ATG Conditioning Research Articles

Favorable Outcomes in Severe Aplastic Anemia Patients Undergoing Allo-HSCT with Fludarabine, Cyclophosphamide, and ATG Conditioning

Favorable Outcomes in Severe Aplastic Anemia Patients Undergoing Allo-HSCT with Fludarabine, Cyclophosphamide, and ATG Conditioning

Bone Marrow Versus Peripheral Blood in HLA-Matched Sibling Transplantation: Does the Stem Cell Source Matter in Severe Aplastic Anemia?

Background Selecting a graft source is critical in HLA-matched sibling donor hematopoietic stem cell transplantation (MSD-HCT) for severe aplastic anemia (SAA). Since graft-versus-host-disease (GVHD) is the major cause of mortality after MSD-HCT, and SAA patients do not need the graft-versus-leukemia effect, the selection of a suitable source of stem cells becomes critical. Although bone marrow (BM) is the preferable stem cell source based on the registry-based studies, mobilized peripheral blood stem cells (PBSC) may offer faster engraftment with rapid immune reconstitution. Moreover, the recent advances in conditioning regimens have significantly reduced transplantation-related complications. Here, we compared the long-term outcomes of BM vs PBSC as a graft source in adult patients with SAA who received MSD-HCT with the reduced intensity conditioning (RIC) regimen. Method We conducted a retrospective analysis of 171 consecutive SAA adult patients who received MSD-HCT with Flu6/Cy2/ATG4 RIC regimen (Flu, 30 mg/m 2 IV for 6 days: Cy, 50 mg/kg IV for 2 days; rabbit ATG, 2.5 mg/kg IV for 4 days) or total nodal irradiation (TNI) of 750 cGy combined with ATG as GVHD prophylaxis (TNI-750/ATG) from March 2002 to March 2021 at Seoul St. Mary's Hospital, Seoul, Korea. BM was the primary graft source, but PBSC was used in donors who refused BM harvest, were unsuitable for general anesthesia, or received TNI-750/ATG conditioning. The primary endpoint of this study was to compare the overall survival (OS), and the secondary endpoints included speed of hematologic recovery, primary or secondary graft failure (GF), and failure-free survival (FFS) between the BM and PBSC group. Results The median patient age in the BM group was 38 years (range, 15 - 59) and 44 years (range, 19 - 64) in the PBSC group (p=0.005). There were no differences of baseline characteristics between the two groups except for a donor-to-recipient sex discrepancy (female to male, BM vs. PBSC; 16.4% vs. 30.9%, p=0.029). The majority of BM group (114/116, 98.2%) received the Flu6/Cy2/ATG4 RIC regimen, while 38.2% (21/55) of PBSC group received TNI-750/ATG for the conditioning (p<0.001). The PBSC contained a median 4.71 (range, 1.22 - 17.03) x 10 6/kg CD34 + cells and 368.82 (range, 25.12 - 1234.70) x 10 6/kg CD3 + cells which were about 1.7-fold (BM 2.80 x 10 6/kg CD34 + cells; range, 0.25 - 10.05) and 10-fold (BM 39.61 x 10 6/kg CD3 + cells; range, 1.34 - 463.68) higher than the BM graft (p<0.001). Consequently, the PBSC group exhibited faster engraftment of neutrophils (median 13 vs. 11 days, p<0.001) and platelets (median 18 vs. 12 days, p=0.012). However, there were no differences in the incidence of secondary GF (19.0% vs. 12.7%, p=0.386) and donor-type aplasia (4.3% vs. 10.9%, p=0.178) between two groups. Furthermore, the incidence of acute grade I-II (8.6% vs. 10.9%, p=0.619) and moderate to severe chronic graft-versus-host disease (GVHD) (3.5% vs. 5.5%, p=0.542) were also not significantly different between two groups. However, the BM group has shown more CMV reactivation (44.0% vs. 24.9%, p=0.005), and higher incidence of respiratory infection (14.7% vs. 3.6%, p=0.037) than those of the PBSC group. The incidences of transplantation-related complications between the BM and PBSC group are summarized in Table. The 10-year OS (93.8% vs. 98.1%, p=0.246) and FFS (65.7% vs. 69.0%, p=0.455) were comparable (Figure). Moreover, there was no significant difference in FFS between BM and PBSC group by potential risk factors of patient and donor age at MSD-HCT, sex incompatibility, ABC mismatch, PNH clone presence, HCT-CI ≥3, infused CD34 + cell count, interval from diagnosis to transplant, prior IST history, heavy transfusion before allo-HCT, or severity of AA. Conclusions Our results suggest that survival outcomes of MSD-HCT showed no difference between BM and PBSC as graft sources, and using PBSC may be an efficient alternative offering faster engraftment without significant difference in graft failure or transplantation-related adverse events such as GVHD, and survival. Current analysis and results were drawn from the Asian population with a fludarabine-based conditioning regimen and therefore, need to validate in the Western population. In addition, well-designed prospective studies in a larger cohort are required to confirm our findings.

Allogeneic Stem Cell Transplant for High-Risk Transfusion Dependent Thalassemia [Tdt] - a Comparative Analysis of Flu/Bu/Cy/ATG Versus Flu/Thio/Treo Regimen

Background & Significance: Allogeneic stem cell transplantation (allo-SCT) is the curative treatment for transfusion dependent thalassemia (TDT), until the gene therapy is readily accessible, which could revolutionize the treatment approach. 1 Later, Mathews et al. identified additional factors affecting outcomes, leading to the recognition of a specific very high-risk subgroup within Class III, referred to as Vellore high risk (HR) or Class IIIB. 2, 3 Following the identification of Class IIIB, treosulfan (Treo) was introduced as a treatment option due to its favorable low hepatic toxicity profile. 4, 5 Several studies have reported the use of Treo-based conditioning in the high-risk subset, showing treatment-related mortality (TRM) rates ranging from 7 to 21%. 6 - 8 Here, we present our (allo-SCT) experience for transfusion-dependent thalassemia (TDT) using the Flu/Bu/Cy/rabbit ATG or Flu/Thio/Treo as a conditioning regimen in a high risk cohort. Study design & Methods: We did a retrospective analysis of all transfusion dependent thalassemia [TDT] patients who underwent allogeneic stem cell transplant [allo-SCT] with Flu/Bu/Cy/ATG or Flu/Thio/Treo as a conditioning regimen. TDT patients who have received conditioning other than this were excluded from the study. The objective of the study is the assess the rate of thalassemia free survival [TFS], thalassemia GVHD free survival [TGFS], overall survival [OS], graft rejection and non-relapse mortality [NRM]. Results: We included a total of 64 patients in our study. Forty-one patients in Flu/Bu/Cy/ATG group and 23 patients were in Flu/Thio/Treo group. The median age of the patients was 9 years (range: 8 -14) and 10 years (8 - 15) in Flu/Bu/Cy/ATG and Flu/Thio/Treo group respectively. Upon presentation, the distribution of patients among Class I, II, and III were 2(4.9%) v/s 1(4.4%), 27(65.8%) v/s 11(47.8%), and 12(29.3%) v/s 11(47.8%) respectively. The graft source was bone marrow in 38 (92.7%) patients all in Flu/Bu/Cy group, whereas 3 (7.3%) v/s 23(100%) in peripheral blood stem cells. The mean CD34 stem cell dose administered was 3.4 × 10^6/kg (range: 0.7 - 9.8) v/s 5 × 10^6/kg (1.65 - 6.1). Neutrophil and platelet engraftment occurred at a median of 17 days (range: 11 - 26) v/s 15.5 days (range: 12 - 20) and 18 days (11 - 47) v/s 15 (9 - 41) respectively. Table:1 Primary rejection was in 3 (7.3%) whereas 2 (4.8%) were secondary rejection in Flu/Bu/Cy group only. Mixed chimerism was observed in a higher percentage of patients in the Flu/Bu/Cy/ATG group 17 (41.5%) compared to the Flu/Thio/Treo group 4 (17.4%). Veno-occlusive disease was reported in 14 (34.1%) v/s 4 (17.4%) patients, with [6 (14.6%) v/s 0] cases being mild, [6 (14.6%) v/s 3 (13%)] cases moderate, and [2 (4.8%) v/s 1 (4.3%)] case severe. Acute and Chronic graft-versus-host disease occurred in patients [7 (17.1%) v/s 10 (43.5%)] and [1 (2.4%) v/s 7 (30.4)] patients respectively. Fortunately, there were no cases of treatment-related mortality in Flu/Bu/Cy/ATG whereas 3 (13.3%) TRM was observed in Flu/Thio/Treo group. The overall survival at a median follow-up of 25 months were 100% v/s 87% [CI: 64.8 - 95.6] was statistically significant (p=0.02). Thalassemia GVHD free survival at a median follow-up was highly significant with a p value of 0.024 {83.7% [CI: 66.9 - 92.5] v/s 55% [CI: 31.3 - 73.5] (p = 0.024)} in Flu/Bu/Cy and Flu/Thio/Treo group respectively. Fig:1 Conclusion: In conclusion, the study suggests that the Flu/Bu/Cy/ATG conditioning regimen resulted in better overall survival [OS] and thalassemia GVHD free survival [TGFS] compared to Flu/Thio/Treo regimen in a high-risk transfusion dependent thalassemia [TDT] patients undergoing allo-SCT. However, it is essential to consider the limitations of the study, such as its retrospective nature and the relatively small sample size, before making any definitive conclusion. Further research with large sample sizes and prospective study designs may be needed to confirm these finding.

Myeloproliferative neoplasms – a global view

Myeloproliferative neoplasms – a global view

Steroid-resistant intestinal aGVHD and refractory CMV and EBV infections complicated by haplo-HSCT were successfully rescued by FMT and CTL infusion

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) produces similar survival outcomes as HLA-matched sibling donor allogeneic HCST in younger patients with acquired severe aplastic anemia (SAA). This study reported a 29-years-old man with SAA and intracranial hemorrhage who underwent haplo-HSCT with a modified BU/CY + ATG conditioning regimen. Neutrophil and platelet engraftment were both achieved on day 14 after HSCT. The patient developed grade IV acute graft-versus-host disease (aGVHD) on day 20 and acquired cytomegalovirus (CMV) and Epstein–Barr virus (EBV) infections on day 47. After the failure of methylprednisolone, basiliximab, ruxolitinib, and antiviral treatment, the patient was diagnosed with steroid-resistant grade IV aGVHD and refractory CMV and EBV infections. We performed fecal microbiota transplantation and infused CMV- and EBV-specific cytotoxic T lymphocytes. After that the stool volume and frequency gradually decreased, and viral DNA was undetectable on day 80. This report provides helpful clinical experience for treating steroid-resistant aGVHD and refractory viral infections.

Effectiveness of T-Cell Replete Haploidentical Hematopoietic Stem Cell Transplantation for Refractory/Relapsed B Cell Acute Lymphoblastic Leukemia in Children and Adolescents.

Background: The prognosis of refractory/relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) remains dismal owing to acquired resistance to chemotherapeutic agents. This study aimed to evaluate the efficacy of T-cell replete HLA haploidentical hematopoietic stem cell transplantation (TCR-haplo-HSCT) for pediatric refractory/relapsed BCP-ALL (RR-BCP-ALL).Methods: Nineteen pediatric patients with RR-BCP-ALL underwent TCR-haplo-HSCT between 2010 and 2019 at the Fukushima Medical University Hospital. The disease status at TCR-haplo-HSCT included complete remission (CR) in eight patients and non-CR with active disease in 11 patients. Total body irradiation-based, busulfan-based, and reduced-intensity conditioning regimens were employed in 11, 6, and 2 patients, respectively. Low-dose anti-thymocyte globulin (thymoglobulin, 2.5 mg/kg) was used in all patients. Graft-vs.-host disease (GVHD) prophylaxis was administered with tacrolimus, methotrexate, and prednisolone.Results: All patients received peripheral blood stem cells as the stem cell source. The HLA disparities in graft vs. host directions were 2/8 in one, 3/8 in five, and 4/8 in 13 patients. Among 18 patients who achieved primary engraftment, acute GVHD occurred in all 18 evaluable patients (grade II, 9; grade III, 8; grade IV, 1), and chronic GVHD was observed in 10 out of 15 evaluable patients. Three patients died because of transplant-related mortality. The 3-year overall survival (OS) and leukemia-free survival rates were 57.4 and 42.1%, respectively. Compared to patients older than 10 years in age (N = 10), those younger than 10 years in age (N = 9) showed an excellent OS rate (3-year OS rate: patients < 10 years old, 100%; patients > 10 years old, 20% [95% confidence interval, 3.1–47.5]; p = 0.002).Conclusions: We suggest that TCR haplo-HSCT with low-dose ATG conditioning has the potential to improve the transplantation outcomes in patients with RR-BCP.

Fludarabine/Treosulfan/Thiotepa/ATG Conditioning for Related Transplantation in b Thalassaemia Major Leads to Early and Sustained Engraftment with Low Incidence of VOD and GvHD but a High Rate of Long-Term Mixed Chimerism

Haemopoietic stem cell transplantation is a well-established treatment modality for b thalassaemia. The challenge is to minimise graft failure due to the expanded haemopoietic compartment whilst reducing the risk VOD due to the effects of iron load. From 2011 to 2016 thirty-nine consecutive sibling transplants were conditioned with fludarabine 160 mg/m2, treosulfan 42 g/m2, thiotepa 10 mg/kg and ATG (Thymoglobulin) 7.5 mg/kg (FTTA). Endogenous haemopoiesis was suppressed pre-transplantation with hypertransfusions for a minimum of 8 weeks. GvHD prophylaxis was provided with ciclosporin and MMF. The median age was 5 years (2 - 19). The source of stem cells was BM in 33 patients and mixed cord blood and BM in 6 patients. The median cell dose was 3.88 x108 TNC/kg (range 0.23 - 8.51, including patients having red cell depletion for major ABO incompatibility) and 7.19 x 106 CD34+/kg (range 2.05 - 14.43) for the patients receiving bone marrow only; 1.55 x 108 TNC/kg (range 1.27 - 3.9) and 3.32 x 106 CD34+/kg (range 2.06 - 8.79) for BM and 2.47 x 107 TNC/kg (range 0.8 - 7.32) and 0.44 x 106CD34+/kg (range 0.06 - 1.8) for cord blood for the patients receiving a combination of cord blood and bone marrow. The median survival was 25.8 months (2.6-65.9). Patients were Pesaro class I or II (Pesaro class III patients were intensively chelated pre-transplantation to reduce the stage of fibrosis and return to class I or II). All patients were evaluated with liver biopsy pre-transplantation and defibrotide prophylaxis given if Ishak stage ≥3.All patients engrafted and achieved evidence of donor haemopoiesis on day +28, and all but two patients achieved transfusion-independence and donor haematological values. Two patients had secondary graft failure: one patient had progressive reduction of donor haemopoiesis with reestablishment of ineffective thalassaemic haemopoiesis and transfusion dependence on day +313, another patient had reestablishment of ineffective thalassaemic and myelodysplastic clonal haemopoiesis with 7del on day +532; both patients established normal haemopoiesis following a second BMT. There was one death (2.7%), on day +89 due to idiopathic pneumonia syndrome. Acute GvHD ≥ grade 2 occurred in 5 patients (12.8%). Chronic limited GvHD occurred in 6 patients (15.4%) and extensive in 2 patients (5.1%). Chronic GvHD was only present at 18 months in one patient (2.7%). VOD occurred in one patient (2.7%, day +9) and responded to standard measures and defibrotide treatment. The median neutrophil engraftment was 11 days (range 9 to 21). The median platelet engraftment >20 x109/L was 27 days (range 16 to 73) and >50 x109/L was 32 days (range 19 to 73). The median time to cessation of immunosuppression was 160 days (105-309).Chimerism studies on day +28 demonstrated 97.4% in whole blood (WB) and 76.7% in T cells (T) >95%, 2.6% WB and 3.3% T >90-95%, 0% WB and 20% T >50-89%; day +90: 83.8% WB and 57.1% T >95%, 2.7% WB and 14.3% T >90-95%, 10.8% WB and 22.9% T >50-89%, 2.7% WB and 5.7% T <50%; day +180: 50% WB and 44.8% T >95%, 30% WB and 17.2% T >90-95%, 10% WB and 37.9% T >50-89%, 10% WB and 0% T <50%; and day +365: 55.6% WB and 42.3% T >95%, 7.4% WB and 19.2% T >90-95%, 14.8% WB and 18.2% T >50-89%, 22.2% WB and 19.2% T <50%.In conclusion, the 3 years' overall survival was 97.4% and the disease-free survival 90.3%. Following a second procedure for the two cases suffering secondary graft failure, the disease-free survival increased to 97.4%. Hence, FTTA leads to early and sustained engraftment with low rate of graft failure, and minimal occurrence of VOD and IPS, whilst the incidence of GvHD is low, though there is a high rate of mixed chimerism, the large majority which is stable. Further improvements to minimise unstable cases will focus in changes to serotherapy. Disclosuresde la Fuente:bluebird bio: Consultancy; Jazz Pharmaceuticals: Honoraria; HCA: Consultancy.

Allogeneic Hematopoietic Cell Transplantation for Acute Myeloid Leukemia and Myelodysplastic Syndrome: Effect of Transplant Conditioning Regimen Intensity on Outcomes

The objective of this study was to examine the effect of commonly used conditioning regimens on transplantation outcomes for acute myeloid leukemia (AML; n=1258) and myelodysplastic syndrome (MDS; n=951) transplanted in the US between 2009 and 2014. The median age of the study population was 58 years (18 - 83). Patients with AML were in first or subsequent remission and those with MDS had refractory anemia with unilineage or multilineage dysplasia, 5q- syndrome or refractory anemia with excess blasts at transplantation. Eighty-one percent of patients with AML had intermediate disease risk index (DRI). In contrast, 53% of patients with MDS had high DRI and 22%, very high DRI. Fifty-eight percent of patients reported performance scores of 90 or 100, and 48% had HCT-comorbidity score of 2 or less. A third of patients (35%) received grafts from HLA-matched siblings, 55% from HLA-matched and 10% from HLA-mismatched adult unrelated donors. Peripheral blood was the predominant stem cell source (90%) regardless of donor type. Graft-versus-host disease (GVHD) prophylaxis included a calcineurin inhibitor with methotrexate or mycophenolate; 73% received tacrolimus with methotrexate and 19%, tacrolimus with mycophenolate. All patients received non-irradiation transplant conditioning regimens, 57% being myeloablative (MAC), and 43% were reduced intensity (RIC). ATG was the predominant agent (98%) for in vivo T cell depletion. Cox regression models were built to study the effect of conditioning regimens on transplant outcomes after adjusting for other factors significantly associated with outcomes (Table 1). Fludarabine/busulfan with/without ATG was the predominant conditioning regimen (64%). Treatment failure (relapse or death; inverse of relapse-free survival), relapse and overall survival differed by conditioning regimen. Treatment failure was higher after RIC fludarabine/busulfan (Flu/Bu2) with/without ATG than after MAC fludarabine/busulfan (Flu/Bu4) without ATG conditioning due to higher relapse risks. However, compared to Flu/Bu4 with ATG, treatment failure did not differ with Flu/Bu2 with ATG (p=0.17) or without ATG (p=0.84). Although relapse risks were higher after Flu/Bu4 with ATG than without ATG, there were no differences in mortality or treatment failure risks. Treatment failure, relapse and overall survival were not different after Flu/Bu4 and busulfan/ cyclophosphamide (MAC) . But when ATG was added to Flu/Bu4, treatment failure (HR 1.22, p=0.05), relapse (HR 1.47, p=0.003) and overall mortality (HR 1.27, p=0.02) were higher compared to busulfan/ cyclophosphamide . Fludarabine/melphalan (Flu/Mel) with or without ATG accounted for only 12% of regimens and all were RIC regimens. Relapse (HR 2.32, p<0.0001) and treatment failure (HR 1.33, p=0.01) were higher with Flu/Bu2 compared to Flu/Mel. The addition of ATG also led to higher relapse (HR 2.13, p=0.004) with Flu/Bu2 compared to Flu/Mel but there were no differences in treatment failure (p=0.24) or overall survival (p=0.73). Among patients who received Flu/Mel, the addition of ATG was associated with higher mortality (HR 1.49, p=0.03). The 3-year probabilities of relapse-free and overall survival, adjusted for age, performance score, HCT-comorbidity score, disease and DRI are shown in Figure 1 and 2, respectively. In conclusion, Flu/Bu4 without ATG or busulfan/ cyclophosphamide regimens are optimal for patients with AML in remission and MDS. RIC regimens should be reserved for patients unable to tolerate MAC. Flu/Mel without ATG appears to be the optimal RIC regimen.Funded in part by a research grant from medac GmbH, Wedel, Germany [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure of immunosuppressive therapy

In 87 patients with aplastic anemia who failed to respond to immunosuppressive treatment, we determined the minimal dose of total body irradiation (TBI) required when added to antithymocyte globulin (ATG, 30 mg/kg x 3) plus cyclophosphamide (CY, 50 mg/kg x 4) to achieve engraftment of unrelated donor marrow. TBI was started at 3 x 200 cGy, to be escalated or deescalated in steps of 200 cGy depending on graft failure or toxicity. Patients were aged 1.3 to 53.5 years (median, 18.6 years). The interval from diagnosis to transplantation was 3 to 328 months (median, 14.6 months). Donors were HLA-A, -B, -C, -DR, and -DQ identical for 62 patients, and nonidentical for 1 to 3 HLA loci at the antigen or allele level for 25. The dose-limiting toxicity was diffuse pulmonary injury. The optimum TBI dose was 1 x 200 cGy. Nine patients did not tolerate ATG and were prepared with CY + TBI. Graft failure occurred in 5% of patients. With a median follow-up of 7 years, 38 (61%) of 62 HLA-identical, and 10 (40%) of 25 HLA-nonidentical transplant recipients are surviving. The highest survival rate with HLA-identical transplants was observed at 200 cGy TBI. Thus, low-dose TBI + CY + ATG conditioning resulted in excellent outcome of unrelated transplants in patients with aplastic anemia who had received multiple transfusions.

Immunophenotyping and Function of Peripheral Blood Mononuclear Cells in Patients Undergoing Unrelated Allogeneic Transplantation with Post-Transplantation Cyclophosphamide in Combination with ATG Anti-Graft Versus Host Disease Prophylaxis

Post-transplant cyclophosphamide (PTCy) effectively prevents alloreactive responses from unmanipulated grafts, but its effect on subsequent immune reconstitution remains largely undetermined. A total of 32 patients (pts) (ages &gt; 18) who underwent allogeneic hematopoietic stem cell transplantation (HCT) mainly for acute leukemia and myelodysplastic syndrome were evaluated. Blood samples were collected at 15, 30, 60, 90, 180 and 270 days post-HCT. Cell phenotype was assessed on peripheral blood mononuclear cells (PBMC) using multiparametric flow cytometry. The proliferative and activation T cell capacity in response to mitogenic stimulation was determined. Pts who received graft versus host disease (GVHD) prophylaxis with PTCy/ATG combination (n=8) were compared with pts who received ATG (Grafalon®, Neovii) alone (n=13), and pts undergoing HCT with no ATG (n=11). Five healthy controls served for the functional assays. First, CD4+ and CD8+ T cell differentiation was evaluated assessing CD62L, CD45RA, and CCR7 expression. A smaller percentage of effector cells was detected in the PTCy/ATG group at days 30, 60 and 90 after HCT, compared to the no-ATG and ATG cohorts. Accordingly, the percentage of central memory and effector memory cells was significantly increased in PTCy/ATG pts at the same time points. Next, immuno-modulatory cell populations were assessed. A transient increase in the percentage of CD4+CD25+CD127- Treg cells was detected in both ATG and PTCy/ATG cohorts in the early post-HCT period, reaching maximal frequency of 21% Treg out of the CD4+ subset (range 14-42%) in the ATG group and 19% (range 12-30%) in the PTCy/ATG group on day 21, suggesting that Treg cells are relatively spared by Cy. However, a distinct expression pattern of checkpoint molecules was revealed in pts receiving PTCy/ATG. The percentage of PD1-positive CD4+ and CD8+ cells was comparable in the no-ATG and ATG cohorts (44% vs 45% and 35% vs 41%, respectively). Yet, PD1 expression was significantly increased on both CD4+ (61%, p&lt;0.05) and CD8+ cells (60%, p&lt;0.02) from pts receiving PTCy/ATG. Similarly, an increased frequency of LAG-3+ CD8+ cells was observed in the PTCy /ATG pts, in comparison to the no-ATG and ATG groups. Concomitantly, HLA-DR expression on CD56+ NK subset was decreased in PTCy/ATG pts versus the ATG and non-ATG groups (47% vs 61% vs 67%, respectively, p&lt;0.05), indicating reduced activation of NK cells. Furthermore, a significant increase in the percentage of CD28-CD127- cells out of CD8+ cells was detected in PTCy/ATG pts compared to the ATG and no-ATG groups on day 60 post-HCT (82% vs 56% vs 38%, respectively, p&lt;0.02). CD57 up-regulation combined with CD28 down-regulation on T cells reflected an exhausted/senescent phenotype; therefore, we further analyzed the frequency of CD28-CD57+ T cells. Significantly elevated expression of CD57 on CD8+CD28- cells was detected in the PTCy/ATG cohort, in comparison to the ATG and no-ATG groups (65% vs 43% vs 21%, respectively, p&lt;0.01). Exhausted/senescent T cells showed functional impairment, which manifested as reduced potential for cytokine production and poor proliferative capacity. Notably, both CD4+ and CD8+ cells from ATG and PTCy/ATG cohorts demonstrated diminished proliferation in response to ex-vivo αCD3/αCD28 stimulation, when compared to the no-ATG cohort and healthy controls (p&lt;0.01). Moreover, significantly reduced production of IFNγ in response to stimulation was detected in PTCy/ATG and ATG pts (p&lt;0.01). PBMC from the no-ATG cohort displayed inducible IFNγ production levels comparable with healthy controls. Finally, PTCy/ATG-treated patients demonstrated altered phenotypic and functional recovery of the CD8+ cells. While CD4+ cells gradually downregulated PD1 expression and up-regulated co-stimulatory molecules CD28 and CD127 on days 180 and 270, notably CD8+ cells retained exhausted/tolerogenic phenotype as late as 270 days post-HCT. Our results demonstrate that the acquisition of senescent/exhausted phenotype by cytotoxic CD8+ cells is a distinctive feature of HCT with ATG conditioning. PTCy further compromises recovery of CD8+ cells and impairs NK activation, resulting in a sustained immunosuppression. Hopefully, these results will not only lead to a better understanding of the tolerance mechanisms behind PTCy/ATG anti GVHD prophylaxis but will also assist in the development of novel clinical treatments. Disclosures No relevant conflicts of interest to declare.

Stem Cell Transplantation in Pediatric Patients with Myelodysplastic Syndrome at a Single Institution

AbstractIn MDS, the bone marrow produces abnormal, immature blood cells called blast cells. Imprecise, in half of pediatric MDS, blast count is normal. A retrospective observational study was conducted to review the outcome of our HCT in pediatric patients with MDS. Record of 35 MDS patients after BMT, 1993-2016, were reviewed. Median age at transplant was 4 yrs (0.8-14.8) and median time to transplant from diagnosis 8.1 (2.3-102.5) months. TRM was 17.1% (6); [low risk (LR) = 5 (19.2%) and high risk (HR) = 1 (11.1%)] MDS group succumbed within first 100 days. The rest were fully engrafted; [low risk = 21 (72.4%) and high risk = 8 (27.6%)]. Primary and secondary graft failure was observed in one patient each (2.9%). VOD was seen in 2 patients (5.7%) and 5 (14.3%) had hemorrhagic cystitis. With a median follow-up of 112.4 months and 12 events of mortality, 3-years OS was 68.1% ± 8.0%. No significant risk factor including age, time to transplant, disease risk group, gender, conditioning regimen, source of stem cells, or a GvHD through uni- or multi-variable analyses were found to be associated with OS. Bu/Cy/±ATG conditioning regimen showed a trend of superiority for OS and EFS in our small series. The relapse incidence in our cohort was 11.5% in LR MDS.

Allogeneic Hematopoietic Cell Transplantation for Severe Idiopathic Aplastic Anemia Older Than 40y

▪Upfront allogeneic hematopoietic cell transplantation (alloHCT) as first line therapy for older than 40 or 50 years is not usually recommended for severe aplastic anemia patients even though there are suitable matched sibling donors because they usually have poor outcomes after alloHCT. Therefore, first line immune suppression therapy (IST) is recommended. However, current outstanding alloHCT outcome can make it possible to try upfront alloHCT even in older AA patients. The purpose of this retrospective study is to determine the transplantation-related results in AA patients older than 40 years. This study collected data retrospectively for older AA patients. Congenital bone marrow failure was excluded from this study. alloHCT was divided as upfront and second alloHCT according to prior IST.Total 129 patients were enrolled in this study from 2001 to 2017. Age at diagnosis and at alloHCT were 25 to 63 (median 48.0) years and 40.3-64.9 (median 49.1) years, respectively. Median time from diagnosis to alloHCT was 5.2 (range, 1-234.1) months. Upfront and second alloHCT were 42 and 87 patients, respectively. Upfront alloHCT received more stem cells from related donors, more BM stem cells and more fludarabine conditioning compared with second alloHCT (83.3% vs. 58.6%, p=0.005; 52.4% vs. 74.7%, p=0.011; 52.4% vs. 30.2%, p=0.015, respectively). However, ABO mismatching (p=0.747), TBI conditioning (p=0.547), cyclophosphamide conditioning (p=0.114), ATG conditioning (p=0.483) were similar between upfront and second alloHCT. Any engraftment failure, neutrophil engraftment failure and platelet engraftment failure were similar between upfront and second alloHCT (28.6% vs. 28.7%, p=0.985; 19.0% vs. 18.4%, p=0.928; 19.0% vs. 34.5%, p=0.072). Hepatic SOS, acute GvHD and chronic GvHD were also similar between upfront and second alloHCT (4.8% vs. 5.7%, p=0.817; 28.6% vs. 36.5%, p=0.376; 19.0% vs. 19.5%, p=0.947). Survival rates at 1Y, 2Y, 3Y and 5Y were 90.7, 82.2, 73.5 and 64.3%, respectively. Survival rates at 5 years in upfront and second alloHCT were 76.2 and 54.1%, respectively (p=0.059). Survival rates at 5 years (5YSR) in age 40-50y, 50-60y, and older than 60y were 64.1, 62.4 and 50.0%, respectively (p=0.349). alloHCT from matched related donor or other donors had similar survival rates (p=0.404). However, upfront alloHCT showed superior survival rate (5YSR 76.5% vs. 53.2%, p=0.114) without statistical significance compared with second alloHCT even in matched related donor subgroup. This trend is similar in alternative donor subgroup (5YSR 75.0% in upfront alloHCT vs. 54.9% in second alloHCT, p=0.459).alloHCT in older AA showed promising results even in patient older than 60 years although upfront alloHCT showed marginal statistical superiority. In conclusion, upfront alloHCT in older AA needs further confirmation by prospective studies. DisclosuresNo relevant conflicts of interest to declare.

Fludarabine/Treosulfan/Thiotepa/ATG Conditioning for Related Transplantation in β Thalassaemia Major Leads to Early and Sustained Engraftment with Low Incidence of VOD and GvHD

▪Haemopoietic stem cell transplantation is a well-established treatment modality for β thalassaemia. The challenge is to minimise graft failure due to the expanded haemopoietic compartment whilst reducing the risk VOD due to the effects of iron load. From 2011 to 2016 thirty-five consecutive sibling transplants were conditioned with fludarabine 160 mg/m2, treosulfan 42 g/m2, thiotepa 10 mg/kg and ATG (Thymoglobulin) 7.5 mg/kg (FTTA). Endogenous haemopoiesis was suppressed pre-transplantation with hypertransfusions for a minimum of 8 weeks. GvHD prophylaxis was provided with ciclosporin and MMF. The median age was 5 years (2 - 16). The source of stem cells was BM in 30 patients and mixed cord blood and BM in 5 patients. The median cell dose was 4.17 x108 TNC/kg (range 0.23 - 8.51, including patients having red cell depletion for major ABO incompatibility) and 7.28 x 106 CD34+/kg (range 2.05 - 14.43) for the patients receiving bone marrow only; 1.64 x 108 TNC/kg (range 1.22 - 3.9) and 4 x 1 06 CD34+/kg (range 2.06 - 8.79) for BM and 3.8 x 107 TNC/kg (range 0.8 - 7.32) and 0.53 x 106CD34+/kg (range 0.06 - 1.8) for cord blood for the patients receiving a combination of cord blood and bone marrow. The median survival was 21.1 months (0.4-50.7). Patients were Pesaro class I or II (Pesaro class III patients were intensively chelated pre-transplantation to reduce the stage of fibrosis and return to class I or II). All patients were evaluated with liver biopsy pre-transplantation and defibrotide prophylaxis given if Ishak stage ≥ 3.All patients engrafted and achieved evidence of donor haemopoiesis on day +28, and all but one patient achieved transfusion-independence and donor haematological values. Two patients had secondary graft failure: one patient had progressive reduction of donor haemopoiesis with reestablishment of ineffective thalassaemic haemopoiesis and transfusion dependence on day +313, another patient had reestablishment of ineffective thalassaemic and myelodysplastic haemopoiesis with 7 deletion on day +532; both patients established normal haemopoiesis following a second BMT. There was one death (2.9%), on day +89 due to idiopathic pneumonia syndrome. Acute GvHD ≥ grade 2 occurred in 4 patients (11.4%). Chronic limited GvHD occurred in 5 patients (14.3%) and extensive in 1 patient (2.9%). Chronic GvHD was only present at 18 months in one patient (2.9%). VOD occurred in 2 patients (5.7%, days +7 and +9 respectively) and responded to standard measures and defibrotide treatment. The median neutrophil engraftment was 11 days (range 9 to 21). The median platelet engraftment >20 x109/L was 28 days (range 16 to 73) and >50 x109/L was 32 days (range 19 to 73). The median time to cessation of immunosuppression was 160 days (105-309).Chimerism studies on day +28 demonstrated 97.1% in whole blood (WB) and 76.9% in T cells (T) >95%, 2.9% WB and 3.8% T >90-95%, 0% WB and 19.2% T >50-89%, and 0% WB and 4.8% T <50%; day +90: 81.3% WB and 56.7% T >95%, 3.1% WB and 13.3% T >90-95%, 12.5% WB and 23.3% T >50-89%, 3.1% WB and 6.7% T <50%; day +180: 48.1% WB and 42.3% T >95%, 33.3% WB and 19.2% T >90-95%, 11.1% WB and 38.5% T >50-89%, 7.4% WB and 0% T <50%; and day +365: 56% WB and 41.7% T >95%, 8% WB and 20.8% T >90-95%, 16% WB and 20.8% T >50-89%, 20% WB and 16.7% T <50%.In conclusion, the 3 year's overall survival was 97.1% and the disease-free survival 89.2%. Hence, FTTA leads to early and sustained engraftment with low rate of graft failure, and minimal occurrence of VOD and IPS, whilst the incidence of GvHD is low, though there is a high rate of mixed chimerism, the large majority which is stable. Further improvements should focus on minimising unstable cases. DisclosuresNo relevant conflicts of interest to declare.

Fludarabine/Treosulfan/Thiotepa/ATG Conditioning for Matched and Single Antigen Mismatched Unrelated Bone Marrow Transplantation in Haemoglobinopathies Is Feasible and Leads to Early and Sustanined Engraftment with No Long-Term Toxicity or GvHD

Related haemopoietic stem cell transplantation is a well-established treatment modality for haemoglobinopathies, but it is limited by the availability of related donors. The results of unrelated transplantation have been variable with historical variable results, concern about GvHD and usually restricted to 10/10 matches. From 2011 to 2016 twelve consecutive unrelated bonemarrow transplants were conditioned with fludarabine 160 mg/m2, treosulfan 42 g/m2, thiotepa 10 mg/kg and ATG (Thymoglobulin) 11.25 mg/kg (FTTA). Endogenous haemopoiesis was suppressed pre-transplantation with hypertransfusions for a minimum of 8 weeks. GvHD prophylaxis was provided with ciclosporin and MMF. 7 patients were transplanted for β thalassaemia major, one of α thalassaemia major and 4 for sickle cell disease. The median age was 9.5 years (2 - 17). The source of stem cells was BM in all patients: five 10/10 matched, six 9/10 matched and one 11/12 matched. The median cell dose was 3.01 x 108 TNC/kg (range 1.53 - 11.07) and 4.27 x 106 CD34+/kg (range 1.16 - 27.41). The median survival was 9.9 months (6.9-44.1). Patients with thalassaemia were Pesaro class I or II (Pesaro class III patients were intensively chelated pre-transplantation to return to class I or II). Patients with sickle cell disease were transplanted for stroke or recurrent vaso-oclusive crises and/or acute chest syndrome not responding to hydroxycarbamide. All patients were evaluated with liver biopsy pre-transplantation and defibrotide prophylaxis given if Ishak stage ≥3.All patients engrafted and achieved evidence of donor haemopoiesis on day +28 and achieved transfusion-independence and donor haematological values. No patient suffered primary or secondary graft failure. There was one death, one on day +257 due to idiopathic pneumonia syndrome in a patient with b thalassaemia major. Acute GvHD ≥ grade 2 occurred in 5 patients (41.7%). Chronic limited GvHD did not occur in any patient, but extensive occurred in the 5 patients developing acute GvHD. None of the patients had chronic GvHD at 18 months. VOD occurred in 4 patients (33.3%) and responded to standard measures and defibrotide treatment. The median neutrophil engraftment was 13 days (range 9 to 22). Patient with sickle cell disease had the platelet count maintained >50 x 109/L at all times. The median platelet engraftment >50 x 109/L was 37 days (range 15 to 86) and >50 x 109/L was 32 days (range 15 to 111). The median time to cessation of immunosuppression was 134 days (68-219).Chimerism studies on day +28 demonstrated 100% in whole blood (WB) and 88.9% in T cells (T) >95%, and 0% WB and 11.1% T >50-89% [n=11]; day +90: 91.7% WB and 91.7% T >95%, 8.3% WB and 0% T >50-89%, 0% WB and 8.3% T <50% [n=12]; day +180: 91.7% WB and 91.7% T >95%, and 8.3% WB and 8.3% T >50-89% [n=12]; and day +365: 50% WB and 50% T >95%, 25% WB and 25% T >90-95%, and 25% WB and 25% T >50-89% [n=4].In conclusion, unrelated bone marrow transplantation for all haemoglobinopathies is feasible and the overall survival and disease-free survival is 90.9%. FTTA leads to early and sustained engraftment with low rate of graft failure, and whilst the occurrence of VOD despite adequate chelation pre-transplantation and the incidence of GvHD was significant, these complications resolved with standard therapies and long-term outcomes approach those of related transplantation with no treatment required by 18 months. DisclosuresNo relevant conflicts of interest to declare.

Fludarabine/Treosulfan/Thiotepa/ATG Conditioning for Related Transplantation in Haemoglobinopathies Leads to Early and Sustanined Engraftment with Low Incidence of VOD and GvHD

Haemopoietic stem cell transplantation is a well-established treatment modality for haemoglobinopathies. The challenge is to minimise graft failure due to the expanded haemopoietic compartment whilst reducing the risk VOD due to the effects of iron load. From 2011 to 2015 fifty-four consecutive sibling transplants were conditioned with fludarabine 160 mg/m2, treosulfan 42 g/m2, thiotepa 10 mg/kg and ATG (Thymoglobulin) 7.5 mg/kg (FTTA). Endogenous haemopoiesis was suppressed pre-transplantation with hypertransfusions for a minimum of 8 weeks. GvHD prophylaxis was provided with ciclosporin and MMF. 33 patients were transplanted for b thalassaemia major and 21 for sickle cell disease. The median age was 7 years (2 - 16). The source of stem cells was BM in 49 patients and mixed cord blood and BM in 5 patients. The median cell dose was 3.48 x108 TNC/kg (range 0.23 - 8.51) and 6.73 x106 CD34+/kg (range 1.3 - 14.72) for the patients receiving bone marrow only; 1.64 x108 TNC/kg (range 1.22 - 3.9) and 4 x106 CD34+/kg (range 2.06 - 8.79) for BM and 3.8 x107 TNC/kg (range 0.8 - 7.32) and 0.53 x106 CD34+/kg (range 0.06 - 1.8) for cord blood for the patients receiving a combination of cord blood and bone marrow. The median survival was 14.4 months (1-41.9). Patients with thalassaemia were Pesaro class I or II (Pesaro class III patients were intensively chelated pre-transplantation to return to class I or II). Patients with sickle cell disease were transplanted for stroke or recurrent vaso-oclusive crises and/or acute chest syndrome not responding to hydroxycarbamide. All patients were evaluated with liver biopsy pre-transplantation and defibrotide prophylaxis given if Ishak stage ≥ 3.All patients engrafted and achieved evidence of donor haemopoiesis on day +28, and all but one patient achieved transfusion-independence and donor haematological values. One patient (1.9%) had progressive reduction of donor haemopoiesis with reestablishment of ineffective thalassaemic haemopoiesis and transfusion dependence on day +313. There were two deaths, one on day +89 due to idiopathic pneumonia syndrome in a patient with b thalassaemia major and one on day +189 due to an intracranial haemorrhage caused by refractory immune thrombocytopenia off immunosuppression in a patient with sickle cell disease and moya moya. Acute GvHD ≥ grade 2 occurred in 6 patients (11.1%). Chronic limited GvHD occurred in 7 patients (13%) and extensive in 3 patients (5.6%). Chronic GvHD was only present at 18 months in one patient (1.9%). VOD occurred in 2 patients (3.7%, days +7 and +9 respectively) and responded to standard measures and defibrotide treatment. The median neutrophil engraftment was 12 days (range 8 to 21). The median platelet engraftment >20 x109/L was 27 days (range 15 to 73) and >50 x109/L was 32 days (range 15 to 73). The median time to cessation of immunosuppression was 160 days (91-538).Chimerism studies on day +28 demonstrated 92.3% in whole blood (WB) and 69% in T cells (T) >95%, 5.8% WB and 4.8% T >90-95%, 1.9% WB and 21.4% T >50-89%, 0% WB and 4.8% T <50%; day +90: 83.3% WB and 60.4% T >95%, 4.2% WB and 8.4% T >90-95%, 10.4% WB and 20.8% T >50-89%, 2.1% WB and 10.4% T <50%; day +180: 59.4% WB and 45% T >95%, 19% WB and 17.5% T >90-95%, 16.7% WB and 35% T >50-89%, 4.8% WB and 2.5% T <50%; and day +365: 59.4% WB and 53.4% T >95%, 12.5% WB and 13.3% T >90-95%, 15.6% WB and 20% T >50-89%, 12.5% WB and 13.3% T <50%.In conclusion, FTTA leads to early and sustained engraftment with low rate of graft failure, and minimal occurrence of VOD whilst the incidence of GvHD is low. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic and Functional Characteristics of NK Cells Associated with Cytomegalovirus (CMV) Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation (HCT)

s / Biol Blood Marrow Transplant 20 (2014) S128eS150 S146 virus (EBV) is a gammaherpes virus that is reactivated from latent to lytic cycle and can cause aggressive lymphomas known as post transplant lymphoproliferative disorder (PTLD) in immunocompromised hosts. We attempted to distinguish immune reconstitution profiles in double UCBT recipients who developed EBV viremia from those who did not. Thirty-one patients with hematologic malignancies received dUCBT with melphalan, fludarabine, ATG conditioning and tacrolimus plus sirolimus for GvHD prophylaxis. During the first 12 months after dUCBT, 14 of 31 (45%) patients developed EBV viremia and four (13%) developed PTLD. At one month after dUCBT, patients with EBV viremia displayed higher numbers of CD19+ B cells (p1⁄40.04) and CD4+CD25+ Treg cells (p1⁄40.03) compared with patients who never became viremic. Surprisingly, EBV viremia correlated with increased numbers of CD3+ (p1⁄40.04), CD4+ (p1⁄40.015) and CD8+ (p1⁄40.021) T cells, but this response was ineffective at controlling the virus. One explanation for this unexpected result might be skewing towards a late effector memory T cell phenotype, a stage in which T cells are incapable of mounting protective immune responses. We examined naive vs. memory T cell distribution and determined that patients who developed EBV reactivation had higher numbers of memory cell subsets (CD4+CD45RO+, p1⁄40.023; CD8+CD45RO+, p1⁄40.019) at two months after dUCBT. Analysis of repertoire diversity by deep-sequencing on PCR-amplified CDR3 regions of the TCRb gene using the ImmunoSEQ assay showed a more diverse TCR repertoire, as determined by higher entropy (p1⁄40.03) and lower clonality (p1⁄40.03), among patients who did not develop EBV reactivation. Assessment of SCF and IL-7, critical regulators of hematopoietic stem cell and naive T cell homeostasis, respectively, showed that patients without EBV viremia had higher levels of SCF (p1⁄40.0016) and IL-7 (p1⁄40.046) compared with patients who developed viremia and PTLD. Our findings suggest that control of EBV reactivation after dUCBT might be linked to the support of naive T cell homeostasis, which enables maintenance of a diverse TCR repertoire.

Post-Transplant Lymphoproliferative Disorder Is Common after Reduced-Intensity ATG Conditioning and Haplo-Cord Transplantation

Post-Transplant Lymphoproliferative Disorder Is Common after Reduced-Intensity ATG Conditioning and Haplo-Cord Transplantation

Comparable Allogeneic Hematopoietic Cell Transplantation Outcome Of Haplo-Identical Family Donor With Matched Unrelated/Mismatched Family Donor In Adult Aplastic Anemia

Allogeneic hematopoeitic cell transplantation (alloHCT) is a curative therapy for severe/very severe aplastic anemia (sAA) in adult. alloHCT from haplo-identical family donor (HD) is an alternative approach while alloHCT from matched sibling donor (MSD) is the choice of therapy. Our previous study suggested that alloHCT from matched unrelated donor (MUD) were comparable with MSD when pre-transplantation conditions were well-matched. It means poor outcome of MUD might come from poor pre-transplantation clinical factors such as delayed alloHCT. Same will be applied to HD. In this study we compared HD with alternative donors (AD; MUD or mismatched family donor) by matched case study.We selected AD cases from KSBMT2007-01 study population who had comparable pre-transplantation clinical factors with HD by propensity-score matching. Pre-tranplatation clinical factors such as age, ATG conditioning or ABO-compatibility were matched. Therefore 48 AD cases were selected for the comparison with 16 HD cases.Male (p=0.009) and female to male transplantation (p=0.002) were more frequent in HD. AD received prior immune suppression therapy (IST) frequently (p=0.092). Cyclophosphamide (p<0.001) and total body irradiation (p=0.055) were frequent in AD while fludarabine (p=0.006) and busulfan (p<0.001) were common in HD as a conditioning regimen. Bone marrow was used frequent in AD (60.4%; p<0.001) and peripheral blood was used in all HD (100%; p<0.001) as stem cell source. Despite this differences, ATG as prior IST (p=0.769), ABO compatibility (p=0.111), T-cell ex vivo depletion (0%, both), age at diagnosis (p=0.622), age at alloHCT (p=0.400), time from diagnosis to alloHCT (p=0.822), unit of red blood cell transfusion prior to alloHCT (p=0.655), unit of platelet cell transfusion prior to alloHCT (p=0.604) and infused CD34+ cell dose (p=0.087) were similar. All transplantation outcomes except for extensive chronic graft versus host disease (GvHD) were similar. The frequencies of hepatic sinusoidal obstruction syndrome (p=1.000), acute GvHD (p=0.769), grade 3/4 acute GvHD (p=0.258), chronic GvHD (p=0.173), extensive GvHD (p=0.099), primary neutrophil engraftment failure (p=1.000), secondary graft failure (p=1.000), platelet engraftment failure (p=0.505), all graft failure (p=0.745) were similar (Table 1). Time to neutrophil engraftment was faster in HD (p=0.003) while cumulative incidence of platelet engraftment (p=0.505), acute GvHD (p=0.378), grade 3/4 acute GvHD (p=0.255), chronic GvHD (p=0.202), extensive GvHD (p=0.918) were similar. Overall survival was also similar between AD and HD (p=0.730; Figure 1). [Display omitted] In conclusion, alloHCT from HD in sAA is comparable with alloHCT from AD when pre-transplantation clinical factors were similar although extensive chronic GvHD seemed frequent in HD. Therefore alloHCT from sAA could be alternative approach of alloHCT from MUD in adult sAA. Disclosures:No relevant conflicts of interest to declare.

Transplantation Of TcRαβ/CD19 Depleted Stem Cells From Haploidentical Donors In Children: Current Results

▪T-cell depletion of the graft is an effective method to prevent or completely avoid Graft-versus-Host Disease (GvHD) in haploidentical stem cell transplantation. In order to increase the T-cell depletion efficacy while maintaining the anti-tumor and anti-infectious properties of the graft, we have investigated a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, Natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of posttransplant EBV-associated lymphoproliferative disease. The CliniMACS system was used for manipulation of peripheral stem cell grafts from full haplotype mismatched family donors in 35 patients. ResultsThe overall depletion of αβ+ T-cells was highly effective with 4.6 log (range 3.8–5.0). Patients received a median number of only 14 x 103/kg residual αβ+ T-cells. Recovery of CD34+ stem cells was 72%, and the median number of infused CD34+ stem cells was 12 x 106/kg (range 5-38 x 106/kg). Additionally, the patients received 2 types of potential antileukemic effector cells: 107 x 106/kg (range 35 -192 x 106/kg) CD56+ NK-cells and 11 x 106/kg (range 5–30 x 106/kg) γδ+ T-lymphocytes. Diagnoses wereALL (n=20), AML/MDS/JMML (n=9), nonmalignant diseases (n=4), solid tumors (n=2); disease status: CR2-CR6 (n=17), active disease (n=18). 23 patients received a second or third SCT (65%). A toxicity reduced conditioning regimen (fludarabin 40mg/m² or clofarabin 50mg/m² (day -8 to d -5), thiotepa 10mg/kg (d -4), melphalan 70mg/m² (d -3 and d -2) was used. The anti CD3 specific OKT3 antibody was used as rejection prophylaxis from day -8 to day -1 without affecting cotransfused effector cells because of its short half-life period in the first 7 patients. However, due to its restricted availability, the substance was substituted since 2011 by a reduced ATG-F dose (15mg/kg) given at start of the conditioning regimen in order not to impair NK and γδ+ T-cells of the grafts (1 mg/kg d -12, 4 mg/kg d -11, 5 mg/kg d -10 and -9; n=28 patients). Short course MMF (until day +30) was given in 25 patients. Graft rejection occurred in 14% of the patients. However, after reconditioning and second stem cell donation, final engraftment was achieved in all patients. The median time to reach neutrophil and platelet recovery in patients with primary engraftment was 10 and 11 days respectively. All patients showed a rapid immune reconstitution with 250 (OKT3 conditioning) and 273 (ATG conditioning) CD3+ T-cells/µl, 30 (OKT3) and 47 (ATG) CD3+4+/µl and 300 (OKT3) and 382 (ATG) CD56+ NK-cells/µl at day +30 posttransplant. γδ+ T-cells started to expand faster than αβ+ T-cells in the early post-transplant period (156 vs. 82 cells/µl at day +30) whereas at day +90, αβ+ T-cells were predominant (170 vs. 134 cells/µl). Acute GvHD grade 0-I occurred in 25 patients (71%); 6 patients had GvHD II (17%), 3 patients had GvHD III (9%) and one patients experienced GvHD grade IV (3%). 3 patients experienced chronic GvHD (8%). Incidence of acute GvHD was not influenced by the number of residual T cells or by the type of serotherapy. 1 year EFS for patients with acute leukemias was 66% (any CR) and 14% (active disease).TRM at 1 year was 20%. ConclusionsThese data indicate that transplantation of TcR αβ+/CD19 depleted cells from a haploidentical donor results in sustained engraftment, remarkably fast immune reconstitution and low incidence of both acute and chronic GvHD. OKT3 could be substituted by ATG without negative effects. The anti-leukemic efficacy of this approach in comparison to other methods of T-cell depletion needs to be evaluated with a longer patient follow-up. Disclosures:No relevant conflicts of interest to declare.

Reconstitution Of EBV-Specific Immunity In Adult Recipients Of Double Cord Blood Transplantation Depends On SCF and IL-7 Levels and Maintenance Of a Diverse TCR Repertoire

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells (HSC) for allogeneic HSC transplantation but reconstitution of T cell immunity remains problematic even with double UCB transplantation (dUCBT). This is related to the small T cell numbers of the UCB graft, the naïve nature of these T cells and often the use of ATG as part of the conditioning regimen. Consequently, UCBT recipients are susceptible to viral infections that require intact T cell immunity for their containment. Epstein Barr virus (EBV) is a gammaherpes virus that is transmitted orally. EBV initially replicates in a lytic cycle in the epithelium of the oropharynx and then establishes a latent state in memory B cells. Reactivation of EBV from latent to lytic cycle is mediated by physiologic stress of the memory B cell pool and by defects in antiviral T cell immunity, and can cause aggressive lymphomas known as post transplant lymphoproliferative disorder (PTLD) in immunocompromised hosts. The specific immunological correlates of EBV reactivation in adult UCBT recipients have not been examined. In the present study we attempted to distinguish immune reconstitution profiles in UCBT recipients who developed EBV viremia from those who did not, in order to better understand the immune profile of UCBT recipients who control gammaherpes virus reactivation. Thirty-one patients with hematologic malignancies received dUCBT with melphalan, fludarabine, ATG conditioning and tacrolimus plus sirolimus for GvHD prophylaxis. EBV viral load was determined on a weekly basis after dUCBT. Immunophenotype of peripheral blood lymphocytes, serum cytokine levels and T cell receptor excision circle analysis (TREC) values, were examined prior to and at 1, 2, 3, 6 and 12 months after UCBT and were compared between EBV viremic and non-viremic patients using the Wilcoxon-rank sum test. During the first 12 months after dUCBT, 14 of 31 (45%) patients developed EBV viremia and four (13%) developed PTLD. At one month after dUCBT, patients who developed EBV viremia displayed higher numbers of CD19+ B cell numbers (p=0.04) and CD4+CD25+ T regulatory cells (p=0.03) compared with patients who never became viremic. Surprisingly, development of EBV viremia correlated with increased numbers of CD3+ (p=0.04), CD4+ (p=0.015) and CD8+ (p=0.021) T cells. This finding was counterintuitive, as one might expect better quantitative peripheral T cell reconstitution in patients with recovering immunity and no EBV reactivation. One mechanism of impaired immune reconstitution after UCBT is skewing towards a late effector memory T cell phenotype, a stage in which T cells are incapable of mounting protective immune responses. We examined whether development of EBV viremia was associated with altered naïve versus memory T cell distribution. We determined that patients who developed EBV reactivation had higher numbers of memory cell subsets (CD4+CD45RO+, p=0.0023; CD8+CD45RO+, p=0.019) at two months after dUCBT. Neither development nor control of EBV viremia correlated with TREC recovery, which occurred after the time period of EBV viremia. We were unable to identify EBV-specific T cells in any patient group due to the very low T cell numbers during the time of viremia. However, analysis of repertoire diversity by deep-sequencing on PCR-amplified CDR3 regions of the TCRb gene using the ImmunoSEQ assay showed a more diverse TCR repertoire, as determined by higher entropy (p=0.03) and lower clonality (p=0.03), among patients who did not develop EBV reactivation, compared with those who developed EBV viremia and PTLD. Assessment of serum levels of SCF (a c-kit ligand), IL-7, thrombomodulin, VEGF and angiopoetin-1 showed that patients without EBV viremia had significantly higher levels of SCF (p=0.0001) and IL-7 (p=0.05), at 1 and 2 months after dUCBT, compared with patients who developed EBV reactivation and PTLD. SCF-mediated signaling via c-kit is integral to the homing and longevity of uncommitted hematopoietic stem cells whereas IL-7 is a critical factor for survival and homeostasis of naïve T cells. Together our findings suggest that control of EBV reactivation after dUCBT might be linked to the support of naïve T cell homeostasis, which enables maintenance of a diverse TCR repertoire.▪ Disclosures:No relevant conflicts of interest to declare.