HLA-matched Hematopoietic Stem Cell Transplantation Research Articles

Post-Transplant T Cell Clonotype Diversity Is Associated with Survival in Patients with TP53-Mutated Acute Myeloid Leukemia

Background TP53 mutations are associated with unfavorable outcomes in various cancer types and present an obstacle to achieving sustained remission in acute myeloid leukemia (AML). Even with allogeneic hematopoietic stem cell transplantation (HSCT), the risk of relapse approximates 80%, with dismal long-term survival. This could imply methods of immune escape in TP53 mutated leukemias that allow them to evade the graft-versus-leukemia (GVL) effect, which is a critical mechanism of disease control with HSCT. This study investigates the dynamics of immune reconstitution at single-cell resolution in TP53-mutated AML at pre- and multiple post-HSCT timepoints in patients who either remain in long-term remission or relapse following HSCT. Methods We collected 26 longitudinal bone marrow aspirates and 1 peripheral blood sample from 12 patients with TP53-mutated AML who underwent HSCT at the Dana-Farber Cancer Institute between 2015-21. All patients received HLA-matched T-cell replete HSCT in morphologic remission, with standard tacrolimus/methotrexate-based prophylaxis and peripheral blood stem cells. We categorized the patients into two cohorts based on their treatment response: long-term remission (>3.5 years, cohort 1, n=4) or relapse (cohort 2, n=8). We performed paired 5' single-cell RNA and T cell receptor (TCR) sequencing on sorted viable mononuclear from each sample and CD3+ T cells from selected samples using 10x Genomics procedures. In total, we analyzed 44 samples (27 mononuclear, 17 CD3+), resulting in 250,640 high-quality single-cell transcriptomes, including 137,649 T cells and NK cells. Data analysis was performed using Cell Ranger Multi (10x Genomics) and R 4.3 with the tidyverse 2.0, Seurat 5.1, and scRepertoire 1.1 packages. Results We focused our initial analysis on the 2-6-month post-HSCT period that is critical for immune reconstitution. This included eight samples from seven patients in the two cohorts (1: remission, 2: relapse, pink box in Figure 1). Of these 7 patients, 4 were in the relapse cohort, with these relapses occurring >4 months post-HSCT. No significant difference in T-cell chimerism was seen between these 7 patients in this 2-6 month period. Based on canonical gene expression signatures, we annotated cell types and selected T cells for further evaluation (Figure 2). First, we found that the CD4/CD8 ratio differed between the cohorts, indicating a relative CD8+ T cell expansion in the relapse cohort (cohort 1: 1.69, cohort 2: 0.55, P<0.0001). Second, we found lower TCR diversity in the relapse cohort (inverse Simpson index, cohort 1: 234.6, cohort 2: 83.0, P<0.0001). This difference remained significant when subsampling the same number of T cells per patient and when restricting all samples to the 3 to 4-month timepoint. Considering the disparity in CD8 T cell numbers between the cohorts, we also analyzed repertoire diversity, specifically in CD8+ T cells, and found that the observed significance persisted. To further investigate TCR diversity, we overlaid T cell clonotypes (cells that share the same TCR sequence) in a UMAP visualization, which highlighted increased hyperexpanded CD8+ T cells in the relapse cohort (Figure 2). Finally, we found that greater clonotype expansion was correlated with the expression of key T cell genes, including CD8A, CD8B, PRF1, NKG7, and GZMA. These results suggest that cytotoxic CD8+ T cells link lower TCR diversity to the likelihood of disease relapse. Conclusions By performing deep analysis of single-cell gene expression linked to TCR clonotypes, we discover new insights into the biology of hematopoietic reconstitution after HSCT of patients with TP53-mutated AML. Despite having analyzed a limited number of samples, we find that patients who remain in long-term remission have significantly higher CD8+ TCR diversity between 2-6 months post-HSCT, while those who relapsed between 5-18 months post-HSCT showed lower TCR diversity and hyperexpanded CD8+ clones at the same timepoints. This work suggests that early post-HSCT TCR diversity could be a biomarker associated with GVL and warrants further study. In ongoing and future analyses, we will mine the current dataset for further insights into the factors influencing survival in TP53-mutated AML patients. We also plan to extend the measurement of TCR diversity as a predictor of survival for validation in a larger cohort.

CR1 Favors PTCy-haplo over UCB in Allogeneic Stem Cell Transplantation for Adult Patients with AL

[Introduction] HLA-haploidentical stem cell transplantation using post-transplant cyclophosphamide (PTCy-haplo) and umbilical cord blood transplantation (UCB) are alternative to HLA-matched hematopoietic stem cell transplantation (HSCT). However, advantage of PTCy-haplo versus UCB has not been determined in patients with acute lymphoblastic leukemia (ALL). Thus, we compared post-HSCT outcomes of PTCy-haplo and UCB in adult patients with ALL to determine patient subgroups who benefit from PTCy-haplo. [Methods] We analyzed data on adult patients (≥16 years) with ALL who underwent HSCT with PTCy-haplo or single-unit UCB between January 2013 and December 2021, using the Japanese nationwide registry database. Endpoints of the analysis included overall survival (OS), disease-free survival (DFS), cumulative incidence of relapse, non-relapse mortality (NRM), engraftments, acute and chronic graft-versus-host disease (GVHD), and infections. In addition to multivariate analysis, propensity score matching was performed as a sensitivity analysis. [Results] We analyzed 1999 patients (PTCy-haplo, 330; UCB, 1669). Median age at HSCT was 44.5 years (range, 16-73) in PTCy-haplo and 45 years (range, 16-74) in UCB group. The proportion of patients who underwent HSCT in the first complete remission (CR1) (PTCy-haplo, 50.3%; UCB, 48.8%) and median time from diagnosis to HSCT (PTCy-haplo, 7.8 months; UCB, 7.7 months) were comparable between the groups. Regarding conditioning, the proportion of those who received myeloablative conditioning (PTCy-haplo, 50.6%; UCB, 54.8%), or conditioning with total body irradiation (PTCy-haplo, 82.4%; UCB, 79.3%) were comparable between the groups. Multivariate analyses showed that PTCy-haplo recipients had comparable 3-year OS (54.1% vs. 52.3%; adjusted hazard ratio [aHR], 0.93; p = 0.51) and DFS (49.7% vs. 47.1%; aHR, 1.06; p = 0.60) with UCB recipients. PTCy-haplo recipients had a significantly higher 3-year relapse rate (32.2% vs. 26.4%; aHR, 1.47; p = 0.01), but had a significantly lower 3 -year NRM (18.1% vs. 26.5%; aHR, 0.70; p = 0.03) as compared to UCB along with significantly faster neutrophil (median, 16 days vs. 20 days) and platelet (median, 30 days vs. 42 days) engraftments, and lower incidence of viral, fungal and bacterial infections. There was no significant difference in cumulative incidence of acute GVHD (30.9% vs. 36.4%, grade II-IV at 100 days; p = 0.15), or chronic GVHD (22.5% vs. 20.9% at 3 years; p = 0.85) between the two groups. These comparable OS and DFS between the two groups were confirmed after minimizing differences in patient characteristics with propensity score matching analysis (Figure 1A). Then, to identify patient subgroups who benefit from PTCy-haplo rather than UCB transplant, we performed subgroup analyses of OS. Significantly favorable effects of PTCy-haplo over UCB were observed in patients with CR1 at HSCT (HR, 0.66; p = 0.03). Indeed, propensity score matching analysis showed that PTCy-haplo recipients had a significantly higher 3-year OS (76.4% vs. 67.6%; HR, 0.57; p = 0.01) and DFS (70.5% vs. 60.6%; HR, 0.66; p = 0.03) than UCB recipients, along with a significantly lower NRM (12.9% vs. 26.3%; HR, 0.39; p < 0.01), and a comparable relapse rate (16.6% vs. 13.1%; HR, 1.41; p = 0.25) with UCB. These results suggest that the low NRM in PTCy-haplo transplant outweighs potential concerns of relapse among patients transplanted in CR1 (Figure 1B). By contrast, in patients with ≥ 2 nd CR or non-CR at HSCT, survival benefits of PTCy-haplo were not observed. [Conclusions] Our results suggest that while both PTCy-haplo and UCB are suitable alternative donor sources for adult ALL patients without HLA-matched donors, in patients with CR1, selection of PTCy-haplo rather than UCB improves post-transplant prognosis.

Relevance of vitamin D in patients undergoing HLA matched allogeneic stem cell transplant for acute leukemia

BackgroundIn hematopoietic stem cell transplant (HSCT), vitamin D deficiency has been variably associated with increased complications, primarily graft versus host disease (GvHD), with a potential impact on survival. Results from various studies however, have not been consistent. This analysis was conducted to study the impact of peri-transplant vitamin D levels on transplant outcomes in patients with acute leukemia (AL) who underwent HLA matched (related/unrelated) HSCT. MethodsThis was a single center retrospective study. Patients of AL including Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML) or Mixed Phenotypic Acute Leukemia (MPAL) who underwent fully matched or 9/10 transplants (related/unrelated) between 2008 and 2019 were included. Vitamin D deficiency was defined as serum 25-hydroxy vitamin D3 levels ≤20 ng/ml. Those with deficiency received replacement with oral vitamin D at a dose of 60,000 IU weekly for 8 weeks followed by maintenance with daily vitamin D (800 IU/day). Vitamin D levels were repeated at 4 months from start of replacement. For patients who received correction, repeat levels >20 ng/ml were considered replete. Based on vitamin D levels in the peri-transplant period (within 120 days of transplant), patients were categorised as either vitamin D replete (> 20 ng/ml) or deplete (≤ 20 ng/ml). Peri-transplant vitamin D status was correlated with transplant outcomes. ResultsOf the 133 patients included, 31 were deplete (median vitamin D 15.0 ng/ml) and 102 were replete (median vitamin D 34 ng/ml) at time of transplant. Both groups were matched for age, diagnosis, EBMT score and disease risk index (DRI). There were no differences in time to neutrophil or platelet engraftment, CMV reactivation, acute GvHD (aGvHD) or chronic GvHD (cGvHD) between the two groups. Relapse rate, Progression Free Survival (PFS) and Overall Survival (OS) were also comparable between the 2 groups. ConclusionThe incidence of vitamin D deficiency was high in our patient cohort. Patients who were vitamin D deficient at the time of transplant did not have inferior outcomes, suggesting a limited role of vitamin D in influencing transplant outcomes.

Post-Transplantation Cyclophosphamide-Based Graft-versus-Host Disease Prophylaxis

BackgroundIn patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT), a calcineurin inhibitor plus methotrexate has been a standard prophylaxis against graft-versus-host disease (GVHD). A phase 2 study indicated the potential superiority of a post-transplantation regimen of cyclophosphamide, tacrolimus, and mycophenolate mofetil.MethodsIn a phase 3 trial, we randomly assigned adults with hematologic cancers in a 1:1 ratio to receive cyclophosphamide–tacrolimus–mycophenolate mofetil (experimental prophylaxis) or tacrolimus–methotrexate (standard prophylaxis). The patients underwent HSCT from an HLA-matched related donor or a matched or 7/8 mismatched (i.e., mismatched at only one of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci) unrelated donor, after reduced-intensity conditioning. The primary end point was GVHD-free, relapse-free survival at 1 year, assessed in a time-to-event analysis, with events defined as grade III or IV acute GVHD, chronic GVHD warranting systemic immunosuppression, disease relapse or progression, and death from any cause.ResultsIn a multivariate Cox regression analysis, GVHD-free, relapse-free survival was significantly more common among the 214 patients in the experimental-prophylaxis group than among the 217 patients in the standard-prophylaxis group (hazard ratio for grade III or IV acute GVHD, chronic GVHD, disease relapse or progression, or death, 0.64; 95% confidence interval [CI], 0.49 to 0.83; P=0.001). At 1 year, the adjusted GVHD-free, relapse-free survival was 52.7% (95% CI, 45.8 to 59.2) with experimental prophylaxis and 34.9% (95% CI, 28.6 to 41.3) with standard prophylaxis. Patients in the experimental-prophylaxis group appeared to have less severe acute or chronic GVHD and a higher incidence of immunosuppression-free survival at 1 year. Overall and disease-free survival, relapse, transplantation-related death, and engraftment did not differ substantially between the groups.ConclusionsAmong patients undergoing allogeneic HLA-matched HSCT with reduced-intensity conditioning, GVHD-free, relapse-free survival at 1 year was significantly more common among those who received cyclophosphamide–tacrolimus–mycophenolate mofetil than among those who received tacrolimus–methotrexate. (Funded by the National Heart, Lung, and Blood Institute and others; BMT CTN 1703 ClinicalTrials.gov number, NCT03959241.)

Triple-Drug Graft-versus-Host Disease Prophylaxis after HLA-Matched Unrelated Donor Nonmyeloablative Allogenic Hematopoietic Stem Cell Transplantation

Adding sirolimus to graft-versus-host disease (GVHD) prophylaxis with cyclosporin and mycophenolate mofetil (MMF) reduced the risk of grade II-IV acute GVHD after nonmyeloablative (NMA) allogenic hematopoietic stem cell transplantation (HSCT) with an HLA-matched unrelated donor in a randomized clinical trial. We analyzed real-life data to investigate the impact of implementing the triple-drug regimen with cyclosporin, MMF and sirolimus as standard GVHD prophylaxis after NMA HSCT with an HLA-matched unrelated donor at our institution. We studied all adult patients (age ≥18 years) who underwent NMA HSCT with an HLA-matched unrelated donor at Rigshospitalet, Copenhagen University Hospital, Denmark between 2018 and 2021 and received GVHD prophylaxis with cyclosporin, MMF and sirolimus (triple-drug group [TDG]). Comparisons were made with a historical cohort who received tacrolimus and MMF as GVHD prophylaxis after HLA-matched unrelated donor NMA HSCT between 2014 and 2017 (control group [CG]). Outcomes were grade II-IV and grade III-IV acute GVHD, chronic GVHD, relapse, nonrelapse mortality (NRM) andoverall survival (OS). A total of 264 patients were included (TDG, n = 137; CG, n = 127). Median age was 66 years (interquartile range [IQR], 58 to 69 years) in the TDG and 63 years (IQR, 57 to 68 years) in the CG. Acute myeloid leukemia and myelodysplastic syndrome were the most frequent indications for HSCT in both groups (TDG, 33% and 23%, respectively; CG, 36% and 22%, respectively). The cumulative incidence at day +110 of grade II-IV GVHD was 17% (95% confidence interval [CI] 11% to 23%) in the TDG versus 29% (95% CI, 21% to 37%) in the CG (P = .02, Gray's test) and that of grade III-IV acute GVHD was 3% (95% CI, 0 to 6%) versus 5% (95% CI, 1% to 8%), respectively (P = .4, Gray's test). In a Cox regression model adjusted for age, donor age and female donor to male recipient the risk of grade II-IV acute GVHD was lower in the TDG compared to the CG (hazard ratio [HR], .51; 95% CI .30 to .86; P = .01). The 2-year OS was 77% (95% CI, 70% to 84%) in the TDG and 69% (95% CI, 61% to 77%) in the CG (P = .04), and this difference remained significant after adjustment for age and Karnofsky Performance Status (HR, .65; 95% CI, .42 to .99; P = .04). The 2-year cumulative incidences of chronic GVHD, relapse and NRM were 60% (95% CI, 51% to 69%), 21% (95% CI, 13% to 28%), and 12% (95% CI, 6% to 17%), respectively, in the TDG and 62% (95% CI, 54% to 71%), 27% (95% CI, 19% to 35%) and 14% (95% CI, 8% to 20%), respectively, in the CG. Multivariable analyses revealed no difference in the risk of chronic GVHD (HR, .91; 95% CI, .65 to 1.26; P = .56), relapse (HR, .70; 95% CI, .42 to 1.15; P = .16) or NRM (HR, .56; 95% CI, .31 to 1.05; P = .07). After changing the standard GVHD prophylaxis in patients undergoing NMA HSCT with an HLA-matched unrelated donor from tacrolimus and MMF to cyclosporin, MMF and sirolimus, we observed a reduction in the incidence of grade II-IV acute GVHD and improved 2-year OS.

Adverse impact of a high CD4/CD8 ratio in the allograft may be overcome by methotrexate- but not mycophenolate- or posttransplant cyclophosphamide-based graft versus host disease prophylaxis.

A high CD4/CD8 T cell ratio in hematopoietic stem cell transplant (HSCT) allografts was observed to predict graft-versus-host disease (GVHD) and non-relapse mortality (NRM) but has not been comparatively examined in settings of various GVHD-prophylaxis regimens. This retrospective monocentric study included all consecutive HSCT performed with peripheral blood stem cells (PBSC) between January 2000 and June 2021. The impact of the graft CD4/CD8 ratio was analyzed in three cohorts with different GVHD-prophylaxis platforms. In the cyclosporine/mycophenolate-mofetil (CSA/MMF) cohort (n=294, HLA-matched HSCT), a high (> 75th percentile) CD4/CD8 ratio was associated with increased overall mortality (HR 1.56; P=0.01), increased NRM (HR 1.85; P=0.01) and GVHD-associated mortality (HR 2.13; P=0.005). In the post-transplant cyclophosphamide (PTCy)/tacrolimus/MMF cohort (n=113, haploidentical-related or mismatched-unrelated HSCT), a high CD4/CD8 ratio was associated with increased overall mortality (HR 2.07; P=0.04) and aGVHD3-4 (HR 2.24; P=0.02). By contrast, in the CSA/methotrexate (CSA/MTX) cohort (n=185, HLA-matched HSCT) the CD4/CD8 ratio had no significant impact on any of the investigated endpoints. A high CD4/CD8 ratio in the allograft has an adverse impact on GVHD and survival in CSA/MMF- and PTCy-based HSCT, while MTX-based prophylaxis may largely alleviate this important risk factor.

Curing Systemic Lupus Erythematosus and Severe Aplastic Anemia with an Allogeneic Transplant: A Case Report and Brief Literature Review

Systemic lupus erythematosus (SLE) is an autoimmune disease that can affect many organs, including the bone marrow. Aplastic anemia (AA) is a rare but serious complication of this condition. Patients with AA secondary to SLE are usually treated with immunosuppression but less is known about hematopoietic stem cell transplantation (HSCT) as a therapeutic option. We present the case of a 22-year-old Latin-American woman with a known diagnosis of SLE referred to our center due to pancytopenia. She presented joint pain, fatigue, oral ulcers, a non-scarring alopecia, and anemic syndrome. A diagnostic workup revealed severe aplastic anemia (sAA). She received immunosuppressive therapy with corticosteroids, cyclosporine, danazol, and rituximab, which led to a transient partial response. An outpatient HLA-matched related allogeneic HSCT was performed. She received 5.9 x 106 CD34+ cells/kg after conditioning with high-dose cyclophosphamide plus fludarabine and anti-thymocyte globulin. She achieved a complete response and continues in remission with no signs of graft-versus-host disease (GVHD) or SLE activity at 38 months of follow-up. Even though allogeneic hematopoietic stem cell transplantation is not defined as a first-line treatment for severe AA in SLE, the procedure resulted in the complete remission of both related autoimmune diseases in this patient.

Long-Term Outcome of Low Dose of Anti-t-Lymphocyte Globulin in the Prophylaxis of Graft-Versus-Host Disease in Unrelated Myeloablative Hematopoietic Stem Cell Transplantation

INTRODUCTION In vivo T-cell depletion with Anti-T-lymphocyte globulin (ATLG; Grafalon) is one of the strategies used in the graft-versus-host disease (GVHD) prevention in the setting of hematopoietic stem cell transplantation (HSCT). One open-label randomized trial in HLA-matched unrelated myeloablative HSCT have reported the reduction of cGVHD with the incorporation of ATLG (60mg/Kg), however progression-free survival (PFS) and overall survival (OS) were lower than in the placebo group1. Other randomized trial in related myeloablative HSCT, using an inferior dose of ATLG (30mg/Kg), have reported this reduction in GVHD results without increasing transplantation-related mortality (TRM) or disease relapse (DR)2. Based on this, the policy in our center is to use a lower dose of ATLG (21mg/Kg) in unrelated myeloablative HSCT. The aim of this study is to analyze the outcome of this strategy in terms of GVHD results, PFS, OS, TRM and DR. METHODS We conducted a retrospective study in all patients who underwent a HLA-matched unrelated myeloablative HSCT at our institution between 2013 and 2021. Patients received a total dose of 21mg/Kg of ATLG divided into three doses of 7mg/Kg on days -3, -2 and -1. The primary endpoints were cumulative incidence (CI) of aGVHD, CI of moderate-severe cGVHD, PFS and OS. Secondary endpoints were TRM, CI of DR and toxicity. RESULTS Sixty-one patients were included. The median age was 51 years. Thirty-seven (60,7%) were men. Twenty-three (37,7%) patients had an acute myeloid leukemia (AML). Eighteen (29,5%) patients had an HLA-mismatched donor. Forty-two (68,8%) receive a Bu-Flu conditioning. Cyclosporine combined with mycophenolate mofetil (MMF) or short course of methotrexate (MTX) as GCVH prophylaxis was used in 43 patients (70,5%). Clinical characteristics of patients are showed in table 1. The median follow-up of the alive patients was 36 months. A median value of 0.1 x 106/L lymphocytes was observed on the first day of infusion. In 35 patients (57,4%) a grade 1 to 2 infusion reaction was reported and in 3 (4,9%) a grade 3 to 4. Those three patients did not complete all infusions, the rest of patients did. Median time to neutrophil engraftment (3 consecutive days of >500/μL) was 15 days (IR, 13-18) and to platelet engraftment (3 consecutive days of self-supported platelets >20000/μL) was 17 days (IR, 15-23). CI of aGVHD grade 2 to 4 within 180 days after HSCT was 39,5% (95% CI, 27,1% to 51,6%) and CI of aGVHD grade 3 to 4 was 11,6% (95% CI, 5,0% to 21%), CI of moderate-severe cGVHD at two years was 20,9% (95% CI, 11,4% to 32,4%). PFS at two years was 59,6% (95% CI, 47% to 71%) and OS at two years was 71,8% (95% CI, 59% to 83%). The cGVHD relapse free survival (cGRFS) at two years was 36,2% (95 CI, 24% to 48%). The CI of RD at two years was 23,3% (95% CI, 13% to 35,3%) and the TRM at two years was 17,1% (95% CI, 8,7% to 27,9%). Median time to cyclosporine or tacrolimus withdrawal was 6,7 months (interquartile range, 5-8 months) and median of use of any type of immunosuppressors (i.e corticosteroids) from the day of HSCT was 6,9 months (interquartile range, 5-10 months). In 4 (6,6%) patients a primary graft failure was reported, and two patients developed post-transplantation lymphoproliferative disease. CONCLUSSIONS The use of ATLG at dose of 21 mg/Kg in HLA-matched unrelated myeloablative HSCT offers promising results of OS and PFS with a low TRM, despite being related to a higher incidence of aGVHD and cGVHD than previously trials were the ATLG dose was higher. It showed a better profile of tolerability and adverse effects. More investigation should be undertaken to determine the appropriate dose of ATLG in this type of HSCT. REFERENCES 1. Soiffer RJ, Kim HT, McGuirk J, Horwitz ME et al. Randomized, Double-Blind, Phase III Clinical Trial of Anti-T-Lymphocyte Globulin to Assess Impact on Chronic Graft-Versus-Host Disease-Free Survival in Patients Undergoing HLA-Matched Unrelated Myeloablative Hematopoietic Cell Transplantation. J Clin Oncol. 2017 Dec 20;35(36):4003-4011. doi: 10.1200/JCO.2017.75.8177. Epub 2017 Oct 17. PMID: 29040031; PMCID: PMC8462523. 2. Kröger N, Solano C, Wolschke Cet al. Antilymphocyte Globulin for Prevention of Chronic Graft-versus-Host Disease. N Engl J Med. 2016 Jan 7;374(1):43-53. doi: 10.1056/NEJMoa1506002. PMID: 26735993. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal Dynamics after Haematopoietic Stem Cell Transplantation Using Genome-Wide Somatic Mutations

Background Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment received by over 40,000 patients/year. Despite being an established treatment for 50 years, fundamental questions remain about the dynamics of long-term stem cell engraftment and immunological reconstitution in humans. How many of the >108 transplanted CD34+ cells contribute to long-term hematopoiesis? How does increasing age of donor and recipient reshape clonal dynamics of HSCT? Are the same HSCs responsible for both myeloid and lymphoid reconstitution? Does HSCT increase mutation rates or clonal evolution? Methods We used spontaneous somatic mutations occurring genome-wide in single hematopoietic stem and progenitor cells (HSPCs) to reconstruct high-resolution lineage trees of the hematopoietic system after HSCT. We analysed blood samples from both the donor (D) and recipient (R) for 10 HLA-matched sibling HSCTs - the duration of time elapsed since transplant ranged from 9 to 31 years, meaning that we could study genuinely long-term reconstitution in the recipient, compared to the unperturbed hematopoiesis in the donor. For each individual (D and R), peripheral blood CD34+ HSPC-derived colonies were grown on methylcellulose medium. Whole genome sequencing was performed on 100-300 colonies per individual, generating a final dataset of 3,413 genomes. Somatic mutations from each colony's founder cell were identified and used to build phylogenies for each D/R pair (Figure). For 7/10 pairs, we then used ultra-deep targeted sequencing to assess clonal contributions in sorted mature D/R cell subsets, including monocytes, B-cells and T-cells. Approximate Bayesian computation was used to estimate numbers of engrafting HSCs. RESULTSIn most cases, 10,000 - 50,000 HSCs engrafted and contributed to long-term reconstitution during HSCT. However, grafts from older donors, or those with lower CD34+ cell doses tended to have lower estimates, some as low as <1,000 cells.Recipients had decreased clonal diversity compared to their donors, equivalent to 8-10 years of additional ageing.We detected 93 individual clones that contributed >1% of the population of at least one mature cell type. Each of these expanded clones was detectable in both the donor and the recipient, confirming that they had originated prior to HSCT.Only ~10% of these expanded clones carried mutations in genes known to contribute to clonal hematopoiesis, suggesting that there are many other genes that can influence stem cell dynamics after HSCT.The sizes of these expanded clones often varied dramatically between donors and recipients, up to 25-fold differences in some instances.The population bottleneck caused by HSCT was not sufficient to explain the differences in clonal composition between recipient and donor, suggesting that there are additional HSCT-specific selective pressures during stem cell harvest, in vitro manipulation, infusion, homing and engraftment.While large proportions of mature myeloid and B-cell populations usually derived from the identified clones, this was not true for T-cell populations. This suggests that following HSCT, T-cells are largely derived from stem cells that do not contribute substantially to myeloid reconstitution.HSPC mutation burdens in recipients were similar to their donors, contradicting previous hypotheses that the proliferative stress of HSCT would lead to excess mutations. CONCLUSION Our findings reveal that tens of thousands of HSCs typically engraft during HSCT, but with significant loss of clonal diversity in recipients compared to their unperturbed donors. HSCT-specific selective pressures shape dynamics of hematopoietic reconstitution in the recipient, with substantial differences in clonal composition between myeloid and T-lymphoid populations. This study shows the power of using somatic mutations as clonal marks for lineage tracing in real-world HSCT in humans, an approach we believe will be a new gold standard for understanding the impact of different stem cell sources and transplant protocols. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell RNA-Seq Reveals an Interferon-Driven Inflammatory CD4 Naïve T Cell Subpopulation at Day 100 in Hematopoietic Stem Cell Transplant Patients That Ultimately Develop Chronic Gvhd

Background: Chronic GVHD (cGVHD) is the most significant cause of long-term morbidity and mortality after hematopoietic stem cell transplant (HCT). Improving patient outcomes requires a better understanding of biological mechanisms that underly disease, in order to develop novel risk-assignment biomarkers and therapeutic strategies. Naïve T cells (TN) are a prevailing risk factor for cGVHD, with mouse studies and clinical trial data demonstrating that graft depletion of TN cells decreases the frequency of cGVHD. Nevertheless, little is known about how TN cells progress to alloreactivity and, ultimately, to clinical cGVHD. In this study, we compared single-cell RNA sequencing (scRNA-Seq) from patients receiving unrelated donor transplantation (URD HCT) who developed mod-severe cGVHD, versus patients who were free from cGVHD, to identify disease-specific subpopulations of T cells that preceded disease. Methods: This analysis was designed to identify immune profiles in blood at Day 100 that could distinguish patients who ultimately developed cGVHD from those who remained free of this disease. Blood was drawn from patients with hematologic malignancies undergoing 8/8 HLA-matched URD HCT after myeloablative conditioning, and who received CNI/MTX GVHD prophylaxis. We focused on Day 100 samples from patients with Gr 0-2 acute GVHD who did not require systemic steroids as our starting population, and then divided these patients into those who subsequently developed moderate-severe cGVHD ('cGVHD' group, n=8; median cGVHD onset = 181 days) and patients who never developed mod-severe cGVHD through at least 2 years post-HCT (termed 'operationally tolerant' patients, 'OpTol', n = 24). Flow cytometry of longitudinal samples was performed using 16 ten-color panels, characterizing T cell reconstitution, activation, exhaustion, and naïve/memory subpopulations. Bulk RNA sequencing was performed on flow-sorted CD4 T cells (within 3h of collection) at Day 100 on patients from two central lab sites (cGVHD = 7, OpTol = 9). Statistical modeling was performed using DESeq2, followed by differential expression and Gene Set Enrichment Analysis (GSEA) with clusterProfiler. scRNA-Seq was performed on flow-sorted CD4 T cells using the 10X platform (cGVHD = 7, OpTol = 5). scRNA-Seq data was batch corrected with Harmony, annotated with SignacX and analyzed with Seurat. Results: Flow cytometry identified a higher proportion of CD4TN but not CD8TN cells in cGVHD patients that persisted longitudinally through Day 100 (25.9% vs. 10.0% at Day 100, p=0.03). Bulk transcriptome GSEA of Day 100 CD4 T cells confirmed enrichment of TN cells in cGVHD patients, but also revealed additional gene sets relating to interferon signaling (Fig. 1A). In contrast, OpTol patients revealed gene signatures relating to memory T cells and quiescence. scRNA-Seq enabled the discovery of CD4 T cell types/states enriched in patients who would develop mod-severe cGVHD. We profiled >100,000 CD4 T cells from Day 100 samples from cGVHD and OpTol patients. Unsupervised clustering identified two populations that were enriched in cGVHD vs. OpTol patients (Fig. 1B): interferon-stimulated CD4TN cells and interferon-stimulated CD4 memory cells. Both subsets show significantly higher expression of an interferon-response signature relative to all other cells (TN = 0.93, Tmem = 1.61, others = 0.14, p < 0.0001). The CD4TN subset possessed a signature for IFN-α-related stimulation (including IFI6, ISG15, IFI44L, EPSTI1) as well as IFN-γ-related genes (including STAT1, MX1). These findings further substantiate the IFN gene signature identified by bulk RNA-Seq at day 100 in cGVHD vs. OpTol patients. Conclusions: These data identified an inflammatory CD4TN cell subpopulation at day 100 in patients that go on to develop cGVHD, and suggest that CD4TN cells may harbor pathogenic cells that could predispose patients to allo-inflammatory complications. scRNA-Seq revealed a strong type I and type II interferon signaling signature in this CD4TN subset, and also identified a novel population of interferon-stimulated memory cells which may indicate a more differentiated population of alloreactive cells. These data highlight an interferon-driven transcriptome signature preceding the onset of cGVHD. Continued exploration of these cells may yield new risk-assignment biomarkers and nominate therapeutic approaches to prevent cGVHD in high-risk patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ex Vivo Generation and Infusion of Anti-Minor Histocompatibility Antigen Expanded T Cells for Patients Who Relapse after Allogeneic HLA-Matched Stem Cell Transplantation

Rationale Long-term clinical remissions of hematologic malignancies after allogeneic hematopoietic stem cell transplantation (HSCT) largely rely on the graft-versus-leukemia effect (GVL). In a fully HLA-matched setting, the GVL effect is mediated primarily by T cell immune responses against host minor histocompatibility antigens (MiHAs) found on the surface of malignant cells. Upon relapse of leukemia following HSCT, donor lymphocyte infusions (DLI) have unfortunately failed to exert efficient GVL activity. This is likely due to the insufficient number of anti-MiHA T cells within DLIs and to the risk of graft-vs-host disease (GVHD) that prevents the administration of high numbers of T cells. To generate potent and selective anti-leukemia activity, we have developed a strategy for the ex vivo selection and expansion of T cells targeting MiHAs preferentially expressed on hematopoietic cells (Perreault, Leukemia 2016). Thus, these cells should display augmented GVL immune reactivity while harboring a low risk of causing GVHD. To our knowledge, this is the first clinical study targeting several different molecularly defined MiHAs and on different HLA molecules. Patients and Methods In an open-label, multi-center phase 1 clinical trial (CR-MIHA-001; NCT 03091933), patients with hematologic malignancies who relapsed after an allogeneic HLA-matched HSCT were treated with sibling donor-derived, ex vivo- expanded T cells selected and expanded for reactivity toward tissue-restricted recipient MiHAs (GLIDE: Guided Lymphocyte Immunopeptide Derived Expansion). After donor cell collection, patients received fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2 for 3 days followed by infusion of GLIDE, consisting of donor T-lymphocytes expanded ex vivo following exposure to dendritic cells coated with immunogenic peptides (MiHAs) present on the surface of host hematopoietic cells, at a single target dose of 4x107 viable T-cells/m2. A second dose of GLIDE, aiming at 3-5 times the original dose, could be administered to patients at a minimum of 6 weeks after the first infusion. GLIDE cell characteristics were evaluated using multi-parameter flow cytometry and TCR sequencing. Patients were followed up for one year post-GLIDE infusion for toxicity and clinical outcome measures. Results Ten patients, median age of 58 (range 33-69), 4 females/6 males with AML (n=7), NHL (1), CLL (1), and myelofibrosis (1) were treated with GLIDE. These patients had relapsed at a median of 12 months (range 5-126 mo) after allogeneic HSCT. GLIDE cells were personalized to target single MiHAs in 9 patients and two MiHAs in 1 patient. Overall, GLIDE cell products were directed at 9 different MiHAs on 6 different HLA-class 1 molecules (HLA-A02:01, A24:02, B07:02, B18:01, B44:02, and B44:03). A first dose of GLIDE was administered at 4.0x107 CD3+ cells/m2 to 8 patients, and 2 patients received slightly lower doses (2.6 and 3.7 x 107 CD3+ cells/m2). Final GLIDE products consisted mainly of T-cells (median: 98,7%), with CD8>CD4>NKT>NK cells. Most CD8+ T cells were effector memory and central memory T cells (Figure 1). At the end of the expansion period, CD8+ T cells expressed activation markers (CD25+ = 67%) with low levels of PD1+, PDL1+, CD57+ and KLRG1+ as markers of exhaustion. Not surprisingly, GLIDE cells had a more restricted TCR β chain profile than initial lymphopheresis cells. Six patients received a second dose of GLIDE (mean: 12 x 107 CD3+ cells/m2) at 53 days after the first infusion (median, range: 48-118 days). There were no infusional toxicities, and cells were well-tolerated by patients. Only one patient developed thrombocytopenia and subsequent chronic GVHD. There were no other adverse events potentially related to GLIDE. According to TCR β sequencing data, T cells present in the adoptively transferred anti-MiHA GLIDE product persisted in peripheral blood up to one year after infusion. Survival at one year was observed in 4 patients with 3 patients in sustained remission (Figure 2). Conclusion This pilot study demonstrates the feasibility of anti-MiHA T cell ex vivo generation (GLIDE) and safety of its administration to patients relapsing after allogeneic HSCT, as well as the clinical potential of these cells. The persistent remissions and low occurrence of GVHD support further development of GLIDE to target acute myeloid leukemia and other hematologic malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The effect of intensity of conditioning regimen on the outcome of HSCT in children with sickle cell disease.

Allogeneic hematopoietic stem cell transplantation (HSCT) provides a cure for patients with sickle cell disease (SCD). This study describes the effect of conditioning regimen intensity on HSCT outcomes among children younger than 14 years with SCD. Transplants from HLA-matched related donors (MRD) and unrelated donors (MUD) using either myeloablative conditioning (MAC) regimens or reduced intensity conditioning (RIC) regimens were considered. Event-free survival (EFS) was the primary endpoint. Secondary endpoints included overall survival (OS) and occurrence of GVHD. 48 SCD patients underwent HSCT, 45 (93.8%) patients had MRD, 1 (2.1%) had 9/10 related donor, and 2 (4.1%) had MUD. The median age at transplant was 8.6 years (range, 3.1-13.8). Conditioning regimens were myeloablative (MAC) in 41 (85.4%) patients and of reduced intensity in 7 (14.6%) patients. EFS at 2 years was 100% among MAC group compared to 29% in the RIC group (p<.001). The median follow-up was 43.4 months (range 26.8-134). All events in the RIC group were secondary graft failure. However, OS was 100% in both groups at 2 years. Acute GVHD II-IV was diagnosed in 2 (4.1%) patients. Chronic GVHD occurred in 2 (4.1%) patients. GVHD did not occur in patients who underwent MUD HSCT. MAC in children with SCD is well tolerated and associated with an excellent outcome for HLA-matched HSCT in SCD. There was a high rate of secondary graft failure with the use of RIC. Future studies are needed to optimize RIC regimens in HSCT of children with SCD.

Outcomes in Hematopoietic Stem Cell Transplantation for Congenital Amegakaryocytic Thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare, inherited bone marrow failure syndrome. Hematopoietic stem cell transplantation (HSCT) is considered a curative treatment option, but existing descriptions of patient and transplant characteristics and outcomes after related and unrelated donor HSCT are sparse. We describe outcomes after HSCT for congenital amegakaryocytic thrombocytopenia (CAMT; n = 86) from 2000 to 2018. We conducted an analysis of data collected by the Center for International Blood and Marrow Transplant Research on patients with CAMT receiving therapeutic allogeneic HSCT. The predominant donor type was HLA-matched or mismatched unrelated donors (n = 58, 67%). The remaining included HLA-matched sibling (n = 23, 27%) and HLA-mismatched relative (n = 5, 6%). The predominant graft types were bone marrow (n = 53, 62%) and cord blood (n = 25, 29%). The median age at transplantation was 3 years, with 82 of 86 patients being transplanted aged ≤10 years. The 5-year graft failure-free and overall survival were 83% (95% confidence interval [CI], 74-90) and 86% (95% CI, 78-93), respectively. An examination for risk factors confirmed mortality was higher after HLA-mismatched relative and mismatched unrelated donor HSCT compared to HLA-matched sibling and matched unrelated donor HSCT (hazard ratio 3.52, P = .04; 75% versus 93%). The 1-year incidence of graft failure was 19% after HLA-mismatched HSCT (n = 32) compared to 7% after HLA-matched HSCT (n = 54, P = .15). Day-100 grade II-IV acute graft-versus-host disease was 13%, 26%, and 30% after HLA-matched sibling, HLA-matched and mismatched unrelated donor HSCT. The 5-year incidence of chronic graft-versus-host disease was 33% with 24 of 28 patients having received grafts from HLA-matched (n = 13) and mismatched unrelated (n = 11) donors. Although HLA-matched donors are preferred, HLA-mismatched donors also extend survival for CAMT.

Mismatch in SIRPα, a regulatory protein in innate immunity, is associated with chronic GVHD in hematopoietic stem cell transplantation

AbstractRecent compelling evidence showed that innate immune effector cells could recognize allogeneic grafts and prime an adaptive immune response. Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily receptor that is expressed on myeloid cells; the interaction between SIRPα and its ubiquitously expressed ligand CD47 elicits an inhibitory signal that suppresses macrophage phagocytic function. Additional studies showed that donor-recipient mismatch in SIRPα variants might activate monocytic allorecognition, possibly as the result of non-self SIRPα-CD47 interaction. However, the frequency of SIRPα variation and its role in hematopoietic stem cell transplantation (HSCT) remains unexplored. We studied 350 patients with acute myeloid leukemia/myelodysplastic syndrome who underwent HLA-matched related HSCT and found that SIRPα allelic mismatches were present in 39% of transplantation pairs. SIRPα variant mismatch was associated with a significantly higher rate of chronic graft-versus-host disease (GVHD; hazard ratio [HR], 1.5; P = .03), especially de novo chronic GVHD (HR, 2.0; P = .01), after adjusting for other predictors. Those with mismatched SIRPα had a lower relapse rate (HR, 0.6; P = .05) and significantly longer relapse-free survival (RFS; HR, 0.6; P = .04). Notably, the effect of SIRPα variant mismatch on relapse protection was most pronounced early after HSCT and in patients who were not in remission at HSCT (cumulative incidence, 73% vs 54%; HR, 0.5; P = .01). These findings show that SIRPα variant mismatch is associated with HSCT outcomes, possibly owing to innate allorecognition. SIRPα variant matching could provide valuable information for donor selection and risk stratification in HSCT.

Early and Profound Exhausted Phenotype of Central Memory and Memory Stem T Cells in the Bone Marrow of AML Patients Characterizes and Predicts Relapse after Allo-HSCT

Background: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse.Patients and Methods: We longitudinally analysed T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA-matched (20 pts) or HLA haploidentical (Haplo, 12 pts) donors. BM and PB samples were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained, at 1 year (CR; 16 pts). 10 healthy donors (HD) were used as controls. Samples were analysed by polychromatic flow cytometry for the expression of inhibitory receptors (IR) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression. Results were also analysed with BH-SNE, an unbiased dimensionality reduction algorithm. To evaluate T-cell function and specificity, CD107a expression, cytokine profiles and killing of autologous blasts were quantified.Results: After Haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were expressed in BM and PB T cells at higher percentage when compared to HD and independently from the clinical outcome. Conversely, after HLA-matched HSCT, patients who relapsed displayed a higher frequency of BM-infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p<0.05) and HD (p<0.01). When investigating each T-cell subset, in both HD and CR pts the IR expression was confined to effector memory and effectors; at relapse, instead, PD-1, 2B4, KLRG1 and Tim-3 were more commonly expressed in BM-infiltrating central memory (TCM) and memory stem T cells (TSCM, p<0.01). Interestingly, at relapse leukemia expressed PD-L1 (9/9 cases) and Galectin-9 (6/9) and the latter linearly correlated with Tim-3+ BM CD8 T-cell percentages, suggesting a preferential role for this immunomodulatory axis after HSCT. Based on phenotype similarities, the BH-SNE algorithm separated HD and HSCT samples in bi-dimensional maps and identified T cell clusters that associated with relapse (32) and were characterized by T cells co-expressing high levels of multiple IRs.Consistent with the exhausted phenotype, BM CD8 T-cells at relapse displayed lower degranulation ability and IL-2 production compared to CR (p<0.05). The ability to degranulate and to produce pro-inflammatory cytokines of BM-T cells expressing at least 1 inhibitory receptor (IR+) could be recovered in five patients upon polyclonal stimulation in vitro with high doses of IL-2, suggesting that T-cell dysfunction can be efficiently reversed. Upon challenge with autologous leukemic blasts, higher proportion of IR+ lymphocytes upregulated HLA-DR (p<0.05), granzyme A and B (p<0.001) and killed blasts (p<0.05) compared to IR- T cells. In addition, IR+ but not IR- cells were efficiently expanded by stimulation with matched Leukemic Antigen-Presenting Cells (fold increase 146 and 3, respectively).Interestingly, we observed that, at day 60 after HLA-matched HSCT, BM CD8 TSCM and TCM cells from patients who shall subsequently relapse already expressed PD-1, Tim-3 and LAG3 at higher percentage when compared to patients who shall maintain long-term CR (p<0.05). Co-expression of at least 3 IR on TSCM and TCM T-cells, but not on effectors, or TEM, at this early time-point was associated with significantly decreased disease free survival.Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing patients includes PD-1, CTLA-4, 2B4 and Tim-3, expressed on early differentiated T cells. Exhausted BM-T cells are preferentially enriched in specificities directed against leukemic blasts. Taken together, these results highlight a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Interestingly, the analysis on early time points suggests that the exhausted phenotype may be predictive of leukemia relapse. [Display omitted] DisclosuresBondanza:TxCell SA: Research Funding; Menarini Biotech: Consultancy; Molmed Spa: Research Funding. Bonini:MolMed: Consultancy.

Highly Variable Frequencies of Donor Antigen-Specific Naive Cells May Dictate the Immunogenicity of Minor Histocompatibility Antigens after Hematopoietic Stem Cell Transplantation

In unrelated HLA-matched hematopoietic stem cell transplantation there are thousands of mismatches of non-synonymous SNPs between donor and patient, some of which result in polymorphic peptides presented in MHC context, known as Minor Histocompatibility Antigens (MiHAs). As these peptides are foreign to the donor immune cells they may drive allogeneic immune response, causing both graft-versus-host disease and beneficial graft-versus-tumor effect. But due to the phenomenon of immunodominance some of MiHAs are much more immunogenic than the others. We propose that antigenic dominance could be at lest partially explained by the frequencies of precursor T cells in the donor immune system and thus in the graft. As donor lymphocytes have never encountered polymorphic human peptides, before transplantation MiHA specific T cells reside exclusively in naïve T cell (TN) subpopulation. To test the proposed hypothesis we compared the frequencies of naïve T cells, specific for highly immunogenic minor antigen HA-2 and less immunogenic ACC-1Y in individuals negative for these antigens.We have selected a panel of healthy volunteers bearing HLA-A*02:01 and HLA-A*24:02 which can present HA-2 and ACC-1Y respectively. The panel was genotyped for SNP encoding these antigens using rapid multiplex MiHA genotyping approach developed in our laboratory. We have chosen donors having HA-2 -/- and ACC1Y -/- genotypes as sources of MiHA specific TN cells and HA-2 +/- and ACC1Y +/- donors as negative controls. Dendritic cells were derived from donor peripheral blood monocytes, loaded with appropriate peptides and co-cultured with autologous TN cells in extreme limiting dilution assay, which allowed us to calculate TN frequencies.We have determined that frequency of specific TN was substantially higher for immunodominant HA-2 antigen than for subdominant ACC-1Y (5 x 10-6 vs. 2 x 10-7, p=3 x 10-9), which correlates with immunodominance of HA-2 and falls in line with the initial hypothesis. The variation of frequencies of TN specific to various MiHAs could be explained either by the peptide properties or by the influence of the negative selection on initial T cell repertoire. ACC-1Y has the alternative allelic variant (ACC-1C) presented in the thymus, which leads to elimination of all cross-reactive clones, whereas alternative allelic variant of HA-2 is cleaved by proteasome and is not presented to the immune system. Further studies of antigen-specific TN frequencies would facilitate creating a complex model of MiHA immunogenicity, which could be used to predict allogeneic immune response after transplantation. Funding was provided by Russian Science Foundation grant 17-15-01512 DisclosuresNo relevant conflicts of interest to declare.

Post-Transplant Cyclophosphamide Contributes to Impaired Graft-Versus-Leukemia Effects and Regulates Graft-Versus-Host Disease in the Murine Stem Cell Transplant Model

IntroductionPost-transplant cyclophosphamide (PTCy) for controlling alloreactivity after allogeneic hematopoietic stem cell transplantation (HSCT) is an effective prophylaxis for acute and chronic graft-versus-host disease (GVHD). HLA-haploidentical HSCT using PTCy is a promising option for patients with hematological malignancy who require allo-HSCT and lack an immediately available HLA-identical related or unrelated donor. In contrast, a relatively high relapse rate was reported in HLA-haploidentical HSCT using PTCy with non-myeloablative conditioning compared to HLA-matched related or unrelated HSCT using conventional GVHD prophylaxis. The effects of GVHD have been shown to correlate partially with those of graft-versus-leukemia (GVL), and therefore, PTCy treatment might contribute to the increase in relapse. However, the rationale for the high relapse rate in HSCT using PTCy compared to conventional HSCT remains poorly understood. To address this issue, we explored the effect of PTCy on survival, leukemia progression and immune reconstruction in a murine transplant model.Material & MethodsTwelve-week old C57BL/6 (B6) mice were irradiated with 5 Gy in one fraction on day 0 and transplanted with 1.0 × 107 spleen cells and 1.0 × 107 bone marrow cells from Balb/c mice. All mice were purchased from Japan SLC Inc. and maintained in a pathogen-free environment. Mice consumed normal chow and autoclaved hyperchlorinated water (pH 4) after the transplantation. Mice were not treated (control) or received cyclophosphamide 100 mg/kg at days 3 and 4 intraperitoneally. A leukemia cell line that was derived from the B6 bone marrow and transformed with the BCR/ABL fusion gene (BA-1) was used for leukemogenic experiments. One million BA-1 cells were intravenously infused into the recipients 25 (±4) days after transplantation with no additive treatment. We continuously examined weight, GVHD score, peripheral blood cell counts, and peripheral blood cell prolife of the recipient mice, and finally investigated spleen and bone marrow cells. The chimeric balances among host-residual H-2Db+ cells and donor graft-derived H-2Dd+ cells in CD3+ T cells were monitored. To evaluate the function of activated T cells in the spleen, CD62L+, CD44+, and PD-1+ marker levels in CD8+ and CD4+ cells were quantitatively monitored at various time points. GVHD scoring was based on weight loss, posture, activity, fur texture, and skin integrity (severity score 0 to 2 for each variable, maximum index 10) as previously reported. Leukemia progression was defined by the presence of 0.05% BA-1 cells in peripheral blood mononuclear cells as detected by GFP expression. All experimental procedures were approved by an institutional review committee of the Osaka City University.ResultsControl B6 recipients developed severe weight loss and GVHD after transplantation and died of GVHD at around day 60, whereas the recipients with PTCy had significantly improved weight, GVHD score, and survival compared to control mice (figure1). These results were comparable to those of previous reports. In the leukemogenic experiment, the control recipients showed significantly longer survival than the ones with PTCy (figure2). Recipients with PTCy had a higher rate of leukemia progression compared to the control at day 30 after BA-1 injection, although there were no significant differences in the chimeric balances among donor graft-derived H-2Dd+ T cells both before and at day 7, 14, 21, 28, and 35 after BA-1 injection. Prior to BA-1 injection, CD62L+ CD44- naïve CD8+/CD4+ T cell number was low, and CD62L- CD44+ effector memory CD8+/CD4+ T cell number was high in the spleen of the control recipients compared to that in PTCy recipients. Moreover, the numbers of CD44+ PD-1+ memory CD8+/CD4+ T cells were also overwhelmingly high in the spleen of control recipients. These results showed that use of PTCy after allo-HSCT promoted T cell tolerance to alloantigens by eliminating antigen-exposed T cells and reduced the probability of rejection in leukemia as a consequence.ConclusionOur data indicated that PTCy treatment after allo-HSCT contributes to impaired GVL effects by reducing the number of antigen-exposed T cells and improves GVHD regulation in a murine stem cell transplant model. [Display omitted] DisclosuresHino:Novartis: Research Funding.

Allogeneic hematopoietic stem cell transplantation from family members other than HLA-identical siblings over the last decade (1991-2000)

AbstractThe reported outcome of hematopoietic stem cell transplantation (HSCT) from HLA-mismatched family members has been inconsistent. The object of this study was to evaluate the true impact of HLA-mismatch by using recent data from a homogenous population, excluding HSCT procedures that used graft manipulations, and by considering genotypic matching. Clinical data of 2947 patients who underwent allogeneic HSCT for leukemia or myelodysplastic syndrome were extracted from the database of the Japan Society for Hematopoietic Cell Transplantation. The main outcome measures were survival and the incidence of graft-versus-host disease (GVHD). The presence of serologic HLA-mismatch, higher age, and high-risk disease were identified as independent risk factors for both shorter survival and the development of grade III to IV acute GVHD. The impact of HLA-mismatch on survival was more relevant in standard-risk patients. These findings persisted when we used genotypic HLA matching. Survival after one-locus–mismatched HSCT was equivalent to that after HLA-matched unrelated HSCT. We concluded that when a one-locus–mismatched family donor is available for high-risk patients, immediate HSCT using this donor is warranted. In standard-risk patients, however, survival after one-locus–mismatched HSCT is significantly shorter than that after HLA-matched HSCT, and the indications for HSCT should be considered carefully.

Survival in Patients Who Underwent HLA-Haploidentical Stem Cell Transplantation in Two Centers in Colombia

Introduction: Allogeneic hematopoietic stem cell transplantation (HCT) is a potentially curative treatment for several malignant and non-malignant hematological diseases. However, sometimes it is challenging to find human leukocyte antigen (HLA)-matched related or unrelated donors, especially in minority populations such as Hispanics. Transplantation of T cell replete HLA haploidentical graft (HaploHCT) followed by a high dose post-transplantation cyclophosphamide (PTCy) to eradicate alloreactive T cells is an option for populations with low donor availability. HaploHCT has emerged as an effective and safe strategy in this population (Luznik L et al. BBMT 2008), but data in Hispanics is scarce. Objective: The aim of our study was to describe the clinical characteristics and assess overall survival at 100 days, 1- and 3-years of patients who underwent haploHCT in two Colombian reference centers. Methods: An observational retrospective study was conducted at two tertiary referral centers in Colombia. Patients who underwent haploHCT at Clinica FOSCAL and Centro Medico Imbanaco between January 2013 and January 2020 were selected. Demographic and clinical characteristics where analyzed using descriptive statistics. The Kaplan-Meier method was used to assess overall survival (OS) rates and the log-rank test was used to compare survival rates between groups. All data were analyzed using R statistical software®. Results: Seventy-six patients were included. Mean age at transplantation was 34 years (range 18-60). Forty-two (57.89%) patients were female. Average body mass index was 19.86kg/m2. The majority of patients (57%) had a pre-transplantation ECOG performance status of 2. The most common indication for haploHCT was acute lymphoblastic leukemia (55.26%), followed by acute myeloid leukemia (23.64%). A sibling was the donor in 69.74% of the cases. Forty-seven (61.84%) patients were ABO-matched group. Peripheral blood stem cells were the graft source in 96% of the patients. The average number of infused CD34+ cells was 11.15 x 106/kg. Fludarabine-melphalan was the most commonly used conditioning regimen (57%), followed by fludarabine-busulfan-thiotepa (39%). Graft-versus-host disease (GVHD) prophylaxis consisted of PTCy (50 mg/kg/day) on days 3 and 4 after HCT and a calcineurin inhibitor plus mycophenolate mofetil from day 5 to day 35 post-HCT. Median time to neutrophil engraftment (neutrophils &amp;gt; 0,5x109/L) was 15 days (range 10-33), while platelet engraftment, defined as as the second day of unsupported platelet count of ≥20 × 109, occurred at a median time of 13 days (range 5-38) post-transplantation. On day 28 post-HSCT, 93.4% of the patients had achieved 100% chimerism. OS was 91% (95%CI 84-97.5) at 100 days, 81% (95%CI 72-90) at 1-year, and 77% (95%CI 68-88) at 3-years after HaploHCT. C onclusion: The results of our haploHCT retrospective study in a Hispanic population are comparable to several other cohorts that have reported similar outcomes of haploHCT compared to HLA-matched HCT. The OS rates reported in our study suggest that haploHCT is a viable option in patients without an HLA-matched related or unrelated donor available, especially in populations with lower rates of suitable donors. Acknowledgements: We deeply thank Gonzalo Gutiérrez García, M.D. for his leadership and guidance implementing the haploHCT program in Clinica FOSCAL. Disclosures Peña: Roche: Honoraria. Salazar:Janssen: Honoraria; Novartis: Honoraria. Sandoval-Sus:MorphoSys US: Consultancy; Janssen: Consultancy; Massive Bio: Consultancy; Celgene: Speakers Bureau. Sossa:Novo: Honoraria; Takeda: Honoraria; Astellas: Honoraria; Roche: Honoraria.

Naive T-cell depletion in stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (HCT) is curative in many patients with advanced hematopoietic malignancies. Donor T cells not only facilitate engraftment and protect against opportunistic pathogens and residual disease, but can also cause graft-versus-host disease (GVHD), with significant morbidity and mortality. Complete T-cell depletion can not only substantially reduce GVHD rates but can also delay immune reconstitution and increase rates of opportunistic infections and relapse. Murine models have shown that naive T cells (TNs) consistently cause severe GVHD, whereas memory T cells cause milder or no GVHD and have critical graft-versus-tumor function. Informed by experiments performed in murine models of HCT, clinical trials are being conducted to evaluate TN-depleted peripheral blood stem cell (PBSC) grafts. These trials are showing very low rates of chronic GVHD and of serious acute GVHD in the HLA-matched HCT setting, with lower frequencies of opportunistic infections than after fully T-cell-depleted HCT and no apparent increase in relapse rates. Randomized clinical trials are ongoing, comparing standard unselected HCT with TN-depleted PBSCs and other promising GVHD-reduction strategies. Correlative laboratory studies will clarify how antitumor function is retained in TN-depleted HCT and inform strategies to further augment graft-versus-leukemia in patients at a high risk of relapse. TN depletion of donor lymphocyte infusions and of haploidentical stem cell grafts is also being investigated.