Peripheral Blood Stem Cell Grafts Research Articles

Peripheral blood stem cell versus bone marrow graft for patients ≥60 years undergoing reduced intensity conditioning haploidentical transplantation for acute myeloid leukemia in complete remission: An analysis of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

In the context of T-cell replete haploidentical stem cell transplantation (Haplo-SCT) using post-transplantation cyclophosphamide (PT-Cy), it is still unknown whether peripheral blood (PB) or bone marrow (BM) is the best graft source. While PB is associated with a higher incidence of graft-versus-host disease (GVHD), it may induce a stronger graft-versus-leukemia effect compared to BM, notably in acute myeloid leukemia (AML). From the EBMT registry database, we compared T-cell replete PB (n = 595) versus BM (n = 209) grafts in a large cohort of 804 patients over the age of 60 years who underwent Haplo-SCT with PT-Cy for an AML in first or second complete remission. The risk of acute GVHD was significantly higher in the PB group (Grade II-IV: HR = 1.67, 95% CI [1.10-2.54], p = 0.01; Grade III-IV: HR = 2.29, 95% CI [1.16-4.54], p = 0.02). No significant difference was observed in chronic GVHD or non-relapse mortality. In the PB group, the risk of relapse was significantly lower in the PB group (HR = 0.65, 95% CI [0.45-0.94], p = 0.02) and leukemia-free survival was significantly better (HR = 0.76, 95% CI [0.59-0.99], p = 0.04), with a trend toward better overall survival (HR = 0.78, 95% CI [0.60-1.01], p = 0.06). We conclude that in the specific context of Haplo-SCT with PT-Cy, PB grafts represent a valid option to decrease the risk of relapse and improve outcome of older AML patients who usually do not benefit from conditioning intensification.

Treatment-Responsive Acute Graft-versus-Host Disease after Post-Transplantation Cyclophosphamide-Based Prophylaxis: Incidence and Clinical Outcomes

Post-transplant cyclophosphamide (PTCy) following hematopoietic cell transplantation (HCT) has emerged as standard of care for graft-versus-host disease (GVHD) prevention in adult patients without increasing malignant relapse. We previously defined acute GVHD (aGVHD) treatment response categories as either corticosteroid-sensitive (SS), dependent (SD), or resistant (SR) based on response to first-line corticosteroids and reported their clinical outcomes following non-PTCy based prophylaxis. Over one-third of patients developed aGVHD requiring systemic therapy. Cases were predominantly SR with a 14% overall incidence of SR aGVHD. The incidence and clinical outcomes of these three distinct aGVHD treatment response groups following PTCy-based prophylaxis is not well described. The objective of this retrospective, single-institution, cohort study was to assess the incidence and clinical outcomes of SS, SD, and SR aGVHD following HCT with PTCy-based prophylaxis using a prophylactic regimen of PTCy, tacrolimus, and mycophenolate mofetil (MMF). We included 196 consecutive (2017-2021) adult and pediatric patients receiving allogeneic HCT for malignant and non-malignant disorders at the University of Minnesota. Patients received PTCy on days +3 and +4 plus tacrolimus and mycophenolate mofetil prophylaxis. Bone marrow (BM) and peripheral blood stem cell (PBSC) graft sources and related and unrelated donors were included. Recipients received myeloablative (MAC) or reduced-intensity conditioning (RIC) regimens. In 196 allografts, 54 (28%) developed aGVHD before day +180 with median time to onset of 50 days (IQR 34-71 days). Of those, 32 patients (16% overall) developed maximum grade II-III aGVHD requiring systemic corticosteroids with the following response: 13 (41%) SS, 10 (31%) SD, and 9 (28%) SR. Overall incidence of SR aGVHD was 4.6%. Only 12 patients (6%) developed maximum grade III aGVHD while none had grade IV aGVHD. 2-year overall survival analyzed from 80 days after initiation of systemic treatment was similar in the SS and SD groups (77 and 75%, respectively), comparable to those without aGVHD (81%), but was lowest in the SR group (20%) with GVHD being the primary cause of death. Non-relapse mortality (NRM) was highest in the SR group. MN high risk and higher GVHD grade at onset were risk factors for developing SR aGVHD. Overall, we report a low incidence (16%) of aGVHD requiring systemic corticosteroids with PTCy-based prophylaxis. Acute GVHD cases were predominantly SS aGVHD with a lower incidence of SD and SR aGVHD. Our findings suggest that PTCy-based prophylaxis reduces rates of treatment resistant aGVHD. Patients with SR aGVHD had the worst clinical outcomes and poorest survival. Those with SS and SD aGVHD had similar clinical outcomes, both better than SR aGVHD.

Post-transplant cyclophosphamide versus anti-thymocyte globulin after reduced intensity peripheral blood allogeneic cell transplantation in recipients of matched sibling or 10/10 HLA matched unrelated donors: final analysis of a randomized, open-label, multicenter, phase 2 trial

The use of post-transplantation cyclophosphamide (PTCy) for graft-versus-host disease (GVHD) prophylaxis is not established after reduced intensity conditioning (RIC) hematopoietic stem cell transplantation (HSCT) from fully matched donors. This was a randomized, open-label, multicenter, phase 2 trial. All patients received a RIC regimen with fludarabine, intravenous busulfan for 2 days (Flu-Bu2), and a peripheral blood stem cell (PBSC) graft from a matched related or 10/10 HLA-matched unrelated donor. Patients were randomly assigned to receive anti-thymocyte globulin (ATG) 5 mg/kg plus standard GVHD prophylaxis or PTCy 50 mg/kg/d at days +3 and +4 plus standard GVHD prophylaxis. The primary endpoint was the composite endpoint of GVHD- and relapse-free survival (GRFS) at 12 months after HSCT. Eighty-nine patients were randomly assigned to receive either PTCy or control prophylaxis with ATG. At 12 months, disease-free survival was 65.9% in the PTCy group and 67.6% in the ATG group (P = 0.99). Cumulative incidence of relapse, non-relapse mortality, and overall survival were also comparable in the two groups. GRFS at 12 months was 54.5% in the PTCy group versus 43.2% in the ATG group (P = 0.27). The median time to neutrophil and platelet count recovery was significantly longer in the PTCy group compared to the ATG group. Except for day +30, where EORTC QLQ-C30 scores were significantly lower in the PTCy compared to the ATG group, the evolution with time was not different between the two groups. Although the primary objective was not met, PTCy is effective for GVHD prophylaxis in patients receiving Flu-Bu2 conditioning with a PBSC graft from a fully matched donor and was well tolerated in term of adverse events and quality of life. This trial was registered at clinicaltrials.gov: NCT02876679.

Dual T cell depletion for graft versus host disease prevention in peripheral blood haploidentical hematopoietic cell transplantation for adults with hematological malignancies.

The ideal immunosuppressive agents to complement post-transplant cyclophosphamide (PTCy) in PBSC-based haploidentical hematopoietic cell transplantation (haplo-HCT) remain debated. This study looks at our experience with ATG-PTCy-Cyclosporine (CsA) prophylaxis in PB haplo-HCT since 2015. Between October 2015 and December 2021, 157 adults underwent haploidentical hematopoietic cell transplantation (haplo-HCT) using a GVHD prophylaxis regimen comprising rabbit-ATG, PTCy, and CsA. Among these patients, 76.4% received a total ATG dose of 4.5 mg/kg, and 23.5% received 2 mg/kg. T-cell replete peripheral blood stem cell (PBSC) grafts were infused on day 0. The study reported a median follow-up of 32 months (range 0.3-61.64) for survivors. The cumulative incidence of grade II-IV and grade III-IV acute GVHD at day +100 was 26.3% and 9.5%, respectively. Moderate/severe chronic GVHD at 1 year was 19.9%. The 2-year overall survival (OS) was 49.4%, with a relapse-free survival (RFS) of 44.6%. In multivariate analysis, older patients, and those with high/very-high disease risk indices (DRI) were at higher risk for worse OS and higher non-relapse mortality (NRM). The study confirms that using PTCy and ATG (4.5 mg/kg), alongside CsA is safe and effective in preventing GVHD when using peripheral blood as the stem cell source in haploidentical hematopoietic cell transplantation (haplo-HCT).

The role of graft T-cell size in patients receiving alemtuzumab serotherapy for non-malignant disorders: results of an institutional protocol

Although graft T cells assist in engraftment, mediate antiviral immune-reconstitution, and cause graft-versus-host disease, graft size is not determined by T-cell content of the graft. The conventional method of graft size determination based on CD34+ cells with alemtuzumab serotherapy is associated with delayed immune reconstitution, contributing to an increased risk of viral infections and graft failure. Alemtuzumab, a long half-life anti-CD52 monoclonal antibody is a robust T-cell depleting serotherapy, and relatively spares memory-effector T cells compared to naïve T cells. We therefore hypothesized that graft size based on T-cell content in patients receiving peripheral blood stem cell graft with alemtuzumab serotherapy would facilitate immune-reconstitution without increasing the risk of graft-versus-host disease. We retrospectively analysed twenty-six consecutive patients with non-malignant disorders grafted using alemtuzumab serotherapy and capping of graft T cells to a maximum of 600 million/kg. The graft T-cell capping protocol resulted in early immune-reconstitution without increasing the risk of severe graft-versus-host disease. Graft T-cell content correlated with CD4+ T-cell reconstitution and acute graft-versus-host disease. The course of CMV viraemia was predictable without recurrence and associated with early T-cell recovery. No patient developed chronic graft-versus-host disease. Overall survival at one year was 100% and disease-free survival was 96% at a median of 899 days (range: 243–1562). Graft size determined by peripheral blood stem cell graft T-cell content in patients receiving alemtuzumab serotherapy for non-malignant disorders is safe and leads to early T-cell immune-reconstitution with excellent survival outcomes.

Severity and organ distribution of chronic graft-versus-host disease with posttransplant cyclophosphamide-based versus methotrexate/calcineurin inhibitor-based allogeneic hematopoietic cell transplantation.

The reduced risk of chronic graft-versus-host-disease (GVHD) with posttransplant cyclophosphamide (ptCy) in the setting of haploidentical related donor and more recently, with HLA-matched related and matched and mismatched unrelated donor allogeneic transplantation has been established. There is, however, paucity of data to show if ptCy impacts chronic GVHD pathogenesis, its phenotype and evolution after HCT regardless of the donor status. We examined the differences in chronic GVHD incidence and presentation in 314 consecutive patients after receiving their first allogeneic transplantation (HCT) using ptCy-based GVHD prophylaxis (ptCy-HCT; n = 120; including 95 with haploidentical related donor) versus conventional calcineurin inhibitor-based prophylaxis (CNI-MUD; n = 194) between 2012 and 2019. The 1-year cumulative incidence of all-grade chronic GVHD and moderate/severe chronic GVHD was 24% and 12%, respectively, after ptCy-HCT and 40% and 23% in the CNI-MUD recipients (p = 0.0003 and 0.007). Multivariable analysis confirmed that use of CNI-based GVHD prophylaxis and peripheral blood stem cell graft as the risk factors for chronic GVHD. The cumulative incidence of visceral (involving ≥1 of the following organs: liver, lungs, gastrointestinal tract, serous membranes) chronic GVHD was significantly higher with CNI-MUD vs. ptCy-HCT (27% vs. 15% at 1 year, p = 0.009). The incidence of moderate/severe visceral chronic GVHD was 20% in CNI-MUD group vs. 7.7% in the ptCy-HCT group at 1 year (p = 0.002). In addition, significantly fewer ptCy-HCT recipients developed severe chronic GVHD in ≥3 organs (0.8%) vs. 8.8% in the CNI-MUD group at 1-year posttransplant (p = 0.004). There was no significant different in relapse, non-relapse mortality, and relapse-free and overall survival between the two groups. Further investigation is needed to confirm that reduced risk and severity of chronic GVHD, less visceral organ distribution with ptCy-HCT leads to improved quality of life.

Alemtuzumab, Dual Graft-versus-Host Disease Prophylaxis, and Lower CD3+ T Cell Doses Equalize Rates of Acute and Chronic Graft-versus-Host Disease in Pediatric Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation with Matched Unrelated Donor Peripheral Blood Stem Cells or Bone Marrow Grafts

Data comparing hematopoietic stem cell transplantation (HSCT) using bone marrow (BM) or peripheral blood stem cell (PBSC) grafts in children after alemtuzumab-based conditioning are lacking. We investigated whether in vivo T cell depletion using alemtuzumab could reduce the risk of severe acute graft-versus-host disease (aGVHD) and chronic GVHD (cGVHD) after HSCT with matched unrelated donor (MUD) BM or PBSCs. This retrospective multicenter study included 397 children (BM group, n = 202; PBSC group, n = 195) who underwent first MUD HSCT at 9 pediatric centers in the United Kingdom between 2015 and 2019. The median age at transplantation was 7.0 years (range, .1 to 19.3 years), and the median duration of follow-up was 3.1 years (range, .3 to 7.5 years). The 3-year overall survival was 81% for the entire cohort (BM group, 80%; PBSC group, 81%). The incidence of grade II-IV aGVHD was significantly higher in the PBSC group (31%) compared to the BM group (31% versus 19%; P = .003), with no difference in the incidence of grade III-IV aGVHD (BM, 7%; PBSC, 12%; P = .17). CD3+ T cell dose >5 × 108/kg and the use of PBSCs were independent predictors of grade II-IV aGVHD. When considering CD3+ T cell dose and GVHD prophylaxis, PBSC transplantation with a calcineurin inhibitor (CNI) and mycophenolate mofetil (MMF) and a CD3+ T cell dose ≤5 × 108/kg had a comparable grade II-IV aGVHD to BM transplantation plus a CNI (20% versus 18%; P = .52). PBSC transplantation was associated with a lower incidence of cGVHD compared to BM transplantation (6% versus 11%; P = .03). Within the limits of this study, we identified a potential strategy to reduce the risk of severe GVHD in pediatric PBSC recipients that includes a combination of in vivo T cell depletion using alemtuzumab and dual GVHD prophylaxis (with a CNI and MMF) and limiting the CD3+ T cell dose to ≤5 × 108/kg.

MAIT Cell Frequencies within PBSC Grafts Are Associated with Donor CMV Serostatus and Age: An Initial Analysis from the DKMS and NMDP Graft Composition Study

Introduction We have established an international collaboration between the DKMS (Germany) and the National Marrow Donor Program (NMDP; USA) to characterize more than 2000 donor peripheral blood stem cell (PBSC) grafts. We aim to correlate graft immunophenotype with patient outcomes and perform additional analyses on biobanked aliquots of selected samples to deepen our understanding of the relationships between features of the donor graft and key patient outcomes, such as graft-versus-host disease, relapse, and overall survival. Here, we present an interim analysis, focused on the CD8 T cell compartment, describing relationships between donor characteristics and graft composition. We have developed a novel computational clustering method to integrate cytometry data from both the US and German analytical laboratories. Methods 300 donor PBSC graft samples were collected, processed, freshly stained (34-color panel for immunophenotyping of lymphocytes and hematopoietic stem cells), and analyzed using an Aurora Cytek instrument at the NMDP contract laboratory (Roswell Park, Buffalo, NY). 53 additional samples were processed in the DKMS laboratory in Dresden, Germany (equivalent antibody panel and instrument). We have developed a novel analytical pipeline for integrating these data. First, we group samples by facility, and by month run for computational convenience, to form “batches” for initial clustering. We perform Leiden clustering, and subclustering, to generate a large number of clusters per batch. To integrate data from all batches, we then cluster all subcluster centroids from all batch-specific clusterings. This gives a global clustering for each stream ( e.g. the CD8+ T cells). Cluster frequencies per sample provide features for quantifying links between immunophenotype and donor metadata ( e.g., CMV serostatus, age, and sex). Our approach offers an important complement to traditional gating and allows full exploration of the data in an unbiased fashion. Results 353 donor products were included in the analysis. The median donor age was 28 years (range 18 - 60), 59% were female and 65% were CMV positive. Initial metaclustering demonstrated that the 53 DKMS samples and the 300 NMDP samples are each well represented in all clusters, and that site-driven batch effects, while present, are mild. We initially focused on the CD8 T cell compartment (UMAP in Fig 1A) and discovered several phenotypic differences when we compared CMV seronegative and seropositive donors. CD8 T cell clusters 10 and 11 (both TCRαβ+ CD45RA+ CCR7+ HLA-DR+ CD57+; cluster 10 CCR9+; cluster 11 CCR9-) were present at higher frequency in CMV+ donors (n = 116) compared with CMV- donors (n = 220; p <0.0001; Fig 1B). Conversely, clusters 9 and 12, enriched for mucosal-associated invariant T (MAIT) cells, were significantly reduced in CMV+ donors (p <0.001). Of note, CMV serostatus was not significantly associated with age (median age in the CMV+ group was 29.4 years; vs 28.4 years in CMV- donors). We also observed an association between cluster 8 (TCRαβ+ CD45RA+ CCR7+ HLA-DR+ CD38+; consistent with an activated phenotype) and age (p = 0.0009; Fig1B). There was a trend toward decreased MAIT frequencies with age (Cluster 9; p = 0.058). When we analyzed the MAIT cell frequency as defined by traditional flow cytometry gating (CD3+ CD8+ CD161+ Vα7.2+), we observed a statistically significant relationship between younger donor age and increased MAIT cell frequency (p = 0.0002), consistent with the trend seen in cluster 9. Sex at birth was not linked with immunophenotype in our analyses thus far. Conclusions In this initial analysis of our multi-center observational study of PBSC graft products, we demonstrate the development and application of new computational tools for large-cohort flow cytometric data analysis. CMV-positive serostatus and younger donor age are both associated with an increased frequency of MAIT cells in the graft. We also found differences in CD8 T cell activation status in association with age and CMV. In the future, we will assess the relationships between these populations and patient outcomes, as well as explore cluster frequencies in association with key patient outcomes including GVHD, relapse, and overall survival. Sample collection and analysis are ongoing at both US and German sites as an ongoing collaborative effort between the NMDP and the DKMS.

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.

Hematopoietic Stem Cell Transplantation for Fanconi Anemia: Outcome and Prognostic Factors for Survival and Subsequent Neoplasms

Introduction Fanconi anemia (FA) is an inherited disorder of impaired DNA repair, associated with progressive bone marrow failure (BMF), leukemia, and cancer predisposition. Hematopoietic cell transplantation (HCT) may be curative for FA associated BMF, and changes in HCT practice over the past decades may have impacted outcomes in FA patients. Methods We used CIBMTR database to perform a retrospective cohort study of children and adults undergoing first allogeneic HCT for FA BMF (patients with MDS/AML excluded) from 2000-2018. We hypothesized that contemporary HCT approaches, including decreases in radiation and increase in use of serotherapy and ex-vivo graft manipulation, would associate with improved outcomes in FA patients receiving HCT. The primary endpoints were overall survival (OS) and early subsequent malignant neoplasm (SMN; by 5 yr post-HCT). Secondary endpoints included event free survival (EFS; death, SMN or graft failure), treatment related mortality (TRM), graft failure, and acute (grade II-IV) and cGVHD. Univariate and multivariate Cox regression models were created to evaluate the impact of conditioning, donor and graft source, treatment era, and patient characteristics on outcomes, adjusting for other significant covariates. Due to confounding factors, we were not able to create a single multivariate model that included radiation, ex-vivo graft manipulation, and donor and graft source; therefore, we performed subgroup univariate analysis for patients receiving unrelated donor bone marrow (BM) or peripheral blood stem cell (PBSC) HCT. Results We analyzed 652 HCTs from 2000-07 (n=353) and 2008-18 (n=293). 223 (35%) patients had a matched related donor (MRD) with any graft type, 244 (38%) had an unrelated donor BM or PBSC grafts, 123 (19%) had an unrelated cord blood graft, and 44 (7%) had a related haploidentical donor with any graft type. Between the two treatment eras, 5-yr OS has dramatically improved (78.8% 2008-18 v 62.5% 2000-07, p<0.01; figure 1) driven by lower 5-yr TRM (15.2% v 25.5%, p=0.004) and lower rates of primary (7.9% v 12.9% at 42 days, p=0.038) and secondary (4.1% v 8.9% at 1 year, p=0.014) graft failure. No statistically significant change was observed for aGVHD, cGVHD, or early SMN. Assessing the entire cohort, use of total body irradiation (TBI) >300cGy was associated with lower OS (HR=1.75, 95% CI 1.24-2.47) and increased TRM (HR=3.36, 95% CI 1.54-7.34; figure 2) on multivariable analysis. For patients that received unrelated BM or PBSC HCT, use of TBI was associated with inferior OS survival (HR=1.93, 95% CI 1.18-3.17), EFS (HR=1.78, 95% CI 1.13-2.80), and TRM (HR=2.31, 95% CI 1.20-4.46) on univariate analysis. Among all patients, CD34 selection or T-cell depletion ( ex vivo depletion) were not associated with better OS (95% HR=1.014, 95% CI 0.84-1.56), EFS (HR=1.163, 95% CI 0.86-1.58), or TRM (HR=0.79, 95% CI 0.52-1.19) but was associated with less aGVHD (HR=0.35 95% CI 0.22-0.57) and lower but not-statistically significant cGVHD (HR=0.54, 95% CI 0.29-1.01) on multivariable analysis. Within the unrelated donor setting, univariate analysis showed reduced aGVHD (HR=0.39 95% CI 0.23-0.68) and cGVHD (HR=0.47 95% CI 0.28-0.79) but did not have a significant effect on OS, EFS, or TRM. Early SMN occurred in 3.2% of participants by 5-years post-HCT and was associated with cGVHD occurrence (HR=2.14, 95% CI 1.06-4.33) and older age (>18 v <9 years, HR=6.35, 95% CI 2.66-15.14), but not ex-vivo depletion or use of ATG or alemtuzumab. Early SMN risk was not impacted by the use of TBI (HR=1.60, 95% CI 0.77-3.34), but secondary analysis demonstrated a trend towards a relationship with doses >300 cGy (HR=2.24, 95% CI 0.99-5.08). Conclusions Outcomes for patients with FA BMF have improved significantly in more recent HCT eras, driven by reductions in TRM and graft failure risk. Although radiation doses >300cGy were associated with greater toxicity, doses of 100-300cGy were not associated with inferior outcomes in our cohort. Use of CD34 selection or T-cell depletion may reduce the risk of GVHD in transplant FA patients, but does not appear to impact 5-yr OS. Risk for early SMN development in FA patients receiving HCT associates with older age and cGVHD.

Outcomes after Bone Marrow Versus Peripheral Blood Haploidentical Hematopoietic Stem Cell Transplantation Using Post-Transplant Cyclophosphamide-Based Gvhd Prophylaxis

Background: Patients who do not have a human leukocyte antigen (HLA)-matched donor can undergo hematopoietic stem cell transplantation (HSCT) by utilizing haploidentical (haplo) family donors and post-transplant cyclophosphamide (PTCy) for Graft-versus-host disease (GVHD) prophylaxis. Originally, Haplo transplants utilized a bone marrow (BM) source because of concern of higher incidence of GVHD with peripheral blood stem cells (PBSC) graft. Recent years have seen an increase in PBSC graft utilization. Some published studies have reported comparable survival between the two graft sources. These studies also reported lower rate of chronic GVHD and higher quality of life with BM and a lower relapse rate and faster engraftment with PBSCs. We aimed to investigate the outcomes following BM versus PBSC haplo HSCT using (PT-Cy)-based GVHD prophylaxis. Methods: In this single-center retrospective study, we included all (n=176) adult patients who underwent haplo HSCT from August 2016 to July 2021 and had at least one year of follow-up. Bivariate analyses, using the chi-square and t-test, were performed. Kaplan-Meier and regression analyses were conducted. Data were analyzed using SPSS version 28. Statistical significance was considered at p<0.05. Results: The study included 176 haplo transplants including 65% peripheral blood and 35% bone marrow grafts. The median age was 54 (18-74) years and 68% of recipients were males. Ethnic minorities comprised 30% of recipients. Hematologic diagnoses included myeloid disease (65%), lymphoid disease (29%), and others (6%). 31 % of the patients underwent a Myeloablative transplant. Hematopoietic cell transplantation comorbidity index of 3 or higher was noted in 53% of patients. With a Median follow-up 21 (0-73) months, Overall survival (OS) and disease-free survival (DFS) was not reached in either BM or PBSC haplo HSCT group. 1-year OS of 75% and 74% (p-0.898) and 1-year DFS of 63% and 63% (p-0.994) were noted in BM and PBSC group respectively. In PBSC versus BM haplo recipients, an earlier neutrophil engraftment (17 days vs 18 days, p=0.022) was seen. Similar incidences of grade II-IV acute GVHD (50% vs 51%, p=0.875), relapse (22% vs 26%, p=0.579), non-relapse mortality (NRM, 17% vs 20%, p=0.682), platelet engraftment (28 vs 31 days, p<0.092) and 1- year chronic GVHD (35% vs 38%, p=0.410) were noted. Conclusions: In this study, Haploidentical allogeneic hematopoietic stem cell transplant recipients demonstrated similar rates of overall and disease-free survival, relapse, non-relapse mortality, acute and chronic GVHD, and platelet engraftment regardless of graft source. Neutrophil engraftment was observed one day earlier in peripheral stem cell recipients. This supports the use of either PBSC or Bone marrow as the preferred source of Haploidentical transplants.

Lymphocyte Reconstitution after Naïve T Cell-Depleted Hematopoietic Cell Transplantation

Background: Allogeneic hematopoietic cell transplantation (HCT) is curative for many high-risk hematological malignancies. However, graft-versus-host disease (GVHD) causes morbidity, mortality, and reduced quality of life after HCT. Removal of all T cells from hematopoietic cell grafts (pan-T cell depletion; pan-TCD) significantly reduces GVHD but is associated with impaired immune reconstitution and increased non-relapse mortality due partly to opportunistic infections. We developed a novel strategy to remove CD45RA + naïve T cells (T N) from peripheral blood stem cell grafts (PBSC) that is associated with a low incidence of serious acute GVHD (aGVHD) and an exceptionally low incidence of chronic GVHD (cGVHD) (Bleakley, JCO 2022). Although CD45RA-depletion removes T N and other CD45RA-expressing effector T cell populations from PBSC along with most NK and B cells, T N-depletion (T ND) does not appear to increase infections or have a major negative impact on immune reconstitution. Understanding subtle differences in lymphocyte population dynamics after T ND compared to T cell-replete (TR) HCT will enable further graft engineering advances and may provide broader insights into human HCT immunobiology. Objective: To evaluate lymphocyte reconstitution after T ND HCT. Methods and Results: We compared blood lymphocyte populations in T ND HCT recipients (n=47) to those in TR HCT controls (n=19) at days 28, 56, 90, 180, and 360 post-HCT using multiparametric flow cytometry. To complement standard Boolean analysis, we employed a metaclustering approach to simultaneously evaluate ~20 cell surface markers for each class of lymphocytes (CD8 + and CD4 + T cells, NK cells, and B cells) over scores of batches of recipient and control samples. Absolute numbers of total CD8 + T cells were similar in T ND compared to TR at all timepoints, whereas total CD4 + T cell numbers were consistently lower in T ND until 1 year post-HCT. Using conventional gating and high-dimensional clustering analyses, we identified several key differences in T cells after T ND. CD45RA-expressing populations (T N and effector memory re-expressing CD45RA [T EMRA]) in CD8 + and CD4 + T cells were rare in CD45RA-depleted PBSC and remained decreased in T ND HCT recipients until around 1 year (CD8 +, Fig. 1). Activated CD8 + and CD4 + central and effector memory (T CM, T EM) T cells were increased proportionally and in absolute numbers in T ND early post-HCT (CD8 +, Fig. 1). Absolute numbers of regulatory T cells (Treg) were decreased in T ND; consequently, ratios of activated CD8 + and CD4 + to Treg were both markedly increased early post-HCT, despite a similar incidence of aGVHD in T ND and TR recipients. Although most NK cell populations were removed from CD45RA-depleted PBSC, absolute numbers of NK cells in all compartments, including both activated and inhibited, were increased in T ND recipients early post-HCT compared to TR. Like NK cells, most B cells except for plasmablasts and plasma cells were removed by CD45RA-depletion of PBSC. Immature, naïve, and memory B cells were reduced very early post-T ND HCT, but substantial B cell recovery occurred within the first 2-3 months. Conclusions: We present the first detailed description of lymphocyte reconstitution after T ND HCT. Despite the major effect CD45RA-depletion has on graft composition-removing all T N and T EMRA from the T cell compartment and most NK and B cells-NK and B cell recovery occurred early, and by 1 year post-HCT there was little difference in lymphocyte populations between T ND and TR. This is consistent with hematopoietic progenitors, not mature lymphocytes, as the source of B and NK cell reconstitution. Among T cells, the most striking findings were an increase in activated T CM and T EM CD4 + and CD8 + T cells early after T ND HCT, and increased ratios of activated T cells to Treg. Although elevated blood T effector/Treg ratios have been associated with cGVHD, cGVHD was uncommon and steroid-responsive after T ND HCT. Whether increased activated T cells reflect a reduction in Treg-mediated suppression after HCT, bystander T cell activation, activation by antigen (e.g., alloantigen, latent herpes viruses, microbiota), or multiple factors are questions we are now investigating. We are also exploring associations between lymphocyte populations early post-T ND HCT and clinical outcomes; these could serve as biomarkers to facilitate early intervention to prevent or treat GVHD, relapse, or infection.

A Phase 2, Single-Arm, Multicohort, Open Label, Multicenter Trial of Off-the-Shelf Natural Killer Cells (SAR445419) in Patients with High-Risk Myeloid Malignancies Undergoing Allogeneic HSCT

Background and Significance: Relapse remains the leading cause of treatment failure for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although the risk depends on parameters such as age and conditioning intensity, relapse is experienced in 30-50% of patients after conventional HSCT in high-risk AML/MDS. Initial safety and post-HSCT relapse risk reduction results were reported in a phase I study of haploidentical (haplo) donor-derived natural killer (NK) cells expanded ex vivo on feeder cells expressing IL-21 and 4-1BBL in conjunction with haplo-HSCT (Ciurea et al, Leukemia, 2022). This study supported exploring SAR445419 (SAR'419), an off-the-shelf (OTS) product, derived from universal donor NK cells and expanded ex vivo in a feeder cell-free system using plasma membrane particles bearing membrane-bound IL-21 and 4-1BBL. Here, we aim to test whether SAR'419 can mediate an effective anti-leukemia response and reduce post-transplant relapse rate when administered in the peri-transplant period. Study Design and Methods: In this multicenter, multicohort, phase 2, single-arm trial (NCT05726682), patients with high-risk AML/MDS planned for allo-HSCT from matched sibling, matched unrelated or haplo donors will be enrolled in 2 cohorts. Cohort A will comprise patients aged 18‒65 years undergoing myeloablative conditioning with hematopoietic cell transplantation comorbidity index ≤3 and cohort B will comprise patients aged 18‒75 years undergoing reduced intensity conditioning. Patients with prior allogeneic transplantation or AML beyond first complete remission will be excluded from the study. Patients will undergo HSCT conditioning starting on Day ‒7 and will receive a peripheral blood stem cell graft on Day 0. Prophylaxis for Graft-versus-host disease (GVHD) using cyclophosphamide, mycophenolate mofetil, and tacrolimus will start on Day +3. SAR'419 will be administered as adjunctive therapy to the HSCT on Day ‒2, Day +7, and Day +28. To confirm the anticipated safety profile of SAR'419, a safety run-in will be conducted in 6‒12 patients in each cohort prior to open enrollment. Patients will be enrolled until 51 and 44 patients in cohorts A and B, respectively, receive at least the first 2 doses of SAR'419 at the target dose confirmed during safety run-in. In total, approximately 107 patients will be enrolled. The primary endpoint, relapse free survival (RFS), will be assessed at 12 months post-HSCT. Additional primary endpoints include frequency of graft failure, life-threatening infusion-related reactions or cytokine release syndrome, tumor lysis syndrome, immune cell-associated neurotoxicity syndrome, acute GVHD, overall mortality at 100 days post-HSCT, non-relapse mortality, and cytomegalovirus reactivation/infection. Secondary endpoints are safety, tolerability, and the impact of SAR'419 on HSCT outcomes, stem cell engraftment, GVHD, infection, and quality of life. Exploratory assessments include evaluation of the impact of SAR'419 on immune reconstitution, cytomegalovirus seroconversion, minimal residual disease, resource utilization, SAR'419 immunogenicity and persistence and their impact on outcomes in whole blood (including serum and plasma) and bone marrow aspirates. RFS will be reported as the percentage of patients who are relapse free and alive at 12 months from the date of HSCT and who received at least the first 2 planned doses of SAR'419. Adverse events will be graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v5.0 and will be summarized using descriptive statistics.

Analysis of the Impact of Whole Blood and T-Cell Donor Chimerism after Allogeneic Stem Cell Transplant

Background: Allogeneic stem cell transplant (alloSCT) is one of the few currently available therapies to cure acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Positive outcomes are dependent on engraftment of donor cells to provide hematopoiesis as well as a graft-versus-tumor effect. Measurement of whole blood (WB) and T-cell (TC) chimerism at routine intervals post alloSCT allows for monitoring of engraftment. Development of mixed donor chimerism (MC) may indicate impending graft failure or disease relapse. The ideal frequency for chimerism measurements as well as the role of WB versus TC chimerism remains unclear. In this study, we analyzed the impact of WB versus TC mixed chimerism on survival outcomes in patients with AML and MDS. Furthermore, we studied baseline factors associated with the development of MC. Methods: In this retrospective analysis, adults with AML or MDS who received an alloSCT between January 1, 2016 and February 1, 2022 that had at least 1 donor chimerism measured on Day 30 (D30), Day 60 (D60), Day 90 (D90) at Juravinski Hospital and Cancer Centre (Hamilton, ON, Canada). Baseline demographic and transplant-related characteristics were collected. AML and MDS diagnoses were based on WHO 2016 classification of myeloid neoplasm and acute leukemia. Conditioning intensities were classified according to CIBMTR definitions. A total of 271 transplants were completed during the study period for AML or MDS. 128 were excluded from the analysis (chimerism testing not performed (n=121), second transplants (n=7)). We analyzed a total of 143 patients with median age of 63 years (19 - 76) who were transplanted for AML (n=105) and MDS (n=38). Donors were HLA-matched siblings (MRD, n=26), unrelated donors (MUD (8/8 or 7/8), n=85) or haploidentical related donor (MMRD, n=32). All patients except one received peripheral blood stem cell grafts. For alloSCT, 20 % of patients received myeloablative conditioning (MAC) and 80% received reduced-intensity conditioning (RIC). Complete chimerism (CC) was defined as ≥95%, whereas MC was defined as ≤95%. All chimerism measurements were measured using a PowerPlex® 16 System (Promega) (Hamilton Regional Laboratory Medicine Program, Hamilton, ON). Results: In our cohort, OS at 2 years was 57% and RFS 49%. Of the baseline factors, only disease-risk based on ELN 2022 for RFS with a hazard ratio (HR) of 1.43, 95% CI [0.83, 2.49] for adverse risk and HR 0.53 95% CI [0.23, 1.03] (p=0.03) for favourable risk. WB MC was detected in 22%, 22%, 23% of patients on D30, D60, and D90, respectively. TC MC was detected in 48%, 46%, and 43% of patients on D30, D60, and D90, respectively. WB MC had a strong negative impact on RFS, with a HR of 2.81, 95% CI [1.69, 4.66; p<0.001] on D30, 3.07, 95% CI [1.67, 5.66; p<0.001] on D60 and 4.77, 95% CI [2.47, 9.22; p<0.001] on D90. The influence of TC MC on RFS was also significant at all time points. Of 101 patients who were alive and did not have a relapse prior to D90 and had D30 chimerism and D90 chimerism evaluated, the patients who had a mixed WB and TC chimerism on D90 had a HR of 6.13, 95% CI [2.23, 16.85] and HR 2.73, 95% CI [1.24, 5.98] for RFS, which was statistically significant even after adjusting for known information of D30 WB/TC chimerism status (Table 1). WB MC had a strong negative impact on OS with a HR of 2.67, 95% CI (1.54, 4.64; p=0.001) on D30, 2.81, 95% CI (1.47, 5.38; p=0.002) on D60 and 4.78, 95% CI (2.47, 9.24; p<0.001) on D90. Again, the later time points remained statistically significant, when adjusting for known D30 chimerism status. The influence of TC MC on OS was also significant at all time points, however only a non-significant trend remained at later time points when adjusting for D30 TC chimerism (Table 2). We analyzed baseline factors predictive for D30 chimerism. For WB chimerism, only donor source remained statistically significant (p=0.038) with MMRD having a lower HR for MC 0.39, 95% CI [0.11, 1.41] and MRD having a higher HR for MC 2.33, 95% CI [0.9 - 5.99]. Conclusions: Our data confirm that a mixed chimerism as early as D30 post-alloSCT can have a detrimental impact on outcomes (RFS and OS) in alloSCT. Patients receiving haploidentical alloSCT had a lower risk to develop mixed chimerism in our cohort. These dismal outcomes of patients with mixed chimerism in our cohort provides a strong rationale to study interventions such as novel maintenance strategies or donor-lymphocyte infusions in this patient group.

A Comprehensive Model for Predicting 1-Year Survival after Allogeneic Hematopoietic Cell Transplantation in Older Patients

Background: As the number of elderly patients referred to and undergoing allogeneic hematopoietic cell transplantation (alloHCT) continues to rise, there is a growing need for a comprehensive risk assessment model to accurately predict transplantation outcomes in this specific patient population. The widely used Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) does not fully capture factors influencing transplant outcomes in elderly patients. This study aims to enhance risk stratification for elderly patients undergoing alloHCT by integrating additional functional, nutritional, and clinical indicators. Methods: This is a retrospective study utilizing the data registry of HCT patients who received their treatment at UF Health Cancer Center. The inclusion criteria included all patients age > 65 years, who underwent their first alloHSCT between January 2012 and December 2022, regardless of graft type, conditioning regimen, donor type, or graft source. Patient data were extracted through chart review. The primary endpoint was 1-year Overall Survival (OS). Prognostic significance of each variable was assessed using Cox proportional hazard models. We subsequently used all variables to build a comprehensive prediction model to assess 1-year survival outcomes in elderly patients undergoing alloHCT. The predictive model's performance was measured by the Concordance-statistic (c-statistic). Results: A total of 69 patients satisfied the inclusion criteria and were included in the analysis. Median age was 69 years (range 66-81) and 65.2% were male. Unrelated donors were used in 66.6% of patients, while the remainder received grafts from related donors. Most patients received peripheral blood stem cell grafts (91%). Myeloid malignancies comprised the most common indication for alloHCT in this group of patients (89.8%). 59.4% had abnormal cytogenetics. 35 variables were considered in the analysis. In the univariate analysis, variables with impact on 1-year OS post-transplant included creatinine (HR 3.35, CI 1.62-6.91, p=0.001), blood urea nitrogen (HR 1.03, CI 1.01-1.06, p=0.007), estimated glomerular filtration rate (HR 0.93, CI 0.89-0.93, p=0.001), hemoglobin (HR 0.8, CI 0.66-0.97, p=0.02), abnormal cytogenetics (HR 2.27, CI 1.20-4.31, p=0.012) and high and very high disease risk index (HR 0.51, CI 0.27-0.98, p=0.04). Notably, both hemoglobin and cytogenetics demonstrated a c-statistic greater than 0.6 in the univariate analysis. In the multivariate analysis, variables impacting 1-year OS included hemoglobin (HR 0.69, CI 0.48-0.99, p=0.04), remission status at the time of transplant i.e., not reached (NR) vs complete remission 1 (CR1) (HR 0.05, CI 0.00-0.71, p=0.027), and abnormal cytogenetics (HR 7.55, CI 2.32-24.58, p=0.001). Interestingly, the HCT-CI did not significantly impact 1-year OS in the univariate analysis (c-statistic 0.51, HR 0.99, CI 0.85-1.15, p=0.896). Our prediction model showed good performance with a mean area under the receiver operating characteristic curve of 0.82. Conclusion: We developed a comprehensive prediction model to assess 1-year OS in elderly patients undergoing alloHCT to overcome some of the limitations of the HCT-CI in this unique age group. This model holds potential to enhance decision-making regarding candidacy for elderly patients undergoing alloHCT.

Post-Transplant Cyclophosphamide Equalizes Clinical Outcomes of Allogeneic Hematopoietic Cell Transplantation across Racial and Ethnic Populations

Background Despite being the only curative option for many hematologic malignancies, allogeneic hematopoietic cell transplant (HCT) has been plagued by health disparities due to differences in access, donor availability, and outcomes across racial and ethnic minority populations (REM) (Gragert, NEJM, 2014). Even when a matched donor is available, HCT outcomes for REM patients are historically worse compared to non-Hispanic white (NHW) patients. (Landry, Stem Cell Investigation, 2021). The development of post-transplant cyclophosphamide (PTCy) for graft-versus-host disease (GVHD) prophylaxis has led to increased use of HLA haploidentical (haplo) or mismatched unrelated donors (MMUD) with comparable outcomes to HLA matched HCT (McCurdy, Advances in Hematology, 2015). While PTCy allows for almost every transplant-eligible REM patient to have a suitable donor, there is limited data evaluating whether this transplant platform results in similar outcomes for REM patients compared to NHW patients. The aim of this study was to investigate if disparities in transplant outcomes still exist between REM patients and NHW patients who received an HLA mismatched transplant with PTCy. Methods We retrospectively analyzed 302 consecutive adult patients who underwent haplo or MMUD HCT with PTCy at Moffitt Cancer Center between 2014 and 2022. All endpoints were compared between four groups: NHW, Hispanic-White, Black, and Other (Asian, biracial, or multiracial.) Results In this cohort, 59% (n=178) of patients identified as NHW, 14% (n=41) as Hispanic-White, 16% (n=48) as Black, 7% (n=21) as biracial or multiracial, and 4% (n=11) identified as Asian. Most patients were male (57%). The median age of all patents was 58. The most common indication for transplant was acute myeloid leukemia (48%), myeloablative conditioning (MAC) was utilized in 42%, and 89% received peripheral blood stem cells grafts. REM patients were more likely to be <65 years of age (60% vs 66-90%, p<0.001), female (36% vs 50-54%, p=0.04), and receive MAC conditioning (33% vs 40-71%, p<0.001). Median follow-up was 21 months. In all clinical outcomes, no significant differences were observed between NHW and REM patients. At day 100, the cumulative incidence of Grade II-IV aGVHD was 37% [95% CI 30-44%] for NHW patients, 32% [95% CI, 18-46%] for Hispanic-White patients, 27% [95% CI, 15-40%] for Black patients, and 26% [95% CI, 13-41%] for patients of Other races (p= 0.38). The cumulative incidence of moderate to severe cGVHD at 2 years was 21% [95% CI 15-28%] for NHW patients, 25% [95% CI, 13-39%] for Hispanic-White, 17% [95% CI, 7.8-29%] for Black, and 27% [95% CI, 13-42%] for patients of Other races (p = 0.80). At 2 years, the probability of relapse was 25% [95% CI 19-32%] for NHW patients, 24% [95% CI, 12-39%] for Hispanic-White patients, 27% [95% CI, 15-40%] for Black patients, and 18% [95% CI, 7.1-33%] for patients of Other races (p = 0.97). During the same time interval, non-relapse mortality (NRM) for NHW patients, Hispanic-White patients, Black patients, and patients of Other races were 19% [95% CI, 13-25%], 15% [95% CI, 5.9-27%], 19% [95% CI, 9.4-32%], and 24% [95% CI, 11-40%] (p = 0.73), respectively. At 2 years, relapse free survival (RFS) was 56% [95% CI 49-64%] for NHW patients, 61% [95% CI, 47-79%] for Hispanic-White patients, 54% [95% CI, 41-71%] for Black patients, and 58% [95% CI, 43-78%] for patients of Other races (p = 0.77). Comparably, OS at 2 years for NHW, Hispanic-White patients, Black patients, and patients of Other races was 64% [95% CI, 57-72%], 72% [95% CI, 60-88%], 53% [95% CI, 40-71%], and 63% [95% CI, 49-83%] respectively after two years (p = 0.92). In multivariate analysis, race/ethnicity had no significant impact on incidence of Grade II-IV aGVHD (p= 0.40), moderate/severe cGVHD (p = 0.80), rates of relapse (p = 0.86), NRM (p= 0.77), RFS (p = 0.69), or OS (p = 0.64) (Table). Conclusions These results show that the use of HLA mismatched donor HCT with PTCy achieves comparable clinical outcomes between REM and NHW patients. Our study suggests that PTCy effectively contributes to equitable healthcare in the setting of allogeneic HCT for REM patients.

Post Transplant Cyclophosphamide (PTCY) in Peripheral Blood Stem Cell Transplant Results in Successful Engraftment and Substantially Low Frequency of Chronic Graft Versus Host Disease (GvHD), without Impaired Cellular Immune Reconstitution

The Post-Transplant Cyclophosphamide (PTCY) approach was first studied in the haploidentical transplant setting, and it resulted in high percentages of engraftment and reduced frequency of acute and mainly chronic GvHD as well as non-relapsed mortality. PTCY is now increasingly being used in HLA-partially and fully matched unrelated, as well as matched related donors. The aim of this study was to investigate the impact of PTCY on the incidence of chronic GvHD, disease relapse and infections because of possible cellular immune impairment. Fifty-one consecutive patients who underwent allogeneic hematopoietic stem cell transplant from an unrelated donor were studied. PTCΥ was administered on days +3 and +4 post-transplant at a dose of 50 mg/kg/day. Additional GvHD prophylaxis included tacrolimus and mycophenolate mofetil starting on day +5. In patients with no evidence of GvHD, immunosuppression was gradually tapered, and eventually stopped by day +90. We evaluated the safety and efficacy of PTCY, focusing on toxicity, engraftment, aGvHD and cGvHD frequency, relapse, non-relapse mortality, survival and immune reconstitution. We performed 51 unrelated donor transplants with PTCY from January 2021 to May 2023 in patients with a median age of 52 (18-69) years. The indication for transplant was AML/MDS (n=36), NHL/CLL, (n=2), pMF (n=2), CML (n=2) και ALL (n=9). Disease risk index (DRI) was intermediate for most patients (34/51), 7 had low and 10 had high DRI. Thirty-seven patients received myeloablative conditioning and 14 reduced-intensity. Overall, three patients were transplanted from a matched sibling, 36 from an 8/8 matched unrelated donor and 12 from an 7/8 HLA-matched unrelated donor. All patients received a peripheral blood graft with a median CD34+ cell dose of 6.97×10e6/kg (3.27-15.82). The most common toxicities observed were mucositis (n=34), diarrhea (n=40), vomit (n=12), liver toxicity (n=3) and acute kidney injury (n=4) - which in all cases were grade Ι-ΙΙ. Engraftment, defined as neutrophils >500/μl, was achieved in 46 cases in a median of 16 (13-27) days. Three patients died before day +28 due to NRM and two additional patients with myelofibrosis and MDS respectively had primary graft failure. The cumulative incidence of aGvHD, grade ΙΙ-IV at 100 days was 46.7% (95%CI: 34.1-63.8). However, aGvHD, grade ΙΙΙ-IV was only 3.9% (95%CI: 1.0-15.5). The cumulative incidence of cGvHD at 18 months was 13,4% (95%CI: 5.8-31.0), with only one case of extensive cGvHD. In a median follow-up of 14.5 (1-28) months, disease free (DFS) was 76,8% (95%CI: 65.5-90.0) at 18 months and overall survival (OS) was 76.3% (95%CI: 64.9-89.8) and 71.6% (95%CI: 58.6-88.3) respectively. There was no statistically significant difference in survival rates for patients who received an HLA mismatched and those who received an HLA-matched unrelated-donor transplant. cGvHD-Relapse-Free Survival was 63.5% (95%CI: 50.3-80.1). Two patients died of their disease and non-relapse mortality was 19.1%. Specifically, 6 patients died of Infections, 1 due to primary graft failure, 1 due to heart failure, and 1 due to Idiopathic Pulmonary Syndrome. Immune reconstitution at month 6 was 420 cells/ul (20-2225) and 179 (13-594) for CD8 and CD4 respectively. At month 12 CD8 and CD4 were 552 (55-3002) and 364 (60-787). 31 viral infections were documented in 28 patients (Respiratory viruses n=13, BKV n=11, CMV n=7). In regard with opportunistic infections, 2 patients had fungal infections and 1 patient atypical mycobacterium. We used PTCY for GvHD prophylaxis in mismatched, full-matched unrelated and matched sibling donor transplants with peripheral blood stem cell graft. This approach was well tolerated. Engraftment was documented in most patients, with a low incidence of grade III-IV aGvHD. Importantly cGvHD, which is the most important barrier in unrelated donor transplant, was substantially low with this method. Cellular immune reconstitution was adequate for CD8 but somewhat impaired for CD4. However, the incidence of viral and opportunistic infections was not increased. Overall, there are implications that PTCY may revert the negative impact of HLA mismatch on the outcomes of unrelated donor transplantation, while it is also a viable option for MUD and MSD transplant.

Updated Findings of a Phase II Study of Ruxolitinib Pre-, during- and Post-Hematopoietic Stem Cell Transplantation for Patients with Primary or Secondary Myelofibrosis

Background: Despite tremendous progress in the treatment of myelofibrosis in the last decade, the only curative therapy remains allogeneic hematopoietic cell transplantation (HCT). There are now three FDA-approved JAK inhibitors (JAKi) to treat symptomatic splenomegaly and constitutional symptoms in myelofibrosis, although these agents do not prevent disease progression. Discontinuation of JAKi before (pre) HCT is challenging as patients can experience return of symptoms. Thus, ruxolitinib is often continued during HCT in an off-label fashion; however, little is known about the safety and efficacy of this approach. Furthermore, there is increasing use of ruxolitinib after (post) HCT, following its approval in treating refractory graft-versus-host disease (GVHD). Methods: We previously reported the interim results of a phase II, multicenter, investigator-initiated trial investigating ruxolitinib given pre-, during- and for 1-year post-HCT for patients with primary or secondary MF (NCT03427866). Here we present the results of the study with full accrual of 43 patients. The primary endpoint was 1-year GVHD-free, relapse-free survival (GRFS). Secondary endpoints included overall (OS) and progression free survival (PFS), engraftment, and incidence of acute and chronic GVHD. Patients were treated with reduced intensity conditioning with fludarabine (30mg/m 2/d x 5 days) and melphalan (100 or 140mg/m 2 x 1). Patients received peripheral blood stem cell grafts from either 7/8 or 8/8 HLA-matched donors. GVHD prophylaxis consisted of standard tacrolimus and methotrexate. Results: 44 patients with myelofibrosis were enrolled and 43 underwent HCT between 9/2018 and 5/2023. Median age was 66 (range 46-75), 37% were female (n=16), and most patients had DIPSS intermediate-2/high risk (n=36), 60% (n=27) were receiving ruxolitinib prior to enrolling on the study. Most (n=35) received 8/8 matched unrelated transplants and had measurable splenomegaly by either ultrasound or CT (n=40) prior to HCT. At baseline, 25 patients had JAK2 mutation, 8 had CALR and 5 had MPL mutations, 13 patients had ASXL1 mutations (Figure 1). No differences in outcomes were observed by pre-HCT mutational status. At day 100, all but one patient had undetectable mutations by next generation sequencing. The most common grade 3/4 hematologic adverse events (AE) were anemia (n=12), thrombocytopenia (14), neutropenia (n=11). Non-hematologic grade 3/4 events included hypertriglyceridemia (n=3). Median time to neutrophil engraftment was 15 days (range 4-38), one patient did not engraft. Median time to platelet (>20x10 9) engraftment was 25 days (range 11-145). At day 30, 2 patients had a donor-cell chimerism below 90% (one ultimately relapsed) and at day 100, 2 patients had chimerism below 90 (one ultimately relapsed). With a median follow up among survivors of 13 months (range 0.7 - 26), 1-year GRFS was 74%. OS, PFS and cumulative incidence of NRM and disease relapse were, 86%, 79%, 10%, and 10%, respectively (Figure 2). The cumulative incidence of grade III-IV acute GVHD at 6 months was 2.4%. Only one patient had grade IV acute GVHD. One year moderate/severe chronic GVHD was 11%In univariable analysis, thrombocytopenia of <40 x 10 9/L pre-HCT was associated with worse OS (1-yr OS: 64% vs 92%, p=0.008). Splenomegaly (defined as >17 cm) was associated with worse PFS (1-yr PFS: 100% vs 64%, p=0.046). There was an improvement in OS in patients that received ruxolitinib and achieved response pre-HCT compared to those that did not but this did not achieve statistical significance (1-yr OS 100% vs 74%, p=0.054). Discussion: This study is the first, multicenter trial to test ruxolitinib use pre-, during- and post-HCT in patients with myelofibrosis. Our results demonstrate safety and feasibility of this approach. Favorable rates of engraftment were observed with ongoing ruxolitinib administration even through hematopoietic nadir. We continue to demonstrate favorable outcomes of PFS, OS and GRFS with a low incidence of severe acute and chronic GVHD. Our results strongly support the use of ruxolitinib in the peri- and post-transplant setting for patients with myelofibrosis undergoing HCT.

Phase II Trial of Reduced-Intensity Fludarabine, Melphalan, and Total Body Irradiation Conditioning Results in Favorable Outcomes with Haploidentical Donor Peripheral Blood Stem Cell Transplant

Introduction High risk hematologic malignancies are curable with allogeneic hematopoietic cell transplant (HCT). Haploidentical (haplo) HCT with post-transplant cyclophosphamide (PTCy) has improved access to transplant in patients lacking an HLA matched donor. However, traditional reduced-intensity conditioning (RIC) with fludarabine/cyclophosphamide/total body irradiation (TBI) for haplo HCT with PTCy is marred by high rates of relapse. This study seeks to optimize the RIC haplo HCT with PTCy platform in older adults with a slight intensification in conditioning and the use of peripheral blood stem cell (PBSCT) grafts in lieu of bone marrow grafts (BMT). Additionally, tacrolimus was substituted by sirolimus based on prior phase II data suggesting favorable rates of graft-versus-host disease (GVHD) after haplo PBSCT with PTCy (Bejanyan, Blood Adv. 2021). We hypothesized that this approach could successfully reduce the risk of relapse and improve disease-free survival (DFS) without substantially increasing non-relapse mortality (NRM). Methods: This single arm, single center phase II trial of haplo PBSCT used a RIC regimen consisting of Melphalan 70 mg/m 2 IV on day -6, fludarabine 30mg/m 2 IV daily on days -6 to -2, and TBI 200 cGy on day -1 (NCT04191187). Subjects received haplo PBSCT with a target dose of 5 x 10 6 CD34+ cells/kg and capped at 7 x 10 6 CD34+ cells/kg. GVHD prophylaxis consisted of PTCy 50 mg/kg on days +3 and +4, mycophenolate mofetfil (MMF) 1 gram three times daily from days +5 to +35, and sirolimus initiated at a target trough of 8-14 ng/mL on day +5 and tapered off between days +100 and +180. Eligibility criteria required a hematologic malignancy as the indication for allogeneic HCT and either age >55 years or a hematopoietic cell transplant comorbidity index (HCT-CI) > 3. The primary endpoint of this study is DFS with a hypothesis that the experimental regimen would have an 18 month DFS of 60% compared to a historic DFS of 40% and 90% power to reject the null hypothesis. Results: A total of 34 subjects were treated on the trial. The median age was 66 years (range: 31-74 years). The HCT-CI score was > 3 in 41%. Acute myeloid leukemia (AML, 44%) was the predominant indication for transplant followed by myelodysplastic syndromes (MDS, 29%). Nine patients (26%) were from underrepresented racial/ethnic minorities (those other than non-Hispanic white). Five patients (15%) received tacrolimus for GVHD prophylaxis before a protocol amendment changed the regimen to sirolimus for the remaining 29 patients (85%). The median follow-up time for 25 surviving patients was 28.5 months . The trial met the primary endpoint with 18-month DFS of 70.5% (90% CI > 59.1%, p=0.002). We observed no primary graft failure events. Neutrophil engraftment by day 30 was achieved in 30 patients (88%), and platelet engraftment by day 60 was achieved in 28 patients (82%). The cumulative incidence of grade II-IV acute GVHD at day 100 was 11.8% (95% CI: 3.6-25.1%). The cumulative incidence of moderate to severe chronic GVHD at 18 months was 9.1% (95% CI: 2.2-22.0%). At 18 months, the cumulative incidence of NRM was 17.7% (95% CI: 7.0-32.2%) and of relapse was 11.9% (3.7-25.4%). GVHD-free relapse-free survival at 18 months was 61.4% (95% CI: 42.9-75.5%) and the overall survival at 18 months was 73.3% (95% CI: 54.9-85.1%). Conclusion: In this completed phase II trial of fludarabine, melphalan 70 mg/m 2, TBI with haplo PBSCT and PTCy/sirolimus/MMF, the primary endpoint was met with an 18-month DFS that was superior to historical control. This was attributable to low rates of relapse without excessive NRM. Acute and chronic GVHD rates were favorable. Pharmacogenomic and pharmacokinetic correlative data is pending and will be included in the final presentation. These promising results merit further investigation to potentially establish this platform as a new standard approach to RIC haplo PBSCT.

Total Marrow and Lymphoid Irradiation (TMLI) -Based Conditioning Regimen in Patients with Acute Leukemia Undergoing Allogeneic Hematopoietic Cell Transplant with Measurable Residual Disease

Relapse of primary hematologic malignancy remains the leading cause of treatment failure afterallogeneic Hematopoietic cell transplantation (allo-HCT). Multiple studies have shown that presence of measurable residual disease (MRD), at the time of HCT, irrespective of the detection method, is associated with lower overall survival (OS) and relapse-free survival (RFS). In a recent meta-analysis of 11,151 patients with AML, Short et al., reported 5-year disease-free survival (DFS) of 25% for patients who have detectable MRD pre -HCT, compared to 64% for those without MRD. The 5-year OS was 34% and 68% for patients with or without MRD at HCT (JAMA Oncology 2020). In AML patients with genomic evidence of MRD before allo-HCT, myeloablative conditioning (MAC) regimens result in improved survival when compared to reduced intensity conditioning (RIC); (Hourigan et al, JCO 2020). Our center has pioneered and developed Total marrow and lymphoid irradiation (TMLI) that allows for delivery of higher dose of radiation to the sites with high disease burden (bone marrow and lymphoid organs), while sparing healthy tissues. In an ongoing Phase 2 trial (NCT02094794), we delivered TMLI (20Gy) with high dose chemotherapy to patients with high-risk leukemias (ALL or AML) undergoing allo-HCT from an HLA matched or mismatched donor. Here, we report on the results of our prospective analysis on a subset of patients of NCT02094794 who underwent allo-HCT with morphologic remission but had detectable MRD status in their pre HCT bone marrow biopsy (n=30). Conditioning regimen comprised of TMLI on days -9 to -5 (total of 20Gy), etoposide (60 mg/kg) on day -4, and cyclophosphamide (100mg/kg) on day -2. Unmanipulated peripheral blood stem cells (PBSC) graft from 10/10 HLA matched related or unrelated donor was infused on day 0 and graft-versus-host disease (GVHD) prophylaxis was tacrolimus and sirolimus-based. TMLI was delivered by intensity modulated radiation therapy (IMRT). MRD assay was done by either University of Washington flow cytometry with 0.01% - 0.1 % sensitivity, FISH/conventional cytogenetics, or NGS-based assays. Patient and HCT characteristics are summarized in Table 1. Briefly, the median age at the time of HCT was 36 years (range: 16-59) with the majority of patients receiving PBSC grafts (87%). Donors were match related (67%) or match unrelated (33%). The underlying diagnosis was AML (n=22; 73%), ALL (n=7; 23%), or CML-BC (n=1; 3%). Karnofsky performance status was 80-100 in 90% of patients and HCT-CI ≥3 was seen in 35% of patients. Pre-HCT MRD + status was documented by Flow cytometry MRD assay in 17 patients [AML (n=14) and ALL (n=3)], with mean MRD positivity of 2.9 % (range: 0.02-5.6%) for AML and 0.4% (range: 0.003-1.7%) for ALL patients. Thirteen additional patients had MRD positive status confirmed by FISH/cytogenetics (n=6 patients) or NGS (n=7). With a median duration follow up of 22.3 months (range: 6.0-81.1), the 1 and 2-year OS were 68% (95% CI: 0.47-0.82) and 59% (95% CI: 0.38-0.75), respectively. The 1 and 2- year disease-free survival (DFS) were 55% (95% CI: 0.34-0.71) and 45% (95% CI: 0.26-0.63), while the cumulative incidence (CI) of relapse at 1 and 2 years were 28% (95% CI: 0.13-0.46) and 33% (95% CI: 0.16-0.51), respectively. At day 100 post-HCT, the CI of NRM was 7%, grade 2-4 acute GVHD was 40% and Grade 3-4 acute GVHD was 13%. The CI of NIH moderate- severe chronic GVHD at 1- year was 35% (95% CI: 0.17-0.52). No difference in outcomes were noted in patients who were MRD positive by UWFC vs FISH/NGS testing. In conclusion, TMLI based conditioning regimen in patients undergoing allo-HCT with MRD+ disease from matched donors is associated with improvement in 2-year RFS/OS and is associated with low NRM compared to historic controls. The current phase 2 study is enrolling patients at our center.