E. N. Oliva 3,*

1 UOC Ematologia e Trapianto, AORN San Giuseppe Moscati, Avellino; 2 UOC Oncoematologia, Azienda Ospedaliera Pedemontana,ASL 7, Bassano del Grappa (VI); 3 U.O.C di Ematologia, Grande Ospedale Metropolitano Bianchi Melacrino Morelli, Reggio di Calabria; 4 Dipartimento di Ematologia, Policlinico S. Orsola Malpighi, Bologna; 5 Dipartimento di Ematologia e trapianti di CSE, Fondazione Policlinico Universitario IRCCS A. Gemelli, Roma; 6 UOC di Ematologia e Trapianti di Cellule Staminali Emopoietiche, AOU San Giovanni di Dio e Ruggi d’Aragona, Salerno; 7 U.O. Ematologia, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano, Italy

Background: Paroxysmal Nocturnal Hematuria (PNH) is a rare acquired hematopoietic stem cell disorder with non-specific clinical features and onset prevalent at young adult age. Main symptoms are fatigue (80%) and dyspnea (64%). Treatment for symptomatic and/or predominantly haemolytic PNH patients is associated with an improvement in survival and quality of life (QoL). To date, cancer-specific patient-reported outcome (PRO) measures (PROMS) have been used in PNH. A novel PNH-specific PROM, EORTC QLQ-AA/PNH, consisting of 54 items has undergone initial Phase 2-3 evaluation. We present preliminary results of the first 31 cases enrolled.

Aims: The aim of this observational non-interventional prospective multicenter Italian study is to evaluate whether PROMs can identify patients’ biological response to treatment and factors associated with health-related QOL. The primary endpoint is differences in EORTC QLQ-C30 scores between patients with versus without hemolysis. Secondary endpoints include further validation of EORTC QLQ-AA/PNH Italian version, differences in scale scores between patients experiencing hemolysis versus those not experiencing hemolysis and factors associated with PROs by EORTC QLQ-AA/PNH.

Methods: 110 adult patients age ≥ 18 years with PNH will be included. Exclusion criteria consists of bone marrow failure, history of treatment for cancer within the last 5 years and clinically significant comorbidities that impact PROs. Patients complete both PROMs on the day of visit to the clinic for the scheduled treatment and re-test after 2 weeks. For EORTC QLQ-AA/PNH higher scores identified higher impact or symptoms. Demographics and disease features are collected at baseline. Hemolysis is defined by a lactate dehydrogenase (LDH) ≥ 1.5 times upper limit of normal (ULN) at baseline.

Results: Patient characteristics are shown in the Table 1. The EORTC QLQ-C30 scores did not distinguish patients with or without hemolysis, probably due to the small sample size. Baseline Global Function was good with median score 83 (IQR 50-100). Fatigue and dyspnea were not frequently reported with median scores 33 (IQR 11-33) and 0 (0-33), respectively. The EORTC QLQ-AA/PNH showed good reliability with Chronbach alpha standardized scores for all domains except Body Image (BI), Infections (IN) and Social Support (SS), confirmed at retest. There were strong correlations between the 2 questionnaires. QLQ-AA/PNH Cognitive Functioning (CF) median score was 0 (IQR 0-25), Emotional Functioning 20 (IQR 5-35), Fatigue (FA) 20 (IQR 10-30), Illness Intrusiveness (II) 11 (IQR 4-32), other symptoms (OS) 11 (0-21), Physical Functioning (PF) 17 (0-33), Role Functioning (RF) 14 (9-32), Stigmatisation (ST) 12 (0-25) and Fear of Progression (PAF) 123 (4-29). Hemoglobin levels correlated with QLQ C30 physical function (R=0.505, P=0.006), role function (R=0.450, P= 0.014), Fatigue (R=-0.520, P=0.004), Dyspnea (R=-0.410, P=0.027) and Pain (R0-0.409, P0 0.028). They correlated with EORTC QLQ-AA/PNH PF (R=0.499, P=0.006), ST (R=0.395, P=0.034) and FA (r=-0.517, p=0.004).

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Summary/Conclusion: The preliminary analysis confirms the validity of the EORTC QLQ-AA/PNH as a PROM for PNH. Hemoglobin is an important factor which correlates with many PRO domains. Further analyses at study completion will inform on the impact of PNH in patients’ lives.

P1726: NON-PHARMACOLOGICAL INTERVENTIONS FOR SYMPTOM MANAGEMENT AND QUALITY OF LIFE IN PATIENTS WITH HEMATOLOGICAL MALIGNANCY: A SYSTEMATIC REVIEW OF RANDOMIZED TRIALS

M. Pedersen 1,* , M. Schaufuss Engedal 1 , B. Kornblit 1 , M. Torp Larsen 1 , K. Lomborg 2,3 , M. Jarden 1,3

1 Department of hematology, Rigshospitalet, Copenhagen; 2 Steno Diabetes Centre, Copenhagen University Hospital; 3 Clinical medicine, University of Copenhagen, Copenhagen, Denmark

Background: Non-pharmacological interventions have been developed and tested in patients with hematological malignancy as patients experience debilitating and persistent symptoms affecting everyday life.

Hematological malignancies are a diverse and complex group of diseases and treatment regimens vary from aggressive chemotherapeutic treatments, to daily or weekly treatment regimens, or ‘watch and wait’ strategies. Therefore, for many patients the diagnosis is an incurable chronic condition characterized by multiple exacerbations, functional impairment and a high symptom burden affecting quality of life with negative consequences for optimal treatment, survival, return to work, and daily activities. As pharmacology is not always possible or optimal in managing symptoms there is a need for non-pharmacological interventions targeted this population. However, evidence collating the impact of non-pharmacological interventions is sparse.

Aims: To identify and provide a summary of types of non-pharmacological interventions that measure symptoms and quality of life investigated in randomized controlled trials in patients with hematological malignancies.

Methods: A protocol of this study was registered at the international register of systematic reviews PROSPERO (Registry no. CRD42021237344). A systematic search for articles was conducted until April 20, 2021 in Medline, Embase, Cinahl, APA PsycInfo and Central Cochrane. This systematic review followed the PRISMA guidelines, and applied Cochranes Risk of Bias tool (Rob2).

Results: There were 14.610 studies identified through the initial search. After removal of duplicates (n=2223), 12.387 studies remained. Sixty-four articles were included after full-text screening. The non-pharmacological interventions were divided into five types across 12 hematological diagnosis or treatment groups; i) Exercise (n=36), ii) Mind/body (n=9), iii) Clinical consultation-based (n=4), iv) Music and Art (n=6) and v) Tele-health and App-based (n=9) measuring symptom and quality of life outcomes along the disease and treatment trajectory.

Overall patients with leukemia were dominant within mind/body interventions, whereas HSCT patients were mainly tested in music/art and exercise interventions. Fatigue was improved significantly in patients testing tele health care (n=3), mind/body interventions (n=2) and several exercise interventions. Depression and anxiety decreased following clinical palliative consultation care (n=2), music/art (n=2) and mind/body interventions (n=3). Except for one study (music/art) all the interventions found improved patient quality of life.

Studies included had divergent quality assessments assed with ROB2.

Summary/Conclusion: Different types of non-pharmacological interventions had varying effects on managing symptoms and in improving quality of life. Symptoms of fatigue, anxiety and depression and quality of life were most frequently investigated, and significant positive effects of non-pharmacological interventions were found on several studies.

P1727: EPIDEMIOLOGICAL TRENDS OF HAEMATOLOGICAL MALIGNANCIES IN BELGIUM 2004-2018: OLDER PATIENTS SHOWED THE GREATEST IMPROVED SURVIVAL OVER TIME

K. Henau 1 , T. Tambuyzer 1 , B. Van Gool 1 , L. Van Eycken 1 , H. A. Poirel 1,*

1 Belgian Cancer Registry, Brussels, Belgium

Background: Haematological malignancies (HM) are common (11% of the total cancer burden in Belgium in 2018) and, at the same time, they represent a heterogeneous group of rare malignancies, which are distinguished by specific clinico-biological characteristics, behaviour and outcome.

Aims: We aim to analyse in more depth the epidemiological trends of HM in Belgium over a recent 15-year period, with a particular focus on the impact of age at diagnosis on the incidence and the survival trends by type of HM.

Methods: The Belgian Cancer Registry (BCR) is a population based national cancer registry, collecting data since incidence year 2004. We studied the 23 main HM types (see Figure 1), representing 98% of all HM cases (N = 92.202), diagnosed between 2004 and 2018. Patients follow-up was available until 01/07/2020.

We estimated age-standardised (WSR) incidence, incidence trends (Average Annual Percentage Change or AAPC), projections up to 2025, 5- & 10-year relative survival (RS), conditional RS and RS trends for the 23 main types of HM.

Results: During the period 2004-2018, the WSR incidence rates of HM increased in Belgium (AAPC: 1.2%) and mainly in the older population (70+), while they remain stable in children. The greatest increase is observed for BCR/ABL1-negative chronic myeloid neoplasms.

The 10-yr RS varies by age group and HM type. ALL shows the greatest age variation, from >90% in children under age 10 to <20% in adults 70+.

For each type of HM, outcome gets progressively worse with age. The greatest decrease in 5yr-RS with age is observed for aggressive HM, such as AML, ALL/LL and BL: from 67%, 90% and 97% respectively in children and adolescents (0-19 yr) to 2%, 12% and 16% in adults 80+, respectively.

The 5-yr RS of all HM has increased over the 15 years with 5 percentage point (pp) from 64% in 2004-08 to 69% in 2014-18 (Table 1). The greatest improvement is seen in ALL (+14 pp), followed by IPD and HDCN (+11 pp). No clear improvement is observed for most myeloid neoplasms except CML.

Analysis by age group reveals larger increases in survival over time. The most impressive increases are seen in the older patients (80+), with +45 pp in HL (from 21% in 2004-2008 to 66% in 2014-2018), followed by IPD (+33 pp), FL (+28 pp), CML (+16 pp), PMF (+13 pp), CLL/SLL (+12pp) and MCL (+11pp). A strong improvement (+22 pp) is also observed for ALL/LL in the 60-79 age group, even if the 5y-RS remains low (37% over the 2014-18period). Young adults (20-39 yr) with BL showed great improvement (+21%) with survival progressing towards the level seen in children. PNK/TCL and MDS showed the greatest increase (+14 pp and +13pp respectively) in adults (20-59 yr), and AML (+17 pp) in children.

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Summary/Conclusion: The increase in incidence and the improvement in survival during the period 2004-2018 can be partly explained by the diagnostic and therapeutic innovations over the 2 last decades. This real-world population-based study provides useful information on patients usually excluded from clinical trials (those with advanced age, comorbidities, other malignancies).Older patients showed the greatest improvement over time. However, this study also highlights entities that need more attention for improvement, such as AML and MDS.

P1728: THE FUTURE OF MYELODYSPLASTIC SYNDROME – PATIENT PRIORITIES AND OUTCOMES THAT MATTER

M. Poynton 1,* , C. Hamilton Gilmour 1 , I. Dale-Harris 1 , E. Clarke 1 , S. Stanworth 2 , M. Murphy 2 , N. Roy 1

1 Oxford University Hospitals NHS Trust; 2 NHS Blood and Transplant, Oxford, United Kingdom

Background: Without a definitive curative option available to many patients, learning to live with myelodysplastic syndrome (MDS) and manage symptoms effectively becomes a priority in their care. Anaemia is an almost universal feature of MDS but other cytopenias are common and may be linked to bleeding and infection. Individuals suffer differently and better individualisation of care is needed. Most MDS patient information offers scant appreciation for disease heterogeneity, variable response to treatment and each patient’s likely trajectory.

Aims: We undertook to discuss what matters most to people living with MDS and provide feedback on discussions in patient support programmes. The aim was to inform lived experience views on research prioritisation in MDS.

Methods: We conducted a 2-part interactive online workshop using Zoom and the online platform Miro, with the assistance of a skilled technical facilitator, to replicate a face-to-face environment (eg white board, post-it notes and sticky dots for voting). Patients were invited to generate questions about their condition which they felt should be addressed by research or a change in how their care is delivered. These were then grouped into themes, and reframed to create individual non-overlapping topics. Using a model inspired by the James Lind Alliance Priority Setting Partnerships, patients voted on the importance of each topic, creating a final “prioritised” list of issues.

Results: 14 participants of varying age and experience took part raising 56 unique questions under the themes of: Prognosis; End of life; Treatment; Supportive Care; Medical Staff Training; Diagnosis; Communication; Organisations; resource sharing.

Using the voting system, patients prioritised 14 key outcomes (shown in Figure 1). These reflect the symptoms of MDS, improving quality of life (QoL), communication, access to healthcare professionals and individualising care.

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Summary/Conclusion: There are few studies in MDS where QoL is the primary outcome, hence the paucity of evidence around best practice when prioritising this. Given the pervasiveness of anaemia in MDS, managing this, and subsequent fatigue is of vital importance.

Although haemoglobin (Hb) levels have been found to be correlated to QoL, it is widely reported that other factors are important in determining QoL and need for transfusions can vary despite a stable Hb level. Indeed, a key finding from patient discussions showed that Hb levels and the need for transfusions is not comparable between different patients and even non-comparable over time meaning that the maximal benefit and timing of transfusions cannot be determined from Hb level alone.

Better communication with patients can ensure transfusions are given to improve symptoms. Previously, evaluation of success of transfusion and the clinical decision to transfuse has been determined only by the effect on Hb levels. This workshop highlighted patient dissatisfaction with the ‘numbers-led’ approach and the need for an alternative method to determine when to transfuse. While studies show using liberal transfusion targets improve QoL there are conflicting results showing a prolonged increase in Hb requires more transfusions which would in turn require more time in hospital.

Further research is required to define optimal transfusion policies and improved use of blood products alongside other key outcomes highlighted in this workshop. There is growing evidence that treatment needs to be individualised and with the growing use of lifestyle monitoring devices such as smartwatches, this wealth of information could be utilised to determine transfusion need.

P1729: COMPARING THE PSYCHOSOCIAL IMPACTS OF THE COVID-19 PANDEMIC BETWEEN HAEMATOLOGICAL CANCER AND MYELOMA PATIENTS: FINDINGS FROM AN ONLINE SURVEY

S. J. Quinn 1,* , L. Anderson 2 , L. Lohfeld 1 , C. McShane 1

1 Centre for Public Health, Queen’s University Belfast, Belfast; 2 Aberdeen Centre for Health Data Science, University of Aberdeen, Aberdeen, United Kingdom

Background: Haematological cancers are risk factors for severe COVID-19, and multiple myeloma (MM) patients may have further risk due to older age and susceptibility to infections.

Aims: This study aims to understand the pandemic’s psychosocial impact and investigate whether MM patients experience extra stress compared to non-MM blood cancers.

Methods: The IMPaCCT survey is an international, longitudinal online survey, open to cancer patients. The first survey captured the period April-July 2020 and used quantitative and qualitative measures of quality of life (QoL), including validated tools (WHOQOL-BREF and EQ-5D-5L).

Results: Overall, 944 blood cancer patients responded (173 MM, 93 leukaemia, 117 lymphoma and 561 with a myeloproliferative neoplasm). Most were female, resided in the UK and did not live alone. Patients with MM were older (p<0.001) and less likely to be in employment (p<0.001), with more having received their cancer diagnoses in the last 5 years (p<0.001) than non-MM. Only three had a COVID diagnosis, with none having MM. Both groups reported high levels of concern about COVID, and this level was higher in MM; 50% of MM patients reported they were “extremely concerned”. In both groups, most said they would be less concerned about COVID if they didn’t have cancer.

While 70% reported their QoL as good/very good across both groups, QoL was lower in physical, psychological and social domains than healthy pre-pandemic UK norms. Patients reported more satisfaction with environmental factors, such as living arrangement and access to services, however.

There were high rates of anxiety/depression (67%, severe in 5%) and MM patients reported more issues with pain, mobility and engaging in their normal activities. Of the overall 84% who reported COVID had changed their lives, 63% found this hard to manage.

Patients’ most trusted sources for information about COVID-19 were healthcare providers and scientists for both groups (90% and 86% reported high levels of trust, respectively). Interestingly, MM patients had more trust in their friends and family for COVID information (58% with high trust in MM, vs. 41% in non-MM; p<0.001).

Regarding services, 43% had used telemedicine, with more MM patients using this service (61% of MM, 39% of non-MM). Most reported a positive experience. Health appointments were changed in format for most, and more often in MM patients (Changes: 69% of MM, 52% of non-MM) –with oncologist/haematologist visits most affected. In MM, treatments were changed, delayed or cancelled for 34%, which was significantly higher than non-MM (11%).

Summary/Conclusion: This study summarises the self-reported impacts of COVID on psychosocial health and care access during the early pandemic in vulnerable patient groups; MM patients are also highlighted as especially vulnerable. Understanding the needs of these patients will enable healthcare providers to properly support them.

P1730: IMPLEMENTATION OF PRO-CTCAE QUESTIONNAIRE IN PROM PROGRAM COLLECTS DIFFERENT ADVERSE EVENTS PROFILE THAN PHYSICIANS AND REDUCE UNSCHEDULED HEALTHCARE IN PATIENTS WITH LYMPHOMA RECEIVING IV THERAPY

S. RAMOS 1,* , E. CASTILLO 2 , N. BARRERAS 2 , M. RIESCO 3 , A. HERRERO 4 , A. PASCUAL 1 , A. LOPEZ 1 , M. A. PEREZ 1 , D. MORILLO 1 , E. ASKARI 1 , E. PRIETO 1 , P. LLAMAS 1 , J. BECARES 2 , C. BARRIOCANAL 5 , M. DEL OLMO 5 , J. SHORT 5 , J. ARCOS 5 , R. CORDOBA 1,5

1 HEMATOLOGY; 2 PHARMACY; 3 ONCOHEALTH INSTITUTE; 4 SYSTEMS AND ICTs, FUNDACION JIMENEZ DIAZ UNIVERSITY HOSPITAL; 5 CLINICAL AND ORGANIZATIONAL INNOVATION UNIT (UICO), QUIRONSALUD PUBLIC HOSPITALS NETWORK, MADRID, Spain

Background: PROMs have been included in cancer clinical trials, but it is yet to come in daily clinical practice. Different PROMs exist for assessing patients’ quality of life (QoL), physical functioning and symptom burden. Integrating PROMs in the healthcare of patients with cancer has the potential to improve their care delivery and outcomes.

Aims: We hypothesize that patients subscribed to a PROMs program may benefit from a better self-perception of health status and might require fewer hospital admissions and emergency room visits, which might also allow a reduction in the costs of treatment.

Methods: Patients (pts) with diagnosis of any type of lymphoma in the need of starting therapy in our hospital were included in our study between 1st Jan 2019 and 31st Dec 2020. Inclusion in “E-Res Salud”, the value-based healthcare program using PROMs was offered to patient starting intravenous treatment since 2020. Pts who started treatment in 2019 and those who refused to participate in 2020 were considered the control arm of our study. The principal endpoint was to compare physician and patient-reported adverse events through a standardized questionnaire such as PRO-CTCAE. Secondary endpoints included association between inclusion in PROMs program and reduction of hospital admissions and emergency room visits. Here, we present the preliminary results from answers given at the beginning of treatment.

Results: A total of 142 pts were included in our study; 76 of them (53.5%) reported outcome in the PROMs program. There were no differences in pts characteristics with regards of age or sex. Most frequent diagnoses were diffuse large B-cell, follicular and Hodgkin lymphoma. Adverse events (AEs) most frequently reported by physicians were hematological (76.1%), general (73.9%) and gastrointestinal symptoms (59.9%) and infections (43.7%). When patients were asked to report AEs of higher intensity the most frequently reported were general (31.6%), genitourinary (26.3%), gastrointestinal (23.7%), neurological (15.8%) and cutaneous (13.2%) symptoms (Figure 1). Patients included in the PROMs program reported fewer general (64.5% vs. 84.8%; p<0.05 ) and infectious (30.3% vs. 59.1%; p<0.05 ) AEs than those in the control arm. Moreover, inclusion in the PROMs program was associated with fewer number of visits to Emergencies, with 34.2% of pts versus 60% of pts not enrolled in the PROM program (p=0.003). They only group of symptoms which would itself increase the risk of visiting Emergencies was cutaneous AEs (p=0.027). It is important to note that those patients reporting symptoms of higher intensity, frequency or impact in their QoL AEs had more visits than those reporting AEs of lower grade (p<0.05). None of the different types of AEs were associated to an increase in the number of admissions or outpatient consultations. With such a short follow-up we have not found association between any type of AE and survival.

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Summary/Conclusion: Accurate assessment of patient-reported symptoms allows physicians to help cancer patients manage these issues and thereby, improve the survivorship experience and QoL. Our study establishes the association of PROMs with healthcare utilization among patients with different types of lymphoma. A longer follow-up and changes in PROMs during the treatment will be further analyzed.

P1731: IBRUTINIB PLUS RCHOP VERSUS RCHOP ONLY, IN YOUNG PATIENTS WITH ACTIVATED B CELL-LIKE DIFFUSE LARGE B-CELL LYMPHOMA (ABC-DLBCL): A COST EFFECTIVENESS ANALYSIS

H. Rho 1,* , I. J.-H. Jeong 2 , A. Prica 2

1 Medicine, University of Toronto; 2 Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada

Background: The standard chemoimmunotherapy for Diffuse Large B Cell Lymphoma (DLBCL) is RCHOP (Rituximab, Cyclophosphamide, doxorubicin Hydrochloride, vincristine/Oncovin, and Prednisone), improving progression free and overall survival. However, many patients require subsequent treatment after refractory/relapsed disease.

Molecular B-cell differentiation subtypes of DLBCL have prognostic implications, with the ABC subtype being associated with inferior prognosis. As the pathophysiology of ABC-DLBCL involves a chronically active B-cell antigen receptor pathway, it was hypothesised that higher-risk ABC-subtype population may benefit from a Bruton’s tyrosine kinase inhibitor, such as ibrutinib. Thus, the PHOENIX trial exploring the outcomes of adding ibrutinib to RCHOP (I-RCHOP) for patients with ABC-DLBCL was developed. Overall, it was found that I-RCHOP yielded favourable outcomes for patients younger than 60-years, as compared to older patients, whose event free survival did not improve and had worse outcomes due to toxicity.

Aims: As addition of ibrutinib may improve survival for younger patients, our study aimed to inform clinical decision-making using a cost-effectiveness decision analysis model, and to compare R-CHOP, with and without ibrutinib for patients with ABC-DLBCL.

Methods: A comprehensive Markov decision analysis model was designed (TreeAge Pro Healthcare) to compare I-RCHOP to RCHOP alone in a hypothetical cohort of patients younger than 60-years and newly diagnosed with ABC-DLBCL. The patients underwent either treatment pathway and survived, experienced adverse events (e.g., febrile neutropenia), relapse, or died. Available data on salvage treatment, autologous stem cell transplantation and novel therapies such as chimeric antigen receptor T-cell (CAR-T) for relapsed/refractory disease, were incorporated. The probabilities of transitioning between states were evaluated on a 3-month cycle for a lifetime horizon of 30 years. Costs were obtained from a Canadian public health payer’s perspective (CAN$1=US$0.78) and inflation-adjusted for 2022. Data on health state utilities were incorporated, and sensitivity analyses were performed for key variables. An annual discount rate of 1.5% to both quality-adjusted life-expectancies (QALEs) was utilised.

Model variability and uncertainty were assessed through probabilistic sensitivity analyses over 10,000 simulations.

Results: Probabilistic analyses showed that, for a willingness-to-pay (WTP) threshold of CAN$150,000/QALE, the addition of ibrutinib to RCHOP chemotherapy in younger patients with ABC-DLBCL was more cost-effective. I-RCHOP had greater QALE (7.9 years [SD: 0.04]) and the incremental cost-effectiveness ratio (ICER; cost gained per QALE) was CAN$16,478.84/QALE compared with RCHOP only strategy (QALE: 5.3 years [SD: 0.02]). Sensitivity analyses included key variables, including probability of adverse events, mortality associated with relapse and next-line managements, and showed that the model was robust.

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Summary/Conclusion: Our results informed us that I-RCHOP is more costly given the currently high market price for ibrutinib in Canada, however suggested that the preferred cost-effective treatment strategy for younger patients with ABC-DLBCL is RCHOP with ibrutinib. This approach was analysed to be dominant, when considering the high costs of salvage therapies such as stem cell transplantation and CART therapy, along with adverse events, overall survival, and higher quality of life. This decision-analytic model, though not absolute, provided relevant and robust results to help inform clinical decision making.

P1732: EVALUATION OF A DIGITAL SYSTEM LEVERAGING MOBILE TECHNOLOGY AND ARTIFICIAL INTELLIGENCE FOR DIGITALIZATION, REMOTE ANALYSIS AND SUPPORTED DIFFERENTIAL CELL COUNT OF BONE MARROW ASPIRATES.

J. Martínez López 1,2,* , S. Rueda Charro 3 , D. Bermejo Peláez 3 , A. Mousa Urbina 3 , R. Trelles Martínez 4 , M. García Roa 4 , A. Bobes Fernández 5 , M. Hidalgo Soto 6 , M. L. Morales Fernández 1 , A. Ortiz Ruiz 1 , A. Rodríguez García 1 , R. García Vicente 1 , A. Blanco Sánchez 7 , R. Ayala Diaz 1 , L. Lin 3,8 , E. Álamo García-Donas 3 , E. Dacal Picazo 3 , M. Luengo Oroz 3 , M. Linares Gómez 1,9

1 Department of Translational Hematology, Research Institute Hospital 12 de Octubre (i+12); 2 Hematological Malignancies Clinical Research Unit, H120-CNIO, CIBERONC; 3 Spotlab, Madrid; 4 Department of Hematology, Hospital Universitario Fundación Alcorcón, Alcorcón; 5 Department of Hematology, Hospital Clínico San Carlos, Madrid; 6 Vall Hebron Institute of Oncology (VHIO), Barcelona; 7 Department of Hematology, Hospital Universitario 12 de Octubre; 8 Biomedical Image Technologies, ETSI Telecomunicación, Universidad Politécnica de Madrid; 9 Department of Biochemistry and Molecular Biology, Pharmacy School, Universidad Complutense de Madrid, Madrid, Spain

Background: Microscopic examination of bone marrow aspirates (BMA) is still, in almost every organization world-wide, a task that is performed with conventional microscopes relying on an in-person diagnostic process. Being these samples left outside the digital world, it is not possible to leverage tools such as Artificial Intelligence (AI) algorithms to support diagnosis in hematological medical images. Concerning the BMA analysis, the differential cell count (DCC) remains non-automated, being a tedious and time consuming task that is frequently affected by considerable interobserver variability.

Aims: To support the work of hematologists in the analysis of BMA samples by implementing a simple digitization system to develop and validate an AI algorithm for the automatic recognition of different bone marrow cell lineages. This system aims at decreasing the interobserver variability associated with bone marrow DCC and improving the efficiency due to the increased speed of cell quantification.

Methods: 100 BMA samples from 95 patients were digitized by photographing 20 fields for each sample (1000x magnification) by hematology researchers. The digitization equipment consists of a 3D printed device to align the smartphone to the ocular lens of a conventional microscope; an acquisition app, and a web-based telemedicine platform to remotely visualize and analyze the images. Resulting images were used to train and validate an AI algorithm for the recognition of different bone marrow cell lineages.

Subsequently, a total of 32,229 cells were identified and each of them was remotely classified and tagged by a panel of 3 different hematologists (randomly selected from the 6 who participated in the study) according to 6 different lineages: Myeloid, Erythroid, Monocytic, Lymphoid, Blasts and Plasma cells.

The AI algorithm was trained only with cells in which all 3 experts agreed on the class (n=28,058). Validation of the algorithm was performed on an independent test set composed by 7,063 cells not included in the training set. Interobserver agreement was calculated for all possible pairs of experts and mean agreement was calculated for each lineage class. 95% confidence intervals (CI) were properly calculated.

A two-stage cell detection and lineage classification deep learning-based algorithm was developed to automatically identify and differentiate the aforementioned 6 lineage classes. Ethics approval for the study was obtained from the Clinical Research Ethics Committee.

Results: The detection algorithm achieved high overall accuracy for detecting nucleated cells in microscopy images (sensitivity and precision of 91.6% and 91.3% respectively). Identified cells were further classified by the algorithm into 6 different lineage classes with an overall accuracy of 92.6%.

Additionally, and for comparative purposes, interobserver agreement was also calculated. Average interobserver agreement was 88.1%. The Figure shows the 95% CI for AI performance and agreement between observers for each lineage class independently. As it can be shown, the AI algorithm demonstrated similar performance to that of a hematologist.

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Summary/Conclusion: It is possible to digitize BMA samples in a standardized way just relying on smartphones and a simple 3D printed device, transforming BMAs’ DCC in a digital, asynchronous and ubiquitous process. A reliable AI algorithm has been generated to perform an automatic bone marrow DCC with comparable performance to that of a human expert. This AI algorithm can be embedded in any smartphone and has the potential to run in any lab in the world, increasing efficiency and objectivity.

P1733: NON-HEMATOLOGICAL MALIGNANCIES IN IDIOPATHIC MULTICENTRIC CASTLEMAN DISEASE PATIENTS: A MATCHED COHORT ANALYSIS USING A HEALTH CLAIMS-BASED DATASET

S. Mukherjee 1,* , K. Kanhai 2 , D. Kauffman 3 , R. Martin 4 , J. Paige 3 , A. Ghosh 3 , H. Kannan 3 , F. Shupo 2 , D. Fajgenbaum 5

1 Department of Hematology and Medical Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, United States of America; 2 EUSA Pharma, Hemel Hempstead, United Kingdom; 3 EVERSANA, Milwaukee; 4 EUSA Pharma, Burlington; 5 Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States of America

Background: Idiopathic multicentric Castleman disease (iMCD) is a rare lymphoproliferative disorder characterized by a hyperinflammatory syndrome driven primarily by elevated cytokines, particularly interleukin-6 (IL-6). Numerous studies provide evidence that inflammation increases the risk of several cancers. Elevated inflammatory mediators such as cytokines and chemokines can alter the cellular micromilieu and promote tumorigenesis. A systematic literature review of iMCD reported a three-fold increased prevalence of malignancy in iMCD patients (19%) compared to age-matched controls (6%). However, emergence and prevalence of malignancies in iMCD warrants further investigation to inform evidence-based management guidelines.

Aims: The primary objective of this study was to evaluate the odds of developing non-hematological malignancies and cancer types in iMCD patients.

Methods: An iMCD cohort was identified using an administrative health claims dataset that enrolled 30.7 million US patients between January 1, 2017 and December 2, 2020. This updated analysis followed a similar methodology to our previously published work ( Mukherjee et al, Blood Advances 2022). iMCD patients were identified by CD-specific ICD-10 diagnosis code (D47.Z2), negative HHV-8 and HIV status, and diagnostic or laboratory claims for ≥2 minor criteria required to diagnose iMCD. We conducted a matched cohort analysis where odds of developing non-hematological malignancies (breast, carcinoma of unknown primary, colon, head and neck, lung, prostate, skin, and thyroid cancers) in iMCD patients, were compared to a non-iMCD cohort. iMCD and non-iMCD cohorts were matched (1:50) by age group (0-17, 18-44, 45-54, 55-64, >65 years), sex, insurance type, history in database, and region. Malignancies were also identified by ICD-10 diagnosis codes. We performed subgroup analyses by three age groups - 0-44, 45-60, and >60 years.

Results: We identified 271 individuals likely to have iMCD (mean age: 51.8 years; 59.4% female), including 191 patients previously reported (ASH, 2021). We found significantly higher odds of non-hematological malignancies (OR= 2.8, 95% CI= 2.0, 3.9) in iMCD patients (18%) compared to matched non-iMCD patients (7%). Among non-hematological malignancies, there was 36 times higher likelihood of carcinoma of unknown primary (OR= 36.0, 95% CI= 17.3, 75.1), 10 times higher likelihood of lung cancer (OR= 9.8, 95% CI= 5.4, 17.9), 6 times higher likelihood of thyroid cancer (OR= 6.1, 95% CI= 2.7, 13.6), 6 times higher likelihood of head and neck cancer (OR= 6.0, 95% CI= 1.4, 26.3), and 3 times higher likelihood of colon cancer (OR 3.2, 95%CI= 1.4,7.3) in iMCD patients compared to non-iMCD matched patients. Odds of other solid tumors such as breast, skin, and prostate were not significantly elevated in iMCD patients. When analyzed by age groups, we observed a significantly higher likelihood of developing non-hematological malignancies in iMCD patients compared to non-iMCD matched patients across all three age cohorts.

Summary/Conclusion: We report significantly higher odds of having non-hematological malignancies in iMCD patients when compared to the non-iMCD cohort. As iMCD is a challenging diagnosis and claims data can include incorrect codes, it is possible that some patients in the iMCD group could have been incorrectly diagnosed or a malignancy incorrectly coded. Further studies are needed to investigate the biological mechanisms of these observed associations, potential management implications of these findings, and whether IL-6 directed therapies can mitigate these outcomes.

P1734: LESSONS FROM HIV- HAEMOPHILIA CRISIS AND CURRICULAR LEARNING IN POST COVID TIMES

M. Sekhar 1,2,* , C. Merrion 3 , S. Jadhav 4

1 Haematology, Royal Free London NHS Trust; 2 Haematology, University College London Hospital; 3 MBBS programme, Royal Free campus, University College London Medical School; 4 Division of Psychiatry, Faculty of Brain Sciences, University College London, London, United Kingdom

Background: The COVID19 pandemic has posed challenges to clinical decision-making in the context of prolonged uncertainties in clinical, scientific and social areas. The HIV pandemic of 1980s represents a similar period with data and insights acquired over years. Haematologists are familiar with the notion of uncertainty. In particular, haemophilia care during the early 1980s posed specific and unique problems in clinical decision-making. An understanding of professional and personal concerns faced by doctors at the time can contribute to learning and continued professional development.

Aims: Compile, structure and analyse audio interviews from physicians involved in haemophilia care in the 80s to generate learning material for curricular learning.

Methods: Between June and November 2019 oral history interviews were conducted using a semi-structured questionnaire with 16 senior haemophilia doctors (13 male, 3 female) from Australia, Canada, France, Italy, Netherlands. 28 person-hours of interview material was analysed using pre-established criteria 1 . Audio recordings and transcripts from UK Infected Blood Inquiry 2 held between October 2020 and January 2021 were analysed for content. 18 oral evidences comprising approximately 286 person-hours were examined to extract themes addressing difficulties and dilemmas in clinical decision-making. Following ethical approval, an audio-podcast series 3 was produced to address specific domains and themes.

Results: Audio material was compiled into segments of audio clips that identified topics across four areas of practice: diagnostic tests, clinical decision making, shared axes of care and personal experiences of adverse outcomes. Material from 6 oral history interviews (Italy, France, Netherlands) was compiled to illustrate cross-national themes and personal dilemmas. These themes were mapped on relevant curricular domains defined by the GMC. (Table 1) The compilation was interspersed with an audio commentary by the first author and published as a podcast series comprising 8 podcasts over approximately 4 hours. Access was via free public platforms. Between November 2021 and February 2022, 472 downloads were documented on one of four platforms. Six medical students and six faculty provided triangulation including feedback. Their feedback confirmed the pedagogic relevance of these podcasts for undergraduate students across years 1 to 5 as part of curricular learning. Feedback from postgraduate trainees in Haematology, other specialities and consultants confirmed the relevance of this material in their continued professional development.

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Summary/Conclusion: •Our study generates data that contribute to future research on physicians exposed to prolonged uncertainties in clinical and laboratory practice.

•Lessons from haemophilia-AIDS crisis are relevant for some aspects of COVID19pandemic.

•Recent history of medical events provides a valuable resource to enhance teaching.

•Oral history interviews and oral evidence submitted at an inquiry differ in nature but provide complimentary insights.

•Podcasts can provide an effective platform for curricular and self-directed learning for graduate and postgraduate learning, worldwide.

•A structured platform to catalogue difficult decisions can provide haematologists and others a frame of reference in making difficult decisions. Future research could address the scope and applicability of this pragmatic approach to history of medicine.

Ref: 10.1111/hae1.3967

https://www.infectedbloodinquiry.org.uk/

https://thebitterpillpodcasts.libsyn.com

P1735: IMPROVEMENTS IN PATIENT-REPORTED OUTCOMES IN MITAPIVAT-TREATED PATIENTS WITH PYRUVATE KINASE DEFICIENCY: A DESCRIPTIVE ANALYSIS FROM THE PHASE 3 ACTIVATE TRIAL

K. H. Kuo 1,* , R. F. Grace 2 , E. J. van Beers 3 , W. Barcellini 4 , A. Glenthøj 5 , S. Holzhauer 6 , P. Patel 7 , V. Beynon 7 , J. Li 7 , E. Zagadailov 7 , H. Al-Samkari 8

1 Division of Hematology, University of Toronto, Toronto, Canada; 2 Dana-Farber/Boston Children’s Cancer and Blood Disorder Center, Harvard Medical School, Boston, United States of America; 3 Van Creveldkliniek, Department of Internal Medicine, University Medical Center Utrecht, Netherlands; 4 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 5 Department of Hematology, Rigshospitalet, Copenhagen, Denmark; 6 Department of Pediatric Hematology and Oncology, Charité University Medicine, Berlin, Germany; 7 Agios Pharmaceuticals, Inc., Cambridge; 8 Division of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, United States of America

Background: Pyruvate kinase (PK) deficiency is a rare, inherited, non-spherocytic hemolytic anemia associated with acute and long-term complications, as well as a spectrum of signs and symptoms including jaundice, fatigue, and dyspnea. PK deficiency has a profound wide-ranging impact on health-related quality of life (HRQoL). Mitapivat (AG-348), a first-in-class, oral, allosteric activator of PK, was shown to improve hemoglobin (Hb), hemolysis, and hematopoiesis in a global, phase 3, randomized, placebo (PBO)-controlled trial evaluating mitapivat efficacy and safety in adults with PK deficiency who were not regularly transfused (ACTIVATE; NCT03548220 ). Furthermore, significant improvements in patient-reported outcomes (PROs) (measured by disease-specific PRO instruments: the PK deficiency diary [PKDD] and the PK deficiency impact assessment [PKDIA]) were demonstrated in patients (pts) receiving mitapivat compared with PBO. The PKDD is a self-administered, 7-item daily diary to assess the signs and symptoms of PK deficiency (including jaundice, tiredness, and shortness of breath), while the PKDIA is a 12-item weekly measure assessing the impacts of PK deficiency (pts rated the frequency of occurrence or difficulty of various activities).

Aims: To describe PKDD and PKDIA outcomes for the subset of pts in the ACTIVATE trial who achieved the primary endpoint of Hb response, defined as a ≥1.5 g/dL increase in Hb from baseline (BL), sustained at ≥2 scheduled assessments at weeks (wks) 16, 20, and 24.

Methods: Eighty pts were randomized 1:1 to receive mitapivat (5/20/50 mg twice daily) or PBO for 24 wks in the ACTIVATE trial. Change from BL to wk 24 in PKDD and PKDIA scores were prespecified secondary endpoints. For this post hoc analysis, changes from BL in PKDD weekly mean scores and PKDIA scores measured at scheduled visits (wks 4, 8, 12, 16, 20, and 24) were summarized for Hb responders, the overall mitapivat treatment arm, and PBO, using mean and 95% confidence intervals (CIs). For both the PKDD and the PKDIA, a lower score represents a lower disease burden.

Results: In the ACTIVATE trial, 16/40 (40%) pts receiving mitapivat met the primary endpoint of Hb response compared with none for PBO (0/40; 0%). At wk 24, mean (95% CI) change from BL in PKDD weekly mean score was ‒7.12 (‒10.98, ‒3.27) for pts who achieved Hb response, ‒5.43 (‒7.47, ‒3.40) for the overall mitapivat treatment arm, and ‒1.86 (–4.04, 0.32) for the PBO arm (Figure 1a). At wk 24, mean (95% CI) change from BL in PKDIA score was ‒8.07 (‒11.05, ‒5.08) for pts who achieved Hb response, ‒4.82 (‒7.18, ‒2.46) for the overall mitapivat treatment arm, and ‒1.06 (‒3.70, 1.59) for the PBO arm (Figure 1b). Across both PRO instruments, improvements among mitapivat pts who achieved Hb response were sustained over time.

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Summary/Conclusion: In the ACTIVATE trial, mitapivat-treated pts demonstrated significant improvements in signs, symptoms, and impacts based on PK deficiency-specific PRO instruments, compared with PBO. This analysis further reveals that improvements were greater in the subset of mitapivat-treated pts who achieved the primary endpoint. Together, these data indicate that mitapivat has the potential to improve HRQoL in pts with PK deficiency who are not regularly transfused.

P1736: CHARACTERISTICS, HEALTHCARE UTILIZATION, AND COSTS ASSOCIATED WITH INOTUZUMAB OZOGAMICIN, BLINATUMOMAB, OR OTHER AGENTS FOR THE TREATMENT OF RELAPSED/REFRACTORY ACUTE LYMPHOBLASTIC LEUKEMIA

A. Russell-Smith 1,* , R. Shah 2 , A. Silverstein 3 , A. Petrilla 3 , B. Leinwand 3 , D. Norris 3 , A. Schroeder 4

1 Pfizer R&D UK Ltd, Sandwich, United Kingdom; 2 Pfizer Inc., New York; 3 Inovalon; 4 Avalere Health, Washington, United States of America

Background: Adults with relapsed/refractory (R/R) B-cell precursor ALL treated with inotuzumab ozogamicin (ino, anti-CD22 monoclonal antibody [mAb]) and blinatumomab (blin, CD19-directed CD3 T-cell engager mAb) showed improved clinical outcomes over chemotherapy in the INO-VATE and TOWER trials, respectively. Although both novel agents are in use in the United States (US), data on healthcare resource utilization (HCRU) and costs in the real-world among patients receiving these treatments is limited.

Aims: This study identified such data, alongside patient characteristics, for Medicare beneficiaries with R/R ALL initiating ino, blin, or other agents (chemotherapeutic, tyrosine kinase inhibitor [TKI], or mAb).

Methods: A retrospective study was conducted utilizing Medicare enrollment and claims data. Patients were eligible for the study if they were diagnosed with R/R ALL between January 1, 2017 and June 30, 2019 and initiated ino, blin, or other agents at/after diagnosis (first observed treatment = index date). Claims for blin were identifiable in the inpatient and outpatient settings. Claims for ino/other agents were only visible in outpatient and pharmacy settings so patients were identified from there; the proportion of these who had received inpatient administration prior to outpatient was unknown. The B- and T-cell status was not captured. Patients receiving T-ALL therapy (nelarabine) were excluded. Data analyzed included characteristics and HCRU during 6 months prior to treatment initiation (pre-index) and HCRU and costs per patient per month (PPPM; costs in 2021 USD) during the variable follow-up period (≥3 months until death, disenrollment, or study end).

Results: 2,306 initiated an ALL treatment in the study period. This accounted for less than 15% of Medicare beneficiaries with R/R ALL (15,586). 1,329 patients qualified for inclusion in the study: 55 (4%) received ino, 209 (16%) blin (73% initiated blin during hospital stay), and 1,065 (80%) other agents. The mean age was 62 among the ino and blin cohorts and 66 for the other agents group; a larger proportion of the ino cohort qualified for Medicare with non-age-related factors (disability) than those initiating blin or other agents. Treatment groups were comparable by sex and race. The ino cohort had higher prevalence of congestive heart, pulmonary, and renal diseases (35%, 31%, 38%) than blin (19%, 25%, 19%) and other agents (23%, 30%, 21%), respectively; diabetes prevalence was lower for ino than for blin and other agents (20%, 34%, 36%, respectively).

There was variability in exposure to chemotherapies over the 6-month pre-index period: 76% of both the ino and blin cohorts, and 66% of the other agents cohort, were hospitalized for any reason. Duration of follow-up varied by cohort: 8.0 months for ino, 12.8 months for blin, and months for 17.8 other agents, during which blin patients had higher PPPM rates of all-cause and disease-related hospital stays, longer stays (9.3 days blin, 6.0 ino, 3.9 other agents), and higher mean (SD) total cost of care: $65,437 ($155,664) blin, $55,995 ($36,688) ino, and $17,520 ($22,720) other agents.

Summary/Conclusion: Around 20% of patients who received an active treatment for their R/R ALL received a novel CD-targeted therapy. There were differences between treatment cohorts with respect to comorbidities. Mean costs PPPM during the follow-up period from the date of identified treatment administration were highest for patients receiving blin; costs for patients receiving ino were 14% lower than blin, whilst other agents were 73% lower than blin.

P1737: QUALITY OF LIFE IN CHILDREN WITH BETA-THALASSEMIA MAJOR: A CROSS-SECTIONAL STUDY IN CHINA

Z. Song 1,* , X. Liu 1 , X. Wu 2 , L. Qiu 3 , G. Zhang 4 , J. Chen 1 , D. Wei 4 , T. Cheng 1

1 Center for Information and Resources, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin; 2 Department of Paediatrics, Nanfang Hospital,Southern Medical University, Guangzhou; 3 Beijing AngelMom Charity Foundation; 4 EdiGene Inc., Beijing, China

Background: Thalassemia is a hereditary hemolytic anemia disease. Most Chinese thalassemia patients can be diagnosed in initial episode, due to the fact that the prevention and treatment of thalassemia has achieved remarkable effects in recent two decades. The research of new therapies have also made considerable progress. Patients with transfusion-dependent thalassemia, especially those with β -Thalassemia Major( β -TM), require regular blood transfusions and iron chelation therapy. This places a large burden on patients and their families. The burden is not only physical pain, but also includes psychological and social dimensions. The unmet needs of the patient population are also worth studying. However, the overall QoL of Chinese thalassemia patients and its affecting factors remain unclear.

Aims: The aim of this study was to investigate the level of QoL and ascertain its influencing factors in children with β -TM in China.

Methods: The study data came from a nationwide and cross-sectional survey of patients with thalassemia in China (NCSTC, Ethical ID: IIT2021045-EC-1). Participants were recruited from 9 provinces and 1 municipality. Recruitment was based on the Beijing AngelMom Charity Foundation. Multistage stratified cluster sampling method was used for the sampling. The study used a web-based questionnaire to collect the QoL of children and the burden level of caregivers. The QoL was assessed using the Pediatric Quality of Life Inventory (PedsQL4.0), which contained 23 items in 4 dimensions (physical, emotional, social, and school functioning). The caregiver’s burden level was assessed by Zarit Burden Interview-22 (ZBI-22). The multiple linear regressions were used to analyze.

Results: A total of 519 children (aged 2-17 years) with β -TM were obtained from NCSTC. The mean of PedsQL total score was 65.7 (standard deviation, 18.1). In the multi-adjusted linear regression model, participants with younger age (2-4 years= 72.1; 5-7 years= 67.8; 8-12 years = 64.3; 13-17 years= 60.5), without complications (with complications vs. no complications: 61.6 vs. 75.4), shorter course of disease (< 8 years vs. s 8 years: 67.6 vs. 63.8), without iron chemotherapy (with iron chemotherapy vs. without chemotherapy: 64.9 vs. 68.8), with lower caregiver burden level (0-20: 79.3, 21-40: 68.5, 41-60: 57.7, 61-80: 46.1), and receive transplantation treatment (with HLA transplantation vs. without HLA transplantation: 71.9 vs. 64.0) have higher PedsQL total score. The younger age, without complications, with shorter course of disease, with lower caregiver burden level, and receive transplantation treatment were all related to higher performance of physical, emotional, social, and school functioning (Figure 1).

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Summary/Conclusion: The health-related QoL assessed by PedsQL scores in Chinese children with β -TM were lower than that in previous studies. Multiple factors were associated with the QoL of Chinese children with β -TM, and should be comprehensively paying attention to physical, emotional, social, and school functioning in β -TM patients.

P1738: A SEER-MEDICARE ANALYSIS OF THE ECONOMIC BURDEN AMONG ELDERLY PATIENTS WITH ACUTE MYELOID LEUKEMIA TREATED WITH HYPOMETHYLATING AGENTS

A. McBride 1,* , D. Huggar 1 , R. Copher 1 , Z.-Y. Zhou 2 , M. L. Zichlin 2 , A. Anderson 2 , N. Downes 2 , A. M. Brunner 3

1 Bristol Myers Squibb, Princeton; 2 Analysis Group; 3 Massachusetts General Hospital, Boston, United States of America

Background: The economic burden of relapse among Medicare patients with newly diagnosed acute myeloid leukemia (AML) is high, with >50% receiving ≥1 cycle of a hypomethylating agent (HMA) (Tabah A, et al. Blood 2020:136(suppl1):45).

Aims: To assess healthcare resource utilization (HCRU) and costs associated with AML phases among elderly patients who received only HMA monotherapy during induction, and then achieved remission.

Methods: A retrospective analysis of Medicare claims, parts A, B, and D (2007–2016), and cancer diagnoses (2007–2015) in the Surveillance, Epidemiology, and End Results-Medicare database was conducted. Eligible patients had an AML diagnosis, were ≥65 years of age at diagnosis date, had initiated only HMA in the outpatient (OP) setting during the first induction cycle post-diagnosis, and had evidence of remission post-initiation of induction therapy. Patients with another blood malignancy, who had received hematopoietic stem cell transplantation, or were enrolled in a clinical trial were excluded.

The induction therapy period was from first HMA initiation post-diagnosis (index date) to end of the cycle during which remission occurred. The baseline period was the 6-month period prior to index date. The post-remission phase ended at the earliest relapse or end of follow-up (ie, death, end of eligibility or of available data [Dec 31, 2016]). The post-relapse phase was from the date of first AML relapse after remission to the end of follow-up.

Baseline patient characteristics were summarized descriptively. HCRU and costs (adjusted to 2019 US dollars) associated with induction and post-remission therapy were assessed during a treatment cycle. Average per-patient monthly (PPM) HCRU and costs were reported for the induction, post-remission, and post-relapse phases.

Results: A total of 71 patients (azacitidine: n=31; decitabine: n=40) were included. Median age at AML diagnosis was 78.8 years, 50.7% of patients were male, and 85.9% were White. The mean ± standard deviation (median) time from index date to the end of follow-up was 16.0 ± 12.3 (14.0) months. Mean number of total treatment days was 181.3 and 153.0 for the induction and post-remission phases, respectively. A total of 63.4% of patients (n=45) received post-remission therapy. Among all patients, 43.7% relapsed and 85.9% had died by the end of follow-up. During the induction, post-remission, and post-relapse phases, 91.5%, 77.8%, and 77.4% of patients received a blood transfusion, respectively. OP visits were the most common visits across all phases, with 95.6% of patients having ≥1 visit during post-remission therapy and 83.9% during the post-relapse phase. The proportion of patients with inpatient (IP) visits was highest in the post-relapse phase, with 77.4% having ≥1 visit. Mean PPM healthcare costs were highest for the post-relapse phase, followed by post-remission therapy, and induction (Table). Costs in the OP setting were the greatest contributor to induction costs (48.9%), while costs in the IP setting drove costs in the post-remission therapy (56.2%) and post-relapse (72.8%) phases.

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Summary/Conclusion: The economic burden of AML treated with HMA was highest in the post-relapse phase, approximately 1.7 and 1.6 times the PPM costs during the induction and post-remission therapy phases, respectively. In addition, IP costs made up nearly two-thirds of total PPM costs in the post-relapse phase, up from approximately 44% and 56% of the induction and post-remission therapy phases, respectively. Treatment options extending the post-remission phase would reduce the high economic burden associated with AML relapse.

P1739: HEALTH-RELATED QUALITY OF LIFE IN PATIENTS WITH TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA TREATED WITH IBERDOMIDE PLUS DEXAMETHASONE IN A PHASE 1B/2A OPEN-LABEL DOSE ESCALATION STUDY

N. W. van de Donk 1,* , S. Guo 2 , L. Shi 2 , J. Lord-Bessen 3 , D. Tang 3 , L. Karlin 4 , K. Weisel 5 , K. Ramasamy 6 , P. C. Abad 3 , A. Amin 3 , S. Amin 3 , S. Lonial 7

1 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Department of Hematology, Cancer Center Amsterdam, Amsterdam, Netherlands; 2 Evidera, Waltham, MA; 3 Bristol Myers Squibb, Princeton, NJ, United States of America; 4 Centre Hospitalier Lyon Sud/Hospices Civils de Lyon, Pierre-Benite, France; 5 University Medical Center of Hamburg-Eppendorf, Hamburg, Germany; 6 Department of Clinical Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; 7 Winship Cancer Institute, Emory University, Atlanta, GA, United States of America

Background: Patients with relapsed/refractory multiple myeloma (RRMM) who are triple-class exposed (TCE) have limited treatment options and experience poor health-related quality of life (HRQoL). Iberdomide (IBER), a novel cereblon E3 ligase modulator (CELMoD ® ) compound, has shown an overall response rate of 26% in this population when administered with dexamethasone (DEX) in the open-label, single-arm, phase 1b/2a dose-escalation CC-220-MM-001 clinical trial ( NCT02773030 ). Here, we assess the exploratory endpoint of HRQoL for patients enrolled in this clinical trial.

Aims: To assess HRQoL in patients with RRMM who received IBER + DEX in cohorts D and I of the CC-220-MM-001 trial.

Methods: In this pooled analysis of cohorts D and I, cohort D patients had RRMM; had received ≥3 prior lines of therapy, including lenalidomide, pomalidomide, a proteasome inhibitor, a glucocorticoid, and an anti-CD38 monoclonal antibody; had experienced disease progression within 60 days of last myeloma therapy; and were triple-class refractory. Patients who had received prior anti-B-cell maturation antigen (BCMA) therapy were excluded from cohort D but included in cohort I. In both cohorts, IBER (1.6 mg) was given orally on days 1–21, in combination with DEX (40 mg; 20 mg if >75 years) on day 1, 8, 15, and 22 of each 28-day cycle. The European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire Core 30 (QLQ-C30), EORTC QLQ-Multiple Myeloma Module (QLQ-MY20), and EuroQoL 5 dimensions 5 levels (EQ-5D-5L) were administered before treatment, on day 1 of each treatment cycle, and at end of treatment. The HRQoL-evaluable population included all patients who received IBER + DEX and who had evaluable assessments of a given HRQoL measure at baseline (cycle 1 day 1) and at ≥1 post-baseline time point. Minimally important differences were prespecified for each domain to interpret whether a mean within-group change from baseline was meaningful. Overall, least square mean HRQoL score changes from baseline were estimated from a mixed-effects model for repeated measures. Proportions of patients who experienced clinically meaningful HRQoL deterioration and improvement were calculated using prespecified responder definitions.

Results: A total of 133 patients received IBER + DEX; 99 patients were included in the QLQ-C30-evaluable population (TCE n=79, TCE + anti-BCMA n=20). Median (range) age was 63 (44–82) years, 57% were male, and 95% had ≥4 prior MM regimens (median 6 prior MM regimens). Completion rates for most time points were ≥70%. Mean HRQoL scores were generally stable over time during treatment in all the HRQoL domains, including fatigue, pain, disease symptoms, and side effects. However, the EORTC QLQ-MY20 body image showed clinically meaningful improvement from baseline (Table). At individual level, most patients (≥60%) experienced either clinically meaningful improvement or no change while on treatment in all domains.

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Summary/Conclusion: HRQoL scores among IBER + DEX patients in cohorts D and I were generally stable in all domains across 3 HRQoL measures. Most patients experienced either clinically meaningful improvement or no change in each domain. This suggests the IBER + DEX treatment maintained HRQoL in heavily pretreated patients with TCE RRMM, while demonstrating a promising clinical response.

P1740: HEALTH-RELATED QUALITY OF LIFE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM) TREATED WITH IDECABTAGENE VICLEUCEL VS BELANTAMAB MAFODOTIN: A MATCHING-ADJUSTED INDIRECT COMPARISON STUDY

N. Shah 1,* , D. Ayers 2 , S. Cope 2 , K. Karampampa 3 , K. Towle 2 , J. Braverman 3 , A. Mojebi 2 , C. Caisip 2 , D. Dhanda 3

1 University of California San Francisco, San Francisco, CA, United States of America; 2 PRECISIONheor, Vancouver, BC, Canada; 3 Bristol Myers Squibb, Princeton, NJ, United States of America

Background: Patients (pts) with RRMM who are triple-class exposed (TCE; to an immunomodulatory imide drug [IMiD ® agent], proteasome inhibitor [PI], and anti-CD38 monoclonal antibody [mAb]) have poor clinical outcomes and poor health-related quality of life (HRQoL). The B-cell maturation antigen (BCMA)-directed CAR T cell therapy idecabtagene vicleucel (ide-cel, bb2121) received regulatory approval in the US for pts with RRMM with ≥4 prior lines of therapy (LOT), including an IMiD agent, PI, and anti-CD38 mAb, and in Europe and Japan for pts with RRMM with ≥3 prior LOTs including an IMiD agent, PI, and anti-CD38 mAb. Ide-cel showed frequent, deep, and durable responses in TCE pts with RRMM in KarMMa, a phase 2, single-arm trial ( NCT03361748 ). Clinically meaningful improvements in HRQoL outcomes vs baseline were observed. The BCMA-targeted antibody-drug conjugate belantamab mafodotin (BM) was approved in the US and Europe for pts with RRMM with ≥4 prior LOTs including an IMiD agent, PI and anti-CD38 mAb, based on data from the phase 2, single-arm DREAMM-2 trial ( NCT03525678 ). There is a paucity of evidence comparing HRQoL for different treatment options approved in RRMM.

Aims: To compare HRQoL outcomes for TCE pts with RRMM treated with ide-cel vs BM.

Methods: Data were analyzed using unanchored matching-adjusted indirect comparisons. Between-study differences in pt characteristics were adjusted using individual-level pt data from KarMMa to align with summary-level pt characteristics from DREAMM-2. The ide-cel-treated population (N=128; median follow-up 15.4 months) from KarMMa (of whom, 54 pts received the target dose of 450×10 6 CAR+ T cells) and the intention-to-treat population (N=97; median follow-up 13.0 months) from DREAMM-2 who received the 2.5 mg/kg dose of BM (Popat et al. HemaSphere 2020;4:S1. EP1746) were analyzed. Predefined outcomes of interest were differences in mean change from baseline scores for the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire C30 (QLQ-C30) pain, fatigue, and global health/QoL domains, and the QLQ-Multiple Myeloma (MY20) pain domain. Analyses were performed at 2, 3, 4, 6, 9, and 12 months of follow-up. Given differences in time points of evaluation between KarMMa and DREAMM-2, the nearest neighbor approach was used. Pt characteristics included as covariates in the propensity model included: age, sex, race, years from initial diagnosis, disease stage, and number of prior treatments. Sensitivity analyses included additional covariates: high-risk cytogenetics and extramedullary disease.

Results: Ide-cel was associated with improved HRQoL vs BM at all evaluated time points for the QLQ-C30 global health status/QoL domain (Table). Improved HRQoL scores were statistically significant for ide-cel vs BM at 6 and 9 months for the QLQ-C30 fatigue domain (HRQoL scores were numerically better for ide-cel vs BM at all other time points). Similarly, HRQoL scores favored ide-cel vs BM at all evaluated time points for the QLQ-C30 pain and QLQ-MY20 pain domains, with statistically significant findings at 4, 6, and 9 months for QLQ-C30 and 3, 4, and 6 months for QLQ-MY20. Sensitivity analyses were consistent with the base case analyses.

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Summary/Conclusion: While this analysis was limited by the availability of aggregate data from DREAMM-2, which may influence the outcomes of interest, results suggest that one-time treatment with ide-cel offers improved HRQoL vs BM across the QLQ-C30 pain, fatigue, and global health status/QoL domains, as well as the QLQ-MY20 pain domain.

P1741: EXAMINING THE BURDEN OF ILLNESS ASSOCIATED WITH TRANSFUSION-DEPENDENT ΒETA-THALASSEMIA FROM THE PATIENT’S PERSPECTIVE

R. L. Knoth 1,* , S. Gupta 2 , K. Perkowski 2 , H. Costantino 2 , B. Inyart 2 , L. Ashka 2 , K. Clapp 2 , A. McBride 1

1 Bristol Myers Squibb, Princeton, NJ; 2 Cerner Enviza, Malvern, PA, United States of America

Background: β-thalassemia is a rare genetic blood disorder affecting approximately 1 in 100,000 people globally. Typically diagnosed in childhood, β-thalassemia is marked by the inability to produce hemoglobin, which results in chronic anemia. Transfusion-dependent β-thalassemia (TDβT) is the most severe form of this disorder, with treatment requiring frequent red blood cell transfusions (RBCTs). Recognized complications of the frequent RBCTs include alloimmunization, iron overload, resultant cardiac and liver disease, and reported poor health-related quality of life (HRQoL).

Aims: To examine the burden of illness and HRQoL in patients diagnosed with TDβT.

Methods: Potential study participants were recruited with the assistance of the Cooley’s Anemia Foundation. Participants were ≥18 years of age, had a self-reported physician diagnosis of β-thalassemia, and had received ≥1 RBCT in the past 6 months. After providing informed consent, patients completed an institutional review board-approved 30-minute online survey that included both validated patient-reported outcome instruments, and other questions written expressly for the purpose of this study. Data were collected in May 2021. Patients who completed the survey earned an incentive payment of 75 US dollars.

Results: A total of 100 patients completed the survey; mean age was 36.0 years (standard deviation [SD] 10.4) and 35% of patients were male. Patients had been diagnosed with TDβT for a mean of 34.4 years (SD 10.3) and experienced a mean of 9.6 (SD 4.3) RBCT events in the past 6 months. As an indication of severity, 66% of patients reported requiring >1 RBCT per month, 31% each month, and 3% every other month. Side effects of RBCT included iron overload (in 81% of patients), rashes/hives (74%), fatigue (61%), and pain/bruising at injection site (52%). Mean time required for an RBCT was 7.4 hours (SD 11.3), including transfusion preparation time. Travel and wait times were an additional 7.1 hours. Among all patients, 70% reported a “moderate” to “extremely high” burden of RBCT. On HRQoL, 71% of patients reported “wondering what life would be like without β-thalassemia,” 86% reported “feeling fatigued” leading up to RBCT, and 42% reported “not being as social with friends/family due to the time needed for RBCT.” On validated psychological instruments, 52% of patients reported mild-to-severe depression (Patient Health Questionnaire [PHQ-9]) and 53% reported mild-to-severe anxiety (Generalized Anxiety Disorder Questionnaire [GAD-7]), over the previous 2 weeks.

Summary/Conclusion: The survey results suggest there is a substantial time, side-effect, and HRQoL burden associated with treatment by RBCTs in patients diagnosed with TDβT. This includes considerable time devoted to preparing for and receiving transfusions, transfusion-related side effects, and increases in depression and anxiety. These findings contribute to the understanding of the humanistic and psychological burden of RBCT dependency in patients with β-thalassemia. New treatment options that can improve outcomes in this population are needed.

P1742: SYSTEMATIC LITERATURE REVIEW OF THE BURDEN OF ILLNESS AND OUTCOME ANALYSES OF PATIENTS WITH NON-TRANSFUSION DEPENDENT BETA-THALASSEMIA

Y. Aydinok 1,* , A. Yucel 2 , S. Deshpande 3 , S. Purushotham 3 , M. Dixit 3 , R. Raorane 3 , O. Agbeleye 3 , M. Glassberg 4

1 Faculty of Medicine, Ege University, Izmir, Turkey; 2 Bristol Myers Squibb, Princeton, NJ, United States of America; 3 Evidera, London, United Kingdom; 4 Bristol Myers Squibb, Summit, NJ, United States of America

Background: Beta-thalassemia is a hereditary blood disorder characterized by impaired red blood cell (RBC) production resulting in chronic anemia, which may lead to serious complications if untreated. Patients with non-transfusion dependent beta-thalassemia (NTDT) do not require regular, lifelong RBC transfusions for survival but may need occasional transfusions. Although NTDT implicates a less severe form of beta-thalassemia, morbidities in NTDT cause a substantial disease burden that negatively impacts health-related quality of life (HRQoL).

Aims: To understand the clinical, epidemiologic, economic, and humanistic burden of illness in adult patients with NTDT.

Methods: A systematic literature review (SLR) was conducted in June 2021 to identify articles on patients with NTDT, defined by beta-thalassemia intermedia status and frequency of RBC transfusions received. Search and screening followed the methodology of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Cochrane.

Results: In total, 3294 records were reviewed, with 308 publications identified for full-text review. Of those, 39 publications met the eligibility criteria and were included in the final analyses (Figure). In the 25 studies that reported clinical outcomes, patients with NTDT experienced elevated serum ferritin (SF) levels and increased liver iron concentration (LIC) requiring iron chelation therapy. Baseline mean SF level in patients with NTDT ranged from 518 to 4314.06 ng/mL; baseline mean LIC ranged from 2.1 to 31.8 Fe/g. Twenty-four of 25 epidemiologic studies reported morbidity outcomes. Patients with NTDT reported high morbidity burden such as diabetes (prevalence across studies range, 1.4–44.0%), heart failure (2.1–6.7%), pulmonary hypertension (10.0–33%), hypothyroidism (1.4–83.3%), hypogonadism (1.4–83.3%), cholelithiasis (13.8–100.0%), osteoporosis (13.3–83.3%), osteopenia (11.0–83.3%), extramedullary hematopoiesis (0.6–40.0%), hepatosplenomegaly (20.0–37.7%), hepatitis C virus infection (0.3–55.0%), thrombosis (6.7–26.7%), and liver complications (7.5–32.1%). In the 5 studies that reported HRQoL, patients with NTDT had significantly worse HRQoL outcomes than patients with transfusion-dependent beta-thalassemia (TDT), as shown by mean Short Form-36 (SF-36) total scores (66.5 [standard deviation (SD) 16.1] NTDT vs 75.8 [SD 18.8] TDT; P =0.021). Mean data from the various scales of SF-36 indicated significantly worse outcomes in physical health (66.2 [SD 16.8] NTDT vs 77.1 [SD 18.1] TDT; P =0.008), mental health (47.4 [SD 8.4] NTDT vs 51.6 [SD 7.2] TDT; P =0.009), and general health domains (41.9 [SD 8.2] NTDT vs 46.3 [SD 10.0] TDT; P =0.018). A higher burden of anemia was associated with lower mean health state utility values in the NTDT population (low burden of anemia 0.79 [SD 0.34] vs high burden of anemia 0.63 [SD 0.4]).

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Summary/Conclusion: This SLR demonstrated that patients with NTDT experience poor clinical and HRQoL outcomes and have significantly worse HRQoL than patients with TDT; however, the limited literature identified provide an incomplete picture. Future studies on the epidemiology, morbidity, mortality, and economic burden associated with NTDT are needed to understand the clinical and economic impact experienced by these patients and society.

P1743: IDENTIFYING DUTCH HOSPITALIZED PATIENTS AT HEMATOLOGY WARDS WITH A LIMITED LIFE EXPECTANCY: A SUBGROUP ANALYSIS OF A FLASH MOB STUDY

A. van der Velden 1,* , A. de Heij 1 , C. Hazenberg 2 , A. Reyners 1 , P. de Graeff 1

1 Expertise Center Palliative Care; 2 Hematology, UMCG, Groningen, Netherlands

Background: Disease trajectories and perspectives differ considerably between various subtypes of hematological malignancies, whereas determination of the transition point from a curative to palliative approach to care can be challenging. It is not known how many in hospital patients with hematological diseases have a decreased life expectancy and if these patients receive palliative care.

Aims: This study aimed to investigate how many inpatients with a probable limited life expectancy were hospitalized in Dutch hospitals on a given day, if a palliative care team (PCT) was involved or involvement was considered desirable.

Methods: A flash mob study was conducted in Dutch hospitals on the 16 th of April 2021 at all wards, except the pediatric and obstetric wards, emergency and day care departments. For all hospitalized patients, nurses and doctors independently answered the surprise question (SQ): “Would you be surprised if this patient died within the next 12 months?”, estimated life expectancy of less than three months and involvement of the palliative care team (PCT). In addition, categorized baseline characteristics and treatment limitations were registered. In this study we report a subgroup analysis for hematology wards in university medical centers.

Results: All eight academic hospitals in the Netherlands participated in the flash mob study. Surveys were completed for 2178 patients, of which 210 patients were admitted to hematology wards. The SQ was negatively answered in 40% of cases by doctors and 36% by nurses, respectively. Agreement for the SQ between doctors and nurses was moderate (kappa 0.41). In comparison, the life expectancy of inpatients was estimated to be less than 12 months in approximately 49% at medical oncology wards and approximately 37% at internal medicine wards according to the doctors in academic centers. Life expectancy was estimated to be less than three months in 8.6% and 7.1% of patients by doctors and nurses, respectively (kappa 0.58) for in hospital patients at hematology wards. Treatment limitations were recorded in 17.1% of all patients, and in 25% of patients with an estimated life expectancy of less than 12 months. The PCT was involved in 1.6% of cases, and involvement was desirable in 3.3% according to doctors, increasing to 11.1% and 4.2% when life expectancy was estimated to be less than three months.

Summary/Conclusion: In more than one-third of patients admitted to hematology wards, the SQ was answered negatively, suggesting a limited life expectancy and possible palliative care needs. PCT involvement was considered of added value by healthcare professionals in 4.9% of all admitted patients at hematology wards. Further studies should focus on exploration of palliative care needs of hospitalized patients and possible benefits of timely implementation of specialized palliative care.

P1744: ACCURACY OF PREDICTING LONG-TERM SURVIVAL OF CHIMERIC ANTIGEN RECEPTOR (CAR) T CELL THERAPIES IN LARGE B-CELL LYMPHOMA (LBCL)

E. J. Verburg-Baltussen 1,* , A. Stewart 2 , F. F. Liu 3 , C. Parker 4 , E. F. Peterse 1 , M. Treur 1 , B. Malcolm 4 , S. Klijn 3

1 OPEN Health, Rotterdam, Netherlands; 2 OPEN Health, Oxford, United Kingdom; 3 Bristol Myers Squibb, Princeton, United States of America; 4 Bristol Myers Squibb, Uxbridge, United Kingdom

Background: In health economic evaluations of CAR T cell therapies in LBCL, long-term survival predictions have been based on pivotal overall survival (OS) study data. Due to several reasons including the immaturity of these studies, various survival models have been used to extrapolate OS. Selection of the survival model may be guided by statistical goodness-of-fit criteria (GOFC), which provide a relative measure of fit to the observed data. However, GOFC are not necessarily indicative of predictive accuracy beyond the study follow-up.

Aims: The primary objective was to retrospectively examine the predictive accuracy of different survival models for estimating OS of CAR T cell therapies in LBCL using data cutoffs with different follow-up durations from the respective studies. The secondary objective was to assess the appropriateness of using GOFC as guidance for survival model selection.

Methods: OS data for tisagenlecleucel, axicabtagene ciloleucel, and lisocabtagene maraleucel were sourced from JULIET ( NCT02445248 ), ZUMA-1 ( NCT02348216 ), and TRANSCEND NHL 001 ( NCT02631044 ), respectively. A broad range of survival models (standard parametric, cubic spline, mixture cure, and mixture models) were fit to multiple data cutoffs, with varying follow-up durations for each study. Goodness-of-fit was assessed using the Akaike Information Criterion and Bayesian Information Criterion. Predictive accuracy of each model was calculated as the mean absolute error (MAE) relative to OS observed in the most mature data cutoff. The correlation between GOFC and predictive accuracy was assessed using linear regression.

Results: The importance of selecting an appropriate survival model was demonstrated by the large variations seen in the 5-year survival estimates of the different extrapolation models, which ranged between 0%–45%, 10%–55%, and 0%–47% over all data cutoffs of JULIET, ZUMA1, and TRANSCEND NHL 001, respectively. For all studies, cubic spline and mixture cure models provided the best predictive accuracy for the least mature data cutoffs (MAE, 0.014–0.128 and 0.013–0.085, respectively), meaning that those models were closest to the observed data of the most mature data cutoff. The predictive accuracy of the standard parametric and mixture models showed larger variations (MAE, 0.024–0.162 and 0.013–0.176, respectively). With increasing maturity of the data, cubic spline and mixture cure models continued to have high predictive accuracy (MAE, 0.014–0.036 and 0.015–0.050, respectively). Mixture models showed an increase in predictive accuracy when longer follow-up data was available. Standard parametric models provided poor predictive accuracy, even with longer follow-up. Furthermore, for the least mature data, the correlation between GOFC and predictive accuracy was low, demonstrated by a coefficient of determination < 20%, for all studies. With increasing data maturity, an increase in correlation between GOFC and predictive accuracy was observed.

Summary/Conclusion: Based on the data currently available, cubic spline and mixture cure models can achieve higher predictive accuracy for OS in LBCL compared with standard parametric and mixture models, even when follow-up is short. As such, these methods should be considered when modeling OS in LBCL. GOFC demonstrated a low correlation with predictive accuracy; thus, limited weight should be assigned to GOFC when selecting the optimal survival model in LBCL.

P1745: ADHERENCE TO TREATMENT OF HEMATOLOGICAL MALIGNANCIES PATIENTS WITH ANXIETY AND DEPRESSION

D. E. Vybornykh 1,* , T. Moiseeva 1 , E. Gemdzhian 1 , T. Gaponova 1 , D. Novikova 1

1 NATIONAL RESEARCH CENTER FOR HEMATOLOGY, Moscow, Russia

Background: The issue of factors influencing adherence to treatment of patients with hematological malignancies (HM) has not been sufficiently studied.

Aims: To establish the features of the influence of anxiety and depressive disorders on treatment adherence, as well as to clarify the factors associated with it in HM patients.

Methods: The study included 117 patients: 51 men and 66 women, aged 19 to 67 years, with Hodgkin’s lymphoma (HL) - 88, acute lymphoblastic leukemia (ALL) - 16 and aplastic anemia (AA) - 13 patients. Patients were examined by psychiatrist using the Brief Psychiatric Rating Scale, as well as some psychometric methods.

Results: Anxiety and depressive spectrum disorders were detected in 36 (40.9%) patients with HL and 8 (50%) with ALL, in the AA group there were three (23.1%) of such patients. It was found that the average adherence to treatment was in 2/3 of patients, low and high - in the remaining 1/3 of patients. The adherence to treatment it is significantly higher in patients older than 45 years. Signs of depression that negatively correlated with adherence to treatment, were pessimism (Fig.1) and disruption of social ties (Fig.2). Adherence to treatment significantly positively correlates with the following types of attitudes towards the disease: anosognosic, hypochondriac and egocentric, and significantly negatively correlates with the following types of attitudes towards the disease: anxious, melancholic and dysphoric. With medium and low adherence to treatment, the risk of adverse events increases by an average of 1.7 times (Fig.3).

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Summary/Conclusion: Anxiety/depressive disorders contribute to reduced adherence of HM patients to treatment. Their correction and increased adherence should be carried out jointly by hematologists and mental health professionals.

P1746: LONG-TERM NON-HODGKIN LYMPHOMA SURVIVORS IN CÔTE D’OR: HEALTH-RELATED QUALITY OF LIFE AND LIVING CONDITIONS.

S. Wasse 1,* , S. dabakuyo-yonli tienhan 2 , C. rossi 3 , J. adnet 4 , S. gauthier 1 , S. boulanger-girard 1 , A. kueshivi midodji 1 , E. assogba 2 , M. maynadie 5

1 Registry of Hematological Malignancies of Côte d’Or, Dijon-Bourgogne University Hospital; 2 Epidemiology and Quality of Life Research Unit, Breast and Gynaecologic Cancer Registry of Côte d’Or, Georges François Leclerc Comprehensive Cancer Centre; 3 Clinical hematology unit, Dijon-Bourgogne University Hospital; 4 Methodology biostatistics and data-management unit, Georges François Leclerc Comprehensive Cancer Centre; 5 University Bourgogne-Franche_Comté, INSERM, U1231,Registry of Hematological Malignancies of Côte d’Or, Dijon-Bourgogne University Hospital, Dijon, France

Background: Non-Hodgkin lymphoma (NHL) is the largest group of hematological malignancies and represented 12% of all new cancer cases in metropolitan France in 2018. The survival outcomes of NHL patients have improved due to important therapeutic advances. Age-standardized 5-year net survival from 2010 to 2015 in France was 86% for follicular lymphoma (FL) and 61% for diffuse large B cell lymphoma (DLBCL), which are above the average survival rates in Europe (FL 72% and DLBCL 51%). In this context, the question of quality of life in NHL patients is garnering increasing interest. To the best of our knowledge, few data from France have addressed the issue of living conditions of long-term NHL survivors at the scale of the general population.

Aims: To identify the clinical and social determinants of long-term health related quality of life (HRQoL) in NHL survivors in the general population and to describe their socio-professional reintegration, socio-economic status, sexual well-being and the impact of COVID.

Methods: All patients were registered in the population-based cancer registry specialized in hematological malignancies in the Côte d’Or area (A French Department with a total of 532,901 residents in 2019). We identified patients diagnosed with DLBCL or FL according to the third edition of the International Classification of Diseases for Oncology (ICD-O-3), from January 1 st 2010 to December, 31 st 2017, and who were still alive on March, 1 st 2021, with an updated address. Patients under 18 years old and adults unable to provide consent were not eligible. In March 2021, patients completed standardized self-report questionnaires for HRQoL (SF-12), anxiety and depression (HADS), social support (SSQ6), socio-economic deprivation (EPICES). Reminders were sent to non-responders after one month. The determinants of HRQoL were identified using a generalized linear model.

Results: Among 436 patients diagnosed, 248 were alive at the study endpoint, of whom 157 (FL 51% and DLBCL 49%) completed the questionnaires, yielding a response rate of 63.3%, the median of time since diagnosis was 76 months [39-133]. The mean age of participants was 67.3 years (SD = 12.4), 55% were men, 74% Ann Arbor stage III-IV, 78% were treated by chemotherapy and immunotherapy, with 99% in the DLBCL group, 11% relapsed after treatment, 64% had no comorbidities and 62% did not have socio-economic deprivation, 27% were employed at the time of the survey, 60% of survivors had not received information about sexuality, 29% reported a negative impact of the disease on their professional activities, 54% reported an impact of the COVID crisis on their life. This impact was socio-economic for 77% and psychological for 23% of respondents. The main factors associated with a negative impact on HRQoL were depression, anxiety, and loss of sexual desire.

Summary/Conclusion: Six years after diagnosis, clinical parameters did not have a major influence on HRQoL, except for relapse. The main determinants of HRQoL identified were psychological and social factors. All these elements are potential targets for specific interventions by the social system to improve HRQoL in NHL patients.

P1747: A REAL-WORLD ANALYSIS OF HEALTHCARE RESOURCE UTILIZATION AMONG PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA WHO RECEIVED TREATMENT WITH RAVULIZUMAB

J. Fishman 1,* , S. Kuranz 2 , M. Yeh 1 , K. Brzozowski 2 , H. Chen 2

1 Apellis Pharmaceuticals, Waltham; 2 TriNetX, LLC, Cambridge, United States of America

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and potentially life-threatening disease involving complement-mediated hemolysis and thrombosis. C5 inhibition (C5i) therapy for PNH with ravulizumab (RAV) has led to hemoglobin (Hb) stabilization (avoiding a ≥2 g/dL decrease in Hb levels from baseline) and some decreases in transfusion dependence. However, international guidance recommends assessing PNH treatment response with Hb normalization (e.g., Hb levels ≥12 g/dL; Debureaux et al ., Bone Marrow Transplant . 2021 Oct;56(10):2600-2602), and evidence showing Hb normalization with RAV is scarce. Anemia-related complications, including transfusions, contribute significantly to PNH-associated costs of care, thus highlighting the need to understand Hb level changes and healthcare resource utilization (HCRU) in patients with PNH.

Aims: To evaluate Hb level changes in patients with PNH and corresponding HCRU after 6 months of RAV treatment.

Methods: Data were extracted from patients with PNH aged ≥12 years between 2010-2021 from the TriNetX Dataworks USA Network, a federated electronic medical record (EMR) data network of >68 million de-identified patients. Patients were required to have a PNH diagnosis based on the International Classification of Diseases 9 th or 10 th Revision code, a medical visit recorded ≥6 months before index/first RAV treatment, and no record of myasthenia gravis, atypical hemolytic uremic syndrome, or neuromyelitis optica spectrum disorder. RAV-treated patients were analyzed as a whole and stratified by C5i treatment history (C5i naïve or prior C5i/eculizumab). Patients were categorized by Hb levels using changes in Hb levels from baseline (within 365 days pre-index) to the final Hb value recorded between 91-180 days post-index. Patients were excluded if they were missing baseline Hb levels, Hb values at 91-180 days or received a transfusion between 0-180 days post-index. HCRU values were evaluated through 180 days post-index for inpatient visits (total number of events post-index) and length of stay ([LOS] total days in the inpatient setting post-index, calculated among all patients regardless of hospitalization history).

Results: Data from 36 RAV-treated patients were extracted from the EMR network (Table). Before starting RAV treatment, 24 (66.7%) patients were naïve to C5i, and 12 (33.3%) patients had prior C5i treatment. Following 6 months of RAV, many patients displayed decreased or no change in Hb levels (77.8%, n=28). Patients with decreased or no change in Hb levels after 6 months of RAV experienced more inpatient visits (mean visits [SD]: 1.3 [2.0]) compared to patients with increased Hb levels (mean visits [SD]: 0.9 [1.5]). RAV-treated patients with decreased or no change in Hb levels also demonstrated a longer mean LOS (mean days [SD]: 2.2 [6.7]) compared to patients with increased Hb levels (mean days [SD]: 0.2 [0.4]). Findings were similar among all three analysis groups (All RAV; RAV, C5i naïve; RAV, prior C5i), but slightly more C5i naïve patients with decreased or no change in Hb levels following RAV treatment (79.2%, n=19) had higher HCRU values for inpatient visits and LOS (Table).

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Summary/Conclusion: The majority of RAV-treated patients did not display Hb improvement after 6 months of treatment, and patients lacking Hb improvement also demonstrated increased HCRU. This suggests improvements in Hb may be an important driver of morbidity and costs among patients with PNH. These findings also highlight the utility of improved Hb levels as a metric for assessing the PNH treatment response.

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PB1748: DIFFERENTIAL IRON HOMEOSTASIS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA IN VITRO MODELS

M. M. Capelletti 1,* , M. D’Angiò 1,2 , G. Fazio 2 , P. Sara 1 , M. Villa 1 , A. Piperno 3 , C. Giovanni 1,2

1 School of Surgery and Medicine; 2 Centro Ricerca Tettamanti Pediatric Department, Università di Milano Bicocca; 3 Centre for Rare Diseases, Disorder of Iron Metabolism, ASST-Monza, San Gerardo Hospital, Monza, Italy

Background: Iron is an essential element which plays several physiological activities in different human organs. Current studies suggest that dysregulation of iron proteins and subsequent accumulation of iron are associated with the response of leukemia to treatments. In particular, it has been reported that low levels of ferroportin-1 (FPN1), lead to an increased sensitivity to chemotherapy in acute myeloid leukemia.

Aims: We aimed at analyzing the gene expression profile of iron-related genes in childhood B acute lymphoblastic leukemia (ALL) and characterize the iron metabolism in vitro models.

Methods: Iron related genes (Ceruloplasmin ( CP ), Ferroportin-1 ( SLC40A1 ), Transferrin Receptor 1 ( TFR1 ) and Ferritin Light Chain ( FTL )) were analyzed by qRT-PCR in BM samples of 87 childhood ALL and 8 healthy donors. Cases were selected to be representative of t(12;21), t(4;11), t(9;22) or t(1;19) subgroups, as well as MRD-based standard-risk (SR), medium-risk (MR) and high-risk (HR) response. Furthermore, we evaluated the cellular viability in REH, RS4;11, 697 and TOM-1 cell lines after exposure to iron as ferric ammonium citrate (FAC 100 to 400 µM), deferoxamine (DFO 25 µM) and FAC+DFO (100 µM + 25 µM), using MTT assay at 24-48 and 72h. TFR1 and FTL mRNA levels were assessed at 8h, 16h, 24h, 48h. Labile Iron Pool (LIP) was measured by flow-cytometry using Calcein-AM technique after the exposure to FAC (100-200-400 µM) at 48h.

Results: TFR1 , SLC40A1 and CP were up-regulated in HR group vs. controls. SLC40A1 and CP were down-regulated in all genetic subgroups, except for CP in t(9;22). TFR1 was slightly expressed in all subgroups. In the t(1;19) subgroup all the iron genes were downregulated, suggesting that this subgroup may not be responsive to iron, as also confirmed by our in vitro data. Iron supplementation by FAC [100 µM] resulted in a boost of viability for TOM-1, REH and RS4;11. TOM-1 and REH viability decreased with FAC 200-400 µM, while RS4;11 proliferation was stimulated by all concentrations. Together with low transcriptional levels of iron genes, 697 viability did not change with different FAC treatments. DFO reduced cell viability in a time-dependent manner. At 48h the viability started to drop for all cell lines (TOM-1 80% - RS4;11 66% - REH 71% - 697 51%) and the co-administration of iron restored the viability to levels similar or even higher than the untreated (TOM-1 100%-RS4;11 80% -REH 92% - 697 81%). FTL expression was significantly up-regulated in all cell lines, while the TFR1 was down-regulated at 8, 16 and 24h vs . untreated. Still, some differences were detected within the cellular models: RS4;11 showed highest level of FTL expression, while in 697 the transcript was slightly upregulated. In 697 cell line, the LIP increased with higher dosages of FAC, suggesting that this cell line was not able to handle high amount of free iron. TOM-1, RS4;11 and REH presented LIP levels higher than the untreated condition, still no significant differences were noticed between the concentrations.

Summary/Conclusion: SLC40A1 and CP high levels of HR group potentially suggest that their refractory behavior may be correlated with ability to keep regulate iron intracellular concentration. TOM-1, REH and RS4;11 behave as resistant cell lines, whilst the 697 appear as not sensitive to iron metabolism. In conclusion, leukemia cell lines can be considered as trustable in vitro models for further studies in ALL to investigate whether iron metabolism can be determinant for survival and resistance, and consequently a target for treatment.

PB1749: APOPTOTIC EFFECT OF FLUMATINIB COMBINED WITH BCL2 INHIBITOR ON PH+ B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Y. Gu 1 , C. Song 2,3 , Z. Ge 1,*

1 Department of Hematology, Zhongda Hospital, Medical School of Southeast University, Institute of Hematology Southeast University, Nanjing 210009, China; 2 Hershey Medical Center, Pennsylvania State University Medical College, Hershey, PA17033; 3 Division of Hematology, The Ohio State University Wexner Medical Center, the James Cancer Hospital, Columbus, OH 43210, United States of America

Background: Philadelphia chromosome (Ph+) is the most common genetic abnormality of B-cell acute lymphoblastic leukemia (B-ALL), and also an independent prognostic high-risk marker. In addition to allogeneic hematopoietic stem cell transplantation, now tyrosine kinase inhibitor (TKI) has greatly improved the prognosis of patients with Ph+ B-cell ALL. Flumatinib is a kind of TKI originally developed in China and has been approved for chronic myeloid leukemia (CML). ABT-199, a selective Bcl-2 inhibitor, was effective in reducing leukemic burden in vitro and in vivo in B-ALL. However, there is no report about the anti-tumor effect of flumatinib in combination with Bcl-2 inhibitor in B-ALL.

Aims: We explore the effect of flumatinib combined with Bcl-2 inhibitor on cell proliferation arrest and apoptosis in SUP-B15 human Ph+ B-cell ALL cells.

Methods: CCK-8 assay was used to analyze the proliferation of SUP-B15 cells treated with flumatinib only, ABT-199 only, combination group, and vehicle control. All groups were treated for 48h at the same time. Apoptosis assay was performed with Annexin V staining following flow cytometry analysis. Expression of the apoptosis-associated gene and protein level was detected by real-time PCR and western blot, respectively.

Results: Flumatinib and ABT-199 were sensitive to SUP-B15 in both single or combined treatment in a dose-dependent manner (Fig. 1A). The IC50 of flumatinib was 2.4uM, and ABT-199 5.5nM in the cells treated for 48 hours. Combination of various doses of flumatinib with 5.5nM (IC50) ABT-199 showed significantly higher proliferation arrest than that of either single drug, and CalcuSyn analysis showed a synergistic effect of the two drugs on the cell proliferation arrest (Fig. 1B). The combination of flumatinib with ABT-199 also significantly increased the apoptosis rate of SUP-B15 (39.2±1.2%) compared to either flumatinib (25.3±0.3%) or ABT-199 (10.6±0.7%) (p<0.05) (Fig. 1C). Furthermore, the combination treatment showed significantly higher expression of apoptosis-related proteins, Bax, Bad, Caspase-3 and Caspase-9 compared to either single drug in mRNA level by qPCR (p<0.05) (Fig. 1D) and protein level by western blot (Fig. 1E).

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Summary/Conclusion: Both single drugs of flumatinib and ABT-199 are sensitive to SUP-B15 human Ph+ B-cell ALL cells. The combination of flumatinib with ABT-199 has a synergistic effect on cell proliferation arrest and apoptosis of the cells, revealing the potential of the novel combination for therapy of Ph+ B-cell ALL.

PB1750: REARRANGEMENTS OF T-CELL RECEPTOR (TCR) LOCI IN CHILDREN WITH T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

L. Lizcova 1,* , E. Prihodova 1 , L. Pavlistova 1 , K. Svobodova 1 , L. Hodanova 1 , E. Mejstrikova 2 , O. Hrusak 2 , I. Janotova 3 , L. Sramkova 3 , J. Stary 3 , Z. Zemanova 1

1 Center of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine, Charles University in Prague; 2 CLIP - Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology; 3 Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, Prague and University Hospital Motol, Prague, Czechia

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a clinically and genetically heterogeneous disease that constitutes 10%–15% of newly diagnosed pediatric ALL cases and is caused by the accumulation of genetic abnormalities that alter the mechanisms controlling normal T-cell development. The most common structural aberrations involve rearrangements of the T-cell receptor (TCR) loci – TRA/TRD (14q11) and TRB (7q34), which are found in ~25% of patients. These aberrations are the main factors initiating the events in T-ALL carcinogenesis and juxtapose TCR genes and protooncogenes that encode pivotal transcription factors, leading to their aberrant expression. The outcomes of children with T-ALL significantly improved on intensified protocols, however, ~20% of patients relapse and die owing to acquired therapy resistance. Moreover, survivors also endure the acute and long-term effects of intensive toxic chemotherapy.

Aims: To analyze TCR loci rearrangements and associated genetic abnormalities in children with T-ALL treated according to BFM-based protocols, and to correlate our findings with the clinical features and the treatment responses.

Methods: The bone marrow samples of 66 children diagnosed between 1996-2017 (46 boys, 20 girls, age median 7.9 years, follow-up median 86.4 months) were analyzed with cytogenomic methods. For the detection of TCR loci rearrangements and other recurrent aberrations of the genes TLX3 , CDKN2A/CDKN2B and ABL1 interphase FISH (DAKO, Abbott Molecular) was used. MLPA analysis with ALL-IKZF1 probemix (MRC-Holland) was performed to detect deletions/amplifications of additional genes. Complex karyotypes were analyzed with multicolor FISH (MetaSystems) or array CGH/SNP (Agilent). Differences in OS and EFS were assessed using Kaplan-Meier method and the Mantel-Cox test.

Results: Rearrangements of TCR loci were detected in 18/66 (27%) patients. Translocations involving the TRA/TRD locus were demonstrated in nine children, the TRB locus in five and the simultaneous occurrence of these aberrations in two cases, respectively. In 10/18 patients, known recurrent chromosomal translocations affecting the oncogenes TAL1 (3x), LMO2 (3x), TLX1 (2x), TAL2 (1x) and MYC (1x) were identified. In the remaining cases, TCR rearrangements were cryptic or were a part of complex karyotype. Additional chromosomal aberrations were detected in all but one patient with the deletion of CDKN2A gene being the most frequent one. Immunophenotypic data classified the patients into cortical-T (12x) and mature-T (5x) groups (1x no data). Children with aberrations of TCR loci had a significantly better prognosis - EFS (p=0.011) and OS (p=0.0074), all patients are living in the first complete remission.

Summary/Conclusion: Aberrations of T-cell receptors form a genetically heterogeneous group of rearrangements, leading to aberrant expression of oncogenes involved in T-cell maturation and proliferation. They mostly occur with abnormalities of genes involved in the regulation of the cell cycle and/or signaling pathways, confirming the multistep process of T-ALL pathogenesis. In our cohort, patients with TCR rearrangements had an excellent prognosis regardless of the presence of other aberrations. Although data on larger series are clearly required, we suppose, that these children could benefit from less-intensive therapy because they may experience fewer therapeutic consequences of toxic treatment.

Supported by MH CZ-DRO-VFN64165.

PB1751: ALTERATIONS IN THE GUT MICROBIOME IN PEDIATRIC, ADOLESCENT, AND YOUNG ADULT PATIENTS WITH ACUTE LEUKEMIA

M. Park 1,* , J. Jung 2 , H.-Y. Jin 1 , J. A. Lee 1 , E. Lee 2 , H. Lee 2 , H. S. Eom 2 , H. J. Park 1

1 Department of Pediatrics, Center for Pediatric Cancer; 2 Center for Hematologic Malignancy, National Cancer Center, Goyang, South Korea

Background: Gut microbiome is involved in the progression of various diseases and may affect the human immune system. The composition of gut microbiome and its change upon anti-leukemic therapy in pediatric, adolescent, and young adult patients with acute leukemia is poorly understood.

Aims: We performed a longitudinal analysis of gut microbiome composition to assess associations between gut microbial dynamics and clinical outcomes in pediatric, adolescent, and young adult patients with acute leukemia during chemotherapy and/or hematopoietic cell transplantation (HCT). And also, we aimed to compare the gut microbiome composition of leukemia patients with their healthy sibling controls.

Methods: The bacterial 16S rRNA gene was analyzed by high-depth sequencing to determine the diversity and composition of the microbiome. Data collected between August 2020 and September 2021. The median age of patients was 17.1 years (range from 3.5 years to 34.1 years). Seventy-nine stool samples from 15 patients (10 acute lymphoblastic leukemia and 5 acute myeloid leukemia) and 9 stool samples from 9 siblings were analyzed in this study. Relative abundance, overall richness, various diversity indexes were calculated.

Results: At diagnosis, microbial diversity index of the patient group was lower than that of their siblings. Clostridia and Lachnospiraceae were reduced in patients, but Actinobacteria were more abundant in patients compared to sibling controls. The Firmicutes/Bacteroidetes (F/B) ratio was not different between patients and their siblings. In patients, microbial diversity decreased after induction chemotherapy, but it recovered to a level similar to that of sibling controls after consolidation chemotherapy. The F/B ratio significantly increased after induction, but it decreased to a similar value at diagnosis after consolidation. Microbial diversity index at diagnosis was significantly lower in patients who experienced fever and/or infection during induction compared to patients who did not. A baseline abundance of Proteobacteria predicted fever and/or infection during induction chemotherapy. In patients underwent HCT, compared to the values before HCT, the number of identified species has decreased to about half at day 28 after HCT and decreased further at day 56 after HCT. However, it recovered to pre-HCT level at day 90 after HCT. Diversity index significantly decreased at day 0 of HCT compared to that of pre-HCT, but it recovered to a level similar to that of pre-HCT at day 90 after HCT. At day 0 of HCT, patients who experienced fever and/or acute graft versus host disease (aGVHD) within day 100 of HCT showed abundant Bacteroidetes with median F/B ratio of 0.1, while patients who did not showed low level of Bacteriodetes with median F/B ratio of 1.4. Diversity index at day 0 of HCT was not different between patients who experienced fever and/or aGVHD and patients who did not.

Summary/Conclusion: In pediatric, adolescent, and young adult patients undergoing therapy for acute leukemia, decreased microbial diversity and abundance of Proteobacteria at diagnosis were associated with fever and/or infection during induction chemotherapy. Abundance of Bacteroidetes at day 0 of HCT predicted subsequent fever and/or acute GVHD. Better understanding of the gut microbiome could provide actionable information in guiding precision medicine.

PB1752: ANALYSIS OF HLA-PEPTIDE BINDING POCKETS IN PATIENTS WITH ACUTE LEUKEMIA UNDERGOING UMBILICAL CORD BLOOD TRANSPLANTATION

W. Boukouaci 1,* , M. M. Rivera Franco 2 , F. Volt 2 , C.-L. Wu 1 , H. Rafii-Elayoubi 2 , G. M. Scigliuolo 2,3 , B. Cappelli 2,3 , C. Kenzey 2 , E. Gluckman 2,3 , R. Tamouza 1

1 Univ Paris Est Créteil, INSERM U955, IMRB; 2 Eurocord, Hôpital Saint Louis APHP, Institut de Recherche de Saint-Louis (IRSL) EA3518, Université de Paris, Paris, France; 3 Monacord, Centre Scientifique de Monaco, Monaco, Monaco

Background: Despite a better understanding of the genetic heterogeneity of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), relapse rates remain high and long-term survival is suboptimal. In this context, the association between these malignancies and the human leukocyte antigens (HLA) has been widely studied. The highly polymorphic HLA loci have significance and relevance for disease susceptibility thanks to their peptide-binding grooves-dependent antigen presentation to effector cells. Such processes trigger an algorithm of immune responses based on multiple fine-tuned combinations of amino acid sequence interactions. Thus, the HLA genetic diversity has been a focus of interest in acute leukemia settings as exemplified by studies showing the implication of both HLA class I and II polymorphisms. However, there is scarce data regarding the diversity of peptide-binding pockets per se especially in the context of umbilical cord blood transplantation (UCBT).

Aims: To analyze whether variants of HLA-DQB1 and -DRB1 peptide-binding groove pockets 4 and 9 and/or their amino acid positions are associated with the diagnosis of ALL or AML among patients undergoing UCBT.

Methods: A retrospective analysis was performed including 849 patients with ALL and AML undergoing an UCBT. Demographics and genotyping data were obtained from the Eurocord database. Pockets and amino acids were imputed from the four-digit HLA genotypes of patients. Easy-HLA was used for haplotype inference when needed. Descriptive statistics were applied. Risk ratios (RRs) and 95% confidence intervals (CI) were estimated by applying log-binomial regression to compare the allelic frequencies between both types of leukemia.

Results: ALL and AML were diagnosed in 50.2% and 49.8%, respectively. The median age at UCBT was 8.7 and 17.9 years. Overall, they were mostly pediatric patients (61.8%), males (52%), and in first complete remission (58.7%). From the ten variants analyzed in pocket 9 of the HLA-DQB1 allele, only the variant YYVSY, corresponding to the allele HLA-DQB1*05:02/04, was more frequent in ALL when compared to AML (RR=2.12, 95% CI: 1.30-3.44; p=0.001). Also, within the 21 analyzed pocket 4 variants of the HLA-DRB1 allele, RFDRAY, corresponding to DRB1*16:01/02, was the only variant with a significant higher frequency among ALL patients versus AML patients (RR=3.26, 95% CI: 1.81-5.99; p=0.001). No differences were observed when comparing ALL and AML at either pocket 4 of HLA-DQB1 allele or pocket 9 of HLA-DRB1 allele. Further amino acid analysis revealed that the frequency of serine 57 of the HLA-DQB1 pocket 9 was higher in ALL (RR=2.03, 95% CI: 1.26-3.27; p=0.037). None of the analyzed amino acid positions in HLA-DRB1 pockets was statistically enriched neither in ALL or AML.

Summary/Conclusion: Our analyses suggest that specific variants in terms of HLA pockets and amino acids might be unique to ALL. As a result of expressing HLA molecules on their surface, leukemic cells may participate to the disease development and/or to immune evasion. Thus, the immune and genetic expression profile of the leukemic microenvironment has been described as a potential useful biomarker in guiding therapeutic approaches. Interestingly, peptide binding to HLA class II molecules is frequently viewed as a merger of detached anchor residue preferences for pockets 4 and 9. In our study, these associations, at a genotypic level, might be a stepping stone towards analyzing results in acute leukemia after UCBT as there is robust evidence supporting the impact of HLA on important post-transplant outcomes.

PB1753: SYNERGISTIC ANTITUMOR EFFECT OF CHIDAMIDE WITH VENETOCLAX IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA BY SUPPRESSING PI3K/AKT SIGNALING

D. Song 1 , C. Song 2,3 , Z. Ge 1,*

1 Department of Hematology, Zhongda Hospital，Medical School of Southeast University, Institute of Hematology Southeast University, Nanjing, China; 2 Hershey Medical Center, Pennsylvania State University Medical College, Hershey; 3 Division of Hematology, The Ohio State University Wexner Medical Center, the James Cancer Hospital, Columbus, United States of America

Background: Chidamide, an oral histone deacetylase (HDAC) inhibitor of the benzamide class that selectively inhibits HDAC1, 2, 3, and 10, is currently used for patients with peripheral T-cell lymphoma. ABT-199 (Venetoclax), BH3-mimetic, is a highly specific inhibitor of the anti-apoptotic protein Bcl-2. HDAC inhibitors have been shown to synergize effects with ABT-199 in acute myeloid leukemia (AML), cutaneous T-cell lymphoma (CTCL), and mantle cell lymphoma (MCL). Notably, T-ALL patients treated with ABT-199-based combination regimens showed promising efficacy. However, there are no reports about the combination of HDAC inhibitor with ABT-199 in T-ALL.

Aims: This study is to examine the synergistic antitumor effect of chidamide in combination with ABT-199 in T-ALL cells.

Methods: CCK-8 assays were employed to evaluate the synergistic effects of chidamide and ABT-199 on cell proliferation arrest in CEM and Molt4 T-ALL cells treated with vehicle control, chidamide, ABT-199, and chidamide+ABT-199 for 48 hours. Apoptosis was measured by cell staining with Annexin V+ propidium iodide (PI) following flow cytometry analysis. The distribution of the cell cycle was detected by flow cytometry with a PI staining assay. The gene expression upon the drug treatment is evaluated by qPCR and western blot.

Results: Both chidamide and ABT-199 significantly inhibited the cell proliferation of CEM and Molt4 cells in a dose-dependent manner. The combination of chidamide with ABT-199 significantly increases the rate of cell proliferation arrest (Fig. 1A), and apoptosis (Fig. 1B) compared to the single drug control in both cell lines. Also, the chidamide alone or combined treatment significantly induced the G0/G1 phase arrest in the cells (Fig. 1C). Moreover, the Combination of chidamide with ABT-199 induced the up-regulation of apoptotic proteins, Bax and cleaved caspase-3, and down-regulation of anti-apoptotic protein, Mcl-1 compared to that of either single drug control (Fig. 1D). Similarly, chidamide alone or combined treatment induced the up-regulation of cell cycle regulatory protein, p21 in the cells (Fig. 1E). We also analyzed the pathway enrichment based on network pharmacology from T-ALL treated with chidamide and ABT -199 and found the PI3K/AKT is significantly enriched upon the drug treatment (Fig. 1F). Western blot results also showed that the combination treatment significantly down-regulated expression of PI3K and AKT, also the AKT phosphorylation (Fig. 1G).

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Summary/Conclusion: The combination of chidamide with ABT-199 exerted a synergetic effect on cell proliferation arrest by suppressing PI3K/AKT signaling pathway. Our data also highlight the combination therapy in clinical application in T-ALL.

PB1754: TELOMERE LENGTH IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA

E. Vakonaki 1,* , P. Iordanis 2 , M. Tzatzarakis 1 , A. Alegkakis 1 , M. Lourou 2 , N. Katzilakis 2 , A. Tsatsakis 1 , E. Stiakaki 2

1 Laboratory of Toxicology, Medical School, University of Crete; 2 Department of Pediatric Hematology-Oncology and Laboratory of Blood Diseases and Childhood Cancer Biology, University Hospital of Heraklion, Medical School, University of Crete, Heraklion Crete, Greece

Background: Acute Lymphoblastic Leukemia (ALL) is the most common neoplastic disease in childhood with an excellent prognosis, accounting for 80% of all childhood malignancies. In almost 90% of ALL cases, numerical chromosomal as well as structural abnormalities, resulting in disruption of the expression of genes involved in hematopoiesis can be detected. As a result cells proliferate uncontrollably, leading to a rapid cell aging. A marker of the cellular senescence is the shortening of the telomere length. Telomeres have a protective role from chromosome degradation, recombination and fusion. In each cell division a reduction of telomeres occurs, until the Hayflick limit is reached and the cell is led to apoptosis.

Aims: The purpose of the study was the evaluation of cellular aging in children and adolescents with ALL, determined by the telomere length measurement, as well as the possible correlation with the response to treatment, the risk of relapse and ultimately the overall survival.

Methods: A total of 38 children, adolescents and young adults with the diagnosis of ALL were included in the study. The age range was from 1.5 to 18 years old and 25 were males. Regarding the lineage type of ALL, 33 were diagnosed with B-cell ALL and the remaining with T-ALL. A bone marrow sample was obtained at diagnosis and when remission was achieved. In case of relapse (3 children) additional samples were obtained. The telomere length was determined by qPCR methodology.

Results: The mean telomere length in the lymphoblast specimens obtained at diagnosis was 1.97 ± 0.9 Kb, while at the time of remission the mean length was higher (4.35 ± 1.75 Kb). Two of the patients showed longer telomere length in lymphoblasts compared to normal lymphocytes at remission. Four of the patients had no difference in the mean telomere length between diagnosis and remission and these are the only with disease relapse. In these patients samples were also collected at relapse and no difference in telomeres length was noted as well.

Grouping the patients according to the type of ALL, patients with T-ALL presented longer telomere length compared to those with B-ALL (3.27 ± 1.22 Kb versus 2.48± 1.87 Kb, respectively). High-risk group patients had longer telomere lengths compared to the intermediate-risk counterpart (2.89 ± 1.24 Kb and 2.41 ± 2.05 Kb respectively). Finally, comparing the telomeres length based on the karyotype analysis, individuals with 46 chromosomes had median telomere length of 2.73 ± 1.75 Kb, which is longer than those with a karyotype with > 46 chromosomes (2.33±2.14).

Summary/Conclusion: The decrease of the telomere length observed at diagnosis seems to be reversible and could be attributed to the uncontrolled lymphoblastic proliferation. The telomeres length measurement could be correlated with disease progression and serve as a useful biomarker of ALL disease.

PB1755: CASPASE 8, CASPASE 10 AND FAS GENE POLYMORPHISMS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA

M. Lourou 1,* , E. Kampouraki 1 , M. Zervou 2 , I. Pelagiadis 1 , N. Katzilakis 1 , G. Goulielmos 2 , E. Stiakaki 1

1 Pediatric Hematology-Oncology and Laboratory of Blood Diseases and Childhood Cancer Biology, University Hospital of Heraklion, Medical School, University of Crete; 2 Section of Molecular Pathology and Human Genetics, Department of Internal Medicine, School of Medicine, University of Crete, Heraklion Crete, Greece

Background: Acute lymphoblastic leukemia (ALL) is the most common type of childhood leukemia accounting for one third of all pediatric malignancies. Over the last decade major progress has been made in understanding the biology of ALL, genetic basis of leukemogenesis and response to treatment. Epidemiological studies have shown that various genetic factors play a crucial role in leukemogenesis. Abnormal regulation of apoptosis and the deregulation of expression or function of caspase antagonists’ pathways seem to play an important role in the pathogenesis and progression of hematological malignancies. Polymorphisms in the apoptotic genes may enhance the risk of disease, as implicated by the strong link between genetic diversity and susceptibility to ALL. Caspases are a family of cysteine proteases whose functions are strongly associated with programmed cell death. Caspase 8 plays an important role in apoptosis and several studies have been performed to clarify the relationship between polymorphisms in Casp8 gene and different types of cancer. Caspase 10 is the only caspase that shares homologous death–effector domains with Caspase 8, while somatic mutations in Casp10 gene have been observed in many cancers. Fas is a transmembrane molecule belonging to the tumor necrosis factor (TNF) receptor superfamily and its ligation by FasL induces programmed cell death in several lymphoid cell lines. Studies suggest that polymorphisms in the FAS gene may affect cancer risk.

Aims: The purpose of this study was the investigation of the incidence of rs1045485 single nucleotide polymorphism (SNP), located in exon 10 of the Casp8 gene, rs13006529 SNP, located in exon 10 of the Casp10 gene and rs2234767 SNP, located in the promoter of FAS gene, in children and adolescents with ALL, compared to healthy individuals.

Methods: The patient group consisted of 119 children and adolescents (1 to 18 years) with ALL and the control group of 121 healthy individuals. The genomic DNA was isolated from the peripheral blood or the bone marrow and the genetic regions harboring the SNPs under investigation were amplified with Polymerase Chain Reaction (PCR). The PCR product was subsequently digested with the appropriate restriction enzymes ( Bst UI for rs1045485 and rs2234767, Ssp I for rs13006529). Statistical analysis was performed with GraphPad Prism application (GraphPad Software, San Diego, CA, USA).

Results: The frequencies of the minor alleles of rs1045485, rs13006529 and rs2234767 SNPs of Casp8, Casp10 and FAS genes, respectively, were significantly increased in ALL patients compared to controls. In particular, there was an association between ALL diagnosis and the aforementioned alleles of these three genes studied (G for Casp8 , T for Casp10 , G for FAS). Likewise, both the homozygotes for the minor alleles as well as the heterozygotes (CG/GG for Casp8 , AT/TT for Casp10 and AG/GG for FAS ) were associated with ALL in children and adolescents (Table 1).

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Summary/Conclusion: Rs1045485, rs13006529 and rs2234767 SNPs of Casp8 , Casp10 and FAS genes, respectively, were strongly associated with the diagnosis of ALL in the studied population. Further investigation would be of great interest in an attempt to clarify the functional role of these polymorphisms in the development of the disease.

PB1756: INOTUZUMAB OZOGAMICIN IN THERAPY RELAPSE/REFRACTORY B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (R/R B-ALL): A SINGLE CENTER EXPERIENCE

A. Antipova 1,* , O. Baranova 2 , A. Ibragimov 1 , G. Petrova 2 , G. Tumian 1 , E. Osmanov 1

1 Hematology; 2 Transplantation, Federal State Budgetary Institution National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia, Moscow, Russia

Background: The current modern chemotherapy (CHT) protocols for ALL, taking into account prognostic factors, including assessment and monitoring of MRD, in the general group of patients (pts) allow achieving complete remission (CR) in 80–90% cases. In adult pts with primary B-cell ALL CR rates of 80–90% are routinely achieved with standard multidrug chemotherapy, however, 5-year disease free survival is only around 40%. The prognosis of pts with relapses or refractory disease are dismal, 5-y overall survival after treatment failure is about 10%. Therapy of r/r B-ALL remains a challenge. Intensification of CHT has not been successful secondary to high toxicity rates. Each subsequent line of therapy reduces the response rate and the duration of the achieved remission, which complicates the sequential implementation of allogeneic hematopoietic cell transplantation (alloHCT). Novel targeted therapies have been shown to improve outcomes in pts with adult B-ALL. Inotuzumab ozogamicin (InO), a new antibody–drug conjugate, has shown significant activity in adult patients with r/r B-ALL.

Aims: To evaluate the efficacy and safety immunotherapy with inotuzumab ozogamicin in pts r/r B-ALL.

Methods: In the Hematology Department of N.N. Blokhin National Medical Research Center of Oncology (Russia, Moscow) 5 pts were treated with InO between September 2021 and February 2022. There were 3 females and 2 males. Median age at start of InO was 39 years (range 23-62).Bone marrow infiltration was on the average 48% blasts.Lymphoblasts for all pts expressed CD22. All pts were Ph-negative. Cytogenetic findings were obtained in different pts: KMT2A rearrangement, hyperdiploidy, del(IGH ), complex karyotype, trisomy of chromosome 10. There was no clinically significant leukocytosis, anemia, thrombocytopenia, extramedullary involvement including central nervous system. Four of five pts had a relapse of B-ALL and one had refractory disease. These 4 pts were primary treated according ALL-2009 protocol (Russian ALL-Study Group). In relapses they received intense programs like HyperCVAD,FLAG, 1 patient had undergone prior alloHCT. One patient with refractory disease received only 1 course dose adjusted HyperCVAD. InO was given as monotherapy in all pts in dose 1,8 mg/m 2 first course and 1,5 mg/m 2 in subsequent (and in 1 case 1,8 mg/m 2 during 2nd cycle without response), but in 1 pt the first course was in combination with mini-hyper-CVD regimen. Candidates for alloHCT (4 pts) received 2 cycles of InO therapy in 3 cases and 3 cycles in one. Also, all pts received standard triple intrathecal prophylaxis CNS involvement. The median follow-up is 6 months.

Results: All 5 pts achieved CR or CR with incomplete hematologic recovery (CRi) after first (4pts) and second (1 pt) courses. 4 of 5 pts reached MRD-negativity by flow cytometry and cytogenetic remission by standard karyotyping or FISH. No severe toxic complications were observed. Hematological toxicity was registered, such as thrombocytopenia (grade 3/4 in 2 pts), neutropenia (grade 3/4 in 3 pts), leucopenia (grade 3/4 in 3 pts). Patients also noted fatigue, fever. To date 2 responders underwent alloHCT. Also, the search for an unrelated donor was initiated for other two patients.

Summary/Conclusion: Inotuzumab ozogamicin has shown high efficacy in adult patients with r/r B-ALL. All patients achieved complete remissions with a minimal spectrum of toxicity, which is important when performing alloHCT. Further studies in our center with a larger number of patients and a longer follow-up period will be conducted to evaluate the safety and efficacy of inotuzumab ozogamicin therapy.

PB1757: VARIANT PHILADELPHIA CHROMOSOME IN ALL

N. Bouayed Abdelmoula 1 , B. Abdelmoula 1,* , A. Sbai 1 , M. Ben ayed 1 , S. Aloulou 1

1 Genomics of Signalopathies at the service of Medicine, Medical University of Sfax, Sfax, Tunisia

Background: The Philadelphia chromosome (Ph) is detected in 10–20% of adult patients with B-cell acute lymphoblastic leukemia (B-ALL). At the cytogenetic level, it usually arises from the balanced (9;22)(q34;q11) translocation. In 50–70% of the Ph-positive ALL patients, the BCR gene breaks between exons 1 and 2, known as m-bcr region. The BCR-ABL chimeric gene encodes the p190 fusion protein. In approximately 5% of these patients, the Ph originates through other rearrangements than the classic t(9;22). These variant translocations involve others chromosomes in addition to chromosomes 9 and 22.

Aims: To contribute to the description of patients’ clinical characteristics and prognosis with variant Ph chromosome-positive leukemia, we report a Ph chromosome-positive ALL characterized by an unusual variant Ph chromosome.

Methods: A 34-year-old woman with B-ALL was assessed at the genetic level. Cytogenetic and molecular exploration were conducted using bone marrow and blood samples. Cytogenetic analysis was performed on lymphocytes and bone marrow cells. The cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Multiplex reverse transcription-PCR (RT-PCR) was used to identify the BCR-ABL transcript. RT-PCR was performed according to European recommendations, as described previously.

Results: Cytogenetic analysis showed 45 chromosomes with a masked variant Ph translocation: 45,XX,der(9)t(9;20;22)(q34;q?,q11), der(20) t(9;20;22)(q34;q?,q11),-20,-22. At the molecular level, the patient’s BCR-ABL fusion gene was confirmed as e1a2 transcript. FISH procedures were performed using an bcr-abl probe that showed a double fused gene signal on interphase nuclei.

Summary/Conclusion: Unrelatedly to age and breakpoint location, Ph-positive ALL are characterized by poor prognosis. A unique case of similar complex chromosome 9, 20, and 22 rearrangements in acute lymphoblastic leukemia has been reported. This variant Ph chromosome involving three chromosomes was associated with two markers leading to a duplication of BCR and ABL sequences. Further FISH analysis using the whole chromosome 9, 20 and 22 painting probe will be significant to delineate the complex chromosomal rearrangement involved in our B-ALL case.

PB1758: TWENTY-ONE YEARS’ EXPERIENCE OF THE EORTC 58951 PROTOCOL FOR THE TREATMENT OF CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA IN THE SOUTH OF TUNISIA

I. Frikha 1,* , N. Siala 1 , I. Turki 1 , M. Mdhaffar 2 , I. Ben Amor 1 , M. Charfi 1 , N. Louati 3 , Y. Fakhfakh 1 , F. Kallel 1 , L. Khalifa 1 , O. Kassar 1 , S. Hadijji 1 , H. Mnif 1 , M. Elloumi 1

1 Department of Hematology; 2 Hedi Chaker Hospital, Hedi Chaker Hospital; 3 Blood center transfusion, Sfax, Tunisia

Background: Childhood acute lymphoblastic leukemia (ALL) is a hematologic malignancy with high rate of cure.

Aims: We report the experience of the clinical hematology department of Sfax-Tunisia for the treatment of childhood ALL with the EORTC 58951 protocol.

Methods: From January 2000 to December 2020, we retrospectively studied the outcome of childhood ALL aged less 16 years, treated with the EORTC 58951 pediatric protocol. For those patients we studied the leukemia characteristics (sex ratio, white blood cell counts WBC, blast’s phenotype, cytogenetic abnormalities) and response to treatment: response to prophase, remission rate, risk group stratification, treatment related mortality (TRM) (induction and post induction death) and survival (overall survival OS and event free survival.

Results: From January 2000 to December 2020, 284 children were treated with the EORTC 58951 protocol. Medium age was 7 years (range: 1 to 15 years). Sex ratio M/F was 1.53. WBC counts more than 100 G/L were observed respectively in 41, 44 and 15% of cases. The blast’s phenotype was B in 70%. Cytogenetic abnormalities were noted in 37% of cases. A good response to prophase was noted in 80% and complete remission in 92% of cases. The EORTC risk group stratification was Low Risk (LR) in 5%, Average Risk1 (AR1) in 39%, Average Risk2 (AR2) in 25% and Very High Risk (VHR) in 31%. TRM was 10%: induction rate death was 6% and post-induction rate death was 4%. Twenty-eight patients from VHR group with sibling familial donor underwent allograft. The relapse rate was 29% of all patients in remission; 4% for LR group, 39% for AR1 group, 18% for AR2 group and 39% for VHR group. At a follow up of 126 months, OS and EFS at 10 years were respectively 61% and 56%.

Summary/Conclusion: At diagnosis, childhood ALL in our study had poor characteristics particularly higher rate of T phenotype of blasts, higher rate of leukocytosis more than 100 G/L, higher rate of VHR therapeutic group and higher rate of cortico-resistant regarding occidental series. Despite the acceptable results concerning remission rate but survival rates remain lower than those observed in the literature, this is explained mainly by the high rate of relapse (29% vs ≃15%). It can be improved our results by detecting high risk patient with MRD study especially for AR1 risk group and doing allograft for VHR patients.

PB1759: TWENTY-ONE YEARS’ EXPERIENCE OF ADOLESCENTS AND YOUNG ADULTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA TREATED WITH PEDIATRIC PROTOCOL: MONOCENTRIC STUDY

I. Frikha 1,* , N. Siala 1 , I. Turki 1 , M. Mdhaffar 1 , I. Ben Amor 1 , N. Louati 2 , Y. Fakhfakh 1 , F. Kallel 1 , O. Kassar 1 , M. Charfi 1 , S. Hadijji 1 , H. Mnif 1 , M. Elloumi 1

1 Department of hematology, Hedi Chaker Hospital; 2 Regional blood Trasfusion, Sfax, Tunisia

Background: Several retrospective studies have confirmed that adolescents and young adults (AYA) with acute lymphoblastic leukemia (ALL) treated with pediatric protocols have better outcomes than similarly aged patients treated with adult protocols.

Aims: We reported results and feasibility of a pediatric-based protocol (EORTC 58951) in adolescents and young adults.

Methods: From January 2000 to December 2020, 95 patients aged 16 to 30 years with newly diagnosed ALL were treated, in the department of hematology department of Hedi Chaker Hospital, according to the EORTC 58951 pediatric protocol. Further leukemia characteristics (Sex, White Blood cell count, Blasts phenotype, Cytogenetic results), we studied the protocol results: response to prophase), risk group stratification (average: AR1 and AR2, very high: VHR), complete remission rate (CR), death rate, relapse rate and 5 years survivals (overall OS and event free EFS).

Results: Ninety-five AYA ALL were treated with the pediatric protocol. Medium age was 20 years (range: 16 to 30 years). The patients were 60 males and 35 females (SR=1.71). A WBC> 100 G/l was noted in 28%. A T blast phenotype was noted in 48% of cases. Cytogenetic abnormalities were noted in 40% of cases. Fourteen patients (14%) were treated according AR1 arm, 50 patients (53%) according AR2 arm and 31 patients (33%) according VHR arm. CR rate was 81% after one course and 89% after 2 courses. Induction death was noted in 7% and post-induction death was noted in 14%. Twenty-eight patients from VHR group with familial donor underwent allograft. Thirty-one patients treated with VHR arm protocol were eligible for allogenic stem-cell transplantation (SCT), among them 21 patients had a familial donor and 15 patients were allograft (48%). Relapse was observed in 25 patients (26%) among them 4 after allograft. Fifty-four of relapses occurred within a year of CR. The median follow up was 129 months. The five years OS and EFS were respectively 49% and 47%.

Summary/Conclusion: The results of this pediatric based study show that response to therapy and prognostic in adolescent and young adults were better than those treated with adult protocols and tolerability of chemotherapy is acceptable. However, OS and EFS, better than adult ALL treated by adult protocol (OS= 14%, EFS=14% in previous local study) but less than describe in the literature.

PB1760: AZACITIDINE AS TREATMENT OF KMT2A INFANT ACUTE LYMPHOBLASTIC LEUKEMIA: SINGLE CENTER EXPERIENCE

G. Giagnuolo 1,* , A. De Matteo 1 , P. Stellato 1 , F. Petruzziello 1 , R. Cuccurullo 1 , G. Beneduce 1 , F. Cacace 1 , F. P. Tambaro 1 , R. Parasole 1 , G. Menna 1

1 Department of Oncology, AORN Santobono-Pausilipon, Napoli, Italy

Background: KMT2A rearranged (r) infant acute lymphoblastic leukemia (ALL) is characterized by a very poor prognosis, particularly after relapse. Intensive chemotherapy does not improve outcome and novel therapies are under investigation for this dismal disease. Recent studies reported an hypermethylated DNA profile in KMT2Ar ALL, leading to repression and silencing of tumor suppressor genes. Hypomethylating agents (HMA) have been hypothesized to exert an antileukemic activity restoring normal gene expression.

Aims: Here we report the off-label administration of Azacitidine (AZA) in two little patients aged 1 month old and 26 days old respectively, diagnosed with KMT2Ar infant ALL.

Methods: Both patients were treated according to the high risk arm of the AIEOP-BFM ALL 2017 protocol as front-line therapy.

AZA was administered every 21 days (d) for 4 cycles (cy), with the following schedule: 55 mg/m 2 for 5 d, 55 mg/m 2 for 7 d, 75 mg/m 2 for 5 d, 75 mg/m 2 for 7 d, respectively; prednisone 1 mg/m 2 was associated to each cycle.

Results: Patient 1 was diagnosed with infant B ALL t(11;19)/KMT2A-MLLT1r, high-risk (HR) for age, hyperleucocytosis and KMT2Ar. At the end of induction (EOI) he was in hematologic remission with molecular minimal residual disease (MRD) and t(11;19) still detectable. The child received consolidation chemotherapy followed by 2 cy of bispecific CD3-CD19 antibody (ab) Blinatumomab and underwent allogeneic hematopoietic stem cell transplant (HSCT) from matched unrelated donor (MUD) with detectable MRD. Two months after transplant patient relapsed with 68% CD19 CD22 negative blasts in bone marrow. Due to early relapse and no target therapy available, AZA was administered. Grade 4 cytopenia and pneumonia were main adverse events. Patient achieved MRD complete remission (CR) and underwent to haploidentical HSCT from mother, followed by 3 courses of 40 mg/m 2 AZA maintenance for 5 d, every 21 d. Patient is alive, in MRD negative CR3, 3 months after SCT.

Patient 2 was diagnosed with infant B ALL t(4;11)/KMT2A-AFF1, HR for age, hyperleucocytosis and KMT2Ar. At the EOI bone marrow showed blasts 25% detected by flow cytometry. Patient received one courses of chemotherapy according to the HR protocol, followed by 2 cy of Blinatumomab. The child achieved MRD negative CR and underwent HSCT from MUD. Thirty two days after HSCT, patient relapsed with 60% BM blasts detected by flow cytometry. Due to early relapse after HSCT, he received 2 cy of anti-CD22 ab Inotuzumab, achieving CR without platelet recovery (CRi). Nevertheless, t(4;11) was positive again at the end of the 2 nd cy. Patient then received AZA as 3 th -line therapy achieving CRi after 2 nd cy. However, due to severe cytopenia and gastrointestinal toxicity, patient received 3 out of 5 days treatment of the 3 th cy and the 4 th cy was delayed. Patient relapsed after the 4 th course.

Summary/Conclusion: In our experience, single agent AZA alone was effective and well tolerated in heavily pretreated patients. To our knowledge, single agent HMA in KMT2Ar ALL has been reported only in one patient. Preclinical studies have revealed heterogeneous results when HMA are combined with cytotoxic drugs; results of two pilot trials of AZA combined with chemotherapy are still ongoing. Further clinical trials could clarify the optimal schedule, timings and dosage of HMA.

PB1761: ACUTE LEUKEMIA STATISTICS IN ARMENIA DURING 2010-2020

L. Harutyunyan 1,* , L. Sahakyan 1 , Y. Hakobyan 1

1 Armenian Hematology Association, Armenian Hematology Association, Yerevan, Armenia

Background: Acute leukemia (AL) is a group of hematological malignancies, the types of which include lymphoblastic and myeloblastic variants. In USA 60530 new cases of leukemia are diagnosed during 2020, of which about 30% is Acute myeloid leukemia (AML). The prevalence of AML in USA is 4.3:100000 per year during 2013-2017, while death rate is 2.8:100000 during 2014-2018. Compared to AML, Acute lymphoblastic leukemia (ALL) in 80% cases occurs in children. Being a malignant transformation and proliferation of lymphoid progenitor cells in the bone marrow, blood and extra medullary sites, ALL represents a devastating disease when it occurs in adults. The incidence of ALL follows a bimodal distribution, with the first peak occurring in childhood and a second peak occurring around the age of 50. While dose intensification strategies have led to a significant improvement in outcomes for pediatric patients, prognosis for the elderly remains very poor. Despite a high rate of response to induction chemotherapy, only 30–40% of adult patients with ALL will achieve long-term remission.

Aims: The aim of the current study was a comprehensive analysis of AL, i.e., their prevalence and incidence in Armenia for the period of 2010-2020.

Methods: The main data source for this study was outpatient and dispensary cards, hospitalization journals, as well as clinical data from the Register of Blood Diseases of the Hematology Center after. prof. R. Yeolyan. This information has been supplemented by the data from National Oncological Center after V.A. Fanarjyan as well as from death registration service at Ministry of Justice RA. Patients with newly diagnosed AL who were older than age 18 years, considered fit for intensive therapy, and diagnosed and treated in Armenia from January 1, 2010, until December 31, 2020, were included. Demographic indicators were derived from the database of Statistical Committee of Armenia ( https://www.armstat.am/en/ ).

Results: ALL incidence did not significantly change, however AML incidence significantly increased in women, with estimated annual percentage change (EAPC) of 2.6% during the whole period, and in men since 1998, with EAPC of 3.2%. Mortality rates were stable for both lymphoid and myeloid leukemia in both sexes. The median number of AL is 88.7 in Armenia during 2010-2020, making the highest number of patients 113 in 2015. The prevalence in Armenia was 2.6:10 4 during 2010-2012, and 2.7:10 4 for the next year. In 2014 it was 3.3:10 4 . In 2016, 2017, 2018, 2019 the prevalence was 2.9:10 4 , 3.2:10 4 , 3:10 4 , 2.8:10 4 , respectively. The highest prevalence was admitted in 2015, making 3.8:10 4 , otherwise the lowest was in 2020, making 2.4:10 4 . Median prevalence for 10 years is 2.9:10 4 in Armenia. The median number per year of acute promyelocytic leukemia (APL) patients is 3 during 2010-2020, and the APL median prevalence for the same span is 0.1:10 4 . 10-year median prevalence of ALL and AML is 0.6:10 4 and 1.12:10 4 , respectively.

Summary/Conclusion: Compared to USA, there are lower incidence trends in Armenia, however in 2015 rates were similar. The lack of declines of leukemia incidence and mortality could be attributed to late introduction of optimal therapies. As currently the most up-to-date diagnostics and treatments are available and covered by health insurance, we expect more favorable trends in the future.

PB1762: PONATINIB VERSUS IMATINIB WITH REDUCED-INTENSITY CHEMOTHERAPY IN PATIENTS WITH NEWLY DIAGNOSED PHILADELPHIA CHROMOSOME–POSITIVE (PH+) ACUTE LYMPHOBLASTIC LEUKEMIA (ALL): PHALLCON STUDY

E. Jabbour 1,* , G. Martinelli 2 , M. Vignetti 3 , H. Kantarjian 4 , D. Gomez-Almaguer 5 , Y. Minami 6 , H. Dombret 7 , M. Hennessy 8 , A. Vorog 8 , B. Wang 8 , J.-M. Ribera 9

1 The University of Texas, MD Anderson Cancer Center, Houston, TX, United States of America; 2 IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori”- IRST S.r.l, Meldola; 3 GIMEMA Research Foundation, Rome, Italy; 4 The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America; 5 Hospital Universitario, UANL, Monterrey, Nuevo Leon, Mexico; 6 National Cancer Center Hospital East, Kashiwa, Japan; 7 Institut de Recherche Saint-Louis, Université de Paris, Hôpital Sain-Louis, Paris, France; 8 Takeda Development Center Americas, Inc., Lexington, MA, United States of America; 9 Institut Català d’Oncologia-Hospital Germans Trias i Pujol, Josep Carreras Research Institute, Badalona, Spain

Background: Ponatinib (PON) is active against native BCR::ABL1 and all identified single-resistance mutations, including T315I. In newly diagnosed patients with Ph+ ALL treated with first- or second-generation tyrosine kinase inhibitors (TKIs), development of secondary resistance mutations in BCR::ABL1 is strongly associated with disease progression. Use of first-line PON in Ph+ ALL may decrease likelihood of these mutations and provide deeper, more durable responses. A Phase 2 study of PON in combination with chemotherapy in patients with newly diagnosed Ph+ ALL reported improved long-term outcomes with a 5-year event free survival and overall survival of 67% and 71%, respectively.

Aims: This study aims to compare efficacy and safety of first-line PON versus imatinib with reduced-intensity chemotherapy in patients with newly diagnosed Ph+ or BCR::ABL1 –positive ALL (p190/p210 transcript type).

Methods: PhALLCON ( NCT03589326 ) is a Phase 3, open-label, parallel-assignment, randomized study. Patients remain on treatment until relapse from complete remission (CR), have progressive disease, have unacceptable toxicity, withdraw consent, proceed to hematopoietic stem cell transplant (HSCT), complete study (20 cycles), are deceased, or sponsor terminates study. The trial will enroll ≈230 evaluable patients (aged ≥18 years) with ECOG performance status ≤2, randomized 2:1 to PON 30 mg/d or imatinib 600 mg/d orally, with reduced-intensity chemotherapy in induction (Cycles 1–3), consolidation (Cycles 4–9), and maintenance (Cycles 10–20). Enrollment is defined as randomized to study drug. Primary endpoint is minimal residual disease (MRD)-negative ( BCR::ABL1/ABL1 ≤0.01%) CR at end of induction (≈3 months). Key secondary endpoint is EFS, defined as dates of randomization until death (any cause), failure to achieve MRD-negative CR by end of induction, or relapse from CR. Other secondary endpoints include CR/incomplete blood count recovery rates, molecular response rates, MRD-negative CR duration, primary induction failure, overall response rate, CR duration, time to treatment failure, overall survival, and safety, including arterial occlusive/venous thromboembolic events. After induction, durations will be measured from first report. Other analyses include a subgroup analysis of patients with/without HSCT and an exploratory analysis by mutation status.

Results: First patient was randomized into the study in January 2019 with enrollment ongoing in 18 countries at 117 initiated sites; ≈226 patients were randomized as of February 2022.

Summary/Conclusion: This is the only registrational RCT Phase 3 study in Ph+ ALL, and it compares efficacy and safety of the only third-generation pan-inhibitor BCR::ABL1 TKI PON with first-generation imatinib in combination with chemotherapy in the first-line setting for adult patients with Ph+ ALL.

This abstract is an encore from the Society of Hematologic Oncology 2021 Annual Meeting.

PB1763: A PHASE 1/2 STUDY TO EVALUATE PONATINIB WITH CHEMOTHERAPY IN PEDIATRIC PATIENTS WITH RELAPSED/RESISTANT/INTOLERANT PHILADELPHIA CHROMOSOME–POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA OR WITH T315I MUTATION

L. Gore 1,* , M. Loh 2 , C.-H. Pui 3 , Y. Matloub 4 , M. J. Hanley 4 , J. Du 4 , M. Hennessy 4 , M. Granier 5 , A. Biondi 6 , L. Silverman 7

1 University of Colorado Cancer Center and Children’s Hospital, Aurora, CO; 2 University of Washington, Seattle, WA; 3 St. Jude Children’s Research Hospital, Memphis, TN; 4 Takeda Development Center Americas, Inc., Lexington, MA, United States of America; 5 Incyte Biosciences Intl Sarl, Morges, Switzerland; 6 Clinica Pediatrica Universita Milano-Bicocca, Monza, Italy; 7 Dana Farber/Boston’s Children’s Hospital, Boston, MA, United States of America

Background: Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) is associated with improved outcomes when tyrosine kinase inhibitors (TKIs) are added to chemotherapy; current 5-year event-free survival (EFS) and overall survival (OS) for newly diagnosed patients (pts) are 60% and 86%, respectively. Ponatinib (PON) is a potent third-generation TKI active against BCR::ABL1 and all identified single resistance mutations, including the gatekeeper T315I mutation that confers resistance to other TKIs. PON is approved for adults with chronic/accelerated/blast-phase chronic myeloid leukemia or Ph + ALL whose disease is resistant/intolerant to other TKIs or has the T315I mutation.

Aims: This study in pediatric pts with relapsed/resistant Ph+ ALL or with the T315I mutation aims to establish the recommended Phase 2 dose (RP2D) of PON and assess the pharmacokinetics, safety, and efficacy of PON in combination with chemotherapy.

Methods: This Phase 1/2, single-arm, open-label, multicenter study will enroll pts aged ≥1‒21 years with Ph+ ALL, Ph+ mixed-phenotype acute leukemia, or Ph-like ALL (United States only) who have a targetable kinase-activating lesion and whose disease has relapsed or is resistant/intolerant to ≥1 prior therapy with a BCR::ABL1 –targeted TKI or has a BCR::ABL1 T315I mutation. Phase 1 has a rolling 6 design; the first cohort will include up to 12 pts (≥30 kg and able to swallow tablets) for the dose confirmation. Another cohort of 6 pts will be enrolled to assess the age-appropriate mini-tablet formulation at the RP2D. Phase 2 will enroll ≈68 pts, including those enrolled in Phase 1 at the RP2D. Pts aged >16 years must have a Karnofsky performance status of ≥50%; pts aged ≤16 years must have a Lansky Play Scale score of ≥50%.

In Phase 1, the primary endpoint is the RP2D of PON in combination with chemotherapy. Secondary endpoints are complete response (CR) rate at the end of the reinduction block and characterization of BCR::ABL1 domain mutations before and after PON treatment. In Phase 2, the primary endpoint is the CR rate at the end of the reinduction block. Secondary endpoints include the proportion of pts in continued CR or who achieve CR at the end of consolidation, EFS, progression-free survival, and OS.

Results: The study is currently open and enrolling pts and will include ≈70 study sites in ≈16 countries.

Summary/Conclusion: This study will establish the RP2D of PON and assess pharmacokinetics, safety, and efficacy of PON in combination with chemotherapy in pediatric pts with relapsed/resistant Ph+ ALL or with T315I mutation.

This abstract is an encore from the Society of Hematologic Oncology 2021 Annual Meeting.

PB1764: THE CHARACTERISTICS, TREATMENT PATTERNS, AND SURVIVAL OF ADULT ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) TREATED BY GRAALL OR GRAAPH PROTOCOL

Y. Fakhfakh 1 , F. Kammoun 1,* , I. Frikha 1 , M. Charfi 1 , M. Chaari 2 , N. Louati 3 , N. Abdeljalil 4 , O. Kassar 1 , F. Kallel 1 , I. Ben Amor 1 , L. Khalifa 1 , M. Medhafar 1 , S. Hadiji 1 , H. Elleuch 2 , H. Mnif 3 , T. Ben Othman 4 , M. Elloumi 1

1 Clinical Hematology Depatment; 2 Biological Hematology Department, Hedi Chaker Hospital; 3 Flow Cytometry Unit, Regional Blood Transfusion Center, Sfax; 4 Allograft Department, National Bone Marrow Transplant Center, Tunis, Tunisia

Background: The clinical features, treatments, and survivals of adult ALL patients are very different from the pediatric ALL patients. However, since the use of pediatric-like protocols for Philadephia (Ph) negative ALL (GRAALL), and treatments based on tyrosine kinase inhibitor (TKI) for Ph + ALL (GRAAPH), great improvements in adult ALL therapy have resulted.

Aims: Our study aims to evaluate the characteristics, treatment patterns, as well as therapeutic results of ALL adult patients treated by GRAALL or GRAAPH protocol in a Tunisian center.

Methods: Our study is retrospective about newly diagnosed ALL adult patients (>30 years old) treated between 2004 and 2020 at the hematology department of Hedi Chaker Hospital, Sfax, Tunisia, according to the GRAALL protocol for ph negative ALL, and the GRAAPH protocol (TKI containing treatment) for ph+ ALL. Imatinib was the first line TKI used. Allogenic hematopoietic stem cell transplantation (Allo-HSCT) was indicated for Phi+ and high-risk phi negative patients aged less than 50 years, and in complete remission. It was done in the national bone marrow transplant center.

Results: A total of 70 patients were enrolled. Fifty-five patients were phi negative ALL treated with GRAALL protocol, and 15 were ph+ ALL and treated with GRAAPH protocol. We did not note any secondary ALL in our study. The clinical features, treatment patterns, and clinical outcomes are reported in Table 1.

The realization of the A-HSCT was a predictive adverse prognostic factor for OS and PFS in both groups (p=0.015).

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Summary/Conclusion: Our study is consistent with clinical trial findings of adult ALL reported in the literature, showing that compared with Phi negative patients, Phi + patients present more hyperleukocytosis forms and neurological involvement, and are more cortiocoresistant. However, they have higher complete remission rates and better survival due to the TKI- containing treatments. Moreover, despite the better therapeutic results in this group, our results are still lower than that reported in international studies, which incites us to a better involvement of A-HSCT, the only curative treatment for these patients.

PB1765: EFFICACY OF A NEW INDUCTION REGIMEN IN THE TREATMENT OF EARLY T-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA.

M. Wang 1 , J. Rao 1 , Q. Wen 1 , G. Chen 1 , X. Tan 1 , Y. Ma 1 , L. Gao 1 , L. Gao 1 , P. Kong 1 , C. Zhang 1 , X. Zhang 1,*

1 Medical center of hematology, Xinqiao hospital, Army medical university, Chongqing, China

Background: There is no standard induction chenotherapy for adult acute lymphoblastic leukemia. Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has poor response to standard intensive chemotherapy, high risk of recurrence and poor prognosis.

Aims: To explore the appropriate induction program to improve the remission rate and prolong the survival period.

Methods: A total of 21 newly diagnosed patients with ETP-ALL who received induction chemotherapy with different regimens in the Second Affiliated Hospital of Chongqing Army Military Medical University (Xinqiao Hospital) from January 2017 to December 2020 were retrospectively analyzed for efficacy and main adverse reactions.The initial diagnosis of ETP-ALL was based on the results of bone marrow cytology and flow cytometry immunophenotype.

Results: The 21 patients included 14 males and 7 females, with a median age of 27(16-57) years. Among them, 7 patients received PCIOD program and 14 patients received VTLP like program.Among the 7 patients who received PCIOD, 5 patients achieved cytological response (71.43%), and 2 patients were MRD negative (28.57%).In VTLP-like regimen, 7 patients (50%) had cytological remission, of which 1 patient (7.14%) was MRD negative.Major adverse reactions: bone marrow suppression, granulocytopia with fever and septicemia were the major adverse reactions after chemotherapy.Among the 7 patients with PCIOD, 3 patients developed pulmonary infection, including 1 patient with sepsis (klebsiella pneumoniae), while 5 patients with VTLP-like regimen developed pulmonary infection, 3 patients with sepsis, and 2 patients with acute pancreatitis.It did not increase the incidence of major adverse events compared with conventional regimens.There was no treatment-related mortality in any of the patients.

Summary/Conclusion: ETP-ALL induced remission rate is low, malignant degree is high, prone to early recurrence and short survival.PCIOD regimen can improve the complete response rate and MRD conversion rate of ETP-ALL patients, and does not increase the treatment-related mortality. However, due to the small number of cases, it needs to be further confirmed by multi-center, randomized controlled studies.

PB1766: PROGNOSTIC SIGNIFICANCE OF ACTIVIN A IN ADULT EGYPTIAN PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA.

M. Azzazi 1,* , H. Hegab 1 , A. EL-Ghammaz 1 , V. Shawky 1 , H. Hafez 1 , R. Helal 1

1 Hematology and Bone marrow transplantation, internal medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Background: Activin A belongs to the transforming growth factor-beta (TGFβ) superfamily of cytokines which exert a plethora of biological functions, including developmental differentiation, sex determination, control of cellular proliferation, migration and immune responses. Activins are dimeric glycoproteins that play a significant role in reproduction and in endocrine active tumors, although inhibins and activins have primarily been described in human gonads and identified as modulators of follicle-stimulating hormone production of the pituitary gland, they have also been detected in several solid tumor types, including endocrine-responsive endometrial, ovarian, and breast carcinomas. Their differential expression has suggested their important role in malignant cell transformation, as well as possible roles in cancer differentiation, proliferation, and growth tumors

Aims: The aim was to assess expression of Activin A in the serum of adult patients with acute lymphoblastic leukemia and its influence on remission and survival of ALL patients.

Methods: Serum Activin A was measured using ELISA in 30 acute lymphoblastic leukemia patients recruited from Hematology and Bone Marrow Transplantation Unit, Ain Shams University Hospitals and followed for 1 year in comparison to 15 healthy controls

Results: Serum level of Activin A was elevated in acute lymphoblastic leukemia patients (ALL) in comparison to control group with a statistically significant difference (P<0.001).A statistically significant negative correlation was detected between age of the patients and Activin A level(P= 0.035). The comparison between different outcomes of the patients (Remitted, Relapsed and died patients) above and below the mean level of activin A (265.667 ng/ml ),was statistically significant (P<0.001). A statistically significant negative correlation was detected between Activin A level in ALL patients and overall survival (OS) (P <0.001) and by using the log-rank test, a statistically significant difference (P <0.001) was detected in ALL patients above and below the mean level of Activin A. However, statistically nonsignificant difference was detected between the mean Activin A level in Philadelphia chromosome (Ph) positive patient and Ph negative patients (P= 0.839)

Summary/Conclusion: Activin A can be a useful poor prognostic biomarker in acute lymphoblastic leukemia patients, also it can be used as a predictor for aggressiveness of the disease, resistance and survival.

PB1767: OUTCOME OF RELAPSED CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA IN THE SOUTH OF TUNISIA SINGLE CENTER EXPERIENCE

N. Siala 1,* , I. Frikha 1 , M. Medhaffar 1 , I. Ben Amor 1 , I. Turki 1 , N. Ben Abdejallil 2 , Y. Fakhfekh 1 , N. Louati 3 , O. kassar 1 , M. Charfi 1 , F. Kallel 1 , L. Khelifa 1 , S. hadiji 1 , H. mnif 3 , T. Ben Othmen 2 , M. Elloumi 1

1 Department of Hematology, Hedi Chaker Hospital, Sfax; 2 National Bone Marrow transplantation, Tunis; 3 Blood center transfusion, Sfax, Tunisia

Background: Relapsed acute lymphoblastic leukemia (ALL) has remained challenging to treat in children, with survival rates lagging well behind those observed at initial diagnosis. Although there have been some improvements in outcomes over the past few decades, only ∼50% of children with first relapse of ALL survive long term, and outcomes are much worse with second or later relapses.

Aims: In this study we describe clinical characteristics and therapeutics results of first relapse of childhood ALL.

Methods: Our study included children under 16 years on first relapse of ALL treated according to the EORTC 58951 protocol between January 2000 and December 2020 in the Hematology department of Hedi Chaker Hospital of Sfax. The marrow relapse was defined as the reapparition in the peripheral smears and/or the increase of blasts (>20%) in bone marrow (BM) after a period of complete remission (CR). A combined relapse included another extra medullary location, neurological (N) or gonadal (G). Relapse treatment was based on the COOPRALL 1997 protocol and since 2008, COPRALL 2007 protocol or the VHR group of the 58951 EORTC protocol.

Results: Among the 262 Childs in CR, 81 was relapsed (31%) after a medium follow-up of 127 months. There are 27 females and 54 males. Median age was 7 years and 6 months. Relapse’s frequencies in the groups low risk, average risk 1, average risk 2 and high risk were respectively 4, 39, 18 et 39% of cases. Median of relapse timing was 12 months (2 to 83). Cumulative incidence of relapse at 1, 2 et 3 years after complete remission (CR) were respectively 51, 67 et 84%. Three relapses were occurred after 5 years of CR. Timing of relapse was not correlated with treatment group (p=0.8). The relapse was only medullary in 72% of cases, only neurological in 5% of cases and combined in 19% of cases (M+N= 6%, M+G= 9% et M+N+G= 4%). Second course was delivered to 57% of these patients. Protocol followed was the COOPRALL 1997 in 12 cases (26%), COOPRALL2007 in 28 cases (61%) and EORTC VHR in 6 cases (13%). The second CR (CR2) was get in 55% (44 patients) of treated patients, among them only 6 were allograft (24%). Toxic death before CR had occurred in 19% of treated cases. Among relapsed patients without curative treatment, only one was vivant (neurological relapse). Survivors after first relapse were less than 20%.

Summary/Conclusion: In our cohort, relapse’s rate was more than rates reported in other literature series (31% vs 10 to 15%). Relapse’s prognostic factors validated by the literature were the ALL’s phenotype, cytogenetic abnormalities (phi +), timing of relapse, combined relapses (medullary is more serious) and intensity of treatment. Those factors were not been find in our cohort because of the little number of cases. CR2 rates was less than literature (55 vs 80%); the revision of followed relapse’s protocol is to be discussed. As well, the survival of our patients after ALL relapse was less then reported rates (less than 20% vs 35% at three years). This is due to the abstention of treatment in patient in precocious relapse haven’t a sibling familial HLA donor.

PB1768: IS NATIVE E-COLI OR PEG-ASPERAGINASE FORMULATION A HIGHER RISK FOR VTE DEVELOPMENT IN PATIENTS WITH POST TREATMENT ACUTE LYMPHOBLASTIC LEUKEMIA? A META-ANALYSIS

J. T. Seki 1 , E. Atenafu 2 , H. Sibai 3,*

1 Leslie Dan Faculty of Pharmacy, University of Toronto; 2 Department of Biostatistics, Princess Margaret Cancer Centre, University Health Network; 3 Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada

Background: Thrombosis, is one of the serious complications of asparaginase-containing chemotherapy in Adult Acute Lymphoblastic Leukemia (AALL), reeks considerable morbidity and mortality. A large meta-analysis of 17 studies reported that symptomatic VTE in children is low (about 5%), post asparaginase-chemotherapy (Caruso 2006). Prospective screening of asymptomatic children revealed that the VTE rate can reach as high as 32% post native Asparginase (NEA) treatment (Mitchell LG 2003). Given the paucity of data in AYA, ALL patients receiving PEG-a, Underwood B. 2020 et al, reported an amazingly high VTE rate of 41%, with the cumulative incidence rate of 25% after an initial dose within 30 days. As age advance from the teen years, to adolescent and young adult (AYA), to older than 30 years, the VTE rates picked up momentum rapidly from 20%, 25% to 42% respectively (Grace RF et al in 2011). However, in a similar age group, Stock et al 2019 revealed a mere 5% VTE in patients with PEG-a experience.

Aims: Given the seemingly high symptomatic VTE incidence, especially in PEG-a recipients, we would like to get a better understanding of which asparaginase formulation instigating thrombosis more intensely while under the influence of pharmacological prophylaxis (LMWH) in the reduction of VTE rates.

Methods: Review of studies and abstracts from PubMed, Medline, published between 2006 and 2021 with study designs (excluding case reports) on VTE incidences reported as primary or secondary objective. Thrombosis incidences from the published studies using either the NEA or the PEG formulation in the treatment of AALL are identified. The VTE incidence from each of the NEA and PEG involved studies are abstracted. The significance of these is compared. Intervention with the use of LMWH prophylaxis, although in limited number of studies, for the prevention of VTE rates are compared for effectiveness between NEA and PEG-a groups.

Results: In this meta-analysis, the data showed that while LMWH as thrombo-prophylaxis is effective and beneficial in the NEA recipients [47/191 (24.61%) vs. 24/200 (12.00%), p=0.0012]. However, in spite of that, LMWH prophylaxis did not have significant impact on the development of VTE rates in the PEG-a treated patients (22/120 (18.33%) vs. 7/23 (30.43%), p=0.1861). This may be due to the fact that the number of patients receiving LMWH prophylaxis is low in adult patients receiving PEG-a containing protocol. A larger sample size and more prophylaxis containing PEG-a studies are needed to show a potential difference in the impact of LMWH in these patients.

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Summary/Conclusion: In this meta-analysis, the data showed that while LMWH as thrombo-prophylaxis is effective and beneficial in the NEA recipients (24.61% vs. 12.00%, p=0.0012). However, in spite of that, LMWH prophylaxis did not have significant impact on the development of VTE rates in the PEG-a treated patients (22/120 (18.33%) vs. 7/23 (30.43%), p=0.1861). This may be due to the fact that the number of patients receiving LMWH prophylaxis is low in adult patients receiving PEG-a containing protocol. A larger sample size and more prophylaxis containing PEG-a studies are needed to show a potential difference in the impact of LMWH in these patients.

PB1769: REAL WORLD DATA OUTCOMES IN A SINGLE MEXICAN PUBLIC INSTITUTION. ADOLESCENTS AND YOUNG ADULTS (AYAS) WITH NEWLY DIAGNOSED OF ACUTE LYMPHOBLASTIC LEUKEMIA:COMPARATIVE RESULTS OF DIFFERENT PROTOCOLS

G. Sotomayor Duque 1 , W. Nava Gutiérrez 1,* , S. Baltazar Arellano 1 , M. L. Guajardo Leal 1 , D. G. Cruz Contreras 1 , J. L. Cedillo de la Cerda 1 , H. P. Sorkee Davila 1 , J. A. Carrizales Villarreal 1 , M. P. Pequeño Luevano 1 , I. Borjon Cabada 1 , C. Quirino Marquez 1 , A. Mecott Estudillo 1 , G. Avila Contreras 1 , D. C. Aldama Gutiérrez 1 , D. Garza Escobar 1 , Y. Garcia Cerda 1 , V. Valerio Bugarin 1 , K. Machuca Adame 1 , L. O. Gudiño Cobos 1 , R. Saldaña Vazquez 1 , R. E. De León Cantu 1 , R. Hernadez Valdez 1

1 Hematology, IMSS, Monterrey, Mexico

Background: Acute lymphoblastic leukemia (ALL) is a hematopoietic neoplasm with arrest in lymphoid lineage differentiation. Adolescents and young adults (AYA) account for 50-67.3% of cases. Pediatric chemotherapy has increased survival from 48 to 70% in High income countries.

Aims: Primary objective is to compare Overall survival (OS) and event-free survival (EFS) in AYA with ALL treated with pediatric chemotherapy protocol versus adult chemotherapy protocol. Prognostic factors were evaluated as a secondary objective.

Methods: A Retrospective analytical observational study was conducted from January 1, 2018, to December 31, 2020 in Hospital IMSS UMAE 25 Monterrey, Mexico. OS and EFS were analyzed using Kaplan Meier method. Prognostic factors were evaluated with univariate and multivariate logistic regression (Odds Ratio, OR) and Hazard Ratio (HR) with Cox regression, p value of <0.05 (C 95%) was deemed statistically significant. The complete statistical analysis was conducted using SPSS version 20.0 (IBM, NY, USA).

Results: We include 61 patients (female 59%, male 41%) with median age 25 years (15-38). Rest of demographics is described in Table 1.

Median follow-up was 12 months (8.7-15.1). Global OS and EFS at 2 years was 32 % and 19% respectively. Thirty seven patients (64%) were treated with pediatric protocol (BFM-like); OS and EFS at 2 year was 38% and 22 % respectively, with median follow up of 10 months(5-5-19.5). Twenty four patients (36%) were treated with HyperCVAD protocol; OS was 22% and EFS at 2 years was 16 %, with median follow up of 8.8 months (8.7- 17.1).There were no significant differences in OS and EFS between the groups compared.

In univariate and multivariate analysis of prognostic factors the following results were found: hiperleukocytosis, OR 4.7 (IC 95% 0.9-23.0); positive BCR/ABL, OR 1.6 (IC 95% 1.04-1-3,); central nervous system infiltration, OR 1.9 (IC 95% 1.1-1.35); extramedullary infiltration, OR 1.9 (IC 95% 1.1-1.35). Induction failure (IF) was the only statistically significant risk factor with an OR 8.0 (IC 95% 1.6-39.3), p value 0.025.

When stratified by risk factors twenty-two patients (36.1%) were found to be standard risk and 2-year OS was 45.5% with median 28.5 months (4.3-52.7); with a single risk factor the 2-years OS was 30% with median survival of 14 months (8.6-19.4); two or more risk factors the 2- year OS was 5% with median of 5.9 months (4.1-11). (Log Rank p =<0.001).

Two year OS in IF patients was 8 % compared to 55% in those who achieved remission (HR 14.95, p =<0.001). Patients who achieved remission with pediatric regimens had better OS at 2 years compared with those who were treated with HyperCVAD (62% vs 34%, p =0.007).

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Summary/Conclusion: In conclusion, no significant difference was found in OS with none of the protocols; however, those patients who achieve remission with the pediatric regimen had better two-year survival compared with patients receiving HyperCVAD.

Response rates in our population, are lower compared to high income countries reports, which could suggest that biology of the disease in Latin American patients may have adverse prognostic markers that confer greater aggressiveness and chemo-resistance in our entity. We cannot forget to mention the great difference on supportive therapies and access to new, more effective drugs, that are practically impossible to access in low- and middle-income countries as ours, and have a direct impact on survival.

PB1770: REASONS FOR CHANGES OF BONE FORMATION PROCESSES IN CHILDREN WITH ALL LIVING IN RADIOACTIVELY CONTAMINATED TERRITORIES OF UKRAINE

K. Bruslova 1,* , N. Tsvietkova 1 , L. Lyashenko 2 , T. Pushkareva 1 , I. Tryhlib 1 , L. Honchar 1 , S. Iatsemyrskyi 2

1 Radiation Hematology for Children; 2 State Institution “National Research Center for Radiation Medicine of NAMS of Ukraine”, Kyiv, Ukraine

Background: The state of bone structures, stromal microenvironment of the bone marrow and their hormonal regulation can be affected by iron metabolism. The mechanisms of increased bone density (IBD) in children with ALL deserve special attention.

Aims: Identify the reasons of elevated bone density in children with ALL, who living in radiatioactively contaminated areas (RCA).

Methods: 96 children aged 2 to 18 years with common-B ALL were examined after completion of maintenance therapy according to the protocol. The children lived at the RCA of Ukraine and were examined from 2012 to 2021. Data were studied in a comparative aspect in ALL children of 2 groups: Group I (n = 36) - with elevated IBD, above 100 units (117.4 ± 10.2 units), Group II (n = 60) - with standard IBD (100-85 units, 87.4 ± 9.1 units). Bone fractures in the anamnesis, dental caries, obesity, biochemical parameters of blood (amino acids in urine, serum ferritin (SF), serum alkaline phosphatase (AF), cholesterol, calcium, vitamin D, free thyroxine, thyroid-stimulating hormone, pituitary hormone, cortisol) as IBD were evaluated. The doses of bone marrow irradiation in patients according to their length of stay on RCA were calculated. Conducted statistical processing of materials (t-Student, Spearman’s correlation coefficient, U-Test).

Results: The distribution of children by age in both groups did not differ. Boys (52 out of 60) predominated in Group I and in 30% of patients the levels of SF exceeded 500 ng / ml (728.8 ± 12.3 units). In children of both groups, a direct correlation was found between IBD and SF (p <0.001 and p <0.01, respectively). In children of Group I there was a direct relationship between IBD and fractures frequency (p <0,01). There were more children with low levels of total serum protein and glycine (p <0.01) in Group I, as number of children with high levels of alkaline phosphatase and cholesterol were less (p <0.05). IBD was inversely correlated with cortisol levels (rs = -0.73; p <0.001) and alkaline phosphatase activity (rs = -0.21; p <0.05). Dental caries in children in Group I was less frequent than in Group II (p <0.05). Survival of children was directly correlated with IBD (p <0.001). Irradiation doses in patients of Groups I and II ranged from 0.03 to 5.51 mSv (average 4.01 ± 0.17 mSv and 3.66 ± 0.18 mSv, respectively). In children, regardless of the surveillance group, there was a direct correlation between radiation dose, age of the child and the presence of obesity (p <0.001).

Summary/Conclusion: The state of bone homeostasis in children with ALL depends on anabolic and catabolic metabolic processes that provide energy substrate for bone formation, the level of iron stores in the body and hormonal regulation.

PB1771: THE SIGNIFICANCE OF MRD DETECTION BY FLOW CYTOMETRY IN TRACING R/R B-ALL PATIENTS RECEIVING CD-19-CART BRIDGING TO ALLO-HSCT

M. chen 1 , W. zhao 2 , M. fu 3 , H. Wang 4,*

1 Hebei Yanda Lu Daopei Hospital, Langfang, sanhe; 2 Beijing Lu Daopei Hospital; 3 beijing Lu daopei hospital, beijing; 4 clinical lab, Hebei Yanda Lu Daopei Hospital, Langfang, sanhe, China

Background: Minimal Residual Disease(MRD) detection by multicolor flow cytometry(MFC) is an important parameter for monitoring therapy effect in acute leukaemia. However, MFC panels for MRD detection after CD19-CAR-T treatment should change because the expression of CD19 will lose or partially lose.

Aims: To discuss the significance of MRD detection by MFC with cytoplastic CD79a(cCD79a) gating panel in relapsed or refractory B-cell acute lymphoblastic leukemia(r/r B-ALL) and evaluate its value for prognosis in CD19-CAR-T bridging to allogeneic-hematopoietic stem cell transplantation (allo-HSCT).

Methods: 37 r/r B-ALL patients who received CD-19-CAR-T infusion bridging to allo-HSCT were enrolled at Hebei Yanda Lu Daopei Hospital from January 2019 to July 2019. Patients had not been treated with CAR-T and/or allo-HSCT before. MFC is used to monitor the patient’s MRD and the dynamic of B cells. The median follow-up time of 37 patients after treatment was 890 days (104-1062). Binary logistic regression was used to retrospectively analyze the data.

Results: 8 patients (8/37, 21.62%) died and 29 (29/37, 78.38%) survived. when using death/survival as the end point in the dependent variable, MFC-MRD-positive recurrence ( P=0.0006 ), extramedullary infiltration ( P=0.047 ), and chromosomal karyotype changes ( P=0.047 ) showed significant differences between the two groups, as well as the remission rate of MRD on 15d ( P=0.011 ), the remission rate of the MRD on the 28d ( P=0.002 ), and the time from CD19 positive recovery to CAR-T before allo-HSCT ( P=0.020 ) after CAR-T reinfusion to allo-HSCT. P values of these indicators were all less than 0.05. the rest factors were not significant between the two groups. In the binary Logistic regression model of related factors during treatment and disease outcome, FCM-MRD remission on 28d can be used as a potential indicator to evaluate disease death or survival outcome, while other factors are excluded from the equation. The area under the ROC curve for the analysis of disease prognosis affecting factors was 0.762 ( P=0.026 ), OR=12.667, the sensitivity was 0.625, and the specificity was 0.897.

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Summary/Conclusion: The cCD79a antibody gated MFC-MRD monitoring can play a certain guiding significance in the prognosis judgment of the disease and the selection of the type of CART.

PB1772: IN-VITRO APOPTOTIC EFFECT OF NATURALLY OCCURRING COMPOUNDS, PIPERINE AND ASCORBIC ACID ON ACUTE MYELOID LEUKAEMAI (AML) CELL LINE.

R. Aljohani 1,* , C. Munialo 2 , S. Bokaee 2 , U. Osadolor 2 , S. Hewamana 3 , T. Lin 2

1 Faculty of applied medical sciences, Tabuk University, Tabuk, Saudi Arabia; 2 Coventry university, Coventry; 3 Leicester Royal Infirmary, Leicester, United Kingdom

Background: AML is a haematological malignancy, which is almost always fatal without treatment with survival ranging from few days to a few weeks. Myeloid cell formation, proliferation and apoptosis are disrupted in AML, caused by a range of genetic and epigenetic abnormalities. Piperine and Ascorbic acid are naturally occurring organic substances with potential anticancer capabilities. In addition various dietary and vitamin supplements are used during anti-cancer treatment.

Aims: We evaluated the in-vitro apoptotic effect of naturally occurring organic substances Piperine and Ascorbic acid in comparison to commonly used chemotherapeutic drug Fludarabine on an AML cell line (HL-60 leukaemia cells).

Methods: HL-60 cells were cultured in RPMI-1640 media supplemented with 10% (v/v) FBS, 1% (w/v) penicillin/streptomycin at 37°C with 5% CO 2 . Viability was assessed using trypan blue exclusion. Final concentration of 1 × 10 6 cells/well achieved using haemocytometer and annexin V-FITC/PI apoptosis was assessed by flow cytometry. Percentage apoptosis calculated at 24h time point using Piperine, Ascorbic acid and Fludarabine alone and in combination.

Results: Piperine (50 μM), Ascorbic acid (250 μM) and Fludarabine (50 μM) induced apoptosis in HL-60 cells at 24h time point compared to controls. The combination of Piperine and Ascorbic acid showed higher percentage apoptosis but the percentage apoptosis was significantly lower when Fludarabine was combined with either Piperine (P=0.0003) or Ascorbic acid (P=0.002).

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Summary/Conclusion: Our data shows interactions of naturally occurring organic compounds and chemotherapeutic agents in inducing in-vitro apoptosis. However, further studies are needed to access the clinical impact of taking naturally occurring organic compounds and chemotherapeutic agents together.

PB1773: MYBLOCK: A MYELOID BLOOD CANCER INITIATIVE

L. Cairns 1,* , K. Clarke 2 , M. Catherwood 2 , K. Mills 1

1 Patrick G. Johnston Centre for Cancer Research, Queen’s University Belfast; 2 Haematology Department, Belfast City Hospital, Belfast, United Kingdom

Background: MyBLOCk is a myeloid blood cancer initiative that encompasses a range of myeloid malignancies including Acute Myeloid Leukaemia (AML), Myelodysplastic Syndromes (MDS) and Myeloproliferative Neoplasms (MPN). These are a complex group of blood cancers that typically have poor overall survival. They result from genetic and epigenetic alterations that perturb key processes such as self- renewal, proliferation and differentiation. Despite improvements in overall survival, relapse rates in myeloid malignancies remain high. Standard of care therapies have not changed for many decades and often this intense chemotherapy is intolerable for older patients. Alongside this, there is an inherent complexity of these diseases. Over 70 different mutations have been identified in myeloid malignancies, patients typically display multiple mutations all of which contribute to the aggressiveness of the disease and the potential response to therapies.

Aims: The overall aim of the MYBLOCk initiative is to adopt a personalised medicine approach for the treatment of myeloid malignancies. Using existing standard of care therapies alongside novel agents we aim to identify the most effective combination of compounds for an individual patient, at time of diagnosis. The ideal drug combination can then be mapped to the mutational spectrum of these patients to gain further insight into the clinical picture.

Methods: We have created a custom panel of 48 compounds covering a range of different drug classes, many of which are already used in the treatment of myeloid malignancies. We want to investigate all possible pairwise combinations of these 48 compounds to determine the most effective combination for each patient, at diagnosis. Using a multiplex screening protocol that we have established in the lab, the compounds are grouped into pools of 5 compounds per well covering all potential pairs, this reduces the number of wells from 1128 to 172. Alongside this, a single agent screen of the 48 compounds is also performed. Peripheral blood mononuclear cells (PBMC) are added to the drug screen and incubated for 72 hours, the biological readout for this experiment is CellTox™ Green Express Cytotoxicity Assay.

Results: For each of the 172 5-compound combinations a Z-Score is calculated based on normalised relative fluorescence values. Combinations with a Z-Score>2 are taken forward for further investigation. A 5-compound combination is then deconvoluted into 10 pairwise combinations and 10 triple combinations, to identify a more clinically feasible combination for each patient. Alongside this screening strategy, each patient has undergone RNA sequencing at diagnosis to determine their mutational spectrum. By comparing the mutational landscape of each patient to their response to the MYBLOCk screening protocol we can investigate the relationship between molecular data and sensitivity to combination therapy. Multiple myeloid patient samples have been examined using this approach and have demonstrated a potential therapeutic backbone. Triple combinations involving a backbone of idarubicin and pracinostat with the addition of patient specific third agent has shown sensitivity.

Summary/Conclusion: Moving forward with the MYBLOCk initiative we will build a database of mutational information alongside sensitivity to the MYBLOCk panel to allow the rapid identification of personalised therapeutic options highlighting more effective, and novel, combinations for a wider range of patients at diagnosis.

PB1775: DEVELOPMENT OF ASSAY CALIBRATION MEHTHODOLOGY FOR CEPHEID XPERT® NPM1 MUTATION PROTOTYPE ASSAY

Y. Liu 1,* , M. Kao 1 , B. Saatian 1 , S. Kayhan 1 , Y. Zhao 1 , A. Aslam 1 , K. Shridhar 1 , L. Yuan 1

1 Oncology, Cepheid, Sunnyvale, United States of America

Background: Currently, no International Standards are available for the quantitation of the NPM1 mutation transcript for AML, posing a challenge when developing a quantitative assay to monitor the NPM1 transcript levels.

Aims: Implementation of a control material, containing in vitro transcribed RNA (IVT-RNA) of ABL and three major NPM1 subtypes (A, B and D), for standardization of ABL and NPM1 quantifications through copy number (CN) was used to develop an assay calibration methodology at Cepheid. This process generated an in-house control material for assay calibration and standardization of the Xpert NPM1 Mutation Prototype Assay.

Methods: A background comparison study was performed by testing contrived clinical samples in EDTA whole blood lysate and PBS lysate to demonstrate the background equivalency for assay performance. Titration studies were performed with Cepheid’s IVT-RNA, containing three subtypes of NPM1 (A, B and D) and ABL, to generate a 6-member control material of A20%, A5%, A1%, A0.2%, B1% and D1% in a PBS lysate background. The NPM1/ABL % value per member was obtained by the IVT-RNA copy number spiked in. A lot-specific curve of delta Ct vs NPM1/ABL % value was generated from the control material for the NPM1 Mutation Prototype Assay, providing an Efficiency Value ( E ▫▫Ct ) and a lot-specific scaling factor ( SF ). The NPM1/ABL% value of each clinical sample was calculated with E ▫▫Ct and SF . A 3-assay comparison study was performed to validate the assay calibration methodology with contrived clinical samples.

Results: The background comparison demonstrated the equivalency of assay performance in EDTA whole blood lysate and PBS lysate. The E ▫▫Ct and SF of 5 lots of the NPM1 Mutation prototype assay were generated with control material. NPM1/ABL % values of contrived clinical samples in EDTA whole blood from the NPM1 Mutation Prototype Assay were consistent with those from two other comparator assays, RT-ddPCR, and Ipsogen NPM1 MutaQuant assay(Table 1).

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Summary/Conclusion: Implementing a control material of IVT-RNA allowed linkage of known RNA CN to corresponding Ct, thus providing a methodology to assign an NPM1/ABL % value to any given clinical samples. A lot-specific E ▫▫Ct and SF for the NPM1 Mutation Prototype Assay were generated and utilized to calculate the NPM1/ABL % value for a clinical sample. Data from the 3-assay comparison suggested that we established a successful assay calibration methodology with IVT-RNA in PBS lysate background as control material and the NPM1/ABL % value reported by the NPM1 Mutation Prototype Assay was compatible with other comparator assays.

PB1776: ABC TRANSPORTERS AFFECT THE SENSITIVITY OF ACUTE MYELOID LEUKEMIA BLASTS TO CHEMOTHERAPY

E. Cerovska 1,2,* , C. Salek 1 , D. Kundrat 1 , A. Pesek 1 , M. Belickova 1 , H. Remesova 1

1 Institute of Hematology and Blood Transfusion; 2 Faculty of Science, Charles University, Prague, Czechia

Background: Approximately 40 % of acute myeloid leukemia (AML) patients does not achieve a complete hematologic remission (CR) after the first cycle of 3 + 7 induction therapy and thus face an adverse prognosis. Mechanisms of chemoresistance are still poorly understood, but drug export from cytosol plays a crucial role. 7 ABC transporters (ABCA2, ABCA5, ABCB1, ABCB6, ABCC1, ABCC3, ABCG2) were selected from literature for expression, methylation, genotype and efflux activity analysis.

Aims: The aim of this study is to clarify the role of selected ABC transporters in reduced sensitivity to chemotherapy in AML.

Methods: Peripheral blood was collected at diagnosis (day 0) and at the third day of 3 + 7 therapy (day 3). Leukemic blasts were separated on MACS (CD34+ or CD117+, Miltenyi Biotec). Gene expression was measured on StepOne Plus (Applied Biosystems) using TaqMan assays, B2M and YWHAZ were used as reference genes (n=85). Promotor, exons and splicing regions were sequenced on MiSeq (V2, Illumina) using Roche reagents (n=109). Methylation analysis was performed on Pyromark Q24 (Qiagen) for 85 CpGs in promoters of ABCA2 , ABCC1 and ABCG2 (n=51). Efflux activity was assessed with Efflux-ID kit (Enzo) (n=12). For data analysis, we divided patients by primary response status into responding and refractory group based on a CR achievement after the first cycle of 3 + 7 therapy. Data analysis was performed in GraphPad Prism 9.1.2 and R software 4.0.0.

Results: Higher expression of ABCA2 (p=0.005), ABCA5 (p=0.016), ABCC1 (p<0.0001) and ABCG2 (p=0.001) at day 0 was associated with primary treatment failure. ABCA2 (p=0.018) and ABCC1 (p=0.008) remained significant in the Cox regression analysis adjusted to age, leukocyte count at diagnosis and ELN risk stratification. Moreover, higher number of concurrently overexpressed ABC transporters (threshold defined by Cutoffinder) was predictive of failure in CR achievement (p=0.006) by Cox regression. We detected a significant rise of expression at day 3 in ABCA5 (p=0.003), ABCB6 (p=0.001) and ABCC3 (p<0.0001). Significantly higher rise of ABCC3 (p=0.029) expression was detected in responders compared to refractory patients.

In terms of a CR achievement, we identified 6 unfavorable DNA variants (rs2301837, rs8077268, rs17563146, rs1373970189, rs113369901, 9:137022522-137022522), and 3 favorable DNA variants (rs556735807, rs2235048, rs1407267536). The risk variant rs113369901 correlated with higher expression of ABCC1 (p=0.039), which was also associated with chemoresistance. Cox regression analysis confirmed prognostic relevance for four variants: rs2301837 (p=0.032), rs8077268 (p=0.033), 9:137022522-137022522 (p=0.003) and rs1373970189 (p=0.039).

Higher methylation of ABCG2 promoter correlated with its lower expression (p=0.0001). Promoters of ABCA2 and ABCC1 were basically unmethylated, especially ABCC1 , which had the highest expression among the studied genes.

The greatest contribution to cell efflux had transporters inhibited by MK-571 inhibitor, following by Verapamil. These influence activity of ABCC1 and ABCB1.

Summary/Conclusion: We described differences in expression levels and genetic variants of selected ABC transporters in association with treatment response in AML. ABCC1 seems to contribute substantially as illustrated by expression, genotyping, DNA methylation as well as activity data. With such detailed knowledge of chemoresistance mechanisms in leukemic cells and an emergence of new drugs, a different therapeutic strategy for chemoresistant patients may be considered in the future.

Supported by MHCR (00023736, IHBT).

PB1777: DEVELOPMENT OF NEXT GENERATION SEQUENCING PANELS FOR SCREENING FUSION GENES IN LEUKEMIA

S. Çiğdem 1,* , M. Arikan 1,* , E. Keskin 2

1 Biotechnology Institute; 2 Faculty of Agriculture, Department of Fisheries and Aquaculture, Ankara University, Ankara, Turkey

Background: Diagnosis of hematologic malignancies is based on multidisciplinary approaches including cytological, cytogenetic, and molecular genetic analyzes. Advances in cancer genomics have enabled the identification of recurrent mutations with the apparent prognostic value for various cancers. To date, hundreds of fusion genes resulting from chromosomal rearrangements have been identified in acute leukemia. Testing such mutations in leukemias is essential for personalized treatments. Many methods used in conventional clinical diagnosis could not fully detect fusion genes.

Aims: To overcome the limitations of existing techniques, targeted next generation sequencing approach was tested in this study.

Methods: The libraries of the regions containing the junctions of the fusion genes, formed by multiplex PCR-based panels, was deep-sequenced using Illumina platform, thereafter the results were analyzed by bioinformatics approaches.

Results: Using AmpliSeq (Illumina) fusion panel adapted for 20 KMT2A fusion transcripts and DNA fragments mimicking these fusion transcripts, 19 amplicons could be obtained with mPZR. With NGS procedures and bioinformatics analyses, significant results were obtained for 17 amplicons, while few junction reads that precisely describe the fusion event were obtained for the remaining 3. In titration studies, it was shown that the fusion panel can detect the template DNA fragments in trace amounts up to 1 pg.

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Summary/Conclusion: These results show that the fusion panel can be used in clinical diagnosis for the fusion transcripts in its scope, after testing on clinical specimens.

PB1778: NB4 CELLS CAN SURVIVE DURING ATO TREATMENT BY REGULATING THE NUMBER OF MITOCHONDRIA

S. Ding 1,* , J. Wang 1 , Y. Wang 1 , S. Cui 1 , Z. Li 2 , X. Yin 2 , J. Xu 1 , K. Liu 1 , R. Xu 1

1 hematology department, Affiliated hospital of shandong university of TCM; 2 The 1st clinical college, ShanDong University of TCM, Jinan, China

Background: Defects in mitochondrial dynamics are associated with many human diseases including leukemia.Therefore, targeting mitochondrial dynamic proteins is a strong candidate target for future treatments.

Aims: We aimed to explore whether mitochondrial dynamics play a role in Arsenic Trioxide(ATO)-induced apoptosis in acute promyelocytic leukemia (APL) NB4 cells and its mechanism.

Methods: (1) We cultured human APL NB4 cells. (2)The cells were treated with different concentration of ATO for 24/48 h, and the cell viability was measured by cell counting kit-8(CCK-8) to determine the IC50 of ATO.(3) The apoptotic rate was measured by Annexin V-FITC/PI double staining flow cytometry(FCM).the number of mitochondria was measured by Mitotracker Deep Red staining flow cytometry(FCM). (4) The mRNA expression levels of Drp1, Opa1, Mfn1, and Mfn2 are measured by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR).

Results: We found that during ATO-induced apoptosis, the mitochondrial abundance of surviving cells increased, the mitochondrial division gene Drp1 was up-regulated, and the expression of mitochondrial fusion genes Opa1, Mfn1, and Mfn2 was down-regulated.

Summary/Conclusion: Our results indicate that NB4 cells can survive during ATO treatment by regulating the number of mitochondria. It is mediated by regulating the expression of mitochondrial dynamic proteins Drp1, Opa1, Mfn1 and Mfn2.

PB1779: DEVELOPING NOVEL SYNERGISTIC DRUG COMBINATIONS WITH VENETOCLAX IN ACUTE MYELOID LEUKAEMIA

R. Harrigan 1,* , E. Johnston 1 , K. Matchett 1 , T. Bjourson 1

1 School of Biomedical Sciences, Ulster University, Derry, United Kingdom

Background: The treatment of AML remains a challenge due to complex genomic heterogeneity. In recent years, advancements in genomic technologies and the development of targeted therapies have changed the treatment paradigm, offering targeted treatments for patients with specific genomic vulnerabilities (1). The orally available BCL-2 inhibitor venetoclax is well tolerated and has recently received FDA approval in combination with hypomethylating agents (HMAs) for the treatment of newly diagnosed AML patients unfit for standard induction chemotherapy. Recent clinical trials have shown complete remission in around 70% of newly diagnosed AML cases with venetoclax and HMA regimens (2,3). However, relapses are common and there is still an unmet need for effective therapies with long-lasting remissions. The development of novel combinations allows for dual targeting of molecular drivers in AML. Recent studies have investigated novel venetoclax-based combination regimens to further improve anti-leukemia efficacy, evade drug resistance, reduce toxicities, and promote deep durable remissions. Venetoclax in combination with IDH1/2 inhibitors, FLT3 inhibitors, CDK inhibitors, and MEK inhibitors are among the most promising novel combination regimens currently being investigated (4–7).

Aims: This investigation aims to develop novel synergistic drug combinations with venetoclax via high throughput screening of >2000 compounds to improve venetoclax safety, efficacy and overcome resistance.

Methods: Venetoclax dose response experiments, Western blot analyses of BCL-2 protein expression and qPCR analyses of BCL2 gene expression were performe in a panel of 8 AML cell lines. A drug library comprised of 1,971 FDA approved compounds and 755 clinical compounds will be screened for anti-leukemia efficacy at 3 concentrations, in 3 AML cell lines at 3 timepoints. A secondary screen will combine successful candidate compounds with venetoclax and assess synergistic anti-leukaemic effect and ability to overcome resistance.

Results: Venetoclax IC50 values in the 8 AML cell lines range from <0.01µM to >5µM highlighting differential response among the panel. Western blot and qPCR analyses showed diverse BCL-2 protein and BCL2 gene expression in the 8 cell lines which may account for variation in response.

Summary/Conclusion: Further experiments will be performed to characterize mechanisms of induced cell death including target engagement analysis, Annexin V/ Propidium iodide staining. Candidate venetoclax-based combinations will be assessed for efficacy in a larger panel of cell lines and patient samples.

PB1780: AN INNOVATIVE INTERACTIVE CASE-BASED ONLINE EDUCATION TOOL SUBSTANTIALLY INCREASES KNOWLEDGE AND COMPETENCE OF CLINICIANS MANAGING HIGH-RISK AML

V. Harvey-Jones 1,* , S. Koekkoek 1 , G. Adair 1 , C. Craddock 2

1 Medscape, Global Education, London; 2 Centre for Clinical Haematology, University Hospitals Birmingham NHS Trust, Birmingham, United Kingdom

Background: Advances in digital technology platforms present the opportunity to develop new educational activities accessible to large numbers of health care professionals unable to leave their frontline clinical roles. Novel diagnostics and therapeutics are transforming treatment strategies in high-risk acute myeloid leukaemia (AML) and it is of vital importance that these breaking advances are communicated to clinicians as quickly and as effectively as possible. Up to 20% of AML cases may be secondary- or treatment-related (sAML, t-AML) to prior cancer therapy and are associated with worse outcomes. In order to achieve optimal outcomes, physicians caring for patients with AML need to have a solid understanding of the criteria for classifying newly diagnosed patients with these higher-risk subsets of disease, the factors involved in determining suitability for intensive treatment, and data supporting use of newer therapies for these patients so that they can make appropriate treatment decisions.

Aims: This study aimed to determine whether online continuing medical education could improve the knowledge, competence, and confidence of hematologists/oncologists (hem/oncs) in diagnosing and managing patients with high-risk AML, using a case-based approach.

Methods: Hem/oncs participated in a 22-minute segmented online multi-media activity consisting of videos portraying realistic physician–patient interactions followed by expert commentaries. Educational effect was assessed using a repeated-pair design with pre-/post-assessment. 3 multiple choice questions assessed knowledge/competence, and 1 question rated on a Likert-type scale assessed confidence. A paired samples t-test was conducted for significance testing on overall average number of correct responses and for confidence rating, and a McNemar’s test was conducted at the question or learning objective level (5% significance level, P <.05). Cohen’s d with correction for paired samples estimated the effect size of the education on number of correct responses (<.20 modest, .20-.49 small, .59-.79 moderate, ≥.80 large). Data were collected from 27/09/21 to 12/20/21.

Results: Overall, significant improvements in hem/onc knowledge and competence were seen after participation in the education (average correct response rate: 74% pre-assessment vs 87% post-assessment; P<.001, Cohen’s d = 0.58, N=95). 65% more hem/oncs answered all questions correctly after the education (43% pre, 71% post). Specifically, there were significant improvements in knowledge regarding the clinical trial data that should inform evidence-based decision-making for patients with newly diagnosed high-risk AML (61% pre-assessment vs 76% post-assessment; P<0.01), and competence related to the selection of the most appropriate treatment approach for the individual patient (81% pre-assessment vs 93% post-assessment; P<0.001). 42% had a measurable increase in confidence (P<0.001), resulting in 54% who were mostly or very confident in selecting the most appropriate treatment for patients with high-risk AML post-education (27% pre-education). Finally, 95% (N=77) reported the education will improve their performance and 89% that it would improve patient outcomes.

Summary/Conclusion: This study demonstrates the ability of innovative online, interactive case-based models to transform access to new educational opportunities and improve knowledge and competence of hem/oncs in diagnosing and managing patients with high-risk AML. This could lead to earlier diagnosis and improvements in the management of high-risk AML.

PB1781: THERAPEUTIC POTENTIAL OF DNA DAMAGE RESPONSE IN ACUTE MYELOBLASTIC LEUKEMIA

B. Lapa 1,2,3 , M. I. Costa 1 , J. Jorge 1,2,3,4 , R. Alves 1,3,4,5 , A. B. Sarmento-Ribeiro 1,3,4,5,6,* , A. C. Gonçalves 1,3,4,5

1 Laboratory of Oncobiology and Hematology, Faculty of Medicine, University of Coimbra; 2 Coimbra Institute for Clinical and Biomedical Research (iCBR)—Group of Environmental Genetics of Oncobiology (CIMAGO), Faculty of Medicine of University of Coimbra; 3 Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra; 4 Clinical Academic Center of Coimbra (CACC); 5 Coimbra Institute for Clinical and Biomedical Research (iCBR)—Group of Environmental Genetics of Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra; 6 Clinical Hematology Service, Centro Hospitalar e Universitário de Coimbra (CHUC), Coimbra, Portugal

Background: Genome integrity is of utmost importance for cell survival and homeostasis maintenance. However, DNA integrity is threatened by endogenous and exogenous sources. DNA lesions can block genome replication and transcription, and if unrepaired or repaired incorrectly can lead to mutations and genomic instability. Hence, cells have developed a variety of mechanisms to protect genome integrity and repair damaged DNA, collectively termed the DNA damage response (DDR). These mechanisms comprise DNA damage recognition, damage tolerance, signal transduction, transcriptional regulation, cell cycle control, and DNA repair. Key DDR elements include sensor proteins that detect damage (e.g. PARP1 and 2), mediators and signaling transducers (e.g. DNA-PK) that regulate downstream targets, and effector molecules (e.g. CHK1 and 2). Increased DNA damage and altered DDR are critical features of genetic instability presumably implicated in the pathogenesis of acute myeloblastic leukemia (AML). AML is a clonal malignant disease of hematopoietic stem and/or progenitor cells characterized by a block in myeloid differentiation and increased proliferation. This neoplasia is considered the most common acute leukemia in adults and is characterized by recurrent genetic abnormalities. Recently, specific recurrent chromosomal translocations and genetic mutations typical of AML have been associated with DNA repair defects.

Aims: Our goal is to assess the potential of DDR as a therapeutic target in AML, using in vitro models, in order to identify new therapeutic approaches.

Methods: Five AML cell lines of different molecular and cellular subtypes were used (HEL, HL-60, K-562, LAMA-84, and NB-4). Cells were incubated in the absence and presence of increasing concentrations of two DDR inhibitors, CCT245737 (CHK1 inhibitor) and Niraparib (PARP1/2 inhibitor). Cell density and viability were assessed, for 72 hours, by trypan blue assay. Afterward, resorting to flow cytometry, the cell cycle was assessed using propidium iodide/RNAse, and the cell death was determined using Annexin V/7-AAD double staining as well as by optic microscopy (May-Grünwald-Giemsa staining). Results were statistical analyzed, considering a significance level of 95%.

Results: CCT245737 reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. NB-4 was the most sensitive cell line, with an IC 50 at 48 hours of 54 µM. In contrast, LAMA-84 was the most resistant cell line with an IC 50 at 48 hours of 203 µM, 3.7-fold higher than NB-4. PARP1/2 inhibition also reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. Once again NB-4 was the most sensitive cell line, with an IC 50 at 48 hours of 25 µM and LAMA-84 was the most resistant with an IC 50 at 48 hours of 66 µM, 2.7-fold higher than NB-4. Both inhibitors induced cell death by apoptosis since an increase in the percentage of cells in late apoptosis was observed. This result is in agreement with the observed by optic microscopy. We also detected a cytostatic effect since a cell cycle arrest in the S phase was observed after treatment with both inhibitors.

Summary/Conclusion: AML cell lines have different sensibilities to DDR inhibitors, particularly CCT245737 and niraparib, with the NB-4 cell line appearing to be the most sensitive to DDR inhibition (namely CHK1 and PARP1/2 inhibition), whereas LAMA-84 appears to be the most resistant to this inhibition. In conclusion, this work may help to identify new therapeutic approaches that could eventually improve AML patients’ outcomes.

PB1782: ZINC IN THE MODULATION OF THE ANTIPROLIFERATIVE AND CYTOTOXIC EFFECTS OF OLAPARIB IN ACUTE MYELOID LEUKEMIA

M. I. Costa 1 , B. Lapa 1,2,3 , J. Jorge 1,2,3,4 , R. Alves 1,2,3,4 , A. B. Sarmento-Ribeiro 1,2,3,4,5,* , A. C. Gonçalves 1,2,3,4

1 Laboratory of Oncobiology and Hematology; 2 Coimbra Institute for Clinical and Biomedical Research (iCBR)—Group of Environmental Genetics of Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra; 3 Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra; 4 Clinical Academic Center of Coimbra (CACC); 5 Clinical Hematology Service, Centro Hospitalar e Universitário de Coimbra (CHUC), Coimbra, Portugal

Background: Micronutrients are cofactors and substrates of several DNA repair enzymes. Among them, zinc (Zn) is particularly relevant in the regulation of oxidative stress, apoptosis, cell cycle, DNA synthesis and DNA damage response (DDR). DDR comprises a variety of mechanisms that protect genome integrity and repair damaged DNA. Acute myeloid leukemia (AML) is a clonal malignant disease of hematopoietic stem and/or progenitor cells characterized by a block in myeloid differentiation and increased proliferation, which displays DDR defects. Additionally, patients usually present decreased levels of serum Zn, which impact in the DDR is not completely understood. Further, studies have shown a role for Zn in the modulation of anticancer therapy in some malignancies. However, its effects in the modulation of therapeutic compounds in AML is not well understood.

Aims: The aim of this work was to investigate and characterize the potential of zinc sulfate (ZnSO 4 ) in the modulation of genotoxic compounds (cytarabine; ara-C) and DDR inhibitors (olaparib; Ola) in cell lines of several AML subtypes.

Methods: The AML cell lines, HEL, NB-4, and K-562 cells, were incubated in the absence and presence of ara-C and Ola in monotherapy and in combination with ZnSO 4 (IC 25 ) for 72h. Cell density and viability were evaluated by trypan blue exclusion test at each 24h. Cell death was assessed by flow cytometry (FC), using the Annexin V and 7-AAD double staining, and by optical microscopy through May-Grünwald-Giemsa staining. Cell cycle was analyzed by FC using PI/RNAse. Results were statistical analyzed, considering a significance level of 95%.

Results: Ola in monotherapy decreased cell density in a dose- and time-dependent manner (p<0.05), while viability varied depending on the cell line. HEL cells were the most sensitive to Ola. The estimated IC 50 of Ola at 48h were of 51μM, 173μM, and 180μM for HEL, K-562, and NB-4, respectively. The effect of ara-C in monotherapy was also cell line dependent. The most sensitive cell lines were HEL and NB-4, and the least were K-562 cells (IC 50 at 48h: 2μM, 2μM, and 23μM, respectively). ZnSO 4 increased the cytostatic and cytotoxic effects of Ola and ara-C in all cell lines (p<0.05). Combination of Ola and ZnSO 4 reduced 2.9-fold, 1.6-fold, and 7.5-fold the IC 50 in HEL, NB-4, and K-562 cell lines, respectively, after 48h. Similarly, combination of ara-C and ZnSO 4 also decreased the IC 50 4.8-fold, 19-fold, and 19.2-fold the IC 50 in HEL, NB-4, and K-562 cell lines (after 48h). Combinations with ZnSO 4 increased the percentage of cells in apoptosis, comparatively to monotherapy (p<0.05). The morphological analysis also showed that ara-C and ola in monotherapy as well as in combination with ZnSO 4 induced cell death mainly by apoptosis. Moreover, the association of Zn with ara-C increased the cytostatic effect in HEL and K-562 cells, leading to cell cycle arrest in G 0 /G 1 (p<0.05). Similarly, combination of Ola with ZnSO 4 induced G 0 /G 1 arrest in HEL cells (p<0.05).

Summary/Conclusion: The results suggest that Zn synergize with ara-C and Ola in AML cells, increasing the cytostatic and cytotoxic effects of these compounds. The considerable reduction of the IC 50 of the ara-C and Ola in the presence of Zn may result in more efficient therapeutic responses.

PB1783: IDENTIFICATION OF RAMAN SPECTROSCOPIC SIGNATURE OF IDH MUTATIONS IN AML IN VITRO MODEL

P. Laskowska 1,* , A. Nowakowska 2 , S. Orzechowska 2 , A. Borek-Dorosz 2,3 , J. Stolarska 2 , A. Adamczyk 2 , P. Leszczenko 2 , M. Zasowska 1 , A. Szlachetka 1 , M. Szydłowski 1 , E. Białopiotrowicz 1 , P. Juszczyński 1 , M. Barańska 2,3 , K. Majzner 2,3 , P. Mrówka 1,4

1 Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Warsaw; 2 Faculty of Chemistry, Jagiellonian University; 3 Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow; 4 Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Warsaw, Poland

Background: Current acute myeloid leukaemia (AML) laboratory diagnostics is based on genotyping and immunophenotyping. Although these methods revolutionized the way we understand, diagnose and treat myeloid malignancies, it does not free from disadvantages such as cost or lack of functional information on cells state. Raman spectroscopy, the label-free method based on the specificity of light inelastic scattering, has potential to overcome those limitations and enrich diagnostic process with new data. Raman spectra serve as ‘molecular fingerprints’ characterizing biochemical composition of complex cellular structures. We utilized Raman microscopy to analyse the influence of single mutation on Raman spectral images of leukemic cells. Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are recurrently identified, actionable driver events in adult patients with AML. The mutant form of IDH enzymes convert alpha-ketoglutarate to 2-hydroxyglutarate. Accumulation of the latter impairs histone demethylation and leads to the interruption of gene expression. These properties make IDH mutants an ideal target for attempting the label-free identification using Raman spectroscopy.

Aims: The aim of the study was to identify Raman spectroscopic signature of IDH oncogene-driven molecular changes in in vitro AML models.

Methods: IDH1/R132H, IDH2/R140Q, and IDH2/R172K constructs were retrovirally transduced into HEL and HL-60 cell lines. Cells were imaged using the confocal Raman system WITec Alpha 300. Wild-type forms of genes and empty pMIG plasmid served as control samples. Collected Raman maps were processed with K-means cluster analysis (KMCA) followed by Principal Component Analysis (PCA) and Partial Least Squares–Discrimination Analysis (PLS-DA) performed on averaged spectra of cells and subcellular components.

Results: PCA analysis revealed that IDH1/2 mutations (R132H, R140Q and R172K) carrying cells show subtle but significant differences in protein and nucleic acids composition in comparison to control cells. The most significant influence on Raman spectra was observed for IDH1 R132H mutation. Raman features in PCA loadings were also related to proteins and nucleic acids. Using the PLS-DA method, we built a model for distinguishing transgenic IDH1/2-mutated models from IDH wild-type cells.

Summary/Conclusion: The results confirm the utility of Raman spectroscopy/microscopy to identify the changes between mutated and wild type forms of IDH1/2 suggesting the potential of optical methods for studying point-mutations in cancer cells and subsequently screening of leukaemia. Further studies to improve and optimise the method and collect more data for machine learning are warranted.

PB1784: CYTOGENETIC LANDSCAPE OF 87 MOROCCAN ACUTE MYELOID LEUKEMIA PATIENTS

E. M. Mahtat 1,* , H. MOUSSA BOUH 1 , S. JENNANE 1 , M. ABABOU 1 , S. AHCHOUCH 1 , A. HAMMANI 1 , H. EL MAAROUFI 1 , H. EDDOU 1 , K. DOGHMI 1

1 Clinical Hematology, Hôpital Militaire Mohamed V Rabat, Rabat, Morocco

Background: Cytogenetics and molecular biology are the most important diagnostic tools for determining prognosis in acute myeloid leukemia (AML). In our country, access to molecular biology tests is limited compared to conventionnal cytogenetics. Still data about cytogenetics in AML moroccan patients are scarce.

Aims: Describe cytogenetic landscape of patients diagnosed with acute myeloid leukemia treated in our center.

Methods: We conducted a descriptive retrospective analysis of 87 adult patients diagnosed with acute myeloid leukaemia treated in our unit.

Results: 87 patients diagnosed with acute myeloid leukmemia were included. Median age was 45 years and 25% of patients were older than 60 years. The cytogenetic analysis failed in 13 patients (15%). Prognostic group distribution according to the ELN 2017 was found to be favorable in 17 patients (19,5%) with 6 cases of t (8; 21) and 7 cases of inv (16), 4 patients had t(15;17). Intermediate group was predominant (48,5%), 28 patients (32%) had normal karyotype.15 patients (17%) had unfavourable cytogenetics including 9 patients with complex karytype and 3 patients with del7q. Intermediate risk cytogenetics were predominant in patients under 60 years old whereas Unfavourable karyotypes were more frequent in older patients.

Summary/Conclusion: The landscape of karyotype abnormalities in our population found a predominance of intermediate risk group enhancing the need of molecular biology to refine prognosis and guide the treatment of AML patients in Morocco.

PB1785: DISCOVERY OF APPROVED LOW-TOXICITY AGENTS DISRUPTING ACUTE MYELOID LEUKEMIA STEM AND PROGENITOR CELLS

J. Meilstrup 1,* , C. Leitch 2,3 , M. Kriegbaum 1 , V. Andresen 2 , B. T. Gjertsen 2,4 , K. Wennerberg 1

1 Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark; 2 Centre of Cancer Biomarkers CCBIO, Department of Clinical Science, University of Bergen; 3 KinN Therapeutics AS; 4 Department for Medicine, Haematology Section, Haukeland University Hospital, Bergen, Norway

Background: Acute myeloid leukemia (AML) is a disease driven by leukemia-initiating cells (LIC) that resemble early myeloid progenitors. The standard treatment of AML is intensive chemotherapy (IC) along with stem cell transplantation. However, with the average age of AML diagnosis being approximately 70 years, many AML patients are ineligible to receive IC. Instead, elderly patients are primarily treated with low-toxicity chemotherapy, originally, hypomethylating or low-dose cytotoxic agents and more recently, venetoclax in combination with hypomethylating agents. These treatments, however, are generally inadequate to eradicate the LICs and the disease almost invariantly recurs with poor overall survival. As such, there is an ongoing need to identify novel options of higher efficacy and lower toxicity treatment for elderly AML patients.

In this context, many generally safe non-hematologic drugs exhibit sporadic adverse effects on bone marrow function, including cytopenias and bone marrow aplasia. This suggests that these low toxicity drugs affect myeloid progenitor cells and potentially target LICs in AML.

Aims: To explore whether low toxicity drugs with known adverse effects on bone marrow function may be able to target AML initiating cells, and hence suggest their potential to be repositioned for treatment of elderly AML patients ineligible for IC.

Methods: Experiments were carried out on primary AML patient samples and flow cytometry was used to identify subpopulation specific responses. Both short (5 days) and long-term (up to 28 days) assays were performed to explore the effects of drug treatment on the AML growth and cell renewal capacity.

Results: Seven AML patient samples were screened in a 5-day ex vivo assay using a library of 21 low toxicity approved drugs with known myeloid adverse effects. Flow cytometry analysis identified four drugs; chloramphenicol, mefloquine, phenylbutazone and pyrimethamine, that target AML cells and decreased viability in a dose-dependent manner. These four drugs presented with tendency of low toxicity by preferentially targeting the AML cells rather than lymphocytes in the patient samples. This was found to be statistically significant for two of four drugs (P<0.05).

Single treatments of the drugs were tested in primary AML sample long-term cultures and showed significant inhibition of the sample expansion as compared to control. Sequential flow cytometric analyses of the AML subpopulations showed that the drugs targeted the least differentiated subpopulations, in which the LICs are expected to reside. Such observations would support the treatments ability to target cells at the top of the AML hierarchy, which subsequently result in a loss of regeneration capacity and slower diminish of the leukemic bulk cells.

Next, four AML samples were screened to observe for synergistic interactions between the drugs. We identified chloramphenicol-phenylbutazone combination of interest for continued investigation in long-term culture and in vivo settings.

Summary/Conclusion: There is a potential of non-hematologic low toxicity drugs to be repurposed for AML treatment. Approved drugs reported to cause bone marrow failure may have an impact on the hematopoietic progenitors and if the drugs likewise affect the stem and progenitor cells in leukemia this could be translated into a therapeutic effect. By investigating some of these drugs on primary AML patient samples, we observed that several could selectively target leukemic progenitor populations. Taken together, this emphasizes the importance for further investigation of repurposing drugs for novel treatments toward AML.

PB1786: COMPARISON OF OPTICAL GENOME MAPPING WITH STANDARD OF CARE TESTING TO INVESTIGATE GENOMIC ABNORMALITIES IN LEUKAEMIAS: THE EXPERIENCE OF THE CENTRAL AND SOUTH GENOMIC LABORATORY HUB, UK

M. Muraru 1,* , A. Skowronska 1 , S. Dyer 1 , A. Beggs 2 , J. Woodley 1 , C. Poxon 2 , J. Mason 1

1 West Midlands Regional Genetics Laboratory, Birmingham Women’s and Children’s NHS Foundation Trust; 2 Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom

Background: Here we present the Central and South Genomic Laboratory Hub experience of verifying Optical Genome Mapping (OGM). OGM a has great potential to become the new gold standard for detecting structural and copy number variations (SVs and CNVs) identified by cytogenetic techniques for routine use in a diagnostic laboratory environment. A range of haematological referrals were analysed by OGM and results were compared with standard of care testing consisting of G-band metaphase analysis, Fluorescence in Situ Hybridisation (FISH) and SNP array analysis. The use of OGM in various research and diagnostic labs is being validated and implemented around the world.

Aims: Our objective was to compare the workflows in terms of processing and analysis times and concordance of results.

Methods: This next generation cytogenomic technique consists of isolating ultra-high molecular weight DNA, genome wide labelling and imaging of the molecules using the Bionano Saphyr system. This allows the acquisition of big amount of data in parallel followed by analysis using the Bionano Access software. Bionano Genomics Rare Variant Pipeline (RVP) was used for somatic variant detection.

Results: We show the concordance between standard of care tests and OGM in the analysed cohort and also present details of additional aberrations detected by OGM, due to its superior resolution and sensitivity compared to G-band analysis.

Summary/Conclusion: With improvements to capacity on the Saphyr instrument, and automation of key processes, we believe that OGM has the potential to replace G-band chromosome analysis currently used as a global screen in routine diagnostics for haematological malignancy.

PB1787: GADD45A IS OVER-EXPRESSED IN THE BONE MARROW OF PATIENTS WITH DE NOVO ACUTE MYELOID LEUKEMIA REFRACTORY TO INDUCTION CHEMOTHERAPY

A. Bouchla 1,* , G. Deliconstantinos 1 , T. Loupis 2 , F. Kontsioti 1 , V. Mpakou 1 , M. Delatola 3 , M. Pagoni 3 , K. Theodorakopoulos 1 , S. Papageorgiou 1 , V. Pappa 1

1 Second Department of Internal Medicine and Research Institute, Attikon University General Hospital; 2 Haematology Research Lab, Clinical, Experimental and Translational Research Center, Biomedical Research Foundation, Academy of Athens; 3 Hematology-Lymphomas Department and BMT Unit, Evangelismos General Hospital, Athens, Greece

Background: Acute myeloid leukemia (AML) is a life-threatening malignant myeloid disorder with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment.

Aims: The aim of this study was to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and verify the findings in AML patient samples at diagnosis before the onset of chemotherapy.

Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT 2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression was evaluated and analyzed through the RT 2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of lymphoma patients, used as controls, and from AML patients at diagnosis who subsequently received chemotherapy with the 7 + 3 regiment. Response to first-line chemotherapy was recorded. Total RNA was isolated (RNeasy plus Mini Kit, Qiagen) and quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Green-based real-time RT-PCR (QuantiTect ® Primer Assay, Qiagen). Statistics were performed using linear regression.

Results: The following genes were over two-fold up-regulated in live cells from leukemic cell lines after treatment with either agent: PPP1R15A and CDKN1A in both cell lines, GADD45A and GADD45G in live MV4-11 cells and EXO1 in live Kasumi cells. These genes were selected for relative quantification; 25 control samples and 38 AML samples (28 responders and 10 non-responders to treatment) were evaluated. GADD45A in AML compared to controls was found 2-fold up-regulated in treatment responders (ns) and 9-fold up-regulated in non-responders [p=0.01]. In addition, GADD45A was overexpressed in non-responders compared to responders [p=0.02]. PPP1R15A in AML compared to controls was 12-fold up-regulated in responders [p=0.001] and 16-fold up-regulated in non-responders [p=0.001]. (Figure 1)

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Summary/Conclusion: GADD45A controls the balance between apoptosis and autophagy via the p38 and JNK/MAPK pathways and PPP1R15A participates in growth arrest and apoptosis in response to DNA damage by attenuating the translational elongation of key transcription factors. Both genes are likely to play an important role in the pathogenesis of AML. Furthermore, GADD45A overexpression appears to be related with resistance to induction treatment, which may have significant prognostic and therapeutic implications.

PB1788: LOW EXPRESSION OF C-TYPE LECTIN-LIKE MOLECULE-1 AND REDUCED OXIDATIVE STRESS DEFINE A SUBSET OF ACUTE MYELOID LEUKAEMIA CELLS WITH ‘STEM-LIKE’ PROPERTIES

K. Sharp 1,* , E. Ladikou 1,2 , T. Burley 1 , E. Kennedy 1 , A. Pepper 1 , C. Pepper 1

1 Brighton and Sussex Medical School; 2 Department of Haematology, Brighton and Sussex University Hospital Trust, Brighton, United Kingdom

Background: Leukaemic stem cells (LSCs) are an important therapeutic target in acute myeloid leukaemia (AML) as failure to fully eradicate these cells is thought to drive disease relapse. Therapeutic targeting of LSCs has been hindered by the controversy surrounding their phenotypic characterisation. So, in this study, we built a multi-colour immunophenotyping panel, which included C-type lectin-like molecule-1 (CLL-1), to address the possible shortcomings of using CD34 and CD38 alone as LSC markers. CLL-1 is preferentially expressed on the majority of AML cells, but not on haematopoietic stem cells. Here we identified heterogeneity of CLL-1 expression within the CD34 + CD38 - compartment of the KG1a AML cell line, which led us to explore whether this marker could more accurately identify LSCs.

Aims: We set out to phenotypically and functionally characterise the ‘stem-like’ sub-population in the KG1a cell line. Ultimately, we aim to determine whether we can preferentially target these ‘stem-like’ cells for therapeutic benefit.

Methods: We used the AML KG1a cell line as it contains a sub-population of CD34 + CD38 - ‘stem-like’ cells. A 9-colour immunophenotyping panel (comprised of CD34, CD38, CD47, CD71, CD90, CD117, CD123, HLA-DR & CLL-1), was used to look for heterogenous expression of ‘stemness’ markers in KG1a cells. Subsequently, we used fluorescence activated cell sorting (FACS) to purify phenotypically distinct sub-populations and compared their growth kinetics, relative viabilities and cell cycle distributions in hypoxia and normoxia. We also compared their metabolic activity using the redox-sensitive probe CellROX to assess relative levels of reactive oxygen species (ROS) as a readout of oxidative stress.

Results: Most of the immunophenotypic markers in our flow cytometry panel showed homogeneous expression patterns in KG1a cells. In contrast, we observed heterogeneity in CLL-1 expression, particularly in the CD34 + CD38 - sub-population. CD34 + CD38 - CLL-1 - cells were smaller in size than their CD34 + CD38 - CLL-1 + counter-parts and represented approximately 2% of the entire CD34 + CD38 - subset. Cell sorting allowed us to isolate highly purified CD34 + CD38 - CLL-1 - & CD34 + CD38 + CLL-1 + sub-populations (Figure 1). After four days in culture, we found a 5.5-fold preferential expansion of the CD34 + CD38 + CLL-1 + cells relative to the CD34 + CD38 - CLL-1 - cells (P<0.0001). However, both subsets expanded and remained viable in culture. The lower growth rate observed in the CD34 + CD38 - CLL-1 - sub-population was associated with a significantly higher percentage of G₀/G 1 cells than the CD34 + CD38 + CLL-1 + sub-population (91% vs 49%; P=0.0001), respectively. We then went on to show that this subset of CD34 + CD38 - CLL-1 - cells were metabolically distinct from the bulk tumour. Purified CD34 + CD38 - CLL-1 - cells contained significantly lower levels of ROS than CD34 + CD38 + CLL-1 + cells (P=0.048; Figure 1).

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Summary/Conclusion: We identified a CLL-1 - ROS low sub-population within the CD34 + CD38 - compartment of the KG1a cell line that is both phenotypically and functionally distinct from the remaining tumour cells. These cells were more quiescent and showed reduced oxidative stress suggesting that they possess LSC properties.

PB1789: WT1, HMGA2, EVI1, BAALC AND PRAME MRNA LEVELS IN PATIENTS BLOOD SAMPLES WITH ACUTE LEUKEMIA

M. Stolyar 1,2,* , A. Gorbenko 1 , I. Olkhovskiy 1,2 , M. Mikhalev 3 , V. Bakhtina 3 , T. Kaporskaya 4 , V. Lyskova 4

1 Department of Health, Krasnoyarsk branch of the National Research Center for Hematology; 2 Federal State Budgetary Scientific Institution Federal Research Center Krasnoyarsk Scientific Center of the Siberian Branch of the Russian Academy of Sciences; 3 Regional Clinical Hospital, Krasnoyarsk; 4 State-financed health care institution, a winner of the “Mark of the Honor” award, Irkutsk regional clinical hospital, Irkutsk, Russia

Background: An increase in the level of mRNA expression of a number of embryonic activity genes in blood samples from patients with acute leukemia is known. Among them, mRNAs of the WT1, HGMA2, EVI1, BAALC, and PRAME genes are most frequently mentioned, but previous publications do not discuss the possible diagnostic value of individual combinations of activity of these genes.

Aims: To determine the profile of mRNA expression of these genes in blood samples from patients with acute leukemia

Methods: The study included anonymous venous blood samples from patients with acute leukemia, sent to the laboratory for molecular genetic studies. A total of 129 blood samples from patients with initially diagnosed acute leukemia were examined. As a comparison group, 64 samples of patients with an altered ratio of blood cells, but without oncohematological diseases, were used. Testing was carried out by multiplex PCR using kits developed in the Krasnoyarsk branch of the Federal State Budgetary Institution National Research Center for Hematology. Statistical calculations were performed in the R.

Results: The results indicate a high heterogeneity of indicators, while the levels of individual mRNAs did not correlate with each other, as well as with the level of leukocytosis or the proportion of blast cells. Maximum diagnostic efficiency (AUC 0.958; P<0.001) with a sensitivity of 83% and a specificity of 97% for distinguishing between samples of patients with acute leukemia and the comparison group was observed in the total assessment changes two mRNA of WT1 and BAALC genes (Figure).

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Summary/Conclusion: The high diagnostic efficiency of a routine PCR test for evaluating mRNA activity in acute leukemia suggests that it can be used at the initial stage of diagnosis, especially in the absence or low level of blast cells in the venous blood of patients. In addition, the analysis of the obtained data confirms the hypothesis of a possible independent prognostic role of individual transcriptome profiles.

The work was financed within the framework of the state assignment No. АААА-А20-120021890168-5.

PB1790: ABCB1 AS A POTENTIAL BENEFICIAL TARGET OF MIDOSTAURIN IN ACUTE MYELOID LEUKEMIA

S. Sucha 1,* , A. Sorf 1 , M. Svoren 1 , D. Vagiannis 1 , F. Ahmed 1 , B. Visek 2 , M. Ceckova 1

1 Department of Pharmacology and Toxicology, Charles University, Faculty of Pharmacy in Hradec Kralove, Hradec Králové; 2 4th Department of Internal Medicine - Hematology, University Hospital Hradec Kralove, Hradec Kralove, Czechia

Background: In AML, therapeutic outcome of anthracycline-based induction therapy often can be compromised by a resistant phenotype associated with overexpression of ABCB1 transporters. Novel anti-AML agent, midostaurin, was designed to aim at multiple targets including FLT3 mutation. Besides, it was also identified as ABCB1 inhibitor.

Aims: To evaluate ABCB1 efflux activity and expression in primary AML samples in relation to multiple clinical parameters and investigate ABCB1 regulation by microRNA.

Methods: ABCB1 gene expression was absolutely quantified in 28 primary AML samples collected prior to any treatment. Efflux activity was examined using combination of mitoxantrone (ABCB1 substrate) and midostaurin (ABCB1/FLT3 inhibitor). Results were compared in molecular classes defined by CD34 marker, FLT3-ITD and NPM1 mutations, ELN risk classification and (un)achieved complete remission (CR). Enhancement of daunorubicin proapoptotic effect mediated by midostaurin was determined in HL-60 wt and HL-60 overexpressing ABCB1. ABCB1 regulation by microRNAs (miR-9, miR-27a, miR-331) was evaluated by ddPCR and correlated with ABCB1 efflux activity.

Results: High ABCB1 expression was associated with unachieved CR, adverse ELN risk and CD34 positivity. ABCB1 efflux activity was observed in a functional study when mitoxantrone accumulation increased as a direct consequence of midostaurin in CD34+ AML and patients with unachieved CR. ABCB1 efflux activity and expression were found to be linked to miR-9 downregulation

Summary/Conclusion: We propose therapeutic value of midostaurin for drug-resistant patients beyond the FLT3 inhibition based on ABCB1 expression and CD34 positivity. We also propose miR-9 as a predictive ABCB1-related biomarker that could be helpful in identifying ABCB1-resistant AML phenotype.

PB1791: ON THE ISSUE OF HOUSEKEEPING GENE PRIMER DESIGN FOR ADEQUATE ASSESSMENT OF THE EXPRESSION OF THE CHIMERIC TRANSCRIPTS.

I. Fevraleva 1,* , O. Glinshchikova 1 , Y. Sidorova 1 , I. Yakutik 1 , A. Sudarikov 1

1 Department of Molecular Hematology, National Research Center for Hematology, Moscow, Russia

Background: Chimeric transcripts, which are the markers of hematological malignancies, is often assessed by expression level relative to the control housekeeping gene. The critical step for the analysis is RNA isolation and subsequent cDNA synthesis. Routine RNA isolation methods do not guarantee the absence of genomic DNA impurities, which may interfere with cDNA amplification. This results in the bias in control gene expression and underestimation of the relative expression. The use of sophisticated RNA isolation procedures or treatment with RNase-free DNAses significantly complicates and increases the cost of analysis, and can also lead to degradation of target sequences by endogenous nucleases. Here we describe a simple and effective solution to this problem.

Aims: To optimize the RT-PCR approach for quantitative assessment of the chimeric transcripts gene expression relative to the control ABL1 gene, for the insensitivity to genomic DNA impurities.

Methods: Total RNA was isolated from peripheral blood leukocytes or bone marrow cells as described by Chomczynski (1987). RT-PCR was performed using reagents and primers from Synthol (Moscow, Russia).

Results: For ABL1 mRNA amplification primers flanking 2 gene introns were designed (see Table 1). This precludes PCR amplification of the corresponding (139708bp long) genomic sequence. At the same time, the cDNA of this fragment is only 100 nucleotides long. The primers recommended by Europe Against Cancer Association (EAC) and widely used (including as part of commercial diagnostic kits) flank 687bp ABL1 gene DNA fragment. In this case, the admixture of genomic DNA is co-amplified with the cDNA transcript in PCR. We have compared CBFB::MYH11 chimeric transcript relative expression levels in patients with AML, obtained with ABL -EAC primers and those proposed by us ( ABL -N). We have tested 40 cDNA samples obtained from diverse RNA isolation routines, including 8 archival samples stored in the freezer for more than 6 months. The threshold cycles of amplification (ΔCt) for the samples from freshly isolated RNA were mostly the same with ABL -EAC and ABL -N primers. For archival RNA, cDNA, and RNA isolated from frozen cells, ΔCt with ABL -N primers drops significantly while ΔCt with ABL -EAC primers is preserved. This may indicate degradation in archival RNA and cDNA samples, while the level of genomic DNA decreases slightly. Thus, usage of ABL -EAC primers may lead in some circumstances to a significant bias in chimeric transcript evaluation. While primers we offer ( ABL -N) are still effective even in the cases with compromised sample quality.

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Summary/Conclusion: Genomic DNA amplification does not tamper with fusion gene transcripts RT-PCR measurement due to the absence of an appropriate target in the genomic DNA. While primers for measuring housekeeping genes expression (even those widely used) may have these targets and therefore amplify genomic DNA along with cDNA. Here we propose primers for measuring ABL1 control gene expression allowing reliable calculation of chimeric transcripts relative expression, regardless of RNA sample purity, in contrast to primers recommended by the EAC.

PB1792: NGS VS PCR FOR THE DETECTION OF NPM1 GENE MUTATIONS IN ACUTE MYELOID LEUKEMIA.

N. Severina 1,* , Y. Sidorova 1 , N. Risinskaya 1 , B. Biderman 1 , A. Pshenychnyy 1 , N. Ryzhikova 1 , I. Lukianova 2 , A. Kashlakova 2 , A. Sudarikov 1

1 Department of Molecular Hematology; 2 National Research Center for Hematology, Moscow, Russia

Background: NPM1 gene mutations are found in 30% of acute myeloid leukemia (AML) cases. It is a reliable marker for the evaluation of therapy outcome and minimal residual disease (MRD) monitoring. The most often mutations are 4bp insertions in exon 12, affecting 2 functionally important tryptophan residues (W288 and W290) in the C-terminal region of nucleophosmin and causing a frameshift. As a result, a leucine-rich motif (L-xxx-V-xx-V-x-L) is formed, which is a signal sequence for protein export from the nucleus to the cytoplasm. Commonly NPM1 gene mutations are detected by fragment analysis (FA) of PCR fragment of 12 exon for the presence of 4bp insert. Alternatively, allele-specific polymerase chain reaction (AS-PCR) to detect inserts belonging to the A, B, or D type (representing up to 90% of all inserts) is used. However, about 10% remaining cases are non-standard inserts and intron polymorphisms that require more precise evaluation.

Aims: Evaluation of diverse approaches for efficient NPM1 gene mutation analysis and their robustness for the diagnostics.

Methods: The study included 80 DNA samples from AML patients admitted to the National Research Center for Hematology in 2019-2021 that were positive for NPM1 gene mutations detected by FA performed with the Nanophor-5 genetic analyzer (Synthol, Russia). The type of insert was assessed by AC-PCR. Samples in doubt were evaluated by targeted next-generation sequencing (NGS) on MySeq (Illumina, USA).

Results: In 62 out of 80 samples, the type of insert (A, B, or D) was reliably determined by AS-PCR. In 9 cases type of insert was not detected. In further 9 cases, amplification ΔCt between wild-type and mutant allele was out of reliable range. These 18 samples were examined by NGS, and 2 out of 18 showed an intron deletion, which, presumably, does not affect the function of the protein, but leads to the appearance of an additional peak on FA. In 16 out of 18 samples, non-standard inserts were detected (Table 1). Type A inserts were found in 48 out of 78 cases (61.5%), type B - in 2 out of 78 (4.5%), and type D - 15.4% (12 out of 78). We have detected atypical inserts in 16 out of 78 cases (20.5%), which is more than reported in the literature. 14 out of 16 atypical inserts result in a leucine-rich motif, as do standard inserts. In 4 out of 16 cases, one of the 2 functionally important tryptophan residues was retained (W280), therefore possibly conserving nuclear localization signal. The effect of partial preservation of the nucleolar localization of nucleophosmin on prognosis in AML has not been studied yet.

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Summary/Conclusion: FA is still a golden standard for screening NPM1 gene mutations in AML. AS-PCR could be effectively used for monitoring cases with standard inserts. However, NGS greatly facilitates non-standard inserts identification, appropriate allele-specific primers design for MRD analysis, and avoiding false-positive FA results for cases with intron localization of the insert.

PB1793: NEUTROPHIL TO LYMPHOCYTE RATIO AS A PROGNOSTIC FACTOR IN AML AND MDS

V. Van Oorschot 1,* , M. Beckers 2

1 KU Leuven; 2 Hematology, UZ Leuven, Leuven, Belgium

Background: The neutrophil to lymphocyte ratio (NLR) reflects the balance between innate and adaptive immunity and has been studied as a prognostic biomarker in several solid and some hematological malignancies. In the tumor micro-environment, lymphocytes have an anti-tumor activity, while neutrophils possess a pro-tumor activity. Several studies show that a higher NLR is associated with an increased risk of mortality. Only a few studies are available on NLR in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

Aims: The aim of this study is to investigate if the NLR can be a prognostic marker in AML and MDS. The effect of age and cytogenetic risk profile on NLR is also investigated.

Methods: In this single-center, retrospective study 261 AML patients and 105 MDS patients treated at the University Hospitals of Leuven were included. The patients were divided into two subgroups by defining NLR=2.0 as the cut-off point. The NLR was calculated by the ratio of the amount of neutrophils to lymphocytes in the peripheral blood (PB). Further division was made based on treatment: stem cell transplant, hypomethylating agents or supportive therapy. Kaplan-Meier analyses were used to assess overall survival (OS). Survival predictors were analyzed using Cox regression with uni- and multivariate analyses. The correlation between age, cytogenetic risk profile and NLR was analyzed using independent-samples median tests.

Results: AML patients with low NLR (<2.0) had a longer OS than those with high NLR (≥2.0). NLR, age, hemoglobin, neutrophil count, and lymphocyte count in PB had a significant effect on OS in all AML patients. NLR was no longer significant in multivariate analysis. Furthermore, a high NLR before receiving stem cell transplant was associated with longer OS. Regarding MDS, no significant difference in OS was found between patients with low (<2.0) and high NLR (≥2.0). Finally, in AML patients higher age was associated with an increase in NLR.

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Summary/Conclusion: NLR at the time of diagnosis may be predictive for OS in AML patients but not in MDS patients. However our findings need to be confirmed in larger cohorts.

PB1794: EFFECT OF HYPOXIA ON CD47 EXPRESSION IN HUMAN ACUTE MYELOID LEUKEMIA CELL LINES

C. Wang 1,* , Y. Liu 1

1 Hematology, Chongqing University cancer hospital, Chongqing, China

Background: To investigate the effect of hypoxia on hypoxia-inducible factor 1α(HIF-1α) and CD47 expression in human acute myeloid leukemia (AML) cell lines.

Aims: To investigate the effect of hypoxia on hypoxia-inducible factor 1α(HIF-1α) and CD47 expression in human acute myeloid leukemia (AML) cell lines.

Methods: The detection of CD47 expression on U937、HL-60 and K562 cells were evaluated by flow cytometry. U937、HL-60 and K562 cells were all divided into 2 groups, including hypoxia-treated group and conventional oxygen group as control. The U937、HL-60 and K562 cells in hypoxia-treated group were treated with 1%O 2 , while the cells in control group were cultured in conventional oxygen condition (20%O 2 ), then the cells in 2 groups were collected after treatment for 24 hours. Real time PCR was used to examine the mRNA changes of CD47 . Western blot assay was applied to detect the protein expression of HIF-1α and CD47.

Results: The expression of CD47 in U937、HL-60 and K562 cells were 98%(98%±0.03), 99%(99%±0.05) and 75%(75%±0.11) respectively. The real time PCR showed that the mRNA expression of CD47 in U937 and HL-60 cells of hypoxia group were upregulated ( P <0.05). However, the mRNA expression of CD47 in K562 cell of hypoxia group was not upregulated ( P> 0.05). The results of Western blot showed that after treatment of U937、HL-60 and K562 cells with hypoxia, the protein levels of HIF-1α and CD47 were increased compared with those in the control group ( P <0.05).

Summary/Conclusion: The hypoxia can up-regulate the expression of CD47 in acute myeloid leukemia cells, which may be related to HIF-1α.

PB1795: BIO-INSPIRED NANOMEDICINE FOR TARGETED ACUTE MYELOID LEUKEMIA IMMUNOTHERAPY

X. Wang 1,* , R. Huang 1 , K. Fang 1 , X. Zhang 1

1 Medical Center of Hematology, Xinqiao Hospital, Chongqing, China

Background: Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy linked to molecular alterations and disease remission requires multiple chemotherapies. In spite of the recent approval of new promising targeted therapies, the clinical outcome of AML patients remains suboptimal with high relapse rate, prompting the search for synergistic therapeutic approaches. In recent years, the rise of nanotechnology provides new horizons in AML treatment by using versatile and safe nano systems.

Aims: To achieve the targeted AML treatment by virtue of MnO2 nanostructure-based immune- and chemotherapy synergy.

Methods: A bio-inspired nanomedicine (LHMD) nanomedicine was first designed by coating AML cell membrane onto DOX-loaded hollow MnO2 nano-carrier. The nanostructure of LHMD was observed and characterized. The cell viability, cell injury behavior, homologous targeting effect and immune activation after LHMD nanomedicine treatment were investigated in vitro . In vivo BM homing properties and anti-AML efficacy were also analyzed in the AML mice model.

Results: We developed LHMD to specifically target in the BM and deliver chemotherapeutic agents, DOX, for potent immune therapy and chemotherapy against AML. After cell internalization of nanomedicine, LHMD was disassembled owing to the high level of GSH and endosomal acid in tumor cells, which mediated the release of DOX and Mn 2+ . DOX destroyed dsDNA breakage to further induce cellular immunogenic death (ICD), thus killing leukemia cells. Mn 2+ triggered the STING pathway activation in macrophage and sensitize the cGAS to recognize of dsDNA, thus further boosting innate immunity.

Summary/Conclusion: A bio-inspired nanomedicine was constructed by integrating Mn2+ mediated STING pathway activation for synergized acute myeloid leukemia chemo- and immunotherapy.

PB1796: DETERMINATION OF THE RELATIONSHIP BETWEEN NEXT GENERATION SEQUENCING AND FLOW CYTOMETRY RESULTS IN NEWLY DIAGNOSED ACUTE MYELOBLASTIC LEUKEMIA

I. Yavasoglu 1,* , A. H. E. küçükdiler 1 , I. erdoğdu 1 , Z. bolaman 1

1 hematology, Adnan Menderes university, Aydin, Turkey

Background: Flow cytometry is one of the classical diagnostic tools used in the diagnosis of AML. In particular, the use of these two methods is essential for minimally measurable disease. According to our current knowledge, there is only one study in the literature that evaluated the expression of surface markers detected by both NGS and flow cytometry. In this study, NPM1, FLT3 (TKD and ITD), KRAS and NRAS signal mutations were found to be associated with high CD33 levels

Aims: In this study, we aimed to determine the relationship between flow cytometrically determined surface markers in newly diagnosed AML patients whose genetic profile was determined by the NGS method

Methods: Atotal of 120 patients diagnosed with AML by histochemical methods and flow cytometric analysis were evaluated retrospectively. Flow cytometric examination was performed using Beckman Coulter Navious 3L10C device (10 colors). NGS was performed with bone marrow aspiration samples taken at the time of diagnosis by the Molecular Pathology unit. Sequence analysis was performed using the Illumina MiniSeq next-generation sequencing system and supporting automated platforms and the QIAseq

Targeted Myeloid Neoplasms DNA panel (DHS-003Z) containing 141 genes and 5887 primers for testing. The obtained raw data were first transferred to the QCI-A software where variants would be examined for bioinformatics analysis in the same laboratory. Variants transferred to QCI-Interpret, phenotype-related molecular hemato-pathological findings determined after bioinformatics interpretation were reported. The conformity of the quantitative data to the normal distribution was tested with the Shapiro-Wilk test and graphical examinations. Independent groups t-test was used for comparisons between two groups of normally distributed quantitative variables, and Mann-Whitney U test was used for comparisons between two groups of non-normally distributed quantitative variables. Statistical significance was considered as p<0.05.

Results: ASXL1, ANKRD26, ATRX, BCR, BLM, BRCA1, BRCA2, CEBPA, CTCF, DNMT1, FLT3, KMT2A, KRAS, MSH6, NSD1, PTEN, RUNX1, SRSF2, SUZ12, TET2, U2AF1 in bone marrow aspiration samples and a significant correlation was found between WRN and high values of surface markers detected by flow cytometry. The some results are shown in Table 1.

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Summary/Conclusion: The effects of CD 36 and 9 on platelet functions and cell signal interactions are well known. In this context, the relationship between the positivity of both surface markers and the ANKRD26, ATRX, KMT2A, MSH6, NSD1, RUNX1 and SUZ12 mutations is important. The correlation of CD 7, which is aberrantly expressed surface markers in AML, with CEBPA and KRAS was also remarkable. However, a significant correlation was found between high CD 33 values and BLM, TET2 and WRN positivity. These results are critical for future targeted therapies, and multi-center studies with larger numbers of patients are needed.

PB1797: ZNO NANOPARTICLES PROMOTES HUMAN ACUTE MYELOID LEUKEMIA (THP-1) CELL APOPTOSIS BASED ON MITOCHONDRIAL FISSION PATHWAY

X. Yin 1 , Z. Li 1 , L. Chunyi 1 , W. yan 2 , J. Wang 2 , Y. Si 1 , S. Cui 2 , R. Xu 2,*

1 Shandong University of Traditional Chinese Medicine; 2 Shandong University of Traditional Chinese Medicine Affiliated Hospital, Jinan, China

Background: AML is a malignant tumor caused by changes in myeloid hematopoietic stem cells and abnormal changes in the proportion of primitive and immature myeloid cells. ZnO NPs have unique optical, chemical, electrical conductivity, antibacterial and anti-tumor properties, and are widely used in antibacterial, wound healing and tumor treatment. Mitochondria are a highly dynamic organelle that is important for many biological processes. At present, the effect of ZnO NPs on mitochondrial division and on human THP-1 cell apoptosis is still unclear.

Aims: The present study aimed to elucidate the underlying mechanism of ZnO NPs induced apoptosis in acute myeloid leukemia (AML) cells by regulating mitochondrial division.

Methods: The inhibitory effect of ZnO NPs on THP-1 cell proliferation was monitored by the CCK-8 method, and the THP-1 cells were incubated with different concentrations (i.e., 0, 10, 20, 40 μM) ZnO NPs for 24 h. The expression of mitochondrial division related factor Drp-1, apoptosis related factor Bcl-2, Bax mRNA and protein was detected by Q-PCR and WB, respectively. Moreover, the effects of ZnO NPs on the levels of reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), apoptosis and ATP generation in leukemia cells were measured, and the effect of Drp-1 inhibitor Mdivi-1 and ZnO NPs on THP-1 cells was further detected. In addition, the relationship between Drp-1-mediated mitochondrial division and ZnO NPs-induced apoptosis in human leukemia cells was also explored.

Results: The results showed that ZnO NPs could significantly inhibit cell proliferation and facilitate the apoptosis of THP-1 cells in a time- and dose-dependent manner. Meanwhile, the results also showed that ZnO NPs could significantly reduce the Δψm and ATP levels and induce the ROS production in THP-1 cells. The expression of Drp-1 and Bax at mRNA and protein levels was elevated whereas the mRNA and proteins levels of Bcl-2 were significantly decreased after the cells exposure to 10.0, 20.0, 40.0 μM of ZnO NPs for 24 h (all P <0.05). After intervention with different concentrations (0, 10.0, 20.0, and 40.0 μM, respectively) of Drp-1 inhibitor Mdivi-1, the Drp1 mRNA level was significantly reduced. Furthermore, the results also showed that the expression of Bax was significantly reduced, and the expression of Bcl-2 was increased after intervention with different concentrations of Drp-1 inhibitor Mdivi-1 (all P <0.05). Meanwhile, the apoptosis level of ThP-1 cells was significantly increased after exposure to ZnO NPs, whereas the apoptosis level was significantly reduced after the cell exposure to Drp-1 inhibitor Mdivi-1.

Summary/Conclusion: The human AML cell survival rate decreased with the increment of ZnO NPs concentration and incubation time, accompanied by a dose-and time-dependent manner. ZnO NPs can reduce the cell Δψm and ATP levels, induce the ROS production in THP-1 cells, suggesting that the mitochondrial function was impaired or decreased. The levels of mitochondrial division and apoptosis of AML cells increased after exposure to ZnO NPs, indicating the activation of apoptosis pathway induced by ZnO NPs in human AML cells may be related to mitochondrial division. However, the apoptotic level was significantly reduced after intervention of Drp-1 inhibitor, confirming that ZnO NPs can induce the apoptosis of THP-1 cells by regulating mitochondrial division. Overall, ZnO NPs may provide a new basis and idea in treating human acute myeloid leukemia in clinical practice.

PB1798: EVALUATION OF EXPRESSION OF PLODS FAMILY GENES (PLOD1, PLOD2, PLOD3) IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

F. Zaker 1,* , A. qorbanpour 2

1 haematology; 2 iran university of medical sciences, tehran, Iran

Background: AML is a multifactorial disease and different factors play a role in its occurrenc, timely diagnosis and appropriate treatment. On the other hand, due to the importance of PLOD family genes (PLOD1, PLOD2, PLOD3) that lysine hydroxylase enzyme is directly involved in collagen synthesis and extracellular matrix regeneration and their vital role in migration, invasion and angiogenesis in many cancers.

Aims: Since the expression of PLOD family genes in AML patients have not been studied so far, also due to the importance of these two risk factors, namely invasion and angiogenesis and the relationship between the expression of these genes and the prognosis of patients, the expression status of PLOD family genes were studied in patients with acute myeloid leukemia.

Methods: In this study, the expression of PLOD family genes (PLOD1, PLOD2, PLOD3) in 54 newly identified AML patients and 53 healthy controls was evaluated by qRT-PCR. Then, the obtained results were interpreted using statistical analysis.

Results:

In this study, 54 patients with AML were studied. 35% of the patients were female and 65% were male. The mean age of patients was 42.05 years. Hematological findings showed that the number of white blood cells and platelets as well as hemoglobin in patients was significantly different from healthy individuals (P <0.05). Classification of patients based on FAB subgroups showed that most patients (50%) belong to M4 subgroup. Gene expression studies showed that PLOD1 and PLOD3 gene expression was significantly increased in AML patients compared to healthy controls (P value = 0.0002 and P value = <0.0001, respectively). But there was no significant difference in PLOD2 gene expression in AML patients compared to normal control group (P value = 0.3).

Summary/Conclusion: Expression of PLOD1 and PLOD3 genes in patients with acute myeloid leukemia has significantly increased compared to the healthy control group and may be used as a prognostic factor in AML patients. There was no significant difference in PLOD2 gene expression in AML patients compared to the normal control group. However, due to the heterogeneity of AML, further studies are needed in different AML subgroups based on FAB and cytogenetic classification.

PB1799: KMT2A-REARRANGED PEDIATRIC ACUTE MYELOID LEUKEMIA

E. Zerkalenkova 1,* , S. Lebedeva 1 , A. Borkovskaia 1 , Y. Menchits 1 , M. Gaskova 1 , A. Itov 1 , E. Matveev 1 , M. Kazanov 1 , Y. Olshanskaya 1

1 Laboratory of Cytogenetics and Molecular genetics, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia

Background: Histone-lysine N-methyltransferase 2A gene rearrangements ( KMT2A -r) are common genetic events in acute leukemia. They demonstrate great molecular heterogeneity with more than 90 partner genes and multiple KMT2A breakpoints involved. These characteristics challenge both the initial molecular diagnostics and MRD monitoring in ALs with KMT2A -r. On the other hand, the profile of accompanying mutations is relatively quiet in this type of leukemia due to strong oncogenic potential of KMT2A rearrangement itself.

Aims: Our recent study aims to characterize KMT2A-rearranged acute myeloid leukemia in children in Russian Federation both in terms of partner genes involved and in accompanying mutations study.

Methods: A cohort of 313 KMT2A -positive AMLs in children aged 4 days to 18 y.o. (median 2 y.o.; 162 boys and 151 girls) was analyzed using G-banded karyotyping and FISH together with real-time and nested RT-PCR. Transcripts were Sanger sequenced. Patients with rare or unknown translocations were subjected to long-distance inverse PCR (LDI-PCR), anchored multiplex PCR (ArcherDX, CO, USA) or custom targeted KMT2A enrichment panel (Illumina, CA, USA; Meyer et al., 2019). Additional events were characterized by fragment analysis, Sanger sequencing and NGS (Qiagen Myeloid Neoplasms panel, Hilden, Germany) in 113 cases included in Russian AML registration study and AML-MRD-2018 local treatment protocol.

Results: The incidence of KMT2A -rs was the highest in children under 2 y.o. inclusive (n=120, 38%) and goes down with age. A total of 26 different KMT2A -rs were found in 296 patients (94.6%). The prevailing translocations were as follows: t(9;11)(p21;q23.3)/ KMT2A-MLLT3 (n=129, 41.21%), t(10;11)(p12;q23.3)/ KMT2A-MLL10 (n=71, 22.68%), t(11;19)(q23.3;p13.1)/ KMT2A-ELL (n=24, 7.67%), t(6;11)(q27;q23.3)/ KMT2A-AFDN (n=15, 4.79%), t(11;19)(q23.3;p13.3)/ KMT2A-MLLT1 (n=13, 4.15%). Other translocation partners were much rarer, for 15 partner genes only single cases were found. Three novel KMT2A -rs were identified, namely t(11;16)(q23;q23.3)/ KMT2A-USP10 , t(X;11)(q22.1;q23.3)/ KMT2A-BTK and t(X;11)(q26.3;q23.3)/ KMT2A-CT45A .

KMT2A breakpoint location was analyzed in 274 patients (87,54%). It was found in introns 7 to 12, which is narrower than in ALL, and was most often situated in introns 9 (n=131, 47.81%) and 10 (89 cases, 32.48%) as numbered by Meyer et al. [Meyer et al., 2006]. Intron 9 was also predominant in rearrangements with all but MLLT1 frequent translocation partners and most rare translocation partners. KMT2A break outside the main cluster was found in intron 7 on 4 cases of KMT2A-MLL10 . The achieved data on KMT2A translocation partners and breakpoint locations was used for subsequent patient-specific MRD monitoring.

The molecular profile of myeloid neoplasms-associated gene was studied in 113 samples. One to 9 additional pathogenic somatic events (median 4) with variant allele frequency above 1% were found in the studied cohort with most of them being subclonal (VAF<10%). Only 62 samples contained additional mutations with VAF greater above 10%. Among them NRAS (n=11 of 62, 17.74%), KRAS (n= 8, 12,90%) and PTEN11 (4, 6,45%) were prevalent. Accompanying mutations did not exhibit translocation partner preference.

Summary/Conclusion: Molecular profiling of KMT2A -rearranged AML in Russian Federation reveals the highest incidence of KMT2A-MLLT3 fusion gene and KMT2A intron 9 involvement. Our data also confirms the relatively quiet landscape of accompanying mutations in KMT2A-rearranged AML with RAS pathway most frequently involved.

PB1800: DEEPDECON: A DEEP-LEARNING METHOD FOR DETECTING MINIMAL RESIDUAL DISEASE OF ACUTE MYELOID LEUKEMIA

J. Zhong 1,* , A. Stucky 1 , F. Sun 1 , J. Huang 1

1 University of Southen California, Los Angeles, United States of America

Background: Acute myeloid leukemia (AML) is a heterogeneous disease that the myeloid lineages of hematopoietic progenitor cells (blasts) lose the ability of normal differentiation and build up in the bone marrow. The ability to identify minimal residual disease (MRD) below the morphology-based 5% blast threshold has significant impact in clinical outcome. Flow cytometry and real-time quantitative polymerase chain reaction [qPCR]) are the most common methods for MRD detection while next-generation sequencing (NGS) is emerging as a more sensitive method.

Aims: Here, we report DeepDecon, a deep neural network model that utilizes NGS RNAseq data to detect MRD of AML.

Methods: DeepDecon makes use of the non-linear transformation among the latent layers and learns to extract high-order representations of the input gene expression. Meanwhile, DeepDecon doesn’t need to select a subset of genes. The neural network will optimize and assign different weights to all genes to minimize the prediction error. A challenging issue with deep learning models is the need for large data sets of diverse examples for model training to obtain robust and good performance. With single-cell RNAseq data, we digitally create a large number of bulk RNAseq samples with known AML fraction for model training.

Results: When applied to clinical data TARGET-AML from GDC Data Portal ( https://portal.gdc . cancer.gov/), DeepDecon demonstrated better performance than other machine learning algorithms, including MuSic, NNLS, Bisque, RNA-Sieve, and Scaden, for detecting MRD of AML.

Summary/Conclusion: Deepdecon can be applied to clinical NGS data for accurate and sensitive detection of MRD in AML treatment. It will significantly improve the clinical outcomes of current AML therapy.

PB1801: CHROMOSOMAL ABNORMALITIES DURING THE CLINICAL COURSE OF ADULT ACUTE MYELOID LEUKEMIA

O. Zotova 1,* , A. Lukianova 2 , M. Valchuk 1 , Y. Karol 3 , O. Shalay 1 , V. Loginsky 1

1 SI «Institute of Blood Pathology and Transfusion Medicine, NAMS of Ukraine», Lviv; 2 Medical Biology Centre «Genom», Kyiv; 3 Communal Noncommercial Enterprise «Municipal Clinical Hospital No 5», Lviv, Ukraine

Background: Using conventional cytogenetics, an abnormal karyotype can be detected in 60-70% of adult acute myeloid leukemia (AML) patients. Taking into consideration the identified cytogenetic abnormalities AML patients are currently classified into three risk groups: favorable, intermediate and adverse. These genetic abnormalities are the most important factors in determining response to chemotherapy as well as outcome in AML.

Aims: Aim of this study was to detect the diagnostic and prognostic significance of chromosomal abnormalities in leukemic cells at different phases of AML.

Methods: Cytogenetic investigations of bone marrow and/or peripheral blood cells from 140 adult patients were performed during clinical evolution of AML: in newly diagnosed patients, in remission and at relapse. The methods of conventional cytogenetics (GTG) and fluorescence in situ hybridization (FISH) were used. Cytogenetic methods were performed using standard techniques and karyotypes were described according to the International System for Human Cytogenetic Nomenclature.

Results: Cytogenetic investigations of 140 adult AML patients were performed in newly diagnosed patients, in remission and at relapse. Structural (del(5q), del(7q), rearrangements of 3q, 12p, 17p and 11q23, t(8;21)(q22;q22), t(9;22)(q34;q11), t(15;17)(q22;q21), t(16;16)(p13;q22), inv(16)(p13q22), marker and ring chromosomes, acentric structures and others) and numerical (-5, -7, -Y, +8, monosomal karyotype and others) chromosomal abnormalities in leukemic cells were found. Some genetic abnormalities ( BCR/ABL1, PML/RARa, AML1/ETO, CBFβ/MYH11 fusion genes) were detected by molecular genetic methods (FISH). Spectrum of cytogenetic abnormalities had an important diagnostic and prognostic significance. Diagnosis of AML was possible due to the presence of specific genetic markers that can confirm some types of AML (AML with t(8;21)(q22;q22), t(9;22)(q34;q11), t(15;17)(q22;q21), t(16;16)(p13;q22)/inv(16)(p13q22), 3q21q26 rearrangements, BCR/ABL1, PML/RARa, AML1/ETO, CBFβ/MYH11 fusion genes). Taking into consideration the identified cytogenetic abnormalities AML patients were classified by risk groups: the group of patients with adverse cytogenetic markers (3q21q26 rearrangements, -5, -7, t(9;22)(q34;q11), BCR/ABL1 fusion gene, multiple changes (≥3), monosomal karyotype), the intermediate risk group without significant prognostic markers and the group with favorable prognostic factors (t(8;21)(q22;q22), t(15;17)(q22;q21), t(16;16)(p13;q22)/inv(16)(p13q22), AML1/ETO, PML/RARa, CBFβ/MYH11 fusion genes). In remission cytogenetic investigations showed a normal karyotype without cytogenetically visible changes in all patients. Cytogenetic investigations at relapse were performed in 5 of 140 AML cases. Additional/secondary chromosomal aberrations in leukemic cells or new cells clones with multiple structural and numerical changes at relapse was not detected.

Summary/Conclusion: Cytogenetic investigations had an important significance for diagnosis, prognosis and selection the optimal treatment strategy of AML. Besides the analysis of chromosome banding patterns it is necessary for patients with AML to apply molecular genetic studies, namely FISH and polymerase chain reaction (PCR). Taking into consideration the identified cytogenetic abnormalities AML patients were classified by risk groups. Presence of the additional/secondary chromosomal aberrations in leukemic cells or new cells clones with multiple structural and numerical changes at AML relapse is the one sign of clonal evolution and progression of disease.

PB1802: ACUTE PROMYELOCYTIC LEUKEMIA AND THROMBOSIS: HOW TO IDENTIFY THE HIGH-RISK PATIENTS?

M. `Ben Salah 1,* , M. Bchir 1 , R. Berred 1 , R. Kharrat 1 , Y. Ben Abdennebi 1 , L. Aissaoui 1 , R. Ben Lakhal 1 , H. Ben neji 1 , B. Meddeb 1

1 Hematology department, Tunis El-Manar University, Faculty of medicine of Tunis, Aziza Othmana Hospital, Tunis, Tunisia

Background: Acute promyelocytic leukemia (APL) is frequently associated with a disseminated intravascular coagulopathy (DIC), leading to severe bleeding complications. It’s thus a diagnostic and therapeutic emergency. Nevertheless, the thrombotic risk is not to be neglected and is an interesting issue to highlight.

Aims: The aim of this study was to describe the thromboembolic events (TE) associated with APL and to evaluate the associated clinical and biological factors.

Methods: We conducted a retrospective single-center study that included patients diagnosed with APL between 2010 and 2019 in our department, treated with all-trans retinoic acid (ATRA) and anthracycline. Descriptive data related to the TE was collected. In order to identify the risk factors of TE, univariate and multivariate analysis were performed.

Results: This study included 90 patients. A TE occurred in 10 patients (11%), with a median age of 48 years [20-62 years], and a sexe-ratio of 1. None had a previous history of thrombosis. Cardiovascular risk factors were found in 5 patients with TE (50%).

An arterial TE developed in half of the cases: 2 had cerebrovascular accident (CVA, 20%), 2 had pulmonary embolism (PE, 20%), and 1 had myocardial infarction (MI, 10%). The venous TE encompassed deep vein thrombosis (DVT, 40%), and Budd-Chiari syndrome (10%).

The TE was an initial symptom of APL for 4 patients (40%). It occurred during induction, and consolidation for 4 and 2 patients respectively. None of these patients had a differentiation syndrome or severe bleeding at the time of the TE. DIC was present in 2 patients (20%).

As potential predictors of TE, we analyzed the following parameters: gender, age, Body Mass Index, cardiovascular risk factors, white blood cell (WBC) count greater than 10 G/L, hemoglobin (Hb), platelet count, fibrinogen less than 2g/l, prothrombin time (PT) less than 60%, DIC at diagnosis, Sanz score, cytological type, presence of CD2, CD15, additional cytogenetic abnormalities, PML-RARa variant, and the occurrence of a differentiation syndrome during induction. The following parameters were related to a statistically significant higher incidence of TE: Cardiovascular risk factors (p=0,011; 50% vs 29%), PT < 60% (p=0.02; 72% vs 32.5%), differentiation syndrome during induction (p=0.05; 40% vs 15%). The independent risk factors of TE, identified through the multivariate analysis, were: age (p=0.015, HR=7.7, IC 95%: [1.5 – 40]), and PT < 60% (p=0.019, HR= 7.5, IC95% [1.4 – 40.3]).

Summary/Conclusion: The occurrence of TE in APL is a risk that should not be underestimated. The therapeutic strategy remains a challenge to overcome due to the associated DIC and thrombocytopenia. A better understanding of the risk factors is crucial to improve the management and the outcome of TE. Would a prophylaxis strategy be beneficial for APL patients with high risk of thrombosis?

PB1803: EARLY DEATH IN ACUTE PROMYELOCYTIC LEUKEMIA: DEFINING HIGH RISK PATIENTS

M. `Ben Salah 1,* , M. Bchir 1 , R. Berred 1 , R. Kharrat 1 , L. Aissaoui 1 , Y. Ben Abdennebi 1 , R. Ben Lakhal 1 , H. Ben neji 1 , B. Meddeb 1

1 Hematology department, Tunis El-Manar University, Faculty of medicine of Tunis, Aziza Othmana Hospital, Tunis, Tunisia

Background: Acute promyelocytic leukemia (APL) is a rare type of leukemia characterized by its good prognosis, thanks to the introduction of all-trans retinoic acid (ATRA). However, early mortality (death within 30 days of the start of treatment) remains a challenge to undertake.

Aims: The objective of this study was to identify the causes and risk factors of early death (ED) in APL patients treated with ATRA and anthracycline.

Methods: We retrospectively collected the data relative to the patients diagnosed with APL in our department between 2010 and 2019, who received an induction treatment consisting of ATRA and Idarubicin, according to the PETHEMA protocols: LPA 99 (2010-2013) and LPA2005 (2014-2019). A statistical analysis was performed, aiming to define ED risk factors.

Results: A total of 90 patients were included in our study. ED was observed in 17 patients (19%), characterized by a sex ratio of 0.89 and a median age of 40 (4 - 71 years). The main cause of ED was represented by severe hemorrhage (11 patients, 65%), followed by differentiation syndrome (3 patients, 18%). The ED was due to a septic choc, a thrombotic event, and a leucostasis, for one patient each.

The correlation between certain clinical and biological parameters and ED was analyzed. The clinical parameters studied were: age, gender, delay between onset of symptoms and treatment, Performance Status (PS), treatment protocol, Sanz Score, clinical presentation, severe bleeding during induction, thromboembolic events (TE), and differentiation syndrome (DS). The biological parameters studied were: White Blood Cell (WBC) count, platelets, hemoglobin, LDH level, creatinine clearance, prothrombin time (PT), fibrinogen, presence of Disseminated Intravascular Coagulation (DIC) at diagnosis, cytological type, PML/RARA variant, and the presence of additional cytogenetic abnormalities.

A statistically significant difference was found for the following risk factors: PS higher than 1 (p<0.0001; 71% vs 14%), Sanz score (p=0.005; 71% at high risk vs 29%), WBC count(p<0.0001; median of 26.03G/L in the ED group versus 2.5G/L), LDH level(p=0.001; a mean of 732 versus 372), prothrombin time (p=0.011; a median of 55% versus 70%), fibrinogen level less than 1g/L (p=0.05; 35% vs 15%), presence of a TE at diagnosis (p=0.004; 18% vs 1.4%), and occurrence of severe bleeding complication during induction (p<0.0001; 61% vs 39%). The independent risk factors of ED identified through the multivariate analyses were: PS (p=0.001, OR=41.6, IC95% [4.7-363], and severe bleeding during induction (p<0,0001, OR=71.5, IC95% [7.1 - 716.3]).

Summary/Conclusion: Our results are comparable to those of the literature. ED mainly due to severe bleeding, is a major obstacle in the management of APL. The identification of its risk factors allows us to improve the therapeutic strategies. The implementation of a supportive therapy, as soon as the diagnosis is suspected, seems to be an adequate measure to tackle this challenge.

PB1804: CLINICAL AND DEMOGRAFIC RELATIONS OF BAALC GENE OVEREXPRESSION IN AML PATIENTS - A SINGLE-CENTER OBSERVATIONS

E. Aleksov 1,* , M. Jagurinoski 1 , I. Tonev 1 , K. Venkov 1 , M. Genova 1 , G. Balatzenko 1 , Y. Lazarova 1 , G. Arnaudov 1 , B. Spasov 1

1 Hematology, NSBALHZ, Sofia, Bulgaria

Background: Impaired regulation of the gene expression of BAALC (brain and acute leukemia, cytoplasmic) gene, which plays a role in the regulation of myeloid differentiation, leads to changes in functional gene activity and probably acts as a component of the leukemogenesis in acute myeloid leukemia (AML).

Aims: To analyze the frequency and clinical significance of overexpression of the BAALC gene in patients with AML.

Methods:: A total of 67 samples of patients (33% men and 34 % women) with an overt AML with >20% blast cells, excluding acute promyelocytic leukemia, were included in this study. The diagnosis was made according to the criteria of WHO classification (2016). BAALC gene expression was assessed by polymerase chain reaction after reverse transcription (RT-PCR). Risk stratification was done according to ЕLN.

Results: The overexpression of the BAALC gene was found in 33/67 (49.3%) AML patients of whom 14/33 (42.4%) male and 19/33 (55.9%) female with median age 56.4 years (range, 23-77 years). BAALC -positive cases were distributed as follows: AML - not otherwise specified (AML NOS) 11/33 (33.3%); AML with MDS-related changes 10/33 (30 %); AML with recurrent genetic abnormalities 6/33 (18.1%) and therapy related AML 6/33 (18.1%).

In the analysis of the main hematology and clinical parameters, we observed a tendency for higher BAALC overexpression in the following groups of AML patients:

• AML patients with WBC ≤10.5x10^9/l 11/19 (57.8%), vs patients with WBC ≥10.5x10^9/l 14/33 (42.4%)

• AML patients with neutropenia <2.1x10^9/l 19/33 (57.6%), vs patients with neutrophilia >6.4x10^9/l 8/20 (40%).

• AML patients with thrombocytopenia <50G/l 17/29 (58.6%), vs patients with platelets count >50G/l 10/28 (35.7%);

• AML with MDS-related changes/therapy related AML 17/26 (65.4%), vs other types 16/41 (39%)

• AML in the groups with an unfavorable karyotype 12/16 (75%) vs 18/39 (46.1%) with good/intermediate.

• AML patients who failed to achieve remission 24/34 (70.5%) vs patients who achieved remission - 9/26 (34.6%).

The mean overall survival (OS) was poorer in BAALC (+) compared to BAALC (-) patients (respectively 11.7 months vs. 18.6 months, (p = 0.62).

Summary/Conclusion: Our single-center observations confirmed previous publications that BAALC gene overexpression is a frequent genetic abnormality in AML patients. There is a tendency for a correlation between the BAALC overexpression and some unfavorable prognostic indicators, as well as a trend to shorter OS. Our data show that future studies of the clinical significance and prognostic role of BAALC gene would be worth.

PB1805: INSIGHTS FROM ORAL AZACITIDINE EARLY ACCESS PROGRAM IN FRANCE: PATIENT POPULATION WITH ACUTE MYELOID LEUKEMIA RECEIVING ORAL-AZA FOR MAINTENANCE OF COMPLETE REMISSION AFTER INDUCTION CHEMOTHERAPY

E. Raffoux 1,* , S. De Botton 2 , L. Ades 1 , M. Carré 3 , T. Cluzeau 4 , C. Saillard 5 , M. Heiblig 6 , F. Beckerich 7 , L. Gastaud 8 , A. Dupuis 9 , U. Richez 9 , P.-Y. Dumas 10

1 Hematology, APHP St-Louis, Paris; 2 Hematology, Gustave Roussy, Villejuif; 3 Hematology, Grenoble Alpes University Hospital, Grenoble; 4 Hematology, Nice University Hospital, Nice; 5 Hematology, Institut Paoli Calmettes, Marseille; 6 Hematology, Lyon Sud hospital, Lyon; 7 Hematology, Henry Mondor hospital, Créteil; 8 Medical Oncology, Centre Lacassagne, Nice; 9 Medical, Bristol Myers Squibb, Rueil Malmaison; 10 Hematology, Haut-Leveque-Bordeaux University Hospital, Bordeaux, France

Background: While many older patients with acute myeloid leukemia (AML) reach complete remission (CR) with intensive chemotherapy (IC), ~80% will ultimately relapse. Treatment options in relapse settings are limited and outcome is usually poor. The randomized, placebo-controlled, phase 3 QUAZAR AML-001 trial evaluated oral azacitidine (Oral-AZA; formerly CC-486), a hypomethylating agent, in patients with AML in first remission (CR1) after IC, who were not eligible for hematopoietic stem cell transplantation (HSCT). Unprecedentedly in AML, maintenance treatment with Oral-AZA was associated with significantly longer overall survival and relapse-free survival compared with placebo. Therefore, Oral-AZA received a marketing authorization in the EU as maintenance therapy in adult patients with AML who achieved CR or CR with incomplete blood count recovery (CRi) following induction therapy with or without consolidation treatment and who are not candidates for, including those who choose not to proceed to, HSCT. Also, French health Authority enabled an early access to Oral-AZA in this indication via a Temporary Authorization for Use (ATU) that started in June 2021.

Aims: The aim of the study was to describe the characteristics of patients with newly diagnosed AML who received Oral-AZA for maintenance of CR/CRi after IC and consolidation treatment, who were not eligible for HSCT and were enrolled into ATU in France.

Methods: ATU is a French early access allowing administration of the drug under a Therapeutic Use Protocol approved by health authorities. Data presented in this abstract were collected on a declaratory basis and were not monitored. A total of 52 patients were included in 32 centers across France and treated with Oral-AZA during the period from 09-Jun-2021 to 14-Sep-2021. Oral-AZA monotherapy was administered at 300mg once daily for 14 days per 28-days cycle.

Results: Overall, 52 treated patients were included with a median age of 67.5 years [range: 35-85]. Regarding Performance Status, 22/52 (42.3%) had an ECOG 0, 25/52 (48.1%) 1 and 5/52 (9.6%) ≥2. The majority (43/52 [82.7%]) had a diagnosis of de novo AML, and the genetic risk (according to ELN2017 criteria) was favorable for 13/52 (25.0%), intermediate for 22/52 (42.3%) and adverse for 17/52 (32.7%) of the patients. Most of the patients had received 1 cycle of IC induction (45/52 [86.5%]). The induction regimen was 3 + 7 for 42/52 (80.8%) patients, among those, 3 (5.8%) received midostaurin along with the 3 + 7. Also 7/52 (13.5%) received CPX-351 as induction regimen. The median number of consolidation cycles was 2 [range: 0-6]; 8/52 (15.4%) patients did not receive any consolidation and 6/52 (11.5%) had >3 cycles of consolidation. The majority of patients (27/52 [61.4%]) received IDAC consolidation. The median time between AML diagnosis and Oral-AZA ATU request approval was 6.8 months [range: 1-98]. Because of the limited time frame of the ATU, follow-up is available for 14/52 patients, and the median follow-up is 21.8 days [range: 1-35]. During the follow-up period, one patient had a temporary interruption of Oral-AZA treatment due to grade 4 neutropenia. There was no reported permanent discontinuation of treatment related to an adverse event.

Summary/Conclusion: The characteristics of the patients included in the Oral-AZA Early Access Program in France are in line with European label. These real-life results bring new insights in this population of patients who are not eligible to HSCT with high unmet need to prolong CR1. No unexpected safety signal was registered. Updated results will be presented at the meeting.

PB1806: AZACITIDINE + VENETOCLAX IN PATIENTS WITH ACUTE MYELOID LEUKEMIA, POTENTIAL CANDIDATES TO ALLOGENIC HEMATOPOIETIC STEM CELL TRANSPLANT – RESULTS FROM A HOSPITAL CENTER

C. Barros Lima 1,* , S. Marini 1 , A. Ribeiro 1 , D. Luís 1 , M. Pereira 1 , R. Ferreira 1 , E. Cortesão 1

1 Hematology Department, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal

Background: Azacitidine + Venetoclax (Aza/Ven) is indicated for the treatment of Acute Myeloid Leukemia (AML) in patients not fit for intensive regimens. Although this combination is not approved as first line for younger patients, it may be an alternative is situations where chemoresistant disease is predictable.

Aims: To evaluate the results of the use of Azacitidine + Venetoclax in patients with Acute Myeloid Leukemia, potencial candidates to allogenic hematopoietic stem cell transplant (alloHSCT) treated at the Centro Hospitalar e Universitário de Coimbra in Portugal, a country where CPX-351 is not available.

Methods: We included the patients aged 18-60 years old who were diagnosed with AML, potencial candidates to alloHSCT, and treated with Azacitidine + Venetoclax as first line treatment or following relapse post chemotherapy, between 2019 and 2021.

We evaluated the characteristics of the disease at diagnosis, the risk classification according to the ELN 2017 classification, the response to Aza/Ven, the complications of the treatment and how many patients were able to proceed to transplant.

Results: 13 patients were included, 61% female, median age 46.2 yo [19-60], 3 secondary AML and 10 de novo including 1 myeloid sarcoma.

At diagnosis 9 patients (69.2%) presented with high risk disease, 2 with intermediate risk and 2 with low risk disease.

9 patients underwent chemotherapy with the 3 + 7 regimen as first line, with 55.6% (5) being primarily refractory. 2 underwent re-induction with FLAG-IDA remaining unresponsive.

The 5 patients refractory to first/second induction were treated with Aza/ Ven as 2 nd or 3 rd line. 3 patients achieved complete response (CR), 2 with negative minimal residual disease (MRD -) and 1 with positive minimal residual disease (MRD +) by Immunophenotyping (IFT). 2 had progressive disease and died. Only 1 patient was able to proceed to alloHSCT.

The remaing 4 had CR to first induction and proceeded to consolidation therapy but replapsed. They were treated with Aza/Ven as 2 nd line.

4 patients were treated with Aza/Ven as first line treatment, median age 46.25 yo, 3 with high risk and 1 with intermediate risk disease at diagnosis. 3 patients achieved CR, 2 with MRD – by IFT and 1 with MDR + by MB and 2 have received alloHSCT and 1 is waiting for transplant. 1 died with progressive disease.

The most common complication of the treatment with Aza-Ven was febrile neutropenia, 53.8%, with septic shock in 2 patients.

5 patients (38.5%) were able to proceed to alloHSCT: 1 refractory to induction and had a high risk genetic profile (TP53) and is currently one month post trasplant, 1 with theraphy related AML, 18 months post AlloHSCT and assymptomatic, 1 with MRC AML died with post transplant complications and 1 with MRC AML relapsed after transplant and died with progressive disease. 1 patient is currently in CR and waiting for transplant (MPS secondary AML).

The global mortality rate was 61.5% (8), 4 with progressive disease (1 after transplant), 3 with infectious complications and 1 with transplant complications.

Summary/Conclusion: This results show that Azacitidin + Venetoclax is a valluable tool for the treatment of young patients with AML allowing the achievement of complete responses in patients with refractory disease to induction, including in patients candidates to CPX-351 or as first line treatment, and their referral to a potencially curative alloHSCT.

We need more data to evaluate Aza/Ven as first line regimen in young patients with predictable chemoresistant disease.

PB1807: OUTCOME OF CORE BINDING FACTOR ACUTE MYELOID LEUKEMIA IN CHILDREN AND ADOLESCENTS: A SINGLE CENTER EXPERIENCE

N. Ben Sayed 1,* , G. Abichou 1 , E. Bouselema 1 , H. Regaieg 1 , Y. Ben Youssef 1 , A. Khelif 1

1 Hematology departement, Farhat Hached university hospital, Sousse, Tunisia

Background: Core binding factor acute myeloid leukemia (CBF-AML) is a subgroup of AML, mainly defined by two gene rearrangements affecting CBF;t (8; 21)/RUNX1-RUNX1T1 and inv (16)/CBFB-MYH11. CBF-AML is considered as a favorable cytogenetic group. However, one-third of these patients relapse succeeding treatment.

Aims: The main of this study is to evaluate the clinical characteristics and the outcome of this group of patients in our center.

Methods: This retrospective study included all CBF-AML diagnosed between 2002 and 2020 in the hematology department of the University Hospital of Sousse. Patients were aged 20 years or less. The diagnosis was confirmed according to the WHO classification of hematological malignancies based on cytology and immune-phenotyping. An initial karyotype during diagnosis was performed in all the cases to classify the cases according to the prognostic MRC classification. Patients were treated by the standard chemotherapy regimen. Promyelocytic leukemia and secondary leukemia were excluded. Statistical analysis was performed using the Kaplan Meier method.

Results: Among the 66 children and adolescents diagnosed with AML 15 had CBF AML (23%). 60% were male. The median age was 9 years (3-16). WBC at diagnosis was<4000 in 5 cases, 4000-10000 in 2 cases, between 10000-50000 in 7 cases, and >50000 in 1 case. Disseminated intravascular coagulation was noted in 2 cases. Twelve patients had t(8,21) and 3 patients had inv(16). 75% achieved complete remission after induction. 2 patients had a refractory disease (12,5%). Treatment-related mortality was 12,5% and meantime to relapse was 6,2 months in 5 patients (45%). Three patients responded to salvage chemotherapy (60%) and 2 died (40%). Hematopoietic stem cell transplantation (HSCT) was performed in only 2 patients. For the 3 others, HSCT was not feasible because of the absence of compatible matched related donors. The 5-year overall survival and relapse-free survivals were 55,7% and 58,3% respectively.

Summary/Conclusion: Compared to the literature, the frequency of CBF-AML and epidemiological features were very similar. Furthermore, the majority had t (8, 21) in karyotype. Complete remission rate was slightly lower in our study (73% vs 85-96%), Treatment-related mortality was higher (12% vs 3-10%) which may be explained by the absence of protective isolation in the earlier years of the study. Regrettably, PFS was inferior to data from the literature (70-80%). The early use of stem cell transplants could improve our results.

PB1808: LANGERHANS CELL HISTIOCYTOSIS IN ADOLESCENT PATIENTS: A SINGLE-CENTER RETROSPECTIVE STUDY

H. Cai 1,2,* , J. Chen 1 , T. Liu 1 , H. Cai 1,2 , M. Duan 1,2 , J. Li 1,2 , D. Zhou 1,2 , X. Cao 1,2

1 Department of Hematology; 2 State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China

Background: Langerhans cell histiocytosis (LCH) is a myeloid dendritic cell disorder frequently affecting children and adults, however the clinical characteristics and genetic profiles of adolescent LCH remain elusive.

Aims: This research focused on the clinical and genetic features of adolescent LCH and aimed to address the knowledge gap.

Methods: We performed a single-center retrospective study of 36 adolescent LCH patients aged between 14 and 17 years.

Results: At the time of diagnosis, 10 patients were classified as unifocal single system LCH (27.8%), 2 patients had pulmonary single system LCH (5.6%), 5 patients had multifocal single system LCH with bone involvement (13.9%), and 19 patients had multisystem LCH (52.8%). The most prevalent involvement in multisystem patients was pituitary gland (78.9%), followed by the bone (42.1%), lung (42.1%), and lymph nodes (42.1%). Eight (42.1%) patients had risk organ involvement. BRAF N486_P490 was detected in 50% of patients who underwent next generation sequencing. One patient died and thirteen patients relapsed during the follow up. The estimated 5-year OS rate and EFS rate were 94.7% and 59.0%, respectively. Adolescent patients resembled previously reported adult LCH series in organ involvements, prevalent BRAF deletion mutations and clinical outcomes.

Summary/Conclusion: As the largest adolescent LCH cohort to date, our study found that the clinical characteristics of adolescent LCH patients may be close to adult LCH.

PB1809: CLINICAL TRAJECTORY OF LONG-TERM SURVIVORS OF ACUTE MYELOID LEUKEMIA TREATED WITH INTENSIVE INDUCTION CHEMOTHERAPY

G. Heering 1,* , M. Sasson 1 , D. Dominissini 1 , A. Shimoni 1 , A. Avigdor 1 , A. Nagler 1 , J. Canaani 1

1 Sheba Medical Center, Givatayim, Israel

Background: Notwithstanding the advent of a new era of targeted therapy and innovative molecular breakthroughs seen in acute myeloid leukemia (AML), long-term survival is realized only in a minority of patients. This speaks to the need to thoroughly characterize AML patients achieving long-term survival.

Aims: To characterize long-term survivors in terms of baseline clinical, laboratory, cytogenetic, and molecular data and determine whether these parameters differed to a significant degree from a subset of AML patients who died within two years of initial diagnosis.

Methods: A single center retrospective analysis of all patients with newly diagnosed AML who were treated with intensive induction chemotherapy from July 1, 2008, through July 31, 2016, and who were alive at least five years from the time of initial diagnosis. Clinical, laboratory, cytogenetic, and molecular data were extracted from the institution’s electronic medical records system.

Results: The long-term cohort consisted of 93 patients treated at our institution between the years 2007-2016 with a median follow-up duration of 7.7 years (range 5-13.6 years). Median patient age was 47 years, with 90% presenting with de novo AML. European LeukemiaNet (ELN) 2017 favorable risk patients accounted for 60% of the cohort. All long-term survivors achieved remission following induction chemotherapy. Multivariate analysis showed that compared with 132 patients experiencing death within 2 years of diagnosis, long term survivors were more likely to be of younger age [odds ratio (OR), 0.92; 95% confidence interval (CI), 0.9-0.95; P <.001], have a lower initial WBC count (OR, 0.58; 95% CI, 0.43-0.79; P =0.0004), higher platelet count (OR, 2.19; 95% CI, 1.34-3.57; P =0.0016), undergo an allogeneic stem cell transplantation (OR, 7.95; 95% CI, 3.07-20.59; P <.0001), and harbor favorable risk cytogenetics (OR, 0.03; 95% CI, 0.006-0.23; P =0.0004).

Summary/Conclusion: Long-term survival of AML is seen in a distinct demographic and biologic patient subset.

PB1810: OUTCOME OF ACUTE MYELOID LEUKEMIA RELAPSE AFTER ALLOGENIC STEM CELL TRANSPLANT: A SINGLE INSTITUTION RETROSPECTIVE STUDY

C. Castoldi 1,* , Y. Hicheri 1 , R. Devillier 1 , M.-A. Hospital 1 , E. D’Incan 1 , S. Garciaz 1 , V. Maisano 1 , C. Saillard 1 , J. Rey 1 , A. Granata 1 , S. Harbi 1 , F. Legrand 1 , S. Furst 1 , D. Blaise 1 , N. Vey 1

1 Institut Paoli Calmettes, Marseille, France

Background: With the advances in allogenic stem cell transplant (Allo-SCT), disease relapse has replaced transplant-related mortality (TRM) as main cause of treatment failure in acute myeloid leukemia (AML). Relapse risk after allo-SCT is about 30%, but is extremely variable depending on clinical and genomic features. The prognostic is very poor, with a 2-year survival rate reported to be less than 20%.There is no standard of care in this situation given the variability in patient’s and disease’s characteristics. In patients with good performance status, intensive chemotherapy can be considered, with subsequent cellular therapy such as donor lymphocyte infusion (DLI) or 2nd allo-SCT. For unfit patients, the main goal is to provide acceptable quality of life.

Aims: Our aim is to evaluate the outcome of AML post-transplant relapse.

Methods: We presented here a single-institution series of 118 AML patients who relapsed after allo-SCT between January 2009 and December 2019.

Results: Median age was 53 (20 – 69) years. 22% of the patients had FLT3 mutation and 53% had adverse genetic risk according to ELN. 79% have been transplanted in CR and 21 % with active disease. Conditioning regimen were myeloablative (MAC) in 39% and reduced intensity (RIC) in 61%. 45% of patients received an allograft from a matched related donor (MRD), 22% from an unrelated donor (MUD), 18% from a haplomismatch related donor, 10% from cord blood cells and 5% from a mismatched unrelated donor (MMURD). 18 patients received prophylactic DLI. 22 developed grade >1 aGVHD, and 23 cGVHD (all grades included).

Median time from allo-SCT to relapse was 4.7 months (0.43-88.6). Intensive salvage therapy was used for 28% of the cohort, with a CR rate of 54% and an early mortality of 18%. 64% of patients received non intensive salvage therapy (i.e. demethylating agents, targeted therapies, DLIs…) with a CR rate of 18%. Four of these patients are still alive in CR, all after an association of Azacytidine plus Sorafenib with or without DLI. 8% received supportive care only.

Seventeen patients of the cohort underwent a 2 nd allo-SCT, 14 in CR (13 after intensive salvage, 1 after non intensive salvage) and 6 with active disease. Conditioning regimen were MAC in 35% and RIC in 65% and 90% of patients had a different donor. Donor was haplomismatch related (60%), MRD (20%), MUD (10%), MMUD (10%). The aGVHD (grade≥1)) and cGVHD (all grades included) rates were respectively 25% and 15%. Early TRM after 2 nd allo-SCT was 25%. Four patients (25%) are alive in CR, after intensive salvage treatment and a 2 nd allo-SCT in CR.

In the whole study group, 1year OS from relapse was 25%, and median OS 5.1months. Factors influencing OS in univariate analyses were Sorror comorbidity score ≥3 at 1 st Allo-SCT (1 year OS 17% vs 32%, p=0.036), time to relapse after Allo-SCT < 6months (1 year OS 16% vs 35%, p=0.020), and intensive salvage treatment (1 year OS 33% vs 24%, p=0.019). By multivariate analysis, only PS at relapse (2-3 vs 0.1, HR 1.96, p=0.01) independently influenced OS. Median follow-up (patients alive) was 70.2 (46.3-83.5) months.

Summary/Conclusion: Our data are in line with published studies, and confirm the poor outcome of post-transplant relapse. Intensive salvage therapy shows reasonable response rate, but subsequent cellular therapy seems necessary to provide long term disease-free survival. Azacytidine in association with targeted therapy and/or DLI could be an alternative option to achieve remission.

PB1811: BIOLOGIC AND CLINICAL SIGNIFICANCE OF CD7 ABERRANT EXPRESSION IN ACUTE MYELOID LEUKEMIA: A POOR PROGNOSIS MARKER

H. Charfi 1,* , N. Louati 2 , G. Baccar 2 , O. Kassar 1 , N. Siala 1 , Y. Fakhfakh 1 , M. Chaari 3 , M. Ellloumi 1 , J. Gargouri 2 , H. Menif 2 , T. Rekik 2 , I. Ben amor 2

1 hematology, Hedi Chaker Hospital; 2 hematology, regional blood transfusion center; 3 hematology laboratory, Hedi Chaker Hospital, Sfax, Tunisia

Background: CD7, a T-cell lineage associated antigen, is the most commonly expressed lymphoid antigen in acute myeloid leukemia (AML). There have been conflicting reports in the literature on its expression’s frequencies in AML and whether it may impact treatment response and survival.

Aims: To identify the clinical and biological features of CD7+ AML, and evaluate the association of CD7 aberrant expression with response rates and survival parameters.

Methods: We retrospectively reviewed data from patients diagnosed with de novo AML in our laboratory during january 2013 and december 2020. Acute promyelocytic leukemia was not included in our study. CD7 expression was established by flow cytometry (bone marrow or peripheral blood samples) using a cut-off of > 20%, firstly on Epics XL cytometre (BC) then on FACS Canto II (BD).

Patients demographics, AML characteristics, response to treatment and follow up data were collected. Only patients under the age of 60 were intensively-treated in our center. End points were complete remission (CR), overall survival, event free survival (EFS) and relapse rate. Survival statistical analysis performed with Kaplan Meyer method.

Results: A total of 138 patients were included, of whom 41 (29%) expressed CD7 (9 children and 32 adults). The median age was 7 years and 44 years respectively, with male/female ratio of 1.5. CD7 aberrant expression was significantly associated with an inferior white cells count at diagnosis (9.5 vs 29 G/L, p=0.018), the presence of tumoral syndrome such as adenopathies (24% vs 9%, p=0.02), a FAB M2 classification (34% vs 20%, p=0.028), and the expression of immaturity antigen CD34 (80% vs 54%, p=0.05). Eleven percent (vs 37%, p=0.018), 68% (vs 58%) and 21% (vs 15%) of the cases belonged to the favourable, intermediate (of which a normal karyotype was found in 69% of cases (vs 62%, p=0.03)) and unfavourable cytogenetic categories respectively. Lymphoid cytogenetic abnormalities performed (11q23, t(9;22), t(1;19)) were not detected. CD7 positivity adversely affected both the OS and EFS at 5 years (26% vs 43%, p=0.05 and 27% vs 49% p=0.04 respectively), with an inferior CR rate to induction (43% vs 65% p=0.1) and a higher relapse rate (37% vs 21%, p=0.2). By multivariate cox regression, CD7 expression (HR 1.6; p=0.08) represent an independent predictor of mortality.

Summary/Conclusion: Our results were similar to those of the literature with regard to the frequency of aberrant expression of CD7 in AML (10 to 40%), the frequency of association of CD7+ AML with type M2 and its association with immature antigen CD34 but not with lymphoid cytogenetic abnormalities. The expression of this lymphoid marker in our AML was correlated with prognosis in terms of response to treatment and survival. CD7 expression may be so useful for therapeutic decision algorithms.

PB1812: CORE BINDING FACTOR ACUTE MYELOID LEUKEMIA IN PEDIATRIC POPULATION: THE RESULTS OF A MONOCENTRIC EXPERIENCE

H. Charfi 1,* , O. Kassar 1 , M. Charfi 1 , Y. Fakhfakh 1 , H. Senanna 2 , S. Menif 3 , F. Kallel 1 , I. Frikha 1 , I. Ben Amor 1 , L. Khalifa 1 , M. Medhaffer 1 , S. Hadiji 1 , M. Elloumi 1

1 hematology, Hedi Chaker Hospital, sfax; 2 cytogenetics, Farhat Hached Hospital, Sousse; 3 hematology laboratory, Pasteur institute, Tunis, Tunisia

Background: The core binding factor acute myeloid leukemia (CBF-AML) is characterized by t(8;21) and inv(16)(p13q22)/t(16;16)(p13;q22) cytogenetic abnormalities. It is associated with a good prognosis.

Aims: In this study, we aimed to assess the treatment outcome of the pediatric CBF- AML in comparison with non CBF-AML treated in our center.

Methods: We retrospectively reviewed data of patients under the age of 20 diagnosed with de novo AML in our center during january 2005 and december 2020. The treatment protocol includes: a course of induction (Cytarabine 200mg/m2/day associated with Daunorubicine (DNR) 60 mg/m2/day or Novantrone 12mg/m2/day). If a complete remission (CR) is achieved the chemotherapy is followed by: a cure of ADE (Cytarabin, DNR, Etoposide) associated with 2 cures of high dose of Cytarabine (12g/m2) before 2011 and a cure of ADE associated with 3 cures of high dose of cytarabine (18g/m2) after January 2011. Allogeneic stem cell transplantation (ASCT) from matched sibling donors was limited to patients who are in CR1, having an HLA-identical family donor before January 2011 and for patients who are in CR2 after January 2011. Endpoints were complete remission (CR), overall survival(OS), event free survival (EFS) and relapse rate. Survival statistical analysis performed with Kaplan Meyer method.

Results: A total of 52 patients were included, of whom 16 (30%) presented CBF genes. The median age was 10years. The median white blood cells count at diagnostic was 28G/L. Distribution according to the FAB classification was as followed: 1(6%)M1, 8(50%) M2, 6(38%) M4 and 1(6%) M6.the most frequent subgroup was M2 (vs M1 in non CMF-AML, p=0.001). Cytogenetic analysis showed the Inv 16 (p13;q22) in 3 cases (18%) and t(8;21) (q22;q22) were observed in 11 cases (68%). Two patients had both CBFB–MYH11and AML-ETO rearrangement. Six cases (38%) had additional cytogenetic abnormalities. The correlation between cases with and without additional cytogenetic abnormalities did not conclude to any significent difference in terms of CR rate, OS and EFS.

We observed a comparatively higher CR rate (87% vs 51% p=0.018) and also a better OS rate (83% vs 35%, p=0.02) at 5 years between CBF-AML and non CBF-AML. In contrast, there was no statisticly significent difference in terms of the EFS (67%vs 62%, p=0.4) and the relapse rate (31% vs 43%, p=0.3) between the 2 groups.

Relapse occurred in 5 cases (31%) at a median of 13 months. A second remission was obtained in 2/5 patients.

ASCT was performed in only 4 cases (3 after RC1 and 1 after relapse).

Summary/Conclusion: The frequency of core binding AML in our study is higher than what has been reported in the literature (30 vs 15%) with predominance of The cytogenetic t(8,21) (68%). The additional cytogenetic abnormalities has no prognostic value. Long term survival in our series compared favorably with results reported by cooperative groups. It can be concluded that ASCT in CR1 for pediatric CBF-AML is no longer indicated.

PB1813: EFFICACY OF AZACITIDINE AND VENETOCLAX IN AML PATIENTS MAY BE LIMITED BY INTERACTION WITH POSACONAZOLE AND LACK OF STANDARDISED TREATMENT PROTOCOL. REAL WORLD DATA FROM 3 MAJOR CENTRES IN SINGAPORE.

D. Christopher 1,* , N. A. Abdul Halim 2 , C. A. Gallardo 1 , G. H. Lim 2 , W. Y. Jen 3 , C. Y. Lin 1 , E. F. Bingwen 1 , M. Ooi 3 , Y. L. Chee 3 , V. S. Sampath 1 , G. C. Wong 2

1 Haematology, Tan tock seng hospital; 2 Haematology, singapore general hospital; 3 Haematology, National university hospital, singapore, Singapore

Background: Acute myeloid leukaemia (AML) is a disease of the elderly, with the median age at diagnosis being 68 years. However, both curative and palliative treatment options for elderly AML are limited, with dismal five-year survival outcomes. In Singapore, hypomethylating agents (HMA), particularly azacitidine, is the most used treatment in this cohort of patients. Consistent with results from the phase 3 trial, our experience with azacitidine only showed modest response at best, with complete response (CR) rate of 14.2% and median overall survival (OS) of 9.8 months.

Aims: Here, we report our experience with azacitidine and venetoclax in both newly diagnosed (ND) and relapsed/refractory (R/R) AML. We further evaluate the impact of heterogeneity in both patients and clinical practices on outcome with this novel treatment combination.

Methods: We included 100 consecutive patients with ND and R/R AML, treated with azacitidine and venetoclax in three tertiary hospitals in Singapore from February 2017 to March 2020. These patients were deemed unfit for induction chemotherapy in view of their age/comorbidities. Azacitidine was dosed at 75mg/m 2 for seven days per cycle. The target dose of venetoclax was 400mg once a day. For those on concomitant strong CYP3A4 inhibitor, reduced doses were given at the physicians’ discretion. The treating physicians also decided on the timing of bone marrow assessment as well as dose interruptions and modifications for toxicities. Minimal residual disease (MRD) was assessed via multiparameter flow cytometry (MFC) with a sensitivity of 10 -4 .

Baseline demographics data, disease characteristics and treatment details were obtained from electronic health records of the respective hospitals, after obtaining IRB approval.

Results: In ND patients, the overall response rate (ORR) was 51.9%: 16.7% achieved complete response (CR), 27.8% CR with incomplete count recovery (CRi), and 7.4% morphologic leukaemia-free state (MLFS). Amongst responders, MRD was undetectable in 13 (46%) patients. The median time to best response was 1.8 months (1.1-3.5 months) and median DOR was 4.8 months. Red cell transfusion independence was achieved in 38.2% of patients, and platelet transfusion independence in 41.8%. Median OS for ND patients was 8.4 months (4.6 months -NR). Responders had significantly longer median OS than non-responders (20 vs 3.55 months, p=<0.001).

For R/R patients, the ORR was 48.8% with 11.1% achieving CR, 33.3% CRi, and 4.4% MLFS. 41.9% of the responders had undetectable MRD. The median time to best response was 1.8 months (1.6-2.1 months) and median DOR was 4.9 months. 38.1% achieved red cell transfusion independence and 38.1% was platelet transfusion independent. The median OS for R/R patients was 8.6 months (4.9 months – NR), with the median OS for responders being significantly longer than non-responders (NR vs 4.93 months, p=0.006).

13 patients (Five ND and eight R/R) proceeded to allogeneic SCT following response to azacitidine and venetoclax. Five (38.4%) had undetectable MRD prior to proceeding to transplant. 11 are still alive and in remission.

Summary/Conclusion: In summary, our analysis demonstrated that azacitidine and venetoclax show high response rates and improved OS amongst patients who responded to treatment. However, survival and haematological improvement may be hampered by early discount intuition due to interaction with posaconazole and lack of adherence or availability of a standardised dose modification protocol. Alternative treatment strategies such as continuation of azacitidine as a single agent should also be explored.

PB1814: RARE PHENOTYPE WITH CD33 ABSENT IN ACUTE MYELOID LEUKEMIA MIMICKING MRD POSITIVITY: A CASE REPORT

L. O Marani 1,* , A. Costa 1,2 , F. B Silva 1 , M. I. A Madeira 1 , P. S Scheucher 1 , J. L S Schiavinato 1 , A. S. G Lima 1 , K. B B Pagnano 3 , B. K Duarte 3 , S. Freeman 4 , E. M Rego 5 , F. Traina 1 , L. L Figueiredo-Pontes 1

1 Hematology Division, Department of Medical Images, Hematology, and Clinical Oncology, Ribeirao Preto Medical School at University of Sao Paulo, Ribeirao Preto, Brazil; 2 Division of Hematology and Oncology, Department of Medicine, UAB Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, United States of America; 3 Hemocentro, University of Campinas, Campinas, Brazil; 4 Institute of Immunology and Immunotherapy, University of Birmingham, Birminham, United Kingdom; 5 Internal Medicine, Medical School of the University of Sao Paulo, Sao Paulo, Brazil

Background: Treatment of acute myeloid leukemia (AML) remains difficult to this day, with high rates of relapse that lead to poor long-term prognosis and low cure rates and are usually driven by residual leukemic cells that can be identified as measurable residual disease (MRD). Multiparametric Flow Cytometry (MFC) has been a fast and effective tool for detection of MRD, but disease heterogeneity makes MRD assessment a clinical challenge, especially in cases with rare phenotype, needing clinical and technical expertise to be accurately performed.

Aims: To show a rare case of AML with absent CD33 expression and the importance of accurately interpreting MDR in AML.

Methods: We report a clinical case of AML with absent CD33 assessed for FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype and an 8-color panel immunophenotyping (Table 1). Empty gating strategy was used to identify LAIPs (Leukemia Associated-Immunophenotype), different from normal and leukemic stem cells, compared to healthy BM from diagnosis and follow up. Assays were performed at the Hematology Laboratory of Ribeirão Preto the Medical School, University of São Paulo, in accordance to Local Ethical Boards.

Results: A 21-year-old male with AML without maturation, presented anemia (7.9 g/dL), thrombocytopenia (26 000/µL) and a total white blood cell count of 5710/μL with 84% of blasts in BM. MFC revealed CD45 dim and low side scatter, CD117 + , CD34 + , HLA-DR + , CD38 + , CD123 + , CD11c + , CD64 + , CD15 + , CD133 + , CD14 - , CD7 - , CD11b - , CD4 - , CD56 - , CD19 - , CD41a - , NG2 - and, unexpectedly, completely negative CD33. No metaphases or molecular abnormality was found. Treatment consisted of 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day). Consolidation consisted of 1 or 2 cycles of 6 days of cytarabine 1 g/m2 twice a day. BM were obtained at diagnosis, after 1 st and 2 nd induction, and 3, 9, 12 and 15 months after consolidation of chemotherapy. A specific phenotype (CD117+/CD34+/CD33wk/CD13+ ≥ 0.1%) was preserved from diagnosis to all follow up points: 0,506%, 0,681%, 0,402%, 0,101%, 0,158% and 0,122% (Figure 1). Complete remission was achieved by day 30 after 1 st induction and no change in the outcome was reported, even though an altered maturational phenotype persisted with complete absent CD33 expression, during and after treatment, suggesting a very rare polymorphism of CD33 receptor that mimicked MRD positivity.

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Summary/Conclusion: Post-analytical phase of MFC is the most vulnerable to wrong interpretations. CD33 is a myeloid antigen expressed on malignant blasts in AML and had been reported as a potential target therapy. CD33 low blasts are associated to a more mature AML and its high expression is related to adverse landscapes in AML, highlighting the importance of its evaluation. Standard care for AML enrolls MRD monitoring and failure to achieve an MRD-negative, CR or detected MRD during or after therapy is associated with relapse. Still, MRD analysis must embrace a set of standards in order to prevent post-analytic errors. Here we present a case report of AML with persistent absent CD33 expression since diagnosis that we found to be mimicking MRD positivity. Our data emphasizes the importance of clinical and technical expertise in MFC analysis and correlation of diagnostic and follow-up interpretation, especially in a heterogeneous disease such as AML, bringing up the need of standardization and constant training of the team.

PB1815: T(8;16)(P11;P13): A RARE CASE OF ACUTE MYELOID LEUKEMIA

R. Cuccurullo 1,* , G. Aloj 1 , A. De Matteo 1 , G. Giagnuolo 1 , F. Cacace 1 , M. L. Conelli 2 , L. Bufalo 2 , F. P. Tambaro 1 , G. Menna 1

1 Oncology, AORN Santobono- Pausilipon; 2 Pediatric, Università degli Studi di Napoli Federico II, Naples, Italy

Background: Acute myeloid leukemia (AML) with t(8;16) is a rare AML subtype (0.2% in adult setting) that represents a distinct clinical entity, often therapy-related. t(8;16)(p11;p13) involves MYST histone acetyltransferarse 3 (MYST3) gene on chromosome 8p11 and cAMP response element binding protein (CREBBP) gene on chromosome 16p13. Both genes code for proteins with histone acetyltransferase activity crucial for cell cycle control. Pediatric t(8;16) AML may be associated with extramedullary disease, high rate of Disseminated Intravascular Coagulation (DIC), FAB M4 or M5 morphology. In neonatal cases spontaneus regression is frequently observed.

Aims: To report on a pediatric case of the rare AML with t(8;16)(p11;p13).

Methods: A 12 year-old female was hospitalized for hypermenorrhea, drop attack, fatigue and leukocytosis. Clinical examination showed: paleness, diffuse pain, petechiae to the oral mucosa. No organomegaly was detected. At peripheral blood cells count she had serum hemoglobin of 5 g/dL, 78.900/mm 3 White Blood Cells (WBC) and 96.000/mm 3 platelets. Fibrinogen was diminished (32 mg/dL; range 150-400 mg/dL) while D-Dimers were elevated (30.644 ng/ml; Range 0-500) as laboratory signs of DIC. Peripheral blood smear showed myeloid blasts. Platelets and plasma transfusions were performed.

Myeloid blast combined to DIC suggested acute promyelocitic leukemia presumptive diagnosis but t(15;17) was not found at PCR rapid test. Bone marrow observation confirmed AML-M5 diagnosis with monocytoid blasts infiltrate. Central Nervous System involvement was ruled out by lumbar puncture. The patient was enrolled in AIEOP LAM 2013-01 protocol. Before first chemotherapy administration, spontaneous WBC reduction was observed (6.730/mm 3 ). The hemorrhagic diathesis worsened drammatically during the second day of chemotherapy with bleeding from the central venous catheter insertion site. The blood tests revealed rapid decrease of WBC to 1.240/mm 3 , platelet and fibrinogen respectively to 34.000/mm 3 and 57 mg/dL, while D-Dimers raised to 113.304 ng/ml and INR became 2.04.

During the following days of chemotherapy, the performance status of the patient improved and the bleeding episodes decreased. WBC count progressively declined while platelet count and fibrinogen level gradually raised to better values; D-Dimers level and INR progressively recovered too.

Bone marrow molecular biology revealed the presence of t(8;16)(p11;p13). In addition, a complex karyotipe (45XX,t(8;16)(p11;p13),-9[4]/46XX,t(8;16)(p11;p13),-9,+21[1]/46XX[1]) was found during the cytogenetic tests. According to protocol LAM 2013-01, both these features are predictive of poor prognosis, therefore the patient was enrolled in the high risk therapeutic outline, receiving four cycles of chemotherapy (ICE, ICE, AVE, HAM) and bone marrow transplantation. She achieved hematological complete remission after the first cycle of chemotherapy, maintaining that until an HLA identical sibling bone marrow transplant was performed.

Results: Actually, 7 months after the trasplant, the patient is in complete remission.

Summary/Conclusion: t(8;16) AML is a rare pediatric leukemia, characterized by M4/M5 morphology, high frequency of extramedullar involvement, complex karyotipe and DIC. Spontaneous remission often occur in neonatal cases. In our case, the presence of t(8;16) could explain the WBC reduction before the administration of chemotherapy. The knowledge of this entity is importat to quickly recognize the correct diagnosis and to begin the proper therapy as soon as possible, avoiding major complications due to coagulopathy.

PB1816: CHEMOTHERAPY FREE APPROACH IN A PEDIATRIC PATIENT WITH VERY LATE CUTANEOUS RELAPSE OF ACUTE PROMYELOCYTIC LEUKEMIA.

V. Crisci 1,* , E. Rossitti 2 , G. Aloj 3 , P. Stellato 3 , G. Menna 3 , R. Cuccurullo 3

1 Pediatric, Università degli Studi di Salerno, Salerno; 2 Pediatric, Università degli Studi di Napoli Federico II; 3 Oncology, AORN Santobono- Pausilipon, Naples, Italy

Background: Acute Promyelocytic Leukemia (APL) is a very rare leukemia in children. At diagnosis it is frequently fatal but at the same time highly curable through the combination of all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy. Extramedullary involvement by APL can be observed in about 3-5% of cases, mainly during the relapses, and the most affected sites are the central nervous system, the skin, and the testes.

Aims: To report on the use of arsenic trioxide (ATO) combined with ATRA in a pediatric patient with very late APL extramedullary relapse.

Methods: A.M., a 14-year-old female, was admitted to our hospital with post-traumatic ecchymosis and metrorrhagia. Trilinear cytopenia and coagulopathy were present at diagnosis. Bone marrow aspirate showed an infiltrate of immature myeloid cells with features of promyelocytes. Molecular analysis by RT-PCR showed expression of the PML/RARa fusion protein. Standard Risk APL was diagnosed. The patient was treated according to protocol AIEOP ICC-APL 01 SR as front-line therapy. At the end of the induction the first complete haematological remission was achieved, while the first complete molecular remission was obtained at the end of the first consolidation cycle.

Three years and seven months after the onset of the disease, a skin lesion appeared on her back. A biopsy was performed and a promyelocytic infiltrate was found. The diagnosis was confirmed by the immunohistochemical analysis. Bone marrow aspirate, lumbar puncture and total body Computed Tomography did not show any further site of relapse. Thus, an Isolated Extramedullary Very Late Relapse (more than 36 months after the onset of the disease) of APL was diagnosed.

Although the best therapeutic option for APL relapses was still controversial at that time, the patient was treated with the most updated approach, receiving an induction cycle of 30 days with the association of ATRA at the dose of 25 mg/m 2 /die and ATO at the dose of 0.15 mg/Kg/die and a consolidation with 4 cycles of ATO (5 days per week, 4 weeks on and 4 weeks off) and 7 cycles of ATRA (7 days per week, 2 weeks on and 2 weeks off). The treatment was well-tolerated, except for sporadic occurrence of headache and an isolated episode of generalized seizures.

Results: The patient completed the treatment 8 months later and actually, 5 years after the relapse, is in complete haematological and molecular remission.

Summary/Conclusion: Several large multicenter studies have shown that regimens combining upfront ATRA with chemotherapy lead to high remission rates in APL. However, relapses still occur in 15% to 25% of cases. Extramedullary involvement is extremely rare at relapse, especially in pediatrics. Salvage therapy should be chosen considering the previous front-line therapy and is followed or not by auto or alloHSCT depending on the achievement of molecular remission.

The presented pediatric case did not receive the association ATO-ATRA in front-line because it was not available in that era. Therefore, she received the association ATO-ATRA in second line, obtaining the second complete remission with a chemo-free regimen.

This case shows that the association ATO-ATRA as single therapy in pediatric patients is safe and effective also for relapses and that the best second line therapeutic regimen should be chosen considering the treatment history of each patient.

PB1817: PROGNOSTIC SIGNIFICANCE OF CD200 ON ADULT PATIENTS WITH ACUTE MYELOID LEUKEMIA AND ITS MECHANISM

X. Quan 1,* , D. Huang 1 , J. Li 1 , T. Yang 1 , Y. Liu 1

1 Department of Hematology-Oncology, Chongqing University Cancer Hospital, Chongqing, China

Background: The prognostic significance of CD200 expression in adult AML (non-M3) patients is still unknown.

Aims: To analyze the prognostic significance of CD200 expression in newly diagnosed adult patients with non-M3 acute myeloid leukemia (AML) in our department, and to explore the effect of CD200 expression on lymphocyte subsets of patients.

Methods: The subject was 86 adult patients with AML (non-M3) who were newly diagnosed from April 2018 to December 2020. Clinical information of these patients was collected, and their survival station was followed up to analyze the connection between CD200 and 1-year overall survival (OS) rate. Patients who received chemotherapy were further evaluated, analyzing the relationship between CD200 expression and other clinical characteristics, as well as the relationship between CD200 and complete response (CR) after the first chemotherapy. And the effect of CD200 expression on lymphocyte subsets of patients was analyzed.

Results: Of our study of 86 patients, 60 patients were given chemotherapy. These 60 patients were divided into CD200 + AML and CD200 - AML group according to the CD200 expression. There were no differences between CD200+AML group and CD200-AML group in parameters such as gender, age, white blood cell count and proportion of bone marrow blast cells. The difference of CR rate between two groups was statistically significant (p = 0.025). Univariate and multivariate logistic analysis of potential influencing factors for CR rate showed that CD200 was an independent influencing factor for CR rate after the first course of chemotherapy, and the probability of obtaining CR in the CD200 negative group was 4.15 times that of the CD200 positive group (P =0.018). Among them, 30 patients were stratified to moderate risk according to 2021 NCCN guidelines of AML. The CR rate between CD200 negative and CD200 positive subgroups was compared, and the CR rate in the CD200 negative subgroup was higher than that in the CD200 positive subgroup, but there was no statistical significance (P=0.056). There was no difference in 1-year OS rate between the two groups. Among patients included in the study, there was no significant difference in the percentage of T lymphocytes, regulatory T lymphocytes, NK cells and other subsets in WBC between the CD200+AML group and the CD200-AML group.

Summary/Conclusion: In summary, our data suggest that CD200 expression in adult AML (non-M3) patients indicated poor CR rate after the first course of chemotherapy, in the follow-up data of this study so far, CD200 had no prognostic significance for 1-year OS rate. There was a correlation between CD200 expression and CR rate in patients of moderate risk, but there was no statistical significance. Moreover, our data suggest that CD200 expression had no significant effect on lymphocyte subsets in patients.

PB1818: INFLUENCE OF BODY MASS INDEX ON THE OUTCOME IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

A. Elazab 1,* , M. Denewer 2 , M. Mabed 3

1 Oncology Center, Mansoura University; 2 Oncology Center, Mansoura University; 3 Oncology Center Mansoura University, Mansoura, Egypt

Background: Previous studies have demonstrated an association between high body mass index (BMI) and acute myeloid leukemias (AML). However, the results remain controversial.

Aims: The aim of our study was to assess the impact of BMI on the outcome in adult patients with newly diagnosed AML

Methods: Our study enrolled 140 patients with AML diagnosed between 2015 and 2020. The data were collected from Oncology Center, Mansoura University (OCMU) electronic medical record (eMR). They are further classified according to BMI into patients with normal BMI (40 patients), overweight (42 patients) and obese patients (58 patients)

Results: No significant association is found between BMI and laboratory studies including CBCs, LDH (95% confidence interval [CI], P=0.645) or blast percentages in bone marrow aspirates (95% CI, p=0.587). As regard Complete Remission (CR) rates after induction chemotherapy with 3 + 7 protocol (three days of anthracycline+ seven days of cytrabine), our study concluded that CR rates are higher in patients with normal BMI when compared to overweight and obese patients (95% CI, p=0.004). BMI does not significantly affect disease-free survival (DFS) (95% CI, p=0.408). However, Higher BMI is associated with decreased overall survival (OS) (95% CI, p=0.004).

Summary/Conclusion: In the treatment of acute myeloid leukemia in adults, overweight and obesity were associated with decreased complete response rates. However, overweight and obesity dont have a significant impact on disease free survival. A larger, prospective randomized trial would be required to confirm these results.

PB1819: CHARACTERISTICS AND OUTCOME OF PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA IN SOUTHERN TUNISIA

O. kasser 1,* , A. koubaa 1 , M. charfi 1 , Y. fakhfakh 1 , F. kallel 1 , I. frikha 1 , I. ben amor 1 , L. khalifa 1 , M. medhaffer 1 , S. hdiji 1 , M. elloumi 1

1 hematology, university of Sfax, Sfax, Tunisia

Background: All-trans retinoic acid (ATRA) is the major advance in the treatment of acute promyelocytic leukemia (APL). Its introduction in the treatment of APL has revolutionized the outcome of this disease.

Aims: The current study reports the clinical features and treatment outcome of patients with acute promyelocytic leukemia (APL) treated at the hematology department of Sfax from Tunisia.

Methods: Our study was retrospective. It concerned all patients with the diagnosis of APL and followed in the hematology department of Hedi Chaker Hospital Sfax, from January 2000 until December 2019. The diagnosis of APL is based on morphological, cytogenetic and molecular study. The specific treatment is similar to that of the French protocol APL93 until 2013, and then similar to that of Spanish protocol LPA2005 since January 2014. We evaluate the rates of remission, death and overall survival (Kaplan-Meier method).

Results: We collected 79 patients. The sex ratio was 1.3 with a median age of 34 years (extreme: 2-75 years). Hyperleukocytosis (GB> 10000 / mm3) was noted in 29 patients (37%). The average white blood cell count was 21900 / mm3. The disseminated intravascular coagulation (DIC) was found in the majority of cases (72% of cases). The t(15,17) was found in 89% of cases. The PML-RARA transcript was positive in 100% of cases. Eight patients (10%) died before treatment with hemorrhage. Thirty three patients were treated by the APL 93 (group1) and thirty eight patients were treated by the protocol LPA2005 (group 2). Fifteen patients (22%) died early during the induction course before evaluation. Causes of death during treatment were multiple: respiratory disease due to the differentiation syndrome, a severe haemorrhagic syndrome and or septic shock. Complete remission was obtained in 55 patients (98% of the evaluable patients and 77% of the treated patients) and one patient failure (2%). No deaths were reported during consolidation or maintenance treatment. A late bone marrow relapse was observed in 8 patients (14,5%). At five years of follow up, overall survival and event-free survival are 66% and 64%, respectively, and relapse-free survival is 77%.

Summary/Conclusion: In our series, the DIC rates are correlated with that of the literature, as well as early death rate. The complete remission and overall survival remain lower than that of the literature. This is explained by a high rate of early mortality (during induction) by hemorrhage, of the higher frequency of hyper-leukocyte forms and the delay in diagnosis and management for our patients.

PB1820: CD47-BLOCKER TTI-622 COMBINED WITH AZACITIDINE IN PATIENTS WITH TP53-MUTATED ACUTE MYELOID LEUKEMIA (AML) AND WITH AZACITIDINE + VENETOCLAX IN ELDERLY OR UNFIT PATIENTS WITH TP53-WILDTYPE AML

N. Daver 1,* , M. Maris 2 , R. Ramchandren 3 , D. Bixby 4 , K. Doucette 5 , R. Mawad 6 , D. Egan 6 , D. Stevens 7 , J. Zonder 8 , N. Molloy 9 , A. Scheuber 9 , I. Bruns 9 , I. Mantzaris 10 , M. Konopleva 1 , A. D. Goldberg 11

1 University of Texas MD Anderson Cancer Center, Houston; 2 Colorado Blood Cancer Institute and Sarah Cannon Research Institute, Denver; 3 University of Tennessee, Knoxville; 4 University of Michigan, Ann Arbor; 5 Georgetown University Hospital, Washington, DC; 6 Swedish Cancer Institute, Seattle; 7 Norton Healthcare, Louisville; 8 Karmanos Cancer Institute/Wayne State University, Detroit; 9 Trillium Therapeutics, a Pfizer company’, Cambridge; 10 Einstein College of Medicine; 11 Memorial Sloan Kettering, New York, United States of America

Background: CD47 is an innate immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a “don’t eat me” signal to suppress macrophage phagocytosis. TTI-622 is a fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG4. It is designed to enhance phagocytosis and antitumor activity by preventing CD47 from delivering its inhibitory signal as well as generating a moderate pro-phagocytic signal via IgG4 Fc. Importantly, unlike many CD47-blocking agents, TTI-622 does not bind to human red blood cells. Increased expression of CD47 is an adverse prognostic factor in acute myeloid leukemia (AML) due to its association with reduced event-free and overall survival, and blockade of CD47 has shown pre-clinical and clinical activity in AML. In vitro, both azacitidine and venetoclax significantly enhanced the pro-phagocytic effect of TTI-622 on macrophages.

Aims: To characterize the safety and preliminary efficacy profile of TTI-622 in combination with azacitidine in newly diagnosed TP53-mutated AML and in combination with azacitidine + venetoclax in elderly or unfit, newly diagnosed TP53-wildtype AML. Pharmacokinetics and immunogenicity of TTI-622 combined with azacitidine ± venetoclax will also be investigated. Primary endpoints include dose-limiting toxicities, incidence of adverse events (AEs) and complete response (CR). Minimal residual disease status will also be evaluated.

Methods: As part of an ongoing dose-escalation and expansion trial of TTI-622 ( NCT03530683 ), patients with AML meeting the below key entry criterion are enrolled. Cohort A includes patients with newly diagnosed TP53-mutated AML. Cohort B includes patients with newly diagnosed TP53-wildtype AML who are either elderly ( 3 75 years) or unfit. Unfit patients include those < 75 years with one or more of the following comorbidities: Eastern Cooperative Oncology Group (ECOG) performance status of 2, clinically significant heart or lung comorbidities, contraindication to anthracycline therapy, or other comorbidity the investigator regards as incompatible with intensive remission induction chemotherapy. All patients from both cohorts must have documented bone marrow or peripheral blast counts 3 20% and must have received no prior systemic therapy for AML nor hypomethylating agents for any antecedent hematologic disorders prior to transformation to AML. Patients must have no extramedullary disease or leukocytosis >25,000/mL. Renal function must be 3 30 mL/minute based on Cockcroft-Gault estimate. There are no restrictions based on neutrophil or platelet counts, or hemoglobin level. Patients with AML secondary to antecedent hematologic disorders or therapy induced will not comprise more than 50% of either Cohort. Up to 30 patients will be enrolled into each cohort.

In 28-day cycles, patients in both Cohorts will receive TTI-622 8 mg/kg intravenous (IV) infusions weekly combined with azacitidine 75 mg/m 2 administered either IV or subcutaneously for 7 consecutive days, repeated approximately every 4 weeks. Patients in Cohort B will also receive venetoclax 100 mg and 200 mg orally on Days 1 and 2, respectively, followed by 400 mg (or equivalent adjusted for azoles) daily thereafter. Safety monitoring includes clinical laboratories and assessments of AEs based on CTCAE v 4.03. Response assessments of both bone marrow and peripheral blood are performed at the end of Cycles 1, 2 and 4; and then every 3 cycles thereafter. Response assessment is per the ELN2017 criteria.

Results: Results N/A (trial-in-progress)

Summary/Conclusion: Conclusion N/A (trial-in-progress)

PB1821: COMBINATION OF BCL-2 INHIBITOR, HYPOMETHYLATION AGENTS AND LOW-DOSE CYTARABINE AS A SALVAGE THERAPY FOR ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES

X.-N. Gao 1,* , X. Lou 2 , J. Lin 2 , Y.-F. Su 1 , J. Wang 1 , L. Xu 1 , H. Yu 2 , A. Wang 1 , Y.-Z. Wang 1 , D.-H. Liu 1

1 Senior Department of Hematology, the Fifth Medical Center; 2 Chinese PLA General Hospital, Beijing, China

Background: Although studies are ongoing to evaluate the efficiency and safety of venetoclax-based therapy, alone or in combination with hypomethylation agent or low-dose cytarabine, in relapsed/refractory acute myeloid leukemia, data are scarce and heterogenous. Herein, we report our experience of a new venetoclax-based triple-drug combination regimen in acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) in the salvage setting.

Aims: To assess safety and response to venetoclax + hypomethylation agent + low-dose cytarabine regimen after minimum 1 cycle of therapy in patients with AML or MDS, who developed morphologic or molecular relapse, or had refractory disease or persistent positive minimal residual disease (MRD) at the end of consolidation.

Methods: This is a retrospective, observational cohort study enrolled in 11 consecutive patients with AML or MDS with excess blasts (MDS-EB). Venetoclax was given at a dose of 400 mg/day for 28 days per cycle. Decitabine was given at a dose of 20 mg/m 2 /day for 5 days (n=3) or azacytidine (n=8) was given at a dose of 75 mg/m 2 /day for 7 days at the discretion of the treating physician. Cytarabine was given at a dose of 10 mg/m2 twice daily for 7 days. This study was approved by the Ethics Committee of the Chinese PLA General Hospital, and signed informed consents were obtained from all patients prior to treatment in accordance with principles of Declaration of Helsinki.

Results: As shown in Figure 1, overall response rate was 72.7%. Three of the 6 patients with relapsed and refractory diseases achieved MRD-negative complete remission. Remarkably, all of the 5 patients with persistence or relapse of MRD achieved durable MRD-negative remission. Majority of patients (10/11) were heavily pretreated with an Eastern Cooperative Oncology Group score of 2-3, whereas no early mortality (death within 90 days of therapy initiation) was recorded and disease relapse was the only cause of death. The estimated 1-year overall survival and relapse-free survival rates were 72.9% (95%CI, 27.6%-92.5%) and 58.3% (95%CI, 7.7%-89.3%), respectively.

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Summary/Conclusion: This study highlights the promising activity and safety of the venetoclax-based triple-drug regimen in patients with relapsed/refractory AML or MDS unfit for intensive chemotherapy and patients with persistent MRD.

PB1822: A CASE OF EXTRAMEDULLARY ACUTE MYELOID LEUKEMIA SUCCESSFULLY TREATED WITH NOVEL AGENTS

V. Gianfelici 1,* , L. Levato 1 , D. Magro 1 , E. Piro 1 , C. Santamaria 1 , L. Scalise 1 , P. Marinaro 1 , D. Loiacono 1 , A. Mannella 1 , A. Russo 1 , L. Tucci 2 , M. Rossi 1,3

1 Department of Hematology-Oncology; 2 Department of Pathological Anatomy, “Pugliese-Ciaccio” Hospital; 3 Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy

Background: Extramedullary acute myeloid leukemia (eAML) is a relatively rare clinical presentation, that carries a poor prognosis and poses therapeutic dilemmas. Treatment intensification with allogeneic hematopoietic cell transplantation (alloHCT) is often recommended for concurrent extramedullary and bone marrow (BM) involvement. In the last years, novel drugs have been approved for AML treatment, though their efficacy in eAML needs to be clarified. We here describe a case of therapy-related AML (tAML) with NPM1 mutation and cutaneous localization that achieved complete remission (CR) with CPX-351. Unfortunately, she developed a meningeal relapse followed by medullary relapse with acquired FMS-like tyrosine kinase 3 (FLT3) internal tandem duplications (ITD) mutation.

Aims: To define the genetic profile and the efficacy of novel drugs in eAML.

Methods: The diagnosis of AML was established according to the WHO. Flow cytometry analysis was performed by FACSCanto Instrument on BM and cerebrospinal fluid (CSF). Cytogenetic analyses (G-banding) were performed on BM at diagnosis and relapse. Molecular analysis at diagnosis and relapse included NPM1, FLT3-ITD and -TKD. NPM1 monitoring was performed after induction and consolidation. Cutaneous involvement was documented by biopsy.

Results: A 56 year-old woman, previously treated for choriocarcinoma, presented to our Institution because of leukocytosis, anemia and diffuse cutaneous lesions. BM examination was consistent with a diagnosis of AML with monocytic differentiation, while cutaneous biopsy showed a dermic infiltration of leukemic cells. Chromosome analysis showed a normal karyotype, but molecular analysis revealed the NPM1 mutation with wild-type conformation of FLT3 gene. Even with a favorable molecular profile, she was considered a high-risk patient as of tAML and leukemia cutis, thus eligible for alloHCT. She underwent induction and 2 cycles of consolidation with CPX-351. After induction, she obtained a complete hematological and molecular response with complete disappearance of cutaneous nodules. Three months after CR, she complained headache, diplopia and paralysis of the right eyes. CSF examination showed a meningeal relapse of AML while BM biopsy confirmed the haematologic and molecular remission. She was treated with intrathecal methotrexate and cytarabine (ITC) given bi-weekly until achievement of morphological and cyto-fluorimetric CSF-remission. After the XII ITC, the patient developed anemia and leukopenia. BM and CSF examination revealed concomitant BM and CSF relapse. Rescue therapy with high dose cytarabine was promptly started. Subsequently, molecular analysis documented NPM1 mutations in 4% of cells and the acquisition of FLT3-ITD in 40% of cells. Based on these data, second generation FLT3 inhibitor gilteritinib was chosen due to previous reports of crossing hemato-encephalic barrier. The drug was started from day 8 at 120 mg/day. Treatment was well-tolerated. She obtained second CR and at time of writing she was waiting for a familiar alloHCT. Targeted next-generation sequencing by Illumina MiSeq is ongoing.

Summary/Conclusion: This is the first report of a positive effect of CPX-351 in cutaneous localization of AML, while few cases have been reported about the effect of gilteritinib in meningeal sarcoma. Furthermore, the case here reported underlines the importance of FLT3 retesting at relapse. Overall, these data suggest to design ad hoc studies in eAML to clarify the genetic profile and the effect of novel drugs in this setting.

PB1823: SECONDARY ACUTE MYELOID LEUKEMIA AND DE NOVO ACUTE MYELOID LEUKEMIA WITH MYELODYSPLASIA-RELATED CHANGES DISTINCT LANDSCAPE, OR THE SAME DISEASE?

M. Jagurinoski 1,* , E. Aleksov 2 , M. Stojcov-Jagurinoska 2 , I. Tonev 2 , R. Vladimirova 1 , Y. Davidkova 1 , G. Balatzenko 3 , B. Spassov 2 , M. Guenova 1

1 Laboratory of Hematopathology & Immunology; 2 Department of Clinical Hematology; 3 Laboratory of Cytogenetics & Molecular Biology, Specialized Hospital for Active Therapy of Hematological Diseases, Sofia, Bulgaria

Background: Acute myeloid leukemia (AML) with myelodysplasia-related changes is a heterogeneous category in the WHO classification. It includes both AML occurring de novo which harbors myelodysplastic morphological and/or cytogenetic features (dn-AML-MDS), and “secondary” AMLs after a previous myeloid neoplasm, e.g., myelodysplastic syndrome (MDS) or myeloproliferative neoplasms or aplastic anemia (s-AML-MDS). There is still uncertainty about the affiliation of these categories to a common pathogenetic and clinical course.

Aims: To perform a comparative analysis between patients with AML secondary to MDS and dn-AML-MDS.

Methods: A total of 63 patients (58.7% men and 41.3% women), mean age 62 years (range, 21-85 years), divided into two groups, s-AML-MDS (n=30) and dn-AML-MDS (n=33), were analyzed. Diagnosis was based according to WHO classification criteria (2016).

Results: Comparative analysis showed no differences in demographic characteristics, hematologic parameters, and % of blasts in bone marrow or peripheral blood between the groups. Morphological analysis revealed dysplasia in more than one cell lineage in all patients, with significantly higher incidence of granulocytic dysplasia in the dn-AML-MDS 30/32 (93.8%) vs. s-AML-MDS 16/27 (59.3%) (p<0.001). The phenotypic profile showed no differences between the groups, either in terms of early markers CD34, CD117 or aberrant expression of lymphoid markers. We observed a higher incidence of aberrant karyotypes in dn-AML-MDS vs. s-AML-MDS (71.4% vs. 40%, p=0.02). Patients with dn-AML-MDS had a higher frequency of abnormalities on chromosome 7 and chromosome X in contrast to s-AML-MDS (respectively, 32.1% and 21.4% vs. 8% and 0.0%, p=0.031 and р=0.014). Molecular analysis also showed differences, as the FLT3-ITD mutation, was observed in only one case (4.2%) with dn-AML-MDS while in 30% with s-AML-MDS, respectively (p=0.02). No differences were found with respect to mutations in the NPM1 gene and EVI1 overexpression. During the follow-up period, s-AML-MDS showed a 13.8% survival rate compared to 33.3% in dn-AML-MDS patients, and the median survival in both groups was very short, 5.9 mo. (SE 0.85, p>0.05).

Summary/Conclusion: AML-MDS occurring de novo or as a “secondary” disease unites categories with extremely unfavorable prognosis that show similarities but also differences in biological features. The accumulation of knowledge in the field of genomic, epigenetic changes determining its course, could lead to the construction of new algorithms for more accurate diagnosis, risk assessment and personalized therapeutic approach.

PB1824: IMPACT OF DAY 15 BONE MARROW ASPIRATION ON PREDICTION OF COMPLETE REMISSION AND RELAPSE IN ACUTE MYELOID LEUKEMIA

R. Kharrat 1,* , O. Charef 1 , R. Berred 1 , A. Chabaane 1 , S. Sammoud 1 , M. Bchir 1 , F. Ben Lakhal 2 , S. Feki 2 , W. Borgi 2 , E. Gouider 2 , A. Amouri 3 , W. Ayed 3 , H. Ben Neji 1 , B. Meddeb 1

1 clinical hematology; 2 Laboratory Hematology, Aziza Othmana hospital; 3 Laboratory of Histology and Cytogenetics, Institut Pasteur de Tunis, tunis, Tunisia

Background: Although all the progress made in hematology in the last decades, the treatment of acute myeloblastic leukemia (AML) is still disappointing with long term survival which does not extend 40%.

Aims: The aim of our study is to assess the impact of early bone morrow blast (BMB) clearance in prediction of complete remission and relapse risk in acute myeloid leukemia

Methods: We conducted a retrospective study including patients with AML treated with standard induction chemotherapy (3 days of idarubicine 12mg/m 2 and 7days of cytarabine 200mg /m 2 ) at the department of hematology at Aziza Othmena Hospital in Tunisia during 2020 - 2021.

Results: Thirty four patients were included. The median age was 41 (range 17-57) and sex ratio was 1.5 (22M/12F).

At the time of diagnosis, the median of white blood cells count was 57.8G/L (range 0.76-300G/L) and cytogenetic data were available for 91% of patients.

According to cytogenetic stratification, our patients were classified as favorable in 29%, intermediate in 45.1% and unfavorable risk in 25.8% of cases.

At the end of induction course, complete remission (CR) was achieved in 64% of patients. Induction related mortality was 8.8%. The most common cause of death was infection (66%).

Bone morrow aspiration at day 15 was performed for all our patients. Only one patient had re-induction at day 18.

Fifteen patients (44%) had more than 5% BMB in day 15.

53% of patients with more than 5% BMB did not achieve a CR compared to 18% with less than 5% BMB. (p=0.05)

Induction related mortality was not affected by the results of BMB in day 15.

However, the relapse rate was significantly higher in the group of patients with more than 5% BMB at day 15 compared with the other group (66% vs 0%; p=0.002).

Summary/Conclusion: Our study showed a significant statistical relation between BMB at day 15 and relapse risk. Thus, the kinetic of bone morrow blast clearance could be correlated to the minimal residual disease at the end of induction and so could predict the risk of relapse.

PB1825: TREATMENT PATTERNS AND SURVIVAL OUTCOMES OF ACUTE MYELOID LEUKEMIA PATIENTS MANAGED IN THE COMMUNITY SETTING IN CROATIA

I. Krecak 1,* , M. Moric Peric 2 , I. Zekanovic 2 , H. Holik 3 , N. Zivkovic 4 , T. Marketin 2 , B. Coha 3 , V. Gveric-Krecak 1

1 Department of Internal Medicine, General Hospital of Sibenik-Knin County, Sibenik; 2 Department of Internal Medicine, General Hospital Zadar, Zadar; 3 Department of Internal Medicine, Dr Josip Bencevic, Slavonski Brod; 4 Emergency Department, General Hospital of Sibenik-Knin County, Sibenik, Croatia

Background: In Croatia, patients with acute myeloid leukemia (AML) who are considered eligible for allogeneic bone marrow transplantation are usually treated at academic centers with high-intensity chemotherapies; however, no data exist regarding clinical outcomes of patients who remain treated in the community setting.

Aims: To analyze treatment patterns and survival outcomes of AML patients treated in the community setting in Croatia.

Methods: This was a multicenter study conducted at three community hospitals in Croatia. Clinical and laboratory data regarding AML patients who were considered unfit for allotransplant (due to advanced age and/or comorbidities) was retrospectively collected through the medical chart review. Best suportive care (BSC) included transfusion support and/or cytoreduction with hydroxycarbamide/thioguanine. Lower-intensity chemotherapies included low-dose cytarabine (LDAC) and azacytidine (AZA) with or without venetoclax (VEN), whereas higher-intensity chemotherapies included the co-administration of cytarabine and anthracyclines. Antimicrobial prophylaxis considered the administration of fluoroquinolones/co-trimoxazole. Treatment response was defined according to 2017 European Leukemia Net (ELN) guidelines. Overall survival (OS) was measured as the time from diagnosis until death or the last follow-up.

Results: A total of 117 AML patients were included; median age was 76 years (56-91), 47% were females, and the median Charlson Comorbidity Index (CCI) was 5 (2-12). Seventy-two patients (61.5%) had de-novo AML, 41 (35%) secondary AML and four (3.4%) therapy-related AML.

Fifty (42.7%), 43 (36.8%), and 24 (20.5%) of patients received BSC, lower-intensity (AZA=31, AZA/VEN=4, LDAC=8) and higher-intensity chemotherapies (3 + 7=12, 1 + 5=7, 2 + 5=2, 3 + 5=1, 1 + 4=1) as front-line treatments, respectively. Ninty-one patients (77.8%) prophylactically received antibiotics. Seventy-one patients (60.6%) had data available for response assessment. There was a similar proportion of CR (29.4% vs 27.8%) but more PR in the lower-intensity group when compared to a higher-intensity group (41.2% vs. 22.22%).

The median OS for the entire cohort was 3 months (95% CI 2-5) and early death rate (<1 month after the diagnosis) was substantial (40.2%). Patients receiving lower-intensity treatments had longer OS (median 12 months) when compared to BSC (median OS 1 month) and higher-intensity treatments (median OS 6 months, p<0.001), as shown in the Figure 1A. Similarly, patients achieving CR (median OS 13 months) or PR (median OS 12 months) had better outcomes when compared to refractory patients (median OS 2 months, p<0.001), as depicted in Figure 1B. Other variables univariately associated with an inferior OS were: age ≥75 years (p<0.001), CCI>5 (p=0.004), no antimicrobial prophylaxis (p<0.001), total leukocyte count >3x10e9 (p=0.003), hemoglobin ≤85g/L (p=0.003) and platelets ≤66x10e9 (p=0.049). In the sex- and AML-type-adjusted Cox regression model, BSC/high-intensity treatments (p<0.001), no antimicrobial prophylaxis (p=0.006) and leukocyte count>3x10e9 (p=0.041) were independent predictors of an inferior OS.

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Summary/Conclusion: Our real-life data shows that younger age, lower comorbidity burden, hypoproliferative disease, azacytidine/LDAC, treatment response and antimicrobial prophylaxis are associated with an improved OS of AML patients treated in the community setting in Croatia. Nevertheless, high early mortality and poor AML-related survival highlight the urgent need for clinical trials focused at this patient population.

PB1826: A NEW WIDELY APPLICABLE MODEL COMBINED MOLECULAR AND CLINICAL PROGNOSTIC INDEX FOR OVERALL SURVIVAL AND RISK STRATIFICATION IN ADULTS WITH ACUTE MYELOID LEUKEMIA

J. Ma 1,2 , Z. Ge 1,*

1 Department of Hematology, Zhongda Hospital, School of Medicine, Southeast University, Institute of Hematology Southeast University, Nanjing; 2 Department of Hematology, Xuzhou Central Hospital, Xuzhou, China

Background: The 2017 European LeukemiaNet (ELN) risk stratification combines cytogenetic abnormalities and genetic mutations to provide risk-stratifying optimal therapies for patients with acute myeloid leukemia (AML). However, due to the lack of full cytogenetic and genetic mutation files at diagnosis, the ELN-2017 applicability in low- and middle-income countries is limited. Additionally, many clinical prognostic factors are not included in the risk stratification.

Aims: This study is to develop a widely applicable prognostic model by combining molecular and clinical characteristics based on the real-world data.

Methods: The Adapted ELN guideline ([AELN]) was used by adjusting some molecular genetic abnormalities and allowing the missing cytogenetic or molecular data to improve the performance and expand adaptability of the guidelines. The training cohort of 183 de novo adult AML (non APL) patients was conducted at Zhongda Hospital of Southeast University. The validation cohort of 103 AML patients was obtained from Xuzhou Central Hospital. Univariate Cox, multivariate Cox, Best Subset Regression (BSR) and LASSO regression analyses were used to determine the significant factors that influenced overall survival (OS). We chose the final model that yielded the largest AUC or the smallest AIC. The receiver operating characteristic curve (ROC) was used to evaluate the efficiency of the prognostic model. Kaplan-Meier (K-M) survival curve with Log Rank analysis were performed in the study.

Results: Median follow-up was 39.6 (95%CI: 32.9-48.9, reverse K-M) and 33.6 (22.7-45.8) months for training and validation cohort, respectively. Median OS was 15.7 (12.9-15.9, K-M) and 16.9 (11.9-46.6) months, respectively. AELN risk classification was statistically significant for OS in both training (P<0.0001) and validation cohorts (P=0.016). AELN prognostic classification was more superior in sensitivity and accuracy than ELN-2010 in distinguishing different risk group categories with the AUC value of 0.724 vs. 0.631 respectively in the training cohort. The AUC of BSR (0.75) or LASSO (0.75) regression analysis was higher than univariate Cox analysis (0.737). In this study, we optimized the modeling methods, and LASSO combined with multivariate Cox analysis were selected eventually. On the basis of age, WBC count, and AELN risk classification, patients were classified into low-risk (LR, ≤1.57) and high-risk (HR, >1.57) groups according to the AML OS score model [AML OS score =age (years) × 0.03 + WBC count (× 10 9 /L) ×10 -9 × 0.004 + 1.15 (if AELN poor, otherwise 0)] with median OS of 46.3 months and 31.4 months in LR and HR groups, respectively (P<0.0001; Fig. 1A). C-statistic in training cohort was 0.706 (95% CI: 0.66-0.76). Meanwhile, in validation cohort, the OS was significantly different between LR and HR (P=0.015, Fig. 1B) with the median OS of 87.5 and 12.3 months, respectively. Compared with AELN risk classification, AML OS score model was more superior in both sensitivity and applicability with the AUC value in AML OS vs. AELN alone of 0.716 vs. 0.693, 0.702 vs. 0.531 in training cohort and validation cohort, respectively (Fig. 1C, D).

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Summary/Conclusion: AELN is a more applicable genetic risk classification tool for low-income groups. AML OS , the integral risk stratification system developed by integrating AELN and the clinical prognostic factors, is a widely applicable prognostic model for treatment guidance in adult patients with AML.

PB1827: ACUTE PROMYELOCYTIC LEUKEMIA IN THE ELDERLY

I. Marques 1,* , C. Sol 1 , A. L. Pinto 1 , E. Aguiar 1 , M. L. Amorim 1 , M. J. Soares 1 , R. Bergantim 1 , P. Gomes 1 , F. Trigo 1

1 Hematologia Clínica, Centro Hospitalar Universitário São João, Porto, Portugal

Background: Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia characterized by the fusion of the PML-RARA. Its incidence in the population aged ≥60 years is lower than that observed in other AML, leading to little knowledge about this pathology in this type of population with specific characteristics

Aims: Descriptive analysis of elderly patients with APL

Methods: Retrospective review of APL patients, aged ≥60 years followed up in a tertiary hospital (2005-2021). Data analysis was performed using the program IBM SPSS Statistics 26®

Results: It was included 47 patients [42,6% male (n=20), 57,4% female (n=27)], median age at diagnosis of 70 years (61-81), median Eastern Cooperative Oncology Group performance status (ECOG PS) 1 (0-2) and median Charlson Comorbidity Index (CCI) 6 (2-9). Considering the Sanz risk score, 29,8% patients (n=14) had low risk, 38,3% patients (n=18) had intermediate risk and 31,9% patients (n=15) had high risk. There was expression of aberrant markers in 36,2% of patients (n=17), namely CD56 (63,8%, n=30), CD34 (63,8%, n=30) and CD2 (4,3%, n=2). Additional karyotype alterations were observed in 14,9% patients (n=7) [addition of chromosome 8 (n=3), complex karyotype (n=2), t(1;5) (n=1) and t(1;3) (n=1)]. PML-RARA breakpoint was type BCR1/2 in 23 patients (48,9%) and type BCR3 in 21 patients (44,7%). Treatment was selected according protocols used in the service [AIDA/GIMENA (n=35, 74,5%), PETHEMA-LPA (n=10, 21,2%), or no treatment (n=2, 4,3%)]. During induction treatment, 14 patients (29,8%) died in a median of 11 days (1-53), due to cerebral hemorrhage/ischemia (n=8), sepsis/infection (n=3), cardiogenic shock (n=2) and ATRA differentiation syndrome (n=1). Considering the patients who died from a cerebral hemorrhage/ischemia event, in only 1 patient the event occurred during induction and in the remaining patients it was already present at the time of disease presentation. 33 patients achieved a complete response to induction treatment. Disease relapse was seen in 7 patients. To determine factors with impact on overall survival and response to therapy, a univariate logistic regression analysis was performed, and no association was found with age at diagnosis, ECOG PS, ICC, hemoglobin, leukocytes, platelets, fibrinogen, D-dimers, DHL, creatinine, albumin, additional karyotype changes, PML-RARA breakpoint type and aberrant marker expression. The OS was not reached and PFS was 13 months [median follow-up time 22 months (0-157)] and this results was similar in the patient aged <70 and ≥70 years (OS not reached in both groups, PFS was 12 and 13 months in patient aged <70 years and ≥70 years, respectively)

Summary/Conclusion: Real-life studies evaluating elderly population of APL patients are scarce, however the characteristics of the population studied in this study do not differ from previously published series. Death of the patients studied during induction treatment occurred in a significant proportion of patients mainly due to cerebral hemorrhage/ischemia event, in which in almost all cases it was already present at the time of diagnosis. Although no correlation has been identified between the characteristics of patients, analytical parameters and molecular, cytogenetic and immunophenotypic features and the response to treatment and OS, early death may be due to clinical and biological aspects of the elderly patient not included in this assessment. Future studies in this population should include a detailed characterization of comorbidities and additional analytical parameters of these patients and not just the use of comorbidity assessment scales

PB1828: CADENZA: A PIVOTAL STUDY OF PIVEKIMAB SUNIRINE (IMGN632) IN PATIENTS WITH UNTREATED/FRONTLINE BPDCN

N. Pemmaraju 1,* , G. Martinelli 2 , E. Todisco 3 , C. Tarella 3 , P. Montesinos 4 , A. A. Lane 5 , H. P. Erba 6 , K. L. Sweet 7 , R. B. Walter 8 , E. Deconinck 9 , E. S. Wang 10 , A. Aribi 11 , M. L. Ulrikson 12 , M. Y. Levy 13 , D. Lebon 14 , G. Marconi 15 , D. J. DeAngelo 16 , D. Rizzieri 17 , M. Konopleva 18 , C. M. Sloss 19 , J. Wang 19 , K. A. Malcolm 19 , P. A. Zweidler-McKay 19 , N. G. Daver 18

1 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, United States of America; 2 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. e A. Seràgnoli”, Bologna; 3 Division of Onco-Hematology, European Institute of Oncology IRCCS, Milano, Italy; 4 Hospital Universitari i Politècnic La Fe, Valencia, Spain; 5 Department of Medical Oncology, Dana-Farber/Boston Children’s Cancer and Blood Disorders Ctr, Boston; 6 Division of Hematologic Malignancies and Cellular Therapy, Duke University, Durham; 7 Department of Malignant Hematology, H. Lee Moffitt Cancer Center and Research Institute, Tampa; 8 Fred Hutchinson Cancer Research Center, Seattle, United States of America; 9 Clinical Hematology, Besançon University Hospital, Besançon, France; 10 Roswell Park Comprehensive Cancer Center, Buffalo; 11 Gehr Family Center for Leukemia Research, City of Hope, Duarte; 12 Banner Health MD Anderson Cancer Center, Gilbert; 13 Texas Oncology-Baylor Charles A. Sammons Cancer Center, Dallas, United States of America; 14 Department of Clinical Hematology and Cellular Therapy, Amiens University Medical Center, Amiens, France; 15 Istituto Seragnoli, Bologna, Italy; 16 Department of Medical Oncology, Harvard Medical School, Dana-Farber Cancer Institute, Boston; 17 Department of Medicine, Division of Hematologic Malignancies and Cellular Therapy, Duke University Medical Center, Durham; 18 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 19 ImmunoGen, Inc., Waltham, United States of America

Background: Overexpression of CD123 occurs in multiple hematological malignancies, including blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and others. With limited expression on normal hematopoietic progenitor cells, the CD123 antigen is an attractive target for new therapeutics. IMGN632 is a CD123-targeting antibody-drug conjugate (ADC) comprising a novel anti-CD123 antibody coupled, via a peptide linker, to a unique DNA-alkylating cytotoxic payload of the IGN (indolinobenzodiazepine pseudodimer) class. Preclinically, IMGN632 has demonstrated potent activity against BPDCN, AML, and ALL models, with a wide therapeutic index in animal models, as well as 150-fold differential cytotoxicity in AML patient-derived samples compared to normal hematopoietic progenitors (Kovtun. Blood Adv (2018) 2(8):848, Angelova. Haematologica (2019) 104(4):749). In particular, remarkable sensitivity of BPDCN patient-derived xenografts to IMGN632 was demonstrated (Zhang Blood (2018) 132:3956). The clinical safety profile of IMGN632 in BPDCN is characterized by low-grade peripheral edema and infusion reactions, and reversible veno-occlusive disease in < 5%; there have been no capillary leak syndrome and no treatment-related deaths. Data in relapsed or refractory BPDCN led to FDA granting of Breakthrough Therapy Designation for IMGN632 (October 2020). We have previously reported three of three frontline BPDCN patients achieved clinical complete responses (CRc) with duration of response (DOR) of up to 10.7 months without transplant (Pemmaraju. ASH 2021 Abstract #1284).

Aims: This study is designed to determine the safety, tolerability, and activity of IMGN632 when administered to patients with frontline BPDCN.

Methods: The dose escalation phase identified a recommended phase 2 dose of 0.045 mg/kg given intravenously every 3 weeks. Expansion cohorts include a pivotal cohort for frontline/untreated BPDCN (no prior systemic therapy), and exploratory cohorts for patients with relapsed or refractory BPDCN. Patients with central nervous system disease are not eligible in the frontline BPDCN cohort.

Results: N/A

Summary/Conclusion: Enrollment continues in the pivotal cohort for frontline/untreated BPDCN patients.

CADENZA, https://BPDCNtrial.com , NCT03386513

PB1829: ARGENTINEAN REAL WORLD EXPERIENCE ON MIDOSTAURIN PLUS INTENSIVE CHEMOTHERAPY VS INTENSIVE CHEMOTHERAPY TO TREAT FLT3 POSITIVE ACUTE MYELOID LEUKEMIA PATIENTS

A. Navickas 1,* , J. González 2 , M. M. Rivas 3 , M. Clavijo 4 , N. Carnelutto 5 , M. E. Funes 6 , M. J. Mela Osorio 7 , R. Mariano 8 , M. L. Rapan 9 , M. Moirano 10 , I. Rey 11 , S. Cranco 12 , J. E. Giunta 13 , R. Ramirez 14 , M. B. Castro 15 , A. Enrico 16 , A. Gimenez Conca 17 , C. Belli 18

1 Hematologia, Hospital el Cruce, Florencio Varela; 2 Hematología, Hospital Durand, CABA; 3 Hematología, Hospital Austral, Pilar; 4 Hematología, Hospital Aleman; 5 Hematología, Hospital de Clinicas, CABA; 6 Hematologia, Sanatorio Britanico, Rosario; 7 Hematologia, Fundaleu, CABA; 8 Hematologia, Hospital San Martin, Parana, Entre Rios; 9 Hematologia, Sanatorio Sagrado Corazon, CABA; 10 Hematologia, Hospital San Martin, La Plata, Buenos Aires; 11 Hematologia, Hospital Ramos Mejia, Buenos Aires; 12 Hematologia, Instituto Alexander Fleming, CABA; 13 Hematologia, Instituto Privado de Hematología y Hemoterapia, Parana, Entre Rios; 14 Hematologia, Instituto Conciencia, Neuquen; 15 Hematologia, Hospital Privado, Cordoba; 16 Hematologia, Hospital Italiano, La Plata, Buenos Aires; 17 Hematologia, Hospital Italiano Buenos Aires, CABA; 18 Hematologia, Academia Nacional de Medicina, Buenos Aires, Argentina

Background: Midostaurin has been approved by EMA and FDA in combination with intensive chemotherapy (CMT) for FLT3 positive AML based on improved survival. In Argentine was included among therapy strategies in 2018. The real-world experience in day-to-day practice is limited.

Aims: To examine the impact of introduction of midostaurin within the backbone to treat FLT3+ AML patients in an Argentinean cohort.

Methods: This is a retrospective analysis of 120 FLT3+ AML patients intensively treated with or without midostaurin excluding APL- and CBF-AML, from the Argentine Society database of 1200 AML diagnosed between March-13/Dec-21 including 20 centers. Responses were evaluated according to the ELN 2017 criteria for intensive chemotherapy (CMT). Statistical analysis included Chi2/Fisher’s exact test, Kaplan-Meier survival analysis, censoring or not at HSCT, to last follow-up or death.

Results: FLT3 variants were 103 (86%) ITD, 15 (13%) TKD and 2 (1%) ITD/TKD. Seventy-two patients received first line treatment with standard 7 + 3 CMT and 48 added midostaurin. Sex ratio M/F was 0.6 (47/73) with a median age of 50.7 years (15.1-76.4), being 25% >60 years old, 26% an ECOG>1, 17% a Charlson’s Index (CCI)>2, 95 (97%) intermediate MRC karyotypes (23 not available), 43 (43%) NPM1+ (21 not available). No differences were observed between both populations regarding basal characteristics.

The median time to treatment initiation was 3 days, and with a median follow-up of 8.2 months, 84 (68%) died with an early mortality at 28 days of 9.6% (1/11 received midostaurin). Regarding responses on those treated with CMT 64% (n=46) obtained Complete Remission (CR), subsequently 37 pts (51%) received consolidation with high doses of CMT, 13 pts relapsed, and 26 received HSCT. Within the MDT cohort, 79% (n=38) achieved CR, 37 (77%) were consolidated, 7 pts relapsed, and 25 received HSCT.

The inclusion of MDT to CMT improved the outcome of our patients. The overall survival censoring at HSCT was 7.0 vs 4.7 (95%CI 1.7-12.4 vs 3.1-6.4, p=0.002), not censoring 12.1 vs 7.7 (95%CI 0.4-23.8 vs 5.5-9.9, p=0.008). Among those who achieved CR, the OS was 24.8 vs 10.3 (95%CI 7.9-41.7 vs 6.7-13.9, p=0.046) and 11.9 vs 6.4 (95%CI 9.5-14.4 vs 5.4-7.4, p=0.015), without differences regarding relapse free survival, evaluated to last follow-up (median not reached, p=0.800). No statistical differences were observed regarding survival after HSCT (9.6 vs 10.3, p=0.773). In our media FLT3 inhibitors are not approved as maintenance therapy after HSCT.

Summary/Conclusion: Our results in day-to-day practice confirm that midostaurin associated with CMT improved the outcome of FLT3 positive AML patients.

PB1830: HIGH EARLY MORTALITY RATES IN OLDER PATIENTS WITH ACUTE MYELOID LEUKEMIA TREATED WITH INTENSIVE CHEMOTHERAPY IN TWO ACADEMIC CENTERS IN BRAZIL

F. Lisboa Nogueira 1,2,3,* , F. Rodrigues Mendes 1,4 , R. da Costa Bandeira de Melo 4 , W. Fernandes da Silva Junior 4 , A. B. Firmato Glória 2,5 , S. Guerra Xavier 6 , I. Bendit 1 , E. Maranhão Fagundes 2,5 , E. Deolinda Rodrigues Pereira Velloso 1,4 , E. Magalhães Rego 1,4

1 Laboratory of Medical Investigation in Pathogenesis and Targeted Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo; 2 Grupo Oncoclínicas; 3 Hospital das Clínicas da Universidade Federal de Minas Gerais, Belo Horizonte; 4 Division of Hematology, Instituto do Cancer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de Sao Paulo, São Paulo; 5 Unidade Funcional Hematologia e Oncologia, Hospital das Clínicas da Universidade Federal de Minas Gerais; 6 Departamento de propedêutica complementar, Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Background: Most patients with acute myeloid leukemia (AML) are 60 years old or more at diagnosis. They represent a challenging to treat population due to their heterogeneity, exemplified by different levels of fitness and comorbidities. Data from younger patients treated in middle-income countries with intensive chemotherapy point out to higher early death rates, mainly owing to infections. The outcomes of older patients treated in the same setting, however, are largely unknown. Moreover, the determination of factors driving early death in these countries is expected, since it can provide guidance on measures to reduce it, including better patient selection to intensive induction.

Aims: To access the early mortality rate in older AML patients in two teaching hospitals in Brazil and describe the risk factors.

Methods: Patients aged 60 years old or older, diagnosed with AML from 2011 to 2019 and treated with intensive chemotherapy at the Instituto do Câncer do Estado de São Paulo (ICESP) or the Hospital das Clínicas da Universidade Federal de Minas Gerais (HC-UFMG), were retrospectively included in the cohort. Those testing positive for BCR-ABL1 were excluded. Diagnosis was performed as per WHO criteria. Cytogenetic and molecular analysis for RUNX1-RUNX1T1, CBFβ/MYH11, FLT3-ITD, NPM1 were routinely performed. Patients were classified according to the Adapted Genetic Risk (AGR) as previously described by Silveira DRA et al. Clinical data and basal laboratory values were retrieved from medical charts. Early death was defined as mortality within 30 days of the beginning of induction chemotherapy. The study was approved by each institution`s review board.

Results: Ninety-five patients, 65 (68.4%) from ICESP and 30 (31.6%) from HC-UFMG fulfilled the study criteria. The median follow-up was 24.2 months (follow-up rate of 95.8%). The median age was 65 years old (range 60-74) and 41 (43.2%) were female. Patients from ICESP were slightly older (median 66 vs. 65; p=0.041). The Charlson comorbidity index (CCI), which scores points for age and some co-diagnosis yielded a median value of 2, with 57 (60.0%) patients without any comorbidity scored on CCI. Cytogenetics returned no metaphasis on 14 (14.7%) patients. According to the AGR, 28 (34.6%) were assigned to favorable, 28 (34.6%) to intermediate and 25 (30.9%) to the adverse risk category, while 14 (14.7%) patients were not classified due to missing data. Eighty-eight (92.6%) received 7 + 3 and 4 (4.2%) FLAG. Thirty-eight (40.0%) patients achieved complete remission and 16 (16.8%) were refractory to the first induction cycle. No patient has undergone hematopoietic stem cell transplantation. Thirty-nine (41.1%) patients died within 30 days of the start of induction. On multivariate analysis, CCI above 2 (HR 5.52; p=0.006), higher creatinine (HR 2.84; p=0.043), lower albumin (HR 0.34; p<0.001) and higher total bilirubin (HR 7.25; p=0.034) were associated with early death. Sixty-two (65.3%) were admitted to intensive care unit during induction, 12 (12.6%) started hemodialysis and 29 (30.5%) had a diagnosis of invasive fungal infection. Median overall survival was 3.7 months. According to AGR, median overall survival was 6.8 months on favorable and 1.2 month on adverse risk group (p=0.012).

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Summary/Conclusion: Early death rates proved to be exceedingly high despite the favorable characteristics of the cohort, which included mainly patients aged less than 70 years old and with a low comorbidity burden. In this adverse scenario, predictive factors can help to identify who benefits the most from intensive induction.

PB1831: UTILITY OF FLT3 INHIBITORS IN PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) AND T(6;9)(P22;Q34)

F. Ong 1,* , T. M. Kadia 1 , N. J. Short 1 , M. Yilmaz 1 , Y. Alvarado 1 , S. Pierce 1 , G. Garcia-Manero 1 , C. DiNardo 1 , G. Borthakur 1 , M. Konopleva 1 , N. Daver 1 , H. Kantarjian 1 , F. Ravandi 1

1 Leukemia, The University of Texas MD Anderson Cancer Center, HOUSTON, United States of America

Background: Efficacy of FLT3 inhibitors in patients (pts) with AML and t(6;9)(p22;q34)/DEK-NUP214 is not well established.

Aims: We compared overall survival (OS) and event-free survival (EFS) of AML pts with t(6;9) who received FLT3 inhibitor (FLT3-i) in frontline (FL) or salvage (SL) setting, regardless of their FLT3 mutation status.

Methods: We retrospectively evaluated pts with AML or high-risk Myelodysplastic Syndrome (HR-MDS) who presented to our center between 1995-2021. Among 9936 pts, 55 pts (0.5%) had t(6;9) (48 AML, 7 HR-MDS). In FL setting, pts were divided according to their induction regimen with (n:5) or without FLT3-i (n:48). In SL setting, 35 pts who relapsed or were refractory to FL regimen were recategorized according to their SL regimen with (n:16) or without FLT3-i (n:17). Four pts were excluded from analysis due to incomplete data.

Results: Among 47 pts with available molecular data, 35 (74%) was FLT3 -ITD+, 5 (10.6%) with both ITD and TKD mutation. ITD allelic ratio (AR) was known for 28 (80%) pts (AR range: 0.01-0.95) with 11 (31%) pts having an AR ≥0.5. Age was well-balanced across all cohorts with median of 41 years (yr). Most pts had ECOG performance status of 0-1 (92%) on presentation. Pts with FLT3 -ITD mutation were more likely (83%) to receive intensive chemotherapy (IC) compared to pts without (50%, p=0.02). Most pt in FL cohort with or without FLT3-i received IC (80% and 77%, respectively). There was no significant different in FLT3 -ITD positivity between FL cohort with or without FLT3-i (p=0.3). Sorafenib was used in 62% of cases, followed by Quizartinib (24%). All pts in FL cohort with FLT3-i achieved CR/CRi with 60% MRD negative rate as compared to 52% in non-FLT3-I, and 60% of FLT3-i cohort had stem cell transplantation (SCT) in CR1 (vs. 21% of non-FLT3-i). Median number (No.) of therapy cycle was 1 to reach CR/CRi in all FL cohorts. Outcomes are shown in Figure 1. With median follow up of 47 months (mos), 2-yr OS and EFS were higher in FLT3-i than non-FLT3-i cohort (80% and 60% vs. 41% and 20%, respectively), although not statistically significant (p=0.2 for OS and 0.1 for EFS). Among pts with known mutation status, there were non-significant lower 2-yr OS and EFS in FLT3 -ITD+ pts than wild-type FLT3 ( FLT3 -wt) pts (44% and 22% vs. 58% and 42%, respectively; p=0.4 for OS and 0.3 for EFS), regardless of whether FLT3-i was included in the induction regimen. CR/CRi rate was similar between FLT3 -ITD+ (57%) and FLT3 -wt pts (58%) but FLT3 -wt pts was more likely to receive SCT in CR1 (42%) than FLT3 -ITD+ pts (23%). In SL setting, 75% pts in FLT3-i cohort received low intensity therapy as compared to 12% pts in non-FLT3-i cohort (p<0.01) and 69% pts in SL cohort without FLT3-i were FLT3 -ITD+ (p=0.03). Pts were heavily treated in both group (67% pts were in salvage 2+), but never received FLT3-i as part of their prior salvage regimen. No. of prior therapies and proportion of pts with prior SCT and FLT-i- containing induction regimen were similar across cohort. CR/CRi rate was higher in FLT3-i cohort (69%) than non-FLT3-i cohort (41%) after median No. of 1 therapy cycle with similar proportion of pts who went to SCT post salvage regimen. With median follow up of 47 mos, 2-yr OS and EFS were poor in both FLT3-i cohort and non-FLT3-i cohort (27% and 12% vs. 12.5% and 6%, respectively, p=0.3 for OS and 0.7 for EFS).

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Summary/Conclusion: FLT3 mutation status does not impact survival in pts with t(6;9). Addition of FLT3-i to FL and SL regimen in subset of pts with FLT3 -ITD mutation is beneficial and should be further evaluated.

PB1832: ARTERIAL THROMBOSIS AND ACUTE MYELOID LEUKEMIA – IS THERE A CONNECTION?

N. Pantic 1,* , M. Virijevic 2,3 , Z. Pravdic 2 , N. Sabljic 2 , Z. Bukumiric 4 , M. Cvetkovic 5 , J. Rajic 2 , A. Vidovic 2,3 , M. Todorovic-Balint 2,3 , N. Suvajdzic-Vukovic 2,3 , D. Antic 2,3 , M. Mitrovic 2,3

1 Department of mixed hematological diseases; 2 Clinic of Hematology, University Clinical Center of Serbia; 3 Department of Internal Medicine; 4 Institute for Medical Statistics and Informatics; 5 Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Background: Arterial thrombotic events (ATE) are important cause of noncancer-related deaths among patients with cancer. It is estimated that the prevalence of ATE among those patients is between 2-5%. However, data regarding acute myeloid leukemia (AML) related ATE are scarce and far less available than those related to venous thrombotic events.

Aims: To determine the incidence of ATE in nonM3-AML patients and to underline the potential risk factors for ATE development.

Methods: The single center, retrospective, cohort study was carried out in University Clinical Center of Serbia. Adult patients, who were diagnosed with nonM3-AML between January 2009. and December 2021. were included. In all patients the occurrence of ATE (e.g. a heart attack, a stroke, critical limb ischemia) was assessed during the active treatment and the three months following the last chemotherapy session. Diagnosis of ATE was established using clinical, laboratory and radiological methods. Patients who experienced venous thromboembolism during the treatment period were excluded. Demographic data, presence of obesity, smoking status, history of thrombosis, baseline laboratory findings (complete blood count, fibrinogen, D-dimer, PT, aPTT, LDH), leukemia-related parameters (cytogenetics (including ELN risk stratification), flow cytometry), Khorana score, ECOG PS, HCT CI score, concurrent COVID-19 were collected from patients’ health records. The methods of descriptive (mean ± standard deviation, median (range), frequency (%)) and analytic statistics (Student’s t-test, chi-squared test) were used.

Results: A total of 545 patients (293 males (53.8%)) were included in the study. Median age of the study population was 58 (range: 18-81) years. ATE was noted in 18/545 (3.3%) subjects with following distribution: ischemic stroke 12/18 (66.7%), myocardial infarction 5/18 (27.8%), and acute lower extremity arterial thrombosis 1/18 (5.5%). ATE was diagnosed most commonly during the induction (8 (44.4%) patients), reinduction (3 (16.7%) patients) and consolidation (4 (22.2%) patients) cycles. However, cases of ATE were noted at diagnosis (1 (5.6%) patient), after transplantation (1 (5.6%) patient) or at relapse (1 (5.6%) patient) as well.

ATE were significantly more frequent among patients with previous history of thromboembolic events (p = 0.016). Moreover, ATE were more common in patients with adverse cytogenetic abnormalities (p < 0.001). Other examined parameters did not significantly differ between those with and without ATE.

Summary/Conclusion: The incidence of arterial thrombosis in our group was 3.3% which is in accordance with the previously published studies. Since the great number of already known risk factors for the arterial thrombosis are modifiable (e.g. smoking, diet, physical activity, excessive drinking…) it is important to actively work on the reduction on those risk factors, especially if the patient has the history of previous thromboembolic event and/or suffer from high risk AML. Prophylactic therapy with antiplatelet agents is aggravated due to the lack of firmer evidences and the presence of thrombocytopenia. Therefore further studies regarding this issue are needed.

PB1833: FUNCTIONAL POLYMORPHISMS OF TLR2-4 & C2 DO NOT PLAY A CRUCIAL ROLE IN INCIDENCE OF INFECTIONS IN A GREEK COHORT OF PATIENTS WITH ACUTE MYELOID LEUKEMIA.

C. Papadopoulos 1,* , G. Tsinti 2 , S. Matthaios 2 , P. Euthimia 3 , D. Katerina 4 , N. Giannakoulas 1

1 Department of Hematology; 2 Department of Immunology and Histocompatibility; 3 Department of Microbiology; 4 Department of Hygiene and Epidemiology, University of Thessaly, Larissa, Greece

Background: Patients with Acute Myeloid Leukemia (AML) display long periods of neutropenia due to marrow failure and chemotherapy, which make them particularly susceptible to infections from a wide range of pathogens. The incidence of infections in AML patients under treatment may also delay subsequent treatments and also increases the morbidity and mortality rates. Innate immune system is the first line of defense against microbial pathogens, while at the same time it is the orchestrator of adaptive immunity. Disorders in the major molecules of innate immunity such as Toll Like Receptors (TLRs) or in individual components of other system such as the complement, might be aggravating factors for the already immune-compromised patient. The role of common functional innate immunology genes in the incidence in patients with AML remains unclear as previous studies provide conflict results.

Aims: In the present study, the scientific question was whether the D299G (rs4986790) & T299I(rs4986791) polymorphisms of the TLR4 gene, the R753Q (rs57443708) polymorphism of the TLR2 gene, and the most common deletion of the C2 (rs9332736) gene are prognostic factors for the development of microbial infections in 107 AML Caucasian patients from Greece.

Methods: I) Clinical data (type of infections, survival, etc.) of 107 patients with AML (73 males, 34 females, 68 mean age) were recorded. All patients or their families had given consent for their genetic material processing and analysis. The isolated pathogens were separated into four groups (Table 1B) (Gram-Positive Bacteria, Gram-Negative Bacteria, Fungi and Clamydia).

II) The sample type consisted of peripheral blood and bone marrow. For DNA extraction PureLink®Genomic DNA kit (Qiagen & Invitrogen) was used.

III) Genetic polymorphisms TLR4(D299G, rs4986790), TLR4(T399I, rs4986791), TLR2(R758Q, rs57443708) and C2(rs9332736, C2-c.841_849 + 19del28) detection, was performed as previously described in detail (Johnson et al., 1992; Liadaki et al., 2011).

IV) For the comparison of our cohort’s genotype frequency with the genotype frequency of worldwide population, was used gnomAD database.

V) The results assessed with Fisher Exact test. Statistical significance level was 0.05. Statistical analysis was performed with SPSS 2017.

Results: The results of molecular detection are described on Table 1A. None statistical significant were observed between polymorphisms of immune genes (TLR2, TLR4, C2) and the frequency of appearance of isolated infectious agents (P-value > 0,05).

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Summary/Conclusion: As mentioned above, there are conflicting results in the literature considering the role of the analyzed polymorphisms in the incidence of infections and sepsis in patients with AML. In our study, our patients did not have any statistically significance between polymorphisms (TLR2, TLR4 and C2) and underlined infections. Further studies and also meta-analysis of all related published studies are necessary in order to provide conclusive remarks.

PB1834: CLEVO: A NON-INTERVENTIONAL STUDY TO INVESTIGATE CLONAL EVOLUTION OF FMS-LIKE TYROSINE KINASE 3 (FLT3) GENE MUTATIONS DURING DISEASE PROGRESSION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

P. Vyas 1,* , A. Upadhyay 2 , A. Houweling 3 , I. Wouters 4 , D. Elsouda 5

1 University of Oxford, Oxford; 2 Astellas Pharma Europe Ltd, Addlestone, United Kingdom; 3 Astellas Pharma B.V, Leiden, Netherlands; 4 Astellas Pharma B.V, Vilvoorde, Belgium; 5 Astellas Pharma Inc., Northbrook, IL, United States of America

Background: Acute myeloid leukemia (AML) is clinically heterogeneous, with differing response to treatment and patient survival reflecting the diverse cytogenetic abnormalities and genetic mutations that give rise to the disease. Clonal evolution means that genetic characteristics of the leukemic cells and the proportions of individual cell clones can change because of disease progression and selection by therapy. Fms-like tyrosine kinase 3 ( FLT3 ) gene mutations are found in approximately 30% of the general AML population and are often secondary events associated with leukemic transformation. These mutations are known to either develop or become undetectable during the disease course, and the mutant-to-wild-type allelic ratio also typically changes. This clonal evolution is not well understood but may have clinical consequences.

Aims: The aim of this study is to characterize FLT3 mutations at AML diagnosis and chart the clonal evolution of FLT3 mutations (gain or loss) through treatment.

Methods: This non-interventional, descriptive study will prospectively enroll approximately 650 adults (aged ≥18 years) with AML diagnosed within 30 days of enrollment at oncology or oncology/hematology specialist sites in Belgium, France, Germany, Israel, Italy, Spain, the UK and the USA, with follow-up for 36 months. Patients will be excluded if they have relapsed or refractory AML at enrollment, acute promyelocytic leukemia, or clinically active central nervous system leukemia. Patients will undergo treatment according to local standard of care and treatment guidelines, at the treating physician’s discretion. Patients will undergo routine FLT3 genetic testing at diagnosis and at relapse/refractory disease, and all AML treatments (including hematopoietic stem cell transplantation) received will be recorded. The primary objective is to determine the proportion of relapsed/refractory AML patients with FLT3 clonal evolution (gain/loss of FLT3 mutations) from diagnosis. Secondary objectives include a description of the: frequency, allelic ratio, length and location of FLT3 -internal tandem duplication mutations and frequency of other FLT3 mutations at diagnosis and at relapse/refractory disease; frequency of FLT3 clonal evolution of FLT3 mutations at relapse/refractory disease by mutation type and treatment(s) received; patient survival (overall 3-year survival rate, disease-free survival and event-free survival) in relation to FLT3 mutation status at diagnosis, FLT3 clonal evolution and treatment received; frequency of composite complete remission in relation to FLT3 mutation status at diagnosis, FLT3 clonal evolution (relapsed patients only) and treatment received; and frequency and characteristics of other AML-associated genetic mutations at diagnosis and relapse/refractory disease. Interim analyses are planned for ~1 and ~3 years following study initiation to provide an estimate of FLT3 mutation frequencies in the study population, and potentially an early estimate of the rate of clonal evolution. The first patient was enrolled in September 2020.

Results: The trial is in progress.

Summary/Conclusion: The information generated by this study will help to better understand the clonal evolution of FLT3 throughout the course of the disease for AML patients.

PB1835: THE ISTH-DIC SCORE PREDICTS 30-DAYS OUTCOME IN NON-M3 ACUTE MYELOID LEUKEMIA PATIENTS

G. Paterno 1,* , R. Palmieri 1 , V. Forte 1 , F. Bonanni 1 , L. Guarnera 1 , F. Mallegni 1 , M. R. Pascala 1 , B. Elisa 1 , F. Moretti 1 , A. Savi 1 , L. Maurillo 1 , F. Buccisano 1 , A. Venditti 1 , M. I. Del Principe 1

1 Department of Biomedicine and Prevention, Tor Vergata University, Rome, Italy

Background: The International Society of Thrombosis and Haemostasis (ISTH) disseminated intravascular coagulation (DIC) score is widely used to predict mortality in critically ill - typically septic - patients. DIC is an acquired condition characterized by systemic activation of coagulation, intravascular fibrin degeneration, consumption of platelets and coagulation factors along with fibrinolytic imbalance. The intravascular coagulation may disseminate and cause a variety of severe and potentially fatal clinical manifestations including major bleedings and thromboembolic events. Patients with hematological malignancies (HM) can present with DIC as defined by the evidence of coagulative disorders, in the absence of thrombo-hemorrhagic manifestations. Despite several studies have already investigated the association between HM, including Acute Myeloid Leukemia (AML) and DIC in adults, which factors and their specific degree of contribution to a worse outcome is still a matter of debate.

Aims: The aim of the present analysis was to investigate whether the ISTH DIC score can be used to predict the 30-day mortality from disease presentation in patients with non-promielocytic (non-M3) AML.

Methods: In this monocentric retrospective study, we included consecutive adult patients (≥18 years of age) who were diagnosed with non-M3 AML between 2010 and 2020. Patients were stratified according to European Leukemia Net 2017 genetic/cytogenetic risk stratification. Patients with documented sepsis at AML presentation were excluded from the study. The ISTH DIC 2018 score was calculated at AML presentation as follows: platelet count (≥100 x 10 9 /L = 0; 50-99,999 x 10 9 /L = 1; <50 x 10 9 /L = 2), fibrinogen level (≥100 mg/dL = 0; <100 mg/dL = 1), PT prolongation above upper limit of normal (ULN) (<3 seconds = 0, 3-6 seconds = 1, >6 seconds = 2), and D-dimer (<3 µg/ml = 0, 3-7 µg/ml = 2, >7 µg/ml = 3). A score sum ≥ 4 identified high-risk patient.

Results: A total of 300 patients were included in this study (39% female; median age 64 years, range 21-90 years). Of them, 174 were treated with intensive chemotherapy, 57 with low intensive therapy and 69 received only supportive care. Seventy-five patients (25%) presented with DIC score ≥ 4 at AML onset. Several factors were significantly associated with DIC score ≥ 4, including diabetes (p=0.038), FLT3 and NPM1 mutations (p=0.024 and p=0.017, respectively), elevated serum LDH (p<0.001) and a leukocyte count above 50x10 9 /L (p=0.002). The 30-days mortality rate was 14,7% (44/300), with 16 patients dying from hemorrhagic or thrombotic complications. A DIC-score ≥ 4 was associated with a significantly higher 30-day mortality rate (28% vs. 9,8%, HR 3.307 95%CI 1.818-6.015; p < 0.001, Figure 1). The independent role of DIC score (0=0.017) and leukocytosis above 50x10 9 /L (p=0.001) in influencing 30-day mortality rate was confirmed in multivariable analysis.

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Summary/Conclusion: The ISTH-DIC score is easy to assess, and this study suggests that it accurately predicts 30-day mortality risk of AML patients, regardless of age and cytogenetic risk. A potential role of DIC score is to select patients who are at high-risk of fatal bleeding and therefore in need of aggressive transfusion support. Further studies on larger population are needed to confirm the findings, and to incorporate DIC score in prognostic models.

PB1836: ACUTE MYELOID LEUKEMIA IN OLDER ADULTS IN RESOURCE CONSTRAINT SETTING: SINGLE CENTER DATA FROM SOUTH INDIA

M. Paul 1,* , L. Abraham 2 , C. M. Jeswin 1 , A. Ajithkumar 1 , C. O. D’souza 2 , M. Abraham 2

1 Clinical Haematology & Haemato-oncology; 2 Clinical Pathology, Rajagiri Hospital, Alwaye, India

Background: Acute myeloid leukaemia (AML) is an aggressive haematological malignancy and is more common in the older adults. The outcome of AML remains highly unsatisfactory in the elderly due to disease-related and patient-related differences between younger and older patients. Data of AML in older patients from the developing nations like India are scarce in the literature. The management of AML in India still remains a challenge due to lack of financial resources and inaccessibility to health care facilities. Most of the elderly patients are denied treatment considering the overall poor outcome and financial toxicity. We analysed the clinico-hematological profile and treatment outcome of patients above 60 years diagnosed with AML during a period of four years in a single centre.

Aims: The objective of the study was to analyse the clinical, haematological and molecular genetic profile of AML in older adults and to assess the treatment outcome.

Methods: This was a retrospective cross-sectional study including all newly diagnosed AML patients above 60 years of age during the period from January 2017 to December 2020 in a tertiary care teaching hospital. Clinical, haematological, cytogenetic and molecular profile were analysed. The patients were stratified according to the International Prognostic Scoring system (IPSS) cytogenetic categories, molecular risk groups and the European Leukemia Net (ELN) risk categories. They were followed up for a minimum period of one year.

Results: Our series included forty four patients with a male preponderance (63.6 %) and age ranging from 60 to 91 years with a mean age of 70. At the time of presentation, 68% patients had a haemoglobin (Hb) level of less than 10g/dL and 43 % had severe thrombocytopenia.

Majority of the cases (38) were de novo AML including two cases of acute promyelocytic leukemia (APML) and one case of pure erythroid leukemia. There were six cases of secondary AML including a single case of therapy related AML. Most of the patients(77%) belonged to IPSS intermediate cytogenetic category.

Of the thirty eight patients tested, six cases were NPM1 mutated and four were FLT3- ITD mutated. The majority of the patients(41%) belonged to ELN intermediate risk group.

Sixteen males and ten females opted for treatment and all of them were given hypomethylating agent (HMA) based chemotherapy. Response assessment marrow was done in four cases, which showed complete remission (CR) in one case and complete remission with incomplete hematologic recovery (CRi) in two cases. Cumulative survival of patients who underwent treatment compared with those who did not (Figure1) showed an obvious survival benefit in the former group(Log Rank{Mantle-Kox}p<0.001).

Antifungal prophylaxis was given in twenty patients. Five had reactivation of infection while on antifungal prophylaxis.Nineteen patients had at least one episode of neutropenic fever admissions of which ten patients had more than two episodes.

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Summary/Conclusion: There is limited data concerning the treatment and survival of elderly patients with AML from developing countries. This cohort gives a glimpse of clinico-hematological profile and treatment outcome of AML in older patients in South India. Our study has shown a definite survival benefit for patients who have taken treatment with HMA when compared to others who opted for palliaton. Larger population based studies with follow up data are required for standardized assessment of patient characteristics and to individualize treatment decisions.

PB1837: ADDITION OF A THIRD, NON-TARGETED, AGENT TO CYTARABINE- AND ANTHRACYCLINE-BASED INDUCTION CHEMOTHERAPY FOR ACUTE MYELOBLASTIC LEUKEMIA INCREASES COMPLETE REMISSIONS AND TREATMENT-RELATED MORTALITY

M. Pereira 1,* , G. Marques 2 , A.-B. Sarmento-Ribeiro 1

1 Clinical Hematology Department; 2 Clinical Pathology Department, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal

Background: Non-promyelocytic acute myeloblastic leukemia (AML) associates with high mortality rates, with a dismal prognosis; treatment options were unchanged over almost four decades, and recent attempts at improving the outcome of AML patients have included risk-adjusted post-remission consolidation protocols, anthracycline and cytarabine dosing adjustments, the addition of a third active agent to the standard anthracycline-cytarabine doublet protocol and, over the last decade, the addition of targeted agents directed at disease biology. However, even when the addition of a third drug results in improved disease-related outcomes, these improvements may be offset by the increase in treatment-related morbidity and mortality.

Aims: To determine whether there is a benefit in terms of early disease control, early toxicity and long-term survival in the addition of a third, non-targeted, drug to anthracycline-cytarabine doublet-based first line induction chemotherapy in adults with AML.

Methods: We performed a systematic review and meta-analysis of the published literature; the validity of the analytical method of comparing the addition of any third drug to the same standard treatment has been previously described by the Lung Cancer Group of the Cochrane Collaboration . The methodological quality of each study selected for inclusion was assessed according to the Jadad Scale; publication bias was investigated through Begg’s and Egger’s methods. The pooled risk ratio for the effect size of the triplet protocol compared to the doublet protocol, with a pooled 95% confidence interval, was calculated for each of the outcome variables; both a fixed effects model and a random effects model (for heterogeneous data) were used.

Results: Our meta-analysis showed that non-targeted triplet-based regiments induced marginally higher complete remission (CR) rates (RR=1.08, 95% CI: 1.04-1.12), with lower rates of primary refractoriness to treatment (RR=0.78, 95% CI: 0.70-0.87), although inducing higher rates of treatment-related mortality (RR=1.11, 95% CI: 0.95-1.30), with a lack of improvement in overall survival and disease-free survival.

Summary/Conclusion: There is a very fragile balance between disease-specific survival and treatment-specific survival with the current doublet protocols, due to their high intrinsic toxicity; decreases in disease-related death through the addition of a third non-targeted drug are often accompanied by increases in treatment-related death secondary to cumulative toxicity.

We suggest that the decision to use a non-targeted triplet-based regimen should be patient-adapted, with a preference for doublet protocols in patients at high risk for toxicity and with low risk for refractory disease, and a preference for triplets in patients at low risk for toxicity and high risk for refractory disease.

PB1838: TOXICITY AND OUTCOME OF HIGH DOSE CYTARABINE (18 G/M2) FOR AML CONSOLIDATION.

L. Lopes 1 , M. Nucci 1 , R. Portugal 1,*

1 Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Background: The combination of an anthracycline and cytosine arabinoside has been the standard induction therapy in adults with acute myeloid leukemia (AML). After complete remission (CR), a post-remission therapy is necessary. CALGB study in 1994 established high dose cytarabine as one of the standard regimens. However, the current 2017 European LeukemiaNet recommendations for AML discourage the use of HIDAC, because intermediate doses of cytarabine (1-1.5g/m 2 ) may be as effective. Hereby we present retrospective data of AML patients treated with HIDAC after the first CR (CR1).

Aims: The main purpose of this study was to determine the toxicities rates of patients receiving HIDAC.

Methods: Adult patients with a diagnosis of AML, who received at least one cycle of HIDAC after achieving first complete remission (CR1) were included. The study was conducted at University Hospital Clementino Fraga Filho, Universidade Federal do Rio de Janeiro (UFRJ), Brazil. Complete remission (CR) was defined using standard criteria. Patients in CR1 received consolidation chemotherapy consisting of cytarabine 3 g/m 2 given by 3-hour IV infusion every 12 hours on days 1, 3, and 5 (18g/m 2 per course). A minimum of 2 HIDAC courses and a maximum of 4 courses were usually planned.

Results: From April 2008 to September 2020, 29 AML patients in CR1 received a total of 67 courses of HIDAC. The median age was 36 years (range, 18 to 62), and (41%) were females. Normal karyotype was present in 11 patients; five cases of CBF AML were identified: four t(8;21) and one t(16;16) with trisomy 22. The median number of cycles of consolidation was 2; 4 patients (14%) received 4 cycles, 10 received 3 cycles, 10 received 2 cycles and 5 received just 1 cycle. The overall survival was 16 months in patients who received 1 or 2 cycles (15 patients) and not reached in patients who received 3 or 4 cycles (14 patients). No significant differences in survival were observed according to the number of cycles (P=0.095). Granulocyte-colony stimulating factor (G-CSF) was given after 65 cycles (95.5%). The most common side effect was febrile neutropenia 38 (56.7%). Neurotoxicity was documented after 2 cycles. One patient presented with seizures on day 2 of the first cycle, and another patient presented with diplopia, abnormal coordination (dismetry) and visual blurring on the 10 th day of the first cycle. Other significant side effects included: severe bleeding 8 (11.9%); mucositis 10 (15%), severe mucositis 1 (1.5%); nausea or vomiting 16 (23,9%); diarrhea 8 (11.9%), short duration diarrhea 5 (7.5%), diarrhea with dehydration 2 (3%), diarrhea with necessity of treatment 1 (1.5%); neutropenic enterocolitis 3 (5.5%); renal failure 3 (4.5%), hemodialysis 1(1.5%). Two deaths occurred before any documentation of relapse (TRM of 6.8%). One death occurred in the first cycle (day 15) due to Candida tropicalis candidemia and another after neutropenic enterocolitis (day 20). Discharge shortly after HIDAC was possible in 45 cycles (67.5%) and no readmission was required in 14 (20.9%). Readmission was necessary after 31 cycles (46.2%) and the median days at home was 5.8 (range, 3 to 8).

Summary/Conclusion: HIDAC (18 g/m 2 ) can be safely given to select AML patients after CR1. Our analysis demonstrates febrile neutropenia was the most common adverse event, neurologic toxicities were reversible and treatment related mortality occurred during early cycles. Hospital discharge after a HIDAC cycle is safe with the use of G-CSF.

PB1839: INDEPENDENT PROGNOSTIC VALUE OF A PLATELET COUNT OVER 300X109/L IN ACUTE MYELOBLASTIC LEUKEMIA PATIENTS TREATED WITH AN INTENSIVE REGIMEN.

I. Ródenas Quiñonero 1,* , A. Jeréz 1 , B. Ferrer 2 , E. Orna 3 , J. T. Navarro-Ferrando 3 , J. M. Raya 4 , T. Martin-Santos 4 , M. Ricard 5 , A. Bermejo 6 , R. Morales 7 , C. Prats 7 , M. Navarrete 8 , V. Roldán-Galiacho 9 , M. L. Amigo 1 , V. Vicente 1 , F. J. Ortuño 1

1 H. Morales Meseguer, Murcia; 2 H. Clínico Universitario Valencia, Valencia; 3 H. Germans Trias i Pujol/ICO-Badalona., Badalona; 4 H.Universitario de Canarias, Tenerife; 5 H.Universitario Fundación Alcorcón; 6 H.Universitario de Fuenlabrada., Madrid; 7 H.Universitario Virgen del Rocío, Sevilla; 8 H.Vall d’Hebron, Barcelona; 9 H. Universitario Cruces, Barakaldo, Spain

Background: Acute myeloblastic leukemia (AML) usually presents with cytopenias, although its degree is variable and in some cases it is non-existent. We herein highlight the existence of “de novo” AML patients that debut with a normal or elevated platelet count.

Aims: Our aim was to evaluate the prognostic impact of a platelet count over >300 x10 9 /L at diagnosis.

Methods: One hundred and twenty-eight adult patients from nine tertiary centers of Spain, with a diagnostic of non-promyelocytic AML between 2000 and 2021, were included.

Two groups were defined based on platelet count at diagnosis: those who debuted with a platelet count over >300 x10 9 /L and those who debuted with a lower platelet count. Patients were also distributed in two different groups according to whether they received intensive chemotherapy treatment.

We used the Kaplan-Meier and the Cox method to analyze overall survival (OS), with a two-sided P value ≤ 0.15 for a factor in the univariate analysis to be included in the multivariate regression, where a P value ≤ 0.05 was considered statistically significant.

Statistical analysis was performed using SPSS software (IBM SPSS Statistics 21, IBM Corporation, Chicago, IL).

Results: A total of 128 patients were included. The median age at diagnosis was 65 y.o (25-89). Eighty patients (62.5%) had received an intensive regimen.

Patients were considered upon platelet count: higher or less than 300 x10 9 /L. We found significant differences in the white blood cells (WBC) count (19.9x10 9 /L vs 5.3x10 9 /L, p= 0.047), the percentage of patients who responded to treatment and in those who achieved complete remission, (56.0% vs 87.0%, p=0.004 and 44 .0% vs 80.4%, p=0.002, respectively).

Figure 1 shows how presenting a platelet count over 300 x10 9 /L was an adverse prognostic factor only in the group that received an intensive regimen. Within this group, only the age (p=0.079), the blast count (%) in bone marrow (p=0.05) and belong to intermediate-adverse European LeukemiaNet-2017 (ELN2017) risk group (p=0.008), were significantly associated with a shorter OS in the univariate analysis

When confronting these variables with the presence of a platelet count at diagnosis over 300x10 9 /L, only intermediate-adverse ELN2017 risk group and the platelet count remained significant for a shorted OS (p=0,023, HR 2.17, 95%CI 1.11-4.24 and p=0.025, HR 3.50, 95% CI 1.16-10.54, for platelet count and ELN risk group, respectively).

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Summary/Conclusion: In “de novo” AML patients treated with an intensive regimen, an unusually high platelet count is related to a worse prognosis. In those patients, a specific molecular study focused on megakaryopoiesis could be of interest.

PB1840: MONOCENTRIC EXPERIENCE OF VENETOCLAX-BASED REGIMENS FOR ACUTE MYELOID LEUKEMIA

M. Sciumè 1,* , A. Bosi 1,2 , G. Ceparano 1,2 , C. De Magistris 1,2 , F. Serpenti 1,2 , P. De Roberto 1 , G. Galassi 1 , F. Onida 1,2 , N. S. Fracchiolla 1

1 Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2 Università degli Studi di Milano, Milan, Italy

Background: Combination of venetoclax and hypomethylating agents (HMAs) has become a standard of care in adult patients with acute myeloid leukemia (AML) who are aged≥75 years or who have comorbidities that preclude use of intensive induction chemotherapy. Few promising data are available in the setting of relapsed/refractory (R/R) AML, while the utility of venetoclax in the post-transplant setting remains poorly investigated.

Aims: To investigate clinical outcome and safety of venetoclax-based combinations in AML patients treated outside of clinical trials.

Methods: We conducted a monocentric retrospective analysis on adult patients affected by treatment-naïve AML not eligible for standard induction therapy or R/R AML, who started venetoclax combination regimens between November 2017 and December 2021. AML genetic risk stratification and response criteria were assessed according to 2017 European Leukemia Net (ELN) consensus. Venetoclax was administered at the dose of 400 mg daily after a short ramp-up. The venetoclax dosage was reduced to 50 or 100 mg daily in case of concomitant strong CYP3A4 inhibitors (e.g. posaconazole) and 200 mg daily in case of moderate CYP3A4 inhibitors (e.g. isavuconazole). Disease free survival (DFS) and overall survival (OS) were assessed using the Kaplan-Meier method and compared between groups using the log-rank test; p values <0.05 were considered significant.

Results: A total of 60 consecutive AML patient treated with venetoclax-based combinations were considered in the study. Twenty-three patients (38%) were affected by treatment-naïve AML and 37 patients (62%) by R/R AML. The median age was 70 years and 67% of the patients were ≥65 years old. The majority of patients showed an intermediate ELN genetic risk, while adverse ELN risk was found in 39% of treatment-naïve patients and 32% of R/R group. Among patients with R/R AML, 62% were treated in the salvage-2 setting or later and 37% had received a prior allogenic stem cell transplantation (allo-SCT). In combination with venetoclax, 51 patients (85%) received azacitidine 75 mg/m2 days 1-7. Eight patients (13%) added venetoclax to HMAs after progression during hypomethylating treatment previously administered for pre-leukemic conditions. Antifungal prophylaxis and subsequent venetoclax dose reduction were performed in 32 patients (53%). Overall response rate was 60% with a complete remission (CR) rate of 53% (78% for treatment-naïve group and 49% for R/R group, p 0.017). The best response was achieved after a median of one months. Median OS was 130 days for R/R patients and 269 days for treatment-naïve patients; median DFS was 199 days for treatment-naïve patients and 145 days for R/R cohort. Genetic adverse risk had a significant worst survival (p 0.02, Figure 1). Among patients who were treated after allo-SCT venetoclax lead to a CR rate of 36%. Overall, 11 patients (18%) received an allo-SCT after venetoclax combinations (7 as consolidation of a first line therapy). Most common grade 3 or 4 adverse events were hematological or infectious toxicities (31% and 64%). One event of severe tumor lysis syndrome was reported.

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Summary/Conclusion: The results of this real-life monocentric study confirmed that venetoclax-based regimens are a viable and safe option as first-line therapy for AML unfit patients and a valid rescue therapy in the R/R setting. Furthermore, the association of venetoclax and hypomethylating agents proved to be a feasible bridging therapy to transplantation in elderly AML patients.

PB1841: A NEW APPROACH IN ACUTE MYELOID LEUKEMIA (AML): SAMATYA-PREDICTING SCORE

I. Serin 1,* , B. ORHAN 2 , D. SONMEZ 2 , T. A. CINLI 1 , H. GOZE 1 , H. SERIN 2 , O. YOKUS 1

1 Department of Hematology; 2 Department of Medical Biochemistry, University of Health Sciences, Istanbul Training and Research Hospital, Istanbul, Turkey

Background: Since it is not possible to access all methods simultaneously in determination of prognosis, it is important to use different methods together in acute myeloid leukemia (AML).

Aims: We aimed to examine the efficiency for prediction of prognosis and response to induction therapy in non-APL AML cases of the “Samatya-predicting score” that we developed using clinical, cytogenetic and flow cytometric features

Methods: A total of 213 patients diagnosed between January 2010-December 2020 were examined. Mortality and response to induction therapy (complete, incomplete, failure) were recorded. The subgroups were formed as follows: Responder: Complete response (CR); non-responder: Incomplete or failure. Patients who were not eligible for the study were excluded.

Results: Of the 158 patients included in the study, the median value of risk score was determined as 2,5. The area under the curve for the median risk score of 2,5 in ROC analysis was 0,635 (0,541-0,729; 95% confidence interval, p=0,006) for exitus; while it was 0,605 (0,517-0,692; p=0,024) for being responder. The sensitivity for mortality was 88%, the specificity 42%, the positive predictive value (PPV) 90,1%, and the negative predictive value (NPV) was 24,7%. In terms of being non-responder to induction therapy, the sensitivity was 90,1%, the specificity 25,3%, the PPV 89,8%, and the NPV was 32%. OS was shorter in those with high risk scores.

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Summary/Conclusion: This practical and usable scoring system we developed can be supported also by new parameters.

PB1842: EARLY DEATH IN NEWLY DIAGNOSED ADULT ACUTE MYELOID LEUKEMIA

N. Siala 1,* , Y. Fakhfekh 1 , I. Ben Amor 1 , F. Kallel 1 , I. Frikha 1 , O. Kassar 1 , M. Chaari 2 , N. Louati 3 , H. Sennana 4 , L. Khelifa 1 , M. Charfi 1 , S. Hadiji 1 , M. Medhaffar 1 , M. Elloumi 1

1 Hematology; 2 hematology laboratory, Hedi Chaker Hospital; 3 Regional blood transfusion center, Sfax; 4 Cytogenetic laboratory, Farhat Hached Hospital, sousse, Tunisia

Background: Recent advances have led to higher response rates and improved survival in patients with newly diagnosed acute myeloid leukemia (AML). However, early death, remain important challenges.

Aims: The aim was to explore risk factors for early mortality in adult AML patients in Southern Tunisia.

Methods: Our study is retrospective over 15 years (2005-2019). Patients included (age: 20- 60 years old) were diagnosed with de novo AML, diagnosed in the clinical hematology department of Hedi Chaker Sfax Hospital / TUNISIA. Secondary leukemia and FAB m3 were excluded. We assessed the one-month mortality in these patients.

Results: One hundred seventy-seven patients with newly AML were in indication of treatment with intensive CT. Thirty-seven died early (21%). The factors that significantly influence the rate of ED are age, gender, delay in diagnosis and the presence of disseminated intravascular coagulation (DIC) (Image/Table).

Death before the start of CT was found in 6 cases (16%) with a median time between diagnosis and death of 5 days [2-28]. Respiratory distress was the only cause of death before treatment, which was caused by intra-alveolar hemorrhage, pulmonary leukocytosis and hypoxemic infectious pneumonitis in 3, 2 and 1 case respectively.Thirty-one patients (84%) died during the induction cure with a median time between the start of the cure and death of 17 days [2-29]. The causes of death during the induction cure were multiple. Death by respiratory distress in 15 cases (48%) originating from: intra-alveolar hemorrhage, PAO and hypoxemic infectious pneumonitis in 7, 4 and 4 cases respectively. Severe sepsis was the cause of death in 11 cases (35%). In the remaining 5 cases the cause of death was: compressive pericardial effusion (2 cases), cerebral hemorrhage (1 case), hyperosmolar coma (1 case) and status epilepticus (1 case).

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Summary/Conclusion: The ED rate in our series is higher than those described in the literature (21% vs 5-10%). Advanced age, gender, delay in diagnosis and initial DIC were the risk factors for ED in our study. Death most commonly occurs from infectious or bleeding complications related to cytopenia. Early diagnosis of AML and improve supportive care is essential to reduce the ED rate and improve overall survival by establishing early treatment

PB1843: ACUTE MYELOID LEUKEMIA WITH PHILADELPHIA CHROMOSOME: EXPERIENCE OF SFAX HEMATOLOGY DEPARTMENT

N. Siala 1,* , L. Khelifa 1 , I. Frikha 1 , F. Kallel 1 , H. Sennana 2 , S. Menif 3 , N. Ben abdeljalil 4 , S. Laddeb 4 , Y. Fakhfekh 1 , I. Ben Amor 1 , O. Kassar 1 , S. Hadiji 1 , M. Charfi 1 , T. Ben othmen 4 , M. Medhaffar 1 , M. Elloumi 1

1 Hematology, Hedi Chaker Hospital, Sfax; 2 Cytogenetic laboratory, Sousse; 3 Hematology laboratory -Pasteur Institut; 4 National bonne marrow transplantation centre, Tunis, Tunisia

Background: Acute myeloid leukemia with Philadelphia chromosome (AML Ph+) is recently been listed in the 2016 revised World Health Organization (WHO) classification of myeloid malignancies as a provisional entity.

Aims: A rare entity whose treatment is not yet established. we are studying the characteristics of a series of patients with AML Phi+.

Methods: It’s a retrospective study over 15 years (2005-2019) which includes patients with AML Phi+, diagnosed and treated in the clinical hematology department of Hedi Chaker Sfax Hospital / TUNISIA. We are interested in studying the clinical and biological characteristics, the different treatments received and overall survival (OS). Known patients followed for Chronic Myeloid Leukemia (CML) and who are transformed into blast phase are excluded.

Results: Among 264 cases of newly diagnosed AML, we collected 5 cases of AML Phi+ (1.8%). The average age was 43 years (range: 34 to 54). Sex-ratio was 1.5. On initial examination, splenomegaly was found in all cases. WBC counts less than 10 G/l, from 10 to 100 G/L and more than 100 G/L were observed respectively in 20, 40 and 40% of cases. Myelemia was found in 4 cases (80%) and basophilia greater than 2% was noted in 3 cases (60%). Cytogenetic study showed the presence of a t (9.22) in all cases, which was isolated in 1 case. The BCR-ABL transcript was of the p190 type in 2 cases and p210 in 2 cases, while the molecular study was not done in the remaining case. Four patients were treated with tyrosine kinase inhibitor (TKI) alone with the achievement of complete remission (CR) in 3/4 of the cases while one patient was treated with the combination TKI and chemotherapy with good evolution. Allograft was not done for all patients, in 2 cases for the absence of an HLA compatible siblings donor. The median follows up was 108 months. Relapse was observed in 2 patients with CR2 in both cases. The five years OS was 80%.

Summary/Conclusion: Phi+ AML in our series is higher than that reported in the literature (1.8 vs <1%) this could be explained by the underestimation of the total number of AML in our country because some patients with AML (>55 years old) are not treated. The major question that arises in front of each phi+ AML is: is it a newly phi+ AML or a CML diagnosed at a blast phase? Previous abnormalities of the blood count, significant myelemia, blood basophilia >2% and the presence of associated cytogenetic abnormality tends towards CML diagnosed at a blast phase while the BCR-ABL transcript type is non-specific. By referring to these data, we can deduce that for our series most probably it is a newly phi+ AML in 2 cases. The major problem posed in the different series is the absence of standardized treatment due to the rarity of the pathology.

PB1844: THROMBOTIC EVENTS DURING REMISSION INDUCTION OF ACUTE MYELOID LEUKEMIA – FREQUENCY AND RELATED FACTORS

W. Silva 1,* , R. Melo 1 , F. Mendes 1 , E. Velloso 1 , V. Rocha 1 , E. Rego 1

1 Hematology, Instituto do Cancer do Estado de Sao Paulo, Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, Brazil

Background: Thrombotic events in newly diagnosed patients with acute myeloid leukemia (AML) have been considered rare and have not received a great deal of attention. These events may occur at the diagnosis, during the remission induction, or later. The incidence of thromboembolism in AML varied markedly among studies, ranging from 2 to 13%, and are usually divided between arterial and venous events. Limited evidence points that using markers of disseminated intravascular coagulation (DIC) might predict thrombosis in AML, and the Khorana score is not useful for this purpose.

Aims: In this work, we evaluated a cohort of intensively treated adults with non-promyelocytic AML in order to assess the thrombosis rate and baseline factors that might be associated with this event.

Methods: This is a retrospective cohort encompassing newly diagnosed AML adult patients (pts) treated at Instituto do Cancer do Estado de Sao Paulo, Brazil, between June 2011 and June 2020. All patients received the “7 + 3” regimen as the frontline regimen, and all thrombotic events until day 60 from diagnosis were captured. Logistic regression was used to find risk factors for thrombosis. Cutoffs for numeric variables were defined by Akaike’s information criteria.

Results: Overall, 204 pts were included, with a median age of 54 years (range,17-74). The main baseline features of this cohort are summarized in Table 1. Overall, 24 thrombotic events were registered – the thrombotic rate of 11.9% (95% CI 7.9-17.4%). Most events were catheter-related (9/24), followed by upper (10) and lower (3) limb events. Two patients presented spontaneous jugular vein thrombosis. DIC score (Taylor et al., 2001) was calculated for 107 patients with laboratory data fully available and resulted in overt DIC (≥ 5) in 45.2%. By univariate analysis, morphologic subtype, AML genetic classification, fibrinogen, d-dimer, and DIC score were not associated with thrombosis during induction in AML. Excluding those catheter-related cases, we noticed a higher monocyte count in patients who developed thrombosis (15.8 vs. 4.7x10 9 /L, p=0.009). Incidence of thrombosis in patients with monocyte count >8.5x10 9 /L was 16% vs. 6.2%. The overall 60-day mortality was 25.8%, with no death related to thrombosis. DIC score was not associated with early mortality during AML induction (p=0.19). Remaining coagulation parameters when individually assessed were also not linked with thrombosis or mortality during induction.

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Summary/Conclusion: In this study with intensively treated newly diagnosed AML, we noticed a similar incidence of thrombosis to that previously reported. While most events reported in this cohort were related to catheter devices, spontaneous thrombosis was associated with a higher baseline monocyte count. While monocytic AML may present with DIC at the diagnosis, monocytic cells are a major source of blood tissue factor, pointing it as an independent marker for thrombosis in AML. Our data, although limited, highlights that the understanding of the thrombosis landscape in AML is complex, and the sole use of already recognized markers of coagulation activation might not be sufficient for prediction.

PB1845: ACUTE MYELOID LEUKEMIA IN THE ELDERLY: A MULTICENTER EXPERIENCE IN COLOMBIA, ON BEHALF OF ACHO’S RENEHOC INVESTIGATORS

C. Sossa-Melo 1,* , V. Abello 2 , M. Solano 2 , A. Peña 3 , M. Rosales 4 , G. Quintero 5 , W. Mantilla 6 , L. Salazar 3 , H. Idrobo 7 , J. Reyes 8 , L. Gaviria 9 , C. Bermudez 10 , R. Gomez 11 , M. Correa 12 , J. Herrera 13 , P. Guerrero 13 , K. Galvez 14 , D. Martínez-Cuadrón 15 , M. A. Sanz 15 , P. Montesinos 15

1 Departamento de Hematología, Clínica Foscal, Universidad de Valencia, Universidad Autónoma de Bucaramanga, Floridablanca; 2 Departamento de Hematología, Hospital San José, Facultad de Medicina, Fundación Universitaria de Ciencias de la Salud, Bogotá; 3 Departamento de Hematología, Clínica Foscal, Universidad Autónoma de Bucaramanga, Floridablanca; 4 Departamento de Hematología, Clínica Foscal; 5 Departamento de Hematología, Fundación Santa Fé, Asociación Colombiana de Hematología, Facultad de Medicina Universidad de los Andes, RENEHOC; 6 Departamento de Hematogía y Oncología, Fundación Cardio Infantil, RENEHOC, Bogotá; 7 Departamento de Hematología, Facultad de Medicina Universidad del Valle, Centro Medico Julian Coronel, Cali; 8 Departamento de Hematología, Clínica los Nogales, Bogotá; 9 Departamento de Hematología, Hospital Universitario San Vicente Fundación, Universidad de Antioquia, Medellin; 10 Departamento de Hematología, Centro de Investigaciones Clínica San Diego, CIOSAD, Bogotá; 11 Departamento de Hematología, Hematooncólogos SAS, Universidad Libre Seccional Cali, Facultad Ciencias de la Salud; 12 Departamento de Hematología, Clínica Nuestra Señora de los Remedios; 13 Departamento de Hematología, Clinica Imbanaco, Quirónsalud, Cali; 14 Departamento de Hematología, Hospital Pablo Tobon Uribe, RENEHOC, Medellin, Colombia; 15 Departamento de Hematología, Instituto de Investigación Sanitaria la Fe (ISLAFE), Hospital Universitario La Fe, Valencia, Spain

Background: The overall survival (OS) of acute myeloid leukemia (AML) decreases as age increases among patients. There are clinical and biological differences in AML in older patients. Even with actual treatments, 70% of patients older than 65 years will die of their disease within 1 year of diagnosis.

Aims: Our aim was to characterize the elderly patients (>65 years old) with acute myeloid leukaemia (AML), who were attended in 13 health institutions in Colombia, from 2009 to 2021, including RENEHOC (online platform) and PETHEMA date base (Spanish Program for Hematology Treatments).

Methods: An ambispective registry of elderly AML patients, treated in approved centers over the last 12 years. Descriptive statistics were used for patient’s demographic and clinical characteristics. The Kaplan-Meier method was used to assess RFS and OS.

Results: A total of 175 patients were included. 54,86% were male and the median age at diagnosis was 73 years old. Most of the patients were classified according to the Eastern Cooperative Oncology Group (ECOG) as ECOG 0-1 (52,7%), ECOG 2 (25 %) and ECOG > 2 (22.3%). 24,26% presented as secondary AML, from these 34% had previous history of myelodysplasic syndrome. At presentation, fever was the main symptom (39,16), followed by bleeding (18,56%). The most frequent comorbidities were hypertension in 20% and diabetes in 9,4%. 24.34% of the patients had one comorbidity while 21.6% of the population had 3 or more. For treatment: 32,9% of the patients received hypomethylating agents, 23,2% received intensive chemotherapy with cytarabine and idarrubicine, 14,1 % had FLUGA (fludarabine low dose and cytarabine) and supportive therapy in 16,7% of the patients. After induction, complete remission (CR) was achieved in 16,9% of patients; CR with incomplete hematologic recovery (Cri) in 7% and 20,9% had primary refractory disease. Mortality during in induction therapy was 12,9%.

The median age of patients in the intensive chemotherapy group was 66 years old (66-70), with ECOG 0-2 in 63,8%. The OS was 36.1% (95%CI: 27.0%–45%) at 1 year and 0 % at 5 years of follow-up, the relapse-free survival (RFS) was 38.3 % (95%CI: 27.4%- 49.1%) at 1 year and 0% at 5 years.

According to the ECOG. the OS at 1 year was 50,9 % (95%CI: 36.1%–63.9%) for patients with ECOG <2, while the OS at 1 year was 22.7% (95%CI: 12.7%–34.4%) for ECOG >2 with p<0.001.

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Summary/Conclusion: Management of AML in older patients remains a medical challenge, not only because of the age, but also because older adults are more likely to have impaired functional status and comorbidities that limit treatment options. More refined assessments tools and scales are needed to differentiate between fit and fragile older adults. This multicenter report analyzing real world data from AML patients in Colombia confirmed the impact of clinical factors: age, ECOG on OS and RFS. Challenges includes access to newer treatments with novel, targeted and less intensive therapies.

Keywords : acute myeloid leukemia, survival, elderly

PB1846: ALTERATIONS IN PRIMARY HEMOSTASIS AS ADDITIONAL RISK FACTORS OF BLEEDING IN PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA (AML): UPDATED PROSPECTIVE ANALYSIS.

Z. Stupakova 1,* , D. Iryna 1 , M. Zoya 1 , S. Anna 1 , O. Karnabeda 2 , U. Melnyk 3 , O. Novosad 4

1 Radiation Hematology, State Institution “National Research Center for Radiation Medicine of NAMS of Ukraine”; 2 Bohomolets National Medical University; 3 Kyiv City Clinical Hospital №9; 4 National Cancer Institute, Kyiv, Ukraine

Background: Hemorrhagic complications in patients with AML are usually associated with thrombocytopenia, infections, concomitant disorders and coagulation abnormalities. Von Willebrand factor could well be considered as a link in hemorrhagic events because of its unique features in primary hemostasis as well as further role in factor VIII binding and stabilizing role.

Aims: The primary endpoints were a qualitative and quantitative functions of plasma von Willebrand factor (vWF) which include measurements of vWF activity (vWF:ristocetin cofactor [vWF:RCo]), vWF level (vWF:antigen [vWF:Ag]), factor VIII coagulation activity regarding to vWF(vWF:FVIII) and FVIII activity (FVIII) as an additional bleeding risk factor in newly diagnosed AML patients.

Methods: 34 patients with newly diagnosed AML (excluded APL) were included in the study group (17 males and 17 females; median age: 50 years). The diagnosis of AMLestablished by FAB and WHO classification systems and the plasma samples were collected before the beginning of induction therapy. The grade (G) of bleeding events was assessed according to Modified WHO Bleeding Scale (WHO BS). Control group included 20 age-matched and sex-matched healthy individuals. Levels of vWF:Ag and vWF:RCo tested onautomated latex particle-enhanced immunoassay ACL Elite Pro and ACL TOP 300 (Instrumentation Laboratory). vWF:FVIII and FVIII were measured using a clotting assay with factors deficient plasma by fully automated coagulometer ACL Elite Pro (Instrumentation Laboratory).

Results: Seventeen patients (50 %) in the study group were experienced bleeding events (G1-4) before and during the first course of treatment and other half of patients had no bleeding episodes (G0). There were no bleeding episodes in the control group (G0). The median platelets in the study group was 39,5 versus 260 in the control group (p<0,05).

In seven patients (20,5%) with AML we observed levels of vWF:RCo below normal ranges. Among them the most frequent bleeding events were G2 (4/7) and G4 (2/7), two of them died during the first course of chemotherapy due to gastrointestinal bleeding. Therefore we found astatistically significant association between a decreased levels of vWF:RCo below normal rangesand the occurrence of severe bleeding episodes (G2-G4)in patients with AML compared with the group that had normal ranges of vWF:RCo and no bleeding episodes (G0) (p>0,001). Despite decreasing in vWF:RCo below normal ranges in the control group as an accidental finding, bleeding events have not emerged.

Low levels of vWF:Ag had been observed in seven patients with AML (20%) and was associated with the occurrence of severe bleeding in patients with AML compared with the patients who had normal vWF:Ag levels and no bleeding episodes (p=0.03).

It is important to mention, that vWF:Ag levels (median 142,5) were elevated in most cases (56%) in the main group and are associated with a compensatory mechanism for stopping bleeding compared with the control group.

Low vWF:FVIII and FVIII levels in patients with AML and bleeding episodes G2-G4 as well as other values had statistic difference to the patients with no bleeding events (G0) (p=0,03).

Summary/Conclusion: Despite the rapid development of treatment options we steel need better understanding of underlying causes of haemorrhagic complications due to high mortality rates from bleeding. According to received results activity of vWF together with quantitative values may become a promising purposes for further investigation and creation of bleeding risk scale in patients with AML.

PB1847: ACUTE MYELOID LEUKEMIA – IS IT MORE BESIDES GENETIC ABNORMALITIES?

M. Urbano 1,* , B. Marques 1,2,3 , M. Peixinho 4 , R. Sousa 1 , C. Nora 1 , C. Almeida 1 , D. Silva 1 , P. Moura 1 , S. Marini 1 , N. Silva 4,5 , A. Roque 1,6 , E. Cortesão 1,2,3,7 , C. Geraldes 1,2,3,8

1 Clinical Hematology, CHUC; 2 Laboratory of Oncobiology and Hematology, FMUC; 3 Environment Genetics and Oncobiology (CIMAGO), Coimbra Institute for Clinical and Biomedical Research (iCBR); 4 Pathology department, CHUC; 5 Pathology department; 6 Institute of Physiology, FMUC; 7 Clinical Academic Center of Coimbra, CHUC; 8 Clinical Academic Center of Coimbra, FMUC, Coimbra, Portugal

Background: Acute myeloid leukaemia (AML) still carries a poor prognosis, despite novel aproaches. Age and previous myelodysplastic syndrome (MDS) are important risk factors. Environmental factors: professional benzene exposure, residential background and exposure to chemotherapy (CT)/radiotherapy (RT) have also been implicated.

Aims: To evaluate environmental factors of AML patients, its genetic characteristics and overall survival (OS), in a tertiary centre.

Methods: We retrospectively revised newly diagnosed AML in our centre between 2018 - 2020. Clinical and laboratory data were obtained by clinical files review

Results: We included 235 patients, 47.2% (n=111) male. Median age 69 (19-97) years. (67 [28.5%]≤60 years old. – YOUNG and 168 [71.5%] with >60 years old - OLD).

The most common WHO 2016 category was AML with myelodysplasia-related changes (MRC) (37.0%; n=87), followed by AML NOS (21.3%; n=50); 15 (6.4%) patients had acute promyelocytic leukaemia with PML―RARA , 21 (8.9%) therapy-related myeloid neoplasm and 17 (7.2%) post-myeloproliferative neoplasm (post-NMP) AML.

The main category within YOUNG patients was AML with mutated NPM1 (19.4%) and in the OLD group was AML-MRC (45.2%).

Genomic classification (GC) (Papaemmanuil, 2016) was available for 117 cases (49.8%): AML with no detected driver mutations (n=28; 23.9%); AML with mutated chromatin, RNA-splicing genes, or both (n=26; 22.2%). Considering YOUNG vs OLD patients, the main category was AML with mutated NPM1 (25%) vs AML with no detected driver mutations (32.3%).

Regarding ELN risk stratification: 20.0% were low-risk, 27.1% intermediate risk and 52.9% high-risk category.

OS for total cohort was 4.2 months, with 177 events (75.3% of included patients). The YOUNG patients had a median OS of 18,7 months while the OLD had median OS of 2,6 months, with P<0,001 (HR 2,31).

Ninety nine (42.1%) patients lived in industrial regions. We found no correlation between living place and dysplasia, even after adjusting for age, ELN risk or GC. We found an apparent association of WHO subtype with living place (p=0.013), as post-MPN AML was more common in non-industrial (10.3 vs 3.0%) and AML with mutated NPM1 in industrial areas (11.1 vs 7.3%), but they were lost after adjusting for age. There was no OS difference between industrial and non-industrial places even after adjusting for age.

Regarding professional exposure (available for 85 patients), 7.1% were exposed to benzene, 2.4% to agricultural activities and 31.8% to other chemicals, 58.8% with no significant exposure, with no differences in WHO classification, GC nor age.

Smoking appeared associated with longer OS (6.6 vs 34.9%; p=0.026; HR 0.53), but this effect is lost after adjusting for age, as smoking was more common in YOUNG (65.7 vs 25.0%). There was no correlation with AML subtype or risk.

Concerning other risk factors like alcohol consumption, obesity, family history of cancer, previous solid neoplasm, previous chemotherapy or previous radiotherapy none of them had impact in AML subtype, ELN risk or OS (p=NS).

Summary/Conclusion: In our cohort, the majority of patients were over 60 years old and presented with AML-MRC, as described in the literature. Concerning GC, the dominant category was AML with no detected driver mutations.

Looking at environmental factors, in our cohort only residential background correlated with AML subtype, despite non-significance impact in prognosis.

PB1848: NGS AS A FUNDAMENTAL TOOL FOR THE DEVELOPMENT OF NEW TARGETED THERAPIES

P. Velarde 1,* , M. Sole 1 , R. Zapata 1 , E. Gil 1

1 Hematología y Hemoterapia, Hospital Juan Ramon Jimenez, Huelva, Spain

Background: Sequenced patients with Acute Mieloblastic Leukemia “AML” using second-generation sequencing “NGS” has notably increased in recent years. The development of targeted agents aims to achieve an improvement in clinical outcomes through personalized medicine.

Aims: To analyze the frequency of mutations detected by NGS in adult patients with non-secondary AML diagnosed in a single center in the last 5 years. Disease profile at diagnosis and sample evolution in terms of overall survival (OS) and progression-free survival (PFS) were analyzed.

Methods: Retrospective descriptive study. Data was collected through medical records review. Characteristics of 32 patients (≥18 years) with non-promyelocytic/non-secondary AML who received intensive chemotherapy (+/- Midostaurin and Gentuzumab Ozogamicin “GO”) were reported. Samples were analyzed by NGS in a different center. The gene panel includes 26 genes (ASXL1, CALR, CBL, CEBPA, DNMT3A, EZH2, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MLL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1). SPSS program (Statistical Package for Social Sciences) was used to analyze collected data.

Results: 32 patients were reported: 41% male, 59% female. Patient characteristics and frequency of mutations detected by NGS are referred in Image 1. ELN risk; favorable (21%) intermediate (53%), adverse (21.9%). Induction regimen; Standard 3 + 7 (87.5%), 3 + 5 (3%), Vyxeos (CPX-351) (3%). FLT3+ (31%) received Midostaurin (day 8 to 21), CBF+ patients (3%) received GO. 37% were not trasplantated, 9% received Autologous Hematopoietic Stem Cell Transplantation, and 34% received Allogeneic Hematopoietic Stem Cell Transplantation. 77% achieved complete remission (CR) post-Induction and 12% were resistant. 28% achieved negative Minimal Residual Disease “MRD” and 53% remained positive MRD post-Induction. 25% patients relapsed once and 12.5% ​​twice. 42% patients were exitus. OS (global sample) was 56% median of 35 months 95% CI (26-44), PFS was 75% median of 43 months 95% CI (35-52). 100% of NMP1+/FLT3- patients achieved CR post-Induction compared to 80% NMP1+/FLT3+. OS was 62% in NPM1+, median time of 38 months 95% CI (20-57) compared to 54% in NPM1- patients, median time of 31.7 months IC 95% (22-40).

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Summary/Conclusion: Our results in terms of frequency of mutations in patients remains very similar with previous published data: FLT3+ (28% in our sample compared to 25-39% reported), NMP1 (25% compared to 25-29%), DNMT3A (28% compared to 31%), IDH1 and IDH2 (6%, 15%) compared to 7% and 20%, NRAS (15% compared to 5-22%), WT1 (15% compared to 13%), ASXL1 (6% compared to 7%).

NPM1 mutation is associated with higher CR rates and better OS. Our sample showed OS 62% vs 54% in NPM1+ compared to NPM1- patients proving published data, however statistical significance was not reached probably due to the insufficient sample. NPM1+/FLT3- genotype represents a favorable prognostic marker with higher CR rates and better OS. In fact, our sample showed 100% of CR in NPM1+/FLT3-ITD- patients compared to 80% of NMP1+/FLT3ITD+ patients.

Due to NGS, patients can easily be stratified according to the ELN risk scale, however the limited number of patients was not enough to achieve significant results in terms of OS and PFS referred to the ELN. This study confirms the most frequent molecular alterations with should be mainly approached for development of new agents in order to achieve an improvement in clinical outcomes through personalized medicine.

PB1849: TRIAL IN PROGRESS: PHASE IB/II STUDY OF SIREMADLIN IN COMBINATION WITH VENETOCLAX + AZACITIDINE IN PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) WHO ARE INELIGIBLE FOR INTENSIVE CHEMOTHERAPY

N. G. Daver 1,* , A. H. Wei 2 , E. M. Stein 3 , D. J. DeAngelo 4 , D. Pathak 5 , Y. Xu 6 , S. Grzesiak 5 , A. Venditti 7

1 Department of Leukemia, Division of Cancer Medicine, University of Texas MD Anderson Cancer Center, Houston, United States of America; 2 Department of Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Australia; 3 Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York; 4 Dana-Farber Cancer Institute, Boston, United States of America; 5 Novartis Pharmaceuticals AG, Basel, Switzerland; 6 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 7 Hematology, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy

Background: Treatment options for patients with AML who are not candidates for intensive chemotherapy (IC) are limited. The addition of venetoclax to hypomethylating agents demonstrated improved remission rates and survival; however, relapse rates are high, and most patients remain measurable residual disease (MRD) positive (DiNardo CD, N Engl J Med , 2020). Murine double minute-2 (MDM2) is a key negative regulator of tumor protein 53 (p53). Inhibition of the p53-MDM2 interaction results in up-regulation of the tumor-suppressive effect of p53. Siremadlin is a selective inhibitor of the p53-MDM2 interaction. Siremadlin + venetoclax has shown durable antitumor responses in p53-wild-type AML patient-derived xenograft models (Wang Y, Cancer Res , 2019). Findings from a first-in-human Phase I study with siremadlin demonstrated a tolerable safety profile and preliminary antitumor activity in patients with AML (Stein EM, Clin Cancer Res, 2021; NCT02143635 ). Preliminary results from the ongoing Phase Ib study exploring siremadlin in combination with venetoclax showed promising antileukemic activity in patients with relapsed/refractory AML (Wei AH, ASH 2021; NCT03940352 ).

Aims: Here we describe the study design of a Phase Ib/II study to assess the safety and efficacy of the novel combination of siremadlin + venetoclax + azacitidine in patients with AML who are unfit for IC ( NCT05155709 ).

Methods: The study will enroll approximately 55 adult patients with AML who are unfit for IC due to older age (≥75 y) or presence of comorbidities (cardiac or pulmonary, Eastern Cooperative Oncology Group performance status of 2 or 3). Patients with prior myelodysplastic syndrome, myelofibrosis, essential thrombocythemia, polycythemia, or therapy-related AML may also be eligible. Patients with TP53 mutation or del17p are excluded. Two subpopulations will be evaluated in 2 arms: patients with prior suboptimal response (no complete remission [CR], CR with partial/incomplete hematologic recovery [CRh/CRi], or morphologic leukemia-free state) after 2-4 cycles of first-line venetoclax + azacitidine (Arm 1) and patients with newly diagnosed AML with high-risk clinical features that confer a low likelihood of response to venetoclax + hypomethylating agents (adverse genetic risk stratification per European Leukemia Network, 2017; Arm 2). The study will consist of a safety run-in (Part 1) and expansion phase (Part 2; Figure). The safety run-in will evaluate safety across 2 siremadlin dose levels (20 or 30 mg) from Day (D)1 to D5 in each 28-d treatment cycle in combination with venetoclax and azacitidine. In Arm 1, pts will continue receiving their venetoclax and azacitidine doses that were evaluated as tolerable prior to study enrollment. In Arm 2, venetoclax will be ramped up over 3 days to a target daily dose of 400 mg and azacitidine (75 mg/m 2 ) will be administered from D1–D7 or from D1–D5, and D8 and D9). Upon confirmation of the recommended dose for expansion (RDE, determined for each arm separately), enrollment for the expansion phase (Part 2) will open; patients will be treated with siremadlin at the RDE in combination with venetoclax + azacitidine. The primary endpoints are incidence of dose-limiting toxicities (Arm 1 and Arm 2 separately) and CR rate (Arm 1 only). Secondary endpoints include safety and tolerability; CR rate (Arm 2 only); CR/CRh and CR/CRi rates; duration of CR, CRi, and CRh; overall survival; early mortality; MRD; and pharmacokinetics.

Results: None, trial in progress.

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Summary/Conclusion: None, trial in progress.

PB1850: A DOSE FINDING AND EXPANSION OPEN LABEL, MULTICENTER STUDY OF IADADEMSTAT AND GILTERITINIB IN PATIENTS WITH RELAPSE/REFRACTORY ACUTE MYELOID LEUKEMIA WITH MUTATED FLT3: THE FRIDA STUDY

A. Fathi 1,* , A. Molero 2 , S. Gutierrez 2 , M. Arévalo 2 , H. Yang 2 , C. Buesa 2 , A. Limon 2

1 Leukemia Department, Massachusetts General Hospital, Boston, United States of America; 2 Oryzon Genomics SA, Cornella de Llobregat, Spain

Background: Despite improvements in therapy for acute myeloid leukemia (AML), relapses remain a common challenge, contributing to the death of more than 50% of AML patients (pts). Approximately a third of pts with newly diagnosed AML harbor mutations in the fms-like tyrosine kinase 3 (FLT3) gene. Current guidelines recommend molecular testing for FLT3-mut at diagnosis and at relapse, for earlier incorporation of targeted agents FLT3 inhibitors (FLT3i). The approval of the FLT3i midostaurin with induction therapy in FLT3mut AML, and of a more specific potent FLT3i, gilteritinib, as monotherapy for relapsed/refractory (R/R) pts have resulted in improved outcomes. Nevertheless, the duration of remission achieved with single-agent gilteritinib in R/R disease is transient and often brief. Iadademstat (iada) is a highly selective and potent covalent inhibitor of LSD1/KDM1A (lysine demethylase 1) that induces differentiation of AML cells in vitro and decreases leukemic stem cell survival in preclinical models. More than 100 subjects have been treated with iada, including R/R AML pts with monotherapy and naïve AML pts in combination with azacitidine, revealing a manageable safety profile and encouraging response rates. The purpose of the FRIDA clinical trial is to evaluate the safety and effect of iada in combination with the standard-of-care gilteritinib for the treatment of R/R FLT3-mut AML pts. The preclinical rationale supporting the combination is based on: 1) a potent synergistic activity in FLT3-AML in several preclinical models, and 2) the anti-leukemic activity of iada, mediated by preventing the interaction between LSD1 and the GFI1 transcription factor and modifying the epigenetic marks of genes resulting on macrophage/monocytic differentiation. These data, together with iada’s favorable ADME and low DDI risk, warrants further exploration of this combination with the aim of improving outcomes for FLT3-mut R/R AML pts.

Aims: The FRIDA study will establish the safety, tolerability and determine the recommended phase 2 dose (RP2D), and preliminary activity for the combination of iada and gilteritinib in pts with FLT3mut R/R AML.

Methods: Adult pts with a body weight >50Kg and ECOG 0-2, with relapsed or refractory disease, after 1 or 2 prior lines of therapy, and with FLT3-mutations will be enrolled. In a 3 + 3 escalation phase, up to 18 pts would receive iada at doses of 75 to 150 ug, orally, on 5 days ON -2 days OFF schedule, with continuous gilteritinib PO, at 120 mg/day. In the expansion phase, up to 14 pts at the selected safe and pharmacologically active dose/s will be enrolled. Primary endpoints of the study are safety and RP2D determination (based on PK, PD, safety, tolerability, and preliminary activity). Bayesian efficacy monitoring will be performed with cumulating enrollment to ensure futility boundaries are not crossed and to evaluate the activity of the combination. Secondary endpoints include Overall Survival, Event-Free-Survival, Overall Response Rate, Time to Response, Duration of Response and Transfusion rate. Exploratory endpoints include measurable residual disease and gene mutational analysis.

Results: This is a clinical trial in progress. No results available.

Summary/Conclusion: Study recruiting in Q2 2022 in US.

PB1851: EARLY DEATHS IN PATIENTS WITH NEWLY DIAGNOSED ACUTE PROMYELOCYTIC LEUKEMIA: A SINGLE-CENTER RETROSPECTIVE STUDY FROM A GENERAL HOSPITAL

B. Yohannan 1,* , F. Cervoni-curet 1 , A. Rios 1

1 Hematology and Oncology, University of Texas Health Science Center, McGovern Medical School, Houston, United States of America

Background: Acute promyelocytic leukemia (APL) is a rare and distinct subtype of acute myeloid leukemia characterized by t(15;17) translocation. Although APL is considered a highly curable disease, early deaths (EDs, death within 30 days) due to coagulopathy and bleeding diathesis remain a significant challenge. ED rates in APL can be as high as 30% in a general hospital. We reviewed all cases of APL treated at Memorial Hermann Hospital (MHH) Texas Medical Center.

Aims: To evaluate the ED rate and causes of EDs in newly diagnosed APL patients

Methods: We retrospectively analyzed adult patients (≥18 years old) with newly diagnosed APL treated at MHH between January 2009 and December 2021. The consecutive list of patients with APL was prepared using the pharmacy electronic medical records of MHH Cancer Center. The following data were collected: Baseline demographic variables, WBC, hemoglobin, platelet count on admission, coagulation parameters, presence/absence of disseminated intravascular coagulation, bone marrow or peripheral blood molecular analysis, immunohistochemistry, cytogenetics, Sanz score and risk stratification, timing of initiation of all trans retinoid acid (ATRA), complete response (CR) rate, major adverse events during induction, and ED rate.

Results: We identified 25 patients with newly diagnosed APL. All patients had flow cytometry, immunohistochemistry, cytogenetics, and molecular testing for t(15;17). The median age was 40 years (range, 20-67 years), and 52% were men. Subject races included 10 whites, 4 African Americans, 7 Hispanics, and 4 Asians. Based on Sanz risk stratification, 5 patients had low-risk, 5 had intermediate-risk, and 15 had high-risk disease. Sixteen patients presented with bleeding diathesis: 8 with intracranial hemorrhage (ICH), 6 with gastrointestinal or genitourinary bleeding, and 1 each with retinal bleed and abdominal wall hematoma. The median time to initiation of ATRA was 12 hours. Patients with high-risk APL (WBC >10,000) received idarubicin or gemtuzumab ozogamicin. Three patients died before response assessment. All 22 response-evaluable patients treated with ATRA and arsenic trioxide induction had a CR. The most common adverse events during induction therapy were febrile neutropenia (n=6), differentiation syndrome (n=5), and respiratory failure requiring mechanical ventilation (n=6); 12 required ICU admission, 4 had acute kidney injury, with 1 requiring dialysis support. Of the 8 patients who presented with ICH, 4 required emergent craniotomy, and 4 were managed conservatively. ED occurred in 3 (12%) patients: 2 died within 48 hours of admission and another between days 8 and 15. Two patients died of ICH, and one patient with subdural hematoma died from pseudomonas sepsis and multiorgan failure. Five patients developed thrombotic events: 2 each had pulmonary embolism and stroke and 1 had splenic and renal infarct.

Summary/Conclusion: In our series of 25 patients, the ED rate was relatively low despite 8 of the patients presenting with ICH. Early diagnosis, timely administration of ATRA, correction of coagulopathy, critical care, and neurosurgery support are pivotal in reducing ED in APL.

PB1852: AZACITIDINE COMBINED WITH HAG REGIMEN FOR ELDERLY PATIENTS WITH ACUTE MYELOID LEUKEMIA:PRIMARY ANALYSIS OF A RETROSPECTIVE COHORT

W. Zheng 1 , S. CUI 1,* , J. Wang 1 , Y. Wang 1 , J. Xu 1 , Y. Zhou 1 , Z. Liu 1 , R. Xu 1

1 hematology department, Affiliated Hospital of Shandong University of traditional Chinese Medicine, Jinan, China

Background: Retrospective analysis of azacytidine combined with HAG regimen in elderly patients with acute myeloid leukemia

Aims: To investigate the clinical efficacy and safety of azacytidine combined with HAG regimen in the treatment of senile acute myeloid leukemia.

Methods: 21 elderly patients with acute myeloid leukemia (AML) hospitalized in The Affiliated Hospital of Shandong University of Traditional Chinese Medicine from January 2019 to January 2022 were retrospectively analyzed, including 10 males and 11 females, with a median age of 67 years (60-75 years).Among them, 14 patients were newly treated and 7 patients were relapsed and refractory. Among the newly treated patients, 6 patients were in the poor prognosis group, 6 patients were in the moderate prognosis group and 2 patients were in the good prognosis group. Treated with Azacitidine combined with HAG regimen (azacytidine 75mg/(m 2 ·d), iH D1-7+ homoharringtonine 1mg/(m 2 ·d) D2-8+ cytarabine 10mg/m 2 iH, D2-8+ G-CSF 200ug / (m 2 ·d) iH D1-8). and observed the complete response rate (CR), total response rate (ORR) and the occurrence of adverse reactions in 21 patients,after 1 course of induction chemotherapy.

Results: The total effective rate (ORR) and complete response rate (CR) of azacitidine combined with HAG regimen in the treatment of elderly acute myeloid leukemia were 85.7% and 61.9% respectively.For the newly treated patients, The ORR was 78.57%, and the CR rate was 64.29% (9/14). For the recurrence refractory patients, the ORR was 100%, but the CR rate was low, accounting for 28.57%.Among the newly treated patients, the CR rates of the good prognosis group, the medium prognosis group and the poor prognosis group were 100%, 66.67% and 50%, respectively, and the differences were statistically significant.The main adverse reactions during the treatment were myelosuppression, of which the incidence of grade 3-4 myelosuppression was 21.43% in newly treated patients and 28.57% in refractory patients.One patient died early in the initial treatment, due to infection in the myelosuppression stage, which induced heart failure.

Summary/Conclusion: Azacitidine combined with HAG regimen is effective in the treatment of acute myeloid leukemia (AML) with early treatment in elderly patients and refractory relapse, with few adverse reactions and good tolerability.

PB1853: IN-HOSPITAL SURVIVAL OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND CONCOMITANT CORONAVIRUS INFECTION DEPENDING ON VARIOUS CLINICAL FACTORS.

E. Baryakh 1 , E. Misyurina 1 , E. Zhelnova 1 , K. Yatskov 1 , O. Kochneva 1 , Y. Polyakov 1 , D. Ivanova 1 , K. Kaluzhskaya 1 , T. Tolstykh 1 , T. Chudnova 1 , I. Samsonova 1 , M. Lysenko 1

1 SBHI Municipal Clinical Hospital No. 52 of the Moscow Department of Health, Moscow, Russia

Background: Acute myeloid leukemia (AML) is characterized by rapid progression and requires highly intensive therapy. Complete remission is achieved in 60 to 80% of young patients. The disease can be cured in only 1/3 of patients who have achieved remission. Many patients die from infectious complications at different stages of treatment. COVID19 infection significantly worsens the prognosis of AML patients who are not in remission.

Aims: To assess overall in-hospital survival of AML patients and concomitant coronavirus infection, as well as to assess survival taking into account various clinical factors.

Methods: From April 2020 to August 2021, 102 AML patients aged 18-91 years: 44 men and 58 women, average age 58 years underwent treatment for coronavirus infection in the Department of Hematology of Municipal Clinical Hospital No. 52. In several cases, coronavirus infection developed during a course of chemotherapy or in the periods between the treatment courses, and therefore these patients were transported to the department via Emergency Medical Services (EMS) from other medical facilities. We assessed overall survival, as well as survival depending on age, disease status, the extent of lung damage based on the computed tomography (CT) results, neutrophil level at admission, chemotherapy performed before the onset of coronavirus infection, and at its outcome.

Results: Overall survival was 44.1%. The highest mortality was in the groups of patients over 65 years of age (73.7%) with newly diagnosed disease (72.4%), with the highest extent (grade 4) of lung damage based on CT results at admission (100%). The death rate among patients with agranulocytosis at admission was 2-fold higher than among patients without agranulocytosis (66% versus 33%). The most unfavorable prognosis was found in the group of patients who received low-dose Cytosar (LDC) before the development of coronavirus infection (63.6%). Among the patients on chemotherapy at the outcome of coronavirus infection, the highest mortality was observed with LDC and hydroxyurea therapy (77.8% and 87.5%, respectively). The prognosis in these groups was probably influenced by the predominantly elderly age of patients (over 60 years), and the aggressive course of the viral infection.

Summary/Conclusion: Coronavirus infection is a significant adverse factor in elderly patients with newly diagnosed AML.

PB1854: NEXT GENERATION SEQUENCING APPROACHES IN CHRONIC LYMPHOCYTIC LEUKAEMIA: FINDINGS FROM EXTERNAL QUALITY ASSESSMENT

A. Cartwright 1,* , H. Whitehouse 1 , D. Travis 1 , H. J. Clouston 1 , S. Scott 1 , L. Whitby 1

1 UK NEQAS for Leucocyte Immunophenotyping, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom

Background: Chronic lymphocytic leukaemia (CLL) represents a clinically heterogeneous, lymphoproliferative neoplasm resulting in chronic B-lymphocyte proliferation. Historically, large cytogenetic aberrations detected by fluorescent in situ hybridisation were identifiable in patients, enabling prognostic assessment. With the development of next generation sequencing (NGS), variants have been identified in several genes that are recurrently mutated in CLL, including TP53 , ATM , BIRC3 , NOTCH1 and SF3B1 , with the prognostic implications of variants in these genes being reported. However, only TP53 variant status has been incorporated into routine clinical diagnostics to guide prognosis, risk stratification and appropriate therapeutic interventions based on findings indicating decreased survival and resistance to immunochemotherapy.

Aims: The aims of this study were to assess NGS gene panel content and design approaches in CLL to determine levels of harmonisation amongst laboratories.

Methods: The United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping has provided external quality assessment for use of NGS technologies in CLL since 2018. The programme routinely requests NGS panel information (gene content and exon coverage) and other methodological data from participating laboratories. Data returns from 2018-2021 relating to NGS gene content and panel analysis were evaluated.

Results: In total, 127 genes were analysed across 34 laboratories, ranging from 1-82 genes analysed in the context of CLL per laboratory (median of 12 genes). Of the 34 laboratories evaluated, five analysed TP53 only, with four of these utilising a non-disease specific myeloid NGS panel. The most commonly analysed genes were TP53 (100% laboratories), SF3B1 (73.5%) and NOTCH1 (70.6%). For these genes, there was a high-level of standardisation in sequencing approaches; with 91.2% of laboratories sequencing coding exons 2-11 in TP53 and 100% laboratories sequencing ‘hotspot’ exon 34 of NOTCH1 , accounting for >80% variants in NOTCH1 associated with CLL. For SF3B1 , 87% laboratories sequenced exons 14-16, which are hotspots for variants. Other frequently analysed genes included MYD88 (61.8% laboratories), BTK (58.8%), ATM and PLCG2 (52.9%).

Summary/Conclusion: The number of genes analysed in the context of CLL amongst laboratories was highly variable. Use of NGS genetic testing approaches in haemato-oncology aids in accurate diagnosis and prognosis, however, the cost of developing these panels is not insubstantial and as such, laboratories often design panels to encompass multiple lymphoid/haematological neoplasms. Whilst recommendations outline the need for TP53 testing in CLL, the lack of technical and clinical guidelines on minimal NGS panel content and bioinformatic analysis that provide additional prognostic information has resulted in a wide variation in bioinformatic analysis approaches. This inevitably results in wide disparity of data generated and subsequent clinical interpretation. Guidelines are urgently needed for the identification of minimal NGS panel content in CLL and in other mature lymphoid neoplasms, outlining key genes directly involved in the diagnostic, prognostic and theranostic outputs in order to standardise patient testing and care.

PB1855: LACK OF MUTATIONS IN TNFRSF13B (TACI) IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

N. Giannakoulas 1,* , E. Georgiadi 1 , S. Makri 2,2 , G. Tsinti 2 , F. Kalala 2 , M. Speletas 2

1 Hematology Department, University Hospital of Larissa, university of thessaly; 2 Deparment of Immunolgy and Histocompatibility, Medical School, University of Thessaly, Larissa, Greece

Background: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world. Many molecular mechanisms and functionally relevant biological programs seem to be of importance in the pathogenesis of the disease, with emphasis on the antigenic drive within a specific microenvironment along with the apoptotic pathways failure that may have eventually a role in the survival of CLL cells. Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) is a major regulator in the immune responses and is encoded by TNFRSF13B gene. TACI is mainly expressed by mature B cells, inhibits B cell expansion and promotes the differentiation and survival of plasma cells. TNFRSF13B germline mutations contribute to the pathogenesis and phenotype of common variable immunodeficiency (CVID), a disease entity that usually complicated by B cell malignanices.

Aims: The purpose of the present study was to analyze the mutational status of the TNFRSF13B gene, since the majority of mutations are presented in these exons.

Methods: Fifty CLL patients (30 men and 20 women) were included in the study. The median age at diagnosis was 70 years. (range 40-80). Twenty-four patients (48%) were treated naïve, while the remaining twenty-six patients (52%) have already received at least one prior therapy. Four out of thirty one patients (13%) were at high risk group according to available molecular data. The molecular analysis of TNFRSF13B genes was performed by was PCR sequencing.

Results: There was no pathogenic mutation detected in the samples analyzed. We identified the common intronic polymorphism, g.28376A> C, c.445 + 25A> C, rs2274893 intron variant (30 patients in the 50 studied) and the silent mutation rs 35062843 (p.P97=, in 3 patients).

Comparing our results with data of the Exac database (displaying the prevalence of polymorphisms in European general population), no significant differences were observed (p> 0.05). Thereafter, we did not investigate our hypothesis in a bigger sample of CLL patients since no mutations were found in our initial sample.

Summary/Conclusion: Mutations of TNFRSF13B gene do not seem to participate in the pathogenesis and phenotype of CLL patients.

PB1856: DEMETHYLATING AGENTS AND HISTONE DEACETYLASE INHIBITORS AS NEW THERAPEUTIC STRATEGIES IN CHRONIC LYMPHOCYTIC LEUKEMIA

J. Jorge # 1,2,3,4,* , T. Gaspar # 1 , M. I. Costa 1 , B. Lapa 1,2,3 , R. Alves 1,2,3,4 , A. C. Gonçalves 1,2,3,4 , A. B. Sarmento-Ribeiro 1,2,3,4,5

1 Laboratory of Oncobiology and Hematology; 2 Coimbra Institute for Clinical and Biomedical Research (iCBR)—Group of Environmental Genetics of Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra; 3 Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra; 4 Clinical Academic Center of Coimbra (CACC); 5 Clinical Hematology Service, Centro Hospitalar e Universitário de Coimbra (CHUC), Coimbra, Portugal

Background: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is characterized by the accumulation of long-lived resting clonal B lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. This disease has a notable heterogeneous clinical course, with different prognosis and distinct treatment approaches. However, the exact etiology is still unknown. From global hypomethylation and hypermethylation of single-gene promoters, to altered regulation of histone modifying enzymes, epigenetic dysregulation plays an important role in CLL development and progression. Due to reversible nature of epigenetic alteration, modulation of these mechanisms might constitute a successful therapeutic application in CLL.

Aims: With this work we intended to evaluate the therapeutic potential of four epigenetic modulators, two demethylating agents (DNMTis), azacytidine (AZA) and decitabine (DAC), and two histone deacetylase inhibitors (HDACis), vorinostat (SAHA) and Panobinostat (PAN), in monotherapy and in combination, in an in vitro model of CLL.

Methods: To this propose, the CLL cell line HG-3 was incubated in the absence and presence of epigenetic modulators, in monotherapy and in combination (0.1µM AZA+0.1µM SAHA, 0.1µM AZA+0.1nM PAN, 0.5µM DAC+0.1µM SAHA and 0.5µM DAC+0.1nM PAN) for 72h. Metabolic activity was evaluated by resazurin assay. Cell death was assessed by flow cytometry (FC), using the Annexin V and 7-AAD double staining, and by optic microscopy using May-Grünwald-Giemsa staining. Cell cycle analysis was evaluated by FC using a PI/RNAse solution. The statistical analysis was performed, considering a significance level of 95%.

Results: Our results showed a decreased metabolic activity induced by all the epigenetic modulators in a time- and dose-dependent manner. After 48h, the IC 50 was 2.1μM for AZA, 2.9μM for DAC, 0.5μM for SAHA and 5.5nM for PAN. The metabolic activity decrease was even more relevant in the combination scheme of small doses of DNMTis plus HDACis (0.1µM AZA+0.1µM SAHA, 0.1µM AZA+0.1nM PAN, 0.5µM DAC+0.1µM SAHA and 0.5µM DAC+0.1nM PAN), reaching a 75% decrease in metabolic activity. DNMTis induced a cytotoxic effect mediated by apoptosis, confirmed by morphology analysis, namely the presence of apoptotic blebbings. When in combination with HDACis, DAC induced a higher percentage of apoptotic cells comparing to combinations with AZA. All epigenetic modulators, excepting PAN, induced a cytostatic effect, with cell cycle arrest in G 0 /G 1 phase. Therapeutic combinations also induced cell cycle arrest in G 0 /G 1 phase, with exception to AZA+SAHA.

Summary/Conclusion: These results suggest that epigenetic modulators may constitute a new therapeutic strategy for chronic lymphocytic leukemia treatment. This is even more relevant when combining DNA methyltransferase inhibitors with histone deacetylase inhibitors. Studies including primary cell culture from CLL patients should be performed to confirm these results.

# equally contributing authors.

PB1857: NRF2 MODULATORS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: A POSSIBLE NEW THERAPEUTIC APPROACH

J. P. Carda # 1,2,3,4,5,* , R. Gomes # 1 , J. Jorge 1,2,3,4 , A. C. Gonçalves 1,2,3,4 , B. Marques 1,2,3,5,6 , R. Alves 1,2,3,4 , A. B. Sarmento-Ribeiro 1,2,3,4,5

1 Laboratory of Oncobiology and Hematology; 2 Coimbra Institute for Clinical and Biomedical Research (iCBR)—Group of Environmental Genetics of Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra; 3 Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra; 4 Clinical Academic Center of Coimbra (CACC); 5 Clinical Hematology Service, Centro Hospitalar e Universitário de Coimbra (CHUC); 6 Center for Innovative Biomedicine and Biotechnology (CIBB), Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal

Background: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of resting clonal B lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. This disease has a notable heterogeneous clinical course, with different prognosis and distinct treatment needs. Oxidative stress (OS) is one of the mechanisms important in CLL development and progression. OS state consists in an unbalance between the production of reactive oxygen species (ROS) and the ability to neutralize them by antioxidant defenses. The nuclear factor erythroid 2–related factor 2, NRF2, is a transcription factor that regulates the expression of several antioxidants and detoxification systems and works as a protective mechanism in normal cells against malignant transformation. However, during the tumorigenic process, NRF2 protects tumor cells from OS effects compromising the therapeutic efficacy.

Aims: The study aims to evaluate the therapeutic potential of two NRF2 modulators, Brusatol and ML385, in B cells from CLL patients.

Methods: Peripheral blood mononuclear cells (PBMCs) from 13 CLL patients (stage initial, Binet A/B, n=8; advanced stage, Binet C, n=5) were isolated by density gradient using Ficoll-Paque Premium. PBMCs where immediately incubated in absence and presence of Brusatol and ML385. The cytotoxic effect was evaluated 24h and 48h after incubation by fluorometric microculture cytotoxicity assay. After 24h incubation with Brusatol (100nM) and ML385 (100μM) cells were stained with anti-CD5-PerCP, anti-CD19-APC and Annexin V, to assess cell death by flow cytometry. The statistical analysis was carried out in the total CLL patient group and in risk groups. The results were considered statistically significant when p<0.05.

Results: CLL patients presented 93,3% (±3.7%) of neoplastic B cells (CD5+/CD19+), 3,7% (±3,0%) of normal B cells (CD5-/CD19+) and 2,7% (±1,2%) of normal T cells (CD5+/CD19-), with no statistical differences regarding staging groups. Brusatol induced a decreased survival rate in CLL cells comparing to ML385, being this effect dependent on the dose and disease stage. Brusatol induced a higher reduction of cell viability in PBMCs from advanced disease patients (Binet C). ML385 did not induced a significant decrease in cell survival, nevertheless its effect is more marked in advanced stage patients at 48h post incubation. IC 50 was only achieved at 48h in advanced stage patients and corresponds to 65nM for Brusatol and to 114µM for ML385. NRF2 modulators induced a cytotoxic effect mainly mediated by apoptosis. Cytotoxicity induced by both drugs was more evident in neoplastic B cells, with no significant cytotoxicity in normal B and T cells. In PBMCs incubated with Brusatol an increase of neoplastic B cells in apoptosis about 68% was observed (Brusatol: 91,9% ± 7,1%; Control: 23,7% ± 4,3%), and of 34% in cells incubated with ML385 (ML385: 57,9% ± 10,2%; Control: 23,7% ± 4,3%). No differences of apoptotic cells were observed in advanced stage groups.

Summary/Conclusion: Our results showed that neoplastic cells from CLL patients were sensitive to NRF2 modulators, suggesting that these drugs may constitute a new therapeutic approach in CLL patients, particularly in CLL patients in advanced stage. We also demonstrated the low cytotoxic effect of these modulators in normal lymphocytes and its higher efficacy in neoplastic cells from advanced stage patients. Altogether, these results show that these NRF2 modulators have an effect directed to the neoplastic cell and suggest a possible involvement of NRF2 on CLL development and progression.

# equally contributing authors.

PB1858: INFLUENCE OF INFLAMMATION ON ANGIOGENIC FACTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA

O. Mitrović Ajtić 1,* , T. Subotički 1 , M. Diklić 1 , D. Đikić 1 , M. Vukotić 1 , T. Dragojević 1 , E. Živković 1 , D. Antić 2 , V. Čokić 1

1 Department for Molecular Oncology, Institute for medical research, National Institute of Republic of Serbia, Belgrade, Serbia; 2 Clinic of Hematology, University Clinical Center of Serbia, Belgrade, Serbia

Background: Several studies have shown that angiogenic factors are increased in patients with chronic lymphocytic leukemia (CLL). B-CLL cells secrete high levels of major angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and hypoxia inducible factor 1 alpha (HIF1α). However, it remains unclear how these angiogenic factors are self-controlled during inflammation.

Aims: The aim of the study is to assess the role of inflammation in activation of endothelial nitric oxide synthase (eNOS), HIF1α and VEGF angiogenic factors via proliferation related signaling pathways and VEGF self-control by negative feedback.

Methods: We isolated mononuclear cells (MNC) and CD19 + cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory Interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-kβ (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR.

Results: The angiogenic factors eNOS, VEGF and HIF1α have significantly higher frequencies in MNC of CLL in comparison to healthy controls (p<0.001), while they were reduced in CD19 + immunoreactive cells compared to MNC. IL-6 increased the quantity of HIF1α (p<0.05), while IL-6 increased the number of VEGF immunoreactive cells in the presence of JSH23 (p<0.01). HIF1α gene expression was significantly increased by VEGF (p<0.05), while eNOS gene expression was significantly decreased after VEGF and/or IL6 treatment (p<0.01) in MNC of CLL. VEGF stimulation of eNOS protein expression was prevented by inhibition of JAK1/2 and mTOR signaling in MNC of CLL (p<0.05). Immunocytochemistry revealed that VEGF significantly (p<0.001) increased the number of HIF1α positive MNC of CLL. Treatment with RUXO, Ly294002 and Rapamycin significantly decreased the number of HIF1α and eNOS immunoreactive cells compared to control and VEGF treated MNC of CLL (p<0.001). VEGF stimulated activation of SMAD (p<0.05) and STAT5 (p<0.01) signaling, prevented by Ruxolitinib and Rapamycin (p<0.01) in MNC of CLL. In addition, VEGF activation of STAT5 and SMAD signaling were prevented by Ly294002 and LDN-193189, respectively (p<0.01).

Summary/Conclusion: We demonstrated increased level of angiogenic factors in CLL, while pro-inflammatory IL-6 and angiogenic VEGF preferentially stimulated HIF1α. Further studies with clinical data of CLL patients will indicate their potential role in diagnosis, progression, and response to therapy.

PB1859: DEFINING T-CELL POPULATIONS IN CHRONIC LYMPHOCYTIC LEUKAEMIA

A. Mora 1,2,3,* , P. Jara 1,2,3 , M. Argüello 1,2,3 , N. Albiol 3 , J. Sierra 1,2,3 , M. E. Moga 4 , C. Moreno 1,2,3

1 Laboratory of Oncology/Hematology and Transplantation, Biomedical Research Institute Sant Pau-UAB; 2 José Carreras Leukemia Research Institute (IIB-Sant Pau); 3 Department of Hematology; 4 Servei Immunologia, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

Background: Chronic Lymphocytic Leukaemia (CLL) is a B-cell lymphoproliferative disorder in which the survival of leukemic cells highly depends on the tumour microenvironment. Indeed, abnormalities in the T-cell compartment have been described many years ago although their role role in supporting or controlling leukemic cells is still unclear. More recently, some studies suggested that pathway inhibitors including BTKi (i.e., ibrutinib) may act as immunomodulatory drugs by restoring at least in part the T-cell compartment and other immune circulating cells.

Aims: We aimed to further characterize the differentiation and maturation status and in vitro activation capability of T-cells at different stages of CLL (stable and progressive) and to analyze the changes induced on these cells by the BTKi Ibrutinib given as single agent in patients who were in clinical response.

Methods: T-cells populations from whole blood samples were analyzed by multicolour flow-cytometry in patients with stable CLL (n=10), progressive CLL (n=10) and patients in clinical response under Ibrutinib treatment (n=10) [(median time on treatment 2.06 years (0.69-4.37)]. Healthy donors’ values were used as reference. We further analysed the immunological synapses by coculturing in vitro autologous T-cells and CLL B-cells after stimulation, assessed by flow cytometry and Western Blot assays (CD3e and Granzyme B).

Results: We observed an increase of CD4 + :CD8 + ratio with a predominance of Th1 cells, decreased of Tregs and the expansion of effector memory CD8 + T-cells in stable and progressive CLL Furthermore, the terminally differentiated effector memory CD8 + T-cells re-expressing CD45RA (T EMRA ) were significantly reduced in progressive CLL compared to stable CLL (5.60% vs.12.30%, p≤0.05). In sample patients under ibrutinib, however, CD4 + :CD8 + ratio decreased and Th1:Th2 ratio increased due to Th1 polarization. In addition, the T EMRA cell population were normalized.

We further observed that under ibrutinib the percentage of expression of PD1 significantly decreased mainly in CD4 + T-cells as compared to that seen in progressive CLL (Figure 1A). Interestingly, this observation was correlated with the fact that synapse capability was only partially recovered in CD4 + T-cells but not in CD8 + T-cells (Figure 1B). These changes resulted in an effective activation of TCR with an increased expression of CD3e and Granzyme B.

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Summary/Conclusion: In conclusion, our results confirm that in CLL there is an imbalance of T-cell subpopulations (i.e CD4 + :CD8 + ratio, Tregs, T EMRA, ) and an anergic status which are more evident when the disease progresses. Treatment with ibrutinib may overcome T-cell dysfunction by not only controlling leukemic cells but also promoting antitumoral T-cell responses mediated by promoting Th1 polarization, restoring CD4 + : B-cell interactions, and inducing CD8 + T-cells cytotoxicity.

PB1860: CLONALITY RELATEDNESS AND MOLECULAR CHARACTERISTICS OF RICHTER SYNDROME

Y. Sha 1,2,* , R. Jiang 1,2 , Y. Miao 1,2 , T. Qiu 1,2 , S. Qin 1,2 , J. Qiu 2 , H. Mi 2 , W. Wu 1,2 , C. Qiao 1,2 , Y. Wu 1,2 , Y. Xia 1,2 , L. Wang 1,2 , L. Fan 1,2 , W. Xu 1,2 , J. Li 1,2 , H. Zhu 1,2

1 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University; 2 Department of Hematology, Pukou CLL Center, Pukou division of Jiangsu Province Hospital, Nanjing, China

Background: Richter Syndrome (RS) is defined as the development of an aggressive lymphoma from chronic lymphocytic leukaemia/Small lymphocytic leukemia (CLL/SLL). RS clonally derived from CLL and RS unrelated to the original CLL display distinct pathological and biological entities. Clonally related RS DLBCL patients have extremely pooor prognosis even in the era of novel agents. Therefore, determination of clonality of RS to the underlying CLL is critical for proper management of RS.

Aims: To explore the clonality relatedness, clinical and molecular characteristics of patients with RS.

Methods: This study retrospectively analyzed 18 RS patients diagnosed in the First Affiliated Hospital of Nanjing Medical University (Pukou CLL center) from January 2019 to December 2021. The immunoglobin heavy variable (IGHV) gene usage and IGHV-D-J rearrangement pattern of diagnosed CLL/SLL and transformed diffuse large B-cell lymphoma (DLBCL) were compared in order to determine the clonality relatedness. Clinical and molecular characteristics of patients when newly diagnosed CLL/SLL and transformed DLBCL were collected to explore the risk factors of RS.

Results: The median age of RS was 56.5 (41-75) years old. 17 patients transformed to DLBCL and 1 transformed to Hodgkin lymphoma (HL). Among 17 patients transformed to DLBCL, 15 were clonality related to CLL/SLL while 2 showed clonality unrelated to CLL/SLL. Next-generation sequencing (NGS) analysis of 11 paired initially diagnosed treatment-naïve CLL/SLL and RS DLBCL found that, EGR2、TP53 and NOTCH1 were among the most frequently mutated genes both in treatment-naïve CLL/SLL and in RS DLBCL. The median sample tumor mutational burden (TMB) were 2-hit in treatment-naïve CLL/SLL and 3-hit in RS DLBCL. Certain mutations were acquired or lost during Richter transformation in some patients, indicating clonal evolution. Among 10 patients prior exposure to BTK inhibitors before RS, 4 patients acquired BTK mutation. Above mentioned mutations might be the high risk factors of transformation; Moreover, TP53 and EGR2 mutation in RS DLBCL tissue might be associated with poor prognosis after Richter transformation even in patients treated with the combination of novel drugs.

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Summary/Conclusion: This study is the first one in China who introduce the methods to analyze the clonality relatedness between RS DLBCL and initial CLL/SLL. Among 17 RS DLBCL patients diagnosed in our center, 15 were clonality relatedness while 2 were clonality unrelatedness. We recommend all qualified centers to evaluated clonality relatedness of RS DLBCL patients. The mutational landscape between initially diagnosed treatment-naïve CLL/SLL and transformed DLBCL showed certain heterogeneity. The molecular mechanism underlying RS remains to be explored further.

PB1861: ACALABRUTINIB IN THE TREATMENT OF RICHTER SYNDROME

T. Byalik 1,* , N. Kokosadze 2 , E. Paramonova 3 , D. Semichev 3 , ?. Odzharova, A. Semenova 3 , G. Tumyan 3 , E. Osmanov 3 , T. Byalik 1

1 Federal State Autonomous Educational Institution of Higher Education I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University); 2 pathomorphology; 3 oncohematology; 4 radiology, «N.N. Blokhin Medical Research Center of Oncology», Moscow, Russia

Background: Richter syndrome is a transformation of chronic lymphocytic leukemia into DLBCL, occurring in about 2-15% of patients (pts) with CLL. These patients have a very poor prognosis with an unsatisfactory response to immunochemotherapy (R-XT) and a short overall survival. B-cell receptor signaling through Bruton tyrosine kinase (BTK) is one of the essential growth pathways for CLL. Acalabrutinib (highly selective BTK inhibitor) has shown significant efficacy in relapsed/refractory and untreated CLL.

Aims: improving the results of treatment of patients with Richter syndrome

Methods: we reported 4 cases of Richter syndrome treated with acalabrutinib + R-СТ.

Results: Table 1 shows the characteristics of 4 CLL pts who developed biopsy-proven DLBCL. The median time to transformation from CLL was 40,75 months (range 4-66). Clonality was not available testing in any pts. At the time of transformation, 2 pts were initially treated with R-CHOP with a suboptimal response, then acalabrutinib was added, 1 pt received 6 cycles of R-CHOP+ acalabrutinib and 1pt - 5 cycles R-CVP + acalabrutinib. The overall response (OR) was 75%:1 pt had a CR and 2 pts - PR. The pts who achieved OR are currently receiving acalabrutinib, 1 pt had progression of DLBCL after 15 months of acalabrutinib therapy. Acalabrutinib was well-tolerated; no patient required discontinuation as a result of adverse events.

At the time of CLL diagnosis (table1)

At the time of DLBCL diagnosis (Table 1- continuation)

Summary/Conclusion: our experience with these patients suggests that acalabrutinib in combination with immunochemotherapy has potential as a novel therapeutic approach for patients with Richter syndrome.

PB1862: BRUIN CLL-313: A PHASE 3 OPEN-LABEL, RANDOMIZED STUDY OF PIRTOBRUTINIB VS BENDAMUSTINE+RITUXIMAB IN UNTREATED PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTIC LYMPHOMA (TRIAL IN PROGRESS)

W. Jurczak 1 , C. Dartigeas 2 , M. Coscia 3 , P. Ganly 4 , G. Al-Jazayrly 5 , C. Wang 6 , K. Bao 7 , C. C. Leow 7 , S. Shahda 7 , P. L. Zinzani 8,*

1 Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 2 Centre Hospitalier Universitaire de Tours, Hôpital Bretonneau, Tours, France; 3 Molecular Biotechnology and Health Sciences, Division of Hematology, A.O.U. Città della Salute e della Scienza di Torino and Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy; 4 Christchurch Hospital, Christchurch; 5 Hollywood Presbyterian Medical Tower, Los Angeles; 6 Elli Lily and Company, Indianapolis; 7 Loxo Oncology at Lilly, Stamford, United States of America; 8 Institute of Hematology “Seràgnoli” University of Bologna, Bologna, Italy

Background: Covalent Bruton’s Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but these treatments are not curative and the majority of patients will require additional treatment. Covalent BTKi share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage, for example in rapidly proliferating tumors with high BTK protein turnover such as accelerating CLL/SLL, ultimately manifesting as acquired resistance in some patients.

Aims: To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. In a phase 1/2 BRUIN study, pirtobrutinib achieved pharmacokinetic exposures that exceeded its BTK IC96 at trough, was well tolerated, and demonstrated promising efficacy in CLL/SLL patients regardless of prior therapy, number of prior lines of therapy, or BTK C481 mutation status.

Methods: BRUIN CLL-313 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus bendamustine plus rituximab (BR) in treatment naïve CLL/SLL patients with retained 17p. Approximately 250 patients will be randomized 1:1. Randomization will be stratified by IGHV mutation status (mutated vs unmutated), and Rai stage (low/intermediate vs high). Patients in the BR arm are eligible to crossover to pirtobrutinib monotherapy if they experience progressive disease per iwCLL 2018 and confirmed by an independent review committee (IRC).

Eligible patients are adults with confirmed diagnosis of CLL/SLL and who require therapy per iwCLL 2018 criteria. Key exclusion criteria include CNS involvement by CLL/SLL, Richter transformation to DLBCL, prolymphocytic leukemia or Hodgkin lymphoma any time pre-enrollment, presence of 17p deletion, prior systemic therapy for CLL/SLL, and significant cardiovascular disease.

The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an independent review committee. Secondary endpoints include investigator-assessed PFS, overall survival (OS), overall response rate (ORR), duration of response (DoR), safety and tolerability, and patient reported outcomes. The global study is currently enrolling patients.

Results: This study is a Trial in Progress. The results will be presented at a later date.

Summary/Conclusion: This study is a Trial in Progress. The conclusions will be presented at a later date.

PB1863: BRUIN CLL-322: A PHASE 3 OPEN-LABEL, RANDOMIZED STUDY OF FIXED DURATION PIRTOBRUTINIB+VENETOCLAX AND RITUXIMAB VS VENETOCLAX AND RITUXIMAB IN PREVIOUSLY TREATED CLL/SLL (TRIAL IN PROGRESS)

A. R. Mato 1 , W. G. Wierda 2 , J. M. Pagel 3 , M. S. Davids 4 , P. L. Zinzani 5 , Y. Lu 6 , H. Liu 3 , S. Shahda 3 , C. C. Leow 3 , C. S. Tam 7 , J. A. Woyach 8 , T. A. Eyre 9,*

1 Memorial Sloan Kettering Cancer Center, New York; 2 University of Texas MD Anderson Cancer Center, Houston; 3 Loxo Oncology at Lilly, Stamford; 4 Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States of America; 5 Institute of Hematology “Seràgnoli”, University of Bologna, Bologna, Italy; 6 Elli Lily and Company, Indianapolis, United States of America; 7 Peter MacCallum Cancer Center, Royal Melbourne Hospital, and University of Melbourne, Melbourne, Australia; 8 The Ohio State University Comprehensive Cancer Center, Columbus, United States of America; 9 Oxford University Hospitals NHS Foundation Trust, Churchill Cancer Center, Oxford, United Kingdom

Background: Covalent Bruton’s Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but these treatments are not curative and the majority of patients will require additional treatment. The MURANO study established the time-limited combination of 2 years venetoclax plus rituximab as a clinically important regimen for patients with R/R CLL/SLL. However, that trial almost exclusively enrolled patients who were never treated with a covalent BTKi, a population less relevant in the context of today’s standard of care.

Aims: Pirtobrutinib is a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency. In a phase 1/2 BRUIN trial, pirtobrutinib achieved pharmacokinetic exposures that exceeded its BTK IC96 at trough, was well tolerated, and demonstrated promising efficacy in CLL/SLL patients regardless of prior therapy, number of prior lines of therapy, or BTK C481 mutation status. Therefore, adding fixed duration pirtobrutinib to the time-limited MURANO regimen may allow for even deeper and more prolonged disease control, and generate a clinically relevant dataset in a BTK-pretreated CLL/SLL population.

Methods: BRUIN CLL-322 is a randomized, open-label, global phase 3 study comparing fixed duration pirtobrutinib plus venetoclax and rituximab (PVR) versus venetoclax and rituximab (VR) in patients with CLL/SLL who have received prior therapy. To ensure relevance in the modern therapy context, a minimum of 80% of patients must have had a prior covalent BTKi. Approximately 600 patients will be randomized 1:1. Randomization will be stratified by 17p deletion (yes/no) and prior BTKi experience (discontinuation due to progressive disease vs due to other reasons vs no prior BTKi exposure).

Eligible patients are adults with a diagnosis of CLL/SLL and requirement for therapy per iwCLL 2018 criteria who have received prior therapy that may or may not include a covalent BTKi. Unlimited number of lines of prior therapy are allowed. Key exclusion criteria include CNS involvement by CLL/SLL, Richter transformation at any time pre-enrollment, history of allogeneic stem cell transplant (SCT) or autologous SCT or chimeric antigen receptor (CAR) T-cell therapy within 60 days and prior therapy with a BCL2 inhibitor or non-covalent BTKi.

The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an independent review committee. Secondary endpoints include overall response rate (ORR), overall survival (OS), time to next treatment (TTNT), event-free survival (EFS), safety and tolerability, and patient-reported outcomes. This global study is currently enrolling patients ( NCT04965493 ).

Results: This study is a Trial in Progress. The results will be presented at a later date.

Summary/Conclusion: This study is a Trial in Progress. The conclusions will be presented at a later date.

PB1864: BRUIN CLL-321: A PHASE 3 OPEN-LABEL, RANDOMIZED STUDY OF PIRTOBRUTINIB VS INVESTIGATOR’S CHOICE OF IDELALISIB+RITUXIMAB OR BENDAMUSTINE+RITUXIMAB IN BTKI PRETREATED CLL/SLL (TRIAL IN PROGRESS)

J. P. Sharman 1 , W. Jurczak 2,* , C. C. Coombs 3 , M. Hill 4 , D. Wang 4 , N. C. Ku 4 , A. Guntur 4 , S. Shahda 4 , C. C. Leow 4 , P. Ghia 5 , A. R. Mato 6

1 Willamette Valley Cancer Institute and Research Center, US Oncology Research, Eugene, United States of America; 2 Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland; 3 University of North Carolina at Chapel Hill, Chapel Hill; 4 Loxo Oncology at Lilly, Stamford, United States of America; 5 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 6 Memorial Sloan Kettering Cancer Center, New York, United States of America

Background: Covalent Bruton Tyrosine Kinase (BTK) inhibitors (BTKi) have transformed the management of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but these treatments are not curative and many patients will require additional treatment. Covalent BTKi share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that collectively may lead to suboptimal BTK target coverage especially in rapidly proliferating tumors with high BTK protein turnover such as accelerating CLL/SLL, ultimately manifesting as acquired resistance in some patients.

Aims: To address these limitations, pirtobrutinib, a highly selective, non-covalent BTKi that inhibits both wild type (WT) and C481-mutated BTK with equal low nM potency was developed. In the phase 1/2 BRUIN study, pirtobrutinib achieved pharmacokinetic exposures that exceeded its BTK IC96 at trough, was well tolerated and demonstrated promising efficacy in CLL/SLL patients regardless of prior therapy, number of prior lines of therapy, or BTK C481 mutation status. The purpose of this randomized phase 3 study is to demonstrate the superiority of continued BTK pathway inhibition with pirtobrutinib compared to other available therapies in patients with BTKi-treated CLL/SLL.

Methods: BRUIN CLL-321 is a randomized, open-label, global phase 3 study comparing pirtobrutinib monotherapy versus investigator’s choice of idelalisib plus rituximab or bendamustine plus rituximab in patients with CLL/SLL who have been treated with a prior covalent BTKi. Prior therapy with venetoclax is permitted. Approximately 250 patients will be randomized 1:1. Randomization will be stratified by 17p deletion (yes/no) and prior venetoclax (yes/no). Patients receiving investigator’s choice are eligible to crossover to pirtobrutinib monotherapy if they experience progressive disease per iwCLL 2018 (determined by IRC).

Eligible patients are adults aged ≥18 years with a diagnosis of CLL/SLL who require therapy per iwCLL 2018 criteria and who have received prior covalent BTKi. Unlimited number of lines of prior therapy are allowed. Key exclusion criteria include CNS involvement by CLL/SLL, Richter transformation any time pre-enrollment, a major bleeding event on prior covalent BTKi and history of allogeneic stem cell transplant (SCT) or autologous SCT or chimeric antigen receptor (CAR) T-cell therapy within 60 days of randomization.

The primary endpoint is progression-free survival (PFS) per iwCLL assessed by an independent review committee. Secondary endpoints include overall survival (OS), overall response rate (ORR), duration of response (DoR), safety and tolerability, and patient reported outcomes. The global study is currently enrolling patients ( NCT04666038 ).

Results: This study is a Trial in Progress. The results will be presented at a later date.

Summary/Conclusion: This study is a Trial in Progress. The conclusions will be presented at a later date.

PB1865: SINGLE-COUNTRY, MULTICENTER, RETROSPECTIVE, OBSERVATIONAL, DESCRIPTIVE COHORT STUDY OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS TREATED WITH VENETOCLAX IN ARGENTINA (VENARG).

C. Pagano Vilar 1,* , M. A. Pavlovsky 2 , R. K. Tannuri 2 , A. Enrico Mattos 3 , M. Zerga 1 , L. Guanchiale 4 , R. Custidiano 5 , A. Miroli 6 , M. Higashigata 7 , F. Figueroa 8 , J. Pose Cabarcos 9 , J. Real 10 , L. Giordano 11 , M. Miodosky 1 , G. De Stefano 1 , A. Federico 12 , E. Klimovsky 12 , M. S. Cugliari 1

1 Hematology, Instituto de Oncología A. Roffo; 2 Hematology, FUNDALEU; 3 Hematology, Hospital Italiano de La Plata, Buenos Aires; 4 Hematology, Hospital Privado de Córdoba, Córdoba; 5 Hematology, Instituto Alexander Fleming; 6 Hematology, Complejo Médico Churruca-Visca y Policlínico del Docente., Buenos Aires; 7 Hematology, Sanatorio Simes. Sanatorio Nosiglia, Misiones; 8 Hematology, Hospital Pte Néstor Kirchner, Tucumán; 9 Hematology, Sanatorio Otamendi; 10 Hematology, CEIM Investigaciones Médicas, Buenos Aires; 11 Hematology, Hospital Luis Lagomaggiore, Mendoza; 12 K&H Latam Consulting, Buenos Aires, Argentina

Background: Venetoclax (VEN), an oral BCL-2 inhibitor, has shown efficacy and good tolerability in pivotal clinical trials for patients (pt) with Chronic Lymphocytic Leukemia (CLL). However, real world data is limited and no local nor regional information from Argentina has been reported.

Aims: To describe the epidemiological characteristics, effectiveness of therapy, and safety and accessibility outcomes in CLL pt treated with VEN in the real-world setting in Argentina.

Methods: Study including pt ≥18 years old with CLL according to the iwCLL criteria treated with VEN as per label, in monotherapy or in combination to Anti CD20 prior to 31 March 2021. Descriptive statistics and Kaplan Meier curves were utilized, using the Statistical Package for Social Sciences (SPSS).

Results: Fifty seven CLL pt treated with VEN from 13 sites were included, with a median age of 58 years (38-80) at diagnosis; 35% were female. At baseline, 94.5% (52/55) were ECOG 0-1, 43.8% (25/57) intermediate and 33.3% (19/57) high RAI stage. From 46 pt tested, 24 were found to have deletion 17 and 15/46 deletion 11q. Comorbidities were experienced by 38pt (66.7%) being high blood pressure the most frequently reported (32pt, 56.1%) and CIRS score was >6 in 27pt (47.4%). Twenty one pt were at high risk of tumor lysis syndrome (TLS) (36.8%).

VEN was received as monotherapy by 24pt (42.1%) and as first line therapy by 15pt (26.3%).

In this cohort 94,7% had health insurance coverage. As to accessibility, 11pt (19.3%) were supplied with drug after >2 months, including 10/54 covered with health insurance. Statistically significant differences were observed in access to the drug according to coverage (p:0.015).

With a median follow up of 18,3 months (4-39) ORR was 91.2% (CR 24.6% and PR 66.7%) as per iwCLL criteria (32/38 pt were not performed a bone-marrow procedure for response assessment).

No statistically differences were observed in terms of response rates between pt with VEN monotherapy vs VEN plus Anti CD20 (Chi2:2,737; p:0.434) either pt treated in first line vs subsequent line (Chi2:1.948; p:0.583).

Progression-free survival (PFS) reported was 91.2%, with 5pt presenting disease progression. Estimated PFS at 24 months was 90.8% (95% CI 82.0%-99.6%) (Figure). Overall survival (OS) was 89.5%, with 6 deaths (2 due to CLL progression) and estimated OS at 24 months was 90.5% (95% CI 81.0%-99.9%).

Related to VEN safety profile, 4 drug-related serious adverse events were observed (hospitalizations due to infections, pneumonia, perianal abscess, disseminated herpes zoster). Twenty grade 3 CTCAE (15pt) were reported, being leukopenia and neutropenia the most common.

TLS was reported in 2pt (1 high-risk pt with clinical and laboratory lysis/1 low-risk pt only lab tests). None of them result in therapy discontinuation. Mean duration of hospitalization during ramp-up was 5.4 days (range 1-11) in the 21pt with high risk of TLS and 4,8 days (range 0 – 16) for pt at intermediate risk (24p).

Nine pt (15.8%) discontinued treatment (2 due to safety reasons/4 due to progression); 9pt temporarily discontinued treatment. Dose was reduced due to safety reasons in 11pt.

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Summary/Conclusion: VENARG study included a cohort of unselected naïve and previously treated Argentinian CLL pt, receiving VEN in monotherapy or combination with Anti CD20. Despite the high percentage of pt with high cytogenetic risk and differences in drug access in this cohort, outcomes analyzed with the use of VEN in the real-world setting in Argentina suggest an effectiveness comparable to that reported in pivotal clinical trials and no unexpected adverse events.

PB1866: RESPONSE TO NOVEL ANTI CD20 ANTIBODY AND BCL2 INHIBITOR IN A PATIENT WITH CLL AND MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS ASSOCIATED WITH LEUKEMIC KIDNEY INFILTRATION AND REVIEW OF LITERATURE

J. G. Goanta 1,* , I. Rotaru 1 , C. I. Popa 1 , A. M. Patrascu 1

1 Hematology, University of Medicine and Pharmacy, Craiova, Romania

Background: Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder with mature B cell, characterized by a progressive accumulation of monoclonal B lymphocytes in the peripheral blood, bone marrow, lymphatic tissue and rarely in other organs. Although renal impairment occurs in approximately 80-90% of patients with CLL, nephrotic syndrome is rare and occurs in less than 2% of patients. In the literature this condition is reported in a maximum of 16 patients with CLL, and no cases described with Membranoproliferative glomerulonephritis (MPGN) treated with Obinutuzumab and Venetoclax. Renal failure due to leukemic infiltration in chronic lymphocytic leukemia is extremely rare condition.

Aims: To evaluate the response of renal impairment due to leukemic infiltration and due to MPGN as well as disease response, after specific treatment, compared with literature data.

Methods: We present a 58-year-old male diagnosed with B-CLL stage A (0) in November 2020, under hematologic regular monitoring with no specific therapy since November 2021. This year he presented with important lower and upper extremities edema, oliguria (1100ml / 24h), minor increase of lymph nodes and spleen; laboratory test showed: haemoglobin: 11.4 g / dl, white blood cells: 186.020 / mm3, platelet count 207.000 / mm3, urea: 126 mg / dl, creatinine: 1.94 mg / dl; urinary protein excretion: 10 g / 24 h, serum proteins: 4.7 g / dl, serum albumin: 1.9 g / dl. We contacted the Nephrology Department, where renal biopsy was performed with histopathological and immunohistochemical examination. Findings were consistent with MPGN secondary type and moderate involvement with leukemic cells CD20 +, CD5 +, CD23 +. Treatment with novel anti CD20 (cluster of differentiation) antibody Obinutuzumab and Venetoclax has been initiated with tumor lysis syndrome prophylaxis.

Results: After first cycle of Obinutuzumab and Venetoclax (OV) regimen, renal function was normal, proteinuria has been improved decreasing to 1g / 24h, the edema was remitted and the blood count returned to normal, white blood cell count was 4.200 / mm3. Until today the patient followed 4 cycles of OV with complete remission of CLL and complete response of his underlying MPGN and leukemic renal infiltration.

Summary/Conclusion: Of all the cases in the literature, our approach to this condition was different, choosing Obinutuzumab and Venetoclax therapy, obtaining the complete response after the first cycle.

PB1867: PURINE ANALOGS ON HAIRY CELL LEUKAEMIA TREATMENT

G. Gomes 1,* , I. Gonçalves 1 , L. Rodrigues 1 , F. Miranda 1 , F. Mousinho 1 , T. Mendes 1 , A. Jorge 1 , C. Claudino 1 , P. Lupi 1 , A. Reichert 1 , C. Afonso 1

1 Department of Hematology, Centro Hospitalar Universitário de Lisboa Ocidental, Lisboa, Portugal

Background: Hairy cell leukaemia (HCL) is a rare indolent neoplasm of small mature lymphoid cells. Most patients present with splenomegaly, cytopenias, characteristically monocytopenia, blood and marrow infiltration by neoplastic cells and infection susceptibility. Treatment with purine analogs is the first line standard of care with very good response rates. Cladribine (Cld) and Pentostatin (Pt) are described to induce similar response and relapse rates and safety profile. There continue to be though few therapeutic options after relapse.

Aims: The aim of the study was to perform a comparative analysis of the treatment outcomes with Cld and Pt in a group of patients with HCL.

Methods: A single-centre retrospective study of 22 patients was performed. Data was collected from clinical records.

Results: Our study included 22 patients, 73% (16) male, with a median age at diagnosis of 62 years [33-80] and with 86% (19) with an Eastern Cooperative Oncology Group (ECOG) of 1 or less at diagnosis. All patients started treatment after diagnosis. 64% (14) received Pt as first line treatment and 36% (8) received Cld. Overall response rate was obtained in 86% (12) of patients treated with Pt - 5 with complete remission, 7 with partial remission and 2 with no response. An overall response was obtained in 100% of patients receiving Cld as first line treatment, with all patients achieving complete remission. Relapse was observed in 41% (9) of all patients. The relapse rate was of 41% (5) in patients treated with Pt and 50% (4) in patients treated with Cld. Median time until treatment after first line was of 38 months [9-232] – 37 months in the group treated with Pt and 39 months in the group treated with Cld. From the relapsed patients 7 received Cld as second line treatment, 1 received Pt and 1 Pt plus rituximab. Overall response was observed in all retreated patients.

Summary/Conclusion: Our data confirms the well-recognised efficacy of purine analogs in the treatment of HCL in a real-world population of patients, presenting very good response rates both in first and subsequent treatment lines. Response rates were similar between Cld and Pt, as described in literature. However, Cld induced higher complete remission rates, although of similar duration, compared to Pt. Additional studies are needed for adequate stratification and identification of patients with higher risk of relapse in order to further improve response monitoring and treatment of these patients.

PB1868: EXPERIENCE IN THE TREATMENT OF HAIRY CELL LEUKEMIA IN KAZAKHSTAN.

N. Kemelbekov 1,* , S. Gabbasova 1 , Z. Dushimova 1 , R. Ramazanova 1 , Z. Amankulov 1 , B. Nassipov 1 , G. Sagyndikov 1 , A. Jazyltayeva 1 , A. Koshkarbayeva 1 , M. Donenbaeva 1

1 Hematology, kazakh Scientific reserch Institute of oncology and radiology, Almaty, Kazakhstan

Background: Hairy cell leukemia is a rare oncohematological disease that manifests itself in damage to the bone marrow and spleen. One of the characteristic changes in the bone marrow are large cells, a rounded nucleus and thin outgrowths - “hairs” of the cytoplasm. The standard of care are drugs: cladribine and purine analogues-pentostatin. The complexity of therapy lies in the high risk of infections due to prolonged cytopenia during therapy.

Aims: The aim of the study was to analyze the detection of hairy cell leukemia in Kazakhstan and the effectiveness of the cladribine regimen.

Methods: In Kazakhstan 30 patients were diagnosed over the last 5-year period. All patients underwent cytological, histological examination of the bone marrow, bone marrow immunophenotyping. Thirty patient s were treated with cladribine intravenous infusion 0.1 mg/kg per day for 7 days. The average age was 54.6years (from 29 to 80 years), men-10, women-20. With a median follow-up of 4 years, the complete response (CR) was -25 patients, progression of disease – 3 patients, two patients die from infection.

Results: Median duration of response was 38 months (range, 0.0+ to 52.0+). Median overall survival was not reached in all patients with CR. All patients had hematological complications in the form of grade 4 leukopenia - 30 patients, grade 4 thrombocytopenia - 28 patients, grade 3 thrombocytopenia – 2, patients grade 2 anemia - 9 patients. Emetic syndrome in the form of vomiting of the 3rd degree was in one patient, diarrhea of the 2nd degree in one patient. Eleven patients had febrile neutropenia. Two patients had a fatal outcome due to the development of pneumonia and subsequent sepsis.

Summary/Conclusion: Treatment of patients with hairy cell leukemia with a purine analogue showed high efficiency, but was accompanied by prolonged cytopenia and frequent secondary infection. It is necessary to consider the issue of vaccination of this category of patients with anti-pneumococcal vaccine and also to conduct continuous monitoring. Ten patients with CR were vaccinated against coronavirus infection.

PB1869: IGHV REPERTOIRE FEATURES IN KAZAKHSTAN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

A. Klodzinskiy 1 , L. Turgunova 2,3 , M. Butyugina 2,3,* , M. Kakhanova 2,3 , I. Pivovarova 3

1 Hematology Department, Hematology Center LLP, Nur-Sultan; 2 Department of Internal Diseases, Karaganda Medical University; 3 Hematology Department, Hematology Center LLP, Karaganda, Kazakhstan

Background: The immunoglobulin heavy chain gene (IgHV) mutation is one of the most reliable predictive biomarkers in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy. The incidence of CLL in Asia is about 10 times lower, than in Western countries and there are potential different IgHV genes mutations and stereotypy between Eastern and Western CLL. For instance, Chinese patients with CLL have higher percentage of mutated subgroup compared with European patients (55%) and different clinical presentation (Yi Xia et al., 2013; Marilisa Marinelli et al., 2016). The incidence of CLL in Kazakhstan is similar to that of the Western world, but to date nothing is known on the IgHV gene representation and mutations.

Aims: The aim of the present study was to evaluate IgHV Repertoire Features In Kazakhstan Chronic Lymphocytic Leukemia Patients.

Methods: The study included samples and medical records from 35 patients (19 male and 16 female) from the different regions of Kazakhstan. Participants’ ages ranged from 35 to 84 years (Median = 61). According to the European Research Initiative on CLL recommendations, IgHV mutational status was determined by amplifying the IgHV transcript by PCR, sequencing the gene through Sanger sequencing, and comparing the transcript to known germline genes available in immunoglobulin databases. We use the 2% cut-off to discriminate between mutated and unmutated rearrangements. Descriptive statistics for discrete variables included counts and frequency distributions.

Results: Patients were divided into the four prognostic groups proposed by the CLL-IPI as follows: 6 (17.2%) at low risk, 16 (45.7%) at intermediate risk, 10 (28.6%) at high risk, and 3 (8.6%) at very high risk. The median observation time was 2.5 years. Eleven patients (31.4%) have no indications for treatment. 21 (60%) patients were treated with chemoimmunotherapy as first line and only 3 (12.5%) patients with 17p deletion received Ibrutinib.

20 (57.1%) had unmutated IGHV genes (U-CLL), 13 (37,1%) patients had mutated genes (M-CLL) and 2 (5.7%) cases carried double in-frame rearrangements. Prevalence of U-CLL which is consistent with their poor clinical outcome was close to Italian population (51%) and almost two times higher than among the Chinese patients (34%). The most of patients in this study were Caucasian (29; 83%) and only 6 patients (17%) were identified as Asians. In U-CLL group the most common IgHV genes were IGHV1-69 (8; 40%), IgHV1-2 (5; 25%), IgHV3-15 and IgHV4 (2; 10%). IgHV1-69 gene in the vast majority of cases was expressed in an unmutated state. In M-CLL most common IgHV genes were IGHV4, IgHV3 and IgHV2 without predominance of one of the subgroups and there were not any cases with IgHV1-69. Gender ratio in this study was 1:1 among patients with IgHV1-69 and all of them were Caucasian. During the study 4 participants with U-CLL died and two of them had IgHV 1-69 rearrangement.

Summary/Conclusion: Hence, we conclude that Kazakhstan CLL patients show unique IgHV repertoire which is close to patients from western countries. Remarkably, IgHV1-69 was the most prevalent IgHV subgroup in Kazakhstan U-CLL patients (40%). IgHV1-69 gene is more associated with ethnicity, but not a gender.

PB1870: COVID - 19 IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: HOSPITAL MORALITY PREDICTORS

O. Kochneva 1,* , E. Baryakh 1 , E. Misyurina 1 , E. Zhelnova 1 , K. Yatskov 1 , Y. Polyakov 1 , K. Kaluzhskaya 1 , T. Chudnova 1 , T. Tolstykh 1 , D. Ivanova 1

1 Moscow City Clinical Hospital 52, Moscow, Russia

Background: Patients with chronic lymphocytic leukemia (CLL), as the disease progresses and the tumor load increases, are characterized by high susceptibility and severe course of viral infections. The depth of immune dysfunction can be influenced by factors such as age, comorbidity, disease status and previous treatment. The detection of SARS-COV2 in this cohort of patients made it possible to analyze the course of COVID-19, as well as to develop a strategy for the disease management of these patients.

Aims: To assess the risk factors for the severe course of COVID-19, as well as to identify predictors of hospital mortality.

Methods: 179 patients with verified CLL and COVID-19 were followed up by the hematology service of Moscow City Clinical Hospital No.52 (Moscow) from April 21, 2020 to November 31, 2021. Anthropometric, anamnestic, clinical and laboratory data were evaluated, treatment tactics of both CLL and COVID-19 were analyzed in 179 patients (69 women and 110 men, median age - 66 years). A statistical multivariate analysis of hospital mortality, factors of unfavorable prognosis of COVID-19 (complications, humoral response, hospital mortality) was carried out.

Results: Age was a significant adverse factor for men: the age of a man from 73 years and more, increases the risk of death of the patient by 3.0 times (p<0.05). For women, age was not a significant adverse factor. Assessment of the effect of comorbidity: patients with 6 or more comorbidity scores according to Charlson have 2.2 times greater risk of death; the presence of cardiovascular diseases increases the risk of death of the patient by 2.3 times. The presence of 1 or more lines of chemotherapy in the anamnesis increased the risk of hospital mortality by 1.5 times. Patients who had previously undergone chemotherapy were treated until the moment of discharge statistically significantly longer than patients who had not undergone antitumor therapy. Relapse/progression of the disease was a factor of unfavorable prognosis for the survival of patients with CLL (mortality was 47% compared to 14.7% in patients in remission). A significant unfavorable prognosis is also the Binnet stage C (p<0.05). A valid factor in the failure of COVID-19 (p<0.05) was ICU therapy and the development of secondary bacterial complications (the use of 2nd-line antibacterial therapy). The assessment of the humoral response in 59 patients shows the presence of an increase in IgG on the 10th-14th days of the disease in 44% of patients, among whom no deaths were detected, 1/3 were vaccinated patients. The absence of a humoral response was a statistically significant adverse outcome factor in 25% of patients (p<0.05).

Summary/Conclusion: Progression/recurrence of chronic lymphocytic leukemia is a factor in the severe course of COVID-19, and, as a result, increases the risk of hospital mortality. The absence of a humoral response determines the persistent course of COVID-19 and increases the risk of death by 25%. The study of the course of COVID-19 in patients with CLL allows us to identify key aspects affecting the choice of management tactics for this category of patients: additional booster vaccination (stimulation of the humoral response), early detection (screening) of SARS-COV2 for virus-neutralizing antibodies therapy, timely hospitalization of patients.

PB1871: PROGNOSTIC SIGNIFICANCE OF THE NOTCH1 GENE MUTATION AND OTHER LABORATORY FACTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA

D. Krauchanka 1,* , S. Arcady 2 , M. Viktor 2

1 Hematology; 2 RRC RM&HE, Gomel, Belarus

Background: An important stage in the management of patients with CLL is the prediction of the course of the disease in order to assess the need for an earlier onset of therapy and the use of alternative therapy on an individual basis, depending on the detectable prognostic markers.

Aims: To determine the interrelationship of clinical-laboratory (as well as molecular-genetic) prognostic markers with various courses of CLL.

Methods: We have examined 107 patients diagnosed with CLL in SI «RRC RM&HE» from 2017 to 2020. This cohort was divided into 2 groups: 1st group (49 patients) without signs of progression of CLL, and the 2nd group (58 patients) with clinical signs of disease progression after 3 years of observation. The median age in the 1st group was 61 years, in the 2nd - 62 years. The material for the study was venous blood. To determine the immunophenotype of tumor cells we used the flow cytofluorimeter FacsCanto II. Detection of β2-mg, TK and TPA was performed using the chemiluminescent analyzer. To determine the mutations of the NOTCH1 gene (34th exon), SSCP-PCR was used, followed by direct sequencing of DNA samples with conformational polymorphism. We used nonparametric statistics methods calculated in the Statistica 10.0. Statistically significant differences were considered for p<0.05.

Results: Patients in group 1 differed from patients in group 2 in levels of β2-mg-1.8 and 3.5 μg/L (p<0.001) and LDH-220 and 326 U/L (p<0.002). TK levels (p<0.001) and TPA (p<0.011) were also significantly higher in group 2 (7.7 and 49.8 IU/L) compared with patients in group 1 (4.2 and 34.3 IU/L). According to immunophenotyping data, statistically significant differences were obtained on the Zap70 marker: in the 1st group the median was 10.1%, in the 2nd group - 21.6%. As a result of molecular genetic analysis, mutations in the NOTCH1 gene were identified in 14 patients. It was found that patients without progression at the time of inclusion in the study in the presence of mutations in the NOTCH1 gene had a statistically significantly shorter progression-free survival (mean 33.4 months (95% CI [22.0-44.8]) compared with patients without mutations (59.2 months (95% CI [54.1–64.3]) (p=0.018), which indicates the association of NOTCH1 mutations with a poor prognosis for disease progression in patients with CLL.

Summary/Conclusion: It was found that markers such as LDH, β2-mg, TK, TPA, ZAP70 and NOTCH1 gene mutation have certain prognostic significance, which was confirmed by comparing their levels in patients with and without CLL progression after 3 years of observation. We can use NOTCH1 gene mutation as an additional factor in predicting the course of CLL. Knowledge of reliable prognostic markers may allow to initiate in-time treatment or to adjust therapy for patients with high-risk CLL with a probable increase in their life span.

PB1872: ANTIBODY RESPONSES AFTER COVID-19 VACCINATION IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKAEMIA, EITHER TREATED OR NAÏVE. A MONOCENTRIC EXPERIENCE.

I. Innocenti 1,* , G. Benintende 2 , L. Stirparo 2 , A. Tomasso 2 , A. Mosca 2 , A. Fresa 2 , E. Galli 1 , F. Autore 1 , L. Laurenti 3

1 Sezione di Ematologia, Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario A. Gemelli IRCCS; 2 Sezione di Ematologia, Dipartimento di Scienze Radiologiche ed Ematologiche, Università Cattolica del Sacro Cuore; 3 Sezione di Ematologia, Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy

Background: The efficacy of spike messenger RNA vaccines against SARS-COV2 in immunocompetent population is about of 94-95%, while in immunocompromised patients no precise value has been established. Most of patients with Chronic Lymphocytic Leukaemia (CLL) present a consistent immunological defect, which increases the risk of mortality and morbidity from infections, including SARS-COV2. Moreover, they have shown a highly heterogeneous response to vaccination, depending on many factors including baseline features, disease and treatment status, types of therapy, vaccine administered, and immunoglobulin level at time of vaccination.

Aims: We tried to evaluate the efficacy of vaccine, stratifying them according to treatment status (naïve, on treatment, off therapy) and of type of treatment, including chemo-immunotherapy (CIT), BTK or anti-Bcl2 inhibitors, monoclonal antibodies (MoAb).

Methods: This monocentric study included 100 CLL patients who received two/three dose of mRNA vaccine (BNT162b2 or mRNA-1273) between January 2021 and January 2022. Median age at time of vaccination was 69 years and 51% were males. 44% patiets presented unmutated IgHv, 7% del17p, 13% del11q. 61% patients suffered significant comorbidities as hypertension, diabetes, BPCO. 71% pts received at last one prior CLL therapy, including CIT or inhibitors; 44% were actively treated at the time of vaccination. CLL treatment was not held or modified at the time of vaccination. All patents were vaccinated: BNT162b2 was administered to 80%, mRNA-1273 to 14%, two BNT162b2 and mRNA-1273 booster to 6%. Baseline demographics, treatment history and laboratory parameters including lymphocyte count and IgG levels prior to first dose of COVID-19 vaccine were collected and patients were tested for SARS-COV2 anti-spike Ig at different times from vaccination (Table 1).

Results: No CLL patient experienced SARS-COV2 infection. Median IgG level at baseline was 733 mg/dL, 78% of patients had IgG levels above 500 mg/dL. At first serologic testing, at a median of 131 days after the 2 nd dose, 53% patients tested negative and 47% positive. Among patients who did not develop immunity, 58% were on treatment with CIT (26%) or inhibitors, alone (62%) or in association with MoAb (12%). Among patients who developed immunity, 38% were naïve while 30% were off therapy. All patients who received MoAb in the previous six months failed to develop immunity; among patients who received MoAb from more than 6 months, 55% did not develop immunity, while 45% did. At second serologic testing, at a median of 44 days after the booster, 80% patients tested positive, with a 67% rate of seroconversion, and 20% patients tested negative (Table 1).

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Summary/Conclusion: Our monocentric experience confirms a low humoral response in CLL, due to both disease and therapy related factors. Patients who, at the time of vaccination, were either naïve or off therapy yielded better results, probably due to a lower disease burden. The timing of MoAb administration strongly influences the response rate. Drugs that block the BTK receptors impair the humoral response of vaccination to any pathogen and behave accordingly for SARS-COV2. Data show that, in these patients, the third dose confers higher rates of response and a wider protective effect. In conclusion, vaccination is strongly recommended to all CLL patients. Due to the higher infectious risk, caused by immune dysregulation typical of CLL, it is important to evaluate the timing of vaccination and maintain safety measures at any time.

PB1873: EFFECTS OF LENALIDOMIDE MAINTENANCE IN CHRONIC LYMPHOCYTIC LEUKEMIA: A TRIAL-SEQUENTIAL ANALYSIS

C.-H. Lee 1,* , C.-L. Ho 2

1 Hematology and Oncology, Tri-Service General Hospital/National Defense Medical Center; 2 Hematology and Oncology, Tri-Service General Hospital, Neihu Dist, Taiwan

Background: Chronic lymphocytic leukemia (CLL) is the most common lymphoproliferative disease in adults. Despite durable responses to front-line therapy and sustained remission rates in patients with CLL, CLL is still incurable within standard therapy and relapse eventually. Although kinase inhibitors can improve outcomes for some patients with relapsed and refractory disease, not all patients have access to these novel drugs. Maintenance therapies aim to reach a deep remission and extend the period of disease quiescence. However, the efficacy and safety of lenalidomide as a maintenance therapy are inconsistent within individual studies and still under debate.

Aims: To assess the effect and safety of lenalidomide maintenance in patients with chronic lymphocytic leukemia.

Methods: Eligible randomized controlled trials published before January 2022 were retrieved from databases. Primary outcomes were progression free survival (PFS) and overall survival (OS). Trial sequential analysis was adopting to examine the power of analyses in primary outcomes. Secondary outcomes were grade 3-4 neutropenia, treatment discontinuation (TD), serious adverse events (SAE) and fatal adverse events (FAE). We calculated the hazard ratio (HR) and odds ratios (ORs) with 95% confidence intervals (CIs).

Results: Four articles, involving 1,118 patients, met the selection criteria. Compared to observation, lenalidomide maintenance was associated with a statistically significant effect in prolongation of PFS (HR, 0.43; 95% CI, 0.28–0.68; I 2 = 57%), and TSA confirmed the statistical power. Compared to observation, lenalidomide maintenance was no difference in OS (HR, 0.62; 95% CI, 0.29–1.30; I 2 = 52%), and TSA showed that the analysis would be inconclusive. In secondary outcomes, compared to observation, lenalidomide maintenance was associated with a higher proportion of SAE (OR 4.64; 95% CI 2.96–7.26; I 2 = 0%). There was no significant difference between lenalidomide maintenance therapy and observation with regard to grade 3-4 neutropenia (OR 2.30; 95% CI 0.84–6.28; I 2 = 81%), TD (OR 0.76; 95% CI 0.29–1.99; I 2 = 84%), and FAE (OR 0.86; 95% CI 0.28–2.63; I 2 = 0%).

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Summary/Conclusion: In our meta-analysis, lenalidomide maintenance therapy could be an effective treatment option for patients with CLL in prolongation PFS and relatively acceptable in safety concerns. Further studies would be warrant to justify the conclusion of OS.

PB1874: BTK GENE MUTATIONS IN RUSSIAN PATIENTS TREATED WITH IBRUTINIB: AS-PCR VERSUS NGS FOR ALLELIC LOAD ASSESSMENT

E. Likold 1,* , B. Biderman 1 , I. Fevraleva 1 , N. Severina 1 , E. Dmitrieva 2 , E. Nikitin 2 , V. Ptushkin 2 , A. Sudarikov 1

1 National Research Center for Hematology; 2 S.P.Botkins’s City Clinical Hospital, Moscow, Russia

Background: Ibrutinib is highly effective for CLL treatment. It inactivates Bruton’s tyrosine kinase (BTK) by forming a covalent bond in the enzyme catalytic domain at position C481. Ibrutinib resistance in 50-80% of cases is associated with the C481S mutation in exon 15 of the BTK gene (Ahn et al, 2017; Woyach et al, 2017), which corresponds to the c.1441T>A and c.1442G>C substitutions, still, other variants of gene mutations at this position are also reported. Monitoring allelic load of the BTK gene mutations conferring resistance to ibrutinib is extremely important for studying the mechanism of CLL progression during treatment with BTK inhibitors.

Aims: Development and validation of a real-time AS-PCR approach for assessing the relative allelic load of BTK gene mutations in Russian CLL patients treated with ibrutinib.

Methods: The study included DNA samples from 101 patients (pts) with CLL: 39 pts with disease progression on ibrutinib therapy (67% male, median age 64.5 years; 33% female, median age 64 years); 24 pts with progression after FCR or FCR-lite therapy (62% men, median age 62 years; 38% women, median age 67 years); 38 primary pts (76% men, median age 64 years; 24% women, median age 60 years). 89 pts with non-neoplastic hematological diseases were selected as control samples. All DNA samples were tested using a real-time AS-PCR with the following primers and probes: c.1441T (5’-CTGAGTACATGGCCAATGGCT-3’); c.1441T>A (5’-CTGAGTACATGGCCAATGGCA-3’); c.1442G (5’-CTGAGTACATGGCCAATGGCTG-3’); c.1442G>C (5’-CTGAGTACATGGCCAATGGCTC-3’); c.1442G>A (5’-TGAGTACATGGCCAATGGCAA-3’); c.1442G>T (5’-TGAGTACATGGCCAATGGGTT-3’); REV (5’-CTGCTACTTCCACCCCATCA-3’); Probe (5’-R&G-TGCTAGAGATGTGCAAGGATGTCTGTGAAG-RTQ1-3’). All DNA samples from pts treated with ibrutinib were analyzed by NGS on a MiSeq genetic analyzer (Illumina, USA).

Results: Initially, AS-PCR was performed only for the most common mutations c.1441T>A and c.1442G>C, however, after NGS of the BTK gene in pts with progression during ibrutinib therapy, other options were added to the test system. Using AS-PCR, out of 39 Russian pts with CLL progressing on the background of ibrutinib therapy, a single c.1442G>C mutation was detected in 19 (48.7%) pts (including 3 samples with a low mutant load); 2 (5.1%) had a single mutation c.1442G>T (C481F). In 1 (2.6%) patient, two mutations were detected simultaneously: c.1441T>A and c.1442G>C, and in another 1 (2.6%) patient, 3 mutations were detected simultaneously: c.1442G>C, c. 1442G>T (C481F) and c.1442G>A (C481Y). No mutations in the BTK gene were found in 16 (41%) Russian pts. Table 1 shows a good agreement between the results of NGS with AS-PCR when sequencing the BTK gene in this group of pts, both in identifying the type of mutation and in determining the relative mutational load. To determine the presence or absence of mutations in the studied samples, the amplification curves and Ct values of the normal and mutant alleles were compared. Thus, mutations in the BTK gene leading to an amino acid substitution at position C481 were found in 59% of Russian pts with CLL treated with ibrutinib. In groups of pts with CLL without prior treatment, after FCR/FCR-lite regimens, and in the control group of pts with non-neoplastic diseases, no mutations were found.

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Summary/Conclusion: AS-PCR protocol effective for the analysis of the BTK gene mutations leading to an amino acid substitution at position C481 has been developed and validated. Prospective studies of low burden BTK mutations in the pathogenesis of relapse in CLL patients treated with BTK inhibitors have been started using this approach.

PB1875: TREATMENT OF YOUNGER PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA IN TUNISIA: FCR STILL STANDARD FOR FIRST-LINE THERAPY

W. Miled 1,* , J. Dorra 1 , K. Rihab 1 , K. Kacem 1 , B. L. Raihane 1 , M. Raoudha 1 , M. Balkis 1

1 clinical hematology department, Aziza othmana hospital, Tunis, Tunisia

Background: Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of B-cell lymphocytes that typically express CD19+,CD5+,and CD23+.Fludarabine,cyclophosphamide and rituximab (FCR) is recommended as first-line therapy for fit patients who are younger than 65 years and have no 17p deletion/ TP53 mutation.

Aims: The aim of this study was to analyze the characteristics of CLL in younger patients treated with FCR.

Methods: We performed a retrospective study involving young patients with CLL (≤ 65 years old),in the clinical hematology department of Aziza Othmana Hospital,in Tunisia between 2013 and 2020. Patients were considered as fit when CIRS(cumulative illness rating skill) was inferior to six.

Results: Seventy-six patients were included(46 males and 30 females).The median age was 56.3 years with a sex ratio of1.53.Seventy-two patients were FIT for chemotherapy. 85.5% had more than one lymph nodes regions affected. Cytogenetics testing were requested for 41 patients of which 42.1% were FISH method. Only 6 patients had 17 p deletion. In the Binet staging system, 40.8% patients were stage A, 27.6% were B and 31.6 % were C. Thirty patients were on therapeutic abstention initially. Five patients received chlorambucil.10 had CLB-Rituximab and 21 had FCR.

Fifteen patients treated with FCR were in complete remission (71.4%). Four febrile neutropenia episodes happened, two of them was grade 4. Seven patients had neutropenia grade 3 during minimum one cycle however no infection has occurred. The event free survival was 33.3 months for patients who have been seen regularly. Thirty –nine patients were lost to follow –up; thus, we couldn’t evaluate the event or relapse free survival. Death rate was 7.89% (6 patients of which 3 received FCR).

Summary/Conclusion: FCR remains the first-line therapy for fit patients who are younger than 65 years and have no 17p deletion /mutation TP53 in Tunisia.Our patients have a good tolerance to the drugs and few infectious episodes have occurred.

PB1876: ACALABRUTINIB RELATED INFECTIOUS COMPLICATIONS-A SYSTEMATIC REVIEW AND META-ANALYSIS OF PHASE III RCT

K. Parmar 1,* , K. Thein 2 , L. Tijani 1 , S. Ball 3

1 Texas Tech University Health Sciences Center, lubbock; 2 OSHU, Oregon; 3 Moffitt Cancer Center, Tampa, United States of America

Background: Acalabrutinib is a new, selective, irreversible Bruton’s tyrosine kinase inhibitor (BTKi) approved for the treatment of adults with chronic lymphocytic leukemia (CLL). It has shown remarkable efficacy and significantly improved toxicity profile than ibrutinib (a first generation BTKi) in clinical studies. Patients with CLL have inherent immunodeficiency due to disease associated defects in innate and adaptive immunity. Although ibrutinib leads to an immune reconstitution, reports have suggested an increased risk of infections (particularly fungal) with ibrutinib in patients with B cell malignancies. However, the risk of infection with acalabrutinib is unknown in patients with CLL.

Aims: We conducted this systematic review and meta-analysis to estimate the risk of infections associated with acalabrutinib in patients with CLL.

Methods: A comprehensive literature search was performed in PubMed, EMBASE, Clinical trials.gov and meeting abstracts till January 31,2021. All randomized controlled trials comparing acalabrutinib-based regimens with a control group in patients with CLL, with infections reported as adverse effects were included. Pooled risk ratios (RR) with 95% confidence intervals (CI) were calculated using the Mantel-Haenszel method of the fixed-effect model. Heterogeneity of effect size was quantified using the I 2 statistic. Statistical analysis was performed using the Review Manager, version 5.3.

Results: Three phase III RCT with total of 1362 patients were included for our analysis (Figure). Byrd et al., compared acalabrutinib with ibrutinib. The ASCEND trial compared acalabrutinib with investigator’s choice chemotherapy, either idelalisib plus rituximab or bendamustine plus rituximab. In the ELEVATE -TN study the comparator arm included the combination of chlorambucil and obinutuzumab. Acalabrutinib was used at the dose of 100mg twice daily in all 3 studies. All but ELEVATE -TN study included patients previously treated with CLL. The upper respiratory tract infection (URTI) was the most reported type of infection across trials. The risk of total infection (CTCAE any grade) was significantly lower (pooled RR 0.92; 95%CI 0.85-0.99; P=0.03) in patients on acalabrutinib containing regimens; however, there was no difference between CTCAE grade 3+ infections between the two groups (Figure). On further analysis of types of infections, patients on acalabrutinib had a significantly less URTI than those in the control arm (pooled RR 0.76; 95% CI 0.61-0.96; P=0.02) (Figure). There was no difference in grade 3+ URTI, pneumonia (any grade), grade 3 pneumonia or sepsis between the two groups. Looking at infectious etiologies, patients on acalabrutinib containing regimen did not have an increased risk of fungal (pooled RR 0.76; 95% CI 0.32-1.81; P=0.53) or viral infections than other regimens. The rate of treatment discontinuation due to infections in the acalabrutinib group was significantly less compared to control (pooled RR 0.53; 95% CI 0.33-0.87; P=0.01). We noted a substantial heterogeneity across trials included in our analysis.

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Summary/Conclusion: Our meta-analysis demonstrated that patients on acalabrutinib-containing regimens experienced a significantly lower relative risk of infections compared to non-acalabrutinib-based therapies. There were significantly lower risks of URTI and infection related treatment discontinuations with acalabrutinib therapy in patients with CLL.

PB1877: ADJUSTING SURVIVAL DATA FOR TREATMENT CROSSOVER IN THE ELEVATE-TN TRIAL BY USING A HISTORICAL COHORT OF PATIENTS TREATED WITH CHEMOIMMUNOTHERAPY IN FRONT-LINE CHRONIC LYMPHOCYTIC LEUKEMIA

P. Gaitonde 1,* , B. Liljas 1 , B. Shaw 2 , P. Miranda 1

1 AstraZeneca, Gaithersburg, United States of America; 2 AstraZeneca, Cambridge, United Kingdom

Background: Acalabrutinib (A) is a next-generation Bruton tyrosine kinase inhibitor (BTKi) approved for chronic lymphocytic leukemia (CLL) treatment. ELEVATE-TN trial interim data (~47 months median follow-up [FU]) did not show survival benefit for A±obinutuzumab (O) versus chlorambucil+obinutuzumab (C+O) despite significantly superior progression-free survival (PFS). One explanation is C+O-to-A patient crossover (39%), which may have provided added overall survival (OS) benefit to the C+O arm.

Aims: We evaluated OS by adjusting for crossover using a historical C+O cohort.

Methods: The CLL11 trial demonstrated superior PFS and OS for C+O versus C and C+rituximab in first-line CLL prior to BTKi availability. Therefore, OS data for BTKi-naive C+O patients were digitized and compared with A±O from ELEVATE-TN. Individual patient datasets were used to plot Kaplan-Meier (KM) curves and calculate hazard ratios (HR) using Cox regression in R software. To validate our hypothesis (impact of crossovers on relative survival benefit), we compared 28-month FU data from ELEVATE-TN, where we observed lower OS HRs (non-significant) to CLL-11 data until that time period. The CLL11 and ELEVATE-TN trials enrolled patients with similar baseline characteristics; however, this analysis did not account for within-trial heterogeneity.

Results: There was a statistically significant reduction in risk of death with all 3 ELEVATE-TN treatments (47-month FU data) versus CLL-11 C+O: A+O versus C+O CLL11 (HR=0.23 [95% CI=0.12-0.42, p<0.001]); A versus C+O CLL11 (HR=0.43 [95% CI=0.27-0.70, p<0.001]); C+O versus C+O CLL11 (HR=0.44 [95% CI=0.27-0.71, p<0.001]). In the additional analysis with 28-month FU data, comparing ELEVATE-TN arms with C+O from CLL-11, KM curves for C+O overlapped (HR=0.79 [95% CI=0.42-1.47], p>0.05), indicating no difference. However, HRs for A+O versus C+O CLL11 (HR=0.39 [95% CI=0.18-0.84], p=0.02) and for A versus C+O CLL11 (HR=0.48 [95% CI=0.24-0.99], p<0.05) were statistically significant.

Summary/Conclusion: The non-statistically significant OS for A±O versus C+O was likely due to treatment crossover. Conducting a treatment-switching analyses was challenging due to immature OS data; however, this analysis supports the hypothesis that crossover to A bolstered ELEVATE-TN C+O OS.

PB1878: CROSS-TRIAL ANALYSIS OF FIXED-DURATION IBRUTINIB (I) PLUS VENETOCLAX (V) VS FLUDARABINE (F), CYCLOPHOSPHAMIDE (C), AND RITUXIMAB (R) AS FIRST-LINE TREATMENT FOR CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)

J. C. Barrientos 1,* , P. M. Barr 2 , A. R. Mato 3 , C. S. Tam 4 , P. Ghia 5 , C. Moreno 6 , N. E. Kay 7 , T. Siddiqi 8 , E. Szafer-Glusman 9 , C. Zhou 9 , L. Neumayr 9 , G. Krigsfeld 9 , W. G. Wierda 10 , T. Shanafelt 11

1 Zucker School of Medicine at Hofstra/Northwell CLL Research & Treatment Program, Lake Success, NY; 2 Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY; 3 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 4 Peter MacCallum Cancer Center & St. Vincent’s Hospital and the University of Melbourne, Melbourne, VIC, Australia; 5 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 6 Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona, Barcelona, Spain; 7 Mayo Clinic, Rochester, MN; 8 City of Hope National Medical Center, Duarte, CA; 9 Pharmacyclics LLC, an AbbVie Company, South San Francisco, CA; 10 Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX; 11 Stanford University Medical Center, Stanford, CA, United States of America

Background: While continuous single-agent I or I-based therapies are superior to FCR, FCR remains an efficacious fixed-duration regimen for fit patients (pts) with previously untreated CLL. CAPTIVATE (PCYC-1142; NCT02910583 ) is a multicenter phase 2 study of first-line treatment with the all-oral, once-daily, chemotherapy-free regimen of I+V in fit pts with CLL. Primary analysis of the CAPTIVATE Fixed Duration (FD) cohort demonstrated deep, durable responses with fixed-duration I+V (Ghia et al, ASCO 2021).

Aims: We performed a cross-trial analysis to evaluate outcomes with fixed-duration I+V relative to FCR using data from the FD cohort of the CAPTIVATE study and the FCR arm of the recent E1912 study ( NCT02048813 ; Shanafelt et al. NEJM 2019;381:432-43).

Methods: This cross-trial analysis included pts with previously untreated CLL or SLL aged ≤70 y who were treated with I+V in the FD cohort of CAPTIVATE or with FCR in E1912. As pts with del(17p) were not enrolled in E1912, pts with del(17p) from CAPTIVATE were excluded from the analysis. In CAPTIVATE, pts received three 28-d cycles of I, followed by 12 cycles of I+V (I 420 mg/d orally; V ramp-up to 400 mg/d orally). In E1912, pts received six 28-d cycles of FCR (F 25 mg/m 2 on days 1-3; C 250 mg/m 2 on days 1-3; R 50 mg/m 2 on day 1 and 325 mg/m 2 on day 2 in cycle 1, then 500 mg/m 2 on day 1 in cycles 2-6). PFS and OS were estimated by Kaplan-Meier method. Adjusted treatment effects for PFS, OS, and ORR were estimated by inverse probability of treatment weighting (IPTW) using propensity score (PS) which balanced treatment groups for the following baseline prognostic factors: age, sex, ECOG performance status ≥1, CLL/SLL diagnosis, time from diagnosis, advanced Rai stage, absolute lymphocyte count, beta-2 microglobulin >3.5 mg/L, and high-risk genomic features ( TP53 mutated, del[11q], unmutated IGHV). AEs were evaluated using overall incidence and exposure-adjusted incidence rates (EAIRs).

Results: Analysis included 136 pts without del(17p) treated with fixed-duration I+V in the CAPTIVATE FD cohort and 175 pts treated with FCR in E1912; baseline characteristics were generally well-balanced across groups, with notable differences in the proportions of pts aged ≥65 y and with baseline thrombocytopenia (Table). Median follow-up was 38.7 mo with I+V and 33.7 mo with FCR. After IPTW, PFS was improved with I+V relative to FCR (hazard ratio [HR] 0.42; 95% CI 0.25-0.71; log-rank P =0.0018; 36-mo PFS rate: 89% vs 70%), as was OS (HR 0.19; 95% CI 0.05-0.77; log-rank P =0.0084; 36-mo OS rate: 98% vs 90%). Results were generally similar in unadjusted analyses (Table). Adjusted ORRs were 96% for I+V and 85% for FCR (difference 11.2%; 95% CI 5.1–17.3; Chi-square P =0.0011). Median duration of treatment was 13.8 mo for I+V and 4.7 mo for FCR. Treatment was discontinued due to AEs in 5% and 21% of pts, respectively. Overall, grade ≥3 AEs occurred in 62% of pts with I+V and 90% with FCR. Grade ≥3 AEs in ≥5% of pts with I+V were neutropenia (36%), and with FCR were lymphopenia (67%), neutropenia (45%), leukopenia (41%), lymphocytosis (31%), anemia (18%), thrombocytopenia (18%), febrile neutropenia (16%), hypertension (6%), hyperglycemia (6%), and maculopapular rash (5%). EAIRs for grade ≥3 AEs and AEs of clinical interest of any grade were lower with I+V than with FCR (Table).

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Summary/Conclusion: Results of this cross-trial analysis suggest superior PFS with fixed-duration I+V relative to FCR in first-line CLL. The safety profile with I+V appeared favorable relative to the safety profile of FCR, with lower AE rates and fewer discontinuations due to AEs.

PB1879: OVERALL SURVIVAL OF RELAPSED/REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS TREATED WITH IBRUTINIB IN ALBANIA (2018-2022)

E. Calliku 1,* , A. Perolla 1 , A. Cili 1 , P. Pulluqi 1 , T. Caja 1 , A. Ivanaj 1

1 Hematology, UHC “ Mother Teresa”, Tirana, Albania

Background: The survival and the outcome of the patients with refractory/relapsed Chronic Lymphocytic Leukemia, in the beginning has been limited and unsatisfactory, but has significantly improved during the last decades, with the discovery of the pathway inhibitors. The overall survival depends mostly on their response to the lines of treatments used and is patient-related and we have to take in consideration also the numbers of chemotherapies applied. The significant benefit of Ibrutinib approved in 2014 as a target therapy for all the patients with Chronic Lymphocytic Leukemia who has been treated previously with at least one line of therapy has been shown across all the risk groups, as demonstrated in the RESONATE study.

Aims: Our study aims to evaluate the Overall Survival (OS) of all the refractory/relapsed Chronic Lymphocytic Leukemia patients treated with Ibrutinib in the third line of therapy from 2018 until january 2022 and evaluate the correlation of this survivance with the age subgroups and gender.

Methods: We studied all the patients diagnosed in our University Hospital Center “Mother Teresa” in Tirana, Albania with R/R CLL who started the treatment with ibrutinib, in combination or not with immunotherapy from July 2018 until January 2022 from age >18 years old and up. We collected the data from the registries and database of Hematology Clinic. The data were analysed with SPSS variant 25.

Results: From July 2018 until January 2022 we have treated with Ibrutinib third line 46 patients diagnosed with R/R CLL, 32 (69.6%) were males and 14 (30.4%) were females with a ratio M/F of about 2:1 with a median age of 63 ± 7.4 (range 43 – 80) years old. The estimated median Overall Survival resulted of 28 ± 4.9 (95%CI range 18- 37) months. Overall survival estimated according to age subgroups was higher in the 50-59 yo subgroup resulting in 38 ± 7.7 (95%CI range 16- 39) months. The differences of overall survival of the patients with R/R CLL treated with ibrutinib in the third line depending on gender were non significant

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Summary/Conclusion: Chronic Lymphocytic Leukemia is the most common type of leukemia in the western countries and with the median age at the diagnosis of 72 years old and diagnosed more in males than in the females 1 . In 2013 Byjrd JC et al published in N Engl J Med that the response to Ibrutinib of R/R CLL patients previously treated was independent of clinical and genomic factors with an OS 83% at 26 months.

In our study we found a median younger age of the patients at the moment of the diagnostication (median age of 63 years old) compared with the literature and a ratio male:femato 2:1. The median overall survival resulted 28 ± 4.9 (95%CI range 18- 37) months

In our study found also that gender does not influence the overall survival of the patients with R/R CLL treated with ibrutinib in the third line.

PB1880: A MULTICENTER PHASE 1 STUDY EVALUATING THE SAFETY AND TOLERABILITY OF HMPL-760 IN PATIENTS WITH PREVIOUSLY TREATED CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTIC LYMPHOMA OR OTHER NON-HODGKIN LYMPHOMA

B. Kahl 1,* , J. Cohen 2 , R. Pillai 3 , V. Jayaprakash 3 , C. Wang 3 , M. Hahka-Kemppinen 3 , M. Kania 3 , P. Zinzani 4

1 Washington University School of Medicine, St Loius; 2 Emory University School of Medicine, Atlanta; 3 HUTCHMED International Corporation, Florham Park, United States of America; 4 University of Bologna Institute of Hematology, Bologna, Italy

Background: Covalent Bruton’s tyrosine kinase (BTK) inhibitors for the treatment of B-cell malignancies bind irreversibly to BTK. The most frequent mechanism of acquired resistance to covalent BTK inhibitors in chronic lymphocytic leukemia (CLL) is the development of mutations in the binding site, with a cysteine to serine substitution at position 481 (C481S). HMPL-760 is a potent, selective, and reversible BTK inhibitor which can inhibit B-cell malignant tumor growth of either BTK WT or mutant BTK C481S . In addition to its antitumor activity as a single agent, HMPL-760 showed synergistic and/or additive effects on B-cell lymphoma models when combined with other targeted therapies or chemoimmuno-therapy.

Aims: This is a first in human phase 1, ( NCT05176691 ) multicenter, dose escalation/expansion study to evaluate safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of HMPL-760 in patients with previously treated CLL/small lymphocytic lymphoma (SLL) or non-Hodgkin lymphoma (NHL). The primary objective of dose escalation is to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of HMPL-760 and to evaluate the safety and tolerability in dose expansion. Secondary objectives for dose escalation/expansion are to assess the preliminary antitumor activity, PK, and PD of HMPL-760.

Methods: In dose escalation, cohorts of up to 12 patients each will receive ascending oral doses of HMPL-760. The modified toxicity probability interval scheme-2 (mTPI-2) 1,2 design will be used to perform dose escalation and MTD/RP2D determination, using dose increments from 25 mg to 400 mg once daily. The first 3 patients in each cohort will be administered a single oral dose of HMPL-760 on day 1 of the PK period and serial blood samples will be collected from days 1 to 4 for analysis of HMPL-760 plasma concentration. Dose limiting toxicities (DLTs) will be assessed during the first 28 days of continuous treatment, not including the PK period. Safety assessments will include DLTs, treatment emergent adverse event, serious adverse events, electrocardiograms and clinical laboratory tests. Efficacy assessments will include objective response rate, duration of response, clinical benefit rate, and progression-free survival. In dose expansion, ~25 patients each will be enrolled in the following cohorts: (1) patients with CLL/SLL who have failed prior BTK inhibitor therapy; (2) patients with mantle cell lymphoma who have failed prior BTK inhibitor therapy; (3) patients with marginal zone lymphoma who have failed prior BTK inhibitor therapy; (4) patients with lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia who have failed prior BTK inhibitor therapy; (5) patients with CLL/SLL or other NHL who are intolerant to or failed BTK therapy and not meeting the definition for cohorts 1 to 4 if approved by the Sponsor; (6) patients with NHL not otherwise specified, inclusive of diffuse large B-cell lymphoma (DLBCL), DLBCL arising from follicular lymphoma (FL), Richter’s transformation, high grade B-cell lymphoma, or FL who have failed at least 2 prior systemic therapies, for which no curative therapy exists, and for whom treatment with a BTK inhibitor may be deemed appropriate. No formal hypothesis testing is planned for this study.

1. Guo et al. Contemp Clin Trials . 2017;58:23-33

2. Yang et al. Contemp Clin Trials . 2015;45:426-34

Results: N/A

Summary/Conclusion: N/A

PB1881: IBRUTINIB REDUCES THE RISK OF RICHTER’S TRANSFORMATION IN PREVIOUSLY TREATED CLL PATIENTS: A POPULATION-BASED STUDY IN BRITISH COLUMBIA

R. Ramadan 1 , A. Alzaki 1 , H. Leitch 1 , K. Ramadan 1,*

1 Hematology, St. Paul’s Hospital, Vancouver, Canada

Background: Richter Transformation (RT) presents in approximately 5% of Chronic Lymphocytic Leukemia (CLL) patients. Richter transformation is a rare and aggressive complication of CLL, with little information in current literature regarding possible predictive factors. In this study we assessed the possible factors for incidence of Richter’s transformation. This is a population-based study in British Columbia, Canada.

Aims: To evaluate the possibility of Ibrutinib reducing the incidence of Richter transformation in patients with Chronic Lymphocytic Leukemia. Evaluating risk factors for Richter’s transformation can help implement Ibrutinib in widespread usage as a first-line treatment for cases presenting the evaluated risk factors.

Methods: An electronic database search of 1056 patients with confirmed diagnosis of CLL who presented at St Paul’s Hospital, Vancouver, Canada over 30 years and between 1991 and 2021 was performed with 520 having a complete data set. 197 have undergone at least one treatment. 48 received Ibrutinib (Ibr group), and 149 patients received other non-novel therapies (non-Ibr group). Their Transformation-Free Survival (TFS) is then calculated using SPSS and incorporated in the Kaplan Meier Method.

Results: The median age at diagnosis of CLL and RT was 61 y (range 34-86 y) and 64.5 y (range 33-84 y) respectively. The median lymphocyte count at diagnosis was 12 x109/L. The median time to transformation for Richter Patients was 60.1 months. By 5 years, the percentage of CLL patients who transformed in the non-Ibr group is 9%, while for the Ibr-group is 0%. By 10 years, the percentage of CLL patients who transformed in the non-ibr group is 17%, while for the Ibr group is 2% (p = 0.012). Other factors such as age, gender, and lymphocyte count at diagnosis were not significant predictors for RT on univariate analysis. On multivariate analysis, previous ibrutinib therapy retained its significance and lowered the risk of RT.

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Summary/Conclusion: Previous Ibrutinib therapy reduced the risk for RT in CLL patients. Incorporating Ibrutinib based therapy in management of CLL is a reasonable strategy in reducing the risk of RT.

PB1882: RICHTER TRANSFORMATION FEATURES, SURVIVAL AND TREATMENT OUTCOME IN HEMATOLOGY CLINIC OF CRAIOVA, ROMANIA

I. Rotaru 1,* , L. Ocroteala 2 , D. Duta 2 , J. G. Goanta 1 , A. M. Patrascu 1 , C. I. Popa 1

1 University of Medicine and Pharmacy; 2 Hematology Clinic, Craiova, Romania

Background: Richter transformation is a rare but potential lethal complication in patients diagnosed with chronic limphocytic leukemia. The most common histological transformation is of Hodgkin lymphoma or Diffuse Large B-cell lymphoma type.

Aims: The aim is to assess the clinicopathological characteristics survival and treatment in Richter transformation patients diagnosed and treated in Hematology Clinic of Craiova in the last 10 years.

Methods: Between January 2012 and January 2022, 453 patients with chronic lymphocytic leukemia (CLL) were diagnosed and treated in our clinic, 121 female and 332 male.

Results: Median age of the patients was 68 years and the male:female ratio was 2,74. The majority were diagnosed in A stage (52%), 27% in B stage and 21% in C stage. 29% of patients required treatment initiation upon diagnosis. Richter transformation occurs in 1,76 % cases (8/453) of all CLL. The transformation occurred as follows: 5 diffuse large B-cell lymphoma (DLBCL)-type pacients and 3 Hodgkin lymphoma-type pacients. In all cases the mutational status was unmutated, CD38 was positive in 6 patients and 3 patients were positive for TP53 mutation. The time until transformation lasted between 6 and 42 months, measured from treatment initiation. In 6 patients, transformation involved peripheral lymphadenopathy and in 2 patients, intraabdominal lymphadenopathy. The treatment received was ABVD regimen in Hodgkin transformation and R CHOP in DLBCL-type. On average, survival was 16 months in Hodgkin transformation and 7,8 months in DLBCL transformation.

Summary/Conclusion: The Richter transformation is a rare complication of CLL, an indolent lymphoproliferative disorder. Only 8 patients were diagnosed with Richter transformation in our clinic in the last 10 years. Chemotherapy response was poor and survival was short.

PB1883: TIME-LIMITED TREATMENT WITH ACALABRUTINIB PLUS OBINOTUZUMAB IN TREATMENT-NAÏVE CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS INDUCES DEEP REMISSIONS: EARLY RESULTS OF AN ONGOING PHASE 2 TRIAL.

J. Burger 1,* , M. Sivina 1 , E. Kim 1 , G. Issa 1 , W. Wierda 1 , N. Jain 1 , P. Thompson 1 , A. Ferrajoli 1

1 Leukemia, MD Anderson Cancer Center, Houston, United States of America

Background: The Bruton’s tyrosine kinase (BTK) inhibitor acalabrutinib is approved for treatment of chronic lymphocytic leukemia(CLL). Acalabrutinib induces durable remissions in most CLL patients, which mostly are partial remissions (PR), and therefore treatment typically is given as long-term monotherapy. As a potential alternative we developed a time-limited regimen, combining acalabrutinib with obinutuzumab.

Aims: Here, we report early results from 14 treatment-naïve patients with CLL who enrolled in this ongoing phase 2 trial ( NCT04505254 ) since September, 2020 at MD Anderson Cancer Center.

Methods: Patients and Study Design : Treatment-naïve CLL patients requiring therapy as per iwCLL criteria receive acalabrutinib 100 mg orally twice a day for 24 cycles, combined with monthly obinotuzumab for 6 doses, starting in cycle 3. The first dose of obinutuzumab is divided into 100 mg on day 1 and 900 mg on day 2 of cycle 3; 1000 mg are given during subsequent cycles (cycles 4-8). Patients who do not achieve a complete remission (CR) after cycle 8 can receive an additional 6 monthly doses of obinotuzumab during cycles 9 -14. Treatment is discontinued after 24 cycles, and patients will be monitored. The primary objective is to determine the durability of remissions after treatment discontinuation, secondary objectives are to determine clinical and laboratory characteristics that predict for early versus late relapse after time-limited therapy.

Results: The median age of the patients is 70 yrs (range, 40 -83 yrs), 14% had del17p or TP53 mutation, 43% had an unmutated IgHV and 71% advance stage disease (RAI stage III and IV). The median baseline absolute lymphocyte count (ALC) and b2 microglobulin at start of therapy were 39.2x10 9 /L (range: 7.1 – 188.4 x 10 9 /L) and 4.2 mg/L (range: 2.2 – 7.9 mg/L), respectively. After a median follow-up of 7 months (2 – 16 months), 13 (93%) of patients remain on study; one patient died (7%) due from complications from a presumed bacterial (COVID19-negative) pneumonia after 2 months on therapy. The estimated one-year PFS and OS for the cohort is 92.8 %. Seven patients were evaluable for response assessment after 8 months of therapy. No patient has yet discontinued therapy. All patients achieved a PR (one patient with undetectable minimal residual disease/U-MRD in the bone marrow), accounting for an overall responsonse rate of 100%. The median levels of bone marrow infiltration by CLL cells, quantified by flow cytometry, declined from 83.6% (range: 54.3 – 94.0 %) at baseline to 4.1% (range, 0.0 - 63.3%, n=7, p<0.05, see figure) after 6 cycles of combination treatment. Sixty-four percent of patients completed all doses of obinotuzumab, 50% requiered a dose reduction of acalabrutinib to 100 mg per day due to adverse events (AE). Grade 3 3 AE were observed in 4 patients (29%), which included decreased neutrophil counts (n=2), syncope (n=1), and grade 5 lung infection (COVID19 not detected, n=1). The most frequently reported non-serious related AE ( 3 2 patients) were anemia (n=5 [36%]), decreased platelets counts (n=3 [21%]), bruising (n=3 [21%]), limbs edema (n=2 [15%]) and headache (n=2 [15%]). All these events were grade 1. Importantly, no bleeding or atrial fibrillation events were observed.

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Summary/Conclusion: Our preliminary data indicate that combination therapy of acalabrutinib plus obinotuzumab induces remissions with a major reduction in bone marrow disease after 6 months of combination therapy. Longer treatment and follow-up is warranted to determine the durability of responses after therapy discontinuation.

PB1885: THE ROLE OF PET CT IN HAIRY CELL LEUKEMIA

G. itchaki 1 , K. gurevich 2 , M. Gorenberg 2 , T. Tadmor 3,*

1 Hematology, davidoff cancer center rabin medical center, petach tikva; 2 Department of Nuclear Medicine; 3 Hematology, Bnai Zion Medical Center, haifa, Israel

Background: Currently Hairy Cell Leukemia guidelines recommend the routine use of chest X-ray and abdominal ultrasound as imaging workup

PET- CT / CT has become a key tool in assessing disease spread and treatment response of several types of cancer in general and B-cell malignancies in particular.

In HCL, the use of PET-CT has been reported mainly in patients who presents with unusual clinical sites of involvement or manifestation, and are available in the literature only as case reports.

Aims: To characterize the typical baseline assessment of pet uptake in HCL with particular attention to BM involvement and SUV uptake in classical HCL, Extramedullary sites of involvement and Predictive value in response to therapy.

Methods: Clinical laboratory and demographic data were summarized for HCL patients who performed PET CT during follow up in 2 medical centers in Israel.

2 central reviewer experts evaluated all PET CT test using the Deauville criteria.

Results: The study included 8 patients diagnosed and follow up in 2 medical centers in israel with classical HCL.

All patients included performed at least one PET CT with a total of 20 test

median age was 50 (range 34-84) and 7 were males (14%).

All pateinets recived at least 1 line of therapy.

Indication for PET CT included investigation for infection, vasculitis, second cancers or extramiddolary disease.

We will presnet median SUV uptake during active disease in BM, Spleen, and bone.

Uptake in unusual site will be descibe.

Pet results following therapy and correltion with response assesment will be presented.

Summary/Conclusion: This study is pioneer in learning the role of PET CT inHCL. findings suggest that PET/CT may helpfully in identify extramidollary sites and reponse to therapy.

Prospective clinical trials including larger cohorts of patients are needed to conclusively define the role and prognostic impact of PET/CT in HCL patients.

PB1886: LONG-TERM TRENDS IN THE LOSS OF LIFE EXPECTANCY AFTER A DIAGNOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA: A POPULATION-BASED STUDY IN THE NETHERLANDS, 1989-2018

L. Van Der Straten 1,2,3,* , C. C. Maas 1,4 , M.-D. Levin 2 , O. Visser 5 , E. F. Posthuma 6,7 , J. K. Doorduin 8 , A. W. Langerak 3 , A. P. Kater 9 , A. G. Dinmohamed 1,4,9,10

1 Research and Development, Netherlands Comprehensive Cancer Organisation (IKNL), Utrecht; 2 Internal Medicine, Albert Schweitzer hospital, Dordrecht; 3 Immunology, Laboratory Medical Immunology, Erasmus MC; 4 Public Health, Erasmus MC, University Medical Center Rotterdam, Rotterdam; 5 Registration, Netherlands Comprehensive Cancer Organisation (IKNL), Utrecht; 6 Internal Medicine, Reinier The Graaf Hospital, Delft; 7 Hematology, Leiden University Medical Center, Leiden; 8 Hematology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam; 9 Hematology, Amsterdam UMC, University of Amsterdam, Cancer Center Amsterdam, Lymphoma and Myeloma Center Amsterdam; 10 Hematology, Amsterdam UMC, Vrije Universiteit Amsterdam, Cancer Center Amsterdam, Amsterdam, Netherlands

Background: The introduction of chemoimmunotherapy heralded a new era for chronic lymphocytic leukemia (CLL) management. More recently, the chemoimmunotherapy paradigm has shifted towards more novel targeted agents that can further improve progression-free survival and overall survival. Population-based studies have shown that the five-year relative survival in CLL increased markedly since the introduction of chemoimmunotherapy, approximating 90% for patients up to age 70. Seeing the comparatively favorable prognosis of CLL patients in modern times, other survival estimates may be more informative because relative survival does not inform on patient survival across the entire remaining life span.

Aims: Studies estimating the life expectancy of CLL patients are hitherto lacking. Therefore, we assessed trends in the life expectancy of CLL patients from a historical and contemporary perspective.

Methods: We selected all CLL patients diagnosed in the Netherlands between 1989 and 2018—with follow-up for survival up to December 31, 2020—from the Netherlands Cancer Registry. We estimated the loss in expectation of life (LEL)—quantifying the difference between the life expectancy of CLL patients and the general population, the latter being matched to patients by age, sex, and calendar year—using flexible parametric relative survival models. The LEL is interpreted as the average number of life years lost due to a CLL diagnosis. The LEL can vary markedly across ages because life expectancy is age-dependent. Therefore, the proportional LEL (PLEL) was estimated (calculated as the LEL divided by the population life expectancy). These survival measures were presented by year of diagnosis for four age categories at diagnosis (i.e., 50, 60, 70, and 80 years), stratified by sex.

Results: Our cohort included 23,692 CLL patients (median age, 69 years, interquartile range 61-77 years; 61% males) diagnosed in the Netherlands between 1989-2018. The life expectancy of CLL patients increased gradually across all ages between 1989-2018, with a most pronounced increase in patients aged 50 and 60 years at diagnosis (Fig 1A). CLL patients diagnosed in 1990 and 2018 lost 3.1-19.0 and 1.7-8.2 life-years depending on age and sex, respectively (Fig 1A). The increase in the life expectancy of CLL patients was greater than in the general population. Consequently, the LEL (Fig 1B) and PLEL (Fig 1C) of CLL patients across all four age categories decreased over time. Despite the decrease in LEL over time, all studied age groups diagnosed in 2018 had excess mortality, reflected in a LEL ranging from 1.7 to 8.2 life-years, depending on age and sex (Fig 1B). The PLEL estimates showed that the age differential in survival became less pronounced over time and eventually disappeared for female patients, of which the former indicates that older patients (i.e. 70 and 80 years), who generally have fewer life-years left, experience the same improvement in life expectancy as younger patients.

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Summary/Conclusion: The life expectancy of CLL patients diagnosed in the Netherlands between 1989-2018 has steadily increased over time. This increase is likely attributed to the broader application of more efficacious therapies over time. Notwithstanding, continuous population-based surveillance is essential to assess the impact of the rapidly evolving management of CLL on survival since excess mortality is still a threat in contemporary diagnosed and managed patients.

PB1887: NETWORK META-ANALYSIS OF PROGRESSION FREE SURVIVAL IN THE TREATMENT OF RELAPSED OR REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA

A. Chanan-Khan 1,* , T. Liu 2 , K. Yang 2 , A. Cohen 2 , K. Fahrbach 3 , J. Campbell 3 , B. Tang 2

1 Mayo Clinic, Jacksonville; 2 Beigene USA, San Mateo; 3 Evidera, Lexington, United States of America

Background: Zanubrutinib, a next-generation Bruton’s tyrosine kinase inhibitor (BTKi), demonstrated superior overall response rate and a trend toward improved progression-free survival (PFS) as compared to ibrutinib in patients with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) in the phase 3 ALPINE trial ( NCT03734016 ) based on interim analysis. However, its comparative efficacy with other treatments is not known.

Aims: The goal of this study was to estimate the relative efficacy of zanubrutinib compared with standard treatments for R/R CLL through a network meta-analysis (NMA).

Methods: The efficacy of zanubrutinib from ALPINE (study of zanubrutinib and ibrutinib in patients with R/R CLL was compared to ibrutinib, acalabrutinib, bendamustine and rituximab (BR), and venetoclax + rituximab using data from 4 RCTs. Relevant RCTs were identified using a systematic literature review. A network was constructed that composed RCTs (ALPINE, ELEVATE-RR [ NCT02477696 ], ASCEND [ NCT02970318 ], and MURANO [ NCT02005471 ]). Bayesian network meta-analysis (NMA) models were used to simultaneously synthesize hazard ratios (HRs) and 95% credible intervals (CrIs) for investigator-assessed PFS and overall survival (OS) to estimate the relative efficacy of zanubrutinib versus comparators of interest. An assumption of constant hazard ratio was applied in the NMA analysis. Analyses were performed using well-established codes published by the National Institute for Health and Care Excellence (NICE) Decision Support Unit and were implemented with OpenBUGS (version 3.2.3).

Results: The NMA suggested a significant improvement in PFS for zanubrutinib over acalabrutinib (HR = 0.52, 95% CrI [0.30, 0.90]), ibrutinib (0.47, [0.29, 0.76]), and BR (0.13, [0.06, 0.26]). Zanubrutinib had a numerically better PFS than venetoclax + rituximab (0.69, [0.32, 1.46]) while not reaching statistical significance. For OS, NMA results showed a trend favoring zanubrutinib over acalabrutinib (0.75, [0.35, 1.59]), ibrutinib (0.62, [0.31, 1.22]), and BR (0.52, [0.21, 1.24]). The difference was not statistically significant with wide CrIs, suggesting a high uncertainty as inherent limitation of the analysis.

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Summary/Conclusion: This NMA reports the first summary of comparative PFS of available treatments for R/R CLL. Indirect treatment comparisons suggest PFS on zanubrutinib may be superior to other BTKis and immunochemotherapies in R/R CLL patients.

PB1888: EFFICACY OF FIRST-LINE TREATMENT FOR CHRONIC LYMPHOCYTIC LEUKEMIA: A BAYESIANNETWORK META-ANALYSIS.

A. Chanan-Khan 1,* , K. Yang 2 , T. Liu 2 , A. Cohen 2 , K. Fahrbach 3 , Y. Wang 3 , B. Tang 2

1 Mayo Clinic, Jacksonville; 2 Beigene USA, San Mateo; 3 Evidera, Lexington, United States of America

Background: Zanubrutinib is an oral, highly selective, next-generation Bruton tyrosine kinase inhibitor. Efficacy of zanubrutinib was compared with bendamustine + rituximab (BR) in adult patients with treatment-naïve (TN) chronic lymphocytic leukemia (CLL) in the phase 3 SEQUOIA trial ( NCT03336333 ). However, efficacy of zanubrutinib compared with other frontline CLL treatments remains unknown.

Aims: The goal of this network meta-analysis of randomized controlled trials (RCTs) was to estimate the relative efficacy of zanubrutinib compared with standard frontline treatments for CLL.

Methods: A total of 4 relevant RCTs were identified by a targeted literature review conducted throughout January 2022, CLL11 ( NCT01010061 ), ALLIANCE ( NCT01886872 ), MABLE ( NCT01056510 ), and SEQUOIA. A feasibility assessment was performed to ensure that RCTs included within each population strata did not differ with respect to effect modifiers including presence of mutation, age, del11, and del17 status. A network was constructed for the composed 4 RCTs to compare the efficacy of zanubrutinib with bendamustine + rituximab, chlorambucil + obinutuzumab, chlorambucil + rituximab, and ibrutinib in patients with previously untreated CLL. Bayesian NMA models were conducted to simultaneously synthesize hazard ratio (HR) and 95% credible intervals (CI) for investigator-assessed progression-free survival (PFS). A constant hazard ratio was assumed in the NMA analysis. Statistical analyses were performed using codes suggested by the National Institute for Health and Care Excellence.

Results: The efficacy results of the NMA indicated a statistically significant improvement in PFS for zanubrutinib over bendamustine + rituximab (HR = 0.42; [95% CI = 0.27, 0.65]), chlorambucil + obinutuzumab (0.45 [0.23, 0.86]), and chlorambucil + rituximab (0.22 [0.12, 0.41]). Zanubrutinib achieved comparable PFS to ibrutinib (1.07 [0.59, 1.94]).

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Summary/Conclusion: This is the first NMA to compare the efficacy of zanubrutinib with all commonly utilized first-line treatment for TN CLL. Results from this indirect treatment comparison suggested that the PFS for zanubrutinib may be statistically significantly better than immunochemotherapy. Findings from this study require validation with further large scale RCTs with longer follow up time.

PB1889: A CASE OF REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA INDUCED IMMUNE THROMBOCYTOPENIC PURPURA SUCCESSFULLY TREATED WITH VENETOCLAX

B. Yavuz 1,* , A. F. Karatas 1 , E. Erdogan Yucel 1 , O. Seker 1 , S. Guven 1 , M. Kakci 1 , G. H. Ozsan 1 , I. Alacacioglu 1 , F. Demirkan 1

1 Hematology, Dokuz Eylul University, Izmir, Turkey

Background: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. Approximately 2% of patients with CLL develop immune thrombocytopenic purpura (ITP) during the course of the disease. When resistant to steroids, this constitutes as indication for treatment of the CLL. Here we report a patient with refractory ITP secondary to CLL successfully treated with venetoclax.

Aims: To present an interesting case with CLL related refractory ITP treated successfully with novel agent venetoclax.

Methods: Patient data was taken from the patient herself and hospital records. Informed consent to publish the case is obtained from patient. Permission for off-label venetoclax and eltrombopag was obtained from Ministry of Health of Turkey.

Results: 46-year-old female patient presented with lumps on her neck that were present for the last 9 months in November 2020. She has a history of frequent pneumonia and otitis but no constitutional symptoms. Her physical examination reveals multiple 2cm lymphadenopathies on her neck and no organomegaly or other pathological features. Blood work shows mild lymphocytosis (6800/mm 3 ) with no serious cytopenias. Peripheral blood smear, flow cytometry and bone marrow biopsy were all compatible with CLL. She was classified as Binet A CLL and was followed up with no treatment after appropriate vaccinations against capsulated pathogens. In July and August 2021 she received two doses of mRNA vaccination against COVID-19. On 1 st November 2021 she experienced excessive menstrual bleeding and blood work showed platelet count of 23000/mm 3 , she was started on steroids (1 mg/kg/day) and after 4 days platelet count has risen to 55000/mm 3 , she discontinued steroids on her own against medical advice. On 13 th of November she presented with extensive petechiae and purpura and was again started on steroids and was given the courses of intravenous immunoglobulins (IVIG) without any sustained response. She was refractory to platelet transfusions too. She was transferred to our clinic. She was found to have del11q and del13q. She refused bone marrow examination. She was treated with rituximab, steroids, vincristine, IVIG and eltrombopag for ITP without success (Fig. 1). She had a minimal response to IVIG only. She received two courses of bendamustine (90 mg/kg for two days) also without success. Three courses of plasmapheresis yielded no response either. After mild success with immunadsorbtion apharesis she was started on venetoclax plus rituximab with ramp-up. Sustained response was achieved within the first week of venetoclax therapy.

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Summary/Conclusion: Gordon et al. reported 2 CLL cases one with ITP and other with Evans syndrome successfully treated with venetoclax. We think, this treatment should be considered in patients with refractory immune cytopenias secondary to CLL and assessed with prospective clinical trials.

PB1890: ZANUBRUTINIB IN ACALABRUTINIB-INTOLERANT PATIENTS (PTS) WITH B-CELL MALIGNANCIES

M. Shadman 1,* , I. W. Flinn 2 , E. C. Kingsley 3 , B. Freeman 4 , M. Y. Levy 5 , J. Cultrera 6 , C. M. Farber 7 , A. Chaudhry 8 , R. Porter 9 , R. Crescenzo 10 , A. Idoine 10 , X. Zhang 10 , A. Cohen 10 , K. By 10 , J. Huang 10 , J. P. Sharman 11

1 Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA; 2 Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN; 3 Comprehensive Cancer Centers of Nevada, Las Vegas, NV; 4 Summit Medical Group, Florham Park, NJ; 5 Texas Oncology-Baylor Charles A. Sammons Cancer Center, Dallas, TX; 6 Florida Cancer Specialists & Research Institute, Leesburg, FL; 7 Atlantic Hematology Oncology, Morristown Medical Center, Morristown, NJ; 8 Summit Cancer Centers, Spokane, WA; 9 SSM Health Dean Medical Group, Madison, WI; 10 BeiGene (Beijing) Co., Ltd., Beijing, China and BeiGene USA, Inc., San Mateo, CA; 11 Willamette Valley Cancer Institute and Research Center, Eugene, OR, United States of America

Background: Bruton tyrosine kinase inhibitors (BTKi) are highly effective against several B-cell malignancies; however, their use is limited by adverse events (AEs), potentially due to off-target inhibition of other kinases. The next-generation BTKi zanubrutinib was designed to minimize off-target inhibition thereby prolonging duration of treatment with limited AEs. Zanubrutinib is approved in the US for Waldenström’s macroglobulinemia (WM), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL) and in Europe for WM. In phase 3 trials of zanubrutinib versus ibrutinib in WM and chronic lymphocytic lymphoma/small lymphocytic lymphoma (CLL/SLL), zanubrutinib has consistently shown higher tolerability than ibrutinib ( Blood 2020;136(18):2038-50; EHA 2021 LB1900). Previous results from this ongoing phase 2 study, BGB-3111-215 ( NCT04116437 ), show that zanubrutinib was well tolerated in pts who discontinued ibrutinib and/or acalabrutinib due to AEs ( Blood 2021;138[suppl 1]:1410). Here, we report updated results from acalabrutinib-intolerant pts (cohort 2) in BGB-3111-215.

Aims: To assess the safety of zanubrutinib in pts intolerant to acalabrutinib by the recurrence and change in severity of AEs that led to acalabrutinib intolerance.

Methods: In this ongoing, multicenter, single-arm, phase 2 study in the US, eligible acalabrutinib-intolerant pts with CLL/SLL, WM, MCL, or MZL who met protocol-defined acalabrutinib-intolerance criteria and gave informed consent were enrolled in cohort 2. Pts who progressed on prior BTKi therapy were excluded. Pts received zanubrutinib 160 mg twice daily or 320 mg once daily and were evaluated for safety and efficacy, including recurrence of any intolerance events from prior BTKi. Responses were assessed by investigators every 3 cycles based on standard response criteria for each indication using parameters at study entry as baseline ( Blood 2008;131:2745; J Clin Oncol 2012;30:2820; J Clin Oncol 2014;32:3059; Br J Haemtol 2013;160:171).

Results: As of 6 January 2022, 13 pts received zanubrutinib in cohort 2 (9 CLL/SLL; 2 WM; 1 MCL; 1 MZL). Median age was 73 years (range 51-83) and median duration of treatment was 9.2 months (range 0.5-16.0), with a median follow-up of 12.9 months (range 0.8-16.0). Median number of prior therapies was 2; 62% of pts received ibrutinib before acalabrutinib. Acalabrutinib was the most recent therapy for all pts. Ten pts remain on treatment. Three pts discontinued treatment (myalgia, progressive disease and withdrawal; 1 pt each) and withdrew from the study thereafter.

Twenty-two acalabrutinib-intolerance events were reported in 13 pts, most commonly arthralgia (4), myalgia (3), headache (2), and hemorrhage (2). A total of 73% of the acalabrutinib-intolerance events did not recur on zanubrutinib, corresponding to 62% of pts not experiencing a recurrence of any event. Six events recurred: 1 at lower grade, 5 at same grade, and none at higher grade (Figure). One pt discontinued due to recurrence of their acalabrutinib-intolerance event (myalgia; same grade). Three pts who experienced the same intolerance events (pain in extremity, diarrhea, and atrial fibrillation) on ibrutinib and acalabrutinib did not have a recurrence of those on zanubrutinib. Among the 10 pts on zanubrutinib with ≥90 days of follow-up, 80% achieved at least stable disease and 70% achieved a deepening of response.

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Summary/Conclusion: Zanubrutinib may be a viable therapeutic option for pts who are intolerant to acalabrutinib—80% of pts received clinical benefit, and most did not experience recurrence of their prior intolerance events. Enrollment and follow-up are ongoing.

PB1891: VARIANT PHILADELPHIA CHROMOSOME IN CML

N. Bouayed Abdelmoula 1 , S. Aloulou 1,* , B. Abdelmoula 1

1 Genomics of Signalopathies at the service of Medicine, Medical University of Sfax, Sfax, Tunisia

Background: Chronic myeloid leukemia is a clonal malignant disorder of a pluripotent hematopoietic stem cell characterized by the Philadelphia chromosome and the reciprocal translocation t(9:22)(q34;q11). Almost 5 to 10 percent of chronic myeloid leukemia cases demonstrate variant Philadelphia translocations.

Aims: To contribute to the description of patients’ clinical characteristics and prognosis with variant Ph chromosome-positive leukemia, we report a Ph chromosome-positive CML in the chronic phase characterized by an unusual variant Ph chromosome.

Methods: An elderly male Libyan patient was suspected to have chronic myeloid leukemia. Cytogenetic and molecular exploration were conducted using bone marrow and blood samples. Cytogenetic analysis was performed on lymphocytes and bone marrow cells after short-term culture (48 hours). The cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Multiplex reverse transcription-PCR (RT-PCR) was used to identify the BCR-ABL1 transcript. RT-PCR was performed according to European recommendations, as described previously.

Results: Clinical assessment confirmed the first chronic phase of a chronic myeloid leukemia. Chromosomal formula showed an homogeneous structural translocation: t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Molecular investigation confirmed the Ph nature of the chromosomal abnormality by showing the BCR-ABL1 transcript. The patient was treated by using Imatinib molecule with a favorable therapeutic response.

Summary/Conclusion: A new variant Ph chromosome-positive CML in the chronic phase is reported. It is a four-way Ph translocation t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Imatinib, the first-generation TKI treatment was efficient.

PB1892: IMATINIB RESISTANCE: THE ROLE OF PHARMACOGENETIC VARIABILITY IN A SOUTH AFRICAN COHORT

C. De Long 1,* , F. February 1 , N. Mashigo 1,2 , F. Bassa 3 , C. Swanepoel 1,2

1 Department of Pathology, Stellenbosch University; 2 Department of Pathology, National Health Laboratory Services; 3 Department of Medicine, Stellenbosch University, Cape Town, South Africa

Background: Drug resistant cancers are often associated with poor patient outcomes and burdens an already strained healthcare system. The underlying mechanism of resistance is poorly understood, however Chronic Myeloid Leukemia (CML) serves as a useful disease model. For CML, highly effective therapies have been developed, such as tyrosine kinase inhibitors, particularly the first line drug Imatinib. Unfortunately, approximately 20-30% of patients do become resistant to Imatinib. Variability in patient drug response could be due to single nucleotide polymorphisms (SNPs) in genes that encode for Imatinib-metabolizing enzymes and transporters.

Aims: The overall aim of the study is to evaluate the effect selected SNPs located within genes ( CYP3A4/3A5 (Cytochrome P450), OATP1A2 ( SLCO1A2 ), OCTN1 ( SLC22A4 ) and hOCT1 ( SLC22A1 ) that encode selected drug transporters has on drug uptake, particularly Imatinib and to determine if these SNPs contribute to an alternative mechanism leading to Imatinib resistance in a South African cohort. Furthermore, the study aims to investigate whether the identified SNPs can serve as biological predictors that will aid in more efficient treatment plans.

Methods: A maximum of 44 samples from Imatinib resistant CML patients will be analysed. The patient’s treatment response will be monitored using the GeneXpert and the selected SNPs will be analysed using PCR-based genotyping assays. In addition, 44 non-resistant CML patients will be included as controls.

Results: Provisional data for SNPs SLC22A1 (rs628031) and CYP3A4 (rs2740574) suggest that the SNPs are more prevalent in the non-resistant group with 25% and 77.2% of patients having the SNPs opposed to the 7.89% and 68.1% of patients having the SNP in the resistant group, respectively. However, the data should be analysed along with the data of other SNPs in the study to determine poor outcome of imatinib treatment in CML patients.

Summary/Conclusion: The anticipated results would aid in determining whether the selected SNPs contributes to Imatinib resistance and to identify potential biological predictors of outcome which could help improve patient treatment stratification.

PB1893: THE STUDY OF SET GENE AND PROTEIN PHOSPHATASE 2A IN CHRONIC MYELOID LEUKEMIA AND THEIR ROLE IN DISEASE PROGRESSION

H. Fathy 1,* , H. Naenaa 2 , N. Halawani 2 , A. Bassiouny 3 , R. Abo Elwafa 4 , N. Farrag 5

1 Ass. Lecturer of Clinical Haematology, Faculty of Medicine, Kafr El Sheikh University; 2 Prof. of Internal Medicine, Faculty of Medicine, Alexandria University; 3 Prof. of Molecular Theraputics, Faculty of Science, Alexandria University; 4 Ass. Prof. of Clinical and Chemical Pathology, Faculty of Medicine, Alexandria University; 5 Fellow of Haematology, Alexandria University Hospitals, Alexandria, Egypt

Background: Chronic myeloid leukemia (CML) is characterized by three stages, chronic, accelerated and blastic phases. Tyrosine kinase inhibitors (TKIs) delay the progression of CML into blastic phase. However, resistance to TKIs has emerged due to different factors. Protein phosphatase 2A (PP2A) is a tumor suppressor protein that affect many cellular pathways. Inhibition of PP2A by PP2A inhibitors such as SET is one mechanism responsible for CML progression. Moreover, phosphorylation of C-terminal tyrosine residue (Tyr307) on the PP2A catalytic subunit was associated with decreased protein activity and consequently, loss of its tumor suppression function.

Aims: This work studied the role of SET gene expression as well as phosphorylated PP2A expression in CML progression

Methods: The study included 50 CML patients under TKI therapy for at least 18 months, and 50 healthy controls. All study participants were tested for SET gene expression level in peripheral blood by RQ-PCR and PP2A (Tyr307) in serum by ELISA. Moreover, patients were analyzed for complete blood count, BCR-ABL1 in peripheral blood by RQ-PCR and bone marrow examination.

Results: High SET gene expression in patients was found to be significantly associated with advanced disease phase and worse clinical and laboratory findings while low expression was significantly associated with better molecular response assessed by BCR-ABL1 level. High levels of PP2A (Tyr307) was significantly associated with blastic phase. Moreover, significant positive correlations was found between BCR-ABL1 level and both SET gene expression and PP2A Tyr307 levels.

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Summary/Conclusion: SET gene and PP2A are important factors in CML progression and can be used as possible targets for treatment in TKI-resistant cases.

PB1894: MIR-199A EXPRESSION PROFILES IN CHRONIC MYELOID LEUKEMIA: A PRELIMINARY STUDY

R. Frikha 1,* , I. Belguith 2 , K. Leila 2

1 Medical Genetics, University Hospital of Sfax; 2 LGMH, Faculty of Medicine, Sfax, Tunisia

Background: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells. Up to date, although there is a certain number of reports on microRNA expression features, the expression and function of miR-199a in chronic myeloid leukemia remain to be investigated.

Aims: We aimed to investigate miR-199a expression in CML patients and correlate with minimal residual disease (MRD).

Methods: RT-PCR was performed to measure the expression level of miR-199a.

Quantitative assessment of the BCR-ABL transcript was performed using the Cepheid Xpert BCR-ABL ultra-assay.

Results: This study was conducted in 12 CML patients treated with TKI therapy.

MiR-199a is highly expressed in CML patients mainly with a deeper molecular response (Ratio=33.36 vs 5.94 for DMR and MMR respectively).

Summary/Conclusion: Despite the limit of our study, it underscores the importance of MiR-199a expression, as potential marker of optimal molecular evolution in patients under TKI therapy. Data from a larger number of patients are needed to reveal the clinical relevancy of MiR-199a expression in CML Tunisian patients.

PB1895: ANALYSIS REVEALS CANCER-ASSOCIATED GENE MUTATIONS IN CHRONIC MYELOID LEUKEMIA PATIENTS ：A CHINESE MONOCENTRIC RETROSPECTIVE STUDY

J. Huang 1,* , S. Wang 1 , X. Xu 1

1 Department of Hematology, the Fourth Affiliated Hospital, College of Medicine,Zhejiang University, YiWu, China

Background: Recent genetic studies have shown that tyrosine kinase inhibitors (TKI) resistant or intolerant Chronic myeloid leukemia (CML) patients often carry one or more genetic changes related to myeloid malignancies, suggesting that additional mutations other than ABL1 mutation may lead to TKI treatment failure or disease progression.

Aims: We performed sequencing of 22 CML patients using next-generation sequencing (NGS). 23 CML patients tested WT1 expression detection.

Methods: The second cohort (No. 1) tested by second-generation sequencing included 22 CML patients. The second cohort (No. 2) tested by WT1 expression detection included 23 CML patients. The study has been approved by the institutional ethics committee and patients provided written informed consent in accordance with the Fourth Affiliated Hospital of Zhejiang University School of Medicine.Definitions Response was defined as BCR-ABL1 transcript levels according to the International Scale (IS) ≤10%, ≤1% and ≤0.1% at 3, 6 and 12 months after initiating TKI-therapy. Molecular response with 4.0 log reduction (MR4.0) was defined as 0.01%. Molecular response with 4.5 log reduction (MR4.5) was defined as 0.0032% or less BCR-ABLtranscript level. The comparison between groups of measurement data showed that the data with normal distribution were analyzed by one-way ANOVA or t-test.

Results: Overall, a total of 51 mutations involving 25 genes were detected most frequently in the ASXL1 (40.9%), KMT2C (27.3%), DIS3 (18.2%), ATM (13.6%), DNMT3A (13.6%) and NOTCH3 (13.6%) genes. NOTCH3 and RELN mutations were only 3/20 (10%) in those who did not reach 12M-MMR group. Although the difference was not statistically significant (P = 0.214, P = 0.476). The number of median mutant genes in reaching MR4.5 group was 1 (0-3). The number of median mutant genes in not reaching MR4.5 group was 4 (0-6), and the difference was statistically significant (P = 0.033). ASXL1 mutation in reaching MR4.0 group and non reaching MR4.0 group was statistically significant (P = 0.023). With KMT2C mutation group had lower PFS (P = 0.079). There was a potential statistical difference between low WT1 expression group and high WT1 expression group in early EMR after a 3-month treatment (P = 0.089).The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011; the 2019–2024 Academician Workstation of the Fourth Affiliated Hospi-tal of the Zhejiang University School of Medicine; the 2019–2022 Key Medical Dis-cipline (Hematology) Fund of Jinhua, China. *Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China.E-mail: househuang@zju.edu.cn

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Summary/Conclusion: Genomic risk assessment can improve the risk stratification of clinical diagnosis, and can more accurately identify patients with poor effect of TKI treatment in the era of TKI treatment. The treatment effect of CML patients with NOTCH3, RELN and ASXL1 mutations and more than two gene mutations is poor. CML patients with high expression of WT1 at diagnosis have poor therapeutic effect in the early stage.

PB1896: WHOLE EXOME SEQUENCING IDENTIFIES A NOVEL TRANSCRIPTION FACTOR ASSOCIATED WITH FATAL DISEASE PROGRESSION IN CHRONIC MYELOID LEUKEMIA

Z. Iqbal 1,* , M. Absar 2 , T. Akhtar 2 , N. Alanazi 1 , M. Shammas 3 , S. Basit 4 , A. Mahmood 5

1 College of Applied Medical Sciences (CoAMS-A), King Saud Bin Abdulaziz University for Health Sciences / KAIMRC/ SSBMT, Al-Ahsa, Saudi Arabia; 2 Hematology, Oncology and Pharmaco-genetic Engineering Sciences (HOPES) Group, Health Sciences Laboratories (HaSiL), Department of Zoology, Faculty of Biological Sciences, University of the Punajb, Lahore, Pakistan; 3 Dana Farbar Cancer Institute, University of Harvard, Boston, United States of America; 4 Centre for Genetics and Inherited Diseases, Taibah University, Madinah; 5 College of Medicine, King Saud University, Riyadh, Saudi Arabia

Background: Chronic Myeloid Leukemia (CML) primarily develops as a result of t(22;9) that creates Philadelphia chromosome and oncogenic BCR-ABL fusion transcript (1). Discovery of BCR-ABL paved the way for designing signal transduction inhibitor drugs called tyrosine kinase inhibitors (TKIs) that have revolutionized CML treatment in 21st century, by making survival of CML equal to general population (2). Nevertheless, TKIs are minimally effective CML patients progressed to blast crisis (BC-CML). Unfortunately mechanism of CML progression is poorly understood (2).

Various transcription factors (TFs) are associated with different diseases and progression of many cancers (3). Although a large number of TFs are involved in pathogenesis of acute leukemias, very little is known about role of TFs in CML progression.

Aims: Therefore, objective of this study was to find about novel TFs associated with CML progression.

Methods: Patient selection: Whole-exome sequencing (WES) was employed to find out mutations in novel TF-genes associated with accelerated phase (AP-CML) and blast crisis phase CML (BC-CML) patients (Experimental groups 1 & 2, respectively). Chronic phase treatment-naïve CML patients (CP-CML) were used as control 1, CP-CML CML long-term TKI responders (at least 3 continuous years of MMR) as control group 2 and TK-resistant CML patients as control group 3, along with healthy controls.

Sample collection: DNA extraction and Clinical follow-up: 10 ml peripheral blood was collected from all study subjects. DNA was extracted and patient follow-up was carried out during course of this study. All treatment criteria per ENL guidelines were adopted.

Whole Exome Sequencing (WES): WES was carried out using Illumina NGS instrument (HiSeq). bcl files were converted to fastq files by using bcl2fastqtool (4). Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio (4). R package was employed to align specific TF-gene mutants to disease phenotypes (5). TF-genes mutated in all AP/BC-CML patients but not mutated in any of control groups were selected. Variants were confirmed using Sanger sequencing.

Results: We found ZNF208 as a novel TF-gene mutated in all AP/BC-CML but in none of the controls.

Summary/Conclusion: ZNF208, a member of zing-finger binding transcription factor family, has been reported to be play role as tumor suppressor gene in various cancers through distant signaling pathways (3,6,7). Further studies are required to find exact role of ZNF208 in CML progression and its evaluation as potential biomarker and novel drug target in AP-and BC-CML.

References:

1: Schwarz A, Roeder I, Seifert M. Cancers (Basel). 2022 Jan 5;14(1):256.

2: Annunziata M, Bonifacio M, Breccia M, Castagnetti F, Gozzini A, Iurlo A, et al. Front Oncol. 2020;10:883.

3: Hauffe L, Picard D, Musa J, Remke M, Grünewald TGP, Rotblat B, et al. Cell Death Discov. 2022 Feb 28;8(1):91.

4: Hashmi JA, Albarry MA, Almatrafi AM, Albalawi AM, Mahmood A, Basit S. Congenit Anom (Kyoto). 2017 Apr 16. doi: 10.1111/cga.12225. [Epub ahead of print].

5: R Core Team (2012). R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org/

6: Roy Choudhury S, Ashby C, Zhan F, van Rhee F. Cancers (Basel). 2021 Dec 17;13(24):6348.

7: Liu B, Li H, Liu X, Li F, Chen W, Kuang Y, et al. CCell Signal. 2021 Aug;84:110012.

PB1897: DISCONTINUATION OF IMATINIB IN PATIENTS FOLLOWED FOR CHRONIC MYELOID LEUKEMIA IN DEEP MOLECULAR RESPONSE

Y. Bouchakor Moussa 1,* , S. Taoussi 2 , S. Oukid 2 , F. Lamraoui 2 , N. Rekab 2 , K. M. Benlabiod 2 , H. Brahimi 2 , M. T. Abad 2 , M. Bradai 2

1 HEMATOLOGY, EHS ELCC CAC BLIDA Laboratory of Research on Malignant Hemopathies and Hemoglobinopathies, Faculty of Medicine, University Blida1, B.P 270, Route de Soumaa, Blida, Algeria; 2 HEMATOLOGY, EHS ELCC CAC BLIDALaboratory of Research on Malignant Hemopathies and Hemoglobinopathies, Faculty of Medicine, University Blida1, B.P 270, Route de Soumaa, Blida, Algeria, BLIDA, Algeria

Background: Treatment-free remission (TFR) in CML is defined as achieving a deep molecular remission (DMR) corresponding to molecular remissions of grade 4 or >4. Classically, Imatinib allows it to be achieved in nearly 20% of cases.

Aims: We present our experience of stopping Imatib (IMc) (a copy of Imatinib)

Methods: Methods and patients

This was a study of 25 patients (pts): median age at diagnosis 40 years (9-62) including 2 children and 23 adults, at the time of discontinuation 50 years (18- 73), sex ratio M/F=0.92 (12H/13F); the sokal: 11 pts are intermediate risk, 8 low risk and 6 high risk. According to ELTS, 10 pts low-risk, 10 intermediate-risk, and 5 high-risk, all in chronic phase, all Mbcr/abl and received IMc at 400 mg/day, the first since June 2007 and the last in October 2013. The pts were followed by GeneXpert automated PCR, until deep MR [MMR4 and MMR4.5](Xpert BCR-ABL Ultra test) was achieved and maintained for more than 24 months with a hindsight of more than 5 years. Follow-up of discontinuation was done by PCR every month (2 to 3 months during covid-19); resumption of IMc if BCR/ABL transcript > 0.1% and maintenance of IMc discontinuation, if transcript ≤ 0.1% with monitoring. Deep MR (DMR) was completed at a mean of 70 months; 2 pts (8.3%) were in MMR4 and 23 pts (91.7%) in MMR4.5. with a median follow-up of 8.5 years (6 - 12 years). Imatib discontinuation was started in January 2019

Results: Results of discontinuation

CHR was maintained in 24 pts, relapse at 6 months in one pt. TFR was lost in 9 pts (36%): [2 pts (20%) at 1 month, 3 pts (30%) at 2 months, 5 pts (50%) at 6 months]. The median duration of TFR was 23.5 months, 16 pts (64%) remained in DMR (2 children and 14 adults), no progression, withdrawal syndrome noted in 36% (9pts) at one month of stopping, mainly musculoskeletal pain in 28% (7pts), skin rash with pruritus in 12% (3 pts), asthenia in12%, palpebral myositis; among patients who presented these events 66.6% (6 pts) are still in TFR. For relapsed patients, the resumption of IMc was effective in 9 patients, with MMR4 and MMR4.5 achieved on average at 6 months,

Summary/Conclusion: Comments: the FIT rate is 64% in our cohort, the relapses (9/25) occurred very quickly, during the first 6 months, the duration of impregnation in our patients who lost their TFR was on average 9 years (6-12 years), without difference with the patients who maintained the FIT which is 8.7 years (6- 13). Sokal and ELTS scores had no influence on FIT since 44.4% (4pts) of intermediate, 33.3% (3pts) of low and 2 of high risk relapsed vs 37.5% (6pts) of intermediate, 25% (4pts) of low and 4 of high risk in FIT, an influence of gender since 77.7% of relapses were men, age and even depth of MMR are all potential factors for cessation but the results in our cohort. The results in our cohort do not confirm their influence since 88.8% of our relapses were in MMR4.5 with a median age of 45 years (42-69)

Conclusion : The A-STIM and EURO-SKI studies demonstrate that the depth of response before stopping does not play a prognostic factor for a good FIT, which is confirmed by our study; therapeutic impregnation is the only factor ensuring the maintenance of the FIT found in the studies less in our cohort, hence the factors ensuring the maintenance of the FIT in CML are still to be determined, in order to select the eligible patients.

PB1898: BONE MARROW APLASIA – A RARE COMPLICATION OF IMATINIB THERAPY IN CML PATIENTS: ABOUT A CASE

W. chawad 1,* , S. fari firdaous 1 , F. Ibourk Idrissi 1 , S. belkhettab 1 , A. taouch 1 , H. khibri 1 , N. mouatassim 1 , W. ammouri 1 , M. maamar 1 , H. harmouche 1 , Z. Mezalek Tazi 1 , M. adnaoui 1

1 internal Medecine and Clinical Hematology, Ibn Sina’s Hospital, Rabat, Morocco

Background: Chronic myeloid leukemia (CML) belongs to the family of myeloproliferative neoplasms. Tyrosine kinase inhibitors (TKIs) constitute the reference therapeutic family. They have revolutionized the future of patients because of their ability to prevent, in the majority of cases, the once inevitable progression of CML into fatal acute leukemia. Imatinib, a 1st generation TKI, is a drug that is generally well tolerated in the majority of patients. However, a significant number of hematological complications has been reported.

Aims: TKI-induced bone marrow aplasia is a rarely described complication of TKI. We wanted to highlight this poorly recognized entity.

Methods: We report the case of a patient with CML, who developed severe bone marrow failure after treatment with Imatinib.

Results: A 25-year-old man presented himself to the emergency in October 2019. He reports fatigue, weight loss, fever and heaviness in the left hypochondrium. Physical examination noted the presence of cutaneous pallor associated with splenomegaly. Biologically, we found a hyperleukocytosis at 74 giga made up of neutrophils (66 giga), anemia at 5g/dl and a thrombocytosis at 669 giga. The blood smear showed a harmonious myelemia with blasts at 4% rate. The initial analysis of the bown marrow aspirate showed a myeloid hyperplasia at 82% with a blast rate of 1%. The diagnosis of CML was confirmed by the presence of the Philadelphia chromosome and the detection of the bcr-abl fusion transcript at 86%. The patient initially received cytoreductive treatment based on hydroxyurea for 1 week before the introduction of imatinib at a dose of 400 mg/day. One month after, the splenomegaly reduced and the blood count showed a neutropenia at 900, which leaded initialy to an adaptation of the doses of imatinib to 300 mg / day and resuming after that to the optimal dose. Six months after the initiation of the TKI at 400 mg/day, the patient presented a severe bone marrow failure. The blood count found a non regenerative pancytopenia with hemoglobin at 5g/dl, agranulocytosis at 40 elements/mm3 with thrombocytopenia at 6000/mm3 leading to definitive discontinuation of imatinib. The bown marrow aspirate was poor. On karyotype, all mitoses still presented the Phi chromosome. The osteo-medullary biopsy revealed a medullary hypoplasia. In the absence of an HLA-compatible donor and antilymphocyte serum, the patient was treated with thrombopoeitin analogs at a dose of 2 ug/week along with transfusion support, witch showed a good clinical and biological evolution.His last blood count is strictly normal and he has not been transfused in the last 6 months.

Summary/Conclusion: TKIs have revolutionized the treatment of CML. In fact, most of imatinib side effects are moderate and subside over time. However, ITK can also lead to rare complications, such as bone marrow failure. Clinical and hematological monitoring with the study of bone marrow apirate will be required at regular intervals for the early recognition of this phenomenon. Patients will be referred early to an allograft of bone marrow,thus avoiding the fatal consequences of sepsis due to neutropenia or hemorrhage on severe thrombocytopenia

PB1899: DEVELOPMENT AND VALIDATION OF A PREDICTIVE SCORING SYSTEM FOR THE PROGNOSIS IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) IN CHRONIC PHASE RECEIVING FIRST-LINE IMATINIB

M. Bektaş 1 , A. E. Eşkazan 2,* , T. O. Tiryaki 3 , R. Mullai 4 , T. Elverdi 2 , A. Salihoğlu 2 , M. Tiribelli 4 , M. C. Ar 2 , S. Öngören 2 , Z. Başlar 2 , M. Nalçaci 3 , T. Soysal 2

1 Department of Internal Medicine; 2 Department of Internal Medicine, Division of Hematology, Istanbul University-Cerrahpaşa, Cerrahpaşa Faculty of Medicine; 3 Department of Internal Medicine, Division of Hematology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey; 4 Division of Hematology and BMT, University of Udine, Udine, Italy

Background: Since the introduction of tyrosine kinase inhibitors (TKIs), the management of chronic myeloid leukemia (CML) has changed significantly, and in patients (pts) with optimal responses, survival is generally similar to that of age- and sex-matched normal population. World Health Organization stated that, the life expectancy at birth for women is 5 years longer than that of men. With knowing this fact, sex might have an impact on survival in patients with CML. There are 4 risk scores currently in use for the prediction of outcomes and survival in CML including Sokal, Euro-Hasford, European Treatment and Outcome Study (EUTOS), and EUTOS long-term survival (ELTS). But none of these scores contain sex as a parameter.

Aims: In our study, we aimed to propose a new prognostic score for pts with CML in chronic phase (CML-CP), including sex as a parameter.

Methods: Our training cohort (TC) included CML-CP pts diagnosed between 2003-2018 receiving imatinib as a first-line TKI therapy. While generating the new prognostic score – C errahpaşa C ML P rognostic S core (CCPS), uni- and multivariate Cox regression analysis were performed in order to identify factors having an impact on progression and survival. Beta coefficients were used in creating this prognostic score. The area under the receiver operator characteristic curve (AUROC) was used to estimate accuracy of the predictive model. The CCPS had 3 parameters; age, blast %, and sex (Age (years) x 0.085 + Blast (%) x 0.489 + Sex (Male) x 1.449) and 3 risk groups [Low: <6.62, Intermediate: 6.62-7.85, and High: >7.85)]. Progression-free (PFS) and overall (OS) survivals were evaluated by Kaplan-Meier curves. For the validation of this new score, two independent validation cohorts (VCs) were used.

Results: TC consisted of 185 pts, of which 103 (55.7%) were male, and the median age at diagnosis was 47 years (Table 1). First VC (VC1) had 86 pts, and median age was 44 years. All pts in VC1 received imatinib as upfront treatment. Second VC (VC2) consisted of 183 pts, and 62.8% of these cases were male. The median age of VC2 at diagnosis of was 59 years, which was higher than TC and VC1 (Table 1). While 148 (80.9%) had first-line imatinib, the remaining 35 pts (19.1%) received upfront second-generation TKI (2GTKI) therapy (10 pts with dasatinib and 25 pts with nilotinib). In TC, 60.5%, 25.9%, and 13.5% of the cases were in low-, intermediate-, and high-risk groups for the ELTS score, respectively. For the CCPS, the percentages of low-, intermediate-, and high-risk patients were 64.9%, 20.5%, and 14.6%, respectively (Table 1). Distribution of the ELTS and CCPS for VC1 and VC2 are displayed in Table 1. AUROC for the new prognostic score was the highest among others for TC, and for both VC1 and VC2 (Figure 1A-C). In the survival analysis of TC, ELTS score successfully dissected 3 risk groups both for PFS and OS ( p =0.013 and p <0.001, respectively) (Figure 2A-B). Similarly, according to the CCPS, the low-risk group had significantly superior PFS and OS ( p =0.025 and p <0.001, respectively) in TC (Figure 2C-D). For VC2, according to the CCPS, 3 risk groups significantly differed ( p <0.001) for OS, however, although PFS was inferior for the high-risk group, the difference was not significant ( p =0.063) (Figure 2E-F).

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Summary/Conclusion: We developed and validated a score using sex as a parameter for the first time, which successfully predicts PFS and OS in pts with CML-CP receiving frontline imatinib. Although some pts in VC2 received upfront 2GTKIs, the CCPS should further be tested especially in cases receiving 2GTKIs in the first-line setting.

PB1900: COVID-19 IMPACT ON CHRONIC MYELOID LEUKEMIA PATIENTS

S.-M. Galdean 1,* , R. Arbore 2 , R. Ababei 1 , D. Dragancea 1 , R. A. Tomai 1 , A. Trifa 1 , S. Pasca 3 , D. M. Dima 1

1 Hematology, Oncology Institute “Ion Chiricuta” Cluj-Napoca; 2 Farmacy; 3 Hematology, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

Background: The management of CML (chronic myeloid leukemia) patients in COVID-19 era can be a challenge. CML patients are prone to severe infections and an increased mortality risk. There is an unmet need of clear and updated information in CML and COVID-19.

Aims: We present our department’s experience on COVID-19 prevalence and severity in a cohort of CML patients diagnosed and treated at „ Prof. Ion Chiricuta” Oncology Institute, Hematology department. We report the SARS-CoV-2 vaccination rate and the vaccine’s adverse events.

Methods: We analyzed the data on 286 CML patients, from our data base. 51 patients gave information on COVID-19 and vaccintaion status, through phone interview. Statistical data were obtained using R 3.5.1 software. Fisher test was used for contingent tables. Standard normal distribution was obtained using Shapiro-Wilk’s test. P value under 0.05 was considered statistically significant.

Results: The patients were diagnosed with CML from 1996 to 2020. Median age at diagnosis was 48 and 54.9% were males.

62.7% patients had a low Sokal score, 29.4% intermediare Sokal score, 3.9% high Sokal score, respectively 80% patients had a low Eutos score.

74.5% patients were treated with Imatinib, 49.01% with Dasatinib, 41.1% with Nilotinib and 7.8% with Ponatinib, and 5.8% with Bosutinib.

MMR was obtained in 84.3% patients, CHR in 11.7% and 3.9% patients lost their hematologic response.

COVID-19 diagnosis was established by RT-PCR/ rapid antigen test or specific symptoms in 10 patients, during February and May 2021.

24 patients received SARS-CoV-2 vaccination in January to August 2021.

Among COVID-19 patients, 30% discontinued on TKI treatment during COVID-19.

The only parameter correlating with COVID-19 risk was the Hb value at diagnosis.

Among vaccinated patients, 7 presented adverse events: 3 had local pain, 3 fever and myalgia, 1 digestive symptomes. There was no relation of vaccine adverse events and hematologic parameters at diagnosis or TKI treatment.

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Summary/Conclusion: COVID-19 prevalence in CML patients is 0.9%, similare to the general population (0.1%).

In România, the anti-COVID19 vaccination rate with complete doses, at current moment is 27.5%, and in our CML patient cohort the vaccination rate was 47%. The safety profile of vaccine is similar for CML patients and general population.

The COVID-19 risk is not higher in CML patients.

TKI treatment does not negatively influence the COVID-19 risk or response to vaccine.

NCCN, ESMO and local guidelines reccommend for CML patients to vaccinate for SARS-CoV-2.

PB1901: KARYOTYPIC PROFILE OF CHRONIC MYELOID LEUKAEMIA DIAGNOSED AT TERTIARY LEVEL IN AFGHANISTAN.

A. M. Haidary 1,* , S. Noor 2

1 Haematopathology and Blood Bank, French Medical Institute for Mothers and Children; 2 Haemato-oncology, Jumhoriat Hospital, Kabul, Afghanistan

Background: Current guidelines for chronic myeloid leukemia (CML) management include utilization of both conventional cytogenetics as well as advance molecular analysis at diagnosis and while monitoring the therapy. The reason being the fact that karyotype analysis is one of the indispensable tools for identification of additional chromosomal abnormalities of prognostic significance.

Aims: We conducted a descriptive case-series study of 33 patients to demonstrate the profile of karyotype abnormalities in CML.

Methods: A descriptive “case-series” study was conducted from 1st January, 2020 to 31st January. 2021, including 33 patients who were diagnosed with CML, after acquisition of informed consent from patient or next of kin. For inclusion in the study, patients either had to have demonstrable “pathognomonic” clinic-morphological features of splenomegaly, bone marrow and/ or peripheral blood hyperleukocytosis, and bi-modal peak of mature neutrophils with myelocytes, or presence of leucoerythroblastic picture with myeloproliferative features, where conventional karyotyping revealed presence of Ph, into chronic phase (CP), accelerated phase (AP) and blast phase/ crisis (BC) considering the recommendations by World health Organization (WHO) classifications [11]. The statistical analysis was performed using statistical package for social sciences (SPSS), version 25.

Results: Out of 33 patients included in our study, 18 (54.5%) were female and 15 (45.5%) were male. The age at diagnosis ranged between 12 to 75 years with median age at diagnosis being 42 years. 20 patients were permanent residents of Kabul, 3 patients were from Wardak province, two patients were from Ghazni province, while from Badakhshan, Balkh, Bamyan, Jouzjan, Khost, Nangarhar, Paktia and Parwan, there were only one patient each. Considering the disease phase at diagnosis, 30 (90.9%) of patients were in chronic phase (CP), one patient (3%) in accelerated phase (AP) and two patients (6.1%) in blast crisis/ phase (BC). Considering the cytogenetic profile at diagnosis, as shown in Figure 1, 25 (75.7%) patients had only a single Ph chromosome, while in addition to Ph, one patient (3%) had t (11;17), one patient (3%) had t (7;14), one patient (3%) had derivative of chromosome 22 (t (9;22) (q34q11)) resulting in double Ph, one patient had de-novo 5q deletion (5q13.3 to q35.3) that we have reported elsewhere in the literature and two patients who presented with pathognomonic clinic-morphological feature of CML had normal karyotype, thus were advised to proceed either with fluorescent in situ hybridization or Multiplex Qualitative PCR for BCR-ABL1, for disease confirmation. Unfortunately, due to financial constrains the two patients opted to rather consider tyrosine kinase inhibitory (TKI) therapy and achieved complete hematological remission (CHR) within a span of one month.

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Summary/Conclusion: Conventional cytogenetics enables to identify additional cytogenetic abnormalities that can have prognostic significance in CML patients with significant impact upon the management plan. Since most of our patients deferred follow-up, it would be ideal to counsel the patients regarding significance of regular follow-up, to ensure better outcomes.

PB1902: ASC4OPT TRIAL IN PROGRESS: A PHASE 3B OPEN-LABEL OPTIMIZATION STUDY OF ORAL ASCIMINIB IN CHRONIC MYELOGENOUS LEUKEMIA IN CHRONIC PHASE PREVIOUSLY TREATED WITH 2 OR MORE TYROSINE KINASE INHIBITORS

M. Breccia 1,* , A. Turkina 2 , C. Boquimpani 3 , C. Chuah 4 , G. Sharf 5,6 , P. Schuld 7 , M. Hoch 7 , J. Yssel 8 , Y. Zhang 9 , A. Hochhaus 10

1 Division of Hematology, Department of Translational and Precision Medicine, Policlinico Umberto 1, Sapienza University, Rome, Italy; 2 National Research Center for Hematology, Moscow, Russia; 3 HEMORIO, State Institute of Hematology Arthur de Siqueira Cavalcanti, Rio de Janeiro, Brazil; 4 Singapore General Hospital, Duke-NUS Medical School, Singapore, Singapore; 5 Israeli CML Patients Organization, Netanya, Israel; 6 CML Advocates Network, Bern; 7 Novartis Pharma AG, Basel, Switzerland; 8 Novartis Ireland Limited, Dublin, Ireland; 9 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 10 Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany

Background: Tyrosine kinase inhibitors (TKIs) are the standard of care in the treatment of chronic myeloid leukemia (CML). However, patients (pts) may experience resistance or intolerance to successive lines of TKI therapy. Asciminib is the first BCR::ABL1 inhibitor that works by Specifically Targeting the ABL Myristoyl Pocket (STAMP). It was recently approved in the United States for the treatment of adults with Philadelphia chromosome–positive (Ph+) CML in chronic phase (CP) previously treated with ≥2 TKIs (at 40 mg twice daily [BID] or 80 mg once daily [QD]), and also Ph+ CML-CP with the T315I mutation (at 200 mg BID). This was based on the Phase 3 ASCEMBL trial (Rea et al. Blood 2021) and a Phase 1 study (Cortes et al. ASH 2020). The ASC4OPT ( NCT04948333 ) trial was initiated to investigate the potential optimization of treatment with asciminib in adult pts with CML-CP previously treated with ≥2 TKIs (≥3 rd -line).

Aims: ASC4OPT will explore 3 aspects of treatment optimization: 1. Pt population , ASCEMBL inclusion criteria were based on the European LeukemiaNet (ELN) treatment recommendations from 2013. ASC4OPT will re-evaluate asciminib in an expanded ≥3 rd -line population using the 2020 treatment recommendations for warning and failure criteria. 2. Simplified posology , asciminib dosing in ASCEMBL was 40 mg BID. BID dosing may affect optimal compliance for pts and ASC4OPT will investigate both asciminib 40 mg BID and 80 mg QD. 3. Dose escalation , with limited treatment options in the ≥3 rd -line setting, ASC4OPT will explore whether pts not in major molecular response (MMR) at ≥48 weeks (wks) could benefit from a potential dose escalation to 200 mg QD.

Methods: ASC4OPT is an international, multi-center, non-comparative, Phase 3b study in pts aged ≥18 years with CML-CP previously treated with ≥2 TKIs. The trial will enroll approximately 156 pts who are in treatment failure or warning (ELN 2020) or intolerant to the last prior TKI. Intolerant pts must not have achieved MMR at baseline visit. Up to 30 additional pts in MMR at baseline and intolerant to their most recent TKI treatment will also be enrolled; these pts will not be part of the primary endpoint analysis; however all assessments will be performed. Pts are excluded if they have had previous or planned hematopoietic stem cell transplantation, second CP after previous progression to accelerated phase/blast crisis, or known presence of the BCR::ABL1 T315I mutation at any time prior to study entry. Pts will be randomized 1:1 to receive asciminib 40 mg BID or 80 mg QD. In pts not achieving MMR at 48 wks or losing response after wk 48 and up to wk 108, asciminib dose may be escalated to 200 mg QD if in the investigator’s opinion the pt may benefit from the escalation. In addition, for dose escalation there must not be any grade 3 or 4 toxicity while on therapy, or persistent grade 2 toxicity, possibly related to asciminib and unresponsive to optimal management (Figure). The primary endpoint is MMR rate ( BCR::ABL1 IS ≤0.1%) at wk 48 in pts not in MMR at baseline. Secondary endpoints include MMR rate at wks 12, 24, 36, 72, 96 and 144 in pts not in MMR at baseline, MMR rate at wk 48 for pts in MMR at baseline, time to MMR, rate of early responses of BCR::ABL1 IS ≤10% and ≤1% at wks 12, 24, 36 and 48, deep molecular responses (rates of MR 4.0 [ BCR::ABL1 IS ≤0.01%] and MR 4.5 [ BCR::ABL1 IS ≤0.0032%] at wks 12, 24, 36, 48, 72, 96 and 144), rate of cytogenetic response at wk 48 and at end of treatment (up to 144 wks), and patient-reported outcomes and quality of life.

Results: Trial in progress.

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Summary/Conclusion: The study is currently recruiting.

PB1903: TRIAL IN PROGRESS: A MULTICENTER, OPEN-LABEL, PHASE IB/II STUDY TO DETERMINE THE DOSE AND SAFETY OF ASCIMINIB IN PEDIATRIC PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE (CML-CP)

N. Hijiya 1,* , S. Kapoor 2 , M. Hoch 3 , L. Descamps 4 , M. A. Bayar 4 , N. Ramscar 5

1 Columbia University Medical Center, New York; 2 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 3 Novartis Institute for BioMedical Research, Basel, Switzerland; 4 Novartis Pharma SAS, Rueil-Malmaison, France; 5 Novartis Pharma AG, Basel, Switzerland

Background: Tyrosine kinase inhibitors (TKIs) are the standard of care for adult and pediatric patients (pts) with CML-CP. Five TKIs are approved for adults: imatinib, dasatinib, nilotinib, bosutinib, and ponatinib. However, only imatinib, dasatinib, and nilotinib are approved for pediatric pts. Efficacy and safety profiles of TKIs are similar in adult and pediatric pts; however, growth retardation has been reported in the pediatric population, in whom long-term adverse effects are unknown. Therefore, safer and more efficacious options are needed for pediatric pts. Asciminib is a recently approved drug that inhibits BCR::ABL1 by S pecifically T argeting the A BL M yristoyl P ocket (STAMP). In the phase III ASCEMBL trial for adults with heavily pretreated CML-CP, asciminib (40 mg twice daily [BID]) had an improved major molecular response rate (25.5% vs 13.2%) at week 24 and a better safety profile compared with bosutinib (500 mg once daily). Here we present the phase Ib/II trial evaluating asciminib in pediatric pts.

Aims: The primary objectives of this study are to characterize the pharmacokinetic (PK) profile of asciminib in pediatric pts and identify a pediatric formulation dose leading to an asciminib exposure comparable to the adult dose, 40 mg BID.

Methods: This is a multicenter, open-label study ( NCT04925479 ). Eligible pts have Philadelphia chromosome–positive CML-CP treated with ≥1 prior TKI, do not have a T315I mutation, and are aged ≥1 to <18 years, provided informed consent. Adolescent pts aged ≥14 to <18 years and weighing ≥40 kg will be enrolled in the exploratory adult formulation group, receiving the adult dose, until the pediatric formulation is available.

In part 1 (dose-determining cohort), 4 to 6 pts aged ≥1 to <18 years will receive the pediatric formulation (body weight–adjusted dose of asciminib taken with food) to obtain ≥4 participants evaluable for PK data. This evaluation will determine whether median exposure in pediatric pts is comparable to asciminib 40 mg BID taken without food in adult pts. PK and safety will be assessed during the first 28 days after treatment initiation.

In part 2 (expansion cohort), the body weight–adjusted dose determined in part 1 will be used, and the total pts enrolled on pediatric formulation will be increased to 30, including those from part 1 (15 pts aged 1 to <12 years and 15 pts aged 12 to <18 years). Pts receiving the adult formulation may switch to the pediatric formulation in part 2 but will not be counted toward the 30 pts enrolled in part 2. A 2nd interim PK and safety analysis will be conducted after all pts in part 2 have completed 28 days of treatment.

The total duration of the study treatment period is 5 years (Figure). The primary PK endpoints, safety, and pharmacodynamics will be assessed after all pts have been on study for 52 weeks or discontinued earlier. After all pts have been on study for 5 years or discontinued earlier, a final analysis will be performed. Pts who discontinue the study early will be followed up for survival until the study is complete.

Results: The primary endpoints include PK parameters. Secondary endpoints include hematologic and molecular responses, safety, acceptability, and palatability of asciminib.

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Summary/Conclusion: Data from this study will be used to provide a pediatric dose and support a strategy of full extrapolation from adult data to use in the pediatric setting.

PB1904: TRIAL IN PROGRESS: A PHASE III STUDY OF ASCIMINIB VS AN INVESTIGATOR-SELECTED TKI IN PATIENTS WITH NEWLY DIAGNOSED CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE (CML-CP)

T. Hughes 1,* , J. Cortes 2 , N. Takahashi 3 , R. Larson 4 , G. Issa 5 , F. Bombaci 6 , N. Ramscar 7 , S. Kapoor 8 , S. Ifrah 9 , A. Hochhaus 10

1 SA Pathology and South Australian Health and Medical Research Institute, University of Adelaide, Adelaide, Australia; 2 Georgia Cancer Center, Augusta, United States of America; 3 Akita University, Akita City, Japan; 4 The University of Chicago, Chicago; 5 The University of Texas MD Anderson Cancer Center, Houston, United States of America; 6 CML Advocates Network, Turnin, Italy; 7 Novartis Pharma AG, Basel, Switzerland; 8 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 9 Novartis Pharma AG, Paris, France; 10 Universitätsklinikum Jena, Jena, Germany

Background: The main treatment goal for patients (pts) with CML-CP is disease control. Pts may be treated with 1 of 4 tyrosine kinase inhibitors (TKIs) approved for 1st-line (1L) use: the 1st-generation TKI imatinib and the 2nd-generation (2G) TKIs bosutinib, dasatinib, and nilotinib. However, in 1L CML-CP, >50% of pts treated with imatinib develop resistance to or intolerance of therapy, and 30% to 40% of pts treated with a 2G TKI need to change therapy by 5 years. In addition, only 30% to 55% of pts achieve a deep molecular response (DMR), an important criterion for attempting treatment-free remission. While 2G TKIs yield a higher major molecular response (MMR) than imatinib, they are associated with more adverse events (AEs), which can include arterio-occlusive events. Therefore, highly potent and safe treatment options are needed for pts with 1L CML-CP.

Asciminib is a recently approved drug that inhibits the BCR::ABL1 oncoprotein through a novel mechanism of action: S pecifically T argeting the A BL M yristoyl P ocket (STAMP). In the phase III ASCEMBL trial in pts with CML-CP treated with ≥2 prior TKIs, asciminib resulted in improved MMR (25.5% vs 13.2%) at week 24 and fewer treatment discontinuations due to AEs (5.8% vs 21.1%) compared with bosutinib. Here we present a phase III trial evaluating asciminib vs an investigator-selected approved TKI in adult pts with newly diagnosed CML-CP.

Aims: The primary objectives of this study are to assess the efficacy of asciminib vs that of an investigator-selected TKI in the 1L and compare the efficacy of asciminib with that of the stratum of pts receiving investigator-selected imatinib in the 1L via the primary endpoint of MMR at week 48. The study will be positive if either of these objectives is met. Secondary objectives include long-term efficacy outcomes and safety.

Methods: This is a multicenter, open-label, randomized, phase III study ( NCT04971226 ) of asciminib 80 mg once daily (QD) compared with an approved, investigator-selected TKI (imatinib, bosutinib, dasatinib, or nilotinib) in the 1L (expected N=402). Pts must not have received prior TKI therapy for CML except for ≤2 weeks of imatinib therapy and must have Eastern Cooperative Oncology Group performance status of 0 or 1 and provided informed consent. Pts could have received hydroxyurea or anagrelide for urgent disease control. Pts will be randomized 1:1 to the asciminib arm or investigator-selected TKI arm using 2 strata: European Treatment and Outcome Study long-term survival risk score and the control arm TKI selected by the investigator before randomization (Figure). Pts randomized to the investigator-selected TKI arm will receive their selected TKIs at approved doses: imatinib 400 mg QD, bosutinib 400 mg QD, dasatinib 100 mg QD, or nilotinib 300 mg twice daily.

Pts will remain on study for 5 years after the last pt’s 1st treatment has been administered, unless they have discontinued early due to treatment failure, disease progression, intolerance, or investigator/pt decision. Pts who discontinue early will continue to be followed up for survival and disease progression until the end of the study

Results: The primary endpoint is MMR at week 48. Secondary endpoints include MMR at week 96, safety, and DMR; exploratory endpoints include biomarker assessments.

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Summary/Conclusion: This study will assess the efficacy of asciminib in adult pts with newly diagnosed CML-CP vs that of currently approved TKIs.

PB1905: FEASIBILITY OF TREATMENT DISCONTINUATION IN CHRONIC MYELOID LEUKEMIA IN CLINICAL PRACTICE: RESULTS FROM A SERIES OF 19 PATIENTS

F. KACHA 1,* , D. LAMARA 2 , A. BOUDAOUD 1 , A. RABHI 1 , S. ADJA 1 , K. BOUMKHILA 1 , M. SAÏDI 1

1 Unversity of Batna 2 Algeria, Hematology, Batna; 2 Constantine university regional military hospital, Hematology, Constantine, Algeria

Background: The therapeutic of chronic myeloid leukemia (CML) has profoundly changed over the past years. Patients have now almost normal life expectancy with an excellent quality of life

The main goal is to achieve a stable deep molecular response (DMR) and to discontinue medication for treatment-free remission (TFR) for patients with chronic phase CML (CP-CML).

The data from the published literature demonstrate that 40–60% of patients achieve TFR, with relapses risk occurring within the first six months, almost all patients who relapsed regained a molecular response upon retreatment, indicating TKI discontinuation is safe.

The ELN defines DMR as undetectable BCR–ABL1 mRNA transcripts in the blood using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and/or two consecutive high-quality PCR samples with >10 4 sensitivity.

For patients aiming at TFR, the optimal response (at any time) is BCR-ABL1 ≤ 0.01% (MR4) for at least two years. The loss of MMR (BCR-ABL1 > 0.1%) indicates failure after TFR

Aims: to follow patients having CML-CP who have achieve DMR 4.0 or more for at least two years, to allow TKl discontinuation with TFR as an aim.

Methods: This is a retrospective cohort study of patients diagnosed with CML-CP in Hematology department

The study was approved by an ethical review authority of the medical department. All consenting patients with a reported diagnosis of CML-CP between 1 January 2009 and 31 December 2016 and whiling an TKI discontinuation were identified.

Data regarding patients with TKI treatment interruption were gathered as follows: last TKI prior to discontinuation, date of discontinuation, time to DMR and duration of treatment by lmatib before discontinuation, BCR-ABL1 IS (International Scale) percentage prior to at least 2 years beforeTKI discontinuation, if patient had re-initiated TKI treatment, date and reason of re-initiation and if the patient had progress to AP or BP

An intensive monitoring is recommended earlier in the first six months after stopping TKI discontinuation. A peripheral blood monitoring with PCR analysis every 4 weeks for 6 months, then every 8 weeks for 12 months, then every 3 months thereafter.

TKI was re-initiated at any time if loss of MMR

First-line therapy is based on a TKI; imatinib brand is not available in Algeria so we use generics, dasatinib and nilotinib are the only other ITKs but not approved as first line therapy.

Statistical methods were treated by IBM SPSS statistics 26

Results: were included for analysis 19 patients. Date of treatment discontinuation was between January and December 2019, last follow-up was 1 st January 2022 and median follow-up time was 15 months from discontinuation (range 4–34 months). All patients received lmatib as first line therapy at a dose of 400 MG/day and they have all achieved a stable DMR for two (or more) years. All patients who relapsed regained a molecular response upon retreatment, despite one who progressed to AP. 35,8% have maintained a molecular remission after discontinuation.

Summary/Conclusion: Compared to the first prospective proof of concept for stopping TKI treatment, the Stop Imatinib 1 (STIM1) trial which showed that 38% of the patients maintained a molecular remission after a median follow-up of 77 months, we notice in this small cohort that although we did not reach the median of fallow up that our results are quiet similar.

In our country generic imatinib(Imatib) is the cost-effective initial treatment in CML-CP.

PB1906: IS THERE A PLACE FOR IMATINIB IN THE FIRST-LINE TREATMENT OF CHRONIC MYELOID LEUKEMIA CHRONIC PHASE: REAL LIFE DATA FROM UNIVERSITY CENTER AFTER LONG PERIOD OF FOLLOW-UP

D. Lekovic 1,2,* , J. Jovanovic 1 , V. Djordjevic 1 , V. Milosevic 1 , J. Bodrozic 1 , A. Bogdanovic 1,2

1 Clinic of Hematology, University Clinical Center Serbia; 2 Medical faculty, University of Belgrade, Belgrade, Serbia

Background: The introduction of tyrosine kinase inhibitors (TKIs) in the treatment of chronic myeloid leukemia (CML) significantly changed the natural course of the disease and increased the overall survival of these patients. Any of three TKIs (imatinib, nilotinib, dasatinib) can be used in the first treatment line (I TL). So far, no significant survival difference between imatinib and second generation TKIs has been observed. The place of imatinib in first-line therapy of CML is not yet clearly defined.

Aims: of our study was to analyze the cytogenetic and molecular response of patients treated with imatinib 400mg daily in the I TL as well as to identify their corelation with duration of survival.

Methods: This retrospective study included 331 consecutive CML patients (chronic phase) who were diagnosed and treated at Clinic of Hematology, University Clinical Center of Serbia in period of 20 years. Diagnosis of CML was made according to the ELN criteria. Data were extracted from medical records. In all patients Sokal, Hasford, EUTOS and ELTS prognostic scores were analyzed.

Results: The median age of patients at diagnosis was 52 years (range 18-82), with 28.4% ≥ 60 years and 51.2% were male. Hepatomegaly had 25.7% of patients while splenomegaly 71.6%. The cytogenetic response during imatinib treatment was as follows: 72.8% achieved CCyR, 9.1% majorCyR, 3.3% minorCyR while 14.2% had no response. After median follow-up of 88 months, 40.5% was switched to another therapy due to primary resistance (26.6%), secondary resistance mainly incompetence in taking the drug (11.5%) and adverse events (2.4%; 2/6 patients had hepatic toxicity verified by liver biopsy in the first 6 months of imatinib treatment). Patients with primary resistance had significantly higher white blood cell counts (p=0.019), percentage of blasts in peripheral blood (p=0.019), and spleen size (p=0.004) at the time of diagnosis than the patients with secondary resistance. Significant predictors of survival were: achievment of CCyR (p=0.001), percentage of blasts in peripheral blood (p=0.008) and Hasford score (p=0.010). The median survival of patients who achieved CCyR during treatment with imatinib at a dose of 400 mg daily was 218 months (95% CI, 204-232) while in those who did not achieve was 152 months (95% CI, 120-185) (p = 0.001). Patients with CCyR on imatinib therapy achieved MR3.0 in 45,6%, MR4.0 in 27.4%, MR4.5 in 14.5% and MR5.0 in 12.5% of the patients. Overall survival after 5, 10, and 15-years was 95.7%, 92,5%, and 84,2% respectively. Fatal outcome had 17.8% of patients with the cause being the progression of underlying disease in 51% and comorbidities in 49% mostly cerebrovascular stroke (7 pts), heart disease (7 pts) and secondary malignancies (6 pts).nalyzed.

Summary/Conclusion: During the treatment with imatinib 400mg in I TL, 2/3 of patients achieved and maintained CCyR. Patients with achieved CCyR had significantly longer survival and 54% of patients achieved at least deep molecular response (MR4.0). Survival after 10 years is over 90% without fatal adverse events. Due to imatinib resistance during follow-up, 40.5% of patients require the use of newer generation of TKI. TKI selection should be based on treatment goals, age and comorbidities and should take into consideration the adverse event profile of the available drugs.

PB1907: CLINICAL VALUE OF DETECTING ABL KINASE MUTATIONS IN CHRONIC MYELOID LEUKEMIA BASED ON HIGH THROUGHPUT SEQUENCING TECHNOLOGY

Z. LING 1 , L. XIAODONG 1,* , W. XIANWEI 1 , Z. ZHE 1 , W. LINLIN 1 , L. YANGWEI 1

1 Central Lab, Henan Cancer Hospital, Zhengzhou, China

Background: Tyrosine kinase inhibitor (TKI) is effective in the treatment of chronic myeloid leukemia (CML), but about 20% ~ 30% of patients still have TKI resistance, which is mainly due to the drug resistance mutation in ABL kinase region.In order to further optimize TKI treatment and achieve the best treatment response, sensitive and accurate detection of ABL kinase region mutation has important clinical value.Sanger sequencing is the gold standard for detecting mutations in ABL kinase region, but its sensitivity is low, the proportion of mutations that are easy to be missed is less than 15 ~ 20%, and it is a qualitative detection, which can not distinguish the mutated clone structure.In recent years, high-throughput sequencing technology has gradually become a routine clinical diagnostic method for blood tumors. Its sensitivity to detect mutations can reach 1%, and the clonal structure of mutations can be analyzed according to the variant allele frequency (VAF value) of mutation sites to further clarify the clonal structure of mutations.This study compared and analyzed the efficiency of high-throughput sequencing and Sanger sequencing in detecting ABL kinase region mutations, further clarified the clinical value of high-throughput sequencing in detecting low proportion mutations and multipoint mutations in ABL kinase region, and provided basis for TKI precision treatment.

Aims: To compare the efficacy and clinical impact of high throughput sequencing and SS (Sanger sequencing) in detecting ABL kinase domain mutations.

Methods: A total of 198 samples of 147 CML patients from July 2017 to March 2021 in Henan cancer hospital were collected and sequenced by high throughput sequencing and SS to detect the mutation of ABL kinase domain, and relevant clinical data were collected for comparative analysis.

Results: The proportion of mutations and ≥ 2 mutations detected by high throughput sequencing was significantly higher than that by SS ( P =0.046). Of the 22 cases with ≥ 2 mutations, 5 (23%) were found to have compound mutations. In 198 samples, 25 (13%) were low proportion mutations, 33 (17%) were high proportion mutations and 10 (5%) were mixed high and low proportion mutations. In the analysis of related clinical factors, the proportion of mutation detection and the proportion of low mutation in the optimal, failure and warning response were gradually increased (mutation proportion, P =0.016; P =0.038); The proportion of mutation with additional chromosomal abnormalities was also significantly increased ( P =0.009).The mutation rate of patients receiving first-line and second-line treatment was significantly lower than that of patients receiving third-line and fourth line treatment ( P =0.006).According to the VAF value analysis of mutation sites, it is helpful to visually evaluate the clonal evolution status of TKI-resistance in CML.

Summary/Conclusion: High throughput sequencing is more sensitive and accurate than Sanger sequence in mutation detection, which is helpful for accurate and visual evaluation of TKI treatment response and optimization of treatment strategy for CML.

PB1908: COVID-19 INSIGHTS IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA: DIAGNOSIS AND TREATMENT

V. Musteata 1,*

1 Discipline of Hematology, State University of Medicine and Pharmacy “N. Testemitanu”, Institute of Oncology, Chisinau, Moldova

Background: The recent published reports pointed to the increased incidence of COVID-19 in patients with hematologic neoplasms as compared to the apparently healthy population. The growing incidence of COVID-19 and chronic myeloid leukemia (CML) in a workable population may be considered as an important issues of hematooncology and public health.

Aims: The aim of the study was the determination and assessment of the current epidemiological and diagnosis features of COVID-19 in patients with CML in order to adjust the management of this hematologic malignancy

Methods: We realized the analytical, observational and prospective study of a group of 17 patients with CML, who were treated and followed up in the Institute of Oncology from the Republic of Moldova between 2005–2021. COVID-19 was diagnosed between March 2020 – November 2021 only in these 17 patients out of the whole cohort of CML cases treated with tyrosine kinase inhibitors (TKIs). The quantitative reverse transcription polymerase chain reaction (RT-PCR) was accomplished with the aim to determine the expression of the BCR-ABL p210 and p190 transcripts. Nucleic acid amplification testing with a RT-PCR assay was performed in order to detect SARS-CoV-2 RNA from the upper respiratory tract, as the preferred initial diagnostic test for COVID-19. The study assessed the impact of Sokal score on COVID-19 morbidity in CML patients.

Results: These 17 cases constituted 11.9% of 142 patients diagnosed with CML and followed up under the treatment with TKIs. There were 10 (58.8%) males and 7 (41.5%) females, with the male:female ratio 1.4:1. The patients’ age at diagnosis of COVID-19 ranged between 38-74 years (average age 52.3 years). Seven (41.5%) patients were under 50 years of age, 10 (58.8%) patients were over 50 years, and 6 of them were over 60 years of age. According to the WHO classification of COVID-19 severity, mild forms were registered in 4 (23.5%) cases, moderate in 8 (47.1%) and severe in 5 (29.4%) cases. The ECOG score of performance status of CML patients ranged between 0-2 at the time of diagnosis of COVID-19. The ECOG score was 2 only in 2 (12.5%) patients, who experienced severe COVID-19. SARS-CoV-2 affected 8 (50.0%) low-risk patients, 5 (31.3%) intermediate-risk and 3 (18.7%) high-risk patients. COVID-19 developed in 14 (82.4%) patients with complete hematologic response and in 3 (17.6%) patients without stable response to treatment with TKIs. Nine (52.9%) patients were hospitalized for treatment of COVID-19, including all 5 (29.4%) with severe and 4 (23.5%) with moderate forms. Viral pneumonia was diagnosed by radiological examinations in 7 (41.2%) patients, including 5 (29.4%) with severe and 2 (11.8%) with moderate COVID-19. Non-infectious comorbidities were registered in 4 of 5 patients with severe COVID-19 form.

Summary/Conclusion: COVID-19 affected commonly CML patients of male gender. The older CML patients were more susceptible to COVID-19. Sokal score did not influence the COVID-19 morbidity in CML patients. Severe SARS-CoV-2 infection occurred in CML patients with serious non-infectious comorbidities. Patients with accelerated and acute phases exhibited the unfavorable correlative evolution of CML and COVID-19.

PB1909: A STUDY ON TROUGH LEVELS OF GENERIC DASATINIB AND ITS CORRELATION WITH ADVERSE EVENTS AND EFFICACY.

A. Nair 1,* , A. khadwal 1 , A. jain 1 , S. attri 2 , S. pattnaik 3 , S. Naseem 4 , N. verma 4 , A. jandial 1 , D. lad 1 , G. prakash 1 , P. malhotra 1

1 CLINICAL HEMATOLOGY AND MEDICAL ONCOLOGY; 2 pediatric biochemistry; 3 CLINICAL PHARMACOLOGY; 4 Hematology, PGIMER, CHANDIGARH, India

Background: Generic dasatinib has flooded the Indian market since April 2020. It has made dasatinib a feasible option as 1 st or 2 nd line TKI in our patients. Various studies worldwide have shown correlation between Cmin (trough levels) and incidence of pleural effusion. Through this study we try to collect pharmacokinetic data on generic dasatinib in our population.

Aims: To study the relationship between trough levels of generic dasatinib drugs with adverse events and molecular response in patients with Ph+ve ALL and CML.

Methods: This study was a prospective observational study.It was conducted over a period of fourteen months i.e., from November 2020 to December 2021. Patients of Ph+ve ALL and CML were prospectively screened for inclusion (minimum 1 month of drug intake) and exclusion criteria (poor compliance, inappropriate sample collection). Selected 51 patients were enrolled in the study. Blood samples were collected for doing dasatinib trough levels and the patients were followed up over 6 months from the time of sample collection for adverse events and molecular responses. The dasatinib drug levels were measured by Liquid chromatographic mass spectrometry (LCMS) technique.

Results: The median age of overall study cohort was 39 years (range 13-74). There were 15 (29.6%) females and 36 (70.4%) males, with Male: Female ratio of 2.4:1. Among the CML patients, 22 patients (68.75%) were in chronic phase, 3 (9.3%) in accelerated phase and 7 (22.8%) in blast phase with all of them being myeloid blast crisis. One patient was on ATT (1.96 %), fifteen on azoles (30.61 %), fourteen on PPI/H2RB/Antacid (28.57 %), and none on antiepileptics.

Achievement of Early molecular response (EMR), Major molecular response (MMR) or deep molecular response (DMR) was not related to the dose of drug. In ALL patients, there was no statistically significant correlation between dose of dasatinib and achieving MRD negativity (p – 0.794).

The median Cmin in the study population was 1.21 ng/ml (range 0 – 20.5). Cmin was found to be similar across various age groups with a median of 1.17 ng/ml, 1.42 ng/ml and 1.46 ng/ml for age group 20-39 years, 40-59 years and ≥ 60 years respectively (range – 0-22.5, p – 0.346). Median Cmin values were higher in those taking 140mg of dasatinib (3.22 ng/ml), as compared to lower doses but the trough levels for 50 mg and 100 mg were similar (1.46 ng/ml and 1.175 ng/ml) and lowest for those taking 70 mg of dasatinib (0.745 ng/ml), but there was no statistically significant correlation among them.

Pleural effusion was seen in 6 patients (11.76%) with two patients each in grade1, grade 2 and grade 3. Patients with pleural effusion had higher median Cmin values when compared to those without (2.17 ng/ml vs. 1.18 ng/ml, p value – 0.558). Relation between cytopenias and Cmin was analysed in CML patients, amd no statistical significant correlation was found between Cmin and toxicity profile in the overall study cohort. Patients taking azoles, PPI’s, H2-receptor blocker, ATT etc. did not show any statistical significance with their Cmin values as compared to those not taking these drugs.

Summary/Conclusion: In this prospective study of generic dasatinib Cmin levels in patients with Ph+ve leukemias, there was no statistical significant correlation between Cmin values and incidence of pleural effusion which has been demonstrated in DARIA01 and OPTIMDASATINIB study. Efficacy of dasatinib did not correlate with dosage or with Cmin levels. The small sample size and shorter follow up are the main drawbacks of this study.

PB1910: CHRONIC MYELOID LEUKEMIA AND RELATIONSHIP TO SECONDARY SOLID NEOPLASMS

M. J. Ramos 1,* , R. Peixeiro 2 , L. Regadas 2 , C. Pedrosa 2 , P. Seabra 2 , A. Mota 2 , R. Cabral 2 , R. Coutinho 2 , L. Xavier 2 , C. Casais 2 , J. Rodrigues 2 , I. Freitas 3 , C. Gonçalves 2

1 |Oncologia Médica; 2 Hematologia Clinica; 3 Hematologia Laboratorial, CHUP, Porto, Portugal

Background: Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm whose first-line treatment involves Tyrosine Kinase Inhibitors (TKI), that have improved the prognosis of the disease after its introduction, with a gain in the overall survival of these patients. In patients in the chronic phase of the disease, the most common cause of death is associated with other neoplasms, which raises the hypothesis that the observed mortality could be the result of a carcinogenic effect of tyrosine kinase inhibitors. Since studies to date have had contradictory results, this work aims to assess the risk of developing secondary solid neoplasms (NSS) in patients with CML.

Aims: This study aims to assess the risk of developing SSN in patients with CML.

Methods: Patients with positive BCR-ABL transcripts were analysed in a University Hospital Center between 01/01/2008 and 15/02/2022.

Results: Sample of 69 patients (53.6% women) all in the chronic phase at diagnosis. Median age 53 years (23-85 years). In the first therapeutic line, most of them underwent TKI (91.3% underwent Imatinib and 5.7% underwent nilotinib). In the total sample, 69.5% needed only 1 therapeutic line. Eight (11.6%) died, only in 2 cases the death was related to CML.

Nine patients (11.9%) developed SSN, a majority men (n=8, 88.9%) with a median age of 69 years (57-74 years) at diagnosis. All of them were diagnosed between 14 and 121 months. The majority of patients had gastrointestinal cancer (n=4, 44.4%), followed by prostate cancer (n=3, 33.3%), head and neck cancer (n=1, 11.1%) and lung cancer (n=1, 11.1%). All were exposed to TKI and Imatinib was common to all. Two patients died due to SSN, corresponding to 25% causes of death in the total study sample.

When statistically comparing the occurrence of SSN with the time between the diagnosis of CML and the second neoplasm or the duration of the different therapeutic lines, there was no correlation with statistical significance (p>0.05).

Summary/Conclusion: SSN appeared in about 12% of patients with CML. Contrary to what is described in the literature, the sample of patients with SSN is mostly made up of older men. As the risk of developing neoplasms in the general population is greater in the elderly, the development of cancer in these patients can be considered to be similar to that in the general population. The greatest risk for the development of SSN is described in the 1st year after the diagnosis of CML, which was not verified in our study. As limitations of the study the authors highlight the reduced sample as well as the need for a longer follow-up period.

PB1911: A COMPREHENSIVE EHEALTH IMPLEMENTTION GUIDE CONSTRUCTED ON A QUALITATIVE CASE STUDY ON BARRIERS AND FACILITATORS OF THE DIGITAL CARE PLATFORM CMYLIFE, FOR PATIENTS WITH CHRONIC MYELOID LEUKEMIA

L. Verweij 1,* , Y. Smit 1 , N. Blijlevens 1 , R. Hermens 2

1 Hematology; 2 IQ Healthcare, Radboudumc, Nijmegen, Netherlands

Background: Substantial proliferation of eHealth has enabled a move in patient-centred cancer care from the traditional in-person care model to real-time, dynamic, and technology supported on-demand care. However, in general, the uptake of these innovations is low. Studies show that eHealth is helpful in providing patient empowerment through e.g. providing high quality and timely information, enabling self-monitoring and shared decision making, but dropout rates are high and guidance for optimal implementation is lacking.

Aims: To explore barriers to and facilitators for nationwide implementation and consolidation of CMyLife, a multi-component, patient-centred, digital care platform for patients with chronic myeloid leukemia, and to construct a comprehensive implementation guide for digital care platforms in general.

Methods: The first qualitative case study of a digital care platform like CMyLife was performed including five focus group- and eighteen in-depth interviews with stakeholders. Data were collected using a semi-structured interview guide, based on the frameworks of Grol and Flottorp. Transcripts of the interviews were analysed and barriers and facilitators were identified and categorized according to the frameworks. An iterative process including participation of the main stakeholders and using the CFIR-ERIC framework led to creating a comprehensive implementation guide for digital care platforms in general.

Results: In total, 45 barriers and 41 facilitators were identified. Main barriers were lack of connectivity between information technology systems, changing role for both health care providers and patients, insufficient time and resources, doubts about privacy and security of data, and insufficient digital skills of users. Main facilitators mentioned were motivating patients and health care providers by clarifying the added value of use of a digital care platform, clear business case with vision, demonstrating (cost) effectiveness, using an implementation guide, and educating patients and health care providers about how to use CMyLife. Based on these barriers and facilitators a clear and comprehensive implementation guide was developed for digital care platforms in general.

Summary/Conclusion: Several barriers to and facilitators for implementation were identified, a clear overview was presented, and a unique comprehensive implementation guide was developed for lancing future digital care platforms in daily clinical practice. The next step is to validate the implementation guide in other (oncological) diseases.

PB1912: MYELODYSPLASTIC SYNDROME WITH ISOLATED 5Q DELETION AND ASSOCIATED 5Q DELETION TO 13Q DELETION

N. Bouayed Abdelmoula 1,* , B. Abdelmoula 1 , A. Karra 1

1 Genomics of Signalopathies at the service of Medicine, Medical University of Sfax, Sfax, Tunisia

Background: The 5q deletion is the classic chromosomal abnormality in myelodysplastic syndrome (MDS). Critical regions are 5q31 and 5q33, leading to gene haplodeficiency and other pathogeneic mechanisms. 13q deletion occurs in about 2% of MDS cases. This deletion involved band 13q14.

Aims: Here we describe cytogenetic abnormalities in a Tunisian cohort of MDS patients and we shed the light on cases with 5q deletion.

Methods: Cytogenetic analyses of bone marrow cells were performed according to standard procedures for 12 Tunisian patients presenting with MDS. Cytogenetic analysis was performed on lymphocytes and bone marrow cells. Cultured cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature.

Results: Cytogenetic abnormalities were detected in 9 cases and del5q was recorded in four patients (3 patients with refractory anemia (RA) and a fourth with RA and excess blasts-T (RAEB-T)). The del5q was isolated in two RA cases as a del (5) (q13 q33) in a new diagnosed and a treated patients. It was associated in two patients: a RA patient explored during the evolution, harboring a del (5) (q12 q33) associated to a del (13) (q12 q14) and the RAEB-T patient harbouring a del (5) (q21 q31) associated to a clonal hyperploidy.

Summary/Conclusion: The diagnosis of MDS was based on cytomorphology and cytogenetics. However, nowadays, an increasing number of molecular aberrations have been discovered and included in the WHO revised new classification of myeloid neoplasms. The del13q described in MDS seems to be a marker of the evolution of the clonal cells harboring 5q deletion. However, the molecular basis and the implicated genes of the deletion of the long arm of chromosome 13 associated to the 5q deletion clone remain until now not well explored.

PB1913: SYNERGISTIC EFFECTS OF DECITABINE AND APR-246 IN TP53 P.248Q-MUTATED MYELODYSPLASTIC SYNDROME

Y. Fang 1,2,* , C.-K. Chang 1

1 Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital; 2 State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Background: Mutations of TP53 gene confers inferior prognosis in myelodysplastic syndromes. Decitabine (DAC) has been used as the cornerstone drug for MDS treatment. However, less than half of MDS patients maintain a long-term response to DAC, and the maintenance time is usually less than one year.

Aims: It is of great importance to reveal the therapeutic response mechanism of DAC to TP53 -mutated MDS, explore related targets of DAC with short remission period, and optimize combined targeted therapy.

Methods: We investigated the effect of small molecule inhibitor of TP53 mutation (APR-246) in combination with decitabine on the proliferation of MDS/AML cells (SKM-1, NB4 and KASUMI-1) which carries the recurrent homozygous TP53 mutation p.R248Q ( TP53 p.248Q ). We further explore the associated mechanisms of combination therapy.

Results: We found DAC combined with APR-246 had a synergistic effect on tumor cells with TP53 p.248Q mutation in vitro . DAC activated a series of endogenous retroviruses (ERV) and upregulated TLR3 which activated anti-tumor immunity by promoting IFN-α secretion. However, IKK phosphorylation was also increased and induced NF-κB activation. Interestingly, this side-effect can be suppressed by APR-246, thereby synergistically promoting apoptosis of tumor cells.

Summary/Conclusion: These results provided important evidence on optimizing existing therapeutic strategies for MDS/AML with TP53 p.248Q .

PB1914: CYTOGENETIC FINDINGS IN 18 CHILDREN WITH MYELODYSPLASTIC SYNDROMES BY KARYOTYPIC AND FISH ANALYSIS

K. Manola 1,* , P. Diamantopoulou 1 , C. Kelaidi 2 , L. Petrikkos 2 , T. Ioannidou 1 , K. Stefanaki 3 , E. IOANNIDOU 4 , N. Tourkantoni 5 , K. Tsitsikas 2 , M. Kourti 6 , K. Antoniadi 2 , E. Dana 7 , I. Pelagiadis 8 , E. Mantadakis 9 , M. Economou 10 , A. Makis 11 , E. Papakonstantinou 12 , H. Kosmidis 7 , E. Stiakaki 8 , G. Paterakis 13 , A. Kattamis 14 , I. Peristeri 4 , D. Pantelia 15 , M. I. Margariti 1 , P. Nakopoulos 1 , C. Sambani 1 , S. Polychronopoulou 2

1 Department of Biodiagnostic Sciences & Technologies, INRASTES, NCSR DEMOKRITOS; 2 Department of Paediatric Haematology-Oncology; 3 Department of Pathology; 4 Bone Marrow Transplantation Unit; 5 Division of Paediatric Hematology-Oncology, 1st Dpt of Pediatrics, National & Kapodistrian University of Athens, Aghia Sophia Children’s Hospital, ATHENS; 6 Department of Paediatric Oncology, Hippokration General Hospital, Thessaloniki; 7 Pediatric & Adolescent Oncology Clinic, “Mitera” Hospital, ATHENS; 8 Department of Paediatric Hematology-Oncology, University Hospital of Heraklion, Heraklion, Crete; 9 Department of Pediatrics, Democritus University of Thrace, Alexandroupolis; 10 Division of Haematology, 1st Dpt of Paediatrics, Aristotle University of Thessaloniki, Hippokration General Hospital, Thessaloniki; 11 Department of Paediatrics, University Hospital of Ioannina, Ioannina; 12 Departments of Paediatric Oncology, Hippokration General Hospital, Thessaloniki; 13 Department of Immunology, Peripheral General Hospital Athens Giorgos Gennimatas; 14 5.Division of Paediatric Hematology-Oncology, 1st Dpt of Pediatrics, National & Kapodistrian University of Athens, Aghia Sophia Children’s Hospital; 15 Biodiagnostic Sciences & Technologies, INRASTES, NCSR DEMOKRITOS, ATHENS, Greece

Background: Pediatric myelodysplastic syndromes (MDS) are rare disorders characterized by hematopoietic cell dysfunction, an increased risk of AML and a poor prognosis. Although it is known that cytogenetic abnormalities are seen in approximately half of children diagnosed with MDS, their detailed cytogenetic profile has not been elucidated yet due to the rarity of the disease.

Aims: We tried to detect chromosomal abnormalities in pediatric patients with MDS, including submicroscopic aberrations such as gene alterations associated with the disease and possible malignant transformation by karyotypic and molecular cytogenetic analysis (fluorescence in situ hybridization, FISH) to contribute to the elucidation of the cytogenetic profile of pediatric MDS and the investigation of the pathogenesis of the disease.

Methods: In this study 18 children with MDS are included. Karyotypic analysis was performed on 24h, 48h and 72h bone marrow (BM) cultures using GTG-banding. Karyotypic analysis was carried out on 25 at least metaphases. FISH analysis was performed on cultured BM cells using IVD probes for centromere (CEP) of chromosomes 7 and 8 and the chromosomal regions 7q22, 7q31, 8q24(MYC), 11q22.3(ATM), 11q23(MLL), and 17p13(TP53). For each sample at least 200 interphase cells (iFISH) and 5 metaphases (mFISH) were analyzed to increase sensitivity. Normal cut-off was estimated at 3%.

Results: The sex ratio was 1,25 (10 M/8 F) and the mean age was 9,42 years (range 0,4y - 19y). Eleven patients had RCC, 5 MDS-EB and 2 t-MDS. Normal karyotypes were detected in 10 patients (55,6%), 6 patients with RCC, 3 with MDS-EB and 1 with t-MDS. Abnormal karyotypes were found in 8 patients (44,4%), 4 with RCC, 2 with MDS-EB and 1 with t-MDS. Monosomy 7 was observed in 2/18 patients (>70% of metaphases), +8 in 2/18 patients (5-6.6% of metaphases), inv(17)(p11.2q21) in 1/18 patients (18.75% of metaphases), complex karyotypes in 2/18 patients (31.3-73.2% of metaphases including abnormalities of 1q, 2q, 4q, 5q, 16q, 18 and Xp), in one patient after SCT in the transformation of MDS into s-AML and in the other at diagnosis with t-MDS) and chromatid and chromosome breaks in 1/18 patients, (8,6-9,6% of metaphases) in 2 different BM samples taken with an interval of 3 months, which were also found in his brother’s BM karyotype. FISH revealed -7 in 2/18 patients confirming their karyotypes, loss of one p53 allele in 3/18 patients at low frequencies (6,5%, 4,5%, 3,5%) all with hypocellular RCC and transfusion-dependency that underwent SCT, +8 in 2/18 (in 8% and 2,7% of cells) confirming trisomy 8 in their karyotypes and loss of one MLL allele and MLL rearrangement in a patient that was transformed to s-AML in 4,4% of cells. A normal pattern of hybridization was observed for the above probes in the rest cases and for MYC and ATM genes in all the examined cases.

Summary/Conclusion: Karyotypic aberrations were detected in 44,4% of children including -7, +8, inv(17), complex karyotypes and chromatid and chromosome breaks, revealing that other chromosome aberrations except -7/del(7q) and trisomy 8 are characterized this disease. Clonal chromosome abnormalities can be detected at low frequencies in the karyotypes of pediatric MDS patients so except from iFISH, mFISH is also needed especially in cases with small clones or submicroscopic translocations. The low frequency of submicroscopic aberrations such as p53 deletions observed in 3 cases of hypocellular RCC must be confirmed in larger studies by molecular analysis as they may play a potential role in MDS pathogenic evolution.

PB1915: SOMATIC ALTERATIONS IN PATIENTS WITH INHERITED MYELOID MALIGNANCIES SYNDROMES: A SINGLE-CENTER STUDY

M. Santiago Balsera 1,2,3,* , A. Liquori 3 , J. V. Gil 4 , G. Avetisyan 3 , Á. Díaz 1,3 , L. Cordón 3 , B. Martín 3 , E. Mora 1 , A. Villalba 1 , B. Sánchez 5 , I. Gómez 6 , C. Moscardó 7 , B. Ferrer 8 , J. Serrano 9 , E. González 3 , M. Morote 3 , C. Sargas 4 , C. García 3 , C. Martínez 3 , B. Fernández 3 , M. Guaita 3 , A. San Juan 3 , M. Ibáñez 3 , E. Barragán 3,4 , P. Montesinos 1,3 , J. de la Rubia 1,3 , Á. Zúñiga 2 , G. Sanz 1,3 , E. Such 1,3 , J. Cervera 1,2,3

1 Hematology; 2 Genetics; 3 Hematology Research Group; 4 Molecular Biology, Hospital La Fe, Valencia; 5 Hematology, Hospital Vinalopó, Elche; 6 Hematology, Hospital General Albacete, Albacete; 7 Hematology, Hospital General Alicante Dr Balmis, Alicante; 8 Hematology, Hospital Clinico Universitario, Valencia; 9 Hematology, Hospital Reina Sofia, Cordoba, Spain

Background: Clinical heterogeneity, variable latency, and incomplete penetrance characteristic of inherited myeloid malignancies syndromes (HMMSs) suggest that the acquisition of additional alterations are potential drivers of clonality and are required for myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) onset. However, despite advances in the identification of germline variants, predicting the clinical implications of clonal abnormalities in these syndromes is still challenging.

Aims: We aim at exploring the putative associations between germline and somatic molecular signatures found in a cohort of consecutive patients diagnosed with HMMS at Hospital La Fe, in Valencia (Spain).

Methods: We examined paired germline/somatic samples from 43 MDS and 75 AML consecutive patients along with 5 patients selected from our historical cohort (4 MDS and 1 GATA2-deficiency). The median age was 56.5 (range 12-81 years). Thirteen cases were considered therapy-related. The germinal samples included 89 fibroblasts cultured from a skin biopsy, 29 CD3 + lymphocytes isolated from peripheral blood (PB), 1 bone marrow in remission, and 5 PB. We analyzed them, through a targeted next-generation sequencing (NGS) using a capture-based custom gene panel of 180 genes to assess the diagnosis of HMMSs, and two commercial panels to investigate somatic alterations. Conventional cytogenetics was available in 118 (96%) patients and NGS-based somatic profiling in 115 (93.5%).

Results: Sixteen (13.5%) patients from the consecutive cohort and 5 patients from the historical cohort presented (likely) pathogenic germline variants in genes responsible for HMMSs. Findings derived from cytogenetics and molecular characterization in these patients are shown in Figure1. High risk karyotype was higher in patients with solid cancer predisposition compared to the rest of patients of the series (71% vs 29%; p=0.02). Somatic NGS analysis found 42 clinically significant variants in genes commonly altered in myeloid neoplasms. The most recurrently somatic mutated genes were RUNX1 (5 variants) and TP53 (4 variants). The mean number of somatic mutations per patient in group associated with platelet disorders and group associated with cancer solid predisposition (3.3 and 3 respectively) was similar to patients with a sporadic neoplasm of the same cohort (2.9). However, DDX41 group and group associated with organ dysfunction presented a lower number of somatic mutations per patient (1). In addition, 30.7% of therapy-related neoplasms in our series presented (likely) pathogenic germline variants in contrast with 15,4% in sporadic neoplasms (p=ns). They showed a 2.25 mean number of somatic changes and a high-risk karyotype in 3 out of 4 cases.

Figure 1. Somatic acquired alterations in patients with inherited myeloid malignancies syndromes. AML/SMD-t: AML/SMD therapy-related. NK: normal karyotype. CK: complex karyotype. Chr: chromosome. Blue boxes represent variants with allele frequency (VAF) >10%. Yellow boxes represent variants with VAF <10%.

Image:

Иллюстрация — в первоисточнике ↗

Summary/Conclusion: In our series, 13.5 % of patients had a potential HMMS, being the incidence higher in those with a therapy-related neoplasm (30.7%). Patients with solid cancer predisposition presented a higher risk karyotype. Patients with DDX41 mutations and those HMMSs associated with organ dysfunction presented a lower number of somatic mutations per patient. It is necessary to expand this series to find early detection biomarkers of progression in high-risk families.

PB1916: SOMATIC MUTATIONS ASSOCIATED WITH MYELODYSPLASTIC SYNDROME IN RUSSIAN PATIENTS

A. Kohno 1,* , B. Biderman 2 , A. Sudarikov 2 , S. Treglazova 2 , A. Abdullaev 2 , S. Kulikov 3 , Y. Chabaeva 3 , V. Dvirnyk 4 , T. Obukhova 5 , V. Troitskaya 1 , E. Parovichnikova 1

1 National Research Center for Hematology, Moscow, Russia; 2 Department of Molecular Hematology; 3 Biostatistics; 4 Dapartment of Clinical Diagnostics; 5 Department of Cytogenetics, National Research Center for Hematology, Moscow, Russia

Background: Myelodysplastic syndrome (MDS) is characterized by the presence of clonal hematopoiesis, with genetic mutations detected in 90% of cases. Their significance in the pathogenesis, clinical picture, and prognosis of the disease has been actively studied in recent years.

Aims: To study somatic mutations and their prognostic significance in Russian patients with MDS.

Methods: The study included 48 patients with MDS, aged 19-80 years (Me-50), 25M/23F. According to WHO classification (2017), the variants of the disease were identified as MDS with ringed sideroblasts - 7 (14.6%), MDS with multilinear dysplasia - 16 (33.3%), MDS with 5q- - 8 (16.7%), MDS with an excess of blasts - 17 (35.4%). Cytogenetic analysis revealed normal karyotype in 18 cases (37.5%), an abnormal karyotype in 29 (60.4%), and in 1 case (2.1%) no mitosis was observed. A custom panel (Qiagen, USA) was designed for targeted next-generation sequencing (NGS) of SF3B1, U2AF1, SRSF2, ZRSR2, DNMT3A, TP53, ASXL1, RUNX1, TET2, NRAS, GATA2, and EZH2 genes. The results were analyzed using the open-source QIAseq Targeted DNA Variant Calling software (Qiagen, USA). Only somatic mutations were included in the final analysis (based on the VAF value).

Results: In 34 (70.8%) MDS patients somatic mutations were detected. In 17 of 34 (50.0%) mutations were found in 2 or more genes. The distribution by frequency of detection was as follows: ASXL1 - 12 (25.0%), RUNX1 - 10 (20.8%), DNMT3A - 8 (16.7%), TET2 - 8 (16.7%), SRSF2 - 6 (12.5%), SF3B1 - 5 (10.4%), U2AF1 - 4 (10.4%), ZRSR2 - 3 (6.3%), TP53 - 2 (4.2%), NRAS - 2 (4.2%), GATA2 - 2 (4.2%), EZH2 - 0. For 18 patients with normal karyotype, somatic mutations were detected in 13 cases (72.2%) cases. These were mutations in ASXL1 - 3 (23.1%), RUNX1 - 4 (30.8%), DNMT3A - 1 (7.7%), TET2 - 2 (15.4%), SRSF2 - 3 (23.1%), SF3B1 - 4 (30.8%), U2AF1 - 2(15.4%), ZRSR2 - 1(7.7%), TP53 - 1(7.7%), and NRAS - 1 (5.0%) genes. Mutations in a single gene were detected in 8 patients (61.5%) patients; double mutations - in 3 patients (23.1%); 3 or more mutations were found in 2 cases (15.4%). Of the above mentioned, only SF3B1 gene mutations can be associated with a favorable prognosis. When analyzing the mutational profile in MDS patients with 5q- (n=8), mutations were detected in 50.0% of cases and occure in genes: DNMT3A - 3 (37.5%), TET2 - 1 (12.5%), ASXL1 - 2 (25.0%), TP53 - 0. In 2 cases with a combination of mutations in the DNMT3A + TET2 and DNMT3A + ASXL1 genes, progression to AML was noted. In a unifactorial analysis, for all MDS patients progression to AML was significantly associated with mutations in the RUNX1 (p=0.001) and SRSF2 (p=0.0026) genes. While worst survival was associated with mutations in the RUNX1 (p=0.0012), SRSF2 (p= 0.0101) and U2AF1 (p=0.0276).

Summary/Conclusion: The data obtained on 48 patients with MDS demonstrate a high frequency of somatic mutations (70.8%), including those detected in cases with normal karyotype (72.2%). An unfavorable prognosis was associated with RUNX1 , SRSF2 , and U2AF1 gene mutations. Extending of a patient sample is required to assess the predictive value of other somatic mutations in MDS.

PB1917: PROGNOSTIC AND PREDICTIVE SIGNIFICANCE OF ELEMENTS OF THE ADAPTIVE IMMUNITY IN THE MICROENVIRONMENT OF MYELODYSPLASTIC SYNDROMES TREATED WITH 5-AZACITIDINE

Z. Tsakiraki 1,* , S. Papageorgiou 2 , A. Spathis 1 , A. Pouliakis 1 , A. Bouchla 2 , T. Thomopoulos 2 , I. G. Panayiotides 1 , V. Pappa 2 , P. Foukas 1

1 2nd Department of Pathology; 2 2nd Department of Internal Medicine and Research Institute, Hematology Unit, National and Kapodistrian University of Athens School of Medicine, “Attikon” University Hospital, Athens, Greece

Background: Myelodysplastic syndromes (MDS) are an heterogenous group of clonal hematopoietic neoplasms.

Aims: Our objectives were to investigate the immune micro-environment of bone marrow (BM) trephine biopsies from patients with high risk MDS and whether elements of the adaptive immunity predict response to 5-Azacitidine (5-AZA) treatment.

Methods: The study population consisted of 65 patients with intermediate and high-risk MDS (n=57) according to IPSS-R and oligoblastic acute leukemias (n=8), treated with 5-AZA. Sections from BM trephine specimens before (n=65) and during treatment (n=28) (interval: 6-12 months), were immunostained for CD3, CD8, CD20, CD138 and Foxp3, for T cells, cytotoxic T cells, B cells, plasma cells and regulatory T cells, respectively. The slides were digitally scanned and the number of positive cells was counted in five areas with the highest density of CD8(+) cells, a well validated antineoplastic population. Since BM cellularity varied between specimens, all cell counts were normalised to 1mm 2 of tissue area after removing fat and empty spaces. CD4(+) T helper cells were calculated indirectly subtracting cytotoxic from total T cells. The study also included a control group consisting of 20 normal BM trephine biopsies.

Results:

- Patients presented with lower number of FoxP3(+) cells per mm 2 , FoxP3/CD3 ratio and FoxP3/CD8 ratio (p<0.001, Mann-Whitney) compared to controls.

- Patients that had a complete or partial response had higher CD3(+) and CD4(+) T cell density prior treatment (887 vs. 732, p=0.041 and 376 vs 262 p-0.034, Mann-Whitney) and had decreased both populations after treatment (-251 vs. +153 p=0.001 and -148 vs. +58.4, p=0.038, Mann-Whitney).

- In MDS cases before treatment, lower CD3(+) T, CD4(+) T h and CD8(+) T c cell densities, FoxP3/CD3 and FoxP3/CD8 ratios were significantly correlated with decreased overall survival (OS) (p=0.049, 0.045, 0.043, 0.008 and 0.049, respectively). During treatment with 5-AZA, increased levels of CD3(+) T (p=0.012) and CD20(+) B (p=0.012) cells and decreased levels of Foxp3(+) Tregs (p=0.043), FoxP3/CD3 and FoxP3/CD8 ratios (p=0.043, Cox) were significantly correlated with decreased OS.

- In the entire population no significant changes were observed when comparing prior and during therapy cell densities. However, decrease in OS was found when patients retained or increased CD3(+) or CD4(+) cell densities in matched, before and during treatment, samples (p=0.026 and p=0.002, Cox).

- Moreover, higher plasma cell densities before and during treatment were found in patients that transformed to AML earlier (17.9 months vs. 47.5 months, p=0.001 and 22.8 months vs. 50.8 months, p=0.022, Kaplan Meyer).

Summary/Conclusion: Our results indicate that the density of cells of adaptive immunity in the bone marrow specimens may significantly contribute to outcome (overall survival and time to transformation to AML) of high-risk MDS patients and alter response to treatment with 5-AZA.

PB1918: MOLECULAR CHARACTERIZATION AND CLINICAL RELEVANCE OF ERYTHROPOIESIS IN MIELODYSPLASTIC SYNDROMES.

A. Villalba 1,* , E. Such 1 , L. Senent 1 , E. Mora 1 , G. Avetisyan 1 , A. I. Vicente 1 , M. Tormo 2 , A. Jerez 3 , F. Ramos 4 , J. Montoro 5 , T. Bernal 6 , I. lLuna 1 , M. Santiago 7 , A. Diaz 8 , A. Liquori 8 , J. Cervera 7 , G. Sanz 1

1 Hematología y Hemoterapia, Hospital Universitario y Politécnico La Fe; 2 Hematología y Hemoterapia, Hospital Clínico de Valencia, Valencia; 3 Hematología y Hemoterapia, Hospital Morales Meseguer, Murcia; 4 Hematología y Hemoterapia, Hospital Universitario de León, León; 5 Hematología y Hemoterapia, Hospital Universitario Vall d´Hebron, Barcelona; 6 Hematología y Hemoterapia, Hospital Universitario Central de Asturias, Oviedo; 7 Unidad de Genética, Hospital Universitario y Politécnico La Fe; 8 Hematología y Hemoterapia, IIS La Fe, Valencia, Spain

Background: The prognostic relevance of the percentage of erythroid precursors in Myelodysplastic Syndromes (MDS) and its relationship with other clinic-biological characteristics remains largely unknown. Some studies support that the presence of more than 50% and less than 15% of erythroid precursors (EPs) in bone marrow (BM) is a poor prognostic factor in MDS for long-term outcomes. In those cases, a higher frequency of complex karyotypes and monosomies have been observed but the mutational profile associated with the percentage of EPs remains largely unknown.

Aims: Our main aim was to analyze the biological and clinical features of MDS patients according to the percentage of EPs in BM and mutational profile at the time of diagnosis to evaluate its prognostic value on overall survival (OS) and to better understanding the molecular pathogenesis of these disorders.

Methods: We retrospectively analyzed 133 de novo MDS patients from the MDS Spanish Registry. All patients were categorized into two groups according to the percentage of EPs in BM: ≤15% and ≥15%. The molecular study by NGS of the BM samples was performed using MYS (Myeloid SolutionTM) from SOPHiA Genetics. All mutations selected were categorized as benign, likely benign, variant of uncertain significance (VUS), likely pathogenic, or pathogenic by following the “Standards and guidelines for the interpretation of sequence variants” from the American College of Medical Genetics and Genomics. Information from biological databases and in silico was considered. All statistical analysis were performed by the R software.

Results: In Table 1 are displayed the clinical and biological characteristics statistically significant in both groups. The mutation study showed that the most frequently mutated genes in the total series were SF3B1 , which was altered in 39 of the cases analyzed (29%), TET2 in a total of 35 patients (26%) and ASXL1 in 34 patients (26%). Patients with ≤ 15% of EPs showed a statistically significant lower frequency of mutations in SF3B1 (11% vs. 41%; p< 0.001), higher frequency of mutations in DNMT3A (26% vs. 6%; p = 0.003) and increased number of mutations in NRAS (15% vs. 1%; p = 0.003). Patients with ≤ 15% of EPs in BM had a higher frequency of mutations in the signal transduction gene group (5% vs 2%; p = 0.013). The ≤15% EPs group showed a markedly inferior OS (median OS, 24 months versus 69 months in the group with >15% EPs; p < 0.001). However, there were no clear differences between both groups in the risk of transformation to AML. In the multivariate analysis the ≤ 15% of EP group was an independent risk factor on global survival (p = 0,003), as well as, R-IPSS (p = 0.008) and mutations in RUNX1 (p = 0.042) , ASXL (p = 0.004) and TP53 (p = 0.005 ).

Summary/Conclusion: Our study confirms the prognostic impact of EPs percentage in BM on OS and demonstrate notable differences in their mutational profile.

PB1919: S100A9 DEFICIENCY ACCELERATES MDS ASSOCIATED TUMOR ESCAPE VIA PD-1/PD-L1 OVEREXPRESSION

R. Wang 1 , C. Chang 1,*

1 Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China

Background: MDS are mainly characterized by ineffective hematopoiesis contributed to inflammatory signaling or immune dysfunction. Our previous studies have shown that S100a9 expression was higher in low-risk MDS and lower in high-risk MDS.

Aims: In recent studies, the tolerable safety profile and positive BM response suggest a beneficial use of anti-PD-1 agents in the treatment of MDS, but the underlying mechanism is still unknown.

Methods: In this study, skm-1 cells and k562 cells co-cultured with S100a9 acquired apoptosis features. Moreover, we confirmed the inhibition effect S100a9 on PD-1/PD-L1. Importantly, PD-1/PD-L1 blockade and S100a9 can both activate the PI3K/Akt/mTOR signaling pathway. The cytotoxicity was higher in lower-risk MDS-lymphocytes than in high-risk MDS-lymphocytes, and S100a9 partially rescue the exhausted cytotoxicity in lymphocytes.

Results: Our study demonstrated that S100a9 may inhibit MDS associated tumor escape via PD-1/PD-L1 blockade through PI3K/Akt/mTOR signaling pathway activation.

Summary/Conclusion: Our findings indicated the possible mechanisms anti-PD-1 agents may contribute to the treatment of MDS. These insights may provide more accurate treatment as the supplementary therapy for MDS patients with high risk mutations, like TP53, N-RAS or other complex mutations.

PB1920: CLINICOHEMATOLIGICAL CHARACTERISTICS AND CYTOGENETIC PROFILE OF CHRONIC MYELOMONOCYTIC LEUKEMIA; AN EXPERIENCE FROM PAKISTAN

N. ANWAR 1,* , Z. ZIAD 1 , N. FATIMA 2 , A. KHALID 2 , L. MAJEED 2 , M. BORHANY 1

1 HEMATOLOGY; 2 RESEARCH AND DEVELOPMENT, NATIONAL INSTITUTE OF BLOOD DISEASES AND BONE MARROW TRANSPLANTATION, KARACHI, Pakistan

Background: Chronic myelomonocytic Leukemia (CMML) is a clonal stem cell disorder characterized by the presence of sustained (>3 months) peripheral blood monocytosis (≥1 x 10 9 /L; monocytes ≥10% of white blood cell count) along with dysplastic features in the blood/bone marrow. The pathophysiology of CMML is very complex and heterogenous demonstrating overlapping features of myelodysplasia and proliferation.

Aims: The aim of the study was to study baseline clinicohematological characteristics, and cytogenetics profile of this disorder in our population

Methods: A cross sectional study was conducted at National Institute of Blood Diseases and Bone Marrow Transplantation, Karachi Pakistan from 2018-2021. Approval from the Institutional ethics committee was obtained prior to the study. Baseline investigations were done and clinical parameters were recorded. Bone marrow biopsy samples were taken and stained by leishman’s stain. Cytogenetic analysis was performed on overnight, 24-h un-stimulated and 72-h stimulated bone marrow cultures using standard procedures. Descriptive statistics were reported by using SPSS version 23.0

Results: A total of 18 patients were included in the study having median age 63(28-85) years with male predominance 11(61%). Out of total, 15(83%) had no addiction and 12 (67%) were from lower socioeconomic status. Out of 18, 6(33%) had known comorbids, 5(28%) had hepatomegaly, and 11(61%) had splenomegaly. Symptoms at presentation included fever 10(56%), weight loss 9(50%), shortness of breath in 8(44%), bleeding and gastrointestinal discomfort in 3(17%) respectively. The duration of symptoms before presentation was 8 (2-192) weeks. Transfusion dependent anemia at presentation was found in 14(78%) of patients. Out of 18, 16(89%) had normal karyotype. Median (range) hemoglobin (g/dl), platelet countx10 9 /L, total leucocyte count x10 9 /, monocytes x10 9 / and blast (%) was 9.3(5.7-14.2), 2(2-249), 35.2(3.4-145.8), 6.5(1.3-26.1) and 6(1-19) respectively. Out of total, 10 (56%) were alive, 2(11%) were expired and 6 (33%) were lost to follow up at the time of study analysis.

Summary/Conclusion: This was one of the first studies conducted on CMML Pakistani patients. The range duration of symptoms before presentation was found much longer which might represent delayed referrals. Routine follow ups and clinical treatment data would be important addition to the existing data done prospectively. Further longitudinal studies with inferential statistics are needed in this context.

PB1921: A RANDOMIZED, DOUBLE-BLIND, PHASE 3 STUDY OF TAMIBAROTENE/AZACITIDINE VERSUS PLACEBO/AZACITIDINE IN NEWLY DIAGNOSED ADULT PATIENTS SELECTED FOR RARA-POSITIVE HR-MDS (SELECT-MDS-1)

A. DeZern 1,* , G. Marconi 2 , D. Deeren 3 , M. Diez Campelo 4 , M. Lübbert 5 , P. Krishnamurthy 6 , Z. Nagy 7 , G. Basak 8 , A. Jonášová 9 , E. Morozova 10 , K. Geissler 11 , Y. Ofran 12 , H. Kelley 13 , A. Volkert 13 , K. Baker 13 , Q. Kang-Fortner 13 , G. Hodgson 13 , C. Madigan 13 , D. A. Roth 13 , M. Kelly 13 , P. Fenaux 14

1 Johns Hopkins University, Baltimore, United States of America; 2 Romagnolo Institute to Study Tumors “Dino Amadori”, Meldola, Italy; 3 AZ Delta, Roeselare, Belgium; 4 Hospital Universitario de Salamanca, Salamanca, Spain; 5 UniversitätKlinikum Freiburg, Freiburg, Germany; 6 King’s College Hospital, London, United Kingdom; 7 Semmelweis University, Department of Internal Medicine and Hematology, Budapest, Hungary; 8 Medical University of Warsaw, Warsaw, Poland; 9 General University Hospital Prague, Prague, Czechia; 10 Gorbacheva Research Institute, St. Petersburg, Russia; 11 Hospital Hietzing, Vienna, Austria; 12 Department of Hematology, Shaare Zedek Medical Center, Faculty of Medicine Hebrew University of Jerusalem, Jerusalem, Israel; 13 Syros Pharmaceuticals, Inc, Cambridge, United States of America; 14 Hôpital Saint Louis, Paris, France

Background: A novel genomically defined subset of higher-risk MDS (HR-MDS) patients with an actionable target characterized by overexpression of RARA has been identified (McKeown 2017). Approximately 30% of HR-MDS patients are RARA-positive (RARA+) by a blood-based biomarker test (Vigil, 2017). This RARA positivity enables biologically targeted therapy with tamibarotene (formerly SY-1425), an oral selective RARα agonist with potential to provide clinical benefit for HR-MDS patients, irrespective of mutation or cytogenetic risk.

Initial Phase 2 clinical data in RARA+ relapsed/refractory HR-MDS patients treated with single agent tamibarotene showed myeloid differentiation, improved blood counts, and reduced bone marrow blasts, including one patient who achieved marrow complete remission with hematologic improvement (Jurcic 2017). Tamibarotene in combination with azacitidine (aza) demonstrated high complete response rates with rapid onset of response in RARA+ newly diagnosed (ND) unfit AML patients, including those with low blast count (≤ 30%) AML, and a majority of patients achieved or maintained transfusion independence (de Botton 2020). Tamibarotene plus aza was generally well tolerated with no increase in myelosuppression compared to single agent aza (de Botton 2020). HR-MDS and AML have common cytogenetic abnormalities and gene mutations present in the blast populations in each condition. The historical precedent demonstrating similar clinical outcomes among patients with HR-MDS and AML, particularly low blast count AML (Estey 2021), supports further development of the combination of tamibarotene plus aza in the HR-MDS population, with the opportunity to improve upon the clinical outcomes of standard of care treatment with hypomethylating agents (HMAs).

Aims: This is a global Phase 3, multi-center, randomized, double-blind, placebo-controlled study comparing the activity of tamibarotene plus aza, to placebo plus aza in RARA+ patients with ND HR-MDS. The primary objective is to compare the complete remission rate of tamibarotene plus aza vs. placebo plus aza, with secondary objectives to compare objective response rate, event-free survival, overall survival, transfusion independence rate, time to and duration of initial and complete responses, and safety.

Methods: Patients must be RARA+ based on the investigational biomarker test, ND with HR-MDS by WHO classification (Arber 2016), classified by IPSS-R risk category as very high, high, or intermediate risk with a bone marrow blast count > 5% at study entry. Patients suitable for allogeneic HSCT at the time of screening, or who received prior treatment for MDS with any HMA, chemotherapy or allogeneic HSCT are excluded. Aza will be administered at 75 mg/m 2 IV/SC daily on days 1-7 (or days 1-5, 8-9) followed by tamibarotene/placebo at 6 mg BID orally days 8-28 of each 28-day cycle. Response will be assessed per the modified IWG MDS criteria (Cheson 2006).

Results: Approximately 190 patients will be randomized 2:1 and stratified by IPSS-R risk group and geographic region, providing 90% power to detect the difference in CR rates between the experimental and control arms, respectively, with one-sided alpha of 0.025.

Summary/Conclusion: The SELECT-MDS-1 trial opened to enrollment in February 2021, recruitment is ongoing, with sites located in North America, Israel, and Europe ( NCT04797780 ).

PB1922: EFFECTIVENESS OF AZACITIDINE IN FRONTLINE HIGHER-RISK MYELODYSPLASTIC SYNDROME (HR MDS): A RETROSPECTIVE COHORT STUDY OF 382 PATIENTS

N. Rajakumaraswamy 1,* , M. Gandhi 2 , A. H. Wei 3 , D. A. Sallman 4 , N. G. Daver 5 , S. Mo 1 , S. Iqbal 1 , M. Chen 1 , Y. Wang 6 , P. Vyas 7

1 Gilead Sciences, Inc., Foster City; 2 Virginia Cancer Specialists, Fairfax, United States of America; 3 Department of Clinical Haematology, The Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Australia; 4 Moffitt Cancer Center, Tampa; 5 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston; 6 Ontada, The Woodlands, United States of America; 7 Department of Medicine, University of Oxford Radcliffe, Oxford, United Kingdom

Background: 5-azacitidine (AZA) is the frontline standard of care in HR MDS, defined as intermediate-, high-, or very-high-risk MDS per the Revised International Prognostic Scoring System (IPSS-R). Real-world studies reporting on the effectiveness of AZA were often limited by sample size or lacked standardized response assessments based on International Working Group (IWG) criteria.

Aims: To assess the effectiveness of frontline AZA in treatment-naive adult patients with HR MDS in a real-world setting.

Methods: This retrospective cohort study examined outcomes in treatment-naive adult patients with HR MDS who started frontline AZA between 1 Jan 2014 and 1 Jan 2020, identified from the US Oncology Network Electronic Health Record (EHR) database. Key exclusionary criteria were receipt of any prior antileukemic therapy, stem cell transplant within 6 months of AZA initiation, active second malignancies, participation in a clinical trial during the study period, or off-label use of AZA regimens. Data were abstracted by targeted chart review into an electronic case report form from AZA initiation until the earlier of 1 Jan 2021 or death. Effectiveness of AZA was assessed using IWG 2006. Real-world equivalent (based on EHR only) of these endpoints were also derived. All responses were adjudicated by an independent hematologist.

Results: Overall, 932 patients with HR MDS were identified, of whom 382 were eligible. Study population was majority male (243 [63.6%]), had a median age of 74 years, and consisted of IPSS-R intermediate-, high-, and very-high-risk categories in 164 (42.9%), 150 (39.3%), and 68 patients (17.8%), respectively. There were 101 patients (26.4%) with poor cytogenetic risk and 46 (12%) with TP53 mutation.

Overall median duration on the study was 12.9 months, and median duration on AZA was 4.9 months; reasons for AZA discontinuation were: disease progression, 135 (35.3%); adverse event, 55 (14.4%); death, 41 (10.7%); and palliation, 36 (9.4%). Based on IWG 2006, 30 patients (7.9%) achieved complete remission (CR), with 240 (62.8%) achieving an objective response (OR; defined as CR, marrow CR [mCR], partial remission [PR], or hematologic improvement [HI]). IWG 2006–based median (95% CI) duration of OR, progression-free survival, time to next treatment, and overall survival (OS) were 9.5 (7.7-10.6), 9.1 (8.0-10.8), 11.5 (9.5-12.6), and 17.9 (15.5-21.7) months, respectively. Presence of poor cytogenetic risk appeared to reduce OS (Table). Estimates for response categories based on IWG 2006 differed from those based on EHR, but OR rates (ORRs) were similar (Table).

Summary/Conclusion: In this, one of the largest real-world HR MDS cohorts, CR and OS with AZA in treatment-naive patients were limited. Novel agents are urgently needed to improve the CR and survival rates in these patients.

PB1923: PATIENT AND PHYSICIAN CHARACTERISTICS ASSOCIATED WITH HYPOMETHYLATING AGENT USE IN HIGHER-RISK MYELODYSPLASTIC SYNDROMES

A. Zeidan 1,* , V. Divino 2 , M. DeKoven 2 , D. Shah 2 , E. Wang 2 , D. Bey 3 , T. Salimi 3 , R. Epstein 4

1 Hematology, Yale University and Yale Cancer Center, New Haven, CT; 2 Real World Insights, IQVIA, Falls Church, VA; 3 Taiho Oncology, Inc., Princeton, NJ; 4 Epstein Health, Woodcliff Lake, NJ, United States of America

Background: Hypomethylating agents (HMAs) decitabine and azacitidine are recommended standard of care treatment for patients with higher-risk MDS (HR-MDS). However, real-world studies have revealed underutilization of HMAs among HR-MDS patients in clinical practice, leading to suboptimal outcomes.

Aims: This study evaluated patient and physician characteristics associated with use vs non-use of HMA therapy among HR-MDS patients in US clinical practice, in order to better understand trends and potential barriers to optimal HMA utilization.

Methods: This was a retrospective longitudinal cohort study using the IQVIA PharMetrics ® Plus database, which contains adjudicated claims from over 190 million unique patients in the US. Patients included in the study were adults newly diagnosed with HR-MDS from January 1, 2017 to December 31, 2019 who had ≥1 inpatient or ≥2 outpatient claims >60 days apart with diagnosis codes for high grade MDS lesions or refractory anaemia with excess blasts (RAEB). Patients also had to have continuous enrolment for 6 months prior to and following the index date (date of first claim), with variable follow-up (up to 12 months) through June 30, 2020. Logistic regression analysis was used to evaluate predictors of HMA use.

Results: Of 366 included patients, only 128 (35.0%) received HMA therapy, while 238 (65.0%) did not. The HMA group had a numerically higher proportion of male patients (61.7 vs 55.5%), were younger (mean age, 67.5 vs 70.4 years), had lower comorbidity burden (Charlson Comorbidity Index [CCI] ≥3, 29.7 vs 34.5%), and were more likely to be commercially insured (46.1 vs 34.9%) than patients in the no HMA group. Logistic regression analysis demonstrated similar trends (Figure): patients aged 55-64 and 65-74 years had numerically higher odds of receiving HMA therapy (odds ratio [OR] 2.29, p=0.05, and 2.04, p=0.11, respectively) than those aged ≥75 years (OR 1.13, p=0.79; reference group 18-54 years); females were less likely to receive HMA therapy than males (OR 0.65; p=0.09); and patients with a baseline performance status proxy for disability were significantly less likely to receive HMA treatment than those without (OR 0.28, p<0.01). Additionally, patients with red blood cell (RBC) or platelet transfusion were >4-times more likely to receive HMA therapy (OR 4.09, p<0.0001). Comparison of physician characteristics between the HMA non-prescribing vs HMA prescribing groups did not show significant differences in terms of age (54.5 vs 52.2 years; p=0.13), sex (69.7 vs 75.6% male; p=0.34), specialty (hematologist/oncologist, 90.5 vs 86.8; p=0.58), years since graduation (28.2 vs 25.4 years; p=0.09), or region of the US (p=0.74).

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Summary/Conclusion: This real-world analysis confirmed prior observations that among HR-MDS patients in the US, utilization of HMA therapy is low. Patients receiving HMA therapy tended to be younger and with a lower burden of comorbidity or disability, suggesting possible barriers to prescribing HMAs to older, frailer patients. Receipt of RBC/platelet transfusion was also significantly associated with HMA prescribing, suggesting physicians may be influenced by the potential to avoid or reduce the need for RBC/platelet transfusion with HMA therapy.

PB1924: ARE WE TAKING ADVANTAGE OF THERAPEUTIC OPTIONS IN MYELODYSPLASTIC SYNDROME?-REAL WORLD DATA FROM HUNGARY

Z. Miltényi 1,* , A. E. Dr Gál 1 , D. Tóthfalusi 1 , Á. Prof Dr Illés 1

1 Hematology, University of Debrecen, Debrecen, Hungary

Background: The majority of patients with myelodysplasia (MDS) are over 70 years of age, so the only curative treatment, allogeneic stem cell transplantation, can be given to only a few patients. However, treatments available in recent years (lenalidomide, azacitidine, erythropoietin) may improve both quality of life and survival, so it is important to identify and treat these patients where possible.

Aims: Analysis of the characteristics, treatment modalities, and survival outcomes of our MDS patients.

Methods: Data from MDS patients diagnosed and treated at our department were analyzed retrospectively between January 1, 2010 and December 31, 2020. We compared the characteristics of patients treated before and after 2015 and their treatment. Allogeneic transplantation, azacitidine, lenalidomide and erythropoietin treatment were considered active treatments. Transfusions, GCSF, and chelating agents were considered supportive treatments.

Results: A total of 341 patients had confirmed MDS during this period, with a mean age of 69.52 (23-95) years and a median survival of 31.77 (0-385) months. 56% of patients were older than 70 years at diagnosis and only 8% were younger than 50 years. The most common histological subtype (44.57% of patients) wasrefractory cytopenia with multilineage dysplasia. Based on IPSS, 78% of patients were in the low and intermediate 1 risk groups. 78% of patients already required transfusion at diagnosis and 55% of all patients received supportive care only. While 39% of patients diagnosed before 2015 received active treatment alone, 51% of patients in the group diagnosed after 2015 already received active treatment (p=0.061). 40% of patients died and an additional 35% were lost from follow-up. Overall survival of patients in the low and intermedier-1 group was significantly better than the intermedier-2 and high risk group (p=0.000). Until 2015 the survival of actively treated patients was worse, in the period after 2015 an improving trend can already be observed, which is more due to the low and intermedier-1 risk patients thanks to the available active treatment.

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Summary/Conclusion: Although the only curative treatment remains the allogeneic transplantation in MDS, therapies have become available with therapies available since 2015. For this reason, we must strive for the earliest and most accurate diagnosis possible.

PB1925: VENETOCLAX IN COMBINATION WITH AZACITIDINE OR LOW-DOSE CYTARABINE IN PATIENTS WITH HIGH-RISK MDS AND AML - EXPERIENCE OF A CZECH HAEMATOLOGY CENTRE.

L. Minarik 1,2 , T. Stopka 1,2 , Z. Zemanova 3 , A. Jonasova 1,*

1 Clinic of Hematology, General University Hospital and First Faculty of Medicine, Charles University, Prague; 2 1st Faculty of Medicine, BIOCEV, Vestec; 3 Center of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and First Faculty of Medicine, Charles University, Prague, Czechia

Background: The prognosis of older patients diagnosed with high-risk myelodysplastic syndrome (HR-MDS) or acute myeloid leukemia (AML) ineligible of intensive treatment that are progressing on hypomethylating therapy (HMA) remains dismal. Recently two international, randomized, placebo-controlled clinical trials VIALE-A and VIALE-C compared administration of azacitidine (AZA) or low-dose cytarabine (LD-AraC) alone or in combination with BCL-2 inhibitor venetoclax (VTX) in patients diagnosed with de novo AML. Results were promising with significant prolongation of median overall survival (OS) and achieving early remissions in combination cohort groups. Recently was published also trials involving 78 HMA-naïve high-risk MDS patients (Garcia JS et al., 2020) and a similar trial of 44 patients (Zeidan AM et al. 2020).

Aims: To monitor the toxicity of therapy, to identify how many patients received reduced treatment due to cytopenia and to identify markers associated with worse outcome in patients treated with VTX combination therapy.

Methods: Panel of 33 genes frequently mutated in MDS was used for mutation screening.

Description of Cohort: Patients were treated either with combination of AZA-VTX or LD-AraC-VTX. Our cohort consists of 22 patients (males, N=12, females, N=10) diagnosed with MDS-AML (N=16) or de novo AML (N=6) at the time of VTX administration. Median age was 75 years. Average cytogenetic risk was 1.9 according to IPSS-R.

Results: Median total dose of VTX was 1500 mg during standard azacitidine 5-2-2 regimen (75 mg/sqm, 28 day period) or standard LD-AraC regimen (10 mg or 20 mg, 28 day period). The average dose of VTX was 53 mg/day which is lower compared to therapeutic dose used in treatment of other hematologic malignancies due to risk of life-threatening cytopenia. In the group of de novo AML patients we observed ORR of 67% (CR=33%, PR=17%, HI=17%) with median OS on therapy of 6.5 months. In the group of MDS/AML patients we observed ORR of 57% (CR=29%, PR=21%, hematologic improvement (HI)=7%) with median OS on therapy of 5.4 months. We also included 13 patients (59%) after AZA failure as 1 st line of therapy and addition of VTX led to transient remission.

Summary/Conclusion: Venetoclax in combination with azacitidine or LD-AraC provides an effective treatment for patients with de novo AML, MDS-AML or MDS patients after azacitidine failure. Total dose of venetoclax is significantly lower due to cytopenia. Reduction of azacitidine dose during first two cycles was associated with worse overall survival (p=0.0191). Mutation of TP53 was associated with worse overall survival (p=0.0184). Achieving of HI-P during first two cycles was associated with better overall survival (p=0.0341). Failure of combination therapy remains unclear and will be studied further.

PB1926: REAL-WORLD ERYTHROPOIESIS-STIMULATING AGENT (ESA) TREATMENT PATTERNS AND OUTCOMES AMONG US PATIENTS WITH LOWER-RISK MYELODYSPLASTIC SYNDROMES (LR-MDS)

S. Mukherjee 1,* , K. Zimmerman Savill 2 , A. Gajra 2 , J. Kish 2 , C. Brown-Bickerstaff 2 , D. Gentile 2 , A. Falkenstein 2 , T. Miller 2 , J. Laney 2 , A. McBride 3

1 Cleveland Clinic, Cleveland; 2 Cardinal Health Specialty Solutions, Dublin; 3 Bristol Myers Squibb, Princeton, United States of America

Background: ESAs are used in first-line treatment for symptomatic or transfusion-dependent anemia in patients with LR-MDS. Ultimately, the majority of patients with LR-MDS who receive an ESA will experience ESA treatment failure.

Aims: This real-world (RW) study aims to describe patient characteristics, treatment patterns including ESA use, and outcomes in patients with LR-MDS.

Methods: A retrospective, observational, US multisite, cohort chart review of adult patients diagnosed with LR-MDS between January 1, 2015 and June 24, 2020 was conducted. Eligible patients had not received luspatercept or participated in a clinical trial and had ≥ 1 year of follow-up after LR-MDS diagnosis, unless follow-up was < 1 year due to death during this interval. Demographics, clinical characteristics, treatment patterns, and outcomes were summarized using descriptive statistics. Median duration of therapy (DOT) was estimated using the Kaplan-Meier method.

Results: Among 338 eligible patients, median age at LR-MDS diagnosis was 70 years (range: 19–100), 55% were female, 71% were White, 17% had known detection of del(5q), and ring sideroblast status was known to be positive for 7%. Transfusion independence within the 8 weeks prior to diagnosis (TI8) was reported for 48% of eligible patients and 17% had intermediate-1/ intermediate disease risk by IPSS/IPSS-R risk classification. Within the study follow-up time (median of 28.3 months from diagnosis), 76% of patients had received systemic therapy for MDS (median DOT: 29.2 months). An ESA was used in 56% (n = 190) of patients, of whom 38% received red blood cell (RBC) transfusions during ESA treatment. Of those receiving systemic treatment for LR-MDS, 11% were hospitalized for reasons that physicians reported were related to MDS treatment complications. At the time of data collection, 30% of patients had died. Among 38 patients determined to have failed ESA treatment by abstracting physicians, 11% had TI8, 34% had intermediate-1/ intermediate disease risk, 82% received RBC transfusions during ESA treatment, 26% required hospitalization that abstracting physicians reported was related to MDS treatment complications, 32% continued to receive ESA post-failure, and 53% were deceased at data collection.

Summary/Conclusion: Results from this RW study indicate that a higher proportion of patients with LR-MDS who experienced ESA treatment failure according to abstracting physicians had intermediate-1/ intermediate disease risk at diagnosis, were transfusion dependent at diagnosis, required RBC transfusions during ESA treatment, were hospitalized for reasons related to MDS treatment complications, and had poor short-term survival. Nearly a third of patients who experienced ESA treatment failure still received an ESA after initial failure. In summary, findings from this study indicate that patients with LR-MDS who experience ESA treatment failure are likely to have worse outcomes.

PB1928: ALLOGENEIC STEM CELL TRANSPLANTATION FOR ADULT CHRONIC MYELOMONOCYTIC LEUKEMIA IN THE MOLECULAR ERA

T. Zhang 1,*

1 Haematopoietic Stem Cell Transplantation centre, Institute of Haematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

Background: Allogeneic hematopoietic cell transplantation (allo-HSCT) is potentially curative for patients with chronic myelomonocytic leukemia (CMML), however, few data exist regarding prognostic factors and transplant outcomes regarding Chinese.

Aims: To review the updated trends of myeloablative conditioning practice and outcomes in allo-HSCT to treat CMML, we retrospectively evaluated 40 patients who underwent myeloablative conditioning regimen followed by allo-HSCT between January 2009 to December 2021.

Methods: The CMML-specific prognostic scoring system (CPSS) has been validated in subjects receiving non-transplant therapy and was included in our study. From 2009–2021, there were 40 adult subjects who received allo-HSCT for CMML.

Results: After the median duration of follow-up was 36.9 months (range, 3 to 156 months), the overall survival (OS) and disease-free survival (DFS) were 65.0% and 47.5% respectively. The median age at allo-HSCT was 47 years (range, 20 to 59 years). Thirty-five percent of the patients had HLA-haploidentical donors, 62.5% had HLA-identical sibling donors, and 2.5% had matched unrelated donors. The cumulative incidence of grade II-IV acute graft-versus-host disease (aGVHD) was 30.0%, and that of extensive chronic GVHD (cGVHD) was 25.0%. The cumulative incidence of relapse was 32.5%, the non-relapse mortality (NRM) was 20.0%. Eighteen patients harbored at least one gene mutation detected by Next-generation sequencing (NGS) mutation analysis. In addition, according to the number of gene mutations, the patients could be divided into groups with 0, 1-2 and ≥3 gene mutations, and the 3-year DFS of the three groups were 68.2%, 27.3%, and 14.3% respectively (P=0.010). Furthermore, the achievement of a complete remission (CR) before allo-HSCT was associated with longer OS and DFS (3-year OS: 74.2% vs 33.3%, P=0.016; 3-year DFS: 58.1% vs 11.1%, P=0.017). Those with minimal residual disease (MRD + ) by FACS or PCR have a significantly worse prognosis compared to patients with no evidence of MRD (MRD − ) at the time of allo-HSCT (3-year OS: 52.4% vs 78.9%, P=0.026; 3-year DFS: 28.6% vs 68.4%, P=0.009). In multivariate analysis, the depth of remission before allo-HSCT was identified as significant risk factors for OS; Meanwhile, MRD and gene mutation were independent predictors of DFS.

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Summary/Conclusion: Taken together, this retrospective analysis reports updated trends in allo-HSCT for CMML in China and identifies pre-transplantation factors influencing post-transplantation outcomes. Whether prophylactic use of emerging agents, such as azacitidine, can reduce post-transplant relapse rates remains to be further explored in prospective studies.

PB1929: PRELIMINARY STUDY OF EVALUATION IRON OVERLOAD MYOCARDIAL INJURY BY LOAD CT MYOCARDIAL PERFUSION IMAGING

Y. Zhang 1,* , W. Zhu 2 , L.-X. Song 1 , H. Long 2 , C. Guo 1 , C. Xiao 1 , Z. Zhang 1 , Y. Zhao 1 , J. Su 1 , J. Zhao 2 , C. Chang 1

1 Hematology; 2 Radiology, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiaotong University, shanghai, China

Background: Currently, Iron overload cardiomyopathy(IOC) is assumed to be the most common cause of mortality in patients with secondary iron overload and is a major comorbidity in patients with genetic hemochromatosis. At present, there is still a lack of sensitive means to detect the injury of iron overload myocardium. Although ECG, echocardiography and serum ferritin level are simple and cheap methods to detect iron overload cardiomyopathy, they have no specificity for cardiac overload and are not sensitive to the injury caused by iron overload. Although echocardiography can determine the effect of iron on myocardial structure and function, but in patients with hemoglobinopathy and other hematological diseases, left ventricular diastolic dysfunction and decreased LVEF may be masked by the high output state caused by anemia. Recent literature showed CT myocardial perfusion imaging can well evaluate the effect of myocardial amyloidosis on myocardial function. Is it feasible to evaluate iron overload myocardial injury by CT myocardial perfusion imaging? There are few reports in the literature.Is the degree of cardiac iron overload consistent with the degree of myocardial injury? These problems need further study.

Aims: This study investigated the relationship between iron overload(IO) myocardial injury and myocardial iron content

Methods: Quantitative detection of cardiac virtual iron concentration (CVIC) and liver virtual iron concentration (LVIC)by energy spectrum CT.The myocardial blood flow (MBF) and myocardial extracellular volume fraction(ECV) was evaluated simultaneously by adenosine triphosphate (ATP) load CT myocardial perfusion imaging(CTPI), adjusted SF(ASF) were simultaneous detection.To analyze the correlation between myocardial injury and myocardial iron content.

Results: ASF and LVIC is inconsistent with CVIC, patients with high ASF or LVIC but CVIC < 1.8 mg/ml had no myocardial injury. The myocardial injury appear in CVIC≥1.8 mg/ml patients but CVIC≥1.8 mg/ml patients were not all have myocardial injury. Transfusion dependence and iron chelation therapy (ICT) may be the influencing factors of iron overload myocardial injury.

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Summary/Conclusion: IO myocardial injury appear in moderate and severe cardiac iron deposition patient,but the degree of myocardial injury was not inconsistent with myocardial iron content, so independent load CTPI testing is required. Relevant influencing factors need to be further studied.

PB1931: HEMATOLOGICAL COMPLICATIONS OF COVID IMMUNIZATION-CASE SERIES

A. Aribandi 1,* , C. Ranjith Kumar 1 , K. Ashok Kumar 1

1 Haemato-oncology & BMT, American oncology Institute, Hyderabad, India

Background: Effective vaccines are most powerful armamentariums in our fight against global COVID-19 pandemic. Based on available literature, the benefits of vaccination significantly outweigh the low risk of blood clots and other hematological complications reported following vaccination. Here we are reporting 6 such rare complications of COVID immunization which was happened within 4days -42days following vaccination.

Aims: we aimed to study the rare hematological adverse events follwoing the COVID vaccination

Methods: Here we are reporting six cases presented to our institute following COVID vaccination within 4 weeks following vaccination, data were retrieved from MRD and events and outcomes were tabulated.

Results:

Summary/Conclusion: Hematological complications commonly reported following COVID-19 vaccination were thrombosis with thrombocytopenia syndrome following AstraZeneca vaccination. However for severe aplastic anemia literature was scanty and hardly any reports in the literature search.In our paper we reported three severe aplastic anemia and 3 Immune thrombocytopenic Purpura following Astrazenca vaccination. Need high index of suspicion and Bone marrow examination required to confirm the underlying entity appropriately.

PB1932: GENERIC ELTHROMBOPAG WITH STANDARD IMMUNOSUPPRESSIVE THERAPY IN SEVERE APLASTIC ANEMIA

C. Ranjit Kumar 1,* , A. Aribandi 1 , A. K. K 1

1 Haemato-oncology & BMT, American oncology Institute, Hyderabad, India

Background: Acquired aplastic anemia is a result of immune mediated destruction of bone marrow. Triple drug therapy become standard of care in patient who are not having a full matched donor or who are not fit for allogenic stem cell transplantation. Addition of Elthrombopag to standard immunosuppressive therapy improved the response and cure rate, however data with generic Elthrombopag with triple drug therapy is lacking in the literature. In this study we are presenting our data with generic Elthrombopag along with standard immunosuppressive therapy in severe aplastic anemia.

Aims: The data for generic Elthrombopag in severe aplastic anemia (SAA) is lacking, we aimed to study the efficacy and safety of generic Elthrombopag in immunosuppressive therapy regimen in SAA.

Methods: In this study patient were enrolled with confirmed diagnosis of Severe aplastic anemia who are not having full matched donor [10/10 HLA] or not fit for transplant but eligible for immunosuppressive therapy. Patient were enrolled between march 2020 to December 2021.Enrolled patient were ruled out fanconi anemia and other inherited bone marrow failure syndromes. Total 21 patient were enrolled both pediatric and adults. One patient was non Severe aplastic anemia remaining 20 patient were severe aplastic anemia. All except one received triple drug therapy with standard dosing of Horse ATG at 40mg/kg/day along with Cyclosporine from day 5 and Generic Elthrombopag from day 1 of therapy. Elthrombopag dose was 150 mg daily in patients who were 12 years of age or older, at a dose of 75 mg daily in patients who were 6 to 11 years of age, and at a dose of 2.5 mg per kilogram of body weight per day in patients who were 2 to 5 years of age. Hematological response at 3 were calculated for Absolute Neutrophil count, platelet count. All the data were tabulated and analyzed.

Results: Of the total 21 patient enrolled, pediatric age group less than 18(28%) years were 6 and adult age group were 15 (72%). All enrolled patient had bone marrow cellularity <20%. Sex distribution males 15 (72%) and females were 6 (28%). The baseline average hemoglobin was 7.1gm/dl, Average ANC was 430/cmm and Average platelet count was 33,000/cmm. All were transfusion dependent before initiating therapy. ATG dose density was 100% [40mg/kg/day for 4days].Two patient died one 62years old female in less than 4week of therapy due to fungal pneumonia and 2 nd case was 19years old male suicide at 7weeks of therapy. Overall hematological response at 3months was 86% with complete remission was 42%. Side effect profile was documented most common adverse event was elevated aminotransferases in 56%,six patient had (28%) had hyperpigmentation of sclera but their bilirubin was normal, abdominal pain in 36%.

Summary/Conclusion: Generic Elthrombopag is a potential alternative to innovator molecule for treating the severe aplastic anemia triple drug regimen. In our pilot study we observed similar response rate though it was not a randomized data. Generic molecule would potentially reduces the cost with probable similar outcomes. However due to non randomized data we need to confirm this results with much larger randomized trial with innovator molecule.

PB1934: REAL-WORLD OUTCOMES OF ECULIZUMAB TREATMENT IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINUREA: A SYSTEMATIC LITERATURE REVIEW AND EVIDENCE SYNTHESIS

J.-I. Nishimura 1,* , E. Popoff 2 , A. Cheung 2 , A. Wang 3 , I. Tomazos 3 , J. Szer 4

1 Osaka University Graduate School of Medicine, Suita, Japan; 2 Broadstreet HEOR, Vancouver, BC, Canada; 3 Alexion, AstraZeneca Rare Disease, Boston, MA, United States of America; 4 Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, Melbourne, VIC, Australia

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, chronic, hematologic disorder characterized by uncontrolled terminal complement activation, thrombotic events (TEs) and increased morbidity and mortality. The complement component 5 (C5) inhibitor eculizumab has been the standard of care for treating patients with PNH since approval by the US Food and Drug Administration and European Medicines Agency in 2007. Although randomized controlled trials have demonstrated the clinical efficacy and safety of eculizumab, there is a paucity of real-world effectiveness data.

Aims: To describe the real-world effectiveness of eculizumab treatment in patients with PNH.

Methods: A systematic literature review (SLR) and grey literature search was conducted to identify published observational studies that reported outcomes of eculizumab treatment in patients (aged ≥18 years) with PNH. The search was performed using MEDLINE, Embase and the Cochrane Library from database inception to July 2021. Outcomes of interest included lactate dehydrogenase (LDH) levels, transfusion requirement, units of red blood cells (RBCs) transfused, TEs and mortality. A feasibility assessment for meta-analyses was also performed.

Results: The search identified 2,622 unique records, of which 28 were eligible for inclusion (patient sample range: 6–1,807). Of the included studies, 15 (53.6%) utilized a prospective, observational study design and, aside from seven multinational studies, data from 11 unique countries were reported. Meta-analyses were not feasible owing to the considerable heterogeneity in study populations, assessment time points, outcome reporting and definitions. The median age of patients at assessment ranged from 23–56 years and the proportion of male patients was 32–83%. Median PNH disease duration ranged from 2.1–11.0 years (5 studies). Duration of follow-up varied from 11 weeks to 10 years (12 studies). LDH levels following eculizumab treatment were reported in 10 studies and all demonstrated a reduction from baseline levels. The mean (standard error; SE) change in LDH ranged from –338.0 (87.3; mean [SE] baseline LDH = 581.0 [94.9] U/L; n = 11) to –3,101.0 (569.9; mean [SE] baseline LDH = 3,552.0 [584.6] U/L; n = 12) U/L. Amongst studies that had non-zero baseline values, the reduction in transfusion dependence following eculizumab treatment ranged from 15.0% (baseline proportion, 23.0%; n = 231) to 80.0% (baseline proportion, 100.0%; n = 5). The mean (SE) reduction in units of RBCs transfused following eculizumab treatment was reported in six studies and ranged from 1.5 (1.1; n = 6) to 14.3 (1.4; n = 75). TEs following eculizumab treatment were described in 11 studies and ranged from 0.0% (baseline TEs, 0.0–33.3%; n = 5–105) to 9.0% (baseline TEs, 40.9%; n = 22). All-cause mortality among eculizumab-treated patients was reported in six studies and ranged from 0.0% (n = 17–105) to 7.3% (n = 123). These deaths were not related to eculizumab.

Summary/Conclusion: This SLR synthesized real-world outcomes associated with complement C5 inhibition in patients with PNH and demonstrated consistent effectiveness of eculizumab beyond the clinical trial setting. Although the heterogeneity of study populations precluded meta-analyses of outcomes data, consistent improvements in key clinical parameters were widely reported, including reductions in LDH levels, TEs, transfusion dependence and mortality outcomes. This analysis further reinforces the benefits of complement C5 inhibition in patients with PNH.

PB1935: MANAGEMENT OF NON-SEVERE APLASTIC ANEMIA: LABORATORY WORKUP AND TREATMENT PATTERNS.

G. Cassanello 1,* , J. A. Giannotta 2 , L. Cro 2 , M. Barbieri 2 , W. Barcellini 2 , B. Fattizzo 1,2

1 oncology and hemato-oncology, University of Milan; 2 oncology and hemato-oncology, IRCCS Policlinico di Milano, Milan, Italy

Background: Non severe aplastic anemia (NSAA) is a rare and heterogeneous disease characterized by different cytopenias and variable severity and possible evolution into myeloid neoplasms. Data on clinical workup and therapeutic strategies adopted are largely lacking due to the rarity of the disease and the limited number of dedicated clinical trials.

Aims: To outline disease features, management and adverse events in 25 NSAA patients followed at a single tertiary hematologic center

Methods: We evaluated all patients diagnosed with NSAA according to Camitta criteria (hypocellular bone marrow with peripheral blood values not meeting criteria for severe AA) at a single tertiary hematologic center in the last 20 years. We collected baseline hematologic features, bone marrow biopsies, flow-cytometry (FCM) of marrow aspirates and mutational status by next generation sequencing (NGS) for 69 genes involved in myeloid neoplasms. Different management strategies were registered retrospectively and categorized in 1) steroids +/- cyclosporin (CyA), 2) eltrombopag + CyA and 3) watch & wait. Responses were evaluated according to 2009 Marsh criteria

Results: We included 25 patients, mainly females (64%) and with a median age of 48 years (17-91) (Table 1). The most frequent cytopenia was thrombocytopenia (N=22) and 1/3 of cases were transfusion dependent. Bone marrow samples showed a median cellularity of 15% (range 5-30), with dyserythropoiesis in 44%, dysmyelopoiesis in 16%, and reticulin fibrosis (MF-1) in 16% of patients. Bone marrow microenvironment by FCM appeared enriched for lymphocytes (22% of the total cellularity, range 7-58), particularly T lymphocytes (median 72%, range 58-97). A minor infiltrate of monocytes (median 3.3%, range 0.6-5) and mast-cells (median 0.5 %, range 0.02-2) was also detected. NGS for mutations in myeloid genes showed that 7/9 had negative results, whilst 1 patient harbored NF1 and U2AF1 mutations (VAF 4.7% and 4.9%, respectively), and one harbored a germline SBDS mutation (VAF 48%). The latter had a concurrent familiar history of myeloid neoplasm and the mutation was confirmed on cutaneous biopsy.

During a median follow up of 21 months (range 1-187), 16/25 patients received treatment. These patients showed more profound cytopenias and a higher frequency of small PNH clones (p=0.02) as compared to untreated cases (Table 1). Eight patients were treated with CyA and the other 8 with eltrombopag+CyA. The overall response rate (CR+PR) in the former group at 3, 6 and 12 months were 66%, 100% and 100%, respectively. In the second one, the corresponding response rates were 57%, 49% and 100%, with no significant differences between treatment groups.

Seven patients (43%) experienced one or more adverse events (AEs), mostly grade 1 or 2 according to CTCAE. Grade 3/4 toxicities were all registered in the eltrombopag+CyA and consisted in retinal thrombosis, gastroenteritis, diarrhea and acute hepatitis. The sudden rise of transaminases and bilirubin levels was registered in 3 patients and led to treatment discontinuation. Single agent eltrombopag was subsequently readministered due to loss of platelet response in 2/3 patients, with no further toxicity to date.

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Summary/Conclusion: In conclusion we observed that NSAA required treatment in about 2/3 of cases characterized by more severe cytopenias. Treatment with CyA+/- eltrombopag led to a significant clinical improvement. Occurrence of G3 liver toxicity in patients receiving combination therapy deserves attention and further studies. FCM showed a prominent T-cell infiltrate reflecting the prominent cell mediated autoimmunity of the disease.

PB1936: PAROXYSMAL NOCTURNAL HEMOGLOBINURIA AND THROMBOSIS: A MULTICENTRIC RETROSPECTIVE STUDY

S. Chatzileontiadou 1,* , N.-E. Loutsidi 2 , I. Asimakopoulos 3 , T. Chatzikonstantinou 4 , P. Zikos 5 , A. Bouchla 6 , Z. Bezirgiannidou 7 , E. Kouvata 8 , A. Apsemidou 1 , C. Frouzaki 1 , E. Hatjiharissi 1 , P. Chaloudis 1 , D. Sotiropoulos 4 , V. Douka 4 , A. Syrigou 4 , E. Mandala 9 , M. Psyllaki 10 , H. Papadaki 10 , T. Marinakis 11 , N.-A. Viniou 12 , S. Kokori 13 , G. Vassilopoulos 8 , I. Kotsianidis 7 , V. Pappa 6 , M. Pagoni 2 , C. Lalayanni 4 , A. Anagnostopoulos 4 , I. Baltadakis 2 , S. Delimpassi 2 , M. Angelopoulou 3 , M. Papaioannou 1

1 1st Department of Internal Medicine, Hematology Unit, University Hospital of Thessaloniki AHEPA, Aristotle University of Thessaloniki, Thessaloniki; 2 Hematology - Lymphomas Department and Bone Marrow Transplant Unit, Evangelismos General Hospital, Athens; 3 Department of Hematology, National and Kapodistrian University of Athens, Laikon General Hospital, Athens; 4 Department of Hematology, Bone Marrow Transplantation Unit, G. Papanicolaou Hospital, Thessaloniki; 5 Department of Hematology, General Hospital of Patras “Aghios Andreas”, Patras; 6 Second Department of Internal Medicine and Research Unit,Hematology Unit, University General Hospital “Attikon”, Athens; 7 Department of Hematology, Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupolis; 8 Department of Hematology, University Hospital of Larissa, Larissa; 9 Forth Department of Internal Medicine, “Hippokration” Hospital, Aristotle University of Thessaloniki, Thessaloniki; 10 Department of Hematology, University of Crete School of Medicine, University Hospital of Heraklion, Heraklion; 11 Department of Hematology, “G. Gennimatas” General Hospital, Athens; 12 Hematology Unit, First Department of Internal Medicine, National and Kapodistrian University of Athens, Laikon General Hospital; 13 Hematology Laboratory, University General Hospital “Attikon”, Athens, Greece

Background: INTRODUCTION Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired clonal hematopoietic stem cell disorder, potentially life-threatening, manifesting with chronic hemolytic anemia, thrombosis, and various degrees of bone marrow failure. Thrombosis is the most common cause of death in PNH with 29% - 44% of patients experiencing at least one thromboembolic event during the course of the disease. Although thrombosis can affect any anatomical site, the most frequent sites include intra-abdominal and cerebral veins. The mechanism of thrombosis in PNH is complex and continues to be of great research interest.

Aims: OBJECTIVE-PATIENTS The aim of the study is the retrospective analysis of thrombotic episodes of 38 patients with PNH from 13 hematology centers in Greece.

Methods: -

Results: RESULTS Of the 38 patients, 17 were women (45%) and 21 men (55%) with a median age at diagnosis of 38 years (range 11 - 82 years). Twenty (52.6%) patients were diagnosed with the classic form of PNH while 16 (42.1%) patients developed PNH in the setting of another bone marrow disorder. The main symptoms recorded were weakness, abdominal pain, hemoglobinuria, back pain, headache, dysphagia and erectile dysfunction. Anemia was present in 92.10% at diagnosis with 60.52% requiring red blood cell transfusion, while thrombocytopenia was observed in 25 patients (65.79%) with 15.7% requiring platelet transfusion. In total, 15 (39.47%) patients experienced at least one episode of thrombosis, eight (21.05%) at diagnosis and the rest during the course of the disease. Five patients experienced episodes of thrombosis at multiple sites. The most common vascular sites of thrombosis were hepatic (Budd-Chiari syndrome; n=6 patients), portal (n=2) and mesenteric veins (n=4) and ocular arteries (n=2). Of the eight patients who developed thrombosis at diagnosis, six received eculizumab and to-date they have not had a new thrombotic episode [median time of f/u 22 months (range 12 - 179)]. Of the 15 patients without a thrombotic episode who received eculizumab due to hemolytic anemia, two developed thrombosis during the course of the disease (one 14 months after eculizumab initiation with underlying primary myelofibrosis, and the second after five years without identifying any other factor for thrombosis). The antithrombotic therapy that was used was acenocoumarol following a short course of low molecular weight heparin. In none of the 15 patients the thrombotic event was fatal.

Summary/Conclusion: CONCLUSIONS Thrombotic events occur in a significant percentage of patients with PNH, often in unusual anatomical sites. Ongoing treatment with complement inhibitors has significantly contributed to the reduction of thrombotic events and consequently the increase in patient survival.

PB1937: UTILITY OF STERNAL BONE MARROW CELL MORPHOLOGY EVALUATION FOR DIAGNOSTIC CATEGORIZATION IN PATIENTS WITH ACQUIRED HYPOCELLULAR BONE MARROW FAILURE SYNDROMES

J. Chen 1,* , T. Qin 1 , S. Qu 1 , P. Zhang 1 , L. Pan 1 , B. Li 1 , Z. Xiao 1 , Z. Xu 1

1 Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China

Background: Bone marrow failure syndromes (BMFS) are a group of heterogeneous diseases characterized by pancytopenia due to dysfunctional bone marrow elements. Approximately 15%-20% of patients with MDS presenting with marrow hypoplasia for age would be incorrectly categorized as AA, which the pathobiology and prognosis are distinct from hypoplastic MDS (hypo-MDS). The hematopoiesis of sternal bone marrow is typically more active than the ilium.

Aims: To assess the diagnostic value of sternal bone marrow cell morphology in patients with acquired hypocellular bone marrow failure syndromes (BMFS) characterized by normal cytogenetics and develop a predictive scoring system to identify these patients that needed to perform sternal bone marrow evaluation.

Methods: Between June 2014 and January 2019, 194 eligible patients with pre-sternum diagnosis of acquired hypocellular BMFS in our center were retrospectively reviewed. Binary logistic regression was used to develop a predictive scoring system.

Results: In 152 patients (78.4%) with pre-sternum diagnosis of AA, sternal bone marrow evaluation confirmed a post-sternum diagnosis of AA, hypo-MDS, idiopathic cytopenia of undetermined significance (ICUS), and clonal cytopenia of undetermined significance (CCUS) in 50% (76/152), 42.8% (65/152), 3.3% (5/152) and 3.9% (6/152), respectively. In 29 patients (14.9%) with pre-sternum diagnosis of ICUS, a post-sternum diagnosis of ICUS and hypo-MDS were identified in 75.9% (22/29) and 24.1% (7/29) patients, respectively. In 13 patients (6.7%) with pre-sternum diagnosis of CCUS, sternal bone marrow evaluation confirmed a post-sternum diagnosis of CCUS and hypo-MDS in 69.2% (9/13) and 30.8% (4/13) patients.

Disease-related risk of 76 patients with a post-sternum diagnosis of hypo-MDS were assessed by the revised version of International Prognostic Scoring System (IPSS-R), included 30 (39.5%) with low risk, 44 (57.9%) with intermediate, and 2 (2.6%) with high risk. Patients with post-sternum diagnosis of hypo-MDS showed a significantly difference in five-year survival compared with patients with post-sternum diagnosis of AA (98.6% vs 86.1%, p=0.005). Patients with ICUS/CCUS showed no difference in overall survival (OS) compared with AA and hypo-MDS (p=0.095 and p=0.480, respectively).

A 4-items predictive scoring system to identify hypocellular BMFS patients that needed to perform sternal bone marrow evaluation was developed, including age>60 years (OR=6.647, 95%CI 1.954-22.611, p=0.002, 2 points), neutrophil alkaline phosphatase (NALP) score≤160 (OR=2.654, 95%CI 1.214-5.804, p=0.014, 1 point), abnormal erythroid markers evaluated by flow cytometry on iliac bone marrow (OR=6.2, 95%CI 1.165-32.988, p=0.032, 2 points), and DAT (DNMT3A, ASXL1, TET2) genes mutation (OR=4.809, 95%CI 1.587-14.572, p=0.005, 1 point). The AUC was 0.730 (95%CI 0.643-0.816) when the total scores ≥2 points.

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Summary/Conclusion: Patients with a pre-sternum diagnosis of acquired hypocellular BMFS characterized by normal cytogenetics may not reach accurate diagnostic categorization without sternal bone marrow cell morphology evaluation, which could be considered a diagnostic tool for these patient population. We developed a predictive scoring system and when the total scores≥2 points, sternal bone marrow evaluation should be performed for accurate diagnostic categorization that is critical to optimal patient care.

PB1938: 4WHIM: EVALUATING MAVORIXAFOR, AN ORAL CXCR4 ANTAGONIST, IN PATIENTS WITH WHIM SYNDROME VIA A GLOBAL PHASE 3, RANDOMIZED, PLACEBO-CONTROLLED TRIAL WITH OPEN-LABEL EXTENSION

D. C. Dale 1,* , L. Alsina 2 , A. Azar 3 , R. Badolato 4 , Y. Bertrand 5 , A. Deya 2 , K. E. Dickerson 6 , N. Ezra 7 , H. Hasle 8 , H. J. Kang 9 , S. Kiani-Alikhan 10 , T. Kuijpers 11 , A. Kulagin 12 , D. Langguth 13 , C. Levin 14 , O. Neth 15 , J. Peake 16 , C. E. Rutten 17 , A. Shcherbina 18 , T. K. Tarrant 19 , M. G. Vossen 20 , C. A. Wysocki 21 , A. Belschner 22 , D. Cadavid 22 , Y. Hu 22 , H. Jiang 22 , R. MacLeod 22 , W. Tang 22 , M. Tillinger 22 , J. Donadieu 23

1 Department of Medicine, University of Washington, Seattle, United States of America; 2 Allergy and Clinical Immunology Department, Hospital Sant Joan de Déu Barcelona, Institut de Recerca Sant Joan de Déu, Barcelona, Spain; 3 Division of Allergy and Clinical Immunology, Johns Hopkins University, Baltimore, United States of America; 4 Department of Pediatrics, Angelo Nocivelli Institute of Molecular Medicine, University of Brescia, Brescia, Italy; 5 Institute of Pediatric Hematology and Oncology (IHOPe), Civil Hospital of Lyon, Lyon, France; 6 Department of Pediatrics, Division of Hematology and Oncology, UT Southwestern Medical Center, Dallas; 7 California Dermatology Institute, Thousand Oaks, United States of America; 8 Department of Paediatrics, Aarhus University Hospital, Aarhus, Denmark; 9 Department of Pediatrics, Seoul National University College of Medicine, Seoul National University Cancer Research Institute, Wide River Institute of Immunology, Seoul National University Children’s Hospital, Seoul, South Korea; 10 Department of Immunology, Barts Health NHS Trust, London, United Kingdom; 11 Department of Paediatric Immunology, Rheumatology and Infectious Diseases, Amsterdam University Medical Center, Amsterdam, Netherlands; 12 RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia; 13 Immunology Department, Sullivan Nicolaides Pathology Auchenflower, Queensland, Australia; 14 Pediatric Hematology Unit, Emek Medical Center, Afula and the Ruth and Bruce Rappaport Faculty of Medicine Technion, Haifa, Israel; 15 Pediatric, Infectious Diseases, Rheumatology and Immunology Department, Hospital Virgen del Rocío, Institute of Biomedicine of Seville (IBIS), Seville, Spain; 16 Department of Allergy and Immunology, Queensland Children’s Hospital, University of Queensland, South Brisbane, Queensland, Australia; 17 Department of Hematology, Amsterdam University Medical Center, Amsterdam, Netherlands; 18 Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia; 19 Division of Rheumatology and Immunology, Department of Medicine, Duke University, Durham, United States of America; 20 Division of Infectious Diseases and Tropical Medicine, Department of Medicine, Medical University of Vienna, Vienna, Austria; 21 Division of Allergy and Immunology, Departments of Pediatrics and Internal Medicine, UT Southwestern Medical Center, Dallas; 22 X4 Pharmaceuticals, Boston, United States of America; 23 Service d’Hémato-Oncologie Pédiatrique, Hôpital Trousseau, Paris, France

Background: Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome is a primary immunodeficiency characterized by retention of leukocytes in the bone marrow (myelokathexis), resulting in neutropenia, leukopenia, and in some cases hypogammaglobulinemia. WHIM syndrome is classically caused by gain-of-function mutations in CXCR4, with the resulting hyperactivation of the CXCR4-CXCL12 pathway. Patients diagnosed with WHIM syndrome have a heterogeneous phenotype yet typically experience recurrent sinopulmonary infections and unusual susceptibility to human papillomavirus (HPV), resulting in predisposition to warts and malignancy. There are no approved medications for WHIM syndrome. To date, all management strategies are symptomatic only, failing to address the underlying mechanism of WHIM syndrome, and are mostly ineffective for HPV infections. Mavorixafor is an investigational, oral CXCR4 antagonist that directly inhibits CXCR4-enhanced signaling in WHIM syndrome pathogenesis, and has been shown to increase white blood cell counts, decrease annualized infection rate, and reduce cutaneous warts in an open-label phase 2 clinical trial ( NCT03005327 ) for patients with WHIM syndrome.

Aims: Here, we describe the design, baseline characteristics, and status of a global phase 3, double-blind, placebo-controlled, randomized trial (4WHIM, NCT03995108 ) with open-label extension evaluating the safety and efficacy of mavorixafor in patients with WHIM syndrome.

Methods: Eligibility for enrollment included age ≥12 years, diagnosis of WHIM syndrome, confirmed CXCR4 mutation, and absolute neutrophil count (ANC) ≤400 cells/µL without clinical evidence of active systemic infection. Patients were randomized in a 1:1 ratio to receive mavorixafor (adults, 400 mg; adolescents weighing ≤50 kg, 200mg; adolescents weighing >50 kg, 400mg) or placebo orally once daily. The primary endpoint is defined as number of hours above ANC threshold (500 cells/µL) over a 24-hour period, assessed every 3 months for 52 weeks. Secondary endpoints include the composite end point of infection and warts, infection rates and severity, change from baseline in cutaneous warts, and number of hours above absolute lymphocyte count (ALC) of ≥1000 cells/µL over a 24-hour period, and patient-reported quality-of-life assessment using age-appropriate questionnaires.

Results: Enrollment for the trial has been completed, with a total of 31 patients enrolled globally. Demographics of the enrolled population include: 48% adolescents (age 12-18 years), 42% male, 71% with warts, 81% with nonsense CXCR4 mutations, and 19% with frameshift CXCR4 mutations. Mean and median ANC and ALC at screening visit were 220 cells/µL and 180 cells/µL, and 515 cells/µL and 440 cells/µL respectively.

Summary/Conclusion: 4WHIM is the first double-blind, placebo-controlled, randomized trial in patients with WHIM syndrome; top-line clinical results are expected in Q4 2022. The study enrolled children (aged ≥ 12 years) and adults, all with genetically confirmed CXCR4 variants consisting of both nonsense and frameshift mutations. The patients all presented with severe neutropenia and lymphopenia and a high percentage of the patients had warts. This study represents an important next step in the development of the novel, orally bioavailable, targeted therapy mavorixafor for the treatment of patients with WHIM syndrome.

PB1939: CLINICAL AND GENETIC CHARACTERISTICS OF PATIENTS WITH GAUCHER DISEASE: A SINGLE CENTER EXPERIENCE

S. Elsayed 1,* , R. Shawky 1 , A. Abdelkalek 2

1 Medical Genetics Department; 2 Pediatrics Department, Faculty of medicine, Ain Shams University, cairo, Egypt

Background: Gaucher disease is a rare lysosomal storage disease due to deficiency of β-glucosidase and autosomal recessive inheritance. Clinical presentation includes organomegaly, skeletal and variable neurological manifestations with two main types: neuropathic type I and non-neuropathic type 3.

Aims: To describe the main clinical and genetic features of Gaucher patients (GD) in the Genetics Department, Ain Shams University

Methods: The study included 27 patients with Gaucher disease confirmed by both enzyme and molecular testing. History, clinical data and laboratory and imaging investigations were brought from patients’ files.

Results: Patients ages ranged between 3 months and 41 years. Male to female ratio was 1.7:1. Their age ranged between 3 months and 41 years. Parental consanguinity was present in 81.5 %. Two thirds of patients were type III. Age at presentation was significantly lower in type III patients (1.6±2.4 years) compared to 14.3±12.3 in patients with type 1. The most common presenting symptom was abdominal distention (96.3%) followed by developmental delay, while growth retardation was the least. Huge splenomegaly and low hemoglobin level was significantly present in type III, while thrombocytopenic platelet level showed no significant difference between the two types. The most common pathogenic variant was L444P present in homozygous state in all type III patients. All patients received enzyme replacement therapy (ERT) with improvement of growth, organomegaly, cytopenias and severity score and limited improvement of neurological manifestations. Mortality was present in 12 patients (due to pneumonia), all were type III.

Summary/Conclusion: Although beta-thalassemia is the most common cause of chronic anemia in Egypt especially if present with organomegaly, the presence of thrombocytopenia should alert the physician to exclude GD. ERT is effective in improving visceromegaly with limited effect on neurological manifestations. Type III GD is the most common type in Egypt with one common pathogenic variant which could be used as tool of national wide carrier screening program.

PB1940: ACQUIRED APLASTIC ANEMIA: THE CHALLENGE OF TREATMENT IN MEXICO. SINGLE HEMATOLOGY CENTER EXPERIENCE

M. L. Guajardo Leal 1 , G. Sotomayor Duque 1 , A. J. Mecott Estudillo 1,* , D. Cruz Contreras 1 , M. P. Pequeño Luevano 1 , H. P. Sorkee Dávila 1 , J. A. Carrizales Villarreal 1 , J. L. Cedillo 1 , S. Baltazar 1 , R. E. De León Cantú 1 , R. Hernandez Valadez 1 , I. Borjón Cabada 1 , C. Quirino Marquez 1 , J. D. Garza Escobar 1 , Y. B. García Cerda 1 , W. Nava Gutiérrez 1 , D. C. Aldama Gutiérrez 1 , L. G. Ávila Contreras 1 , V. Valeugarín 1 , K. M. Machuca Adame 1 , L. O. Gudiño Cobos 1

1 Hematology, IMSS, Monterrey, Mexico

Background: Aplastic anemia is a T-cell mediated autoimmune pathology that results in apoptosis of CD34 + progenitor cells, resulting in pancytopenia. In Mexico, most patients receive first-line immunosuppressive treatment, the international literature reports a response of 94% and survival rates of 97%.

Aims: Establish the outcomes to immunosuppressive treatment (IST) and hematopoietic stem cell transplant (HSCT) in patients with aplastic anemia in our center.

Methods: Observational, retrospective, cross-sectional and analytical. Patients with Aplastic Anemia, older than 16 years, from January 2014 to December 2020. We analyze the data with absolute frequencies, percentages and measures of central tendency. The qualitative variables were analyzed with chi squared and the quantitative variables Student’s T. Survival was analyzed with Kaplan-Meier curves. The statistical package SPSS version 21.0 will be used.

Results: We include 36 patients, 23 patients (64%) was male and female 13 patients (36%) with median age of 46 years (17-84) and median follow-up of 28.6 months (4.8-80.1). Classification by degree of severity were: Non severe 9 patients (25%), severe 13 patients (36%), and very severe 14 patients (38%).

Response Rates: The first-line therapy was cyclosporine (CsA) mono therapy in 52.7%, CsA and rabbit thymoglobulin (rATG) in 38.8% and CsA/danazole in 8.3%. Four patients (11.1%) achieved a complete response, 2 patients with CsA, 1 with rATG and 1 with CsA/danazole and partial response in eight patients (22%). Global response was 32%. After 6 months of treatment, those patients who did not achieve a response and without rATG in the first-line who were unable to undergo a HSCT were given a course of rATG of second line therapy (11 patients). Four patients (36.3%) achieved a partial response and two patients (18.1%) with a complete response. Two patients started with second-line danazol (5.5%), one patient obtained a partial response. A total of 10 patients (27.7%) underwent a HCST achieving a response a partial responsen in 30% and complete response in 50%.

Survival and mortality: Overall survival (OS) at 3, 4, and 5 years was 45%, 30%, and 24% respectively. Patients who achieved a response had a 4-year OS of 47% versus 17% for those who did not achieve a response (p = 0.001). Patients who underwent HSCT had a 4-year overall survival of 45% with a median post-transplant survival of 20.7 months. Compartive results of patients with >40 years old vs < 40 years old in OS was 75 % vs 35% at 18 months. The principal cause of death was infection (27%).

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Summary/Conclusion: The IST has a lower response rate in our center compared to what has been published internationally. Transplantation continues to be the gold standard treatment, especially in patients under 40 years of age, so it is important to procure an HLA donor in this group of patients and to do without delay.

PB1941: PRELIMINARY EXPLORATION OF PROLONGED USE OF ELTROMBOPAG IN PATIENTS WITH NAÏVE SEVERE APLASTIC ANEMIA IN THE REAL WORLD: DURATION AND EFFICACY

R. Li 1 , Z. Liu 1 , X. Chen 2 , Q. Long 2 , Y. Yang 3 , S. Lin 4 , J. Jia 5 , G. He 1,* , J. Li 1

1 Hematology, the First Affiliated Hospital of Nanjing Medical University; 2 Hematology, the Second People’s Hospital of Nanjing, Nanjing; 3 Hematology, the First Bethune Hospital of Jilin University, Changchun; 4 Hematology, Zhejiang Province Hospital of Traditional Chinese Medicine, Hangzhou; 5 Hematology, Peking University People’s Hospital, Beijing, China

Background: Eltrombopag (E-PAG) plus the immunosuppressive therapy (IST) consisted by antithymocyte immunoglobulin (ATG) and cyclosporin (CsA) has been reported with better efficacy in both refractory and naïve severe aplastic anemia (SAA) patients than IST alone. E-PAG was used up to 6 months in clinical trials. Patients with refractory SAA who remained on E-PAG with a median duration of 12 months showed further hematological improvement.

Aims: To explore whether patients with naïve SAA could benefit from continuous use of E-PAG beyond 6 months and when E-PAG should be withdrawn in patients with no response.

Methods: From February 2018 to November 2021, we collected 57 Chinese adult patients with naïve SAA who were treated with rabbit-ATG based IST (r-ATG, 3.5mg/kg/d × 5d and CsA 3-5 mg/kg/d) combining with E-PAG at dose of 75mg per day in the China Eastern Cooperation Group for Anemia (Clinical trials: ChiCTR2100045895). All patients were treated with E-PAG at least 6 months, and then whether to continue treatment was decided by the attending doctor according to patients’ situation. The dosage of E-PAG would be tapered gradually at 3 months after complete remission (CR) or 6 months after stable partial remission (PR).

Results: Clinical and laboratory characteristics of patients were listed in Table 1. The median time from diagnosis to treatment was 3 weeks (1 week to 291 weeks). The median follow-up time was 17 months (1 month to 44 months) and overall survival rate was 98%.

The overall response rates were 35% (20 of 57), 67% (36 of 54), 84% (42 of 50) and 95% (41 of 43) at 1, 3, 6 and 12 months, and CR rates were 17% (9 of 54), 24% (12 of 50) and 35% (15 of 43) at 3, 6 and 12 months. The first response was observed at 2 weeks after treatment, and the median time to remission during the first 6 months was 11 weeks (2 weeks to 25 weeks). 11% of patients achieved efficacy between 7 and 12 months. In patients with NR after continuous use of E-PAG for 12 months, no more patients met criteria for PR by extending the treatment of E-PAG. The cumulative effect curve showed that 88 percent of all 12 months responses occurred within 6 months, and the efficacy reached a peak at 6 months (Figure 1).

11 patients (22%, 11 of 50) who achieved CR within 6 months were under steady state with the exception of 1 patient converting to paroxysmal nocturnal hemoglobinuria at 12 months after treatment. In 30 patients achieved PR at 6 months, 5 patients (17%, 5 of 30) discontinued E-PAG after 6 months and 25 patients had a continuous exposure to E-PAG, while the efficacy of 13 patients (52%, 13 of 25) were improved to CR within median extended usage time of 9 months (2 to 31 months) of E-PAG, and the dose of E-PAG was tapered gradually in 6 patients (24%, 6 of 25), maintained at 75mg per day in 7 patients (28%, 7 of 25). The other 12 patients (48%, 12 of 25) were in stable PR.

In 8 patients (16%, 8 of 50) were ineffective at 6 months, 1 patient died early at 1 month; 2 patients achieved PR at 12 months by E-PAG continuously; 5 patients treated with CsA alone latter; and 2 patients achieved CR at 12 months, 3 patients still were in NR in CsA alone group.

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Summary/Conclusion: It was suggested that E-PAG was used at least 6 months. Continuous administration of E-PAG could improve the hematological response of PR patients to CR. No patient with NR taking effect after 12 months, new options should be considered with caution for them.

PB1942: HEPATIC MANIFESTATIONS OF CHILDHOOD HEMOPHAGOCYTIC LYMPHOHISTOCYTOSIS

M. El Sayed 1 , F. Ebeid 1 , S. M. Abd El Kader 1 , F. El-Shorbay 1 , I. Ragab 1,*

1 Pediatric Hematology Oncology, Ain Shams University, Cairo, Egypt

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare hyper-inflammatory disorder caused by benign systemic overgrowth of macrophages in the reticulo-endothelial system leading to cytokine storm. The main features of HLH are; fever, splenomegaly, bi/pancytopenia; hyper-ferritinemia, hyper-triglyceridemia and hypo-fibrinogenemia; which if not diagnosed and early treated progresses to disseminated intravascular coagulation (DIC), multi-organ dysfunction with dismal outcome. Hepatic manifestations are not well recognized primary presentation in pediatric patients with HLH. This presentation mandates high levels of suspicious for early diagnosis.

Aims: To study the clinical, laboratory, and pathological hepatic manifestations in patients with HLH at diagnosis and upon reactivation.

Methods: A 6 month retrospective cohort study included 35 patients with genetically confirmed HLH divided into familial HLH by its types, X linked lymphoproliferative syndrome and HLH with partial albinism; following at pediatric hematology/oncology clinic, Ain Shams University. In the studied patients, detailed review of patient’s clinical, laboratory data of HLH, hepatic transaminases and synthetic liver functions were done at time of presentation, at week 2, 8 from treatment start and at time of reactivation; Liver biopsy results and genetic analysis were recorded. Biochemical liver involvement was considered when alanine aminotransferase was 3 or more the upper level of normal value at presentation. Overall and reactivation free survival were analyzed according to liver involvement.

Results: Thirty five patients with HLH were recruited with age range of 2-108 months at presentation. Twenty patients (62.9%) had genetically confirmed HLH; Twelve (34.3%) of them had MUNC13D mutations, Three (8.6%) had STXBP2 mutation, Five (14.3%) had RAB27A mutation while four (11.4%) had secondary HLH. Overall survival at 12 month was 40% with median event free survival of 3 months.Among studied patients; 29 (82.9%) had hepatomegaly at diagnosis with hepatic reactivation in 51.4%. Eight (22.8%) patients had biochemical liver involvement with transaminase level 6 times above normal at presentation. There was no significant difference in demographic data, clinical presentation, type of mutant gene, and overall or reactivation free survival according to liver involvement.

Summary/Conclusion: Variable transaminitis and synthetic liver dysfunction might be the presenting manifestation of HLH and upon reactivation. Significant biochemical liver involvement is an under recognized presentation of HLH. Hepatic involvement did not impact response to treatment and disease outcome.

PB1945: CLINICAL AND MOLECULAR CHARACTERISTICS OF CHILDREN WITH DIAMOND-BLACKFAN ANEMIA (DBA): A SINGLE CENTER EXPERIENCE IN ARGENTINA

M. F. Mininni 1,* , A. Albero 1 , V. J. Avalos 1 , C. Cervio 1 , A. C. Candás 1 , L. Diaz 1 , D. A. Chaves 1 , B. Milanesio 1 , C. M. Pepe 1 , G. Sciuccati 1

1 Hematologia y Oncologia, Hospital de pediatría “Prof. Dr. J. P. Garrahan”, Buenos Aires, Argentina

Background: DBA is an inherited bone marrow failure characterized by erythroblastopenia due to an intrinsic defect in erythropoietic progenitors. Half the patients (pts) present with physical malformations. To date 20 ribosomal proteins (RP) genes associated with DBA and 4 non-RP genes related to non-classical DBA ( GATA1, TSR2, EPO, ADA2 ) have been described.

Aims: To describe clinical and molecular characteristics and outcomes of a pediatric cohort with DBA admitted at a single tertiary pediatric center in Argentina

Methods: Patients diagnosed with DBA between 2004 and 2020 were retrospectively analyzed. DBA diagnosis was based on constitutional normocytic/macrocytic anemia, reticulocytopenia, paucity of bone marrow erythroid precursors and congenital anomalies.

Demographic, clinical, and laboratory data, genetic evaluation and outcomes were recorded. Pts were screened for DNA changes by using a custom designed NGS panel with 19 genes related to DBA ( RPS19, RPL11, RPL15, RPL18, RPL27, RPL31, RPL35, RPL35A, RPL5, RPS10, RPS15A, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 and GATA1 ). All identified variants were classified according to the American College of Medical Genetics guidelines.

Results: Fifteen unrelated pts with DBA were analyzed, 10 (66%) males, median age at diagnosis 3,7months (25days-6yrs). Twelve pts (80%) had physical malformations.

All pts showed paucity of bone marrow erythroid precursors, and only two presented with compromise of other linages.

Thirteen pts (87%) received treatment with prednisone, 8 (62%) achieved transfusion independence and half of them achieved complete treatment independency, while five pts (33%) did not respond to steroids and are under transfusion regimen. These 5pts developed liver iron overload and are under chelation therapy. No therapy was required in two pts with variants in GATA1 and RPL5 . Median follow-up was 7.1years (1.7-16.7 years), no pts received HSCT and no pts died.

Molecular variants were detected in 10/15 pts (66%), 8/10 were novel. Eight variants were classified as ¨pathogenic¨ or ¨likely pathogenic” and two as “of uncertain significance”. Three pts (30%) presented variants in RPS19, two (20%) in RPS26 , four (40%) in other RP genes and one pt with non-classical DBA, presented a variant in GATA1.

Phenotype and molecular results of the pts are summarized in Table1.

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Summary/Conclusion: In this cohort of pts with DBA, molecular diagnosis was achieved in the majority. RPS19 variants were the most common. Interestingly, all pts with RPS19 variants presented with malformations, one of them had also pancytopenia at onset.

As the penetrance of DBA is incomplete, the molecular diagnosis is important for detecting silent phenotype carriers in the family that will allow for an accurate selection of stem cell donors, genetic counseling and tailored treatment.

PB1946: ACQUIRED AMEGAKARYOCYTIC THROMBOCYTOPENA (AAMT), A SINGLE CENTER EXPERIENCE

S. Mohamed 1,* , S. Kohla 1,2 , K. Ahmed 1 , A. Amer 2 , K. Obeidat 1,3 , D. Soliman 2 , F. Ibrahim 2

1 hematology; 2 Department of Laboratory Medicine and pathology, NCCCR, Doha, Qatar; 3 Medicine, John Stroger hospital, Chicago, United States of America

Background: Acquired Amegakaryocytic Thrombocytopenia (AAMT) is a very rare hematological condition. There are few cases reported in the literature. It is characterized by severe thrombocytopenia with complete absence or severe reduction of megakaryocytes in the bone marrow, with otherwise normal hematopoiesis. The underlying mechanism for AAMT is not fully understood but is suggested to be related to anti-thrombopoietin (TPO) antibodies. The diagnosis of AAMT can be challenging for both clinicians and pathologists. On many occasions, it goes undiagnosed or misdiagnosed as Immune thrombocytopenic purpura (ITP).

Aims: Describe patients (pts) or cases of AAMT diagnosed in our national center for cancer care and research (NCCCR) in Qatar.

Methods: We retrospectively collected pts aged ≥ 18 years (yrs.) with AAMT based on bone marrow finding who had been diagnosed and/or treated between January 2011 and December 2021 at the (NCCCR) in Qatar.

Results: Seven cases were identified; the median age was 43 years (25-55). Four patients were males and all of the patients were from Asian ethnicity. Complete blood count (CBC) showed HB with median of 9.2 gm/dl (6.6-11.8), white blood cells of 6.8 x10^3/ul (2.1- 9) and platelets 13 x10^9/L (5-20). Bone marrow biopsies showed suppressed or absence of megakaryocytic components in all patient with variable degrees of marrow cellularity. potential underlying etiologies identified in the cases were autoimmune disease, HBV, B12 deficiency and solid malignancy. Three patients progressed to frank aplastic anemia. Steroids plus intravenous Immunoglobulin (IVIGg) were the backbone of treatment of all patients since they were diagnosed as ITP first before bone marrow sampling. The addition of the thrombopoietin receptor agonist eltrombopag and immunosuppressant Cyclosporine improved the platelets’ response in five patients. Transplant was considered for patients who developed aplastic anemia however, not done due to improvement in counts or due to patients’ social reasons

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Summary/Conclusion: In summary, AAMT is rare disease characterized by thrombocytopenia resulting from a marked decrease in bone marrow megakaryocytes. It’s very important to consider it in some cases of ITP as an alternative diagnosis specially in atypical cases or cases resistant to treatment. eltrombopag and Cyclosporine improved patients’ blood counts. There are no guidelines currently for the management of this rare condition

PB1947: ELTROMBOPAG COMBINED WITH IST AS A MULTICENTER STUDY OF THE FIRST LINE TREATMENT OF CHILDREN WITH SAA.

L. Yang 1,* , H. He 2 , Y. Fang 3 , Y. Yang 4 , S. Lin 5 , G. He 6 , N. Wang 1

1 Department of pediatric, the second affiliated hospital of Anhui medical university, hefei; 2 Department of hematology, Children’s Hospital of Soochow University, suzhou; 3 Department of hematology, Children’s Hospital of Nanjing medical University, nanjing; 4 Department of hematology, The first hospital of Jilin University, jilin; 5 Department of hematology, The First Affiliated Hospital of Zhejiang Chinese Medical University,Zhejiang Provincial Hospital of TCM, hangzhou; 6 Department of hematology, The First Affiliated Hospital of Nanjing Medical University，Jiangsu Province Hospital, nanjing, China

Background: Eltrombopag and Improved Hematopoiesis in Refractory Aplastic was published in the New England Journal of Medicine in 2012 The research found that Eltrombopag monotherapy showed significant hematologic reaction in adult patients with refractory SAA, of which 44%(11/25) patients had clinically significant hematologic performance. Important progress has also been made in the treatment of SAA with Eltrombopag combined with IST: A nonrandomized, historically-controlled study funded by the NHLBI intramural Research Program of the National Institutes of Health compared the efficacy and safety of conventional IST therapy with Eltrombopag in combination with IST for SAA. The research results were published in Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia in 2017 in the New England Journal of Medicine. In this study, a total of 92 SAA patients aged 2 years and above were enrolled, and the results showed that the overall effective rate and complete response rate of Eltrombopag combined with IST in the treatment of SAA were higher than that of traditional IST standard treatment. However, there is no research on Eltrombopag therapy for children SAA in China. The aim of this study was to improve the hematologic response rate and quality, and to prevent late complications such as recurrence and clonal progression, by adding Eltrombopag to standard IST therapy.

Aims: To evaluate the efficacy and safety of attriboma combined with IST as first-line treatment for SAA patients, and to study the clonal transformation and the incidence of PNH cloning in attriboma combined with IST patients.

Methods: This study was a non-randomized, multi-center, historical controlled study, with traditional IST standard therapy as the historical control. All patients in the experimental group received “IST+ Attribopal” combined therapy, in which ATG dose was 3.5mg. Kg -1 .d -1 for a total of 5 days. The initial dose was 1.25 mg · kg -1 · d -1 for 2-5 years old children, 37.5mg · d -1 for 6-11 years old, 75mg · d -1 for 12-18 years old, and continued for 6 months. A total of 31 children were included in the Altrupalpa group and 136 in the historical control group.

Results: There were no differences in gender, severity of disease, co-infection before ATG, co-infection within 1 month after ATG, incidence of serum disease, CD3, CD4, CD8, TREG cell count, hemoglobin level, platelet count, neutrophil count and ferritin level between the attripopa group and the historical control group. In terms of the effective rate, the effective rate of attriboma group was 62.5% vs 68.8% (P= 0.548) in June and 86.4% vs 76%(P=0.285) in September, showing no statistical significance. Among them, neutrophil count recovery was better in the attriboma group than in the historical control group at 1 month, 3 months, 6 months, 9 months and 12 months after the application of ATG, the difference was statistically significant (P < 0.05), while platelet and hemoglobin recovery was not different. 2-year EFS 80.4% VS 83.4% (P=0.799) and 2-year OS 93.3% VS 91.3% (P=0.548) in the attriboma group and the historical control group. None of the patients with clone transformation or PNH clone were found in all the follow-up of attriboma group.

Summary/Conclusion: There was no difference in infection rate, serum disease rate and overall effective rate between the two groups within 1 month, but the neutrophil count level of the attriboma groups was better than that of the historical control group.

PB1949: ELTROMBOPAG IN CHINESE PATIENTS WITH REFRACTORY OR RELAPSED SEVERE APLASTIC ANEMIA: A NON-RANDOMIZED, OPEN-LABEL, MULTICENTER, PHASE II STUDY ASSESSING THE SAFETY AND EFFICACY OF ELTROMBOPAG

H. Chang 1 , G. He 2 , R. Fu 3 , F. Li 4 , B. Han 5 , T. Li 6 , L. Liu 6 , H. Mittal 6 , H. Jin 6 , F. Zhang 7,*

1 West China Hospital of Sichuan University, Chengdu; 2 Jiangsu Province Hospital, Nanjing; 3 Tianjin Medical University General Hospital, Tianjin; 4 The First Affiliated Hospital of Nanchang University, Nanchang; 5 Peking Union Medical College Hospital; 6 Novartis Pharma Co., Ltd.; 7 Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China

Background: Severe aplastic anemia (SAA) is a life-threating bone marrow failure disorder characterized by pancytopenia and hypocellular bone marrow. SAA that does not respond to or relapses after first-line immunosuppressive therapy (IST) is known as refractory or relapsed (R/R) SAA. For Chinese patients with R/R SAA, there is no established standard of care other than transfusion support and treatment of infections. Evidence suggests that, even with support, a substantial proportion of patients with R/R SAA die within 5 years of diagnosis. There is evidence that eltrombopag can improve response rates in patients with R/R SAA. Eltrombopag is approved in the United States and Europe for the treatment of SAA following an insufficient response to IST. Eltrombopag is not currently approved for the treatment of SAA in China.

Aims: To assess eltrombopag efficacy and safety in Chinese patients with R/R SAA.

Methods: This Phase II, non-randomized, multicenter, open-label study ( NCT03988608 ) enrolled Chinese adult patients (≥18 yrs) with R/R SAA. The starting dose of eltrombopag was 25 mg/day, which increased by 25 mg/day every 2 wks based on the total platelet count, up to a maximum dose of 150 mg/day. The primary endpoint was hematologic response (HR) rate at Wk 26, defined as the proportion of patients meeting any of the International Working Group criteria for changes in platelet, hemoglobin, or neutrophil count. Secondary endpoints included HR rate at Wk 13 (and 1 yr; data not shown); time to HR and its duration; changes in transfusion need; safety outcomes; and the rate of clonal evolution.

Results: A total of 20 patients were enrolled. The median (range) age was 37.5 (18-69) yrs, 75% were male, and all (100%) patients were Asian. The median (range) duration of exposure to eltrombopag was 258.5 (69-443) days, and the median (range) daily dose was 122.8 (26-138) mg/day. The primary endpoint (HR at Wk 26) was met in 70% (14/20) of patients (7/14 bi-lineage; 2/14 tri-lineage) (Fig. 1). The secondary endpoint (HR at Wk 13) was met in 65% (13/20) of patients (4/13 bi-lineage; 1/13 tri-lineage) (Fig. 1). The median (90% confidence interval) time to HR was 10 (8-12) wks. Of the 14 patients who were red blood cell (RBC) transfusion-dependent at baseline, 10 (71%) and 8 (57%) had reduced RBC transfusion or were transfusion-independent vs baseline at Wks 13 and 26, respectively. Of the 10 platelet transfusion-dependent patients at baseline, 7 (70%) and 8 (80%) had reduced platelet transfusion or were transfusion-independent at Wk 13 and 26, respectively. Adverse events (AEs) occurred in 19/20 patients (95%), with 70% (14/20) of patients experiencing a treatment-related AE (TRAE) and 15% (3/20) of patients having a grade ≥3 TRAE. The most common all-grade AE was upper respiratory tract infection (30%). There were 2 cases of treatment-related severe AEs (osteonecrosis [n=1]; groin pain [n=1]). One death from gastrointestinal bleeding (not considered study drug-related) was reported 45 days after last eltrombopag dose. One case of transient clonal evolution (abnormal karyotype present at Wk 13; absent at Wk 26) was reported.

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Summary/Conclusion: Overall, 70% of patients met the primary endpoint of HR at Wk 26, with more than half of patients reporting reduced transfusion need during the study. Safety findings were consistent with the established safety profile of eltrombopag. Although the sample size was small, this is the first prospective study to show that eltrombopag is efficacious and has a favorable safety profile in a Chinese population with R/R SAA.

PB1950: MASS CYTOMETRY ANALYSIS IDENTIFIES T CELL IMMUNE SIGNATURE FOR APLASTIC ANEMIA AND PREDICTS RESPONSE TO CYCLOSPORINE

L. Zhang 1 , J. Mao 1 , Y. Lian 1 , Q. Liang 1 , H. Pan 1 , Z. Gao 1 , W. Li 1 , J. Zhao 1 , L. Fang 1 , Y. Chu 2 , W. Yuan 2 , J. Shi 1,*

1 Regenerative Medicine Clinic; 2 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Aplastic anemia (AA) is a bone marrow failure syndrome attacked by autologous T cells. Cyclosporine is often taken for non-severe AA which is accounting for 80% of AA. Although effective, the response rate to cyclosporine is only around 50%. Emerging data support that T cell subpopulations, expression of transcription factors and immune checkpoint molecules are critical to regulate pathophysiology of AA. It’s also verified that T cell egress and activation play an important role in autoimmune diseases.

Aims: We hypothesis that defining T cell immune signature will facilitate the development of predicting cyclosporine response and exploring novel immune-based therapeutics.

Methods: Here, we performed mass cytometry for phenotypic and functional immune analysis on clinical samples from AA patients receiving cyclosporine treatment and demonstrated a significant heterogeneity on expression of inhibitory molecules, T cell trafficking and cytokines.

Results: T-cell expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) was upregulated following response to cyclosporine and a lower level of CTLA-4 on CD8 + T cells was correlated to better treatment response. Moreover, higher expression of sphingosine-1-phospharylation receptor 1 (S1P 1 ) on naïve T cells and higher level of interleukin-9 (IL-9) on T helpers predicted a worse response to cyclosporine. Therefore, this immune signature has a strong predictive value for clinical response.

Summary/Conclusion: Collectively, our study suggests that immune-based analysis may predict clinical response to cyclosporine and provide a therapeutic strategy to improve the treatment of AA.

PB1951: INVESTIGATION OF THE RELATIONSHIP BETWEEN CD56 AND CD117 EXPRESSIONS AND CLINICAL AND LABORATORY FINDINGS IN MULTIPLE MYELOMA PATIENTS AT DIAGNOSIS

D. M. Akkurd 1,* , H. Haydaroglu Sahin 1 , E. Yilmaz 1 , V. Okan 1

1 Haematology department, Gaziantep University Sahinbey Research And Practice Hospital, Gaziantep, Turkey

Background: Multiparametric flow cytometry-based immunophenotyping is a useful tool for the diagnosis and monitoring of multiple myeloma.Multiparametric flow cytometry can distinguish malignant plasma cell populations from the reactive/normal plasma cells in samples effectively.

Aims: We planned to analyze the relationship between demographic data, biochemical parameters, survival times of CD56 and CD117 positive or lamps in MM patients who were treated at the center and present it as the experience of a center.

Methods: In our study, 94 Multiple Myeloma patients who were diagnosed and treated and followed up in Gaziantep University Hospital Hematology Clinic between April 2004 and October 2018 were included. Normal distribution tests of numerical variables were performed using Kolmogorov-Smirnov and Shapiro Wilk tests. Survival analyzes were performed by Kaplan Meier analysis. Analyzes were carried out with the help of SPSS 22.0 program. A significance level of p <0.05 was chosen.

Results: The median age of the patients was 62.5. 57.4% of the patients were male and 42.6% were female. 6.3% of the patients were IgM type, 72.3% IgG, 13.8% IgA, 7.4% light chain type. Kappa chain type was present in 55.3% of the patients, while lambda type was detected in 44.6% of the patients. The CD56 expression rate of the patients at the time of diagnosis was 42.5%, and the CD117 expression rate was 30.9%. Double negativity was calculated as 53.1%, while double positivity was calculated as 26.5%. A statistically significant relationship was found between the detection of CD56 positivity at the time of diagnosis and early stage disease according to the ISS classification (p = 0.01). When hemogram and biochemical parameters were examined separately, it was shown that creatinine was numerically significantly lower in CD56 and C117 positive patient groups (p = 0.01) (p = 0.04), but no statistically significant difference was found between the two groups in terms of renal involvement. The median overall survival (OS) duration was 44 months (25.8-62.1) and the 5-year OS rate was 40.2%.

Summary/Conclusion: The results of the study show that, no statistically significant relationship was found between the presence of CD56 expression and OS (p = 0.404). In the group with negative CD117 expression, the median OS duration was 50.1 months (32.2-67.7), and the 5-year OS rate was 39.5%. In the group with positive CD117 expression, the median OS was 61.7 months (17.3-106) and the 5-year OS rate was 50%. When looking at the general results, a statistically significant relationship was not found between the presence or absence of CD117 expression and OS (p: 0.853).

PB1952: EFFECTS OF KNOCKDOWN OF NUPR1 GENE ON THE PROLIFERATION AND APOPTOSIS OF MULTIPLE MYELOMA CELLS AND ITS MECHANISM

C. Zeng 1,* , C. Xiao 1 , Y. Nan 1 , L. Zhang 1 , Y. Xiang 1 , Y. Liu 1

1 Department of Hematology-Oncology, Chongqing University Cancer Hospital, Chongqing, China

Background: Multiple myeloma is easy to relapse and incurable. It is necessary to further explore the pathogenesis to find new drugs

Aims: To construct nuclear protein 1-short hairpin RNA (NUPR1-shRNA) expressing lentiviral vector targeting NUPR1 gene and investigate the effect of NUPR1 gene silencing on the proliferation and apoptosis of human multiple myeloma U266 and RPMI8226 cells and its related mechanisms.

Methods: The normal human bone cells were used as control and the mRNA expression level of NUPR1 in multiple myeloma cell lines (U266 and RPMI8226) and MM patient specimens were detected by quantitative real-time PCR (qRT-PCR). Then the NUPR1 gene target shRNA plasmid was constructed and screen for the best target for transfected into U266 and RPMI8226 cells. The transfection efficiency was detected by flow cytometry. The interference effect of NUPR1 gene was detected by qRT-PCR and Western blotting. The cell proliferation was analyzed by CCK-8 assay. The cell apoptosis and cell cycle were determined by flow cytometry. Quantitative RT-PCR and Western blot were used to detect the expression of proliferation and apoptosis-related genes and proteins.

Results: The mRNA expression levels of NUPR1 in MM cell lines (U266 and RPMI8226) and MM patient specimens were higher than that in normal bone cells. NUPR1-shRNA-expressing lentiviral vector was successfully constructed, and flow cytometry results shows that the transfection efficiency of both cell line reached over 80%. Quantitative RT-PCR and Western blot showed that NUPR1-shRNA2 lentiviral vector most effectively and as the best target. With NUPR1 gene interference, cell proliferation were significantly inhibited in U266 and RPMI8226 cells infected with the NUPR1-shRNA lentiviral compared with the control group. Compared with the control group, the apoptosis rate of U266 and RPMI8226 in shNUPR1 group were significantly increased ( P <0.05). Flow cytometer indicated that the U266 and RPMI8226 cells in shRNA group were arrested in G0/G1 phase ( P < 0.01). Compared with the control group, qRT-PCR and Western blot showed that the levels of Bcl-2 and PCNA were significantly down-regulated, and the level of PTEN was significantly increased in shNUPR1 group. Meanwhile, the expression of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 protein were significantly higher in U266 and RPMI8226 cells infected with the NUPR1-shRNA lentiviral compared with the control group.

Summary/Conclusion: The mRNA expression levels of NUPR1 in U266 and RPMI8226 cells were higher than that in normal bone cells, and the NUPR1-shRNA-expressing lentiviral vector was successfully constructed. Down-regulate the expression of NUPR1 in U266 and RPMI8226 cells can inhibit cell proliferation and promote its apoptosis.

PB1953: ASSESSMENT OF THE EFFICACY OF BORTEZOMIB-CONTAINING THERAPY REGIMENS DEPENDING ON MAGE-C1 GENE EXPRESSION IN MULTIPLE MYELOMA PATIENTS.

M. Eleonora 1,* , M. Larisa 1 , S. Vadim 1 , S. Maxim 1 , F. Maia 1 , G. Irina 1 , K. Sergey 1 , S. Anton 1 , M. Elisabeth 1 , A. Alexandra 1

1 Federal State-Funded Institution «National Research Center for Hematology» of the Ministry of Healthcare of Russia, Moscow, Russia

Background: The treatment of multiple myeloma (MM) has undergone significant changes over the past decade due to the emergence of new therapeutic agents and the development of personalized approaches. The search for factors to predict the course of the disease remains relevant. MAGE-C1 gene expression is considered as a possible marker of refractoriness to bortezomib-containing therapy regimens.

Aims: Determine MAGE-C1 gene expression by real-time polymerase chain reaction (RT-PCR). Evaluate the effectiveness of bortezomib-containing regimens in MM patients depending on MAGE-C1 gene expression.

Methods: The prospective study from February 2019 to November 2021 included 81 MM patients (37 men, 44 women), aged 35 to 82 years (Me 59). The level of MAGE-C1 gene expression by RT-PCR in bone marrow samples, enriched with CD138+ cells, was determined in all patients. The diagnosis of MM was made according to the IMWG (2014). The ISS stage was determined as I in 24 (20%) patients, II in 21 (67%), III in 36 (13%). Plasma cell content ranged according to myelogram from 1 to 89% (Me 26%), paraprotein secretion was 0.1 to 85 g/L, Bens-Jones protein in urine (from trace values to 12.54 g/day) was determined in 54 patients. Bone plasmacytomas were documented in 42 patients, myeloma nephropathy in 19 cases, high risk cytogenetic in 25. Induction of remission was carried out by three-component regimens including bortezomib, 30 patients required two or more lines of therapy with lenalidomide or daratumumab. The number of courses was 4 to 10 (median 7). The anti-tumor response after 2-6 courses of therapy with bortezomib-containing regimens was evaluated in 68 patients according to the IMWG (2016). Anti-tumor ≥partial response (PR) was achieved in 52, refractory to bortezomib MM was found in 16 patients. As a control, similar bone marrow cells of 7 healthy donors were investigated.

Results: The maximum value of MAGE-C1 gene expression in donors was 0.06 (2ΔCt). The MAGE-C1 gene expression value >0.06 (2ΔCt) in patients was accepted as increased. The group with normal MAGE-C1 gene expression (0.003 to 0.05, 2ΔCt) consisted of 25 patients and the group with increased expression (0.07 to 14.12, 2ΔCt) consisted of 56 patients.

The probability of achieving ≥PR in patients with normal MAGE-C1 gene expression was 87.8% and in patients with increased expression- 72.8%. The median time to achieve ≥PR in patients with an expression value ≤0.06 (2ΔCt) was 1.77 months, and in patients with an expression >0.06 (2ΔCt) was 3.06 months, p=0.03 (Fig. 1). In addition, we were able to determine that the expression values of the studied gene were significantly lower in patients with ≥PR compared to patients who were refractory to bortezomib: median 0,15 vs 0.27 (2ΔCt), p=0.05.

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Summary/Conclusion: In a single-factor analysis, we were able to determine that patients with normal MAGE-C1 gene expression were significantly more likely to achieve an overall response (≥PR) compared to patients whose MAGE-C1 gene expression was increased. Achievement of ≥PR on bortezomib-containing therapy regimens was documented twice as fast in patients with normal expression of the investigated gene. Further study of the MAGE-C1 gene may allow to determine the tactics of induction therapy already at the diagnostic stage.

PB1954: COMPARING STR PROFILES OF DNA ISOLATED FROM DIVERSE TUMOR SITES IN MULTIPLE MYELOMA

M. Firsova 1,* , N. Risinskaya 2 , E. Nikulina 2 , T. Obukhova 3 , T. Abramova 3 , M. Solovev 1 , A. Sudarikov 2 , L. Mendeleeva 1

1 Department of high-dose chemotherapy of paraproteinemic hemoblastoses; 2 Department of Molecular Hematology; 3 Laboratory of karyology, National Research Center for Hematology, Moscow, Russia

Background: Multiple myeloma (MM) course is often complicated by extramedullary lesions - plasmacytomas, which pathogenesis is still not completely understood. Genome instability underlies dissemination in various neoplasias, including MM. An important manifestation of genetic instability is the loss of heterozygosity, i.e., the loss of the second allele in the regions of the genome featuring inherited polymorphisms. STR profiling is a method that allows simultaneously assessing the degree of DNA degradation and giving an integral assessment of the stability of the tumor genome. To assess the heterogeneity of MM, not only the tumor substrate in the bone marrow and plasmacytoma but also in circulating cell-free DNA (cfDNA) present in the peripheral blood plasma could be effectively analyzed.

Aims: To compare STR profiles of DNA isolated from various anatomical locations (plasma, bone marrow, plasmacytoma) in MM patients.

Methods: The prospective study included 21 patients with newly diagnosed MM (11 women, 10 men) aged 39 to 82 years (median 53 years). MM was diagnosed according to the IMWG-2014 criteria. In 15 patients, MM was complicated by plasmacytoma, in 6 of them a tumor biopsy was performed and tumor DNA was isolated. In all patients, plasma circulating cfDNA and control DNA were isolated from buccal epithelium using a commercial kit (Qiagen, Germany) according to the manufacturer’s protocol. In 11 patients, positive immunomagnetic selection of CD138+ bone marrow cells was performed using a monoclonal antibody to CD138 (Miltenyi Biotec, Germany). STR-profiling using COrDIS Plus panel (Gordiz LLC, Russia) of isolated DNA samples (21 plasma circulating cfDNA samples, 11 CD138+ DNA samples of bone marrow cells, 6 plasmacytoma DNA samples) was performed. STR profiles were analyzed using GeneMapper v.4 software (Applied Biosystems, USA).

Results: In 10 out of 11 patients, analysis of STR profiles in CD138+ bone marrow cells revealed loss of heterozygosity (LOH) variants with different allelic load, suggesting either a deletion/copy number neutral LOH, or a duplication of one of the two alleles affecting from one to six STR loci for every patient. In all patients for whom plasmacytoma DNA was isolated (n=6), various variants of LOH were also identified (from 1 to 6 loci) (Table 1). Of the 21 plasma circulating cfDNA samples, LOH variants were detected in ten cases (48%) (from 1 to 4 loci), in the other 10 cases, no tumor DNA was detected, the samples matched the control DNA from the buccal epithelium; only in one case, the result turned out to be non-informative due to a significant degree of degradation of the cfDNA. STR profiles of DNA isolated from different tumor sites of the same patient in most cases were identical. In one case aberrant STR loci in plasma circulating cfDNA and bone marrow CD138+ cells were different (patient No. 20). In this patient, MM was complicated by plasmacytomas of various anatomical locations (Th7, 8th rib). One can speculate that the discrepancy between plasma circulating cfDNA and bone marrow CD138+ STR profiles indicates the presence of various MM clones in different tumor loci.

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Summary/Conclusion: Evaluation of plasma circulating cfDNA might be an effective approach for molecular diagnosis in MM. In 48% of MM patients, it is possible to detect tumor cfDNA in the plasma at the onset of the disease. Comparison of DNA STR profiles from different lesions might reveal the diversity of tumor clones in MM.

PB1955: THE AGENDA FOR FUTURE MULTIPLE MYELOMA RESEARCH ACCORDING TO PATIENTS, CAREGIVERS, AND CLINICIANS

S. Fowler 1,* , L. McLaughlin 1 , S. Bridges 1 , M. Robichaud 2 , B. Ridgway 3 , D. Reece 3,4 , K. Song 3,5 , L. Dalrymple 3 , R. Sully 3 , S. Nason 3 , S. Rowland 3,4 , T. MacDonald 3 , W. Paine 3 , A. Reiman 6,7,8

1 Maritime SPOR SUPPORT Unit, Horizon Health Network, Saint John; 2 Maritime SPOR SUPPORT Unit, Université de Moncton, Moncton; 3 Canadian Myeloma Priority Setting Partnership Steering Group, Montreal; 4 Cancer Clinical Research Unit, Princess Margaret Cancer Centre, Toronto; 5 Leukemia/Bone Marrow Transplant Program, Vancouver General Hospital, Vancouver; 6 Oncology, Horizon Health Network; 7 Biological Sciences, University of New Brunswick; 8 Medicine, Dalhousie University, Saint John, Canada

Background: Multiple myeloma, a hematological malignancy characterized by the abnormal accumulation of plasma cells in the bone marrow, is increasing in incidence worldwide. Yet despite its status as the second most common hematological malignancy, much remains unknown about this incurable disease. The insufficiency of scientific knowledge related to the diagnosis, treatment, and management of multiple myeloma has real-world implications for patients and those who care for them. These individuals often have unanswered questions about living with or treating multiple myeloma. By addressing stakeholder questions, researchers can simultaneously advance the uptake of their research into practice and improve the quality of life of those with the disease.

Aims: The purpose of this study was to empower patients, caregivers, and clinicians to set the agenda for the future of multiple myeloma research, thereby leading to research that is meaningful to those impacted by the disease and the improved translation of evidence into practice.

Methods: We followed the rigorous and transparent methodology of the James Lind Alliance (JLA) to set the priorities for future research. The iterative priority-setting process began with a national survey to elicit questions from the multiple myeloma community ( N = 594). The resulting questions were formed into summary questions and subsequently compared to systematic reviews and clinical practice guidelines to ensure that they were unanswered by prior research. The remaining unanswered questions were included in an interim prioritization survey, wherein participants ( N = 651) selected and ranked their top questions. We used the results of this survey to inform the selection of a shortlist of questions for inclusion at the final consensus-building workshop. At the event, JLA facilitators used nominal group techniques to help the participants ( N = 23) rank the questions and produce a Top 10 list of priorities.

Results: Through this process, we distilled 3,042 questions from the myeloma community into 66 summary questions. We compared these questions to 608 systematic reviews and 321 clinical practice guidelines and determined that seven questions were answered. After consultation with our steering group of patients, caregivers, and clinicians, we included 45 questions in the interim prioritization survey. Quantitative analysis of survey responses identified 18 questions for ranking at the final workshop.

The Top 10 list is predominantly about improving pharmacological treatments for multiple myeloma. Beyond this threshold, there were also important questions about wide-ranging topics, including physical activity, genetic testing, cognitive and immune functioning, minimal residual disease, and familial screening (see Table).

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Summary/Conclusion: By moving beyond the Top 10 research priorities, we can appreciate the breadth of questions posed by the multiple myeloma community. These questions demonstrate that research is needed in diverse areas related to multiple myeloma to inform a holistic care approach and contribute to improved quality of life for patients. Therefore, we call upon research funders, researchers, and service providers across disciplines to recognize the perspectives and experiences of multiple myeloma patients and caregivers and invest in answering these questions.

PB1956: EVALUATION OF AN INNOVATIVE NEW METHOD FOR QUANTITATION OF PLASMA CELLS ON CD138 IMMUNOHISTOCHEMISTRY

E. Gantana 1,* , N. Mashigo 1 , I. Abdullah 1 , E. Musekwa 1 , R. Lohlun 1 , E.-M. Nell 1 , C. Chetty 1 , M. Ntobongwana 1 , Z. Chapanduka 1

1 Haematology Pathology, National Health Laboratory Service, Cape Town, South Africa

Background: The estimation of plasma cells in bone marrow biopsies using CD138 immunohistochemistry, as proposed by the World Health Organization (WHO), has become popular over the last few years. The determination of the plasma cell percentage plays a vital role in categorising patients into different diagnostic and prognostic groups at diagnosis and evaluating response to treatment during follow-up, which guides management protocols. There is currently no reference method (gold standard) for the estimation of plasma cells. The most commonly used method involves scanning of slides and expression of plasma cell numbers as a percentage of all nucleated cells (overview estimate).

Aims: To compare the frequently used CD138 immunohistochemistry- based method of plasma cell quantitation, to a proposed new method, using interobserver and intraobserver concordance parameters.

Methods: Archival CD138 immunohistochemically stained slides made from paraffin- embedded bone marrow biopsies of 33 patients with a confirmed diagnosis of multiple myeloma were used. Light microscopic examination was performed using low magnification lenses (10×) for both the overview estimation method (method A) and the new method (method B), and high magnification lenses (50×), for method B only. For method B, reviewers selected three areas with low, intermediate and high plasma cell densities using 10× lenses. Using a well- defined technique, the 50× lens was then used to count plasma cells as a percentage of all nucleated cells. After blinded relabelling of all the slides, the nine reviewers repeated the plasma cell quantitation using both methods. The plasma cell counts were obtained, and the review times were recorded.

Results: Overall intraobserver concordance was comparable for method A (concordance correlation coefficient (CCC)=0.840) and method B (CCC=0.733). Interobserver concordance for method A (intraclass correlation coefficient (ICC)=0.793 and 0.713) and method B (ICC=0.657 and 0.658) indicated high similarity between reviewers. Method A showed poor interobserver concordance (ICC=0.105) at low plasma cell densities.

Summary/Conclusion: The new method is comparable to the frequently used overview estimation method in terms of intraobserver and interobserver concordance, and cost. The new method has superior interobserver concordance at low plasma cell densities. The new method appears more amenable to digital scanning and analysis.

PB1957: CIRCULATING MIRNA-16 AND MIRNA -21 EXPRESSION LEVELS IN MYELOMA; PRELIMINARY RESULTS.

A.-I. Gkioka 1,* , M. Tsota 2 , M. Papadatou 1 , A. Gkiokas 1 , A. Alexandropoulos 1 , V. Bartzi 1 , G. V. Dedousis 2 , M.-C. Kyrtsonis 1

1 Laikon University Hospital Athens Hematology Section - 1st Department Of Propaedeutic Internal Medicine Athens, National and Kapodistrian University of Athens, Greece, National and Kapodistrian University of Athens; 2 Nutrition and Dietetics School of Health Sciences and Education, Harokopeio University, Athens, Greece

Background: MicroRNAs (miRNAs) are short, noncoding RNAs of 19–22 nucleotides that participate in gene expression regulation. Many studies have focused on circulating miRNAs which might be potential biomarkers. In Multiple Myeloma (MM) they are involved in bone marrow microenvironment, methylation, immune regulation, genomic instability, and drug resistance. Their exact role has not been fully explored yet.

Aims: We investigated hsa-miR-16-5p miRNA-16 and miRNA-21 expression levels in MM patients’ serum, their possible prognostic role and correlations with disease parameters.

Methods: Thirty-seven MM patients were studied, of whom 22 were at diagnosis and then all 37 at relapse before lenalidomide-dexamethasone treatment (RD). We analyzed miRNA-16 and -21 expression in serum of 49% were women and 51% men with median age 69 yrs (range 49-84), 46% were ISS 1, 19% ISS 2 and 35% ISS 3. Ig type was IgG in 62%, IgA in25%, Light-Chain in 11% and other in 2%. Median OS was 75 mo. Clinical characteristics and laboratory tests, as well as sera samples, at diagnosis and before RD treatment were collected after patients’ informed consent. Frozen sera from patients and 14 healthy individuals (HI) were used for miRNA detection processed by Real–Time PCR. MiRNA expression was compared to exogenous has-miRNA-39-5p. Two-Fold change (x2) expression (upregulation) of miRNA was considered as overexpression. Statistical analysis was performed with the SPSS v.25 software. We analyzed thereafter the compared levels of miRNA-16 -21 termed as hsa-miR-16 and hsa-miR-21. Upregulation by one fold was used as cutoff value for survival analysis plotted by Kaplan Mayer.

Results: Median hsa-miR-16 expression at diagnosis was 0.23 and 0.64 at relapse. Median hsa-miR-21 expression was 0.63 at diagnosis and 1 at relapse. In HI, median expression levels were 13.8 for hsa-miR-16 and 13.3 for hsa-miR-21. The difference between HI and patients was significant, with regard to hsa-miR-16 at diagnosis (p= 0.003) and at relapse (p=0.007), as well as to hsa-miR-21 at diagnosis (p=0.041) and relapse (p=0.001). A correlation between hsa-miR-16 and hsa-miR-21 was found at diagnosis (p=0.0003, r =0.706) and before RD initiation (p =0.02 , r = 0.438). Hsa-miR-16 at diagnosis correlated with abnormal Calcium (p=0.05, r =-0.424), Creatinin (p=0.01, r =-0.534), while hsa-miR-21 expression levels at diagnosis were negatively correlated with abnormal Creatinin (p=0.04, r =0.438). No correlation was observed between miRNA serum levels and age and sex at diagnosis and at RD initiation. Hsa-miR-21 before RD treatment correlated with median bone marrow infiltration ( r =0.549, p=0.02), RISS ( r =0.769, p=0.005) and the type of relapse to lenalidomide (clinical or biochemical) ( r = -617, p=0,02), while hsa-miR-16 correlated with abnormal LDH ( r = 0.365, p=0.03), number of previous treatment lines ( r =- 0.334, p=0.04) and negatively with creatinin ( r = - 0.427, p=0.01).

Decreased has-miR-16 expression at diagnosis was related to better survival (p=0.16).

Summary/Conclusion: We observed correlations between factors of MM disease activity and hsa-miR-16 and hsa-miR-21 while in addition hsa-miR-16 expression levels predicted survival. Larger studies shall confirm these results.

First Authors; Gkioka A-I *, Tsota M*, *equal contribution,

Signing Authors; Kyrtsonis MC § , Dedousis VG § § equal contribution

PB1958: IN SILICO ANALYSIS OF SYNONYMOUS AND NON-SYNONYMOUS SNPS OF THE HUMAN CEREBLON (CRBN) GENE: AN IMPORTANT GENE IN IMMUNOMODULATORY DRUGS (IMIDS)-BASED TREATMENT OF MULTIPLE MYELOMA PATIENTS

M. Güler 1,* , A. Macauda 1 , F. Canzian 1

1 Genomic Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany

Background: Immunomodulatory drugs that target cereblon, a component of the E3 protein ligase complex encoded by the CRBN gene, are used to treat multiple myeloma (MM) and myelodysplastic syndrome. Because the CRBN gene is so important in IMiD metabolism, it is of interest to assess the effects of single nucleotide polymorphisms (SNPs) on cereblon, which can potentially affect IMiD treatment response in MM patients

Aims: This study aims to detect deleterious SNPs (i.e. disrupting cereblon function) in CRBN by using bioinformatics tools based on protein-IMiD structure, nucleotide and protein sequence, and post-translational modifications (PTMs).

Methods: The CRBN SNPs were collected from dbSNP with filtering validation status by ALFA option. The nucleotide sequence of CRBN and amino acid sequence were downloaded from NCBI and UniProt respectively. The structure of the CRBN-pomalidomide complex was downloaded from RCSB PDB and the CRBN structure was obtained from AlphaFold. Genetic variants were analyzed with different tools for variant prioritization. The missense variants were scored by 12 different tools (PANTHER, SIFT, PolyPhen, CADD, BayesDel, ClinPred, LRT, M-CAP, MetaLR, MetaSVM, REVEL, fathmm-XF) and PTM position. The potential structural effects of amino acid changes were predicted by PremPLI (protein-IMiD complex) and INPS-MD (only protein) tools by prediction of protein stability free energy change (ΔΔG). The non-coding and non-exonic variants were scored by 10 tools (CADD, Deepsea, eigen_PC, fathmm, funseq2, fitcons_Fitness, gerp_Base.RS, gwava, phast, ReMM) and CpG region, TFBS and miRNA binding site. After calculating scores by different tools, we assigned to each SNP deleterious-benign status (0 and 1) for each prediction, then we calculated a Z-score and cumulative distribution function of normal distribution of Z-scores (one-tailed, 95% CI).

Results: dbSNP has 12611 CRBN SNPs validated by ALFA: 267 deletions, 1394 deletion-insertions, 78 insertions, and 10872 single nucleotide variants. After removing duplicates, we found 314 non-synonymous and 8602 synonymous and non-coding SNPs. The experimentally detected PTM sites are extremely conserved, as only eight of them have SNPs, all of them being rare in all populations (minor allele frequency between 0-0.00002639). The 27 of 314 non-synonymous SNPs passed nominal p-value (<0.05), but after Bonferroni correction (0.05/314) none of them were significant. However, PremPLI and INPS-MD tools-based prediction of protein stability free energy change (ΔΔG) values were significantly different between these 27 SNP and the others. The INPS-MD predicted ΔΔG mean in significant SNPs is -1.048kcal/mol (min -3.659-max 0.645), while in the non-significant group the mean was -0.376kcal/mol (min -3.361-max 1.617), t-Test p=1.61x10 -6 . The PremPLI-predicted ΔΔG mean in the top 27 SNPs was 0.415kcal/mol (min 0.01-max 0.76), while in non-significant group the mean was 0.309kcal/mol (min -0.93 - max 0.9), t-Test p=0.041. The 107 of 8602 synonymous and other non-coding SNPs passed p-value threshold after multiple test correction (0.05/8602). The gnomAD global allele frequency of these SNPs is ranging from 1.16x10 -4 to 8.34x10 -6 .

Summary/Conclusion: As a result, the PTM sites of cereblon are clearly functionally significant. Therefore, malfunctions in proteins involved in the post-translational modification of cereblon should have an impact on its activity. Furthermore, protein stability ΔΔG predicts also deleteriousness of SNPs. Even though significant SNPs are very rare, they should be tested to assess their functional effect in MM cells.

PB1959: ESTABLISHMENT OF A NEW MULTIPLE MYELOMA (MM) RESEARCH MODEL USING MAGNETICALLY EMPOWERED TUMOR CELLS AND NK CELLS

Z. Wang 1 , R. Hou 2 , L. Tan 3 , J. Zhang 1 , D. Zhang 1 , D. Pan 1 , Q. Chen 1 , L. Tang 2 , Y. Hu 2 , X. Kong 2 , Q. Jiang 1,*

1 Department of Hematology; 2 Nanfang Hospital, Southern Medical University; 3 the first clinical medical school，Southern medical university, Guangzhou, China

Background: Multiple myeloma (MM) is different from leukemia on pathogenesis, clinical features, and treatment prognosis, it remains a type of incurable cancer although there are such treatment as hematopoietic stem cell transplantation and CAR-T, etc . One of the important characteristics is that MM cells are focally distributed within the bone marrow.

Aims: In order to mimic the clinical characteristics of local distribution of MM cells, we try to establish such in vitro and in vivo model by the technology of magnetically empowered tumor cells (MET) and magnetically empowered NK cells (MENK).

Methods: 1) Myeloma cells (NCI-H929) labeled with green fluorescent protein (GFP) and luciferase (Luc) dual reporter genes were constructed. 2) To simulate the MM situation with an in vitro model, the magnetically GFP + Luc + NCI-H929 was inoculated as MET into 6-well plate, cell islands were formulated under according tiny magnetisms, then MENK was added to observe its treatment effect. 3) By injecting the magnetically GFP + Luc + NCI-H929 as MET into the right femur under a special-made magnetism, the in vivo nude mouse tumor model was established. Then the MENK was added to kill the tumor formed within the femur. The efficacy was evaluated by X-ray (or micro-CT), monoclonal protein, as well as morphology, immunology, cytogenetics and molecular biology (MICM) analysis. 4) To explore the possibility of establishing other kinds of MET tumor models.

Results: 1) We established the in vitro model with locally distributed MET, and the MENK can efficiently eliminate the MM cells. 2) GFP + Luc + NCI-H929 specifically located in the femur in nude mouse model, the model was a quite successful and ideal in vivo model for MM study since it has cluster of MM cells in injected femur and localized bone destruction. 3) Other different kinds of tumor models were successfully established. It illustrated that MET technology can be applied on multiple kinds of tumor cells.

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Summary/Conclusion: We established the in vivo and in vitro models for MM with the technology of MET/MENK which can be used not only for mechanism research but also for the therapy evaluation, such as CAR-NK and CAR-T.

PB1960: M6A-REGULATED LNCRNA SNHG15 MODULATES HISTONE H3K36ME3 METHYLATION AND HETEROCHROMATIN REDISTRIBUTION IN MULTIPLE MYELOMA

Y. Lan 1 , H. Mei 1 , L. Tingting 1 , Y. Hua 1 , W. Qiuling 1,*

1 Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, wuhan, China

Background: Our previous results indicated that the m6A demethylase ALKBH5 acted on lncRNA SNHG15 in multiple myeloma and positively regulated its expression. LncRNA SNHG15 was highly expressed in CD138 + plasma cells from MM Patients and MM cell lines. The overexpression of lncRNA SNHG15 in ALKBH5-knockdown cells rescued the inhibited cell proliferation, the suppressed cell migration, and the accelerated cell apoptosis. Fatherly, we preliminarily found that ALKBH5 regulated histone H3K36me3 modification through SNHG15.

Aims: We aim to further display the potential molecular mechanisms between ALKBH5 regulated-lncRNA SNHG15 and histone-related heterochromatin involved in the progression of MM.

Methods: The expression of lncRNA SNHG15, SETD2, Histone 3 and H3K36me3 were detected by RT-qPCR and western blot.

SNHG15-siRNA lentivirus SNHG15L1-cNDA lentivirus and NC were transfected into MM cells to knock-down and knock-in lncRNA SNHG15 gene.

Chromatin Immunoprecipitation sequencing (CHIP-seq) and CHIP-qPCR were previously accomplished (the results have not yet been published).

Heterochromatin protein-1 alpha (HP1-α) IF (Immunofluorescence) staining was used to show the distribution of heterochromatin.

Results: ALKBH5, lncRNA SNHG15 and SETD2 are all overexpressed in MM primary cells and cell lines, associating with poor prognosis, especially renal injury. Through overexpressing lncRNA SNHG15 in MM PMI8226 ALKBH5 - cells, we display that lncRNA SNHG15 rescued the inhibited cell proliferation, the suppressed cell migration, and the accelerated cell apoptosis caused by ALKBH5 knockdown. Western blot analysis reveals the expression changes of histone H3K36me3 methyltransferase SETD2 among control, ALKBH5 - , ALKBH5 - SNHG15 + group MM RPMI8226 cells. Heterochromatin protein-1 alpha (HP1-α) IF (Immunofluorescence) staining exhibited the heterochromatin redistribution following ALKBH5 knock-down and lncRNA SNHG15 Knock-in. CHIP-seq uncovered that cellular H3K36me3 level has been showed no significant difference, but a redistribution existed in groups above. Besides, Enrichment analysis and western blot experiments revealed that ALKBH5/lncRNA SNHG15 axis mediated cell adhesion/migration through CDH4/CDH9 associated β-catenin signal pathway. CHIP-qPCR verified that the enrichment of H3K36me3 on CDH4 gene body altered following ALKBH5 knock-down and lncRNA SNHG15 Knock-in.

Summary/Conclusion: The ALKBH5/lncRNA SNHG15 axis promotes myelomagenesis and cell migration through β-catenin/CDH4 signal pathway.

m6A-regulated lncRNA SNHG15 alters H3K36me3 distribution and modulates heterochromatin through upregulating SETD2 expression in MM cells.

SETD2 is overexpressed in MM primary cells and cell lines, associating with poor prognosis.

PB1961: ZNO NANOPARTICLES INDUCE MULTIPLE MYELOMA CELL DEATH BASED ON REACTIVE OXYGEN SPECIES AND CASPASE SIGNALING PATHWAY

Z. Li 1 , X. Yin 1 , C. Lyu 1 , S. Cui 2 , J. Wang 2 , Y. Si 1 , R. Xu 2,*

1 Shandong University of Traditional Chinese Medicine; 2 Shandong University of Traditional Chinese Medicine Affiliated Hospital, Jinan, China

Background: Human multiple myeloma (MM) is a malignant and incurable B cell tumor. Zinc oxide nanoparticles (ZnO NPs) have been widely used in biomedical fields including anti-bacterial and anti-tumor. However, the influence of ZnO NPs on MM cells is still unclear.

Aims: The present study aimed to investigate the effect of ZnO NPs on MM cell (a human myeloma-derived RPMI8226 cell line) death in vitro and the underlying mechanism.

Methods: The morphology of ZnO NPs was characterized by transmission electron microscopy (TEM), and the inhibitory and apoptotic effect of ZnO NPs on human MM cells was monitored by a CCK-8 method and an Annexin V-FITC/PI assay. Meanwhile, the morphological change in the cells after exposure to ZnO NPs was observed by a light field microscope. Moreover, the effects of ZnO NPs on the ATP level, reactive oxygen species (ROS) generation, and apoptosis were separately explored by the DCFH-DA fluorescent probe, flow cytometry, and ATP bioluminescence assay. Moreover, the expression of cytochrome C (Cyt-C), Apaf-1, Caspase-9 and Caspase-3 at mRNA and protein levels was further determined by using quantitative PCR (Q-PCR) and western blotting. In the present study, the human peripheral blood mononuclear cells (PBMCs) were used as normal control samples for the relevant experiment.

Results: The results indicated that ZnO NPs could significantly inhibit human MM cell proliferation and cell death in a time- and dose-dependent manner in vitro , and this outcome can be confirmed by cell morphology and apoptosis assay. Meanwhile, the results also showed that ZnO NPs could effectively increase ROS production and decrease ATP levels in human MM cells. ZnO NPs could also significantly elevate the expression of Cyt-C, Apaf-1, Caspase-9 and Caspase-3 at mRNA and protein levels, leading to cell death. By contrast, ZnO NPs showed little cytotoxic influence on PBMCs.

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Summary/Conclusion: ZnO NPs can significantly induce human MM cell death in a time- and dose-dependent manner in vitro , decrease the ATP production and enhance the ROS generation. ZnO NPs can also increase Cyt-C, Apaf-1, Caspase-9 and Caspase-3 expression at mRNA and protein levels in human MM cells, and initiate MM cell apoptosis, indicating that Cyt-C, Apaf-1, Caspase-9 and Caspase-3 play crucial roles in ZnO NPs-induced, mitochondria-mediated apoptosis in human MM cells. Overall, ZnO NPs may be a potential agent in treating human multiple myeloma in clinical practice.

PB1962: COMPARISON OF EXPRESSION OF NSD2, CYCLIN D1 AND C-MAF BY PLASMA CELLS IN BONE MARROW AND BONE PLASMACYTOMA IN PATIENTS WITH PLASMA CELL MYELOMA

E. Mamaeva 1,* , M. Firsova 1 , M. Solovev 1 , A. Abakumova 1 , A. Startsev 1 , E. Makunina 1 , A. Kovrigina 2 , T. Danilina 2 , L. Mendeleeva 1

1 Department of intensive high-dose chemotherapy of paraproteinemic hemoblastoses; 2 Department of Pathology, FSFI National Research Center for Hematology, Ministry of Health of Russia, Moscow, Russia

Background: Bone plasmacytomas forms as a result of destruction of bone structures by tumor proliferate and its escape out of the medullar cavity. According published data, the incidence of bone plasmacytomas at the time of plasma cell myeloma (PCM) diagnosis ranges from 7 to 32,5%, whereas rates are higher in a relapse. Bone plasmacytomas in most cases are composed of mature plasma cells, while immature morphology is mainly characteristic for extramedullary plasmacytomas. It is shown, that plasmacytoma cells are the descendants of bone marrow tumor plasma cells. Due to acquisition of new qualities as a downregulation of adhesion molecules, secretion of growth factors, activation of oncogenes and antiapoptotic factors, new subclones of tumor cells can survive without support from bone marrow microenvironment.

Aims: to compare morphological consistency and immunohistochemical characteristics of PCM substrate in bone marrow trephines and bone plasmacytomas biopsy specimens.

Methods: In retrospective study were included 13 patients with PCM aged from 36 to 67 years (median 55 years). At the time of diagnosis all patients underwent biopsy of bone plasmacytoma. The diagnosis of PCM verified according to IMWG criteria (2014). All patients have been examined with routine laboratory tests, immunochemical analysis of serum proteins and urine, cytologic examination of bone marrow smears, histologic and immunohistochemical examinations of bone marrow trephines and bone plasmacytomas biopsy specimens, instrumental examinations (CT, MRI).

Immunohistochemical staining of bone marrow trephines and bone plasmacytomas biopsy specimens made with using a stainer Leica Bond-Max, reactions with antibodies to CCND1 (clone SPA4, Cell Marque), NSD2 (clone 29D1, Abcam), c-MAF (clone ERP16484, Abcam). The cut-off scores for determining positivity for markers have been taken 5% for CCND1, 10% for NSD2 and c-MAF (by T. Murase et al., 2019). Microscopic examination of histologic and immunohistochemical slides have been done on light microscope Leica 3000.

Results: PCM substrate by histologic examination in bone marrow trepnines and bone plasmacytomas biopsy specimens were presented by mature plasma cells. In two patients (1 and 8) in bone plasmacytoma have been seen proplasmocytes between mature plasma cells, but in bone marrow have been seen only clusters of mature plasma cells.

According to the results of immunohistochemical examination the expression of proteins-products of oncogenes have been revealed in most patients (12 of 13) in plasma cells of bone marrow and bone plasmacytomas, wherein in half of the patients (patients 7-12) the expression of proteins-products of oncogenes coincides in bone marrow and bone plasmacytoma. We have revealed a discordant expression in another patients (tabl. 1). We’ve paid a special attention to the patients 1-3, in whom in bone marrow have been revealed an expression of 1/3 protein, but in bone plasmacytoma – 2/3 (patients 2 and 3) or all of 3 studied proteins (patient 1). This phenomenon is possible to consider as a heterogeneity of clonal composition of bone plasmacytoma as a consequence of tumor evolution.

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Summary/Conclusion: Discordant expression of proteins-products of oncogenes NSD2, cyclin D1 and c-Maf in tumor cells in bone marrow and bone plasmacytoma could be evidence of different tumor clones activation in PCM.

PB1963: A RARE CASE OF MULTIPLE SOLITARY PLASMOCYTOMA

Z. Mlayah 1,* , N. Slama 1 , B. Wiem 1 , B. A. Nouha 1 , B. sarra 1 , L. Adnene 1

1 Hematology, CHU Fattouma Bourguiba Monastir, Monastir, Tunisia

Background: Plasmacytoma is a rare hematological malignancy characterized by extramedullary plasma cell infiltration. It is exceptional in its multiple form. It often affects males with a median age between 55 and 60 years. According to the international myeloma working group, the diagnosis of multiple solitary plasmacytoma is made by objectifying more than one bone or extramedullary plasma cell lesion, absence of monoclonal IgM gammapathy in the blood and urine, absence of spinal cord damage, normal skeletal survey, no end-organ damage. Biologically, monoclonal proteins in serum or urine are detected. Sataging and evaluation are based essentially on the magnetic resonance imaging (MRI). The treatment is based mainly on radiotherapy, chemotherapy and, in some cases, on an autologous hematopoietic stem cell transplant or surgical treatment.

Aims: Given the rarity of plasmocytoma and specially the multiple solitary plasmocytoma, very few cases are published in the literature. In this work, we will report a case of multiple solitary plasmacytoma in order to enrich the bibliography as well as to better define the clinical, biological and radiological characteristics of this hemopathy.

Methods: we encountered only one case of multiple solitary plasmocytoma in our department that we are going to postpone. We used literature searches to identify previously reported cases of multiple solitary plasmocytoma.

Results: The patient is a 61-year-old male with no notable pathological history, smoking at 30 pack-years. As functional signs, there was a weight loss of 10 kg in 6 months and lumbar spine pain. On clinical examination: the performance score was 3, the chest mass was 15 x 8 x 4 cm, absence of anemic syndrome and absence of sensory or motor deficit. The MRI showed a mass of left thoracic soft parts with medullary extent and lumbar and spinal retroperitoneal masses (L3-S1). The biological assessment showed normocytic normochrome anemia at 8.2 g /dL, hyperprotidemia at 108 g / L, absence of hypercalcemia or renal failure. 24-hour proteinuria was 7.21 g, microglobulin β2 was 16.03 mg/L, lactate dehydrogenase levels were normal. The positive diagnosis was made on a biopsy of the chest mass showing tumor cells expressing intensely the CD138 and not expressing the CD3, the CD20 or the CD79a. The osteomedullary biopsy showed the absence of infiltration and the karyotype on marrow by the technique “fluorescence by hybridization in situ” was normal. A monoclonal peak with IgG kappa was objectified in the serum immunofixation. Urinary immunofixation showed the presence of Kappa free light chains and serum free light chain assay showed an increased Kappa/Lambda ratio. The patient was treated with chemotherapy courses based on cyclophosphamide, anthracycline (Doxoribicin) and corticosteroid therapy. Decompressive radiotherapy sessions were indicated for intense spinal pain and sensory-motor deficit installed in both lower limbs. The evolution was marked clinically by the regression of the tumor mass but persistence of spinal pain, biologically by the disappearance of the anemia and the hyperprotidemia.

Summary/Conclusion: To conclude, multiple solitary plasmocytoma is rare. It’s clinical, biological and radiological characteristics are particular. Given it’s rarety, there is no specific protocol for staging and treatment. Thus, it is important to collect a sufficient number of patients to develop a protocol allowing a special care for this type of hemopathy.

PB1964: RELATIONSHIP BETWEEN SIRT1-7 AND SELECTED MIRNAS EXPRESSIONS IN MYELOMA/LYMPHOMA PATIENTS IN DIFFERENT REMISSION STATUS.

M. Nowicki 1,* , A. Wierzbowska 1,2 , E. Stec-Martyna 3 , D. Kulczycka-Wojdala 3 , G. Nowicki 4 , A. Szmigielska-Kapłon 1,2

1 Department of Hematology, Copernicus Memorial Hospital in Lodz Comprehensive Cancer Center and Traumatology, Poland; 2 Department of Hematology; 3 Central Scientific Laboratory, Medical University of Lodz, Łódź, Poland; 4 Georgetown University, Washington, DC, United States of America

Background: Sirtuins are members of the NAD+-dependent class III histone deacetylase group, which participate in the post-translational modification of proteins. MicroRNAs (miRNAs) are a class of small (19-25 nucleotides), endogenous RNA which play a significant role in regulation of gene expressions. Both molecules are engaged in cell proliferation, differentiation, stress response, apoptosis and hematopoietic stem cells (HSC) fate during mobilization and homing after auto-HSCT.

Aims: The aim of the study was to investigate the association between SIRT1-SIRT7 and selected miRNAs on the day of first apheresis and the depth of myeloma/lymphoma response (CR versus not CR before mobilization regimen) in patients with lymphoproliferative malignancies during HSC mobilization for autologous transplantation.

Methods: Forty nine patients were enrolled in the study (25 F, 24 M). The median (Me) age was 60 years. The investigated group consisted of thirty-three multiple myeloma (MM), seven non-Hodgkin lymphoma (NHL) and four Hodgkin lymphoma (HL) patients. Sirtuins and miRNAs (miRNA-15a, miRNA-16, miRNA-34a miRNA-126, miRNA-146a, miRNA-223 and miRNA-155) expression was evaluated in peripheral blood (PB). The blood serum samples were collected at two time points: before hematopoietic stem cell mobilization chemotherapy (day 0) and on the day of the first apheresis (day A). SIRT1-SIRT7 expression was evaluated by Droplet Digital PCR method while the miRNAs level was evaluated by Real Time PCR method.

Results: To evaluate the influence of miRNA expression on the sirtuins level after mobilization, patients were divided into “high” (n=25) and “low” (n=24) expression groups according to median miRNA levels on day A (above and below median). The group of miRNA-34a “high expressors” had upregulated level of SIRT7 than “high expressors” (Me = 189.01 vs. 110.72 copies/200µL, p=0.03). The group of miRNA-223 “high expressors” had upregulated level of SIRT3, than “high expressors” (Me = 357.43 vs. 157.12 copies/200µL, p=0.04).

Patients mobilized with chemotherapy and G-CSF (n = 44) were divided into CR (n = 10) and non-CR (n = 34) groups. On day A, a positive correlation was noticed between SIRT1, SIRT2, SIRT3, and SIRT6 level and miRNA-126 expression in non-CR group (R = 0.35, p=0.04), (R = 0.41, p=0.01), (R = 0.38, p=0.02), (R = 0.36, p=0.04) respectively. Additionally, a positive correlation was found between SIRT1, SIRT2, SIRT3, SIRT6 and SIRT7 concentration and miRNA-223 in non-CR group (R = 0.44, p=0.008), (R = 0.44, p-0.01), (R = 0.48, p=0.004), (R = 0.45, p=0.008), (R = 0.36, p=0.04) respectively. We found no correlation between sirtuins and miRNAs expression in patients in CR remission status.

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Summary/Conclusion: Mutal reactions between sirtuins and miRNAs may influence the hematopoietic landscape during HSC mobilization and differ depending on the depth of myeloma/lymphoma response. Chemotherapy combined with G-CSF stimulation causes inflammation associated with intense angiogenesis and disruption of signaling pathways associated with HSC motility. These processes can be regulated by miRNA-34a, miRNA-126, miRNA-223 and selected sirtuins. The lack of correlation between miRNA expression and sirtuins in the CR group may be associated with a small number of patients.

PB1965: A META-ANALYSIS OF THE RATE OF MALIGNANT PROGRESSION OF MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE: PRELIMINARY FINDINGS FROM A SYSTEMATIC REVIEW

S. J. Quinn 1,* , C. Cardwell 1 , L. Anderson 2 , C. McShane 1

1 Centre for Public Health, Queen’s University Belfast, Belfast; 2 Aberdeen Centre for Health Data Science, University of Aberdeen, Aberdeen, United Kingdom

Background: Monoclonal gammopathy of undetermined significance (MGUS) is an asymptomatic premalignant condition preceding multiple myeloma (MM) and other haematological malignancies (HM). Clinically the rate of progression from MGUS to HM is important; current guidelines from the International Myeloma Working Group (IMWG) report this rate as “around 1% per year”, however variation has been reported by ethnic groups, MGUS type, and clinical features.

Aims: A systematic review of the published literature was conducted to investigate the rate of MGUS progression to support clinical practice.

Methods: Four electronic databases (Embase, Medline, Pubmed and Web of Science) were searched from database inception to 11/12/2019 using key words and MeSH subject headings for “MGUS”, “haematological malignancy” and “progression”. Studies were reviewed against predefined eligibility criteria by two independent reviewers. For studies reporting progression events and person-years of follow-up, a random-effects meta-analysis model 5 was used to produce a pooled rate of MGUS progression to HM and MM. Studies reporting on cumulative rates of progression at specified time-points (1, 5 and 10-year) were included in a narrative synthesis.

Results: Overall, 36 papers met the inclusion criteria; 12 studies (sample size: 114-17,963; follow-up 3.3-42.1 years) were included in the meta-analysis. In preliminary analyses, the annual pooled rate of MGUS progression was 1.1% (95%CI 0.9-1.3) to HM and 0.9% (95%CI 0.7-1.1) to MM.

In total, 18 studies contributed to the narrative synthesis on MGUS progression to HM (sample size: 52-17,963; follow-up 2.1-34.1 years) and 10 studies reported on MGUS progression to MM (sample size: 75-2046, follow-up: 2.1-17 years). These narrative syntheses highlighted the heterogeneity of research, which encompassed key traits like methods, sample sizes and follow-up duration. Studies were also mainly conducted in Europe, being particularly scarce in Asia.

Between meta-analysis and narrative synthesis, only seven studies reported on IgM-MGUS progression to Waldenström’s macroglobinaemia or HM, which could not be clearly synthesised in either analysis due to differing reporting; these papers indicated higher progression rates in IgM-MGUS than non-IgM.

Summary/Conclusion: Preliminary findings from this systematic review support current IMWG guidelines on the rate of MGUS progression to HM. However, as the majority of studies included were limited to geographic areas within Europe and USA, further research reflecting population and clinical diversity are needed.

PB1966: HEMOPOIETIC NICHE IMPAIRMENT IN TREATED MULTIPLE MYELOMA PATIENTS

N. Y. Semenova 1,* , N. I. Enukashvily 1,2 , I. I. Kostorma 1 , L. A. Belik 2 , D. V. Baram 1 , V. A. Balashova 1 , S. V. Gritsaev 1 , S. S. Bessmeltsev 1 , D. I. Ostromyshenskii 2 , I. S. Martynkevich 1

1 Russian research institute hematology and transfusiology; 2 Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, Russia

Background: It has been increasingly recognized that cancer progression is not an autonomous process driven and performed exclusively by cancer cells. Hematopoietic cells live in a complex mesenchymal environment ant this environment influences the cancer progression and vice versa is influenced by cancer cells. The tumor-educated microenvironment plays a role in immunosuppression, angiogenesis, and drug resistance. Being in contact with cancer cells, mesenchymal stem/stromal cells acquire cancer-associated phenotype. It is not known whether the hematopoietic niche is restored in multiple myeloma (MM) patients’ bone marrow after treatment.

Aims: The aim of the work was to study hematopoietic niche in MM patients with different treatment outcome.

Methods: Healthy donors (HD, n=2) and patients (n= 24) with different MRD status after bortesomib based therapy were enrolled in the study. Histological examination was performed on their trephines, bioinformatics analysis of aspirate transcriptomes was performed to analyze non-coding tandemly repeated RNA profile and the level of transcription of genes of WNT family, the results of computational analysis was checked in vitro by qPCR and in situ by DNA-RNA FISH of mesenchymal stem cells (MSC) of patients’ bone marrow. The interaction of of MSC and cancer cells was mimicked in coculture experiment with or without bortezomib.

Results: The histology of MM hematopoietic niche of patients with partial response (PoCR) and non-responders (NR) was different from normal - the level of vessels density and a-smooth muscle actin staining (a marker of MSC/fibroblasts with cancer-associated phenotype) were significantly higher than in HD niche. MSC from MM patients were more resistant to bortezomib treatment and more effective in supporting cancer cells viability during incubation with the drug as compare with HD MSC. Moreover, MM MSC have prominent cancer-associated phenotype and increased transcription of some tandemly repeated sequences. We have found that transcription of several WNT family genes was downregulated in NR and PoCR patients. Our results give an evidence that MM hematopoietic niche is not returned to normal after treatment especially in patients non-responding or partially responding to it. Some of the genes of WNT family are involved in ostegenesis and they are downregulated in treated MM patients.

Summary/Conclusion: MSC from MM patients have a number of features in morphology, genes and non-coding DNA expression profiles persisted after treatment.

The work was supported by by the Ministry of Science and Higher Education of the Russian Federation (project 15.BRC.21.0011, Agreement № 075-15-2021-1063)

PB1967: ROLE OF CYTOKINES IN MULTIPLE MYELOMA: IL-1RN AND IL-4 VNTR POLYMORPHISMS

I. Serin 1,* , Y. OYACI 2 , M. PEHLIVAN 3 , S. PEHLIVAN 2

1 Department of Hematology, University of Health Sciences, Istanbul Training and Research Hospital; 2 Department of Medical Biology, Istanbul University, Faculty of Medicine, Institute of Health Sciences, Istanbul; 3 Department of Hematology, Gaziantep University, Gaziantep, Turkey

Background: Cytokines have important effects on immune regulation and oncogenesis. IL-1 is a cytokine with important physiological roles in cytokine regulation, vascular permeability and sepsis in autoimmune diseases. The IL-1 receptor antagonist (IL-1Ra or IL-1RN) is a member of the IL-1 superfamily that functions as a competitive antagonist of the cell surface IL-1 receptor, thereby regulating various immune and inflammatory responses related to IL-1. IL-1 induces tumor growth and metastasis, while IL-1RN inhibits the secretion of IL-1α and IL-6 in cancer cells. Interleukin-4 (IL-4) is a potent anti-inflammatory cytokine, and its gene is located on the long arm of chromosome 5 (q23-31) along with other cytokine genes. IL-4 can be secreted by many types of immune cells, including basophils, eosinophils, myeloid lineage cells, mast cells, and activated T cells.

Aims: In this study, it was aimed to reveal the effects of IL-1RN and IL-4 VNTR polymorphisms on disease development and survival in patients with MM.

Methods: In this study, 244 patients diagnosed with MM in hematology clinic between January 2010 and January 2021, and 179 healthy individuals were included. The control group did not have any kind of cancers and consisted of a unrelative population permanently residing in Turkey. ASCT was performed for 73% of the patients after 4 courses of VCD (bortezomib, cyclophosphamide, and dexamethasone) with at least a partial remission and then LD (lenalidomide and dexamethasone) was used as a maintenance therapy for the following follow-up period. The 1/1, 1/2, 1/3, 1/4, 2/2, 2/3 and 2/4 genotypes of the IL-1RN VNTR polymorphism were statistically compared before treatment between patients having undergone ASCT and healthy controls, as were the 1/1, 1/2 and 2/2 genotypes of IL-4 VNTR polymorphism. Additionally, the statistically significant effects of these genotypes on survival were examined.

Results: The median age of 244 patients included in the study was 58 old years (range: 29-81). The 5-year and 10-year OS were 76% and 53%. The 5-year and 10-year PFS were 42% and 21%, respectively. The median PFS was 42,3 months. The median follow-up period was 27 months (4,4-155 months). In the statistical analysis of the distribution of IL-1RN VNTR gene variants, 1/3 and 1/4 genotypes were found to be significantly higher in patients with MM compared to the healthy controls (p=0,035). In the statistical analysis of the distribution of IL-4 VNTR gene variants, there was no significant difference between the MM patient group and the healthy controls in terms of genotype distribution (p>0.05, for all). IL-4 VNTR genotype distribution did not have a significant effect on PFS and OS. PFS of patients with IL-1RN VNTR non-2-allele carrier genotypes was significantly shorter, but no significant effect was found on OS (p=0,03, p=0,786, respectively. Patients with IL-1RN VNTR non-2-allele carrier genotypes had 1,718-fold increased risk of shorter PFS.

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Summary/Conclusion: In conclusion, with this study, the effects of IL-1RN VNTR and IL-4 VNTR polymorphisms on MM were evaluated for the first time in the literature. The IL-1RN 1/3 and 1/4 genotypes were significantly more common in patients with MM compared to healthy controls. Patients with IL-1RN VNTR non-2-allele carrier genotypes had 1,718-fold increased risk of shorter PFS, but no significant effect on OS was demonstrated. IL-4 polymorphism, on the other hand, did not have a significant effect on MM development or survival. This study will shed light on ones on cytokine-MM relationship and epigenetic mechanisms.

PB1968: EPIGENETIC AND GENETIC INVESTIGATION OF SOCS-1 GENE IN PATIENTS WITH MULTIPLE MYELOMA

I. Serin 1,* , F. C. TUNCEL 2 , S. PEHLIVAN 2 , Y. OYACI 2 , M. PEHLIVAN 3

1 Department of Hematology, University of Health Sciences, Istanbul Training and Research Hospital; 2 Department of Medical Biology, Istanbul University, Faculty of Medicine, Institute of Health Sciences; 3 Department of Hematology, Gaziantep University, Istanbul, Turkey

Background: The suppressor of cytokine signaling-1 (SOCS-1) functions to induce an appropriate immune response and is an essential physiological regulator of interferon signaling. DNA methylation, an epigenetic regulatory mechanism studied in multiple myeloma (MM) and many tumors, involves the addition of a methyl group to the carbon 5 position of cytosine.

Aims: In this study, besides comparing SOCS-1 gene hypermethylation between patients with MM and healthy controls, it was aimed to demonstrate the effect of SOCS-1 gene distribution and additionally, the effect of hypermethylation of SOCS-1 on progression free survival (PFS) and overall survival (OS).

Methods: In this study, 120 patients diagnosed with MM between January 2018 and 2020 and 80 healthy individuals were included. The distribution of the SOCS-1 genotypes was statistically compared between patients and healthy controls. Additionally, the statistically significant effects of these genotypes on survival were examined.

Results: CA/CA genotype of SOCS-1 was found to be statistically significantly higher in healthy controls (p=0,001), while Del/Del genotype was significantly higher in patients with MM (p=0,034). The percent methylated reference (PMR) value of the SOCS-1 gene was found to be significantly higher in the healthy controls (median: 43,48, range: 2,76-247,75; p=0,001). The SOCS-1 genotype distribution had no effect on PFS and OS (for PFS, p=0,229; for OS, p=0,763). Patients with a PMR value of <43,48 had a significantly longer PFS (for <43,48 %56, for ≥43,48 %43; p=0,027).

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Summary/Conclusion: The PMR value of the SOCS-1 gene was found to be significantly higher in the healthy control group. Patients with a PMR value of <43,48 were found to have a significantly longer PFS. The effects of SOCS-1 polymorphisms on the pathogenesis of MM and SOCS-1 methylation will shed light on the literature.

PB1969: FUNCTIONAL CHARACTERIZATION OF SUSCEPTIBILITY LOCI TO MULTIPLE MYELOMA

M. Shokouhi 1,2,* , A. Macauda 1 , F. Canzian 1

1 Genomic Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany; 2 Natural Science, Tor Vergata University, Rome, Italy

Background: Multiple myeloma (MM) is the second most common blood malignancy, caused by an uncontrolled growth of plasma cells in the bone marrow, accounting for 20% of all newly diagnosed hematological cancers. Although the current 5-year survival rate is ranging between 40-60%, MM is still considered an incurable disease since most of the patients eventually relapse. While the causes of MM are incompletely understood, several genome-wide association studies (GWAS) have been conducted to identify germline variants that predispose to MM. Up to date, a total of 24 loci were found to be associated with MM risk, but very little information is available about their functional role.

Aims: The principal goal is to explore in silico the function of the germline variants associated with MM risk. As we do not know if the GWAS-identified SNPs are the causal variants or just markers of risk, the functional characterization of the causal risk variants would lead to a better understanding of disease development.

Methods: GWAS design takes advantage of the linkage disequilibrium (LD) structure of the human genome, thus the main GWAS findings are single-nucleotide polymorphisms (SNPs) that show the strongest association with MM risk (measured as the lowest p-values), but they are not necessarily the functionally causal variants. In this project, we used bioinformatics tools (GTEx, HaploReg v4.1, Roadmap, LDlinke, SNPnexus, RegulomeDB 2.0.3, SNP2TFBS, miRNASNP v3, GeneMANIA) to perform fine mapping of all GWAS-identified loci and to prioritize in each locus the polymorphism with the highest chance of being functionally relevant. In particular, we focused on the loci with the smallest number of SNPs in high LD (r 2 >0.8) in order to maximize the probability to capture the casual variant.

Results: Four of the 24 MM risk loci had a relatively small number of SNPs in high LD and within them we found that the locus located at chromosome 16 contained the greatest number of functionally annotated SNPs. Of particular functional interest was rs3747481 (chr16:30666367 C/T) due to the following reasons: it is a missense variant (protein change: P359L), it is located in the PRR14 gene, that contributes to chromatin hierarchical organization and has a role in gene regulation, has a high CADD PHRED score (22.1). Additionally, according to GTEx portal, rs3747481 is associated with the expression level of the RNF40 gene (which plays a central role in histone code and gene regulation) in whole blood cells (p=3.02 -12 ). It has a score of “1d” in RegulomeDB (eQTL+ TF binding + any motif + DNase peak), meaning that this variant has a high likelihood to affect binding of transcription factors. Some other SNPs (like rs35629860 and rs67128646) in the same LD block show the co-occurrence of H3K4me3 and H3K27me3 histone marks (associated with gene activation and repression, respectively) in promoters and enhancers in B lymphocytes. rs6565197 is predicted to affect the binding of the KLF4 and KLF5 transcription factors which play key roles in cell cycle regulation.

Summary/Conclusion: Through a fine mapping of MM risk loci by bioinformatics tools, we found a variant in the locus 16p11.2 that shows in silico a very high probability to have biological role in the risk disease.

PB1970: AS2O3 INDUCE IMMUNOGENIC CELL DEATH IN MULTIPLE MYELOMA VIA NLRP3/CASPASE SIGNALING PATHWAY

Y. Si 1 , L. Yu 2 , X. Yin 1 , R. Xu 3 , Z. Li 1 , S. Cui 3,*

1 Shandong University of Traditional Chinese Medicine; 2 The Third Affiliated Hospital of Shandong First Medical University; 3 Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China

Background: Multiple myeloma (MM) is a common plasma cell malignant disease, the clonal evolution of myeloma cells and the immune escape caused by it are an important mechanism of refractory drug resistance.

Aims: To explored the relationship between NLRP3 / Caspase signaling pathway and immunogenic cell death (ICD) in the intervention of As 2 O 3 on MM cell (a human myeloma-derived KM3 cell line), and explained the inhibitory effect of As 2 O 3 on the proliferation of myeloma cells.

Methods: The inhibitory and apoptotic effect of As 2 O 3 on human MM cells was monitored by a CCK-8 method. Meanwhile, the effects of different concentrations of As 2 O 3 on MM cell apoptosis were detected was further determined by flow cytometry. Moreover, the expression of BCL-2, BAX, PARP, HSP70/90, MHC-I, and NLRP3 at mRNA was determined by using quantitative PCR (Q-PCR). The effects of different concentrations of As 2 O 3 on the expression of apoptosis related proteins Bcl-2, Bax, Caspas-3 and PARP were detected by ELISA. Moreover, the expression of ICD related HSP70/90 and MHC-I proteins, as well as the expression of NLRP3 and caspase-1 proteins were explored.

Results: The results indicated that As 2 O 3 could significantly inhibit human MM cell proliferation and cell death in a time- and dose-dependent manner in vitro. Meanwhile, the results also showed that As 2 O 3 could effectively increase the expression of Bax, PARP, HSP70/90, MHC-I, and NLRP3 mRNA levels, and decrease the Bcl-2 mRNA level in human MM cells. Moreover, As 2 O 3 could also significantly elevate the expression of NLRP3, caspase-1 proteins and ICD related proteins, leading to increase the expression of apoptosis related proteins Bax, Caspas-3, PARP.

Summary/Conclusion: As 2 O 3 can significantly induce human MM cell death in a time- and dose-dependent manner in vitro, and activate NLRP3 / caspase signaling pathway in human MM cells. As 2 O 3 can also increase the expression level of ICD related molecules and apoptosis related molecules Bax, Caspas-3, PARP, and initiate MM cell apoptosis, indicating that As 2 O 3 induce immunogenic cell death in multiple myeloma via NLRP3/Caspase signaling pathway. Overall, As2O3 may be a potential agent in treating human multiple myeloma in clinical practice.

PB1971: FREQUENCY AND CHARACTERISTICS OF DEL(6Q) IN PATIENTS WITH MULTIPLE MYELOMA FROM LATIN-AMERICAN COUNTRIES. A REAL-WORLD STUDY

F. Stella 1,2 , P. Leone 3 , E. Pedrazzini 4 , C. Galvano 5 , S. Zurita 6 , M. Coccé 7 , M. Gallego 7 , N. Schultz 8 , G. Alfonso 9 , J. Canelos Moreno 10 , A. Noboa 10 , F. Huamán Garaicoa 11 , C. Paz y Miño 12 , M. Ziembar 13 , P. Ochoa 14 , P. Duarte 15 , M. Quatrin 16 , F. Colombi Martínez 17 , D. Fernández 18 , S. Cugliari 18 , F. Uturbey 19 , C. Peña 20 , I. Slavutsky 5,*

1 Área de Genética, Hospital Posadas; 2 Universidad de Morón, Buenos Aires, Argentina; 3 Laboratorio de Genética, SOLCA-Quito, Quito, Ecuador; 4 Laboratorio de Genética; 5 Genética de Neoplasias Linfoides, Instituto de Medicina Experimental, CONICET-Academia Nacional de Medicina; 6 Laboratorio de Genética, Hospital Posadas; 7 Laboratorio de Citogenética y Citogenética Molecular; 8 Sección Hematología, Hospital Italiano de Buenos Aires; 9 Servicio de Hematología, Hospital Posadas, Buenos Aires, Argentina; 10 Hematología; 11 Patología, SOLCA-Guayaquil, Guayaquil; 12 Academia Ecuatoriana de Medicina, Quito, Ecuador; 13 Laboratorio de Análisis Clínicos y Biología Molecular; 14 Hematología, Instituto Fleming; 15 Hematología, CEMIC, Buenos Aires; 16 Laboratorio Bionet; 17 Hospital Rodolfo Rossi, La Plata; 18 Hematología, Instituto Angel Roffo, Buenos Aires, Argentina; 19 Laboratorio de Genética y Biología Molecular, Facultad de Medicina, Universidad de La República, Montevideo, Uruguay; 20 Hospital del Salvador, Santiago, Chile

Background: Multiple myeloma (MM) is a clonal B cell malignancy characterized by the presence of multiple structural and numerical genetic alterations, including gains, losses and rearrangements of different chromosomal regions. Among them, the partial deletion of the long arm of chromosome 6 [del(6q)] is a common recurrent abnormality in MM, observed in 20-35% of patients. Despite this, there is no data about this alteration from MM patients in Latin America (LA).

Aims: To evaluate frequency, distribution and clinical characteristics of MM patients with del(6q) in a multicenter study from the GELAMM (Grupo de Estudio Latinoamericano de MM).

Methods: We retrospectively reviewed MM patients from Argentina, Chile, Ecuador and Uruguay, whose specimens were assessed by conventional cytogenetics and tested using interphase FISH between 2010 and 2020. Conventional cytogenetic analysis was performed on unstimulated bone marrow (BM) cells cultured. G-banded technique was used. FISH analysis was performed on unstimulated BM samples or on isolated plasma cells. The MM DNA probe panel was used. To compare clinical characteristics, a reference group of 105 MM patients without cytogenetic and FISH alterations (w/alt) (57 males; R-ISS: 1: 10.6%, 2: 50.3%, 3: 39.4%) was included. The study was approved by the local Ethics Committees of each Institution, in compliance with the Declaration of Helsinki.

Results: A total of 220 patients with abnormal karyotypes were detected; 55 (25%) of them showed del(6q) (28 males; R-ISS: 1: 27.5%, 2: 31.1%, 3: 41.4%). Cytogenetic analysis showed simple karyotypes (SK; one or two alterations) in 51% of cases and complex karyotypes (CK; 3 or more alterations) in 49%. The distribution of modal number was: hypodiploid (33% of cases), hyperdiploid (30%), hypertriploid (11%), hyperhaploid (8%), pseudodiploid (7%), hypotriploid (7%) and hypotetraploid (4%). The most frequent breakpoints were 6q23 and 6q25 (32.7% each), followed by: 6q21 (29.1%), 6q13 (18.2%), 6q15 (16.4%) and 6q27 (10.9%). Most common alterations associated to del(6q) involved chromosomes: 1 (38.7% of cases), 13 (32.7%), 11 (30.9%), 3 (27.3%), 14, 15 and 19 (25.5% each). By FISH analysis, patients with CK showed high frequency of del(17p) (63.5%) and chromosome 1 alterations (61.5%). Translocations t(11;14) and t(4;14) were observed in 15% of cases each and t(14;16) in 10% of patients. Cases with del(6q) were older (61.4 years) than those w/alt (57.6 years) (p=0.0216). Analysis of clinical parameters showed increased lactate dehydrogenase and serum monoclonal M protein levels in patients with del(6q) than those w/alt (p<0.0001 and p=0.0353, respectively). Cases with CK also had higher creatinine levels (p=0.0276). Seventy percent of patients received proteasome inhibitor and immunomodulatory agents at induction. No differences in overall survival (OS) between patients with SK (82.2 months) compared to those w/alt (78.2 months) was seen, but a significant short OS in cases with del(6q) and CK (36.1 months) (p=0.003) was observed.

Summary/Conclusion: To our knowledge, this is the first analysis of del(6q) in MM patients from LA. Our cohort had a similar frequency of del(6q) than published series, with particular involvement of 6q23 and 6q25 breakpoints. Interestingly, patients with del(6q) showed high frequency of chromosome 1 alterations, particularly in the context of CK, associated to poor clinical outcome. Our study reinforces the importance of conventional metaphase karyotyping, and its contribution to a better stratification and biologic characterization of MM patients.

PB1972: PAEONIFLORIN ALLEVIATES BIPN BY REDUCING IL6 LEVELS AND MODULATING PARKIN-MEDIATED MITOPHAGY

R. Sun 1,* , Q. Sun 2 , F. Zhang 2 , X. Cui 2

1 College of Traditional Chinses Medicine, Shandong University of Traditional Chinses Medicine; 2 The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China

Background: Multiple myeloma (MM)is a common hematological malignant tumor disease. Recent years, the emergence of the proteasome inhibitor bortezomib (BTZ) has greatly improved the remission rate and survival rate of patients with MM. However，BTZ neurotoxicity limits the clinical dosage and continuity of medication, and seriously affects the application and efficacy of BTZ. Paeoniflorin (PF) has proven to have a wide range of anti-inflammatory, painful and immunomodulatory effects. It is reported that paeoniflorin has great potential in neuroprotection, which can reduce neuronal damage by reducing the secretion of IL6 and other inflammasomes. Unfortunately, the research on the improvement of paeoniflorin on Bortezomib-induced peripheral neuropathy (BIPN) is still relatively rare. Therefore, we conducted experiments to explore the therapeutic effect and mechanism of paeoniflorin on Bortezomib-induced peripheral neuropathy to explore its potential as a novel clinical treatment for BIPN.

Aims: To explore the therapeutic effect of paeoniflorin on BIPN and explore its possible mechanism.

Methods: The mechanism of paeoniflorin intervention in peripheral neuropathy was predicted by bioinformatics analysis. The level of IL-6 in plasma of MM patients was detected and the relationship between IL-6 level and BIPN grade was analyzed. BIPN cell models were constructed to evaluate cell mitochondrial function by cell viability, mitochondrial quality and Mito Tracker Green. Mice were given intraperitoneal injection of BTZ (1 mg/kg on days 1, 4, 8 and 11) to establish a BIPN model. The nerve injury of BIPN mice was observed by measuring ethology, motor nerve conduction velocity, HE staining, electron microscope and ROS level. Western blot was used to detect the expression of autophagy related indexes.

Results: 1. The prediction of bioinformatics suggested that paeoniflorin might improve PN by intervening in inflammation levels（IL-6) and mitochondrial autophagy. (Figure 1 A-B) 2. IL-6 levels in MM patients were positively correlated with PN levels. (Figure1 C) 3. Compared with the control group, paeoniflorin could significantly improve cell viability and mitophagy level in BIPN cell model, and improved the damage to mitochondria. (Figure1 D-E) 4. Paeoniflorin could effectively improve the behavior of nerve injury in BIPN mice, improve the sciatic nerve damage in mice, and increase the expression of LC3 Ⅱ/I, beclin1 and Parkin proteins related to autophagy in the sciatic nerve of mice. (Figure1 H-I)

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Summary/Conclusion: Paeoniflorin could significantly reduce peripheral neuropathy caused by bortezomib, which might be achieved by reducing IL6 levels to regulate PARKIN-mediated mitochondrial autophagy and mitochondrial damage.

PB1973: INHIBITION OF LACTATE EXPORTERS INDUCES DIRECT ANTI-TUMOR EFFECTS AND COUNTERS IMMUNE SUPPRESSION IN MULTIPLE MYELOMA

A. Van der Vreken 1,* , I. Oudaert 1 , A. Maes 1 , E. De Bruyne 1 , K. De Veirman 1 , K. Vanderkerken 1 , E. Menu 1

1 Hematology and immunology (HEIM), Vrije universiteit Brussel, Brussels, Belgium

Background: Multiple myeloma (MM) is a cancer characterized by the accumulation of malignant plasma cells in the bone marrow (BM). The BM is an immune suppressive environment, which hampers the efficacy of modern immune therapeutic strategies. Immune suppression is partially orchestrated by the hypoxic nature of the BM. Due to this hypoxia, cancer cells are pushed towards aerobic glycolysis, during which pyruvate is converted to lactate. Lactate can be imported and exported by monocarboxylate transporters (MCTs). Lactate in the BM milieu contributes to an immune suppressive environment by several mechanisms including driving differentiation and attraction of myeloid derived suppressor cell (MDSC).

Aims: In this study, we aimed to evaluate the effects of MCT inhibition on MM cell viability and the immune suppressive character of the BM.

Methods: Correlation of MCT-expression with survival was investigated using the Mulligan cohort. In vitro, several MM cell lines (LP-1, RPMI-8226, 5TGM1) were used. Syrosingopine was used as dual inhibitor of MCT1 and MCT4, AZD0095 as MCT4 inhibitor and AZD3965 as MCT1 inhibitor. The effect on apoptosis was quantified by AnnexinV/7-AAD staining. For MDSC differentiation, BM was isolated from naïve mice. CD11b + cells were selected by MACS and cultured with conditioned medium (CM). CM was prepared from 5TGM1 cells treated with DMSO or syrosingopine, for 48 hours in hypoxia (1% O 2 ). The effect on MDSC differentiation was evaluated by western blotting.

Results: High expression of MCT1/MCT4 genes correlated with a significant lower overall survival in MM patients. Increased expression of MCT1 and MCT4 was observed in MM cell lines after 48 hours of hypoxia. Syrosingopine treatment resulted in intracellular lactate accumulation but did not induce apoptosis. Neither did single inhibition of MCT1 or MCT4. MM cells could however be sensitized to syrosingopine by treatment with metformin, a complex I inhibitor, resulting in apoptosis. AZD0095 and AZD3965 were less potent to induce cell death in combination with metformin, compared to dual MCT inhibition. The effects of the combination treatment on metabolism and cellular pathways are currently investigated. Extrinsic effects of export inhibition were evaluated by using CM of MM cells treated with syrosingopine on MDSCs. Both lactate and MM-CM promoted MDSC differentiation in vitro, while CM deprived of lactate resulted in a clear decrease of differentiation markers such as p-stat3 and arginase. This indicates a less suppressive status of the MDSC’s.

Summary/Conclusion: Dual inhibition of MCT1 and MCT4 is not sufficient to induce apoptosis in MM cells; however, they can be sensitized to syrosingopine with metformin. Blocked lactate export from MM cells resulted in impaired MDSC differentiation. Targeting lactate metabolism could be a new approach to hamper MM survival and create a less suppressive immune environment.

Arne Van der Vreken and Inge Oudaert contributed equally to this work.

PB1974: MULTIVARIATE SPECTRAL BIOTYPING OF EXTRAMEDULLARY MULTIPLE MYELOMA

M. Vlachová 1,* , J. Gregorová 1 , S. Adamová 1 , L. Pečinka 2,3 , L. Moráň 2,4 , V. Porokh 1,2 , M. Štork 5 , L. Pour 5 , J. Havel 2,3 , P. Vaňhara 1,6 , S. Ševčíková 1,7

1 Babak Myeloma Group, Department of Pathophysiology, Faculty of Medicine, Masaryk University; 2 International Clinical Research Center, St. Anne’s University Hospital Brno; 3 Department of Chemistry, Faculty of Science, Masaryk University; 4 Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute; 5 Department of Internal Medicine, Hematology and Oncology, University Hospital Brno; 6 Department of Histology and Embryology, Faculty of Science, Masaryk University; 7 Department of Clinical Hematology, University Hospital Brno, Brno, Czechia

Background: Multiple myeloma (MM) is the second most common hematological malignancy of the elderly. The bone marrow is infiltrated by malignant plasma cells. MM may progress into so-called extramedullary disease (EMD) - EMD occurs when a subclone of malignant plasma cells migrates out of the bone marrow and infiltrates soft tissues. Despite recent progress, pathogenesis of EMD still needs to be clarified.

Aims: We focused on the analysis of low molecular weight molecules in peripheral blood of 20 MM and 20 EMD patients using MALDI-TOF mass spectrometry to create a tool for identification of MM and EMD, and discrimination of individual types.

Methods: Matrix-Assisted Laser Desorption/Ionization Time-of Flight Mass Spectrometry (MALDI-TOF MS) has become an indispensable research tool, which is used for analysis of biomolecules and various organic molecules. Artificial Neural Networks (ANN) are components of artificial intelligence inspired by biological neural networks. Using ANN, we can model complex non-linear systems, as previously published. In our previous study, we recorded mass spectra of MM and healthy donor samples. ANN specifically predicted MM samples with high sensitivity, specificity and accuracy. The same approach as applied on MM and EMD.

Results: The RStudio was used for statistical analysis, where the data were evaluated using Principal Component Analysis (PCA) and Partial least squares discriminant analysis (PSL-DA). Using MALDI-TOF MS, it was possible to distinguish between samples of MM patients and healthy donors, as well as MM and EMD patients. Informative patterns in mass spectra served as inputs for ANN that specifically distinguished between healthy donors and patients.

Summary/Conclusion: We demonstrated that using MALDI-TOF MS coupled with ANN is a useful tool that can distinguish between healthy donors and MM/EMD patients. Thus, it can be used as a fast alternative to conventional analyses.

This study was supported by grant of the Ministry of Health of the Czech Republic, NU21-03-00076.

PB1975: THE RESEARCH ON THE MECHANISM OF RRAD GENE IN MULTIPLE MYELOMA

C. Yi 1 , Y. feng 2,*

1 The third Hospital of Nanchang, Nanchang; 2 The Affiliated Hospital of Zunyi Medical College, zunyi, China

Background: Multiple myeloma is the second most common hematological malignancy with a high incidence. The pathogenesis of multiple myeloma is still unclear, and specific gene expression abnormalities may play a key role in the pathogenesis of MM.

Aims: The research on the mechanism of RRAD gene in multiple myeloma

Methods: The study collected bone marrow specimens and 9 healthy volunteers from 20 newly diagnosed MM patients admitted to the Affiliated Hospital of Zunyi Medical University from January 2019 to June 2020.The RT-qPCR method was used to detect the transcription level of RRAD in 20 newly diagnosed MM patients, 9 healthy donors and human MM cell lines; RRAD overexpression cell lines were constructed in SKO-007 and H929 by lentivirus infection, and flow cytometry Cytometry, RT-PCR and Western Blot verified the efficiency of overexpression; CCK8 method was used to study the effect of RRAD on cell proliferation; flow cytometry was used to detect cell apoptosis and cycle.Decitabine was used to treat MM cell lines, and RT-qPCR was used to detect changes in RRAD gene expression; CCK8 was used to detect the effect of cell proliferation; flow cytometry was used to detect cell apoptosis and cycle; MSP method was used to detect RRAD methylation of MM cells Level.

Results: Compared with 9 healthy donors, the expression level of RRAD was lower in 20 newly diagnosed MM patients (P = 0.017).Compared with 9 healthy donors, the expression level of RRAD in the MM cell line is low.The ROC curve results show that the expression level of RRAD has a significant diagnostic value for the diagnosis of MM (P <0.001).RRAD gene overexpression can promote MM cell apoptosis and block cell cycle into S phase.DAC can inhibit the proliferation of SKO-007, H929, U266 cells in a dose-dependent manner.Promote the apoptosis of SKO-007, H929, U266 cells, block the cell cycle into S phase or G2 phase and increase the expression of RRAD.After SKO-007, H929, and U266 cells were treated by DAC, the RRAD methylation level of SKO-007 cells decreased, but the RRAD methylation level of H929 and U266 cells did not change significantly.

Summary/Conclusion: 1. RRAD expression in MM is low.2. The expression level of RRAD is closely related to the presence or absence of bone disease, which affects the patient’s quality of life and prognosis assessment.3. The expression level of RRAD has significant diagnostic value for the diagnosis of MM.4. Overexpression of RRAD can block the cell cycle in S phase and promote MM cell apoptosis.5. Hypermethylation of RRAD in MM leads to epigenetic inactivation, which reduces the expression of RRAD and reduces MM Apoptosis of cells loses its anti-tumor effect, which is a new molecular mechanism for the occurrence and development of MM.

PB1976: CRISPR-CAS9 KNOCKOUT SCREEN IDENTIFIES RNA MODIFICATION ENZYMES ESSENTIAL FOR CELL SURVIVAL IN MULTIPLE MYELOMA

D. Zhao 1,* , C. Pauli 1,2 , C. Rohde 1 , M. Kienhöfer 1 , A. L. Grab 1 , S. Huhn 1 , M. Raab 1 , N. Weinhold 1 , M. Frye 2 , C. Müller-Tidow 1

1 Department of Medicine V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital; 2 German Cancer Research Center—Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany

Background: RNA modifications play a critical role in regulating gene expression and determining cell fate. Evidence is emerging that dysregulation of RNA modification pathways is involved in the development of cancer. However, the function of RNA modifying enzyems in multiple myeloma (MM) and its mechanisms are largely unknown.

Aims: To identify RNA modification enzymes essential for cell survival and proliferation in myeloma.

Methods: For functional analysis, we performed a focused CRISPR knock-out screen to identify RNA modifying enzymes essential for cell survival and proliferation in two myeloma cell lines, OPM2 and RPMI8226. We used a CRISPR-Cas9 library consisting of 1700 sgRNAs (~8 sgRNAs/gene) targeting around 150 RNA modifying enzymes and collected samples at day 1, 8 and 21. MAGeCK software was used to analyze the results.

Results: The focused CRISPR/Cas9-screening identified six writers/erasers/readers of RNA modifications that were essential for myeloma cell survival. Among these hits, gRNAs directed against either TRMT112, YRDC, MEPCE, RBM15, EIF2 and ALKBH5 consistently inhibited cell growth.

Summary/Conclusion: Several of the enzymes regulating these RNA modifications are essential for myeloma cell lines.

PB1977: SPECTRUM OF INFECTIOUS COMPLICATIONS FOLLOWING TRANSPLANTATION OF AUTOLOGOUS BLOOD STEM CELLS IN PATIENTS WITH MULTIPLE MYELOMA

A. Abakumova 1,* , L. Mendeleeva 1 , G. Klyasova 2 , M. Solovev 1 , M. Firsova 1 , E. Makunina 1 , E. Mamaeva 1 , A. Startsev 1

1 Department of intensive high-dose chemotherapy of paraproteinemic hemoblastoses; 2 Department of Clinical Bacteriology, Mycology and Antimicrobial Therapy, FSFI National Research Center for Hematology, Ministry of Health of Russia, Moscow, Russia, Moscow, Russia

Background: Transplantation of autologous blood stem cells (auto-HSCT) is one of the key methods in multiple myeloma (MM) treatment recommended to patients under 65 years of age and good somatic status. The early post-transplantation period is associated with the development of deep myelotoxic agranulocytosis, disruption of mucous membranes of gastrointestinal tract and high risk of infectious complications.

Aims: Determine the incidence of infectious complications and the spectrum of pathogens in MM patients after autologous transplantation.

Methods: Our study includes 111 MM patients (51 male) aged 19 to 65 (median age 53) who performed an auto-HSCT between 2018 and 2020. In 99 patients Durie-Salmon stage was considered as III, in 1 - as II and in 11 - as I. Myeloma nephropathy was observed in 23 patients, programmed hemodialysis in 4. Prior to the auto-HSCT 42 patients had reached the CR, 48 - VGPR, 15 - PR, 4 - stabilisation, 2 - progression. Hematopoietic stem cells were mobilized and 2,1-23,15x106/kg CD34+ cells (median 7,7) collected. Conditioning was performed with melphalan 200mg/m2 (n=93) and 140 mg/m2 (n=15), due to myeloma nephropathy and hemodialysis. Agranulocytosis lasted from 6 to 97 days (median 12).

The colonization of non-sterile compartment was investigated before and in agranulocytosis. In case of the febrile fever, Bactec blood culture systems and chest CT were performed, in case of the oral mucositis - microbiological mucus examination from tonsils and the throat back wall. If pneumonia was established by CT, fibrobronchoscopy was performed with microbiological, molecular-biological and PCR examinations of bronchoalveolar lavage (BAL). Viral blood profile was investigated by PCR for CMV, HHV-6, Herpes simplex viruses. During neutropenia all patients were given antiviral prophylaxis by valaciclovir. Due to infection developing, broad-spectrum antibiotics were prescribed.

Results: Infectious complications in the early post-transplant period was noted in 94.6% of MM patients. The most frequent were oral mucositis (95.5%) and necrotic enteritis (85.6%).

Microbiological analysis of non-sterile compartment in 101 patients revealed 52 cases of E.coli without ESBL , 35 - with E.coli ESBL . In agranulocytosis, 93 cases were detected: E.coli without ESBL (n=32), E.coli ESBL (n=19).

Bacteriemia was identified in 18 patients: 7 cases of gram-positive and 11 of gram-negative. Pneumonia, confirmed by chest CT, was detected in 17 (15.3%) patients, and in 8 cases fibrobronchoscopy was performed. The pathogens etiology was established in 5 patients - gram-positive (n=3) and gram-negative (n=2). Viral infection associated with HHV-6 was found in 8%.

Analysing non-sterile compartment before the therapy and in case of bloodstream infection, they matched in 7 cases, a difference was found in 4 cases ( C.koseri without ESBL, P.aeruginosa, C.putigena ). In 110 patients all infections were successfully cured and patients were discharged satisfactorily. Only one patient died at +9 day of auto-HSCT from bilateral pneumonia caused by S. aureus and bacteriemia associated with P. aeruginosa and S. aureus .

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Summary/Conclusion: In our study, the most frequent infectious complications in patients with MM in the early post-transplantation period, were oral mucositis and necrotic enteritis, mainly I-II degree of severity. Complications, such as pneumonia and bacteriaemia, developed in 32%. Viral infections were detected in 8% of patients and associated with HHV6. Early post-transplant mortality was 0.9%.

PB1978: AUTOLOGOUS STEM CELL TRANSPLANTATION AND MAINTENANCE IMPROVES SURVIVAL, IN COLOMBIAN MULTIPLE MYELOMA PATIENTS. ACHO’S RENEHOC REGISTRY REPORT

V. Abello 1,2,3,* , C. Sossa-Melo 3,4,5,6 , H. Idrobo 7,8,9 , I. Munevar 3,10 , G. Quintero 3,11,12 , W. Mantilla 3,13 , Y. Borjas 3,14 , K. Galvez 15,16 , R. Gomez 3,17 , J. D. Saavedra 16,18 , L. M. Gaviria 3,19 , J. Reyes 3,20 , C. Bermudez 3,21 , J. M. Herrera 9,22 , H. Martinez 3,23

1 Servicio de Hematología, Hospital de San José; 2 Facultad de Medicina, Fundación Universitaria de Ciencias de la Salud; 3 RENEHOC, Asociación Colombiana de Hematología y Oncología (ACHO), Bogota; 4 Facultad de Medicina, Universidad Autonoma de Bucaramanga, Bucaramanga; 5 Departamento de Hematología, Fundación Oftalmológica de Santander-Clinica Carlos Ardila Lulle (FOSCAL); 6 Programa para el tratamiento de enfermedades hematológicas de Santander (PROTEHOS), Floridablanca; 7 Facultad de Medicina, Universidad Del Valle; 8 Servicio de Hematología, Centro Medico Julian Coronel; 9 RENEHOC, Asociación Colombiana de Hematología y Oncología (ACHO), Cali; 10 Hematoncologos Asociados; 11 Facultad de Medicina, Universidad de los Andes; 12 Servicio de Hematología, Fundacion Santa Fe de Bogota; 13 Servicio de Hematología, Fundacion Cardioinfantil, Bogota; 14 Servicio de Hematología, Clinica Astorga; 15 Servicio de Hematología, Hospital Pablo Tobon Uribe; 16 RENEHOC, Asociación Colombiana de Hematología y Oncología (ACHO), Medellin; 17 HematoOncologos SA, Cali; 18 Servicio de Hematología, Clinica Vida; 19 Servicio de Hematología, Hospital Universitario San Vicente Fundacion, Medellin; 20 Servicio de Hematología, Clinica Los Nogales; 21 Servicio de Hematología, CIOSAD, Bogota; 22 Servicio de Hematología, Cl nica imbanaco Quiron Salud, Cali; 23 Servicio de Hematología, Instituto Nacional de Cancerología, Bogota, Colombia

Background: Multiple myeloma (MM) represents 1% of all newly diagnosed neoplasms, Autologous Stem Cell Transplantation (ASCT) has become the standard of care for fit MM patients after completion of induction therapy. Maintenance treatment after transplant has also proven to positively impact event free survival in multiple clinical trials.

Aims: RENEHOC is a multicenter database established in January 2018. Its primary aim is to provide local information on diagnosis, treatment and outcomes of MM and other hematologic neoplasms, in Colombia. This report aims to characterize the MM population in our registry treated with ASCT.

Methods: An ambispective registry of adult MM patients, treated in approved centers over the last 10 years. Descriptive statistics were used for patient’s demographic and clinical characteristics. The Kaplan-Meier method was used to assess DFS and OS, comparing ASCT vs No-ASCT and Maintenance vs No-Manteinance. Hazard Ratios (HR) using Cox proportional hazards regression modeling was estimated.

Results: Until February 2022, 1230 patients have been registered, ASCT were performed in 366 (29.7%). MM transplanted patients median age at diagnosis was 63 (IQR 57-68) years, 139(38%) were older than 65 y; 190(51.9%) were male. At diagnosis 73 (19.9%) had renal failure, 84(22.9%) plasmacytomas and 147(40.1%) bone fractures. Most patients were in an advance stage by Durie Salmon at diagnosis (DS IIIA or IIIB: n=207, 56.6%), and had high risk ISS (ISS 3: n=113, 46.7%; 124 unknown). 296 (80%) underwent transplantation after the first line treatment. Median cycles in first line before transplant were 5 (IQR 4-6) and time from last cycle to transplant was 9 weeks (IQR 5-17).

The most common induction regimen were Cyclophosphamide-Bortezomib-Dexamethasone (CyBorD)(n=136, 37.1%), Bortezomib-Thalidomide-Dexamethasone (VTD)(n=101, 27%), Bortezomib-Dexamethasone (VD)(n=42, 11.5%), 22 (6%) Bortezomib-Doxorubicin-Dexamethasone (PAD) and 50 (13.4%) other regimens. At transplantation 196 (53.3%) were in VGPR or better and 97 (26.5%) in partial response.

268 (73.2%) received maintenance after transplant, 168 (62.7%) with Lenalidomide, 43 (16%) with Talidomide and 57 (21.3%) with other agents. 214 (80%) received continued maintenance until progression or is still receiving it.

There were no statistically significant differences between groups characteristics and treatment (ASCT, no-ASCT, maintenance, no maintenance).

Mean follow-up for the 1230 MM patients registered was 29.11 months (SD 29.79 CI 27.42-30.82), for no-ASCT group 22.66 months (SD 25.71, CI 20.90-24.41) and for the 366 patients in ASCT group was 44.22 months (SD 33.07, CI 40.76-47.68). The median DFS was significantly higher in ASCT patient vs no-ASCT (108 vs 43 months, p=0.0001); the estimated 5 years DFS was 73% vs 41%. Maintenance also improved DFS (149 vs 82 months in non-maintenance group, p=0.0084); estimated 5 years DFS 77% vs 64%. Median OS was also significantly longer in ASCT group (149 vs 69 months, p=0.0001), estimated 5 years OS 81 vs 53%; without significant difference for maintenance vs no maintenance (not-reached both groups, p=0.11; estimated 5 years OS 83 vs 75%.

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Summary/Conclusion: The results In this analysis provide insights into the real-world benefits of ASCT and maintenance treatment in MM, with similar efficacy to that reported in controlled clinical trials. These and other data provided by RENEHOC will allow for health pharmaco-economic, epidemiologic and outcomes research in Colombia and Latin America.

PB1980: MULTIPLE MYELOMA: ADOPTING MRD BY NEXT GENERATION FLOW CYTOMETRY (NGF) AND FUNCTIONAL IMAGING GUIDED CONSOLIDATION INTO CLINICAL ROUTINE

D. Boeckle 1,* , P. Tabares 1,2 , X. Zhou 1 , S. Schimanski 1 , M. J. Steinhardt 1 , M. Bittrich 1 , E. Seebacher 1,2 , M. Ulbrich 1,2 , A. Wilnit 1,2 , C. Metz 3 , A. Heidemeier 3 , T. Bley 3 , R. Werner 4 , A. Buck 4 , H. Einsele 1 , M. Kortüm 1 , A. Beilhack 1,2 , L. Rasche 1,5

1 Department of Internal Medicine II, Division of Oncology and Hematology; 2 Interdisciplinary Center for Clinical Research Laboratory; 3 Department of Radiology; 4 Department of Nuclear Medicine; 5 Mildred Scheel Early Career Center, University Hospital Würzburg, Würzburg, Germany

Background: Minimal residual disease (MRD) detection by NGF and next generation sequencing gains is gaining increasing importance as an endpoint in clinical studies to determine deep responses beyond traditional immunohistochemical complete remission. However, little is known about the value of combining NGF with functional imaging and its role for MRD based consolidation strategies in clinical routine.

Aims: The aim of the study was to investigate the role of MRD and functional imaging in clinical routine in MM. In addition, we report our real-world experience with an MRD-guided consolidation strategy.

Methods: We included 102 patients with both newly diagnosed (ND) (n=57) and relapsed/refractory (RR) MM (n=45) that achieved serological response of VGPR or better. Among NDMM patients, 92% (n=52) underwent stem cell transplantation (SCT) after induction and 27% of RRMM patients received a salvage transplantation. MRD assessment by NGF was available in all patients. Bone marrow samples were collected between July 2019 and May 2021 and analyzed according to the Euroflow guidelines at a sensitivity level of 10 -6 . We performed functional imaging in 78 patients with either FDG positron emission tomography or diffusion-weighted magnetic resonance imaging within a median of 1 day after NGF assessment.

Results: 51 patients (50%) were negative on MRD assessment by NGF. We observed higher rates of MRD-negativity in subjects with a serological response of CR/sCR or better versus VGPR (59% versus 34%, p=0.01), OR=2.8). Furthermore, we combined NGF results with functional imaging: MRD-negativity on both NGF and functional imaging (double-negativity) was achieved in 45% of patients. 8% and 40% of patients were negative on either NGF or imaging respectively. In some patients, imaging was the only technique applied to detect residual disease. We observed imaging positivity despite negativity on NGF more often in heavily pretreated patients (≥ 4 previous lines) compared to NDMM (p<0.01). In detail, we found a number needed to scan for imaging-only positive patients of 40, 8 and 3 in NDMM, relapsed, and heavily pretreated RRMM, respectively. MRD-triggered consolidation therapy was initiated in 29 patients with 72% responding to treatment and a MRD conversion rate of 51%. Interestingly, MRD-triggered consolidation (mostly after SCT) led to a superior progression free survival (Figure 1) when compared to standard Lenalidomide maintenance on MRD or imaging positive patients (p=0.04). Subjects achieving double-negativity after standard of care treatment were excluded from this analysis.

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Summary/Conclusion: Our data indicate that combining NGF with functional imaging is beneficial particularly in heavily pretreated MM patients to rule out residual disease. Furthermore, our real-world experience gives evidence that MRD-based consolidation may be used to improve PFS of patients being MRD positive following SCT. Further studies investigating these issues in a randomized prospective fashion are highly warranted.

PB1981: EFFICACY AND SAFETY OF DENOSUMAB IN PATIENTS WITH MULTIPLE MYELOMA

S. Bougherira 1,* , S. Bentorki 1 , F. Grifi 1,1

1 Hematology, University hospital, Annaba, Algeria

Background: Multiple Myeloma (MM) bone disease can lead to skeletal complications, including skeletal-related events, bone pain, and hypercalcemia. These complications can seriously affect the quality of life of patients and be life-threatening. Usually, the prevention of these complications uses Bisphosphonates (BP). Denosumab, a fully human monoclonal antibody, binds to RANKL to inhibit the formation, function and survival of osteoclasts, the cells responsible for bone resorption. A therapeutic alternative to BPs, Denosumab has become the preferred agent over Zoledronic acid because it is approved for use in people with kidney failure. We report our experience with the use of this new therapeutic class.

Aims: The aim of this study is to determine the efficacy of Denosumab in patients with MMand evaluate the tolerance of this molecule

Methods: This is a descriptive retrospective study for 4 years from 2015 to 2019, having collected 62 patients, 30% of all patients with MM, treated in the Hematology Department, CHU Annaba during the same period. The majority, 79% of patients were newly diagnosed. Data were collected from medical records, comorbidities and concomitant medications, clinical examination (bone events, Performans Status), with all the diagnostic workup. Denosumab was prescribed at a dose of 120 mg subcutaneously every 4 weeks, in combination with daily calcium and vitamin D supplementation. Adverse events were assessed according to the WHO rating.

Results: The patients are between 33 and 92 years old, the average age is 65, with a female predominance, the sex ratio is 0.82. The most frequent comorbidities are represented by arterial hypertension and diabetes. End-stage renal failure is present at diagnosis in 5 patients. The clinical picture is dominated by bone manifestations, anemic syndrome (81%) and renal failure. Protein electrophoresis most often revealed the gamma peak with the immunofixation of the IgG Kappa isotype. Symptomatic hypercalcemia (≥ 3 mmol/l) was found in 9 patients. Renal function is impaired in 50 patients (81%) including 13 at the stage of end-stage chronic renal failure. The majority of patients (71%) have an ISS III score. All patients are eligible for chemotherapy with a predominance for the VTD protocol. Denosumab is indicated both in newly diagnosed patients and in those (15 patients) previously treated with BP with an average of 6 monthly injections. The efficacy of Denosumab is assessed clinically (absence of bone pain) and no new radiological lesions, serum calcium dosage (spectacular efficacy) and renal function. All patients are evaluable for toxicity with grade ½ adverse effects. Grade ¾ hypocalcemia was noted in 4 of the 24 patients, and one case of osteonecrosis of the jaw (ONJ) ​​requiring definitive discontinuation of Denosumab. After a median follow-up of 17.23 months, 12 patients died, the major cause of which was progression of MM.

Summary/Conclusion: Denosumab is a new weapon in the prevention and treatment of bone disease. Its use is simpler (subcutaneous route), of similar efficacy and also more manageable depending on the renal insufficiency with a significant improvement in the quality of life

PB1982: DEEPENING OF RESPONSE IN PATIENTS WITH MULTIPLE MYELOMA AFTER AUTOLOGOUS STEM CELL TRANSPLANTATION: A MULTICENTRIC STUDY IN A LATINO POPULATION

C. Bourlon 1 , A. Leal-Alanis 1,* , C. Vargas-Serafin 1 , K. Teran-de-la-Sancha 1 , L. Tarin-Arzaga 2 , D. Gomez-Almaguer 2 , P. Colunga-Pedraza 2 , A. Gomez-de Leon 2 , G. Ruiz-Arguelles 3 , I. Murrieta-Alvarez 3 , M. Alvarado-Ibarra 4 , J. M. Perez-Zuñiga 4 , L. F. Amador-Medina 5 , C. Garcia-Castillo 6 , G. Cardoso-Yah 6

1 Haematology, INCMNSZ, Mexico; 2 Haematology, Universidad Autónoma de Nuevo León, Facultad de Medicina y Hospital Universitario Dr. José Eleuterio González, Monterrey; 3 Haematology, Centro de Hematología y Medicina Interna de Puebla, Puebla; 4 Haematology, Centro Médico Nacional 20 de Noviembre, ISSSTE, Mexico; 5 Haematology, Hospital Regional de Alta Especialidad del Bajío, Guanajuato; 6 Haematology, Hospital Central Militar, SEDENA, Mexico, Mexico

Background: Over the last decades Multiple myeloma (MM) outcomes have shown a continuous improvement as novel drugs and maintenance therapy have been incorporated. However, availability of these resources is still limited in low- and middle-income countries (LMIC), where autologous stem cell transplant (ASCT) continues to be the best strategy to achieve better response and survival.

Aims: Our aim was to analyze the deepening of response after ASCT and its impact over survival outcomes in newly diagnosed MM (NDMM) patients.

Methods: Multicenter retrospective study that included patients ≥ 18 years of age with NDMM who received upfront ASCT as consolidation between 2015 and 2020 at six transplant centers in Mexico. Response after ASCT was defined as any improvement of response according to IMWG criteria. Novel agents were considered: proteasome inhibitors, immunomodulatory drugs, other than thalidomide, and monoclonal antibodies.

Results: A total of 126 patients were included. The median age at diagnosis was 53.1 years (range 38-68) and 55% were male. Most common isotype was IgG (60.3%) and extramedullary disease was present in 12.2%. Regarding risk, 72.7% were classified as ISS ≥2 and 18.7% of patients evaluated had high-risk cytogenetics (n=64). Induction therapy with novel agents was administered to 77 patients (61.2%). Pre-ASCT, 73.1% had a very good partial response (VGPR) or better.

Conditioning regimen was melphalan-based in 96%, with 15.9% being oral melphalan. Five patients received a tandem ASCT (4.0%) and 86 (68.3%) maintenance therapy, being thalidomide-based in 54.6%. Response after ASCT was evaluated in 86.5% (n=109), with an improvement of response in 27.5%. Patients with ≥VGPR were 82.5%.

The median overall survival (OS) of the cohort was 119 months (90.0 – 149.34) and progression-free survival (PFS) was 67.1 months (61.3 - 72.8). Deepening and maintenance of response after ASCT were associated with a better OS, 119.7 vs. 63.0 months (p=0.017) (Figure 1).

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Summary/Conclusion: This is the first multicenter study of a Latino population that reports the impact of transplant on response deepening and survival of NDMM patients. ASCT improved responses in almost 30% of our patients, being this related to an increased OS. It is of utmost importance to increase access to novel drugs in low- and middle-income countries, albeit ASCT continues to be the standard of care in patients with NDMM and demonstrated to be the best consolidation strategy to improve response and survival in our population and therefore its use should be promoted and expanded in LMIC.

PB1983: TRIAL-IN-PROGRESS: PHASE II STUDY OF PHE885, A B-CELL MATURATION ANTIGEN-DIRECTED CHIMERIC ANTIGEN RECEPTOR T-CELL THERAPY, IN ADULTS WITH RELAPSED AND/OR REFRACTORY MULTIPLE MYELOMA

N. Munshi 1,* , A. Spencer 2 , M. S. Raab 3 , A. Masood 4 , M. Martinez-Prieto 4 , J. Chu 4 , S. Iida 5 , S. Lonial 6 , M. A. Dimopoulos 7

1 Dana-Farber Cancer Institute, Harvard University, Boston, United States of America; 2 Monash University, Melbourne, Australia; 3 Universitätsklinikum Heidelberg, Heidelberg, Germany; 4 Novartis Pharmaceuticals Corporation, East Hanover, United States of America; 5 Nagoya City University, Mizuho-ku, Nagoya City, Japan; 6 Winship Cancer Insitute, Emory University, Atlanta, United States of America; 7 National and Kapodistrian University of Athens, Athens, Greece

Background: New therapies for multiple myeloma (MM) such as immunomodulatory drugs (IMiD), proteasome inhibitors (PI), and CD38-targeted monoclonal antibodies (anti-CD38 mAb) enhance the overall survival (OS) of patients (pts) with relapsed and/or refractory (r/r) MM. However, MM remains incurable, with insufficient rates and duration of response, and progression-free survival (PFS). B-cell maturation antigen (BCMA) is expressed in MM cells and is essential for maintenance of long-lived plasma cells. It is a clinically validated target in therapies that have been developed to address unmet needs in MM. T-Charge™ is a novel platform that retains the naive/T scm immunophenotype of the input leukapheresis and reduces the manufacturing process time to <2 days. This simplified manufacturing process may reduce the need for bridging therapy, and the preserved T-cell stemness has the potential to provide high rates of durable responses. A preliminary Phase I study of PHE885, a fully human BCMA-directed CAR-T cell therapy manufactured using T-Charge™, in 15 pts showed a 93% overall response rate (ORR) across all dose levels and a manageable safety profile, suggesting robust efficacy.

Aims: To determine efficacy and safety of PHE885 in adults with r/r MM who have failed ≥3 prior lines of therapy.

Methods: This is a Phase II, open-label, single-arm, multicenter trial of PHE885 in pts with r/r MM ( NCT05172596 ). The trial population includes adults aged ≥18 y with r/r MM who failed ≥3 different prior lines of therapy, including an IMiD, a PI, and an anti-CD38 mAb, and who have measurable disease at enrollment per International Myeloma Working Group (IMWG) criteria. Patients should not have received a prior anti-BCMA therapy. No MM-specific therapy is allowed prior to eligibility confirmation. After obtaining informed consent, eligible pts will undergo leukapheresis for PHE885 manufacture. Following receipt of the final product, lymphodepletion (LD) chemotherapy will commence 5 days before PHE885 infusion as a 3-day cycle unless the pt has significant cytopenia. Optional bridging therapy can be given to pts who have significant worsening of clinical status prior to PHE885 infusion. After LD chemotherapy, PHE885 will be infused (in the outpatient or inpatient setting per physician discretion) as a single dose of 5.0×10 6 (range, 4×10 6 to 5×10 6 ) CAR-positive viable T cells. Each pt will be monitored 24 months post-infusion. Pts who will complete their Month 24 visit before the study ends will be followed yearly until the end of the study. Pts will be followed for up to 15 years after infusion in a separate protocol.

Results: The primary endpoint is the best overall response (BOR) per independent review committee using the IMWG criteria. The summary measure is ORR, defined as the proportion of pts in the efficacy analysis set with a BOR of PR or better. Secondary endpoints include complete response rate, duration of response, PFS, OS, rate and durability of minimal residual disease negativity, safety, and cellular kinetics. An exploratory objective of the study is the assessment of CRS symptoms using a non-invasive biosensor patches (made of lightweight, water-resistant material that continuously take several vital sign parameters).

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Summary/Conclusion: This Phase II trial of PHE885 will help further determine efficacy and safety outcomes in pts with r/r MM who failed ≥3 prior lines of therapy. The trial will be initiated in February 2022 and is estimated to treat 100 pts across 40 trial sites.

PB1984: ELOTUZUMAB PLUS LENALIDOMIDE AND DEXAMETHASONE IN RELAPSED/REFRACTORY MULTIPLE MYELOMA: EXTENDED 3-YEAR FOLLOW-UP OF AN ITALIAN, MULTICENTER, EXPERIENCE OUTSIDE OF CONTROLLED CLINICAL TRIALS

A. Bruzzese 1,* , D. Derudas 2 , M. Galli 3 , E. A. Martino 4 , S. Rocco 5 , C. Conticello 6 , C. Califano 7 , N. Giuliani 8 , S. Mangicavalli 9 , G. Farina 10 , A. Lombardo 11 , M. Brunori 12 , E. Rossi 13 , E. Antonioli 14 , R. Ria 15 , R. Zambello 16 , N. Di Renzo 17 , G. Mele 18 , G. Marcacci 19 , G. Pietrantuono 20 , G. Palumbo 21 , N. Cascavilla 22 , C. Cerchione 23 , A. Belotti 24 , C. Criscuolo 25 , G. Uccello 26 , P. Curci 27 , E. Vigna 4 , F. Mendicino 4 , E. Iaccino 28 , S. Mimmi 28 , C. Botta 29 , D. Vincelli 30 , N. Sgherza 27 , A. Bonalumi 31 , L. Cupelli 32 , R. Stocchi 33 , M. Martino 30 , S. Ballanti 34 , D. Gangemi 35 , A. Gagliardi 36 , B. Gamberi 37 , A. Pompa 38 , G. Tripepi 39 , F. Frigeri 10 , U. Consoli 28 , S. Bringhen 40 , E. Zamagni 41 , F. Patriarca 33 , V. De Stefano 42 , F. Di Raimondo 43 , S. Palmieri 5 , M. T. Petrucci 44 , M. Offidani 45 , P. Musto 46 , M. Boccadoro 40 , M. Cavo 41 , A. Neri 38 , F. Morabito 47 , M. Gentile 4

1 Azienda Ospedaliera of Cosenza, Cosenza; 2 Businco Hospital, Cagliari; 3 Azienda Socio-Sanitaria Territoriale-Papa Giovanni XXIII, Bergamo; 4 Azienda Ospedaliera di Cosenza, COSENZA; 5 Ospedale Cardarelli, Napoli; 6 University of Catania, Catania; 7 Ospedale Umberto I, Nocera Inferiore; 8 Parma University Hospital, Parma; 9 IRCCS Fondazione Policlinico San Matteo, Pavia; 10 AORN “Sant’Anna e San Sebastiano”, Caserta; 11 Azienda Ospedaliera Santa Maria of Terni, Terni; 12 Ospedale S. Croce, Azienda Ospedaliera Ospedali Riuniti Marche Nord, Presidio of Fano, Fano; 13 Università Cattolica, Fondazione Policlinico Gemelli IRCCS, Rome; 14 Careggi Hospital, Firenze; 15 University of Bari “Aldo Moro” Medical School, Internal Medicine “G. Baccelli”, Policlinico, Bari; 16 Padua University School of Medicine, Padova; 17 Hematology Unit, Lecce; 18 Hematology Unit, Brindisi; 19 Istituto Nazionale Tumori, Fondazione ‘G. Pascale’, IRCCS, Napoli; 20 IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture; 21 Hospital University Riuniti, Foggia; 22 IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo; 23 IRCCS - Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola; 24 A.O. Spedali Civili, Brescia; 25 AO S.G. Moscati, Aversa; 26 G. Garibaldi Hospital, Catania; 27 AOUC Policlinico, Bari; 28 University “Magna Graecia” of Catanzaro, Catanzaro; 29 Internal Medicine and Medical Specialties, University of Palermo, Palermo; 30 Great Metropolitan Hospital “Bianchi-Melacrino-Morelli”, Reggio Calabria; 31 University of Verona, Verona; 32 S. Eugenio Hospital, Roma; 33 DISM, University of Udine, Udine; 34 Ospedale Santa Maria della Misericordia, University of Perugia, Perugia; 35 Fabrizio Spaziani Hospital, Frosinone; 36 S. Maria di Loreto Nuovo Hospital, Napoli; 37 AUSL-IRCCS, Reggio Emilia; 38 Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano; 39 National Research Institute of Biomedicine and Molecular Immunology, Reggio Calabria; 40 University of Torino, AOU Città della Salute e della Scienza di Torino, Torino; 41 Seràgnoli Institute of Hematology and Medical Oncology, University of Bologna, Bologna; 42 Università Cattolica, Fondazione Policlinico Gemelli IRCCS, Roma; 43 Hematology Section, University of Catania, Catania; 44 Sapienza University of Rome, Roma; 45 AOU Ospedali Riuniti di Ancona, Ancona; 46 ”Aldo Moro” University School of Medicine, Bari; 47 Biotechnology Research Unit, AO di Cosenza, Cosenza, Italy

Background: The combination of elotuzumab, lenalidomide, and dexamethasone (EloRd) enhanced the clinical benefit over Rd with a manageable toxicity profile in the ELOQUENT-2 trial, leading to its approval in relapsed/refractory multiple myeloma (RRMM).

Aims: The present study is a 3-years follow-up update of a previously published Italian real-life RRMM cohort of patients treated with EloRd.

Methods: This revised analysis entered 319 RRMM patients accrued in 41 Italian centers.

Results: After a median follow-up of 36 months (range 6-55), 236 patients experienced disease progression or died. Median progression-free survival (PFS) and overall survival (OS) were 18.4 and 34 months, respectively. The updated multivariate analyses showed a significant reduction of PFS and OS benefit magnitude only in cases with ISS stage III.

Major adverse events included grade 3/4 neutropenia (18.5%), anemia (15.4%), lymphocytopenia (12.5%), and thrombocytopenia (10.7%), while infection rates and pneumonia were 33.9% and 18.9%, respectively. No new safety signals with longer follow-up have been observed.

Of 319 patients, 245 (76.7%) reached at least a partial remission. A significantly lower response rate was found in patients previously exposed to lenalidomide.

Summary/Conclusion: In conclusion, our study confirms that EloRd is a safe and effective regimen for RRMM patients, maintaining benefits across multiple unfavorable subgroups.

PB1985: CHEMO OR CHEMO-FREE REGIMENS IN HEAVILY PRETREATED MULTIPLE MYELOMA? ROLE OF BENDAMUSTINE-BORTEZOMIB-DEXAMETHASONE (BVD) IN NOVEL AGENTS’ ERA

C. Cerchione 1,* , D. Nappi 1 , A. E. Pareto 2 , S. Ronconi 1 , D. Cangini 1 , M. Ceccolini 1 , G. Musuraca 1 , L. Catalano 1 , G. Martinelli 1

1 Hematology Unit, Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST IRCCS, Meldola (FC); 2 Hematology Unit, Ospedale San Giuliano, Giugliano in Campania, Italy

Background: The treatment of heavily pretreated Multiple Myeloma continues to be considered as an important unmet clinical need. Bendamustine is an old bi-functional alkylating agent which has proved to be effective in relapsed, refractory and in new diagnosed Multiple Myeloma (MM).

Aims: Thus, aiming to provide further insights in this field, also in novelagents’era, we present here a retrospective, real-life analysis of patients with relapsed/refractory MM (rrMM), who had received salvage therapy with bendamustine in combination with bortezomib and dexamethasone (BVD).

Methods: 81 patients (44 M/37 F), with rrMM, median age at diagnosis 59.4 years (r. 36-82), median age at start of treatment 63.6 years (r.37-86) treated with several lines of treatments (median 6, r. 2-11), every refractory to all the drugs previously received (also Bortezomib), received BVD (B 90 mg/sqm days 1,2; V 1.3 mg/sqm days 1,4,8,11, D 20 mg days 1,2,4,5,8,9,11,12, Pegfilgrastim day +4) every 28 days, until progression. All patients had previously received bortezomib-based and IMIDs-based treatments, and 32% (26/81) had also received radiotherapy. 69% (56/81) had undergone single or double autologous and three (2%) allogeneic stem cell transplant. All patients were relapsed and refractory to last therapies received before BVD.

Results: Bendamustine was well tolerated, with grade 3-4 transfusion-dependent anemia in 56% (46/81) of patients, and 43% (35/81) grade 3-4. Bendamustine was well tolerated, with grade 3-4 transfusion-dependent anemia in 56% (46/81) of patients, and 43% (35/81) grade 3-4 neutropenia (no ospedalization was required, no septic shocks wereobserved). No severe extrahematologic toxicity was observed, only grade 1 gastrointestinal side effect (nausea), treated by common antiemetic drugs. According to IMWG, ORR was 63% (51/81: 7 CR, 18 VGPR, 15 PR, 11 MR) with11 PD and 19 patients in SD, which can be considered as an impressive resultin this subset of rrMM patients. In particular, for 11 patients, BVD was, after having achieved at least a PR, a bridge to second auSCT, and for two patients a bridge to alloSCT. Eight patients have surprisingly achieved a notable PRafter failure of novel agents (i.e. Carfilzomib, Daratumumab and Pomalidomide). Median time to response was 1.3 months (r.1-3), median OS from diagnosis was 67.3 months (r.6-151), median OS from start of Bendamustine was 9.6 months (r.2-36).

Summary/Conclusion: The triplet Bendamustine-Bortezomib-Dexamethasone has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, and, in particular cases, it could be considered as a bridge to a second autologous or allogenic SCT,also after failure of novel agents.

PB1986: ADDITION OF CARFILZOMIB AS A THIRD AGENT IN LENALIDOMIDE-REFRACTORY MULTIPLE MYELOMA: SWITCHING FROM DOUBLET TO TRIPLET

C. Cerchione 1,1,1,* , D. Nappi 1 , A. E. Pareto 2 , S. Ronconi 1 , D. Cangini 1 , M. Ceccolini 1 , G. Musuraca 1 , L. Catalano 1 , G. Martinelli 1

1 Hematology Unit, Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST IRCCS, Meldola (FC); 2 Hematology Unit, Ospedale San Giuliano, Giugliano in Campania, Italy

Background: Carfilzomib is an epoxyketone proteasome inhibitor of secondgeneration, proved to be effective and safe in relapsed and refractory Multiple Myeloma (rrMM), in combination with dexamethasone or lenalidomide and dexamethasone.

Aims: In this retrospective observational trial, it has been evaluated efficacy and safety of carfilzomib, in combination with lenalidomide-dexamethasone (KRD) as salvage regimen in patients with rrMM, refractory to lenalidomide, where lenalidomide-based regimens have no proven efficacy.

Methods: 41 patients (23 M/18 F), with rrMM, median age at diagnosis 63.7years (r. 43-82), median age at start of treatment 67 years (r. 48-84)previously treated with several lines of treatments (median 3, r. 2-1),underwent to KRD regimen (ASPIRE trial schedule) for a median treatmentcycles of 8 (r 2-18). ISS was equally distributed, and all patients hadpreviously been treated with bortezomib and IMIDs, and were refractory to this agents. 61% (19/31) of them had undergone at least to a single ASCT.

Results: According to IMWG criteria, after a median follow-up of 9 months (r.2-18), ORR was 68,2% (28/41: 9 CR, 12 VGPR, 7 PR) with 5 progressive diseases (PD) and 8 patients in stable disease (SD): this can be considered as an impressive result in this subset of rrMM patients, refractory to lenalidomide. In particular, for 11 patients, KRD was, after having achieved at least a PR, a bridge to second/third autologous SCT. Median time to response was 1.3 months (r.1-4), median OS from diagnosis was 62 months (r. 9-170), median OS from start of Carfilzomib was 11 months (r. 2-18). Carfilzomib was well tolerated, with grade 2 anemia in 39% (16/41) of patients,successfully managed by ESAs, without necessity of blood transfusions; 29%(12/41) grade 3-4 neutropenia (pegfilgrastim in primary prophylaxis wasgiven, no ospedalization was required, no septic shocks were observed); 34%(14/41) grade 2, 21% (9/41) grade 3 and 12% (5/41) grade 4 thrombocytopenia, without hemorrhagic events and transfusion-dependency. Moreover, it was observed pneumonia in 39% (16/41) of patients, treated by common antibiotic drugs and always solved. A cardiac monitoring was performed for all patients: hypertension (grade 2-3) in 34%(14/41) of patients; fatigue in 39% (16/31) of patients.

Summary/Conclusion: Carfilzomib-Lenalidomide-Dexamethasone has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, also lenalidomide, and it could be considered as a bridge to a second autologous or allogenic SCT.

PB1987: DOMESTIC OPPORTUNITY IN HEAVILY PRETREATED MULTIPLE MYELOMA NOT ELIGIBLE TO HOSPITAL-BASED TREATMENT: ROLE OF POMALIDOMIDE-DEXAMETHASONE

C. Cerchione 1,1,1,* , D. Nappi 1 , A. E. Pareto 2 , S. Ronconi 1 , D. Cangini 1 , M. Ceccolini 1 , G. Musuraca 1 , L. Catalano 1 , G. Martinelli 1

1 Hematology Unit, Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST IRCCS, Meldola (FC); 2 Hematology Unit, Ospedale San Giuliano, Giugliano in Campania, Italy

Background: Many patients affected by heavily pre-treated Multiple Myeloma could be not eligible to hospital-based treatment: in this context domestic opportunities should be considered.

Aims: Pomalidomide is a new generation IMID, with a very good compliance, thanks to oral administration, which can be used also in heavily pretreated patients, in a domestic setting.

In this retrospective observational trial, It has been evaluated efficacy and tolerance of pomalidomide plus dexamethasone (PD) as salvage regimen in heavily pretreated patients with relapsed and refractory MM (rrMM), whose prognosis is particularly severe.

Methods: 57 patients (31 M/26 F), with rrMM, median age at diagnosis 69 years (r. 52-86), and median age at start of treatment 76 years (r.56-90) treated with several lines of treatments (median 7, r. 2-11), every refractory to all the drugs previously received (also Bortezomib, Thalidomide and Lenalidomide), received Pomalidomide-Dexamethasone (Pomalidomide 4 mg for 21 days, Dexamethasone 40 mg days 1,8,15,22, pegfilgrastim day +8) every 28 days, until progression. ISS was equally distributed, and cytogenetic at relapse was evaluable in 14 patients. All the patients had previously been treated with schedule containing bortezomib and IMIDs. 63% (36/57) had undergone at least to a single ASCT. All patients were relapsed and refractory to last therapies received before PD.

Results: Pomalidomide was well tolerated, with grade 3-4 transfusion-dependent anemia in 58% (33/57) of patients, 44% (23/57) grade 3-4 neutropenia (pegfilgrastim in primary prophylaxis was given, no hospitalization was required, no septic shocks were observed), 40% (23/57) grade 3-4 thrombocytopenia without hemorrhagic events and transfusion-dependence. No severe extra-hematologic toxicity was observed. According to IMWG, ORR1 (≥PR) was 47.3% (27/57: 5 CR, 11 VGPR, 7 PR, 4 MR), but, considering that we are evaluating a cohort of heavily pretreated patients, with poor prognosis, another parameter should be considered, ORR2 (≥SD), considering stable disease as a successful result in progressive MM. ORR2 was 77.1% (17 SD). These can be considered as impressive result in this subset of patients. Oral treatment gives a really good compliance, in frail and unfit patients, and response, when present, is always really fast (median time to response: 2 months (r.1-6)), median OS from diagnosis was 94 months (range 21-234), median OS from start of pomalidomide was 9 months (range 1-25).

Nine patients have surprisingly achieved a notable response (3 VGPR, 4 PR, 2 MR) after failure of novel agents (i.e. Carfilzomib, Daratumumab and Pomalidomide).

Summary/Conclusion: Pomalidomide-dexamethasone has shown significant efficacy and a very good compliance, thanks to oral administration, in a particularly severe setting of heavily pretreated patients, relapsed and refractory to all available therapeutic resources, also after failure of novel agents.

PB1988: AL CARDIAC AMYLOIDOSIS: A CASE SERIES OF 13 PATIENTS AND LITERATURE REVIEW

S. Chadli 1,* , M. Maamar 1 , H. Khibri 1 , S. Belkhettab 1 , N. Moatassim 1 , W. Ammouri 1 , H. Harmouche 1 , Z. Tazi Mezalek 1

1 Internal medicine and clinical hematology, Ibn Sina University Hospital Center, Rabat, Morocco

Background: Amyloidosis is a systemic disease caused by the extracellular deposition of misfolded proteins in various tissues and organs. Cardiac involvement is commonly observed and has a major impact on prognosis. The light chain immunoglobulin is the most severe subtype involved, associated to a poor outcome.

Aims: The objective of our study is to describe the clinical profile and outcome of patients with AL cardiac amyloidosis, and compare our results to the literature.

Methods: We retrospectively conducted a monocentric and descriptive study, including the medical records of 56 patients admitted in our department for a systemic amyloidosis, between 2015 and 2022. A cardiac involvement was confirmed among 18 patients. 13 cases of AL cardiac amyloidosis were enrolled.

Results: Patients were mostly males with a sex ratio (M/F) of 2.2, and a mean age of 59 ± 13 years. Two patients were previously diagnosed with a multiple myeloma, one patient had a T lymphoma in complete remission, and one patient had a monoclonal gammapathy of undetermined significance. Patients symptomatology consisted mostly in dyspnea and peripheral edema (54%), with a mean duration of 12 ± 4 months. On cardiovascular examination, signs of right and global heart failure were found in respectively 50% and 15% of the cases. Were observed simultaneously: mucocutaneous (46%), renal (30%), neurologic (30%), digestive (23%), articular (15%), muscular (15%), and ocular (8%) involvements. The main electric abnormalities were: low-voltage (38%), atrioventricular block (23%), and right bundle branch block with repolarization abnormalities (15%). On chest x-ray, a cardiomegaly was found in half of the cases. Transthoracic echocardiography revealed: left ventricular hypertrophy (100%), septal and valvular hypertrophy (70%), sparkling myocardium (60%), pericardial effusion (54%), bull’s eye strain pattern (46%), diastolic dysfunction (46%), dilated cavities (46%), pulmonary hypertension (23%), and intracardiac thrombosis (8%). Systolic function was generally conserved, with a mean of 50 ± 6 %. On cardiac MRI, the most frequent features were: left ventricular hypertrophy (100%), delayed myocardial enhancement (50%), and abnormalities in myocardium nulling (40%). Biological findings included a median troponin of 0.95 ng/ml [0,10; 67,12] and NT-proBNP of 5640 pg/ml [2577; 14 825]. Bence Jones proteinuria was positive in 30% of the cases. All patients presented a low plasma cell infiltration (4 to 8%), except one (25%). The (4;14) translocation was positive in one case. The monoclonal light chain involved was lambda (62%) and kappa (38%), with a median dFLC of 429 [34; 3053]. Patients were all put under chemotherapy: Daratumumab-Velcade-Cyclophosphamide-dexamethasone (30%), Velcade-Cyclophosphamide-dexamethasone (23%), Melphalan-dexaméthasone (23%), Cyclophosphamide-Thalidomide-dexamethasone (8%). A complete remission was obtained in one patient, and a partial remission in two patients. A death was recorded in six cases, following a cardiogenic shock (n=2) and arrhythmias (n=4).

Summary/Conclusion: Cardiac AL amyloidosis is a life-threatening condition. Because of its rapid progression and nonspecific clinical features, it is often misdiagnosed or discovered lately during the disease course. The AL subtype is linked to a poor survival rate because of the frequent malignant nature of the underlying disease and the high cardiac toxicity of the monoclonal light chains. We thus believe that a screening of patients at risk to develop AL amyloidosis could lead to an early diagnosis and better prognosis.

PB1989: LIGHT CHAIN AMYLOIDOSIS (AL): THE EXPERIENCE OF A TUNISIAN CENTER

F. Kallel 1,* , A. Chalbaoui 1 , O. Kassar 1 , I. Saguem 2 , M. Charfi 1 , I. Aguerbi 2 , Y. Fakhfekh 1 , L. Khalifa 1 , I. Frikha 1 , I. Ben Amor 1 , S. Hdiji 1 , T. Boudawara 2 , M. Ben Hmida 2 , M. Mdhaffar 1 , M. Elloumi 1

1 Hematology department, Hedi cheker hospital; 2 histology department, Habib bourguiba hospital, sfax, Tunisia

Background: The AL amyloidosis is one of the most common types of amyloidosis that is derived from the immunoglobulin light-chain. The clinical presentation varies widely depending on which organs are involved. In this work, we report the experience of the hematology department of Sfax in the management of patients followed for AL amyloidosis.

Aims: Our study aims to evaluate the clinico-biological, therapeutic and evolutionary characteristics of al amyloidosis in our patients.

Methods: Our study is retrospective, it concerned AL amyloidosis patients, followed in the hematology department of Hedi Chaker Sfax hospital between January 2010 and December 2021.

The treatment was based on different chemotherapy protocols: the Cybor-D protocol (Bortezomib – Cyclophosphamide – Dexamethasone), the BorTD protocol (bortezomib – thalidomide – dexamethasone) and the M-DEX regimen (melphalan – Dexamethasone).

Results: Our series included 20 patients with a male predominance (sex-ratio = 1.8) and a mean age of 58 years. 85% of the patients were followed for a multiple myeloma which was then complicated by amyloidosis. The initial symptomatology was polymorphic: cutaneous-mucosal involvement, cardiac symptomatology and neurological symptomatology were noted in 50%, 30% and 25% of cases respectively. The diagnosis was made on 2 different sites in 11 patients (55% of cases). Anemia was found in 55% of cases, and renal failure was reported in 50% of thes cases. Monoclonal gammopathy was found in all patients with demonstration of the Lambda and kappa chain in 50% of cases each. Transthoracic ultrasound revealed signs of myocardial amyloid infiltration, such as wall thickening greater than 15 mm, in 11 patients. Cardiac involvement, labial amyloidosis and renal involvement were diagnosed in 60%, 55% and 25% of cases respectively. According to the classic Mayo Clinic prognostic score, 4 patients were at stage I, 4 others were at stage II and 12 patients were at stage III.

Regarding the treatment, 9 patients (45% of cases) received the Cybor-D protocol, 6 patients were treated with the BorTD protocol, 4 patients received the M-Dex regimen, and 1 patient died before starting treatment. This first-line treatment resulted in objective response rate (VGPR and CR) at 42% of cases (n=8), partial response at 16% (n= 3), Progression at 16% (n=3), toxic death at 10% (n=2) and 3 patients were lost to sight before assessment. This objective response was obtained by protocols based on Bortezomib. For patients with amyloid cardiomyopathy (12 patients), 5 were progressing at the end of treatment despite the fact that 4 of them were in hematological CR, and 2 died from cardiac decompensation.

A relapse occured in 6 patients out of 11(CR+VGPR+RP) after a median time of 29 months.

Median overall survival was 18 months.

Summary/Conclusion: Through this work, we notice the polymorphism of the amyloidosis clinico-biological presentation as well as the concept of multiorgan involvement. The light chain was Lambda in 50% of cases, whereas it is predominant (80%) in the literature (1), and cardiac involvement was the leading cause of morbidity and mortality as similar as what is reported (1). Treatment is based on Bortezomib combined with other chemotherapy molecules (2). With this protocol, we obtained an objective response rate and a median survival rate a bit lower than the rates described in literature (1).

PB1990: MULTIPLE MYELOMA IN THE ELDERLY: A MONOCENTRIC EXPERIENCE

H. Charfi 1,* , L. Khalifa 1 , F. Kallel 1 , M. Medhaffer 1 , O. Kassar 1 , M. Charfi 1 , Y. fakhfakh 1 , I. Frikha 1 , I. Ben Amor 1 , S. Hadiji 1 , M. Elloumi 1

1 hematology, Hedi Chaker Hospital, Sfax, Tunisia

Background: Multiple myeloma (MM) is a common hematological malignant neoplasm and it is primarily a disease of the elderly. Development of newer therapeutic agents and improving supportive care over the last decades has significantly improved outcome of MM in younger patients. However, most studies suggest that improvements are marginal in elderly patients.

Aims: The aim of the study is to report the clinical, paraclinical and therapeutic features of the elderly multiple myeloma (age >65 years) in our center.

Methods: We retrospectively reviewed data of patients aged over 65 years diagnosed with MM and treated at the hematology department of Hedi Chaker Hospital Sfax between January 2012 and December 2019. Response to treatment is evaluated according to the International Myeloma Working Group (IMWG). these patients are considered ineligible for autologous stem cell transplantation (ASCT). The induction regimen is based on the combination melphalan-prednisone-thalidomide (MPT) or melphalan-prednisone (MP). Clinical outcomes included: reason for treatment discontinuation, overall response rate (ORR), overall survival (OS), progression-free survival (PFS), and adverse events (AE).

Results: A total of 40 patients (sex ratio M/F=1.35) were included in our study. Median age was 73 years [65-93 years]. 10 patients (25%) aged ≥80 years. Performance status (PS) was ≥2 in 29 patients (73%). sixty percent of the cases had at least one comorbidity. The median Charlson Comorbidity Index (CCI) was 4 [2–7]. Bone pain was the most common presenting complaint observed in 65% of the cases. Monoclonal IgG was the predominant protein M type’s (70%). According to the Durie-Salmon Staging System, IIIA was the main stage noted in 64% of the cases followed by IIIB in 23% of the cases.

MPT and MP regimens were received in 57% and 17/40 respectively.

ORR was 23%, including, VGPR 5 %, and PR 18%. None of the cases acheived complete remission. Stable disease occurred in 30 % and progressive disease in 7.5%. The remain cases were non evaluable (15/40). Treatment was discontinued in 14 cases (35%) after a median of 2 courses due to either progression or toxicity.

Main adverse events were infectious in 25% (10 cases) and hematological in 22% (9 patients).

Median Follow up was 77 months. Median survival was 24 months. OS and PFS were 47% and 44% at 2 years and 27% and 23% at 5 years respectively.

Summary/Conclusion: Despite the fact that one third of our patients had no comorbidity, MPT and MP regimens in our study had insufficient results and correlated with a lower ORR and median survival comparatively to the literature (24 vs 48 months). It can be explained by the low rate of patients receiving Thalidomide. The combination of bortezomib-lenalidomide-dexamethasone for elderly patients may improve our results. In addition, structured frailty assessment is required in order to devise individualized treatment plan able to improve the clinical outcomes and the Quality of life.

PB1992: HISPANIC ETHNICITY AS A STRONG PREDICTOR OF POOR RESPONSE TO INDUCTION THERAPY AND ASCT IN TRANSPLANT ELIGIBLE MULTIPLE MYELOMA PATIENTS

I. Chineke 1 , E. Lu 2 , H. Win 1 , A. Larsen 3 , G. Yao 2,3 , F. Chilton 4,5 , K. Gowin 1,6,*

1 Division of Hematology and Oncology; 2 Department of Molecular and Cellular Biology; 3 Arizona Cancer Center; 4 School of Nutritional Sciences and Wellness, College of Agriculture and Life Sciences; 5 BIO5 Institute; 6 Department of Bone Marrow Transplant and Cellular Therapy, University of Arizona, Tucson, AZ, USA, Tucson, United States of America

Background: Attempts have been made to use machine learning analysis of multidimensional data to determine personalized therapeutic approaches in multiple myeloma. Outcomes of disparities in Hispanic multiple myeloma treatment have been attributed in part to inadequate access to novel therapies and autologous stem cell transplant (ASCT). We sought to utilize a machine learning approach to identify clinical and demographic features associated with patient responses to multiple myeloma induction therapy and ASCT

Aims: We aimed to employ machine learning analysis in the identification of clinical and demographic characteristics associated with patients’ response to multiple myeloma induction therapy and ASCT

Methods: A retrospective chart review of patients with a documented diagnosis of multiple myeloma seen at the University of Arizona Cancer Center between January 2017 and April 2021 was performed. RedCapp was utilized to organize demographic, clinical, laboratory, therapeutic, and outcome data. A patient’s best response (per the IMWG myeloma response criteria) following induction therapy prior to ASCT was classified into the following categories: progression of disease, less than PR (partial response), partial response (PR), very good partial response (VGPR), complete response (CR), and stringent complete response (sCR). This response was then compared to the response at 100 days post-transplant and labeled as ‘1’ if the response improved or ‘0’ if the response stayed the same or worsened. A clinical decision tree model was generated by recursive partitioning and minimizing cross-validation error, which outperformed other tested models (e.g elastic net and LASSO regression) in accuracy. Critical indices that best stratified patient’s response (1 vs 0) were then identified based on the tree model.

Results: Of 112 patients reviewed, 48 were transplant-eligible and had complete data for analysis. Among available input features derived from RedCapp, (including demographic and clinical baseline factors and the choice of first induction regimen), ethnicity (Hispanic/non-Hispanic) was identified as the central node in the tree model that stratified patient responses. Among Hispanic patients (23% in this cohort), only 18% showed any improvement in their documented best response when comparing induction therapy and 100 days after transplant. In contrast, among non-Hispanic patients (77% in this cohort), 68% exhibited an improvement. Furthermore, serum creatinine (sCr) was identified as the key risk factor in non-Hispanic patients, with those having sCr ≥ 1.6 (mg/dL) showing a much smaller chance of improvement (17%) on day 100 post-transplant compared with patients with sCr < 1.6 (79%).

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Summary/Conclusion: Utilizing a machine learning-enabled analysis of multidimensional data from 48 patients, we identified Hispanic ethnicity and renal dysfunction at the time of diagnosis (sCr ≥ 1.6) as key factors that may impact patient responses between the end of induction therapy and at 100 days post-transplant. Although limited to a single institution and a small sample size, these findings warrant further evaluation in larger patient cohorts, and raise important questions regarding the pathobiology and corresponding therapeutic strategies for multiple myeloma in Hispanic populations. This study further illustrates the utility of data-driven approaches such as machine learning to shed light on complex issues such as cancer care and related disparities as well as the pathobiology and therapy choice of the complex disease of multiple myeloma.

PB1993: MULTIPLE MYELOMA BRAZILIAN REGISTER- HOW ABOUT THE TRANSPLANT ELIGIBLE PATIENTS?

E. Crusoe 1,2,* , G. Ribeiro 3 , K. Zanella 4 , L. Perobelli 5 , J. C. Saraiva FIlho 6 , R. Magalhaes 7 , R. Bittencourt 8 , E. Souza 9 , R. Centrone 10 , N. Hamerschlak 11 , R. Gaiolla 12 , B. Gusmao 13 , F. HIgashi 14 , J. Farley 15 , M. Capra 16 , W. Braga 17 , A. Hallack Neto 18 , J. Vaz P Neto 19 , C. Bonamin 20 , C. Silva 21 , J. T. Souto Filho 22 , G. Martinez 23 , E. Mattos 24 , N. Castro 25 , L. da Silva 26 , V. Farnese 27 , R. Tavares 28 , J. Bigonha 29 , J. Lima 30 , E. Ribeiro 31 , R. Cunha 32 , F. Nucci 33 , M. Almeida 34 , L. Cruz 35 , A. Maiolino 7 , V. Hungria 36

1 Hematology and Bone Marrow Transplantation, Federal University of Bahia- University hospital; 2 Rede D’or Oncologia, Salvador; 3 Clínica Hematologica, Belo Horizonte; 4 Clinica Viver- CEPHON, Florianopolis; 5 Hospital de Transplantes Euryclides de Jesus Zerbini, Sao Paulo; 6 Hospital Ophir Loyola, Belem; 7 Universidade Federal do Rio de Janeiro, RIo de Janeiro; 8 , University Hospital of the Federal University of Rio Grande do Sul, Porto Alegre; 9 Hospital das CLinicas UFMG, Belo Horizonte; 10 Instituto Hemomed; 11 Hospital ALbert Einstein, sao paulo; 12 Hospital Das Clínicas UNESP, Botucatu; 13 Beneficiencia Portuguesa; 14 Irmandade Santa Casa de Misericordia de Sao Paulo, Sao Paulo; 15 Liga Norte-Riograndense Contra o Câncer, Natal; 16 Hospital do Cancer Mae de Deus, Porto Alegre; 17 Hospital Sao Paulo- UNIFESP, Sao Paulo; 18 Universidade Federal de Juiz de Fora, Juiz de Fora; 19 CETTRO, Brasilia; 20 HC - UFPR, curitiba; 21 IPSEMG, Belo Horizonte; 22 Faculdade de Medicina de Campos, Campos de Goytacazes; 23 Hospital das Clinicas da Faculade Medicina da USP, sao paulo; 24 Fundação Hospital Amaral Carvalho, Jau; 25 Hospital de CAncer de Barretos, Barretos; 26 Federal University of Alagoas, Maceio; 27 ; Federal University of Uberlandia, Uberlandia; 28 Hospital das CLinicas da UFG, Goiais; 29 Faculdade de Medicina do ABC, Santo Andre; 30 Instituto de Oncologia e Hematologia de Curitiba, Curitiba; 31 Hospital Santa Lúcia Centro, Brasilia; 32 Oncoclinica RJ, Rio de Janeiro; 33 Hospital Universitário Antônio Pedro, Niteroi; 34 Santa Marcelina, Sao Paulo; 35 HOSPITAL NAPOLEÃO LAUREANO, Joao Pessoa; 36 Clinica Sao Germano, Sao Paulo, Brazil

Background: The Brazilian Multiple Myeloma Group (GBRAM) developed an electronic database platform with the intention of prospectively registering the multiple myeloma (MM) cases diagnosed in the Brazilian healthcare system and analyzing the disease characteristics, treatments patterns and the outcomes.

Aims: Describe characteristics, treatment and outcomes from MM transplant eligible patients belonging to GBRAM prospective database plataform.

Methods: This is a prospective, multicenter data-based platform. This analysis was focused on the MM transplant eligible patients (TE). The MM patients diagnosed after January 1, 2018 have been included. The eligibility criteria were: intent-to-treat (ITT) MM patients, aged over 18 years and under care in any healthcare system (private and public). All clinical and laboratory data, prognostic profiling, treatment patterns and responses, adverse events and survival were compiled. The data were analyzed with the NCSS® 2020 software. This project is registered in the Brazilian study platform (CAAE-05340918.3.1001.8098).

Results: A total of 915 patients were registered with transplant intent-to-treat. The median age was 59 (25 - 80) years and 475 (51.9%) were male. A total of 413 (45.1%) of the patients were nonwhite. According to the (ECOG) rating at diagnosis: 0 = 217 (32.9%), 1 = 222 (33.7%), 2 = 110 (17.6%), 3 = 71 (10.85%) and 4 = 39 (5.9%). The ISS 1, 2, and 3 were 209 (25.7%), 228 (28.1%) and 303 (37.2%), respectively, and 72 (8.9%) not available (NA). The respective isotypes were: 466 (52.5%) IgG, 168 (18.9%) IgA, 158 (17.8%) free-light chain, 15(1.7%) non-secretor and 68 (7.7%) NA. Stratifying the intent-to-treat TE patients according to healthcare system, 178 (19.5%) and 737 (80.5%) were from the private and public systems, respectively. Among the patients who underwent the transplant, 89 (66.4%) belonged to the private system and 154 (32.6%), the public. The induction chemotherapy treatments in the public system were: 366 (59%) based on thalidomide, 226 (36.5%) based on bortezomib and 21 (3.4%) based on daratumumab. In the private system the induction treatments were: 7 (4.2%) based on thalidomide, 135 (75.8%) based on bortezomib and 31 (18.8%) based on daratumumab. After a median follow-up of 18.4 months, the complete response (RC) rate post transplant was 13.5% for the public and 28.7% for the private system, respectively (p < 0.01).

Summary/Conclusion: This prospective register enrolled patients diagnosed since January 2018 and is of a nationwide scope. Our register, in addition to identifying the epidemiological characteristics of the Brazilian multiple myeloma patients, demonstrates the treatment standards at the public and private institutions. Patients eligible for transplant have greater access to the procedure at private institutions than at public ones (p < 0.001), as well as to better chemotherapy induction protocols. The complete remission (CR) rate following transplant was greater in the private system than in the public and, with a longer follow-up, it will be possible to demonstrate the impact of the treatments on the survival in patients eligible for transplant.

PB1994: REAL WORLD DATA OF PATIENTS WITH MULTIPLE MYELOMA WITHOUT ACCESS A NOVEL AGENT IN FIRST LINE THERAPY IN PUBLIC HEALTH HOSPITAL IN MEXICO

D. Cruz 1,* , G. SOTOMAYOR 1 , L. G. AVILA 1

1 HEMATOLOGY, Mexican Institut of Healt, Monterrey, Mexico

Background: Real World data of patients with Multiple Myeloma without access a novel agent in first line therapy in public Health hospital in Mexico

Aims: To expose the 5-year OS and PFS of patients with multiple myeloma who did not use Bortezomib as first line therapy and to compare its use after first line.

Methods: Observational, longitudinal and retrospective study. Measures of central tendency, Kaplan-Meier curves with Log-Rank as well as Cox regression were performed for survival analysis, with HR report, ranges were obtained considering significant a value of p <0.05 with 95% CI. 0.1 amplitude with 5% error

Results: 265 patients, 126 (47.5%) male and 139 (52.5%) female, with a median age of 62 years (34-90). The 5-year OS was 47%. In PFS 55% at 1 year and 9% at 5 years. In comparison OS of subsequent line with Bortezomib versus absence, OS of 59% was reported in the Bortezomib arm versus 43% with HR of 1.819 (95% CI 1.235-2.677) with a P value of P=0.002.

Summary/Conclusion: The absence of proteosome inhibitor as first line negatively impacts OS and PFS; its use even as subsequent line therapy may improve prognosis

PB1995: SAFETY OF SUBCUTANEOUS DARATUMUMAB IN MULTIPLE MYELOMA: A RETROSPECTIVE MULTI-CENTER REAL LIFE EXPERIENCE

D. De Novellis 1,* , A. Carobene 1 , R. Fontana 1 , L. Mettivier 1 , F. Trastulli 2 , S. Rocco 2 , S. Palmieri 2 , M. Di Perna 3 , A. Pagano 3 , V. Giudice 4,5 , C. Califano 3 , F. Ferrara 2 , C. Selleri 1,5

1 Hematology and Transplant Unit, University Hospital “San Giovanni di Dio e Ruggi d’Aragona”, Salerno; 2 Division of Hematology, Cardarelli Hospital, Napoli; 3 Division of Hematology, Andrea Tortora Hospital, Pagani; 4 Clinical Pharmacology Unit, University Hospital “San Giovanni di Dio e Ruggi d’Aragona”, Salerno; 5 Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana” University of Salerno, Baronissi, Italy

Background: Daratumumab, an anti-CD38 monoclonal antibody, is approved for Multiple Myeloma (MM) treatment at an intravenous (IV) dosage of 16 mg/kg with high rates of infusion-related reactions (IRR; 45-56%), especially at the first infusion. However, the subcutaneous (SC) formulation has shown comparable efficacy with lower IRR risk and with shorter administration time (3-5 minutes) compared to IV daratumumab as reported in PAVO ( NCT02519452 ), COLUMBA ( NCT03277105 ) and PLEIADES ( NCT03412565 ) clinical trials. Approved SC daratumumab dosage is 1800 mg, independently from patient variables including renal failure or body weight.

Aims: Here, we investigate safety of SC daratumumab in different MM regimens in a multi-center real-life experience

Methods: A total of 29 MM patients (Table 1) who received at least one dose of SC daratumumab at 1800 mg in any association regimen since December 2021 was included in this retrospective study. Enrolled patients were daratumumab-naïve starting directly with SC formulation combined with any association regimen displayed in Table 1. Primary end point was SC daratumumab safety, especially for IRR incidence and severity.

Results: All patients received premedication with dexamethasone, paracetamol and antihistamine according to current guidelines and montelukast was used in 23 out of 29 (79%) cases. Median number of SC administrations for single patient was 3 (range 1-10), and administration time ranged from 3 to 5 minutes. One patient (3%) experienced grade III IRR in two consecutive administrations, and grade I hematological and grade I gastroenteric toxicities (diarrhea) were observed in 3 (10%; 2 thrombocytopenia and 1 neutropenia) and 1 (3%) cases

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Summary/Conclusion: In our multicenter retrospective experience, SC daratumumab showed high safety with a very low (3%) IRR rate, even inferior to that reported in PAVO and PLEIADES trials, despite we included in our study patients with renal failure, different body weights or without montelukast premedication. Of note, IV daratumumab induced IRR especially during the first exposition, while we reported a very low IRR rate with SC daratumumab also during first administrations (median number of cycles, 3).

The only patient with grade III IRRs weighted 65 kg, did not have renal failure and did not receive montelukast premedication. The patient was switched to IV daratumumab without further IRR suggesting an SC formulation excipient-related intolerance rather than daratumumab-induced reaction. However, IRR incidence was low to analyze hypothetical associations between predisposing factors and IRR insurgence.

In conclusions, our real-life findings showed that SC daratumumab is very manageable with an excellent safety profile and very short administration time. Further validation on larger and prospective clinical studies are needed

PB1996: TREATMENT PATHWAYS AT FIRST RELAPSE POST-ASCT IN MULTIPLE MYELOMA: IMPACT OF MAINTENANCE THERAPY ON TREATMENT OPTIONS

C. Duane 1,* , C. Comerford 1 , P. Murphy 1,2 , P. Thornton 1,2 , J. Sargent 1 , S. Glavey 1,3 , J. Quinn 2,4

1 Department of Haematology, Beaumont Hospital Dublin; 2 School of Medicine; 3 Department of Pathology; 4 Department of Haematology, Royal College of Surgeons in Ireland, Dublin, Ireland

Background: The introduction of new agents and treatment combinations has led to significant improvements in outcomes for multiple myeloma (MM) patients. In particular, lenalidomide maintenance therapy (LenM) following autologous stem cell transplant (ASCT) in younger/fitter MM patients has now become standard given the survival benefits associated with it. However, most MM patients will relapse post-ASCT and many effective relapse regimens have a lenalidomide backbone (e.g. KRD: carfilzomib, lenalidomide, dexamethasone, DRD: daratumumab-RD, IRD: ixazomib-RD) making choice of regimen at first relapse increasingly difficult in patients progressing during LenM therapy. Real world data reflecting these choices is needed to inform treatment decisions and consensus guidelines.

Aims: We carried out an audit with an aim to: 1. Evaluate treatment and outcomes in MM patients undergoing ASCT from 2010-2020 2. Assess the impact of LenM post-ASCT and therapy choice after relapse.

Methods: We carried out a retrospective audit of all patients with MM attending an Irish hospital who underwent ASCT from 2010-2020. Data was collected by chart/electronic record review. Endpoints included no. of ASCT/year and type of induction/maintenance therapy, relapse rates post-ASCT and choice of post-relapse therapy.

Results: 71 patients at our centre underwent ASCT for MM between 2010-2020 (24 female,47 male) with median age of 67 and most common isotype IgG Kappa (n=33). The most frequently used induction was cyclophosphamide(C), bortezomib(V) and dexamethasone(D) (>60%, VCD). Use of maintenance therapy increased markedly over this decade, with 0% receiving maintenance in 2010, 44% in 2014 and 83% in 2020. LenM was most common, >75% (n=28).Other maintenance therapies included bortezomib n=4, daratumumab (dara)n=3 and lenalidomide + bortezomib (RV) combination n=4. First relapse treatment data was available for 36 patients. Regimens included carfilzomib/dexamethasone (KD) n=16 +/- lenalidomide (KRD) n=1, pomalidomide (PD), n=1, ixazomib (ID) n=1, dara (n=3) and dara combinations, including daraVD, n=1, and daraPD, n=1. 26 patients did not receive maintenance containing lenalidomide and the majority of these patients received ASCT pre-2015. There were n=13 relapses in this group: n=4 received lenalidomide regimens (RVD/VCD) at relapse. KD n=4, dara n=1, and ID n=1 were also used. First relapse therapy for 17 patients who progressed during LenM included: KD(n=11) 64.7%, dara(n=2) 11.7%, pomalidomide(n=1) 5.8%, and RV(n=2) 11.7% and VCD(n=1) 5.8%. Overall, carfilzomib-based therapy (without lenalidomide) was the most commonly prescribed therapy on first relapse. We observed a clear increase in its use from 2015-2020, in parallel with increased use of LenM. We also noted reducing rates of lenalidomide-based relapse therapies in later years, correlating with increased LenM. Importantly, neither DRD or PVD combinations were reimbursed for prescription in Irish healthcare during the period of audit and are notable absences from the relapse treatment regimens used.

Summary/Conclusion: This audit highlights the treatment pathway and outcomes for all patients with MM undergoing ASCT over 10 years in our centre. We have noted significant changes in choice of therapy upon relapse, with carfilzomib prescription rates increasing and lenalidomide-based regimen use decreasing. Our real world data provides direct insight into the effect of widespread LenM therapy on choice of agent upon relapse and highlights the need for consensus treatment guidelines to reflect this evolution in treatment for MM patients.

PB1997: DARATUMUMAB, LENALIDOMIDE, BORTEZOMIB, AND DEXAMETHASONE FOR TRANSPLANT-ELIGIBLE NEWLY DIAGNOSED MULTIPLE MYELOMA: OUTCOME AND SAFETY PROFILE FROM A REAL-WORLD SINGLE CENTER EXPERIENCE

H. Elsabah 1,* , N. Kassem 2 , A. Gulied 2 , R. Taha 1 , H. Elomri 1 , H. Cherif 1

1 Hematology; 2 Pharmacy department, Hamad medical corporation, Doha, Qatar

Background: lenalidomide, bortezomib, and dexamethasone (RVd) followed by autologous stem cell transplant is standard frontline therapy for transplant eligible newly diagnosed multiple myeloma (MM).The addition of daratumumab (D) to RVd improve the depth of response (Stringent complete remission and MRD negativity). RVd-D combination is now being suggested as first-line treatment. Real-word data is lacking.

Aims: We aim to share our experience regarding clinical response and safety profile of D-RVd for treatment of newly diagnosed transplantation eligible MM patients.

Methods: We conducted a retrospective study with medical review of all patients diagnosed with multiple myeloma and treated with D-RVd at The National Center for Cancer Care and Research (NCCCR) in Qatar between January 2019 to December 2021. Overall response rate and safety profile were evaluated. All analyses were descriptive and exploratory in nature.

Results: Fifteen newly diagnosed MM patients had received D-RVd in the time period of the study. The median age of patients was 58 year (range, 36-68 years) and the majority 12 (80 %) were male. The immunoglobulin (Ig) subtypes were IgG 9 (60%), IgA 3 (20%), free light chain 3 (20%). At diagnosis, 14 patients had bone lesions and 4 had extramedullary plasmacytoma. Anemia, hypercalcemia (serum calcium > 2.6 mmol/L) and/or renal impairment (serum creatinine > 133µmol/L) was present in 66%, 66% and 33% of patients respectively. Bone marrow plasmacytosis more than 60% on bone marrow examination was reported in 11/14 (78%) of patients. The proportion of patients at ISS stage I, II, and III were 1 (7%), 6 (40%), 8 (53%) respectively. Cytogenetic abnormalities detected via karyotyping and/or FISH were present in 9 of 12 evaluated patients.

The average number of D-RVd were 4 cycles. The overall response rate (ORR) post induction therapy was evaluated in 14 patients (one patient was not evaluated for response due to early death), 9 (64%) patients had a complete response (CR), 4(29%) had very good partial response (VGPR) and one had partial response (PR). No myeloma progression while receiving induction therapy occurred.

So far, seven patients have undergone autologous stem cell transplant. Median CD 34+ cell yield was 5,2 million cells per kg body weight, plerixafor were used in 5 patients and 5 patients required more than one apheresis session. Survival data to be collected and reported after a longer follow up period.

The most common adverse events (AEs) were hematological toxicities; grade 3/4 neutropenia was detected in 6 patients (40%), thrombocytopenia in 3 patients (20%), lymphopenia in 4 patients (27%) and leukopenia in 2 patients (13%). Infusion reactions occurred in 2 patients (13%), one of them experienced anaphylactoid reaction that required infusion interruption. Non-hematological AEs where uncommon and included grade 2 hepatotoxicity in one patient and venous thrombosis in one patient. None of the AEs lead to treatment discontinuation. One death occurred early during the second cycle due to streptococcus pneumoniae meningitis.

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Summary/Conclusion: Our real-world results show a high response rate (CR/VGPR) to D-RVd as first-line treatment in transplantation eligible patients with MM. However, the feasibility and safety of this treatment regimen needs to be confirmed in larger prospective studies.

PB1998: WEEKLY OPPOSED TO BI-WEEKLY BORTEZOMIB AS PART OF INDUCTION CHEMOTHERAPY IN NEWLY DIAGNOSED MULTIPLE MYELOMA IS BETTER TOLERATED AND EQUALLY EFFICIENT: REAL-WORLD DATA FROM A RETROSPECTIVE AUDIT

L. Faulkner 1,* , M. Garg 1 , C. Suresh 1

1 University Hospitals Leicester, Leicester, United Kingdom

Background: The VTD (Bortezomib, Thalidomide, Dexamethasone) triplet chemotherapy regime is frequently used as induction chemotherapy in newly diagnosed multiple myeloma prior to autologous stem cell transplant. The manufacturer’s protocol recommends a twice-weekly dosing schedule. Adverse effects are common, most notably neuropathy and cytopenias. These can be disabling and are often permanent, with significant impacts on quality of life. This limits drug tolerance with management confined to dose reduction or discontinuation. A once-weekly dosing schedule has been widely adopted, but to date, no clinical trials have been conducted to compare the efficacy and side effect profile of this dosing frequency in transplant-eligible myeloma.

Aims: In order to investigate prescribing practice locally, we conducted a retrospective audit in Leicester Royal Infirmary, a tertiary Haematology centre in the UK. Primary outcomes were response to induction therapy, and the development of neuropathy (peripheral or autonomic) or serious adverse events occurring whilst on VTD. Secondary outcomes included cumulative Bortezomib dose, number of Bortezomib cycles and Thalidomide tolerance.

Methods: Real-world data was collected consecutively from all patients treated in the intensive treatment arm with VTD as first-line chemotherapy between January 2015 and November 2020. Patient received Bortezomib 1.3mg/m 2 subcutaneously on days 1,4,8 and 11 of a 21-day cycle (bi-weekly) or days 1,8,15 and 22 of a 35-day cycle (weekly). The trust protocol was altered in December 2020, increasing the bi-weekly regime to a 28-day cycle

Results: We demonstrate a trend of lower incidence of neuropathy, both peripheral and autonomic, with the weekly regime (PN≥2: weekly 8.7% bi-weekly 19.6% AN: weekly 8.7% bi-weekly 26.5%). There was also a trend of fewer serious adverse events with lower rates of hospital admissions due to infection (weekly 33.3% bi-weekly 47.8%). Patients on the weekly regime received more VTD cycles and a higher cumulative dose (p=0.044). The initial therapeutic response between the two regimes were similar (weekly: ORR 87% ≥VGPR 78.3%, bi-weekly: ORR 86.3% ≥VGPR 60.8%).

Preliminary data from patients treated with bi-weekly Bortezomib in a 28-day cycle shows lower rates of peripheral neuropathy compared to the 21-day cycle.

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Summary/Conclusion: Here we demonstrate that weekly Bortezomib is better tolerated with equal efficacy in terms of initial therapeutic response. We believe that delivery of Bortezomib through a weekly regime facilitates patients being able to maintain on Bortezomib longer and receive higher cumulative doses. This may have important implications in those later deemed unsuitable or transplantation and for future treatment as intolerance or refractoriness limits the ability to obtain Bortezomib in further treatment lines. Preliminary data shows the cycle length may also be important in determining the risk of side of side effects and we intent to continue to monitor patients treated with this regime.

PB1999: ATG-010 PLUS LOW-DOSE DEXAMETHASONE (SD) IN CHINESE RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM) PATIENTS PREVIOUSLY RECEIVED CHIMERIC ANTIGEN RECEPTOR T-CELL (CAR-T)

W. Fu 1,* , Z. Xia 2 , C. Fu 3 , W. Chen 4 , C. Chang 5 , B. Fang 6 , G. An 7 , Y. Wei 8 , Z. Cai 9 , S. Gao 10 , J. Weng 11 , L. Chen 12 , H. Jing 13 , F. Li 14 , Z. Liu 15 , X. Chen 16 , J. Liu 17 , Y. Yu 18 , A. Wang 18 , Y. Fei 18 , L. Qiu 7

1 Shangai Changzheng Hospital, Shanghai; 2 Sun Yat-sen University Cancer Center, Guangzhou; 3 The First Affiliated Hospital of Soochow University, Suzhou; 4 Beijing Chaoyang Hospital, Beijing; 5 Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai; 6 Henan Cancer Hospital, Zhengzhou; 7 Institute of Hematology and Blood Diseases Hospital, Tianjin; 8 Nanfang Hospital, Guangzhou; 9 The First Affiliated Hospital,Zhejiang University School of Medicine, Hangzhou; 10 the First Affiliated Hospital of Jilin University, Changchun; 11 Guangdong Provincial People’s Hospital, Guangzhou; 12 Jiangsu Province Hospital, First Affiliated Hospital of Nanjing Medical University, Nanjing; 13 Peking University Third Hospital, Beijing; 14 the First Affiliated Hospital of Nanchang University, Nanchang; 15 Shengjing Hospital of China Medical University, Shenyang; 16 Xi Jing Hospital affiliated to the Fourth Military Medical University, Xi’An; 17 the Third Xiangya Hospital of Central South University, Changsha; 18 Antengene Therapeutics Ltd., Shanghai, China

Background: There are limited treatment options for multiple myeloma (MM) patients who have a disease progression after CAR-T therapy. ATG-010 (selinexor) is a novel, oral selective inhibitor of nuclear export, inhibiting exportin 1. US FDA has approved selinexor plus low dose dexamethasone (Sd) to treat patients (pts) with penta-refractory MM. MARCH study, a single arm, Phase 2, registrational study evaluating Sd in Chinese RRMM pts, achieved an overall response rate (ORR) of 29.3% (95% CI: 19.7, 40.4), rejecting the null hypothesis of the study. Given its unique and novel mechanism of action, Sd preserves anti-tumor activity regardless of specific prior therapies. In the MARCH study, encouraging activity was demonstrated in a small group of Chinese RRMM pts previously exposed to CAR-T therapy.

Aims: To evaluate efficacy and safety of Sd in Chinese RRMM pts previously treated with CAR-T.

Methods: The study enrolled 82 pts previously exposed and refractory to a proteasome inhibitor (PI), an immunomodulatory agent (IMiD), and last line of therapy. Among them, 10 had received lymphodepleting conditioning followed by CAR-T cell therapy before study screening. ATG-010 (80mg) plus dexamethasone (20mg) was administered orally twice weekly. Response was assessed by an independent review committee.

Results: Among 10 pts, 8 were male and 2 were female. Median age was 58.5 years. Median duration from MM initial diagnosis was 5.2 years. A total of 6 pts (60.0%) had high-risk cytogenetic abnormalities, including 4 pts (40.0%) with del (17p). Three pts had baseline plasmacytoma. Five pts (50%) experienced very rapid disease progression as indicated by a median of 46.2% increase of tumor burden from screening to Cycle 1 Day 1. Patients were heavily pre-treated with a median of 9.5 prior regimens (range: 5-12), with 8 receiving more than 6 regimens. Four pts were exposed to daratumumab (triple-class exposure). ORR was 50% including 1 very good partial response and 4 partial responses. Disease control rate defined as SD and above was 70%. As of 10 th Feb 2022, all pts had disease progression, 5 pts are still under survival follow-up. Median duration of response was 1.4 months (mo) (95% CI: 0.96, NE). Median progression free survival was 1.9 mo (95% CI: 0.93, 3.74). Median overall survival was not reached and estimated 12-mo OS rate was 70%. After disease progression (PD) after Sd treatment, 4 pts received CD38 antibody based regimen, 4 pts received pomalidomide based regimen, 4 pts had cytotoxic therapy, 1 pt received 2 nd CAR-T therapy, and 2 pts had no chance to receive any treatment due to death after rapid PD. Of note, 1 pt received selinexor plus lenalidomide based regimen and obtained a prolonged disease control.

Adverse events were consistent with those events previously reported with Sd regimen in RRMM patients. The most common grade≥3 treatment emergent adverse events (TEAEs) included anemia, thrombocytopenia, neutropenia and nausea. Most events were manageable with appropriate supportive care or dose modification. Four pts (40%) experienced TESAEs, including anemia, pneumonia, neutropenia, and upper gastrointestinal hemorrhage. There were no TEAEs leading to treatment discontinuation or death.

Summary/Conclusion: Sd was able to induce an encouraging response with a manageable safety profile for a group of Chinese RRMM patients desperately needing treatment after failing CAR-T therapy. This suggests that selinexor is a highly potent anti-MM therapy and further investigation is warranted, including using selinexor in combination with other anti-MM therapies in earlier lines of treatment.

PB2000: LESSONS LEARNED FROM CERBA RESEARCH CHIMERIC ANTIGEN RECEPTOR T-CELL (CAR T) MULTIPLE MYELOMA RUNNING TRIALS

R. Gaspo 1,* , N. Selliah 1 , F. Clottens 1 , I. Modi 1 , J. Sallette 1

1 Science, Cerba Research, Ghent, Belgium

Background: The pipeline has 359 liquid tumor trials that are CAR T driven where Cerba Research (CR) understands that there is a demand to provide innovative specialty testing, such as flow cytometry, sequencing and downstream biomarker analysis. Herein, CR examined 38 of our active multiple myeloma (MM) trials since 2016 with a focus on CAR T therapy. We identified 15 of those trials where CR participated as a central laboratory.

Aims: The aim of this evaluation is to identify gaps within current CAR T MM trial endpoints and to improve for the future of our liquid tumor trials.

Methods: We analyzed internal trial matrix database and examined other documents such as laboratory specification documents, trial design, protocol, and schedule of activities. We used descriptive statistics to illustrate our specialty testing involvement and to identify gaps in our offerings.

Results: CR currently has 38 running MM trials, in which 15 (39%) have a CAR T therapy. Sixteen (42%) of our MM trials are phase I/first-in-human (FIH), whereas 14 (37%) and 7 (18%) are phase II and III, respectively. Within our MM CAR T trials, we perform flow cytometry (FCM) in 87% of cases. Our involvement in FCM is exploratory in nature, and the design of the panels is performed with continued scientific discussions with sponsors. Some of the flow cytometry panels performed in our CAR T trials are:

- Enumeration of CAR T cells in whole blood and bone marrow aspirates, including PK evaluation

- Immunophenotyping of CAR T cells (memory, regulatory, activation status, exhaustion status)

- Minimal residual disease (MRD)

CR is often involved across full CAR T programs starting at FIH up to registration trials. We have robust upstream capabilities where we perform peripheral blood mononuclear cell (PBMC) isolation (60%), bone marrow mononuclear cell (BMMC) isolation (53%), DNA extraction (47%), cell sorting (53%), serum and urine protein electrophoresis/immunofixation (47%), and histopathology (decalcification, embedding, sectioning) (53%). We are also involved in routine testing such as hematology (27%), biochemistry (33%) and coagulation (27%). However, except for FCM (87%), immunohistochemistry (IHC 47%) and FISH (47%), we identified a gap in requests for sequencing within MM CAR T trials (0%.) Next generation sequencing (NGS), which was mostly for MRD assessment, was performed by referral labs. We also identified up to 11 referral labs participating in MM trials which significantly adds to a trial operation complexity.

Summary/Conclusion: CR currently has strong FCM fit-for-purpose design and execution capabilities as demonstrated by our participation in CAR T programs as a central laboratory. Using FCM in CAR T trials is important as it can aid in determining effectiveness of therapy, any potential side effects, and general health of the immune system by performing longitudinal monitoring of the immune system. We also frequently isolate PBMCs/BMMCs, extract DNA and perform histopathology on a global scale for clinical trial purposes. Herein, we determined that we have good downstream efforts with FCM, IHC and FISH and plan on growing NGS offerings by creating a new MRD NGS oncopanel and compare it with FCM for validation and concordance in multiple myeloma.

PB2001: A LOOK INTO THE REAL WORLD: PICTURE OF NEWLY DIAGNOSED MULTIPLE MYELOMA TRANSPLANT INELIGIBLE TREATED PATIENTS IN THE FRONTLINE SETTING. CARINAE STUDY

F. de Arriba de la Fuente 1,* , M. T. Hernández Garcia 2 , J. A. Soler Campos 3 , H. R. Susana 4 , M. J. Moreno Belmonte 5 , M. González Pardo 6 , M. Gironella Mesa 7 , M. Casanova Espinosa 8

1 Hematology, Hospital General Universitario Morales Meseguer, Murcia; 2 Hematology, Complejo Hospitalario Universitario de Canarias, Santa Cruz de Tenerife; 3 Hematology, Consorci Corporació Sanitària Parc Taulí, Sabadell; 4 Hematology, Hospital Universitario Basurto, Bizcaia; 5 Hematology, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia; 6 Medical, Medical Department, Janssen-Cilag, Madrid; 7 Hematology, Hospital Universitari Vall d’Hebron, Barcelona; 8 Hematology, Hospital Costa del Sol, Málaga, Spain

Background: Multiple myeloma (MM) is the second most common blood cancer, with an estimated 5-year prevalence of nearly 230,000 cases worldwide. MM is most frequently diagnosed among people >65 years of age and the median age at onset in Europe is 72 years.Despite the significant proportion of older patients with newly diagnosed multiple myeloma (NDMM), most clinical trials (CT) driving therapeutic decisions in routine practice include younger and presumably healthier patients than those in the real-world. Furthermore, longitudinal studies transplant-ineligible (TNE) elderly patients with MM showed limited data describing the benefit from new anti-MM agents.

Aims: To try to respond to this need in the real practice of MM treatment, we share for the first time a new observational study called Carinae, that describes the clinical practice in TNE-NDMM in Spain. These results aim to help clinicians in optimizing treatment approaches for patients with MM.

Methods: Observational, ambispective, descriptive study with 110 TNE-NDMM patients who started antineoplastic treatment within daily clinical practice. To describe the efficacy and safety of monoclonal antibodies (mAb) in daily clinical practice, the results collected in two patient groups before and after the availability of the DVMP, the first and only mAb approved for the treatment of TNE-NDMM in Spain. Group A: N =53 starts treatment between Sep/01/2018 and Aug/31/19 with a combination of ≥2 drugs. Group B: N =57 starts treatment between Sep/01/19 and Nov/30/20 with daratumumab in combination with bortezomib, melphalan and prednisone (daratumumab-VMP). This analysis shows a representative population of the patients in real clinical practice in Spain and analyzes the similarities and differences in the efficacy and safety of the treatments under study (with and without monoclonal antibody), for the first months. Since the follow-up of the patients has not yet been completed, deeper analysis will be presented in the future.

Results: 110 NTE MM patients have been registered in Carinae study. Table 1 describes demographic and multiple myeloma at diagnosis characteristics. Updated data on effectiveness and tolerability will be presented during the Congress.

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Summary/Conclusion: New treatments have been incorporated into the therapeutic scenario of TNE-NDMM in the last years. For the first time, a large real-world evidence study describes their incorporation in clinical practice in Spain. Our aim through the Carinae study is to provide meaningful insights about standard of care regimens with monoclonal antibodies as DVMP for TNE-NDMM patients in the real-world practice in Spain.

PB2002: OBSERVATIONAL PROSPECTIVE STUDY TO INVESTIGATE THE RELATIONSHIP BETWEEN DURATION OF TREATMENT AND RESPONSE IN PATIENTS WITH MULTIPLE MYELOMA OR AL AMYLOIDOSIS: THE DORIANT STUDY (NCT: 04659798)

M. Grande 1,2 , A. Naves 1 , J. Aparicio 1 , M. Gironella 3,*

1 Medical, Takeda Farmacéutica España, Madrid; 2 Universidad de Alcalá, Alcalá de Henares, Madrid; 3 Medical, Vall d’Hebron, Barcelona, Spain

Background: The introduction of new drugs with a lower toxicity profile and the increasing evidence regarding the benefits of continuous over fixed-duration therapy have progressively shifted the therapeutic approach for patients with multiple myeloma (MM) from the traditional fixed duration strategy to continuous regimens. Patients with MM often discontinue treatment in clinical practice before the foreseen time. Treatment duration may depend on multiple factors, including patient’s baseline characteristics, available drugs, treatment response, progression, or toxicity. Despite the rising evidence regarding the influence of treatment duration on therapeutic outcomes, the determinants of treatment duration and its relationship with survival in real-life practice have been little investigated, including reasons for treatment discontinuation. Moreover, this research on the influence of treatment duration and outcomes such as survival has mainly focused on MM, while it has hardly been investigated in monoclonal gammopathies treated with similar therapeutic approaches, such as systemic AL amyloidosis.

Aims: To investigate the relationship between duration of treatment according to selected treatment strategy (fixed vs. continuous) and response to treatment among patients diagnosed with MM and/or AL amyloidosis. We provide an update regarding the characteristics of the population currently enrolled.

Methods: Prospective, non-interventional, multi-center study in real-life practice in 14 Spanish hospitals. The study included subjects aged 18 or older diagnosed with MM and/or AL amyloidosis who started treatment up to 12 months before the inclusion. The study is planning to enroll 250 patients (220 with MM and 30 with AL amyloidosis).

Results: Of the 181 patients enrolled, 164 (90.6%) were included in the analysis; 145 (88.4%) of them were diagnosed with MM, 11 (6.7%) with AL amyloidosis, and 8 (4.9%) with both diseases. The median age was 74.0 (68.0-79.0) years for MM patients, 76.0 (70.0- 83.0) years for AL amyloidosis patients, and 67.5 (56.5- 70.5) years for patients with both diseases.

Among patients diagnosed with MM therapy strategy data (n=116), 32 (27.6%) received fixed-duration therapy, and 84 (72.4%), continuous therapy. Among patients diagnosed with AL with therapy strategy data (n=11), 1 (9.1%) received fixed-duration therapy and 10 (90.9%) continuous therapy. Of the 7 patients with both diseases and therapeutic strategy, 4 (57.1%) received fixed-duration therapy, and 3 (42.9%), continuous therapy. Of the patients diagnosed with MM (n=116), 62 (53.4%) were on first line (1L), 30 (25.9%) on second line (2L), 16 (13.8%) on third line (3L), and 8 (6.9%) on fourth line (4L). Among 11 patients diagnosed with AL amyloidosis, 6 (54.5%) were on 1L and 5 (45.5%) on 2L, and over 8 patients with both diseases, 3 (37.5%) were on 1L, 3 (37.5%) on 2L, 1 (12.5%) on 3L, and 1 (12.5%) on 4L. Over 94 MM patients with treatment type data, 14 (14,9%) received consolidation and/or maintenance treatment. Of these patients (n=14), 1 (7.1%) received consolidation; 9 (64.3%), maintenance, and 4 (28.6%), consolidation and maintenance. No patients with AL amyloidosis received consolidation or maintenance treatment.

Summary/Conclusion: In the patients analyzed to date, continuous treatment was the most common therapeutic strategy, regardless of the disease. This study will provide insights into determinants of treatment duration and its relationship with survival in real-life practice, which might help clinicians manage MM and AL amyloidosis.

PB2003: REAL-WORLD DURATION OF USE AND DOSING FREQUENCY OF DARATUMUMAB IN PATIENTS WITH MULTIPLE MYELOMA IN THE U.S.

R. Fonseca 1,* , E. Chinaeke 2 , N. Gupta 2 , A. Fu 2 , T. Ran 2 , S. Kaila 2

1 Mayo Clinic, Phoenix; 2 Janssen Scientific Affairs, LLC, Horsham, United States of America

Background: Daratumumab (DARA) is an anti-CD38 monoclonal antibody approved for treatment of patients with newly diagnosed and previously treated multiple myeloma (MM). Hence, it is possible that patients initiating DARA may continue to use it as backbone of their treatment even if their line of therapy (LOT) or treatment regimen changes, sometimes with interruptions.

Aims: This study examines the continuous duration of DARA treatment and dosing frequency across all LOTs using real-world data from MM patients in the U.S.

Methods: This is a retrospective observational study conducted using Optum Clinformatics Data Mart database consisting of some Medicare and commercially insured MM patients utilizing DARA, between 11/1/2015-3/31/2021. Duration of DARA use was defined as the time interval between first initiation and discontinuation of DARA spanning multiple LOTs as a time-to-event outcome using Kaplan-Meier method. A gap of >60 days between two consequent DARA claim dates was defined as DARA discontinuation. Dosing frequency was calculated as the average number of DARA doses during defined time periods. Compliance ratios were calculated as observed dosing frequency divided by the dosing frequency for FDA approved regimens (DARA-Lenalidomide-Dexamethasone [DRd] and DARA-Pomalidomide-Dexamethasone [DPd]). All results were calculated for patients using DARA across multiple LOTs regardless of treatment regimen.

Results: A total of 2125 patients initiating DARA therapy were included in this analysis with mean age (SD) of 70.9 (9.8) years, 51.3% males, and 64.8% white. The median length of DARA use spanning multiple LOTs, was 16.6 months. During the first year of DARA use, 90.5%, 83.2%, 72.6% and 63.5% of the patients continued DARA at 3, 6, 9, and 12 months, respectively. At years 2 and 3 of DARA use, 33.1% and 14.5% of the patients, respectively, continued DARA treatment (Figure 1). The mean dosing frequency in real world was similar to the dosing frequency on the approved label (Table 1). The compliance ratio was 0.98 over the period of 36 months.

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Summary/Conclusion: Patients initiating DARA remain on the therapy for a median of 16.6 months with one-sixth continuing DARA beyond 3 years. The dosing frequency observed in the real-world was consistent with the approved label.

PB2004: BCMA-DIRECTED THERAPY FOR MULTIPLE MYELOMA: EFFECTIVENESS OF ONLINE EDUCATION IN IMPROVING KNOWLEDGE AND COMPETENCE IN PRACTICAL APPLICATION

V. Harvey-Jones 1,* , S. Koekkoek 1 , J. Vandenbroucque 1 , M.-V. Mateos 2

1 Medscape, Global Education, London, United Kingdom; 2 University Hospital of Salamanca, Salamanca, Spain

Background: With BCMA-targeted therapies becoming available for multiple myeloma, it is important that physicians are knowledgeable and competent in how to use these novel agents. Furthermore, physicians need to gain confidence in understanding and managing associated adverse events (AEs) to optimize patient risk-benefit and individualize therapy.

Aims: To determine whether online continuing medical education (CME) could improve the knowledge, competence and confidence of clinicians regarding clinical aspects of BCMA-directed therapies.

Methods: Two 30-minute online video panel discussions with synchronized slides were launched for physicians outside the USA during 2021 to a target audience of hematologist/oncologists (hem/oncs) and ophthalmologists. Educational effect was assessed for each activity using a repeated-pair design with pre-/post-assessment. 3 multiple choice questions (Qs) assessed knowledge/competence, and 1Q rated on a Likert-type scale assessed confidence. A paired samples t-test was conducted for significance testing on overall average number of correct responses and for confidence rating, and a McNemar’s test was conducted at the Q or learning objective level (5% significance level, P <.05). Cohen’s d with correction for paired samples estimated the effect size of the education on number of correct responses (<.20 modest, .20-.49 small, .59-.79 moderate, ≥.80 large). Data were subsequently combined and analyzed to provide a summative overview of the effect of the education across the combined knowledge-, competence- and confidence-based Qs from the two programs. Data were collected from April–July 2021.

Results: Between 47–76 hem/oncs (N=123) and 58–176 ophthalmologists (N=234) completed pre- and post-activity Qs. A statistically significant effect was seen for hem/oncs across both activities (P<0.05–P<0.001) with improvements in knowledge/competence ranging from 34–37%. Furthermore, there was an overall improvement in confidence, ranging from 30–57%. The education had less impact on ophthalmologists; while some improvements were seen in single activities, in the summative overview the greatest improvement was seen in knowledge and competence in managing ocular toxicities. The summative overview showed that hem/oncs had significant improvements in knowledge of trial data with BCMA-directed therapies (average correct response rate: 24% pre- vs 43% post-CME; P<0.05) and competence in managing BCMA-therapy associated AEs (17% pre- vs 36% post-CME; P<0.01). Relative changes of 9% (NS) and 13% (NS) were observed in knowledge of AE management and competence in treatment initiation, respectively. Ophthalmologists showed significant improvements in knowledge of AE management (21% pre- vs 52% post-CME; P<0.001) and competence in AE management (67% pre- vs 76% post-CME; P<0.001). Relative changes of 32% (NS) and -9% (NS) were observed in knowledge of trial data and competence in treatment initiation, respectively. In combined self-assessed confidence Qs, 46% of hem/oncs increased in and 51% maintained confidence. 50% of ophthalmologists increased in and 46% maintained confidence. All confidence changes were significant (P<0.01/ P<0.001). In the single activities, ~50% of participants stated they would modify treatment plans and ~80% were committed to those changes.

Summary/Conclusion: These two educational activities resulted in significant educational impact, particularly for hem/oncs. Online CME is valuable in improving knowledge, competence and confidence, as well as identifying areas of future educational need and potentially improving patient outcomes.

PB2005: BORTEZOMIB, LENALIDOMIDE AND DEXAMETHASONE IN NEWLY DIAGNOSED MULTIPLE MYELOMA PATIENTS: A REAL-WORLD STUDY OF CHINA

X. Jingyu 1,* , Y. Wenqiang 1 , F. Huishou 1 , L. Jiahui 1 , L. Lingna 1 , Q. Lugui 1 , A. Gang 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Blood Diseases Hospital & Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Multiple myeloma (MM) is a hematological malignancy characterized by the malignant proliferation of monoclonal plasma cells. In recent years, a series of new drugs such as proteasome inhibitors, immunomodulatory drugs and autologous hematopoietic stem cell transplantation (ASCT) have been widely used in MM patients and have prolonged the survival of them. Nowadays Bortezomib/lenalidomide/dexamethasone (VRD) is considered as the first-line induction therapy for newly diagnosed multiple myeloma (NDMM) in most clinical guidelines. However the data of VRD in China is insufficient.

Aims: To evaluate the efficacy of VRD in NDMM patients as well as the effect of the regimen on the stem cell collection and on the long-term prognosis in the real world.

Methods: The clinical characteristics, survival rates, response rates and minimal residual disease (MRD) of 87 NDMM patients at Institute of Hematology & Blood Diseases Hospital from January 1, 2013 to January 1, 2020 were retrospectively analyzed and the impact of lenalidomide on stem cell collection was evaluated. Response was evaluated after 2 courses of induction therapy, before ASCT and at day100 after ASCT. The best response was defined as the deepest response during the follow-up. MRD was evaluated by multiparameter flow cytometry (MFC) with 10 -4 sensitivity.

Results: The median age of the patients was 56 years old (36-78) and males and females accounted for 58.6% and 41.4%, respectively. The overall response rate (ORR) was 95.9% after 2 courses of induction therapy, with 13.5% achieving the deep response (complete response (CR) or better) and 51.3% of patients achieving a very good partial response (VGPR) or better. After 4 courses of induction therapy, the ORR achieved 95.2%, and the proportions of the deep response and VGPR or better grew up to 46.0% and 77.7%. According to the treatment, the patients were divided into transplantation group and non-transplantation group. After the induction therapy, 88.8% of patients in the transplantation group achieved VGPR or better, and 55.5% reached the deep response. After the transplantation, the proportion increased to 97.2% ( P =0.174) and 77.2% ( P =0.055), respectively, with the rate of undetectable MRD increasing from 44.4% to 77.8% ( P =0.004). In the non-transplantation group, 78.9% of patients achieved VGPR or better after 4 courses of induction therapy, 44.7% of the patients achieved deep response and the rate of undetectable MRD was 42.4%. Compared with the non-transplantation group, transplantation was associated with a higher rate of response ( P ＜0.001) and a lower rate of MRD detection(78.4% vs 57.1%, P =0.053). The median follow-up time of all patients was 26.3 months (6.2-61.4). The median PFS and OS were not reached. The three-year PFS and OS rates were 82.1% and 87.0%, respectively. None of the standard-risk group, the high-risk group, the transplantation group and non-transplantation group achieved the median PFS and OS. The number of CD34 + positive cells tended to decrease after more than 4 courses of VRD induction therapies.

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Summary/Conclusion: VRD regimen is highly effective and results in a substantial survival benefit. VRD followed by ASCT is associated with higher rate of deep response, lower rate of undetectable MRD and longer survival.

PB2006: CHARACTERISTICS AND OUTCOMES OF PATIENTS WITH PLASMA CELL LEUKEMIA (PCL): A SINGLE-CENTER STUDY FROM TUNISIA

Y. Fakhfakh 1 , F. Kammoun 1,* , O. Kassar 1 , F. Kallel 1 , M. Chaari 2 , N. Louati 3 , L. Torjeman 4 , M. Charfi 1 , I. Frikha 1 , I. Ben Amor 1 , L. Khalifa 1 , M. Medhafar 1 , S. Hadiji 1 , H. Elleuch 2 , H. Mnif 3 , T. Ben Othman 4 , M. Elloumi 1

1 Clinical Hematology Depatment; 2 Biological Hematology Department, Hedi Chaker Hospital; 3 Flow Cytometry Unit, Regional Blood Transfusion Center, Sfax; 4 Autograft Department, National Bone Marrow Transplant Center, Tunis, Tunisia

Background: Plasma cell leukemia (PCL) is a rare form of leukemia and plasma cell dyscrasia defined by the presence of greater than 2×10 9 /L peripheral blood plasma cells or plasmacytosis accounting for more than 20 % of the differential white cell count. PCL can be divided into primary PCL (PCL) and secondary PCL (sPCL) following previously diagnosed multiple myeloma (MM). It has an aggressive clinical course.

Aims: In this study, we aim to identify the main clinical and laboratory disease findings, treatment patterns, and evolution in patients with PCL in a Tunisian center.

Methods: Our study is a review of all cases of PCL, diagnosed and treated between 2000 and 2021 at the hematology department of the hospital of Sfax, Tunisia.

Results: Nine cases of PCL were identified: 8 primary PCL cases, and only one sPCL case. The mean age was 53 years [range 31-64], and there was a female predominance (6:3). Most presenting symptoms were bone pain noted in 5/9 patients, asthenia in 3/9 patients, and bleeding was noted in only one case. Physical examination revealed extramedullary involvement in 4 patients: 2 had splenomegaly, one had hepatomegaly and one had lymphadenopathy. None of our cases showed skin infiltrates. Standard radiologic surveys showed bone osteolysis in all 9 patients, generally in the skull, and MRI showed epidural space infiltration and spinal cord compression in 2 patients. Normocytic, normochromic anemia was noted in almost all patients with mean hemoglobin of 6.9 g/dl. Rouleaux formation and a leukoerythroblastic picture were evident on almost all the peripheral blood smears. The mean leukocytosis was 15.3x10 9 /l [range 3.4-100] and consisted of 20% to 88% of plasma cells. Thrombocytopenia with platelets < 100x10 9 /l occurred in 50% of patients. Immunophenotyping by flow cytometry was done only in 2 cases with difficult morphology and showed expression of CD38 and CD138. Hypercalcemia was noted in almost all patients (8/9), and renal failure was present in 4 cases. Immunofixation revealed an even distribution between IgG and IgA, and light chain in 3 cases. The frequency of the Kappa chain was higher. Serum albumin levels were low in all patients. For treatment, 4 patients received VTD (Bortezomib, Thalidomide, and Dexamethasone), 2 received VAD (Vincristine, Adriamycin, and Dexamethasone), 2 were on Thalidomide Dexamethasone, and one patient received Revlemid Dexamethasone. Only one patient of these four patients who received VTD underwent autologous stem cell transplant (ASCT) after attaining CR but relapsed after 2 years into multiple myeloma and is currently in PR receiving MPT. All other patients died of progressive and refractory disease. The median survival was 9 months with a duration ranging from 2 to 63 months.

Summary/Conclusion: As reported internationally, our study showed that patients with PCL were younger than myeloma patients, and presented with more aggressive clinical and laboratory features at diagnosis especially more bone lesions than classically described, and a high frequency of hypercalcemia. Besides, it indicated that even after the use of novel drugs, response to treatment is poor and PCL still has a poor prognosis.

PB2007: FACTORS ASSOCIATED WITH EARLY RELAPSE IN MYELOMA PATIENTS AFTER AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION

A. Karatas 1,* , B. Yavuz 1 , E. Erdogan Yucel 1 , M. Kakci 1 , G. H. Ozsan 1 , I. Alacacioglu 1 , F. Demirkan 1

1 Hematology, Dokuz Eylul University, IZMIR, Turkey

Background: Multiple myeloma (MM) is a clonal disease characterized by a neoplastic proliferation of plasma cells that make up approximately 10% of hematologic malignancies. Autologous hematopoietic stem cell transplantation (ASCT) after an induction regimen using combination of immunomodulatory drugs, proteasome inhibitors and dexamethasone is considered the standard treatment of newly diagnosed multiple myeloma in eligible patients.

Aims: To demonstrate the pre-transplant factors associated with early relapse in MM patients undergoing ASCT.

Methods: 27 adult MM patients who underwent ASCT at The Stem Cell Transplant Center of Dokuz Eylul University Hospital between October 2011 and August 2021 and who relapsed within 18 months of transplantation were included in this study. The data were compiled from the Dokuz Eylul Hematology archive and the Hospital Information Management System at Dokuz Eylul University Hospital and analyzed with SPSS v.24 (Statistical Package for Social Sciences). Mann-Whitney U test is used to compare kappa/lambda ratios, Kaplan Meier test is used to analyze survival and log-rank test is used to test significant difference between survival of two groups.

Results: The median age at diagnosis is 57 (42-68) and 52% of patients were male (n=14). Patients received a median one line of treatment before ASCT (1-3). According to the ISS staging system, 9 patients were stage I, 9 patients were stage II and 9 patients were stage III. In 43% of patients (n=12), extramedullary (EM) disease involvement was present at the diagnosis. 33% had hypercalcemia (n=9), 19% had kidney involvement (n=5), 57% had anemia (n=16), and 75% had bone (n=21) at the time of diagnosis.

Two patients achieved complete remission (CR) after 2 courses of induction therapy. But they lost this response pre-ASCT and they relapsed in 5 th and 6 th month after ASCT respectively. The median time to relapse post-ASCT was 6 (2-17) months. In 63% of these patients, clinical relapse was observed, treatment was started due to aggressive biochemical relapse in the rest. 25% (n=7) had EM disease involvement in relapse. In this patient group, the median survival after ASCT is 32 months (5-120). In 3 out of 10 patients with biochemical relapse, EM disease was not observed despite being present at the time of diagnosis. In 9 of the 17 patients with clinical relapse and EM disease at diagnosis, 7 patients relapsed with EM disease. Median Kappa/Lambda ratios at the time of diagnosis were 13.84 and 13.1 at the time of relapse. There was no statistically significant difference in kappa/lambda rates at the time of diagnosis and relapse of patients with biochemical and clinical relapse. Median progression-free survival (PFS) was 5 months in those with clinical relapse and 8 months in patients with biochemical relapse. Median overall survival (OS) was 19.2 months in those with clinical relapse and it was not reached in those with biochemical relapse (p=0.001) (Fig. 1).

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Summary/Conclusion: It is known that in multiple myeloma, ASCT prolongs PFS. Early loss of response in patients who achieved CR after 2 courses of treatment may be a prognostic clue. OS and PFS are shorter in those with clinical relapses while biochemical relapses are more indolent. The fact that 43% of our patients have EM disease involvement in diagnosis indicates that this feature may be associated with early relapse. Outcomes of single and tandem ASCT in these patients should be examined in further studies.

PB2008: CUTANEOUS HEMORRHAGIC SYNDROME IN PATIENTS WITH AL-AMYLOIDOSIS

V. Khyshova 1,* , I. Rekhtina 1 , N. Zozulia 1 , V. Dvirnyk 1 , L. Mendeleeva 1

1 National Research Center for Hematology, Ministry of Health of Russia, Moscow, Russia

Background: Hemorrhages on the skin and mucosa are seen in 30-40% of patients with systemic AL amyloidosis (AL-A). The pathogenesis and clinical significance of this syndrome are poorly understood.

Aims: To analyze the relationship of cutaneous and mucous hemorrhagic syndrome in AL-A patients with the hemostatic system parameters and the leading clinical syndromes.

Methods: A prospective study included 49 patients with newly diagnosed systemic AL-A. 18 (37%) patients were diagnosed with stage I, 16 (33%) with stage II, and 6 (12%) and 9 (18%) with stage III and stage IV, respectively (Mayo 2004/Europian). Activated partial thromboplastin time, thrombin time, fibrinogen concentration, XIIa-dependent fibrinolysis time, antithrombin III (ATIII), D-dimer, factors VIII (FVIII), X (FX) and Von Willebrand factor (VWF) activity were determined before therapy. Patients with cutaneous hemorrhagic syndrome were included in the Group №1, and those without cutaneous hemorrhages in the Group №2.

Results: Cutaneous and mucosal hemorrhagic syndrome was observed in 23 (47%) patients. Hypercoagulable syndrome (increase of fibrinogen, D-dimer, FVIII, VWF, and decrease of ATIII) was registered in 18 (78%) patients, significantly more frequently than in Group №2 patients (11 patients - 42%; p=0,01). Eight (35%) patients with skin hemorrhages developed venous and arterial thrombosis before treatment, which were absent in the control group (p=0.002). Nephrotic syndrome was present in 19 (82%) group №1 patients, nephrotic syndrome was 2 times less frequent in group №2 (10 (38%) patients, p=0,001). Patients with hemorrhages had higher urinary protein loss (5.7 g/day vs 2.4 g/day; p=0.03) and lower serum albumin values (23 g/l vs 33 g/l; p=0.001). In patients from the first group, the development of hydropericardium, hydrothorax and anasarca was more often observed (p=0.02).

We didn’t find significant correlation between cutaneous hemorrhagic syndrome and involvement of other organs, content of free light chains and cardiac markers.

Summary/Conclusion: Cutaneous hemorrhagic syndrome is a clinical manifestation of hypercoagulable disorders in the hemostasis system in AL-A patients and associated with a high frequency of thrombosis development. Skin hemorrhages were more often associated with severe nephrotic syndrome, which may indicate a single pathogenetic mechanism in the form of small vessel lesions.

PB2009: OPPORTUNITIES OF THERAPY IN PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA WITH DIALYSIS-DEPENDENT RENAL IMPAIRMENT

I. Kliuchagina 1,* , A. Semenova 2 , E. Gromova 2 , L. Kuznetsova 2 , O. Kolomeytsev 2 , G. Tumyan 2 , D. Osmanov 2

1 Sechenov First Moscow State Medical University (Sechenov University); 2 Federal State Budgetary Institution «N.N. Blokhin National Medical Research Center of Oncology» оf the Ministry of Health of the Russian Federation (N.N. Blokhin NMRCO), Moscow, Russia

Background: Renal impairment (RI) is one of the most frequent complications of multiple myeloma (MM). At the diagnosis time RI is detected in 20-40% of patients, in 2-4% of patients with newly diagnosed MM develop dialysis-dependent RI, which is associated with early mortality and a high complications’ incidence.

Aims: Compare PFS and OS in patients with newly diagnosed MM with dialysis-dependent renal impairment depending on the achievement of renal response and autoHSCT.

Methods: The study included 39 patients with newly diagnosed MM III B stage (Durie-Salmon) with dialysis-dependent RI for the period from 2000 to 2020. 24 (61.5%) of patients were male, the median age is 58 years (range 36 to 76). The diagnosis was established in accordance with the criteria of IMWG-2014. Cytogenetic examination was performed in 17 (43.6%) patients. 4 (10.2%) patients have high–risk translocation t (4;14). At the disease onset the median creatinine level was 631 mkmol/L (range 448 to 2138), median CKD-EPI was 7 ml/min/1.73 m2 (ranged 2 to 10 ml/min/1.73 m2). The following programs were used as induction therapy: CyBorD - 10 (25.6%), VCP - 6 (15.4%), CP – 14 (35.9%) VAD – 7 (17.9%), VMCP – 2 (5.1%). Bortezomib-containing regimens were received by 16 patients (41%). 30 (77%) patients were the candidates for autoHSCT. AutoHSCT was performed in 8 (20.5%) patients, in 3 (7.7%) cases - tandem autoHSCT. An antitumor response was evaluated after 3-4 courses of induction therapy and on the 100th day after autoHSCT. The results of the study were subjected to statistical processing using the Statistica program (version 10.0). The survival analysis was carried out using the Kaplan–Meier method.

Results: 25 patients (64.1%) during induction therapy achieved renal response: complete response - 1 (2.6%) patient, partial response - 10 (25.6%) patients, minimal response -14 (35.9%) patients. 14 (35.9%) patients didn’t have any response. The achievement of any renal response was associated with an improvement 3-year PFS 61.1% (95%CI 43.7, 76.1) vs. 18.9% (95% CI 11.8, 23,9) (p = 0.045), 3-year OS 72.2% (95%CI 58.2, 86,1) vs. 38.1% CI (27.6, 48,2) (p = 0.049) respectively.

3-year PFS in patients who underwent autoHSCT was 48,6% (CI 35.8, 67,2), 3-year OS - 67,2% (CI 46.8, 78.9) to compare with patients who were the candidates for autoHSCT, but didn’t undergo autoHSCT: 3-year PFS 17,9% (CI 9.7, 31,2), 3-year OS – 33,7% (CI 16.7, 57.3) (p=0,036). There wasn’t transplantation related mortality.

Summary/Conclusion: Achieving at least a minimal renal response as well as performing autoHSCT improve the PFS and OS in patients with newly diagnosed MM with dialysis-dependent RI.

PB2010: RETROSPECTIVE STUDY OF RELAPSED/REFRACTORY MYELOMA PATIENTS FROM AN INDIAN TERTIARY CENTRE: RESPONSE MATTERS!!

R. R. Lavu 1,* , L. Kumar 1 , S. Konda 2 , R. M. Velagapudi 3

1 Medical Oncology, All India Institute of Medical Sciences, Delhi, India; 2 Department of Epidemiology and Biostatistics, University of Illinois, Chicago, United States of America; 3 Internal Medicine, Pandit Madan Mohan Malviya Hospital, Delhi, India

Background: Treatment paradigm of relapsed multiple myeloma (MM) has changed in the last decade. We report treatment outcomes among relapsed MM patients in the era of proteasome inhibitors (PI) and newer immunomodulatory drugs (IMiDs) in an Indian cohort.

Aims: The primary objective was to determine the progression free survival (PFS) of relapsed patients. Secondary objectives include complete response rates (CR) & objective response rates (ORR), (≥ partial response (PR)).

Methods: The study is a retrospective review of relapsed MM patients treated at a North Indian tertiary cancer referral center between January 2014 to December 2019 and were subsequently followed till October 2021 for outcomes. Patients who relapsed after atleast one prior line of therapy were included. Records of 300 patients were screened and 195 (65%) patients were included in the study. 105(35%) patients with incomplete treatment details and significant missing data were excluded.

Results: Median age was 58 years (range; 51 to 65 years). 108 (55.4%) patients had an IgG subtype. High risk features observed were renal failure in 16% patients, extramedullary disease in 9% patients. Bortezomib based triplet in 27.6% of patients followed by combination of bortezomib with IMiD based triplet in 25.1% patients followed by pomalidomide based regimen (without PI) in 15.3% and doublet regimen were used in 15.3%. Responses were evaluable in 176 patients. ORR in the entire cohort was seen in 98/176 (55.6%) patients. Among the individual regimens, it was 66% with bortezomib based triplet, 58% with bortezomib & IMiD based regimen. Complete response (CR) was seen in 19/176 (10.8%) patients. After a median follow up of 11.5 months (range; 5.5-25.6 months), 143 (73.3%) patients progressed. Median PFS of the entire cohort was 8 months (range, 0-55.0 months). Median PFS did not differ by type of salvage treatment used (p-value= 0.7). Multivariate cox proportion analysis suggested that patients achieving CR had better PFS (median 31.2 months [95% CI 10.1-55.9] vs 6·9 months [1.61, 36.2]; hazard ratio (HR) 3.07 [95% CI 1.34,7.01], two-sided p=0·005). Similarly achieving a response 5.09 v 11.4 months, ([HR], 95% CI; 15.1 (5.59, 41.0), p value <0.001). Median OS was not reached during the study duration. Toxicities were managable with serious adverse events noted in 2% of patients.

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Summary/Conclusion: In relapsed myeloma patients we suggest that depth of response, and acheiving a CR irrespective of drugs used in salvage regimen predict a better PFS. To date this is the largest retrospective series from an Indian center. However, these findings require validation by a randomized control trial.

PB2011: CHIDAMIDE COMBINED WITH THALIDOMIDE, CYCLOPHOSPHAMIDE AND DEXAMETHASONE (TC2D) AS AN ORAL QUADRUPLET REGIMEN FOR RELAPSED/REFRACTORY MYELOMA PATIENTS: INITIAL RESULTS OF A PHASE IIA, MULTICENTER TRIAL

Y. Li 1 , J. He 1 , X. Han 1 , G. Zheng 1 , E. Zhang 1 , Q. Chen 1 , Y. Yang 1 , D. He 1 , Y. Zhao 1 , J. Fu 2 , L. Shou 3 , W. Jiang 4 , W. Jiang 5 , S. Qian 6 , H. Hu 7 , H. He 1 , Z. Cai 1,*

1 Bone Marrow Transplantation Center, The First Affiliated Hospital,School of Medicine, Zhejiang University, Hangzhou; 2 Hematology, Shaoxing People’s Hospital, Shaoxing; 3 hematology, Huzhou Central Hospital, Huzhou; 4 Hematology, Taizhou First People’s Hospital, Taizhou; 5 Hematology, Shaoxing Shangyu People’s Hospital, Shaoxing; 6 Hematology, Affiliated Hangzhou First People’s Hospital, Zhejiang University school of Medicine, Hangzhou, Hangzhou; 7 Hematology, Jinhua Municipal Central Hospital Medical Group, Jinhua, China

Background: Research has showed histone deacetylase (HDAC) is overexpressed in plasma cells. Panobinostat was the first HDAC inhibitor (HDACi) approved by FDA for patients with relapse and refractory multiple myeloma (RRMM) in 2015. Chidamide is an oral subtype-selective HDACi, independently developed in China and approved for peripheral T-cell lymphoma. Preclinical findings have demonstrated the anti-myeloma activities of chidamide in vitro and in vivo.

Aims: Here we report the initial efficacy and safety of this prospective, phase IIa, multicenter clinical trial with the chidamide based oral quadruplet regimen in patients with RRMM (ChiCTR2000035100).

Methods: The TC2D regimen (thalidomide 100mg on days 1-28, chidamide 5mg on days 1-24, cyclophosphamide 50mg on days 1-24, dexamethasone 20mg on days 1,8,15,22) was administered to patients with RRMM of a 28-day cycle. Overall response rate (ORR) was based on patients whose response ≥MR and clinical beneficial rate (CBR) was calculated in patients completed at least 3 cycles and reached ≥SD.

Results: A total of 32 patients were enrolled as of June 30, 2021, with a median age of 63.5 years (range 49–74). 82% (19/23) of which had unfavorable cytogenetics (defined by the presence of del (17p) or p53 mutation, t (4:14), t (14:16), t (14:20), amp (1q)). 75% were double refractory to bortezomib and lenalidomide. 43% (14/32) were heavily pre-treated (>3 lines). 59% (19/32) of patients completed ≥ 3cycles at the cut-off date (Table 1).

With the median follow-up of 6 months (range 0.7–19.5), the median progression free survival (PFS) was 3.3 months and the 6-mon PFS rate was 27.5% (Fig 1a). Median overall survival (OS) has not reached yet, 6-mon survival rate was 82%, and the estimated 12-month survival rate was 65% (Fig 1b). Study end points were death (8 cases, 25%) and disease progression (24 cases, 75%). Patients who received ≥3 cycles of treatment had a superior median PFS (3.8 vs 1.4 months, P =0.01) than < 3 cycles. Of the 28 patients whose follow-up time more than 3 months, ORR was 25% and CBR was 46.4%, respectively. Better CBR (56.3% vs. 33.3%) and ORR (31.3% vs. 16.7%) were observed in patients received less than 3 prior lines, although with no significant statistical difference. Stratified analysis of PFS and OS showed no significant differences in sex, age, clinical classification, cytogenetic risk, prognosis score (DS and ISS staging system), and the status of ASCT.

Grade 3/4 adverse events were mainly hematological toxicity (neutropenia 28%, anemia 34%, thrombocytopenia 15.6%), which could be tolerated. Fatigue was reported in 68.8% (22/32) of the patients. The incidence of all-cause infection was 37.5% (12/32). There were no treatment related deaths observed.

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Summary/Conclusion: To our knowledge, this is the first report of chidamide-based oral quadruplet regimen for patients with RRMM. Preliminary results suggested efficacy and relatively high safety, and different groups of patients could benefit from this regimen. A longer treatment period may strengthen this effect. The most common adverse events were hematological toxicity which could be controlled. Choices of treatment are also influenced by physical and emotional impact of hospitalization or frequent hospital visits, which reflects the patient’s ability to continue their treatment and quality of life. Hospitalization and intravenous fluids are not involved in this regimen, which bring far more convenience and could be a cost-effective alternative for patients. Updated results will be presented at the following trial.

PB2012: A PHASE 2 MULTIARM STUDY OF MAGROLIMAB COMBINATIONS IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

B. Paul 1,* , M. Liedtke 2 , D. S. Siegel 3 , J. Khouri 4 , J. Hillenglass 5 , R. Rifkin 6 , M. D. Gandhi 7 , A. Kin 8 , M. Y. Levy 9 , A. Mohrbacher 10 , W. Schmidt 11 , R. Silbermann 12 , F. Cottini 13 , D. W. Sborov 14 , R. Walker 15 , M. Murphy 15 , L. Gu 15 , A. Chen 15 , N. Rajakumaraswamy 15 , S. Z. Usmani 16

1 Levine Cancer Institute, Charlotte; 2 Stanford Cancer Institute, Stanford; 3 John Theurer Cancer Center, Hackensack University Medical Center, Hackensack; 4 Cleveland Clinic, Cleveland; 5 Roswell Park Comprehensive Cancer Center, Buffalo; 6 The US Oncology Network, Denver; 7 Fairfax Office, Virginia Cancer Specialists, Fairfax; 8 Barbara Ann Karmanos Cancer Institute, Detroit; 9 Baylor University Medical Center, Dallas; 10 University of Southern California, Los Angeles; 11 Bend Memorial Clinic, Bend; 12 Oregon Health & Science University, Portland; 13 The Ohio State University Comprehensive Cancer Center, Columbus; 14 Huntsman Cancer Institute, University of Utah, Salt Lake City; 15 Gilead Sciences, Inc., Foster City; 16 Memorial Sloan Kettering Cancer Center, New York City, United States of America

Background: Magrolimab is a first-in-class humanized monoclonal antibody that blocks the immune checkpoint CD47, a “do not eat me” signal, overexpressed on tumor cells. Binding of magrolimab to CD47 leads to phagocytosis of cancer cells in human solid tumors and hematologic malignancies. CD47 is highly expressed in multiple myeloma (MM), and overexpression contributes to MM pathogenesis. Blocking of CD47 induces immediate activation of macrophages and elimination of myeloma cells. Hence, a blockade of CD47 with magrolimab may have therapeutic benefit in patients with MM. Additionally, in vitro studies have shown synergy between magrolimab and daratumumab in MM cell line phagocytosis, and complimentary mechanisms suggest potential enhanced efficacy in other combinations.

Aims: To evaluate the safety, tolerability, and efficacy of magrolimab in combination with commonly used myeloma therapies in patients with relapsed/refractory MM (RRMM).

Methods: This phase 2 open-label multiarm study includes patients aged ≥18 years currently requiring treatment for RRMM; all participants must have received ≥3 previous lines of therapy for MM, including an immunomodulatory drug and a proteasome inhibitor. This study includes safety run-in and corresponding dose-expansion phases with the following combinations: magrolimab + daratumumab, magrolimab + pomalidomide + dexamethasone, magrolimab + carfilzomib + dexamethasone, and magrolimab + bortezomib + dexamethasone (Figure). Magrolimab + bortezomib + dexamethasone may be initiated based on the preliminary safety and efficacy of magrolimab + carfilzomib + dexamethasone, and, if initiated, only requires 1 prior line of therapy. The primary objectives of the safety run-in phase are to evaluate the safety and tolerability of magrolimab and determine the recommended phase 2 dose (RP2D), with incidence of dose-limiting toxicities (DLTs), adverse events, and laboratory abnormalities as the primary endpoint. The dose-expansion phase will evaluate efficacy, with objective response rate as the primary endpoint. Secondary endpoints include duration of response, progression-free survival, and overall survival. Minimal residual disease negativity, biomarker changes from baseline, immune cell signaling alterations, and the mutational profile of myeloma cells will also be explored. Magrolimab will be administered intravenously as an initial priming dose to mitigate on-target anemia, followed by a maintenance dose on days 8, 15, 22, and 29 during cycle 1 (35 days); days 1, 8, 15, and 22 during cycle 2 (28 days); and days 1 and 15 from cycle 3 (28 days) onward (maximum of 8 cycles in patients who previously received bortezomib). Daratumumab, pomalidomide, dexamethasone, carfilzomib, and bortezomib will be administered according to manufacturer’s prescribing information. Dose de-escalation may occur based on DLTs per protocol in the safety run-in cohorts; magrolimab doses will not be reduced in the dose-expansion cohorts. Patients will continue treatment until unacceptable toxicity, progressive disease, or patient/investigator choice to discontinue. Planned enrollment is approximately 153 patients, with up to 27 in each safety run-in cohort and 72 in the dose-expansion cohorts following completion of the safety run-ins.

Results: Trial in progress.

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Summary/Conclusion: Patient recruitment is ongoing. Clinical trial information: NCT04892446 .

PB2013: TECLISTAMAB COMPARED WITH REAL-WORLD THERAPIES IN PATIENTS WITH TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA

A. Krishnan 1,* , A. K. Nooka 2 , A. Chari 3 , A. Garfall 4 , T. Martin 5 , S. Nair 6 , X. Lin 7 , K. Qi 8 , A. Londhe 8 , L. Pei 9 , E. Ammann 10 , R. Kobos 9 , J. Smit 11 , T. Parekh 12 , M. Slavcev 10 , S. Usmani 13

1 City of Hope Comprehensive Cancer Center, Duarte, CA; 2 Winship Cancer Institute, Emory University, Atlanta, GA; 3 Mount Sinai School of Medicine, New York, NY; 4 Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 5 University of California, San Francisco, CA, United States of America; 6 Janssen Pharmaceutica NV, Beerse, Belgium; 7 Janssen Global Services, Horsham, PA; 8 Janssen Research & Development, Titusville, NJ; 9 Janssen Research & Development; 10 Janssen Global Services, Raritan, NJ; 11 Janssen Research & Development, Spring House, PA; 12 Janssen Research & Development, Bridgewater, NJ; 13 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America

Background: Teclistamab is a B-cell maturation antigen × CD3 bispecific antibody that promotes T cell activation by redirecting CD3+ T cells, resulting in lysis of BCMA-expressing myeloma cells. MajesTEC-1 ( NCT04557098 ) is an open-label, single-arm, phase 1/2 trial of teclistamab in patients with relapsed/refractory multiple myeloma (RRMM) who had received at least 3 prior lines of therapy (LOT) and were triple-class exposed (TCE) to a proteasome inhibitor, an anti-CD38 monoclonal antibody, and an immunomodulatory agent.

Aims: Because MajesTEC-1 did not include a control arm, comparative effectiveness of teclistamab versus treatment regimens using an external control arm from a real-world database was evaluated.

Methods: The external control arm was created using eligible patients in the nationwide Flatiron Health multiple myeloma database, which was derived from de-identified electronic health records. Patients comprising the external control arm had received ≥3 prior LOT, started a new LOT per physician’s choice after triple-class exposure between January 2011 and August 2021, and met key MajesTEC-1 eligibility criteria. Individual patient data were included from patients who had been treated with 1.5 mg/kg weekly teclistamab in the MajesTEC-1 at the Sep 7, 2021 clinical cutoff. Imbalances in baseline covariates of prognostic significance (refractory status, number of prior LOT, progression on last LOT, International Staging System stage, cytogenetic risk, time since diagnosis, age, and hemoglobin), were adjusted by using inverse probability of treatment weighting (IPTW). Progression-free survival (PFS), time to next treatment (TTNT), and overall survival (OS) were outcomes of interest, and were analyzed as time-to-event data utilizing IPTW adjusted Kaplan-Meier estimates and a weighted Cox proportional hazards model. Several sensitivity analyses were also performed.

Results: The two cohorts were comparable for baseline characteristics after IPTW. Teclistamab improved PFS (hazard ratio [HR] 0.43; 95% confidence interval [CI] 0.32–0.59; P <0.0001), TTNT (HR 0.42; 95% CI 0.31–0.58; P <0.0001), and OS (HR 0.73; 95% CI 0.48–1.09; P =0.13) compared with real-world treatments. Sensitivity analyses showed results were also consistent with these findings.

Summary/Conclusion: In patients with TCE RRMM who received at least 3 prior LOT, teclistamab showed improved effectiveness for PFS, TTNT, and OS versus real-world treatments, highlighting the therapeutic potential of teclistamab for patients with RRMM.

PB2014: TECLISTAMAB VS SELINEXOR-DEXAMETHASONE IN PATIENTS WITH TRIPLE-CLASS EXPOSED RELAPSED/REFRACTORY MULTIPLE MYELOMA: A MATCHING-ADJUSTED INDIRECT COMPARISON

L. Rosinol 1,* , S. Z. Usmani 2 , N. Bahlis 3 , A. Y. Krishnan 4 , A. K. Nooka 5 , A. Oriol 6 , M. Delforge 7 , A. L. Garfall 8 , N. W. van de Donk 9 , P. Rodríguez Otero 10 , T. G. Martin 11 , J. Diels 12 , S. Van Sanden 12 , L. Pei 13 , E. Ammann 14 , R. Kobos 13 , M. Slavcev 14 , J. Smit 15 , A. Londhe 16 , P. Moreau 17

1 Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain; 2 Memorial Sloan Kettering Cancer Center, New York, NY, United States of America; 3 Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, AB, Canada; 4 City of Hope Comprehensive Cancer Center, Duarte, CA; 5 Winship Cancer Institute, Emory University, Atlanta, GA, United States of America; 6 Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain; 7 University of Leuven, Leuven, Belgium; 8 Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America; 9 Amsterdam University Medical Center, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; 10 University of Navarra, Pamplona, Spain; 11 University of California, San Francisco, CA, United States of America; 12 Janssen Pharmaceutica NV, Beerse, Belgium; 13 Janssen Research & Development; 14 Janssen Global Services, Raritan, NJ; 15 Janssen Research & Development, Spring House, PA; 16 Janssen Research & Development, Titusville, NJ, United States of America; 17 Hematology Clinic, University Hospital Hôtel-Dieu, Nantes, France

Background: Teclistamab, is a B-cell maturation antigen × CD3 bispecific antibody. MajesTEC-1 ( NCT04557098 ), a single-arm, phase 1/2 study evaluating teclistamab in patients with RRMM who were exposed to at least 3 lines of therapy (LOT), comprising a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 monoclonal antibody. Though there is currently no standard of care for patients with triple-class exposed (TCE) RRMM, selinexor-dexamethasone (sel-dex) is a recently approved, novel therapeutic option.

Aims: An unanchored matching-adjusted indirect comparison (MAIC) was conducted to compare efficacy results of patients treated with teclistamab in MajesTEC-1 with those treated with sel-dex in the single-arm, phase 2b STORM Part 2 study ( NCT02336815 ).

Methods: Individual patient-level data (IPD) collected from 150 patients treated with 1.5 mg/kg weekly teclistamab from MajesTEC-1 (Sep 7, 2021 data cut) and published summary-level data from 122 patients treated with sel-dex in the STORM Part 2 study were used in the unanchored MAIC. Eligibility criteria from STORM Part 2 (triple-class refractory, penta-exposed, and refractory to last LOT) were applied to patients in the MajesTEC-1 trial. The resulting IPD from 69 patients from the MajesTEC-1 trial were then weighted to match the aggregated baseline characteristics of patients from STORM Part 2. Baseline characteristics of prognostic significance were cytogenetic profile, presence of extramedullary disease, refractory status, revised International Staging System stage, and number of prior LOT; all were adjusted for in the analysis. Overall response rate (ORR), complete response or better (≥CR) rate, progression-free survival (PFS), duration of response (DOR), and overall survival (OS) were estimated to compare efficacy between teclistamab and sel-dex. For binary endpoints (ORR and ≥CR rate), odds ratio (OR) and 95% confidence interval (CI), derived from a weighted logistic regression, were used to estimate relative effects of teclistamab vs sel-dex. A weighted Cox proportional hazards model was used to estimate time-to-event endpoints (PFS, OS, and DOR).

Results: Baseline characteristics were balanced between the 2 cohorts. The effective sample size (ESS) of the MajesTEC-1 cohort was 37 after adjustment. Improved ORR (OR 3.14; 95% CI 1.48–6.69; P =0.0029), ≥CR rate (OR 16.3; 95% CI 3.5–77.1; P =0.0004), PFS (HR 0.58; 95% CI 0.30–1.11; P =0.1007), DOR (hazard ratio [HR] 0.04; 95% CI 0.01–0.10; P <0.0001), and OS (HR 0.52; 95% CI 0.28–0.95; P =0.0344) were observed in patients treated with teclistamab versus those treated with sel-dex. The majority of outcomes in favor of teclistamab were significant ( P <0.05), despite a reduced ESS which limited power to detect statistically significant differences.

Summary/Conclusion: Teclistamab significantly improved efficacy over sel-dex for all outcomes except PFS (though numerically in favor of teclistamab) in this MAIC. These findings highlight the potential of teclistamab as a highly effective treatment option for patients with TCE RRMM who received at least 3 prior LOT.

PB2015: YOUNG ADULTS WITH MULTIPLE MYELOMA – A SINGLE CENTER EXPERIENCE

I. Dos Santos Marques 1,* , C. Sol 1 , M. Henriques 1 , P. Baptista 1 , A. Airosa Pardal 1 , P. Oliveira Monteiro 1 , E. Aguiar 1 , M. J. Soares 1 , P. Gomes 1 , R. Bergantim 1 , F. Trigo 1

1 Hematologia Clínica, Centro Hospitalar Universitário São João, Porto, Portugal

Background: Multiple myeloma (MM) is a plasma-cell disorder characterized by clonal proliferation of malignant plasma cells in the bone marrow with monoclonal protein in the serum and/or urine, and associated organ dysfunction. It mainly affects the elderly population and is uncommon in younger than 40 years and extremely rare before 30 years. Since this is an incurable disease, it is important to understand its behavior in young adult patients for a better therapeutic decision and surveillance.

Aims: Description of presenting features and outcomes of MM patients ≤45 years.

Methods: Retrospective review of MM patients, aged ≤45 years followed up in a tertiary hospital, between 2008 and 2021. Data analysis was performed using the statistical program IBM SPSS Statistics 26®. The overall survival (OS) and progression-free survival (PFS) was estimated by the Kaplan‐Meier method.

Results: It was included 28 patients [71,4% male (n=20), 28,6% female (n=8)], with a median age at diagnosis of 41 years (36-45). M-proteins consisted of IgG (n=12, 45%), IgA (n=9, 31,1%), light-chain (n=4, 14,3%) and non-secretory disease (n=1, 3,6%). According to the International Staging System (ISS), 35,7%, 35,7%, and 17,9% of the patients had stage I, II, and III disease at the time of diagnosis, respectively. The most common ‘CRAB’ symptom was bone disease (n=17, 60,4%), followed by anemia (n=10, 35,7%), hypercalcemia (n=5, 17,9%) and kidney failure (n=3, 10,7%); 12 patients (42,9%) had two or more ‘CRAB’ symptoms. Fluorescence in situ hybridization (FISH) was done in 57,1% patient specimens, and cytogenetic high-risk abnormalities [del(17p), t(4;14), t(14;16), t(14;20), gain 1q, or p53 mutation] were detected in 6 cases. Other chromosomal abnormalities, namely del(13q) and t(11;14) were found in 2 and 3 patients, respectively. Patients received induction treatment according local protocols [Vd (n=11), VTd (n10), CBd (n=3), VAd (n=2), TD (n=1), VTd-PACE (n=1)] followed by autologous stem cell transplant (ASCT). No patients had allogeneic stem cell transplant. Median follow-up time 68 months (7-165) with relapse was seen in 16 patients (57,1%) and the estimated median time until progression was 52 months. OS was not reached. Using univariate logistic regression analysis no association was found between overall survival or response to therapy, and disease staging, CRAB symptoms, cytogenetic abnormalities, immunophenotypic features or treatment strategy.

Summary/Conclusion: Most data regarding the impact of age on survival mainly come from clinical trials developed before the current therapy choices were in use. However, some real-life studies evaluating young population of MM patients has been conducted to identify patient and disease features may have impact on this group of patients. The results of these studies are heterogeneous. On the other hand, in our population, ISS, CRAB symptoms, cytogenetic data, immunophenotypic features and treatment was not shown to have an impact on clinical course of the disease or survival. This study has limitations, particularly attributable to its retrospective nature, small number of patients within a single institution and lack of detailed information regarding imaging techniques, and comorbidities.

PB2016: COMPARISON BETWEEN DRD VS KRD AS SALVAGE THERAPY FOR MULTIPLE MYELOMA PATIENTS IN FIRST RELAPSE: THE REAL LIFE EXPERIENCE OF RETE EMATOLOGICA PUGLIESE (REP)

A. Mele 1,* , E. Prete 1 , S. Citiso 1 , G. Mele 2 , D. Pastore 2 , N. Sgherza 3 , P. Curci 3 , P. Musto 3 , A. P. Falcone 4 , N. Cascavilla 4 , C. Germano 5 , T. Giuseppe 5 , G. Reddiconto 6 , N. Di Renzo 6 , G. Palazzo 7 , P. Mazza 7 , B. Rossini 8 , A. Guarini 8 , G. Palumbo 9 , L. M. A. Melillo 9 , V. Pavone 1

1 Hematology and Bone Marrow Transplant, Hospital Cardinale G.Panico, Tricase; 2 Hematology, Hospital Perrino, Brindisi; 3 Hematology and Bone marrow Transplant, Policlinico University of Bari, Bari; 4 Hematology and Bone marrow Transplant, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo; 5 Hematology, ASL BT, Barletta; 6 Hematology, Hospital Vito Fazzi, Lecce; 7 Hematology and Bone marrow Transplant, Hospital G.Moscati, Taranto; 8 Hematology, Hospital IRCCS Oncologico, Bari; 9 Hematology, Hospital University Riuniti, Foggia, Italy

Background: Daratumumab (DRD) and Carfilzomib (KRD), in combination with lenalidomide and dexamethasone, performed well in patients with relapsed or relapsed/refractory multiple myeloma (RRMM). The lack of randomized and/or real-life trials comparing the two triplets prompted us to assess their effectiveness and safety in RRMM in first relapse.

Aims: To analyzed effectinness and safety of Drd and Krd in first line relapse Multiple Myeloma

Methods: 176 RRMM patients, including 107 DRD and 69 KRD, entered this non-randomized comparison.

Results: Baseline characteristics and details of the previous therapies are analyzed (Table 1). KRD cohort accounted for a higher incidence of III Durie-Salmon staging (71 vs 23%, p 0,001). Instead, a higher number of patients with refractory disease were treated with DRD (29 vs 13%, p 0,01). Moreover, median age of patients, elevated LDH, III ISS stage, cytogenetic risk categories, type of previous therapy, were equally distributed between the two therapy arms. Half of them (43%) relapsed after a previous ASCT, without differences in KRD and DRD group of patients.

The overall response rate (ORR) was 78% (n=83), with 31% complete response (CR; n= 33) in DRD. In patients treated with KRD, the ORR was 74% (n=51), with 42 % CR (n=29),p 0,31. Median time to best response was shorter in KRD patients (2,8 vs 4,3 months, p 0,03). The probability of CR+VGPR response was significantly higher in patients with normal LDH and treated with DRD (41 vs 22%, p 0,02). Elevated LDH didn’t influenced the probability of response in KRD cohort (45 vs 42%, p 0,89). Response was better in late relapse (64 vs 21%, p 0.001) and in patients relapsed after a prior autologous transplant (61 vs 33%, p 0,02) in KRD arm. After a median follow-up of 18 months, median PFS was NR in DRD and 31 months in KRD, p 0,0001. The 2y-PFS was 72% and 57% in DRD and KRd, respectively. PFS was longer in patients achieving a Very good partial response (VGPR) with median PFS of 28 months and NR, in DRD and KRD, respectively (p 0, 001). Better PFS was obtained in patients with normal LDH treated with DRD as KRD. The treatment discontinuation rate due to adverse events (AEs) was 13% and 22% in DRD and KRD, respectively (p 0, 001). No differences in hematologic and non-hematologic AEs were observed between the two triplets.

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Summary/Conclusion: With limitations characteristic to any retrospective analysis, this real-life study demonstrated similar ORR in patients treated in first relapse with DRD or KRD,shorter time to best response with KRD but longer PFS with DRD. PFS was longer in patients achieving a VGPR after DRD as KRD. Patients who had relapsed after a previous transplant appeared to benefit more from KRD than DRD. Unfortunately, high LDH values ​​at the first relapse had a negative impact on PFS beyond the treatment used. These observations may help the daily clinical practice.

PB2017: YOUNG PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA AND RENAL IMPAIRMENT: A TUNISIAN SINGLE-CENTER EXPERIENCE

F. Kallel 1 , K. Mezghani 1,* , N. Siala 1 , Y. Fakhfekh 1 , L. Khalifa 1 , S. Hadiji 1 , O. Kassar 1 , I. Frikha 1 , I. Ben Amor 1 , M. Charfi 1 , D. Balloumi 2 , L. Torjmen 2 , K. Kammoun 3 , M. Ben Hamida 3 , T. Ben Othmen 2 , M. Medhaffar 1 , M. Elloumi 1

1 Clinical Hematology, Hedi Chaker Hospital, Sfax; 2 National Bone Marrow Transplant Center, Tunis; 3 Nephrology, Hedi Chaker Hospital, Sfax, Tunisia

Background: Patients (pts) with newly diagnosed Multiple Myeloma (NDMM) face a high risk of developing a renal impairment (RI). It is one of the most feared complications in this population due to its severe immediate consequences and its poor outcome on the prognosis and the overall survival. We report our retrospective single-center study of NDMM in young pts with RI.

Aims: This study aims to identify the incidence of renal failure (RF) in NDMM and analyze the renal response (RR), hematological response (HR), the overall survival (OS) and the prognostic impact after induction therapy.

Methods: This retrospective single-center review over 4 years (between June 2016 and December 2020) includes young pts under the age of 65 years old with NDMM with RF eligible for autologous stem cells transplantation (ASCT). RF was defined by a serum creatinine higher than 177 μmol/L and the serum Creatinine Clearance (CC) was calculated according to the MDRD formula. All pts with RF in this study were treated with Bortezomib, Thalidomide and Dexamethasone (DT) as an induction regimen.

RR and HR were defined according to the International Myeloma Working Group (IMWG14) criteria.

In addition to antimyeloma treatment, pts with RI received symptomatic treatment such as intravenous hydration, corticosteroids, correction of hypercalcemia and in some cases dialysis.

In this study, we identify the incidence of RF in NDMM and analyze the RR, HR, OS and the prognostic impact after induction therapy.

Results: We included in our study 58 pts with NDMM. The incidence of NDMM with RF at diagnosis was 48,3% (n=28), the serum creatinine level median was 213μmol/L and the serum creatinine clearance (CC) median was 23.5 ml/min.

According to the CC staging, the renal function was severely decreased in 11 cases (39%) and in the kidney failure stage in 5 cases (17%).

The MM with RF immunoglobulin type was Light chain in 11 cases (39%), IgG in 12 cases (43%) and IgA in 5 cases (18%).

After receiving symptomatic treatments of RF, 35% (n=10) of NDMM with RF pts had their renal function recovered whereas 64%(n=18) did not and 10% (n=3) needed dialysis.

After induction therapy, the serum creatinine level median was 95μmol/L and the serum CC median was 73.5ml/min

Thus, the RR was: complete in 64% (n=18), partial in 7%(n=2), minor in 18% (n=5), no response in 2 cases (7%) and one patient died before assessment.

HR after induction regimen was 74% (n=20): complete response (CR) in 4 cases (14%), very good partial response (VGPR) in 8 cases (29%) and partial response (PR) in 8 cases (29%). Among the 18 pts with complete RR, 6 pts (33%) underwent ASCT.

Pts with RF at diagnosis had a poorer OS median (18.5 months) comparing to those with normal renal function (OS median=21.5 months). The OS was significantly influenced by the degree of RF and the recovery of normal renal function (p<0.05).

Summary/Conclusion: Our single center study showed that RI occurs in 48% of the cases in NDMM at diagnosis, which is significantly higher to what is found in the literature (5-30%). It also revealed that NDMM with RI has poorer HR and OS than pts with normal renal function when both treated by a Brotezomib induction regimen followed by ASCT. The RR after supportive care measurements and antimyeloma therapy was 74%, which is slightly lower to what is found in the literature (77%). Thus, early diagnosis and rapid initiation of a triplet Brotezomib based regimen for this high-risk group is essential and improves renal outcomes in RI pts.

PB2018: MULTIPLE MYELOMA WITH CENTRAL NERVOUS SYSTEM RELAPSE IN THE ERA OF NOVEL DRUGS - A MULTICENTER RETROSPECTIVE STUDY FROM THE CROATIAN COOPERATIVE GROUP FOR HEMATOLOGICAL MALIGNANCIES (KROHEM)

Z. Mitrovic 1,2,* , J. Batinic 2,3 , D. Radic Kristo 4 , I. Krecak 5 , H. Holik 6 , V. Perisa 7 , T. Valkovic 8 , H. Matijaca 2,9 , M. Pirsic 1 , S. Basic Kinda 3 , I. Aurer 2,3

1 Departmenf of Medicine, University Hospital Dubrava; 2 School of Medicine University of Zagreb; 3 Departmenf of Medicine, University Hospital Center Zagreb; 4 Departmenf of Medicine, University Hospital Merkur, Zagreb; 5 Departmenf of Medicine, General Hospital Sibenik, Sibenik; 6 Departmenf of Medicine, General Hospital dr. Josip Bencevic, Slavonski Brod; 7 Departmenf of Medicine, University Hospital Center Osijek and School of Medicine University of Osijek, Osijek; 8 Departmenf of Medicine, University Hospital Center Rijeka and School of Medicine University of Rijeka, Rijeka; 9 Departmenf of Medicine, University Hospital Center Sisters of Mercy, Zagreb, Croatia

Background: Central nervous system (CNS) relapse in multiple myeloma (MM) is a rare complication with dismal prognosis. It primarily affects transplant-eligible patients, and the incidence seems to be higher in the era of novel antimyeloma drugs.

Aims: We aimed to assess clinical characteristics, treatment choices, and survival of these patients.

Methods: This is a retrospective study by the Croatian Cooperative Group for Hematological malignancies (KROHEM). Patients with MM treated with novel agents prior to CNS relapse were included.

Results: From April 2018 to October 2021, 13 patients from eight centers were identified. There were 8 men and 5 women, with a median age of 61 years (range 47-70 years). A median time from diagnosis to CNS progression was 3 years (ranging from 4 months to 7.5 years). Patients were treated with 1 to 5 lines (median 3) of systemic therapy prior to CNS progression. The patients progressed during treatment or maintenance with bortezomib-based regimen (3 pts), carfilzomib (2 pts), lenalidomide (3 pts), daratumumab (2 pts), pomalidomide (1 pt), bendamustine (1 pt), or in treatment-free remission (1 pt). Six patients had >10% plasma cells in bone marrow, whereas only a single patient had plasma cell leukemia. Nine patients had plasma cells in cerebrospinal fluid (CSF) and were treated with intrathecal therapy – median time to CSF clearance was two to three weeks. Nine patients had osteodural myeloma mass and 7 were irradiated. Systemic treatment was administered in 12 patients with CNS penetrating agents such as bendamustine + IMiDs, daratumumab +/- IMiDs, or other chemotherapy such as DT-PACE. At the time of analysis, three patients are alive with survival 3.5, 4.5, and 8 months from CNS relapse, respectively. Two of them were autografted with BEAM or melphalan/thiotepa conditioning. The median overall survival for all patients was 3 months (1-38 months). The longest survival had two patients treated with systemic therapy and local irradiation who died 11 and 38 months after CNS relapse, respectively (image).

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Summary/Conclusion: CNS relapse in our cohort occurred as a late event; after a median of 3 years from diagnosis and a median of 3 prior therapy lines. Importantly, 7 of 13 CNS relapses occurred during treatment with CNS-penetrating agents. Consistent with previous reports, the median overall survival was only 3 months. The optimal treatment is unknown. Nevertheless, patients with localized osteodural infiltration amenable to radiotherapy and those autografted may have better survival. IMiDs in combination with bendamustine or daratumumab were the most frequently used systemic agents in our study.

PB2019: EFFICACY AND SAFETY OF IXAZOMIB PLUS LENALIDOMIDE AND DEXAMETHASONE FOLLOWING INJECTABLE PROTEASOME INHIBITOR-BASED THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

M. Sasaki 1,* , K. Suzuki 2 , Y. Abe 3 , S. Ito 4 , K. Nishiwaki 5 , H. Handa 6 , T. Chou 7 , J. Soeda 8 , I. Mori 8 , T. Shinozaki 9 , N. Takezako 10

1 Division of Hematology, Department of Internal Medicine, Juntendo University School of Medicine; 2 Division of Hematology, Myeloma and AL Amyloidosis Center, Japanese Red Cross Medical Center; 3 Division of Hematology, Japanese Red Cross Medical Center, Tokyo; 4 Division of Hematology and Oncology, Department of Internal Medicine, Iwate Medical University School of Medicine, Iwate; 5 Department of Clinical Oncology/Hematology, Kashiwa Hospital, The Jikei University School of Medicine, Tokyo; 6 Division of Hematology, Department of Internal Medicine Medical Center, Gunma University Hospital, Gunma; 7 Niigata Kenshin Plaza, General Incorporated Foundation, Health Medicine Prevention Association, Niigata; 8 Japan Medical Affairs, Japan Oncology Business Unit, Takeda Pharmaceutical Company Limited; 9 Department of Information and Computer Technology, Faculty of Engineering, Tokyo University of Science; 10 Division of Hematology, Japan Association for Development of Community Medicine, Nerima Hikarigaoka Hospital, Tokyo, Japan

Background: Long-term proteasome inhibitor (PI)-based therapy improves outcomes in multiple myeloma (MM). However, physical, social, and geographic access to health services and treatment toxicity may interfere with long-term PI therapy in clinical practice.

Aims: To evaluate the efficacy and safety of treatment with the oral PI, ixazomib (IXA), lenalidomide (LEN) + dexamethasone (DEX) [IRd] in patients with relapsed/refractory MM (RRMM) initially treated with an injectable PI-based therapy.

Methods: Nationwide, multicenter, open-label, single-arm study in Japan. Patients aged ≥20 years with RRMM (including those not refractory to bortezomib (BOR), carfilzomib (CFZ), or LEN who provided written informed consent were enrolled. Patients received BOR, LEN + DEX (VRd), or CFZ, LEN + DEX (KRd) for 3 cycles. Patients with at least a minor response to VRd or KRd transitioned to IRd. After transition, patients received 4 mg IXA on days 1, 8, and 15, 25 mg LEN on days 1 through 21, + 40 mg DEX on days 1, 8, 15, and 22 in 28-day cycles until progressive disease (PD) or unacceptable toxicity. The primary endpoint was the 12-month progression-free survival (PFS) rate from the start of injectable PI-based therapy, with dropouts, missing response data, PD, and death at 12-month included as events. Secondary endpoints included PFS with dropouts treated as censoring (Kaplan-Meier [KM] method), overall survival (OS), overall response rate (ORR), percentage of patients with very good partial response (VGPR) or better, healthcare resource utilization, time to next treatment (TTNT), and safety.

Results: Forty-five RRMM patients were enrolled, 36 of whom achieved minor response or better after 3 cycles of VRd (n=6) or KRd (n=30) and received IRd, and followed up for at least 12 months after the last patient startedinjectable PI-based therapy. Mean age was 70.7 (SD±9.2) years, the median (min, max) number of prior lines of therapy was 2 (1, 4), and 95.6% had an ECOG PS of 0−1. At a median follow-up of 20.8 (95% CI: 17.4-23.7) months, the 12-month PFS rate (primary endpoint) was 48.9% (22/45, 90% CI: 35.9−62.0) with 11 events of PD/death, 8 dropouts, and 4 missing response data, which exceeded the threshold of 36% but did not meet significance (p=0.0518). As secondary endpoints, the 12-month PFS rate (dropouts as censoring) by KM analysis was 74% (95% CI: 56−86). Median PFS and TTNT were 29.0 (95% CI: 21.3−NE) months and 32.3 (95% CI: 14.9−35.4) months, respectively; median OS was not estimable. The ORR was 73.3% (95% CI: 58.1−85.4) and 42.2% (95% CI: 27.7−57.8) of patients had a VGPR or better. The length of hospital stay, and outpatient visits during the initially 3 cycles of injectable PI-based treatment and following IRd treatment were 5.3 vs 1.0 days per person-month, and 4.2 vs 1.9 visits per person-month, respectively. The safety profile of IRd was similar and consistent to that previously reported. Frequent (≥10% incidence) Grade ≥3 treatment emergent adverse events were neutrophil count decreased (n=7 [15.6%]) and platelet count decreased (n=7 [15.6%]); the only SAE with a ≥10% incidence was pneumonia (n=5 [11.1%]). Two deaths occurred during the study, one during KRd treatment (pneumonia, bacterial, n=1 [2.2%]) and one during IRd treatment (pneumonia, n=1 [2.2%]), which was related to IRd.

Summary/Conclusion: Long-term treatment with the oral PI, IXA, + LEN and DEX after an injectable PI-based treatment was tolerable and had favorable efficacy in patients with RRMM in the real-world setting.

PB2020: MAJESTEC-3: RANDOMIZED, PHASE 3 STUDY OF TECLISTAMAB PLUS DARATUMUMAB VERSUS DPD OR DVD IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

M. V. Mateos 1,* , N. Bahlis 2 , L. Costa 3 , A. Perrot 4 , L. Pei 5 , M. Rubin 6 , K. Lantz 6 , W. Sun 7 , M. Jaffe 5 , R. Kobos 5 , A. Nooka 8

1 Hospital Clínico Universitario de Salamanca, Salamanca, Spain; 2 Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Canada; 3 University of Alabama at Birmingham, Birmingham, United States of America; 4 Centre Hospitalier Universitaire de Toulouse, Service d’Hématologie, Toulouse, France; 5 Janssen Research & Development, Raritan; 6 Janssen Research & Development, Spring House; 7 Janssen Research & Development, Los Angeles; 8 Winship Cancer Institute, Emory University, Atlanta, United States of America

Background: Treatment of relapsed/refractory multiple myeloma (RRMM) in patients (pts) previously treated with a proteasome inhibitor (PI) and lenalidomide is challenging. Although daratumumab plus pomalidomide and dexamethasone (DPd), and bortezomib and dexamethasone (DVd) are approved for RRMM, further disease control is still needed. Thus, newer therapeutic options with diverse modes of action are warranted. Teclistamab (tec; JNJ-64007957), a B-cell maturation antigen (BCMA) x CD3 bispecific antibody (Ab), induces T cell activation and subsequent lysis of BCMA-expressing myeloma cells. Results from the phase 1 MajesTEC-1 study ( NCT03145181 ) demonstrated that teclistamab was generally well-tolerated with encouraging efficacy in pts with RRMM who were heavily pretreated. In the phase 1b TRIMM-2 study ( NCT04108195 ), teclistamab plus daratumumab (tec-dara) provided promising efficacy and was also well tolerated.

Aims: To describe MajesTEC-3 ( NCT05083169 ), a phase 3, multicenter, open-label, randomized study comparing the efficacy of tec-dara and investigator’s choice of therapy (DPd or DVd) in pts with RRMM.

Methods: Eligible patients (pts) are ≥18 years of age with documented MM based on the International Myeloma Working Group (IMWG) criteria and have measurable disease, an Eastern Cooperative Oncology Group (ECOG) performance status 0–2, previous exposure to 1–3 lines of treatment (tx) which may include a PI and lenalidomide (of note, pts with 1 prior tx line must be lenalidomide-refractory), and progressive disease status on or following their last tx (or within 60 days of completing lenalidomide). Informed consent form will be signed before the first study-related activity is conducted. Pts who are refractory to an anti-CD38 monoclonal antibody or had prior BCMA-directed tx are excluded. Pts (560 planned) will be randomly assigned (1:1) to receive 28-day cycles of tec-dara or investigator’s choice of DPd or DVd (stratified by investigator’s choice of DPd or DVd, ISS stage, and number of lines of prior tx). Pts will receive dara, DPd, and DVd tx per the approved schedules and will be treated until disease progression, death, withdrawal of consent, intolerable toxicity, or end of study, whichever occurs first. Assessment of responses will be based on 2016 IMWG criteria. The primary endpoint will be progression-free survival (PFS), and secondary endpoints will include overall response rate, complete response or better, MRD negativity, PFS on next-line tx (PFS2), overall survival, and incidence and severity of adverse events (AEs). All reported AEs will be graded as per the Common Terminology Criteria for AEs (CTCAE) v5.0. Immune effector cell-associated neurotoxicity syndrome and cytokine release syndrome will be graded per the American Society for Transplantation and Cellular Therapy (ASTCT) guidelines. MajesTEC-3 opened in October 2021 and is currently enrolling pts.

Results: Not applicable

Summary/Conclusion: Not applicable

PB2021: QUALITY OF LIFE AND CLINICAL OUTCOMES IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA (RRMM) TREATED WITH IXAZOMIB-LENALIDOMIDE-DEXAMETHASONE (IRD): RESULTS OF A REAL-WORLD EVIDENCE STUDY

T. Ionova 1,* , O. Vinogradova 2 , Y. Kochkareva 2 , E. Ado 2,3 , K. Kaplanov 4 , M. Shirokova 4,5 , T. Shelekhova 6 , A. Levanov 6 , A. Kopylova 7 , O. Li 8 , T. Mitina 9 , O. Rukavitsyn 10 , P. Simashova 10 , L. Anchukova 11 , E. Babich 12 , D. Dasheeva 13 , M. Demchenkova 14 , S. Dubov 15 , T. Esenina 16 , L. Ivanova 14 , T. Kravchuk 17 , E. Rimashevskaya 18 , N. Saraeva 19 , M. Savinova 20 , S. Volkova 21 , N. Porfirieva 1 , T. Nikitina 1

1 Multinational Center for Quality of Life Research, Saint-Petersburg; 2 Hematology Center of Botkin Hospital; 3 State-financed Institution of Moscow city «Scientific Research Institute of Healthcare Organization and Medical Management of Moscow Department of Healthcare»; 4 S.P. Botkin City Clinical Hospital, Moscow; 5 Regional Clinical Oncological Dispensary, Volgograd; 6 Department of Occupational Pathology, Haematology and Clinical Pharmacology, Saratov State Medical University named after V.I. Razumovsky, Saratov; 7 Lipetsk City Hospital №3 “Svobodny Sokol”, Lipetsk; 8 Sakhalin Region Hospital Clinic, Yuzhno-Sakhalinsk; 9 Moscow Regional Research and Clinical Institute (“MONIKI”); 10 N.N. Burdenko Main Military Clinical Hospita, Moscow; 11 Regional Clinical Hospital, Vologda; 12 Regional Clinical Hospital, Khanty-Mansiysk; 13 Zabaikalsky Regional Oncology Center, Chita; 14 Regional Cancer Center, Irkutsk; 15 Regional Clinical Hospital № 2, Vladivostok; 16 Amur Regional Clinical Hospital, Blagoveshchensk; 17 Goldberg Research Institute of Pharmacology and Regenerative Medicine, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk; 18 Russian Medical Academy of Continuous Professional Education, Moscow; 19 Regional Clinical Hospital, Irkutsk; 20 Сity Clinical Hospital №16, Kazan; 21 Federal State Budgetary Educational Institution of Higher Education «Privolzhsky Research Medical University» of the Ministry of Health of the Russian Federation, Nizhny Novgorod, Russia

Background: Comprehensive evaluation of new treatment regimens in RRMM patients both from physician’s and patients’ perspective is worthwhile.

Aims: We aimed to evaluate clinical and patient-reported outcomes during IRd treatment as ≥ 2nd line in RRMM patients in a multicenter real-world evidence study.

Methods: Adult patients with RRMM who have been assigned IRd as ≥2nd line treatment were enrolled in 18 centers of Russian Federation from April 2019 till May 2020. Treatment response was evaluated by IMWG 2011 criteria. For assessment of adverse events (AEs) NCI CTCAE v. 4.0 was used. Patients filled out RAND SF-36 and ESAS-R questionnaires at baseline, at 1 and 3 mos, and thereafter every 3 mos till 18 mos after IRd treatment onset. Statistical analysis of patient-reported outcomes was conducted using GEE with adjustment to age, gender and baseline quality of life (QoL). Duration of response (DOR), progression-free (PFS) and overall survival (OS) from the start of IRd treatment were evaluated using Kaplan-Meyer method.

Results: In total, 40 patients with RRMM were enrolled into the study: median age – 64 years (range, 33–80), 35% males. Durie–Salmon stage at study entry: II/III – 40/60%, ECOG status 0/1 – 70%, 2/3 – 30%. Median time since initial MM diagnosis – 55 mos (range, 2.0–99.0). Median number of lines of prior therapy – 3 (range, 1–7). Comorbidities were revealed in 65% patients; median Charlson Comorbidity index – 2 (range, 0–5); 95% patients had bone complications. The median duration of IRd treatment – 7.5 mos (IQR, 3.9-18.0). Two-thirds of the patients (28/39) responded to therapy. The overall response rate was 46.2% (95%CI: 30.6-61.8), median DOR – 16.3 mos (95%CI: 15.4–17.3). Among them 3 patients achieved complete response, 1 – stringent complete response, 2 – very good partial response, 12 – partial response. Ten patients had minor response. Clinical benefit rate – 71.8% (95%CI: 57.7-85.9). Six patients (15.4%) had stable disease and 4 (10.3%) progressed upon therapy. Median PFS was 10.6 mos (95%CI: 6.3-16.3). During the entire period of the study 5 deaths were registered: 3 were related to progression, 2 – because of COVID-19. Мedian OS was not reached. One-year OS rate was 85.2% (95%CI: 71.0–99.0). AEs were revealed in 55% patients: grades 1-2 AEs – 15 patients; grades 3-4 AEs – 7 patients; SAEs – 3 patients (neurological toxicity, gastric bleeding, hypotension and diarrhea). Baseline QoL was dramatically impaired by the majority of SF-36 scales; 42% patients experienced severe/critical QoL impairment. At baseline all the patients experienced symptoms; 85% with moderate-to severe symptoms (≥4 scores on the scale from 0 to 10). The most prevalent and severe symptoms were tiredness (98%), drowsiness (90%), pain (82%) and shortness of breath (80%). During IRd treatment QoL was stable or improved. Physical and role physical functioning, general health, vitality and mental health significantly improved as compared to baseline (GEE, p<0.05). Twice increase of Integral QoL Index was observed – 0.27 at baseline vs 0.48 at 18 mos (p<0.05). Severity of pain, tiredness and nausea meaningfully decreased during IRd treatment as compared to baseline (GEE, p<0.05). Total ESAS-R score decreased by 10 points at 18 mos of therapy as compared to baseline – 31 vs 21 (GEE, p<0.05).

Summary/Conclusion: In summary, results obtained in a real-world evidence study confirmed RCTs data that IRd regimen is an effective treatment in RRMM patients. This treatment is accompanied with definite improvement of QoL. Our results demonstrate benefits of IRd, both from physician’s and patient’s perspective.

PB2022: EXPLORING ALTERNATIVE DOSING REGIMENS OF SINGLE-AGENT BELANTAMAB MAFODOTIN ON SAFETY AND EFFICACY IN PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA: DREAMM-14

M. Hultcrantz 1,* , D. Kleinman 2 , P. Ghataorhe 3 , A. Mckeown 3 , W. He 4 , T. Ling 3 , R. C. Jewell 5 , J. Byrne 6 , L. Eliason 6 , E. Scott 6 , J. Opalinska 6

1 Memorial Sloan Kettering Cancer Center, New York; 2 University of Rochester, Rochester, NY, United States of America; 3 GlaxoSmithKline, London, United Kingdom; 4 GlaxoSmithKline, Cambridge, MA; 5 GlaxoSmithKline, Research Triangle Park, NC; 6 GlaxoSmithKline, Upper Providence, PA, United States of America

Background: Belantamab mafodotin (belamaf: BLENREP) is a first-in-class, monomethyl auristatin F (MMAF)-containing, B-cell maturation antigen (BCMA)-directed antibody–drug conjugate (ADC). In the DREAMM-2 study, belamaf showed deep responses with a manageable safety profile in patients with relapsed/refractory multiple myeloma (RRMM). At 13 months of follow-up, the median duration of response was 11 months and overall survival was 13.7 months at the 2.5 mg/kg Q3W dose.

Corneal events are common and expected with belamaf and other MMAF-containing ADCs. In DREAMM-2, corneal events were managed with dose modifications. Clinical responses were observed even with prolonged dose holds, suggesting alternative dosing regimens may lower corneal event rates without compromising efficacy.

Aims: The DREAMM-14 study ( NCT05064358 ) will investigate if an improved benefit/risk profile of single-agent belamaf can be achieved by modifying the dose, schedule, or both, relative to the approved dosing regimen (2.5 mg/kg Q3W).

Methods: This Phase II, randomized, open-label study will include adults with RRMM who had ≥3 prior lines of therapy (LOT), including an anti-CD38 monoclonal antibody, an immunomodulatory agent, and a proteasome inhibitor. Patients with corneal epithelial disease (except nonconfluent superficial punctate keratitis) or with prior exposure to BCMA-targeted therapies, or ADCs will be excluded.

Patients will be randomized into Arms A–D (n=40 each) and Arm E (n=20) in parallel and stratified by the International Staging System (I vs II vs III) and prior LOT (3 vs ≥4). Belamaf will be administered as follows—Arm A: 2.5 mg/kg Q3W; Arm B: 1.9 mg/kg Q3W; Arm C: 2.5 mg/kg Q6W; Arm D: 1.9 mg/kg Q6W; Arm E: 1.9 mg/kg Q6W with dose modifications based on oncology staff assessment of ocular symptoms (patient-reported symptoms using the Ocular Surface Disease Index), and visual acuity (Snellen chart or equivalent) in addition to corneal findings assessed by an eye care specialist. Patients in all Arms will have response assessments, safety assessments, and ophthalmic exams performed by an eye care specialist Q3W regardless of dosing schedule. Ocular event-related dose modifications (except in Arm E) will be guided by a modified Keratopathy and Visual Acuity scale.

The primary endpoint will be incidence of ocular events. Key secondary endpoints include ocular safety and tolerability, overall safety and tolerability, pharmacokinetics, and efficacy outcomes. Follow-up for progression-free survival will be Q3W until progressive disease, start of new anticancer therapy, withdrawal of consent, end of study, or death.

Results: N/A

Summary/Conclusion: First patient first visit is currently targeted for 04Mar22.

PB2023: UPDATE AND FOLLOW-UP OF NEWLY DIAGNOSED MULTIPLE MYELOMA (NDMM) WITH HIGH-RISK CYTOGENETIC (HRC).

M. I. Nuevo López 1,* , B. Merchan Muñoz 1 , M. A. Marta 1 , P. A. Rocio 1 , G. P. angela 1 , G. G. Helga 1 , D. M. L. Dunia 1 , G. Nuria 1 , V. R. alejandro 1

1 Hematology, Hospital, Guadalajara, Spain

Background: Despite therapeutic strategies improvement, there is still a subgroup of NDMM patients (pts) that pose a clinical challenge, whose represents a <20%, we refer to HRC. HRC Pts are associated with a poor prognosis and aggressive course, although, recent studies propose different clinical evolution according to the cytogenetic alteration (CA). IMWG identified a 4-year progression free survival (PFS) of 12% and OS of 35%.

Aims: Describe our clinical experience and therapeutic management, also the clinical-biochemical alterations at diagnosis in a heterogeneous NDMM HRC`s cohort. Evaluate survival curves according to HRC.

Methods: Descriptive and retrospective analysis, using clinical and analytical NDMM HRC patient’s data from 2009 to 2022 at Guadalajara University Hospital. 61/201 pts were selected in the MM treatment (tto) protocols. FISH results were not found in <30% NDMM, due to lack of metaphases or no request. Pts with t(4;14), t(14;16), gain 1q, t(14;20), plasmablastic leukemia and/or del(17p13) were classified as HRC. Survival data and Pearson (P) correlation were used. P value <0.05 was considered significant.

Results: Total HRC NDMM 61 pts were analyzed. 21 of 61 pts (35%) were diagnosed with stage monoclonal gammopathy of undetermined significance (MGUS) prior to HRC NDMM, with a mean 4 years (y) (IR 2-10 y), greater % representation of MGUS were del17p (38), gain 1q (48), t (14;16) (4,5) and del17p + gain1q (9,5). Clinical characteristics data at diagnosis according to HRC are shown in Table 1.

Pts receiving 1 st line were 100% (61), 90,2% of the pts received bortezomib (V) - based induction, of them 29% (n=16) were treated with alkylating agents, 49,1% (n=27) received IMIDs and 18,2% (n=10) V with dexamethasone (d), plus two of these pts received regimens composed of 3xVCd + 3xVPd and the other dara-VRd. 25 pts (40,98%) were transplant eligible (TPH). 2 nd line, 32 pts (52,5%), (n=3) VTD-PACE, (n=6) daratumumab (n=1 alone, 3 R, 1 V, 1 K), (n=1) Kdexa, (n=1) Pocydex and (n=9) V plus 3 IMIDs, 2 alkylating, 2 P or 2 alone and (n=12) IMIDs and alkylatings in combination. 5 pts (8,2) were TPH (1 alogenic). 3 rd line, 16 pts (26,2), just 3 pts were TPH. 4 th line, 7 pts (11,4), 1 pts were TPH. 5 th line, 4 pts (6,5). 6 th line, 2pts (3,3). In the last lines, the use of triplets with pomalidomide, karfilzomib or intensive chemotherapy prevail.

After a median follow-up of 3 y (IR 1,6-6,2) from diagnosis, pts had relapsed at least one time (60,6%) and more than 3 times (11,5%), and 25 had died (40,9%), 16 of them due to infections (14 bacteremia, 2 COVID), 5 cardiorespiratory arrest and 3 due to progression. (Image 1) represents PFS y OS for only 5 CA, as data were no representative in other CA.

Correlation between ISS and ISS-R data were only able to execute in HRC gain1q, due to lack of sample in other CA. We found a P coefficient of 0.568399 or 56.83% (p- <0.00578, CI 95%).

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Summary/Conclusion: Our case series continues with a longer survival curve compared to those commented in other studies. As cytogenetic abnormalities (t(4;14), t(14;16), t(14;20) and gain1q), similar % of representation are described as Kumar. Nat Rev Clin Oncol. 2018, except for del(17p), 23% vs 10%. From the second line, the probability of receiving a new line, the duration of tto and the interval without tto decreased with each line of tto. As treatment lines progress, therapeutic combinations are more heterogeneous and less concordant. A further longer follow-up and higher HRC NDMM pts`s recruitment will be necessary to clarify the response to tto regimens based on individual cytogenetic groups.

PB2024: PREVALENCE OF TYPE 1 GAUCHER DISEASE IN PATIENTS WITH MULTIPLE MYELOMA: FIRST INTERIM ANALYSIS OF A PROSPECTIVE, MULTICENTER, OBSERVATIONAL STUDY

M. Offidani 1,* , C. Zizzo 2 , S. Rupoli 1 , I. Federici 1 , A. Bossi 1 , S. Morè 1 , V. M. Manieri 1 , M. T. Petrucci 3 , T. Caravita di Toritto 4 , M. Brunori 5 , A. Gozzetti 6 , A. Tordi 4 , F. Fazio 3 , C. Lisi 3 , E. Morsia 1 , L. Corvatta 7 , A. Olivieri 1 , G. Duro 2

1 Clinica di Ematologia, Ospedali Riuniti di Ancona, Ancona; 2 Istituto per la Ricerca e l’Innovazione Biomedica CNR-Palermo, Palermo; 3 Azienda Policlinico Umberto I, Sapienza Università di Roma; 4 UOSD Ematologia ASL Roma 1, Roma; 5 UOS Ematologia Ospedale Santa Croce Fano, Fano; 6 Ematologia, Policlinico Santa Maria alle Scotte, Siena, Siena; 7 UOC Medicina Ospedali Profili, Fabriano, Fabriano, Italy

Background: Type I Gaucher disease (GD1) is an autosomal recessive lysosomal storage disease, caused by deficiency of the enzyme glucocerebrosidase, that degrades glycosphingolipids. In GD1, aberrant macrophage activation and immune dysregulation are associated with increased cancer risk. The association of GD1 and cancer was examined in the International Collaborative Gaucher Group (ICGG) Registry comprising 2,742 eligible patients [Rosenbloom BE et al, 2005]. This study found no increase of overall cancer risk (0.79, 95% CI 0.67–0.94). Nevertheless, there was a 5.9-fold (95% CI 2.8–10.8) increased risk of Multiple Myeloma (MM). However, cancer risk was likely underestimated in this study because the younger age distribution of the study population and incomplete ascertainment because the ICGG is an observational registry to track responses to treatment (ClinicalTrails.gov NCT 00358943 ). Therefore, we thought it was useful to prospectively investigate the prevalence of GD1 in a large population of patients affected by MM

Aims: The primary objective of this study was to determine the prevalence of Dried Blood Spots (DBS) test positivity in a large prospective MM population. Patients with DBS test positivity were then purposed for genetic test to confirm the diagnosis GD1. If the observed prevalence will be significantly higher than predicted, the identification of potential risk predictors in the selected population will be carried out

Methods: This is an observational, prospective, cross-sectional, multicenter study. Considering that no data are available about the effective prevalence of GD1 in patients affected by MM, the sample size has been determined considering clinically relevant a prevalence of the condition > 0.5% for defining as “high risk” the selected population. To test this hypothesis with an alpha error of 5% and a statistical power of 95%, we will enroll approximately 1000 patients in 46 hematology centers overall. All patients, according to the inclusion criteria and provided a signed written informed consent, with a confirmed diagnosis of MM will be screened for GD1 undertake through DBS sampling technique. The DBS test will be centralized at the Istituto per la Ricerca e l’Innovazione Biomedica CNR-Palermo. This is the first interim analysis planned when the first posivity DBS test was happened

Results: Two hundred and sixty-eight patients were enrolled, so far. Median age was 67 years (range 37-90) and 58% of patients were male. No patients was of Jew etnicity, one was asiatic and one was black, Caucasian the remaining. Fifty-eight percent had newly diagnosed MM while 42% had relapsed-refractory MM. SMM or MM was diagnosed in 10% and 90% of patients, respectively. Monoclonal component was IgG in 54%, IgA in 25%, light chain in 11%. Median Ferritin was 322 ng/ml (range 17-1236) and median Alkaline Phosphatase was 81 U/L (range 29-355). Data of DBS test found with mutations in GBA gene, were pictured in the Table 1. DBS test was considered positive in 1 patient; so the prevalence was 1/268 (0.4%), so far.

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Summary/Conclusion: Despite the trial enrolment is just in the first quarter of the whole planned sample size, our preliminary data showed an interesting prevalence of DBS test positivity (0.4%). Data updates are needed to define the real incidence of DBS test positivity in MM. Likewise, pathological genetic test in DBS positive patients is needed to confirm the diagnosis of GD1 and to demonstrate the relationship of this disease and plasma cell disorders.

PB2025: MANAGEMENT AND PATIENT ESTIMATION OF AMYLOID LIGHT-CHAIN (AL) AMYLOIDOSIS IN PORTUGAL: RESULTS FROM A PHYSICIANS’ SURVEY

S. Santos 1 , R. Jaime 2 , F. Pereira 3,* , H. Pedrosa 3 , M. Pinto 3 , S. Miguel 1

1 Market Access; 2 Medical Affairs, Janssen Portugal; 3 Consulting, IQVIA, Porto Salvo, Portugal

Background: Light-chain (AL) amyloidosis is a clonal, non-proliferative plasma cell disorder in which fragments of immunoglobulin light or heavy chain are deposited in tissues in the form of amyloid fibrils. According to an incidence-to-prevalence model from 2019, the estimated incidence in Portugal is 12.50 cases per million individuals, with an equal 5-year prevalence of 35.41 cases per million individuals.

Aims: Assess the AL Amyloidosis patient journey, understand treatment strategies in Portugal as well as unmet needs. Estimate AL Amyloidosis patient numbers, including potential underdiagnosis, according to epidemiology, public hospital Diagnosis Related Group (DRG) database and key opinion leaders’ insights.

Methods: Market research study supported by qualitative interviews with 4 hematologists key opinion leaders (KOL’s). Total AL Amyloidosis patients were estimated through epidemiologic data available for Portugal and supported by Hospital production quantified using the National Health Service (NHS) hospital Diagnosis Related Group (DRG) database.

Results: Most patients begin their medical process through General Practice. After referral, patients are ultimately transferred to Hematology for final diagnosis and follow-up. HCPs report a time to diagnosis of up to one year with multiple referrals needed and limited treatment options. Patients eligible for transplant may reach a median overall survival of 4-5 years, while non-eligible patients tend to live 1 to 2 years after diagnosis. Only 10% of patients are eligible for Autologous Stem Cell Transplant (ASCT), with the preferred choice of treatment being Cyclophosphamide, bortezomib, dexamethasone (VCD). On the other hand, most patients (~90%) are not eligible for ASCT, where VCD triple combination or combinations of proteasome inhibitors are reported Standard of Care (SoC) for frontline treatment. Since effective therapies for this disease are lacking, such results may lead to a widespread use of Daratumumab, as suggested in the recently published Mayo Clinic treatment guidelines. A forecast model was developed, considering a medium value for AL Amyloidosis prevalence in MM patients of 12.5%, and assuming a MM prevalence of 2 262 patients in Portugal. Between 2017 and 2018, only 68 patients were treated with an official diagnosis of AL Amyloidosis in Portuguese hospitals, which is lower than the reported prevalence in the literature. Considering NHS hospital DRG database and assuming the 12.5% prevalence of AL amyloidosis in MM patients, there is an estimate of 351 AL amyloidosis patients in Portugal, from which only 19.4% are properly diagnosed, while 80.6% are potentially miscoded or undiagnosed.

Summary/Conclusion: AL amyloidosis pathway is not well established, and hematologists report a long time to diagnosis and treatment. Physicians highlight an urgent need for well-tolerated therapies that can tackle organ involvement, with less frequent adverse effects and less severe toxicity. The lack of awareness in early detection, as well as patients developing AL Amyloidosis after a MM diagnosis that are not classified with the second pathology, are the main reasons claimed by physicians that lead to a possible underdiagnosis in Portuguese hospitals. The undeniable gap between the estimated prevalence of AL amyloidosis patients in Portugal and treated patients with an AL amyloidosis diagnosis coding in Portuguese public hospitals, reinforces the pressing need to adopt health policies that may prevent misdiagnosis/delayed diagnosis.

PB2026: CONSENSUS STATEMENT: IMPORTANCE OF TIMELY ACCESS TO MULTIPLE MYELOMA DIAGNOSIS AND TREATMENT IN CENTRAL AMERICA AND THE CARIBBEAN

M. Pimentel 1,* , O. D. F. Espinal Jaquez 2 , F. Godinez 3 , F. Jimenez 4 , D. Martinez 5 , N. Mendoza 6 , A. Quintana 7 , J. Richmond 4 , E. Romero 8

1 Hematology, INCART Instituto Nacional de Cáncer Rosa Emilia Tavares, Santo Domingo; 2 Hematology, Sociedad Dominicana Hematología, santiago, Dominican Republic; 3 Hematology, INSTITUTO GUATEMALTECO DE SEGURIDAD SOCIAL, Guatemala, Guatemala; 4 Hematology, CAJA COSTARRICENSE DE SEGURO SOCIAL, San Jose, Costa Rica; 5 Hematology, Instituto Hondureño de Seguridad Social, Tegucigalpa, Honduras; 6 Hematology, CHOP Centro Hemato Oncológico Panamá; 7 Hematology, Complejo Hospitalario Arnulfo Arias, Panama, Panama; 8 Hematology, Hospital Salvador B Gautier, Santo Domingo, Dominican Republic

Background: Background: In Central America and the Caribbean, multiple myeloma (MM) patients face significant barriers to diagnosis and treatment.

Aims: Aims: To describe the current situation of MM in the region, discuss the current barriers to timely diagnosis and proper treatment, and develop consensus recommendations to address these issues.

Methods: Methods: Nine experts from five countries took part in a virtual consensus meeting on MM in Central America and the Caribbean. During the meeting, experts analyzed the disease burden, the current conditions for disease management, and access to treatment in the region. The participants reached a consensus on the extent of the problem and the necessary measures.

Results: Results: Hard evidence on the incidence and prevalence of MM in the region is scarce, but the experts perceive an increase in MM cases. The lack of data on the direct and indirect costs at the local and regional levels obscures the impact of the disease and limits awareness among decision-makers. Most patients are diagnosed late, and face long waiting times and geographical barriers to access treatment. Access to efficacious innovative therapies that increase survival time is limited due to access barriers within health systems.

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Summary/Conclusion: Conclusion: There was consensus on five recommendations: 1) to generate evidence; 2) to educate the public; 3) to increase timely diagnosis and facilitate access to treatment; 4) to promote interaction, collaboration, and participation among all sectors involved in the decision-making process; and 5) to guarantee timely access to new therapies.

PB2027: PROGNOSTICATION IN THE EVERYDAY MYELOMA CLINIC. A SYSTEMATIC REVIEW OF PROGNOSTIC MODELS VALIDATED IN REAL WORLD POPULATIONS

L. Redder 1,2,3,* , T. Rosenberg 1 , N. Hyldig 1 , S. Möller 2,4 , N. Abildgaard 1,2,3

1 Department of Hematology, Odense University Hospital, Denmark; 2 Department of Clinical Research, University of Southern Denmark; 3 Academy of Geriatric Cancer Research (AgeCare); 4 Open Patient data Explorative Network - OPEN - Registry og Statistics, Odense University Hospital, Denmark, Odense, Denmark

Background: Multiple Myeloma (MM) is a severe malignancy with changing treatment possibilities and generally improved survival. However, prognosis is variable, and solid prognostic models for use in the daily clinic are warranted. Several new prognostic models and frailty scores have been introduced, but it can be difficult to keep track of their performance and validation.

Aims: To help clinicians prognosticate patients with newly diagnosed MM in the daily clinic, we performed a systematic review of studies validating prognostic models in general MM populations.

Methods: A review was performed according to the Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines (PRISMA-P) and pre-registered in the International Prospective Register of Systematic Review (PROSPERO) – identification number: CRD42021230604 https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=230604 .

Search strategy

Louise Redder, Tine Rosenberg, and Niels Abildgaard created the search strategy in collaboration with a Health Sciences Librarian from the University of Southern Denmark. A broad search string was set up with four search blocks: the first included a validated search filter for finding prognostic and diagnostic predictions studies. The second block searched all MeSH terms for MM. The third block contained an explorative search for progression free survival (PFS) and overall survival (OS), as these were our comparative outcomes and an inclusion criterion. In the fourth block, we attempted to target MM populations as close to the real world as possible, through an explorative search for population, cohort studies, multicenter or single center, and registry.

Two systematic literature searches were conducted on the 18 th of September 2020 and the 15 th of December 2021, using the electronic bibliographic databases, MEDLINE, EMBASE, and the Cochrane Library.

Inclusion & exclusion criteria

Studies validating or replicating prognostic models predicting OS and/or PFS in a population-based cohort of patients with newly diagnosed MM were included. Cohorts composed by fulfilling inclusion and exclusion criteria in clinical trials were excluded. Further, we excluded cohorts based on specific ethnicities, socioeconomic status, somatic diseases or disabilities, and populations representing other plasma cell dyscrasia, such as plasma cell leukaemia or smouldering MM.

A prognostic model was defined as a combination of at least two separate variables predicting patient outcome. The model had to predict OS/PFS at the time of diagnosis.

The design could be cohort, nested case-control or case-cohort studies as they are recommended for prognostic model development and validation. Prospective as well as retrospective studies were included, but had to include at least 50 participants and to reflect current treatment. Data had to be collected in 2005 or later.

Results: In total 7,128 studies were identified, whereof 1,475 duplicates were removed in Covidence. By screening titles and abstracts, 5,109 studies were found to be irrelevant. Full texts were obtained for 544 studies, and 438 were excluded due to the reasons explained in figure 1. Thus, 106 studies were included in this systematic review. Several studies validate ISS and R-ISS, but also models like the IMWG frailty score, R-MCI, MRP, CCI, IPSI, HCT-CI, KFI, CONUT score, and mSMART were represented in the literature search. See firgure 1.

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Summary/Conclusion: The work is ongoing. By the end of May 2022, we will be able to present detailed results of our systematic review of prognostic models predicting PFS or OS at the time of diagnosis.

PB2028: TREATING MULTIPLE MYELOMA IN A RESOURCE-LIMITED SETTING: REAL-WORLD OUTCOMES

G. Martinez 1 , F. Seguro 1,2,* , M. Jacomassi 2 , H. Visnadi 1 , M. Atanazio 1 , R. Szor 1 , P. Neffá 1 , T. Pereira 1 , W. Silva 1 , P. Dorlhiac-Llacer 2 , R. Velasques 1 , L. Bassolli 2 , V. Rocha 2

1 Hematology, Instituto do Câncer do Estado de São Paulo; 2 Hematology, Hospital das Clinicas, Faculdade de Medicina, Universidade de São Paulo, Sao Paulo, Brazil

Background: Over 70% of the Brazilian population (> 150 million people) do not have access to private health care. Alkylating agents, thalidomide, and autologous stem cell transplantation (ASCT) are the mainstay of newly diagnosed multiple myeloma (NDMM) treatment in the public health care system. Therefore, real-life data from resource-limited countries are scarce and essential to optimize the incorporation of new technologies into this scenario.

Aims: Describe characteristics of patients with NDMM, access to therapy, and outcomes, including overall survival (OS) and progression-free survival (PFS).

Methods: Retrospective chart study including all patients with NDMM treated at Hospital das Clinicas de São Paulo Complex between 2009 and 2019.

Results: A total of 816 patients with NDMM were included. Mean age was 63 years and 55% were male. At the diagnosis, 91% of patients had bone lesions, 56% anemia, 41% hypercalcemia, and 26% presented with impaired renal function. Regarding the clinical stage, 88% were Durie Salmon III clinical stage, and 46% were ISS III stage. Median follow-up time was 5 years. A triplet regimen (cyclophosphamide, dexamethasone, and thalidomide) was the most prescribed in the frontline (446/792, 56%), followed by alkylating agent + dexamethasone regimens (281/792, 27%). Only three patients (0.4%) in this cohort received bortezomib upfront. 379 (46%) were referred to ASCT for consolidation, and 257 (25%) proceeded with it in the first-line treatment. 36% of the patients received three or more lines of therapy. Patients who underwent ASCT had fewer comorbidities by Charlson’s index, lower ECOG at diagnosis, and fewer ISS stage III patients (31%) (p<0.001 for all), as shown in Table 1. Only 16% of the patients over 60 years underwent ASCT. Median time from diagnosis to transplant was ten months. Early mortality (≤ 120 days after diagnosis) was 9.7%, mainly due to infection. For the whole cohort, the overall response rate (ORR) after the first induction was 408/647 (63%), and 144 (22%) had disease progression. PFS and OS were 1.7 years (95% CI 1.6-1.9) and 3.7 years (95% CI 3.4-4.2), respectively. Median OS for patients that received ASCT was 5.9 (95%CI 5.1-7.8) years.

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Summary/Conclusion: This real-world cohort demonstrated that the available therapy in a resource-limited setting is far from the current gold standard for NDMM. Alkylating-based therapy results in suboptimal response rates, while the ASCT procedure is not timely available for all patients. In addition, poor clinical status and advanced disease may impact the choice of not using triplet regimens upfront. Early diagnosis, incorporating proteasome inhibitors and newer agents into the frontline, and ASCT availability are the major initiatives to decrease the disparity between rich and developing countries.

PB2029: MULTIPLE MYELOMA AND SPINAL CORD COMPRESSION: THE EXPERIENCE OF THE HEMATOLOGY DEPARTMENT OF SFAX

F. Kallel 1 , N. Siala 1,* , K. Mezghani 1 , M. Charfi 1 , L. Ghorbel 2 , H. Ben Salah 2 , Y. Fakhfekh 1 , O. Kassar 1 , L. Khelifa 1 , S. Hadiji 1 , I. Frikha 1 , I. Ben Amor 1 , J. Daoued 2 , M. Medhaffar 1 , M. Elloumi 1

1 Hematology, Hedi Chaker Hospital; 2 Radiotherapy, Habib Borguiba Hospital, Sfax, Tunisia

Background: Spinal cord compression (SCC) is the most serious complication in Multiple myeloma (MM). An emergency that must be diagnosed and treated promptly to preserve neurological functions and save the patient’s live.

Aims: We studied the clinical, biological, and radiological features of patients with MM from southern Tunisia complicated by SCC and the different modalities of treatment.

Methods: It is a retrospective study over 18 years (2003-2020) which included young patients (pts) (<65years old) with MM presented SCC and treated at the hematology department of Hedi Chaker Hospital Sfax, Tunisia. Here we are interested in studying the characteristics (clinical, biological, and radiological) of affected pts, the various revealing symptoms of SCC, the possible therapeutic modalities and the outcome of patients.

Results: Among 173 young pts followed with MM, 18 had SCC (10%) which was inaugural in 5 pts (27% of cases). The average age was 53 years (36- 63 years) and the sex ratio M/F= 10/8. The MM immunoglobulin type was IgG in 6 cases, IgA in 7 cases, Ig D in 1 cases and light chains in 4 cases. Symptoms of SCC included pain, motor defects, sensory deficits and bowel and bladder dysfunction in respectively 72, 11,100 and 2% of cases. While in 2 cases the SCC was asymptomatic (11%). The spinal MRI showed a cervical compression in 2 cases (11%), a dorsal compression in 8 cases (44.5%) and lumbar compression in 8 cases (44.5%). In 17 cases, lesions were first treated by radiotherapy (which did not produce regression of the compression). Decompressive laminectomy was carried out one time. All patients underwent high doses of corticosteroids. Pts were subsequently treated by different regimens of systemic therapy (DT in 12 cases, MPT in 3 cases and VTD in 3 cases). In 17 cases a definite and steady regression of the neurological symptoms was achieved. Median overall survival was 34 months after identification of SCC. At the date of last news, around 66% of the pts were dead.

Summary/Conclusion: The frequency of different localizations of compression in our studies are comparable to the literature, however, the SCC rate in our series is higher than those described in the other series in literature (10% vs 5%) the same for the rate of different symptoms at diagnosis (in literature more of asymptomatic form) which could be explained by the delay in the diagnosis of MM and by the neglect of minimal symptoms by patients and their consultation only after the installation of deficient signs.

PB2030: CLINICAL, LABORATORY PROFILE, TREATMENT AND OUTCOME OF PRIMARY AMYLOIDOSIS IN THE ERA OF NOVEL AGENTS: AN EXPERIENCE FROM A TERTIARY CARE HOSPITAL IN INDIA

Y. Sivaprakasam 1,* , J. Kotwal 1 , A. Saraf 1 , P. Gupta 2 , N. Gupta 1

1 Hematology; 2 Histopathology, Sir Ganga Ram Hospital, New Delhi, India

Background: Diagnosis of AL amyloidosis requires demonstration of amyloid in affected tissues along with clonal plasma cells in bone marrow or presence of monoclonal light chains in blood. With increasing awareness among physicians and availability of proper diagnostics, more cases of AL amyloidosis are being diagnosed. Here we present our experience of AL amyloidosis diagnosis and treatment in the era of modern diagnostics and therapy with novel agents.

Aims: We aimed to describe the clinical presentations, laboratory features and outcomes of patients with AL amyloidosis in a single center using standard diagnostic tests and treatment with novel agents.

Methods: A retrospective analysis of AL amyloidosis patients, diagnosed in our hospital, a tertiary care center in India from January 2016 to December 2021. The data was collected from departmental database. All statistical analyses were done by SPSS version 17.

Results: Diagnosis of AL amyloidosis was done in 27 patients. Median age of presentation was 59 years. 22 (81.5%) were males. Major symptoms were pedal edema (37%), shortness of breath (22.2%), frothy urine (11.1%) and fatigue (11.1%). Twenty two (81.5%) presented with ECOG PS ≥ 2. Most common system involved was renal in 16 (59.2%), followed by cardiac in 13 (48.1%) and gastro-intestinal in 9 (33.3%). Fifteen (55.6%) had two or more system involvement while 12 (44.4%) had single system involvement. Lambda monoclonal light chain was present in 22/27 (81.5%) and kappa monoclonal light chain was present in 5/27 (18.5%). Median Hb was 11.6 g/dl (range 6.7-14.8 g/dl), median M-protein was 0.69 g/dL (range 0-2 g/dL) and median bone marrow plasma cells were 7% (range-1-18%). Fourteen patients were treated; cyclophosphamide, bortezomib and dexamethasone (CyBORD) in 10/14 (71.4%) and bortezomib + dexamethasone in 4/14 (28.6%). Among 14 patients followed up with median follow up of 13 months (range 6-60 months), 5 expired; 3 due to COVID, one due to cardiac arrhythmia (during first cycle) and one due to relapse and rest 9 were alive. Among the 9 patients who were alive 6 were in complete hematological response and 3 were in partial response after 6 cycles of therapy.

Summary/Conclusion: Our study presents the spectrum of clinical manifestations, management and outcomes of primary amyloidosis in Indian context. There is a need to increase the awareness among the physicians about amyloidosis so that early diagnosis can be made and timely treatment can be done with novel agents to improve the dismal historical results.

PB2031: CD269, CD319 EXPRESSION ON MALIGNANT PLASMA CELLS AND SOLUBLE BCMA, SLAMF7 PROTEINS COMPARATION IN NEWLY DIAGNOSED MULTIPLE MYELOMA PATIENTS

A. Startsev 1,* , M. Solovev 1 , V. Dvirnyk 1 , M. Panasenko 1 , I. Galtseva 1 , N. Kapranov 1 , M. Firsova 1 , E. Makunina 1 , A. Abakumova 1 , E. Mamaeva 1 , L. Mendeleeva 1

1 National Medical Research Center for Hematology, Moscow, Russian Federation (Russia), Moscow, Russia

Background: Malignant plasma cells (PC) immunophenotype is well understood and represented by a large group of antigens expressed with varying frequency. Clusters of differentiation are both diagnostic and prognostic factors. Some of these serve as targets for monoclonal antibody and genetically engineered T-cells with chimeric antigen receptors in multiple myeloma (MM) patients. These molecules are SLAMF7 (Signaling lymphocytic activation molecule factor - CD319) and BCMA (B-cell maturation antigen - CD269). Membrane subunits of these molecules can be cleaved by enzymes (f.ex. γ-secretase) and released to the plasma as soluble proteins and can be easily detected using readily available commercial enzyme-linked immunosorbent assays (ELISA). SLAM family of receptors is a self-regulating and stimulates tumor proliferation. Therefore, soluble BCMA detection is one of the predictors of refractory disease.

Aims: To compare the CD269, CD319 expression on malignant PCs and sBCMA, sSLAMF7 protein concentrations in serum of newly diagnosed multiple myeloma patients.

Methods: The prospective study included 18 patients (8 women, 10 men) aged 26 to 70 years (median 54) with NDMM between June and September 2021. They received VRD induction therapy (2-4 cycles, median 4). Malignant PCs immunophenotyping was determined by multicolor flow cytometry (MFC) (CD319; CD269; CD138; CD38; CD19; CD45; CD81; CD27; CD20; CD56; CD200; CD117). Concentration of soluble BCMA, SLAMF7 in serum was detected using commercial ELISA (RayBio® Human CRACC/SLAM7 ELISA Kit, RayBio® Human TNFRSF17 ELISA Kit). Statistical analyses were performed using SPSS.

Results: The results of the CD269, CD319 and sSLAMF7, sBCMA protein concentrations in the serum of NDMM patients are presents in Table 1.

According to MFC of bone marrow malignant PCs, the presence of CD319 is detected in 90% of patients (n=16). sSLAMF7 protein is detected in the serum of 39% of patients (n=7) in the range of 1.14 to 5.43 ng/ml (median 2.9), in 61% of cases (n=11) sSLAMF7 wasn’t detected. In 56% of cases sSLAMF7 wasn’t detected, but CD319 was detected on the surface of malignant PCs. No cases of sSLAMF7 without the presence of CD319 on the malignant PC were detected in this study.

CD269 was detected on malignant PCs in 45% of patients (n=8) and in 55% of cases (n=10) malignant PCs was CD269 negative. sBCMA was detected in 100% of patients (n=18). The concentration ranged from 122.33 to 11617.32 pg/ml (median 2139.7). In the group of CD269 positive patients, sBCMA concentration was significantly (p=0.001) higher than in CD269 negative group: medians were 5334.12pg/ml (1933.6-11617.3) vs 318.87pg/ml (122.3-3962.2), respectively.

After induction therapy response was complete response in 16.5% (n=3), very good partial response in 12% (n=2), partial response in 66% (n=12), and progression disease in 5.5% (n=1). However, statistically significant correlations were not found between the depth of response, the presence of CD319, CD269 on the malignant PCs or/and the concentration of sSLAMF7, and sBCMA.

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Summary/Conclusion: The high frequency of CD319-positive patients was not accompanied by detection of sSLAMF7 in 61% of patients. Whereas, 100% sBCMA detection in NDMM patients was not followed by CD269 detection on malignant PC in half of the patients. These findings may indicate both the heterogeneity of MM and the necessity for further studies of sBCMA and sSLAMF7 to determine their prognostic significance.

PB2032: MAGNETISMM-9: AN OPEN-LABEL, MULTICENTER, NON-RANDOMIZED PHASE 1/2 STUDY OF ELRANATAMAB IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

R. Fonseca 1,* , J. Kuroda 2 , T. Ishida 3 , R. Popat 4 , J. S. Huang 5 , A. Yver 6 , E. Vandendries 7 , M. Elmeliegy 8 , W. D. Ma 9 , D. W. Sborov 10

1 Division of Hematology/Oncology, Mayo Clinic Hospital, Phoenix, United States of America; 2 Division of Hematology and Oncology, Kyoto Prefectural University of Medicine, Kyoto; 3 Japanese Red Cross Medical Center, Shibuya, Japan; 4 NIHR UCLH Clinical Research Facility, University College London Hospitals NHS Foundation Trust, London, United Kingdom; 5 Division of Hematology, National Taiwan University Hospital, Taipei, Taiwan; 6 Pfizer Inc, Paris, France; 7 Pfizer Inc, Cambridge; 8 Pfizer Inc, La Jolla; 9 Pfizer Inc, New York; 10 Division of Hematology & Hematologic Malignancies, Huntsman Cancer Institute, Salt Lake City, United States of America

Background: Elranatamab (PF-06863135) is a humanized bispecific antibody that targets both B cell maturation antigen (BCMA) on multiple myeloma (MM) cells and CD3 on T cells resulting in T cell- mediated cytotoxicity. In preclinical studies, elranatamab has demonstrated anti-myeloma activity and delayed disease progression. In the ongoing phase 1 MagnetisMM-1 study, elranatamab has demonstrated clinical efficacy and a manageable safety profile in patients with relapsed/refractory MM (Bahlis et al, J Clin Oncol 2021). Elranatamab maximum tolerated dose has not yet been reached. Subcutaneous elranatamab at the recommended dose of 76 mg once weekly on a 28-day cycle with a 2 step-up priming dose regimen administered during the first week is currently being evaluated in a phase 2 study, MagnetisMM-3 (Lesokhin et al, Blood, 2021).

Aims: To evaluate an alternative priming regimen to further mitigate cytokine release syndrome incidence and severity, and to evaluate subcutaneous elranatamab every 2 weeks and every 4 weeks in patients with relapsed/refractory MM.

Methods: MagnetisMM-9 (start date: October 2021; estimated completion date: April 2025) is an open-label, multicenter, non-randomized phase 1/2 study. In part 1 of the study, patients will receive premedications and 2 step-up priming doses on cycle 1 day 1 (4 mg) and day 4 (20 mg), followed by elranatamab 76 mg once weekly for 6 cycles, and a dose every 2 weeks thereafter if partial response or better is achieved and maintained. In part 2, higher doses of elranatamab (>76 mg) at increased intervals between doses will be evaluated. The primary endpoint is grade ≥2 cytokine release syndrome rate during cycle 1. Secondary endpoints include dose-limiting toxicities (part 2 only), objective response rate, duration of response, progression-free survival, overall survival, minimal residual disease negativity rate, safety, and pharmacokinetics. Eligible patients should be 18 years of age or older with a MM diagnosis and measurable disease according to International Myeloma Working Group criteria, refractory to at least one proteasome inhibitor, one immunomodulatory drug, and one anti-CD38 antibody, relapsed/refractory to their last treatment regimen, and have an Eastern Cooperative Oncology Group performance status ≤1. Key exclusion criteria are smoldering MM, active plasma cell leukemia, amyloidosis, POEMS syndrome, stem cell transplant within 12 weeks of enrollment, active, uncontrolled bacterial, fungal, or viral infections, or any other active malignancy within 3 years prior to enrollment (except for adequately treated basal cell or squamous cell skin cancer, or carcinoma in situ), or previous treatment with an anti-BCMA bispecific antibody. The study is open at centers in the USA, UK, Japan, and Taiwan. ClinicalTrials.gov ID: NCT05014412 .

Results: Results not available (trial-in-progress)

Summary/Conclusion: Conclusion not available (trial-in-progress)

PB2033: INFLUENCE OF INDUCTION THERAPY WITH LENALIDOMIDE ON MAINTENANCE THERAPY AND PROGNOSIS AFTER AUTOLOGOUS STEM CELL TRANSPLANTATION IN PATIENTS WITH MULTIPLE MYELOMA

Q. Sun 1 , J. Liu 1 , B. Huang 1 , J. Gu 1 , X. Li 1 , J. Li 1,*

1 Department of hematology, FAH of SYSU, Guangzhou, China

Background: We have finished a single-center, prospective study registered at www.chictr.org.cn (ChiCTR1900021558). 60 newly diagnosed multiple myeloma (NDMM) patients were included from 2017 to 2018, which were divided into lenalidomide, liposomal doxorubicin, and dexamethasone (RAD) group (n=30) or bortezomib, liposomal doxorubicin, and dexamethasone (PAD) group (n=30). Patients received 4-6 cycles of RAD or PAD induction therapy every 4 weeks, with subsequent autologous stem cell transplantation (ASCT) and maintenance therapy. This prospective study showed that the failure rate of stem cell mobilization in the RAD group was significantly higher than that in the PAD group. The hematopoietic reconstruction time in the RAD group was longer than that in the PAD group.

Aims: Comparison of the process of maintenance therapy, efficacy, and prognosis between RAD induction and PAD induction in patients in previous clinical studies.

Methods: Patients who completed induction therapy, ASCT, and received maintenance therapy through the previous clinical trial in our center were retrospectively analyzed. We divided enrolled patients into two groups according to the induction regimens, and compared maintenance treatment discontinuation, efficacy, time to progress (TTP), and overall survival (OS) between the RAD group and the PAD group.

Results: 21 patients in the PAD group and 12 patients in the RAD group were enrolled in the study, the median follow-up time was 44.6 months. 28 patients received thalidomide and 5 received lenalidomide at the beginning of maintenance therapy. The rate of maintenance treatment discontinuation was higher in the RAD group compared to the PAD group (41.7% vs. 4.8%, P =0.016), but the interval of maintenance treatment discontinuation was not statistically different (1.0 month vs. 0.9 months, P =0.917). During maintenance treatment, patients in the RAD group had a lower median count of white blood cells than the PAD group (2.96×10 9 /L vs. 3.38×10 9 /L, P =0.033), and so did the median count of platelets (102×10 9 /L vs. 150×10 9 /L, P =0.011). The incidence of grade 3/4 hematologic adverse events (AEs) was also higher in the RAD group than in the PAD group (66.7% vs. 28.6%, P =0.033). The ratio of patients who achieved CR and MRD-negative in the RAD group was lower than those in the PAD group ( P =0.047 and 0.016, respectively). The median TTP and median OS were not reached in both groups, but the median TTP and OS in the RAD group were shorter than those in the PAD group ( P =0.032 and 0.022, respectively). Reinfusion of CD34 + cells <2×10 6 /L was an independent prognostic factor for TTP ( P =0.009); minimum count of platelets <50×10 9 /L might be an independent prognostic factor for OS ( P =0.050).

Summary/Conclusion: Patients who received the RAD induction had worse efficacy and prognosis after receiving maintenance therapy compared to the PAD group, possibly due to discontinuation of maintenance therapy caused by a higher incidence of grade 3/4 hematologic AEs.

PB2034: CAMMA 1: A MULTICENTER PHASE IB TRIAL EVALUATING THE SAFETY, PHARMACOKINETICS AND ACTIVITY OF CEVOSTAMAB-CONTAINING REGIMENS IN PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA

R. Vij 1,* , H. Schade 2 , S. Trudel 3 , A. C. Chang 4 , J. Huang 4 , D. Samineni 4 , T. Sumiyoshi 4 , J. Tsai 4 , T. Wong 4 , S. J. Harrison 5

1 Washington University, St. Louis, MO; 2 Colorado Blood Cancer Institute, Denver, CO, United States of America; 3 Princess Margaret Cancer Centre and University of Toronto, Toronto, ON, Canada; 4 Genentech, Inc., South San Francisco, CA, United States of America; 5 Peter MacCallum Cancer Center and Royal Melbourne Hospital, and Sir Peter MacCallum Dept of Oncology, University of Melbourne, Melbourne, VIC, Australia

Background: Treatment of relapsed/refractory (R/R) multiple myeloma (MM) is challenging, especially in later lines where drug resistance reduces therapeutic options and remission duration. Prognosis is poor (estimated survival: <1 year) for patients with MM who have received >3 prior lines of therapy and are triple refractory to immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs) and anti-CD38 agents (Gandhi et al. 2019). Thus R/R MM constitutes a significant unmet medical need. Fragment crystallizable receptor-like 5 (FcRH5) is expressed on myeloma cells with near 100% prevalence (Li et al. 2017), constituting a novel therapeutic target. Cevostamab is an IgG1-based T-cell-engaging bispecific antibody engineered to target the most membrane-proximal domain of FcRH5 on myeloma cells and cluster of differentiation 3 (CD3) on T-cells, resulting in T-cell killing of myeloma cells. Clinical data from the first-in-human Phase I study (GO39775) suggest that cevostamab monotherapy is highly active in heavily pre-treated patients with R/R MM, with an overall response rate of 56.7% at the 132–198mg dose level (Trudel et al. ASH 2021 Oral presentation). Thus, cevostamab’s activity and safety profile support further development. Due to their stimulatory effects on T-cell activity, combination of cevostamab with anti-myeloma agents (pomalidomide [P] or daratumumab [D]) may be synergistic, offering the potential to further improve efficacy.

Aims: To evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of cevostamab-containing combination regimens (Arm B: cevostamab plus P and dexamethasone [d] [Pd]; Arm C: cevostamab plus Dd) in patients with R/R MM. A modified weekly schedule for cevostamab is also under investigation (Arm A: cevostamab monotherapy).

Methods: CAMMA 1 ( NCT04910568 ) is an open-label, multicentre Phase Ib trial in patients with R/R MM. Patients must be aged ≥18 years, have an Eastern Cooperative Oncology Group Performance Status of 0 or 1 and a life expectancy of >12 weeks. Patients in all arms have R/R MM; Arms B and C include patients with prior IMiD and PI exposure. Patients with prior CAR-T therapy may enroll with a washout period of 12 weeks post-CAR-T infusion. Cevostamab is administered by intravenous infusion q1w (C1–2)/q2w (C3–6)/q4w (C7–13) in Arm A, q2w (C1–6)/q4w (C7+) in Arm B, and q3w (C1–8)/q4w (C9+) in Arm C. Each arm consists of a safety run-in and an expansion cohort. The primary objective is to evaluate the safety and tolerability of cevostamab plus Pd, cevostamab plus Dd and cevostamab monotherapy. Secondary objectives include assessment of activity, PK, immunogenicity, and pharmacodynamic biomarkers.

Results: Enrolment for Arm A is ongoing, with patients receiving up to 13 treatment cycles. Arms B and C are planned; patients will receive treatment until disease progression or unacceptable toxicity. All patients must provide informed consent.

Summary/Conclusion: CAMMA 1 is an ongoing study of cevostamab plus Pd, cevostamab plus Dd and cevostamab monotherapy in patients with R/R MM. Further study details will be presented.

PB2035: CAMMA 3: A MULTICENTER PHASE IB TRIAL EVALUATING THE SAFETY, PHARMACOKINETICS, AND ACTIVITY OF SUBCUTANEOUS CEVOSTAMAB MONOTHERAPY IN PATIENTS WITH RELAPSED OR REFRACTORY MULTIPLE MYELOMA

S. Delimpasi 1,* , H. Quach 2 , M. Cavo 3 , P. J. Ho 4 , C. H.-S. Lee 5 , A. Santoro 6 , R. Schots 7 , P. Vlummens 8 , D. H. Yoon 9 , S.-S. Yoon 10 , C. D. Santos 11 , D. Samineni 11 , J. Huang 11 , K. Wehrman 11 , U. Patil 11 , S. Sheikh 12 , M. A. Dimopoulos 13

1 Department of Hematology and Lymphoma, Evangelismos Hospital, Athens, Greece; 2 Faculty of Medicine, University of Melbourne, Department of Haematology, St. Vincent’s Hospital, Melbourne, VIC, Australia; 3 IRCCS Azienda Ospedaliero‐Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy; 4 Institute of Haematology, Royal Prince Alfred Hospital & University of Sydney, Camperdown, NSW; 5 The Queen Elizabeth Hospital and Royal Adelaide Hospital, SA, Australia; 6 Cancer Center in Humanitas Research Hospital, Milan, Italy; 7 University Hospital Brussels, Brussels; 8 Department of Clinical Hematology, Ghent University Hospital, Ghent, Belgium; 9 Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine; 10 Seoul National University College of Medicine, Seoul, South Korea; 11 Genentech, Inc., South San Francisco, CA, United States of America; 12 F. Hoffmann-La Roche Ltd, Basel, Switzerland; 13 Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

Background: Multiple myeloma (MM) remains an incurable disease. Although new treatment paradigms have increased survival, most patients relapse and treatment in later lines remains a challenge. Prognosis for patients refractory to immunomodulatory drugs, proteasome inhibitors and anti-CD38 antibodies is extremely poor, with an estimated survival of <1 year (Gandhi et al. 2019). Therefore, patients with relapsed/refractory (R/R) disease represent a high unmet need, and new targets and treatment modalities are needed. Cevostamab is an IgG1-based T-cell-dependent bispecific antibody engineered to target the most membrane-proximal domain of fragment crystallisable receptor-like 5 (FcRH5) on myeloma cells, and cluster of differentiation 3 (CD3) on T cells. This dual binding results in efficient immunological synapse formation and T-cell-mediated killing of myeloma cells. In the ongoing first-in-human Phase I GO39775 study, intravenous (IV) administration of cevostamab monotherapy continues to show clinically meaningful activity and durable responses in patients with heavily pre-treated R/R MM (Trudel et al. ASH 2021), and uses Cycle (C) 1 step-up dosing for the mitigation of cytokine release syndrome (CRS). Subcutaneous (SC) delivery of antibody therapies has been shown to be effective and well tolerated and offers several advantages over IV administration in regards to improved healthcare utilization, including ease of administration, reduced treatment burden and reduced hospitalization. The slower absorption rate observed with SC versus IV antibody therapies may also support the potential for SC cevostamab to provide a further improved CRS profile (Bartlett et al. ASH 2021).

Aims: To evaluate the the safety, tolerability, pharmacokinetics (PK) and preliminary activity of SC cevostamab monotherapy in patients with R/R MM.

Methods: CAMMA 3 (GO43227; ISRCTN26168155) is an open-label, multicenter Phase Ib dose-escalation and dose-expansion trial in patients with R/R MM. For inclusion, patients must be aged ≥18 years and must have R/R MM for which no established therapies are available or appropriate. Cevostamab is administered by SC injection in 28-day cycles, with step-up dosing in C1, q2w dosing in C2–6, and q4w dosing in C7–13. Patients may receive up to 13 cycles unless there is disease progression or unacceptable toxicity. Patients who respond to cevostamab but develop recurrent or progressive disease after 13 cycles may be eligible for cevostamab re-treatment. Primary objectives are to evaluate the safety and tolerability (including the maximum tolerated dose and dose-limiting toxicities) of SC cevostamab and to identify a recommended Phase II dose. Secondary objectives include assessment of PK, activity, and immunogenicity, and identification of biomarkers associated with response and resistance.

Results: Enrolment for CAMMA 3 is underway. All patients must provide informed consent.

Summary/Conclusion: CAMMA 3 is a study of SC cevostamab monotherapy in patients with R/R MM. Further study details will be presented.

PB2036: RISK AND PATTERN OF INFECTIONS IN NEWLY DIAGNOSED PATIENTS OF MULTIPLE MYELOMA VACCINATED UPFRONT AGAINST PNEUMOCOCCUS & INFLUENZA

A. VA 1,* , A. Jain 1 , P. Malhotra 1 , A. Jandial 1 , A. K 1 , D. Lad 1 , G. Prakash 1 , S. S 2 , A. A 3 , R. Aggarwal 4

1 Clinical Hematology & Medical Oncology; 2 Hematology; 3 Microbiology; 4 Immunopathology, PGIMER, Chandigarh, India

Background: Infections contribute to an early mortality risk of 15 percent in newly diagnosed multiple myeloma(NDMM) cases. There is a limited literature on the type of infections in fully vaccinated NDMM patients.

Aims: To study epidemiology, clinical profile and predictors of infection in NDMM who are immunised against pneumococci and influenza.

Methods: NDMM patients were prospectively studied for 6 months for the pattern of infections. All patients were vaccinated with pneumococcal and Influenza vaccine at diagnosis. PJP prophylaxis and fluconazole prophylaxis was given for patients receiving high dose steroids while acyclovir was given to all. Infections were classified as microbiologically defined, clinically defined and fever of unknown focus according to definitions published by the International Immunocompromised Host Society. Severity of infections were graded according to the NCI CTCAE Ver5.

Results: Forty-eight NDMM patients with a median age 55 years comprising of 26 males and 22 females were enrolled. Renal involvement was noted in 42% of enrolled patients and two third of them required renal replacement therapy. ISSIII and R–ISS III were 70.8 % and 62.5 % respectively. 85% had poor performance status(ECOG ≥2) at baseline. RVD was the most common regimen (37%)used. 6 patients received daratumumab based regimen. Treatment response of atleast VGPR was seen in 97 % of NDMM patients.

A total of 19 episodes of infections were observed during 6 months. All episodes of infections were reported in the first 45 of myeloma diagnosis(Median 6 days; Range 0–45). Ten of these episodes of infection were diagnosed during the initial evaluation for myeloma defining events. Microbiological diagnosis was possible in 63 %. Commonest infectious agent was COVID 19(n=8) followed by Gram negative bacteria (n=5) viz E.coli and Klebsiella pneumoniae . None of the eight patients who developed COVID 19 infection had received COVID vaccine as they antedated the operationalisation of national guidelines for immunisation. Respiratory and the urinary tract were the most common focus of infection. All critically ill COVID patients succumbed to progressive respiratory failure and all patients with mild and moderate COVID illness recovered uneventfully. Early mortality in our cohort of forty eight patients was twenty percent(n=10). Three fourths of infections in our cohort were Grade≥3 severity. A total of seven deaths were attributable to infectious diseases in this cohort of NDMM patients. Imune paresis was seen in eighty four percent of patients at diagnosis. On follow up at 6 months; immune paresis had persisted in only thirty seven percent. Regression analysis of variables with odds of infection is shown in Table 1

Baseline BMI<18.5 kg/m2; albumin<3g/dl and ISS or R–ISS stage ≥ 2 was found to be have statistically significant odds of predicting infection risk in the cohort of patients. The choice of myeloma regimen, presence of high risk cytogenetics and response to therapy did not correlate with increased odds of infection in our cohort.

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Summary/Conclusion: Conclusion

In this prospective study of NDMM patients vaccinated against pneumococci and influenza at baseline; infection attributable early mortality was 14.5 %. Advanced stage of presentation, hypoalbuminemia and baseline BMI < 18.5 kg/m2 correlated with increased odds of infection. COVID vaccination and COVID appropriate behavioural practices may mitigate COVID related outcomes including deaths in myeloma patients.

PB2037: RESULTS FROM PIMMS, A UK MYELOMA RESEARCH ALLIANCE (UKMRA) STUDY OF OUTCOMES AND TOXICITY OF PANOBINOSTAT WITH BORTEZOMIB AND DEXAMETHASONE IN PATIENTS WITH RELAPSED MYELOMA IN A REAL WORLD SETTING

J. Waters 1,* , K. Boyd 2 , S. Moore 3 , J. Marawi 1 , P. Bassett 4 , C. Parrish 5 , R. Benjamin 1

1 Haematology, King’s College Hospital; 2 Haematology, Royal Marsden Hospital, London; 3 Haematology, Royal United Hospitals, Bath; 4 Statsconsultancy Ltd, London; 5 Haematology, Leeds Teaching Hospital, Leeds, United Kingdom

Background: Following the PANORAMA-1 trial, Panobinostat was approved in 2016 for the treatment of relapsed/refractory multiple myeloma (RRMM) in combination with bortezomib and dexamethasone (Pan-Bor-Dex) following at least 2 prior regimens including bortezomib and an immunomodulatory imide drug (IMiD). Since then, there have been several new drug approvals for RRMM and the treatment landscape has therefore changed significantly with Pan-Bor-Dex being used at later stages of the disease. It is therefore important to evaluate the efficacy and toxicity of this regimen in a real world setting given that the patient population being treated may be quite different to that in the PANORAMA-1 trial. Furthermore dosing schedules and dose modifications vary considerably in practice and may play an important part in minimising toxicity, preventing drug discontinuation and potentially impacting on duration of response.

Aims: The primary objective of this study was to determine the progression free survival (PFS) of RRMM patients in the UK receiving Pan-Bor-Dex. Secondary objectives were to assess overall response rate (ORR), depth of response, time to best response, duration of response, time to next treatment and overall survival as well as to assess incidence of grade ≥3 adverse events (AEs), dose modifications and impact on health care resource utilisation.

Methods: This was a UKMRA retrospective, observational, multi-centre study conducted at hospitals across England and Wales. All patients who received at least 1 cycle of Pan-Bor-Dex at each site between 01 Jan 2016 and 01 Jan 2018 were enrolled in the study. 01 Jan 2020 was the data cut-off date and patients who had not progressed by this date were censored at this timepoint.

Results: 219 patients were enrolled across 26 University and district general hospitals. The median age of the cohort was 69yrs (IQR 60-75) with 56% (n=106) aged ≥70yrs. The median number of prior treatment lines was 4 (IQR 3-5). 22% were refractory to a proteosomal inhibitor, 68% to an ImiD and and 5% to an anti-CD38 antibody. 54% had received an autologous stem cell transplant. Median number of cycles of Pan-Bor-Dex completed was 4 with 7.3% (n=16) of patients achieving the maximum 16 cycles approved by the National Institute of Health and Care Excellence (NICE).

ORR was 61% (n=133) with 37% achieving a partial response, 19% (n=44) a very good partial response and 5% (n=7) a complete response. Median PFS was 4.2 months (95% CI, 3.5-6) (see Figure). Initial sub-group analyses showed a significant difference in PFS between age groups. Older patients ≥70yrs achieved a median PFS of 6 months compared to 3.5 months in patients <70yrs (p value 0.02).

Toxicity led to discontinuation in 18% (n=38) of patients. Most frequent grade≥3 AEs included thrombocytopenia (59%), anaemia (39%), neutropenia (32%), diarrhoea (12%) and fatigue (12%).

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Summary/Conclusion: In this UK real world study of RRMM patients from 2016-2018 we have shown that the Pan-Bor-Dex regimen despite being used, on average, as fifth line treatment resulted in an ORR of 61%, comparable to that seen in the PANORAMA-1 Trial where patients were less heavily pre-treated. The overall median PFS was 4.2 months, not dissimilar to other drug regimens in advanced stage myeloma. Further data will be presented at the meeting including results of multivariate analysis to identify baseline patient factors that correlate with PFS.

PB2038: LENALIDOMIDE OR BORTEZOMIB AS MAINTENANCE TREATMENT OVERCOMES INFERIOR IMPACT OF HIGH-RISK CYTOGENETIC ABNORMALITIES IN TRANSPLANT-INELIGIBLE PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA IN CHINA

Z. Zhuang 1 , Y. Tian 2 , L. Shi 3 , D. Zou 4 , R. Feng 5 , W.-W. Tian 6 , H. Yu 7 , F. Dong 8 , A. Liao 9 , Y. Ma 10 , Q. Liu 11 , S. Liu 1 , H. Jing 8 , R. Fu 7 , L.-M. Ma 6 , H. Liu 5 , W. Sun 4 , L. Bao 3 , Y. Wu 2 , W. Chen 2 , J. Zhuang 1,*

1 Department of Hematology, Peking Union Medical College Hospital; 2 Department of Hematology, Beijing Chao-Yang Hospital of Capital Medical University; 3 Department of Hematology, Beijing Jishuitan Hospital; 4 Department of Hematology, Xuanwu Hospital of Capital Medical University; 5 Department of Hematology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Science, Beijing; 6 Department of Hematology, Shanxi Bethune Hospital of Shanxi Medical University, Taiyuan; 7 Department of Hematology, Tianjin Medical University General Hospital, Tianjin; 8 Department of Hematology, Peking University Third Hospital, Beijing; 9 Department of Hematology, Shengjing Hospital, China Medical University, Shenyang; 10 Department of Hematology, The Second Hospital of Shanxi Medical University, Taiyuan; 11 Department of Hematology, The First Affiliated Hospital of Anhui Medical University, Hefei, China

Background: Maintenance (MT) after front-line therapy is recommended in guidelines for multiple myeloma. However, no adequate data has shown the current situation of MT in China yet, especially in transplant-ineligible patients with newly diagnosed MM (NDMM).

Aims: We conducted this retrospective real-world study on efficacy and safety of the mainstream maintenance regimens in non-transplant NDMM patients—lenalidomide (L-MT), bortezomib (B-MT) and thalidomide (T-MT).

Methods: Clinical data were collected from 11 centers of North China MM Registry from January 2010 to July 2021. Patients achieving partial remission (PR) or better after induction therapy were included. The progression-free (PFS) and overall survival (OS) from MT, and drug toxicities were compared in L-MT, B-MT and T-MT groups. Thalidomide 75-150mg/day was administrated in T-MT group. L-MT group received lenalidomide as 25mg every other day or 10mg daily, on days 1–21 in a 28-day cycle. Bortezomib (1.3 mg/m 2 s.c.) was administered every 2 weeks or 4 vials every 3 months. Dexamethasone was given along with T, L or B in some patients.

Results: A total of 350 patients were enrolled including 155 in T-MT, 141 in L-MT and 54 in B-MT. At baseline, the gender ratio, paraprotein isotype, ISS and R-ISS stage were comparable. Patients on L-MT were significantly older than the other groups. Meanwhile, greater proportions of patients in L-MT and B-MT group had high-risk cytogenetic abnormalities (HRCA), defined as amplification 1q21, deletion 17p, t(4,14), t(14,16).

The median follow-up duration since maintenance was 42.5, 22.0 and 26.4 months (m) in T-MT, L-MT and B-MT groups, respectively. Disease progression was recorded in 101 patients (65.2%) with T-MT, 58 (41.1%) with L-MT and 22(40.7%) with B-MT. While mortality was 51(32.9%), 23(16.3%) and 5(9.3%), respectively. The median PFS was 23.7m in T-MT, as compared with 27.4m in L-MT and 29.7m in B-MT (p=0.52). Median OS was 90.7m in T-MT, whereas not reached (NR) in L-MT and B-MT groups (p=0.46).

Patients achieving complete response (CR) or stringent CR (sCR) before MT had prolonged PFS compared to those with VGPR or less in T-MT (40.1m vs 17.2m, p＜0.01), while comparable in L-MT (27.4m vs 26.9m, p=0.09) or B-MT group (NR vs 29.7m, p=0.34). Meanwhile, patients in each group had similar OS despite of different response before MT, their PFS and OS were also comparable despite of different induction regimens. Long-term duration of maintenance which was defined as over 24 months translated into significant better PFS in all groups (T-MT 57.2m vs 12.1m, L-MT 54.3m vs 19.2m, B-MT 37.3m vs 16.7m, p＜0.01), as well as better OS.

For patients with high-risk cytogenetic abnormalities, T-MT resulted in impaired PFS (12.2m vs 21.2m, p=0.04) and OS (54.9m vs NR, p＜0.01). In contrast, PFS and OS were both comparable in patients with L-MT or B-MT no matter with HRCA or not.

The main reason of maintenance withdrawal was disease progression. Adverse event related discontinuation was seen in 8.5%, 5.1% and 8.3% of the patients in T-MT, L-MT and B-MT, respectively.

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Summary/Conclusion: In this multi-centered real-world maintenance study, thalidomide, lenalidomide or bortezomib after front-line therapy in non-transplant patients with NDMM has similar PFS and OS. However, patients on L-MT and B-MT have greater proportion of HRCAs, which drags down survival in T-MT, while L-MT and B-MT reverses the negative effect. Clinicians in the real practice prefer to select lenalidomide or bortezomib as maintenance in patients with HRCAs, while thalidomide is still an option for patients with standard risk.

PB2039: CLINICAL FEATURES AND NEXT-GENERATION SEQUENCING LANDSCAPE OF ESSENTIAL THROM-BOCYTHEMIA，PREFIBROTIC PRIMARY MYELOFIBROSIS, AND OVERT FIBROTIC PRIMARY MYELOFIBROSIS: A CHINESE RETROSPECTIVE STUDY

J. Huang 1,* , L. Zhang 1 , X. Ye 1

1 Department of Hematology, the Fourth Affiliated Hospital, College of Medicine,Zhejiang University, YiWu, China

Background: Since prefibrotic primary myelofibrosis (pre-PMF) was recognized as a separate enti-ty in the 2016 revised classification of MPN differed from essential thrombocythemia (ET) or overt fibrotic primary myelofibrosis (overt PMF), it has been a subject of de-bate among experts due to its indefinite diagnosis.

Aims: We conducted a retrospective study of patients with myeloproliferative neoplasms (MPNs) in China, including 56 ET pa-tients, 19 pre-PMF patients, and 43 overt PMF patients.

Methods: We conducted a retrospective study of patients with BCR-ABL-negative MPN from October 2014 to May 2021 in the Fourth Affiliated Hospital of Zhejiang University School of Medicine. Patients who were diagnosed with ET, pre-PMF or overt PMF according to the 2016 WHO revised Classification of MPN were included. Patients with incomplete information from laboratory tests and medical records were excluded. This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Zhejiang University School of Medicine in line with the principles of the Declaration of Helsinki.Statistical analyses were performed using SPSS statistics 26.0 and GraphPad Prism 8.0 software.

Results: Pre-PMF patients exhibited higher leukocyte counts[14.2(6.0-28.1)×109/L vs 9.6(4.0-55.0)×109/L, P=0.003], LDH values[307(233-479)U/L vs 241(129-1182)U/L, P<0.001], onset ages[67(32-76)years vs 50(16-79) years, P=0.006], a higher frequen-cy of splenomegaly(47.4% vs 16.7%, P=0.018) and hypertension(57.9% vs 23.2%, P=0.005) than ET patients. On the other hand, pre-PMF patients had higher platelet counts[960(500-2245)×109/L vs 633(102-1720)×109/L, P=0.017], haemoglobin levels[152(115-174)g/L vs 119(71-200)g/L, P=0.003], lower LDH val-ues[307(233-479)U/L vs 439(134-8100)U/L, P=0.007] and a lower frequency of splenomegaly(47.4% vs 75.6%, P=0.031) than overt PMF patients. Next-generation sequencing landscape was performed in 50 patients, revealed the frequency of EP300 mutations was significantly increased in pre-PMF patients compared with ET and overt PMF patients (60% vs 10% vs 15.79%, P=0.033), and WT1 was more often overexpressed (WT1/ABL1 copies≥1.0%) in patients with overt PMF than in those with ET or pre-PMF(54.55% vs 16.67% vs 17.65%, P=0.009). In terms of outcome, male sex, along with symptoms including MPN10, anaemia (haemoglobin<120g/L), thrombocytopenia (platelet count<100×109/L), leucocytosis (leukocyte counts>13×109/L), high LDH value (>350U/L), splenomegaly, WT1 overexpres-sion(WT1/ABL1 copies≥1.0%), KMT2A, ASXL1 and TP53 mutations, indicated a poor prognosis for PMF patients.

The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011; the 2019–2024 Academician Workstation of the Fourth Affiliated Hospi-tal of the Zhejiang University School of Medicine; the 2019–2022 Key Medical Dis-cipline (Hematology) Fund of Jinhua, China.

*Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China.

E-mail: househuang@zju.edu.cn

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Summary/Conclusion: In summary, the results of this study indicated that comprehensive evaluation of BM features, clinical phenotypes, haematologic parame-ters, and molecular profiles is needed for the accurate diagnosis and treatment of ET, pre-PMF, and overt PMF patients.

PB2040: INTERRELATION BETWEEN JAK2 V617F MUTATION AND THE INCIDENCE OF THROMBOTIC EVENTS IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS

D. Nikolova 1,2,* , A. Yordanov 3 , V. Damyanova 1 , A. Radinov 3

1 Department of medical genetics, Medical University - Sofia; 2 Laboratory of Genetics; 3 Department of Clinical hematology, University Hospital “St.Ivan Rilski”, Sofia, Bulgaria

Background: Thrombotic incidents are the major cause of death for patients with Philadelphia negative myeloproliferative neoplasms (MPNs). During years, several studies have investigated the role of JAK2 V617F mutation as a risk inferring factor for thrombotic complications, but with ambiguous and contradictory findings.

Aims: Our study aims to clarify the role of JAK2 V617F mutation in MPN in a large cohort of Bulgarian patients with previous medical history of vascular incidents.

Methods: 231 patients with a confirmed clinical diagnosis of various MPNs (essential thrombocytemia/ET, polycythemia vera/PV and primary myelofibrosis/PMF) were screened for the presence of V617F mutation in JAK2 gene by molecular-genetic testing. The data was interrelated to the previously collected history of thrombotic events of the patients. The results were statistically analyzed.

Results: The overall prevalence of thrombotic complications in our patients’ group was 25,01%. Venous thromboembolism occurred in 15 patients (25.9%), while brain/myocardial/splenic infarctions in 74.1% (43 patients). In patients from different groups, thrombotic events were distributed as follows: 39% in PV, 23.4% in ET and 40.6% in PMF. JAK2 V617F was found more frequently in patients with manifested thrombotic incidents (67.2%). There was no statistically significant difference in hematocrit, thrombocyte and hemoglobin level between patients with/without thrombotic incidents in different diagnostic groups.

Summary/Conclusion: Based on these findings, initial screening for the JAK2 mutation and careful monitoring for thrombotic events should be performed in patients with MPN.

PB2041: EVALUATION OF THE PROGNOSTIC VALUE OF THROMBOPHILIA MARKERS IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS

T. Makarik 1,* , K. Sabirov 2 , E. Kitsenko 3 , S. Treglazova 1 , E. Stepanova 1 , I. Fevraleva 1 , S. Kulikov 4 , A. Sudarikov 1

1 Department of Molecular Hematology; 2 Surgery department, National Research Center for Hematology; 3 National Research Center for Surgery; 4 Biostatistics, National Research Center for Hematology, Moscow, Russia

Background: The clinical course of myeloproliferative neoplasms (MPN) is often complicated by impaired microcirculation, arterial and venous thrombosis. According to the literature, common for MPNs JAK2 gene mutations may contribute to the development of venous thrombosis. Even in the absence of overt MPN, a significant proportion of patients with recurrent cerebral and/or portal vein thrombosis carry the JAK2 V617F mutation. It is known that hereditary thrombophilic conditions, such as congenital deficiency of anticoagulants (antithrombin, protein C, and protein S) and genetic mutations (factor V Leiden, prothrombin G20210A , etc.), play an important role in the pathogenesis of venous thrombosis. However, limited data concerning the potential contribution of these mutations to the thrombotic risk in MPNs have been provided so far.

Aims: Evaluation of the prognostic value of thrombophilia markers testing in patients with J AK2 V617F positive myeloproliferative neoplasms for the choice of treatment tactics.

Methods: The study included 50 patients with MPNs (diagnosed according to WHO, 2016) admitted to the National Research Center for Hematology (Moscow, Russia) for surgical assistance due to bleeding from varicose veins of the stomach and esophagus. The control group consisted of 72 donors of blood and its components. DNAs were isolated from 5-10 ml of blood or bone marrow using standard salt extraction procedure or AmpliSens reagent kit (InterLabService, Moscow, Russia). DNA concentration and purity were assessed spectrophotometrically. The JAK2 V617F mutation was quantified using the Reagent Kit from Synthol (Moscow, Russia) according to the manufacturer’s instructions. In-house multiplex allele-specific real-time polymerase chain reaction (AS-PCR-RT) protocol was used to detect polymorphic alleles G1691A in the F5 gene (Leiden mutation), G20210A and T165M in the F2 gene, С677T and A1298C in the MTHFR gene, and (-675)4G/5G in the PAI-1 gene.

Results: V617F JAK2 gene mutation was found in 86% of patients with MPN (group #1). In the control group (#2), no mutations in the JAK2 gene were found. The frequency distribution of mutations G1691A in the F5 gene (Leiden mutation), G20210A and T165M in the F2 gene, C677 T and A1298C in the MTHFR gene, and (-675)4G/5G in the PAI-1 gene in groups #1 and #2 is presented in Table 1. In group #1, the frequency of heterozygous mutation in the F5 gene was 8.9%. This is more than 6 times higher than the similar value in group #2. The frequency of the homozygous mutation (-675)4G/5G in the PAI-1 gene in group #1 was 1.74 times higher than in group #2. The frequency of the heterozygous mutation T165M in the gene F2 in patients from group #1 and donors from group #2 were 28.9% and 4.4%, respectively. Thus, the mutation rates of the F5, F2, and PAI-1 genes are statistically significantly different between patients with MPN-associated thrombosis and donors of blood and its components.

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Summary/Conclusion: Information about the presence of markers of hereditary thrombophilia may be of prognostic value for assessing the risk of thrombotic complications in patients with JAK2 V617F positive MPNs.

PB2042: CLINICAL AND MOLECULAR CORRELATION OF PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA: A RETROSPECTIVE ANALYSIS FROM A TERTIARY CARE CENTER IN INDIA.

R. Balasubramanian 1,* , V. Arya 1 , J. Kotwal 1 , D. Gupta 1 , N. Gupta 1

1 Hematology, Sir Ganga Ram Hospital, New Delhi, India

Background: Approximately 50–70% of patients of ET harbor JAK2 mutation while about 20–30% patients carry CALR mutations. Data from developed countries has shown that CALR mutated patients have higher platelet count and lower thrombotic risk compared to JAK2 mutated patients. In India, testing for CALR and MPL mutation and next generation sequencing has become available only in last 5 years. There is lack of data regarding mutation status and its clinical correlation. We studied clinical features of ET patients presented to our hospital according to their mutation status.

Aims: To study the molecular mutations and correlate them with the clinical features in ET patients

Methods: We did the retrospective analysis of the patients diagnosed as ET from January 2016 to December 2021. Allele Specific PCR and fragment analysis was done to detect JAK2 (V617F), MPL (W515L/K) and CALR exon-9 mutations. Risk stratification done as per revised IPSET (International Prognostic Scoring for thrombosis in Essential Thrombocythemia) score. Therapy was started in all the patients according to their risk score with hydroxyurea or aspirin as the standard of care.

Results: A total of 39 diagnosed cases of ET were identified. Median age was 55 years (30-83), 20/39 males (51.3%) and 19/39 females (48.7%). JAK2 mutation was found in 25.6% (10/39), CALR in 38.5% (15/39) and MPL in 5.1% (2/39) cases. Triple negativity was found in 30.8% (12/39) cases. Very low, low, intermediate and high-risk patients as per IPSET score were 53.9% (21/39), 10.2% (4/39), 20.5% (8/39) and 15.4% (6/39) respectively. The median age was statistically significantly lower in CALR positive cases compared to the JAK2 positive cases (45.9 vs 60.9years, p=0.011). The median Hemoglobin and platelet counts were slightly higher in CALR positive group (12.3 g/dL and 10.15x10 9 /l) compared to JAK2 mutated and triple negative groups but the difference was not statistically significant. Three patients (7.7%) presented with thrombosis; two with cerebral venous thrombosis both CALR positive, one with arterial stroke and MPL gene mutation. Ten (25.6%) patients were lost to follow up. One (2.6%) died to pulmonary infection. Median follow up period was 24 months. None of the patient transformed to acute myeloid leukemia or myelofibrosis. None of the patient developed thrombotic event after diagnosis and appropriate treatment.

Summary/Conclusion: We observed that CALR was the most common mutation in our study. The CALR mutated patients were younger in age but the median platelet counts, WBC count and Hemoglobin were not statistically different among different mutation groups. In contrast to western studies, thrombosis was observed to be higher in CALR mutated group than others in our patient population. We recommend larger multicenter study for further elucidation of these observations.

PB2043: ERYTHROCYTOSIS IN PATIENTS WITH TIBETAN EGLN1C127S MUTATION: IS IT A POLYMORPHISM IN PATIENTS LIVING AT SEA LEVEL?

G. Biagetti 1,* , A. Benetti 1 , F. Polese 1 , E. Cosi 1 , I. Bertozzi 1 , G. Ceolotto 1 , M. L. Randi 1

1 DIDAS, University of Padua, Padova, Italy

Background: Absolute erythrocytosis is characterized by persistently raised Haemoglobin (Hb) and hematocrit (Ht) levels. Some congenital erythrocytosis (CE) are caused by mutations in a variety of genes, including those involved in the Oxygen-sensing pathway (OSP) (VHL, HIF2a, PHD2) and erythropoietin-receptor (EPO-R) gene. We recently focused our attention on patients with PHD2 mutations, having encountered a number of them affected by erythrocytosis. The EGLN1 C127S is considered a polymorphism being very common in Tibetan individuals: in these subjects it facilitates adaptation to high altitude and therefore it is not associated with a phenotype of erythrocytosis.

Aims: To study if there is erythrocytosis in PHD2 patients people living at sea altitude like our patients.

Methods: We have chosen 12 patients with a PHD2 mutation previously studied with the Sanger method and we re-evaluated them again with NGS panel specific for genes involved in erythrocytosis: JAK2, EGLN1 (PHD2), EPOR, FTL, FTH, ASXL1, HFE, HFE2, TFR2, HAMP, SLC40A1, SLC11A2, VHL, BPMG, EPAS1 (HIF-2α). We then collected their hemoglobin and hematocrit at diagnosis, treatment for erythrocytosis and presence of other relatives with erythrocytotic phenotype. We arbitrarily used phlebotomies to maintain Ht under 50%, or antiplatelet agents to prevent thrombotic complications.

Results: The main data of our patients are summarized in the table.

The median basal Hb and Ht levels of both patients with Tibetan or other mutations are similar.

Two third of patients (66%) needed phlebotomies and 2 patients antiplatelet agents: 6 (85.7%) patients with Tibetan mutation and 4 (80%) of the others with different mutations needed treatment. Three patients had associated mutations: 1 EPO-R A99V, 1 VHL P25L and 1 EPAS1 T766P genes. All of them underwent phlebotomies.

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Summary/Conclusion: This research shows how most patients with PHD2 mutated erythrocytosis needed a treatment regardless of the mutation. Therefore, we can conclude that EGLN1 C127S genotype is associated in our patients with a phenotype of erythrocytosis and necessity of treatment, given the fact that they live at sea level.

While the Tibetan mutation facilitates adaptation to chronic reduced oxigen pressure which decreases erythrocytes production in subjects living at high altitude, at the normoxia condition occurring at the sea level the normal process is altered and the erythropoietin gene is activated through not degraded HIF-2 alpha by PHD2.

Congenital erythrocytoses are very rare conditions, and those due to PHD2 mutations are only a small part of them. Our study shows an intriguing aspect that needs larger and collaborative researches.

PB2044: TRIAL IN PROGRESS: OBSERVATIONAL STUDY IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS TREATED IN ITALY WITH MIDOSTAURIN (OVIDIO)

F. I. Grifoni 1,* , F. Mannelli 2 , C. Papayannidis 3 , M. Triggiani 4 , R. Bini 5 , D. Valsecchi 5 , R. Zanotti 6

1 Hematology Unit, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico, Milan; 2 CRIMM, Azienda Ospedaliera Universitaria Careggi, Florence; 3 IRCCS, Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna; 4 Division of Allergy and Clinical Immunology, Department of Medicine, University of Salerno, Salerno; 5 Novartis Oncology, Novartis Farma SpA, Origgio; 6 Hematology Unit, Department of Medicine, Azienda Ospedaliera Universitaria Integrata di Verona, Verona, Italy

Background: Mastocytosis is a group of diseases resulting from the accumulation of clonal mast cells (MC) in one or more organ systems. The World Health Organization (WHO) classifies the disease into cutaneous mastocytosis (CM), systemic mastocytosis (SM), and localized mast cell tumors. SM is further divided into indolent/smoldering SM and advanced SM (AdvSM), that includes aggressive SM (ASM), SM with associated hematological neoplasm (SM-AHN) and mast cell leukemia (MCL) (Valent 2022). Treatment with midostaurin, the first orally multikinase/KIT inhibitor approved for AdvSM in both the United States and the European Union in 2017, resulted in a relevant clinical activity with an acceptable safety profile as demonstrated by two single-arm trials (Gotlib 2016, DeAngelo 2018). After approval, few real-world evidence (RWE) reports have been published on the safety and efficacy of midostaurin. Indeed, sound evidence from a large real-world setting confirming the impact of midostaurin on disease burden, efficacy parameters, quality of life and safety is lacking. Also, reports on efficacy data according the International Working Group–Myeloproliferative Neoplasms Research and Treatment–European Competence Network on Mastocytosis (IWG-MRT-ECNM) response criteria are limited (Tzogani 2019, Szudy-Szczyrek 2021).

Aims: The non-interventional OVIDIO study aims to describe a cohort of patients with AdvSM treated with midostaurin according to local label and a cohort of patients with AdvSM non treated with midostaurin in clinical practice, to fill the knowledge gap between the clinical experience from registration trials and routine patient’s management in Italy.

Methods: OVIDIO is a multicenter, non-randomized, observational study in adult male and female patients with AdvSM. The study includes two cohorts: 1) patients with AdvSM treated with midostaurin starting from Jan 2016. The data collection period is of 60 months from the start of midostaurin treatment; 2) patients with AdvSM not treated with midostaurin (diagnosis between Jan 2010 - Dec 2015). The data collection period is of 60 months from the initial diagnosis of AdvSM. Patients’ data will be entered in the clinical database after all necessary local approvals from Ethics Committees and following the patient’s signed informed consent, if applicable. The study is conducted at approximately 25 sites located in Italy and it will enroll up to 80 patients in the midostaurin-treated cohort and up to 50 patients in the midostaurin-not treated cohort. The study co-primary objectives are: i) to describe the profile and disease burden of a cohort of patients with AdvSM treated with midostaurin according to approved local label; ii) to describe the profile and disease burden of a cohort of patients with AdvSM not treated with midostaurin. Secondary objectives include the characterization of clinical management in both cohorts and, specifically for midostaurin cohort, the evaluation of the effectiveness according to IWG-MRT-ECNM criteria, the impact on the quality of life (QoL) and the safety of midostaurin in AdvSM. As exploratory objective, the study will also compare the overall survival (OS) between patients treated and not treated with midostaurin.

Results: Trial in Progress. First interim data will be made available by the end of 2022.

Summary/Conclusion: As of February 2022, 64 and 18 patients have been enrolled in the midostaurin treated and not treated cohort respectively. Enrollment is ongoing.

PB2045: POLYCYTHEMIA VERA IN YOUNG SUBJECTS: ABOUT A COHORT OF 11 CASES

S. Bougherira 1,* , S. Bentorki 1 , F. Grifi 1

1 Hematology, University hospital, Annaba, Algeria

Background: Polycythemia Vera (PV) is a typical Philadelphia-negative (Ph _ ) myeloproliferative disease. It is secondary in 95% of cases to the JAK 2 mutation on exon 14. The median age at diagnosis is 65 years. PV is unusual in young patients, so that little information is available on long-term clinical evolution in this particular group.

Aims: The aim of this study is to describe the clinical presentation, biologic features, treatment approaches, and long-term outcome especially the risk of thrombosis.

Methods: Of all the 64 patients followed in our department for PV, we report 11 cases aged less than 55 years. The inclusion criteria were a diagnosis satisfying the PVSG criteria and, from 2016 on, the WHO criteria. Secondary causes of erythrocytosis were carefully ruled out in all patients. At diagnosis, cytogenetic and molecular analysis were not done in our patients. Search for JAK2 mutation and serum EPO dosage were performed in all cases.

Results: PV in young patients represents 17% of all PV followed. Median age of our population is 44 years, range 32-52, with 27% of patients under 40 years old. slightly more often in men with (sex ratio 1.75). Arterial hypertension is the most common comorbidity and it is a cardiovascular risk factor in the majority of patients. A thromboembolic event revealing PV is found in 2 patients. The main clinical signs were significant burning sensation in fingers and palms due to the increased blood viscosity, splenomegaly which observed in 63% of patients, and the occurrence of severe itching on contact with warm bath. Thromboembolic events occurred in 3 patients during the evolution. All patients had the diagnosis of absolute erythrocytosis with moderate hyperleukocytosis and thrombocytosis. Bone marrow biopsy showed hypercellular bone marrow with erythroid and megakaryocytic lineage hyperplasia. The JAK2 V617F mutation was positive with an average allelic load high at 63.1% [22.9 - 93.6]. The dosage of serum EPO was low with an average of 1 mU/ML. During the follow-up period, no patient had progression into acute leukemia or myelofibrosis with myeloid metaplasia, thus no death. The median survival is 49 months [3-99] and the survival without thromboembolic event is 42 months [3-99]. The majority of patients were treated by phlebotomy with 1 or 2 units per month, before starting hydroxy urea. The use of the latter in association with Aspirin (n=10 pts) reduced the use of bloodletting. The thrombotic events required treatment with antithrombotic drug. No patient was treated with Ruxolitinib (treatment not available).

Summary/Conclusion: PV remains rare in patients under 55 years of age (17% in our cohort). This has an impact on overall survival and thromboembolic event-free survival with a good prognosis in the young population.

PB2046: SHORT-TERM EFFICACY OF DECITABINE IN COMBINATION WITH LOW-DOSE CHEMOTHERAPY IN THE TREATMENT OF JMML: A RETROSPECTIVE STUDY

Y. Cai 1,* , J. Zhang 1 , M. Yi 1 , W. Zhang 1 , X. Liu 1 , X. Zhang 1 , Y. Wan 1 , L. Chang 1 , L. Zhang 1 , X. Chen 1 , Y. Guo 1 , Y. Zou 1 , Y. Chen 1 , J. Li 1 , Y. Zhang 1 , W. Yang 1 , X. Zhu 1

1 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Background: Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myeloproliferative disease, but several newly diagnosed patients cannot tolerate hematopoietic stem cell transplantation (HSCT) at diagnosis.

Aims: n the present study, we aimed to explore the short-term effect of decitabine in combination with low-dose chemotherapy on improving the condition of JMML patients before HSCT.

Methods: A total of 10 JMML patients were enrolled in this retrospective analysis. All the patients were treated with decitabine supplemented with low-dose chemotherapy with a interval of 4 weeks, bridging to HSCT after three~four courses.

Results: The median treatment course was three (one-five) cycles, and the response rate was 70.0% after one cycle and 71.4% after three cycles. The WBC and monocyte counts were significantly decreased after chemotherapy, and the spleen volume showed a decreasing tendency. The 12-month progression-free survival rate was 80.0±12.6%.

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Summary/Conclusion: Decitabine in combination with low-dose chemotherapy was favorable in reducing tumor burden and improving the clinical status.

PB2047: MOLECULAR DIAGNOSIS OF CONGENITAL ERYTHROCYTOSIS IN A SINGLE HOSPITAL CENTER.

L. Castilla Garcia 1,* , J. M. Aspa Cilleruelo 1 , G. Lopez de Hontanar Torres 1 , P. A. Rodriguez Barquero 1 , R. Sánchez Perez 2 , R. Ayala Diaz 2 , P. Ropero Gradilla 3 , J. Martínez Nieto 3 , A. González Fernández 3 , M. Argüello Marina 1 , M. Lopez Rubio 1

1 Hematology, Hospital Principe de Asturias; 2 Hematology, Hospital 12 de Octubre; 3 Hematology, Hospital Clínico San Carlos, Madrid, Spain

Background: The etiologic diagnosis of erythrocytosis is sometimes frustrating since about 70% are idiopathic. In primary erythrocytosis the only known form of congenital erythrocytosis is associated with erythropoietin receptor defects caused by mutations in the EPOR gene and has an autosomal dominant inheritance pattern. It is a rare entity that accounts for only 12% of erythrocytosis, with decreased EPO. Secondary congenital erythrocytosis present elevated EPO levels and normal erythroid precursors. They are mainly caused by mutations in genes of regulatory proteins of oxygen (O2) sensing pathways such as VHL, PHD2 (EGLN1) and HIF2A (EPAS1); mutations in α- or β-chain globin genes (HBB, HBA2, HBA1), or by mutations causing 2,3-bisphosphoglycerate mutase (BPGM) deficiency with 2,3-BPG deficiency, all of them presenting an increased affinity of Hb for O2.

Aims: To identify idiopathic erythrocytosis under follow-up in the hematology department and to study possible hereditary causes by means of molecular studies in order to be able to subsequently carry out a family screening.

Methods: Retrospective descriptive study of 13 patients under follow-up in the hematology department of the Hospital Príncipe de Asturias. By reviewing the clinical history, the personal and family history were gathered, alongside laboratory values at the time of diagnosis and the diagnostic tests used to rule out secondary and primary causes such as polycythemia vera (abdominal ultrasound, chest X-ray, blood gas analysis with cooximetry, etc.). All patients were tested for hemoglobinopathies at the Hospital Clínico San Carlos using high-performance liquid chromatography, capillary electrophoresis and DNA analysis. On the other hand, the Hospital 12 de Octubre performed a genetic study by next-generation sequencing (NGS), and the demographic and analytical characteristics are shown in the table.

Results: From a total of 13 patients we have found a mutation causing erythrocytosis in 6 of them, among which there are two family clusters. The first group consists of three patients, two of them (father and son) with mutations in EPAS1 and TET2, and the third (uncle) in TET2 and DNMT3. The second familial cluster consists of a mother and her child with Syracuse Hb. There is one patient with mutation in the EPO receptor. Among the patients with no pathogenic alteration identified, we have found one patient with EPO above normal levels and two others with alterations in P50 in arterial blood gases (one with elevated P50 and the other decreased). In 80% of our patients, the therapeutic management has done been by means of periodic bleeding to obtain a hematocrit <45%, which has been interrupted on occasions due to secondary iron deficiency anemia. In 6 patients, who also had associated cardiovascular risk factors, antiplatelet treatment with salicylic acetic acid was associated with antiplatelet therapy (100 mg per day). None of our patients had thrombotic events or other incidences.

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Summary/Conclusion: The diagnosis of erythrocytosis is a challenge because the clinical features may overlap with different etiologies, and it is not possible to distinguish them by conventional biochemical and molecular techniques; sometimes these are pathologies with mild or even non-existent clinical features, which means a delay in the diagnosis of these patients. Since targeted NGS provides rapid and accurate mutation analysis, this technique could be applied directly to cases of erythrocytosis in which a genetic cause is suspected in order to improve patient and family counseling and initiate early treatment.

PB2048: EFFICACY AND SAFETY OF RUXOLITINIB IN PATIENTS WITH MYELOFIBROSIS: A RETROSPECTIVE MULTICENTER STUDY

C. Chen 1 , S. Ma 1,* , F. Yan 1 , H. Zhao 2 , X. Chu 3 , H. Zhang 4 , X. Deng 5 , Z. Liu 6 , D. Li 7 , T. xiao 8 , C. Yuan 9 , Z. Wang 10 , G. Liu 11 , M. Hou 1 , Y. Yu 1

1 Qilu Hospital of Shandong University, Jinan; 2 Affiliated Hospital of Qingdao University, Qingdao; 3 The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai; 4 Linyi People’s Hospital, Linyi; 5 Weihai Municipal Hospital, Weihai; 6 Shandong Provincial Hospital of Traditional Chinese Medicine; 7 Central Hospital Affiliated to Shandong First Medical University, Jinan; 8 Liaocheng People’s Hospital, Liaocheng; 9 Qilu Hospital of Shandong University Dezhou Hospital, Dezhou; 10 Zibo Central Hospital, Zibo; 11 Shengli qilfield Central Hospital, Dongying, China

Background: Ruxolitinib is a Janus kinase (JAK) 1/2 inhibitor currently used in the treatment of Myelofibrosis (MF). Ruxolitinib confirmed improvements in splenomegaly, MF-related symptoms and survival benefit in COMFORT and JUMP studies. At present, the real-world data on the efficacy and safety of ruxolitinib in the treatment of MF in China is still insufficient.

Aims: The aim of this study was to evaluate the efficacy and safety of ruxolitinib in patients with MF and to provide guidance for the usage of ruxolitinib in MF in China.

Methods: This was a retrospective, multicenter study of MF patients who received ruxolitinib treatment in Shandong province from August 2012 to December 2021. Data were analyzed using SPSS. Overall survival (OS) and Event-free survival (EFS) were estimated using the Kaplan– Meier method.

Results: A total of 130 MF patients were recruited, among which 97 (74.6%) primary MF patients and 33 (25.4) secondary MF patients were involved. Most patients started at dose of 20 mg bid ( n =44, 33.1%), while other patients started ruxolitinib treatment at lower doses, including 15 mg bid ( n =29, 21.8%), 5 mg bid ( n =26, 19.5%) and 10 mg bid ( n =23, 17.3%). The most common adverse events (AEs) were anemia (23.1%), thrombocytopenia (15.4%), lung infection (9.2%) and elevated aminotransferase (5.4%). Overall, Ruxolitinib led to remarkable reductions in spleen length and MF-related symptoms in most patients. 74.7% (71/95) of patients experienced an improvement in splenomegaly at any time after medication with a median reduction of 33.3% (Fig. A). At week 24, 73.8% (45/61) of the evaluated patients had a ≥25% decrease in spleen length, therein 45.9% (28/61) reached a ≥50% decrease (Fig. B). 87.5% (70/80) of patients experienced decreased MPN-SAF TSS with a median of -63.4% (Fig. C). The median reduction of more than 50% from baseline in all MF symptoms was observed in 53 assessed patients (Fig. D). The estimated OS probability of Low/ Int-1 patients was 96.7% at 24 months. The estimated OS probability of Int-2/ High patients was 83.9% at 24 months (Fig. E). Primary causes of death on study included MF ( n =7), lung infection ( n =1), cerebrovascular accident ( n =1) and AML ( n =1). The median EFS of Low/ Int-1 patients was 21 months, while the median EFS of Int-2/ High patients was 8 months (Fig. F).

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Summary/Conclusion: In summary, Chinese MF patients receiving ruxolitinib showed improved splenomegaly and MF-related symptoms. Besides no new safety concerns were found. Less adverse events were observed in lower risk of MF patients during ruxolitinib treatment. Generally, the efficacy and safety profiles of ruxolitinib in the treatment of Chinese MF patients were basicly consistent with other studies.

PB2049: MEXICAN REGISTRY OF MYELOPROLIFERATIVE NEOPLASMS. INITIAL REPORT

J. A. De La Peña Celaya 1,* , A. L. Tapia Enriquez 2 , E. P. Paredes Lozano 1 , R. Bates Martin 1 , A. Villela Peña 1 , O. Teomitzi Sanchez 1 , R. Ovilla Martinez 3 , N. Ramirez Muñiz 3 , A. Cuevas Juarez 3 , A. M. Lopez Flores 3 , J. E. Medina Coral 4 , A. S. Garcia Camacho 5 , C. Garcia Castillo 6 , M. M. Bahena Garcia 6 , H. Romero Rodelo 7 , J. Aguilar Luevano 8 , P. E. Baez Islas 9 , J. L. Alvarez Vera 1 , J. M. Perez Zuñiga 1 , E. Hernandez Ruiz 1 , L. L. Arana Luna 1 , M. E. Espitia Rios 1 , L. Montoya Jimenez 1 , M. Alvarado Ibarra 1

1 Hematology, ISSSTE, Mexico; 2 Hematology, ISSSTE, Chihuahua; 3 Hematology, Hospital Angeles Lomas, Mexico; 4 Hematology, ISSSTE, Culiacan; 5 Hematology, IMSS; 6 Hematology, SEDENA, Mexico; 7 Hematology, ISSSTE, Tijuana; 8 Hematology, IMSS, Queretaro; 9 Hematology, Hospital Angeles Lomas, Hermosillo, Mexico

Background: The abnormal proliferation of one or more myeloid cell lines in peripheral blood gives rise to a heterogeneous group of diseases called myeloproliferative neoplasms (MPN). In Mexico the lack of records prevents knowing the exact incidence of this entities, however there have been some efforts to register, such as the one carried out by Ruiz-Argüelleset al in wich are described 24 patientsof one medical center conluding that these pathologies are a rare condition in the mestizo population of our country. Other works by Martínez-Flores et al in 2016 with 36 patients describe some of the clinical and prognostic implications of the JAK2 mutation in this population. Despite this efforts it is unknown the frequency and impact of these pathologies in our population.

Aims: To report the frequency of the myeloproliferative neoplasms in the Mexican population.

Methods: Multicenter, retrospective, observational report of myeloproliferative neoplasms in 13 mexican medical centers. The frequencies are expresed in median and porcentages.

Results: Eighty-six patients from 12 centers throughout the country were included. The baseline demographic characteristics, health status prior to the begining of symptoms and diagnosis are shown in Table 1. Of the total number of patients, bone marrow biopsy was performed in 76% all assessed by general pathologists, 75% of the centers had mutational tests available for clinical diagnosis, the rest based their protocols on clinical criteria and general studies. Of the baseline characteristics of the disease, the most frequent symptom was asthenia/adynamia (45%), 17% had a history of thrombosis at the time of diagnosis and 20% described bone marrow dysplasia. The most frequent clinical diagnosis according to WHO criteria was essential thrombocythemia (53%) which is not consistent with that described in pathology results where primary myelofibrosis was the most frequent (30%) (Fig.1). Disease progression was reported in 5% of patients (4 patients) and death in 4% (3 patients). The most used treatment was cytoredyuctyve therapy in 54%, follow by JAK inhibitor in 27% cases. A correlation between progression and mortality with the clinical diagnosis, the presence of blasts or dysplasia was sought and no statistical significance was found.

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Summary/Conclusion: To our knowledge this is the most extensive descriptive report of mieloproliferative neoplasias in our country. The heterogeneity of health systems in our country is reflected in the heterogeneity of the results obtained, the great differences in resources of each medical center complicate objective comparisons in order to correlate events with elements of the disease or patients. Comparative analyzes were carried out with the data obtained, however the number of patients did not allow a robust result. However this report should be understood as a first effort for a much-needed national registry that would be of great importance to homogenize the study and management of this disease in Mexico.

PB2050: EVALUATION OF NON-CLONAL ERYTHROCYTOSIS: SINGLE CENTRE EXPERIENCE

A. Šumah 1 , N. Vodošek Hojs 1,2 , M. Dreisinger 3,*

1 Faculty of Medicine, University of Maribor; 2 Department of Nephrology, Clinic for Internal Medicine; 3 Department of Haematology and Haematologic Oncology, Clinic for Internal Medicine, University Medical Centre Maribor, Maribor, Slovenia

Background: Secondary erythrocytosis has a broad differential diagnosis that includes hypoxic lung disease, cyanotic congenital heart disease, kidney disease, medications and erythropoietin-producing malignant disorders. Aetiology remains unknown in some patients, they have the so-called idiopathic erythrocytosis (IE). Among the latter, patients with inherited/congenital erythrocytosis can be found, especially if they have a positive family history.

Aims: The aim of our study was to analyse the characteristics of non-clonal erythrocytosis in patients referred to our Haematology department in a 5-year period (2016 – 2021) and to compare the IE group to the group with known causes.

Methods: We included all individuals who were Janus kinase 2 ( JAK2 ) mutation negative and had erythrocytosis. Data was retrospectively collected with the help of the hospital database and statistically analysed with SPSS.

Results: The study included 250 patients, 176 (70%) men and 74 (30%) women, with mean age 58.8 years (21 – 94 years). Their average haemoglobin values within a 1-year time period were 182.5 g/L for men and 169 g/L for women. Average haematocrit levels were 0.53 for men and 0.50 for women. Elevated erythropoietin (EPO) levels were found in 10.5% patients, 83.4% had normal and 6.1% decreased values. Symptoms were reported by 48% of the patients, mostly itchiness (16.4%), sweating (12%) and other (30%) such as tinnitus, myalgia, joint pain and snoring. The most probable reasons for secondary erythrocytosis were smoking/chronic obstructive pulmonary disease (COPD) (32%), IE (29%), dehydration (7%), nephrological aetiology (6%), obesity (4%), sleep apnoea (4%), asthma (4%), infection (4%), heart or pulmonary disease hypoxia (4%), possible hemochromatosis (3%), medications (2%) and high altitude/training (1%). Patients with IE had an average age of 57.2 years (24 – 83 years), 57 (78%) were men and 8 (11%) had a positive family history. EPO levels in this group were elevated in 7.8%, normal in 88.2% and decreased in 3.9%. Past thrombotic events were reported by 23.3% of individuals with IE and 20.3% of patients with known secondary cause. There was no statistical difference in age, gender, EPO, haemoglobin levels and past thrombotic events between the IE group and group with known causes. Patients with IE significantly differed from the group with known causes in haematocrit levels (p=0.0219) with lower levels in IE patients.

Summary/Conclusion: Among 250 patients with non-clonal erythrocytosis the majority had acquired aetiology. The most common cause was hypoxia caused by smoking or COPD. Patients with IE significantly differed from the group with known causes in haematocrit levels, while there were no significant differences between groups in age, gender, EPO levels, haemoglobin levels and past thrombotic events.

PB2051: PRIMARY MYELOFIBROSIS IN YOUNG PATIENTS ≤50 YEARS: CLINICOPATHOLOGICAL CHARACTERISTICS, GENETIC LANDSCAPE AND DISEASE OUTCOME. A SINGLE CENTER EXPERIENCE

S. Elkourashy 1,2,* , D. Soliman 3,4 , P. Chandra 5 , R. Taha 1 , H. Elomri 1

1 1Department of Hematology & BMT, NCCCR; 2 Clinical Medicine, Weill Cornell Medical College; 3 2Department of laboratory medicine and pathology, NCCCR, Doha, Qatar; 4 National Cancer Institute, Cairo University, Cairo, Egypt; 5 3Medical Research Center, Hamad Medical Corporation, Doha, Qatar

Background: Primary Myelofibrosis (PMF) is a rare myeloproliferative neoplasm, often diagnosed in the 6th and 7th decade. Approximately 5 and 17% of the patients are diagnosed before the age of 40 and 50 years, respectively.

Aims: Here, we aim to analyze PMF patients by age group, comparing young patients (≤50 years) to older age group, in terms of clinical, pathologic, genetic profile, treatment history and outcomes.

Methods: This is a single center retrospective study carried out in National Center for Cancer Care and Research, Hamad Medical Corporation over the period 2008-2021.

We identified WHO-defined PMF patients and collected their demographic data, bone marrow findings, driver mutations, clinical and hematologic characteristics. DIPSS-Plus score was calculated as well.

Time to progression, was considered as the time to acquisition of ≥1 of the following: Hemoglobin < 10 g/dl, Leukocyte count >25 x109/L, Platelet count < 100 x109/L, Circulating blast cells ≥1%, transfusion dependency. Overall survival (OS) and progression free survival (PFS) were assessed using Kaplan-Meier method.

Results: A total of 61 patients identified, from which 33(54.1%) were ≤50 years at the time of diagnosis. The median age at diagnosis was 39 years (range 24-50). 51.5% had prefibrotic MF. Females accounted for 51.5%.

DIPPS-Plus at diagnosis was calculated, 12 patients (36.4%) were low, 11(33.3%) intermediate-1, 8(24.2%) intermediate-2, and 2(6.1%) high-risk. Driver mutations; JAK2 mutation was detected in 19/33(57.6%), CALR in 10/26 (38.5%), MPL in 1/6 (16.7%) and only 1 was triple-negative. 30 patients had cytogenetic information. 29/30(96.7%) showed favorable Karyotype (of which 27 had normal Karyotype).

Around half of patients showed disease progression and two third (23 patients) need treatment eventually mainly Cytoreduction 18/23 (78.3%) then Ruxolitinib 11/23 (47.8%), 1 underwent Bone marrow transplant (BMT). Complications include thrombosis in 21.2% and refractory anemia 33.3% with transfusion need in 21.2%, but no leukemic transformation. 28 patients (84.8%) were alive over follow up period.

Comparison between 2 studied groups were summarized in Table 1.

Three patients died in the age group >50 years and one patient died in age group ≤50 years. No statistically significant difference observed in median OS between age ≤50 years (median 276.9 months, 75 th percentile 276.8 months) and > 50 years (median 315.9 months, 75 th percentile 108.7 months) (Log rank test P-value 0.494). The median PFS was observed to be higher in patients with age ≤50 years (median PFS 249.8 months, 75 th percentile 249.8 months) compared to patients with age >50 years (median PFS 105.3 months, 75 th percentile 93.04 months), however this observed difference was statistically insignificant (Log rank test P-value 0.172).

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Summary/Conclusion: PMF is rarely diagnosed in patients ≤ 50 years old (estimated < 20%), but surprisingly it constitutes > 50% of our cohort. Young patients showed female predominance, more favorable cytogenetics, lower risk-score categories, better disease outcome and no AML transformation. Interestingly, the young group displayed higher median Lactate dehydrogenase level which is marker of cell turnover, and corelated as well with higher median leucocyte and peripheral blast count, along with more frequent splenomegaly and constitutional symptoms indicating prominent clonal myeloproliferation.

No difference between the two groups regarding the degree of anemia, platelets counts and disease complications.

The prognosis in these patients seems more favorable with higher median PFS over the observed period of follow up.

PB2052: CLINICOPATHOLOGIC VARIABLES AFFECTING DISEASE OUTCOME IN PRIMARY MYELOFIBROSIS. A SINGLE CENTER EXPERIENCE.

S. Elkourashy 1,2,* , D. Soliman 3,4 , R. Taha 1 , H. Elomri 1

1 1Department of Hematology & BMT, NCCCR; 2 Clinical Medicine, Weill Cornell Medical College; 3 2Department of laboratory medicine and pathology, NCCCR, Doha, Qatar; 4 National Cancer Institute, Cairo University, Cairo, Egypt

Background: Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm. The prognosis of PMF is variable and clearly worsens with evolution of specific factors which accelerate disease progression.

This is a single center retrospective study carried out in National Center for Cancer Care and Research, Hamad Medical Corporation over the period 2008-2021.

In this study, we included all patients given a diagnosis of World Health Organization-defined PMF (prefibrotic and overt fibrotic). We collected demographic data, pathologic bone marrow, driver mutations, clinical and hematologic characteristics of the study patients and correlated with disease progression. Dynamic International Prognostic Scoring System – Plus (DIPSS Plus) was used as well but was further grouped into two (low and high risk groups) instead of four groups.

Aims: Here we aim to study different clinicopathologic and genetic parameters that affect the disease course in PMF patients along with their treatment history and disease outcome, in order to find out the most significant factor/s.

Methods: Our Cohort included 61 patients diagnosed with PMF. Variables studied were diagnosis (prefibrotic and overt fibrotic), age, anemia, DIPSS scoring as modified into 2 groups (low/ intermediate-1 vs intermediate-2/high), cytogenetic (favorable and unfavorable karyotype), J AK2V617F mutation, need to treatment, complications mainly thromboembolic complications.

Time to progression, was considered as the time to progression, was defined as the time to acquisition of ≥1 of the following: Hemoglobin < 10 g/dl, Leukocyte count >25 x10 9 /L, Platelet count < 100 x10 9 /L, Circulating blast cells ≥1%, transfusion dependency. Overall survival (OS) and progression free survival (PFS) were assessed using time-dependent, competitive risk analysis.

Kaplan-Meier method was used for survival analysis. Results were considered statistically significant when P values were less than 0.05

Results: The most significant factors adversely affected overall survival are refractory anemia (p. value 0.006), fibrotic stage of the disease (p. 0.030), higher risk group according to DIPSS scoring system (p.0.03), unfavorable karyotype (p.0.02) and eventually AML transformation (P.0.00).

For factors that had a significant impact on disease progression, again included refractory anemia (p.0.000), patients with higher DIPSS score (p.0.00), patient with PMF secondary to other myeloproliferative neoplasms had worse disease outcome compared to primary PMF (p.0.026), patients who needed treatment had shown disease progression compared to those who did not need treatment (p. value 0.001). Disease progression was also significantly associated with patients who received Ruxolitinib compared to those who did not (p.value 0.000), and patients who needed cytoreduction therapy (p. 0.029)

Regarding DIPPS-Plus, 39(63.9%) patients were encountered in low to Intermediate-1, while 22(36.1%) in Intermediate-2 to high risk groups. For disease stage, 32 (52.4%) were prefibrotic, but 29(47.5%) were fibrotic. 44(72.1%) patients needed treatment, while 17(27.86%) did not.

On the other hand, disease progression and the overall survival were not affected by age, molecular genetics findings, hematologic counts, driver mutations or thromboembolic complications.

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Summary/Conclusion: Among all factors studied we found that refractory anemia, unfavourable karyotype and higher risk group according to DIPSS scoring are the most important factors affecting the disease progression and overall survival with statistically significant difference.

PB2053: PRIMARY ARTERIAL HYPERTENSION IN MYELOPROLIFERATIVE NEOPLASMS: A SYSTEMATIC REVIEW

M.-A. Gaman 1,2,* , A. M. Gaman 3,4 , C. C. Diaconu 1,5

1 Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy; 2 Hematology Department, Centre of Hematology and Bone Marrow Transplantation, Fundeni Clinical Institute, Bucharest; 3 Pathophysiology Department, University of Medicine and Pharmacy of Craiova; 4 Clinic of Hematology, Filantropia City Hospital, Craiova; 5 Internal Medicine Clinic, Clinical Emergency Hospital of Bucharest, Bucharest, Romania

Background: Patients diagnosed with myeloproliferative neoplasms (MPNs) are prone to experience thrombotic events and often experience a high burden of cardiovascular comorbidities. Primary arterial hypertension (HTN) remains one of the most common comorbidities in the elderly. However, the impact of HTN on MPNs is unclear.

Aims: Report on the occurrence of HTN in MPNs. Describe its epidemiology, influence on development of thrombosis, HTN treatment strategies.

Methods: Systematic search in PubMed/Medline, Web of Science and SCOPUS, from the inception of these databases to retrieve relevant English-written articles. Eligibility criteria: 1. Confirmed MPNs diagnosis, 2. Confirmed HTN diagnosis, 3. Studies reported on the epidemiology of HTN in MPNs, impact of HTN on the development of thrombosis or on the therapeutic options used to treat HTN in MPNs. We excluded studies investigating secondary HTN, e.g., drug-induced HTN.

Results: The systematic search resulted in 590 potentially relevant manuscripts. After removal of duplicates and screening of abstracts/titles (n=503), 87 manuscripts were selected for full-text review. After applying the eligibility criteria and full-text evaluation, 25 articles were excluded and the remaining 62 were entered into the qualitative/quantitative analysis. HTN emerged as the most common comorbidity in MPNs, affecting 41-63% of primary myelofibrosis (PMF), 40-64% of essential thrombocythemia (ET), 46-90% of polycythemia vera (PV) cases. In PV, the presence of HTN was associated with decreased serum free epinephrine and aldosterone levels, reduced retinal microperfusion, elevated kidney arteries resistance, JAK2-V617F mutation and high-risk PV. In PMF, it was linked with high uric acid concentrations and elevated risk of thrombosis (OR=1.96). PV subjects with HTN were non-dippers, suffered from systolic/diastolic dysfunction as assessed by cardiac ultrasound, displayed elevated cell-free hemoglobin and nitrite/nitrate ratio, and an increased risk of thrombosis (HR=1.77). HTN in ET was associated with overall thrombosis, especially arterial thrombosis (HR=1.91-3.8). The cardio-IPSET score was superior in predicting thrombosis in ET patients with HTN. Individuals with MPNs and HTN exhibited signs of carotid artery injury, end-organ damage, increased albumin-to-creatinine ratio, P-selectin concentrations and were prone to develop kidney dysfunction. Moreover, stroke was more common in MPNs with concomitant HTN (HR=4.24) and HTN was a predictor of ischemic stroke recurrence in MPNs. The risk of thrombosis in MPNs was higher if HTN was also associated with other cardiovascular risk factors, i.e., dyslipidemia, diabetes or smoking. The risk of cerebral venous sinus thrombosis was lower in MPNs with co-occurring HTN. In terms of treatment options for patients with HTN and MPNs, angiotensin-converting enzyme inhibitors (ACEI), e.g., lisinopril, and calcium-channel blockers (CCB - nifedipine for hypertensive emergencies) have been successfully used. The recommendation to avoid the use of diuretics in patients with elevated blood viscosity, e.g., PV, remains unaltered.

Summary/Conclusion: Our findings suggest that HTN is the most common comorbidity in MPNs and is associated with development of thrombosis, end-organ damage, i.e., retinal and kidney dysfunction. Integration of HTN in the current scores employed to predict the development of thrombosis and stratify MPNs subjects in warranted. Screening for HTN in MPNs is urgently needed. Accurate management strategies based on the use ACEI and CCB need further exploration in MPNs with associated HTN.

PB2054: HIP AND KNEE OSTEOARTHRITIS IN PHILADELPHIA CHROMOSOME-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS

H. Holik 1,2,* , I. Krečak 3 , I. Samardžić 4 , D. Pilipac 5 , B. Coha 1 , V. Gverić-Krečak 3 , I. Vučinić Ljubičić 1 , M. Lucijanić 6 , I. Rajkovača Latić 7,8 , L. Vinković 1 , B. Miškić 9,10 , S. Zupančić Šalek 8,11,12

1 Department of Hematology and Oncology, General Hospital dr. Josip Benčević, Slavonski Brod; 2 School of Medicine, University of Osijek Josip Juraj Strossmayer, Osijek; 3 Internal medicine department, General Hospital Sibenik- Knin county, Šibenik; 4 Department of Orthopaedic Surgery, General Hospital dr. Josip Benčević, Slavonski Brod; 5 Department of Orthopaedic Surgery, General Hospital Sibenik- Knin county, Šibenik; 6 Hematology Department, University hospital Dubrava, Zagreb; 7 Department of gastroenterology, General Hospital dr. Josip Benčević, Slavonski Brod; 8 School of Medicine, University of Osijek Josip Juraj Strossmayer, Osijek; 9 Internal medicine department, General Hospital dr. Josip Benčević, Slavonski Brod; 10 Faculty of Dental Medicine and Health Osijek, University of Osijek Josip Juraj Strossmayer, Osijek; 11 School of Medicine, University of Zagreb; 12 Department of Hematology and coagulation, University Hospital Holy Spirit, Zagreb, Croatia

Background: Hip and knee osteoarthritis (OA) affect approximately 10-15% of persons older than 60 years of age. Considering that Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are typically diagnosed in older age and characterized by chronic inflammation, bone marrow remodeling and frequent osteoporosis, we hypothesized that OA may be highly prevalent in MPNs.

Aims: To investigate the prevalence of hip and knee OA in MPNs and its associated risk factors.

Methods: This is an ongoing prospective two-center study being conducted in Croatia (dr. Josip Bencevic General Hospital, Slavonski Brod and General Hospital of Sibenik-Knin County, Sibenik). Consecutive patients with essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) diagnosed according to 2016 World Health Organization criteria are being prospectively included. All participants with suspected hip and knee OA (asymmetry, pains, swelling, crackling, or reduced flexibility) are being evaluated by an orthopedic surgeon and undergo X-ray imaging of the symptomatic joint(s) with OA being defined as grade ≥1 according to the radiological Kellgreen-Lawrence classification; grades 3-4 represent moderate and severe OA, respectively. Relevant clinical and laboratory data, as well as the MPN Symptom Assessment Form (MPN-SAF), are being recorded at the time of study entry. High-risk ET and PV patients are defined as those having age >60 years and/or history of thrombosis. Cardiovascular risk factors considered were arterial hypertension, diabetes mellitus, hyperlipidemia, and smoking. The study was approved by the Ethical Committees from the participating centers. The chi-square, the Mann-Whitney U, and the t-test were used. MedCalc Statistical Software® was used for all analyses.

Results: Fifty MPN patients (ET=18, PV=20, MF 12) were recruited; mean age was 61.5 years (±13.17), 26 (52%) were females, 44 (88%) had symptoms suggestive of OA and 35 (70%) had radiologically confirmed OA, with no statistically significant difference in the prevalences of OA with respect to disease phenotype (61.1% vs. 83.3% vs. 70% for ET, PV and MF, respectively; p=0.428). The prevalence of hip OA was similar among all three disease entities (61.1% vs. 60% vs. 66.7%), whereas knee symptomatic OA was more prevalent in PV (65%) and MF (58.3%) than in ET (22.2%), as shown in Figure 1. Moderate to severe (grade 3-4) hip OA and knee OA were present in 13 (26%) and 9 (18%) MPN patients, respectively, with no statistically significant differences among different disease subtypes (p=0.778 for hip- and p=0.716 for knee OA). Expectedly, the presence of symptomatic hip OA correlated with the presence of knee OA (p=0.002). The presence of symptomatic OA in MPNs was generally associated with high-risk disease (p=0.020), older age (p=0.046), higher body mass index (p=0.015), presence of cardiovascular risk factors (p=0.030), systemic autoimmune disorders (p=0.004), hemoglobin ≤125g/L (p=0.045), hyperuricemia (p=0.076) and high (>8) MPN-SAF (p<0.001).

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Summary/Conclusion: Out preliminary data suggest that symptomatic OA may have strikingly high prevalence (60-80%) in MPNs, with a substantial proportion of MPN patients having moderate to severe OA (18-26%). Finally, the presence of symptomatic OA in MPNs seems to be predominantly associated with older age, higher body mass index, cardiovascular risk factors, and parameters indicative of a higher degree of inflammation. The accrual is ongoing.

PB2055: A RARE CASE OF HIGH-RISK HEREDITARY THROMBOPHILIA COMBINED WITH PRIMARY MYELOFIBROSIS

S. Khoduleva 1,* , D. Novik 2 , D. Blizin 2 , I. Novikova 1 , A. Silin 3 , I. Malishevskaya 4 , A. Demidenko 2 , I. Romashevskaya 2 , E. Mitsura 2 , L. Karataeva 4

1 Hematology department, Gomel State University; 2 Hematology department; 3 Molecular Genetics Laboratory; 4 Clinical Laboratory, Republican Research Center for Radiation Medicine and Human Ecology, GOMEL, Belarus

Background: The risk of venous thrombosis in high-risk hereditary thrombophilia reaches 80%. Among the numerous acquired thrombogenic factors, Ph-negative myeloproliferative diseases (MPD) deserve special attention, which are manifested by thrombosis already at the initial diagnosis in 20% of cases. The risk of thrombosis in MPD is associated with thrombocytosis, plethoric syndrome, and the presence of the JAK2V617F mutation. There is no doubt that the combination of hereditary thrombophilia and MPD significantly increases the risk of thrombotic complications.

Aims: to present a clinical case of high-risk thrombophilia combined with primary myelofibrosis.

Methods: The analysis of the clinical course of primary myelofibrosis in a 28-year-old patient with high-risk thrombophilia was carried out: double heterozygous: Leiden mutation and mutation of the prothrombin G20210A gene. The diagnosis of primary myelofibrosis was verified in accordance with the WHO criteria, revised in 2016. The diagnosis of thrombophilia included an assessment of antithrombin III activity, proteins C and S, and homocysteine levels. Thrombogenic mutations were assessed by polymerase chain reaction (PCR).

Results: The patient complained of spontaneous thrombosis of the portal and splenic veins, and two episodes of miscarriage at 6 and 10 weeks. Primary diagnosis: Portal vein thrombosis. Portal hypertension. Varicose veins of the esophagus of the 3rd degree complicated by bleeding. Condition after endoscopic ligation of varicose veins of the esophagus. Splenomegaly. Ascites. Examination revealed dilatation of the saphenous veins of the chest and abdomen, enlargement of the spleen up to 6 cm below the edge of the costal arch. During dynamic observation the general blood test revealed the following states: leukocytosis up to 17x109/l; thrombocytosis (800-1100x109/l); anemia of mild severity, normocytic, normochromic, normoregenerative (hemoglobin - 98g/l). The level of lactate dehydrogenase in the patient’s blood serum was 400 U/l (reference values - 115-300 U/l). Molecular genetic studies using polymerase chain reaction for the presence of genes-potential markers of Ph-negative MPDs (JAK2V617F, CALR and MPL) revealed a mutation in the JAK2V617F gene. The image of the bone marrow according to trepanobiopsy: the bone marrow is sharply hypercellular. Adipose tissue less than 10%. Red lineage in the form of medium-sized focal clusters, without megaloblastoidity, without redistribution, not expanded. The granulocytic lineage with maturation is sharply hyperplastic. Megakaryocytes in the amount of more than 50 in the lacuna, polymorphic “ugly”, with lobular nuclei, located diffusely (with a tendency to paratrabecular location), with the formation of “loose” and “dense clusters, with the phenomena of emperiopolesis. Diffuse fibrosis In the course of a diagnostic study under the thrombophilia program, mutations of the following genes were found: FII G20210A(G/A); FV G1691A (G/A); PAI-I (5G/4G); MTHFR C677T (C/T). Based on the above clinical examination data and indicators of laboratory and instrumental studies, the main clinical diagnosis was determined: Peripheral myelophibrosis, prefibrotic stage Hereditory thrombophilia: double heterozygous: Leiden mutation and mutation of the prothrombin G20210A gene.

Summary/Conclusion: The described case demonstrates the importance of screening patients with newly diagnosed PMD for high-risk hereditary thrombophilia. On the one hand, this will allow optimizing the antithrombotic prophylaxis regimen, on the other hand, it may be an additional indicator in determining the risk group.

PB2056: MYELOFIBROSIS IN PATIENTS OLDER THAN 70 YEARS: IS TREATMENT WITH RUXOLITINIB WORTH IT?

M. I. Montero 1,*

1 Hospital Virgen del Rocío, Seville, Sevilla, Spain

Background: Myelofibrosis is an agressive myeloproliferative neoplasm with a higher incidence in older patients who have worse prognosis. In last years, ruxolitinib, a JAK1/2 inhibitor that may control myelofibrosis (MF)-related splenomegaly and symptoms, han been used in this disease regardless of age.

Aims: Fifty three patients treated with ruxolitinib between April 2012 and March 2021 from a clinical database of 149 patients were studied in order to assess the effect of age in survival and toxicities

Methods: Diagnosis of primary MF (PMF) an post-Essential Thrombocytemia /post-Polycithemic MF was made according to the WHO 2016. Molecular tests for detection for JAK2, MPL and CALR mutations were performed. Ruxolitinib starting dose was decided accordingly to prescribing information.Survival analysis was performed by means of Kaplan-Meier curves and differences between KM curves were evaluated using the Log-rank test. P values less than 0.05 were considered satistically significant. Analyses were performed with SPSS software v.15.

Results: Median age was 64 years (range 36–88); median haemoglobin was 110 g/l (range, 60–157) and 48,1% patients had a transfusion-dependent anemia. Median platelet an leucocyte counts were respectively 311 × 10 9 /l (range, 52–1887) and 14,8 × 10 9 /l (range, 52–1887). 54,5% patients have a PMF diagnosis. JAK2 V617F was present in 69,8% patients, CALR mutations in 15,4% and MPL W515K/L in 5,3%; 9,5%of the patients were triple negatives. Leucocytes <15x10e9/ and JAK2 V617F were correlated with better survival. At the moment of diagnosis, 32,7% of patients were older than 70 years. In this group, the median of survival from the diagnosis was 70 months. Causes of death were, specifically: progression of myelofibrosis (22,2%), evolution into acute leukemia (22,2%), second neoplasias (22,2%), heart disease (33,3%).

Summary/Conclusion: 1.-Elderly patients in our group treated with ruxolitinib have a median survival of 70 months which is a clear advantage with respect to historic cohorts of MF. 2.-Ruxolitinib was well tolerated but does not avoid clonal evolution and JAK1 inhibition could worsen tumor inmmunosurveillance.

PB2057: REAL -WORLD OVERALL SURVIVAL OF MYELOFIBROSIS PATIENTS ON RUXOLITINIB – A 6 YEAR ANALYSIS IN ALBANIAN POPULATION

A. Perolla 1,* , P. Pulluqi 1 , A. Cili 1 , E. Calliku 1 , T. Caja 1 , A. Ivanaj 1

1 Hematology, UHC “ Mother Teresa”, Tirana, Albania

Background: The survival and the outcome of the patients with Myelofibrosis has been prolonged with the use of Janus kinase inhibitors. The long term follow -up of the patients with primary /secondary Myelofibrosis treated with ruxolitinib demonstrated through several clinical trials an improvement in overall survival and in the quality of life of those patients. The use of IPSS risk classification in Myelofibrosis has played an important role in the prognostic stratification of the patients predictioning also the survival.

Aims: Our study aims to evaluate the Overall Survival (OS) of all the patients diagnosed with Myelofibrosis and treated with Ruxolitinib from 2015 until january 2022. We also aimed to evaluate the role of IPSS risk group classification in the outcome and survival of MF patients in the Albanian population.

Methods: We selected retrospectively all the patients diagnosed in our University Hospital Center “Mother Teresa” in Tirana, Albania with primary and secondary Mielofibrosis who started the treatment with ruxolitinib from 2015 until January 2022. We collected the data from the registries and database of Hematology Clinic based on the first availability of ruxolitinib in Albania since 2015. The data were analysed with SPSS Statistics 25.01.

Results: From 2015 until January 2022 we have treated with Ruxolitinib 103 patients diagnosed with Myelofibrosis, 87 (84.5%) were primary MF and 16 (15.5%) were secondary MF. The median age resulted of 64 (range 31 – 88) years old. According to the IPSS risk group classification 1(!%) resulted IPSS Low risk, 21 (20.4%) were IPSS Int-1 risk, 49 (47.6%) resulted IPSS Int-2 risk, and 32(31.1%) were IPSS High risk. Based on the presence of 0 (low risk), 1 (intermediate risk-1), 2 (intermediate risk-2) or greater than or equal to 3 (high risk) of IPSS, we delineated 4 risk groups with no overlapping in their survival curves and the respective median survivals were 75, 40, 33, and 24 months

We classified as IPSS Low risk all the patient with IPSS low and Int-1 risk, and as High risk all the patients with IPSS Int-2 and High risk. We identified 22 (21.4%) low risk patients and 81(78.7%) high risk patients. We compared the median overall survival between the Low risk and High risk MF groups and concluded that median OS in Low risk MF resulted 47 months (95% Confidence interval (CI): 11.2-82.7) and in High risk MF resulted of 32 months (95% Confidence interval (CI): 3,9 – 39,7) with P<0.01.

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Summary/Conclusion: Myelofibrosis (MF) is a chronic myeloproliferative neoplasm who can develop de novo (primary MF) or secondary in patients with antecedent polycythemia vera (PV) or essential thrombocythemia (ET). The mean age at the moment of diagnosis is 65 year old. In our study me median age at the moment of the diagnosis resulted 64 years old. In our study we compared the median overall survival between the Low risk and High risk MF groups and concluded that median OS in Low risk MF resulted 47 months (95% Confidence interval (CI): 11.2-82.7) and in High risk MF resulted of 32 months (95% Confidence interval (CI): 3,9 – 39,7) with P<0.01Despite the established fact on efficacy of Ruxolitinib in MF patients, real -world data still are limited. Our objective was to analyse the overall survival of our patients with Mielofibrosis under treatment with ruxolitinib, however additional data are needed.

PB2058: IMPACT OF COMORBIDITIES AND RESPONSE TO TREATMENT ON THE MPN SAF ASSESSMENT OF PHILADELPHIA CHROMOSOME NEGATIVE MYELOPROLIFERATIVE SYNDROMES.

B. Mohamed 1,* , H. Asma 1 , R. Amina 1 , S. A. Nadjet 1 , Z. Zahia 1

1 Hematology Department, Unviversitary Hospital Of Sidi Bel Abbes, Sidi Bel Abbes, Algeria

Background: Ph chromosome negative myeloproliferative syndromes (MPN) are a heterogeneous group of hematological malignancies including essential thrombocythemia (ET), polycythemia (PV) and primary myelofibrosis (PMF). The MPN SAF or MPN 10 (Myeloproliferative Neoplasm Symptom Assessment Form) questionnaire is a validated tool for assessing the symptoms presented by patients with MPN, and their evolution during treatment.

Aims: To assess the impact of comorbidities and response to treatment on the symptomatology of patients with MPN assessed by MPN SAF.

Methods: This is a study carried out in our hematology department, which involved patients monitored and treated for MPN. We used the MPN SAF questionnaire wich includes 10 items, rated from 0 to 10, in relation of the MPN symptoms burden. Comorbidities were assessed using the CIRS score (Cumulative Illness Rating Scale). Statistically, ANOVA and log rank tests were used to study the impact of comorbidities and response to treatment on MPN SAF score. The tests are significant if p < 0.05.

Results: 67 patients participated in this study, 31 women and 36 men, with a median age of 65 years [22-86]. 36 (54%) patients had PV, 25 (37%) ET, and 6 (9%) PMF. 16 (24%) patients had splenomegaly, bulky in half. The mean MPN SAF score was higher in patients with PMF (47) than in those with ET (26) or PV (21) (p=0.04). 43 (64%) patients had at least one associated disease, with a CIRS ≥ 6 in 17 (25%). The MPN SAF score was well correlated with the comorbidity score of the patients (p<0.001): patients with CIRS < 6 had a mean MPN score of 48 vs 18 in patients with CIRS ≥ 6. Depending on the response to treatment and disease control, the MPN SAF score was higher in patients refractory to treatment (mean: 35), compared to those with complete or partial remission (17, 22 respectively, p=0.02).

Summary/Conclusion: Our study concludes to a worsening of MPN symptoms when associated to comorbidities. It is very likely that associated diseases maintain a chronic inflammatory state, which is added to that of chronic myeloid neoplasia, mainly when it is refractory to treatment.

PB2059: SUCCESSFUL OUTCOME OF A PATIENT WITH ADVANCED SYSTEMIC MASTOCYTOSIS TREATED WITH AZACYTIDINE AND AVAPRITINIB: A CASE REPORT

C. Saha 1,* , V. Stables 2 , E. Dimitriadou 2 , J. Schofield 3 , A. Green 4 , D. Radia 1

1 Hematology, Guy’s and St Thomas’s Hospitals NHS Foundation Trust, London; 2 Hematology; 3 Histopathology, Maidstone Hospital, Maidstone; 4 Cellular Pathology, Guy’s and St Thomas’s Hospitals NHS Foundation Trust, London, United Kingdom

Background: Systemic mastocytosis (SM) with an associated hematologic neoplasm (AHN) comprise approximately 70% of patients with Advanced SM (AdvSM). >80% of patients with SM-AHN have associated myeloid neoplasms: CMML being common. The median overall survival of patients with SM-CMML is reported to be 15 months. Current algorithms for patients with SM-AHN direct treatment to each of the components tailored to the patient. Avapritinib,a C-KIT tyrosine kinase inhibitor, approved by the FDA in 2021 for patients with AdvSM has an overall response rate of 70% as reported in early phase trials.

Aims: To report successful treatment of advanced SM: SM+CMML, both components requiring treatment simultaneously.

Methods: A retrospective review of a case (with patient consent).

Results: A 58yr old previously fit male was seen in July 2019 with urticaria pigmentosa, recent significant weight loss & mild hepatosplenomegaly. Initial blood counts:hemoglobin(Hb) 123g/L;leukocytes(WBC) 32.6x10 9 /L;platelets(PLT) 123x10 9 /L;neutrophils 20.5x10 9 /L;lymphocytes 3.3x10 9 /L;monocytes 4.2x10 9 /L;eosinophil 0.7x10 9 /L and normal biochemistry. His MC Tryptase was 54.4µg/L. JAK2 V617F & C-KIT D816V mutations was detected. A diagnostic bone marrow trephine(BM) in July 2019 was >90% cellular with marked granulocytic proliferation, hypolobated megakaryocytes, peritrabecular spindle shaped mast cells(10%) positive for CD117 and CD25 immunostains &focal fibrosis of WHO grade 0-1,with diagnosis of SM with CMML. Cytogenetics were normal. Myeloid gene panel detected high risk mutations ( TET2 x2, SRSF2 ). He initially declined treatment. By October 2020, he had intense pruritus, further weight loss, night sweats, bone pain, progressive hepatosplenomegaly, small volume generalised lymphadenopathy, mild ascites & progressive cytopenias (Hb 60g/L & PLT 43x10 9 /L), leucocytosis(WBC 48.9x10 9 /L) & MC tryptase of 105µg/L. Repeat BM showed MC burden of 15-20% (MC tryptase+, CD117+, CD25+) in a hypercellular marrow with predominant increase in granulopoiesis with 20% monocytic cells(CD14+), no increase in CD34+ cells: consistent with evidence of progression of both components. He was treated with azacytidine to debulk the CMML component due to progressive cytopenias and leucocytosis. His PLT count of < 50 x 10 9 /L precluded eligibility onto the PATHFINDER trial due to a risk of intracranial haemorrhage. After 3 cycles, his blood counts improved (Fig 1) and correction of thrombocytopenia (PLT 282 x10 9 /L) allowed treatment of the SM component by avapritinib via Compassionate Access Programme and he started avapritinib 200mg daily. He had rapid symptom improvement with resolution of ascites, hepatosplenomegaly & lymphadenopathy. BM post 4 cycles of avapritinib showed complete remission of SM with persistent CMML-0. MC tryptase normalised to 7.4 µg/L with WBC slowly rising again off azacitidine(Fig 1). He is currently asymptomatic on 100mg of avapritinib.

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Summary/Conclusion: This patient needed treatment of symptoms predominantly due to SM; thrombocytopenia precluded treatment with avapritinib. The CMML component was treated first to improve thrombocytopenia with azacytidine followed by avapritinib for the SM component. The outcome was excellent with complete remission of his SM component & marked clinical improvement. The patient can be considered for an allogeniec bone marrow transplant as a curative treatment. International trials with sequential/combination therapy for patients with AdvSM in the new era of targeted C-KIT inhibitors will inform better treatment decisions for patients with SM-AHN.

PB2060: THE MDS-SPECIFIC COMORBIDITY INDEX (MDS-CI) PREDICTS SURVIVAL INDEPENDENTLY FROM DIPSS AND MIPSS70 IN PATIENTS WITH OVERT MYELOFIBROSIS NOT ELIGIBLE FOR ALLOGENEIC STEM CELL TRANSPLANTATION.

K.-L. Koster 1 , N.-M. Messerich 2 , T. Volken 3 , S. Cogliatti 4 , T. Lehmann 1 , A. Holbro 5 , R. Benz 6 , L. Graf 7 , I. Demmer 4 , W. Jochum 4 , T. Silzle 1,*

1 Clinic for Medical Oncology and Hematology; 2 Departement of Intensive Care, Cantonal Hospital St. Gallen, St. Gallen; 3 Institute of Public Health, ZHAW School of Health Sciences, Winterthur; 4 Institute of Pathology, Cantonal Hospital St. Gallen, St. Gallen; 5 Division of Hematology, University Hospital Basel, Basel; 6 Clinic for Hematology and Oncology, Spital Thurgau AG, Münsterlingen; 7 Centre for Laboratoty Medicine, St. Gallen, Switzerland

Background: For several hematological malignancies, both number and severity of comorbidities are prognostically relevant. The MDS-specific comorbidity index (MDS-CI) offers a comprehensive tool to assess the prognostic impact of comorbidities of the major organ systems (see fig A). Its usefulness in patients with overt myelofibrosis (MF) is currently unknown.

Aims: To assess the prognostic utility of the MDS-CI in patients with overt MF.

Methods: We performed a retrospective chart review of all patients diagnosed with overt MF at our institution between 2000 and 2020 and collected clinical data from hospital records as documented at diagnosis. Patients receiving hematopoietic stem cell transplantation were excluded. For cases lacking next generation Sequencing (NGS) during diagnostic work-up, NGS was performed retrospectively (Oncomine Myeloid Research Assay, Thermo Fisher Scientific, Waltham, MA, USA), if DNA was available from diagnostic samples.

Results: 71 patients were evaluable (male n=38, female n=33; age median 73 years, range 28-87; primary MF n=52, MF post ET or PV n=19). During follow-up (median 40 months, range 0-184) 36 patients (51%) died. Median overall survival (OS) was significantly different between the single MDS-CI subgroups: low (n= 38) 100 months (95% CI 65-137), intermediate (n=26) 50 months 95% CI 25-74) and high (n=7) 8 months (95% CI 0-19), p<0.001; see fig. B.

The Dynamic International Prognostic Scoring System (DIPSS) was available for all patients (low: n=8, intermediate-1: n=30, intermediate-2: n=25, high: n=8). Since no death occurred in the DIPSS-low-risk group, we split the cohort into a DIPSS lower-risk group (DIPSS low and intermediate-1, n=38) and a DIPSS higher risk group (DIPSS intermediate-2 and high, n= 33) for further analyses. After inclusion of the MDS-CI as a categorical variable (reference group low-risk) into a Cox regression model together with the DIPSSdich, the MDS-CI retained its independent prognostic value (MDS-CI intermediate HR 2.05, 95% CI 1.0-4.15, p= 0.046 and MDS-CI high HR 13.2 (95% CI 4.0-43.1, p<0.001).

The Molecular International Prognostic Scoring System 70 (MIPSS70) was available for 57 patients (MIPSS70-low n=2, MIPSS70-intermediate n=42, MIPSS70-high n=13). Within the MIPSS70-intermediate group, the MDS-CI subgroups were associated with significant differences of the median OS: MDS-CI low [n=23]: 115 months [95% CI 76-154], MDS-CI intermediate [n=14]: 89 months [95% CI 50-128] and MDS-CI high [n=5] 11 months [95% CI NA], p=0.007; see fig. C.

Despite the low patient numbers, significant differences regarding the median OS were observed for patients within the MIPSS-high risk group as well: MDS-CI low (n=2): median OS 44 months [95% CI NA], MDS-CI intermediate (n=9): median OS 25 months [95% CI 13-37] and MDS-CI high (n=2): median OS 1 months [95% CI NA], p=0.001.

Since no death occurred in the MIPSS70-low risk group, we dichotomized the cohort into a MIPSS70-lower group (MIPSS70-low- and -intermediate, n=44) and a MIPSS70high risk group (n=13) for further analysis. The MDS-CI added prognostic information after inclusion into a multivariate model together with the MIPSS70dich (MDS-CI high-risk HR 18.7, 95% CI 4.64-75.6, p<0.001; MDS-CI-intermediate HR 1.66, 95% CI 0.68-1.06, p=0.2674).

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Summary/Conclusion: The analysis of our small and retrospective cohort suggests that the MDS-CI represents a useful tool in daily clinical practice to assess the prognostic impact of comorbidities in patients with overt MF. However, analyses of larger cohorts are desirable.

PB2061: A RARE CASE OF CALR MUTATION IN JAK2-NEGATIVE PATIENT WITH POLYCYTHEMIA

T. Subbotina 1,2,* , I. Maslyukova 1 , D. Kurochkin 1 , M. Mikhalev 3 , M. Osadchaya 4 , V. Khorzhevskyi 5 , E. Dunaeva 6 , K. Mironov 6

1 Siberian Federal University; 2 The Federal State-Financed Institution «Federal Siberian Research Clinical Centre under the Federal Medical Biological Agency»; 3 Regional Clinical Hospital; 4 Regional State Budgetary Healthcare Institution «Krasnoyarsk Interdistrict Polyclinic No. 1»; 5 Krasnoyarsk State Regional Bureau of Pathology, Krasnoyarsk; 6 Central Research Institute of Epidemiology of The Federal Service on Customers’ Rights Protection and Human Well-being Surveillance, Moscow, Russia

Background: JAK2 mutations can be associated with any phenotypic form of Ph-myeloproliferative neoplasia (Ph-MPN), while MPL and CALR mutations occur, as a rule, in cases of essential thrombocythemia and primary myelofibrosis and they are not observed by polycythemia vera (PV). However, the cases of CALR mutations in the absence of JAK2 mutations when PV or idiopathic erythrocytosis is diagnosed are reported in the literature.

Aims: To describe a clinical case about the presence of CALR mutation in a JAK2 -negative PV patient.

Methods: DNA molecular genetic examination included the analysis of all driver mutations associated with Ph-MPN according to the WHO recommendations. The Real-Time PCR method was used to analyze the p.V617F mutation in exon 14 of the JAK2 gene. Heteroduplex analysis followed by electrophoresis in polyacrylamide gel was used to screen for mutations in exon 12 of the JAK2 gene and exon 9 of CALR gene. Sanger sequencing was used to confirm the identified mutation. The allele burden level was determined by pyrosequencing.

Results: In January 2018, a 36-year-old man had changes in the hemogram for the first time (Table 1). Afterwards in June 2018 a moderate plethoric syndrome, hematologic syndrome, an increase in the number of white blood cell and profound thrombocytosis were revealed. At the same time a diagnostic ilium bone marrow trephine biopsy was made. Microscopic examination showed increased bone marrow cellularity, megakaryocyte lineage hyperplasia with a wide range of abnormal megakaryocytes, erythroid lineage hyperplasia and expansion (granulocytic lineage within the normal range). By the results of pathomorphological study it was suggested that the observed clinical aspect and clinical laboratory data referred to myeloproliferative neoplasia – PV, MF-0. Thus, taking the plethoric syndrome, pancytosis, pathologist’s report into consideration and according to the clinical recommendations of 2016 the final diagnostic decision was myeloproliferative neoplasia: PV, MF-0. From June 2018 to the present day, the patient has been receiving cytostatic and antiplatelet therapy. No mutations in JAK2 exons 12 and 14 as well as in MPL were revealed in the patient. CALR mutation was revealed in the patient during the initial mutations screening by the presence of heteroduplexes on the electrophoregram and further confirmed and identified by the Sanger’s sequencing as insertion c.1154_1155insGTGTC; p.E386fs*46. The allele burden level in the DNA sample as of June 2018 was 20%. The information about the mutation we revealed is available in COSMIC database and in general there are few cases of this mutation described in the literature. This mutation с.1154_1155insGTGTC, p.E386fs*46 is very similar to the most frequent CALR mutation of type 2 с.1154_1155insTTGTC, p.K385fs*47 and also leads to a longer CALR protein with a modified C-terminus that becomes the basic one. We cannot preclude the possibility of a biclonal disease caused by mutation of CALR and some other unknown Ph-MPN-association mutation. However we can suppose that the revealed CALR mutation с.1154_1155insGTGTC, p.E386fs*46 plays its role in our patient’s polycythemia phenotype.

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Summary/Conclusion: In this research we describe a clinical case about the presence of CALR mutation in a JAK2 -negative PV patient. We can suppose that the revealed CALR mutation с.1154_1155insGTGTC, p.E386fs*46 plays its role in our patient’s polycythemia phenotype.

PB2062: EPIDEMIOLOGICAL, CLINICAL AND THERAPEUTIC FEATURES OF REFRACTORY HODGKIN’S LYMPHOMA.

R. Ben Sghaier 1 , Z. kmira 1 , E. bouslama 1,* , M. zaier 1

1 clinical hematology, Farhat Hached Hospital, Sousse, Tunisia

Background: Approximately 10 to 15 percent of patients have refractory Hodgkin’s lymphoma (HL) that either does not respond to initial therapy or progresses after an initial partial response. Refractory HL poses a major problem of care, especially since there is currently no consensus regarding second or third line chemotherapy.

Aims: The objective of our study is to report the epidemiological, clinical and therapeutic features of patients with refractory HL to first-line chemotherapy.

Methods: This is a retrospective study including 32 patients with refractory HL treated during the period from 2015 to 2020. The patients received the first line chemotherapy according to the national MDH 2015 protocol.

Results: The median age of our patients was 33 years old (17-64years) with a sex ratio of 0.88. B symptoms at diagnosis were present in 53% of cases. Localized stage (II) and extented stages (III and IV) were observed in 25% and 75%, respectively. Tumor bulky (≥7cm) was present in 34.5% of cases. The International Prognostic Score (IPS) ≥3 was present in 31% of patients. Frontline treatment was ABVD chemotherapy in 4 patients with PR in 3 cases, progression in 1 case and BEACOPP-R chemotherapy in 28 patients with PR in 15 cases, failure in 8 cases and progression in 5 cases.

First line salvage therapy was DHAP chemotherapy in 13 patients with CR in 2 patients, PR in 1 patient, failure in 3 patients, progression in 4 patients and toxic death in 3 patients. For patients refractory to DHAP (8patients), 7 patients received the second line salvage therapy which was IGEV in 2 patients with obtaining a PR in 1 patient and progression in the other patient and Bendamustin-Brentuximab (BB) in 5 patients with obtaining a CR in 3 patients and progression in 2 patients.

First line salvage therapy was IGEV chemotherapy in 16 patients with CR in 4 patients, PR in 3 patients and progression in 9 patients. For patients refractory to IGEV (12patients), the second line therapy was DHAP with obtaining a CR in 2 patients, toxic death in 4 patients and failure in 4 patients who received (BB) as third line therapy without response.

First line salvage therapy was BB chemotherapy in 3 patients with obtaining a CR in 1 patient and death during chemotherapy in 2 patients. For good responders after salvage therapy (11 /32 patients), autologous stem cell transplant (ASCT) was performed only in 3 patients. The 5 years overall survival and relapse free survival were 49% and 18%, respectively.

Summary/Conclusion: Our results are much lower than the literature with a mediocre survival of these refractory forms encouraging the introduction of BB as protocol of salvage therapy and the respect of the ACSP in the good responders.

PB2063: CHARACTERISTICS AND THERAPEUTIC RESULTS OF PATIENTS WITH REFRACTORY/RELAPSED HODGKIN LYMPHOMA IN A TUNISIAN CENTER

F. Kammoun 1,* , F. Kallel 1 , M. Charfi 1 , I. Ben Amor 1 , N. Toumi 2 , H. Ben Salah 3 , L. Torjemane 4 , D. Baloumi 4 , I. Frikha 1 , L. Khalifa 1 , Y. Fakhfakh 1 , O. Kassar 1 , M. Medhafar 1 , A. Khanfir 2 , J. Daoued 3 , T. Ben Othman 4 , S. Hadiji 1 , M. Elloumi 1

1 Clinical Hematology Department, Hedi Chaker Hospital; 2 Oncology Department; 3 Radiotherapy Department, Habib Bourguiba Hospital, Sfax; 4 Autograft Department, National Bone Marrow Transplant Center, Tunis, Tunisia

Background: Hodgkin lymphoma (HL) is a hematologic malignancy with a high rate of curability. However, 5 to 20% are primary refractory to treatment or relapse after achieving a complete remission (CR) and present a therapeutic challenge.

Aims: Our study aims to evaluate the clinical characteristics, treatment patterns, as well as therapeutic results in patients with refractory/ relapsed HL (R/R HL) in a Tunisian center.

Methods: It is a retrospective study about adult patients (<60 years old) with R/R HL, treated between January 2008 and Mars 2020 at the hematology, oncology, and radiotherapy departments of the hospital of Sfax, Tunisia. The first-line treatment was based on ABVD in favorable early stages and BEACOPP regimens in advanced stages and localized stages with a bulky mediastinal mass. The standard of care for R/R HL was salvage chemotherapy followed by autologous stem cell transplantation (ASCT) in the national bone marrow transplant center in Tunis. The date of the point is in July 2021.

Results: A total of 55 patients were enrolled, which represents 25% of our series of HL managed in this period. The median age was 30 years (range 17-59), and the sex ratio was 1.3. Twenty-five patients (45%) presented with refractory disease. Among the 30 patients (55%) who relapsed, 19 patients (63%) relapsed within 12 months after achieving CR. At relapse, advanced stage, anemia, and B-symptoms were respectively recorded in 60%, 33%, and 53% of cases. Two patients died before salvage chemotherapy (CT), so 53 patients were treated. Second-line CT regimens used were Cytarabine-based regimens (DHAC/DHAP) in 74% of cases, Gemcitabine-based regimens (IGEV) in 19%, and ICE in 7% of cases respectively. IGEV was the most effective regimen to mobilize peripheral blood stem cells (69% vs 30% with Cytarabine-based regimens). A CR was noted in 61% of cases. Radiotherapy was associated with CT in 35% of cases. Only 13 patients (42% of eligible patients) benefited from ASCT. The 5-year overall survival estimate for all patients was 46%, and 5-year PFS was 41%. Chemoresistance to salvage therapy, ≥ 2 lines of salvage therapy, non-realization of ASCT, and insufficient response after treatment were predictive adverse prognostic factors (p<0.001).

Summary/Conclusion: Our study showed that CR and feasibility of ASCT were lower than that reported in international studies (61% vs 76%, and 46% vs 69 to 100% respectively), which incites us to a better involvement of ASCT. Besides, the association of new therapeutic agents such as Brentuximab Vedotin should be considered in our patients with chemoresistant HL.

PB2064: RELAPSED/REFRACTORY HODGKIN LYMPHOMA PATIENTS TREATED WITH RADIOTHERAPY

F. Kallel 1 , F. Kammoun 1,* , H. Ben Salah 2 , L. Ghorbel 2 , N. Toumi 3 , M. Charfi 1 , I. Ben Amor 1 , I. Frikha 1 , L. Khalifa 1 , Y. Fakhfakh 1 , O. Kassar 1 , M. Medhafar 1 , S. Hadiji 1 , A. Khanfir 3 , J. Daoued 2 , M. Elloumi 1

1 Clinical Hematology Department, Hedi Chaker Hospital; 2 Radiotherapy Department; 3 Oncology Department, Habib Bourguiba Hospital, Sfax, Tunisia

Background: Relapsed and refractory Hodgkin lymphoma (RR-HL) challenges clinicians to devise treatment strategies that are effective and safe. Besides salvage systemic therapy, the use of consolidative stem cell transplantation, new biologics, and immunotherapeutic approaches, radiation therapy remains an integral component of treatment for many patients and must be used effectively and judiciously.

Aims: The purpose of this study is to describe the indications and the modalities of radiotherapy (RT) for patients with RR-HL in our center, as well as its therapeutic results.

Methods: It is a retrospective study about adult patients with RR-HL, who were treated with salvage treatment including radiotherapy, at the university hospital of Sfax, Tunisia, between January 2008 and Mars 2020. Chemotherapy was given to patients in the hematology or oncology departments, and radiation therapy was given in the radiotherapy department, using linear accelerators or cobalt machines. Partial response (PR) was defined as a greater than 50% reduction in the product of the largest diameter and its perpendicular of measurable disease, lasting at least 1 month after radiotherapy.

Results: Among the 55 patients having RR-HL during the study period, 19 underwent chemotherapy followed by RT. We did not note any case treated by radiotherapy alone. Eleven patients presented with refractory disease and 8 patients with relapse. No relapse in previously irradiated areas was noted. RT was palliative in 11 patients and consolidative for a residual mass in 8 patients. All patients with residual mass received a total of 36 Gy, except one patient who received 40 Gy. The radiation doses given were daily fractions of 2 Gy, 5 days per week. The majority of cases (5/8) had a residual mediastinal mass, but only one of them had a mediastinal bulky at the initial diagnosis of the disease. In one patient, RT was consolidative following autologous hematopoietic cell transplantation (ASCT). For palliative radiotherapy, it was received after a median of 2 lines of salvage chemotherapy (range 1- 4 lines), with a classic schedule of daily 2 Gy in almost all cases (9/11), and higher doses of 3 Gy/day in 2 cases. Involved-field radiotherapy was adopted in all 11 patients. Therapeutic response analysis showed that the majority of patients (88%) had at least a partial response. For patients with a chemoresistant disease and in indication of palliative RT, this alternative allowed for a partial response in half of the patients.

Summary/Conclusion: Although concerns about the toxicities of RT will always be relevant to decision-making, our study showed that RT used as salvage therapy after standard-dose chemotherapy enhances local disease control in sites of relapsed or refractory HL in the majority of patients with residual disease, and in half of chemoresistant patients for whom palliative therapy was indicated.

PB2065: IMPORTANCE OF PET AS A PROGNOSTIC MARKER IN TREATMENT STRATEGY CHOICE IN PATIENTS WITH HODGKIN LYMPHOMA

O. Novosad 1,* , I. Pastushenko 1 , T. Kadnikova 1 , I. Tytorenko 1 , A. Martynchyk 1 , O. Gorbach 1 , A. Ashyhmin 2 , Y. Kmetyuk 3 , O. Karpova 3 , L. Mykhalska 4 , O. Kindrakevych 4 , M. Novikov 5 , O. Oliinichenko 6 , I. Kriachok 1

1 Oncohematology; 2 Radiology, National Cancer Institute; 3 Radiology; 4 Oncohematology, Clinical Hospital “Feofaniya”; 5 Radiology, Oncology Hospital “LISOD”; 6 Radiology, Center of Nuclear Medicine KMCOC, Kiev, Ukraine

Background: The systematic use of PET in staging is based on international guidelines. The accuracy of PET/CT over CT scanning (with a sensitivity and specificity higher than 90%) has been compared in several works in order to assess the extent of the disease, leading to modifying the staging in 15%-40% of cases with an impact on the therapeutic strategy in approximately 15%-20% of cases.

Aims: The primary endpoints were to assess a correlation between PET findings and response to therapy, type of regimen and clinical outcome (relapse/death), as well as whether there is a difference when patient treatment is intensified or not intensified depending on PET findings.

Methods: This substudy was performed within the framework of a wider prospective multicenter study on the predictive value of PET response assessment (249 patients with HL up to 65 years of age or younger). Patients received a treatment with ABVD or ABVD/AVD; BEACOPP-14/esc or “switched-regimens” (ABVD+BEACOPP-esc/14, BEACOPP-esc/14+ABVD). Metabolic PET imaging was performed to routine protocols using Deauville criteria for response assessment.

Interim PET (PET2) was performed at 15.5 ±3 days (range, 5-26) after the 2-3 cycle of treatment.

Results: 9 patients of the trial group had the 1 st stage, 127 people were stage 2, 47 and 66 had stage 3 and 4, respectively. Most patients had stage 2A recorded, 35% of patients (86/249). 75.9% (189/249) and 24% (60/249) had the IPS factors of risk£2 and 3 3, respectevly (p>0.05). 63% (157/249) of patients went through an ABVD regimen treatment; only 9 patients of this group were switched to an ABVD/ABV course of treatment after receiving CMR, in accordance to PET.

The ORR was 85% (212/249): where 91% of patients (195) achieved CMR and 9% were with PMR. In total, the disease progression occurred in 18% (45/249). In patients with PMR (4-DS) after 2 courses of treatment, the relapse frequency was significantly higher (47%) compared to the CMR (8%), р>0.05.

5-year event free survival (EFS) rate and overall survival (OS) for study group were 70% and 88%, respectively.

We confirmed a significant difference between the 5-year EFS rate for patients with PET2+ vs PET2-. Thus, PET2- patients with early and advanced stages had an EFS level much higher than the PET2+ patient group (80% vs 30% respectively, Log-rank test, p<0.0001). However, when comparing the EFS level in patients with 4-DS vs 5-DS, we did not find any statistically relevant difference (р=0.2).

EFS level in patients with PET-negative (PET3-) end of treatment was also much better compared to patients with PET-positive (PET3+) one, (83% vs 20%, respectively Log-rank test, p<0.0001).

The 5-year OS findings in patients with PET2- and PET3- are significantly better compared to PET2+ and PET3+ (98% vs 35% and 97% vs 50%, р=0.04).

Summary/Conclusion: Our findings have confirmed one more time that a good response to therapy achieved early enough leads to an increase of general relapse-free survival. This way, the issue of customizing treatment currently remains quite an important questions.

PB2066: THE STUDY OF GSTP1 AND MDR1 GENES POLYMORPHISMS IN HODGKIN’S LYMPHOMA AND THEIR LINK WITH CHEMOTHERAPY RESPONSE.

M. Voytko 1 , Y. Shebunyaeva 1,* , O. Goreva 2 , T. Pospelova 1 , A. Grishanova 2 , V. Vavilin 2

1 Hematology, Novosibirsk State Medical University; 2 Molecular biology, Institute of Molecular Biology and Biophysic FRC FTM, Novosibirsk, Russia

Background: Glutathione-S-transferase P1 and ATP-dependent transporter P-glycoprotein are enzymes of the second phase of xenobiotic metabolism that participate in biotransformation of agents of standard protocol of Hodgkin’s Lymphoma chemotherapy. The polymorphism of the GSTP1 and MDR1 genes encoding these proteins causes changes in their activity, which can provide an individual patients’ response to chemotherapy. In this regard, the search for predictive genetic factors for the effectiveness of antitumor therapy is an important task.

Aims: to demonstrate the association between polymorphic variants of GSTP1 (A313G, C341T) and MDR1 (C1236T, G2677T, C3435T) genes in patients with Hodgkin’s lymphoma.

Methods: Overall, 77 Hodgkin’s lymphoma patients aged 18-74 years who were treated by BEACOPP and ABVD chemotherapy protocols in Oncohematological Departments of City Clinical Hospital №2 and Novosibisk State District clinical Hospital were enrolled in this study. The effectiveness of chemotherapy was evaluated after 4-6 cycles of chemotherapy in accordance with clinical guidelines. Depending on the achieved results of treatment, all patients were divided into two groups: patients with complete/partial remission and patients with progressive/refractory or relapsed lymphoma. Polymorphisms of GSTP1 (A313G, C341T) and MDR1 (C1236T, G2677T, C3435T) genes were defined by PCR method with real-time detection in the samples of peripheral blood in the Centre of Collective Usage «Proteomic analysis» FRC FTM. The odds ratio (OR) used to evaluate the link between genotypes with efficacy of chemotherapy.

Results: We found the following frequencies of mutant alleles in Hodgkin’s Lymphoma patients: GSTP1313G – 31.17%, GSTP1 341T – 9.7%, MDR1 1236T – 50.00%, MDR1 2677T – 48.70%, MDR13435 ТОНН – 58.44%. Patient’s genotypes were similar to described genotypes of healthy Europeans. There was no significant difference in distribution of frequency alleles and GSTP1 A313G (χ2=0,89, p=0,34), GSTP1 C341T (χ2=0,11 R=0,74), MDR1 C1236T (χ2=0,01, p=0,93), MDR1 G2677T (χ2=0,37, p=0,54), MDR1 C3435T (χ2=0,01, р=0,97) genotypes between group of Hodgkin’s lymphoma patients with complete/partial remission and group with progressive/refractory or relapsed lymphoma. At the same time we found the significant correlation between GSTP1 A313G heterozygotes and formation of progressive/refractory and prolapsed course of disease (OR = 10.80; CI 95% = 1.23 – 496.52). There was no significant association between efficacy of chemotherapy and other studied genotypes, namely GSTP1 C341T, MDR1 C1236T, MDR1 G2677T, MDR1 C3435T genotypes.

Summary/Conclusion: The observed correlation between GSTP1 A313G gene polymorphisms and formation progressive/refractory or prolapsed course of disease needs to be studied to provide safe and effective specific chemotherapy.

PB2067: THE REALM STUDY PROTOCOL: A MULTICENTER, NON-INTERVENTIONAL, RETROSPECTIVE STUDY TO DESCRIBE THE TREATMENT PATHWAYS, OUTCOMES, AND RESOURCE USE IN PATIENTS WITH CD30+ LYMPHOMA FROM CHINA.

W. Xu 1 , L. Fan 1 , L. Wang 1 , H. Zhu 1 , J. Wang 2,* , J. Li 1

1 Jiangsu Province Hospital, The First Affiliated Hospital With Nanjing Medical University, Nanjing, Jiangsu; 2 Takeda Medical Affairs, Takeda (China) International Trading Co, Ltd, Shanghai, China

Background: Current scientific evidence on the demography, disease, and distribution characteristics of CD30+ lymphoma subtypes focus on patients from Western Countries. Although some differences have been reported between CD30-expressing lymphoma populations in Western Countries and East Asia, there is limited information on Chinese patients. New targeted therapies such as CD30 monoclonal antibodies have been approved in China for the management of CD30-expressing lymphoma. Analyzing the treatment patterns and outcomes for patients with CD30-expressing lymphoma in a real-world setting will bridge the existing knowledge gap.

Aims: Our study aims to investigate the treatment pathways and define CD30-expressing lymphoma patients’ demographic, clinical characteristics, treatment response, survival rate, and healthcare cost and utilization in the Chinese population.

Methods: The study represents a multicenter, non-interventional, retrospective study of newly diagnosed or relapsed/refractory CD30-expressing lymphoma patients who received treatment in China between January 1, 2018, and March 31, 2021, with survival follow-up until September 30, 2021. The study will include 1000 patients with classical Hodgkin lymphoma (cHL) and 1100 ~1800 with non-Hodgkin lymphoma (NHL), inclusive of all subtypes, aged ≥ 18 years from 14 hospitals with Haematology or Oncology departments across China. Based on their diagnosis, patients will be assigned to one of the following cohorts: (i) (cHL); (ii) (NHL) with a minimum observation period of 6 months; however, eligible patients who died or were lost to follow-up within 6 months will also be included. The planned study design is shown in Figure 1. The demographic, diagnostic, number of refractory/relapsed, clinical, laboratory parameters, cost, and resources utilized data will be retrieved from hospital information system/electronic medical records/Laboratory information management systems. Clinical characterization will be based on Ann Arbor staging, WHO/EORTC staging of mycosis fungoides/Sézary syndrome, B symptoms, subtype, mass, and ECOG performance status. Patients whose demographics and clinical features are not available from the medical records will be excluded. All data will be analyzed using descriptive statistics. Real-world overall survival (rwOS) and progression-free survival (rwPFS) will be analyzed using Kaplan-Meier estimates at a 95% confidence interval. All statistical procedures will be analyzed using the SAS software, version 9.4.

Results: Based on the preliminary analysis of medical records, a total of 2100 to 2800 patients will be included. The study outcomes will be assessed based on subgroups. The study will describe the treatment pathways among adult patients as a primary endpoint. Patient demographics/clinical characteristics, real-world objective response rate, overall survival, progression-free survival, healthcare cost, and resource utilization will be assessed as secondary endpoints. First-line treatment pathways and their related efficacy will be evaluated in newly diagnosed CD30-expressing lymphoma patients in China.

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Summary/Conclusion: The results of this study may elucidate effective treatment strategies for adult Chinese patients with newly diagnosed or relapsed/refractory CD30-expressing lymphoma. The study will also enable a better understanding of demographic, clinical, pathological characteristics, and the related economic burden of adult Chinese patients with lymphoma.

PB2068: RETROSPECTIVE ANALYSIS OF HAIRY CELL LEUKEMIA (HCL) PATIENTS (PTS) TREATED WITH DIFFERENT MODALITIES AS FIRST LINE: REAL-LIFE EXPERIENCE OVER 20 YEARS IN KUWAIT.

A. Alhuraiji 1,* , M. Ram 1 , A. Kunhikrishnan 1 , S. Alshemmari 1 , A. Hamadah 1 , A. R. Shah 1 , F. Khadadah 1 , R. Pandita 1

1 Hematology/BMT, Kuwait Cancer Control Center, jaber alahmad, Kuwait

Background: Introduction: Hairy cell leukaemia (HCL) is a rare chronic lymphoproliferative disorder

Aims: We retrospectively analysed all HCL cases diagnosed and treated at the Department of Hematology at Kuwait Cancer Control Center over a 20 year period.

Methods: Baseline parameters collected included patient and disease demographics including bone marrow findings, as well as treatment protocols, and complications arising from treatment or relapse. Overall survival (OS) and progression free-survival (PFS) were calculated using a Kaplan-Meier analysis.

Results: Forty-one patients with a median age of 51 (range 31–78) years were enrolled in the study over the time period 1994-2020. Diagnosis was established according to the WHO criteria for HCL. Clinical characteristics are shown in Table 1. Almost one third of the pts were Kuwaiti which reflects the demographic distribution of the country’s population. All pts were male (n=40) except for 1 pt. Treatment was initiated in 39 pts. In first-line, 28 pts were treated with cladribine (2CDA) alone, five received 2CDA in combination with rituximab, and four received interferon. The overall response rate (ORR) was 87.8%. In pts who received 2CDA-based therapy, the ORR was 97%. After a median follow-up of 92 months (3-266), median OS was not reached and median PFS was 7.7 years (95% CI, 5yrs-NR) (Figure 1). Sixteen pts (39%) had disease progression and were retreated mostly with 2CDA. Therapy was well tolerated with no reported treatment-related mortality. Febrile neutropenia was 22%.

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Summary/Conclusion: This is the first report of HCL characteristics and outcomes from Kuwait. HCL pts have outcomes that are comparable to those in the published literature.

PB2069: A CASE OF HAIRY CELL LEUKEMIA PRESENTED AS HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

C. Almeida 1,* , R. Costa e Sousa 1 , C. Afonso 1 , A. Roque 1 , D. Luis 1 , L. Araujo 1 , C. Geraldes 1

1 Hematology Department, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal

Background: Hairy cell leukemia (HCL) is an indolent and uncommon chronic B cell lymphoproliferative disorder. The clinical picture is mainly dominated by cytopenias and splenomegaly, with some patients only receiving a watch and wait approach. It has generally a very favorable prognosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of uncontrolled inflammation that occurs due to severe systemic immune activation, mainly secondary to infection, autoimmune diseases, and malignancies (mostly aggressive T/NK-cell or B-cell lymphomas) and carries a dismal prognosis.

Aims: To present a case of a newly diagnosed HCL that manifests as HLH.

Methods: Clinical registry review.

Results: Female, 62 years old, ECOG-PS 0, with an unremarkable medical history. She presented to the emergency department with a two-week history of constitutional symptoms (fever, marked weight loss and night sweats) and skin lesions. Physical examination revealed hepatosplenomegal and no adenomegalies. Blood tests showed bicytopenia (hemoglobin 10.8 g/dL normocytic/normochromic and leukocyte 2.1x10^9/L), an elevated C-reactive protein (14 mg/dl) and an elevated erythrocyte sedimentation rate (85 mm/h); lactate dehydrogenase (LDH), kidney function and liver function analysis were unremarkable. She was then admitted as an inpatient to the hospital for additional etiologic investigation. An extensive screening for infectious agents and autoimmune diseases was performed but showed no alterations. Skin lesion biopsy was not diagnostic, for two consecutive times, suggesting the possibility of erythema nodosum or vasculitis, with normal immunophenotyping.

Bone marrow trephine (dry tap for aspirate) showed a small focal invasion by B-cells with HCL phenotype (CD20+/Annexin A+/ CD23+/Cyclin D1+).

PET/CT scan showed infra-diaphragmatic hypermetabolic adenopathies (maxSUV 15.1) and splenic involvement.

Intraabdominal lymph node biopsy showed a diffuse involvement by HCL clonal B-cells (CD20+/CD10-/CD25+/CD5-/Annexin A+). New peripheral blood immunophenotyping showed 0.4% B-clonal cells with HCL phenotype. The BRAF V600E gene mutation was positive in both products. The diagnosis of HCL (classical) was performed.

Throughout the next month as she remained at the hospital for investigation, she had been febrile with no germen identification and no improvement despite multiple antibiotic courses.

Despite the usage of corticosteroids and later life-saving rescue with cladribine 0.12 mg/kg 5 days, she developed severe multiorgan dysfunction - pancytopenia (hemoglobin 6.9 g/dL, leukocyte 0.6x10^9/L, platelet 10x10^9/L), liver dysfunction (bilirubin 24.7 mg/dL, gamma-glutamyl transferase 1071 U/L, alkaline phosphatase 707 U/L, LDH 1771 U/L), kidney failure (creatinine 3.14 mg/dL) and coagulopathy (fibrinogen 111.0 mg/dL). Markedly elevated serum ferritin (10271 ng/mL), triglycerides (618 mg/dL) and soluble CD25 (5119 pg/ml,) supported a diagnosis of HLH (6 of 8 HLH-2004 criteria and HScore 268 points) the patient immediately started treatment with dexamethasone 10 mg/m 2 per day. Due to respiratory failure, she was admitted in ICU, where she unfortunately died due to Klebsiella pneumoniae pneumonia, a month and a half after the beginning of the clinical symptoms.

Summary/Conclusion: Conclusion: HCL is usually associated with an indolent clinical picture and with a good prognosis, while HLH is mainly associated with aggressive lymphoma. This case illustrates a case of HCL with a difficult diagnosis and highlights the importance of considering HCL as a triggering factor for HLH

PB2070: EFFICACY OF RITUXIMAB MAINTENANCE THERAPY IN ADVANCED-STAGE FOLLICULAR LYMPHOMA: A SINGLE-CENTER RETROSPECTIVE STUDY.

C. Amorós 1,* , O. Mompel 1 , P. López-Menargues 1 , C. Játiva 1 , A. Cámara 1 , R. García-Serra 1 , M. J. Lis-Chulvi 1 , M. E. López-Pavía 1 , C. Villegas-Da Ros 1 , M. Linares-García 1

1 Hematology, Consorcio Hospital General Universitario de Valencia, Valencia, Spain

Background: Previous studies have established that 2-year rituximab (RTX) maintenance after induction treatment improved progression-free survival (PFS) in patients with advanced follicular lymphoma, although without evidence of effects on overall survival (OS). However, the effectivity in routine clinical practice should be confirmed.

Aims: The primary goal of the present study was the assess the effect of RTX maintenance on clinical outcomes (OS and PFS) and study its prognostic impact in patients with advanced disease.

Methods: We conducted a comprehensive single-center retrospective analysis of outcomes of 88 advanced FL patients, treated between January 2005 and February 2021. We selected patients that had obtained a complete (CR) or partial response (PR) after induction therapy regardless of follow-up with RTX maintenance. Baseline data collected included the following: age, sex, FLIPI stage, hemoglobin level, lymphocyte and platelet count, serum LDH and beta-2 microglobulin level. Continuous variables were compared with Mann-Whitney U test, and categorical using Chi-square or Fisher´s exact tests. PFS and OS were estimated using the method of Kaplan-Meier and compared with the log-rank test. Prognostic factors for PFS and OS were determined per Cox regression analysis.

Results: Patients´ median age was 60,5 years; 58% were female. 63 patients received induction therapy with R-CHOP, 8 with R-CVP and 17 with bendamustine-R. Overall, 54 of the selected patients started rituximab maintenance and 34 continued follow-up only with clinical observation. The rates of CR and PR were 81.5% and 18.5% in the maintenance group and 73.5% and 26.5% in the observation group. There were no significant differences among the prognostic variables at diagnosis (Table 1). In the observation group, more patients progressed (58.2% vs 24.7% in maintenance group) and more received second-line treatment (44,1% vs 24.1%). After a median follow-up of 60 months, 19 patients (21,6%) had died. RTX maintenance therapy showed a significant benefit in both PFS and OS, compared with observation (Table 2). Multivariant analysis showed that age (HR 1.063; 95% CI: 1.015-1.105; p=0.010), lymphocyte count (HR 1,049; CI 1.01701-1.083; p=0.003), LDH (HR 1.001; CI 1.001-1.002; p<0,001) and RTX maintenance (HR 0.193; CI 0.061-0.612; p=0.005) were independent prognostic factors for OS. Regarding PFS, the only independent variables were response to induction-therapy CR vs PR (HR 0.084; CI 0.038-0.187; p<0.001) and RTX maintenance (HR 0.251; CI 0.110-0.577; p=0.001).

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Summary/Conclusion: RTX maintenance following induction therapy provided a significant long-term PFS, concordant with previous studies. Meanwhile, respect the OS, it has shown significant benefit unlike previously published, probably due to differences in the characteristics of our study: older patients, more advanced-stage patients, and lower intensity second-line therapy.

PB2071: INTRALESIONAL RITUXIMAB IN THE TREATMENT OF PRIMARY CUTANEOUS B-CELL LYMPHOMA

M. Callejas Charavia 1 , L. Castilla García 1 , J. Aspa 1,* , P. García Ramírez 1 , M. Argüello Marina 1 , C. Martínez Vázquez 1

1 Hematology, Hospital Príncipe de Asturias, Alcalá de Henares, Spain

Background: Cutaneous B-cell lymphomas can be classified into two categories: primary cutaneous lymphomas (PCL) and secondary lymphomas. PCL are restricted to skin infiltration and are indolent. The two most prevalent PCL subtypes are primary cutaneous marginal zone lymphomas (PCMZL) and primary cutaneous follicle center lymphomas (PCFCL). The first line treatment recommended both by EORTC and ISCL, is surgery (excisional biopsy) associated or not to local radiotherapy. Nevertheless, these recommendations are based on retrospective studies including few patients. There is a lack of randomized clinical studies comparing the different available therapies for primary cutaneous lymphomas. Among the alternative therapies to the classical first line of treatment, the guideliness of EORTC and ISCL consider the use of intralesional rituximab. The advantages of this treatment are its tolerability, absence of secondary aesthetic effects, accessibility, and easy administration. On the contrary, radiotherapy is less accessible in certain hospitals and presents a greater incidence of secondary cutaneous adverse effects. It causes both acute and chronic reactions, such as alopecia, erythema, peeling, hyperpigmentation and, in cases of repeated doses, even ulceration. Similarly, the need for appropriate margins of excision in surgical procedures entails a more aggressive approach with worse esthetic results that can even lead to infection or ulceration, and it also requires more resources, both material and human.

Aims: To present our experience in the management of PCL.

Methods: Out of a total of 30 patients diagnosed between April 2009 and June 2018, 14 patients (46,7%) were treated with intralesional rituximab, 15 with the standard therapies, and 1 with both. We evaluated treatment efficacy, security, and tolerability of both approaches. Treatment response was assessed following our own criteria which categorizes the response as: complete (100% of skin bleaching), partial (50-99% size reduction), stable disease (<25% increase to <50% decrease in size of the treated injuries), and progression (> 25% increase of the treated injuries). The rituximab dosing schedule was 10mg/injury (intralesional) or 10-40 mg in the case of PCFCL, 3 times per week, for one week. If the lesion persists, the dose can be repeated, until it disappears.

Results: Both groups of patients were demographically comparable. The median age of patients was 60 years (interquartile range: 49-75 years). 52% were women. The median number of injuries was 1. The most frequent locations were the face (41%, including the frontal region, jaw and temple) followed by the back (14%).

In the group treated with rituximab, the median PFS was 75 months (ICR: 38,6-99 months) (with a median follow-up of 86 months). As the only adverse effect, 3 patients showed a flu-like reaction the day after the injection without need of hospital assistance. Two patients (14%) suffered from a relapse of the skin lesions and were retreated with intralesional rituximab achieving CR. One of the patients showed persistent lesions after 3 doses, reaching CR after the sixth dose.

In the standard of care group, the median PFS and median follow up were both 118 months. Ten patients (67%) presented adverse effects related with local radiotherapy.

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Summary/Conclusion: In our experience, intralesional rituximab is a safe, accessible, and effective therapeutic option. Therefore, we consider that it should be included as a first line treatment at the same level as the classic alternatives, especially in cases where the esthetic consequences of the treatment are particularly relevant.

PB2072: EFFICACY AND SIDE EFFECTS OF IBRUTINIB IN RELAPSED AND REFRACTORY MANTLE CELL LYMPHOMA

A. Basturk 1,* , M. Bagci 2 , M. S. Dal 3 , T. Hacibekiroglu 4 , S. Kabukcu Hacioglu 5 , I. Aydogdu 6 , M. Kizil Cakar 3 , C. Sunu 4 , G. Akgun Cagliyan 5 , A. Kaya 6 , M. H. Dogu 7 , S. Celik 8 , H. Uskudar Teke 9 , M. A. Erkurt 10 , O. Ekinci 11 , B. Turgut 12 , A. Tekinalp 13 , S. Korkmaz 14 , T. Ulas 15 , F. Altuntas 3

1 Hematology Department, Konya City Hospital; 2 Hematology Department, Selcuk Univercity Faculty of Medicine, Konya; 3 Hematology Department, Ankara Oncology Training and Research Hospital, Ankara; 4 Hematology Department, Sakarya University, Sakarya; 5 Hematology Department, Pamukkale University, Denizli; 6 Hematology Department, Celal Bayar University, Manisa; 7 Hematology Department, Istinye University, Istanbul; 8 Hematology Department, Erciyes University, Kayseri; 9 Hematology Department, Eskisehir Osmangazi University, Eskisehir; 10 Hematology Department, Inonu University, Malatya; 11 Hematology Department, Gazi Yasargil Training and Research Hospital, Diyarbakir; 12 Hematology Department, Namik Kemal University, Tekirdag; 13 Hematology Department, Necmettin Erbakan University, Konya; 14 Hematology Department, Kayseri City Training and Research Hospital, Kayseri, Turkey; 15 Hematology Department, Near East University, Nicosia, Cyprus

Background: Mantle cell lymphoma constitutes 6–8% of all B-cell lymphomas. Due to its efficacy and reliability, ibrutinib has been used to treat patients with mantle cell lymphoma who experience a relapse after transplantation, similar to patients with other B-cell malignancies.

Aims: The purpose of the present study was to evaluate the efficacy and side effects of ibrutinib in patients with relapsed and refractory mantle cell lymphoma.

Methods: This retrospective study included 37 patients with relapse/refractory mantle cell lymphoma who were over the age of 18 years and who were treated in 16 different healthcare centers. The patients’ responsiveness to the administration of ibrutinib, the duration of the response, the prognostic factors affecting the response, and the side effects associated with ibrutinib were analyzed retrospectively.

Results: The median follow-up period was eight months. The total response rate was 58% (19.3%, CR). The mean life expectancy was 31.3 months. The mean period to achieve complete remission was four months. While 51.6% of patients responded three months after the initiation of ibrutinib treatment, this rate increased to 56.5% at six months, and to 66.7% at 12 months. In 40.5% of the patients, the duration of treatment was longer than 12 months, and in 13.5% of the patients, it was longer than 24 months.

Summary/Conclusion: Our study confirmed the findings of previous studies that the use of ibrutinib in the treatment of R/R MCL was effective and reliable.

PB2073: LACK OF AGREEMENT ON EVENT-FREE SURVIVAL DEFINITION IN MARGINAL ZONE LYMPHOMA

C. Bommier 1,2,3,* , J. Lambert 4 , G. Nowakowski 5 , E. Zucca 6 , C. Thieblemont 7

1 Hematology/Oncology; 2 INSERM U1153 - ECSTRRA Team, St Louis Hospital; 3 Université de Paris Cité; 4 INSERM U1153 - ECSTRRA Team, St Louis Hospital, Paris (France), Paris, France; 5 Hematology, Mayo Clinic, Rochester, MN, United States of America; 6 Hematology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 7 Hematology/Oncology, DMU DHI, St Louis Hospital, Paris, France

Background: A recent systematic review reported that trials involving patients with marginal zone lymphoma (MZL) show marked heterogeneity both in the choice and definitions of primary and secondary endpoints, thus hampering comparability between trials (1).

Aims: The main objective of this study was to reach consensus, through a Delphi process, on the definitions of four time-to-event endpoints in MZL trials, by surveying clinicians and methodologists involved in the conduct of clinical trials including patients with MZL.

Methods: We polled a panel of leading international experts involved in MZL trials, after the 2021 IELSG meeting, by means of self-administered sequential questionnaires. Of these 105 experts, 62 responded to the Round 1 questionnaire regarding the definitions of Progression-free survival (PFS), Event-free survival (EFS), Time-to-failure (TTF) and Time-to-next-treatment (TTNT). Afterwards we therefore focused the Round 2 and 3 questionnaires among principal investigators, coinvestigators and trial methodologists. Consensus was reached when there was a >80% agreement on all potential events (11 choices) of each endpoint.

Results: Participants in our survey reached consensus on 3 of the 4 time-to-event endpoints definitions. Consensus was reached on the definitions of PFS and TTNT after Round 1, of TTF after Round 2 and was not reached for EFS after Round 3. Although experts agreed on the inclusion of the addition of new therapy as an event, the experts differed on the criterion of treatment discontinuation (53% vs 47%). Since there was no trend for or against this criterion, it was decided to stop the Delphi process at this point (see Figure).

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Summary/Conclusion: The main interest of our study was to elicit investigators interest in the importance of consistently defining endpoints in MZL trials and to highlight that composite endpoints should not be encouraged. Fifteen years after the last consensus statement on time-to-event endpoints definitions issued in Lugano (2007), both the review of literature and survey of international investigators agree on the inconsistency of endpoints definitions used within the MZL community. Hopefully, revised standardized definitions of endpoints shall be provided at the upcoming International Conference on Malignant Lymphoma in 2023.

(1) Bommier C, Ruggiu M, Monégier A, Zucca E, Thieblemont C, Lambert J. Systematic review reveals urgent need to homogenize endpoints choices and definitions in marginal zone lymphomas trials. Leukemia & Lymphoma . Published online February 24, 2022:1-12. doi:10.1080/10428194.2022.2032038

PB2074: IBRUTINIB AS A PROMISING TREATMENT FOR WALDENSTRÖM MACROGLOBULINEMIA

O. Ilhan 1,* , G. Cengiz Seval 1 , F. Avcu 2 , B. Ferhanoglu 3 , M. Ozcan 1 , G. Cetin 4 , E. Kurtoglu 5 , H. Yilmaz 1 , F. Atalay 6 , E. Gulturk 7 , M. Nalcaci 8 , M. Beksac 1

1 Hematology, Ankara University School of Medicine; 2 Hematology, Private Memorial Hospital, Ankara; 3 Hematology, Koc University School of Medicine; 4 Hematology, BezmiAlem University Schhol of Medicine; 5 Hematology, Kolan International Hospital; 6 Hematology, Baskent University; 7 Hematology, Kartal Lutfu Kirdal Education and Research Hospita; 8 Hematology, Istanbul University School of Medicine, İstanbul, Turkey

Background: Waldenström Macroglobulinemia (WM) remains an incurable disease with a heterogeneous course, relapse following initial treatment happens in virtually all patients. Ibrutinib, a Bruton Tyrosine Kinase inhibitor, is a promising drug used in WM, chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone lymphoma, and chronic graft versus host disease. Ibrutinib first received FDA approval in WM as a monotherapy in January 2015 and expanded the approved use of ibrutinib with rituximab in August 2018. Ibrutinib-related prior data in Waldenström Macroglobulinemia (WM) remain sparse, particularly outside of clinical trials.

Aims: Herein, we share a real-life experience using off-label ibrutinib in the treatment of heavily pretreated WM.

Methods: This multicenter retrospective study conducted in eight different sites included 16 adult patients diagnosed with WM and used off-label ibrutinib monotherapy or with rituximab from April 2016 to January 2022. Disease-refractoriness to prior therapy was defined as progression on therapy or within 2 months of treatment interruption. The response rates were assessed by the modified Third International Workshop on WM Response Criteria.

Results: Sixteen RRWM patients received ibrutinib monotherapy for active/symptomatic WM during the study period; median follow up of the entire cohort was 29 months for the subset that received ibrutinib as salvage therapy. The median time on therapy for the entire cohort was 12.5 months. WM IPSS score was calculated intermediate in five patients and high in eleven patients. MYD88 mutation status was assessed in eight patients and detected positive at the time of WM diagnose. Before therapy, nine of the 16 (56.3%) had a serum level of 3000 mg per deciliter or higher; after treatment, at the time of best response, three of the 16 patients (18.8%) had a serum IgM level of 3000 mg per deciliter or higher. ECOG performance score was 1 in 75% of our cohort. The median lines of prior therapy for the RRWM were 3 (range: 2-8). Only two patients had received autologous stem cell transplantation (ASCT) prior to ibrutinib monotherapy. Responses included a complete response in 4 patients, a very good response in 4 patient and a partial response in six patients, representing overall response of 83.3%. The remaining two patients discontinued ibrutinib due to progressive disease. No several adverse events due to ibrutinib were observed in our cohort. At the time of data collection, one patient had deceased.

Summary/Conclusion: Ibrutinib is an effective drug, associated with durable responses, and safety for both previously-treated and newly diagnosed WM. Our study has several limitations that should be considered when reviewing the results, including the retrospective design and inclusion of a small number of patients. Nevertheless, because of the rarity of the diseases, our data provide useful information on the use of ibrutinib in patients with heavily pretreated WM.

PB2075: CRYSTAL STORING HISTIOCYTOSIS PRESENTING AS HEAD AND NECK TUMORS

E. P. De La Flor Weiss 1,2,* , A. Tupac-Yupanqui 3

1 Hematopathology, VAMC, West Palm Beach, Florida, United States of America; 2 Pathology; 3 Hematology and Internal Medicine, Laboratorio de Medicina y Patologia, Lima, Lima, Peru

Background: Crystal storing histiocytosis (CSH) is a benign proliferation of histiocytes ladden with crystals of immunoglobulin, usually containing kappa light chain in association with a clonal lymphoplasmacytic disorder. Clinical manifestations of CSH may overshadow the underlying lymphoproliferation, usually an indolent lymphoma. Diagnosis is made by tissue examination in the proper clinical, imaging, and serologic context.

Aims: This report highlights the importance of considering the diagnosis of CSH in the differential of H&N lesions to reach the diagnosis with additional sytemic evaluation to establish extent of involvement for both CSH and lymphoma. In addition, we emphasize that persistence of CSH mass lesions does not imply failure of lymphoma treatment.

Methods: Clinical features, imaging, and pathology are reviewed in three patients who presented with head and neck masses: palate mucosa, tonsil, and lacrimal gland respectively. Preliminary pathologic diagnosis was oncocytic neoplasia or histiocytosis NOS in two cases. The third patient was initially diagnosed as CSH. Further testing including Ig levels and molecular studies were also diagnostic and contributory for monitoring of disease status and response to therapy.

Results: Additional clinical hematology/hematopathology evaluation including bone marrow examination (figure 1) revealed systemic involvement by low grade lymphoma as well as involvement of bone marrow and lymph nodes with CSH. One patient had been diagnosed with Myd88 negative Waldentrom’s lymphoplasmacytic lymphoma 9 years earlier and treated with anti Cd20 and radiation therapy with apparent good response and remained asymptomatic until two months before presenting with tonsil mass Current evaluation disclosed a similar lymphoma with Myd88 negative clone and CSH.

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Summary/Conclusion: Initial diagnostic evaluation of oncocyte-like histiocytic lesions of H&N, however benign or bland in appearance, raise the differential of lymphoma and CSH, and require additional work-up and perhaps systemic evaluation, including bone marrow examination. Lack or decrease in size/volume of CSH lesions after treatment for lymphoma does not indicate failure of therapy but requires alternate monitoring criteria.

PB2076: PEDIATRIC-TYPE FOLLICULAR LYMPHOMA - A NEW PARADIGM IN DIAGNOSIS?

L. Fonseca 1,* , C. Marques 2 , H. Barroca 3 , J. Lobo 4 , R. Henrique 4

1 Serviço de Sangue e Medicina Transfusional, Centro Hospitalar Tondela-Viseu, Viseu; 2 Serviço de Patologia Clínica; 3 Serviço de Anatomia Patológica, Centro Hospitalar Universitário São João; 4 Serviço de Anatomia Patológica, IPO-Porto, Porto, Portugal

Background: Recognized as an entity in the 2016 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues, Pediatric-Type Follicular Lymphoma (PTFL) is a rare nodular follicular lymphoma that affects primarily children and young adults. The clinical presentation is characterized by the sudden appearance of an isolated lymphadenopathy, with a predilection for the head and neck region, without systemic symptoms. The incidence is higher in men. It has an excellent prognosis with the excision of the affected ganglion. By definition, diagnosis is histological, immunocytochemical and molecular. There are no known risk factors or any described association with immunodeficiency or viral infections.

Aims: We report two clinical cases.

Methods: Case 1 - a previously healthy 18-year-old boy with an isolated, non-painful, cervical lymphadenopathy of approximately 20 mm, which was incidentally found.

Case 2 – a 13-year-old boy without relevant personal history, who, after the second dose of vaccination against COVID-19, developed multiple adenomegalies that spontaneously regressed. However, one month later, a right submandibular adenomegaly appeared. It was analysed by ultrasound and was described as suspicious.

In both cases, a fine-needle lymph node biopsy was performed for cytological diagnosis and material was sent for immunophenotyping by flow cytometry and molecular cytogenetics by FISH.

Results: Immunophenotyping suggested a large-cell B-lymphoma with a phenotype compatible with Burkitt’s Lymphoma (BL). In the cytology of both cases, the population observed was more consistent with diffuse large B cell lymphoma (DLBCL) or possibly high-grade follicular lymphoma (FL). In the FISH study, no rearrangements in the MYC, BCL2, BCL6 or IRF4 genes were detected in the samples of the two cases. The lymphadenopathies were excised with a probable diagnosis of PTFL or DLBCL. Histological examination confirmed the PTFL diagnosis.

Summary/Conclusion: We did not find in the literature any reference to clear causal relationship between vaccination against COVID-19 and the onset of lymphoproliferative diseases.

The cytological/immunophenotypic/molecular approach of this entity in both cases seems to define a characteristic pattern, which may eventually allow, in a first approach, to suspect this diagnosis.

More extensive studies will be needed to establish the role of these methodologies in the diagnosis of this pathology.

PB2077: POD12 AND POD24 – A NEW SURROGATE MARKER IN MANTLE CELL LYMPHOMA TREATMENT?

J. Freitas 1,* , D. Pereira 1 , C. Moreira 1 , S. Chacim 1 , N. Domingues 1 , A. Espírito Santo 1 , Â. Martins 1 , I. Oliveira 1 , L. Viterbo 1 , I. Moreira 1 , J. Mariz 1

1 IPO Porto, Porto, Portugal

Background: Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin’s lymphoma (NHL) with a generally aggressive, albeit heterogeneous, clinical course. It can present either with asymptomatic monoclonal lymphocytosis or non-bulky nodal/extra nodal disease with minimal symptoms or with a progressive generalized lymphadenopathy, cytopenia, splenomegaly and extra-nodal disease.

Despite the paradigm shift in the treatment landscape, there is still a subset of patients with a worse prognosis. Novel surrogate markers had been emerging in other entities such as POD24 (progression of disease within 2 years) in follicular lymphoma, which is not still studied in MCL.

Aims: Evaluate the prognostic factor of POD within 12months (POD12) and 24months (POD24) after first line treatment in MCL patients

Methods: We retrospectively evaluated 118 patients (pts) with MCL between Jan/2010 and Dec/2020. Survival analysis was evaluated using Kaplan-Meier curves and the identification of predictive factors by the Cox regression method.

Results: 83 (70.3%) pts were male and the median age at diagnosis was 67 years [39-89]. 114 (96.6%) pts had stage III/IV at diagnosis, 85.6% ECOG <2, 81.4% bone marrow involvement, 79.5% diffuse lymphadenopathies, 45.8% B symptoms, 33.9% splenomegaly, 30.5% leukemic phase, 25.4% extra-nodal disease, 25.4% gastrointestinal involvement and 23.7% bulky disease (>5cm). The median LDH at diagnosis was 212U/L. According to the MIPI score, 82pts (69.5%) were low risk, 13 (11.9%) intermediate and 22 (18.6%) high risk. The median lines of therapy was 2 [1-5] and 32pts (27.1%) underwent autologous stem cell transplant after first line therapy. Overall survival (OS) at 2 and 5 years was 81% and 56.6%. POD12 and POD24 occurred in 39 dts (33.1%) and 56 (47.5%), respectively. Patients with POD12 had a 2‐year OS of 44.5% and 5‐year OS of 18.4% compared to 98.7% and 75% for patients without early POD12, respectively. Regarding POD24, 5‐year OS of 20.9% compared to 84.4%, respectively, for patients without early POD24.The Kaplan‐Meier curve showed an inferior OS for patients with early POD compared to those not relapsing within 12 months and/or 24 months (P < 0.001) [figure ​1 and 2]. Univariable analysis showed that ECOG ≥2, leukemic phase, elevated LDH, MIPI score, POD12 and POD24 were associated with inferior OS. In multivariable analysis, clinical factors predictive of inferior OS were POD12, POD24, high risk MIPI, elevated LDH and ECOG ≥2.

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Summary/Conclusion: POD12 and POD24 are associated with unfavorable outcomes and worse OS. These results support the potential to use these criteria as primary surrogate endpoints. Survival analysis did not allow us to conclude which of the endpoints have the greatest discriminative power in OS, so prospective studies with larger sampling will be necessary.

PB2078: A RETROSPECTIVE ANALYSIS OF THE EFFICACY OF LOW-DOSE METRONOMIC CYCLOPHOSPHAMIDE FOR TREATMENT IN PATIENTS WITH LOW GRADE NON-HODGKIN LYMPHOMA

B. Han 1,* , B. J. Kim 1 , H. Y. Kim 1 , D. Y. Zang 1 , H. J. Kim 1

1 Internal medicine, Hallym university sacred heart hospital, Anyang-Si, South Korea

Background: Low-dose metronomic cyclophosphamide chemotherapy is an emerging strategy offering a potentially less toxic yet effective treatment modality.

Aims: We evaluated the efficacy and safety of low-dose metronomic cyclophosphamide in patients with low grade non-Hodgkin lymphoma (NHL) by retrospectively reviewing the data.

Methods: The patients received oral cyclophosphamide (50 mg per day) and/or oral methotrexate (2.5 mg twice weekly) until disease progression or unacceptable toxicity was noted.

Results: Of the 36 patients, 17 (47.2%) were male, median age was 66.4 years (range, 37-91 years), and subtypes of NHL were mucosa associated lymphoid tissue lymphoma (55.6%), small lymphocytic lymphoma (13.9%), follicular lymphoma (11.1%), mantle cell lymphoma (8.3%), nodal marginal zone lymphoma (4.5%), splenic marginal zone lymphoma (2.8%), and lymphoplasmacytic lymphoma (2.8%). The stage I, II, III, IV were 38.9%, 13.9%, 11.1%, 36.1%, respectively and 55.6% of patients received this regimen as the first-line treatment, mainly due to frailty, refusal to standard chemotherapy.

The overall best response rate (ORR) was 73.5% (12 complete responses, CRs and 13 partial responses, PRs), with 29.1 months of the median duration of response and the disease control rate was 88.2%. The median treatment duration was 8.8 months (range, 0.1-38.4 months); the median progression-free survival (PFS) was 43.5 months, and the median overall survival (OS) was not yet reached. Especially, the ORR in patients who were treated with metronomic cyclophosphamide as the first line treatment and as more than the second line treatment were 78.9% (10 CRs, 5 PRs) and 66.6% (2 CRs, 9 PRs), respectively. The median PFS were not reached in the first line cyclophosphamide treatment group and 26.5 months (range, 5.7-47.2 months) in the other group (p=0.110), and similarly, the median OS were not reached and 64.4 months (0-135.7 months) (p=0.058), respectively.

The regimen was generally well tolerated, with small numbers of grade 3-4 toxicities; neutropenia (2%), anemia (3%), thrombocytopenia (3%), fatigue (4%), and anorexia (1%).

Summary/Conclusion: We concluded that low-dose metronomic cyclophosphamide represents efficacious and well-tolerated treatment for low-grade NHL patients.

PB2079: INTERIM REPORT OF BENDAMUSTINE PLUS RITUXIMAB AS FIRST-LINE INDUCTION THERAPY WITH OR WITHOUT LENALIDOMIDE MAINTENANCE THERAPY IN CHINESE PATIENTS WITH INHL OR EMCL: A MULTI-CENTER PROSPECTIVE TRIAL

H. Wang 1 , Q. He 1 , R. Zhao 1 , L. Xie 1 , J. Ma 1 , C. Liu 1 , K. Lu 1 , H. Wei 1 , D. Liu 1 , L. Xing 1 , X. Deng 2 , Z. Sun 3 , X. Chu 4 , N. Gao 5 , Y. Zhong 6 , Y. Sun 7 , H. Zhang 8 , H. Zhang 9 , W. Xu 10 , Z. Li 1,*

1 Department of Hematology, Shandong Cancer Hospital and Institute, Jinan; 2 Department of Hematology, Weihai Municipal Hospital, Weihai; 3 Department of Hematology, Jining First People’s Hospital, Jining; 4 Department of Hematology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai; 5 Department of Hematology, Binzhou Medical University Hospital, Binzhou; 6 Department of Hematology, Qingdao Municipal Hospital, Qingdao; 7 Department of Hematology, Weifang People’s Hospital, Weifang; 8 Department of Hematology, Linyi People’s Hospital, Linyi; 9 Department of Hematology, Affiliated Hospital of Jining Medical University, Jining; 10 Department of Hematology, The First Affiliated Hospital of Shandong First Medical University, Jinan, China

Background: Bendamustine plus rituximab (BR) was established as a preferred first line therapy for patients with previously untreated indolent non-Hodgkin’s lymphoma (iNHL) and elderly MCL (eMCL) based on the BRIGHT and STiL trials. But the data is rare from Chinese patients because the unavailability of bendamustine. Furthermore, the safety and efficacy of maintenance therapy with rituximab remained contradictory after BR induction therapy, due partly to the durable immuno-supression of bendumustine. On the other hand, lenalidomide, as a proven effective agent for iNHL and MCL, have immune modulatory function. So, as maintenance therapy after BR induction, lenalidomide may increase response rate and prolong progression free survival (PFS) for iNHL and eMCL.

Aims: To assess the efficacy and safety of BR regimen for Chinese patients with iNHL and eMCL. And more importantly, to assess the efficacy and tolerability of lenalidomide as maintenance therapy after BR.

Methods: We conducted a multi-institutional, prospective, non-randomized clinical trial for patients with iNHL and eMCL. The patients received standard six cycles of BR regimen as induction therapy, and then received lenalidomide (10mg, qd) maintenance vs. no maintenance, from December 2020 to February 2022. Patients with iNHL and elderly MCL who received at least two cycles of BR were evaluated. The primary endpoint of the study was progression-free survival (PFS).

Results: Overall, 61 patients enrolled in the study, baseline characteristics of cohort were shown in Table 1. Median age at BR initiation was 55 years (range 24-74), 73.8% had ECOG 0-1, and 26.2% ECOG 2. Follicular lymphoma (FL) (62.3%) and marginalzone lymphoma (MZL) (24.6%) were the main subtypes of iNHL. The proportion of elderly MCL was 9.8%. Most patients were in stage 3 (9.8%) and stage 4 (72.1%). All patients have indication for systemic therapy. Till the date of February 10, 2022, a total of 85.2% completed 4 cycles induction therapy. Overall response was 94.2%, with 82.7% complete response (CR) and 11.5% partial remission (PR), (Figure 1). Progression during induction occurred only in one MCL patients. And another two patients with FL was suspected progressive disease during observation (no lenalidomide maintenance). After 4 cycles induction therapy, PET/CT detected in 40 patients (76.9%), while CT or MRI detected in 12 patients (19.7%). Among the 39 patients with complete remission assessed by PET-CT, 8 patients (20.5%) of them had residual lymph nodes with diameter > 1cm. So the CRR by CT is 67.3%, which is much higher than that reported in STiL study and BRIGHT study. The safety profile of BR regimen was shown in Table 2. Hematological toxicities was the main adverse events, with grade 3-4 neutropenia was recorded in 21.3%, febrile neutropenia in 8.2%, grade 3-4 anemia in 3.3%, and grade 3-4 thrombocytopenia in 1.6% of patients. The rate of fatigue, nausea/vomiting, skin rash and infections were less than 15%. A total of 40.0% patients proceeded to lenalidomide maintenance. However, 10% patients discontinued because of rash or other adverse reaction to lenalidomide.

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Summary/Conclusion: BR regimen was feasible, safety and got high CRR for Chines iNHL and eMCL patients by our clinical trial. The efficacy and safety of maintenance lenalidomide after BR induction are still under follow-up. This trial was registered at http://www.chictr.org.cn . Clinical trial information: ChiCTR2200056288.

PB2080: EFFICACY AND SAFETY OF BENDAMUSTINE-BASED REGIMENS AS FIRST-LINE TREATMENT FOR INDOLENT B-CELL NON-HODGKIN LYMPHOMA: A PROSPECTIVE, MULTICENTER, REAL-WORLD STUDY IN CHINA

S. Li 1 , S.-J. Wen 1 , J. Ma 2,*

1 Department of Lymphoma, Affiliated Cancer Hospital of Xinjiang Medical University, Urumqi; 2 Department of Hematology & Oncology, Harbin Institute of Hematology & Oncology, Harbin, China

Background: Indolent non-Hodgkin lymphoma is typically responsive to initial immune-chemotherapy, but it is also prone to persistent recurrence. Bendamustine, an alkylating agent with properties of a purine analog, has been approved for first-line treatment of indolent non-Hodgkin lymphoma in Japan. Although China has approved the second-line treatment of bendamustine for indolent B-cell non-Hodgkin lymphoma in 2019, evidence of bendamustine as first-line treatment for this patient population in China is still scanty currently.

Aims: To evaluate the efficacy and safety of bendamustine-based regimens as first-line treatment for Chinese patients with indolent B-cell non-Hodgkin lymphoma in real-world setting.

Methods: Treatment-naïve patients with indolent B-cell non-Hodgkin lymphoma were enrolled in this prospective, multicenter, single-arm, real-world study (ChiCTR2000039605). Eligible patients will be given 4-6 cycles (4 weeks/cycle) of bendamustine-based regimens including bendamustine monotherapy (90 mg/m 2 on Day 1-2), bendamustine (90 mg/m 2 on Day 1-2) plus rituximab (375 mg/m 2 on Day 1), bendamustine (70 mg/m 2 on Day 1-2) plus rituximab and cytarabine (500 mg/m 2 on Day 2-4), or bendamustine (90 mg/m 2 on Day 1-2) plus rituximab and bortezomib (1.6 mg/m 2 on Day 1, 8 and 15). Tumor response was assessed by independent central review according to Lugano 2014 response criteria. Primary endpoint was objective response rate (ORR). Secondary endpoints were disease control rate (DCR) and safety.

Results: Between December 2019 and February 2022, 29 patients were enrolled in this study. The median age of the 29 patients was 63.0 years (range, 35.0-83.0 years). Majority of patients were female (17/29, 58.6%), had stage II-IV disease (23/29, 79.3%), had an International Prognostic Index (IPI) score of 0-2 (20/29, 69.0%), and had an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0-1 (21/29, 72.4%). Sixteen patients (55.2%) completed 6 cycles of treatment, and 22 patients (75.9%) completed 4-6 cycles of treatment. Of the 29 patients, 19 (65.5%) achieved a complete response, 5 (17.2%) achieved a partial response, 1 (3.5%) had a stable disease, and 4 unknown (13.8%). The ORR and DCR were 82.8% (95% CI, 64.2-94.2%) and 86.2% (95% CI, 68.3-96.1%), respectively. Hematological and non-hematological adverse events were reported in 75.9% and 17.2% of patients, respectively.

Summary/Conclusion: Bendamustine-based regimens as first-line treatment were effective and well-tolerated in patients with indolent B-cell non-Hodgkin lymphoma, suggesting that this first-line regimen might offer a potential therapeutic option for this patient population in China.

PB2081: TREATMENT PERSISTENCE AND ADHERENCE TO IBRUTINIB IN PATIENTS WITH WALDENSTRÖM MACROGLOBULINEMIA: A GERMAN CLAIMS DATA ANALYSIS

C. Buske 1,* , G. Hess 2 , N. Bahar 3 , K. Yang 4 , B. Tang 4 , A. Imhof 5 , K. Vinz 5 , E. Herweijer 6 , M. Herold 7

1 Institut für Experimentelle Tumorforschung, Universitätsklinikum Ulm, Ulm; 2 III. Med. Klinik | UCT Mainz | Universitätsmedizin Mainz, Mainz, Germany; 3 BeiGene Switzerland GmbH, Basel, Switzerland; 4 BeiGene USA, Inc., San Mateo, United States of America; 5 BeiGene Germany GmbH, München; 6 Ingress-Health HWM GmbH, Wismar; 7 Helios Klinikum Erfurt, Erfurt, Germany

Background: Waldenström macroglobulinemia (WM) is an incurable condition characterized, in most patients, by symptomatic recurrences that affect the quality of life. Until recently, ibrutinib, a first-generation Bruton tyrosine kinase inhibitor, was the only agent approved in the EU for the treatment of WM. Previous research suggests that adherence to this orally-administered agent is directly related to clinical outcomes. Even if adherence to ibrutinib was reported to be high in particularly monitored clinical trial settings, the rate of ibrutinib adherence and persistence in real-world populations has hardly been investigated so far.

Aims: This study aimed to describe treatment persistence and adherence to ibrutinib in German patients with WM.

Methods: This retrospective study was conducted using anonymized claims data covering the period January 1, 2010, to June 30, 2020, provided by a regional German statutory health insurance fund (AOK PLUS) that insured approximately 3.4 million individuals in Saxony and Thuringia.

Individuals were included in the analysis if at least one inpatient diagnosis with WM (ICD-10-GM code C88.0) and/or two confirmed outpatient WM diagnoses made in different quarters were observed between January 1, 2011, and June 30, 2020. To ensure incident disease, patients had to show a diagnosis-free period of ≥12 months prior to the first observable WM diagnosis. Patients were followed from the first ibrutinib prescription (ATC-code: L01XE27) until either death, loss to follow-up, or June 30, 2020.

Non-persistence (NP) was defined as a supply gap of >90 days, with supply provided by each prescription being derived based on the defined daily dose (DDD by WHO/WIdO) of ibrutinib. Adherence was assessed by calculating the proportion of days covered (PDC) in the period where a patient was generally considered to still continue treatment with ibrutinib (i.e., no supply gap >90 days). A patient was considered non-adherent if the PDC was <80%. Adherence and persistence were assessed under the assumptions that a patient was stockpiling in case of overlapping prescriptions and that there was ongoing drug coverage during a hospitalization (base case; sensitivity scenario analysis regarding different assumptions was conducted). The sensitivity analysis also considered different gap definitions to account for the uncertainty of the concordance between the prescribed daily dosage (treating physician) and DDD (WHO/WIdO definition related to an average patient).

Time to NP was assessed using the Kaplan-Meier estimation. The PDC and rate of non-adherence were analyzed by means of descriptive statistics.

Results: A total of 483 patients with incident WM were identified during the study period, of which 23 patients initiated ibrutinib treatment (mean age: 71.8 years, females: 47.8%). Patients had an average follow-up of 321.5 days after treatment initiation. Based on the Kaplan-Meier estimates, 77.1% (95%-CI: 34.5-93.9%) of patients were still on treatment after one year. The mean PDC was 77.4% (95%-CI: 68.7-86.1%), and the proportion of patients considered non-adherent to ibrutinib accounted for 42.9% (95%-CI: 19.8-65.9%; see the base case in Table 1).

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Summary/Conclusion: This study suggests that a substantial portion of WM patients have limited adherence and persistence to orally administered ibrutinib. Further nationwide studies with larger sample sizes may be required to confirm the results.

PB2082: POPULATION-WIDE PATTERNS OF CARE IN MANTLE CELL LYMPHOMA IN AUSTRALIA: AN ANALYSIS OF THE PHARMACEUTICAL BENEFITS SCHEME DATASET

C. Tam 1,* , F.-L. Zhao 2 , T. Liu 3 , R. Gauba 2 , S. C. Li 4 , B. Tang 3

1 Peter MacCallum Cancer Centre, St. Vincent’s Hospital, and University of Melbourne, Melbourne; 2 BeiGene AUS PTY Ltd., Sydney, Australia; 3 BeiGene USA, Inc., San Mateo, United States of America; 4 University of Newcastle, Callaghan, Australia

Background: The treatment landscape in patients with mantle cell lymphoma (MCL) is changing with the introduction of Bruton’s tyrosine kinase inhibitors (BTKis) and bendamustine in Australia.

Aims: We sought to analyze the practice impact of the introduction of publicly funded novel agents in MCL. The objective of this study was to describe the evolving treatment patterns of Australian patients with MCL over the last 10 years using population-wide prescription records.

Methods: Patients who initiated a treatment for MCL between 01/01/2011 and 07/31/2021 were extracted from the Services Australia 10% Pharmaceuticals Benefits Scheme (PBS) dataset, which includes the dispensing records for 10% of the Australian population. This dataset captures all publicly funded treatments in Australia. The index date was defined as the commencement of any drug for MCL. First-line (1L) therapy was defined as the first treatments prescribed for MCL. A patient was defined as relapsed/refractory (R/R) if they had commenced a drug which was in a different therapeutic category, or if they re-started the same regimen after a gap of more than 180 days. Descriptive analyses were conducted to examine the use of treatment regimens for the overall 10-year population by line of therapy. Analyses by calendar year were also performed to assess the changes in the treatment patterns.

Results: Overall, 241 patients with MCL were identified over the study period. The majority of patients were male (68.4%), age > 60 years (84.9%), and most being aged 70-79 years (42.1% of total). Many patients were receiving comedications at baseline, including antihypertensives (44.1%), anticoagulants (14.5%), and/or antipsychotics or antidepressants (12.5%). In the overall population (2011-2021), the majority of patients had received 1L treatment with bendamustine-rituximab (BR, 53.9%), rituximab plus other regimens (27.6%), or rituximab monotherapy (11.2%). The most commonly used regimens in R/R patients at any subsequent episode of treatment included BTKi (66.3%), rituximab monotherapy (52.8%) or rituximab plus other regimens (31.5%). A trend in adoption of novel agents was observed throughout the years following their PBS listing. Analysis by calendar year showed that from 2011 to 2020 for 1L therapy, the use of BR increased from 0% to 50% and use of rituximab-containing regimen except BR decreased from 100% to 16.7%; use of BTKi increased in R/R patients from 0% to 74.7%.

Summary/Conclusion: MCL treatment patterns have significantly changed in Australia since the introduction of BTKis and bendamustine-containing regimens. The use of rituximab-containing regimens except BR in 1L MCL has decreased and use of BTKis in R/R patients has increased.

PB2083: STUDY CONDUCT AND PATIENT SAFETY DURING THE COVID-19 PANDEMIC: OBSERVATIONS IN PATIENTS WITH PREVIOUSLY UNTREATED FOLLICULAR LYMPHOMA FROM THE GAZELLE TRIAL OF OBINUTUZUMAB SHORT DURATION INFUSION

M. A. Canales 1,* , T. A. Buchholz 2 , T. Ishikawa 3 , L. M. Fogliatto 4 , J. Bortolini 5 , F. J. Capote Huelva 6 , A. Salar 7 , D. Tucker 8 , E. Vorozheikina 9 , D. Klingbiel 10 , S. Pokala 10 , J. Parreira 10 , S. Nick 10 , K. Hübel 11

1 Hospital Universitario La Paz, Madrid, Spain; 2 Scripps MD Anderson Cancer Center, San Diego, United States of America; 3 Kobe City Medical Center, General Hospital, Hyogo, Japan; 4 Hospital de Clínicas de Porto Alegre, Porto Alegre; 5 Centro de Pesquisas Oncologicas, Florianopolis, Brazil; 6 Hospital Universitario Puerta del Mar, Cádiz; 7 Hospital del Mar, Barcelona, Spain; 8 Royal Cornwall NHS Trust, Truro, Cornwall, United Kingdom; 9 IQVIA, Medical and Scientific Services, Moscow, Russia; 10 F. Hoffmann-La Roche Ltd, Basel, Switzerland; 11 Uniklinik Köln, Köln, Germany

Background: Obinutuzumab is routinely administered as a 3–4-hour infusion in patients (pts) with follicular lymphoma (FL). A 90-minute short duration infusion (SDI) of obinutuzumab is being evaluated in GAZELLE ( NCT03817853 ), a Phase IV study in pts with previously untreated FL. Earlier analyses showed that response rates with obinutuzumab SDI were in line with previous studies of obinutuzumab administered at the standard rate and no new safety signals were reported (Canales, et al. 2021). In addition, >95% of healthcare providers preferred obinutuzumab SDI to the standard infusion, mainly due to clinic and pt time savings (Trask, et al. 2021). The time savings associated with obinutuzumab SDI could be particularly beneficial during the coronavirus disease 2019 (COVID-19) pandemic.

Aims: To investigate the effect of the COVID-19 pandemic on the GAZELLE study conduct and to describe safety in vaccinated and unvaccinated pts in an exploratory analysis.

Methods: Pts with previously untreated FL received obinutuzumab (1000mg) intravenously on Days (D) 1, 8 and 15 of Cycle (C) 1, and on D1 thereafter, plus chemotherapy (bendamustine; cyclophosphamide, doxorubicin, vincristine and prednisone [CHOP]; or cyclophosphamide, vincristine and prednisone [CVP]) for 6–8 cycles (induction phase). Pts received a standard infusion (3–4 hours) of obinutuzumab in C1. Pts without a Grade ≥3 infusion-related reaction during C1 received obinutuzumab SDI (90-minute infusion) from C2 onwards. Pts with a complete or partial response received maintenance with obinutuzumab SDI for 2 years or until disease progression (maintenance phase). All pts provided informed consent prior to study entry.

Results: At the clinical cut-off date of January 18, 2022, 35/113 (31.0%) pts in the overall population had received a COVID-19 vaccine. Demographic and baseline characteristics were generally similar in the vaccinated and non-vaccinated groups; however, the vaccinated group had a greater proportion of pts who were aged ≥65 years (n=17/35, 48.6% vs n=27/78, 34.6%, respectively). The most common vaccine received was Pfizer BioNTech (n=22/35, 62.9%), and most pts received 2 vaccine doses (n=25/35, 71.4%). Fewer study discontinuations were reported in the vaccinated versus the non-vaccinated group (n=1 vs n=21, Figure). Five pts in the vaccinated group had adverse events (AEs) ≤7 days after vaccination (no serious AEs reported). None of these AEs were considered study drug related and all resolved without changes to study treatment. COVID-19 infection was reported in 5 pts (induction phase, n=1; maintenance phase, n=4). Of these, 1 pt had been vaccinated (Moderna, 2 doses; >6 months prior to infection) and 3 pts received concomitant medication to treat COVID-19 infection. Three of the 5 pts recovered, and 2 pts (both unvaccinated) died due to respiratory complications following COVID-19 infection. Low total leukocyte count (an indicator of COVID-19 disease severity) was observed in 4 pts with a COVID-19 infection (7 episodes of Grade 1/2; 3 episodes of Grade 3). Overall, 19/113 (16.8%) pts had ≥1 major protocol deviation related to the COVID-19 pandemic, the most common being delayed (n=8) or missed (n=7) study drug infusion.

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Summary/Conclusion: The conduct of the GAZELLE study was not substantially impacted by the COVID-19 pandemic. Documented COVID-19 infections occurred in 5 pts and fatal outcomes were reported in 2 of these pts (both unvaccinated). Up to 7 days after COVID-19 vaccination, no AEs considered related to obinutuzumab or other study treatments were observed.

PB2084: FIRST-LINE TREATMENT IN ADVANCED STAGE FOLICULAR LYMPHOMA: A SINGLE-CENTER EXPERIENCE WITH R-CHOP VS BENDAMUSTINE-RITUXIMAB.

O. Mompel 1,* , C. Amorós 1 , P. López-Menargues 1 , C. Játiva 1 , A. Cámara 1 , R. García-Serra 1 , M. J. Lis 1 , M. T. Orero 1 , S. Ortiz 1 , M. Linares 1

1 Hospital General Universitario de Valencia, Valencia, Spain

Background: R-CHOP and bendamustine-rituximab (B-R) have been the most widely used induction regimens in recent years in advanced-stage follicular lymphoma (FL). B-R in comparison with R-CHOP has shown in 2 randomized studies a higher rate of complete remissions and better progression-free survival (PFS) in one of them, although without improving overall survival (OS).

Aims: To review our experience and compare the efficacy of R-CHOP vs B-R in advanced-stage FL.

Methods: We reviewed the medical records of 92 patients diagnosed in our hospital with advanced-stage FL, who had received R-CHOP or B-R as first-line treatment between June 2005 and June 2021. We assessed the clinical and biological data at diagnosis, including FL International Prognostic index (FLIPI), response to induction treatment, incidence of major infectious events requiring hospitalization, PFS and OS. Quantitative variables were compared with Mann-Withney U-test and qualitative variables using Chi-square or Fisher’s exact tests. Survival was calculated using Kaplan-Meier and the differences between groups were studied using the Log-rank test.

Results: No differences were observed as per the variables with prognostic influence between the two groups, and although age was higher in the BR group, the differences were not significant, neither the risk distribution according to FLIPI. We observed a high response rate with both regimens, with a slightly higher percentage of complete responses with B-R, yet the differences were not significant (Table). Regarding the number of admissions for infection during treatment, it was higher in patients treated with R-CHOP although there were no significant differences (Table). With a median follow-up of 62 months, no significant differences were observed in OS between the two regimens, and median survival was not achieved in neither group (see Table and Figure). As for PFS, no differences were observed between the two regimens (Table and Figure). When patients older than 60 years were analyzed separately (44 patients), no significant differences were observed in PFS and OS, as in the complete series of patients (data not shown). 8 patients received rituximab maintenance after B-R (44%), and 48 patients following R-CHOP (64,95%). Only 2 patients in each treatment group underwent high-dose therapy followed by autologous stem-cell transplantation (ASCT).

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Summary/Conclusion: Our experience does not demonstrate greater efficacy regarding depth of response neither in PFS with the B-R regimen in comparison with R-CHOP as has been previously reported, although it must be considered that the limited number of patients included may have influenced these results. Regarding the similarity in OS with both regimes, our study confirms the findings observed in previous studies. Hence, based on our experience, both regimens are a valid option for the treatment of patients with advanced-stage FL. However, given the higher incidence of admissions due to infectious events in patients treated with R-CHOP, we consider the use of B-R seems to be a preferable option for patients with greater frailty and susceptibility to infection.

PB2085: UPFRONT TREATMENT OF MANTLE CELL LYMPHOMA (MCL) WITH R-HDAC (RITUXIMAB-HIGH DOSE CYTARABINE + DEXAMETHASONE) IN YOUNG, HIGH RISK PATIENTS IS WELL TOLERATED AND LEADS TO DURABLE RESPONSES.

G. U. Naylor 1,* , S. Mant 1 , C. Bedford 2 , D. Turner 3 , M. Furtado 2 , D. Lewis 1

1 Haematology, University Hospitals Plymouth NHS trust, Plymouth; 2 Haematology, Royal Cornwall Hospitals NHS Trust, Truro; 3 Haematology, Torbay and South Devon Hospitals NHS Trust, Torbay, United Kingdom

Background: MCL commonly presents as aggressive disease with a low cure rate and multiply relapsing/remitting course. Treatment of classical MCL with upfront intensive chemoimmunotherapy (CIT) consolidated with an autologous stem cell transplant (ASCT) is considered best practice in younger patients. There is no gold standard intensive CIT regimen. Common approaches include alternating RCHOP/RDHAP (Nordic) and R-Hyper-CVAD.(1) The addition of high dose Cytarabine (HDAC) has been shown to improve time to treatment failure (TTF), complete response (CR) rates and progression free survival (PFS).

Aims: There is little published on the efficacy of R-HDAC in treatment-naive MCL, however a 2013 case series (Forbes et al) reported good early efficacy (CR in 16/18), with no detriment to harvest success. We describe tolerability and efficacy of upfront R-HDAC in a high risk cohort of younger patients with MCL.

Methods: We identified 28 patients who received upfront R-HDAC in 3 UK centres from 2010–2021. Data were collected, anonymized and transferred centrally for analysis using Kaplan-Meier survival curves.

Results: For baseline characteristics see Table 1. Treatment regimen: Dexamethasone 40mg d1-d4, Rituximab 500mg d1, Cytarabine 3000mg/m 2 BD d2-d3 in 21day cycles. 22/28 (79%) completed their planned quota of 4-6cycles; 6/28 (21%) did not. Of these n=5 had progressive disease (PD), n=1 ceased with toxicity. 4/28 incurred cycle delays. 1 patient developed PD after completing R-HDAC.

Haematological toxicity ≥ Grade 3 CTCAE: Anaemia 71%; Thrombocytopenia 93%; Neutropenia 93%. Fatigue (57%), Nausea 25%, Vomiting 7%, Constipation 21%, Diarrhoea 11%, Cardiac toxicity 11%, Pulmonary toxicity 18%, Rash 11%. 20/28 (71%) underwent stem cell transplant consolidation of which 19 had an ASCT. The median CD34 + stem cell harvest was 8.3x10 9 ^L/Kg (2.56-40.9) with no harvest failures. One patient underwent an allogeneic SCT. Of those who didn’t undergo transplant: 1/8 declined; 1/8 was ineligible; 6/8 had PD and switched to 2 nd line therapy.

Overall Response Rate (ORR) (RECIST) was 78%, CR 41%; PR (Partial response) 37%; and PD 22%. The median PFS was 3.39years (95% CI 0-7.937). 5y OS was 64.3% (95% CI 44.9-83.7%), median OS 8.7years (95% CI 2.634 – 14.894).

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Summary/Conclusion: Our cohort showed higher rates of haematological toxicity, but lower gastrointestinal toxicity than those reported with the Nordic approach.(1) There were no incidents of treatment related mortality (TRM). More intensive regimens e.g. R-Hyper-CVAD have documented TRM rates of up to 8%. Only 1 patient in our cohort ceased treatment because of toxicity. Whilst our ORR is lower than those published in trial data on commonly used regimens (78% vs MCL Younger trial ORR: RCHOP arm 90%/Cytarabine arm 94%) our cohort’s baseline demographics are comparably much higher risk: MIPIb High 53% vs 25/20%; Blastoid disease 25% vs 9/8%. Of those with PD in our cohort, 4/6 had high MIPIb and blastoid disease. Despite this disparity, R-HDAC retains a comparable CR rate (41% vs 38%) and 5y OS (64.3% vs 65%). Whilst this observational retrospective study has limitations, it has shown that R-HDAC is an option for younger patients with high risk MCL. Using fewer CIT drugs could reduce toxicity and reserve more agents for use in future relapse. References: 1. Hermine, O. et al. Addition of high-dose cytarabine to immunochemotherapy before autologous stem-cell transplantation in patients aged 65 years or younger with mantle cell lymphoma (MCL Younger): a randomised, open-label, phase 3 trial of the European Mantle Cell Lymphoma Network. Lancet. 2016;388(10044):565-75.

PB2086: CHRONIC LYMPHOPROLIFERATIVE DISORDER OF NK CELLS WITH LIVER INFILTRATION

V. Otasevic 1,* , D. Antic 1,2 , V. Vukovic 1 , V. Djurasinovic 1,2 , K. Tomic 1 , B. Mihaljevic 1,2

1 Lymphoma Center, Clinic for Hematology, University Clinical Center of Serbia; 2 Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Background: Chronic lymphoproliferative disorder of natural killer (NK) cells (CLPD-NK) represents exceptionally rare and heterogeneous indolent disorder, which has various clinical manifestations. Infrequently CLPD-NK can present with several non-indolent features, potentially mimicking more aggressive clinical entities (such as aggressive NK cell leukemia).

Aims: We report the case of CLPD-NK with liver infiltration, extreme splenomegaly and direct Coombs negative hemolytic anemia.

Methods: The patient was thoroughly examined both in ambulatory and hospital conditions. The pleiad of diagnostic tools and procedures was used, ranging from variety of laboratory examination of blood samples, radiological diagnostic procedures (for instance, computerized tomography) and invasive diagnostics (bone marrow biopsy, lymph node biopsy and blind percutaneous liver biopsy).

Results: A 42 years-old male patient presented with B symptoms that lasted for 3 months. After exclusion of infective and immunologic etiology of the condition, the patient was referred to hematologist. Complete blood count (CBC) demonstrated normocytic anemia (Hb 79 g/L, MCV 81 fL), leucocytosis (11.4 x10 9 /L) with lymphocytosis (72%) and NK cells (14%) in peripheral blood smear, accompanied with presence of spherocytes and reticulocytosis. Negative direct Coombs test was observed together with indicators of hemolytic anemia (indirect hyperbilirubinemia, unmeasurable haptoglobin level and elevated level of lactate dehydrogenase). By both physical examination and ultrasound, hepatomegaly (CC 17cm) and extreme splenomegaly (CC diameter 25cm) was revealed, together with axillary lymphadenopathy (up to 26mm). By bone marrow examination lymphocytic infiltration was excluded. Immunophenotyping of peripheral blood showed that 91% of circulating lymphocytes (9.88x10 9 /L) were NK-lymphocytes with distinctive immunophenotype profile (CD2, CD7, CD16) + /(CD3, CD56) - (Figure 1). Additionally, axillar lymph node biopsy was performed and pathohistological analysis showed sinus histiocytosis. Because of extreme splenomegaly and non-homogenous structured hepatomegaly, the patient was examined by gastroenterologist which indicated and afterwards performed blind liver biopsy. The pathohistological analysis of the liver biopsy indicated that the most favourable diagnosis is liver infiltration of CLPD-NK, with possible differential diagnosis of hepatosplenic T-cell lymphoma and EBV negative aggressive NK cell leukemia. Considering this finding, the fact that patient was corticosteroid resistant and that the patient’s condition and CBC parameters were significantly deteriorating, he was administered with one cycle of CHOP chemotherapy regimen. The therapy was complicated by febrile neutropenia which demanded hospital treatment, with prolong neutropenia grade II-III and the persistence of all previously mentioned symptoms and signs of disease activity. Following this, the peroral cyclophosphamide and prednisolone combination therapy was initiated and resulted in complete remission inside of 4 months from the treatment initiation.

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Summary/Conclusion: CLPD-NK is highly uncommon and indolent disorder, with wide variety of clinical presentation. The backbone of the diagnostic approach and consequent adequate treatment is the differentiation the condition from more aggressive entities.

PB2087: EFFICACY OF A 3RD COVID VACCINE, INCLUDING DURING A BTKI PAUSE, IN INDIVIDUALS WITH WALDENSTRÖM MACROGLOBULINEMIA AND FOLLICULAR LYMPHOMA

K. Rankin 1,2,* , B. Beaton 3,4 , S. Sasson 5,6 , J. Raedemaecker 3 , A. Wong 3 , P. Hastak 5 , C. Phetsouphanh 5 , A. Warden 7 , M. Patel 3 , V. Klemm 5 , C. M. L. Munier 5 , A. Carey Hoppe 5 , F. Tea 8 , A. Pillay 8 , A. Ospina 5 , A. Aggarwal 5 , O. Lavee 9 , I. D. Caterson 10 , S. Turville 5 , A. D. Kelleher 5 , F. Brilot 8,11,12 , J. Trotman 3,4

1 Haematology Department, Calvary Mater Hospital Newcastle, Waratah; 2 Haematology, NSW Health Pathology, Newcastle; 3 Haematology Department, Concord Repatriation General Hospital; 4 Concord Clinical School, Faculty of Medicine and Health, The University of Sydney; 5 The Kirby Institute, The University of New South Wales; 6 Immunology Department, Westmead Hospital; 7 WMozzies: Australian Patient Support Group for Waldenström’s Macroglobulinemia; 8 Sydney Institute of Infectious Disease, Faculty of Medicine and Health, The University of Sydney; 9 Haematology Department, St Vincent’s Hospital; 10 COVID Vaccination Hub, Sydney Local Health District; 11 Brain Autoimmunity Group, Kids Neuroscience Centre, The Children’s Hospital at Westmead; 12 The School of Medical Sciences, The University of Sydney, Sydney, Australia

Background: Patients (pts) with indolent lymphomas are at increased risk of severe COVID-19 infection. We have shown limited seroconversion and live viral neutralisation (VN), but preserved COVID-specific T cell responses after 2 doses of mRNA COVID-19 vaccination in such pts. (Beaton, B ASH 2021, 149348). A 3 rd vaccine dose to complete primary vaccination has since been recommended.

Aims: To assess humoral & cellular immune responses to a 3rd COVID-19 (mRNA) vaccination in pts with follicular lymphoma (FL) & Waldenström Macroglobulinemia (WM), including assessment of response after pausing BTKi therapy in WM pts.

Methods: Patients with WM, FL & healthy controls (HC) were enrolled in a prospective observational study to measure immune responses 21-28 days after a 3rd mRNA COVID-19 vaccine. Immune response was measured by mean fluorescence intensity (MFI) of anti-SARS-CoV-2 spike antibodies (ASAb) obtained using a high-sensitivity live cell assay, live VN to a panel of SARS-CoV-2 variants of concern, and CD4+ & CD8+ antigen-specific T cell responses. The associated TRIBECA (TReatment Interruption of BTKi to Enhance COVID-19 Antibody response) study sought to determine if a superior immune response could be gained by pause of BTKi therapy prior to and up to 4 weeks after 3 rd vaccine dose. Patients were closely monitored during the BTKi pause with weekly clinical, full blood count and IgM assessments. WM pts receiving a 3 rd dose while continuing on BTKi served as a control. Statistical analysis of medians between cohorts were compared by the non-parametric Mann-Whitney (Graphpad Prism). Comparison of medians between paired grouped data was assessed by 2-way ANOVA.

Results: To date, 56 of 125 pts had their ASAb measured following 3 rd vaccine dose administered between October 2021 and January 2022: 28 WM pts (including 6/9 WM pts on the BTKi pause sub-study), 24 FL pts and 4 HC. Median age was 68 years with 21 females and 35 males. Median follow up from 2 nd dose was 140 days (range: 79-170 days). In antibody responders, median MFI fell from 163042 (IQR 82663-249934) 28 days post 2 nd dose to 52117 (IQR 19942-60973) (p<0.0001) immediately before the 3 rd dose. Median MFI in all FL & WM pts pre- 3 rd dose vaccine was 17111 (IQR 0-52650), rising significantly post 3 rd dose to a median of 86730 (IQR 0-221937). Only 4/20 pts without measurable ASAb prior to the 3 rd dose (2 WM, 2 FL) developed measurable ASAb following the 3 rd dose: 16/20 patients (8 WM, 8FL, all treated) had no ASAb response. The median MFI in pts who underwent a BTKi treatment pause rose from 9151 (IQR 1671-21232) pre-3 rd dose to 87720 (IQR 2785-152195) post 3 rd dose, significantly higher than the median MFI in WM pts who did not pause their BTKi, which rose from 16769 (IQR 218-22447) pre- to 20252 (IQR 168-114262) post 3 rd dose, (p = 0.016). Of the 5/125 with COVID infection in the study to date, only one patient (without measurable ASAb) in this 3-dose cohort had COVID, requiring intensive care support.

Summary/Conclusion: Most WM & FL pts who responded to a 2nd dose COVID vaccine showed a decline in ASAb titre over time which increased following a 3 rd mRNA vaccine. Only 20% of pts without detectable ASAb pre-3rd dose showed improvement post 3 rd dose, highlighting the importance of other COVID protection strategies in these pts. Although initial numbers are small, there may be a higher increment in ASAb when BTKi therapy is paused around the time of vaccination. Comprehensive immune analysis, including VN and T-cell response on the entire FL & WM cohort will be presented at the EHA congress.

PB2088: IMMUNE PROFILE OF PATIENTS WITH FOLLICULAR LYMPHOMA ASSESSED BY FLOW CYTOMETRY IN PERIPHERAL BLOOD: CHARACTERISTICS AT DIAGNOSIS AND AT RELAPSE OF THE DISEASE

A. Rivero Arango 1,* , P. Mozas 1 , J. Correa 1 , A. Rivas-Delgado 1 , F. Araujo-Ayala 2 , K. Guinetti 1 , A. Bataller 1 , M. Condom 1 , A. Gaya 1 , J. Delgado 1,2,3 , E. Giné 1,2,3 , P. Perez-Galán 2 , E. Campo 2,3,4,5 , A. Vlagea 6 , E. Matutes 4 , A. López-Guillermo 1,2,3,5 , N. Villamor 2,3,4 , L. Magnano 1,2

1 Hematology, Hospital Clinic de Barcelona; 2 Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Barcelona; 3 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Madrid; 4 Hematopathology Unit, Department of Pathology, Hospital Clinic de Barcelona; 5 University of Barcelona (UB); 6 Immunology, Hospital Clinic de Barcelona, Barcelona, Spain

Background: Follicular lymphoma (FL) is the indolent lymphoma more frequently diagnosed in Western countries and is characterized by a heterogeneous clinical course. The biologic importance of the tumour microenvironment in FL pathogenesis is well established. However, detailed information on the immune profile in peripheral blood (PB) has not been previously investigated. In the current era of CAR-T cell therapy and bispecific antibodies, the characterization of the immune cellular populations in the blood becomes very relevant.

Aims: Characterize the immune populations in PB of patients with FL at diagnosis and at relapse, and compare them with healthy controls. In addition, a subanalysis was performed on the relapse group to identify associations between immune populations and POD24.

Methods: We collected samples of FL patients either at diagnosis (n=35) or at relapse (n=34) from March 2019 to December 2021 (median age: 61 years [27-81]; 40M/29F). Sixteen of the 34 relapsed patients were POD24. Moreover, 15 PB samples from healthy donors were also obtained (median age: 31 years [28-65]; 5M/10F). Baseline characteristics of the FL patients were collected. All individuals signed informed consent to participate in the study. The identification of different immune populations (major subsets of T-cells, B-cells, NK cells, monocytes, neutrophils, eosinophils, basophils, dendritic cells and myeloid suppressor cells) was performed by multiparameter flow cytometry. Euroflow methodology was used to set up the Omnicyt cytometers. 150.000 events were the minimum acquired by the Omnicyt™ flow cytometer. All data were analysed using Infinicyt™ software version 2.0 (Cytognos, Salamanca Spain). The Mann-Whitney U test was used to compare means between FL patients and healthy controls, and the chi-squared test was used to assess the association of immune populations with POD24. Immune populations were categorized using the mean percentage value of healthy controls as cut-off.

Results: Detailed data of immune populations of FL patients and healthy controls is shown in the table. Compared with healthy controls, patients with FL showed a lower CD4 + /CD8 + ratio due to an increase in CD8 + lymphocytes and a decrease in total CD4 + lymphocytes. In addition, FL patients had a significantly lower percentage of naive T lymphocytes, a higher percentage of effector memory and effector lymphocytes, both CD4 + and CD8 + , and a higher percentage of activated CD4 + and CD8 + lymphocytes (p<0.0001 in all cases). Of note, FL PB samples had increased percentage of memory and activated regulatory T lymphocytes (Treg) (p= 0.01). These differences were maintained when immune populations were compared at diagnosis and at relapse (see table). Finally, patients with FL at diagnosis showed a decrease percentage of myeloid dendritic cells (p= 0.04). No differences were observed in the percentage of neutrophils, monocytes, eosinophils or total B lymphocytes. Interestingly, POD24 events were found in patients who had higher CD8 + effector and/or lower CD8 + central memory percentages in comparison with no POD24 patients (p= 0.004).

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Summary/Conclusion: FL patients show an immunological profile in PB that is significantly different from that observed in healthy controls. The high CD8 + effector and/or low CD8 + central memory profile is significantly associated with early relapse patients. Knowledge of these immune alterations may contribute to the understanding of its pathogenesis and behaviour and might help to devise new immunotherapeutic strategies adapted to the patient´s immunological profile.

PB2089: ALGORITHM FOR SEVERE COVID-19 RISK STRATIFICATION DEPENDING MONOCLONAL ANTI-CD20 ANTIBODIES TREATMENT DURATION IN NON-HODGKIN LYMPHOMA PATIENTS.

K. Sychevskaya 1 , S. Kravchenko 1,* , F. Babaeva 1 , A. Misyurina 1 , A. Korneeva 1 , A. Abdulpatahov 1 , R. Abdurashidova 1 , A. Keniaykina 1 , Z. Sarakaeva 1 , E. Fastova 1 , L. Gorenkova 1 , E. Nesterova 1 , Y. Mangasarova 1 , M. Bagova 1 , O. Margolin 1 , A. Magomedova 1

1 National Research Center for Hematology, Moscow, Russian Federation (Russia), Moscow, Russia

Background: Optimization of Non-Hodgkin’s lymphomas (NHL) treatment during the COVID-19 pandemic is urgently needed. Monoclonal anti-CD20 antibody (MA) treatment has been recently defined as an independent factor of unfavorable prognosis of COVID-19. The negative role of MA treatment during COVID-19 is related to combined B-cell and T-cell depletion after MA administration. This is especially critical for those lymphomas that require long-term MA maintenance therapy (MT). However, we believe not only the fact of MA administration but mostly prolonged MA therapy is associated with severe COVID-19 course. If true the safety and rationality of prolonged MA treatment and, in particular, MT in NHL patients during the ongoing COVID-19 pandemic should be revised.

Aims: To analyze association between COVID-19 course and the fact of MA treatment and duration and time points of initiation and completion of MA therapy as well in NHL patients.

Methods: From April 2020 to February 2022 a total of 83 NHL patients with COVID-19 were observed. Informed consent was obtained from all patients. Statistical analysis was performed in STATISTICA 12.0 software (TIBCO Software, Inc., USA).

Results: As was shown before combined immunodeficiency is formed after 6 months, on average, after MA therapy initiation. Additionally restoration of humoral and cellular immunity occurs, on average, 6 months after the end of MA therapy. Therefore, we supposed more than a 6-month duration of MA treatment and less than a 6-month interval from the last MA injection to COVID-19 are critical features for a severe COVID-19 course. The algorithm considering both of these characteristic was proposed (Figure A).

As our NHL cohort was heterogeneous, there were patients after chemotherapy without MA inclusion at all (a total of 13 cases, mostly T-cell lymphomas patients). This group initially was analyzed separately (Figure B). We identified that 2-year OS after COVID-19 manifestation is significantly decreased in “high-risk” MA patients (p=0.02, Log-rank test), exactly, 52% vs 76% in “low-risk” MA group. OS in “no MA” group was mostly similar to that in “low-risk” MA group, so these cohorts were merged (Figure C). Two-year OS in these combined group was 74%, that remained to significantly exceed OS in “high-risk” MA patients (p=0.03, Log-rank test).

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Summary/Conclusion: As we initially supposed, our results established both the duration of previous MA therapy and the time before the manifestation of COVID-19 are important for following COVID-19 infection course in NHL patients. Prognosis of patients after chemotherapy without MA administration is comparable with those patients who had MA treatment of less than 6 months duration or had shortly begun MA treatment before COVID-19. However, this fact needs to be refined as groups were not comparable in terms of lymphomas type. Additionally we were not able to evaluate different chemotherapy regimens influence on COVID-19 course, as cohort was extremely heterogeneous. Nevertheless we believe our results prove prolonged MA in NHL patients is not safe and excluding MA from MT is rational during the ongoing COVID-19 pandemic.

PB2090: TREATMENT OUTCOME OF ORBITAL MARGINAL ZONE B CELL LYMPHOMA -- TEN YEARS FOLLOW UP IN A SINGLE INSTITUTION

T.-D. Tan 1,* , L.-W. Chiou 1 , H.-C. Kau 2

1 Hematology and Medical Oncology; 2 Ophthalmology, Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan

Background: Marginal zone B cell lymphoma (MZBCL) is one of indolent B cell lymphoma including nodal, extra-nodal, and splenic types. The extra-nodal MZBCL includes gastric, bronchial, bowel, salivary gland, thyroid, and orbital MALT lymphoma. As the indolent in character, we know that durable survival is expected but relapse could be not unusual.

Aims: To analyze the treatment modality and outcome in real world experence.

Methods: Between 2009 and 2018, 10 patients of orbital MZBCL and we analyzed the treatment modality and outcome in our institution.

Results: Between 2009 and 2018, 10 patients of orbital MZBCL with median age 43 (range 33 to 63) and male/female was 6/4. Seven patients were in stage I (70%), one in stage II (10%), and 2 in stage IV (20%). Five patients underwent radiotherapy alone, two underwent chemo-immunotherapy (R-CVP) followed by radiation, and three of them underwent chemo-immunotherapy without radiation. Three of them got lymphoma relapsed including one had large B cell transformation and second complete remission after salvage chemo-immunotherapy. All ten patients are survived at the time of report.

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Summary/Conclusion: High overall survival (100%) and progression free survival (70%) in our cohort and two of the relapsed patients were stage IV disease and another patient had large localized tumor.

PB2091: INFLUENCE OF HISTOLOGICAL GRADE IN CLINICAL CHARACTERISTICS AND LONG-TERM OUTCOME OF FOLICULAR LYMPHOMA

I. Zamanillo 1,* , M. Poza 2 , R. Iñiguez 2 , S. Barrio 2 , P. Sarandeses 2 , A. Martin 2 , A. Rodriguez 2 , T. Baumann 2 , L. Rufian 2 , J. Martinez-Lopez 2 , A. Jimenez-Ubieto 2

1 Hematology; 2 Hospital Universitario 12 de Octubre, Madrid, Spain

Background: Despite extensive studies over the years, follicular lymphoma grade 3A (FL G3A) remains poorly characterized in terms of long-term outcome and optimal clinical approach. While FL grades 1 and 2 and 3B present differential traits and disease course (grades 1 and 2 behave as a slowly progressive disease with multiple relapses after different lines of treatment, and grade 2B resembles a diffuse large B-cell lymphoma), studies disagree weather G3A FL behaves similarly to low grade FL or if it presents a more aggressive clinical course.

Aims: To analyse the impact of histological G3A in clinical characteristics and evolution of patients with FL in comparison with grades 1 and 2 FL.

Methods: This is a retrospective analysis of a cohort of 241 patients with newly diagnosed FL between the years 2006 and 2020. Data regarding histological grade was unavailable in 10 patients. Patients with FL grade 3B (18) and patients with a single extranodal area affected (13) were excluded from the analysis. Finally, a total of 200 patients were included in the study. SPSS® was used for all statistical analysis. Chi square test was used for comparisons between groups and Kaplan-Meier method and log rank test for the survival analysis.

Results: 173 patients (86.5%) were diagnosed with low-grade FL (G1-2 FL), and 27 (13.5%) with G3A FL. Median age at diagnosis was 66 years and it did not differ in both groups. 52% of the patients were female.

25.7% of the patients presented a high-risk FLIPI2 in the G1-2 FL group vs 45% in the G3A FL group, although this difference was not statistically significant (p=0.1). Lymphoma characteristics at diagnosis were in the main similar in both groups: Ann Arbor stage, bone marrow infiltration, extranodal involvement and presence of bulky mass did not differ in both groups. However, G3A FL patients more frequently presented elevated LDH at diagnosis (27 vs 52%; p=0.027) and B symptoms (25 vs 61%; p=0.001).

Regarding the evolution, 157 patients required treatment at any point during the evolution, 80% of patients with G1-2 FL and 89% of patients with G3A FL (p=0.26). First line therapy was most commonly immune-chemotherapy, the most used regimens were R-CHOP (59.2%) and bendamustine-rituximab (24.8%). 67.5% of patients achieved complete response (CR), and 87% partial response or better to first line therapy; the proportion was similar in both groups.

Relapses occurred in 40% of the patients who received a first-line treatment. There were no differences in percentage of relapses (35.5% for G1-2 FL vs 42.3% por G3A FL) or POD depending on histological grade.

With a median follow-up of 58 months, there were no differences in Progression Free Survival (Estimated 3-year PFS G3A 62% vs G1-2 68%; p=0.75) or Overall Survival (Estimated 5-year OS G3A 95% vs G1-2: 91%; p=0.38) (Figure 1). G3A FL showed in PFS curve no relapses after 6 years.

21 patients transformed into a high-grade lymphoma and 14 developed a second neoplasm during follow-up. Finally, 35 patients died during this time, 13 due to lymphoma progression and 27 due to non-disease related causes, with no difference between groups.

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Summary/Conclusion: G3A follicular lymphoma presents mostly similar characteristics and outcome as low-grade follicular lymphoma. However, it may present some more aggressive traits, such as more frequent B symptoms and elevated LDH. Our results indicate similar long-term outcomes in terms of progression-free survival and overall survival in grades 1-2 and 3A. No relapses were observed in G3AFL group after 6 years. These findings should be confirmed in prospective studies.

PB2092: PRIMARY BONE DIFFUSE LARGE B CELL LYMPHOMA: A SINGLE CENTER EXPERIENCE

G. Abichou 1,* , M. Charfi 1 , F. Kallel 1 , Y. fakhfekh 1 , L. Ghorbel 2 , I. Saguem 3 , W. Feki 4 , H. Ben salah 2 , T. Boudawara 3 , Z. Mnif 4 , J. Daoud 2 , M. Elloumi 1

1 hematology department, university hospital hedi chaker; 2 radiotherapy department; 3 pathology department, university hospital habib bourguiba; 4 radiology department, university hospital hedi chaker, sfax, Tunisia

Background: Primary bone lymphoma (PBL) is a rare disease and represents 5% of extranodal lymphomas and less than 2% of Non-Hodgkin Lymphomas. Primary bone diffuse large B-celllymphoma (PB-DLBCL) is the most common pathological type, comprising about 80% of PBL. This entity has a good prognosis and its treatment options are very limited, especially for elderly patients.

Aims: The aim of this study is to describe characteristics of patients with PB-DLBCL and to assess the outcome of a cohort of patients in south of Tunisia.

Methods: Our retrospective study involved all patients diagnosed with PB-DLBCL at the hematology department of Sfax between January 2008 and December 2019.We studied the diagnostic characteristics of these patients: age, sex andclinical features. The diagnosis was confirmed by histopathological examination according to the 2008 and the 2016 World Health Organization (WHO) classification of hematologic malignancies. Staging was estimated according to Ann Arbor system. Depending on the location of the lesion and skeletal radiographs, computed tomography (CT) scans and magnetic resonance imaging (MRI) were analyzed.We used the age adjusted International Prognostic Index (aaIPI) to evaluate the prognosis. Patients were treated with CHOP-like regimens with Rituximab when available. Autologous stem cell transplantation (ASCT) was indicated for patients aged 60 and younger with aaIPI>1. Radiotherapy was performed as a planned consolidation therapy after immunochemotherapy Kaplan Meier method was used to estimate overall survival and event-free survival

Results: Twenty five patients with PB-DLBCL were identified during the study period. The mean age at diagnosis was 53,7 years (range 17-78). Sex ratio M/F was 1,5. The mean consultation delay was 3,8 months (1-12). The revealing symptom was bone pain in the majority of the cases (40%). B symptoms occurred in 19 cases (76%). Performans status was <=1 in 14 cases (56%). The most common tumor sites was the vertebrae (44%) followed by femur (12%), pelvis (12%) and rib cage (12%).

Sixteen patients had elevated LDH (64%) and 2 patients had hypercalcemia at diagnosis. According to the Ann-Arbor classification, the stage of the disease was I in 5 patients (20%) and IV in 20 patients (80%). The aaIPI was > 1 in the majority of thes cases (64%).

Three patients died before treatment (12%) and 22 patients received chemotherapy. Consolidative radiation therapy was administered in 36% of cases and ASCT was performed only in 3 patients.

Complete remission was achieved in 8 patients (36%) and 3 patients were refractory to first-line chemotherapy (12%). Relapses were observed in 2 patients (8%) 2 and 12 months after completion of treatment. These 2 patients received platinum based salvage therapy and died from disease progression. With a median follow-up of 80 months (17-161), 5-year Overall survival and event-free survival were respectively 67% and 65%.

Summary/Conclusion: PB-DLBCL is a rare disease. The non-specific initial presentation often leads to a late diagnosis. In our study, epidemiological and clinical characteristics were similar to the literature except for the stage of the disease, which was localized in 40-50% of the cases in the literature; this may be explained by the important consultation delay. Staging of the disease and response evaluation were not optimal due to non-availability of PET-CT and difficulty to realize MRI on time. Our CR rates were inferior to the literature (80-85%). EFS was inferior to those reported in other studies (70-100%) too. Radiotherapy and ASCT may improve our results.

PB2093: DIFFUSE LARGE B CELL LYMPHOMA IN THE ELDERLY

G. Abichou 1,* , M. Charfi 1 , O. Kassar 1 , F. Kallel 1 , I. Saguem 2 , W. Feki 3 , I. Ben amor 1 , W. Ben kridis 4 , I. Frikha 1 , L. Khelifa 1 , Y. fakhfekh 1 , N. Toumi 4 , M. Medhaffar 1 , S. Hdiji 1 , T. Boudawara 2 , Z. Mnif 3 , M. Elloumi 1

1 hematology department, university hospital hedi chaker; 2 pathology department, university hospital habib bourguiba; 3 radiology department, university hospital hedi chaker; 4 oncology department, university hospital habib bourguiba, sfax, Tunisia

Background: Diffuse large B cell lymphoma (DLBCL) primarily affects older patients. In fact, quarter of all DLBCL are over 75 years. The majority of these patients is unfit for aggressive treatment protocols and the lack of randomized trials in this category makes it difficult to choose the best regimen.

Aims: The aim of this study is to evaluate the outcome of DLBCL in this category of patients in our center.

Methods: We conducted a single-center retrospective study. Newly diagnosed DLBCL patients over 60 years in 2008-2019 treated in the hematology department of Sfax were included. The diagnosis was confirmed according to the WHO Classification of Hematological malignancies. We defined the disease stage according to Ann Arbor staging system. Patients were treated according to the last 2 versions of the national protocol (LNH2008, LNH 2013): patients aged ≤70 years and ≤75 years since 2013 were treated with 6-8 courses of CHOP+/-R and patients aged >70 years (>75 since 2013) were treated with 6 courses of miniCHOP+/-R. We evaluated the response with the International Working Group (IWG) Response Criteria for NHL 1999. We estimated overall survival (OS) and event-free survival (EFS) with Kaplan Meier.

Results: A total of 66 elderly patients with DLBCL aged >60 years were identified. Mean age was 71,2 years(61-87). 59% were male. Mean time from diagnosis was 7,7 months. B symptoms were present in 71% of the cases. An altered performans status (≥2) was noted in 50% of the cases. 42 patients had a raised LDH level. The stage of the disease was advanced in 59%. aaIPI was high (≥2) in 27 cases (41%). Nine patients died before treatment (14%). 37 Patients received R+/-CHOP regimen and 20 patients received R+/-miniCHOP regimen. An overall response was noted in 34 cases (60%) with a complete remission in 70% and a partial remission in 30%. Nineteen patients did not achieve the treatment (33%) because of treatment related mortality (TRM) in 10 cases (15%). A hematological toxicity with a grade III/IV neutropenia was noted in 16 patients. Four patients were refractory (7%). Nine patients relapsed (26%). Mean delay from end of treatment was 29 months. Five patients received a platinum-based second-line chemotherapy and 4 patients were unfit for an intensive chemotherapy. Median follow-up of the study was 94 months. 3-year OS and EFS were respectively 60% and 55%.

Summary/Conclusion: DLBCL is an aggressive lymphoma of the elderly. The age limit should not be an obstacle for prescribing a chemotherapy. Evaluating the elderly must be using objective tools to distinguish the category of patients unfit for an intensive treatment. In our study, CR rates were lower than the values reported in the literature (56-70%). EFS in our study was 55%, it was comparable to the literature (45-71%). TRM was slightly higher in our study (15% vs <10%). The availability of liposomal Doxorubicin, oral CHOP regimen and Dexrazoxane could change the outcome of this group of patients.

PB2094: PRIMARY MEDIASTINAL B-CELL LYMPHOMA: EXPERIENCE OF THE HEMATOLOGY DEPARTMENT OF SFAX

M. Charfi 1 , G. Abichou 1,* , I. Frikha 1 , L. Khelifa 1 , L. Ghorbel 2 , I. Saguem 3 , W. Feki 4 , H. Ben salah 2 , T. Boudawara 3 , Z. Mnif 4 , J. Daoud 2 , M. Elloumi 1

1 hematology department, university hospital hedi chaker; 2 radiotherapy department; 3 pathology department, university hospital habib bourguiba; 4 radiology department, university hospital hedi chaker, sfax, Tunisia

Background: Primary mediastinal B-cell lymphoma (PMBCL) is a rare lymphoma subtype that usually occurs in adolescents and young adults. Its clinical features resemble classical Hodgkin lymphoma. The prognosis of most patients with PMBCL is very good. Thus, there is no standard of care for this group of lymphoma.

Aims: The aim of this study is to describe the epidemiological, clinical and therapeutics characteristics of this disease in our center.

Methods: This retrospective study involved untreated patients with PMBCL diagnosed between January 2008 and December 2019 in the Hematology department of Sfax in Tunisia. Diagnosis was made on the basis of histopathological examination with mandatory immunohistochemical staining. Ann Arbor system was used for staging. Age adjusted international Prognostic Index (aa-IPI) score was used to evaluate prognosis. Patients were treated with immunotherapy combined with chemotherapy (CHOP+/-R, ACVBP+/-R). For patients aged 60 and younger with aaIPI>1, a consolidative therapy by autologous stem cell transplantation (ASCT) was indicated. We evaluated the response with the International Working Group (IWG) Response Criteria for NHL 1999. Kaplan Meier method was used to estimate overall survival (OS) and event-free survival (EFS).

Results: Twenty-nine patients with PMBCL were identified during the study period. The median age was 34 years (range:17-64) and Sex-ratio was 0,36. The most common revealing symptoms were dyspnea (45%) followed by cough (21%). B symptoms were reported in 62% of cases. Approximately half of the patients (48%) presented with upper vena cava syndrome and bulky mediastinal disease was noted in 90 % of cases (n=26 patients). 30% of our patients had a performance status>1, 76 % had elevated Lactates Dehydrogenases and 48% had advanced stage. aaIPI was high (>1) in 45% of the cases. All the patients were treated with an immuno-chemotherapy (R-CHOP=23, R-ACVBP=6). The overall response rate was 86% and complete remission was noted in only 38% of cases. ASCT was indicated for 13 patients (45%) but only four patients (31%) underwent the procedure. Eight patients had a radiation therapy to mediastinal residual mass after chemotherapy. 14% of patients had refractory disease and 11% relapsed. OS and EFS at 5 years were 76% and 70%, respectively.

Summary/Conclusion: Our epidemiological and clinical data are globally similar to reported studies except for systemic symptoms affecting only 20 % of cases and advanced stages, which are less frequent (20-30%) in the literature. Complete remission rate in our study is insufficient compared to data from the literature (80 %).The response was unfortunately assessed by computed tomography because of non-availability of PET-CT until 2020. Five years OS and EFS were lower than the values reported in other series in literature (OS: 80-90 %; EFS: 78-89%). These results may be improved by the assessment of the response by PET-CT, using more intensified regimens and the availability of ASCT especially in high-risk patients and in patients who achieved only PR.

PB2095: RELAPSED AND REFRACTORY DIFFUSE LARGE B CELL LYMPHOMA: EXPERIENCE OF A LOW-INCOME CENTER IN TIME OF NEW DRUGS

M. Charfi 1,* , G. Abichou 1 , F. Kallel 1 , I. Ben amor 1 , L. Khelifa 1 , I. Frikha 1 , Y. Fakhfekh 1 , O. Kassar 1 , M. Medhaffar 1 , S. Hdiji 1 , M. Elloumi 1

1 hematology department, university hospital hedi chaker sfax, sfax, Tunisia

Background: Refractory/relapsed (RR) disease is the main cause of morbidity and mortality in Diffuse Large B cell Lymphoma (DLBCL). Unfortunately, one-third of patients with diffuse large B-cell lymphoma continue on to relapsed or refractory disease despite the improvement of its management.

Aims: The aim of this study is to evaluate our management of RR disease in our low-income center in the era of immunotherapy and monoclonal antibodies.

Methods: This is a single-center retrospective study. We included all the cases of DLBCL diagnosed in University Hospital of Sfax between 2008-2019 that were refractory and/or relapsed. Clinical and biological characteristics at diagnosis were reported. A second biopsy was indicated in all cases when feasible. Refractory disease was defined as the absence of criteria of remission (complete or partial). Relapse was defined as the appearance of new lesions or the increase of at least 50% from nadir in the SPD from any previously involved site after a complete remission. Early relapse was defined as a relapse happening 12 months after end of treatment. The patients were treated with a second-line platinum based chemotherapy if fit. Autologous stem cell transplantation (ASCT) was indicated for patients who respond to second line treatment (partial or complete remission) and are fit for ASCT. Kaplan Meier system was used to estimate survival.

Results: Among the 150 DLBCL diagnosed and treated during the study, 46 (31%) were relapsed/refractory: 13 (9%) were refractory to first line chemotherapy and 33 experienced relapse (22%). At the diagnosis, RR-patients are aged of 60 and less in 69% of the cases. A poor performans status≥2 was found in 30% of the cases. We found raised LDH levels in 53%. The disease was localized in 31%. aaIPI was at a minimum of 1 in 87% of the cases (1: 42%, 2: 24%, 3: 21%). We identified 3 predictive factors of refractory disease: Performans status (p=0,007), aaIPI (p=0,036) and raised LDH (p=0,019). For patients who relapsed, an early relapse was noted in 51,5% of the cases. Five patients relapsed after ASCT. Mean time from diagnosis was 20 months (2-72). Twenty-six patients were eligible for chemotherapy: 20 were treated with DHAP and 6 with ICE regimen. Five patients were in complete remission after salvage chemotherapy and 3 were in partial remission. Only 1 patient underwent ASCT after relapse and 3 patients after refractory disease. 1-year and 5-years Post-RR survival were respectively 24% and 14%.

Summary/Conclusion: Even in the Rituximab era, patients continue to experience relapse and refractory disease. Our results are comparable to the literature where one third of DLBCL are either refractory (10-15%) or relapse (20-30%) and most relapses happen within the first 2 years after treatment. Predictive factors of RR identified in our study are the same than the factors reported in the literature in addition to age, B symptoms, advanced stage of the disease and bulk. Post-RR survival was poor in our study as well as in reports from the literature. The poor survival post-RR could be improved with new drugs regrettably not available in our country.

PB2096: A PHASE 1B/2 STUDY OF GB5121, A NOVEL, HIGHLY SELECTIVE, POTENT, AND CNS-PENETRANT BTK INHIBITOR FOR RELAPSED/REFRACTORY PRIMARY/SECONDARY CNS LYMPHOMA AND PRIMARY VITREORETINAL LYMPHOMA

C. Soussain 1,* , C. Grommes 2 , R. Ward 3 , C. Peterson 3 , M. Cravets 3 , A. Mathias 3 , J. Sosa 3 , B. Kirby 3 , Z. Ding 3 , I. Yusuf 3 , M. Rose 3 , M. Steinberg 3 , H. Tun 4

1 Institut Curie, Saint-Cloud, France; 2 Memorial Sloan Kettering Cancer Center, New York, NY; 3 Gossamer Bio, Inc., San Diego, CA; 4 Mayo Clinic, Jacksonville, FL, United States of America

Background: Bruton’s tyrosine kinase (BTK) plays an important role in B cell receptor and Toll-like receptor signaling pathways, which are constitutively active in primary CNS lymphoma, and represents an excellent therapeutic target. Ibrutinib, a first-generation BTK inhibitor (BTKi), has been evaluated in two phase 1/2 trials for relapsed/refractory primary/secondary CNS lymphoma (R/R PCNSL, SCNSL) and primary vitreoretinal lymphoma (PVRL) showing limited survival outcomes. Improved BTKis may achieve better therapeutic outcomes. GB5121 is a novel, orally available, covalent BTKi, which has shown superior specificity, CNS penetration, and CNS target occupancy in preclinical testing compared to other BTKis including ibrutinib. It is well-suited for further evaluation in CNS lymphoma. We will therefore conduct a phase 1b/2 open-label, international study of GB5121 in adult subjects with R/R PCNSL, SCNSL, or PVRL ( NCT05242146 ).

Aims: The objective of the phase 1b dose-escalation portion of the study will be to evaluate safety, tolerability, pharmacokinetic/pharmacodynamic profile, dose-limiting toxicity (DLT), maximum tolerated dose, and preliminary therapeutic activity to determine the optimal biological dose that will inform the preliminary recommended phase 2 dose (pRP2D). The phase 1b expansion study will further explore the therapeutic activity and characterize the safety and tolerability of GB5121 at the pRP2D. The efficacy, safety, and tolerability of GB5121 at the pRP2D established in the phase 1b dose escalation study will be investigated in phase 2.

Methods: The study will be conducted in three parts: phase 1b dose-escalation, phase 1b expansion, and phase 2. Eligibility criteria for phase 1b dose-escalation and expansion (N ≈ 30 for each part) include ≥ 18 years of age, ECOG ≤ 2, R/R PCNSL, R/R SCNSL with CNS-only relapse, or R/R PVRL. Patients with newly diagnosed PCNSL who cannot tolerate standard high-dose methotrexate-based therapies are also eligible. Subjects who had prior allogeneic stem cell transplant or previously received a BTKi will be excluded. A Bayesian optimal interval design will be employed to perform dose escalation with the primary objective of determining the preliminary recommended phase 2 dose. In the absence of DLT, subsequent dose levels will increase sequentially according to a modified Fibonacci approach. The phase 2 portion of the study will initiate following the determination of the RP2D based on phase 1 results and is a single-arm, open-label study of GB5121 in subjects with R/R PCNSL. Adverse events will be graded per Common Terminology Criteria for Adverse Events (CTCAE v5.0) to establish a toxicity profile. Therapeutic response (overall response rate, complete response, partial response) will be determined according to the criteria of the International Primary CNS Lymphoma Collaborative Group. Progression-free survival and overall survival will be evaluated. This clinical trial is anticipated to start enrollment in May 2022.

Results: N/A (trial in progress)

Summary/Conclusion: N/A (trial in progress)

PB2097: ZANUBRUTINIB PLUS RITUXIMAB FOR THE TREATMENT OF ELDERLY UNFIT PATIENTS WITH PREVIOUSLY UNTREATED DIFFUSE LARGE-B CELL LYMPHOMA

J.-F. Bai 1 , J. T. Li 1 , R. Feng 1 , L. Qian 1 , J. Yin 1 , H. Liu 1,*

1 Beijing Hospital, National Center of Gerontology, P.R. China., Beijing, China

Background: Diffuse Large B-cell lymphoma (DLBCL) is common in the elderly patients, Some elderly patients can not tolerate the standard dose of R-CHOP due to age, poor physical condition, and severe complications. The overall response rate of elderly unfit DLBCL patients who received a reduced dose R-CHOP regimen was only 60%. It is extremely important to explore new therapeutic options. Zanubrutinib is a Bruton tyrosine kinase (BTK) inhibitor which was still in an exploratory phase in patients with DLBCL.The efficacy of Zanubrutinib plus Rituximab in elderly unfit Patients with DLBCL is still unknown.

Aims: This study was conducted to investigate the efficacy and safety of Zanubrutinib plus Rituximab in elderly unfit Patients With Previously Untreated Diffuse large-B cell Lymphoma in China.

Methods: In the present study, a prospective, single-arm,single-center study was conducted (CHiCTR-2000039342). patients who fulfil the inclusion criteria were enrolled. Inclusion criteria: patients aged ≥65 years, pathologically confirmed DLBCL,and assessed unfit by Comprehensive Geriatric Assessment(IACA). Unfit patients defined as follows: i.e. IADL score ≤7, or age >75 years, or CCI score ≥3, or serum albumin <3.4 g/ml. Patients enrolled were planed to treat with Zanubrutinib combined with rituximab (Zanubrutinib 160 mg orally twice daily and rituximab 375 mg/m 2 day1 iv, every 28 days, ZR regimen), for a total of eight courses. After induction therapy, maintenance treatment with Zanubrutinib 160mg orally twice daily was given until disease progression or unacceptable toxicity. Efficacy was evaluated by PET/CT or enhanced Computed Tomography scan every two months, treatment-related adverse effects were evaluated in every one month. This study was approved by the Ethics Committee of Beijing Hospital, National Center of Gerontology, Beijing, China.

Results: From July 2020 to December 2021, a total of eight elderly unfit DLBCL patients were enrolled. one patient was 72 years old with IADL score of 6, CCI score of 4. the other seven patients were aged over 80 years. the most common comorbidities were cardiovascular disease, hypertension, diabetes.The female to male ratio was 6:2. four patients were classified as GCB pathological subtype, six patients were diagnosed with stage III-IV. At a median follow up of 8 months (2-19months). two patients achieved complete response(CR), and remained in CR at months 8. four patients achieved partial response(PR), with one patient disease progression(PFS 8 months), one patient achieved stable disease (SD) after 2 cycles and have disease progression after 4cycles, with a progression-free survival (PFS) of 4 months. one patient was refractory to treatment and withdrew after 2 cycles. One patients died due to disease progression, with overall survival(OS) of 11 months, ZR regimen was well-tolerated, with grade 2 urinary tract infections in 4/7 patients, grade 2 respiratory infections in 2/7, grade 1 Petechial hemorrhages in 3/7, grade 2 skin rash in 2/7, and grade 2 deep vein thrombosis in 1/7. No grade 4 or higher adverse events were observed. Seven patients had a dose reduction due to toxicity. The regular doses of Zanubrutinib can be tolerated was 160 mg qdA1-80mg qdP1. Two patients had discontinued Zanubrutinib due to adverse events for one to three weeks.

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Summary/Conclusion: Zanubrutinib in combination with Rituximab may be a well-tolerated and effective regimen in elderly unfit DLBCL patients. Dose reduction may be required for some patients to improve tolerability. A large sample size clinical trial is needed to further validate this conclusion in the future.

PB2098: ‘REAL-WORLD’ TREATMENT PATTERNS AND PATHWAYS OF SECOND LINE DIFFUSE LARGE B-CELL LYMPHOMA PATIENTS IN UK, CANADA, FRANCE, GERMANY, ITALY AND SPAIN USING A PROSPECTIVE SURVEY OF PHYSICIANS

A. Bailey 1 , N. Milloy 1 , I. Sanderson 1 , J. Butcher 1 , C. Spooner 2 , D. Kilic 2 , S. Vadgama 2,*

1 Oncology, Adelphi Real World, Bollington; 2 Kite, a Gilead company, Uxbridge, United Kingdom

Background: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent form of Non-Hodgkin Lymphoma, representing 25% to 35% of cases annually 1 . Despite this, there is a lack of research into treatment patterns of real-world patients, especially in the 2 nd line (2L) setting.

Aims: To determine the ‘real-world’ treatment patterns and treatment pathway of DLBCL patients treated at the second line, with a focus on patients who are ‘fast progressors’ as defined below.

Methods: In this study ‘real-world’ data were drawn from the Adelphi DLBCL Disease Specific Programme™ (DSP), a point-in-time survey of clinicians and their next 6 presenting patients with DLBCL. The survey was conducted in France, Germany, Italy, Spain, the United Kingdom (UK) and Canada between Jan-May 2021.

Patients who were refractory/relapsed <12 months from 1 st line (1L) therapy, eligible for a stem cell transplant (SCT) at initial relapse/refractory, had an ECOG performance score of 0-1 and had no history of hepatitis B or C or CNS events were studied.

Results: 47 patients met the criteria. Mean age of the patients was 58.0 (SD: 11.7) years, 68% were male and 26% were retired. At 1L, 94% of patients received R-CHOP with a median of 171.2 days (IQR: 61.0-247.5) between the end of their 1L and the start of their 2L treatments.

All patients received salvage chemotherapy (SC) in the form of R-ESHAP, R-DHAP or R-GDP in most cases (Figure 1). 49% of the patients had a complete response (CR) to SC, 21% had partial response (PR), 30% stable/no response. In total 26 (55%) patients received high-dose therapy-SCT (HDT-SCT). Figure 1 shows the response to 2L treatments across patients and indicates why some patients did not progress to SCT despite being previously eligible. Patients receiving SCT had a median time from 2 nd line SCT to relapse/refractory of 189.0 (IQR: 94.0-333.0) days.

54% of patients received chimeric receptor antigen T-cell (CAR-T) therapies at 3L (Figure 1), with the majority having previously received SCT. CAR-T use was higher in UK (67%), Canada (63%) and Germany (60%) than in other regions.

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Summary/Conclusion: Despite all patients with DLBCL observed being SCT eligible at their first relapse, only half of these patients went on to receive an SCT. Unacceptable response to SC, progressive disease and patient choice were common reasons for not going on to receive SCT. Despite seemingly favourable responses to SCT, these patients progressed quickly and were likely to receive a CAR-T treatment in 3L, indicating an unmet need and highlighting potentially high resource use and substantial impacts to quality of life.

PB2099: A FIRST-IN-HUMAN PHASE 1 TRIAL OF NX-1607, A FIRST-IN-CLASS ORAL CBL-B INHIBITOR, IN PATIENTS WITH ADVANCED MALIGNANCIES INCLUDING RICHTER TRANSFORMATION DLBCL

A. Sharp 1,2,* , A. Williams 3 , S. Blagden 4 , R. Plummer 5 , D. Hochhauser 6 , M. G. Krebs 7,8 , S. Pacey 9 , J. Evans 10 , S. Whelan 11 , S. Nandakumar 11 , S. Rogers 11 , K. L. Jameson 11 , F. G. Basile 11 , J. de Bono 12 , H.-T. Arkenau 3

1 The Institute of Cancer Research, London, United Kingdom; 2 Royal Marsden Hospital, Wallington, United States of America; 3 Sarah Cannon Research Institute (SCRI), London; 4 University of Oxford, Oxford; 5 Northern Centre for Care, Newcastle upon Tyne; 6 University College London (UCL), London; 7 The Christie NHS Foundation Trust; 8 University of Manchester, Manchester; 9 Cambridge Cancer Centre (CCC), Cambridge; 10 University of Glasgow, Glasgow, United Kingdom; 11 Nurix Therapeutics, Inc., San Francisco, United States of America; 12 The Institute of Cancer Research, London, London, United Kingdom

Background: Standard of care treatment for diffuse large B-cell lymphoma type Richter transformation (DLBCL-RT) is combination chemo-immunotherapy but is limited by poor response rates and lack of durability. Recent studies have shown some clinical benefit with PD-1 blockade treatment in DLBCL-RT including deep responses, highlighting that T-cell targeted immunotherapy may improve outcomes. Casitas B-lineage lymphoma proto-oncogene B (CBL-B) is an E3 ubiquitin ligase expressed in multiple immune cell lineages, which in contrast to cell surface immune checkpoints, acts as a regulator of both T and NK cell activation. Inhibition of CBL-B enhances T-cell response to suboptimal priming and restores response in exhausted T cells. In addition, NK cells deficient in CBL-B have increased cytokine production upon activation. Thus, CBL-B is a promising immune-oncology target and may overcome challenges seen with other T-cell directed therapies.

NX-1607 is an oral small molecule inhibitor of CBL-B that has demonstrated anti-lymphoma activity in murine models as both a single agent, and in combination with anti-CD19 monoclonal antibodies. In addition to increased anti-tumor T-cell activity, NX-1607 also potentiates the anti-tumor cytotoxic and antibody-dependent cellular cytotoxicity (ADCC) activity of NK cells. Therefore, NX-1607 may be effective as a single agent or it may significantly enhance efficacy of other anti-tumor agents in a subset of hematologic malignancies.

Aims: The main objective is to establish the safety and tolerability, characterize the pharmacokinetics (PK) and pharmacodynamics (PD), and determine the early clinical activity of NX-1607 in advanced solid malignancies including DLBCL-RT.

Methods: NX-1607-101 is a first-in-human, multicenter, open-label, Phase 1 dose escalation and expansion trial evaluating NX-1607 in a variety of indications including DLBCL-RT. It will be conducted at approximately 20 sites in the UK and US.

NX-1607 will be given orally once daily at doses ranging from 5 to 100 mg in up to 6 dose levels. After determining the expansion dose, up to 8 cohorts in Phase 1b will be investigated in patients with subsets of advanced cancers including DLBCL-RT ( n = 16). Key eligibility criteria include patients with metastatic or unresectable disease that have progressed after prior therapy and for whom standard therapy with proven clinical benefit does not exist, is no longer effective, or is not appropriate. Prior treatment with CAR-T cells with washout is allowed, but a history of active autoimmune disease is not.

The primary endpoints are safety and maximum tolerated dose (Phase 1a) and objective response rate (Phase 1b) of NX-1607. Secondary endpoints (Phase 1a and Phase 1b, unless otherwise indicated) include PK/PD, objective response rate (Phase 1a), duration of response, disease control rate, progression-free survival, overall survival, and safety (Phase 1b).

Results: Dose escalation is ongoing.

Summary/Conclusion: Accrual is ongoing. Clinical trial information: NCT05107674 .

PB2100: MANAGEMENT OF RELAPSED / REFRACTORY (R/R) B-CELL NON HODGKIN LYMPHOMA (B-NHL) WITH ANTI-CD20XCD3 BISPECIFIC ANTIBODY GLOFITAMAB. A SINGLE CENTER EXPERIENCE.

C. Giatra 1,* , E. Souravla 1 , I. Darmani 1 , A. Roumelioti 1 , K. Kaouranis 1 , P. Kosmas 1 , V. Bampali 1 , D. Gardeli 1 , M. Papageorgiou 1 , Z. Mellios 1 , F. Karaolidou 1 , I. Tsonis 1 , M. Karatza 1 , K. George 2 , L. Marinos 3 , D. Karakasis 1 , T. Karmiris 1 , M. Bakiri 1 , M. Bouzani 1

1 Hematology and Lymphoma, BMTU; 2 Pathology Department; 3 Hematopathology Department, Evaggelismos General Hospital, Athens, Greece

Background: Prognosis of r/r B-NHL is detrimental. Potentially curative therapeutic approaches, such as autologous stem cell transplantation and innovative CAR-T cell therapy, require maximum disease control to achieve optimal results. Glofitamab is a new bispecific antibody, with a unique 2:1 molecular configuration resulting in superior potency compared with other CD20xCD3 bispecific antibodies with a 1:1 format.

Aims: Based on these encouraging results, we included 5 heavily pretreated patients in the early access program of Glofitamab, available in our country.

Methods: We collected the data of 5 consecutive patients with r/r B-NHL, who were treated with Glofitamab in our department during the last 15 months.

Results: Three men and 2 women, median age of 57 years (38-62), were resistant to 4 (n = 3) and 5 (n = 2) previous lines of treatment. The underlying lymphoma was Richter’s transformation of CLL after allogeneic transplantation (alloHSCT), transformed follicular lymphoma (tFL), primary mediastinal B-cell lymphoma (PMBCL), r/r diffuse large B-cell lymphoma (DLBCL) after CAR-T therapy and gray zone lymphoma (GZL) transformed to DLBCL. The median number of Glofitamab cycles administered was 3 (2-7). All 5 patients responded early to treatment, which became apparent immediately after the first dose of 2.5 mg.

The patient with Richter’s syndrome achieved metabolic remission after the 4th cycle and underwent second alloHSCT after the 7th cycle. Unfortunately, he passed away 8 months after alloHSCT due to disseminated atypical mycobacterial infection, remaining however disease free. The patient with tFL also achieved metabolic remission, but the drug was discontinued after the 7th cycle due to COVID-19 infection. He died two months after Glofitamab interruption due to disease progression and CMV encephalitis. The patient with PMBCL, responded partially after Glofitamab and had mediastinal radiotherapy as bridging therapy to CAR-T therapy. As the latter was delayed due to CMV reactivation and CMV enteritis, our patient deceased due to progressive disease. The patient with DLBCL after CAR-T therapy had initial clinical response after two Glofitamab cycles. Due to severe COVID-19, we decided to hold Glofitamab. COVID-19 and disease progression led to his death, a few weeks after COVID-19 diagnosis. Finally, the patient with transformed GZL had Glofitamab administered as bridging therapy prior to CAR-T treatment. After 3 cycles, while she was prepared to proceed to CAR-T therapy, she was diagnosed with invasive aspergillosis. She is currently been treated with antifungal agents, whereas disease is still active. Cytokine release syndrome (CRS) occurred in 3 out of 5 patients. In all cases it was grade 1-2 and manifested at the first administration of the drug, after 4, 32 and 10 hours respectively, from infusion initiation. CRS was managed with antipyretics and steroids, whereas none patient required Intensive Care Unit support. Only one patient required tocilizumab. No Immune effector cell-Associated Neurotoxicity Syndrome (ICANS) was observed.

Summary/Conclusion: Glofitamab is effective in treating patients with r/r aggressive B-cell NHL. Efficacy makes it an appropriate bridging tool to autologous, alloHSCT or CAR-T therapy. Nevertheless, relapse remains a challenge for r/r disease. Adverse events, such as CRS, were generally manageable. Given the fact that it was administered to heavily pretreated patients, caution to opportunistic pathogens should be paid. Indeed, toxicity profile may be proven to be more favorable if the agent is being administered earlier in therapeutic algorithms.

PB2101: TREATMENT OF AGGRESSIVE B-CELL LYMPHOMA WITH DA-EPOCH-R, A SINGLE CENTRE EXPERIENCE

E.-K. Dimitraki 1,* , C. Giatra 1 , M. Delatola 1 , A. Roumelioti 1 , L. Ligdi 1 , P. Kosmas 1 , V. Drakopoulou 1 , E. Souravla 1 , I. Darmani 1 , G. Kanelis 2 , L. Marinos 2 , K. Sakellariou 1 , D. Karakasis 1 , M. Bakiri 1 , M. Bouzani 1

1 Hematology and Lymphoma, BMTU; 2 Hematopathology Department, Evaggelismos General Hospital, Athens, Greece

Background: Dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin and rituximab (DA-EPOCH-R) is a 96-hour continuously infused regimen, aiming to improve first-line treatment of aggressive B-cell lymphoma. Prolonged exposure to chemotherapeutic drugs and dose intensification target to reduce tumor resistance, however toxicity remains a barrier for the broad use of this regimen.

Aims: To reveal the experience of our Centre on the treatment of aggressive B-cell lymphoma with DA-EPOCH-R

Methods: We retrospectively collected data from 54 consecutive patients treated with DA-EPOCH-R in our Department.

Results: Thirty one women and 23 men with median age of 37 years (range 18-76) were diagnosed between 2013 and 2021, with a High Grade B-cell Lymphoma, HGBCL (24 cases) and Primary Mediastinal B cell Lymphoma, PMBCL (30 cases). The Ann Arbor stage was I (16 cases), II (14 cases), III (5 cases) and IV (19 cases). The majority (85%) of patients had good (0-1) ECOG performance status. All but 2 patients were HIV positive. All patients were planed to be treated with 6 cycles of DA-EPOCH-R. Ten patients changed treatment plan due to disease progression (5 patients) and toxicity (5 patients). Among patients with HGBCL, 19 achieved a complete response (CR), 3 manifested resistant disease (RD) and for 3 patients response assessment was not possible due to disease related early death. Among patients with PMBCL, 24 showed CR, 1 patient partial response and 4 patients a RD. With a median follow up of 23 months (range 1- 86), the median progression free survival (PFS) and overall survival (OS) of all patients were not reached. Comparing survival between HGBCL and PMBCL, the latter had significantly better PFS and OS.

Among the 42 patients who completed 6 cycles of treatment, only 3 reached the level 5 of dose escalation, 12 the level 4, 13 the level 3, 7 the level 2 and 4 remained at the level 1. Seven cases underwent 1 step of dose increase, 14 patients 2 steps 13 patients 3 levels and 3 patients 4 levels. The administration of high dose methotrexate in 11 patients, hampered the dose escalation. In 7 subjects, level deescalation was required (1 level for 6 patients and 2 levels for 1patient).

Toxicity was manifested in 51 out of 54 patients. Haematological toxicity with neutropenia was the most common side effect: grade 4 in 88%, grade 3 in 7%, grade 1 in 5%. Severe thrombocytopenia was manifested in 7 cases. All patients received erythropoietin to treat anaemia. Thirty five subjects (65%) suffered from febrile neutropenia. Fever was related to bacterial infection in 14 cases (37%) and viral infection in 7 cases (20%). Neuropathy was observed in 11% of patients, stomatitis in 24%, gastrointestinal side effects in 13% and grade 3 or greater hepatic toxicity in 11%. Eight patients died, all due to disease relapse or progression. No death was attributed to treatment toxicity.

Summary/Conclusion: DA-EPOCH-R is an effective treatment for aggressive B-cell lymphoma limited by tolerability. Its administration should be confined to subgroups where the efficacy of this regimen is clearly superior.

PB2102: PHASE 2 RANDOMIZED, OPEN-LABEL, MULTICENTER STUDY TO EVALUATE THE EFFICACY AND SAFETY OF PLAMOTAMAB COMBINED WITH TAFASITAMAB (TAFA) + LENALIDOMIDE (LEN) VS TAFA+LEN IN RELAPSED OR REFRACTORY DLBCL

J.-M. Michot 1,* , Y. Koh 2 , P. Lee 3 , J. Jin 4 , R. Clynes 5 , S. Kye 6 , M. Chiarella 6 , K. Patel 7

1 Hematology and Innovative Drugs, Institut de Cancérologie Gustave Roussy, Villejuif, France; 2 Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea; 3 Clinical Operations; 4 Biostatistics, Xencor, Inc.; 5 Translational Biology, Xencor, Inc; 6 Clinical Development, Xencor, Inc., San Diego; 7 Center for Blood Disorders and Stem Cell Transplantation, Swedish Cancer Institute, Seattle, United States of America

Background: Plamotamab (XmAb13676) is a humanized bispecific antibody that binds both CD20 and CD3 and thereby recruits cytotoxic T cells to kill CD20-expressing malignant cells. In a Phase 1 trial in B-cell malignancies, plamotamab was generally well tolerated, with no Grade ≥ 3 cytokine release syndrome events at the recommended Phase 2 dose (RP2D) regimen, and other safety events being generally mild or moderate in severity. The RP2D demonstrated clinical activity in subjects with relapsed/refractory (R/R) non-Hodgkin lymphoma. The objective response rate (ORR) in follicular lymphoma (FL) was 100% (4/4), with a complete response rate of 50% (2/4). The ORR in diffuse large B-cell lymphoma (DLBCL) was 40% (2/5). Subjects were heavily pretreated with a median of 4 prior lines of therapy (range 2-8) for FL and a median of 5 lines of therapy for DLBCL (range 4-10); all DLBCL subjects received prior CAR-T (ASH 2021 Abstract 2494).

TAFA is a humanized CD19-directed cytolytic monoclonal antibody that contains an IgG1/2 hybrid Fc domain with 2 amino acid substitutions to modify the Fc-mediated functions of the antibody and mediates B-cell lysis through apoptosis and immune effector mechanisms. TAFA is indicated for use in the US and EU in combination with LEN for treating adults with R/R DLBCL who are not eligible for autologous stem cell therapy (ASCT). Addition of the CD3xCD20 plamotamab to the TAFA+LEN doublet offers the potential for improved efficacy through recruitment of distinct T cell and Fc-mediated cytotoxic mechanisms while also overcoming tumor resistance that might otherwise arise from single antigenic loss of either CD19 or CD20. These characteristics support a study of the combination of plamotamab, TAFA, and LEN.

Aims: This trials-in-progress abstract details eligibility criteria and trial design of the Phase 2 XmAb13676-03 trial.

Methods: This randomized, multicenter, open-label, 2-part study is intended to compare the safety and efficacy of plamotamab+TAFA+LEN vs TAFA+LEN in adult subjects with DLBCL who have relapsed or are refractory to ≥ 1 prior line of therapy and are ineligible for or refuse ASCT. Prior therapy must include multiagent chemoimmunotherapy that includes an anti-CD20 mAb. The trial consists of 2 parts, performed sequentially; Part 1 is a 2-cohort safety run-in that serves to identify the optimal dose of plamotamab (20 or 50 mg) to be administered with TAFA+LEN (shown in figure). Part 2 randomizes 200 subjects in a 1:1 ratio to the 2 treatment arms (with dosing regimen selected from Part 1), stratified by international prognostic index risk score, number of lines of prior therapy, and primary refractory (y/n). Eligible subjects must have documented diagnosis of DLBCL, not otherwise specified, CD20+ and CD19+ lymphoma, and relapsed or primary refractory disease. Exclusion criteria include central nervous system (CNS) lymphoma or secondary CNS involvement. Subjects with prior CAR-T are eligible if current biopsy is CD19+.

Results: The primary objectives are safety and efficacy. The primary endpoint in Part 1 is safety and the primary endpoint in Part 2 is progression-free survival with disease assessed at regular intervals. Response, overall survival, and duration of response are secondary endpoints. Subjects continue study treatment until progression and are followed for long-term survival.

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Summary/Conclusion: The XmAb13676-03 trial is registered at EudraCT (2021-003658-22) and anticipates enrolling in centers in the US, EU, and Asia.

PB2103: CAN WE ACCURATELY PREDICT SURVIVAL IN DIFFUSE LARGE B CELL LYMPHOMA IN THE RITUXIMAB ERA? RETROSPECTIVE COMPARISON OF IPI, AAIPI, R-IPI AND NCCN-IPI

P. Chorão 1,* , M. Henriques 1 , P. Baptista 1 , I. Carvalhais 1 , A. L. Pinto 1 , P. Silva 1 , A. M. Carneiro 1 , F. Ferreira 1 , F. Trigo 1

1 Clinical Hematology Department, Centro Hospitalar Universitário São João, Porto, Portugal

Background: In diffuse large B cell lymphoma (DLBCL), the value of the International Prognostic Index (IPI) and age-adjusted IPI (aaIPI) changed with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) as standard first line. The revised (R-IPI) and enhanced (NCCN-IPI) indices have emerged to improve prognostic ability. An analysis from Surrogate Endpoints for Aggressive Lymphoma (SEAL) database suggests NCCN-IPI better discriminates prognosis.

Aims: In light of data scarcity and future changes in second line therapy for relapsed/refactory patients, we aimed to analyse the suitability of 4 survival prognostic indices in a DLBCL cohort.

Methods: Single centre analysis of 169 patients diagnosed with de novo DLBCL from 2008 to 2016 and treated with R-CHOP. Primary central nervous system DLBCL or upfront stem cell transplant were exclusion criteria. IPI, aaIPI, R-IPI and NCCN-IPI scores at diagnosis were calculated. Analysis for 5-year overall survival (5y-OS) and progression free survival (5y-PFS) were performed using Kaplan-Meier curves and compared using the log-rank test. For each prognostic index, C-index test from receiver operating characteristic curves was calculated and area under the curve comparisons between prognostic indices were made using DeLong’s test.

Results: Median age was 61 years (interval 18-82), 55% of patients had >60 years and 54% were male. At diagnosis, 23% had an ECOG performance status ≥2, 68% had Ann-Arbor stage III-IV, 63% had increased lactic dehydrogenase levels and 77% had extranodal disease (24% >1 site). Median follow-up was 71 months and 5y-OS and 5y-PFS were 74% and 67%, respectively.

For the whole cohort, C-index for 5y-OS was 0.701, 0.629 and 0.751 for IPI, R-IPI and NCCN-IPI, respectively, and R-IPI was outperformed by IPI and NCCN-IPI (p=0.007 and p<0.001, respectively). For 5y-PFS C-index was 0.673, 0.695 and 0.689 for IPI, R-IPI and NCCN-IPI, with no statistically significant differences between indices. NCCN-IPI managed to discriminate the intermediate low (IL) and intermediate high (IH) risk groups for both 5y-OS (IL 89% versus (vs) IH 64%, p=0.001) and 5y-PFS (IL 77% vs IH 60%, p=0.029), contrary to IPI (5y-OS IL 69% vs IH 74%, p=0.552; 5y-PFS IL 63% vs HI 64%, p=0.925).

The cohort was divided into age groups so that R-IPI and NCCN-IPI could be compared with aaIPI. For patients with ≤60 years, C-index for 5y-OS was 0.722, 0.585 and 0.724 for aaIPI, R-IPI and NCCN-IPI, respectively, and R-IPI was outperformed by NCCN-IPI but not by aaIPI (p=0.023 and p=0.077, respectively). For 5y-PFS C-index was 0.705, 0.622 and 0.625 for aaIPI, R-IPI and NCCN-IPI, with no statistically significant differences between indices. IL and IH risk groups could not be differentiated neither by aaIPI (5y-OS IL 85% vs IH 82%, p=0.725; 5y-PFS 76% vs 69%, p=0.556) nor NCCN-IPI (5y-OS IL 85% vs IH 70%, p=0.162; 5y-PFS IL 70% vs IH 70%, p=0.905), although in the last index there were no high-risk patients.

Summary/Conclusion: NCCN-IPI seems the most robust prognostic tool for OS with frontline R-CHOP. Our results are in accordance with the SEAL analysis, although our sample was significantly smaller and SEAL included other immunochemotherapy regimens. However, improvements are needed for PFS prognostication, especially in younger patients within intermediate risk groups, and all indices performed similarly. Accurate prediction of PFS will ultimately allow for early benchmarking of patients for advanced or more aggressive therapies, and future research will be needed from bigger cohorts.

PB2104: DOSE-DENSE CHEMOTHERAPY WITH R-CHOP-14 IN OLDER ADULTS AGED ≥80 YEARS WITH DIFFUSE LARGE B-CELL LYMPHOMA

K. Christofyllakis 1,2,* , Z. G. Dilbaz 1,2 , S. Denker 3,4 , C. Ankermann 3 , A. Kunte 5 , V. Poeschel 1,2 , M. Bewarder 1,2 , D. Kaddu-Mulindwa 1,2 , S. Stilgenbauer 1,2,6,7 , L. Thurner 1,2 , I.-K. Na 3,4,8,9 , J.-T. Bittenbring 1,2

1 Department of Oncology, Hematology, Rheumatology and Clinical Immunology, Saarland University Medical Center; 2 Saarland University Medical School, Homburg; 3 Medical Department, Division of Hematology, Oncology and Tumor Immunology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin; 4 Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin; 5 Department of Stem Cell Transplantation, Hamburg-Eppendorf University Medical Center, Hamburg; 6 Department of Internal Medicine III, Ulm University Hospital; 7 Comprehensive Cancer Center Ulm, Ulm; 8 ECRC Experimental and Clinical Research Center, Charité, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin; 9 German Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype. Approximately 29% of patients with DLBCL are ≥75 years old. Treatment with the RCHOP regimen is standard of care and can be administered either every 3 weeks (RCHOP-21) or dose-dense biweekly (RCHOP-14). The RICOVER-60 trial showed excellent outcomes with RCHOP-14 in patients aged 60-80 years. In patients ≥80 years, attenuated dose regimens are preferred. Insufficient data are available concerning the use of RCHOP-14 in patients aged ≥80 years.

Aims: To evaluate dose dense RCHOP-14 in patients with DLBCL aged ≥80 years.

Methods: We performed a single center retrospective analysis of patients with DLBCL aged ≥80 years treated with RCHOP-14 at Saarland University Medical Center from 2005 to 2017. Baseline patient characteristics, dose intensity and treatment outcomes were extracted from hospital medical electronic records. Relative dose intensity (RDI) was calculated as the percentage of the dose intensity achieved divided by the intended dose intensity. Primary endpoints were overall (OS), progression-free (PFS) and event-free survival (EFS), defined as time from diagnosis to death, death or progression/relapse, progression/relapse or treatment discontinuation. Statistical analyses included descriptive statistics, Cox regression and Kaplan Meier estimates.

Results: We identified 58 patients. Of them, 19 patients were excluded (primary CNS lymphoma: n=5, no rituximab: n=3, only palliative therapy: n=5, bendamustin: n=1, RCHOP-21: n=2, death during prephase treatment: n=2, missing documentation: n=1). In total, 39 patients (67%) received at least one cycle of RCHOP-14 and were selected for further analysis. Median age was 83 years (80-91) and 61.5% of patients were female. Eastern cooperative oncology group performance score (ECOG-PS) was ≥2 in 54.1% of patients. Charlson’s comorbidity index (CCI) was ≥2 in 38.5% of patients. International prognostic index (IPI) was ≥3 in 54% of patients and 67% had stage III-IV disease. The overall response rate was 93.3%. Rates of complete remission (CR)/unconfirmed CR were 80% and partial remission were 13.3%. With a median follow-up of 21 months, the 2-year OS, PFS and EFS estimates were 73%, 59% and 38% respectively. Treatment-related mortality was 7.6%. Treatment was discontinued early due to toxicity in 11 patients (28%) with a median number of 6 (2-6) chemotherapy cycles in the overall cohort. Mean RDI was 80.3%, and RDI did not correlate with OS (p=0.32). In the multivariate analysis, there were significant associations of OS with CCI (p=0.043, relative risk [RR] 0.36, 95% confidence interval [CI]= 0.13-0.97) and age (p=0.048, RR 0.60, 95%CI= 0.36-0.97). Female sex and higher ECOG-PS were associated univariately with worse EFS (p= 0.003, RR 22.1, 95%CI= 2.79-175.7 and p=0.001, RR 4.14, 95%CI= 1.75-9.89). A serum albumin level of ≥3.5 g/dl was associated univariately with improved PFS (p=0.043).

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Summary/Conclusion: This report demonstrates feasibility of RCHOP-14 in selected octogenarians with DLBCL. Despite relative high treatment discontinuation rates, adequate RDI levels were achieved and TRM did not exceed that reported for attenuated regimens in previous prospective trials. Better outcomes were achieved in male patients without comorbidities, a serum albumin level of ≥3.5 g/dl and a good performance status. Even though this is a retrospective single center analysis, the excellent outcome reported here suggests that advanced age alone does not preclude application of dose dense chemotherapy with curative intent in selected patients of this high-risk group.

PB2105: REAL WORLD EXPERIENCE WITH POLATUZUMAB IN COMBINATION WITH BENDAMUSTINE AND RITUXIMAB FOR R/R DLBCL IN 3 ACADEMIC CENTERS IN MADRID, SPAIN.

M. Corrochano 1,2,* , S. Nistal 1,2 , V. Pradillo 2 , A. Alonso 2,3 , C. Alaez 1,2 , M. Tercero-Mora 2,3 , I. Delgado 2 , J. M. Sanchez 2 , E. Marti 2 , A. Segado 2,3 , G. Benzo 2 , A. Escudero 2 , J. M. Fernández-Rañada 2 , C. Martinez Chamorro 2

1 Hematology, Hospital HLA Universitario Moncloa; 2 Hematology, Hospital Universitario QuirónSalud Madrid; 3 Hematology, Complejo Hospitalario Ruber Juan Bravo, Madrid, Spain

Background: On 2019, the FDA and later the EMA granted approval to polatuzumab vedotin-piiq, a CD79b-directed antibody-drug conjugate indicated in combination with bendamustine and rituximab (P-BR) for adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS), after at least two prior therapies. P- BR has demonstrated ( NCT02257567 ) better overall response rates (complete and partial responses) compared with BR alone (63% vs 25%) and response durations of at least 12 months in 48% of the patients. The most common adverse reactions with P-BR (incidence at least 20%) included cytopenias (most common reason for treatment discontinuation), peripheral neuropathy, fatigue, diarrhea, pyrexia, decreased appetite and pneumonia. Serious adverse reactions occurred in 64%, most often from infection.

Aims: To analyze results in terms of efficacy and safety of the P-BR regimen in real life conditions.

Methods: Observational, retrospective study in 3 academic centers. Adult patients (≥ 18 years old) diagnosed with DLBCL NOS R/R who received P-BR between July 2019 and December 2021 were included in the analysis.

Results: 11 patients were treated with P-BR. The mean (SD) age was 70.1 (8.2) years (Range 57-81 years). Cell of origin was informed in 9/11 cases, 6 of them were activated B-cell (ABC) subtype. No double-/triple-hit lymphomas were confirmed. The median number of prior lines of therapy before P-BR was 2, with most patients (63%) refractory to the last treatment. All patients had received anti-CD20 (Rituximab) on prior treatments and only 2 (18%) Bendamustine. Baseline characteristics are shown in Table 1.

Efficacy

Seven patients were evaluated by PET-CT after 3 cycles, 4 (57%) achieved CR and 3 PR (43%).

Five patients achieved CR by PET-CT at the end of treatment. One of these patients is still in CR after 12 months of follow up and three of them after 24 months from the start of P-BR. One patient relapsed after 19 months.

Of the patients achieving CR, all of them had responses >12 months. Only 3/5 completed the 6 cycles scheme, 1 patient received 5 cycles (treatment was interrupted due to an invasive fungal infection) and 1 patient received only 2 cycles as bridge therapy for and autoHCT and achieved CR after transplantation.

1 patient was refractory to treatment and progressed after 2 cycles.

Toxicity:

All patients were evaluated for toxicity. 63% (7/11) of them presented hematological toxicity, mainly neutropenia which required GCSF administration and 71% RBC transfusion. Two patients required hospital admission because of neutropenic fever.

There were 3 documented cases of SARS-CoV-2 infection. Two patients had moderate disease with bilateral pneumonia (vaccinated) after the 2º cycle of treatment which is temporarily interrupted. One patient completed 6 cycles but died of severe SARS-CoV2 infection (unvaccinated) before being assessed for response at end of treatment.

Two patients interrupted treatment definitely because of toxicity: severe cytopenia and invasive fungal infection. No other extra hematological toxicities were reported.

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Summary/Conclusion: The P-BR regimen provides sustained good results for patients with R/R DLBCL who have failed treatment with prior therapies. Cytopenias were the most frequent form of toxicity and were easily addressed in most cases. In our experience, SARS-CoV2 infection has been a challenge due to delay in treatment and high morbidity and mortality.

PB2106: REDUCTION OF METHOTREXATE (MTX) LEVELS AFTER GLUCARPIDASE IN PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) AT RISK OF CENTRAL NERVOUS SYSTEM (CNS) INVOLVEMENT WHO RECEIVE HDMTX: PHASE 2

A. De La Fuente 1,* , F. J. Peñalver 2 , P. Herrera 3 , R. Cordoba 4 , C. Martinez Chamorro 5 , M. T. Olave 6 , I. Jarque 7 , M. B. Navarro 8 , M. Estévez 1

1 Hematology, MD Anderson CC Madrid; 2 Hematology, H. U. Fundación Alcorcón; 3 Hematology, H. U. Ramon y Cajal; 4 Hematology, H. Fundación Jimenez Diaz; 5 Hematology, H Quironsalud Madrid Pozuelo, Madrid; 6 Hematology, H. U. Lozano Blesa, Zaragoza; 7 Hematology, H. U. Politecnico La Fe, Valencia; 8 Hematology, H. U. Puerta de Hierro, Madrid, Spain

Background: CNS relapse in patients with Diffuse Large B Cell Lymphoma (DLBCL) is usually an early complication and with poor prognosis. R-CHOP, the standard treatment for DLBCL plays a minimal role preventing leptomeningeal and/or brain parenchyma relapse. Risk factors have been previously identified for this complication and high dose intravenous methotrexate (HDMTX) has been suggested as an effective strategy to prevent CNS lymphoma relapse (GELTAMO guidelines Haematologica 2017 and British guidelines BJAEM 2020). Unfortunately the administration of HDMTX is related with risk of renal, hepatic and haematological adverse events.

Aims: Primary objective is:

To describe the reduction of MTX levels after systematic administration of reduced Glucarpidase doses (2,000 units) 12 hours following start of HDMTX infusion in DLBCL patients at high risk for CNS involvement, receiving multiple cycles of HDMTX.

Secondary objectives are:

To analyze the prophylactic effect of Glucarpidase administered after 12 hour of high-dose MTX (HDMTX) on the incidence and severity and duration of MTX related-toxicity (renal toxicity, mucositis, liver toxicity, neutropaenia, thrombocytopaenia).

To assess the incidence of CNS relapse during one year

To assess the length of hospital stay (LOS) during HDMTX therapy

To analyze the appearance of antibodies against Glucarpidase

To analyze the appearance of neutralizing antibodies against Glucarpidase

The study is currently enrolling. ClinicalTrials.gov Identifier: NCT05022797

Methods: This is an open-label, interventional, non-randomized, phase 2, pilot, multicenter study in Diffuse large B-cell lymphoma (DLBCL) patients at high risk of CNS relapse to assess of prophylactic effect of 2,000 unit of Glucarpidase in MTX related-toxicity administered after 12 hours of HDMTX. This study will be performed in the context of in-patients setting for receiving the three HDMTX courses.

Criteria for CNS involvement are concordant with those of the last approved version of the GELTAMO guideline for the diagnostic, prevention, and therapeutical management of Central Nervous System involvement in patients with diffuse large cell B Lymphoma (revised 2017) [1].

Inclusion criteria: Age 18 year and above, diagnosis with diffuse large B-cell lymphoma at high risk of CNS involvement and adequate haematological, renal and hepatic function.

Criteria to consider risk of CNS involvement were:

>=2 extranodal site involved plus an elevated LDH and/or lymphoma involvement of the

testis, breast, adrenal gland or kidney

In order to include patients without CNS involvement, patients will be included in the study with a negative result of the CSF cytometry flow test, which will be performed according to normal clinical practice.

Results: Trial ongoing

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Summary/Conclusion: Trial ongoing

PB2107: CLINICAL MANIFESTATIONS AND EFFICACY ANALYSIS OF 71 CASES OF AIDS-RELATED LYMPHOMA

J. Liu 1,* , W. Zhang 1 , T. Yang 1 , Q. Li 1 , J. Li 1 , Y. Xiang 1 , Y. Liu 1

1 Department of Hematology-Oncology, Chongqing University Cancer Hospital, Chongqing, China

Background: Patients with AIDS-related lymphoma (ARL) is difficult to accept standard treatment for various reasons.

Aims: To summarize the clinical characteristics of AIDS-related lymphoma (ARL) patients admitted to Chongqing Cancer Hospital and improve the understanding of the disease.

Methods: The clinical data of 71 ARL patients admitted from January 2008 to January 2021 were retrospectively analyzed.

Results: The ratio of male to female in 71 ARL patients was 6.1:1, and the median age was 50 years. Fifty-one percent of patients had no other HIV-related manifestations and were only found at the time of lymphoma diagnosis. 20% of patients were diagnosed with lymphoma within 1 year of AIDS diagnosis and antiviral therapy. At the time of diagnosis of lymphoma, 35 cases (49.3%) with CD4+ cell count>200×10^6/L, 22 cases (30.9%) with CD4+ cell count 100-200×10^6/L, and CD4+ cell count<100×10^6/L and 6 cases of <50×10^6/L (accounting for 8.5

), and 2 other cases are unknown. The most common pathological type in the 71 patients was diffuse large B-cell lymphoma (38 cases, accounting for 53.5%), Burkitt lymphoma (13 cases, accounting for 18.3), and the types also include Hodgkin lymphoma, high-grade B-cell lymphoma, plasmablastic lymphoma, follicular lymphoma, marginal zone B-cell lymphoma tumor, CLL/SLL, T lymphoblastic lymphoma and Castleman disease. Thirty patients (42.3%) had extranodal involvement at the initial diagnosis, including lung, kidney, intestinal, pancreas, breast, testis, skin and soft tissue involvement. Ann-Arbor staging included 56 patients (78.8%) with stage III-IV, and 19 patients (26.8%) with B symptoms. Survival analysis showed that patients with central involvement, elevated LDH, and intermediate-high-risk IPI scores had shorter progression-free survival, and patients with large masses, intermediate-high-risk and high-risk IPI scores had shorter overall survival. Both CHOP and EPOCH-based chemotherapy regimens are used in AIDS-related non-Hodgkin lymphoma, but the efficacy varies greatly. ABVD treatment of AIDS-related Hodgkin lymphoma is effective, but extranodal involvement may be a poor prognostic factor.

Summary/Conclusion: ARL has diverse clinical manifestations, frequent extranodal involvement, late clinical stage, high malignancy, and poor clinical efficacy.

PB2108: PREVALENCE AND CLINICAL IMPLICATIONS OF CYCLIN D1 POSITIVE DIFFUSE LARGE B-CELL LYMPHOMA IN CHINA

X. Liang 1,* , R. Hu 1 , C. Wang 1 , Q. Li 1 , T. Yang 1 , B. Guo 1 , C. Xiao 1 , Y. Liu 1

1 Department of Hematology-Oncology, Chongqing University Cancer Hospital, Chongqing, China

Background: Cyclin D1 protein expression in lymphocytes is classically associated with mantle cell lymphoma. It has been ralely reported in patients with diffuse large B-cell leukemia (DLBCL), and studies have been few and generally small, and they were no obvious clinical implications attributable to cyclin D1 expression in China.

Aims: To investigate clinical implications attributable to cyclin D1 expression n patients with diffuse large B-cell leukemia (DLBCL) in China.

Methods: he authors reviewed 541 patients who were diagnosed with DLBCL treated with R-CHOP (rituximab with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone,R-CHOP) in 3 years. Consortium Program and performed clinical, immunohistochemical, and genetic analyses with a focus on cyclin D1. All patients who were cyclin D1-positive according to immunohistochemistry were also assessed for rearrangements of the cyclin D1 gene (CCND1) using fluorescence in situ hybridization. Clinical and immunohistochemical features were performed to compare patients who had DLBCL with and without cyclin D1 expression.

Results: In total, 11 patients (2.0%) who had DLBCL that expressed cyclin D1 and lacked CCND1 gene rearrangements. Patients with cyclin D1-positive DLBCL had a median age of 59.45±3.10 years (ranged 48-80 years). There were 7 males and 4 females. B-symptoms were present in 18.2% of patients. 90.9% patients were regarded as IV stage. The International Prognostic Index was high-intermediate and high (>3) in 81.8% of patients. Two or more extranodal sites were involved in 36.4% of patients. CD20 was positive in all cases. These cases showed a strong nuclear positivity for cyclin D1 in the DLBCL, but no expression of CD5 and SOX11 was detected. All cases were consistently negative for CD10, BCL6 was positive in 8 of 11 cases (72.2%), and MUM1 was positive in 9/11 cases (81.8%). The majority of cases (90.9%) showed expression of MYC. All cases showed a proliferation rate above 80%. Patients with cyclin D1-positive DLBCL patients had more Ki-67 proliferation, elevated serum lactate dehydrogenase and more advanced stage (P<.05). The other clinical characteristics, such as sex, age, extraodal site, IPI score, and β macroglobulin level did not differ significantly (P>.05). However, patients with cyclin D1-positive DLBCL had shorter PFS and OS than cyclin D1-negative patients (P<0.05). Cyclin D1-positive DLBCL patients with MYC or MYC/BCL2 coexpression showed poorer prognosis than double protein lymphoma (DPL) patients, but did not significantly (P>.05).

Summary/Conclusion: Compared with patients who had cyclin D1-negative DLBCL, cyclin D1-positive DLBCL patients had later staging and greater tumor load with more Ki-67 proliferation. There were no other significant differences in clinical presentation, pathologic features. Cyclin D1-positive DLBCL patients showed poorer overall survival and progression-free survival than cyclin D1-negative patients.

PB2109: REALMIND: A PROSPECTIVE, MULTICENTER, OBSERVATIONAL STUDY OF PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA STARTING SECOND/THIRD-LINE THERAPY AND NOT RECEIVING A STEM CELL TRANSPLANT

C. R. Flowers 1,* , J. M. Burke 2 , M. Vukcevic 3 , S. Snodgrass 4 , K. Saverno 4 , M. A. A. Lumiqued 3

1 Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston; 2 US Oncology Hematology Research Program, Rocky Mountain Cancer Centers, Aurora, United States of America; 3 MorphoSys AG, Planegg, Germany; 4 Incyte Corporation, Wilmington, United States of America

Background: Although R-CHOP in the first-line (1L) setting is curative for >60% of diffuse large B-cell lymphoma (DLBCL) patients, up to 40% will have relapsed/refractory (R/R) disease. Treatment options in the R/R setting for patients not eligible for autologous stem cell transplant (ASCT) include the anti-CD19 antibody tafasitamab plus lenalidomide, the anti-CD79b Ab-drug conjugate polatuzumab plus bendamustine plus rituximab, the anti-CD19 antibody-drug conjugate loncastuximab tesirine, the nuclear export inhibitor selinexor, and CD19 CAR T-cell therapies. Sequencing these options and identifying the optimal choice of treatment for individual patients can be challenging for practicing oncologists.

Aims: realMIND ( NCT04981795 ) will assess real-world treatment patterns, effectiveness measures, and patient-reported outcomes among patients in the United States (US) with R/R DLBCL not receiving ASCT.

Methods: This Phase IV, prospective, multicenter, observational study is enrolling patients ≥18 years old with histologically confirmed DLBCL at initial diagnosis and R/R status after at least one prior systemic therapy, including ASCT. Sites are being targeted across the US with the intent of enrolling a diverse patient population representative of the underlying US population with R/R DLBCL regarding age, ethnicity, and race. Patients may enroll in the study (a) after completing 1L therapy and before starting second-line (2L) therapy, (b) within 30 days after starting 2L therapy, and (c) after completing 2L and before receiving third-line therapy (Figure). Patients who are eligible or candidates for ASCT may be enrolled in the study if they are not receiving ASCT at the time of enrollment. Patient history data on therapies received before study enrollment will be collected from patient records; data generated from time of enrollment onwards will be collected prospectively. Real-world effectiveness measures will include complete and objective response rate, duration of response, event-free survival, and overall survival; safety endpoints include serious adverse events and adverse events leading to treatment discontinuation, interruption, or dose modification. Patient-reported outcomes will include general and lymphoma-specific measures of health-related quality of life assessed by the EORTC Quality of Life Questionnaire-Core 30 and Functional Assessment of Cancer Therapy – Lymphoma, and work productivity by the Work Productivity and Activity Impairment Questionnaire. The end of the study is expected to occur approximately 79 months after the first patient is enrolled to ensure a minimum of 24 months’ follow-up after last enrollment. All analyses will be descriptive, and time-to-event analyses will be conducted using Kaplan–Meier methods. realMIND is actively recruiting.

Results: Results for this study are not yet available: outcome data as described above will be presented in due course.

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Summary/Conclusion: realMIND will provide further understanding and characterization of the R/R DLBCL patient journey with respect to treatment patterns, clinical and patient-reported outcomes. The study population will reflect a representative distribution of patients within the US, facilitating investigation of outcomes in a patient population reflecting real-world clinical practice and producing data to support healthcare decision-making.

PB2110: PHARMACOKINETICS AND PHARMACODYNAMICS IN FIRST-MIND: A PHASE IB, OPEN-LABEL, RANDOMIZED STUDY OF TAFASITAMAB ± LENALIDOMIDE + R-CHOP IN PATIENTS WITH NEWLY DIAGNOSED DIFFUSE LARGE B-CELL LYMPHOMA

D. Belada 1,* , K. Kopeckova 2 , J. M. Bergua Burgues 3 , D. Stevens 4 , G. S. Nowakowski 5 , M. Waldron-Lynch 6 , N. Hadar 6 , J. Weirather 7 , C. Lässig 7 , D. Blair 7 , M. Dreyling 8

1 4th Department of Internal Medicine - Haematology, Charles University, Hospital and Faculty of Medicine, Hradec Králové; 2 Department of Oncology of the 2nd Faculty of Medicine, Charles University and University Hospital in Motol, Prague, Czechia; 3 Department of Hematology, Hospital San Pedro de Alcantara, Cáceres, Spain; 4 Norton Cancer Institute - St. Matthews Campus, 3991 Dutchmans Lane, 40207, Louisville; 5 Division of Hematology, Mayo Clinic, Rochester; 6 MorphoSys US, Inc., Boston, United States of America; 7 MorphoSys AG, Planegg; 8 Medical Clinic III, Ludwig-Maximilians-University Hospital, Munich, Germany

Background: The combination of tafasitamab + lenalidomide (LEN) has been granted accelerated approval in the United States (2020) and is conditionally approved in Canada and Europe (2021) for R/R DLBCL in

autologous stem-cell transplant-ineligible adult patients. A tolerable safety profile for rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) + tafasitamab ± LEN in patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) in the Phase Ib First-MIND study ( NCT04134936 ) was previously reported, with clinically meaningful efficacy for R-CHOP + tafasitamab + LEN compared to R-CHOP + LEN (ASH 2021; #3556).

Aims: To report pharmacokinetics, pharmacodynamics, and immunogenicity in patients with newly diagnosed DLBCL after adding tafasitamab ± LEN to R-CHOP as first-line treatment.

Methods: Eligible patients were ≥18 years with treatment-naïve DLBCL, International Prognostic Index 2–5, and Eastern Cooperative Oncology Group performance score 0–2. Patients were randomized 1:1 to either six 21-day (D) cycles (C) of R-CHOP (R-CHO, D1; P, D1–5) + tafasitamab (12 mg/kg IV, D1, 8, 15) (Arm A) or R-CHOP + tafasitamab + LEN (25 mg orally, D1–10) (Arm B). Secondary endpoints included tafasitamab serum concentration and number and percentage of patients developing anti-tafasitamab antibodies. Exploratory endpoints included natural killer (NK) cell, T-cell, and B-cell count in peripheral blood.

Results: In total, 83 patients were screened; 66 were randomized (Arm A, n=33; Arm B, n=33). The data cut-off was 13 March 2021 for the safety analysis, including ≥1 month follow-up after end of treatment (EoT) visit for all patients, and 15 September 2021 for the efficacy analysis.

Tafasitamab serum concentrations reached steady state by C3 (geometric mean trough concentrations: Arm A, 186.40–216.55 µg/mL; Arm B, 171.77–201.54 µg/mL) and steadily declined after treatment completion. Anti-tafasitamab antibodies were detected in 1/65 (1.5%) patients. This patient showed pre-existing anti-tafasitamab antibodies at baseline, which decreased during treatment.

Median NK cell counts decreased from baseline at C1 D8, but were at baseline or higher levels by EoT visit (Arm A) and C1 D15 (Arm B; Table). T-cell counts decreased from baseline at C1 D8 in both arms but were at baseline level or higher by C1 D15 (Arm A) and EoT visit (Arm B). Median B-cell counts decreased from baseline to 0 cells/μL (Arm A, C1 D15; Arm B, C1D8); at 6-month follow-up after EoT visit, B-cell counts had recovered to measurable levels in ~50% of patients.

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Summary/Conclusion: Tafasitamab serum concentration reached and maintained a therapeutic dose level in this first-line regimen and declined in line with known tafasitamab half-life (~16 days) after treatment completion; tafasitamab levels were comparable between the two treatment arms. No patients developed treatment-induced or treatment-boosted anti-tafasitamab antibodies. Median NK, T-cell, and B-cell counts were comparable between treatment arms in all cycles. NK and T-cell counts recovered to baseline levels by EoT; further follow up on B-cell recovery will be performed.

PB2111: SUBGROUP ANALYSIS IN RE-MIND2, AN OBSERVATIONAL, RETROSPECTIVE COHORT STUDY OF TAFASITAMAB + LENALIDOMIDE VERSUS SYSTEMIC THERAPIES IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA

G. S. Nowakowski 1,* , D. H. Yoon 2 , E. Joffe 3 , P. L. Zinzani 4,5 , L. Sabatelli 6 , E. E. Waltl 7 , C. G. Alvero 8 , G. Hess 9 , P. Riedell 10 , K. Kim 11,12 , D. Brixner 13 , G. Salles 3

1 Division of Hematology, Mayo Clinic, Rochester, United States of America; 2 Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea; 3 Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, United States of America; 4 IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli; 5 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy; 6 Incyte Biosciences International Sàrl, Morges, Switzerland; 7 MorphoSys AG, Planegg, Germany; 8 MorphoSys US Inc., Boston, United States of America; 9 Department of Hematology, Oncology and Pneumology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany; 10 Division of Hematology and Oncology, University of Chicago Medicine, Chicago; 11 Department of Pharmacotherapy, College of Pharmacy, University of Utah, Salt Lake; 12 Department of Pharmacy, Systems, Outcomes and Policy, University of Illinois at Chicago, Chicago; 13 Department of Pharmacotherapy, College of Pharmacy, University of Utah, Salt Lake City, United States of America

Background: Tafasitamab (tafa) + lenalidomide (LEN) demonstrated efficacy in adult patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) ineligible for autologous stem-cell transplant in the pivotal Phase II study L-MIND ( NCT02399085 ). The combination received accelerated approval in the United States in 2020 and conditional marketing authorization in Europe and Canada in 2021 in this setting. RE-MIND2 ( NCT04697160 ), an observational, retrospective cohort study compared patient outcomes from L-MIND with matched patient cohorts treated with other NCCN/ESMO recommended therapies. Prolonged overall survival (OS) with tafa + LEN was detected compared to a cohort of systemic therapies for DLBCL pooled (STP), bendamustine + rituximab (BR), rituximab + gemcitabine + oxaliplatin (R-GemOx), polatuzumab vedotin + BR (pola-BR), and rituximab + LEN (R2). Comparable OS was detected versus CD19 CAR-T therapies (CAR-T). However, limited information is available on the comparative effectiveness of tafa + LEN in specific subpopulations.

Aims: To inform on the relative effectiveness of tafa + LEN versus other treatment regimens for R/R DLBCL in clinically relevant patient subgroups in the absence of head-to-head trials.

Methods: Data were collected from health records of eligible patients aged ≥18 years with histologically confirmed DLBCL, who had received ≥2 systemic therapies for DLBCL (including ≥1 anti-CD20 therapy). Patients from L-MIND were matched with patients from the RE-MIND2 observational cohort using estimated propensity score-based 1:1 nearest neighbor matching, balanced for nine or six covariates. Hypothesis-generating analyses were conducted for patient subgroups of number of extranodal sites (ENS) (0–1 vs ≥2) and elevated lactate dehydrogenase (LDH) (yes vs no) in matched cohorts of patients receiving tafa + LEN vs STP, pola-BR, R2, and CAR-T. The primary endpoint was OS.

Results: Of 3,454 patients enrolled, 961, 106, 106, and 149 were treated with STP, pola-BR, R2, and CAR-T, resulting in 76, 24, 33, and 37 matched pairs for patients receiving tafa + LEN, respectively. Hazard ratios (HR) for OS show a trend toward favoring tafa + LEN in most patient subgroups (Table).

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Summary/Conclusion: These analyses suggest that tafa + LEN may be associated with improved OS vs selected systemic therapies for certain patients with high-risk disease and may further inform physicians’ treatment choices for patients with R/R DLBCL. These analyses are not powered for statistical comparison; small sample sizes in some subgroups result in wide confidence intervals and, although supported by several sensitivity analyses, the observed trends must be interpreted with caution. Additional analyses including other patient subgroups, endpoints, and cohort balancing approaches will be presented.

PB2112: MINDWAY: A PHASE IB/II DOSE OPTIMIZATION STUDY TO ASSESS SAFETY AND PHARMACOKINETICS OF TAFASITAMAB + LENALIDOMIDE IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA

R. Greil 1,* , K. Kopeckova 2 , L. Arcaini 3 , A. Amin 4 , K. de Graaf 4 , A.-M. Jegg 4

1 Paracelsus Medical University, Salzburg Cancer Research Institute-Center for Clinical Cancer and Immunology Trials (SCRI-CCCIT); Cancer Cluster, Salzburg, Austria; 2 Department of Oncology of the 2nd Faculty of Medicine, Charles University and Motol University Hospital, Praha, Czechia; 3 Division of Hematology, Fondazione IRCCS Policlinico San Matteo and Department of Molecular Medicine, University of Pavia, Pavia, Italy; 4 MorphoSys AG, Planegg, Germany

Background: Tafasitamab is an Fc-enhanced, humanized, anti-CD19 monoclonal antibody which, in combination with the immunomodulatory drug lenalidomide, has been granted accelerated FDA approval and EU conditional marketing authorization based on results from L-MIND ( NCT02399085 ) for the treatment of relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) in adult patients ineligible for autologous stem cell transplant. Treatment schedule optimization aims to reduce the frequency of hospital visits and decrease treatment burden and healthcare utilization while maintaining clinical benefits.

Aims: MINDway ( NCT05222555 ) will investigate if administration of tafasitamab at levels higher than the approved 12 mg/kg dose at a reduced dosing frequency can maintain the benefit-risk relationship of tafasitamab + lenalidomide treatment for patients with R/R DLBCL.

Methods: MINDway is an open-label, multicenter, Phase Ib/II study in patients aged 18‒80 years with histologically confirmed DLBCL and R/R status after at least one, but no more than three, prior systemic regimens, including CD20-targeted therapy. Patients previously treated with CD19-targeted therapy or with immunomodulatory imide drugs such as lenalidomide are excluded. The modified tafasitamab dosing regimen will be investigated in a stepwise design with two sequential dose-finding cohorts (planned n=6 each) followed by an expansion cohort (planned n=39; Figure). Lenalidomide 25 mg will be administered in all cohorts on Day (D) 1‒21 of each 28-D cycle for up to 12 cycles. After Cycle 12, patients will continue tafasitamab monotherapy at the assigned dosing regimen until progression. Following a review of the safety and pharmacokinetic data from Cohorts 1 and 2, patients will be enrolled in an expansion cohort to receive tafasitamab + lenalidomide at the tafasitamab dose of 24 mg/kg or 30 mg/kg for further evaluation. No intra-patient dose escalation from 24 mg/kg to 30 mg/kg is permitted.

The primary endpoint is the incidence and severity of treatment-emergent adverse events; key secondary endpoints include tafasitamab serum concentrations after 3 and 12 treatment cycles and best objective response rate, duration of response, and progression-free survival by investigator assessment. All endpoints will be analyzed using descriptive statistics; no formal statistical tests will be performed.

MINDway is currently enrolling, with a planned sample size of 51 patients.

Results: Results for this study are not yet available: outcome data as described above will be presented in due course.

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Summary/Conclusion: MINDway will optimize the tafasitamab dosing regimen to reduce the frequency of hospital/clinic visits for tafasitamab treated R/R DLBCL patients. Reducing the overall frequency of clinic visits by half is expected to reduce the patient burden and support long-term treatment compliance. Furthermore, given the severity of the disease, reduced hospital visits may result in less exposure to hospital infections in this already susceptible population.

PB2113: FRONTMIND: A PHASE III, RANDOMIZED, DOUBLE-BLIND STUDY OF TAFASITAMAB + LENALIDOMIDE + R-CHOP VS R-CHOP ALONE FOR NEWLY DIAGNOSED HIGH-INTERMEDIATE AND HIGH-RISK DIFFUSE LARGE B-CELL LYMPHOMA

U. Vitolo 1,* , G. S. Nowakowski 2 , J. M. Burke 3 , C. P. Fox 4 , M. Trneny 5 , A. Chiappella 6 , M. Waldron-Lynch 7 , N. Hadar 7 , A. Pachori 7 , G. Lenz 8

1 Candiolo Cancer Institute, FPO-IRCCS, Candiolo (Torino), Italy; 2 Division of Hematology, Mayo Clinic, Rochester; 3 Department of Hematology, US Oncology Research and Rocky Mountain Cancer Centers, Aurora, United States of America; 4 Department of Clinical Hematology, Nottingham University Hospitals NHS, Nottingham, United Kingdom; 5 1st Department of Medicine, First Medical Faculty, Charles University and General University Hospital, Prague, Czechia; 6 Division of Hematology & Stem Cell Transplant, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy; 7 MorphoSys US, Boston, United States of America; 8 Department of Medicine A – Haematology and Oncology, University Hospital Münster, Münster, Germany

Background: Up to 40% of patients relapse or are refractory (R/R) to R-CHOP, the current standard of care for diffuse large B-cell lymphoma (DLBCL), indicating an unmet need, particularly in patients with high-intermediate and high-risk disease (International Prognostic Index [IPI] 3–5). POLARIX ( NCT03274492 ) evaluated a modified regimen substituting polatuzumab vedotin for vincristine (pola-R-CHP), demonstrating modest improvement in progression-free survival (PFS) with no overall survival (OS) benefit between treatment arms. The addition of lenalidomide (LEN) to R-CHOP improved both PFS and OS in the Eastern Cooperative Oncology Group (ECOG)-ACRIN E1412 trial but not in the Phase III ROBUST study, in which a lower dose of LEN was used, despite a positive trend toward 2-year PFS in a subgroup analysis.

The chemo-free immunotherapy tafasitamab + LEN, has received accelerated approval in the United States (2020) and conditional marketing authorization in Europe and Canada (2021) in adult patients with R/R DLBCL ineligible for ASCT based on the pivotal L-MIND trial ( NCT02399085 ). The primary analysis of First-MIND ( NCT04134936 ), a Phase Ib randomized safety study of R-CHOP + tafasitamab ± LEN in patients with previously untreated and newly diagnosed DLBCL, demonstrated that adding tafasitamab + LEN does not impair dosing and scheduling of R-CHOP, with toxicities similar to those expected with R-CHOP alone (ASH 2021; #3556).

Aims: frontMIND ( NCT04824092 ) will investigate the efficacy and safety of R-CHOP + tafasitamab + LEN vs R-CHOP alone in previously untreated patients with high-intermediate and high-risk DLBCL.

Methods: frontMIND is a Phase III, multicenter, randomized, double-blind, placebo-controlled study (Figure). Approximately 880 patients are planned to be randomized at approximately 350 centers in North and South America, Europe, and Asia-Pacific. Eligible patients aged 18‒80 years with previously untreated local biopsy-proven, CD20-positive DLBCL with IPI score 3–5 (age-adjusted IPI 2–3 if ≤60 years) and ECOG performance score 0–2 will be enrolled. Patients with transformed lymphoma (except double or triple hit lymphoma) are excluded. Patients will be randomized 1:1 to receive six 21-day (D) cycles of either R-CHOP + tafasitamab (12 mg/kg intravenously, D 1, 8, and 15) + LEN (25 mg orally, D1–10) or R-CHOP plus tafasitamab + LEN placebos. Patients will be followed for up to 5 years after the end of treatment. The primary endpoint is investigator-assessed PFS. Secondary endpoints include investigator-assessed event-free survival, OS, safety, and tafasitamab serum concentration (trough and Cmax levels). Sensitivity and specificity of minimal residual disease for early detection of disease progression is an exploratory endpoint; further minimal residual disease parameters may also be investigated.

Results: Results for this study are not yet available: outcome data as described above will be presented in due course.

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Summary/Conclusion: There remains a high unmet need to improve treatment options for newly diagnosed patients, especially with high-intermediate and high-risk DLBCL (IPI 3–5). The combination of tafasitamab, LEN and R-CHOP may have synergistic potential. Preliminary data from the First-MIND study suggest that tafasitamab ± LEN + R-CHOP may be tolerable in patients with treatment-naïve DLBCL. The Phase III frontMIND study will provide further evaluation of clinical benefits and safety in patients with newly diagnosed high-intermediate and high-risk DLBCL.

PB2114: TREATMENT RESULTS IN PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA FROM HIGH-RISK GROUP

K. Filonenko 1,* , I. Kriachok 2 , A. Martynchyk 2 , I. Titorenko 2 , Y. Stepanishyna 3 , O. Aleksyk 4 , K. Ukianchenko 3 , I. Pastushenko 3 , Y. Kushchevyy 3 , O. Novosad 3 , O. Karnabeda 5 , I. Diagil 6 , V. Kozlov 7

1 Clinic of Hematology and Transplantology, University Clinical Centre, Gdańsk, Poland; 2 Department of chemotherapy of haemoblastoses and adjuvant treatment methods; 3 Oncohematology department with the sector of adjuvant treatment methods; 4 Chemotherapy of haemoblastoses and adjuvant treatment methods, National Cancer Institute; 5 Hematology department, Clinic “Innovation”; 6 Department of Radiation Hematology, National Scientific Center of Radiation Medicine, Kyiv; 7 Hematology department, Odessa Regional Clinical Hospital, Odessa, Ukraine

Background: The “real-life” treatment approaches in the “difficult cases” vary depending on еру financial support of health-care system and local preferences in different countries. Such a kind of case is the patient with high risk DLBCL, in whom the standard treatment still does not allow to achieve the results of highest efficiency.

Aims: Aim of our study was to compare efficacy and toxicity of different treatment regimens in patients with DLBCL from high-risk group.

Methods: Prospective cohort study was conducted in four Ukrainian centers in 2014-2017. Patients with newly diagnosed DLBCL and ≥ 3 risk factors according to International Prognostic Index were treated according to “investigators decision” with 6-8 cycles of CHOP-like (1st group), R-CHOP (2nd group) or R-DA-EPOCH regimens (3rd group). Hear we present the results from the updated analysis of the study.

Results: Totally 140 patients were included into analysis, 68 males (48.6 %), 72 females (51.4 %), in the age 23-86 years old, median age 63 years (95 % CІ 60;65). Observation period was 1-82 months, median – 56 months. Patients treated with CHOP-like regimens were included into the 1st group (53 patients, 37.9%), treated with R-CHOP – into the 2nd group (60 patients, 42.9%) and 27 patients (19.2%) treated with R-DA-EPOCH – into the 3rd group. Significant difference between the groups was observed by the age (younger patients in the 3rd group, p=0.042) and stages distribution (early stages were more common in the 1st group, p=0.05).

Overall response rate was 61.5% in the 1st group, 52.5% in the 2nd group and 83.3 % in the 3rd group (p=0.01). Complete response rate was 17.3%, 42.5% and 83.3%, respectively (p<0,001). 3-year event-free survival (EFS) was 41,3±7.3% in the 1st group, 49.9±7.2% in the 2nd group and 83,9±7.4% in the 3rd group (р=0,005). 3-year overall survival (OS) was 55.4±7.7%, 55.0±7.2% and 87.0±7.1%, respectively (р=0,008).

The rates of hematological complications, thrombocytopenia and hepatotoxicity were comparable in three groups. Cardiotoxicity was less common in the group treated with R-DA-EPOCH (p<0,01). Neurotoxicity was more frequent in this group (p=0.043).

Summary/Conclusion: The level of treatment response was significantly higher in the R-DA-EPOCH group. 3-year EFS was significantly higher in the R-DA-EPOCH group. Presented analysis also showed the significantly higher 3-year OS in the R-DA-EPOCH group, that was not shown in the final analysis performed in 2017. Toxicity was acceptable in all groups. We did not observe significant differences in the hematological toxicity, that may be explained by the features of local supportive care. The cardiotoxicity was less common and neurotoxicity was more frequent in the R-DA-EPOCH group. Despite the limitations of the study, we suggest the R-DA-EPOCH regimen could be considered as the most efficient treatment regimen in patients with DLBCL from high-risk group.

PB2115: TRIAL IN PROGRESS: A RANDOMIZED PHASE II STUDY OF MB-CART2019.1 COMPARED TO STANDARD OF CARE THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY DLBCL INELIGIBLE FOR AUTOLOGOUS STEM CELL TRANSPLANTATION

P. Borchmann 1,* , P. Vandenberghe 2 , A. Urbano 3 , C. Haioun 4 , F. Lemonnier 4 , L. Griškevicius 5 , S. Maury 4 , S. Holtkamp 6 , B. Friedrichs 6 , G. Zadoyan 6 , L. Hanssens 6 , C. Brillant 7 , U. Bethke 7 , M. Assenmacher 7 , I. Bürger 7 , B. Philippe 7 , T. Overstijns 6 , U. Jäger 8 , M. J. Kersten 9

1 Department of Internal Medicine I, University Hospital of Cologne, Cologne, Germany; 2 Department of Hematology, University Hospital Leuven, Leuven, Belgium; 3 Institut Clinic de Malaties Hematologiques i Oncologiques (ICMHO), Hospital Clinic de Barcelona, Barcelona, Spain; 4 AP-HP, Henri Mondor University Hopital, Créteil, France; 5 Santaros Klinikos, Vilnius University Hospital, Vilnius, Lithuania; 6 Miltenyi Biomedicine GmbH; 7 Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany; 8 Department of Hematology and Hemostaseology, University Hospital Vienna, Vienna, Austria; 9 Department of Hematology, Amsterdam University Medical Centers, Amsterdam, Netherlands

Background: A tandem CAR T-cell product (MB-CART2019.1) targeting CD20 and CD19 antigens has been developed and has shown superior efficacy as compared to each single-CAR in pre-clinical models. MB-CART2019.1 is manufactured in a 13-day process and consists of autologous CD4 and CD8 enriched T-cells, which are transduced with a lentiviral vector that encodes the CAR construct for both CD20 and CD19 single chain variable fragments. The CAR incorporates 4-1BB and CD3ζ signaling domains. The safety of MB-CART2019.1 was investigated in a Phase I dose finding study ( NCT03870945 ) in mainly elderly patients with relapsed or refractory (r/r) B-cell lymphoma. It demonstrated encouraging safety with a low incidence of neurotoxicity and cytokine release syndrome accompanied by first evidence of efficacy, which provide the rationale of this Phase II trial.

Aims: This study is a pivotal Phase II randomized, multi-center, open-label study with two arms evaluating the efficacy and safety of MB-CART2019.1 compared to standard of care (SoC) therapy in participants with r/r diffuse large B-cell lymphoma (DLBCL) who are not eligible for high-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT).

Methods: The primary endpoint of the trial is progression-free survival. Key secondary endpoints include event-free survival, best complete response rate, duration of response and overall survival. Secondary endpoints in the experimental arm include persistence of MB-CART2019.1, phenotype and immune cell compositions, and anti-MB-CART2019.1 antibodies. Safety endpoints include frequency and severity of adverse events as well as the use of tocilizumab and/or high-dose steroids. PET-CTs are performed for response assessment by an Independent Review Committee according to the Lugano 2014 criteria. A total of 168 adult patients with r/r DLBCL will be randomized in a 1:1 ratio to receive MB-CART2019.1 or SoC therapy. In the experimental arm, patients are treated with a dose of 2.5 × 10 6 CAR+ T-cells per kg body weight based on the recommended dose in the Phase I trial. MB-CART2019.1 is infused as a fresh product (day 0) after a lymphodepleting chemotherapy regimen consisting of fludarabine 30 mg/m 2 body surface area (BSA) and cyclophosphamide 300mg/m 2 BSA (each from day -5 to day -3 prior to MB-CART2019.1 infusion). The comparator treatment consists of either R-GemOx (rituximab, gemcitabine and oxaliplatin) or BR (rituximab and bendamustine) plus polatuzumab vedotin, pre-defined to a maximum of 10% of enrolled patients. Main criteria for inclusion are i) histologically proven DLBCL and associated subtypes, according to the WHO 2016 classification ii) patients with either refractory disease after first-line chemoimmunotherapy or relapsed disease within ≤ 12 months from the completion of first-line therapy, and iii) patients ineligible to receive HDC followed by ASCT due to age ≥ 65 years and/or documented organ dysfunction.

Results: Final results regarding the primary endpoint are expected by end of 2023.

Summary/Conclusion: This trial evaluates the superiority of 2 nd -line CAR T-cell treatment with MB-CART2019.1 compared to SoC in an elderly high-risk population of patients with r/r DLBCL and early relapse. It is planned to be performed in up to 50 clinical trial sites in 10 countries in Europe. The study is actively enrolling patients since August 2021. Clinical trial information: NCT04844866 .

PB2116: CLINICAL PRESENTATION AND TREATMENT OUTCOME OF CHILDREN WITH B-CELL LYMPHOMA; 3-YEAR STUDY AT CHILDREN’S WELFARE TEACHING HOSPITAL (2016-2018)

H. Ghali 1,* , M. Al-Jadiry 1 , R. Haitham 2 , A. Fadhil 2 , R. Majid 2 , S. Faraj 2 , A. Hatem 2 , S. Abdulrazzaq 2 , S. Al-Hadad 1

1 Pediatric hematology/oncology, College of Medicine, University of Baghdad / Children Welfare Teaching Hospital, Medical City; 2 Pediatric hematology/oncology, Children Welfare Teaching Hospital, Medical City, Baghdad, Iraq

Background: Lymphoma is an aggressive tumor with promising survival rate; indeed, it needs supportive care to reach this goal which is not available in developing countries that make survival much lower compared with the developed countries.

Aims: To study the clinico-pathological presentation of B-cell non-Hodgkin’s lymphoma and whether the outcome has been changed in comparison with previous studies done at the same hospital.

Methods: A descriptive analytic study on 92 children less than 14 years of age with B-cell non-Hodgkin’s lymphoma who were admitted to Oncology Unit/ Children Welfare Teaching Hospital /Medical City campus from January 2016 to December 2018. They were managed according to the modified FAB LMB 96 protocol. Duration of data collections was from January 2021 to January 2022.

Results: Fifty-two (56.5%) patients were below 5 years, male to female ratio was 2.28:1 and 72.8% had duration of symptoms less than 60 days. The abdomen represents the major primary site seen in 68 (73.9%) patients, advanced stages (III&IV) form 95%. Tissue diagnosis seen in 40.2% while cytology in 59.7% of cases, Burkitt’s lymphoma was the commonest type seen in 44 (47.8%). Management was carried out by modified FAB LMB 96 protocols; 7 patients were not eligible for outcome analysis. From 85 patients received COP-reduction phase, response was poor in 9 (10.6%) patients, 9 (10.6%) died, 57 patients assigned for group B and 19 for group C. Three-year Event Free Survival was and Overall Survival were equal (62%) and the most common cause of deaths were presumptive sepsis followed by tumor lysis syndrome. Variables that contribute to survival were LDH level and PS score before starting treatment and response to COP phase after treatment ensued.

Summary/Conclusion: Therapy related mortality remained high and EFS and OS has a trend for improvement compared with previous published papers from the same centre. Proper risk stratification and staging with Reduction of the intensity of chemotherapy to accommodate the lack of supportive care in resource-limited settings is appreciated.

PB2117: TEN-YEAR SINGLE CENTER EXPERIENCE TREATING PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA: CAN RADIOTHERAPY BE OMITTED?

I. L. Goncalves 1,* , G. Gomes 1 , L. Rodrigues 1 , P. Lupi 1 , C. Claudino 1 , F. Miranda 1 , F. Mousinho 1 , T. Mendes 1 , A. Reichert 1 , A. Jorge 1 , C. Afonso 1

1 Hematology, Centro Hospitalar de Lisboa Ocidental, EPE - Hospital de São Francisco Xavier, Lisboa, Portugal

Background: Primary central nervous system lymphoma (PCNSL) is a rare form of lymphoma confined to the central nervous system (CNS) that is characterized by an aggressive course, high rates of treatment resistance and short overall survival.

In recent decades, the treatment of PCNSL has undergone significant changes; its standard treatment consists of combined immunochemotherapy regimens that should include high-dose methotrexate (HD-MTX) and/or cytarabine, followed by consolidative autologous hematopoietic cell transplantation (AHCT). HD-MTX–based chemotherapy followed by consolidative whole brain radiotherapy (WBRT) has, historically, been the standard of care. In elderly and comorbid patients who are not candidates for AHCT, maintenance therapy with procarbazine or temozolomide is often used.

Aims: We aim to describe a single center’s ten-year experience treating PCNSL.

Methods: Retrospective study of patients with PCNSL treated in a single center from 2011 to 2021, describing the demographic, clinical features and outcomes. Data was collected from clinical records and analyzed with IBM SPSS 25.0.

Results: Twenty patients, 11 (55%) female, with a median age of 61 years (range 44-77) were identified. 65% had an Eastern Cooperative Oncology Group performance status (ECOG) of 0, 25% of 1, 5% had an ECOG of 2 and 5% had an ECOG of 4. International Extranodal Lymphoma Study Group (IELSG) score was 0-1 in 7/35%, 2-3 in 11/55% and 4-5 in 2/10%; 17/85% presented with parenchymal disease, 2/10% with leptomeningeal disease only, and 1/5% with both; only one patient had eye involvement at presentation. Frontline therapy consisted of chemotherapy only (with or without rituximab) in 14 patients while 6 had chemotherapy plus WBRT; all patients received methotrexate (MTX)-based regimens (Ferreri, MATRix, DeAngelis, R-MP and R-MPV-A). Overall response was obtained in 12/60% patients (complete response in 10/50% and partial response in 2/10%). 8/40% patients relapsed, at a median time of 4.2 months after completion of therapy; 7/87.5% relapsed with parenchymal disease and 1/12.5% with mixed parenchymal and leptomeningeal disease.

Median follow up was 12.1 months, median overall survival (OS) was 25.3 months and median progression-free survival (PFS) was 4.4 months. In univariate analysis (log rank), age, sex, ECOG, presence of deep brain lesions and IELSG score did not significantly impact OS and PFS, whereas radiotherapy use was significantly associated with improved OS (63.7 vs. 13.0 months; p-value 0.002) and PFS (36.1 vs. 3.3 months; p-value 0.016; Image ). Univariate Cox regression analysis showed significant PFS improvement with radiotherapy use (HR 0.12; 95% CI 0.02-0.92, p-value 0.042), but no statistically significant OS improvement; in our cohort, no other variables significantly impacted survival. All surviving patients who underwent WBRT have shown cognitive impairment in neuropsychological tests.

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Summary/Conclusion: Based on our analysis, age, ECOG and IELSG score did not significantly impact OS and PFS, whereas WBRT use was significantly associated with improved PFS, suggesting that the omission of WBRT may not be associated with improved survival, although its use should be weighed against potentially limiting neurotoxicity.

PB2118: PHASE 3 RANDOMIZED STUDY OF LONCASTUXIMAB TESIRINE IN COMBINATION WITH RITUXIMAB (LONCA-R) VERSUS IMMUNOCHEMOTHERAPY IN PATIENTS WITH R/R DLBCL (LOTIS-5)

M. Hamadani 1,* , Y. Linhares 2 , M. D. Gandhi 3 , M. Chung 4 , H. Adamis 5 , D. Ungar 6 , C. Carlo-Stella 7 , E. Kingsley 8 , J. Depaus 9 , S. Snauwaert 10 , M. Kwiatek 11 , J. López-Jiménez 12

1 Cancer Center - Froedtert Hospital, Medical College of Wisconsin, Milwaukee; 2 Miami Cancer Institute, Baptist Health, Miami; 3 Virginia Cancer Specialists, Gainesville; 4 The Oncology Institute of Hope and Innovation, Downey, United States of America; 5 ADC Therapeutics SA, Epalinges, Switzerland; 6 ADC Therapeutics America, Inc., Murray Hill, United States of America; 7 Humanitas Clinical and Research Center - IRCCS, Humanitas University, Milan, Italy; 8 Comprehensive Cancer Centers of Nevada, Las Vegas, United States of America; 9 Department of Hematology, Centre Hospitalier Universitaire (CHU) Université Catholique de Louvain (UCL) Namur Site Godinne, Yvoir; 10 Algemeen Ziekenhuis Sint-Jan Brugge-Oostende – Campus Sint-Jan, Brugge, Belgium; 11 Centrum Medyczne Pratia Poznań, Skorzewo, Poland; 12 Hospital Universitario Ramón y Cajal, Madrid, Spain

Background: Patients (pts) with refractory or relapsed (R/R) diffuse large B-cell lymphoma (DLBCL) have poor outcomes with standard treatment. Loncastuximab tesirine (loncastuximab tesirine-lpyl; Lonca), an antibody-drug conjugate (ADC) comprising a humanized anti-CD19 monoclonal antibody conjugated to a pyrrolobenzodiazepine dimer toxin, is approved in R/R DLBCL based on data from the phase 2 LOTIS-2 trial (Caimi et al. Lancet Oncol 2021). Rituximab (R) is part of standard immunochemotherapy for DLBCL, both as frontline therapy and in subsequent treatments. Preclinical evidence suggests that the addition of rituximab to anti-CD19 ADC therapy may result in prolonged tumor control (Ryan et al. Blood 2017).

Aims: LOTIS-5 aims to evaluate Lonca-R vs. standard immunochemotherapy of R + gemcitabine + oxaliplatin (R-GemOx) in pts with R/R DLBCL.

Methods: This is a phase 3 randomized open-label, two-part, two-arm multicenter study of Lonca-R in pts with R/R DLBCL ( NCT04384484 ). A review of safety data from the nonrandomized safety run-in (Part 1), comparing the safety of Lonca-R to previous Lonca safety data, was completed in January 2022. The trial is now continuing to the randomized phase (Part 2). In Part 2, approximately 330 pts will be randomized 1:1 to receive Lonca-R or R-GemOx. The primary objective of the study is to evaluate the efficacy of Lonca-R versus R-GemOx. The primary endpoint is progression-free survival by independent central review (ICR).

Secondary endpoints include overall survival, overall response rate (by ICR using 2014 Lugano classification), complete response rate by ICR, duration of response by ICR, frequency and severity of adverse events, changes from baseline in safety laboratory and clinical variables, concentration and pharmacokinetic parameters of Lonca (conjugated and total antibody and unconjugated warhead), immunogenicity, and changes in patient-reported outcomes. The time to event endpoints will be analyzed based on the intent-to-treat population using a stratified log-rank test. The dosing regimen for Lonca-R: Lonca at 0.15 mg/kg + rituximab at 375 mg/m 2 every 3 weeks (Q3W) for 2 cycles and then Lonca at 0.075 mg/kg + rituximab at 375 mg/m 2 Q3W for up to 6 cycles. The dose regimen of R-GemOx: rituximab at 375 mg/m 2 , gemcitabine at 1000 mg/m 2 , and oxaliplatin at 100 mg/m 2 every 2 weeks for up to 8 cycles.

Key inclusion criteria include age ≥18 years, pathologic diagnosis of DLBCL (including pts with DLBCL transformed from indolent lymphoma) or high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, ≥1 line of prior systemic therapy, not being a candidate for stem cell transplantation, and measurable disease per the 2014 Lugano classification.

Results: The randomized part of LOTIS-5 commenced in January 2022, and recruitment is ongoing.

Summary/Conclusion: N/A

PB2119: LONG-TERM SURVIVAL PROJECTIONS OF LONCASTUXIMAB TESIRINE-TREATED PATIENTS IN RELAPSED OR REFRACTORY (R/R) DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL)

M. Hamadani 1,* , C. N. Graham 2 , L. Liao 3 , K. H. Zhang 2 , H. Strat 2 , D. Ungar 4 , W. Ai 5 , L. Chen 4 , C. Carlo-Stella 6

1 Medical College of Wisconsin, Milwaukee; 2 RTI International, Research Triangle Park; 3 ADC Therapeutics America, Inc; 4 ADC Therapeutics America, Inc., Murray Hill; 5 Division of Hem/Onc, University of California, San Francisco, United States of America; 6 Humanitas Clinical and Research Center – IRCCS, and Humanitas University, Milan, Italy

Background: Loncastuximab tesirine (loncastuximab tesirine-lpyl; Lonca) is an FDA approved CD19-directed antibody-drug conjugate for R/R DLBCL. From the LOTIS-2 trial primary data cut (April 6, 2020), overall response rate was 48.3% and median overall survival (OS) was 9.9 months. The OS Kaplan-Meier (KM) plot displayed a survival plateau suggesting the presence of long-term survivors (LTS).

Aims: Survival analyses were conducted on a more mature data cut (March 1, 2021; median follow-up = 1.7 years, follow-up completeness at median = 81%) to estimate the percentage of LTS and expected lifetime survival (mean OS) for Lonca-treated patients.

Methods: Consistent with studies of other R/R DLBCL treatments, identified through a literature review, parametric and mixture cure models were fit utilizing multiple distributions. Flexible cubic spline (hazard scale 1-3 knots) and non-mixture cure analyses were also conducted. Age- and gender-matched United States life table hazards were used in projections for LTS and to ensure modeled hazards were not less than the general population. Best-fit models were determined through fit statistics, KM and fitted curve overlays, and clinical plausibility. The best-fit model from each method was a candidate for overall best fit. A hybrid model following the best-fit parametric/spline model to a defined time point and switching to general population mortality was also constructed.

Results: Mixture and non-mixture cure models fit best (individual best fits gamma and Weibull, respectively). Parametric and spline models (individual best fits log-normal and 2 knot models, respectively) did not fit the observed data well nor fit the clinical expectation of long-term survival. Due to better fit, spline models were used in the hybrid model. LTS from the mixture cure and non-mixture cure models were 24-26%. Mixture cure, non-mixture cure, and hybrid model with a 2-year switch point were consistent in survival predictions (6.11 to 6.23 years). In a sensitivity analysis with 3-year switch point in the hybrid model, the estimated survival was shorter (4.96 years) due to the switch point being below the observed survival plateau. Table 1 presents full survival results and fit statistics.

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Summary/Conclusion: The observed survival plateau suggests Lonca-treated patients may include LTS. Mixture cure, non-mixture cure, and hybrid models fit the trial data well and align on survival projections (6.11-6.23 years). Additional survival follow-up may assist in refining the switch point of the hybrid model and confirm presence of LTS.

PB2120: HIV AND MALIGNANT HEMOPATHIES

H. Khibri 1,* , M. Maamar 1 , M. El Amani 2 , N. Jait 1 , M. Bouaouad 2 , S. Haidouri 2 , N. Benlachgar 2 , N. Mouatassim 1 , W. Ammouri 1 , H. Harmouche 1 , M. Adnaoui 1 , Z. Tazi Mezalek 2

1 Internal Medicine; 2 Clinical Hematology, Ibn Sina Hospital, Rabat, Morocco, Rabat, Morocco

Background: Hematological malignancies remain a major cause of morbidity and mortality in patients living with the HIV virus (PLHIV). Their training would be linked to several factors.

Aims: The objective of our study is to describe the main types of blood diseases malignancies and their epidemiological characteristics in PLHIV followed up internal medicine department – ​​clinical hematology of the CHU Ibn Sina in Rabat.

Methods: This is a retrospective and descriptive observational study of PLHIV having an associated hematological malignancy.

Results: Among the 1035 PLHIV followed, we collected 11 patients with blood disease malignant (1.06%). The average age was 40.6 years (29 – 73 years). The F/M sex ratio was 0.1. 90% of patients were in stage C. Heterosexual transmission was the

main mode of contamination (100%). They had as

antecedent: chronic smoking 60%; pulmonary tuberculosis 30%; drug addiction 10%. The regimens of ARVs received corresponded to international recommendations: 90% received triple therapy of the TNF+FTC+EFZ type and 10% received DTG+TNF+3TC. HIV infection was discovered incidentally in 73% of PLHIV during the

pre-therapeutic assessment of the management of hemopathy. their viral status revealed an average viral load of 6.2 log (4.3 – 6.5 log). the mean CD4 count was estimated at 148 (78 – 243 cells/mm3). 27% of patients had an average follow-up time of

4 months. they had an undetectable viral load and persistently low CD4 count (< 200 /mm3). We identified 9 cases (82%) of NHL:

2 DLBCL (22%), 2 plasmablastic lymphomas (22%), 3 Burkitt’s lymphomas (34%), 1 T-cell lymphoma angioimmunoblastic (11%), 1 T/NK lymphoma (11%), 1 case of classic HL at

mixed cellularity (9%), and 1 case of multiple myeloma (9%).

The other syndromes lymphoproliferative or myeloproliferative were not found in our series. The advanced Ann Arbor stage (III – IV) was found in 90% of PLHIV. All

patients received first-line poly-chemotherapy: 40% of patients

received an R-DAEPOCH, 20% CHOEP, 20% R-CHOP, 10% BEACOPP, 10% CTD. 10% of patients required a second line of therapy (R-DHAOX) then a third line (Ciclosporine). The evolution was favorable with remission complete in 30% of patients. A negative viral load was obtained in

80% of PLHIV. We mourn 6 deaths: 5 deaths were related to hematological malignancy, and 1 was related to opportunistic infections complicating HIV after poor adherence to ARVs.

Summary/Conclusion: Our series is mainly distinguished by a low frequency of the HIV association – hematological malignancy, but also by the diversity of these hemopathies. The persistence of a low CD4 count in PLHIV on ARVs should encourage look for possible hematological malignancies. Early introduction of ARVs is mandatory before starting any chemotherapy. The prognosis mainly depends type and stage of hemopathies.

PB2121: EFFECT OF EDUCATIONAL ACTIVITY ON THE KNOWLEDGE OF CLINICIANS REGARDING THE LATEST CLINICAL TRIAL DATA INVESTIGATING NOVEL EPIGENETIC AGENTS FOR B- AND T-CELL LYMPHOMAS

V. Harvey-Jones 1,* , S. Koekkoek 1 , N. Dorkhom 1 , S. Horwitz 2

1 Medscape, Global Education, London, United Kingdom; 2 Member at Memorial Sloan Kettering Cancer Center; Attending Physician at Memorial Hospital; Professor of Medicine, Weill Cornell Medical College, New York, United States of America

Background: While immunotherapeutic approaches are now offering new treatment options for many patients with certain lymphomas, advances in the understanding of key disease pathways have the potential to bring much-needed novel therapies to patients. Several early trials are showing promise with novel epigenetic agents for B-cell and T-cell lymphomas. Thus, the increase in clinical data available, and the variety of new epigenetic therapies in development, requires physician education as to their relevance as potential therapeutic approaches to lymphoma treatment.

Aims: This study aimed to determine whether online continuing medical education could improve the knowledge of hematologists/oncologists (hem/oncs) in the latest clinical developments with novel epigenetic agents for patients with B- and T-cell lymphomas.

Methods: This educational activity consisted of a 30-minute expert commentary with synchronized slides summarizing the presentations from a Medscape Live Virtual Townhall in June 2021. Educational effect was assessed using a repeated-pair design with pre-/post-assessment. 3 multiple choice questions assessed knowledge, and 1 question rated on a Likert-type scale assessed confidence. A paired samples t-test was conducted for significance testing on overall average number of correct responses and for confidence rating, and a McNemar’s test was conducted at the question or learning objective level (5% significance level, P <.05). Cohen’s d with correction for paired samples estimated the effect size of the education on number of correct responses (<.20 modest, .20-.49 small, .59-.79 moderate, ≥.80 large). Data were collected from 25/6/21 to 9/12/21.

Results: Overall significant improvements in hem/onc knowledge were seen after participation in the education (average correct response rate: 39% pre-assessment vs 50% post-assessment; P<.001, Cohen’s d = 0.41, N=73). Around 16% of hem/oncs improved and 64% reinforced (confirmed) their knowledge regarding the most recent data with epigenetic agents in patients with B-cell lymphomas, with 81% knowing this information post-education (P=0.251). While there were significant improvements in knowledge regarding the latest data with epigenetic agents for T-cell lymphomas, with 29% improving and 25% reinforcing knowledge (P<0.001), 65% still did not know this information after education. In addition, as a result of the education, 37% of hem/oncs had a measurable improvement in their confidence regarding integrating epigenetic-based therapy into clinical practice, resulting in 16% of oncologists being mostly or very confident after the education.

Summary/Conclusion: This study analysis demonstrated that an online education consisting of a 30-minute expert commentary with synchronized slides summarizing the presentations from a Medscape Live Virtual Townhall, effectively improved the knowledge and confidence of hem/oncs regarding the latest clinical trial data investigating novel epigenetic agents for B- and T-cell lymphomas. However, gaps in knowledge do remain, particularly for T-cell lymphomas, which warrants further education to help address these needs and continue supporting the improvement of patient outcomes with these hematological malignancies.

PB2122: THE EFFICACY OF THE STANDARD AND INTENSIVE CHEMOTHERAPY IN ELDERLY PATIENTS WITH DOUBLE- AND TRIPLE-EXPRESSOR LYMPHOMA.

M. Fujita 1,* , H. Itamura 1 , M. miyazaki 1 , K. Ishii 1 , A. Sugihara 1 , H. Sano 1 , S. Okamoto 1 , M. yoshimura 1 , H. Katsuya 1 , H. Ureshino 1 , T. Ando 1 , S. Kimura 1

1 Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga University, Saga, Japan

Background: Overexpression of c-myc and bcl-2 and/or bcl-6 in diffuse large B-cell lymphomas, defined as double-expressor lymphoma (DEL) and triple-expressor lymphoma (TEL), are considered to have a poor prognosis. These patients using intensified chemotherapy have been tried to achieve therapeutic efficacy and prolong their prognosis. Although several clinical data have been reported, the requirement of intensive chemotherapy, including dose-adjusted R-EPOCH (DA-R-EPOCH), for DEL and TEL is still controversial. Primarily, there seems to be insufficient analysis in elderly patients over 70 years old, which is the preferred age for malignant lymphoma.

Aims: To reveal the efficacy of dose-intensifying regimens in managing DEL and TEL, we analyzed data comparing the outcomes of R-CHOP and DA-R-EPOCH as frontline therapy.

Methods: We retrospectively analyzed the 37 of DEL and TEL patients diagnosed from January 2017 to December 2021 using R-CHOP versus more intensified treatment, DA-R-EPOCH. Formalin-fixed and paraffin-embedded tissues were constructed for immunohistochemistry (IHC). Statistical analyses were performed using the Kaplan-Meier method and log-rank test. Univariate and multivariate analyses were conducted via the Cox proportional hazards model, and it took p values lower than 0.05 to be significant.

Results: The median age in the patients with DEL and TEL was 71.3 (range, 56-88). Moreover, the patients aged 70 years old over were 56 %. Twelve patients were diagnosed in DEL, and twenty-five patients were TEL, respectively. In DEL and TEL patients, twenty-one patients (60%) are received R-CHOP, and 14 patients (40%) were treated DA-R-EPOCH, respectively. The patients in Stage III-IV were 73%. GCB types were constituted 18 cases (48.6%). Fifteen patients (40.5%) diagnosed with DEL and TEL were received central nervous system (CNS) prophylaxis.

The progression-free survival (PFS) was equivalent when comparing the TEL and DEL patients, respectively. Two-years PFS with R-CHOP vs DA-R-EPOCH for the overall population were 52.6% (95% confidence interval [CI], 21.7-76.2) vs 49.6% (95% CI, 14.8-77.3), respectively. The overall response rates and complete response rates for R-CHOP were 93% and 46%, and for DA-R-EPOCH were 90% and 60%, respectively (Figure A). Especially, over 70 years old patients with DA-R-EPOCH showed poor prognosis than DA-R-EPOCH in younger patients, although the PFS with R-CHOP has no difference between aged patients or not (Figure B). Furthermore, the patients with the drug discontinuation or dose reduction in DA-R-EPOCH treatment were tended to indicate poor PFS outcomes than the rest of the patients (Figure C).

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Summary/Conclusion: Our finding is consistent with the fact that R-CHOP and R-EPOCH showed a similar prognosis in patients with DEL, and the association of aged TEL with intensive therapy confers earlier relapses of this variant DLBCL, requiring more appropriate treatment. It might be considered about the therapeutic approach for DEL and aged TEL patients whether required the intensive therapies or not.

PB2123: MANAGEMENT OF PATIENTS WITH CONCOMITANT LIVER CIRRHOSIS AND AGGRESSIVE LYMPHOMA – A LITERATURE REVIEW

J. Jelicic 1,* , T. Stauffer Larsen 2 , A. Dam Fialla 3 , B. Andjelic 4

1 Vejle Hospital, Vejle, Denmark, Department of Hematology, Vejle; 2 Department of Hematology; 3 Department of Gastroenterology, Odense University Hospital, Odense, Denmark; 4 Department of Haematology, Blackpool Victoria Hospital, Lancashire Haematology Centre, Blackpool, United Kingdom

Background: Lymphoma and liver cirrhosis represent significant public health issues. The rising incidence of both diseases increases the risk of having cirrhosis as a comorbidity in a significant number of lymphoma patients. However, the management of such patients is challenging due to the lack of clear recommendations.

Aims: This study aimed to summarize papers that analyzed patients with liver cirrhosis who were diagnosed with aggressive lymphoma.

Methods: We performed a comprehensive literature search in PubMed and Embase among 1631 papers published between 2000 and January 2022. We used the following search terms: liver, cirrhosis, and lymphoma. Only articles that reported patients with liver cirrhosis as comorbidity and lymphoma with enough data on each patient were included in the study.

Results: The final list consisted of 84 papers (case reports/series, abstracts, short communications). A total of 134 patients with liver cirrhosis and aggressive lymphoma were found. There were inconsistencies regarding available data across the studies. The median age of patients was 65 years (range 32-85 years) with a predomination of males (72, 65.5%).

The most frequent cause of liver cirrhosis was hepatitis C infection diagnosed in 92 patients (68.6%), followed by hepatitis B infection (19 patients, 14.1%) and alcohol disease (12 patients, 8.9%), while other causes were less frequent. Decompensated liver cirrhosis (Child-Pugh B and C, ascites, jaundice, bleeding varices) was diagnosed in 35/76 patients (46.1%). Ann Arbor’s lymphoma stage was as follows: stage I/II had 34/75 patients (45.3%), III/IV had 41/75 (54.7%). B symptoms were present in 37/63 patients (58.7%). Standard immunochemotherapy (R)-CHOP/CHOP-like (Rituximab, cyclophosphamide, doxorubicin, oncovine, prednisone) was given to 58 patients (43.2%). Treatment modification and reduction (including palliative and less intensive treatment) were performed in 41/82 patients (50.0%), primarily due to liver disease (19/35 patients, 54.3%), while comorbidities and poor performance status were the cause of reduction in 9 patients (25.7%). Other causes were registered in 7 patients (20.1%). Side effects following treatment were recorded in 75 patients, including liver toxicity in 36 (48.0%) patients.

Patients with decompensated liver cirrhosis received dose-reduced therapy more often than patients with compensated liver cirrhosis (72.7% vs. 27.3%, χ 2 =6.942, p=0.008, n=41). Complete and partial remission (CR/PR) was recorded in 48/64 patients (75.0%). Of 126 with known vital status, 63 were alive (50.0%). More deaths occurred among patients who received treatment reduction/modification than standard treatment (55.6% vs. 18.8%, χ 2 =9.72, p=0.002, n=68).

The median survival was 25 months. Survival of patients with decompensated liver cirrhosis was significantly lower compared to patients without decompensation (5 months vs. median not reached, Log Rank=4.45, p=0.035). Patients who received reduced treatment had inferior survival (median 5 months) compared to patients treated with standard treatments (median not reached, Log Rank=7.150, p=0.007). Patients who did not achieve CR/PR had poorer survival than patients who achieved favorable treatment response (median 5 months vs. median not reached, Log Rank=42.69, p<0.0001).

Summary/Conclusion: Due to liver disease, lymphoma patients tend to receive reduced/modified treatment regimes and have poorer outcomes. Management of patients with aggressive lymphoma and cirrhosis is challenging and requires a multidisciplinary approach.

PB2124: MYC EXPRESSION ANALYSIS AND PROGNOSIS VALUE IN TRANSFORMED FOLLICULAR LYMPHOMA

J. Liang 1,* , K.-X. Du 1 , W.-T. Wang 1 , J.-Y. Li 1 , W. Xu 1

1 the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China

Background: Follicular lymphoma (FL) is the most common indolent lymphoma in the Western World, accounting for approximately 20-25% of all non-Hodgkin lymphomas. A subset of patients happened to high-grade transformation (HT) to the more aggressive diffuse large B cell lymphoma (DLBCL), associated with inferior clinical outcomes. MYC is an oncogene that occupies an important position in proliferation, apoptosis, and regulation of microRNA. Many human neoplasms show unscheduled MYC expression and it is important in pathogenesis of some lymphomas. Several RNA expression studies have identified MYC up-regulation as an important pathway in transformation of FL.

Aims: This study aimed to investigate and quantify MYC expression in a series of FL with subsequent transformation. Furthermore, we tried to identify the role of MYC expression in transformed follicular lymphoma (tFL) and its prognosis value.

Methods: We retrospectively analyzed a cohort of 414 patients newly diagnosed with FL treated at the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital) from June 2009 to April 2021. In addition, we performed RNA sequencing on 6 paired biopsies from 3 patients with FL with subsequent transformation to double-hit lymphoma (DHL). We combined our sequencing data with GSE142334 . The dataset GSE142334 and GSE16131 were downloaded from Gene Expression Omnibus database (GEO). GSE142334 was gene expression profiling in paired tFL. GSE16131 were analyzed by survival data using the Kaplan–Meier method.

Results: 414 FL patients were retrospectively analyzed in our study. Among them, 48 cases were stained for MYC in immunohistochemistry with 16 tFL. These 48 patients were divided into 4 groups, including MYC+ with transformation, MYC+ without transformation, MYC- with transformation and MYC- without transformation. The prevalence of MYC+ with transformation was 27.08%, found significant difference between MYC expression and transformation applied by chi-square test (Figure 1 A , P=0.002). The 4 groups were analyzed by the Kaplan–Meier method for the survival curves, which MYC+ with transformation associated with worse PFS (Figure 1 B , P=0.001). The MYC expression was significantly higher in tFL than in the corresponding FL (Figure 1 C ). It is worth noting that three cases showed lower MYC expression in the tFL sample, suggesting that MYC independent mechanisms are involved in transformation. From there, we took further analysis on survival data with GSE16131 . MYC high expression contributed to adverse outcomes for OS (Figure 1 D , P = 0.031).

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Summary/Conclusion: MYC expression was uniformly high in tFL but much more heterogeneous and MYC high expression was associated with adverse outcomes in FL.

PB2125: LYMPHOMATOID PAPULOSIS-LIKE (LYP-LIKE) AFTER BRENTUXIMAB VEDOTIN-CHP (BV-CHP) TREATMENT FOR ALK-NEGATIVE ANAPLASTIC LARGE CELL LYMPHOMA (ALCL)

P. Lopez Pereira 1,* , O. Servitje 2 , F. Climent 3 , E. Domingo-Domenech 1 , A. Oliveira 1 , C. Muniesa 2 , A. Sureda 1 , E. González-Barca 1

1 Hematology, Institut Catalá d’Oncologia, Hospital Duran i Reynals; 2 Dermatology; 3 Pathology, Hospital Universitario Bellvitge, Hospitalet de Llobregat, Spain

Background: Lymphomatoid papulosis is an indolent form of CD30 primary cutaneous lymphoproliferative disorder characterized for recurrent crops of self-healing papular nodular lesions. Histological findings may overlap with those in primary cutaneous ALCL so clinical features and evolution determine the appropriate therapeutic approach. Most cases of LyP don’t require any treatment, but PUVA or low-dose oral methotrexate can reduce skin lesions. LyP-like is uncommon after a systemic ALCL. BV-CHP has become the new standard front-line treatment for ALCL in the last years; some aspects of the biological and clinical behavior of the disease with this new therapy are still under research.

Aims: NA

Methods: NA

Results: Patient 1: 45 years female, diagnosed in January/20 with ALK-negative ALCL stage IV-A, IPI 3, involving multiple supra- and infradiaphragmatic lymphadenopathies, spleen, skin, bone and bone marrow. The diagnosis was performed by skin biopsy of an extensive ulcerated mass in the right chest area. FISH showed no rearrangements of DUSP22 and TP63. BV-CHP was started in January/20, achieving PR by PET-CT after 3 cycles. She completed 6 cycles. Four weeks after the last dose of chemotherapy she developed several papular skin lesions in the trunk and extremities. The skin biopsy was reported as ALCL. PET-CT showed metabolic CR at that time. After 3 weeks, the skin lesions spontaneously disappeared and she underwent ASCT conditioned with BEAM in July/20. Two months later, she developed new skin lesions and a new biopsy showed nonspecific findings: perivascular lymphocytic dermatitis with epidermal spongiotic changes. PET-CT showed two enlarged axillary lymphadenopathies with increased activity. A watch and wait strategy was adopted, until September/21 when she relapsed involving multiple lymphadenopathies, bone, spleen and new papulo-necrotic skin lesions. Skin biopsy showed a CD30+ lymphoprolipherative disorder. She received 3 cycles of ESHAP achieving PR, and an haploidentical stem cell transplantation was performed in January/22. Two months after transplant, the patient has no signs of disease and no skin lesions.

Patient 2: 62 years male, diagnosed in August/21with systemic ALK-negative ALCL stage IV-B, IPI 3, with supra- and infradiaphragmatic lymphadenopathies and extensive colonic involvement. DUSP22-IRF4 and TP63 rearrangements were assessed, showing no abnormalities. Bone marrow and skin were not affected. He started front-line treatment with BV-CHP in September/21, with metabolic CR in the interim PET-CT after 4 cycles. He completed 6 cycles with good tolerance until late December/21. One month later, he developed papular and papulo-necrotic skin lesions in different evolutionary stages, affecting trunk, extremities and scalp. Skin biopsy was performed, showing a CD30+ lymphoprolipherative disorder. Another PET-CT demonstrated systemic CR. Clinicopathological findings were consistent with lymphomatoid papulosis. The patient underwent an ASCT as consolidation treatment. Currently, the skin lesions have involuted spontaneously.

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Summary/Conclusion: These two similar cases illustrate an unusual scenario after systemic ALK-negative ALCL, both developed within weeks after frontline chemotherapy with BV-CHP. Diagnosis and therapeutic decisions are challenging. Further experience with this new regimen will reveal whether the addition of BV may play a role in the development of this condition, and whether the clinical course is the same as primary cases of LyP or precedes a relapse of systemic lymphoma.

PB2126: R-CPOP AS FIRST LINE TREATMENT FOR DLBCL PATIENTS WITH SIGNIFICANT REDUCED LVEF OR HIGH RISK OF ANTHRACYCLINE INDUCED CARDIOTOXICITY

K. Lorenz 1,* , T. Strüßmann 1 , M. Trepel 2 , G. Illerhaus 3 , H. Pelz 4 , C. Scholz 5 , J. Duyster 1 , R. Marks 1

1 Department of Hematology and Oncology, Universitätsklinikum Freiburg, Freiburg; 2 Department of Hematology and Oncology, Universitätsklinikum Augsburg, Augsburg; 3 Department of Hematology, Oncology, and Palliative Medicine, Klinikum Stuttgart, Stuttgart; 4 Onkologie Offenburg, Offenburg; 5 Department of Hematology and Oncology, Vivantes Klinikum Am Urban, Berlin, Germany

Background: R-CHOP chemotherapy remains the standard of care in treatment of fit patients without comorbidities suffering from diffuse large B-cell lymphoma (DLBCL), even in elderly patients up to 80 years. Anthracyclines are essential in treatment however they are as well as liposomal anthracyclines contraindicated for patients with impaired cardiac function. No standard treatment for cardiac frail patients exists. Pixantrone is an aza-anthracenedione showing reduced cardiotoxicity in vitro compared to standard anthracyclines. Data by Herbrecht et al. (Ann Oncol, 2013, 24:2618) comparing R-CHOP as first line treatment for DLBCL with R-CPOP (substituting doxorubicin with pixantrone, 88 mg/m 2 ) resulted in similar PFS with reduced grade 3 CHF rates.

Aims: We aimed to investigate efficacy and safety of first line R-CPOP in DLBCL patients with significant cardiac morbidity or high risk of anthracycline induced cardiotoxicity.

Methods: This trial was a mixed prospective and retrospective analysis. The prospective non-randomized, multicenter phase 2 trial recruited patients in 5 investigational sites in Germany. Patients were aged >18 years with untreated DLBCL and impaired cardiac function with a left ventricular ejection fraction (LVEF) >40% and ≤ 50%. In addition, we conducted a retrospective analysis from patients with untreated DLBCL and significant reduction in LVEF or high risk of anthracycline induced cardiotoxicity who were not eligible for the prospective trial. Patients received up to six 21-day cycles of R-CPOP (rituximab 375 mg/m 2 i.v. day 0 or day 1, cyclophosphamide 750 mg/m 2 i.v. day 1, pixantrone 88 mg/m 2 i.v. day 1 (= 150 mg/m 2 pixantrone dimaleate), vincristine 1.4 mg/m 2 (maximum dose 2 mg) i.v. day 1, prednisone 100 mg orally day 1-5) followed by additional 2 cycles of rituximab every 21 days. The primary endpoint of the prospective trial was the rate of CR after induction therapy as assessed by PET-CT at the end of treatment. Secondary endpoints were OS and PFS.

Results: A total of 28 patients were treated between 2016 and 2021 and included in our analysis (10 patients in the prospective multicenter trial, 18 patients were retrospectively analyzed from our center). Median age was 76 years (range 51-85) with 75% male patients. 71% had an advanced Ann Arbor stage (III/IV), 71% had an IPI ≥3. The median LVEF was 45% (range 25-63%), 79% patients had a LVEF ≤ 50%. Patients with LVEF >50% had echocardiographic signs of cardiac morbidity like severe heart valve disease, history of significant cardiac impairment or prior treatment with anthracyclines. The median number of R-CPOP cycles delivered was 5 (range 1-6). Overall response rate was 82.1%, with 17 patients (60.7%) achieving CR after induction. With a median follow up of 24.7 months, estimated 2y- PFS and OS both were 71.9%. Concerning the cardiac parameter NT-ProBNP there was no significant change of the median NT-ProBNP from start of induction (685 pg/ml, range 103-1598) to end of treatment (655 pg/ml, range 96-2199) in 15 patients which received at least 4 cycles R-CPOP and achieved a CR.

Summary/Conclusion: With this mixed prospective and retrospective analysis we could show not only feasibility but also encouraging efficacy of R-CPOP in DLBCL patients with high risk of anthracycline induced cardiotoxicity. Therefore, one could envision additional clinical trials to establish R-CPOP as standard first line treatment in this patient cohort.

PB2127: NO IMPACT OF STATIN USE ON RESPONSE AND SURVIVAL RATES IN NEWLY DIAGNOSED DLBCL PATIENTS TREATED WITH R-DA-EPOCH

A. Sabljic 1 , M. Sedinic 1 , R. Kusec 1 , V. Pejsa 1 , M. Lucijanic 1,*

1 Hematology, University hospital Dubrava, Zagreb, Croatia

Background: There are controversial clinical data on potential benefits of statin use concomitantly with immunochemotherapy in the first line treatment of agressive Non Hodgkin lymphoma (NHL) patients. Majority of information is based on R-CHOP treated cohorts. Recent reports indicate that statins might improve chemosensitivity of tumor cells to doxorubicine and diffuse large B cell lymphoma (DLBCL) patients concomitantly treated with statins in addition to R-CHOP might experience improved response rates and improved survival.

Aims: We aimed to evaluate whether concomitant statin use resulted in improved outcomes in our cohort of newly diagnosed DLBCL patients with unfavorable disease features treated with R-DA-EPOCH regimen.

Methods: We retrospectively evaluated all consecutive first line DLBLC patients who presented to our institution in period 2005-2019 and who were treated with R-DA-EPOCH regimen. All patients were Caucasians and had unfavorable disease features (Ki67+ ≥80% and/or International Prognostic Index (IPI) ≥2 points). Electronic and written medical records were analyzed for statin use at the time of immunochemotherapy. Clinical and laboratory features, as well as outcome measures (response rates, progression free and overall survival) were compared between patients with and without concomitant statin use.

Results: A total of 130 newly diagnosed DLBLC patients were evaluated. Median age was 62 years IQR (48-71), there was a similar proportion of male (73/130 (56.2%)) and female patients (57/130 (43.8%)). Median R-IPI score was 3 points IQR (2-3). A total of 17/130 (13.1%) patients received statins concomitantly with immunochemotherapy (atorvastatin (11/17, 65%), rosuvastatin (4/17, 24%), simvastatin (2/17, 12%)).

There were no significant differences in baseline demographic and clinical characteristics between patients concomitantly exposed and non-exposed to statins. Also, there were no significant differences in number of cycles with R-DA-EPOCH dose escalation, reduction, anemia, thrombocytopenia, neutropenia, febrile neutropenia and septic complications. Also, there was no significant difference in proportion of patients achieving complete (53% vs 66%) or partial response (24% vs 19%) between patients exposed and non-exposed to statins. Median follow-up of our cohort was 48 months. Statin use had no significant association with neither OS (P=0.480) nor PFS (P=0.891) as shown in Figure.

To account for potential age, sex and disease biology differences between patients with and without statin exposure we evaluated statin use in a logistic regression model assessing predictors of response to therapy where only lower R-IPI score was significantly associated with achieving the response (OR 0.44 95% CI (0.21-0.94), P=0.035), whereas statin use (P=0.909), age (P=0.119) and sex (P=0.291) had no significant associations. In a similar Cox regression model assessing predictors of PFS, achieving response to therapy was statistically significant (HR 0.08 95% CI (0.03-0.21), P<0.001) and there was a tendency for higher R-IPI (HR 1.36 95% CI (0.99-1.87), P=0.058) to be associated with PFS, whereas there were no significant associations of statin use (P=0.674), age (P=0.597) and sex (P=0.151) with PFS.

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Summary/Conclusion: Our data on first line DLBLC patients with unfavorable disease features treated with R-DA-EPOCH regimen do not confirm previously reported observations of improved response rates nor improved PFS with concomitant statin use. Differences in immunochemotherapy regimens and underlying disease biology might play a role in observed differences.

PB2128: CELL OF ORIGIN ACCORDING TO HANS LOGARITHM IS AN INDEPENDENT RISK FACTOR FOR DLBCL PATIENTS TREATED WITH R-CHOP: A REAL WORLD MOROCCAN EXPERIENCE

E. M. Mahtat 1,* , I. Ait Filali 1 , M. Allaoui 2 , S. Jennane 1 , H. El Maaroufi 1 , M. Oukabli 2 , K. Doghmi 1

1 Clinical Hematology; 2 Pathology, Hôpital Militaire Mohamed V Rabat, Rabat, Morocco

Background: Diffuse large B cell lymphoma is classified into two molecular prognostic subgroups: germinal center B cell like (GCB) and activated B cell like (ABC), based on cell of origin (COO). The Hans algorithm based on immunohistochemistry (IHC) analysis helps classifying DLBCL into GCB and non-GCB subtypes in clinical practice. The non-GCB subtype has been correlated with inferior overall survival in the pre-rituximab era. The use of chemoimmunotherapy has shown variable results.

Aims: We compared retrospectively the outcomes of DLBCL patients treated with R-CHOP in our center according to the COO.

Methods: We retrospectively reviewed patients diagnosed with DLBCL and treated with R-CHOP from January 2010 to December 2020. Only patients with the COO obtained according to the Hans alborithm were include in the analysis. Demographic, prognostic and therapeutic data were collected. Overall survival (OS) and progression free survival (PFS) were estimated by the Kaplan-Meier method and compared by the log rank test. The COX model was used for the multivariate analysis.

Results: A total of 140 patients with DLBCL treated with R-CHOP were analysed. Sixty-one had their COO identified by IHC. Median age was 58 years. 72% were male. 43 (70%) had advanced stage. According to Hans algorithm, 19 (31%) GCB origin, 42 (68%) were non-GCB. The median OS and PFS time were not reached for the total population. The OS wasn’t reached for the GCB and was estimated at 42 months for the non-GCB (p=0.039).The median progression free survival time was 24 months for non-GCB and not reached for GCB (p=0.049).When adjusted for other prognostic scores (IPI, R-IPI, NCCN-IPI, CNS-IPI) the GCB sub-type was significantly associated with longer OS HR=3,7 (95% CI, 1,03 -13,3) and PFS, HR=3,7 (95% CI, 1,13 - 12,1).

Summary/Conclusion: In our local DLBCL population treated with immunochemotherapy the COO according to the Hans algorithm, despite the small number, was suprinsingly found to be a significant independent prognostic factor. This should be confirmed in future prospective studies in our region.

PB2129: COULD LYMPHOPENIA PREDICT OUTCOME OF PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA?

B. Merchan Muñoz 1,* , D. Subirá Pérez 1 , M. I. Nuevo Lopez 1 , M. Mora Argumanez 1 , R. Pérez Alonso 1 , A. Vazquez Ramo 1 , A. Gil Perez 1 , H. Guillen Garcia 1 , J. Arbeteta Juanis 1 , A. B. Santos Montero 1 , N. Golbano Lopez 1 , S. Herrero Lopez 1 , M. D. Morales Sanz 1 , D. De Miguel Llorente 1

1 Haematology, Hospital Universitario Guadalajara, Guadalajara, Spain

Background: The international prognostic index (IPI) is the standard clinical tool used to predict the outcome of patients diagnosed with diffuse large B-cell lymphoma (DLBCL). However, there is considerable heterogeneity within the IPI groups and immunochemotherapy (ICT) response. Surrogate markers of host immunity and more sensitive techniques to identify extranodal involvement could potentially be added to the IPI model to further improve it.

Aims: To investigate whether absolute lymphocyte count (ALC), and peripheral blood and bone marrow (BM) infiltration using multiparametric flow cytometry immunophenotyping (MFC) may offer valuable information to predict prognosis in DLBCL.

Methods: Eighty-eight patients diagnosed with DLBCL between 2014 and 2021 at a single centre were included in this study. Sixty percent of patients (n=53) had an intermediate-high or high IPI score-risk and 40% (n=35) showed low IPI score-risk. All ICT regimens included Rituximab. Median age of patients at diagnosis was 71 years (range, 24-90 years). After a median follow-up of 23.4 months (range, 2.8-99.5) complete remission (CR) without relapse was achieved by 66% of patients (n=58) and refractory disease or relapsed (R/R) was exhibited by 34% of patients (n=30). No differences were observed in median age, IPI or ICT administered in these two groups of patients.

ALC was registered from complete blood cell count at diagnosis and after 2 months of ICT-ending, considering lymphopenia as ALC≤1´10 9 /L in two determinations. Eight-color MFC analysed circulating lymphoma cells and BM infiltration. Chi square, Fisher’s exact test and t-test were used to compare patient’s characteristics, considering p<0.05 as statistically significant.

Results: As it could be expected, IPI score showed impact on CR (p=0.013).

Lymphopenia at diagnosis was associated with increased R/R DLBCL incidence (p=0.0001; figure 1). Besides, low ALC at diagnosis identified 9 patients within the low IPI group who did not achieve CR (p=0.001). This association was not observed in patients with high IPI score (p=0.102).

Lymphocyte subtypes were studied by MFC in 41 patients: 83% (n=34) presented low B-cell counts and 17% (n=7) presented only low T and/or NK-cell counts.

MFC detected circulating lymphoma cells in 17 out of 52 patients studied (33%). No correlation between the quality of response achieved and circulating lymphoma cells was recognized (p=0.141).

BM infiltration was noticed in 31 patients (35%) at diagnosis: 6 were only detected by histopathology and 12 were only detected by MFC, showing minimal BM involvement (median 0.3%, range 0.01-3.3%). Three out of the 12 patients only detected by MFC (25%) would change to a higher IPI score-risk.

Following the completion of first-line ICT, ALC normalization was observed in 43 patients (58%). Relapse and progression risk was significantly higher in patients with persistent low ALC (p=0.0037).

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Summary/Conclusion: 1. Lymphopenia at diagnosis is an additional, inexpensive and easy value to predict relapse or refractory disease in DLBCL with low IPI score.

2. High resolution MFC improved the detection of BM infiltration, potentially affecting IPI score. However, circulating lymphoma cells did not offer additional prognosis information.

3. Persistent lymphopenia during routine follow-up after ICT-ending helps clinicians to identify patients who are more likely to relapse.

PB2130: RATIONAL FOR METHOTREXATE USAGE FOR THE TREATMENT AND PREVENTION OF CNS INVOLVEMENT IN PATIENTS WITH HIGH-GRADE B-CELL LYMPHOMAS.

A. Misyurina 1,* , S. Kravchenko 1 , A. Magomedova 1 , A. Kovrigina 2 , T. Obukhova 3 , K. Sychevskaya 1 , F. Babaeva 1

1 Intensive high-dose chemotherapy of hemablastosis; 2 Pathology department; 3 Laboratory of cariology, National Research Center for Hematology, Moscow, Russia

Background: The role of methotrexate-containing prophylaxis (MTX-CP) still to be discussed due to high-rate of primary resistance of high-grade B-cell lymphoma (HGBL) for intensive and conventional treatment. In several cases HGBLs are represented by transformed low-grade lymphomas (LGL) (follicular or marginal zone). Despite of high-dose methotrexate (HD-MTX) has better pharmacodynamics in cerebrospinal fluid than endolumbar methotrexate given with endolumbar triplet injections (ETI), it couldn’t be prescribed in some cases due to old age and comorbidities.

Aims: To characterise patients with CNS-involvment and to estimate the role of MTX-CP.

Methods: We had data about CNS involvement relating 91 pts with HGBLs (48 pts with HGBL NOS, 22 – c-MYC/BCL2 HGBL DH, 6 - HGBL TH, 15 - c-MYC/BCL6 HGBL) which were treated in National Research Center for Hematology, Moscow, Russia in the period from 2008 till 2022 years. The majority of pts (71/86 (83%)) had IPI score 3-5. Till 2018 year we used Burkitt-like regimens similar as CODOX-M/IVAC or Hyper-CVAD in young and fit patients to treat HGBL DH or NOS. Since 2018 year it was initiated randomized prospective trail «R-Burkitt Lymphoma-Moscow-04 Versus R-(DA)-EPOCH and Autologous Stem Cells Transplantation in Patients With High-Grade B-cell Lymphoma Double-hit (HGBL DH) and High-Grade B-cell Lymphoma Not Otherwise Specified (HGBL NOS)», NCT03479918 . We suggested an integral transformation index (ITI) indicating the probability of HGBL development from LGL ( EHA-1767 ). Statistical analysis was performed in STATISTICA 12.0 software. Compassion of groups were carried out using χ2 test. Differences were considered as significant at p-value < 0.05.

Results: The majority of pts had CNS prophylaxis/treatment with HD-MTX (1,0-1,5 g/m 2 )) which was administered in 51/89 of pts (57%), 26/89 (29%) of pts had both HD-MTX and ETI; 22/89 (25%) of pts had ETI only; 18/89 (20 %) of pts had no prophylaxis, p<0,001. CNS-IPI score was high in 12/18 (67%) of pts without CNS-prophylaxis, in 31/51 of pts (61%) with HD-MTX-containing prophylaxis, in 16/22 of pts (72%) with ETI (p>0,05). CNS involvement at the time of disease onset was revealed in pts received HD-MTX-containing therapy in 10/51 of pts (20%), in 6/22 of pts with ETI (27%) (5 pts had leptomeningeal involvement and one elderly patient had foci in brain and ECOG 3). ITI score at least 1 point was revealed in 24/25 of pts (96%) with CNS involvement and 43/68 of pts (63%) without, p=0,002. CNS involvement was strongly associated with bone marrow infiltration, p<0,001.

Rate of CNS involvement due to relapse or disease progression was following – 4/51 (8%) of pts in the HD-MTX-group, 1/22 (5%) of pts – in the ETI-group, 5/17 (29%) of pts without any prophylaxis. Despite HD-MTX prophylaxis failure in 8 % of pts it decreased significally the probability of CNS relapse, p=0,02. ETI also showed benefit in treatment of leptomeningeal involvement and prophylaxis of relapse into CNS compared with absence of any prophylaxis, p=0,04.

Summary/Conclusion: CNS involvement was strongly associated with bone marrow infiltration and probability of HGBL development from low-grade lymphoma. MTX-CP showed benefit in treatment of CNS involvement in HGBLs. Cases of relapses could be explained by methotrexate-resistance due to intrinsic mechanisms of tumor development.

PB2131: CLINICAL CHARACTERISTICS AND TREATMENTS OUTCOMES OF PRIMARY GASTROINTESTINAL LYMPHOMA:RETROSPECTIVE SINGLE CENTER STUDY FROM MOROCCO

E. Nora 1,* , E. mahtat 1 , S. jennan 1 , H. elmaaroufi 1 , K. doghmi 1

1 hematology, military hospital RABAT MOROCCO, rabat, Morocco

Background: Primary gastrointestinal lymphoma represents non-Hodgkin’s lymphoma(NHL) is the most common extra-nodal lymphoma.

Aims: The aim of this work is to collect all of primary gastric lymphomas, in order to specify the characteristics epidemiological, clinical, paraclinical and therapeutic strategies.

Methods: This is a descriptive retrospective study conducted in the clinical hematology department at the RABAT military hospital over a period of 20 years (2001 – 2021), We included all patients with primary digestive NHL (LPTD) with histological evidence

Results: Our study focused on 74 cases of primary digestive NHL, an average of 3.7 cases/year, with a male predominance of 69% (sex ratio: 2.2) whose average age was 56 years with extremities of 15 at 80 years old. Performance status (PS) at diagnosis was between 0 and 1 in 64%.The functional symptomatology was dominated by abdominal pain; essentially gastric seat (86%) followed by transit disorders (28.5%) and digestive hemorrhage (27%). clinical examination found epigastric tenderness in 34% and tumor syndrome in 24%.General signs were present in 40%. The stomach was the predominant localization for LPTD (67.5%), followed by the colon (11%), the small intestine (7%) and the esophagus (3%) and finally the rectum and the liver (2.5%).).biological investigations found a microcytic hypochromic anemia in 54% of cases and a disturbance of the liver assessment in 11% of cases. The endoscopic appearance was an ulcero-budding process in 59.5% of cases, gastric infiltration (9.5%) and a large folds or polyploid mass (7%). Aggressive lymphoma was dominant in 80% of which large B-cell lymphoma presented the predominant histological form (67.5%), followed by marginal zone lymphoma (21.5%), Burkitt’s lymphoma (5.5%), mantle (7%), and 2 cases of T-cell lymphoma. The Hp status was determined in all patients, it was positive in 35% of cases. Extension assessment: it was localized forms (IE, II1E) in 50% of cases and disseminated forms (Ii2E to IV) in 47.5% of cases. 11% of patients were complicated by stenosis and/or occlusion.Therapeutically: Treatment based on immuno-chemotherapy with RCHOP (Rituximab- Cyclophosphamide- Doxorubicin- Oncovin- Prednisone) was the most used in first line (73% of cases), with an eradication treatment of Hp in 74% of cases, 10 patients required intensification by autograft, 21patients Were operated, and radiotherapy was indicated only in 2 patients for decompressive purposes. Treatment-related toxicity was mainly infectious (39%) and hematological (24%). At the end of our study, 63.5% of cases were in complete remission after 1st line treatment, 38% of patients died (75% by toxicity and 20% by progression), and 2 patients are undergoing treatment. Overall survival after median follow-up of 35 months was 124 months

Summary/Conclusion:

Primary digestive NHL is a rare lymphoma with a poor prognosis.

epidemiological and clinical characteristics of our series are similar to those described in the literature

PB2132: COPANLISIB PLUS RITUXIMAB-BENDAMUSTINE FOR RELAPSED-REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA: RECRUITMENT UPDATE ON AN ONGOING PHASE II TRIAL OF THE FONDAZIONE ITALIANA LINFOMI (FIL_COPA-RB)

M. Novo 1,* , A. Castellino 2 , A. Chiappella 3 , G. Ciccone 4 , M. Balzarotti 5 , A. Arcari 6 , E. Scarpa 7 , A. Tucci 8 , N. Di Renzo 9 , G. Tarantini 10 , G. Gini 11 , M. Moretti 12 , D. Mannina 13 , A. Di Rocco 14 , M. Spina 15 , U. Vitolo 1

1 FPO-IRCCS Candiolo Cancer Insitute, Candiolo (Torino); 2 Hematology Unit, Santa Croce e Carle Hospital, Cuneo; 3 Division of Hematology and Stem Cell Transplantation, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milano; 4 Unit of Clinical Epidemiology and CPO, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, Torino; 5 Unit of Hematology, Humanitas Clinical and Research Hospital IRCCS, Rozzano (Milano); 6 Unit of Hematology and Bone Marrow Transplantation, Guglielmo da Saliceto Hospital, Piacenza; 7 Ca Foncello Hospital, Treviso; 8 Division of Hematology, Spedali Civili, Brescia; 9 Hematology and Transplant Unit, Ospedale Vito Fazzi, Lecce; 10 Haematology and BMT Unit, Ospedale Monsignor R. Dimiccoli, Barletta; 11 Division of Hematology, Azienda Ospedaliera Universitaria Ospedali Riuniti, Ancona; 12 Università degli Studi di Perugia, Azienda Ospedaliera Santa Maria di Terni, Terni; 13 Azienda Ospedaliera Papardo-UOC di Ematologia, Messina; 14 Department of Translational and Precision Medicine, Università La Sapienza, Roma; 15 Division of Medical Oncology A, National Cancer Institute, Aviano, Italy

Background: Diffuse large B cell lymphoma (DLBCL) is curable in 50-60% of cases with frontline RCHOP. Relapsed/refractory (R/R) DLBCL can be cured with salvage treatments in roughly 40-50% of cases with standard available treatments which include high dose-chemotherapy plus autologous stem cell transplant (ASCT) in fit patients, and chimeric antigen receptor T-cells (CAR-T), permitted as third or subsequent line in patients with no major comorbidities. The outcome of R/R DLBCL unresponsive or ineligible to ASCT or CAR-T is highly unfavorable and new treatment options are needed. Rituximab plus bendamustine (RB) is active in R/R DLBCL with overall response rates (ORR) of 40-60% but generally short-lasting. Copanlisib is an intravenous pan-class I PI3K inhibitor with predominant activity against PI3K-α and -δ isoforms, active as single agent in DLBCL with ORR 25% ( NCT02391116 ). The combination of copanlisib plus RB (Copa-RB) has showed a safe toxicity profile in R/R indolent lymphomas (iNHL) ( NCT02626455 ).

Aims: To assess the safety and efficacy of Copa-RB followed by copanlisib maintenance in R/R DLBCL not eligible or relapsed after ASCT or CAR-T.

Methods: FIL Copa-RB is a multicentric open-label single arm, phase II trial. An estimated 81 patients will be enrolled, from 30 centers in Italy. Main inclusion criteria are patients aged ≥ 18 affected by DLBCL (including de-novo DLBCL, DLBCL transformed by iNHL and high-grade lymphoma) or FL grade IIIb R/R after ≤ 3 previous lines of therapy, ineligible or R/R to ASCT or CART, ECOG PS ≤ 2. Patients with primary mediastinal B cell lymphoma histology, significant organ dysfunction, uncontrolled hypertension and hyperglycemia (HbA1c> 8.5%) are excluded. Treatment consists of an induction phase with six 28-day cycles of Copa-RB (copanlisib 60 mg intravenously on day (D) 1, D8 and D15, rituximab 375 mg/m2 on D1, bendamustine 90 mg/m2 on D1-D2) followed by a maintenance phase of twelve 28-day cycles of copanlisib 60 mg given on D1 and D15. The primary endpoint is progression free survival (PFS) defined as the time between the date of enrolment and the date of disease progression, relapse or death from any cause, whichever occurs earlier, with an expected improvement of 12-month PFS from 20% to 35%. Secondary endpoints include overall survival, ORR, complete response (CR) rate, duration of response and rate of response improvement during maintenance. Exploratory analysis will include association between outcome and cell of origin, MYC, BCL2 and BCL6 overexpression and rearrangements, and mutational analysis of selected genes possibly involved in predicting response to copanlisib. The expected study duration from first patient’s first visit to last patient’s last visit is approximately 4 years.

Results: The recruitment started in November 2020; at March 1st, 27/81 patients have been enrolled. Results for this study are not yet available.

Summary/Conclusion: The treatment of R/R DLBCL ineligible or R/R to ASCT or CAR-T is still an unmet clinical need and new therapeutic strategies are warranted. Copanlisib as single agent has been already shown a moderate activity in DLBCL and the association Copa-RB plus copanlisib maintenance could be a new treatment option to improve the outcome of these patients. Biomarkers studies may further elucidate patients who are more likely to respond to this treatment.

Funding: The study is sponsored by the Fondazione Italiana Linfomi; Bayer partly supported the study and kindly provided Copanlisib; Sandoz kindly provided Rituximab.

PB2133: ASSESSING SAFETY, TOLERABILITY, AND EFFICACY OF SUBCUTANEOUS EPCORITAMAB IN NOVEL COMBINATIONS WITH ANTI-NEOPLASTIC AGENTS IN PATIENTS WITH NON-HODGKIN LYMPHOMA IN A PHASE 1B/2, OPEN-LABEL STUDY

L. H. Sehn 1,* , I. Avivi 2 , J. Brody 3 , D. H. Yoon 4 , B. M. Elliott 5 , S. R. Siddani 6 , Y. Bai 6 , A. Parikh 6 , M. M. Seliem 6 , M. H. Dinh 6 , A. P. Skarbnik 7

1 BC Cancer Centre for Lymphoid Cancer, The University of British Columbia, Vancouver, Canada; 2 Department of Hematology, Tel-Aviv Sourasky (Ichilov) Medical Center, Tel Aviv, Israel; 3 Icahn School of Medicine at Mount Sinai, New York, United States of America; 4 University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea; 5 Genmab, Princeton; 6 AbbVie Inc., North Chicago; 7 Lymphoma and CLL Program, Novant Health Cancer Institute, Charlotte, United States of America

Background: The recent evolution of newer treatment options for patients with B-cell non-Hodgkin’s lymphoma (NHL) are encouraging, yet there remains a need for more effective and better tolerated therapies. Epcoritamab is a subcutaneously administered bispecific antibody that binds to CD3+ T cells and CD20+ B cells, inducing CD3+ T cells to kill CD20+ tumor B cells through a unique mechanism of action (MOA). In a dose-escalation study, epcoritamab monotherapy demonstrated a manageable safety profile and anti-tumor activity in pts with heavily pretreated R/R B-cell NHL (Hutchings et al, Lancet 2021). Promising preliminary results from the ongoing EPCORE NHL-2 trial (NTC04663347) were previously presented with epcoritamab in combination with other anti-neoplastic agents: epcoritamab + R-CHOP in previously untreated DLBCL (Belada et al, ASH 2021), and epcoritamab + rituximab and lenalidomide in R/R Follicular Lymphoma (Linton et al, ASH 2021). Novel combinations with epcoritamab may offer additional options for pts with B-cell NHL. An ongoing study ( NCT02077166 ) evaluating ibrutinib, rituximab, and lenalidomide in R/R DLBCL has shown encouraging results; ibrutinib and lenalidomide may enhance T-cell activity and further potentiate the effects of epcoritamab in R/R DLBCL, particularly post-CAR-T. Investigation of pola-R-CHP compared to R-CHOP in pts with previously untreated DLBCL demonstrated an improvement in progression-free survival in the pola-R-CHP group (Tilly et al, NEJM 2022). Combining epcoritamab with the anti-neoplastic agents used in these studies, which have different MOAs and non-overlapping toxicities, may offer enhanced clinical benefit to pts with DLBCL.

Aims: To characterize the safety, tolerability, and preliminary efficacy profiles, and determine the recommended dose of epcoritamab co-administered with other anti-neoplastic agents as novel combinations in pts with B-cell NHL.

Methods: This open-label, global, multicenter study (EPCORE NHL-5) will evaluate subcutaneous administration of epcoritamab in novel combinations with other anti-neoplastic agents: Arm 1) epcoritamab + lenalidomide in R/R DLBCL; Arm 2) epcoritamab + ibrutinib and lenalidomide in R/R DLBCL; Arm 3) epcoritamab + pola-R-CHP in treatment-naïve DLBCL. Each arm will include a dose-escalation, followed by a dose-expansion phase. The primary objectives include assessment of dose-limiting toxicities and identification of the recommended dose for further investigation. Secondary endpoints include ORR, CR rate, DOR, PFS, TTR, and OS. Additional endpoints are safety, pharmacokinetics, and biomarker assessments. Pts must be ≥18 years old with an ECOG performance status 0 – 2. Other inclusion criteria are measurable disease by PET-CT and diagnosis of CD20+ DLBCL, including i) DLBCL, not otherwise specified; ii) “double-hit” or “triple-hit” DLBCL (high-grade B cell lymphoma), and iii) FL grade 3B. Pts must not have prior treatment with epcoritamab or other bispecific antibodies targeting CD3 and CD20 and no autologous SCT ≤3 months before screening.

Results: NA

Summary/Conclusion: Enrollment is planned to open in May 2022 in the United States and Israel, with additional countries in North America, Europe, and Asia for a total of ~100 sites.

PB2134: THE ROLE OF ADDITIONAL CARBOGEN GAS MIXTURE AND NICOTINAMIDE ON R-CHOP CHEMOTHERAPY IN DLBCL PATIENTS WITH TUMOR HYPOXIA

D. Rizky 1,* , E. A. Pangarsa 1

1 Hematology & Medical-Oncology, Dr. Kariadi General Hospital, Kota Semarang, Indonesia

Background: Tumor hypoxia increases tumor aggressiveness, immunosuppression, & decreases sensitivity to chemoradiotherapy. The role of tumor hypoxia in DLBCL remains unclear. Hypoxia-inducible factor 1-alpha (HIF-1α) is an oxygen-dependent transcriptional factor that activates several tumor survival pathways. Hypoxia & inflammation have a positive correlation, one is defined by the HIF-1α & NFkB relationship. NFkB involves in cytokine regulations such as TNF-α & sIL-2R. Carbogen gas mixture (95% O2 and 5% CO2) & nicotinamide (an amide of vitamin B3) have been recently studied as a radiosensitizer in solid tumors & also improve tumor oxygenation level. We compared tumor size changes, TNF-α, & sIL-2R serum in DLBCL patients who received carbogen + nicotinamide during chemotherapy R-CHOP versus those who did not.

Aims: The primary endpoint was tumor size reduction. Secondary endpoints were changes of TNF-α & sIL2R serum pre-& post-chemotherapy. Tumor MRI was used for examining the size reduction.

Methods: This trial is an ongoing single-center, randomized controlled trial. Eligible DLBCL patients who had HIF-1α tissue expression ≥10% were included. Subjects were randomly assigned to the study group (nicotinamide 40 mg/kgBW + carbogen 10 liter/minute during R-CHOP) or control group (R-CHOP) for 1 cycle. Tumor MRI and blood parameters examinations were performed before and 7-days after interventions.

Results: Between April-December 2021, 20 patients were enrolled (median age 56.5 years; min & max 21-65 years). For the baseline data, median tumor volume were 152.8 cm3 (4.3-1,542.5 cm3) & 172.2 cm3 (6.4-2,349.7 cm3), median sIL-2R serum were 304 U/mL (24-1,625 U/mL) & 417 U/mL (13-1,897 U/mL), median TNF-α serum were1.95 pg/mL (1.2-4.2 pg/mL) & 1.65 pg/mL (0.8-6.7 pg/mL) in control and subject group respectively, there were no statistically significant differences in these results. Post-chemotherapy, median tumor volume were 34.54 cm3 (1.86-297.5 cm3) & 112 cm3 (1.2-862.98 cm3), there were no significant differences of median Δ tumor size reduction in both groups (-118 cm3 & -85 cm3; p = 0.762) control & subject group respectively. Median sIL-2R serum in control group was increased; 519 U/mL (24-1,557 U/mL) & decreased in subject group; 157 U/mL (13-464 U/mL) although there were no statistically significance (p = 0.314 & p = 0.109 respectively). Median TNF-α serum were 2.1 pg/mL (0.8-6.9 pg/mL) & 1.8 pg/mL (0.4-3.3 pg/mL) in control & subject group respectively (p = 0.671 & p = 0.766). There was a positive correlation between Δ tumor size & Δ sIL-2R serum (p = 0.04 & r = 0.463). 2 patients complaint minor rash & itchy skin.

Summary/Conclusion: We found no difference in tumor size reduction between two groups. However, we found a clinical reduction trend of serum sIL-2R in the subject group although it was not statistically significant, this is because of the incomplete sample collection. Serum sIL-2R level also had a positive correlation with tumor size reduction which showed an interesting field of study for the upcoming research.

PB2135: BURKITT LYMPHOMA INTERNATIONAL PROGNOSTIC INDEX (BL-IPI): IS IT EFFICIENT ENOUGH IN ADULT BURKITT LYMPHOMA PATIENTS?

B. Sayinalp 1,* , I. Barista 2 , Y. Buyukasik 3

1 Internal Medicine; 2 Internal Medicine, Division of Medical Oncology; 3 Internal Medicine, Division of Hematology, Hacettepe University School of Medicine, Ankara, Turkey

Background: Burkitt lymphoma is a rare but clinically severe subtype of non-Hodgkin lymphoma. An ideal risk scoring system specific to Burkitt lymphoma had not been available for many years, however Burkitt Lymphoma International Prognostic Index (BL-IPI) has been recently proposed by Olszewski et al. BL-IPI is consisted of following risk factors; age ≥ 40 years, performance status ≥ 2, serum lactate dehydrogenase (LDH) ≥ 3X upper limit of normal (ULN), and central nervous system (CNS) involvement. Accordingly, patients are grouped as low risk (zero risk factor) disease, intermediate risk (one risk factor) and high risk (≥2 risk factors).

Aims: Our aims were to examine the basic characteristics and survival rates of Burkitt lymphoma patients that are being treated in our institution, to determine the prognostic factors which affect survival and to evaluate the efficacy of BL-IPI and other risk scoring systems in our patient population.

Methods: Patients who were 18 years or older and diagnosed with Burkitt lymphoma between 2000-2021 at Hacettepe University Hospital were included in our study. Patient, disease and treatment characteristics were reviewed through hospital electronic health records. Disease free survival (DFS) and overall survival (OS) were determined as the primary endpoints. Variables that have prognostic impact were identified through univariate and multivariate analyses. Finally, the efficacy of BL-IPI was tested in our patient population. Statistical analyses were conducted using SPSS version 25.

Results: A total of 71 patients whose median age of diagnosis was 40 (IQR: 29-55) and median follow-up time was 11,4 months (minimum 0,33, maximum 243,67 months) were included in our study. 1 year OS was estimated as 58%, 3 year OS as 53% and 3 year DFS as 64%. Factors affecting survival were examined and in univariate analyses; performance status ≥ 2, stage 4 disease, bone marrow involvement, CNS involvement, serum LDH > ULN, hypoalbuminemia, anemia and thrombocytopenia were associated with lower 3-year OS (p<0,05); while stage 4 disease, bone marrow involvement, anemia and thrombocytopenia were associated with lower 3-year DFS (p<0,05). In multivariate analysis, only performance status ≥ 2, stage 4 disease, bone marrow involvement and thrombocytopenia caused a statististically significant increase in death rate (p<0,05). CNS involvement caused a near significant increase (p=0,067). 25,4% of the patients had low risk, 31% of the patients had intermediate risk and 34,6% of the patients had high risk disease according to BL-IPI. 3-year OS rates were 82%, 65% and 27% respectively, and 3-year PFS rates were 81%, 71% and 36% respectively.

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Summary/Conclusion: Our study supports the fact that BL-IPI predicts survival efficiently, however taking our aforementioned results into consideration, we propose that instead of “CNS involvement”, “CNS involvement and bone marrow involvement” or “stage 4 disease” could be added to the score as a risk factor. In the study conducted by Olszewski et al., 58% of the patients in the low risk group had advanced stage disease, however only 12% had bone marrow involvement, which is a relatively low ratio compared to literature. Studies involving larger cohorts who have higher bone marrow involvement ratios consistent with the literature are undoubtedly needed to support this proposition.

PB2136: PLANNED-USE GLUCARPIDASE FOR OUTPATIENT HIGH DOSE METHOTREXATE (HD-MTX) ADMINISTRATION IN PATIENTS WITH CNS LYMPHOMA (CNSL) DURING THE COVID-19 PANDEMIC

L. Schaff 1,* , M. Lobbous 2 , D. Carlow 3 , L. DeAngelis 1 , L. B. Nabors 2 , C. Grommes 1

1 Neurology, Memorial Sloan Kettering Cancer Center, New York City, NY; 2 Neurology, University of Alabama at Birmingham, Birmingham, AL; 3 Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York City, NY, United States of America

Background: First-line treatment for central nervous system lymphoma (CNSL) consists of high-dose methotrexate (HD-MTX) combination therapy, a potentially curative strategy. Administration of HD-MTX typically requires inpatient hospital admission for monitoring and aggressive hydration to prevent toxicity. During the height of the COVID-19 pandemic, patients were reluctant to present for this potentially curative chemotherapy out of concern for increased risk of COVID exposure during hospital admission.

Aims: This study explores the feasibility of planned-use glucarpidase, a recombinant bacterial enzyme that rapidly reduces serum MTX levels, to facilitate the outpatient administration of HD-MTX for patients with CNSL.

Methods: Eligible adult patients had lymphoma isolated to the CNS, normal renal function, and had previously tolerated standard inpatient HD-MTX. After informed consent was obtained, MTX 3.5 g/m 2 was administered in the outpatient setting along with intravenous hydration. Patients returned 24 hours after MTX administration for glucarpidase 2000u and additional hydration. MTX level was determined by high pressure liquid chromatography (HPLC) 48 hours following start of administration.

Results: Twenty outpatient treatments of HD-MTX with glucarpidase were administered to a total of 7 patients (men=3, women=4). Patients had a median age of 71 (range, 47-73) and baseline Karnofsky Performance Status of 90 (range 70-90). They received either one (n=1), two (n=3), three (n=2), or seven (n=1) treatments on study. MTX levels were reduced to <100 nmol/L at 48 hours following 20/20 treatments (100%). Adverse events at least possibly attributed to glucarpidase included grade 1 and 2 fatigue (n=3). Events possibly attributed to MTX included grade 1 and 2 fatigue (n=2), nausea (n=2), and vomiting (n=1). Five treatments were associated with an elevated creatinine, one of which was grade 2 and resolved to baseline following additional hydration. There was one grade 3 decreased lymphocyte count attributed to MTX. Remaining lab abnormalities were grade 1 or 2, also attributed to MTX, and included elevated alanine aminotransferase (n=3), elevated aspartate aminotransferase (n=3), anemia (n=1), hypocalcemia (n=1), hypokalemia (n=3), and decreased lymphocyte count (n=4). Out of an abundance of caution, all patients received leucovorin prior to documentation of MTX clearance. None of the patients required hospital admission during treatment. Anti-glucarpidase antibody data from enrolled patients is being analyzed.

Summary/Conclusion: This study demonstrates feasibility of outpatient HD-MTX administration with planned-use glucarpidase for a select patient population during the COVID-19 pandemic. We are currently enrolling to a larger study of planned-use glucarpidase for repeated doses of outpatient HD-MTX ( NCT03684980 ).

PB2137: INFLUENCE OF PREVIOUS RADIOTHERAPY ON THE EFFECT OF CART CELL THERAPY IN AGGRESSIVE B-CELL LYMPHOMA

F. Shaomei 1,*

1 haematology, Beijing BOREN Hospital, China, China

Background: In previous studies, radiotherapy has an effect on the number and function of lymphocytes. In recent years, with the advent of CART cell therapy, the PFS and OS of relapsed and refractory highly aggressive B-cell lymphoma have been significantly improved. Whether previous radiotherapy is effective for CART cell therapy The impact is inconclusive.

Aims: To investigate the influence of the radiotherapy history before CART cell therapy on the culture, expansion and treatment effect of CART cells in patients with aggressive B-cell lymphoma.

Methods: A retrospective study from December 2018 to September 2021 in adult patients with aggressive B-cell lymphoma treated with CART cells at our center who underwent radiotherapy before lymphocyte collection. The patient’s previous radiotherapy dose, radiotherapy distance from lymphocyte collection time, peripheral blood lymphocyte count before lymphocyte collection, CART cell culture dose, reinfusion dose, and curative effect statistics of 3 months after CART cell reinfusion were counted. Statistical methods such as logistic regression and spearman correlation analysis were used for analysis.

Results: 16 males and 18 females were eligible, with a median age of 45.5 years (18-86 years). These included 2 Burkitt lymphoma, 26 unspecified diffuse large B-cell lymphoma, 5 primary mediastinal B-cell lymphoma, and 1 primary central lymphoma. The median time from lymphocyte collection to previous radiotherapy was 151 (5-4772) days, and the median radiation dose was 46 (4-66) Gy. The absolute value of lymphocytes before the collection of lymphocytes was 0.79 (0.14-2.58)×10^9/L, which was lower than the normal reference value. The median culture dose of CART cells was 0.837(0.1-7.03)×10^6/kg, and the median infusion dose was 0.837(0.1-4.2)×10^6/kg. After CART cell infusion, the peak CART cell expansion was 18.6(0-919)×10^6/kg, 3-month CR: 20.59%, 3-month ORR: 44.12%. The time interval between previous radiotherapy and lymphocyte collection had a significant effect on 3-month CR, P=0.0417. The effect on 3-month ORR was not statistically significant, P=0.1388. There was no significant effect of previous radiotherapy dose on CR/ORR at 3 months, with P values ​​of 0.9188/0.5078, respectively. The effect of previous radiotherapy dose on the CART cell culture dose and the maximum value of CART cell expansion was not found to be statistically significant. CR and ORR at 3 months after reinfusion were significantly correlated with the reinfusion dose, with P values ​​of 0.0102 & 0.0121, respectively. There was no significant relationship between the absolute value of lymphocytes and the proportion of lymphocytes, the culture dose and the CR/ORR of the curative effect.

Summary/Conclusion: Radiotherapy affects the number and cell quality of lymphocytes, the median culture dose is significantly lower than the 2×10^6/kg reported in the literature. In patients with aggressive B-cell lymphoma who had a history of radiotherapy, the CR/ORR at 3 months after CART cell therapy was significantly lower than that reported. This adverse effect was independent of radiation dose. The CR rate is affected by the time interval between radiotherapy and lymphocyte collection. The longer the interval, the greater the adverse effect.

PB2138: VITALIZE TRIAL: A PHASE 2B, OPEN-LABEL, MULTICENTER, RANDOMIZED PARALLEL-GROUP, TWO-STAGE, STUDY OF MAVEROPEPIMUT-S, PEMBROLIZUMAB, WITH OR WITHOUT INTERMITTENT LOW-DOSE CYCLOPHOSPHAMIDE, IN R/R DLBLC

E. Lee 1,* , M. Matasar 2 , R. Conlon 3 , K. Mody 3 , S. Fiset 4 , J. Graff 3

1 Compassionate Cancer Care Group, Fountain Valley; 2 Memorial Sloan Kettering Cancer Center, Montvale, United States of America; 3 IMV inc., Dartmouth; 4 IMV inc., Quebec City, Canada

Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin’s lymphoma in adults, and up to 40% of patients will be refractory to front-line therapy or experience a relapse. In the relapsed/refractory (r/r) setting, novel treatment options are available, but many patients will relapse following or be ineligible for such treatment options, due in part to the safety profile. Thus, more effective, better tolerated therapeutic options are needed.

Maveropepimut-S (MVP-S, previously DPX-Survivac) leverages the DPX platform, a non-aqueous, lipid-based delivery system, to educate a specific and persistent T cell-based immune response to 5 HLA-restricted peptides from survivin, a cancer-associated protein commonly upregulated in advanced, malignancies including DLBCL. In a prior clinical study in patients with r/r DLBCL (SPiReL: NCT03349450 ), MVP-S in combination with pembrolizumab and low dose, intermittent cyclophosphamide (CPA) led to durable complete and partial responses as well as persistent, survivin-specific T cells, particularly in patients with PD-L1+ disease. Importantly, this combination was well-tolerated.

Aims: Further exploration of the regimen in r/r DLBCL is warranted to confirm and extend these encouraging results and to clarify the relative contributions of the constituent components of the combination regimen.

Methods: The VITALIZE trial ( NCT04920617 ) is a phase 2b, open-label, randomized trial of MVP-S and pembrolizumab, with or without intermittent low-dose CPA. Eligible patients with r/r DLBCL (including transformed indolent B cell lymphomas and high-grade lymphoma) have persistent or progressive disease following ≥ 2 lines of prior systemic therapy and are ineligible for, or have relapsed after, ASCT and/or CAR-T. Using a Simon’s Two-Stage Design, patients will be randomized (1:1) to: Arm 1: MVP-S, pembrolizumab and CPA or, Arm 2: MVP-S and pembrolizumab, and will be treated for up to 2 years. The primary objective of the study is centrally assessed objective response rate (ORR) (Lugano 2014), and secondary endpoints include PFS, DoR, DCR, OS and safety. Exploratory objectives include characterization of outcomes based on PD-L1 expression, and survivin-specific immunogenicity in baseline and on-treatment blood and tissue samples. This study will enroll up to 92 eligible subjects at 65 global study sites. Enrollment has begun, with one subject enrolled as of abstract submission date.

Results: Trial in progress

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Summary/Conclusion: Trial in progress

PB2139: A REAL-WORLD OUTCOME OF NEWLY DIAGNOSED PRIMARY MEDIASTINAL B-CELL LYMPHOMA TREATED WITH 6 CYCLES OF R-CHOEP-14 AND 2 CYCLES OF DHAP FOLLOWED BY AN AUTOLOGOUS STEM CELL TRANSPLANT

J. Tan Tsen Chuen 1,* , K. W. Ho 1 , P. L. Low 1 , L. C. Chow 1 , G. Kasinathan 1 , C. Ng 1 , S. Shahnaz Alkudsi 1 , S. M. Tan 1

1 Department of Haematology, Hospital Ampang, Selangor, Malaysia

Background: Historically, Primary Mediastinal B-Cell Lymphoma (PMBL) treated at our centre with 6 cycles of R-CHOP-21 appears to have a poorer outcome compared to the conventional DLBCL. There were few prospective randomized studies to look specifically at the optimal treatment of PMBL. Some physicians advocated more intensified regimens such as R-DA-EPOCH, R-HYPERCVAD, VACOP-B/MACOP-B or the BFM ALL protocol. A prospective phase 2 study by Dunleavy et al. in 2013 showed an impressive outcome with R-DA-EPOCH and had become the preferred regimen at our centre. However in real-world practice, adhering to this intensified regimen was difficult and doses were rarely escalated beyond level 2. Logistic problems at our centre with arranging central venous access, monitoring counts and timely admissions result in delays in administration.

Aims: To evaluate the efficacy and tolerability of 6 cycles of R-CHOEP-14 followed by 2 cycles of DHAP and an autologous stem cell transplant.

Methods: At the treating physician’s discretion, PMBL patients diagnosed between August 2019 - Sept 2021 were offered 6 cycles of R-CHOEP-14 followed by 2 cycles of DHAP and an autologous stem cell transplant with standard BEAM conditioning. Etoposide was given orally whenever possible and prophylactic GCSF was given routinely. The primary treatment was done in an outpatient setting whenever possible. Patients also received consolidation radiotherapy to the mediastinum at the physician’s discretion.

Results: At data cut off, a total of 16 patients have completed their treatment. 87.5% (14) of patients were male, and 12.5% (2) of patients were female. Median age was 33.5 years (25-42). All patients had bulky disease > 7cm, B symptoms and Stage ≥2. 93.8% (15) had elevated serum LDH. One patient received 2 cycles of high dose methotrexate & cytarabine instead of DHAP as he was deemed at high risk of CNS relapse. 37.5% (6) of patients also received radiotherapy at the treating physician’s discretion either prior to or after transplant. The mean duration between the R-CHOEP cycles were 18 days, with a median of 16 days and mode of 14 days. 37.5% (6) of patients had a PET/CT done either prior or after the transplant and among these, 83.3% (5) achieved a complete metabolic remission. With a median follow up of 48 weeks (12-129), progression free survival (PFS) was 100% and the overall survival (OS) was 93.8%. There was one unexpected treatment related mortality (TRM) due to massive pulmonary haemorrhage which may be related to patient’s pulmonary stent removal prior to his 6 th R-CHOEP chemotherapy. Serious adverse events were rare, one patient developed CMV retinitis which resolved upon treatment. No patient had progressed or relapsed.

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Summary/Conclusion: 6 cycles of R-CHOEP-14 followed by 2 cycles of DHAP and an autologous stem cell transplant is feasible and well tolerated with limited toxicity. The primary regimen can be done in an outpatient setting, does not require central venous access, easier to adhere to and can be completed within a shorter time frame. The early PFS and OS appears comparable to historical real-world outcome of intensified regimens such as R-DA-EPOCH. Longer follow up will be desirable to evaluate long term outcome and toxicity.

PB2140: ALLOGENEIC STEM CELL TRANSPLANTATION IN RARE T-NHL SUBTYPES: FOCUS ON AITL, MF/SS, HSTCL. A SINGLE BMT-UNIT EXPERIENCE.

T. Tzenou 1 , A. Roumelioti 1,* , N. El Gkotmi 1 , P. Christoforou 1 , N.-E. Loutsidi 1 , A. Apsemidou 1 , I. Tzannou 1 , T. Ioannis 1 , C. Giatra 1 , F. Karaolidou 1 , Z. Mellios 1 , B. Maria 1 , S. Gigantes 1 , I. Baltadakis 1 , D. Karakasis 1

1 BMT-UNIT, EVAGGELISMOS GENERAL HOSPITAL, ATHENS, Greece

Background: Angioimmunobastic Lymphoma (AITL) and T-γδ-hepatosplenic lymphomab(HSTCL) consist two rare, aggressive T-NHL subtypes while patients with Mycosis Fungoides/Sézary (MF/SS) syndrome may have a more indolent disease course but are rarely cured with conventional therapy. AutoHSCT may offer prolonged survival in selected cases of AITL but relapses are common. Despite the significant innovation in therapeutics of hematological malignancies, little progress is made in these lymphomas management. Due to the rarety as well as to the fact that at least for AITL and MF/SS median age at diagnosis is greater than 60 years old, there is limited number of reports on allogeneic stem cell transplant outcomes and data from prospective studies are missing

Aims: To describe our experience on allogeneic transplant outcomes in a small cohort of relatively young patients with AITL (3), HSTCL (3), MF/SS (8)

Methods: Records of 14 patients (6 men, 8 women), who underwent consecutively allogeneic stem cell transplant from 01/01/2008 to 31/12/2019, were retrospectively studied. Survival status was censored on 15 th February 2022. All had consented to the use of their data before conditioning initiation

Results: Median age was 51.5, 34 and 49 years old in MF/SS, HSTCL and AITL respectively. All donor types had been used. All MF/SS had been submitted to RIC alloHSCT with FLU MEL +/-ATG and no MF/SS patient was on CR at the time of alloHCT (3 PR, 2SD, 3PD). 1 patient with AITL was in CR at the time of alloHSCT and remains alive, relapse and GVHD free 28 months after alloHSCT while 1 patient in PR at the time of alloHSCT is alive in CR, relapse/GVHD free 6 years later. 1 patient with HSTCL is alive relapse/GVHD free 12 years after HSCT. There were no early deaths before day 30 in the whole cohort, while 3 patients died before day 100 – 1 with AITL due to primary graft failure, 2 with MF/SS due to refractory disease. 2 patients with HSTCL died due to early disease relapse, 4 and 6 months post-transplant respectively, while 2 patient with MF/SS died 7 and 15 months post-transplant due to EBV positive PTLD and severe gastrointestinal PTLD complicated by CMV-colitis. At the date of data censoring 4 patients with MF/SS were alive in CR and relapse/GVHD free, while 1 patient was alive but in PR after the second relapse treated with pembrolizumab 39 months after alloHSCT

Summary/Conclusion: Due to the rarity of the studied lymphoma subtypes our cohort was small and proper statistical analysis of data could not be done safely. However, it becomes clear that younger patients with MF/SS -even with relapsed/refractory disease - may benefit of prolonged survival with allogeneic stem cell transplant. For patients with HSCTL, allogeneic stem cell transplant is considered “sine qua non” for survival and cure, while fit patients with AITL should be considered candidates for alloHSCT. Immunological manipulation may contribute to relapse management.

PB2141: CLINICOPATHOLOGICAL FEATURES AND OUTCOME OF CD5 POSITIVE DIFFUSE LARGE B CELL LYMPHOMA AND THE SIGNIFICANCE OF CD5 EXPRESSION STRENGTH: 10-YEAR SINGLE CENTER EXPERIENCE

V. Vukovic 1,* , T. Karan-Djurasevic 2 , V. Otasevic 1 , S. Sarac 1 , N. Sabljic 1 , D. Antic 1,3 , V. Djurasinovic 1,3 , K. Tomic 1 , B. Mihaljevic 1,3

1 Clinic for Hematology, Clinical Center of Serbia; 2 Institute of Molecular Genetics and Genetic Engineering; 3 Medical Faculty, University of Belgrade, Belgrade, Serbia

Background: CD5 positive (CD5+) diffuse large B cell lymphoma (DLBCL) is a rare subtype of DLBCL encompassing 5-10% of patients (pts) with DLBCL, and it is characterized by poor outcome.

Aims: To identify clinicopathological features of CD5+ DLBCL in a cohort of pts managed at Clinic for Hematology, Clinical Center of Serbia during a 10-year period (2012-2021).

Methods: We retrospectively analyzed immunohistochemistry (IHC) results of biopted tumor tissue of 537 pts with DLBCL NOS. Survival analysis was performed using Kaplan-Meier method and log-rank test.

Results: Out of a total of 537 pts, in 211 (39.3%) CD5 was not analyzed by IHC, while in the remaining 326 (60.7%) was. Among them, 27 (8.3%) were found to be positive for CD5, out of which 10 pts were designated at the pathology report as CD5 +/- (5 pts) or CD5 -/+ (5 pts), while the rest 17 pts were clearly CD5+.

In CD5 positive cohort, 52% of pts were men and median age at diagnosis was 63 years. ECOG performance status (PS) was poor (≥2) in 8 (29.6%) pts. One third of pts had advanced stage disease (Ann Arbor III/IV), while the rest two thirds were limited stage (I and II). Eleven out of 26 pts (42.3%) presented with B symptoms, 3/26 (11.5%) expressed bulky disease, while two thirds presented with extranodal disease. More pts (13/23 or 56.5%) were of nonGCB subtype according to IHC, while 4 out of 11 pts (36.4%) analyzed for c-myc protein expression were double expressors. Median Ki-67 proliferative index was 80%. Almost half of pts (47.6%) had increased initial lactate dehydrogenase (LDH) activity, while median beta-2 microglobulin (b2m) level was 3.38 mg/L. Nine out of 26 pts (34.6%) were IPI>2, while the rest 17 pts were IPI 0-1. Vast majority (25/27, 92.6%) were treated with chemoimmunotherapy in the first line, most of them with anthracycline-based protocols, 1 patient received cyclophosphamide, vincristine, prednisone (COP), and 1 patient succumbed before treatment commencement. Among treated pts with known outcome after first line, 18 (72%) pts achieved complete remission, 4 (16%) achieved partial response, while 3 (12%) pts did not respond to treatment.

Median progression-free survival (PFS) after first treatment line for all pts in CD5 positive cohort was 25 months (95%CI 13.76-36.24), while median overall survival (OS) was not reached. Age at diagnosis, presence of B symptoms, and extranodal disease did not significantly influenced PFS and OS. Patients with bulky disease, nonGCB subtype, and double expressors experienced significantly shorter PFS (p=0.03, P=0.043, and p=0.025, respectively), but these features did not impact OS. On the other hand, b2m levels >3.38 mg/L, LDH >1x upper limit normal, and IPI >2 were significantly associated with shorter OS (p=0.014, p=0.01, and p=0.015, respectively), while they were not significant regarding PFS. ECOG PS >1 was significantly associated with both poor PFS and OS (p=0.005 and p=0.002, respectively).

Intrestingly, pts designated as CD5+ at pathology reports experienced median PFS of 20 months in contrast to 52 months in pts descripted as CD5+/- and CD5-/+ (p=0.021). However, the difference between these two subgroups regarding OS was not observed.

Summary/Conclusion: In our CD5+ DLBCL cohort, bulky disease, nonGCB, and double expressors were associated with shorter PFS, higher b2m, LDH, and IPI correlated with shorter OS, while higher ECOG PS was associated with shorter both PFS and OS. Median PFS after 1 st treatment line was around 2 years, while median OS was not reached. Strenght of CD5 expression was significantly associated with PFS. CD5 should be mandatory within IHC for DLBCL.

PB2142: CLINICAL CHARACTERISTICS AND OUTCOMES OF NEWLY DIAGNOSED PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUS-ASSOCIATED LYMPHOMA ADMINISTERED HIGHLY ACTIVE ANTIRETROVIRAL THERAPY

C. Wang 1,* , J. Liu 2 , H. Lei 2 , Y. Li 2 , J. Wu 2 , B. Guo 1 , R. Hu 2 , T. Liu 2 , J. Wu 2 , Y. Ding 2 , C. Hu 1 , S. Liang 1 , C. Xiao 1 , X. Liang 1 , D. Huang 1 , T. Yang 1 , W. Zhang 2 , Z. Yang 2 , J. Li 2 , Y. Nan 2 , Q. Li 1 , Y. Xiang 2 , Y. Wu 2 , Y. Liu 1

1 Hematology; 2 Chongqing University cancer hospital, Chongqing, China

Background: HIV-associated lymphoma is the most common subtype of HIV-associated cancers, with a very low incidence. To date, there are only few retrospective studies analyzing the clinical features and prognosis of HIV-associated lymphoma. Therefore, we performed a study to analyze the clinical characteristics and outcomes of newly diagnosed HIV-associated lymphoma patients in our center, as the largest HIV-associated lymphoma cohort in China so far.

Aims: Herein, we analyzed the clinical characteristics and prognosis of 107 newly diagnosed HIV-associated lymphoma patients, as the largest cohort reported in China to date.

Methods: Totally 107 newly diagnosed HIV-associated lymphoma patients in Chongqing University Cancer Hospital, Southwest China, from July 2008 to August 2021, were analyzed.

Results: In the 107 HIV-associated lymphoma cases, median age was 49 years (range: 23–87 years), and more patients were male (87.9%). Most patients had elevated lactate dehydrogenase (LDH) (78.5%), elevated β 2 -MG (91.6%), advanced Ann Arbor stage (73.8%) and high IPI score (IPI score: 3-5) (62.7%) at diagnosis. Totally 46 patients (43%) had one or more sites of extranodal involvement, of whom 24 (22.4%) possessed two or more extranodal sites. Median CD4 + T cell count at diagnosis was 191/μl (range: 4-1022); 84 patients (78.5%) were on combination antiretroviral therapy (cART) at lymphoma diagnosis. Of the 72 patients evaluated at the end of treatment, the overall response rate (ORR) was 81.9% (59/72); 29 (40.3%) cases achieved complete response (CR), 30 (41.7%) had partial response (PR) and 9 (12.5%) experienced progressive disease (PD). In 99 HIV-associated lymphoma cases with complete follow-up data (median follow-up of 44.5 months [range: 2-160 months]), median PFS and OS were 36.0 months and not reached, respectively. The overall 5-year PFS and OS rates were 46.9% and 53.4%, respectively. Compared with individuals not administered anti-lymphoma treatment or administered less than 3 cycles anti-lymphoma therapy, those who received standardized anti-lymphoma therapy (≥3 cycles) showed improved PFS ( p =0.0006) and OS ( p <0.0001). In patients administered systemic anti-lymphoma therapy, the overall 2-year PFS rates for chemotherapy and R-chemotherapy were 29.5% and 67.1%, respectively (HR=0.44, 95%CI 0.210-0.927; p =0.020 ). The overall 2-year OS rates for chemotherapy and R-chemotherapy were 51.9% and 73.1%, respectively (HR=0.42, 95%CI 0.183-0.969; p =0.042 ).

Summary/Conclusion: Overall, these data indicated that HIV-associated lymphoma has aggressive characteristics, including advanced Ann Arbor stage, high IPI score, more extranodal involvement and elevated LDH. Survival is significantly improved due to the wide use of cART and chemoimmunotherapy. Standardized anti-lymphoma therapy is significantly associated with superior outcomes.

PB2143: CLINICAL CHARACTERISTICS AND PROGNOSTIC FEATURES OF 63 HIV-ASSOCIATED DIFFUSE LARGE B-CELL LYMPHOMA: A SINGLE-CENTER REAL-WORLD STUDY IN CHINA

C. Wang 1,* , J. Liu 1 , X. Liang 1 , Y. Liu 1

1 Hematology, Chongqing University cancer hospital, Chongqing, China

Background: To date, only a few studies have assessed the clinical features of Chinese HIV-associated lymphoma patients. Meanwhile, Chongqing University Cancer Hospital is the only cancer center in Western China that treats HIV-associated lymphoma patients. So far, more than 130 patients have been diagnosed in our center. Herein, we analyzed the clinical characteristics and prognosis of 63 newly diagnosed HIV-associated diffuse large B cell lymphoma(DLBCL) patients, as the largest cohort reported in China to date.

Aims: To explore the clinical characteristics and prognosis of patients with Human immunodeficiency virus (HIV)-associated diffuse large B cell lymphoma(DLBCL).

Methods: Retrospective analysis the clinical data of 63 HIV-infected patients with DLBCL diagnosed at Chongqing University Cancer Hospital from July 2008 to August 2021. Survival curves were calculated with Kaplan-Meier method, and comparison of survival among the groups used log-rank test method. Cox proportional hazards model was used for multivariate analysis.

Results: In 63 patients with HIV-associated DLBCL, 90.5% (57/63) were male,and the median age was 49(23-87) years. The main pathological subtype is the germinal center B-cell-like lymphoma(GCB)(74.6%), 46.0% (29/63) were combined with extranodal lesions. 27.0%(17/63)occurred large masses(≥7.5cm). 31.7% (20/63) of patients had B symptoms.The median CD4 + T cell count was 203(4-1022)×10 9 /L. 49%(25/51) patients had CD4 + cell count <200×10 6 /L. 56.9%(33/58) of patients with a high (3-5) International Prognostic Index (IPI) scores.78% (46/59) were Ann Arbor stage III/IV patients. 25.4% (16/63) patients gave up treatment. 22.2% (14/63) of patients received less than 4 cycles chemotherapy. 52.4% (33/63) of patients received 4 or more cycles chemotherapy. Among patients receiving chemotherapy, 61.7% (29/47) received R-CHOP-like regimens,and 38.3% (18/47) of patients used CHOP-like regimens.The 1-year, 2-year, 3-year and 5-year OS rates were 65.0%, 53.8%, 47.1% and 43.5%, respectively. Univariate analysis showed that age ≥60 years ( P =0.012), ECOG-PS physical performance 2-4 points ( P =0.043), IPI score 3-5 points ( P =0.001), β2-Mg elevation(≥5.5 mg/L) ( P =0.007), systemic chemotherapy cycles less than 4 times ( P <0.001) were the adverse prognostic factors affecting the overall survival of patients. Cox multivariate analysis showed that age ≥60 years ( HR =2.272, 95% CI 1.110-4.651, P =0.025), IPI score 3-5 points( HR =3.562, 95% CI 1.794-7.074, P <0.001), ECOG-PS score 2-4 points ( HR =2.675, 95% CI 1.162-6.153, P =0.021) and the number of cycles of chemotherapy<4 ( HR =0.290, 95% CI 0.176-0.479, P <0.001) were independent risk factors for adverse prognosis of OS.

Summary/Conclusion: HIV-associated DLBCL is the most common HIV-associated tumor, most commonly seen in males, with a high one-year fatality rate. Chemotherapy combined with antiretroviral therapy can significantly improve patient prognosis.

PB2144: PEARL: A STUDY TO ASSESS THE EFFICACY AND SAFETY OF PENPULIMAB PLUS LENALIDOMIDE, RITUXIMAB, GEMCITABINE AND OXALIPLATIN IN PATIENTS WITH RELAPSED/REFRACTORY DIFFUSE LARGE B CELL LYMPHOMA

X. Wei 1,* , L. Wang 1 , J. Liang 1 , H. Shen 1 , J. Li 1

1 Department of Hematology, the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China

Background: Relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) patients have limited treatment strategies and are associated with poor prognosis, so it is necessary to explore the treatment options for these patients. Penpulimab, an anti-PD1 antibody of IgG1 isotype, has demonstrated high response rate and very good tolerance, and is now approved in R/R classic Hodgkin lymphoma. The immunomodulatory agent lenalidomide has demonstrated direct anti-tumor effects in lymphoma. The regimen of rituximab, gemcitabine and oxaliplatin is used for the treatment of R/R DLBCL, which is recommend by current National Comprehensive Cancer Network guideline.

Aims: The PEARL study aims to assess the efficacy and safety of penpulimab Plus lenalidomide, rituximab, gemcitabine and oxaliplatin (Pen-R 2 -GemOx) in Patients With R/R DLBCL.

Methods: PEARL ( NCT05186558 ) is a Phase II, multicenter, single-arm, open label study (Figure 1). An estimated 54 patients will be enrolled. Eligible patients are aged 18 to 80 years with relapsed or refractory after one prior treatment, CD20-positive DLBCL, and Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. All patients will be treated with Pen-R 2 -GemOx scheme (penpulimab, 200 mg, q2w, d0; lenalidomide, 10 mg, d1-7; rituximab, 375 mg/m2, d0; gemcitabine, 1000 mg, d1; oxaliplatin, 100 mg/m2, d1; 14d/cycle) during induction therapy, and they will be assessed the efficacy after four cycles by computed tomography (CT). Patients who achieve stable disease/progressive disease (SD/PD) will exit the study. Patients who achieve complete response/unconfirmed complete response/partial response (CR/CRu/PR) will receive Pen-R 2 -GemOx again for two cycles and be assessed the efficacy by positron emission tomography-CT (PET-CT). Afterwards, patients who achieve PD will withdraw the study. Patients who achieve CR/CRu/PR and are eligible for autologous stem cell transplantation (ASCT) will undergo ASCT, while patients who are not eligible for ASCT will directly receive penpulimab and lenalidomide as maintenance treatment. During maintenance treatment, the combination of penpulimab and lenalidomide will be used for 6 months, followed by lenalidomide monotherapy for 18 months. The primary endpoint is CR rate. Secondary endpoints include progression-free survival (PFS) rate, overall survival (OS) rate of 2 years and toxicity. Patients will be followed up from their end-of-induction treatment visit until the end of the study, including clinical evaluation every 3 months in the first year and every 6 months in the second year for patients undergoing ASCT and maintenance treatment. The expected study duration from first patient’s first visit to last patient’s last visit is approximately 3 years. Enrollment for the study will begin in Q2 of 2022.

Results: Results for this study are not yet available.

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Иллюстрация — в первоисточнике ↗

Summary/Conclusion: Conclusion for this study is not yet available.

PB2145: REAL-WORLD THERAPY LINES IN DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) – A POPULATION-BASED ANALYSIS

J. Duell 1 , S. Weinand 2 , T. Kaworski 3 , T. Schubert 2,*

1 Universitätsklinikum Würzburg, Würzburg; 2 LinkCare GmbH, Stuttgart; 3 Incyte Biosciences Germany GmbH, Martinsried, Germany

Background: Real-world data on patients suffering from diffuse large B-cell lymphoma (DLBCL) in Germany treated in 1 st -and 2 nd -line therapies are limited.

Aims: The aim of this study was to investigate the number of DLBCL pateints treated in 1 st -and 2 nd -line therapies in Germany. Further patients deceased or being in remission and potentially therapy-related complications were considered.

Methods: A database of German health administrative anonymized claims data of statutory health insurances – comprising a total of 5,166,845 insured persons from 2015 to 2016 – was used for analyses. Newly diagnosed DLCBL-patients were defined as having at least one confirmed outpatient or inpatient main diagnosis (ICD-10-GM code C83.3), a co-existing DLCBL-specific treatment (claims for chemo-/antibody-therapy, stem cell transplantation or radiation therapy) and no DLCBL diagnosis one year before. Rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) was defined as 1 st -line therapy, other therapies than R-CHOP were defined as 2 nd -line therapy. Patients neither deceased nor receiving a 2 nd -line therapy within 3 years after 1 st -line therapy were defined as patients in remission. Potential therapy-related complications were defined based on the following ICD-10-GM codes: renal insufficiency (N14, N17-N19, N99.0), heart insufficiency (I50), liver insufficiency (K71-K77), polyneuropathy (G62.0). Only newly emerged ICD-10-GM coding’s were assessed as being potentially treatment related. Burden of comorbidities was assessed using the Charlson-comorbidity-index (CCI).

Results: We identified 445 DLBCL-patients (2015-2016) out of them 89.4% (n=398) were treated with R-CHOP in 1 st -line therapy. Patients were on average 66 years old and mean CCI was 5.15. Out of 398 these patients, 21.1% (n=84) received a 2 nd -line therapy [Ø-age: 65 y / Ø-CCI: 4.98], 23.9% (n=95) died without proceeding to 2 nd -line therapy [Ø-age: 75 y / Ø-CCI: 5.89] and 55.0% (n=219) were in remission after 1 st -line therapy [Ø-age: 62 y / Ø-CCI: 4.86]. Out of 398 DLCBL-patients treated with R-CHOP, 23% developed renal insufficiency, 13% heart insufficiency, 10% liver insufficiency, and 9% polyneuropathy. Deceased patients (n=95) suffered significantly more often (p<.01) from a renal (33% vs. 19%) and heart insufficiency (20% vs. 9%) than patients in remission (n=219).

Summary/Conclusion: 23.9% of patients treated with 1 st -line therapy R-CHOP deceased within 3 years. 10.6% of newly diagnosed DLBCL-patients were not treated with R-CHOP. Further research is need to get more insights in treatment patterns of DLBCL-patients (3 rd -line therapies or use of CAR-T-cells).

PB2146: A PHASE 2, OPEN-LABEL STUDY OF LONCASTUXIMAB TESIRINE IN COMBINATION WITH RITUXIMAB (LONCA-R) IN PREVIOUSLY UNTREATED UNFIT/FRAIL PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) (LOTIS-9)

J. Westin 1,* , J. M. Burke 2 , A. E. Chapman 3 , T. Kilavuz 4 , C. Xu 4 , Y. Shmuely 4 , J. Radford 5

1 Department of Lymphoma and Myeloma, MD Anderson Cancer Center, Houston; 2 US Oncology Hematology Research Program, Rocky Mountain Cancer Centers, Aurora; 3 Sidney Kimmel Cancer Center, Jefferson Health, Philadelphia; 4 ADC Therapeutics America, Inc, Murray Hill, United States of America; 5 NIHR Clinical Research Facility, the Christie NHS Foundation Trust and University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom

Background: Rituximab in combination with chemotherapy (R [rituximab]-CHOP [cyclophosphamide, doxorubicin, vincristine, and prednisone]) is the standard first-line therapy for patients (pts) with DLBCL. With an aging population, unfit or frail pts who may not tolerate R-CHOP represent an increasing unmet need. There is also significant heterogeneity in how fitness for therapy is assessed. The simplified geriatric assessment (sGA) identifies three distinct categories (fit, unfit, and frail) based on age, activities of daily living (ADL), instrumental activities of daily living (IADL), and the Cumulative Illness Rating Scale for Geriatrics (CIRS-G; Merli et al. J Clin Oncol 2021). Loncastuximab tesirine (loncastuximab tesirine-lpyl; Lonca), an antibody-drug conjugate comprising a humanized anti-CD19 monoclonal antibody conjugated to a pyrrolobenzodiazepine dimer toxin, is approved in relapsed or refractory (R/R) DLBCL based on data from the phase 2 LOTIS-2 pivotal trial (Caimi et al. Lancet Oncol 2021). The safety and efficacy of Lonca-R in combination is also being studied in pts with R/R DLBCL versus immunochemotherapy in an ongoing, separate study (LOTIS-5; NCT04384484 ).

Aims: To determine the safety and efficacy of Lonca-R in previously untreated unfit/frail pts, as characterized by the sGA, in the LOTIS-9 clinical trial ( NCT05144009 ).

Methods: This is a phase 2, open-label, response-adapted study of Lonca-R in previously untreated unfit (Cohort A) or frail (Cohort B) pts with DLBCL. Key inclusion criteria include diagnosis of DLBCL (including DLBCL transformed from indolent lymphoma), high-grade B cell lymphoma, or grade 3b follicular lymphoma; Eastern Cooperative Oncology Group performance status of 0-2; and measurable disease (2014 Lugano Classification). Each cohort will enroll 40 pts, with fitness (Cohort A) and frailty (Cohort B) assessed using the sGA. Primary objectives are to assess efficacy (Cohorts A and B) and tolerability (Cohort B) of Lonca-R. The primary endpoint will be the complete response (CR) rate (Cohorts A and B) and tolerability defined by the percentage of patients completing a total of 4 cycles of therapy (Cohort B). Lonca-R treatment consists of R intravenously [IV] 375 mg/m 2 on day 1 of cycles 1-4 (subcutaneously allowed starting at C2), Lonca 150 µg/kg IV on day 2 of cycle 1 and day 1 of cycle 2, and Lonca 75 µg/kg IV on day 1 of cycles 3 and 4. After completion of three Lonca-R cycles, pts who achieve CR or partial response (PR) will continue to receive 1 or 3 additional cycles of Lonca-R, respectively. Pts in Cohort A who do not achieve a CR or PR will discontinue treatment on study. Pts in Cohort B who achieve stable disease and derive clinical benefit, per the treating physician, may continue to receive an additional 3 cycles of Lonca-R. All pts will be followed every 12 weeks for 1 year, then every 24 weeks for up to 3 years, and then annually up to 5 years.

Results: The study will open for recruitment in 2022.

Summary/Conclusion: N/A

PB2148: EFFICACY OF SMALL MOLECULE TARGET THERAPIES IN REFRACTORY OR RELAPSED DIFFUSE LARGE B-CELL LYMPHOMA AND MOLECULAR SUBTYPING: A MULTI-CENTER REAL-WORLD EVIDENCE

Y. Zhang 1 , Y. Hou 2 , X. Zhou 3 , L. Sun 4 , W. Wang 5 , Y. Wo 6 , Y. Sun 6 , Q. Xu 6 , P. Liu 1,*

1 Department of Hematology, Zhongshan Hospital, Fudan University; 2 Department of Hematology and Oncology, Minghang Hospital, Fudan University, Shanghai; 3 Department of Hematology, Wuxi People′s Hospital Affiliated to Nanjing Medical University, Wuxi; 4 Department of Hematology, Zhongshan Hospital Qingpu Branch, Fudan University; 5 BeiGene, Shanghai; 6 Canhelp Genomics Research Center, Hangzhou, China

Background: Diffuse large B-cell lymphoma (DLBCL) shows remarkable molecular heterogeneity that can demonstrates different clinical outcomes. The two major cell-of-origin (COO) subtypes, germinal center B cell-like (GCB) and activated B cell-like (ABC) lymphomas, have been transcriptionally defined. Small molecule target therapies such as lenalidomide and Bruton’s tyrosine kinase (BTK) inhibitor have been widely used for refractory or relapsed DLBCL (R/R DLBCL) in the chemo-free manner or in combo therapy together with salvage chemotherapy.

Aims: To reveal the efficacy and administration scenario of lenalidomide and BTK inhibitor in R/R DLBCL with molecular subtyping of COO.

Methods: Real-world data of R/R DLBCL patients exposure to lenalidomide and BTK inhibitor in the chemo-free manner or in combo therapy together with salvage chemotherapy were extracted into the current study from the national medical center as well as regional medical centers. Archived tumor samples of included R/R DLBCL patients were submitted to COO subtyping with DLBCL-COO assay (Canhelp Genomics, Hangzhou, China).

Results: Ninety-one patients were registered into current study, 43 (47.3%) of which received BTK inhibitors (ibrutinib, acalabrutinib, or zanubrutinib) and/or lenalidomide chemo-free therapies (chemo-free therapy) and 48 (52.7%) of which received BTK inhibitors (ibrutinib, acalabrutinib, or zanubrutinib) and/or lenalidomide combo therapy with cytotoxic regimen (combo therapy). The most common administrated cytotoxic regimen with BTK inhibitors and/or lenalidomide were: R2 (rituximab plus lenalidomide) in 11 patients, lenalidomide in 10 patients and R2-GEMOX in 9 patients. The median age was 64.5, male to female ratio was 1.08:1. The proportion of patients receiving second-line and further lines of therapy in “combo cohort” and “chemo-free cohort” were 82% and 18% vs. 62% and 38%, respectively. Less lines of prior systemic therapies was seen in “combo cohort”. Overall response rate was 48.4% with 19.8% complete remission rate. The median progression free survival was 4 months. 35.2% patients was GCB subtype and 41.8% patients was ABC subtype. There was no significant difference regarding ORR between GCB and ABC subtype in the whole population (43.8% vs. 60.5%, P=0.23). However, significantly higher ORR was identified in patients with ABC subtype receiving “combo therapy” (GCB: 26.7% vs. ABC: 65.2%, P=0.044), which was not observed in “chemo-free therapy” (GCB: 58.8% vs. ABC: 53.3%, P=1). Other clinical-pathologic parameters were not associated with ORR.

Summary/Conclusion: Small molecule target therapies are more commonly administrated with cytotoxic regimen in the real world, and its efficacy is different from the one in the clinical trial. A considerable proportion GCB subtype R/R DLBCL received BTK inhibitor and/or lenalidomide. Molecular subtyping predicts the clinical benefit for patients with combo therapies, instead of the chemo-free therapies, in which heavier prior treatment may significantly impact the clinical outcome.

PB2149: ZANUBRUTINIB IN COMBINATION WITH LENALIDOMIDE AND RITUXIMAB VERSUS R-MINICHOP IN UNFIT OR FRAIL PATIENTS WITH NEWLY DIAGNOSED DIFFUSE LARGE B-CELL LYMPHOMA AGED OVER OR EQUAL TO 70 YEARS

P. Xu 1,2,3,4,* , Z. Shi 1,2,3,4 , L. Wang 1,2,3,4 , S. Cheng 1,2,3,4 , W. Zhao 1,2,3,4,5

1 Shanghai Institute of Hematology; 2 National Research Center for Translational Medicine at Shanghai; 3 Ruijin Hospital; 4 Shanghai Jiao Tong University School of Medicine; 5 Pôle de Recherches Sino-Français en Science du Vivant et Génomique, Shanghai, China

Background: The optimal treatment for elderly diffuse large B-cell lymphoma (DLBCL) patients needs to be further explored due to their comorbidities, intolerability of standard immunochemotherapy, and unfavorable genetic features. The balance of efficacy and safety is a major issue in the treatment of elderly patients. Rituximab in combination with attenuated CHOP (R-miniCHOP) is the standard treatment for unfit or frail elderly patients. However, the toxicity of chemotherapeutic drugs is still inevitable. In a phase 2 trial ( NCT04460248 ), 40 patients aged 75 years or older with newly diagnosed DLBCL received zanubrutinib in combination with lenalidomide and rituximab (ZR2) regimen, and achieved similar treatment response rate as those of attenuated immunochemotherapy (manuscript in preparation). The promising preliminary results of ZR2 make the combination a potentially chemo-free option versus immunochemotherapy in unfit or frail elderly patients.

Aims: PEARL study ( NCT05179733 ) is a multicenter, prospective, randomized, open-label, controlled, non-inferiority trial to evaluate the efficacy and safety of ZR2 versus R-miniCHOP in the treatment of unfit or frail newly diagnosed DLBCL patients aged older than or equal to 70 years.

Methods: Key eligibility criteria include patients aged ≥ 80 or 70-79 years with comprehensive geriatric assessment stratified as unfit or frail, histologically-confirmed DLBCL without central nervous system involvement, Eastern Cooperative Oncology Group performance status 0-3, and at least one measurable site of disease. Two hundred and eighty patients will be randomized 1:1 to receive either ZR2 or R-miniCHOP for 6 cycles. The primary endpoint is progression-free survival. Secondary endpoints include complete response rate, overall survival, safety, and quality of life. Response will be evaluated before treatment and repeated after 3 cycles and at the end of treatment. Disease response will be assessed by investigator according to Lugano 2014 criteria. Patients’ enrollment is ongoing.

Results: N/A

Summary/Conclusion: N/A

PB2150: RELATIONSHIP BETWEEN P16 AND P53 CO-EXPRESSION AND SURVIVAL IN PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA

E. Vaneeva 1,* , A. Turubanova 1 , M. Sarpova 1 , D. Dyakonov 1 , V. Rosin 1 , S. Samarina 1

1 ФГБУН КНИИГиПК ФМБА России, ФГБУН КНИИГиПК ФМБА России, Kirov, Russia

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive non-Hodgkin’s lymphoma. The p16 protein is a representative of the family of inhibitors of cyclin-dependent kinases (INK4; Cdk). It is involved in the regulation of the cell cycle by destroying Cdk4/Cdk6 complexes with cyclins D, blocking the transition of the cell from G1 to S phase. The protein is encoded by the tumor suppressor gene CDKN2A (9p21), aberrations of which are detected in 15-30% of DLBCL cases. The p53 protein is a transcription factor. Its activation leads to cell cycle arrest, replication of damaged DNA, or cell apoptosis. Mutations in the TP53 gene encoding this protein occur in 20% of patients. It is known that high isolated expression of p16, p53 is associated with low overall (OS) and progression-free (PFS) survival of patients with DLBCL. However, there are no data on the significance of the combined expression of these proteins.

Aims: Assesment of relationship between the co-expression p16 and p53 proteins and the survival of patients with DLBCL.

Methods: Tumor tissue biopsies (paraffin blocks) obtained from 66 patients with newly diagnosed DLBCL (median age - 58 years) were used as material for the study. All patients received R-CHOP chemotherapy. The determination of the relative number of tumor cells expressing p16, p53 was carried out using immunohistochemical and morphometric methods. The threshold value of p16, p53 expression was calculated by enumeration method of data using a log-rank test when comparing survival curves with different threshold values. Quantitative values ​​of protein expression were converted into binary indicators. For p16, the threshold was 9% of positive tumor cells, for p53 - 35%. According to the established criteria, all subjects were divided into groups with high (+) [≥9% for p16; ≥35% for p53] and low (-) [<9% for p16; <35% for p53] protein expression. The five-year OS and PFS were calculated using the Kaplan-Meier method (log-rank test). The relationship of different variants of p16 and p53 co-expression with the course of DLBCL was analyzed.

Results: The median 5-year survival of patients with DLBCL was 43 months. The highest 5-year OS (86%) was recorded in the group of patients with simultaneously low expression of markers (p16-/p53-; n=25). In subjects with high expression of one of the proteins (p16+/p53- and p16-/p53+; n=34), it was 21.6%. Patients with a simultaneous combination of high expression of markers (p16+/p53+; n=7) did not survive to the end of the observation period (0%). Using Cox regression analysis, it was found that in patients with p16+/p53+, the risk of developing an adverse event was 13.6 times higher than in the group of subjects with p16-/p53- (HR=13.6; 95% CI=10.2 -37.7). Intergroup differences in the analysis of PFS did not reach statistical significance (p=0.087).

Summary/Conclusion: Сo-expression of p16+/p53+ biomarkers is associated with lower OS in DLBCL patients compared to other combinations of their expression.

PB2151: EVALUATION OF BONE MINERAL DENSITY IN LYMPHOMA PATIENTS WITH USING PET/CT AND THE ROLE OF BCL-2

V. Okan 1,* , H. Haydaroglu Sahin 1 , H. Gün 1 , D. M. Akkurd 1

1 Haematology department, Gaziantep University Sahinbey Research And Practice Hospital, Gaziantep, Turkey

Background: With the advances in treatment, osteoporosis becomes an important morbidity and mortality problem in lymphoma patients. Identification of opportunistic osteoporosis screening without the need for additional examination and identification of effective factors in the development of osteoporosis will provide an advantage in complication management.

Aims: In our study,we evaluate the guidance of routine PET/CT in the diagnosis of osteoporosis, which is one of the chemotherapy-related complications in NHL and HL patients, with a time-and cost-effective understanding, without requiring additional examination and it was intended to investigate the possible relationship between BCL-2, an antiapoptotic protein, and osteoporosis in NHL patients with bone marrow involvement.

Methods: A total of 108 patients, 55 of whom were HL and 53 were NHL, who were treated with the diagnosis of HL and NHL at the Hematology clinic of Gaziantep University Faculty of Medicine, were followed up. Demographic data such as age, gender, stage, laboratory parameters, BCL-2 results and PET/CT images were analyzed retrospectively. Three PET/CT scans were selected; at the time of diagnosis PET/CT1, in the mid-response evaluation of chemotherapy PET/CT2, and taken at least six months after the completion of chemotherapy PET/CT3. In the evaluation of the presence of osteoporosis by PET/CT, Hounsfield Unit (HU) measurement was used in the lumbar spine (L1-L4) and CT imaging of trabecular bones. HU threshold value was determined as 160 based on previous studies.

Results: Bone mineral density in PET/CT2 was significantly decreased in all patients diagnosed with HL and NHL compared to PET/CT1 (p = 0.001, p = 0.001). In patients diagnosed with early stage HL and in patients diagnosed with early stage NHL, bone mineral density in PET/CT 3 increased significantly compared to PET/CT2 (respectively; p = 0.001, p = 0.017). Bone mineral density in PET/CT3 was significantly reduced in all patients with NHL and in patients with advanced NHL diagnosis compared to PET/CT2 (respectively; p=0.001 and p=0.002). In all patients with HL, in patients with early stage HL, in all patients with NHL, in patients with early-advanced stage NHL, it was found that bone mineral density in PET/CT3 decreased significantly compared to PET/CT1 (respectively p = 0.004, p = 0.049, p =0.001, p= 0.005, p= 0.001). Bone mineral density in female gender PET/CT1 and PET/CT2 was significantly lower in the NHL patient group (p = 0.004, p = 0.001,respectively). In our study; when both groups compared; BCL-2 positive patients with bone marrow involvement in PET CT(n=20) vs. BCL-2 negative patients without bone marrow involvement(n=14) in PET/CT, results was not found as a risk factor for the development of osteoporosis(p>0.05).

Summary/Conclusion: In our study; A significant decrease in bone mineral density was found at the time of diagnosis and treatment of HL and NHL patients. In post-treatment follow-up, it was determined that negative effects on bone disappear earlier in patients with early stage HL and NHL. The results of the study show PET/CT used in diagnosis and follow-up, can be safely used in opportunistic osteoporosis screening with a cost-effective approach without using additional examination. The disadvantage of this study was the small number of patients for evaluating bcl-2 and osteoporosis relationship.

PB2152: BIOLOGICAL AND CLINICAL CORRELATES OF GALECTIN-3 (GAL-3) EXPRESSION IN DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL)

S. Basic-Kinda 1 , I. Ilic 2 , A. Duletic-Nacinovic 3,4 , N. Jonjic 4,5 , I. Radman 1 , T. Valkovic 3 , R. Serventi-Seiwerth 1 , D. Dujmovic 1 , B. Dreta 1 , F. Santek 6,7 , I. Aurer 1,7,*

1 Division of Hematology, Department of Internal Medicine; 2 Department of Pathology, University Hospital Centre Zagreb, Zagreb; 3 Division of Hematology, Department of Internal Medicine, University Hospital Centre Rijeka; 4 Medical School, University of Rijeka; 5 Department of Pathology, University Hospital Centre Rijeka, Rijeka; 6 Department of Oncology, University Hospital Centre Zagreb; 7 Medical School, University of Zagreb, Zagreb, Croatia

Background: Gal-3 is the only known member of the group of so-called chimeric galectins. It has 3 structural domains, a NH2 terminal domain with serine phosphatase activity, a repetitive collagen-like domain and a COOH terminal carbohydrate-recognizing domain. Gal-3 is thought to be involved in crucial cellular processes, including regulation of cell proliferation, apoptosis and angiogenesis. Gal-3 promotor contains NF-κB binding sites (Kadrofske et al, 1998). Gal-3 shares the BH1 domain with BCL2 and has antiapoptotic activitiy, but seems to affect different intracellular structures than BCL2 (Akahani et al, 1997). Normal memory B-cell, but not germinal-center cells, express gal-3. More than 50% of DLBCLs express gal-3, but none of Burkitt, follicular, marginal zone and small lymphocytic lymphomas (Hoyer et al, 2004). While preliminary data suggest that gal-3 + DLBCLs have inferior prognosis, its relation to other immunohistochemical markers, cell of origin (COO) and clinical characteristics of lymphomas are unknown

Aims: To compare biological and clinical characteristics of gal-3 + and gal-3 – DLBCLs and thus gain insight into its biological function and clinical importance in this tumor type.

Methods: We analyzed clinical characteristics (age, sex, IPI factors, response to treatment, event-free survival (EFS) and overall survival (OS)), immunohistochemical expression of gal-3, BCL2, VEGF and NF-κB (p65), and cell of origin (COO) as determined by Hans’ algorithm, in newly diagnosed DLBCL patients treated with R-CHOP or similar regimens. All markers were evaluated using commercially available monoclonal antibodies.

Results: 123 patients were included in the study with a median follow-up of survivors of 68 months. Gal-3 was positive in 69 (56.1%), negative in 51(41.4%) and undetermined in 3. Patients with higher IPI, non-GC subtype and NF-κB + expressed gal-3 more frequently (Table). In univariate analysis gal-3 expression was not a statistically significant prognostic factor for OS and EFS in the whole group of patients (p=0.16 for EFS and p=0.19 for OS). However, BCL2 + patients fared significantly worse if they coexpressed gal-3 (EFS at 2 y 44% vs 73% (p=0,002) OS at 2 y 50% vs 74% (p=0,006)). Gal-3 expression had no influence on the outcome of BCL2- patients. In multivariate analysis of influence of immunohistochemical markers on EFS and OS, BCL2 was the only significant factor, while gal-3 showed a strong trend (p=0.068 for EFS and 0.093 for OS). If IPI was introduced into the model, gal-3 expression lost its significance.

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Summary/Conclusion: Our results are in accordance with experimental data indicating that NF-κB stimulates gal-3 expression and that gal-3 potentiates the antiapoptotic effect of BCL2. Gal-3 is more frequently expressed in advanced-stage DLBCLs and those of non-GC subtype, consistent with its putative proliferative/antiapoptotic effect and its expression in memory B- but not germinal-center cells.

PB2153: SURGICAL SPECIMENS OF INDOLENT AND AGGRESSIVE B-NHLS SHOW DIFFERENCES IN THEIR FLOW-CYTOMETRY PROFILE OF PLOIDY, PROLIFERATING FRACTION AND PROPORTION OF TUMOR-ASSOCIATED MYELOID AND T-CELL SUBSETS

D. Azoulay 1,2,* , T. Tapuchi 2 , H. I. Cohen 3 , L. Akria 1,2 , G. Stemer 1,2 , O. Ronen 2,4

1 Hematology, Galilee Medical Center, Nahariya; 2 Azrieli Faculty of Medicine, Bar-Ilan University, Safed; 3 Pathology Unit and Laboratories; 4 Otolaryngology Head and Neck Surgery, Galilee Medical Center, Nahariya, Israel

Background: Multiparameter flow cytometry (FC) is an important and valuable diagnostic tool for specimens collected via minimally invasive techniques, when only a limited tissue sample is available and tissue architecture is inadequate.

Aims: Here we examined and compared the incidence of various cellular components in specimens of patients with aggressive versus indolent B-cell non-Hodgkin’s lymphomas (B-NHLs).

Methods: The data were retrospectively collected from surgical specimens collected via fine-needle aspiration or core biopsies that were submitted to our FC laboratory for a routine lymphoma diagnostic workup. The data included: ploidy status and incidences of cells in the proliferative fraction, lymphocytes, CD64 + monocytes, immature (iGr) and mature granulocytes (mGr) in the total CD45 + cellular component, clonal B cells, CD3 + T cells, CD3/CD56 + NK cells in the total lymphocyte population, CD4 + T cells, CD8 + T cells, CD4 + /CD8 + DP T cells, CD4 - /CD8 - DN T cells and CD3 + /CD56 + NKT cells in the total T cell component. Pearson Chi-squared or two-sided t test analysis was performed to compare the profiles of specimens of patients with aggressive versus indolent B-NHLs as determined by histopathological analysis.

Results: A total of 40 specimens were included in this study (F:M [%] 62.5:37.5, age [mean ± SD, range] 67.87 ± 14.73 years, 27.4-92.6). Histopathological analysis confirmed the diagnosis of aggressive B-NHL in 23 (20 DLBCL, 2 MCL and 1 ALC) (57.5%) specimens, and indolent B-NHL in 17 (11 FL, 6 MZL) (42.5%) specimens. Specimens from aggressive B-NHLs showed an increased rate of aneuploidy (56.52% vs. 11.76% prob>chiSq =0.0025), increased incidences of cells in the proliferative fraction ([mean % ± SD] 13.03 ± 10.52 vs. 3.21 ± 1.42 p = 0.0005), CD64 + monocytes (3.89 ± 3.88 vs. 0.93 ± 0.75 p = 0.0038 ), iGr (4.43 ± 4.47 vs. 0.96 ± 0.86 p = 0.0033 ), CD8+ T cells (33.16 ± 16.23 vs. 18.54 ± 6.44 p = 0.001 ), DN T cells (6.90 ± 4.34 vs. 3.47 ± 1.74 p = 0.004 ) and DP T cells (6.74 ± 5.94 vs. 2.38 ± 1.26 p = 0.005 ), and reduced incidences of lymphocytes (72.71 ± 24.26 vs. 89.22 ± 13.33 p = 0.016 ), CD4+ and T cells (65.67 ± 15.86 vs. 78.68 ± 7.24 p = 0.003 ), as compared to specimens of indolent B-NHLs.

Summary/Conclusion: These preliminary data confirm our previous report and suggest the proliferating fraction and the incidences of tumor-associated myeloid cells and T cells as flow cytometry-identifiable biomarkers that could assist in the differentiation between aggressive vs. indolent B-NHLs in specimens collected via minimally invasive sampling.

PB2154: TP53 GENE MUTATION DIVERSITY IN RUSSIAN PATIENTS WITH B-CELL LYMPHOMAS

B. Biderman 1,* , N. Severina 1 , D. Koroleva 1 , N. Gabeeva 1 , A. Belyaeva 1 , S. Tatarnikova 1 , F. Babaeva 1 , E. Nesterova 1 , Y. Mangasarova 1 , O. Margolin 1 , M. Bagova 1 , A. Magomedova 1 , S. Kravchenko 1 , T. Obukhova 1 , E. Zvonkov 1 , A. Sudarikov 1

1 National Medical Research Center for Hematology, Moscow, Russia

Background: The TP53 gene plays a key role in cell cycle control, DNA repair, initiation of apoptosis, and other critical cellular processes. Mutations of this gene are found in almost all types of malignant neoplasms and the vast majority of cases are associated with poor prognosis. Among hematological malignancies, TP53 gene lesions are most actively studied in CLL, while for other lymphomas fewer data are available so far.

Aims: To study the frequency, variation, and allelic load of TP53 mutations in Russian patients with B-cell lymphomas.

Methods: The study included 145 DNA samples from patients with B-cell lymphomas admitted to the National Medical Research Center for Hematology (Moscow, Russia) in 2020-2021: 53 patients with DLBCL (29F, 24M, age 25-87 years, median 54 years), 47 patients with MCL (17F, 30M, age 42-73 years, median 59 years) and 45 patients with FL (28F, 17M, age 30-72 years, median 49 years), 16 of them with transformation to DLBCL. Mutations in the TP53 gene (exons 4-10) in most samples were detected using NGS according to Pavlova et al., or Sanger sequencing (7 patients). The clinical significance of the detected variants was assessed using the Seshat online tool based on the UMD TP53 database.

Results: TP53 gene mutations were detected in 24 DLBCL patients (45.4%). Out of them were found 21 missense mutations (2 cases in the “hot spots” p.R249M and p.R282W and 2 cases in the same position p.G266), 2 splicing site mutations, and 1 insertion. Also, 16 variants were identified as (likely) pathogenic, 13 of which had a mutation load (VAF) greater than 15%. Of the 8 mutations of unclear significance, 4 had a VAF<10%. Del17p was simultaneously detected in 7 patients with TP53 mutations (out of 11 examined). In the MCL patients mutations were found in 19 cases (40%). 18 of them were missense mutations (4 in the “hot spot” p.R248 and 2 in the same position p.V173) and 1 splicing mutation. 16 variants were identified as (likely) pathogenic and had a VAF>14%. All 3 variants of unclear significance were minor. Chromosome 17 abnormalities were found in 7 MCL patients with TP53 mutations out of 11 studied. In the FL patients, TP53 mutations were detected in 12 cases (26.7%). Out of 8 missense mutations, 4 were in hotspots p.R175H, p.R248Q, p.R249M, p.R273H. Also in this group 2 nonsense mutations, 1 splicing mutation, and 1 deletion were observed. Of the 9 variants recognized as (likely) pathogenic, only 1 was minor, as were 2 variants of unclear significance. Only 1 patient with TP53 mutation out of 6 examined had del17p. All results are presented in Table 1.

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Summary/Conclusion: The frequency of mutations in the TP53 gene in patients with B-cell lymphomas in our sample is significantly higher than according to the literature data. One can explain this bias by patient selection. A significant portion of DLBCL and FL patients admitted to our center in the state of progression or relapse. Most MCL cases were primary, however, MCL patients with advanced stages of the disease are more often admitted to the National Medical Research Center for Hematology. At the same time, the occurrence of “hot spots” mutations in MCL and FL is also higher than expected. Further studies concerning the type of mutations, their allelic load, and their combination with 17th chromosome lesions in association with disease course are on the way.

PB2155: B-CELL NON-HODGKIN’S LYMPHOMAS ASSOCIATED WITH AMYLOIDOSIS

M. Navrátilova 1,* , P. Flodrova 1 , D. Holub 2 , T. Pika 3 , J. Minarik 3 , P. D. Petr Dzubak 2 , P. Flodr 1

1 Department of Clinical and Molecular Pathology, FH and FMD Palacký University Olomouc, Department of Clinical and Molecular Pathology, FH and FMD Palacký University Olomouc; 2 Department of Molecular and Translational Medicine, FMD Palacký University Olomouc, Department of Molecular and Translational Medicine, FMD Palacký University Olomouc; 3 Department of Hemato-oncology, FH Olomouc, Department of Hemato-oncology, FH Olomouc, Olomouc, Czechia

Background: Different types of B-NHLs (MZL, LPL, B-CLL/SLL, FL, MCL, DLBCL) may show plasmacytic differentiation and may produce AL amyloid locally or as a part of systemic amyloidosis. Case reports of coincidence of B-NHL and non-AL amyloidosis were also reported, therefore correct diagnostic workflow to type amyloid deposits should include three main steps 1. special histological staining (Congo red and/or Saturn red), 2. immunoprofiling and proteomic analysis (LMD-LC/MS). Nomenclature classification distinguishes 36 amyloidogenic proteins and more are expected (ISA 2020).

Aims: Analysis of amyloid deposits irrespective of origin and localization is appealing for diagnostic and experimental precising which brings new insights in pathogenesis, classification and correct therapy due to a possibility of combination or hybrid amyloid deposits, and concurrent B-NHL may be detected.

Methods: Amyloid deposits were detected in variable FFPE tissues stained with Congo red and/or Saturn red with consequent immunohistochemical analysis (IHC) and proteomic analysis with the use of laser capture microdissection and liquid chromatography/tandem mass spectrometry (LMD-LC/MS/MS).

Concurent B-NHLs (excl. multiple myeloma, plasmocytoma and MGUS) were diagnosed due to characteristic morphology (HE, PAS, Giemsa), immunoprofile (primary antibodies with appropriate dilution - CD3, CD5, CD10, CD20, CD21, CD23, CD30, CD138, Bcl-2, Bcl-6, MUM1/IRF4, PAX5, cyclin D1 and/or SOX11, c-Myc, Ki67) and detected clonal rearrangement of IgVH (BIOMED-2).

Results: In our file with 329 amyloid positive specimens concurent B-NHLs were found in 14 specimens. 7 specimens were signed as highly suspected for B-NHL with plasmacytic differentiation and immunoglobulin light chain restriction but without evidence of IgVH clonal rearrangement. The most common B-NHL associated with localised amyloidosis was ENMZL MALT-type (10 cases, 1 case with hybrid amyloidosis AL kappa/AH IgG1), in 4 specimens LPL was detected. In all cases with IHC and/or LCM-LC/tandem MS analysis AL type of amyloid was detected including 2 cases with hybrid amyloidosis AL/AH and AL/AApoAIV, and in 1 case local AL amyloid deposition was produced after a therapy for B-NHL.

Summary/Conclusion: M-protein produced by B-NHL may be low or high according to a localised or systemic AL involvement resp., localised amyloid deposition is commonly associated with ENMZL MALT-type, both localised and systemic amyloidosis may be found out in LPL. Our category of highly suspected B-NHL with plasmacytic differentiation is literary sign as “AL amyloidosis with a localized B-cell neoplasia of undetermined significance” or as “AL amyloidosis with a an associated predominant kappa or lambda light chain expressing plasma cell population without evidence for clonality”. In our file (7 cases) only kappa/lambda immunohistochemically detected monotypy was recorded without an evidence of IgVH clonal rearrangement (somatic hypermutation of IgVH may be expected). The possibility of senescence evolution in B-NHL after a therapy may also bring local AL deposits (1 case in our cohort). Coincidence or association of non-AL amyloidosis (ATTRwt, ATTRv, AA and other) with B-NHL was described mainly in single case reports and extends the differential diagnosis. In a such cases both methods (IHC and LCM-LC/tandem MS) are suitable for correct amyloid typing, although LMD-LC/tandem MS brought more promising results than IHC.

PB2156: ANALYSIS OF POSSIBLE CLONAL ORIGIN OF LARGE B-CELL DIFFUSED LYMPHOMAS ARISING IN PATIENTS WITH PRE-EXISTING HODGKIN’S LYMPHOMA

P. Masciopinto 1 , E. Bellitti 2 , G. Opinto 3 , E. Arcuti 1 , L. Mastracci 4 , S. Ciavarella 3 , G. Cazzato 2 , A. Guarini 3 , E. Maiorano 2 , P. Musto 1,5 , G. Ingravallo 2 , F. Gaudio 5,*

1 Hematology Section, Department of Emergency and Organ Transplantation (DETO), University of Bari; 2 Pathology Section, Department of Emergency and Organ Transplantation (DETO), University of Bari; 3 Hematology Unit, Giovanni Paolo II IRCCS Cancer Institute Oncology Hospital, Bari; 4 Department of Surgical Sciences and Integrated Diagnostics, University of Genoa, Genoa; 5 Unit of Hematology and Stem Cell Transplantation, AOUC Policlinico, Bari, Italy

Background: Lymphomas are a variegated group of lymphoproliferative diseases featuring differences in cellular origin, biological development, clinical signs and symptoms and outcome. In fact, they derive from lymphocytes at various stages of development, and the characteristics of the specific subtype of lymphoma mirror those of the cell from which they originated. However, in some cases, a synchronic or metachronic onset of two different lymphomas in the same patient is possible.

Aims: This retrospective study was focused on 269 patients with newly diagnosed classic Hodgkin lymphomas treated at Bari University Hospital (Italy) between 2007 and 2020, with the aim of identifying patients who developed DLBCL and verifying a possible common origin.

Methods: All patients were clinically staged according to the Ann-Arbor system, recording the medical history, complete physical examination, blood counts, biochemical profile, chest X-ray films, PET/CT total body, computed tomography of the chest, abdomen and pelvis, unilateral bone marrow biopsy. Median age at diagnosis was 35 years (range 15-83), 90 pts (33%) had advanced stage (III-IV), 110 (41%) bulky disease, 123 (46%) presented B symptoms, and 71 (26%) extranodal disease.

In addition to the routine pathological examination, PD-L1 was assayed as a critical marker to differentiate Primary mediastinal large B-cell lymphoma from other entities. CD3 and CD5 expression, as well as that of PD-L1, was evaluated to identify some characteristics of the tumor microenvironment (TME).

Cell-of-Origin (COO) was analyzed according to the Hans algorithm and with gene expression profile. With the Nanostring Lymphoma Subtype (LST) Assay we evaluated the expression profile of 20 genes within the LST signature, 15 target genes and 5 reference genes.

Results: Over the study period, 269 patients were diagnosed with cHL; in 4 patients (1.5%), DLBCL developed after a median time of 53 months.

The mean age at presentation with cHL was 28 years (range 18-76 years), and the mean age when DLBCL was diagnosed was 35 years (range 21-79).

Morphological review of histological slides, as well as immunohistochemical evaluation, confirmed the previous diagnosis. All cHL cases were positive to PD-L1 against a non-neoplastic inflammatory background, while only two patients with DLBCL revealed positivity in a high percentage of neoplastic cells

Using Hans’s algorithm, all DLBCL cases showed a non-GBC subtype. None of the DLBCLs showed EBV infection detected by EBER.

The gene expression profile evaluated by the Nanostring Lymphoma Subtype Assay revealed two cases of the GCB molecular subtype, while the others were not classified. Regarding the T cell background, only two cases showed a diffuse and prominent CD3+/CD5+ population, while the other cases showed sporadic T cell infiltration.

Summary/Conclusion: The origin of the second neoplastic cell, in patients with DLBCL with a previous history of cHL, remains controversial, although the different immunophenotypic characteristics suggest that it may mainly arise de novo in a subject with a possible individual predisposition to develop lymphoid neoplasms. Large studies are necessary to verify the distinct roles of environmental factors, individual predispositions and constitutional or immune dysfunction consequent to previous treatment.

PB2157: TREATMENT WITH DIPHENYLENEIODONIUM CHLORIDE EXHIBITS CELL GROWTH INHIBITORY EFFECTS ON AGGRESSIVE LYMPHOMA CELLS

S. Haingartner 1,* , S. Jantscher 1 , K. Pansy 1 , M. Szmyra 1 , P. Neumeister 1 , H. Greinix 1 , A. Deutsch 1

1 Division of Hematology, Medical University of Graz, Graz, Austria

Background: Non-Hodgkin lymphoma (NHL) represent the most common hematologic malignancies in the world. Especially the incidence of diffuse large B-cell lymphoma (DLBCL) as one aggressive subtype increases and up to 40% of patients relapse or do not attain complete remission. Thus, there is an urgent need to develop new drugs and employing them in novel therapeutic strategies. Transcription profiling identified a DLBCL subgroup that exhibits increased expression of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) and which is insensitive to B-cell receptor inhibition.

Aims: Therefore, the aim was to investigate whether OXPHOS inhibition could be therapeutically targeted.

Methods: Six different lymphoma cell lines (Karpas422 and SuDHL4 as germinal center B-cell-like (GCB) DLBCL models, RI-1 and U2932 as activated B-cell-like (ABC) DLBCL models, BL2 as Burkitt lymphoma model and Jurkat as model for T-cell NHL) were used and cultured with Diphenyleneiodonium chloride (DPI) – an OXPHOS inhibitor. To analyse the in vitro effects of DPI cell-growth assays as well as cell-cycle analysis and apoptosis assays were performed.

Results: After 72 hours of DPI exposure, a concentration-dependent growth inhibition in all investigated cell lines was detected by MTS assay. Especially, DLBCL cell lines exhibited low IC 50 values ranging from 12nM to 40nM, whereas IC 50 values for BL2 (149nM) and Jurkat (640nM) cells were higher (p=0.071). Furthermore, DPI treatment (250nM, 50nM and 10nM) of SuDHL4 and BL2 cells induced a cell cycle arrest in the S phase in comparison to untreated cells after 24 hours. In Jurkat and U2932 cells, a change in cell cycle distribution was also observed but to a lower extend. Interestingly, in none of these four cell lines (SuDHL4, BL2, Jurkat and U2932) apoptotic effects were detected.

Summary/Conclusion: These preclinical data indicate that DPI treatment causes cell growth inhibition, which is mediated by changes in cell cycle distribution. Thus, pharmacologic inhibition of OXPHOS might serve as a novel therapeutic approach for further DLBCL therapies and the mechanism behind it should be deciphered in more detail.

PB2158: EXPLORING THE BIOLOGICAL ROLE OF LILRA4 IN MANTLE CELL LYMPHOMA

M. K. Blum 1,* , S. V. Hansen 1 , O. Cédile 1,2 , J. Haaber 1 , L. Chen 3 , M. Kassem 3 , N. Abildgaard 1 , M. H. Hansen 1 , C. G. Nyvold 1,2

1 Hematology-Pathology Research Laboratory, Research Unit for Hematology and Research Unit for Pathology, University of Sothern Denmark and Odense University Hospital; 2 OPEN, Odense Patient data Explorative Network, Odense University Hospital; 3 Molecular Endocrinology, University of Southern Denmark, Odense, Denmark

Background: Mantle cell lymphoma (MCL) is a heterogeneous, rare, and aggressive subtype of B cell non-Hodgkin’s lymphomas, with a generally poor prognosis. In a previous study, we found the leukocyte-immunoglobulin-like receptor subfamily A member 4 ( LILRA4 ) gene to be upregulated in 3 of 4 MCL patients at both diagnosis and relapse using RNA sequencing of sorted MCL cells from bone marrow (BM) and peripheral blood (PB). Furthermore, LILRA4 expression was investigated in diagnostic PB samples from a larger cohort of patients with MCL and chronic lymphocytic leukemia using qPCR, showing that LILRA4 was overexpressed in one-half of the patients, displaying a bimodal expression profile. LILRA4 is normally expressed in monocytes, B cells, and in plasmacytoid dendritic cells. It has proven to be involved in immunomodulating processes by binding to its only known ligand, bone marrow stromal cell antigen 2 ( BST2).

Aims: LILRA4 remains uncharacterized in a MCL context, and the goal of this study was thus to investigate its biological role in MCL.

Methods: LILRA4 mRNA expression was investigated in diagnostic samples from 33 MCL patients (26 PB and 10 BM samples) and 10 healthy donors (PB) using qPCR. Of these, 17 MCL and donor samples have previously been published (Hansen et al., 2020). Differential expression for BM and PB was evaluated using the Mann-Whitney U test (α = 0.05). Clinical information was extracted from patient files and compared to LILRA4 expression levels.

GRANTA-519, a MCL cell line, were transfected with siRNA targeting LILRA4 and investigated by RNA sequencing using cells transfected with scrambled siRNA as a negative control.

Results: LILRA4 transcripts showed a bimodal expression pattern for MCL PB and BM samples with high expression in half of the cases. Interestingly, the LILRA4 expression levels were significantly higher for blood compared to bone marrow (p<0.05). The LILRA4 expression level did not correlate with the degree of MCL burden in the samples, Ki67 proliferation index, mantle cell lymphoma prognostic impact (MIPI) score, or disease progression.

Transcriptome sequencing was performed on LILRA4 knockdown cells to investigate a potential biological role. A stable knockdown of ~75 % was obtained at 48 to 120 hours and was confirmed by both qPCR and RNA sequencing at 72 hours post-transfection. Gene set enrichment analysis suggested a high expression of genes related to lymphomagenesis and cell cycle regulation in the MCL cell line (3.4*10^-10≤p≤1.5*10^-5, 1.7*10^-8≤FDR≤7.48*10^-5, Hallmark gene sets). A twofold expressional reduction of macrophage migration inhibitory factor, MIF , a pro-inflammatory cytokine, was observed following LILRA4 knockdown, suggesting an immunomodulating role of LILRA4 in MCL.

Summary/Conclusion: In conclusion, we confirmed the bimodal LILRA4 transcript expression pattern in MCL samples at the time of diagnosis. We also demonstrated a significantly higher expression of LILRA4 in PB compared to BM, suggesting tissue-specific expression. The preliminary results from knockdown of LILRA4 indicated a potential immunomodulating role of LILRA4 through interaction with MIF, although the specific biological function of this gene in this setting remains to be elucidated.

References

Hansen, M. H., Cédile, O., Blum, M. K., Hansen, S. V., Ebbesen, L. H., Bentzen, H. H. N., . . . Nyvold, C. G. (2020). Molecular characterization of sorted malignant B cells from patients clinically identified with mantle cell lymphoma. Exp Hematol, 84 , 7-18.e12. doi:10.1016/j.exphem.2020.03.001

PB2159: FREQUENCY OF PRACTICE-CHANGING FINDINGS IDENTIFIED BY COMPREHENSIVE GENOMIC PROFILING IN NON-MYELOID HEMATOLOGIC MALIGNANCIES

C. Lin 1,* , K. Zhou 2 , M. Green 3 , B. Caughey 4 , M. Datto 3 , J. Strickler 4 , M. McKinney 1

1 Division of Hematologic Malignancies & Cellular Therapy; 2 Department of Medicine; 3 Department of Pathology; 4 Division of Medical Oncology, Duke University, Durham, United States of America

Background: Comprehensive genomic profiling (CGP) is increasingly used to guide management of myeloid and advanced solid malignancies, but its role in non-myeloid hematologic malignancies is less clear. Studies have found a high rate of potentially actionable variants by CGP in this population, but these do not always translate into clinical practice changes.

Aims: To determine the rate at which variants found on CGP changed clinical practice.

Methods: We retrospectively reviewed a cohort of 101 consecutive patients with non-myeloid hematologic malignancies at Duke, comprising a total of 105 samples that were sent for CGP by FoundationOne Heme (104) or HemeComplete (1) in 2014–2021. Variants were classified into evidence levels A–D according to the AMP/ASCO/CAP 2017 guidelines. For therapies, level A: FDA-approved / guidelines; B: expert consensus; C: clinical trial / FDA-approved in different tumor type; D: preclinical data. For diagnostic or prognostic significance, level A: guidelines; B: expert consensus; C: small studies. We further identified documented changes in clinical practice that occurred in direct response to CGP results.

Results: Commonly cited reasons for CGP included guiding therapy selection (27), identifying resistance mutations (19), refining prognosis (14), and clarifying diagnosis (11). Of the 105 samples sent for sequencing, 38 (36%) were from bone marrow, 37 (35%) peripheral blood, 7 (7%) skin, 5 (5%) lymph node, and 18 (17%) other. Ninety-two samples (88%) yielded at least one pathogenic or likely pathogenic variant. CGP resulted after death in 12 patients and within 30 days of death in another 11 patients.

Seventy-three out of 101 patients (72%) had at least one variant with therapy sensitizing, diagnostic, or prognostic significance (levels A–C) or associated with therapy resistance (levels A/D). Consistent with prior studies, no level A therapy sensitizing variants were identified. While 61 patients (60%) had a level B–C therapy sensitizing variant, only 6 patients (10%) were offered a biomarker-directed therapy. Offered therapies included ibrutinib for DLBCL with a CD78B mutation, romidepsin for T cell lymphoma with a TET2 mutation, and ruxolitinib for T cell lymphoma with JAK2 fusion. In contrast, the presence of a resistance mutation led to discontinuation of current therapy or influenced future therapy selection in 9 out of 13 patients (69%). Identified resistance mutations included alterations in BTK , PLCG2 , and BCL2 genes, which led to changes in therapy from a BTK inhibitor to venetoclax or vice versa. The absence of a resistance mutation influenced choice of therapy in another 4 patients. Among patients whose therapy was changed based on a sensitizing variant or resistance mutation, the median time from CGP results to a change in therapy was 26.5 days (range, 1-105). Sequencing results also helped clarify the diagnosis in 4 patients (e.g., truncated SOCS1 in DLBCL and ETV3-NCOA2 fusion in indeterminate cell histiocytosis) and led to medical genetics referrals in 3 patients ( BRCA2 , MSH6 , and VHL mutations).

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Summary/Conclusion: Comprehensive genomic profiling of non-myeloid hematologic malignancies identified variants of clinical significance in 72% of patients and led to changes in practice in 22% of patients. CGP was often sent late in the clinical course.

PB2160: ASSOCIATION OF BCL-2 EXPRESSING TUMOR CELLS WITH RESPONSE TO FRONTLINE CHEMOTHERAPY IN NODULAR SCLEROSIS HODGKIN LYMPHOMA

D. Diakonov 1,* , E. Perfilova 1 , M. Minaev 2

1 PATHOMORPHOLOGY LABORATORY, KIROV SCIENTIFIC RESEARCH INSTITUTE OF HEMATOLOGY AND BLOOD TRANSFUSION OF THE FEDERAL MEDICAL AND BIOLOGICAL AGENCY, Kirov; 2 Hematology department, Kaliningrad Region Central City Clinical Hospital, Kaliningrad, Russia

Background: The BCL-2 protein belongs to the family of intracellular protein factors that control the processes of apoptosis. The process of regulation of programmed cell death depends on the balance between pro- and anti-apoptotic proteins of this family. In most cases, activation of BCL-2 helps suppress apoptosis and increase the lifespan of tumor cells, as well as increase their resistance to chemotherapy. The ability to block apoptosis is a hallmark of many malignant neoplasms, including hematological ones.

Classical Hodgkin lymphoma (cHL) refers to a tumor of the hematopoietic system and has a relatively good response to treatment. However, in 15-20% of cases there is a lack of response to standard first-line chemotherapy. The data available in the relative literature regarding the relationship between BCL-2 expression in tumor cells and the effectiveness of treatment are contradictory. A number of authors confirms that the detection of this protein in Hodgkin and/or Reed-Sternberg cells (HRS) is an independent factor in the adverse outcome of cHL. But the inconsistency of the data indicates the advisability of further study of the association of the expression of the specified protein factor of apoptosis with the response to 1st line therapy.

Aims: To study the relationship of BCL-2 expression with the response to frontline therapy in nodular sclerosis classical Hodgkin lymphoma (NSCHL).

Methods: We used formalin-fixed paraffin-embedded (FFPE) lymph node samples of 37 patients with newly diagnosed NSCHL who were treated at the clinic of KRIHBT in the period from 2006 to 2018. Depending on the response to the 1st line therapy according to the BEACOPP-14 regimen, the subjects were divided into 2 groups: group 1 (n=17) included patients who achieved complete remission (CR). Group 2 (n=20) included patients with refractoriness to the first and subsequent lines of chemotherapy or minimal response to it, or patients who received autologous or allogeneic hematopoietic stem cell transplantation.

To identify BCL-2+ tumor cells an immunohistochemical staining method was used. Pathomorphological analysis and relative number counting of BCL-2+ tumor cells were performed in sequential fields of view in 100 HRS cells for each sample.

Results: According to the response to 1st-line chemotherapy, differences in the relative number of BCL-2+ tumor cells were established. In patients with a partial response (PR) to treatment, the number of positive cells was statistically significantly higher than in patients with CR: 80% (58-88) vs. 24% (8-42), respectively, p<0.001. The optimal threshold value for the proportion of BCL-2+ tumor cells was 46%. Based on the established threshold, all subjects were divided into two categories: with high (≥46%) and low (<46%) BCL-2 expression. Patients with a BCL-2+ cell count ≥46% are 18.4 and 59.5 times more likely to develop a PR and an adverse course, respectively, than those with a low threshold (p<0.05). The frequency of occurrence of BCL-2+ cells above the threshold level was detected statistically significantly more often in patients with frontline treatment failures of in relation to patients with CR: 17 (81%) versus 4 (19%) patients (p<0.001).

Summary/Conclusion: A high relative content of BCL-2+ tumor cells (≥46%) is associated with treatment failures in patients with NSCHL. Determining the proportion of neoplastic cells marked with this antibody could be used as an additional morphological criterion for predicting the course of the disease already at the stage of diagnosing patients in order to apply a personalized approach to therapy with BCL-2 inhibitors.

PB2161: CHARACTERISTICS OF AGGRESSIVE B-CELL LYMPHOMAS WITH MYC REARRANGEMENT

A. Ortuzar Pasalodos 1,* , M. Chicano 2 , F. Díaz-Crespo 3 , P. Muñiz 2 , R. M. Martín-Rojas 1 , I. Gómez-Centurión 1 , J. Menarguez 3 , M. Kwon 1 , J. L. Diez-Martín 1 , I. Buño 2 , C. Martínez-Laperche 1,2 , M. Bastos-Oreiro 1

1 Hematology, Gregorio Marañón General University Hospital; 2 Hematology, Gregorio Marañón Health Research Institute (IiSGM); 3 Pathology, Gregorio Marañón General University Hospital, MADRID, Spain

Background: MYC rearrangements ( MYC -R), genetic hallmark of Burkitt lymphoma (BL), have been identified in 5-15% of DLBCL and 30-60% of high-grade B-cell lymphomas (HGL). MYC -R in BL always involve an immunoglobulin (IG) gen ( IGH, IGK , IGL ), whereas in DLBCL and HGL non-IG partners are frequent. The prognostic significance of the MYC partner gene in non-BL is controversial .

Aims: We aimed to describe the histobiological features of the aggressive B-cell lymphomas (aBCL) with MYC -R identified in our center and explore their clinical and outcome differences within the context of MYC partner gene ( IGH vs non -IGH ).

Methods: We retrospectively collected data from all adult patients with a MYC -R aBCL diagnosed in our center between 2010-2021 . Diagnostic samples were reviewed by 2 expert hematopathologists. Fluorescence in situ hibridation (FISH) studies were performed either on paraffin tissue sections or bone marrow smears using Vysis LSI MYC, BCL2, BCL6 Dual color break apart probes and Vysis IGH/MYC/CEP 8 Tri-Color Dual Fusion probes (Abbott Molecular, USA). The study was approved by the local ethics committee.

Results: We identified 61 cases of MYC-R aBCL . Tissue sections were available for MYC-IGH FISH analysis in 57/61 cases, MYC partner was evaluable in 52 patients . The morphologic distribution was: 34 (64.5% ) cases with diffuse large B-cell histology, 3 (5.8% ) with intermediate-cell or high-grade features and 15 (29% ) with Burkitt histology . According to the current WHO classification (Table 1a) 28/34 large-cell morphology cases corresponded to double/triple hit (DH/TH) HGL because of additional BCL2 and/or BCL6 -R and 6/34 were DLBCL NOS. 2/3 intermediate-cell cases were DH/THL and 1/3 was a HGL NOS. 14/15 cases with Burkitt histology were diagnosed as BL and one case was reclassified into DHL due to concurrent BCL6-R.

Excluding BL, 38 cases (6 DLCBL NOS and 32 HGL ) were selected for further analysis. Among this subset MYC-IGH -R was detected in 17/38 (44.7% ) cases: 14 DH/THL and 3 DLBCL NOS (Table 1a). Noteworthy, 14/31 (45.2%) of all DH/THL and 3/6 (50%) of LBDCG NOS had an IGH partner. Considering cell of origin (COO) by Hans algorithm, MYC-IGH fusion was detected in 40% of Germinal center-derived (GC) lymphomas and in 62.5% of non-GC. Comparing clinico-biological data and outcome depending on MYC partner (Table 1b) our results show that patients with MYC-IGH fusion were significantly younger (p=0.019 ) and more likely to have multiple (≥2) extranodal (EN) sites (p=0.023). No other variable was significant. Referring to treatment, 33 patients (92%) received R-CHOP or R-EPOCH regimens, with no differences between groups. First line complete remission (CR) rates were 52% in both MYC-IGH and MYC-non-IGH groups . With a median follow up of 62 months (37-114), no significant association was observed between MYC -R partner and duration of response (p=0.784) , event free survival (EFS ) (p=0.933) or overall survival (OS ) (p=0.706). At last follow-up 15 patients remain alive in CR.

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Summary/Conclusion: Excluding BL, most patients with MYC-IGH rearrangement had diffuse large B-cell histology (93.8%) and were classified as DH/THL (82.3%). Among all DH/THL cases, 14/31 (45.2%) had IGH partner. MYC-IGH patients were younger and had more EN site involvement as MYC-non-IGH , but no differences in EFS and OS were noted. We are working to incorporate molecular techniques for a more precise analysis of BCL2, BCL6 and MYC translocations and their partners that permit a better understanding of the behavior of patients with aBCL.

PB2162: INCREASED OXIDATIVE STRESS IN DIFFUSE LARGE B-CELL LYMPHOMA

D. Antic 1,2,* , D. Djikic 3 , V. Otasevic 1 , O. Mitrovic-Ajtic 3 , V. Vukovic 1 , T. Suboticki 3 , V. Djurasinovic 1,2 , K. Tomic 1 , B. Mihaljevic 1,2 , V. Cokic 3

1 Lymphoma Center, Clinic for Hematology, University Clinical Center of Serbia; 2 Faculty of Medicine, University of Belgrade; 3 Department of molecular oncology, Institute for Medical Research, National Institute of Republic of Serbia, University of Belgrade, Belgrade, Serbia

Background: Oxidative stress is caused by imbalance between excessive production of reactive oxygen species and decreased capabilities of antioxidant system, and it is recognized as a feature in cancerogenesis, as well in hematologic malignancies. Previous studies have shown increasing expression of oxidative stress markers and antioxidant enzymes in lymph nodes progressing to aggressive lymphomas.

Aims: The aim of our study was to assess the clinical and prognostic significance of oxidative stress markers in patients with untreated diffuse large B-cell lymphoma (DLBCL).

Methods: We analysed 64 patients diagnosed with DLBLC during 2018 and 2019, while 27 healthy volunteers (51.9% males) served as a control group. The plasma sample and laboratory analyses were obtained prior to initiation of specific hematologic treatment. After completion of the therapy, the patients were followed up for up to 4 years and for each of them progression free survival (PFS) and overall survival (OS) were calculated. Malondialdehyde (MDA) and protein carbonyl (PC) were used as markers of oxidative stress in plasma of patients and volunteers, while catalase is used as an antioxidant marker.

Results: The mean patients’ age was 56.2 years (range, 20–87); 51.6% were males. Majority of patients were analysed before 1L therapy (n=61; 95,3%), and had following clinical stages: Ann Arbor stage I 23.4%, stage II 37.5%, stage III 15.6% and stage IV 23.4%. Majority of the patients had satisfactory performance status (73.5% had ECOG PS ≥1), bulky tumorous mass was present in 34.4% of patients, whereas 70.3% had extranodal localisation of lymphoma. MDA (6.66±2.7 nmol/ml) was significantly increased (p<0.001, 2.6-fold), while PC (4.29±2.7 nmol/mg) was also significantly increased (p=0.0027, 5-fold) in patients with DLBCL compared to healthy volunteers. In opposite, antioxidant catalase (0.194±0.06 IU/ml) was significantly reduced (p=0.0034, 1.9-fold) in patients with DLBCL. MDA was in significant (p<0.05) negative correlation with hemoglobin (r 2 =0.586) and LDH (r 2 =0.59) levels before chemotherapy. MDA was in significant (p<0.01) positive correlation with the age (r 2 =0.769) of patients with DLBCL at diagnosis. Moreover, MDA was in significant positive correlation with overall survival (p<0.01, r 2 =0.736) and progression-free survival (p<0.01, r 2 =0.736) of patients with DLBCL. PC was in negative correlation with the clinical stage (r 2 =0.103, p=0.146) and therapy response (r 2 =0.136, p=0.091) of DLBCL but did not reach significance.

Summary/Conclusion: The elevated oxidative markers and reduced antioxidant support oxidative stress in patients with DLBCL, while MDA can be a prognostic marker of overall survival.

PB2163: SCREENING FOR SUSPECTED AUTOIMMUNE LYMPHOPROLIFERATIVE SYNDROME (ALPS) PATIENTS IN CHILDREN WITH CHRONIC CYTOPENIAS AND NON-MALIGNANT LYMPHADENOPATHY: A STEP FOR BETTER STRATIFICATION.

L. Saleh 1,* , N. El-Masry 2 , E. El-Sayed 3 , M. Sarhan 4 , H. Abdel-ghaffar 1

1 Hematology section-Clinical pathology department, Mansoura University, Mansoura; 2 Damietta oncology institute, Damietta oncology institute, Damietta; 3 clinical microbiology section, Clinical pathology department, Mansoura University; 4 Pediatric Hematology, Mansoura University Children Hospital, Mansoura, Egypt

Background: Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune disorder characterized by chronic, benign lymphoproliferation, autoimmunity, mainly cytopenis, and an increased risk of lymphoma due to a genetic defect in the FAS derived apoptotic pathway. Accumulation of CD3+TCRab+CD4-CD8- double negative T-cells (DNT) in peripheral blood and tissue samples is a hallmark of the disease. Approximately 10-20% of ALPS patients are at highly risk of secondary malignancies. The exact prevalence of ALPS is still unknown. Additionally, it is often misdiagnosed due to variable phenotypic expressions. ALPS cases probably remain undiagnosed in our locality due to variable phenotypic expression, overlapping of symptoms with many other hematological disorders and limited availability of genetic screening.

Aims: This has been a trigger to start a registry for suspected ALPS patients for future genomic characterization. Through establishing a simple, cost effective preliminary diagnostic workup to find out the frequency of suspected ALPS patients among children attending Mansoura University Children Hospital (MUCH).

Methods: We investigated CD3+TCRab+CD4-CD8- DNT by multi colour flowcytometry in 36 patients with non-infectious, non-malignant lymphadenopathy.

Results: The suspected ALPS cases were 9/36 patients representing 25% with median CD3+TCRab+CD4-CD8- of 3.2% significantly different from the other group of patients with non-infectious, non-malignant lymphadenopathy 1.7%, P value <0.0001 . Those suspected ALPS patients showed different forms of cytopenias (55% anemia, 33% neutropenia and 11% thrombocytopenia).

Summary/Conclusion: our study identified 25% of patients to be categorized as suspected ALPS cases by fulfilling the primary criteria of diagnosis with association of accessory criteria which trigger in depth molecular investigation. Our results help the clinician to identify this group of patients to avoid diagnostic dilemmas and allow them to modify their therapeutic decisions.

PB2164: PROGNOSTIC IMPACT OF ONCOGENE DEK IN DIFFUSE LARGE B-CELL LYMPHOMA

R. Alonso Alonso 1 , M. Rodríguez 1 , L. M. Privette Vinnedge 2 , J. Serrano 3 , Y. Jiménez de las Pozas 4 , S. G Pedrero 5 , D. Laínez 5 , A. García Raso 6 , J. M. Alonso Dominguez 6 , A. López García 6 , R. Córdoba 6 , M. A. Piris 1 , P. Llamas Sillero 6 , J. Sánchez García 3 , J. Serrano Lopez 5,*

1 Pathology, Fundación Jiménez Díaz, Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain; 2 Oncology, Cancer and Blood Diseases, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, United States of America; 3 Hematology, Reina Sofía University Hospital/Maimonides Biomedical Research Institute of Córdoba (IMIBIC)/University of Córdoba, Cordoba; 4 Immunology, Hospital Universitario Fundación Jiménez Díaz UAM; 5 Experimental Hematology Lab, IIS-Fundacion Jimenez Diaz, UAM; 6 Hematology, Fundacion Jimenez Diaz University Hospital, Health Research Institute IIS-FJD, UAM, Madrid, Spain

Background: Diffuse Large B-Cell Lymphoma (DLBCL) is an aggressive hematologic malignancy that represents 25-30% of non-Hodgkin’s lymphomas (NHL). DLBCL can be subdivided according to its cell of origin (COO) into germinal center B-cells (GCB), activated B-cell (ABC) and non-classifiable (NC) subtypes, with the ABC subtype having the worst prognosis. Previous studies by our group describe that in normal hematopoiesis DEK knockout mice had fewer mature B cells than controls. In some hematological neoplasms, DEK is considered a biomarker for normal and malignant plasma cells, such as in Multiple Myeloma and Chronic Lymphocytic Leukemia, where the use of fludarabine and Nutlin-3 decreased DEK expression levels significantly in those patients with normal TP53 function.

Aims: Since the DEK oncogene has not been studied in DLBCL previously, the aim of this work is to study the prognostic impact of DEK mRNA expression levels in DLBCL.

Methods: This study included a total of 66 FFPE samples from DLBCL patients, 41 were collected from the HURS and 25 from the HUFJD. The COO categorization was carried out on the Nanostring platform applying the Lymphoma subtyping test(LST) algorithm. Using qRT-PCR, we quantified DEK gene expression and the median expression was used as the cut-off point. p53 status was analyzed by immununohistochemistry using an anti-TP53 antibody (clone: DO-7). Analysis statistics: χ 2 test to study associations. Correlation analyses were obtained by GEPIA. Kaplan-Meier to calculate survival curves. Multivariate analysis by Cox regression and Binary logic regression.

Results: Mean patient age was 68.5 years (range 40-89 years). 29 patients were classified as GCB subgroup, 22 patients in ABC and 15 as NC. High levels of DEK expression ( DEK High ) were found in 38 patients, and low expression levels in 28 ( DEK Low ). TP53 protein was positive in 15 patients, negative in 25 and not studied in 26. All p53+ patients had high DEK levels (TP53+/ DEK High ); however, 16 of the TP53- patients had low DEK expression levels (TP53-/ DEK Low ). GCB subgroup had a significant association between p53 status and DEK expression. However, in the ABC subgroup, in addition to the association between DEK and p53, significant associations with stage, response to treatment, relapse and death were also found. DEK expression correlates with the ABC subtype markers IRF4 and LIMD1 (R=0.58), BCL family protein markers (BCL6/BCL2)(R=0.49 and 0.41), poor prognosis markers (MYC/CD5)(R=0.54 and 0.31), and with nuclear transcription factor RELA(R=0.65), but does not correlate with NFkB pathway activation markers (IL2/IL6) and GCB subtype. DEK expression also correlated with TP53 (R=0.59). Kaplan-Meier survival curves show lower overall survival in DEK High patients in the entire cohort. This association was significant by the Log Rank test (p=0.02). Overall survival (OS) in ABC patients was significantly lower in DEK High and TP53+/ DEK High patients (p=0.005 and p=0.05 respectively); however, no significance was found in the GCB subgroup. Finally, DEK High at diagnosis was an independent marker to predict standard treatment response (OR=4.17, IC=1.18-14.73) and a risk factor for OS (HR=3.39, IC=1.21-9.52).

Summary/Conclusion: Patients diagnosed with DLBCL with high levels of DEK oncogene expression have worse responses to treatment and lower overall survival compared to patients with lower levels of expression. These associations were especially strong in the ABC subtype, where DEK expression also correlated with poor prognosis markers and was an independent predictor of treatment response.

PB2165: BIOINFORMATIC TOOL FOR IMMUNOMODULATING THE ONCOGENIC TRANSCRIPTOME OF ABC-DLBCLS

M. Rodríguez 1 , J. Cornago 2 , J. Serrano 3 , R. Alonso Alonso 1 , D. Morillo 2 , M. A. Perez Saenz 2 , J. M. Alonso Dominguez 2 , M. A. Piris 1 , J. Sánchez García 3 , P. Llamas Sillero 2 , J. Serrano Lopez 4,*

1 Pathology, IIS-Fundación Jiménez Díaz, Centro de Investigación Biomédica en Red de Cáncer (CIBERONC); 2 Hematology, Fundación Jimenez Diaz University Hospital, Health Research Institute IIS-FJD, UAM, Madrid; 3 Hematology, Reina Sofía University Hospital/Maimonides Biomedical Research Institute of Córdoba (IMIBIC)/University of Córdoba, Cordoba; 4 Experimental Hematology Lab, IIS-Fundación Jiménez Díaz, UAM, Madrid, Spain

Background: Activated B-cell (ABC) lymphomas emerged as a distinct molecular subtype within Diffuse Large B-Cell Lymphoma (DLBCL). Canonical and non-canonical constitutive activation of nuclear factor kappa B (NF-kB) signaling pathway has been widely reported in the ABCs, and mainly implicates gain-of-function mutations in many players downstream of the BCR receptor. This molecular entity is normally associated with poor clinical outcome after standard immunochemotherapy. The discouraging results obtained in several clinical trials using proteasome inhibitors, tyrosine kinase inhibitors, or immunomodulators, lead to an intense search for new, potentially druggable biomarkers in DLBCL. Immunosuppressive microenvironment is a hallmark in many solid tumors as well as in DLBCLs.

Aims: Herein, we searched for bioinformatic tools aim to repurpose drugs capable of modulating immune cells from transcriptomics dataset generated by RNA-seq analysis.

Methods: RNA-seq was applied to RNA samples from FFPE blocks of DLBCL patients, 4 of whom were previously classified as GCB and the remaining 4 as ABC, based on NanoString-LST. All ABC were negative for the MYD88L265P mutation (ABC-MYD88 non-L265P ) determined by allele-specific PCR (AS-PCR). Median of age was 66.5 years (range 40-82) and 71.5 years (range 53-85) for GCB and ABC, respectively. Indexed samples were run together on a NextSeq 500 instrument (Illumina Inc.) with a 2 × 150-bp paired-end protocol. An average of 25.8 million mapped reads was generated per sample. Next, bioinformatic tool DREIMT was utilized to predict drugs for intratumor immunomodulation. DREIMT employ FGSEA ES and Pairwise ES calculations to calculate the enrichment score (ES). P-value estimation is calculated by adaptive multi-level split Monte-Carlo scheme and is P-adjusted using Benjamin correction. DREIMT apply a drug prioritization score (T, tau, ranging +100 to -100) following the approach described by LINCS L1000 team. To consider strong hypotheses for the drug profile-immune gene expression associations, we considered tau> 99 and selected top 26 candidates ranked by tau score. In the same way, we examined tau> -99 and chosen top 26 candidates ranked by tau score

Results: We identified 79 differentially expressed genes (DEGs) obtained after RNA-seq analysis. GSEA showed two main enriched routes implicated in metabolism and immune pathways in the ABC lymphomas, tumor suppressor genes (TSGs) as MAPK10 and GRHL1 were downregulated while anti-inflammatory such as ALOX15B was upregulated. Triggers of Th2 response as CHIT1 and CHI3L-1 were upregulated, like serine protease inhibitors SERPINA1 and SERPINA5 . The proto-oncogene FRG was upregulated. DREIMT applied on the 79 DEGs interestingly showed its capacity to selected drugs involved in augmenting anti-tumor immune response. Among them, Bucladesine, Labetalol or Glimepiride boost Th1 response and macrophages M1. On the other hand, Moracizine and Voriconazole inhibit of expression of genes encoding inflammatory molecules in monocytes

Summary/Conclusion: RNA-sequencing-derived transcriptome described new tumor-resistant genes dysregulated in the ABC-MYD88 non-L265P which promote the maintenance of tumor cells. DREIMT enables us to propose a prioritized list of drugs as the best theoretical therapeutic candidates to treat these patients in order to immunomodulating the oncogenic transcriptomic of ABC-DLBCLs. Of note, most of the proposed drugs are not being yet considered in the clinical practice of this cancer subtype opening up the approach of preclinical studies and new treatment possibilities

PB2166: TET ENZYMES PROTECT AGAINST FATAL LYMPHOPROLIFERATION

S. Spoeck 1,* , N. Kinz 1 , A. Villunger 1 , V. Labi 1

1 Department of Developmental Immunology, Medical University of Innsbruck, Innsbruck, Austria

Background: The most common epigenetic modification in eukaryotic genomes is methylation of cytosine. TET enzymes, of which TET2 and TET3 are expressed throughout the immune system, facilitate DNA demethylation via oxidation of 5-methylcytosine in DNA to 5-hydroxymethylcytosine and iterative oxidative species. These oxidized variants ameliorate chromatin accessibility and transcriptional activity by enabling DNA demethylation or serving as epigenetics marks on their own.

In the immune system and beyond, TET enzymes serve to establish and adapt cell identity programs during lineage specification. In human leukemias and lymphomas, TET2 is one of the most frequent mutated genes, and TET3 gene variants are associate with the most common autoimmune disorder, systemic lupus erythematosus (SLE). Thus, in humans, loss of TET activity associates with different pathologies of the hematopoietic and immune systems. In mice, TET2 loss-of-function promotes inflammation and boosts oncogene-driven lymphomas and leukemias. However, the kinetic and nature of molecular and cellular changes upon TET loss-of-function remain ill-defined.

Aims: Ultimately, our work aims to identify therapeutic entry points for hematologic and immune pathologies with impaired TET activity.

Methods: In our lab, we employ cell culture systems and pre-clinical mouse models to elucidate pathophysiological mechanisms that underlie TET loss-of-function driven pathologies originating from B lymphocytes.

Results: We have established that TET2 and TET3 synergize in stabilizing DNA methylation patterns and transcriptional programs that guard cell identity while B lymphocytes transit through developmental stages, allowing the generation of functional B lymphocytes. Furthermore, the joint deletion of TET2 and TET3 specifically in antigen-activated B lymphocytes drives combined immunodeficiency and autoimmunity already in young mice. When aging, a fatal lymphadenopathy with 100% penetrance arises, which also involves TET-proficient immune cells such as T lymphocytes.

Summary/Conclusion: Our data suggest that diminished TET activity in antigen-activated B lymphocytes critically determines optimal humoral immune responses and predisposes to fatal lymphoproliferation.

PB2167: NGS CLONALITY TESTING FOR A DIFFERENTIAL DIAGNOSIS OF MYCOSIS FUNGOIDES

Y. Sidorova 1,* , L. Gorenkova 2 , B. Biderman 1 , N. Ryzhikova 1 , A. Sudarikov 1

1 Department of Molecular Hematology; 2 Department of Hematology and High-dose Chemotherapy, National Research Center for Hematology, Moscow, Russia

Background: Mycosis fungoides (MF) is a primary epidermotropic lymphoma of the skin, accounting for up to 75% of all skin lymphoproliferative diseases. Diagnosis of MF in the initial stage (Ia-Ib; IIa) or its erythrodermic forms (EMF) is a difficult task. In most cases, neither histological/immunohistochemical examination, nor PCR methods for determining clonality give a definite conclusion. It is known that in the early stages, the PCR method with fragment analysis (PCR-FA) makes it possible to detect T-cell clonality in 75% of cases. On the other hand, in non-tumor dermatoses (ND), clonal products are detected in 10-15% (false-positive result). However, next-generation sequencing (NGS) of the TCRG and TCRB genes may provide more accurate identification of the T cell clone and quantification of the clonal product at the lesion site.

Aims: To compare the robustness of NGS and PCR-FA methods for T-cell clonality evaluation in MF and ND.

Methods: Skin biopsy specimens and peripheral blood of patients with initial stages of MF (n-7), EMF (n-3), and ND (n-17) were included in the study. Evaluation of clonality for the TCRG and TCRB genes was carried out in parallel by the PCR-FA and NGS. Data processing and T-cell clone fraction/frequency calculation were performed according to the MiXCR protocol ( https://github.com/milaboratory/mixcr ).

Results: PCR-FA and NGS gave the same results in all samples tested. T-cell clonality was detected in 6 out of 10 patients with MF and EMF. In these patients, the fraction of the dominant TCRG clone ranged from 5.5 to 83.4% (mean 32.3%), for TCRB - 6.1 to 47.9% (mean 20.8%). In 2/7 patients with MF and 2/3 with EMF, clonality was not detected, while the fraction of the dominant T-cell clone did not exceed 4.2%. In two patients with MF and no T-cell clonality, skin samples were examined at disease progression at 6 years and 3 years from the initial biopsy, and a dominant tumor clone was found. These dominant clones were represented by only 2.2% ( TCRG ) and 4.2% ( TCRB ) in the initial biopsy for one patient, and 0.26% for the second. In 3/7 patients with the initial stages of MF, a tumor clone was detected in the blood in the amount of 0.022-0.054%. All 3 patients with the minimal disseminated disease at the onset, rapidly progressed to the advanced stages of the disease. In the control group (ND) dominant TCRG clone fractions were 0.49-4.8% (mean 1.93%), for TCRB - 0.9-6.1% (mean 3.11%). The results are shown in Figure 1.

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Summary/Conclusion: Despite a small sample of patients, the following trends were noted: 1 - patients with the initial stages of MF with similar clinical and histological features, are diverse in the number of tumor T-lymphocytes infiltrating the skin (from 0.26% to 83.4%); 2 - the initial stage of MF with the minimal disseminated disease, might have an unfavorable outcome; 3 - comparing to PCR-FA NGS might not provide further improvement in the specificity and sensitivity of T-cell clonality evaluation for the differential diagnosis in MF.

PB2168: BRUSATOL INDUCES APOPTOSIS IN AGGRESSIVE LYMPHOMA CELLS IN VITRO AND SYNERGIZES WITH VENETOCLAX

M. Szmyra 1,* , S. Jantscher 1 , S. Haingartner 1 , K. Pansy 1 , B. Uhl 1 , H. Greinix 1 , P. Neumeister 1 , M. Dengler 2 , B. Klösch 3 , A. Deutsch 1

1 Division of Hematology; 2 Division of Oncology, Medical University of Graz; 3 Ludwig Boltzmann Institute for Arthritis and Rehabilitation, Graz, Austria

Background: Aggressive lymphomas include Burkitt lymphoma, follicular lymphoma (grade 3), mantel cell lymphoma and the most frequent type - diffuse large B-cell lymphoma (DLBCL). These types of lymphoma represent the most common lymphoid malignancies in adults and their incidence rate is still increasing. Despite chemoimmunotherapy (R-CHOP: Rituximab, Cyclophosphamide, Doxorubicin Hydrochloride, Vincristine Sulfate and Prednisone) more than one-third of patients experiences treatment failure, due to primary refractory disease or relapse, thus indicating the great need for the development of novel therapeutic strategies. One of the investigated drugs for these disease entities is Venetoclax (FDA approved Bcl-2 inhibitor), which shows an overall response rate of 44% in a phase 1 trial. In order to improve the effectiveness of the available treatment, we tested a flavonoid named Brusatol, that has anti-cancer activity in human cancer cells including leukemic cells as previously shown.

Aims: Here, we investigated the potential of Brusatol to treat aggressive lymphomas as a single agent or in combination with Venetoclax.

Methods: Seven cell lines (BL2, Jurkat, Karpas422, Raji, RI-1, SuDHL4 and U2932) representing different types of aggressive lymphomas (Burkitt lymphoma, germinal-center B-cell-like DLBCL, activated B-cell DLBCL) and T-cell leukemia, were treated with increasing concentrations of Brusatol (up to 10µM) and IC50 values were determined. After 24 and 48 hours of treatment with Brusatol (50nM and 250nM), induction of apoptosis (Annexin V staining, Caspase-3 cleavage, PARP cleavage) and changes in cell cycle were assessed. Furthermore, samples from untreated and Brusatol-treated cells were collected for Western blot analysis. Moreover, co-treatment of Brusatol and Venetoclax for 24 hours was performed in four cell lines (BL2, SuDHL4, RI-1 and U2932) and induction of apoptosis was analyzed by Annexin V staining.

Results: In all seven cell lines, Brusatol caused cell growth inhibition in a concentration-dependent manner. However, they could be grouped into more (BL2, Jurkat, Raji, SuDHL4) and less (Karpas422, RI-1, U2932) Brusatol-sensitive cell lines in the apoptosis assays. Moreover, cell cycle analysis revealed an increase of cells in sub-G1 phase – indicative of cell death induction – in the more sensitive cell lines. Interestingly, Western blot analysis of Brusatol-sensitive cell lines showed decreased protein levels of pro-survival apoptosis-regulating proteins Bcl-2, Bcl-XL, and Mcl-1. Furthermore, we detected reduced p53 and Myc protein expression in Brusatol-sensitive cell lines upon treatment. Notably, the protein expression profile of untreated cells indicates that cell lines with higher Myc-levels are more sensitive to Brusatol treatment. Interestingly, the combination of Brusatol with the Bcl-2 inhibitor Venetoclax synergistically enhanced the cell killing, both in more (SuDHL4, BL2) and less sensitive (RI-1, U2932) cell lines to Brusatol.

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Summary/Conclusion: Our data indicate that Brusatol is able to efficiently induce cell death in aggressive lymphoma cells by reducing the expression of pro-survival proteins. Interestingly, cells with higher Myc-levels were especially sensitive to this flavonoid. Additionally, the combination of Brusatol with Venetoclax results in enhanced induction of apoptosis. Thus, our study suggests that Brusatol, alone or in combination with Venetoclax, represents a very interesting agent to further develop regarding novel anti-lymphoma therapies.

PB2169: METHYLATION OF P53-RESPONSIVE ONCOSUPPRESSIVE MICRORNA GENES IN DIFFUSE LARGE B-CELL LYMPHOMA TUMOR TISSUE

E. Voropaeva 1,* , T. Pospelova 2 , M. Churkina 2 , O. Berezina 2 , A. Gurazheva 1 , V. Maximov 1

1 Research Institutе of Internal and Preventive Medicine - Branch of the Federal State Budget Scientific Institution The Federal Research Center Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences; 2 State Medical University, Novosibirsk, Russia

Background: Hsa-miR-203, hsa-mir-129, hsa-miR-34a, hsa-miR-34b and hsa-miR-34c are oncosuppressive microRNAs through which the antitumor activity of p53 is realized.

Aims: To identify the frequency, specificity and clinical significance of MIR-34B/C, MIR-34A, MIR-203 and MIR-129-2 genes methylation in Diffuse Large B-cell Lymphoma (DLBCL).

Methods: Methylation status of the genes in tumor tissue (n=73) was studied by methyl-specific PCR and methyl-sensitive analysis of high-resolution melting curves. DNA isolated from lymph node biopsies with reactive polyclonal B-cell proliferation (n=11) was used to control the tumor-specificity of the detected methylation. The quantitative analysis of the combined methylation of the studied genes was carried out with the calculation of the one-sided Fisher exact criterion (p-value) and the frequency of false discoveries (FDR) (q-value) was calculated using the Benjamin-Hochberg procedure.

Results: Aberrant methylation of the promoters of the studied genes can serve as a significant mechanism for reducing the miR-34B/C, miR-34A, miR-203 and miR-129 micro-RNAs expression in the tumor tissue of DLBCL. It is occurs in combination and is tumor-specific. Thus methylation of MIR-129-2, MIR-203, MIR-34A and MIR34B/C in lymphoma samples occurred with a frequency of 67%, 66%, 27% and 62%, respectively, and there was no reactive lymph node tissue. Combined methylation of MIR-203, MIR-129-2 and MIR-34B/C genes (p<0.013, q<0.020), as well as pair of MIR-34B/C and MIR-34A genes (p=0.010, q=0.029) was detected.Methylation of MIR-34A was associated with an unfavorable prognosis according to the International Prognostic Index (p=0.002), and methylation of MIR-34B/C (p=0.026) and MIR-203 (p=0.011) was associated with a Ki-67 expression level of more than 45% in tumor tissue.

Summary/Conclusion: Aberrant methylation of oncosuppressive microRNA genes associated with underlying p53 signaling pathways is a potentially useful molecular biomarker in the diagnosis, prognosis of tumors and the development of a strategy for targeted therapy of DLBCL.

PB2170: HIGH EXPRESSION OF AP2M1 CORRELATES WITH WORSE PROGNOSIS BY REGULATING IMMUNE MICROENVIRONMENT AND DRUG RESPONSE TO R-CHOP IN DLBCL

X.-D. Liu 1 , Y.-Z. Liang 1 , Y.-S. Piao 2 , L. Wang 1,*

1 Hematology; 2 Pathology, Beijing Tongren Hospital, Capital Medical University, Beijing, China

Background: DLBCL (diffuse large B cell lymphoma) is the most common subtype of non-Hodgkin lymphoma (NHL). After anthracycline-based chemotherapy, more than 70% of DLBCL patients can get complete response or partial response, but only 50% - 60% of patients can survive for a long time. Adaptor Related Protein Complex 2 Subunit Mu 1 (AP2M1) encodes a subunit of the hetero tetrameric coat assembly protein complex 2 (AP2), which required for the activity of a vacuolar ATPase and may also play an important role in regulating the intracellular trafficking and function of CTLA-4 protein. There are few reports on the relationship between DLBCL and AP2M1. Meanwhile, it is unclear whether it is related to the pathogenesis of DLBCL.

Aims: Here, in this study, the relationship between DLBCL and AP2M1 and its effect in DLBCL chemotherapy were studied.

Methods: Using public datasets of DLBCL from both GEO and TCGA databases, we analyzed the role of AP2M1 in mediating chemoresistance to R-CHOP and its correlation with various clinical parameters and prognosis. By using various R packages, we evaluated the role of AP2M1 on regulating tumor immune microenvironment. Moreover, FFPE samples of DLBCL from Beijing Tongren Hospital were used to valiate our findings.

Results: We found that the expression of AP2M1 was significantly increased in DLBCL, which was correlated with poor prognosis and a variety of clinical indicators. On the basis of enrichment analysis, it was found that AP2M1 may be related to intracellular receptor signaling pathway. Through immune analysis and drug prediction, we found that the expression of AP2M1 affected the immune environment and drug response of DLBCL, which further revealed the important role of AP2M1 in DLBCL. Using FFPE DLBCL samples from patients treated in our center, we validated the above findings that high expression of AP2M1 correlated with inferior survival outcomes and affected sensitivity to R-CHOP treatment.

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Summary/Conclusion: In this study, we found that the expression of AP2M1 could affect the prognosis of DLBCL patients by affecting the immune environment, and the expression of AP2M1 affected the response of many drugs in DLBCL treatment, indicating AP2M1 as a potential therapy target in DLBCL.

PB2171: THE NOVEL CSN5/JAB1 INHIBITOR CSN5I-3 SUPPRESSES CELL GROWTH AND UPREGULATES GENE EXPRESSION OF TYPE-I INTERFERONS (IFN) IN ANAPLASTIC LARGE CELL LYMPHOMA (ALCL)

I. Xagoraris 1 , G. Kokaraki 1 , K. Stathopoulou 1 , F. X. Claret 2 , E. Drakos 3 , G. Z. Rassidakis 1,*

1 Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden; 2 Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America; 3 Department of Pathology, University of Crete Medical School, Heraklion Crete, Greece

Background: The CSN5/Jab1 (cJun activation domain-binding protein 1), initially discovered as a cJun coactivator, represents the fifth component of an evolutionary highly conserved 8 subunit protein complex, named COP9 signalosome (CSN5). Accumulating evidence suggests that the CSN5/Jab1 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via CDK inhibitors. We have previously shown that CSN5/Jab1 protein is overexpressed in T-cell lymphomas with the highest frequency of positive tumors being observed in both ALK+ and ALK- anaplastic large cell lymphomas (ALCL). Targeting CSN5/Jab1 activity in cancer is now possible since a novel inhibitor, namely CSN5i-3 (Novartis), has been developed for clinical use. The potential role of CSN5/Jab1 in modulation of anti-tumor immune responses in cancer, and particularly in ALCL, is unknown to date.

Aims: To investigate the effects of the selective CSN5i-3 inhibitor in cell growth and type-I interferon - associated anti-tumor immune responses in ALCL.

Methods: The ALCL in vitro system included 5 cell lines (3 ALK+ and 2 ALK- ALCL cell lines). Cell growth and viability were assessed by trypan blue and MTT assays. The effects of the CSN5i-3 on G1-S cell cycle progression were assessed by BrdU incorporation assay and on apoptosis by Annexin V/AAD staining and flow cytometry. Expression of various cell cycle regulating proteins at baseline and experimental conditions were analysed by Westem blot analysis. Gene expression of type I interferons, including interferonbeta (IFN-β), CXCL10 and interferon gamma (IFN-y), as well as, STING and a control gene (GAPDH) at the RNA level, were analysed with qRT-PCR. The ALCL cell lines were treated with the specific CSN5i-3 inhibitor at different concentrations. Silencing of the CSN5/Jab1 gene was performed using transient transfection with specific siRNA construct using the Amaxa Nucleofector System (Lonza).

Results: Treatment of ALCL cell lines with increasing concentrations of CSN5i-3 resulted in decreased protein levels of CSN5/Jab1, which was associated with significantly decreased cell growth but slight increase in apoptosis (10-15%). The effects on cell growth linked to downregulation of cell cycle progression were associated with upregulation of the CDK inhibitors p21, p27 and p57. Similarly, CSN5/Jab1 gene silencing resulted in decreased cell growth associated with increased expression of similar cell cycle inhibiting proteins. In addition, inhibition of CSN5/Jab1 activity by CSN5i-3 resulted in increased IFN-β and IFN-y mRNA levels in ALK+ ALCL cells, and CXCL10, IFN-y and STING mRNA levels in ALK- ALCL cells. Similarly, knocking down CSN5/Jab1 gene by transient transfection with siRNA led to upregulation of type-I interferons, particularly IFN-β and CXCL10 in ALCL, suggesting that the effects of CSN5i-3 can be attributed to specific inhibition of CSN5/Jab1 activity.

Summary/Conclusion: The novel inhibitor CSN5i-3 sufficiently downregulates expression of CSN5/Jab1 oncogene, inhibits cell cycle progression and contributes to type-I interferon - associated anti-tumor immune response in ALK+ and ALK- ALCL. These preclinical findings suggest a potential role of CSN5i-3 in molecular therapeutics and immunotherapy in T-cell lymphomas.

PB2172: IRON OXIDE NANOPARTICLES INHIBIT DIFFUSE LARGE B LYMPHOMA CELL SURVIVAL AND INVASION

L. Yan-Li 1,* , H. Qi-Tang 2 , H. Quan-Quan 2 , W. Zhao-Feng 2

1 Pathology, Anhui Medical University, The Second Affiliated Hospital of Anhui Medical University; 2 Pathology, Anhui Medical University, Hefei, Anhui, China

Background: Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma world wide. It is a phenotypically and genetically heterogeneous disease, accounting for 30-40% of all cases. 50%-70% of patients can be cured by the R-CHOP regimen, but nearly one-third of patients develop relapsed or refractory disease. Until now, the Food and Drug Administration (FDA)-approved iron supplement ferumoxytol and other iron oxide nanoparticles have been used for treating iron deficiency, as contrast agents for magnetic resonance imaging and as drug carriers. But recently, more studies have revealed a hidden intrinsic therapeutic effect of ferumoxytol on tumors, but the role and mechanism in DLBCL has not been reported yet.

Aims: The purpose of this study is to investigate the effect of iron oxide nanoparticles on DLBCL cells proliferation and migration by in vitro and in vivo.

Methods: Different concentrations of iron oxide nanoparticles were applied to DLBCL cell lines, such as SUDHL-2 and SUDHL-4 cells, and control cells were treated with equal amounts of PBS. The proliferation, migration, apoptosis and reactive oxygen (ROS) were detected by CCK8, transwell, flow cytometry (FC) and Multifunctional Fluorescence Enzyme Marker, respectively in vitro. The engraftment, tumor growth, dissemination and survival time were observed in BALB/c nude mice and NOD-SCID mice.

Results: Iron oxide nanoparticles inhibited DLBCL cells proliferation, migration and anti-apoptosis ability. The mechanism involved in the proliferation and migration of DLBCL cells by promoting the production of ROS. Furthermore, administration of iron oxide nanoparticles decreased tumor growth and dissemination of DLBCL cells, and increased survival time in the xenograft model.

Summary/Conclusion: Our study demonstrates that iron oxide nanoparticles cound inhibit DLBCL cells proliferation, migration, and promote apoptosis. The supplement of iron may, therefore, be a potential target for anticancer therapy in DLBCL.

PB2173: IDENTIFICATION OF POTENTIAL THERAPEUTIC TARGETS OF BAICALIN INTERVENING GASTROINTESTINAL ACUTE GRAFT-VERSUS-HOST DISEASE BASED ON NETWORK PHARMACOLOGY AND WHOLE TRANSCRIPTOME SEQUENCING

J. fu 1,* , J. lin 2 , L. xingyan 2 , C. xing 2

1 Shandong University of Traditional Chinese Medicine; 2 The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China

Background: Acute graft-versus-host disease (aGVHD) is one of the major complications of allo-HSCT 1 . Up to 60% of patients have gastrointestinal (GI) involvement, which may result in secretory diarrhea and abdominal pain 2 . Corticosteroid therapy is most often used as first-line treatment in patients, while the treatment is very limited 3 . Baicalin is an extract from huangqin, which exerts various protective effects on the intestinal system in modern studies 4 . However, its chemical and pharmacological mechanisms remain to be elucidated. Therefore, we try to characterize the potential molecular mechanism of baicalin intervening aGVHD with the combination of network pharmacology prediction with high-throughput sequencing.

Aims: To identify potential therapeutic targets of baicalin intervening aGVHD and reveal its pharmacological mechanisms.

Methods: The potential targets of baicalin were obtained from the TCMSP database, Swiss Target Prediction databaicalinse and Pharm Mapper. 4 serum samples from aGVHD patients and 4 control samples were sent for high-throughput sequencing. Then genes with significant differences (P<0.001 and |logFC|>2) were selected and intersected with therapeutic targets of baicalin. Potential targets were identified to establish interaction networks by STRING and Cytoscape. GO enrichment and KEGG pathway analyses were performed and visualized in R software. Molecular docking was performed to visualize the patterns of interactions between the baicalin and associated targeted proteins. Subsequently, transfer of mononuclear cell suspension (bone marrow + spleen) from Balb / c mice into CB6F1 mice irradiated with 60co X-ray via tail vein injection. Model group and treatment group were given 0.9 sodium chloride and baicalin by gavage respectively. Observation of pathological sections and immunohistochemistry (IHC) of core targets were executed based on the result of network pharmacology analysis.

Results: 1. Targets Identification of baicalin on aGVHD

In total, 293 targets of baicalin were obtained and 1025 differently expressed genes were identified (Fig 1). Then, 23 overlapping genes were extracted with 17 up-regulated genes and 6 down-regulated genes (Tab 1).

2. Network and Pathway Enrichment Analysis

MMP9 (degree=9) and CCNA2 (degree=7) showed significant importance (Fig 2A). MAPK14(degree=14) and SRC (degree=12) were enriched in multiple pathways (Fig 2B). The biological process was mainly associated with inflammation, including regulation of inflammatory response (GO:0050727), positive regulation of cytokine production (GO:0001819) (Fig 3A). KEGG enrichment analysis revealed that the IL-17 signaling pathway was closely associated with baicalin intervention for aGVHD (Fig 3B).

3. Molecular docking between the core genes and baicalin

Four core targets (MAPK14, SRC, MMP9 and CCNA2) were choosed to dock with baicalin (Fig 4) and MMP9 bound to baicalin best.

4. Experimental Validation

Pathological observation showed that the mice with acute graft versus host disease in treated group had more complete intestinal mucosa (Fig 5A, B). Baicalin treatments decreased MMP9 protein levels in small intestinal tissues detected by IHC (Fig 5C, D).

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Summary/Conclusion: Our study suggested baicalin conspicuously demonstrates multiple curative effects in aGVHD correlating with intricate and innumerable mechanisms and signaling pathways, mainly by mediating immune response pathways induced by inflammation.

PB2174: ON THE ROBUSTNESS OF THE BUCCAL EPITHELIUM FOR ASSESSING RECIPIENT DNA PROFILE IN PATIENTS AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION

N. Risinskaya 1,* , A. Yushkova 1 , F. Shikhveledova 1 , M. Drokov 1 , L. Kuzmina 1 , A. Sudarikov 1 , E. Parovichnikova 1

1 National Medical Research Center for Hematology, Moscow, Russia

Background: Сommonly DNA from the buccal epithelium of patients is used to confirm the germinal nature of mutations, as well as the reference of the recipient DNA for monitoring chimerism after hematopoietic stem cell transplantation (HSCT). We came across the cases where mutation load calculated for DNA isolated from the buccal epithelium did not correspond to either the heterozygous or homozygous variant. In addition, we accidentally noticed in some patients after BMT a significant admixture of donor DNA in the samples isolated from the buccal epithelium. These facts prompted us to investigate post-transplant chimerism in the buccal epithelium.

Aims: To evaluate the relevance of the buccal epithelium swab as a reliable source for a reference of the recipient DNA profile in patients after HSCT.

Methods: The study included 63 patients admitted to the National Research Center for Hematology for allogeneic HSCT from 2007 to 2022, with complete donor chimerism in the blood. Buccal epithelium swab was taken an OLDANS plastic-viscose probe-tampon (Russia). Cells were suspended and lysed in 20 μl of buffer containing 0.5% NP40, 50 mM Tris (pH 8.3), 75 mM KCl, and 20 μg proteinase K. Short tandem repeats (STR) profiling was carried out by polymerase chain reaction using a lyophilized CorDIS Plus multiplex kit (19 polymorphic STR markers and the human amelogenin X\Y locus), followed by separation of PCR products by capillary electrophoresis. Chimerism was calculated based on the percentage of unique donor and recipient markers in informative STR loci using GeneMapper v.4-0 software.

Results: Of 63 patients, only 14 (22%) had a 100% recipient STR profile in the buccal epithelium. Mixed donor-recipient chimerism was found in the buccal epithelium of 49 patients with the proportion of own recipient DNA ranging from 11% to 98%. A re-examination of the buccal epithelium was performed a month later for 12 patients. Of these, in three patients with previously mixed chimeras 100% recipient STR profile was detected. In two patients the balance in the mixed chimera shifted towards the recipient. One patient lost 100% recipient STR profile and become chimera with 43% of the recipient. In six cases, the balance of donor and recipient DNA in the buccal epithelium remained almost the same for two consecutive measurements (Figure 1).

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Summary/Conclusion: We believe that depending on the condition of the buccal epithelium and the technique of taking a swab from patients after HSCT, lymphocytes of donor origin may contaminate the sample. Therefore, the usage of buccal epithelium swab as the only reference material for the recipient STR profile or for confirming the germinal nature of mutations should be avoided in patients after HSCT.

PB2175: TRYPAN BLUE AND FLOW CYTOMETRY CELL VIABILITY TESTING OF PACKED OR DILUTED CRYOPRESERVED AT -80ºC HEMATOPOIETIC STEM CELLS SHOW SIMILAR RESULTS

I. Tonev 1,* , S. Simeonov 1 , M. Ilieva 1 , M. Muhtarova 1 , R. Vladimirova 1 , N. Andreinski 1 , T. Yancheva 1 , M. Guenova 1 , M. MIncheff 1

1 National Specialized Hospital for active treatment of hematological diseases, Sofia, Bulgaria

Background: Trypan blue exclusion and flow cytometry were compared for testing of cell viability of hemopoietic stem cells (HSCs) cryopreserved at -80ºC. Stem cells were frozen and stored with 5% final DMSO concentration with different degree of cell packing.

Aims: We wanted to evaluate the difference in viability results with folw cytometry and trypan blue exclusion with different degree of cell packing.

Methods: We analyzed 47 samples stored at -80ºС in a cryoprotectant solution with final concentrations of 5% DMSO, 3.6% hydroxyethyl starch (HES 450 000 mw) and 3% of human serum albumin. The cell concentration in the frozen suspension ranged between 69х10 6 /ml and 665х10 6 /ml (median 310,25х10 6 /ml). The cell viability was tested simultaneously by trypan blue exclusion test and by flow cytometry with 7 -aminoactinomycin D (7-AAD) without lysing solution.

Results: Both viability tests gave similar results. When tested by trypan blue, the viability was 95,5% (80-99%), and by flow cytometry was 95,92% (89,1-98,4%). The additional statistical Spearman’s Rho analysis does not show statistically significant correlation between viability results, performed with either test, and the degree of cell packing.

Summary/Conclusion: Flow cytometry and trypan blue tests show similar viability results. Since trypan blue testing is well established, has lower cost, and can be performed faster, we accepted it as a routine for our further analyses of cell viability of packed prior to freezing stem cell concentrates. HSC viability after storage at -80ºС with 5% final DMSO concentration is very good and is not affected by higher cell packing thus allowing the reduction of infused DMSO and the side effects (cardiac, neurologic, respiratory, etc.) during transplantation.

PB2176: AN AUSTRALIAN/NEW ZEALAND RANDOMIZED TRIAL OF POST-TRANSPLANT CYCLOPHOSPHAMIDE IN MATCHED RELATED PERIPHERAL BLOOD STEM CELL TRANSPLANTS FOR ACUTE LEUKAEMIA OR MYELODYSPLASIA – ALLG BM12

D. Curtis 1,* , S. Patil 1 , D. Purtill 2 , D. Ritchie 3 , D. Yeung 4 , C. Lewis 5 , E. Wong 6 , J. Moore 7 , T. Perera 8 , M. King 9 , R. De Abreu Lourenco 10 , O. Morrissey 11 , G. Hill 12 , J. Reynolds 13 , S. Tey 14 , D. Gottlieb 15

1 Clinical Haematology, Alfred Health, Melbourne; 2 Haematology Service, Fiona Stanley Hospital, Perth; 3 Clincial Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne; 4 Haematology, Royal Adelaide Hospital, Adelaide, Australia; 5 Haematology, Auckland City Hospital, Auckland, New Zealand; 6 Haematology, Austin Health, Melbourne; 7 Haematology, St Vincents Health Network, Sydney, Australia; 8 Haematology, Wellington Blood and Cancer Centre, Wellington, New Zealand; 9 School of Psychology, University of Sydney; 10 Centre for Health Economics, University of Technology Sydney, Sydney; 11 Infectious Diseases, Alfred Health, Melbourne, Australia; 12 Medicine, Fred Hutch, Seattle, United States of America; 13 Biostatistics Platform, Monash University, Melbourne; 14 Haematology and Bone Marrow Transplantation, Royal Brisbane and Women’s Hospital, Brisbane; 15 Haematology, Westmead Hospital, Sydney, Australia

Background: Graft versus host disease (GVHD) remains a major clinical problem in allogeneic peripheral blood stem cell (PBSC) transplantation, even when matched-siblings are used as donors. The cumulative risk of clinically significant acute (grade III-IV) and chronic (moderate-severe) GVHD is 40% after PBSC matched-sibling transplant with calcineurin inhibitor-based prophylaxis. Post-transplant cyclophosphamide (PTCy) is increasingly used for matched-sibling PBSC transplants despite the absence of randomized data. While retrospective data suggest PTCy show low rates of chronic GVHD, potential disadvantages of PTCy include increased risks of CMV reactivation, veno-occlusive disease (VOD), haemorrhagic cystitis and exposure of donor haematopoiesis to high-dose alkylator resulting in DNA damage and potential for secondary myeloid neoplasia. Finally, PTCy may suppress graft-versus-leukemia effects in the setting of matched donor cells, potentially increasing the risk of relapse. Therefore, randomized trials are needed before widespread use in the matched-sibling setting can be recommended. The randomised BMT CTN 1703 study comparing Tacrolimus and Methotrexate (MTX) to PTCy/Tacrolimus and MMF is ongoing but does not include myeloablative conditioning (MAC) regimens.

Aims: Perform a prospective randomized trial to compare PTCy with calcineurin inhibitor-based GVHD prophylaxis in matched-sibling PBSC transplants for acute leukemia in CR or MDS.

Methods: The BM12 Australasian Leukaemia Lymphoma Group (ALLG) trial (ACTRN12618000505202) funded by the Australian Government Medical Research Future Fund (APP1152188) will randomize 134 adult patients with acute leukemia or MDS to either conventional GVHD prophylaxis (CsA and MTX) or PTCy (50 mg/kg) on Days +3 and +4 followed by CsA from Day +5. Both MAC and reduced intensity conditioning (RIC) regimens are allowed. Regimens are the same across the two arms apart from TBI-based MAC, where there was lack of safety data for the use of PTCy following CyTBI. In this setting, Fludarabine plus TBI is used for the PTCy arm. The primary endpoint is GVHD and relapse free survival (GRFS), with GVHD defined as grade 3 or greater acute GVHD and NIH-defined moderate or severe chronic GVHD. The study has an 80% power for a 2-sided log-rank test (a=0.05) to detect a hazard ratio of 0.54, which corresponds to 24-month GRFS of 28% and 50% in the two arms. Analysis of the primary endpoint (GRFS) will take place after 86 events are known to have occurred. An interim analysis (IA), to enable consideration of early stopping either for overwhelming evidence of an efficacy difference or for futility, will take place when approximately half of the quota of 86 events has occurred. Secondary endpoints include engraftment, rates of acute and chronic GVHD, relapse, non-relapse mortality, overall survival, VOD, quality of life and health economic impact. Correlative studies include impact of PTCy on measurable residual disease, immune activation, immune reconstitution and CMV immunity.

Results: The trial is open at 8 Australian and 2 New Zealand sites, with 73 patients randomised up to 5 th January 2022. Early stopping rules for unexpectedly high rates of non-relapse mortality, VOD, haemorrhagic cystitis and engraftment failure in the first 30 patients in either arm have to date not been triggered. Recruitment is expected to be completed in 2023.

Summary/Conclusion: This prospective randomized trial will provide evidence for the use of PTCy for GVHD prophylaxis in matched-sibling PBSC transplants for acute leukemia in CR and MDS.

PB2177: ANTITHYMOCYTE GLOBULIN AS TREATMENT OF ACUTE GRAFT VERSUS HOST DISEASE - 10 YEARS OF EXPERIENCE AT A SINGLE-CENTER

C. Dantas Rodrigues 1,2,* , C. Afonso 1,3 , P. Coelho 1 , G. Brás 1 , L. Leite 1 , R. Branca Ferreira 1 , C. Pinho Vaz 1 , A. Campos 1

1 Bone Marrow Transplantation, Instituto Português de Oncologia do Porto Francisco Gentil, Porto; 2 Hematology, Centro Hospitalar Tondela Viseu, Viseu; 3 Hematology, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal

Background: Acute graft versus host disease (aGVHD) is the leading cause of morbidity and non-relapse mortality, following allogeneic hematopoietic stem cell transplant (AHSCT). It affects particularly the skin, liver, and gastrointestinal tract (GI). The recommended first-line treatment is glucocorticoid therapy. However, 35-50% of patients (pts) become refractory to steroid therapy (SR-aGVHD). There is still no consensus regarding subsequent treatment, and outcomes remain poor. Antithymocyte globulin (ATG) is an alternative therapy. Although leading to an initial response, especially in pts with skin-only GVHD, studies have suggested that it may not be an effective option since it does not prolong OS (5.5 months) and it is associated with high mortality rates due to infection.

Aims: To characterize pts with SR-aGVHD treated with ATG 2mg/kg or thymoglobulin 1.5mg/kg in alternate days for 5 administrations.

Methods: Forty-three pts were treated between August 2010 and April 2021. Diagnosis and classification were based on clinical, laboratory and histological data. Response criteria was defined as: complete response (CR); Very Good Partial Response (VGPR); Partial Response (PR); No Response (NR). Endpoints were overall survival (OS) and response after 28 days of treatment.

Results: Patient characteristics are shown in Table 1. Median time of follow-up was 4 months (1-129). aGVHD was diagnosed at a median of 23 days (7-228) and immunosuppressive therapy was started immediately in all cases except for 11pts with grade < II. Before any treatment, median clinical stage score was II (range II–IV). Initial organ involvement was skin only (n=22), GI only (n=5) or multiorgan (n=16). Median time from steroid treatment to ATG was 23 days (5–213) and from AHSCT to ATG was 57 days (28-249). At day one of ATG, more than 50% had multiorgan involvement (n=24), followed by GI only (n=10), skin only (n=8) and liver only (n=1). ATG was second-line therapy in 40pts and third line in 3pts, with a median of 5 doses (1-5). Response to ATG was assessed on a weekly basis, but for the purpose of the study, we considered the assessment after 28 days of the first dose. 26pts had NR; 8 VGPR; 7 achieved PR and 2 CR. Most of the pts that were refractory to ATG had multiorgan involvement (n=17), followed by GI only (n=6), liver only (n=1) and skin only (n=1). Of those who achieved at least a PR, 7pts only had the skin affected, 6 had multiorgan and 4 had GI only. Adverse events included virus reactivation in 16pts (62.5% had cytomegalovirus) and bacterial infection in 8pts. A total of 33pts died: 23 from infection, 10 from other complications directly related to aGVHD. The NR group had more deaths (25 vs 9 pts). Median OS was of 5 months (3.6–6.3). Those who responded to ATG had a significantly better OS (12 vs 4 months, p=2.8x10 -5 ). There is a correlation between the organs affected and the response to ATG ( χ 2 , p=0.011): when the skin is the only organ affected, pts are more likely to respond than when compared to multiorgan involvement.

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Summary/Conclusion: Except for ruxolitinib, no treatment has shown superiority over other treatments and no new drugs have been approved either as first-line or second-line treatment for aGVHD in the past 30 years. Our study showed that ATG can be a good alternative in pts with less extensive GHVD limited to single organs, particularly the skin alone, and that in more extensive disease it led to infrequently sustained responses and consequently poorer survival. A high rate of major complications, mainly infectious, was observed after treatment with ATG.

PB2178: OUTCOMES OF REDUCED DOSE TEAM (THIOTEPA, ETOPOSIDE, CYTARABINE, MELPHALAN) PRIOR TO AUTOLOGOUS STEM CELL TRANSPLANTATION FOR HODGKIN AND NON-HODGKIN LYMPHOMA: A MONOCENTRIC EXPERIENCE

S. Di Folca 1,* , V. Marafioti 1 , S. barbato 1 , S. Avilia 1 , F. Grimaldi 1 , A. Severino 1 , M. Picardi 1 , L. Luciano 1 , G. Battipaglia 1 , F. Pane 1

1 Ematologia, AOU Federico II, napoli, Italy

Background: Combination of carmustine, etoposide, cytarabine, melphalan (BEAM) is a commonly used regimen for autologous stem cell transplantation (auto-SCT) in patients with Hodgkin (HL) or non-Hodgkin lymphoma. Due to both a shortage of carmustine in the past decade and to reduce its pulmonary toxicity, this agent has been replaced in several centers by thiotepa (TEAM). However, BEAM and TEAM are associated with mucosal toxicities. Therefore, in our center since 2015 we reduced cytarabine and etoposide doses to reduce conditioning toxicities.

Aims: Study is designed to explore whether halving the doses of cytarabine and etoposide can reduce transplant-related toxicity while maintaining the antitumor response.

Methods: We retrospectively analyzed transplant outcomes of adult patients undergoing auto-SCT conditioned with reduced TEAM (thiotepa 5mg/kg/12h on day -7; etoposide 100 mg/m2/12h and cytarabine 100 mg/m2/12h on days -6 to -3 (instead of the commonly used 200 mg/m2/12h); melphalan 140 mg/m2 on day -1).

Results: Included were thirty-nine patients (males, n=24; females, n=15) transplanted during the period 2015-2021. Median age was 43 (range 22-65) years. Most patients were transplanted for refractory (n=10) or relapsed (n=15) disease, while 12 patients were transplanted after first line treatment. The most frequent histology was diffuse large B-cell lymphoma (n= 15) followed by HL (n=10), follicular lymphoma (n=5), mantle cell lymphoma (n= 5) and primary mediastinal B-cell lymphoma (n=4). The median number of chemotherapy lines prior to transplant was 2 (range 1-5), with a median interval from diagnosis to transplant of 16 months (range 5-218). Disease status at auto-SCT was complete response (CR) in 37 (CR1=22, CR2=14, >CR2=1) and partial response (PR) in 2 patients. Eight patients needed plerixafor for CD34 mobilization, with a median dose of CD34+ collected of 3.36 (range 0.70-17.00) x 10 6 /kg.

All but 1 patient engrafted, with a median time to neutrophil and platelet engraftment of 11 (range 8-16) and 19 (range 9-40) days, respectively. The median duration of hospitalization was 19 days (range 15-27). Regimen related toxicities included mucositis in 32 patients (82%; grade 1-2, n=29; grade 3, n=3; no grade 4 mucositis) and febrile neutropenia in 26 patients (67%). Blood cultures were positive for Gram-positive or Gram-negative bacteria in 3 and 8 patients, respectively. One patient developed resolutive pneumonia.

Five patients (13%) underwent maintenance therapy after transplantation (brentuximab, n=3; rituximab, n=1; ibrutinib, n=1) while twelve patients relapsed with a median time after auto-SCT of 5.5 (range 3-19) months, with a 1-year cumulative incidence of relapse of 38% [95% CI 20-56]. Two patients underwent allogeneic SCT with a median time after auto-SCT of 11 and 23 months, respectively. The 100-day and one-year non-relapse-mortality (NRM) was 2.5% [95% CI 0.2-11]. Eight patients died: one 5 days after auto-SCT due to septic shock; 5 due to disease progression and 2, after disease relapse, due to suicide and cerebral hemorrhage, respectively. At last follow-up, 29 patients (81%) were in CR. With a median follow-up of 21 (range3-77) months, 2-year progression-free and overall survival were 59%[95% CI 38-75] and 81% [95% CI 74-88]. Median PFS was 14 months (range 0-77) and median OS was 17 months (range 0-77).

Summary/Conclusion: Reduced TEAM is a feasible and valid regimen prior to auto-SCT, with low toxicity and NRM and acceptable survival rates.

PB2179: RETROSPECTIVE ANALYSIS OF THE EFFICACY AND SAFETY OF RUXOLITINIB IN CHRONIC GRAFT VERSUS HOST DISEASE (CGVHD). A SINGLE-INSTITUTION EXPERIENCE.

Ó. Muñiz 1,* , T. Torrado Chedas 1 , A. Nieto Vazquez 1 , A. Santamaria Lopez 1 , E. Winsnes 1 , L. González Rodríguez 1 , C. Albo Lopez 1

1 Hospital Álvaro Cunqueiro, Vigo, Spain

Background: GvHD still remains the main cause of transplant-related morbidity and mortality. While the first line treatment is corticosteroids, 50% of patients will not achieve and adequate response. At this moment, there is no consensus about which treatment should be used as second line and beyond. Because of this, the use of ruxolitinib has been positioning itself as a salvage therapy due to the latest results in different studies as well as the safety profile that offers compared with other alternative therapies.

Aims: Evaluate the overall response rate (ORR) at 6 and 12 months and the adverse eventes (AEs) of ruxolitinib used as salvage therapy for cGvHD.

Methods: In this single-center retrospective analysis we reviewed the charts of patientes who received ruxolitinib for cGvHD between January 2017 and December 2021 in Hospital Álvaro Cunqueiro, Vigo. Statistical analysis have been made by using SPSS Statistics. Staging of cGvHD was based on National Institutes of Health (NIH) disease severity score. AEs were grading according to Common Terminology Criteria (CTCAE) version 5.0.

Results: Our cohort comprised 14 patients. Median patient age was 54 years and 78.6% (n=11) were male. Most common diagnosis for undergo hematopoietic stem cell transplantation (HSCT) was myelodysplastic syndrome (35.7%, n=5). Conditioning regimen was reduced intensity in 78.6% (n=11) and myeloablative in 24.6% (n=3). The average onset of cGvHD was 21 months post-HSCT, and the mediant time to ruxolitinib initiation was 44 months. Almost all patients had moderate (71.4%, n=10) or severe (21.4%, n=3) disease. Most common involved organ was skin in 64.3% (n=9). On average, patients received 3 previous therapies before ruxolitinib. Among previous treatments, predonisone (92.9%, n=13), tacrolimus (50%, n=7), sirolimus (50%, n=7) and photoapheresis (35.7%, n=5) were the most frequently used. 57.1% (n=8) of patients were able to reduce (n=2) or stop (n=4), and stop other inmunosupresor (n=2).

After 6 months, we observed an ORR of 64.3% (n=9) with complete response (CR) in 35.7% and partial response (PR) in 28.6%. After 12 months, ORR was observed in 42.8% (n=6), with CR in 35.7% and PR in 7.1%. At 1 year, treatment failure was observed in 35.7% (n=5). Causes of failure were progression of cGvHD (n=4) and death in 1 of them.

The most common AEs were cytopenias, affecting 71.4% of patientes (n=10), but only 21.4% were grade 3-4. The most frequent cytopenia was anemia in 42.8% (n=6), followed by neutropenia (35.7%, n=5) and thrombocytopenia (14.2%, n=2). 42.8% (n=6) developed a total of 9 infectious complications requiring admissión, most commonly pneumonia. The infectious complications caused death in 14.3% (n=2). Other AEs were: urinary infections not requiring admissión (n=2), cytomegalovirus viremia (n=1), herpes simplex virus reactivation (n=1), deep venous thromboembolism (n=1), diarrhea (n=1), skin atrophy (n=2) and folliculitis (n=2). None of those AEs caused ruxolitinib discontinuation.

Summary/Conclusion: In our analysis, the ORR obtained have been greater than 50%, maintaining this response at one year of therapy. Besides its effectiveness, ruxolitinib favors dose reducing or discontinuation in corticodependent patientes. Ruxolitinib is a well-tolerated drug with a good safety profile. Globally, 2 patientes died of complications (14.3%), which is a low mortality rate in fragile and heavily pre-treated patients.

PB2180: EPIDEMIOLOGICAL CHARACTERS OF ADULT EGYPTIAN STEM CELL TRANSPLANT PATIENTS: A SINGLE CENTER EXPERIENCE

M. Elbogdady 1,* , S. Shamaa 1 , M. Nasr 1 , M. Elzaafarany 2 , T. Elkhodary 2 , M. Abd Elhamid 2 , E. Azmy 1 , M. Awad 2 , H. Ghazy 2 , M. Elbayomy 2 , M. Taalab 1 , S. Saber 1 , Z. Emarah 2 , M. Denwar 1 , A. Elsergany 2

1 Hematology; 2 Medical Oncology, Oncology center Mansoura university - Mansoura university - Egypt, Mansoura, Egypt

Background: Stem cell transplanrt is an integral part of the treatment of hematologic disorders and especially malignant ones. Egypt which has a population around One hundred and five million people has a limited number of stem cell beds not exceeding 100 beds allover the country which is very low compared to international standards.

Aims: The aim of this study is to describe the epidemiological situation of adult Stem cell transplant patients in Mansoura, Egypt as basedon retrospective analysis of our electronic clinical records at hematology unit Oncology center mansoura university(OCMU) between November 2019 and February 2022 in our freshly established stem cell unit which started operating on November 2019.

Methods: Adult patients who underwent stem cell transplant were recruited from stem cell transplant unit at OCMU from November 2019 to February 2022.Demographic characteristics, clinical history, and laboratory and treatment data were collected.

Results: The study included 55 recorded patients who underwent Stem cell transplant at OCMU from November 2019 to February 2022. All cass came from delta region 70 % from Dakahlia governorate. Male patients represented 47.3% (26) while female 52.7% (29). We Had only 2 cases of Allogenic stem cell transplant while the remaining 53 were Autologus.The mean age undergoing SCT was 34.7 years with the minimum and the maximum being 22 & 66 years. 40% (22) had NHL, 29.1% (16) had HD, 27.3% had MM and 1.8 % (1) had AML & ALL respectively. Most cases were transplanted in CR2 or more for Lymphoma except for 3 in CR1 being highly refractory or aggressive. MM cases were at least in VGPR and 2 cases of plasma cell leukemia were transplanted. Acute leukemia who underwent allogenic were in CR2 for ALL & CR3 for AML. The used conditioning regimen was BeEAM for lymphoma, High dose melphalan for MM, Bu/Cy for Allogenic.

Summary/Conclusion: Stem cell transplant is still not feasible to all leigable candidates due to shortage of facilities and trained personnel especially in developing countries. establishement of new SCT units wil help provide a more convinient obtion for those who require such a procedure

PB2181: DOES SARCOPENIA INFLUENCE THE SUCCESS OF HEMATOPOIETIC STEM CELL TRANSPLANTATION?

E. Erdogan Yucel 1,* , I. Alacacioglu 1 , A. F. Karatas 1 , B. Yavuz 1 , M. Kakci 1 , O. Seker 1 , G. H. Ozsan 1 , F. Demirkan 1

1 Hematology, Dokuz Eylul University, Izmir, Turkey

Background: Sarcopenia has been widely studied in solid tumors. However, its presence and prognostic significance in hematological malignancies and transplantation cases are not clear enough.

Aims: In this study, we aimed to evaluate the presence of sarcopenia and its effects on transplantation success in patients undergone stem cell transplantation.

Methods: Twenty-four patients who had autologous or allogenic stem cell transplantation between May 2021 and January 2022 were included in this study. Patients who had a secondary non-hematological malignancy, neuropathy or paralysis, chronic liver/kidney disease, glomerular filtration rate <60 ml/min, congestive heart failure were excluded. Patients with a body mass index (BMI) <20.0 kg/m2; 20.0 - 24.9 kg/m2; 25.0 -29.9 kg/m2; > 30 kg/m2 were considered as underweight; normal weight; overweight and obese respectively. Low muscle mass was evaluated with the Bioelectrical Impedance Analysis (BIA) method. TANITA-SC-240MA device was used to measure body composition by BIA method. The low skeletal muscle mass index (SMI) was considered as ≤10.76 kg/m2 in men and ≤ 6.76 kg/m2 in women. The muscle strength was evaluated by using a handgrip dynamometer (TAKEI 5401 Hand Dynamometer, 100 kg). The average of 3 measurements were calculated and <30 kg in men and <20 kg in women were considered as low muscle strength. Patients were evaluated by bioelectrical impedance analysis (BIA), hand grip test, sit-stand test within 1 week before administration of preparative regimens for stem cell transplantation. The neutrophil and trombocyte engraftment days, presence of bacteremia, need for thrombocyte infusion and hospitalization time were analyzed due to the presence of sarcopenia.

Results: 24 patients were included in our study. The median age of the patients was 48 (23-67), and 16 patients (66%) were female. Three patients (12.5%) were 65 years or older. Two patients had autologous stem cell transplantation previously and were undergone allogeneic transplantation due to the relaps. Among all patients, 5 patients (20.8%) were obese, 5 patients (20.8%) were overweight, 9 patients were (37.5%) normal weight and 5 patients were (20.8%) underweight. 4 patients (16.6%) were determined as sarcopenic. According to the BMI values of the sarcopenic patients, 3 were underweight and 1 was obese. Muscle strength and SMI were significantly lower in patients with sarcopenia (p: 0.035; p: 0.01, respectively) (Table 1). There was no significant relationship between the presence of sarcopenia and gender, age, BMI, diagnosis, neutrophil and platelet engraftment days. Although the length of hospitalization in patients with sarcopenia seemed to be longer, it was not statistically significant (p=0.83) (Table 1). The administration of parenteral nutrition was more frequent in patients with diabetes mellitus. When the patients were evaluated in terms of mortality on the 100th day, this value was found to be 50% in the group with sarcopenia.

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Summary/Conclusion: Sarcopenia is more common in patients who are candidates for allogenic transplantation compared to autologous transplantation. However, since the number of patients included in our study was low, the effect of sarcopenia on transplantation success could not fully clarified. Prospective studies with larger number of patients are needed on this subject.

PB2182: CHARACTERISTICS THAT INFLUENCE THE STEM CELL COLLECTION BY APHERESIS IN PATIENTS WHO ARE CANDIDATES FOR AUTOLOGOUS STEM CELL TRANSPLANTATION

K. Garzón 1,* , E. Fernández 2 , K. Bermúdez 1

1 Instituto Nacional de Cancerologia, México city; 2 Private Practice, Mexico city, Mexico

Background: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a consolidation treatment for several hematologic malignancies such as multiple myeloma (MM), Hodgkin lymphoma (LH) and non-Hodgkin lymphomas (diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), NK/T- cell lymphoma (NKTL), etc.).

However, between 5-40% of patients fail to obtain an adequate collection of hematopoietic progenitor cells (HPCs), this is known as mobilization failure. This failure can be due to multiple factors that may be associated with the previous treatment of the disease (number of treatment lines, type of chemotherapy or radiotherapy), the patient (age, bone marrow cellularity) or the collection procedure and other biochemical parameters like pre-count CD34+ cell/ul, pre-harvest hemoglobin and platelet count.

Aims: -To determine the clinical characteristics of patients and the haracteristics of apheresis procedures in patients who had fail to obtain an adequate collection of HPCs by apheresis of peripheral blood and who are candidates for ASCT.

Methods: A retrospective study was carried out, in patients aged between 18 and 65 years old, with hematological onco diseases who went to the Blood Bank of the National Cancer Institute for the collection of HPCs from peripheral blood by apheresis for ASCT, between January 2017 and December 2020. A review of the electronic medical record of each patient was carried out, as well as the records of the procedure for collecting HPCs.

The nominal variables were compared by chi-square test and the quantitative variables with Student´s t-test, a value less than 0.05 was considered statistically significant.

Results: The study included 127 patients, who underwent HPCs collection for ASCT; 15% (19 patients) had failure to mobilize (63% men; mean age 48 years). The most frequent diagnosis was MM 47.4%, followed by non-Hodgkin lymphoma 42.1% (37.5% DLBCL, 25% MCL, 37.5% NKTL, follicular lymphoma and peripheral T-cell lymphoma) and Hodgkin lymphoma with 10.5%.

The most common used method of mobilization was chemotherapy with filgrastim (57.8%), followed by filgrastim.

In the pre-harvest studies, 26% had anemia and 42% thrombocytopenia; pre-count CD34+ less than 20/ul was presented in 94%. More than 50% required two or more days for collection. During the process to obtain HPCs, the median duration of this procedure was 273 minutes (range 193 to 311 minutes), with an average processing speed of 81.1 ml/h. While the mean volume extracted was 16 879 ml (range 4 087 to 20 463ml) with an average number of complete cycles of 14.6.

It was determined that age >50 years, two lines of treatment, mobilization with chemotherapy and filgrastim, pre-count CD34+ less than 20/ul and more days of harvesting were associated with HPCs harvest failure. While other factors such as sex, bone marrow status, radiotherapy site, pre-harvest hemoglobin, platelet count did not have a statistically significant association.

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Summary/Conclusion: In our study, harvest failure (15%) is within the range reported in the literature and the factors associated with this failure have also been documented in other studies. With these data obtained, it could be established that patients at high risk of failure to harvest, such as those with multiple myeloma, are considered to be harvested receiving a smaller number of lines of treatment and consider stimulation with other stimulants such as plerixafor.

PB2183: RELAPSE PREDICTS POOR OUTCOMES IN FLT3-MUTATED ACUTE MYELOID LEUKEMIA POST ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION

E. Gavriilaki 1,* , A. Papalexandri 1 , D. Mallouri 1 , I. Batsis 1 , A. Vardi 1 , Z. Bousiou 1 , C. Demosthenous 1 , A. Panteliadou 1 , N. Spyridis 1 , G. Karavalakis 1 , T. Touloumenidou 1 , M. Masmanidou 1 , G. Pistiolas 1 , F. Mouladeniou 1 , C. Vadikoliou 1 , C. Lalayanni 1 , E. Yannaki 1 , A. Anagnostopoulos 1 , I. Sakellari 1

1 G. Papanicolaou Hospital, Thessaloniki, Greece

Background: Allogeneic hematopoietic cell transplantation (alloHCT) is the only curative option for patients with FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplication) acute myeloid leukemia (AML). Although sorafenib has been recommended as maintenance treatment, data on risk factors and outcomes in these patients are limited.

Aims: We investigated the risk factors and outcomes in patients with FLT3 -mutated AML that underwent alloHCT.

Methods: We enrolled consecutive adult patients with FLT3-mutated AML that underwent alloHCT at our JACIE-accredited center over the last two decades based on EBMT standards. The following factors were recorded and analyzed: age, disease, phase at transplant, type of donor and transplant, grade II-IV acute and moderate/severe chronic graft-versus-host disease (GVHD), relapse, treatment-related mortality (TRM), disease-free (DFS) and overall survival (OS).

Results: We studied 43 patients with FLT3-mutated AML who received alloHCT at a median age of 45 (18-62) years from sibling (21), matched (16) or mis-matched (3) unrelated, and haploidentical donors (3). Phase at transplant was first complete remission/CR1 (31 patients), CR2 (9) or relapsed/refractory disease (3).

With a median follow-up of 26.5 (1.2-239) months, relapse was observed in 16 patients, 4 of whom presented only with isolated extramedullary disease. Five-year cumulative incidence (CI) of relapse was 41.7% and was independently associated with previous lines of treatment (p=0.015), disease phase at transplant (25.7% for CR1, 50% for CR2, and 100% for relapsed/refractory disease, p=0.007), and chronic GVHD (p=0.004). Relapse was treated with chemotherapy (15 patients), donor lymphocyte infusions (7), azacytidine (6), or gilteritinib (3). No patient received sorafenib either as maintenance or treatment. Only one relapsed patient survived after treatment of isolated extramedullary disease.

Five-year CI of TRM was 17.4% and was associated only with disease phase at transplant (p<0.001). Five-year DFS and OS were 46.5% and 52.5%, respectively. The only significant predictor of OS was relapse (beta=0.303, p<0.001).

Summary/Conclusion: r study confirms that relapse post alloHCT is an imminent feature of FLT3 -mutated AML, associated with poor outcomes. Disease phase at transplant is an important risk factor for alloHCT outcomes. Nevertheless, relapse incidence remains high, even in patients transplanted in CR1, highlighting the necessity of maintenance therapy with FLT3 inhibitors.

PB2184: COURSE OF CORONAVIRUS INFECTION IN ONCOHEMATOLOGICAL PATIENTS IN THE POST-TRANSPLANT PERIOD

E. Grishina 1,* , D. Gagloeva 1 , E. Zhelnova 1 , E. Karimova 1 , E. Zotina 1 , E. Paramonova 1 , E. Baryakh 1 , E. Misyurina 1

1 City Clinical Hospital №52, Moscow, Russia

Background: In March 2020, specialists in the field of oncohematology faced the problem of severity of coronavirus infection in patients after high-dose course of chemotherapy and autologous or allogeneic bone marrow transplantation. This required a revision of a number of issues related to the selection of patients for bone marrow transplantation (BMT), the development of new preventive and therapeutic tactics aimed at the treatment of infectious and immunological complications in this category of patients, depending on the nature of the underlying disease, the status of the disease and the timing of the treatment.

Aims: To assess the severity, the most typical complications and the COVID 19 severity aspects in patients in early and late post-transplant periods to develop the most optimal tactics for the prevention and treatment of COVID 19 in this group of patients.

Methods: An analysis was made of patients after HSCT with active coronavirus infection from 2020 to 2021, hospitalized in the hematology department of the Moscow multidisciplinary hospital (the hospital was completely redesigned to work on the COVID19 profile). A total number of hospitalized patients after HSCT was 25: 4 patients after allogeneic transplantation, 21 - after autologous. According to the timing of HSCT, patients were divided into 2 groups - early post-transplant period (ETP) (2-90 days after HSCT) - 14 patients, and late post-transplant period (LTP) (3-24 months) - 11 patients. According to nosology, patients were divided into following groups: lymphomas - 72%, MM - 12%, AA and CML 8% each. All patients were admitted with a positive PCR test (0-7 days of COVID). COVID therapy was carried out according to the protocols adopted in the Russian Federation using antiviral drugs, biological therapy, corticosteroids, anticoagulants. If necessary, the required supportive therapy was carried out for this period of HSCT.

Results: Severe COVID19 (CT 3-4 severity) was more often observed in patients in ETP (100%) than in LTP (45%) (p=0.021). The incidence of respiratory failure is 70% and 36% in ETP and LTP, respectively. According to the analysis in ETP, agranulocytosis was observed in 65% of cases, for LTP group - in 18% (p = 0.022). Development of severe infectious complications (bacterial, fungal and viral) was detected in 100% of patients in ETP, in 45% of patients in LTP (p = 0.002). Antifungal therapy was required in 100% of cases in ETP, and only in 27% of the LTP group (p=0.001). 90% of patients in both groups required biological therapy. Mortality in the group of patients in ETP was 35%, while no deaths were registered in the group of patients in LTP (p=0.027). Median of hospitalization period for ETP and LTP patients was 20 and 13 days, respectively.

Summary/Conclusion: Patients in the early period after HSCT have a higher risk of developing lower respiratory tract infection, more likely to require antifungals, reserve group antibiotics, and have a greater risk of death from COVID-19. Biological therapy is not contraindicated in case of leukopenia and agranulocytosis in this group of patients.

PB2185: MINIMAL RESIDUAL DISEASE AND THE NUMBER OF CD34+ CELLS CIRCULATING IN THE PERIPHERAL BLOOD OF MULTIPLE MYELOMA PATIENTS ON THE DAY OF THE FIRST SESSION OF LEUKOCYTAPHERESIS DURING AUTOGRAFT HARVESTING

I. Kostroma 1,* , Z. Chubukina 1 , Z. Sidorova 1 , R. Sabitova 1 , T. Glazanova 1 , S. Gritsaev 1

1 Bone marrow transplantation, Russian Scientific-Research Institute of Haemotololy and Transfusiology, St. Petersburg, Russia

Background: The efficiency of autograft harvesting, i.e. the number of CD34+ cells in terms of the patient’s weight is determined by factors characterizing the intensity of the mobilization regimen, describing the type and duration of therapy preceding the collection of hematopoietic stem cells (HSC), and also assessing the activity of the disease. The significance of the volume of residual myeloma cells on the quantitative composition of the autograft of patients with multiple myeloma (MM) is not clear. Therefore, the question of the appropriateness of choosing the appropriate mobilization regimen depending on the status of minimal residual disease (MRD) remains open.

Aims: To evaluate the possible influence of MRD status on the mobilization HSCs from the bone marrow into the peripheral blood in patients with MM.

Methods: A retrospective analysis of the results of HSC mobilization in 64 patients with MM was performed. Mobilization of HSCs was carried out by regimens with introduction of cyclophosphamide 3 g/m/2 (n=14), vinirelbine 35 mg/m/2 (n=30), granulocyte colony-stimulating factor (G-CSF) (n=16) and plerixafor (n=4). When using cyclophosphamide, vinorelbine and plerixafor, patients additionally prescribed G-SCF. The efficiency of mobilization was assessed by the number of CD34+ cells circulating in one mkL of peripheral blood on the day of the first session of leukocytapheresis. To initiate leukocytoapheresis, this indicator had to be at least 20. Thus, cases of unsuccessful mobilization were excluded from the analysis. The status of MRD was assessed by the results of flow cytometry performed before the start of the mobilization regimen. An indicator of >0,01% was considered positive.

Results: There were 20 and 44 patients with MRD (-) and MRD (+) status in the study, respectively. The number of CD34+ cells circulating in the peripheral blood on the day of the first session of leukacytapheresis in the patients in the group did not differ: 154 and 88.5/mkL, respectively; p=0.164. The difference was also not significant depending on the mobilization regimen used, despite the trend towards a negative effect of less intensive courses in the case of a significant volume of residual cells. So if in the group of cyclophosphamide CD34+ cells in 1 mkL of blood of patients with MRD (-) status and MRD (+) status were 97 and 90, respectively; p=0.540, then when prescribing vinirelbine 276 and 109, respectively; p=0.116 and 117 and 47, respectively, with the introduction of G-SCF; p=0.173.

Summary/Conclusion: The potential of the used mobilization regimens does not depend on the MRD status of patients with MM. However, the trend towards a decrease in the number of CD34+ cells circulating in the peripheral blood on the day of the first session of leukacytapheresis with the use of less intensive schemes (G-CSF, vinorelbine with G-CSF) gives reason to take into account the status of MRD when choosing a mobilization regimen.

PB2186: INTENSITY OF THE CONDITIONING REGIMEN AND TIMING OF AUTOGRAFT ENGRAFTMENT IN PATIENTS WITH MULTIPLE MYELOMA

I. Kostroma 1,* , R. Sabitova 1 , Z. Sidorova 1 , T. Zamotina 1 , S. Gritsaev 1

1 Bone marrow transplantation, Russian Scientific-Research Institute of Haemotololy and Transfusiology, St. Petersburg, Russia

Background: One of the ways to increase the intensity of conditioning regimen is the adding of additional drug to melphalan. Supposed that combination of drugs has more potential to reduce the myeloma cells content, which translates into improved progressive-free survival. On the other hand, the development of prolonged cytopenia with extended hospital stay is not exclude.

Aims: Comparative assessment of graft engraftment time in patients with multiple myeloma (MM) after autologous stem cell transplantation (AutoSCT) using four conditioning regimens: Mel200, Mel140, melphalan with thiotepa (Mel/Thio) and melphalan with carfilzomib (Mel/Carfil).

Methods: The data of 53 patients with MM analyzed. The number of patients who received Mel200, Mel140, Mel/Tio and Mel/Carfil regimens prior to infusion of thawed cell suspension was 24, 13, 10 and 6, respectively. Eleven AutoSCT (20.8%) were the second ones. The conditioning regimen with reduced dose of melphalan (Mel140) was prescribed predominantly for the second transplantation: 6 (46.2%) vs 3 (12.5%) for the first AutoSCT; p=0.022. In the posttransplant period. granulocyte colony-stimulating factor (G-SCF) was received by 20 (83.3%), 12 (92.3%), 8 (80.0%) and 5 (83.3%) patients in the respective groups.

Results: The median achievement by the absolute number of neutrophils at the level 0.5x10/9/L and more in the groups was 10 (6-15), 13 (9-19), 10 (9-11) and 14 (10-18) days, respectively. The difference in enraftment time was significant between the Mel/Thio and Mel/Carfil groups; p=0.012. There were no difference in the number of patients with episodes of febrile neutropenia: 6 (25.0%), 2 (15.4%), 3 (30.0%) and 1 (16.7%), respectively. Three (12.5%) patients with Mel200 regimen and 1 (10.0%) patient with Mel/Thio regimen did not have thrombocytopenia below 20x10/9/L. In other patients, the median of reaching platelet level 20x10/9/L and more did not differ: 14 (17-19), 14 (9-19), 13 (10-15) and 15 (12-16) days, respectively. The median number of infused platelets doses did not differ either: 2.5 (1-5), 3 (2-6), 2.5 (1-4) and 2 (1-2), respectively. The length of stay of patients in the hospital from the day of infusion of thawed cell suspension to discharge was as follow: 17.5 (12-21), 19 (14-26), 16 (15-20) and 18 (14-20) days, respectively, differences no.

Summary/Conclusion: The intensification of conditioning regimen is haracterized by acceptable toxicity, regardess of the type of drug used, and does not lead to a lengthening of the hospital stay.

PB2187: VIABILITY AND PROLIFERATIVE POTENTIAL OF AUTOGRAFT HEMATOPOIETIC STEM CELLS

V. Balashova 1,* , N. Semenova 1 , Z. Chubukina 1 , I. Kostroma 1 , R. Sabitova 1 , S. Gritsaev 1

1 Bone marrow transplantation, Russian Scientific-Research Institute of Haemotololy and Transfusiology, St. Petersburg, Russia

Background: Autograft cellularity is a fundamental condition for the timing of hematopoiesis recovery in patients with multiple myeloma (MM) after autologous hematopoietic stem cell transplantation (AutoSCT). Taking the number of CD34+ cells in a volume of 2x10/6/kg of the patient’s weight as a suboptimal value that provides fast and reliable graft engraftment, it remains unclear whether this number will be sufficient for subsequent transplantation, if tandem AutoHSCT assumes an interval of 6 months, and repeated - not earlier 36 months.

Aims: Determination of the preservation of colony-forming (CFU) and CD34+ cells in the autograft of MM patients depending on the terms of their cryopreservation.

Methods: A retrospective analysis of the results of the study of 127 samples of the product of leukocytapheresis was performed. The prepared cell suspension was placed in disposable CryoMacs Freesing bags and mixed with dimethyl sulfoxide to its final concentration of 7.5%. Freezing was carried out in a Cryo 560-16 Planer RLC programmed freezer. Containers with cell suspension were stored in liquid nitrogen. To assess the effect of the duration of the cryopreservation period on CFU, the numerical values of each sample were converted into percentages: the primary CFU values from each sample were taken as 100%, and the CFU values after the cryopreservation period were compared with them.

Results: Depending on the terms of cryostorage, 3 groups were formed. Group 1: 83 samples, shelf life from 1 to 5.9 months. Group 2: 34 samples, shelf life from 6 to 10 months. Group 3: 10 samples, shelf life over 10 months. A trend towards a decrease in the number of colonies was revealed as the duration of the cryopreservation period increased. Thus, the number of CFU-totals decreased from 66% recorded after 1–5.9 months of storage, to 60% after 6–10 months and to 44% of the original number when stored for more than 10 months; p=0.131. The decrease in the number of colony-forming cells of granulomonocytopoiesis was distinct in cell suspension samples stored for more than 10 months, but also did not reach significant values: 64%, 58% and 37%, respectively; p=0.451. On the contrary, the decrease in the number of burst-forming cells of erythrocytopoiesis (BFU-E) after 10 months of cryostorage was significantly lower than at earlier periods of cell suspension thawing. So if the content of BFU-E after 1–5.9 and 6–10 months of storage was 65% and 57% of the initial amount, respectively, then after 10 months it was only 26%; p=0.01.

When analyzing the preservation of CD34+ cells, a tendency was also found to decrease in their percentage as the duration of cryopreservation increased, but the difference did not reach a significant value: 77% during thawing after 1–5.9 months of cryopreservation, 49% after 6–10 months and 39% in case of cell suspension thawing after 10 months of storage; p=0.465. Despite the fact that in some patients with the maximum terms of cell suspension cryopreservation, the need for a larger number of doses of donor erythrocytes was recorded and there were longer terms for platelet recovery than in patients in the other two groups, the median values of the analyzed parameters did not differ.

Summary/Conclusion: When planning the first and subsequent, repeated, AutoHSCT in MM patients, it is advisable to prepare an autograft with a greater than suboptimal cellularity due to a possible decrease in the number of CFU during long-term cryostorage of the cell suspension.

PB2188: APPLICATION PROSPECT OF LOW DOSE PORCINE ANTITHYMOCYTE GLOBULIN IN HLA MATCHED SIBLING DONOR TRANSPLANTATION

X. chen 1,* , R. zhang 1 , W. zhai 1 , Q. ma 1 , A. pang 1 , D. yang 1 , Y. he 1 , J. wei 1 , S. feng 1 , M. han 1 , E. Jiang 1

1 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical Colleg, Tian Jin, China

Background: To investigate the preventive effect of low-dose porcine antithymocyte globulin (P-ATG) on graft versus host disease (GVHD) in HLA matched sibling donor transplantation.

Aims: To investigate the preventive effect of low-dose porcine antithymocyte globulin (P-ATG) on graft versus host disease (GVHD) in HLA matched sibling donor transplantation.

Methods: The clinical data of 60 patients with hematological malignancies who received MSD-HSCT from July 2020 to October 2021 were analyzed retrospectively. These patients / donors were older than 40 years old or the donors were female. Of these, 30 patients received Low-dose porcine ATG as a part of conditioning regimen (P-ATG group), while the other 30 patients didn’t receive ATG (No-ATG group). In addition to the fact that the ages of the patients and donor were older in P-ATG group, all patients’ other baseline characteristics and donor condition of the two groups were similar.

Results: All patients engrafted in two groups. There were significant differences in the incidence of aGVHD ([23.3 (10.1-39.7)%] vs [50.0 (30.8-66.5)%], P = 0.0276), grade III-IV aGVHD ([16.7 (5.94-32.1)%] vs [40.0 (22.4-57.0)%], P = 0.0491) and cGVHD ([22.4 (6.03-45.1)%] vs [69.0 (43.4-84.8)%], P = 0.0009) between the P-ATG and No-ATG groups. But there was no significant difference in terms of 1-year OS ([79.9 (60.5-90.4)%] vs [84.0 (61.2-94.0)%], P = 0.4411) or RR ([13.6 (2.73-33.2)%] vs [15.2 (4.40-32.1)%], P = 0.7416). And there was also no significant difference with regard to the incidence of severe bacterial infection (P = 0.612), invasive fungal disease (P = 1.000) or CMV infection (P = 0.2976). 80% (4 / 5) of patients in P-ATG group died of secondary infection after grade III-IV aGVHD, and 16.7% (2 / 12) in No-ATG group.

Summary/Conclusion: The application of low-dose P-ATG in the conditioning regimen of MSD-HSCT can significantly reduce the incidence of aGVHD, grade III-IV aGVHD and cGVHD, doesn’t increase the risk of recurrence after transplantation in patients / donors older than 40 years old or female donors. However, the patients in P-ATG group were older and had high mortality caused by secondary infection after grade III-IV aGVHD. Therefore, there was no significant difference in OS between the two groups.

PB2189: HAPLOIDENTICAL HEMATOPOIETIC STEM CELL TRANSPLANTATION IN PATIENTS WITH SEVERE CONGENITAL NEUTROPENIA:A CASE REPORT OF 13 YEARS OLD PATIENT

J. Wang 1,* , K. Liu 1 , S. Cui 1 , X. Dong 1 , W. Zheng 1 , Z. Liu 1 , R. Xu 1 , Y. Wang 1 , J. Xu 1 , F. Kong 1

1 Hematology Department, SHANDONG UNIVERSITY OF TCM, JINAN, China

Background: Haploidentical hematopoietic stem cell transplantation in patients with severe congenital neutropenia:A case report of 13 Years old patient

Aims: Severe congenital neutropenia (SCN) is a heterogeneous group of diseases progressing with arrest of maturation in bone marrow. It is characterized by recurring skin, lung, and deep tissue infections from the first months of life. The use of G-CSF therapy has radically changed these patients’ outlook. However, a minority of patients is refractory to G-CSF, and some undergo malignant transformation.

Methods: Allogeneic hematopoietic stem cell transplantation (HSCT) appears to be the only curative treatment for such patients. We report a 13 years femal patient who presented in years of life with severe neutropenia with a mutation in ELANE. This patient is allergic to a variety of antimicrobial drugs, difficult to treat, low quality of life, and has a high possibility of conversion to leukemia. In order to improve the quality of life, reduce the risk of conversion to leukemia and prolong its survival time, the transplantation treatment procedure was started through communication with patients and their families.Since the patient has no sibling full donor, the donor chooses the non consanguineous half matched donor sought by the bone marrow bank. The patient received conditioning regimens used for leukemia (myeloablative regimens),including CY (120–200 mg/kg) and BU (16–20 mg/kg or 560 mg/m2 administered orally, generally divided into 16 doses). In addition to the BU and CY combination, the agents used for conditioning included ATG.

Results: The transplantation process was smooth, with granulocyte implantation on day + 14 and platelet implantation on day + 22. The hemogram returned to normal after 48 days. The STR-PCR was detected on + 30d, + 60d, + 90d, + 180d and + 270d respectively to evaluate the evidence of implantation and the chimeric body showed complete chimerism after transplantation. So far, the patient has survived without events for 20 months. After transplantation, the patient’s congenital gene ELANE mutation disappeared.

Summary/Conclusion: Adolescents with SCN who long term dependence on G-CSF can receive haploidentical allogeneic hematopoietic stem cell transplantation.

PB2190: HLA-HAPLOIDENTICAL PERIPHERAL BLOOD STEM CELL TRANSPLANTATION WITH POST-TRANSPLANT CYCLOPHOSPHAMIDE AFTER REDUCED-INTENSITY CONDITIONING

J. Kim 1,* , Y. Park 2 , N. Yun 1 , J.-A. Kim 1

1 Hematology, St. Vincent Hospital, College of Medicine, The Catholic University of Korea, Paldal-gu, Suwon-si, South Korea; 2 Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington D.C, United States of America

Background: Allogeneic peripheral blood stem cell transplantation (PBSCT) is a potentially curative treatment for patients with hematological malignancies. However, the availability of HLA-matched, related or unrelated donors is always in question. Haploidentical related donors are alternative donor options for patients who lack an HLA-matched donor but are in urgent need of transplantation. However, haploidentical PBSCT (haploPBSCT) poses significant challenges because of the HLA mismatch, which often increases the risk of graft-versus-host disease (GVHD) and non-relapse mortality (NRM). A recent study led by a group at Johns Hopkins pioneered the use of post-transplant cyclophosphamide (PTCY) in the haploPBSCT setting. In the haploPBSCT, the use of PTCY in combination with tacrolimus and mycophenolate mofetil (MMF) have been found to lower the rates of acute and chronic GVHD, as well as NRM.

Aims: We conducted this prospective study to further evaluate the safety and efficacy of PTCy-haploPBSCT.

Methods: Eleven patients diagnosed with hematologic disease (AML, ALL, MDS, and aplastic anemia; 4, 1, 4, and 2, respectively) were enrolled in this study. The median age was 44 (range, 24 to 65). The conditioning regimen consisted of fludarabine (150 mg/m 2 ), busulfan (6.4 mg/kg), and total body irradiation (TBI, 3Gy). High-dose cyclophosphamide (50 mg/kg/day on days 3 and 5), cyclosporine, and MMF were used for GVHD prophylaxis. The median numbers of CD34 + cells of PBSCs were 11.6 × 10 6 /kg (range, 6.1–24.9 × 10 6 /kg).

Results: Overall survival (OS) and relapse-free survival (RFS) at 1-year were 79% and 69%, respectively. Two patients relapsed. One patient was AML and another relapsed with RAEB. Neutrophil engraftment was achieved in 91% of patients with a median of 14 days (range, 13–15 days). The cumulative incidence rates of grades I–IV and III-IV acute GVHD at 100 days were 36% and 9%, respectively. The cumulative incidence rates of mild and moderate chronic GVHD at 1-year were 9% and 18%, respectively. No patients developed severe chronic GVHD. The cumulative incidence rates of NRM, relapse and GVHD-free/RFS at 1-year were 21%, 31% and 62%, respectively.

Summary/Conclusion: Our results indicate that PTCy-haploPBSCT is a valid and safe strategy for preventing severe acute and chronic GVHD. In addition, it may also provide better clinical outcomes in long-term disease control. However, our study suggests that relapse still remains as a major cause of treatment failure. Therefore, there is a need for a modified conditioning regimen that can enhance anti-leukemic effects in aggressive diseases such as acute leukemia.

PB2191: CENTRAL VENOUS ACCESS AND PERIPHERALLY INSERTED CENTRAL CATHETERS IN HEMATOPOIETIC STEM CELL TRANSPLANTATION: A RETROSPECTIVE COMPARISON OF POSSIBLE COMPLICATION RATES

A. Mele 1,* , E. Prete 1 , R. De Francesco 1 , G. Greco 1 , A. Greco 1 , L. Tonialini 1 , S. Sibilla 1 , F. Donatelli 1 , F. Martella 1 , A. Cannoletta 1 , S. Citiso 1 , F. Ciardo De Marti 1 , M. R. Morciano 1 , R. Giulia 1 , A. Orlando 1 , V. Pavone 1

1 Hematology and Bone marrow Transplant, Hospital Cardinale G.Panico, Tricase, Italy

Background: Central Venous access devises (CVAD) and peripherally inserted central catheters (PICC) are essential for the managing of patients undergoing hematopoietic stem cell transplantation (HSCT).

Aims: The aim of this retrospective study is to evaluate the risk factors and incidence of complications associated to vascular accesses in terms of catheter-related blood stream infections (CRBSI) and catheter related thrombotic complications (CRTC) in CVAD and PICC.

Methods: A total of 127 patients, undergoing HSCT at our Hospital between January 2019 and December 2021, were analyzed. Vascular accesses were successfully insered in all patients. As a policy of our center, the dual-lume PICC was implanted only in patients with autologous transplant. The dual-lume Broviac access was used in autologous as in allogeneic transplant. SPSS™ v22 was used to perform the descriptive analysis of the main variables using a confidence interval of 95%. The student’s t-test was used to compare the means of two independent samples assuming unequal variances.

Results: The baseline characteristics of patients, underling disease and different phases of the HSCT are listed in Table 1. The Broviac and dual-lume PICC was implanted in 85 and 42 patients, respectively. The clinical characteristics of the two cohorts are not balanced because the patient populations are different due to the different policy of the center in the use of venous devices. In fact, PICC has been used in autologous patients (93%); 38 of these were multiple myeloma patients. Broviac was placed in both allogenic (53%) and autologous (47%) HSCT. The catheter-related blood stream infections (CRBSI) occurred in 17% (n=14) and in 5% (n=2) of Broviac and PICC, respectively (p= 0,04). The different pathogens associated to CRBSI were reported in Table 1. Broviac and PICC were removed for infections in 5% (n=4) and in 3% (n=1) of patients, respectively. No death for CRBSI occurred. The catheter related thrombotic complications (CRTC) was recorded only in PICC (n=7; 17%), p 0,003. None of the catheters was removed early due to thrombotic episodes. After the thrombotic episode, all patients were treated with low-molecular-weight heparin (LMWH) while the catheter remained in place; in case of PLTs <50×10^9/l low dose LMWH were used. No fatal event related to the CRTC was observed. Broviac and PICC remained without problems throughout the HSCT procedure in 91% (n=73) and 93% (n=39), respectively. Univariate analyses of variables associated to vascular complications were analyzed. A lower number of CD34 infusion (<4x10^6/Kg) resulted significantly associated to a higher incidence of PICC-CRTC, p 0,03. Mucositis was statistacally predictive of CRBSI in patients with Broviac (100% vs 80%, p 0,01). No other risk factors appear to have had a statistically significant impact.

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Summary/Conclusion: In our experience, Broviac was associated with an increased risk of bloodstream infections. Thrombotic episodes were observed in PICC. No fatal events related to CRBSI or CRTC were observed. Despiste, vascualr accesses remained without problems throughout the HSCT procedure. Further prospective studies and homogeneous populations are needed to confirm these data.

PB2192: THE IMPACT OF DENTAL DISEASE ON THE MANAGEMENT OF HAEMATOLOGICAL DISEASE: A REVIEW OF TWO CASES

E. Miles 1,* , R. Wali 1 , C. Bryant 1 , J. Patel 1 , S. Nayani-Low 2 , S. Moosajee 2

1 Oral Surgery; 2 Special Care Dentistry, King’s College Hospital, London, United Kingdom

Background: Significant numbers of haematology patients are seen on a clinic dedicated to the management of patients with medically complex backgrounds within the Dental Institute of King’s College Hospital. Many are referred for dental assessment and treatment prior to haematopoietic stem cell transplant (HSCT), our clinic aims to provide this service in a timely manner in order to avoid dental concerns delaying transplant. The objective of pre-HSCT dental assessment is the identification and elimination of possible source of dental infection which could otherwise cause significant morbidity or mortality during the transplant process. For most of the patients found to require dental treatment this will involve the extraction of teeth which are non-vital, infected or considered to pose a future risk of infection. Our team includes dental consultants, specialty trainees and hospital grade dentists who work closely with haematology colleagues within our hospital and others.

We report two cases in which concerns about oral health identified at dental assessment resulted in planned HSCT being significantly delayed or abandoned. The impact on the patients’ haematological management will be discussed with the aim of highlighting the importance of effective pre-transplant dental assessment and the effects of poor dental health on HSCT.

Aims: - To identify patients whose medical outcome was directly adversely affected by their dental health.

- To discuss the cases in which findings at dental assessment resulted in significant delay to HSCT.

Methods: From the dedicated database of patients managed within our medically complex dental clinic over the last five years, those referred for pre-HSCT dental assessment whose oral health contraindicated proceeding with HSCT around the time this was planned were identified and data pertaining to them extracted.

Results: Since 2017 our clinic received more than 600 referrals for pre-HSCT dental assessment from haematology colleagues. The vast majority of patients were deemed dentally fit for transplant either at presentation or after the successful completion of dental treatment. Two patients who could not proceed to transplant due to oral contraindications were, however, identified.

Patient 1 had been diagnosed with Hodgkin’s lymphoma 2 years earlier, previous treatment included zolendronic acid. Unfortunately, no dental assessment was undertaken before this bisphosphonate therapy was commenced. He presented with a large, spontaneous osteonecrosis of the left maxilla and a neglected dentition; all remaining teeth required extraction. The patient’s haematologist believed that the presence and extent of the necrotic bone contraindicated HSCT so alternative, less effective treatment, was pursued instead.

Patient 2 had received chemotherapy to treat his follicular lymphoma. During pre-HSCT dental assessment a squamous cell carcinoma of the right tongue was noted and later confirmed on biopsy; he subsequently underwent hemi-glossectomy and right neck dissection with free flap reconstruction. HSCT had to be postponed but was successfully completed 3 months after his surgery.

Summary/Conclusion: The cases presented highlight the value of pre-HSCT dental assessment and how oral health can impact significantly on wider aspects of healthcare. Although a rare occurrence dental assessment may identify patients who should not proceed to transplant as planned and in doing so may help to prevent adverse outcome of this intervention.

PB2193: IS HYPERHYDRATION NECESSARY FOR HIGH DOSE MELPHALAN CONDITIONING REGIMEN IN AUTOLOGOUS HAEMATOPOIETIC STEM CELL TRANSPLANTATION?

M. Mora Argumánez 1,* , A. Vázquez Ramo 1 , B. Merchán Muñoz 1 , R. Pérez Alonso 1 , I. Nuevo López 1 , Á. Gil Pérez 1 , H. Guillén García 1 , A. B. Santos Montero 1 , J. Arbeteta Juanis 1 , M. D. Morales Sanz 1 , N. Golbano López 1 , S. Herrero 1 , D. De Miguel Llorente 1

1 Hospital Universitario de Guadalajara, Guadalajara, Spain

Background: Single-agent Melphalan or combined with Bortezomib is often used as a conditioning regimen in autologous haemopoietic stem cell transplantation (HSCT) for multiple myeloma (MM) and primary amyloidosis (AL). In order to prevent nephrotoxicity, Melphalan is often administrated with hyperhydration (HH) consisting of 4-6 Litres (L) 0.9% saline which has a potential fluid overload risk, mainly in elderly patients.

Given the potential morbility of HH, some institutions use a less intensive hydration regimen consisting of 2-3L 0.9% saline the day Mephalan is administrated.

Aims: This study aimed to retrospectively analyze the safety of not using hyperhydration (NHH) in our center.

Methods: This study used a retrospective cohort design, reviewing patients from Guadalajara University Hospital diagnosed with MM or AL who underwent autologous HSCT between January 2017 and December 2021. Cohorts were defined by HH (6L/24h 0.9% saline) or NHH (2L/24h 0.9% saline) the day Melphalan was administrated.

The primary endpoint of this study was the incidence of acute renal impairment, the secondary endpoints were fluid overload and other adverse effects.

Comparisons were assessed using Student t-test or Fisher’s exact test, considering a p-value <0.05 statistically significant.

Results: The sample included 50 patients: 11 with HH (100% MM) and 39 NHH (87.18% MM, 12.82% AL). Patient’s gender (HH 45.45% women; NHH 61.54% women) was similar in both cohorts. Median age in HH cohort was 71, while in NHH it was 61.

The prevalence of kidney disease due to MM at diagnosis was higher in NHH cohort (HH 9.1%, NHH 17.9%; p=0.48) but not statistically significant. Baseline creatinine (HH 0.79, NHH 0.78; p=0.92) and the prevalence of cardiovascular risk factors (HH 63.6%, NHH 66.7%; p=0.31) were comparable.

The incidence of acute renal failure (HH 18.1%, NHH 7.7%; p=0.31) was higher in HH but statistically non-significant, while median creatinine increase from Melphalan administration to day 7 after haemopoietic stem cell infusion was similar in both groups (HH 0.38, NHH 0.18; p=0.08). All patients who developed acute renal failure had a history of chronic kidney disease or a baseline creatinine >1 before HSCT and it was always reversible. Likewise, all patients developed mild kidney injury (AKIN stage 1) except for one (1/5) from the HH cohort (AKIN stage 3).

Concerning the presence of clinical fluid overload, it was significantly more frequent in the HH cohort (36.6%) in opposition to the NHH cohort (7.7%) (p = 0.02) as expected due to the greater volume administrated, but no patients developed acute pulmonary edema.

Regarding other adverse effects as hepatic toxicity (HH 0%, NHH 17.9%; p=0.13), rash (HH 9.1%, NHH 5.1%; p=0.63) or grade IV mucositis (HH 81.8%, NHH 64.1%; p=0.07), they were similar in both cohorts with no statistically significant difference between both groups.

Neutrophil engraftment was reached at day +10 in HH group and at day +14 in NHH group. This difference is probably due to the elimination of G-CSF from the protocol in the last years in order to avoid engraftment syndrome.

There were limitations in this study such as the small sample size and retrospective analysis.

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Summary/Conclusion: Hyperhydration did not demonstrate to protect the kidney compared to non-hyperhydration regimes, with a significantly greater incidence of fluid overload. Consequently, HH may not be necessary for melphalan conditioning regimen in autologous haemopoietic stem cell transplantation as our protocol demonstrates an adequate safety profile.

PB2194: ISCHEMIC STROKE IN PATIENTS WITH THROMBOCYTOPENIA AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

M. Pérez Raya 1,* , I. Sánchez Bazán 1 , A. I. Gallardo Morillo 1 , M. I. Muñoz Pérez 1

1 Hematology and Hemotherapy, Hospital Regional Universitario de Málaga, Málaga, Spain

Background: Hematopoietic stem cell transplantation (HSCT) is a critical strategy in the treatment of hematopoietic diseases, but it is not free of complications. Those occurring in the central nervous system (CNS) exhibit potential severity and poor prognosis, the most frequent being infection and hemorrhagic stroke secondary to thrombocytopenia. Up to 3% of transplant recipients develop a stroke; however, despite the relationship between thrombocytopenia and bleeding at any level, ischemic strokes have also been described in patients with thrombocytopenia.

Aims: The aim of our study was to review the development ischemic stroke following allogeneic HSCT and to identify possible associated risk factors.

Methods: A total of 4 patients developed ischemic stroke after allogeneic HSCT in a single center in 2019, 75% male and 25% female, aged between 43 and 70 years. Fifty percent had cardiovascular risk factors prior to transplantation (smoking, dyslipidemia, obesity and hypertension), without previous structural heart disease, arrhythmias, thrombotic or ischemic pathology. Only one patient had a positive lupus anticoagulant.

All received allogeneic HSCT from related donors (50% haploidentical, 50% identical) as consolidation treatment for malignant disease without CNS infiltration. The source of progenitors was bone marrow 50% and peripheral blood 50%. 75% were performed with reduced-intensity conditioning and 25% myeloablative. None received total body irradiation.

Results: Seventy-five percent of the episodes took place in the immediate post-transplantation period (first 100 days), only one occurred later (day +2142). Seventy-five percent had localized ischemic stroke and 25% had cerebral diffuse microangiopathy (Fazekas 3). 75% had no graft-versus-recipient disease (GVHD) at the time of the event, whereas 25% suffered from chronic grade 3 GVHD with pulmonary, renal, and cutaneous involvement. 50% presented the episode in the setting of thrombotic microangiopathy, or suspicion of it. Only 50% were receiving immunosuppressive treatment based on calcineurin inhibitors or corticosteroids. In 75% the episode was followed by a concurrent hemorrhagic event (hemorrhagic cystitis, sublingual hematoma). Concomitant infections were detected in 50% of the patients, of which 50% presented pneumonia caused by Aspergillus spp and Respiratory Syncytial Virus, 25% bacteremia caused by S. Parasanguinis and 25% pneumococcal pneumonia. In half of the cases there was coexistence of more than one microorganism at pulmonary level. Cytomegalovirus was positivized in blood in 50% of the cases, with no evidence of proven disease.

Thrombocytopenia was present in all patients, with under 47x10 9 /L platelets in 50% and under 20x10 9 /L platelets in 25%. Hemoglobin was under 9 g/dL in 75%.

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Summary/Conclusion: In summary, the development of ischemic stroke after allogeneic HSCT is infrequent; however, there are factors that favor their appearance, such as cardiovascular risk factors prior to transplantation, treatments used and intercurrent complications, including thrombotic microangiopathy, which can cause ischemia due to induced endothelial damage. In most cases the events occur in an infectious background.

Thrombocytopenia does not in itself constitute a protective factor. In addition, long-term or acute bleeding has been related to thrombotic and ischemic phenomena. However, given the scarcity of case reports, further studies are needed to establish the relationship between allogeneic HSCT and the presence of ischemic events in CNS.

PB2195: MOBILIZATION WITH CYTARABINE AND GRANULOCYTE COLONY STIMULATING FACTOR (G-CSF) GIVES BETTER YIELD THAN MOBILIZATION WITH G-CSF ALONE OR COMBINED WITH CYCLOPHOSPHAMIDE

S. Simeonov 1 , I. Tonev 1,* , V. Denchev 2 , M. Stoichov 1 , Y. Davidkova 1 , E. Aleksov 1 , T. Zhivkov 1 , M. Stoyanova 1 , M. Donchev 1 , G. Arnaudov 1 , M. Mincheff 2

1 National Specialized Hospital for active treatment of hematological diseases; 2 Medical University, Sofia, Bulgaria

Background: Patients with multiple myeloma are poor mobilizers and often require multiple cytapheresis procedures. Most popular mobilization regimens are with G-CSF given as a sole treatment or in combination with cyclophosphamide. Recently, several reports indicated that higher yields could be obtained following chemomobilization with cytarabine and G-CSF.

Aims: Our aim was to compate the effectiveness of chemomobilization with cytarabine and G-CSF with the other regimens which we have used.

Methods: Patients received 2x400mg/m 2 cytarabine for two days, followed by 5mcg/kg G-CSF two days after for at least nine days. Cytapheresis was initiated with WBC count above 5x10 9 /L, PLT count above 30x10 9 /L and CD34+ cell count above 20/µl. Results of this group of patients were compared with results from a historic group of myeloma patients harvested between 2018 and 2020.

Results: The new regimen showed markedly higher initial counts of CD34+ cells resulting in significantly better average yields (12.6x10^6/kg for the cytarabine + G-CSF group; 3.98 x10^6/kg for the G-CSF group and 4.998 x10^6/kg for the Cyclophosphamide+ G-CSF group).

Summary/Conclusion: Compared with other hematological malignancies, patients with multiple myeloma are hard to mobilize for collection of sufficient CD34+ cells. Possible explanations are advanced age and bone marrow involvement. Chemomobilization with cytarabine and G-CSF seem to overcome poor mobilization and collection yields for this patient group.

PB2196: EQUATOR: A PIVOTAL PHASE 3 DOUBLE-BLIND, RANDOMIZED, PLACEBO-CONTROLLED STUDY OF ITOLIZUMAB IN COMBINATION WITH CORTICOSTEROIDS FOR THE INITIAL TREATMENT OF ACUTE GRAFT-VERSUS-HOST DISEASE

J. Koreth 1,* , L. Kean 2 , L. Chinn 3 , L. M. Acevedo 3 , O. Bandman 3 , J. Rothman 3 , S. Connelly 3 , D. Thomas 3 , C. Cutler 1

1 Division of Stem Cell Transplantation and Cellular Therapy; 2 Department of Pediatric Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston; 3 Equillium, Inc., La Jolla, United States of America

Background: Acute graft-versus-host disease (aGVHD) remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplant (allo-HSCT); however, there is still no approved first-line treatment and corticosteroids (CS) remain standard of care. Itolizumab is a humanized IgG1 monoclonal antibody that binds CD6 and blocks interaction with activated leukocyte cell adhesion molecule (ALCAM) to inhibit T effector cell activity and trafficking to target organs. This mechanism of action represents a promising therapeutic approach to treat aGVHD, as suggested by safety and efficacy results from EQUATE ( NCT03763318 ), an open-label Phase 1b/2 study that evaluated itolizumab in combination with CS as treatment for newly diagnosed aGVHD. The benefit-risk profile currently observed in EQUATE for doses of 0.8 and 1.6 mg/kg supports continued evaluation of itolizumab in a pivotal Phase 3 study.

Aims: EQUATOR is a pivotal Phase 3, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of itolizumab in combination with CS as initial treatment for subjects with aGVHD. This global study will enroll approximately 200 subjects ≥12 years old who are diagnosed with Grade III-IV or Grade II with lower gastrointestinal involvement aGVHD following initial allo-HSCT.

Methods: To be eligible, subjects must start study treatment within 72 hours of starting CS treatment for aGVHD (Day 1 CS dose must be 2 mg/kg/day). Eligible subjects will be randomized 1:1 to receive 7 IV doses of itolizumab (1.6 mg/kg on Day 1 and 0.8 mg/kg for the subsequent 6 doses) or placebo every two weeks. Subjects will be followed for one year (Figure). The selected dosing regimen is expected to rapidly decrease CD6 from the surface of T cells and maintain low surface CD6 throughout the dosing interval. An interim analysis for futility and efficacy will be conducted when approximately 50% of subjects complete through Day 29.

Results: The primary endpoint is the complete response (CR) rate at Day 29 and key secondary endpoints include Day 29 overall response rate and durable CR rate at Day 99. This will enable assessment of speed and sustainability of symptom improvement and resolution. Additional secondary efficacy endpoints include the evaluation of duration of response, survival outcomes, CS use, and chronic GVHD incidence. Safety endpoints include the incidence of treatment-emergent adverse events (AEs), serious AEs, infections and CMV and EBV reactivation.

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Summary/Conclusion: Here we present the rationale and design of EQUATOR, a pivotal Phase 3 study that will evaluate itolizumab in combination with CS and the ability to induce rapid and durable CR rates for the initial treatment of aGVHD.

PB2197: PRE-HAEMATOPOIETIC STEM CELL TRANSPLANT DENTAL ASSESSMENT: A SERVICE EVALUATION

R. Wali 1,* , E. Miles 1 , C. Bryant 1 , J. Patel 1 , S. Moosajee 2 , S. Nayani-Low 2

1 Oral Surgery; 2 Special Care Dentistry, King’s College Hospital, London, United Kingdom

Background: Complications of haematopoietic stem cell transplant (HSCT) may result from or be exacerbated by inadequate oral preparation of patients prior to transplantation. It is essential that patients referred for HSCT undergo a thorough dental assessment and are deemed “dentally-fit” prior to transplant.

Aims: This service evaluation aims to review the referral-to- “dentally-fit” timeline for haematology patients referred for dental assessment prior to planned HSCT at King’s College Hospital. We aim to evaluate the factors that may impact on the timely delivery of haematological care for this cohort of patients.

Methods: Retrospective data was extracted from a dedicated database of medically compromised patients and filtered for those referred for the purpose of pre-HSCT dental assessment. Data relating to the demographics, referral to assessment time, dental findings, dental treatment needs and time from referral to establishing a “dentally-fit” status was recorded for pre-HSCT patients with any haematological disease seen in our clinic between 1 st January and 31 st December 2021. Patients whose HSCT was delayed because of concerns about their oral health were identified, the duration of the delay calculated and the reasons for this identified.

Results: 125 patients with haematological disease were referred for pre-HSCT dental assessment between January-December 2021. Gender distribution was 58% (n=72) male and 42% (n=53) female. Age at dental assessment ranged from 19-75 years, with a mean age of 57 years. The haematological diagnoses of this cohort were myeloma 46% (n=58), lymphoma 24% (n=30), leukaemia 18% (n=22), myelodysplastic syndromes 6% (n=8), sickle cell disease 3% (n=4), aplastic anaemia 2% (n=2) and red cell aplasia 1% (n=1). Referrals were made with a provisional HSCT date in 83% of cases. The mean time between referral and dental assessment was 27 days. 43 patients (34%) were diagnosed with dental disease requiring pre-HSCT treatment, 36 (84%) of these required dental extraction. The mean time from referral to acquiring a “dentally-fit” status was 33 days, an additional 10-day healing period was necessary for patients requiring dental extractions. Four (3%) patients had their provisional HSCT date delayed for dental reasons, two were referred late and the 10-day post-extraction healing period of two others extended beyond the date planned.

Summary/Conclusion: Very few of our patients experienced a delay in HSCT because of their dental treatment needs, demonstrating the value of timely referral for pre-HSCT dental assessment, which must factor in the need for post-extraction healing periods. To avoid delays in HSCT for future patients, the implementation of a fast-track service within our current patient pathway may improve the management of cases where time to transplant is limited.

For the majority of patients our service is effective in providing dental assessment and treatment prior to their proposed HSCT date. Improvements could be made by encouraging our haematology colleagues to refer patients earlier where this is possible and highlighting the need to consider post-extraction healing periods when making a dental referral and planning transplant dates.

PB2198: EFFECTS OF NOVEL CORONAVIRUS VACCINATION ON THE COLLECTION OF PERIPHERAL BLOOD HEMATOPOIETIC STEM CELLS FROM HEALTHY DONORS

Z. Wang 1 , S. Cui 1 , J. Xu 1 , X. Tan 1 , Y. Tian 1 , X. Tang 1 , Y. Wang 1,*

1 Department of Hematology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China

Background: With the development and marketing of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccine, vaccination has become an effective way to block the novel coronavirus disease 2019 (COVID-19) the most efficient way to spread. Hematopoietic stem cell transplantation (HSCT) is one of the important means for the treatment of aplastic anemia, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, autoimmune diseases and other hematological diseases. Peripheral blood hematopoietic stem cell (PBSC) transplantation has the advantages of convenient collection, small impact on donors, good tolerance of donors and recipients, fast and durable recovery of hematopoietic function and immune function of patients after transplantation, and low transplantation-related mortality. However, it is unknown whether the novel coronavirus vaccine has any effect on the collection of peripheral blood hematopoietic stem cells from transplant donors.

Aims: To compare the effects of unvaccinated and vaccinated novel coronavirus vaccine on the collection of peripheral blood hematopoietic stem cells from healthy donors.

Methods: A retrospective analysis was made on the 112 times of peripheral blood hematopoietic stem cell apheresis conducted by 59 healthy donors who were not vaccinated with the new crown vaccine and 41 healthy donors who were vaccinated with the new crown vaccine in the Affiliated Hospital of Shandong University of traditional Chinese medicine from February 6, 2017 to February 11, 2022, and the quality of hematopoietic stem cell collection products was evaluated by MNC count and CD34 + count.

Results: There was no significant difference in gender, age and collection times between the two groups (P>0.05). The MNC count collected in the unvaccinated group was (7.99±2.38) ×10 8 /kg, and the CD34 + count was (3.24±0.69) ×10 6 /kg. The MNC count collected in the vaccinated group was (7.97±3.71)×10 8 /kg, and the CD34 + count was (3.41±0.59) ×10 6 /kg. There was no significant difference between the two groups (P>0.05).

Summary/Conclusion: In this preliminary study, we found that the novel coronavirus vaccine had no effect on MNC and CD34 + counts in peripheral blood hematopoietic stem cell collection products from healthy donors. However, these findings require further investigation in larger randomized studies to be validated.

PB2199: A REAL WORLD STUDY OF PEG-RHG-CSF ON HEMATOPOIETIC RECOVERY AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

J. Xu 1,* , F. Kong 1 , S. Cui 1 , K. Liu 1 , Z. Liu 1 , J. Wang 1 , W. Zheng 1 , Y. Zhou 1 , R. Xu 1

1 Hematology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China

Background: The data of PEG-rhG-CSF on hematopoietic recovery after allogeneic hematopoietic stem cell transplantation is few in China.

Aims: To observe the efficacy and safety of PEG-rhG-CSF on hematopoietic recovery after allogeneic hematopoietic stem cell transplantation（allo-HSCT）in the real world study.

Methods: 103 patients after allo-HSCT were enrolled in this study from January 2019 to October 2021. PEG-rhG-CSF(xinruibai) was used in the hematopoietic recovery and given in the dose of 100μg/kg on day +1and day +8. Hematopoietic stem cell implantation time and recovery of white blood cells (WBC) and platelet (PLT)，implantable syndrome and Infectious fever were observed until day +60. The patients were divided into Aplastic Anemia group and hematological malignancy group for further study.

Results: All patients got neutrophil implantation successfully and the median neutrophil implantation time was 12 (7-25) d. 3 patients were failed to complete platelet implantation and the platelet implantation rate is 97%. The median platelet implantation time of the other 101 evaluable patients was 17 (10-47) d. The incidence of implantable syndrome and infection was low and there was no related death case. There were no significant differences of the neutrophil implantation time［11.5（7-25）d vs 12（9-23）d，P＞0.05］and platelet implantation time［17.5（10-43）d vs 17（11-47）d，P＞0.05］between Aplastic Anemia and hematological malignancy groups respectively. There was no significant difference in the incidence of fever and duration of fever between two groups(P＞0.05)．

Summary/Conclusion: The efficacy of PEG-rhG-CSF in promoting hematopoietic recovery after allogeneic hematopoietic stem cell transplantation is good, without severe untoward effects. There is no obvious difference in hematopoietic recovery in Aplastic Anemia and hematological malignancy.

PB2200: CLONAL HEMATOPOIESIS AT THE CROSSROADS OF INFLAMMATORY BOWEL DISEASES AND HEMATOLOGICAL MALIGNANCIES: A BIOLOGICAL LINK?

C. Cumbo 1 , F. Tarantini 1 , A. Zagaria 1 , L. Anelli 1 , C. F. Minervini 1 , N. Coccaro 1 , G. Tota 1 , L. Impera 1 , E. Parciante 1 , M. R. Conserva 1 , I. Redavid 1 , P. Carluccio 1 , M. Delia 1 , A. Giordano 1 , C. Longo 1 , T. Perrone 1 , A. Russo Rossi 1 , G. Specchia 2 , P. Musto 1 , F. Albano 1,*

1 Department of Emergency and Organ Transplantation (D.E.T.O.) - Hematology and Stem Cell Transplantation Unit; 2 School of Medicine, University of Bari, BARI, Italy

Background: Inflammatory bowel diseases (IBDs) are a group of chronic relapsing conditions of the gastrointestinal tract. Nationwide studies have revealed a higher risk of hematological malignancies (HMs) but not colorectal cancer in IBD patients. Clonal hematopoiesis (CH) is a premalignant condition defined by the presence of an acquired somatic mutation characterized by a variant allele frequency (VAF) of ≥2% in a gene frequently associated with HMs. A growing body of evidence suggests a correlation between inflammation and CH.

Aims: To assess CH possible co-occurrence in patients with an IBD associated with HMs, we performed a targeted next-generation sequencing analysis in a cohort of 13 patients who were referred to our center from February 2011 to May 2021 with IBD associated with HMs.

Methods: A customized panel, encompassing 26 target genes frequently mutated in myeloid malignancies, was performed on genomic DNA from bone marrow (BM) (cases #3 - #13) or peripheral blood (cases #1, #2) samples. Only variants (non-intronic, non-synonymous, with >1% global MAF in the healthy population) affecting the CH-associated genes, with ≥2% VAF, and with a depth of coverage >500x were considered.

Results: Overall, 20 variants affecting 9 genes ( DNMT3A , ASXL1 , JAK2 , GATA2 , TET2 , ETV6 , SF3B1 , EZH2 , KIT ) were detected in our cohort; 11/13 (85%) patients showed one or more mutations, with a VAF ranging from 2.6% to 53.0%. Moreover, cases #6 and #7 showed variants of CH-associated genes but in the absence of a BM lymphoma localization. Five variants (25.0%) showed a VAF<10%, whereas, in the 15 others identified (75.0%), the clone was dominant. In accordance with previously published data in the IBD context, DNMT3A was the most frequently mutated gene (4/20, 20.0%) (13,14), with ASXL1 (4/20, 20.0%), followed by JAK2 (3/20, 15.0%). All mutations are single nucleotide variants (SNV) with different functions: missense variants (13/20, 65.0%), nonsense variants (4/20, 20.0%), and unknown variants affecting a splice site or an untranslated region (3/20, 15.0%). Five cases (45.5%) carried just one variant, whereas the other six (54.5%) showed two or more mutations.

Recent works demonstrated that chronic infection, ulcerative colitis (UC) and atherosclerosis, can drive the gene loss of function associated with CH. In particular, chronic infection and UC may promote the selection of the DNMT3A gene mutation associated with CH by the IFNγ signaling induced in the course of these disorders. It is worthy of note that four (36.4%) cases in our series bore DNMT3A gene mutations. According to data previously shown in UC patients, this fact may be considered to demonstrate that the mechanisms at the basis of the inflammatory environment potentially select for the growth of CH specific mutations. Thus, CH as a possible biological link, may be fed by the IBD inflammatory stimuli, and in turn, feed the IBD inflammation. Particularly, the low VAF of DNMT3A gene in cases #6 and #7, both patients without evidence of BM lymphoma involvement, is strongly suggestive of CH promoted by IBD. Furthermore, cases #1, #2, #8, #10, #11 showed CH-associated gene variants usually rare in chronic myeloid and lymphoid leukemia.

Summary/Conclusion: Our report suggests that CH may be seen as a biological link at the crossroads of IBDs and HMs, whose role is yet to be fully elucidated. Nonetheless, if these data are confirmed, IBD patients screened and positive for CH should undergo hematologic follow-up to assess the risk of developing HM. Further studies are warranted to characterize the biological relationship between IBDs, CH, and HMs.

PB2201: THYMUS DEVELOPMENT IN CONGENITAL HEART DISEASES

I. Bódi 1 , D. Belina 2 , Z. Prodan 1 , A. Škrtić 3 , D. Heckel 4 , D. Grčević 5 , M. Antica 4,*

1 Semmelweis University, Budapest, Hungary; 2 Rebro Clinical Hospital; 3 Merkur Clinical Hospital; 4 Ruder Boskovic Institute; 5 University of Zagreb Faculty of Medicine, Zagreb, Croatia

Background: Congenital alterations of thymic development or thymectomy have a clear immunological impact on T cell development and function. The current thymus reconstitution strategies are based on enhancing the endogenous thymus regeneration or transplantation of thymic tissue, thymic epithelial pluripotent cells or thymic organoids. Most of the work relies on thymic tissue removed during cardiac surgery to provide access to the heart and major cardiac vessels. Although, these samples were considered healthy thymic tissues, we recently captured an interesting new phenotype of the thymus tissue in children with atrial septal defect (ASD). The thymic endodermal epithelial cells develop from the third pharyngeal pouches, induced by signalling network from the mesenchymal cells, which have neural crest origin.

Aims: During thymus development the Foxn1 transcription factor is crucial for the development, function, and maintenance of the mature thymus. Foxn-1 influences the migration of neural crest-derived cells, their differentiation into mesenchymal cells, the transformation process in which they become fibroblasts of the capsule, septae and several cells in the medulla. The presence of neural crest cells is substantial in the functional development of the thymic anlage to take place to the epithelio-mesenchymal interactions. In the absence of neural crest cells, the epithelio-mesenchymal interaction is obstructed and therefore epithelial cells develop abnormally. We hypothesize that the septal defect is caused by the defective regulation of signalling processes in the neural crest cells, which can also influence thymus development.

We aimed to dissect expression of crucial regulatory and signalling factors in thymus tissue samples and interpret them in the context of known healthy thymus characteristics.

Methods: Immunohistochemistry, Confocal microscopy and 3D cell cultures

Results: We compared EPCAM, FOXN1, DLL1 and cytokeratin expression by immunohistochemistry in the thymic tissue we recovered as medical waste from children with different congenital cardiac diseases. We found a clear cell compartment differences between the thymi recovered from surgery that so far were considered as healthy, especially in FOXN1 expression and TEC marker eypression.

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Summary/Conclusion: Our results shed new light on human thymus development and regeneration possibilities from thymic epithelial precursor cells.

By this research, we can get closer to understanding, recognizing and treating diseases as a result of immunodeficiency.

PB2202: FEATURES OF COLONY FORMING ABILITY OF BONE MARROW IN PATIENTS WITH NEUTROPENIA

M. Butolina 1,* , E. Poponina 1 , O. Tselousova 1 , E. Vaskina 1 , A. Pestrikova 1

1 The Federal State-Financed Scientific Institution Kirov Research Institute of Hematology and Blood Transfusion under the Federal Medical Biological Agency (KRIHBT), Kirov, Russia

Background: To study the proliferative and differentiating ability of hematopoietic precursors and to determine the hypoplastic variant of neutropenia, a cultural method is used. A special semi-liquid nutrient medium based on methylcellulose is used in this study. A certain amount of nucleated bone marrow cells is incubated for a fixed period. Colony forming units (CFU) make colonies in depth and on the surface of the medium. The number of colonies is proportional to the amount of the corresponding colony-forming cells in cell suspension. The colonies of each hematopoietic cell lines have characteristic morphological features which make it possible to distinguish and count them.

Aims: Evaluating the colony-forming ability of the bone marrow using a culture method as part of the differential diagnosis of neutropenia of unknown origin.

Methods: The colony-forming ability of the bone marrow was studied in 20 patients of children’s department of hematology clinic, surveyed in 2016-2021 (13 boys, 7 girls). Age ranged from 1.2 to 17 (median 13) years. The criterion for the neutropenia was the absolute number of neutrophils less than 1.5 thousand in 1 μl of peripheral blood.

For cultivation, a nutrient medium based on methylcellulose (MethoCult Human No. 4434, StemCell Technologies, Canada) was used, inoculation was performed according to the standard method. Cultures are incubated at 37°C in 5% CO 2 .The proliferative potential and differentiation ability of bone marrow cells was determined by counting the number of corresponding colonies in a 14-day culture. The norm was considered for granulocyte-erythrocyte-macrophage-megakaryocytic colonies (CFU-GEMM) - 0-8 per 10 5 myelokaryocytes, granulocytic (CFU-G) - 7-35, macrophage (CFU-M) - 1-18, eosinophilic (CFU-Eo) – 0-20, respectively. The total number of colonies is normally 49-166.

Results: The total number of colonies ranged from 6 to 158 (median 55). Of these, within the reference values - 55.0% of patients. The number of CFU-G was determined in the range from 1 to 45 (median - 4), in a number of patients the granulocytic cell line of the bone marrow was not weakened: in 25.0% of cases, a normal number of CFU-G was found, in 10.0% - increased.

The number of CFU-M ranged from 3 to 67 (median 21.5); an increased number of macrophage precursors was observed in 60.0% of neutropenic patients. A direct correlation between the number of CFU-G and CFU-M was determined, Spearman’s coefficient (r) 0.64 (p=0,003). The increased activity of the granulocytic cell line of hematopoiesis was accompanied by a high number of CFU-M.

In all patients, the number of CFU-GEMM (0-1, median - 0) and CFU-Eo (0-10, median - 1.5) was determined within the normal range.

Summary/Conclusion: Neutropenia is a group of conditions with different etiology and pathogenesis. The culture method can be used for the differential diagnosis of neutropenia of unknown origin. Correlation of the number of granulocytic and macrophage precursors in the bone marrow in patients with neutropenia can be due to active hematopoiesis maybe because of cytokine stimulation.

PB2203: AGEING OF LONG-TERM ALLOGENEIC HEMATOPOIETIC CELLS RECIPIENTS COMPARED TO THEIR DONORS

M. Czarnogórski 1,* , J. Sakowska 2 , M. Maziewski 3 , M. Zieliński 2 , J. M. Witkowski 3 , A. Piekarska 1 , I. Obuchowski 4 , M. Młyński 1 , P. Trzonkowski 2 , M. Dutka 1 , A. Sadowska-Klasa 1 , E. Zarzycka 1 , M. Bieniaszewska 1 , A. Hellmann 1 , J. M. Zaucha 1

1 Hematology and Transplantology; 2 Department of Medical Immunology; 3 Department of Physiopathology; 4 Intercollegiate Faculty of Biotechnology, Medical University of Gdańsk, Gdańsk, Poland

Background: Ageing is a complex phenomenon that leads to decreased proliferative activity, gradual loss of function of the cells, and cellular senescence. Specifically, ageing of the immune system exacerbates individual’s immune response, both humoral and cellular but increases the frequency of infections.

Aims: We hypothesized that a physiological aging occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at a faster rate when compared to their respective donors since the small number of donor transplanted cells undergo immense proliferative stress restoring the whole recipient’s hematopoiesis. To verify our hypothesis we compared molecular characterizations of ageing between recipients and donors of allo-HCT including telomeric length, proinflammatory cytokines concentration and immunophenotypic changes in main lymphocyte subsets – CD4 + , CD8 + , CD19 + and CD56 + .

Methods: Telomeric length was measured by qPCR technique and proinflammatory cytokines concentrations as well as immunophenotype were assessed by flow cytometry.

Results: Median telomeric length (TL) of CD8 + lymphocytes was significantly longer in donors of 2,1kb compared to recipients 1,7kb (p = 0,02). Similar trends were observed for CD4 + and CD19 + although the results have not reached statistical significance. We have also found differences however not significant in the immunophenotype between recipients and donors in the subpopulations of CD4 + (Naïve and Effector Memory), CD8 + Eomes + and B-lymphocytes (B1 and B2) which resembled physiological ageing-associated quantitative changes. When grouped according to imfection status, there were differences between recipients in the TNF- and Il-4 concentrations. They were higher in the recipients with lower infection risk status p=0,05 and p=0,003 respectively. Lower infection risk recipients had also a significantly greater percentage of NK cells (22,3%) than high-risk patients (9,3%) p=0,04.

Summary/Conclusion: The strong difference between recipients and donors in CD8 + population may result from faster reconstitution of CD8 + lymphocytes population in a respective recipient compared to CD4 + population after allo-HCT. Quantitative changes in immunophenotype of the recipients in some lymphocyte subsets are similar to those found in the elderly, whereas proinflammatory cytokines concentrations differ only among recipients grouped according to infection risk status which seems to resemble “inflammaging”. Lower infection risk recipients also seem to have increased innate immune response. The results of our study imply that long-term allo-HCT recipients differ from their respective donors. Those molecular differences are similar to those found in physiological ageing process.

PB2204: THE ROLE OF HES6 IN NORMAL HUMAN HEMATOPOIESIS

T. De Vos 1,* , T. Putteman 1 , M. Lavaert 2 , A. Kuchmiy 3 , J. Philippé 1 , T. Taghon 1

1 Diagnostic sciences, UGent, Gent, Belgium; 2 Laboratory of genome integrity, National cancer institute, Bethesda, United States of America; 3 eTheRNA Immunotherapies NV, Niel, Belgium

Background: The Notch signaling pathway is an important regulator of normal hematopoiesis and is often involved in leukemia. Some members of the HES gene family of transcription factors, like HES1 and HES4, are known Notch targets and have important roles in hematopoiesis. HES6, on the other hand, does not seem to be Notch dependent and has been poorly studied in the hematopoietic system. Interestingly, in the context of neurogenesis, it has been shown that HES6 can inhibit HES1 function, raising the hypothesis that HES6 might influence canonical Notch signaling activity. Within the hematopoietic system, HES6 has been described to be overexpressed in BPDCN, a very rare and aggressive hematological malignancy of plasmacytoid dendritic cells (pDCs). Also other studies have suggested the involvement of HES6 in cancer, including glioma, colorectal cancer, prostate cancer and uveal melanoma, and this mostly relates to a possible role for HES6 in cell proliferation.

Aims: Given that HES6 is poorly studies within the hematopoeitic system, this study aims to unravel the role of HES6 in normal human hematopoiesis.

Methods: We analyzed published datasets to examine the expression pattern of HES6 in different blood cell types. Through shRNA-mediated knockdown, we studied the impact of HES6 knockdown on the differentiation of CD34+ hematopoietic stem cells from cord blood to various hematopoietic lineages using in vitro stromal co-cultures and colony-forming unit assays. These experiments were analyzed using flow cytometry for the evaluation of differentiation, and cell counts for the evaluation of cell proliferation.

Results: HES6 is highly expressed in megakaryocyte-erythroid progenitors (MEPs) and to a lesser extent in NK cells, pDCs and T cells. When exploring the expression in developing T cells, we observed that HES6 expression is inversely correlated with the expression of HES1. Considering the high HES6 expression in MEPs, we particularly studied the role of this gene during erythroid/megakaryocyte development. In our stromal cultures, HES6 expression remained high in developing erythroblasts and was low in megakaryocytes. Upon HES6 knockdown in CD34+ cells, a significant reduction in the number of megakaryocytes (50-85%) and erythroblasts (75-85%) was observed. The effect of HES6 knockdown in the was significant, with a 55-80%.reduction of colony formation. The number of cells per colony was also reduced by approximately 50%. HES6 knockdown during the development of other lineages also appears to impact cell proliferation but this effect is less obvious and needs further exploration.

Summary/Conclusion: Consistent with the high expression of HES6 in MEPs, we observed significantly reduced cell proliferation upon HES6 knockdown during erythroid/mekaryocyte differentiation, with the most distinct effect on erythroblasts. HES6 knockdown also seems to impact the differentiation potential of precursor cells towards the myeloid lineages, as shown through colony-forming assays. Although the impact of HES6 on other hematopoietic lineages needs to be explored further, our results do provide the first clear insights into the functional role of HES6 in human hematopoiesis.

PB2205: ANTI-INFLAMMATORY AND REGENERATIVE EFFECTS OF UMBILICAL CORD BLOOD ENDOTHELIAL PROGENITOR CELL-DERIVED EXTRACELLULAR VESICLES ON ENDOTHELIAL CELLS IN VITRO

S. Ben Fraj 1 , A. Aouida 1 , G. Uzan 2 , H. Houda 1,*

1 Department of Biological and Chemical Engineering, National Institute of Applied Sciences and Technology, Carthage University, LR18ES40, Inflammation environment and signalization pathologies, Tunis, Tunisia; 2 INSERM U1197, Paul Brousse Hospital, Villejuif, France

Background: Circulating endothelial progenitor cells (EPCs) play a pivotal role in vascular regeneration and the repair of diseases in which angiogenesis is required including ischemia and tissue transplantation. Although a potentially valuable cell therapy tool their clinical use remains limited due to suboptimal storage conditions and especially, immune rejection. EPC-derived extracellular vesicles (EPC-EVs) may be an attractive alternative given their role in cell-cell communication and expression of the same parental markers.

Aims: In this study, we investigated the regenerative effects of umbilical cord blood EPC-EVs on endothelial cells in vitro

Methods: EPCs were obtained from human umbilical cord blood (UCB) and characterized by flow cytometry. Briefly, mononuclear cells were isolated by Ficoll and seeded on collagen-coated plates. After 7-20 days, colony-forming cells were amplified and cultured in a medium containing an EVs-depleted serum. Then the conditioned medium (1L) was used to isolate the EVS with Tangential Flow Filtration (MWCO 500KDa). The EVs were characterized by Nanoparticle Tracking Analysis (NTA) and Western blot. The angiogenic effects of EPC-EVs on endothelial cells were investigated with transwell migration, wound healing and tube formation assays. We also analyzed the expression of pro and anti-inflammatory markers and NO production by flow cytometry. In all the experiments, several EV doses were assessed.

Results: Our study shows that purified EPC-EVs (179,6 nm diam) enhance the migration and tube formation of endothelial cells. Moreover, they have an anti-inflammatory effect and do not change the NO level.

Summary/Conclusion: Our isolated EPC-EVs have the same beneficial properties (proangiogenic, anti-inflammatory) than their parent cells. In the next step, we shall confirm these results in vivo .

PB2206: FULL SPECTRAL FLOW CYTOMETRY ANALYSIS OF THE BONE MARROW IMMUNE MICROENVIRONMENT IN PATIENTS WITH MYELODYSPLASTIC SYNDROME

M. chen 1 , M. fu 2 , H. Wang 3,*

1 Hebei Yanda Lu Daopei Hospital, Langfang, sanhe; 2 Beijing Lu Daopei Hospital, beijing; 3 clinical lab, Hebei Yanda Lu Daopei Hospital, Langfang, sanhe, China

Background: The bone marrow (BM) microenvironment is actively studied in patients with myelodysplastic syndrome (MDS). Full-spectral flow cytometry (FSFCM) is a novel technique that makes it possible to simultaneously study the immune system and malignant cells.

Aims: to study the microenvironment and immune checkpoint of MDS by FSFCM.

Methods: Retrospective analysis of BM samples from 22 MDS patients and 22 healthy donors who received treatment at the Hebei Yanda Lu Daopei Hospital between January 2020 and September 2020 using 26 parameter FSFCM in a single tube. Multiple immune subsets, myeloblasts and their expression of CD96 and CD200 were also analyzed.

Results: The percentage of multiple lymphocytic subsets and number of plasmacytoid dendritic cells (PDCs) were significantly significantly lower in the samples from MDS patients compared to the samples from non-malignant controls.Additionally, expression of the CD96 surface marker were significantly lower on T-cell subsets and CD56bri NK cells,and was significantly higher levels on PDCs in the MDS patient samples compared to the healthy control samples. in the MDS samples CD200 expression on immune cells and myeloblasts were significantly higher in the MDS samples compared to those from healthy controls. The overexpression of CD200 on CD56dim natural killer (NK) cells was an independent risk factor for MDS.

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Summary/Conclusion: The percentage of immune cells and expression of immune checkpoint proteins on immune cells from BMs of MDS were distinct from that of healthy control samples.

PB2207: IN VITRO VALIDATION OF DENDRIMER-BASED APPROACH FOR MICRO-RNA DELIVERY INTO LEUKEMIA CELLS

N. Knauer 1,2,3,* , E. Pashkina 1 , O. Boeva 4 , A. Aktanova 1 , V. Arkhipova 2 , M. Meschaninova 2 , R. Gomez 5 , J. Sánchez-Nieves 5 , A.-C. Nickel 3 , U. Kahlert 6 , J.-P. Majoral 7 , V. Kozlov 1 , E. Apartsin 7

1 Laboratory of clinical immunopathology, Research Institute of fundamental and clinical immunology; 2 Laboratory of RNA chemistry, Insitute of chemical biology and fundamental medicine, Novosibirsk, Russia; 3 Department of preclinical studies, Heinrich Heine University, Düsseldorf, Germany; 4 Faculty of medicine, Novosibirsk State University, Novosibirsk, Russia; 5 Department of Organic and Inorganic Chemistry, University of Alcala, Alcala de Henares, Madrid, Spain; 6 Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery, Medical Faculty and University Hospital Magdeburg, Magdeburg, Germany; 7 Laboratory of coordination chemistry, CNRS, Toulouse, France

Background: microRNAs are known to be important players in the epigenetic regulation of many cell processes. Some of

them demonstrate anti-tumor properties (e.g. miR-34), others are associated with the enhancing of the tumor growth (e.g. miR-21), therefore being prospective effectors and targets for the gene therapy. For this aim, synthetic microRNAs or microRNA inhibitors can be used. We suggest to use dendrimers - highly symmetric hyperbranched polymers - as a novel type of nanocarriers for therapeutic RNAs delivery and gene therapy.

Aims: In this work we assessed the effects of microRNA-34a (miR-34a) and microRNA-21 inhibitor (anti-miR-21) on

Jurkat and 1301 cells (human T-cell leukemia) upon the dendrimer-mediated delivery.

Methods: As carriers for microRNAs, amphiphilic second- and third generation triazine-carbosilane dendrons (DG2

and DG3 respectively) and fully symmetrical 3rd generation carbosilane (BDEF33) and phosphorus (AE2G3) dendrimers have been taken. Oligonucleotides were complexed onto the surface of dendrimer vesicles by means of electrostatic interactions, forming dendriplexes. To prove the dendriplexes can be sufficiently internalized, we performed FACS analysis after 3 h of incubating cells with FITC-labelled microRNA.

Cells were cultivated with free dendrimers or complexes (25, 50, 100, 150 nM RNA, 10-fold excess of cations) in the complete cell medium for 72 h. We investigated cell viability (MTT assay, Annexin V/PI apoptosis assay) in comparison with non-tumor cells (PBMCs), IL-10 secretion and expression of several markers, characterizing interaction of tumor with immune microenvironment - PD-L1, TIM-3, CD47. Statistical analysis was done by using Mann-Whitney and Wilcoxon tests, differences between groups were considered significant if p<0.05.

This work was supported by RFBR grant No.18-33-20109, by the grant of the President of the Russian Federation No.2278.2019.4, by the HHU STIBET Scholarship grant support and based upon work from COST Action CA 17140 “Cancer Nanomedicine from the Bench to the Bedside” supported by COST (European Cooperation in Science and Technology).

Results: Free dendrimers have their own dose-dependent toxic effects, which is slightly higher for tumor cells, than for PBMCs. Treatment by free dendritic molecules led to increasing of expression of PD-L1, TIM-3 and CD47 on Jurkat cells, but decreased the IL-10 secretion.

Dendriplexes can be efficiently internalized into tumor cells, transport activity of dendrimers was higher than for Lipofectamine 3000 or was comparable. Dendron-based dendriplexes (DG2, DG3) had pro-apoptotic effect on leukemia cells. PD-L1 expression did not change after treatment by dendriplexes.

Summary/Conclusion: Dendrimers not onle have their own toxic effect per se, but also they can be used as effective

nanocarriers for therapeutic microRNAs. Using of complexes, containing microRNAs with anti-tumor activity, leads to induction of apoptosis of leukemia cells. Treatment by free dendrimers, but not by dendriplexes, increases expression of PD-L1, TIM-3, CD47 on Jurkat cells, but IL-10 secretion was found to be decreased.

We suppose, that dendrimer-based approach can be useful and perspective tool for delivery of therapeuthic microRNAs into tumor cells. At the same moment, characterization of their effects on interactions between tumor and immune microenvironment demands the further studies.

PB2208: CAR-T TREATMENT POST FIRST ALLO-HSCT FAILURE MAY HAVE DIFFERENT PROCESSION COMPARING TO CAR-T BEFORE ALLO-HSCT

D. wang 1,* , L. qiu 1 , H. Wang 1

1 Hebei Yanda Lu Daopei Hospital, Langfang, sanhe, China

Background: chimeric antigen receptor(CAR) T cell therapy achieved a great success in the treatment of B cell acute lymphoblastic leukemia(B-ALL). However, CAR-T post first allogeneic hematopoietic stem cell transplantation (allo-HSCT), donor cells would involve to patients’ immune system, even if same donors’ lymphocytes were used to produce CAR-T cells. To analyze the potential influence of first allo-HSCT, the expansion of CAR-T cells, the clearance of CD19+ cell, and expression of 24 cytokines related to T cell life cycle and graft versus host disease (GVHD) were measured in this study.

Aims: To figure out the promising biomarkers may influence the process of CAR-T treatment of HSCT relapsed patients.

Methods: 60 patients with B-ALL received CD19-CAR-T treatment in Hebei Yanda Lu Daopei Hospital in 2020 were randomly selected. For 30 patients, CAR-T therapy was performed after the first allo-HSCT failure(group1), and another 30 cases who received CAR-T before allo-HSCT(group2). 22 were females, and 38 were males, with median age 17 (from 2 to 61). All the patients were collected EDTA anti-coagulated PB samples to test CAR-T cells expansion and CD19+ cells clearance by flow cytometer(FCM), and non-anti-coagulated serum to detect 24 kinds of cytomkines by FCM microbeads on before CAR-T(d0+), d4, d7, d11, d15, d20, and d30 after CAR-T.

Results: As showed in Figure 1A and 1B, CAR-T cells expanded on d4 and reached the peak on d11, and then decreased and went to smooth from d15 in both group. However, a difference was observed between two groups. The peak was higher and the decrease curve was sharp in group2, and the peak was lower and a plateau was formed from d7 to d15 in group1. CD19+ cells clearance formed similar tracking lines in both groups. The CD19+ cells could not be detected on d14 in both group. The clearance rate might be smoother in group2 than that in group1. However, there were no significant differences based on statistic analyze which might result the bigger variable coefficient and less cases.In Figure 1C, within 24 kinds of cytokines, IL-6, sCD25, MCP-1, REG3a, Elafin, ST-2 and TNFRI were observed higher in group1 than group2 with statistic differences.To avoid the effects of allogeneic cells, all the patients in group2 were treated with autologous CD19 CAR-T cells, and patients in group1 with CD19-CAR-T cells from the same donor. Even though CAR-T expansion and target clearance between two groups were lack of statistic differences, the cytokines related to GVHD were higher in grou1 as expected, which might be the results of absent of anti-rejection drugs for CAR-T expansion. Furthermore, general markers, skin related markers, and GI tract related markers were all increased in group1, which might be partially explain why higher rate of unexpected erythra or gastrointestinal hemorrhage in group1 after CAR-T.

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Summary/Conclusion: Most CAR-T clinical trials were targeting refractory or relapsed patients, and more markers should be observed not only related to CRS or inflammation, but also related to GVHD during CAR-T treatment especially for those who relapsed after first HSCT. Based on our data, clinicians should be alert about GVHD markers while treating HCT relapsed patients with CAR-T therapy.

PB2209: HIGH RISK MYELODYSPLASTIC SYNDROME DEVELOPING IN A PATIENT AFTER CHIMERIC ANTIGEN RECEPTOR (CAR) T- CELL THERAPY FOR RELAPSED DIFFUSE LARGE B CELL LYMPHOMA

E. Accorsi Buttini 1,* , M. Farina 1 , A. Turra 1 , E. Morello 1 , N. Polverelli 1 , F. Colnaghi 1 , C. Almici 2 , E. Ferrari 2 , A. Bianchetti 2 , A. Leoni 1 , F. Re 3 , K. Bosio 3 , S. Bernardi 3 , M. Malagola 1 , A. Re 4 , D. Russo 1

1 Unit of Blood Diseases and Bone Marrow Transplantation, Cell Therapies and Hematology Research Program, Department of Clinical and Experimental Science; 2 Stem Cell Laboratory, Section of Hematology and Blood Coagulation, Clinical Chemistry Laboratory, Diagnostics Department; 3 Research Center Ail (CREA), Chair of Hematology Department of Clinical and Experimental Science; 4 Hematology Unit, Spedali Civili di Brescia, Brescia, Italy

Background: Chimeric antigen receptor (CAR)-T cell therapy showed exciting results in relapsed/refractory DLBCL, PML and B-ALL. Cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), transient cytopenias and hypogammaglobulinemia are the main toxicities. We report here a case of high risk myelodysplastic syndrome (HR-MDS) after CAR-T cell treatment for relapsed diffuse large B cell lymphoma (DLBCL).

Aims: To describe a case of HR-MDS occurred after CAR T-Therapy.

Methods: NA

Results: A 57 year-old woman was diagnosed in August 2019 with germinal-center type DLBCL, IVs-A, IPI score 3, CSN-IPI 4, BCL6 and cMYC negative was treated with 4 cycles of R-CHOP and one more cycle of R-MAD, achieving clinical and PET scan complete remission (CR). At this time, bone marrow (BM) biopsy revealed hyperplastic erythropoiesis with hypoplastic megakaryo-granulopoiesis, without lymphoma infiltration. A second course of intensification with HD-AraC and autologous stem cell transplantation (ASCT) was performed on April 2020.

Unfortunately, on December 2020 the patient relapsed. She was evaluated for CAR-T cell therapy and the leukapheresis (T-Ly) was performed on January 2020. After two courses of R-ESHAP no response was achieved and Tisagenlecleucel was administrated on April 2021.

The abdominal ultrasound 30 days after CAR-T cell therapy and TC scan on day 90 showed a partial remission (PR); neither CRS nor ICANS were observed but a progressive pancytopenia (grade II anemia, grade IV thrombocytopenia, grade II leukopenia according to CTCAE). Six month later CAR T-cell infusion, the patient was in clinical and CT- PET scan CR but, the pancytopenia worsened, requiring recombinant human G-CSF, erythrocyte and platelet support. On January 2022, a bone marrow biopsy ruled out lymphoma infiltration but revealed a multilineage dysplasia without excess of blasts and the karyotype showed a chromosome 7 deletion. A diagnosis of Intermediate-2 IPSS risk or high revised-IPSS risk myelodysplastic syndrome (MDS) was made and the patient started azacitidine as bridge allogeneic hematopoietic stem cell transplantation (alloHSCT). Bone marrow NGS analysis before ASCT and after CAR-T cell therapy is ongoing in order to evaluate clonal hematopoiesis and mutational profile.

Summary/Conclusion: Prolonged cytopenias after CAR-T cell treatment for DLBCL have been reported both in the Juliet and ZUMA-1 trial. Outside clinical trials, sustained myelosuppression after BCMA-CAR-T therapy for relapsed myeloma was successfully treated with back up of autologous stem cells and a case of bone marrow failure after axicabtagene-ciloleucel for DLBCL was submitted to allo-HSCT. This first case of HR-MDS developed after CAR-T cell infusion in patient with DLBCL rises several questions: should the candidates to CAR T-cell be evaluated for the presence of a clonal hematopoiesis before the procedure? Is there a rationale for performing BM NGS mutational profile in all patients receiving CAR T-therapy especially in those previously submitted to ASCT? When should myelodysplastic or myeloproliferative disease be suspected after CAR T-cell therapy? May CAR T therapy itself play a role in developing a clonal hematopoiesis or myeloid diseases? Further informations are needed to give us conclusive answers to these questions.

PB2210: FAVORABLE OUTCOME AFTER SECOND CD19 CAR T INFUSION OF AN IDENTICAL PRODUCT: HOST ENVIRONMENT AND DISEASE CHARACTERISTICS DETERMINE THE OUTCOME OF TREATMENT

L. Frölich 1,2,* , V. Blumenberg 1,2,3 , K. Rejeski 1,2,3 , C. Schmidt 1 , M. Winkelmann 4 , F. Schöberl 5 , L. von Baumgarten 6 , W. Kunz 4 , V. Bücklein 1,3 , M. Subklewe 1,2,3

1 Department of Medicine III, University Hospital, University of Munich, Munich; 2 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg; 3 Laboratory for Translational Cancer Immunology, Gene Center, LMU Munich; 4 Department of Radiology; 5 Department of Neurology; 6 Department of Neurosurgery, University Hospital, LMU Munich, Munich, Germany

Background: CD19-targeted CAR T cell therapy has revolutionized treatment options for patients with relapsed/refractory large B-cell lymphoma (r/r LBCL). However, a significant proportion of patients are refractory or relapse after treatment. So far, no consented treatment recommendations for patients failing CAR T cell therapy exist. Patients who relapse after initial response to therapy may be candidates for a second infusion, but data on the efficacy of this approach is still limited (Gauthier et al., Blood 2021).

Aims: We sought to analyze differences in CAR T associated parameters that contribute to a favorable outcome after 2 nd CAR T infusion of the identical CAR T product in a patient with r/r LBCL.

Methods: Here, we present the clinical course of a 39-year-old patient with r/r LBCL, transformed from splenic marginal zone lymphoma (sMZL), who relapsed 11 months post their first CAR T cell infusion (CART1; October 2019) and received a second CAR T cell infusion afterwards (CART2; November 2020).

Results: LBCL relapse was confirmed by histology, and IHC demonstrated positivity for CD19 and PD-L1. After lymphodepletion, the patient received a second dose (CART2; November 2020) of Tisagenlecleucel (Tisa-cel). Prior (day -27) and post (day 20) CART2 the patient received pembrolizumab (200 mg). Toxicity during CART1 was substantial, with ASTCT grade III CRS, grade I ICANS, and prolonged cytopenia (ANC <500/µl at day 90). After CART2 infusion, neither CRS nor ICANS were observed, and no longer-lasting cytopenias occurred. The patient achieved a CR following CART2, which has been ongoing for >15 months.

Although CART1 and CART2 were identical products, derived from the same apheresis and production run, baseline proinflammatory markers (Ferritin) and tumor volume (LDH, MTV) differed between the two CART transfusions. The proinflammatory markers were significantly higher prior to CART1 vs CART2 (Ferritin 1582 vs 529 ng/ml) and the same trend was observed for tumor volume (LDH 262 vs 133 U/l; STLV 23,3 vs 2,6 cm 3 ).

Summary/Conclusion: Second CAR infusions represent a feasible salvage strategy in patients with late relapse after first CAR T infusion. The case illustrates that pretreatment factors, including patient inflammatory state and tumor volume may determine CAR T outcome in this setting. Peri-CAR-T checkpoint blockade may ameliorate tumor-intrinsic resistance mechanisms.

PB2211: PLASMA REG3Α MAY PREDICT PROGNOSIS IN PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA TREATED WITH CAR-T BRIDGING TRANSPLANTATION

M. chen 1 , W. zhao 2 , H. Wang 1,*

1 clinical lab, Hebei Yanda Lu Daopei Hospital, Langfang, sanhe; 2 beijing Lu daopei hospital, beijing, China

Background: Although ST-2, Reg3a, and Elafin can serve as potential prognosis biomarkers for post-transplantation aGVHD, the relationship between serum cytokines and outcomes in chemiric antigen receptor(CAR)-T cell therapy or CAR-T bridging transplantation treatment remain uncleared.

Aims: To investigate the inner connection between clinical outcomes and plasma cytokines level in aGVHD.

Methods: 14 acute lymphoblastic leukemia (ALL) patients (2 T-ALL and 12 B-ALL, 9 males and 5 females) who received CD7 or CD19 CAR-T therapy bridging allogeneic hematopoietic stem cell transplantation(allo-HSCT) were enrolled from September 2019 to January 2022 in Beijing Lu Daopei hospital. The median last time of cytokine detection before CART- therapy was +29d. The levels of cytokines were determined both after CART and one month before the transplantation. The median follow-up time was 997d(374-1239). 4 out of 14 patients died due to aGVHD(28.6% of total, 1 in intestine, 1 in lung and liver, 1in lung and 1 in skin, intestine, and liver). The rest 10 patients were alive in the period of the research.

Results: cytokine levels before CAR-T related with outcomes. The levels of IL-8, IL-10, TNF-α, IFN-γ, sCD25, MIP-1α, MCP-1, Elafin, ST-2, and TNFRI in the death group were higher than those in the survival group, while the serum IL-4 level was higher in the survival group. But the difference was not statistically significant (P>0.05). The serum Reg3α level in the death group was higher than that in the survival group, and the difference was statistically significant (P<0.05). Analysis of serum Reg3α levels at different time points before transplantation showed that the average Reg3α level in the death group was higher than that in the survival group two months before all o-HSCT , indicating that the patients in the death group had a higher level of Reg3α protein pre-existing in the body before treatment. Before treatment, with the use of the induction regimen, the levels of Reg3α protein in the death group and the survival group were decreased (Figure 1).

According to the ROC curve between the concentration of Reg3α and disease outcome in the two groups about one month before transplantation, the optimal critical value was calculated as 1200.57 pg/mL, and the AUC was 0.938 (95% confidence interval was 0.792-1.000, P<0.05). The specificity was 75.0%, the sensitivity was 100%, the PPV was 60.0%, and the NPV was 100% 14 patients were divided into a high Reg3α group and a low Reg3α group by the optimal critical value of Reg3α in patients before transplantation (1200.57pg/mL) obtained from the ROC curve. Started on the date of the allogeneic HSCT, the survival analysis showed that the average survival period of the low Reg3α group was 472 days, and the average survival of the high Reg3α group was 94 days, and the difference between the two groups was statistically significant (P<0.05)

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Summary/Conclusion: High serum Reg3α level in patients after CART treatment before hematopoietic stem cell transplantation is associated with poor prognosis, which can be used as a marker to prompt clinical selection of appropriate therapy.

PB2212: VARIANT HAEMOGLOBIN ASSESSMENT AND CORRELATION WITH PHENOTYPIC PRESENTATION AT DIAGNOSIS IN SICKLE CELL DISEASE: A SINGLE CENTRE RETROSPECTIVE ANALYSIS OF CENTRAL GUJARAT COHORT

S. Bhatwadekar 1,* , S. Deshpande 2 , P. Bhanvadia 1 , R. Shah 1 , A. Shrivastava 1 , D. Desai 3

1 Haemato Oncology, Haemato Oncology Care Centre; 2 Medicine, GMERS Medical College Gotri; 3 Haemato Pathology, Neuberg Labcore Laboratory, Vadodara, India

Background: Sickle cell disease (SCD) is caused by heritable point mutation at codon 6 of the human beta-globin gene widely distributed around the world. According to 2011 census, in India, there is a large tribal community of about 180 million. Among them18 million people carry the sickle cell trait and ~140,000 suffer from SCD. Gujarat State of India has a population of about 62 million, there are ~8.9 million tribal populations, among whom ~9, 00,000 people with Sickle cell Trait and ~70,000 people with SCD are expected. Though phenotypic heterogeneity of SCD is well known across the India, there is limited data on phenotypic presentation with variant haemoglobin correlation on large cohort from Central Gujarat.

Aims: To correlate variant haemoglobin pattern with phenotypic presentation at diagnosis in SCD.

Identify laboratory and clinical parameters to predict risk of complications in SCD.

Methods: Five Hundred and seventy three patients registered at Haemato Oncology Care Centre (HOCC), Vadodara, Gujarat between August 2012 to December 2021, and diagnosed to have SCD were included in this study. This cohort represents tribal belt covering Bharuch,Narmada,Vadodara,Panchmahal,Dahod,Sabarkantha and Banaskantha districts of Central Gujarat. Diagnosis was based on clinical profile, complete blood counts (CBC) and peripheral smear evaluation along with variant haemoglobin assessment by High Performance Liquid chromatography / Capillary electrophoresis. Relevant Biochemistry investigations were done along with radiological assessment of hip joints in patients with specific symptoms.Osteonecrosis of Femur head (ONFH) was diagnosed according to Ficat and Arlet classification for ONFH. Recurrent Vaso occlusive episode(R-VOE) was labelled if there are >3 VOE/yr. Occasional VOE (O-VOE) labelled if <3 VOE/yr.Blood transfusion dependency was labelled if > 3 transfusion /yr required to maintain HB > 7 gm%.Splenomegaly was noted as palpable spleen below costal margin.

Results: A total 573 SCD patients were evaluated. The median age was 20 years (range: 1-74) with 328 (57%) males and 245 (43%) females.HB was < 10 gm% in 378 (66%),HB <7 gm% in 78(14%),between7gm% to 10 gm% in 300(52.4%).315/378 (83%) had HBA <5%,Only 5/378 (1.32%)had Ferritin <20ng/ml.None had high indirect bilirubin with low HB. Transfusion dependency was found in 28(5%),HBA was 0%,<5%,6 to 10%,>10% in 12/28 (43%),7/28 (25%),6/28(21%),3/28(11%) respectively. R-VOE observed in 207 (36%),O-VOE in 259(45%),No VOE in 107(19%).Splenomegaly was found in 268(47%).HBF <10% in 86 (15%),10 to 20% in 258(45%)>20% in 229(40%).All Patients with no VOE 107(19%) had HBF >10% with palpable splenomegaly.ONFH was diagnosed on imaging in 108 (19%).101/108(93.5%) had HBF >10%.,25/108 (23%)had palpable splenomegaly.

Summary/Conclusion: In our cohort High HBF with Splenomegaly was associated with no VOE.

No protection for ONFH seen with high HBF or palpable spleen

Regional increased prevalence of coinheritance of Thalassemia in SCD play important role in influencing clinical presentation and progression of complication like transfusion dependency and ONFH.

In LMIC, Variant haemoglobin assessment with CBC, Retic count values can be used to predict probable coinheritance of Thalassemia; however DNA Mutation analysis on larger cohort can be done for validation.

Region specific risk adapted optimization of treatment can modify natural history of disease

PB2213: EFFECTIVENESS OF CRIZANLIZUMAB IN REDUCING VASO-OCCLUSIVE CRISIS IN PATIENTS WITH SICKLE CELL DISEASE IN BRAZIL: STUDY PROTOCOL FOR AN OBSERVATIONAL, RETROSPECTIVE, MULTICENTER, NATIONAL STUDY

C. Bueno 1,* , L. Campos 1 , A. Ritter 2 , C. Zampirolli 2 , R. Watanabe de Oliveira 2

1 Novartis; 2 IQVIA, São Paulo, Brazil

Background: Sickle cell disease (SCD) is a genetic condition resulting in a structurally abnormal hemoglobin, that leads to a cascade of physiologic consequences, including erythrocyte rigidity, vaso-occlusion, chronic anemia, hemolysis, and vasculopathy. Its management can be intensive, time-consuming, and costly. Few effective treatment options are currently available for these patients, specially to control the vaso-occlusive crisis (VOC). In March 2020, crizanlizumab, a monoclonal antibody drug, was approved by the Brazilian Health Regulatory Agency (ANVISA) to reduce the frequency of VOCs in patients with SCD. Crizanlizumab has been provided through Novartis Managed Access Program (MAP), classified in Brazil as Expanded Access Program (EAP), as an alternative therapy for SCD patients with an immediately life-threatening condition or serious disease. So far, limited evidence is available on crizanlizumab effectiveness and safety profile in Brazil and no real-world evidence study was conducted in the country. In this scenario, real-world studies present an essential role on complementing efficacy and safety data from pivotal and randomized studies in a much broader population profile.

Aims: To describe the study design and rationale for investigation for the first real-world study assessing crizanlizumab in SCD patients in Brazil. The study aims to describe the effectiveness and safety profile of crizanlizumab treatment in patients with SCD in Brazil.

Methods: This is an observational, retrospective, multi-center study based on secondary data of SCD patients treated with crizanlizumab in Brazil. In total, 98 patients treated with crizanlizumab within the Novartis MAP (EAP) will be screened for the study. Patients aged ≥ 16 ≤ 70 years old, diagnosed with SCD and with documented history of at least one VOC episode up to abstraction data initiation will be included in the study. Patient known to be participating in an interventional study at any point of the two-year study period and/or on a chronic transfusion program will be excluded. Data will be retrospectively abstracted from medical chart, considering one-year prior to crizanlizumab onset and up to one year of crizanlizumab treatment to assess the pattern of VOCs. The annual rate of VOC experienced by the patient and leading to a healthcare visit within the preceding 12-months prior to crizanlizumab initiation and 12-month after will be assessed and compared as the primary outcome. Secondary outcomes include the overall effectiveness of crizanlizumab over 1 year of treatment; time to next VOCs in SCD patients; safety aspects – including the frequency of adverse events of interest, e.g.: arthralgia, nausea, back pain, pyrexia, abdominal pain and infusion-related reactions in SCD patients during crizanlizumab treatment; clinical parameters in SCD management as well as demographics and clinical characteristics of SCD patients who were treated with crizanlizumab.

Results: Study in progress. Results not yet available.

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Summary/Conclusion: This is the first real world study to assess the effectiveness and safety of crizanlizumab in Brazilian SCD patients. It is expected that the assessment of SCD patients under MAP in Brazil will provide valuable information closer to what is expected in the real-world scenario. The availability of more robust evidence can support decision-making process, from clinical assessment by providers, to implementation of public healthcare policies.

PB2214: SCD POPULATION SURVEY IN ALBANIA, A PROSPECTIVE NON-INTERVENTIONAL MULTI-CENTER STUDY EVALUATING DISEASE AND PATIENTS CHARACTERISTICS OF SICKLE CELL DISEASE IN ALBANIAN SCD POPULATION

A. Cili 1,* , E. Petrela 2 , A. Zaka 3 , A. Hatellari 4 , P. Pulluqi 1 , T. Caja 1 , A. Perolla 1 , E. Calliku 1 , M. Kreka 5 , E. Dollaku 6 , X. Ngjela 7 , A. Memini 8 , A. Kaleci 9 , A. Ivanaj 1

1 Service of Hematology; 2 Universitary Hospital Centre “ Mother Teresa”, Tirana; 3 Lushnja Hospital, Lushnje; 4 Durres Hospital, Durres; 5 University of Medicine, Tirana Albania, Tirana; 6 Fier Hospital, Fier; 7 Vlora Hospital, Vlore; 8 Kavaja Hospital, Kavaje; 9 Novartis Pharma Services Inc, Albania, Tirana, Albania

Background: SCD is a group of inherited disorders affecting hemoglobin (Hb) synthesis. Sickle cell erythrocytes have a mutant form of Hb, sickle Hb (HbS), which turns normally pliable erythrocytes into rigid, sickle-shaped cells. We conducted a survey project for generation of local data on SCD, to deeper understanding disease burden, patient journey treatment pathways and QoL and get insights on SCD epidemiology on national level, in our country

Aims: The study answered the following main research question: What is the frequency (number per year) of vaso-occlusive crises (VOCs) and their overall QoL in patients with Sickle Cell Disease within routine treatment regimens in the real-world setting in Albania and different regions among permanent resident SCD patients?

Methods: This study was a cross-sectional primary data collection NIS focused on disease that enrolled 242 patients with confirmed SCD diagnosis who signed patient consent forms. 10 selected physicians from Albanian Association of Hematology from 7 centers across the country fulfilled a questionnaire (survey) for each SCD patient, based on patient medical history (retrospective data) and interviewing the patient live (collecting primary data-EQ-5D instruments).

Results: The study demonstrated in fatigue, 11% of patients reported “severe” and “very severe”; in pain/discomfort, 8% reported “severe” and “very severe”, while in mobility only 3.7% reported “severe” and “very severe”; in self-care, the majority of patients reported “no”, “mild” or “moderate” (respectively 86%, 11%, 2.5%); and usual activity dimensions, most of patients reported “no”, “mild”, “moderate” (respectively 64%, 26%, 7.4%). The data we received from our study corrected with the data from literature.

Our study reported 37% of patients reported more than 2 VOCs requiring medical care (hospitalization or other medical facility visit and treatment with oral or parenteral narcotic agents or parenteral NSAIDs), while 67% of patients reported more than 2 VOCs in last 12 months managed at home.

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Summary/Conclusion: This is the first study conducted in Albania in SCD disease area. The study demonstrated the local burden of SCD, the impact of the disease on QoL of SCD patients, one overall of prevalence, geographical distribution of SCD patients, clinical characteristics of sickle cell patients, treatment landscape and clinical practice in Albania. According to our results we concluded that in Albania the burden is considered moderate. The study concluded also some very important findings about the role of treatment and quality of life of SCD patients and the need for other treatment options to improve the outcomes of SCD patients.

PB2215: GLOBAL EPIDEMIOLOGY OF SICKLE CELL DISEASE: A SYSTEMATIC LITERATURE REVIEW

R. Colombatti 1,* , C. Birkegård 2 , M. Medici 2

1 University of Padova, Padova, Italy; 2 Novo Nordisk A/S, Søborg, Denmark

Background: Sickle cell disease (SCD) has a high prevalence and social impact worldwide, with a high mortality within the first three years of life. A high prevalence has been reported in sub-Saharan Africa, the Mediterranean, the Middle East, and India. With global migration, prevalence is also increasing in other geographic regions, but there are no definitive data on global prevalence and mortality of SCD.

Aims: To perform a systematic literature review (SLR) of available evidence on the epidemiological burden (prevalence, birth prevalence and mortality/life expectancy) of SCD.

Methods: A comprehensive literature search of bibliographic databases and proceedings from key conferences was performed to identify peer-reviewed studies reporting on SCD epidemiology, from 1 January 2010, to 30 December 2020 (congress abstracts 2018 to 2020 only). The SLR protocol followed PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Two independent reviewers screened titles and abstracts based on predefined inclusion and exclusion criteria, including reporting of relevant population data and at least one outcome of interest. Multiple data elements were extracted, including number of people screened and diagnosed, prevalence, birth prevalence, mortality/survival/life expectancy, geographic region, years of data collection, genotype and age category. Study quality was assessed with a version of the GRADE (Grading of Recommendations, Assessment, Development and Evaluations) framework, adapted to observational studies. Meta-analyses using a binomial normal random-effects model were carried out to estimate the prevalence and birth prevalence globally and regionally (when sufficient data were available).

Results: Of 1581 journal articles and 482 abstracts screened, 115 publications met the inclusion criteria; 43 included data on birth prevalence, 24 on prevalence and 33 on mortality or life expectancy.

Birth prevalence of SCD (children ≤1 year old), mostly measured through new-born screening, was highest in several sub-Saharan Africa countries (500–2000/100,000), South America and Caribbean Islands (20-1000/100,000); in USA and European countries found in the SLR, birth prevalence was ≤500/100,000.

Overall findings on regional prevalence indicated that sub-Saharan and North-East Africa, India and the Middle East are global SCD “hotspots”. However, prevalence data were too sparse to provide consistent prevalence numbers per region through meta-analysis.

Mortality appeared particularly high in low-resource settings, including Africa and India; although, different methods of data presentation and source populations make comparisons across studies difficult.

There are limitations to this SLR due to gaps in the published literature, including inconsistent reporting of SCD genotypes, diagnosis criteria and study settings. In addition, there was paucity of peer-reviewed publications on life expectancy and prevalence from European countries with assumed high prevalence. A search of global and local registries is ongoing to supplement the published literature.

Summary/Conclusion: This SLR has provided some insights into the global epidemiology of SCD, confirming earlier studies of areas with a high prevalence. However, this SLR confirms that there is a great need for resources for additional studies with uniform data collection on prevalence and mortality to ensure an increased awareness of SCD among public health policy makers worldwide.

PB2216: VACUOLES APPEAR IN RED BLOOD CELLS TRANSFUSED TO PATIENTS WITH SICKLE CELL DISEASE-RELATED HYPOSPLENISM

L. Dumas 1,* , A. Sissoko 1 , A. Fricot 1 , N. Salama 1 , L. Joseph 2 , S. Manceau 2 , S. Dokmak 2 , A. Michel 3 , B. Maitre 3 , C. Gachet 3 , M. Cavazzana 2 , C. Roussel 2 , P. Buffet 1

1 Université de Paris, UMR1134; 2 Assistance publique des hôpitaux de Paris (APHP); 3 Université de Strasbourg, UMR 1255, Paris, France

Background: Defective spleen function (hyposplenism) affects subjects with sickle cell disease (SCD) and is associated with complications. Red blood cell (RBC) alterations are very accurate markers of spleen function. Vacuole-containing RBC (pocked RBC) circulate in high proportions (10 to 70%) in the blood of hyposplenic subjects while their proportion does not exceed 5% of RBC in healthy subjects. Vacuoles in pocked RBC are generally considered as remnants of organelles in erythroblasts.

Aims: The aim is to determine the origin of vacuoles

Methods: Patients with SCD who benefit from an exchange transfusion program, have not only their own HbSS RBC but also transfused HbAA RBC in circulation. In vitro sickling “Emmel test” discriminates HbSS RBC that sickle, from HbAA RBC that do not sickle.

We perfused a mixture of HbSS RBC and HbAA RBC (from exchange transfusion left-overs) into human spleens ex-vivo. We then analyzed spleen sections and quantified vacuoles in RBC containing hemoglobin polymers or not.

Results: Using Emmel Test, we observed vacuoles in HbSS RBC but also in transfused HbAA RBC. Vacuoles in HbAA RBC were more frequent in transfused SCD patients than in healthy subjects (25.1±4.41% vs. 2.8±2.1%).

In our experimental ex vivo perfusion, most RBC without hemoglobin polymers is HbAA RBC. We observed a high rate of vacuoles in both polymerized and non-polymerized RBC (29.8±3.7% vs. 17.9±5.8%).

Summary/Conclusion: Taken together, these results strongly suggest that vacuoles are created de novo in transfused HbAA RBC. Counting vacuoles in RBC labeled with anti-HbS and anti-HbA antibody is ongoing to validate these observations. When the spleen is dysfunctional, these pocked RBC stay in circulation hence their validity as a spleen function marker. De novo appearance of vacuoles in transfused HbAA RBC likely impacts the kinetics of reappearance of pocked RBC in circulation following regular or exchange transfusion of hyposplenic subjects, and opens interesting prospects on cell processes involved in their genesis.

PB2217: SICKLE CELL DISEASE IS A RISK FACTOR FOR THE PROGRESSION AND PROGNOSIS OF COVID-19

V. Goyal 1,* , A. Singh 1 , Y. Memon 1 , N. Thakre 1 , P. Sahu 1 , R. Goyal 1

1 Sanjeevani CBCC Cancer Hospital, RAIPUR, India

Background: Since December 2019, COVID-19 has aroused global attention. Studies show the link between many comorbid condition and severe outcome incoronavirus disease 2019 (COVID-19). There are limited number of studies regarding the association between sickle cell disease and COVID-19 outcomes.

Aims: To figure out whether sickle cell disease is a risk factor influencing the progression and prognosis of 2019 novel coronavirus disease (COVID-19).

Methods: A total of 152 consecutive patients confirmed with COVID-19 were studied. Demographic data, medical history, symptoms and signs, laboratory findings, chest computed tomography (CT) as well the treatment measures were collected and analysed.

Results: We found that COVID-19 patients without other comorbidities but with sickle cell disease (n = 18) were at higher risk of severe pneumonia, release of tissue injury-related enzymes, excessive uncontrolled inflammation responses and hypercoagulable state associated with sickle cell pathology. Furthermore, serum levels of inflammation-related biomarkers such as IL-6, C-reactive protein, serum ferritin and coagulation index, D-dimer, were significantly higher (P < .01) in sickle cell patients compared with those without, suggesting that patients with sickle cell disease are more susceptible to an inflammatory storm eventually leading to rapid deterioration of COVID-19.The frequency of death (sickle: 23.9% vs. nonsickle: 13.8%), ICU admission (sickle: 27.8% vs. nonsickle:14.71%), and ventilator requirement (sickle: 35.93% vs. nonsickle: 20.63%) were significantly higher in sickle patients than in nonsickle patients (P < 0.001).

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Summary/Conclusion: Our data support the notion that sickle cell disease should be considered as a risk factor for a rapid progression and bad prognosis of COVID-19. More intensive attention should be paid to sickle cell disease patients having COVID-19 infection.

PB2218: ASSESSMENT OF CYSTATIN C IN PEDIATRIC SICKLE CELL DISEASE AND B-THALASSEMIA AS A MARKER OF SUBCLINICAL CARDIOVASCULAR DYSFUNCTION: A CASE-CONTROL STUDY

M. Beshir 1 , D. Hanna 1,* , N. Khalifa 1 , E. Baz 2 , A. Elhewala 1

1 Pediatric Hematology and Oncology; 2 Zagazig University Hospital, Zagazig, Egypt

Background: Cardiovascular complications represent the main determinant of survival in patients with hemoglobinopathies. Serum cystatin C is a well-known marker of nephropathy in sickle cell disease (SCD) and b-thalassemia patients that has recently emerged as a strong predictor of cardiovascular dysfunction in patients with and without kidney disease.

Aims: We performed a case control study to determine the role of cystatin C as a predictor of subclinical cardiovascular dysfunction in SCD and b-thalassemia patients.

Methods: We enrolled 40 SCD patients with a mean age of 12.4 years, 40 b-thalassemia patients with a

mean age of 11.4 years and 40 age and sex-matched controls. We assessed hematological profile, serum ferritin, urinary albumin–creatinine ratio (UACR), serum cystatin C, echocardiography and carotid intima media thickness (CIMT).

Results: UACR, cystatin C and CIMT were higher in SCD and b-thalassemia patients compared to controls

(p < .001). Significantly higher cystatin C levels were observed in SCD and b-thalassemia patients with nephropathy or left ventricular systolic dysfunction (shortening fraction <30%, or ejection fraction

<55%; p < .001). Moreover, SCD patients with pulmonary hypertension had significantly higher cystatin C levels. Cystatin C levels were positively correlated with CIMT in SCD (p = .02) and b-thalassemia

patients (p < .001) while negatively correlated with ejection fraction and shortening fraction (p < .001). The cutoff values of cystatin C ≥ 16.03 and ≥ 13.2 (ng/mL) could detect subclinical cardiac dysfunction risk among SCD and b-thalassemia patients respectively.

Summary/Conclusion: Cystatin C appears to be a promising marker for subclinical cardiovascular dysfunction in SCD and b-thalassemia patients.

PB2219: A REAL-WORLD EXPERIENCE WITH HEALTHCARE RESOURCE UTILIZATION PATTERNS AND RELATED COSTS IN PATIENTS WITH SICKLE CELL DISEASE IN NORTH LEBANON

A. Inati 1,* , M. Sabbagh 2 , C. Al Alam 3 , A. Al Kheir 4 , Z. Massouh 5 , R. Demachkie 6 , C. Ojaimy 5 , N. Dani 7 , A. Siddiqui 8 , R. Rajat Adhikary 9 , B. Lynch 10 , A. Srivastava 9

1 School of Medicine, Lebanese American University; 2 Novartis, Beirut, Lebanon; 3 American Center for Psychiatry and Neurology, Abu Dhabi, United Arab Emirates; 4 Saudi German Hospital, Dammam, Saudi Arabia; 5 Department of Pediatrics, NINI Hospital, Tripoli; 6 School of Medicine, Lebanese American University, Byblos, Lebanon; 7 Novartis; 8 Novartis Pharma AG, Basel, Switzerland; 9 Novartis, Hyderabad, India; 10 Novartis, Dublin, Ireland

Background: Sickle cell disease (SCD) remains associated with high healthcare resource utilization (HRU), despite a reduction in morbidity and mortality associated with the use of disease-modifying therapies, mostly because of vaso-occlusive crises (VOC). In Lebanon, there are limited national estimates on HRU, cost of medical care, and treatment expenditures for patients with SCD. A better understanding of these estimates may provide new perspectives to improve access to high-quality cost-effective health care services.

Aims: Aim of our study is to evaluate patterns of HRU and related cost in a cohort of patients with SCD receiving care in a Comprehensive SCD referral center at NINI hospital in North Lebanon.

Methods: An observational, non-interventional, retrospective study of 136 patients (54.4% females) with confirmed diagnosis of SCD was conducted in North Lebanon. Data on HRU, including ED visits, ambulatory visits, and hospitalizations, was collected in a patient health information (PHI)-anonymized format from patients’ paper and electronic medical files, as well as hospital and ED files, from May 1, 2018 to April 30, 2020. Uncomplicated VOCs were defined as pain crises, whereas complicated VOCs were defined as acute chest syndrome, acute splenic sequestration, acute hepatic sequestration or priapism. The annual rate of HRU visits was calculated as a ratio of the total number of visits to the total number of years of follow-up across all patients. Based on the available data, the average yearly cost was determined as the ratio of the total cost of these visits to the total number of years of follow-up.

Results: The median age of patients in this study was 10.8 years (IQR 5.3 to 19.6 years) at the time of investigation. Majority of patients were diagnosed with SS (72.1%) and Sβ0 (21.3%) genotype across all age groups. The most prevalent SCD-related complications were pain crisis (90.4%), fever (43.4%), acute chest syndrome (33.1%), and acute splenic sequestration (22.8%). Most of hospitalizations, ICU visits, and ED visits in adult patients (age >16 years) were due to uncomplicated VOCs. The average annual costs of HRU were 8,270,920 Lebanese pounds (LBP) amounting to 5,514 USD (per patient per year) of which 92.1% was for hospitalizations, 4% for ED visits and 3.9% for ambulatory visits. Uncomplicated VOCs had the highest annual costs for ED, ICU and hospitalizations. Costs related to medications, diagnostics, non-hematologist medical consults, uncompensated care and lost productivity are not included in this analysis. Analgesics, folic acid and hydroxyurea were the most commonly prescribed drugs across all age categories, with hydroxyurea being prescribed to 84% of patients.

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Summary/Conclusion: SCD and associated complications resulted in significant acute HRU, according to this real world study. VOCs continues to be most important factor for resource use, ICU admission and costs, with hospitalizations accounting for majority of annual costs. Despite the high HRU rate in this relatively young SCD cohort, the cost of HRU in a comprehensive setting with effective outpatient management as that in North Lebanon appears affordable for the health care system. However when additional SCD related and non SCD related costs of care are factored in, the economic burden of SCD care is likely to be far higher than the figures presented here.

PB2220: MORTALITY IN PATIENTS WITH HEMOGLOBINOPATHIES AND (SARS-COV-2) COVID-19 INFECTION: SAUDI ARABIA EXPERIENCE

H. Ezzat 1,* , O. Kashari 2 , D. Alissa 1 , R. Almasuood 1 , A. Al-jedai 1

1 Ministry of Health, Riyadh; 2 Ministry of Health, jeddah, Saudi Arabia

Background: Hemoglobinopathies are a group on inherited red blood cell disorders that include Sickle Cell Disease (SCD) and Thalassemia. Prevalence of hemoglobinopathy disorders in Saudi Arabia is high. However, information on clinical manifestations and mortality among patients with SARS-CoV-2 (COVID-19) infection in SCD and thalassemia is scarce.

Aims: To explore patients’ characteristics, associated comorbidities, clinical manifestations, received treatment, and the main causes of death among in-hospital patients with COVID-19 infection who have hemoglobinopathies in Saudi Arabia during 2021.

Methods: The study is a retrospective review of the Ministry of Health (MOH) COVID- 19 mortality database. The analysis included all reported deaths with hemoglobinopathy disorders and positive COVID-19 infection.

Results: The study included 35 patients, 19 (54.3%) were males. Their age ranged from 16 to 63 years (Mean±SD: 40.5±13.2 years). A total of 25 patients (71.4%) were with sickle cell anemia, 6 (17.1%) with sickle cell trait, while each of a-thalassemia trait, b-thalassemia trait, b-thalassemia major and sickle b-thalassemia affected only one patient (2.9% each). Almost three-fourths of patients (26, 74.3%) were admitted to the intensive care units. Exchange transfusion was done for one patient (2.9%), top up blood transfusion (RBCs) was done for 18 patients (51.4%), fresh frozen plasma infusion for 6 patients (17.1%), platelets transfusion for 5 patients (14.3%), cryoprecipitate infusion for 4 patients (11.4%) and intravenous immunoglobulin infusion for two patients (5.7%). Clinical manifestations for COVID-19 among hemoglobinopathy patients were manifested as agitation (34, 97.1%), chest pain (33, 94.3%), diarrhea (31, 88.6%), confusion (29, 82.9%), cough (20, 57.1% each), oxygen desaturation (20, 57.1% each), fever (17, 48.6%), dyspnea (9, 25.7%), headache (2, 5.7%), abdominal colic (2, 5.7%), or vomiting (2, 5.7%). The main associated comorbidities were hypertension (8, 22.9%), diabetes (7, 20%), or chronic kidney diseases (7, 20%). The main received medications were anticoagulants, such as Enoxaparin (22, 62.9%), Heparin (14, 40%), norepinephrine (16, 45.7%), epinephrine (9, 25.7%), inotropes (20, 57.1%), Vasopressin (6, 17.1%), antivirals, such as Favipiravir (15, 42.9%), immunosuppressives, e.g., Methylprednisolone (8, 22.9%), dexamethasone (7, 20%), Tocilizumab (5, 14.3%) and antibiotics, such as Azithromycin (16, 45.7%), Ceftriaxone (8, 22.9%), Vancomycin (5, 14.3%), and Tazocin (3, 8.6%). The main causes for death among patients reported to the mortality database with hemoglobinopathies were septic shock (12, 34.3%), acute respiratory distress syndrome (8, 22.9%), multiple organ failure (5, 14.2%), renal failure (5, 14.2%), acute chest syndrome (5, 14.3%), pulmonary embolism (3, 8.6%), vaso-occlusive crisis (3, 8.6%), or heart failure (2, 5.7%), or disseminated intravascular coagulation (1, 2.9%).

Summary/Conclusion: The mortality rate among patients with hemoglobinopathies is approximately 0.38 percent of all deaths reported with COVID-19 to the database. The main clinical manifestations for COVID-19 among hemoglobinopathy patients were agitation, chest pain, diarrhea, confusion, cough, oxygen desaturation, fever, and dyspnea. Causes of death are variable. Most of deaths were indirectly related to COVID-19 infection. The main received medications are anticoagulants, inotropes, antivirals, and antibiotics. Causes of mortality were mainly septic shock, multiple organ failure, renal failure, or acute chest syndrome.

PB2221: A SYSTEMATIC LITERATURE REVIEW DESCRIBING PROTECTIVE EFFECTS OF HBF IN SICKLE CELL DISEASE OUTCOMES

O. Mitelman 1,* , J. Barry 1 , J. Bernhard 1 , P. Bruno 1 , C. Morabito 1 , S. Snedecor 2 , S. Ronnebaum 2

1 Fulcrum Therapeutics, Inc., Cambridge; 2 OPEN Health Evidence & Access, Bethesda, United States of America

Background: Patients with sickle cell disease (SCD) are vulnerable to a variety of clinical complications, including pain crises, acute chest syndrome (ACS), splenic sequestration, leg ulcers, stroke, and mortality. HbF (fetal hemoglobin) reduces disease severity and decreases the risk of SCD complications, but the degree of HbF’s protective effects has not been systematically explored.

Aims: To characterize the clinical benefit of elevated HbF levels on patients with SCD.

Methods: A systematic literature review (SLR) was performed in Embase, MEDLINE, and the Cochrane Library to identify all evidence describing HbF levels and/or longitudinal changes versus adverse outcome occurrence among patients with SCD in observational or interventional studies. Citations were screened and extracted by dual independent review. Weighted linear regression was performed to identify data trends where applicable. Variability in factors such as study design, outcomes analyses, and reporting methods may have contributed to the heterogeneity of results.

Results: The SLR identified 2997 citations, from which 148 studies were selected for investigation. Within the 148 studies, 901 distinct analyses detailed the impact of HbF levels on SCD patient outcomes. Given the differences in study design, 4 different approaches were described in evaluating the impact of HbF levels:

1.) Average adverse outcome rates based on study-level HbF averages were investigated in 735 analyses. Weighted linear regression demonstrated a directional trend and supported HbF’s protective impact on outcomes such as among hospital admissions, ED visits, and hospital days.

2. The associations between patient-level HbF and adverse outcome occurrence were detailed in 109 analyses (see image). Higher HbF levels led to lower risk of adverse outcome occurrence (i.e., were protective) in 91 analyses (36 analyses were statistically significant and 55 were not significant or significance was not reported).

3. The average adverse outcome occurrence among patients segmented by HbF ranges/thresholds were described in 40 analyses. There was a lower rate of adverse outcomes in patients in the highest HbF range compared to patients in the lowest HbF range in 30 out of the 40 comparisons (10 were statistically significant, 13 were not significant, and 7 were not reported).

4. Study-level comparisons of average HbF levels of patients that experienced an adverse outcome vs. those that did not were described in 17 analyses. In 14 comparisons, patients not experiencing the outcome showed higher average HbF (9 were statistically significant, 5 were not significant), with the difference between the 2 groups ranging from 1.0%-7.1% of total Hb.

Summary/Conclusion: The vast majority of studies demonstrated that elevated HbF values have a clinically meaningful benefit on SCD patient outcomes. Relatively small changes in HbF appear to protect patients against relevant adverse clinical outcomes.

PB2222: THE PROTECTIVE EFFECT OF THE SPLEEN IN SICKLE CELL PATIENTS. A COMPARATIVE STUDY BETWEEN PATIENTS WITH ASPLENIA AND HYPERSPLENISM.

S. Peretz 1,2,* , L. Livshits 1,3 , E. Pretorius 4 , A. Makhro 3 , A. Bogdanova 3,5 , M. Gassmann 3 , A. Koren 1 , C. Levin 1,2

1 Pediatric Hematology Unit, Emek Medical Center, Afula; 2 The Ruth and Rappaport Faculty of Medicine, Technion–Israel Institute of Technology, Haifa, Israel; 3 Red Blood Cell Research Group, Vetsuisse Faculty, Institute of Veterinary Physiology, University of Zurich, Zürich, Switzerland; 4 Department of Physiological Sciences, Faculty of Science, Stellenbosch University, Stellenbosch, South Africa; 5 The Zurich Center for Integrative Human Physiology (ZIHP), Zürich, Switzerland

Background: Sickle cell disease (SCD) is caused by a point mutation in the beta globin gene. SCD is characterized by chronic hemolytic anemia, vaso-occlusive events leading to tissue ischemia and progressive organ failure. A chronic inflammatory state is part of the pathophysiology of SCD. Patients with SCD have extremely variable phenotypes, from a mild disease to severe complications including early age death.

The spleen is commonly injured in SCD. Early splenic dysfunction and progressive spleen atrophy are common. Splenomegaly and hypersplenism, can also occur with loss of the crucial splenic function. Acute, life-threatening spleen-related complications in SCD are well studied. The implications of the spleen status including asplenia and splenomegaly/ hypersplenism in vaso- occlusive crisis and long-term complications in SCD remain to be determined.

Aims: In this study, we compared blood parameters and RBC properties of SCD patients and compared the results between patients with asplenia, to patients with splenomegaly/hypersplenism. In addition, we implemented novel, non-routinely used methods for the evaluation of the functional status of the spleen in those patients.

Methods: We evaluated the association between the spleen status with clinical and laboratory parameters in 31 SCD patients divided into two groups: Group a) Patients with asplenia (N=22) (including auto-splenectomy and patients that were splenectomized) vs. Group b) patients with Splenomegaly and or Hypersplenism (N=9). Laboratory studies included: CBC, reticulocyte count, iron metabolism parameters, CRP, Hb variant distribution and D-dimer. Metabolic and morphological red blood cell (RBC) studies included: density gradient (by Percoll), glucose consumption, lactate release and K + leakage; fetal RBC (F-Cells) and F Reticulocytes, annexin V+, CD71+, oxidative stress measured by GSH presence in RBC and finally HJB count were all analyzed by FC. SEM analysis of RBC was also performed.

Results: Patients in group (a) showed significantly higher WBC, platelet, HCT, hemoglobin S, CRP, D-dimer, GGT, cholesterol and transferrin levels than patients in group (b). The percent of hyperchromic RBCs, F-Reticulocytes were lower in patients with asplenia. Important differences were also found between the groups in the studied RBCs metabolic parameters.

Summary/Conclusion: Further studies are required to elucidate the effect of the spleen including hyper and hypo-splenia in vaso-occlusive crisis, vascular pathology, and long-term complications of SCD. The benefits and risks of splenectomy compared to chronic transfusion need to be evaluated in clinical trials and the standard approach to manage hypersplenism in SCD patients should be re-evaluated.

PB2223: COST OF ILLNESS, EPIDEMIOLOGY AND HEALTHCARE RESOURCE UTILIZATION FOR SICKLE CELL DISEASE IN THE GULF “CESGU”

D. Soliman 1,* , J. Altooq 2 , S. Alkindi 3 , H. Dewedar 4 , A. Ibrahim 5 , S. Elsawy 6

1 Novartis Middle East, Dubai, United Arab Emirates; 2 Salmaniyah Medical Complex, Manama, Bahrain; 3 Sultan Qaboos University, Muscat, Oman; 4 Dubai health Authority; 5 Accsight, Dubai, United Arab Emirates; 6 Accsight, Cairo, Egypt

Background: Sickle cell disease SCD is the most predominant form of haemoglobinopathy worldwide. The disease presents challenges to healthcare systems, both in terms of patients’ morbidity, mortality and in terms of healthcare costs. In the middle east world, SCD is prevalent and patchy. As currently there are insufficient information published on the financial burden of SCD in the GCC region, there is a growing need to calculate the cost of the disease across the region.

Aims: The aim of our study is to estimate the financial burden from payor’s perspective through calculation of direct-medical costs (DMC) of SCD in Oman (OMN), Bahrain (BHR) and United-Arab-Emirates (UAE).

Methods: A prevalence-based probabilistic model was used to calculate the DMC associated with SDC within different treatment sites across the GCC region. Aggregate input was gathered from hospital record databases of adult SCD patients managed in year 2019. Data was captured on Microsoft© Excel. Additionally, Case Report Forms (CRFs) were filled. DMCs included treatment, hospitalization, consultations, diagnostics, interventions and costs related to managing SCD complications.

Results: Aggregate data for 2,600 SCD patients in BHR, 2,000 in OMN and 350 in UAE were collected from hospital records data bases. The total DMC per patient per year was estimated at $15,300, $10,000 and $15,800 in BHR, OMN and UAE, respectively. The costs incurred by payers for “VOCs management” were identified as major cost driver including hospitalization and acute medications administered during managing the event, contributing to 63%, 39% and to a lesser extent 14% of the total DMC of SCD in UAE, BHR and OMN. A sub analysis of sever SCD patients with ≥5 VOC in a year revealed a significant burden across all sites, contributing to 89%, 75% and 45% of the total SCD burden in UAE, OMN and BHR sites, respectively.

Summary/Conclusion: The financial burden of individuals affected by SCD in GCC region is on rise with highest burden in Bahrain, followed by UAE then Oman. Severe SCD patients with ≥5 VOCs represent a significant burden on payers. Shedding the light on the incurred costs by payers for SCD management can inform decision makers about efficient medical interventions that would help optimize the use of resources and secure access od SCD to better treatment options.

PB2224: IMPACT OF COVID-19 PANDEMIC ON PATIENTS WITH SICKLE CELL DISEASE: RESULTS FROM THE REAL WORLD ASSESSMENT SURVEY FOR SICKLE CELL DISEASE IN SAUDI (ROARS)

H. Ezzat 1,* , A. Al Zayed 2 , H. Al Saeed 2 , M. AlDarwish 2 , M. Albagshi 3 , H. Malhan 4 , A. Tarawa 5 , A. AlManea 6 , I. AlHazmi 6 , M. Qari 7 , A. Al Jefr 8 , A. Alzahrani 9 , M. Alzahran 10 , W. Soliman 11 , M. Arsalan 11 , W. Jastaniah 12

1 Johns Hopkins Aramco Healthcare, Dahran; 2 Qatif Central Hospital, Qatif; 3 Almana General Hospital-Al Ahsa, Hofouf; 4 Prince Mohamed ben Naser, Jizan; 5 King Salman Medical City Al Madinah, Madinah; 6 King Fahad Armed Forces Hospital; 7 King Abdulaziz University Hospital, Jeddah; 8 King Faisal Specialist Hospital; 9 Prince Sultan Military Medical City; 10 King Abdulaziz Medical City; 11 Novartis, Riyadh; 12 King Faisal Specialist Hospital & Research Center (Gen. Org.), Jeddah, Saudi Arabia

Background: Sickle cell disease (SCD) is a chronic hemolytic disease with multisystem involvement leading to significant morbidities and mortalities. SCD confers a significant burden on patients (pts), including a high impact on quality of life (QoL). The impact of the COVID-19 pandemic on pts with SCD has not been well characterized.

Aims: The Real World Assessment Survey for Sickle Cell Disease in the Kingdom of Saudi Arabia (KSA) (ROARS) is a cross-sectional survey of pts with SCD and treating healthcare professionals (HCPs), which was designed to understand several aspects of the burden of SCD in KSA. In this study, we examined the impact of the COVID-19 pandemic on SCD pts based on findings from ROARS.

Methods: Between September and December 2021, pts with SCD (age: 5-65 years) and HCPs (>2 years SCD experience, SCD load >50 pts, >50% of clinical practice time spent treating SCD pts) were surveyed (in person or via caregiver if pts were between 5-14 years of age) using the structured, bilingual ROARS questionnaires (English/Arabic) through personal quantitative web-assisted telephone interviews. ROARS was developed by SCD experts from KSA and Novartis, and includes the global health domain of Patient-Reported Outcomes Measurement Information System (PROMIS), a validated tool for assessing QoL among SCD pts and caregivers of different age ranges. Questions covered topics such as disease impact on QoL, symptoms, disease complications, treatment goals, treatment strategies, and COVID-19. Descriptive statistics were used to summarize the results.

Results: A total of 1011 pts (pediatric: n=504; adult: n=507) and 64 HCPs (hematologists: n=31; non-hematologists: n=33) completed the survey. Data were collected from 11 cities, with 90% of respondents from Eastern and South-Western provinces of KSA where the SCD prevalence is highest. The top 5 complications reported by both pediatric and adult pts were fever (52%), joint issues (49%), swollen and/or painful fingers/toes (47%), infections (37%), and tight chest pain (35%). A high proportion of pts (80%) felt that the pandemic had an impact on their medical care (Figure) (adults: 85%;[PM(1] pediatric: 76%). The impact of COVID-19 on medical care was particularly evident in pts treated with hydroxyurea compared to untreated pts (adults: 91% and 79%; pediatric: 84% and 74%). When survey results were stratified by the number of vaso-occlusive crises (VOCs), 85% of adult pts reported an impact of the pandemic on their medical care, regardless of the VOC frequency (<5 or ≥5); among pediatric pts, 72% with ≥5 VOCs and 83% with <5 VOCs reported an impact. Mental/emotional impact of the pandemic was reported by 66% of pts (adults: 68%; pediatric: 64%). The majority of pts missed or cancelled their appointments due to COVID-19 (adults: 81%; pediatric: 74%). Overall, 39% of the pts reported the impact of the pandemic as severe (Figure). From the HCPs’ perspective, 86% felt that COVID-19 affected their medical service, and 78% thought it affected their pts’ mental/emotional well-being. Overall, 16% of HCPs thought the pandemic significantly impacted their pts.

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Summary/Conclusion: The ROARS survey among Saudi pts with SCD found that the majority of pts, especially adults, felt the COVID-19 pandemic had an impact on their medical care and emotional well-being. The impact of COVID-19 was also reflected in a decline in pt care, as reported by HCPs. In future, it would be interesting to understand the impact of the pandemic on vaccinated vs unvaccinated SCD pts.

PB2225: ENVIRONMENTAL MODIFIERS OF SEVERITY IN CHILDREN WITH SICKLE CELL DISEASE: A FEASIBILITY PILOT STUDY

J. Strouse 1,* , U. Obdodo 2 , H. Stapleton 3 , D. Thompson 1

1 Hematology, Duke University, Durham; 2 Medical School, Lincoln Memorial University Debusk School of Osteopathic Medicine, Harrogate; 3 Environmental Science and Policy, Duke Nicholas School of the Environment, Durham, United States of America

Background: Sickle cell disease is a severe hemoglobinopathy that affects approximately 40,000 children in the United States and over 300,000 infants worldwide each year, mostly in Africa and India. Environmental factors including air and other sources of pollution and weather are potentially modifiable factors that are associated with disease severity in children and adults with SCD in ecological studies of exposure. Our overarching hypothesis is environmental factors such as air pollution, weather, and chemical exposures modify disease severity in children with sickle cell disease.

Aims: To evaluate the feasibility of measuring individual exposure to air pollution and other toxins and morbidity in children with sickle cell disease

To describe the prevalence of exposure to brominated flame retardants, organophosphates, polyaromatic hydrocarbons, polychlorinated biphenyls, pesticides, and phthalates in children with sickle cell disease.

Methods: We enrolled participants with sickle cell disease between 5 and 18 years of age in a prospective study of environmental exposures. We collected information on exposure by questionnaire, biospecimens (saliva, venous blood), and silicone wrist bands worn by the participants for 5 days. We extracted polycyclic aromatic hydrocarbons and other semi-volatile organic compounds from the silicone wrist bands and characterized the compounds by gas chromatography-mass spectrometry. We measured salivary cotinine using a highly sensitive quantitative enzyme immunoassay (Salimetrics, Carlsbad, CA). We collected information on acute health utilization [ED visits, hospitalizations retrospectively (2 years) and prospectively (6 months)] and laboratory results from the electronic health record.

Results: We successfully consented 30 (86%) of the 35 patients approached for the study. Our 30 participants were 60% female with a mean age of 12.5 ±3.6 years and hemoglobin of 8.7 ± 1.5 g/dL. Most (84%) had homozygous sickle cell (HbSS), 13% were compound heterozygotes for HbS and C (HbSC) and 3% had sickle beta null thalassemia. Almost all (90%) were treated with hydroxyurea, 10% with voxelotor, and 3% with crizanlizumab. We successfully collect blood and saliva specimens on all 30 participants and completed surveys on 17 (57%) of participants. We identified salivary cotinine levels consistent with environmental tobacco smoke exposure (1 – 20 ng/ml) in 11 participants (44%) and primary tobacco use in 1 (4%). We received 25 of 30 silicone bands from the initial visit (83%) and identified a high prevalence of exposures to multiple chemicals.

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Summary/Conclusion: This study confirms the feasibility of individual level measures of indoor and outdoor chemical exposures using saliva and silicone bands in children with sickle cell disease. We identified substantial levels of exposure to tobacco smoke and multiple classes of chemicals from other consumer products, furniture, housing materials, and air pollution that may influence the severity of sickle cell disease. We plan to assess the relationship among these exposures and severity in future studies.

PB2226: THE ROLE OF COMPLEMENT IN THE IMMUNE RESPONSE OF SICKLE CELL DISEASE PATIENTS AFER COVID-19 VACCINATION

C. Varelas 1,* , E. Gavriilaki 1 , I. Sakellari 1 , P. Klonizakis 2 , E.-E. Koravou 1 , I. Christodoulou 2 , I. Mavrikou 1 , A. Kourelis 3 , F. Chatzopoulou 3 , D. Chatzidimitriou 3 , T. Touloumenidou 1 , A. Papalexandri 1 , A. Anagnostopoulos 1 , E. Vlachaki 2

1 Hematology & HCT Unit, General Hospital “George Papanikolaou”, Thessaloniki, Greece; 2 Adult Thalassemia Unit, 2nd Department of Internal Medicine, Aristotle University of Thessaloniki, Hippokration General Hospital; 3 Microbiology Department, Aristotle University of Thessaloniki, Thessaloniki, Greece

Background: Healthcare systems around the world are still under considerable pressure from the constant threat of the coronavirus disease-19 (COVID-19) pandemic. Although adult sickle cell disease (SCD) patients were vaccinated as a high-risk group, due to their coexisting immunosuppression and possible development of acute thoracic syndrome, there are no data on the following immune response.

Aims: We investigated the immune response of our SCD patients to COVID-19 vaccination.

Methods: We prospectively studied adult SCD patients monitored at our Center who received at least one dose of COVID-19 vaccination until August 2021. We detected neutralizing antibodies (nAbs) against SARS-CoV-2 in patients’ sera (ELISA, cPass™ SARS-CoV-2 nAbs Detection Kit; GenScript, Piscataway, NJ, USA), 21 days after each vaccination dose. Levels ≥30% are considered as positive, while levels ≥50% are considered highly protective against severe disease. We investigated possible predictive factors, including age, usage of anticoagulants, pulmonary hypertension, previous episodes of vascular strokes, hydroxycarbamide administration, regular transfusions, renal damage and complement activation. Complement activation was detected in patient’s sera by measuring soluble C5b-9 with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Quidel, upper normal limit: 245 ng/ml), before vaccination and 21 days after the second dose.

Results: Among 53 adult SCD patients monitored at our Center, 27 were studied after their first dose (24 received BNT162b2, 2 mRNA-1273, and 1 patient JNJ-78436735). Their median age was 40 years. Sixteen were female and 23 had the S/β genotype. NAbs ≥30% were detected in 14/27 patients, 11 of whom (84%) had already increased complement activation. On the contrary, among the 13 patients who did not develop nAbs after the first dose, only 5 (38.4%) were found with increased levels of soluble C5b-9 (p=0.042). The patient that received the single-shot JNJ vaccine did not develop adequate nAbs, despite increased C5b-9 baseline levels. Interestingly, 2/27 patients who had suffered from a vascular stroke in the past, also did not develop adequate nAbs (p=0.002). No other significant associations between nAbs and studied parameters were found. Clinically important SARS-CoV-2 inhibition and protection against severe disease indicated by nAbs ≥50% was detected only in 11/27 patients (40.7%) after the first dose.

Twenty-one days after the second dose we were able to study 22 patients. NAbs significantly increased in all patients (p<0.001) at levels ≥50%, even though 12 patients had levels of <30% after the first dose. Similarly, a significant increase of C5b-9 levels was also detected after the second dose (p=0.009). This increase was above the upper normal limit in the majority of patients (16/22), although 13/22 had normal baseline levels. No complications of COVID-19 vaccinations were reported.

Summary/Conclusion: Our study demonstrates a satisfactory immune response of adult SCD patients, primarily after the second vaccination dose. Complement activation seems to play an important role in the establishment of adequate immunogenic response. Patients suffering from serious complications, such as stroke, appear to develop clinically significant nAbs, only after the second dose. More data are needed regarding the duration and adequacy of immune responses in SCD patients in order to evaluate the necessity of additional doses.

PB2227: HEALTH-RELATED QUALITY OF LIFE OVER TIME IN PEDIATRIC PATIENTS WITH SICKLE CELL DISEASE ADMITTED FOR VASO-OCCLUSIVE CRISIS.

C. Vuong 1,* , I. Moussa 1 , L. Haverman 2 , E. Rettenbacher 1 , H. Heijboer 1 , J. Twisk 3 , K. Fijnvandraat 1 , C. L. de Groot-Eckhardt 1

1 Pediatric Hematology; 2 Child and Adolescent Psychiatry and Psychosocial Care, Amsterdam University Medical Centers, Amsterdam Medical Center; 3 Clinical Epidemiology and Biostatistics, Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, Amsterdam, Netherlands

Background: Sickle cell disease (SCD) is the most common inherited hemoglobinopathy characterized by chronic hemolytic anemia, vaso-occlusive crises (VOCs), multi-organ damage and an impaired health-related quality of life (HRQoL). Previous studies have shown that the occurrence of VOCs affects the HRQoL substantially in children with SCD. However, there is limited evidence about the HRQoL changes over time following hospitalization for VOC.

Aims: The aim of this study is to evaluate the longitudinal impact of hospital admission for VOCs on the HRQOL in pediatric patients with SCD.

Methods: In this retrospective cohort study, children between the age of 8 and 18 years with SCD treated at the Amsterdam University Medical Centers were included. As part of standard care, these children complete once a year Patient Reported Outcomes measures (PROMs) on the KLIK PROM portal ( www.hetklikt.nu ). HRQoL was measured with the Pediatric Quality of Life Inventory (PedsQL) (score ranging from 0-100, a higher score indicates better QoL). Data were collected from the medical files of patients, who filled out at least two PedsQL questionnaires between 2012 and 2021. The effect of the occurrence and frequency of hospital admissions for VOCs on the HRQoL was evaluated using a linear mixed model 3, 6, 9 and 12 months after hospital admission, adjusted for age and SCD genotype.

Results: In total, 109 children were included with a median age of 11.8 years (IQR: 9.2 – 14.2), including 72 patients (66%) with the severe genotype: HbSS or HbSβ0. During follow-up, 45 patients (41%) were admitted to the hospital for a VOC. If hospitalization occurred 6 and 12 months prior to the PedsQoL measurement, a decrease of 3.61 (p=0.049) and 3.80 points (p=0.007) in HRQoL respectively, was seen compared to patients without hospitalization. As for the frequency of VOC admission in the preceding 12 months, a decrease of 2.28 points in HRQoL was observed for every additional admission (p=0.012).

Summary/Conclusion: The negative longitudinal association between hospital admission for VOC and HRQoL in pediatric patients with SCD gets worse and persists up to 12 months. These findings highlight the importance of monitoring using PROMS in clinical practice and extra attention and support for the negative consequences of the hospital admission for VOCs, affecting the HRQoL.

PB2228: PRECISION MEDICINE IN THALASSEMIA: 30-YEAR EXPERIENCE AT SINGLE INSTITUTE FROM SAUDI ARABIA

S. Al Jaouni 1,2,*

1 Hematology Department, Faculty of Medicine,King Abdulaziz University, Jeddah, King Saudi Arabia; 2 Hematology Department, King Abdulaziz University, Jeddah, Saudi Arabia

Background: The potential for precision medicine a wellness vision and improve the quality of life of thalassemia.

Treatment of thalassemia disorders is complex, expensive and requires a multi-disciplinary approach. Disease complication is common due to excessive iron overload and sub-optimal chelation.

Aims: A new comprehensive and integrated approach to wellness

Proper diagnostic of disease complication

Early detection of disease risk

Prevention of disease complication

Individualized treatment plans

Methods: Retrospective Record Review for all thalassemia patients Treated at King Abdulaziz University Hospital from the year 2000 until 2021.

Assess disease complication including Chronic pain and disease outcome for wellness and happiness

Results: Serum ferritin is a poor indicator of myocardial and hepatic iron overload in thalassemia (T.M. &T.I.)

The clinical and all the conventional tests were insufficient

to detect excessive iron overload in the liver and the heart.

Magnetic resonance imaging using T2-star (MRIT2*) is a reliable and safe non-invasive method for accurate assessment of iron overload in the heart and the liver

Splectomy increase the risk of morbidity and morbidity in thalassemia (TM & T I)

Endocrine complication is common problem in thalassemia with increased in the prevalence of glucose dysregulation

Experienced chronic pain more than 10 years 71.1% (n=138)

Experience chronic pain more than 5 years 37.6% (n=73)

Musculoskeletal pain 66.6% (n=92)

Joint pain and bone pain (30.1%; n=22), (16.4%; n=12)

Chronic headaches 33.3% (n=46)

Summary/Conclusion: Comprehensive care by multi-disciplinary approach for early detection and recognition of disease complication with appropriate transfusion regimen, chelation therapy are the keys for preventing disease complication and improve quality of life on thalassemia.

Chronic pain is common complication in thalassemia patients, unfortunately not addressed well with comprehensive care.

Increase the challenges of disease complication duo to inappropriate care and shortages of essential drugs.

We recommend to have Stem cell transplantation for all thalassemic with HLA match donor at younger age

PB2229: NECESSITY FOR A ROBUST INFRASTRUCTURE FOR TREATING ALLIED COMPLICATIONS OF AGEING HAEMOGLOBINOPATHY POPULATION IN MIDDLE INCOME COUNTRIES

T. Imran 1,* , H. Altaf 1 , L. Khan 2

1 Surgery, Shifa International Hospital; 2 Antomy, Rifah Medical College, Islamabad, Pakistan

Background: Haemoglobinopathies are the most common genetic disorders in Pakistan. With the significant advancement in therapeutic modalities the overall survival of this affected population has increased significantly. This improved survival has highlighted several associated sequalae awaiting considerations by advanced medical centers with expert faculty that are lacking in developing countries. The most common complications observed are cardiovascular disease, endocrine disorders, osteoporosis, hepatic pathology, renal dysfunction, and tumors

Aims: Advanced centers are mandatory to treat the emerging complications of ageing population especially beta-thalassemia’s and sickle cell syndromes patients. Intensive research projects under supervision by west are needed to cater this ageing haemoglobinopathy population to lead a normal life expectancy in Pakistan

Methods: The study is conducted at Jamila Sultana Foundation (JSF) Pakistan. It is a cross sectional study conducted from 2016 till date

Results: Total patients at JSF are 954, Deceased 125, 828 are under treatment by conventional transfusion and chelation approach while BMT is done for only 5 patients. Out of 954, 592 detected beta thalassemia major, 304 Thalassemia trait, 24 Thalassemia intermedia 24, 9 sickle cell,12 sickle beta thalassemia,7 E Beta thalassemia and 6 D beta thalassemia patients. Patient age ranges from o1year to 47 years, gender distribution 215 females and 744 males. Among dead 106 patients were of thalassemia major, 16 thalassemia intermedia and 3 sickle cell anemia. Major cause of death was cardiovascular 32, 4 due to thrombosis, respiratory infection affected 3 and 3 died due to liver syndromes. Data showed that all these patients were not getting appropriate advance care availability at large at national. However better quality of care was received by only those patients who were educated and wealthy and could afford international consultations both online and personal visits.

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Summary/Conclusion: Changing attitude is required at national health regulatory authority level for long term project development and sending local doctors for training abroad. This project updates must be shared with all the ageing patients’ for their opinion in decision-making for the advance centers. Educate and permit ageing patients to support and strengthen their role in monitoring their own disease.

PB2230: EFFICACY AND SAFETY OF SINOPHARM VACCINE FOR SARS-COV-2 AND BREAKTHROUGH INFECTIONS IN IRANIAN PATIENTS WITH HEMOGLOBINOPATHIES: A PRELIMINARY REPORT

M. Karimi 1,* , T. Zarei 1 , S. Haghpanah 1 , A. Azarkeivan 2 , M. Naderi 3 , S. Matin 4 , A. Bazrafshan 1 , Z. Zahedi 1 , A. Shirkavand 5 , P. Pishdad 6 , V. De Sanctis 7

1 Hematology Research Center, Shiraz University of Medical Sciences, Shiraz; 2 Zafar Adult Thalassemia Clinic, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine; 3 Iranian Blood Transfusion Organization Research Center, Thalassemia Clinic, Tehran; 4 Pediatric Department, Jahrom University of Medical Sciences, Jahrom; 5 Laser Medicine Research group, MLRC, Yara institute, ACECR; Medical physicist, Pardis Noor Medical Imaging center, Tehran; 6 Medical Imaging Research Center, Department of Radiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; 7 Coordinator of ICET-A Network (International Network of Clinicians for Endocrinopathies in Thalassemia and Adolescence Medicine) and Pediatric and Adolescent Outpatient Clinic, Quisisana Hospital, Ferrara, Italy

Background: The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to high morbidity and mortality worldwide. Vaccination against SARS-CoV-2 is a leading strategy to change the course of the COVID-19 pandemic.

Aims: Our aim was to investigate the efficacy and side effects of Sinopharm vaccine in patients with hemoglobinopathies in Iran, and the frequency of breakthrough infection after a full course of vaccination.

Methods: A multicenter cross-sectional study of 434 patients with hemoglobinopathies (303 β-thalassemia major, 118 β-thalassemia intermedia, and 13 sickle-thalassemia) were conducted from March to July 2021 in IRAN. All patients have received the first dose of China Sinopharm vaccine and received the second dose of the vaccine 28 days apart. Detection of immunity after vaccination was evaluated by commercial enzyme-

linked immunosorbent assay (Pishtazteb ELISA commercial kit), including a surrogate virus

neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total

neutralizing antibody (NAb).

Results: The mean age of patients was 35.0± 8.5 (from 18 to 70) years and 55.6% were positive for antibody. Overall, 48.2% of the studied population had at least one side effect after vaccination. The most frequent side effects were fever and chills, dizziness, and body pain.

A total of 90 (20.7%) vaccinated patients developed breakthrough infections after full Sinopharm

vaccination. Disease severity was recorded, and it was classified as mild in 77.8%, moderate in

13.6%, and severe in 7.4% of patients. One 28-year-old woman with β- thalassemia major died

eight days after the diagnosis of breakthrough SARS-CoV-2 infection.

Summary/Conclusion: No safety concerns were identified in patients who received two doses of Sinopharm vaccine. Its efficacy was not optimal which might be due to lack of its effect on new variations of virus. However, our data show that it can prevent from severity of COVID-19 infection in patients with hemoglobinopathies. The frequency of breakthrough infections after full Sinopharm vaccination support the evolving dynamic of SARS-CoV-2 variants requiring special challenge, since such infection may represent a risk for vulnerable patients.

PB2231: RATIONALE FOR THE USE OF COMBINATION CHELATION THERAPY IN PATIENTS WITH THALASSEMIA SYNDROMES OR SICKLE CELL ANEMIA: A SYSTEMATIC LITERATURE REVIEW

A. Kattamis 1,* , J. Kwiatkowski 2 , F. Tricta 3 , E. Igboekwe 4 , C. Fradette 3 , A. Piga 5 , S. Sheth 6

1 National and Kapodistrian University of Athens, Athens, Greece; 2 The Children’s Hospital of Philadelphia and Perelman School of Medicine of the University of Pennsylvania, Philadelphia, United States of America; 3 Chiesi Canada Corporation, Toronto, Canada; 4 Chiesi USA, Inc., Boston, United States of America; 5 University of Turin, Turin, Italy; 6 New York Presbyterian Hospital/Weill Cornell Medical College, New York, United States of America

Background: Patients with thalassemia syndromes or sickle cell disease receiving regular blood transfusions may experience iron overload. Currently, 3 iron chelators are approved for clinical use: deferoxamine administered parenterally (DFO), oral deferiprone (DFP), and oral deferasirox (DFX). Combination therapy of DFP was approved by the European Medicines Agency in 2016, and recent British Society for Haematology guidelines state that DFP+DFX is highly effective. Combination therapy is commonly used in patients who do not adequately respond to monotherapy, have increased iron overload in the heart or liver, or have dose-dependent toxicity, but there is a need to understand which combination therapy to initiate, and when to do so.

Aims: A systematic literature review was conducted to explore the use-rationale of 3 combination therapies (DFP+DFO, DFX+DFO, and DFP+DFX) in patients with thalassemia syndromes or sickle cell disease.

Methods: A systematic search was performed of the Embase and MEDLINE databases to identify trials related to use of 3 combination iron chelation therapies (DFP+DFO, DFX+DFO, or DFP+DFX) in patients with thalassemia syndromes or sickle cell disease. The literature search was conducted in May 2021 and was not restricted to a specific start date. The search strings were established by logical association of appropriate words and MESH terms. Publications were screened based on a priori exclusion and inclusion criteria (eg, case-report exclusion). Use rationales were extracted from trial-inclusion criteria and patient-history data.

Results: Of 1725 publications found in the search, 71 (prospective, 48 studies; retrospective, 23 studies) assessed combination chelation therapies in patients with transfusional iron overload. Half (35 studies) explicitly described the rationale for combination chelation therapy: DFP+DFO, 24 studies, 33.8%; DFX+DFO, 6 studies, 8.5%; and DFP+DFX, 5 studies, 7.0%. Reasons for initiating DFP+DFO included patients having elevated serum ferritin despite previous DFO therapy (9 studies, 12.7%); poor compliance to DFO (7 studies, 9.9%); elevated cardiac or liver iron content (5 studies, 7.0%); cardiac disease/complications (4 studies, 5.6%); and decreased left ventricular ejection fraction, assessment of sequential dosing, adverse events with DFP, and inadequate chelation with DFP (1 study each, 1.4%). Reasons for initiating DFX+DFO included elevated serum ferritin or severe liver or cardiac iron load despite previous use of monotherapy (DFP, DFO, or DFX) or combination therapy (DFP+DFO, DFX +DFO) (5 studies, 7.0%); adverse events due to elevated doses of unspecified monotherapy, agranulocytosis due to DFP in DFP+DFO combination, end-organ injury, and compliance issues with DFO (1 study each, 1.4%). Reasons for initiating DFP+DFX included severe serum iron load despite previous monotherapy (5 studies, 7.0%), of which 3 studies reported patients having elevated liver or cardiac iron and 2 studies noted DFP, DFO, or DFX monotherapy at the maximum tolerated dose.

Summary/Conclusion: As expected, this systematic literature review of iron chelation combination therapy captured studies on DFP+DFO, with recent publications focusing on DFP+DFX. Most studies did not explicitly report the rationale for use. The most common reasons for combination therapy were severe iron overload despite previous chelation therapy, followed by compliance or toxicity concerns. A meta-analysis of available clinical evidence may provide insights to help standardize clinical practice for combination chelation therapy.

PB2232: TRIAL IN PROGRESS: A PHASE 2, OPEN-LABEL STUDY EVALUATING THE SAFETY AND EFFICACY OF THE ERYTHROCYTE PYRUVATE KINASE ACTIVATOR ETAVOPIVAT IN PATIENTS WITH THALASSEMIA OR SICKLE CELL DISEASE

A. Lal 1,* , R. C. Brown 2 , T. Coates 3 , T. Kalfa 4 , J. Kwiatkowski 5 , J. Brevard 6 , C. Trenor 6 , K. Wood 6 , S. Sheth 7

1 University of California San Francisco, San Francisco; 2 Children’s Healthcare of Atlanta, Atlanta; 3 University of Southern California Keck School of Medicine, Los Angeles; 4 Cincinnati Children’s Hospital Medical Center, Cincinnati; 5 Children’s Hospital of Philadelphia and Perelman School of Medicine, Philadelphia; 6 Forma Therapeutics, Inc., Watertown; 7 Weill Cornell Medicine, New York, United States of America

Background: In sickle cell disease (SCD), a single β-globin gene mutation results in sickle hemoglobin (HbS) that polymerizes upon deoxygenation, causing red blood cells (RBCs) to sickle leading to various complications. In thalassemia, RBCs have an imbalance in the ratio of α/β globin chains and aggregated, unpaired globin chains increase metabolic demands. These stresses result in ineffective erythropoiesis and shorten RBC lifespan, leading to chronic anemia. The resultant anemias, exacerbated by impaired RBC health, are associated with lower ATP levels than in healthy RBCs. Supportive care and agents like hydroxyurea are used most in treating SCD, with some patients (pts) on regular transfusions. Regular or episodic transfusions, with their own set of complications, are the mainstay of treatment for thalassemia.

Etavopivat, an investigational, once-daily, selective, erythrocyte pyruvate kinase (PKR) activator increases ATP and decreases 2,3-DPG. 1,2 In a Phase 1 study, etavopivat 300-600 mg once daily in pts with SCD not regularly transfused was well tolerated, improved hematologic markers, decreased hemolysis and improved markers of RBC health. 1,2 Etavopivat 200 and 400 mg once daily (doses predicted to provide desired pharmacodynamic response profiles) are being evaluated in a Phase 2/3 study of pts with SCD not on chronic transfusions (The Hibiscus Study, NCT04624659 ).

Aims: Describe the design of a Phase 2, open-label, multicenter study ( NCT04987489 ) evaluating efficacy and safety of etavopivat in pts with SCD on chronic transfusions (Cohort A), transfusion-dependent thalassemia (Cohort B) and non-transfusion-dependent thalassemia (Cohort C).

Methods: Key eligibility criteria and study design are outlined in the Figure. Pts will receive etavopivat 400 mg once daily for 48 wks. Pts will provide written informed consent.

Baseline assessments will include medical, disease, transfusion and medication histories. Transfusions received during the study (every ~3-5 wks) will be recorded and include Hb values before and ≥15 min after transfusion, transfusion dates, number of RBC units, volume of packed RBCs and hematocrit of the transfused unit (if available). If a pt has an increase ≥1.0 g/dL in pre-transfusion Hb vs baseline, the investigator may delay transfusion 1 wk or reduce the number of RBC units transfused. RBC-exchange transfusions may also be performed in pts with SCD.

The primary endpoints are outlined in the Figure. Secondary and exploratory endpoints include the proportion of pts with a reduction in transfusions ≥33% and ≥50%, respectively, over 12 wks; reduction in transfusions over 12, 24 and 48 wks (Cohorts A/B); Hb response at Wks 24 and 48; and change from baseline in Hb over 12, 24 and 48 wks (Cohort C). Additional endpoints will be assessed in all cohorts: change from baseline in quality of life (using SF-36 and PROMIS); change from baseline in serum ferritin level at 12, 24 and 48 wks; liver iron at 48 wks; 2,3-DPG and ATP; pharmacokinetics; and safety. Primary endpoints will be analyzed using a 1-sided test at α=0.025.

Results: Results are not yet available for this trial in progress. Planned enrollment includes ≤20 pts (aged 12-65 y) in each of the 3 cohorts (Figure).

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Summary/Conclusion: Etavopivat is a novel, investigational, once-daily, selective PKR activator with potential to improve RBC health and lifespan. This Phase 2 study will assess the safety of etavopivat and its impact on Hb levels and transfusion burden in pts (12-65 y) with SCD or thalassemia.

1 Brown et al, Blood 2021.

2 Kalfa et al, Blood 2021.

PB2233: SYSTEMATIC LITERATURE REVIEW OF HEALTH-RELATED QUALITY OF LIFE BURDEN IN PATIENTS ACROSS THE SPECTRUM OF THALASSEMIA

K. M. Musallam 1,* , L. Vinals 2 , K. Gilroy 3 , M. Rizzo 4 , L. Lombard 5

1 Thalassemia Center, Burjeel Medical City, Abu Dhabi, United Arab Emirates; 2 Health economics and outcomes research, Cytel Inc., Montreal, Canada; 3 Medical affairs, Agios Pharmaceuticals, Cambridge, United States of America; 4 Health economics and outcomes research, Cytel Inc., London, United Kingdom; 5 Health economics and outcomes research, Agios Pharmaceuticals, Cambridge, United States of America

Background: Recent systematic literature reviews (SLRs) have highlighted the health-related quality of life (HRQoL) burden in transfusion-dependent thalassemia (TDT), particularly β-thalassemia (β-thal). However, less is known about HRQoL in non–transfusion-dependent thalassemia (NTDT) and alpha-thalassemia (α-thal).

Aims: To conduct an SLR on the real-world HRQoL burden across the entire spectrum of thalassemia and report evidence gaps.

Methods: Searches (2010 to September 2021) were conducted in MEDLINE, Embase, Cochrane Database of Systematic Reviews, Health Technology Assessment Database, NHS Economic Evaluation Database, and EconLit for real-world studies reporting on HRQoL, utilities, and patient-reported outcomes in adult and pediatric patients with thalassemia. Conference abstracts from 2017 to September 2021 were also searched. References were screened at title/abstract and then full text by two independent reviewers. Data extraction was undertaken by one reviewer and validated by a second independent reviewer. Results were grouped by genotype (β-thal/α-thal) and transfusion phenotype (TDT/NTDT) to support comparisons.

Results: A total of 2,541 records were screened at title/abstract, 441 at full-text and 7 references were identified through other sources. A total of 126 publications reporting on 116 study populations were included. Most evidence was published since 2015 (n=95) and sample sizes ranged from 22 to 1240. The number of publications by transfusion phenotype (TDT/NTDT) and thalassemia genotype (β-thal/α-thal) is shown in Table 1. Overall, most TDT/NTDT studies were conducted in β-thal or did not report genotype. Four studies included patients with α-thal but did not report HRQoL results separately for this subgroup. Across 52 studies reporting on TD β-thal, most showed that both adult and pediatric patients have reduced HRQoL. One study found EQ-VAS utility values to be poorer in adults than in children with TDT (mean [SD]: 75.04 [17.86] for >18 years vs. 84.97 [15.16] for ≤18 years; P<0.001). Four studies in pediatric patients noted the negative impact of TDT on caregivers’ HRQoL. Very few studies assessed HRQoL for NTDT patients vs healthy controls, and none were in adult population. One pediatric study found NTDT patients had worse total and domain PedsQOL scores (all P<=0.05) than healthy controls. Several studies compared NTDT and TDT patients. Using the SF-36, one study found significantly poorer HRQoL in NTDT than in TDT adults (mean total score [SD]: 66.5 [16.1] vs. 75.8 [18.8]; P=0.021). A prospective, non-interventional study found significantly lower SF-36 mental component scores at baseline in NTDT compared to TDT adults (mean [SD]: 47.4 [8.4] vs. 51.6 [7.2]; P <0.01), and NTDT patients had worse HRQoL change from baseline at 24 weeks than TDT patients. In pediatric patients, one study found poorer HRQoL in TDT compared to NTDT (mean PedsQL total score [SD]: 42.25 [20.61] vs. 67.76 [18.31]; P=0.0001). However, both NTDT and TDT patients had poorer HRQoL than healthy controls (83.45 [15.6]); P<0.0001).

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Summary/Conclusion: This SLR highlights the negative impact of thalassemia on HRQoL. Adding to previously published SLRs, it shows limited evidence is available for NTDT patients; however, studies suggest that HRQoL burden may be worse in NTDT than TDT patients, particularly in adults. Characterizing the HRQoL burden of α-thal is a clear evidence gap. High-quality, real-world studies in NTDT and α-thal are needed to better understand the HRQoL burden and the potential impact of new treatments on HRQoL.

PB2234: HEALTH LITERACY LEVEL ASSOCIATED WITH THALASSEMIA AMONG TUNISIAN’S YOUTH AND PROSPECTS FOR PRIMARY PREVENTION

F. Maaoui 1,2,* , I. Moumni 1 , F. Arboix-Calas 3 , I. Safra 1 , S. Mnif 1

1 Laboratory of molecular and cellular hematology, Pasteur Institute of Tunis; 2 ISEFC, Virtual University of Tunis, Tunis, Tunisia; 3 Faculty of Education, Montpellier University, Montpellier, France

Background: Thalassemia remains a public health problem in Tunisia. The difficulties in setting up a prevention program can be explained in part by negative social representations and the general public’s ignorance of this disease.

Aims: This study examined the health literacy (HL) of Tunisian secondary school students regarding thalassemia as well as their emotional and behavioral attitudes towards this disease and preventive measures.

Methods: This is a quantitative study; a cross-sectional survey was conducted among Tunisian youths (n = 182; male 23,3 %, female 76,7 %). Data collection took place in two public secondary schools. Students aged 17 to 19 completed a paper and pencil questionnaire and described their knowledge level about Thalassemia, their affective and behavioral attitudes as well as their estimates of hereditary transmission risks and usefulness of prevention measures. Statistical software (SPSS, v.25.0) was used to analyze the data.

Results: The results indicate low HL scores concerning Thalassemia. Lack of knowledge and confusion about the cause, symptoms, and diagnosis of Thalassemia is a common finding at all school levels surveyed.

48,9 % has not identified the hereditary nature of diseases and confuse it with dietary iron anemia 30,7%.

Although 70,5% of respondents recognized severe anemia as a symptom however, 57,4% did not link jaundice to thalassemia, almost 30% said this disease causes rash, fever, dry cough and sore throat.

The results show low levels in relation with assessment of the hereditary transmission risks on the one hand and the prenuptial screening usefulness on the other hand. 24,4% do not consider it necessary to inform their partner of their carrier status. 45% do not see the benefit of genetic counseling before marriage and prenatal diagnosis even if the family of one of the two partners has a history with disease.

Summary/Conclusion: Primary prevention of thalassemia depends on a comprehensive strategy of promotion and health education for Tunisian’s youth. This survey reveals the difficulties of youths to consider the risks of hereditary transmission of thalassemia and to understand the importance of screening and genetic counseling. These results can be explained by the deficit of communication about disease severity and the lack of adequate educational content in Life Sciences programs.

PB2235: COMPLETE DELETION OF THE CACCC BOX OF THE HBB GENE PROMOTER: A NEW MECHANISM LEADING TO A SILENT PHENOTYPE OF BETA THALASSEMIA

E. Mondesert 1 , M. GIANSILY-BLAIZOT 1 , J. DELAGE 2 , F. DANTON 1 , P. AGUILAR-MARTINEZ 1,*

1 Biological Haematology Department, CHU de Montpellier; 2 Service d’hématologie clinique, Clinique du Parc, Montpellier, France

Background: Beta-thalassemias result from defects in the production of beta globin chains. They are inherited in an autosomal recessive manner. Heterozygotes, who are clinically asymptomatic, can typically be diagnosed by the presence of a microcytic hypochromic anemia associated with elevated HbA2 (above 3.5%), HbF being inconsistently increased. The spectrum of molecular defects is broad and heterogeneous. Exonic, intronic and flanking regions of the HBB gene, as well as distant regulatory areas on the beta globin locus control region (LCR) may be affected.

Aims: We describe for the first time a complete deletion of the CACCC box of the beta globin gene promoter, which lead to a silent beta thalassemia phenotype and compared this presentation with Single Nucleotide Variants at this box and with mouse models.

Methods: The proband was referred to our specialized consultation to explore an isolated increase in HbA2 (3.8 and 4.1% on 2 different controls) found at hemoglobin studies, in the context of a medically assisted procreation (MAP). The blood count was normal (Hb 156 g/L, MCV 86 fL). The patient was not taking any medication. Ferritin, thyroid hormone, vitamin B12 and B9 levels were normal. After informed written consent, molecular analysis of the beta and alpha globin gene clusters was performed, including HBB, HBA1 and HBA2 gene sequencing by the Sanger method and searching for large rearrangements (SALSA MLPA Probemix P140 HBA, and SALSA MLPA Probemix P102 HBB, MRC Holland). Family members were also examined.

Results: DNA sequencing revealed an undescribed 15-base pair deletion in the HBB gene promoter: [HBB:c.-145_-131del] in the heterozygous state. This deletion removes the entire proximal CACCC motif, a binding site for the erythroid Kruppel-like factor transcription factor (EKLF/KLF1). We ruled out the coexistence of alpha-thalassemia, which would have reduced the imbalance of the alpha and beta chains, by checking the absence of pathogenic sequence variations within the alpha globin genes, as well as of large rearrangement of the alpha globin gene cluster. Family screening showed that the variant was inherited from the proband’s mother who had increased Hb A2 and normal CBC, while the father had a strictly normal profile. Genetic counseling was offered to the proband and affected family members.

Summary/Conclusion: The HBB gene promoter has two CACCC motifs: a distal one located at positions c.-155_-151, where a single sequence variation, associated with silent beta-thalassemia, has been described and a proximal one, at positions -140_-136, where 7 beta + thalassemia point mutations have been reported. EKLF, which normally binds the deleted region, is involved in the regulation of several genes through its C2H2 zinc finger domain recognizing canonical CCNCNCCC sites. It plays a critical role in erythroid differentiation, and EKLF -/- mice display a beta-thalassemia phenotype and die early from anemia. However, the in vitro deletion of the CACCC box of the HBB gene shows that EKLF may also be involved in the regulation of the beta globin gene independently of the proximal CACCC motif, a hypothesis that suggests the role of other upstream CACCC elements on the LCR. The absence of anemia and/or microcytosis in the proband also contrasts with the complete loss of the proximal CACCC motif. The complete deletion of the proximal CACCC motif, a natural variant described here for the first time in the heterozygous state, in a patient with a silent beta thalassemia phenotype, confirms that this motif is not essential for the action of the erythroid transcription factor EKLF/KLF1.

PB2236: HUMORAL RESPONSE TO SARS-COV-2 VACCINATION WITH BNT162B2 COVID-19 MRNA IN PATIENTS WITH HAEMOGLOBINOPATHIES

I. Giatagantzidou 1,* , D. Pantelidou 2 , D. Papadopoulou 2 , E. Sidiropoulou 1 , S. Dionysopoulou 1 , P. Zebekakis 3 , L. Skoura 1

1 Microbiology Department; 2 1st Department of Internal Medicine THALASSEMIA UNIT; 3 1st Department of Internal Medicine, AHEPA UNIVERSITY HOSPITAL, Thessaloniki, Greece

Background: Thalassemia and sickle cell disease are globally amongst the most common inherited hemoglobin disorders, both resulting in defective hemoglobin production and chronic hemolytic anemia. Patients with sickle cell disease and transfusion-dependent thalassemia (TDT) are considered in high risk of developing severe SARS-CoV-2 infection, due to the several co-morbidities related to the underlying disease, as well as complications of chronic transfusions. These include cirrhosis, diabetes, nephropathy, coagulopathy, anatomic and functional asplenia.

Aims: Even though vaccination is encouraged for these patients, data on the efficacy of anti-Sars-CoV-2 vaccines in this specific population remain limited, due to its exclusion from clinical trials.

Methods: In the following prospective observational study, serum antibody titers for SARS-CoV-2 after vaccination were measured in a total of 63 TDT and SCD patients and 63 healthy controls matched for age and sex. Informed consent was obtained from all participating individuals. Both groups received two doses of Pfizer-BioNTech mRNA COVID-19 vaccine (BNT162b2). Serum samples were collected from both groups 15 to 30 days after the second dose of the vaccine and tested for the quantitative determination of anti-spike IgG antibodies to SARS-CoV-2. Antibody titers were measured by Abbott SARS-CoV-2 IgG II (Semy-Quant) immunoassay with the Alinity i system. Results are reported as Arbitrary Units (AU)/ml and the cut-off defining non- response, as set by the manufacturer, is <50 AU/ml.

Fifty-three patients suffered from TDT and 10 patients had SCD. None reported COVID-19 infection before the vaccination. Patients’ mean age was 46 years (range 28-75), 41% of them were male and 59% were female.

Results: All patients achieved a serological response to vaccination (>50AU/ml). Antibody titers were significantly higher in the patients’ group (p=0,001) (mean 17.676,63 AU/ml ± 13286,01, median 14.536,60, range 489,4-40.000) compared to the control group (mean 13.244,69 ± 9837,91, median 11.304,40, range 451,3-40.0000). In particular patients with TDT had a significantly elevated mean antibody titer (18.848,4 AU/ml) compared to the healthy population, while those with SCD had a slightly lower one (11.466,3 AU/ml). Age wise the youngest subjects in both groups (<30 years old) had the poorest serological response in terms of antibody titers. Among the TDT patients those who developed the highest titers (>30.000 AU/ml) had particularly low serum ferritin levels (<500ng/ml). In the patients’ group the ferritin levels above 1000ng/ml were related with lower but not statistically significant antibody titers (18.931,93 vs 14213,41 p=0,652). Response to vaccination was not affected by the spleen status (anatomic and functional asplenia vs spleen presence) or the immunoglobulins’ serum levels. With a median follow-up of 6 months, only one patient with TDT and Common Variable Immunodeficiency (CVID) developed a COVID-19 infection.

Summary/Conclusion: In conclusion, the BNT162b2 anti-SARS-Cov-2 mRNA vaccine demonstrated efficacy in our cohort of TDT and SCD patients. Antibody titers in TDT patients without hemosiderosis were significantly higher than in the control group. The immunological stimulation as a result of multiple blood transfusions could increase nonspecifically the antibody producing cells in TDT without iron overload. Further observations are needed to assess the humoral response, efficacy and possible factors influencing this finding in TDT patients.

PB2237: HEMATIMETRIC DETECTION OF THE TRIPLICATION OF ALPHA-GLOBIN GENES. THE IMPORTANCE OF YOUR DIAGNOSIS

P. Ropero 1,2,* , F. A. González 1 , J. M. Nieto 1,2 , S. Rochas 1,2 , M. Gómez 1 , B. Colás 1 , H. Martín 1 , P. Estival 1 , A. Villegas 1 , C. Benavente 1 , G. E. de Eritropatología 3

1 Hematology, Hospital Clínico San Carlos; 2 Instituto de Investigación Sanitaria HCSC; 3 SEHH, Madrid, Spain

Background: The triplication of alpha-globin genes (ααα) caused by homologous recombination between duplicated alpha-globin genes, rarely presents with detectable clinical symptoms since hematometric parameters appear normal. There are two types of α-globin gene triplication, ααα anti3.7 and ααα anti4.2 . ααα anti4.2 is very common among Asians, whereas ααα anti3.7 is more common in African, Middle Eastern, and Mediterranean populations. The number of α genes plays a very important role in the thalassemia phenotype. The co-heritability of a triplication of alpha genes (ααα anti3.7 ) is considered an important factor in the severity of β-thalassemia, exacerbating its phenotypic expression, by causing more imbalance between globin chains, behaving like a β -thalassemia intermedia that in some cases require regular transfusions. Thus, detection of ααα anti3.7 carriers would be of great help in preventing severe cases of thalassemia.

Aims: Triplications are considered rare because the de novo crossover events that generate them occur infrequently. In our laboratory, approximately 85% of the alterations in the alpha globin chain are due to large deletions, the most frequent being -α 3.7 . However, reviewing the results of the alterations in the alpha genes in our laboratory, we have observed that triplications are not as infrequent as previously thought, either alone or associated with β-thalassemia. To this end, we consider hematometric characterization of the heterozygous alpha gene triplication (ααα anti3.7 ).

Methods: Retrospective case/control study from 2013 to 2020. Fifty healthy controls and 48 cases were carriers of the alpha gene triplication (ααα anti3.7 ) in the heterozygous state. Hematological parameters and reticulocyte count were determined on a Coulter LH750 analyzer. Hb A2 and Hb F levels were measured by ion-exchange HPLC (VARIANTTM). Patients with elevated Hb A2 compatible with β-thalassemia or increased Hb F were discarded. Only were selected the subjects did not present abnormal hemoglobins by zonal capillary electrophoresis and ion exchange HPLC. Molecular characterization was carried out by multiplex PCR with the commercial Alpha-Globin StripAssay kit and confirmed by MLPA (SALSA Probemix P140 HBA).

Results: A total of 7625 samples have been analyzed in 8 complete years and a total of 121 triplications have been identified, 73 associated with β-thalassemia, the rest 48 alone. The incidence was 1.58% and taking into account that the incidence of α-thalassemia in Spain is 4.79%, it is one-third of the incidence of α-thalassemia. The hematological data appear collected in the table.

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Summary/Conclusion: The heritability of an alpha gene triplication is in many cases underdiagnosed as it does not present clinical complications. This study shows that the α-globin gene triplication presents Hb levels, MCV, MCH, and MCHC decreased concerning controls, with Hb A2 levels close to 3%. Although there is an overlap of the red blood cell indices at the low limit of normality, a triplication of alpha genes should be suspected, especially when HbA2 levels are at the high limit of normality. Above all, in cases where genetic counseling is requested because the partner has heterozygous β-thalassemia and the transmission of both alterations would aggravate the clinical picture.

PB2238: ΒETA-THALASSEMIA INTERMEDIA: INTERACTION OF ΑLPHA-GLOBIN GENE TRIPLICATION WITH ΒETA-THALASSEMIA HETEROZYGOUS IN SPAIN

P. Ropero 1,2,* , F. A. González 1 , J. M. Nieto 1,2 , W. M. Torres-Jiménez 3 , B. Colas 1 , P. Estival 1 , A. Calo 1 , S. Escribano 1 , S. Rochas 1,2 , A. Villegas 1 , C. Benavente 1 , G. E. de Eritropatología 4

1 Hematology, Hospital Clínico San Carlos; 2 Instituto de Investigación Sanitaria HCSC, Madrid; 3 Hematology, Hospital Virgen de la Luz, Cuenca; 4 Grupo Español de Eritropatología, España, Spain

Background: The spectrum of β-thalassemias is broad, ranging from β-thalassemia major (β-TM), to mild forms known as β-thalassemia minor, and intermediate forms [β-thalassemia intermediate (β-TI)]. Although β-TI lacks specific molecular correlates and diagnosis remains largely clinical, a genotype/phenotype association has been described such that genetic modulators determine the genetic basis of phenotypic diversity. Other genetic modulators are those that are directly involved in the imbalance of the β-globin chains. Homozygosity or double heterozygosity with overexpression of γ-globin chains, either because one or both alleles correspond to a δβ-thalassemia, or because they are associated with molecular alterations in the same or a different one, result in increased γ-globin chain synthesis. Also, the increased production of α-globin chains by a triplication or quadruplication of α genes (ααα/αα or αααα/αα) associated with a heterozygous β-thalassemia is a β-TI.

Aims: To verify with hematimetric data that the diagnosis and clinical grade of β-TI can be established when a triplication of alpha genes (αααanti 3.7) and heterozygous β-thalassemia coexist.

Methods: Retrospective study from January 2010 to December 2019 and involved 73 patients of Caucasian origin with triplication or quadruplication of α-genes and β-thalassemia. Screening the most frequent α-thalassemia mutations as well as gene triplication (αααanti 3.7) was carried out with a commercial Alpha-Globin StripAssay kit and confirmed by MLPA. Molecular diagnosis of β-thalassemia was performed by automatic Sanger sequencing.

Results: The genotypes have been classified into three groups according to the number of α globin genes and the severity of the alteration in the β globin gene. All had a mutation in the HBB gene (β 0 -thalassemia, severe β + -thalassemia, and mild β + -thalassemia). Group I patients who have coherent 6 α genes and groups II and III with 5 α globin genes. In group III, the patients were carriers of mutations affecting the β and δ globin genes [(δβ) 0 -Spanish Thalassemia]. The most significant hematological parameters were hemoglobin levels, MCV, RDW, and the percentage of Hb F (Table).

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Summary/Conclusion: This study has provided the largest series of patients with heterozygous β-thalassemia associated with α-globin gene triplication. These data showed that the effect of the association of the α-globin gene triplication with a heterozygous β-thalassemia mutation is highly variable. In group I, regardless of the distribution of the 6 α-globin genes, homozygous triplication (ααα/ααα) or heterozygous quadruplication (αααα/αα), association with heterozygous β-thalassemia results in anemia of moderate to severe that may or may not require transfusion therapy, it is the severity of the HBB gene mutation that would determine the clinical variation. Group II patients behaved phenotypically as mild thalassemia intermedia. Finally, group III patients behaved as a thalassemia trait since all of them were carriers of mutations that increase the overexpression of γ genes. The phenotype could be established through the most significant hematological parameters 1) hemoglobin levels, 2) MCV, 3) RDW and 4) the percentage of Hb F. But molecular diagnosis and genetic counseling should continue to be used because, although the heritability of a triplication of alpha genes, in many cases underdiagnosed, does not present clinical complications, it can be inherited with another pathology such as heterozygous β-thalassemia and aggravate the clinical picture.

PB2239: DOES SIZE MATTER? THREE NEW DELETIONS IN THE HBB GENE CAUSE Β0-THALASSEMIA

P. Ropero 1,2,* , F. A. González 1 , J. M. Nieto 1 , V. Recasens 3 , Á. Montañés 3 , M. J. Murúzabal 4 , M. Sarasa 5 , C. Fernández 5 , A. Calo 1 , M. Gómez 1 , S. Escribano 1 , H. Martín 1 , S. Rochas 1,2 , A. Villegas 1 , C. Benavente 1 , G. E. de Eritropatología 6

1 Hematology, Hospital Clínico San Carlos; 2 Instituto de Investigación Sanitaria HCSC, Madrid; 3 Hematology, Hospital Miguel Servet, Zaragoza; 4 Hematology, Hospital Universitario Marqués de Valdecilla, Santander (Cantabria); 5 Hematology, Hospital Comarcal de Laredo, Laredo (Cantabria); 6 SEHH, Madrid, Spain

Background: Most β-thalassemias are caused by mutations involving one or a limited number of nucleotides within the gene or its adjacent regions. They can be substitutions or deletions, in these cases, the loss ranges from a single nucleotide to 44 nucleotides, so we wonder if the phenotype is due to the size of the deletion or the location of the mutation.

Aims: Is it the size of the deletion that determines the phenotype of β 0 -thalassemia? To elucidate this, we present three new deletions in the β-globin gene that cause β 0 -thalassemia.

Methods: The hematological parameters were determined with an automated cell counter; The Hb A 2 and Hb F levels were measured by performance liquid chromatography. Hemoglobins were analyzed by capillary zone electrophoresis (Sebia Capillarys Flex system) and ion exchange HPLC (BioRad Variant II β-thalassemia Short Program). Molecular characterization was performed by automatic Sanger sequencing. The screening of common α-thalassemia point mutations and deletions in the world (21 in total) were carried out using multiplex PCR followed by reverse-hybridization with a commercial Alpha-Globin StripAssay kit

Results: Three new mutations have been characterized: 1) 1 bp deletion [CD61 / 62 (–G)] [HBB: c.186_187delG]; 2) 25 bp deletion [HBB: c.93-22_95del] and 3) 105 bp deletion [IVS-2-nt767-CD111] [HBB: c.316-84_333del].

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Summary/Conclusion: These three mutations present a phenotype consistent with β 0 -thalassemia, supported by the hematological parameters that correlate the degree of β globin chain synthesis reduction. Thus, all of them presented anemia with hemoglobin levels between 8.3 g/dL and 13 g/dL, the most severe corresponding to the girl with hepatitis A, with this greater severity probably being related to the hepatitis. Microcytosis evaluated by mean corpuscular volume was more marked in the family carrying the 105 bp (57.8 fL) deletion, while the single base deletion resulted in a MCV of 65.8 fL. About to the mean corpuscular hemoglobin levels to determine hypochromia, the values furthest from the normal are observed to coincide with the most extensive deletion, even though in all patients the MCH was lower than usual for heterozygous β-thalassemia, while Hb A2 levels were higher, close to 6%, although similar in all three types of mutation. Nonetheless, the values do not differ enough between them to think that the size of the deletion determines the phenotype.

These three new mutations show that it is not the size of the deletion that determines the phenotype in β-thalassemia, but rather the location of the deletion, and which process it affects among the stages of gene expression.

Two of the three mutations have been identified in non-Spanish patients (Dominican and Moroccan). This confirms the increase in patients with this type of pathology as well as the use of health services as a reflection of immigration for work reasons along with refugees due to war conflicts, in addition to adoptions of children from areas where these pathologies are frequent.

PB2240: SILENT GLOBIN MUTATIONS. 10 YEARS REFERRALS FOR GENETIC COUNSELING.

S. Theodoridou 1,* , A. Teli 2 , E. Boutou 3 , E.-E. Delaki 3 , E. Yfanti 3 , V. Efthimia 4 , E. Zarkada 1 , A. Balassopoulou 3

1 Thalassemia Prevention Unit, Blood Bank Centre; 2 Thalassemia Unit, Hippokration Hospital, THESSALONIKI; 3 Thalassemia National Centre of Greece, Laiko General Hospital, ATHENS; 4 Thalassemia Unit, Hippokration Hospital, THESSALONIKI, Greece

Background: Haemoglobinopathies, constitute the most frequent monogenic disorders worldwide and in Greece. Silent β and α globin mutations may be missed during screening programs. The identification of combinations of silent defects with severe thalassemia mutations is essential for the genetic counseling of at-risk couples.

Aims: We report the results of the molecular analysis of 241 subjects with borderline hematologic indices or HbA2 levels respectively, without iron deficiency, during a ten year period in order to provide the appropriate genetic counseling for couples at-risk.

Methods: The screening methodology of identifying the carriers is simple and low cost based on determination of red cell indices (RBC, Hb, Ht, MCV, MCH, RDW) and quantification of HbA, F, A 2, S, C, and other haemoglobins with the HPLC (Bio-Rad Variant Haemoglobin Testing System) and Capillary electrophoresis (Minicap). The practice guidelines are similar throughout the prevention network laboratoties of the country. The screening methods used for DNA analysis, all performed in the National Thalassemia Centre, were denaturating gradient gel electrophoresis (DGGE), allele-spesific oligonucleotide (ASO) analysis, high resolution melting analysis and other polymerace chain reaction directed methods and DNA sequencing.

Results: 162 subjects (67,2%) were found carriers of a silent thalassemic mutation. Among the subjects with MCH<27,5 pg, we found 147(89%) with a silent mutation. Most common was the -α 3.7, -101(C>T) (68,7%). Other α deletions were found and some unstable variants as Ηb Icaria, Hb Adana, Hb Agrinio, Hb Constant Spring, triplication of α genes and Termination Cd +6 C-G in the 3’ untranslated region (3’UTR of the β-globin gene (+1480 C→G) and silent Cap+1570(T>C) (HBB:c*96T>C),.

Among 76 subjects with borderline Hb A2 values (3,1-3,9%), 29 (39%) had a molecular defect in the β or α-globin genes. No molecular defects were found in the remaining 47 individuals. Among the positive samples the most common silent mutation was the-101(C>T)(n=15). In our study group the -101 (C>T) mutation shows an elevated Hb A2, Term+ 6 and silent Cap+1570(T>C) (HBB:c*96T>C), have a phenotype closer to normal. The -α3,7 deletion can be suspected when MCH is below 27,5 and triplication of a genes (ααα anti–3.7/αα) is often silent.

Summary/Conclusion: Current application of molecular techniques to the study of borderline heamatologic measurements is important in the field of thalassemia diagnosis and the genetic counseling of couples at risk.

PB2241: CHARACTERISTICS AND OUTCOME OF WARM AUTOIMMUNE HEMOLYTIC ANEMIA (WAIHA) IN ADULTS: A RETROSPECTIVE ANALYSIS OF 110 TUNISIAN PATIENTS

E. Bouslama 1,* , A. Dhib 1 , K. Zahra 1 , H. Regaieg 1 , N. Bensayed 1 , W. bouteraa 1 , M. Zaier 1 , Y. ben youssef 1 , A. khelif 1

1 hematology, farhat hached hospital, SOUSSE, Tunisia

Background: Warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease caused by increased erythrocyte destruction by immunoglobulin G (IgG) autoantibodies, with or without complement activation.

Aims: To study the clinical characteristics, outcomes, and survival of warm-type autoimmune hemolytic anemia (wAIHA) patients (pts).

Methods: This study was a retrospective study from 2001 to 2020. Clinical data of wAIHA pts from a single referral center in Tunisia were reviewed and analyzed.

Results: One hundred and ten pts (59.1% females) were included.

The mean age at the time of wAIHA onset was 54 (range 19 to 98).

All the pts had a positive DAT. Results of DAT showed IgG in 77 pts (70%) and IgG plus C3d in 33 pts (30%).

Primary wAIHA was found in 58 pts (52.7%). Fifty-two (47.3%) of wAIHA cases were secondary: lymphoproliferative diseases (30 patients), connective tissue disease (14 patients: 5 systemic lupus, 3 vitiligo, 2 rheumatoid arthritis, 1 anti-phospholipid syndrome, 1 ankylosing spondylitis, 1 Hashimoto’s thyroiditis, 1 Basedow’s Disease), Evan’s syndrome (5 patients), cancer (3 pts).

Corticosteroids were the first line therapy with an overall initial response rate of 73%.

Thirty-six (32,7%) of the pts were corticosteroid-dependant and 20 pts (18.1%) required at least one second-line treatment including Rituximab (3), splenectomy (5) and Cyclophosphamide (12).

After a mean follow-up of 44 months, 53 pts (48.2%) were in remission and off treatment.

Twenty-seven (24.5%) had died. Infection was the most common cause of death (11 pts). Other causes of death were: 1 ischemic stroke and 5 progression of hematologic malignancies or cancer.

Secondary wAIHA were associated with reduced response to corticosteroids and increased need for second-line therapy.

Summary/Conclusion: This study confirms gender distribution in wAIHA and shows a high proportion of secondary wAIHA. Most patients have responded initially to steroids. The mortality rate was high, with sepsis as a leading cause of death.

PB2242: MANAGEMENT OF AUTOIMMUNE HEMOLYTIC ANEMIA.RETROSPECTIVE STUDY OF A SINGLE CENTER

I. Hortensia-Marioara 1,* , I. Ionita 1 , I. Claudiu 1 , D. Calamar Popovici 1 , M. Ionita 1 , D. Oros 1

1 Spitalul Clinic Municipal de Urgenta, Timișoara, Romania

Background: Autoimmune hemolytic anemia (AIHA) is characterized by the production of autoantibodies to red blood cell surface antigens with destruction of red blood cells by complement and reticuloendothelial system; usually idiophatic, it is also associated with infection, lymphoproliferative disorders, autoimmune diseases, and some drugs.

Aims: To evaluate in this study the clinical, biological and therapeutic aspects of AIHA.

Methods: We presented this retrospective study about 110 cases of AIHA observed in the our hematology clinic, over a period of 10 years (I 2009- I 2020). We present the clinical aspects of AIHA and evaluate the management of AIHA.

Results: There were 43 men and 69 women with a mean age of 47 years [19-82]. Regarding the medical history, 10 patients were with hipertension of whom 3 were receiving Methyldopa, 15 patients were diabetic, 11 had thyroid dysfunction and 20 had a history of autoimmune disease. The clinical aspects of discovery were an anemic syndrome in 83 of patients, mainly due to paleness and asthenia. Physical examination revealed icterus in 85 causes, splenomegaly in 51 causes, hepatomegaly in 17 cases, lymph node in 14 cases and fever in 31 cases. The blood tests reveald that anemia was normocytic in 43 cases and macrocytic in 64 cases. There were biological signs of hemolysis hyperbilirubinemia in 70 patients, high LDH rate in 79 patients. Direct Coombs test was positive for IgG in 77 cases, C 3 in 14 cases, IgG+C 3 in 19 cases. AIHA was idiophatic in 58 cases and secondary to lymphoproliferative desorders in 4 cases, autoimmune disorders in 25 cases,3 cases were secondary to Methyldopa. The therapeutic consisted of transfusion in 61 cases and, the patients underwent a corticosteroid treatment in addition to folic acid theraphy in 74 cases and etiological treatment in the non idiopathic cases.

The patients that were resistant or relapse therapy were treated by splenectomy, immunosupresive drugs (azathioprine, cyclophosphamide) and anti CD20 monoclonala antibody

Immunosuppressive therapy was prescribed in 24 patients, anti-CD20 monoclonal antibody were prescribed in 10 patients, the splenectomy was permormed in 25 patients and intravenous immunoglobulins in 15 patients.

Summary/Conclusion: Glucocorticoids and/or intravenous immunoglobulins are the mainstay of the treatment in the majority of patients with warm AIHA. When these treatments fail, patients often require cytotoxic drugs or splenectomy.

PB2243: TREATMENT OF TWO JORDANIAN SIBLINGS WITH CONGENITAL DYSERYTHROPOEITIC ANEMIA - TYPE 1BUSING INTERFERON-ALPHA-2A

E. Khattab 1,* , M. abu shanap 1 , R. rihani 1 , H. hashem 1 , I. Sultan 1

1 pediatrics, king Hussein cancer center, Amman, Jordan

Background: Congenital dyserythropoietic anemias (CDAs) are a group of rare hereditary congenital anemias characterized by marked ineffective erythropoiesis and characteristic morphologic abnormalities of the erythroblasts. Three major types have been described based on characteristic morphologic abnormalities of the erythroblasts, and the molecular approach for the causative genes. CDA type 1 is inherited as autosomal recessive disorder caused mainly by mutation in CDAN1 gene encoding the codanin-1 protein. More than 100 cases have been described, the severity of anemia varies considerably. Nonhematologic dysmorphologies may be present. Therapy includes redcell transfusion,iron chelation therapy, interferon alpha (INF alpha) and allogenic stem cell transplantation.Congenital dyserythropoietic anemias (CDAs) are a groupof rare hereditary congenital anemias characterized bymarked ineffective erythropoiesis and characteristicmorphologic abnormalities of the erythroblasts. Threemajor types have been described based on characteristicmorphologic abnormalities of the erythroblasts, and themolecular approach for the causative genes. CDA type 1 isinherited as autosomal recessive disorder caused mainlyby mutation in CDAN1 gene encoding the codanin-1protein. More than 100 cases have been described, theseverity of anemia varies considerably. Nonhematologicdysmorphologies may be present. Therapy includes redcelltransfusion, iron chelation therapy, interferon alpha(INF alpha) and allogenic stem cell transplantation.

Aims:

Methods: We are reporting two transfusion dependent siblings diagnosed with CDA type 1, including their treatment with IFN and outcome. We are reporting two transfusion dependent siblingsdiagnosed with CDA type 1, including their treatment withIFN and outcome.

Results: We are reporting 2 boy siblings with CDA type 1 who were referred for bone marrow transplantation at our center at the ages of 11 and 12 years. They have first-degree consanguineous parents and were born with low birth weight (1677 gm and 1500 gram, respectively) and have been transfusion dependent since birth. They were diagnosed using genetic testing and were found to be homozygous at C150RF41, consistent with a diagnosis of CDAN1B. They both had short stature and mild splenomegaly but no dysmorphism. Their ferritin levels at time of referral was 1500 ng/ml and 1220 ng/ml respectively. Bone marrow evaluation showed erythroid precursors are quantitatively increased. The erythroid maturation is megaloblastoid and show binucleation in 10% to 15% of the erythroid cells. They were started on interferon-Alfa-2a as one million unit weekly, with excellent response, no blood transfusion requirement since starting therapy, and iron chelation has been stopped. Due to shortage in the interferon-alpha 2a, a once monthly Pegylated interferon alpha was started for both patients with excellent response without toxicity.

Summary/Conclusion:

We report 2 Jordanian siblings with transfusion dependent CDA-1 caused a homozygous mutation of C150RF41 gene. They both showed dramatic response to IFN-alpha treatment which was maintained for at more than 2 years with no toxicity.

PB2244: THE VALUE OF ANEMIA FOR ORGAN DAMAGE EVALUATION IN SYSTEMIC SCLEROSIS PATIENTS:A MULTI-CENTER RETROSPECTIVE STUDY

Z. Li 1,2,* , D. Xu 1 , T. Li 1 , R. Mu 1

1 Rheumatology and Immunology, Peking University Third Hospital; 2 Rheumatology and Immunology, Peking University People’s Hospital, Beijing, China

Background: Systemic sclerosis (SSc) is a systemic autoimmune disease with multiple organ damage. Scleroderma Clinical Trials Consortium (SCTC) has developed the Damage Index (DI) in SSc to quantify organ damage in SSc. While the relationship between the SCTC-DI and basic clinical and laboratory features remains unclear.

Aims: To describe the SCTC-DI in SSc patients from Peking University Third Hospital SSc cohort (PKUTH-SSc) and Peking University People’s Hospital SSc cohort (PKUPH-SSc). And to develop models to discriminate higher risk of organ damage and predict new-onset organ manifestation within 5 years.

Methods: Patients were recruited from PKUTH-SSc and PKUPH-SSc cohort between January 2001 to July 2021. All clinical and laboratory results were identified at initial visit and at follow-up timepoint. And extracted from electronic medical records. SCTC-DI was calculated as previously reported. LASSO algorithm was performed to select and sort the statistically significant clinical features. Logistic regression and Cox survival analysis were used for developing the evaluation model of SCTC-DI risk and prediction model for new-onset organ damage respectively.

Results: A total of 155 and 278 SSc patients fulfilled selection criteria were enrolled in our study from PKUTH-SSc and PKUPH-SSc cohort respectively. Using LASSO-Logistic regression, we developed a model included underweight, age, duration since first symptom onset, steroids usage at baseline, positive anti-RNP/Sm autoantibody, ESR level and anemia at baseline to assess the high-risk SCTC-DI score. ROC analysis revealed a good discriminatory capability of the SCTC-DI risk group with ROC area under the curve of 0.75 (95% CI 0.70 to 0.79) using this model. Furthermore, in our follow-up study, anemia at baseline was independently related to 5-year organ damage progression (HR=1.67, 95% CI 1.11 to 2.53, p =0.014). As for the typical organ damage profile, patients with anemia at baseline were more likely to develop musculoskeletal and skin (60.6% vs 39.5%, p <0.001), gastrointestinal (62.3% vs 44.6%, p <0.001), cardiovascular (16.6% vs 10.1%, p =0.046) and renal (3.4% vs 0.0%, p =0.004) system damage compared to patients without anemia. While during follow-up, progression of the cardiovascular system damage was more common in anemia patients (14.5% vs 5.6%, p =0.031).

Summary/Conclusion: This study based on the PKUTH-SSc and PKUPH-SSc cohort is the first retrospective study describe the SCTC-DI of SSc patients in external cohorts. Our results highlight the value of anemia at initial visit in discriminating high SCTC-DI score and predicting 5-year organ damage progression, demonstrating the regular and careful monitoring of these patients may help to improve the prognosis of organ damage in SSc.

PB2245: CLINICAL CHARACTERISTICS AND RISK FACTORS OF ANEMIA IN PATIENTS WITH SYSTEMIC SCLEROSIS FROM THE PKUPH-SSC COHORT

Z. Li 1,2,* , D. Xu 1 , T. Li 1 , R. Mu 1

1 Rheumatology and Immunology, Peking University Third Hospital; 2 Rheumatology and Immunology, Peking University People’s Hospital, Beijing, China

Background: Systemic sclerosis (SSc) is a heterogeneous disease with diverse clinical phenotypes. Anemia has been implicated as an independent prognosis factor for mortality and organ complications in systemic sclerosis (SSc).

Aims: To determine the characteristics of SSc patients with anemia in a population of patients from Peking University People’s Hospital SSc cohort (PKUPH-SSc), and associated risk factors.

Methods: Patients were recruited from PKUPH-SSc between January, 1999 and January, 2020. The diagnosis of anemia was based on the reduced hemoglobin (Hb) concentrations (<120g/L for female and <130g/L for male) during follow-up. The demographics, clinical characteristics, laboratory parameters, autoantibodies and medication were collected. Logistic regression analysis and Cox regression analysis was applied to identify the risk factors independently associated with anemia in SSc.

Results: A total of 302[Office1] patients were enrolled in PKUPH-SSc cohort, 150 (49.8%) patients developed anemia, containing more female (n=136, 90.7%) [Office2] patients. Normocytic-mild anemia[Office3] (n=115, 76.7%) was the most common type of anemia in SSc. According to the age-adjusted logistic regression analysis, gender of female (OR=4.77, 95%CI 2.09-10.92, p<0.001), SSc with overlap syndrome (OR=3.42, 95%CI 1.55-7.53, p=0.002), severe skin thickness (OR=3.04, 95%CI 1.44-6.41, p=0.004), ESR elevation (OR=2.22, 95%CI 1.20-4.11, p=0.011), CRP elevation (OR=2.20, 95%CI 1.03-4.69, p=0.042) and hypocomplementemia (OR=3.58, 95%CI 1.90-6.75, p<0.001) were independent risk factor determining the SSc patients with anemia[Office4]. In addition, as shown in figure, age at onset ≥ 49 years (HR=2.54, 95%CI 1.51-4.27, p<0.001) and SRC (HR=3.27, 95%CI 1.37-7.79, p=0.008) were proved to be the independent predictors to determine the 20-year incidence of anemia in SSc, while positive anti-centromere proteins (HR=0.42, 95%CI 0.24-0.76, p=0.003) seemed to be a protective factor. In terms of anemia subgroups, positive anti-Ro52 was a potential risk factor for microcytic anemia, but a protective factor for normocytic anemia.

Summary/Conclusion: Anemia is associated with distinct clinical behaviors and disease progression in SSc. Age at disease onset ≥ 49 years, negative anti-centromere proteins antibody and SRC were independent predictors for 20-year incidence of anemia in SSc patients. Monitoring anemia in these patients was a potential management to evaluate the disease progression of SSc.

PB2246: FEATURES OF THE SECRETION OF HEPCIDIN, A SOLUBLE TRANSFERRIN RECEPTOR DEPENDING ON THE TYPE OF ANEMIA IN PATIENTS WITH MALIGNANT NEOPLASMS

V. Sakhin 1,* , E. Kryukov 2 , S. Kazakov 3 , A. Sotnikov 4 , A. Gordienko 4 , N. Gulyaev 5 , O. Rukavitsyn 6

1 Intensive care unit, “3 Central Military Clinical Hospital named after A.A. Vishnevsky” of the Ministry of Defense of the Russian Federation, Krasnogorsk; 2 Russian Military Medical Academy of the Ministry of Defense of Russia, St. Petersburg; 3 center for clinical laboratory diagnostics, Main Military Hospital n.a. N. Burdenko, Moscow; 4 Department of Hospital Therapy, Russian Military Medical Academy of the Ministry of Defense of Russia, St. Petersburg; 5 Cardiology Center, “3 Central Military Clinical Hospital named after A.A. Vishnevsky” of the Ministry of Defense of the Russian Federation, Krasnogorsk; 6 Hematology Center, Main Military Hospital n.a. N. Burdenko, Moscow, Russia

Background: Anaemia of chronic disease or anaemia of chronic inflammation is the most common cause of anaemia in admitted patients. This anemia can be a consequence of several states such as malignancies, chronic infections and auto-immune diseases, thereby indicating the multiplicity in pathogenetic pathways.

Aims: to study the secretion of hepcidin a soluble transferrin receptor (sTfR) in patients with malignant neoplasms with anemia of chronic diseases (ACD), iron deficiency anemia (IDA) and an ACD/IDA combination and to investigate their effect on the development of anemia

Methods: 106 patients with stage II-IV of solid malignant neoplasms were examined: 84 with anemia (55 men, 29 women, 67.1±9.9 year olds), 22 without anemia (17 men, 5 women, 60.2±14.9 year olds). According to Van Santen and Worwood criteria , by determining the transferrin saturation index (TSI), ferritin, C-reactive protein (CRP), patients were divided into 4 groups: group 1 - ACD, 31 (20/11 patients (hemoglobin 99.2[IQR,87-118], erythrocytes 3.5[IQR,3.1-3.9], TSI 19.9[IQR,12.6-22], ferritin 443.4[IQR,361.9-574.9], CRP 137.1[IQR,89.7-155.2]), group 2 – ACD/IDA, 28 (18/10) patients (hemoglobin 111[IQR,91-128], erythrocytes 4.02[IQR,3.3-4.5], TSI 12.7[IQR,7.4-20.5], ferritin 220.5[IQR,102.7-370.6], CRP 53.4[IQR,14.5-64.5]), group 3 - IDA, 25(17/8) patients (hemoglobin 107 [IQR,96.5-121.5], erythrocytes 4.1[IQR,3.7-4.5], TSI 7.3[IQR,4.2-12.5], ferritin 19.3[IQR,10-24.1], CRP 14[IQR,6.6-23.9]), group 4(control) - 22 patients without anemia (hemoglobin 135.3[IQR,125-150], erythrocytes 4.4[IQR,3.9-4.9], TSI 16.8[IQR,12.4-21.6], ferritin 111[IQR,25.7-189.2], CRP 11.4[IQR,3.3-16.8]). The significance of differences between several unrelated groups was determined using the Kruskal-Wallis test at a significance level (p) of less than 0.05. To assess the relationship between the variables, the Spearman correlation coefficient (r) was calculated.

Results: the ACD group had the highest concentrations of CRP, ferritin in comparison with the other groups (p<0.05). The TSI in the ACD group was higher compared to the IDA and ACD/IDA groups (p<0.05), and did not differ from the control group (p>0.05). The ACD group had the maximum concentration of hepcidin (47.3[IQR,29.7-60.5]) compared with the ACD/IDA group (34.6[IQR, 26.6-50]) (p<0.05), the IDA group (6.16[IQR,1.8-10]) (p<0.05) and the control group (23.5[IQR,4.7-45.4) (p<0.05). The concentration of sTfR in patients with anemia did not differ (p>0.05) and amounted to 26.1[IQR,19.8-28.3] in ACD, 23[IQR,13.3-21.1] in ACD/IDA, 28.4[IQR,16.1-34.4] in IDA, 16.8[IQR,13.7-18.8] in the control group. In all groups with anemia, the concentration of sTfR was higher compared to the control group (p<0.05). A correlation was found between the number of erythrocytes and the concentration of hepcidin (r=-0.57), as well as between the concentration of hemoglobin and sTfR (r=-0.57).

Summary/Conclusion: Patients with solid malignancies may have IDA, ACD, or a combination of both. The high concentration of hepcidin in patients with ACD, the presence of a correlation between it and erythrocytes confirms its importance in the development of anemia and the possibility of using it for the diagnosis of ACD. The absence of differences in the concentration of sTfR between patients of the three groups with anemia indicates a low value of this indicator for the differential diagnosis of ACD and IDA. The correlation between sTfR and hemoglobin reflects the importance of sTfR in the pathogenesis of anemia in cancer patients.

PB2247: PHARMACOKINETIC MODELING AND SIMULATION TO SUPPORT MITAPIVAT DOSE SELECTION USED IN PEDIATRIC PHASE III STUDIES

O. Yin 1,* , K. Baron 2 , J. Mondick 3 , M. Little 1 , P. D. Tyler 1 , V. Beynon 1

1 Agios Pharmaceuticals, Inc., Cambridge, MA; 2 Metrum Research Group, Tariffville, CT; 3 Metrum Research Group, Tariffville, United States of America

Background: Mitapivat (AG-348) is a first-in-class, oral, small molecule, allosteric activator of the red blood cell pyruvate kinase (PK) enzyme (PKR). The positive benefit-risk profile of mitapivat has been demonstrated in two recently completed phase 3 studies in adults with PK deficiency who are not regularly transfused (ACTIVATE, NCT03548220 ) or regularly transfused (ACTIVATE-T, NCT03559699 ). Currently, two phase 3 studies are being initiated to evaluate the efficacy and safety of mitapivat in pediatric subjects aged 1 to <18 years (yrs) with PK deficiency who are not regularly transfused (ACTIVATE-Kids, NCT05144256 ) or regularly transfused (ACTIVATE-KidsT, NCT05175105 ).

Aims: Conduct a pharmacometrics analysis to support the mitapivat dose selection in the phase 3 studies in pediatric subjects.

Methods: A three compartment population pharmacokinetic model with allometrically scaled parameters (clearances and volume of distribution) was applied. Dose simulations were conducted for various age subsets and body weight ranges using data from the National Health and Nutrition Examination Survey (NHANES) database. For subjects aged 1 to <2 yrs, in addition to allometric scaling, maturation factors (Salem F et al. Clin Pharmacokinet . 2014;53:625–36. Salem F et al. Clin Pharmacokinet . 2015;54:671) accounting for changes in cytochrome P450 (CYP) 3A activity were implemented. This approach was employed because mitapivat is metabolized primarily by CYP3A, and maturational changes in CYP3A ontogeny are known to occur during the first few yrs of life. The selected pediatric dose for each age subset and weight range was based on a dose that was predicted to achieve mitapivat exposure (ie, the area under the plasma concentration-time curve [AUC] during a dosing interval at steady state) similar to that achieved in adults receiving the recommended clinical dose for PK deficiency (dose titrations occurring every 4 weeks, from 5 mg twice daily [BID] to 20 mg BID, and then to the maximum recommended dose of 50 mg BID).

Results: The proposed dosing scheme is shown in the Table. For subjects aged 12 to <18 yrs who weigh ≥40 kg, the recommended dose used in adults would provide a similar mitapivat AUC to that achieved in adults; the predicted difference is <20%, and therefore is not considered to be clinically meaningful. For subjects aged 12 to <18 yrs who weigh <40 kg, or subjects aged 2 to <12 yrs, dosing according to body weight (≥40 kg, ≥20 to <40 kg, and <20 kg) would yield similar mitapivat AUC ranges between pediatric and adult subjects. For subjects aged 1 to <2 yrs, the proposed doses shown in the Table are predicted to provide <5% difference in mitapivat AUC as compared with adults.

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Summary/Conclusion: Population pharmacokinetic modeling and simulation was utilized to identify candidate doses for evaluation in mitapivat pediatric phase 3 studies. The selected doses for each pediatric age subset and body weight range are projected to provide mitapivat AUC similar to that achieved in adults receiving the recommended clinical dose.

PB2248: CONCOMITANT HEREDITARY SPHEROCYTOSIS AND PYRUVATE KINASE DEFICIENCY IN A SPANISH FAMILY WITH CHRONIC HEMOLYTIC ANEMIA. CONTRIBUTION OF LASER EKTACYTOMETRY TO CLINICAL DIAGNOSIS

J.-L. Vives Corrons 1,* , E. Krishnevskaya 1 , L. Montllor 1 , V. Leguizamón 1 , M. Garcia Bernal 2

1 Red Blood Cell and Hematopoietic Disorders, Josep Carreras Leukaemia Research Institute (IJC), Badalona; 2 Pediatric Hematology Department, University Hospital Mútua Terrassa Terrassa, Barcelon, Spain

Background: Hereditary Spherocytosis (HS) and Pyruvate Kinase deficiency (PKD) are the most common causes of hereditary chronic hemolytic anemia. Here we describe clinical and genetic characteristics of a Spanish family with concomitant b-spectrin (SPTB) c.647G>A variant and pyruvate kinase (PKLR) c.1706G>A variant

Aims: The objective of our study is to evaluate the possible modulator effect of Pyruvate Kinase deficiency (PKD) on the clinical expression of Hereditary Spheroctosis (HS) in a Spansh family with coinheritance of HS and PKD

Methods: Hematological investigation, hemolysis tests and specific red cells studies were performed in a family of 11 members according to conventional procedures.The ektacytometric study was performed using the osmoscan module of the Lorca ektacytometer (MaxSis. RR Mechatronics). The presence of the SPTB and PKLR variants was confirmed by t-NGS

Results: The t-NGS genetic characterization of the 11 family members showed the presence of a heterozygous mutation for the b-spectrin (SPTB; c.647G>A) in 7 members with HS, 3 of them co-inherited the PKLR variant c.1706G>A. In the remaining 4 members, no gene mutation was found. (FIGURE)

Ektacytometry allowed a clear diagnostic orientation of HS, independently from the PKLR variant

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Summary/Conclusion: This family study allows to conclude that the SPTB mutation, (c.647G>A) previously described as Likely Pathogenic (LP), has to be classified as Pathogenic (P) according to the HGVS variation nomenclature. In addition, after the 6 years of clinical follow-up of the patients with HS it can infer that the chronic hemolytic anemia may be attributable to the SPTB mutation only, without influence of the concomitant PKLR. Moreover, only the family members with the SPTB mutation exhibited an ektacytometric profile characteristic of HS

PB2249: SUCROSOMIAL IRON SUPPORT IN PATIENT WITH IRON DEFICIENCY ANEMIA IS HIGLY EFFECTIVE IN IMPROVING ANEMIA REDUCING ERYPTOSIS AND M1 MACROPHAGES POLARIZATION

G. giordano 1,* , M. napolitano 2 , A. lucchesi 3

1 MEDICINE - HEMATOLOGY SERVICE, REGIONAL HOSPITAL “ANTONIO CARDARELLI” CAMPOBASSO, CAMPOBASSO; 2 hematology, university of palermo, palermo; 3 hematology, irst emilia romagna, meldola, Italy

Background: Sucrosomial iron is a macroassembly in wich a shield of polisaccarides protects a liposome that contains iron pyrophosphate.

Sucrosomial iron allows a better iron absorbtion than iron sulphate, bypassing DMT1 and ferroportin.

Iron support works improving effective erythopoiesis. It is due also to a reduction of eryptosis. A marker of eryposis is the expression of phosphatidyl serine(PS) on the external layer of erythrocyte membrane.

Pro-inflammatory macrophages, increasing inflammations, impairs iron absorbtion and bioavailability.

Aims: Aim of this study is to assess if sucrosomial iron support is effective in improving anemia and in reducing eryptosis by PS reduction on external erythrocyte membrane and M1 macrophages polarization.

Methods: This study is a multicentric randomized1:1:1 study with informed consent. In group A100patients received sucrosomial iron containing phosphatidyl coline,in group B100patients received sucrosomial iron containing phophatidyl serine,in group C100patients received iron sulphate.In each group patients received 60mg of elemetal iron.

Inclusion critheria were age>18years,no other causes of anemia,Hb<10g/dl, ferritin<30ng/ml,TSAT<20%, 1FOBT+/3 with EGDS and PCS normal,no neoplastic hypermenorrhea and/or menometrorrhagia,gastric bleeding(ulcer, gastritis, angiodysplasia),small intestine bleeding(angiodysplasia),Colon bleeding (diverticulitis, angiodysplasia, hemorroides).

Exclusion critheria were:Loss of 1g Hb/dl in a time<7days,hyperthyroidism, Severe CHF,Autoimmune disease,IBD,ischemic/hemorragic enterocolitis,neoplasia with or without treatment,elmintic infestation, pregnancy/breastfeeding,severe organ failure,intolerance to iron sulphate.

To measure PS expression we used a two-step flow cytometry procedure to quantitatively measure not only the externalPS but also the intracellular and shedPS. In this method,PS is first bound to fluorescent annexinV,and then the residual nonbound annexin is quantified by binding toPS exposed on apoptotic cells.

M1 macrophages were counted by two tube cytoflurimetry(iNOS high /CD163 low/CD14+).

Patients carachteristics were superimposable except for patients receiving sucrosomial iron in which start Hb level was lower (p<0.05). Hb median level in group Awas 8 g/dl,7.8 inB and 8.5 inC.

Follow-up was6 months.

Results: Median Hb concentration(g/dl) was at 4wks 9.5inA,8.6inB,8.8inC,at8wks 10.9inA,10.7inB,10inC,at 12wks 12.3inA,12.5inB,10.5inC.

Median ferritin level(ng/ml) was at 0mo inA3,inB10,inC7;at3mo inA20,inB10,inC3;at6mo inA150,inB300,inC70.

Expression of median %RBC with PS on external membrane was at 1wks 2.5inA,2.7inB,2.4inC,4wks 2inA,2.2inB,2.1inC,at8wks 0.8inA,1.1inB,1.5inC,at 12wks 0.7inA,0.8inB,1.6inC.

Expression of median %M1 was at 1wks 1.5inA,1.2inB,1.3inC,4wks 1.1inA,1.3inB,1.2inC,at8wks 0.9inA,1inB,1inC,at 12wks 0.5inA,0.8inB,1.1inC.

Summary/Conclusion: Sucrosomial iron formulation is more effective than iron sulphate in increasing Hb level in sideropenic patients increasing effective erythropoiesis and reducing eryptosis, by reducing expression of PS on the external membrane of RBC and reducing proinflammatory M1 macrophages.

PB2250: GENETIC REGULATION IN IRON OMEOSTASIS IN SIDEROPENIC COVID19 DISEASE AFFECTED PATIENTS WITH NEW IRON ORAL FORMULATION: LESSONS FROM A DIFFERENT PERSPECTIVE

G. giordano 1,* , L. alessandro 2 , M. napolitano 3

1 MEDICINE - HEMATOLOGY SERVICE, REGIONAL HOSPITAL “ANTONIO CARDARELLI” CAMPOBASSO, CAMPOBASSO; 2 hematology, irst-emilia romagna, meldola; 3 hematology, universita degli studi di palermo, palermo, Italy

Background: Coronaviruses are able to upregulate gene chop/gadd153. This gene induces CD8 lymphocyte apoptosis and stimulates hepcidin production. Covid19 is also able to induce Asp-1 gene expression that degrades asparagine blocking NO production, impairing immune response and antithrombotic activity.

We know that iron deficiency is linked to an increase incidence of infection and impaired immune response, and anemia impact negatively on covid 19 survival.

Aims: To show how covid19 interferes in iron metabolism of sideropenic patients upregulating expression of genes as chop/gadd153 and arginase-1 and how sucrosomial oral iron support might change that.

Methods: We enrolled 60 patients with sideropenic anemia due to gastrointestinal bleeding, of whom 40 affected by documented COVID19 infection only with mild form with fever, diarrhea, bone or muscle pain or flu-like syndrome.

This study is a multicenter, retrospective study. The time of enrollment was 20 months. 20 Patients received 60 mg/die of elemental iron of Sucrosomial® iron (20 patients – group A). 20 patients received no iron support (group B). We enrolled also 20 patients without COVID19 infection, but with gastrointestinal bleeding, as control group (group C).

Charachteristics of patients were superimposable in the three groups.

Whole blood was taken from the 60 COVID-19 patients and 20 healthy individuals as control group, and after RNA extraction and complementary DNA (cDNA) synthesis of Arg1, hepcidin and chop/gadd153 gene, expression of genes was measured by real-time PCR and was referred to gene expression of control group.

The circulating M1 proinflammatory macrophages (iNOS high /CD163 low/CD14+) and M2 antiinflammatory (iNOSlow/CD163high /CD14-) were analyzed by cytofluorimetry.

Data were collected at 0 and 3 months.

Results: At 0mo Chop gene expression was 2inA,4inB,1inC; Arg-1gene expression was 2inA,3inB,1inC, M1 expression was 0.4%inA,1%inB,0.4%inC,M2 expression was 0.6%inA,0.2%inB,0.5%inC.

At 3mo Chop gene expression was 1inA,1.5inB,1inC; Arg-1gene expression was 1inA,1inB,1inC, M1 expression was 0.4%inA,0.8%inB,0.4%inC,M2 expression was 0.5%inA,0.4%inB,0.5%inC.

Summary/Conclusion: Patients with gastrointestinal bleeding with mild covid 19 disease show respect to patients with gastrointestinal bleeding and without covid19 disease an increased activation of chop gene and asp-1 gene, that leads to lymphopenia, immunodepression, functional iron deficiency, henanced m1 macrophage polarization with an high inflammation, an higher transfusion need, an high number of hospitalized patients with a lenght of hospital stay higher. And these facts are evident mainly in the first phase of the mild disease.

When patients are supported with oral sucrosomial iron, the expression of chop and asp-1 gene and hepcidin gene are lower than we can see in sideropenic patients not supported. The level of inflammation, M1 macrophages proinflammatory polarization and functional iron deficiency is lower in patients supported, with no transfusion need, a slight level of anemia, no patients hospitalized. Also in this case differences are evident mainly in the early phase of disease.

Probably new oral iron formulation, as sucrosomial iron, are important to influence expression of genes as chop and asp-1, mainly in the early phase of covid19 disease, stopping functional iron deficiency and allowing a restart of a normal immune system functioning.

PB2251: TWO POLYCYTHEMIA VERA PATIENTS WITH A DIAGNOSIS OF CONCOMITANT HEREDITARY HEMOCHROMATOSIS

H. D. Kiper Unal 1,* , T. Cetintepe 1 , S. Solmaz 1 , A. Subasioglu 1 , K. Payzin 1

1 Katip Celebi University Medicine School Ataturk Training Hospital, IZMIR, Turkey

Background: Hereditary Hemochromatosis (HH) is an inherited iron metabolism disorder characterized by excessive iron accumulation in the liver, heart, pancreas and other endocrine organs. Although it is often associated with mutations in the HFE gene, it can also be caused by mutations associated with HJV, TFR-2 and ferroportin. JAK2 V617F (+) Polycythemia Vera (PV) is the most common cause of acquired primary erythrocytosis and has been reported very rarely in patients with HH.

Aims: We presented two polycythemia vera patients with a diagnosis of concomitant hereditary hemochromatosis.

Methods: Presence of HFE gene mutation and jak-2 V617F mutation was determined by PCR test

Results: Case 1:

N.G, a 67-year-old male patient with a history of hypertension, insulin-dependent diabetes mellitus and cerebrovascular accident was diagnosed with PV in another center in 2007. The patient was JAK2 V617F (+) and the tests performed in our laboratory after phlebotomies were EPO: 7.8 mIU/ml, ferritin: 34 ng/dl, transferrin saturation: 49%. Bone marrow aspiration biopsy revealed hypercellularity with erythroid and megakaryocytic hyperplasia. Although Hb/Hct levels were within the targeted range with hydroxyurea 500 mg 2x1 treatment, he was followed up with frequent phlebotomies due to persistent pruritus. After phlebotomy, there was a significant improvement in his pruritus. He was using clopidogrel as antiaggregant treatment. During follow-up, there was deterioration in blood glucose regulation and an increase in insulin requirement. In June 2018, he developed widespread hyperpigmentation on his skin. Due to unregulated diabetes, hyperpigmentation, and high ferritin levels despite frequent phlebotomy, he was examined for HH. He was found to be heterozygous positive for the HFE gene H63D mutation. The patient is still being followed up with HU 2X1 and phlebotomy without any problem.

Case 2:

E.H, a 57-year-old male patient with a history of hypertension and type 2 diabetes mellitus was diagnosed with PV in 2015. He was JAK2 V617F(+) and at diagnosis EPO:3.7 mIU/ml, ferritin:1403 ng/ml, transferrin saturation:63%. Bone marrow aspiration biopsy revealed hypercellularity and erythroid hyperplasia. Since he was a low-risk patient under the age of 60 and had no history of thrombosis, he was followed up with ASA 100 mg/day and intermittent phlebotomies. Similar to the first case, persistent pruritus was present despite the normal Hb/Hct level. Since hyperferritinemia was not expected in polycythemia vera and the patient was diabetic, he was examined for HH. In the HFE gene analysis, the H63D mutation was found to be heterozygous. Due to the frequent need for phlebotomy, the patient is considered to start treatment with hydroxyurea, and the follow-up still continues.

Summary/Conclusion: The association of HH and PV has been rarely reported in the literature, and its clinical significance is not clearly known. In the presence of unexplained high ferritin levels in PV, even if the clinical findings are unclear, screening for HH may be recommended as an appropriate approach. The role of HFE genotypes in the development of myeloproliferative diseases is unknown. Clinical data accumulation is needed to elucidate iron metabolism and prognostic features in PV patients with HFE mutations.

Keywords: polycythemia vera, hereditary hemochromatosis, JAK2 V617F, HFE mutation

PB2252: TREATMENT OF ANEMIA IN PATIENTS WITH GASTROINTESTINAL AND CHRONIC LIVER DISORDERS BASED ON PREDOMINANT PATHOGENETIC FACTOR.

O. Rybina 1,* , V. Sakhin 2 , A. Gubkin 1 , E. Kryukov 3 , O. Rukavitsyn 4

1 hematology, Central Clinical Hospital of Russian Railways, Moscow; 2 hematology, Third Central Military Clinical Hospital named after A.A. Vishnevsky of the Ministry of Defense of the Russian Federation, Krasnogorsk; 3 therapy, 3- Military medical academy of S.M. Kirov, Saint-Petersburg; 4 hematology, 4-FSBI «Main Military Clinical Hospital named after N.N. Burdenko of the Ministry of Defence of Russia», Moscow, Russia

Background: Anemia is one of the most common complications of pathological gastrointestinal and chronic liver conditions. This blood disorder can have a negative effect on the underlying disease and reduce patients’ quality of life. The etiology of anemia is often multifactorial. In most cases iron deficiency anemia (IDA), anemia of chronic diseases (ACD) and its combination are diagnosed. There are several guidelines for the diagnosis and management of anemia, however, they still show insufficient effectiveness in some patients.

Aims: Тo evaluate the feasibility and effectiveness of different types of anemia treatment for the persons with gastrointestinal and chronic liver disorders; to create approaches to personalized therapy for these patients.

Methods: We performed retrospective single-center study lasting from 2015 to 2022, including patients with diagnosed gastrointestinal and chronic liver disorders complicated by IDA or ACD. Patients were divided into two groups: IDA group and ACD group. In both groups we evaluated the effectiveness of different types of anemia treatment (treating the underlying disease only, oral iron, intravenous iron, erythropoiesis-stimulating agents (ESA), B vitamins), by comparing hemoglobin and erythrocyte values, red blood cell (RBC) indices before and after treatment.

Results: Overall 112 patients (45 women and 67 men) with diagnosed gastrointestinal and chronic liver disorders complicated by IDA or ACD were included in the study. The median age was 58 years (r: 21-90). 65 patients (58%) had a gastrointestinal and chronic liver disorders complicated by IDA, 47 patients (42%) had a gastrointestinal and chronic liver disorders complicated by ACD. Median follow-up was 10,2 (6-45) days from the start of treatment to the control of blood counts.

35 patients (31%) had a statistically significant (P=0.01) increases in RBC, RBC indices, hemoglobin (Hb) levels after intravenous iron supplementation. 25 patients (22%) with IDA were treated with oral iron. Clinical evidence of the effectiveness of this therapy was not obtained (P>0.05). Treatment with ESA as well as the treatment of the digestive system disorders only, was not carried out in this group.

10 patients (9%) with ACD who received ESA demonstrated a statistically significant improvement in Hb and RBC levels (p=0,01).

Therapy focused only on treating the underlying disease in 21 patients (19%) did not lead to an increase in Hb (p> 0.05), although patients subjectively noted an improvement in well-being.

Iron replacement therapy that’s prescribed for 12 patients (11%) with ACD did not have a positive effect on Hb, RBC, RBC indices. Moreover 3 patients noted the appearance of symptoms of dyspepsia after treatment. 9 patients (8%) from both groups were prescribed B vitamin supplements which had no effect on the red hematopoietic germ (p>0.05). Most likely, this is due to the fact that the “point of application” for this treatment does not coincide with the pathogenesis of anemia in these patients.

Summary/Conclusion: Improvement in Hb and RBC levels was demonstrated in patients with IDA, who received intravenous iron, as well as in patients with ACD after ESA treatment. Other patients did not have positive effect after treatment. Therapy focused on blocking the main mechanism of anemia, will increase hemoglobin level in a shorter period, as well as avoid multiple side effects from therapy, which, in general, will improve patient’s quality of life.

PB2253: NEUTROPHIL TO LYMPHOCYTE RATIO (NLR) AS A PREDICTOR OF SEVERE COVID-19 ON ADMISSION

M. Ababou 1,* , M. Meziane 2 , C. Chouikh 2 , M. Ababou 2 , J. Laoutid 3 , K. Doghmi 1

1 Clinical Hematology; 2 Anesthesia and Intensive Care Unit, Military hospital Mohammed v, Rabat, Morocco, Rabat; 3 Anesthesia and Intensive Care Unit, Military Hospital Moulay Ismail, Meknes, Morocco

Background: In December 2019, Wuhan City became the center of a pneumonia epidemic caused by a virus of the Coronaviridae family, SARS-CoV-2, which was later designated by the WHO as COVID-19 (Corona Virus Disease-2019). The clinical spectrum of COVID-19 appears to be very broad, ranging from asymptomatic infection to severe viral pneumonia with respiratory failure and even death. Today, there are more than 4.5 million deaths due to this infection with a worldwide case fatality rate of 6.9%.

Aims: The aims of our work is to identify the predictive factors of a severe evolution of the disease at the admission of the patients

Methods: This is a monocentric cross-sectional procepective cohort study conducted at the polyclinic hospital of the National Social Security Fund of Tangier, Morocco, between July and September 2020. We included all patients hospitalized for management of symptomatic COVID-19 infection with a positive PCR test on oropharyngeal or nasopharyngeal swab. Their epidemiological, clinical, biological and radiological characteristics at admission as well as the therapeutic modality and clinical evolution were collected. The main event of interest was the occurrence of death. The descriptive and analytical analysis was done using the JAMOVI software (version 1.6). We used the t-student, Mann-Whitney, χ2 (or Fisher exact test) to compare differences between survivors and decedents. To explore factors predictive of death, we used univariate and multivariate logistic regression models

Results: Our study included 113 patients with a median age of 58 years [46-68], a sex ratio=1.69, 27.4% of patients were diabetic. The main symptoms at admission were fever (63.1%), dyspnea (60.2%) and cough (53.1%). The mortality rate was 12.4%, i.e. 14 patients with a median age of 68 years [57-70.8]. Our study found a significant difference between the patients who died and those who survived in terms of age (p=0.024), obesity (p=0.006), presence of cough (p=0.011) and dyspnea (p=0.001), degree of temperature (p=0.026), heart rate (<0.001) and arterial oxygen saturation level (p<0.001) at admission, white blood cell count (p=0.012), neutrophils (<0.001), lymphocytes(p=0.028), eosinophils(p=0.026), neutrophil to lymphocyte ratio NLR (p<0.001), D-dimer(p=0.011), CRP(p=0.001), procalcitonin (p=0.001), LDH (p<0.001), degree of lung parenchymal involvement (p<0.001), use of corticosteroids (p<0.001), antibiotics (p=0.027) and use of preventive or curative thromboprophylaxis (p=0.003). Given the low number of deaths, we chose to include only the biological parameters at admission in univariate and multivariate analysis. The main biological parameters predictive of a severe form of COVID-19 at admission revealed in our univariate regression analysis were the level of D-dimer (OR: 1; 95% [1-1.001]; p=0.033), the level of CRP (OR: 1.009; 95% [1. 004-1,016]; p=0.001), LDH level (OR:1.004; 95%[1.001-1.019]; p<0.001) and NLR ratio (OR: 1.18; 95%[1.07-1.3]; p<0.001), including these four parameters in multivariate analysis, we find that LDH level (OR: 1.007;95%[1.002-1.011];p=0.005), and NLR ratio (OR:1.47;95%[1.16-1.87];p=0.001) at admission are predictive of mortality.

Summary/Conclusion: On admission of a patient with COVID19, calculation of the neutrophil to lymphocyte ratio (NLR) is important, a high NLR could be a predictor of a severe disease course to which clinicians should be alerted for better management of the patient.

PB2254: ASSOCIATION BETWEEN TNF-ALPHA LEVELS AND TNF-ALPHA 238 ALLELES POLYMORPHISMS AND SEVERITY OF ANEMIA AMONG SUDANESE CHILDREN WITH FALCIPARUM MALARIA

K. A. Mohamedahmed 1,*

1 Hematology and Immunology, University of Gezira, Wad Medani, Sudan

Background: Falciparum malaria is the most leading cause of mortality and morbidity in the world especially in Africa among infants and young children due to severe malaria and malaria anemia. TNF-α is plays a central role in malaria pathogenicity either in the cure or complication of malaria. TNF-α levels overproduction and promoter polymorphisms at TNF-α 238 alleles may play a central role in reduced red cell production and malaria-related anemia through suppression of bone marrow erythropoiesis and dyserythropoiesis.

Aims: The purpose of this study was to evaluate the TNF-α 238 alleles polymorphism and its association with TNF-α levels in the severe malaria anemia among Sudanese children in Gezira State.

Methods: A cross-sectional hospital-based study was included 200 Sudanese children (mean age 8.43 ± 3.80 years; 53% boys; 47% girls). 100 children with severe falciparum malaria (mean age 8.63 ± 3.40 years; 61% boys; 39% girls) and 100 children with uncomplicated falciparum malaria (mean age 8.83 ± 4.20 years; 45% boys; 55% girls). RBCs parameters were determined using the Sysmex XP 300 N automated hematology analyzer. ELISA was further processed for TNF-α level using Human TNF-α ELISA MAX™ Deluxe Sets. DNA extraction was done using G-DEX™IIb Genomic DNA Extraction Kit. PCR and gel running system were used for detecting TNF-α 238 alleles polymorphism (A allele/ G allele). Obtained data were analyzed by SPSS (V 20.0) and Stat disk (V 13.0).

Results: TNF-α 238A allele was a common allele (66.8%) among Sudanese children with falciparum malaria, and the G allele was rare (33.2%). While TNF-α 238 GA, AA, and GG account for (54.5, 39.5, and 6% respectively). Falciparum malaria-related anemia accounted for 32%, commonly in SM (55%) compared to UM (9%) giving highly statistically significant differences in TNF-α levels between falciparum malaria-related anemia and falciparum malaria without anemia (P value 0.000). Otherwise, The average of TNF-α levels in mild, moderate, and severe anemia were (190.75 ± 102.55, 189.70 ± 80.35, and 299.75 ± 82.27 pg/ml respectively) giving highly significant differences and strongly significant positive correlated with the severity of anemia (r + 0.309; P value 0.000). TNF-α 238 GA, AA, and GG account for (58, 36, and 6% respectively) in UM; while (51, 43, and 6% respectively) in SM. The TNF-α 238 A allele accounts for 83.6% of malaria anemia (P value 0.000) and 100% severe anemia (P value 0.000); children who have TNF-α 238 A allele, they have ability to acquire falciparum malaria-related anemia with susceptibility to 3.13 folds risk.

Summary/Conclusion: Overproduction of TNF-α is essential for the elimination and clearance of falciparum parasite but may be associated with severity of malaria and malaria anemia. Overproduction of TNF-α in children with TNF-α 238 A allele may result in falciparum malaria-related anemia among children (mainly severe anemia). The findings of this study will assist clinicians in diagnosing and better managing severe malaria cases.

PB2255: ANTIMICROBIAL SUSCEPTIBILITY TO MICROORGANISMS IN HEMATOLOGICAL MALIGNANCIES; A NEED TO REVIEW INSTITUTIONAL PROTOCOLS

N. ANWAR 1,* , N. FATIMA 2 , V. KUMAR 1 , L. MAJEED 2 , A. KHALID 2 , M. BORHANY 1

1 HEMATOLOGY; 2 RESEARCH AND DEVELOPMENT, NATIONAL INSTITUTE OF BLOOD DISEASES AND BONE MARROW TRANSPLANTATION, KARACHI, Pakistan

Background: Febrile neutropenia is a medical emergency in patients with hematological malignancies. The severity of neutropenia and the duration of infection have an important implication on primary treatment outcome. Previously assessed institutional review revealed gram negative bacteria to be the most common microorganisms, and leading cause of febrile neutropenia. However, the predominance of gram positive bacteria had also increased in later past years. In high risk patients, infections caused by multidrug resistant bacteria worsen treatment complications and thus at times empirical use of antibiotics like colomycin is required; otherwise the prognosis may be jeopardized. Regular surveillance of local flora and susceptibility pattern of antimicrobial is must to elude the antimicrobial resistance.

Aims: The study was conducted to evaluate the prevalence of microorganisms and to observe the sensitivity pattern of antimicrobials as an institutional protocol to establish antimicrobial policies.

Methods: A cross sectional study was conducted at National Institute of blood diseases and bone marrow transplantation, PECHS campus from August 2021 - January 2022. Febrile neutropenia was defined as a single reading of oral temperature of ≥38 °C on two or more occasions in 12 h in the presence of absolute neutrophil count of less than 1000cells/mm. Blood cultures were performed using the BACTEC blood culture system. Organisms were identified according to routine bacteriological procedures and disc diffusion method was used for interpreting antibiotic susceptibility. Data was analyzed by SPSS version 23.0. Frequencies and percentages were calculated for categorical variable and mean (standard deviation) was computed for quantitative variables. Association between categorical variables was observed using the chi-square test or Fisher’s exact test. P value of <0.05 was considered statistically significant.

Results: A total of 310 cultures were observed in 305 patients. The cultures were taken from blood, peripheral line, hickman line, red line, pus, and urine. Out of total, 213 (69%) were found to be gram negative and 97 (31%) were gram positive. The most prevalent gram negative microorganism were Ecoli in 87 (41%) followed by Klebsiella pneumonae 36 (17%), Pseudomonas species 25 (12%) and Pseudomonas aeruginosa in 24(11%) cultures. However, in gram positive; Staphylococcus epidermidis 49 (51%) was more prevalent. Moreover, in gram negative organisms, Acinetobacter species, Burkholderia cepacia, Citrobacter species and Moraxella Species were identified substantially which were not previously previously prevalent. However, in gram positive organisms, Micrococcus species was reported for first time. Gram negative were mostly reported in blood cultures 61 (20%) while gram negative was found in urine cultures 80 (26%). The association of sensitivity and resistance with gram negative and positive isolates was found statistically significant with Amikacin, Vancomycin, Fosfomycin and Ciprofloxacin

Summary/Conclusion: Gram negative isolates were found prevalent in our study. The sensitivity of Meropenem, Amikacin and Colistin was high than other regimens in gram negative while the sensitivity of Fosfomycin, and Linezolid was found in gram positive isolates. Few emerging isolates were observed in the study as well. The regular review of microbial pattern and susceptibility evaluation is imminent for prevention of microbial resistance and assessment of institutional practices.

PB2256: BACTERIAL BLOODSTREAM INFECTIONS AND ANTIMICROBIAL SUSCEPTIBILITY PATTERN IN PATIENTS WITH ACUTE LEUCAEMIA

N. Balshova 1,* , I. Iskrou 2 , I. Lendina 1

1 Hematology, State Institution “Minsk Scientific and Practical Centre for Surgery, Transplantology and Haematology”; 2 State Educational Institution “Belarusian Medical Academy of Postgraduate Education “, Minsk, Belarus

Background: Currently, there is a trend towards an increasing proportion of Gram-negative microorganisms, including Enterobacterias. Escherichia coli and Klebsiella pneumoniae constitute the major part in the structure of bloodstream pathogens. One important mechanism of Enterobacterias’ resistance is the production of extended-spectrum beta-lactamases (ESBL), AmpC β-lactamases and carbapenemases.

Aims: Determination of the antibiotic resistance profile of bloodstream pathogens in patients with acute leukaemia and evaluation of the efficacy of starting antibiotic therapy regimens.

Methods: The study included 69 cases of bloodstream infections, confirmed by bacteriological blood testing in patients with acute leukaemia between January 2020 and January 2022, 46.4% of which were male and 53.6% were female, with a median age of 44 years. All patients had myelotoxic agranulocytosis at the time of infectious complications.

Results: During the study period and according to the bacteriological examination of blood, 58 strains of E. coli and Kl. pneumoniae were detected. The antibiotic sensitivity pattern is shown in the figure.

Bloodstream infection caused by E. coli was detected in 23 cases (32.8%), herewith ESBL production was detected in 47.8% of E. coli strains.

In the scope of the empirical antibiotic therapy, the 4th generation cephalosporin (cefepime) was used in 39.1% of cases or carbapenem (meropenem) in 60.9% of cases in combination with the 3rd generation aminoglycoside (amikacin). Herewith the empirical antibiotic therapy was considered as rational in 78.3% of cases, whereas in the remaining cases escalation was essential. The use of the 4th generation cephalosporin (cefepime) in the scope of the empirical therapy was ineffective in 55.6% of cases. Due to the therapy in 95.7% of cases the patient’s condition was stable, and in 4.3% of cases led to a fatal outcome.

Bloodstream infection caused by Kl. pneumoniae was detected in 35 cases (50%), and resistance to carbapenems was reported in 68.6% of strains of the Kl. pneumoniae.

The share of ESBL-producing Kl. pneumonias trains was 40%, while the proportion of Enterobacteriaceae strains, susceptible to extended-spectrum cephalosporins and carbapenems, reached only 8.8%. During the therapy in 72.4% of cases the patient’s condition stabilised, and in 27.6% of cases a fatal outcome was recorded. In all cases of fatality сarbapenem-resistant strains, including panresistant strains (5.9%) acted as a causative agent.

Summary/Conclusion: The most frequent causative agents of bloodstream infections in acute leukaemia patients during myelotoxic agranulocytosis are the Gram-negative bacteria E. coli and Kl. pneumoniae.

The empirical antibiotic therapy regimens including 3rd generation cephalosporins (cefepime) was ineffective in 55.6% of cases, as 47.8% of isolated E. coli strains were generating ESBL. This increases the relevance of using carbapenems.

Resistance to carbapenems was reported in 68.6% of strains of Kl. pneumoniae. This indicates a high prevalence of carbapenem-resistant strains in the haematological hospital, as well as the low efficacy of empirical antibiotic therapy, including both cephalosporins and carbapenems.

PB2257: SARS-COV-2 VARIANTS DETECTED IN EMPLOYEES OF ONE MEDICAL CENTER

B. Biderman 1,* , O. Gavrilina 1 , O. Glinstchikova 1 , T. Makarik 1 , I. Fevraleva 1 , D. Tikhomirov 1 , T. Tupoleva 1 , A. Sudarikov 1

1 National Medical Research Center for Hematology, Moscow, Russia

Background: March 2020 strict measures were introduced at the National Research Center for Hematology (Moscow, Russia) in order to prevent the spread of SARS-CoV-2: the mandatory wearing of masks, screening for the presence of symptoms of acute respiratory viral infections, regular PCR testing of all staff and patients, a ban on visiting patients, and cancellation of all face-to-face meetings of the employees. Nevertheless, in the fall of 2020, an outbreak of the disease among employees was detected in the institution.

Aims: To study the variety of SARS-CoV-2 variants infecting employees during the outbreak and to reveal possible transmission routes.

Methods: The study included 42 employees infected during the 09/17/2020 - 01/01/2021 outbreak, which corresponded to the 2nd wave of COVID-19 worldwide. PCR testing for the detection of SARS-CoV-2 RNA in nasopharyngeal samples from the staff of the center was performed using the PCR-RV-2019-nCov kit (Syntol, Russia). Sequencing of SARS-CoV-2 samples was performed by the NGS method using the QIAseq SARS-CoV-2 Primer Panel kit (Qiagen, USA); the raw data were analyzed according to the HAVOC protocol (DOI 10.1186/s12859-021-04294-2).

Results: Only 4 (10%) out of 42 employees had a positive PCR test result while having COVID-19 symptoms, the rest - during routine screening. The course of disease was characterized as asymptomatic in 2 (5%), mild in 28 (86%), moderate in 3 (7%), severe in 1 (2%), extremely severe was not detected. Based on sequencing data, we identified 5 different variants of SARS-CoV-2 found in 2 or more samples. Four of them were found each within only one clinical department and did not occur outside of it. Variant 1 was first detected in an asymptomatic carrier on 10/29/2020 and again on 11/2/2020 simultaneously in 8 employees during routine testing (a total of 13 cases were registered in one department). This type was distinguished by the presence of 5 missense mutations in the Spike region, which may have been the reason for its high contagiousness. Among these 13 employees, 1 (7%) had a severe infection, 2 (14%) had a moderate infection, 9 had a mild course (1 had a cutaneous form), and 1 (7%) had an asymptomatic infection. Variant 2 was detected in 2 carriers, variants 3, 4 - in 3, with an interval of a week. Variant 5 was identified in 4 employees from 2 departments (2 people in each from 10/8/2020 to 10/26/2020). For these employees, the course of COVID-19 was characterized as mild. Also, 3 pairs of similar variants were identified, however, the lack of complete similarity and a large time interval raises doubts about their connection. All other 11 SARS-CoV-2 nucleotide sequences were unique.

Summary/Conclusion: Anti-epidemic measures taken at the center have shown effectiveness in reducing the spread of nosocomial infection. The identified clustering of virus variants indicates the predominant infection of employees within departments. The obtained clinical data do not make it possible to identify associations of a particular SARS-CoV-2 variant with the course of the disease. Sequencing of SARS-CoV-2 samples allowed for an epidemiological study and identification of a variant that is likely to be highly contagious.

PB2258: MANAGEMENT OF FEBRILE NEUTROPENIA IN LEUKEMIC PATIENTS: A MONOCENTRIC STUDY

W. Boufrikha 1,* , R. Rakez 1 , Z. Mlayah 1 , A. Cheffai 1 , N. Slama 1 , S. Boukhris 1 , M. A. Laatiri 1

1 Hematology, Fattouma Bourguiba University Hospital, Monastir, Tunisia

Background: Febrile neutropenia (FN) is one of the major complications of chemotherapy, which requires urgent broad-spectrum antibiotics in order to minimise the high rate of morbidity and mortality.

Aims: The aims of our study are to describe the demographic characteristics of febrile neutropenic episodes in leukemic patients and to evaluate the antibiotic prescriptions.

Methods: This is a retrospective study conducted at the Haematology Department of Monastir, Tunisia, from January 2020 to December 2020. We included patients hospitalized for acute leukaemia treatment (induction or consolidation course) complicated by febrile neutropenia.

Results: The twenty-six patients enrolled in the study were classified as high risk according to the Multinational Association of Supportive Care in Cancer (MASCC) score. Eighty-two febrile neutropenic episodes were studied. The median age of our population was 31 years (3-58 years) with a slight male predominance (sex-ratio: 1,16). The most frequent malignancy was acute myeloid leukaemia.

In our study, 14 patients (53,8%) had at least 2 febrile neutropenic episodes and only 4 patients (15,4%) presented with one febrile episode. Fever of unknown origin (FUO) and clinically documented infection were found respectively in 36,6% and 28% of cases. Microbiologically documented infections (MDI) were noted in 29 cases of which 90% were monomicrobiol blood stream infections (BSI). Gram-positive Bacteria accounted for 75% of the isolated strains, Coagulase-negative Staphylococci (87,5%) being the most frequent. Gram-negative Bacteria were isolated in 25% of all strains with predominance of Escherichia coli (75%). Neither extended-spectrum beta-lactamases nor carbapenemase-producing microorganisms were detected (table 2).

Empirical antibiotic therapy was appropriate according to the European Conference on Infection in Leukaemia (ECIL) and the Infectious Diseases Society of America (IDSA) guidelines in 61% of cases. Favorable response to first-line antibiotherapy was seen in 74,4% of cases. Septic shock complicated the course of 2 MDI due to Coagulase-negative Staphylococci.

Summary/Conclusion: FN in leukemic patients is an emergency and the onset of treatment should, in no way, be delayed. Demographic characteristics of our study consist with those reported in the literature and therapeutic prescriptions are in agreement with the international guidelines.

PB2259: MONOCYTE’S INVOLVEMENT IN INFLAMMATORY RESPONSE IN COVID-19 INFECTION IN CHRONIC RENAL DISEASE

V. Bumbea 1,2 , H. Bumbea 2,3,* , L. Ardelean 1 , L. Radulescu 1 , L. Damian 4 , I. Dumitru 2,5 , C. Lambert 6 , V. Popov 7 , C. Tomuleasa 8,9 , A.-M. Vladareanu 2,10

1 Dyalisis, Emergency Hospital Bucharest; 2 University of Medicine and Pharmacy Carol Davila Bucharest; 3 Hematology Blood and Marrow Transplant Unit; 4 Nephrology; 5 Blood Unit, Emergency University Hospital Bucharest, Bucharest, Romania; 6 CHU de Saint Etienne, Saint Etienne, France; 7 Hematology, Clinical Colentina Hospital Bucharest, Bucharest; 8 Hematology, Institute of Onclology Cluj Napoca; 9 University of Medicine and Pharmacy Iuliu Hateganu, Cluj Napoca; 10 Hematology, Emergency University Hospital Bucharest, Bucharest, Romania

Background: COVID-19 infection is known to associate an important inflammatory response with high risk for organ damages and thrombotic events. Monocytes are found to play an important role in inflammatory response against various antigens and are classified in three subsets: Classical , Non-classical , Intermediate .

Aims: The aim of our study is to analyse monocyte subsets in patients with advanced renal failure and COVID-19 infection.

Methods: We present a case-control study with paired samples: 5 patients from 15 suffering from advanced renal failure treated by hemodialysis, before, during developing a medium severity form of COVID-19 infection, and in follow-up (mean time 10 months), in which we analyzed monocytes subsets distribution and activation markers by flowcytometry from whole blood samples. Monoclonal antibodies used: CD14, CD16, CD300e, HLA-DR, CD64, CD38, CD45. Statistical analysis was performed with the Jasp and NCSS software.

Results: Decreasing number of leukocytes (p=0,023), lymphocytes (p=0,029) and monocytes (p=0,167) during the infection was found, decreasing of the percentage of classical monocytes subset (p=0,295), and nonclassical monocytes subset (p=0,001), increasing in intermediate monocytes subset (p=0,115). HLA-DR and CD300e during the disease generally was increased without statistical significance (p=0,119, p=0,625), conventional monocytes had a lower CD300e (p=0,188) and HLA-DR expression is in fact unchanged with a slight tendency to increase (p=0,745), nonclassical monocytes had a higher percentage of HLA-DR (p=0,033) and CD300e (p=0,654), and intermediate monocytes had a slight elevated percentage of HLA-DR expression (p=0,332) and CD300e expression is in fact unchanged with a slight tendency to decrease (p=0,690). The percentage of classical monocytes increased (p=0,05), with a slight tendency of increasing in CD300e and HLA-DR expression after versus before infection. Nonclassical monocytes and intermediary monocytes percentage decreased after COVID-19 infection (p=0,001 respective p=0,049), but intermediary monocytes show a higher level of HLA-DR expression (p=0,012) versus before infection. CI was 95%. Boxplots in figure.

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Summary/Conclusion: Apparently, advanced renal failure treated by hemodialysis does not dramatically change the inflammatory response medium form of COVID-19 infection. Distribution of inflammatory response consist in decreasing of percentage of classical and nonclassical monocytes, similar to severe forms of COVID-19 infection but the percentage of intermediate monocytes subset is increased. Elevation of HLA-DR and CD300e expression seems to occur especially in nonclassical monocytes, suggesting that this subset of monocytes is more activated. These changes suggest a higher inflammatory activity in nonclassical monocytes despite their lower percentage and a higher percentage of inflammatory and phagocytic intermediate monocytes. After a mean time of one year from infection resolution classical monocytes increased, and other two subtypes decreased, in nonclassical monocytes with the loss of inflammatory activity, but in intermediary monocytes with the persistence of important inflammatory activity similar during the infection, suggesting that the inflammatory response is more durable in intermediary monocytes after COVID-19 infection. The total monocytes expression of CD300e and HLA-DR after COVID-19 infection is statistically significant increase than before infection suggesting that the monocytes remain with a significant proinflammatory status after COVID infection.

PB2260: CIDOFOVIR FOR THE TREATMENT OF ADENOVIRUS INFECTION IN PEDIATRIC PATIENTS UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT): A SINGLE CENTER EXPERIENCE

F. Cacace 1,* , V. Caprioli 1 , M. R. D’amico 1 , G. De Simone 1 , F. Muriano 2 , M. Paparo 3 , I. Ricciardi 3 , M. Grimaldi 4 , F. P. Tambaro 1

1 BMT UNIT, AORN Santobono Pausilipon; 2 Hematology, AOU Federico II; 3 Microbiology, AORN Santobono Pausilipon; 4 UOC Microbiology and Virology, AORN Ospedale dei Colli, Naples, Italy

Background: Adenovirus (ADV) infection is usually self-limited and asymptomatic in immunocompetent individuals but is a cause of morbidity and mortality in immunocompromised pediatric patients undergoing HSCT. In particular, impaired T-cell immunity following HSCT increases the risk of viral reactivation and/or infection. Patients developing ADV-associated disease present with a broad variety of clinical manifestations ranging from mild gastroenteric or respiratory symptoms to severe hemorrhagic enteritis, cystitis, hepatitis, pneumonia, encephalitis, myocarditis or multiple organ involvement. Currently, no specific drugs are approved for the treatment of ADV infection. Cidofovir, a nucleotide analogue inhibiting viral DNA replication, has potential for therapeutic activity against ADV.

Aims: We retrospectively evaluated efficacy and safety of cidofovir in pediatric patients undergoing HSCT with ADV infection.

Methods: We identified cases of ADV infection following HSCT in pediatric patients treated in our Institution since December 2015. Patient characteristics are summarized in the Table. ADV-DNA was monitored in peripheral blood twice weekly in the first 100 days post-HSCT, then once monthly in the first year after HSCT. Cidofovir was started when blood ADV-DNA was > 1000 copies/mL in symptomatic patients.

Results: Eight patients out of 76 who underwent HSCT (11 from haploidentical donors) between December 2015 and February 2022 developed ADV infection: 7were male; median age was 8.5 years (range2-15 years). Pre-transplant diagnosis was severe aplastic anemia for 3 patients, the remaining 5 patients were diagnosed with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Cooley disease, Fanconi anemia and hyper IgM X-linked immunodeficiency. One patient received HLA identical SCT from a family donor, one patient underwent matched and another mismatched unrelated donor SCT, 5 patients received haploidentical SCT: oneunmanipulated with post-transplant cyclophosphamide and 4 with B- T α/β depleted graft. Median time to ADV infection was 90 days (range 23-170 days). ADV-DNA was isolated by RT–PCR in blood in 7/8 patients; ADV-DNA was isolated by RT-PCR in bronchoalveolar lavage fluid and pericardial fluid in 4 and one patient who developed severe interstitial pneumonia and pericardial effusion, respectively. None of the patients received probenecid for kidney protection. In our cohort, cidofovir was given at the dose of 1 mg/kg/three times a week, in order to prevent kidney injury. Patients received a median of 9 (range 8-14) cidofovir doses. 7/8 patients cleared ADV viremia and resolved pericardial and pulmonary manifestations.

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Summary/Conclusion: ADV infection is a potentially life-threatening complication after HSCT, with an increased incidence in pediatrics and in patients undergoing haploidentical HSCT, consistent with our experience, with no approved standard treatment. Cidofovir represents a valid therapeutic option. Our treatment schedule at the dose of 1 mg/kg/3 times a week showed 87.5% resolution with no major hematological or renal toxicities requiring dose adjustment for concomitant potentially nephrotoxic agents or treatment discontinuation. Our schedule was well-tolerated and feasible for outpatient administration, resulting in a median disease resolution of 3 weeks. Although very encouraging, these results should be confirmed by larger cohorts of patients.

PB2261: LINEZOLID AS A SAFE ANTIMICROBIAL AGENT IN THROMBOCYTOPENIC PATIENTS UNDERGOING CHEMOTHERAPY FOR HEMATOLOGIC MALIGNANCIES

F. Angotzi 1,* , A. Cellini 1 , S. Marinello 2 , A. Cattelan 2 , L. Trentin 1 , C. Gurrieri 1 , F. Lessi 1

1 Hematology and Clinical Immunology Unit, Department of Medicine, University of Padua; 2 Infective and Tropical Disease Unit, Padua University Hospital, Padua, Italy

Background: Febrile neutropenia (FN) is a common event in patients undergoing high-dose chemotherapy (HD-CT) for hematologic malignancies. Linezolid (LZ) is the first-in-class oxazolidinone antimicrobial agent active against gram-positive bacteria, often administered during FN as treatment for hospital-acquired pneumonia, gram-positive bacteriemia and soft-skin tissue infection. LZ induced thrombocytopenia or delayed platelet recovery are adverse events of concern in patients receiving HD-CT. As of today, few data are available on the safety of LZ in this specific setting.

Aims: To retrospectively analyze a cohort of thrombocytopenic patients treated with LZ for FN in a single center, evaluating bleeding risk and impact on PLT recovery time.

Methods: The analysis included patients diagnosed with Acute Leukemia (AL), High Grade Lymphomas (HGL) and Multiple Myeloma (MM) treated at our institution, between 2016 and 2021, with LZ for a FN episode during induction chemotherapy or ASCT conditioning. To be enrolled, PLT count at the start of LZ therapy had to be < 50×10 9 /L.

Results: A total of 40 FN episodes treated with LZ were identified. 22 occurred during induction therapy for AL and 18 during ASCT conditioning regimens. The median PLT count at the start of LZ therapy was 19×10 9 /L in both groups (range 5-49×10 9 /L in the AL cohort, 5-35×10 9 /L in the ASCT group), while the median treatment duration was 8 days in both groups (range 2-15 days in the AL cohort, 3-12 days in the ASCT cohort). Only one clinically relevant bleeding event was observed in the AL group, while 0 bleeding events were observed in the ASCT group. Resolution of infection (defined as fever resolution, normalization of CRP and blood culture negativization) after the introduction of LZ therapy was observed in 17 (77%) AL patients and 15 (83%) ASCT patients. In the AML group PLT recovery (defined as a stable PLT count ≥20×10 9 /L without the need for PLT transfusion) was observed in 18 (81%) patients with a median time to recovery of 22 days from the start of the chemotherapeutic regimen (range 18-41). PLT recovery was observed in 10 (71%) ASCT patients with a median time to recovery of 35 days from the stem cell infusion (range 13-136). 4 out of 18 patients in the ASCT group started LZ therapy after PLT recovery, and 3 out of 18 patients in the same group died due to the infectious complication that prompted the start of LZ therapy. Median PLT recovery time was not significantly different between patients enrolled in this study and historical controls from our center (AML: median 20 days, p>0.1. ASCT: median 22.5 days, p>0.1).

Summary/Conclusion: Our observations support the use of LZ in this setting, as only one relevant bleeding event was observed among the two groups, and no impact on PLT recovery time was noted after comparison with historical data. Our study suggests that LZ is a safe and effective antimicrobial therapy in patients undergoing HD-CT for hematological malignancies. This data needs to be prospectively confirmed in a larger cohort of patients in a multicenter study.

PB2262: PEGFILGRASTIM IN THE SUPPORTIVE CARE OF HEAVILY PRETREATED MULTIPLE MYELOMA IN POMALIDOMIDE-BASED TREATMENT

C. Cerchione 1,1,* , D. Nappi 1 , A. E. Pareto 2 , S. Ronconi 1 , D. Cangini 1 , M. Ceccolini 1 , G. Musuraca 1 , L. Catalano 1 , G. Martinelli 1

1 Hematology Unit, Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST IRCCS, Meldola (FC); 2 Hematology Unit, Ospedale San Giuliano, Giugliano in Campania, Italy

Background: Pegfilgrastim is a pegylated long-acting recombinant form of G-CSF that extends the half-life and allows for once-per-cycle dosing, requiring less frequent dosing than nonpegylated G-CSF.

Aims: The objective of this study was to compare the efficacy and safety of pegfilgrastim in patients affected by heavily pretreated MM, treated with pomalidomide-dexamethasone, in order to determine whether a single subcutaneous injection of pegfilgrastim is as effective as daily injections of standard filgrastim, in terms of haematological toxicity, febrile neutropenic episodes, antibiotic usage and hospedalization duration.

Methods: 57 patients (31 M/26 F), median age at diagnosis 69 years (r. 52-84), and median age at start of treatment 76 years (r.56-90) treated with several lines of treatments (median 7, r. 2-12), every refractory to all the drugs previously received, received Pomalidomide-Dexamethasone (P 4 mg for 21 days, D 40 mg days 1,8,15,22, pegfilgrastim day +8) every 28 days, until progression. Since first course, patients performed blood counts once weekly and received, from day +8 to day +19, prophylactic oral chinolonic antibiotics and anti-fungal drugs. During neutropenia after first cycle, Filgrastim (5 μgr/kg/day for 3 days) was given if neutrophils count was <1500 x 10^9 cells/L.

Results: Median number of filgrastim administrations was 4.6 (r. 3-6); nadir neutropenia was registered after a median of 10.4 days (r. 7-14); median of nadir neutrophil count was 1.13 x 10^9 cells/L (r.0.3–1.5), with maximum duration of 14 days. From the second course, all patients switched to prophylaxis with pegfilgrastim (6 mg), injected subcutaneously with a single administration on day +3 independently from the neutrophil count at that time. During pegfilgrastim, neutropenia was never longer than 8 days, with a consequent reduction of neutropenia-related infections. Median nadir neutrophil count, evaluated for every patients for at least three courses of therapy (r. 3-6) registered at day +11, was 1.28 (r.0.9-2.2). Only 4 patients needed a supplement of 3 administrations of filgrastim.

Summary/Conclusion: Pegfilgrastim was well tolerated in all patients: main side effects in our patients were mild fever and bone pain (21.2%). In patients affected by heavily pretreated MM treated with pomalidomide-dexamethasone, pegfilgrastim seems to reduce the incidence of severe neutropenia and infections and may increase the possibility to maintain the scheduled time of treatment.

PB2263: SUPPORTIVE CARE IN LOW-RISK MYELODYSPLASTIC SYNDROMES: ROLE OF ERYTHROPOIESIS STIMULATING AGENTS (ESAS)

C. Cerchione 1,* , G. Musuraca 1 , B. Giannini 1 , D. Nappi 1 , F. Alfinito 2 , G. Martinelli 1

1 Hematology Unit, Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” - IRST IRCCS, Meldola (FC); 2 Hematology Unit, AOU Federico II, Napoli, Italy

Background: Erythropoiesis stimulating agents (ESAs) are the frontline treatment in low-risk anemic MDS patients and an employment of this therapy in the earlier stage of the disease can delay the need for RBC transfusion, hypothetically by slowing the disease course. It’s matter of debate whether the clinical response is a result of proliferation and maturation of the dysplastic clone or stimulation of residual normal erythropoiesis by ESAs.

Aims: Macrocytosis is one of the cytological hallmarks of dyserithropoiesis in MDS: an analysis of the erythropoietic response to ESAs therapy in a cohort of anemic non trasfusion-dependent MDS patients, enrolled in a retrospective register, RECAMDS, subgroup of Italian register, was performed.

Methods: 183 patients, treated with standard-dose ESAs, have been retrospectively analyzed. Data analysis was performed, according to IWG 2006 criteria, at the baseline, after 3 and 6 months of continuous treatment, with a subanalysis of the patients according to WHO and R-IPSS risk stratification. ESAs were started at mean Hb concentration of 9.31 g/dl, mean serum EPO concentration: 51 mU/L, after a mean time from diagnosis of 6 months (r.1-118).

Results: ORR was 83.6% (153/183), no difference among WHO and IPSS subgroups was found: 132/183 (72.1%) achieved response after 3 months of treatment, while other 21/183 (11.2%) after 6 months. 19 patients with stable disease

(non-responders, according to IWG criteria), in which treatment was continued, achieved response after 9 months. In the macrocytic-responders group 83.2% exhibits again macrocytosis after 3 months, while 16.8% become normocytic. In the normocytic-responders group 89.8% exhibits again normocytosis, while 10.2% become macrocytic: in these patients, after 3 months, there was a contemporary worsening in neutropenia and thrombocytopenia, with transfusion-dependence, regarded as first signs of progression of disease. Non-responders were 30/183 (16.3%): in the macrocytic non-responders group 89% exhibit again macrocytosis after 3 months, while 11% become normocytic; in the normocytic group 76% exhibits again macrocytosis, while 24% become normocytic.

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Summary/Conclusion: These preliminary data can suggest that, in the majority of MDS patients responsive to ESAs, the increase of Hb concentration occurs mainly stimulating erythroid production in MDS clones; in the minority of patients probably it happens recruiting residual polyclonal erythropoiesis.

It is interesting to note that stimulating effects of ESAs last even when the expression of dysplasia progresses.

PB2264: A REVIEW OF MORPHOLOGICAL ABNORMALITIES ON PERIPHERAL BLOOD FILM: BEYOND ROUTINE ANALYSIS IN THE COVID-19 POPULATION IN THE FIRST WAVE OF THE DISEASE.

H. Fernandez Leyva 1,*

1 Haematology, NHS, Licoln, United Kingdom

Background: Vaccination is considered the most promising approach for ending or containing the coronavirus disease 2019 (COVID-19) pandemic. Available vaccines have proven highly safe and effective. The morphology appearance of the blood film is an excellent tool to analyse the disease severity. In late February of 2021, a prothrombotic syndrome was observed in a small number of individuals who received the ChAdOx1 CoV-19 vaccine (AstraZeneca, University of Oxford, and Serum Institute of India), an adenoviral vector-based vaccine. This syndrome has been designated vaccine-induced immune thrombotic thrombocytopenia (VITT) with a particular and intriguing characteristic.

Aims: An examination of peripheral blood smear findings in COVID-19 patients was performed in 50 consecutive patients at the first wave of the disease and prior to the full vaccination program instituted with current-acute respiratory syndrome coronavirus 2 (SARS-CoV-2) confirmed disease (PCR)- Pneumonia

Methods: A morphology analysis was aimed at identifying the principal abnormalities in patients diagnosed with COVID-19. Interestingly (but not surprisingly) the most severe forms were associated with a more prominent abnormalities, including leukoerythorblastic features and secondary hemophagocytic lymphohistiocytosis, leading to an often fatal outcomes. In this study, absolute lymphopenia (despite non constant) were the predominant feature despite its not specific to COVID-19. A spectrum of variant lymphocytes is seen in COVID-19 cases, albeit constituting less than 1% prolymphocytes in most cases. Plasmacytoid lymphocytes were more common as well as acquired Pelger-Huët were significantly observed (but not representative).

Results: A laboratory findings reported in association with COVID-19 included leukopenia, lymphopenia, monocytosis, neutrophilia, eosinopenia, These abnormalities mainly highlight the severe, transitory and reversible perturbation of myelopoiesis, especially in the form of accelerated and disordered granulopoiesis, in patients with COVID19 in severe symptomatic phase. The hypothesis of quantitative and qualitative abnormalities can be related to the cytokine storm and hyperinflammation, which is a pathogenic factor in the evolution of COVID19 pneumonia.

Summary/Conclusion: An early identification of severe lymphoid abnormality could represent a high risk factor for poor outcomes with COVID-19. A decreased number of mature lymphocytes, and eosinophils in peripheral blood smear were observed in the severe stage patients (p <0.05). The limitation of the study is in the small numbers of the analysed population and the absent of the control comparative group. The subsequent analysis after patient received at least two dose of the vaccination shows a less severe lymphopenia (P< 0.05) and disease severity

PB2265: OUTCOMES OF COVID-19 INFECTION IN PATIENTS WITH B AND PLASMA CELL MALIGNANCIES DURING THE THIRD WAVE IN ARGENTINA: A SINGLE CENTER EXPERIENCE

O. Gelo 1,* , L. Korin 1 , L. Ayala 1 , S. Cranco 1 , E. Babuin 1 , A. Vitriu 1 , P. Ochoa 1 , M. L. Fuente 1 , N. Tartas 1 , M. Alderete 2 , S. Flagel 3 , C. Sosa 4 , M. A. Gazzaneo 4 , M. Zylberman 3 , F. Díaz Couselo 3 , C. Irrazábal 5 , M. D. R. Custidiano 1 , J. Sánchez Ávalos 1 , M. C. Foncuberta 1

1 Hematology; 2 Infectology; 3 Internal medicine; 4 Urgent care; 5 Intensive care, Instituto Alexander Fleming, Ciudad Autónoma de Buenos Aires, Argentina

Background: The global COVID-19 pandemic caused by SARS-CoV-2 is still ongoing. The third wave arrived in Argentina in mid-December 2021 with Omicron variant as the dominant strain representing 84% of sequenced cases. Patients (pts) with hematologic malignancies (HM) have an increased risk of severe COVID-19 due to their immunosuppressed condition secondary to both underlying disease and subsequent treatment, and lower rates of seroconversion after vaccination. Omicron variant shows an unprecedented degree of neutralizing antibody escape and may require a higher level of anti-spike antibodies to prevent severe COVID-19.

Aims: To describe characteristics and outcomes of pts with HM and COVID-19 treated in our hospital during the third wave.

Methods: Observational, descriptive and retrospective study of adult pts with chronic lymphocytic leukemia, non-Hodgkin B cell lymphoma and multiple myeloma with confirmed COVID-19 infection between 15th Dec 2021 and 7th Feb 2022. We included pts with active disease or who had received anticancer therapy within the last year of COVID-19 infection. Complete vaccination schedule was defined as tree doses or two if the last shot was applied between 14 days and 5 months of COVID-19 infection. IgG levels against SARS-CoV-2 spike protein were analyzed with Abbott SARS-CoV-2 IgG II Quant-test with ≥50 AU/ml as a threshold for positivity. Pts received convalescent plasma according to physician criteria.

Results: A total of 38 pts were included. Mean age was 63 years, 60.5% were males and 55% had ≥1 comorbidity. Eleven pts had chronic lymphoid leukemia, 10 indolent B cell lymphoma, 7 aggressive B cell lymphoma and 10 multiple myeloma. Concerning disease status, 39.5% were in complete or partial response, 42.1% had progressive or stable disease and 18.4% were on watch and wait. Nearly half of the pts had received a B-cell-depleting monoclonal antibody-based regimen: Rituximab (n=13), Daratumumab (n=4) or Obinutuzumab (n=1); the other half: other anti-myeloma therapy (n=5), chemotherapy (n=3), Ibrutinib (n=3), Venetoclax (n=2) or were untreated (n=7). Twenty-eight were fully vaccinated, 8 had incomplete vaccination and only one was unvaccinated. Heterologous vaccine regimens were used in 21 pts, most frequently vector-based plus mRNA vaccine (n=12). Regarding the last shot, the majority received vector-based vaccine (AstraZeneca Oxford n=21; Sputnik V n=4), followed by mRNA vaccine (BioNTech/Pfizer n=7, Moderna n=4) and inactivated vaccine (Sinopharm n=1). The median time from the last dose and COVID-19 diagnosis was 77 days (IQR 40.5-177). At the time of COVID-19 diagnosis, IgG anti-spike antibodies were measured in 16 pts, 4 were no responders and the median level for those who did response was 176,6 AU/ml (IQR 86,6-879,8). Fourteen required hospital admission, among which 8 needed oxygen support and 5 were admitted to ICU. Twelve pts were treated with convalescent plasma, 9 received corticosteroids and 1 tocilizumab. With a median follow up of 42 days, the overall mortality rate was 7.9% (n=3). Follow-up swabs were performed in 21 pts, 18 converted to negative after a mean of 23.4 days (SD +/- 8,4) from the first nasal swab and in 3 it still remained positive after 37, 40 and 45 days respectively.

Summary/Conclusion: This report sheds light into outcomes of pts with HM infected with Omicron variant. Our findings show lower fatality rate than reported in previous waves, but still higher than the general vaccinated population. This might be explained because even if fully vaccinated, responsive patients fail to achieve protective levels of anti-spike antibodies.

PB2266: INFECTIVE ENDOCARDITIS IN NEUTROPENIC PATIENTS: A SYSTEMATIC REVIEW

M. Guaraná 1,* , E. Beteille 2 , M. Nucci 1

1 Universidade Federal do Rio de Janeiro, Rio de Janeiro; 2 Universidade Federal de Santa Catarina, Santa Catarina, Brazil

Background: Infective endocarditis (IE) is a serious infection in non-neutropenic patients. We have published a low incidence of IE caused by viridans streptococci in neutropenic patients.

Aims: To evaluate the frequency of IE caused by pathogens other than viridans streptococci in neutropenic patients and characterize the clinical presentation of IE in such patients.

Methods: We performed a computerized search of MEDLINE (PubMed version) from May 1948 through October 2021 using the keywords combinations “(neutropenia or neutropenic) endocarditis” and “endocarditis leukemia”. Additional hand searching in reference lists was performed in all articles that met eligibility criteria. All studies in English language that included patients with documented neutropenia during the episode of IE were selected

Results: Among 570 studies, 21 were selected, reporting only 22 episodes of IE caused by bacteria and fungi (11 cases each). Cases of IE caused by bacteria involved mostly left heart valves while cases caused by fungi involved the right heart. A new cardiac murmur was the most frequent clinical presentation and the majority of patients with IE had no pre-existing valve abnormalities.

Summary/Conclusion: Given the rarity of IE in neutropenic patients, routine investigation with echocardiography seems not indicated.

PB2267: COVID-19 OUTCOMES ON HAEMATOLOGY PATIENTS WHO RECEIVED RITUXIMAB: SINGLE CENTRE STUDY

L. Hariharan 1,* , M. Champion-Homfray 1 , E. Hassan 1

1 Haematology, Russells Hall hospital, NHS foundation trust, Brimingham, United Kingdom

Background: Rituximab is a monoclonal antibody, targeting CD20 on surface of B lymphocytes. It is used in the treatment of various of haematologic conditions. Rituximab treatment is associated with rapid and prolonged B-cell depletion, reduced humoral response and secondary hypogammaglobulinemia. Recovery from which may take up to 12 months, therefore patients who receive rituximab are at increased risk of infectious complications for a long period of time after treatment.

Aims: To evaluate the potential impact of prior rituximab therapy on clinical outcomes of COVID-19 infection.

Methods: We conducted a retrospective study on haematology patients who received rituximab from December 2019 to October 2021 and were diagnosed with COVID-19 pneumonia. Indications of rituximab were treatment of lymphoma, CLL, and autoimmune haematological disorders.

Results: 139 were included in the study. Of these 139 patients, 13 got admitted to the hospital. The reason for hospitalization of patients was severe COVID-19 pneumonia and its complications. Median age was (71.5) years. 5 patients (38.4%) developed severe COVID-19 that ultimately led to their deaths. Mean admission days was (10.5) days. Median time between rituximab and COVID-19 infection was 3.9 months. 5 patients had ITU admissions, 8 had oxygen support, one was discharged on home oxygen. 2 of the patients who died received anti-SARS-CoV-2 monoclonal antibodies which did not seem to improve the condition.

Summary/Conclusion: There is no doubt that COVID-19 pandemic presents unprecedented challenge to haematologist and patients receiving rituximab.Our observed mortality in hospitalised patients indicates that a significant proportion of haematology patients recover and survive despite recent rituximab treatment. Further studies including larger cohort of patients are essential to validate that rituximab can be safely delivered for haematology patients. Dose modification regimens, and effects on long term disease control remain to be clarified.

PB2268: IMPACT OF HEMATOLOGICAL DISORDERS ON MORTALITY AND SERIOUS OUTCOMES IN SARS-COV-2 INFECTION: A MULTICENTER COHORT STUDY

A. P. Hernandez Perez 1,* , E. I. Apodaca-Chavez 1 , I. A. Borjon-Cabada 2 , R. E. Leon-Cantu 2 , Y. S. Baltodano-Martinez 3 , K. L. Osorno-Rodríguez 4 , B. Guzman-Beltran 5 , B. Lafarga-Amao 6 , M. Mendoza-Oliva 7 , A. Almeida-Arvizu 8 , L. E. Hernandez-Torres 4 , M. R. Demichelis-Gomez 1

1 Hematology, Instituto Nacional de Nutricion Salvador Zubiran, Mexico city; 2 Hematology, Centro Médico Nacional del Noreste, UMAE Hospital de Especialidades #25, Monterrey; 3 Hematology, Hospital General de México “Dr. Eduardo Liceaga”, Mexico city; 4 Hematology, Hospital Universitario “Dr. José Eleuterio González”., Monterrey; 5 Hematology, Hospital Civil de Guadalajara “Fray Antonio Alcalde”, Guadalajara; 6 Hematology, UMAE Hospital de Especialidades #71, Torreon; 7 Hematology, Hospital General de México “Dr. Eduardo Liceaga”.; 8 Internal Medicine, Instituto Nacional de Nutricion Salvador Zubiran, Mexico city, Mexico

Background: Presence of hematologic diseases has been associated with worse outcomes in patients with SARS-CoV2 virus infection, including higher mortality rates than patients without these comorbidities. This cohort study included patients with and without prevalent hematologic diseases.

Aims: To determine the impact of hematologic disorders on 30-day mortality and other severe outcomes in patients with SARS-COV-2 infection.

Methods: A prospective cohort study was conducted in 6 COVID-19 reference centers in Mexico between March 1, 2020 and January 31, 2021. The main objective of our study was to determine the impact of hematologic disease status on 30-day mortality and on a composite outcome of severe complications(AKI, ARDS, and liver failure). Using data from patients treated in the largest center, those with COVID-19 and hematologic diseases were compared to non-hematologic COVID-19 controls. Pairing was performed according to COVID-19 treatment, age, sex, BMI, and CDC-defined variables of high mortality risk. As a secondary objective, a multi-center analysis was performed to determine risk factors for 30-day mortality in hematologic disease patients, determined by univariate and multivariate analysis.

Results: For the main objective, 59 patients were recruited(matched 1:1). In the 30-day mortality analysis, we found no differences between the two groups: HR 1.08 (95% CI 0.52-2.24) p =0.833, OR 1.08 (95% CI 0.46-2.48) p =0.872. Regarding the composite outcome of severe complications, we found a HR 1.03, (95% CI 0.59-1.80) p =0.896. There was no difference in the 30-days survival between the two groups( p =0.832)(Figure 1).

For our secondary objective, 158 patients were recruited: 46.6% women, median age 45 years, BMI 26.3 (IQR 23.2-29.7), 19.0% diabetes, 14.6% hypertension and 18% smokers, and 83.5% with hematologic malignancies. Supplementary oxygen was used in 81.6%, in hospital-treatment was necessary in 84.1%, and 41.4% required ICU admission. 30-day mortality was 42.4%. Complications during hospitalization included respiratory failure in 84.5%, of which only 48% received VMI; reasons for not receiving VMI included a non-intubation order issued by the patient or family members and lack of infrastructure. Regarding COVID-19 treatment, 30% received dexamethasone 6 mg/day and 30% received thromboprophylaxis.

In the univariate analysis, the following were predictive factors of mortality: all types of malignancy HR 2.64 (95% CI 1.06-6.59) p =0.030, lymphoid malignancy HR 2.52 (95% CI 1.06-6.95) p =0.035, last treatment <3months HR 2.3 (95% CI 1.15-4.61) p =0.018, and palliative treatment HR 3.01 (95% CI I.35–7.72) p =0.007. Complete response was a protective factor HR 0.229 (95% CI 0.093–0.53) p =0.001. The use of dexamethasone was not a protective factor in this population HR 1.32 (95% CI 0.80-2.18) p =0.273. In the multivariate analysis, palliative status HR 4.8 (95% CI 1.07-22.18) p =0.040 and complete response HR 0.22 (95% CI 0.062-0.815) p =0.023 were risk and protective factors, respectively.

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Summary/Conclusion: After careful pairing according to clinical characteristics, including CDC variables predictive of high mortality risk, we observed survival didn’t differ between groups, concluding hematologic disease did not impact 30-day mortality in COVID-19 patients. This is striking, since previous reports have described a higher mortality risk among patients with hematologic diseases, although the pairing process hadn’t been as thorough as ours. Also of note, use of dexamethasone was not associated with lower 30-day mortality in patients with hematologic diseases.

PB2269: HUMORAL AND T-CELL RESPONSES TO SARS-COV2 VACCINATION IN PATIENTS WITH MULTIPLE MYELOMA MANAGED IN THE COMMUNITY SETTING

H. Holik 1,2,* , I. Krečak 3 , I. Vučinić Ljubičić 1 , L. Cvetko Krajinović 4 , A. Požgain 2,5 , I. Čabraja 6 , L. Vinković 1 , B. Halassy 4 , S. Mitrović 4 , B. Coha 1

1 Department of Hematology and Oncology, General Hospital dr. Josip Benčević, Slavonski Brod; 2 School of Medicine, University of Osijek Josip Juraj Strossmayer, Osijek; 3 Internal medicine department, General Hospital Šibenik- Knin county, Šibenik; 4 Research Department, University Hospital for Infectious Diseases “Dr Fran Mihaljevic”, Zagreb; 5 Microbiology Department, Institute of Public Health Osijek-Baranja County, Osijek; 6 Department of Infectious Diseases, General Hospital dr. Josip Benčević, Slavonski Brod, Croatia

Background: Multiple myeloma (MM) is a plasma cell neoplasm with an impaired humoral and cellular immune system, mostly due to underlying disease and treatment. Approximately one-third of MM patients dies from coronavirus disease 2019 (COVID-19). Vaccines can provide significant protection against COVID-19 but seroconversion and protection in MM patients with MM is lower than that in the healthy population. For this reason, there is an unmet need to identify MM patients at high risk of COVID-19-related morbidity and mortality.

Aims: To investigate specific humoral and T-cell response patterns to SARS-CoV2 vaccination in MM patients treated in the community setting.

Methods: This was a single-center study conducted at dr. Josip Benčević General Hospital, Slavonski Brod, Croatia. Consecutive patients with multiple myeloma vaccinated against COVID-19 were prospectively included in the study. Humoral and celullar responses were determined by live virus neutralization test (with a negative result being <4 IU/mL) and QuantiFERON SARS-CoV-2 Ag1/Ag2, Qiagen (test being positive or negative), respectively. The chi-squared and the Mann-Whitney U tests were used. Statistics were performed with MedCalc Statistical Software® (Ostend, Belgium, v. 20.014)

Results: A total of 17 patients with active MM were included; median age was 65 years (range 53-85), 35.3% were female and 5 (29.4%), 52.9% and 3 (17.6%) of patients had International Staging System (ISS) stages of 1, 2, and 3, respectively. Eleven patients (64.7%) were screened during first line/maintenance treatment (mostly bortezomib-based=10) and six (35.2%) in the relapsed setting (mostly daratumumab-based=4). Ten patients (56%) were previously autotransplanted, six using a tandem approach. Three patients (17.6%) previously recovered from COVID-19.

Eleven patients (64.7%) received at least two doses of mRNA-based vaccines and six (35.3%) were fully vaccinated with vector-based vaccines. A total of eleven (64.7%) and six (33.3%) patients had humoral and T-cell responses, respectively; however, responses were not correlated (p=0.408). On the other hand, more mRNA-vaccinated patients achieved humoral (81.8% vs. 33.3%; p=0.050) response, whereas there was no difference with respect to cellular response (27.3% vs. 33.3%; p=0.799). Age, sex, ISS stage, line of treatment and prior SARS-CoV2 infection were not associated with humoral or cellular responses (p>0.050 for all analyses). Interestingly, both humoral (72.7% vs. 27.3%; p=0.031) and cellular responses (100% vs. 0%; p=0.014) were more frequent in transplanted patients. The median follow-up was two months and two patients (11.7%) developed COVID-19, both had no humoral (p=0.048) or cellular (p=0.345) response to previous vaccination.

Summary/Conclusion: This data acquired in the community setting demonstrate low T cell response rates and better humoral response rates to SARS-CoV2 vaccination in actively treated MM patients. Better responses were seen with mRNA-based vaccines and in transplanted patients, indicating that mRNA vaccines and stem cell transplantation may provide higher stimuli to the immune system and provide higher disease control and better immune reconstitution, respectively. Two patients without humoral and T cell responses contracted COVID-19, emphasizing the need for vaccination against SARS-CoV2 in order to protect this highly vulnerable population. Limitations of the study are its small sample size and single-center design.

PB2270: CLEARANCE OF PERSISTENT SARS-COV2 INFECTION WITH ANTIBODY CONTAINING PLASMA IN LEUKEMIA AND LYMPHOMA PATIENTS

M. Janssen 1,* , A. Leo 2 , C. Wolf 2 , R. Bartenschlager 3 , A. Schmitt 1 , I. Haberbosch 1 , J. Brandt 1 , S. Sauer 1 , M. Zeier 4 , S. Meuer 2 , M. Schmitt 1 , P. Dreger 1 , R. F. Schlenk 1,5,6 , C. Müller-Tidow 1 , C. M. Denkinger 7

1 Department of Internal Medicine V, Heidelberg University Hospital; 2 Institute for Clinical Transfusion Medicine and Cell Therapy Heidelberg; 3 Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital; 4 Division of Nephrology, Heidelberg University Hospital; 5 NCT-Trial Center, National Center of Tumor Diseases, Heidelberg University Hospital; 6 German Cancer Research Center; 7 Division of Tropical Medicine, Department of Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany

Background: Patients with hematological malignancies or other reasons for inadequate immune response are at high risk for poor outcome of COVID-19 disease. In addition, a significant fraction of patients unable to mount an antibody response fail to clear SARS-Cov2 virus putting them at risk for clinical deterioration. Also, persistent COVID-19 infection impedes further treatment of the underlying disease. So far, no treatment has been established for persistent COVID-19 infection in immunocompromised patients.

Aims: Here, we treated eight immunocompromised patients with active SARS-COV2 infection with plasma from convalescent or/and vaccinated donors in a case series of individual treatments.

Methods: Patients were eligible for treatment if active SARS-Cov2 infection was confirmed at least 14 days after initial detection. In two out of eight patients sequencing results confirmed the omicron variant. Therapies before plasma treatment included antivirals and monoclonal antibodies. Underlying diseases were B-ALL (n=3; including one patient after allogeneic stem cell transplantation), multiple myeloma (n=2), lymphoma (n=2) and immunosuppression after kidney transplantation (n=1). Median age was 48 years (range 7 – 78).

Antibody containing plasma was obtained from healthy donors either after confirmed SARS-COV2 infection or after mRNA vaccination. Overall, 22 treatment cycles were administered in 8 patients (median 2, range 1 to 7). For each patient the available plasma with the highest titers of neutralizing antibodies and matching blood type was administered.

Results: Neutralizing antibody titers in plasma were median 1:320 (range 1:40 to 1:2560). Titers of neutralizing antibodies in convalescent plasma (n=10 treatments; median titer: 1:80) were lower than in vaccinated donor plasma (median 1:320; p=0.058, Mann-Whitney U test) (n=12 treatments).

Median Ct-values of viral load before treatment were 20.5 (range 14.9 to 37.3). After treatment (median 5 days), the Ct-values changed to median 26.9 (range 19.5 to non-detectable). The median fold decrease in viral load was estimated to be 127-fold with a range from 0.8 to >2x10 4 (p<10 -4 , paired t-test). Similar reductions in viral load were achieved after administration of either plasma from convalescent (median 109) or vaccinated donors. SARS-COV2 virus was cleared in 7 of 8 (87.5%) patients with ongoing treatment in one patient. No specific adverse events were observed.

Figure 1: Fold reduction in viral load after a single treatment with anti-SARS-COV2 containing plasma.

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Summary/Conclusion: Plasma with high titers of neutralizing SARS-COV2 antibodies can clear persistent infection in patients with hematological malignancies and immunocompromised patients. Plasma obtained either from convalescent patients or vaccinated donors might constitute an effective and inexpensive treatment in this situation.

PB2271: GERMLINE LOSS OF RHOG IDENTIFIES ITS ROLE AS AN ESSENTIAL REGULATOR OF HUMAN LYMPHOCYTE CYTOTOXICITY

A. Kalinichenko 1,* , G. P. Casoni 2 , L. Dupré 3 , L. Trotta 4 , J. Huemer 1 , D. Galgano 2 , Y. German 3 , B. Haladik 1 , J. Pazmandi 5 , M. Thian 1 , Ö. Yüce Petronczki 1 , S. C. Chiang 2 , M. Taskinen 6 , A. Hekkala 7 , S. Kauppila 7 , O. Lindgren 7 , T. Tapiainen 7 , M. J. Kraakman 1 , K. Vettenranta 8 , A. J. Lomakin 1 , J. Saarela 4 , M. R. Seppänen 8 , Y. T. Bryceson 2 , K. Boztug 1

1 St. Anna Children’s Cancer Research Institute (CCRI), Vienna, Austria; 2 Karolinska Institutet, Stockholm, Sweden; 3 INFINITy (Toulouse Institute for Infectious and Inflammatory Diseases), Toulouse, France; 4 Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland; 5 Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria; 6 8Oulu University Hospital and University of Oulu; 7 Oulu University Hospital and University of Oulu, Oulu; 8 Rare Disease and Pediatric Research Centers, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

Background: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of severe hyper-inflammation characterized by immune dysregulation and massive, aberrant activation of cytotoxic T cells and macrophages. While the molecular pathologies underlying secondary HLH are diverse, the primary or monogenic form of HLH is caused by the defects selectively disrupting perforin-mediated cytotoxicity in human lymphocytes.

Aims: In our study we investigated the genetic etiology of and molecular pathomechanisms of the disease in a patient with for early-onset HLH.

Methods: We applied a combination of state-of-the-art genetic and -omics technologies, including whole-exome sequencing (WES) and pharmacological screening combined with a wide range of cellular and molecular techniques to elucidate pathways underlying HLH pathology.

Results: We investigated a patient with early-onset HLH who fulfilled all diagnostic criteria who was wildtype for mutations in any of the known HLH genes. We identified biallelic germline mutations in RHOG . Using genetic ablation of the RHOG in a model cell line and primary cytotoxic T lymphocytes (CTLs) from healthy individuals, we confirmed its crucial role in lymphocyte cytotoxicity. To decipher the underlying molecular pathomechanism, we defined the molecular partners of RhoG in human lymphocytes and discovered that RhoG interacts with the regulator of cytotoxic granule (CG) release Munc13-4 inducing docking of Munc13-4-positive CGs to the plasma membrane. This step is required for subsequent fusion of the membranes to release cytolytic cargo toward target cells. Using a pharmacological screening approach, we defined the molecular pathways perturbed in RhoG deficient lymphocytes and assessed in detail their impact on the cytoskeleton. Employing high-resolution microscopy, we showed that RhoG is essential for the reorganization of the synaptic F-actin and control of immune synapse architecture. We also demonstrated that cytoskeletal defects in RhoG deficient lymphocytes could be pharmacologically reconstituted. Thus, our results suggest a dual role of RhoG for CG exocytosis through regulation exocytosis and cytoskeletal machinery.

Summary/Conclusion: Collectively, our work discovers a novel Mendelian disease affecting human immune function and homeostasis, potentially representing familial HLH type 6; defines molecular pathomechanisms of the discovered disorder; and identifies a novel layer of exocytosis and cytoskeleton regulation in human lymphocytes.

PB2272: THERAPY AND HOSPITAL MORTALITY PREDICTORS IN HEMATOLOGICAL PATIENTS WITH 4 GRADE NEUTROPENIA AND CONCOMITANT CORONAVIRUS INFECTION

K. Kaluzskaya 1,* , Y. Polyakov 1 , E. Baryakh 1,2,3 , E. Gemdzhian 4 , E. Misyurina 1 , E. Zhelnova 1 , K. Yatskov 1 , O. Kochneva 1 , T. Chudnova 1

1 Moscow City Clinical Hospital 52; 2 Russian Medical Academy of Continuous Medical Education; 3 Federal State Budgetary Educational Institution of Higher Education “Russian National Research Medical University named after N. I. Pirogov” of the Ministry of Health of Russia; 4 National Research Center for Hematology, Moscow, Russia

Background: According to published data, cancer patients are 5 times more likely to contract coronavirus infection (COVID-19) than non-cancer patients.

Aims: To assess and identify predictors of hospital mortality in hematological patients with grade 4 neutropenia and concomitant coronavirus infection.

Methods: A prospective study included 130 patients (55% women, 45% men) aged 18 to 91 years (median 59) with acute leukemia (AL) and lymphoproliferative diseases (LPD) who had grade 4 neutropenia and coronavirus infection (COVID-19) and were observed from April 2020 to December 2021 at Moscow City Clinical Hospital 52 (Russia). The study also included a reference group of 130 patients matched by sex, age and diagnosis, with AL/LPD and COVID-19, but without grade 4 neutropenia. The statistical analysis was an intention to treat analysis with adjustment for false discovery rate.

Results: AML prevailed in the AL group (80.6%), aggressive non-Hodgkin’s lymphomas in the LPD group. Distribution by disease status: 62 patients (47.7%) on chemotherapy (CT), 19 (14.6%) in remission, 23 (17.8%) with disease progression, 19 (14.6%) with newly diagnosed disease, 7 (5.3%) with a relapse. The patients had a high NEWS score (7.5) and a high Charlson comorbidity index (5). The majority had moderate or severe lung tissue damage (CT2 in 44 (33%), CT3 in 25 (20%), CT4 in 13 (10%)). There were increases in the levels of C-reactive protein (> 100 g/L in 55% of patients, mean 241.5 g/L), LDH (506 U/L), D-dimer (1866), procalcitonin (> 0.5 ng/mL in 73%). IL-6 and IL-1b blockers were used for hypercytokinemia; the dose of IL blockers was reduced in view of grade 4 neutropenia. Empiric antibiotic therapy for complications was used in 91.5% of patients. Antifungal therapy was carried out in 65% of cases. Sepsis developed in 63 (48.5%) patients. The median duration of hospitalization in patients with grade 4 neutropenia was 18 days. Transfer to the ICU was required in 78 (60%) patients in the total group and in 41 patients (52.5%) among those older than 60 years. Mortality in ICU patients: 80.7%, mortality in patients older than 60 years: 92.7%. In non-ICU patients with AL and LPD younger than 60 years, the mortality rate was 0%. Mechanical ventilation was used in 48.5% of cases. Mortality was lower in vaccinated patients than in unvaccinated ones (40% vs 51%). The mortality rates in the main group (AL 48.6%, LPD 51.7%, Fig. 1) and the reference group (AL 37.5%, LPD 13.8%, Fig. 2) showed that the presence of grade 4 neutropenia increased the risk of death almost twofold: the relative risk was 1.9 (95% CI 1.3–2.6), p=0.0001. According to the results of multivariate analysis (logistic regression), the predictors of hospital mortality in the main group were ICU stay (odds ratio 12.1 (1.4–24.0), p=0.0001), sepsis (3.0 (1.1–7.7), p=0.001), and age > 65 (3.0 (0.9–7.5), p=0.001). Severe disease progression/relapse is also a predictor of mortality (close to statistical significance).

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Summary/Conclusion: Coronavirus infection with severe neutropenia (caused by tumor progression and/or combination chemotherapy) is a significant adverse factor of hospital mortality in patients with hematological cancers. Predictors of hospital mortality are age over 60 years, sepsis, ICU stay, and the absence of remission.

PB2273: ANALYSIS OF THE HAEMATOLOGICAL PARAMETERS AND BLOOD PICTURE FINDINGS AND THEIR IMPACT ON THE DURATION OF STAY IN A COHORT OF PATIENTS WITH COVID 19 INFECTION IN A TERTIARY CARE HOSPITAL IN SRI LANKA

C. Kariyawasan 1,* , D. Gunaratne 1 , U. Samarasekara 1 , T. Fernando 1 , C. Ranatunga 1

1 Haematology, Sri Jayewardenepura General Hospital, Pannipitiya, Sri Lanka

Background: COVID-19 an infectious disease caused by the SARS-CoV-2 virus reveal a multi-systemic involvement, including the hemopoietic system. The haematological manifestations include blood count anomalies notably lymphopenia and neutrophilia which are of prognostic significance. Common viral infections often cause a leucopenia and frequently , thrombocytopenia. B acterial infections are typically associated with a neutrophil leukocytosis, and raised CRP. Studies on morphological changes in peripheral blood smears in COVID-19 and their impact on clinical severity are limited. FBC and a peripheral blood smear are inexpensive, easily performed, rapid tests which could add some novelty as effective markers of disease progress.

Aims: To analyze the full blood count (FBC) parameters and blood picture findings in patients with Covid 19 infection and identify the association of these parameters with the hospital stay of these patients.

Methods: A retrospective analytical study done during a period January 2021 – January 2022 at the haematology department of Sri Jayewardenepura General hospital. 154 Covid PCR positive patients were randomly selected and their data in relation to age, sex, FBC parameters, CRP, D dimers, blood picture findings and days of hospitalization were obtained from the hospital records and entered into a data extraction sheet and statistically analyzed using SPSS version 20 and Pearson’s Correlation with a P value of <0.05 considered statistically significant. Ethical clearance wa s Obtained from the hospital ethical committee

Results: The patients were in the age range of 15 to 95 years with a male preponderance of 2:1. The WBC count ranged from 2500/cu mm to 36,000/cu mm and counts < 4,000/cu mm were 12.3%. WBC counts were normal in 63.6% and high in 24%. Neutrophils were predominant but normal in 48.1% and 33.1% had neutrophilia. 96.8% revealed a lymphocyte count of <3,500/cu mm of which 52.6% had counts <1,500/cu mm. Thrombocytopenia was seen in only 38.3%. CRP was in the range of 10 -200 mg/L in 72% of patients. 53.8% revealed D dimer values ranging from100-5000 ng/ml with 13.6% having values greater than 5000ng/ml. 31.2% had no D dimer values. The blood pictures revealed a moderate rouleaux formation in 65.6%, toxic neutrophils in 85%, myelocytes in 37% and reactive lymphocytes in 55.8%. In 68.8%, comment was a bacterial infection and viral infection in 5.2%. Regards to hospital stay 3 and 5 days was of the highest frequency (25% each). A positive correlation noted between the duration of stay and age of patient (P value 0.05) with a negative correlation between the stay and lymphocyte count (P value -0.024). A positive correlation observed between WBC count and neutrophils (P value 0.00), with both demonstrating a positive correlation with platelets, CRP and D dimer, having P values of, 0.00,0.00 and 0.012/0.010 respectively with a negative correlation between WBC and lymphocytes (P value -0.025).

Summary/Conclusion: Our study revealed, the FBC, blood picture and other parameters in COVID 19 depict a bacterial infection with predominance of neutrophils, lymphopenia, high CRP and normal to high platelet counts. The negative correlation between hospital stay and lymphopenia was significant, which could be considered a poor prognostic marker.

PB2274: A PROGNOSTIC MODEL TO PREDICT MORTALITY IN ICU DEPARTMENT OF HEMATOLOGY CLINIC

A. Luchinin 1,* , A. Lyanguzov 1

1 The Federal State-Financed Scientific Institution Kirov Research Institute of Hematology and Blood Transfusion under the Federal Medical Biological Agency (KRIHBT), The Federal State-Financed Scientific Institution Kirov Research Institute of Hematology and Blood Transfusion under the Federal Medical Biological Agency (KRIHBT), 610027, Kirov city, Russia

Background: The ICU department’s mortality rate is one of the most important indicators of quality of care. The prognosis of mortality is a part of clinical, social, and economic workflows in any clinic, but its measure is a very difficult task that depends on several factors.

Aims: Based on real clinical data from the years 2018-2021 to create a prognostic model of mortality for patients with blood diseases in the ICU.

Methods: The study included 202 patients in total. The median age was 57 (19-82) years. There were 112 males and 90 females with the following distribution by diagnosis: Hodgkin lymphoma - 12 patients, non-Hodgkin lymphoma and CLL - 40, multiple myeloma - 24, MDS - 12, acute lymphoblastic leukemia - 18, acute myeloid leukemia - 82, chronic myeloproliferative diseases - 6 and other - 2. In all cases, patients were placed in the ICU due to severe conditions infection during disease progression or chemotherapy. In order to calculate the odds ratio (OR), we used a binomial logistic regression approach. A total of 4444 variables were processed for a matrix representation of both the training and the test dataset (80/20 split). The training data included 21 predictors: sex, age, fever, respiratory rate, systolic blood pressure, diastolic blood pressure, mean blood pressure, heart rate, need to inotropic support (Yes/No), blood hypoxemia, HGB level, PLT level, WBC level, creatinine level, CRP level, serum total protein level, serum albumin level, blood total bilirubin, blood procalcitonin, bloodstream infection detection (Yes/No) and Glasgow scale (<>15 scores). The dependent variable was death during the hospitalization in the ICU. All statistical analyses were undertaken using R version 3.4.2 and the “missForest”, “glmnet”, “caret”, “pROC” packages.

Results: The absolute risk of death (mortality rate) was 67 from 202 (33%), odds – 0.496. We performed all steps according to assumptions for logistic regression including missing data imputation, multicollinearity test, regularization for feature selection and log numeric data with outliers (log base 2). Our model for “high-risk of death” detection classified patients in the test dataset was with AUC 0.816 accuracy (95%CI 0.679-0.912, McNemar’s Test p-value = 0.5), with sensitivity 0.80 and specificity 0.82. We received a pretty accurate and balanced model with 3 predictors: Glasgow scale (<>15), blood PLT level, and serum total protein level. The equation of the model is Y(probability of death) = 14.13 - 0.28*log2(PLT) -2.37*log2(total protein) + 2.98*1 (if Glasgow score <15 scores) / 0 (if Glasgow score = 15). The odds of death in ICU grow up to ~2 times if the patient has a Glasgow score of less than 15. Also, the odds of death increase by 1.3 and 10.7 times of PLT, or serum total protein level decreases by 2 times accordingly.

Summary/Conclusion: Our research revealed the most important predictors of mortality in patients with blood diseases. They are Glasgow score (represents the level of consciousness), PLT level on CBC (represents the bone marrow failure due to blood disease or chemotherapy), and serum total protein level (represents liver function and overall metabolism in the body). The information received can be used in clinical practice and in developing clinical decision support systems.

PB2275: IMPACT OF EARLY FEVER ONSET AND NOVEL ANTIBIOTICS PROLONGED FEBRILE NEUTROPENIA OUTCOME FOLLOWING AUTOLOGOUS STEM CELL TRANSPLANTATION. SINGLE CENTER 10-YEAR RETROSPECTIVE STUDY

U. Markovic 1,2,* , G. A. Milone 1 , V. Iachelli 3 , P. Scire 1 , G. Amato 1 , F. Galbo 1 , E. Martorana 4 , D. Carco 3 , G. Moschetti 1

1 Division of Oncohematology and BMT, Istituto Oncologico del Mediterraneo, Viagrande; 2 Department of Biomedical, Dental, Morphological and Functional Imaging Sciences, University of Messina, Messina; 3 Unità Operativa di Laboratorio Analisi Chimco Cliniche; 4 IOM Ricerca: unità di genomica e oncologia molecolare, Istituto Oncologico del Mediterraneo, Viagrande, Italy

Background: Even though early transplant-related mortality following autologous stem cell transplantation (ASCT) is extremely low, infections represent one of the leading causes of death in the first 30 days. Prolonged period of neutropenia and engraftment with granulocyte colony stimulating growth factors can also cause fever even in the absence of infectious episode.

Aims: The aim of our study was to identify predictive factors of fever outcome in patients that had febrile neutropenia following ASCT that lasted more than 72 hours, had a documented infection and/or was unresponsive to empirical antibiotics.

Methods: We retrospectively evaluated data records from 482 patients that underwent ASCT from January 2011 until October 2021 in our Institute. Two hundred twenty-seven patients (47%) had an episode of febrile neutropenia during hospitalization, with 88 patients having fever for more than 72 hours of or documented infection site, that were analyzed as the study cohort.

Results: Median age of the study population was 56 years (range 19-70), with 64% being male. Patients were mainly affected by multiple myeloma (40%) and lymphoma. Median treatment line prior to ASCT was two (range 1-5) with 42 patients being in first line, while around 40% was treated with rituximab previously. Twenty-six patients (30%) had previous infectious episode, while thirty-eight (43%) had very good partial response or better to baseline disease prior to ASCT.

Median day of fever onset following ASCT was five (range 1-12), with median fever duration of four days (range 1-16). Seven patients had positive rectal swab for carbapenem-resistant enterobacteriaceae (CRE) at hospital admission. Half of the population had grade 3 or higher of mucositis, nearly two-third of patients had more than 39°C of body temperature at least once, and median peak number of daily fever spikes was three (range 1-5). In thirty-four episodes of febrile neutropenia at least one blood culture resulted positive, respectively 28 and 6 for gram negative and gram positive bacteria. In sixteen patients with positive blood culture the same agent was isolated previously, while three patients had CRE in the blood culture.

Median peak C-reactive protein (CRP) value was 20.1 mg/dL, and CRP value greater than 10 mg/dL was present in a median of 4.5 days. CT scan evidenced either signs of pulmonary consolidation, ileotyphlitis or sinusitis in 35% of the cohort. Novel antibiotic agents, namely ceftolozane/tazobactam and ceftazidime/avibactam, were available in half of the population during fever episode and it was used in twenty-one patients (24%). Eleven patients suffered from septic shock (13%), with seven patients (8%) passing away.

Wilcoxon Rank-Sum test demonstrated association between overall survival and both prolonged fever duration (p=0.0002) [Figure 1A] and number of days with CRP greater than 10 mg/dL (p=0.004). Fever duration was impacted by ceftolozane/tazobactam and ceftazidime/avibactam treatment (p=0.04), and documented infection site on CT scan (p=0.009). Finally, prolonged fever duration was associated early fever onset following ASCT [Figure 1B] and number of days with CRP greater than 10 mg/dL using Spearman correlation analysis.

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Summary/Conclusion: Although fever onset following ASCT was present in half of the patients in our population, in less than 40% duration was more than 72 hours. Clinical and laboratory characteristics, such as early fever onset following ASCT, can be of aid in identifying potentially life-threatening infections. The availability of novel antibiotics could improve the outcome in this setting of patients.

PB2276: EARLY ADMINISTRATION OF REMDESIVIR PLUS CONVALESCENT PLASMA THERAPY IS EFFECTIVE TO TREAT COVID-19 PNEUMONIA IN B-CELL DEPLETED PATIENTS WITH HEMATOLOGICAL MALIGNANCIES

L. I. Pinczes 1,* , F. Magyari 1 , E. Páyer 1 , K. Farkas 1 , S. Ujfalusi 2 , Á. Diószegi 2 , A. Deme 3 , M. Sik 4 , G. Nagy 5 , Z. Hevessy 6 , B. Nagy Jr. 6 , Á. Illés 1

1 Division of Hematology, Department of Internal Medicine; 2 Division of Endocrinology, Department of Internal Medicine; 3 Department of Internal Medicine; 4 Department of Radiology; 5 Department of Emergency Medicine; 6 Department of Laboratory Medicine, University of Debrecen, Debrecen, Hungary

Background: Patients with hematological malignancies are at a higher risk of developing severe form and protracted course of COVID-19. Remdesivir, an inhibitor of RNA polymerase was identified early as a promising therapeutic regimen for COVID-19, while convalescent plasma (CP) therapy with anti-SARS-CoV-2 antibodies can aid the treatment of COVID-19 in B-cell depleted patients.

Aims: Here we investigated whether the combination of externally administered immunoglobulins (Ig) via CP therapy and specific inhibition of viral replication might be sufficient to effectively treat B-cell depleted patients with COVID-19.

Methods: We enrolled a series of 20 consecutive patients who suffered from various hematological malignancies with profound B-cell lymphopenia and COVID-19 pneumonia between December 2020 and May 2021. These patients demonstrated a profound B-cell lymphopenia based on flow cytometry analysis of peripheral blood lymphocyte subpopulations. Consequently, they showed undetectable baseline anti-SARS-CoV-2 Ig levels before CP therapy according to anti-SARS-CoV-2 nucleocapsid- and S1-RBD-specific total Ig tests. Each patient received a complete course of remdesivir and at least one unit (200 ml) of AB0 compatible convalescent plasma during their treatment for COVID-19.

Results: Previous anti-CD20 therapy resulted in a more prolonged SARS-CoV-2 PCR positivity compared to other causes of B-cell lymphopenia (p=0.004). Timing of CP therapy showed a significant impact on the clinical outcome. Simultaneous use of remdesivir and CP reduced time period for oxygen weaning after diagnosis (p=0.017), length of hospital stay (p=0.007), and PCR positivity (p=0.012) compared to patients who received remdesivir and CP consecutively. In addition, time from the diagnosis to CP therapy affected the length of oxygen dependency (p<0.001) and hospital stay (p<0.0001). In those cases where there were at least ten days from the diagnosis to plasma administration, oxygen dependency was prolonged vs. patients with shorter interval (p<0.001).

Summary/Conclusion: Via combined administration of remdesivir and CP, we experienced an overwhelming survival rate in COVID-19 patients with hematological malignancies. Inhibition of viral replication with passive immunization proved to be efficient and safe. Our results suggest the clear benefit of early administration of CP to avoid protracted COVID-19 disease in patients with B-cell lymphopenia.

PB2277: ANALYSIS ON POLLUTION STATUS AND INFLUENCING FACTORS OF NURSES’ UNIFORMS IN HEMATOLOGY GENERAL WARD

X. Du 1 , L. Wang 2 , W. He 3 , Q. Li 4 , L. Tang 5 , X. Feng 1 , P. Zhou 1 , N. Zhao 1 , X. Zhang 1 , C. Song 6,*

1 Medical center of hematology; 2 Nursing department; 3 Department of gastroenterology; 4 Internal Medicine-Neurology; 5 Operation room; 6 Department of President Office, Xinqiao hospital Army Medical University, Chongqing, China

Background: In clinical work, nurses are the direct caregivers of patients, and they need to cooperate with doctors to observe patients’ conditions and carry out a number of clinical operations. Therefore, the contamination of nurses’ uniforms should be taken into account by hospital infection control workers. Contaminated nurses’ uniforms will reduce the quality of nursing and affect the treatment and rehabilitation of diseases, especially the hematological tumors patients who with low immunity. According to the study, nurses should change cleaning.

Aims: Investigate and analyze the contamination of nurses’ uniforms after different working hours in hematology general ward, in order to providing some references to improve the quality of hospital infection management.

Methods: From July to November 2021, front-line clinical nurses in the hematology general ward of a tertiary hospital in Chongqing were selected as the research objects by convenience sampling method. Samples were taken from different parts of the nurse at 8 different times, which including when changing clean uniform, and 1h, 3h, 6h, 2d, 3d, 4d and 5d after changing clean uniforms.

Results: In this study, a total of 264 qualified specimens were collected from 33 nurses’ uniforms. Descriptive analysis showed that the bacterial colony counts of cuff, abdomen and front chest at the 6th hour after changing clean uniforms were (11.30±3.56) CFU/cm 2 , (8.79±2.33) CFU/cm 2 , and (11.06±2.71)CFU/cm 2 and the qualified rates of cleanliness were 0, 9.1% and 3.0% respectively. Multiple linear regression analysis showed that, at the 6th hour after changing clean uniforms, the main influence factors of the bacterial colony count on the cuffs were work times, environmental humidity and the height of nurse, the fore chests were work times and the height of nurse, and the abdomens were work times, the total number of patients and the number of first-grade patients (all P <0.05). After 5 days, the main influence factors of the bacterial colony count on the cuffs were work times and the total number of patients, the fore chests were work times and the height of nurse, and the abdomens were work times and the total number of patients (all P <0.05).

Summary/Conclusion: It is necessary for nurses in general hematology department to increase the frequency of changing nurses’ uniforms and improve the cleanliness to ensure the nursing quality and patient safety. For nursing managers, it is essential to optimize the allocation of human resources further and provide necessary infection protection equipment to reduce the risk of hospital-acquired infection caused by the serious pollution of nurses’ work clothes.

PB2278: INVESTIGATION ON THE STATUS OF INFECTION IN PATIENTS WITH MULTIPLE MYELOMA

D. Lan 1 , L. Shi 1 , L. Gao 1,*

1 Medical center of hematology, Xinqiao hospital Army Medical University, Chongqing, China

Background: multiple myeloma; infection; clinical features; predisposing factors

Aims: To preliminarily explore the clinical features, distribution of infection sites and susceptibility factors of infection in patients with multiple myeloma.

Methods: he clinical data of 505 patients with multiple myeloma who were treated in our hospital from January 2015 to October 2021 were retrospectively analyzed, and they were divided into infection group and control group according to whether infection occurred. The baseline data, clinical characteristics, infection site, and blood biochemical indexes during infection were collected and analyzed.

Results: Among the 505 patients, 109 patients had infection, and the infection rate was about 21.6%. Lung was the main infection site in 53 cases, followed by 6 cases of urinary system infection, 3 cases of skin infection, 2 cases of perianal infection, and 45 cases with no clear infection foci. In the distribution of pathogenic bacteria, a total of 13 gram-negative bacteria, 9 gram-positive bacteria, 13 fungi and 1 virus were detected. Males and patients with education below high school were more susceptible to infection (P<0.05).

Summary/Conclusion: The incidence of infection in patients with multiple myeloma is about 21.6%. The lung is the main infection site, and gram-negative bacteria are the main infection bacteria. Male and low education are the susceptibility factors for infection.

PB2279: REDUCED ABSOLUTE COUNT OF MONOCYTES IN PATIENTS CARRYING HEMATOLOGICAL NEOPLASMS AND SARS-COV2 BREAKTHROUGH INFECTION

A. Romano 1,2,* , C. Conticello 2 , S. Filetti 2 , A. Bulla 2 , A. Chiarenza 2 , V. Del Fabro 2 , S. Leotta 2 , U. Markovic 2 , G. Motta 2 , M. Parisi 2 , F. Stagno 2 , G. A. Palumbo 2 , F. Di Raimondo 2

1 Dipartimento di Chirurgia e Specialità Medico Chirurgiche, Università degli Studi di Catania; 2 UOC Ematologia, AOU Policlinico Rodolico San Marco, CATANIA, Italy

Background: Clinical course of COVID-19 depends on several patient-specific risk factors, including immune function, that is largely compromised in cancer patients.

Aims: To identify predictors of outcome in COVID-19 breakthrough infection in patients carrying hematological neoplasms.

Methods: We prospectively evaluated clinical outcome of 120 consecutive adult patients (including 34 cases of COVID-19 breakthrough after two full doses of BNT162b2 vaccine) with underlying hematological malignancies and a SARS-CoV-2 infection.

Results: The median value of anti-SARS-CoV-2 antibodies (IgG) titer was 4.7 (range 1.1–38.7 BAU), as measured at one month from the documented viral clearance in 66/86 unvaccinated patients, showing that 42/66 (64%) patients were seroconverted.

Among the fully vaccinated patients, the anti-SARS-CoV-2 antibodies (IgG) titer was 5.2 (range 0–13.1 BAU) in 24/34 tested patients, showing that none of them achieved a protective titer (>40 BAU) at one month after two doses of BNT162b2 vaccine.

Among fully vaccinated patients the achievement of viral clearance by day 14 was more frequent than in unvaccinated patients. Increased 30-day mortality was associated with presence of active/progressing disease and absolute monocyte count lower than 400 cells/uL.

In unvaccinated patients of our series, the median OS was 10.4 months (95% CI. 9.6–11.3), which was affected in univariate analysis, by presence of: active disease ( p = 0.003), pre-infection absolute count of monocytes less or equal to 400 cells/mmc ( p = 0.04), hospitalization due to COVID-19 ( p < 0.0001), lack of viral clearance at 14 days ( p = 0.005) and lack of seroconversion ( p = 0.04). The difference in survival was consistent throughout all subgroups tested, independent of whether the patients were aged below or above 70 years, of female or male sex, or suffered from myeloid, lymphoid or plasma cell neoplasm.

Following PSM there was no statistical difference in 30-day mortality, rate of hospitalization for intensive cure and viral clearance at 14 days between fully vaccinated and unvaccinated patients, where adjusted odds ratios (AOR) and 95% confidence intervals (CI) have been reported. Increased 30-day mortality was associated with the presence of active/progressing disease and absolute monocyte count lower than 400 cells/uL (presence of non-active disease versus active/progressing disease: AOR −0.64, 95% CI: −1.09–0.2; absolute monocyte count (AMC) less than 400 cells/uL versus AMC ≥ 400 cells/uL, AOR 0.68, 95% CI: 0.14–1.21).

Results of multivariable analysis of OS showed that the pre-infection absolute count of monocytes less or equal to 400 cells/mmc ( p = 0.008), active or progressive disease of the underlying hematological malignancy ( p = 0.009), COVID-19 severity identified by hospitalization requirement ( p = 0.004) and lack of viral clearance at 14 days ( p = 0.03) were independent predictors of 1-year OS in unvaccinated patients affected by hematological neoplasms tested positive for SARS-CoV-2 infection.

Summary/Conclusion: Taken together, our results indicate that absolute monocyte count determined one month before any documented SARS-CoV-2 infection could identify patients affected by hematological neoplasms with increased risk of inferior overall survival.

PB2280: COVID-19 INFECTION IN PATIENTS WITH ACUTE LEUKEMIA

M. Shapetska 1,* , I. Iskrov 1 , I. Stoma 2

1 Minsk Scientifical and Practical Centre of surgery, transplantology and hematology, Minsk; 2 Gomel State Medical University, Gomel, Belarus

Background: Due to disorders in the immune system associated with the manifestations of the oncohematological disease and specific therapy, against the background of the development of secondary immunodeficiency syndrome, patients with acute leukemia are at high risk of infection during the COVID-19 pandemic. Patients with hematological malignancies tend to have higher rates of COVID-19 complications and higher mortality (30% to 62%) compared to patients in the general population.

Aims: Evaluate overall survival in patients with acute leukemia and COVID-19 infection.

Methods: Prospective cohort study of 26 patients with acute leukemia and COVID-19 infection confirmed by real-time PCR (nasopharyngeal and oropharyngeal swab). Indications for diagnosis of COVID-19: fever, lung symptoms (cough, dyspnea),screening. The severity of the infectious process was defined as: asymptomatic; mild - pneumonia without oxygen support; severe - severe pneumonia with SpO2

<92% and oxygen support; extremely severe - ARDS, the need for mechanical ventilation, sepsis, organ failure. In accordance with the clinical and hematological status for acute leukemia, all patients were divided into 2 groups: remission and no remission. Outcomes are defined as alive and dead.

Results: The observation group consisted of 26 patients with a confirmed diagnosis of acute leukemia and COVID-19 infection in period from April 2020 to October 2021. Men - 18 (69.2%) and women 8 (30.8%), ratio 2.3:1. The median age was 62 years. The main oncohematological pathology is represented by ALL - 6 (23.1%); AML - 16 (61.5%), mixed type acute leukemia - 4 (15.4%). In 4 (15.3%) patients, COVID-19 infection was asymptomatic, 7 (26.9%) patients had mild, severe in 6 (2.31%) and extremely severe in 9 (34.7%) patients. In accordance with the clinical and hematological status for acute leukemia at the time of the infectious process, 11 (42.3%) patients were in remission, 15 (57.7%) patients had no remission. Polychemotherapy in terms from several days to several months was carried out in 22 (84.6%) patients, not in 4 (15.4%) patients.Of the 26 patients with acute leukemia and COVID-19 infection, 9 (34.6%) died, which accounted for almost 1/3. A greater number of patients with an unfavorable outcome were in AML - 9 (56.2%) patients out of 16, while in ALL all 6 (100%) patients were alive.There was a statistically significant decrease in overall survival in group no remission in patients with COVID-19 and acute leukemia (log-rank test, p=0.022).

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Summary/Conclusion: 1) More than 50% of patients with acute leukemia have severe or extremely severe COVID-19.2) Patients with AML are more susceptible to adverse outcome of COVID-19 infection compared to patients with ALL.3) Overall survival in patients with acute leukemia and COVID-19 infection in remission is statistically significantly higher compared to patients in non-remission status (log-rank test, p=0.022).

PB2281: HOW TO AVOID FALSE-NEGATIVE AND FALSE-POSITIVE COVID-19 PCR TESTING

I. Fevraleva 1,* , O. Glinshchikova 1 , A. Sudarikov 1

1 Department of Molecular Hematology, National Research Center for Hematology, Moscow, Russia

Background: Since early 2020 the most demanded molecular genetic test is real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2. However, up to 40% of test results for COVID-19 in the presence of clinical manifestations of the disease might be negative. A false-negative analysis means untimely treatment of the patient and the possible spread of infection from a non-isolated person. Despite the fact that there are many highly sensitive RT-PCR test systems for COVID-19 on the market, the search for new reliable approaches to increase the robustness of testing is still relevant.

Aims: To develop a PCR testing approach featured to minimize the risk of the false-negative results and to increase assay sensitivity and specificity by the use of multiple targets.

Methods: A cotton swab taken from the nasopharynx was placed in an Eppendorf tube with 300 μl of phosphate-buffered saline. The resulting suspension (100 μl) was taken for RNA isolation using the Riboprep Amplisens kit (InterlabService, Russia). Primer sequences for RT-PCR are shown in Table 1.

Results: The reason for a false-negative result might originate from any stage of the analysis: poor-quality or empty swab, poor RNA isolation, inactivation of reverse transcriptase or Taq polymerase in the test. To control the entire process usually control DNA or an RNA-containing virus (eg bacteriophage MS-2) is added to the sample before RNA isolation. This is sufficient to control RNA isolation, reverse transcription, and PCR amplification. However, improper swab application, handling, transporting, or even attempts to falsify the sample, cannot be excluded this way. Here we propose to include primers for the detection of human ABL1 gene RNA to control swab quality and integrity. Designed primers work with the cDNA of the ABL1 gene, not genomic DNA. Therefore achieved ABL1 gene amplification ensures that enough material containing host RNA was taken by the swab, RNA was nicely isolated, reverse transcribed, and PCR-amplified. For SARS-CoV-2 detection, we propose to use primers targeted to the conserved region of the nucleocapsid gene and the less conserved region of the spike gene (currently specific for the Omicron strain). The simultaneous appearance of three signals corresponding to the nucleocapsid, spike, and ABL1 gene indicates infection with the Omicron strain. The amplification of ABL1 gene and nucleocapsid only indicate other than Omicron infection. The appearance of ABL1 amplification only indicates a true negative result for SARS-CoV-2. All other variants are null and void.

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Summary/Conclusion: A system has been developed for multiplex PCR diagnostics of SARS-CoV-2, which makes it possible to eliminate errors leading to false-negative and false-positive results at all stages of analysis. This is accomplished by the presence of specific primers for human RNA, controlling proper swab application, handling, and all the stages of RT-PCR. Targeting diverse regions of SARS-CoV-2 increases the reliability of the results and makes it possible to identify the virus strain.

PB2282: HERPESVIRUSES REPLICATION DURING COVID-19 IN NON-HODGKIN’S LYMPHOMA PATIENTS.

K. Sychevskaya 1,* , S. Kravchenko 1 , D. Tikhomirov 1 , A. Korneeva 1 , A. Abdulpatahov 1 , R. Abdurashidova 1 , A. Keniaykina 1 , Z. Sarakaeva 1 , T. Tupoleva 1 , A. Misyurina 1 , F. Babaeva 1

1 National Research Center for Hematology, Moscow, Russian Federation (Russia), Moscow, Russia

Background: At the moment, the association between COVID-19 and herpesvirus infection has been shown for herpes simplex virus-1 and 2 (HSV 1-2) and herpes zoster (HZ). However, publications are limited to clinical cases of herpes clinical manifestation before or in period of acute COVID-19. So the problem is not sufficiently disclosed. Activation of other types of herpes viruses, as well as the frequency of asymptomatic replication have not been studied at all.

Aims: To study the probability of herpesviruses reactivation during COVID-19 via analyzing the frequency of HSV1-2, cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpes virus 6 (HHV6) replication in patients with PCR-proven COVID-19 infection and concomitant non-Hodgkin’s lymphomas (NHL).

Methods: The study included 15 NHL patients with PCR-proven COVID-19. During the period of positive DNA SARS-CoV-2 test preservation the PCR replication of herpesviruses DNA (HSV1-2, CMV, EBV, HHV6) in biological fluids (blood, saliva, urine) was studied.

Results: In all patients, COVID-19 manifested after chemotherapy cycle. Our cohort was treatment heterogeneous. The most frequent treatment options were R-DA-EPOCH or high-dose pulse immunochemotherapy. The frequency of herpesvirus DNA detection in at least one biological material was HSV1-2 – 20%, CMV – 13%, EBV – 20%, HHV6 – 47%, respectively. Total activation frequency of at least one herpesvirus was 80%. The clinical signs, mainly of viral stomatitis, were present only in 47% patients. In the same patient cohort herpesviruses replication in the history (before COVID-19), mainly during previous chemotherapy, was HSV1-2 33%, CMV 20%, EBV 33%, HHV6 67%, and overall 73%, respectively.

Summary/Conclusion: According our results the frequency of herpesviruses replication in NHL patients during COVID-19 is as high as 80%. However, this was comparable with 73% herpesviruses persistence during previous chemotherapy. Also, there was no significant difference between DNA copies number in the same patients before and during COVID-19. Therefore, the presented data do not indicate the direct causal relationship between COVID-19 and herpesviruses reactivation since almost in all cases there was a history of herpesvirus infection before. In addition, we should take in account the external chemotherapy trigger, equally potentiating both COVID-19 infection and herpesviruses reactivation. At the same time, our data are valuable as indicate high probability of parallell manifestation (for one reason or another) of COVID-19 and latent herpesvirus infection reactivation in NHL patients. So this fact should be considered in management of NHL patients with concomitant COVID-19. As we demonstrated subclinical herpesviruses replication without concomitant clinical signs, indicating underestimation of the real frequency herpesviruses reactivation in NHL patients with COVID-19, complex diagnostic approach is required including DNA herpesviruses evaluating. Neurological complications common after COVID-19 are predominantly referred to the direct neurotoxic effect of SARS-CoV-2. However, in the light of the presented data, we should not forget the possible concomitant herpetic neuropathy as the cause of neurological failure.

PB2283: COVID-19 (SARS-COV-2 INFECTION) IN LYMPHOMA PATIENTS

I. Tytorenko 1,* , I. Kryachоk 1 , O. Alexik 1 , T. Kadnikova 1 , A. Martynchik 1 , O. Novosad 1 , Y. Pastyshenko 1 , Y. Stepanishina 1 , N. Shokun 1 , K. Filonenko 1

1 National cancer institute, Kyiv, Ukraine

Background: Observations from initial case series suggested that cancer patients in general might have an unfavorable outcome following coronavirus disease 2019 (COVID-19), due to their underlying conditions and cytotoxic treatments. Lymphoma patients may be especially vulnerable, due to the immunodeficiency and immune dysregulation caused by the lymphoma itself and the antitumor treatments.

Aims: analysis of the flow of coronavirus disease (COVID-19) in cases of the patients with non-Hodgkin’s lymphoma and Hodgkin’s lymphoma on the background of specific treatment. Patients with lymphoma appear particularly vulnerable to SARS-CoV-2 infection, only partly because of the detrimental effects of the anti-neoplastic regimens (chemotherapy, pathway inhibitors, monoclonal antibodies) on the immune system.

Methods: The National Cancer Institute conducted a retrospective analysis of the course of the disease on COVID-19 from March 2020 to October 2021. The study included 87 patients. The average age of the patients was 30.4 years (from 23 to 75 years old). 60 patients had non-Hodgkin’s lymphoma, 19 patients had Hodgkin’s lymphoma, and 8 had multiple myeloma.

Results: Coronavirus disease in 85 % of the patients was confirmed by PCR, in 15% - by IFM (detected IgM). Severe cases of coronavirus disease were observed in 19.23 %, moderate severity - in 53.8% of the patients, light- in 26.9%. Covid-19 pneumonia was diagnosed in 74.71% of the patients. The duration of coronavirus disease ranged from 7 to 60 days, on average of 26.56 days, in cases of 22 patients (25.28 %) - more than 30 days. 18 patients (20.68%) died due to complications of coronavirus disease. Fatalities were more common among patients with DBCL - 29.16 %, and among patients with recurrent and refractory forms of the disease - 33.33 %. The type of anticancer therapy affected the outcome of the disease, mortality was more common among patients receiving chemotherapy RB and RdaEPOCH regimens, respectively 31.57 % and 40%. Among of toxicity after continued special treatment in 11.5 % patients were recorded complications - pulmonitis, 8 patients with thromboembolic complications, 1 patient developed pulmonary embolism. Patients after infection before the start of special treatment required a recovery period, which averaged 21.7 days, ranging from 7 to 60 days.

Summary/Conclusion: COVID-19 infection is of particular concern for morbidity and mortality of patients with lymphoma due to their immunodeficiency status and 11.5% caused by the disease itself and / or its treatment. Patients with lymphoproliferative diseases are more likely to have moderate COVID-19 disease, mortality from complications is higher than in cases of the population and makes up 20.68%.

PB2284: PROTECTION OF PATIENTS WITH HEMATOLOGIC MALIGNANCY FROM SEVERE INFECTION DURING PANDEMIA: ESTONIAN TWO CENTRES EXPERIENCE

I. Vaide 1,2,3,* , V. Aabrams 4 , E. Laane 2,3

1 Internal Medicin, Pärnu Hospital, Pärnu; 2 Internal Medicin, Sa Kuressaare Haigla, Kuressaare; 3 Hemato-Oncology Department, Tartu University. Institute for Clinical Medicine. Hematology-Oncology Clinic, Tartu; 4 Internal Medicin, Sa Pärnu Haigla, Pärnu, Estonia

Background: In the course of lymphoproliferative diseases, an immunodeficiency of humoral or cellular origin has been described. Infectious complications of common or atypical origin alter patients’ lifestyles or result in severe, life-threatening hospitalizations. Hematology patients are prone to opportunistic infections after viral infection. In Estonia, chemotherapy for haematological patients is mainly covered in two regional centers in Tartu (University Hospital) and in Tallinn. The outpatient consultations are also covered in other parts of Estonia. In times of pandemic, when lockdowns are changing the situation and it has been difficult to travel to large centers, local hematology care needs for high-risk hematology elderly patients are growing.

Aims: In the case of lymphoproliferative diseases, a secondary immune deficiency with severe infections can develop during the course of therapy, which is objectively discovered by immunoglobulin testing (IgG), and replased by immunoglobulin substitution. According to Kreuth IV, the value for prophylactic IgG replacement therapy is below 4g/l

Methods: Review of the 20 case records of the two hospitals in Estonia, in Pärnu and in Kuressaare.

Results: There are in regulare treatment or watching in Pärnu center and in Kuressaare center about 80 patients with lymphoproliferative disease. From them needed according to IgG status below 7(ref 7-16) 20 patients. The mean age of patisents is 70.8 (range 38-89). The mean basic IgG (ref 7-16) in patients in Immunoglobulin treatment is 4,49 (range 3.3-6).The intervall for prophylactic replasment was 4.6 or 8 weeks in 20 patients. From those 8 patients had SARS-Cov2 during this period, no patient was dying because of SARS-Cov2 infection and in 4 patients there was SARS-COV2 IgG pos later, in one patients SARS-COV2 IgG was negative afther the severe infection, in 3 patients is SARS-COV2 IgG not known afther frecovery from disease. Two patents died: one of secondary Pulm tumor related infection and one of sigmoidal ca, who had 6 months earlyer COVID19 severe infection. From 20 patients 9 diceided to have vaccination and 6 got no SARS-COVIgG antibodyes afther that. All patients had during 01.06.2019- 01.02. 2022 years in some period immunosupressive treatment.

Summary/Conclusion: We recommend the individualized protection of vulnerable hematologyc diseases patients with long-term illness. Secondary immunodeficient patients need during pandemic when hospital beds are overgrown immunoglobulin replasmenttherapy to prevent their unneccesary hospitalization.

PB2285: CLINICAL EXPERIENCE WITH CEFTAZIDIME-AVIBACTAM FOR THE TREATMENT OF BACTEREMIA DUE TO CARBAPENEM-RESISTANT GRAM-NEGATIVE BACTERIA IN HEMATOLOGIC PATIENTS

S. Zhen 1,* , S. Feng 1 , W. Zhai 1

1 Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, tianjin, China

Background: Experience on effectiveness of ceftazidime-avibactam (CAZ-AVI) for bacteremia caused by carbapenem-resistant gram-negative bacteria in hematologic patients is scare.

Aims: We conducted this cohort to evaluate the effectiveness and safety of CAZ-AVI for this population, and also, to explore the predictors of clinical outcome.

Methods: We performed a retrospective, observational study at a blood diseases hospital in China for patients who received ≥ 72 h of CAZ-AVI for bloodstream infections due to carbapenem-resistant Enterobacteriacea and Pseudomonas aeruginosa between November 2018 and June 2021. Clinical and microbiological characteristics of these patients were collected; clinical and microbiological outcomes, occurrence of adverse effect were assessed. We compared the survival between transplant and nontransplant patients, also between patients who did or did not undergo granulocyte recovery within 14 days. We used Kaplan-Meier survival estimates to generate survival curves. To identify predictors of survival and clinical success, univariate and multivariate analysis was performed.

Results: Forty-seven patients were eligible for our study. Forty (85.1%) patients received combination therapy, while seven received CAZ-AVI monotherapy. All patients infected with MBL (metallo-β-lactamase)-CRE (carbapenem-resistant Enterobacteriaceae ) (n=23) received combination therapy with aztreonam. The survival at 28 days was 83.0% in 47 patients, and was 91.3% (21/23) in patients with MBL-CRE bacteremia. Clinical success was observed in 35/47 (74.5%) patients. Multivariate analysis revealed the most significant factor of clinical success was the granulocyte recovery within 14 days.

Summary/Conclusion: CAZ-AVI-containing regimen is an effective option for the bacteremia due to carbapenem-resistant Gram-negative bacteria among hematologic patients, even in those infected with MBL-CRE bacteremia. Appropriate combination therapy is important after acquiring the information of enzymes and susceptibility to CAZ-AVI in vitro. Granulocyte recovery within 14 days may be a predictor of clinical success.

PB2286: INFLUENCE OF ANTI-CD20 MONOCLONAL ANTIBODY THERAPY ON THE OUTCOME OF CORONAVIRUS INFECTION COVID-19 IN PATIENTS WITH LYMPHOPROLIFERATIVE DISEASES

E. Zotina 1,* , E. Zhelnova 1 , E. Karimova 1 , E. Grishina 1 , E. Paramonova 1 , D. Gagloeva 1 , E. Baryakh 1 , E. Misyurina 1

1 Hematology, City Clinical Hospital №52, Moscow, Russia

Background: The use of monoclonal anti-CD20 antibodies in patients with B-cell non-Hodgkin lymphoma causes a deep and prolonged depletion of the number of B-lymphocytes in the peripheral blood and the suppression of the humoral immune response. In the context of the pandemic of COVID-19, the influence of anti-CD20 monoclonal antibody therapy on the outcome of a coronavirus infection is urgent.

Aims: To study the effect of anti-CD20 monoclonal antibody therapy on the outcome of coronavirus infection in patients with lymphoproliferative diseases.

Methods: The study included 210 patients with lymphoproliferative diseases treated at City Clinical Hospital №52 in Moscow from March 2020 to November 2021 for COVID-19. There were 117 men (56%) and 93 women (44%). The median age was 58 years (range, 20-92). Eighty-nine per cent of patients had B-cell non-Hodkin lymphoma. At the time of hospitalization 47% of patients were in a complete or partial remission of lymphoma, 18% had a relapse/refractory disease, 24% were at the stage of therapy, a lymphoproliferative disease was detected for the first time in 11%.

Results: One hundred sixty-two patients (77%) received anti-CD20 monoclonal antibody therapy before hospitalization, 92% of them received therapy with rituximab and 8% received therapy with obinutuzumab, 80% received anti-CD20 monoclonal antibody therapy for the last 12 months before hospitalization. It was found that among patients who received anti-CD20 monoclonal antibody therapy for the last 12 months, 35 patients (27%) died; 3 (9%) and 6 (13%) patients died, respectively (p=0,020) among ones who received anti-CD20 monoclonal antibody therapy over 12 months ago and who had not previously received this regimen of chemotherapy. In the group of patients treated with obinutuzumab, 6 (46%) died, and in the group of patients treated with rituximab, 28 (19%) died (p=0,004). When comparing the number of deaths in the groups that received anti-CD20 monoclonal antibodies as induction chemotherapy regimens (22%) and as maintenance therapy (19%), statistically significant differences were not found (p>0,05). Forty-five per cent of patients who received anti-CD20 monoclonal antibody therapy for the last 12 months had a longer persistence (more than 3-4 weeks) of the virus SARS-CoV-2 compared with the patients who received anti-CD20 monoclonal antibody therapy more than 12 months ago (22%) (p=0,038). The median hospital stay was 16 days (range, 2-53). During hospitalization 44 patients (21%) with lymphoproliferative diseases died. The 30-day in-hospital survival in patients who received anti-CD20 monoclonal antibody therapy within the last 12 months was 62%, in patients who received anti-CD20 monoclonal antibody therapy more than 12 months ago this was not achieved (p=0,042).

Summary/Conclusion: An increase in the number of deaths from COVID-19 and a longer persistence of the virus SARS-CoV-2 are experienced among patients with lymphoproliferative diseases who have received anti-CD20 monoclonal antibody therapy within the last 12 months before hospitalization; all this negatively affect in-hospital survival.

PB2287: END OF LIFE TRANSFUSIONS IN PATIENTS WITH AN ONCOLOGICAL DIAGNOSIS

L. Duque 1,* , A. P. Brome Uribe 1 , C. Durango Sánchez 2 , V. Vasquez Estrada 1 , L. M. Martínez Sánchez 1 , A. Morales Montoya 1 , J. F. Vargas Silva 3 , L. Herrera Almanza 1 , M. Fonseca Gúzman 1 , M. Saavedra Valencia 1 , S. M. Molina Naranjo 3

1 Universidad Pontificia Bolivariana; 2 Universidad Pontificá Bolivariana; 3 Hospital Pablo Tobón Uribe, Medellín, Colombia

Background: Cancer patients in terminal stages present symptoms related to anemia, other cytopenias and coagulopathies that deteriorate the quality of life. These symptoms can be multicausal and do not improve in most cases with a single medical intervention. Cancer patients in terminal stages present symptoms related to anemia, other cytopenias and coagulopathies that deteriorate the quality of life.1,2 These symptoms can be multicausal and do not improve in most cases with a single medical intervention.

Aims: To describe the use of blood transfusions in the 6 months prior to death as a palliative measure, in patients with an oncological diagnosis in the city of Medellin during the period 2013-2018.

Methods: Retrospective cohort, in patients with cancer diagnosis who received transfusions in the last 6 months of life, a non-probabilistic sampling of consecutive cases was performed. The analysis executed in SPSS, they were estimated; absolute and relative frequencies and median with interquartile ranges.

Results: Out of 3,254 medical records of cancer patients, 151 were included, of which 78 (51.7%) were male, with a median age of 64 years (IQR 52-75). The time to oncological diagnosis had a median of 2 years (IQR 1 and 3). Of the patients included in the study, 62.3% (94) had outpatient management and 37.7% (57) were hospitalized. Palliative treatment with blood products was used in the study patients during the 6 months prior to their death, with a median of 261.5 cubic centimeters (cc) (IQR 242 - 293) of doses of blood products and a median of 1 unit (IQR 1-2) transfused per patient.

Summary/Conclusion: The findings of this research allow to appreciate the current panorama in the institutions in which palliative care medical services are provided and the considerations taken into account when transfusing blood products to a patient at the end of life as a therapeutic measure in the context of a human being cared for by professionals specialized in palliative care.

PB2288: THE ABO SYSTEM POLYMORPHISM IN ALGERIAN BLOOD DONORS

F. talbi 1,* , D. damerdji 2

1 hematology, military hospital; 2 hematology, PTS BOUFARIK, algiers, Algeria

Background: knowledge of the prevalence of ABO-Rhesus globular antigens ensures the stock of the blood bank allows the rational use of blood and its derivatives.

Aims: assess the phenotypic frequencies of the ABO, Rhesus (D, C, c, E, e) and Kell systems in our population

Methods: ambidirectional study over 11 years (2008-2018), n= 10,977 voluntary blood donations in 08 establishments. Bio-rad® reagents used for ABO, RH, K.

Results: the number of blood donations is stable according to the quarter, p=0.25 (NS). The average age =34 years ±9.75 [18-71]. The most represented age group = 21-30 years: HR =1.94, p=.000 (s). Male donors n=9,778 (89%), sex ratio (M/F) =8.15. Compensation donors and occasional donors are evenly distributed between the sexes p=0.41 p=0.17 (NS) respectively. On the other hand, it is female donors who present themselves regularly for a blood donation as opposed to the male sex: (n=649 M, n=102 F): p=0.009 (S). We observed a trend increasing (different types of donations over time): HR=0.38, p=.000 (S). Blood donations are collected in 6 units, n=8136 by fixed teams and n=2839 by mobile fixed units. The phenotypic prevalences were: group O n=4666 (42.5%), group A (3825/35%), group B (1720/15.66%) and group AB (556/05%), the unidentified group n=210 cases. RHD (+) phenotype = 87%, Ag C (67%), c (65%) and e (36%), E (+) antigen = 18%, K (+) phenotype 10%. The predominant phenotype in our region is CceeK(-) then ccee-k(-)

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Summary/Conclusion: The data of our study agree with those of previous studies carried out in different national centers. The use of these data allows us to better manage our stock of blood products and the formation of alloantibodies against minor antigens in polytransfused patients.

PB2289: TRENDS IN ANTIGEN-NEGATIVE RED BLOOD CELL DISTRIBUTIONS ABOUT TEN THOUSAND THREE HUNDRED WHOLE BLOOD DONATIONS

F. Talbi 1,1,* , D. Djamel 2 , D. djameli 3

1 hematology, military hospital, algiers; 2 hematology, PTS Boufarik, Blida; 3 hematology, pts boufarik, algeria, Algeria

Background: The overall number of red blood cell (RBC) units distributed to hospitals throughout the world has significantly decreased lately.

Aims: The aim of this study is to determine the prevalence of rhesus D negative in the population of whole blood donors in a region of central AlgiersWe carried out a retrospective study over a period of 10 years (2008-2017) including blood donors listed at the Boufarik PTS. We looked for a significant association of the D phenotype with the following variables: sex, age group and blood group.

Methods: We carried out a retrospective study over a period of 10 years (2008-2017) including blood donors listed at the Boufarik PTS. We looked for a significant association of the D phenotype with the following variables: sex, age group and blood group. An antigen-negative unit was defined as a unit with a specific RBC phenotype that had been specially ordered as such by a hospital.

Results: Out of n=10,300 blood donations, n=210 bags (NP grouping, carried out at the level of the reception structure), the median age is 23 ±5.19 years, group O represents 41%, group A: 35 %, group B=16% and group AB:05%.

n=1129 RHD negative (11%), p<.000 (s): the distribution by sex (féminin = female, Masculin = Male) finds a non-significant threshold with the RHD neg: p=.25 (fig 1) and age: p=. 45 (fig 2) contrary to blood group: p<.000 (s) (fig 3)

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Summary/Conclusion: The present study reveals that the negative RHD represents a non-negligible sample (11%) with a significant association with certain blood groups (p<.000) and emphasizes the potential risk of alloimmunization since the methods Current blood typing tests do not identify “variant” RhD phenotypes.

PB2290: SEROPREVALENCE OF HEPATITIS B AND C VIRUSES, HIV-1/2 AND SYPHILIS AMONG BLOOD DONORS IN THE CENTRE REGION OF ALGERIA

F. talbi 1,* , D. djameli 2

1 hematology, military hospital, algiers; 2 hematology, pts boufarik, algeria, Algeria

Background: Prevention of blood transfusion-transmitted i infections i includes blood donor i screening i and effective infectious i markers screening for i donated blood.

Aims: This study i had two i main objectives: i to determine seroprevalence i of HBV, HCV, HIV and syphilis in i blood i donors of i Boufarik i post transfusion and to study i sociodemographic i factors of blood donors.

Methods: A iretrospective study i of consecutivei blood donors’ irecords from January 2008 ito December 2018 wasi conducted. Seroprevalence of hepatitis Bi, hepatitis Ci, humani immunodeficiencyi viruses iand isyphilis were determined in 10.300 wholei blood donationsi collected ini the centre region Algeriani

Results: The prevalence of serological markers HIV, HBS, HCV and syphilis is respectively 0%, 24%, 0.67 and 23%. Taking two comparative periods: 2008-2012 (n=4139 donations) and 2013-2017 (n=6161 donations): we note an increase in the number of positive cases for the hepatitis B virus: n=07 (2008- 2012) versus n=18 (2013-2017): RR =.69 (.37-1.30), p=.13 (NS) fig.1. An increase in the number of positive cases for the hepatitis C virus: n=02 (2008-2012) vs n=05 (2013-2017): RR =.89 (.21-3.73), p=.45 (NS) fig.2. A stable number of positive cases for the syphilis virus: n=12 (2008-2012) versus n=12 (2013-2017): RR =1.49 (.66-3.31), p=.16 (NS) fig. 3

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Summary/Conclusion: These results show that the prevalence of viral infections is stable over time among blood donors in our region. This is due to the establishment of a quality assurance system, the choice of collection site, the selection of donors and finally the improvement of the quality of screening which has contributed to guaranteeing optimal transfusion safety.

PB2291: STATUS OF IRREGULAR ANTIBODIES IN THE COVID ERA: A NEW PRESSURE FOR THE TRANSFUSION DEPARTMENT?

H. Fernandez Leyva 1,*

1 Haematology, NHS, Licoln, United Kingdom

Background: Despite the introduction of robust detection programs into clinical practice, alloimmunisation remains a problem, particularly in the context of increased transfusion requirements and RhD pregnancy-induced alloimmunisation. Antibody identification was performed only on patients who tested positive in the screening test. From the total number of patients who were screened for unexpected antibodies (1.01%) tested positive. The Rh blood group system antibodies were the most frequent, particularly anti-D. Alloimmunization to red cell antigens is still a current problem in our transfusion practice. It is obvious that the additional testing of blood donors for Rh and Kell antigens should be implemented as routine to prevent as far as possible the incidence of alloimmunization. It would also be cost-effective, bearing in mind the additional laboratory testing necessary to provide compatible blood for alloimmunized patients.

Aims: This research aims to assess the prevalence of unexpected antibodies and their correlation with several clinical conditions. Due to the high prevalence of acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the pressure of the transfusion department raised proportionally. Evaluation of ABO, RhD and blood group antigen frequencies in blood donors as well as the incidence of alloimmunization in transfused patients. The need for routine typing of certain blood group antigens in addition to ABO and RhD was also evaluated.

Methods: In this cross-sectional prospective study we evaluated data from 542 ABO/Rh and Kell typed blood donors undertaken from January 2020 to January 2022, as well as the data from pre-transfusion testing included ABO, RhD grouping, cross-matching, irregular red blood cell antibody screening and compatibility testing identification in the mention period. All tests were performed by the DiaMed tube gel system.

Results: The frequencies of ABO antigens were as follows: O (48.5%), A (29.9%), B (17.2%), AB (7.4%). The frequencies of Rh antigens were as follows: D positive. (85.3%), D negative. (13.6%), C (28.3%), c (32.4%), E (19.3%), e (37.8%). We found the following frequencies of Kell phenotypes: K+ k- (0.25%), K+ k+ (6.18%), and K- k+ (25.6%) with the total frequency of K antigen of 4.4%.

Antibody screening and/or cross-match were positive in the sera from 64 transfused patients. In 42 sera the following 81 antibodies were identified: anti-K (36), -E (5), -e (1), -C (2), -c (2), -C(w) (2), -k (1), -Fy (a) (3), -Fy(b) (1), -Jk(a) (3), -Lu(b) (1), -Le(b) (2), -Le(a) (1), -M (3), -P1 (1). The most frequent alloantibody was anti-K with 32%, and anti-E with 11.3% of all identified antibodies.

Summary/Conclusion: There was also a significant correlation between the unexpected antibodies and history of transfusion, pregnancy, and autoimmune diseases as P ≤ 0.05. The most prominent unexpected antibodies in the study belong to the Rh system (Anti-D). Extended blood typing is regularly implemented for some polytransfused patients. This strategy is a step forward to improve the safety of blood transfusion with optimal blood grouping. More to the point, because of the strong correlation between the unexpected antibodies as well as the history of transfusion, pregnancy, and autoimmune diseases, the highest safety criteria must be followed during the transfusion of blood to patients with these clinical conditions.

PB2292: AMSAN VARIANT OF GUILLAIN BARRE SYNDROME COMPLICATING THE RECOVERY PHASE OF COVID-19 RELATED DISEASE

A. M. Haidary 1,* , S. Noor 2

1 Haematopathology and Blood Bank, French Medical Institute for Mothers and Children; 2 Haemato-oncology, Ali Abad Hospital, Kabul, Afghanistan

Background: The novel coronavirus, since its first identification in China, in December 2019, has shown remarkable heterogeneity in its clinical behavior. It has affected humans on every continent. Clinically, it has affected every organ system. The outcome has also been variable, with most of the older patients showing grave outcomes as compared with the younger individuals. Here we present a rare and severe variant of Guillain-Barre syndrome that complicated the disease in recovery phase.

Aims: To inform about a rare and life-threathening complication of COVID19 that can be treated with timely institution of IVIG/ Plasmapheresis

Methods: Case reporting

Results: A 60-year-old Afghan man, who had been recovering from symptoms related to novel coronavirus associated disease, presented with sudden onset of progressive muscle weakness and oxygen desaturation. Electrophysiological workup confirmed the diagnosis of Guillain-Barre syndrome, and early institution of intravenous immunoglobulin resulted in complete resolution.

Summary/Conclusion: In our case report, we presented the rare and most severe variant of GBS, AMSAN, that complicated COVID-19-related disease in its recovery phase. To our knowledge, this was the first case of AMSAN complicating COVID-19. Clinical suspicion and prompt diagnosis led to early intervention, leading to complete recovery. While the world is entering the second wave of COVID-19, clinicians, particularly who manage old-age individuals, need to have insight into this dreadful but manageable complication of COVID-19-related disease.

PB2293: HARNESSING ACCELERATED SEQUENTIAL EVANS SYNDROME

M. Almakadi 1,* , D. Aldhafeeri 1

1 King Faisal Specialist Hospital and Research Center, Madinah, Saudi Arabia

Background: Evan’s syndrome (ES) is a rare autoimmune disorder characterized majorly by the co-occurrence of immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) with or without immune neutropenia. The ITP and AIHA commonly develop simultaneously, or sequentially within at least 8 months apart. The exact mechanism remains unclear; however, it is crucial to rule out secondary causes when formulating treatment plan. ES tends to be less responsive to therapy than isolated ITP or AIHA, and is usually associated with multiple relapses. Here we present a rare case of “accelerated” sequential ES.

Aims: To outline the features of a unique time course of sequential ES, and how early novel therapy can lead to better outcome.

Methods: Our 65-year-old male patient developed petechiae and wet purpura due to severe thrombocytopenia (platelets count 2 x10 9 /L). ITP was suspected, and secondary causes were ruled out. He was refractory to corticosteroids and gamma globulins (Figure solid curves ). On Day 21, he developed severe neutropenia with a nadir of 0.1 x10 9 g/L and was initiated on granulocytes-colony stimulating factor (data not shown), which triggered the diagnosis of ES. He was started on romiplostim, that was escalated weekly (Figure solid curves ). He subsequently developed severe autoimmune hemolytic anemia with hemoglobin nadir of 49 g/L almost 6 weeks into his presentation. Corticosteroids’ dose was increased, and rituximab at 375 mg/m 2 was added. It is interesting to note that both ITP and autoimmune neutropenia began to recover as the AIHA was progressing (Figure solid curves ). Expectedly, our patient relapsed with ITP seven years later (Figure dashed curves ). All secondary causes were ruled out, again. This prompted early initiation of romiplostim and rituximab. The patient did not develop autoimmune neutropenia and AIHA at his relapse. Unfortunately, he suffered gastrointestinal and intracranial bleedings that were managed conservatively. The ITP began to recover around 8 days earlier than initial presentation (Figure dashed curves ).

Results: Learning from our patient’s initial presentation, the strategy we adopted at relapse “harnessed” this patient’s ES triad by preventing the development of autoimmune neutropenia and AIHA, and shorted the time to platelets recovery, which we believe was delayed due to bleeding occurrences that will usually lead to platelets consumption.

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Summary/Conclusion: ES results in variable cellular compromises with heterogenous time courses. It is associated with poorer response to therapy than isolated ITP, AIHA, or autoimmune neutropenia, which results in worse morbidity and mortality. Our case exhibits a very rare phenotype of accelerated sequential ES with corticosteroids refractoriness that was managed successfully with early addition of romiplostim and rituximab. The lack of response to corticosteroids shall induce introduction of novel therapies sooner in the treatment course of suspected ES cases.

PB2294: CHEMOTHERAPY INDUCED THROMBOCYTOPENIA AND ITS ASSOCITAION WITH COAGULOPATHY; A SINGLE CENTRE EXPERIENCE

N. ANWAR 1,* , N. FATIMA 2 , A. KHALID 2 , L. MAJEED 2 , M. BORHANY 1

1 HEMATOLOGY; 2 RESEARCH AND DEVELOPMENT, NATIONAL INSTITUTE OF BLOOD DISEASES AND BONE MARROW TRANSPLANTATION, KARACHI, Pakistan

Background: Chemotherapy induced thrombocytopenia is perilous and may complicate disease treatment resulting in extended hospitalisation, bleeding, platelet transfusion and increase economic burden on the patients. Typically patients with hematological malignancies are at high risk of thrombosis and hemorrhage which may lead to poor outcome and overall survival. As coagulopathy in hematological malignancies is a predictor of thrombotic and bleeding complications, by adding the intellectual observation of platelet count, we may segregate patients and identify the susceptibility to either thrombosis or bleeding apparently. Our center is a referral institute for management of blood disorders with huge representation of patients with hematological malignancies.

Aims: The study was done to observe the association of chemotherapy induced thrombocytopenia with coagulopathy in patients with hematological malignancies.

Methods: This was a retrospective cohort study done at NIBD PECHS campus from 2020-2021. All the patients diagnosed with hematological malignancies receiving chemotherapy during the study period were included. Patients who had thrombocytopenia before chemotherapy were excluded. PT, APTT and platelet count, post chemotherapy was assessed. Chi square test and Fisher exact test was applied to observe the association. P-value <0.05 was considered to be statistically significant.

Results: A total of 34 chemotherapy cycles for hematological malignancies were analyzed. Median and range of age of patients was 36(9-75years) with predominant representation of male patients. Median platelet count post chemo was 39x10 9 /L (4-398). The overall incidence of chemotherapy induced thrombocytopenia was 18%. Overall PT and APTT were deranged in 21(61.8%) and 24(70.6%) cases respectively. The association between chemotherapy induced thrombocytopenia and PT and APTT was observed however it was found out that deranged PT and APTT results were not associated with thrombocytopenia and found to be statistically non-significant (P-value >0.05).

Summary/Conclusion: In conclusion, the association of chemotherapy induced thrombocytopenia with coagulopathy was not found statistically significant. However, the findings might suggest the identification of high risk cohort at risk of bleeding and/or thrombosis. Longitudinal studies with larger sample size are needed in this regard.

PB2295: EVANS SYNDROME AND PHENOTYPIC HETEROGENEITY OF SASH3 GERMLINE LOSS-OF-FUNCTION MUTATIONS BEYOND X-LINKED IMMUNODEFICIENCY

J. Berner 1,2,3,* , W. Novak 2 , R. Jimenez-Heredia 1,3,4 , L. Kager 1,2 , K. Boztug 1,2,3,4

1 St.Anna Childrens Cancer Research Institute Vienna; 2 St. Anna Children’s Hospital, Department of Pediatrics, Medical University Vienna; 3 Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases; 4 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

Background: “Evans syndrome (ES)” denotes a heterogeneous disease entity, defined by two or more autoimmune cytopenias, due to mostly unknown pathogenic mechanisms. Further, ES is increasingly recognized in pediatric patients with defined inborn errors of immunity (IEIs) including pathogenic variants in CTLA4 , STAT3 , TNFRSF6 , PIK3CD , and KRAS . Loss-of-function mutations in Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3 ) have recently been reported as a novel IEI entity in four patients with X-linked combined immunodeficiency with immune dysregulation including immune cytopenias. SASH3 is an X-chromosomal encoded adaptor protein that is exclusively expressed in lymphocytes. Although knowledge of the exact molecular function of this protein is scarce, humans and mice lacking expression of SASH3 show defects in proximal and distal T-cell receptor signalling.

Aims: We aimed to dissect aberrant immune cell functionality in SASH3-deficient patients to further enhance our understanding of pathogenic mechanisms in patients with ES.

Methods: We performed whole exome sequencing, deep clinical phenotyping and immunophenotyping in a patient with ES and his clinically asymptomatic brother.

Results: Our index patient presented with severe ES and recurrent infections at the age of 8 years. Immune phenotyping of his peripheral mononuclear cells (PBMCs) showed transient CD4+ T- and B-cell lymphopenia and reduced T-cell proliferation upon PHA and SEB stimulation. Furthermore, the patient had hypogammaglobulinemia, and the ‘transitory CD21 low B-cells’ were significantly increased, reflecting impaired lymphocyte maturation possibly causing the autoreactive phenotype of the disease. Interestingly, the patient developed large hemorrhagic splenic cysts during ITP associated bleeding diathesis, and thus underwent splenectomy, which led to a complete remission of ES.

Using whole-exome sequencing, we discovered a germline nonsense mutation in SASH3 (c.862C>T;p.Arg288Ter) in a now 16 years-old patient with ES. Surprisingly, this maternally inherited mutation was also identified in the hitherto clinically inconspicuous younger brother. The identified mutation is identical to the one reported in two males in the discovery of SASH3 deficiency. Divergently, the now 8-year-old brother who is also carrying the same variant has no clinically apparent phenotype until now. Notably, disease onset occurred earlier (i.e., between age 2 and 5 years) in the previously reported patients compared to our patients.

Summary/Conclusion: We present the 5th and 6th patient with germline hemizygous deleterious variants in SASH3 . Thus, this study expands the clinical phenotype and underlines a marked phenotypic heterogeneity as illustrated in siblings bearing the same mutation. Future studies will need to address comprehensively the phenotypic spectrum of the disease and genetic and non-genetic modifiers of clinical phenotypes and severity.

PB2296: THE PROGNOSTIC ROLE OF NEUTROPHIL TO LYMPHOCYTE RATIO AND PLATELET TO LYMPHOCYTE RATIO IN COVID-19 PATIENTS: A MONOCENTRIC STUDY

W. Boufrikha 1,* , R. Mezriqui 2 , N. Slama 1 , S. Chouchene 2 , S. Abidi 2 , Z. Mlayah 1 , A. Benabdeljelil 2 , M. Ftita 2

1 Hematology; 2 Hematology Laboratory, Fattouma Bourguiba University Hospital, Monastir, Tunisia

Background: Several studies described numerous factors to affect the prognosis and predict the severity of COVID-19. Inflammatory markers like neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) have been used to prognosticate cancers and inflammatory diseases such as COVID-19.

Aims: To describe and identify the predictive factors of intensive care unit admission in COVID-19.

Methods: This is a retrospective study conducted in Fattouma Bourguiba University Hospital of Monastir, Tunisia, from April 2020 to February 2022. Our study concerned patients diagnosed with COVID-19 during this period. NLR and PLR were investigated for each patient at the first day of hospitalization and all patients were followed in order to notice who required intensive care unit (ICU).

Results: We enrolled 569 patients. The median age was 62 years old (ranged 1-96) and we had male predominance (sex ratio 1.63). Seventy one per cent of our population had lymphopenia and polynucleosis was found in 45.7% of cases. The median NLR and the median PLR were respectively 6.37 and 232.05. NLR >3, NLR >9 and PLR >180 accounted for 77%, 35% and 57.9% respectively. Twenty three per cent of our population required hospitalization in ICU and this condition was significantly associated with elevated NLR and elevated PLR in univariate analysis (Table 1).

In multivariate analysis, elevated NLR (>9) (hazard risk [HR] 2.83, 95% confidence interval [CI] 1.75–4.57) was retained as only factor associated with intensive care hospitalization.

Summary/Conclusion: Many factors can modify the clinical outcome in COVID-19 patients. Our study supports the fact that elevated NLR (>9) may be a prognostic biomarker.

PB2297: THE PREDICTIVE VALUE OF PLATELET SPECIFIC ANTIBODIES FOR THE FIRST-LINE TREATMENT EFFECT IN NEWLY DIAGNOSED PEDIATRIC ITP

S. Dong 1,*

1 Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, China, Beijing, China

Background: Immune thrombocytopenia (ITP) is an autoimmune mediated hemorrhagic disease characterized by thrombocytopenia. Platelet specific antibodies play a key role in platelet destruction.

Aims: This study was aimed to determine whether platelet specific antibodies (including anti-GPIIb/IIIa, anti-GPIb/IX and anti-GPIa/IIa antibodies) are related to the first-line treatment effect of newly diagnosed pediatric ITP.

Methods: This study retrospectively analyzed 154 newly diagnosed ITP patients from October 2016 to September 2021 in Beijing Children’s Hospital. These patients were treated with glucocorticoids and/or intravenous immunoglobulin (IVIG). And platelet counts were detected one week and one month after treatment. Platelet specific antibodies were detected by PAKAUTO.

Results: When the antibody levels were divided into positive and negative, children with positive anti-GPIb/IX antibody responded worse than those with negative anti-GPIb/IX antibody at both one week and one month (Z=-2.565, P =0.010; Z=-2.579, P =0.010). When the antibody titer ratio was used to reflect the antibody levels, there was no significant difference between platelet specific antibodies and the first-line treatment effect in newly diagnosed pediatric ITP.

Summary/Conclusion: Anti-GPIb/IX antibody is a sign of worse response in newly diagnosed pediatric ITP.

PB2298: THE RELATIONSHIP BETWEEN PLATELET SPECIFIC ANTIBODIES AND THE SEVERITY OF NEWLY DIAGNOSED PEDIATRIC ITP

S. Dong 1,*

1 Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, China, Beijing, China

Background: Immune thrombocytopenia (ITP) is an autoimmune mediated hemorrhagic disease characterized by thrombocytopenia. Platelet specific antibodies play a key role in platelet destruction.

Aims: This study was aimed to determine whether platelet specific antibodies (including anti-GPIIb/IIIa, anti-GPIb/IX and anti-GPIa/IIa antibodies) are related to the severity of newly diagnosed pediatric ITP.

Methods: This study retrospectively analyzed 154 newly diagnosed ITP patients from October 2016 to September 2021 in Beijing Children’s Hospital. Children who used only glucocorticoids during hospitalization were defined as Severity 1. Children who used glucocorticoids combined with intravenous immunoglobulin (IVIG) and/or single rhTPO were defined as Severity 2. Platelet specific antibodies were detected by PAKAUTO.

Results: When the antibody levels were divided into positive and negative, children with positive anti-GPIIb/IIIa antibody are more severe than children with negative anti-GPIIb/IIIa antibody in newly diagnosed ITP ( c 2 =3.969, P =0.046). When the antibody titer ratio was used to reflect the antibody levels, there was no significant difference between platelet specific antibodies and the severity of newly diagnosed pediatric ITP.

Summary/Conclusion: Anti-GPIIb/IIIa antibody is a sign of higher severity in newly diagnosed pediatric ITP. anti-GPIb/IX and anti-GPIa/IIa antibodies were not found to be related to the severity of ITP.

PB2299: CLINICAL AND LABORATORY CHARACTERISTICS OF CHILDREN WITH INHERITED THROMBOCYTOPENIA IN A SINGLE EGYPTIAN UNIVERSITY HOSPITAL

Y. El Chazli 1,* , N. S. Badawy 2 , R. Abo Elwafa 3 , M. Salama 1

1 Hematology/Oncology Unit, Pediatric Department, Alexandria University; 2 Pediatrics, Ministry of Health Hospitals; 3 Clinical Pathology, Alexandria University, Alexandria, Egypt

Background: Inherited thrombocytopenias (IT) are rare bleeding disorders, which cause a deficiency of platelets in early infancy. This group of disorders is sometimes associated with abnormal phenotypes. ITs are being recognized with increasing frequency. The study of IT in children is difficult due to the heterogeneity of the patients and the rarity of each disease. To the best of our knowledge, the study of the epidemiology of such a hematological finding in our area has not been previously published.

Aims: To report the clinical and laboratory characteristics of children with inherited thrombocytopenia.

Methods: The files of all children with thrombocytopenia followed at the Hematology clinic of Alexandria University Children’s Hospital, Alexandria, Egypt, were retrospectively analysed. Patients who joined the clinic until the 31 st of December 2021 were included. IT was considered in patients with suggestive clinical features as young age of presentation, abnormal physical features, positive family history, no response to ITP drugs. The blood film was revised for assesment of platelet size and morphology. Flowcytometry of platelet glycoproteins was perfromed.

Results: Forty patients were identified as suspected IT. The median age of patients was 65 months, ranging from 9 months to 17 years at the time of the study. Thrombocytopenia was first discovered at a median age of 20 months. Fifty-five percent were males. In 55% of patients, there was a history of thrombocytopenia in a family member, and 42% had consanguineous parents (first-degree cousins). The most common bleeding symptoms were purpura and ecchymoses, followed by mucosal bleeding. The median platelet count at diagnosis was 30 ×10 3 cell/uL, while the lowest was 9 ×10 3 cell/uL. The initial recorded diagnosis was ITP in 65% of patients, followed by Glanzmann thrombasthenia (7.5%), CAMT and WAS (5% each). After revision of the diagnosis, BSS was confirmed in 20% of patients, WAS in 12.5%; some rare disorders were identified like MYH9-RD and Glanzmann thromboasthenia with macrothrombocytopenia. In 35% of patients the diagnosis was not defined yet. Seven patients were missing for further workup, and seven patients were sent for genetic testing.

Summary/Conclusion: Inherited thrombocytopenias are rare disorders that are frequently overlooked. A detailed family and disease history, examination of the blood film by an expert, and assessment of the response to usual ITP treatments are all simple methods that help pick up suspected cases. Simple tests like platelet glycoprotein flowcytometry or MYH9 staining can confirm the diagnosis in a substantial number of patients. Genetic tetsing is the final step for undiagnosed cases.

PB2300: COMPLEMENT ACTIVATION NEGATIVELY AFFECTS THE PLATELET RESPONSE TO THROMBOPOIETIN RECEPTOR AGONISTS IN PATIENTS WITH IMMUNE THROMBOCYTOPENIA: A PROSPECTIVE COHORT STUDY

A. Åkesson 1,* , J. B. Bussel 2 , M. Martin 1 , A. M. Blom 1 , J. Klintman 1 , W. Ghanima 3 , E. Zetterberg 1 , L. Garabet 3,4

1 Lund University, Malmö, Sweden; 2 Cornell University, New York, United States of America; 3 Østfold Hospital, Grålum; 4 Akershus University Hospital, Lørenskog, Norway

Background: Increased platelet destruction is central in the pathogenesis of immune thrombocytopenia. However, impaired platelet production is also relevant, and its significance underlies the rationale for treatment with thrombopoietin receptor agonists (TPO-RAs). Previous studies have associated enhanced complement activation with increased disease severity. Additionally, treatment refractoriness has been demonstrated to resolve by the administration of complement-targeted therapeutics in a subset of patients.

Aims: To investigate the association between complement activation and platelet response to TPO-RA therapy.

Methods: In this study, blood samples from patients with chronic immune thrombocytopenia (n = 15) were prospectively collected before and 2, 6 and 12 weeks after the initiation of TPO-RA therapy. Levels of complement degradation product C4d and soluble terminal complement complexes were assessed.

Results: Patients with significantly elevated baseline levels of terminal complement complexes had more often undergone splenectomy ( p = .08), exhibited more often an inadequate platelet response ( p = .04) and were exclusively subjected to rescue therapy with intravenous immunoglobulin ( p = .02) during the study period. C4d showed a significant ( p = .01) ability to distinguish samples with significant terminal complement activation, implying engagement of the classical complement pathway.

Summary/Conclusion: Elevated levels of complement biomarkers were associated with a worse TPO-RA treatment response. Larger studies are needed to confirm these results. Biomarkers of complement activation may prove valuable as a prognostic tool to predict which patients that potentially could benefit from complement-inhibiting therapy in the future.

PB2301: PEDIATRIC IMMUNE THROMBOCYTOPENIA; A GLANCE AT PATIENTS AND CURRENT THERAPEUTIC CHALLENGES

M. Al-Jadiry 1 , H. Ghali 1,* , I. Aref 2 , A. Hatem 2 , S. Faraj 2 , S. Abdulrazzaq 2 , R. Majid 2 , A. Fadhil 2 , S. Al-Hadad 1

1 Pediatric hematology/oncology, College of Medicine, University of Baghdad / Children Welfare Teaching Hospital, Medical City; 2 Pediatric hematology/oncology, Children Welfare Teaching Hospital, Medical City, Baghdad, Iraq

Background: Immune thrombocytopenia (ITP) during childhood is usually characterized by acute onset of thrombocytopenia with around 75% of children having spontaneous remission within 6 months. Most children experience only mild bleeding in the form of bruising and Petechiae, with severe bleeding being rare even in children with persistent disease

Aims: To study demography, clinical, laboratory and treatment options of patients who were newly registered cases of immune thrombocytopenia referred to Children Welfare Teaching Hospital, Medical City, Baghdad.

Methods: A retro-prospective descriptive study was done on pediatric patients with ITP who were referred to Children’s Welfare Teaching Hospital (CWTH) / Medical City / Baghdad from January 1, 2020 to August 31, 2021. The period of data collection and follow up started in May 2020 till October 2021.

Results: A total of 138 patients were identified as having a presumptive newly diagnosed Immune Thrombocytopenia. Sixteen patients were excluded after being diagnosed as secondary ITP or other causes of thrombocytopenia during observation period. The analysis was limited to 122 patients. The mean age was 4.75 years (range 2 months-13.1 years). There were 63 (51.6%) boys and 59 (48.4%) girls, with a M:F ratio was 1.06:1. The mean duration of symptoms is 13.6 days. Seventy-five (61.5%) patients were managed as an outpatient only.

Sole cutaneous bleeding was reported in 57 (46.7%) patients while epistaxis was the leading cause of mucosal bleeding occur in 30 (25%) patients followed by gum bleeding in 15 (12.5%) patients. According to grading, grade I/II was seen in 57 (46.7%). The mean platelet count was 20.2x10 9 /L, Platelet’s count of <30 x10 9 /L in 95 (77.8%) patients.

Ninety-three patients (76.2%) reached CWTH without receiving any treatment while 29 (23.8%) patients received treatment elsewhere. The total number of patients kept under watchful waiting strategy was 56 (45.9%).

The mean and median platelets count at both time of diagnosis and time of last contact in the observation group were higher than that of the treatment group at time of diagnosis (26 vs 15.4 x 10 9 /L) with a significant statistical correlation, and comparable at time of last follow up (200.7 vs 210.4 x 10 9 /L) with negligible statistical correlation.

There was a significant statistical correlation between both groups of patients in regard to the grades of bleeding, the observed group showed grade I\II as the major type of bleeding while the treated group showed grade III as the major type of bleeding.

Summary/Conclusion: The result of this study confirms the result of two previous studies in the same center which encourages the successful management of ITP by observation only and support the consensus that ITP is a benign and self-limited condition for the majority of affected children. The incidence of intracranial bleeding was 0.8%. The study observations support the emerging trend toward reserving drug therapy for ITP patients with major bleeding manifestations or troublesome impairment of quality of life.

PB2302: HEPARIN-INDUCED THROMBOCYTOPENIA ANTIBODIES CAN MAINTAIN PLATELET-ACTIVATING PROPERTIES AFTER TWO SESSIONS OF THERAPEUTIC PLASMA EXCHANGE

V. Gkalea 1,* , P. Tseke 2 , T. Pappas 1 , A. Kotsiauti 1 , V. Petrou 1 , E. Psimenou 2 , C. Matsouka 1

1 Hematology departement; 2 Nephrology departement, Alexandra General Hospital, Athens, Greece

Background: Therapeutic plasma exchange (TPE) has been used as a salvage therapy in a small subset of heparin-induced thrombocytopenia (HIT) patients (severe or refractory HIT, before heparin re-exposure during cardiac surgery, in bleeding patients and in autoimmune HIT). Existing data show that HIT antibodies can lose platelet-activating properties after one or two TPE sessions, while more sessions may be needed to decrease the antibody titer. However, results may be assay dependent. The utility of Heparin-Induced Multiple aggregometry (HIMEA) in this setting has not been evaluated.

Aims: To assess the effect of TPE on pathogenic HIT antibodies using HIMEA before an emergency surgery for gynecologic malignancy.

Methods: TPE was performed in a 72-year-old patient necessitating emergency surgery for a voluminous uterine sarcoma in the acute phase of HIT (10 days after diagnosis). TPE aimed to reduce post-operative thrombotic risk by decreasing pathogenic HIT antibodies in a patient presenting a high risk of post-operative thrombotic complications. Two consecutive sessions of TPE using Spectra Optia ® Apheresis System were performed immediately before surgery. During each plasma exchange 2 L of plasma were removed and replaced by 5% human albumin. Acid-Citrate-Dextrose (ACD) was used as anticoagulant. Blood samples were taken before and after each TPE session (TPE1 pre, TPE1 after, TPE2 pre, TPE2 after). Removed patient’s plasma collected to the plasmapheresis bag was also tested. IgG anti-PF4/heparin antibodies tested with Enzyme Immunosorbent Assay (EIA) (Hyphen ZYMUTEST™ HIA IgG, Hyphen Biomed) and Heparin-Induced Multiple aggregometry (HIMEA) (Multiplate, Roche) in the absence of heparin (HIMEA saline), in the presence of 1UI/ml unfractionated heparin (UFH) (HIMEA low) and in the presence of 100IU/ml UFH (HIMEA high) were assessed.

Results: Patient’s 4T’s score was 7 (2 for platelet count fall, 2 for timing, 2 for catheter associated venous thrombosis and 1 for other causes), strongly positive IgG anti-PF4 antibodies (OD: 2.43, cut-off<0.3 units) and a HIMEA profile favoring HIT (typical sigmoid curve at HIMEA low, >50% decrease of AUC at HIMEA high, absence of platelet aggregation at HIMEA saline). She was treated with fondaparinux at 1.5 mg/ day due to sepsis-associated acute kidney injury requiring hemodialysis. Platelet count (PLT) were 168 G/l before surgery. After two TPE sessions antibody titers remained unchanged (TPE1 pre: 2.63 units, TPE1 after:2.19 units, TPE2 pre:2.51 units, TPE2 after:5.52 units). Moreover, platelet reactivity remained also strong as demonstrated by high area under the curve (AUC) values in HIMEA low (figure 1). Plasma from the collection bag showed strong EIA and platelet activation test reactivity. No thrombosis nor thrombocytopenia were observed in one month follow-up.

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Summary/Conclusion: Two TPE sessions failed to reduce platelet-activating properties of HIT antibodies as assessed by HIMEA in a cancer patient. TPE could be considered as a therapeutic intervention that can reduce thrombotic risk in patients necessitating an urgent non-cardiac surgery in the acute phase of HIT. However, more data is needed on the clinical benefit and the optimal number of TPE sessions in this clinical setting. Functional assays assessing Fcγ receptor mediated platelet activation may help guide the timing of the surgery.

PB2303: THROMBOCYTOPENIA IN COVID-19 PATIENTS ON ECMO SUPPORT

C. Jones 1,* , K. Chen 1 , V. Narendran 2

1 Internal Medicine, University of Miami / Palm Beach Regional GME Consortium, West Palm Beach; 2 Hematology/Oncology, JFK Medical Center, Atlantis, United States of America

Background: Thrombocytopenia is a common complication of COVID-19 (coronavirus disease 2019). The possible mechanisms include decreased platelet production, increased platelet destruction, and consumption. According to studies, thrombocytopenia indicates a poor prognosis and increased mortality in COVID-19 patients. Meanwhile, thrombocytopenia is often complicated in ECMO (extracorporeal membrane oxygenation) due to shearing force in the circuit and heparin-induced thrombocytopenia (HIT). COVID-19 patients who are managed on ECMO are at high risk of developing thrombocytopenia and life-threatening bleeding.

Aims: Venous thromboembolism prevention is critical in managing COVID-19 patients. The balance between bleeding secondary to thrombocytopenia and thromboembolic prophylaxis is challenging. ECMO support also increases the risk of thrombocytopenia. Our retrospective observation study reports the incidences of thrombocytopenia and major bleeding events in twenty-three COVID-19 patients who were managed with ECMO.

Methods: We retrospectively reviewed the data of twenty-three patients who were diagnosed with COVID-19 and managed on ECMO. Thrombocytopenia is defined by platelet level lower than 150,000/uL. Incidences of thrombocytopenia and related complications were recorded and analyzed.

Results: Twenty-three adult patients who were diagnosed with COVID-19 received ECMO support. The mean age of patients was 45-year-old and mean body mass index (BMI) was 31.29. Seventeen patients were males, and eleven patients had at least one of the following pre-ECMO comorbidity: ten (43.5%) patients had hypertension, eleven (47.8%) patients had type 2 diabetes and four (17.3%) patients had hyperlipidemia. None of the patients were active smokers or had chronic lung disease when admitted. Anticoagulation was initiated on the day of admission.

Twenty-one (91.3%) patients developed thrombocytopenia and HIT was excluded. Eighteen (78.3%) patients had hemorrhage requiring transfusion, with the gastrointestinal tract being the most common site. Other bleeding sites included brain, chest, tracheostomy area and cannulation site. Thromboembolic prophylaxis was held for active bleeding or platelet count threshold of < 30,000/uL. The overall mortality rate was 70.0%.

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Summary/Conclusion: Based on our data, the incidence of thrombocytopenia was 91.3% in ECMO-managed COVID-19 patients; 78.3% patients developed hemorrhage requiring transfusion. Given the high risk of thromboembolic events in COVID-19 patients, anticoagulation is recommended to all hospitalized COVID-19 patients unless there is active bleeding, previous bleeding history within three days or platelet count is lower than 30,000 cells/uL. However, anticoagulation further increases the risk of bleeding in the setting of thrombocytopenia, which may lead to sudden deterioration and death. Further investigation into the mechanisms, implications, and management of thrombocytopenia will lead to significantly improved outcomes and prognosis for COVID-19 patients who are managed on ECMO.

PB2304: THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP): A SINGLE-CENTER EXPERIENCE

F. Kammoun 1,* , Y. Fakhfakh 1 , M. Charfi 1 , O. Kassar 1 , M. Chaari 2 , L. Bouaoun 3 , M. Bahloul 4 , I. Frikha 1 , F. Kallel 1 , I. Ben Amor 1 , L. Khalifa 1 , M. Medhafar 1 , S. Hadiji 1 , H. Elleuch 2 , H. Mnif 3 , M. Bouaziz 4 , M. Elloumi 1

1 Clinical Hematology Depatment; 2 Biological Hematology Department, Hedi Chaker Hospital; 3 Plasmapheresis Unit, Regional Blood Transfusion Center; 4 Medical Reanimation Department, Habib Bourguiba Hospital, Sfax, Tunisia

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare and life-threatening thrombotic microangiopathy characterized by microangiopathic hemolytic anemia, consumption thrombocytopenia, and organ injury. TTP pathophysiology is based on a severe ADAMTS13 deficiency, the specific von Willebrand factor (VWF)-cleaving protease.

Aims: In this study, we aim to report our experience in patients with TTP, identify the main clinical and laboratory disease findings, treatment patterns, and evolution in these patients.

Methods: This is a retrospective study of all adult patients (> 15 years) diagnosed and treated between 2000 and 2021 with TTP, at the hematology department of the university hospital of Sfax, Tunisia.

Results: A total of 11 patients were enrolled in this study period. The median age was 38 years [range 22-72] and there was a female preponderance (2.6). At presentation, the majority of patients (8/11) had asthenia and neurological symptoms: confusion in 4 cases, convulsive seizure in 2 cases, and cerebrovascular accident in 2 cases. Five patients had fever, 4 had jaundice, and 3 presented with bleeding. Microangiopathic hemolytic anemia was noted in all patients with mean hemoglobin of 7.3 g/dl [5-8 g/dL], and 5% to 20% of schistocytes. Thrombocytopenia with platelets < 100x10 9 /l occurred in 10 of 11 patients with mean platelet of 14 x10 9 /l. Three patients had high serum creatinine levels, proteinuria and hematuria were present in 3 and 2 other cases respectively. The causal investigation showed that TTP was related to viral infection in 3 cases, newly discovered neoplasia in 2 cases, pregnancy in 3 cases, an immunologic disorder in one case, and idiopathic in 2 cases. Of the 3 pregnancy cases, 2 had a plasma activity of ADAMTS13 lower than 5%, and TTP was thus related to Upshaw-Schulman syndrome. For treatment, plasmapheresis associated with corticosteroids was promptly started in 9 patients, and 2 patients received corticosteroids alone due to hemodynamic instability. Four patients had also Rituximab as salvage therapy, and for the 2 patients with neoplasia, adequate chemotherapy was initiated. Four patients died due to shock with neurological involvement, respiratory failure, or multiple organic failure. The other 7 patients achieved remission, but two of them relapsed.

Summary/Conclusion: Our study is consistent with clinical trial findings reported in the literature, showing that TTP has heterogeneous modes of presentation and not all patients have the complete typical features present. Since it has a fatal outcome, rapid diagnosis and management are recommended. Finally, the causal diagnosis should be made to initiate the appropriate curative treatment.

PB2305: EFGARTIGIMOD: CLINICAL DEVELOPMENT OF A NOVEL FCRN ANTAGONIST IN THE TREATMENT OF AUTOIMMUNE DISEASES

P. Knoebl 1,* , H. de Haard 2 , W. Parys 2 , P. Ulrichts 2 , F. Rocca 2 , A. Guglietta 2 , J. Ayguasanosa 2

1 Medical University of Vienna, Vienna, Austria; 2 argenx, Ghent, Belgium

Background: Immunoglobulin G (IgG) autoantibodies play a key role in the pathogenesis of various autoimmune diseases. The neonatal Fc receptor (FcRn) is the central regulator of IgG homeostasis, rescuing IgG (including pathogenic autoantibodies) and albumin from lysosomal degradation and is responsible for the long half-life of IgG. Therapeutic blocking of FcRn leads to reduction of all IgG subtypes without reducing other immunoglobulin types, making it a possible target for symptomatic treatment of autoimmune disorders, while maintaining normal immunological responses. Efgartigimod (EFG), an FcRn antagonist, is a human IgG1-derived Fc-fragment that outcompetes endogenous IgG binding, reduces IgG recycling, and increases IgG degradation. EFG is approved in the US and Japan for treatment of generalized myasthenia gravis (MG) in adult patients.

Aims: This presentation provides an overview of EFG as a molecule and summarize the clinical development program to date.

Methods: Studies in healthy volunteers and patients with autoimmune diseases are already completed. Phase 3 studies in immune thrombocytopenia (ITP; ADVANCE and ADVANCE SC) and phase 2/3 studies in chronic inflammatory demyelinating polyneuropathy (ADHERE), pemphigus vulgaris and foliaceus (ADDRESS), bullous pemphigoid (BALLAD), and myositis (ALKIVIA) are ongoing.

Results: In a Phase 2 trial in patients with ITP, EFG (10 mg/kg given intravenously) reduced total IgG levels by 63.7% (from mean [SD] 10.6 g/L [5.1] at baseline to 4.1 g/L [2.0]) 3 days after the fourth weekly infusion. No changes in IgG levels were observed in patients treated with placebo during that time. These reductions led to clinically relevant increases in platelet counts; 46% of EFG patients vs 25% on placebo achieved a platelet count of ≥50 x109/L on ≥2 occasions. In a Phase 3 trial in MG (ADAPT), EFG rapidly reduced IgG without impacting IgM, IgA, or albumin levels while participants recorded statistically significant functional neurological improvements. In all studies to date, EFG was well tolerated, and adverse events were mainly mild to moderate.

Summary/Conclusion: FcRn inhibition by EFG is a promising potential therapeutic option for several autoimmune diseases mediated by pathogenic IgG autoantibodies.

PB2306: RELAPSE OF IMMUNE THROMBOCYTOPENIA AND MYOCARDITIS

F. Kapsouli 1,* , E. Sanidas 2 , M. Mantzourani 1,2

1 First Department of Internal Medicine, Laiko Hospital, Athens, National and Kapodistrian University of Athens; 2 Department of Cardiology, Laiko Hospital, Athens, Greece

Background: Immune thrombocytopenia (ITP) is an acquired thrombocytopenia caused by autoantibodies against platelet antigens. ITP most often occurs within a few weeks following a viral infection, but it is also associated with other infections, drugs, hematopoietic malignancies and autoimmune diseases. Myocarditis is an inflammatory disease of cardiac muscle caused by a variety of infectious and noninfectious conditions. Autoimmune mechanisms have been implicated in the pathogenesis of myocarditis with or without a virus trigger.

Aims: Our aim is to present an unusual case of myocarditis in a 17-year-old male, with a history of ITP presenting simultaneous relapse of ITP.

Methods: A 17-year-old male with a history of ITP presented at the Emergency Department complaining of gingival bleeding, left shoulder pain and fever. Clinical examination showed extremity petechiaes and tachycardia, there was an ST elevation in V2-V6 leads on ECG and laboratory data included PLTs 2000/μL and elevated high sensitivity troponin (1.240pg/mL). He was treated immediately as a relapse of ITP with corticosteroids and he received b-blockers due to persistent tachycardia. Viral and immunological tests were negative.

Results: Gradually, during hospitalization, there was an elevation of PLT count and a decrease in serum troponin levels with improving and finally normal ECG findings. The patient was discharged and continued treatment with prednisolone and b-blocker. He was reassessed after one month and troponin levels, PLT counts and ECG were normal. However, the cardiac Magnetic Resonance Imaging (MRI) revealed fibrosis with signal intensity and gadolium enhancement in the anterolateral and inferior segments. After three months a follow up cardiac MRI showed reduction of fibronitic tissue size.

Summary/Conclusion: Autoimmunity plays a major role in the pathogenesis of both ITP and myocarditis. It seems that they share common autoimmune pathways and further research of which is important for understanding pathophysiology behind these clinical entities and improving their treatment.

PB2307: A MULTICENTER, RANDOMIZED, OPEN-LABEL STUDY OF ROMIPLOSTIM PLUS DEXAMETHASONE VS DEXAMETHASONE IN PATIENTS WITH NEWLY DIAGNOSED PRIMARY IMMUNE THROMBOCYTOPENIA

M. E. Mingot Castellano 1,* , C. A. Bradbury 2 , C. Rosso Fernández 3 , I. Thomas 4 , M. T. Alvarez-Román 5 , M. Canaro 6 , I. S. Caparrós Miranda 7 , M. Cappechi 8 , M. Carpenedo 9 , G. Evans 10 , J. T. González-Lopéz 11 , J. R. González-Porras 12 , I. Jarque Ramos 13 , M. L. Lozano-Almela 14 , M. F. Lopez Fernandez 15 , G. Lowe 16 , H. Lyall 17 , C. Pascual Izquierdo 18 , S. Pavord 19 , R. Rayment 20 , P. Roberts 21 , P. Rosique Cortina 22 , B. Sánchez-González 23 , C. Santoro 24 , K. Talks 25 , D. Valcarcel-Ferreiras 26 , F. Zaja 27

1 Hematology, Hospital Universitario Virgen del Rocio, Sevilla, Spain; 2 Bristol Haematology and Oncology Centre, University of Bristol, Bristol, United Kingdom; 3 Clinical Trials Unit Hospital Universitario Virgen del Rocío, Sevilla, Spain; 4 Centre for Trials Research, Cardiff University, Cardiff, United Kingdom; 5 Hematology Department, Hospital Universitario La Paz, Madrid; 6 Hematology Department, Hospital Son Espases, Mallorca; 7 Hematology Department, Hospital Universitario Virgen de la Victoria, Málaga, Spain; 8 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and University of Milan, Milan; 9 U.O Ematologia e Trapianto ASST Ospedale San Gerardo di Monza, Monza, Italy; 10 East Kent Hospitals University NHS Foundation Trust, Kent, United Kingdom; 11 Hematology Department, Hospital Universitario de Burgos, Burgos; 12 Hematology Department, Hospital Universitario de Salamanca, Salamanca; 13 Hematology Department, Hospital Universitario La Fe, Valencia; 14 Hematology Department, Hospital Universitario Morales Meseguer, Murcia; 15 Hematology Department, Complexo Hospitalario Universitario A Coruña, A Coruña, Spain; 16 University Hospitals Birmingham, Birmingham; 17 Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, United Kingdom; 18 Hematology Department, Hospital Universitario Gregorio Marañon, Madrid, Spain; 19 Hematology Department, John Radcliffe Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford; 20 University Hospital of Wales, Cardiff; 21 Torbay and South Devon NHS Foundation Trust, Devon, United Kingdom; 22 Hematology Department, Hospital Virgen de la Arrixaca, Murcia; 23 Hematology Department, Hospital del Mar, Barcelona, Spain; 24 Sapienza University, Roma, Italy; 25 Newcastle Haemophilia Comprehensive Care Centre, The Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle, United Kingdom; 26 Hematology Department, Hospital Val de hebrom, Barcelona, Spain; 27 Università degli Studi di Trieste/University of Trieste, Trieste, Italy

Background: High dose glucocorticoids are recommended as initial treatment for adults with Immune primary thrombocytopenia (ITP). In the majority of patients (85%), dexamethasone is effective at increasing platelet counts, but within 6 months 50% will relapse. Romiplostim is a peptibody designed to activate the TPO receptor, leading to platelet responses in 79-95% of ITP patients as second line treatment. There is accumulating evidence that the combination of immunosuppressive drugs and TPO analogues (TPOa) may improve ITP responses.

Aims: Primary objective

To evaluate the superiority of romiplostim plus dexamethasone vs dexamethasone alone in patients with newly primary immune thrombocytopenia (ITP) in terms of sustained response off any ITP treatment and without WHO grade 2 or more bleeding

Secondary objectives

• To compare the efficacy of dexamethasone plus romiplostim vs dexamethasone in terms of complete response (CR), response (R), global response (GR), SR at 6 and 12 months and other efficacy-related endpoints

• Safety and tolerability

• Quality of life

• Cost-effectiveness analysis comparing dexamethasone plus romiplostim vs dexamethasone (Health-resource utilization, HRU)

Methods: This is an open label, randomized (1:1), phase 3 study designed to compare romiplostim plus dexamethasone vs dexamethasone alone (See figure 1). 126 patients will be enrolled in 30 study sites from Spain, United Kingdom and Italy.

• Arm 1 (Romiplostim plus dexamethasone): dexamethasone 40 mg daily for 4 days only in the first cycle and subcutaneous romiplostim weekly for up to 12 months (ROM + DEX arm). Romiplostim will be tapered when platelets are ≥ 50x109/L for more than 4 weeks (see figure 2).

• Arm 2 (Dexamethasone): dexamethasone 40 mg daily for 4 days for up to 3 cycles every 14 to 28 days (DEX arm).

Results: Patients will be followed for a minimum of 12 months from randomization and a minimum of 6 months after discontinuing treatment with either romiplostim or dexamethasone. The following assessments will be done in these visits: platelets count, bleeding score (ITP-BAT, WHO criteria), number and characteristics of bleeds, adverse events (CTCAE version 4.03), study medication, rescue medication and concomitant medication, HRU associated with the ITP or its treatment and loss of productivity and quality of life (EQ-5D, 36-Item Short-Form Health Survey (SF-36) version 2, Investigating Choice Experiments Capability Measure–Adults (ICECAP-A) questionnaire, version 2; fatigue as measured on the Functional Assessment of Chronic Illness Therapy–Fatigue (FACIT-F) scale, version 4).

Intravenous immunoglobulins (IVIg), platelet transfusion or prednisone will be considered rescue treatment. Treatment failure definitions:

• In the ROM + DEX arm, treatment failure is defined as a platelet count <30x109/L after 4 weeks of romiplostim at 10 mcg/kg/week or if rescue medication is needed to get this level of platelets despite romiplostim 10mcg/kg/week for 4 weeks.

• In DEX arm, treatment failure is defined as a platelet count <30x109/L after 3 cycles of dexamethasone or if rescue medication is needed to get this level of platelets despite the 3 cycles of dexamethasone.

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Summary/Conclusion: This study will evaluate dexamethasone and romiplostim combination in comparison to dexamethasone alone for first line ITP treatment in terms of response, sustained response off any ITP treatment (SROT), bleeding, adverse events, need for rescue treatments and quality of life.

PB2308: QUALITY OF LIFE AND CLINICAL OUTCOMES IN ADULT PATIENTS WITH CHRONIC IMMUNE THROMBOCYTOPENIA (CITP) DURING TREATMENT WITH ROMIPLOSTIM IN A REAL-WORLD SETTING

T. Ionova 1,* , O. Vinogradova 2 , E. Markova 2 , L. Mukha 2 , N. Novitskaya 2 , M. Pankrashkina 2 , T. Shelekhova 3 , D. Sherstnev 3 , A. Proydakov 4 , E. Lyurova 4 , T. Pospelova 5 , T. Babaeva 5 , N. Bulieva 6 , G. Kuchma 7 , E. Andreevskaya 8 , M. Frolova 9 , E. Zinina 10 , K. Trizna 11 , I. Shestopalova 12 , T. Shneyder 13 , L. Khvorostenko 14 , A. Kuchin 15 , I. Mulina 16 , S. Volkova 17 , S. Zakharov 18 , N. Porfirieva 1 , T. Nikitina 1 , S. Gritsaev 19

1 Multinational Center for Quality of Life Research, Saint-Petersburg; 2 Hematology Center of Botkin Hospital, Moscow; 3 Saratov State Medical University named after V.I. Razumovsky, Saratov; 4 Komi Republican Oncology Center, Syktyvkar; 5 Federal State Budgetary Educational Institution of Higher Education «Novosibirsk State Medical University» of the Ministry of Health of the Russian Federation, Novosibirsk; 6 Regional Clinical Hospital of the Kaliningrad Region, Kaliningrad; 7 Regional Clinical Hospital, Orenburg; 8 Regional Clinical Hospital №1, Chita; 9 Regional Hospital № 1, Vologda; 10 Surgut Regional Clinical Hospital, Surgut; 11 Regional Clinical Hospital, Tomsk; 12 Kray Clinical Hospital, Khabarovsk; 13 Leningrad Regional Clinical Hospital, Saint-Petersburg; 14 Regional Clinical Hospital, Tyumen’; 15 Republican Clinical Hospital, Petrozavodsk; 16 Republican Clinical Hospital №1, Yakutsk; 17 Federal State Budgetary Educational Institution of Higher Education «Privolzhsky Research Medical University» of the Ministry of Health of the Russian Federation, Nizhny Novgorod; 18 Moscow Regional Research and Clinical Institute (“MONIKI”), Moscow; 19 Russian Research Institute of Hematology and Transfusiology, Saint-Petersburg, Russia

Background: Chronic primary immune thrombocytopenia (cITP) is a rare autoimmune disease which needs lifelong therapy to prevent bleeding and is accompanied with impaired quality of life (QoL) due to hemorrhagic syndrome, psychological concerns and limitations in patients’ daily life. Fatigue with its negative impact on QoL is of tremendous concern for patients with cITP.

Aims: We aimed to explore QoL and fatigue changes as well as to examine platelet response in patients with cITP during treatment with romiplostim in a real-world setting.

Methods: Adult patients with cITP who have been assigned romiplostim treatment were enrolled in multicenter observational prospective study. Romiplostim was administered in accordance with prescription. Patients filled out RAND SF-36 and FACT-Th6 questionnaires for QoL assessment and FACIT-Fatigue tool for fatigue assessment at baseline and at 3, 6 and 12 months after romiplostim treatment start. Fatigue level was categorized according to the quartiles of the FACIT-Fatigue scores as follows: low (Q4, 40-52), low-medium (Q3, 27-39), medium-high (Q2, 14-26), or high (Q1, 0-13). For statistical analysis of patient-reported outcomes (PRO) Mann-Whitney test, GEE and MacNemar test were applied. Adjustment to age, gender, severity of cITP and baseline QoL/fatigue for GEE was made. Kaplan-Meyer method was used to measure time till the first platelet response from romiplostim treatment start.

Results: In total, 60 patients with cITP from 17 centers in Russia were enrolled in the study: mean age – 51.9±15.4 (range, 25-85); 70% were females; 71.7% patients had comorbidities. Four patients were splenectomized (6.7%). Median of disease duration was 25.1 months (range, 12.6-765.1). Majority of patients experienced hemorrhagic syndrome (96.7%). Median platelet count before romiplostim was 20 х10 9 /l. Median number of treatments before romiplostim was 1 (range, 0-6). At baseline cITP patients experienced meaningful QoL impairment by all SF-36 scales excluding pain as compared to healthy controls (p<0.05). Integral QoL index was 0.285 for patients vs 0.432 for healthy controls (p<0.001); 39% patients had critical/severe QoL impairment before romiplostim onset. Mean FACT-Th6 score was 11.2±5.5 (range, 0-22). Мean FACIT-Fatigue score was 33.0±10.2 (range, 7–49); 22.4% of patients had high/medium-high level of fatigue. At median follow-up of 9.7 months (IQR, 7.2–12), 98.3% patients achieved overall platelet response; median time to first platelet response was 4 weeks (95%CI: 3–5). Median platelet count at 3 months of treatment was 104 х10 9 /l. During 12 months of romiplostim treatment dramatic meaningful QoL improvement by all SF-36 scales (GEE, p<0.001) and by FACT-Th6 score (GEE, 11.4 at baseline vs 17.1 at 12 months, p<0.001) was demonstrated. Fatigue severity reduced during romiplostim treatment: FACIT-Fatigue score increased from 33.1 at baseline to 42.7 at 12 months (GEE, p<0.001); the number of patients with high/medium-high level of fatigue significantly decreased from 22.4% at baseline to 6.8% after 3 months of treatment (p=0.01).

Summary/Conclusion: The results obtained demonstrated high response rates to romiplostim treatment in cITP patients and significant positive changes in QoL and fatigue level during therapy in a real-world evidence study. Benefits of romiplostim treatment was confirmed both from physician’s and patient’s perspective.

PB2309: EVALUATION OF MORBIMORTALITY FACTORS OF PATIENTS WITH ACQUIRED THROMBOTIC THROMBOCYTOPENIC PURPURA

M. I. Pinto 1,* , B. Marques 1,2 , D. Coelho 2 , P. Martinho 2 , T. Fidalgo 2 , J. P. Carda 1,2,3 , A. B. Sarmento Ribeiro 2,4,5,6

1 Faculty of Medicine, University of Coimbra; 2 Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra; 3 Coimbra Institute for Clinical and Biomedical Research (iCBR); 4 University Clinic of Hematology and Laboratory of Oncobiology and Hematology, Faculty of Medicine of University of Coimbra; 5 Coimbra Institute for Clinical and Biomedical Research (iCBR) Research Area of Environment Genetics and Oncobiology (CIMAGO), FMUC, and Center for Innovative Biomedicine and Biotechnology (CIBB); 6 Clinical Academic Center of Coimbra (CACC), University of Coimbra, Coimbra, Portugal

Background: Acquired Thrombotic Thrombocytopenic Purpura (aPTT) is a rare disease caused by the action of antibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), with high mortality when not treated properly. Formerly, it was defined by the pentad of fever, microangiopathic hemolytic anemia, thrombocytopenia, neurological symptoms and severe acute kidney injury. Currently, it is characterized by the presence of microangiopathic hemolytic anemia, thrombocytopenia and variable target organ lesion. Despite its rarity, a prompt diagnosis is essential and the overlapping symptoms with other thrombotic microangiopathies, as well as the multiplicity of nonspecific manifestations, makes its diagnosis highly challenging.

Aims: To study in detail the natural history of aPTT and possible morbimortality factors associated on the moment of diagnosis and its evolution on the first 30 days of disease.

Methods: Retrospective evaluation of patients diagnosed with aPTT between

2009 and 2019 in a terciary center.

Results: There were 26 patients included in this study, with a median age of 41 years and of which 58% (n=15) were female. At the time of diagnosis, 16 patients (62%) presented neurological manifestations, but only 2 (11%) of the patients submitted to CE-CT had imagiological findings. Cardiac involvement, defined by troponin’s elevation, was observed on 14 patients of the 16 whom have measured it (54%). Other manifestations of the disease included haemorrhage, mainly petechiae and ecchymosis (n=11; 42%), fever (n =10; 39%) and renal dysfunction (n =7, 27%). Only 1 patient presented with the classic pentad. On blood analysis, 73% of the patients presented with haemoglobin levels below 9 g/dL (median: 8,2g/dL) and 77% with a platelet count below 30x10 9 (median: 15x10 9 /L).Five patients (19,5%) had a previous diagnose of a comorbid autoimmune disease. Most patients were treated with plasmapheresis (85%, n=22) and/or corticotherapy (89%, n=23). From the patients treated with plasmapheresis, 19 (73%) started the treatment on the day of admission or first day of hospitalization. The mortality in this sample was 27% (n=7): 3 patients (12%) died due to acute aPTT and the remaining 4 from other causes (3 from an oncological disease and 1 from septic shock). Progression free survival (PFS) and Global survival (GS) were 77% and 81% on the first year, and 60% and 73% at five years, respectively. Patients treated with plasmapheresis only on the beginning of the 2nd day after admission showed significant worse GS compared to those who started the same treatment sooner (HR =8,44, p=0.022, IC 1,35 = [1,35-52,65]. Older age (M=61,67, DP=19,21) was associated with marginally significance to therapeutic refractoriness (p=0.089). The presence of neurological symptoms, fever, high troponin levels, low hemoglobin, a raise of LDH and low platelet counts did not interfere significantly on the PFS and GS.

Summary/Conclusion: aPTT is a rare and severe disease that should be promptly diagnosed, since time until the beginning of treatment is determinant on the disease mortality. Given the wide range of differential diagnosis and the variability of symptoms, the presence of microangiopathic hemolytic anemia associated with thrombocytopenia should raise our clinical suspicion. Although organ dysfunction has been proved to have an impact on prognosis, this association was not demonstrated in our sample, probably due to the early initiation of treatment.

PB2310: VALUE OF PLATELET SURFACE GP IIB-IIIA AND BLEEDING QUESTIONNAIRE IN IDENTIFICATION OF EGYPTIAN PATIENTS WITH GLANZMANN’S THROMBASTHENIA

L. Saleh 1,* , D. Ali 2 , M. Sarhan 3 , H. Azzam 1

1 Hematology section-Clinical pathology department, Mansoura University; 2 Blood Bank – Dekirnis general Hospital, Dekirnis general Hospital; 3 Pediatric Hematology, Mansoura University Children Hospital, Mansoura, Egypt

Background: Patients with inherited platelets disorders are characterized by a prolonged clinical history of mucocutaneous bleeding. Glanzmann’s Thrombasthenia (GT) is an autosomal recessive inherited platelet disorder, characterized by prolonged bleeding time, normal platelet count, and absence of platelet aggregation in response to all platelet agonists except for ristocetin. Although Glanzmann’s thrombasthenia is usually considered to be a rare disorder, it can be almost as frequent as hemophilia and von Willebrand disease in regions where consanguinity is common, such as Indians, Iraqi Jews, Arabs and Iranians. GT has been subclassified into types I, II, and III. Patients with less than 5% of normal GPIIb/IIIa are classified as type I and 5–20% as type II. Type III variants usually have dysfunctional receptors with near-normal GPIIb/IIIa levels. Bleeding severity is unpredictable in GT. Some patients never have serious bleeding and others are free of bleeding complications. Although, it is clear that GT is associated with significant bleeding, the relation between types of GT and bleeding score remains unclear.

Aims: this study was designed to evaluate glycoproteins IIb-IIIa levels (CD41and CD61) by flowcytometry on the platelet surface of patients with GT in addition to bleeding score for proper typing and bleeding assessment of Egyptian patients with GT.

Methods: All the patients and their parents/guardians were subjected to a bleeding questionnaire to assess their bleeding score developed by ISTH/SSC-BAT (Bleeding assessement tool) and were investigated for platelet surface receptor GPIIb-IIIa (CD41 and CD61) by using flow cytometry.

Results: Our study included 16 pediatric patients with GT, 31% of GT patient families have consanguineous marriages. On subgrouping our patients according to CD41 and CD61 levels, most of our patients exist in the type 1 group 11 patients (68.8 %) with absent or extremely reduced levels (<5%) of CD41 and CD61. Three (18.8%) patients were categorized as type II with median CD41 levels of 11.3% and median CD61 levels of 28.6%. Two (12.4%) patients exhibited a normal or near-normal amount of CD41 and CD61 (median: 95%). The analysis of data showed that most of our patients with GT had mild and moderate bleeding episodes (62.5 and 31.3%, respectively), while severe bleeding only represents 6.2%. The commonest form of bleeding in our patient was epistaxis and cutaneous bleeding representing 94% followed by oral cavity bleeding, tooth extraction bleeding and bleeding after surgery representing 50% then bleeding from minor wound, gastrointestinal bleeding and hematuria (25, 19 and 6% respectively), no patients had menorrhagia.

Summary/Conclusion: flow cytometry should be used as an important tool in the screening and typing of GT patients. Type I GT is the most common type in our patients with lowered mean CD41 expression in comparison with CD61.No significant correlation was found between different forms of bleeding and percentage CD41,CD61 and double expressing platelets.

PB2311: LOW-DOSE PREDNISON AND ELTROMBOPAG COMBINATION AS FRONTLINE TREATMENT IN NEWLY DIAGNOSED ITP PATIENTS

S.-Y. Liu 1 , C.-Y. Yuan 2 , X.-H. Sui 1,3 , X. Liu 1,3 , Y. Li 1,3 , N.-N. Shan 1,3,*

1 Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan; 2 Department of Hematology, Dezhou Hospital Affiliated to Qilu Hospital of Shandong University, Dezhou; 3 Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by isolated low platelet counts due to platelet autoimmune destruction and megakaryocytogenesis defects. The most routine first-line treatments are glucocorticoid and IVIG, while patients with severe refractory ITP are often resistant to glucocorticoid or IVIG treatment and respond to TPO-RAs. And the increase of ineffective megakaryocytes may be due to insufficient circulating TPO. Eltrombopag is approved as an orally available TPO-RAs for ITP, which efficiently induces recovery of platelet counts by stimulating the production of megakaryocytes at the level of the hematopoietic stem cell.

Aims: In order to further explore the combined application of eltrombopag and first-line drugs, the efficacy and recurrence rate of the combination of eltrombopag and prednisone in the treatment of ITP were evaluated in our experiment.

Methods: We performed a retrospective study to assess the response to prednison (dose 0.5-1 mg/kg/day with taper) in combination with eltrombopag (dose 25 mg/day) in 11 patients with newly diagnosed ITP (age ≥ 17 years, the median age was 45.2 years, nine patients (81.8%) were women). None of the patients had been treated with glucocorticosteroids prior to recording. Participants should be excluded if they have drug-related thrombocytopenia, are seropositive for HCV, HIV, and HBV, have an active infection, or have other malignant diseases. Early response was defined as an increased platelets response at 1 week. (R, platelets ≥ 30 × 10 9 / L, with at least doubling baseline; CR, platelets ≥ 100 × 10 9 / L)

Results: Eleven consecutive patients were enrolled, and the therapeutic effect was shown in Table 1. The median basal platelet account at diagnosis was 11 × 10 9 /L (range 2-37). All patient achieved early response (R or CR) with prednison and eltrombopag at 8 days, eight (72.7%) achieved CR, and three (40%) achieved R. CR at 1 months (platelets≥100×10 9 / L) was achieved in 81.8% of patients. Approximately 88.9% of patients, excluding two unrecorded persons, achieved CR beyond 3 months. Duration of prednisone was 69.1 days (range 43-150). No obvious adverse reactions occurred during the treatment. Five patients (45.5%) had achieved treatment-free remission (TFR) and median follow-up was 5 months (range 1-10), one patient relapsed to follow-up 1 month after response evaluation. Currently, nine (81.8%) patients remain in CR, one (9.1%) in R, with an additional 9.1% relapsing.

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Summary/Conclusion: The combination of low-dose prednison and eltrombopag have the benefit of a higher early response (R or CR) rate inducing a more rapid increase in platelet count than corticosteroids alone, and usually demonstrate a more durable response. The response was also relatively lasting in follow-up after drug withdrawal. Low-dose prednisone have fewer side effects and are particularly suitable for women and older adults who cannot tolerate excessive corticosteroids. And low-dose eltrombopag also have high safety and tolerability. Thus, prednison/eltrombopag has been used as frontline therapy in ITP.

PB2313: DARATUMUMAB AS A TREATMENT FOR ADULT IMMUNE THROMBOCYTOPENIA: A PHASE II STUDY WITH SAFETY RUN-IN (THE DART STUDY)

G. Tsykunova 1,2,3,* , P. A. Holme 3,4 , H. Thi Thyuet Tran 5 , T. H. A. Tvedt 4 , L. Munthe 6,7 , M. Michel 8 , H. Frederiksen 9 , J. Bussel 10 , D. Kuter 11 , W. Ghanima 1,3

1 Departments of Medicine, Hematology-Oncology, and Research, Østfold Hospital Trust, Kalnes; 2 Division of Hematology, Department of Medicine, Haukeland University Hospital, Bergen; 3 Institute of Clinical Medicine, University of Oslo; 4 Department of Hematology, Oslo University Hospital; 5 Department of Hematology, Akershus University Hospital; 6 K.G.Center for B-cell malignancies, Institute for Clinical Medicine,University of Oslo; 7 Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway; 8 Service de Médecine Interne 1, Centre de Référence des Cytopénies Auto-Immunes de l’Adulte, Centre Hospitalo-Universitaire Henri Mondor, Créteil, France; 9 Department of Hematology, Odense University Hospital, Odense, Denmark; 10 Department of Pediatrics, Weill Cornell Medicine, New York; 11 Division of Hematology, Massachusetts General Hospital,Harvard Medical School, Boston, United States of America

Background: Immune thrombocytopenic purpura (ITP) is an autoimmune condition.

We hypothesize that persistent antibody-producing plasma cells can be the source of the treatment failure in some ITP patients and treatment that directly targets plasma cells may overcome resistance to treatment.

Daratumumab is an anti-CD38 antibody approved for the treatment of multiple myeloma and has been successfully used to treat autoimmune hemolytic anemia and ITP in children.

Aims: We initiated a multicenter, open-label total dose-escalating phase II study with a safety run-in to evaluate the safety and efficacy of daratumumab in patients with ITP ( NCT04703621 ). The first 3 patients were included in the safety run-in between January and May 2021. We present results of a follow-up on patients treated in the safety run-in phase.

Methods: Main inclusion criteria: adults (age ≥18) with primary ITP and platelet count of ≤30X10 9 /L who experienced failure of corticosteroid therapy and at least one second-line therapy including rituximab and/or TPO- RA. A platelet count of 15-30×10 9 /L was required for entry into the safety run-in.

For the safety run-in phase, 3 patients were to receive 4 weekly subcutaneous daratumumab injections followed by a 4-week observational period. Enrollment of the next patient in this phase occurred only after the previous patient have had completed treatment and the observational period.

In the subsequent phases of the study, 9 patients (Cohort 1) will receive 8 weekly injections. If no serious safety issues appear, the study will proceed to Cohort 2 in which 9 subsequent patients will receive 8 weekly daratumumab injections followed by 2 injections administered every other week. All patients will receive standard premedication before each injection with antihistamine, corticosteroid (methylprednisolone or equivalent) and paracetamol. Participants must remain on stable dose of steroids and TPO-RAs during the 2 weeks preceding the inclusion without dose escalation during the study.

The primary endpoint is response defined as a platelet count ≥50×10 9 /L in 2 measurements 12 weeks after treatment initiation for safety run-in and Cohort 1, and 16 weeks for Cohort 2, in the absence of rescue therapy after week 8. Safety will be assessed by the incidence and severity of adverse events. Secondary endpoints are duration of response, defined as duration of sustained platelet count ≥50×10 9 /L between the end of treatment and end of study (duration does not consider the 4 weeks following the last daratumumab injection in the study); time to treatment failure defined as the time with platelet count ≥50×10 9 /L from 4 weeks after the last daratumumab injection to the first platelet count <30×10 9 /L.

Results: As of February 01, 2022, all 3 patients have completed treatment in the safety run-in. 2/3 responded to study treatment at week 12, one relapsed by study week 24. There was only a slight decrease of immunoglobulin G levels. No serious adverse events or adverse events >grade 2 were reported in this group. Results of follow-up data on platelet response is presented in Figure 1. Enrollment in Cohort 1 is ongoing with 5 patients included so far.

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Summary/Conclusion: Treatment with daratumumab in ITP patients shows promising results and seems safe.

PB2314: OLIPUDASE ALFA FOR ADULTS WITH ACID SPHINGOMYELINASE DEFICIENCY: IMPROVEMENTS IN CROSSOVER PLACEBO PATIENTS AND FURTHER IMPROVEMENTS IN ORIGINAL OLIPUDASE ALFA PATIENTS AFTER 2 YEARS IN ASCEND TRIAL

J. Villarrubia 1,* , M. Wasserstein 2 , A. Barbato 3 , R. C. Gallagher 4 , R. Giugliani 5 , N. B. Guelbert 6 , J. B. Hennermann 7 , C. Hollak 8 , T. Ikezoe 9 , R. Lachmann 10 , O. Lidove 11 , P. Mabe 12 , E. Mengel 13 , M. Scarpa 14 , E. Senates 15 , M. Tchan 16 , B. L. Thurberg 17 , A. Yarramaneni 18 , A. M. Rawlings 18 , Y. Kim 19 , M. Kumar 18

1 Hematology, Hospital Universitario Ramón y Cajal, Madrid, Spain; 2 Children’s Hospital at Montefiore, Albert Einstein College of Medicine, Bronx, NY, United States of America; 3 Clinical Medicine and Surgery, Federico II University Hospital, Naples, Italy; 4 Pediatrics, University of California, San Francisco, CA, United States of America; 5 Med Genet Serv & DR BRASIL Research Grp, HCPA, Dept Genetics, UFRGS & INAGEMP, Porto Alegre, Brazil; 6 Reina Fabiola University Clinic, Córdoba, Argentina; 7 Pediatric and Adolescent Medicine, Villa Metabolica, University Medical Center, Mainz, Germany; 8 Endocrinology and Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands; 9 Hematology, Fukushima Medical University, Fukushima, Japan; 10 Charles Dent Metabolic Unit, National Hospital for Neurology and Neurosurgery, London, United Kingdom; 11 La Croix St Simon Hospital, Paris, France; 12 Clinica Santa Maria, Santiago, Chile; 13 Clinical Science for LSD, SpinCS, Hochheim, Germany; 14 Regional Coordinator Centre for Rare Diseases, University Hospital of Udine, Udine, Italy; 15 Istanbul Medeniyet University, Istanbul, Turkey; 16 Genetic Medicine, Westmead Hospital, Sydney, Australia; 17 Sanofi, Cambridge, MA; 18 Sanofi, Bridgewater, NJ, United States of America; 19 Sanofi, Paris, France

Background: Acid sphingomyelinase deficiency (ASMD) is a rare debilitating lysosomal storage disease characterized by pulmonary dysfunction, hepatosplenomegaly, thrombocytopenia/bleeding issues, and dyslipidemia. Disease awareness is low and diagnostic delays are common. Undiagnosed patients may be referred to hematologists for thrombocytopenia/bleeding issues and splenomegaly. Olipudase alfa, intravenous-recombinant-human ASM, is in late-stage development (Sanofi Genzyme) for non-central-nervous-system manifestations of ASMD. The ASCEND study ( NCT02004691 ), a phase 2/3 placebo-controlled trial in 36 adults with ASMD, has completed its primary analysis. At Year 1, olipudase alfa patients compared to placebo patients (1:1 randomization) had statistically significant increases in %predicted diffusing capacity of carbon monoxide (DL CO ) and decreases in spleen and liver volume. Thirty-five patients continued in an open-label trial extension: 17/18 placebo patients, 18/18 olipudase alfa patients.

Aims: We report Year 2 results for the former placebo patients after 1 year of olipudase alfa (crossover group) and for the continuing olipudase alfa patients after 2 years of olipudase alfa (original olipudase alfa group).

Methods: Patients underwent dose-escalation to 3.0 mg/kg every 2 weeks for ~14 weeks when starting olipudase alfa. Changes from baseline are presented as least-square (LS) mean ANCOVA % change ±standard error of the mean for %-predicted DL CO , spleen volume, liver volume, platelet count, liver function, and lipid profile. Change in lung high-resolution computed tomography (HRCT) scores for ground glass appearance is presented as LS mean ANCOVA absolute change from baseline.

Results: Thirty-three of 35 patients completed Year 2. In Year 2, improvements for crossover patients paralleled the olipudase alfa group in the primary analysis and improvements continued for the original olipudase alfa group (Table). In the crossover group, spleen volume decreased by 36.0±3.0% (n=11); %-predicted DL CO increased by 28.0±6.2% (n=10); spleen volume decreased by 36.0±3.0% (n=11); liver volume decreased by 30.7±2.5% (n=11), and platelet count increased by 21.7±6.4% (n=15). In the original olipudase alfa group, %-predicted DL CO increased by 22.2±3.4% (n=17) at Year 1 and 28.5±6.2% at Year 2 (n=10); spleen volume decreased by 39.5±2.4% (n=17) at Year 1 and 47.0±2.7% (n=14) at Year 2; liver volume decreased by 27.8±2.5% (n=17) at Year 1 and 33.4±2.2% (n=14) at Year 2, and platelet count increased by 16.6 ±4.0% at Year 1 (n=18) and 24.9±6.9% (n=13) at Year 2. HRCT ground glass appearance score decreased by 0.30±0.5 (n=14) at Year 2 for the crossover group and decreased by 0.45±0.13 (n=18) at Year 1 and 0.48±0.07 (n=16) at Year 2 for the original olipudase alfa group. Improvements in dyslipidemia, liver function, liver sphingomyelin clearance, and plasma lyso-sphingomyelin in crossover patients paralleled those seen in the original olipudase alfa patients in Year 1; original olipudase alfa patients maintained these benefits in Year 2. Overall, 99% of treatment-emergent adverse events (AEs) were mild or moderate, with one treatment-related serious AE. No patient discontinued due to an AE.

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Summary/Conclusion: Crossover patients had the same magnitude and time course of clinical improvement seen in patients receiving olipudase alfa for 1 year, while 2-year olipudase alfa patients had sustained or further improvements. Olipudase alfa reduces manifestations of chronic ASMD in adults and has sustained efficacy. Increased awareness of ASMD among hematologists is important for timely diagnosis and disease management.

PB2315: EFFICACY AND SAFETY OF TWO DIFFERENT LOW-DOSE RITUXIMAB REGIMENS FOR CHINESE CHILDREN PATIENTS WITH PRIMARY IMMUNE THROMBOCYTOPENIA

X. Zhu 1,*

1 Hematology department, Beijing Children‘s Hospital affiliated to Capital Medical University, Beijing, China

Background: Rituximab is one of the second-line treatments for ITP. At present, there are few studies on low-dose rituximab, lacking of a large number of prospective and randomized trials to support the efficacy and safety of low-dose rituximab, especially in children’s ITP. Influenced by COVID-19, we used two low-dose rituximab regimens before and after March 2020 for second-line treatment of children’s ITP.

Aims: To compare the efficacy and safety of two different regimens for low-dose rituximab of children patients with chronic /refractory ITP, so as to provide basis for clinical treatment.

Methods: 83 children patients were enrolled in this study and non-randomly assigned to receive 100mg/200mg (body weight 30kg) rituximab weekly for 4 weeks (group A, 53 cases) or a single dose of 375mg / m2 rituximab (group B, 30cases). The study was follow-up for at least half a year.

Results: The baseline data of group A and B were the same. For group A: Overall and complete response (OR and CR) rates were 35.8% and 15%, respectively; the side effects rate is 3.8%. In responders, the median time to response was 4 (1 -12) weeks, with a median follow-up time of 12 (6 ~ 36) months, 6 of 19 responders (31.6%) relapsed. For group B: OR and CR rates were 36.7% and 23%, respectively; the side effects rate is 10%. In responders, the median time to response was 1 (1 ~ 4) weeks, with a median follow-up time of 11.5 (6 ~ 17) months, 4 of 11 responders (36.4%) relapsed. No significant difference in the OR, NR, relapse free survival and incidence of side effects was observed in patients between the two groups.

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Summary/Conclusion: The two low-dose rituximab regimens in the treatment of ITP in children both are safe and effective; The single-agent scheme is more recommended because of easier use and not increasing safety events.

PB2316: EFFICACY AND SAFETY OF ELTROMBOPAG IN PEDIATRIC PATIENTS WITH PRIMARY IMMUNE THROMBOCYTOPENIA AND ITS EFFECT ON CELLULAR IMMUNE FUNCTION

?. 吴 1

1 安徽医科大学第二附属医院, 安徽医科大学第二附属医院, 合肥市, China

Background: eltrobopag is the only TPO-RA approved by the US FDA for the treatment of ITP in children, and foreign prospective multicenter randomized controlled clinical studies have found that atropopa has a good efficacy in the treatment of chronic ITP in children, and some children still maintain the efficacy after stopping the drug, but there are few reports on the application of atropopa in children with persistent/chronic ITP in China. The clinical data of the Treatment of Persistent/Chronic Pediatric ITP in the Second Affiliated Hospital of Anhui Medical University are retrospectively analyzed to evaluate the efficacy and safety of eltrobopag in the treatment of chronic/persistent ITP in children, and to explore the relevant factors that may affect the efficacy of Erlopapa in children, in order to better guide the clinical application of eltrobopag.

Aims: To evaluate the efficacy and safety of eltrobopag in children with persistent /chronic ITP and to explore its effect on cellular immune function.

Methods: The clinical characteristics of 28 pediatric persistent or chronic ITP patients who received eltrombopag from October 2019 to June 2021 were retrospectively analyzed.

Results: 1、Efficacy: in total,20 patients (71.4%) achieved overall response, 12 (42.8%) achieved complete response, and 8 (28.5%) achieved partial response. And the clinical bleeding scores were significantly decreased after treatment (P < 0.05). In children with ITP who had a hematologic response, the median onset time was 14 d. 2、Safety: Eltrombopag was well tolerated, the common adverse events included elevated transaminase (5 cases) and serum bilirubin (1 case); None of the 28 children experienced serious adverse drug reactions during the treatment period. 3、 Platelet responses were not different irrespective of age, sex, disease duration, and bone marrow megakaryocyte count levels (P > 0.05). 4. Effects of eltrombopag on cellular immune function: there were no differences in the percentages of Treg cells， B lymphocytes, Th1, and Th2 cells between before and after treatment。But the group with higher pretreatment levels of Treg cells had better hematologic responses than the group with low levels of Treg cells (P < 0.05).

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Summary/Conclusion: Eltrombopag is safe and effective in pediatric patients with primary ITP ，It can effectively reduce the bleeding risk of children with persistent / chronic ITP and improve the quality of life of children with ITP，and the level of Treg cells in children may be a predictor of eltrombopag response rate.

PB2317: COINHERITANCE OF COMBINED FACTOR VII AND FACTOR XIII DEFICIENCY IS ASSOCIATED WITH A MILD BLEEDING PHENOTYPE IN PATIENT WITH NON-TRANSFUSION DEPENDENT THALASSEMIA

O. Alshareef 1,* , B. Allahyani 1 , E. Alnasser 1

1 Pediatrics, King Abdul Aziz Hospital, Jeddah, Saudi Arabia

Background: The coinheritance of FVII and FXIII is extremely rare. FVII and FXIII deficiency was common in the Saudi population. However, double heterozygous have not been described in hypercoagulable states such as thalassemia. This is a case of combined FVII and FXIII deficiency in a non-transfusion-dependent thalassemic presented with hemarthrosis.

Aims: This report aims to describe a case of combined coagulation factor VII and factor XIII deficiency in non-transfusion dependent thalassemia.

Methods: We conducted a retrospective chart review.

Results: An 8-year-old boy known to have non-transfusion dependent thalassemia presented with painful knee swelling after significant trauma. He was pale, jaundiced, with absent palpable hepato-splenomegaly. Soft tissue ultrasound suggested hemarthrosis. Coagulation factors assays showed normal results with a notably combined deficiency of coagulation factors VII (<50% activity) and FXIII (<10% activity).

During infancy, he had a history of prolonged oozing after circumcision. Since then, the child has had a history of multiple bleeding episodes post minor trauma that was controlled by fresh frozen plasma (FFP) transfusion. Moreover, with a significant history of traumatic subgaleal hematoma, he was admitted and received FFP and packed red blood cells (PRBCs).

Summary/Conclusion: We have identified a previously unreported mild phenotypic combined FVII and FXIII deficiency in patients with hemoglobinopathy disorder. Hypercoagulable state in hemoglobinopathies might mitigate the bleeding phenotype of factor deficiency, though bleeding history should be thoroughly investigated in such cases.

PB2318: ACQUIRED WILLEBRAND SYNDROME REVEALING ASYMPTOMATIC MULTIPLE MYELOMA: A CASE REPORT

W. Chawad 1,* , A. Laabichi 1 , Y. Moussadik 1 , Y. chhih 1 , F. ibourk idrissi 1 , A. taouch 1 , S. el fari firdaous 1 , H. talebi 1 , H. hanani 1 , S. belkhettab 1 , M. bouaouad 1 , N. mouatassim 1 , H. khibri 1 , W. ammouri 1 , M. maamar 1 , H. harmouche 1 , Z. tazi mezalek 1 , M. adnaoui 1

1 internal Medecine and Clinical Hematology, Ibn Sina’s Hospital, Rabat, Morocco

Background: Acquired Willebrand syndrome (AvWS) is a rare hemorrhagic syndrome, clinically and biologically identical to constitutional Willebrand syndrome (vWS). It predominantly affects subjects with no personal or family history of hemorrhage, at an average age of 60. Its prevalence has been estimated by some authors at 0.04%, and over 300 cases have been reported since its discovery in 1968. The pathologies responsible for acquired Willebrand syndrome are diverse; including lymphoproliferative and myeloproliferative syndromes,autoimmune and cardiovascular diseases, solid tumors, and even certain medications. The physiopathology is complex and leads to an accelerated clearance of Willebrand factor. A Willebrand anti-factor antibody is preferentially found during lymphoid proliferation, while the mechanical destruction of high molecular weight multimers is observed during heart problems.

Aims: To emphasize the pre-existing link between acquired Willebrand disease and multiple myeloma, as well as show the complexity of this relation.

Methods: We report the case of a patient in whom the diagnosis of acquired Willebrand syndrome allowed to reveal an asymptomatic multiple myeloma.

Results: A 50-year-old patient was admitted for the etiological assessment of an iron deficiency anemia due to a hemorrhagic symptomatology made up of rectal bleeding and epistaxis. There was no family or personal history of hemorrhage, especially during circumcision and dental extractions. On the biological report, we found microcytic anemia with iron deficiency. The hemostasis assessment revealed an elongated partial thromboplastin time at 2.94 with a correct prothrombine ratio at 100%. Factor XIII has collapsed to 5%. FWV antigen was at 9.4%, and its activity at 3%. This biological profile was in favor of Willebrand disease type 3. On the etiological assessment, protein electrophoresis objectified a monoclonal peak with the presence of an immunoglobulin G kappa at immunofixation. The diagnosis of multiple myeloma was retained due to the presence of 28% of monotypic plasma cells for kappa light chains. CRAB criteria were absent except for anemia which has been attributed to chronic bleeding. ISS score was at 1. FISH was not carried out. The diagnosis of an AVWS on multiple myeloma was retained. Despite the absence of myeloma treatment criteria, the indication for chemotherapy has been made. The patient received 2 courses of chemotherapy combining bortezomib, thalidomide and dexamethasone. The hematological reassessment after 2 cures has shown a partial response on the monoclonal gammopathy. However, the absence of response on Willebrand’s disease was noted: the FWV antigen was at 3% and its activity at 11.5%, which resulted on the interruption of the chemotherapy protocol. The patient was put on rituximab for his von Willebrand’s disease according to the following protocol: 1g on Day 1, and on Day15. The cessation of the hemorrhagic syndrome was noted during the reassessment.

Summary/Conclusion: Acquired von Willebrand syndrome is a very heterogeneous hemorrhagic syndrome, usually characterized by mild to moderate hemorrhagic symptoms, which can be severe and life-threatening in some cases. During the investigation of a hemorrhagic syndrome in the elderly, it is crucial to think of the AWVS. We recommend screening for AvWS in patients followed for onco-haematological pathologies when unexplained abnormal bleeding occurs.the treatment of the monoclonal gammapathy can allow the regression of the hemorrhagic manifestations in some cases.

PB2319: ACQUIRED COAGULOPATHY: FACTOR X DEFICIENCY AT DIAGNOSIS OF MULTIPLE MYELOMA WITHOUT ASSOCIATED AMYLOIDOSIS

M. De La Torre De La Paz 1,* , G. Figaredo García Mina 1 , A. Guerrero Díez 1 , A. Rodríguez Alén 1 , M. D. L. O. Abío Calvete 1 , N. Rollón Simón 1 , J. Cuesta Tovar 1

1 Haematology, Hospital Universitario de Toledo, Toledo, Spain

Background: Plasma cell dyscrasias, including Multiple Myeloma (MM), are frequently associated with alterations in hemostasis tests and to a lesser extent with the presence of coagulopathy. Acquired factor X deficiency is usually associated with the presence of amyloidosis (AL) and very rarely appears in patients with MM without AL.

Aims: The aim of this work is to report the presence of acquired coagulopathy at the debut of Multiple Myeloma, specifically the presence of an acquired factor X deficiency.

Methods: Description of a clinical case diagnosed in our haematology service about the acquired FX deficiency at diagnosis of a patient with MM without AL

Results: A 77-year-old patient was diagnosed with MM IgG Lambda. Initial laboratory studies revealed an alteration in the hemostasis tests, not previously present, evidencing an acquired origin. She presented with prolonged PT and aPTT measurements, which corrected after incubation with normal plasma. Thrombin Time (TT) did not show alterations. FX was 19%. Given the frequent association between this deficit and AL, abdominal fat pad biopsy was performed, which was negative for Congo red staining. Treatment was started correcting the coagulopathy during this period. In 2021, she suffered an episode of epistaxis and she needed hospital admission. A relapse of his underlying disease and secondary worsening of the coagulopathy were observed, with a FX level of 8%. Furthermore, the TT was prolonged, the Clauss

fibrinogen decreased, and there was a mild multifactorial deficit. Treatment with Vitamin K, CCP and fibrinogen was started with temporary improvement in factor deficits. It was decided to perform BMO: 5% of pathological PCs. Congo red staining was negative. Cardiac MRI ruled out AL. The patient started a new line of treatment and a few days later, the haemorrhagic symptoms improved and the patient was discharged. Two months later, she was readmitted due to suspected sepsis abdominal. Abdominal CT shows nonspecific recto sigmoiditis and partial occlusion of the suprahepatic veins. The patient died a few days after admission.

Summary/Conclusion: Conclusions:

Although classic manifestations of MM (CRAB) are the most common at diagnosis of the disease, it is important to keep in mind the haemostatic alterations and haemorrhagic diathesis in these patients.

Although we do not know the exact pathophysiological mechanisms, it seems that the monoclonal protein interferes at different points of coagulation, which can lead to a fibrin clot with an abnormal structure, factor deficiencies, hypo / dysfibrinogenemia or hyperfibrinolysis.

Acquired factor X deficiency is widely described in the literature associated with amyloidosis (AL). However, its presence in MM patients without AL is extremely rare and there are few cases described in the literature.

We must suspect factor X deficiency in the presence of a TP and APTT prolongation that is corrected with mixing tests.

Treatment of acquired factor X deficiency is the treatment of the cause that originates it.

In the prevention and treatment of acute bleeding, administration of exogenous factor X is indicated. If not available, CCP or PFC can be used.

PB2320: AN INDICTION OF TREATING ADULT HEMOPHILIA IN A DEVELOPING COUNTRY: A RETROSPECTIVE ANALYSIS OF A SINGLE CENTER EXPERIENCE “PART I DESCRIPTIVE EPIDEMIOLOGY”

M. Elbogdady 1,* , S. Shamaa 1 , M. Nasr 1 , E. Azmy 1 , M. Elzaafarany 1 , T. Abo Zeid 1 , M. Taalab 1 , N. Eissa 1 , S. Saber 1 , M. Denwar 1 , H. Elkerdawy 1 , Y. Essam 1 , M. Atef 1 , B. Atef 1 , A. Mohsen 1 , E. Jamal 1 , M. Saafan 1 , A. Aly 1 , A. Talaat 1 , A. Elazab 1 , A. Elshamy 1 , S. Dheim 1 , Y. Hamouda 1 , R. Ibrahim 1 , M. Awad 1 , D. Ashraf 1 , N. Elbayomy 1 , A. Mokhtar 1 , A. Sasy 1 , E. Ebrahim 1

1 Hematology, Oncology center Mansoura university - Mansoura university - Egypt, Mansoura, Egypt

Background: Hemophilias are the most frequent Inherited bleeding disorders. In Egypt which has a population of approximately(80 million) consanguineous marriage are frequent, there fore recessive coagulation disorders reach a higherprevalence than in many other countries.

Aims: The aim of this study was to describe the epidemiological situation of adult hemophilia in Mansoura, Egypt as basedon retrospective analysis of our electronic clinical records at hematology unit Oncology center mansoura university (OCMU) between 2006 and 2021.

Methods: Adult patients complaining of hemophilia were recruited from hematology unit at OCMU from 2006 to 2021.Demographic characteristics, clinical history, and laboratory and treatment data together with long term complications were collected.

Results: The study included 117 recorded cases of Hemophilia (93 A & 24 B). Almost all the cases were previously diagnosedand were either on follow up with a pediatric unit and got their service transferred to our unit or came with ableeding emergency or for preoperative assessment. Male were 105 with 12 females with the females being 10 in A &only 2 in B. The mean age of first follow up at OCMU was 24.7 years with the minimum and the maximum being 17 & 54 years. The severity of hemophilia was distributed as follows 22% severe, 30 % moderate and the remaining 48%mild. HCV infection was found in 38 % of cases due to increased plasma transfusion being the main line of treatment.

Summary/Conclusion: Dealing with adult hemophilia in a developing country is hard due to limited access to factor and poor patientcompliance and the transfusion related infections and complication are very hazardous & frequent with poor controlof bleeding episodes and joint affection leading to loss of patient’s adherence and increased adverse events

PB2321: A COMPLEX GENOTYPE THAT TRANSLATES INTO A BLEEDING PHENOTYPE

L. Fonseca 1,* , F. Martins Pereira 1 , A. Brito 1 , F. Lobo 1 , M. Costa 1

1 Serviço de Sangue e Medicina Transfusional, Centro Hospitalar Tondela-Viseu, Viseu, Portugal

Background: Epistaxis is one of the most common complaints at emergency room and is recurrent among children. Most cases are of benign origin. Trauma, inflammation, vascular alterations, drugs or coagulopathies are the most frequent causes. In this latter case, it may be the first or even the only symptom.

Aims: To describe a clinical case.

Methods: A 9-year-old girl was referred to a coagulopathies specialist due to frequent and profuse epistaxis and gingivorrhagias. She had no bleeding complication during tooth extractions or vaccination. In family history, only her father and brother had recurrent episodes of epistaxis. No surgical history or medication were reported. The results of coagulation screening tests were: prolonged prothrombin time (PT) of 14.2 seconds, normal activated partial thromboplastin time (aPTT) of 34.2 seconds and low fibrinogen levels (1.8 g/L). The initial study also included a complete blood count and C-reactive protein, which were normal. Coagulation factor levels were measured and FVIII and FVII were normal, but FV was slightly decreased (41%). The VWF:Ag was 80.5% and VWF:Rco was 79.3% (VWF:RCo/VWF:Ag ratio 0,98). Because of the reduced plasma levels of FV, genetic tests were required. A panel of 43 genes was made in Next-Generation Sequencing (NGS).

Results: NGS showed 3 genetic alterations: functional polymorphism on F5 gene associated with decreased FV levels; mutation in heterozygosity on VWF gene (autosomal dominant variant) characterized by increased affinity of VWF for platelet GPIBa, suggestive of von Willebrand Disease (VWD) type 2B; mutation in heterozygosity on NBEAL2 gene associated with macrothrombocytopenia.

Summary/Conclusion: The study in this patient suggests that she has VWD type 2B. Although, the co-inheritance of the other 2 variants may be contributing to the patient hemorrhagic phenotype. Additional studies are essential to elucidate the genotype-phenotype correlation.

PB2322: TTP AFTER COVID 19

D. Gagloeva 1,* , E. Zhelnova 2

1 Hematology ahd high dose chemotherapy, State Moscow City Hospital #52, Moscow; 2 Гематология ahd высокодозная химиотерапия, Государственная Московская городская больница № 52, Москва, Russia

Background: It is widely known that viruses can cause autoimmune disorders. Since 2019 medical community has been convinced, that SARS COV2 can also initiate such autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, antiphospholipid syndrome, etc. In our hospital we faced a clinical case of thrombotic thrombocytopenic purpura (TTP) after vaccination and SARS COV2 – infection

Aims: to clarify more possible autoimmune disorders induced by COVID 19 and complications after vaccination against SARS COV2

Methods: A 72-year-old man was admitted to hematological department in August 2021 because of rapidly worsening general condition, hemolytic anemia (hemoglobin 6.82 mmol/l, hematocrit 1,28), and thrombocytopenia (28 Gpt/l) for unknown reasons. Additionally, he was found to have an elevated lactate dehydrogenase (63.42 μmol/l/s) and bilirubin (86 μkmol/l/s). Direct and indirect Coombs test were negative. He was also presented with cognitive defects and neurologic disorders. It is known from the case history, that he had been vaccinated against SARS COV2 with one of the Russian vaccines one months before admitting to our clinic. After that he was admitted to the hospital with SARS COV2 –associated pneumoniae. He underwent a course of therapy, including anticoagulants and IL-6 inhibitors. When pneumonia regressed, he was discharged. But one week later he started to feel worse. We suggested autoimmune hemolytic anemia and gave him steroid pulse therapy according to vital indications, with no improvement. The differential diagnostic was continued among hemolytic-uremic syndrome, TTP, cancer, autoimmune disorder and viral infections. We detected extremely low level of protease ADAMTS13 (5%) and diagnosed TTP. We started daily plasma exchange #15, which ended with complete remission of the disease – total regress of cognitive defects and neurologic disorders, rapid raise of thrombocyte level and hemoglobin.

Results: Our patient was completely cured. We have 6 months of observation data on him. There were no relapses of TTP during this time. The cause of manifestation of the TPP in 72 year- old patient remained unclear. To date, there are more and more cases of autoimmune diseases after vaccination and COVID 19. In particular, one similar case of TTP after Pfizer vaccine injection was already described.

Summary/Conclusion: Thus, previous vaccination and coronavirus infection, could be the possible causes of TTP in this case. However, another equally important question still remains open – what exactly contributed to autoimmunization - vaccination or COVID 19. Further research is required. That is why more careful selection of patients for vaccination is required.

PB2323: ACQUIRED HEMOPHILIA: A CASE REPORT

N. Haddad 1,* , S. ABDI 1

1 Central laboratory, CHU Blida, Blida, Algeria

Background: Acquired hemophilia A (AHA) is a rare disease resulting from autoantibodies (inhibitors) against endogenous factor VIII (FVIII) that can lead to life-threatening bleeding. It may be secondary to an underlying disease or idiopathic. The annual incidence is 1.5 cases per million per year, with two peaks in frequency: in postpartum women (2 to 15%) and in patients aged over 60 (75%). 80% of patients have extensive unexplained bruising requiring urgent laboratory exploration. Mortality due to misfortune is between 8 and 22%.

Aims: we discuss a case of acquired hemophilia A, presenting as spontaneous bruising in an elderly patient.

Methods: This is an 80-year-old man, with a history of type I diabetes and hypertension, referred to our laboratory for exploration of extensive bruising, which appeared spontaneously.

The questioning did not reveal any personal or family history of abnormal bleeding, or the use of any anticoagulant or antiplatelet medication.

Results: Hemostatic tests of the patient revealed isolated prolonged aPTT to 59 sec (control 29 sec) with a normal PT. The measurement of the FVIII activity revealed a level of 4.5 % (normal value 60–150%). Accordingly, factor VIII inhibitors were detected at 04 Bethesda units (BU).

Recombined FVIIa have been used in the treatment of bleeding episode. Immunosuppressive treatment with rituximab was administered in order to remove the factor VIII inhibitor. After 04 cures of rituximab, the aPTT wase normalised (patient 30 sec/control 30 sec) which means a total removal of factor VIII inhibitor.

Summary/Conclusion: Diagnosis of Acquired hemophilia A is considered a challenge given its nonspecific presentation and low prevalence worldwide.

PB2324: SAFE ADMINISTRATION OF SECOND DOSE OF MRNA COVID-19 VACCINE FOLLOWING DEVELOPMENT OF IDIOPATHIC THROMBOCYTOPENIC PURPURA

A. Hajizadeh Barfejani 1,* , S. Prasad 1 , D. Henry 2

1 Royal College of Surgeons in Ireland, Dublin, Ireland; 2 Hematology and Oncology, University of Pennsylvania, Philadelphia, United States of America

Background: Multiple cases of immune thrombocytopenia (ITP) occurring after SARS-CoV-2 mRNA vaccines have recently reached public attention. It has been reported in patients with previous ITP or other autoimmune diseases and in individuals with no associated past medical history. The management, and the recurrence of ITP with booster doses are still not well investigated and remains a challenge for clinicians.

Aims: To report potential benefit of Thrombopoietin receptor agonist (TPO) following immune thrombocytopenia (ITP) associated with COVID-19 vaccination.

Methods: Case report.

Results: A 36-year-old previously healthy woman presented with a 5-day history of menorrhagia, epistaxis, gingival bleeding, and petechial rash in extremities. The patient received her first dose of the BNT162b2 COVID-19 Vaccine two weeks before symptom onset. Laboratory workup on admission revealed a platelet count of less than 4,000/ml, hemoglobin of 14.7 g/dL, and white blood cell count of 10,900/mL. A peripheral smear confirmed severe thrombocytopenia with no schistocytes. Additional studies showed normal prothrombin time, partial thromboplastin time, basic metabolic panel, folate, B12, and Thyroid-stimulating hormone. Given the clinical and laboratory findings, the patient was diagnosed with Idiopathic thrombocytopenic purpura (ITP). She was started on a three-day course of Intravenous immune globulin (IVIG) 1 g/kg/d and methylprednisolone 60 mg/d, after which her symptoms improved and the platelet count increased to 80,000/ml. The patient was discharged three days after admission on a four-day prednisone taper plan. She was started on Eltrombopag 50 mg daily two weeks after discharge and received her second dose of the COVID-19 vaccine three weeks after discharge without any adverse reaction. Her platelet count was 356,000/ml at the time of initiation of Eltrombopag and increased to 668,000/mL after the completion of four weeks of treatment (three weeks after the second dose of vaccination). In a follow-up appointment three weeks after discontinuation of Eltrombopag, she denied any recurrent symptoms of bleeding or menorrhagia, and her platelet count was normal (371,000/ml).

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Summary/Conclusion: Multiple cases of thrombocytopenia have been reported in recipients of both BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) COVID-19 Vaccines so far, with a reported incidence rate of 0.80 per million doses for both vaccines. Based on an annual incidence rate of 3.3 ITP cases per 100,000 adults, the observed number of all thrombocytopenia cases following administration of mRNA COVID-19 vaccines does not exceed the number of expected ITP cases. Symptomatic ITP is a severe condition that carries the risk of fatal hemorrhage and is generally treated with corticosteroids and/or IVIG. In refractory disease, splenectomy, or administration of TPO agonists can be considered. In patients with vaccine associated ITP, using TPO agonists earlier might be considered to avoid the use of immunosuppressants shortly after vaccination, which might impair the intended immunity against SARS-CoV-2.

PB2325: APPLICATION AND COMPARISON OF TWO PHARMACOKINETIC DETECTION METHODS IN CHILDREN WITH HEMOPHILIA A—AN INSPECTION REPORT FROM SINGLE CENTER

C.-G. Li 1,* , Y. Wang 1 , H.-R. Mai 1 , X.-M. Fang 1 , Q.-L. Long 1 , G.-C. Zhou 1 , C.-Y. Wang 1 , S.-X. Liu 1 , X.-Y. Fu 1 , Q.-R. Shu 2 , L.-S. Cai 3 , Z.-P. Chen 4 , R.-H. Wu 4

1 hematology and oncology; 2 Dept.of Clinical Research, shenzhen children’s hospital; 3 hematology, shenzhen 2nd people’s hospital, shenzhen; 4 Hematology Oncology Center, Beijing Children’s Hospital, Beijing, China

Background: Personalized prophylaxies for moderate and severe hemophilia A and B are based on personal pharmacokenetics (PK), half-life(T1/2). While classical method for personal PK needs multiple blood collections, at least 5 points collections for children and 11points for adults, and expensive for testing and leads low compliance. Popular PK based on pupolar data can have a less blood collections but a rough data(range) is recommended for reference. One-chamber model may also have a less blood collection 2-3 collections for PK detection.

Aims: Objective: To analyze and compare the half-life (T 1/2 ) results of Factor VIII (FVIII) calculated by modified one-chamber model by “CAI’s” hemophilia pharmacokinetic calculation tool and WinNolin software (five-point sampling), non-av model, and to explore the reliability and clinical application value of “CAI’s” hemophilia pharmacokinetic calculation tool.

Methods: A total of 30 patients with moderate and severe hemophilia A were treated with FVIII (50IU/Kg) after the 72-hour elution period. Peripheral blood samples were collected at five time points before and after FVIII injection (0h, 1h, 9h, 24h, 48h), and FVIII activity was detected by one-stage method. The T 1/2 of FVIII was calculated by WinNolin software. Two of the three time points of FVIII activities (9h~24h, 9h~48h, 24h~48h) were used to calculate the FVIII T 1/2 by “CAI’s” hemophilia A pharmacokinetic calculation tool, and the results were compared with those of WinNolin software, and the correlation analysis was conducted.

Results: 1. 30 cases of T 1/2 of FVIII calculated by WinNolin software was from 5.55 hs to 12.5 hs.

2. Compared with WinNolin software (five-point method, the results of T 1/2 of FVIII caculated by “CAI’s” hemophilia A pharmacokinetic calculation tool, (the modified one-chamber model method) at 9h~24h, 9h~48h and 24h~48h showed that the detection periods with highist consistent T 1/2 were 9h~48h, 24 out of 30, 80.0%, while 9h~24h 6 out of 30, 20.00%;

3. Correlation analysis shows that there is a good correlation between the T 1/2 of the modified one-chamber model and the results of WinNolin’s calculation in all three time periods, but the correlation/consistency is the highest in the period of 9h~48h (see fig 1);

4. Analysis of the FVIII T 1/2 results of 9h~24h, 9h~48h and 24h~48h we found that there were 8 out of 30 cases (26.67%) of T1/2 were basically stable at the same level, while 53.33% (16/30) of the children had significant changes at each time period.

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Summary/Conclusion: 1. Compared with the T 1/2 results by WinNolin’s method and modified ond-chambor modle (”CAI’s” tool), the most consistency of T 1/2 of blood sampling was 9h~48h. However, for those T 1/2 short than 7~8hs, the detection time should be adjusted (the later blood sample taking should be adjust to earlier);

2. Nearly 3/4 of the 30 cases, the T 1/2 results fluctuated among three periods (9h~24h, 24h~48h, 9h~48h) in one case, suggesting a standardized test procedures such as specimen collection, sample transportation and detection ect needs to set up to ensure the stability and reliability of test results.

3. The classical pharmacokinetic test requires at least 5 points of blood sample collections, which is difficult to be popularized and applied in clinical practice. If a standardized and standardized testing process is established, the one-chamber model method is relatively simple and easy to be accepted and popularized by patients.

Key words: hemophilia A, pharmacokinetics, children, one-chamber model, non-av-model

PB2326: SAFE AND EFFECTIVE PROPHYLAXIS WITH PLASMA-DERIVED FACTOR X CONCENTRATE IN AN ELDERLY PATIENT WITH HEREDITARY FACTOR X DEFICIENCY

H. Patel 1,* , S. Peltier 2 , K. Clark 3 , M. T. Reding 2

1 Bio Products Laboratory Ltd. UK, Elstree, United Kingdom; 2 University of Minnesota Medical Center, Minneapolis; 3 Bio Products Laboratory USA, Inc., Durham, United States of America

Background: Coagulation factor X, human is a plasma-derived factor X concentrate indicated to treat and prevent bleeding in individuals with hereditary factor X deficiency (HFXD). The ages of patients in whom data was collected in the initial registration trials and post-marketing studies ranged from 0-58 years. To date, there have been no reports on treatment of elderly (>65 years) patients with plasma-derived factor X (pdFX) concentrate.

Aims: To report the experience with pdFX concentrate in the prophylactic treatment of HFXD in a patient >65 years.

Methods: Data was collected on a 71-year-old female with HFXD receiving prophylactic treatment with pdFX.

Results: Although suspected to have some type of bleeding disorder since age 2, this individual was not diagnosed with HFXD until age 20. Initial treatment included fresh frozen plasma on-demand for acute bleeding episodes. Subsequent treatment included three-factor prothrombin complex concentrate (PCC), starting in the 1990s. Prophylactic treatment was started at age 57 after she developed a spontaneous subdural hematoma. She was transitioned from PCC to pdFX prophylaxis in November 2019 to more directly address her factor X deficiency and to avoid increasing other clotting factor levels above the normal range. She is currently receiving pdFX 21 units/kg weekly, which has been well tolerated and she has experienced no bleeding issues since starting this therapy.

Summary/Conclusion: This is the first reported experience of prophylactic treatment of an elderly patient with pdFX. Additional details of this case including laboratory data and updates from her recent clinic visit(s) will be presented. The data supports the successful prophylactic treatment of elderly patients and the need to continue to collect data in this population of patients with this rare bleeding disorder.

PB2327: BONE MARROW STUDY IN THROMBOPENIA

P. López De Ugarriza 1,* , I. Hernández de Castro 1 , A. Porro Camarero 1 , J. C. Iglesias Lopez 1 , G. Flórez Meana 1 , C. Rodríguez de Miñón 1 , A. Fonseca Mourelle 1 , T. Arias Fernández 1

1 Hematología, Hospital Universitario Central de Asturias, Oviedo, Spain

Background: Bone marrow (BM) aspirated and biopsy are invasive procedures with potential hemorrhagic complication. In classic primary immune thrombocytopenia (ITP) guidelines it was recommended to assess the study of BM in patients older than 60 years, refractory to first-line treatment or before splenectomy. Also prior to the start thrombopoietin receptor agonists (TPO-RA) due to the occurrence of medullary fibrosis (MF). However, in more current guidelines these indications for the study of BM are not so clear.

Aims: Describe the findings of BM studies performed for thrombopenia at our center.

Methods: Patients with BM studies performed at our center for suspected thrombopenia between January 2017 and May 2021 were retrospectively reviewed. Both aspirates and biopsies of BM were included and those patients who had an BM study prior to the date of this review were excluded. The data obtained were analyzed using the PSPP statistical program (version 1.2.0-g0fb4db).

Results: A total of 52 patients were reviewed, of which 48% were women and the age range reached was 2 – 89 years. Biopsy was only made in 23% of cases. In patients in whom only medullary aspiration was performed, 36% had thrombopenias < 30,000 x10⁹/L (considering biopsy as unsafe), 61% it was not requested and 2.5% it was not technically possible. The diagnosis of peripheral mechanism thrombopenia (PMT) occurred in 62% of cases, myelodysplastic syndrome (MDS) in 5.8%, chronic myelomonocytic leukemia (CMML) in 3.8%, and 2% of medullary aplasia, medullary fibrosis, dysplasia without MDS criteria or monoclonal gammopathy of uncertain significance, respectively. The BM study was considered normal or reactive in 10%. In both patients diagnosed with MDS and CMML, abnormalities were observed in peripheral blood smears (PBS) performed prior to the BM study, while 84% of patients diagnosed with PMT did not have atypia in the PBS reviewed. In 53% of PMT treatment with TPO-RA was initiated after the BM study. No significant bleeding complications were described in the studies conducted.

Summary/Conclusion: Patients with other diagnoses than PMT already had findings in the PBS that made these diagnoses suspect. Last evidence suggests that the risk of MF of reticulin secondary to treatment with TPO-RA is low (5%) because of this, BM study before or during treatment with TPO-RA is no longer recommended. Greater consensus is needed to avoid unnecessary invasive testing for the diagnosis and follow-up of patients with ITP.

PB2328: CHRONIC LYMPHATIC LEUKAEMIA (CLL) ASSOCIATED TO ACQUIRED FACTOR V INHIBITOR. A CASE REPORT.

A. Porro Camarero 1,* , I. Hernandez de Castro 1 , J. C. Iglesias 1 , P. López de Ugarriza 1 , C. Rodríguez de Miñón 1 , G. Flórez Meana 1 , S. Alonso Álvarez 1 , Á. Bernardo Gutiérrez 1 , I. Soto Ortega 1

1 Hematology, HUCA, Oviedo, Spain

Background: Acquired inhibitors against Factor V (FV) are a very unusual, being its incidence: 0.09-0.29 cases/10 6 inhabitants/year, although it may be underestimated. A non-negligible percentage of cases are related to autoimmune diseases, pregnancy, malignant diseases, infections, as well as antibiotics and idiopathics causes. FV inhibitors may debut as thrombosis or be discovered incidentally in blood clotting tests. Antibodies against FV are usually IgG-type, although IgM-type may also occur. Management involves both controlling hemostasis and eradication of the inhibitor.

Aims: Describe the finding and management of an inhibitor against clotting FV in an CLL diagnosed patient.

Methods: Analysis of a clinical case by basic coagulation study, factors dosing by coagulative methods, as well as inhibitors identification by Bethesda tests according to ISTH recommendations.

Results: A 83-year-old pluripathologic male was diagnosed of CLL in 2019 as an occasional finding without treatment criteria. CLL progressed through 2020 and is currently in stage C of Binet, RAI 4 with 13q delection (CLL IPI 4 high risk group). During hospital admission due to chronic kidney disease exacerbation he developed an Atrial Fibrillation and initiated treatment with low molecular weight heparin (LMWH). A month after discharge he suffered from severe epistaxis with poor local haemostatic control and an elongation of TTPAr (3.96) was objected. Clotting factors dosage was normal except for FV: 0.10% with a FV inhibitor of 19.45 UB. Corticotherapy was initiated at doses of 1mg/kg plus Cyclophosphamide obtaining the results and progression summarized in Table 1, ending with negativization and obtaining haemostatic levels of FV.

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Summary/Conclusion: A review of the literature showed that 16% of patients with anti-VF inhibitors had an underlying malignant disease, including haematological diseases such as multiple myeloma, myeloproliferative neoplasms and mantle lymphoma. However, no information has been found on its association with CLL, which presents autoimmune complications in 25% of cases. We expose a case of anti-FV associated to CLL, which after a timely diagnosis and treatment, follows favourable evolution.

PB2329: ACQUIRED HEMOPHILIA-POTENTIALLY FATAL IF NOT TREATED PROMPTLY

S. Stankovikj 1,* , S. Stojanovska 1,2 , N. Ridova 1 , T. Ristevska 1 , M. Cvetanoski 1

1 Hemato-oncologyy; 2 University Clinic of hematology, SKOPJE, North Macedonia, Republic of

Background: A rare but potentially life-threatening hemorrhage can occur as a result of development of autoantibodies against coagulation factors. Generally the autoantibodies are directed against factor VIII, so it`s a question of acquired hemiphilia A.

Aims: To present a patient with acquired hemiphilia A and delayed diagnosis.

Methods: We present a 66-year old male with a clinical presentation of a huge hematoma in his left leg and right chest. Both his medical and family history were negative for clotting disorder at any point before. The hematoma in his leg occured two months earlier and it was treated as a muscle rupture.

Results: On the day of admitance to the hospital the blood tests revealed hemoglobin level 76g/L, WBC 12x10(9)/L and platelet count 693x10(9)/L. Liver and renal functional tests were in normal range as well as the glucose level. Screening hemostasis revealed prothrombin and thrombin time in normal range but signficantly prolonged APTT (activated partial thromboplastine time) 59sec (normal range 26-34). The d-dimers were 3393microg/L. Factor VIII level was 0.7% and the Bethesda assay confirmed inhibitor titer of 10.2 BU. The bleeding was treated with a by-passing agent rFVIIa (NovoSeven). Immunosuppressive treatment was started with Cyclophosphamide 100mg and Prednisone 100mg daily. On the 8-th day of hospitalisation rFVIIa was stopped as factor VIII level increased to 6.2% and the inhibitors dropped to 2 and 1.1BU subsequently. However, the next day a new hemorrhage appeared in both legs and in left arm with a drop in hemoglobin level and development of a hemorrhagic shock. It was a life-threatening condition and the patient was promptly treated with packed red cell transfusions and additional supportive therapy. A new treatment with rFVIIa was started. Patient`s clinical condition was improving slowly and he was discharged from the hospital on the 25-th day of hospitalisation with an ongoing treatment with Cyclophosphamide and Prednisone with tapered doses.

Summary/Conclusion: This case report illustrates a case with acquired hemophilia A with delayed diagnosis and development of a life-threatening condition where the treatment was prolonged and the outcome uncertain. Whenever we have a patient with unexpected massive bleeding, without history of a bleeding disorder and prolonged APTT, we should consider acquired hemophilia and start treatment promptly.

PB2330: CORONARY ARTERY DISEASE (CAD) AND XBAI GENE POLYMORPHISM (RS693) IN EGYPTIAN PATIENTS

A. Almenshawy 1,* , R. El-Farahaty 1 , A. Osman 2 , N. Saeed 1 , K. El-sayed 1

1 Clinical Pathology; 2 Cardiology, Faculty of medicine - Mansoura University, Elmansoura, Egypt

Background: Coronary artery disease (CAD) is a multifactorial disease leading to coronary arteries occlusion in the form of ischemic heart events. Although cardiovascular diseases (CVD) mortality have been decreasing in the developed countries along the last decade, it is still rising in the developing countries(Popkin, Adair, & Ng, 2012).Egypt has the largest population in middle east and north Africa with significant high CAD mortality rate. In 2014, about 23.14% of all deaths in Egypt were caused by CAD. This made Egypt ranked 23 rd in the world(El-Moselhy et al., 2018; Ralapanawa & Sivakanesan, 2021).

Aims: To determine the relationship between the presence of XbaI gene polymorphisms and coronary arteries occlusion in Egyptian CAD patients.

Methods: This is a case-control study conducted on 200 CAD Subjects (153 men and 47 women) with a median age of 57.5 ± 8.73 years old and 200 control subjects (127 men and 73 women) with a median age of 56.06 ± 6.56 years. The patients were selected from Mansoura University Hospitals clinics. DNA was extracted from leukocytes using spin column (Qiagen Co. Germany). DNA sequence containing Apo B gene was amplified by PCR using set of primers (Metabion, Germany). The amplified sequence was digested at 37 C with 20 U of XbaI restricton enzyme. Digests were run on 2 % agarose gel at 50 V in 1X Tris–borate- EDTA (TBE) buffer. It resulted in fragments of 741 and 201 bp in the presence of the restriction site (X+ allele). In the absence of the XbaI cutting site, the only produced band on the agarose gel was the 942 bp sequence.

Results: X+X- genotype of XbaI (rs693) polymorphism was the most common among patients (48.5%) and controls (44%). There were no significant difference regarding genotypes and alleles of both studied polymorphisms between CAD group and controls. However, X+X+ genotype of XbaI (rs693) polymorphism had non-significant increase (approaching significance) in controls compared to CAD ( P =0.056).

No significant differences were found between multivessel CAD patients versus single vessel CAD patients regarding genotypes and alleles of XbaI (rs693), however non-significant higher frequency of X+X+ genotype were found in single vessel vs. multi-vessel CAD patients (12.8 vs. 8.4%; OR=0.645).

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Summary/Conclusion: XbaI ApoB gene polymorphisms showed no significant difference between CAD and control groups in the studied Egyptian patients. There is also no significant difference between uni-vessel and multi-vessel patients groups.

PB2331: IN FRONT OF A DEEP VENOUS THROMBOSIS, LOOK AT THE HEMOGLOBIN!

S. Fendri 1,* , B. Mariem 1 , B. S. Raida 2 , J. Kamel 1 , B. Zouheir 2

1 laboratory of biochemistry; 2 internal medecine departement, Hedi Chaker Hospital, Sfax, Tunisia

Background: Venous thromboembolic disease is a public health problem whose diagnosis calls for adequate management, this management includes pathologies associated with thrombosis including anemia. In the course of our work, we propose to screen for cases of anemia in patients with deep vein thrombosis.

Aims: In the course of our work, we propose to screen for cases of anemia in patients with deep vein thrombosis.

Methods: This is an observational study involving patients under the age of 60 admitted to the internal medicine department for the management of deep vein thrombosis confirmed by imaging of unknown etiology. A complete blood count was taken for all patients included in the study performed by the sysmex XN1000 analyser.

Results: Forty-seven patients were included in our study, mean age 40.8 ± 10.5 years with extremes of 18 and 59 years. The group consisted of 27 men (57.5%) and 20 women (42.5%). The sex ratio was 1.35. Fifteen patients had a body mass index exceeding 25 kg / m2. Venous thrombosis of IM was the most frequent localization observed in 34 patients (72.3%). Twelve patients (25.5%) were anemic. The anemia observed in both sexes was in 50% normochromic normocytic, and in 50% of cases microcytic. Hemoglobin level was significantly lower in folate deficiency (p = 0.05). Four patients (33%) had folate deficiency and hyperhomocysteinemia with normocytic anemia. All these patients received vitamin supplementation.

Summary/Conclusion: Adequate management of deep vein thrombosis prompts a full etiological investigation and screening for anemia with possible vitamin deficiency requiring supplementation

PB2332: PREVALENCE OF HYPERHOMOCYSTEINEMIA IN PATIENTS WITH DEEP VEIN THROMBOSIS

B. Mariem 1 , S. Fendri 1,* , B. S. Raida 2 , J. Kamel 1 , B. Zouheir 2

1 laboratory of biochemistry; 2 internal medecine departement, Hedi Chaker Hospital, Sfax, Tunisia

Background: Deep vein thrombosis is a public health problem for which the etiological research is an essential step in the management. Numerous studies have shown an association between hyperhomocysteinemia and venous thrombosis.

Aims: In the course of our work, we set out to study the relationship between deep vein thrombosis and homocysteine.

Methods: This is an observational case-control study comparing 47 patients admitted to the internal medicine department for the management of deep vein thrombosis confirmed by radiological examination, with 47 healthy controls. These two groups were matched in age, sex and body mass index. The homocysteine ​​assay was done by an enzymatic technique.

Results: The mean age of patients and controls was 40.8 ± 10.5 years with extremes of 18 and 59 years. The two groups consisted of 27 men (57.5%) and 20 women (42.5%) with a sex ratio of 1.35. Homocysetine was significantly (p <0.001) in patients (17.42 ± 8.5 µmol / L) compared to controls (9.41 ± 3.1 µmol / L), with a prevalence of hyperhomocysteinemia (defined by a homocysteine ​​level> 15 µmol / L) of 61.7% in the patients against 4% in the controls. HHC was found to be significantly correlated with the occurrence of deep vein thrombosis with an odds ratio of 3.54 with a 95% confidence interval [1.76 - 16.4] compared to the control group.

Summary/Conclusion: Homocysteine ​​is an intermediate sulfur amino acid in the metabolism of methionine and cysteine, the increase in which has been shown to be associated with the risk of developing deep vein thrombosis.

PB2333: BURDEN OF HOSPITALIZATION, COSTS AND CO-MORBIDITIES IN MEDICARE PATIENTS WITH CHRONIC GRAFT VERSUS HOST DISEASE: ANALYSIS OF NATIONAL IN-PATIENT HOSPITAL DATABASE

S. Aggarwal 1,* , S. Kumar 1 , A. Bela 1 , O. Topaloglu 2

1 Health Economics and Outcomes Research; 2 Systematic Literature Review, NOVEL Health Strategies, Chevy Chase, United States of America

Background: Chronic graft-versus-host disease (cGvHD) is a severe complication of allogeneic hematopoietic stem cell transplantation that affects various organs leading to a reduced quality of life.

Aims: To analyze trends in hospital length of stay, total costs and co-morbidities in Medicare patients with Chronic Graft Versus Host Disease (CGVHD).

Methods: The National Inpatient Sample (NIS) data set from the Healthcare Cost and Utilization Project was utilized in order to determine the number of hospital admissions for Chronic Graft Versus Host Disease. Thirty co-morbidities were assessed using Elixhauser scoring. The outcomes assessed included length of stay (LOS), total charges, co-morbidity score, mortality rate and emergency room visits.

Results:

There were an estimated 5950 hospitalizations with a diagnosis of CGVHD, with 44% of the patients being covered by Medicare. The mean age was 57 (SD 14.54) years and 41% of the patients were female. The mean hospital length of stay for Medicare patients with CGVHD was 9.29 (SD 15.17) days (Median was 5 days). The mean hospital charges were $108,875 (SD $245171). The mean comorbidity score was 3.74 (SD 1.88, Median score was 4). The in-hospital mortality rate was 6.38%. The vast majority of the patients were White (79%), followed by Hispanic (9%), Black (7%) and Asian (2%). The top five co-morbid conditions were Fluid and Electrolyte Disorders (45%), Hypertension (39%), Depression (26%), Chronic Pulmonary Disease (25%) and Renal Failure (24%). The top 3 primary diagnosis codes were for Sepsis, Complications of stem cell transplant and Pneumonia.

Summary/Conclusion: Chronic Graft Versus Host Disease poses significant burden on Medicare with high hospital charges and long LOS. There is a need for new treatment options for cGVHD patients.

PB2334: BURDEN OF HOSPITALIZATION, COSTS AND CO-MORBIDITIES IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: ANALYSIS OF NATIONAL IN-PATIENT HOSPITAL DATABASE

S. Aggarwal 1,* , S. Kumar 2 , A. Bela 2 , O. Topaloglu 3

1 Health Economics and Outcomes Research, NOVEL Health Strategies, Chevy Chase; 2 Health Economics and Outcomes Research; 3 Systematic Literature Review Group, NOVEL Health Strategies, Columbia, United States of America

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. It’s a type of cancer that starts in cells that become certain white blood cells in the bone marrow.

Aims: To analyze trends in hospital length of stay, total costs and co-morbidities in Medicare and Private Insurance patients with Chronic Lymphocytic Leukemia (CLL).

Methods: The National Inpatient Sample (NIS) data set from the Healthcare Cost and Utilization Project was utilized in order to determine the number of hospital admissions for Chronic Lymphocytic Leukemia. Thirty co-morbidities were assessed using Elixhauser scoring. The outcomes assessed include length of stay (LOS), total charges, co-morbidity score, mortality rate and emergency room visits.

Results:

There were an estimated 74,355 hospitalizations with a diagnosis of CLL, with 82% and 15% of the patients being covered by Medicare and Private Insurance, respectively. The mean age was 77.96 (SD 8.88) and 64.25 (SD 10.4) years for patients covered by Medicare and Private Insurance, respectively. The mean hospital length of stay was 5.81 (SD 6.01) and 5.91 (SD 7.18) days, for Medicare and Private Insurance patients, respectively. The mean hospital charges were $58,144 (SD $77558) and $66,721 (SD $91299) for Medicare and Private Insurance patients, respectively. The mean comorbidity score was higher for Medicare versus Private Insurance (Mean 4.22 vs 3.69, p<0.05). The in-hospital mortality rate was same 5.03% in both Medicare and Private Insurance patients. The top 5 co-morbid conditions in Medicare patients were Hypertension (45%), Cardiac Arrhythmias (39%), Fluid and Electrolyte Disorders (39%), Congestive Heart Failure (30%) and Chronic Pulmonary Disease (29%). Top 3 primary diagnoses were Sepsis, Pneumonia and Acute kidney failure. Approximately, 70% of Medicare patients were admitted to the hospital via the emergency room visit.

Summary/Conclusion: Chronic Lymphocytic Leukemia patients pose significant burden on Medicare and Private Insurance with high hospital charges and long LOS. There is a need for new treatment options for CLL patients.

PB2335: BURDEN OF HOSPITALIZATION, COSTS AND CO-MORBIDITIES IN MEDICAID PATIENTS WITH SICKLE CELL DISEASE (SCD): ANALYSIS OF NATIONAL IN-PATIENT HOSPITAL DATABASE

S. Aggarwal 1,* , S. Kumar 2 , H. Topaloglu 2 , A. Bela 2 , O. Topaloglu 3

1 Health Economics and Outcomes Research, NOVEL Health Strategies, Chevy Chase; 2 Health Economics and Outcomes Research, NOVEL Health Strategies, Columbia; 3 Systematic Literature Review Group, NOVEL Health Strategies, Chevy Chase, United States of America

Background: Sickle cell disease (SCD) is a group of inherited red blood cell disorders. SCD affects approximately 100,000 Americans.

Aims: To analyze trends in hospital length of stay, total costs and co-morbidities in Medicaid patients with Sickle Cell Disease (SCD).

Methods: The National Inpatient Sample (NIS) data set from the Healthcare Cost and Utilization Project was utilized in order to determine the number of hospital admissions for Sickle Cell Disease (SCD). Thirty co-morbidities were assessed using Elixhauser scoring. The outcomes assessed include length of stay (LOS), total charges, co-morbidity score, mortality rate and emergency room visits. The SCD ICD-10 codes used were ‘D57.0’, ‘D57.1’, ‘D57.2’, ‘D57.4’ and ‘D57.8’.

Results: There were an estimated 125,405 hospitalizations with a diagnosis of SCD, of which 53% were covered by Medicaid. The mean age was 24.17 (SD 13, Median was 24) years and 53% of the patients were female. The mean hospital length of stay for Medicaid patients with SCD was 5 (SD 6.48, Median was 3 days). The mean hospital charges were $36048 (SD $58044). The mean comorbidity score was 1.33 (SD 1.53). The in-hospital mortality rate was 0.3%. The vast majority of the patients were Black (91%), Hispanic (5%) and White (1%). The top five listed co-morbid conditions were were Chronic Pulmonary Disease (26%), Fluid and Electrolyte Disorders (18%), Hypertension (11%), Drug Abuse (11%) and Depression (9%). The second most common listed diagnosis was for asthma (J45909). The top procedure codes were for blood transfusion and Infusion Device into Superior Vena Cava. Approximately, 79% of Medicaid patients were admitted to the hospital via the emergency room visit.

Summary/Conclusion: Medicaid patients with Sickle Cell Disease pose significant burden on hospitals with relatively long length of stay. Due to comorbidities of COPD, asthma and drug abuse, these patients might need special treatment management to improve overall outcomes.

PB2336: BURDEN OF HOSPITALIZATION, COSTS AND CO-MORBIDITIES IN MEDICAID PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA (ALL): ANALYSIS OF NATIONAL IN-PATIENT HOSPITAL DATABASE

S. Aggarwal 1,* , S. Kumar 1 , H. Topaloglu 1 , A. Bela 1 , O. Topaloglu 2

1 Health Economics and Outcomes Research, NOVEL Health Strategies, Chevy Chase; 2 Systematic Literature Review Group, NOVEL Health Strategies, Columbia, United States of America

Background: Acute lymphocytic leukemia (ALL) is a type of cancer of the blood and bone marrow. ALL is the most common type of cancer in children.

Aims: To analyze trends in hospital length of stay, total costs and co-morbidities in Medicaid patients with Acute Lymphoblastic Leukemia (ALL).

Methods: The National Inpatient Sample (NIS) data set from the Healthcare Cost and Utilization Project was utilized in order to determine the number of hospital admissions for Acute Lymphoblastic Leukemia. Thirty co-morbidities were assessed using Elixhauser scoring. The outcomes assessed include length of stay (LOS), total charges, co-morbidity score, mortality rate and emergency room visits.

Results: There were an estimated 18,575 hospitalizations with a diagnosis of ALL that were covered by Medicaid. The mean age was 17.35 (SD 15.59) years and 42% of the patients were female. The mean hospital length of stay for Medicaid patients with ALL was 8.8 (SD 13.17) days (Median was 4 days). The mean hospital charges were $122,626 (SD $259,709). The mean comorbidity score was 1.31 (SD 1.44). The in-hospital mortality rate was 1.75%. The vast majority of the patients were Hispanic (45%), followed by White (34%), Black (11%) and Asian (3%). The top five listed co-morbid conditions were were Fluid and Electrolyte Disorders (24%), Coagulopathy (18%), Hypertension (12%, Depression (9%) and Weight Loss (8%). The top 3 procedure codes were for Blood Transfusion, Infusion Device into Superior Vena Cava and Bone Marrow Extraction. Approximately, 72% of Medicaid patients were admitted to the hospital via the emergency room visit.

Summary/Conclusion: Acute Lymphoblastic Leukemia (ALL) covered by Medicaid pose significant hospital burden. There is a need for new treatment options for ALL patients.

PB2337: BURDEN OF HOSPITALIZATION, COSTS AND CO-MORBIDITIES IN MEDICAID PATIENTS WITH VENO-OCCLUSIVE DISEASE (VOD): ANALYSIS OF NATIONAL IN-PATIENT HOSPITAL DATABASE

S. Aggarwal 1,* , S. Kumar 2 , A. Bela 3 , O. Topaloglu 4

1 Health Economics and Outcomes Research; 2 Veno-occlusive Disease Research Group, NOVEL Health Strategies, Columbia; 3 HEOR; 4 Systematic Literature Review Group, NOVEL Health Strategies, Chevy Chase, United States of America

Background: Veno-occlusive disease (VOD), also called sinusoidal obstruction syndrome (SOS), is a potentially life-threatening complication of hematopoietic stem cell transplantation (HSCT) conditioning or high-dose nontransplant chemotherapy.

Aims: To analyze trends in hospital length of stay, total costs and co-morbidities in Medicaid patients with Veno-Occlusive Disease (VOD)

Methods:

The National Inpatient Sample (NIS) data set from the Healthcare Cost and Utilization Project was utilized in order to determine the number of hospital admissions for VOD. Thirty co-morbidities were assessed using Elixhauser scoring.

Results: There were an estimated 190 hospitalizations with a diagnosis of VOD, with 50% of the patients being covered by Medicaid. The mean age was 15 (SD 16.03) years and 58% of the patients were female. The mean hospital length of stay for Medicaid patients with VOD was 53.47 (SD 40.17) days (Median was 48 days). The mean hospital charges were $1,541,016 (SD $1487179). The mean comorbidity score was 3.53 (SD 1.54). The in-hospital mortality rate was 15.79%. The vast majority of the patients were Hispanic (58%), followed by Asian (16%), Black (11%) and White (11%). The top five listed diagnoses were Liver Disease (100%), Fluid and Electrolyte Disorders (68%), Coagulopathy (47%), Cardiac Arrhythmias (21%) and Hypertension (5%). The top 3 procedure codes were for stem cell transplant, ventilation and bone marrow extraction.

Summary/Conclusion: High hospital burden of VOD in Medicaid patients shows there is a need for better treatment management for these patients.

PB2338: QUALITY OF LIFE AFTER ALLOGENIC HEMATOPOIETIC STEM CELL TRANSPLANTATION: IMPACT OF CHRONIC GVHD

D. Belloumi 1,* , R. Ouerghi 1 , N. Ben Abdeljelil 1 , S. Mekni 1 , L. Torjemane 1 , I. Turki 1 , S. Ladeb 1 , T. Ben Othman 1

1 Centre national de greffe de moelle osseuse, Tunis, Tunisia, tunis, Tunisia

Background: Hematopoietic Stem Cell transplantation (HSCT) is a potentially curative treatment for several hematological diseases, but it is often complicated by significant morbidity and mortality. This is particularly related to chronic Graft versus Host Disease (GVHD) that can deeply impact patients’ quality of life (QoL).

Aims: Our purpose was to evaluate patients’QoL after HSCT and to clarify the impact of chronic GVHD.

Methods: We conducted a cross-sectional descriptive study at the National Bone Marrow Transplant Center including patients who underwent HSCT between January 2018 and December 2019. These patients filled in the questionnaire for the quantitative measurement of quality of life using 36-Item Short-Form Health Survey (SF-36) and to the Functional Assessement of Cancer Therapy-Bone Marrow Transplantation (FACT-BMT) during a phone interview.

Results: Of the 52 patients still alive at the time of the survey, 43 patients were contactable and willing to participate (participation rate: 83%). The median age of the participants was 28 years (7-52) with a sex ratio of 1.52. Underlying disease was acute leukemia for 29 patients and aplastic anemia for 14 others. The median time between HSCT and the interview was of 26 months (17-39). At the time of the survey, all patients were in complete remission and 28% among them (12 of 43) had active extensive chronic GVHD.

Results obtained in the different dimensions of the SF-36 and FACT-BMT scales indicated good overall QoL in patients after HSCT. However, using SF-36, patients with chronic active GVHD had significantly lower scores on « role limitation due to their physical condition » (p=0.001), «role limitation due to their psychological condition » (p=0.0001), « vitality » (p= 0.045) and « general health perception » (p=0.027) than the other patients. In addition, the FACT-BMT score revealed a negative impact on chronic GVHD in physical well-being dimension (p=0.009) and the functional well-being dimension (p=0.027).

Apart from chronic active GVHD, a negative impact on QoL was noted with: adult age, peripheral stem cells transplants and GVHD prophylaxis with ciclosporin alone.

Summary/Conclusion: Active chronic GVHD was associated with impared QoL in patients after HSCT hence the need for rapid diagnosis and optimal prophylaxis and treatment of this complication.

PB2339: PROJECTED LIFETIME ECONOMIC BURDEN OF TRANSFUSION DEPENDENT BETA-THALASSEMIA IN THE UNITED STATES

C. Udeze 1,* , K. Maruszczyk 2 , M. Atter 2 , A. Lopez 1

1 Vertex Pharmaceuticals, Boston, United States of America; 2 HCD Economics, The Innovation Centre, Daresbury, United Kingdom

Background: Beta-thalassemia is a rare hereditary blood disorder affecting approximately 2,000 individuals in the United States (US) and is characterized by reduced or absent beta globin production, leading to ineffective red blood cell production and anaemia. The most severe form of the disease is transfusion-dependent beta-thalassaemia (TDT), in which patients are dependent on regular red blood cell transfusions for survival. TDT patients suffer from chronic anaemia and the effects of iron overload despite chelation therapy, both of which result in significant morbidity and lead to premature mortality. The lifetime economic burden of TDT in the US is not well characterized.

Aims: To estimate the per-patient lifetime direct healthcare cost of TDT from a US healthcare payer perspective using an economic model developed based on published model frameworks.

Methods: An individual-level simulation model was used to estimate the lifetime direct healthcare costs for patients with TDT in the US. The model followed a cohort of TDT patients, who were assumed to receive an average of 16.8 blood transfusions per year, from birth until death. Like previously published economic models for TDT, mortality was estimated based on transfusion status and presence of TDT complications. Key complications tracked in the model included cardiac complications, liver complications, and endocrine complications (diabetes and hypogonadism). Cost of complications were derived from literature and the wholesale acquisition costs in the US were used to inform iron chelation therapy prices. Scenario analyses were conducted to determine the impact of lower iron chelation therapy costs (based on estimates from a published retrospective claims analysis) and average annual number of blood transfusions (12 and 20) on lifetime costs. The cost and impact of a recently approved non-curative TDT therapy, luspatercept, was not included in this analysis.

Results: The model followed patients with TDT for an average of 39 years, over which time each patient incurred an average of 686 blood transfusions. The cost of TDT over the lifetime horizon was estimated to be $5.4 million. The majority of costs were for iron chelation therapy (68%, $3.7 million) and blood transfusions (30%, $1.6 million). Using the alternative cost inputs for iron chelation therapy (approximately 33% lower costs) lowered lifetime cost estimates to $4.2 million. Additional scenario analyses demonstrated the lifetime costs of TDT ranged from $5.0 million to $5.7 million when varying the average number of transfusions per year from 12 to 20.

Summary/Conclusion: Model projections show significant lifetime economic burden with TDT in the US. Cost of chelation therapy and frequency of transfusions are the main drivers of modelled lifetime cost estimates. Treatments that can reduce the need for blood transfusions and iron chelation therapy in this population would significantly lower this economic burden.

PB2340: INFLUENCE OF INITIAL CLINICAL FEATURES IN THERAPEUTIC DECISIONS AND DIAGNOSTIC PROCESS OF ELDERLY PATIENTS WITH ACUTE MYELOID LEUKEMIA. A REAL WORLD EXPERIENCE

L. Bolea 1,* , C. Jimenez 1 , K. Velazquez 1 , J. Marquet 1 , M. Piris 1 , F. Martin 1 , P. Palomo 1 , B. De Felipe 1 , L. Perez 1 , B. Astibia 1 , C. Nuñez-Torron 2 , J. Lopez 1 , P. Herrera 1

1 Hematology and Hemotherapy, Ramon y Cajal University Hospital; 2 Hematology and Hemotherapy, Infanta Sofia University Hospital, Madrid, Spain

Background: Acute myeloid leukemia (AML) is considered a disease of older adults, with a median age at diagnosis of 68 years. Those who are not candidate to intensive chemotherapy represents a high-risk group. Numerous factors contribute to this poor outcome as age older than 75 years, frailty, comorbidities, poor performance status (PS) and unfavorable cytogenetics or secondary AML (sAML). Despite of the improvement of survival with new treatments, the prognostic is fateful with a life expectancy measures of months. Due to high frailty at diagnosis, we have to take into consideration that there is a group of patients that only receive best supportive care (BSC). There is currently a growing tendency to treat as many patients as possible because of the favorable impact of treatment on survival.

Aims: Our purpose was to analyze the impact that clinical features at diagnosis could have in therapeutic decisions and diagnostic process. Secondly, we studied the real-world overall survival (OS) in patients treated with non-intensive therapy and BSC.

Methods: We performed a single-center retrospective analysis of 47 patients, older than 65 years and not candidate to intensive chemotherapy, diagnosed AML between 2019 and 2022. We included all patients with ≥ 20% of blast in a peripheral blood film and/ or a bone marrow aspirate, who had received non-intensive treatment or BSC. Owing to the time of recruitment and the policy of our hospital, none of the patients were evaluated by geriatrics or palliative care at diagnosis. Chi-squared test, Fisher’s exact test and Mann-Whitney U test were used to compare the clinical features in both groups. Survival rates and curves were estimated by Kaplan-Meier method.

Results: Among all patients, 20/47 (42.6) received BSC and 27/47 (57.4%) non-intensive treatment. Table 1 show the clinical features of the population at diagnosis. The clinical features with statistically significant association were age and ECOG. The median age was 81 years in the global cohort (range 68 to 95), with a median age of 85.5 and 78 years in the group of BSC and non-intensive treatment respectively. In the group of non-intensive treatment, 26/27 (96.3%) of patients had ECOG 0-1 and 1/27 (3.7%) had ECOG 2-3; compared to 3/20 (15%) with ECOG 0-1 and 17/20 (85%) with ECOG 2-3 in BSC. All patients in the group of non-intensive treatment had a bone marrow aspirate and cytogenetic study at diagnosis, while 7/20 (35%) had these studies in the group of BSC. At a median follow-up of 91 days (range 3 to 943), the median OS was 89.5 days (range 3 to 943). In the group of BSC, at a median of follow-up of 24 days (3 to 894), the median OS was 22 days (range 3 to 541); in non-intensive treatment, at a median of follow up of 165 days (range 28 to 943), the median OS was 165 days (range 60 to 943) (p<0.0001).

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Иллюстрация — в первоисточнике ↗

Summary/Conclusion: We described a small cohort of patients with AML treated in the same hospital. In our cohort is remarkable that more than a half of patient did not receive any treatment at diagnosis. Older age and ECOG>2 at presentation were the significant factors that determined the treatment schedule more than AML characteristics. These clinical features could be also critical factors that guide the diagnostic process. The survival in both groups were fateful, especially in the group of BSC, as we expected. The implementation of geriatric assessment would help identify patients that may be candidate to targeted intervention in order to improve their baseline situation and make it fit to receive non-intensive treatment.

PB2341: MIDAZOLAM AND LOCAL ANAESTHESIA CO-ADMINISTRATION GIVES BETTER PAIN CONTROL DURING THE BONE MARROW ASPIRATION AND BIOPSY PROCEDURE

Z. C. Chapanduka 1,*

1 Haematological Pathology, Stellenbosch University, Cape Town, South Africa

Background: During the bone marrow aspiration and biopsy (BMAB) procedure, patients report pain of varying intensity ranging from complete absence of pain to severe pain. There is limited literature on the patient and procedure related factors associated with the pain. Furthermore, evidence-based guidelines on the best method of pain management during BMAB are lacking. The use of local anaesthesia(LA) only is still widespread although it does not abolish the pain of BMAB. Conscious sedation, other pharmacological agents and non-pharmacological interventions are used to a variable extent. Midazolam is the most commonly used benzodiazepine for conscious sedation and may be used in conjunction with local anaesthesia, other systemic pharmacological agents or as a single agent. Our centre introduced universal midazolam sedation unless there is a contraindication to its use, 4 years ago.

Aims: To assess the impact of the universal use of intravenous midazolam premedication for the BMAB procedure compared to the use of local anaesthesia only and to determine the patient- and procedure-related factors associated with the patient-reported pain.

Methods: A retrospective cross-sectional study was performed on adult patients who had a BMAB in a day procedure operating theatre over the period from 1 st July 2018 to 30 th March 2019. Patient demographics, the body mass index (BMI), indications, diagnosis, the number of times the patient had the BMAB procedure and the experience of the operator were documented. A numerical rating scale pain assessment tool and questionnaire were used in interviews conducted by the principal investigator. Further data were extracted from the National Health Laboratory Service laboratory information system, patients’ hospital files and the operating theatre records.

Results: A total of 182 BMAB procedures were included in the study; 155 procedures performed with midazolam premedication and 27 performed using local anaesthesia only. Pain was reported in all procedures performed under local anaesthesia and only in 29.1% of procedures performed with midazolam administration. Our study showed no influence of age, sex, race, level of educations, indication, diagnosis, experience of the operator on pain. Patients who had previous BMAB experienced less pain. Midazolam dose showed negative correlation with pain.

Summary/Conclusion: Local anaesthesia only is not enough to abolish pain of BMAB. Midazolam conscious sedation used with local anaesthesia consistently reduces the reported pain to acceptable levels. Patients with previous experience of BMAB under midazolam premedication reported less pain. The universal use of midazolam conscious sedation for BMAB under carefully controlled circumstances is strongly recommended.

PB2342: A REVIEW OF BONE MARROW BIOPSIES PERFORMED AT A LONDON TEACHING HOSPITAL: DO BONE MARROWS AID DIAGNOSIS AND AT WHAT COST?

M. Mahbub 1,* , N. Chauhan 1

1 Haematologiy, Royal Free Hospital, London, United Kingdom

Background: Bone marrow aspirate and trephine biopsy (bone marrow biopsy) are an establised test to diagnose haematological disease. In addition to diagnostics, the bone marrow biopsy also aids staging, management and monitoring. However bone marrow biopsies are invasive, require specialist practitioners and can cause pain and other complications to the patient. Analysis has to be performed in a timely manner by specialists alongwith highly complex tests with their own costs. Bone marrow biopsy is therefore an expensive test with a 2020/21 hospital tariff of £964 (1155 EUR, 1285 USD). This is likely to be less than the true charge given the expense of specialist testing, transport to off-site laboratories, analysis and storage of samples in line with UK regulations. At our institution bone marrows are performed only if consultant-approved by a qualified practitioner.

Aims: To review all bone marrows performed at a London teaching hospital across 6 months for their indications, eventual diagnosis and aid to patient care.

Methods: A retrospective audit was performed for all bone marrow biopsies performed by our department over a 6-month period (1st August 2021 and 31st January 2022). Biopsy samples were identified by request and report. Biopsies were checked against the agreed indications from the International Standards for Standardization in Hematology (ICSH) and their concluding diagnosis and aid to patient care assessed

Results: 81 bone marrow biopsies were identified and assessed.Patient demographics showed age ranged from 24 to 91 years (median 65 years) with similar male to female ratio (Male 1.3:1 Female). The majority of biopsy requests came were for outpatients (73%) rather than inpatients (27%). The consultant making the most requests were from our Haematology faculty (84%) with the remaining being largely requested by the Hepatology (5%) and Nephrology (4%) teams and performed with agreement by a Haematologist. All biopsies met the indications agreed by ICSH. The most common indication for biopsy was diagnosis, staging and follow up of malignant haematological disorders (74%) with myeloproliderative neoplasms and plasma cell dyscrasia predominating. 66% of bone marrow biopsies reached a diagnosis, 34% of biopsies were non-diagnostic or non-contributory.

Summary/Conclusion: Despite advances in radiology and peripheral blood analytics, a bone marrow aspirate and trephine examination has a significant role in diagnosis, staging and management of haematological disorders. Our audit suggests that 160 bone marrows would be performed across a 12-month period and would cost commissioners £160,000 (191,000 EUR, 213,000 USD) although diagnostic lab costs have been higher than this in previous years. This audit confirmed that a consultant-led approach means bone marrows meet approved indications every time. Although for the majority of patients a diagnosis is made a third do not reach a diagnosis. A normal bone marrow can be useful where, for example, the patient has undergone a biopsy for an incidental peripheral blood finding. However within this group there will be patients who have undergone an invasive test, with known complications and cost, for no positive change to their care and it these patients where the role of bone marrow biopsy has to be questioned.

PB2343: MONITORING PHYSICAL ACTIVITY OF OLDER ADULTS WITH HEMATOLOGICAL MALIGNANCIES. THE IMPORTANCE OF CONDUCTING AN INDIVIDUALIZED FOLLOW-UP PROGRAM TO PREVENT FUNCTIONAL IMPAIRMENT

L. Cotano Abad 1 , C. González de Villaumbrosia 1,* , C. Miranda 2 , A. Velasco 2 , M. G. Salvatierra 2 , S. Torras 1 , R. Barrera 1 , V. García 1 , S. Gonzalez 1 , G. Olaya 1 , A. Saenz deTejada 1 , C. Valverde 1 , R. Barba 3 , R. Córdoba 4

1 Geriatrics; 2 Hematology; 3 Internal Medicine, Hospital Universitario Rey Juan Carlos, Móstoles; 4 Hematology, Fundación Jiménez Díaz, Madrid, Spain

Background: Currently in Spain, the percentage of the population over 65 years has reached 22.9% and it is estimated that in 2050, 31.4% of the population will be over 65 years and 11.6% will be over 80 years of age. Regular physical activity (PA) helps to improve physical and mental function as well as to reverse some effects of chronic disease to keep older people mobile and independent. Being able to monitor PA in these patients would help us to carry out a more individualized follow-up and prevent functional decline early.

Aims: To describe the adherence and preliminary data of a personalized programme which monitors physical activity in older adults with hematological malignancies by means of wearables.

Methods: This is a prospective, non-randomized, single center (Hospital Rey Juan Carlos, Mostoles, Spain) study. We estimate a sample of 100 patients. It started in December 2020 and it will last up to December 2022. The inclusion criteria were patients over 70 years with hematological malignancies who were referred to the geriatric clinic to perfom a comprehensive geriatric assessment (CGA) after scoring >14 points in the frailty G8 screening tool. The exclusion criteria were life expectancy less than one month, not indication of treatment, absence of smartphone, or program refusal. Initially, a CGA was made jointly between the geriatrician and the geriatric nurse. All those patients who fit the criteria were included in the program. Then, a personalized program is made with nutritional recommendations and physical activity prescription, following the Vivifrail programme and using a wearable to monitor it. The follow-up is carried out by the geriatric nurse through telephone calls at 1,2,4,5 months and face-to-face at 3 and 6 months.

Results: To date, 34 patients have been referred to the geriatric clinic between December 1st 2020-February 20th 2022. The mean age was 77 (Min. 70, max. 93). The main diagnosis were: lymphoma (38%), multiple myeloma (32%), and chronic lymphatic leukemia (21%). 20 patients (59%) were classified as fit, 11 (32%) as pre-fragile, 2 (6%) as fragile, and 1 (3%) as “poor prognosis”. 33% were excluded (3 absence of smartphone, 2 life expectancy < 1 month, 4 refusal, 2 not indication of treatment, 1 for other causes). Of them, 53% of the sample was male. To date, 77.2 % of the patients have successfully completed the program. 55% of the patients synchronized the wearable with their own mobile. The mean number of daily footsteps was 6,678 (Min. 964, max. 14,562) of the total sample. The mean number of footsteps of the robust patients was higher than the footsteps of the prefragile patients (7,321 vs 4,853) but this difference was not statistically significant (p=0.17). No fragile or palliative-care patients reported data of footsteps. The mean minutes of sleep was 323 (Min. 231, max. 507).

Summary/Conclusion: The programme was feasible and satisfactory due to the adherence shown by most of the patients. The average number of daily steps and hours of sleep shows significant variability between patients. The relationship between the phone call and the face-to face visit, and the increase in footsteps in the following days after the treatment provides us some promising data on the effectiveness of the intervention, pending further analysis when the sample size is sufficient.

PB2344: RESILIENCE IN PATIENTS WITH LYMPHOID MALIGNANCIES: THE REPLY STUDY.

G. Dos Santos 1,* , V. Bogliacino 2 , V. Irigoin 1 , V. Toledo 1 , P. Oldan 1 , K. Blanco 1 , P. Specht 1 , A. Montaño 1 , M. Posada 1 , I. Alpire 1 , D. Garrido 1 , A. Alpuin 1 , D. Villalvir 1 , C. Guillermo 1

1 Hematology Department; 2 Psicology Department, Hospital de Clinicas, Montevideo, Uruguay

Background: Resilience, the individual’s ability to maintain or restore psychological and physical functioning despite adversities and stressful life events, has became a matter of intense investigation in cancer research. In fact, resilience has emerged as a key element to understand differences in coping and adaptation during and/or after cancer treatment. Unfortunately, the evidence of its importance in hematologic and specifically in lymphoid malignancies is very scarce.

Aims: The aims of this study were (a) to investigate the levels of resilience in patients with lymphoid malignancies, (b) to explore the relationship between resilience and quality of life and (c) between resilience and mental health outcomes, in a University Hospital in Montevideo, Uruguay.

Methods: A cross-sectional, observational study was performed at the Hospital de Clinicas Dr. Manuel Quintela, assessing resilience in patients with lymphoid malignancies. The instruments Resilience Scale RS-14, Hospital Anxiety and Depression Scale (HADS) and EQ-5D-5L scale were used. Informed consent was obtained in all patients and the study was approved by the institutional ethics committee.

Results: 105 patients were enrolled between July and December 2019: 76 NHL, 16 HL and 13 CLL. The median age was 53 and 53% of the patients were male. A remarkable high level of resilience was observed among the studied population, with a median resilience RS-14 score of 85,42 (IC 95%: 82,48 – 88,36). 95% of patients reported high or very high levels of resilience. No effects on resilience were found for gender, age, type of lymphoma or specific treatment. Resilience is positively correlated with quality of life (Spearman’s rho = 0.494, p< 0,001) and negatively with anxiety (-0,609, p<0,01) and depression (-0,537, p<0,001).

To better study our lymphoma population, we considered our patients as high or low resilients if their RS-14 score was above or below the observed median, respectively. We found that our ‘high resilients’ showed a higher quality of life than our ‘low resilients’ (p<0,0001). Patients that did not report any issues in any of the dimensions of the EQ-5D-5L questionnaire (26,7%), showed a trend towards higher resilience scores (p=0.058).

Summary/Conclusion: Resilience could be considered as a protective factor for patients with lymphoid malignancies. Stimulating resilience through specific interventions could positively impact their quality of life.

PB2345: ADAPTING PATIENT EDUCATIONAL APPROACHES IN PYRUVATE KINASE DEFICIENCY: EUROPE AND NORTH AMERICA FINDINGS FROM THE AAC PATIENT AND CAREGIVER SURVEY

W. Barcellini 1,* , R. Grace 2

1 Hematology Unit, Pathophysiology of Anemias Unit, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2 Dana-Farber/ Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, United States of America

Background: Pyruvate kinase (PK) deficiency is a rare congenital hemolytic anemia affecting approximately 3 people per million globally. As little is known about the burden of this disease and the patient perspective on its management, a patient and caregiver survey was conducted by the PK Deficiency Advocacy Advisory Council (AAC), a group of patients, advocates and physicians focused on supporting and improving care for people with PK deficiency.

While survey respondents were overall satisfied with their hematologist interactions, certain responses indicated underlying differences between subgroups.

The AAC was created and activities funded by Agios Pharmaceuticals, Inc. Medical writing was provided by FleishmanHillard, paid for by Agios.

Aims: The aims of this study were to understand the perception of patient-hematologist communications among people with PK deficiency and their caregivers and investigate the influence of culture and location on care.

Methods: A global web-based survey was conducted in 2021 to assess the (1) information provided at time of diagnosis, (2) treating hematologist’s knowledge of the condition, (3) level of communication with the physician and (4) patient’s or caregiver’s relationship with their hematologist.

Two versions of a questionnaire were developed: one for adult patients and another for caregivers providing unpaid care to a patient. Survey questions were closed-ended, multiple choice, Likert scale and binary choice plus free text. Data were analyzed according to statistical hypothesis testing to determine the significance of results. Test statistics (standard scores) were calculated, and p-values determined using a normal distribution Z-score table.

Participants were recruited via online panels and AAC member channels, including PK deficiency Facebook groups.

Results: Of the 199 adult PK deficiency patients and 74 caregiver respondents (N=273), 95 (35%) were from North America (NAm) and 178 (65%) from Europe (Eu). Most reported hematologists manage the condition well (82%). In terms of what drives positive feelings, NAm respondents were most likely to cite their clinician’s listening skills (52%), while Europeans most often pointed to their hematologist explaining health complications clearly (55%). After interactions, Americans more often reported feeling ‘optimistic’ than Europeans (33% vs 24%, p=0.89) while Americans less often reported feeling reassured than Europeans (8% vs 25%, p=0.97).

However, negative emotions were also expressed, with Europeans (31%) being somewhat more likely to experience a negative emotion after interactions than Americans (24%). Europeans were less likely to report that their hematologist takes their perspective and experience into consideration when creating their disease management plan (81% NAm vs 65% Eu, p=0.08).

Among those who reported suboptimal disease management, Eu respondents were twice as likely to point to a lack of listening from their hematologist, while Americans were more likely to report a lack of proactive management of their condition (54% vs 36%, p=0.82).

While referrals to specialists, patient groups and counseling services were not common, they were less so in NAm than Eu (18% vs 32%, p=0.05). Sharing information about patient support groups is similar in NAm and Eu (36% and 30%, respectively).

Summary/Conclusion: To assess potential regional influence on respondent answers, results were separately described by geography. Findings provide potential indicators on how to tailor communications and approaches in Eu and NAm to optimize PK deficiency care.

PB2346: COMPARISON OF PRESCRIBING ERRORS WITH DIRECT ORAL ANTICOAGULANTS BETWEEN PAPER BASED AND ELECTRONIC BASED PRESCRIBING METHODS AT A REGIONAL BASE HOSPITAL IN NSW, AUSTRALIA

D. Glendinning 1,* , D. Jackson 2

1 Medical Education; 2 Haematology, Northern NSW Local Health Distric, Lismore, Australia

Background: Direct Oral Anticoagulants (DOACs) are used as an alternative to Warfarin for prevention and treatment of stroke, pulmonary embolism and deep vein thrombosis. Anticoagulants are deemed high risk medications due to their narrow therapeutic index, and the risk of significant adverse patient outcomes that can occur due to over or under dosing. In 2018, the local health district replaced paper-based medication charts with an electronic prescribing system (eMeds). eMeds has a medication interaction warning function, but this has currently been disabled.

Aims: Identify errors in prescribing Venous thrombotic events (VTE) prophylaxis or transitions to Heparin/Low molecular weight heparin (LMWH) for patients prescribed a DOAC. Identify any adverse events from these errors. Compare results post electronic prescribing implementation and compare identified errors to previous paper-based prescribing audit. Are there less errors with electronic prescribing?

Methods: Hospital pharmacy provided a list of patients dispensed either Apixaban or Rivaroxaban over 12 months (30/10/2018 – 30/10/2019) following electronic prescribing implementation. Medication charts and progress notes were reviewed for prescribing errors and any subsequent complications. Reason for admission, DOAC indication, and changes to anticoagulation medications were noted. Simple statistical analysis was performed comparing previous audit (paper-based 01/07/2014- 30/05/2015) to post electronic prescribing audit.

Results: 540 patients were dispensed Apixaban (N=383) or Rivaroxaban (N=157) between the study period. 96 charts were analysed as a sample (Apixaban 50, Rivaroxaban 46). Reasons for admission included Atrial Fibrillation or other arrhythmia (16%), General Medical (72%), Embolic Events (1%), Surgical (11%). Indications for DOAC use were Atrial Fibrillation (76%), PE(6%), DVT(9%), other(8%).

One patient was given concurrent DOAC and therapeutic dose of LMWH. One patient was given a double dose of DOAC, changing from Rivaroxaban (once daily dose) to Apixaban (twice daily dosing). There were four incidents where a DOAC dose was unavailabie at pharmacy, and no cover was provided with LMWH.

Comparison to previous study with paper chart showed reduction in duplication of therapy: DOAC + Therapeutic LMWH (4 paper-based vs 1 eMeds) and DOAC + VTE Prophylaxis (4 paper-based vs 0 eMeds). Duplication dose of DOAC (0 paper-based vs 1 eMeds).

Prescribing errors were less overall using eMeds (N=6, 6%) compared to the paper-based system (N=18, 20%). The two cohorts compared had similar demographics, reason for admission and indication for DOAC.

The duplicate dosing of DOAC + Therapeutic LMWH within the eMeds cohort was on a complex oncology patient. The eMeds chart was extensive and required the prescriber to ‘scroll’ through several screens to see all medications charted. The introduction of a function to group anticoagulation medications on the eMeds chart may lead to greater patient safety.

The omission of a dose of DOAC due to unavailability is a systemic supply issue that needs to be addressed within the hospital. In these events, LMWH cover should be provided, which is extensively available.

There were no adverse outcomes documented due to the medication errors.

Summary/Conclusion: The introduction of the electronic prescribing system eMeds appears to have led to a reduction in the frequency of prescribing errors with DOACs compared to paper-based system. Hospitals need to continue to take appropriate measures to minimise the potential risks of adverse patient outcomes from prescribing errors with anticoagulants.

PB2347: GLOBAL REPRESENTATION AMONG JOURNAL EDITORS IN HEMATOLOGY: ARE WE DIVERSE, EQUITABLE, AND INCLUSIVE?

A. Gómez-De León 1,* , A. Noyola-Pérez 1 , L. Gil-Flores 1 , E. Ramos-Barrera 2 , E. Rojas-Guerrero 1 , H. Martínez-Espinosa 1 , V. Rodríguez-González 1 , P. Colunga-Pedraza 1 , L. Tarín-Arzaga 1 , O. Cantú-Rodríguez 1 , H. Gutiérrez-Aguirre 1 , G. Ruíz-Argüelles 3 , D. Gómez-Almaguer 1

1 Hematology, Universidad Autónoma de Nuevo León; 2 Instituto Tecnológico de Estudios Superiores de Monterrey, Monterrey; 3 Centro de Hematología y Medicina Interna. Clinica Ruiz., Puebla, Mexico

Background: Editorial board members are responsible for the quality of content accepted to a scientific journal, ensuring its ethical integrity, appropriate methodology, presentation, novelty, and potential impact, serving as guides throughout the peer-review process. Journal editors (JEs) are selected due to their expertise in a field, publication record, and history of collaboration with a scientific journal. Assessing diversity and inclusion in editorial boards is needed to understand which biases in research may be present. Having a diverse editorial board can balance perspectives and reduce these biases. In the field of hematology, this has not been assessed.

Aims: We aimed to analyze the characteristics of indexed hematology journal (HJs) editors worldwide.

Methods: We used the InCites Journal Citation Reports (JCR) to extract information from all indexed journals in the Hematology category. Each journal´s website was screened, and we extracted data on the composition of the editorial board, including editors-in-chief and associate editors and excluded editorial board members with other roles which are not of editorial capacity. JEs were classified according to their institution’s country of residence, academic position, and sex, and were compared according to each HJs characteristics, including impact factor quartiles, open access availablity, and scope. The countries of JEs were classified according to the World Bank classification and Human Development Index. Information from each journal and JEs was extracted by one author, and cross-checked for accuracy by a second and third reviewer.

Results: We reviewed 93 HJs indexed in the JCR and found a total of 959 persons identified as JEs with their overall characteristics presented in Table 1. One in four JEs were women with this difference present across HJ impact factor quartiles. Only 13 out of 111 editors-in-chief were women (11.7% vs. 88.3%; p<.001). Most JEs work in high income countries (86%) and are affiliated to institutions with a very high index of human development (94.37%), most frequently in the United States (34.9%), followed by Germany (8.9%), Italy (8.3%), and the United Kingdom (5.4%). Only 124 JEs (12.9%) are working in low- and middle-income countries, 17 in the top impact factor quartile (6.6% of all top-tier JEs). Most JEs from LMICs work in upper-middle income economies, being Brazil the country with the highest number of editors (n=36), followed by China (n=35) and only 2 editors (0.2%) working in a low-income country. The average article processing charges among was $2,485 USD.

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Summary/Conclusion: Three quarters of JEs are men which shows a lack of equity of representation. The majority of HJs and their editorial board members come from high income countries, therefore their distribution is not equitable nor correlated with the global burden of hematological disease. Efforts to increase diversity, equity, and inclusion in HJs should be undertaken.

PB2348: THE PATIENT’S PERSPECTIVE ON CLNICAL TRIALS: RESULTS FROM A REGIONAL SURVEY IN HAEMATOLOGICAL CENTERS IN ANTWERP

B. Heyrman 1,* , S. Meers 2 , A. Van De Velde 3 , S. Anguille 3

1 Haematology, ZNA, Antwerp; 2 Haematology, KLINA, Brasschaat; 3 Haematology, UZA, Antwerp, Belgium

Background: Clinical trials are a prerequisite for medical progress. Unfortunately, only a small proportion of patients participates in clinical studies. Implementation of a digital informed consent form (ICF) is another possible barrier in the process of trial inclusion

Aims: Primary objective was to collect information on the attitude and knowledge on clinical trials in haematological patients. Secondary objective was to explore influenceable variables in the decision making process.

Methods: We conducted an anonymous survey in haematological patients during a fixed period of 6 months in 4 hospitals in the province of Antwerp, Belgium. Patients were invited to participate via flyers that were handed out by accredited haematologists or had the possibility to fill out the survey on site on tablets provided in the hospitals. The survey was built by the authors in Qualtrics® which was used for data collection and approved by the ethical committee of the University Hospital Antwerp.

Results: During the 6-month recruitment period, 420 patients filled out the survey, 219 female, 187 male, 14 patients did not answer this question. More than half of the patients (56 %) was over 60 years old. Half of the patients (50.8%) was not or totally not aware of clinical trial concepts, yet 87.9% indicated to have a positive feeling about clinical trials. Most patients (87.3%) trusted in the safety of clinical trials. The majority of patients (67,0%) was more or less willing to participate in a clinical trial when offered (24% previously or actively participated in a clinical trial). A significant relation in willingness to trial-participation and trial-knowledge (p<0.001) and doctor-patient relationship (p=0.007) was noted. Age was not a statistical significant variable for willingness to participate (p=0.64). A large portion (21.0%) was not willing to go to another hospital for trial participation, or only in case there was no other possible treatment (20.4%). A greater part (79.9%) indicated to prefer to know the results of the study following study closure. In case of study participation, information will mainly be obtained from the general practitioner (26.4%) and treating haematologist (39.9%). In the group of trial patients, 73.1% was satisfied with the information in the ICF, 85.9% indicated to have had sufficient amount of time to pose questions and 76.1% would advise study participation to others. Only 24.5% of patients above 60 years indicated to be willing to use a digital ICF procedure vs 57,0% in the group younger than 60 (p<0.001).

Summary/Conclusion: Our results are in line with other publications that the majority of patients is willing to participate in a clinical trial when offered. Implementation of a digital ICF is not preferred by the majority of our patients, who are mainly 60 years or older. Investment in patient information and patient-doctor relation is a key factor to motivate patients for trial participation.

PB2349: LESSONS LEARNED FROM THE MYPAL DIGITAL HEALTH INTERVENTION: CAPITALIZING ON THE PATIENT REPORTED OUTCOMES (PRO) PARADIGM TOWARDS A PARTICIPATORY HEALTHCARE FOR PATIENTS WITH HEMATOLOGICAL MALIGNANCIES

C. Karamanidou 1,* , L. Scarfò 2 , M. Doubek 3 , S. Garani-Papadatos 4 , J. Didi 3 , C. Pontikoglou 5 , E. Kazantzaki 5 , J. Ling 6 , C. Pain 6 , H. Papadaki 5 , R. Rosenquist 7 , N. Stavroyianni 8 , S. Payne 9 , P. Ghia 10 , K. Stamatopoulos 1 , P. Natsiavas 1

1 Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece; 2 Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 3 Department of Haematology and Oncology, University Hospital Brno, Brno, Czechia; 4 Department of Public Health Policy, University of West Attica, Athens; 5 Department of Hematology, University of Crete School of Medicine, University Hospital of Heraklion, Crete, Greece; 6 European Association for Palliative Care, Vilvoorde, Belgium; 7 Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 8 Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece; 9 : International Observatory on End of Life Care, Lancaster University, Lancaster, United Kingdom; 10 Strategic Research Program on CLL, Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy

Background: Chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS) are two of the most frequent hematological malignancies. CLL and MDS are also considerably heterogeneous in terms of clinical course and response to treatment, ranging from relatively indolent to extremely aggressive. Thus, open issues abound regarding the impact of CLL and MDS and their treatment on patients’ quality of life (QoL). Patient-reported outcomes (PROs) have been identified as an emerging paradigm, aiming to capture the patient’s perspective onself-assessed health status. Obviously, these data are critical with regards to the evaluation of the treatment effects and the patients’ QoL, while also enabling the positioning of the patient as a key stakeholder within the healthcare decision making process. Novel methodologies and eHealth approaches can be valuable for the adoption of the PRO paradigm in real-world settings as they can promote richer, less obtrusive and preemptive communication which could facilitate early recognition of potential symptoms of disease or treatment adverse effects (e.g., adverse drug reactions, lack of physical activity, worsening of QoL etc.).

Aims: In this abstract, we present the lessons learned thus far from the implementation of the MyPal project, a Horizon 2020 Research & Innovation Action aiming to foster palliative care for patients with CLL and MDS by leveraging the ePRO paradigm.

Methods: MyPal aspires to empower patients and their caregivers to more accurately capture their symptoms/conditions, communicate them in a seamless and effective way to their healthcare providers (HCPs); and, ultimately, to foster action through advanced methods of identification of important deviations relevant to the patient’s state and QoL. To this end, MyPal developed a technical platform including a mobile app for patients with CLL and MDS, collecting information via standardized questionnaires and other information sources (e.g., wearable sensors), also enabling spontaneous symptoms reporting, educational material provision, motivational messages, discussion guides, notifications etc. A data intensive web-based dashboard platform is also provided for healthcare professionals, providing real-time analytics, enabling a better view of collected PROs and other relevant information on patients’ health status. Currently, a randomised clinical study is being conducted in 4 European countries to evaluate the proposed intervention and its potential impact on patients’ QoL.

Results: Based on this experience, a number of key issues have emerged: (a) while patients are generally positive about the use of eHealth, they are still reluctant about engaging in eHealth clinical studies; (b) digital literacy levels differ across different age groups as well as among different cultural contexts; (c) the COVID-19 pandemic seriously hindered patient recruitment due to the widely adopted recommendations for patients to avoid visits to hospitals unless absolutely necessary but (d) the COVID-19 pandemic also highlighted the potential benefits for HCPs of using eHealth tools in order to deliver patient care in a more decentralized and patient-centric fashion.

Summary/Conclusion: In conclusion, MyPal is likely to provide important new evidence about how digital health systems can be used to improve QoL and facilitate better communication between patients with hematological malignancies and HCPs.

PB2350: SAFETY AND EFFICACY OF HYPODERMOCLYSIS IN PATIENTS WITH HEMATOLOGICAL CANCERS

A. Kodru 1,* , Z. Koloper 1 , V. Gveric-Krecak 1 , I. Krecak 1

1 Department of Internal Medicine, General Hospital of Sibenik-Knin County, Sibenik, Croatia

Background: Hypodermoclysis (HDC) refers to a subcutaneous infusion of fluids which is then absorbed into circulation via diffusion and perfusion. Compared with different intravenous accesses, HDC has a lower incidence of side-effects, such as local pain, infections, bleeding or allergies. In recent years, HDC has been increasingly used for palliation of advanced solid cancers, especially when oral intake is impaired. On the other hand, there is scarce data regarding the safety and efficacy of HDC in patients with hematological cancers (HC) who are intrinsically more prone to infections and/or bleeding. Since 2019, we have been using HDC for palliation of HC at our institution. Here, we report our experience.

Aims: To analyze the safety and efficacy of HDC for palliation of patients with HC.

Methods: This was a retrospective single-center study conducted at the General Hospital of Sibenik-Knin County, Croatia. Clinical and laboratory data regarding patients with HC under palliative care who have been hospitalized and treated with HDC (BD SAF-T-Intima®, Braun) was collected through the medical chart review. Age- and sex-matched patients with advanced solid cancers and those with impaired oral intake and/or poor venous access treated with HDC served as controls. The chi-square and the Mann-Whitney U tests were used. P-value <0.050 was considered statistically significant. MedCalc Statistical Software® (Ostend, Belgium, ver. 20.014) was used for all statistical analyses.

Results: Thirty-two (54.2%) patients with HC under palliative care (non-Hodgkin lymphoma=10, acute myeloid leukemia=9, myelodisplastic syndrome=7, multiple myeloma/plasma cell leukemia=6) and 27 (45.8%) controls (metastatic lung cancer=6, metastatic breast cancer=1, dementia=3, mental retardation=1, other=16) were retrospectively included in the period between January 2019 and January 2022.

Infused drugs included isotonic fluids (5% glucose, 0.9% saline, Ringer’s solution) in 100%, tramadole in 81.25%, parenteral potassium in 78.1%, furosemide in 78.1%, dexamethasone in 18.75%, meropenem in 12.5%, methylprednisolone in 6.25%, diazepam in 6.25%, haloperidol in 6.25%, gentamicin in 6.25%, morphine in 6.25% and ketoprofen in 3.1% of patients with HC.

Even though the duration of HDC in patients with HC was longer (median 6 vs. 3 days; p<0.001), and the fact that HC patients more often needed erythrocyte/platelet transfusions (71.9% vs. 0%; p<0.001), had lower total leukocyte (median 3.5x10 9 /L vs. 9.6 x10 9 /L; p=0.008), absolute neutrophil (1.86 x10 9 /L vs. 7.1 x10 9 /L; p=0.004) and platelet counts (40 x10 9 /L vs. 271 x10 9 /L; p<0.001), as well as a higher proportions of patients with granulocytopenia (<1500/mL, 46.9% vs. 3.7%; p<0.001) and severe thrombocytopenia (<50 x10 9 /L, 53% vs. 0%; p<0.001), HDC-related infections (0% vs. 0%), bleedings (0% vs. 0%), allergies (0% vs. 0%) and pain (9.4% vs. 7.4%; p=0.787) were entirely absent or minimal in both patient groups. Finally, a higher proportion of patients with HC was eventually discharged from the hospital (78.1% vs. 55.7%; p=0.066).

Summary/Conclusion: Our data shows that HDC is safe and effective for palliation of hospitalized patients with HC. Limitations of this study are its retrospective single-center design and the lack of evaluation of other patient-reported outcomes. Nevertheless, this data confirms the excellent safety of HDC for palliation of patients with both solid and HC.

PB2351: ANXIETY AT THE HEMATOLOGIST

S. Trojan 1,* , A. Garma 1 , V. Gveric-Krecak 1 , I. Krecak 1

1 Department of Internal Medicine, General Hospital of Sibenik-Knin County, Sibenik, Croatia

Background: Referral to outpatient hematology clinics may increase the stress and anxiety for the patients, as they may perceive that they are being referred for evaluation of a cancer diagnosis.

Aims: To investigate the severity of anxiety in subjects referred to a hematologist for the first time.

Methods: This prospective single-center study measured the severity of anxiety in an unselected cohort of subjects referred to a hematologist for the first time using a internationally validated anxiety-focused questionnaire, Generalized Anxiety Disorder-7 (GAD-7). Excluded from participation were subjects younger than <18 years of age and those who were already known to have anxiety disorders, depression, or schizophrenia.

The GAD-7 questionnaire is widely used to rapidly screen for anxiety and to measure its severity across different anxiety disorders, as well as in the general population. It evaluates the 7 core anxiety symptoms on a 4-point scale (0-3) with a maximum score of 21. Seven measured symptoms are as follows; (1) feeling nervous, anxious, or on edge; (2) being able to stop or control worrying; (3) worrying too much about different things; (4) trouble relaxing; (5) being restless; (6) becoming easily annoyed or irritable; and (7) feeling afraid as if something awful might happen. Response options are “not at all”, “several days”, “more than half the days”, and “nearly every day”, scored as 0, 1, 2, and 3, respectively. Higher scores reflect greater anxiety severity, and the scores of 5, 10, and 15 represent mild, moderate, and severe anxiety symptom levels, respectively. The cut-off point of ≥10 identifies probable cases of GAD that may require further psychiatric assessment.

Results: A total of 203 subjects were invited to participate in the study, 163 (80.3%) responded and were included in this analysis. The median age was 60 years (range 19-89) and 96 (59%) were female. Median GAD-7 score of all subjects was 5 (range 0-21), with 81 (49.7%), 44 (27%), 31 (19%) and 7 (4.3%) of subjects having no, mild, moderate and severe anxiety symptoms, respectively (Figure 1). Therefore, a total of 82 subjects (50.3%) showed signs of anxiety and 38 patients (23.3%) had GAD-7 score of ≥10, which may even suggest possible cases of GAD. Females had higher GAD-7 scores (median 5, range 0-21) when compared to males (median 3, range 0-20, p=0.008), whereas the GAD-7 score did not correlate with age (rho=0.041, p=0.600). Notably, there was no difference in GAD-7 scores between patients with malignant (15.3%) or benign (84.7%) hematological disorders (p=0.320)

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Summary/Conclusion: Even though the vast majority of referred patients in this study had benign hematological disorders (84.7%), half of them showed signs of anxiety. Furthermore, approximately one-fourth of subjects showed moderate or severe signs of anxiety and may, therefore, even be considered as having possible GAD; this incidence of possible cases of GAD is well above that from the general population (~5%).

Limitations of the study are single-center design, outpatient selection bias, and the lack of evaluation of other medical (i.e., laboratory assays) and non-medical risk factors (i.e., educational level, employment status, marrital status, or income) on the degree of anxiety. Nevertheless, these results show that referrals to hematologists may have a negative impact on the psychological state of a patient. Therefore, future studies should focus on the strategies which may help to alleviate these negative emotions.

PB2352: CONSOLIDATION OF ERN-EUROBLOODNET: THE EUROPEAN REFERENCE NETWORK ON RARE HEMATOLOGICAL DISEASES FIRST 5 YEARS OF IMPLEMENTATION

M. D. M. Mañú Pereira 1,* , V. Gutierrez Valle 1 , M. Pellegrini 2 , M. D. L. Á. Rodríguez Sánchez 3 , L. Margot 2 , B. Gulbis 4 , P. Fenaux 2

1 Cancer and blood diseases in children, Vall d’Hebron Research Institute, Barcelona, Spain; 2 Hematology, Hôpital Saint-Louis, Paris, France; 3 Cancer and blood diseases in pediatrics, Vall d’Hebron Research Institute, Barcelona, Spain; 4 Hematology, Hôpital ERASME/LHUB-ULB, Brussels, Belgium

Background: ERN-EuroBloodNet was established in 2017 as the European Reference Network on Rare Hematological Disorders (RHDs) bringing together nationally recognized centres of excellence with the goal of promoting EU best health care in RHDs. ERN-EuroBloodNet has been recently enlarged integrating 103 HCP from 24 EU-MS.

Aims: ERN-EuroBloodNet was conceived to contribute to innovative, efficient and sustainable health systems and facilitate access to better and safer healthcare for EU citizens while decreasing the cross-border health barriers.

Methods: Since 2017, ERN-EuroBloodNet established the state-of-the art of RHD allowing the implementation of transversal and disease-specific strategies, where actions on very rare RHD were prioritized.

Results: Profile. 182 expert profiles were created freely accessible. Expert centers follow 65,000 RHD patients and treat 5,000 new patients per year, while 24 patients requested support for cross-border health assistance. Expertise. The need to improve access to next-generation sequencing for non-oncological RHD and bone marrow transplantation for sickle cell disease (SCD) was identified. Also, significant disparities in the clinical practice of primary vitreoretinal lymphoma were found and we demonstrated that less than 30% of children with SCD benefit from adequate annual stroke risk monitoring. Guidelines. A repository of 68 Clinical Practice Guidelines (CPG) classified on quality of evidence and consensus approach was created. Recommendations for diagnosis and treatment of methemoglobinemia was published in collaboration with EHA. A CPG on Adult Burkitt Lymphoma is under development. Next topics focus long-term complications in hemoglobinopathies and patients’ pathways&summary. Education. ERN-EuroBloodNet Webinars were launched for professionals with 26 Thursdays Webinars and 3 EBAH accredited Topic on Focus on Cutaneous Lymphoma, Thrombotic Microangiopathies, and Bone Marrow Failures. A collaboration was established for EHA & ERN-EuroBloodNet Spotlight on Castleman Disease. For patients, 3 Topic on Focus were launched for Myelodysplastic syndromes, SCD, and Cutaneous lymphoma. Past webinars are available at EuroBloodNet EDU Youtube channel. Preceptorships on SCD will be launched soon. Telemedicine. 43 complex cases have been inter-professionally discussed in the Clinical Patient Management System with 21 outcome reports delivered. Registries. 184 Registries were identified through the European Rare Blood Diseases Platform (ENROL), endorsed by the EHA. The ENROL project, which includes rare anemias, dendritic cell leukemia and von Villebrand’s disease pilots, aims to collect exhaustive and therefore epidemiological data for RHDs. The final objective is a possibility of EU health planningl and the promotion of research by identifying cohorts of patients. ERN-EuroBloodNet launched the collaborative platform on patients with red blood cells and COVID-19 containing so far 373 patients. Collaborations. collaborative research projects were encouraged like EC-funded projects i.e., genomics and personalized medicine in hematological diseases (GenoMed4All) and the properties and viability of erythrocytes (EVIDENCE), or the International Hemoglobinopathy Research Network (INHERENT) for genomic and phenotypic correlations.

Summary/Conclusion: The implementation of well-defined strategies but above all adapted to the specific and not yet covered needs of RHD has led to the realization of concrete projects. This has laid the foundations to strengthen health systems in the field of RHD and allow them to flourish under the new EU4Health programme.

PB2353: HEALTHCARE RESOURCE UTILIZATION AND COSTS OFF THERAPY WITH FIXED-DURATION VENETOCLAX AMONG CLL PATIENTS

J. N. Allan 1,* , A. P. Skarbnik 2 , N. Emechebe 3 , H. Alhasani 3 , B. S. Manzoor 3

1 Weill Cornell Medicine University Medical Center, New York; 2 Novant Health, Charlotte; 3 AbbVie, North Chicago, United States of America

Background: Treatment with fixed-duration venetoclax regimens are approved in first-line (1L) and relapsed/refractory (RR) settings for patients with chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL). While studies have described fixed treatment duration (FTD) in the context of clinical trials or total cost of care, there’s a paucity of data that characterize the real-world benefits of FTD with venetoclax therapy.

Aims: This study aims to quantify the healthcare resource utilization (HRU) and costs, during and post-completion of therapy, with venetoclax plus obinutuzumab (VO) or venetoclax plus rituximab (VR).

Methods: Patients diagnosed with CLL/SLL between 1/1/2010 – 6/30/2020 that were at least 18 years old and initiated VO in the 1L or VR in RR were identified in Optum Clinformatics database. Patients were required to be continuously enrolled in their health plan one year pre- and post-initiation with VO or VR. The follow-up period was split into two phases: 1) on-treatment phase (time in months from VO/VR initiation to end of treatment) and 2) off-treatment phase (time in months from one day after end of venetoclax treatment to end of continuous enrollment/one day before next line of therapy). Per-patient, per-month (PPPM) all-cause HRU and costs were summarized using mean, standard deviation (SD), median, and interquartile range (IQR) in both on- and off-treatment phases.

Results: A total of 108 patients (VO=46; VR=62) were identified with a median age of 71 years and 30.6% female. In 1L, the median (IQR) duration of the on- and off-treatment phases were 12.3 (11.1 – 14.1) and 7.5 (3.3 – 10.3) months, respectively. Mean PPPM all-cause costs were $18,552 and $2,837 in the on- and off-treatment phases, respectively. The on-treatment costs were largely driven by medication costs ($15,203 PPPM) compared to medical costs ($3,349 PPPM). Conversely, medical costs ($2,124 PPPM) accounted for the majority of total costs off-treatment. The mean PPPM all-cause outpatient visits were slightly higher in the on vs off-treatment phases (3.3 and 2.3 visits), respectively. In RR, the median (IQR) duration of the on- and off-treatment phases were 17.6 (12.2 – 23.7) and 2.3 (0.5 – 6.9) months. Mean PPPM all-cause costs were $17,061 and $1,889 in the on- and off-treatment phases, respectively. Similar to the VO cohort, medication costs ($13,887) accounted for a majority of the total cost in the on-treatment phase while medical costs ($1,273) were the primary drivers of cost in the off-treatment phase. Furthermore, the mean PPPM all-cause outpatient visits were higher in the on-treatment phase (3.2 vs 1.4).

Summary/Conclusion: This study illustrates that in real-world practice settings, reduced HRU and cost burden was observed with venetoclax combination regimens. These preliminary results suggest that fixed treatment duration therapy allows patients to experience off-therapy economic benefits. Further exploration should be undertaken in a larger cohort of patients.

PB2354: THE IMPACT OF ACUTE HEPATIC PORPHYRIA ON MENTAL HEALTH: RESULTS FROM THE PORPHYRIA WORLDWIDE PATIENT EXPERIENCE RESEARCH (POWER) STUDY

D. Nance 1,* , D. Lyon 2 , S. Hegarty 3 , R. Falchetto 4 , J. Barman-Aksözen 4,5 , J. E. Matos 6 , S. Meninger 7 , S. Lombardelli 8 , A. Dickey 9

1 Banner Health, Gilbert; 2 American Porphyria Foundation, Bethesda, United States of America; 3 Global Porphyria Advocacy Coalition, Durham City, United Kingdom; 4 Swiss Society for Porphyria; 5 Stadtspital Zürich, Triemli, Institute of Laboratory Medicine, Zürich, Switzerland; 6 Cerner Enviza, North Kansas City; 7 Alnylam Pharmaceuticals, Cambridge, United States of America; 8 Alnylam Pharmaceuticals, Maidenhead, United Kingdom; 9 Massachusetts General Hospital, Boston, United States of America

Background: Acute hepatic porphyria (AHP) is a group of rare genetic diseases of heme biosynthesis characterised by potentially life-threatening attacks, most frequently occurring in the abdomen. AHP is also associated with psychiatric symptoms, including depression and anxiety. Prior studies on AHP have also documented its psychosocial burden, which can limit patients’ day-to-day activities, including social interactions and ability to work.

Aims: To investigate the burden of AHP on mental health, the study evaluated the effects of the disease on social life, personal life/goals, depression, and anxiety.

Methods: Patients age ≥18 years who experienced >1 AHP attack within the past 2 years or who were receiving intravenous hemin and/or glucose for attack prevention were recruited from the United States, Italy, Spain, Australia, Mexico, and Brazil from January 19 to April 26, 2021. Patients taking givosiran were excluded. Participants were administered an online survey that used standardised questionnaires and validated screening instruments to evaluate the impact of AHP symptoms on social life, personal life/goals, and mental health. The 8-item Patient Health Questionnaire depression scale (PHQ-8; range, 0–24) and the 7-item Generalized Anxiety Disorder scale (GAD-7; range, 0–21) were used to screen patients for depression and anxiety. Current moderate to severe depression was identified with a PHQ-8 cutoff score of ≥10, and threshold scores of 5, 10, and 15 on the GAD-7 identified mild, moderate, and severe anxiety, respectively. Depression and anxiety were evaluated among the overall patient population as well as several subgroups, including those with sporadic attacks (0–5 attacks over 2 years) vs recurrent attacks (≥6 attacks over 2 years), those receiving vs not receiving prophylactic treatment for AHP, and those with an active disease duration of 0–5 years vs those with a disease duration of ≥6 years.

Results: Ninety-two patients with AHP completed the survey. Mean age was 41.1 years; 90% were female. Patients reported substantial impact on social life—76.1% of patients reported that most of their symptoms were hidden and that people in their social circle did not know they had AHP. An equal percentage of patients reported feeling frustrated that people do not understand the challenges they face, because their symptoms are not visible. Similarly, 72.8% of patients reported feeling guilty/upset that their symptoms and disabilities affect others (Figure 1 A ). Patients also reported AHP has impacted their personal life/goals. For example, >80% of patients reported having had to modify or give up goals important to them, more than half reported that the decline in their mental and physical health feels never-ending, and over a third reported they had lost their sense of purpose (Figure 1 B ). PHQ-8 scores indicating moderate to severe depression were reported in more than half of patients with AHP (58.7%) regardless of attack rate or prophylactic treatment status. GAD-7 scores indicating moderate to severe anxiety were reported in 48.9% of patients, and were highest in patients experiencing recurrent attacks (56.8%) (Figure 1 C ).

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Summary/Conclusion: Patients with AHP, regardless of attack rate, treatment received, or duration of active disease, experience a high mental health burden on their personal and social life. This study suggests that approximately half of patients with AHP experience moderate to severe anxiety or depression. These results highlight the importance of mental health monitoring as part of disease management for AHP.

PB2355: EFFECTS OF CHRONIC DISEASE LOCUS INTERVENTION MODEL ON QUALITY OF LIFE AND STRESS IN PATIENTS WITH HEMATOPOIETIC STEM CELL TRANSPLANTATION

A. Sun 1 , J. Tao 1 , F. Wu 1 , L. Gao 1 , C. Zhang 1,*

1 Medical center of hematology, Xinqiao hospital Army Medical University, Chongqing, China

Background: With the continuous improvement of hematopoietic stem cell transplantation technology, donor source system and supportive treatment, the long-term survival rate of patients after hematopoietic stem cell transplantation has been continuously improved, but it is often accompanied by chronic or late complications, including severe chronic graft-versus-host disease (GVHD), osteoporosis, organotoxicity, infertility, etc, It has an adverse impact on the quality of life of patients. According to the stage change of chronic disease trajectory, the chronic disease trajectory model provides continuous and scientific nursing intervention for patients around their disease-related behavior, self-concept behavior and daily life behavior, aiming to improve patients’ self-management ability, control disease symptoms, prevent complications and improve patients’ quality of life.

Aims: To investigate the influence of chronic disease trajectory intervention mode on quality of life and stress in patients with hematopoietic stem cell transplantation.

Methods: A total of 100 hematopoietic stem cell transplantation patients who received hematopoietic stem cell transplantation from October 2019 to December 2020 in a grade III, Grade A hospital in Chongqing were selected as the investigation subjects. Patients admitted on Mondays and Thursdays were selected as the experimental group, and patients admitted on other times were selected as the control group, with 50 patients in each group. The control group was treated with routine nursing, and the experimental group was treated with chronic disease track intervention mode nursing based on routine nursing. The anxiety Self-rating Scale (SDS), self-rating Depression Scale (SAS), quality of life and pressure score of the two groups were compared before transplantation, during bone marrow suppression period, when patients were discharged from the warehouse and 1 month after transplantation.

Results: Before storage, there were no significant differences in the scores of quality of life, depression, anxiety and pressure between the two groups (P>0.05), and the scores of depression, anxiety and pressure in the experimental group were significantly lower than those in the control group in the marrow suppression period, out of storage and 1 month after transplantation (P<0.05). The score of quality of life in experimental group was significantly lower than that in control group at bone marrow suppression stage and 1 month after transplantation (P<0.05). The scores of quality of life in the control group were significantly higher in the marrow suppression period, at the time of warehousing and 1 month after transplantation (P<0.01). There were no significant differences in depression and anxiety scores among the experimental groups at different periods (P>0.05), and the pressure score at 1 month after transplantation was significantly lower than that before storage (P<0.01). The depression, anxiety and stress scores of the control group were significantly higher than those of the control group at the bone marrow suppression stage, the time of discharging and 1 month after transplantation (P<0.01)

Summary/Conclusion: The chronic disease trajectory model can provide early warning according to the disease trajectory from the beginning of HSCT intervention to the continuous attention 1 month after HSCT, which is helpful to relieve the pressure of HSCT patients.

PB2356: EFFECTS OF TARGETED NURSING INTERVENTIONS ON CANCER-INDUCED FATIGUE AND MENTAL HEALTH IN CHEMOTHERAPY PATIENTS WITH LEUKEMIA

S. Xiao 1 , G. Su 1 , D. Lan 1,*

1 Medical center of hematology, Xinqiao hospital Army Medical University, Chongqing, China

Background: The phenomenon of cancer-induced fatigue is one of the research hotspots at this stage in the field of foreign tumors, but the understanding and attention of this symptom in china leukemia field is not enough, which is not conducive to the provision of high-quality care for leukemia patients.

Aims: To investigate the implementation method of targeted nursing intervention program and its application value in the care of chemotherapy patients with leukemia.

Methods: The 100 leukemia chemotherapy patients admitted to our hospital from January to December 2021 were randomly divided into a control group and an observation group of 50 cases each, and the observation group implemented routine nursing intervention and the control group targeted nursing intervention. Before and after nursing interventions, the Cancer Fatigue Scale (CFS) was used to evaluate the state of cancer-induced fatigue, and the Hamilton Depression Scale (HAMD) and the Hamilton Anxiety Scale (HAMA) were used to evaluate the state of mental health.

Results: After the intervention, the CFS scale score of patients in the control group was lower than that in the observation group (P<0.05), and the HAMD score and HAMA score were lower than those in the observation group (P<0.05).

Summary/Conclusion: The implementation of targeted nursing interventions in the clinical care of chemotherapy patients with leukemia can effectively improve the patient’s cancer-induced fatigue state and negative psychology.

PB2357: THE RELATIONSHIP BETWEEN SYMPTOM CLUSTERS AND QUALITY OF LIFE IN PATIENTS WITH HEMATOPOIETIC STEM CELL TRANSPLANTATION DURING EARLY TRANSPLANT: A LONGITUDINAL STUDY

F. Wu 1 , A. Sun 2 , X. Du 2 , L. Gao 2,*

1 Medical center of hematology, Xinqiao hospital Army Medical University; 2 Medical center of hematology, Xinqiao hospital Army Medical University, Chongqing, China

Background: The conditioning regimen of haematopoietic stem cell trans-plantation (HSCT), a treatment common to haematological diseasesand malignancies, involves chemotherapy and/or total body irradiation. These treatments affect the patient profoundly due to theiradverse effects.monitor the dynamic changes of symptoms, analyze the cluster phenomenon, and explore the dynamic changes of symptom groups and their correlation with quality of life(QOL), in order to provide reference for medical staff to formulate efficient symptom management strategies.

Aims: To investigate symptom clusters and in the HSCT patients at different time points in the early stage of transplant,and QOL. the correlation between was analyzed to provide the basis for clinical nurses to implement symptom manangement and improve the QOL.

Methods: A total of 110 patients who received hematopoietic stem cell transplantation from August 2020 to February 2021 in a class Ⅲ hospital were selected as the study subjects.there were four times:on the day of admission,Pretreatment period(-4 to -3d),Myelosuppression(lowest count of WBC),Hematopoietic reconstitution period. The M.D.Anderson Symptom Inventory Blood and Marrow Transplantation (MDASI-BMT) module measured symptom severity. The Functional Assessment of Cancer Therapy-Bone Marrow Transplant (FACT-BMT) measured QOL.

Results: The main symptom clusters of hematopoietic stem cell transplantion patients in the early stage of transplantation mainly included somatic symptoms,gastrointestinal symptoms,psychological and emotional symptoms,and neurological symptoms.the composition of these symptoms was different at each time point.and the patients at different time points had different scores of QOL,the difference was statistically significant(P<0.05).the severity of the four symptom clusters were negatively correlated with the QOL scures at different times.

Summary/Conclusion: In the early stage of transplantation,patients with multiple stable symptom cluster and sykptoms severity score were negatively correlated with QOL score.medical staff should manage the symptoms of patients in the early stage of transplantation,and carry out effective management to improve the QOL of patients.

PB2358: SIGNIFICANT INCREASE IN NEWLY DIAGNOSED HEMATOLOGICAL MALIGNANCIES FOLLOWING LOCKDOWN EASE IN CROATIA

M. Simunic 1,* , A. Dobricic 1 , M. Vujcic 1 , A. Pijuk 1 , S. Madunic 1 , J. Jakelic-Pitesa 1 , D. Lozic 1

1 Hematology (Internal Medicine Clinic), Clinical Hospital Center Split, Split, Croatia

Background: Special epidemiological measures aimed at suppressing SARS-CoV-2 outbreak were introduced in Croatia in March 2020, thus reducing regular work capacity in hematological outpatient and inpatient care. In our hospital, this included relocating the entire Hematology Department to a remote location, reduction of hospital beds in the Hematology Inpatient Unit by approximately 60%, Day Clinic operating at a reduced capacity, and a complete suspension of Hematology Polyclinic during first lockdown.

Aims: Herein we report our observation of unusually high incidence of newly diagnosed malignant hematological diseases following first lockdown ease in May/June 2020.

Methods: We collected data of patients hospitalized in Hematology Department for 4 periods: May 1 - June 15, 2020 for the test arm, and the same calendar period during previous 3 years (May 1 - June 15 of each of the calendar years 2017, 2018 and 2019), for the control arm. The rationale for such design was that a phenomenon of re-establishing regular work capacity, following temporary restriction, was only observed in the test arm. The study included patients of both sexes older than 18 who were diagnosed with either: Hodgkin lymphoma (C81.0 -C81.9 according to the 10th ICD Revision), different types of non-Hodgkin lymphoma (subsections C82.0 - C83.9 and C85.1-C85), as well as multiple myeloma and malignant plasma cell neoplasms (C90.0 - C90.3). Excluded from our study were diagnoses of T/NK cell lymphoma (C84.0- C84.9; C86.0 - C86.6), malignant immunoproliferative diseases (C88.0 - C88.9), leukemias and other specified malignant neoplasms of lymphatic, hematopoietic and related tissues (C91.0 - C96.9) as well as polycythemia vera and non-malignant hematological diseases (D45 and D50 - D89 in ICD-10).

Results: In years 2017-2019, similar numbers of patients were diagnosed with a hematological malignancy in our Department (n=4 for 2017, n=8 for 2018, n=4 for 2019) whereas in 2020, a total of 28 patients were diagnosed during the same calendar period (Hodgkin lymphoma: n=5, NHL n=12, multiple myeloma n=7, CLL/SLL n=4). Statistical analysis revealed a significant increase ( p <0.05) of newly diagnosed hematological malignancies in May and first half of June 2020, when compared to the same calendar periods during previous three years. Further statistical analysis has not established significant differences in outcome (difference in EFS statistically insignificant, p =0.86), as we had expected in the short follow-up period.

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Summary/Conclusion: Facilitating treatment of patients affected by the novel coronavirus represented a welcome change in healthcare system in early 2020, in our country and abroad. At the same time, however, the reduction of tertiary health care capacity aimed at population with hematological diseases presented serious risks for successful diagnosis and treatment outcome, a subject that gained wide attention in literature. It has been reported that, also due to psychological reasons, a fraction of patients delayed seeking medical attention after noticing symptoms. In our study we aimed at analyzing the effects of lockdown ease on the number of newly diagnosed hematological malignancies. We were able to demonstrate the effect of pandemic-related measures on detecting new disease cases. It remains to be clarified if a sudden surge in new diagnoses was due to delayed first physician’s appointments/hospitalizations, as is suggested by available literature. The results of our study suggest that longer follow-up period will be required in order to clarify the effects of possible late diagnoses on the treatment outcome.

PB2359: HEALTHCARE RESOURCE UTILIZATION OF ADULT PATIENTS WITH PYRUVATE KINASE DEFICIENCY: A REAL WORLD STUDY USING US VETERANS HEALTH ADMINISTRATION DATA

E. Zagadailov 1,* , V. Garcia-Horton 2 , S. Higa 1 , L. Shi 3

1 Agios Pharmaceuticals, Inc., Cambridge, MA; 2 Analysis Group, Inc., New York, NY; 3 School of Public Health & Tropical Medicine, Tulane University, New Orleans, LA, United States of America

Background: Pyruvate kinase (PK) deficiency is a rare genetic condition caused by autosomal recessive mutations in the PKLR gene. Given the rarity of the disorder, limited data are available characterizing this population in the real world.

Aims: This study of healthcare resource use (HCRU) is a sub-analysis of a previously presented US Veterans Health Administration (VHA) study of patients with PK deficiency.

Methods: Patients with ≥1 PK deficiency-related diagnosis code (ICD-9: 282.3, 282.9; ICD-10: D55.2, D58.9) and documented diagnosis of PK deficiency from physicians’ notes from January 1995–July 2019 were included (PK deficiency cohort). Patients in the PK deficiency cohort were matched 1:5 by age at index date, sex, and index year (±1 year) to patients from the general US VHA population with no diagnosis codes related to PK deficiency (non-PK deficiency cohort). The index date for the PK deficiency cohort was defined as the date of the first medical record with a diagnosis code related to PK deficiency while the index date for the non-PK deficiency cohort was defined as the date of a random visit during their match’s index year. Patient characteristics were compared at index, and HCRU was analyzed for time periods of 12 months pre- and post-index.

Results: A total of 18 patients met inclusion criteria for the PK deficiency cohort (mean [SD] age: 56.8 [13.6] years, 94.4% male, mean [SD] duration of follow-up: 7.3 [5.2] years) and were matched to 90 individuals in the general population who became the non-PK deficiency cohort (mean [SD] age: 56.8 [13.1] years, 94.4% male, mean [SD] duration of follow-up: 9.2 [5.8] years). During the 12-month period preceding the index date, there were no statistically significant differences in the number of procedures, outpatient visits, and inpatient admissions between patients with and without PK deficiency (Table). During the 12-month follow-up period, patients in the PK deficiency cohort had higher rates of red blood cell transfusions and monitoring-related procedures such as echocardiograms and gallbladder ultrasounds compared to the non-PK deficiency cohort. Patients with PK deficiency also had more than twice as many outpatient visits than those in the non-PK deficiency cohort (mean [SD]: 35.2 [23.6] vs 14.1 [17.4] visits, respectively, p=0.0004). Approximately 16.7% of patients with PK deficiency had at least one inpatient admission over the 12 months of follow-up compared to 2.2% in the non-PK deficiency cohort (p=0.0319). During this time, patients with PK deficiency had an average of 1.8 admissions vs 0.0 admissions in the non-PK deficiency cohort (p=0.0079).

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Summary/Conclusion: This study suggests that patients with PK deficiency have higher HCRU compared to the general VHA population in the US. While the sample primarily represents adult males and may not be generalizable to the broader PK deficiency population, the results provide insight regarding the resource use and management of an otherwise understudied population.

PB2360: QUALITY OF LIFE AND TREATMENT SATISFACTION AMONG PATIENTS UNDER LONG-TERM ANTICOAGULANT THERAPY WITH VITAMIN K ANTAGONISTS

A. Varaklioti 1,2,* , S. Nika 2,3 , A. Papachronis 1 , A. Chanos 1 , S. Gkotsi 1 , T. Pyrpyri 1 , T. Sergentanis 2,4 , M. Gavalaki 1 , O. Katsarou 1

1 Blood Center and National Reference Center for Congenital Bleeding Disorders, Laiko General Hospital, Athens; 2 Faculty of Social Sciences, Deparment of Health Management, Hellenic Open University, Patras; 3 Pathology Laboratory, Laiko General Hospital; 4 Deparment of Biomedical Sciences, University of West Attica, Athens, Greece

Background: Many patients receive long-term treatment with anticoagulants, mainly vitamin K antagonists (VKAs), after atrial fibrillation (AF), mechanical heart valve replacement and thromboembolic disease (deep venous thromboembolism-DVT and/or pulmonary embolism-PE). Although in the past few years, new oral anticoagulants (DOACs) have been introduced, VKAs are still used widely for certain conditions.

Aims: The aim of the present study was to evaluate health-related quality of life (HRQOL) of patients under anticoagulant therapy after DVT, PE or AF and to assess convenience and satisfaction of their anticoagulant treatment with vitamin K antagonists.

Methods: The present study was a cross-sectional study of adult patients who visit our anticoagulation clinic and receive anticoagulant therapy for more than 1 year after DVT, PE or AF. The research was conducted from November 2020 to April 2021. Quality of life was assessed using SF-36 questionnaire, while Convenience and Satisfaction of anticoagulant treatment were assessed based on the Perception of Anticoagulation Treatment Questionnaire 2 (PACT-Q2).

Results: A total of 67 patients were recruited in the study. The mean value of the physical component health score (PCS) of SF36 was 42.13, while the mean value of mental component health score (MCS) was 44.02. Differences were observed in QoL between certain sociodemographic characteristics, namely gender, educational level and employment status. More specifically, male patients showed higher mean values in both PCS and MCS (47.6 and 49.5 respectively) compared to women (36.2 and 42.5) (p<0.01). Patients with higher education level showed higher mean values in MCS (52.96) compared to those with lower educational level (41.2) (p<0.01). Patients whose main employment was housekeeping exhibited significantly lower mean values in both PCS and MCS (29.93 and 27.19 respectively) compared to patients in retirement (45.59 in PCS and 43.56 in MCS, respectively) (p<0.01) or public workers (48.59 in PCS and 54.04 in MCS, respectively).

Regarding convenience and satisfaction of anticoagulant therapy, scores in both Convenience and Satisfaction subscales of PACT-Q2 were high (Convenience score: 71.45 and Satisfaction score: 71.28). Statistically significant differences were observed between male and female patients in both convenience (male: 72.6 vs female: 62.4, p=0.05) and satisfaction scores (male: 60.5 vs female: 48.5, p=0.02). No differences were observed in either Quality of Life or Convenience and Satisfaction of anticoagulant treatment among different types of disease.

Summary/Conclusion: Quality of life of patients under anticoagulant therapy after venous thromboembolism, pulmonary embolism or atrial fibrillation was found to be moderately satisfactory. Gender and employment status appear to adversely affect patients’ quality of life, with women working as housekeepers showing lower scores on both overall physical and overall mental dimension of SF36. Moreover, the high level of education was positively correlated with quality of life, with university-level participants showing higher scores in the overall mental dimension of SF-36. Furthermore, gender is one of the demographic factors that affects the convenience and satisfaction of anticoagulant treatment, with women having lower scores than men.

PB2361: PREDIGA, EDUCATIONAL AND DIAGNOSIS PROJECT IN ACID SPHINGOMYELINASE DEFICIENCY DISEASE (ASMD) AND GAUCHER DISEASE (GD): RESULTS TO THE OBJECTIVE ACHIEVED OF 200 PATIENTS ANALYZED.

J. Villarrubia 1,* , E. Calderon 2 , M. Morado 3 , V. Quintero 4 , I. Vitoria 5 , M. A. Torralba 6

1 HEMATOLOGY, HOSPITAL UNIVERSITARIO RAMON Y CAJAL, MADRID; 2 Internal Medicine, Hospital Universitario Virgen del Rocio, Sevilla; 3 HEMATOLOGY, Hospital Universitario La Paz; 4 Pediatrics, HOSPITAL UNIVERSITARIO RAMON Y CAJAL, MADRID; 5 Pediatrics, Hospital Universitari i Politecnic La Fe, Valencia; 6 Internal Medicine, Hospital Clinico Universitario Lozano Blesa, Zaragoza, Spain

Background: Acid sphingomyelinase deficiency disease (ASMD) and Gaucher disease (GD) are lysosomal disorders caused by deficiency of lysosomal enzymes acid beta-glucosidase and acid sphingomyelinase. These enzyme deficiencies cause accumulation of glucocerebroside and sphingomyelin, respectively, mainly in macrophages, inducing deterioration of the organs in which they accumulate. Theoretical prevalence of ASMD is 0.4 per 100,000 and GD is 1 per 40,000. The absence of epidemiological studies in Spain leads us to believe that ASMD is highly underdiagnosed, and that the incidence of GD data is still to be defined. The consequences of undertreatment and the availability of effective therapies make it increasingly necessary to promote awareness and knowledge of these diseases and their diagnostic algorithms.

Aims: The main objective of this study is to identify patients with idiopathic splenomegaly or splenectomy as well as to promote awareness and knowledge of ASMD and GD and their diagnosis algorithm through a national medical education program. Prevalence of ASMD and GD in patients with idiopathic splenomegaly or splenectomy will be also established.

Methods: Regarding educational program, the participating centers lead clinical sessions as part of the PREDIGA education program. It was planned to impart about 100 clinical sessions within the medical education program and reach 200 patients fulfilling diagnostic criteria for ASMD and GD in the recruitment process.

In addition, PREDIGA project included an epidemiological, clinical, observational, non-interventional, multicenter, retrospective and prospective, cross-sectional with a single cohort study. The study population included adults and children with idiopathic splenomegaly or splenectomy. We performed a retrospective (5 years) and prospective review (2 years) of medical records to detect sustainable patients. In a single visit, the informed consent form was signed and the patient’s clinical data were recorded. We used the usual clinical procedures to collect a blood sample for Dry Blood Spot (DBS) to perform enzymatic, genetic, and biomarkers analyses.

The analysis of the samples was performed in the Archimed laboratory using ArchimedLife platform. The diagnostic algorithm for the analysis of the samples included two steps: first, enzymatic analysis was performed and if the result was positive or doubtful, genetic analysis was performed to confirm the positive result for ASMD or GD. In addition, samples which results were positive for ASMD or GD were subjected to analysis of the biomarkers Lyso-Gl1 or Lyso-SM.

Results: 52 HCPs (22 Hematologists/ 18 Internal Medicine/ 12 Pediatricians) from 34 hospitals had implemented the medical education program and epidemiological searching in their health area of influence for 2 consecutive years. To date, 49 educational sessions, on-site and virtual, have been held by research team with 1000 assistants.

A total of 206 patients’ blood samples have been collected to perform a DBS. Results to date have revealed the diagnosis of 2 ASMD and 4 GD patients derived from the epidemiological program. Thanks to implementation of this educational program 2 ASMD and 1 GD patients were diagnosed. Therefore, in this study the prevalence of ASMD and GD in patients with idiopathic splenomegaly or splenectomy is 0.97% and 1.9% respectively.

Summary/Conclusion: The medical educational plan and the epidemiological study derived of this project have increased awareness of these conditions allowing the diagnosis of new ASMD and GD patients and better define the prevalence of these diseases in this cohort.

PB2362: BEST PRACTICES ON EMPOWERING AND INVOLVING PATIENTS LIVING WITH HEMATOLOGICAL MALIGNANCIES ON THE DEVELOPMENT OF NOVEL PATIENT-REPORTED OUTCOME MEASURES

N. Vrontaras 1,* , D. Kyrou 1 , A. Kapetanakis 1 , D. Tsakona 1 , C. Karamanidou 1 , L. Scarfò 2,3 , P. Ghia 2,3 , K. Stamatopoulos 1

1 Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thermi, Thessaloniki, Greece; 2 Università Vita-Salute San Raffaele; 3 IRCCS Ospedale San Raffaele, Milan, Italy

Background: Empowering patients is prioritized in research and clinical practice for improving patient outcomes. Patient-reported outcome measures (PROMs) have been highlighted as a means of involving people living with hematological malignancies in the decision process and thus helping them shape their care plans. Despite the clinical significance of PROMs, patients are rarely involved in their development.

Aims: Our aim was to assist in overcoming this limitation and offer best practices that empower patients living with hematological malignancies by increasing their involvement in the development of novel PROMs.

Methods: To this end, in the context of the Patient Empowerment Program of ERIC (European Research Initiative on CLL), we developed a Knowledge Questionnaire and a Treatment Preference Questionnaire for patients with Chronic Lymphocytic Leukemia (CLL). Reflections on the processes of their development and best practices from the literature were considered within a multidisciplinary team to develop lessons learned and a concise list of steps. These allow for coherence and consistency across future studies.

Results: The steps include: (i) to identify unmet clinical needs by drawing from the literature, clinical experience and the experience of patients; (ii) to systematically review the literature for existing PROMs (in this or similar area) towards identifying both tools and methodologies of their development; (iii) to bring together a multidisciplinary panel of experts (e.g., hematologists, nurses, psychologists, statisticians), including patients (experts by experience), the public or family/caregivers (as appropriate). A structure (e.g., Delphi process method or focus groups) and a series of meetings are recommended to best address the following: a) critically discuss existing PROMs, b) decide on an aim (e.g., clinical or research oriented PROM) and group, c) decide on methodology (replicate or diverge from existing methodologies), d) review theoretical models to guide the PROM content, e) develop the initial domains, structure, items, f) develop a statistical analysis plan, g) decide on the instructions of the PROM (e.g., rationale of administration); (iv) to complete readability tests, aiming at a final score of 6 th grade and reword accordingly; (v) to format the PROM structure and the overall layout (e.g., spacing, font size) according to the condition (e.g., visual impairments, cognitive abilities) and preferences of the patient group; (vi) to invite a separate patient group to review comprehensibility, ease, and timing of completion, rate the content in terms of relevance and offer any further suggestions around the items (including addition or removal); (vii) to repeat the steps above as appropriate depending on the extent of changes made; (viii) to pilot test the PROM with a third group of patients and run initial analyses; (ix) conduct the full study for further testing (e.g., validity, reliability) of the PROM and its content (e.g., factor analysis, Cronbach’s alpha and kappa).

Summary/Conclusion: The above steps provide a practical outline, which may vary depending on the aims. Nevertheless, empowering patients by solidifying their position as co-developers and drawing from their experiences can be an invaluable part of developing new tools. Increasing their involvement in the process is expected to improve the tools (e.g., measurement accuracy, quantity, and quality of collected data), which in turn can better inform and facilitate clinical practice.

PB2363: REPORTING LANGUAGE IN HAEMATOLOGICAL MALIGNANCIES

S. Watt 1 , S. Ralston 2,* , A. Evans-Tomlinson 1 , J. Burthem 1

1 Haematology, Manchester NHS Foundation Trust; 2 Haematology, Bolton NHS Foundation Trust, Manchester, United Kingdom

Background: With haematology diagnostics being centralised over the past ten years in the UK and increasingly complex diagnostic pathways, it is important that the treating clinicians and reporters are speaking the “same language” in order that optimal treatment for patient can be achieved.

We have reviewed the literature and there are studies looking at language use in general pathology, but to our knowledge nobody has looked at the language used in haemato-pathology reporting, in particular the descriptive terms around diagnoses.

Sometimes the language used by reporters reflects the uncertainty in the diagnosis due to atypical appearances or other tests not being consistent, the fact that tests are incomplete or clinical information is missing from the referral. Service users may prefer more unequivocal language to definitively guide management. Supplementary communication in addition reports was often utilised to contextualise findings.

Aims: We decided to review the language used in our regional haemato-oncology service

Methods: We collected the most commonly used terms from the combined marrow smear and trephine morphology, flow cytometry, molecular and conventional genetics reports at our regional service.

We then designed a survey to determine a percentage score for how certain a diagnosis was if it was preceded by the 8 commonly used descriptive terms: ‘suspicious of’, ‘compatible with’, ‘diagnostic of’, ‘suggestive of’, ‘in keeping with’, ‘consistent with’, ‘features are those of’ and ‘cannot be excluded’. Participants were also asked to rank the terms according to their certainty and to express if the term had more weighting if it was for a positive diagnosis or that of a remission. The was also a free text section for participants to express any terms they particularly liked or disliked. We asked both reporters and service users to complete the survey.

Results: A total of 47 surveys were returned including 33 who described themselves as reporters and 14 who described themselves as service users. The mean percentage certainty was calculated for all responders as well as for the service user and reporter groups. The range was also calculated for these respective samples. We found that “diagnostic of” scored overall highest for degree of certainty at 97.8% and “cannot be excluded” lowest at 29.9%. The most interesting aspect was the large range of results in “suspicious of” and “cannot be excluded” where there was 70 percentage points between the highest and lowest answers. An unpaired t-test showed that for “suspicious of” and “compatible with” there was a statistically significant difference in mean certainty between reporters and service users. The largest difference between reporters and users was “suspicious of” with reporters rating 14% higher, 59% v 45%.

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Summary/Conclusion: The survey shows how perceptions of reports can differ markedly between individual reporters and service users. This demonstrates the importance of the MDT in ensuring pathologists play an active role in the diagnosis and that their reports are not misinterpreted.

Pathology services should try to standardise their language to reduce the amount of ambiguity in their reports.

We would be very interested to see if these differences are replicated in other diagnostic services both within the UK and also in other languages.

The next step in this project is to gather a focus group and gain more in-depth qualitative data. This would be to discuss not only the terms used in the questionnaire, but the wider themes of communication previously described.