Unrelated Transplantation Research Articles

Outcomes and Prognostic Factors of Second Allogeneic Hematopoietic Cell Transplantation in Patients with Acute Lymphoblastic Leukemia: A Study from the Adult Acute Lymphoblastic Leukemia Working Group of the Japanese Society for Transplantation and Cellular Therapy

Background Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option frequently used in patients with acute lymphoblastic leukemia (ALL). However, relapse after allo-HCT remains the main concern because it is associated with poor outcomes, with a long-term survival rate < 10 %. There is no standard treatment for patients with ALL who relapsed after allo-HCT. Second allo-HCT (allo-HCT2) is a curative treatment option for patients with ALL who relapsed after the first allo-HCT (allo-HCT1). However, data on allo-HCT2 in patients with ALL are limited, and the indications and outcomes of allo-HCT2 are unclear. Therefore, this study aimed to investigate the outcomes and prognostic factors of patients who underwent allo-HCT2 after ALL relapse. Methods This study included 425 adult patients with ALL who underwent allo-HCT2 after relapse between January 2001 and December 2020. The primary endpoint was 2-year overall survival (OS), which was evaluated using the log-rank test. Prognostic factors for the primary endpoint were evaluated using the multivariable Cox proportional hazards model. The variables considered in the Cox proportional hazards model are listed in the Table. The impacts of the candidate factors are shown as hazard ratios (HRs) and 95% confidence intervals (CIs). Additionally, we performed an analysis stratified by disease and disease status because Philadelphia chromosome (Ph)-positive B-ALL could affect the treatment and disease status at allo-HCT2 and the outcome. Results The median age at allo-HCT2 was 36 years (interquartile range; 27-48 years) and 220 patients (52%) were male. According to the Eastern Cooperative Oncology Group, 94 patients (22%) had a performance status of > 2. There were 235 patients (55%) with Ph-negative B-ALL, 122 patients (29%) with Ph-positive B-ALL, and 68 patients (16%) with T-ALL. Regarding disease status at allo-HCT2, 217 patients (51%) had any complete response (CR). One hundred forty-eight patients (35%) received myeloablative conditioning regimen. Ninety patients (21%) received related bone marrow (BM) or peripheral blood (PB) transplantation, 137 patients (32%) received unrelated BM or PB transplantation, and 198 patients (47%) received unrelated cord blood (CB) transplantation. The median observation time of survivor after allo-HCT2 was 22.3 (interquartile range; 6.3-82.5) months and 2-year OS was 28.0% (95% CI, 23.7-32.4). After stratified disease and disease status, the 2-year OS was 56.5% (95% CI, 44.6-66.8), 31.7% (95% CI, 24.0-39.7), 21.5% (95% CI, 10.6-34.9), and 13.1% (95% CI, 8.4-18.8) in any CR (Ph-positive B-ALL), any CR (other), active disease (Ph-positive B-ALL), and active disease (other), respectively ( P < 0.001) (Figure). In multivariable analysis, Ph-positive B-ALL (HR, 0.48; 95% CI, 0.36-0.65; P < 0.001), tacrolimus-based GVHD prophylaxis at allo-HCT2 (HR, 0.72; 95% CI, 0.54-0.95; P = 0.022) were identified as significantly positive prognostic factors for 2-year OS, while allo-HCT1 to allo-HCT2 < 12 months (HR, 1.38; 95% CI, 1.02-1.88; P = 0.038), relapse within 12 months after allo-HCT1 (HR, 1.37; 95% CI, 1.01-1.85; P = 0.043), ECOG PS > 2 (HR, 1.89; 95% CI, 1.44-2.49; P < 0.001), active disease at allo-HCT2 (HR, 1.88; 95% CI, 1.46-2.42; P < 0.001), unrelated BM or PB transplantation at allo-HCT2 (HR, 1.78; 95% CI, 1.22-2.59; P = 0.003), and CB transplantation at allo-HCT2 (HR, 1.54; 95% CI, 1.09-2.18; P = 0.013) were identified as significantly negative prognostic factors for 2-year OS. Conclusion In this study, we demonstrate that the 2-year OS was 28.0% in patients with ALL who underwent allo-HCT2 and identified the prognostic factors that may guide patients to benefit from allo-HCT2.

Post-Transplant Cyclophosphamide or ATG As Gvhd Prophylaxis in Mismatched Unrelated Stem Cell Transplantation? - a Registry Analysis By the EBMT Transplant Complications Working Party

There is an increased risk of GVHD and non-relapse mortality (NRM) after allogeneic stem cell transplantations (alloSCT) when mismatched unrelated donors (MMUD) are used. Prophylaxis with either rabbit anti-thymocyte globulin (rATG, also termed anti-T-lymphocyte globulin) or post-transplantation Cyclophosphamide (PTCy) is often used to reduce the risk of NRM and GVHD. However, it is currently impossible to give general recommendations regarding preference for rATG or PTCy since comparative evidence from larger data sets is lacking. To better understand the outcome of these GHVD prophylactic regimens, we analyzed outcomes of rATG vs. PTCy prophylaxis in adult patients with hematologic malignancies, who underwent peripheral blood alloSCT from 9/10 antigen MMUD between Jan 2018 and June 2021 in the EBMT database. We performed multivariate analyses using Cox cause-specific hazard models, adjusting for known risk factors, and variables that showed significant difference between the two comparison groups. Overall 2123 patient were included, treatment- and patient characteristics are shown in Table 1. Patients receiving PTCy (n=583) were younger and had undergone alloSCT more recently compared to patients receiving rATG (n=1540). Median follow up was 2.0 years (PTCy) and 2.4 years (rATG). Outcome graphs are shown in figure 1. All outcomes are given starting from AlloSCT (with 95% CI). NRM (fig 1A) was higher in the rATG arm (2-years NRM: PTCy 18% [14.6-21.6] vs. rATG 24.9% [22.7-27.3]; HR 0.74, p=0.03), Relapse incidence was slightly higher but not significant in the rATG arm (2-years RI: PTCy 22.9% [19.2 - 26.9] vs. rATG 26.2% [23.9 - 28.7]; HR 0.82, p=0.07), Overall survival (fig 1B) was better in the PTCy arm (2-years OS: PTCy 65.7% [61.2 - 69.9] vs. rATG 55.7% [52.9 - 58.3]; HR 0.77, p<0.001), same for Progression free survival (fig 1C) (2-years PFS: PTCy 59.1% [54.4 - 63.5] vs. rATG 48.8% [46.1 - 51.5]; HR 0.78, p=0.001). The incidences of chronic and acute GVHD were not significantly different between the groups; (2-years cGVHD: PTCy 31.7% [27.4 - 36] vs. rATG 30.3% [27.8 - 32.8]; HR 0.95, p=0.67. And 100-days aGVHD grades II-IV: PTCy 29.9% [25.9-34.1] vs. rATG 32.5% [30-34.9]; HR 0.83, p=0.11). In summary, we found significantly higher survival and lower NRM in recipients of peripheral blood alloSCTs from MMUD receiving PTCy as compared to rATG. The current analysis improves the evidence base for decision making on GVHD prophylaxis in MMUD alloSCT.

Lymphoid Malignancies in Shwachman-Diamond Syndrome: Incidence, Clinical Features, and Outcomes from the North American SDS Registry

Introduction: Many marrow failure conditions are associated with myeloid malignancy predisposition and a subset carry an increased risk of both myeloid and lymphoid malignancies. The increased risk of myeloid malignancy is known in Shwachman-Diamond syndrome (SDS), however, the risk of lymphoid malignancy in SDS has not been previously reported. Through the North American SDS Registry (SDSR), we identified patients with SDS diagnosed with lymphoma. We report the clinical features and outcomes in these patients as well as the incidence rates of lymphoma, MDS, and AML in this cohort. Methods: Patients with biallelic SBDS mutations enrolled on the SDSR were included for analysis. Age- and sex-specific incidence rates from the SEER Program (2016-2020) were multiplied by person-years of observation to obtain expected numbers of events. The crude rate per 100,000 (100,000*observed number of malignancies/person-years of follow-up) and the standardized incidence ratio (SIR) were calculated. Results: Out of a total of 217 patients with SDS with biallelic SBDS mutations in the SDSR, we identified 3 subjects who developed lymphoma. Case 1: A male patient with biallelic SBDS mutations (c.258 + 2T>C; c.653G>A, p.Arg218Gln) presented with dyspnea and was diagnosed with primary mediastinal B-cell lymphoma at age 16 years. Bone marrow exam was negative for lymphoma but revealed a new del20q clone. He received 6 cycles of R-CHOP. His course was complicated by febrile neutropenia, pleural effusion, deep vein thrombosis, and decreased cardiac ejection fraction requiring subsequent omission of doxorubicin and carvedilol initiation. He is in remission >3 years from diagnosis. Case 2: A male patient with biallelic SBDS mutations (c.258 + 2T>C; c.183_184TA>CT, p.Lys62*) was diagnosed at age 23 years with diffuse large B-cell lymphoma (DLBCL) of the distal stomach and duodenal bulb. Bone marrow exam was negative for malignancy but demonstrated an abnormal karyotype: 46,XY,t(11;16)(q23;q24), del(20)(q11.2q13.3)[9]/46,XY[11]. He received 5 cycles of R-CHOP therapy, which was complicated by febrile neutropenia and S. aureus sepsis. Four years later, he was diagnosed with relapsed DLBCL of the distal ileum and concurrent AML with 5q deletion. He achieved complete remission of both malignancies after 2 cycles of cyclophosphamide, vincristine, prednisone, obinutuzumab, azacitidine, and venetoclax. Therapy was complicated by B. cereus bacteremia with brain abscesses and endocarditis and C. difficile infection. He underwent a matched unrelated donor hematopoietic stem cell transplant and is in remission >180 days from transplant. Case 3: A male patient with biallelic SBDS mutations (c.258 + 2T>C; c.183_184TA>CT, p.Lys62*) was diagnosed with Stage IVB classical Hodgkin lymphoma at age 12 years after presenting with mediastinal syndrome. Bone marrow biopsy was negative for findings suggestive of MDS or malignancy, however, the initial PET-CT was consistent with medullary involvement. He initiated therapy with steroids, doxorubicin, vinblastine, dacarbazine, and brentuximab vedotin and received focal radiation. His course was complicated by recurrent febrile neutropenia, C. difficile colitis, and cellulitis. Due to infectious complications and delayed count recovery after 2 cycles, despite chemotherapy dose reductions, he was transitioned to brentuximab and nivolumab. He demonstrated complete response by Deauville criteria prior to cycle 5 of brentuximab and nivolumab. Among 217 patients with biallelic SBDS mutations in the SDSR, the median age was 12.8 years (range: 0.3 - 52.8). The observed number, rates, and SIR for lymphoma, MDS, and AML are given in Table 1. A high SIR indicates a higher incidence in the SDSR relative to the SEER population in 2016-2020. The observed rate of lymphoma was 28.5 times higher in SDS than the expected rate in the general population. Conclusions: The incidence of lymphoma among patients with SDS is increased relative to the general population but is lower than the incidence of MDS or AML. Increased toxicity with standard therapies was observed in these patients. Given the increased risk of MDS/AML in SDS, which may be further increased by antecedent chemotherapy, non-genotoxic therapies should be investigated for lymphoma in SDS. Our results show that SDS should be added to the list of marrow failure conditions predisposing to both myeloid and lymphoid malignancies.

Relapse Incidence Post Unrelated Allogeneic Stem Cell Transplantation with Post-Transplant Cyclophosphamide (PTCy) Versus Conventional Anti-Graft Versus Host Disease Prophylaxis in Patients with Acute Myeloid Leukemia: A Study on Behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation

Background: Recently, the BMT CTN 1703 phase III study compared post-transplant cyclophosphamide with tacrolimus with mycophenolate mofetil (PTCy/TAC/MMF) to TAC /methotrexate (MTX) as graft- versus-host disease (GVHD) prophylaxis post allogeneic transplantation (HSCT), demonstrating a lower incidence of severe acute (a) GVHD and chronic (c) GVHD and better GVHD-free, relapse-free survival (GRFS). The control arm did not include anti-thymocyte globulin (ATG), used in many centers for GVHD prophylaxis. Methods: The study aim was to compare PTCy with TAC or cyclosporine A (CSA) and MMF (PTCy/TAC or CSA & MMF) to ATG combined with TAC and MTX (ATG/TAC/MTX) in acute myeloid leukemia (AML) patients (pts) undergoing HSCT from matched siblings (MSD) or 9-10/10 unrelated donor (UD) in first complete remission (CR1). Statistical tests included a multivariate analysis (MVA) adjusting for potential confounding factors using a Cox proportional-hazards regression model for main outcomes. Results: 6050 pts met the inclusion criteria, 402 received PTCy/TAC or CSA & MMF and 5648 received ATG/TAC/MTX as GVHD prophylaxis. Median follow-up was 23.4 (IQR, 20.3-24.9) and 41.8 (IQR, 39.6-43.3) months (p<0.0001). The median year of the transplant was 2018 (2010-2020) and 2016 (2007-2020) (p<0.0001). Pts in the PTCy/TAC or CSA & MMF group were younger, with a median age of 48.7 (range 18-5.6) versus 51.5 (8-77.8) years (p=0.024). The diagnosis was de novo AML in 84.1% vs 85.3% and secondary (s) AML in 15.9% vs. 14.7% (p=0.49). The cytogenetic risk was categorized as intermediate (70.9% vs. 67.1%), adverse (22.2% vs. 25.7 %), and favorable (6.9% vs. 7.2%) for pts in the PTCy/TAC or CSA & MMF and ATG/TAC/MTX groups, respectively (p=0.35) (data missing for 2214 pts). Karnofsky performance status (KPS) did not differ between the groups. There was a higher frequency of pt cytomegalovirus (CMV) seropositivity and female (F) donor to male (M) pt combination in the PTCy/TAC or CSA & MMF versus the ATG/TAC/MTX groups, 77.8% vs. 71.8% (p=0.009) and 18.4% vs. 14.4% (p=0.029), respectively. More pts in the PTCy/TAC or CSA & MMF group received reduced intensity conditioning (RIC) 51.5% versus 41.1% in the ATG/TAC/MMT group, respectively (p<0001). Day 60 neutrophil engraftment (ANC >0.5 x 10 9/L) was 98.7% vs. 98.6% (p=0.84). Day 180 incidence of a GVHD grade II-IV and III-IV was 21.2% vs. 20.4% (p=0.92) and 8.1% vs. 6% (p=0.1), in pts receiving PTCy/TAC or CSA & MMF versus the ATG/Tac/MTX GVHD prophylaxis, respectively. The 2-year (y) total and extensive chronic (c) GVHD were 33.7% vs. 30% (p=0.09) and 10.7 % vs. 11.2% (p=0.81), respectively. GVHD was the cause of death in 11.6% vs. 13.9% of pts who died. In the MVA, both aGVHD (grade II-IV or III-IV) and cGVHD (total or extensive) did not differ between the groups with hazard ratios (HRs) =1.15 (95% CI 0.86-1.53, p=0.35), HR=0.87 (95% CI 0.56-1.34, p=0.52), HR=0.91 (95% CI 0.7-1.18, p=0. 47 and HR=1.51 (95% CI 0.96-2.36, p=0.074). Two-y NRM was significantly lower in pts that received PTCy/TAC or CSA & MMF versus ATG/TAC/MTX for GVHD prophylaxis, HR=1.57 (95% CI 1.07-2.3, p=0.022). Other HSCT outcome parameters did not differ between the groups. The HR for 2-y RI was 0.99 (95% CI 0.77-127, p=0. 93). The HRs for 2-y leukemia-free survival (LFS), overall survival (OS), and GRFS were HR=1.15 (95% CI 0.94-1.42, p<0.18), HR=1.18 (95% CI 0.94-1.49, p=0.16) and HR=1.12 (95% CI 0.93-1.36, p=0.22), respectively. Donor type and conditioning regimen were poor prognostic factors for grade II-IV, III-IV aGVHD, and total and extensive cGVHD. For cGVHD, additional poor prognostic factors were F donor to M pt combination and pt CMV seropositivity. Poor prognostic factors for LFS, OS, and GRFS were 9/10 UD, age (by 10 y), sAML, adverse-risk cytogenetics, lower KPS, and pt CMV seropositivity. For NRM, factors were the same apart from cytogenetics risk which was not a prognostic factor. In addition, time from diagnosis to HSCT was a prognostic factor for NRM and RI. Other poor prognostic factors for RI were lower KPS and pt CMV seropositivity. Conclusions: In this registry-based retrospective analysis, comparing PTCy in combination with TAC or CSA and MMF to ATG in combination with TAC and MTX as GVHD prophylaxis, we observed a similar incidence and severity of both aGVHD and cGVHD. NRM was significantly lower with the PTCy-based GVHD prophylaxis, while all other transplant outcome parameters were similar.

SMC4 Loss of Function Is a New Cause of Inherited Bone Marrow Failure

Inherited bone marrow failure syndromes (IBMFS) are a rare, heterogenous group of syndromes that are characterized by cytopenias, bone marrow hypocellularity, and in many cases an increased risk of transformation to myeloid malignancy. Presentation with severe pancytopenias at birth is reported but occurs very rarely, and more often neonatal pancytopenia is attributed to congenital and acquired infections or other acquired causes. We present the case of a preterm neonate with pancytopenia from birth and bone marrow failure of no known infectious, other acquired, or genetic etiology. Antenatal findings were notable for non-immune hydrops fetalis at 30 weeks of gestation prompting delivery. The initial laboratory evaluation revealed a normocytic, reticulocytopenic, severe anemia; leukopenia with an absolute neutrophil count of 500; and severe thrombocytopenia. Viral testing, including parvovirus, rubella, herpes simplex virus, cytomegalovirus, adenovirus, and enterovirus, was negative. Additional testing, including a chromosomal microarray, mitochondrial genome sequencing, chromosome stress testing, neonatal alloimmune thrombocytopenia panel, and telomere length analysis, was non-diagnostic. Bone marrow biopsy showed a remarkably hypoplastic marrow without evidence of dysplasia or cytogenetic abnormalities. The proband underwent matched unrelated donor hematopoietic stem cell transplant, and died approximately one month later from post-transplantation complications including hepatic veno-occlusive disease, thrombotic microangiopathy, CNS and pulmonary hemorrhages, pseudomonal sepsis, and multi-organ dysfunction. Autopsy was significant for microscopic pancreatic atrophy with lipomatous hypertrophy and osteopenia. The proband and parents underwent clinical exome testing which was non-diagnostic and then consented to research reanalysis of the exome data. Research reanalysis identified the infant was compound heterozygous for predicted deleterious variants in the Structural Maintenance of Chromosomes 4 gene (SMC4), inherited in trans from each unaffected parent (c.2478_2479ins10 splice site variant predicted to alter splicing and c.T591G; p.Y197X). Germline variants in SMC4 have not been reported as a cause of human disease; however, somatic variants are observed in several cancers including in acute myeloid leukemia (AML). SMC4 is a highly conserved protein that forms a heterodimer with Structural Maintenance of Chromosomes 2 (SMC2) in the condensin II protein complex. This complex is important for chromosome condensation, cell division, and DNA repair. Within hematopoietic tissue, SMC4 expression is higher in hematopoietic stem and progenitor cells when compared with mature lineage cells. Given the predicted loss of function from both SMC4 variants, we performed RNAseq on proband and control fibroblasts. This showed a roughly 10-fold decrease in total SMC4 transcript levels (Figure 1A). A significant proportion of the transcript present was comprised of an aberrantly spliced transcript characterized by exon 15 skipping. We therefore hypothesized that loss of SMC4 expression leads to a cell intrinsic defect in hematopoiesis. To test this hypothesis, we used CRISPR-Cas9 gene editing to generate knockout of SMC4 in human cord blood CD34+ hematopoietic stem/progenitor cells (HSPCs) before culturing cells in an all-colony forming unit (CFU) assay. Using two independent small guide RNAs, we were able to achieve approximately 80% knock-down. After 14 days in culture, we observed a striking decrease in CFUs when compared with controls, though the composition of colony types was relatively unchanged (Figure 1B). Using next-generation sequencing, we observed a significant decrease in remaining SMC4 genome edits at the end of the culture period, suggesting a relative disadvantage of SMC4-edited cells for survival and/or proliferation. In summary, we describe a preterm infant with congenital bone marrow failure without an identifiable cause and link this phenotype to biallelic loss-of-function variants in SMC4. This is the first study identifying SMC4 as a novel gene that causes inherited bone marrow failure, and further experiments are underway to validate this finding.

Hold your Horses! An Uncommon Cause of Bacteremia in HCT Recipients

Abstract Background Diagnosis and management of bloodstream infections due to uncommon microorganisms in immunocompromised hosts (ICH) are challenging. Removing central venous catheters (CVC) and combination antimicrobial therapy may be necessary. Case A 2-year-old female with Mucopolysaccharidosis type I on day +125 after matched unrelated donor allogenic transplant presented with 2 days of decreased activity. Two days prior, she had completed 4 weeks of ciprofloxacin and clarithromycin for treatment of Mycobacterium cosmeticum catheter-related bacteremia (CRBSI). The CVC was removed. The patient lived in a rural area but had no direct animal exposure, except for an indoor vaccinated dog. Household contacts were healthy and immunized. The patient was febrile and tachycardic with no evidence of infection around CVL or G-tube. Laboratory results showed pancytopenia and elevated C-reactive protein Blood cultures grew diphtheroid gram-positive bacilli (GPB) and gram-positive cocci (GPC) in clusters, the later identified as S. epidermidis. The GPB could not be identified from the liquid media and was sent to an outside lab. The report was corrected to GPC after growing on solid medium culture. The patient was started on meropenem and vancomycin and the CVC was removed. The GPB was identified as Rhodococcus equi. A chest CT showed new ground glass nodules in the left lower lobe. The patient defervesced and was discharged on a 10-day course of oral linezolid for S. epidermidis, clarithromycin and levofloxacin. She completed one month of clarithromycin and levofloxacin. Chest CT at end of treatment confirmed resolution. Discussion and conclusions Rhodococcus equi is a pleomorphic gram-positive bacteria that presents as cocci in solid media and as a diphtheroid gram-positive rod in liquid media. Horses are the primary hosts, but most herbivores are fecal carriers. Transmission occurs through inhalation of dust from contaminated soil. R.equi frequently presents as pulmonary infection with cavitated nodules. Over half of ICH develop secondary bacteremia. Extrapulmonary infection secondary to dissemination of pulmonary disease can occur but isolated extrapulmonary disease is rare. R.equi can be easily dismissed as a contaminant requiring a high level of suspicion and communication with lab personnel. When isolated from an extrapulmonary location, chest imagining is essential to guide treatment. Treatment should combine two antibiotics, at least one of them with intracellular activity, with fluoroquinolones and macrolides among the preferred options. For device-related infections, removal is recommended. Treatment duration for uncomplicated infections is 10 to 14 days, but cavitary lesions require longer treatment based on the resolution of lesions

Dynamics of Immune Reconstitution after Unrelated Allogeneic Transplantation with Post-Transplantation Cyclophosphamide Compared to Classical Immunosuppression with ATG

Introduction: Post-transplant cyclophosphamide (PTCY), in hematopoietic stem cell transplantation (HSCT), is responsible for the success of haploidentical stem cell transplantation and is also being used in matched transplants such as unrelated transplants, with promising outcomes in graft versus host disease (GVHD). However, we lack data on immune reconstitution with this type of immunosuppression, especially in unrelated transplantation. Immune reconstitution is directly involved with post-transplant morbidity and mortality, being related to GVHD, relapses and infections, such as cytomegalovirus (CMV). Objectives: To evaluate global and CMV-specific immune reconstitution after unrelated donor HSCT, comparing the use of thymoglobulin (ATG) or PTCY protocol. Methods: We prospectively included patients undergoing first unrelated donor HSCT with ATG, methotrexate and tacrolimus or PTCY (50mg/Kg D+3 and D+4), mycophenolate mofetil (MMF) and tacrolimus with patient and/or donor CMV IgG positive. Immune reconstitution analyzes were performed by flow cytometry and CMV-specific recovery by ELISPot technique on days +30, +60, +90 and +180 post HSCT. Results: Thirty-six patients were included in this study, 20 in the ATG group and 16 in the PTCY group (table 1). Only 2 patients were 9x10 match, and the others were 10x10 match. All patients were submitted to preemptive strategy of CMV treatment, without the use of letermovir. Regarding clinical outcomes, neutrophilic engraftment was earlier with ATG (median of 12 days), compared to PTCY (median of 16 days) (p=0.0001). There was no difference between the groups in terms of length of hospital stay (median 33 days in both groups), CMV reactivation (70% in ATG and 75% PTCY, p=0.98), acute GVHD grades II to IV (p=0.26) or grades III-IV (p=0.56), and chronic GVHD (p=0.45), as well as relapse (p=0.3), non relapse mortality (p=0.77) and overall survival (p=0.4). Regarding immune reconstitution we observed an initial expansion of monocytes and NK lymphocytes in both groups and a higher increase in CD8+ T lymphocytes was evidenced, with an inversion of the CD4/CD8 ratio (CD4 14.9% vs 15.5% p=0.9 and CD8 49.7% vs 54.5%p=0.5 in ATG and PTCY respectively). We identified a CD4+/CD8+ double-positive T cell (DP) population, which was higher in PTCY group compared to ATG on days +30 (3.5% vs 10.2%, p=0.009), +90 (3.1% vs 12% p= 0.01) and +180 (5.2% vs 13%, p=0.007, in ATG and PTCY respectively ). On day +30 there were higher proportion of T cells (19.9% vs 9.6% p=0.004), B cells (0.76% vs 0.2%, p=0.006) and NK cells (19.2% vs 8.6% p=0.006) in ATG group compared to PTCY and monocytes were higher in PTCY (31.7 vs 44.5%, p=0.02). On day +180 the proportion of B cell were higher in PTCY (6.1% vs 16.6% p=0.014) and the NK cells were higher in the ATG (22.4% vs 14.7% p=0.021) (Figure 1), with higher CD56dim population in ATG group (46% vs 33.6% p=0.0079), while in PTCY there were a higher NK population CD56high on day +60 (40.3% vs 61%, p=0.014). We identified a peripheral expansion of a CD56negCD16posNKG2Dpos NK cell population, which was higher in the PTCY group on day +180 (3.7% vs 13.2% p=0.017). Regarding CMV-specific immune response, we found no difference between the groups on days +30 (5 vs 5 spots/10 4 PBMC p=0.41), +60 (10 vs 6 spots/10 4 PBMC p=0.38), +90 (13 vs 4 spots/10 4PBMC p=0.33) or +180 (7 vs 6 spots/10 4 PBMC p=0.57). Conclusions: In the context of unrelated HSCT, we found no difference in CMV-specific immune response with the use of PTCY or ATG protocol. Post-transplant immunosuppression with PTCY compared to ATG protocol is related to lower levels of T cells, NK cells and B cells on day +30 and later neutrophil engraftment, but with a higher proportion of double positive T cells on days +30, +90 and +180 and a higher proportion of B cells on day +180. At 6 months after transplantation, a proportionately lower number of NK cells, but higher proportion of CD56negCd16posNKG2D NK population was observed in the PTCY group.

Comparison between Post-Transplant Cyclophosphamide Use and Conventional Prophylaxis of Graft-Versus-Host Disease: A Retrospective Study

Introduction: In order to modify the immune response balance between donor and recipient in the context of haploidentical transplantation, the use of post-transplant cyclophosphamide (PTCy), which reduces the population of recipient alloreactive T cells, has become the preferred strategy for graft-versus-host disease (GVHD) prophylaxis. Recently, this approach has also been tested and validated in HLA-matched related or unrelated transplants, as well as in unrelated transplants with HLA mismatch. However, high doses of PTCy can cause cardiac toxicity, hemorrhagic cystitis, and potentially increase the incidence of viral infections. Therefore, comparative studies are needed to evaluate the outcomes of PTCy use compared to other conventional GVHD prophylaxis strategies. Objective: To evaluate the outcomes of patients undergoing allogeneic peripheral blood stem cell transplantation (PBSCT) and to compare the use of post-transplant cyclophosphamide with other GVHD prophylaxis strategies. Patients and Methods: Retrospective analysis of consecutive patients transplanted between March 2014 and March 2023 at two transplant centers. The median follow-up was 24 months (range: 3-116 months). Results: A total of 118 adult patients undergoing PBSCT were included. There were 70 HLA-matched related donors, 28 HLA-matched unrelated donors, and 20 unrelated donors with one HLA mismatch. The median age was 52 years (range: 18 to 80), with a majority being male (51%). The most common diagnoses were acute myeloid leukemia (37%), acute lymphoblastic leukemia (18%), and myelodysplastic syndrome (18%). The disease risk index (DRI) was considered low or intermediate in 53% of cases, high in 31%, and very high in 16%. The majority of patients (76%) had a low risk of transplant-related complications (HCT-CI). Most patients received reduced-intensity conditioning regimens (78%), and the majority (72%) received some sort of T-cell depletion, with 36 (31%) patients receiving PTCy and 49 (41%) receiving ATG. No significant differences were found between the groups (PTCy vs. non-PTCy) in overall survival at 24 months (65% vs. 61%, p = 0.71, respectively), disease-free survival at 24 months (55% vs. 53%, p = 0.76), non-relapse mortality at 24 months (21% vs. 23%, p = 0.84), relapse rate at 24 months (24% vs. 24%, p = 0.61), neutrophil engraftment at 30 days (89% vs. 93%, p = 0.49), and incidence of grade II-IV acute GVHD at 100 days (31% vs. 29%, p = 0.99), grade III-IV acute GVHD at 100 days (6% vs. 12%, p = 0.22), chronic GVHD at 24 months (42% vs. 50%, p = 0.51), and moderate/severe chronic GVHD at 24 months (19% vs. 23%, p = 0.66). There was also no difference in the incidence of infections, including viral infections and cytomegalovirus reactivation. Conclusions: The use of PTCy demonstrated similar clinical outcomes to other GVHD prophylaxis strategies, without an increased risk of infections or mortality. Therefore, PTCy can be considered a viable, safe, and cost-effective option for GVHD prophylaxis in the context of HLA-matched and HLA-mismatched transplants.

Large-Scale Post-Transplant TCR Deep Sequencing Reveals a Major T Cell Diversity Bottleneck with Post-Transplant Cyclophosphamide with Implications for Both Efficacy and Toxicity: Results of the BMT CTN 1801 Study

Background: BMT CTN 1703 was a Phase 3 trial comparing Tac/MTX (n = 217) to post-transplant cyclophosphamide (PT-Cy)/Tac/MMF (n = 214) GVHD prophylaxis after reduced intensity conditioning unrelated donor hematopoietic cell transplant (HCT). PT-Cy significantly improved GVHD-free Relapse-Free Survival (Bolanos-Meade et al NEJM 2023). However overall survival was not significantly different and the # of Grade 2 infections was higher with PT-Cy. To investigate its biologic underpinnings 1703 was linked to a mechanistic study, BMT CTN 1801, which co-enrolled 324 pts (159 CNI/MTX 165 PT-Cy). The resulting dataset enabled an analysis of post-HCT T cell reconstitution at an unprecedented level of detail. Methods: Samples were collected from the HCT infusion and on D+7, 14, 28, 63, 98, 180, 270, 1- and 2-yr. We extracted DNA (2,225 samples) and performed Adaptive Biotech β-strand T Cell Receptor (TCR) Immunosequencing (TCR-Seq). We profiled 44,077 (median) TCRs/sample resulting in 215,155,719 T cells analyzed. We evaluated multiple TCR diversity measures longitudinally and before/after cGVHD and infections. Each measure evaluates a specific aspect of TCR diversity, including Simpsons Clonality (focuses on the most expanded clones) Diversity Slope (mid-range clones) Richness (both mid-range and less-expanded clones) and Singletons (the TCR-Seq equivalent of naïve T cells). This analysis was robust to the full range (1000-50,000) of down-sampled TCRs sequenced. Results: We identified major differences in TCR reconstitution in Tac/MTX vs PT-Cy that begin early and persist through 2 yr post-HCT. These resulted in a significantly less diverse TCR repertoire with PT-Cy with substantial implications for its efficacy and toxicity. The most salient differences are: (1) PT-Cy caused significant in-vivo T cell depletion early post-HCT (% T cells/total WBC at D+28: 8.2% (Tac/MTX) vs 2.2% (PT-Cy) p=2.2x10 -23). (2) The remaining donor T cells in PT-Cy undergo substantial clonal expansion ( Fig 1) resulting in significantly constrained TCR diversity measured by Clonality, Slope, Richness, Singletons (not shown) and Singletons/Richness ( Fig 2) which began early (D+14) and persisted for 2 yr. (3) Linked T cell depletion and clonal expansion resulted in a TCR diversity bottleneck after PT-Cy ( Fig 1). Due to the bottleneck, while the fraction of T cells originating from the transplant infusion at =/> 1 yr was similar between arms (18% vs 24%, p=0.7) there were significantly fewer Singleton TCRs in PT-Cy, suggesting a reduction in naïve T cells ( Fig 2), an outcome that has critical consequences for both cGVHD and infection. Thus while cGVHD onset with Tac/MTX was linked to a significant drop in TCR diversity due to singleton clonal expansion (median log-2 fold change in singletons with Tac/MTX = -0.25 p = 0.01) this drop was not observed with PT-Cy (log-2 fold change = -0.05, p = 0.1). Along with the significantly decreased # singletons at all time-points in PT-Cy ( Fig 2) this suggests that while cGVHD after Tac/MTX is linked to substantial naïve T cell expansion, the same is not true with PT-Cy, identifying a novel mechanism for cGVHD control with PT-Cy. The substantially fewer singletons in PT-Cy vs Tac/MTX may also increase the risk of Grade 2+ infections. T cell responses to new infections rely on antigen recognition/effector maturation of naïve (singleton TCR) T cells. After infection, the TCR repertoire became less diverse for both Tac/MTX and PT-Cy (log-2-fold singleton change -0.17 and -1.2 p = 0.0006 and 0.01) indicating that singletons in both cohorts were capable of expanding to pathogens. However the markedly decreased # singletons in PT-Cy at all timepoints was associated with significantly fewer singletons prior to infection (mean 3979 at 5K read-depth for Tac/MTX vs 2820 for PT-Cy p<0.001). This singleton deficit would place PT-Cy patients at higher risk of missing the critical mass of TCRs required to respond effectively to infections, consistent with their increased rate of Gr 2 infections. Conclusions: We performed longitudinal TCR-Seq on >200 million T cells from 324 HCT patients, representing the largest post-HCT TCR-Seq analysis in the history of the field, and enabling novel insights into TCR dynamics. These data identify a previously unappreciated global TCR diversity bottleneck with PT-Cy that simultaneously protects patients from cGVHD and places them at higher risk of infectious complications.

Integrative Single-Cell Tracking of Clonal Evolution, Leukemia Phenotypes and Donor Engraftment Following Allogeneic Stem Cell Transplant Using Mitochondrial DNA Mutations

Mitochondrial DNA (mtDNA) mutations can distinguish cells from unrelated individuals and their spectrum per patient sample can evolve following therapeutic bottlenecks. As such, these natural barcodes can potentially enable integrated single cell tracking of chimeric cellular populations and clonal evolution following unrelated allogeneic hematopoietic stem cell transplantation (HSCT). However, the feasibility of mtDNA-based donor-recipient deconvolution or identification of leukemia-specific phenotypes and the extent of co-evolution with somatic mutations remains unknown. If confirmed, post-transplant monitoring using mtDNA mutations could enable detection of early post-HSCT relapse and provide qualitative information on leukemia phenotypes, clonal evolution, or residual donor engraftment via a combined single cell assay. To investigate the extent of co-evolution of somatic nuclear mutations with mtDNA mutations, we generated 33,796 single cell DNA sequencing profiles (Tapestri) from 3 sample pairs of circulating chronic lymphocytic leukemia (CLL) collected before and after HSCT. The samples were characterized using a primer panel (n=67 nuclear and 21 mtDNA) targeting single nucleotide variants previously identified by whole-exome sequencing. We detected 8 mtDNA mutations that defined CLL subclones together with a median of 14 (8-19) somatic mutations from these cases. Clonal shifts defined by somatic mutations discernable pre- or post-HSCT tracked with analogous changes in mtDNA mutations and included clonal replacement, selection of a resistant subclone or skewing of CLL subclones. We thus provide clear evidence of co-segregation between somatic nuclear and mtDNA mutations. To assess the sensitivity of mtDNA-based donor-recipient deconvolution, we performed an in-silico titration and spiked 1-1,000 single cell mtDNA profiles (mtscATAC-seq) from one CLL case ('CLL4') into 7,579 profiles from another ('CLL5'). Overall, we recovered 98.3-100% of CLL4, identifying up to 1 in 7,579 cells (0.13%). Only 123 cells (1%) failed deconvolution, due to insufficient mtDNA coverage. In an in-vitro titration of expanded T cells (donor1) with peripheral blood mononuclear cells (donor2) at ratios of 1:3, 1:30 and 1:300, we recovered 1,449 of 5,345 (27%), 150 of 4,262 (3.5%) and 14 of 4,381 (0.3%) T cells of donor1. In sum, with current sequencing throughput, mtDNA-based donor-recipient deconvolution was assessed to have a sensitivity of ~0.1%. To determine if mtDNA-based deconvolution can track early AML relapse and identify leukemia phenotypes, we analyzed peripheral blood and bone marrow of 4 patients with disease recurrence 87-185 days post-HSCT and subsequent reinstatement of remission 105-184 days after tapering of immunosuppression (IST). We obtained 19,143 ATAC with select antigen profiling by sequencing (ASAP-seq) profiles at incipient relapse (pre-IST) and following response to IST (post-IST). Donor-recipient deconvolution detected recipient-derived cells in progenitor, erythroid and monocytic populations pre-IST but no recipient-derived cells post-IST, consistent with elimination of AML. Overall, chromatin accessibility of donor- and recipient-derived cells was very similar, reflecting that AML recapitulates hematopoietic differentiation states. Nevertheless, we observed differences in accessibility of genes such as IL1B, CD36, chemokine or homeobox protein genes in recipient- compared to donor-derived cells. Together, mtDNA-based donor-recipient deconvolution reflected clinical responses and identified leukemia-specific chromatin accessibility profiles. Finally, we tracked clonal evolution and donor-derived hematopoiesis in two non-responder cases of overt AML relapse following 3-10 cycles of decitabine and ipilimumab (NCT02890329). We defined 12 and 7 mtDNA-based AML subclones and observed contraction and expansion of subclones during treatment. Donor-derived non-T cells were exceedingly rare (12 of 10,708 [0.1%] and 34 of 5,863 [0.6%]), mainly of erythroid origin (12 and 23 cells) and disappeared during treatment. Thus, mtDNA mutations can monitor donor- and recipient-derived hematopoiesis and resolve short-term changes in AML subpopulations during therapy. As sequencing throughput improves, we envision single cell-based post-transplant monitoring as a powerful approach for guiding clinical decision making.

Interindividual HLA Evolutionary Divergence in Single HLA-Mismatched Unrelated Donor Hematopoietic Cell Transplantation for Malignant Hematological Disorders: A Report on Behalf of the Cellular Therapy and Immunobiology Working Party of the EBMT

In allogeneic hematopoietic cell transplantation (allo-HCT), alloreactive T-cell responses (graft versus leukemia - GvL and graft versus host disease - GvHD) are mediated by the interaction between recipient antigens and donor lymphocytes via human leukocyte antigen (HLA) molecules. HLA evolutionary divergence (HED) is a metric recently implemented as a functional proxy for the structural variability between two homologous HLA alleles at their peptide binding sites, mirroring quantitative differences in pathogen and cancer-related immunopeptidomes ( A). 1 Here, we hypothesize that differences in and between donor and recipient HEDs at given loci may modulate the direction and the intensity of alloreactivity, informing on post-transplant outcomes. We tested this hypothesis in onco-hematologic patients undergoing allo-HCT from a single HLA-A, -B, -C, -DRB1, or -DQB1 (9/10) mismatched unrelated donor (MMUD) at EBMT centers. We included patients with selected hematological malignancies, receiving a thymoglobulin-based T cell depletion, for whom complete clinical and HLA information was available in the EBMT database. We deployed individual locus-specific recipient/donor HED (HED-R, HED-D) metrics as previously described 1, and conceptualized two novel interindividual HED scores accounting for functional differences at the mismatched HLA loci ( A). The latter were considered as: 1) DeltaHED, quantifying the relative directional difference between HED-D and HED-R and informing on differences in immunopeptidome sizes, potentially capturing the direction and the intensity of the alloreactive response (GvH or HvG); 2) HEDmismatch, describing the HED of the mismatched locus as a proxy for the qualitative differences in immunopeptidomes between donor and recipient ( B). Using multivariable Cox regression (cause-specific) models adjusted for relevant clinical variables, we investigated the associations of HED-R, DeltaHED and HEDmismatch, analyzed as continuous variables, with relapse free survival (RFS), overall survival (OS), non-relapse mortality (NRM), relapse and grade II-IV acute and chronic GvHD. For each outcome and in each mismatched subgroup, we built separate models for these immunogenetic parameters and checked for proportional hazards assumption (PHA). To account for multiple comparisons, only p-values <0.01 were considered significant. Overall, 4639 adult patients, with available HLA data, received a 9/10 MMUD allo-HCT between 2010 and 2019 for acute leukemia (N=3853, 83%), myelodysplastic syndromes or myeloproliferative neoplasms (N=786, 17%). Median recipient age was 53.5 (IQR 40.2-61.9) with a male/female ratio of 1.28, whilst median donor age was 33.1 (IQR 24.5-40). Majority of patients (90%) received peripheral blood stem cells and were mismatched with their donors for locus A (33%), followed by C (21%), B (15%), DQB1 (14%) and DRB1 (6%). When analyzing the impact of HED-related parameters in each model, we observed that most recurrent associations with outcomes occurred with DRB1-related metrics. In the DRB1-mismatched subgroup (N=276), a time-varying association was found for HEDmismatch of DRB1 locus with higher HEDmismatch leading to worse RFS [HR:1.25 at t=0, (95% CI 1.1-1.42), p<0.001] and lower OS [HR:1.23 at t=0, (95%CI 1.08-1.41), p=0.002]. The strength of this associations declined over time for both endpoints (respectively RFS: HR:0.91, 95% CI 0.86-0.97, p=0.005 and OS: HR:0.93, 95% CI 0.88-0.98, p=0.013). The (directional) DeltaHED DRB1 measure instead did not display any impact on outcomes. In the same subgroup, higher HED-R in the locus C showed also a time-varying association with NRM at transplant [HR:1.89, (95% CI 1.29-2.85), p=0.002], progressively declining over time (HR:0.77, 95% CI 0.65-0.92, p=0.003). Furthermore, DRB1 HED-R modulated the probability of survival in B-mismatched subgroup, being higher scores associated with reduced OS [HR:1.03, (95%CI 1.01-1.06), p=0.009]. Here, we provide an immunogenetic benchmark to explore donor-recipient interactions in MMUD allo-HCT, demonstrating how different HLA configurations within the mismatched loci, in particular for DRB1 genes, can influence post-transplant outcomes. Given the peculiar associations observed, HED may have a role in driving early GvL effects, informing on future donor selection strategies in high-risk patients.

Early Prognostic Biomarkers of Non-Relapse Mortality in Allogeneic Mismatched Unrelated Hematopoietic Cell Transplantation

Introduction A major challenge in allogeneic hematopoietic cell transplantation (HCT) has been advancing outcomes for individuals lacking a fully matched donor who require a mismatched unrelated donor (MMUD) HCT. Recently, the 15-MMUD study demonstrated encouraging results for post-transplant cyclophosphamide (PT-Cy)-based prophylaxis in MMUD recipients (Shaw, Jimenez, JCO 2021). Our retrospective analysis confirmed improvement in MMUD survival with PT-Cy compared to historical anti-thymocyte globulin (ATG)/calcineurin-based regimens (Jimenez, Blood Adv 2022). Elevations of plasma biomarkers such as Stimulation-2 (ST2) have demonstrated increased risk of non-relapse mortality (NRM) and GVHD, however these have not been validated in the MMUD setting. Here, we carried out a pilot study to determine if biomarker analysis of ST2, Chemokine ligand-9 (CXCL-9), and Regenerating islet-derived 3-α (REG3α) at Day 14 (D14) and 30 (D30) can identify patients at higher risk for NRM after MMUD HCT. Methods Patients with banked samples and underwent MMUD HCT with either ATG or PT-Cy as GVHD prophylaxis at the University of Miami between 2016 - 2020 were included. Enzyme-linked immunosorbent assays for ST2, CXCL-9, and REG3α were performed in batches on cryopreserved plasma. Descriptive statistics of demographic variables were compared using chi-squared, Fisher's exact, or Wilcoxon rank sum tests. ST2 values were treated as continuous variables, and CXCL-9 was dichotomized due to frequent values below the lower limit of detection. Cumulative incidences of NRM were analyzed with relapse as a competing risk. Time-dependent receiver operating curves (ROC) were constructed and Area Under the ROC Curve (AUC) calculated. Youden's index was used to select optimum thresholds for each biomarker. Results We included 47 patients (ATG: 23; PT-Cy: 24). The PT-Cy group had more bone marrow graft representation (50% vs 22%, p = 0.04) and more donors mismatched at >1 HLA-locus (29% vs 0%, p<0.01). Median follow up was 746 days (range 44-1968). Point estimate of overall survival at 2 years was 35% (95% CI 20-61) for ATG and 71% (95% CI 55-92) for PT-Cy (p=0.02). Median overall survival was estimated at 413 days among ATG patients and could not be estimated for PT-Cy patients. The cumulative incidence of NRM at 2-years was 30% (95% CI 13-51%) for ATG and 17% (95% CI 5-35%) for PT-Cy (p=0.03). Among all patients, ST2 was prognostic for NRM using time-varying covariates including combined D14 and D30 (D14+30) with a HR of 2.14 (95% CI 1.04-4.39, p = 0.04). The D14+30 ROC to predict NRM at 1-year using ST2 showed an AUC of 0.83 (95% CI 0.66-1). D+14 thresholded ST2 value maximizing Youden's index of ≥79.89 ng/mL was discriminative for 1-year NRM (p=0.002) (Fig. 1). Additionally, combination of CXCL-9 to ST2 could optimize the prognostic value when compared to ST2 alone: HR at D14+D30: 3.82 (95% CI 1.01-14.47, p=0.048) with an improved AUC of 0.9 (95% CI 0.79-1) (Fig. 2). REG3α did not significantly add to the AUC. Although limited by sample size, we explored the association by GVHD prophylaxis (ATG or PT-Cy). First, in the ATG group, the D14 ST2 value that maximized thresholded Youden's index of 61.39 ng/mL was predictive of 1-year NRM (p=0.04). Similarly, the D+30 ST2 values of 148.06 ng/mL was predictive of NRM (p<0.001). The AUC for prediction of 1-year NRM for Second, in the PT-Cy group, elevations of D30 ST2 were prognostic of 1-year NRM with HR 15.05 (95% CI 1.77-27.83, p=0.01). Consequently, the AUC for prediction of 1-year NRM for D30 ST2 was 0.92 (95% CI 0.77-1) and for ST2 + CXCL-9 was 0.96 (95% CI 0.86-1). Conclusions This pilot study suggests that after MMUD HCT, D14 and D30 biomarkers are prognostic for NRM across GVHD prophylaxis platforms. Importantly, we find that ST2 maintains its prognostic capacity in the increasingly popular PT-Cy group. This supplements what we previously reported in the haploidentical and matched unrelated donor PT-Cy setting (Kanakry, Haematologica 2017). Biomarker combination ST2 + CXCL-9 showed improved AUC over ST2 alone, and the composite D14+30 timepoint may have additive utility over a single timepoint. While the optimal time for biomarker collection is unknown, our data suggests D14 had value for ATG-treated patients, whereas D30 may be more important in the PT-Cy-treated group. A validation cohort is being assembled for PT-Cy based MMUD HCT.

The Impact of Post-Transplant Cyclophosphamide (PTCy) with or without MMF in the Prevention of De Novo and Progressive / Relapsing Chronic Graft-Versus-Host Disease after HLA-Matched Donor Transplant

Background: We previously showed that PTCy/Tac ± MMF is associated with a significant reduction in the rate of chronic graft-versus-host disease (cGvHD) as compared to Tac/MTX after HLA-matched sibling (MSD) but not after HLA-matched unrelated (MUD) donor stem cell transplant (SCT). Notably, ATG was used with Tac/MTX in MUD but not in MSD. [ Transplant Cell Ther. 2022;28(10):695] We aimed to further elucidate the epidemiology of cGvHD with the use of PTCy/Tac by comparing the impact of Tac/MTX vs PTCy/Tac ± MMF on de novo and progressive/relapsing cGvHD. Methods: Recipients of a MSD (Tac/MTX no ATG, or PTCy-based) or MUD (Tac/MTX with ATG, or PTCy-based) peripheral blood SCT between 2015-21 at our institution were eligible. To evaluate the impact of prophylaxis on the incidence of de novo and progressive/relapsing cGvHD, we performed two separate landmark analyses starting 3 months (mo) post-SCT among patients who had not and those who had been diagnosed with acute GvHD (aGvHD) within 3 mo post-SCT, respectively. Patients who died or experienced relapse of malignancy (n=135) or were diagnosed with cGvHD within 3 mo post-SCT (n=13) were excluded from the landmark analyses. There was no difference in the rate of early death/relapse or cGvHD by prophylaxis. Results: 1040 patients met the eligibility criteria. Median follow-up in survivors was 28 mo (IQRT: 18, 50). The cumulative incidence (CumInc) of cGvHD in the Tac/MTX, PTCy/Tac without MMF, and PTC/Tac with MMF groups was 39% (34-45, reference), 20% (14-29, p<0.001) and 20% (13-30, p=0.003) respectively in MSD; and 23% (18-30, reference), 17% (12-26; p=0.2), and 19% (14-26; p=0.6) respectively in MUD. Consistent with our previous findings, within the PTCy/Tac group, MMF was not associated with the overall rate of cGvHD in MSD (HR=1.2, 95% CI 0.6-2.1, p=0.6) or MUD (HR=1.3, 95% CI 0.7-2.2, p=0.4). However, in the current study, stratified analyses performed to evaluate the impact of prophylaxis on the risk of de novo vs progressive/relapsing cGvHD revealed a potential role for MMF in the development of de novo cGvHD. Subsequent results are presented separately for the de novo and progressive/relapsing cGvHD risk cohorts (Table). The de novo cGvHD risk cohort included patients (N=442) who had not been diagnosed with aGvHD within 3 mo post-SCT. AML / MDS (60%) and CML / MPD (16%) were the most common diagnoses. 124 cases of cGvHD were diagnosed 3 -36 mo post-SCT with a CumInc of32% (27-37). In multivariate analysis (MVA), compared to Tac/MTX, the use of PTCy/Tac without MMF (HR=0.3, 95% CI 0.2-0.6, p<0.001) was associated with a significant reduction in the incidence of cGvHD. Such reduction was not observed (Figure) with PTCy/Tac with MMF (HR vs Tac/MTX=1.0, 95% CI 0.6-1.5, p=0.9). Additional independent predictors were myeloablative conditioning (HR=1.9, 95% CI 1.3-2.9, p=0.001) and MSD (HR=1.5, 95% CI 1.2-2.6, p=0.003). Notably, progression-free survival (PFS) was similar in PTCy/Tac/MMF (univariate HR: 0.8, 95% CI 0.5-1.4, p=0.4) vs PTCy/Tac. The progressive/relapsing cGvHD risk cohort included patients (N=450) who had been diagnosed with grade 1 (27%), 2 (59%) or 3-4 (14%) aGvHD within 3 mo post-SCT. AML / MDS (64%) and CML / MPD (14%) were the most common diagnoses. 109 cases of cGvHD were diagnosed 3 -36 mo post-SCT with a CumInc of 28% (24-33). In MVA analysis, the impact of prophylaxis differed by donor type. As compared with Tac/MTX, PTCy/Tac ± MMF was associated with significantly lower rate of progressive/relapsing cGVHD in MSD (HR=0.2, 95% CI 0.1-0.4, p<0.001) but not in MUD (HR=0.6, 95% CI 0.4-1.1, p=0.09). This may be related to the use of ATG with Tac/MTX in MUD which was not used in MSD. HCT-CI ≥3 was the only other significant predictor of cGvHD (HR=0.6, 0.4-0.9, p=0.03). PFS was similar in PTCy/Tac ± MMF vs Tac/MTX in MSD (univariate HR: 0.8, 95% CI 0.5-1.3, p=0.3) or MUD (univariate HR: 0.8, 95% CI 0.5-1.2, p=0.3). Conclusions: PTCy/Tac ± MMF prophylaxis slows the development of progressive/relapsing cGvHD in MSD vs (Tac/MTX no ATG), but not in MUD (vs Tac/MTX with ATG). The addition of MMF to PTCy/Tac (vs PTCy/Tac) has no significant impact on the risk of progressive/relapsing cGVHD in MSD or MUD; but is associated with a higher risk of de novo cGvHD in MSD and MUD. Our data suggest that multi-center studies are warranted to determine whether MMF should be excluded from cGvHD prevention regimens in the HLA-matched donor setting with PTCy- based GvHD prophylaxis.

ATG or Post-Transplant Cyclophosphamide to Prevent Gvhd in Matched Unrelated Stem Cell Transplantation? - a Registry Analysis By the EBMT Transplant Complications Working Party

T There is a considerable risk of GVHD and non-relapse mortality (NRM) after allogeneic stem cell transplantations (alloSCT) when matched unrelated donors (MUD) are used. Prophylaxis with either rabbit anti-thymocyte globulin (rATG, also termed anti-T-lymphocyte globulin) or post-transplantation Cyclophosphamide (PTCy) is in standard clinical use. The unresolved question is whether to prefer PTCy or rATG in this setting. So far, two randomized trials, several retrospective studies and a meta-analysis comparing PTCy to rATG in MUD alloSCT gave contradictory results. Current data from large registry cohorts has not been published. To improve the evidence base, we analyzed outcomes of rATG vs. PTCy prophylaxis in adult patients with hematologic malignancies undergoing peripheral blood alloSCT from 10/10 antigen MUD between Jan 2018 and June 2021 in the EBMT database. We performed multivariate analyses using Cox cause-specific hazard models, adjusting for known risk factors, and variables that showed significant difference between the two comparison groups. Overall 8764 patients were included, treatment- and patient characteristics are shown in Table 1. Patients receiving PTCy (n=1039) were younger, had undergone alloSCT more recently, and had a lower disease risk compared to patients receiving rATG (n=7725). Median follow up was 2.1 years in both groups. Outcomes graphs are shown in figure 1. All outcomes are given starting from alloSCT (with 95% CI): NRM (fig 1A) was higher in the rATG arm (2-years NRM: PTCy 12.4% [10.2 - 14.7] vs. rATG 16.1% [15.3 - 17]; HR 0.72, p=0.016), same for Relapse incidence, (2-years RI: PTCy 22.8% [19.9 - 25.8] vs. rATG 26.6% [25.5 - 27.7]; HR 0.87, p=0.046), Overall survival (fig 1B) was better in the PTCy arm (2-years OS: PTCy 73.1% [70 - 76] vs. rATG 65.9% [64.7 - 67.1]; HR 0.82, p=0.001), same for Progression free survival (fig 1C) (2-years PFS: PTCy 64.9% [61.4 - 68.1] vs. rATG 57.2% [56 - 58.5]; HR 0.83, p<0.001), Chronic GVHD was lower in the PTCy arm (2-years cGVHD: PTCy 28.4% [25.2 - 31.7] vs. rATG 31.4% [30.3 - 32.6]; HR 0.77, p=0.012), whereas acute GVHD grades II-IV was not significantly different between the two arms (100-day aGVHD: PTCy 24.1% [21.3 - 27] vs. rATG 26.5% [25.5 - 27.6]; HR 0.85, p=0.11) In conclusion, we found a lower NRM, lower incidence of chronic GVHD as well as higher survival outcomes in adult recipients of peripheral blood alloSCTs from MUD receiving PTCy compared to rATG. The current analysis improves the evidence basis for decision making on GVHD prophylaxis in MUD alloSCT.

The Area Under Tacrolimus Concentrations on Day 14-28 Is the Independent Predictor of the Development of Chronic Gvhd in Patients without Severe Acute Gvhd after Unrelated Allogeneic HSCT

Introduction: In unrelated allogeneic hematopoietic stem cell transplantation (UR-SCT), chronic graft-versus-host disease (GVHD) is a critical complication that affects not only patients' quality of life but also post-transplant outcome. Acute GVHD is a major risk factor for the subsequent development of chronic GVHD. The combination of tacrolimus and short-term methotrexate (TAC+sMTX) has been widely used as a GVHD prophylaxis for UR-SCT in Japan. Previous reports have shown that tacrolimus blood concentrations early after transplantation are associated with the development of acute GVHD, but not with that of chronic GVHD. On the other hand, among UR-SCT recipients who did not develop severe acute GVHD, the relationship between tacrolimus blood concentrations and the development of chronic GVHD remains unclear. Methods: To examine the association between tacrolimus blood concentrations and post-transplant outcomes, the area under the tacrolimus concentration on day 0-14 (AUTC 0-14) and day 14-28 (AUTC 14-28) was calculated simply as the sum of the area of the trapezoid for the corresponding period ( Figure). Tacrolimus was initiated by continuous intravenous administration from day -1 in all patients, and no patients were switched to oral administration prior to day 28. Short-term methotrexate was administered at 10 mg/m 2 on day 1 and 7 mg/m 2 on day 3, day 6, and day 11. Among all 151 adult patients with hematopoietic malignancies who underwent first UR-SCT with GVHD prophylaxis of TAC+sMTX between 2010 and 2022 at the Jikei University Hospital, the following were excluded from the analysis: developing grade III-to-IV acute GVHD, using prednisolone or methyl-prednisolone ≥0.5 mg/kg within day 100, engraftment failure, death before day 100, and relapse before day 100. Results: A total of 68 patients were included in the analysis. Median age was 50.5 (range, 16-68) years. Twenty-seven (40%) patients had acute myeloid leukemia, and 18 (27%) had acute lymphoblastic leukemia. Fifty-five (81%) were in complete remission/chronic phase/early stage. The donor source consisted of 42 (62%) bone marrow transplantation (BMT), 20 (29%) cord blood transplantation (CBT), and 6 (9%) peripheral blood stem cell transplantation (PBSCT). BMT recipients transplanted from 22 (52%) HLA 8/8 matched donor, 19 (45%) HLA 7/8 matched donor, and one (3%) HLA 6/8 matched donor. CBT recipients transplanted from 15 (75%) HLA 4/6 matched donor and 5 (25%) HLA 5/6 matched donor. PBSCT recipients transplanted from 5 (83%) HLA 8/8 matched donor and one (17%) HLA 7/8 matched donor. Fifteen (22%) patients were administered additional low-dose anti-thymocyte globulin (ATG) as GVHD prophylaxis. Fifteen male patients transplanted from female donors (22%). Thirty-nine (57%) patients received myeloablative conditioning regimen. The median time from UR-SCT to neutrophil engraftment was 19 days (range, 11-34). Regarding acute GVHD, 46 patients (68%) developed none, 13 patients (19%) developed grade I, and 9 patients (13%) developed grade II. The median observation period for all 68 patients was 48.2 months (4.0 years). The 4-year cumulative incidence (CI) of chronic GVHD was 51%, of which 24% were moderate-to-severe. The CI of moderate-to-severe chronic GVHD was not associated with age, disease status, donor source, HLA incompatibility, ATG use, female to male, intensity of conditioning regimen, and grade I-to-II acute GVHD development. AUTC 14-28 <200 was the only risk factor (AUTC 14-28<200: 32% vs AUTC 14-28≥200: 11%; p=0.047). Multivariate analysis also showed that AUTC 14-28 <200 was the independent factor for development of moderate-to-severe chronic GVHD ( p=0.04). In all 68 patients, 4-year CI of relapse was 20%, 4-year CI of non-relapse mortality was 10%, 4-year GVHD-free, relapse-free, survival (GRFS) was 50%, and 4-year Overall survival was 80%, all of which did not differ between AUTC 14-28 <200 and AUTC 14-28 ≥200 groups. Conclusion: In UR-SCT, our data showed that AUTC 14-28 <200 may be an independent risk factor for developing moderate-to-severe chronic GVHD, even in patients who did not develop severe acute GVHD.

Post-Transplant Cyclophosphamide Eliminates Disparity in GvHD-Free, Relapse-Free Survival and Overall Survival between 8/8 Matched and 7/8 Mismatched Unrelated Donor Hematopoietic Cell Transplantation in Adults with Hematologic Malignancies

Background: Post-transplant cyclophosphamide (PTCy) has become a standard of care for graft-versus-host disease (GvHD) prophylaxis in recipients of human leukocyte antigen (HLA)-matched and mismatched unrelated donor (MMUD) allografts, generating new opportunities for successful hematopoietic cell transplantation (HCT) regardless of patient ancestry. The National Marrow Donor Program (NMDP)-sponsored 15-MMUD study, which used PTCy-based GvHD prophylaxis (PMID 3390526), demonstrated encouraging overall survival (OS) in adult patients receiving MMUD bone marrow grafts matched at ≤7/8 HLA alleles. Currently, the follow-up ACCESS study (NCT04904588) will determine if similar outcomes can be achieved using MMUD peripheral blood stem cell allografts. As part of its 5-year strategic plan, the NMDP aims to determine the extent of differences in GvHD-free, relapse-free survival (GRFS) between 8/8 and 7/8 URD allografts using PTCy and seeks to develop approaches over time to close the presumed gap in URD outcomes. Methods: The primary objective was to compare GRFS between 8/8 and 7/8 URD HCT using the Center for International Blood & Marrow Transplant Research (CIBMTR) database. After analyzing primary results, secondary objectives were added to compare GRFS, OS, and other clinical outcomes among 8/8 URD, 7/8 URD, and haploidentical-related donor (Haplo) HCT. The study included adult patients (age≥18y) with hematologic malignancies (ALL, AML, MDS) undergoing first URD or Haplo HCT between 2017-2020 using PTCy-based GvHD prophylaxis. Kaplan-Meier curves were used to estimate probability of GRFS and OS. Cox regression examined the independent effect of HLA match controlling for other clinical factors on outcomes referenced to 8/8 URD HCT. A priori clinically selected factors considered as possible confounders in the regression model included disease risk index, comorbidity risk, pre-HCT Karnofsky score, conditioning regimen intensity, graft source, donor/recipient CMV serostatus, year of HCT, donor/recipient age and race/ethnicity. Final models were stratified by factors violating the proportional hazards assumption. Factors were eliminated in a backwards fashion if p-values were >0.10 unless inclusion affected the hazard ratio (HR) of HLA match by more than 10%. Interactions were tested against a p-value of <0.01. Results: Table 1 shows demographics for the three patient cohorts: Haplo (n=2772), 8/8 URD (N=1517), and 7/8 MMUD (N=540). Notable differences among the cohorts included: 8/8 URD recipients had younger, predominantly Caucasian donors and were more likely to receive myeloablative conditioning. Haplo recipients had more donor/recipient CMV seropositivity and received more reduced intensity conditioning. Otherwise patient characteristics were similar. Contrary to our expectations, multivariable regression analysis (MVA) showed no significant differences in either GRFS or OS between recipients of 8/8 and 7/8 URD HCT at 1, 2 and 3-years post-HCT ( Figure 1). Outcomes, including relapse and NRM, between 8/8 and 7/8 URD HCT were similar irrespective of conditioning intensity. However, 8/8 URD HCT associated with lower two-year moderate/severe chronic GvHD than 7/8 URD [1.30 (1.00-1.68), p=0.049]. We next compared GRFS and OS among 8/8 and 7/8 URD and Haplo at 3y after HCT. On MVA, GRFS [HR 1.15 (1.04-1.26), p=0.005] and OS [HR 1.15 (1.03-1.29), p=0.013] were significantly better in 8/8 URD compared to Haplo HCT. Differences between 8/8 URD and Haplo appeared to be driven by significantly higher NRM [HR 1.46 (1.22-1.74), p<0.001)] and two-year moderate/severe chronic GvHD [HR 1.28 (1.07-1.54), p=0.007] in Haplo. No significant differences were seen in GRFS or OS at 3 years when comparing 7/8 URD to Haplo. Causes of death among donor types were similar with primary disease relapse/progression, infection and organ failure being the most frequent. Conclusions: Use of PTCy-based GvHD prophylaxis seems to have resolved previously observed differences in GRFS and OS between 8/8 and 7/8 URD HCT. HCT using an 8/8 URD HCT, if available, continues to be the best option for patients lacking a matched sibling donor. Further studies comparing Haplo to 7/8 URD HCT are warranted, but will require larger numbers of 7/8 URD HCT. Both mismatched donor sources substantially increase access to HCT for patients regardless of their ancestry.

Outcomes after Bone Marrow Versus Peripheral Blood Stem Cell Matched Unrelated Donor Hematopoietic Stem Cell Transplantation

Background: Matched unrelated donors (MUD) have become an acceptable alternative donor for patients requiring potentially curative hematopoietic stem cell transplantation (HSCT) and lacking a matched sibling donor. Patients may undergo a bone marrow (BM) or peripheral blood stem cell (PBSC) graft, with previous studies showing similar survival outcomes. However, BM grafts have shown a lower incidence of chronic graft-versus-host disease (GVHD) but a higher relapse rate and slower engraftment. We aimed to compare the outcomes of BM versus PBSC in patients undergoing MUD HSCT. Methods: For this retrospective single-center study, we included all (n=276) adult MUD HSCT recipients at the University of Kansas Medical Center who had at least one year of follow-up (August 2016 to July 2021). We conducted a bivariate analysis using chi-squared and ANOVA tests. The Kaplan-Meier and regression analyses were also performed. Data analyses were conducted using SPSS version 28. Statistical significance was considered at p<0.05. Results: Our study included 276 MUD patients including 110 PBSC (40%) and 166 BM (60%) grafts. The median age was 55 (range 21-77) years, and 158 patients (57%) were males. Eighteen patients (6%) were ethnic minorities, and 89 (32%) patients had a Karnofsky performance status of ≥90%. 118 (43%) patients had a hematopoietic cell transplantation-specific comorbidity index of 3 or higher. Hematologic diseases included acute myeloid leukemia (n=119, 43%), myeloid disorders (n=77, 28%), acute lymphoblastic leukemia (ALL) (n=35, 13%), Lymphoma (n=15, 5%), and others (n=30, 11%). Sixty-two percent of patients were in complete remission at the time of transplantation, and patients not in remission included myelodysplastic syndrome, myelofibrosis, and lymphoma patients. Reduced-intensity conditioning (RIC, n=162, 58%) and myeloablative conditioning (MAC) (n=114, 41%) were used. The median follow-up was 55 (21-73) months. Overall survival (OS) was not reached in either BM or PBSC MUD HSCT group, while a median disease-free survival of 45 and 49 months was noted in PBSC and BM groups respectively (p=0.915). We observed similar rates of relapse (25% vs 27%, p=0.413), one-year overall survival (75% vs 72%, p=0.679), one-year disease-free survival (63% vs 62%, p=1.00), and one-year non-relapsed morality (NRM, 23% vs 21%, p=0.779) after PBSC or BM graft. Bone marrow graft recipients had delayed engraftment of neutrophils (20 days vs 16 days, p<0.001) and platelets (>20 K/uL: 24 days vs 16 days, p<0.001; >50 K/uL: 28 days vs 18 days, p=0.006) compared to PBSC recipients. Bone marrow graft recipients had similar rates of grade II-IV acute GVHD (50% vs 52%, p=0.714) and grade III-IV GVHD (12% vs 11%, p=0.847), but lower rates of 1-year chronic GVHD (39 % vs 55%, p=0.014) compared to PBSC recipients. In the subgroup analysis stratified by conditioning intensity, faster engraftment of neutrophils (median 16 vs 21 days, p<0.001) and platelets (>20 K/uL: 18 days vs 27 days, p<0.001; >50 K/uL: 18 days vs 33 days, p<0.001) was observed with PBSC as compared to BM graft in myeloablative transplant recipients, while there was no statistically significant association with the rates of acute or chronic GVHD, NRM, Relapse, DFS, and OS after a PBSC or BM graft. Among the RIC transplant recipients, faster neutrophil engraftment (mean 17 vs 20 days, p<0.001) and higher 1-year extensive chronic GVHD (52% vs 32%, p=0.015) were observed with PBSC compared to BM graft whereas no statistically significant difference was noted in platelet engraftment, acute GVHD, NRM, Relapse, DFS, and OS after a BM or PBSC graft. Conclusions: Allogeneic matched unrelated donor hematopoietic stem cell transplant recipients had similar rates of overall and disease-free survival, relapse, non-relapse mortality, and acute GVHD after a bone marrow or peripheral blood stem cell graft; however, delayed neutrophil and platelet engraftment with a lower incidence of chronic GVHD was noted after a bone marrow graft compared to peripheral blood stem cell graft.

Reverse Autologous Peripheral Blood Stem Cell Transplantation after Allogeneic Transplantation to Restore Immune Function and Pancytopenia

Despite advances in multiple myeloma treatment the disease remains challenging to treat with repeated relapses in most. For high-risk younger patients, allogeneic transplants are occasionally performed. While curative in some, complications can include the development of opportunistic infections and secondary malignancies. Furthermore, patients relapsing after an allogeneic transplant may have poor bone marrow reserve limiting the ability to use subsequent therapies. We describe two patients who underwent a “reverse” autologous peripheral blood stem cell rescue using stem cells banked prior to allogeneic transplantation. The outcomes were successful and led to full engraftment and correction of their immunodeficiencies and pancytopenia respectively. Case 1: A 37-year-old woman was diagnosed with multiple myeloma during routine testing for an in-vitro fertilization assessment. She had ISS stage 2 disease and was treated with CyBorD, followed by an autologous stem cell transplantation. Lenalidomide plus dexamethasone was used as maintenance. She relapsed after just one year then progressed through single agent carfilzomib and then bendamustine. After responding to 2 cycles of DCEP she had a 10/10 matched unrelated donor CD34-selected allogeneic transplant. The patient was EBV positive, but the donor was not. Four months post-transplant she developed tonsillar PTLD with a rising EBV DNA titer and was treated with rituximab. Her CD4 count was 46. Her multiple myeloma relapsed thereafter necessitating treatment with daratumumab. She then developed rituximab-resistant monomorphic PTLD which then responded to brentuximab-vedotin and donor lymphocyte infusions. The multiple myeloma relapsed again and was treated with daratumumab + pomalidomide + dexamethasone. After a rising EBV DNA titer, brentuximab-vedotin was restarted but the lymphoma progressed again with a 5 x 3 cm anterior mediastinal mass biopsy proven to be monomorphic PTLD. R-CHOP was given for 4 cycles but resulted in only a partial response. Due to subsequent rapidly symptomatic and progressive disease a decision was made to use high-dose melphalan (200 mg/m2) followed by a reinfusion of her previously collected autologous stem cells to restore her T-cell immunity and hopefully control her lymphoma. She engrafted uneventfully at Day + 12 with chimerism studies showing autologous hematopoiesis and a CD4 count of 856. Her post-transplant course was complicated by renal thrombotic microangiopathy treated successfully with eculizumab. After further myeloma relapses, she is currently alive and well and in remission while on teclistamab over 4 yrs after her 2 nd autologous PBSC transplant. Her lymphoma has resolved. Case 2: A 37-year-old-male with beta-thalassemia was noted to have an elevated total protein on routine blood testing for a life insurance policy. Work up revealed multiple myeloma with a t(4:14) abnormality and complex cytogenetics. After initial observation he was started on KRD for progressive anemia. A VGPR was obtained and consolidated with an autologous PBSC transplant. He was maintained on Ixazomib+lenalidomide+dexamethasone but relapsed 2 years later with a sacral mass. After progressions though daratumumab+pomalidomide+dexamethasone then carfilzomib and then XRT to a large chest mass followed by DCEP as salvage, he underwent a 9/10 mismatched allogeneic transplant. He developed Grade 2 skin GVH but had disease progression again after just 6 months. Salvage treatment with elotuzumab+pomalidomide+dexamethasone then DCEP and subsequently ciltacabtagene autoleucel were unsuccessful with progression after just 6 weeks from the latter. He was treated again with DCEP but became transfusion dependent after the 2 nd cycle and had progressive disease again. Due to symptomatic myeloma and pancytopenia he underwent a reverse autologous PBSC transplant using banked stem cells. He engrafted uneventfully on Day +11 and despite recurrent multiple myeloma, he is currently in remission while on talquetamab. His beta-thalassemia returned. These cases demonstrate the ability to reverse an allogeneic transplant successfully using previously stored autologous peripheral blood stem cells. The recovery allowed for resumption of effective anti-cancer treatment that became lifesaving. Whether this approach could correct patients with unrelenting GVH issues should be contemplated as well.

Is the Youngest Donor Always the Best Choice to Optimize Outcomes for Matched Unrelated Allogeneic Transplant? Improving Precision Using Novel Statistical Methodology

INTRODUCTION Our recently published research has shown younger donors have superior survival for recipients of allogeneic hematopoietic stem cell transplant (HSCT). But, previously, we had not explored the donor age impact on other outcomes, or the impact of smaller age differences (<4 years) when balanced against other factors. We report results using our recently developed machine learning survival methodology, Nonparametric Failure Time Bayesian Additive Regression Trees (NFT BART). Here we study patient-specific optimization of donor selection for overall survival (OS) and event-free survival (EFS including events of death, relapse, graft failure/rejection, or moderate/severe chronic graft vs. host disease, GVHD). METHODS The patient data was provided by the Center for International Blood and Marrow Transplant Research. Each recipient underwent matched unrelated donor HSCT from 2016 to 2019 and was followed for OS and EFS outcomes up to three years. We trained our NFT BART model on 10,016 patients to provide predictions for OS and EFS. The model was validated on 1,802 patients among whom 699 had archived unrelated donor search records available from the National Marrow Donor Program. We examined donor age, sex/parity, CMV status, DPB1 nonpermissive mismatching, and DQB1 matching, while adjusting for relevant patient characteristics. We further restricted donor factors to those with a >1% positive impact on 3-year OS and/or EFS. Using archived search data for each patient, we examined three optimal strategies for donor selection: OS only, EFS only and a 2:1 weighted average between OS and EFS (denoted OS2:EFS1). The OS2:EFS1 effectively prioritizes optimizing OS while still allowing EFS consideration when the difference in OS predictions between two potential donors is minimal. We calculated predicted OS and EFS outcomes for each patient under all three optimal donor strategies. These predicted outcomes were benchmarked against observed practice by subtracting the predicted outcomes from the actual donor used for the transplant. RESULTS Among donor features, only age (for OS and EFS) and sex (for EFS) had clinically important impacts on outcomes. For OS, the youngest donor provided the best result regardless of patient characteristics: see the waterfall plot presented in Figure 1. As shown, the older the donor, the greater the impact of selection by age. Compared to donors 34+, use of an 18 year-old donor is associated with a substantial increase in OS where all of the recipient OS differentials are above 0.01. Compared to donors 22 to 30, the impact of an 18 year-old donor is negligible. For EFS, donor sex was the dominant factor, with males having a median 4% improvement in EFS at 3 years compared to females, while the difference for 42 vs. 18 was <2%. Optimizing for OS led to selection of the youngest donor regardless of sex, while optimizing EFS led to selection of the youngest male donor. Optimizing OS2:EFS1 generally picked the youngest donor except for selections of a male donor that may be older than the female when the age difference is small (median is 1 year older: Q1=0, Q3=2). In said cases, the OS detriment was minimal (median=-0.0005, Q1=-0.0008, Q3=-0.0003) with a substantial improvement in EFS (median=0.038, Q1=0.031, Q3=0.042) gained by switching to the male donor. Individual patient differences (OS2:EFS1 vs. actual donor) in OS vs. EFS are plotted in Figure 2. CONCLUSION Using our novel NFT BART methodology, we showed that donor selection to optimize HSCT outcomes can be improved by considering both OS and EFS. This weighted optimization strategy balances outcomes, prioritizing OS while also allowing for consideration of EFS when OS is likely similar between donors. Additionally, this supports previous research showing that choosing among donors from 18 to 30 with resultant minimal differences in outcomes that are negligible allowing for more flexibility in donor selection.

HLA Class I Genotypes Predict the Survival after Hematopoietic Stem Cell Transplantation in Immune Aplastic Anemia

Introduction Immune aplastic anemia (AA) is a bone marrow failure syndrome mediated by cytotoxic T cells, leading to the loss of HLA class I allele expression in hematopoietic stem and progenitor cells. In unrelated hematopoietic stem cell transplantation (HSCT), we recently reported an association between HLA loss and a short time to transplantation (TTT), consistent with an abrupt onset of immune AA. In addition, patients who lost HLA-A*02:06 or HLA-B*40:02 showed better survival outcomes after HSCT than those with other HLA allele losses. To clarify the clinical significance of the loss of specific HLA class I alleles in AA, we correlated loss-prone HLA class I allele genotypes with HSCT outcomes in patients with a short TTT, assuming the frequent absence of these alleles in this subgroup. Methods We conducted a nationwide retrospective analysis of patients with acquired AA ≥16 years old who underwent their first allogeneic HSCT in Japan between 2006 and 2020. Patient data were obtained from the Transplant Registry Unified Management Program (TRUMP ®), a comprehensive Japanese HSCT registry. Results From 1025 patients with acquired AA, we excluded patients with drug-induced AA and those missing 2-field HLA allele information and TTT information, leaving 874 patients for the analysis. The median patient age at HSCT was 34 years old, and the median TTT was 14 months. When focusing on the most frequently absent HLA class I alleles in Japanese patients with AA, including HLA-B*40:02, HLA-A*02:06, HLA-A*02:01, HLA-B*54:01, and HLA-A*31:01, all 5 alleles were associated with higher survival rates than in 220 patients without these alleles (Figure 1A). As expected, this association was more pronounced with a shorter TTT. For instance, among the 424 patients with a TTT <14 months, the 5-year survival rates were 79%, 78%, 85%, 78%, and 81% in patient groups with the respective HLA class I alleles above, while it was 55% in the other 101 patients (Figure 1B). Of note, having any of the 5 alleles (5-allele +) proved to be the best predictor of a survival out of 242792 combinations of 2 to 5 HLA-A, HLA-B, and HLA-DRB1 alleles. To adjust for potential confounders, such as age and graft sources, we performed propensity score matching and confirmed that the overall survival was significantly lower in patients without any of the 5 alleles (5-allele -) than in 5-allele + patients ( P = .00018). A multivariable Cox regression analysis including age, AA severity, prior IST, graft sources, HLA disparity, and HLA-B leader peptide dimorphism as covariates further demonstrated that the 5-allele genotype was an independent predictor of the survival. The consistent effect of the 5 alleles on the survival has been internally validated across various subgroups. In contrast, the 5 HLA class I alleles did not affect the survival in patients with hematological malignancies, including acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome. Regarding other post-HSCT complications, 5-allele - AA patients had a higher incidence of primary graft failure (GF) than 5-allele + AA patients, even when deaths before engraftment were censored and when compared to the propensity score-matched patients. Nevertheless, the 5 HLA alleles continued to stratify the survival among patients who achieved engraftment ( P < .0001). There were no significant differences in the incidence of acute and chronic graft-versus-host disease, late GF, or cause of death between 5-allele -and 5-allele + AA patients. To further stratify the survival rate in 5-allele - AA patients with a short TTT, we examined the impact of other HLA class I alleles and found that patients carrying HLA-B*07:02 or HLA-B*48:01, in which HLA loss had been previously detected in some AA patients, showed a particularly poor survival (Figure 1C; P = .0082). This association remained significant even after propensity score matching ( P = .031). Conclusions Significant associations between specific loss-prone HLA class I alleles and diverse survival outcomes after HSCT in AA patients with short TTT indicate the relevance of HLA information in patient selection for allogeneic HSCT. These findings also support the existence of a distinct immune pathogenesis affecting the clinical manifestations of AA differently, as observed in IST-treated patients. Further investigations in other ethnic groups are required to validate these observations.