GvHD prophylaxis with tacrolimus, sirolimus, and mycophenolate mofetil after reduced intensity conditioning hematopoietic stem cell allogeneic transplantation.

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Introduction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option for many patients with hematologic malignancies [ 1 ]. Although the outcome of allo-HSCT has improved over the years [ 2 ], non-relapse mortality (NRM) still limits its use. In this setting, graft-versus-host disease (GvHD) continues to be the main cause of morbidity and mortality [ 3 , 4 ].

Different GvHD prophylaxis are currently available in the context of reduced intensity conditioning (RIC) [ 5 – 18 ]. One of the most commonly used approaches has been the combination of a calcineurin inhibitor (CNI) and mycophenolate mofetil (MMF) or methotrexate with or without anti-thymocyte globulin (ATG) [ 17 , 19 ]. Since the development of these combinations, our understanding of the GvHD pathophysiology has greatly improved.

In this regard, sirolimus binds to cytosolic FK-binding protein 12 (FKBP12) proteins. The sirolimus/FKBP12 complex inhibits mammalian target of rapamycin (mTOR). In contrast to CNIs or other immunosuppressants, sirolimus preserves Tregs lymphocytes. The addition of sirolimus enhances the activity of CNIs reducing cytotoxic cell generation and precursor frequency during in vitro alloactivation, while CNIs amplify the inhibitory effect of sirolimus on IL-2 and IL-6 proliferation. Both our group and others have published promising results with this combination [ 11 , 15 , 20 – 22 ].

A randomized phase 3 trial was published in which 168 patients received a non-myeloablative allo-HSCT from matched-unrelated donors (MUD) and GvHD prophylaxis with cyclosporine + MMF with or without sirolimus [ 14 ]. The triple-drug combination was associated with a lower risk of grade 2-4 acute GvHD (aGvHD) (26% vs. 52%, p = 0.0013) and 1- and 4-years NRM (4%/16% vs. 16%/32%, p = 0.021), with improvements in overall survival (OS) at 1 and 4 years (86%/64% in the triple therapy group vs. 70%/46% in the standard group, p = 0.035) [ 14 ]. More recently, Kornblit et al. described the results of 76 patients receiving allo-HSCT from mismatched-URD (MMUD) with the triple-drug prophylaxis and showed a low incidence of aGvHD, which translated into superior OS, compared to historical controls [ 12 ].

Until August 2019, GvHD prophylaxis was based on the combination of tacrolimus plus sirolimus in the context of RIC allo-HSCT (allo-RIC) in the two centers involved in the current study. Thereafter, based on the results from Sandmaier et al. [ 14 ], we switched to triple-drug combination with tacrolimus plus sirolimus plus MMF (Tacro-Siro-MMF). Herein, we report the largest prospective real-life experience to date with this triple prophylaxis in the context of allo-RIC from MRD, MUD, and MMUD.

Material and methods

Patients and HSCT procedures

This is an observational study of a cohort of adult patients who underwent allo-HSCT from either related or unrelated donors after RIC and received Tacro-Siro-MMF as GvHD prophylaxis in two transplant centers. The study was designed prospectively before patient inclusion, with data collection structured from the time of patient enrollment. All consecutive allo-RIC allograft recipients between September 2019 and December 2022 were included in the study. Peripheral blood (PB) was used as graft source.

Tacrolimus was administered orally twice daily at a dose of 0.05 mg/kg or intravenously at a dose of 0.02 mg/kg starting 3 days before HSCT and adjusted to maintain concentrations between 4 and 10 ng/ml, and, in the absence of GvHD, tapered from day 100 to day 180; MMF was given orally three times daily at a dose of 15 mg/kg (maximum 1 g) orally or intravenously every 8 h from day 0 until day 30, then twice daily until day 40 and then discontinued. Sirolimus was started 3 days before HSCT, taken orally at 4 mg once daily, and adjusted to maintain concentrations between 4 and 10 ng/ml through to day 180, and (in the absence of GvHD) tapered off by day 240. Close monitoring of tacrolimus and sirolimus levels was performed (at least twice a week during hospital admission, at least weekly until day +100, and every 2–4 weeks thereafter).

RIC regimens were mainly based on thiotepa, fludarabine, and busulfan (TBF-RIC) or busulfan and fludarabine (Bu-Flu-RIC): fludarabine (150 mg/m 2 ) and busulfan (8 mg/kg) with or without thiotepa (10 mg/kg).

Patients were nursed in HEPA-filtered rooms. Supportive care, antibiotic prophylaxis and detection and treatment of CMV were provided according to local institutional guidelines. The Ethics Committee of the Salamanca Health Area approved the study (CEIM Salamanca; IRB code: 0009322). All patients signed the approved informed consent. In addition, the study was performed in accordance with the Declaration of Helsinki.

Elegibility criteria and donor selection

Patients were eligible for enrollment if they met the following criteria: (1) age > 16 and <75; (2) high-risk hematological malignancy; (3) ECOG ≤ 2 without uncontrolled infections, pregnancy, or organ dysfunction >grade 2 as defined by NCI criteria (Common Toxicity Criteria version 5); (4) written informed consent.

For donor selection, the first option was a MRD, whenever available. Sibling donors were 6/6 match for HLA-A and HLA-B at intermediate or higher resolution and for DRB1 alleles at high resolution, were willing to donate mobilized PB, and met institutional criteria for donation. If not, a MUD search (identical at HLA-A, B, C, DRB1 and DQB1 loci by high resolution techniques) was started. Unrelated donors were 7/8 (i.e., mismatched at only one of the HLA-A, HLA-B, HLA-C and HLA-DRB1 loci) or 8/8 matched at HLA-A, HLA_B, HLA-C, and HLA-DRB1 at high resolution on DNA-based typing, were willing to donate mobilized PB, and were medically eligible according to the Spanish National Marrow Donor Program criteria. Haploidentical transplants were not included in the study.

Study endpoints and definitions

The primary endpoint was the cumulative incidence (CuIn) of aGvHD grades 2-4. Secondary endpoints included CuIn of aGvHD grades 3-4 at day +100 and +180, chronic GvHD (cGvHD) at 1 and 3 years, NRM at +100, +1 year and overall NRM, OS at 1 and 3 years, progression-free survival (PFS) at 1 and 3 years and GvHD-free relapse-free survival (GRFS) at 1 and 3 years.

Myeloid recovery was defined as the first day of an absolute neutrophil count of 0.5 × 10 9 /l lasting for 3 consecutive days. Platelet recovery was defined as the first day of a platelet count of 20 × 10 9 /l, without transfusion support for 7 consecutive days.

aGvHD was diagnosed and scored according to Mount Sinai Acute GvHD International Consortium (MAGIC) criteria [ 23 ]. cGvHD diagnosis was performed and graded following the NIH criteria [ 24 ]. Disability related to cGvHD was evaluated as previously described [ 25 ].

Patients who had evidence of engraftment were evaluable for aGvHD. Sinusoidal obstruction syndrome (SOS) [ 26 ], CMV DNAemia and disease [ 27 ], Epstein-Barr virus [ 28 ] and adenovirus DNAemia [ 29 ], invasive fungal infection [ 30 ], transplantation-associated thrombocytic microangiopathy (TA-TMA) [ 31 ] and hemorrhagic cystitis [ 32 ] were diagnosed according to consensus criteria. Toxic effects were measured according to the Common Terminology Criteria for Adverse Events (CTCAE version 5), with the exception of SOS and TA-TMA, which were graded using European Society for Blood and Marrow Transplantation and Cellular Therapy (EBMT) criteria [ 26 ] and EBMT, American Society for Transplantation and Cellular Therapy, Asia-Pacific Blood and Marrow Transplantation Group and Center for IBMTR criteria, respectively [ 31 ].

OS was defined as the time from transplantation to death (any cause) or last follow-up. PFS was defined as time from transplantation to second transplantation, relapse, or death (whichever ocurred first). NRM was defined as death without previous occurrence of relapse or progression. Relapse incidence was calculated from the date of HSCT to the date of relapse or progression. The composite endpoint GRFS was defined as survival without grades 3-4 aGvHD, cGvHD that requires systemic immunosuppression, relapse, or death from any cause.

Deaths due to infection in the context of active GvHD were considered to be GvHD-related.

Statistics

Quantitative variables were expressed with median and range and qualitative variables with frequency and percentage. The CuIn of NRM and GvHD were estimated using the Gray test. OS, PFS, relapse and GRFS were analyzed using the non-parametric test Kaplan-Meier estimator including 95% confidence interval (95% CI).

Competing risk data were considered as follows [ 1 ]: for myeloid and platelet engraftment, early death or second HSCT with no evidence of engraftment [ 2 ]; for aGvHD, relapse before aGvHD or death before day 100 [ 3 ]; for cGvHD, death without development of cGVHD [ 4 ]; for NRM, relapse [ 5 ]; for progression or relapse, death with no previous relapse; or [ 6 ] for other post-transplant events, death or relapse with no previous event.

Comparisons of time to event endpoints were performed using cause-specific Cox models. Time-dependent covariates were included as time-dependent variables to analyze their impact on the different endpoints. Clinical variables included in the analysis of risk factors for aGvHD, cGvHD, NRM, PFS, GRFS and OS were as follows: donor type, diagnosis, age, gender, disease status at transplantation, ECOG, age-adjusted HCT-specific comorbidity index (HCT-CI), disease risk index (DRI), previous HSCT, CMV serology, ABO blood group mismatch, number of CD34 + cells infused and conditioning regimen. In addition, for survival endpoints, acute and cGvHD, TA-TMA, SOS, CMV disease, and fungal infection were also included in the analysis. Variables which were significantly correlated to different endpoints in the univariate analysis were entered into the multivariate analysis, and only those with p < 0.05 were retained. For analysis related to cGvHD, a cut-off was fixed at day 100 after transplantation. SPSS (IBM, SPSS Statistics for Windows, Version 21.0. Armonk, NY, USA) and R software v4.3.1 ( www.r-project.org ) were used for the analysis.

Twenty patients received treatment with ixazomib within a clinical trial (NTC03225417) from day +100 as prophylaxis of cGvHD, so these patients were not included in the cGvHD and GRFS analysis. None of the patients received post-transplantation therapy to prevent relapse.

Results

Patients’ characteristics

159 patients were consecutive candidates to allo-RIC and were assigned to triple-drug combination for GvHD prophylaxis. Pretransplant demographics and transplant characteristics are shown in Table 1 . Median age was 61 years (range, 22–72). Intermediate and high/very high DRI represented 89.3% of the series (n = 142), whereas 57.2% (n = 89) had HCT-CI ≥ 3 and 21.4% had received a prior HSCT (28 autologous and 6 allogeneic HSCT). Sixty-one recipients were allografted from MUD (38.4%) and 33 from MMUD (20.8%). The median follow-up time for surviving patients at the time of analysis was 20 months (range, 3–40). Table 1 Pretransplantation demographics and transplant characteristics. Characteristic Total n = 159 Age at transplant, years, median (range) 61 (22–72) Donor age, years, median (range) 49 (19–74) Male, n (%) 98 (61.6) HCT- CI, % 0 3 (1.9) 1,2 65 (40.9) ≥3 91 (57.2) Diagnosis, n (%) Acute myeloid leukemia 53 (33.3) Myelodysplastic syndrome (MDS) 39 (24.5) Hodgkin lymphoma 9 (5.7) Non-Hodgkin Lymphoma 22 (13.8) Acute lymphocytic leukemia 3 (1.9) Chronic myelomonocytic leukemia (CMML) 8 (5.0) MDS/MNP 6 (3.8) Primary myelofibrosis 10 (6.3) Multiple myeloma/Plasma cell leukemia 2 (1.3) Others a 7 (4.4) Previous transplantation, n (%) Autologous transplantation 28 (17.6) Allogeneic transplantation 6 (3.8) Disease risk index, DRI, n (%) Very high/High 80 (50.6) Intermediate 62 (39.2) Low 16 (10.1) CMV status, n(%) Donor+/recipient+ 101 (63.5) Donor+/recipient- 15 (9.4) Donor-/recipient+ 30 (18.8) Donor-/recipient- 13 (8.2) Donor recipient ABO match n(%) Major mismatch 30 (19.1) Minor mismatch 25 (15.9) Major and minor mismatch 3 (1.9) Donor-recipient gender match, n (%) Female—Male 40 (25.2) Male—Female 37 (23.3) Related donor 98 Identical HLA 95 (59.7) 1 mismatched b 3 (1.9) Unrelated donor 61 Identical HLA 28 (17.6) 1 mismatched b 33 (20.8) Conditioning regimen, n (%) Flu-Bu 129 (81.1) Flu-Bu-Thio 26 (16.5) Others 4 (2.5) GVHD prophylaxis, n (%) Tacro + sirolimus + MMF 159 (100) Graft source, n (%) Peripheral blood 159 (100) CD34+ (×10 6 /kg) infused, median (range) 6.21 (3.4–13.1) CMN (×10 8 /kg) infused, median (range) 7.38 (0.4–63.0) a Others: blastic phase of chronic myeloid leukemia (n = 1), T prolymphocytic leukemia (n = 2), chronic lymphocytic leukemia (n = 2) and secondary aplastic anemia (n = 1) and secondary haemofagocytic syndrome (n = 1). b Antigen and/or allele mismatches.

Engraftment

Except for 2 graft failures (1.25%), all patients achieved sustained engraftment. Additionally, 2 patients developed a secondary graft failure (1.25%). Patients who developed graft failure had MDS-EB-2 (n = 1), CMML (n = 1) and myelofibrosis (n = 2), all with active disease at the time of transplant, high/very high DRI and HCT-CI > 3 (n = 4) and received transplantation from MMUD (n = 2). The remaining patients achieved stable neutrophil and platelet engraftment at a median of 16 days (range, 3–75, IQR 15–19) and 12 days (range, 7–210, IQR 12–15), respectively (Table 2 ). Table 2 Transplantation outcomes: engraftment and GvHD. Engraftment n (%)/median (range) Primary graft failure: 2 (1.3) Secondary graft failure: 2 (1.3) Median time to neutrophil engraftment 16 (3–75, IQR 15–19) Myeloid engraftment, CuIn at 30 days 99.3 Median time to platelet engraftment 12 (7–210, IQR 12–15) Platelet engraftment, CuIn at 30 and 60 days 94.2/99.2 Acute GvHD n (%)/median (range) Median onset acute GvHD (days) 32.5 (8–399, IQR 18–78) aGvHD grades 2-4, CuIn at 100 and 180 days 29.2/30.8 aGvHD grades 3-4, CuIn at 100 and 180 days 11.3/13.4 Chronic GVHD n (%)/median (range) Median onset chronic GvHD (days) 233 (93–966, IQR 169–576) Overall cGvHD, 1-year and 3-year CuIn 23.3/41.1 cGvHD moderate to severe 1-year and 3-year CuIn 13.2/26.0

CuIn of neutrophil recovery at 30 days was 99.26% (95% CI: 91.56–100%), while the 30 and 60 days CuIn of platelet engraftment was 94.19% (95% CI: 88.27–97.17) and 99.17 (95% CI: 90.28–100%), respectively (Supplementary Fig. 1 ).

CuIn of complete chimerism by day 28 and 60 in bone marrow was 63.61% (95% CI: 50.36–74.21) and 79.75% (95% CI: 67.13–87.94).

One patient received donor lymphocyte infussion for disease relapse, without developing subsequent GvHD.

Graft-versus-host disease

aGvHD of any grade occurred in 74 (46.5%) patients at a median time of 32.5 days (range, 8–399, IQR, 18–78). Maximum grades of aGvHD were: grade 1 in 14 (8.8%), grade 2 in 37 (23.3%), grade 3 in 18 (11.3%), and grade 4 in 5 (3.1%). The skin was involved in 30 patients, gut in 55 patients and liver was involved in only 6 patients. The CuIn of aGvHD 2-4 at day +100 and +180 was 29.15% (95% CI: 22.1–36.6) and 30.77% (95% CI: 23.5–38.3), including 40% of cases with upper gastrointestinal tract involvement, which resolved with topical treatment with beclomethasone (Table 2 , Fig. 1a ). The CuIn of aGvHD 3-4 at day +100 and +180 was 11.34% (95% CI: 6.6–17.5) and 13.43% (95% CI: 8.1–20.2) (Table 2 , Fig. 1b ). In multivariable analysis, MMUD was associated with a higher risk of grade 2-4 (HR 6.99, CI: 2.99–16.29, p < 0.001) and 3-4 aGvHD (HR 6.66, CI: 2.70–16.45, p < 0.001) (Supplementary Tables 1 , 2 and Supplementary Fig. 2 .) In addition, the development of TA-TMA was also independently associated with an increased risk of grade 2-4 (HR 4.33, CI: 1.23–15.23, p = 0.023) and grade 3-4 (HR 6.50, CI: 1.74–24.32, p = 0.005) aGvHD (Supplementary Tables 1 , 2 ). Fig. 1 Cumulative incidence of Graft-versus-Host Disease (GvHD). a Cumulative incidence of grade 2 to 4 at day +180. b Cumulative incidence of grade 3-4 acute GVHD at day +180. c Cumulative incidence of overall chronic GvHD at 3 years. d Cumulative incidence of moderate-to-severe chronic GvHD at 3 years.

Overall, cGvHD occurred in 42 (32.8%) patients at a median time of 233 days (range, 93–966; IQR, 169–576). Maximum grade of cGvHD was mild in 16 patients (12.5%), moderate in 23 (18%), and severe in 3 (2.3%). CuIn of cGvHD at 1 and 3 years was 23.25% (95% CI: 16.2–31.1) and 41.05% (95% CI: 30.8–51.0) and for moderate/severe cGvHD were 13.22% (95% CI: 7.9–19.9) and 26.59% (95% CI: 17.6–36.4) (Table 2 , Fig. 1c, d ). Mouth (n = 30), followed by skin (n = 26) was the most common sites of cGvHD involvement. Only 1 patient developed bronchiolitis obliterans. The CuIn of patients off immunosuppresive therapy at 3 years was 62.72% (95% CI: 43.8–76.9). In the multivariable analysis (Supplementary Table 3 ) development of TA-TMA was independently associated with a higher risk of overall cGvHD (HR 3.82, CI: 1.45–10.10, p = 0.007). In addition, a female donor for a male recipient was also independently associated with an increased risk of overall (HR 2.46, CI: 1.29–4.67, p = 0.006) and moderate/severe cGvHD (HR 2.66, CI: 1.21–5.85, p = 0.015), Supplementary Tables 3 , 4 .

Other toxicities

The most common toxicity was oral mucositis, with 32% being grade 3-4. The incidence of any grade of hemorrhagic cystitis was 7.5% (n = 12) with only 3 patients displaying grade ≥2. Nine patients (5.6%) developed TA-TMA (2.5% high-risk TA-TMA) at a median time of 51 days (range, 25–213). The median levels of tacrolimus and sirolimus at the time of TA-TMA diagnosis were 6.95 ng/ml (2.20-10.3) and 7.8 ng/ml (2.40-13.40), respectively. Six patients (3.8%) developed SOS at a median time of 48 days (range, 12–417).

CMV DNAemia occurred in 22 recipients (13.8%) at a median time of 73 days (range, 4–727). Three patients developed CMV disease. EBV DNAemia was detected in only one patient (0.6%) and resolved after one dose of rituximab. Cases of EBV-associated lymphoproliferative disease or adenovirus DNA-emia were not observed.

Other toxicities are summarized in Table 3 . Table 3 Other toxicities. Toxicities Total n = 159 n (%) Mucositis grade 3-4 51 (32) SOS 6 (3.8) Mild 1 (0.6) Moderate 1 (0.6) Severe 4 (2.5) Very severe 0 TA-TMA a /high risk TA-TMA 9 (5.6)/4 (2.5) Hemorrhagic cystitis 12 (7.5) Grade 1 9 (5.6) Grade 2 0 Grade 3 3 (1.3) Grade 4 0 Infections ≥ grade 3 by Day 100 9.1 (CI: 5.5–14.1) CMV DNA-emia CMV Reactivation by Day 100 22 (13.8) 7.6 (CI: 4.2–12.4) CMV disease (gastro-intestinal disease) 3 (1.3) EBV DNA-emia 1 (0.6) EBV-associated lymphoproliferative disease 0 Adenovirus DNA-emia 1 (0.6) Varicella Zoster virus reactivation 4 (1.3) Hepatitis B virus reactivation 1 (0.6) Respiratory viruses (grade 3) 9 (5.6) Respiratory syncitial 3 (1.9) Influenza 2 (1.3) Parainfluenza 1 (0.6) Metapneumovirus 1 (0.6) SARSCoV2 6 (3.8) Fungal infection (grade ≥ 3) 16 (10) Candida 2 (1.3) Pulmonary aspergillosis (probable/proven) 13 (8.0) Mucormycosis 1 (0.6) Bacteriemia/bacterial infection (grade ≥ 3) 24 (18.9) Escherichia Coli 8 (5.0) Klebsiella pneumoniae 5 (3.1) Staphilococcus epidermidis 4 (2.5) Staphilococcus aureus 1 (0.6) Staphilococcus haemolyticus 1 (0.6) Pseudomonas aeruginosa 3 (1.9) Enterococcus faecium 3 (1.9) Streptococcus pneumoniae 1 (0.6) Campylobacter jejuni, Clostridium difficile 1 (0.6)/1 (0.6) Pneumocystis/Enterococcus pneumoniae 1 (0.6)/1 (0.6) Secondary malignancies 4 (2.5) (Basal cell, urothelial, esophageal or thyroid Carcinoma) Other grade 3-4 adverse events Deep-vein thrombosis 4 (2.5) (3 catheter related) Stenosing diverticulitis 1 (0.6) Fibrosing hepatopathy 1 (0.6) Acute coronary syndrome 1 (0.6) Renal and urinary disorders 4 (2.5) Haemorrage (lung, gut) 2 (1.3) a At diagnosis, all patients had therapeutic tacrolimus and sirolimus levels except two with supratherapeutic levels. In the 2 weeks prior to TA-TMA diagnosis, three patients had supratherapeutic levels (tacrolimus n = 3, sirolimus n = 2).

NRM, causes of death, relapse, PFS, OS, and GRFS

Fifteen patients (9.43%) died without prior relapse at a median time of 2.5 months after transplantation (range 0–14). The causes of NRM are detailed in Table 4 and were aGvHD-related complications with or without infection (n = 5), infectious diseases (n = 6), hepatic SOS (n = 1) and TA-TMA (n = 1). In addition, one patient who had previously relapsed, commited suicide and another patient died due to progressive respiratory insufficiency in the context of previous pulmonary disease. The CuIn of NRM at day +100 was 5.1% (95% CI: 2.4–9.3). After 1 and 3 years, NRM increased to 8.6% (95% CI: 4.8–13.9) and 10.37% (95% CI: 6.1–16.0) (Fig. 2 ). In multivariable analysis (Supplementary Table 5 ) development of SOS (HR 6.10, CI: 1.17–31.87, p = 0.032) and grade 2-4 aGvHD (HR 4.84, CI: 1.46–16.10, p = 0.010) were independently associated with a higher NRM. Table 4 Causes of death. Causes of death Total n = 53 (33.3%) Recurrence or persistence of disease 36 (22.6) Acute GvHD +- infections 5 (3.1) SOS 1 (0.6) TA-TMA 1 (0.6) Infection 6 (3.8) Bacteriemia/shock 4 (2.5) SARSCoV2 (+- influenza A) 2 (1.3) Suicide 1 (0.6) Organ failure 1 (0.6) Fig. 2 Cumulative incidence of non-relapse mortality (NRM). Cumulative incidence of NRM at 3 years.

Fifty-two patients (32.7%) relapsed at a median time of 97 days (range, 54–132). The CuIn of relapse was 31.10% (95% CI: 23.9–38.5) and 35% (95% CI: 26.5–56.1) at 1 and 3 years. Median PFS was 35 months, with 1- and 3-year PFS of 59.9% (95% CI: 51.4–67.0) and 48.0% (95% CI: 31.9–60.7) (Fig. 3a ). Finally, 91 patients (57.2%) were alive and disease-free at last follow-up. In the multivariable analysis (Supplementary Table 6 and 7 ) high/very high DRI (HR 3.14, CI: 0.97–10.24, p = 0.05) (Supplementary Fig. 3 ) and grade 2-4 aGvHD (HR 1.99, CI: 1.22–3.26, p = 0.006) were independently associated with a lower PFS, whereas any grade of cGvHD was a protective factor (HR 0.03, CI: 0.15–0.63, p < 0.001). Fig. 3 Survival outcomes: progression-free survival (PFS), GvHD-free, relapse-free survival (GRFS), and overall survival (OS). a Kaplan-Meier estimate of PFS. b Kaplan-Meier estimate of GRFS c Kaplan-Meier estimate of OS.

Median GRFS was 8 months. The GRFS at 1 and 3 years was 44.3% (95% CI: 35.3–51.2) and 32.5% (95% CI: 23.1–40.6), respectively (Fig. 3b ). In the multivariable analysis (Supplementary Table 8 ) MMUD (HR 2.85, CI: 1.83–4.43, p < 0.001) (Supplementary Fig. 4 ) and TA-TMA (HR 2.18, CI: 1.00–4.76, p = 0.05) were independently associated with a lower GRFS. Median OS was not reached, with 1 and 3 years OS of 70.3% (95% CI: 61.5–76.4) and 60.1% (95% CI: 50.2–68.7%), Fig. 3c . Patients with high/very high DRI (HR 3.75, CI: 1.08–13.00, p = 0.038) (Supplementary Fig. 5 ), with SOS (HR 12, CI: 3.89–44.70, p < 0.001) or grade 2-4 aGvHD (HR 2.18, CI: 1.23–3.88, p = 0.008) were independently associated with a lower OS, whereas any grade of cGVHD was a protective factor (HR 0.21, CI: 0.09–0.50, p < 0.001), Supplementary Table 9 .

Outcomes according to donor type

Sixty-one recipients were allografted from MUD (38.4%) and 33 from MMUD (20.8%). Pretransplant characteristics were well balanced between patients receiving transplantation from a MRD, MUD or a MMUD, except donor age and the conditioning regimen (Supplementary Table 10 ). The use of MMUD had a negative impact on the incidence of grades 2-4 aGvHD (at +100 and +180, 24.33% and 27.16% in matched donors vs 46.68% and 51.91% in MMUD; p = 0.016) and grades 3-4 aGvHD (at +100 and +180, 3.42% and 6.72% in matched donors vs 37.3% and 40.66% in MMUD; p < 0.001). This translated into a higher risk of GRFS (at 1 and 3 years 51.2% and 37.7% for matched donors vs 9.1% for MMUD; p < 0.001) and OS in patients with MMUD (at 1 and 3 years 72.3% and 64.6% for matched donors vs 59.7% and 44.2% in MMUD; p = 0.035), although type of donor did not retain its negative impact on survival in the multivariable analysis (Supplementary Table 1 and Supplementary Fig. 6 ). Posttrasplant events according to donor type are summarized in (Supplementary Tables 11 and 12 ).

Discussion

Along the last few years, different studies have challenged the classical combination of a calcineurin inhibitor plus antimetabolite for GvHD prophylaxis [ 33 ]. In this regard, the EBMT recommends the use of ATG based on several prospective randomized trials which demonstrate a significant decrease in the risk of GvHD [ 34 ], although none of these studies have reported a favorable impact on OS [ 35 ]. This is in contrast to the study by Sandmaier et al., which described that the triple combination of CNI plus MMF and sirolimus in the context of non-myeloablative HSCT from URD, translated into a better OS as compared to CNI plus MMF [ 14 ]. The current study shows, in a real-world population, that addition of MMF to sirolimus plus tacrolimus is a feasible, effective, and tolerable GvHD prophylaxis in the allo-RIC setting. Despite the use of PBSC across all types of transplant, a relatively low rate of aGvHD, as well as moderate to severe cGvHD were observed, which translated into very low rates of NRM in a high-risk and frail population.

Our cohort included both HSCT from related and unrelated donors. In this setting, outcomes of allo-HSCT from MUD were broadly equivalent to those of MRD. However, allo-HCT from MMUD had worse outcomes. Of note, we included a high percentage of patients receiving an HSCT from a MMUD (21%). This type of transplantation is greatly underrepresented in the study from Sandmaier et al. (4% with a single-allele HLA mismatch in triple-drug group) [ 14 ]. By contrast, in a subsequent study, Kornblit et al. described 76 patients receiving allo-HSCT from MMUD and the triple-drug prophylaxis with a low incidence of aGvHD [ 12 ]. It is worth pointing out that patients receiving HSCT from MMUD in our series do not seem equivalent, but rather at higher risk to those of the aforementioned trial, especially in terms of DRI. In addition, the more intensive conditioning regimen in our series could be a factor that partly explains the differences in the incidence of GvHD. In any case, in our experience, allo-RIC from MMUD is a group that might benefit from other prophylaxis strategies. In this regard, post-transplantation cyclophosphamide (PTCy) has emerged as a promising platform to overcome HLA barriers in the setting of haploidentical HSCT, and more recently in matched and mismatched donor transplants [ 5 – 9 , 36 – 39 ]. Bolaños-Meade et al. reported a phase 3 trial comparing PTCy-tacrolimus-MMF vs tacrolimus-methotrexate [ 5 ]. Although the overall and disease-free survival, relapse and NRM did not differ substantially between the two groups, the GRFS at 1 year was significantly better among those who received PTCy. Of note, the number of MMUD transplants in this study was underrepresented, since only 4% of patients received HSCT from a single-allele HLA MMUD. In this regard, Shaw et al. demonstrated favorable outcomes in a cohort of 40 patients who underwent an allo-RIC from MMUD and prophylaxis using PTCy, sirolimus, and MMF [ 39 ].

In our experience, the triple combination Tacro-Siro-MMF was safe and well tolerated. The vast majority of patients engrafted early with a CuIn of neutrophil recovery at 30 day of 99.26% and a 30 days CuIn of platelet engraftment of 94.19%. These results compare favorably with those of the cyclophosphamide-based platform, where the CuIn of neutrophil recovery and platelet recovery at day 100 were 90.3% [ 5 ]. In addition, in this study we found no evidence that this triple combination increases the risk of developing severe SOS and TA-TMA. This low incidence was likely due, at least in part, to a particular attention to the risk factors (such as tacrolimus and sirolimus serum levels) in patients receiving this protocol. Unlike in cyclophosphamide-based prophylaxis platforms, in our series only 1.3% of patients developed grade 2-3 hemorrhagic cystitis and no patient developed grade 4. Regarding infections, CMV reactivation was low, despite letermovir not being available in Spain during most of the period included in this analysis. This could have been caused by the specific antiviral activity of sirolimus and/or by lower steroid use due to the reduced incidence of aGvHD [ 40 ]. There were also no warnings regarding other infections. In this regard, a higher overall incidence of infections was noted with cyclophosphamide-tacrolimus-MMF in the Bolaños-Meade trial, with a grade 2-3 infection rate of 39.4% [ 5 ]. Of note, no other safety concerns, such as cardiac disorders, also frequent with cyclophosphamide based prophylaxis [ 5 ], were observed.

The population included in the current study is elderly and frail. To begin with, the median age in our trial was 61 years. Moreover, 56% had an HCT-CI ≥ 3, higher than that reported by Bolaños-Meade et al. (30%) [ 5 ]. Despite this, the NRM rate was remarkably low (NRM at 1 year was 8.6%), similar to that reported in the aforementioned clinical trial (with 12.3% in the experimental arm) [ 5 ]. In addition, our population was at high risk of relapse, as indicated by the high percentage of patients with a high or very high DRI (50.3% vs. 32.7% in PTCy-arm of Bolaños-Meade trial) [ 5 ]. Moreover, inclusion criteria also differ from those described by Bolaños-Meade, since they excluded second allo-HSCT, AML with more than 5% of blast in BM and chemo-refractory lymphomas [ 5 ]. Thus, it is not a surprising that our relapse rate was higher (31% vs 20.8% at 1 year) and our PFS was slightly lower (PFS at 1- and 3-year of 60% and 49% vs PTCy in Bolaños trial of 67% at 1 year). In spite of this, OS at 1 and 3 years was 70.3% and 61%, respectively, only 5% lower than the group treated with PTCy if we focus on HLA-matched transplants. In fact, low and intermediate DRI patients had excellent OS rates (80.8% and 80.8% at 1 and 3 years for low and 86.7% and 78.3% at 1 and 3 years for intermediate DRI patients). In addition, GRFS reached 44% at 1 year in our series, 51.2% if we exclude MMUD, similar to the experimental arm in the trial by Bolaños-Meade et al. [ 5 ].

One of the advantages attributed to PTCy prophylaxis is the reduction of GvHD, especially severe acute and chronic forms. In this regard, our grade 2-4 aGvHD rate at day +180 was 30.3%, although it should be emphasized that this includes 40% of upper gastrointestinal tract involvement, which resolved with topical treatment. In fact, the CuIn of grades 3-4 aGvHD at +180 was 13% and decreases to 6.72% if we focus on HLA-identical transplant. These rates compare favorably with those from PTCy, with 2-4 and 3-4 aGvHD at day +180 of 53.8% and 6.3% [ 5 ]. In the same line, CuIn of cGvHD at 1 and 3 years were 23.2% and 41% and for moderate/severe were 13.2% and 26.6%.

To our knowledge, our study is the largest real-world evidence study showing that combining sirolimus, tacrolimus, and MMF as GvHD prophylaxis in the RIC setting is safe and effective. The favorable survival results seem to be driven by the reduced incidence of GvHD and NRM. These findings warrant prospective comparisons of Tacro-Siro-MMF vs. other platforms including cyclophosphamide or ATG-based platforms for GvHD prophylaxis.

Supplementary information

Supplementary material

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Supplementary information

The online version contains supplementary material available at 10.1038/s41409-025-02562-w.

Acknowledgements

We thank all the hematology and transplant staff from our two hospitals for their contribution to this study and patient care. We thank all the patients and their families because, without them, this study would not have been possible. This study was supported by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union (Next Generation EU), through the RICORS programs TERAV (RD21/0017/0006, RD21/0017/0021) and TERAV+ (RD24/0014/0034, RD24/0014/0027).

Author contributions

LLC and JAPS designed the study; CBG, EPL, FMMD, ACM, NRT, MCC, ETI, MLAA, RPP, MBG, CVT, SSE, AAP, PAM, AGB, ERA, MLP, JFFG, ANB, LVL, VEG, and FSG cared for patients; LLC, CBG, and CRM collected and analyzed the data; LLC and JAPS drafted the manuscript. All authors contributed to manuscript editing and agreed in the final version.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

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