HLA-haploidentical Hematopoietic Stem Cell Transplantation Research Articles

Effect of Immunopeptidome Gap Caused By Recipient-Donor HLA Mismatch in Umbilical Cord Blood Transplantation and HLA-Haploidentical Hematopoietic Stem Cell Transplantation

Introduction The immune response by alloreactive T cells plays a crucial role in allogeneic hematopoietic stem cell transplantation (allo-HCT), causing not only graft-versus-leukemia but also harmful graft-versus-host disease (GVHD). Human leukocyte antigens (HLA), which are highly polymorphic and differ widely among individuals, are the key molecules that present antigens to T cells. This individual variation in antigen-presenting capacity, the immunopeptidome, is thought to be assessable as HLA evolutionary divergence (HED) between alleles. Several autoimmune diseases are explained by HED, and HED is reported to be associated with outcome in HLA-matched allo-HCT. Thus, HED can be a useful indicator of potential immune reactivity. HLA-mismatched allo-HCT, including umbilical cord blood transplantation and HLA-haploidentical allo-HCT (Haplo-HCT), has been increasing in recent years. The combination of HLA mismatch alleles varies from case to case, thus speculating that it causes individual differences in immune responses is easy. HLA mismatches have long been known to be associated with recurrence and GVHD, but a few risk assessment studies have focused on the immunopeptidome gap due to each HLA mismatch in individual cases. Aim This study aimed to examine the prognostic impact of the gap of immunopeptidome in mismatched HLA between recipient and donor in HLA-mismatched allo-HCT. Methods Allo-HCT was performed on 317 patients at our institution from 2006 to 2022. Of the 272 patients with evaluable allele data, 123 patients who underwent more than 3 alleles HLA-mismatched allo-HCT were retrospectively analyzed. Graft sources included cord blood (n=69, 56.1%) and haploidentical stem cells derived from peripheral blood (n=54, 43.9%). The median age at allo-HCT was 44 (range: 16-67) years and the median observation period was 545 [8-5551] days. Diagnoses include acute myeloid leukemia (n=61, 49.6%), acute lymphoblastic leukemia (n=24, 19.5%), malignant lymphoma (n=24, 19.5%), myelodysplastic syndrome (n=10, 8.1%), and others (n=4, 3.2%). The conditioning regimens was non-myeloablative (n=89, 72.4%). GVHD prophylaxis for 54 Haplo-HCT was anti-thymocyte globulin in 27 (50%) and post-transplant cyclophosphamide in 27 (50%) patients. The immunopeptidome gap of HLA mismatch (HED-mis) was evaluated between each mismatched allele. Grantham distance, which predicts amino acids from HLA allelic information and attempts to measure evolutionary distance based on the chemistry of the two amino acids, was used to calculate HED-mis. HLA allele data were drawn from clinical records. Tobias Lenz et al. produced the calculator program (https://hladiv.net/). HED-mis for each HLA class I: HLA-A (HED-mis A), HLA-B (HED-mis B), and HLA-C (HED-mis C) were calculated to evaluate acute and chronic GVHD (aGVHD and cGVHD), cumulative relapse rate (CIR), and overall survival (OS). Result The median [range] of HED-mis for 123 patients was 0.13 [0.00-13.70] for HED-mis A, 7.15 [0.00-11.25] for HED-mis B, and 4.19 [0.00-7.58] for HED-mis C. Patients were divided into 4 groups at each HED-miss value quartile: Q1, Q2, Q3, and Q4. Non-Q4 (Q1, Q2 and Q3 with the lower gaps) and Q4 with the highest gap of HED-mis A, B, and C were compared to confirm the effect of gap size. Univariate analysis revealed that non-Q4 of HED-mis B was associated with increased aGVHD 2-4 up to 100 days after all-HCT, compared to Q4 (non-Q4 49.8% vs. Q4 25.0%, p=0.017), but was unrelated to the incidence of severe aGVHD 3-4 ( p=0.58). Q4 of HED-mis A demonstrated no difference in aGVHD 2-4 compared to non-Q4 ( p=0.20) but was associated with increased severe aGVHD 3-4 (non-Q4 of 0.06% vs. Q4 of 20.7%, p=0.019, Table and Figure). Similarly, multivariate analysis adjusted for each HED-mis, graft, and conditioning revealed that Q4 of HED-mis A was a risk factor for severe aGVHD 3-4 (hazard ratio [HR]: 3.85, p=0.015), and non-Q4 of HED-mis B was a risk factor for developing aGVHD 2-4 (HR: 3.64, p=0.017). HED-mis C was unrelated to any grade aGVHD, but Q4 of HED-mis C may have a better 2-year CIR in univariate (20.3%, vs. in non-Q4, 46.2%, p=0.055) and multivariate analyses (HR: 0.34, p=0.051). No association was found with each HED gap and each quartile for cGVHD and OS. Conclusion Gaps in the immunopeptidome of HLA-mismatched alleles, especially in HLA-A, should be considered in severe aGVHD development during donor selection.

Efficacy and safety of allogeneic hematopoietic stem cell transplantation in the treatment of 28 patients with hepatitis-related aplastic anemia

Objective: To evaluate the efficacy and safety of HLA-haploidentical hematopoietic stem cell transplantation (allo-HSCT) for hepatitis-related aplastic anemia (HRAA) patients. Methods: Retrospective analysis was performed on hepatitis-associated aplastic anemia patients who received haplo-HSCT at our center between January 2012 and June 2022. October 30, 2022 was the final date of follow-up. Results: This study included 28 HRAA patients receiving allo-HSCT, including 18 males (64.3% ) and 10 females (35.7% ), with a median age of 25.5 (9-44) years. About 17 cases of severe aplastic anemia (SAA), 10 cases of very severe aplastic anemia (VSAA), and 1 case of transfusion-dependent aplastic anemia (TD-NSAA) were identified. Among 28 patients, 15 patients received haplo-HSCT, and 13 received MSD-HSCT. The 2-year overall survival (OS) rate, the 2-year failure-free survival (FFS) rate, the 2-year transplant-related mortality (TRM) rate, the 100-day grade Ⅱ-Ⅳ acute graft-versus-host disease (aGVHD) cumulative incidence rate, and the 2-year chronic graft-versus-host disease (cGVHD) cumulative incidence rate were 81.4%, 81.4% (95% CI 10.5% -20.6% ), 14.6% (95% CI 5.7% -34.3% ), 25.0% (95% CI 12.8% -45.4% ), and 4.2% (95% CI 0.6% -25.4% ), respectively. After transplantation, all patients had no significant liver function damage. Compared with the MSD-HSCT group, only the incidence of cytomegaloviremia was significantly higher in the haplo-HSCT group [60.0% (95% CI 35.2% -84.8% ) vs 7.7% (95% CI 0-22.2% ), P=0.004]. No statistically significant difference in the Epstein-Barr virus was found in the 2-year OS, 2-year FFS, 2-year TRM, and 100-day grade Ⅱ-Ⅳ aGVHD cumulative incidence rates and 2-year cGVHD cumulative incidence rate. Conclusion: Allo-HSCT is safe and effective for HRAA, and haplo-HSCT can be used as a safe and effective alternative for newly diagnosed HRAA patients who cannot obtain HLA-matched sibling donors.

HLA-haploidentical hematopoietic stem cells transplantation with regulatory and conventional T-cell adoptive immunotherapy in pediatric patients with very high-risk acute leukemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is still needed for many children with very high-risk acute leukemia. An HLA-haploidentical family donor is a suitable option for those without an HLA-matched donor. Here we present outcomes of a novel HLA-haploidentical HSCT (haplo-HSCT) strategy with adoptive immunotherapy with thymic-derived CD4+CD25+ FoxP3+ regulatory T cells (Tregs) and conventional T cells (Tcons) performed between January 2017 and July 2021 in 20 children with high-risk leukemia. Median age was 14.5 years (range, 4-21), 15 had acute lymphoblastic leukemia, 5 acute myeloid leukemia. The conditioning regimen included total body irradiation (TBI), thiotepa, fludarabine, cyclophosphamide. Grafts contained a megadose of CD34+ cells (mean 12.4 × 106/Kg), Tregs (2 × 106/Kg) and Tcons (0.5-1 × 106/Kg). All patients achieved primary, sustained full-donor engraftment. Only one patient relapsed (5%). The incidence of non-relapse mortality was 15% (3/20 patients). Five/20 patients developed ≥ grade 2 acute Graft versus Host Disease (aGvHD). It resolved in 4 who are alive and disease-free; 1 patient developed chronic GvHD (cGvHD). The probability of GRFS was 60 ± 0.5% (95% CI: 2.1-4.2) (Fig.6), CRFS was 79 ± 0.9% (95% CI: 3.2-4.9) as 16/20 patients are alive and leukemia-free. The median follow-up was 2.1 years (range 0.5 months-5.1 years). This innovative approach was associated with very promising outcomes of HSCT strategy in pediatric patients.

Updated Analysis on the Outcomes of Children with Acute Leukemia (AL) Receiving an Alpha/Beta T and B-Cell Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation (TBdepl-haploHSCT)

Background: We previously reported (Locatelli et al., Blood 2017) that TBdepl-haploHSCT is a suitable and effective option for children with AL lacking an HLA-compatible donor and/or needing an urgent allograft. This type of transplant is characterized by a low incidence of both acute and chronic graft-versus-host disease (GvHD) and by low non-relapse mortality (NRM), translating into survival outcomes comparable to those of patients transplanted from an HLA-compatible donor. Here we report an update on a cohort of 213 children with AL of our study, with a minimum follow-up of 180 days after the allograft. Patients and methods: Between October 2010 and January 2022, 213 children with AL received TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù in Rome, Italy. All patients were in morphological complete remission (CR) and were prepared to TBdepl-haploHSCT using a fully-myeloablative conditioning regimen including a combination of total body irradiation (TBI) and/or cytotoxic drugs. All patients received also anti-T-lymphocyte globulin (ATLG) before transplantation (12 mg/kg total dose, from days -5 to day -3) to modulate bi-directional donor/recipient alloreactivity and rituximab (200 mg/sqm) on day -1 to prevent post-transplantation EBV-induced lymphoproliferative disorders (PTLD). No patient received any post-transplant pharmacological GvHD prophylaxis. Data were analyzed as of July 15, 2022. Results: Table 1 reports details on characteristics of patients included in the analysis. The median follow-up of surviving patients is 47.6 months (range: 6 months - 11.7 years). Two hundred and eight patients (98% of the total number) engrafted with a median time to neutrophil and platelet recovery of 13 (range 9-24) and 11 (range 7-23) days, respectively. Only 5 patients experienced graft failure; all were rescued with a second allograft. Eleven out of the 213 patients (5%) died of transplant-related complications (4 because of idiopathic pneumonia, 2 each because of disseminated adenovirus infection and thrombotic microangiopathy and 1 each of cardiac insufficiency, combined CMV/rhinovirus pneumonia and sepsis from Pseudomonas aeruginosa), the 5-year cumulative incidence of NRM being 5.2% (95% CI, 2.8-8.8). Forty-four patients relapsed at a median time of 192 days (range 56-1711) after HSCT, the 5-year cumulative incidence of relapse being 22.7% (95% CI, 16.9-29.2). Cumulative incidence of grade II-III acute GvHD was 14.7% (95% CI 10.6-20.3). Three patients and 1 developed gut and liver GvHD, respectively, while for all other patients skin was the sole organ involved. Fourteen out of the 193 patients at risk developed chronic GvHD, in all cases of limited severity, the cumulative incidence of this complication being 8.1% (95% CI 4.8-13.3). The 10-year probability of overall and leukemia-free survival (LFS) were 75.4 (95% CI 68.6-80.9) and 71.6% (95% CI 64.4-77.6), respectively. In univariate analysis, disease status at transplantation (CR1 vs CR2 vs CR>3), use of TBI during the preparative regimen and age at transplant above the median value (9.5 years) were associated with better patient's outcome, mainly because of a reduced incidence of relapse. Notably, among TBI-based regimens, the use of thiotepa and fludarabine was associated with an improved DFS as compared to other combinations (82.4% vs 63.9%, p=0.01). In a multivariable model for LFS including also gender and type of disease, only TBI (hazard ratio (HR) 0.38 (95% CI, 0.17-0.85, p=0.01) and disease status at HSCT (HR 1.72 (95% CI, 1.05-2.79, p=0.02) remained statistically significant. The 10-year GvHD/relapse-free survival was 65.1% (95% CI 57.7-71.4). The median CD3+ cell count on day +30, +90, +180 and +360 were 140, 185, 533 and 1058 /mcl, respectively. Conclusions: TBdepl-haploHSCT is becoming a widespread haplo platform for pediatric patients affected by both malignant and non-malignant disorders. This cohort of homogeneously treated patients, the largest reported so far with a median follow-up of almost 4 years, confirm once more the effectiveness and safety of this approach. Furthermore, this analysis identified subgroups of patients (e.g., those in CR>3) for whom post-transplant strategies aimed at reducing the risk of relapse (the main cause of treatment failure) are desirable. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Debating the Future of Sickle Cell Disease Curative Therapy: Haploidentical Hematopoietic Stem Cell Transplantation vs. Gene Therapy.

Hematopoietic stem cell transplantation (HSCT) is a well-established curative therapy for patients with sickle cell disease (SCD) when using a human leukocyte antigen (HLA)-matched sibling donor. Most patients with SCD do not have a matched sibling donor, thereby significantly limiting the accessibility of this curative option to most patients. HLA-haploidentical HSCT with post-transplant cyclophosphamide expands the donor pool, with current approaches now demonstrating high overall survival, reduced toxicity, and an effective reduction in acute and chronic graft-vs.-host disease (GvHD). Alternatively, autologous genetic therapies appear promising and have the potential to overcome significant barriers associated with allogeneic HSCT, such as donor availability and GvHD. Here the authors each take a viewpoint and discuss what will be the future of curative options for patients with SCD outside of a matched sibling transplantation, specifically haploidentical HSCT vs. gene therapy.

Acute rhabdomyolysis in hepatitis-associated aplastic anemia patient undergoing allogeneic hematopoietic stem-cell transplantation: case report and literature review

BackgroundHepatitis-associated aplastic anemia (HAAA) is a specific type of aplastic anemia, and hematopoietic stem-cell transplantation (HSCT) is recommended as the first-line. Acute rhabdomyolysis (AR) during hematopoietic stem-cell transplantation (HSCT) is a rare, serious complication, with only 10 cases reported in the world so far.Case presentationHerein, we present a case of AR developing during HLA-haploidentical HSCT in a 55-year-old man who suffered from HAAA. On day 7 after stem cell transfusion, the patient reported a muscle pull in thigh and complained of muscle swelling, pain and change in urine color. Despite the timely diagnosis (based on the levels of myoglobin and creatine kinase, and muscle MRI findings, etc.) and rapid hydration and alkalization, the situation progressed dramatically, and the patient died of multi-organ failure during the preparation for continuous renal replacement therapy (CRRT). Five days after his death, the whole-exome sequencing result confirmed that the patient had a germline missense mutation in SCN4A I 1545 V and ACTN3 R577X.ConclusionAR is a rare but threatening complication during HSCT, especially in cases with kidney dysfunction. The creatine kinase level may not truly and completely reflect the severity and prognosis for cases with localized lesion. We suggest that genetic analysis should be performed for better understanding the pathological changes of AR during HSCT, especially for patients with bone marrow failure.

Severe acute interstitial lung disease after BNT162b2 mRNA COVID-19 vaccination in a patient post HLA-haploidentical hematopoietic stem cell transplantation

Severe acute interstitial lung disease after BNT162b2 mRNA COVID-19 vaccination in a patient post HLA-haploidentical hematopoietic stem cell transplantation

Comparison of porcine ALG and rabbit ATG on outcomes of HLA-haploidentical hematopoietic stem cell transplantation for patients with acquired aplastic anemia

ObjectiveTo evaluate the efficacy and safety of P-ALG (porcine anti-lymphocyte globulin) and R-ATG (rabbit anti-thymocyte globulin) in the conditioning regime for patients with acquired aplastic anemia who underwent HLA-haploidentical hematopoietic stem cell transplantation (halpo-HSCT).MethodsA total of 91 patients with acquired aplastic anemia who received haplo-HSCT at our center between January 2014 and December 2020 were retrospectively reviewed. Twenty-eight patients were in the P-ALG group while sixty-three patients were in the R-ATG group.ResultsThe median time was 11 versus 13 days (P = 0.294) for myeloid engraftment and 12.5 versus 15 days (P = 0.465) for platelet engraftment in the P-ALG and R-ATG groups, respectively. There were no significant difference in 5-year overall survival (74.83% ± 8.24% vs 72.29% ± 6.26%, P = 0.830), GVHD-free, failure-free survival (71.05% ± 8.65% vs 62.71% ± 6.22%, P = 0.662), failure-free survival (74.83% ± 8.24% vs 66.09% ± 5.84%, P = 0.647) and transplantation-related mortality (25.17% ± 8.24% vs 26.29% ± 6.22%, P = 0.708) between the two groups. The incidence of aGVHD (acute graft versus host disease) (65.39% ± 9.33% vs 62.71% ± 6.30%, P = 0.653), II–IV aGVHD (38.46% ± 9.54% vs 35.64% ± 6.24%, P = 0.695), III–IV aGVHD (19.23% ± 7.73% vs 10.53% ± 4.07%, P = 0.291), cGVHD (chronic graft versus host disease) (22.22% ± 12.25% vs 22.31% ± 6.30%, P = 0.915), and moderate to severe cGVHD (5.56% ± 5.40% vs 9.28% ± 4.46%, P = 0.993) were not significantly different. Similar outcomes were observed between the P-ALG and R-ATG groups for severe bacterial infection (17.9% vs 25.4%, P = 0.431), invasive fungal diseases (3.6% vs 9.5%, P = 0.577) and graft rejection (0% vs 9.5%, P = 0.218). However, the incidence of cytomegalovirus infection and Epstein-Barr virus infection was significantly lower in the P-ALG group (46.4% vs 71.4%, P = 0.022; 3.6% vs 25.4%, P = 0.014).ConclusionThe efficacy and safety of P-ALG were similar with R-ATG in the setting of haplo-HSCT for patients with acquired aplastic anemia patients. P-ALG could be an alternative for R-ATG.

Thrombospondin-1, Platelet Factor 4, and Galectin-1 Are Associated with Engraftment in Patients with Sickle Cell Disease who Underwent Haploidentical Hematopoietic Stem Cell Transplantation

Sickle cell disease (SCD) is an inherited red blood cell disorder that leads to significant morbidity and early mortality. The most widely available curative approach remains allogeneic hematopoietic stem cell transplantation (HSCT). HLA-haploidentical (haplo) HSCT expands the donor pool considerably and is a practical alternative for these patients, but traditionally with an increased risk of allograft rejection. Biomarkers in patient plasma could potentially help predict HSCT outcome and allow treatment at an early stage to reverse or prevent graft rejection. Reliable, noninvasive methods to predict engraftment or rejection early after HSCT are needed. We sought to detect variations in the plasma proteomes of patients who engrafted compared with those who rejected their grafts. We used a mass spectrometry-based proteomics approach to identify candidate biomarkers associated with engraftment and rejection by comparing plasma samples obtained from 9 engrafted patients and 10 patients who experienced graft rejection. A total of 1378 proteins were identified, 45 of which were differentially expressed in the engrafted group compared with the rejected group. Based on bioinformatics analysis results, information from the literature, and immunoassay availability, 7 proteins-thrombospondin-1 (Tsp-1), platelet factor 4 (Pf-4), talin-1, moesin, cell division control protein 42 homolog (CDC42), galectin-1 (Gal-1), and CD9-were selected for further analysis. We compared these protein concentrations among 35 plasma samples (engrafted, n=9; rejected, n=10; healthy volunteers, n=8; nontransplanted SCD, n=8). ELISA analysis confirmed the significant up-regulation of Tsp-1, Pf-4, and Gal-1 in plasma samples from engrafted patients compared with rejected patients, healthy African American volunteers, and the nontransplanted SCD group (P < .01). By receiver operating characteristic analysis, these 3 proteins distinguished engrafted patients from the other groups (area under the curve, >0.8; P < .05). We then evaluated the concentration of these 3 proteins in samples collected pre-HSCT and at days +30, +60, +100, and +180 post-HSCT. The results demonstrate that Tsp-1 and Pf-4 stratified engrafted patients as early as day 60 post-HSCT (P < .01), and that Gal-1 was significantly higher in engrafted patients as early as day 30 post-HSCT (P < .01). We also divided the rejected group into those who experienced primary (n=5) and secondary graft rejection (n=5) and found that engrafted patients had significantly higher Tsp-1 levels compared with patients who developed primary graft rejection at days +60 and +100 (P < .05), as well as higher Pf-4 levels compared with patients who developed primary graft rejection at post-transplantation (PT) day 100. Furthermore, Tsp-1 levels were significantly higher at PT days 60 and 100 and Pf-4 levels were higher at PT day 100 in engrafted patients compared with those who experienced secondary graft rejection. Increased concentrations of plasma Gal-1, Tsp-1, and Pf-4 could reflect increased T regulatory cells, IL-10, and TGF-β, which are essential players in the initiation of immunologic tolerance. These biomarkers may provide opportunities for preemptive intervention to minimize the incidence of graft rejection.

Post-Transplantation Cyclophosphamide After HLA Identical Compared to Haploidentical Donor Transplant in Acute Myeloid Leukemia: A Study on Behalf of GETH-TC

Post-transplantation cyclophosphamide (PTCY) effectively prevents graft-versus-host disease (GVHD) after unmanipulated HLA-haploidentical hematopoietic stem cell transplantation (HSCT) and achieves low rates of GVHD in HLA-identical transplantation. To compare the outcomes of haploidentical versus HLA identical HSCT in patients undergoing HSCT for acute myeloid leukemia (AML) using PTCY. We conducted a retrospective study of 229 patients undergoing first HSCT for AML using PTCY with additional immunosuppression, 99 from matched sibling or unrelated donor (MSD/MUD) performed in 3 hospitals and 130 from haploidentical donors (haplo group) performed in 20 hospitals within the Spanish Group of Hematopoietic Stem Cell Transplantation and Cellular Therapy. Peripheral blood stem cells were used as graft in 89% of patients; myeloablative conditioning was used in 56%. There were significantly more patients with active disease (5% versus 20%, P = .001), high/very high disease risk index (DRI) (32% versus 67%, P = .000) and prior auto-HSCT (2% versus 11%, P = .010) in the haplo group. Median follow-up was 27 and 62.5 months for MSD/MUD and haplo, respectively. At 2 years, no significant differences were observed in overall survival (OS) (72% versus 62%, P = .07), event-free survival (EFS) (70% versus 54%, P = .055), cumulative incidence of relapse (19% versus 25%, P = .13), non-relapse mortality (14% versus 19%, P = .145), and the composite endpoint of GVHD and relapse-free survival (49% versus 42%, P = .249). Multivariate analysis identified only age and active disease as significant risk factors for OS and EFS; reduced-intensity conditioning, high/very high DRI, and haplo donor were nearly statistically significant for these outcomes. Grade II-IV acute GVHD was lower in MSD/MUD (14% versus 47%, P = .000). Cumulative incidences of grade III-IV acute GVHD (4% versus 9%, P = .14) and moderate-severe chronic GVHD (22% versus 19%, P = .28) were similar. Limitations of our study include limited sample size, differences between haplo and MSD/MUD groups and heterogeneous additional immunosuppression and PTCY timing in MSD/MUD. The use of an HLA-identical donor with PTCY in patients with AML showed lower incidence of clinically significant grade II-IV acute GVHD compared to haplo donors. Further studies with larger sample sizes should be performed to establish a possible benefit of HLA-identical donor on survival.© 2022 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Mother Donors Improve Outcomes after HLA Haploidentical Transplantation: A Study by the Cellular Therapy and Immunobiology Working Party of the European Society for Blood and Marrow Transplantation

Transplacental trafficking of maternal and fetal cells during pregnancy establishes long-term reciprocal microchimerism in both mother and child. Consequently, the maternal immune system may become sensitized to paternal histocompatibility antigens. It has been hypothesized that mother's "exposure" to paternal HLA haplotype antigens during pregnancy may affect the outcome of hematopoietic stem cell transplantation (HSCT) when the mother serves as a donor for the child. In T cell-depleted HLA haploidentical HSCT, maternal donors have been associated with improved transplantation outcomes. The present retrospective multicenter study, conducted on behalf of the Cellular Therapy and Immunobiology Working Party of the European Society of Blood and Marrow Transplantation, involved 409 patients (102 pediatric and 307 adult) with acute leukemia who underwent HLA-haploidentical HSCT. The goal of the study was to evaluate the role of maternal donors in a large cohort of haploidentical transplantation recipients. Transplantation from maternal donors was associated with a lower relapse incidence in T cell-depleted HSCTs (hazard ratio [HR], 2.13; 95% confidence interval [CI],1.16 to 3.92; P=.018) as well as in a limited series of unmanipulated in vivo T cell-depleted HSCTs (HR, 4.15; 95% CI, 0.94 to 18.35; P=.06), along with better graft-versus-host disease/relapse-free survival (GRFS) in T cell-depleted HSCT (HR, 1.67; 95% CI, 1.02 to 2.73; P=.04). These results indicate that the mother is the preferred donor to provide better GRFS in T cell-depleted HLA-haploidentical HSCT for acute leukemia.

Outcome of Children with Wiskott-Aldrich Syndrome (WAS) Given TCR Alpha-Beta/CD19 Depleted Hematopoietic Stem Cell Transplantation (HSCT) from an HLA-Haploidentical Relative

Background: WAS is a rare X-linked recessive disorder, characterized by thrombocytopenia with low platelet volume, recurrent infections, eczema, autoimmunity, vasculitis and increased incidence of malignancies. Patients with classical WAS have chronic morbidities, severely impaired quality of life and a decreased life expectancy. Allogeneic HSCT is the only well-established curative treatment, gene therapy being still an experimental approach. Excellent outcome have been reported in patients transplanted at early age from either matched related and unrelated donors, with 5-year overall survival (OS) exceeding 90%. However, experience with HLA-haploidentical HSCT is limited and has been historically associated with inferior results. Haploidentical HSCT after selective depletion of α/β+ T-cells and CD19+ B-cells (TBdepl-haploHSCT) was shown to be safe and effective in children with multiple types of non-malignant disorders (Merli et al, Blood Adv 2021). To further optimize this approach and accelerate the recovery of adaptive immunity, we conducted a phase I/II trial evaluating the safety and efficacy of post-transplant infusion of a titrated number of donor T-cells transduced with the inducible caspase-9 (iC9) suicide gene (BPX-501, or rivogenlecleucel, cells) in children with either malignant or non-malignant disorders (ClinicalTrials.gov identifier: NCT02065869). We report the outcome of a cohort of 12 children affected by WAS who received a TBdepl-haploHSCT at our Center.Patients and methods: Between 2014 and 2021, 12 patients affected by WAS received a TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù in Rome, Italy. Patients and transplant characteristics are depicted in Table 1. All patients received a myeloablative conditioning regimen, combining pharmacokinetic-adjusted busulfan with Thiotepa and Fludarabine. Pre-transplant anti-thymocyte globulins (from day -4 to day -2) were given to modulate bi-directional donor/recipient alloreactivity, while rituximab was administered on day -1 to prevent PTLD. No post-transplant pharmacological GvHD prophylaxis was employed. Eight subjects, enrolled in NCT02065869 trial, additionally received post-transplant infusion of BPX-501 cells (dose: 1x10 6 cells/kg) at a median time of 16 days after HSCT (range 12-20).Results: 11 patients achieved primary donor cell engraftment, while one patient experienced secondary graft failure (GF), likely triggered by CMV reactivation. Median time to neutrophil and platelet recovery was 15 (range 8-33) and 10 days (range 9-16), respectively. The patient with secondary GF was successfully re-transplanted with a second TBdepl-haploHSCT from the same donor. Grade I/II skin acute GvHD (aGvHD) occurred in 3 patients, the cumulative incidence of aGvHD being 25.9% (95% CI 0-47.7). None of these 3 patients required activation of iC9 with rimiducid and no cases of chronic GVHD (cGvHD) were observed. No patient died. With a median follow-up of 58 months (range 1 - 78), the 5-year probability of OS and event-free survival is 100% and 90.7% (95% CI 50.8-98.7), respectively. Considering the successful second allograft, the 5-year disease-free survival is 100%. Platelet recovery is reported in Figure 1A. Two months after the allograft, all evaluable subjects had a platelet count above 50.000/microL (median 202.000, range 53.000-353.000). All subjects but one have full donor chimerism at last follow-up, the remaining patient having stable mixed chimerism (40% of donor cells) without any WAS manifestation. All subjects with a follow-up of at least 6 months are independent of immunoglobulin replacement. Details on reconstitution of lymphocytes subsets are reported in Figure 1B,C,D.Conclusions: TBdepl-haploHSCT after Bu-based conditioning regimen is an highly-effective curative option for children with WAS, being characterized by high-engraftment rate with fast recovery of both neutrophils and platelets, low incidence of aGvHD and no occurrence of cGVHD. Given the prognostic impact of age in determining HSCT outcome in WAS and prompt availability of haploidentical family donors, our data suggest that this approach should be offered without delay to those patients with WAS who lack a matched donor. Infusion of BPX-501 cells contribute to accelerate the recovery of adaptive T-cell immunity, further increasing the safety of the procedure. [Display omitted] DisclosuresMerli: SOBI: Consultancy; JAZZ: Consultancy. Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyl: Honoraria.

Alphabeta T and B-Cell Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation (TBdepl-haploHSCT) in Children with Myelodysplastic Syndromes

Background: Pediatric myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal disorders, accounting for less than 5% of childhood hematologic malignancies. Usual indications to HSCT are: MDSs with excess of blasts, MDSs secondary to previously administered chemoradiotherapy and RCC associated with monosomy 7, complex karyotype, severe neutropenia, or erythrocyte/platelet transfusion dependence [Locatelli & Strahm, Blood 2018]. We previously demonstrated that TBdepl-haploHSCT is a suitable option for children with acute leukemia, with outcomes comparable to those reported in studies using either an HLA-identical sibling or an unrelated volunteer as donor. Here we present the results of this approach in children with MDSs.Patients and methods: Between February 2013 and February 2021, 23 children with MDSs other than juvenile myelomonocytic leukemia received TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù, Rome, Italy or at IRCCS Fondazione Policlinico San Matteo, Pavia, Italy as part of a prospective study (#NCT01810120). All patients were prepared to the allograft using a fully-myeloablative conditioning regimen including a combination of cytotoxic drugs and/or total body irradiation (TBI). Anti-T-lymphocyte globulin (ATLG) was used before transplantation (12 mg/kg total dose, from days -5 to day -3) to modulate bi-directional donor/recipient alloreactivity. Rituximab (200 mg/sqm) was administered on day -1 to prevent post-transplantation EBV-induced lymphoproliferative disorders (PTLD). No patient received any post-transplant pharmacological GvHD prophylaxis.Results: Characteristics of patients enrolled in the study are shown in Table 1 (which reports also donor and graft characteristics). Median follow-up of surviving patients is 4.2 years (range: 0.5 - 8.5 years). Seventeen children were affected by refractory cytopenia of childhood (RCC) (2 cases occurring in the context of inherited bone marrow failure syndromes: one had GATA2 deficiency and the other SAMD9L mutation), while 1 and 5 were affected by MDS with excess of blasts 1 (EB1) and EB2 (one had GATA2 deficiency), respectively. Median time to neutrophil and platelet recovery was 14 (range 10-19) and 11 (range 9-14) days, respectively, with four patients (3 with RCC and 1 with EB2) experiencing primary graft failure, the cumulative incidence of this complication being 17.3% (95% CI 0.3-31.5). All these 4 patients were rescued with a second TBdepl-haploHSCT from the same or the other parent. Cumulative incidence of grade II-III acute GvHD was 11.4% (95% CI 0-25.2). One patient developed skin and gut GvHD after the second TBdepl-haploHSCT, while for all other patients skin was the sole organ involved; no case of grade IV GvHD was observed. One patient developed moderate chronic GvHD [cumulative incidence 5.2% (95% CI 0-14.8)], which completely resolved with low-dose steroids and ruxolitinib. Notably, no patient died for transplant-related complications. Six patients experienced CMV, 2 HHV-6 and 1 adenoviral infection/reactivation; one patient developed lung aspergillosis, which resolved with specific treatment. One patient affected by EB2, not in remission at time of transplant, relapsed 27 months after HSCT, the 5-year cumulative incidence of relapse being 7.1% (95% CI, 0-19.7); she eventually died after failing a second HSCT. The 5-year probability of overall and event-free survival were 92.3% (95% CI 56.6 -98.9) and 76.3% (95% CI 51.3-89.6) (Figure 1A and B), respectively. Five-year disease-free-survival was 90% (95% CI 47.3-98.5). Because of the low number of events, no prognostic factor related to OS and EFS was found. In particular, the MDS variant did not influence the patient's outcome. The median CD3+ cell count on day +30, +90, +180 and +360 were 113, 171, 558 and 1307/mcl, respectively.Conclusions: These data indicate that TBdepl-haploHSCT is a safe and effective transplant option also in children with MDS. Indeed, the low risk of both non-relapse mortality and acute/chronic GvHD makes this approach particularly attractive in the pediatric setting. Moreover, this haplo strategy compares favorably with T-cell replete approaches [Suo et al., 2020]. [Display omitted] DisclosuresMerli: JAZZ: Consultancy; SOBI: Consultancy. Locatelli: Miltenyl: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.

Outpatient Haploidentical Stem Cell Transplantation Using Post-Transplant Cyclophosphamide Is Feasible. Experience in a Single Latin-American Center

Introduction: Only a minority of patients can find a fully matched donor for hematopoietic stem cell transplantation (HSCT). HLA-haploidentical HSCT is an attractive option since the likelihood of identifying a donor is high. Stimulated by economic constraints in developing countries and the significant expenses related to HSCT, in an effort to lower costs and resources, our center has performed an outpatient-based haplo-HSCT program since 2012. In the present study, we describe the characteristics of those patients and analyze the factors relating to the performance of a successful ambulatory procedure.Methods: Adult patients who underwent haplo-HSCT in an outpatient-intended basis were eligible. All were required to have a Karnofsky score ³70%, serum creatinine <2 mg/dL, residence near the hospital, an adequate caregiver, with appropriate educational level and treatment adherence. The conditioning regimen was delivered in all individuals as outpatients and consisted of fludarabine 25 mg/m2/day (day -5 to -3), cyclophosphamide (Cy) 350 mg/m2/day (day -5 to -3), and oral busulfan 4 mg/kg/day (day-2 to -1) or melphalan 50-100 mg/m2 (day -2 to -1). GVHD prophylaxis consisted of Cy (50 mg/kg/day, days 3 and 4), cyclosporine 6 mg/kg, and mycophenolate mofetil 1 g/day. After infusion, patients stayed at home and were seen in the outpatient clinic every day for the first 72 hours, every other day until hospitalization requirement or engraftment; afterwards weekly and bi-weekly through day +100, and thereafter according to physician criteria. Patients were instructed to report the presence of fever among other adverse effects at any hour of the day. Hospital beds were available for those needing admission.Results: Thirty-six consecutive patients from February 2013 to May 2017 were eligible for analysis. Median follow-up was 12 months (4-40). Median age was 23 years (range, 15-63); 13 were female. Acute leukemia (13 lymphoblastic and 5 myeloblastic) was the most common indication for HSCT, followed by Hodgkin's lymphoma (5), non-Hodgkin's lymphoma (4), chronic myeloid leukemia (3), aplastic anemia (3), chronic lymphocytic leukemia (2), and chronic myelomonocytic leukemia (1). Median PBSC CD34+ cells/kg was 7x106 (range, 2-16). Neutrophil engraftment was achieved at a median of 16 days (range, 11-23), with platelet recovery at a median of 16 days (range, 13-100). Seven patients (19%) were followed in a fully outpatient basis while the other 29 (81%) required hospitalization. The median length of stay (LOS) was 10 days (0-35). Seven patients (19%) stayed ≤7 days in hospital, while 22 patients >7 days. Febrile neutropenia was the most common reason for hospitalization (65.5%). Other reasons included mucositis (27.5%) and hemorrhagic cystitis (6.9%). The median day of admission was day +3 (1-14). The development of febrile neutropenia (P=0.004) and a slower neutrophil engraftment (P=0.023) were significantly associated with the need for hospital admission. Eight of the 18 patients admitted for febrile neutropenia had a rapid response and no source of infection evidenced, thus LOS was ≤10 days in 9 patients. Age, gender, mucositis, and acute graft versus host disease were not associated with hospitalization need. The 2-year overall survival (OS) was 50% for patients with a completely ambulatory transplant vs. 28% for those requiring hospitalization (log rank P=0.08).Conclusion: Currently conventional transplantation is unaffordable for the majority of patients living in the developing world. Almost 20% of our patients did not require hospital admission. Although 62% of the patients were admitted, most of them had a short LOS. Our outpatient haploidentical HSCT program is feasible, potentially resulting in cost savings and perhaps a higher quality of life. [Display omitted] DisclosuresGomez-Almaguer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Outcome of Children with Acute Leukemia Given Allogeneic HSCT Either from an Unrelated Donor or from an HLA-Partially Matched Relative after αβ-T Cell/B-Cell Depletion: A Multicenter, Retrospective, Comparative Analysis of the AIEOP Network

IntroductionFor patients in need of an allograft and lacking an HLA-identical sibling, hematopoietic stem cell transplantation (HSCT) from an unrelated donor (UD) is the standard of care. In the recent few years, αβ T-cell and B-cell depleted HLA-haploidentical HSCT (αβhaplo-HSCT) is becoming a widely employed alternative to UD-HSCT in patients with acute leukemia. We, thus, decided to conduct a retrospective analysis on the outcome of patients given either αβhaplo-HSCT or UD HSCT in the same time period, namely October 2010-December 2015, in one of the 13 centers affiliated with the Italian-HSCT pediatric network.Methods: Included in the study were 97 αβhaplo-HSCT recipients and 244 patients receiving UD-HSCT in the same period. Six centers performed both αβhaplo-HSCT and UD-HSCT, while only this latter procedure was performed in the remaining 7 centers. All children were transplanted in morphological complete remission (CR) after a fully myeloablative conditioning regimen. The UD was selected using high-resolution typing for the HLA-loci A, B, C, DRB1. The UD was 8/8 HLA-matched with the recipient in 52% of cases, while the remaining 48% of patients were transplanted from a donor with either 1 or 2 HLA-disparities. Details on patient characteristics of the 2 groups are shown in the Table; recipients of αβhaplo-HSCT were transplanted in more advanced phase and received more frequently a TBI-based regimen than UD-HSCT patients. Patients given αβhaplo-HSCT did not receive any post-transplantation pharmacological prophylaxis of graft-versus-host disease (GvHD), while the combination of anti-T lymphocyte globulin (ATLG), cyclosporine-A and short-term methotrexate was employed for preventing GvHD occurrence in all UD-HSCT recipients. ATLG was also infused before transplantation in all children treated with αβhaplo-HSCT to prevent both graft rejection and GvHD.Results: Two (2%) and 4 (2%) patients experienced graft failure in the αβhaplo-HSCT and UD-HSCT groups, respectively. Median time to neutrophil and platelet recovery was shorter in children given αβhaplo-HSCT (13 and 11 days vs. 19 and 23 days, respectively, P < 0.001 in both cases). The cumulative incidence (CI) of grade II-IV and grade III-IV acute GvHD in patients given αβhaplo-HSCT was 16% and 0%, as compared to 39% and 12% in UD-HSCT recipients (P < 0.001 and < 0.0005, respectively). Children treated with αβhaplo-HSCT benefited also from a lower incidence of both overall and extensive chronic GvHD (6% and 1%, respectively, vs 20% and 7% in UD-HSCT recipients, P < 0.01 and < 0.05, respectively), especially when compared with 7/8 and 6/8 UD-HSCT recipients. Forty-eight patients died for transplant-related complications: 9 (9%) and 40 (16%) in the αβhaplo-HSCT and UD-HSCT group, respectively, the CI of non-relapse- mortality (NRM) being 9% and 17% (P = N.S.). While the probability of NRM is superimposable in αβhaplo-HSCT and 8/8 UD-HSCT, 7/8 and 6/8 UD-HSCT had a higher risk of mortality (25% and 37%, respectively). Seventy-three children relapsed at a median time of 190 days (range 40-1603) after the allograft; no statistically significant difference for the CI of disease recurrence was observed between the 2 groups (25% vs 20%, respectively). With a median follow-up of 3.3 years (range 0.5-5 years for surviving patients) the 3-year probability of overall survival for patients given either αβhaplo-HSCT or UD-HSCT is 68% and 64%, respectively (P = N.S.), while that of event-free survival is 63% and 62%, respectively (P = N.S.). The 3-year chronic GvHD-free/relapse-free survival (GRFS) in the 2 groups is 59% and 48%, respectively (P = 0.03). GRFS of αβhaplo-HSCT or 8/8 UD-HSCT recipients was superimposable.Conclusions: Patients givenαβhaplo-HSCT had a faster ANC/PLT recovery and a lower probability of acute GvHD in comparison to children transplanted from an UD donor. Moreover, they also had a lower risk of NRM and better probability of GRFS in comparison to patients transplanted from an UD with 1 or 2 HLA-disparities. αβhaplo-HSCT is associated with a CI of disease recurrence comparable to that of children transplanted from an UD. Altogether, these data indicate that αβhaplo-HSCT is a competitive alternative to UD-HSCT, especially in the absence of a fully-matched donor. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Salvage HLA-haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide for graft failure in non-malignant disorders

Graft failure requires urgent salvage HSCT, but there is no universally accepted approach for this situation. We investigated T-cell replete haploidentical HSCT with post-transplantation cyclophosphamide following serotherapy-based, radiation-free, reduced intensity conditioning in children with non-malignant disorders who had rejected their primary graft. Twelve patients with primary or secondary graft failure received T-cell replete bone marrow grafts from haploidentical donors and post-transplantation cyclophosphamide. The recommended conditioning regimen comprised rituximab 375 mg/m2, alemtuzumab 0.4 mg/kg, fludarabine 150 mg/m2, treosulfan 20–24 g/m2 and cyclophosphamide 29 mg/kg. After a median follow-up of 26 months (7–95), eleven of twelve patients (92%) are alive and well with complete donor chimerism in ten. Neutrophil and platelet engraftment were observed in all patients after a median of 18 days (15–61) and 39 days (15–191), respectively. Acute GVHD grade I was observed in 1/12 patients (8%) and mild chronic GVHD in 1/12 patients (8%). Viral reactivations and disease were frequent complications at 75% and 42%, respectively, but no death from infectious causes occurred. In summary, this retrospective analysis demonstrates that a post-transplantation cyclophosphamide-based HLA-haploidentical salvage HSCT after irradiation-free conditioning results in excellent engraftment and overall survival in children with non-malignant diseases.

Haploidentical age-adapted myeloablative transplant and regulatory and effector T cells for acute myeloid leukemia

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment in eradicating high-risk acute myeloid leukemia (AML). Here, we present data from a novel HLA-haploidentical HSCT protocol that addressed the 2 remaining major unmet medical needs: leukemia relapse and chronic graft-versus-host disease (cGVHD). Fifty AML patients were enrolled in the study. The conditioning regimen included total body irradiation for patients up to age 50 years and total marrow/lymphoid irradiation for patients age 51 to 65 years. Irradiation was followed by thiotepa, fludarabine, and cyclophosphamide. Patients received an infusion of 2 × 106/kg donor regulatory T cells on day −4 followed by 1 × 106/kg donor conventional T cells on day −1 and a mean of 10.7 × 106 ± 3.4 × 106/kgpurified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GVHD prophylaxis was administered posttransplantation. Patients achieved full donor–type engraftment. Fifteen patients developed grade ≥2 acute GVHD (aGVHD). Twelve of the 15 patients with aGVHD were alive and no longer receiving immunosuppressive therapy. Moderate/severe cGVHD occurred in only 1 patient. Nonrelapse mortality occurred in 10 patients. Only 2 patients relapsed. Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGVHD/relapse-free survival was 75% (95% confidence interval, 71%-78%). A novel HLA-haploidentical HSCT strategy that combines an age-adapted myeloablative conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy resulted in an unprecedented cGVHD/relapse-free survival rate in 50 AML patients with a median age of 53 years. This trial was registered with the Umbria Region Institutional Review Board Public Registry as identification code 02/14 and public registry #2384/14 and at www.clinicaltrials.gov as #NCT03977103.

Identification of New Soluble Factors Correlated With the Development of Graft Failure After Haploidentical Hematopoietic Stem Cell Transplantation.

Graft failure is a severe complication of allogeneic hematopoietic stem cell transplantation (HSCT). The mechanisms involved in this phenomenon are still not completely understood; data available suggest that recipient T lymphocytes surviving the conditioning regimen are the main mediators of immune-mediated graft failure. So far, no predictive marker or early detection method is available. In order to identify a non-invasive and efficient strategy to diagnose this complication, as well as to find possible targets to prevent/treat it, we performed a detailed analysis of serum of eight patients experiencing graft failure after T-cell depleted HLA-haploidentical HSCT. In this study, we confirm data describing graft failure to be a complex phenomenon involving different components of the immune system, mainly driven by the IFNγ pathway. We observed a significant modulation of IL7, IL8, IL18, IL27, CCL2, CCL5 (Rantes), CCL7, CCL20 (MIP3a), CCL24 (Eotaxin2), and CXCL11 in patients experiencing graft failure, as compared to matched patients not developing this complication. For some of these factors, the difference was already present at the time of infusion of the graft, thus allowing early risk stratification. Moreover, these cytokines/chemokines could represent possible targets, providing the rationale for exploring new therapeutic/preventive strategies.

Comparison of Fixed Vs ALC-Based Doses of Thymoglobulin® (ATG) in Pediatric Patients with Acute Leukemia Given αβhaplo-HSCT: Impact on Immune Reconstitution at Day 90

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the treatment of choice for pediatric patients with high risk or refractory leukemia. In the absence of related or unrelated HLA-matched donors, alternative approaches such as HLA-haploidentical HSCT have been implemented. Our group has developed one such approach, αβ T-cell/CD19 B-cell depletion (αβhaplo-HSCT), and demonstrated its clinical efficacy (Bertaina A, Blood 2018). In αβhaplo-HSCT, anti-thymocyte globulin (ATG) is used for preventing graft rejection and graft-versus-host disease (GvHD). However, the optimal dose still needs to be elucidated. Here, we present the first analysis comparing 2 different Thymoglobulin® ATG doses: one fixed at 3.75 mg/Kg, as established in previous European studies with the closely related ATG Grafalon®, and one based on a newly developed algorithm that also integrates absolute lymphocyte count (ALC). METHODS: Between March 2017 and April 2020, 27 pediatric patients (median age 12 years) with hematological malignancies were transplanted at Stanford University's Lucile Packard Children's Hospital. Importantly, 60% of these patients were in CR2 or greater and 2/27 had active disease at the time of HSCT. All children received a fully myeloablative conditioning regimen. On days -9/-8/-7 before αβhaplo-HSCT, patients were given a regimen of Thymoglobulin® ATG. No patient received post-HSCT pharmacological GvHD prophylaxis. The fixed dosage was given to 14 patients in cohort 1 (ATG: 3.75 mg/Kg) and the novel ALC-based dosage to 13 in cohort 2 (ATG ranging between 3-6 mg/Kg). All patients enrolled in this study (BMT Protocol 179 and 351 approved from our IRB) had a minimum follow-up of 100 days, evaluated at Day 30 and 90. Following Admiraal R (Lancet Hematology 2015), we defined CD4 immune reconstitution (IR) as more than 50 CD4 T-cells/ul twice within the first 100 days after HSCT. PBMC were enriched by Ficoll-Hypaque (Sigma Aldrich) density gradient centrifugation. Flow analyses were performed on fresh cells resuspended in PBS 2% FBS on Cytek DxP 10 flow-cytometer. At least 5x104 events of total cells were acquired and analyzed using FloJo software. RESULTS: With a median follow-up of 555 and 124 days for cohort 1 and 2 respectively, 12 patients (85.7%) in cohort 1 and 7 patients (54%) in cohort 2 achieved CD4 immune reconstitution. In cohort 2, CD3 αβ T cells were significantly lower at both Day 30 and Day 90 (P=0.0003, Figure 1A). At Day 90, both the CD4 and CD8 subpopulations were significantly depressed (P=0.01 and P=0.056, respectively, Figure 1C). In both subpopulations, the memory compartment was the most reduced (Figure 1D). The absolute numbers of CD3 γδ T cells did not differ between the cohorts at either Day 30 or 90 (Figure 1B). Viral reactivations were higher in cohort 1 (10/14, 71%) than in cohort 2 (6/13, 46%, P=NS). Half of the reactivations were CMV reactivations, but no patient developed organ disease. There was no statistically significant difference in overall survival and the incidence of relapse in the two cohorts. Three patients developed grade III-IV aGvHD: 2 in cohort 1 (14%) and 1 in cohort 2 (8%, P=NS). Remarkably, the only patient, who developed grade IV aGvHD in cohort 2, did not experience symptoms until Adenovirus reactivation 138 days after HSCT. CONCLUSION: Our analysis confirms that the optimal dose of ATG Thymoglobulin® before αβhaplo-HSCT remains elusive. There were no significant clinical differences between the 2 ATG regimens. However, the ALC-based regimen resulted in the more pronounced reduction of donor-derived memory T cells. Our analysis suggests two intriguing explanations for the observed pattern of results. First, the selective depletion of the memory compartment in both CD4 and CD8 T cells may well be due to a priming effect of ATG Thymoglobulin® on the few αβ T cells left over in the graft. Second, the equivalent reconstitution of naive T cells in the 2 cohorts is likely because the ATG has no impact on the thymus-dependent IR. Remarkably, in our overall cohort, 70% of the patients achieved CD4 IR by 90 days after αβhaplo-HSCT. This result is superior to the best results from other ex vivo T-cell depleted approaches 54%, recently reported by Van Roessel (Cytotherapy 2020). In vivo studies of the pharmacokinetics of ATG in αβhaplo-HSCT recipients and a comparison with the use of Grafalon® are required to shed more light on this crucial topic. Disclosures No relevant conflicts of interest to declare.