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Graft-versus-Host Disease Prophylaxis with Cyclophosphamide and Cyclosporin. BACKGROUND: Allogeneic peripheral-blood stem-cell transplantation (SCT) from a matched related donor after myeloablative conditioning is the preferred curative treatment for patients with high-risk blood cancers. The combination of a calcineurin inhibitor and an antimetabolite remains standard care for graft-versus-host disease (GVHD) prophylaxis in these patients. Data from two randomized trials have suggested that post-transplantation cyclophosphamide can reduce the risk of GVHD after SCT from a matched donor when it is added to or replaces the antimetabolite. However, the effects of post-transplantation cyclophosphamide specifically after SCT from a matched related donor remain uncertain, and effects in the context of myeloablative conditioning are unclear. METHODS: We randomly assigned adults who were undergoing SCT from a matched related donor after myeloablative or reduced-intensity conditioning to receive either post-transplantation cyclophosphamide-cyclosporin (experimental prophylaxis) or cyclosporin-methotrexate (standard prophylaxis). The primary end point was GVHD-free, relapse-free survival. RESULTS: Among 134 patients who underwent randomization, 66 were assigned to receive experimental prophylaxis and 68 to receive standard prophylaxis. GVHD-free, relapse-free survival was significantly longer with experimental prophylaxis (median, 26.2 months; 95% confidence interval [CI], 9.1 to not reached) than with standard prophylaxis (median, 6.4 months; 95% CI, 5.6 to 8.3; P<0.001 by a log-rank test). GVHD-free, relapse-free survival at 3 years was 49% (95% CI, 36 to 61) with experimental prophylaxis and 14% (95% CI, 6 to 25) with standard prophylaxis (hazard ratio for GVHD, relapse, or death, 0.42; 95% CI, 0.27 to 0.66). The cumulative incidence of grade III to IV acute GVHD at 3 months was 3% (95% CI, 1 to 10) in the experimental-prophylaxis group and 10% (95% CI, 4 to 19) in the standard-prophylaxis group. At 2 years, overall survival was 83% and 71%, respectively (hazard ratio for death, 0.59; 95% CI, 0.29 to 1.19). The incidence of serious adverse events was similar in the two groups in the first 100 days after SCT. CONCLUSIONS: The combination of post-transplantation cyclophosphamide and a calcineurin inhibitor led to longer GVHD-free, relapse-free survival than standard prophylaxis after transplantation from a matched related donor with either reduced-intensity or myeloablative conditioning in patients with blood cancers. (Funded by the Australian Government Medical Research Future Fund and others; ALLG BM12 CAST Australian-New Zealand Clinical Trials Registry number, ACTRN12618000505202.).

Allogeneic peripheral blood stem cell transplantation (PBSCT) using a matched related donor (MRD) and myeloablative conditioning is the preferred curative treatment for patients with high-risk blood cancers. However, PBSCT carries a major risk of graft-versus-host disease (GVHD), where the donor immune cells attack recipient tissue1. GVHD affects 20–50% of patients and is a leading cause of both morbidity and mortality1-3. Peripheral blood stem cells (PBSCs) from a human leucocyte antigens (HLA)-matched related donor are the preferred donor source, using myeloablative conditioning where possible, to reduce the risk of relapse. The use of PBSCs and myeloablative conditioning both increase the risk of GVHD compared with bone marrow cells and reduced intensity conditioning4,5. The combination of a calcineurin inhibitor (CNI; cyclosporin or tacrolimus) and an anti-metabolite (methotrexate or mycophenolate) for transplantation using a MRD has formed the backbone of GVHD prophylaxis for four decades6,7. In many European and Australasian centers, anti-thymocyte globulin (ATG) is added to reduce GVHD risk for PBSCT using a MRD based upon two randomized trials8,9, but its use in the U.S. is uncommon, following a randomized trial suggesting it may lower progression-free and overall survival10.

hylaxis for four decades6,7. In many European and Australasian centers, anti-thymocyte globulin (ATG) is added to reduce GVHD risk for PBSCT using a MRD based upon two randomized trials8,9, but its use in the U.S. is uncommon, following a randomized trial suggesting it may lower progression-free and overall survival10. The addition of high-dose cyclophosphamide 3 and 4 days after stem cell infusion (post-transplant cyclophosphamide, PTCy) to tacrolimus-mycophenolate (triple GVHD prophylaxis) revolutionized GVHD prophylaxis by making haploidentical transplantation feasible11. PTCy has subsequently been explored using matched donors. PTCY-alone was not sufficient for preventing GVHD using PBSCs12,13 but gave similar GVHD-outcomes compared with tacrolimus-methotrexate when bone marrow stem cells were used14-16. The BMT CTN 1703 trial assessed triple GVHD prophylaxis in recipients of PBSCs from a matched related or unrelated donor receiving reduced-intensity or non-myeloablative conditioning17. Post hoc analysis of the MRD subgroup suggested benefit for GVHD-free, relapse-free survival (GRFS). Meanwhile, single arm and retrospective studies suggested that dual GVHD prophylaxis, where PTCy replaces the anti-metabolite, may perform as well as triple prophylaxis in PBSCT using matched donors18-20. This was confirmed by the HOVON-96 randomized trial, which found reduced GVHD with dual GVHD prophylaxis (PTCy-cyclosporin) compared to standard prophylaxis (cyclosporin-mycophenolate). However, this study predominantly involved matched unrelated donors and exclusively used non-myeloablative conditioning21. A randomized trial comparing PTCy with ATG in patients receiving reduced-intensity conditioning before PBSCT from a matched related or unrelated donor did not show significant differences in rates of GVHD22. As a result, the benefit of PTCy for PBSCT using a MRD remains uncertain, and unknown in the context of myeloablative conditioning, which was excluded from both the BMT CTN 1703 and HOVON-96 trials. Here, we present the results of the phase 3 randomized ALLG BM12 CAST trial, which aimed to establish the benefit of the dual GVHD prophylaxis regimen (PTCy-cyclosporin) for PBSCT using a MRD in the context of either reduced-intensity or myeloablative conditioning.

The Australasian Leukaemia & Lymphoma Group (ALLG) BM12 CAST trial is a prospective, open-label, two-arm randomized phase 3 trial comparing cyclosporin and methotrexate (control arm) with PTCy and cyclosporin (investigational arm) for GVHD prophylaxis. The primary objective was to compare GRFS between the two GVHD prophylaxis regimens. The second author was responsible for trial concept and the protocol was designed and written by the first two authors with input from other authors as part of the Stem Cell Transplantation Working Party of the Australasian Leukaemia & Lymphoma Group (ALLG). The full protocol is available at NEJM.org. The trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12618000505202) and approved by the Human Research Ethics Committee at each participating site. All patients provided informed consent to participate. The ALLG sponsored, coordinated, and monitored the trial through a designated Trial Management Committee and its Safety and Data Monitoring Committee. All site investigators (Table S1) and the ALLG confirm adherence to the trial protocol and the accuracy of the site data. The first and third author (senior trial statistician) confirm the accuracy of the data analysis. The first and third authors wrote the first draft of the manuscript. All authors contributed to subsequent revisions. No one who is not an author contributed to writing the manuscript. There was no confidentiality agreement of the data between the sponsor and the authors or institutions. The first and second author contributed equally to this work.

ote the first draft of the manuscript. All authors contributed to subsequent revisions. No one who is not an author contributed to writing the manuscript. There was no confidentiality agreement of the data between the sponsor and the authors or institutions. The first and second author contributed equally to this work. We enrolled adult patients (age 18 to 70 years) undergoing PBSCT using peripheral blood stem cells from MRDs at 8 Australian and 2 New Zealand transplant centers. Indications for transplantation were acute leukemia in first or second remission or myelodysplastic syndrome with less than 20% blasts in the bone marrow. Exclusion criteria included acute leukemia beyond second remission and those with significant organ dysfunction. Related donors were a minimum of 6/6 match (HLA-A, HLA-B and HLA-DRB1) with the recipient. Full inclusion and exclusion criteria can be found in the Study Protocol.

h less than 20% blasts in the bone marrow. Exclusion criteria included acute leukemia beyond second remission and those with significant organ dysfunction. Related donors were a minimum of 6/6 match (HLA-A, HLA-B and HLA-DRB1) with the recipient. Full inclusion and exclusion criteria can be found in the Study Protocol. The choice between myeloablative and reduced-intensity conditioning was decided by the transplant physician before randomization to enable stratification. Intensity of conditioning was guided by age-adjusted comorbidity score and Disease Risk Index23,24. Conditioning regimens are described in Supplementary Methods. The only difference in conditioning between the two arms was in the TBI-based myeloablative cohort, where high-dose cyclophosphamide was replaced by fludarabine in the investigational arm due its proven efficacy with PTCy25 and the lack of safety data for cyclophosphamide-TBI 12Gy. Palifermin and ATG were not permitted before transplant. The target CD34+ cell dose was between 3x106/kg and 10x106/kg body weight of the recipient.

dose cyclophosphamide was replaced by fludarabine in the investigational arm due its proven efficacy with PTCy25 and the lack of safety data for cyclophosphamide-TBI 12Gy. Palifermin and ATG were not permitted before transplant. The target CD34+ cell dose was between 3x106/kg and 10x106/kg body weight of the recipient. The control GVHD prophylaxis consisted of methotrexate (intravenous bolus of 15 mg per meter squared on day 1 and 10 mg per meter squared on days 3, 6 and 11) and cyclosporin (dosing and monitoring levels as per institutional practice) starting 1 to 3 days before transplant and tapered not before day 90. The investigational GVHD prophylaxis consisted of cyclophosphamide (50 mg per kilogram on days 3 and 4 after transplant followed by cyclosporin (dosing and monitoring levels as per institutional guidelines) starting on day 5 after transplant and tapered not before day 90. Uroprophylaxis with Mesna was used according to institutional practice. For both arms, supportive care including use of growth factors and anti-microbial prophylaxis was permitted according to institutional practice. Weekly serial monitoring of CMV viral loads was recommended until 90 days after transplant. As letermovir was not available, institutions used either a pre-emptive approach or prophylactic high-dose acyclovir or low dose (val)ganciclovir to minimize CMV disease. Donor lymphocyte infusions were permitted only in the setting of falling donor chimerism or documented measurable residual disease.

after transplant. As letermovir was not available, institutions used either a pre-emptive approach or prophylactic high-dose acyclovir or low dose (val)ganciclovir to minimize CMV disease. Donor lymphocyte infusions were permitted only in the setting of falling donor chimerism or documented measurable residual disease. The primary endpoint was time-to-event GRFS measured from the date of stem cell transplantation until the date of the earliest occurrence of any one of grade III or greater acute GVHD using MAGIC criteria (Table S2)26, NIH-defined moderate or severe chronic GVHD27, morphological relapse or death from any cause. Secondary endpoints examined neutrophil and platelet engraftment, hepatic veno-occlusive disease, acute GVHD, chronic GVHD, disease relapse, non-relapse mortality, overall survival, quality-of-life and cost-effectiveness. Impact of PTCy on quality-of-life endpoints and cost-effectiveness will subsequently be published. Timing and details of unblinded study assessments can be found in the Study Protocol.

ve disease, acute GVHD, chronic GVHD, disease relapse, non-relapse mortality, overall survival, quality-of-life and cost-effectiveness. Impact of PTCy on quality-of-life endpoints and cost-effectiveness will subsequently be published. Timing and details of unblinded study assessments can be found in the Study Protocol. Statistical analysis followed the pre-specified statistical analysis package (available online) and additional details are provided in the Supplementary Methods. A total of 134 patients were randomized to the two treatment arms using a 1:1 allocation ratio, following the decision to transplant but not more than 28 days before transplant admission to minimize dropout before PBSCT. Randomization was stratified by age (dichotomised at 50 years) and intensity of the conditioning regimen (myeloablative or reduced intensity). A sample size of 134 patients, guided by results of the single arm PTCy-cyclosporin study18, was selected to achieve an 80% power for a two-sided log-rank test (a=0.05) to detect a hazard ratio of 0.54, corresponding to 86 total primary events. An interim analysis of the primary efficacy endpoint was performed in November 2022 after 47 GRFS events. Early stopping rules for efficacy and futility (p-value for the log-rank test less than 0.005 or greater than 0.592 respectively) were not met and the trial continued to full recruitment.

o 86 total primary events. An interim analysis of the primary efficacy endpoint was performed in November 2022 after 47 GRFS events. Early stopping rules for efficacy and futility (p-value for the log-rank test less than 0.005 or greater than 0.592 respectively) were not met and the trial continued to full recruitment. Statistical analysis was performed, unless otherwise specified, using the Intention to Treat (ITT) principle with all randomized subjects analyzed according to the assigned treatment arm. Analyses of time-to-event endpoints used the Kaplan-Meier method to estimate survival distribution in a group of patients. Estimation of a hazard ratio was based on a Cox proportional hazards (PH) regression model. Formal hypothesis testing was restricted to the unadjusted analysis of the primary endpoint (GRFS). Statistical analyses of the secondary efficacy endpoints used an intention-to-treat or where appropriate, a modified subset that excluded patients who did not proceed to transplant. For secondary endpoints, estimates and their associated 95% confidence interval are reported and as they are not adjusted for multiplicity, they should not be used for hypothesis testing. The proportions, in each arm, of acute GVHD within 6 months and cGVHD at 12 and 24 months were explored using estimates of cumulative incidence functions allowing for competing events. The censor date was the earliest of the last dates of disease assessment for all patients remaining on study who were not known to have relapsed or died or experienced a competing event.

We enrolled adult patients (age 18 to 70 years) undergoing PBSCT using peripheral blood stem cells from MRDs at 8 Australian and 2 New Zealand transplant centers. Indications for transplantation were acute leukemia in first or second remission or myelodysplastic syndrome with less than 20% blasts in the bone marrow. Exclusion criteria included acute leukemia beyond second remission and those with significant organ dysfunction. Related donors were a minimum of 6/6 match (HLA-A, HLA-B and HLA-DRB1) with the recipient. Full inclusion and exclusion criteria can be found in the Study Protocol.

The choice between myeloablative and reduced-intensity conditioning was decided by the transplant physician before randomization to enable stratification. Intensity of conditioning was guided by age-adjusted comorbidity score and Disease Risk Index23,24. Conditioning regimens are described in Supplementary Methods. The only difference in conditioning between the two arms was in the TBI-based myeloablative cohort, where high-dose cyclophosphamide was replaced by fludarabine in the investigational arm due its proven efficacy with PTCy25 and the lack of safety data for cyclophosphamide-TBI 12Gy. Palifermin and ATG were not permitted before transplant. The target CD34+ cell dose was between 3x106/kg and 10x106/kg body weight of the recipient. The control GVHD prophylaxis consisted of methotrexate (intravenous bolus of 15 mg per meter squared on day 1 and 10 mg per meter squared on days 3, 6 and 11) and cyclosporin (dosing and monitoring levels as per institutional practice) starting 1 to 3 days before transplant and tapered not before day 90. The investigational GVHD prophylaxis consisted of cyclophosphamide (50 mg per kilogram on days 3 and 4 after transplant followed by cyclosporin (dosing and monitoring levels as per institutional guidelines) starting on day 5 after transplant and tapered not before day 90. Uroprophylaxis with Mesna was used according to institutional practice.

prophylaxis consisted of cyclophosphamide (50 mg per kilogram on days 3 and 4 after transplant followed by cyclosporin (dosing and monitoring levels as per institutional guidelines) starting on day 5 after transplant and tapered not before day 90. Uroprophylaxis with Mesna was used according to institutional practice. For both arms, supportive care including use of growth factors and anti-microbial prophylaxis was permitted according to institutional practice. Weekly serial monitoring of CMV viral loads was recommended until 90 days after transplant. As letermovir was not available, institutions used either a pre-emptive approach or prophylactic high-dose acyclovir or low dose (val)ganciclovir to minimize CMV disease. Donor lymphocyte infusions were permitted only in the setting of falling donor chimerism or documented measurable residual disease.

The primary endpoint was time-to-event GRFS measured from the date of stem cell transplantation until the date of the earliest occurrence of any one of grade III or greater acute GVHD using MAGIC criteria (Table S2)26, NIH-defined moderate or severe chronic GVHD27, morphological relapse or death from any cause. Secondary endpoints examined neutrophil and platelet engraftment, hepatic veno-occlusive disease, acute GVHD, chronic GVHD, disease relapse, non-relapse mortality, overall survival, quality-of-life and cost-effectiveness. Impact of PTCy on quality-of-life endpoints and cost-effectiveness will subsequently be published. Timing and details of unblinded study assessments can be found in the Study Protocol.

Statistical analysis followed the pre-specified statistical analysis package (available online) and additional details are provided in the Supplementary Methods. A total of 134 patients were randomized to the two treatment arms using a 1:1 allocation ratio, following the decision to transplant but not more than 28 days before transplant admission to minimize dropout before PBSCT. Randomization was stratified by age (dichotomised at 50 years) and intensity of the conditioning regimen (myeloablative or reduced intensity). A sample size of 134 patients, guided by results of the single arm PTCy-cyclosporin study18, was selected to achieve an 80% power for a two-sided log-rank test (a=0.05) to detect a hazard ratio of 0.54, corresponding to 86 total primary events. An interim analysis of the primary efficacy endpoint was performed in November 2022 after 47 GRFS events. Early stopping rules for efficacy and futility (p-value for the log-rank test less than 0.005 or greater than 0.592 respectively) were not met and the trial continued to full recruitment. Statistical analysis was performed, unless otherwise specified, using the Intention to Treat (ITT) principle with all randomized subjects analyzed according to the assigned treatment arm. Analyses of time-to-event endpoints used the Kaplan-Meier method to estimate survival distribution in a group of patients. Estimation of a hazard ratio was based on a Cox proportional hazards (PH) regression model. Formal hypothesis testing was restricted to the unadjusted analysis of the primary endpoint (GRFS).

arm. Analyses of time-to-event endpoints used the Kaplan-Meier method to estimate survival distribution in a group of patients. Estimation of a hazard ratio was based on a Cox proportional hazards (PH) regression model. Formal hypothesis testing was restricted to the unadjusted analysis of the primary endpoint (GRFS). Statistical analyses of the secondary efficacy endpoints used an intention-to-treat or where appropriate, a modified subset that excluded patients who did not proceed to transplant. For secondary endpoints, estimates and their associated 95% confidence interval are reported and as they are not adjusted for multiplicity, they should not be used for hypothesis testing. The proportions, in each arm, of acute GVHD within 6 months and cGVHD at 12 and 24 months were explored using estimates of cumulative incidence functions allowing for competing events. The censor date was the earliest of the last dates of disease assessment for all patients remaining on study who were not known to have relapsed or died or experienced a competing event.

From 4 April 2019 to 30 January 2024, the ALLG enrolled 134 patients on the prospective, open-label, two-arm, randomized BM12 CAST trial. The median age was 55.7 years, 55.2% were male, the most common indication for transplant was acute myeloid leukemia and 44% received myeloablative conditioning. Baseline characteristics for the two arms are displayed in Table 1 and further details provided in Table S1. Overall, the two arms were relatively well-balanced although the proportions of patients with acute lymphoblastic leukaemia and low comorbidity scores (Score 0-2) were numerically higher in the investigational arm. Sixty-eight patients were assigned to the control cyclosporin-methotrexate arm and 66 patients to the investigational PTCy-cyclosporin arm. Five patients randomised to the investigational arm did not proceed to transplant: four were withdrawn from the study before an event while one patient died while still on study. Details of how these patients were accounted for the intention-to-treat analysis are provided in Table S3. All other patients proceeded to transplant and were followed according to protocol for 5 years, death or withdrawn consent. Disposition of patients by treatment arm at the time of database snapshot is shown in Figure 1. Details of the five patients receiving donor lymphocyte infusions (three in the control arm and two in the investigational arm) are provided in Table S4.

ollowed according to protocol for 5 years, death or withdrawn consent. Disposition of patients by treatment arm at the time of database snapshot is shown in Figure 1. Details of the five patients receiving donor lymphocyte infusions (three in the control arm and two in the investigational arm) are provided in Table S4. The final event-driven analysis occurred after 87 GRFS events using a censor date of 4 Dec 2024. Transplanted event-free patients (n=43) had at least 13 months of follow-up for GRFS. Reported first events are summarised in Table 2. Estimated median potential follow-up was 36.1 months (interquartile range, IQR 26.5-43.5 months). GRFS was significantly improved in the PTCy group with median GRFS of 26.2 months (95% CI:9.1-not reached) with PTCy-cyclosporin and 6.4 months (95% CI:5.6-8.3) in the control arm (Log-Rank test p-value <0.0001) (Table S5). The estimated hazard ratio (HR) was 0.419 (95% CI:0.267-0.656) and Kaplan-Meier curves are presented in Figure 2A. The randomization strata of age and conditioning intensity appeared to be unrelated to GRFS and in the pre-specified adjusted analyses the inferences about the treatment effect were consistent with the primary (unadjusted) analysis (Figure S1A). Pre-specified adjusted analyses indicated no apparent relationships of female-donor to male-recipient, recipient CMV status, comorbidity-age score or disease risk index with GRFS. Kaplan-Meier curves are presented in Figure S1B.

t the treatment effect were consistent with the primary (unadjusted) analysis (Figure S1A). Pre-specified adjusted analyses indicated no apparent relationships of female-donor to male-recipient, recipient CMV status, comorbidity-age score or disease risk index with GRFS. Kaplan-Meier curves are presented in Figure S1B. Cumulative incidences for grade III-IV acute GVHD displayed Figure S2 are suggestive of a difference in treatment arms. At 3 months, the cumulative incidences were 10.4% (95% CI: 4.5-19.0) and 3.2% (95%CI: 0.6-10.0) for control and investigational arms, respectively (Table 3). Worst grades of acute GVHD in transplanted patients are shown in Table S6. Three (4.4%) patients developed grade IV acute GVHD in the control arm compared with no transplanted patient in the investigational arm.

(95% CI: 4.5-19.0) and 3.2% (95%CI: 0.6-10.0) for control and investigational arms, respectively (Table 3). Worst grades of acute GVHD in transplanted patients are shown in Table S6. Three (4.4%) patients developed grade IV acute GVHD in the control arm compared with no transplanted patient in the investigational arm. The cumulative incidences of moderate-severe cGVHD shown in Figure S2 are suggestive of a difference between the treatment arms. The cumulative incidences of moderate-severe cGVHD at 12 months were 23.9% (95%CI: 14.4-34.8) and 14.5% (95%CI: 7.1-24.5) with control and investigational prophylaxis, respectively (Table 3). Worst grades of chronic GVHD are shown in Table S6. Nine patients (13.2%) developed severe chronic GVHD in the control arm compared with one patient (1.6%) in the investigational arm. In a post hoc exploratory analysis, the proportions of patients at 12 months that were in remission and free of systemic immunosuppression (see Supplementary Appendix for definition) were 30.9% (95%CI: 20.2-43.3) and 54.5% (95%CI: 41.8-66.9) in the control and investigational arms, respectively (Table 3). The cumulative incidences for relapse and death are shown in Figure S2 and Table 3 and did not appear to be different. Overall, the difference in the primary endpoint of GRFS between the prophylaxis arms appeared to be driven primarily by differences in the incidences of grade III-IV acute and moderate-severe chronic GVHD (as seen also in Table 2).

relapse and death are shown in Figure S2 and Table 3 and did not appear to be different. Overall, the difference in the primary endpoint of GRFS between the prophylaxis arms appeared to be driven primarily by differences in the incidences of grade III-IV acute and moderate-severe chronic GVHD (as seen also in Table 2). For the analyses of both relapse-free-survival (a post hoc endpoint) and overall survival (a secondary endpoint), last dates of follow-up for both endpoints ranged from 11 Jul 2024 to 31 Mar 2025 and accordingly a study censor date of 11 Jul 2024 was implemented. This resulted in five relapses being excluded from the analysis of relapse-free survival (two from the control arm and three from the investigational arm) and one death being excluded from the control arm in the overall survival analyses. The estimated median potential follow-up for relapse-free survival was 29.2 months (IQR 16.4-38.9) and 28.1 months (IQR 14.3-41.6) for overall survival. At 2 years, 74.5% of the experimental arm patients and 59.3% of control arm patients were relapse-free (estimated HR 0.547; 95% CI: 0.299-1.001) and the Kaplan-Meier curves are shown in Figure 1B. At 2 years, overall survival was 83.1% in the experimental arm and 70.6% in the control arm (HR 0.591; 95% CI:0.294–1.188) (Table 3) and the Kaplan-Meier curves are shown in Figure 1C. Primary causes of all deaths according to treatment arm are shown in Table S7.

001) and the Kaplan-Meier curves are shown in Figure 1B. At 2 years, overall survival was 83.1% in the experimental arm and 70.6% in the control arm (HR 0.591; 95% CI:0.294–1.188) (Table 3) and the Kaplan-Meier curves are shown in Figure 1C. Primary causes of all deaths according to treatment arm are shown in Table S7. The rates of neutrophil engraftment by Day 90 were similar at 94.1% and 89.4% with control and investigational prophylaxis, respectively (Table 3). Two of 67 (3.0%) patients in the control arm that survived to Day 28 failed to achieve neutrophil engraftment by Day 28. All 59 transplanted patients in the investigational arm that survived to Day 28 had neutrophil engraftment. The median times to neutrophil engraftment were 18 days (interquartile range, IQR 15.5-22.0) and 19 days (IQR 16.0-22.0) respectively. The rates of platelet engraftment at any time were also similar at 95.6% and 87.9%, respectively (Table 3). However, the median time to platelet recovery appeared to be longer with investigational prophylaxis (21.5 days: IQR 17.0-27.25) than with control prophylaxis (18.0 days: IQR 14.25-21.75). Given the frequency of adverse events following PBSCT, only adverse events of specific interest were reported for the first 100 days after transplant Specific adverse events included ICU admission, veno-occlusive disease, hemorrhagic cystitis, CMV reactivation requiring treatment and any non-hematologic grade 4-5 event according to CTCAE Version 4.03. See Study Protocol for more details of definitions of adverse events.

ported for the first 100 days after transplant Specific adverse events included ICU admission, veno-occlusive disease, hemorrhagic cystitis, CMV reactivation requiring treatment and any non-hematologic grade 4-5 event according to CTCAE Version 4.03. See Study Protocol for more details of definitions of adverse events. CMV reactivation requiring treatment was the most common adverse event reported: 10 patients in the control arm and 14 patients in the investigational arm required treatment. One death from CMV pneumonitis occurred in the control arm. One case of veno-occlusive disease was reported, which occurred in the control arm and led to death. No cases of Grade 3-4 hemorrhagic cystitis were reported. In the control arm, 22 (32.4%) patients had at least one Grade 3 or worse adverse event reported including 5 (7.4%) with Grade 5 (Table S8). In the investigational arm, 12 (19.7%) had at least one grade 3 or worse event reported including 4 (6.6%) with Grade 5 (Table S9). The most common events reported in both arms were sepsis or infections and infestations. Twenty (16%) of the 129 transplanted patients were admitted to intensive care in the first 100 days after transplant: 11 in the control arm and 9 in the investigational arm. Details of these 20 patients are provided in Table S10. No obvious increased toxicity was noted as a function of age over or under 50 years.

The final event-driven analysis occurred after 87 GRFS events using a censor date of 4 Dec 2024. Transplanted event-free patients (n=43) had at least 13 months of follow-up for GRFS. Reported first events are summarised in Table 2. Estimated median potential follow-up was 36.1 months (interquartile range, IQR 26.5-43.5 months). GRFS was significantly improved in the PTCy group with median GRFS of 26.2 months (95% CI:9.1-not reached) with PTCy-cyclosporin and 6.4 months (95% CI:5.6-8.3) in the control arm (Log-Rank test p-value <0.0001) (Table S5). The estimated hazard ratio (HR) was 0.419 (95% CI:0.267-0.656) and Kaplan-Meier curves are presented in Figure 2A. The randomization strata of age and conditioning intensity appeared to be unrelated to GRFS and in the pre-specified adjusted analyses the inferences about the treatment effect were consistent with the primary (unadjusted) analysis (Figure S1A). Pre-specified adjusted analyses indicated no apparent relationships of female-donor to male-recipient, recipient CMV status, comorbidity-age score or disease risk index with GRFS. Kaplan-Meier curves are presented in Figure S1B.

Cumulative incidences for grade III-IV acute GVHD displayed Figure S2 are suggestive of a difference in treatment arms. At 3 months, the cumulative incidences were 10.4% (95% CI: 4.5-19.0) and 3.2% (95%CI: 0.6-10.0) for control and investigational arms, respectively (Table 3). Worst grades of acute GVHD in transplanted patients are shown in Table S6. Three (4.4%) patients developed grade IV acute GVHD in the control arm compared with no transplanted patient in the investigational arm. The cumulative incidences of moderate-severe cGVHD shown in Figure S2 are suggestive of a difference between the treatment arms. The cumulative incidences of moderate-severe cGVHD at 12 months were 23.9% (95%CI: 14.4-34.8) and 14.5% (95%CI: 7.1-24.5) with control and investigational prophylaxis, respectively (Table 3). Worst grades of chronic GVHD are shown in Table S6. Nine patients (13.2%) developed severe chronic GVHD in the control arm compared with one patient (1.6%) in the investigational arm. In a post hoc exploratory analysis, the proportions of patients at 12 months that were in remission and free of systemic immunosuppression (see Supplementary Appendix for definition) were 30.9% (95%CI: 20.2-43.3) and 54.5% (95%CI: 41.8-66.9) in the control and investigational arms, respectively (Table 3).

gational arm. In a post hoc exploratory analysis, the proportions of patients at 12 months that were in remission and free of systemic immunosuppression (see Supplementary Appendix for definition) were 30.9% (95%CI: 20.2-43.3) and 54.5% (95%CI: 41.8-66.9) in the control and investigational arms, respectively (Table 3). The cumulative incidences for relapse and death are shown in Figure S2 and Table 3 and did not appear to be different. Overall, the difference in the primary endpoint of GRFS between the prophylaxis arms appeared to be driven primarily by differences in the incidences of grade III-IV acute and moderate-severe chronic GVHD (as seen also in Table 2).

For the analyses of both relapse-free-survival (a post hoc endpoint) and overall survival (a secondary endpoint), last dates of follow-up for both endpoints ranged from 11 Jul 2024 to 31 Mar 2025 and accordingly a study censor date of 11 Jul 2024 was implemented. This resulted in five relapses being excluded from the analysis of relapse-free survival (two from the control arm and three from the investigational arm) and one death being excluded from the control arm in the overall survival analyses. The estimated median potential follow-up for relapse-free survival was 29.2 months (IQR 16.4-38.9) and 28.1 months (IQR 14.3-41.6) for overall survival. At 2 years, 74.5% of the experimental arm patients and 59.3% of control arm patients were relapse-free (estimated HR 0.547; 95% CI: 0.299-1.001) and the Kaplan-Meier curves are shown in Figure 1B. At 2 years, overall survival was 83.1% in the experimental arm and 70.6% in the control arm (HR 0.591; 95% CI:0.294–1.188) (Table 3) and the Kaplan-Meier curves are shown in Figure 1C. Primary causes of all deaths according to treatment arm are shown in Table S7.

The rates of neutrophil engraftment by Day 90 were similar at 94.1% and 89.4% with control and investigational prophylaxis, respectively (Table 3). Two of 67 (3.0%) patients in the control arm that survived to Day 28 failed to achieve neutrophil engraftment by Day 28. All 59 transplanted patients in the investigational arm that survived to Day 28 had neutrophil engraftment. The median times to neutrophil engraftment were 18 days (interquartile range, IQR 15.5-22.0) and 19 days (IQR 16.0-22.0) respectively. The rates of platelet engraftment at any time were also similar at 95.6% and 87.9%, respectively (Table 3). However, the median time to platelet recovery appeared to be longer with investigational prophylaxis (21.5 days: IQR 17.0-27.25) than with control prophylaxis (18.0 days: IQR 14.25-21.75).

Given the frequency of adverse events following PBSCT, only adverse events of specific interest were reported for the first 100 days after transplant Specific adverse events included ICU admission, veno-occlusive disease, hemorrhagic cystitis, CMV reactivation requiring treatment and any non-hematologic grade 4-5 event according to CTCAE Version 4.03. See Study Protocol for more details of definitions of adverse events. CMV reactivation requiring treatment was the most common adverse event reported: 10 patients in the control arm and 14 patients in the investigational arm required treatment. One death from CMV pneumonitis occurred in the control arm. One case of veno-occlusive disease was reported, which occurred in the control arm and led to death. No cases of Grade 3-4 hemorrhagic cystitis were reported. In the control arm, 22 (32.4%) patients had at least one Grade 3 or worse adverse event reported including 5 (7.4%) with Grade 5 (Table S8). In the investigational arm, 12 (19.7%) had at least one grade 3 or worse event reported including 4 (6.6%) with Grade 5 (Table S9). The most common events reported in both arms were sepsis or infections and infestations. Twenty (16%) of the 129 transplanted patients were admitted to intensive care in the first 100 days after transplant: 11 in the control arm and 9 in the investigational arm. Details of these 20 patients are provided in Table S10. No obvious increased toxicity was noted as a function of age over or under 50 years.

In this randomized trial of PBSCT using MRDs, 3-year GRFS was significantly improved with PTCy-cyclosporin. This improvement appeared to be primarily driven by reductions in both acute and chronic GVHD, including the most severe grades that are associated with increased mortality and long-term morbidity28,29. In line with prior reports that PTCy can prevent GVHD without increasing relapse risk30, the relapse rate with PTCy-cyclosporin did not appear to be increased, and, moreover, the post hoc analysis of relapse-free survival was suggestive of a favourable outcome. The investigational arm of PTCy-cyclosporin was based upon a Phase II single-arm study that reported low rates of acute and chronic GVHD using a mixture of PBSCs from MRD and matched unrelated donors with myeloablative conditioning18. Similar efficacy has been reported in non-randomized studies of the combination of PTCy and tacrolimus19,20. The randomized HOVON-96 trial showed a benefit of PTCy-cyclosporin using PBSCs but compared it to control treatment with cyclosporin and mycophenolate, which is not standard of care for myeloablative conditioning. In addition, the conclusions from HOVON-96 are limited by the small number of PBSCTs using MRDs and the use of non-myeloablative conditioning, which is only relevant for older and less fit patients.

it to control treatment with cyclosporin and mycophenolate, which is not standard of care for myeloablative conditioning. In addition, the conclusions from HOVON-96 are limited by the small number of PBSCTs using MRDs and the use of non-myeloablative conditioning, which is only relevant for older and less fit patients. The BMT CTN 1703 trial found benefit for improving GRFS with the triple GVHD prophylaxis regimen of PTCY-tacrolimus-mycophenolate compared with tacrolimus-methotrexate. In a post-hoc analysis of the MRD subgroup, the triple regimen improved 1-year GRFS from 31.0% to 50.5%. Although the trials significantly differ (CTN 1703 included only reduced-intensity conditioning in an older population and used tacrolimus rather than cyclosporin), the results of replacing the anti-metabolite with PTCy in the CAST trial - 1-year GRFS improvement from 32.9% to 58.1% – are favorable. While there are no randomized trials of PBSCT comparing PTCy-based prophylaxis with and without the anti-metabolite, a single center retrospective comparison found lower rates of acute GVHD with the omission of mycophenolate31. Apart from the favorable efficacy seen with PTCy-cyclosporin, this regimen avoids the adverse effects of the anti-metabolite including gut toxicity and increased infection risk. Unlike the CTN 1703 study which reported numerically more toxic deaths with PTCy-tacrolimus-mycophenolate, we observed no increase in non-relapse mortality, ICU admissions or CMV infection, which is consistent with the known association of mycophenolate with impaired survival and pathogen-specific immunity32.

ion risk. Unlike the CTN 1703 study which reported numerically more toxic deaths with PTCy-tacrolimus-mycophenolate, we observed no increase in non-relapse mortality, ICU admissions or CMV infection, which is consistent with the known association of mycophenolate with impaired survival and pathogen-specific immunity32. Some limitations of CAST should be noted including the non-blinded nature of the study that could bias GVHD assessments and the lack of adverse events collected beyond the first 100 days after transplant, which might miss later complications of PTCy such as infection, cardiac events or secondary malignancies. In addition, we can’t be certain that PTCy-tacrolimus (the preferred CNI in the U.S.) would give the same results as PTCy-cyclosporin although a randomized trial in MRD transplantation showed tacrolimus is superior to cyclosporin for preventing acute GVHD33. Finally, the control arm of CAST did not include ATG, which is considered standard of care in many European and Australasian centers34. While GRFS was not reported in the major European study of cyclosporin-methotrexate with or without ATG9, the Chinese study of ATG with the triple drug regimen of cyclosporin, methotrexate and mycophenolate reported a 3-year GRFS of 37.8%8. The 3-year GRFS of 49.3% reported here with a simplified two-drug regimen is thus encouraging. Results from the MoTD randomized trial comparing ATG with PTCy-based regimens may help address this question further, although only for PBSCTs using reduced-intensity conditioning35.

henolate reported a 3-year GRFS of 37.8%8. The 3-year GRFS of 49.3% reported here with a simplified two-drug regimen is thus encouraging. Results from the MoTD randomized trial comparing ATG with PTCy-based regimens may help address this question further, although only for PBSCTs using reduced-intensity conditioning35. The results reported for PTCy-cyclosporin can rapidly change clinical practice, particularly in low-resource centers and those not using ATG, because: (1) the results simplify treatment and are directly relevant to PBSCT using MRDs, the preferred donor source; (2) we show benefits for myeloablative conditioning, which is used wherever possible for high-risk blood cancers and to date, no randomized data had been generated for PTCy-based GVHD prophylaxis; (3) we show that PTCy can effectively replace mycophenolate, a drug that causes significant early transplant morbidity; and (4) the favorable safety profile and significant improvement in GRFS with reduction in the highest grades of GVHD that cause mortality and long-term morbidity. In summary, dual GVHD prophylaxis with PTCy plus cyclosporin significantly improved long-term GRFS without increasing toxicity in PBSCT using MRD with either myeloablative or reduced-intensity conditioning.