Graft-versus-host Disease Prophylactic Regimens Research Articles

B-Cell Acute Lymphoblastic Leukemia: Donor Matters in Allogeneic Stem Cell Transplant Outcomes of Hispanic Patients

Introduction: Hematopoietic stem cell transplantation (HSCT) improves long term overall survival (OS) and disease-free survival (DFS) in those with intermediate or high-risk B- cell ALL. Historically, matched related donors were the ideal choice, but with improved GVHD prophylactic regimens and supportive care, haploidentical donors are being increasingly used. Due to their increased graft-versus-leukemia (GVL) effect, they may be an option for Hispanic patients, for whom matched donation may be more challenging. Methods: This is a retrospective chart review of B- cell ALL patients that received allogeneic HSCT at Norris Comprehensive Cancer Center (NCCC) from 2012 to 2023. Mismatch-unrelated donors were excluded. For OS, DFS, and cumulative incidence of relapse (CIR), match sibling donors (MSD) and match unrelated donors (MUD) were grouped. However, for GVHD outcomes including graft-versus-host disease (GVHD)-free relapse-free survival (GRFS), incidence of grade 3-4 acute GVHD (aGVHD), and incidence of severe chronic GVHD (cGVHD) requiring immunosuppression MSD and MUD were separated. Incidence outcomes were evaluated using competing risk regression (Fine-Gray method), with death as a competing outcome, while survival outcomes were analyzed using Cox proportional hazards model. Results: One hundred eighteen B- cell ALL patients were included with a median age at transplant of 43 years (range: 21-69). Donor types included 46 (40.0%) MSD, 45 (38.1%) haploidentical, and 27 (22.9%) MUD. Most were Hispanic (N= 92, 78.0%) and our median follow-up time was 23.6 months. The majority of patients achieved CR1 (N=79, 66.9%) and MRD negativity by flow (N=106, 89.8%), pre-transplant. The 3-year OS and DFS for the cohort were 82.1% (95% CI 73.8-91.3%) and 66.2% (95% CI 57.2-76.6%), respectively, while the CR1 subgroup was 90.5% (95% CI 83.3-98.3%) and 77.1% (95% CI 67.8-87.7%) respectively. When compared to haploidentical HSCT, patients who received matched donors had significantly worse CIR (HR = 2.42; 95% CI 1.06-5.51; P = 0.036) and DFS (HR = 2.14; 95% CI 1.03-4.44; P = 0.041), along with a non-significant trend towards worse OS (HR = 2.54; 95% CI 0.83-7.80; P=0.10). Additionally, pretransplant minimal residual disease (MRD) positivity demonstrated worse OS (HR = 5.01; 95% CI 1.73-14.5; P = 0.003), DFS (HR = 3.25; 95% CI 1.48-7.16; P = 0.003), and CIR (HR = 2.05; 95% CI 0.78-5.39; P = 0.15). The 1-year GRFS, 1-year incidence of severe cGVHD, and 100-day incidence of grade 3-4 aGVHD was 52.6% (95% CI 43.5-63.6%), 29.5% (95% CI 20.9-38.6%), and 7.74% (95% CI 3.79-13.5%), respectively. Compared to haploidentical donors, GRFS was worse in the MUD (HR = 2.07; 95% CI 1.08-3.96; P = 0.028) but similar in MSD. This is likely driven by differences in survival and relapse, and there were no differences in the incidence of severe cGVHD or aGVHD between donor types. Multivariate analysis, which included age, MRD status pretransplant, and stage prior to transplant, demonstrated the improved predictive effect of haploidentical donors. Matched donors exhibited worse DFS (HR = 2.25; 95% CI 1.04-4.90; P = 0.04) and CIR (HR = 2.47; 95% CI 1.09-5.60; P = 0.03), with a trend towards worse OS (HR = 2.84; 95% CI 0.92-8.79; P = 0.07). Given the heterogeneity in donor-type outcomes, we conducted a subgroup analysis of the 45 haploidentical HSCT patients on the GRFS and DFS composite outcomes. After controlling for patient age, older donor age analyzed as a continuous variable was a significant predictor of worse GRFS (HR = 1.05; 95% CI 1.00-1.11; P = 0.039) but did not affect DFS. Conclusion: In a predominantly Hispanic population of ALL patients, haploidentical donors demonstrate improved OS and CIR. In those haploidentical patients, younger donor age confers improved GRFS.

212P Dual T lymphocyte suppression with antithymocyte globulin and post-transplant cyclophosphamide as graft-versus-host disease prophylaxis in haploidentical hematopoietic stem cell transplant for acute leukemias and myelodysplastic syndrome

Allogeneic stem cell transplantation (SCT) offers the chance of cure for various hematologic malignancies, but graft-versus-host disease (GVHD) remains a major impediment. Especially, with increasing numbers of haploidentical SCT in light of donor’s immediate availability, the interest in better GVHD prophylactic regimens is also on the rise. Antithymocyte globulin (ATG) has traditionally been used for prophylactic T cell depletion and GVHD prevention. Recently, the success of post-transplant cyclophosphamide (PTCy) has been reported by several groups.

Busulfan-containing conditioning regimens in allogeneic hematopoietic stem cell transplantation for acute lymphoblastic leukemia: A Taiwan observational study.

BackgroundAllogeneic stem cell transplantation (allo‐HSCT) is the ultimate cure for acute lymphoblastic leukemia (ALL).AimThis study was performed to compare the outcomes of ALL patients receiving busulfan (Bu) with cyclophosphamide (Cy)‐based or total body irradiation (TBI)‐based regimen in a Chinese population.MethodsWe enrolled 224 adult patients with ALL who received allo‐HSCT at National Taiwan University Hospital between 1997 and 2016.ResultsThe median age at transplantation was 33 years. Before allo‐HSCT, 75.9% of patients attained first or late complete remission. A total of 141 patients (62.9%) received Bu/Cy‐based conditioning, either myeloablative (MA) or reduced‐intensity stem cell transplantation (RIST), and 83 patients received a TBI‐based regimen (MA‐TBI). Patients receiving the MA‐Bu regimen had longer relapse‐free survival (RFS) than those receiving the MA‐TBI regimen (median, 24.1 vs. 6.7 months, p = .044). There was no difference in overall survival (OS, MA‐Bu vs. MA‐TBI vs. RIST‐Bu: 39.4 vs. 28.2 vs. 13.1 months, p = .276), treatment‐related mortality (TRM), or incidences of grade 3–4 acute graft‐versus‐host disease (GvHD). Among patients receiving identical GvHD prophylactic regimens, there was no difference between MA‐Bu and MA‐TBI groups regarding the incidence of grade 3–4 acute GvHD, grade 2–4, and all‐grade chronic GvHD. In subgroup analysis, patients receiving oral busulfan had comparable RFS and OS to the intravenous busulfan group (p = .436 and p = .236, respectively), but a higher TRM (25% vs. 9.8%, p = .016). In the multivariable analysis, disease status before allo‐HSCT was the only risk factor impacting RFS and OS.ConclusionIn summary, patients receiving Bu/Cy‐based or TBI‐based regimens as conditioning had similar results in terms of OS, TRM, and acute GvHD, whereas the use of myeloablative Bu/Cy resulted in a better RFS. A Bu‐based regimen could be an alternative conditioning choice for patients who are ineligible to receive TBI. Prospective and randomized controlled trials are warranted to validate the long‐term outcomes.

Area-under-the-Curve-Based Mycophenolate Mofetil Dosage May Contribute to Decrease the Incidence of Graft-versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation in Pediatric Patients.

Acute graft-versus-host disease (GvHD) remains the second leading cause of death, after disease relapse, in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). The medical records of 112 pediatric patients who underwent allo-HSCT from matched unrelated and haploidentical donors were analyzed. Patients were divided into two groups, according to the GvHD prophylactic regimen used. In the control group, GvHD prophylaxis consisted of cyclosporine A (CsA) and methotrexate (MTX) or CsA and mycophenolate mofetil (MMF) at a standard daily dose of 30 mg/kg. All subjects in the study group received tacrolimus (FK506) and MMF. In this group, MMF was subjected to therapeutic drug monitoring (TDM) through mycophenolic acid (MPA) area under the curve AUC0–12. We found a statistically significant difference in both overall acute GvHD (p < 0.0001) and overall chronic GvHD (p < 0.05) incidence between the study and the control group. The initial daily MMF dose and the age at transplant in the study group proved to be inversely correlated (r = −0.523, p < 0.0001). The children under six years of age required a significantly higher daily MMF dose (p < 0.008). This study showed that pharmacological monitoring of MPA AUC0–12 concentration allowed a reduction in the incidence of acute and chronic GvHD. MMF showed age-dependent pharmacokinetics due to greater drug clearance in younger children.

Long-term Outcome of Allogeneic Hematopoietic Stem Cell Transplantation From Unrelated Donor Using Tacrolimus/Sirolimus-based GvHD Prophylaxis: Impact of HLA Mismatch.

While tacrolimus and sirolimus (T/S)-based graft-versus-host disease (GvHD) prophylaxis has been effective in preventing acute GvHD post hematopoietic cell transplantation (HCT), its efficacy and long-term outcome in matched (MUD) and mismatched unrelated donor (mMUD) setting is not well defined. Herein, we evaluated a consecutive case-series of 482 patients who underwent unrelated donor HCT (2005-2013) with T/S-based GvHD prophylaxis. With a median follow-up of 6.2 years (range = 2.4-11.3), the 5-year overall survival (OS) and relapse/progression-free survival were 47.5% (95% confidence interval [CI]: 43.0-52.0) and 43.6% (95% CI: 39.1-48.1), respectively; and the 5-year cumulative incidence of nonrelapse mortality (NRM) and relapse were 24.9%, and 31.5%, respectively. In this cohort, mMUD was associated with worse OS (39.0% versus 50.7% at 5 y; P = 0.034), primarily due to greater risk of NRM (33.5% versus 21.7%; P = 0.038). While rates of relapse, acute (II-IV or III-IV) or chronic GvHD (limited or extensive) were not different, death caused by chronic GvHD (20.8% versus 12.8%; P = 0.022) and infection (33.0% versus 18.1%; P < 0.01) were significantly greater in mMUD. In multivariable analysis, high-risk disease (hazard ratio [HR] = 2.21, 95% CI: 1.16-4.23; P < 0.01) and mMUD (HR = 1.55, 95% CI: 1.15-2.08; P = 0.004) were independent predictive factors for OS. T/S-based GvHD prophylaxis is an effective and acceptable GvHD prophylactic regimen. However, survival after mMUD remained poor, possibly related to the severity of chronic GvHD.

Is immunological recovery clinically relevant at 100 days after allogeneic transplantation?

Background/AimsImmune reconstitution following allogeneic hematopoietic stem cell transplantation (HSCT) is affected by multiple variables during the transplantation.MethodsWe assessed the clinical factors contributing to immune function reconstitution at 100 days post-allogeneic HSCT in 114 patients receiving fludarabine-based conditioning. Immunophenotypic analysis using flow cytometry was performed to evaluate the percentage and the absolute numbers of T-cell subsets, natural killer cells, and B-cells as clinical outcomes.ResultsTacrolimus-based graft-versus-host disease (GVHD) prophylaxis, T-cell depletion, and acute GVHD were significantly associated with delayed immune reconstitution of T-cell subsets. The incidence of chronic GVHD was significantly increased in the normal recovery group compared to the abnormal group (p = 0.01). Epstein-Barr virus reactivation was more frequently observed in the abnormal group of T-cell subsets (p = 0.045). All viral reactivation events including cytomegalovirus reactivation appeared to be more frequent in the abnormal group of T-cell subsets.ConclusionsThe immune recovery status post-allogeneic HSCT was affected by GVHD prophylactic regimens, especially in cases receiving tacrolimus-based GVHD prophylaxis, T-cell depletion, and possibly those manifesting acute GVHD. Delayed immune reconstitution might increase the morbidity due to viral reactivation. Treatment strategies are needed to prevent infectious complications and enhance immune reconstitution based on the immune recovery status following allogeneic HSCT with fludarabine-based conditioning.

Outcomes Following Intolerance to Tacrolimus/Sirolimus for Graft-Versus-Host Disease Prophylaxis in Allogeneic Hematopoietic Stem Cell Transplant

INTRODUCTION: Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplant (HCT) resulting in significant morbidity and mortality. The combination of tacrolimus and sirolimus (TAC/SIR) has emerged as a GVHD prophylaxis platform preferred by many institutions given its association with rapid engraftment and acceptable transplant-related toxicity. However, overlapping toxicities between the two drugs can lead to intolerance and premature discontinuation in some patients. There is limited literature describing outcomes and subsequent management of such patients. The goal of this study is to investigate the clinical outcomes of patients becoming intolerant to the combination of TAC/SIR prophylaxis. METHODS: We retrospectively evaluated consecutive adult patients (n=100) at the Moffitt Cancer Center who received allogeneic HCT with TAC/SIR for GVHD prophylaxis in 2018. TAC/SIR intolerance was defined as discontinuation due to the toxicity of either TAC or SIR before post-transplant day 100. Survival analyses were estimated from the time of transplant with the Kaplan-Meier method and compared using the log-rank test. Patients intolerant of this prophylaxis regimen were compared to patients who completed &amp;gt;100 days of therapy, using Mann-Whitney U test for continuous variables and Pearson Chi-square tests for categorical variables. All statistical analyses were performed using SPSS v25 and NCSS v11. RESULTS: Demographics and clinical characteristics of all patients are summarized in Table 1A. TAC/SIR intolerance occurred in 25% (24 discontinued TAC, 1 discontinued SIR) of patients at a median duration of therapy of 19 days (range 4-92). The most common TAC/SIR toxicity (Table 1B) was acute kidney injury (AKI, n=11, 44%), followed by thrombotic microangiopathy (TMA, n=3, 12%). Baseline metabolic and clinical variables including creatinine, liver function, and conditioning intensity were not predictive of TAC-SIR intolerance. At a median follow-up of 10 months, the median overall survival (OS) for patients intolerant of TAC/SIR was 10 months versus was not reached for the patients without intolerance (HR 5.42; 95% CI 1.71-17.14; p&amp;lt;0.001). The 1-year PFS was 16% (95% CI 0% - 42%) vs 75% (95% CI 65% - 86%) and OS was 35% (95% CI 8% - 63%) vs. 79% (95% CI 68% - 90%) for patients who were TAC/SIR-intolerant compared to those who were TAC/SIR-tolerant (p&amp;lt;0.01) (Figure 1A). The cumulative incidence (CuI) of non-relapse mortality (NRM) at 1 year in patients intolerant of TAC/SIR was 47% (95% CI: 28% - 81%) and in patients tolerant of TAC/SIR was 4.4% (95% CI: 1.5% - 14%), (p&amp;lt;0.001). The Cul of relapse at 1 year was 45% (95% CI: 20% - 100%) in patients who were TAC/SIR-intolerant compared to 18% (95% CI: 10% - 30%) in patients who tolerated TAC/SIR (p=0.07) (figure 1B). Overall, 31 patients (31%) developed grade II-IV acute GVHD (aGVHD). The Cul of grade II-IV aGVHD at 100 days in patients who were TAC/SIR-intolerant was 29% (95% CI 15% - 58%) compared to 17% (95% CI 10% - 29%) in patients who tolerated TAC/SIR, (p=0.38). The Cul of cGVHD at 1 year in patients who were TAC/SIR-intolerant was 44% (95% CI: 25% - 79%) compared to 52% (95% CI: 40% - 68%) in patients who were TAC/SIR-tolerant (p=0.89). CONCLUSIONS: Outcomes for patients completing over 100 days of TAC/SIR for GVHD prophylaxis following allogeneic HCT are favorable. However, early intolerance of TAC/SIR GVHD prophylaxis occurred in 25% of allogeneic HCT in 2018 alone and predicted a poor prognosis with increased NRM and overall mortality, largely from drug-related toxicities. Notably, premature discontinuation of TAC/SIR did not contribute to higher subsequent risks of GVHD. Strategies to mitigate the risks of TAC/SIR toxicity are warranted. Future studies are also needed to identify the optimal GVHD prophylactic regimen for patients after TAC/SIR intolerance. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Bejanyan:Kiadis Pharma: Other: advisory board.

A Pharmacokinetic and Pharmacodynamic Study of Maraviroc as Acute Graft-versus-Host Disease Prophylaxis in Pediatric Allogeneic Stem Cell Transplant Recipients with Nonmalignant Diagnoses

Maraviroc is an allosteric small molecule antagonist of chemokine receptor type 5 (CCR5) and has been used in adult allogeneic hematopoietic stem cell transplant (HSCT) recipients to prevent acute graft-versus-host disease (GVHD) of the gastrointestinal (GI) tract and liver. The goal of this study was to establish feasibility and pharmacokinetic and pharmacodynamic profiles of maraviroc in pediatric HSCT recipients. Children ages 2 to 12 years were enrolled and maraviroc was added to standard GVHD prophylaxis, which included a calcineurin inhibitor and either steroids or mycophenolate mofetil. Maraviroc was started on day -3 and administered at a dose of approximately 300 mg/m2 orally twice daily until day +30 after stem cell infusion. On days 0 and day +10, samples for pharmacokinetic analysis were collected before the dose and 1, 2, 4, 6, 8, and 12 hours after maraviroc administration. Additional trough concentrations were collected on days +7, 14, and 21. Patients were followed until day +100 for acute GVHD. Functional blockade of CCR5 was assessed in a pharmacodynamic assay by flow cytometry. Thirteen patients, median age of 4 years (range, 2 to 11 years), were prospectively enrolled. Underlying diagnoses included a primary immune deficiency (n = 6), hemoglobinopathy (n = 4), metabolic disorder (n = 1), and bone marrow failure syndrome (n = 2). Patients received either a myeloablative preparative regimen (n = 7) or a reduced-intensity conditioning regimen (n = 6). Cyclosporine and methylprednisolone (n = 7) was the predominant GVHD prophylactic regimen, followed by tacrolimus and mycophenolate mofetil (n = 4) and tacrolimus and steroids (n = 2). Two formulations of maraviroc (150-mg tablets and 20-mg/mL solution) were used on study. Mean (± SD) area under the concentration-time curve from 0 to 12 hours was 4805 ± 3265 hour * ng/mL on day 0 and 5917 ± 4048 hour * ng/mL on day +10. Four patients developed grade 1 or 2 acute skin GVHD before day +100 and were successfully treated. Two patients developed grade 3 acute GI GVHD on days +23 and +24 after HSCT and both had discontinued maraviroc before development of GI GVHD. No adverse effects attributable to maraviroc were observed and administration by enteral tubes was well tolerated by children and accepted by parents. All evaluable patients demonstrated functional CCR5 blockade on day 0. Administration of maraviroc is feasible in most pediatric HSCT recipients with good safety and tolerability profile.

Short Course of Post-Transplantation Cyclophosphamide and Bortezomib for Graft-versus-Host Disease Prevention after Allogeneic Peripheral Blood Stem Cell Transplantation Is Feasible and Yields Favorable Results: A Phase I Study

An effective graft-versus-host disease (GVHD) preventative approach that preserves the graft-versus-tumor effect after allogeneic hematopoietic stem cell transplantation (HSCT) remains elusive. Standard GVHD prophylactic regimens suppress T cells indiscriminately and are suboptimal. Conversely, post-transplantation high-dose cyclophosphamide selectively destroys proliferating alloreactive T cells, allows the expansion of regulatory T cells, and induces long-lasting clonal deletion of intrathymic antihost T cells. It has been successfully used to prevent GVHD after allogeneic HSCT. Bortezomib has antitumor activity on a variety of hematological malignancies and exhibits a number of favorable immunomodulatory effects that include inhibition of dendritic cells. Therefore, an approach that combines post-transplantation cyclophosphamide and bortezomib seems attractive. Herein, we report the results of a phase I study examining the feasibility and safety of high-dose post-transplantation cyclophosphamide in combination with bortezomib in patients undergoing allogeneic peripheral blood HSCT from matched siblings or unrelated donors after reduced-intensity conditioning. Cyclophosphamide was given at a fixed dose (50 mg/kg on days +3 and +4). Bortezomib dose was started at .7 mg/m2, escalated up to 1.3 mg/m2, and was administered on days 0 and +3. Patients receiving grafts from unrelated donors also received rabbit antithymocyte globulin. The combination was well tolerated and allowed prompt engraftment in all patients. The incidences of acute GVHD grades II to IV and grades III and IV were 20% and 6.7%, respectively. With a median follow-up of 9.1 months (range, 4.3 to 26.7), treatment-related mortality was 13.5% with predicted 2-year disease-free survival and overall survival of 55.7% and 68%, respectively. The study suggests that the combination of post-transplantation cyclophosphamide and bortezomib is feasible and may offer an effective and practical GVHD prophylactic regimen. The combination, therefore, merits further examination.

Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on Graft-Versus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation

Objective: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic bone marrow transplantation (allo-BMT). While multipotent mesenchymal stromal cells (MSCs) demonstrate alloresponse in vitro and in vivo, they also have clinical applications toward prevention or treatment of GVHD. The aim of this study was to investigate the ability of MSCs to prevent or treat GVHD in a rat BMT model. Materials and Methods: The GVHD model was established by transplantation of Sprague Dawley rats’ bone marrow and spleen cells into lethally irradiated (950 cGy) SDxWistar rat recipients. A total of 49 rats were randomly assigned to 4 study and 3 control groups administered different GVHD prophylactic regimens including MSCs. After transplantation, clinical GVHD scores and survival status were monitored. Results: All irradiated and untreated control mice with GVHD died. MSCs inhibited lethal GVHD as efficiently as the standard GVHD prophylactic regimen. The gross and histopathological findings of GVHD and the ratio of CD4/CD8 expression decreased. The subgroup given MSCs displayed higher in vivo proportions of CD25+ T cells and plasma interleukin-2 levels as compared to conventional GVHD treatment after allo-BMT. Conclusion: Our results suggest that clinical use of MSCs in both prophylaxis against and treatment of established GVHD is effective. This study supports the use of MSCs in the prophylaxis and treatment of GVHD after allo-BMT; however, large scale studies are needed. Conflict of interest:None declared.

Effect of Antithymocyte Globulin Dosage for Prophylactic Gvhd On Epstein-Barr Virus Reactivation and Diseases in Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 4486 Background:Antithymocyte globulin (ATG) is one of the main risk factors for Epstein-Barr virus (EBV) reactivation and disease in allogeneic hematopoietic stem cell transplantation (allo-HSCT), however, whether there is a correlation between ATG dose and EBV reactivation is unsure. Aim of this single-center prospective study is to explore the relationship between ATG dose and EBV reactivation in allo-HSCT. Methods:Ninety-nine patients with hematologic malignancies underwent allo-HSCT and administration of ATG for graft versus host disease (GVHD) prophylaxis were enrolled in this study in Nanfang hospital from February 2008 to February 2012. Sixty-one patients were unrelated donor transplants, thirty-eight were HLA-mismatched related donor transplants. GVHD prophylactic regimen was cyclosporine A+Methotrexate+ATG. According to donors’ HLA matching, we chose three dosage groups of prophylactic ATG: low dose group with ATG dose of 5.0∼6.0mg/kg (n=28), medium dose group with ATG dose of 7.0∼8.0mg/kg (n=55), and high dose group with ATG doses of ≥10mg/kg (n=16). The levels EBV-DNA in plasma were regularly monitored by quantitative real-time polymerase chain reaction (RQ-PCR). Results:With a median follow-up of 21 (range, 1–50) months post allo-HSCT, the cumulative incidence of EBV viremia and EBV-associated diseases was 17.6% (5/28) and 17.6% (5/28) in low dose group, respectively, with 32.7% (18/55) and 29.1% (16/55) in medium dose group, respectively, with 50.0% (8/16) and 31.3% (5/16) in high dose group, respectively. There were statistical significances of the incidence (χ2□□9.555, P=0.008). Logistic regression models showed that ATG prophylaxis was one of the main risk factors for EBV infection (RR=16.728, P=0.000) while bivariate correlation analysis presented that the incidence of EBV reactivation was positively correlated with ATG prophylaxis dosage (rpearman = 0.452, P = 0.000). The cumulative incidence of high degree (II∼IV°) aGVHD was 35.7% (10/28) in low dose group, with 39.6% (21/53) in total and 50.0% (6/12) in relapsed leukemia with HLA-mismatched related transplantation in medium dose group, with 68.8% (11/16) in high dose group. There were not statistical significances of the incidence (χ2□□6.971, P=0.137). Conclusion:EBV reactivation might be positively correlated with ATG prophylaxis dosage. According to donors' HLA matching, reducing ATG prophylaxis dose appropriately could prevent EBV reactivation in allo-HSCT without increasing high degree aGVHD. Disclosures:No relevant conflicts of interest to declare.

Sirolimus-Containing Graft-Versus-Host Disease Prophylaxis and High- Resolution HLA Typing Improves the Outcome of Mismatched Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) from unrelated donors can cure a variety of hematologic diseases; however, decreased survival and graft versus host disease (GVHD) are associated with mismatches at HLA class I and class II loci. Beginning in 2004, we have employed a modified three-drug sirolimus-based GVHD prophylactic regimen (Antin JH et al., Blood 2003; 102:1601) for all patients undergoing unrelated donor allogeneic HSCT with any antigen or allele mismatch at HLA A, B, C, DR, or DQ in the GVHD direction. Outcomes of 22 patients undergoing mismatched unrelated donor (MMUD) allogeneic HSCT (sirolimus(+) group) were retrospectively reviewed in comparison to 14 patients receiving MMUD allografts between 2000–2004 who received a tacrolimus-based two-drug GVHD prophylactic regimen (sirolimus(−) group).Median age (45 vs. 51), disease status, donor gender, and type of transplant conditioning (reduced-intensity, 9 vs. 8) were similar between the two groups. Seven of 14 patients in the sirolimus (−) group received bone marrow grafts whereas all patients in the sirolimus + group received peripheral blood stem cells. Nine patients in the sirolimus – group had antigen-level typing at HLA A, B, C and DQ loci and allele level typing at HLA DRB1 and 5 patients had allele-level typing at all loci. Patients in the sirolimus+ group had allele-level typing at all loci. One subject was mismatched at HLA DRB1 and DQ at the allele level; the remaining subjects had a single allele or antigen mismatch. GVHD prophylaxis for the sirolimus(−) group included tacrolimus and mycophenolate (n=11) or tacrolimus and low-dose methotrexate x 3 doses (n=3). GVHD prophylaxis for the sirolimus (+) group included sirolimus, tacrolimus and post- transplant methotrexate for 2 or 3 doses, totaling 15mg/m2.Median follow-up for surviving patients was 1921 days (1781–2295) in the sirolimus-group and 447 days (53–1122) in the sirolimus(+) group. The day +100 cumulative incidence of grades II-IV and III-IV acute GVHD were, respectively, 71% and 43% in the sirolimus(−) group, and 14% and 5% in the sirolimus(+) groups (p=0.002; p=0.02). Kaplan-Meier estimates of the probability of chronic GVHD or late onset acute GVHD at two years were 51% in the sirolimus(–) group and 70% in the sirolimus(+) group.Graft failure occurred in 2 patients each in both groups and was successfully treated in two patients by retransplantation or pentostatin plus donor lymphocyte infusion. Adverse effects of sirolimus included 8 patients with renal dysfunction (36%), 4 with thrombotic microangiopathy (TMA) (18%), and 3 with dyslipidemia requiring medical therapy (14%). Deaths in the sirolimus(−) group were due to disease recurrence (n=3), graft failure (n=1), and GVHD (n=7). Deaths in the sirolimus(+) group were due to disease recurrence (n=2), TMA (n=1) and GVHD (n=4). The Kaplan-Meier estimates of disease free (DFS) and overall survival (OS) at 3 years were 21% and 21% for the sirolimus(−) group and 53% and 56% for the sirolimus(+) group. The Kaplan-Meier estimates of non-relapse mortality (NRM) at day +100 and day +365 were 22% and 50% for the sirolimus (−) group and 5% and 13% for the sirolimus(+) group.Summary: High-resolution HLA typing and GVHD prophylaxis with sirolimus, tacrolimus, and low-dose methotrexate in patients undergoing MMUD allogeneic HSCT is associated with low rates of severe acute GVHD and non-relapse mortality. Renal dysfunction and TMA appear more common with sirolimus, and, as in prior studies with this regimen, a lower rate of acute GVHD did not result in less chronic GVHD. Nonetheless, DFS and OS appear to compare favorably to recently reported registry data for MMUD allograft recipients (Lee SJ et al., Blood;110:4576), and a prospective trial has been initiated to further study this regimen in MMUD recipients.

Sirolimus and Mycophenolate Mofetil as Graft-Versus-Host Disease Prophylaxis in Myeloablative, Matched Related Donor Hematopoietic Cell Transplantation

We investigated a novel graft-versus-host disease (GVHD) prophylactic regimen, sirolimus and mycophenolate mofetil (MMF), in patients with advanced hematologic malignancies receiving myeloablative hematopoietic cell transplantation (HCT) from matched related donors (MRD). The sirolimus and MMF combination was chosen based on intriguing evidence of sirolimus, and less dramatically MMF, conserving the inhibition of T cell proliferation compared to cyclosporine (CSP) in an AGVHD murine model as well as multiple reports supporting sirolimus' preservative or augmentative effects on the T regulatory cell population (CD4+CD25+, FoxP3+) in animals and humans in comparison to cyclosporine (CSP) and a theoretical reduction in the incidence and severity of AGVHD. In addition, this regimen may cause less severe toxicities compared with standard methotrexate and/or calcineurin inhibitor (CI)-containing GVHD prophylactic regimens. Sirolimus was begun as an oral medication on day -3 with a 12 mg loading dose followed by 4 mgs daily, dose adjusted to maintain serum trough levels of 3–12 ng/ml. MMF was begun on D0 at 15 mg/kg IV every 12 hours. Mycophenolic acid (MPA) pharmacokinetics were conducted on days 2 and 21 post-HCT. Sirolimus and MMF were scheduled to be tapered simultaneously beginning 100 days post-HCT. FoxP3+ cell populations in the peripheral blood (PB) were analyzed before and after HCT in the patients receiving sirolimus and MMF GVHD prophylaxis as well as in 15 control patients with similar disease categories and preparative regimens receiving CI-containing GVHD prophylaxis. Based on pre-study stopping rules of expected grade II–IV AGVHD incidence, the trial was closed to accrual after enrollment of 11 patients. The median age of the 11 patients was 51 years (26–59). The diagnoses of the study patients included myelodysplasia (5), acute myelogenous leukemia (3) and nonhodgkin lymphoma (3). Seven patients received busulfan, etoposide and cyclophosphamide (CY); three patients received carmustine, etoposide and CY and one patient received total body irradiation, etoposide and CY as the preparative regimens. Sirolimus was discontinued in 4 patients due to concern for toxicity-related events including severe sinusoidal obstructive syndrome (1), altered mental status (1), portal vein thrombosis (1) and risk of poor wound healing after abdominal exploratory surgery (1) 9 days, 9 days, 61 days and 39 days post-HCT, respectively. Six of 11 patients developed grade II-IV AGVHD a median of 15.5 days post-HCT (range 11–25 days), 3 of the 6 with grade IV AGVHD. Two of the 3 patients with grade IV AGVHD required discontinuation of sirolimus 9 days post-HCT due to toxicity. Five of the 6 AGVHD patients had skin-only and one had liver, gut and skin involvement. All patients responded to AGVHD therapy and there were no AGVHD-related deaths. One patient died 54 days post-HCT due to complications of colonic ulceration believed related to oral MMF therapy. Two patients died of relapsed disease at 103 and 304 days post-HCT. At a median follow-up of 416 days (328–652 days), 8 of 11 patients were alive without disease, 5 with active chronic GVHD. The median MPA area under the curve (AUC) on day 2 was 12.8 mcg*hr/mL (6.4–17.4) and day 21 was 17.9 mcg*hr/mL (8–26.6). We measured a statistically significant increase in the percentage of CD4+FoxP3+ cells per total CD4+ cells in the sirolimus/MMF study group compared to the CI historical control group with a mean of the individual patient means over the first 100 days post-HCT of 16% versus 6%, respectively (p.0005). Despite the increased percentage of CD4+FoxP3+ cells per total CD4+ cells identified in the PB of the sirolimus/MMF cohort, the incidence of grade II-IV AGVHD was similar to the CI cohort (6 of 11 versus 7 of 15, respectively). However, all but one patient had skin-only AGVHD and there were no GVHD-related deaths in the sirolimus/MMF group. All patients requiring early withdrawal of sirolimus had received the busulfan-containing preparative regimen with two of these patients developing severe AGVHD. In a subsequent GVHD prophylaxis trial including sirolimus and MMF, the busulfan-containing preparative regimen will be eliminated to optimize sirolimus and MMF administration with the continued goal of reducing the incidence and severity of AGVHD.

Non-T-Cell-Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation for Children with Hematological Malignancies: Single-Center Pilot Study

Allogeneic hematopoietic stem cell transplantation is the only curative approach for a number of patients with hematological malignancies. For patients who do not have an HLA-identical related or unrelated donor, a related haploidentical transplantation is an alternative option. Recent advances in effective T-cell depletion of stem cells have significantly decreased graft-versus-host disease (GVHD) and early transplantationrelated mortality (TRM). However, the problems related to delayed immune reconstitution causing infectious complications and relapse remain, limiting the efficacy of haploidentical transplantation. Here, we report the results of ten children with hematological malignancies who received non-T-cell-depleted haploidentical hematopoietic stem cell transplantation. Malignancies included acute lymphoblastic leukemia (ALL, n = 6), acute myeloid leukemia (n = 2), chronic myeloid leukemia (n = 1) and Epstein Barr virus (EBV)-associated peripheral T cell lymphoma (n = 1). Median patient age was 5 years, and all patients presented with clinical and biologic features that indicated a very high risk of relapse with conventional chemotherapy. At the time of transplantation, seven patients were in remission, and two of these were urgent cases with primary graft failure of the first cord blood transplantation. The remaining three patients were not in remission. HLA disparity was two loci mismatches in one case and three loci mismatches in nine cases. The conditioning regimen consisted of the myeloablative method in five cases and reduced intensity conditioning in five cases. GVHD prophylaxis was conducted with tacrolimus and short-term methotrexate (sMTX) in the initial two cases and the remaining eight cases were given a combination of tacrolimus, sMTX and prednisolone (PSL). The stem cell source for the initial two cases was peripheral blood stem cells mobilized with G-CSF, and that for the remaining eight cases was bone marrow. Nine (90%) of the ten patients engrafted showed myeloid and platelet recovery on days +15 and +31 (median), respectively. All of these patients achieved full donor chimerism by day +30, and it persisted, except in one patient who relapsed. Acute GVHD of Grades II–IV and III–IV occurred in six (66.7%) and two (22.2%) patients, respectively, all of which responded to temporary augmentation of PSL. Chronic GVHD occurred in five (62.5%) patients, three of whom had extensive GVHD. Although one of these developed steroid refractory chronic GVHD, it was not difficult to control the symptoms of GVHD of the remaining patients. Infectious complications, including cytomegalovirus (CMV) antigenemia (n = 2), interstitial pneumonia associated with CMV (n = 1), temporary elevation of EBVDNA (n = 1), zoster (n = 1), invasive aspergillus infection (n = 1) and sepsis (n = 1), were observed. TRM occurred in two patients, and one patient with ALL relapsed on day +472 after transplantation. The Karnofsky performance scales of six of the eight survivors were 100%, and more than half of the evaluable patients were expected to discontinue immunosuppressive therapy within 2 years after transplantation. Because only one patient had relapsed by the time of the last follow-up, we believe that our GVHD prophylactic regimen suppresses the incidence of both acute and chronic GVHD to an acceptable level, while preserving the graft-versus-leukemia effect. Median follow-up of the eight patients who survived event-free was 34 months (range, 3 to 95 months). The 5-year probability of overall survival and event-free survival (EFS) were 77.6% and 66.6%, respectively. In the analysis of disease status at transplantation, encouraging results were obtained in patients who received the transplantation while in remission, with the rate of engraftment and 5-year probability of EFS at 100% and 80%, respectively. Although the number of patients in this series was small, the results described here indicate the feasibility of non-T-cell-depleted haploidentical stem cell transplantation for children with hematological malignancies who do not have an HLA-identical donor.

Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on Graft-Versus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation.

The major obstacle to successful bone marrow transplantation (BMT) is graft versus host disease (GVHD). Multipotent mesenchymal stromal cells (MSC) have shown immunosupressive effects. The aim of this study was to investigate the effect of MSC on the prevention of GVHD in a rat BMT model. We prepared rat GVHD model using allogeneic BMT+4x108 donor splenocyte infusion (DSI). Allogeneic BMT were performed from Spraque Dawley (SD) rats to female Wistar rats by applying 1x108 mononucleer cells (MNC) via tail vein. The recipient animals were conditioned with a myeloablative regimen consisted of 950 cGy total body irradiation prior to BMT. GVHD prophylactic regimen was performed 3 mg/kg/day cyclosporine-A (CsA)+0.25 mg/kg methotrexate (Mtx) IP +1., +3., +6. days. The rats were divided into three control group (CG) and four study group (SG) (n=7). CG-I; only allogeneic BMT+DSI (GVHD model), CG-II; only myeloablative regimen, CG-III; only inspection. GVHD model was performed in all SGs and then SG-I receiving only GVHD prophylactic regimen, SG-II receiving only MSC, SG-III receiving GVHD prophylactic regimen+MSC, SG-IV receiving MSC after GVHD findings developed. Gross (diarrhea, lose weighting, skin findings), histopathological, and immunophenotypical (CD4, CD8, CD25 and plasma IL-2 levels) examinations of GVHD following allogeneic BMT, and the survival analysis of all groups were performed. The survival following allogeneic BMT was significant longer (p<0.05) in SG-I, SG-II and SG-IV than in CG-I (only GVHD model). However, the survival of SG-III (both GVHD prophylactic regimen and MSC administration) was not significant longer than in CG-I (only GVHD model). Gross and histopathological findings of GVHD was significant lower (p<0.05) in SG-I and SG-II than in CG-I (only GVHD model). The expression of CD25 was showed significant increase (p<0.001) in all SGs and CG-I (only GVHD model) than in CG-III (healthy control). Plasma IL-2 levels were showed significant increase in SG-I (p=0.032), SG-II (p=0.018) and SG-IV (p=0.032) than in CG-I (healthy control). This increase was very prominent (p<0.001) in CG-I (only GVHD model) than in CG-III (healthy control). The ratio of CD4/CD8 expression was showed significant lower in SG-I (P=0.008) than in CG-III (healthy control). In contrast, this expression was showed significant increase in SG-II and SG-IV (p=0.014 for both) than in CG-III (healthy control). Finally, clinical use of MSC in both prophylactic and after GVHD developed was as effective as GVHD prophylactic regimen in preventing GVHD. However, MSC combined with GVHD prophylactic regimen was caused strong immunosuppression, and therefore this combination may results in early mortality. This study may form the basis for use of MSC in the prophylaxis and the treatment of GVHD. Our results are encouraging in the use of MSC in GVHD further to clinical trials.

Acute Graft Versus Host Disease (GVHD) Is Increased in Patients Receiving Cyclosporine/Cellcept as Frontline Prophylaxis Against GVHD in Pediatric Patients Undergoing Allogeneic Stem Cell Transplantation.

Traditionally, pediatric patients are prophylaxed against GvHD with cyclosporine (CSA) plus either methotrexate (Mtx) or methylprednisolone (Pred). Mtx worsens the severity of mucositis and renal insufficiency while pred causes muscle wasting, hypertension, hyperglycemia and increased infections. Cellcept (myophenolic acid) is a better tolerated, less toxic agent with synergistic immunosuppressive effects when combined with cyclosporine or tacrolimus. We explored its use in combination with cyclosporine in 63 pediatric patients (49% male, 74% Caucasian, 36% CMV+) undergoing allogeneic transplantation. Patients with both malignant (n=24) and non-malignant (n=39) conditions ranging in age from 2 weeks to 26 years were transplanted after myeloablative preparative regimens with either related bone marrow or cord blood (n=11) or unrelated cord blood (n=52), using cellcept and cyclosporine for GVHD prophylaxis. Cellcept and CSA were administered intravenously through the first 40–60 days post transplant using cellcept at a dose of 15mg/kg/dose IV q8H beginning on day -2 or -3 and CSA in traditional fashion targeting trough levels of 200–450 (infusion) and 150–350 with intermittent dosing. The patient then transitioned to oral therapy at the same dose and schedule until day 100–180 for cellcept and day 270 for cyclosporine and then tapered if no active GVHD was present. There were no acute toxicities attributable to cellcept observed in patients on IV or oral therapy. Despite the lack of steroids in the GVHD prophylactic regimen, all patients required treatment with antihypertensive therapy. Grossly, infection rates did not vary from those previously observed in conventionally treated patients at our center. The cumulative incidence (CINC) of neutrophil engraftment by day 42 was 79.4% (95% CI 64.7–91.2) which is comparable to previous reports. Two patients engrafted after a second unrelated cord blood transplant. Of the six patients who did not engraft, 3 died of infectious complications, two died of VOD and multi-system organ failure and one died of congenital pulmonary hypertension. Of the 59 patients evaluable for GVHD, follow-up ranged between 52 and 994 days (median 224 days). The CINC of acute GVHD grades II–IV by day 100 was 64.1% (95% CI 47.8–82.1) and the CINC of acute GVHD grades III– IV by day 100 was 6.3% (95% CI 0–17.0). Follow-up is too short to comment on the incidence of chronic GvHD, but to date, 5 of 14 patients followed for >100 days have developed mild chronic GvHD. The incidence of grade II acute GVHD was higher than that previously reported in pediatric patients receiving CSA/Pred while the incidence of grade III–IV GVHD was comparable. While this data is early, it appears that the incidence of acute Grade II GVHD is increased compared to previous series in patients treated with cellcept instead of Mtx (related bone marrow transplantation) or pred (unrelated cord blood transplantation). Engraftment in UCBT recipients was not compromised with the use of IV cellcept administered in the peritransplant period. Overall, despite the use of steroids to treat aGVHD, there was overall steroid sparing with this regimen. Additional patients will need to be tested with this regimen and longer follow-up is necessary to fully define the risks or benefits of this therapy.

Association between Regimen Related Toxicity and Acute-Graft-Versus-Host Disease.

Tissue injury resulting from preparative therapy for transplantation is integral to the development of acute graft-versus-host disease (GVHD) according to current theory. If toxicity to normal tissues is a critical factor in the pathophysiology of GVHD, greater degrees of regimen related toxicities should be associated with a higher incidence and greater severity of GVHD. We analyzed 438 patients who underwent allogeneic transplantation from related (n=360) or unrelated (78) donors and who survived > 100 days following transplantation. Patients had received preparative regimens of BuCy (n=340) or BuCyVP16 (n=98). Median age was 36 (range 4–66). There were 263 males and 175 females. This cohort survived a median of 35 months (range 3 months to 20 years).Sixty-eight of these patients had developed (Bearman) grade 3-4 regimen related toxicities. These patients had a 50% incidence of acute GVHD > grade II and a 26% incidence of developing GVHD ≤ grade II, compared to significantly lower incidences of 33% (P=.01) and 14% (P=.02) respectively in the group experiencing < grade 3 regimen related toxicity. Exclusion of patients whose GVHD prophylactic regimens were significantly altered because of toxicity did not significantly influence these results.This data suggest that patients who develop severe regimen related toxicity are significantly more likely to develop severe acute GVHD, supporting a potential pathophysiologic role for tissue injury in the development of acute GVHD.

CD31 mismatching affects marrow transplantation outcome

Graft-versus-host disease (GVHD) complicating allogeneic bone marrow transplantation (BMT) is often attributed to mismatched minor histocompatibility antigens (mHags), which are poorly defined in humans. CD31 is a candidate human mHag relevant to acute GVHD, but reports disagree about its level of significance, the role of HLA restriction, and the relative importance of different polymorphic codons within the molecule. We therefore examined in greater detail the impact of CD31-matching on BMT outcome in a prospective study from a single institution. Samples of recipient and donor DNA were collected pretransplantation for all patients receiving unmanipulated bone marrow from an HLA-identical sibling over a 45-month period at our institution. CD31 DNA typing of alleles at the 3 polymorphic codons 125 (L or V), 563 (N or S), and 670 (R or G) was performed for 118 patient-donor pairs plus 2 additional pairs who had codon 125 typing only. Donor-recipient CD31 nonidentity was tested for correlation with BMT clinical outcome measures of severe acute GVHD, chronic GVHD, relapse, and survival. Gene frequencies of approximately 0.5 for each allele at all 3 codons were comparable to previous reports. Because complete association was seen for 563N with 670G and for 563S with 670R, nonidentity for those codons was analyzed as a single genetic marker designated codon 563/670. Donor-recipient CD31 nonidentity was a significant risk factor for overall survival, both at codon 563/670 (hazard ratio [hr] = 2.58, P = .005) and at codon 125 (hr = 1.07, P = .036). Similar results held for disease-free survival. Nonidentity at codon 563/670 was also a significant risk factor (odds ratio [OR] = 11.15, P = .011) for severe (grades III, IV) versus no (grade 0) acute GVHD. Nonidentity at codon 125 posed less but still significant risk (OR = 9.30, P = .030). When the comparison group without severe acute GVHD was expanded to include grade I as well as grade 0 patients, the risk from CD31 nonidentity increased for both codon 563/670 (OR = 12.31, P = .010) and codon 125 (OR = 11.24, P = .011). CD31 nonidentity remained a significant independent risk factor for survival and for severe acute GVHD when tested in multivariate analysis with the covariates of adulthood, recipient-donor sex difference, ethnic group, disease, pretransplantation risk category, HLA-A2 type, B44-like types, and GVHD prophylactic regimen. CD31 nonidentity showed a trend but failed to achieve statistical significance as a risk factor for relapse and for chronic GVHD. In conclusion, donor-recipient CD31 nonidentity is a significant risk factor for survival and for severe acute GVHD in HLA-identical sibling BMT. The stronger associations with codon 563/670 suggest that polymorphism may be more important than the linked polymorphism at codon 125.

Cytomegalovirus seropositivity adversely influences outcome after T‐depleted unrelated donor transplant in patients with chronic myeloid leukaemia: the case for tailored graft‐versus‐host disease prophylaxis

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after haematopoietic stem cell transplantation from matched unrelated donors (MUD). The role of T-cell depletion (TCD) as a strategy to prevent GVHD is controversial because of the associated increased risk of leukaemic relapse, graft failure and delayed immune reconstitution. The demonstration that donor lymphocyte infusion (DLI) is effective salvage therapy if patients relapse after transplantation for chronic myeloid leukaemia (CML) prompted us to examine the proposal that TCD may be a form of GVHD prophylaxis particularly suited to this disease in patients undergoing MUD transplantation. We analysed the outcome of 106 consecutive patients with CML in first chronic phase who underwent MUD transplantation. Patients were conditioned with cyclophosphamide and total body irradiation (TBI), and received in vivo TCD, using CD52 monoclonal antibody, as GVHD prophylaxis. Donor lymphocytes were infused at the time of leukaemic relapse. The projected survival at 5 years for all patients was 52.6%. The probability of developing severe acute GVHD (grade 3 or 4) was 14.5%. The only significant predictor of overall survival in univariate and multivariate analysis was patient cytomegalovirus (CMV) serostatus: in CMV-negative patients survival at 5 years was 60% vs. 42% in CMV-positive patients (P = 0.006). The use of TCD was associated with an increased risk of relapse (62% probability at 5 years after transplant), but 80% of patients who received DLI achieved molecular remission that was durable in all but two cases. In vivo TCD, in conjunction with DLI at relapse, is a valuable GVHD prophylactic regimen in CMV-seronegative recipients of MUD allografts, but in CMV-seropositive patients this approach is associated with an increased non-relapse mortality. Consequently, GVHD prophylactic regimens in MUD transplantation should be tailored according to the patient and donor pretransplant characteristics.

Phase I/II trial of recombinant human granulocyte-macrophage colony- stimulating factor following allogeneic bone marrow transplantation

Forty-seven patients with hematologic neoplasia received recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) by daily 2-hour infusion following allogeneic bone marrow transplantation from HLA-identical sibling donors in a phase I-II dose-escalation trial. Dose levels ranged from 30 to 500 micrograms/m2/d. At doses at or below 250 micrograms/m2/d, toxicity felt to be caused by rhGM-CSF was negligible. However, three of five patients treated with 500 micrograms/m2/d had unacceptable side effects caused by rhGM-CSF. Two different graft-versus-host disease (GVHD) prophylactic regimens were administered. Twenty-seven evaluable patients were administered regimens that did not contain methotrexate (MTX) (Group I) and reached an absolute neutrophil count of 1,000/microL by a median of day 14. In contrast, 18 patients who received GVHD prophylactic regimens containing MTX (Group II) reached an absolute neutrophil count of 1,000/microL on a median of day 20. Patients in Group I had fewer febrile days and, of those discharged, had shorter initial hospitalizations than patients in Group II. The overall incidence of severe acute GVHD (grade 2 or greater) in the rhGM-CSF-treated patients was 28% and was similar to that in historical “good risk” patients who did not receive rhGM-CSF. These preliminary data suggest rhGM-CSF is unlikely to exacerbate GVHD in HLA-identical sibling donor transplants and indicate the need for randomized trials of rhGM-CSF in allogeneic marrow transplant patients.