Autologous Reconstitution Research Articles

Transplantation for immune dysregulatory disorders: current themes and future expectations.

Primary immune regulatory disorders (PIRDs) are an increasing indication for hematopoietic stem cell transplant (HCT) in pediatric patients. Here, we provide an updated overview of HCT for PIRDs, and discuss future avenues for improvement in outcomes. There are now more than 50 described monogenic PIRDs, which impact all aspects of immune tolerance, regulation, and suppression. Disease characteristics are highly variable, and HCT remains the only option for cure. We review advances in targeted therapies for individual PIRDs, which have significantly improved outcomes and the ability to safely bridge to transplant. Additionally, advances in GVHD prevention, graft manipulation, personalized conditioning regimens, and supportive care have all increased survival after HCT. The high inflammatory state increases the risk of nonengraftment, rejection, and autologous reconstitution. Therapy to reduce the inflammatory state may further improve outcomes. In addition, although younger patients with fewer comorbidities have better outcomes, the clinical courses of these diseases may be extremely variable thereby complicating the decision to proceed to HCT. HCT for PIRDs is a growing consideration in cell therapy. Yet, there remain significant gaps in our understanding of which patients this curative therapy could benefit the most. Here, we review the current data supporting HCT for PIRDs as well as areas for future improvement.

An international learning collaborative phase 2 trial for haploidentical bone marrow transplant in sickle cell disease

AbstractIn the setting of a learning collaborative, we conducted an international multicenter phase 2 clinical trial testing the hypothesis that nonmyeloablative–related haploidentical bone marrow transplant (BMT) with thiotepa and posttransplant cyclophosphamide (PTCy) will result in 2-year event-free survival (no graft failure or death) of at least 80%. A total of 70 participants were evaluable based on the conditioning protocol. Graft failure occurred in 8 of 70 (11.4%) and only in participants aged <18 years; all had autologous reconstitution. After a median follow-up of 2.4 years, the 2-year Kaplan-Meier–based probability of event-free survival was 82.6%. The 2-year overall survival was 94.1%, with no difference between children and adult participants. After excluding participants with graft failure (n = 8), participants with engraftment had median whole blood donor chimerism values at days +180 and +365 after transplant of 100% (n = 58), respectively, and 96.6% (57/59) were off immunosuppression 1 year after transplant. The 1-year grade 3 to 4 acute graft-versus-host disease (GVHD) rate was 10%, and the 2-year moderate–severe chronic GVHD rate was 10%. Five participants (7.1%) died from infectious complications. We demonstrate that nonmyeloablative haploidentical BMT with thiotepa and PTCy is a readily available curative therapy for most adults, even those with organ damage, compared to the more expensive myeloablative gene therapy and gene editing. Additional strategies are required for children to decrease graft failure rates. The trial was registered at www.clinicaltrials.gov as #NCT01850108.

Anti-thymocyte globulin exposure in patients with diffuse cutaneous systemic sclerosis undergoing autologous haematopoietic stem cell transplantation.

Autologous haematopoietic stem cell transplantation improves event-free survival and lung function and reduces skin thickening in patients with progressive diffuse cutaneous systemic sclerosis. Anti-thymocyte globulin is a key lymphoablative constituent of conditioning protocols and is administered in a weight-based dosage. However, whether anti-thymocyte globulin exposure contributes to response to autologous haematopoietic stem cell transplantation and lymphocyte reconstitution in diffuse cutaneous systemic sclerosis patients is unknown. We aimed to explore the relationship between anti-thymocyte globulin exposure, lymphocyte reconstitution and treatment response in diffuse cutaneous systemic sclerosis patients undergoing autologous haematopoietic stem cell transplantation. A retrospective cohort of 15 diffuse cutaneous systemic sclerosis patients undergoing autologous haematopoietic stem cell transplantation was performed. Clinical characteristics and routine laboratory results were retrieved from electronic medical records. Anti-thymocyte globulin concentrations were measured in cryopreserved plasma samples at four time points (day 1 and week 1, 2 and 4) after stem cell reinfusion. Anti-thymocyte globulin exposure was estimated using a validated population pharmacokinetic model. During a median follow-up of 45 months (interquartile range 19-66), 11 (73%) patients had a treatment response, and 4 (27%) were non-responders. Although all patients received the same weight-based anti-thymocyte globulin dosage, 7.5 mg/kg divided over 3 days, anti-thymocyte globulin exposure varied. Anti-thymocyte globulin exposure was higher in responders than in non-responders (163 AU*day/mL (interquartile range 153-183) and 137 AU*day/mL (interquartile range 101-149), respectively, p = .026). Anti-thymocyte globulin exposure was not correlated with lymphocyte reconstitution or infection rate. Weight-based dosing of anti-thymocyte globulin results in variable anti-thymocyte globulin exposure and treatment response across individuals.

CD3+TCRαβ/CD19+-Depleted Mismatched Family or Unrelated Donor Salvage Stem Cell Transplantation for Graft Dysfunction in Inborn Errors of Immunity

A minority of children experience significant graft dysfunction after an allogeneic hematopoietic stem cell transplant (HSCT) for inborn errors of immunity (IEI). The optimal approach to salvage HSCT is unclear with respect to conditioning regimen and stem cell source. This single-centre retrospective case series reports the outcomes of salvage CD3+TCRαβ/CD19 depleted mismatched family or unrelated donor stem cell transplantation (TCRαβ-SCT) between 2013 - 2022 for graft dysfunction in 12 children with IEI. Outcomes of interest were overall survival (OS), event free survival (EFS), graft-versus-host disease (GvHD)-free and event-free survival (GEFS), toxicities, GvHD, viremia and long-term graft function. A retrospective audit of patients who underwent second CD3+TCRαβ/CD19 depleted mismatched donor graft using Treosulfan-based reduced toxicity myeloablative conditioning. Median age at first HSCT was 8.76 months (range, 2.5 months - 6 years) and at second TCRαβ-SCT was 3.6 years (1.2 - 11 years). Median interval between first and second HSCT was 1.7 years (3 months - 9 years). The primary diagnoses were: severe combined immunodeficiency (SCID) (n=5) and non-SCID IEI (n=7). The indications for second HSCT were: primary aplasia (n=1), secondary autologous reconstitution (n=6), refractory aGVHD (n=3) and secondary leukemia (n=1). Donors were either haploidentical parental donors (n=10) or mismatched unrelated donors (n=2). All received TCRαβ/CD19-depleted-PBSC with a median CD34+ cell dose of 9.3 × 106/kg (2.8-32.3 × 106/kg) and a median TCRαβ+ cell dose of 4 × 104/kg (1.3-19.2 × 104/kg). All engrafted with median days to neutrophil and platelet recovery of 15 (12-24) and 12 (9-19). One developed secondary aplasia and one had secondary autologous reconstitution, but both underwent a successful third HSCT. Four (33%) had grade II aGvHD and none had grade III-IV aGvHD. None had cGvHD but one developed extensive cutaneous cGVHD after third HSCT using PBSC and ATG. Nine (75%) were noted to have at least one episode of blood viremia with HHV6 (n=6, 50%), adenovirus (n=6, 50%), EBV (n=3, 25%) or CMV (n=3; 25%). Median duration of follow-up was 2.3 years (range: 0.5 - 10 years) and the 2-year OS, EFS and GEFS were 100% (95% confidence interval, 0-100%), 73% (37-90%) and 73% (37%-90%) respectively. TCRαβ-SCT from mismatched family or unrelated donors, using a chemotherapy only regimen, is a safe alternative donor salvage transplant strategy for second HSCT in patients without a suitably matched donor.

Outcomes of Non-Myeloablative HLA-Haploidentical Bone Marrow Transplant with Thiotepa and Post-Transplant Cyclophosphamide in Children and Adults with Sickle Cell Disease, a Phase II Trial: Vanderbilt Global Haploidentical Transplant Learning Collaborative (VGC2)

Introduction: Related reduced-intensity HLA-Haploidentical bone marrow transplant (haplo-BMT) expands the donor pool to approximately 90% of children and adults with sickle cell disease (SCD). In 2013, we developed the multi-institutional Vanderbilt Global Haploidentical Learning Collaborative to optimize haplo-BMT for SCD. In a Phase II clinical trial, we developed a protocol focused on improving engraftment rates by augmenting the original Hopkins haplo-BMT approach (Bolaños-Meade et al. Blood 2012) with thiotepa. We initially demonstrated durable engraftment in 93% (14/15) of participants (including 2 with previous graft rejection) and a 100% overall survival after a median follow-up of 13.3 months (de la Fuente et al. BBMT 2019). We present the results of our phase-II trial (ClinicalTrials.gov identifier NCT01850108). Material and methods: The trial opened in October 2013, and each site has a standalone Institutional Review Board-approved protocol with institutional-specific eligibility criteria and a data-sharing agreement with Vanderbilt University Medical Center. A DSMB assessed the aggregate results for participant safety and clinical trial futility. Collaborators met via videoconferencing initially weekly and then bi-weekly to discuss best practices. The common conditioning regimen included: thymoglobulin 4.5 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, cyclophosphamide 29 mg/kg and TBI 2Gy. Graft-versus-host disease (GVHD) prophylaxis included post-transplant cyclophosphamide 100 mg/kg, mycophenolate mofetil, and sirolimus. Bone marrow was the donor source and was primed with filgrastim (5-10 μg/kg/d x5 days) in 11% (9/80) of haploidentical donors. Primary and secondary graft failure was defined as <5% donor myeloid/lymphoid whole blood chimerism at Day>28 and >Day+28 (after initial engraftment), respectively. Primary endpoints were 1-year and 2-year EFS. Primary or secondary graft failure and death were considered events. Safety stopping rules were implemented, independent of age, based on graft failure, death, grade III or IV acute GVHD, or moderate or severe chronic GVHD. Local treating physicians classified the severity of chronic GVHD. Results: At the interim analysis of May 1, 2022, we report results from patients enrolled from 8/14/2014 - 2/28/2022. Final enrollment is a total of 80 participants from 7 sites, 45% were male. Donors were siblings (42.5%), parents (53.8%), daughters (2.5%) and cousins (1.3%); 83.8% (n=67) had sickle cell trait. Participants clinical and laboratory data are listed in table 1. Graft failure occurred in 12.5% (10/80) of the participants, with 4 primary and 6 secondary graft failures, table 2. All graft failures were in participants <18 years of age, and all had autologous reconstitution. For engrafted participants, the median whole blood chimerism values for D+180 and D+365 post-transplant was 100% (n=67; IQR: 99.8 - 100; Range 30.0 - 100) and 100% (n=50; IQR: 100 - 100; Range 95.0 - 100), respectively. Among engrafted participants, 97.7% (43/44) were off immunosuppression at D+365 post-transplant. The incidence of grades III-IV acute and moderate/severe chronic GVHD were 8.8% (7/80) each. The median follow-up time was 1.4 years, based on the time of graft failure, death, or May 1, 2022. Mortality was 5.0% (4/80), Table 2. A Kaplan-Meier analysis for survival found no difference by age group <18 years or >18 years of age (p=0.760). Kaplan-Meier based probability of overall survival was 96.7% (95% CI 87.1% - 99.2%) at one year and 94.3% (95% CI 83.0% - 98.2%) at two years. Results of the final analysis of primary and secondary endpoints (1- and 2-year EFS) will be submitted for publication after all participants have been followed for a minimum of one year (anticipated 2/23). Conclusion: Our Vanderbilt Global Haploidentical learning collaborative is a successful and efficient approach to sharing best practices and implementing curative therapy for SCD. After 8 years, we have shown low mortality rate and 100% engraftment in adults using our haplo-BMT regimen. Three new research questions have emerged in the consortium: 1. What strategies can we develop to address the high graft failure in children? 2. How can we optimize supportive care practices and minimize severe infectious complications in adults? 3. What are the heart, lung, kidney, and other late-health effects after curative therapy? Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Prospective Phase 2 Study Testing High Dose Post-Transplant Cyclophosphamide As Sole Ghvd Prophylaxis after Matched Allotransplant Using Baltimore-Based Reduced-Intensity Conditioning Regimens and PBSC As Source of Graft

Introduction: The use of high-dose post-transplant cyclophosphamide (PTCY) has revolutionized graft-versus-host disease (GVHD) prophylaxis and allowed to successfully reconsider haplotransplant in recent years. As this strategy significantly reduces the incidence of both acute and chronic GVHD, PTCY has been thereafter considered not only in matched settings but also as sole GVHD prophylaxis, at least when considering myeloablative allotransplant using matched sibling (MSD) or unrelated (MUD) donors and bone marrow as source of graft. Here, PTCY, as a sole GVHD prophylaxis, was tested in a reduced-intensity conditioning (RIC) setting, using peripheral blood stem cells (PBSC) as source of graft considering that this platform is currently broadly used worldwide in adults.Methods: This prospective monocentric phase 2 study was designed with the main objective to demonstrate the feasibility and safety of using only PTCY (without cyclosporine A nor mycophenolate mofetyl after transplant) in adults (18-70 years old) eligible for a RIC PBSC transplant with MSD or MUD. The Baltimore platform with 2 days of PTCY 50mg/kg/day on days 3 and 4 post infusion was considered as conditioning regimen, using fludarabine for lymphoid disease or clofarabine for myeloid disease. The primary objective was to appreciate the incidence of corticosteroid-resistant acute grade 3-4 GVHD (CR 3-4 GVHD) within 100 days post-transplant. According to statistical rules, patients have to be included in a step by step fashion (3, 3, 6, 15, 15 and 17 patients) for a total of 59 evaluable patients (meaning having received PTCY), in order to stop the protocol soon enough in case of excessive rate of deleterious severe acute GVHD (graded according to Mount Sinai International Consortium). Thus, the trial had to be stopped in case of documentation of > 2 CR 3-4 GVHD for the first 3 patients, >3 CR 3-4 GVHD for the first 6 patients, > 4 CR 3-4 GVHD for the first 12 patients, > 6 3-4 CR GVHD for the first 27 patients, > 8 CR 3-4 GVHD for the first 42 patients and finally as soon as > 9 CR 3-4 GVHD for the last included patients. All patients gave informed consent. The trial was registered at ClinicalTrials.gov Identifier: NCT03263767.Results: The results of the first 27 first patients (males n=17 and female n=10; median age: 59 years old (yo), range: 26-70) are reported here. They were included between February 2018 and November 2020. Diagnoses were AML (N=8), MDS (N=5), CMML (N=2), myelofibrosis (N=5), CML (N=1), DLBCL (N=1), T-cell lymphoma (N=1), Philadelphia positive B-ALL (N=1), CLL (N=1), lymphoblastic lymphoma (N=1) and mixed phenotype acute leukemia (N=1). Donors were MSD in 10 cases and MUD in 17.Only one primary graft failure was documented in a 61 yo MDS patient with active disease at transplant. He is however still alive in response after autologous reconstitution. With a median follow-up of 17.6 months (range: 10-42) for alive patients at the time of analysis (July 2021), 1-year and 2-year survivals were 80.9+7% and 74.7+9%, respectively, for both OS et DFS. GVHD-free/relapse-free survival (GRFS) at 1-year and 2-year was 58.7+9% and 52.2+10%, respectively. Three relapses (11%) and 6 deaths occurred. Deaths were due to acute GVHD in 4 patients (including 1 with sepsis and 1 with SARS-COVID 19 infection) and relapse in 2. Grade 2, 3 and 4 acute GVHD occurred in 11, 1 and 4 patients, respectively, for a total of 59% of grade 2-4 acute GVHD. CR 3-4 GVHD was observed in all of 5 patients with acute grade 3-4 GVHD and 4 died related to GVHD. Moderate/severe chronic GVHD occurred in 5/22 (22.7%) evaluable patients, including 4 still on immunosuppressive therapy at 40, 28, 25 and 16 months post-transplant. Overall non-relapse mortality (NRM) was 14.8% and related to acute GVHD. However, the number of cases conducting to stop the protocol was not reached.Conclusion: PTCY as a sole GVHD prophylaxis is here demonstrated as possible and relatively safe for adults receiving a matched PBSC Baltimore-based RIC allograft. The very good survivals reported here may be related to a strong GVL effect associated with the high incidence of acute GVHD. However, because of this high incidence and the fact that NRM was related to GVHD after this first analysis, we have now made an amendment to test the addition to PTCY of one day of anti-thymoglobulin (ATG) 2.5 mg/kg on day-2 for the next 32 patients to be included. This second cohort receiving PTCY+ATG as a sole prophylaxis is ongoing. DisclosuresMoreau: Celgene BMS: Honoraria; Sanofi: Honoraria; Abbvie: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Oncopeptides: Honoraria.

Risk Factors and Outcomes According to Age at Transplant with an HLA Identical Sibling for Sickle Cell Disease

Background: Hematopoietic stem cell transplant (HSCT) from an HLA identical (HLAid) sibling donor is a well-established curative therapy for sickle cell disease (SCD). However, the ideal age to perform HSCT in SCD patients remains controversial. We report the outcomes after HLAid sibling HSCT for SCD according to patient's age at the time of HSCT as well as their pre-transplant characteristics.Methods: We retrospectively analyzed 727 HLAid sibling HSCTs performed between 1986 and 2015 in 98 EBMT centres. Patients were divided in three groups according to their age at the time of HSCT: <5 year-old (n=176): group 1, 6-15 year-old (n=431): group 2 and >15 years old (n=120): group 3. Primary endpoint was probability of 3-year overall survival (OS) according to age group.Results: Median follow-up was 46 (range: 0.5-346) months. Median age at HSCT was 4 (range: 0.9-5) years in patients <5 years, 10 (range: 6-14) years in patients 6-15 years and 17 (range: 15-39) years in patients >15 years. Gender distribution did not significantly differ among the three groups. The most common SCD genotype was homozygous (SS) in the three groups (95% vs 92% vs 84%, p=0.004). Positive cytomegalovirus (CMV) serology was less frequent in group 1 (63% vs 79% vs 83%, p<0.001. Also, patients in group 1 were less treated with hydroxyurea (37% vs 60% vs 77%, p<0.001), received less than 20 red blood cell units transfusions (32% vs 43% vs 58%, p=0.002) and presented less than 3 SCD complications before HSCT (11% vs 19% vs 39%, p<0.001). For all age groups, vaso occlusive crisis (VOC), acute chest syndrome (ACS) and cerebral vasculopathy were the most reported SCD complications, and the main indications for HSCT were VOC, cerebral vasculopathy, ACS and nephropathy.The proportion of patients with Karnofsky performance status (KPS) <80 did not differ between groups. Graft source distribution was bone marrow (BM) in 74%, cord blood (CB) in 23% and peripheral blood (PBSC) in 3% of cases in group 1; BM in 85%, CB in 11% and PBSC in 4% of cases in the group 2; BM in 79%, CB in 1% and PBSC in 20% of cases in group 3 (p<0.001). Conditioning regimen was myeloablative in 97% of cases in group 1, 96% in group 2 and 83% in group 3 (p<0.001), and included in vivo T cell depletion, mostly antithymocyte globulin (ATG), in 74%, 86% and 90% of the cases, respectively (p<0.001). In all groups, graft versus host disease (GvHD) prophylaxis was mainly cyclosporine- A with methotrexate±mycophenolate mofetil. ABO and gender match between donor and recipient did not significantly vary among groups.Neutrophil engraftment at day +60 was 96%+1 in the first group, 98%+1 in the second group and 95%+2 in the third group (p=0.009). Four patients in group 1, 10 patients in group 2 and 6 patients in group 3 had primary graft failure. The first group experienced less acute GvHD (aGvHD) within 100 days after HSCT (9%+2 vs 18%+2 vs 17%+4, p=0.022) and less chronic GvHD (cGvHD) within 3 years after HSCT (9%+2 vs 12%+2 vs 20%+4, p=0.006). Three-year OS was significantly better in group 1 (99%+1 vs 95%+1 vs 88%+3, p<0.001) as well as 3-year event free survival (EFS) (96%+2 vs 92%+1 vs 84%+4, p=0.001). In a subset of patients with available chimerism data (n=405), in group 1 and group 2 65% were full donor, 32% mixed chimeras and 3% autologous reconstitution whereas in group 3 46% were full donor, 49% mixed chimeras and 5% autologous reconstitution. A univariate risk factor survival analysis was performed for group 3 (only group with sufficient events for analysis), but no patient, donor or HSCT risk factors influencing OS were identified.Conclusion: Patients transplanted at a young age have a better 3-year OS and 3-year EFS, with lower incidence of aGvHD and cGvHD. These findings outline the importance of early referral to HSCT for SCD patients. Strategies to further evaluate pre HSCT, HSCT and donor factors that may influence survival and adverse outcomes could help improve HSCT results. Disclosuresde la Fuente:Jazz Pharmaceuticals: Honoraria; bluebird bio: Consultancy; HCA: Consultancy. Bader:Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding.

Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Polycythemia Vera or Essential Thrombocythemia Transformed to Myelofibrosis or Acute Myeloid Leukemia: a Single Center Experience

In myelofibrosis (MF) which median overall survival (OS) is about six years allogeneic hematopoietic stem cell transplantation (HSCT) is the sole therapeutic procedure with a curative potential. Recent retrospective and prospective studies have reported that MF patients who receive a related donor graft have a clinical outcome better than that of patients who receive an unrelated donor graft. Thus, in order to verify this issue and identify patient- and HSCT-specific variables that influence this different clinical outcome we conducted this retrospective analysis in 31 pts with chronic and advanced-phase MF who underwent HSCT at our Institution. This series included 6 (19.3%) females and 25 (80.7%) males with a median age of 54.9 years (range 30.2-63.8) whose median follow-up was 1.7 years (range 0-11.8). Fifteen pts were classified as primary MF, 9 as secondary MF and 7 as AML post-MF. From a molecular point of view, 17 pts presented the V617F JAK2 mutation, 7 a CALR mutation, 2 a MPL mutation and 2 none of these mutations (“triple negative”). Eleven pts (35.5%) received the transplant from an identical sibling, 17 (54.8%) from a matched unrelated donor, 3 (9.7%) from a mismatched relative. The graft source was the bone marrow in three pts (9.7%) and the peripheral blood (PBSC) in 28 (90.3%). The median number of CD34+ cell infused was 7.3x106/kg. Neutrophil engraftment occurred in 27 pts (87.0%) at a median time of 16.5 days (range 11-34) and platelet engraftment occurred in 21 pts (67.8%) at a median time of 17 days (14-98). In these pts splenomegaly was associated with a significant delay in neutrophil and platelet recovery. Considering the remaining 4 pts, all with a relevant splenomegaly, 2 did not engraft and 2 experienced an autologous reconstitution. Considering all the cohort, death occurred in 13 pts (41.9%) and the 1-year cumulative incidence of nonrelapse mortality (NRM) was 32.9% (95& CI: 16.3%-50.6%). The 3-year overall survival (OS) was 59.4% (95% CI: 38.1%-75.5%) and the 5-year OS was 47.2% (95% CI: 25.3%-66.4%). The disappearance of the molecular marker present at the onset of the disease on subsequent molecular tests occurred in 10 pts (32.2%) who are still in clinical and molecular complete remission. Relapse occurred in 5 pts (16.1%) and 6-month and 3-year cumulative incidence of relapse (CIR) were 3.5% (95% CI: 0.3%-15.1%) and 19.3% (95% CI: 7.0%-36.1%). Eight pts (27.6%) experienced acute GVHD within 100-day (95% CI: 13.1%-44.3%) and 12 (38.7%) developed chronic GVHD which 6-month, 1-year, 3-years cumulative incidences were 25.2% (95% CI 11.1%-42.2%), 33.1% (95% CI 16.6%-50.6%) and 48.2% (95% CI: 27.3%-66.3%). When the clinical outcome of related donor graft group was compared to that of unrelated donor graft group, the 1-year NRM was 18.2% (95% CI: 2.9%-44.2%) for the first group of pts versus 45.5% (95% CI: 20.5%-67.6%) for the second group (p=0.07); median OS and median progression-free survival (PFS) were not reached for the first group of pts versus 2.2 and 0.7 years respectively for the second group of pts (p=0.05 and p=0.05). In addition, a borderline significance in 2-year OS was noted: 81.8% (95% CI 44.7%-95.1%) versus 51.0% (95% CI: 25.4%-71.8%) (p=0.071). The 100-days cumulative incidence of grade II-IV acute GVHD was 10% (95%CI: 0.6%-35.8%) for the first group of pts versus 43.8% (95% CI: 19.8%-65.6%) for the second group with a similar incidence of chronic GVHD. Re-transplantation of the two pts with graft failure did not result in hematopoietic reconstitution as was followed by rejection of the PBSC infused. One pt with an autologous reconstitution achieved a full donor chimerism after the third transplant from a haplo-identical donor. In conclusion, this retrospective study confirms that HSCT is a potentially curative option for MF patients despite the high relapse incidence and non-relapse mortality. DisclosuresNo relevant conflicts of interest to declare.

Diagnostic and therapeutic caveats in Griscelli syndrome.

Griscelli syndrome (GS) is a rare autosomal recessive disease with characteristic pigment distribution, and there are currently 3 types according to the underlying genetic defect and clinical features. We present the case of a girl born from consanguineous parents who presented with predominant neurologic symptoms, silvery hair and granulomatous skin lesions. Cerebral magnetic resonance revealed diffuse white matter lesions, and central nervous system (CNS) lymphocytic infiltration was suspected. The patient underwent haematopoietic stem cell transplantation with graft failure and autologous reconstitution. She developed elevated liver enzyme with a cholestatic pattern. Multiple liver biopsies revealed centrilobular cholestasis and unspecific portal inflammation that improved with immunomodulatory treatment. She was revealed to have an impaired cytotoxicity in NK cells and a decreased expression of RAB27A. However, no variants were found in the gene. All types of GS present with pigment dilution and irregular pigment clumps that can be seen through light microscopy in hair and skin biopsy. Dermic granulomas and immunodeficiency with infectious and HLH predisposition have been described in GS type 2 (GS2). Neurologic alterations might be seen in GS type 1 (GS1) and GS type 2 (GS2), due to different mechanisms. GS1 presents with neurologic impairment secondary to myosin Va role in neuronal development and synapsis. Meanwhile, GS2 can present with neurologic impairment secondary to SNC HLH. Clinical features and cytotoxicity might aid in differentiating GS1 and GS2, especially since treatment differs.

Incidence, Management, and Prognosis of Graft Failure and Autologous Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation.

BackgroundThis study presents outcomes of management in graft failure (GF) after allogeneic hematopoietic stem cell transplantation (HCT) and provides prognostic information including rare cases of autologous reconstitution (AR).MethodsWe analyzed risk factors and outcomes of primary and secondary GF, and occurrence of AR in 1,630 HCT recipients transplanted over period of 18 years (January 2000–September 2017) at our center.ResultsPrimary and secondary GF occurred in 13 (0.80%), and 69 patients (10-year cumulative incidence, 4.5%) respectively. No peri-transplant variables predicted primary GF, whereas reduced intensity conditioning (RIC) regimen (relative risk [RR], 0.97–28.0, P < 0.001) and lower CD34+ cell dose (RR, 2.44–2.84, P = 0.002) were associated with higher risk of secondary GF in multivariate analysis. Primary GF demonstrated 100% mortality, in the secondary GF group, the 5-year Kaplan-Meier survival rate was 28.8%, relapse ensued in 18.8%, and AR was observed in 11.6% (n = 8). In survival analysis, diagnosis of aplastic anemia (AA), chronic myeloid leukemia and use of RIC had a positive impact. There were 8 patients who experienced AR, which was rarely reported after transplantation for acute leukemia. Patient shared common characteristics such as young age (median 25 years), use of RIC regimen, absence of profound neutropenia, and had advantageous survival rate of 100% during follow period without relapse.ConclusionPrimary GF exhibited high mortality rate. Secondary GF had 4.5% 10-year cumulative incidence, median onset of 3 months after HCT, and showed 5-year Kaplan-Meier survival of 28.8%. Diagnosis of severe AA and use of RIC was both associated with higher incidence and better survival rate in secondary GF group. AR occurred in 11.6% in secondary GF, exhibited excellent prognosis.

Outcome After Cord Blood Transplantation Using Busulfan Pharmacokinetics-Targeted Myeloablative Conditioning for Hurler Syndrome

We report the outcomes of cord blood transplantation (CBT) with a busulfan (Bu) pharmacokinetics-targeted myeloablative conditioning regimen in 97 children with Hurler syndrome (HS) performed between 2004 and 2016. The median age at CBT was 10.8 months (range, 0.23 to 63.2 months). The median duration of follow-up for surviving patients was 4.2 years (range, 1.0 to 12.8 years). Five-year overall survival (OS) and engrafted survival (ES) were 88% and 79%, respectively. OS was 95% in patients who received Bu/fludarabine (Flu)/antithymocyte globulin (ATG) conditioning, 90% in those who received Bu/cyclophosphamide (Cy)/ATG, and 74% in those who received Bu/Cy/alemtuzumab (P = .02). ES was 84% for recipients of Bu/Flu/ATG conditioning, 83% for recipients of Bu/Cy/ATG conditioning, and 65% for recipients of Bu/Cy/alemtuzumab conditioning (P = .34). Receipt of washed CB units (P = .03) and HLA matching ≤6/10 (P = .02) were associated with significantly lower ES. The 1-year cumulative incidence of graft failure was 11% (95% confidence interval, 6% to 21%). Five patients (5%) had grade III-IV acute GVHD, 5 patients had limited chronic GVHD, and 1 patient had extensive GVHD. The incidence of veno-occlusive disease was higher in patients conditioned with Bu/Cy compared with those conditioned with Bu/Flu (19% [n = 10] versus 5% [n = 2]: P = .03). Of the 11 patients with graft failure, 8 (73%) were aplastic, and 3 (27%) had autologous reconstitution. Of 11 patients with graft failure, 9 underwent a second CBT, and 8 (89%) survived. Full donor chimerism was observed in 89% patients after first CBT and in all patients after second CBT. Survival after CBT for HS has improved, but better strategies are still needed to improve graft outcomes.

New Insights on the Role of the Mesenchymal-Hematopoietic Stem Cell Axis in Autologous and Allogeneic Hematopoiesis.

Cytoreductive protocols are integral both as conditioning regimens for bone marrow (BM) transplantation and as part of therapies for malignancies, but their associated comorbidities represent a long-standing clinical problem. In particular, they cause myeloablation that debilitates the physiological role of mesenchymal stem and precursor cells (MSPCs) in sustaining hematopoiesis. This review addresses the damaging impact of cytoreductive regimens on MSPCs. In addition, it discusses prospects for alleviating the resulting iatrogenic comorbidities. New insights into the structural and functional dynamics of hematopoietic stem cell (HSC) niches reveal the existence of "empty" niches and the ability of the donor-derived healthy HSCs to outcompete the defective HSCs in occupying these niches. These findings support the notion that conditioning regimens, conventionally used to ablate the recipient hematopoiesis to create space for engraftment of the donor-derived HSCs, may not be a necessity for allogeneic BM transplantation. In addition, the capacity of the MSPCs to cross-talk with HSCs, despite major histocompatibility complex disparity, and suppress graft versus host disease indicates the possibility for development of a conditioning-free, MSPCs-enhanced protocol for BM transplantation. The clinical advantage of supplementing cytoreductive protocols with MSPCs to improve autologous hematopoiesis reconstitution and alleviate cytopenia associated with chemo and radiation therapies for cancer is also discussed.

Treatment of CD40 ligand deficiency by hematopoietic stem cell transplantation: a survey of the European experience, 1993-2002

AbstractCD40 ligand (CD40L) deficiency causes recurrent sinopulmonary infection, Pneumocystis carinii pneumonia, and Cryptosporidium parvum infection. Approximately 40% to 50% of patients survive to the third decade: long-term survival is unclear. Hematopoietic stem cell transplantation (HSCT) is curative. We present a retrospective analysis of 38 European patients undergoing HSCT for CD40L deficiency in 8 European countries between 1993 and 2002. Donor stem cell source included 14 HLA-identical siblings, 22 unrelated donors, and 2 phenotypically matched parental stem cells (12 T-cell depleted). Of the patients, 34 engrafted and 26 (68%) survived; 3 had autologous reconstitution, 22 (58%) were cured, and 1 engrafted but has poor T-cell immune reconstitution. There were 18 evaluated patients who responded to vaccination. Of the patients, 12 (32%) died from infection-related complications, with severe cryptosporidiosis in 6. Grades 2 to 4 graft-versus-host disease (GvHD) associated with infection occurred in 6 of 12 fatal cases. HSCT cured 58% of patients, 72% of those without hepatic disease. Early T-cell function following whole marrow HSCT may limit cryptosporidial disease, but survival was similar after T-cell-depleted HSCT. Preexisting lung damage was the most important adverse risk factor. Further studies will determine optimal timing and type of HSCT. (Blood. 2004;103:1152-1157)

Outcomes of Non-Myeloablative HLA-Haploidentical Bone Marrow Transplant with Thiotepa and Post-Transplant Cyclophosphamide in Children and Adults with Severe Sickle Cell Disease, a Phase II Trial: Vanderbilt Global Haploidentical Transplant Learning Collaborative (VGC2)

Introduction: HLA-Haploidentical bone marrow transplant (haplo-BMT) is a curative approach for individuals with severe sickle cell disease (SCD) who lack an HLA-identical sibling donor. Haplo-BMT expands the donor pool among adults and children with SCD to &amp;gt; 90% and is a less toxic treatment option than myeloablative matched related donor transplant, gene therapy or gene editing. In 2013, our research team developed a multi-institution learning collaborative with the main objective of optimizing haplo-BMT. In a Phase II clinical trial, to improve engraftment rates, we augmented the original Hopkins haplo-BMT protocol (Bolaños-Meade et al. Blood 2012) with thiotepa (10 mg/kg). The trial was conducted at 3 sites and showed durable engraftment in 93% (14/15) of participants (including 2 with previous graft rejection), and a 100% overall survival after a median follow-up of 13.3 months (de la Fuente et al. BBMT 2019). We present results of extension of this ongoing trial comprising additional sites and participants. Material and methods: The trial was approved by the Institutional Review Board (IRB) at each participating site and opened in October 2013 (ClinicalTrials.gov identifier NCT01850108). The global learning collaborative includes 22 sites, in 10 countries, meets weekly via conference calls for sharing of SCD Haplo-BMT knowledge and patient related questions. Each site has a standalone institutionally IRB protocol, with similar eligibility criteria, and data sharing agreement with Vanderbilt University Medical Center. The agreements for aggregate REDCap data sharing and DSMB assessment of the outcomes with stopping rules. Common conditioning regimen included: rabbit ATG 4.5 mg/kg, thiotepa (10 mg/kg), fludarabine 150 mg/m2, cyclophosphamide 29 mg/kg and TBI 2Gy. GVHD prophylaxis included posttransplant cyclophosphamide (PTCy) 100 mg/kg, mycophenolate mofetil and sirolimus. Bone marrow mobilized with filgrastim (5-10 μg/kg/d x5 days) was initially used, 27% (11/41), but later stopped at several sites. Primary graft failure was defined as &amp;lt;5% donor myeloid chimerism or &amp;lt; 5% donor cells by whole blood chimerism by day +42 posttransplant. Secondary graft rejection was defined &amp;lt;5% donor myeloid chimerism or &amp;lt; 5% donor cells by whole blood chimerism with prior documentation of &amp;gt; 5% donor (myeloid) cells by day +42. The primary endpoint was 1-year EFS. Primary or secondary graft rejection, stroke, and death were considered events. Safety stopping rules were implemented, independent of age, using graft failure, death, grade III or IV acute GVHD or moderate or severe chronic GVHD. Results: We enrolled 41 consecutive participants from 8/14/2014 - 6/30/2020. Participant genotype included 39 patients with HbSS, 1 patient each with HbSC and Sβ0 thalassemia. Median age was 16.9 years (range 1.3 to 43), and 51% (21/41) of participants were &amp;lt;18 years of age. Donors included siblings (14/41, 34%), parents (27/41, 61%), and 88% (36/41) had sickle cell trait. Median TNC dose was 5.8 x 108 /kg (4.2, 8.0), and median CD34+ and CD3+ cell doses were 3.9 x 106/kg (2.6, 6.1) and 32.3 x 108/kg (27.1, 50.8), respectively. A total of 93% of participants engrafted. Median times to neutrophil (&amp;gt; 500/mcL) and platelet (&amp;gt;50 x 109/L) counts were 20 days (17.0, 24.2) and 32 days (27.0, 36.8) respectively. Incidence of grades III-IV acute and moderate chronic GVHD were 9.7% (4/41) and 12.1% (5/41), respectively. No participant had severe GVHD. Among durably engrafted participants, 81.8% (27/33) were off immunosuppression at 1-year posttransplant. Kaplan-Meier probabilities of EFS and OS were 75.5% (95% CI 61.3%, 92.9%) and 87.9% (95% CI 75.5%, 100.0%) at one year, respectively (Figure). Mortality was 7.3% (3/41). Graft failure was 12% (5/41; 3-primary and 2-secondary). All graft failures were in participants &amp;lt;18 years of age, and all had autologous reconstitution (Table). Conclusion: Our novel global learning collaborative effort for haplo-BMT for SCD is a scalable and efficient approach to sharing knowledge and implementing curative therapy for SCD. Importantly all haplo-BMT candidate had donors. An unexpected outcome in this trial, not likely to occur by chance, is that all graft rejections occurred in young participants (&amp;lt; 18 years), and all older participants (&amp;gt; 18 years) engrafted. The trial will add new stopping rules by age strata (&amp;lt;18, &amp;gt; or = to 18 years) and will modify the conditioning regimen in pediatric stratum. Disclosures Black: HRSA: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Micelle BioPharma: Research Funding; NHLBI: Research Funding. DeBaun:Novartis: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics (GBT): Membership on an entity's Board of Directors or advisory committees.

Granulocyte Colony-Stimulating Factor Is Safe and Well Tolerated Following Allogeneic Transplantation in Patients with Sickle Cell Disease

Background: Granulocyte colony-stimulating factor (G-CSF) is used after hematopoietic cell transplantation (HCT) to enhance neutrophil recovery in patients rendered neutropenic by the agents used in the preparative regimen (PMID: 9187067). G-CSF is contraindicated in patients with sickle cell disease (SCD) due to reported life-threatening complications ascribed to sickle vasculopathy, presumably secondary to leukocytosis and granulocyte activation (PMID: 11880644). Complications include severe vaso-occlusive pain crises, splenic engorgement, rupture, and death (PMID: 19513902). Individuals with sickle trait tolerate G-CSF without toxicity (PMID: 29743574, PMID: 27167356).The safety/toxicity of using G-CSF to enhance neutrophil recovery following HCT for SCD has not been previously described in the context of vasculopathy that is present in recipients but whose hematologic milieu is altered by transfusions and donor product infusion. Aim/Method: The hypothesis that G-CSF use would be safe in SCD patients following HCT was tested in a multicenter trial (NCT 03128996). Patients underwent HCT from matched or one-antigen mismatched (at HLA-A, -B, -C, -DRB1 if marrow; -A, -B, -DRB1 loci if cord) related or unrelated donor products between 2004 and 2019 following reduced intensity conditioning (RIC) which included hydroxyurea, alemtuzumab, fludarabine, melphalan +/- thiotepa. The regimen results in short-term marrow suppression followed by recovery. GVHD prophylaxis included tacrolimus, short-course methotrexate or mycophenolate or prednisone, and since 2018, abatacept. All patients underwent transfusions prior to commencing conditioning to reduce Hemoglobin S (Hb S) levels to &amp;lt;45%. G-CSF was commenced on day +7 post-HCT and continued until the absolute neutrophil count (ANC) was &amp;gt;1.5x103 cells/mL on 3 successive days. The clinical course, outcomes, and toxicities in the first 100 days post-HCT were evaluated as best representing the period of G-CSF influence on SCD recipients. Results: Sixty-four patients with SCD were evaluated post-HCT. The median age at HCT was 10.67 years (range, 1-21). HCT was performed for stroke/increased transcranial doppler velocity (N=32), acute chest syndrome (N=24) and vaso-occlusive episodes (VOE) (N=38). Twenty-nine and 35 patients received related and unrelated donor HCT respectively; 24 were mismatched at one antigen or allele locus. Graft sources included marrow (46), cord (12), peripheral blood (3), marrow + cord (2) and CD34 selected graft with T cell add-back (1). G-CSF was administered for a median of 9.4 days (range 6-33 days) post HCT. Neutrophil engraftment occurred at a median of 14.35 days (range 10-38), platelets at median of 25.2 days (range 12-89). Three patients recovered platelets beyond day 100. Seven patients had primary graft rejection and autologous reconstitution of hematopoiesis. Chimerism analysis in the remaining patients included a median of 93% (range 41-100% in the myeloid lineage; N=30), 89% (range 26-100% in the lymphoid lineage; N=36) and 93% (range 48-100% in whole blood; N=45) on day 100. The incidence of grade I-II and III-IV acute GVHD was 23.4% and 17% respectively during first 100 days. A total of 23 episodes of bloodstream and 7 non-bloodstream sites of bacterial infection were observed in 22 patients. No patient developed SCD related symptoms following post-HCT G-CSF administration. Organ toxicities noted in 17 of the 64 patients were variable [most common were hypertension (4), posterior reversible encephalopathy (3), and renal dysfunction (3)] and expected as part of the transplant process. Conclusion: In this cohort of SCD patients who underwent HCT from different donor sources following RIC and G-CSF to enhance neutrophil recovery, we noted no toxicities attributable to G-CSF. Specifically, no SCD related symptoms or extended hospitalization were encountered in any patient attributable to G-CSF administration. Commencing conditioning at a Hb S level &amp;lt;45% (the pre-alemtuzumab level) and chemotherapy induced destruction of native sickle hematopoiesis may be important factors in mitigating G-CSF related SCD complications post-HCT despite persistent vasculopathy and an inflammatory state. In this setting, G-CSF can be safely administered to SCD patients post-HCT. Disclosures No relevant conflicts of interest to declare.

Germline TET2 loss of function causes childhood immunodeficiency and lymphoma

AbstractMolecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.

Autologously Humanized Mice for Immune-Oncologic Studies.

With the rapid approval of immune checkpoint inhibitors for lung, melanoma, breast, genitourinary, and hematological malignancies, the hematopoietic cells in the tumor microenvironment (TME) are now considered an important, if not essential, consideration for cancer scientists. In many instances, syngeneic murine models have not been highly predictive for responsiveness in clinical trials. Our limited understanding of the human TME have, therefore, precluded a rational translation of immunotherapeutic combinations. This has led to the adoption of hematopoietic humanized murine models for the study of human tumor immunology in vivo. However, concerns about chimerism rates, HLA mismatching, and incomplete reconstitution of the innate immune system have driven a quest for improvements in these allogeneic humanized murine systems. Presented in this article is a completely autologous xenotransplantation method for reconstituting the human tumor immune microenvironment in vivo without the use of a patient's peripheral blood which is known to be associated with low engraftment rates. With this new approach, the current limitations of allogeneic humanized models are avoided by using matched bone marrow cells (BMCs) and derived tumor xenoplants (PDXs) from solid tumors in cancer patients. This autologous system provides a platform for studying endogenous lymphocytic and myeloid cell infiltration into the human tumor in vivo. © 2020 Wiley Periodicals LLC. Basic Protocol: Autologous reconstitution of human tumors Support Protocol 1: Transduction of BMCs and/or tumor cells prior to autologous reconstitution Support Protocol 2: Modeling immunotherapeutic agents in an autologously humanized model.

T-Cell Replete Haploidentical Bone Marrow Transplantation and Post-Transplant Cyclophosphamide for Patients with Inborn Errors

Introduction Matched donor hematopoietic stem cell transplantation (HSCT) cures most patients with inborn hematopoietic disorders, but haploidentical HSCT was historically associated with graft rejection, GVHD and infections. In leukemia, in vivo depletion of alloreactive T-cells with post-transplant cyclophosphamide (pTCy) results in outcomes comparable to those with matched related or unrelated donors. Objectives To assess safety and efficacy of haploidentical HSCT with pTCy in patients with inborn errors of hematopoiesis who lack a suitable matched donor or have failed a previous matched donor HSCT. Methods Nineteen patients (10 primary immunodeficiencies, 7 hemoglobinopathies, 1 congenital erythropoietic porphyria, 1 bone marrow failure) with a median age of 9 years (0-22) were treated prospectively on an institutional protocol with a conditioning regimen of alemtuzumab 0.4mg/kg, rituximab 375mg/m², fludarabine 150mg/m², treosulfan 42g/m², thiotepa 10mg/kg and cyclophosphamide 29mg/kg followed by haploidentical bone marrow transplantation, pTCy and tacrolimus/MMF. In four patients who were in bone marrow aplasia after non-engraftment of a first HSCT, thiotepa was omitted and treosulfan reduced to 24g/m². Results After a median follow-up of 42 months (3-87), overall survival is 95% (18/19). One patient died on day +7 from suspected fludarabine neurotoxicity. Two patients experienced autologous reconstitution, one of whom had a successful second HSCT. Two patients have stable mixed chimerism predominantly of donor origin and remain disease free. All others exhibit complete donor chimerism. This results in event free survival of 84% (16/19) and disease free survival of 89% (17/19). Engraftment occurred after 17days (6-61) for neutrophils and 28 days (14-191) for platelets. The incidences of acute GVHD II-IV° and of chronic GVHD were 0%, respectively. Two patients (11%) developed GVHD I° of the skin.Viral reactivations were observed in 53% (10/19) of patients (8 CMV, 1ADV, 1 VZV), but only one patient developed overt disease (ADV). Immune reconstitution was swift with mean CD3+ and CD19+ counts of 793/µl and 271/µl on day +100. Other complications were bacterial bloodstream infections in 5 patients (26%) and moderate severity VOD in one (5%). No renal or hepatic toxicities greater than CTCAE grade 2 were observed. No patient developed hemorrhagic cystitis. Four patients had anti-donor HLA antibodies pre-HSCT, three of whom were treated with plasmapheresis; all engrafted. Conclusion Chemotherapy based conditioning and haploidentical HSCT with pTCy was safe and resulted in good disease free survival in this cohort of patients with inborn errors in the absence of relevant GHVD. These results compare well with outcomes achieved with matched donors in similar cohorts of patients, but larger multi-center studies are warranted to further evaluate this approach.

Second Transplant for Primary Graft Failure after Haematopoietic Stem Cell Transplant

<h3>Introduction</h3> Primary graft failure after hematopoietic stem cell transplantation (HSCT) is a life threatening complication. The incidence is reported to be as high as 11%. It is very challenging as the patient is high risk having been pancytopenic for weeks, may have active infections and a poor performance status. There is also tremendous financial and emotional burden on the family. Here, we describe the results of 12 patients that received a second rescue transplant for primary graft failure. <h3>Aim</h3> To assess the incidence of primary graft failure and the outcome of second rescue transplant. <h3>Methods</h3> A total of 721 Allogenic transplants were performed between 2010-2019 at our centre; 14(1.94%) had primary graft failure. Out of these 12 that failed to engraft or had autologous reconstitution underwent a second rescue transplant. The patient and transplant characteristics are given in Table 1. <h3>Results</h3> Out of 12 second transplant; 9 patients achieved neutrophilic engraftment. The median neutrophil and platelet engraftment was 14(range 9-24) and 19(9-34) days. Two patients had Acute and 3 developed chronic GvHD (one limited and two extensive). CMV reactivation was seen in 4 patients. Seven out of 12 (58%) patients expired (Gram negative sepsis in 6 and chronic liver GvHD in one). At a median follow up of 190 days (-1 to 3003); 42% of patients are alive and disease free. <h3>Conclusion</h3> Outcome of primary graft failure is dismal. A second rescue transplant in our study showed a successful engraftment and long term survival of 42%. Thus a second transplant should be attempted as a life saving measure.

Use of Ex Vivo Graft Manipulation and Posttransplant Cyclophosphamide Result in Low GvHD Rates and Acceptable Engraftment after RIC Regimens in Pediatric Mismatched SCT

Introduction: There are currently two strategies to prevent Graft-versus-Host Diseases (GvHD) mainly applied in haploidentical transplantation. One is ex-vivo T-cell depletion of TcRa/b T-cells and the other is the T-replete approach, in which the donor T-cells remain in the graft and are tolerized in vivo by post-transplant cyclophosphamide (pCy). The ex-vivo depletion strategy does not require post-transplant immune suppression for GvHD prevention, whereas T-replete transplants require intensive immune suppression. A major obstacle for engraftment is the persistence of patients' T-cells despite intensive and myeloablative condition regimens, thus probably leading to rejection of the graft. We hypothesized that both methods could be combined in a setting of Reduced Conditioning setting (RIC). The ex-vivo T-cell depletion would allow to omit post-transplant immunosuppression and the pCy given at day +3 and +4 could induce in-vivo tolerance of the residual patients' T-cells not eliminated by RIC. Therefore, we applied this strategy in patients who were not eligible based on their poor clinical condition and who were considered to endure only a very reduced conditioning regimen. Results: We report on a cohort of 6 pediatric patients who were not eligible for myeloablative condition regimens due to preexisting organ dysfunctions (lungs, gut or liver) but were in urgent need of an SCT from matched unrelated (n=2) or haploidentical family donors (n=4). Diagnoses were: immune deficiencies (n =4; CARMIL 2, STAT 1, ICF 2, 1 not classified), relapsed metastatic ependymoma, refractory Burkitt´s lymphoma. All patients received a non-myeloablative conditioning regimen (ATG (Thymoglobin) 2mg/kg d-9 to d-7, fludarabine 30mg/m² d-6 to d-2, TBI 4Gy d-1, cyclophosphamide 50mg/kg d+3, d+4; adapted from Aversa, Reisner et al. Blood Adv. 2017). One patient additionally received thiotepa 2x5mg/kg on d-2. The CliniMACS® device was used for TCRab/CD19 depletion of peripheral stem cells; a median number of 14x10E6 CD34+ cells/kg bw with 6.4x10E3/kg bw residual TCRa/b T-cells was infused without any further posttransplant immune suppression. Four patients received a single add back of CD45 RA depleted donor T-cells at d+7. Dosages of 1x10E5/kg, 1x10E6/kg or 5x10E6/kg were administered. Two patients received an additional T-cell depleted stem cell boost after application of pCy Engraftment occurred in 4/6 patients; 2 patients rejected their haploidentical grafts and showed complete autologous reconstitution. Median time to reach ANC&gt;500 was 19 days (range 15-23). Four patients had no signs of GvHD; 1 patient had grade I; the patient who had received the highest dose of CD45RA depleted DLI developed grade III but could be treated successfully. No cGvHD occurred. Immune recovery was rapid. Median numbers of CD3+ T-cells, CD3/CD4+ T-cells, CD19+ B cells and CD56+ NK cells at d30 and d100 were 120/µl, 9/µl, 0/µl, 140/µl and 205/µl, 60/µl, 67/µl and 206/µl, respectively. 3 patients are alive and well with a median follow up of 824 days (43-1100). Last observed donor chimerisms were 95-100%. Causes of death in 3 other patients were: MAS/sepsis (STAT 1 deficiency, d 264) and progression in both patients with malignancies (d282 and d73). The patient with relapsed ependymoma showed a transient tumor regression for 3 months posttransplant whereas the patient with refractory Burkitt´s lymphoma had only a short response for 4 weeks. Conclusions: The combination of TCRa/b depletion and pCy allowed to use a very reduced conditioning regimen which could be administered in pediatric patients even with preexisting significant organ dysfunctions without severe side effects. GvHD could be effectively prevented (except in one patient who received a high number of DLI) together with an acceptable engraftment rate provided by post cy. Thus, this method might offer the possibility to establish a donor-derived hematopoiesis without using pharmacological myeloablation and with minimal toxicity and might be the basis for future strategies to further reduce the conditioning regimen, especially for patients with non-malignant diseases. Disclosures No relevant conflicts of interest to declare.