Poor Applicability of Currently Available Prognostic Scoring Systems for Prediction of Outcome in KIT D816V-Negative Advanced Systemic Mastocytosis.

1. Introduction

Systemic mastocytosis (SM) is a rare myeloid neoplasm with an accumulation of neoplastic mast cells (MC) in various tissues, most often in bone marrow (BM), skin and the gastrointestinal tract. The disease is subcategorized into indolent SM (ISM), smoldering SM (SSM), bone marrow mastocytosis (BMM) and advanced SM (AdvSM), the latter comprising SM with an associated hematologic/myeloid neoplasm (SM-AHN/SM-AMN), aggressive SM (ASM) and mast cell leukemia (MCL) [ 1 ]. ISM is associated with a normal life expectancy whereas survival in AdvSM is dependent on subtype and ranges between 1.5 and 4 years [ 1 ].

Mast cells play an important role in the immune system and are directly involved in allergic reactions, stress and tumor growth and their degranulation may lead to a plethora of different symptoms [ 2 , 3 ]. Their pro- and antitumor potential in general is widely discussed, and a complete depletion of mast cells is regarded as a life-threatening condition. The clonal constitutive activation of mast cells in SM may also lead to a multitude of symptoms affecting different organ systems which sometimes makes the diagnosis challenging.

Cytopenias (anemia < 10 g/dL, platelets < 100 × 10 9 /L, neutrophils < 1 × 10 9 /L), hepatomegaly with impaired liver function (hypoalbuminemia, elevated alkaline phosphatase, portal hypertension, splenomegaly with hypersplenism, ascites), malabsorption with weight loss and large osteolytic lesions constitute the so-called C-findings indicating organ damage. C-findings are diagnostic criteria for ASM but not for SM-AHN and MCL, but they are in fact identified in the majority of patients with SM-AHN and MCL. Additional somatic mutations are present in 60–80% of patients with AdvSM of which mutations in SRSF2 , ASXL1 , RUNX1 , NRAS and DNMT3A have been associated with poor prognosis [ 4 , 5 , 6 ]. Several prognostic scoring systems have recently been established variably comprising clinical (age, organomegaly), laboratory (anemia, thrombocytopenia, beta-2-microglobulin and alkaline phosphatase) and genetic (additional somatic mutations) characteristics [ 4 , 5 , 6 , 7 , 8 ].

Depending on the SM subtype, mutations in the receptor tyrosine kinase KIT , typically KIT D816V, are observed in 80% to over 90% of patients. KIT D816V neg. SM/AdvSM comprises alternative mutations at position 816 (e.g., D816H or Y, KIT D816H/Y pos. ) and the absence of any mutation in the coding sequence of KIT ( KIT neg. ) [ 9 , 10 , 11 ]. Across all SM subtypes, KIT neg. SM is identified at a frequency of 5–10%. However, its prevalence is higher in rare subtypes including MCL (10–20%), well-differentiated systemic mastocytosis (WDSM) (approximately 60–70%) [ 12 , 13 ], myelomastocytic leukemia (> 90%) and mast cell sarcoma (MCS, approx. 90%) [ 14 , 15 ]. Due to limited knowledge, we sought to evaluate the clinical, genetic and prognostic characteristics of patients with KIT D816V neg. AdvSM and MCS.

2. Patients and Methods

All patients were registered with the “German Registry on Disorders of Eosinophils and Mast cells” (GREM) and gave written informed consent. Detailed information on clinical, morphological and laboratory parameters are presented in Table 1 a,b and Appendix A , Table A1 . All patients were diagnosed and subtyped as SM according to the 2016 WHO classification. The study design adhered to the tenets of the Declaration of Helsinki and was approved by the responsible institutional review boards.

2.1. Cytomorphology and Histomorphology

All biopsies were evaluated by a reference pathologist (M.R., H.-P.H, K.S.) of the ‘European Competence Network on Mastocytosis’ (ECNM). Mast cell morphology was analyzed in BM aspirate smears stained with May–Grünwald–Giemsa (Carl Roth GmbH, Karlsruhe, Germany) and toluidine blue (Merck/Sigma-Aldrich, Darmstadt, Germany). BM trephine biopsy specimens were decalcified for 8 h, fixed in 4% formalin (Carl Roth GmbH, Karlsruhe, Germany) and paraffin-embedded. Sections were stained with hematoxylin (Carl Roth GmbH, Karlsruhe, Germany) and eosin (Carl Roth GmbH, Karlsruhe, Germany), Giemsa (Carl Roth GmbH, Karlsruhe, Germany), Gömöri´s silver impregnation (Merck/Sigma-Aldrich, Darmstadt, Germany) and naphthol AS-D chloracetate esterase (Merck/Sigma-Aldrich, Darmstadt, Germany).

2.2. Immunohistochemistry

Immunohistochemistry was performed on the fully automated Ventana Benchmark platform (Roche Diagnostics, Mannheim, Germany) using the Ultraview DAB IHC detection kit (Roche Diagnostics, Mannheim, Germany) for visualization. The following antibodies were used: CD117 (Agilent, Santa Clara, CA, USA), CD25 (Leica, Deer Park, TX, USA), CD2 (Leica), MZT (Zytomed, Berlin, Germany), CD30 (Thermo Fisher Scientific, Waltham, MA, USA), CD20 (Agilent, Santa Clara, CA, USA), CD138 (Zytomed, Berlin, Germany), Kappa (Agilent, Santa Clara, CA, USA), Lambda (Agilent, Santa Clara, CA, USA) and CD5 (Leica Biosystems, Nussloch, Germany).

2.3. Microdissection

To exclude false negative KIT mutation analysis in patients with a low mast cell burden (SM-AHN, patients #15–#19), we performed microdissection of mast cell aggregates from paraffin-embedded BM biopsy samples for 4/5 (#15, #16, #17, #19) patients.

Prior to manual dissection from the slides, the tissue was deparaffinized and stained with H & E (Carl Roth GmbH, Karlsruhe, Germany). For manual microdissection, compact mast cell infiltrates were identified by a pathologist and the region of interest of the FFPE tissue was scraped into Eppendorf tubes for subsequent DNA/RNA isolation. This method enriched tumor cells, and the tumor cell content was at least 70%. Following the manufacturer’s protocol, we prepared a DNA library using a hybrid capture-based TruSight Oncology 500 DNA/RNA NextSeq Kit (Illumina, San Diego, CA, USA). Using the unique molecular identifiers (UMIs) in the TruSight Oncology 500 (TSO 500) (Illumina, San Diego, CA, USA), we determined the unique coverage of each position and reduced background noise. We analyzed sequencing data for genomic alterations, including SNVs, CNVs and fusions. SNVs and small indels with a variant allele frequency (VAF) of less than 2% were excluded.

2.4. Mutation Analysis

Quantitative assessment of the expressed allele burden (EAB) at the RNA level was performed by allele-specific reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), as previously described [ 9 ]. Qualitative assessment of KIT D816H/Y was performed by Sanger sequencing of KIT exon 17 from peripheral blood (PB) or BM according to standard procedures.

For analysis of alternative KIT and additional somatic mutations, Next-Generation Deep Amplicon Sequencing (NGS) by 454 FLX amplicon chemistry (Roche, Penzberg, Germany) or library preparation based on the TruSeq Custom Amplicon Low Input protocol (Illumina, San Diego, CA) followed by sequencing on the MiSeq instrument (Illumina, San Diego, CA, USA) was performed in 18/19 patients. The sequencing panel includes a standard myeloid gene panel covering 18 recurrently mutated genes in myeloid neoplasms. Gene mutations were annotated using the appropriate reference sequence (Ensembl release 85: July 2016).

From 12/12 patients without a mutation in KIT , gDNA isolated from PB and/or BM (including patient #18) was used for NGS analysis of all KIT exons. The mean coverage was 1500 reads (400–8819). In two patients, whole genome and whole transcriptome sequencing was applied. DNA sequencing was performed using library preparation by ‘TruSeq DNA PCR-Free’ and sequencing by NovaSeq6000 (Illumina, San Diego, CA, USA). RNA sequencing was performed by RNA-Seq (Illumina TruSeq Stranded Total RNA KIT; Unique Dual Indices; Illumina, San Diego, CA, USA). DNA reads were aligned against the human reference genome (hg19); RNA reads were used to detect potential fusion transcripts (Arriba, Manta, STAR-Fusion).

2.5. Comparison to Control Group

For classification, risk stratification and evaluation of prognostic factors among KIT neg. and KIT D816H/Y pos. patients, we utilized a control group of KIT D816V pos. AdvSM patients ( n = 118; SM-AHN, n = 89; MCL-(AHN), n = 29) with known clinical, laboratory and genetic characteristics ( Table 1 a,b and Table 2 ).

2.6. Statistical Analyses

All statistical analyses were performed using IBM ® SPSS statistics (version 25.0, IBM-Corporation, Armonk, NY, USA) or GraphPad Prism (version 8, GraphPad, San Diego, CA, USA). Survival probabilities were calculated by the Kaplan–Meier method and were determined from the date of diagnosis to death or the date of last contact (if alive). Overall survival (OS) was defined as the time from AdvSM diagnosis to death from any cause. In patients undergoing allo-SCT, progression-free survival (PFS) was defined as the time from allogeneic stem cell transplantation (SCT) to disease progression or death from any cause. A p < 0.05 was considered as statistically significant.

3. Results

From almost 300 advSM patients collected in the GREM, we identified 19 patients without a KIT D816V mutation. Seven patients carried an alternative mutation on locus KIT D816 (D816H, n = 4; D816Y, n = 3), whereas no mutation in the KIT gene was detected in 12 patients ( KIT neg. ). While almost all (6/7, 86%) patients of the KIT D816H/Y pos. cohort were diagnosed with MCL, the KIT neg. subgroup included MCL (7/12, 58%) and SM-AHN (5/12, 42%) patients ( Figure 1 ).

3.1. KIT D816H/Y pos. Patients

Clinical features. The median age of KIT D816H/Y pos. patients ( n = 7) was 58 years (range 37–69, m/f: 3/4). The median BM MC infiltration was 50% (range 20–75%); other notable BM features included eosinophilia (6/7, 86%) and fibrosis (4/7, 57%; grade 2, 2/7, grade 3, 2/7). Blood counts revealed mild leukocytosis > 10 and <16 × 10 9 /L (2/7, 29%) and anemia < 10 g/dL (4/7, 57%, one patient transfusion dependent), while thrombocytopenia < 100 G/L was absent in all seven patients. Eosinophilia > 0.4 × 10 9 /L was frequent (5/7, 82%; median 1 × 10 9 /L, range 0–39) while monocytosis > 1 × 10 9 /L was rare (1/7; 14%, median 0.23 × 10 9 /L, range 0.03–1.3). Other C-findings included ascites (5/7, 71%), elevated alkaline phosphatase (4/7, 57%) and hypoalbuminemia (1/7, 14%). Involvement of the gastrointestinal tract and skin (0/7) was rare (1/7, 14%). The median serum tryptase level was 193 µg/L (range 90–1300). Consequently, morphologic subtypes included MCL ± AHN (6/7, 86%; +MCS, 2/7, 29%; with WDSM phenotype, 1/7, 14%) and SM-AHN (1/7, 14%). Clinically relevant anaphylaxis (1/7, 14%) was rare and no other relevant MC-triggered symptoms were noted.

3.1.1. Molecular Findings

Additional somatic high-risk mutations (HRM) in SRSF2 , ASXL1 , RUNX1 ( S / A / R gene panel) [ 6 ], NRAS or DNMT3A [ 4 , 16 ] were identified in 2/7 (29%) patients ( SRSF2 , n = 1; NRAS , n = 1) and other somatic mutations ( TET2 , JAK2 , and CBL ) [ 5 ] were observed in two patients (29%; CBL/TET2 , 1/7; JAK2 V617F, 1/7). Due to the paucity of leukocytosis ≥ 16 × 10 9 /L (0/7), skin involvement (0/7) and HRM (2/7), almost all patients were low/intermediate risk according to current prognostic scoring systems for AdvSM (IPSM, 7/7; MARS, 6/7; MAPS, 5/7; GPS, 6/7) ( Figure A1 ).

3.1.2. Treatment

Treatment comprised midostaurin (5/7, 71%) with or without sequential cladribine (3/7, 43%) and supportive care only (2/7, 29%; G-CSF, local radiation). The response to treatment was only partially effective and of short duration. Allogeneic SCT was performed in 2/7 (29%) patients who relapsed early and deceased at months +10 and +12 after transplant ( Figure 2 ). Despite a predominantly low/intermediate risk profile, the median OS was only 22 months (range 12–49).

3.2. KIT neg. Patients

Due to significant clinical and morphological differences, two subgroups were considered ( Table 3 ): MCL ± AHN (7/12, 58%) and SM-AHN (5/12, 42%).

3.2.1. Cytomorphology and Histomorphology

The BM aspirates of patients with KIT neg. MCL exhibited atypical MC with round nuclei rather than a spindle-shaped morphology ( Figure 3 A). Two cases presented a well-differentiated phenotype with prominent cytoplasmic granulation, and only a few neoplastic MCs displayed aberrant expression of CD25. In all BM biopsies, multifocal dense infiltrates with aggregates of ≥15 MC were detected, often localized paratrabecularly. Reticulin fibers were increased, consistent with a myelofibrosis grade 1 (MF1) in the majority of cases. Of the five cases diagnosed with KIT neg. SM-AHN, two patients were classified as ISM with associated MDS and multilineage dysplasia (MDS-MLD), one patient was diagnosed with SM- acute myeloid leukemia (AML) and one patient fulfilled criteria for each associated myelofibrosis (SM-MF) and MDS/MPN, respectively. Striking features included: (a) WDSM with a solitary dense infiltrate, diffuse interstitial MC hyperplasia and normal immunophenotype in one patient (case 15) and (b) atypical, spindle-shaped MC exhibiting aberrant CD25 expression without formation of dense clusters in one patient (case 18) ( Figure 3 B).

3.2.2. Molecular Findings

Additional somatic high-risk mutations (HRM) in the S / A / R gene panel [ 6 ], NRAS or DNMT3A [ 4 , 16 ] were identified in 4/12 (33%) patients ( ASXL1 , n = 2; SRSF2 , n = 1; DNMT3A , n = 1; RUNX1 , n = 1). Other somatic mutations ( TET2 , JAK2 , KRAS , CSF3R , U2AF1 , CBL , FLT3 -ITD) [ 5 ] were observed in 6/12 (50%) patients.

3.2.3. Treatment

Treatment comprised TKI (MCL, 6/7 and SM-AHN, 2/5; midostaurin 6/7 and 1/5; imatinib, 3/7 and 1/5) with or without sequential cladribine (6/7 and 0/5) and intensive chemotherapy (0 and 1/5) without significant or durable response. Following allogeneic SCT (3/7 and 1/5), all patients died (months +5, +6, +7, +24) due to progressive disease ( Figure 2 ). According to risk scoring systems of AdvSM, the majority of patients were low or intermediate (IPSM, 2/7 and 5/5; MARS, 5/7 and 5/5; MAPS, 5/7 and 4/5; GPS, 4/7 and 5/5) ( Figure A1 ). The median OS was significantly different (0.9 versus 4.5 years, p = 0.037).

3.3. Commonalities and Differences between the Two Cohorts of KIT neg. AdvSM

The median age (56/62 years, range 27–69) and the m/f ratio were not different (5/7 and 3/5). Significant differences were identified for BM MC infiltration (median 80% versus 20%, p = 0.0013), serum tryptase levels (median 451 µg/L versus 34 µg/L, p = 0.0025), hemoglobin < 10 g/dL (57% versus 0%; median 9.3 g/dL versus 13 g/dL, p = 0.01) and additional somatic mutations (43% and 100%: HRM 2/7 and 2/5; other somatic mutations 2/7 and 4/5). See Figure 4 and Appendix A , Table A2 for detailed molecular features. Although present in some patients, no significant differences between the two subtypes were found for leukocytosis, monocytosis, eosinophilia, alkaline phosphatase (AP), albumin and ascites.

3.4. Analysis of Three MCL Cohorts

Based on registry data, the two MCL cohorts ( KIT D816H/Y pos. , n = 6; KIT neg. MCL ± AHN, n = 7) were compared with a KIT D816V pos. MCL cohort ( n = 29). The most notable significant differences ( KIT D816V pos. versus KIT D816H/Y pos. versus KIT neg. MCL ± AHN) included age (median 68 versus 55 versus 56 years, p < 0.02), vitamin B12 levels (median 1597 versus 300 versus 3091 µg/L, p < 0.04), HRM (66% versus 33% versus 29%, p = 0.03) and survival (2.6 versus 1.7 versus 0.9 years; p = 0.04). One patient (#3) had chronic MCL. There were no significant differences regarding BM MC infiltration, serum tryptase levels and blood counts ( Table 2 and Figure 5 ).

4. Discussion

The pathogenetic driver mutation KIT D816V is detected in >90% of SM patients and is therefore of utmost relevance for the diagnosis of this heterogeneous disease. Its presence and variant allele frequency (VAF) in BM and PB are of paramount importance in assessing the phenotype and overall burden of involvement of MC, but often also non-MC lineages in SM-AHN. Currently available PCR assays are capable of detecting KIT D816V VAF with a sensitivity of up to 0.003%, allowing quantification in patients with very low disease burden at diagnosis and also monitoring response and residual KIT D816V mutational burden during or after treatment with KIT inhibitors, chemotherapy or allogeneic SCT [ 9 , 17 ].

In contrast, very little is known about the clinical phenotype of adult KIT D816V neg. SM. It has been primarily associated with MCL, WDSM and MCS [ 18 ]. Alternative mutations in KIT are recurrently identified at position 816, e.g., D816H, D816N or D816Y [ 12 , 19 ]. In isolated cases, other acquired imatinib-resistant mutations have been identified in coding regions of TK domains 1 and 2 (exons 13 to 18), e.g., I817V in WDSM, D820G in ASM and N822K in SM-AHN [ 18 ]. Potentially imatinib-sensitive mutations between exons 8 and 10 may correspond to germline mutations, e.g., S451C, K509I or F522C, which often show a familial aggregation pattern [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ].

Of 298 patients with AdvSM included in the GREM, we identified 19 KIT D816V neg. patients, corresponding to a 94% prevalence of KIT D816V in AdvSM. Approximately 35% of KIT D816V neg. patients were positive for alternative KIT D816 mutations. In seven patients, we identified only D816H and D816Y, which are known recurrent mutations, whereas D816G, D816I, D816T and D816N were identified only in single cases [ 18 ]. Similar to what has been described in the literature [ 18 ], all but one of the seven patients in our series had an MCL phenotype. Of note, despite the low prevalence of poor prognostic markers such as cytopenias, other C-findings and additional somatic mutations that placed the vast majority of patients in low/medium risk categories in most available prognostic scoring systems, prognosis was poor with a median survival of only 1.8 years. Application of the IPSM risk score delivered the best results: here, the majority of patients were classified into the AdvSM-3 risk group. According to Sperr et al. [ 8 ], patients within the AdvSM-3 and AdvSM-4 exhibit a significantly worse OS compared to AdvSM-1 and AdvSM-2 patients. However, none of the patients were classified into the AdvSM-4 subgroup. These findings suggest a potentially better discrimination of non-molecular scoring systems such as the IPSM but also show that patients with a lack of cytopenia(s) are rarely stratified into the high-risk subgroups of molecular PSS and or AdvSM-4, which overall show the worst OS.

In the remaining 12 patients without a mutation at position 816, no alternative mutations were detected in the complete coding sequences of KIT . From a clinical perspective, two distinct subgroups emerged. Patients with MCL had a very high MC burden and a poor prognosis despite the absence of additional somatic mutations and a predominantly good/intermediate prognostic risk score using molecular annotated risk scores. Again, IPSM was the best discriminator of OS in this subgroup ( Appendix A , Figure A1 ). All MCL patients were aleukemic with three of seven patients presenting with an immature MC morphology, which was predictive of an inferior OS in multivariate analysis in a retrospective study by Pardanani and colleagues [ 28 ].

In contrast, patients in the second cohort were diagnosed with SM and an associated hematological neoplasm (SM-AHN) according to the WHO classification or an associated myeloid neoplasm (SM-AMN) according to the ICC. Interestingly, no cases of chronic myelomonocytic leukemia were diagnosed, despite it being the most common SM-associated AHN based on previous studies [ 29 , 30 ]. Patients from our cohort mainly presented with MDS, MDS/MPN overlap or MPN as associated AHN. In two cases, the diagnosis could only be made through a thorough integration of clinical data and elevated tryptase levels, as either a major histomorphological criterion was lacking or only one minor criterion was present. This highlights the necessity for meticulous morphological analysis supplemented by additional immunohistochemistry and interdisciplinary collaboration when faced with KIT neg. SM cases. All patients had a dominating AHN with only a low MC burden and all patients exhibited additional somatic mutations. Nevertheless, the prognosis was significantly better than in patients with the MCL phenotype.

The diagnosis of all subtypes of SM can be notably challenging when the fraction of diagnostic neoplastic cells in the BM is low. Consequently, the application of sensitive diagnostic methods becomes imperative, often necessitating a comprehensive approach involving multiple techniques, such as thorough clinical evaluation, histomorphology, immunohistochemistry and molecular genetics. The updated WHO classification and ICC require the fulfillment of one major criterion and two minor criteria, or alternatively, three minor criteria for the diagnosis of SM [ 14 , 15 ]. One of the minor criteria involves proving the clonality of the MC through the detection of a KIT mutation, making the diagnosis challenging in cases lacking this mutation. In an unknown proportion of patients, the presence of two clonally independent diseases seems possible, similar to the recently reported cohort of patients with recurrent concurrent presence of KIT D816V pos. SM and JAK2 V617F pos. MPN in the same individual [ 31 ].

In a large series of 92 MCL patients of the ECNM, 73% were KIT D816V pos. , 11% exhibited alternative KIT mutations and 17% were KIT neg. [ 32 ]. In other series, positivity for KIT D816V ranged from 23% to 68% [ 19 , 33 , 34 ]. We compared the two KIT D816H/Y pos. / KIT neg. MCL groups with a larger cohort of 29 registry patients with KIT D816V pos. MCL. Although patients with KIT D816V pos. MCL were median >10 years older and had a significantly higher incidence of HRM, median OS was significantly better. This suggests that yet unknown mechanisms beyond MC burden, phenotype and genetic profile contribute to the poor prognosis of the different MCL subtypes. The 13 KIT neg. / KIT D816H/Y pos. MCL patients did not show a significant and durable response to midostaurin or imatinib, and all five allogeneic transplanted patients died within two years after transplantation. KIT D816V neg. MCL is therefore a high-risk condition regardless of established prognostic scoring systems, especially molecular annotated risk scores. There are no data yet on the efficacy of newly developed tyrosine kinase inhibitors (TKIs) such as avapritinib or bezuclastinib in KIT D816V neg. patients.

Important limitations of our analyses include that elaborate statistical analyses and validations in independent control cohorts could not be performed due to the rarity but also heterogeneity of KIT D816V neg. AdvSM. Notwithstanding, most of the few available case reports or small series in the literature lack a comparable thorough clinical, morphological and molecular work-up. The data clearly show that KIT D816V neg. AdvSM is similarly heterogeneous to KIT D816V pos. AdvSM and that a distinct differentiation between the various subtypes, e.g., MCL and SM-AHN, is of utmost relevance for prognostication and treatment decisions.

5. Conclusions

In conclusion, the diagnosis of KIT neg. AdvSM demands a high level of expertise and close collaboration between hematologists and hematopathologists to avoid diagnostic pitfalls and ensure optimal patient therapy. The integration of multiple diagnostic modalities and molecular studies is essential to achieve accurate SM diagnosis and provide patients with the most effective treatment options. KIT neg. AdvSM can be subdivided into (i) patients with other mutations at codon 816 such as KIT D816H/N/Y, most frequently associated with an MCL phenotype, (ii) KIT neg. MCL with a very poor prognosis despite the absence of C-findings and HRM (iii) KIT neg. SM-AHN with low MC burden and dominating AHN, making SM-specific treatment dispensable and prognosis mainly triggered by AHN. Our data highlight the poor applicability of the majority of current prognostic scoring systems for KIT neg. MCL.

Acknowledgments

The technical advice by Helga Kleiner and Susanne Brendel is acknowledged.

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Author Contributions

Conceptualization, N.N., M.R., J.S. and A.R.; methodology, N.N., J.L., M.R., K.S., G.M. and J.B.; formal analysis, A.F., N.C.P.C. and W.-K.H.; investigation, N.N. and J.S.; data curation, N.N. and J.S.; writing—original draft preparation, N.N., J.S., M.R and A.R.; writing—review and editing, H.-P.H., A.F., W.-K.H., G.M., D.C., N.C.P.C. and J.P.; visualization, M.R., J.L. and N.N.; supervision, A.R. and J.S.; project administration, N.N. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of the Medical Faculty of Mannheim, Heidelberg University (Heidelberg, Germany) (protocol code 2020-593N, 15 September 2020).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data presented in this study are available on request from the corresponding author (accurately indicate status).

Conflicts of Interest

J.S. received honoraria and research support from Novartis Pharma and Blueprint; A.R. received consultancy honoraria, travel reimbursement and research support from Novartis Pharma, Blueprint, Incyte and Deciphera; H.-P.H. received consultancy honoraria from Novartis, Deciphera and Blueprint; K.S. received honoraria and travel support from Novartis; W.-K.H. received research funding from Novartis; N.C.P.C. received honoraria from Novartis, Incyte and Ascentage and research support from Novartis. The other authors declare no conflict of interest.

Appendix A

References

1. Lim K.H. Tefferi A. Lasho T.L. Finke C. Patnaik M. Butterfield J.H. McClure R.F. Li C.Y. Pardanani A. Systemic mastocytosis in 342 consecutive adults: Survival studies and prognostic factors Blood 2009 113 5727 5736 10.1182/blood-2009-02-205237 19363219

2. Bellomo R.G. Stress, Depression, and Dementia Contribute to Neurodegeneration Eur. J. Neurodegener. Dis. 2023 12 15 20

3. Pandolfi F. The Impact of Mast Cells in Neuroimmunology and Cancer Eur. J. Neurodegener. Dis. 2023 12 62 70

4. Munoz-Gonzalez J.I. Alvarez-Twose I. Jara-Acevedo M. Zanotti R. Perkins C. Jawhar M. Sperr W.R. Shoumariyeh K. Schwaab J. Greiner G. Proposed global prognostic score for systemic mastocytosis: A retrospective prognostic modelling study Lancet Haematol. 2021 8 e194 e204 10.1016/S2352-3026(20)30400-2 33508247

5. Pardanani A. Shah S. Mannelli F. Elala Y.C. Guglielmelli P. Lasho T.L. Patnaik M.M. Gangat N. Ketterling R.P. Reichard K.K. Mayo alliance prognostic system for mastocytosis: Clinical and hybrid clinical-molecular models Blood Adv 2018 2 2964 2972 10.1182/bloodadvances.2018026245 30413432 PMC6234360

6. Jawhar M. Schwaab J. Hausmann D. Clemens J. Naumann N. Henzler T. Horny H.P. Sotlar K. Schoenberg S.O. Cross N.C. Splenomegaly, elevated alkaline phosphatase and mutations in the SRSF2/ASXL1/RUNX1 gene panel are strong adverse prognostic markers in patients with systemic mastocytosis Leukemia 2016 30 2342 2350 10.1038/leu.2016.190 27416984

7. Jawhar M. Schwaab J. Alvarez-Twose I. Shoumariyeh K. Naumann N. Lubke J. Perkins C. Munoz-Gonzalez J.I. Meggendorfer M. Kennedy V. MARS: Mutation-Adjusted Risk Score for Advanced Systemic Mastocytosis J. Clin. Oncol. 2019 37 2846 10.1200/JCO.19.00640 31509472 PMC6823885

8. Sperr W.R. Kundi M. Alvarez-Twose I. van Anrooij B. Oude Elberink J.N.G. Gorska A. Niedoszytko M. Gleixner K.V. Hadzijusufovic E. Zanotti R. International prognostic scoring system for mastocytosis (IPSM): A retrospective cohort study Lancet Haematol. 2019 6 e638 e649 10.1016/S2352-3026(19)30166-8 31676322 PMC7115823

9. Erben P. Schwaab J. Metzgeroth G. Horny H.P. Jawhar M. Sotlar K. Fabarius A. Teichmann M. Schneider S. Ernst T. The KIT D816V expressed allele burden for diagnosis and disease monitoring of systemic mastocytosis Ann. Hematol. 2014 93 81 88 10.1007/s00277-013-1964-1 24281161

10. Kristensen T. Vestergaard H. Møller M.B. Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay J. Mol. Diagn. 2011 13 180 188 10.1016/j.jmoldx.2010.10.004 21354053 PMC3279709

11. Orfao A. Garcia-Montero A.C. Sanchez L. Escribano L. Recent advances in the understanding of mastocytosis: The role of KIT mutations Br. J. Haematol 2007 138 12 30 10.1111/j.1365-2141.2007.06619.x 17555444

12. Garcia-Montero A.C. Jara-Acevedo M. Teodosio C. Sanchez M.L. Nunez R. Prados A. Aldanondo I. Sanchez L. Dominguez M. Botana L.M. KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: A prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients Blood 2006 108 2366 2372 10.1182/blood-2006-04-015545 16741248

13. Valent P. Sotlar K. Sperr W.R. Escribano L. Yavuz S. Reiter A. George T.I. Kluin-Nelemans H.C. Hermine O. Butterfield J.H. Refined diagnostic criteria and classification of mast cell leukemia (MCL) and myelomastocytic leukemia (MML): A consensus proposal Ann. Oncol. 2014 25 1691 1700 10.1093/annonc/mdu047 24675021 PMC4155468

14. Arber D.A. Orazi A. Hasserjian R.P. Borowitz M.J. Calvo K.R. Kvasnicka H.M. Wang S.A. Bagg A. Barbui T. Branford S. International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: Integrating morphologic, clinical, and genomic data Blood 2022 140 1200 1228 10.1182/blood.2022015850 35767897 PMC9479031

15. Khoury J.D. Solary E. Abla O. Akkari Y. Alaggio R. Apperley J.F. Bejar R. Berti E. Busque L. Chan J.K.C. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms Leukemia 2022 36 1703 1719 10.1038/s41375-022-01613-1 35732831 PMC9252913

16. Pardanani A. Lasho T.L. Reichard K.K. Hanson C.A. Tefferi A. World Health Organization class-independent risk categorization in mastocytosis Blood Cancer J. 2019 9 29 10.1038/s41408-019-0189-5 30833549 PMC6399221

17. Jawhar M. Schwaab J. Naumann N. Horny H.P. Sotlar K. Haferlach T. Metzgeroth G. Fabarius A. Valent P. Hofmann W.K. Response and progression on midostaurin in advanced systemic mastocytosis: KIT D816V and other molecular markers Blood 2017 130 137 145 10.1182/blood-2017-01-764423 28424161

18. González-López O. Muñoz-González J.I. Orfao A. Álvarez-Twose I. García-Montero A.C. Comprehensive Analysis of Acquired Genetic Variants and Their Prognostic Impact in Systemic Mastocytosis Cancers 2022 14 2487 10.3390/cancers14102487 35626091 PMC9139197

19. Jawhar M. Schwaab J. Meggendorfer M. Naumann N. Horny H.P. Sotlar K. Haferlach T. Schmitt K. Fabarius A. Valent P. The clinical and molecular diversity of mast cell leukemia with or without associated hematologic neoplasm Haematologica 2017 102 1035 1043 10.3324/haematol.2017.163964 28255023 PMC5451335

20. Alvarez-Twose I. Matito A. Morgado J.M. Sánchez-Muñoz L. Jara-Acevedo M. García-Montero A. Mayado A. Caldas C. Teodósio C. Muñoz-González J.I. Imatinib in systemic mastocytosis: A phase IV clinical trial in patients lacking exon 17 KIT mutations and review of the literature Oncotarget 2017 8 68950 68963 10.18632/oncotarget.10711 28978170 PMC5620310

21. Broderick V. Waghorn K. Langabeer S.E. Jeffers M. Cross N.C.P. Hayden P.J. Molecular response to imatinib in KIT F522C-mutated systemic mastocytosis Leuk. Res. 2019 77 28 29 10.1016/j.leukres.2018.12.010 30612056

22. Chan E.C. Bai Y. Kirshenbaum A.S. Fischer E.R. Simakova O. Bandara G. Scott L.M. Wisch L.B. Cantave D. Carter M.C. Mastocytosis associated with a rare germline KIT K509I mutation displays a well-differentiated mast cell phenotype J. Allergy Clin. Immunol. 2014 134 178 187 10.1016/j.jaci.2013.12.1090 24582309 PMC4125511

23. Lanternier F. Cohen-Akenine A. Palmerini F. Feger F. Yang Y. Zermati Y. Barète S. Sans B. Baude C. Ghez D. Phenotypic and genotypic characteristics of mastocytosis according to the age of onset PLoS ONE 2008 3 e1906 10.1371/journal.pone.0001906 18404201 PMC2292130

24. Wang H.J. Lin Z.M. Zhang J. Yin J.H. Yang Y. A new germline mutation in KIT associated with diffuse cutaneous mastocytosis in a Chinese family Clin. Exp. Dermatol. 2014 39 146 149 10.1111/ced.12225 24289326

25. Zhang L.Y. Smith M.L. Schultheis B. Fitzgibbon J. Lister T.A. Melo J.V. Cross N.C. Cavenagh J.D. A novel K509I mutation of KIT identified in familial mastocytosis-in vitro and in vivo responsiveness to imatinib therapy Leuk. Res. 2006 30 373 378 10.1016/j.leukres.2005.08.015 16183119

26. Arock M. Sotlar K. Akin C. Broesby-Olsen S. Hoermann G. Escribano L. Kristensen T.K. Kluin-Nelemans H.C. Hermine O. Dubreuil P. KIT mutation analysis in mast cell neoplasms: Recommendations of the European Competence Network on Mastocytosis Leukemia 2015 29 1223 1232 10.1038/leu.2015.24 25650093 PMC4522520

27. Bibi S. Langenfeld F. Jeanningros S. Brenet F. Soucie E. Hermine O. Damaj G. Dubreuil P. Arock M. Molecular defects in mastocytosis: KIT and beyond KIT Immunol. Allergy Clin. N. Am. 2014 34 239 262 10.1016/j.iac.2014.01.009 24745672

28. Pardanani A. Tefferi A. Singh A. Al-Kali A. Patnaik M.M. Hogan W.J. Begna K. Elliott M.A. Khera N. Palmer J.M. Reappraisal of Mast Cell Leukemia Based on a Single Institution Review of 16 Cases: Role of Mast Cell Morphology in Determining Clinical Outcome Blood 2022 140 6872 6873 10.1182/blood-2022-167720

29. Pardanani A. Lim K.H. Lasho T.L. Finke C. McClure R.F. Li C.Y. Tefferi A. Prognostically relevant breakdown of 123 patients with systemic mastocytosis associated with other myeloid malignancies Blood 2009 114 3769 3772 10.1182/blood-2009-05-220145 19713463

30. Patnaik M.M. Rangit V. Lasho T.L. Hoversten K.P. Finke C.M. Ketterling R.P. Hanson C.A. Gangat N. Tefferi A. Pardanani A. A comparison of clinical and molecular characteristics of patients with systemic mastocytosis with chronic myelomonocytic leukemia to CMML alone Leukemia 2018 32 1850 1856 10.1038/s41375-018-0121-1 29712989

31. Naumann N. Lübke J. Shomali W. Reiter L. Horny H.P. Jawhar M. Dangelo V. Fabarius A. Metzgeroth G. Kreil S. Clinical and histopathological features of myeloid neoplasms with concurrent Janus kinase 2 (JAK2) V617F and KIT proto-oncogene, receptor tyrosine kinase (KIT) D816V mutations Br. J. Haematol. 2021 194 344 354 10.1111/bjh.17567 34060083

32. Kennedy V.E. Perkins C. Reiter A. Jawhar M. Lübke J. Kluin-Nelemans H.C. Shomali W. Langford C. Abuel J. Hermine O. Mast Cell Leukemia: Clinical and Molecular Features and Survival Outcomes of Patients in the ECNM Registry Blood Adv. 2022 7 1713 1724 10.1182/bloodadvances.2022008292 PMC10182174 36094848

33. Georgin-Lavialle S. Lhermitte L. Dubreuil P. Chandesris M.-O. Hermine O. Damaj G. Mast cell leukemia Blood 2013 121 1285 1295 10.1182/blood-2012-07-442400 23243287

34. Jain P. Wang S. Patel K.P. Sarwari N. Cortes J. Kantarjian H. Verstovsek S. Mast cell leukemia (MCL): Clinico-pathologic and molecular features and survival outcome Leuk. Res. 2017 59 105 109 10.1016/j.leukres.2017.05.018 28599188 PMC5573652